Pesticide Residues in Food: 2001 Evaluations Part 2 Toxicological (Pt. 2)

Pesticide residues in food—2001 Toxicological evaluations

Sponsored jointly by FAO and WHO with the support of the International Programme on Chemical Safety (IPCS) Joint meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group Geneva, 17-26 September 2001

The summaries and evaluations contained in this book are, in most cases, based on unpublished proprietary data submitted for the purpose of the JMPR assessment. A registration authority should not grant a registration on the basis of an evaluation unless it has first received authorization for such use from the owner who submitted the data for JMPR review or has received the data on which the summaries are based, either from the owner of the data or from a second party that has obtained permission from the owner of the data for this purpose.

WHO Library Cataloguing-in-Publication Data Joint Meeting of the FAO Panel of Experts on Pesticides Residues in Food and the Environment and the WHO Core Assessment Group (2001 : Geneva, Switzerland) Pesticide residues in food : 2001 : toxicological evaluations / Joint Meeting of the FAO Panel of Experts on Pesticides Residues in Food and the Environment and the WHO Core Assessment Group, Geneva, 17-26 September 2001.

* 2.No-observed-adverse-effect level S.Food contamination 1.Pesticide residues - toxicity I.FAO Panel of Experts on Pesticide Residues in Food and the Environment II.WHO Core Assessment Group on Pesticide Residues ISBN 92 4 166517 3

(NLM Classification: WA 240)

This report contains the collective views of two international groups of experts and does not necessarily representthe decisions northe stated policy of the Food and Agriculture Organization of the United Nations or the World Health Organization. The preparatory work for the toxicological evaluations of pesticide residues carried out by the WHO Expert Group on Pesticide Residues for consideration by the FAO/WHO Joint Meeting on Pesticide Residues in Food and the Environment is actively supported by the International Programme on Chemical Safety (IPCS) within the framework of the Inter-Organization Programme for the Sound Management of Chemicals. The International Programme on Chemical Safety (IPCS), established in 1980, is a joint venture of the United Nations Environment Programme (UNEP), the International Labour Organisation (ILO), and the World Health Organization (WHO). The overall objectives of the IPCS are to establish the scientific basis for assessing the risk to human health and the environment from exposure to chemicals, through international peer-review processes as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemicals. The Inter-Organization Programme for the Sound Management of Chemicals (IOMC) was established in 1995 by UNEP, ILO, the Food and Agriculture Organization of the United Nations, WHO, the United Nations Industrial Development Organization, and the Organisation for Economic Co-operation and Development (Participating Organizations), following recommendations made by the 1992 United Nations Conference on the Environmfent and Development to strengthen cooperation and increase coordination in the field of chemical safety. The purpose of the IOMC is to promote coordination of the policies and activities pursued by the Participating Organizations, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment. IPCS gratefully acknowledges the assistance of Mrs E. Heseltine, St Leon-sur-Vezere, France, for editing these monographs. The World Health Organization welcomes requests for permission to reproduce or translate its publications, in part or in full. Applications and enquiries should be addressed to the Office of Publications, World Health Organization, Geneva, Switzerland, which will be glad to provide the latest information on any changes made to the text, plans for new editions, and reprints and translations already available. © World Health Organization 2002 Publications of the World Health Organization enjoy copyright protection in accordance with the provisions of Protocol 2 of the Universal Copyright Convention. All rights reserved. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Secretariat of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, nor concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. Printed in Malta

TABLE OF CONTENTS Page List of participants Abbreviations

v vii

Introduction

viii

Toxicological evaluations Carbaryl (addendum) Diazinon (addendum) Diflubenzuron Fenpropimorph (addendum) Imazalil (addendum) Imidacloprid* Methomyl (addendum) Methoprene and S-methoprene Phosalone (addendum) Prochloraz Spinosad*

3 31 37 61 65 79 101 111 135 141 183

Annex 1. Reports and other documents resulting from previous Joint Meetings of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and WHO Expert Groups on Pesticide Residues

229

* First full evaluation

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2001 Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group Rome, 20-29 September 1999 PARTICIPANTS Toxicological Core Assessment Group1 Professor A.R. Boobis, Section on Clinical Pharmacology, Division of Medicine, Imperial College School of Medicine, London, United Kingdom (Rapporteur) Professor B.-H. Chen, School of Public Health, Shanghai Medical University, Shanghai, China Dr A. Moretto, Dipartmento Medicina Ambientale e Sanita Pubblica, Universita di Padova, Padova, Italy (Chairman) Dr B.G. Priestly, Scientific Director, Chemicals and Non-prescription Medicines Branch, Therapeutic Goods Administration, Commonwealth Department of Health and Family Services, Woden, ACT, Australia FAO Panel of Experts on Pesticide Residues in Food and the Environment Dr U. Banasiak, Federal Biological Research Centre for Agriculture and Forestry, Kleinmachnow, Germany Dr E.Dutra Caldas, Central Laboratory of Public Health, Brasilia, Brazil (Rapporteur) Dr S. Funk, Health Effects Division, Environmental Protection Agency, Washington DC, USA Mr D. J. Hamilton, Animal and Plant Health Service, Department of Primary Industries, Brisbane, Queensland, Australia (Vice-Chairman) Dr B.C. Ossendorp, Centre for Substances and Risk Assessment, National Institute of Public Health and the Environment, Bilthoven, The Netherlands Secretariat Dr M.C. Alonzo Romanelli, Departamento de Salud Ambiental y Seguridad Quimica, Ministerio de Salud Publica, Montevideo, Uruguay (WHO Temporary Adviser) Dr A. Bartholomaeus, Therapeutic Goods Administration, Commonwealth Department of Health and Aged Care, Woden, ACT, Australia (WHO Temporary Adviser) Dr I.C. Dewhurst, Pesticides Safety Directorate, Ministry of Agriculture, Fisheries and Food, York, United Kingdom (WHO Temporary Adviser) Dr W.H. van Eck, Division of Public Health, Ministry of Health, Welfare and Sport, The Hague, Netherlands (Chairman, Codex Committee on Pesticide Residues) Dr H. Galal-Gorchev, Environmental Health Inc., Chevy Chase, MD, USA (WHO Temporary Adviser)

1

Unable to attend: Professor J.F. Borzelleca, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Medical College of Virginia, Richmond, VA, USA; Dr V.L. Dellarco, Office of Pesticide programs, Health Effects Division, Environmental protection Agency, Washington DC, USA; Dr H. Hakansson, Division of Risk Assessment and Organohalogen Pollutants, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden

-V-

Dr J.L. Herrman, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (WHO Joint Secretary) Mrs E. Heseltine, Communication in Science, St Leon-sur-Vezere, France (WHO Editor) Mrs P. van Hoeven-Arentzen, Centre for Substances and Risk Assessment, National Institute of Public Health and the Environment, Bilthoven, The Netherlands (WHO Temporary Adviser) Mrs F. Jallow-NDoye, National Environment Agency, Banjul, The Gambia (WHO Temporary Adviser) Dr D. MacLachlan, Australian Quarantine and Inspection Service, Department of Agriculture, Forestry and Fisheries, Kingston, ACT, Australia Dr T.C. Marrs, Food Standards Agency, London, United Kingdom (WHO Temporary Adviser) Dr J. Maskeliunas, Joint FAO/WHO Food Standards Programme, Food and Nutrition Division, FAO, Rome, Italy (FAO Food Standards Officer) Dr G. Moy, Food Safety Programme, World Health Organization, Geneva, Switzerland Dr S. Page, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland Dr R. Solecki, Pesticides and Biocides Division, Federal Institute for Health Protection of Consumers and Veterinary Medicine, Berlin, Germany (WHO Temporary Adviser) Dr A. Takagi, Department of Toxicology, National Institutes of Health Sciences, Tokyo, Japan (WHO Temporary Adviser) Dr A.W. Tejada, Pesticide Management Group, Plant Protection Service, Plant Production and Protection Division, FAO, Rome, Italy (FAO Joint Secretary) Dr G. Vaagt, Pesticide Management Group, Plant Protection Service, Plant Production and Protection Division, FAO, Rome, Italy Dr C. Vleminckx, Toxicology Division, Scientific Institute of Public Health, Ministry of Social Affairs, Public Health and Environment, Brissels, Belgium (WHO Temporary Adviser) Dr Y. Yamada, Research Planning and Coordination Division, National Food Research Institute, Tsukuba, Japan

- vi-

Abbreviations used ADI AUC BrdU bw CI Cmax DMSO F F0 F1 p2 FIFRA FOB GLP GST GST-P HPLC IARC IPCS JMPR LC50 LD50 LOAEL M M1 M2 MTD NA NOAEL NOAEC ND NR OECD PCNA PEG ppm QA RfD S9 SMR T3 T4 TSH UGT WAK 3839 w/v

acceptable daily intake area under the curve 5-bromo-2'-deoxyuridine body weight confidence interval maximal concentration dimethyl sulfoxide female parental generation first filial generation second filial generation Federal Insecticide, Fungicide and Rodenticide Act (United States) functional observational battery good laboratory practice glutathione S-transferase glutathione S-transferase, placental form high-performance liquid chromatography International Agency for Research on Cancer International Programme on Chemical Safety Joint FAO/WHO Meeting on Pesticide Residues median lethal concentration median lethal dose lowest-observed-adverse-effect level male N-propyl-N-2-(2,4,6-trichlorophenoxy)ethylurea N-formyl-N'-propyl-N'-2-(2,4,6-trichlorophenoxy)ethylurea maximum tolerated dose not analysed no-observed-adverse-effect level no-observed-adverse-effect concentration not determined not reported Organisation for Economic Co-operation and Develeopment proliferating cell nuclear antigen polyethylene glycol parts per million quality assurance reference dose 9000 x g supernatant fraction of rodent liver standardized mortality ratio triiodothyronine thyroxine thyroid-stimulating hormone UDP-glucuronidyl transferase 1 -(6-chloro-3-pyridylmethyl)-7V-nitroso(imidazolidin-2-ylidene)amine weight per volume

- vii -

Introduction The toxicological monographs and monograph addenda contained in this volume were prepared by a WHO Core Assessment Group that met with the FAO Panel of Experts on Pesticide Residues in Food and the Environment in a Joint Meeting on Pesticide Residues (JMPR) in Geneva, Switzerland, on 17-26 September 2001. Two of the substances evaluated by the Core Assessment Group at this Meeting, imidacloprid and spinosad, were evaluated for the first time. The other nine substances had been evaluated at earlier meetings. For six of these, carbaryl, diazinon, fenpropimorph, imazalil, methomyl and phosalone, only information received since the previous evaluations is summarized, in 'monograph addenda'. Of these, diazinon, methomyl and phosalone were evaluated only for establishment of an acute reference dose. The appropriate earlier documents on the six compounds should be consulted in order to obtain full toxicological profiles of these chemicals. Toxicological monographs were prepared on diflubenzuron, methoprene and S-methoprene and prochloraz, summarizing new data and, where relevant, incorporating information from previous monographs and addenda. Reports and other documents resulting from previous Joint Meetings on Pesticide Residues are listed in Annex 1. The report of the Joint Meeting has been published by the FAO as FAO Plant Production and Protection Paper 167. That report contains comments on the compounds considered, acceptable daily intakes established by the WHO Core Assessment Group, and maximum residue limits established by the FAO Panel of Experts. Monographs on residues prepared by the FAO Panel of Experts are published as a companion volume, as Evaluations 2001, Part I, Residues, in the FAO Plant Production and Protection Paper series. The toxicological monographs and addenda contained in this volume are based on working papers that were prepared by temporary advisers before the 2001 Joint Meeting. A special acknowledgement is made to those advisers. The monographs were edited by Mrs E. Heseltine, St Leon-sur-Vezere, France. The preparation and editing of this volume were made possible by the technical and financial contributions of the lead institutions of the International Programme on Chemical Safety (IPCS), which supports the activities of the JMPR. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Central Unit of the IPCS concerning the legal status of any country, territory, city or area or of its authorities, nor concerning the delimitation of its frontiers or boundaries. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by the IPCS in preference to others of a similar nature that are not mentioned. Any comments or new information on the biology or toxicology of the compounds included in this volume should be addressed to: Joint WHO Secretary of the Joint FAO/ WHO Meeting on Pesticide Residues, International Programme on Chemical Safety, World Health Organization, Avenue Appia, 1211 Geneva 27, Switzerland.

- viii -

TOXICOLOGICAL MONOGRAPHS AND MONOGRAPH ADDENDA

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3

CARBARYL (addendum) First draft prepared by M.E. van Apeldoorn, G. Wolterink, E. Turkstra, M. T.M. van Raaij, P.H. van Hoeven-Arentzen and J. G.M. van Engelen Centre For Substances and Risk Assessment National Institute of Public Health and the Environment, Bilthoven, The Netherlands Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution and excretion Biotransformation Effects on enzymes and other biochemical parameters Toxicological studies Short-term studies of toxicity Long-term studies of toxicity and carcinogenicity Reproductive toxicity Multigeneration studies Developmental toxicity Special studies: Neurotoxicity Observations in humans Comments Toxicological evaluation References

3 4 4 4 5 7 8 8 9 14 14 16 17 22 22 24 27

Explanation Carbaryl was evaluated for toxicological effects by the Joint Meeting in 1963,1965,1966, 1967,1969,1973 and 1996 (Annex preferences 2,3,6,8,12,20 and 7 7). An ADI of 0-0.02 mg/kg bw was established in 1963 on the basis of a 1-year study in dogs, and this ADI was confirmed in 1965, 1966 and 1967. In 1969, a temporary ADI of 0-0.01 mg/kg bw was established, which incorporated an extra safety factor because of concern about effects on the male reproductive system in a 1-year study in rats treated by gavage, with a NOAEL of 2 mg/kg bw per day, and because a dose of 0.12 mg/kg bw per day may have affected renal function in a 6-week study in volunteers. In 1973, the Meeting established an ADI of 0-0.01 mg/kg bw. In 1996, carbaryl was reviewed as part of the periodic review programme of the Codex Committee on Pesticide Residues, and the Meeting established an ADI of 0-0.003 mg/kg bw on the basis of a LOAEL of 15 mg/kg bw per day in a study of carcinogenicity in mice and a safety factor of 5000, which included an extra safety factor of 50 to account for the presence of vascular tumours in male mice at all doses tested. The Meeting stated that the resulting ADI provided an adequate margin of safety, taking into account the LOAEL (3.1 mg/kg bw per day for maternal toxicity) in a study of developmental toxicity in dogs and uncertainties about effects on the male reproductive system. The following information was available to the present Meeting: new studies on metabolism in mice and rats; a 14-day study of effects on some enzyme activities in rats; a 6-month study in p53 knockout mice; a (re-)evaluation of the incidence of bladder tumours in the 2-year study of

CARBARYL 3-29 JMPR 2001

4

toxicity and carcinogenicity in rats; a (re-)evaluation of all slides from the 2-year study of carcinogenicity in mice and the 2-year study of toxicity and carcinogenicity in rats; and an extended and updated epidemiological study. Furthermore, additional studies were available on the neurotoxicity, developmental toxicity, and reproductive toxicity of carbaryl.

Evaluation for acceptable daily intake 1.

Biochemical aspects (a)

Absorption, distribution and excretion Mice

A study was conducted to investigate the possible reasons for the increased incidence of tumours seen at high doses of carbaryl during the final year of a study in CD-I mice. In this study, groups of 10 male CD-1 mice, 4 weeks of age, received a diet containing carbaryl at a concentration of 0, 11, 110, 1100 or 7980 ppm, equivalent to 0, 1.5, 16, 160 and 1100 mg/kg bw per day, for 14 days, followed by a single oral dose of 50 mg/kg bw [14C- naphthalene ringjcarbaryl (radiochemical purity, 100%) by gavage on day 15. Body weights were measured on the day before the first dietary administration and on days 1, 8 and 14 thereafter. After dosing with [14C]carbaryl, urine, faeces and cage washes were collected at 24-h intervals and analysed for radiolabel content. Food consumption was assessed weekly. All animals were killed by exsanguination 168 h after administration of [14C]carbaryl, and blood and carcass samples were analysed for radiolabel. Statements of adherence to good laboratpry practice (GLP) and quality assurance (QA) were included. The radiolabel was eliminated mainly in urine (73-84%, assuming that the radiolabel found in cage washes had also been excreted in urine) and to a lesser extent in the faeces (12-19%). By 168 h after dosing, very small amounts (< 0.8%) of radiolabel were found in the carcass and blood (Table 1) (Valles, 1999). The Meeting noted that much more radiolabel was excreted in the faeces during the first 24 h after treatment by mice given the diet containing carbaryl than by controls that received only the single oral dose of [14C] carbaryl, but the significance of this finding, e.g. decreased absorption or increased excretion via the bile, was not discussed by the study author. Furthermore, the Meeting noted that it can be calculated from data in appendices to the report that the animals at the highest dietary concentration gained almost no weight over the 14 days of treatment, in contrast to the animals at the other doses. Table 1. Cumulative recovery of radiolabel (% dose administered) after a single oral dose of [14C]carbaryl to mice Dose (mg/kg bw)

Time after administration of carbaryl (h)

0

11

110

1100

8000

Urine

0-24 0-48 0-168

58 66 69

45 53 59

55 63 65

55 66 69

58 67 69

Faeces

0-24 0-48 0-168

1.2 11 12

9.8 14 15

16 17 18

15 16 16

17 18 19

Sample

Blood3

168

0.01

0.01

0.01

0.01

0.01

Carcass

168

0.45

0.50

0.82

0.34

0.24

Cage wash

168

3

Total recovery

15

17

16

15

10

96

89

100

101

98

From Valles (1999) Converted by the Meeting from micrograms of equivalents per gram

a

CARBARYL 3-29 JMPR 2001

5

Rats A study was conducted to investigate the role of the metabolism of carbaryl in the causation of tumours in rats in a 2-year study, by qualitative and quantitative comparisons of the metabolic profiles in older rats after a single oral dose or short-term dietary administration of carbaryl. Five male Sprague-Dawley rats aged about 15 months and fed a normal diet received [14C-naphthalene ring]carbaryl at a single dose of 50 mg/kg bw by gavage. Their excreta were collected daily for 168 h after dosing; then, the animals were killed, and tissue samples were collected for determination of radiolabel content. Statements of adherence to GLP and QA were included. Most of the radiolabel was excreted in the urine (86%, assuming that the radiolabel in the cage wash had also been excreted in urine), with 11% in the faeces. Most of the radiolabel was excreted during the first 48 h after treatment. By 168 h, little residue was found in tissues, and 4.3% was present in the skin and fur (see Table 2), probably as a result of contamination of the fur with urine (Totis, 1997). Groups of male Sprague-Dawley rats aged 15 months received a diet containing unlabelled carbaryl at a nominal concentration of 0, 250, 1500 or 7500 ppm, equivalent to 0, 12, 75 and 380 mg/kg bw per day, for 83 days. At the end of this period, five animals per dose received [14Cnaphthalene ring]carbaryl at 2 mg/kg bw per day by gavage for 7 days, and their excreta were collected daily up to 72 h after the last dose. The liver, kidneys, thyroid, urinary bladder and skin with fur were removed from each animal after they were killed by exsanguination and retained for determination of radiolabel content with the residual carcass. Most the radiolabel was found in the urine (85-93%, assuming that radiolabel found in the cage wash had also been excreted in urine), with 7.4-10% in the faeces; < 1% was found in the tissues (see Table 3) (Totis, 1997). (b)

Biotransformation Mice

In the study with CD-I male mice (Valles, 1999), urine samples collected over 0-24, 2448 and 48-96 h were analysed for metabolites. Out of a total of 20 metabolites, four were present in relatively large quantities in the urine (see Table 4). Mice given the diet containing carbaryl at Table 2. Distribution and excretion of radiolabel (% of total administered dose) after a single oral dose of [14C]carbaryl at 50 mg/kg bw to rats Sample

Urine Faeces Cage wash Tissues Skin and fur

Time after dosing (h) 6

24

48

72

96

120

144

27 0.3

50 0.3

63 4.7

67 8.0

68 10

69 11

69 11

168 69 11 17 0.4

4.3

From Totis (1997)

TableS. Distribution and excretion of radiolabel (% of total administered dose) 72 h after 7 daily oral doses of [14C]carbaryl at 2 mg/kg bw to rats Sample

Urine + cage wash Faeces Tissues

Dose (mg/kg of diet)

0

250

1500

7500

92 10

92 7.4

93 9.9

85 10

0.4

0.4

0.4

0.8

From Totis (1997)

CARBARYL 3-29 JMPR 2001

6 Table 4. Metabolites of carbaryl found in mouse urine 0-96 h after treatment (% of administered dose) Metabolite

Dihydro, dihydroxynaphthyl sulfate Hydroxy-carbaryl glucuronide oc-Naphthyl sulfate cc-Naphthyl p-D-glucuronide

Dose (mg/kg of diet)

0

11

110

1100

8000

3.2 14 14 17

3.0 12 11 11

3.7 15 11 13

3.8 14 12. 20

6.8 19 12 20

From Valles (1999). The metabolites were identified by liquid chromatography with mass spectrometry.

8000 ppm had more dihydro, dihydroxynaphthyl sulfate and hydroxy-carbaryl glucuronide metabolites, which may have been formed via epoxidation and conjugation. The Meeting noted that the study author claimed that 21 metabolites of carbaryl were formed. However, one of these metabolites was not detected in any of the samples. Rats In the study with 15-month-old male Sprague-Dawley rats (Totis, 1997), the urine and faecal samples collected from five rats on days 1,2,3,4, 5,6 and 7 were pooled and analysed for metabolites. In the same study, additional groups of 10 male rats received the same treatment (unlabelled carbaryl in the diet at 0,250,1500 or 7500 ppm for 83 days and then [14C]carbaryl at 2 mg/kg bw per day by gavage for 7 days), but were killed by exsanguination 6 h after the last dose, and liver, kidney, spleen, urinary bladder and thyroid were collected for determination of metabolites. As very low concentrations of radiolabel were found in the tissues, the metabolites could be identified only qualitatively. A glucuronide conjugate of naphthol found in kidney and urinary bladder and a sulfonate conjugate of naphthol found in plasma, kidney and urinary bladder were the two main metabolites in tissues. Investigation of the metabolism of carbaryl in urine and faecal extracts revealed the presence of up to 23 radiolabelled components in the urine and up to 20 in the faeces. Overall, the radioalabel in the faeces represented < 1.5-1.7% of the total administered dose in the controls and rats at the low and high doses and < 3.6% in those at the intermediate dose. One of the main analytes was the parent compound. In the urine, most of the recovered radiolabel w,as associated with three metabolites (Table 5). In the control group, the relative quantities of the three main metabolites remained stable over the 7 days of measurement. In the group at 250 ppm, the concentration of the glucuronide of dihydro, dihydroxy-l-naphmyl-W-methylcarbamate tended to increase over the 7 days and those of the sulfonate conjugate of naphthol to decrease as compared with controls, especially on days Table 5. Radiolabelled components in the urine of rats (% daily administered dose) given [14C] carbaryl Metabolite

Glucuronide of dihydro, dihydroxy-l-naphthyl-A'-methylcarbamate

Dose (mg/kg of diet)

0 250 1500 7500

a-Naphthyl P-D-glucuronide

0 250 1500 7500

Sulfonate conjugate of naphthol

0 250 1500 7500

From Totis (1997) ND, not detected

CARBARYL 3-29 JMPR 2001

Day after treatment

1

2

3

4

5

6

7

15 16 21 28 16 16 14 15 24 27 23 12

19 16 20 20 15 15 11 12 23 23 25 18

16 15 19 17 15 15 19 13 26 21 30 13

18 15 21 19 14 15 16 15 22 9 30 ND

16 20 26 21 13 22 14 17 19 ND 30 ND

17 23 27 18 15 22 12 15 24 ND 27 ND

18 23 19 10 15 17 10 12 25 15 25 9

7

4,5 and 6. In the group at 1500 ppm, the concentrations of both the glucuronide and the sulfonate were slightly higher than in the control group on most days. In the group at 7500 ppm, the concentration of the sulfonate conjugate was lower than that in controls, especially on days 4, 5 and 6. Typically, in the groups at 250 and 7500 ppm, the concentrations of the sulfonate conjugate of naphthol were below the limit of detection on days 5 and 6 but were found in relatively large amounts on day 7. It is notable that the metabolite profile of rats at 250 and 7500 ppm was different from that of controls, while that of rats at 1500 ppm was similar. In all groups, the concentrations of a-naphthyl (3-D-glucuronide remained more or less stable. On the basis of the data for day 1, the study author suggested that the metabolism of carbaryl shifted from formation of the sulfonate conjugate of naphthol to the glucuronide of dihydro, dihydroxy-1-naphthyl-W-methylcarbamate after 83 days of pretreatment with carbaryl (Totis, 1997). The Meeting was not convinced. Overall, the quantitative changes in metabolic profiles observed were variable, and the data for rats at 1500 ppm clearly do not support the conclusion of the author. Furthermore, the increased concentration of the glucuronide was observed only on day 1, although that of the sulfonate conjugate remained lower in rats at 250 and 7500 ppm throughout the 7-day period. (c)

Effects on enzymes and other biochemical parameters Rats

A study was conducted to determine whether the induction of hepatic enzymes might account for the formation of tumours in the liver and thyroid of rats at the highest dose (375 mg/kg bw per day) in the long-term study of toxicity and carcinogenicity. In this 14-day study, groups of 10 male and 10 female Sprague-Dawley rats aged about 9 weeks (body weights, 320-350 g for males and 220-240 g for females) received carbaryl (purity, 98.4%) by gavage at a dose of 0,10 or 40 mg/kg bw per day in 0.5% carboxymethylcellulose and 0.1% Tween 80. Five animals of each sex per group were killed on day 4 and the remaining animals on day 15 after an overnight fast. All groups were checked daily for deaths, morbidity and clinical signs; they were weighed on days -1,1,7 and 14, and their food consumption was determined on days 7 and 14. All animals killed on days 4 and 15 were examined macroscopically, and their livers were weighed. Hepatocellular proliferation was measured by proliferating cell nuclear antigen (PCNA) staining, and the livers were examined histologically on days 4 and 15. Additionally, induction of liver enzymes (cytochrome P450s) and triodothyronine and thyroxine UDP-glucuronidyl transferase (UGT) activities were assessed in the livers of animals killed on day 15. Statements of adherence to GLP and QA were included. No deaths were observed. Most of the animals at 40 mg/kg bw per day showed reduced motor activity throughout the study, and tremors, staggering, increased salivation, piloerection, soft faeces and polypnoea were seen sporadically. Males at 40 mg/kg bw per day weighed significantly less than controls on days 7 and 15 (-8.4 and-7.5%, respectively) and consumed less food during the first week. The liver weights were unchanged by treatment, and no macroscopic or microscopic changes were observed. The total cytochrome P450 content was not changed by carbaryl treatment, and the activities of the cytochrome P450 enzymes benzoxyresorufin Odebenzylase and pentoxyresorufin O-depenrylase and of lauric acid hydroxylase were not affected. However, the activity of ethoxyresorufm O-deethylase was increased significantly (5.5 times) in males at 40 mg/kg bw per day, and those of thyroxine-UGT and triiodothyronineUGT were increased significantly in males at 40 mg/kg bw per day and in females at 10 and 40 mg/kg bw per day, with a 1.5-fold increase in thyroxine-UGT activity and a 1.8-fold increase in triiodothyronine-UGT activity in both males and females. A moderate increase in cells in GI phase was observed in males at 40 mg/kg bw per day on day 4 and and an increase in cells in Gj

CARBARYL 3-29 JMPR 2001

8

and S phases on day 15. In females, an increase in cells in G1 and S phases was observed at 10 and 40 mg/kg bw per day on day 15. In view of the increased UGT activities and the increase in cell cycling at 10 and 40 mg/kg bw per day, no NOAEL could be identified (Berthe, 1997). 2.

Toxicological studies (a)

Short-term studies oftoxicity Rats

In the study of Totis (1997), groups of five male Sprague-Dawley rats, about 15 months old, received a diet containing carbaryl at a concentration of 0,250,1500 or 7500 ppm, equivalent to 0,12,75 and 380 mg/kg bw per day, for 90 days. During treatment, the groups were observed for deaths, morbidity (twice a day on week days, once a day at weekends), body weight and food consumption (weekly). One day after treatment, the animals were killed and the liver, kidneys and thyroid were weighed and examined histologically. Liver tissue was also examined biochemically. Statements of adherence to GLP and QA were included. Decreased growth was seen for animals at 1500 and 7500 ppm. Carbaryl induced follicularcell hypertrophy of the thyroid at concentrations > 250 ppm, and pericholangitis, centrilobular hypertrophy of the liver and transitional-cell hyperplasia of the renal pelvis at 7500 ppm (Table 6). Dogs Groups of six beagles of each sex, about 6 months old, were fed diets containing technicalgrade carbaryl (purity, 99.3%) at a concentration of 0,20,45 or 125 ppm (equal to 0,0.6,1.4 and 3.8 mg/kg bw per day for males and 0,0.6,1.5 and 4.1 mg/kg bw per day for females) for 5 weeks. The groups were observed daily throughout treatment for deaths and clinical signs. Body weights and food consumption were recorded weekly, and ophthalmological examinations were performed before and during treatment. Plasma and erythrocyte cholinesterase activity was measured in each dog before treatment and on days 14 and 32, about 2 h after feeding. Complete gross necropsies Table 6. Results of 90-day dietary study in 15-month-old male Sprague-Dawley rats Effect

Dose (mg/kg of diet)

250

0 Mortality rate Body weight (g) Food consumption Dermal reactions Biochemistry in liver tissue Protein (mg/g liver) Glutathione (jamol/g liver) Glutathione (nmol/g protein) GST (^mol/min per g liver) GST (|j,mol/min per g protein) Glutathione peroxidase (lU/g liver) Glutathione peroxidase (IU/g protein) Organ weight (absolute, in g) Liver Spleen Thyroid Histopathologyc Liver Centrilobular hypertrophy Pericholangitis Thyroid Follicular-cell hypertrophy Kidney Transitional-cell hyperplasia From Totis (1997). GST, glutathione 5-transferase Significantly increased b Significantly decreased c Number of animals, out of five, displaying effect a

CARBARYL 3-29 JMPR 2001

No lexicologically relevant effect

820

78

1500

7500

690a

650a

No toxicologically relevant effect No toxicologically relevant effect

100 4.7 47 72 720 220 2100

120 5.8 50 64

540a

220 1900

18 1.2

18 1.1

77a 3.6"

48a 49 640 240

3100a

17 1.4

0.031

0.041

0.052a

0 0

0 0

0 0

0 0

'

89

8.3b

94b 71 810 160 1900

22a 1.6

0.059a

5 3

3

5

5

0

0

2

9

were performed, and brain samples were taken for determination of acetylcholinesterase activity. Statements of adherence to GLP and QA were included. The only finding was inhibition of plasma cholinesterase activity on day 14 in males at 20 and 125 ppm, which was considered incidental and of no toxicological relevance. The NOAEL was 125 ppm, equal to 3.8 mg/kg bw per day, the highest dose tested. (Hamada, 1991). (b)

Long-term studies oftoxicity Mice

A 2-year study with carbaryl in CD-1 mice (Hamada, 1993a) was summarized by the JMPR in 1996 (Annex 1, reference 79). The authors concluded that the lowest concentration tested (100 ppm, equal to 15 mg/kg bw per day) was the LOAEL, on the basis of an increased incidence of vascular tumours in males. The increased incidence was found only for mice killed at the end of the study or which died off schedule; the tumours were located predominantly in the liver and spleen and to a lesser degree in other tissues. An increased incidence of haemangioma and haemangiosarcoma was also seen in females at 8000 ppm (Table 7). Furthermore, at 8000 mgkg of diet, the incidences of renal tubule-cell adenomas and carcinomas were increased in males and the incidences of hepatocellular adenomas and carcinomas were increased in females. The NOAEL for non-neoplastic lesions was 100 ppm, equal to 15 mg/kg bw per day, on the basis of inhibition of erythrocyte and brain acetylcholinesterase activity and histopathological changes in the bladder. As the initial histological examination of tissues of groups of 10 mice of each sex per group at the interim kill at week 53 did not reveal any changes associated with the tumours found at the terminal kill, the slides of the livers and kidneys of controls and mice at the highest dose were reevaluated. Again, no microscopic changes that could be associated with the tumours were seen after 104 weeks (Debruyne & Irisarri, 1996). Subsequently, an independent panel of pathologists (Hardisty, 1996a) re-evaluated all microscopic tissue sections containing proliferative vascular lesions to confirm the accuracy of the original diagnoses (according to the guidelines of the Pathology Workgroup of the USA's Environmental Protection Agency). The slides for 10% of controls and male and female mice at the highest dietary concentration were also re-evaluated to ensure that all proliferative lesions had been identified. Furthermore, all sections of liver and kidneys from all mice in all groups were reexamined to confirm the presence or absence of proliferative changes. The re-evaluation generally confirmed the original evaluation of the study pathologist for the kidneys of male mice and the livers of female mice, as did the re-evaluation of vascular lesions. The few differences between the original diagnosis and that at re-evaluation concerned the classification of vascular neoplasms as benign (haemangioma) and malignant (haemangiosarcoma). The incidences of vascular neoplasms as observed by the panel are shown in Table 8. The re-evaluation did not reveal a significant positive trend or an increased incidence of haemangioma in male mice; the incidence of haemangiosarcoma in male mice at all dietary concentrations was increased significantly, but there was no significant positive trend and the Table 7. Incidences ofhaemangiomas and haemangiosarcomas in various tissues of CD-I mice, by concentration of carbaryl in the diet (mg/kg) Vascular tumours

Total no. of tumours No. of tumour-bearing animals

Males

Females

0

100

1000

8000

2/70 2/70

9/70 6/70

13/70 18/70 10/70 10/70

0

100

1000

8000

5/70 3/70

6/70 3/70

5/70 4/70

10/70 9/70

From Hamada (1993a)

CARBARYL 3-29 JMPR 2001

10 Table 8. Incidences of haemangioma and haemangiosarcoma in CD-I mice treated with carbaryl, by concentration in the diet (mg/kg), in the study ofHamada (1993a), as observed by an independent panel of pathologists Vascular tumours

Males 0

Females 100

1000

8000

0

100

1000

8000

Haemangioma (no. of tumours) Single site (no. of tumour-bearing animals) Multiple sites (no. of tumour-bearing animals)

1/70 1/70 0/70

1/70 1/70 0/70

2/70 2/70 0/70

2/70 2/70 0/70

2/70 2/70 0/70

1/70 1/70 0/70

1/70 1/70 0/70

0/70 0/70 0/70

Haemangiosarcoma (no. of tumours) Single site (no. of tumour-bearing animals) Multiple sites (no. of tumour-bearing animals)

1/70 1/70 0/70

9/70 5/70 1/70

11/70 6/70 2/70

16/70 6/70 2/70

4/70 0/70 2/70

6/70 3/70 1/70

4/70 2/70 1/70

10/70 8/70 1/70

No. of animals bearing both haemangioma and haemangiosarcoma

0/70

1/70

0/70

0/70

0/70

1/70

0/70

0/70

Total no. of vascular neoplasms

2/70

10/70

13/70

18/70

6/70

7/70

5/70

10/70

No. of vascular tumour-bearing animals

2/70

7/70

10/70

10/70

4/70

4/70

4/70

9/70

FromHardisty(1996a)

combined incidence of haemangioma and haemangiosarcoma in male mice was significantly increased only at the two higher concentrations. The incidence of haemangiosarcoma in female mice was significantly increased at the highest concentration, and the trend was statistically significant even at lower concentrations, at which the incidences were similar to those of controls owing to a 'threshold-like' behaviour (Hardisty, 1996a). The Meeting noted that Hardisty conducted statistical analyses of vascular rumour incidences (number of tumour-bearing animals) in a total of 80 mice, whereas the total should have been 70 mice, i.e. those killed at the end of the study plus unscheduled deaths. The 10 mice that were killed after 53 weeks should not have been included. The vascular tumour incidences found in the 2-year study in male CD-I mice at 100 and 1000 ppm were compared with the incidences in controls in previous studies (Klonne, 1995; reported in Annex 1, reference 79). The incidences for males at 8000 ppm were not analysed by Klonne because that concentration exceeded the maximum tolerated dose (MTD). The vascular tumour incidences in liver and spleen were compared with the tumour incidences in the same organs in several databases, because vascular tumours in other organs are rare and are generally not listed or routinely evaluated microscopically. The Meeting noted that, in the paper of Klonne (1995), the percentages of tumours in mice treated with carbaryl were calculated on the basis of a total of 80 animals, which also included the animals killed after 53 weeks, whereas only the unscheduled deaths and animals killed terminally should have been taken into account. In the comparisons shown below, the incidences of haemangioma, haemangiosarcoma and haemangioma and/or haemangiosarcoma were compared with those in databases of controls. Only values that exceeded the range are given. Comparison with database of Cornington Hazleton Virginia (24 studies lasting 78 weeks and one 104-week study, 1988-93): Incidences of haemangiosarcoma in liver and spleen at 1000 ppm and combined incidences of haemangioma and haemangiosarcoma in spleen at 1000 ppm in the study with carbaryl exceeded the range in these controls. Comparison with database of Cornington Hazleton Wisconsin (11 studies lasting 78,91 and 104 weeks, 1986-93 [studies could not be distinguished]): Incidences of haemangiosarcoma in liver and spleen at 100 and 1000 ppm and of haemangiosarcoma in spleen in controls in the study with carbaryl exceeded the range in these controls. The combined incidence of haemangioma and haemangiosarcoma in the liver in animals at 100 and 1000 ppm and in the spleen in animals at 1000 ppm exceeded the range in these controls.

CARBARYL 3-29 JMPR 2001

11 Comparison with database of Charles River Laboratories (unknown number of 24-month studies, 1981-91): Incidences of haemangiosarcoma in spleen at 100 and 1000 ppm exceeded the range in these controls. Haemangiomas in spleen were not listed in this database. Comparison with database of Pharmaco LSR (17 studies lasting 88-104 weeks, 1986-93): Incidences of haemangiosarcoma in spleen at 1000 ppm exceeded the range in these controls. Combined incidences of haemangioma and haemangiosarcoma were not listed. Comparison with database of Malta etal. (1988) (11 studies lasting 104 weeks, 1982-87): Incidences of haemangiosarcoma in both liver and spleen at 100 and 1000 ppm exceeded the mean percentage incidences in these controls. The combined incidences of haemangioma and haemangiosarcoma were not listed. The Meeting noted that the incidences of haemangiosarcomas in liver and spleen of animals given diets containing carbaryl at 100 ppm also exceeded those of controls in an affiliated laboratory in which the 2-year study in mice was performed. Furthermore, the incidence of haemangiosarcoma in spleen in mice at 100 ppm exceeded the range of controls in the laboratories of the animal breeders. Two important phenomena emerge: • As the incidence of spontaneous vascular tumours increased with age, a bias was introduced bycomparing data for controls in a 78-week study with those in a 104-week study . • Comparison of the data from the Charles River laboratories for 1978-85 with those for 198191 showed that the upper end of the range of incidence of vascular tumours had increased by up to four times. Therefore, control data from about the same period should be used in making comparisons. Klonne (1995) stated that the incidence of vascular tumours in males at 100 ppm fell within the range for controls in previous studies in other laboratories. The Meeting noted that, although the incidence of vascular tumours in male controls in the study with carbaryl was low, no data were available on other controls in the same laboratory in studies of the same duration of exposure. Therefore, the possibility that 100 ppm is the LOAEL for vascular tumours in males cannot be excluded. In the 2-year study with carbaryl in CD-1 mice, vascular tumours were observed in males even at the lowest concentration of 100 ppm, equal to 15 mg/kg bw per day. To better understand the carcinogenic potential of carbaryl, especially with regard to vascular rumours, a carcinogenicity study was performed with carbaryl inp53 knockout mice, in which one allele of thep53 tumour suppressor gene has been inactivated. As it is assumed that a mutation at the intactp53 allele is needed to accelerate carcinogenicity, compounds that induce tumours by non-genotoxic mechanisms should be inactive in this model. The model was evaluated with two compounds, the genotoxin urethane, which causes vascular, pulmonary and hepatocellular tumours in standard bioassays for carcinogenicity, and d-limonene, which is not genotoxic and not carcinogenic in mice but is carcinogenic in male rats by a well-described non-genotoxic mechanism. Urethane was given orally by gavage for 180 days to groups of 20 male/>53 knockout mice at a dose of 0, 1, 10 or 100 mg/kg bw per day in 0.5% methylcellulose in sterile water and 0.2% Tween 80. d-Limonene was given at one dose of 250 mg/kg bw in the same vehicle. A control group of wild-type mice was given the vehicle only. None of the controls developed tumours, and one spontaneous tumour of the prostate was seen in the group given d-limonene. On the basis of the results seen with urethane (Table 9), the authors concluded that the/?53 knockout mouse was useful for investigating mechanisms of vascular tumour formation (Bigot, 1999; Carmichael et al., 1999). The Meeting considered that the power of the model to discriminate between genotoxins and non-genotoxins is not as clear as suggested by the sponsor and that for weak genotoxic carcinogens the length of exposure of 6 months used in the protocol was too short to obtain conclusive results. Furthermore, the validation of the model was limited, because:

CARBARYL 3-29 JMPR 2001

12

Table 9. Incidences of vascular proliferative changes in p53 knockout mice treated with urethane Tissue and change

Dose (mg/kg bw per day) 0

1

10

100a

Liver Endothelial hyperplasia Haemangioma, benign Haemangiosarcoma, malignant Combined benign and malignant neoplasms Combined benign and/or malignant neoplasms

0/20 0/20 0/20 0/20 0/20

0/20 0/20 0/20 0/20 0/20

0/20 1/20 0/20 0/20 1/20

2/20 14/20 8/20 4/20 18/20

Spleen Malignant haemangiosarcoma

0/20

0/20

0/20

1/20

Heart Endothelial (haemangioma-like) proliferation Haemangioma, benign

0/20 0/20

0/0 0/0

0/20 0/20

1/20 1/20

0/0

0/0

0/0

Abdomonal cavity Haemangiosarcoma, malignant

1/1

From Bigot (1999) and Carmichael et al. (1999) * Five out of 20 animals were killed when moribund between days 139 and 177, and 12 out of 20 were found dead between day 104 and the end of the study.

• No positive control group consisting of wild-type mice exposed to urethane was included for comparison. • The compound chosen as the negative control was unfortunate, as d-limonene is carcinogenic only in the male rat kidney. • The highest dose of urethane used, 100 mg/kg bw per day, far exceeded the MTD, as indicated by the early deaths of 17/20 mice at this dose. At the intermediate dose of 10 mg/kg bw per day, only one haemangioma in the liver was observed, with no haemangiosarcomas at any site. At the lowest dose, 1 mg/kg bw per day, no vascular tumours were seen. Thus, the range of doses used may not have been well chosen. In a study with carbaryl in thep53 knockout mouse model, groups of 20 males aged 9-11 weeks (weighing 22-29 g) received a diet containing carbaryl (purity, 99%) at a concentration of 0,10,30,100,300,1000 or 4000 ppm, equal to 0,1.7,5.2,18,52,160 and 720 mg/kg bw per day (mean for weeks 1-25), for at least 180 days. All groups were observed twice daily for deaths, and morbidity and clinical signs were recorded at least once per day. Detailed physical examinations were performed at least weekly. Body weights and food consumption were determined weekly for the first 14 weeks and monthly thereafter. All animals were killed on days 181-184 and examined macroscopically. The absolute weights and the organ:body and organ:brain weight ratios of brain, heart, liver, spleen, kidneys, thymus and testes were determined. About 40 tissues from all mice at 0 and 4000 ppm and of all animals that died during the study were examined microscopically. The liver, spleen, thymus, urinary bladder and all lesions seen macroscopically in animals at the intermediate dose were examined microscopically. Adherence to GLP and QA was stated. The deaths seen during the study (one at 10 ppm, two at 30 ppm and two at 300 ppm) and the clinical signs in these animals (reduced motor activity, cold to touch and hunched posture before death) were considered by the authors not to be related to treatment. The food consumption of animals at 4000 ppm was significantly lower than that of controls during the first 8 weeks and was correlated with lower body weights. Some changes in absolute and/or relative organ weights were seen at 1000 and 4000 ppm. The only treatment-related non-proliferative change observed was the presence of intracellular globular deposits in the urinary bladder epithelium at doses > 100 ppm, with a dose-related increase in the severity of the accumulation of globular deposits. No local irritation or hypertrophy of the urinary bladder was observed. No effects were seen at 30 ppm, equal to 5.2 mg/kg bw per day.

CARBARYL 3-29 JMPR 2001

13

Only a few animals at the intermediate dose had spontaneous neoplasms. No neoplastic or preneoplastic changes were observed in the vascular tissue in any organ. The authors concluded that no tumours were induced at any dose, and, further, that these results demonstrated a lack of genotoxic potential of carbaryl (Chuzel, 1999). The Meeting noted that no tumours of the vascular system developed in this 6-month study with carbaryl, in contrast to the results of the 2-year study in CD-I mice. This finding indicates a non-genotoxic mechanism of vascular tumour induction by carbaryl in mice. Donehower (1999) stated, however, that an 8-month study would have provided better assurance of detecting subtle carcinogens. Rats Hamada (1993b; reported in Annex 1, reference 79) conducted a 2-year study in SpragueDawley rats with carbaryl. The initial report stated that neoplasms were observed only at the highest dietary concentration (7500 ppm) in the thyroid, liver and urinary bladder, and 1996 JMPR concluded that carbaryl induced tumours in rats at a dose that exceeded the MTD. The NOAEL for non-neoplastic findings was 250 ppm, equal to 10 mg/kg bw per day, on the basis of inhibition of erythrocyte and brain acetylcholinesterase activity and a decrease in mean body weight. As histological examination of animals at the interim kill revealed no changes associated with the tumours found at the terminal kill, the slides of the target organs (liver, kidney, thyroid gland and urinary bladder) of animals at the 53-week interim kill and at a 57-week kill after a 4week recovery period were re-evaluated. Microscopic changes were found in the urinary bladder (transitional epithelial hyperplasia in males and females, not reversed within 4 weeks), kidney (pelvic urothelial hyperplasia in males, reversed within 4 weeks), thyroid (thyroid follicular hypertrophy in males, reversed within 4 weeks) and liver (hepatocellular hypertrophy in males and females, reversed within 4 weeks) (Debruyne & Irisarri, 1996). Subsequently, an independent panel of pathologists (Hardisty, 1996b) re-evaluated all slides from the 53-week, 57-week and terminal kills (according to the the guidelines of the Pathology Workgroup of the USA's Environmental Protection Agency) to confirm the accuracy of the diagnoses (see Table 10). The results of the independent group confirmed the findings of Debruyne & Irisarri (1996). The mechanism by which the bladder tumours were induced was not investigated, but Cohen (1995) reviewed all the relevant data on the urinary bladder tumours found in this study and concluded that carbaryl causes urinary bladder tumours in rats by a non-genotoxic mechanism. Hyperplastic and neoplastic lesions in the urinary bladders of males and females were induced only at the highest dietary concentration of 7500 ppm, which was far in excess of the MTD. He concluded that carbaryl induces tumours by increasing cell proliferation, probably by a direct mitogenic effect of the parent compound or one or more of its metabolites. He also concluded that the effect may be specific to rats. A similar mitogenic effect on rat bladder urothelium of another aromatic carbamate, propoxur, has been studied extensively (Cohen et al., 1994). Cohen (1995) proposed that systemic toxicity could alter the metabolism and excretion of growth factors involved in bladder proliferation, such as epidermal growth factor or interleukin-6. Studies were therefore conducted with PCNA staining to determine whether cellular proliferation occurred in those organs in which tumours had developed in both rats and mice, i.e. in the liver of female rats, the thyroid gland of male rats and the urinary bladder of male rats, and in the liver and kidney of male and female mice. PCNA staining can be used to identify cells that are actively dividing even though clear increases in mitosis are not seen by conventional histology. The samples preserved at the 52-week interim kill of control animals and those at the high dose were examined. Statements of adherence to GLP and QA were included.

CARBARYL 3-29 JMPR 2001

14

Table 10. Incidences of tumours in Sprague-Dawley rats treated with carbaryl, by concentration in the diet (mg/kg), in the study of Hamada (1993b), as observed by an independent panel of pathologists Males

Time of kill

0 Hyperplasia, papilloma and carcinoma in the urinary bladder 53-week interim Transitional-cell hyperplasia 0/9 57-week recovery Transitional-cell hyperplasia 0/10 104-week terminal kill plus unscheduled deaths 8/71 Transitional-cell hyperplasia 0/71 Transitional-cell papilloma 0/71 Squamous-cell papilloma 0/71 Transitional-cell carcinoma

250

0/10

1500

7500

0/10

7/9

0

1/10

250

1500

0/10

0/10

9/10 3/10

7500

-

-

4/10

0/10

-

-

7/70 0/70 0/70 0/70

11/70 0/70 0/70 0/70

51/71 14/71 2/71 10/71

6/70 1/70 0/70 0/70

4/70 0/70 0/70 0/70

4/70 0/70 0/70 0/70

56/70 8/70 0/70 5/70

0/10

1/10

7/10

0/10

1/10

2/10

1/10

-

-

0/10

1/10

-

-

0/10

10/70 0/70

13/70 0/70

29/70 1/70

22/70 0/70

39/70 0/10

29/70 0/70

21/70 0/70

0/10

0/10

0/10

6/10

0/10

0/10

0/10

7/10

1/10

-

-

0/10

0/10

-

-

0/10

0/70 1/70 0/70

1/70 1/70 2/70

2/70 1/70 3/70

38/70 1/70 1/70

7/70 1/70 0/70

6/70 0/70 0/70

10/70 3/70 0/70

34/70 7/70 0/70

2/10

2/10

0/10

0/10

1/10

1/10

-

0/10

2/70 0/70 0/70

33/70 1/70 0/70

Epithelial hyperplasia and carcinoma in the renal pelvis 53-week interim 1/10 Pelvic epithelial hyperplasia 57-week recovery 2/10 Pelvic epithelial hyperplasia 104-week terminal plus unscheduled deaths Pelvic epithelial hyperplasia 13/70 0/70 Transitional-cell carcinoma Hepatocellular hypertrophy and neoplasms in the liver 53-week interim Hepatocellular hypertrophy 57-week recovery Hepatocellular hypertrophy 104-week terminal plus unscheduled deaths Hepatocellular hypertrophy Hepatocellular adenoma Hepatocellular carcinoma

Females

Follicular-cell hypertrophy, adenoma and carcinoma in the thyroid gland 53-week interim 0/10 Follicular-cell hypertrophy 1/10 57-week recovery 0/10 Follicular-cell hypertrophy 104-week terminal plus unscheduled deaths 2/70 1/70 Follicular-cell hypertrophy 0/70 2/70 Follicular-cell adenoma 0/70 0/70 Follicular-cell carcinoma

-

0/10

0/10

-

1/70 0/70 1/70

9/70 9/70 0/70

3/70 1/70 0/70

4/70 0/70 0/70

FromHardisty(1996b)

In rats, a significant increase in the number of PCNA-positive urothelial cells was seen in the urinary bladder of males (3.3 ± 5.0 compared with 0.3310.71 in controls). No or only a slight increase in the number of cycling cells was observed in the thyroid glands of males (0.56 ±1.7 compared with 0 in controls) and in the livers of females (1.0 ± 2.2 compared with 0.60 ± 1.6). In mice, a trend towards a minimal-to-slight increase in the number of PCNA-positive cortical tubule cells was seen in the kidneys of males and females after 52 weeks at the highest dietary concentration: treated males, 3.9 ± 2.2; control males, 1.2 ± 1.8; treated females, 2.2 ± 1.7; control females, 1.3 ± 0.80. The toxicological or biological significance of this phenomenon is uncertain. No increase in the number of PCNA-positive cells was observed in the livers of male and female mice (treated males, 6.2 ± 4.6; control males, 12 ± 6.0; treated females, 8.3 ± 3.8; control females, 4.6 ± 7.7) (Irisarri, 1996; Debruyne, 1998). (c)

Reproductive toxicity (i)

Multigeneration studies

Rats In a two-generation (one litter per generation) study of reproductive toxicity, groups of 30 Sprague-Dawley CD rats of each sex were fed diets containing carbaryl (purity, 99.1%) at a concentration of 0, 75, 300 or 1500 ppm, equal to 0, 4.7, 24 and 93 mg/kg bw per day for males

CARBARYL 3-29 JMPR 2001

15

and 0, 4.8, 21 and 96 mg/kg bw per day for females, for both generations. At least 70 days after the beginning of treatment, the FQ parents were mated in a 1:1 ratio for 2 weeks, to produce the FI litters. The dams were allowed to rear their young to day 21 postpartum. The F! and F2 litters were culled, if necessary, to a total of five male and five female pups on day 4. After weaning, the FQ parents were killed, and groups of 30 male and 30 female weanlings per dose were used as the FI parents and were given carbaryl by the regimen described above, to produce the F2 litters. The Fj adults and the F2 weanlings were killed at the end of the study. All animals, including pups, were observed daily. The food consumption of the F0 and F1 parents was determined weekly before mating and during gestation and lactation. Body weights were determined weekly, except that the body weights of the females were determined on days 0, 7, 15 and 20 of gestation, on the day of parturition, and on days 4, 7, 15 and 21 after delivery. All parental animals were examined after death, both grossly and histologically. Antibodies against respiratory syncytial and sialodacryoadenitis virus were found at necropsy in five F1 parental animals. The infection had probably occurred near the start of the pre-breeding period and had resolved a number of weeks before mating, although no clinical signs of sialodacryoadenitis were observed. The authors considered that this viral infection had no significant effect on the results. Cholinesterase activity was not measured. Statements of adherence to GLP and QA were included. No treatment-related deaths were observed. Signs of systemic toxicity were seen in F0 and FI parents at 300 and 1500 ppm. The effects at 300 ppm included reduced food consumption, reduced body-weight gain in FI males before breeding (neither statistically significant), and reduced body weights in Ft females during lactation. At 1500 ppm, all parental animals had decreased body weights and food consumption. At necropsy, the FO females at 300 and 1500 ppm had increased absolute and relative liver weights, and F1 females had increased relative liver weights. Treatment had no effect on mating, fertility, pregnancy or gestational indices, numbers of implants, total, live or dead pups per litter, or on the per cent postimplantation loss. No treatment-related gross or histological lesions were found in the reproductive organs of either sex in any generation. The body weights per litter of F1 and F2 pups were reduced at 1500 ppm during lactation. The survival indices and mortality rates of F1 and F2 pups are shown in Table 11. In FI pups, effects on the lactational index and mortality rate were observed at the highest dietary concentration on days 5-21. These effects were not statistically significant. Vaginal opening and preputial separation were delayed by 2.1 and 1.4 days, respectively, in F1 offspring at 1500 ppm, and these effects were related to lowered body weights. F2 pups showed dose-related decreases in 4-day survival and lactational index. The trends were statistically significant, but no statistical significance was reached at 300 or 1500 ppm. The mortality rate was increased in a doserelated fashion on days 0-4. The absolute weights of the thymus and spleen were decreased in most Table 11. Survival indices and mortality rates during lactation of F1 and F2 pups of rats given carbaryl Observation

Dietary concentration (ppm)

From Tyl et al. (2000) Number of surviving pups at 4 days divided by number of live pups at day 0 Number of surviving pups at day 21 divided by number of live pups at day 4

a

b

CARBARYL 3-29 JMPR 2001

16

weanlings of rats at 1500 ppm, to the same degree as body weights. No treatment-related malformations were observed. The NOAEL for parental toxicity based on effects on body and liver weights in parental animals was 75 ppm, equal to 4.7 mg/kg per day. The NOAEL for toxicity in the offspring, based on increased p2 pup mortality, was 75 ppm, equal to 4.7 mg/kg per day (Tyl et al., 2001). (ii)

Developmental toxicity

Rats In a study of developmental toxicity, 25 pregnant Sprague-Dawley CrliCD (SD) BR rats received carbaryl (purity, 99%) in a 0.5% solution of methylcellulose 400 by gavage at a dose of 0, 1, 4 or 30 mg/kg bw per day on days 6-20 of gestation. Statements of adherence to GLP and QA were included. No deaths were observed. Increased salivation was observed at least once in 18/25 dams at 30 mg/kg bw per day within 20 min of treatment, the effect disappearing about 1 h after treatment. This effect was generally observed between days 14 and 20, although one animal showed increased salivation from the first day of treatment. Body-weight gain and food consumption were significantly reduced among dams at 30 mg/kg bw per day. There were no treatment-related effects on pre- or post-implantation loss, number of fetuses per litter or fetal sex ratio. Fetal body weights were significantly reduced at the highest dose. The fetal and litter incidences of incomplete or absent ossification of the seventh cervical centrum, incomplete ossification of the fifth sternebra and non-ossification of the first metacarp were increased at 30 mg/kg bw per day, a maternally toxic dose. There were no treatment-related changes in the incidences of malformations. The NOAEL for maternal toxicity was 4 mg/kg bw per day, on the basis of salivation and reduced body-weight gain and food consumption. The NOAEL for embryo- and fetotoxicity was 4 mg/kg bw per day, on the basis of decreased body weight and delayed ossification (Repetto-Larsay, 1998). The Meeting noted that inhibition of acetylcholinesterase activity in erymrocytes and/or brain was not measured. Rabbits Groups of 22 pregnant New Zealand white rabbits received carbaryl (purity, 99%) in a 0.5% an aqueous solution of methylcellulose by gavage at a dose of 0, 5, 50 or 150 mg/kg bw per day on days 6-29 of gestation. On day 25, about 1 h after dosing, blood was collected, and cholinesterase activity was measured in plasma and erythrocytes. Statements of adherence to GLP and QA were included. One control doe, one doe at 5 mg/kg bw per day and two does at 50 mg/kg bw per day died, but these deaths were considered not to be related to treatment. The body-weight gain of does at the highest dose was reduced. Plasma cholinesterase activity was reduced by 13,46 and 68% and erythrocyte cholinesterase activity by 6, 19 and 27% at 5, 50 and 150 mg/kg bw per day, respectively, the reductions being statistically significant at the two higher doses. As inhibition of plasma cholinesterase activity reflected more closely the expected inhibition in brain than did erythrocyte acetylcholinesterase activity (see studies of neurotoxicity, below), the 19% inhibition of erythrocyte acetylcholinesterase activity seen at the intermediate dose was considered to be toxicologically relevant. There were no treatment-related effects on pre- or post-implantation loss, the number of fetuses per litter or the fetal sex ratio. Fetal body weights were significantly reduced at 150 mg/kg bw per day. There was no treatment-related change in the incidence of malformations. The NOAEL for maternal toxicity was 5 mg/kg bw per day, on the basis of decreased erythrocyte cholinesterase activity at 50 mg/kg bw per day. The NOAEL for embryo- and fetotoxicity was 50 mg/kg bw per day, on the basis of reduced fetal body weight per litter (Tyl et al., 1999).

CARBARYL 3-29 JMPR 2001

17

(d)

Special studies: Neurotoxicity (i)

Single exposure

Rats Groups of 24 Sprague-Dawley rats of each sex received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a single dose of 0,10,30 or 90 mg/kg bw. Six animals of each sex per dose were killed 1, 8, 24 and 48 h after dosing, and blood samples were retained for analysis. Body weights were measured before dosing and before termination of the study. Gross clinical examinations were performed twice daily, and detailed clinical examinations were performed before dosing and just before the terminal kill. At necropsy, the whole brain, the left hemisphere and the frontal cortex, hippocampus, cerebellum and caudate/putamen of the right hemisphere were weighed. Cholinesterase activity was determined in the left hemisphere, as a measure of whole brain activity, in the four brain structures of the right hemisphere and in blood samples. Statements of adherence to GLP and QA were included. The body weight of males at 90 mg/kg bw was significantly reduced 24 h after treatment. No deaths occurred. No clinical effects were observed in animals at 10 mg/kg bw. In animals at 30 mg/kg bw, tremors, salivation and fur staining or wetness around the muzzle were observed 1 h after treatment. In animals at 90 mg/kg bw, tremors, salivation, fur staining and wetness around the muzzle, urogenital and periorbital areas, decreased activity and abnormal breathing sounds were observed 1-48 h after treatment. The severity and frequency of the clinical signs were related to dose and decreased with time. In general, cholinesterase activity was reduced to a similar extent in all the brain regions examined and was reduced in a dose-related fashion 1 h after dosing by about 35,70 and 80% in the groups at 10, 30 and 90 mg/kg bw, respectively. Complete recovery was observed 8 h after treatment with 10 mg/kg bw, whereas the activity was reduced by about 22 and 30% at 30 and 90 mg/kg bw, respectively. By 24 h after dosing, only the group at 90 mg/kg bw showed a reduction in brain cholinesterase activity (by 30%), and by 48 h after dosing the activity had returned to control values in all treated groups. The cholinesterase activity in whole blood, plasma and erythrocytes followed similar patterns. Erythrocyte acetylcholinesterase activity was reduced by 32% 1 h after dosing at 10 mg/kg bw, by 56% and 33% 1 and 8 h after dosing at 30 mg/kg bw and by 65%, 20% and 33% 1, 8 and 24 h after dosing at 90 mg/kg bw. The reduction in plasma cholinesterase activity after 1 h more closely reflected the findings in brain than did erythrocyte acetylcholinesterase activity, the inhibition in plasma cholinesterase activity at 1 h being 38%, 68% and 86% at 10, 30 and 90 mg/kg bw, respectively. On the basis of the effects on brain and erythrocyte acetylcholinesterase activity, the LOAEL was 10 mg/kg bw. No clinical signs were observed at this dose (Brooks & Broxup, 1995a). Groups of two male and two female Sprague-Dawley rats received technical-grade carbaryl (purity, 99.1 %) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a single oral dose of 10, 50, 100, 250, 500 or 1000 mg/kg bw, followed by a 3day observation period. No control groups were used. Body weights were measured twice before dosing and on days 0, 1 and 3. Gross clinical examinations were performed twice daily before dosing. Detailed physical examinations were performed on day 0 before dosing and 0.5,1,2,4 and 8 h and 1, 2 and 3 days after dosing. Cholinesterase activity was not assessed. Statements of adherence to GLP and QA were included. One male and both females at 500 mg/kg bw and all animals at 1000 mg/kg bw died within 24 h of treatment. On the day of treatment, all animals at doses > 50 mg/kg bw showed weight loss, moderate to severe salivation and tremors, lachrymation and/or periorbital staining, staining and/ or wetness of the muzzle and urogenital area, decreased activity and decreased respiration with CARBARYL 3-29 JMPR 2001

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abnormal breathing sounds (except animals at 50 mg/kg bw) and a weakened condition (except at 50 and 100 mg/kg bw). No effects were seen at 10 mg/kg bw (Brooks & Broxup, 1995b). Groups of eight male and eight female Sprague-Dawley rats received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a single dose of 0, 10, 50 or 125 mg/kg bw. Three animals of each sex per dose were tested in a functional observational battery (FOB) before dosing on day 0 and 0.5,1,2,4, 6, 8 and 24 h after dosing, at which time they were killed. Three animals of each sex per dose were killed 0.5,1,2,4 or 8 h after dosing. Blood samples and brains were taken from all animals for determination of cholinesterase activity. Body weights were measured once before dosing and just before termination of the study. Clinical examinations were performed on all animals before dosing and before termination. Statements of adherence to GLP and QA were included. No deaths occurred. Animals at 50 and 125 mg/kg bw had decreased body weights 4,8 and 24 h after treatment. Animals at 50 mg/kg bw showed slight to moderate tremors (0.5-2 h after dosing), slight to severe salivation (0.5-1 h after dosing) and muzzle staining (at all times). In animals at 125 mg/kg bw, slight to severe tremors, slight to severe salivation and muzzle staining (0.5-8 h after dosing) were observed. Piloerection, forepaw staining, urogenital staining, pupil constriction, periorbital staining and discharge were observed occasionally. The animals at 50 and 125 mg/kg bw groups that were subjected to the FOB showed not only the clinical effects described above but also a dose-dependent deterioration of gait, ranging from slight ataxic gait to severe impairment or incapacity 0.5-4 h after dosing. The respiration of these animals was dose-dependently decreased 0.5-2 h after dosing. Activity and arousal were dosedependently decreased at 50 and 125 mg/kg bw, the greatest reductions being seen at 0.5-2 h. Brain cholinesterase activity was decreased in all groups. At 10 mg/kg bw, decreases of 50% and 34% were observed 0.5 and 1 h after dosing, respectively, with almost complete recovery by 2 h after dosing. At 50 mg/kg bw, brain cholinesterase activity was decreased 0.5-8 h after dosing (by 75% 0.5-1 h after dosing and by 36% after 8 h). At 125 mg/kg bw, brain cholinesterase activity was reduced 0.5-24 h after dosing (by 80% 0.5-1 h after dosing and by 34% reduction at 24 h). The timing of the reductions in cholinesterase activity in whole blood, plasma and erythrocytes followed a similar pattern, with maximal reductions 0.5-2 h after dosing. Cholinesterase activity in plasma was reduced to approximately the same extent as that in brain during the first 0.5-4 h after dosing. In erythrocytes, the reduction was slightly smaller, the maximal reductions at 0.5 h being approximately 24%, 40% and 51 % at 10,50 and 125 mg/kg bw, respectively. No consistent differences were found between males and females with regard to the effect of carbaryl on brain or blood cholinesterase activity. On the basis of the effects on brain and erythrocyte cholinesterase activity, the LOAEL was 10 mg/kg bw. No clinical signs were observed at this dose (Brooks & Broxup, 1995c). Groups of 12 Sprague-Dawley rats of each sex received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a single dose of 0,10,50 or 125 mg/kg bw. Body weights were measured before dosing and during behavioural testing. Food consumption was measured weekly. Gross clinical examinations were performed twice daily on all animals, and detailed clinical examinations were performed on days 1, 8 and 15. All animals were subjected to a FOB test and a motor activity test before dosing, and on days 0 (0.5-1.5 h after dosing), 7 and 14. On day 15, six rats of each sex per group were perfused for neuropathological examination, the remaining six rats being subjected to complete necropsy. Cholinesterase activity was not assessed. Statements of adherence to GLP and QA were included.

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No deaths occurred. Males and females at 125 mg/kg bw group showed decreased bodyweight gain and food consumption during the first week after treatment. Animals at 125 mg/kg bw showed staining of the muzzle, lower jaw, urogenital region and ventral surface, females having a higher incidence. A few females also had ocular discharge, opaque eyes, periorbital staining, red liquid in the urine and cervical swelling. Staining of the urogenital region and ventral surface was also found in a few females at 50 mg/kg bw. In the FOB, the animals at 50 and 125 mg/kg bw showed dose-dependent increases in the frequency of tremors, gait disturbance, salivation, passivity, acoustic startle response and hind-limb splay, and dose-dependent decreases in motor and rearing activity, arousal, defaecation, urination, extensor thrust, toe and tail pinch response, grip strength of the fore- and hindlimbs and body temperature, 0.5-1.5 h after dosing. Dosedependent reductions in body temperature were observed in females on days 7 and 14 after treatment, reaching statistical significance in the group given 125 mg/kg bw. The group at 10 mg/kg bw showed no effects in the FOB at any time. No effects on gross appearance or on gross or microscopic neurological appearance were found. The NOAEL was 10 mg/kg bw on the basis of the clinical effects (Brooks et al., 1995). (ii)

Repeated exposure

Rats Groups of 27 Sprague-Dawley rats of each sex received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a dose of 0, 1, 10 or 30 mg/kg bw per day for 13 weeks. Body weights and food consumption were measured weekly. Gross clinical examinations were performed twice daily on all animals, and detailed clinical examinations were performed on day 2 or 3, and weekly thereafter, within 0.5-1 h of treatment. An FOB and a motor activity test were administered to 12 animals of each sex per dose before treatment and in weeks 4,8 and 13 (0.5-1.5 h after dosing). Five animals at each dose were used to determine blood cholinesterase activity before and at the end of the study (week 13). At the end of the study, six rats of each sex per group were perfused for neuropathological examination, the remaining six rats of each sex per group being subjected to complete necropsy. Three subgroups of five animals of each sex per dose were used to determine cholinesterase activity in plasma, erythrocytes and whole blood, in the left hemisphere and in the frontal cortex, hippocampus, cerebellum and caudate/putamen of the right hemisphere of the brain. One subgroup was bled at week 0 and at week 4, a second was bled at week 0 and at week 8 and the third was bled at weeks 4, 8 and 13. Blood samples were collected 1 h after treatment. Before terminal blood collection, the brains were removed. Statements of adherence to GLP and QA were included. No deaths occurred. Rats at 30 mg/kg bw per day showed decreased body-weight gain during the first week of treatment and a slight reduction in food consumption throughout the study. These animals occasionally showed increased salivation and slight to moderate tremors. In the FOB, animals at 10 and 30 mg/kg bw per day had decreased pupil size and increased frequencies of tremors and (in females) reduced rearing activity. Animals at 30 mg/kg bw per day also showed increased salivation and reduced defaecation. The females in this group had reduced tail and toe pinch response and extensor thrust, and the males showed reduced pinna response. The body temperature was dose-dependently decreased, reaching statistical significance in the group at 30 mg/kg bw per day at all times of assessment and occasionally in females at 10 mg/kg bw per day. At 4 and 8 weeks, locomotor activity was decreased in females (not significant in males) at 30 mg/kg bw per day. Cholinesterase activity was significantly decreased by more than 20% in erythrocytes, whole blood, plasma and whole brain and individual brain structures in rats at 10 and 30 mg/kg bw per day at 4, 8 and 13 weeks. In rats at 10 mg/kg bw per day, the reductions in activity did not

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always reach statistical significance. A nonsignificant reduction of 31 % in cholinesterase activity in the hippocampus in females at week 13 was the only finding at 1 mg/kg bw per day, and was due mainly to an extremely low value in one female. As the inhibition seen at the lowest single dose of 10 mg/kg bw was reversed rapidly (within 4 h), as would be expected of a carbamate, the inhibition should not increase markedly with duration of dosing. Thus, at the highest dose, very little change was seen after week 4. Therefore, a statistically nonsignificant reduction of more than 20% was considered not to be a biologically relevant effect. No treatment-related effects on gross appearance or gross or microscopic neuropathological appearance were found. The NOAEL was 1 mg/kg bw per day on the basis of the effects in the FOB and the reductions in cholinesterase activity (Robinson & Broxup, 1996). Groups of 32 mated FO female Sprague-Dawley rats received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1 % Tween 80 by gavage at a dose of 0, 0.1, 1 or 10 mg/kg bw per day from day 6 of gestation to day 10 post partum. Gross clinical examinations of the dams were performed twice daily. Body weights were measured on days 0, 6, 9, 12, 15, 18 and 20 of gestation and on days 0, 4, 7, 11, 13 and 21 post partum. On these days (except day 0postpartum), the animals were subjected to a FOB test 0.5-2 h after dosing. Six animals per dose were bled on day 6 of gestation (before dosing), 1 h after dosing on days 6,15 and 20, on day 4postpartum and terminally on day 10postpartum for measurement of cholinesterase activity. After the terminal blood sample collection, the brains were weighed and analysed. The time of onset and completion of littering and the females' behaviour post partum were recorded. At termination, all females underwent complete necropsy. The numbers of live, malformed and dead F! pups were recorded. Live pups were weighed on days 0, 4, 7, 11, 13 and 21 post partum. The litters were culled to eight pups (four males and four females when possible) on day 4 post partum. From days 4-21, the times of tooth eruption and eye opening were recorded, and the pups were subjected to a FOB and motor activity assessment. One pup of each sex per litter was removed on day 11 post partum, and six of each sex per group were selected for neuropathological and neuromorphological evaluation. The total weights of the brain and brain regions were recorded for the unselected pups. The remaining pups (three males and three females per litter) were weaned on day 21 to form the FI adult generation. Gross clinical examination of the F! adult generation was performed twice daily. Body weights were measured once a week. The days of vaginal opening and preputial separation were recorded. The F! adults were tested for motor activity (day 60), startle habiruation (days 22 and 60), passive avoidance (days 23-24) and activity in a water maze (days 60-65). At termination, six animals of each sex per group were perfused, and their organs were sampled for histological examination. The brains of an additional six animals of each sex at 0 and 10 mg/kg bw per day were retained for neuromorphometric examination. Statements of adherence to GLP and QA were included. No treatment-related clinical signs or deaths were observed in the dams. Those at 10 mg/kg bw per day showed a reduction in body weight on days 6-9 of gestation and also increased incidences of pupil size reduction, slight tremors and disturbed gait in the FOB throughout testing (day 6 of gestation to day 10 post partum). Dams at 10 mg/kg bw showed occasionally decreased cholinesterase activity in erythrocytes (by 28%), whole blood (by 35%) and plasma (by 39%, not significant) from day 20 of gestation to day 10 post partum, and in the brain (42% reduction) at termination. No other treatment-related effects were observed in the dams. A small but statistically significant increase in the number of stillborn pups was observed in the group at 10 mg/kg bw per day, but the cause of death was not established. As the mean number per litter (0.3) was within the range of control groups in other studies (0-0.9 between October 1989 and January 1995), this effect wass considered by the study authors to be of no toxicological significance. The Meeting endorsed that view. No treatment-related effects were

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observed in the F} adult generation. A few changes were observed during neuromorphometric analysis, and some reached statistically significance (Table 12 and see below). Generally, the changes were not seen consistently in males and females, and their direction was inconsistent; the Meeting considered that they were not related to treatment. The NO AEL for maternal toxicity was 1 mg/kg bw per day, based on effects on body weight, observations in the FOB and the reduction in cholinesterase activity. The NO AEL for toxicity in offspring was 10 mg/kg bw per day, the highest dose tested (Robinson & Broxup, 1997). In their review of the final report of the study, the USA's Environmental Protection Agency indicated that "... the bilateral decrease in the length of the cerebellum accompanied by a nonstatistically significant 5% decrease in cerebellar weights in the day 11 females and the bilateral increase in the width of the cerebellum in the day 70 female animals at the highest dose (10 mg/kg bw per day) may possibly be treatment related Further some forebrain measurements may have also been affected." (The Meeting noted that the cerebellar weights of females were decreased [not statistically significantly] on day 11 by 6%, 11% and 5% at the lowest, intermediate and highest doses, respectively.) Therefore, supplemental histomorphometric evaluations were performed. A preliminary study was undertaken in order to examine the morphological changes that occur normally in the developing rat brain, without any treatment. The forebrain (frontal and parietal cortex, left and right side and thickness of corpus callosum) and cerebellum (lobule base thickness and thickness of internal and external granular layers) of 10- and 13-day old pups were compared by histomorphometry. This study indicated that the changes occurring in the right and left forebrain between day 10 and day 13 were similar in male and female pups, and the cerebellar changes observed were consistent with the expected migration of cells from the external towards the internal layer at that time. Additionally, the study showed that morphometric changes in the developing brain of rat pups occur bilaterally in the frontal and parietal cortex and in the cerebellum (Hamelin & Yipchuck, 2001). Table 12. Statistically significant morphometric changes (^m) in right and left sub-locations of the brain in rats in a study of developmental neurotoxicity with carbaryl Morphometric measurement

Females

Males

0

10 mg/kg bw per day

0

10 mg/kg bw per day

Pups, day 1 1 Parietal cortex, right forebrain Parietal cortex, left forebrain Length of right cerebellum Length of left cerebellum

1500 1550 4380 3930

1610* 1560 4730 4710*

1550 1600 4440 4600

1550 1640 3750* 3580**

Adults, day 70 Frontal cortex, right forebrain Frontal cortex, left forebrain Length of right cerebellum Length of left cerebellum Width of right cerebellum Width of left cerebellum

1740 1820 6200 6140 4570 4600

1610 1640* 6720* 6190 4870 4890

1820 1760 62201 6220 4720 4560

1720 1640 6280 6290 4440 4470

Additional evaluation" Length of right cerebellum Length of left cerebellum Width of right cerebellum Width of left cerebellum

6480 6640 5280 4970

6780 6750 4960 4770

5210 6000 4420 4220

5260 5670 5260** 5480***

From Robinson & Broxup (1997). Data rounded to three significant figures because of the degree of inter-animal variation and experimental error *p< 0.05; **p < 0.01; ***p <0.001 (Dunnett's test) a As the slides prepared from the left cerebellum of four of six animals at the highest dose were not suitable for measuring the length of the cerebellum, additional histomorphometric evaluation was performed on the left and right cerebelli of the ani

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In the main study, supplemental histomorphometric evaluations were performed on the existing slides of the cerebelli of six pups and six adults of each sex per dose in the control and highest-dose groups. Further, the bilateral measurements of the parietal cortex in the forebrains of male pups and the frontal cortex in the forebrains of male adults were re-evaluated. The data were presented as the means for the two sides. No statistically significant difference was found between treated and control animals in any of the measures in the cerebellum or in the mean measures in the neocortex. The Meeting concluded that the new morphometric results, which revealed no changes at the highest dose, are in agreement with a lack of treatment-related effects on terminal body weight, brain and cerebellar weights, the results of behavioural and motor activity tests and gross and histopathological appearance of male and female pups and adults (Robinson & Broxup, 2001a,b).

3.

Observations in humans

Pastides (1993; evaluated in Annex 1, reference 79) reported the results of an epidemiological study of employees who had worked in a carbaryl unit and had been hired between the start-up of the unit in 1960 and 1978. Workers in the manufacturing unit, those in maintenance and those in packaging and distribution could have been exposed to carbaryl. Information on vital status and cause of death obtained in 1988 was used. Data on a total of 448 employees representing 7532 person-years were included. Data on employees specifically exposed to carbaryl were not analysed separately. The 25 deaths identified resulted in elevated standardized mortality ratios (SMRs) for cancer of the pancreas, unspecified cancer and cancer of the brain and other parts of the nervous system. The SMRs for cancer of the pancreas and for unspecified cancer showed only slight excesses and were based on only one case each. The SMR for cancer of the brain and other parts of the nervous system was based on only two observed cases with tumours of different histological origins, reducing the probability that the two malignancies were caused by the same exposure. For all categories, the very wide confidence intervals indicated the low precision of the SMR estimates. In a subsequent phase of this study, the analysis was based on the vital status of employees through 1994. The cohort consisted of 765 men who had been employed between 1960 and 1994 (including employees from the previous study who were hired before 1978 and additional employees who were hired after 1978), representing 12 580 person-years. A subcohort from which all men who had been employed for < 1 year were eliminated was also evaluated, resulting in a group of 599 men with 9634 person-years. The SMRs were calculated in relation to the rates for all white males in the USA and to all white males in West Virginia, the production site. Fewer deaths from all causes and from all malignancies than expected were found for both the large cohort and the subcohort. Some of the SMRs slightly exceeded 100 but were well within the 95% confidence interval. This was also the case for SMRs for malignant neoplasms of the brain and other nervous system cancers (Pastides & Zorn, 1997). Comments To elucidate the role of metabolism in the formation of tumours in the long-term studies in mice and rats, metabolite profiles were determined in mice and 15-month-old rats. The kinetics and metabolism of carbaryl in mice and rats were found to be comparable in studies evaluated by the 1996 JMPR. Some evidence was obtained that mice formed more metabolites via epoxidation and conjugation at a high, probably toxic, dietary concentration of carbaryl (about 8000 ppm). In

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the study in 15-month-old rats, no convincing evidence was found for a shift in the urinary metabolite pattern at the high (toxic) dietary concentration of 7500 ppm. The 1996 Meeting could not identify a NOAEL for vascular tumours (haemangiomas and haemangiosarcomas) in male mice. The highest dose in this 2-year study also increased the incidence of renal tubule-cell adenomas and carcinomas in males and the incidences of vascular tumours and of hepatocellular adenomas and carcinomas in females. Re-evaluation of the histological slides from the 2-year study in mice confirmed the original findings of the study pathologists, namely, increased incidences of vascular tumours in males at all doses. Although the incidence of vascular tumours found at the lowest dietary concentration of 100 ppm, equal to 15 mg/kg bw per day, in this study was within the range of incidences in some control groups in 104-week studies performed in other laboratories, it was not possible to exclude the possibility that the incidences were outside the range of values for control groups in similar studies performed in the same laboratory, in the absence of data on such studies. In 1996, the Meeting concluded that carbaryl is not genotoxic. Some support for a nongenotoxic mechanism of vascular tumour formation by carbaryl in mice was found in a newly available 6-month study in the/?53 knockout mouse model, in which tumours are induced readily by genotoxic compounds. In this model, carbaryl did not induce vascular tumours, whereas the genotoxic carcinogen urethane, used as a positive control in the validation study, did. The Meeting concluded that the increased incidence of vascular tumours was likely to be species- and sex-specific, but, in view of the rarity and malignancy of these tumours, they could not be discounted in human risk assessment. In 1996, the Meeting concluded that carbaryl is carcinogenic in rats only at doses that exceed the maximal tolerated dose. Increased incidences of tumours were observed in the thyroid in males, the liver in females and the urinary bladder in males and females at the highest dietary concentration of 7500 ppm. Re-evaluation of the histological slides from the 2-year study in rats confirmed the original findings of the study pathologist that increased incidences of rumours in the thyroid (follicular-cell adenomas and carcinomas in males), liver (hepatocellular adenoma in females) and urinary bladder (transitional-cell papillomas and carcinomas in males and females) occurred at the highest dietary concentration of 7500 ppm. The finding of extensive hyperplasia in the urinary bladder without associated necrosis, inflammation or regeneration suggested that the tumorigenic response was due to cell proliferation associated with a mitogenic effect of carbaryl or one of its metabolites, which appears to be an effect specific to rats. On the basis of this new information, the Meeting reaffirmed the conclusion of the 1996 JMPR. In 1996, the Meeting recommended that a new two-generation study of reproductive toxicity be carried out in rats, with special attention to the male reproductive tract, as effects on this system were observed in some long-term studies of toxicity in rats at doses given by gavage that were significantly lower than those given in the diet in the studies of reproductive toxicity (which suffered from various shortcomings in study design). In the newly available twogeneration study of reproductive toxicity, treatment with carbaryl had effects on the offspring at maternally toxic doses. No effects on reproductive parameters were found in animals of either sex at any dietary concentration up to the highest, equal to 92 mg/kg bw per day. At a concentration of 300 ppm, equal to 21 mg/kg bw per day, effects were seen on the weights of the body and liver of parental animals and on the mortality rate of pups of the F2 generation. The NOAEL for parents and offspring was 75 ppm, equal to 4.7 mg/kg bw per day. On the basis of this new study, and taking into account that all the previously evaluated studies were of limited significance and validity for evaluating reproductive effects, the Meeting concluded that carbaryl does not impair fertility or reproduction and has no adverse effects on the male or female reproductive system. In 1996, the Meeting concluded that carbaryl has developmental toxicity, manifested as deaths in utero, reduced fetal weight and malformations, but only at doses that are overtly maternally toxic. The shortcomings of these studies were such that NOAELs for developmental

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toxicity that could be used for risk assessment could not be identified. In the newly available studies of developmental toxicity in rats and rabbits, treatment with carbaryl caused fetal effects only at maternally toxic doses. In a study in rats, the highest dose of 30 mg/kg bw per day resulted in maternal toxicity, reduced fetal body weights and delayed ossification. The NOAEL for maternal, embryo- and fetotoxicity was 4 mg/kg bw per day. Effects on cholinesterase activity were not determined. In a study in rabbits, doses > 50 mg/kg bw per day were maternally toxic (inhibition of erythrocyte acetylcholinesterase activity). Fetotoxic effects (reduced fetal body weight per litter) were seen at 150 mg/kg bw per day, the highest dose tested. The NOAEL for maternal toxicity was 5 mg/kg bw per day, and that for embryo- and fetotoxiciry was 50 mg/kg bw per day. The compound did not induce irreversible structural effects in either rats or rabbits at doses up to 3 0 and 150 mg/kg bw per day, respectively. On the basis of the new studies and taking into account the limited significance and validity of the previously evaluated studies for evaluating developmental effects, the Meeting concluded that carbaryl is not teratogenic. In studies of neurotoxicity in rats, a single dose > 30 mg/kg bw or repeated administration of doses > 10 mg/kg bw per day for 13 weeks by gavage resulted in dose-dependent clinical effects, including decreased pupil size, tremors, salivation, reduced body temperature and fur staining. A single oral dose of 500 or 1000 mg/kg bw was lethal. Single oral doses > 10 mg/kg bw (lowest dose tested) induced marked, dose-dependent reductions in cholinesterase activity in plasma, erythrocytes, whole blood and brain, which returned to pretreatment values within 4,24 and 48 h after doses of 10, 30 and 90 mg/kg bw, respectively. A NOAEL could not be identified in these studies with single doses. The LOAEL was 10 mg/kg bw. In the 13-week study of neurotoxicity, cholinesterase activity was significantly decreased by more than 20% in erythrocytes, whole blood, plasma, whole brain and individual brain structures at doses of 10 and 30 mg/kg bw at weeks 4, 8 and 13 in a dose-related fashion. The NOAEL was 1 mg/kg bw per day. In a study of developmental neurotoxicity, treatment of pregnant rats from day 6 of gestation to day 10 post partum at a dose of 10 mg/kg bw per day induced clinical effects and reductions in erythrocyte and brain cholinesterase activity. Treatment of the dams had no effect on clinical, developmental or behavioural parameters in offspring up to 4 months of age. The NOAEL for offspring was 10 mg/kg bw per day, the highest dose tested. The NOAEL for neurotoxic effects in the dams was 1 mg/kg bw per day. Two epidemiological studies of carbaryl production workers employed between 1978 and 1994 showed no increase in the mortality rate from cancer when compared with that of unexposed workers. After re-evaluating a 6-week study in volunteers (which was reported in the evaluations of the 1973 and 1996 JMPR), the Meeting concluded that an increased ratio of amino acid nitrogen to creatinine in urine observed at a dose of 0.13 mg/kg bw per day was an equivocal, inconsistent effect and was not relevant for risk assessment.

Toxicological evaluation The Meeting concluded that the existing database on carbaryl was adequate to characterize potential hazards to fetuses, infants and children. The critical effect of carbaryl is inhibition of brain acetylcholinesterase activity. This is a rapidly reversible effect (recovery within 4 h in rats at the lowest single dose of 10 mg/kg bw), which is driven by the peak concentration in plasma rather than by the area under the plasma concentration-time curve (AUC). Therefore, in terms of brain acetylcholinesterase activity, the ADI could be the same value as the acute reference dose (RfD). However, carbaryl was considered to be a non-genotoxic carcinogen in mice, causing vascular tumours in one sex (males) at all doses tested. The Meeting established an ADI of 0-0.008 mg/kg bw on the basis of the LOAEL of 100 ppm, equal to 15 mg/kg bw per day, and a safety factor of 2000, which incorporated an extra

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safety factor of 20 in view of the occurrence of this rare and malignant type of tumour, for which a no-effect level could not be identified. Carbaryl is moderately toxic to rats after a single oral dose (LD50 = 220-720 mg/kg bw). In studies with single and repeated oral doses, the critical effect of carbaryl was of an acute nature, i.e. inhibition of acetylcholinesterase activity. A NOAEL could not be identified for this effect in studies with single doses in rats; the lowest effective dose was 10 mg/kg bw, at which no clinical signs were observed. As dogs and rats have been shown to be equally sensitive to cholinesterase inhibition by carbaryl in short-term and long-term studies, the Meeting established an acute RfD of 0.2 mg/kg bw, on the basis of the NOAEL of 125 ppm, equal to 3.8 mg/kg bw per day, in a 5week study in dogs. A safety factor of 25 was used because the effects were rapidly reversible and were driven by the peak concentration in plasma (see Annex 1, reference 91, Annex 5). Levels relevant to risk assessment Species

Study

Effect

NOAEL

LOAEL

Mouse

2-year study of Carcinogenicity3

Carcinogenicity

_

100 ppm, equal to 15 mg/kg bw per day

Rat

2-year study of toxicity and Carcinogenicity3

Toxicity

250 ppm, equal to 10 mg/kg bw per day 1500 ppm, equal to 60 mg/kg bw per day 1 mg/kg bw per day

1500 ppm, equal to 60 mg/kg bw per day 7500 ppm, equal to 350 mg/kg bw per dayb 10 mg/kg bw per day

Carcinogenicity Neurotoxicity 13- week study of neurotoxicity0 Multigeneration study Parental toxicity of reproductive toxicity3 Offspring toxicity Developmental toxicity0 Acute neurotoxicity0

300 ppm, equal to 21 mg/kg bw per day 300 ppm, equal to 21 mg/kg bw per day 30 mg/kg bw per day 30 mg/kg bw per day 10 mg/kg bw

75 ppm, equal to 4.7 mg/kg bw per day 75 ppm, equal to 4.7 mg/kg bw per day Maternal toxicity 4 mg/kg bw per day Embryo- and fetotoxicity 4 mg/kg bw per day Neurotoxicity -

Rabbit

Developmental toxicity0 Maternal toxicity 5 mg/kg bw per day Embryo- and fetotoxicity 50 mg/kg bw per day

50 mg/kg bw per day 150 mg/kg bw per day

Dog

5 -week study of toxicity8 Toxicity

400 ppm, equal to 10 mg/kg bw per day 400 ppm, equal to 10 mg/kg bw per day

1-year study of toxicity3 Toxicity

125 ppm, equal to 3.8 mg/kg bw per day 125 ppm, equivalent to 3.1 mg/kg bw per day

3

Diet "Above the MTD c Gavage

Estimate of acceptable daily intake 0-0.008 mg/kg bw Estimate of acute reference dose 0.2 mg/kg bw Studies that would provide information useful for continued evaluation of the compound • Study of Carcinogenicity in mice • Studies of the mechanism of formation of vascular tumours in mice1 • Further observations in humans 1

Before such a study is begun, it is recommended that the mechanisms of tumour formation be evaluated within the 'conceptual framework for cancer risk assessment' (developed by an IPCS working group).

CARBARYL 3-29 JMPR 2001

26

List of relevant end-points for setting guidance values for dietary and non-dietary exposure Absorption, distribution, excretion, and metabolism in mammals Up to 95% absorption within 24-48 h Rate and extent of oral absorption Slow in rats, 16-34% at low doses, 1.2-4% at high doses Dermal absorption within 24 h. In humans, 45% within 8 h after application in acetone Uniformly distributed; highest concentrations of residues Distribution in carcass, kidney, and blood None Potential for accumulation Rapid and nearly complete within 24 h at low dose, Rate and extent of excretion within 48 h at high dose. Mainly via urine (90-95%) Extensive, with only 2.9% unchanged in urine. Three Metabolism in animals main metabolic pathways: (i) arene oxide formation with subsequent hydrolysis to dihydrodihydroxycarbaryl and conjugation via the mercapturic acid (ii) carbamate hydrolysis to form 1-naphthol (iii) oxidation of W-methyl moiety (alkyl oxidation) The metabolites formed via these pathways were conjugated with sulfate or glucuronic acid. Parent compound Toxicologically significant compounds Acute toxicity Rat, LD50, oral Rat, LDJO, dermal Rat, LC50, inhalation Skin irritation Eye irritation Skin sensitization Short-term toxicity Target / critical effect Lowest relevant oral NOAEL Lowest relevant dermal NOAEL Lowest relevant inhalation NOAEL Genotoxicity Long-term toxicity and carcinogenicity Target/critical effect

Lowest relevant NOAEL Carcinogenicity

Reproductive toxicity Reproduction target / critical effect Lowest relevant (reproductive) NOAEL Developmental target / critical effect

Lowest relevant developmental NOAEL

CARBARYL 3-29 JMPR 2001

220-720 mg/kg bw (pretreatment with carbaryl increased LD50); cats most sensitive species > 2000 mg/kg bw No data Classification unknown Classification unknown Classification unknown Cholinesterase inhibition; effects on liver 3.8 mg/kg bw per day (5 weeks, dogs) No data 10 mg/m3 (90 days, rats) Weight of evidence suggests no genotoxic concern

Cholinesterase inhibition; liver, kidney, urinary bladder Mice: vascular tumours Rats: thyroid, body weight < 15 mg/kg bw per day (2 years, mice, LOAEL) 10 mg/kg bw per day (2 years; rats) Mice: vascular tumours in males at lowest dietary concentration (15 mg/kg bw per day). At maximum tolerated dose: renal tubular cell-adenoma in males and hepatocellular carcinoma and vascular tumours in females Rats: Thyroid follicular-cell adenoma (males) hepatocellular carcinoma (females), urinary bladder transitional-cell carcinoma (both sexes) at maximum tolerated dose

Deaths of pups in F2 generation at parentally toxic doses (Cholinesterase activity not examined) 4.7 mg/kg bw per day Rats: decreased fetal body weight, delayed ossification at maternally toxic dose (Cholinesterase activity not examined) Rabbits: decreased fetal body weight at maternally toxic dose 4 mg/kg bw per day (rats)

27

Neurotoxicity / Delayed neurotoxicity Acute; NOAEL

Delayed neuropathy

< 10 mg/kg bw; inhibition of cholinesterase activity (rats, single dose) 3.8 mg/kg bw; inhibition of cholinesterase activity (5 weeks, dogs) 1 mg/kg bw per day; inhibition of cholinesterase activity (rats) Negative

Other toxicological studies; observations in humans

No reliable data

Medical data

Several cases of poisoning dominated by symptoms of inhibition of cholinesterase activity

90-day; NOAEL

Summary

Value

Study

Safety factor

ADI Acute reference dose

0-0.008 mg/kg bw 0.2 mg/kg bw

Mice, carcinogenicity (LOAEL) Dogs, 5 weeks

2000 25

References Berthe, P. (1997) 14-day toxicity study in the rat by gavage. Unpublished report No. SA 95515, dated 9 January 1997, from Rhone-Poulenc Agrochimie, Centre de Recherche, Sofia Antipolis, France. Submitted to WHO by Rhone-Poulenc Agrochimie, Lyon, France. Bigot, D. (1999) Validation on p53 knockout mice to predict rodent carcinogenicity. Unpublished draft report No. SA 97040, dated 21 July 1999, from Rhone-Poulenc Agrochimie, Centre de Recherche, Sofia Antipolis Cedex, France. Submitted to WHO by Rhone-Poulenc Agrochimie, Lyon Cedex, France. Brooks, W. & Broxup. B. (1995a) An acute study of the time course of cholinesterase inhibition by orally administered carbaryl, technical grade, in the rat. Unpublished report No. 973 92 from ClinTrials B ioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France. Brooks, W. & Broxup. B. (1995b) An acute benchmark-dose toxicity study of orally administered carbaryl, technical grade, in rats. Unpublished report No. 97387 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France. Brooks, W. & Broxup. B. (1995c) A time of peak effects study of a single orally administered dose of carbaryl, technical grade, in the rat. Unpublished report No. 97388 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France. Brooks, W., Robinson, K. & Broxup. B. (1995) An acute study of the potential effects of a single orally administered dose of carbaryl, technical grade, on behavior and neuromorphology in rats. Unpublished report No. 97389 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France. Carmichael, N.G., Debruyne, E.L.M. & Bigot-Lasserre, D. (1999) The p53 knockout mouse as a model for chemical carcinogenesis in vascular tissue. Unpublished report. Submitted to WHO by Rhone-Poulenc Agrochimie, Lyon Cedex, France. Chuzel, F. (1999) Carbaryl. 6-month carcinogenicity study in p53 knockout mice by dietary administration. Unpublished report from Rhone-Poulenc Agro, Centre de Recherche, Sofia Antipolis Cedex, France. Submitted to WHO by Rhone-Poulenc Agro, Lyon Cedex, France. Cohen, S.M. (1995) Evaluation of the urinary bladder carcinogenicity of carbaryl in rats. Unpublished report from the Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA. Dated 2 November 1995. Submitted to WHO by Rhone-Poulenc Agro, Lyon Cedex, France. Cohen, S.M., Cano, M., Johnson, L.S., St John, M.K., Asamaoto, M., Garland, E.M., Thyssen, J.H., Sangha, O.K. & Van Goethem, D.L. (1994) Mitogenic effects of propoxur on male rat bladder urothelium. Carcinogenesis, 15, 2593-2597. Debruyne, E. (1998) Carbaryl. 52-week toxicity study in the CD 1 mouse: target organs cell cycling assessment.. Unpublished report No. SA 97529, amended report dated 2 December 1998, from Rhone-Poulenc Agro, Centre de Recherche, Sofia Antipolis Cedex, France. Submitted to WHO by Rhone-Poulenc Agro, Lyon Cedex, France.

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Debruyne, E. & Irisarri, E. (1996) Carbaryl technical—Chronic toxicity studies in the rat (HWA study No. 656139) and the mouse (HWA study No. 65 6-13 8) evaluation of histological slides Unpublished report, document No. 601389,studyNo.R&D/CRSA/TOX-HPA-4,dated 19March 1996, from Rhone-Poulenc Secteur Agro. Submitted to WHO by Rhone-Poulenc Agro, Lyon Cedex, France. Donehower, L.A. (1999) ILSI/HESI grant progress report, December 1999. Hamada, N.N. (1991) Subchronic toxicity study in dogs with carbaryl technical. Unpublished report No. HLA 656-152, dated 28 March 1991, from Hazleton Laboratories America Inc., Virginia, USA. Submitted to WHO by Rhone-Poulenc Ag Co., North Carolina, USA. Hamada, N.N. (1993a) Oncogenicity study with carbaryl technical in CD-I® mice. Unpublished report No. HWA 656-138, dated 20 May 1993, from Hazleton, Washington, Inc. Submitted to WHO by Rhone-Poulenc Ag Co., North Carolina, USA. Hamada, N.N. (1993b) Combined chronic toxicity and oncogenicity study with carbaryl technical in Sprague Dawley rats. Unpublished report No. HWA 656-139, dated 6 August 1993 from Hazleton, Washington, Inc. Submitted to WHO by Rhone-Poulenc Ag Co., North Carolina, USA. Hamelin, N. & Yipchuck, G. (2001) Morphometric evaluation of rat brain areas for developmental neuropathology. Unpublished report No. 99579 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France. Hardisty, J.F. (1996a) Pathology working group review report for the oncogenicity study with carbaryl technical in CD-I® mice. Unpublished report EPL project No. 259-011 from Experimental Pathology Laboratories Inc. to Rhone-Poulenc Ag Co.. Submitted to WHO by Rhone-Poulenc Ag Co., North Carolina 27709, USA. Hardisty, J.F. (1996b) Pathology working group review report for the combined chronic toxicity and oncogenicity study with carbaryl technical in Sprague-Dawley rats. Unpublished report EPL project No. 259-012, dated 16 December 1996, from Experimental Pathology Laboratories Inc. to Rhone-Poulenc Ag Co.,. Submitted to WHO by Rhone-Poulenc Agro, Lyon Cedex, France. Irisarri, E. (1996) Carbaryl. 52-week toxicity study in the rat and mouse target organs cell cycling assessment. Pathology report (post-mortem). Unpublished report No. SA 95493, dated 18 March 1996, from RhonePoulenc Agro, Centre de Recherche, Sofia Antipolis Cedex, France. Submitted to WHO by Rhone-Poulenc Agro, Lyon Cedex, France. Klonne, D.R. (1995) Carbaryl—Mouse historical control data position paper. Unpublished report from RhonePoulenc. Dated November 1995. Submitted to WHO by Rhone-Poulenc Agro, Lyon Cedex, France. Maita, K., Hirano, M., Haroda, T., Mitsumori, K., Yoshida, A., Takahashi, K., Nakashima, N., Kitazawa, T., Enomoto, A., Inui, K. et al. (1988) Mortality, major causes of moribundity, and spontaneous tumors in CD1 mice. Toxicol. Pathol, 16, 340-349. Pastides, H. (1993) Standardized mortality ratio analysis of employees exposed to carbaryl at the Rhone-Poulenc Institute, West Virginia Plant. Unpublished report dated 21 January 1997. Submitted to WHO by RhonePoulenc Agro, Lyon Cedex, France. Pastides, H. & Zorn, M. (1997) An evaluation of the mortality experience of carbaryl unit employees at the Rhone-Poulenc Institute, West Virginia plant. Unpublished report. Project No. OORP497, dated 18 June 1997, from University of Massachusetts, Department of Biostatistics and Epidemiology, Amherst, to RhonePoulenc Ag Co., North Carolina, USA. Submitted to WHO by Rhone-Poulenc Agro, Lyon Cedex, France. Repetto-Larsay M. (1998) Carbaryl developmental toxicology study in the rat by gavage. Unpublished report No. SA 98070, dated 21 October 1998, from Rhone-Poulenc Agro, Centre de Recherche, Sofia Antipolis Cedex, France. Submitted to WHO by Rhone-Poulenc Agro, Lyon Cedex, France. Robinson, K. & Broxup. B. (1996) A 13 week study of the potential effects of orally administered, carbaryl technical grade, on behavior, neurochemistry and neuromorphology in rats. Unpublished report No. 97390 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France. Robinson, K. & Broxup. B. (1997) A developmental neurotoxicity study of orally administered carbaryl, technical grade, in the rat. Unpublished report No. 97391 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France. Robinson, K. & Broxup. B. (200la) A developmental neurotoxicity study of orally administered carbaryl, technical grade, in the rat. Unpublished report No. 97391 (Final report amendment No, 1, dated 6 July 2001) from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France. Robinson, K. & Broxup. B. (200Ib) A developmental neurotoxicity study of orally administered carbaryl, technical grade, in the rat. Unpublished report No. 97391 (Final report amendment No. 2, dated 10 July 2001) from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.

CARBARYL 3-29 JMPR 2001

29 Totis, M. (1997) Investigation of the metabolism of [14C]-carbaryl in the 15 month old rat following dietary administration. Unpublished report No. SA 95288, amended 3 October 1997, from Rhone-Poulenc Agrochimie. Submitted to WHO by Rhone-Poulenc Ag Co., North Carolina, USA. Tyl R. W., Marr M.C. & Myers C.B. (1999) Developmental toxicity evaluation (with cholinesterase assessment) of carbaryl administered by gavage to New Zealand white rabbits. Unpublished report No. 65C-7297-200/ 100, dated 3 June 1999, from Research Triangle Institute, Research Triangle Park, North Carolina, USA. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France. Tyl R.W., Myers C.B. & Marr M.C. (2001) Two-generation reproductive toxicity evaluation of carbaryl (RPA007744) administered in the feed to CD® (Sprague-Dawley) rats. Unpublished report No. 65C-07407400, dated 24 May 2001, from Research Triangle Institute, Research Triangle Park, North Carolina, USA. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France. Valles, B. (1999) Carbaryl: Investigation of the metabolism of [14C]-carbaryl following 14 days administration to the male CD1 mouse. Unpublished report study No. SA 97481, dated 16 June 1999, from Rhone-Poulenc Agro. Submitted to WHO by Rhone-Poulenc Ag Co., North Carolina, USA.

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31

DIAZINON (addendum) First draft prepared by T.C. Marrs Food Standards Agency, London, United Kingdom

Explanation Evaluation for acute reference dose Time course of acute inhibition of cholinesterase activity Acute neurotoxicity in rats Studies in volunteers Comments Toxicological evaluation References

31 31 31 32 34 35 35 36

Explanation Diazinon was evaluated by the Joint Meeting in 1963,1965,1966,1970 and 1993 (Annex 1, references 2,3,6,14 and 68). In 1966, an ADI of 0-0.002 mg/kg bw was allocated on the basis of a NOAEL of 0.02 mg/kg bw per day in a study with volunteers and a 10-fold safety factor. Diazinon was last reviewed by the 1993 JMPR, when the ADI of 0-0.002 mg/kg bw was reaffirmed. The present review was undertaken to consider the need for establishing an acute reference dose (RfD).

Evaluation for acute reference dose 1.

Time course of acute inhibition of cholinesterase activity Rats

Diazinon (purity, 88%) was administered as a single dose by gavage to Harlan SpragueDawley rats in order to determine the time course of inhibition of serum, erythrocyte and brain cholinesterase activity. Groups of 15 animals of each sex were given a dose of 0,2.5,150,300 or 600 mg/kg bw. Clinical observations were made immediately before blood sampling from the orbital plexus for determination of serum and erythrocyte cholinesterase activity. Five animals of each sex were killed at 3,5 and 9 h and the remainder at 24 h to obtain brains and spinal cord for determination of cholinesterase activity, the samples being stored at -70 to -90 °C. Acetylcholinesterase activity was measured in the cerebellum, cerebral cortex, striatum and hippocampus and in the thoracic spinal cord by a modification of the method of Ellman et al. (1961). Survival was unaffected by treatment. At the highest dose, clinical signs were seen at 3 h, which were maximal at 9 h in males, with some recovery after 24 h, and maximal after 24 h in females. No significant differences were seen in body weight. Plasma cholinesterase activity was decreased by more than 20% at the lowest dose at 3 and 9 h, maximally so at 9 h in animals of each sex. At 24 h, the cholinesterase activity in animals at the lowest dose was decreased in comparison with that of concurrent controls by 17% in males and 42% in females. Erythrocyte cholinesterase activity was decreased in females at 2.5 mg/kg bw, 9 h after dosing (60% of the control value). At other times in females and at all times in males, the erythrocyte cholinesterase activity was never

DIAZINON 31-36 JMPR 2001

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less than 80% of that of controls. At the higher doses, the activity was depressed to less than 80% of the control activity at all times in animals of each sex. In an analysis of cholinesterase activity in regions of the central nervous system in animals at the lowest dose, that of males was never less than 80% that of controls but in one instance (cerebral cortex at 9 h) it was equal to 80% that of controls. In females at the lowest dose, no substantial decrease in central nervous system cholinesterase activity was observed. At the higher doses, significantly decreased activity was observed in all regions and at all times but was usually greater at 9 and 24 h than at 3 h. The NO AEL for inhibition of brain cholinesterase activity was 2.5 mg/kg bw. No NO AEL could be identified for erythrocyte cholinesterase activity (Potrepka, 1994).

2.

Acute neurotoxicity Rats

Single doses of diazinon (purity, 88%) were administered by gavage to groups of 15 male and 15 female Hsd:Sprague-Dawley rats at a dose of 0, 2.5, 150, 300 or 600 mg/kg bw. Of the 15 animals, 10 were used for neurological testing and five for measurement of cholinesterase activity. Triadimefon (purity, 99%) was administered similarly to groups of 10 male and 10 female rats, as a positive control for neurological effects. On the day of treatment, all the rats were observed before and after dosing and thereafter twice daily, for general appearance, behaviour, signs of toxicity, morbidity and mortality. All animals were examined in detail weekly, including palpation for tissue masses. Body weights were estimated before dosing and weekly thereafter, and food consumption was estimated weekly. A functional observational battery (FOB) test was performed 1 week before administration of the test materials, at the time of peak effect after administration of the test materials (9-11 h for diazinon, 1 h for triadimefon) and 7 and 14 days after administration (of diazinon) on the 10 animals intended for neurological testing. Blood samples were obtained at the estimated time of peak effects and at 14 days from the five animals intended for cholinesterase testing in each group, and plasma and erythrocyte cholinesterase activity was determined, while brain cholinesterase activity was estimated in whole brain samples at termination 14 days after administration. The animals used for neurological examination (both survivors to termination and decedents) were subjected to necropsy. Sections were made at 10 levels of the brain, cervical, thoracic, lumbar and sacral spinal cord with ganglia and right and left sciatic nerves, right and left fibular nerves, right and left tibial nerves and right and left lateral cutaneous sural nerves as well as the Gasserian ganglion. Sections were also taken of skeletal muscle from the right thigh, eyes with the optic nerve and any gross lesions identified. Only the first five animals at the highest dose of diazinon, the controls and those given triadimifon were processed for histopathological examination. Two males and one female died during the study. These deaths were attributed to diazinon, and all occurred at the highest dose. There was a single accidental death in the group of five animals at 300 mg/kg bw and intended for cholinesterase measurement during blood sampling. One control animal was removed from the study as it was thought to have been wrongly dosed, since it had signs of cholinergic poisoning and low cholinesterase activity. The clinical observations included chromodacryorrhoea, reduced activity and tremors at doses > 300 mg/kg bw, while the group at 600 mg/kg bw also had chromorhinorrhoea, diarrhoea and pallor. Significant decreases in body-weight gain were observed in males at doses > 300 mg/kg bw, while no effects were observed on body weight or weight gain in the females. Food consumption was decreased in males at doses > 300 mg/kg bw and in females at doses > 150 mg/kg bw. In both males and females, effects on parameters of the FOB test were seen only at the estimated time of peak effect after administration of the test materials (9-11 h for diazinon, 1 h for triadimefon) and not on day 7 or 14 after exposure.

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The autonomic parameters affected by diazinon at the time of peak effects in males were faecal consistency and soiled fur at doses > 150 mg/kg bw, and these effects were dose-related. Increased salivation, staining of the nose and repeated opening and closing of the mouth were observed at doses > 300 mg/kg bw. Additionally, impaired respiration, lachrymation and staining of the mouth were seen at 600 mg/kg bw. In females, repeated opening and closure of the mouth were observed at doses > 150 mg/kg bw and altered faecal consistency, soiled fur and staining of the nose at doses > 300 mg/kg bw. At the highest dose, impaired respiration and lachrymation were observed. The neuromuscular parameters that were affected in males were: abnormal gait at > 150 mg/kg bw; ataxic gait, impaired righting reflex, impaired hindlimb extensor reflex and decreased hindlimb footsplay at > 300 mg/kg bw and reduced forelimb grip strength at 600 mg/kg bw. In the females, ataxic and abnormal gait was observed at > 150 mg/kg bw and impaired righting reflex at > 300 mg/kg bw. Impaired hindlimb extensor reflex, abnormal hindlimb positioning when held by the tail and reduced forelimb and hindlimb grip strength were observed at 600 mg/kg bw. Central nervous system excitability parameters were also affected. In males, tremors were observed in the home cage and open field at > 300 mg/kg bw, as was twitching or muscle fasciculation. At 600 mg/kg bw, the arousal level was decreased. Females had tremors in the home cage at the highest dose only and in the open field at > 300 mg/kg bw. Twitching in the open field was seen at the highest dose and a lowered arousal level at > 300 mg/kg bw. Touch response was reduced in females at the highest dose, and the tail pinch response was reduced in animals of each sex at this dose. Reduced body temperature was observed in males at > 300 mg/kg bw and in females at > 150 mg/kg bw. Additionally, females at > 300 mg/kg bw were dehydrated. Locomotor activity in the figure-of-eight maze decreased over time in all groups. At the estimated time of peak effect, the activity of males at doses > 300 mg/kg bw and of females at doses > 150 mg/kg bw was decreased. At 7 and 14 days after dosing, the mean total activity counts were similar for all groups. At the time of peak effect after intake of diazinon (9-11 h), plasma cholinesterase activity was diminished in all treated groups; however, no differences were observed between groups 14 days after dosing. Erythrocyte cholinesterase activity was inhibited at doses > 150 mg/kg bw in both males and females at the time of peak effect. In males, the activity was 18%, 17% and 15% of that of concurrent controls at 150, 300 and 600 mg/kg bw, respectively, while in the females the activity was 24%, 23% and 24% that of concurrent controls at the three doses. Partial recovery was seen 14 days after dosing: in males, the activity at 150 mg/kg bw was 91% that of concurrent controls, while it was 66 and 53% that of concurrent controls at 300 and 600 mg/kg bw, respectively; in females, the activity was 89%, 57%, 74% and 65% that of concurrent controls at 2.5, 150, 300 and 600 mg/kg bw, respectively. No significant differences in brain cholinesterase activity were seen among the groups of males. Significantly reduced brain cholinesterase activity at termination was seen in females that had received diazinon at 150 mg/kg bw, in which the activity was 92% that of concurrent controls; however, as the activity in the groups given the higher doses was comparable to that in the concurrent controls, this finding is unlikely to be of biological significance. No gross or microscopic treatment-related abnormalities were seen at necropsy. Triadimefon decreased weight gain and food consumption during week 1 of the study. At the time of peak effect after exposure (1 h), changes in central nervous system excitability parameters (increased incidence of rearing; increased arousal level) were the only changes seen in the FOB test. The NOAEL was 2.5 mg/kg bw on the basis of depressed erythrocyte cholinesterase and behavioural changes at 150 mg/kg bw (Chow & Richter, 1994). Diazinon (purity, 87.9%) was administered by gavage to albino Crl:CD BR/VAF/Plus rats. In phase 1 of the study, five animals of each sex were given 100, 250 or 500 mg/kg bw. Subsequently, in order to identify an NOAEL, groups of five females receiving diazinon at 25 or 50 mg/kg bw were added. In phase 2 of the study, groups of five males received diazinon by gavage

DIAZINON 31-36 JMPR 2001

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at a dose of 0,0.05,0.5,1,10,100 or 500 mg/kg bw, and females received a dose of 0,0.05,0.12, 0.25, 2.5, 25 or 250 mg/kg bw. In phase 1, clinical observations were carried out 1, 2, 4 and 8 h after administration of the test material and daily thereafter. Body weights were determined before treatment and on days 7 and 14 and also on animals that died after 1 day. Cholinesterase activity was not measured in these animals. In phase 2, clinical observations were made 1,2 and 4 h after adminstration of diazinon, while body weights were measured before treatment and on day 1. The surviving animals from phase 1 were killed on day 14 and subjected to gross necropsy; abnormal tissues were retained for possible histopathological examination. Plasma and erythrocyte cholinesterase activity was measured 24 h after treatment in the animals in phase 2, and the animals were then killed and subjected to gross necropsy, abnormal tissues being retained for possible histopathological examination; additionally, the right half of the brain was removed for determination of cholinesterase activity. One female that received diazinon at the highest dose died during phase 1 of the study. There was no treatment-related effect on body weight. Clinical signs were seen in males at 250 and 500 mg/kg bw and in females at doses > 50 mg/kg bw, which included miosis, hypoactivity, absence of the pain reflex, red-stained face, yellow-stained urogenital region and soft stools. No pathological changes were observed that could be attributed to treatment. In phase 2, no deaths occurred. Body-weight loss during 24 h after dosing was greater in males at 500 mg/kg bw and in females at 250 mg/kg bw than in concurrent controls. Clinical signs of toxicity were seen in males at 500 mg/kg bw and in females at 250 mg/kg bw, which included miosis, hypoactivity, absent pain reflex, staggering gait, excessive salivation, red-stained face and yellow-stained and/ or wet urogenital region. The only gross pathological findings of note were yellow staining of the perineum and red paranasal discharge in males at 500 mg/kg bw and in females at 250 mg/kg bw. Plasma cholinesterase activity was reduced in males at doses > 10 mg/kg bw and in females at doses > 2.5 mg/kg bw. A statistically and biologically significant reduction in erythrocyte cholinesterase activity was seen in males at 100 mg/kg bw (51% of concurrent control value) and 500 mg/kg bw (64% of concurrent control value). In females, erythrocyte cholinesterase activity was depressed at 25 mg/kg bw (65% of concurrent control value) and 250 mg/kg bw (55% of concurrent control value). Brain cholinesterase activity was reduced in males at 500 mg/kg bw (31% of concurrent control value) and in females at 250 mg/kg bw (30% of concurrent control). In females at 25 mg/kg bw, the brain cholinesterase activity was 64% that of controls; while this difference was not statistically significant, it may be biologically significant. At necropsy in phase 2, staining of the perineum and red paranasal discharge were seen in animals of each sex at the highest dose. No treatment-related histological lesions were seen. TheNOAEL was 2.5 mg/kg bw, on the basis of inhibition of brain and erythrocyte cholinesterase activity in females at the next highest dose (Glaza, 1993).

3.

Studies in volunteers

A preliminary report was available of a double-blind, placebo-controlled study in which healthy, informed male volunteers were given single, ascending doses of diazinon (purity, 97.8%) in corn oil in gelatine capsules. Some volunteers received only com oil in gelatine capsules. The lowest dose used was 0.03 mg/kg bw, given initially to one volunteer receiving the test material and one the placebo. In the next phase, one volunteer received the placebo, six received diazinon at a dose of 0.03 mg/kg bw and one received a dose of 0.12 mg/kg bw. In the next phase, one volunteer received the placebo, six received diazinon at a dose of 0.12 mg/kg bw and one received a dose of 0.21 mg/kg bw. In the next phase, one volunteer received the placebo, six received diazinon at a dose of 0.21 mg/kg bw and one received a dose of 0.30 mg/kg bw. In the final phase, three volunteers received the placebo and seven received diazinon at a dose of 0.20 mg/kg bw. A complete physical examination was carried out before and 2 and 15 days after dosing, which DIAZINON 31-36 JMPR 2001

35

included an electrocardiogram. Vital signs (respiratory parameters, oral temperature, blood pressure and pulse) were recorded before administration of the test material and 1, 2,4, 6, 8,12, 24 and 48 h and 4,7 and 14 days afterwards. The volunteers were asked to report all adverse events. Blood was taken for clinical chemistry and haematological examination before dosing and 1,2 and 15 days after dosing. Blood was collected 1 and 2 days and immediately before dosing, and plasma and erythrocyte cholinesterase activity was determined by a modification of the method of Ellman et al. (1961). Further samples for cholinesterase determination were taken 1,2,4,6, 8,12,24 and 48 h after dosing and on days 5, 8 and 15 after dosing. Urine samples were collected over 24-h periods before dosing and for 0-6, 6-12, 12-24 and 24-48 h after dosing. The only self-reported adverse event considered to be related to intake of the test material was back pain in a man at the highest dose. No treatment-related effects on haematological or clinical chemical parameters were observed, except for changes in cholinesterase activity. Plasma cholinesterase activity was inhibited by more than 20% at doses > 0.12 mg/kg bw. No significant inhibition of erythrocyte cholinesterase activity was seen at any dose; at 0.21 mg/kg bw, 7% inhibition was observed at 4 h, 4% inhibition at 8 h and 6-7% inhibition at days 5, 8 and 15. At other times, the erythrocyte cholinesterase activity was greater than or equal to that of the group given the placebo. Furthermore, data for the man given the highest dose did not suggest any significant inhibition of erythrocyte cholinesterase activity. The NOAEL was 0.21 mg/kg bw (the highest dose was ignored, as only one man received it). However, the studies in laboratory animals indicated a sex difference in response to diazinon, while this study was performed with male volunteers only (Meyer, 1999).

Comments Diazinon is an organophosphate, and virtually all its toxicological effects are attributable to anticholinesterase activity. The oral LDso of diazinon in mice and rats was > 200 mg/kg bw. Diazinon has been classified by WHO (1999) as 'moderately hazardous'. After preliminary studies to establish the time course of clinical signs and cholinesterase inhibition, a study of acute neurotoxicity in rats was undertaken. This study involved a two-phase protocol. In phase 1, clinical observations were made, but plasma, erythrocyte, and brain cholinesterase activities were not measured. The doses used were 100,250, and 500 mg/kg bw for males and 25,50,100,250 and 500 mg/kg bw for females. In phase 2, wider ranges of doses were used (0.05-500 mg/kg bw for males and 0.05-250 mg/kg bw for females), and plasma, erythrocyte and brain cholinesterase activity were estimated. The NOAEL was 2.5 mg/kg bw in females and 100 mg/kg bw in males on the basis of inhibition of brain cholinesterase activity. The LOAELs were 25 mg/kg bw in females and 500 mg/kg bw in males. A preliminary report was available of a study in male volunteers given ascending single doses of diazinon in gelatine capsules, both the volunteers and the investigators being unaware of who had received diazinon or placebo. Plasma cholinesterase activity was inhibited by > 20% at doses > 0.12 mg/kg bw. Erythrocyte cholinesterase activity was not inhibited. The NOAEL was 0.21 mg/kg bw, the second highest dose studied; the highest dose was ignored, as the group comprised a single volunteer.

Toxicological evaluation After considering the previous evaluation of diazinon and the new data submitted, the Meeting established an acute RfD of 0.03 mg/kg bw. This was based on the NOAEL of 2.5 mg/kg bw in the studies of acute neurotoxicity in rats and a 100-fold safety factor. The study in male

DIAZINON 31-36 JMPR 2001

36

volunteers was considered of limited value in establishing the acute RfD, as the studies in rats provided evidence of a considerable sex difference in sensitivity to the inhibition of acetylcholinesterase activity by diazinon.

References Chow, E. & Richter, A.G. (1994) Acute neurotoxity study with DZN® diazinon MG87% in rats. Unpublished report No. F-00175 from Ciba-Geigy Corp., Plant Protection Division, Farmington, Connecticut, USA. Submitted to WHO by Novartis Crop Protection AG, Basel, Switzerland. GLP Compliant EPA-FIFRA 40 CFR, OECD GLP Principles and MAFF Japan. Guideline USEPA-FIFRA, pesticide assessment guidelines, subdivision F, hazard evaluation, section 81-8. Ellman, G.L., Courtney, K.D., Andres, V. & Featherstone, R.M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical PharmacoL, 7, 88. Glaza, S.M. (1993) Acute oral toxity study with D.z.n® diazinon MG87% in rats. Unpublished report No. HWI 6117-221 from Hazleton Wisconsin Inc., Madison, Wisconsin, USA. Submitted to WHO by Novartis Crop Protection AG, Basel, Switzerland. GLP Compliant EPA-FIFRA 40 CFR 160, OECD GLP Principles Annex 2, C(81)30 final proposed and MAFF Japan. Guideline USEPA 81-1. Meyer, L. (1999) Preliminary summary of a randomized, double-blind ascending, acute, oral dose study of diazinon to determine the no effect level (NOEL) for plasma and RBC cholinesterase activity in normal, healthy volunteers. Unpublished report No. 8373 from Covance Clinical Research Unit Inc., Madison, Wisconsin, USA. Submitted to WHO by Novartis Crop Protection AG, Basel, Switzerland. Consistent with the Declaration of Helsinki, GCP compliant 21 CFR 50,54, 56, 312,314. Not GLP compliant as not audited by Quality Assurance Unit. Potrepka, R.F. (1994) Acute cholinesterase inhibition time course study with D.Z.N® diazinon MG87% in rats. Unpublished report No. F-00185 from Ciba-Geigy Corp., Farmington, Connecticut, USA. Submitted to WHO by Novartis Crop Protection AG, Basel, Switzerland. GLP Compliant EPA-FIFRA 40 CFR 160, OECD GLP Principles Annex 2, C(81)30 and MAFF Japan. WHO (1999) Recommended Classification of Pesticides by Hazard and Guidelines to Classification 1998-1999 (WHO/PCS/98.21/Rev. 1), Geneva, International Programme on Chemical Safety.

DIAZINON 31-36 JMPR 2001

37

DIFLUBENZURON First draft prepared by I. Dewhurst Pesticides Safety Directorate, York, United Kingdom

Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution and excretion Biotransformation Effects on enzymes and other biochemical parameters Toxicological studies Acute toxicity Short-term studies of toxicity Long-term studies of toxicity and carcinogenicity Genotoxicity Reproductive toxicity Multigeneration studies Developmental toxicity Special studies: Toxicity of metabolites Observations in humans Comments Toxicological evaluation References

37 37 38 38 40 43 43 43 43 48 51 51 51 52 53 53 53 55 57

Explanation Diflubenzuron [ 1 -(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea] is an insecticide that acts by disrupting chitin synthesis and deposition. The toxicity of diflubenzuron was evaluated by the JMPR in 1981, 1984 and 1985 (Annex 1, references 36,42 and 44); an ADI of 0-0.02 mg/kg bw was established at the latter Meeting. This ADI was maintained by a 1994 WHO Core Assessment Group that prepared Environmental Health Criteria 184 (WHO, 1996). The present Meeting considered studies performed since the last review and older, re-submitted reports.

Evaluation for acceptable daily intake The studies submitted for this evaluation of diflubenzuron were performed over a period of approximately 30 years. All the studies were considered adequate for their intended purpose unless specifically stated in the text. Studies that contained statements of compliance with good laboratory practice (GLP) or had been subjected to quality assurance (QA) audits are identified in the reference list. Diflubenzuron has been known under a number of synonyms and codes, including DU112307, TH6040, Vigilante and Dimilin. The manufacturing process for diflubenzuron is reported to have been consistent over time (Dykstra, 2001), and the results obtained in the 1970s were considered applicable to the technical-grade material produced currently. DIFLUBENZURON 35-59 JMPR 2001

38

1.

Biochemical aspects (a)

Absorption, distribution and excretion Mice

When mice were given a single oral dose of diflubenzuron at 12,64,200 or 920 mg/kg bw, excretion was almost complete within 48 h. The cumulative percentage of the dose excreted in the urine decreased from 15% at the lowest dose to about 2% at the highest, showing that the relationship between urinary excretion and dose in mice is similar to that in rats (cited in Annex 1, reference 46). Rats The intestinal absorption of diflubenzuron in mammals decreased with increasing dose. Radiolabelled diflubenzuron, with the 14C label uniformly distributed in the anilino moiety, was administered by gavage to rats as a single dose of 4, 16, 48, 128 or 1000 mg/kg bw. Urine was collected every 24 h up to 120 or 144 h. The cumulative excretion in urine as a percentage of the dose decreased from 28% at the lowest dose to only 1 % at the highest, while the recovery remained constant (90%). In rats with cannulated bile ducts, the sum of the excretion in urine and bile decreased from 42% of the dose at the lowest dose to about 4% at the highest. In rats that had received radiolabelled diflubenzuron with the 14C in the carbamoyl group of the benzoyl moiety, 1 % of the radiolabel was found in expired air. In rats, 72 h after administration of a single oral dose of 5 mg/kg bw of double-labelled diflubenzuron (14C in the anilino moiety and 3H in the benzoyl moiety), 1.3% of the 14C and 3.5% of the 3H was retained in the carcass (cited in Annex 1, reference 46). An extensive investigation of the absorption, distribution and excretion of diflubenzuron was reported. Groups of Sprague-Dawley rats received [14C]diflubenzuron uniformly labelled in both phenyl rings (specific activity, 1 jaCi/mg or 19.5 [iCi/mg), by gavage in 1% gum tragacanth. Details of the dosing and sampling are given in Table 1. Samples were retained for subsequent determination of metabolites. Radiolabel was determined by liquid scintillation counting with correction against an external standard after appropriate processing. The results (Table 2) show that diflubenzuron at a dose of 5 mg/kg bw was moderately absorbed, about 30% of the administered dose being excreted in urine and bile, and was less completely absorbed at 100 mg/kg bw. In animals with bile cannulae, reduced excretion was seen in urine (about 7%), indicating some degree of enterohepatic recirculation. 14CO2 in exhaled air represented < 0.1% of the administered dose. The peak concentrations of radiolabel in blood (Cmax) were achieved 4 h after administration. The highest tissue concentrations at 4 h were seen in liver and fat. More radiolabel Table 1. Design of study of absorption, distribution and excretion of [14C] diflubenzuron in rats No. of animals

Dosing schedule

Routine samples2

Terminal samples

5/sex 5/sex

Urine, faeces, air

Fluids, tissues at 1 68 h

5/sex 3/sex 3/sex per time

1 x 5 mg/kg bw 14 x 5 mg/kg bw per day (12C) 1 x 5 mg/kg bw («C) 1 x 100 mg/kg bw 1 x 5 mg/kg bw 1 x 5 mg/kg bw

Urine, faeces, air Urine, faeces, air Blood

Fluids, tissues at 168 h Fluids, tissues at 168 h

3/sex

1 x 5 mg/kg bw

Bile, urine, faeces

Gastrointestinal tract, carcass at 24 h

1 /sex/time

1 x 5 mg/kg bw

-

Autoradiographyb

Fluids, tissues at 4, 14, 48 and 72 h

From Dunsire et al. (1990) a Urine and faecal samples were taken at 8 and 24 h, then every 24 h until 168 h. Air samples were taken at 8 and 24 h. Tail vein blood was taken at 0.25, 0.5, 1, 2, 4, 8, 16, 24 and 48 h. Bile samples were taken hourly for 24 h. b At times corresponding to plasma Cmax, Cmaxf2 ar>d 48 h

DIFLUBENZURON 37-59 JMPR 2001

39 Table 2. Absorption, distribution and excretion of [14C]diflubenzuron in rats (mean values) Dose (mg/kg bw) 14+ 1 >:5

1 x5

Urine (%) 0-8 h 0-24 h Faeces (%) 0-8 ha 0-24 h Total recovery (%)

1 x 100

Males

Females

Males

Females

Males

Females

8 20

9 20

4 14

4 12

1 3

1 2

16 73 97

11 75 99

<1 64 97

11 72 99

66 95 99

35 93 99

4h

4h

Time to blood Cmax/2 (h)

14 h

14 h

ND ND

ND ND

ND ND

ND ND

Plasma (ng equivalents per ml) Cmax Plasma (ng equivalents per ml) Cmax/2 48 h 168 h

695 187 13 3

752 260 •20 3

ND ND ND 4

ND ND ND 5

ND ND ND 0

ND ND ND 10

Liver (ng equivalents per ml) Cmax Liver (ng equivalents per ml) Cmax/2 48 h 168 h

2090

2440 1170

ND ND ND 153

ND ND ND 152

ND ND ND 330

ND ND ND 370

Erythrocytes (ng equivalents per ml) Cmax Erythrocytes (ng equivalents per ml) Cmax/2 48 ha 168 h

548 566 360 169

449 318 398 251

ND ND ND 157

ND ND ND 152

ND ND ND 590

ND ND ND 780

Bile (%) 0-8 h 0-24 h

3 19

3 15

ND ND

ND ND

ND ND

ND ND

Time to blood Cmax (h)

693 336 187

526 151

From Dunsire et al. (1990). ND, not determined a Great inter-animal variation

was found in erythrocytes relative to plasma with increasing duration after dosing. Autoradiography showed a wide distribution of radiolabel, the highest concentrations being found in the gastrointestinal tract. Excretion was relatively rapid, > 90% of the doses of 5 and 100 mg/kg bw being excreted within 24 h. Animals given repeated doses showed evidence of delayed absorption and excretion, but no change in the pattern of excretion or residues was seen at 7 days; it is unclear whether this was an effect of diflubenzuron or of increased age and body weight. The highest concentrations of residues of radiolabel 7 days after administration were found in erythrocytes and liver, but the total residual radiolabel represented < 1% of the administered dose. There were no notable differences between the sexes (Dunsire et al., 1990). The dermal absorption of [anilino-U-14C]diflubenzuron (purity, 97.2%; specific activity, 18 jiCi/mg) was investigated in groups of four male Sprague-Dawley rats (one control per time). A suspension of diflubenzuron (100 (il) in 0.25% gum tragacanth was applied to a 10-cm2 clipped area at a concentration of 0.005 or 0.05 mg/cm2 and kept under semi-occluded conditions. The animals were killed 1,4 or 10 h after application, and the skin site was washed five times with soap solution. Samples of excreta, cage wash, application site and carcass were analysed by liquid scintillation counting after appropriate processing. More than 96% was recovered in all groups, more than 80% of the applied radiolabel being removed in the first wash. The residue at the application site represented 4.5-6% of the applied dose. The mean absorption was < 0.5% in all groups (range, 0.03-1.2%). There was no evidence of increased absorption with longer exposure (Andre, 1996). Rabbits The dermal absorption of [anilino-U-14C]-diflubenzuron (purity, > 99%; specific activity, 11.2 M-g/g) was investigated in a group of eight male New Zealand rabbits. A 10% suspension of

DIFLUBENZURON 35-59 JMPR 2001

40

diflubenzuron in 1% gum tragacanth was applied at 150 mg/kg bw to shaved areas and occluded for 6 h, when the site was washed. Excretion of radiolabel in urine and faeces was determined over 48 h. The concentration of radiolabel in faeces was at the level of determination (< 1% of the applied dose), and there was about 0.1% in urine. Diflubenzuron is thus poorly absorbed through rabbit skin (de Lange, 1979). Cats Diflubenzuron labelled with 14C and 3H was administered orally to cats at a dose of about 7 mg/kg bw on day 10 of a 15-day dosing regimen with unlabelled diflubenzuron. Within 72 h of administration, 9% of the oral dose had been excreted in the urine and 77% of the 14C and 71% of the 3H in the faeces (Hawkins et al., 1980). (b)

Biotransformation

The basic metabolic pathways of diflubenzuron were established in early studies in rats and cows. The main pathway involves hydroxylation of the phenyl moieties of the intact compound. About 80% of the metabolites in rat urine were identified as 2,6-difluoro-3-hydroxybenzuron, 4chloro-2-hydroxydiflubenzuron and 4-chloro-3-hydroxy diflubenzuron. Approximately 20% underwent scission at the ureido bridge, and most was excreted as 2,6-difluorobenzoic acid. 4Chlorophenylurea was not detected in bile or urine in significant quantities (cited in Annex 1, reference 46). The metabolites produced by Sprague-Dawley rats after administration of [14C] diflubenzuron uniformly labelled in both phenyl rings were identified and characterized in samples from the study of Dunsire et al. (1990). Pooled (0-24 h) samples of urine, faeces or bile were prepared from males and females dosed once at 5 or 100 mg/kg bw or 15 times at 5 mg/kg bw per day (see Table 1). After extraction, the metabolites were determined by high-performance liquid chromatography (HPLC) separation, liquid scintillation counting and comparison with standards. The presence of conjugates was determined by the use of a glucuronidase-aryl sulfatase preparation. A specific investigation for the presence of 4-chloroaniline was performed, as routine methods failed to separate this compound from 4-chlorophenylurea. The metabolic profiles were similar for the two sexes. Biliary samples proved difficult to analyse, as 75-80% of the radiolabel was present in a five-component peak. Enzyme hydrolysis of bile increased the proportion of 2-hydroxydiflubenzuron from about 5% to 19%, at the expense of the major multi-component peak. Diflubenzuron represented about 7% of the biliary radiolabel. Initial investigations of faecal samples gave variable recovery rates, with diflubenzuron representing > 93% of the recovered radiolabel. A second method involving enzyme hydrolysis and dilution with control urine was used to investigate samples from animals given the high single dose and repeated doses. Various compounds were identified, the main peaks being diflubenzuron (6575% of recovered radiolabel), 2-hydroxydiflubenzuron (7-10%), 4-chlorophenylurea (5-6%) and 2,6-difluorobenzamide (2-4%). Investigations of urine samples gave reliable recoveries (85105%), and various metabolites were identified (Table 3), though again there was incomplete separation. The results showed that absorbed diflubenzuron was extensively metabolized. Enzyme treatment of the urine samples indicated conjugation of 2 '-hydroxydiflubenzuron and of diflubenzuron. The only notable difference between the groups was the higher concentration of unconjugated 2'-hydroxy diflubenzuron and the presence of peak B in the urine of animals given repeated doses. Specific investigations for 4-chloroaniline did not reveal its presence in bile or urine at a limit of determination of 7.5 ng/ml (Cameron et al., 1990).

DIFLUBENZURON 37-59 JMPR 2001

41

Table 3. Metabolites in urine of rats given [14C]diflubenzuron (% of recovered radiolabel) before and after enzyme hydrolysis Metabolite

Dose group

Males

Diflubenzuron 2,6-Difluorobenzoic acid 2,6-Difluorohippuric acid 2,6-Difluorobenzamidea 4-Chlorophenylurea 2-Hydroxydiflubenzuron Peak Ab Peak Bc a b c

1 4 + 1 x 5 mg/kg bw per day

1 x 100 mg/kg bw

1 x 5 mg/kg bw Females

Males

Females

Males

Females

Before

After

Before

After

Before After

Before After

Before After

Before After

1.5 22 7.4 19 10 6.8 27 0.8

3.8 29 7.4 7.0 11 18 16 3.0

0.4 21 8.7 20 10 7.7 26 1.0

4.0 27 7.9 6.7 12 17 15 3.6

0 30 8.5 25 5.5 4.4 22 0.6

0 28 9.4 27 5.2 2.8 23 0.2

0.3 22 11 21 8.1 12 16 6.1

0.6 25 8.3 20 8.2 12 17 2.6

4.3 29 9.1 6.6 14 15 12 5.0

4.5 29 9.8 6.2 11 15 14 4.0

4.0 27 6.6 7.1 13 17 12 4.4

4.4 25 7.9 8.6 13 19 14 4.1

Probably a two-component peak co-eluting with 2,6-difluorobenzamide standard Five components Mixture of 3'-hydroxydiflubenzuron, 4-nitrochlorobenzene and W-acetyM-chlorophenylurea

The production of 4-chloroaniline and 4-chlorophenylurea was investigated in four male Fischer 344 rats given a single dose of 104 mg/kg bw of [U-14C-anilino] diflubenzuron (specific activity, 9828 dpm/mg; radiochemical purity, 99.9%) in 1% gum tragacanthby gavage. Urine and faecal samples were taken after 20, 48, 72 and 96 h. Pooled 0-20-h samples were analysed by HPLC, liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. Faecal samples were extracted initially with acetonitrile, then sequentially with hexane, acetone and water. Most of the administered dose was excreted within 20 h, with 68% in faeces and 2% in urine. The only compound detected in faeces was diflubenzuron. Three metabolites were identified in significant amounts in urine: 4-chloroaniline-2-sulfate (45% of the total urinary radiolabelled residues), jV-(4-chlorophenyl)-oxamic acid (13%) and 4chloroacetanilide-2-sulfate (2%). Neither diflubenzuron, 4-chloroaniline nor 4-chlorophenylurea nor their Af-hydroxy derivatives were identified in urine. 2-Hydroxydiflubenzuron was present at only < 1 % of the total radiolabelled residues (Wang & Gay, 1999). A proposed metabolic pathway for diflubenzuron is presented in Figure 1. The metabolism of 4-chlorophenylurea, a major plant metabolite of diflubenzuron, was studied in four male Fischer 344 rats that received [U-14C-phenyl]-4-chlorophenylurea (specific activity, 1.9 Ci/mol; radiochemical purity, 97%) in 1% gum tragacanth by gavage at 198 mg/kg bw. Urine and faeces were collected at 20, 48, 72, 96, 120 and 144 h (termination). The total radiolabel in urine, faeces and carcass was determined by liquid scintillation counting after appropriate processing. Pooled 0-20- and 20-48-h faecal and urine samples were examined by HPLC, thin-layer chromatography, liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry and comparison with standards. Metabolites in faeces were identified after sequential extractions with acetonitrile, hexane, acetone and water. Urine samples were also investigated after treatment with glucuronidase or sulfatase. Over 90% of the administered dose was excreted in urine. Four faecal metabolites were identified, all of which were also found in urine. The major faecal metabolite was 4-hydroxyphenylurea (about 3% of the administered dose), with unchanged 4-chlorophenylurea present at 0.6%. Thirteen distinct peaks were obtained from urine samples, the major components being 4-chlorophenylurea-2-sulfate (25% of the administered dose), 4-hydroxyphenylurea (18%), 4-chlorophenylurea-2-glucuronide (17%), 4chlorophenylurea-3-sulfate (8%) andphenylurea-4-sulfate (7%). 4-Chloroaniline was not detected in urine at a limit of detection of < 0.02 ^ig/ml. The metabolic profile of 4-chlorophenylurea showed that it undergoes extensive ring hydroxylation and conjugation with either sulfate or glucuronide (Gay et al., 1999).

DIFLUBENZURON 35-59 JMPR 2001

42

Figure 1. Proposed metabolic pathway for diflubenzuron in rats

Bold arrows, major pathways; light arrows, minor pathways

DIFLUBENZURON 37-59 JMPR 2001

43

There is no clear reason for the marked differences between the findings of Cameron et al. (1990) and Wang and Gay (1999). The overall conclusion from these studies is that the primary steps in the metabolism of diflubenzuron are hydrolysis of the anilino ring, cleavage of the ureido bridge and conjugation (mainly to sulfate). The most recent data (Wang & Gay, 1999), and reputedly the most reliable, indicate that two compounds found as plant residues of diflubenzuron, 4-chloroaniline and 4-chlorophenylurea, might not be formed directly in rats. It is possible that hydroxylation of the anilino ring occurs before cleavage of the ureido bridge. Comparison of the results of Wang and Gay (1999) and Gay et al. (1999) suggests that 4-chlorophenylurea is not formed to a significant extent and that cleavage of diflubenzuron gives rise predominantly to 4chloroaniline derivatives. (c)

Effects on enzymes and other biochemical parameters

The concentrations of methaemoglobin and sulfhaemoglobin and the incidence of Heinz bodies were investigated in animals dosed once with a 25% w/w formulation of diflubenzuron. Groups of 15 male Swiss mice or male Wistar rats received Dimilin at 0 (co-formulants only) or 10 000 mg/kg bw in 1 % gum tragacanth by gavage. Samples of blood were taken 4 and 24 h after dosing. In mice, there were no effects on body weight, sulfhaemoglobin concentration or incidence of Heinz bodies. The methaemoglobin concentrations were increased (p < 0.003) at 4 h but not at 24 h. The increase at 4 h was relatively small (2.2% versus 2.6%), but the ranges (1.32.5% versus 1.5-3.3%) indicated a treatment-related effect. A similar pattern was found in rats, with no effects on body weight, sulfhaemoglobin concentration or incidence of Heinz bodies. The methaemoglobin concentrations were increased (p < 0.01) at 24 h but not at 4 h. The increase at 24 h was relatively small (1.5% versus 1.9%), but there was a small overlap in the ranges (1.21.8% versus 1.6-2.1 %), showing a clear treatment-related effect. A single high dose of diflubenzuron (2500 mg/kg bw) can thus increase methaemoglobin concentrations (Keet et al., 1997a,b).

2.

Toxicological studies (a)

Acute toxicity

The results of studies if acute toxicity show that diflubenzuron (purity, 99.6%) has little acute toxicity when given by the oral, inhalation or dermal route. The LD50 in mice and rats given diflubenzuron in 1 % tragacanth by gavage was > 4600 mg/kg bw (van Eldik, 1973). In rats treated dermally for 24 h, the LD50 was > 10 000 mg/kg bw (Koopman, 1977), and in rats treated by wholebody inhalation of a preparation with a mass median aerodynamic diameter < 5 Jim, the LC50 was > 2.9 mg/1 of air (Berczy et al., 1973). No clinical signs were seen in these studies, although the level of detail in the study reports was inadequate to permit independent confirmation. No haematological investigations were performed. (b)

Short-term studies of toxicity Rats

Groups of 40 Sprague-Dawley rats of each sex (controls, 90 of each sex) received diets containing diflubenzuron (two lots; purity, 96% and 97.2%) at a concentration of 0, 160, 400, 2000, 10 000 or 50 000 ppm. About half the animals were killed at week 7 and the remainder at 13 weeks. Routine clinical investigations were performed. Samples were taken from 10 animals of each sex per group for haematological, clinical chemical and urinary investigations in weeks 7 and 13. All animals were examined grossly, and extensive histological investigations were performed on controls and animals at the highest dose, with more limited examination (but including liver, spleen and marrow) of other groups. DIFLUBENZURON 35-59 JMPR 2001

44

There were no treatment-related effects on the mortality rate, clinical signs or food consumption. Body-weight gain was reduced by about 20% in females receiving 50 000 ppm and in males receiving > 2000 ppm, but with no clear dose-response relationship. Haematology at week 7 showed a range of dose-related alterations in erythrocyte parameters in animals of each sex receiving doses > 400 ppm, with minimal effects at 160 ppm (Table 4). The haematological findings were reproduced at week 13. Alterations in a number of clinical chemical parameters were seen in animals at the two higher doses, but the findings were sporadic, generally occurred in only a few animals in a group and considered to be not biologically significant. The absolute and relative weights of the spleen were increased in males and females at doses > 400 ppm and in males receiving 160 ppm for 7 weeks (Table 4). At week 13, the absolute weight of the liver was increased (by > 10%) in females at the highest dose, and the relative weights were increased (by > 10%) in males at the highest dose and females at doses > 400 ppm. Pathological lesions related to treatment were chronic hepatitis, haemosiderosis and congestion of the spleen and erythroid hyperplasia of the bone marrow in all treated groups, and haemosiderosis in the liver at doses > 400 ppm. The lesions increased in severity with increasing dose and duration of dosing (Table 4). NoNOAEL could be identified, as there were small but statistically significant increases in methaemoglobin concentration, with associated findings in the spleen and bone marrow at the lowest dose, 160 ppm, equivalent to 8 mg/kg bw per day (Burdock et al., 1980; Goodman, 1980). Groups of 10 CrliCD BR (VAF/Plus) rats of each sex received diflubenzuron (purity, 96.7%) in 0.25% aqueous gum tragacanth dermally for 6 h/day for 21 days under semi-occlusive dressings at a dose of 0,20,500 or 1000 mg/kg bw per day on shaved, unabraded sites representing about 10% of the body area at the two higher doses and 1% at the lowest dose. Routine clinical investigations were performed. Blood samples for haematological and clinical chemical analysis were taken before sacrifice. All animals were examined grossly, and liver, kidney and skin from controls and animals at the highest dose were examined histologically. Table 4. Haematological and histopathological findings in rats fed diets containing diflubenzuron for 7 or 13 weeks Sex

Finding

Concentration in feed (ppm)

0

160

Males Females Males Females Males Females Males Females Males Females Males Females

7.7 7.1 16 15 1.7 1.7 0.2 0.2

7.5 6.7 15 15 2.1 2.1 0.3

Males Females Males Females Males Females Males Females Males Females Males Females

0.7 0.5

400

2000

10000 50000

Week?

Erythrocyte count (106/mm3) Haemoglobin (g/dl) Reticulocytes (% RBC) Methaemoglobin (%) Sulfhaemoglobin (%) Spleen weight (g) Week 13 Spleen weight (g) 3

Chronic hepatitis (mean severity) Liver haemosiderosis (%)

3

Spleen haemosiderosis (severity) Spleen congestion (%)

Bone marrow, erythroid hyperplasia (%)

From Burdock et al. (1980) and Goodman (1980) */><0.05 a Control incidence > 90% V<0.01 for trend

DIFLUBENZURON 37-59 JMPR 2001

0.04 0.03

0.7 0.5

0.91 0.77

0 0 1.7 1.3 9 4 0 0

7.0* 6.5*

15* 15

6.5* 5.8*

14* 14*

0.5

0.6

5.4* 5.7* 0.5* 0.5* 0.07 0.06 1.1* 0.8*

0.7 0.5 1.3

0.9* 0.6*

1.1* 0.9*

0.3* 0.08 0.03 0.8*

0.81

0 5 2.7 3.7 21 15 37* 22*

3.0* 3.4* 0.3* 0.4*

0.1 0.06 0.9*

1.4 1.0 45* 60* 3.9 4.3 70* 30* 80* 37*

1.6 1.3 90* 85* 4.2 4.1 95* 70*

6.6* 5.6*

14* 14* 5.6* 5.8* 0.5* 0.5*

1.3* 1.1*

0.1 0.1* 1.2* 0.9*

2.5 1.7 100*

95* 4.2 3.9 80* 85*

100*

100*

79*

82*

5.9* 5.3*

14* 14* 7.9* 9.0* 0.7* 0.7* 0.2* 0.2* 1.7* 1.3* 1.6* 1.2* 2.8b 1.1" 100* 100* 4.1b 4.3b

95* 100*

95* 95*

45

One female at 500 mg/kg bw per day and one at the highest dose died on day 9. As these were isolated findings and there were no subsequent deaths, the relationship to exposure to diflubenzuron is uncertain. Body weight, food consumption, clinical signs, clinical chemical end-points and gross and microscopic appearance were unaffected. There were no signs of irritation at the application site. Reductions in erythrocyte parameters were seen in females at 500 mg/kg bw per day and in animals of each sex at 1000 mg/kg bw per day (Table 5), together with increased incidences and severity of polychromasia, hyperchromasia and anisocytosis in animals of each sex at doses > 500 mg/kg bw per day. Statistically significant increases in leukocyte counts were seen in males at > 500 mg/kg bw per day and a nonsignificant increase in females at the highest dose (Table 5). The findings were linked to increased numbers of segmented neutrophils and lymphocytes, but the association with diflubenzuron is unclear. The NOAEL was 20 mg/kg bw per day on the basis of haematological effects at doses > 500 mg/kg bw per day (Goldenthal, 1996). Groups of 10 Sprague-Dawley rats of each sex were exposed for 6 h/day, 5 days/week for 4 weeks, by nose only, to atmospheres of diflubenzuron (purity, 96.5%), which were generated from a jet mill and contained particles with a mass median aerodynamic diameter of about 2 mm and a gravimetric concentration of 0.2 (control), 12, 34 or 110 mg/m3. The concentrations varied during the study by ± 10%, but homogeneity within the chamber was confirmed. The animals were observed routinely during and after exposure, with a basic neuro-functional assessment. Samples for urinary analysis, haematology and clinical chemistry were taken before sacrifice. All animals were examined grossly. A wide range of tissues from controls and those at the highest concentration were examined microscopically, with kidney, liver and lungs from all other animals. One animal died during the terminal bleeding. Body weight, food consumption, clinical signs, urinary parameters and gross and microscopic appearance were unaffected. A reduction in 'grid count' was evident in the neuro-functional assessment of males and females exposed to 110 mg/m3. Statistically significant (p< 0.05) decreases in some erythrocyte parameters were seen at this concentration, with evidence of a concentration-related effect (Table 6). Slight but statistically significant increases in bilirubin concentrations were seen in both males and females at the highest level of exposure. The NOAEL was 34 mg/m3, approximately 10 mg/kg bw per day, Table 5. Erythrocyte parameters in rats exposed dermally to diflubenzuron for 21 days End-point

Dose (mg/kg bw per day)

3

Erythrocyte count (lOVmm ) Haemoglobin (g/dl) Erythrocyte volume fraction(%) Methaemoglobin (%) Leukocyte count (lOVmm 3 )

Controls

20

500

Males Females

Males Females

Males

Females

Males Females

8.0 16 48 0.1 9.9

8.1 16 49 0.2 11

7.8 15 47 0.2 13*

7.0* 14* 44* 0.2 8.7

7.6 15* 46 0.3* 13*

7.9 15 48 0.1 8.2

7.9 16 48 0.1 7.9

1000

7.1* 14* 45* 0.2* 11

From Goldenthal (1996) */><0.05

Table 6. Erythrocyte parameters in rats exposed by inhalation to diflubenzuron for 4 weeks Concentration (mg/m3 of air)

End-point

6

3

Erythrocyte count (10 /mm ) Haemoglobin (g/dl) Erythrocyte volume fraction (%) Methaemoglobin (%)

Controls

12

34

110

Males Females

Males Females

Males Females

Males Females

8.3 16 49 0.1

8.0 15 47 0.2

8.1 15 46 0.2

7.8 14* 45* 0.2

7.7 15 46 0.1

7.6 15 46 0.2

7.6 15 45 0.1

7.2 14* 44* 0.2

From Newton (1999) *p<0.05

DIFLUBENZURON 35-59 JMPR 2001

46

on the basis of effects on behaviour, haematological end-points and bilirubin concentration at 110 mg/m3 (Newton, 1999). Rabbits Diflubenzuron (purity unknown) was reported not to be irritating to intact or abraded skin of rabbits, although the level of detail in the report was inadequate to permit independent confirmation (Taylor, 1973). Diflubenzuron (purity, 99.6%) was slightly irritating to the eyes of New Zealand white rabbits after instillation of 40 mg (0.1 ml). The findings were similar when the eyes were rinsed 5 min or 24 h after administration (Davies & Liggett, 1973). Guinea-pigs The skin sensitizing potential of diflubenzuron (purity, 95.6%) was investigated in groups of 10 CRL HA BR guinea-pigs exposed in a 'maximization' protocol. For induction, 10% diflubenzuron in maize oil with Freund's complete adjuvant was injected or 30% diflubenzuron in Vaseline was applied topically. The animals were challenged by application of 10% or 30% diflubenzuron in Vaseline. Mild responses were seen after induction by either route. After challenge, mild reactions were seen in one control and two test animals. Diflubenzuron was not a skin sensitizer in this study (Prinsen, 1992). Dogs Groups of three male and three female pure-bred beagle dogs aged 16-23 weeks received diets (400 g/day) containing diflubenzuron (purity unspecified) at a concentration of 0,10,20,40 or 160 ppm for 13 weeks. The achieved intakes were 0, 0.4, 0.8, 1.6 and 6.4 mg/kg bw per day. The animals underwent routine clinical investigations. Samples for haematology were taken before dosing and at weeks 2, 4, 6 and 12. Clinical chemistry (limited), urine analysis and ophthalmological investigations were performed before dosing and at weeks 6 and 12. All animals were examined grossly at sacrifice, and an extensive range of tissues were examined microscopically. The results were presented for the two sexes combined. There were no deaths or adverse clinical signs. Body weights and food and water consumption were not affected by treatment. The body weights fluctuated considerably during the study, but this was to be expected, given the spread of ages, and there was no indication of an association with treatment. The results of urine analysis, ophthalmology and gross and histopathological examinations were similar in all groups. Increases in aspartate aminotransferase activity were seen in animals at the highest dose in weeks 6 and 12, but the finding is of dubious significance as it was not reproduced when the samples were re-tested. Haematological end-points were similar in all groups at week 2, but, by week 4, the haemoglobin concentration of animals at 160 ppm was reduced by about 10%. At week 6, both the haemoglobin concentration and erythrocyte count were reduced and the methaemoglobin and free haemoglobin concentrations were increased in animals at 160 ppm. Haematological end-points were similar in all groups at week 12, although there was a clear increase in the myeloid:erythroid ratio in bone marrow in dogs at 160 ppm (1.2 versus 0.7 in controls). There was an indication of an increase in spleen weight (by about 40%) at 160 ppm, but inter-group variation was high and the ranges in treated and control groups showed considerable overlap. The NOAEL was 40 ppm, equal to 1.6 mg/kg bw per day, on the basis of alterations in haematological end-points and bone marrow at 160 ppm (Chesterman etal., 1974). Groups of six male and six female beagle dogs (controls, 12 of each sex) received gelatin capsules containing diflubenzuron (purity, 97.6%) at a dose of 0, 2, 10, 50 or 250 mg/kg bw per DIFLUBENZURON 37-59 JMPR 2001

47

day. The animals underwent routine clinical investigations. Blood samples for haematology and clinical chemistry were taken before dosing and at weeks 4,7,13,26 and 51. Urine was analysed before dosing and at weeks 7,13,26 and 51. Ophthalmoscopy was performed before dosing and at weeks 26 and 51. All animals were examined grossly and microscopically. One female at the highest dose died from liver failure and one female at 50 mg/kg bw per day died from pneumonia, but neither death was clearly related to treatment. No consistent differences were found between control and test animals in respect of clinical signs, food consumption, body weight, water consumption or urinary parameters. A range of effects related to impaired erythrocytes were seen at 50 and 250 mg/kg bw per day from week 13 onwards. At doses > 50 mg/kg bw per day, signs of haemolytic anaemia, destruction of erythrocytes and compensatory regeneration of erythrocytes were observed. Increases in methaemoglobin and sulfhaemoglobin concentrations were evident at doses > 10 mg/kg bw per day (Table 7). The severity of the haematological alterations appeared to increase with duration of dosing, stabilizing at week 26 and with some evidence of adaptation at week 52. Increased sulfhaemoglobin concentrations seen in animals at the lowest dose in week 26 were considered not to be adverse, as they were not statistically significant and were not seen at 52 weeks. Other findings included increased platelet numbers at doses > 10 mg/kg bw per day in females and > 50 mg/kg bw per day in males; increased lactic dehydrogenase activity from week 7 in animals of each sex receiving 250 mg/kg bw per day and increases in liver (10%) and spleen weights (35%) at doses > 50 mg/kg bw per day. The only notable histopathological findings were in the liver, comprising increased pigmentation of Kupfer cells and macrophages (Table 7). The latter effect in males at the lowest dose was considered not biologically significant as it was graded 'minimal' and the frequency was within the normal range of background findings. The NOAEL was 2 mg/kg bw per day on the basis

Table 7. Findings in dogs given diflubenzuron in capsules for 52 weeks Finding

Week 4 Sulfhaemoglobin (%) Week 13 Methaemoglobin (%) Heinz bodies (no. of animals) Week 26 Haemoglobin (g/dl) Sulfhaemoglobin (%) Methaemoglobin (%) Heinz bodies (no. of animals) Week 52 Haemoglobin (g/dl) Methaemoglobin (%) Sulfhaemoglobin (%) Heinz bodies (no. of animals)

Sex

0

2

10

50

250

Males Females

0.1 0

0 0

0.1 0

0.3*

0.7* 0.4*

Males Females Males Females

1.1 1.4 0 0

1.1

1.2 0 0

1.6 1.8 0 0

1.8 1.5 0 0

4.3* 3.3*

Males Females Males Females Males Females Males Females

17 18 0 0 1.6 1.4 0 0

16 18 0.2 0.4 2.1 1.7 0 0

17 18

17 17*

16* 16*

0.6* 0.8* 2.4*

0.8* 1.0* 4.1* 2.5*

1.5* 1.7* 3.6* 3.4*

0 0

0 3*

5* 5*

Males Females Males Females Males Females Males Females

18 18 1.5 1.3 0 0 0 0 12

17 18 1.4 1.5 0 0 0 0 6

18 18

17 16*

17 17*

3.2* 2.3* 0.2* 0.3*

2.9* 2.4* 0.6* 0.4*

3.6* 3.4* 0.9* 1.2*

0 0 6

0 1 6

4* 3 6

Males Females Males Females Males Females

0 1 0 2 0 3

3* 2 0 0 0 2

0 1 3* 5* 3* 5*

1 1 5* 3 4* 3

1 1 5* 5* 5* 6*

Total no. of tissues/sex per group Pigmented Kupfer cells (minimal) Pigmented Kupfer cells (> mild) Pigmented macrophages in liver (> mild)

Dose (mg/kg bw/day)

2.6

0.2

0 2

From Greenough et al. (1985) *p<0.05

DIFLUBENZURON 35-59 JMPR 2001

48

of significant effects on methaemoglobin and sulfhaemoglobin concentrations, platelet counts and hepatic pigmentation at 10 mg/kg bw per day (Greenough et al., 1985). (c)

Long-term studies oftoxicity and carcinogenicity Mice

Groups of 52 HC/CFLP mice of each sex (controls, 104 of each sex) received diets containing diflubenzuron (purity, 97.6%) at a concentration of 0,16,80,400,2000 or 10 000 ppm for up to 91 weeks. Groups of 24 controls and 12 mice receiving diflubenzuron were killed at weeks 26, 52 and 78 for interim investigations. Routine investigations were supplemented by haematology (weeks 26, 52, 78 and 91), clinical chemistry (weeks 24, 50, 76 and 89) and urine analysis (weeks 25,51,77 and 90). All animals were examined grossly, and a wide range of tissues from all animals were examined microscopically. Table 8. Findings in mice given diflubenzuron in the diet for up to 93 weeks Sex

Finding

Mean intake (mg/kg bw per day) Week 26 Leukocyte count (lOVmm3) 3

Erythrocyte count (lOVmm ) Haemoglobin (g/dl) Mean cell haemoglobin (pg) Methaemoglobin (%) 3

Platelet count ( 1 OVmm ) Liver weight (g) Spleen weight (g) Week 78 Leukocyte count (lOVmm3) 3

Erythrocyte count (lOVmm ) Haemoglobin (g/dl) Mean cell haemoglobin (pg) Methaemoglobin (%) Sulfhaemoglobin (%) Platelet count (lOVmm3) Liver weight (g) Spleen weight (g)

0

16

80

400

2000

10000

Males Females

0 0

1.2 1.4

6.4 7.3

32 35

160 190

840 960

Males Females Males Females Males Females Males Females Males Females Males Females Males Females Males Females

6.5 6.5 7.5 8.7 14 16 19 18 1.6 1.6 890

7.7 5.3 7.8 9.1 15 16 19 17 1.9 1.8 930

8.7 6.4 7.4 8.4 14 16 19 18

8.1 6.7 7.6 8.9 14 16 19 18

7.2 11* 7.1 8.3 14 17* 20* 20*

7.0 8.1 15 17* 22* 21*

2.2* 2.3*

1100

1100

1100

2.9* 3.4* 1200* 1400*

2.6 2.5

2.7 2.3

2.5 2.5

2.6 2.4

4.9* 5.3* 1200* 1400* 3.0*

5.9* 5.9* 1200* 1400 3.0*

0.13 0.15

0.16 0.14

0.14 0.16

0.16 0.22*

2.6 0.22* 0.29*

2.8 0.29*

10 7.1 7.3 7.9 14 15 21 19

10 5.7 7.1 8.1 15 15 21 18

13 5.9 6.8 6.9 12 12 18 18

14 5.5 6.9 7.2 14 14 21 20

9.5 9.4 6.9 7.2 15 14 22 20

13 4.9 6.6

0.66

0.87

1.3 0.1 0.1

1.4 0.2

2.3* 3.6* 4.0* 3.0* 1200

3.2* 5.2* 3.8* 3.5* 1500* 1300

4.6* 6.0* 6.4* 3.7* 1600* 1500

Males Females Males Females Males Females Males Females Males Females Males Females Males Females Males Females Males Females

Week 91 Total no. of tissues/sex per group Hepatocyte enlargement Hepatocyte vacuolation Pigmented Kupfer cells Splenic extramedullary haematopoiesis Splenic siderocytes From Colleyetal. (1984) *p < 0.05

DIFLUBENZURON 37-59 JMPR 2001

Dietary concentration (ppm)

Males Females Males Females Males Females Males Females Males Females

900

11* 8.3*

0.41*

6.3*

16* 15 24* 23*

1000

0.6* 1000

1.4* 1.9* 0.4* 0.6* 1100

590 3.4 2.9

600 3.0 2.9

700 3.9 3.4

930 3.5 3.2

0.22 0.27

0.48 0.22

0.97 0.38

0.52 0.50

0.22 0.55

4.3* 0.33 0.46

104

52

52

52

52

52

36 15 22 33 0 1 27 37 5 15

12 4 17 17 0 0 7 18 1 6

11 5 9 17 1 1 15 19 2 13

23 11 12 17 1 0 21 19 16* 30*

35* 15* 18 12 3 ±4 19 25 32* 31*

31* 29* 26* 26 15* 11* 22* 25 30* 43*

3.6 3.9

3.5

49

The homogeneity and achieved concentrations in the diet were acceptable, the mean achieved intakes covering an approximately 800-fold range (1.2-960 mg/kg bw per day; Table 8). The mortality rates were similar in all groups, survival to week 78 being > 50% except for females receiving 80 or 2000 ppm. Clinical signs (blue-grey colouration of the extremities) were noted at week 1 in animals receiving doses > 2000 ppm and subsequently in animals in all groups receiving doses > 80 ppm. Body weights and food consumption were similar in all groups. Water consumption was increased at some times for females receiving doses > 2000 ppm, with an associated increase in urine volume. Urine analysis did not indicate any impairment of renal function. Statistically significant, dose-related changes were seen in a number of haematological parameters from week 26 onwards, in animals receiving doses > 80 ppm (Table 8). Statistically significant increases in the concentrations of sulfhaemoglobin were seen in animals of each sex at 16 ppm at 52 weeks and in females at week 78. A dose-related increase in the incidence of Heinz bodies was seen consistently in mice at doses > 400 ppm and sporadically in males at 80 ppm. Increased serum alkaline phosphatase and aspartate aminotransferase activities were seen in males and females at the highest dietary concentration at week 26 and in males at this concentration subsequently. Gross pathological examination ahowed increased liver and spleen weights (Table 8) and cyanotic skin as the only consistent findings. Histopathological examination confirmed that the liver and spleen were the primary target organs for non-neoplastic effects (Table 8). No increase in tumour incidence was associated with administration of diflubenzuron. The increased sulfhaemoglobin concentrations at 16 ppm are of questionable biological significance as they occurred in only a proportion of animals in the group, and the frequency was within the range of fluctuating control values seen during the study. The NOAEL was 16 ppm, equal to 1.2 mg/kg bw per day, on the basis of increased methaemoglobin concentrations and incidences of cyanosis at 80 ppm. The NOAEL for carcinogenicity was the highest concentration tested, 10 000 ppm, equal to 840 mg/kg bw per day (Colley et al., 1984). Rats Groups of 60 CD rats of each sex were fed diets containing diflubenzuron (purity unspecified) at a concentration of 0, 10, 20, 40 or 160 ppm for 2 years. All animals were investigated for clinical signs, body weight, food consumption and gross and microscopic appearance of a limited range of tissues. Samples from controls and animals at the highest concentration were taken at 3,6,12,18 and 24 months for determinations of haematological endpoints (10 of each sex), limited clinical chemistry (five of each sex) and urinary parameters (five of each sex). Ophthalmological investigations were performed on controls and animals at the highest concentration at 3, 6, 12, 18 and 24 months. Haematological investigations were performed on animals at 40 ppm (10 of each sex) at 12, 18 and 24 months. The achieved dietary concentrations and homogeneity were not confirmed. The survival rate was unaffected by treatment but was low, < 30% of animals surviving in all groups at termination. There were no treatment-related effects on urinary, clinical chemical or ophthalmic end-points, food consumption or body-weight gain. Statistically significant (p < 0.05) increases in methaemoglobin concentrations were seen at 12 and 18 months in males and females receiving 160 ppm but not in those at 40 ppm. Sulfhaemoglobin was unaffected by treatment, but the free haemoglobin concentration was reduced in groups at the highest concentration at 3 and 12 months. No increase in the incidence of tumours was seen in treated animals, although the poor survival and limited range of tissues examined severely limited the power of this study to detect a rumorigenic compound. The NOAEL was 40 ppm, equivalent to 2 mg/kg bw per day, on the basis of haematological changes at 160 ppm. The NOAEL for carcinogenicity was the highest concentration tested, 160 ppm, equivalent to 8 mg/kg bw per day (Hunter et al., 1976; Colley & Offer, 1977)

DIFLUBENZURON 35-59 JMPR 2001

50

Groups of 50 Sprague-Dawley rats of each sex (controls, 100 of each sex) were fed diets containing diflubenzuron (purity, 97.6%) at a concentration of 0, 160, 620, 2500 or 10 000 ppm for 2 years. All animals were investigated for clinical signs, body weight, food consumption and gross and microscopic appearance of a wide range of tissues. Haematological investigations were performed on 10 rats of each sex per group at 12 and 24 months. No clinical chemical, urinary or ophthalmological investigations were performed, but as these parameters were unaffected in rats at 160 ppm in a previous study, this is considered not to be a significant omission. The achieved dietary concentrations and homogeneity were satisfactory. The survival rate was similar in all groups and was > 50% at termination. Food consumption was similar in all groups, but body-weight gain was reduced by > 10% in females at 25 00 and 10 000 ppm from week 4, achieving statistical significance at week 26. A range of erythrocyte parameters were altered in both males and females at concentrations > 620 ppm at 12 and 24 months, although there was no marked progression with duration of dosing (Table 9). The methaemoglobin and sulfhaemoglobin concentrations were increased significantly in males at the lowest concentration at 12 months, but there was no clear dose-response relationship, and the biological significance of the finding is debatable. Increased numbers of reticulocytes and the extent of bone-marrow hyperplasia indicated the rats were attempting to respond to the effects on erythrocytes. The only gross finding was enlarged spleens, and this was confirmed by the data on organ weights. The main treatmentrelated histopathological findings were pigmented (golden brown) macrophages in the spleen and liver and erythroid hyperplasia of the bone marrow. The background incidence of pigmented Table 9. Findings in rats given diflubenzuron in the diet for up to 104 weeks Finding

Sex

Dietary concentration (ppm)

0 Mean intake (mg/kg bw per day) Week 52 Erythrocyte count (106/mm3) Haemoglobin (g/dl) Erythrocyte volume fraction (%) Reticulocyte count (%) Methaemoglobin (%) Sulfhaemoglobin (%) Week 104 Methaemoglobin (%) Sulfhaemoglobin (%) Myeloid:erythroid cell ratio Spleen weight (g)

160

620

2500

10000

28 37

112 128

472 612

Males Females

0 0

7.1 9.3

Males Females Males Females Males Females Males Females Males Females Males Females

8.7 8.1 17 15 48 46 0.9 1.0 0.2 0.6 0.1 0.1

8.3 7.8 16 15 48 46 0.8 1.1

Males Females Males Females Males Females

0.8 0.7 0.2 0.2 2.5 1.9

1.1 1.3 0.2 0.3 1.6

Males Females

1.0 0.7

2.0*

0.9

7.6*

7.6 15* 14 44 45 1.6 1.8 1.5 1.4

8.0 7.1*

16 14* 46 43 1.7* 3.1* 2.4* 1.9*

7.4* 6.5*

15* 13* 43* 41* 2.1* 5.0* 2.5* 2.2* 1.1*

0.9* 1.0*

0.7 0.1

1.3

1.8* 2.1*

1.8* 2.3*

0.8

0.4

1.1* 1.5*

1.1*

1.1*

0.6 0.6 1.8 1.3

1.3

1.1*

1.0 0.9

1.3 0.9

1.4* 1.1*

1.5* 1.2*

1.3*

0.2

1.6*

0.4

1.5

Histopathological findings Sternum, marrow hyperplasia (%)

Stomach, acanthosis or hyperkeratosis (%)

Males Females Males Females Males Females Males Females Males

Stomach, gastritis (%)

Males

Sternum, erythroid hyperplasia (%) Liver, pigmented macrophages (%) Spleen, pigmented macrophages (%)

From Burdock et al. (1984) *p<0.05

DIFLUBENZURON 37-59 JMPR 2001

16 4 4 1 22 26 66 80 1

10 6 6 2 20 38 84 90 0

8 2 26* 12* 50* 74* 86 100 3

60* 6 38* 22* 82* 80* 88 94 11*

72* 2 30* 26* 72* 86* 74 100 24*

1

0

0

7*

21*

51

macrophages in the spleen was high, however, and there was no clear dose-response relationship. Evidence of irritation in the stomach was seen in males receiving concentrations > 2500 ppm that survived to termination. The overall incidence of tumours was low, with no evidence of atypical findings or any dose-response relationship. No NOAEL for toxiciry could be identified, as the increased methaemoglobin and sulfhaemoglobin concentrations at 160 ppm (equal to 7.1 mg/kg bw per day) were consistent with the pattern of toxicity of diflubenzuron seen at other doses. The NOAEL for carcinogenicity was the highest dietary concentration, 10 000 ppm (equal to 470 mg/kg bw per day) (Burdock et al., 1984). (d)

Genotoxicity

Diflubenzuron has been tested for genotoxicity in an adequate range of studies in vitro and in vivo (Table 10). The overall quality of the studies was acceptable, positive controls giving satisfactorily positive results and the concentration ranges of diflubenzuron being sufficient to induce signs of toxicity or precipitation. Diflubenzuron was not genotoxic in vitro (in the presence or absence of metabolic activation) or in vivo. (e)

Reproductive toxicity (i)

Multigeneration studies

In a two-generation study, groups of 32 male and 32 female Crl:CD(SD)BR (VAF/Plus) rats (28 of each sex in the FI generation) received diets cpntaining diflubenzuron (purity, 97.1%) at a concentration of 0, 500, 5000 or 50 000 ppm, 70 days before mating (1:1). The animals were examined routinely for clinical signs and body weight. Litters were culled, when possible, to four Table 10. Results of assays for genotoxicity with diflubenzuron End-point

Reference

Test system

Concentration or dose

S. typhimurium TA98, TA100,TA1535,TA1537, TA1538

0, 8, 40, 200, 1000 ng/plate 96.9

Negative + S9 Negative - S9

Koorn(1990)

Reverse mutation

5. typhimurium TA98, TA100,TA1535,TA1537, T A 1 5 3 8 ; Saccharomyces cerevisiae D4

0,0.1, 1,10, 500 fig/plate

98.5

Negative + S9 Negative -S9

Brusick&Weir(1977a)

Reverse mutation

S. typhimurium TA98, TA100,TA1537,TA1538

0,10, 100, 1000 ^ig/plate

> 99

Negative + S9 Negative - S9

MacGregoretal. (1979)

Forward mutation

L5 1 78Y mouse lymphoma cells

0,1,5,19,38,75,150, 300 ng/ml

>99

Negative + S9 Negative -S9

MacGregoretal. (1979)

Cell transformation

BALBc/3T3 cells

0,20,40,80,160, 310ng/ml

98.5

Negative

Brusick&Weir(1977b)

Unscheduled DNA synthesis

WI-38 human cells

0, 50, 100, 500, 1000 ng/ml 98.5

Negative +S9 Negative - S9

Brusick&Weir(1977c)

Unscheduled DNA synthesis

Wistar rat hepatocytes (primary culture)

0,1,3,10,33,100, 333 ng/ml

Negative

Enninga(1990)

Chromosomal aberrations

Chinese hamster ovary cells

0, 100, 150, 200, 250 ng/ml 97.6

Negative +S9 Negative - S9

Taalman & Hoorn (1986)

In vivo Dominant lethal mutation

Albino (CR) mice

1 000, 2000 mg/kg bw (in com oil, intraperitoneally)

Negative

Arnold etal. (1974)»

Micronuclei

Swiss mice, bone marrow

0, 1 5, 1 50, 1 500 mg/kg bw > 99 (in corn oil, by gavage)

Negative

MacGregoretal. (1979)

In vitro Reverse mutation

a

Purity (%) Results

96.9

NR

Not validated (IBT study)

DIFLUBENZURON 35-59 JMPR 2001

52

of each sex on day 4. Blood samples for haematological examination were taken from F0 and Fj parents before sacrifice. All pups and parental animals were examined grossly, whereas histopathological examinations were limited to the reproductive organs, liver and spleen of parental animals. Routine investigations of reproductive performance were supplemented with evaluations of developmental end-points including surface righting reflex, startle reflex, air righting reflex, vaginal opening and preputial separation. The homogeneity and achieved concentrations of diflubenzuron in the diet were acceptable. The mean achieved intakes were 0,42,430 and 4300 mg/kg bw per day for males and 0, 36, 360 and 3800 mg/kg bw per day for females. Two deaths occurred, of a male at the highest dose and a female at the lowest, neither of which appeared to be related to treatment. The only clinical sign associated with treatment was pale faeces at the highest dose. Animals at this dose had reduced food consumption at week 1, but not subsequently. Water consumption was increased in all treated groups, but this was considered to be secondary to the high concentrations of diflubenzuron in the diet and not biologically significant. Reproductive parameters and sperm quality and quantity were not impaired by exposure to diflubenzuron. Pup weights were reduced in a dose-related manner in the F0 generation, but not in the F! generation, and the reduction achieved statistical significance (p < 0.05) between day 4 and weaning in animals at 50 000 ppm. The body-weight gain of treated animals after weaning was similar to or greater than that of controls. Attainment of the air righting reflex and startle response was enhanced slightly in diflubenzuron-treated FO litters but unchanged in FI animals. Statistically significant (p < 0.01) alterations in erythrocyte parameters and increases in lymphocyte counts and platelet numbers were seen in all parental groups. The spleen was the primary target organ, with increases in weight, congestion and haemosiderosis at all doses and an increase in the incidence of congested red pulp in F0 animals at the two higher doses. Increased incidences of centrilobular hepatocyte hypertrophy were seen in both generations at concentrations > 5000 ppm, and the prevalence of brown pigmentation of Kupfer cells was increased in all treated groups. The only effect on reproductive outcome and pup development was a reduction in pup bodyweight gain during lactation, with statistically significant decreases in F! pup weights on days 4, 8 and 21 of lactation. The NOAEL for reproductive effects was 50 000 ppm, equal to 4300 mg/kg bw per day, the highest dose tested. The NOAEL for toxicity in offspring was 5000 ppm, equal to 430 mg/kg bw per day, on the basis of reductions in body weight at 50 000 ppm in the F0 generation (although the Meeting noted that haematological end-points and the spleen were not investigated in young animals). No NOAEL could be identified for toxicity in parent animals, as haematological effects consistent with those seen in other studies with diflubenzuron were observed at all doses (Brooker, 1995). (ii)

Developmental toxicity

Rats The developmental toxicity of diflubenzuron (purity, 97.6%) was investigated in groups of 24 timed-mated female Crl:CD(SD)BR rats which received the compound by gavage in 1% tragacanth at 0 or a limit dose of 1000 mg/kg bw per day on days 6-15 of gestation. The dams were killed on day 20 of gestation and examined grossly, and the uterine contents were removed. About 50% of the fetuses were examined by dissection, followed by staining with Alizarin red S for skeletal investigations; the remainder were examined by sectioning and dissection. There were no deaths or evidence of clinical signs in the dams. Maternal body weights and reproductive parameters were unaffected by treatment. The fetal weights and sex ratio were similar in control and treated groups. Two major malformations were seen in the offspring of treated dams: a conjoined twin and a pup with a short body, mal-rotated hind limbs and a horseshoe kidney. Conjoined twins are generally considered to be a pre-implantation event and as such would have

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occurred before the first dose of diflubenzuron. The abnormalities in the other pup had been seen in previous studies in the laboratory, and an isolated incidence was considered not to be related to treatment. The incidences of bilobed thoracic centra and retarded ossification of the third and sixth sternebrae were higher in the treated group than in concurrent controls, but were within the range in contemporary controls mother studies. TheNOAELs for maternal toxicity, embryotoxicity, fetal toxicity and teratogenicity were at the limit dose of 1000 mg/kg bw per day (Kavanagh, 1988a). Rabbits Groups of 16 timed-mated female New Zealand white rabbits received diflubenzuron (purity, 97.6%) by gavage in 1 % tragacanth at 0 or a limit dose of 1000 mg/kg bw per day on days 7-19 of gestation. The does were killed on day 28 of gestation and examined grossly, and the uterine contents were removed. Fetuses were examined by dissection, followed by staining with Alizarin red S for skeletal investigations. One control died, and one treated animal was killed because it decveloped hindlimb paralysis. The latter effect was considered unlikely to be related to treatment, as it was an isolated finding, and no lesions were found in nerves or muscles in other studies with diflubenzuron. Maternal clinical signs, body weights and reproductive parameters were unaffected by treatment. Fetal weights and sex ratios were similar in control and treated groups. The overall incidences of malformations and variations were lower in treated animals than in controls, with no significant increase for any individual finding. The NOAEL for maternal toxicity, embryotoxicity, fetal toxicity and teratogenicity were at the limit dose of 1000 mg/kg bw per day (Kavanagh, 1988b). (/)

Special studies: Toxicity of metabolites

The acute toxicity of 4-chlorophenylurea was investigated in male Fischer 344 rats as part of a study of metabolism. The rats were dosed once with 4-chlorophenylurea (purity, 99.3%) at 190, 220 or 230 mg/kg bw in 1% gum tragacanth. No deaths were seen at the two higher doses, but pronounced clinical signs of central nervous system depression (lethargy, ataxia, loss of righting reflex) were seen within 30 min of dosing. The rat given 190 mg/kg bw showed mild signs of central nervous system depression within 90 min of dosing. Chromodacryorrhoea lasting 3648 h was seen in all animals.

3.

Observations in humans

No reports of adverse effects or poisoning incidents associated with diflubenzuron were found, and no adverse effects have been reported during field use (Dykstra, 2001). Diflubenzuron has been produced in a plant in The Netherlands since 1973, where employees involved in its production and formulation undergo regular medical examinations, including haematological analyses. Although no actual exposures have been described, no disturbances in the health status of these employees were observed that could be linked to exposure to diflubenzuron (Dykstra, 2001).

Comments Diflubenzuron administered orally is absorbed to a limited extent in mice, rats and cats, the extent of absorption decreasing with increasing dose, from about 30% at 5 mg/kg bw to < 5% at 100 mg/kg bw in rats. The rate of absorption is rapid, with peak blood concentrations of radiolabel

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at about 4 h in rats given 5 mg/kg bw per day. The highest concentrations were found initially in liver and fat; at 7 days, significant concentrations of radiolabel remained in liver (170 ng equivalents/g) and erythrocytes (200 ng equivalents/g). The concentration of radiolabel in plasma of rats given 5 mg/kg bw fell from about 700 ng equivalents/g at 4 h to 3 ng equivalents/g on day 7. Excretion was rapid, 80-98% of the dose being excreted within 24 h. Absorbed diflubenzuron was excreted primarily in urine, with involvement of biliary excretion and enterohepatic circulation. Studies of the metabolism of diflubenzuron in rats showed inconsistent results, the reasons being unclear. Unchanged diflubenzuron was the only component excreted in significant quantities in faeces. The results of urinary analyses indicated that absorbed diflubenzuron is metabolized extensively before excretion in the urine. The primary metabolic steps are hydroxylation of the anilino ring, cleavage of the ureido bridge, and conjugation, mainly with sulfate. 4-Chlorophenylurea, a residue of diflubenzuron that occurs in rice, has not been detected in significant quantities in studies of metabolism in rats. Its toxicity was thus not assessed in studies in mammalian systems. After oral administration, 4-chlorophenylurea is both absorbed to a greater extent (about 95%) and is more acutely toxic than diflubenzuron, with an LD^ of 1100 mg/kg bw. The Meeting considered that 4-chlorophenylurea should be considered for inclusion in the residue definition. Another plant metabolite, diflurobenzoic acid, does occur in significant quantities in rats and has acute toxicity after oral administration similar to that of diflubenzuron (LDso, 4600 mg/kg bw). Its toxicity was therefore considered to have been assessed in studies with diflubenzuron. 4-Chloroaniline has been reported to be a minor metabolite of diflubenzuron in plants, but did not appear to be a metabolite in rats in the most recent study. 4-Chloroaniline has been reported to produce tumours in rats and mice at a dose of about 3 mg/kg bw per day and to give positive responses in some assays for genotoxicity in vitro (WHO, 1996). The Meeting concluded that it had insufficient information to assess the toxicity of 4-chloroaniline. The LD50 of diflubenzuron administered orally was > 4600 mg/kg bw in mice and rats; WHO (1999) has classified diflubenzuron as 'unlikely to present an acute hazard in normal use'. Diflubenzuron also had little toxicity in rats exposed dermally, with an LD50 of > 10 000 mg/kg bw, or by inhalation, with an LC50 > 2.8 mg/1 of air. No clinical signs were seen in these studies. A single dose of 10 000 mg/kg bw of a 25% w/w formulation of diflubenzuron given by gavage to mice and rats induced a slight (less than two-fold) but statistically significant increase in methaemoglobin concentration. Those of sulf-haemoglobin and Heinz bodies were unaffected. Diflubenzuron did not significantly irritate the skin or eyes of rabbits and did not irritate the skin of guinea-pigs exposed in a Magnusson and Kligman 'maximization' study. Diflubenzuron showed a consistent profile of toxicity after repeated oral administration to mice, rats, and dogs. In agreement with the studies of the distribution of radiolabel, the primary target of the toxicity of diflubenzuron was erythrocytes. The mechanism of the haematotoxicity of diflubenzuron has not been elucidated, but haematotoxicity is a common finding with pesticides like diflubenzuron that are transformed to halogenated phenylamido derivatives. The most sensitive early end-point was increased concentrations of methaemoglobin and sulf-haemoglobin. The Meeting concluded that, because methaemoglobinaemia and sulfhaemoglobinaemia occurred only after saturation of reduction processes, it was more appropriate to use the statistical significance and dose-response relationships in a study rather than to set a numerical cut-off for adversity. The NOAELs for increased concentrations of methaemoglobin and sulfhaemoglobin were 1.2 mg/kg bw per day for mice, 2 mg/kg bw per day for rats and 2 mg/kg bw per day for dogs. Reduced erythrocyte counts and volume fractions were associated with the increases in methaemoglobin and sulfhaemoglobin. Heinz ^bodies were seen at doses > 32 mg/kg bw per day in mice and > 50 mg/kg bw per day in dogs, but were not seen in rats. The haematological effects showed both time-related and dose-related trends, the NOAEL for methaemoglobinaemia in dogs being > 250 mg/kg bw per day at 4 weeks, 50 mg/kg bw per day at 13 weeks and 2 mg/kg bw per

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day at 26 weeks. The only clinical sign of note was blue-grey extremities in mice after 1 week at doses > 160 mg/kg bw per day and after 6 weeks at > 6.4 mg/kg bw per day. The main gross and histological findings were associated with haematotoxicity. These comprised erythroid hyperplasia of the bone marrow of rats and dogs, enlarged spleens in all three species and increased pigment deposition in the spleens of mice and rats. Increased pigmentation of a variety of cell types was seen in the liver. An increase in the severity of chronic hepatitis was seen in all groups in a 90-day study in rats, the LOAEL being 8 mg/kg bw per day. The NOAELs for pathological findings were 6.4 mg/kg bw per day in mice, 2 mg/kg bw per day in rats and 2 mg/kg bw per day in dogs. In the 13-week study in dogs and a 91 -week study in mice, increased total haemoglobin and reticulocyte counts, bone-marrow hyperplasia and increased erythroid:myeloid ratios indicated that the haematopoietic stem cells were not affected, and that there had been a compensatory response to the effects of diflubenzuron. The carcinogenic potential of diflubenzuron was investigated in one study in mice and two studies in rats. The incidences of tumours were not increased in any of these studies, at dietary concentrations equal to 840 mg/kg bw per day in mice and 470 mg/kg bw per day in rats. The genotoxicity of diflubenzuron was investigated in an adequate battery of tests in vitro and in vivo. Negative results were obtained in all the studies. The Meeting concluded that diflubenzuron is unlikely to be genotoxic. In view of the negative findings in assays for genotoxicity and carcinogenicity, the Meeting concluded that diflubenzuron is unlikely to pose a carcinogenic risk to humans. In a two-generation study of reproductive toxicity in rats, no adverse effects were found on sperm quality or quantity, reproductive performance, litter size or attainment of developmental landmarks at the highest dietary concentration. The NOAEL for reproductive toxicity was the highest dietary concentration, equal to an average intake of 3800 mg/kg bw per day for males and 4300 mg/kg bw per day for females. Reductions in pup weight gain during lactation were seen at the highest dose in the F0 generation, with a NOAEL for toxicity of 360 mg/kg bw per day. Adult animals showed toxic effects consistent with the findings in other studies with repeated doses, including alterations in erythrocyte parameters at all dietary concentrations; the LOAEL was 36 mg/kg bw per day. The absence of a clear NOAEL for general toxicity in adult animals was not considered in assessing diflubenzuron, as NOAELs for the same effects were available from other studies in adult animals. The developmental toxicity of diflubenzuron was investigated in rats and rabbits. There was no evidence of overt maternal toxicity, fetotoxicity, or teratogenicity in either species at the highest dose of 1000 mg/kg bw per day. The Meeting concluded that diflubenzuron is not fetotoxic or teratogenic. Samples from pups and weanling animals in the multigeneration study were not examined forhaematological or histopathological end-points. However, the fact that young animals exposed at about 1000 times the overall NOAEL for haematological effects showed no overt evidence of toxicity provided a degree of reassurance that young animals are not significantly more sensitive to diflubenzuron than adults. Routine medical monitoring (including haematology) of workers in the production and formulation of diflubenzuron over 25 years did not reveal any adverse effects attributable to the compound. Toxicological evaluation The Meeting concluded that the available database on diflubenzuron was adequate to characterize the potential hazards to fetuses, infants, and children The Meeting re-confirmed the previously established ADI of 0-0.02 mg/kg bw, based on the NOAEL for haematological effects of 2 mg/kg bw per day in the 2-year studies in rats and the 52-week study in dogs. DIFLUBENZURON 35-59 JMPR 2001

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The Meeting concluded that, although methaemoglobinaemia is potentially an acute effect, the overall toxicological profile of diflubenzuron indicates that establishment of an acute reference dose is unnecessary. The absorption of diflubenzuron declined with increasing doses, and its excretion was relatively rapid, which would tend to limit systemic concentrations after high acute doses. It is likely that a metabolite, rather than diflubenzuron per se, is responsible for the effects on erythrocytes. Diflubenzuron has very low acute toxicity when given by various routes (oral, dermal and inhalation). Only marginal increases (less than doubling) in methaemoglobin concentrations were seen in mice and rats given 10 000 mg/kg bw of a formulation of diflubenzuron, equivalent to 2500 mg/kg bw, which is above the limit doses used in toxicological tests. No developmental toxicity was seen when diflubenzuron was given at up to a limit dose of 1000 mg/kg bw per day. There was no evidence of neurotoxicity in routine studies of toxicity. Levels relevant to risk assessment Species

Study

Effect

NOAEL

Mouse

9 1 -week study of toxicity and carcinogenicitya

Toxicity

1 6 ppm (equal to 1 .2 mg/kg 80 ppm (equal to 6.4 mg/kg bw per day) bw per day) 10 000 ppmb (equal to 840 mg/kg bw per day

1 04-week study of toxicity and carcinogenicity3' c

Rat

Carcinogenicity Toxicity Carcinogenicity

Two-generation study Parental toxicity of reproductive toxicity3 Reproductive toxicity Offspring toxicity Study of developmental Maternal toxicity toxicity6 Embryo- and fetotoxicity Rabbit

Dog 3 b c d e f

40 ppm (equivalent to 2 mg/kg bw per day) 10 000 ppmb (equal to 470 mg/kg bw per day

Toxicity

160 ppm (equivalent to 8 mg/kg bw per day)

-

-

500 ppmd (equal to 36 mg/kg bw per day)

50 000 ppmb (equal to 3800 mg/kg bw per day) 5000 ppm (equal to 360 mg/kg bw per day) 1000 mg/kg bw per dayb

-

1000 mg/kg bw per dayb

Developmental toxicity6 Maternal toxicity 1000 mg/kg bw per dayb Embryo- and fetotoxicity 1000 mg/kg bw per dayb 52-week study of toxicityf

LOAEL

2 mg/kg bw per day

50 000 ppm (equal to 3800 mg/kg bw per day)

_ 10 mg/kg bw per day

Diet Highest dose tested Two studies combined Lowest dose tested Gavage Capsule

Estimate of acceptable daily intake for humans 0-0.02 mg/kg bw Estimate of acute reference dose Unnecessary Studies that would provide information useful for continued evaluation of the compound • Further observations in humans • Studies on 4-chlorophenylurea

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Summary of critical end-points for diflubenzuron Absorption, distribution, excretion, and metabolism in mammals Rapid, maximum plasma concentration of radiolabel at Rate and extent of oral absorption 4 h; limited, 30% of a 5 mg/kg bw dose in bile and urine; less absorption at 100 mg/kg bw Extensive, highest concentrations in liver and Distribution erythrocytes Generally low, but some potential in erythrocytes and Potential for accumulation liver > 90% in 48 h Rate and extent of excretion Hydroxylation of anilino ring and cleavage of ureido Metabolism in animals bridge Diflubenzuron; 4-chlorophenylurea (plant metabolite) lexicologically significant compounds Acute toxicity Rat, LDso, oral Rat, LD 50 , intraperitoneal Rat, LD50, dermal Skin sensitization

> 4600 mg/kg bw Not applicable > 10 000 mg/kg bw Negative, Magnusson and Kligman

Short-term toxicity Target/critical effect Lowest relevant oral NOAEL

Erythrocytes 2 mg/kg bw per day (52-week study, dogs)

Genotoxicity

No genotoxic potential

Long-term toxicity and carcinogenicity Target/critical effect Lowest relevant NOAEL Carcinogenicity

Erythrocytes 2 mg/kg bw per day (2-year study, rats) No carcinogenic potential

Reproductive toxicity Reproduction target/critical effect Lowest relevant reproductive NOAEL Developmental target/critical effect Lowest relevant developmental NOAEL

None 360 mg/kg bw per day; pup weight gain (rats) None > 1000 mg/kg bw per day (rats and rabbits)

Neurotoxicity

No concern from other studies

Medical data

No adverse effects reported

Summary

Value

Study

Safety factor

ADI Acute RfD

0-0.02 Unnecessary

2-year studies in rats and dogs

100

References Andre, J.C. (1996) Dermal absorption of 14C-diflubenzuron by male Sprague-Dawley rats. Unpublished report No. 6615-95-0275-AM-001 from Ricerca Inc., Ohio, USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP; FIFRA test guideline 85-3. Arnold, D., Kennedy, G.L. & Keplinger, M.L. (1974) Mutagenic study with TH6040 in albino mice. Unpublished report No 622-05068 from Industrial Biotest Laboratories, Inc., USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. (Not validated, but malpractice has not been linked with genotoxicity data from IBT). Berczy, Z.S., Cobb, L.M. & Cherry, C.P. (1973) Acute inhalation toxicity to the rat of DU 112307 technical grade powder. Unpublished report No. PDR74/73849 from Huntingdon Research Centre, England. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA.

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Brooker, A.J. (1995) Diflubenzuron technical. The effect on reproductive function of two generations of the rat. Unpublished report No. PDR 569/932539 from Huntingdon Research Centre, England. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP; OECD test guideline 416 & FIFRA 83-4. Brusick, D. & Weir, R.J. (1977a) Mutagenicity evaluation of diflubenzuron technical batch FL 44/605201. Unpublished report No. 2683 from Litton Bionetics Inc., USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Brusick, D. & Weir, R.J. (1977b) Evaluation of diflubenzuron. In vitro malignant transformation in Balb/3T3 cells. Unpublished report No. 2683 from Litton Bionetics, Inc., USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Brusick, D. & Weir, R.J. (1977c) Evaluation of diflubenzuron. Unscheduled DNA synthesis in WI-38 cells. Unpublished report No. 2688from Litton Bionetics, Inc., USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Burdock, G.A., Serota, D.G., Purvis, D, Andrews, J.P., Alsaker, R.D., Wentz, K.A. & Reno, I.E. (1980) Subchronic dietary toxicity study in rats. Diflubenzuron.. Unpublished report No. 553-119 from Hazleton Laboratories America, Inc., USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP. Burdock, G.A., Wolfe, G.W., Hepner, K.E., Alsaker, R.D., Koka, M. & Phipps, R.B. (1984) Oncogenicity study in rats: Diflubenzuron. Project 553-122. Unpublished report No. 56645/08/1984 from Hazleton Laboratories America, Inc., USA Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP; unspecified FIFRA guideline. Cameron, B.D., Henderson, A. & McGuire, G.M. (1990) The metabolism of [14C] diflubenzuron in the rat: Profiling of radioactivity in urine, feces and bile. Project No. 139768. Unpublished report No. 6255 from Inveresk Research International, Scotland. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP. Chesterman, H., Heywood, R., Barker, M.H., Street, A.E. & Cherry, C.P. (1974) Du 112307. Toxicity in repeated dietary administration to beagle dogs (Repeated administration for 13 weeks). Unpublished report No. PDR169/74157 from Huntingdon Research Centre, England. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Colley, J. & Offer, J.M. (1977). Effects of DU112307 in dietary administration to rats for 104 weeks: revaluated pathology data. Unpublished report No. PDR 171/75945 from Huntingdon Research Centre, England. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Colley, J., Heywood, R., Street, A.E. & Gopinath, C. (1984) The effects of diflubenzuron given by oral administration with the feed on toxicity and tumor development in male and female HC/CFLP mice. Unpublished report No. PDR 3 60/831096/A from Huntingdon Research Centre, England. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP. Davies, R.E. & Liggett, M.P. (1973) Irritant effects of DU 112307 (technical) on rabbit eye mucosa. Unpublished report No. 2170/176D/73 from Huntingdon Research Centre, England. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. US FDA 191.12. Dunsire, J.P., Cameron, B.D. & Spiers, G.C. (1990) The disposition of [14C] diflubenzuron in the rat (study performed to Japanese guidelines), Project No. 139197. Unpublished report No. 4924 by Inveresk Research International, Scotland.. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP; to unspecified Japanese test guideline. Dykstra, A.C. (2001) Diflubenzuron—JMPR answers to a toxicology review. Unpublished report Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. van Eldik, A. (1973) Acute toxicity studies with DU 112307 in mice and rats.. Unpublished report No. 56645/ 14/73 from Philips-Duphar, Netherlands. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Enninga, I.C. (1990) Evaluation of DNA repair inducing ability of diflubenzuron in a primary culture of rat hepatocytes (with independent repeat). Unpublished report No. 002418 from RCC Notox BV Netherlands. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP: OECD test guideline 482 and FIFRA 798.5550. Gay, M.H., Wang, R. & Long, S.K, (1999) Metabolism of [U-I4C-phenyl]-4-chlorophenylurea by male Fisher rats, Project 98203P. Unpublished report from Uniroyal Chemical Co. Inc., CT, USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP? No QA signature. Goldenthal, E.I. (1996) 21-day dermal toxicity study in rats. Unpublished report No. 399-186 from MPI Research, USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP; FIFRA test guideline 82-2.

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59 Goodman, D.G. (1980) Histopathologic evaluation of rats administered diflubenzuron in the diet Unpublished report No. DI- 4279 from Clement Associates Inc., Washington, USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Greenough, RJ. Goburdhun, R., Hudson, R. et al. (1985) Diflubenzuron. 52-week oral toxicity study in dogs. Unpublished report No. 2728 from Inveresk Research International, Scotland. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. QA. Hawkins, D.R., Jackson, A.J.S. & Roberts, N.L. (1980) Excretion of radioactivity after oral administration of 3 H/14C-diflubenzuron to cats. Unpublished report No. PDR302/80443 from Huntingdon Research Centre, England. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Hunter, B., Colley, J., Street, A.E., Heywood, R., Prentice, D.E. & Otter, J. (1976) Effects of DU112307 in dietary administration to rats for 104 weeks. Unpublished report No. PDR 171/75945 from Huntingdon Research Centre, England. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Kavanagh, P. (1988a) Diflubenzuron. Oral (gavage) rat teratology limit study. Unpublished report No. PHD/11/ 87 from Toxicol Laboratories Ltd, England. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP; FIFRA 83-3. Kavanagh, P. (1988b) Diflubenzuron. Oral (gavage) rabbit teratology limit study. Unpublished report No. PHD/ 12/87 from Toxicol Laboratories Ltd, England. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP; FIFRA 83-3. Keet, C.M.J.F. (1977a) The effect of DU112307 WP 25% on the met-haemoglobin, sulph-haemoglobin levels and Heinz body formation after a single oral administration in male mice Unpublished report No. 56645/12/ 77 from Philips Duphar, Netherlands. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Keet, C.M.J.F. (1977b) The effect of DU112307 WP 25% on the met-haemoglobin, sulph-haemoglobin and Heinz body formation after a single oral administration in rats. Unpublished report No. 56645/32/77 from Philips Duphar, Netherlands. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Koopman, T.S.M. (1977) Acute dermal toxicity study with DU 112307 (technical) in rats., Unpublished report No. 56645/7/77 from Philips-Duphar, Netherlands. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. Koorn, J.C. (1990) Study to examine the possible mutagenic activity of diflubenzuron in the Ames Salmonella/ microsome assay. Unpublished report No. 56645/74/90 from Duphar BV, Netherlands. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP. de Lange, N. (1979) Diflubenzuron: Dermal absorption in the rabbit. Unpublished report No. 56654/9/79 from Duphar BV, Netherlands. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. MacGregor, J.T. Gould, D.H., Mitchell, A.D. & Sterling, G.P. (1979) Mutagenicity tests of diflubenzuron in the micronucleus test in E4 mice, the L5178Y mouse lymphoma forward mutation assay and the Ames Salmonella reverse mutation assay. Mutat. Res., 66, 45-53. Newton, P.E. (1999) A 4-week inhalation toxicity study of Dimilin technical in rats. Unpublished report No. 399205 from MPI Research, MI, USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP. Prinsen, M.K. (1992) Sensitization study with diflubenzuron technical in guinea pigs (maximization test). Unpublished report No. B 91-0063/04 from TNO Nutrition and Food Research, Netherlands. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP; FIFRA 81-6. Taalman, R.D.F.M. & Hoorn, A.J.W. (1986) Mutagenicity evaluation of diflubenzuron technical in an in vitro cytogenetic assay measuring chromosome aberration frequencies in Chinese hamster ovary (CHO) cells. Unpublished report No. 56645/36/1986 from Hazleton Biotechnologies, Netherlands. .Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP; FIFRA guideline 84-2. Taylor, R.E. (1973) Primary skin irritation study. Unpublished report No. Doos 136 from Harris Laboratories, USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. US 191.11 WHO (1996) Diflubenzuron (WHO Environmental Health Criteria 184), Geneva. WHO (1999) Recommended Classification of Pesticides by Hazard and Guidelines to Classification 1998-1999 (WHO/PCS/98.21/Rev. 1), Geneva, International Programme on Chemical Safety. Wang, R. & Gay, M.H. (1999) Metabolism of [U-14C-anilino]-diflubenzuron by male Fisher rats, Project 98246P. Unpublished report from Uniroyal Chemical Co. Inc., CT, USA. Submitted to WHO by Uniroyal Chemical Co. Inc., Bethany, CT, USA. GLP? No QA signature; OPPTS 870.7485.

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FENPROPIMORPH (addendum) First draft prepared by Brian G. Priestly Chemicals & Non-Prescription Medicines Branch Therapeutic Goods Administration, Canberra, ACT, Australia

Explanation Evaluation for acute reference dose Acute neurotoxicity in rats Additional studies Comments Toxicological evaluation References

61 61 62 62 63 63 64

Explanation Fenpropimorph was first evaluated by the 1994 Joint Meeting, which established an ADI of 0-0.003 mg/kg bw on the basis of a NOAEL of 10 ppm, equal to 0.3 mg/kg bw per day, in a 2-year study of toxicity and carcinogenicity in rats (Annex 1, reference 71). At its Thirty-third Session, the Codex Committee on Pesticide Residues considered the evaluations of residues and dietary intake of fenpropimorph by the 1999 Joint Meeting and noted that the absence of an acute RfD had precluded completion of an assessment of acute dietary risk. The present Meeting was asked to consider the need to establish an acute reference dose (RfD).

Evaluation for acute reference dose The JMPR in 1994 noted that the LD50 of fenpropimorph after oral administration ranged from 1500 to 3500 mg/kg bw in rats, and that the values in rats after dermal application or inhalation were also low (dermal LD50,4300 mg/kg bw; inhalation LC50, > 3.6 to > 8.5 mg/1 of air). WHO (1999) has classified fenpropimorph as 'unlikely to present acute hazard in normal use'. Studies evaluated by the 1994 JMPR did not show any remarkable early signs of toxicity after repeated doses, even at high levels that eventually elicited signs of liver toxicity, anaemia and growth retardation. In short-term studies in rats (but not dogs) treated in the diet, inhibition of plasma and erythrocyte cholinesterase activity was occasionally seen, but there were no consistent dose- or time-response relationship, nor were the sexes equally susceptible. In the 2-year study in rats treated in the diet, inhibition of brain acetylcholinesterase was seen, males being more severely affected than females. However, these effects were difficult to interpret because brain cholinesterase activity was also depressed in controls at terminal sacrifice and there were no effects on erythrocyte cholinesterase activity at any time. In rabbits treated by gavage on days 618 of gestation, embryotoxicity, fetotoxicity and developmental anomalies were seen only at doses that were clearly toxic to the does (NOAEL, 12 mg/kg bw per day; LOAEL, 36 mg/kg bw per day). Similar findings were made in studies of developmental toxicity in rats, in which the NOAEL was 10 mg/kg bw per day and the LOAEL was 40 mg/kg bw per day. The 1994 JMPR concluded that fenpropimorph is not genotoxic.

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1.

Acute neurotoxicity in rats

In a study of behaviour and neuromorphology performed in accordance with USA's Environmental Protection Agency test guideline 81-8 and complying with good laboratry practice, technical-grade fenpropimorph (purity, 94.3%) was administered by gavage in an aqueous Cremophor suspension to 49-day-old male and female Wistar rats (ChbbiTHOM SPF). The rats were assigned randomly to groups of 10 of each sex and housed singly in steel-wire cages in an air-conditioned environment. The dose administered was 0, 100, 500 or 1500 mg/kg bw. Functional observational testing and motor activity measurements (carried out in separate polycarbonate cages) were conducted 7 days before dosing, on the day of administration and then 7 and 14 days after dosing. Function was measured within the 2-6-h period that was expected to encompass the peak effect. At the end of the study, five rats from each group were killed by perfusion fixation under pentobarbital anaesthesia for histological examination of the central and peripheral nervous systems. Body-weight gain was significantly depressed (by 6.6%) when compared with that of controls after 7 days in males given 1500 mg/kg bw (p < 0.05). The clinical and behavioural signs observed in males and females at 500 and 1500 mg/kg bw included piloerection and decreased rearing activity. Decreased motor activity was observed in males and females at 1500 mg/kg bw, but only in females at 500 mg/kg bw. The effects on rearing and motor activity did not persist beyond the day of dosing, while the piloerection persisted for a few days only in the group at 1500 mg/kg bw. No treatment-related effects were seen in sensorimotor or reflex function. Statistically significant increases in hindlimb grip strength were seen in females at the highest dose on day 14 only and in landing foot-splay tests in males at the intermediate dose on day 7 only, but these findings were considered to be incidental owing to the weakness of the dose- and timeresponse relationships. No gross neuroanatomical or histopathological lesions of the central or peripheral nervous systems were found that could be attributed to treatment. The NOAEL was 100 mg/kg bw (Mellert et al, 1997).

2.

Additional studies

Additional studies in mice and dogs treated with fenpropimorph in the diet for 4-6 weeks were submitted. Although the Meeting considered that they were not relevant to the setting of an acute RfD, they were evaluated as part of this monograph addendum. (a)

Studies of mice treated in the diet for 4—6 weeks

Groups of eight male and eight female CD-1 mice received diets containing fenpropimorph (purity, 92.5%) at a concentration of 0,500,1000,2000 or 4000 ppm, equal to 0,80,170,340 and 670 mg/kg bw per day for males and 0, 83, 200, 380 and 790 mg/kg bw per day for females, for 4 weeks. The highest dose resulted in significant toxicity (piloerection, salivation, hunched posture and lethargy), one animal of each sex died and the deterioration in general health was considered sufficient to justify termination of this dose at 23-24 days. Markedly reduced bodyweight gain was seen at this dose, although statistically significantly reduced weight gain was also seen in males at 2000 ppm. The relative and absolute weights of the liver were significantly increased at all doses except females at 1000 ppm. There were no consistent or dose-related changes in cholinesterase activity in plasma, erythrocyte or brain. No NOAEL could be identified (Hunter et al., 1980).

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Groups of eight male and eight female CD-1 mice received diets containing fenpropimorph (purity, 92.5%) at a concentration of 0,25,50,100 or 200 ppm, equalto 3.3,6.6,13 and24 mg/kg bw per day for males and 0, 4, 8, 15 and 32 mg/kg bw per day for females, for 6 weeks. After 3 weeks, the dietary concentration of the rats at 25 ppm was increased to 400 ppm (equal to 47 mg/kg bw per day for males and 60 mg/kg bw per day for females). There were no deaths, morbidity or effects on weight gain, food consumption or appearance in any group. There were no remarkable findings at autopsy, except that the relative liver weights were increased in both males and females at doses > 100 ppm, and the relative spleen and kidney weights were increased in some treated groups, with no clear consistency by dose or sex. Minimal centrilobular hepatocytic enlargement was seen at 400 ppm. The NOAEL was 100 ppm, equal to 13 mg/kg bw per day, on the basis of the liver changes (Hunter et al., 1979). (b)

Studies of dogs treated in the diet

Groups of four male and four female beagle dogs received diets containing fenpropimorph (purity, 99.1%) at a concentration of 0, 200, 400, 800 or 1000 ppm, equal to 0, 7, 12, 27 and 51 mg/kg bw per day for males and 0,8,15,29 and 62 mg/kg bw per day for females, for 4 weeks. The effects on growth were minimal and not clearly dose-related, although the failure of females at the highest dose to gain weight from a low initial base may have been treatment-related. The absolute weight of the liver was clearly increased in males at 1600 ppm and the relative weight at concentrations > 800 pm, but not in females. The testis weight was reduced in males at the highest dose. The relative weight of the spleen was increased in females, but not in males, at .concentrations > 800 ppm. No histopathological changes were found that were attributable to treatment. The NOAEL was 400 ppm, equal to 12 mg/kg bw (Kirsch et al., 1978).

Comments In a study submitted to the current Meeting, of behaviour and neuromorphology in male and female Wistar rats, a single dose of fenpropimorph administered by gavage at 0, 100, 500 or 1500 mg/kg bw caused a small but significant, depression of body-weight gain only in males at the highest dose. The clinical and behavioural signs in animals of each sex at 500 and 1500 mg/kg bw included piloerection and decreased rearing activity. Decreased motor activity was observed in both males and females at the highest dose and in females at 500 mg/kg bw. The effects on rearing and motor activity did not persist beyond the day of dosing, while the piloerection persisted for a few days in animals at the highest dose. No treatment-related effects on sensorimotor or reflex functions were seen. Statistically significant increases in hindlimb grip strength were seen in females at the highest dose on day 14 only and in landing foot-splay tests in males at the intermediate dose on day 7 only. However, these findings were probably incidental, in view of the weakness of the dose- and time-response relationships. No gross neuroanatomical or histopathological lesions of the central or peripheral nervous system were seen that could be attributed to treatment. The NOAEL was 100 mg/kg bw.

Toxicological evaluation The Meeting considered that the above study was appropriate for assessing the acute hazard of fenpropimorph and used it as the basis for establishing an acute RfD of 1 mg/kg bw, which incorporates a safety factor of 100.

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References Hunter, B., Jordan, J., Gopinath, C, Harrington, S. & Gibson, W.A. (1979) Preliminary assessment of Reg. No. 108 406 toxicity to mice by dietary administration for 6 weeks. Unpublished report dated 31 August 1979 from Huntingdon Research Centre, Huntingdon, Cambridgeshire, England. Submitted to WHO by BASF Aktiengellschaft, Limburgerhof, Germany. Hunter, B., Jordan, J., Heywood, R., Street, A.E. & Gibson, W.A. (1980) Preliminary assessment of Reg. No. 108 406 toxicity to mice by dietary administration for 4 weeks. Unpublished report dated 16 May 1980 from Huntingdon Research Centre, Huntingdon, Cambridgeshire, England. Submitted to WHO by BASF Aktiengellschaft, Limburgerhof, Germany. Kirsch, P., Deckardt, K., Freisberg, K.O., Mirea, D., Schulz, V. & Byrt, B.B. (1978) Study of the toxicity of Reg. No. 108 406 to dogs in a 4-week feeding study. Unpublished report dated 5 December 1978 from BASF Aktiengellschaft, Ludwigshafen, Germany. Mellert, W., Kaufman, W. & Hildebrand, B. (1997) Fenpropimorph—Acute neurotoxicity study in Wistar rats. Unpublished report No. 2C0047/93061 from BASF Department of Toxicology. Submitted to WHO by BASF Crop Protection Division, Limburgerhof, Germany. WHO (1999) Recommended Classification ofPesticides by Hazard and Guidelines to Classification 1998-1999 (WHO/PCS/98.2I/Rev. 1), Geneva, International Programme on Chemical Safety.

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IMAZILIL (addendum) First draft prepared by Timothy C. Marrs Food Standards Agency, Aviation House, London, England Explanation Evaluation for acceptable daily intake Studies of mechanism of action Long-term studies of toxicity and carcinogenicity Comments Toxicological evaluation References

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Explanation Imazalil is used as a human and veterinary pharmaceutical, under the name 'enilconazole'. Imazalil was evaluated by the 1977 JMPR, when a temporary ADI of 0-0.01 mg/kg bw was allocated (Annex 1, reference 28). An ADI of 0-0.01 mg/kg bw was allocated in 1986 on the basis of the NOAEL in a 2-year study in dogs (Annex 1, reference 47). The compound was re-evaluated in 1991, when an ADI of 0-0.03 mg/kg bw was established on the basis of the NOAEL in a new study in dogs and a safety factor of 100 (Annex 1, reference 62). The 2000 JMPR determined that an acute RfD was unnecessary and affirmed the ADI of 0-0.03 mg/kg bw; however, the Meeting was made aware of the existence of a new long-term study in rats (Annex 1, reference 89). The report of this study was supplied, together with those of studies on the mechanism by which imazalil affects the thyroid and liver, to the present Meeting.

Evaluation for acceptable daily intake 1.

Studies of mechanism of action Rats

Groups of 10 male and 10 female specific pathogen-free Wistar Hannover rats were given diets containing technical-grade imazalil (purity, 50%) at a concentration of 0, 800, 1600, 2400 or 3200 ppm, equal to 0,64,130,180 and 250 mg/kg bw per day for males and 0,79,150,240 and 330 mg/kg bw per day for females. The rats were observed daily, and body weights and food consumption were measured weekly. Deaths were recorded. Haematological and clinical chemical measurements were performed on blood samples from all surviving animals before the terminal sacrifice, and the concentration of imazalil was determined by gas chromatography with mass spectrometry (Sterkins, 1996). Urine was analysed before terminal sacrifice. Thyroid-stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4) and testosterone concentrations were measured in serum from blood taken at sacrifice. At autopsy, the animals were examined and weighed, and any gross pathological changes were noted. The liver was examined histopathologically, as were other organs adjudged to be grossly abnormal. Electron microscopy was carried on the livers of two male and two female control animals and two male and two female animals fed the highest dietary concentration of imazalil. Liver microsomes from four rats of each IMAZALIL 65-77 JMPR 2001

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sex per group were assayed for protein and cytochrome P450 content, and the activities of UDPglucuronyltransferase and several monooxygenases were determined (Vermeir, 1996). A significantly lower body weight than that of concurrent controls was found from week 2 of the study in males at the two higher concentrations and from time to time in those at 1600 ppm. The females showed decreased body weight from week 1 at the two higher concentrations and from week 2 at 1600 ppm. The findings for weight gain were similar, except that decreased weight gain by comparison with controls was found in animals of each sex at 2400 and 3200 ppm throughout the study. Sporadic decreases in food consumption were seen in all groups of treated males, particularly frequently at 2400 ppm. Among females, decreased food consumption was observed at concentrations > 1600 ppm from time to time. Sporadic changes in haematological parameters were seen, most of which were not doserelated and did not therefore appear to be related to treatment. However, a decrease in monocyte count was seen in males at dietary concentrations > 1600 ppm. Furthermore, increased erythrocyte volume fraction, haemoglobin and red cell count were seen, with decreases in mean cell volume and increases in mean cell haemoglobin concentration in females at most concentrations, including the lowest. Decreased aspartate aminotransferase activity was seen at all doses in both sexes, and decreased alanine aminotransferase activity and blood urea nitrogen concentration were seen at concentrations > 1600 ppm in males: such changes are not generally considered to be adverse. Decreased serum albumin was seen in females at the two higher dietary concentrations. A decrease in serum calcium and an increase in phosphate concentration was seen in females at 1600,2400 and 3200 ppm. Decreased triglyceride andphospholipid concentrations were observed in males at all dietary concentrations, and a decreased triglyceride concentration was seen in females at the highest dietary concentration. No treatment-related inter-group differences were seen on urinary end-points. The TSH, T3, T4 and testosterone concentrations in serum showed no clear differences between groups. At termination, the body weights of males at concentrations > 1600 ppm were decreased. Decreased absolute lung weights were seen at 2400 and 3200 ppm, and increases in the relative weight of the liver at all concentrations. Other changes in organs weights of males were either not dose-related or reflected changes in total body weight. Similar findings were made in females, which showed a decrease in body weight at dietary concentrations > 1600 ppm. A decrease in absolute lung weight was seen at these concentrations. At examination/?^mortem, gross changes that appeared to be related to treatment were seen only in the liver. The livers of males at 2400 and 3200 ppm were dark, and more pronounced lobulation was seen at dietary concentrations > 1600 ppm. Dark livers were also found in females at 2400 and 3200 ppm and more pronounced lobulation only at 3200 ppm. Histological examination revealed hepatocellular hypertrophy in males at all dietary concentrations, which was particularly pronounced in centrilobular zones. An increased frequency of hepatocytes with small vacuoles was seen at 2400 and 3200 ppm and an increased frequency of fine Oil Red O-positive material at all concentrations. Similar changes were seen in females: they had hepatocellular hypertrophy, which was particularly pronounced in centrilobular zones, at all dietary concentrations. Further, they showed an increased frequency of hepatocytes with small vacuoles at all concentrations and large vacuoles at > 2400 ppm. An increased frequency of fine Oil Red O-positive material was seen at concentrations > 1600 ppm. Histological examination of the thyroid gland revealed no significant intergroup differences. On electron microscopy, the livers of all animals at the four higher dietary concentrations, but not the controls, showed small and/or large lipid droplets in both centrilobular and perilobular areas. Additionally, whorls of smooth endoplasmic reticulum were seen, this change not being present in controls. The protein content of microsomes was increased in males at concentrations > 1600 ppm and in females at > 2400 ppm. The activities of Af-ethylmorphine Af-demethylase, 7-ethoxyresorufm

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deethylase, 7-pentoxyresorufm- and 7-ethoxycoumarin O-dealkylase were significantly induced in animals of each sex at nearly all dietary concentrations. An increase in lauric acid hydroxylase activity was seen more frequently in the livers of males than in those of females but was significant only in males at 2400 ppm. Aniline hydroxylase activity was induced in females at all dietary concentrations. UDP-glucuronyltransferase activity was induced at the two higher dietary concentrations in females. At 800 ppm, the plasma concentration of imazalil was at or below the limit of detection of the assay, which was 1 ng/ml, in males and slightly above the limit of detection in females at 1.3 ng/ml. In the males, the concentrations of imazalil were 3.4.4.4 and 11 ng/ml at 1600,2400 and 3200 ppm, respectively, and the equivalent figures for females were 9.4,6.8 and 17 ng/ml. These values suggest that detoxication pathways may have been saturated at dietary concentrations > 2400 ppm. It was concluded that imazalil is a nonspecific inducer of hepatic enzymes and that it caused morphological changes in the livers of rats. No effect was seen at 3 months either in the concentration of thyroid hormones or the histological appearance of the thyroid. No NOAEL could be identified, as increased liver weight and histopathological changes in that organ were seen at all dietary concentrations. Futhermore, liver enzyme induction, an adaptive change, was seen at all concentrations (vanDeun et al., 1996a; Sterkins, 1996; Vermeir, 1996; van Deun et al., 1999). Groups of 20 specific pathogen-free Wistar rats were given diets containing imazalil (technical grade) at a concentration of 0,200,400 or 800 ppm for 3 months. Over the initial month, these dietary concentrations resulted in intakes of 0,21,42 and 82 mg/kg bw per day for males and 0, 22, 45 and 90 mg/kg bw per day for females; however, over the entire 3 months of the study, the intakes of imazalil were lower, at 0,16, 32 and 64 mg/kg bw per day for males and 0, 19, 38 and 76 mg/kg bw per day for females. Ten animals of each sex at each dietary concentration were killed after 1 month. The rats were observed clinically and for morbidity and mortality at least daily. Ophthalmological examinations were performed at the start of the study and before the interim and terminal sacrifices in the first 10 animals in the control group and those at the highest dietary concentration. Body weight was determined weekly, as was food consumption. Blood was taken at the interim and terminal sacrifice for haematological and clinical chemical investigations; urine was analysed at the same times. Autopsies were performed at both the interim and terminal sacrifices. Selected organs were weighed in each case, and any gross changes were noted. Pieces of liver from four rats of each sex per dietary concentration were taken at both the 1-month and 3-month sacrifices. Liver microsomes were assayed for protein and cytochrome P450 content and for the activities of certain microsomal enzymes, namely aniline hydroxylation, Af-demethylation ofjV-ethylmorphine, 7-ethoxyresorufmO-deethylation, 7-pentoxyresorufin 0-dealkylation, lauric acid hydroxylation, 7-ethoxycoumarin O-deethylation and UDP-glucuronosyltransferase activity towards 4-nitrophenol. The plasma concentrations of imazalil were measured. No deaths were seen. The clinical effects were similar in the various groups, and no ophthalmological abnormalities were found. Body weight was reduced in females at 800 ppm by comparison with concurrent controls at weeks 7 and 9. Body-weight gain was reduced in males at 800 ppm at weeks 1-3 and in females at 400 and 800 ppm in week 1 and at 800 ppm in week 9. No effect was observed on food consumption, except in week 1, when there was decreased consumption by rats of each sex at the highest dietary concentration. At the 1 -month sacrifice, the total leukocyte and lymphocyte counts were decreased at dietary concentrations of 400 and 800 ppm, and the neutrophil and basophil counts were decreased in males at 800 ppm. In females, the total leukocyte and lymphocyte counts were decreased at 200,400 and 800 ppm, the monocyte count at 800 ppm and the mean cell haemoglobin concentration at 200 ppm. At 3 months, males showed a decrease in basophil count at 400 ppm and in erythrocyte volume fraction at 200, 400 and 800 ppm. The haemoglobin concentration was decreased at 400 ppm, the mean cell volume

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at 800 ppm and the mean cell haemoglobin concentration at 200 and 800 ppm. Females had a decrease in mean cell volume at 800 ppm and an increase in mean cell haemoglobin concentration at 200 and 800 ppm. The decrease in mean cell volume in animals of each sex at 800 ppm at 3 months may have been treatment-related. Otherwise, these changes were considered minor and were mostly not dose-related or fell within the reference range for the strain of rats. Some minor changes were seen at lower doses in some clinical chemical parameters, but no treatment-related effects on clinical chemistry or urine were seen at concentrations < 800 ppm. At that concentration, males had increased the serum calcium, phosphate, albumin, cholesterol, triglyceride and blood urea nitrogen concentrations at 1 month, whereas females had decreased calcium, glucose and triglycerides; the total protein was increased. At 3 months, serum calcium concentration was decreased in males and females. The decreases in bilirubin concentration and aminotransferase activities that were observed in the study were considered not adverse, and the inconsistent nature of the other clinical chemical changes casts doubt on their clinical significance. Treatment-related changes were not seen on urinary parameters. After 1 month, the liver microsomal protein content was increased in males at all concentrations and in females at the highest concentration. The liver cytochrome P450 content was increased animals of each sex at the highest concentration. The activities of 7-ethoxyresorufin O-deethylase and 7-pentoxyresorufin O-dealkylase were increased in males at the two higher dietary concentrations, while the activity of 7-ethoxycoumarin O-deethylase was increased at 200 and 800 ppm; UDP-glucuronyltransferase activity was increased at the highest concentration. At 1 month in females, increased activity was seen for TV-ethylmorphine ./V-demethylase, 7ethoxyresorufin O-deethylase, 7-pentoxyresorufm O-dealkylase and 7-ethoxycoumarin Odeethylase at all dietary concentrations and for UDP-glucuronyltransferase activity at the highest concentration. Additional induction was not observed in males at 3 months, and increased activity of 7-ethoxyresorufin O-deethylase was observed at dietary concentrations of 200 and 800 ppm and of 7-pentoxyresorufin O-dealkylase and 7-ethoxycoumarin O-deethylase only at the highest concentration. However, in females, the cytochrome P450 content was further increased. Increased aniline hydroxylation was seen at all dietary concentrations, W-ethylmorphme Ndemethylase activity at the two higher concentrations, 7-ethoxyresorufin O-deethylation and 7pentoxyresorufin O-dealkylase activities at all dietary concentrations and lauric acid hydroxylation at 200 and 800 ppm. Females at 3 months also showed increased activity of 7-ethoxycoumarin Odeethylase at 400 and 800 ppm and of UDP-glucuronosyltransferase at 800 ppm. After 1 month, the plasma concentrations of imazalil ranged from < 2 to 4.4 ng/ml in males and from < 2 to 3.8 ng/ml in females at the three dietary concentrations. At 3 months, the corresponding concentrations were 1.1-2.2 ng/ml in males and 1.0-4.0 ng/ml in females. Increased absolute and relative liver weights were observed after 1 month in males and females at 400 and 800 ppm. Increased relative liver weights were seen in males and increased absolute liver weights in females at 200 ppm. Increased relative thyroid weights were observed in males and increased absolute adrenal weights in females, both at 800 ppm. Decreased thymus weights, both absolute and relative, were seen in males at the highest dietary concentration. At 3 months, males at 800 ppm had increased relative liver and kidney weights, while females had increased relative weight of the kidneys at 800 ppm and increased absolute and relative weights of the adrenals at 400 and 800 ppm. No gross pathological changes were observed that appeared to be related to treatment. Hepatic changes were seen histologically at dietary concentrations > 400 ppm at 1 month; these comprised hepatocyte swelling in males and large vacuoles in females. Adrenal changes (cortical-cell swelling) were observed at 3 months in 2/10 females at 800 ppm and 1/10 females at 400 ppm; the study authors considered that this finding corresponded to the increase in adrenal weights observed. No histopathological differences between groups were seen in the liver at 3 months. At neither 1 nor 3 months were histopathological differences observed in the thyroid (Vermeir, 1995; van Deun et al., 1996b). IMAZALIL 65-77 JMPR 2001

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In a study to investigate the mechanisms underlying the effects of imazalil on the thyroid gland, groups of 50 specific pathogen-free, male Wistar (Hannover substrain) rats were given diets contaning imazalil (purity, 98.8%) at a concentration of 0, 400, 1200 or 3200 ppm for 4 weeks; a further group received a diet containing phenobarbital at 1200 ppm. The dietary concentrations of imazalil resulted in mean intakes of 0, 41, 120 and 340 mg/kg bw per day, while the dose of phenobarbital was 130 mg base equivalent per kg bw. Interim sacrifices were carried out after 1, 2 and 4 weeks of treatment and after a 4-week (week 8) or 9-week (week 13) recovery period, so that the size of the group at each dietary concentration at the time of sacrifice was 10. The rats were observed daily, and body weights and food consumption were measured weekly. Blood was taken at sacrifice for measurement of TSH, T3 and T4 and other clinical chemical end-points, including bilirubin, total protein and alkaline phosphatase and alanine and aspartate aminotransferase activities. The animals were killed at the times described above, five animals from each group being injected 6 h before death with bromodeoxyuridine (BrdU). At sacrifice, a full post mortem was carried out, and macroscopic abnormalities were noted. The thyroid (including parathyroids) and liver were weighed. The thyroid and liver were examined histologically after haematoxylin and eosin staining, and the thyroid, liver and sections of the jejunum from BrdU-treated rats were stained immunohistochemically for BrdU-labelled cells. Liver and thyroid microsomes were prepared from liver pieces and the left thyroid glands (Vermeir, 2000). Induction or inhibition of the hepatic microsomal cytochrome P450 enzymes aniline hydroxylase, Af-ethylmorphine Ndemethylase, 7-pentoxyresorufm O-dealkylase, 5 '-monodeiodinase and T4 glucuronyltransferase was assessed in tissue from the five animals from each group not injected with BrdU; microsomal protein and thyroid peroxidase activity in the thyroid were also measured. No treatment-related deaths were seen, and the only treatment-related clinical effects were seen at the dietary concentration of 3200 ppm, as food wastage. Sedation was noted in the phenobarbital-treated animals. Body weight and body-weight gain were decreased from week 1 to week 8 in animals at 1200 and 3200 ppm, to a greater extent in the latter. Animals given phenobarbital showed decreased weight gain during weeks 5-8. Food consumption was decreased sporadically in animals at 400 ppm, but this was considered not biologically significant. At 1200 ppm, food consumption was decreased during dosing and for the first 3 weeks of the recovery period, while at 3200 food consumption was lower than in the controls during treatment and for 1 week afterwards. In the group given phenobarbital, food consumption was increased during week 2 of dosing. At 400 ppm, no difference from controls in clinical chemical end-points was seen. At 1200 ppm, slight decreases in alkaline phosphatase activity were seen during week 2 and in aspartate aminotransferase activity during weeks 1 and 2. At 3200 ppm, aspartate aminotransferase activity was decreased during weeks 1,2 and 4. animals given phenobarbital showed an increase in total protein content during dosing. TSH concentrations were increased in the phenobarbitaltreated animals and in all imazalil-treated animals at 8 weeks. No statistically significant increases in TSH concentration were seen at other times; nevertheless, the increases at 1 and 2 weeks may have been biologically significant. The T4 concentrations were lower in all imazalil-treated groups after 1 week. After 2 weeks, no differences in T4 concentration were seen, while at 4 weeks the concentrations were increased in rats at 1200 and 3200 ppm, and in those given phenobarbital. At week 8 (i.e. during the recovery period), an increased T4 concentration was seen in the group at 1200 ppm and in that given phenobarbital. An increase was also seen in the group given 3200 ppm at week 13. The T3 concentration was decreased in the group given 3200 ppm at week 4 and in that given phenobarbital at weeks 1,2 and 4. Increased hepatic activities of cytochrome P450, aniline hydroxylase,7V-ethylmorphine jV-demethylase and 7-pentoxyresorufm O-dealkylase were seen in both the imazalil-treated and the phenobarbital-treated groups at weeks 1, 2 and 4. Induction was often found at the lowest dietary concentration. Enzyme induction was largely absent during week 8, indicating that it was a reversible phenomenon. The activity of 5'IMAZALIL 65-77 JMPR 2001

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monodeiodinase was decreased in the group given 3200 ppm of imazalil, and T4 glucuronyltransferase activity was induced in all imazalil-treated groups after 1 week of treatment and also at week 4 in the group given 3200 ppm. Thyroid peroxidase activity was increased in the group at 3200 ppm after weeks 1 and 4 and reduced after 2 weeks of treatment; however, the changes were neither statistically significant nor clearly dose-related. Increases were seen in the relative weight of the thyroid of animals at 3200 ppm at week 2 only, while phenobarbital increased both the relative and absolute weights at this time. At week 2, a positive trend in the relative thyroid weight with dose of imazalil was found. At week 1, the relative and absolute weights of the liver were increased at 400 and 3200 ppm, and the relative weight only at 1200 ppm. At week 2, the relative liver weight was increased at 1200 and 3200 ppm, while at week 4 the relative and absolute weights were increased at 400 and 1200 ppm. The relative liver weight was increased at week 4 at the highest dietary concentration. At 8 weeks, a decrease in absolute but not relative liver weight was observed at the two higher dietary concentrations of imazalil. Phenobarbital increased the absolute and relative weights of the liver at 1,2 and 4 weeks, but this effect had disappeared by 8 weeks. At the terminal kill at 13 weeks, the highest concentration of imazalil had produced a decrease in the absolute but not the in the relative weight of the liver, while phenobarbital had no effect. The only gross findings of interest were in the liver and consisted of swelling in a few animals at 400 and 1200 ppm at week 2 and 4, respectively, and swelling and increased lobulation at 3200 ppm from 1 week onwards. Histologically, hepatic centrilobular hypertrophy was found at concentrations > 400 ppm, and the occurrence of this change appeared to be dose-related. Periportal hypertrophy was found at 1200 and 3200 ppm, while hepatic vacuolation was seen at the highest dietary concentration. All three changes in the liver were also seen with phenobarbital. These changes were not observed 8 and 13 weeks after the start of the study (i.e. during the recovery period). Significant increases in the frequency of thyroid follicular hypertrophy were seen only with phenobarbital. This lesion disappeared during the recovery period. Immunohistochemistry for BrdU-labelled cells did not reveal intergroup differences. The authors concluded that imazalil alters thyroid status by affecting hepatic and thyroid enzymes involved in the synthesis, metabolism and excretion of T4. As histopathological changes were present in the liver at all concentrations, no NOAEL could be identified (Piccirillo, 2000; Verbeek et al., 2000; Vermeir, 2000). The USA's Environmental Protection Agency concluded that these findings may explain the mechanism whereby imazalil induces thyroid metabolism (Khasawinah, 2000). Quantitatively, these studies show that imazalil causes hypertrophy of the liver and, at high doses, some interference with thyroid function. The quantitative differences between the studies may be related to their duration. The results for induction of specific hepatic enzymes were not easy to interpret, as they tended to be inconsistent and dose-effect relationships were not always clear. However, the Meeting concluded that imazalil is a relatively nonspecific enzyme inducer in the liver and its enzyme-inducing effects are reversible.

2.

Long-term studies of toxicity and carcinogenicity Rats

Groups of 50 specific pathogen-free Wistar (Hannover substrain) rats of each sex received diets containing a preparation called 'imazalil 50 premix', which nominally contained 50% imazalil (in fact 49-50%) at a concentration of 0«, 50,200, 1200 or 2400 ppm, equal to intakes of 0,2.4, 9.7, 58 and 120 mg/kg bw per day for males and 0, 3.3, 14, 79 and 160 mg/kg bw per day for females. The animals were observed at least daily. Ophthalmic examinations were carried out in controls and in animals at the highest dose before dosing and towards the end of the treatment.

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Body weight was measured on the first day of dosing, thence weekly and at the end of dosing and additionally in any animal killed in extremis. Food consumption was measured weekly. Haematological and clinical chemical variables were measured in blood samples from all groups at 6, 12 and 18 months and near the end of dosing. At the same time, urine was analysed. Immediately before the terminal kill at 24 months, each rat was examined. A full necropsy was undertaken on all animals, and macroscopic abnormalities were noted. Selected organs were removed and weighed, and samples of tissue were taken and processed for histopathological examination. Blood samples were taken at autopsy for measurement of testosterone, luteinizing hormone, TSH, T4 and T3 by enzyme immunoassay, the assays for TSH and luteinizing hormone being rat-specific. Toxicokinetics was determined for the first four rats at each dietary concentration other than the controls, in blood samples taken on days 180,362 and 726 of the study. Imazalil was measured in these blood samples. The mortality rate was not affected. Increased food wastage was observed for males at the highest dietary concentration and for females at the two higher concentrations. Females at the highest concentration showed a decrease in subcutaneous tissue mass and alopecia. No significant difference was seen between animals at the highest concentration and controls in the ophthalmic examination. The body weights of males at 50 ppm were lower than those of controls at weeks 96, 99,101 and 102, and their weight gain was decreased during weeks 90-94 and weeks 96-104. An increase in weight gain was seen in males at 200 ppm in week 1, but no other effect was seen on body weight or weight gain. Decreased body weight and weight gain were seen in males at 1200 ppm, consistently from week 1 to week 26 and less so thereafter. In males at 2400 ppm, body weight and weight gain were decreased throughout the study (by 15-25%). The body weights of females at 50 ppm were lower than those of controls at weeks 82, 84,85,87,88,100 and 101,and weight gain was decreased in weeks 54, 73, 78, 81-89 and 100-104. A decrease in weight gain was seen in females at 200 ppm in week 34, with no other significant change in weight or weight gain at amy other time. Decreases in body weight were seen in females at 1200 ppm from the third week of the study and in weight gain from the first week. In females at 2400 ppm, body weight and weight gain were decreased throughout the study (by 25-35%). The food consumption of males was decreased in weeks 23, 52, 93 and 96 for those at 50 ppm and in weeks 53,90, 93 and 95 for those at 200 ppm. Decreased food consumption was seen at 1200 ppm intermittently throughout the study and at 2400 almost throughout the study. Total food intake of males throughout the study was decreased at the two higher dietary concentrations. Females at 50 ppm had increased food consumption in week 27 and decreased food consumption at weeks 52,83 and 88. At 200 ppm, increased food consumption was seen in weeks 10,11 and 35-37 and decreased food consumption in weeks 51,83 and 93. The food consumption of females at 1200 and 2400 ppm was decreased almost throughout the study. As for males, total food intake was decreased at the two higher dietary concentrations, and food conversion was increased by 10 and 12% at 1200 ppm and 2400 ppm, respectively. In males at the two higher dietary concentrations, decreased erythrocyte volume fraction, mean cell volume and mean cell haemoglobin were found consistently, with decreased haemoglobin concentration at weeks 78-79 and 104-105. Minor changes, including decreased mean cell volumes at weeks 25 and 52, were seen in males at 200 ppm. The haematological picture in females was more complex: increases were observed in haemoglobin concentration at weeks 25-26 and 78-79 and increases in erythrocyte count at weeks 25-26, 52-53 and 78-79 in animals at dietary concentrations of 200,1200 and 2400 ppm, respectively. Additionally, an increase in erythrocyte count was seen at 104-105 weeks in rats at the highest dietary concentration. Decreased mean cell volume and mean cell haemoglobin concentration were seen at all times in females at 1200 and 2400 ppm and at weeks 25-26 and 52-53 in those at 200 ppm. Thus, imazalil at a dietary concentration of 50 ppm did not affect haematological parameters, minor changes were seen at 200 ppm, and definite changes were seen at 1200 and 2400 ppm. IMAZALIL 65-77 JMPR 2001

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A number of differences between groups were observed in clinical chemical parameters, many of which were transient or inconsistent. The more consistent changes included decreased activity of alkaline phosphatase and aspartate aminotransferase in animals of each sex at all dietary concentrations. Of greater clinical significance were decreases in calcium and total protein concentrations observed at most times in animals at 1200 and 2400 ppm. Increased glucose concentrations were observed at all times in males and females at the highest dietary concentration; at weeks 52-53 and 104-105 in males and at weeks 52-53, 78-79 and 104-105 in females at 1200 ppm; and at all four times in females at 200 ppm. Triglyceride and phospholipid concentrations were decreased in males at 2400 ppm and in females at the two higher dietary concentrations at all times. The blood urea nitrogen concentration was decreased in males at the two higher dietary concentrations at all four times and in females at concentrations > 200 ppm. Decreases in alkaline phosphatase and aminotransferase activity and blood urea nitrogen concentration are generally considered not to be adverse. However, the changes in calcium and total protein, which were observed at most times in animals at 1200 and 2400 ppm, may be clinically significant. Furthermore, the increases in blood glucose concentration seen at 2400 ppm in both sexes, at 200 ppm in females at all times, and from time to time in both sexes at 1200 ppm are likely to be clinically significant. The urine of females showed decreased specific gravity at some times, with increased pH and increased urinary volume at the two higher dietary concentrations. The testosterone and luteinizing hormone concentrations were little altered in male rats, a significant increase in testosterone being observed only at 1200 ppm. The TSH concentrations tended to be higher at 200 and 1200 ppm, and the T4 concentrations were definitely lower in males at 1200 and 2400 ppm, the T3 concentrations being similar in all groups. In females, the T3 concentration was decreased at the two higher dietary concentrations, T4 was decreased at 1200 ppm only, while TSH was not consistently affected. Despite these inconsistent results, it is seems likely that imazalil affected the pituitary-thyroid axis when given at a dietary concentration of 1200 or 2400 ppm. The organ weights of animals killed at term after receiving the higher dietary concentration showed some differences from controls, but these were considered to be a reflection of changes in total body weight, with the following exceptions. The relative weight of the liver was increased in males at the two higher dietary concentrations, with no effect on absolute liver weight. The absolute liver weight was decreased at 2400 ppm, and the relative liver weight was increased at concentrations > 200 ppm in females. At termination, the absolute thyroid weight was increased in males at 1200 and 2400 ppm and the relative thyroid weight at all dietary concentrations, while in females the absolute thyroid weight was decreased at 1200 and 2400 ppm, and the relative thyroid weight was unaffected (Table 1). The sponsor argued that it was appropriate to include decedents in the analysis of organ weights, as the effects on the thyroid were unlikely to be fatal (Tables 2 and 3). The study authors concluded that the NOAEL for changes in organ weights was 200 ppm, presumably on the grounds that the increased relative liver weight observed at 200 ppm Table 1. Thyroid weights of rats killed at termination after receiving diets containing imazalilfor 2 years Thyroid weight

Sex

Dietary concentration (ppm)

50

200

1200

2400

44 84

51 103*

101 202*

76* 154*

67* 145***

42 128

35 112

46 153

31** 112

26*** 104

0 Absolute (mg) Relative (mg/kg)

Male

Absolute (mg) Relative (mg/kg)

Female

From van Deun( 1999) *p < 0.05; **p < 0.01; ***p < 0.001: Mann-Whitney U test (two-tailed)

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Table 2, Thyroid weights of rats kilted before end of study after receiving diets containing imazalil for 2 years Thyroid weight

Sex

Dietary concentration (ppm) 0

50

200

1200

2400

Absolute (mg) Relative (mg/kg)

Male

42 107

34 85

38 92

53 130

39 112

Absolute (mg) Relative (mg/kg)

Female

32 125

28 109

34 138

70 322

26 128

From van Deun (1999)

Table 3. Thyroid weights of rats killed before and at end of study after receiving diets containing imazalil for 2 years Thyroid weight

Sex

Dietary concentration (ppm) 0

Absolute (mg) Relative (mg/kg)

Male

Absolute (mg) Relative (mg/kg)

Female

50

200

1200

2400

44 88

47 98

89 181*

71 148*

64* 142***

38 127

33 111

43 148

38 154

26*** 111

From van deun (1999) *p < 0.05; ***p < 0.001: Mann-Whitney U test (two-tailed)

in females, unaccompanied as it was by a decrease in absolute body weight, was an incidental finding. Further, the increase in relative thyroid weight seen at 50 and 200 ppm in males, in the absence of absolute thyroid weight changes, is not likely to be biologically significant. The pathological changes seen grossly at terminal sacrifice included an increased frequency of liver foci and swollen thyroid glands in males at 1200 and 2400 ppm and decreased adiposity in males at 2400 ppm. The changes seen in females included decreased adiposity and decreased inspissated secretions in the mammary glands at 1200 and 2400 ppm. There was evidence of decreased mammary gland stimulation at all dietary concentrations. An increased prevalence of hepatocellular adenoma was seen in males at the highest dietary concentration, in those killed at termination of the study and when all animals were included in the analysis. Males at 1200 and 2400 ppm also had an increased incidence of follicular-cell neoplasia of the thyroid. In males at 2400 ppm that were killed at termination, the incidences of both follicular-cell adenoma and total follicular-cell neoplasia were increased (Table 4). Addition of animals that died before the end of the study (Table 5) had little effect on the total incidence of the tumours (Table 6). As these tumours are likely to be incidental findings, and not the cause of death, the most appropriate tabulation is probably that in Table 6, showing a clear LO AEL for total follicular-cell neoplasia at 1200 ppm and a NO AEL at 200 ppm. The decreased incidences of Table 4. Incidences of thyroid follicular neoplasia in animals killed at termination of study after receiving diets containing imazalil for 2 years Thyroid follicular neoplasia

Sex

Dietary concentration (ppm) 0

50

200

1200

2400

Total Adenoma Carcinoma

Male

3/40 3/40 0/40

7/35 7/35 0/35

6/38 5/38 2/38

9*/34 7/34 2/34

12*741 10*/41 2/41

Total Adenoma Carcinoma

Female

3/30 3/30 0/30

3/31 3/31 0/31

4/35 3/35 1/35

3/40 3/40 0/40

1/34 1/34 0/34

From van Deun (1999) * p < 0.05 as compared with controls

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Table 5. Incidences of thyroid follicular-cell neoplasia in animals killed or dying before termination of study after receiving diets containing imazalil for 2 years Thyroid follicular neoplasia

Sex

Dietary concentration (ppm) 0

50

200

1200

2400

Total Adenoma Carcinoma

Male

1/10 1/10 0/10

1/15 1/15 0/15

0/12 0/12 0/12

2/16 2/16 0/16

0/9 0/9 0/9

Total Adenoma Carcinoma

Female

4/20 4/20 0/20

2/19 2/19 0/19

0/15 0/15 0/15

1/10 0/10 1/10

0/16 0/16 0/16

From van Deun (1999)

Table 6. Numbers of thyroid follicular-cell tumours in all animals killed or dying after receiving diets containing imazalil for 2 years Thyroid follicular neoplasia

Sex

Total Adenoma Carcinoma

Male

Total Adenoma Carcinoma

Female

Dietary concentration (ppm)

0

50

200

1200

2400

4

8 8 0

6 5 2

11* 9 2

12* 10 2

5 5 0

4 3 1

4 3 1

1* 1 0

4 0 7

7 0

From van Deun (1999) * p < 0.05 as compared with controls

pituitary and mammary gland hyperplasia were probably related to the reduced body weights of animals at the highest concentration in the diet. No differences were found between groups in neoplastic findings. The non-neoplastic findings included decreased incidences of haemorrhagic degeneration of the adrenals in female rats at the highest dietary concentration, in all animals and in the survivors to termination when the results for these animals were analysed separately. The incidence of transitional-cell hyperplasia was increased in all treated males, but with no dose-response relationship; furthermore, this finding was significant only in rats at 50,200 and 1200 ppm when the decedents were included in the analysis. Females at the highest dietary concentration that survived to termination and survivors and decedents taken together had an increased frequency of mineralization of the transitional epithelium of the renal pelvis. In the livers of animals that survived to termination and in all animals, an increased frequency of centriacinar hepatocyte hypertrophy was seen at the two higher concentrations. An increase in the frequency of periacinar hepatocyte hypertrophy was seen in females at the two higher dietary concentrations when the results for all animals were analysed together and at 1200 ppm in survivors only. Changes in the portal tract were discerned in males at the highest concentration, only when all animals were included in the analysis. Clear-cell foci were observed in females at the highest concentration that survived to termination. Decreased frequencies of basophilic foci and foci of hepatocellular alteration were observed in the livers of surviving females at 200 and 2400 ppm and in survivors and decedents together at 2400 ppm. Eosinophilic foci and foci of hepatocellular alteration were seen in surviving males at the two higher concentrations and in all animals at the highest dietary concentration when the results for all animals were analysed together. Fatty vacuolation was found in the livers of surviving males and in survivors and decedents taken together at the two higher concentrations and in female survivors at 1200 ppm at termination. Focal cystic degeneration of the liver was seen in males at 2400 ppm (both survivors separately and all animals), while pigmentladen hepatocytes were seen in females at the three higher dietary concentrations, whether or not IMAZALIL 65-77 JMPR 2001

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the decedents were included in the analysis. In the thyroid gland, cystic focal follicular hyperplasia was seen in male survivors at the two higher concentrations and in those at the highest concentration only when all animals were included in the analysis. Few differences were found between groups with respect to pathological changes in decedents, when the findings were analysed separately; none appeared to be clearly treatment-related, with the possible exception of an increased incidence of transitional-cell hyperplasia in the kidney in males and periacinar hepatocyte hypertrophy and pigment-laden hepatocytes in females at the highest dietary concentration. It might be argued that no NOAEL could be identified in this study, as effects on body weight, weight gain and food consumption were seen at some times in males and females at 50 ppm. However, the findings at 200 ppm suggest that the first two effects were not dose-related, and the LOAEL for body weight and weight gain was likely to be 1200 ppm. In females, increased food consumption was observed at some times at 50 ppm, whereas, in males, the situation was less clear, the decreases observed were small and occurred at only a few times. The NOAEL was therefore 50 ppm, equal to 2.4 mg/kg bw per day, on the basis of minor haematological changes in animals of each sex and increased blood glucose concentration and liver changes (increases in relative liver weight and increased incidence of pigmented hepatocytes) in females at 200 ppm. The NOAELs for tumours in males and females were 200 and 2400 ppm, equal to 9.7 and 160 mg/kg bw per day, respectively (van Deun, 1999).

Comments All the mechanistic studies were carried out in rats and were conducted at dietary concentrations that resulted in daily intakes of up to approximately 350 mg/kg bw per day. These studies consisted of a 3-month dose range-finding study and a 3-month study of the toxicity of imazalil given orally, which included a 1-month interim sacrifice. A 1-month study was also undertaken with repeated oral doses of imazalil, with interim sacrifices at 1 and 2 weeks and recovery periods of 4 and 9 weeks. Hepatocellular hypertrophy and vacuolation were observed, and there was evidence of liver enzyme induction. Some effects were observed on thyroid weight and pituitary and thyroid hormones (increased TSH and decreased T4 concentrations), but the effects were weak and inconsistent, and clear dose-effect relationships were not always found. The Meeting concluded that imazalil is a relatively non-specific enzyme inducer in vivo and its enzyme-inducing effects are reversible. Thus, imazalil may alter thyroid status by affecting hepatic and thyroid enzymes involved in the synthesis, metabolism and excretion of T4. In a long-term study of toxicity and carcinogenicity, rats received diets containing imazalil at a concentration of 0, 50, 200, 1200 or 2400 ppm, providing intakes of 0, 2.4, 9.7, 58 and 120 mg/kg bw per day for males and 0, 3.3, 14, 79 and 160 mg/kg bw per day for females. Consistent decreases in body weights, weight gain and food consumption were seen in males and females at the two higher concentrations. Changes in haematological parameters were seen at concentrations > 200 ppm in both sexes. Increased blood glucose concentrations were observed at the two higher dietary concentrations in animals of each sex and in females at 200 ppm at all times. The changes in thyroid hormone concentrations in serum were inconsistent: in males, the concentrations of TSH tended to be higher and those of T4 definitely lower at the two higher dietary concentrations; in females, the level of T3 was decreased at the two higher dietary concentrations, and that of T4 was decreased only at 1200 ppm. Males at the highest dietary concentration had an increased prevalence of hepatocellular adenoma, and an increased incidence of follicular-cell neoplasia of the thyroid was observed in males at 1200 and 2400 ppm. An increased frequency of pigment-laden hepatocytes was seen in females at the three higher dietary concentrations.

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Effects on body weight, weight gain and food consumption, seen at some times in males and females at 50 ppm, were considered not to be dose-related because they were mild and no effects on body weight or body-weight gain were seen at 200 ppm. The overall NOAEL was 50 ppm, equal to 2.4 mg/kg bw per day, on the basis of minor haematological changes in animals of each sex and increased blood glucose concentrations and hepatic changes (increased relative liver weight and an increased frequency of pigment-laden hepatocytes) in females at 200 ppm. The NOAEL for tumours in males was 200 ppm, equal to 9.7 mg/kg bw per day, whereas that in females was 2400 ppm (the highest dose tested), equal to 160 mg/kg bw per day.

Toxicological evaluation The Meeting concluded that the ADI of 0-0.03 mg/kg bw established by the 1991 Joint Meeting and reaffirmed by the 2000 Meeting is supported by the new data.

References van Deun, K. (1999) Combined oral chronic toxicity/carcinogenicity study in the SPF Wistar rat. Unpublished study No. R023979 dated 8 June 1999. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. Guideline: USEPA guideline for chronic feeding/oncogenicity in the rat, subdivision F, Guideline ref. 83-5 (December 1999). GLP USEPA 40 CFR part 160. van Deun, K., Lammens, L., Benze, J., Coussement, W., Vandenberghe, J., Lampo, A. & van Cauteren, H. (1996a) 3-month dose range finding and mechanistic toxicity study in SPF Wister rats. Unpublished study No. 3672 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP USEPA 40 CFR 160. van Deun, K., Lammens, L., Benze, J., Coussement, W., Vandenberghe, J., Lampo, A. & van Cauteren, H. (1996b) 3-month dose range finding and mechanistic toxicity study with one month interim sacrifice in SPF Wister rats. Unpublished study No. 3514 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP USEPA 40 CFR 160. Guidelines: USEPA FIFRA pesticide assessment guidelines, subdivision F, hazard evaluation (1984); US FDA toxicological principles for the safety assessment of direct food additives and color addtives in food (1982); Ministry of Health and Welfare, Japan; the rules governing medicinal products in the European Community, Volume III. van Deun, K., Lammens, L., Vandenberghe, J., Lampo, A., Jansen, T. & Coussement, W. (1999) 3-month dose range finding and mechanistic toxicity study in SPF Wistar rats: Unpublished amendment No. Nl 17794 to the final report (report No. 3672) dated 30 September 1999 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP USEPA 40 CFR 160. Guideline 21 CFR part 58 (US FDA). Khasawinah, A. (2000) Memorandum to Mark Howard and Betty Shackleford dated 4 October on imazalil, mechnistic study to support the mode of action of thyroid tumors. USEPA. Piccirillo, V.J. (2000) Imazalil: One-month repeated dose oral toxicity study with 1 and 2 week interim sacrifices and 4 and 9 week recovery periods to evaluate thyroid effects. Unpublished report no VJP 5452-00-1 from VJP Consulting Inc., Sterling, Virginia, USA. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. Guidelines: not applicable. GLP USEPA 40CFR 160. Sterkins, P. (1996) The toxicokinetics of imazalil (R023979) in SPF Wistar rats at the end of a three-month doserange-finding and mechanistic toxicity study. Unpublished addendum to study No. 3672 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP USEPA 40 CFR 160. Verbeek, J., Verstyenen, B., Vandenberghe, J., Vinckier, A., de Coster, R., Lampo, A., Jansen, T. & Coussement, W. (2000) One-month repeated dose oral toxicity study in the Wistar rat with 1 and 2 week interim sacrifice and 4 and 9 weeks of recovery. Unpublished study No. 5009 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. Guidelines: not applicable.

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Vermeir, M. (1995) Study on the possible induction and/or inhibition of effects of hepatic drug metabolizing enzymes by imazalil in male and female SPF Wistar rats, after oral adminstration through the diet for one or three consective months at levels of 200, 400 and 800, 1600 ppm. Unpublished study No. FK 1960 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP: USEPA 40 CFR160 and US FDA 21CFR 58. Guidelines: not applicable. Vermeir, M. (1996) Study on the possible induction and/or inhibition of effects of hepatic drug metabolising enzymes by imazalil in male and female SPF Wistar rats, after oral adminstration through the diet for three months at levels of 800, 1600 and 2400 ppm. Unpublished study No. FK 2060 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. GLP: USEPA 40 CFR 160 and US FDA 21 CFR 58. Guidelines: not applicable. Vermeir, M. (2000) Study on the possible induction and/or inhibition of effects of hepatic drug metabolising enzymes and of hepatic 5 '-monodeiodinase and thryoid peroxidase activities in male SPF Wistar rats, after oral adminstration through the diet for one, two and four weeks at levels of 400, 1200 and 3200 ppm. Unpublished study No. FK 3378 from Janssen Pharmaceutica NV, Department of Pharmacokinetics, Beerse, Belgium. Submitted to WHO by Janssen Pharmaceutica NV, Beerse, Belgium. Guidelines: not applicable. GLP USEPA 40CFR 160 (not analysis of samples for 5'-monodeiodinase activity).

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IMIDACLOPRID First draft prepared by Roland Solecki Pesticides and Biocides Division, Federal Institute for Health Protection of Consumers and Veterinary Medicine, Berlin, Germany Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution, and excretion Metabolism Toxicological studies Acute toxicity Short-term studies of toxicity Long-term studies of toxicity and carcinogenicity Genotoxicity Reproductive toxicity Multigeneration studies Developmental toxicity Special studies Neurotoxicity Studies on metabolites Observations in humans Comments Toxicological evaluation References

79 80 80 80 81 84 84 84 87 87 88 88 89 89 89 90 91 91 94 96

Explanation Imidacloprid, l[(6-chloro-3-pyridinyl)methyl]-ALnitro-2-imidazolidinimine, is a new neonicotinoid insecticide which has become an important pest control agent on many crops. The neonicotinoid insecticides are related to nicotine in their structure and action at the nicotinic acetylcholine receptor (Casida, 1998). Imidacloprid is most active against suckling insects because of their unique plant-systemic and translaminar properties. Imidacloprid poisoning in the American cockroach, Periplaneta americana, is characterized, in sequence, by loss of leg strength, leg tremors, body shaking and death. Neurophysiological studies have confirmed that imidacloprid is an agonist at the postsynaptic nicotinic acetylcholine receptor of insects. It appears to act like acetylcholine, by exciting specific nerve cells. There is also some evidence, however, that imidacloprid has multiple agonist and antagonist effects on neuronal nicotinic acetylcholine receptor channels of clonal rat phaeochromocytoma cells (Nagata et al., 1998). The selective toxicity of imidacloprid to insects and not to mammals is attributed to differences in the binding affinity or potency at the nicotinic acetylcholine receptor (Chao & Casida, 1997). The specificity of nicotinoid insecticides appears to be related to receptor subtype, function, neuronal region and developmental stage, and their metabolic lability leads to bioactivated toxicants such as desnitro-imidacloprid in mammals. The selectivity also depends in large part on major structural differences in the neuronal nicotinic acetylcholine receptor binding sites of mammals and insects (Casida, 1998). The selectivity of the chloronicotyl compounds for insects

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as opposed to mammals can be partly explained by differences in the ionization of the pyrolidine nitrogen. Imidacloprid is poorly ionized in neutral media, in contrast to nicotine, and thus passes easily through insect lipophilic barriers (Sone et al, 1994; Roe et al., 1999). Imidacloprid has not previously been evaluated by the JMPR.

Evaluation for acceptable daily intake 1.

Biochemical aspects (a)

Absorption, distribution and excretion

Imidacloprid labelled with 14C in either the methylene or the imidazolidine ring was administered in a physiological saline solution at concentrations of 0.1-2 mg/1 to a total of 50 male and 20 female rats. Groups of five male and five female rats were given a single dose of 1 mg/kg bw intravenously or a single dose of 1 or 20 mg/kg bw orally. Other groups were given 14 doses of of the non-radiolabelled compound at 1 mg/kg bw orally once per day and, 24 h after the last dose, a single oral dose of 1 mg/kg bw as the radiolabelled compound. Radiolabel was determined in plasma and excreta as a function of time and, after sacrifice 48 h after treatment, the concentration of total radiolabel was determined in organs and tissues. A further group of five male rats was given a single oral dose of radiolabelled compound at 20 mg/kg bw, and 14CO2 was measured over 48 h. In an additional test, four groups of five male rats were given a single oral dose of 20 mg/kg bw, killed after 40 min, 1.5, 3 and 6 h, and total radiolabel was determined as a function of time in single organs. A final group of five male rats with bile-duct fistulas was given a single intraduodenal dose of 1 mg/kg bw in order to determine the amount of total radiolabel absorbed and to measure the rate and extent of biliary excretion. The design of the study complied with good laboratory practice (GLP). After oral administration of 1 or 20 mg/kg bw of [pyridinyl-14C-methylene]imidacloprid, the radiolabel was extensively absorbed from the intestinal lumen and readily distributed from the plasma into the body. The radiolabel was also readily eliminated. After intravenous administration of 1 mg/kg bw, about 92% of the recovered radiolabel was excreted in urine and faeces within 48 h. Most of the radiolabel was excreted via the kidneys, with an average ratio in urine:faeces of 4:1. After oral administration, about 96% of the dose was excreted in urine and faeces within 48 h. No difference was found between female and male rats. More than 90% of the renal radiolabel was excreted during 24 h after dosage. The average residual radiolabel in the body, excluding the gastrointestinal tract, at sacrifice was about 0.5%, and that in the gastrointestinal tract was about 0.06% of the administered dose. The rats with bile-duct fistulas excreted only 4.7% of the administered dose with faeces, 56% in urine and about 36% with bile. These findings indicate the existence of enterohepatic circulation of the radiolabel. Significant amounts of radiolabel were not excreted in expired air (CC^). The elimination of total radiolabel from the plasma could be approximated by a combination of two exponential terms, from which elimination half-lives were calculated. The two half-lives varied from 2.6-3.6 to 26-118 h, respectively (Klein, 1987a). The distribution of [pyridinyl-14C-methylene]imidacloprid was investigated in male rats by conventional whole-body autoradiography on X-ray film in a study conducted according to GLP. The rats were given a single oral dose of 20 mg/kg bw, and the distribution of radiolabel was visualized at 1,2,4, 24 and 48 h after treatment. For visualization of the initial distribution, one animal was injected intravenously with 20 mg/kg bw and killed 5 min later. The radiolabel was readily absorbed and rapidly distributed to the tissues and organs. The pattern of distribution showed that the radiolabel could readily permeate tissues: With the exception of fatty tissues, the central nervous system and the mineral part of bone, blackening on

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the autoradiogram was seen on all other parts of the body 5 min after intravenous injection and 1 h after oral dosage. Higher concentrations were seen later in the thyroid and adrenals, but, after 24 h, all other organs and tissues showed only small amounts of radiolabel. The high degree of blackening over the kidney during the first 24 h is a reflection of the high rate of renal excretion of the administered compound. The concentration of radiolabel decreased in organs and tissues with time after administration. The concentrations in the fatty tissues and in the central nervous system were very low throughout the study (Klein, 1987b). Imidacloprid labelled with 14C in the 4 and 5 positions of the imidazolidine ring was administered orally to male and female rats at a dose of 1 mg/kg bw and additionally to male rats at a dose of 150 mg/kg bw, in a study conducted according to GLP. Radiolabel was determined in excreta and in plasma as a function of time, and single organs and tissues were assayed for total radiolabel at sacrifice48 haftertreatment.Thebiokineticsof[imidazolidine-4,5-14C]imidacloprid was similar to that of methylene-14C-labelled compound. The administered radiolabel was extensively absorbed from the intestinal lumen and rapidly distributed in the body. Excretion was rapid, and the renal route predominated (Klein & Brauner, 1991). The nitroso metabolite of imidacloprid, 1 -(6-chloro-3-pyridylmethyl)-Af-nitroso(imidazolidin-2-ylidene)amine (WAK 3839), has been identified as a minor constituent of edible plant commodities but was not found in the excreta of rats in the studies described above. Absorption of imidacloprid and WAK 3839 began immediately after oral administration of 1 mg/kg bw. A comparison of the curves of concentration-time in plasma suggested that the absorption was similarly extensive, and no significant differences in terminal half-times were found after administration of imidacloprid (38 h) and WAK 3839 (47 h). WAK 3839 was eliminated slightly faster, and the concentrations of total radiolabel in the organs were lower than with the parent compound. After administration of a single high dose of imidacloprid, 150 mg/kg bw, to male rats, the ratio of excretion of total radiolabel was the same as after the low dose. Pretreatment with repeated doses also did not affect the pattern of excretion (Klein, 1990). (b)

Metabolism

In the biokinetics study of Klein (1987a), urine was collected separately from each ratunder cool conditions, at intervals of Q~4,4-8,8-24 and 24-48 h, and faeces were collected at intervals of 0-24 and 24-48 h after treatment. The metabolites were extracted from faeces, further isolated by preparative high-performance liquid chromatography (HPLC) and then purified by other HPLC methods. The metabolites were identified by chromatographic comparison with authentic reference compounds in at least two independent chromatographic systems or by ^-nuclear magnetic resonance and mass spectroscopic techniques. A proposed metabolic pathway of imidacloprid in rats is shown in Figure 1. After administration of low doses, no relevant sex dependence was seen in the excretion pattern of the compound or in the metabolic profiles in excreta. All the identified metabolites were found at each dose group and in both sexes. The main metabolites were 6-chloronicotinic acid and its glycine conjugate, which were found only in urine. The amounts of monohydroxylated (5-hydroxyimidacloprid) and olefmic metabolites were similar to those of unchanged parent compound. All other biotransformation products were quantitatively of minor significance. Two main routes of metabolism responsible for the degradation of imidacloprid were identified. The first is oxidative cleavage, yielding 6-chloronicotinic acid, which is conjugated with glycine to form a hippuric acid-type cdhjugate. These two metabolites together represented most of the identified metabolites, or about 30% of the recovered radiolabel. Of minor importance in terms of quantity is dechlorination of the pyridinyl moiety, producing the 6-hydroxy nicotinic

IMIDACLOPRID 79-100 JMPR 2001

82 Figure 1. Proposed metabolic pathway of imidacloprid in rats

acid and its methylmercapturic acid derivative, probably as a degradation product of a glutathione conjugate. The 6-methylmercapto nicotinic acid conjugated with glycine, and the glycine conjugate constituted 5.6% of the recovered radiolabel. The second important biodegradation step starts with hydroxylation of the imidazolidine ring at the 4 or 5 position, and about 16% of the recovered radiolabel was identified as the sum of 4- and 5-hydroxy imidacloprid. The loss of water yields the olefinic compound. These biotransformation products and the unchanged parent

IMIDACLOPRID 79-100 JMPR 2001

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compound were excreted in urine and faeces, while the guanidine compound was a less important metabolite and was eliminated only in faeces. The high excretion rate of the parent compound (average, 14%) indicates rapid passage through the body, as was confirmed by the elimination of > 90% of the recovered radiolabel within 24 h after administration (Klein & Karl, 1990). The distribution of the metabolites in liver and kidney at various times after a single oral dose was investigated in rats under the same test conditions as used by Klein (1987a). The metabolites were extracted from lyophilized organs with water and methanol, further purified by HPLC and thin-layer chromatography and identified by comparative HPLC with authentic reference compounds in at least two independent chromatographic systems and also by mass and ^-nuclear magnetic resonance spectroscopic techniques. The metabolites found in the kidney were identical to those identified in urine. Triazinone was not found in the excreta and may have undergone further biodegradation before elimination via the kidney or the bile. The relative amounts of those biotransformation products formed by oxidative mechanisms (e.g. 6-chloronicotinic acid) increased in the liver during the test period. In kidney, the relative amount of the more polar compounds decreased with time (6-chloronicotinic acid and its glycine conjugate), while the amounts of the olefinic metabolite and the mono-hydroxylated derivative 4-hydroxyimidacloprid showed a relative increase. The proportion of the parent compound decreased slowly as it was metabolized (Karl & Klein, 1992). The metabolic pattern of imidacloprid and its nitroso metabolite in excreta was investigated in male rats after administration of a single oral dose of 1 mg/kg bw of imidacloprid or the nitroso compound and after administration of a high dose bf 150 mg/kg bw imidacloprid. After the low dose of imidacloprid, about 82% of the renal radiolabel was detected. The main renal metabolite (about 30%) was the glycine conjugate of 6-chloronicotinic acid. The parent compound was present at about 12%, the two monohydroxylated biotransformation products (4-hydroxy and 5hydroxyimidacloprid) at about 19% and the olefinic metabolite at about 11%. 6-Chloronicotinic acid represented 7.9% of the renal radiolabel. No nitroso compound was formed under these conditions. In the faeces, imidacloprid, the olefinic metabolite, 6-chloronicotinic acid and its glycine conjugate were identified. These findings are in good agreement with those reported by Klein & Karl (1990). Few metabolites were found in the urine of male rats given the nitroso compound orally. Besides unchanged nitroso compound, only 8% of the renal radiolabel was attributable to the guanidine-type metabolite, some of which was also found in faeces. This finding indicates that the metabolism of the nitroso compound is completely different from that of its parent compound. In order to investigate whether the nitroso compound is formed as a biotransformation product of imidacloprid in vivo, a high single oral dose of 150 mg/kg bw imidacloprid was given to male rats. No nitroso metabolite was formed. In the urine of rats given a diet containing 1800 ppm of imidacloprid for 1 year and then one oral dose of [methylene-14C]imidacloprid, 9.3% of the radiolabel was attributable to the nitroso metabolite, corresponding to 6.8% of the administered dose. The nitroso compound is therefore formed in vivo in rats after long-term intake of imidacloprid. In order to confirm this finding, a direct isotope dilution analysis was conducted with the urine of these rats and also with the urine of mice that had been fed a diet containing 2000 ppm of imidacloprid for about 1 year. Both analyses clearly demonstrated the presence of the nitroso compound in the urine (Klein, 1990).

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2.

Toxicological studies (a)

Acute toxicity

The results of studies on the acute toxicity of imidacloprid are summarized in Table 1. The methods used in these studies complied with OECD guidelines and GLP. Imidacloprid given orally as a single dose was moderately toxic to rats (LD50,3 80-650 mg/kg bw) and mice (LD50, 130-170 mg/kg bw). Behavioural and respiratory signs, disturbances of motility, narrowed palpebral fissures, transient trembling and spasms were seen in rats and mice treated orally at doses > 200 mg/kg bw and > 71 mg/kg bw, respectively. The clinical signs were reversed within 6 days. Imidacloprid given intraperitoneally showed moderate to low acute toxicity in rats, the signs being similar to those after oral administration. Very little toxicity was seen after acute dermal application. The LC50 for a single exposure to an aerosol could not be determined exactly, as rats tolerated inhalation for 4 h of the maximum concentration of dust that could be produced technically (0.069 mg/1 of air) without signs or deaths. Imidacloprid (purity, 94.2%) did not irritate the eyes or skin of HC New Zealand white rabbits (Pauluhn, 1988b,c) and did not sensitize the skin of DHPW guinea-pigs (Ohta, 1988). (b)

Short-term studies of toxicity Mice

Groups of 10 male and 10 female Charles-River B6C3F] mice were given diets containing imidacloprid (purity, 92.8%) at a concentration of 0, 120, 600 or 3000 ppm for up to 107 days, equivalent to 17, 86 and 430 mg/kg bw per day. The study did not comply with GLP. Seven males and seven females at 3000 ppm died; furthermore, several animals displayed a poor general condition, and rough coats were observed frequently. Body-weight gain was reduced and food consumption was increased in males at 600 ppm and males and females at 3000 ppm. At this dose, clinical chemical tests showed significantly decreased urea and cholesterol concentrations in males and lowered alanine aminotransferase activity and glucose concentration in females. Alkaline phosphatase activity was significantly increased in both sexes at 3000 ppm and in females at 120 and 600 ppm. Differences in the weights of the liver, heart, spleen, kidneys, testes and adrenals were observed at 3000 ppm. The NOAEL was 120 ppm, equivalent to 17 mg/kg bw per day (Eiben, 1988a). Table 1. Acute toxicity of imidacloprid Species

Strain

Sex

Route

LD50 (mg/kg bw)

Rat

BorWISW

Male Female

Oral

420 450-480

94.2

Bomann(1989a)

Rat

BorWISW

Male Female

Oral

640 650

96.0

Bomann(1991a)

Rat

BorWISW

Male Female

Oral

500 380

94.3

Bomann(1991b)

Mouse

BonNMRI

Male Female

Oral

130 170

94.2

Bomann(1989b)

Rat

BorWISW

Male Female

Percutaneous

>5000 >5000

94.2

Krotlinger(1989)

Rat

BorWISW

Male Female

Inhalation (aerosol, 4 h)

> 69 >69

95.3

Pauluhn (1988a)

Rat

BorWISW

Male Female

Inhalation (dust, 4 h)

>5300 >5300

95.3

Pauluhn (1988a)

Rat

BorWISW

Male

Intraperitoneal

94.2

Krotlinger(1990)

IMIDACLOPRID 79-100 JMPR 2001

160-170

LC5o 3 Purity (mg/m air) (%)

Reference

85

Rats Groups of 10 male and 10 female Wistar rats [Bor:WISW(SPF-Cpb)] received diets containing imidacloprid (purity, 92.8%) at a concentration of 0, 120, 600 or 3000 ppm for up to 98 days, equal to 11, 57 and 410 mg/kg bw per day for males and 15, 78 and 510 mg/kg bw per day for females. The study did not comply with GLP. Food intake was increased in animals at 3000 ppm, and body-weight gain was decreased at 600 and 3000 ppm. Significantly elevated alkaline phosphatase activity and depressed glucose concentration were found in males and females at 3000 ppm, and the males also showed a reduced cholesterol concentration. Degenerative histological lesions in the epithelium of the testicular tubules were seen in five of 10 males at 3000 ppm, and multifocal group cell necrosis was diagnosed in the liver of one male at this dietary concentration. The NOAEL was 120 ppm, equal to 11 mg/kg bw per day (Eiben, 1988b). Groups of 10 male and 10 female Wistar rats [BonWISW(SPF-Cpb)] received diets containing imidacloprid (purity, 95.3%) at a concentration of 0, 150, 600 or 2400 ppm for up to 96 days, equal to 14, 61 and 300 mg/kg bw per day for males and 20, 83 and 420 mg/kg bw per day for females. Satellite groups consisting of 10 male and 10 female rats received the test substance at a concentration of 0 or 2400 ppm over the same period, and, to study the reversibility of any effects, control diet was provided during the subsequent 4-week post-treatment observation period. The study was conducted according to GLP. In animals at 2400 ppm, feed intake was increased during treatment and recovery. Reduced body-weight gain was observed in males at 600 ppm and in females at 2400 ppm. Slightly longer thromboplastin times and depressed thrombocyte counts were found at 2400 ppm, both of which were only partially reversible. Elevated alkaline phosphatase and alanine aminotransferase activities and depressed protein, albumin, cholesterol and triglyceride concentrations were found in males and females at 2400 ppm. Depressed protein concentrations were also found in males at 150 and 600 ppm. Males at 2400 ppm showed increased incidences of cellular necroses, roundcell infiltration, swollen cellular nuclei and cytoplasmic lesions in the liver, but these hepatotoxic effects were reversible within the subsequent 4-week post-treatment observation period. The NOAEL was 150 ppm, equal to 14 mg/kg bw per day (Eiben & Rinke, 1989). Imidacloprid (purity, 95.3%) was administered in dust form through the nose only to groups of 10 male and 10 female Wistar rats [Bor:WISW(SPF-Cpb)] at analytically determined concentrations of 0,20, 110 and 500 mg/m3 on 5 consecutive days for 6 h/day. The respirability of the test substance at the highest concentration was relatively low; about 20% of the administered mass of the substance was in the form of particles < 5 mg. The study was conducted according to GLP. Body-weight gain was slightly decreased at the two higher concentrations. Elevated Odemethylase and N-demethylase activities were found in a liver homogenate from animals at 110 mg/m3 air. The NOAEL was 20 mg/ m3 air (Pauluhn, 1988a). Imidacloprid (purity, 95.3%) was administered in dust form through the nose only to groups of 10 male and 10 female Wistar rats [BonWISW(SPF-Cpb)] at analytically determined concentrations of 0, 5.5, 30 and 190 mg/m3 for 6 h/day, 5 days per week for 4 weeks. The study was conducted according to GLP. Body-weight gain was decreased in males at the two higher concentrations. Increased mixed-function oxidase activities were found in liver homogenate from females at these concentrations and in males at 190 mg/m3 air. Increased alanine aminotransferase and glutamate dehydrogenase activities were seen in both sexes at the highest concentration, and alanine aminotransferase activity was increased in females at 30 mg/m3 air. The females had increased IMIDACLOPRID 79-100 JMPR 2001

86

alkaline phosphatase activity at the two higher concentrations and increased liver weights at 190 mg/m3 of air. The serum a 1-globulin fraction was reduced in both sexes at the two higher concentrations. At 190 mg/m3 of air, blood coagulation time was increased in females and thrombocyte counts were depressed in males. The NOAEL was 5.5 mg/ m3 air (Pauluhn, 1988d) Rabbits Imidacloprid (purity, 95.0%), mixed to a paste in a physiological saline solution containing 2% Cremophor EL, was applied to the shorn dorsal and flank skin of groups of five male and five female HC New Zealand white rabbits at a dose of 0 or 1000 mg/kg bw for 6 h/day for 15 days. The study was conducted according to GLP. No treatment-related effects were observed. The NOAEL was 1000 mg/kg bw per day at the limit dose tested (Flucke, 1990). Dogs Groups of two male and two female pure-bred beagle dogs received diets containing imidacloprid (purity, 92.8%) at a concentration of 0, 200, 1000 or 5000 ppm for up to 28 days, providing average intakes of 0,7.3,31 and 49 mg/kg bw per day for males and females combined. The study was not conducted according to GLP. Two animals at 5000 ppm died, and the other two were killed in moribund condition. The symptoms observed were ataxia, tremor and occasional vomiting. Reductions in food consumption were recorded at the two higher concentrations, and body weight was markedly reduced at 5000 ppm. The results ofhearing tests, ophthalmic examinations, urine analyses andhaematological examiinations indicated no treatment-related changes at any dietary concentration. Cytochrome P450 activity in the liver was slightly increased in males and females at 1000 ppm. The triiodothyronine concentration in plasma was slightly decreased in one male and one female at 5000 ppm. The weight of the liver was increased in one female at 1000 ppm; one male at this concentration showed slight hepatocellular hypertrophy with slight pigmentation of Kupffer cells, and another had minimal follicular atrophy of the thyroid. The NOAEL was 200 ppm, equal to 7.3 mg/kg bw per day (Bloch et al., 1987). Groups of four male and four female beagles [BorBeag] were given diets containing imidacloprid (purity, 95.3%) at a concentration of 0, 200, 600 or 1800/1200 ppm for 13 weeks. The concentration of active ingredient at the highest level was reduced at week 4 because of low food intake. The study was conducted according to GLP. The food consumption of animals at 600 ppm was reduced. At higher concentrations, the animals showed a clinically emaciated state and transient trembling. Body-weight gain was reduced at 1800 ppm, but, once the active ingredient concentration had been reduced to 1200 ppm, a trend to normalization was apparent. The NOAEL was 200 ppm, equal to 7.5 mg/kg bw per day (Ruf, 1990). Groups of four male and four female pure-bred beagles were given diets containing imidacloprid (purity, 94.9%) at a concentration of 0, 200, 500 or 1250/2500 ppm for 52 weeks. The concentration of 1250 ppm was increased from week 17 onwards. The mean intakes of imidacloprid were equal to 0,6.1,15 and 41772 mg/kg bw per day for males and females combined. The study was conducted according to GLP. Slight, temporary reductions in food intake were seen in both sexes at 1250 ppm and at the increased concentration of 2500 ppm. At 1250/2500 ppm, clinical chemical examination revealed an increased plasma cholesterol concentration in females after 13 and 26 weeks, increased hepatic cytochrome P450 activity and slightly increased liver weights in males and females after 52 weeks. The NOAEL was 500 ppm, equal to 15 mg/kg bw per day (Allen et al., 1989). IMIDACLOPRID 79-100 JMPR 2001

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(c)

Long-term studies oftoxicity and carcinogenicity Mice

Groups of 50 male and 50 female Charles-River B6C3Ft mice received diets containing imidacloprid (purity, 95.3%) at a concentration of 0, 100, 330 or 1000 ppm for 24 months. In a supplemental study to determine the maximum tolerated dose, groups of 50 male and female mice were given diets containing imidacloprid (purity, 90.0%) at a concentration of 0 or 2000 ppm for 24 months. Ten additional mice of each sex were included per dose in both studies for interim sacrifice after 12 months of treatment. The mean intake of imidacloprid was equal to 20, 66,210 and 410 mg/kg bw per day for males and 30,100,270 and 420 mg/kg bw per day for females. The study was conducted according to GLP. Food intake was decreased by 24% in females at 2000 ppm, and water intake was decreased in males and females at this concentration. Mice at 1000 and 2000 ppm had significantly, doserelated reduced weight gains, particularly during the latter half of the study. At 2000 ppm, lower leukocyte counts were found in both sexes. Reduced blood cholesterol concentrations were observed at 2000 ppm after 52 weeks. An increased incidence of low-grade periacinar hepatic-cell hypertrophy was found in males at 2000 ppm. The brains of animals at the highest concentration showed more mineralization of the thalamus than in the control groups. There was no evidence of a carcinogenic effect. The NOAEL was 330 ppm, equal to 66 mg/kg bw per day (Watta-Gebert, 1991a,b). Rats Groups of 50 male and 50 female Wistar rats [BonWISW (SPF-Cpb)] received diets containing imidacloprid (purity, 94.3-95.3%; mixed batch) at a concentration of 0, 100, 300 or 900 ppm for 24 months. In a supplemental study to determine the maximum tolerated dose, groups of 50 male and female Wistar rats were given diets containing imidacloprid at a concentration of 0 or 1800 ppm for 24 months. Ten additional rats of each sex were included per dose in each study for interim sacrifice after 12 months of treatment. The mean intake of imidacloprid was equal to 5.7, 17, 51 and 100 mg/kg bw per day for males and 7.6, 25, 73 and 140 mg/kg bw per day for females. The study was conducted according to GLP. Water intake was reduced by 13% in females at 1800 ppm. Reduced weight gain was noted in males and females at 900 ppm, the decreases reaching a maximum of 11-12% at 1800 ppm. The absolute weights of the liver and kidney in females and of the liver in males were reduced after 12 months at 900 ppm. Histological assessment of these organs afforded no evidence of treatmentrelated lesions. An increased incidence of mineralization in the colloid of the thyroid gland follicles, in comparison with the incidences of this lesion in controls in previous studies, was determined in males at concentrations > 300 ppm and in females at 900 ppm. At 1800 ppm, fewer colloid aggregations and parafollicular hyperplasia were observed. There was no evidence of a carcinogenic effect. The NOAEL was 100 ppm, equal to 5.7 mg/kg bw per day (Eiben, 1991; Eiben &Kaliner, 1991). (d)

Genotoxicity

Imidacloprid was investigated in an adequate range of assays for genotoxicity in vitro and in vivo (Table 2). Tests for reverse mutation in Salmonella typhimurium and for point mutations at the hprt locus in Chinese hamster ovary cells gave negative results. Weak induction of sister chromatid exchange was found in one test in Chinese hamster ovary cells in vitro but not in vivo. Tests for mitotic recombination, DNA damage in the rec assay and unscheduled DNA synthesis also gave negative results. In human lymphocyte cultures, a slight increase in the chromosomal aberration

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Table 2. Studies of the genotoxicity of imidacloprid Test object

Concentration or dose

Purity (%)

Result

Reference

S. typhimurium TA1535, TA100,TA1537,TA98

< 12000ng/plate

95.0

Negative8

Herbold(1989a)

Reverse mutation

S. typhimuriumTA\535, TA100,TA1537, TA98

< 5000 ng/plate

96.0

Negative3

Herbold(1991)

Reverse mutation

5. typhimuriumTA.1535, TA100,TA1537,TA98

< 5000 ng/plate

96.3

Negative3

Herbold(1991)

Reverse mutation

S. typhimurium TA98, TA 1 00, TA 1535, TA 1537

< 5000 ng/plate

97.4

Negative3

Herbold(1992)

Reverse mutation

S. typhimurium TA98, TA100,TA1535,TA1537; E. coll WP2/uvrA

< 5000 ng/plate

93.7

Negative

Watanabe(1991a)

DNA damage

B. subtilismi(rsc+l M45 (rec-)

< 5000 ng/plate

94.7

Negative

Watanabe (1990a)

Point mutation

hprt locus of Chinese hamster < 120, 1200ng/ml ovary cells

95.2

Negative3

Lehn(1989a)

Mitotic recombination

Saccharomyces cerevisiae D7 < 10000ng/ml

95.3

Negative

Herbold(1988a)

Unscheduled DNA synthesis

Rat primary hepatocytes

5.0-750 fig/ml

95.2

Negative

Cifone (1988)

Sister chromatid exchange

Chinese hamster ovary cells

< 5000 jig/ml

95.2

Positive8

Taalman(1988)

Sister chromatid exchange

Chinese hamster ovary cells

< 1200ng/ml

95.2

Negative3

Putman & Morris (1989)

Chromosomal aberrations

Human lymphocytes

< 5200 ng/ml

95.2

Positive1"

Herbold(1989b)

In vivo Micronucleus formation

Chinese hamster bone marrow

2000 mg/kg bw

94.6

Negative

Herbold (1989c)

Micronucleus formation

Mouse

80 mg/kg bw

95.3

Negative

Herbold(1988b)

Sister chromatid exchange

Chinese hamster bone marrow

500, 1000, 2000 mg/kg bw

95.0

Negative

Herbold (1989d)

80 mg/kg bw

94.1

Negative

V61ker(1990)

End-point In vitro Reverse mutation

Cytogenetic damage Mouse germ cells 3 b

With and without metabolic activation With metabolic activation

rate was observed in the range of cytotoxic concentrations in the absence of metabolic activation; no unequivocal findings could be established with metabolic activation. The effect of damaged cellular components on the chromosomes must be considered in assessing this effect. As the results of all tests for chromosomal damage in vivo (micronucleus formation and cytogenetic effects in bone marrow and spermatogonia) were negative, the significance of the clastogenic effect of imidacloprid in human lymphocyte cultures is questionable. (e)

Reproductive toxicity (i)

Multigeneration studies

Rats Groups of Wistar [KfM: Wist] rats received diets containing technical-grade imidacloprid (purity, 94.4-95.3%; mixed batch) at a concentration of 0,100,250 or 700 ppm, equivalent to 6.6, 17 and 47 mg/kg bw per day, for 84 days before mating and throughout mating, gestation and

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lactation of the F la and F lb litters. The Fj parent animals were selected after weaning of the F lb litters on day2\postpartum. The diets were fed for 105 days before breeding of the Fia and p2b litters. Each group of the F0 parent generation consisted of 30 male and 30 female rats, and each group of the FI parent generation consisted of 26 males and 26 females. The study was conducted according to GLP. At 700 ppm, reduced food consumption and depressed body-weight gain were observed in FQ males, reduced food intake in FO and FI females and reduced body-weight gain in FQ females. Increased cytochrome P450,0-detnethylase and N-demethylase activities were found in males at 700 ppm and increased O-demethylase activities in Fj females at 250 and 700 ppm. Reduced body weights and body-weight gains were observed in all generations (Fia, Fib, F2a and F2b) at 700 ppm during lactation. The NOAEL for parental effects was 100 ppm, equivalent to 6.6 mg/kg bw per day, and the NOAEL for reproductive effects was 250 ppm, equivalent to 17 mg/kg bw per day (Suteretal., 1990). (ii)

Developmental toxicity

Rats Imidacloprid (purity, 94.2%) was administered by gavage to groups of 25 mated female Wistar [KfM: Wist] rats at a dose of 0,10,30 or 100 mg/kg bw per day on days 6-15 post coitum. A standard volume of 10 ml/kg bw with daily adjustment to the actual body weight was used. The rats were killed on day 21 post coitum, and the fetuses were removed surgically. The study was conducted according to GLP. Dose-related reductions in food consumption and body-weight gain were observed in dams at 30 and 100 mg/kg bw per day. Skeletal examination showed a slightly increased incidence of wavy ribs at 100 mg/kg bw per day. The NOAEL for maternal effects was 10 mg/kg bw per day, and the NOAEL for developmental effects was 30 mg/kg bw per day (Becker et al., 1988a). Rabbits Imidacloprid (purity, 94.2%) was administered by gavage to groups of 16 female chinchilla [KfmiCHIN] rabbits at a dose of 0, 8, 24 or 72 mg/kg bw per day on days 6-\Spost coitum. A standard volume of 4 ml/kg bw with daily adjustment to the actual body weight was used. The rabbits were killed on day 28post coitum, and the fetuses were removed surgically. The study was conducted according to GLP. Decreased food intake and body-weight gain were observed at doses > 24 mg/kg bw per day, and an increased mortality rate was observed at 72 mg/kg bw per day, two females dying on days 18 and 19 post coitum. A further female in this group aborted on day 26 post coitum., and two females showed total resorption at terminal necropsy. The does at 72 mg/kg bw per day had slightly increased post-implantation loss when only does with live fetuses at termination were considered, which increased to a high loss (32.5%) when females with total resorption or abortion were included. The body weights of the fetuses were reduced, and the incidence of fetuses with retarded ossification was increased at 72 mg/kg bw per day. The NOAEL for maternal effects was 8 mg/ kg bw per day, and that for developmental effects was 24 mg/kg bw per day (Becker et al., 1988b). (f)

Special studies (i)

Neurotoxicity

Rats Imidacloprid (purity, 97.6-98.8%) was administered by gavage to groups of 18 male and 18 female fasted Sprague-Dawley [Sas:CD(SD)BR]/f ats at a single dose of 0,42,150 or 310 mg/kg

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bw. The test substance was suspended in 0.5% (w/v) methylcellulose with 0.4% (w/v) Tween 80 in deionized water and was administered in a volume of 10 ml/kg bw. In a supplemental study, imidacloprid was administered to 12 female rats by gavage at an analytically confirmed dose of 0 or 20 mg/kg bw. Behavioural tests were administered on day 0 of treatment at the time of the peak plasma concentration. Four males and 10 females at the highest dose died, either on the day of treatment or the next day. These deaths were attributed to treatment. A dose-related increase in the incidence and severity of clinical signs was seen, with treatment-related effects in males at 150 or 310 mg/kg bw and in females at the highest dose. In males at 150 mg/kg bw, the effects were limited to tremors and nasal staining, while males at the highest dose also had uncoordinated gait, decreased activity and urine staining and were cool to touch. The treatment-related effects in females at the highest dose consisted of tremors, uncoordinated gait, decreased activity, increased reactivity and red nasal staining. Clinical signs of toxicity were generally observed on day 0 and resolved in surviving males and females within 1-5 days after treatment. Treatment-related effects in a 'functional observational battery' were observed in males and females at the two higher doses. These consisted of an increased incidence of recumbency, tremors and nasal staining in males and tremors in females at 150 mg/kg bw and numerous treatmentrelated effects in males and females at 310 mg/kg bw, consistent with the lethality of this dose within 24 h after treatment. All the toxic effects had resolved in surviving animals by the next observation period, 7 days after treatment. A dose-related decrease in a measure of motor and locomotor activity was observed in both sexes, with reduced activity in males at the two higher doses and in females at all three doses on day 0. As the slightly reduced motor activity in females at 42 mg/kg bw was comparable to that seen before treatment and also 14 days after treatment, this reduction was considered not to be an adverse effect. Habituation was not affected. All clinical signs and neurobehavioural effects showed complete reversal within 7 days of treatment at sublethal doses. Animals at 150 mg/kg bw showed a decrease in serum triglyceride concentration, and animals that survived the highest dose had decreased serum potassium and cholesterol concentrations (females) and decreased serum alanine aminotransferase activity (males and females). Haematological changes were found in females at the highest dose. The NOAEL was 42 mg/kg bw (Sheets, 1994a). Groups of 18 male and 18 female Fischer 344 CDF/BR rats were given diets containing imidacloprid (purity, 97.6-98.8%) at a concentration of 0, 140, 960 or 3000 ppm for 13 weeks, equal to 0, 9.3, 63 and 200 mg/kg bw per day for males and 0, 10, 69 and 210 mg/kg bw per day for females. Twelve rats of each sex per dietary level were used for neurobehavioural evaluation and half of them for neuropathological examination, and six rats of each sex per group were used for observation of clinical effects at interim sacrifice. Body weight and food consumption were reduced by treatment at the two higher concentrations in males and females. In the'functional observational battery', treatment-related effects were seen in males at the highest concentration but not in treated females. Motor activity was not affected in males or females at any concentration. The NOAEL was 140 ppm, equal to 9.3 mg/kg bw per day (Sheets, 1994b). (ii)

Studies on metabolites

Several metabolites of imidacloprid were tested for acute toxicity by oral administration to rats and for their ability to induce point mutations in S. typhimurium. The results of studies on the acute toxicity of imidacloprid metabolites are summarized in Table 3. The methods used complied with OECD guidelines and GLP. The metabolites showed

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Table 3. Acute toxicity of metabolites of imidacloprid given orally to specific pathogen-free rodents Metabolite

Species

Sex

LD50 (mg/kg bw)

Purity (%)

Reference

l-(6-Chloro-3-pyridylmethyl)-2-imidazolidinone

Rat

Male Female

4080 1820

99.9

Ohta(1991a)

l-(6-Chloro-3-pyridylmethyl)-JV-nitro(4-imidazolin2-ylidene)amine (olefmic metabolite)

Rat

Male Female

3500 1100

98.0

Ohta(1991b)

l-(6-Chloro-3-pyridylmethyl)-A^-nitroso(imidazolidin2-ylideneamine (nitroso metabolite)

Rat

Male Female

1980 3560

98.1

Ohta(1991c)

l-(6-Chloro-3-pyridylmethyl)-7V-nitroso(imidazolidin2-ylideneamine (nitroso metabolite)

Rat

Male Female

>600 >600

Not reported

Nakazato(1988a)

1 -(6-Chloro-3 -pyridylmethy l)-jV-nitroso(imidazolidin2-ylideneamine (nitroso metabolite)

Mouse

Male Female

200 200

Not reported

Nakazato (1988b)

l-(6-Chloro-3-pyridylmethyl)imidazolidin2-ylideneamine

Rat

Male Female

300 280

87.0

Nakazato (1991)

moderate acute toxicity after oral administrations, the clinical signs being mydriasis, abnormal gait, sedation, abnormal respiration, salivation and tremor. Groups of 15 male and 15 female Wistarrats [BorWISW (SPF-Cpb)] received an unlimited supply of drinking-water containing nitroso metabolite at a concentration of 0, 100, 300 or 1000 ppm for 12 weeks, providing mean intakes of 13, 35 and 110 mg/kg bw per day for males and 13, 39 and 120 mg/kg bw per day for females. Water intake was decreased in the groups at 1000 ppm. At 300 ppm, higher lymphocyte counts and lower numbers of polymorphonuclear cells were observed. The NOAEL was 100 ppm, equal to 13 mg/kg bw per day (Krotlinger, 1992). All tests for genotoxicity with imidacloprid metabolites in vitro and in vivo gave negative results (Table 4).

3.

Observations in humans

Periodic examinations at the medical department of Bayer AG showed no adverse health effects in employees handling imidacloprid during the production of the active ingredient and formulations (Paul, 1996). In experimental biological testing and field tests with imidacloprid formulations, no adverse effects on the health of operators or workers were reported. No epidemiological studies on exposure of the general population to imidacloprid were available. Mild cases of contact dermatitis have been reported in pet owners after use of a veterinary formulation of imidacloprid (Advantage®). The effect appeared to be due to constituents of the product that are not present in plant protection formulations. No data on symptoms of poisoning or clinical signs were available. A 4-year-old child who ingested four rodlets containing 50 mg of imidacloprid per rodlet, corresponding to about 10 mg/kg bw, showed no signs of poisoning or adverse health effects (Steffens, 2000).

Comments Imidacloprid is rapidly and almost completely absorbed (> 92%) from the gastrointestinal tract of rats, and is eliminated from the organism rapidly and completely, with no indication of bioaccumulation of the parent compound or its metabolites. On average, 75% of an administered dose was excreted in the urine and the remainder in the faeces. Most of that in the faeces originated IMIDACLOPRID 79-100 JMPR 2001

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Table 4. Studies of the genotoxicity of metabolites of imidacloprid Metabolite3

End-point

Test object or dose

Concentration (%)

In vitro Metabolite 1

Reverse mutation

S. typhimurium TA1535, TA100, TA1537, TA98; E. coli WP2uvrA

Metabolite 2

Reverse mutation

Metabolite 3

Purity

Result

Reference

< 5000 u,g/plate 99.9

Negative

Watanabe(1991b)

S. typhimurium TA98, TA100, TA 1535, TA 1537; £ co/i WP2wvrA

< 5000 ng/plate 98.0

Negative

Ohta(1991d)

Reverse mutation

S. typhimurium TA98, TA100, TA1535,TA1537;£. coli WP2«vrA

<2500|ig/plate 87.0

Negative

Watanabe(1991c)

Metabolite 4

Reverse mutation

S. typhimurium TA98, TA100, TA1535,TA1537;£. coli WP2MV/-A

< 5000 ng/plate 98.3

Negative

Watanabe (1990b)

Metabolite 4

Point mutation

hprt locus of Chinese hamster ovary cells

< 2000 jig/ml

94.3

Negative" Lehn (1989b)

Metabolite 4

Point mutation

hprt locus of V79 Chinese hamster cells

<2000ng/ml

98.9

Negative3 Lehn(1989c)

Metabolite 4

DNA damage

B. subtilis HI 7 (rec+), M45 (re
<2000ng/plate 98.1

Negative

Watanabe (199 Id)

Metabolite 4

Unscheduled DNA Rat primary hepatocytes synthesis

5.0-750 jig/ml

98.9

Negative

Fautz(1989)

Metabolite 4

Chromosomal aberrations

V79 Chinese hamster cells

< 1000 tig/ml

98.9

Negative3 Heidemann (1989)

Metabolite 4

Chromosomal aberrations

V79 Chinese hamster cells

1. 0-0.25 mg/ml NR

Negative3 Usami (1988a)

Micronucleus formation

Male BDFi mice

0,40,80,160 96.4 mg/kg bw orally

Negative

Usami (1988b)

Metabolite 4

Micronucleus formation

Male BDFi mice

20, 40, 80 mg/kg 96.4 bw intraperitoneally

Negative

Usami (1988c)

Metabolite 4

Micronucleus formation

Mice

100 mg/kg bw orally

98.9

Negative

Herbold(1989e)

Metabolite 4

Micronucleus formation

Mice

50 mg/kg bw orally

98.9

Negative

Herbold(1989f)

/« v/vo Metabolite 4

NR, not reported 3 Metabolite 1, l-(6-chloro-3-pyridylmethyl)-2-imidazolidinone; metabolite 2, l-(6-chloro-3-pyridylmethyl)-Ar-nitro(4-imidazolin-2ylidene)amine (olefin metabolite); metabolite 3, l-(6-chloro-3-pyridylmethyl)imidazolidi-2-ylideneamine; metabolite 4, l-(6-chloro-3pyridylmethyl)-N-nitroso(imidazolidin-2-ylidene)amine (nitroso metabolite)

from biliary excretion. Peak plasma concentrations of radiolabel were reached within approximately 2.5 h. The radiolabel was rapidly distributed from the intravascular space to the peripheral tissues and organs; the concentrations in tissues after 48 h were very low. Imidacloprid penetrated the blood-brain barrier to only a very limited extent. The metabolism of imidacloprid in rats was rapid, and the amount of unchanged parent compound represented 10-16% of a given dose. The main urinary metabolites were 6-chloronicotinic acid and its glycine conjugate and the two corresponding imidazolidine ring-containing biotransformation products. The two monohydroxylated metabolites (5-hydroxyimidacloprid) and (4-hydroxyimidacloprid) and an unsaturated compound were also detected in urine. Imidacloprid given orally as a single dose was moderately toxic to rats (LDso, 3 80-650 mg/kg bw) and mice (LDso, 130-170 mg/kg bw). Behavioural and respiratory signs, disturbances of motility, narrowed palpebral fissures, transient trembling and spasms were seen in rats and mice treated orally at doses > 200 mg/kg bw and > 71 mg/kg bw, respectively. The clinical signs were reversed within 6 days. Imidacloprid given intraperitoneally showed moderate to low acute toxicity in rats, the signs being similar to those after oral administration. Very little toxicity was seen after acute dermal application. The LCso for acute exposure to an aerosol could not be IMIDACLOPRID 79-100 JMPR 2001

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determined exactly, as rats tolerated inhalation for 4 h of the maximum concentration of dust that could be produced technically (0.069 mg/1 of air) without signs or deaths. Imidacloprid did not irritate the skin or eyes of rabbits and did not sensitize the skin of guinea-pigs in a maximization test. Reduced body-weight gain was the most sensitive toxicological end-point in mice at doses > 86 mg/kg bw per day, in rats at doses > 30 mg/kg bw per day, in rabbits at doses > 24 mg/kg bw per day and in dogs at doses > 22 mg/kg bw per day. Furthermore, decreased body-weight gain was the main effect during lactation in rat pups of dams given doses > 6.6 mg/kg bw per day. In some studies, the lower body weights were accompanied by a simultaneous increase in feed intake. The liver was the other main target organ after repeated administration of imidacloprid to mice, rats and dogs at doses > 410 mg/kg bw per day, > 17 mg/kg bw per day and > 31 mg/kg bw per day, respectively. The spectrum of changes observed ranged from induction of hepatic microsomal enzymes through disturbances of hepatic function to histologically apparent damage to the organ. The initial sign of an effect on the liver was increased activity of cytochrome P450 enzymes, accompanied by slight hepatocellular hypertrophy and necrosis, swollen cell nuclei, round-cell infiltration and increased liver weight. Changes in blood cholesterol, triglyceride, protein and albumin concentrations as well as increased activities of alanine aminotransferase, alkaline phosphatase and galactodehydrogenase in plasma were also observed. Slight follicular atrophy of the thyroid gland and slightly depressed triiodothyronine in plasma were found at the highest dietary concentration, providing an average intake of 49 mg/kg bw per day, in a short-term study in dogs. However, these effects were not found in the 1 -year study in dogs given a dietary concentration providing an average intake of 72 mg/kg bw per day. An increased incidence of mineralization was seen in the colloid of thyroid gland follicles in a longterm study in rats given a dietary concentration equal to 17 mg/kg bw per day, although the plasma concentrations of thyroid hormones remained unchanged. The NOAEL in this study was 5.7 mg/kg bw per day. No evidence of a carcinogenic effect of imidacloprid was found in either mice or rats in the long-term studies of dietary administration. Imidacloprid gave negative results in an adequate range of assays for genotoxicity in vitro and in vivo. Weak induction of sister chromatid exchange was found in one test with Chinese hamster ovary cells in vitro, but not in vivo. The Meeting concluded that imidacloprid is unlikely to be genotoxic or to pose a carcinogenic risk to humans. Imidacloprid was investigated for reproductive toxicity in a two-generation study in rats and in studies of developmental toxicity in rats and rabbits. Reproductive behaviour and outcome were not affected. An increased incidence of wavy ribs was observed at a maternally toxic dose of 30 mg/kg bw per day. Reduced body weights and retarded ossification were found in rabbit fetuses at a maternally toxic dose of 24 mg/kg bw per day. The Meeting concluded that imidacloprid has no teratogenic potential. Several metabolites were tested for acute toxicity by oral administration to rats and for their ability to induce point mutations in Salmonella typhimurium. They were found to be less acutely toxic than the parent compound, and no indications of genotoxic potential were found. In a 12week study in rats in which a nitroso metabolite was administered in drinking-water, the NOAEL was 100 ppm, equal to 13 mg/kg bw per day, on the basis of increased lymphocyte counts and reduced polymorphonuclear cell counts. In a study of acute neurotoxicity in rats, clinical signs and effects on motor and locomotor activity and in a 'functional observational battery' were observed at doses > 150 mg/kg bw 1 day after application. Complete recovery was observed within 7 days. The NOAEL was 42 mg/kg bw. In a 13-week study of neurotoxicity in rats, the NOAEL of 140 ppm, equal to 9.3 mg/kg bw per day, was based on reduced body-weight gain and food consumption at doses > 960 ppm, equal to

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63 mg/kg bw per day. Behavioural effects were observed only in the 'functional observational battery' in males at 3000 ppm, equal to 200 mg/kg bw per day. The results of periodic examinations of employees exposed to imidacloprid showed no adverse health effects. No epidemiological studies of the effects of imidacloprid and no information on symptoms of poisoning or clinical signs were available. A 4-year-old child who ingested about 10 mg/kg bw of a veterinary preparation of imidacloprid showed no signs of poisoning or adverse health effects.

Toxicological evaluation The Meeting concluded that the existing database was adequate to characterize the potential hazards of imidacloprid to fetuses, infants and children. The Meeting established an ADI of 0-0.06 mg/kg bw on the basis of the NOAEL for effects on the thyroid gland of 100 ppm, equal to 5.7 mg/kg bw per day, in the long-term study of toxicity and carcinogenicity in rats and a safety factor of 100. This ADI is supported by the NOAEL for effects on the liver in parental animals in the multigeneration study of reproductive toxicity. The Meeting established an acute reference dose of 0.4 mg/kg bw on the basis of the NOAEL of 42 mg/kg bw in the study of acute neurotoxicity in rats and a safety factor of 100. Levels relevant to risk assessment Species

Study

Effect

NOAEL

LOAEL

Mouse

2-year studies of toxicity and carcinogenicity3

Toxicity

330 ppm, equal to 66 mg/kg bw per day

1000 ppm, equal to 210 mg/kg bw per day

Carcinogenicity

1000 ppm, equal to 210 mg/kg bw per day b

2-year studies of toxicity and carcinogenicity3

Toxicity

100 ppm, equal to 5.7 mg/kg bw per day 300 ppm, equal to 100 mg/kg bw per dayb

300 ppm, equal to 17 mg/kg bw per day

Two-generation study of reproductive toxicity3

Parental toxicity

Developmental toxicity0

Maternal toxicity Embryo- and fetotoxicity Acute neurotoxicityb>c

100 ppm, equivalent to 6.6 mg/kg bw per day 250 ppm, equivalent to 17 mg/kg bw per day 10 mg/kg bw per day 30 mg/kg bw per day

250 ppm, equivalent to 17 mg/kg bw per day 700 ppm, equivalent to 47 mg/kg bw per day 30 mg/kg bw per day 100 mg/kg bw per day

42 mg/kg bw per day

1 50 mg/kg bw per day

8 mg/kg bw per day 24 mg/kg bw per day

24 mg/kg bw per day 72 mg/kg bw per day

500 ppm, equal to 1 5 mg/kg bw per day (52-week study)

600 ppm, equivalent to 22 mg/kg bw per day (13-week study)

Rat

Rabbit

Developmental toxicity6

Dog

13- and 5 2- week studies of toxicitya'd

Carcinogenicity

Offspring toxicity

Maternal toxicity Embryo- and fetotoxicity

a

Diet Highest dose tested c Gavage d Two or more studies combined b

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Estimate of acceptable daily intake for humans 0-0.06 mg/kg bw Estimate of acute reference dose 0.4 mg/kg bw Studies that would provide information useful for continued evaluation of the compound Further observations in humans

List of end-points relevant for setting guidance values for dietary and non-dietary exposure Absorption, distribution, excretion and metabolism in mammals Extensively absorbed (95%) on basis of urinary and Rate and extent of oral absorption biliary excretion Uniformly and rapidly distributed, highest residues (after Distribution 48 h) in liver, kidney, lung, and skin Potential for accumulation No evidence of accumulation Rapidly and completely (-25% in faeces, 75% in urine Rate and extent of excretion within 48 h) Extensively metabolized by oxidative cleavage, Metabolism in animals hydroxylation, conjugation Parent compound and metabolites, including plant nitroso lexicologically significant compounds (animals, plants, metabolite, 1 -(6-chloro-3-pyridylmethyl)-7Vand environment) nitroso(imidazolidin-2-ylidene)amine Acute toxicity Rat, LD50, oral Rabbit, LD5o, dermal Rat, LCso, inhalation Skin irritation Eye irritation Skin sensitization

380-650 mg/kg bw > 5000 mg/kg bw > 0.69 mg/1 of air (4 h, nose only) Not irritating Not irritating Not sensitizing (Magnussen and Kligman)

Short-term toxicity Target / critical effect Lowest relevant oral NOAEL / NOEL

Decreased body-weight gain, liver; thyroid 52 weeks, dog: 500 ppm (15 mg/kg bw per day)

Genotoxicity Long-term toxicity and carcinogenicity Target/critical effect Lowest relevant NOAEL / NOEL Carcinogenicity Reproductive toxicity Reproduction target / critical effect Lowest relevant reproductive NOAEL Developmental target / critical effect Lowest relevant developmental NOAEL Neurotoxicity

NOAEL (acute neurotoxicity) NOAEL (short-term study of neurotoxicity)

No potential for genotoxicity Decreased body-weight gain, liver; thyroid 2 years, rat: 100 ppm (5.7 mg/kg bw per day) No potential for carcinogenicity

Decreased body-weight gain in pups during lactation at parentally toxic doses 250 ppm (17 mg/kg bw per day) Reduced body weight, increased incidence of retarded ossification at maternally toxic doses Rabbit: 24 mg/kg bw per day Clinical signs and neurobehavioural effects ascribed to acute cholinergic toxicity; short-term effects related to general toxicity 42 mg/kg bw 140 ppm (9.3 mg/kg bw per day)

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Other toxicological studies

Studies of kinetics and metabolism, acute toxicity, shortterm effects, and mutagenicity of the plant nitrosometabolite revealed no evidence of particular risk

Human data

No evidence of adverse effects

Summary

Value

Study

Safety factor

ADI Acute reference dose

0-0.06 mg/kg bw 0.4 mg/kg bw

Rat, 2 years Rat, acute neurotoxicity

100 100

References Allen, T.R., Frei, T., Luetkemeier, H., Vogel, O., Biedermann, K. & Wilson, J. (1989) 52-week oral toxicity (feeding) study with NTN 33893 technical in the dog. Unpublished report from Research & Consulting Company AG, report No. R 4856, dated 19 October 1989, GLP, amendment No. R 4856A, dated 3 March 1992. Submitted to WHO by Bayer AG, Mannheim, Germany. Becker, H., Vogel, W. & Terrier, C. (1988a) Embryotoxicity study (including teratogenicity) with NTN 33893 technical in the rabbit. Unpublished report from Research & Consulting Company AG, report No. R 4583, dated 24 November 1988, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Becker, H., Vogel, W. & Terrier, C. (1988b) Embryotoxicity study (including teratogenicity) with NTN 33893 technical in the rat. Unpublished report from Research & Consulting Company AG, report No. R 4582, dated 24 November 1988, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Bloch, I., Frei, T., Luetkemeiner, H., Vogel, W. & Wilson, J. (1987) 28-day oral range-finding toxicity (feeding) study with NTN 33893 tech. in the dog. Unpublished report from Research & Consulting Company AG, report No. R 4196, dated 9 October 1987, non-GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Bomann, W. (1989a) NTN 33893—Study for acute oral toxicity to rats. Unpublished report from Bayer AG, report No. 18594, dated 15 December 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Bomann, W. (1989b) NTN 33893—Study for acute oral toxicity to mice. Unpublished report from Bayer AG, report No. 18593, dated 15 December 1989, GLP, unpublished. Submitted to WHO by Bayer AG, Mannheim, Germany. Bomann, W. (1991a) NTN 33893 AMP (proposed c.n. imidacloprid)—Study for acute oral toxicity to rats. Unpublished report from Bayer AG, report No. 20591, dated 3 September 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Bomann, W. (1991b) NTN 33893 CNS (c.n. imidacloprid (proposed))—Study for acute oral toxicity in rats. Unpublished report from Bayer AG, report No. 20637, dated 20 September 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Casida, J.E. (1998) Cholinergic insecticide toxicology. Crisp Data Base, National Institutes of Health, National Institute of Environmental Health Sciences, Washongton DC, USA. Submitted to WHO by Bayer AG, Mannheim, Germany. Chao, S.L. & Casida, J.E. (1997) Interaction of imidacloprid metabolites and analogs with the nicotinic acetylcholine receptor of mouse brain in relation to toxicity. Pest. Biochem. Physiol., 58, 77-88. Cifone, M.A. (1988) Mutagenicity test on NTN 33893 in the rat primary hepatocyte unscheduled DNA synthesis assay. Unpublished report from Bayer AG, report No. R 4631, dated 21 December 1988, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Eiben, R. (1988a) NTN 33893—Pilot range finding study for cancerogenesis study on B6C3F1 mice (one hundred seven day feeding study). Unpublished report from Bayer AG, report No. 17280, dated 24 October 1988, non-GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Eiben, R. (198 8b) NTN 33893—Pilot range finding study for a chronic toxicity study on Wistar rats (ninety-eight day feeding study). Unpublished report from Bayer AG, report No. 17279, dated 24 October 1988, non-GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Eiben, R. (1991) NTN 33893 (proposed common name: imidacloprid)—Chronic toxicity and cancerogenicity studies on Wistar rats (administration in food'over 24 months)—Supplementary MTD study for two-year study T 1023699). Unpublished report from Bayer AG, report No. 20541, dated 19 August 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany.

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Eiben, R. & Kaliner, G. (1991) NTN 33893 (proposed common name: imidacloprid)—Chronic toxicity and cancerogenicity studies on Wistar rats (administration in food over 24 months). Unpublished report from Bayer AG, report No. 19925, dated 25 January 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Eiben, R. & Rinke, M. (1989) NTN 33893: Subchronic toxicity study on Wistar-rats (administration in the feed for 96 days). Unpublished report from Bayer AG, report No. 18187, dated 14 July 1989. Submitted to WHO by Bayer AG, Mannheim, Germany. Paul, J. (1996) NTN 33893—Occupational medical experience. Unpublished report from Bayer AG, Medical Department, dated March 1996. Submitted to WHO by Bayer AG, Mannheim, Germany. Fautz, R. (1989) Unscheduled DNA synthesis in primary hepatocytes of male rats in vitro with WAK 3839. Unpublished report from Research & Consulting Company AG, report No. R 4746, dated 24 April 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Flucke, W. (1990) NTN 33893 techn.—Study for subacute dermal toxicity in the rabbit. Unpublished report from Bayer AG, report No. 19152, dated 11 June 1990, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Heidemann, A. (1989) Chromosome aberration assay in Chinese hamster V79 cells in vitro with WAK 3839. Unpublished report from Research & Consulting Company AG, report No. R 4849, dated 27 September 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Herbold, B. (1988a) NTN 33893—Test on S. cerevisiae D7 to evaluate for induction of mitotic recombination. Unpublished report from Bayer AG, report No. 16832, dated 27 June 1988,GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Herbold, B. (1988b) NTN 33893—Micronucleus test on the mouse to evaluate for clastogenic effect. Unpublished report from Bay er AG, report No. 16837, dated 27 June 1988, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Herbold, B. (1989a) NTN 3 3 893—Salmonella/microsome test to evaluate for point mutagenic effect. Unpublished report from Bayer AG, report No. 17577, dated 6 January 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Herbold, B. (1989b) NTN 33893—In vitro cytogenetic study with human lymphocytes for the detection of induced clastogenic effects. Unpublished report from Bayer AG, report No. 18092, dated 16 June 1989, GLP, addendum report No. 18092A, dated 24 August 1989. Submitted to WHO by Bayer AG, Mannheim, Germany. Herbold, B. (1989c) NTN 33893—In vivo cytogenetic study of the bone marrow in Chinese hamster to evaluate for induced clastogenic effects. Unpublished report from Bayer AG, report No. 18557, dated 24 November 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Herbold, B. (1989d) NTN 33893—Sister chromatid exchange in bone marrow of Chinese hamster in vivo. Unpublished report from Bayer AG, report No. 18093, dated 16 June 1989, GLP, addendum report No. 18093A, dated 23 November 1993. Submitted to WHO by Bayer AG, Mannheim, Germany. Herbold, B. (1989e) WAK 3839—Micronucleus test on the mouse after oral application. Unpublished report from Bayer AG, report No. 18406, dated 3 October 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Herbold, B. (1989f) WAK 3839 or NTN 37571—Micronucleus test on the mouse after intraperitoneal application. Bayer AG, report No. 18407, dated 3 October 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Herbold, B. (1991) NTN 33893 AMP—Salmonella/microsome test. Unpublished report from Bayer AG, report No. 20090, dated 22 March 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Herbold, B. (1992) NTN 33893 AMP W—Salmonella/microsome test. Unpublished report from Bayer AG, report No. 21775, dated 19 October 1992, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Karl, W. & Klein, O. (1992) [Pyridinyl-14C-methyl] imidacloprid: Distribution of the metabolites in some organs at different times following single oral administration to rats. Unpublished report from Bayer AG, report No. PF 3635, dated 12 March 1992, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Klein, O. (1987a) [14C]-NTN 33893: Biokinetic part of the 'General metabolism study' in the rat. Unpublished report from Bayer AG, report No. PF2889, dated 9 November 1987, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Klein, O. (1987b) [14C]-NTN 33893: Investigations on the distribution of the total radioactivity in the rat by whole body autoradiography. Unpublished report from Bayer AG, report No. PF2891, dated 12October 1987, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany.

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Klein, O. (1990) Imidacloprid—WAK 3839: Comparison of biokinetic behaviour and metabolism in the rat following single oral dosage and investigation of the metabolism after chronic feeding of imidacloprid to rats and mice. Unpublished report from Bayer AG, report No. PF3432, dated 17 July 1990, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Klein, O. & Brauner A (1991) [Imidazolidine-4,5-14C] imidacloprid: Investigation of the biokinetic behaviour and metabolism in the rat. Unpublished report from Bayer AG, report No. PF3629, dated 11 January 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Klein, O. & Karl, W. (1990) Methylene [14C] imidacloprid: Metabolism part of the general metabolism study in the rat. Unpublished report from Bayer AG, report No. PF 3316, dated 30 January 1990, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Krotlinger, F. (1989) NTN 33893 (c.n. imidacloprid (proposed))—Study for acute dermal toxicity to rats. Unpublished report from Bayer AG, report No. 18532, dated 15 November 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Krotlinger, F. (1990) NTN 33893 (c.n. imidacloprid [proposed]—Study for acute intraperitoneal toxicity in rats. Unpublished report from Bayer AG, report No. 19245, dated 19 July 1990, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Krotlinger, F. (1992) WAK 3839—Subchronic toxicological study on rats (twelve week administration in drinking water). Unpublished report from Bayer AG, report No. 21140, dated 2 March 1992, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Lehn, H. (1989a) NTN 33893—Mutagenicity study for the detection of induced forward mutations in the CHOHGPRT assay in vitro. Unpublished report from Bayer AG, report No. 17578, dated 6 January 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Lehn, H. (1989b) WAK 3839—Mutagenicity study for the detection of induced forward mutations in the CHOHGPRT assay in vitro. Unpublished report from Bayer AG, report No. 17757, dated 22 February 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Lehn, H. (1989c) WAK 3839—Mutagenicity study for the detection of induced forward mutations in the V79HGPRT assay in vitro. Unpublished report from Bayer AG, report No. 18281, dated 15 August 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Nagata, K., Song, J.H., Shono, T. & Narahashi, T. (1998) Modulation of the neuronal nicotinic acetylcholine receptor-channel by the nitromethylene heterocycle imidacloprid. J. Pharmacol. Exp. Ther., 285, 731-738. Nakazato, Y. (1988a) NTN 37571—Oral acute toxicity study on rats. Unpublished report from Nihon Tokushu Noyaku Seize report No. RS89007, dated 19 January 1988, non-GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Nakazato, Y. (1988b) NTN 37571—Acute toxicity study on mice. Unpublished report from Nihon Tokushu Noyaku Seizo report No. RS88038, dated 19 October 1988, non-GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Nakazato, Y. (1991) NTN 38014—Acute oral toxicity study on rats. Unpublished report from Nihon Tokushu Noyaku Seizo Report No. RA91018, dated 18 March 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Ohta, K. (1988) NTN 33893 technical—Study for skin sensitising effects on guinea pigs (maximisation test). Unpublished report from Bayer AG, report No. 16533, dated 15 March 1988, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Ohta, K. (1991a) NTN 33519—Acute oral toxicity study on rats. Unpublished report from Nihon Bayer AgrochemK.K.reportNo.RA91023,dated31 May 1991,GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Ohta, K. (1991b) NTN 35884—Acute oral toxicity study on rats. Unpublished report from Nihon Tokushu Noyaku Seizo report No. RA91039, dated 29 November 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Ohta, K. (1991c) WAK 3839—Acute oral toxicity study on rats. Unpublished report from Nihon Tokushu Noyaku Seizo report No. RA91017, dated 11 March 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Ohta, K. (1991d) NTN 35884—Reverse mutation assay (Salmonella typhimurium and Escherichia coli). Unpublished report from Nihon Tokushu Noyaku Seizo reportNo. RA91040, dated 29 November 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Pauluhn, J. (1988a) NTN 33893—Study for acute inhalation toxicity in the rat in accordance with OECD Guideline No. 403. Unpublished report from Bayer AG, report No. 16777, dated 6 June 1988, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany.

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99 Pauluhn, J. (1988b) NTN 33893—Study for irritant/corrosive potential on the skin (rabbit) according to OECD Guideline No. 404. Unpublished report from Bayer AG, report No. 16455, dated 25 February 1988, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Pauluhn, J. (1988c) NTN 33893—Study for irritant/corrosive potential on the eye (rabbit) according to OECD Guideline No. 405. Unpublished report from Bayer AG, report No. 16456, dated 25 February 1988, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Pauluhn, J. (1988d) NTN 33893—Subacute inhalation toxicity study on the rat according to OECD Guideline No. 412. Unpublished report from Bayer AG, report No. 18199, dated 18 July 1988. GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Putman, D.L. & Morris, M.J. (1989) Sister chromatid exchange assay in Chinese hamster ovary cells. Unpublished report from Microbiological Associates Inc., report No. 1149, dated 12 September 1989, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Roe, R.L., Hodgson, E. & Roe, R.M. (1999) Pesticides In: Marquardt, H., Schafer, S.G., McCellan,.R.O. & Welsch, F., eds, Toxicology, San Diego: Academic Press, pp. 663-697. Ruf, J. (1990) NTN 33 893 technical—Subchronic toxicity study on dogs in oral administration (13-week feeding study). Unpublished report from Bayer AG, report No. 18732, dated 2 February 1990, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Sheets, L.P. (1994a) An acute oral neurotoxicity screening study with technical grade imidacloprid (NTN 33893) in rats. Unpublished report from Miles Inc. report No. MOB7221, dated 16 February 1994, GLP, supplement report No. MOB7221, dated 7 June 1994. Submitted to WHO by Bayer AG, Mannheim, Germany. Sheets, L.P. (1994b) A subchronic dietary neurotoxicity screening study with technical grade imidacloprid (NTN 33893) inFischer 344 rats. Unpublished report from Miles Inc., report No. MOB7331, dated 13 June 1994, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Sone, S., Nagata, K., Tsuboi, S. & Shono, T. (1994) Toxic symptoms and neural effect of a new class of insecticide, imidacloprid, on the American cockroach, Periplaneta americana (L.). J. Pest. Sci., 19, 69-72. Steffens, W. (2000) NTN 33983—Personal communication from Bayer AG, Medical Department, dated 26 October 2000. Submitted to WHO by Bayer AG, Mannheim, Germany. Suter, P., Vogel, W., Wilson, T. & Terrier, C. (1990) Multiple generation reproduction study with NTN 33893 technical in rats. Unpublished report from Research & Consulting Company AG, report No. R 5097, dated 21 June 1990, GLP, amendment report No. R 5097A, dated 21 November 1990, amendment report No. R 5097B, dated 3 March 1992. Submitted to WHO by Bayer AG, Mannheim, Germany. Taalmann, R.D.F.M. (1988) Clastogenic evaluation of NTN 33893 in an in vitro cytogenetic assay measuring sister chromatid exchange in Chinese hamster ovary (CHO) cells. Unpublished report from Hazleton Biotechnologies, report No. R 4407, dated 21 April 1988, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Usami, M. (1988a) NTN 37571—In vitro cytogenetic assay measuring chromosome aberrations in CHO-K1 cells—A pilot study. Unpublished report from Nihon Tokushu Noyaku Seizo, report No. RP88008, dated 5 November 1988, non-GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Usami, M. (1988b) NTN 37571—Micronucleus test on the mice after oral treatment—Pilot study. Unpublished report from Nihon Tokushu Noyaku Seizo, report No. RS88040, dated 29 November 1988, non-GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Usami, M. (1988c) NTN 37571—Micronucleus test on the mice after oral treatment—Pilot study. Unpublished report from Nihon Tokushu Noyaku Seizo, report No. RS88041, dated 29 November 1988, non-GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Volker, W. (1990) Mouse germ-cell cytogenetic assay with NTN 33893. Unpublished report from Cytotest Cell Research GmbH, report No. R 5063, dated 22 May 1990, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Watanabe, M. (1990a) NTN 33893—Rec-assay with spores in the bacterial system. Unpublished report from Nihon Bayer report No. RA90016, dated 18 June 1990, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Watanabe, M. (1990b) WAK 3839—Reverse mutation assay (Salmonella typhimurium and Escherichia coli). Unpublished report from Nihon Tokushu Noyaku Seizo report No. RA9003 5, dated 26 November 1990, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Watanabe, M. (1991a) NTN 33893—Reverse mutation assay (Salmonella thyphimurum and Escherichia coli). Unpublished report from Nihon Bayer report No. RA91002, dated 17 January 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany.

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Watanabe, M. (1991b) NTN 33519—Reverse mutation assay (Salmonella typhimurium and Escherichia coli). Unpublished report from Nihon Bayer Agrochem K.K. report No. RA91024, dated 22 July 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Watanabe, M. (1991c) WAK 3839—Rec-assay with spores in the bacterial system. Unpublished report from Nihon Tokushu Noyaku Seizo report No. RA91015, dated 01 March 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Watanabe, M. (199Id) NTN 38014—Reverse mutation assay (Salmonella typhimurium and Escherichia coli). Unpublished report from Nihon Tokushu Noyaku Seizo report No. RA91019, dated 29 March 1991 GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Watta-Gebert, B. (1991a) NTN 33893 (proposed common name imidacloprid)—Carcinogenicity study on B6C3F1 mice (administration in the food for 24 months). Unpublished report from Bayer AG, report No. 19931, dated 28 January 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany. Watta-Gebert, B. (1991b) NTN 33893 (proposed common name: imidacloprid)—Carcinogenicity study in B6C3F1 mice (supplementary MTD testing for Study 5025710 with administration in diet over a 24-month period). Unpublished report from Bayer AG, report No. 20769, dated 24 October 1991, GLP. Submitted to WHO by Bayer AG, Mannheim, Germany.

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METHOMYL (addendum) First draft prepared by Scott Samuels Pesticides Safety Directorate, United Kingdom

Explanation Evaluation for acceptable daily intake Toxicological studies Short-term studies of toxicity Genotoxicity Special studies Reversibility Neurotoxicity Observations in humans Comments Toxicological evaluation References

101 101 101 101 102 103 103 103 105 107 107 108

Explanation Methomyl (5'-methyl-A r -[(methylcaramoyl)oxy]thioacetamidate) was evaluated lexicologically by the JMPR in 1978,1986 and 1989 (Annex 1, references 30,47 and 56). In 1989, an ADI of 0-0.03 mg/kg bw was established on the basis of a NOAEL of 3 mg/kg bw per day in a 2-year study of toxicity in dogs and a 100-fold safety factor. This ADI was maintained by a 1994 WHO Core Assessment Group that prepared Environmental Health Criteria 178 (WHO, 1996). The Meeting was asked to establish an acute reference dose (RfD) for methomyl by the Codex Committee on Pesticide Residues. The present Meeting therefore evaluated data submitted in support of an acute RfD and additional data on the toxicity of repeated doses and of dermal application and on genotoxicity. All the studies submitted complied with the essential elements of the applicable test guidelines, except where noted specifically in the text.

Evaluation for acceptable daily intake 1.

Toxicological studies (a)

Short-term studies of toxicity Rabbits

Methomyl (purity, 98.4%) was applied to the clipped intact skin of groups of New Zealand white rabbits of each sex at a dose of 0 (10 animals), 5,50 (five animals) or 500 (10 animals) mg/kg bw per day for 21 days. The doses were applied in water each day, and the site was occluded for 6 h with a non-porous, plastic dressing. Half the males and females given 0 or 500 mg/kg bw per day and all animals in the other groups were killed at the end of dosing. The remaining animals

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at 0 or 500 mg/kg bw per day were allowed to recover for 14 days and were then killed. Blood samples for measurement of cholinesterase activity were taken after the final exposure, stored on ice and assayed as quickly as possible (usually with 30 min). Brain samples for determination of cholinesterase activity were stored at -70 °C until analysis. The only clinical change was an increased incidence of hyperactivity at 500 mg/kg bw per day. There were no effects on body-weight gain, haematological or biochemical end-points, organ weights or organ histology. Plasma cholinesterase activity was reduced to 36% and 55% of the control mean at day 21 in males and females at the highest dose, respectively. Brain cholinesterase activity was depressed to 48% and 68%, respectively, in males and females at this dose, but erythrocyte cholinesterase activity was not decreased to a biologically significant extent. In males at 50 mg/kg bw per day, plasma cholinesterase activity was depressed to 77%, although most individual values were within the range of the individual control values, and there was no inhibition of erythrocyte cholinesterase activity in animals of either sex. All the values for cholinesterase activity had returned to control values by the end of the 14-day recovery period. The NO AEL was 50 mg/kg bw per day, on the basis of significant inhibition ofbrain acetylcholinesterase activity at 500 mg/kg bw per day (Brock, 1989). Methomyl (purity, 98.6%) was applied to the clipped intact skin of groups of six male and six female New Zealand white rabbits at a dose of 0,15,30,45 or 90 mg/kg bw per day for 21 days. The material was applied in water for 6 h, and the area was covered with a porous, non-occlusive wrapping. The study was intended to define more accurately the NO AEL in the earlier study (Brock, 1989). Standard assessments were made, including observations for deaths and clinical signs of toxicity and measurements of body weight and food consumption. One hour after the final exposure, blood samples were collected for measurement of erythrocyte and plasma cholinesterase activity, after which the animals were killed. Gross necropsy was performed, and the brains were collected and stored frozen for determination of cholinesterase activity. No deaths occurred during the study, and no significant differences in mean body weight, mean body-weight gain, food consumption or food efficiency were noted. No significant signs of toxicity attributable to exposure were seen. No gross lesions were found at necropsy in any treated animals. Slight but statistically significant reductions in mean brain cholinesterase activity were seen in males at doses > 30 mg/kg bw per day, although there was no clear dose-response relationship: the mean values were 99, 90, 88 and 90% of the control value at 15, 30, 45 and 90 mg/kg bw per day. There was some evidence of a pattern in the individual values for males, which were below the control range in 1/6,3/6, 5/6,4/6 males at 15, 30,45 and 90 mg/kg bw per day, respectively. In females, a statistically significant reduction in brain cholinesterase activity was found at the highest dose (94% of the control value). Overall, these reductions in males and females were not biologically significant. There was no statistically significant effect on erythrocyte cholinesterase activity in animals of either sex, although the group mean erythrocyte cholinesterase activities were lower in all treated groups apart from females at 30 mg/kg bw per day; the group mean values for males were 70-87% of the control value and those for females were 88-103%. Comparison of individual values indicated that they were within the range of biological variation. The plasma cholinesterase activities in the treated groups were also generally lower than in controls, although the changes were not statistically significant, nor was there evidence of a dose-response relationship in either group means or individual values. The NO AEL for dermal toxicity was 90 mg/kg bw per day (Finlay, 1997). (b)

Genotoxicity

The results of two newly submitted assays for genotoxicity are shown in Table 1. Methomyl was not genotoxic in vitro or in vivo.

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Table 1. Results of tests for genotoxicity with methomyl End point

Test object

In vitro Reverse mutation

In vivo Micronucleus formation

Concentration

Purity

Results

Reference

S. typhimurium TA97a, 1 0-5000 ng/plate in TA98, TA100, TA1535, dimethyl sulfoxide E. coli WP2 uvrA (pKMlOl)

98.4

Negative with and without metabolic activation

Mathison (1997)

CD-I mice water

98.4

Negative"

Bentley(1995)

3-12 mg/kg bw in

• Single treatment-related death at 12 mg/kg bw

(c)

Special studies (i)

Reversibility

The time necessary for recovery from the inhibition of cholinesterase activity caused by oral administration of methomyl (purity, 98.4%) was investigated in groups of 40 adult Crl:CD BR Sprague-Dawley rats of each sex given methomyl in deionized water once by gavage at a dose of 0 or 3 mg/kg bw. Blood was collected from 10 animals of each sex per group 30 min, 2,3 and 4 h after dosing. Immediately after blood collection, the rats were killed and their brains removed and frozen for subsequent analysis of cholinesterase activity. The sampling times were based on pilot studies that indicated a time-to-peak effect of 0.5 h for both plasma and erythrocyte cholinesterase activity over a dose range of 3-15 mg/kg bw. Clinical signs of toxicity were assessed before collection of blood at each time. Tremors were seen in 17/40 males and 6/40 females treated with methomyl within 30 min of dosing, but no clinical signs were seen later. The treated animals showed marked, statistically significant inhibition of cholinesterase activity 30 min after treatment, the group mean values being 73% of the control mean for plasma, 44% for erythrocytes and 54% for brain in males, and 90,59 and 61 % of the control means in females, respectively. Two hours after treatment, the males showed statistically significant inhibition of blood and brain cholinesterase activities, the group means being 81% of the control mean for plasma, 76% for erythrocytes and 84% for brain. After 3 h, only brain cholinesterase activity in males showed statistically significant inhibition (92% of control value); however, after 4 h, the brain activity was similar to that of controls. In females 2, 3 and 4 h after treatment, the blood cholinesterase activity was not significantly different from that of controls. At 2 and 3 h, there was a minimal but statistically significant reduction in brain cholinesterase activity (92% of control value at both times) (Malley, 1997). (H)

Neurotoxicity

Groups of 52 Crl:CD BR Sprague-Dawley rats of each sex received methomyl (purity, 98.6%) by gavage at a dose of 0,0.25,0.5,0.75 or 2 mg/kg bw in an aqueous solution. Twelve rats from each group were used to evaluate neurotoxicity, and clinical observations, body weights and food consumption were recorded periodically. A 'functional observational battery' (FOB) and motor activity tests were administered before treatment, about 30 min after dosing on day 1 and again on days 8 and 15. Six rats of each sex per group for evaluation of neurotoxicity were killed, and their tissues were fixed in situ by perfusion on day 16. Tissues from the control group and those at the highest dose were processed and examined for neuropathological effects. Forty rats of each sex per group were used to evaluate clinical pathology. Cholinesterase activity in plasma and erythrocytes was measured in the first 10 rats per group before treatment, in order to establish baseline levels. Cholinesterase activity in brain, plasma and erythrocytes was measured again in the same 10 animals per group about 30 min after dosing on day 1 and in another 10 animals per group on day 2. The remaining 20 animals per group used to evaluate clinical pathology were

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evaluated on days 1 and 2; however, as they were not needed for measurement of cholinesterase activity on days 8 and 15, they were removed from the study. Brain samples were kept at - 70 °C until analysis. Body-weight gain was decreased in females at 2 mg/kg bw during days 2-8 but returned to control values during days 8-15. Administration of the FOB on day 1 (30 min after dosing) showed tremors and lachrymation in male rats at 2 mg/kg bw, but no treatment-related effects were seen on days 8 and 15. Although tremors also occurred in females, they occurred in one or two animals in all groups, including controls, indicating no relationship to treatment. There were no effects on forelimb grip strength, hindlimb grip strength or hindlimb foot splay. The motor activity scores of treated rats were unaffected. Clinical signs of toxicity were observed in animals at 2 mg/kg bw, which included tremors (both sexes), salivation (females) and/or lachrymation (both sexes) about 30 min after dosing. No clinical signs of toxicity were observed on day 2. Inhibition of blood and brain cholinesterase activity was detected in males and females at doses > 0.5 mg/kg bw (Table 2) on day 1 about 30 min after treatment. Cholinesterase activity was comparable to that of controls on day 2. Neuropathological evaluation revealed no treatmentrelated adverse findings. The NOAEL was 0.25 mg/kg bw on the basis of statistically significant, > 20% inhibition of brain and erythrocyte acetylcholinesterase activity at doses > 0.5 mg/kg bw (Mikles, 1998a). Groups of 10 male Crl:CD BR Sprague-Dawley rats were given 10 g of feed containing methomyl (purity, 98.4%) at a concentration of 0,30,60,120 or 360 ppm, equal to 1,1.9,3.7 and 10 mg/kg bw. The animals were conditioned to eat within 2 h. During treatment, the animals were observed for clinical signs of toxicity. About 1 h after the end of feeding, each rat underwent an FOB that followed the guidelines of the US A's Environmental Protection Agency, except that grip strength and foot splay were not assessed quantitatively, and the evaluators were aware of which animals had been treated. When the FOB was completed, blood was collected for determination of plasma and erythrocyte cholinesterase activity. The rats were subsequently killed, and brain cholinesterase activity was determined. The mean interval between blood and brain sampling and the start of treatment was 3 h, and the interval before the end of treatment was 1-1.5 h. Blood and brain samples were kept on ice until analysis. No signs of toxicity were observed during treatment. During the FOB, a pattern of changes was seen at concentrations > 60 ppm but was most apparent at 360 ppm. These changes consisted of increased incidences of low arousal, no reaction to approach or touch stimulus and no reaction to tail pinch, although only the last showed a clear dose-related trend at concentrations > 60 ppm. Statistically significant reductions (> 20%) in erythrocyte and brain cholinesterase activity were seen at concentrations > 120 ppm. Plasma cholinesterase activity was also significantly reduced at 120 ppm (by 17%) and 360 ppm (by 37%). However, the individual values were variable, particularly for erythrocyte cholinesterase activity, and there was a poor correlation between the three end-points. The NOAEL was 30 ppm, equal to 1 mg/kg bw, on the basis of the increased incidence of diminished response to tail-pinch at 60 ppm (Filliben, 1999). Table 2. Inhibition of cholinesterase activity (% of control) in rats 30 min after a single dose of methomyl by gavage Tissue

Dose (mg/kg bw per day) Males

Plasma Erythrocytes Brain

Females

0.25

0.5

0.75

2.0

0.25

0.5

0.75

2.0

83 102 94

87 95 81*

77* 70 75*

58* 54* 53*

89 85 96

77 75* 80*

67* 62* 70*

72* 43* 49*

From Mikles (1998a) *p< 0.05

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Groups of 42 Crl:CD BR Sprague-Dawley rats of each sex received diets containing methomyl (purity, 98.6%) at a concentration of 0, 20, 50, 150 or 1500 ppm for about 13 weeks, equal to 0, 1.3, 3.1,9.4 and 95 mg/kg bw per day for males and 0,1.5, 3.9,11 and 110 mg/kg bw per day for females. All animals were observed for clinical changes, body weights and food consumption weekly throughout the study. Twelve rats of each sex per group were evaluated for neurobehavioural changes in an FOB and a motor activity test before treatment (baseline) and again during weeks 4, 8 and 13. Six rats of each sex per group was perfused in situ after week 13. Tissues from all rats were saved, but only those from controls and rats at 1500 ppm were evaluated microscopically. Thirty rats of each sex per group were used to evaluate clinical pathology. Cholinesterase activity in plasma and erythrocytes was measured in the first set of 10 rats of each sex per group before treatment to establish baseline levels. The cholinesterase activities in brain, plasma and erythrocytes were measured again in the same rats during week 4, in the second set during week 8 and in the third set during week 13. Blood samples were kept on ice and brain samples were kept at -70 °C until analysis. The study was possibly compromised, as the animals were fasted before sampling, the duration of food withdrawal not being stated. No treatment-related deaths occurred during the study. Animals of each sex at 1500 ppm showed tremors during the early part of the study, and males in this group also showed increased incidences of aggressive behaviour and hyperreactivity. An increased incidence of alopecia was observed in females in this group. Statistically significant decreases in body weight, body-weight gain, food consumption and food efficiency were seen in male and female rats at 1500 ppm. During the FOB, the animals at 1500 ppm had increased incidences of difficult removal from their home cages, difficult handling, ptosis and abnormal pupillary responses (more dilated than normal for lighting conditions, slow and incomplete reactions to light). The females also had decreased defaecation and urination and increased incidences of low arousal and abnormal gait. Statistically significant decreases in forelimb and hindlimb grip strength were seen in males at 1500 ppm in week 13. Neuropathological evaluation revealed no findings related to treatment. Brain cholinesterase activity was reduced in males and females at 1500 ppm, which was statistically significant only at week 8 in males (81 % of control) and at week 4 in females (90% of control). The reductions at other sampling times were minimal and not statistically significant. There was no evidence of inhibition of erythrocyte cholinesterase activity in any group, although there was considerable variation (without any obvious pattern) between group means and over time. There was some evidence of mild inhibition of plasma cholinesterase activity in males at 1500 ppm during assessment at week 8 (92% of control). The NOAEL was 150 ppm, equal to 9.4 mg/kg bw per day, on the basis of reduced body weight, reduced food consumption, reduced food efficiency, decreased forelimb and hindlimb grip strength, inhibition of brain cholinesterase activity and clinical signs of toxicity at 1500 ppm (Mikles, 1998b).

2.

Observations in humans

In a randomized double-blind study with ascending doses, groups of five healthy male volunteers (four controls) aged 18-40 years received single oral doses of a methomyl formulation, containing 89% methomyl or a placebo (the inert ingredient of the formulation, hydrated silica) in a capsule at a dose of 0, 0.1, 0.2 or 0.3 mg/kg bw. All volunteers were informed of the nature of the test substance, and written informed consent was obtained. The protocol and information were reviewed and agreed by an independent ethics committee. The doses were administered 5 min after a 'standard' breakfast. The men were observed for two nights and attended a followup visit 7 (± 2) days after dosing. Blood samples for analysis of cholinesterase activity were taken -16 h (admission) and-0.5 h before dosing and 0.25,0.5,0.75, 1, 1.25, 1.5,1.75,2,3,4,6,8,12 and 24 h and 7 days (± 2) after dosing, separated immediately and chilled in liquid nitrogen. METHOMYL 101-109 JMPR 2001

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No adverse events requiring treatment with atropine were observed during the study. There were no treatment-related effects on the electrocardiogram, heart rate, pulse, blood pressure, respiratory rate, body temperature, haematological or clinical chemical parameters (excluding cholinesterase activities), urinary end-points or the pupil. The only treatment-related effects were statistically significant decreases in erythrocyte (Table 3) and plasma cholinesterase activity, a single occurrence of mild headache and quantitatively increased salivation, detected by measuring the mass of secretion (Table 4). The biological significance of the increase in salivary secretion is questionable, as, although the men secreted more saliva than before treatment, the mass of secretion was similar to that of controls. Men at 0.1 mg/kg bw showed a notable decrease (19% below baseline value) in group mean erythrocyte cholinesterase activity 1.25 h after dosing (p = 0.072, Student t test), two members of the group having values about 30% lower than their baseline values. However, there were no clear indications of a treatment-related pattern at other times or in the plasma cholinesterase activity in these or other individuals at this dose. Men at 0.2 mg/kg bw had a statistically significant increase in measured salivary secretion 3 h after dosing when outliers were excluded from the data set. Statistically significant depressions of erythroGyte and plasma cholinesterase activities were seen 45 min and 2 h after dosing, but both activities had returned to baseline level within 6 h. Men at 0.3 mg/kg bw showed a statistically significant change from baseline in measured salivary secretion 1 h after treatment when outliers were excluded from the data set. Both erythrocyte and plasma cholinesterase activities were statistically significantly inhibited 15 min to 4 h after dosing. Both activities had returned to baseline levels within 6 h. One man reported mild headache about 1 h after maximal erythrocyte cholinesterase inhibition. The NOAEL was 0.1 mg/kg bw on the basis of statistically significant inhibition of erythrocyte cholinesterase activity and increased salivation at 0.2 mg/kg bw (McFarlane et al., 1998). Table 3. Inhibition of erythrocyte cholinesterase activity (% changefrom baseline)" in male volunteers after a single oral dose ofmethomyl in a capsule Time (h)

Dose (mg/kg bw)

0.25 0.5 0.75 1 1.25 1.5 1.75 2 3 4 6

0.2

0.1

0

0.3

Mean

Min

Max

Mean

Min

Max

Mean

Min

Max

Mean

Min

Max

5.8 -1.8 -2.9 -4.0 -4.3 -0.3 1.8 5.8 12 11 6.2

-3 -10 -16 -30 -8 -4 -4 -A 2 3 -9

20 4 18 8 4 8 10 26 28 24 20

3.1 -9.2 -2.4 -15b -19 -11 -3.7 -8.9 -2.1 5.0 -5.9

-21 -28 -12 -31 -32 -20 -21 -24 -21 -7 -17

27 5 15 0 _y 4 9 1 10 21 22

-1.2 -12 -20* -25** -28** -28** -22** -16* -1.3 -2.3 15

-11 -19 -31 -39 ^1 -39 -28 -23 -7 -12 9

13 _5 4 -15 -18 -21 -16 -10 12 11 25

-19** -32** -35** -27** -27** -23** -22** -16** -13** -5.0* -2.0

-32 -35 -47 -38 -34 -31 -37 -26 -38 -16 -12

1 -26 -21 -17 -15 -15 -12 -8 3 4 5

From McFarlane et al. (1998) defined as mean of values at - 16 h and -30 min p = 0.072 (Student t test), no clear indication of a treatment-related effect at this dose * p < 0.05, **p< 0.01

3 Baseline b

Table 4. Saliva production (g/5 min) in male volunteers receiving a single dose ofmethomyl Dose dosing (mg/kg bw)

30 min before dosing

At time of peak effect after

Mean

Mean

Max

0 0.1 0.2 0.3

2.6 0.9 1.3 1.4

2.6 (4 h) 1.5(6h) 2.3 (8 h) 2.5 (1 h)

4.2 4.1 5.2 3.6

Max 1

4- , 2.1 3.0 2.1

From McFarlane et al. (1998)

METHOMYL 101-109 JMPR 2001

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Comments The acute LDso of methomyl administered orally was approximately 20 mg/kg bw in rats. The compound is classified by WHO (1999) as 'highly hazardous'. Methomyl was tested for genotoxicity in a range of studies in vitro and showed cytogenetic potential in one study in human lymphocytes; it was not cytogenetic in rats in vivo. In studies evaluated by the present Meeting, methomyl did not induce gene mutation in vitro or micronuclei in mice treated in vivo. The Meeting concluded that methomyl is unlikely to be genotoxic. Methomyl is not a reproductive or a developmental toxicant. Rats given methomyl by gavage at a dose of 3 mg/kg bw showed peak neurotoxic effects (clinical signs and inhibition of erythrocyte cholinesterase activity) at 30 min and almost complete recovery by 2 h. In a study with single doses, male rats conditioned to eat within 2 h received methomyl at a single dose of 0, 1, 1.9, 3.7 or 10 mg/kg bw. Significant reductions (> 20%) in erythrocyte and brain cholinesterase activities were seen at doses > 3.7 mg/kg bw. The NOAEL was 1 mg/kg bw on the basis of a dose-related diminution in response to tail pinch at doses > 1.9 mg/kg bw. In another study, rats received methomyl at a dose of 0,0.25,0.5,0.75 or 2 mg/kg bw by gavage in an aqueous vehicle. The NOAEL was 0.25 mg/kg bw on the basis of rapidly reversible inhibition of erythrocyte and brain cholinesterase activity. In a 13-week study of neurotoxicity, groups of rats received diets containing methomyl at a concentration of 0,20,50,150 or 1500 ppm. At 1500 ppm (equal to 95 mg/kg bw per day), several treatment-related effects were seen in animals of each sex, including decreased brain cholinesterase activity, tremors and abnormal pupil responses. The NOAEL was 150 ppm, equal to 9.4 mg/kg bw per day. The Meeting noted that this NOAEL observed after repeated dietary administration was higher than the NOAELs observed in the studies with single doses described above. Male volunteers received single capsules containing methomyl at a dose of 0, 0.1, 0.2 or 0.3 mg/kg bw soon after breakfast. On the basis of dose-related, statistically significant inhibition of erythrocyte cholinesterase activity, by > 20%, and a statistically significantly increase in saliva secretion at doses > 0.2 mg/kg bw, the NOAEL was 0.1 mg/kg bw.

Toxicological evaluation The Meeting allocated an acute RfD of 0.02 mg/kg bw on the basis of the NOAEL of 0.1 mg/kg bw in the study with volunteers. A safety factor of 5 was used because the effects were rapidly reversible and driven by the maximal concentration in plasma (see Annex 1, reference 91, Annex 5). This value was supported by the results of the study of acute neurotoxicity in rats treated in the diet, with a NOAEL of 1 mg/kg bw, the study of acute neurotoxicity in rats treated by gavage, with a NOAEL at 0.25 mg/kg bw, and the absence of any significant sex difference in studies in rats. The Meeting noted that this acute RfD was lower than the current ADI. This is plausible in view of the toxicological profile of methomyl, which shows very rapid recovery from cholinesterase inhibition, such that the NOAELs for dietary intake over 2 h or over 13 weeks were higher than the NOAEL for a single bolus dose. For this reason, setting an acute RfD on the basis of a single meal rather than daily consumption might be justified. Practical implications associated with this situation were noted, as the data on intake do not allow subdivision of daily intake into individual meals. The Meeting concluded that the ADI and acute RfD for methomyl should be based on the same NOAEL and revised the ADI to 0-0.02 mg/kg bw on the basis of the NOAEL of 0.1 mg/kg bw in the study with volunteers and a safety factor of 5.

METHOMYL 101-109 JMPR 2001

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Levels relevant to risk assessment Species Study Rat

Acute neurotoxicity after administration by gavage Acute neurotoxicity after administration in the diet 13-week study of neurotoxicity after administration in the diet

Effect

NOAEL

LOAEL

Inhibition of erythrocyte and brain cholinesterase activity Reduced response to tail pinch

0.25 mg/kg bw

0.5 mg/kg bw

1.0 mg/kg bw

1.9 mg/kg bw

Clinical signs and inhibition of brain cholinesterase activity

150 ppm, equal to 9.4 mg/kg bw per day

15 00 ppm, equal to 95 mg/kg bw per day

0.1 mg/kg bw

0.2 mg/kg bw

Human Single capsule given Inhibition of erythrocyte to male volunteers cholinesterase activity and increased salivation

Estimate of acceptable daily intake for humans 0-0.02 mg/kg bw Estimate of acute reference dose 0.02 mg/kg bw Studies that would provide information useful for continued evaluation of the compound Further observations in humans

References Bentley, K.S. (1995) Mouse bone marrow micronucleus assay of DPX-X1179-394. Unpublished report No. HLR 413-95 from E.I. Du Pont de Nemours & Co., Haskell Laboratory, Newark, Delaware, USA. Submitted to WHO by E.I. Du Pont de Nemours & Co., Wilmington, Delaware, USA. GLP; OECD 474. Brock, W.J. (1989) Repeated dose dermal toxiciry: 21-day study with DPX-X1179-394 (methomyl) in rabbits. Unpublished report No. HLR 387-89 from E.I. Du Pont de Nemours & Co., Haskell Laboratory, Newark, Delaware, USA. Submitted to WHO by E.I. Du Pont de Nemours & Co., Wilmington, Delaware, USA. GLP; OECD 410. Filliben, T. (1999) Acute dietary toxicity study for cholinesterase inhibition with DPX-X1179 in male rats. Unpublished report No. HLR 861-96 from E.I. Du Pont de Nemours & Co., Haskell Laboratory, Newark, Delaware, USA. Submitted to WHO by E.I. Du Pont de Nemours & Co., Wilmington, Delaware, USA. GLP. Finlay, C.A. (1997) Methomyl technical: 21-day repeated dose dermal study in rabbits. Unpublished report No. HL-1997-00913 from E.I. Du Pont de Nemours & Co., Haskell Laboratory, Newark, Delaware, USA. Submitted to WHO by E.I. Du Pont de Nemours & Co., Wilmington, Delaware, USA. GLP; FIFRA 82-2. Malley, L.A. (1997) Reversibility study in rats. Unpublished report No. HL-1997-00641 from E.I. Du Pont de Nemours & Co., Haskell Laboratory, Newark, Delaware, USA. Submitted to WHO by E.I. Du Pont de Nemours & Co., Wilmington, Delaware, USA. GLP. Mathison, B.H. (1997) DPX-X1179 Methomyl: Mutagenicity testing in the Salmonella typhimurium and Escherichia coli plate incorporation assay. Unpublished report No. HL 1997-00043 from E.I. Du Pont de Nemours & Co., Haskell Laboratory, Newark, Delaware, USA. Submitted to WHO by E.I. Du Pont de Nemours & Co., Wilmington, Delaware, USA. GLP;OECD 471 and 472.

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McFarlane, P., Sanderson, J.B. & Freestone, S. (1998) ICR Study 012456, a randomised double blind ascending oral dose study with methomyl to establish a no adverse effect level. Unpublished report No. HLO-199800969 from Inveresk Clinical Research, Edinburgh, Scotland. Submitted to WHO by E.I. Du Pont de Nemours & Co., Wilmington, Delaware, USA. GLP. Mikles, K. A. (1998a) Methomyl technical (DPX-X1179-512): Acute oral neurotoxicity study in rats. Unpublished report No. HL 1998-01080 from E.I. Du Pont de Nemours & Co., Haskell Laboratory, Newark, Delaware, USA. Submitted to WHO by E.I. Du Pont de Nemours & Co., Wilmington, Delaware, USA. GLP; FIFRA 81 8. Mikles, K.A. (1998b) Methomyl technical (DPX-X 1179-512): Subchronic oral neurotoxicity study in rats. Unpublished report No. HL 1998-01639 from E.I. Du Pont de Nemours & Co., Haskell Laboratory, Newark, Delaware, USA. Submitted to WHO by E.I. Du Pont de Nemours & Co., Wilmington, Delaware, USA. GLP; FIFRA 82-7. WHO (1996) Methomyl (Environmental Health Criteria 178), Geneva. WHO (1999) Recommended Classification of Pesticides by Hazard and Guidelines to Classification 1998-1999 (WHO/PCS/98.21/Rev. 1), Geneva, International Programme on Chemical Safety.

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Ill

METHOPRENE and S-METHOPRENE First draft prepared by G. Wolterink, P.H. van Hoeven-Arentzen, andJ.G.MvanEngelen. Centre For Substances and Risk Assessment. National Institute of Public Health and the Environment, Bilthoven, The Netherlands Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution and excretion Biotransformation Toxicological studies Acute toxicity Short-term studies of toxicity Long-term studies of toxicity and carcinogenicity Genotoxicity Reproductive toxicity Multigeneration studies Developmental toxicity Special studies Endocrine activity in mammals Studies on metabolites Comments Toxicological evaluation References

Ill 112 112 112 115 116 116 118 121 122 122 122 123 125 125 125 126 128 130

Explanation Methoprene is the common name for isopropyl-(2£,4£,7/?,S)-ll-methoxy-3,7,lltrimethyldodeca-2,4-dienoate. It is a racemic mixture of two enantiomers (R and S in a ratio of 1:1). The activity of the compound as a juvenile hormone is restricted to the S enantiomer. The identity of the compound was given by the 1984 JMPR in its evaluation of residues (Annex 1, reference 43), and the purity of methoprene was stated to be 92-95%. Methoprene was first evaluated by the 1984 JMPR, when a temporary ADI of 0-0.06 mg/kg bw was established on the basis of aNOAEL of 25 mg/kg bw per day in rats and a NOAEL of 12.5 mg/kg bw per day in dogs, with safety factors of 400 and 200, respectively (Annex 1, reference 42). The 1984 JMPR asked for a 6-month study in dogs treated in the diet, adequate studies of developmental toxicity and a two-generation (two litters per generation) study of reproductive toxicity in rats. No new information became available, but the 1987 JMPR, noting that the data on metabolism and kinetics indicated that the compound was completely metabolized, considered it unlikely that it would reach the conceptus and that long-term studies in dogs would not provide further information. On the basis of these considerations, the 1987 JMPR established an ADI of 0-0.1 mg/kg bw (Annex 1, reference 50). The evaluations of the 1984 and 1987 JMPR were based on the racemic mixture. Methoprene was considered by the present Meeting within the periodic review programme of the Codex Committe on Pesticide Residues. The sponsor that submitted data informed the present Meeting that the methoprene formulations that it markets are based on the biologically METHOPRENE AND ^-METHOPRENE 111-133 JMPR 2001

112

active enantiomer S-methoprene. The Meeting was also informed that other companies continue to market the racemic mixture. The present Meeting reviewed the available database, consisting of the original studies with the racemate and new studies on the kinetics, acute toxicity after oral and dermal administration and inhalation, dermal and ocular irritation, dermal sensitization and mutagenicity with S-methoprene. Three decisions were taken with respect to the database on methoprene: (1) In the reports of studies performed before 1984, it was not clear whether the racemate or 5-methoprene had been tested. Therefore, studies performed before 1980 (the year in which the manufacturing procedure for S-methoprene was established) were considered to have been performed with the racemate. (2) Since the older studies were performed with (technical-grade) racemic methoprene of varying purity (69-96%), the doses used in these studies were corrected accordingly. (3) Studies performed by the Industrial BioTest Laboratory were submitted, but as they had not been validated in accordance with the policy outlined in section 3.1 of the report of the 1981 JMPR (Annex 1, reference 36), they were not evaluated. These studies were also not included in the 1984 evaluation.

Evaluation for acceptable daily intake 1.

Biochemical aspects (a)

Absorption, distribution and excretion Mice

Eight male and two pregnant female mice were given an alcoholic solution of tritiated racemic methoprene (tritium label at the C-10 position; purity unspecified) by gavage at a dose of 1.2 mg/kg bw (7.7 mCi/g bw). Urine and faeces were collected. Individual male mice were killed at 0.5,2,6,12,24,48,72 and 96 h, and two pregnant mice were killed at 6 and 96 h. Sections were prepared for autoradiography. Of the total administered radiolabel, 64% was recovered within 24 h in urine and 12% in faeces. After 96 h, a total of 82% of the administered radiolabel was recovered (68% in urine and 14% in faeces), while 18% was unaccounted for. Elimination of radiolabel in expired air was not measured. The autoradiographs showed large amounts of radiolabel in the stomach and small amounts in the liver and kidney 0.5-2 h after administration. Six hours after administration, radiolabel was found primarily in the small intestine, descending colon and rectum. No radiolabel appeared to have been transferred across the placenta 6 or 96 h after administration. By 12 h after administration, very little radiolabel was detected in the body by autoradiography. At this time, only 60% was recovered in urine and faeces (Cohen & Trudell, 1972). Rats The kinetics of racemic methoprene was studied in a series of experiments in rats given [5C]racemic methoprene (purity, > 99%) in a single dose of 25 mg/kg bw by gavage. In the first experiment, excretion of radiolabel was measured in the urine, faeces and expired air of four rats of each sex every 24 h for 5 days. Within 24 h, 26% of the administered radiolabel had been excreted in expired air (CO2), 13% in urine and 5.2% in faeces. By 48 h, the proprotions were 33%, 17% and 12%, respectively. After 120 h, 76% of the administered radiolabel had been excreted, with 39% in expired air, 20% in urine and 18% in faeces, while 17% was retained in the carcass. There were no apparent differences by sex. The excretion appeared to be biphasic, with rapid elimination during the first 24 h and much slower elimination thereafter. The study authors calculated half-lives of 10 h for 60% of the radiolabel and 107 h for 15%. 14

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In a second experiment, three rats (sex not specified) were equipped with cannulated bile ducts, and bile, urine and faeces were collected for 48 h. During this time, 27% of the administered radiolabel was excreted in bile, 5.9% in urine and 12% in faeces. In view of the percentage excreted in the faeces of rats without bile-duct cannulas, it is likely that most of the radiolabel excreted in bile is reabsorbed. In a third experiment, with three rats of each sex, the plasma concentration of radiolabel peaked after about 6 h, the total amount of radiolabel corresponding to 1.6% of that administered, followed by a relatively slow decline with a half-life of about 48 h. In a fourth experiment, after oral administration of radiolabelled methoprene to eight male rats, peak concentrations in well-perfused organs were reached after 6-12 h. Six hours after administration, the highest concentrations found were 1.7% of the dose per gram wet tissue in liver, 0.58% in kidneys and 0.52% in lungs. In less well-perfused tissues such as fat and muscle, the peak concentrations were reached 12 h after administration. In fat, the concentrations peaked at 0.73% of the dose per gram wet tissue and thereafter very slowly declined to 0.63% at 288 h. The concentrations of radiolabel in the adrenal cortex also remained high up to 288 h after administration. The study authors suggested that this was due to incorporation ofbiotransformation products of methoprene in these tissues. Autoradiographs showed that much of the radiolabel was located in organs involved in absorption, biotransformation and excretion, i.e. the liver, kidneys and lungs. A relatively high concentration of radiolabel was still present after 48 h in the adrenal cortex, lachrymal glands and adipose tissue. A statement of quality assurance was included (Chasseaud et al., 1974; Hawkins et al, 1977). Groups of 24 male Sprague-Dawley rats were fasted for 16 h and then received a single dose of [5-14C]/S-methoprene (purity, 99%) either intravenously at 10 mg/kg bw or by gavage at 10 or 100 mg/kg bw. After intravenous administration, three rats were killed 0.5,1,2,3,4, 5,6 and 7 h after dosing. After oral administration, three rats at each dose were killed 1,2,3,4,5,6,7 and 8 h after dosing. At termination, blood and fat samples were collected for analysis. After intravenous administration, the concentration of radiolabel declined steadily over the 7-h period. After oral administration, the concentration of unchanged methoprene in blood peaked after 2 h, when about 12% of the total radiolabel in blood represented intact methoprene. The concentration of methoprene, as determined by thin-layer chromatography, declined faster than the concentration of total radiolabel. The half-life of intact methoprene in blood was 1.2 h. In fat, the concentration of unchanged methoprene reached a plateau 3-4 h after intravenous and 4-6 h after oral administration and subsequently very slowly declined (Table 1). Almost all the radiolabel in fat represented unchanged methoprene, the proportions being about 95% after intravenous and 49-86% after oral administration, methoprene comprising a greater percentage of radiolabel in fat at later times. Use of a pharmacokinetics model indicated that repeated Table 1. Concentrations of unchanged methoprene (ppm) in fat of groups of three rats given [14C]S-methoprene Time (h)

10 mg/kg bw intravenously

0.5 1 2 3 4 5 6 7 8

4.2 6.9 5.9 5.2 8.2 7.6 6.7 8.1

10 mg/kg bw orally

100 mg/kg bw orally

0.3 1.1 1.7 2.3 3.0 3.4 2.9 2.4

1.1" 15 23 45 38 42 39 47

' Average for two animals

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administration of methoprene does not result in accumulation of methoprene in blood or fat (Ekdawi & Yu, 1996). In view of the very slow decline of methoprene concentrations in fat, the Meeting questioned this conclusion. A study of pharmacokinetics in which blood and fat concentrations were measured over a longer period, covering at least one half-life of methoprene in fat, would elucidate the matter. Guinea-pigs One guinea-pig was given [5-14C]racemic methoprene (purity, 96.9%) at a single dose of 49 mg/kg bw by gavage, and urine, faeces and expired CC>2 were collected for 24 h. At that time, the animal was killed, and samples of blood, fat and muscle were taken. The animal excreted 50% of the radiolabel within 24 h, with 24% in urine, 9% in faeces and 17% in expired air. The peak concentration of radiolabel in urine was reached 5.5 h after dosing. By 24 h after treatment, the blood contained 19 mg equivalent per ml, and muscle and fat contained 3.3 and 11 mg equivalent per gram wet tissue, respectively (Chamberlain et al., 1975). Cows A Hereford steer weighing 277 kg, housed in a metabolism room, was given a single oral dose of 2 g of [5-14C]racemic methoprene, equal to 7.2 mg/kg bw, and urine and faeces were collected for 2 weeks. Room air samples were collected every 3 h for the first 2 days, every 6 h for the next 2 days and once a day for the next 10 days. Blood samples were taken regularly. After 14 days, the animal was killed, and samples of tissues were collected for measurement of radiolabel. The steer excreted about 22% of the administered radiolabel in urine and 39% in faeces over 14 days. Although part of the radiolabel was reported to have been expired as 14CO2, the amount was not quantified. The concentration of radiolabel in blood peaked at 72 h and then very slowly declined. After 14 days, the concentration of radiolabel in blood was about half the maximum value. The highest concentrations of radiolabel were found in bile, gall-bladder, liver, kidney, lung, adrenal, spleen and fat. (Chamberlain et al., 1975). A Jersey cow weighing 338 kg was given a single oral dose of 210 mg of [5-14C]racemic methoprene, equal to 0.61 mg/kg bw, and urine, faeces and milk were collected for 7 days. Expired air was sampled continuously for the first 4 h and for 1 h every fourth hour for the next 3 days. Blood samples were taken 6 and 48 h and 7 days after treatment. After 7 days, 73% of the radiolabel had been eliminated, with 20% in urine, 30% in faeces, 15% in expired air and 8% in the milk, indicating that 27% may have remained in the body. The concentrations of radiolabel in expired air, urine, faeces and milk peaked about 24-48 h after treatment. By day 7 after treatment, the highest concentrations of radiolabel were found in bile, gall-bladder, liver, kidney, ovary, lung, spleen and fat (Chamberlain et al., 1975). Poultry Laying hens were given [5-14C]racemic methoprene (purity, 95.9%) as single oral doses of 0.6-77 mg/kg bw. Excreta, separated into 'urine' and faeces, expired air and eggs were collected from three hens for 14 days, and urine, faeces and expired air were collected from eight hens for 48 h, after which time tissue samples were taken for measurement of radiolabel. Chickens given low doses of methoprene (0.6-3.4 mg/kg bw) excreted most of the radiolabel in expired air (3544% within 48 h), while those given higher doses (59-64 mg/kg bw) eliminated most of the radiolabel in urine (34-3 8%) and faeces (17-19%) within 48 h. Over 14 days after administration, up to 19% of the radiolabel was eliminated in eggs, mainly in the yolk. After 48 h, most of the radiolabel was found in liver, kidney, intestines and lungs (Davison, 1976).

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(b)

Biotransformation Rats

Three rats (sex not specified) with cannulated bile ducts received a single oral dose of [5C]racemic methoprene at 25 mg/kg bw. Bile, urine and faeces were collected for 48 h after treatment. At least 12 unidentified metabolites were detected in urine, and two major unidentified metabolites were detected in bile. Intact methoprene was found only in faeces as a small fraction of the total excreted radiolabel; this probably represented unabsorbed compound, as the bile contained no intact methoprene. Since a large proportion of the radiolabel was excreted as 14CO2, methoprene appears to be extensively metabolized (Chasseaud et al., 1974; Hawkins et al., 1977). A statement of quality assurance was included in the study of Chasseaud et al. (1974). 14

Guinea-pigs In the study of Chamberlain et al. (1975) in which one guinea-pig was given [5-14C]racemic methoprene as a single dose of 49 mg/kg bw by gavage, urine and faeces were examined for metabolites. In urine collected 3-6 h after dosing, 95-99% of the recovered radiolabel consisted of glucuronic acid conjugates and other polar compounds. After treatment with glucuronidase, the two main metabolites identified were 1 l-methoxy-3,7,1 l-trimethyldodeca-2,4-dienoic acid and ll-hydroxy-3,7,ll-trimethyldodeca-2,4-dienoic acid, accounting for 75% of the radiolabel in urine. Isopropyl-11 -hydroxy-3,7,11 -trimethyldodeca-2,4-dienoate and 7-methoxy citronellic acid were also identified. No intact methoprene was found in urine. In faeces, 77% of the recovered radiolabel represented intact methoprene; small quantities (3-8%) of 11-hydroxy-3,7,11trimethyldodeca-2,4-dienoic acid, ll-methoxy-3,7,ll-trimethyldodeca-2,4-dienoic acid and isopropyl-11-hydroxy-3,7,1 l-trimethyldodeca-2,4-dienoate were found. Cows In the study of Chamberlain et al. (1975) in which a Hereford steer was given a single oral dose of [5-14C]racemic methoprene at 7.2 mg/kg bw, a large proportion of the radiolabel in faeces represented intact methoprene. In urine, the main metabolites were isopropyl 11 -hydroxy-3,7,11trimethyldodeca-2,4-dienoateand 1 l-methoxy-3,7,11-trimethyldodeca-2,4-dienoic acid; no intact methoprene was found. Part of the radiolabel was expired as 14COi, and part was incorporated in cholesterol and bile acids, indicating extensive metabolism and biodegradation of methoprene (Quistad et al., 1974, 1975a). In the study of Chamberlain et al. (1975) in which a Jersey cow was given a single oral dose of [5-14C]racemic methoprene at 0.61 mg/kg bw, 15% of the radiolabel was eliminated in expired air over 7 days. Radiolabelled acetic acid and cholesterol were present in blood. In milk, radiolabelled fatty acids, lactose, lactalbumin and casein were detected; only 1 % consisted of intact methoprene. The primary metabolites of methoprene were not detected in milk (Quistad et al., 1975b). Poultry In the study of Davison (1976) in which laying hens were given [5-14C]racemic methoprene at single oral doses of 0.6-77 mg/kg bw, faeces collected on the first day contained 39% intact methoprene, while no methoprene was detected in urine or blood. In urine, 5% of the radiolabel represented conjugated 1 l-methoxy-3,7,1 l-trimethyl-dodeca-2,4-dienoic acid, 4% was associated with 11-hydroxy-3,7,ll-trimethyl-dodeca-2,4-dienoic acid, 5% was incorporated in uric acid, and 85% of the compounds containing radiolabel were not identified. In blood at 48 h, < 0.1% of

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the radiolabel represented methoprene or its primary metabolites. About 1% of the radiolabel in blood was incorporated in cholesterol, and 96% of the compounds containing radiolabel were not identified. Muscle and fat contained only trace amounts of methoprene. Most of the radiolabel in egg yolk was incorporated in natural compounds such as cholesterol and fatty acids, and only 2% represented methoprene residues (Quistad et al., 1976).

2.

Toxicological studies (a)

Acute toxicity (i)

General toxicity

The acute toxicity of the racemate and S-methoprene is summarized in Table 2. Methoprene was injected intraperitoneally to Sprague-Dawley rats at doses of 3.0-52 g/kg. The clinical signs included depression, tremors, lachrymation, diarrhoea and distended abdomen. The histopathological findings included fibrinous peritonitis. Rats treated intraperitoneally with 3 g/kg bw on two subsequent days or at an interval of 48 h showed no increase in mortality rate as compared with rats treated once at a dose of 3 g/kg bw (Jorgenson & Sasmore, 1972a). Two male and two female beagle dogs received methoprene by gavage at a dose of 10 g/kg bw. The two females died within 30 and 40 min of treatment, one male died after 109 min. and the other male was killed for humane reasons after 3 h. The clinical signs observed were aggressive behaviour, piloerection, pupil dilatation, salivation, increased respiratory frequency followed by shallow respiration, loss of gait, convulsions, vomiting, opisthotonos and nystagmus. Gross examination revealed congestion of the kidneys, liver, lungs and scleral vessels; telangiectasia was observed in the liver. Two animals had signs of cardiac effects (ecchymosis, ventricular dilatation). Signs of minor congestion were found in the central nervous system, haematopoietic tissues, the reproductive tract and the digestive tract (Hill, 1972a). Groups of one male and one female dog received methoprene at an oral dose of 1,2 or 5 g/kg bw. No clinical signs were observed. After sacrifice on day 21 after dosing, no treatment-related pathological changes were found on gross examination (Hill, 1972b). No deaths occurred when dogs were exposed for 6 h to a mist (particle size, 2-5 Jim) of technical-grade methoprene as a-2% aqueous solution, providing an estimated total dose of 30 mg/kg bw for males, or 29 mg/kg bw when corrected for purity. However, the actual concentration of methoprene in air was not reported. The clinical signs observed during exposure were increased heart rate and respiration frequency, vomiting, salivation and exhaustion. It was Table 2. Acute toxicity of methoprene and S-methoprene Species

Strain

Sex

Route

Composition

Purity (%)

Reference LD50 (mg/kg bw)a

Ratb

Sprague-Dawley

Male

Oral

R/S

68.9

> 24 000

Rat

Sprague-Dawley

Male

Intraperitoneal

R/S

68.9

3300

Dog Dog Dog

Beagle Beagle Beagle

Male and female Male and female Male and female

Oral Oral Inhalation

R/S R/S R/S

68.9 68.9 95.7

<6900 >3400

Rat

Sprague-Dawley

Male and female

Oral

S

90

>5000

Rabbit

New Zealand white Male and female

Dermal

S

90

>2000

0

R/S, racemate; S, S-enantiomer.; NR, not reported LD5o values for the racemate are corrected for purity. Only two animals per dose c Technical-grade material; GLP and QA statements included a

b

METHOPRENE AND 5-METHOPRENE 111-133 JMPR 2001

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Jorgenson & Sasmore (1972a) Jorgenson & Sasmore (1972a) Hill(1972a) Hill(1972b) Saito(1975a) Schindler & Brown (1984a) Brown (1984a)

117

not clear whether these signs were associated with the compound or the procedure. Food intake and body weight were reduced during the first 1-2 days after exposure (Saito, 1975a). No deaths, no clinical signs and no irritation were seen in New Zealand white rabbits treated dermally with technical-grade S-methoprene. Statements of compliance with GLP and QA were included (Brown, 1984a). In one study of oral administration (Hill, 1972c) and two by inhalation (Hiddemen, 1972; Olson, 1972a), the purity of the technical-grade (racemic) methoprene was not reported. These studies were not evaluated. A number of studies were performed with formulations of S-methoprene. The acute toxicity of formulations containing 2% (Blasczak, 1994a,b), 4% (Blasczak, 1994c,d), 5% (Miles & Collins, 1984a,b) or 20% (Schindler & Baldwin, 1991a,b) S-methoprene was low after oral or dermal administration. No deaths occurred after inhalation of a 20% S-methoprene formulation at the highest concentration tested (5.2 mg/1) (Collins & Procter, 1984). (ii)

Ocular irritation

Undiluted technical-grade racemic methoprene (purity unspecified) was administered into the right conjunctival sac of five male New Zealand white rabbits at a volume of 0.1 ml. The eyes were examined and scored for irritation 24,48 and 72 h after administration. No signs of irritation were observed (Hill, 1971). Eight female New Zealand white rabbits received 0.1 ml of technical-grade racemic methoprene (purity unspecified) into one eye. The eyes of five animals were washed with lukewarm saline 5 min after dosing, and the eyes of the remaining three animals were washed similarly 24 h after dosing. Slight erythema of the conjunctivae was seen in all treated eyes 1 h after dosing. No signs of irritation were seen 1, 2, 3 or 7 days after dosing (Hill, 1973a). The conjunctival sacs of the eyes of three male and three female New Zealand white rabbits were instilled with technical-grade S-methoprene (purity, 90%) and examined and scored for irritation according to the method of Draize after 1, 24, 48 and 72 h and 4 and 7 days. Slight irritation (score 1 for erythema and chemosis) was observed in two females and three males after 1 h, but all signs of irritation had subsided within 48-72 h. Statements of compliance with GLP and QA were included (Brown, 1984b). Further studies of ocular irritation were performed with liquid or solid formulations of Smethoprene. In studies with a 2% ground briquet formulation (Blaszcak, 1992e) or a 4% ground pellet formulation (Blaszcak, 1994f), transient, moderate-to-severe ocular irritation was observed, which disappeared within 3-7 days. In the two studies with liquid formulations containing 5% (Hiles & Collins, 1984c) or 20% (Schindler & Baldwin, 1991c) S-methoprene, conjunctival erythema was observed, which disappeared within 1 and 3 days, respectively. (Hi)

Dermal irritation

Racemic methoprene (purity, 68.9%) was applied in a volume of 0.5 ml onto the intact or abraded clipped skin of six female New Zealand white rabbits, and the site was covered with gauze sponges and rubberized cloth for 24 h. No signs of skin irritation were observed immediately or 48 h after removal of the occlusive dressing (Hill, 1972d) The clipped intact or abraded skin of six New Zealand white rabbits was exposed to 0.5 ml of technical-grade racemic methoprene (purity unspecified), and the site was occluded for 24 h. Slight erythema was observed immediately after removal of the dressing at four of six abraded

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sites and two of six intact sites. Barely perceptible erythema was observed next to the abrasions at five of six sites 48 h after removal of the dressing (Hill, 1973b). The clipped intact skin of three male and three female New Zealand white rabbits was exposed for 4 h to 0.5 ml of technical-grade S-methoprene (purity, 90%), covered with a gauze. Skin irritation was assessed according to the scores of Draize. No signs of skin irritation and no toxic effects were observed in any animal 1,24,48 or 72 h after the end of treatment. Statements of compliance with GLP and QA were included (Schindler & Brown, 1984b). A number of studies of dermal irritation were performed with formulations ofS-methoprene. Formulations containing 2% (Blasczak, 1994g), 4% (Blasczak, 1994h), 5% (Hiles & Collins, 1984c) or 20% (Schindler & Baldwin, 199Id) S-methoprene did not irritate the skin. (iv)

Dermal sensitization

The skin sensitizing properties of racemic methoprene (purity, 95.7%) were tested in groups of five male guinea-pigs according to the method of Landsteiner. In a preliminary range-finding test with an intradermal injection of 0.05 ml of an oily solution, the highest concentration of methoprene that did not cause skin irritation was 0.3%. Therefore, for the induction phase, the animals were given a total of 10 intradermal injections every 48 h of a cottonseed oil solution containing 0.03, 0.1 or 0.3% methoprene. The first injection comprised 0.05 ml of solution, and the nine subsequent injections contained 0.1 ml of solution. After 2 weeks, an intradermal challenge injection was given. During the induction phase, mild, temporary erythema was observed in some animals shortly after injection of methoprene or the vehicle, but the intensity of the dermal reactions did not increase with repeated administration. The type and intensity of dermal reactions after the challenge injection of methoprene did not differ from those observed during the induction phase (Nakayoshi, 1975). The Meeting noted that the appearance of reactions both in animals challenged with the vehicle and in those challenged with methoprene complicates evaluation of the results. It should be noted that the test was not performed according to current guidelines, and the number of animals per group may not have been sufficient for a sound statistical evaluation. Three studies were performed on the skin sensitizing properties of formulations of Smethoprene. In a standard Buehler skin sensitization assay, 0.3 ml of a formulation containing 2% or 4% methoprene diluted with 0.3 ml of physiological saline did not induce skin sensitisation (Blaszcak, 1994i,j). However, in a modified Buehler test with a 20% liquid formulation of Smethoprene in groups of five male and five female Hartley albino guinea-pigs, nine of 10 animals showed significant erythema after challenge with undiluted test substance, while none of the uninduced animals showed this response. Under the conditions of this test, the formulation of Smethoprene was classified as an extreme skin sensitizer. Statements of compliance with GLP and QA were included (Schindler & Baldwin, 1991e). (b)

Short-term studies oftoxicity (i)

Oral administration

Rats In a range-finding study, groups of five male and five female young Sprague-Dawley rats weighing 59-73 g received a diet containing technical-grade racemic methoprene (purity, 68.9%) at a nominal concentration of 1000, 5000,10 000,20 000 or 40 000 ppm, equivalent to 100,500,

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1000,2000 and 4000 mg/kg bw per day (or 70,340, 690,1400 and 2800 mg/kg bw per day when corrected for purity) for 2 weeks. After treatment, all animals were returned to normal food for an additional week. All rats receiving methoprene refused their diets on the first day of the study. Those at 1000,5000 and 10 000 ppm resumed their normal feeding habits, whereas those at 20 000 and 40 000 ppm continued to have greatly reduced food consumption and body growth. The effect was ascribed to poor palatability. In the third week, when the rats received basal diet, all animals resumed normal feeding. Gross examination at the end of the third week showed no abnormalities (Jorgenson & Sasmore, 1972a). The Meeting noted, however, that the treatment period was followed by a 1 -week recovery period, during which any treatment-induced gross pathological changes might have disappeared. In a limited study, groups of 15 male and 15 female Sprague-Dawley rats (approximately 28 days old) were fed diets containing technical-grade racemic methoprene (purity, 68.9%) at a nominal concentration of 0,250,500,1000 or 5000 ppm, equivalent to 12,25,50 and 250 mg/kg bw per day (or 9, 17, 34 and 170 mg/kg bw per day when corrected for purity) for 90 days. No treatment-related deaths occurred, and grossly observed behaviour was normal. Body weight, food consumption and haematological end-points at weeks 4, 8 and 13 were comparable to those of controls, and blood chemistry values were not affected in a dose-related pattern at the end of the study. Urine analysis at 13 weeks showed normal values. At termination, animals at the highest dietary concentration showed increased organ:body weight ratios for liver (both sexes) and kidney (males only). Selected tissues from 10 males and 10 females from the control group and that at the highest dietary concentration, and kidney and liver from the remaining five animals of each sex in these groups and from animals at 1000 ppm were examined microscopically. A slightly higher incidence than in controls of a kidney lesion characterized by vacuoles within swollen convoluted tubules was seen in males at 5000 ppm. In addition, renal tubule regeneration, not seen in concurrent controls or in females, was present in three males at 1000 ppm and seven males at 5000 ppm. The kidneys of animals at doses < 1000 ppm were not examined histologically. The minor histopathological changes in the kidneys were considered to be of no toxicological significance (Jorgenson & Sasmore, 1972b). The Meeting noted that the design of the study deviated in several respects from current guidelines. For instance no ophthalmic examinations were performed, and detailed reports of clinical and behavioural examinations were not given. Haematological, clinical biochemical and urinary end-points were investigated in only five animals of each sex per group. Dogs In a range-finding study, groups of three male beagle dogs were fed diets containing technical-grade racemic methoprene (purity, 68.9%) at a nominal concentration of 0,1000,5000, 10 000 or 20 000 ppm, equivalent to 25, 120, 250 and 500 mg/kg bw per day, for 2 weeks. One male at 20 000 ppm was killed at the end of the 2-week period and necropsied, while the remaining animals were returned to basal diet for an additional week. The animals at 10 000 and 20 000 ppm maintained their weight or showed slight weight loss, while those at 1000 and 5000 ppm showed a slight increase in weight, but less than that of the control animals. The food intake of dogs at 10 000 and 20 000 ppm was markedly decreased. On return to a basal diet in the third week, the food consumption in all treated groups increased. Gross examination at 3 weeks showed an increased relative liver weight with increasing concentration of methoprene in the diet. Histological evaluation showed vacuolization and swelling of hepatocytes in livers of animals at 10 000 or 20 000 ppm, whereas sections from dogs at 1000 and 5000 ppm showed no difference from controls (Jorgenson & Sasmore, 1972a).

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The Meeting noted that treatment was followed by a 1 -week recovery period, during which any treatment-induced gross pathological changes might have disappeared. Groups of four male and four female beagles, about 19 weeks old, were fed diets containing technical-grade racemic methoprene (purity, 68.9%) at a nominal concentration of 0,250,500 or 5000 ppm, equivalent to 6.2, 12 and 120 mg/kg bw per day, for 90 days. No deaths occurred. Behaviour, body weight, food consumption and haematological end-points were not adversely affected. Urine analysis at weeks 4,8 and 13 and ocular examinations at termination were reported to show no abnormal findings. Serum alkaline phosphatase activity was elevated in animals of each sex at 5000 ppm at weeks 4, 8 and 13. At terminal sacrifice, the organibody weight ratio of the liver was increased in both sexes at 5000 ppm. Gross pathological examination of all animals and microscopic evaluation of selected tissues, including the liver, from all animals in the control group and at the highest dietary concentration showed no treatment-related changes. The NOAEL was 5 00 ppm, equivalent to 12 mg/kg bw per day (8.6 mg/kg bw per day when corrected for purity), on the basis of the increased liver weight and the increase in alkaline phosphatase activity (Jorgenson & Sasmore, 1972b). The Meeting noted that, in contrast to the current OECD guideline, detailed clinical and behavioural observations, ophthalmic observations and the results of urine analysis were not reported. (ii)

Dermal administration

Rabbits In a 30-day study, technical-grade racemic methoprene (purity, 95.7%) was applied to the clipped, unabraded skin of five male and five female Japanese rabbits, without occlusion. Undiluted methoprene was applied at a nominal dose of 100,300,900 or 2700 mg/kg bw per day over a circular area 10 cm in diameter. It is not clear what treatment the control animals received. The animals were weighed and observed for clinical signs daily. Haematological and blood chemical end-points were measured before treatment and at necropsy. Food and water intake was assessed every 5 days. Urine was analysed weekly. At necropsy, gross and histopathological examinations were performed. Redness of the skin was observed in animals at the highest dose on days 4-29, and erythema was observed occasionally during treatment in some animals at 300 and 900 mg/kg bw per day. No erythema was seen in controls or animals at 100 mg/kg bw per day. Males at doses > 300 mg/ kg bw per day and females in all treated groups showed reduced weight gain or weight loss during the study and an increased neutrophil count at termination. The leukocyte counts were increased in all treated groups at termination. The treated patch of skin was injured by scratching in all treated groups. The absolute and relative weights of the kidney tended to be dose-dependently increased. The absolute and relative liver weights were increased in animals at the highest dose. Gross and histopathological examinations showed that the only compound-related findings were in the treated skin sites. The study authors suggested that the effects on body weight and leukocyte counts in the treated animals were the result of the skin injury. The LOAEL was 100 mg/kg bw per day (97 mg/kg bw per day when corrected for purity) on the basis of effects on body-weight gain, kidney weight and leukocyte count (Nakasawa, 1975). The Meeting noted that the study design did not meet current OECD guidelines. As the treated dermal patch was not occluded, the animals were able to scratch it. Furthermore, the skin was not washed between treatments, so that residual test compound could have built up, and oral uptake of the test substance could have occurred. The study was considered to be of limited value.

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(Hi)

Inhalation

Rats Groups of 10 male and 10 female rats (strain unspecified) were exposed by inhalation to an aerosol of racemic methoprene (purity, 68.9%) at a nominal chamber concentration of 0, 2 or 20 mg/1 air, 4 h/day, 5 days per week for 3 consecutive weeks. None of the animals died. Animals at 20 mg/1 air had a nasal discharge during each exposure. The weekly body weights and terminal haematological values were comparable to those of controls. Gross necropsy and histopathological evaluation of the liver, lung, kidney and trachea showed no treatment-related changes. Although the Meeting in 1984 (Annex 1, reference 43) concluded that the biochemical parameters in blood did not indicate a consistent pattern of toxicity, re-evaluation of the data by the current Meeting indicated that the total blood bilirubin concentrations were dose-dependently and significantly increased in males at both doses (p < 0.001, Wilcoxon). Serum alkaline phosphatase activity was significantly enhanced at 20 mg/1 in both males and females (p < 0.05, Wilcoxon). No other indication of liver damage was found (aspartate and alanine aminotransferase activity, gross or histopathological appearance). In view of the overall pattern of toxicity in other studies in which the liver was the primary target tissue, the NOAEC was 2 mg/1 (1.4 mg/1 when corrected for purity) (Olson, 1972b). The Meeting noted that, in contrast to the current OECD guideline, only two concentrations were used, the duration of exposure was only 4 h/day instead of 6 h/day, and the actual room concentrations and particle size of the aerosol were not determined. This study was considered of limited value. Dogs Groups of three male and three female beagles were exposed by inhalation via the nose only to technical-grade racemic methoprene (purity, 95.7%) in 2% ethanol solution as an aerosol at a nominal dose of 0.012, 0.0250 or 0.062 mg/kg bw per day, given for 3 min, 6 days/week, for 4 weeks. The particle size of the aerosol was 0.5-2.5 Jim. Groups of two males and two females recived the vehicle only or were untreated. There were no deaths. Except for salivation in two animals during exposure on the first day, no compound-related effects on body weight, food or water consumption, haematological, blood chemical or urinary end-points or gross or histopathology were reported. The NO AEC was 0.062 mg/kg bw per day (0.06 mg/kg bw per day when corrected for purity), the highest dose tested (Saito, 1975b). Owing to the very short daily exposure, this study is of limited value for evaluating the toxic potential of repeated exposure by inhalation. (c)

Long-term studies of toxicity and carcinogenicity Mice

Groups of 50 male and 50 female Charles River CD-I mice received diets containing racemic methoprene (purity, 86.9%) at a nominal concentration of 250, 1000 or 2500 ppm, equivalent to 38, 150 and 380 mg/kg bw per day, for 78 weeks. The survival rates of males were 64% of controls, 56% at the lower dietary concentration, 54% at the intermediate concentration and 60% at the highest concentration, while those of females were 52%, 44% and 48%, respectively. As the survival rate of females at the intermediate concentration at week 72 was only 44%, the remaining animals in this group were killed. The survival rate was considered not to have been affected by treatment. Treatment also had no effects on behaviour, appearance, body weight or food consumption. Histopathological examination revealed the presence of an unidentified brown pigment in the livers of animals at 1000 or 2500 ppm, and many mice at 2500 ppm also had

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numerous focal accumulations of macrophages with brownish foamy cytoplasm in the liver, often associated with small necrotic foci and mononuclear inflammatory cells. The NOAEL was 1000 ppm, equivalentto 150mg/kgbwperday (130mg/kgbwper day when corrected forpurity), on the basis of the latter effect. No treatment-related effects on rumour incidence were observed (Wazeter & Goldenthal, 1975a). The Meeting noted that, in contrast to current guidelines, no differential blood count was performed. Furthermore, negative results for carcinogenicity should be based on a survival rate > 50% at 18 months. Although this requirement was not fully met, the Meeting considered the survival rate of controls and mice at the highest dietary concentration to be acceptable. Rats Groups of 50 male and 50 female Charles River CD rats were fed diets containing technicalgrade racemic methoprene (purity, 86.9%) at a nominal concentration of 0,250,1000 or 5000 ppm, equivalent to 0, 12, 50 and 250 mg/kg bw per day, for 2 years. The survival rates of males were 38% of controls and those at the highest dietary concentration, 54% of those at the lowest concentration and 46% of those at the intermediate concentration. About 50% of control males survived up to week 97, and 50% of males at the highest dietary concentration survived up to week 102. In females, the survival rates at 104 weeks were 52%, 60%, 58% and 48% of controls and those at the low, intermediate and high concentrations, respectively. General appearance, behaviour, body weight and food consumption were not adversely affected. No compound-related effects were seen on haematological, biochemical or urinary parameters measured in five males and five females per1 group at five intervals during the study. Ophthalmic examination revealed no changes related to treatment. The absolute and relative weights of the liver were elevated (about 120% of control liver weight) in females at 5000 ppm. Gross examination indicated no pathological findings attributable to treatment. Histopathological evaluation of a wide range of tissues showed an increased incidence of hepatic lesions, such as bile-duct proliferation and portal lymphocyte infiltration, in males at 5000 ppm. No significant difference in the incidence of any particular tumour was found between control and treated groups. The NOAEL was 1000 ppm, equivalent to 50 mg/kg bw per day (44 mg/kg bw per day when corrected for purity) (Wazeter & Goldenthal, 1975b). The Meeting noted that, in contrast to the current OECD guidelines, clinical, haematological and urinary parameters were assessed in only five instead of 20 animals of each sex per group. Furthermore, no satellite group was included. Negative results for carcinogenicity should be based on a survival rate > 50% at 24 months. Although this requirement was not fully met, the Meeting considered the survival rate of controls and mice at the highest dietary concentration to be acceptable. (d)

Genotoxicity

The results of studies on the genotoxicity of the racemate and ^-methoprene are summarized in Table 3. One assay for dominant lethal mutations performed with the racemate in rats in vivo was available (Johnston, 1973), but it was poorly conducted and was therefore not used in the current evaluation. (e)

Reproductive toxicity (i)

Multigeneration studies

Rats Groups of 20 male and 20 female weanling Long Evans rats were fed diets containing technical-grade racemic methoprene (purity, 86.9-87.5%) at a nominal concentration of 0,500 or METHOPRENE AND S-METHOPRENE 111-133 JMPR 2001

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Test object

Concentration

Purity

Results

Reference

In vitro Reverse mutation"

S. typhimurium TA98,

0.2, 2, 20 ng/plate with S9 TA100.TA1535, TA1537,

NR(R/S) (Negative)

Hsiaetal. (1979)

6.3-12 |ig/ml for lOh, 12and25(xg/mlfor20h without S9 15-60 ng/ml for 2 h with S9

98 (R/S)

Negative

Murli (1988)

TA1538 Chromosomal aberrationsb

Chinese hamster ovary cells

Reverse mutation0

S. typhimurium TA98, 10-10 000 ng/plate TA100,TA1535,TA1537, TA1538

90 (S)

Negative ± S9

Stewart & Riccio (1984a)

Mitotic recombination*1, gene conversion, reverse mutation

Saccharomyces cerevisiae 0.1-5% (v/v) D7

90 (S)

Negative ± S9

Stewart & Riccio (1984b)

S, S-enantiomer; R/S, racemate; NR, not reported; S9, microsomal fraction of Aroclor 1254-induced rat liver for metabolic activation " Tests performed in duplicate; positive controls included. As methoprene was not tested at sufficiently high doses (highest dose, 20 fig/plate), no cytotoxicity was observed, and the result is only indicative of a negative effect. b Positive controls included; statements of compliance with GLP and QA included c Two tests performed, each in duplicate; positive controls included. No cytotoxicity observed. Statement of compliance with QA included. d Three tests performed; positive controls included. Statement of compliance with QA included.

2500 ppm, equivalent to 0,25 and 75 mg/kg bw per day, until they were at least 100 days of age, before mating to initiate a three-generation (one litter per generation) study of reproductive toxicity. FI and ¥2 pups were selected to become parents at weaning and, after a 70-day growth and feeding period, were mated to produce successive generations. In the parental generations, no compound-related effects were seen on mortality rate, food consumption during the growth period, maternal growth rate during gestation and lactation, mating performance, pregnancy rate or duration of gestation. Total weight gain during the growth period was slightly decreased in F0 and FI animals of each sex at 2500 ppm. At this concentration, the mean pup weight was reduced in F2 litters on day 21 and in F3 litters on days 14 and 21. Additionally, the mean number of pups born dead per litter was increased in F3 litters of this group. There were no treatment-related effects on other parameters, including litter size (live pups) at birth, survival of pups during lactation, sex ratio of pups and findings in F3 weanlings at necropsy. The NOAEL was 500 ppm, equivalent to 3 3 mg/kg bw per day (or 29 mg/kg bw per day when corrected for purity), on the basis of reductions in weight gain and mean pup weight and the increased mean number of pups born dead per litter (Killeen & Rapp, 1974). The Meeting noted that, in contrast to current OECD guidelines, only two dietary concentrations were used. Furthermore, no histopathological examinations were performed. (ii)

Developmental toxicity

Mice Groups of 30 mated mice of the ICR lineage were intubated with technical-grade racemic methoprene (purity, 95.7%) in olive oil at a nominal dose of 0, 50,200 or 600 mg/kg bw per day on days 7-14 of gestation. The pregnant dams (20-23 mice in each group) were killed on day 18 of gestation, and the fetuses were removed for external, internal and skeletal examination. There were no compound-related deaths. Food and water consumption during the gestation period was comparable in all groups. An increase in body weight (compared with controls) was noted in pregnant dams at both 200 and 600 mg/kg bw per day on day 18. The mean number of implantations and the mean number of live fetuses were both increased at the highest dose. The body weights of female fetuses in all treated groups were increased. No treatment-related effects were seen on the mean number of dead embryos or on the sex ratio of fetuses. No internal or external abnormalities were seen in fetuses of control or treated groups, although information on METHOPRENE AND S-METHOPRENE 111-133 JMPR 2001

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the number of fetuses per control or treated group examined for such abnormalities was not available. Fetuses in all treated groups showed a statistically significant increase in the number of caudal vertebrae, as compared with controls. The effects observed in the fetuses and the increased maternal body weight were considered indicative of modest advancement of development and thus not of toxicological relevance. The NOAEL for maternal toxicity, embryotoxicity and fetotoxicity was 600 mg/kg bw per day (570 mg/kg bw per day when corrected for purity), the highest dose tested (Nakasawa et al., 1975a). In the same experiment, 10-14 pregnant mice were treated as described above, and the dams were allowed to litter and rear their young until weaning. Pups from five litters per dose group were killed at weaning, and those from the remaining litters were observed for 7 additional weeks. No deaths or abnormal signs were seen in the dams. Maternal body-weight change during gestation and after parturition was unaffected. No compound-related effects were seen on the mean number of implantations, duration of gestation, mean litter size or the survival rate of pups at birth or at weaning. No adverse effects were seen on the rate of physical development of pups before weaning, as judged by auricle development, hair growth and opening of the eyelids. In weaned pups, no dose-related effects were seen on the time of descent of testes or opening of the vagina. The behaviour of pups during 10 weeks post partum was normal. Pups necropsied at 3 or 10 weeks of age showed no gross or skeletal abnormalities. At weaning, increases in the absolute weights of the liver, kidney and lung were observed in male pups at 600 mg/kg bw per day. In female pups, the weights of these organs were also increased but the increase reached statistical significance only for lung weight. A non-dose-related decrease in the organ:body weight ratio of the testes was seen in all treated groups killed after 21 days and in those at the two higher doses killed at 70 days. At 70 days, differences from controls in the weights of the spleen, kidney, heart and lung were observed, but these were not dose-related or were seen only at the highest dose. Histological examination of the ovaries and testes of pups reportedly revealed a single case of atrophy of seminiferous tubules at 50 mg/kg bw per day (data not shown). The NOAEL for toxicity to offspring was 200 mg/kg bw per day (190 mg/kg bw per day when corrected for purity), on the basis of effects on organ weights. Methoprene was not teratogenic under the conditions of these experiments (Nakasawa et al., 1975a). The Meeting noted that, in contrast to the current OEGD guideline, dams were not treated on days 6 and 15 of gestation; therefore, the complete period of organogenesis was not covered. Rabbits Groups of 10 pregnant Japanese rabbits were treated by gavage with technical-grade racemic methoprene (purity, 95.7%) in olive oil at a nominal dose of 0,50,200 or 2000 mg/kg bw per day on days 7-18 of gestation. The does were killed on day 28 of gestation, and the fetuses were removed surgically for examination for external, internal and skeletal abnormalities. No deaths or abnormal symptoms were seen. Two does at the highest dose aborted, and maternal weight gain was depressed. Increases in the incidence of fetal deaths (6% in controls; 20.5% at 2000 mg/kg bw group) and in the proportion of female fetuses were also seen at the highest dose. Fetuses at both 200 and 2000 mg/kg showed a non-dose-related decrease in tail length. No compound-related effects were observed with respect to the mean number of implantations, litter size (live fetuses), body weight or body length of fetuses or the frequency of fetal abnormalities. The NOAEL for maternal toxicity was 200 mg/kg bw per day on the basis of reductions in weight gain and abortions. The NOAEL for fetal toxicity was also 200 mg/kg bw per day, on the basis of the increased percentage of fetal deaths. The NOAELs after correction for purity were 190 mg/kg bw per day. Melhoprene was not teratogenic under the conditions of the experiment (Nakasawa et al., 1975b).

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The Meeting noted that, in contrast to the current OECD guideline, the does were not treated on day 6 of gestation; therefore, the complete period of organogenesis was not covered. Furthermore, only 10 instead of 12 females per dose were used. (f)

Special studies (i)

Endocrine activity in mammals

Immature female mice (19-21 days of age) received racemic methoprene (purity unspecified) subcutaneously at a dose of 0.015 or 0.15 mg/kg bw per day for 3 days. No increase in the uterusibody weight ratio was seen. When methoprene was given subcutaneously to 21-day-old castrated male rats at a dose of 0.37 or 3.7 mg/kg bw per day for 7 days, no increase was seen in the organ:body weight ratio of seminal vesicles, ventral prostate or levator ani. In bilaterally adrenalectomized male rats, 21-23 days old, subcutaneous injection of methoprene at 0.9 or 9 mg/kg bw per day for 6 days did not affect the mymus:body weight ratio. The results of these studies suggest that methoprene has no oestrogenic, androgenic, anabolic or glucocorticoid activity (Rooks, undated). (ii)

Studies on metabolites

The results of studies of the acute toxicity of metabolites of methoprene are summarized in Table 4. The methoprene metabolites 1 l-hydroxy-3,7,1 l-trimethyldodeca-2,4-dienoic acid, 11methoxy-3,7,1 l-trimethyldodeca-2,4-dienoicacid,isopropyl-l l-hydroxy-3,7,11-trimethyldodeca2,4-dienoate and 7-methoxy citronellic acid have been found in both plants and animals. 7Hydroxycitronellic acid and 7-methoxycitronellal are found exclusively in plants. The irritation potential of the methoprene metabolite 7-methoxycitronellal (purity, 97.7%) to the eye was assessed in three male and three female New Zealand white rabbits which received 0.1 ml of the substance into the conjunctival sac of the right eye. Signs of irritation were scored according to the method of Draize. The treatment caused transient, mild conjunctival irritation (redness, chemosis and discharge) in all animals and a slight dulling of the corneal surface in one animal. The signs of ocular irritation disappeared within 2-3 days. (Wazeter & Goldentha 1,1973). The clipped intact or abraded skin of six New Zealand white rabbits was exposed to 0.5 ml of the methoprene metabolite 7-methoxycitronellal (purity, 97.7%), and the site was occluded for 24 h. No skin irritation was observed in the animals with intact skin, and very slight erythema and oedema were observed in the animals with abraded skin. No control groups were included. This metabolite is considered not to be a primary skin irritant (Wazeter & Goldenthal, 1973). Table 4. Acute toxicity of methoprene metabolites in male and female rats treated orally Metabolite

Strain

LD50 (mg/kg bw)

Reference

1 l-Hydroxy-3,7,1 l-trimethyldodeca-2,4-dienoic acid" 1 l-Methoxy-3,7,1 l-trimethyldodeca-2,4-dienoic acidb

CD CD

Johnston (1972a) Johnston (1972b)

Isopropyl-1 l-hydroxy-3,7,1 l-trimethyldodeca-2,4-dienoatec

Sprague-Dawley

>6810 > 68 10 (male) 4870 (female) 89 10 (male) 8260 (female) >5000 >5000 > 10 000 (male) 5763 (female)

d

7-Hydroxycitronellic acid 7-Methoxycitronellal d 7-Methoxy citronellic acid e a b c d e

Sprague Dawley Sprague-Dawley NR

Johnston (1972c) Jorgenson (1973 a) Jorgenson(1973b) Olson (1973)

Dissolved in PEG 300. Rats at all doses displayed depression, and salivation was observed at 4.64 and 6.81 g/kg bw. Rats at all doses displayed depression, and salivation and convulsions were observed at 4.64 and 6.81 g/kg bw. Rats at doses > 4.64 g/kg bw displayed depression, paralytic ptosis, prostration and decreased activity. During a 10-day observation period, no deaths and no adverse reactions (not specified) were observed. All animals at 10 g/kg bw displayed behavioural depression.

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Comments The absorption, distribution, excretion and metabolism of racemic methoprene have been studied in mice, rats, guinea-pigs, cows and chickens given single doses. No study of metabolism after repeated doses was available. After administration of single oral doses of methoprene, the radiolabel was relatively rapidly absorbed and excreted in urine, faeces and expired air. Further, about 8% of the radiolabel administered to a cow was excreted in milk 7 days after dosing, and up to 19% of radiolabel administered to chickens was excreted in eggs 14 days after dosing. In most species investigated, the bulk of the radiolabel was excreted within 5 days or less, and the remainder was incorporated into tissues. Substantial enterohepatic circulation occurs in rats, and a small percentage of intact, unabsorbed methoprene was found in faeces, with none in urine or bile, after its administration. Methoprene is probably extensively metabolized in rats, as a large portion of the radiolabel was excreted with CO2. After a single oral dose to rats, the peak plasma concentration, 1.6% of the administered radiolabel, was reached by 6 h; the level declined slowly, with a half-time of about 48 h. Whole-body autoradiography and tissue analysis showed that most of a single labelled dose was located in organs concerned with absorption, biotransformation and excretion. A relatively high concentration of radiolabel was found in adrenal cortex, lachrymal glands and adipose tissue after 48 h. Studies in guinea-pigs, cattle and chickens showed that racemic methoprene was extensively metabolized to polar conjugates (glucuronides), which were excreted in the urine and faeces, and that the CS-labelled molecule underwent rapid a and b oxidation to produce CO2 and acetate, which was incorporated into natural products such as triglycerides, bile acids and cholesterol found in tissues, milk and eggs. The pharmacokinetics of S-methoprene was investigated for 7-8 h in blood and fat of rats given a single oral or intravenous dose. The clearance of-5-methoprene was relatively rapid after intravenous administration of 10 mg/kg bw. After oral administration of 10 or 100 mg/kg bw, S-methoprene was rapidly absorbed, and the maximum concentration of parent compound in blood was reached 2 h after dosing. In fat, the concentration of unchanged methoprene reached a plateau 3-4 h after intravenous and 4-6 h after oral administration, and then very slowly declined. Because of this slow decline, methoprene may build up in fat after repeated dosing. Most of the radiolabel in fat was unchanged methoprene, whereas in blood methoprene was degraded rapidly to other radiolabelled compounds. The racemate and S-methoprene showed little acute toxicity. The LD50 values for Smethoprene were > 5000 mg/kg bw (oral, rat) and > 2000 mg/kg bw (dermal, rabbit), and those of the racemate were > 24 000 mg/kg bw (oral, rat) and > 3400 mg/kg bw (oral, dog). The LD50 for the racemate after intraperitoneal administration in rats was 3300 mg/kg bw. WHO (1999) has classified methoprene as 'unlikely to present acute hazard in normal use'. The racemate and Smethoprene were not irritating to the eye or skin of rabbits. In a limited test in guinea-pigs, the racemate appeared to have no sensitizing properties. In a study with a formulation containing 20% ^-methoprene, skin sensitization was seen; however, it was unclear whether the effect was due to S-methoprene or to another compound in the formulation. Several studies of the toxicity of repeated doses of racemic methoprene given by oral or dermal application or inhalation were available. The design and reporting of these studies did not meet current guidelines, and the studies of dermal application or inhalation were considered inadequate for evaluation. The studies by oral administration could be used to deduce the toxicological profile of methoprene, and, despite their shortcomings, most were considered suitable for use in risk assessment. Studies in which mice (78 weeks), rats (14 or 90 days, 104 weeks), and dogs (14 or 90 days) were given racemic methoprene in the diet showed that the compound has little toxic potential.

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Some effects were found on food intake and body weight, but the main effect was to increase the weight of the liver relative to body weight (in rats at doses > 5000 ppm; in dogs at doses > 1000 ppm). This effect was not always associated with histopathological changes. In the 90-day study in dogs treated in the diet, the NOAEL was 500 ppm, equivalent to 8.6 mg/kg bw per day. In the 2-year study in rats treated in the diet at 5000 ppm, the highest dose tested, increased absolute and relative liver weights and an increased incidence of hepatic lesions such as bile-duct proliferation and portal lymphocyte infiltration were observed in male rats. The NOAEL was 1000 ppm, equivalent to 44 mg/kg bw per day. Minor histopathological changes in the kidneys observed in this study were considered of no significance for human risk assessment. In the 78week study of carcinogenicity in mice, hepatic lesions characterized by pigment deposition in the cytoplasm of parenchymal cells were seen at 1000 and 2500 ppm, with increased incidence and severity at the highest dose. Focal accumulations of macrophages with brownish foamy cytoplasm were found in the livers of survivors of each sex at 2500 ppm, and an increased frequency of amyloidosis of the intestine was seen in females at this dose. No adverse effects (the brownish pigment was considered not to be of toxicological relevance) were observed at 1000 ppm, equivalent to 130 mg/kg bw per day. No increase in the incidence of tumours at any site was seen in either the 78-week study of carcinogenicity in mice or the 2-year study of toxicity and carcinogenicity in rats treated in the diet. Racemic methoprene did not induce chromosomal aberrations in Chinese hamster ovary cells in vitro. No increase in the frequency of reverse mutations in Salmonella typhimurium was observed. The Meeting noted that only a limited range of concentrations were tested. No definitive conclusion can be drawn about the genotoxic potential of the racemate. S-Methoprene did not induce reverse mutations in S. typhimurium or mitotic crossing-over, gene conversion or reverse mutations in Saccharomyces cerevisiae. On the basis of the negative results in a limited range of studies for genotoxicity and the results of the studies of carcinogenicity with methoprene, the Meeting concluded that methoprene was unlikely to pose a carcinogenic risk to humans. In a three-generation study of reproductive toxicity with racemic methoprene in rats, the total weight gain of animals of each sex in the F0 and F t generations during the growth period was slightly decreased, the mean weight of pups in the FI and FS litters was reduced, and the mean number of pups in the F3 litters born dead per litter was increased at a dose of 2500 ppm. The NOAEL was 500 ppm, equivalent to 29 mg/kg bw per day. In a study of developmental toxicity in which mice were treated on days 7-14 of gestation with racemic methoprene, no toxicologically relevant effects were observed in dams or fetuses at any dose; the NOAEL was 570 mg/kg bw per day, the highest dose tested. In the same experiment, several dams were allowed to litter and rear their pups until weaning; the pups of five litters at each dose were killed at weaning, and the remaining litters were observed for 7 additional weeks. Effects on organ weights were observed in pups at the highest dose. The NOAEL for toxicity to offspring was 190 mg/kg bw per day. In a study of developmental toxicity in which rabbits were treated on days 7-18 of gestation with racemic methoprene, the NOAEL for maternal, embryoand fetotoxicity was 190 mg/kg bw per day, on the basis of reduced weight gain and an increased frequency of abortions among the does and an increased percentage of fetal deaths at 1900 mg/kg bw per day, the highest dose tested. The shortcoming of the studies—mice and rabbits were treated for 2 and 1 day less than that required in the relevant test guideline—was not considered critical for evaluating end-points of developmental toxicity. All the developmental effects were seen at very high doses or only postnatally. Therefore, the Meeting considered that additional studies were not necessary and concluded that methoprene is not teratogenic. Several plant metabolites of methoprene showed little acute toxicity when administered orally.

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Evaluation Racemic methoprene The Meeting reaffirmed the basis of the ADI for racemic methoprene established in 1987, but lowered the value to 0-0.09 mg/kg bw to correct for the purity of the racemate tested. The basis for the ADI was the NOAEL of 500 ppm, equivalent to 8.6 mg/kg bw per day (corrected forpurity), in the 90-day study in dogs and a safety factor of 100. The LDso for racemic methoprene given orally was > 2000 mg/kg bw, and no toxic signs were seen at this dose. In studies with repeated oral doses (including studies of teratogenicity), racemic methoprene did not induce signs indicative of acute toxicity. The Meeting concluded that allocation of an acute reference dose was unnecessary. ^-Methoprene No bridging studies with repeated doses were available for S-methoprene. The Meeting therefore made the conservative assumption that, in the absence of any information to the contrary, all the toxicity of the racemate was due to the S-enantiomer. On this basis, the Meeting established an ADI for S-methoprene of 0-0.05 mg/kg bw, equal to one-half the ADI for the racemate (which is a 1:1 mixture of the R and S enantiomers). Levels relevant to risk assessment of racemic methoprene NOAEL3

Species

Study

Effect

Mouse

Developmental toxicity (expanded)

Maternal toxicity 570 mg/kg bw per dayb Embryo- and fetotoxicity 570 mg/kg bw per day6 Offspring toxicity 190 mg/kg bw per day

570 mg/kg bw per day

Rat

Long-term toxicity and Toxicity carcinogenicity Reproductive toxicity Parental and offspring toxicity

Rabbit

Developmental toxicity Maternal toxicity 190 mg/kg bw per day Embryo- and fetotoxicity 190 mg/kg bw per day

1900 mg/kg bw per day 1900 mg/kg bw per day

Dog

90-day study of toxicity

5000 ppm, equivalent to 86 mg/kg bw per day

3 b

1000 ppm, equivalent to 44 mg/kg bw per day 500 ppm, equivalent to 29 mg/kg bw per day

LOAEL3

500 ppm, equivalent to 8.6 mg/kg bw per day

Dose of racemic methoprene corrected for purity when expressed as mg/kg bw per day Highest dose tested

Estimate of acceptable daily intake for humans 0-0.09 mg/kg bw (racemic methoprene) 0-0.05 mg/kg bw (^-methoprene) Estimate of acute reference dose Unnecessary (racemic methoprene and 5-methoprene)

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5000 ppm, equivalent to 220 mg/kg bw per day 2500 ppm, equivalent to 140 mg/kg bw per day

129

Studies that would provide information useful for continued evaluation of both compounds • • • • •

toxicity of repeated doses in rats (S-methoprene) developmental toxicity in rats (S-methoprene) skin sensitization (S-methoprene and racemic methoprene) gene mutation in mammalian cells (racemic methoprene) observations in humans

List of end-points relevant for setting guidance values for dietary and non-dietary exposure" Absorption, distribution, excretion, and metabolism in mammals Rapid and extensive Rate and extent of oral absorption No data Dermal absorption Mainly in organs concerned with absorption, Distribution biotransformation, and excretion Long half-times of total radiolabelled compounds Potential for accumulation13 (metabolites) in blood and of parent in fat Bulk of radiolabel excreted within 5 days; significant Rate and extent of excretion proportion exhaled; remaining radiolabel incorporated into tissues Very extensive: rapid oxidation to CO2 and acetate, which Metabolism in animals is reincorporated into natural products Methoprene Toxicologically significant compounds

Rat, LD50, oral Rat, LDso, dermal Rat, LC50, inhalation Skin irritation Eye irritation Skin sensitization

> 5000 mg/kg bw > 2000 mg/kg bw No data Not irritating Not irritating No reliable data available

Short-term toxicity Target / critical effect Lowest relevant oral NOAEL Lowest relevant dermal NOAEL Lowest relevant inhalation NOAEL

Body-weight gain; effect on liver 8.6 mg/kg bw per day (90 days, dogs) No reliable data available No reliable data available

Genotoxicity0

Weight of evidence suggests no genotoxic concern

Long-term toxicity and carcinogenicity Target/critical effect Lowest relevant NOAEL Carcinogenicity

Body-weight gain; effect on liver 44 mg/kg bw per day (2 years, rats) No carcinogenic potential (mice, rats)

Reproductive toxicity Reproduction target / critical effect Lowest relevant (reproductive) NOAEL Developmental target / critical effect Lowest relevant developmental NOAEL

Reduced pup weight in ¥2 and Fa litters; reduced number of live F3 pups at birth 29 mg/kg bw per day Offspring toxicity 190 mg/kg bw per day (mouse)

Neurotoxicity / Delayed neurotoxicity Acute neurotoxicity; NOAEL 90-day neurotoxicity; NOAEL Delayed neuropathy

No concern from other studies No concern from other studies No concern from other studies

Other toxicological studies; observations in humans

No data

Medical data

No data

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Summary

Value

Study

Safety factor

ADI for methoprene ADI for S-methoprene Acute reference dose

0.09 mg/kg bw 0.05 mg/kg bw Unnecessary

90 days, dogs 0.5 x ADI of racemic methoprene

100

a

Relevant end-points relate to racemic methoprene, unless otherwise stated in a footnote. S-Methoprene c Racemic methoprene and S-methoprene b

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Saito, M. (1975b) Investigation of Altosid toxicity I. Determination of the subacute toxicity to beagle dogs of Altosid inhalation. Study No. NRI-PL-74-2465. Nomura Research Laboratory, Japan. Schindler, J.E. & Baldwin, R.C. (199la) Zoecon sample No. R437N SAN 810120CS: Acute oral toxicity study in male and female rats. Study No. LSC 2673-M032-91. SRI International, Menlo Park, California, USA. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Schindler, J.E. & Baldwin, R.C. (1991b) Zoecon sample No. R437N SAN 810120CS: Acute dermal toxicity study in male and female rabbits. Study No. LSC 2673-M033-91. SRI International, Menlo Park, California, USA. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Schindler, J.E. & Baldwin, R.C. (1991c) Zoecon sample No. R437N SAN 810 I 20CS: Eye irritation study in rabbits. Study No. LSC 2673-M035-91. SRI International, Menlo Park, California, USA. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Schindler, J.E. & Baldwin, R.C. (1991d) Zoecon sample No. R437N SAN 810120CS: Primary skin irritation study in rabbits. Study No. LSC 2673-M034-91. SRI International, Menlo Park, California, USA. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Schindler, J.E. & Baldwin, R.C. (1991e) Zoecon sample No. R437N SAN 810120CS: Skin sensitisation study in guinea pigs. Study No. LSC 2673-M036-91. SRI International, Menlo Park, California, USA. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Schindler, J.E. & Brown, J.M. (1984a) Acute oral toxicity of S-methoprene in rats. Final report No. 33. SRI Project LSC-7182. SRI International, Menlo Park, California, USA. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Schindler, J.E. & Brown, J.M. (1984b) Primary skin irritation of S-methoprene in rabbits. Final report No. 34. SRI Project LSC-7182. SRI International, California, USA. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Stewart, K.R. & Riccio, E.S. (1984a)/n vitro microbiological mutagenicity assays of Zoecon Corporation's compound S-methoprene. SRIProjectLSC-5854. SRI International, Menlo Park, California, USA. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Stewart, K.R. & Riccio, E.S. (1984b)/« vitro detection of mitotic crossing-over, gene conversion, and reverse mutation with Zoecon Corporation's compound S-methoprene. 1984. SRIProjectLSC-5854. SRI International, Menlo Park, California, USA. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Wazeter, F.X. & Goldenthal, E.I. (1973) ZR-1564: Acute toxicity studies in rabbits (322-004). IRDC. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Wazeter, F.X. & Goldenthal, E.I. (1975a) Eighteen month oral carcinogenic study in mice. Report No. 322-003. International Research and Development Corporation, dated 14 March. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill, NSW, Australia. Wazeter, F.X. & Goldenthal, E.I. (1975b) Chronic oral toxicity studies with Altosid technical. Report No. 322001. International Research and Development Corporation, dated 14 March. Submitted to WHO by Novartis Animal Health Australasia Ltd, Pendle Hill,, NSW, Australia. WHO (1999) Recommended Classification of Pesticides by Hazard and Guidelines to Classification 1998-1999 (WHO/PCS/98.2I/Rev. 1), Geneva, International Programme on Chemical Safety.

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PHOSALONE (addendum) First draft prepared by T.C. Marrs Food Standards Agency, London Explanation Evaluation for acute reference dose Toxicological data Acute toxicity Neurotoxicity Genotoxicity Comments Toxicological evaluation Refrences

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Explanation Phosalone was evaluated toxicologically by the JMPR in 1972, 1993 and 1997 (Annex 1, references 18,68 and 80). The 1997 JMPR allocated an ADI of 0-0.02 mg/kg bw. The compound was re-evaluated by the present Meeting to consider, the need to establish an acute reference dose (RfD). Other studies to underpin maintenance of the ADI were submitted and were evaluated by the Committee.

Evaluation for acute reference dose 1.

Toxicological data (a)

Acute toxicity (i)

Inhalation

Albino Sprague-Dawley-derived rats were exposed to an aerosol of a 90% w/w solution of phosalone (purity, 93.1%) in acetone for 4 h; the mass median aerodynamic diameter was 2.42.5 |Lim. The LCso was 2.1 mg/1 in males and 1.3 mg/1 in females (Paul, 1999). (ii)

Oral administration

Phosalone (purity, 93.8%) was administered by gavage in corn oil to one male and one female Crl:CD BR rat at a dose of 60 mg/kg bw. The animals were observed at half-hourly intervals up to 3 h after dosing and hourly up to 8 h. Tremor and piloerection were seen 4 h after dosing, but no signs were seen at 24 h. As no clinical signs were seen until 4 h after dosing, a limited 'functional observational battery' (FOB) was administered from 5 h after dosing. In this part of the study, groups of three males and three females received a dose of 12 or 80 mg/kg bw in corn oil, and the FOB was administered before treatment and at 5, 6, 7 and 8 h. Body weights were recorded daily, and the animals were killed after 7 days. Animals at 12 mg/kg bw showed no clinical signs, and the results of the FOB were not significantly different from those before dosing. However, at 80 mg/kg bw, chewing movements were seen after 5 h in males and chewing

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movements, tremor, exophthalmos and reduced rectal temperature were seen in females. At 6 h, the males had tremors and decreased rectal temperature and the females had the same signs as at 5 h. Females showed salivation and decreased arousal at 7 h. The greatest decrease in rectal temperature in animals at 80 mg/kg bw was seen 7 h after dosing in both sexes. At 8 h, females also showed lachrymation. By 24 h, the males had recovered, whereas the females had not and still had hunched posture, lethargy and staining of the fur and appeared thin. It was concluded that peak effect occurred at 6 h. No control group was included. The NOAEL for changes in the FOB in comparison with that before treatment was 12 mg/kg bw (Hughes, 1999a). Groups of 10 male and 10 female Crl:CD BRrats were given a single oral dose of phosalone (purity, 93.8%) at 0 or 60 mg/kg bw. The animals were observed for clinical signs after treatment, body weights were measured daily, and blood was taken for measurement of plasma and erythrocyte cholinesterase activity before dosing and then in two subgroups of five animals each, one being sampled at 2.5,6 and 24 h and the other at 4 and 8 h. The animals were killed after 7 days. The brains were removed and cut in half, and one-half was used to estimate brain cholinesterase activity. The method of Ellman et al. (1961) was used to measure cholinesterase activity. Clinical signs were seen 5 h after dosing, consisting of salivation, lachrymation, noisy respiration, tremors, unsteadiness, coldness, piloerection and wet urogenital region. The signs continued for up to 2 days, except for the staining and wetness of the fur, which continued longer but had disappeared by the end of day 2 in the case of male rats. In the females, clinical signs including piloerection, hunched posture and staining of the fur were seen on day 3 after dosing. Staining of the fur was still seen in one female on the morning of the fifth day after dosing but not in the afternoon. Weight gain was decreased 24 h after dosing in males by comparison with the controls, while females showed weight loss, weight gain then weight in the normal range but below that of controls throughout the rest of the study. Plasma cholinesterase activity was depressed 2.5 h after dosing and fell further up to 6 h. In males, the activity was 12.5% that of concurrent controls at 4 h and 16% that of controls at 6 h. At 24 h, it was still only 46% that of controls. In females, plasma cholinesterase activity was 11% that of controls at 4 h and 10% at 6 h; recovery was not complete at 24 h, the activity being 34% that of controls. Erythrocyte cholinesterase activity was more variable, but it was 66% that of controls at 4 h, 42% at 6 h and 44% at 24 h in males; and 31 % that of controls at 2.5 h, 59% at 4 h, 72% at 6 h and 49% at 8 h in females. Brain cholinesterase activity at sacrifice at 7 days was 74% that of concurrent controls in males and 69% in females. As effects were observed in the treated group, no NOAEL could be identified (Hughes, 1999b). Phosalone (purity, 93.8%) was given by gavage to groups of 10 male and 10 female Crl:CD BR rats at a dose of 0,10,25 or 60 mg/kg bw. Clinical signs were monitored throughout the study at least daily. Body weight was recorded before treatment on the day of treatment and then weekly. Food consumption was monitored weekly. The rats were subjected to an FOB before treatment and 6 h and 7 and 14 days after treatment. Satellite groups of five males and five females were treated similarly with phosalone at each dose, and blood was obtained for estimation of cholinesterase activity before dosing and 6 and 24 h and 7 and 14 days after dosing. These animals were killed on day 15, and brain cholinesterase activity was estimated in tissue that had been stored at -70 c»C. Cholinesterase was measured by the method of Ellman et al. (1961). All animals were perfused at sacrifice with glutaraldehyde and paraformaldehyde, and samples of brain were taken. Nervous tissues from five animals of each sex at the highest dose and the controls were processed for neuropathological examination. The structures examined were brain (six levels), spinal cord (cervical and lumbar), dorsal root ganglia and dorsal and ventral root fibres as well as sciatic and tibial nerves. Skeletal muscle (gastrocnemius) was also examined. No deaths were observed. Abnormal clinical signs including jaw clonus, abnormal gait and tremor were seen on the day of treatment with the highest dose in both sexes, but not at lower doses. PHOSALONE 135-140 JMPR 2001

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Decreased weight gain was seen at the highest dose, in males only, during the first week; the weight gain in other groups was comparable to that of controls and was similar in all groups of females. Food consumption was comparable in the various groups, except that males at the high dose in the satellite group had decreased food consumption in the first week. Food conversion efficieny was comparable in all groups. Changes in the FOB were seen at the highest dose and, with one exception, at 6 h on day 1 only. The signs consisted of tremors, clonus of the jaws, exophthalmos, piloerection, coldness to touch, hunched posture and abnormal gait. Females also showed increased urination. Decreased activity and rearing counts were seen in females at the highest dose. Tremors were seen 14 days after dosing in males only, and only at the highest dose; as such changes were not seen at 7 days, they are of doubtful significance. At 6 h, both males and females at the highest dose showed decreased locomotor activity. Reduced group mean body temperature was observed in both sexes at the highest dose. Plasma cholinesterase activity was decreased at 6 and 24 h, but not later in the study, in animals of each sex at 60 mg/kg bw, at 6 h in animals of each sex at 25 mg/kg bw and at 24 h at that dose only in males. Plasma cholinesterase activity was diminished at 6 h in animals of each sex at 10 mg/kg. Erythrocyte cholinesterase activity was decreased at 6 h in males at the highest dose (50% that of concurrent controls); in females at that dose, although the activity was only 75% that of concurrent controls, the difference was not statistically significant. At lower doses, no significant depression of erythrocyte cholinesterase activity was observed at 6 h. At 24 h, erythrocytic cholinesterase activity was reduced to 64% that of controls in males and 75% in females at the highest dose, but at no other time or at lower doses. At termination, brain cholinesterase activity was marginally but significantly decreased in animals at the highest dose (82% that of controls in males and 78% in females). Brain cholinesterase activity was not decreased at lower doses. No intergroup differences were found in brain weight, width or length, and no neuropathological findings were present that could be attributed to treatment. The NOAEL was 25 mg/kg bw on the basis of clinical signs and decreased erythrocyte and brain cholinesterase activity at 60 mg/kg bw (Hughes, 1999c). (b)

Neurotoxicity

Groups of 10 Crl: CD BR rats of each sex received diets containing phosalone (purity, 93.8%) at a concentration of 50,150 or 600 ppm for 13 weeks, corresponding to intakes of 0,3.9, 12 and 46 mg/kg bw per day for males and 0,4.4,13 and 56 mg/kg bw per day for females. Satellite groups of 10 rats of each sex were treated similarly for 4 or 8 weeks, with mean intakes of 0, 5.3, 16 and 64 mg/kg bw per day for males in weeks 1-3 and 0, 3.9, 12 and 48 mg/kg bw per day in weeks 4-7, and equivalent figures for females of 0,5.4,16 and 66 mg/kg bw per day in weeks 1-3 and 0,4.6,14 and 58 mg/kg bw per day in weeks 4-7. Groups of similar size received the vehicle only and served as controls. The animals were observed at least daily for signs of ill-health. Body weights were recorded at the start of the study and weekly thereafter. Food consumption was monitored weekly. The first group of animals were subjected to an FOB before treatment and after 4, 8 and 13 weeks of treatment. At termination of the study, half the animals in each main group were killed, and the tissues were fixed by whole-body perfusion for neuropathological examination. Sections were taken of brain (forebrain, midbrain, cerebellum/pons and medulla oblongata), eyes, optic nerves and cervical and lumbar spinal cord, Gasserian ganglion, dorsal root ganglia and fibres and ventral root fibres. Sections were also taken of the gastrocnemius muscle and peripheral nerves (sciatic and tibial). Only controls and animals at the highest dietary concentration underwent neuropathological examination. The remaining animals in each group were used for determination of blood and brain cholinesterase activity. In the satellite groups, blood for estimation of cholinesterase activity was taken at week 4 from five animals in each group, and then

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these animals were killed for estimation of brain cholinesterase activity. In week 8, blood was taken from the remaining satellite animals for determination of cholinesterase activity, and then these animals were killed for brain cholinesterase estimation. Cholinesterase activity was estimated by the method of Ellman et al. (1961). Brain cholinesterase activity was measured in brains that had been stored at -70 °°C. No deaths occurred during the study, and no inter-group differences in clinical signs were detected.. Weight gain was decreased in females at the highest dietary concentration during weeks 0-13 by comparison with concurrent controls, while food consumption was unimpaired; food efficiency was decreased in this group. Body-weight gain, food consumption and food conversion efficiency in the satellite groups were unaffected. Only minor differences were observed between the groups in the FOB, and these were confined to females: at 8 weeks, abnormal gait was seen at the two higher dietary concentrations, and, at 13 weeks, poor grooming and hair loss were seen at the highest concentration. Differences in group mean activity counts and group rearing counts were not observed. Some inter-group differences in erythrocyte but not plasma cholinesterase activity were seen before dosing. Significant reductions in cholinesterase activity were observed during treatment. Plasma cholinesterase activity was reduced at week 4 in both sexes at all dietary concentrations by comparison with concurrent controls (marginally so in males at the lowest concentration). At week 8 and weeks 13-14, plasma cholinesterase activity was diminished in males at the two higher dietary concentrations and in females at all dietary concentrations. Erythrocyte cholinesterase activity was not reduced at week 4 in males but was diminished in all treated groups of females at that time, by 80% that of concurrent controls at 50 ppm, 67% at 150 ppm and 64% at 600 ppm. At week 8, significant reductions were seen in males at the two higher dietary concentrations (63% and 71% that of concurrent controls, respectively). At week 13, reductions were seen in males at the two higher concentrations (51% and 41% that of concurrent controls, respectively). Reduced activity was observed in all treated groups of females, the value being 63% that of controls at 50 ppm, 64% at 150 ppm and 63% at 600 ppm. Brain cholinesterase activity was reduced in males at the highest dietary concentration at 4 weeks (47% that of concurrent controls) and in females at 150 and 600 ppm (72% and 19%, respectively). At week 8, all treated groups of males showed reduced brain cholinesterase activity, at 75% that of concurrent controls at 50 ppm, 64% at 150 ppm and 28% at 600 ppm. At that time, females showed significant reductions only at the two higher dietary concentrations (70% and 23% that of concurrent controls at 150 and 600 ppm, respectively). At weeks 13-14, significant reductions were seen in both sexes at the two higher dietary concentrations, but the decreases in activity were marginal at 150 ppm. At this time, activities that were 84% and 40% that of concurrent controls were seen in males at 150 and 600 ppm, respectively, and 86% and 23% that of concurrent controls in females at these doses, respectively No differences were seen in brain weight, width or length, and no inter-group differences were observed on neuropathological examination. The NOAEL was 50 ppm, equal to 3.9 mg/kg bw per day, on the basis of effects on brain cholinesterase activity at 150 ppm, as the depressed brain cholinesterase activity in males at 50 ppm at 8 weeks was considered to be of doubtful biological significance, not being observed at any other time nor in females and not being accompanied by clinical effects (Hughes, 1999d). (c)

Genotoxicity

In an assay for unscheduled DNA synthesis in rat hepatocytes in vitro, phosalone (purity, 93.1%) was added at concentrations of 0.005-5 ^ig/ml. No effect was seen (Adams & Kirkpatrick, 2000).

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Comments Phosalone is an organophosphate ester, and virtually all its toxic actions are mediated by cholinesterase inhibition. The LD50 in rats treated orally was approximately 150 mg/kg bw, and WHO (1999) has classified phosalone as moderately hazardous. After preliminary studies had shown that peak effects occur at about 6 h, neurotoxicity was studied in rats given phosalone by gavage at a single dose of 0,10,25 or 60 mg/kg bw. No deaths were observed. Abnormal clinical signs were seen on the day of treatment with the highest dose. Changes in the FOB were seen at the highest dose and, with one exception, on day 1 only. The NOAEL was 25 mg/kg bw on the basis of clinical signs and decreased erythrocyte and brain cholinesterase activity at 60 mg/kg bw. The Meeting also reviewed studies that were not relevant to establishment of an acute RfD but which were nevertheless relevant to continuation of the current ADI. In a study of acute toxicity in rats exposed by inhalation for 4 h, the LCso was 2.1 mg/1 in males and 1.3 mg/1 in females. In a 13-week study of neurotoxicity, rats received phosalone in the diet at a concentration of 50,150 or 600 ppm; satellite groups of rats received phosalone at the same dietary concentrations for 4 or 8 weeks. The NOAEL was 50 ppm, equal to 3.9 mg/kg bw per day, on the basis of reduced brain cholinesterase activity at the next highest dose (150 ppm, equal to 12 mg/kg bw per day). A study of DNA repair in rat hepatocytes in vitro gave negative results. The Meeting concluded that these studies supported continuation of the current ADI.

Toxicological evaluation After considering previous evaluations of phosalone and the new data, the Meeting established an acute RfD of 0.3 mg/kg bw on the basis of the NOAEL of 25 mg/kg bw in the study of acute toxicity in rats treated by gavage and a safety factor of 100.

References Adams, K. & Kirkpatrick, D. (2000) Phosalone in vitro DNA repair test using rat hepatocytes. Study No. RNP 659/002569. Huntingdon Life Sciences Ltd, Woolley Road, Alconbury, Cambridgeshire, England. Supplied to WHO by Rhone-Poulenc Agrochimie, Lyon, France. GLP compliant: UK Statutory Instruments 654,1997; 3106,1999; OECDENV/MC/CHEM(98)17; EUDirective 1999/1 I/EC). Data requirements: USEPAFIFRA 84-2, OECD Guideline 482. Ellman, G.L., Courtney, D. & Andres, V. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7, 88-95. Hughes, E.W. (1999a) Phosalone single dose study by oral gavage administration dose range and time to peak effect in rats. Study No. RNP 601/990040, Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, England. Supplied to WHO by Rhone-Poulenc Agrochimie, Lyon, France. Hughes, E.W. (1999b) Phosalone single dose study by oral gavage administration to assess cholinesterase inhibition in rats. Study No. RNP 600/990041, Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, England. Supplied to WHO by Rhone-Poulenc Agrochimie, Lyon, France. Sponsor did not require GLP compliance. Hughes, E. W. (1999c) Phosalone neurotoxicity study by a single oral gavage administration to CD rats followed by a 14-day observation period. Study No. RNP 586/992263. Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, England. Supplied to WHO by Rhone-Poulenc Agrochimie, Lyon, France. GLP compliant (UK Statutory Instrument 654,1997; FIFRA 40 CFR160; OECD ENV/MC/CHEM(98) 17; EU Directives 87/ 18/EEC and 1999/1 I/EC). Data requirement EPA FIFRA 81-8.

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Hughes, E. W. (1999d) Phosalone 13 -week neurotoxicity study in rats by dietary administration. Study No. RNP 587/984768. Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, England. Supplied to WHO by Rhone-Poulenc Agrochimie, Lyon, France. GLP compliant (UK Statutory Instrument 654,1997; FIFRA 40 CFR 160; OECD ENV/MC/CHEM(98)17; EU Directives 87/18/EEC and 1999/1 I/EC). Guidelines: EPA FIFRA Pesticide assessment guidelines, subdivision F, Addendum F, Neurotoxicity (March 1991). Paul, G.R. (1999) Phosalone acute (four-hour) inhalation study in rats. Study No. RNP 600/994096. Huntingdon Life Sciences Ltd, Huntingdon, Cambridgeshire, England. Supplied to WHO by Rhone-Poulenc Agrochimie, Lyon, France. GLP compliant (UK Statutory Instrument 654,1997; FIFRA 40 CFR 160; OECD ENV/MC/ CHEM(98)17; EU Directives 87/18/EEC and 1999/1 I/EC). Guidelines: EPA OPPTS guideline 870.1300. WHO (1999) Recommended Classification of Pesticides by Hazard and Guidelines to Classification 1998—1999 (WHO/PCS/98.21/Rev. 1), Geneva, International Programme on Chemical Safety.

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PROCHLORAZ First draft prepared by C. Vleminckx Scientific Institute of Public Health, Division Toxicology, Brussels, Belgium.

Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution and excretion Biotransformation Effects on enzymes and other biochemical parameters Effect of dog gastric juice or plasma on prochloraz Toxicological studies Acute toxicity Short-term studies of toxicity Long-term studies of toxicity and carcinogenicity Genotoxicity Reproductive toxicity Multigeneration studies Developmental toxicity Special studies Neurotoxicity Mechanistic studies Studies on metabolites of prochloraz Observations in humans Comments Toxicological evaluation References

141 142 142 142 147 150 151 151 151 154 160 165 166 166 167 169 169 169 170 172 173 174 177

Explanation Prochloraz (Ar-propyl-A^-[2-(2,4,6-trichlorophenoxy)ethyl]-l//-imidazole-l-carboxamide) is a broad-spectrum fungicide. It acts by inhibiting ergosterol biosynthesis. Its toxicology was first evaluated by the Meeting in 1983 (Annex 1, reference 40), when an ADI of 0-0.01 mg/kg bw was established on the basis of a NOAEL of 0.9 mg/kg bw per day in a 2-year study in dogs and a NOAEL of 1.3 mg/kg bw per day in a 2-year study in rats. Since that time, several studies have been conducted: on the absorption, distribution, metabolism and excretion of prochloraz, on its ability to irritate the skin and eye, on developmental toxicity in rabbits, and on the toxicity of plant metabolites. Prochloraz was re-evaluated within the periodic review programme of the Codex Committee on Pesticide Residues.

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Evaluation for acceptable daily intake 1.

Biochemical aspects (a)

Absorption, distribution and excretion Mice

Six male and six female mice were given [14C-phenyl-U]prochloraz (radiochemical purity, > 99%) at a single oral dose of 100 mg kg bw. Urine and faeces were collected 24,48,72 and 96 h after dosing. After 96 h, all animals were killed by asphyxiation with CC^. Various tissues were removed and prepared for analysis by liquid scintillation counting. The study was conducted before GLP regulations were issued in the testing facility, but the protocol and results were well reported. Excretion was virtually complete within 72 h. The overall recovery was 104 ± 15%, urinary excretion (63%) being the major route. There were no significant differences between the sexes in the rate or route of excretion or in the concentrations of residues in tissues. The concentrations were highest in liver (5-7 mg/kg) and lowest in muscle, genitalia, eyes, spleen and fat (0.5-1.0 mg/kg). All other tissues contained mean concentrations > 2.5 mg/kg (Needham, 1982a). Rats In a study conducted in compliance with the principles of GLP (with QA certification), five male and five females rats were given [14C-phenyl-U]prochloraz (radiochemical purity, > 98.9%) at a single oral dose of 5 mg/kg bw. Urine, faeces and expired CO2 were collected. After 4 days, all rats were killed by exanguination after ether anaesthesia. Samples of various tissues were analysed by liquid scintillation counting. Excretion of radiolabelled material in the urine and faeces was rapid and complete (overall recovery, about 98%), faecal excretion predominating. A sex-related difference in the route of excretion was apparent, faecal excretion accounting for 59% of the dose in males and 70% in females. The tissue residue concentrations were very low, often below the limit of detection. Only liver samples contained concentrations consistently > 0.1 mg/kg (0.20 ± 0.04 mg/kg) (Challis & Greedy, 1988). In another study conducted in compliance with the principles of GLP (with QA certification), five male and five female rats were given [14C-phenyl-U]prochloraz (radiochemical purity, 97%) at a single oral dose of 100 mg/kg bw. Urine, faeces and expired CC>2 were collected. After 4 days, all rats were killed by exanguination after ether anaesthesia. Samples of various tissues were analysed by liquid scintillation counting. Excretion of radiolabelled material in the urine and faeces was rapid and complete (recovery, 100 ± 3.0% in males and 98 ± 6.9% in females), with a half-life in both sexes of < 1 day. A sex difference in the route of excretion was found, urinary excretion accounting for 65% of the activity in male rats and only 41% in females. The pattern of excretion with time also differed between the sexes: peak faecal excretion was at 48 h in females and 24 h in males. There was no significant excretion of radiolabelled material into expired air. The tissue residue concentrations also showed sex differences, some concentrations in female rats being up to twice those in males. The highest concentrations were found in liver (2.8 ± 0.22 mg equivalent per kg tissue in males and 5.1 ± 0.78 mg equivalent per kg tissue in females) and kidneys (1.5 ± 0.18 mg equivalent per kg tissue in males and 2.1 ±0.18 mg equivalent per kg tissue in females). The concentrations of residues in blood and plasma of both sexes and in lungs and adrenals of females were > 1 mg equivalent per kg tissue. In all other tissues, the residue concentrations were < 1.0 mg equivalent per kg tissue (Dawson, 1989).

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In a study of the elimination of prochloraz, five male Sprague-Dawley rats were given [14Cphenyl-U]prochloraz (radiochemical purity, > 98%) at a single oral dose of 50 or 250 mg/kg bw. Urine and faeces were collected daily for 4 days, and radiolabel was measured. For analysis of urinary metabolites, the rats were given [14C]prochloraz at 250 mg/kg bw, urine was collected for 48 h, and radiolabel in excreta was assayed by liquid scintillation spectrometry. The study was reported in detail in a publication and is considered to provide useful additional information. At both doses, virtually all of the ingested [14C]prochloraz was excreted in the urine or faeces within 96 h, the bulk of excretion occurring between 24 and 48 h after dosing. Urinary elimination accounted for 61 % and 68% of the respective initial doses. Prochloraz was completely metabolized, no unchanged compound being detectable in the urine. The main biotransformation products in urine were 2,4,6-trichlorophenoxyacetic acid and its corresponding alcohol, the latter as the glucuronic acid conjugate. Aromatic hydroxylation also occurred, with small amounts of hydroxy-2,4,6-trichlorophenoxyethanol and hydroxy-2,4,6-trichlorophenoxyacetic acid excreted in urine. 2,4,6-Trichlorophenol and unconjugated2-(2,4,6-trichlorophenoxy)ethanol were identified as minor urinary metabolites (Laignelet et al., 1992). In a study conducted in compliance with the principles of GLP (with QA certification), the absorption of radiolabelled prochloraz after a single oral dose to rats was determined by characterizing its route and rate of excretion in urine, bile and faeces. Male and female rats with cannulated bile-ducts were given [14C-phenyl-U]prochloraz (radiochemical purity, 98.3%) at a nominal dose of 5 mg/kg bw. The animals were maintained in metabolism cages, and urine, faeces and bile were collected for up to 48 h. After that time, the animals were killed, and the radioactive content of the urine, faeces, bile, the gastrointestinal tract and its contents and residual carcass was determined by liquid scintillation counting. The overall mean recovery of radiolabel over 48 h was 95 ± 4.5% in males and 91 ± 5.2% in females. No sex differences in the disposition of prochloraz were apparent. Biliary excretion was an important route of elimination and accounted for an overall mean of 48 ± 20% (range, 1875%) of the dose. Renal elimination (urine plus cage washings) was quantitatively important and accounted for 21 ± 8.9% (range, 15-34%) in males and 23 ± 13% (range, 11-41 %) in females. The radiolabel voided in faeces (primarily within 24 h) represented 22 ± 12% in males and 14 ± 9.4% in females, and that found associated with the gastrointestinal tract was minimal (males, 0.16%; females, 1.1%). The residual carcass retained a very small fraction of the dose (mean, 2.6% in males and 3.0% in females). Thus, the recovery of radiolabel was quantitative, and there were no apparent sex differences. [14C]Prochloraz was well absorbed, a mean of 74% of the dose (range, 60-96%) being detected in bile, urine, cage washings and carcass. Biliary excretion was the major route of elimination (D'Souza, 1995). In a study conducted in compliance with the principles of GLP (with QA certification), the clearances of radiolabelled residues from tissues in rats after a single low or high oral dose of [14Cphenyl-U]prochloraz (radiochemical purity, > 99%) were compared. Groups of 18 males and 18 females were given a dose of either 5 or 100 mg/kg bw. Those at the low dose were necropsied in groups of three males and three females 2,10, 20,40, 56 and 72 h after treatment, and those at the high dose were necropsied in groups of three males and three females 10, 20,40, 56, 72 and 96 h after treatment. These times were set on the basis of the findings of preliminary studies in which the blood Cmax, €3/4, Ci/2 and C1/4 were determined at the low and high doses. At necropsy, blood, plasma, liver, kidney, spleen, heart, lung, brain, muscle, gonads, eyes, adrenals, bone, renal fat, gastrointestinal tract and residual carcass were collected, and residual radiolabel was quantified. At both the low and high doses, radiolabelled residues of prochloraz were cleared rapidly from the tissues, with a marked decrease in concentration by the end of the study. After the low

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dose, the highest tissue concentrations were seen in the gastrointestinal tract, at 24 ± 1.0 and 28 ± 1.7 mg equivalent per kg, and liver, at 8.5 ± 2.3 and 7.3 ± 0.9 mg equivalent per kg in males and females, respectively, 2 h after dosing. By 72 h after dosing, the residues in all tissues except the liver (0.49 mg equivalent per kg), kidney (0.22 mg equivalent per kg) and gastrointestinal tract (0.22 mg equivalent per kg) of males and females and the plasma (0.13 mg equivalent per kg) of males had fallen to > 0.1 mg equivalent per kg tissue and were below the level of quantification in some tissues. After the high dose, the highest concentrations were seen in the gastrointestinal tract (230 ± 73 and 340 ± 37 mg equivalent per kg), liver (65 ± 19 and 90 ± 12 mg equivalent per kg) and kidney (81 ± 4.6 and 74 ± 10 mg equivalent per kg) in males and females, respectively, 10 h after dosing, with high concentrations also seen in the blood (59 and 38 mg equivalent per kg) and plasma (93 and 57 mg equivalent per kg) in males and females, respectively. By 96 h after dosing, the tissue concentrations were still above the limit of quantification but had decreased markedly, by at least one order of magnitude. The tissue distribution ratios indicated maximum distribution of radiolabel 2-10 h after the low dose and 10-20 h after the high dose. The highest values were seen in the gastrointestinal tract, carcass, blood, plasma, muscle, liver and renal fat. The calculated terminal half-lives in blood, plasma, liver and kidney were similar in all these tissues in both sexes at both the high and low dose. In blood, the terminal half-life was 11 h in males and 14 h in females at the low dose and 12 h in males and 17 h in females at the high dose. There was no significant difference in the half-lives in blood, plasma and kidney at either dose. For the liver, the terminal half-life was 17 h in males and 18 h in females at the low dose and 22 h in males and 24 h in females at the high dose. These half-lives indicate that the elimination of radiolabelled residues of prochloraz is fairly rapid (Reynolds, 1995, 1996). Three male and three female rats were given [14C-phenyl]A^-formyl-Ar'-propyl-7V^'-2-(2,4,6trichlorophenoxy)ethylurea (M2), a major plant metabolite of prochloraz, at a single oral dose of 89 mg/kg bw, and the excretion of radiolabel was followed for 96 h, after which the rats were killed and dissected. The study was conducted before GLP regulations were issued in the testing facility, but the protocol and results were well reported. Excretion of radiolabelled material was virtually complete within 72 h. Urinary excretion accounted for 53-63% of the total dose, the remainder being excreted in faeces. The concentration of the compound appeared to decline rapidly in the tissues, the highest concentrations being detected in liver (2.6-3.4 ^ig/g tissue) and kidney (1.3-5.3 [ig/g tissue) and the lowest in muscle and brain (0.05-0.2 |ig/g tissue). The remaining tissues had concentrations > 1 ppm, except for the skin. There were no apparent sex differences (Needham, 1981). Three rats of each sex were given prochloraz at a dose of 90 mg/kg bw, prochloraz manganese chloride complex at 98 mg/kg bw or M2 at 89 mg/kg bw, so that all animals received an equivalent dose, and the amount of 2,4,6-trichlorophenoxyacetic acid (a major urinary metabolite of prochloraz) in urine was determined by gas-liquid chromatography. Excreta were collected at 24-h intervals for 96 h. The study was conducted before GLP regulations were issued in the testing facility, but the protocol and results were well reported. A sex difference was found in the amount of 2,4,6-trichlorophenoxyacetic acid excreted after administration of all three compounds, male rats excreting approximately twice as much in the urine as did females (36-50% compared with 19-29%). The amounts of 2,4,6trichlorophenoxyacetic acid excreted by rats given prochloraz or prochloraz manganese chloride complex were similar, but significantly less was excreted by male rats given M2. No significant differences were seen in the excretion of 2,4,6-trichlorophenoxyacetic acid by female rats (Campbell & Needham, 1981).

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The pharmacokinetics of [3H-phenyl ring]prochloraz (radiochemical purity, 99%) was studied in rats by measuring radioactivity in plasma and tissues after a single oral dose of 100 mg/kg bw and repeated oral doses of 25 mg/kg bw. Radioactivity was measured for 96 h after the single dose and at specified intervals for 20 days after the repeated doses. The study complied with the general principles of GLP (statement of authenticity from the project manager). After the single dose, the plasma concentrations were highest at 24 h (males, 166 jig equivalent per ml; females, 82 ^ig equialent per ml) and declined steadily to consistently low concentrations by 96 h (males, 13 |Lig equivalent per ml; females, 11 jig equivalent per ml). The tissue concentrations showed a similar pattern, being consistently high up to 24 h after dosing and then declining fairly rapidly to low concentrations at 96 h. In general the plasma concentrations in males were appreciably higher than those in females, but the tissue concentrations were similar in the two sexes, with the exception of the kidney, where the concentrations in females were lower than those in males. Apart from the gastrointestinal tract, the highest concentrations of radiolabel were found in liver and kidneys and the lowest in brain, eyes, male gonads and muscle. After repeated exposure, the plasma concentrations were again higher in males than in females. The tissue concentrations showed no obvious sex differences, with the exception of the kidney, in which the concentrations in females were lower. In males, the plasma concentration of radiolabel rose irregularly to a peak (72 jig equivalent per ml) at 15 days and then declined at day 20. In females, the peak plasma concentration (35 jig equivalent per ml) was observed after 7 days and was maintained at about this level for the remainder of the treatment. Four days after the last dose, the plasma concentrations in both sexes had fallen considerably (males, 11 (ig equivalent per ml; females, 14 jag equivalent per ml) and were similar to that measured 4 days after the single dose. The tissue concentrations in both sexes rose significantly for the first 7 days of dosing and rose only slightly for the remainder of treatment. When selected tissues were analysed for radiolabel during repeated dosing, liver and kidney had thb highest concentrations and muscle and fat the lowest. Thus, the phenoxy moiety of prochloraz was rapidly eliminated from the body, leaving no significant concentrations of residues in tissues, and at a comparable rate after repeated and single oral doses. Repeated doses gave rise to residues, the concentration of which reached a plateau after 7-14 days (Boardman, 1979). Dogs Three dogs of each sex were given [14C-phenyl-U]prochloraz (radiochemical purity, > 99%) at a single oral dose of 18 mg/kg bw in a gelatine capsule. Blood samples were collected 1,2,4,6, 8, 12,24,48, 72 and 96 h after dosing. Urine and faeces were collected daily. The dogs were killed 96 h after dosing by exanguination after barbiturate anaesthesia. An extensive range of tissues were removed and prepared for analysis by liquid scintillation counting. The study was conducted before GLP regulations were issued in the testing facility, but the protocol and results were well reported. Within the first 24 h, 60 ± 10% of the administered dose was excreted. The overall recovery was 96 ± 4%, and faecal excretion was the major route, accounting for 64 ± 3% of the total dose. There were no significant differences in the pattern or rate of excretion between the two sexes. The high concentrations found in the bile (19 ± 14 mg equivalent per kg in males and 41 ± 36 mg equivalent per kg in females) indicate that biliary excretion is a significant route of elimination for this compound. The plasma concentrations 96 h after dosing (15 ± 6 mg equivalent per 1) were significantly greater than those in other tissues. The highest concentrations in tissues were found in liver (7.6 ±1.7 mg equivalent per kg) and kidney (5.6 ± 2.3 mg equivalent per kg). Most tissues contained < 3 mg equivalent per kg, and the lowest concentrations were found in bone (0.5 ± 0.4 mg/kg), cerebellum (0.7 ± 0.3 mg/kg) and cerebrospinal fluid (0.3 ± 0.6 mg/1). The curve for plasma concentration-time showed a rapid initial absorption phase, followed by a slow final

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elimination phase with a half-life of approximately 72 h. The peak plasma concentrations (2931 mg equivalent per 1) occurred 8-24 h after dosing (Needham & Campbell, 1982). Goats As straw containing residues of prochloraz may be used as fodder for livestock, residues of prochloraz may occur in milk and meat. [14C-phenyl-U]Prochloraz formulated as a 40% emulsifiable concentrate was applied to field-grown wheat at a rate of 0.98 kg/ha. The wheat was harvested 11 weeks after treatment and analysed for residues of prochloraz. The straw, which contained radiolabel equivalent to 19 mg/kg, was fed to a lactating goat for 4 days, and milk and blood samples were collected twice daily. At the end of treatment, the goat was killed by exsanguination after anaesthesia. Various tissues were sampled and analysed by liquid scintillation counting. The maximum concentrations were 0.079 mg/1 in plasma and 0.006 mg/1 in milk. The concentrations in tissues were highest in liver (0.05 mg/kg), renal fat (0.04 mg/kg) and rumen wall (0.04 mg/kg). All other tissues contained < 0.03 mg/kg. Bile contained 0.12 mg/1, and the rumen contents contained 0.13 mg/1 (Campbell, 1983). In another study, a lactating goat was given [14C-phenyl]M2 in two oral doses of 60 mg 8 days apart. The goat was slaughtered 24 h after the second dose. After the first dose, the concentration of residues in milk was highest at 7 h (0.07 ppm equivalent) and declined to 0.01 ppm by 31 h. The concentration in plasma was highest at 7 h (0.33 ppm) and declined to 0.01 ppm by 96 h. After the second dose, the concentration in plasma was highest 2 h after dosing and declined in a manner similar to that after the first dose. Residues were detectable in milk 2 h after dosing (0.01 ppm), and the concentration at 17 h was consistent with that seen after the first dose (0.04 ppm). A sample of urine collected 21 h after dosing contained 1.9 mg equivalents, representing 3.2% of the original dose. By 24 h after the second oral dose, the highest concentration of tissue residues was found in liver (0.59 ppm). The tissues of the digestive tract contained 0.020.15 ppm, and the concentration in rumen contents was 0.15 ppm. Bile collected at slaughter contained 3.9 ppm. The concentration of residues in muscle was below the limit of sensitivity of the method (< 0.01 ppm). In general, the tissue concentrations of residues resulting from the ingestion of M2 were lower than those resulting from ingestion of an equivalent dose of prochloraz (Campbell & Needham, 1980). Pigs Four sows were treated dermally with radiolabelled prochloraz to determine the extent of absorption and subsequent distribution of radiolabel in the tissues. Two animals were treated with [14C-imidazole ringjprochloraz, while the other two were given [3H-phenyl ring]prochloraz. Both compounds were formulated as 25% emulsifiable concentrates, and each animal received 2 ml of the formulation (about 500 mg of prochloraz). Urine and faeces were collected throughout the study. The animals were killed after 24 h for removal of tissues, while blood samples were taken at intervals throughout the 24-h period. The concentration of radiolabel recovered from the bandage, treated skin and excreta ranged from 76% to 110% of the activity applied. Less than 1.3% was found in the excreta, and the extent of uptake into the bloodstream and tissues was estimated to have been < 2%. With the exception of treated skin, the highest concentrations of residue were detected in bile ([14C], 13 ppm; [3H], 6.6 ppm). Relatively high concentrations were also found in the iris ([14C], 3.4 ppm; [3H], 2.6 ppm), the liver ([14C], 1.4 ppm; [3H], 2.1 ppm) and adrenals ([14C], 1.6 ppm; [3H], 1.5 ppm). The pattern of uptake of radiolabel into the plasma differed according to the label, suggesting that some degradation of the prochloraz molecule had occurred between application onto the skin and distribution in the blood (Hamilton, 1978).

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(b)

Biotransformation Rats

A generalized metabolic pathway for prochloraz in rats is shown in Figure 1. Figure 1. Metabolites of prochloraz excreted in the urine of rats

Metabolite 1, N-propyl-Af-2-(2,4,6-trichlorophenoxy)ethylurea; metabolite 2,2-(2,4,6-trichlorophenoxy)ethanol; metabolite 3,2,4,6-trichlorophenoxyacetic acid; metabolite 4, jV-2-(2,4,6-trichlorophenoxy)ethylurea; metabolite 5, Ar-2-(4-hydroxy-2,6-dichlorophenoxy)ethyl-A^'-propylurea; metabolite 6, Ar-2-(3-hydroxy-2,4,6-trichlorophenoxy)ethyl-./V'-propylurea; metabolite 7 glucuronide (and sulfate), 2-(2,4,6-trichlorophenoxy)ethanol (metabolite 2) glucuronide conjugate; metabolite 8, 2-(3-hydroxy-2,4,6-trichlorophenoxy)ethanol

The biotransformation of [ 14C]prochloraz (radiochemical purity, > 99%), uniformly labelled on the phenyl or imidazole ring, was studied in male and female rats given an oral dose of 100 mg/kg bw as a suspension in gum acacia or dissolved in com oil. A similar dosing regimen was followed in separate experiments with mice and dogs, except that only 18 mg/kg bw was administered to dogs. Urine was collected at 24-h intervals. In another experiment, a female goat was given a single oral dose of 1.5 mg/kg bw of prochloraz, and one urine sample was collected during the first 24 h after dosing. The study was conducted before GLP regulations were issued in the testing facility, but the protocol and results were well reported. Prochloraz was extensively metabolized in rats, with no unchanged compound detectable in the urine. The main metabolites found were 2,4,6-trichlorophenoxyacetic acid (metabolite 3) and 2-(2,4,6-trichlorophenoxy)ethanol glucuronide conjugate (metabolite 7), which accounted for up to 80% of the urinary radiolabel. Five other metabolites were detected: 2-(2,4,6trichlorophenoxy)ethanol (metabolite 2), 2-(3-hydroxy-2,4,6-trichlorophenoxy)ethanol (metabolite

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8),J/V-2-(3-hydroxy-2,4,6-trichlorophenoxy)ethyl-Ar'-propylurea (metabolite 6), Ar-2-(4-hydroxy2,6-dichlorophenoxy)ethyl-7V'-propylurea (metabolite 5) and]V-2-(2,4,6-trichlorophenoxy)ethylurea (metabolite 4). The metabolism proceeded via cleavage of the imidazole ring, giving rise to two-carbon fragments which were incorporated into the general metabolic pool, followed by hydroxylation of the phenyl ring and/or side-chain hydrolysis to form more polar compounds. The urinary metabolic profile of prochloraz showed no significant qualitative differences between male and female rats, mice and dogs and the female goat. The metabolism did differ quantitatively according to sex, female rats excreting more of the most polar metabolites than males, but no new metabolites were seen. The majorplantmetabolites,7V-propyl-A^-2-(2,4,6-trichlorophenoxy)ethylurea (metabolite 1) and 2,4,6-trichlorophenol, were not detected in the urine of dosed rats (Needham, 1982b). In a study conducted in compliance with the principles of GLP (with QA certification), five male and five female rats were given [14C-U-pnenyl]prochloraz (radiochemical purity, > 98.9%) at a dose of 5 mg/kg bw and two rats of each sex were given 100 mg/kg bw, by gavage. Urine and faeces were collected daily. Rats at 100 mg/kg bw were killed by cervical dislocation after 24 h, while those at 5 mg/kg bw were killed 4 days after dosing. The samples were analysed by liquid scintillation counting, thin-layer chromatography and high-performance liquid chromatography. At both doses, prochloraz and its metabolites were rapidly and completely excreted in the urine and faeces, faecal excretion dominating at the low dose and urinary excretion dominating at the higher dose (Table 1). The main metabolic pathway (Figure 1) at both doses involved opening of the imidazole ring and initial loss of small fragments, which, with a considerable quantity of unchanged prochloraz, were the main compounds found in faeces. Further metabolism of metabolite 1 yielded metabolite 4, which was excreted mainly in faeces or further metabolized to metabolite 2 and then metabolite 3. The latter two compounds were excreted mainly in the urine in free or conjugated form. 2,4,6-Trichlorophenoxyacetic acid (metabolite 3) was the main constituent in urine. A small amount of this acid was further metabolized to the trichlorophenol, which was also excreted in urine. A minor metabolic pathway involved aromatic hydroxylation to give metabolite 5 and metabolite 8, which were excreted in small amounts in urine and faeces. Few differences in the metabolic profiles were seen according to dose, but quantitative differences by sex were apparent with respect to the urinary metabolites in animals at the low dose (Challis & Greedy, 1989; Needham, 1997). Male and female rats were given [14C-phenyl-U]prochloraz (radiochemical purity: > 98%) at a single dose of 100 mg/kg bw by gavage. Urine, faeces and expired CO2 were collected. Four days after dosing, the rats were killed, and tissues were removed for analysis by liquid scintillation counting, thin-layer chromatography and high-performance liquid chromatography or gas-liquid chromatography. Prochloraz underwent extensive metabolism, the primary route being opening of the imidazole ring followed by hydrolysis of the alkyl chain. The main metabolites were 3 and 2, Table 1. Excretion of an oral dose of 5 or 100 mg/kg bw prochloraz in 0-24 h urine and faeces of rats Dose (mg/kg bw)

Sex

% excreted in Urine

Faeces

Total

53 ±6.1 64 ± 8.6

83 ± 4.6 87 ±1.7

37 ±15 29 ±10

70 ± 5.0 49 ±17

5

Male Female

30 ±4.4 23 ±6.9

100

Male Female

33 ±10 20 ±7.0

From Chalhs & Creedy (1989) and Needham (1997)

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which was present mainly as a glucuronide conjugate. Aromatic hydroxylation occurred to produce several minor metabolites. No unchanged prochloraz was excreted in the urine. The concentrations of tissue residues 96 h after dosing were generally < 1 mg equivalent per kg tissue. The highest concentrations were found in the liver (2.8-5.1 mg equivalent per kg) and kidney (1.52.1 mg equivalent per kg). The concentrations of residues in female rats were generally slightly higher than those in males. The metabolites were excreted quantitatively within 96 h, > 50% of the administered radiolabel being found in the 0-24-h excreta. Urinary excretion accounted for 65% of the dose in males and 41% in females (Needham & Challis, 1991). Cows In a study conducted in compliance with the principles of GLP (with Q A certification), [ 14Cphenyl-U]prochloraz (radiochemical purity, 98.9%) was administered in gelatin capsules to a cow twice daily for 3 days, at a rate of 1.5 mg/kg bw per day. All milk was collected, and blood samples were taken. The cow was killed 16 h after the final dose, and tissue samples were collected post mortem. The concentrations of total radiolabel in plasma rose at a constant rate throughout the first 8 h, until administration of the second dose and then continued to rise until they plateaud 72 h after the first dose. The profile of plasma metabolites varied with time. The proportions of products of imidazole ring cleavage (M2, Ml and Af-2-(2,4,6-trichlorophenoxy)ethylurea) decreased from 34% of total plasma radiolabel at 8 h to 20% at 72 h (see Figure 2 for structures of Ml and M2). The phenolic metabolites (hydroxy-2,4,6-trichlorophenoxyethanol, 7V-2-(3-hydroxy-2,4,6trichlorophenoxy)ethyl-jV'-propylurea and 2,4,6-trichlorophenol) and the polar components (2,4,6-trichlorophenoxyacetic acid and baseline material) increased correspondingly. The concentrations of total radiolabel in milk rose to a sustained plateau level of about 0.14 jig equivalent per ml by 24 h, i.e. at the time of administration of the third dose. The metabolites in milk were primarily the phenolic compounds hydroxy-2,4,6-trichlorophenoxyethanol (58%) and A^-2-(3-hydroxy-2,4,6-trichlorophenoxy)ethyl-A^'-propylurea (8.7%), with 23% attributable to M2. The highest tissue concentration was found in liver (10 ^ig equivalent per g), with lower concentrations in kidney (1.7 fig equivalent per g), muscle (0.07 \ig equivalent per g) and fat (0.2 jig equivalent per g). In the liver, 81% of the residue was characterized, consisting of Ml (13%), M2 (12%), Ar-2-(2,4,6-trichlorophenoxy)ethylurea (4.9%), hydroxy-2,4,6trichlorophenoxyethanol (8.3%), 7V-2-(3-hydroxy-2,4,6-trichlorophenoxy)ethyl-7V'-propylurea (6.5%), 2,4,6-trichlorophenol (19%), 2,4,6-trichlorophenoxyacetic acid (10%) and polar material (6.9%). The residues in fat comprised mainly M2 (66%) and Ml (19%), the non-polar products of imidazole-ring opening of prochloraz. No parent prochloraz was observed in any of the samples analysed. Samples of rumen and abomasal contents collectedpost mortem contained M2, Ml, N2-(2,4,6-trichlorophenoxy)ethylurea and 2,4,6-trichlorophenoxyacetic acid. The pattern of metabolites in bile was the same as that in the gut contents. However, in urine, 37% of the activity Figure 2. Structures of metabolites Ml and M2 in cows

Ml, A^-propyl-A'-2-(2,4,6-trichlorophenoxy)ethylurea (metabolite 1 in Figure 1); M2, A/-formyl-A^'-propyl-A^'2-(2,4,6-trichlorophenoxy)ethylurea

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was accounted for by the phenolic metabolites (hydroxy-2,4,6-trichlorophenoxyethanol, N-2-(4hydroxy-2, 6-dichlorophenoxy)ethy 1-7V '-propylurea and 7V-2-(3 -hydroxy-2,4,6-trichlorophenoxy)ethyl-W-propylurea), and 22% was polar and unidentified compounds, the remainder comprising the same metabolites as in bile. The dichlorophenyl metabolite Af-2-(4-hydroxy-2,6-dichlorophenoxy)ethyl-AA'-propylurea was observed only in urine, none being present in any of the tissues or milk. Therefore, the metabolites in milk and tissues should break down to two common components when analysed by the standard method for residue analysis. The non-phenolic compounds yield trichlorophenol, and the phenolic compounds yield trichlororesorcinol. The profile of metabolites of prochloraz seen in bovine tissues and milk was very similar to that in rats. No new metabolites were found (Phillips & Swalwell, 1989). (c)

Effects on enzymes and other biochemical parameters

Prochloraz was found to induce hepatic mixed-function oxidases in a series of investigations in rats and mice. Prochloraz was administered to three groups of mice at a dose of 0, 10 or 100 mg/kg bw, by gavage twice daily for 4 days. A separate group of mice received phenobarbital at 80 mg/kg bw by intraperitoneal injection once daily for 4 days. Twenty-four hours after the last dose, all animals were killed, and a post-mitochondrial supernatant, containing microsomal enzymes, was isolated from each liver, and the content of protein and of cytochromes P450 and b5 was determined. At 100 mg/kg bw, prochloraz was a potent enzyme inducer, liver weight and microsomal protein and cytochrome contents all being significantly increased. At 10 mg/kg bw, only the cytochrome P450 and microsomal protein contents were increased. As the Soret peak of the cytochrome P450 difference spectrum was 450 nm, prochloraz is not a polycyclic aromatic hydrocarbon-type inducer, which have a Soret peak of 448 nm, and therefore similar to phenobarbital (Challis & Campbell, 1983). Prochloraz was administered in the diet to groups of male and female mice at a concentration of 0, 80 (14 weeks), 320 (6 weeks) or 1300 (2 weeks) ppm. A separate group of male and female mice received four daily intraperitoneal doses of phenobarbital at 80 mg/kg bw. Twenty-four hours after the last treatment, all animals were killed, and a postmitochondrial supernatant, containing microsomal enzymes, was isolated from each liver and assayed for protein and cytochromes P450 and b5 content. Prochloraz was a potent inducer of the hepatic mixed-function oxidase system when given at 320 or 1300 ppm for 2 weeks, but a marginal effect was seen when it was administered at 80 ppm for periods in excess of 2 weeks, indicating that this concentration is at or close to the threshold dose for induction. There was no significant sex difference. The Soret peak of the cyrochrome P450 difference spectrum was 450 nm (Needham, 1983a). Prochloraz was administered to three groups of six male rats at a dose of 0,10 or 100 mg/kg bw by gavage twice daily for 4 days. A separate group of six males was given phenobarbital at a dose of 1 g/1 in drinking-water for 7 days. Eighteen hours after the last dose, all animals were killed and a post-mitochondrial supernatant, containing microsomal enzymes, was isolated from each liver and assayed for aniline hydroxylase and/?ara-nitroanisole demethylase activities and for the content of cytochromes P450 and b5. The relative weight of the liver and the microsomal protein content were increased at the highest dose. Prochloraz was a potent inducer of the hepatic microsomal enzyme system at 100 mg/kg bw, but a marginal effect was seen at 10 mg/kg bw, indicating that this dose is close to the threshold dose for induction. The spectrum of induction was similar to that caused by phenobarbital, the content of cytochromes P450 being increased at both doses (Needham, 1983b).

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Similar induction of mixed-function oxidases was seen when male rats were fed diets containing prochloraz at a concentration of 2500 ppm for 7 days, whereas only a small increase was found with 100 ppm (Riviere, 1983). The profile of the hepatic mixed-function oxidase system of male rats was examined after treatment with prochloraz and three of its major metabolites: M1,2,4,6-trichlorophenoxy-ethanol and 2,4,6-trichlorophenoxyacetic acid. All animals were dosed twice daily with a solution or a suspension of a molar equivalent of prochloraz or its metabolites in corn oil for 4 days. A first group of rats were given prochloraz and the first two metabolites at a rate equivalent to 100 mg of prochloraz per kg bw, and 2,4,6-trichlorophenoxyacetic acid at a rate equivalent to 50 mg of prochloraz per kg bw, as the higher dose had been shown to have adverse effects. A second group of rats were dosed with all four compounds at a rate equivalent to 50 mg prochloraz per kg bw. Control animals received corn oil only, and another group received phenobarbital in drinkingwater at 0.1% (w/v) for 14 days. A further group received b-naphthoflavone intraperitoneally at 80 mg/kg bw per day for 4 days, and hepatic microsomes were prepared from rats that had been induced with clofibrate by an intraperitoneal dose of 400 mg/kg bw per day for 3 days before the study. At the end of dosing, all animals were killed and a post-mitochondrial supernatant, containing microsomal enzymes, was isolated from each liver and analysed for protein and cytochromes P450 and b5 content and for the activities of 7-ethoxyresorufln-O-deethylase), 7pentoxyresorufm-0-dealkylase, 7-ethoxycoumarin-O-deethylase, aldrin epoxidase and lauric acid hydroxylase. The profile of induction caused by prochloraz reflected the contribution of the individual metabolites. The activity of lauric acid hydroxylase was slightly increased, but aldrin epoxidase was induced by sevenfold and 7-pentoxyresorufin-O-dealkylase by 14-fold. M1 was a phenobarbitaltype inducer, increasing the activity of aldrin epoxidase by 120% and that of 7-pentoxyresorufm0-dealkylase by eightfold. 2-(2,4,6-trichlorophenoxy)ethanol and 2,4,6-trichlorophenoxyacetic acid were both inducers of the clofibrate type, increasing the activity of lauric acid 12-hydrolase. The induction profile of prochloraz was mixed, but the predominant characteristics were those of a phenobarbital-type inducer (Needham et al., 1992). (d)

Effects of dog gastric juice and plasma on prochloraz

The stability of prochloraz and its plant metabolites M2 and Ml in the gastric juice and plasma of dogs was investigated in vitro. While both prochloraz and Ml were completely stable in gastric juice, M2 underwent slow hydrolysis to form Ml (< 10% in 2 h). After incubation in plasma, < 2% of the prochloraz present underwent hydrolysis (to yield M2), and about 20% of M2 was converted to Ml. No 2,4,6-trichlorophenol was detected as a hydrolysis product in any of these experiments. The initial step in the metabolism of prochloraz is therefore likely to occur in the liver (Needham, 1980).

2.

Toxicological studies (a)

Acute toxicity (i)

Lethal doses

The results of studies to establish the LD50 and LCso of prochloraz are summarized in Table 2. Groups of 10 male CD-I mice were given a single oral dose of 0, 400, 800, 1600 or 2400 mg/kg bw by gavage and observed for 7 days. Deaths occurred within 3 days after treatment

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Table 2. Acute toxicity of prochloraz Species

Strain

Sex

Route

Vehicle

LDso LCso (mg/kg bw) (mg/1 air)

Mouse

CD-I

Male

Oral

1 0% acacia solution

2400

Rat

Boots-Wistar

Oral

GLP

CFY

10% aqueous acacia solution 10% aqueous acacia solution

1600-2400

Rat

2400

GLP

Shawetal. (1979a)

Dog

Beagle

Male and female Male and female Male and female Female

Shaw & Carter (1976) Shawetal. (1979a)

GLP &QA

Morgan et al. (1978) Morgan et al. (1977) Jackson & Hardy (1987)

Baboon

Rat

Wistar

Male and female

Rat

SpragueDawley New Zealand white

Male and female Male and female Male

Rabbit

Rat

CD

Oral Oral (gelatin capsules) Oral (gelatin capsules) Inhalation (4 h, wholebody) Dermal

NOAEL: 10 LOAEL: 100 NOAEL: 50 LOAEL: 250 Aerosol

Dermal Intraperitoneal

Com oil

>2.2 a

GLP orQA

>2100

QA

> 3 ml/kg bw

QA

400-800

Reference

Hounsell & Ogle (1987) Kynoch et al. (1979) Smithson & Lancaster (1980)

QA, quality assurance; GLP, good laboratory practice Maximum attainable concentration

a

at doses > 1600 mg/kg bw. No significant abnormality was noted in survivors of the dose of 2400 mg/kg bw, and the alanine aminotransferase activity in all treated mice was within normal limits, indicating that prochloraz was not acutely hepatotoxic at doses up to and including the LDso (Shaw & Carter, 1976). Groups of five male and five female Boots-Wistar and CFY rats were given a single oral dose of 800,1600 or 2400 mg/kg bw, and a control group of each strain received the vehicle, 10% aqueous acacia solution. The rats were observed for 14 days. Overt signs of toxicity were apparent within 30 min at all doses and included piloerection and diarrhoea; rats at the two higher doses showed signs of central nervous system depression, adopted a hunched posture, had closed eyes and increased salivation, felt cool to touch, became ataxic and had slowed respiration. In addition, some animals had increased lachrymation, a wasted appearance and were tremorous and limp to touch; a few were excitable and one had convulsions. Generally, the CFY rats appeared to return to normal before the Boots-Wistar rats. At 1600 mg/kg bw, two Boots-Wistar rats of each sex and one male and two female CFY r,ats died; at 2400 mg/kg bw, three Boots-Wistar rats of each sex and two CFY rats of each sex died. All deaths occurred within 3 days of dosing. At autopsy, macroscopic evidence of gastrointestinal irritation and pale livers were observed. As gastrointestinal irritation was also observed after intraperitoneal administration, this effect may be systemic rather than local (Shaw et al., 1979b). Five male and five female rats were exposed for 4 h (whole body) to an aerosol containing prochloraz at the maximum attainable concentration of 2.2 mg/1 air. The animals were observed for a further 14 days. There were no deaths. The clinical signs observed during exposure were partial closing of eyes, lachrymation, abnormal respiratory rate and/or movements and hunched posture. Brown staining around the snout and jaws, matted body fur and a waxy deposit or accretion on the tail were seen. Other treatment-related effects were transient reductions in body weight and food consumption in males (Jackson & Hardy, 1987). Technical-grade prochloraz was applied at a dose of 2100 mg/kg bw for 24 h under occlusion to the shaved backs of five male and five female rats, and the animals were observed for 14 days. Five male and five female control rats were treated similarly with no test substance. No clinical signs, no adverse effects on body weight and no signs of irritation were observed. No

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deaths occurred during the study, and no abnormalities were seenpost mortem (Hounsell & Ogle, 1987). Two male and two female rabbits was treated dermally with prochloraz at a dose of 3 ml/kg bw, the maximum practical dosage. The material was applied to the shaved skin of each animal, and the site was occluded for 24 h. Moderate to severe dermal reactions characterized by erythema and oedema were observed, which generally began to heal from day 12 after treatment (Kynoch etal., 1979). One male and one female beagle were given a single oral dose of 0, 10, 100 or 250 mg/kg bw. A clinical examination and haematological, biochemical and urinary analyses were conducted twice before dosing and on days 2 and 8 of the study. Body weight, food consumption, faecal appearance and occult blood content were recorded daily for 1 week before dosing and throughout the study. Emesis occurred 2.25-7 h after dosing in both animals given 100 mg/kg bw and in the male given 250 mg/kg bw. During this time, the females in these groups had diarrhoea. The male given 250 mg/kg bw was anorexic on days 1 and 2 and had lost weight by days 2 and 3. Serum alkaline phosphatase activity was elevated on days 2 and 8 in both animals given 250 mg/kg bw, and low potassium concentrations were seen in the male and a high reticulocyte count in the female (Morgan et al., 1978). One female baboon was given single oral doses of prochloraz at 50 and 250 mg/kg bw in gelatin capsules on days 1 and 17, respectively. A female given empty capsules served as control. Body weight, food and water consumption and faecal appearance were recorded for 1 week before dosing and throughout the study. Haematological, biochemical and urinary analyses were conducted before dosing and on days 2,8,18 and 24, and both animals were killed and examined on day 25. Sections of liver and kidney and bone-marrow smears were examined histologically. Emesis and increased salivation were seen in the treated baboon 2-5 h after the higher dose. On day 24, it had a slightly lowered serum potassium concentration. Treatment had no effect on body weight, food or water consumption, faecal appearance or haematological parameters. Examination post mortem revealed no effect on organ weights and no pathological change related to treatment (Morgan et al., 1977). Prochloraz was more toxic when administered parenterally to rats than when given orally, the LD50 after intraperitoneal injection being 400-800 mg/kg bw. Groups of five male Charles River CD rats were given a single intraperitoneal dose of 50, 100, 200, 400, 800 or 1600 mg/kg bw of a solution of prochloraz in corn oil, and a control group received the vehicle alone. Overt signs of toxicity were apparent within 0.5-1 h after dosing at > 100 mg/kg bw. All these rats showed evidence of central nervous system depression (coma, prostration, lethargy, sedation, inactivity); other signs included ataxia, piloerection, partially closed eyes, irregular respiration, limpness and coolness to touch, cyanosis, adoption of a hunched position, walking on tiptoe, increased nasal exudate, increased lachrymation and salivation, dark staining around the eyes, excitability, urine stains around the genital region, diarrhoea, tremorous and/or convulsive movements and straub tail. No overt signs of toxicity were seen in animals given 50 mg/kg bw. Rats given 100 mg/kg bw recovered overnight after dosing, those given 200 mg/kg bw recovered by day 3, those given 400 mg/kg bw recovered by day 5 and the survivor given 800 mg/kg bw had generally recovered by day 4, although piloerection and excitability persisted throughout the 14day observation period. Two rats given 400 mg/kg bw, four given 800 mg/kg bw and all given 1600 mg/kg died within 24 h of dosing. The macroscopic findings at autopsy included evidence of gastrointestinal irritation and an oily liquid, probably the injected solution, surrounding some viscera. At autopsy on day 15, white globules were found within the mesentery in the abdomens of most animals (Smithson & Lancaster, 1980).

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(ii)

Dermal and ocular irritation and dermal sensitization

The dermal irritation potential of technical-grade prochloraz (purity, 96.3%) was tested in three male and three female New Zealand white rabbits to which 0.5 ml of test material was applied under a gauze patch and covered with occlusive tape for 4 h. At the end of this period, the patch was removed, and skin reactions were assessed 48 and 72 h later. No irritation was seen at treated sites at any time during the study. No clinical signs were seen. A quality assurance statement was included (Cuthbert & D'Arcy Burt, 1984a). The ocular irritation potential of technical-grade prochloraz (purity, 96.3%) was tested in three male and three female New Zealand white rabbits to which 0.1 ml of test material was administered onto the lower eyelid of the right eye. The lids were then gently held together for 12 s. The other eye remained untreated to serve as a control. The eyes were examined for irritation under standard illumination, and any ocular reactions were recorded 1, 24, 48 and 72 h after instillation. No corneal or iridal response was seen. Slight conjunctival responses (redness, score 1) were seen only 1 h after instillation. The eyes were normal after 24 h. No clinical signs were seen. A quality assurance statement was included (Cuthbert & D'Arcy Burt, 1984b). The skin sensitization potential of prochloraz (purity unspecified) was assessed in 20 female guinea-pigs by the method of Magnusson & Kligman (1969,1970), which consists of induction with intradermal injections (20% dilution in ethanol) of the test material, followed after 1 week by topical application of undiluted test compound and a challenge 3 weeks after induction with undiluted test compound or a 50% dilution in ethanol. None of the animals showed any dermal reaction either 24 or 48 h after challenge. Therefore, there was no evidence that prochloraz acts as a delayed dermal sensitizer in guinea-pigs. The study was conducted before GLP regulations were issued in the testing facility, but both the protocol and test results were well reported (Shaw, 1979). (b)

Short-term studies oftoxicity Mice

Technical-grade prochloraz (purity, 95.29%) was suspended in 10% aqueous acacia and given by gavage to groups of five male and five female CFLP mice at a dose of 96, 240 or 600 mg/kg bw per day for 21 days; a control group received the vehicle alone. Overt signs of toxicity were recorded daily and body weight once weekly throughout the study. All mice were killed at the end of the dosing period, and plasma samples were collected for estimation of alanine aminotransferase activity; the mice were then dissected and examined macroscopically. The livers of all mice, the duodena from mice showing macroscopic abnormalities in the liver and any organ showing macroscopic abnormalities were examined microscopically. The study was conducted before GLP regulations were issued in the testing facility, but the protocol and results were well reported. The study is considered to provide useful additional information. Overt signs oftoxicity occurred in males given 600 mg/kg bw per day; these included loss of condition, evidence of central nervous system depression (sedation or inactivity), piloerection and coolness to touch. Four males and one female given 600 mg/kg bw per day died during the study, but two of these deaths were attributable to administration accidents. The body-weight gain of males surviving 600 mg/kg bw per day was reduced during the first 2 weeks of the study, but that of females given 240 or 600 mg/kg bw per day was increased throughout dosing. Plasma alanine aminotransferase activity was increased in mice of each sex surviving 600 mg/kg bw per day. Hyperkeratinization of the forestomach was seen in three males and all females at 600 mg/kg bw per day, in one male at 240 mg/kg bw per day and in one female control. No microscopic

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changes that could be related to treatment were observed in the liver, although there was a high incidence of minor background lesions in all groups. The authors concluded that, as the dose of 600 mg/kg bw per day was not tolerated by the males, the appropriate highest dose for the 90day study would be 400 mg/kg bw per day (Lancaster & Shaw, 1980a). In a study conducted in compliance with the principles of GLP (with QA certification), groups of 15 male and 15 female CD-I mice were given diets containing technical-grade prochloraz (purity, 98.2%) at concentrations providing a dose of 6,25,100 or 400 mg/kg bw per day for 13 weeks. A control group comprised 24 mice of each sex. Further groups of 15 male and 15 female controls and mice treated at 400 mg/kg bw per day were kept for 4 weeks after the end of dosing, and, in addition, groups of nine males and nine females were treated for 6 weeks. Solutions of the test material were prepared in corn oil. Overt signs of toxicity were recorded daily, and body weight and food consumption were recorded three times weekly throughout the study. Haematological and blood biochemical analyses were conducted at 6 and 13 weeks and after 4 weeks on control diet. All mice were killed at the end of dosing or the recovery period and were dissected and examined macroscopically, and the main organs were weighed. A comprehensive microscopic examination of tissues from 10 male and 10 female controls and all mice at 400 mg/kg bw per day killed at 13 weeks was carried out, and the livers of all remaining animals killed at 13 weeks and all mice killed after 4 weeks on control diet were examined. No deaths occurred that were attributable to treatment. At 400 mg/kg bw per day, the incidence of piloerection was increased. Weight gain was slightly reduced in both sexes at 400 mg/kg bw per day and in males at 100 mg/kg bw per day. Food consumption was higher in both sexes at the highest dose throughout dosing and during the first week of the recovery period, but thereafter was similar to that of controls. At week 6, the haemoglobin concentration, packed cell volume and erythrocyte count were increased in both sexes at 400 mg/kg bw per day; mean corpuscular haemoglobin was also marginally increased in males, and the leukocyte count was increased in females due to lymphocytosis. These parameters were no longer affected by treatment at week 13 or after 4 weeks' recovery. The total leukocyte count was increased in females at the highest dose at weeks 6 and 13. Plasma alanine aminotransferase activity was increased at week 6 in some mice of each sex at 400 mg/kg bw per day and in some males at 100 mg/kg bw per day; at week 13, it was increased in the majority of females at 400 mg/kg bw per day and in some mice of each sex at 100 mg/kg bw per day; after 4 weeks on control diet, some males at 400 mg/kg bw per day were still affected. At weeks 6 and 13, the plasma albumin concentration and, consequently the albumin:globulin ratio were slightly reduced in both sexes at 400 mg/kg bw per day and in males at 100 mg/kg bw per day. At week 13, the plasma glucose concentration were increased and the blood urea nitrogen concentration slightly decreased in both sexes at 400 mg/kg bw per day. At autopsy at week 6, the weights of the liver was increased in both sexes at 100 or 400 mg/kg bw per day and were slightly increased in females at 25 mg/kg bw per day. The ovaries of some females at the highest dose and in most at 100 mg/kg bw per day were small, and the prostate and seminal vesicles of males at 400 mg/kg bw per day were small. The weight of the spleen was increased in both sexes at 100 mg/kg bw per day and in a few females at 25 mg/kg bw per day, but was low in most females at 6 mg/kg bw per day (this decrease was considered not to be related to treatment). At week 13, the weight of the liver was increased in both sexes at 25,100 or 400 mg/kg bw per day. The ovaries were small in females and the prostate and seminal vesicles small in males at 400 mg/kg bw per day. After 4 weeks' recovery, the liver weight was still increased in both sexes at the highest dose, but to a much lesser extent than at weeks 6 and 13. The livers of some mice at 400 mg/kg bw per day appeared to be enlarged, and the lobular pattern was prominent. Histopathological examination showed treatment-related changes only in the liver. Minimal liver changes, with loss of glycogen and periportal fat droplets, were seen in both sexes PROCHLORAZ 141-182 JMPR 2001

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at the highest dose and in females at 100 mg/kg bw per day after 13 weeks. Some males at the two higher doses also showed centrilobular hepatocyte enlargement. In mice killed after 4 weeks' recovery, none of these changes was present. The NOAEL was 6 mg/kg bw per day on the basis of effects on the liver at the next higher dose. The hepatic effects were reversible (Gale, 1980; Lancaster, 1982; Keene, 1988a; Mallyon, 1988a). Rats Technical-grade prochloraz (purity, 92.7%) was emulsified in aqueous acacia and given by gavage to groups of five male and five female Boots-Wistar rats at a dose of 25,100 or 400 mg/kg bw per day for 30 days; a control group of 10 males and 10 females received the vehicle alone. Overt signs of toxicity were recorded daily and body weight and food consumption three times weekly, throughout the study. Haematological, blood biochemical and urinary analyses were conducted during week 4. All rats were killed at the end of the dosing period, and serum samples were collected for estimation of enzyme activity and electrolyte content; the rats were then dissected and examined macroscopically, and the main organs were weighed. A comprehensive histological examination of tissues from half the controls and all the rats at 400 mg/kg bw per day was completed; in addition, the liver, lymph nodes, spleen and bone-marrow smears from the remaining controls and rats given 25 or 100 mg/kg bw per day were examined. The study was conducted before GLP regulations were issued in the testing facility, but the protocol and results were well reported. The study is considered to provide useful additional information. Increased salivation was seen in both sexes given 100 or 400 mg/kg bw per day, and slight loss of condition was noted in females at 400 mg/kg bw per day. Slight weight loss and decreased food intake were seen at the start of treatment in both sexes at 400 mg/kg bw per day, and food consumption was also marginally reduced in both sexes given 100 mg/kg bw per day. The erythrocyte count was decreased in some rats of each sex at 400 mg/kg bw per day and in some females at 100 mg/kg bw per day; the haemoglobin concentration was also slightly decreased in one rat of each sex at 400 mg/kg bw per day. The mean corpuscular volume was high in some animals at 400 mg/kg bw per day. The leukocyte count was increased due to neutrophilia and lymphocyte sis in females at 400 mg/kg bw per day. Urine flow was increased and the specific gravity before water was withheld was decreased in some rats of each sex at 400 mg/kg bw per day and in one male at 100 mg/kg bw per day. Urinary aspartate aminotransferase activity was slightly increased in females, and the urinary pH was reduced in a few rats at 400 mg/kg bw per day. The weight of the liver was increased in both sexes at 100 and 400 mg/kg bw per day, the females tending to be more severely affected. The weight of the adrenals was increased, and the prostate and seminal vesicles were small in the males at 100 and 400 mg/kg bw per day. The only finding possibly related to treatment observed at microscopic examination was slight haematopoiesis in the spleen in most females at 25 mg/kg bw per day and in both sexes at 100 and 400 mg/kg bw per day, although this change was also seen in one male and three female controls. The authors concluded that, because the dose of 100 mg/kg bw per day was tolerated by both sexes, it would be the appropriate highest dose for the 90-day study. The increased salivation may have been related to the route of administration, as similar effects were not seen after dietary administration (Lancaster & Shaw, 1980b). In a study conducted before GLP regulations were issued in the testing facility but which complied with the general principles of GLP and the final report of which was reviewed for QA, groups of 20 male and 20 female Boots-Wistar rats were given technical-grade prochloraz (purity, 97.5%) at a dose of 6,25 or 100 mg/kg bw per day by gavage in 10% aqueous acacia suspension for 13 weeks; a control group of 20 males and 20 females received the vehicle alone. Further

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groups of 20 male and 20 female controls and rats at 100 mg/kg bw per day were kept for 4 weeks after the end of the dosing period. In addition, groups of 10 male and 10 female controls and rats at 6,25 or 100 mg/kg bw per day were examined after 6 weeks. Overt signs of toxicity, body weight and food consumption were recorded daily throughout the study; haematological, blood biochemical and urinary analyses were conducted at 6 and 13 weeks and after 4 weeks' recovery. All rats were killed at the end of the dosing or recovery period, and serum samples were collected for estimation of enzyme activity and electrolyte content; the rats were dissected and examined macroscopically, and the main organs were weighed. A comprehensive histological examination of tissues from half the controls and all rats at 100 mg/kg bw per day and killed at 13 weeks was carried out; in addition, the livers from all remaining animals were examined. The investigation was extended to quantify the size of liver cells by counting the number of nuclei in 10 periportal and 10 centrilobular fields of the left lateral lobe of the liver from all rats killed at weeks 6 and 13 and after 4 weeks' recovery. Rats of each sex at 25 and 100 mg/kg bw per day and a few at 6 mg/kg bw per day showed increased salivation. The incidence of diarrhoea was increased during the first 4 weeks of dosing in males at 100 mg/kg bw per day and possibly during the first week of dosing in males at 25 mg/kg bw per day. In females, a few instances of diarrhoea were recorded at each dose. Body-weight gain was increased in females at 25 and 100 mg/kg bw per day during dosing. During the first 5 days of the recovery period, females at 100 mg/kg bw per day lost a small amount of weight, the overall weight gain being statistically significantly less than that of the controls. Food consumption was unaffected by treatment except for a slight increase during the recovery period in males at 100 mg/kg bw per day. At week 6, the haemoglobin concentration was slightly decreased in males at each dose and in females at 100 mg/kg bw per day; the leukocyte count was increased, due to lymphocytosis, in all treated males. At week 13, the mean corpuscular volume was slightly decreased in all treated males and in females at 100 mg/kg bw per day, and the haemoglobin concentration was slightly decreased in females at 25 and 100 mg/kg bw per day and in males at 100 mg/kg bw per day after the 4-week recovery period. The serum bilirubin concentration was slightly decreased in all treated rats at week 6, in males at 25 and 100 mg/kg bw per day and in females at 6 and 100 mg/kg bw per day at week 13, and in a few treated rats of each sex after the 4-week recovery period. The serum potassium concentration was slightly increased at week 6 in males at 25 and 100 mg/kg bw per day and in three females at 100 mg/kg bw per day. At week 6, urinary aspartate aminotransferase activity was slightly increased in a few rats of each sex at each dose. At week 13, the specific gravity or urine before water was withheld and sodium excretion were slightly decreased in both sexes at 100 mg/kg bw per day, urinary aspartate aminotransferase activity was increased in males at 100 mg/kg bw per day, urinary pH was reduced in some females at 25 and 100 mg/kg bw per day and the urinary protein content was reduced in all treated females and in males at 100 mg/kg bw per day. After 4 weeks' recovery, potassium excretion and urinary pH were slightly reduced in females given 100 mg/kg bw per day. At autopsy at week 6, the weight of the liver was increased in both sexes at 100 mg/kg bw per day and in females given 6 or 25 mg/kg bw per day, and the kidney weight was increased in males at 100 mg/kg bw per day and in both sexes at 25 mg/kg bw per day. The prostate and seminal vesicles were smaller than usual in males and the ovaries were larger in females at 100 mg/kg bw per day. The thyroid weight was increased in all treated females. At week 13, the weight of the liver was increased in males at 6 mg/kg bw per day and in both sexes at 25 and 100 mg/kg bw per day; the kidney weight was increased in both sexes at 100 mg/kg bw per day, the spleen weight was slightly decreased in all treated males, the ovary weight was increased in all treated females; and the thyroid weight was increased in females at 6 and 100 mg/kg bw per day. After 4 weeks' recovery, the weights of the liver, ovaries and thyroids of the females were slightly greater than the control values. At weeks 6 and 13 and after 4 weeks' recovery, the centrilobular liver cells were larger than the periportal cells in control rats of each sex, reflecting the normal morphology of the

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liver. This difference was also seen in all treated groups, irrespective of changes in liver weight or cell size. At week 6, the liver cell size was increased in females at 6 and 25 mg/kg bw per day and in both sexes at 100 mg/kg bw per day. At week 13, the liver cell size was increased in both sexes given 6, 25 or 100 mg/kg bw per day. After 4 weeks on control diet, the liver cell size was still increased in males but had returned to normal or was even decreased in comparison with that in controls in females. The changes in liver cell size generally correlated closely with increases in liver weight. The only instance in which such a correlation was not apparent was after 4 weeks' recovery, when, in treated males, the liver cell size was still slightly increased, even though there was no change in liver weight, and in treated females, the liver cell size was decreased although the liver weight was slightly increased. Various treatment-related effects were seen in all groups, the most important, apparently reversible, effects being increased liver weight and hepatocyte size. A NOAEL could not be identified (Lancaster & Shaw, 1979; Shaw et al., 1979b; Jackson, 1988a; Markham, 1988a). Dogs Groups of two male and two female beagle dogs were given technical-grade prochloraz (purity, 96.6%) suspended in a 5% aqueous acacia solution and administered by gastric intubation at a dose of 10 mg/kg bw per day in a volume of 0.3 ml/kg bw or 40 mg/kg bw per day in a volume of 1.2 ml/kg bw, for 14 days. A group of two males and two females receiving 5% acacia solution at the higher dose volume served as controls. Body weight, food consumption and faecal appearance as well as occult blood content were recorded daily for 1 week before dosing and throughout the study. Overt signs of toxicity were recorded throughout dosing. Comprehensive haematological and blood biochemical analyses were conducted once before dosing and on day 14; additional blood samples were collected on days 3 and 8 for estimation of bilirubin concentration and the activity of certain serum enzymes. The dogs were killed the day after the final dose, dissected and examined macroscopically. The main organs were weighed, and sections of selected organs and tissues were examined histologically. The study was conducted before GLP regulations were issued in the testing facility, but the protocol and results were well reported. The study is considered to provide useful additional information. Dogs given 40 mg/kg bw per day usually vomited within 1 h after dosing and had diarrhoea within 2 h. Because of the emesis, the daily dose was divided and administered in two equal volumes morning and afternoon to each dog from day 6; thereafter, the incidence of vomiting decreased and, as for the diarrohea, usually occurred only after the morning dose. In addition, increased salivation was seen sporadically in some dogs at both doses. Both males at 40 mg/kg bw per day were intermittently anorexic and lost weight during the study. On day 14, in animals at 40 mg/kg bw per day, low values were recorded for erythrocyte count, haemoglobin concentration and erythrocyte volume fraction in one female, for haemoglobin concentration in the other female and for erythrocyte count in one male. Serum alkaline phosphatase activity increased progressively throughout the dosing period in both males and one female at the higher dose, and two of these animals also had very slight increases in serum leucine aminopeptidase activity. At autopsy on day 15, the livers of all dogs at 40 mg/kg bw per day appeared large and bulbous, and the absolute and relative weights in these dogs and the relative weight in one female at 10 mg/kg bw per day were increased. The prostates of both males at 40 mg/kg bw per day and the testes of one of these appeared small and flaccid; the testes had spermatid giant cells. In addition, copious amounts of mucus were seen in the stomachs of one male at the higher dose and both males at the lower dose, and a cream-grey layer was observed on the mucosal surface of the ileum in one male at each dose; furthermore, one female at the higher dose had microscopic

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evidence of gastritis. Microscopic examination revealed no other change that was considered to be directly related to treatment. The authors concluded that a single oral dose of 40 mg/kg bw per day would be unacceptable in a long-term study and a single dose of 20 mg/kg bw per day would be well tolerated. This dose was chosen as the highest dose for the 90-day study. The NOAEL was 10 mg/kg bw per day on the basis of the increase in alkaline phosphatase activity. Emesis, which was observed in dogs after both single and short-term intake, was considered not to be a relevant end-point, because it was observed after administration of high doses of prochloraz by capsule or gastric intubation. Dogs are known to be sensitive to nonspecific emetic effects that are not necessarily related to a specific test substance but simply to the stress of administration or direct gastrointestinal discomfort (Morgan et al., 1979). In a study conducted before GLP regulations were issued in the testing facility but which complied with the general principles of GLP and for which the final report was reveiewed for QA, groups of four male and four female beagle dogs were given technical-grade prochloraz (purity, 97.9%) suspended in a 10% aqueous acacia solution at a dose of 1,2.5,7 or 20 mg/kg bw per day by gastric intubation for 13 weeks. A group of four dogs of each sex receiving the vehicle served as controls. Additional groups of four male and four female controls and dogs at 20 mg/kg bw per day were retained for 4 weeks with no treatment at the end of dosing. Overt signs of toxicity, body weights, food consumption and faecal appearance were recorded daily throughout the study. At weeks 6 and 13, a clinical examination, including ophthalmoscopy and electrocardiography, and haematological, blood biochemical, faecal and urinary analyses were conducted; the parameters that were affected at week 13 were measured again at the end of the 4-week recovery period. On each occasion, parameters with values outside normal limits were measured again 1 week later, and serum alkaline phosphatase and leucine aminopeptidase activities were estimated in all dogs at week 9. All dogs were killed at the end of the dosing or recovery period, dissected and examined macroscopically, the main organs were weighed, and a comprehensive histological examination was carried out. The only overt signs of toxicity were isolated bouts of emesis and increased salivation in some dogs in all groups, including controls, although these were more prevalent at the highest dose. In addition, all dogs at 20 mg/kg bw per day and one at 7 mg/kg bw per day occasionally produced yellow mucoid and/or liquid faeces. During the recovery period, no overt signs of toxicity were observed. Body-weight gain was comparable in control and treated groups. Reduced food consumption was recorded for two dogs at 20 mg/kg bw per day. Clinical examination at week 13 revealed that four dogs at 20 mg/kg bw per day had apparently small and/or flaccid testes; in two of these dogs that were allowed to recover, the change was still apparent at week 17. No treatment-related effects were detected by ophthalmoscopy or electrocardiography. There was no definite treatment-related effect on haematological parameters. Serum alkaline phosphatase activity was high during dosing in most dogs at 20 mg/kg bw per day and in some at 7 mg/kg bw per day; in those dogs retained for recovery, the activity returned to within normal limits. Serum leucine aminopeptidase activity was very slightly increased at week 6 in four dogs at 20 mg/kg bw per day and in one at 7 mg/kg bw per day. The activity had returned to within normal limits by week 9 or 13. There was no effect of treatment on urinary parameters. At necropsy, all dogs at 20 mg/kg bw per day and three at 7 mg/kg bw per day had large, heavy livers; this effect was apparent in only one treated dog at the end of the recovery period. Low prostate weight was recorded in three dogs at the highest dose and in two other dogs after the recovery period; one of these killed at week 14 also had a low testes weight. In addition, one dog at 7 mg/kg bw per day had a low relative prostate weight. In two females at the highest dose that were killed at week 14 and in one killed at week 18, a clear brown exudate from freshly sectioned

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mammary glands was observed. The only histological finding of note was apparent immaturity of the testes of some dogs at 20 mg/kg bw per day. Pre-terminal clinical examination revealed small and/or flaccid testes in four of the eight dogs. Histological changes that were indistinguishable from immaturity were observed in one of two dogs killed after 13 weeks, and comparable findings were made in another dog which had shown no associated clinical signs. At the end of the recovery period, the testes of all dogs appeared normal, indicating that the change was reversible. Counts of liver nuclei revealed no statistically significant differences in liver cell size between controls and animals at the highest dose. The NOAEL was 2.5 mg/kg bw per day on the basis of effects on alkaline phosphatase and leucine aminopeptidase activity, liver size and weight and prostate and testes weights at the next highest dose. These effects were reversible (Lancaster et al., 1979; Lancaster, 1980; Keene, 1988b; Markham, 1988b; Jackson, 1989). (c)

Long-term studies oftoxicity and carcinogenicity Mice

In a study conducted in compliance with the principles of GLP (with QA certification), groups of 52 male and 52 female CD-I mice were fed diets containing technical-grade prochloraz (purity, 95.4-96.8%) at a concentration of 78,325 or 1300 ppm, corresponding to mean achieved intakes of 7.5,33 and 130 mg/kg bw per day for males and 8.8, 36 and 150 mg/kg bw per day for females. The control group comprised 104 mice of each sex which received a diet containing the vehicle (corn oil) at the same concentration as that admixed with the highest concentration of test compound. Males were treated for 106 weeks and females for 121 weeks. All animals were observed daily, body weight and food consumption were determined weekly, and masses were palpated every 2 weeks. Haematological investigations were carried out at week 52 and at termination and included packed cell volume, haemoglobin and erythrocyte, reticulocyte, total leukocyte and differential leukocyte counts. Treatment was terminated in males when the survival rate of those at 325 ppm was 25% and in females when the survival rate of those at 78 ppm was 27%. All mice were killed by asphyxiation with CO2, and a full post-mortem was carried out. The brain, heart, kidneys, liver, testes and spleen were weighed, and a large number of tissues were selected and preserved in neutral buffered formalin. All tissues were examined microscopically. Some mice of each sex given 1300 ppm and some males given 325 ppm were killed because their abdomens were distended. These animals were subsequently found to have multiple liver tumours. No other overt signs of toxicity considered to be related to treatment were observed. There were no differences in survival rates on the basis of trend analysis in either sex; however, male mice at 325 ppm showed a pairwise increase in mortality rate. The percentage survival was 52%, 50%, 25% and 48% respectively in control males and those at the low, medium and high dietary concentrations and 30%, 27%, 40% and 29% in females, respectively. Mice receiving 1300 ppm gained less weight than control animals (decrease of 24% in males and 11 % in females), but this was statistically significant in males only. Males at 325 ppm also gained less weight (decrease of 12%). Minimally greater food consumption than by controls was recorded for all treated males and females at 78 or 325 ppm. Haematological investigations at week 52 showed no differences between treated and control mice that were considered to be of toxicological significance. At week 121, slightly lower haemoglobin concentrations were recorded in treated female mice, and lower total leukocyte counts due to lower lymphocyte counts were found in some females in all treated groups, particularly those receiving 1300 ppm. A dose-related increase in the incidence of macroscopic liver masses was observed in mice of each sex that died during the study or were killed at termination. Livers heavier than those of controls were seen in mice of each sex receiving 1300 ppm; however, most of these livers PROCHLORAZ 141-182 JMPR 2001

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contained tumours. In males and females at 325 and 1300 ppm, an increased incidence of liver adenomas and carcinomas was observed when compared with controls, and the difference was statistically significant (Tables 3 and 4). The statistical significance, derived from trend tests by the method of Peto et al. (1980) of the number of tumour-bearing animals, wasp < 0.001 for carcinomas and for any liver tumour in males at 325 and 1300 ppm and females at 1300 ppm and p < 0.01 for any liver tumours in females at 325 ppm. There was an associated increase in the numbers of males and females with more than one liver tumour. In some mice at 1300 ppm, multiple tumours contributed to death. In males, the first adenoma appeared at 52 weeks and the first carcinoma at 62 weeks, while in females the first tumours were observed at 89 weeks. The approximate time of onset can be assessed from the median time of death. The data (Table 5) show no indication of an effect on liver tumour latency in either sex. Females at 78 ppm had a slightly higher incidence of liver tumours than controls, although this difference was not statistically significant and the incidence was not significantly different from that expected by linear extrapolation from the dose-response relationship at higher doses. In males at 78 ppm, the incidence of liver tumours was similar to that of controls; however, the incidence of liver tumours in male controls (36%) was unusually high, as the incidences in 24-month-old CD-I male mice at the same laboratory ranged from 0-17% for adenomas and 1.4-14% for carcinomas. The incidence of liver tumours in control females of the same strain in six concurrent studies of 104108 weeks' duration was 0-12%. Thus, the number of liver tumours in females given 78 ppm in the present study could occur spontaneously in CD-I mice. No treatment-related effect was detected on the incidence of rumours at any other site or any of the non-neoplastic changes recorded. Liver-cell tumours contributed to the deaths of more males (20/52) and females (14/52) at 1300 ppm and more males at 325 ppm (13/52) than controls (9/104 males and 1/104 females). Many of these mice were in poor clinical condition when killed. The NOAEL was 78 ppm, equal to 7.5 mg/kg bw per day (Sharp, 1982; Colley et al., 1983, 1988; Offer et al., 1992; Malarkey, 1993a). Rats In a study conducted in compliance with the principles of GLP (with QA certification), groups of 60 male and 60 female Sprague-Dawley (CD) rats were fed a diet containing technicalgrade prochloraz (purity, 95.1-97.0%) at a concentration of 38, 150 or 625 ppm, corresponding to mean achieved intakes of 1.3,5.1 and 22 mg/kg bw per day for males and 1.6,6.4 and 28 mg/kg bw per day for females. The control group comprised 120 rats of each sex; they received a diet containing the vehicle (corn oil) at the same concentration as that admixed with the highest concentration of test compound. Males were treated for 115 weeks and females for 111 weeks. Separate groups of 20 male and female rats were killed after 52 weeks of treatment, and additional groups of controls and rats at the highest concentration, consisting of 10 animals of each sex, were killed after 13 weeks of treatment. All animals were observed daily, body weights and food consumption were recorded weekly, and masses were palpated every 2 weeks. Ophthalmic examinations were performed before treatment and at weeks 6,13,26,51,78 and 104. Laboratory investigations were carried out during weeks 6-7, 13,26, 52, 77-78 and 104, and samples were collected for haematology, blood chemistry and urine analysis. Treatment was terminated at week 52 for animals in the interim kill and for males when the survival rate of those at 38 ppm was 25% and for females when the survival rate in the control group was 23%. All rats were killed by asphyxiation with CO2, a full post-mortem was carried out, and alarge number of tissues were selected and preserved. All tissues were examined microscopically. There were no overt signs of toxicity during the study that were considered to be related to treatment. During weeks 20-21, most rats and during weeks 66 and 67 a few rats in each group showed signs of sialodacryoadenitis infection, which was associated with weight loss and reduced

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Dietary concentration (ppm)

Sex

No. examin rrl

No. mice with tumours Malignant tumours3

All tumours

Interim

0 78 325

1

2

>3

Total

1

2

>3

Total

2 2 5

0 1 3 23

14 9 20 7

9 2 7 4

2 0 5 6

0 1 2 17

11 3

9

12 6 12 5

Female

73 38 31 37

2 3 2 8

0 1 1 2

0 0 0 15

2 4 3 25

1 0 0 0

0 0 0 0

0 0 0 7

1 0 0 7

Male

54 26 14

1 0 1 13

0 3 2 21

23 12 8 0

4 1 1 2

1 0 0 5

0 2 2 7

5 3

4

22 9 5 4

31 14 21 15

3 1 2 5

0 1 4 5

0 0 2 4

3 2 8 14

0 0 1 1

0 0 0

0 0 0 0

0 0 1 2

Male 27

1300

0 78 325 1300

Termination

50 26 38

0 78 325

25

1300

0 78 325

Female

1300

9

1

14 (3 M)

3(1 M)

From Sharp (1982), Colley et al. (1983, 1988), Offer et al. (1992) and Malarkey (1993a). M, metastasizing 'Including mice with more than one tumour, at least one of which was malignant

Table 4. Incidences of liver tumours in mice in the 2-year study of carcinogenicity Dietary concentration (ppm)

Sex

Incidence of liver tumours Adenomas

Carcinomas

Total

Interim kill

Terminal kill

Total

Interim kill

Terminal kill

Total

0 78 325 1300

Male

3/50 6/26* 6/39 6/27*

18/54 9/26 5+13 14/25*

21/104 15/52 11/52 20/52**

11/50 3/26 14/39 17/27***

5/54 3/26 3/13 7/25*

16/104 6/52 17/52** 24/52***

37/104 21/52 28/52* 44/52***

0 78 325 1300

Female

1/73 4/38* 3/31 18/37***

3/31 2/14 7/21* 12/15***

4/104 6/52 10/52** 30/52***

1/73 0/38 0/31 7/37**

0/31 0/14 1/21 2/15

1/104 0/52 1/52 9/52***

5/104 6/52 1 1/52** 39/52***

From Sharp (1982), Colley et al. (1983, 1988), Offer et al. (1992) and Malarkey (1993a). The incidences of liver tumours in females of the same strain in six concurrent studies of 104-108 weeks' duration were 1/60, 1/60, 5/100, 5/51, 3/52 and 0/55 adenomas and 0/60, 0/60, 1/100, 1/51,1/52 and 2/55 carcinomas. *p < 0.05; ** p < 0.01;/? < 0.001, Fisher exact test for comparisons of numbers of tumour-bearing animals in treated groups with those in the control group; one-tailed values

Table 5. Median time to death (weeks) of liver tumourbearing mice in the 2-year study of carcinogenicity Dietary concentration (ppm) 0 78 325

Sex

Adenoma

Carcinoma

Any

Male

108 107 102 107

95 101 93 102

107 107 94 107

Female

123 116 123 116

89 _ 123 115

122 116 123 116

1300

0 78 325 1300

Liver tumour

From Sharp (1982), Colley et al. (1983, 1988), Offer et al. (1992) and Malarkey (1993a)

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food consumption. The survival rate of males given prochloraz was similar to that of controls, while treated females had better rates than controls (23%, 35%, 37% and 48% of controls and those at the low, medium and high dietary concentrations, respectively). The body-weight gain of both sexes given 625 ppm was reduced (by 9% in males, 13% in females) and was marginally lower in males given 150 ppm (by 6%). The food consumption of males at 625 ppm was lower than that of controls throughout the study, while that of females in this group was initially only marginally lower but became markedly lower as the study progressed. At 150 ppm, food consumption was marginally lower throughout the study for males and up to week 52 for females. Ophthalmoscopy revealed no abnormalities that were considered to be related to treatment. Minor changes considered to be sequelae to sialodacryoadenitis were seen in a few rats at week 26. Changes in some urinary, blood chemical and haematological parameters were detected in treated rats throughout the study, but these were minimal and inconsistent and of doubtful toxicological significance. At the interim kill at week 13, the weight of the liver was higher in most females and three males at 625 ppm, and centrilobular hepatocyte enlargement was seen in one male and two females. At the interim kill at week 52, enlarged or swollen livers were observed in one male and three females at 150 ppm and in four males and one female at 625 ppm. Heavier livers and marginally lower pituitary weights were recorded in males and females at the highest dietary concentration. Centrilobular hepatocyte enlargement was seen in 13 females at 625 ppm; periportal glycogen loss and centrilobular fat deposition were also apparent in some treated rats of each sex, particularly at 150 ppm. At the terminal kill and in rats that died during the study, more male rats (18/61) at 625 ppm than controls (24/121) had enlarged or swollen livers. Slightly heavier livers were recorded in females at this concentration. The incidences and distribution of tumours were unaffected by treatment; a single liver carcinoma observed in one male at 625 ppm was considered incidental. A slightly higher incidence of hyperplastic lesions was seen in the livers of both sexes at 625 ppm (15/61 in males and 23/121 in controls; 21/63 in females and 34/120 in controls), but the difference was not statistically significant. The incidences of other non-neoplastic findings were similar in all groups. The original haematoxylin-and-eosin-stained liver sections from the main group of rats were reexamined by an independent pathologist, who confirmed the original conclusion that there was no treatment-related effect on the incidence of liver tumours (Table 6). A statistically significant trend in the incidence of liver carcinomas was found in males, but there were no statistically significant increase in any individual treated group as compared with concurrent controls. Furthermore, the incidence of liver adenomas or carcinomas in the three treated groups was within or very close to the range of incidences in other CD rats in the same laboratory (0-4% for adenomas in both sexes; 0-4% for adenocarcinomas in males and 0-2% in females). Female rats at 625 ppm Table 6. Hepatic lesions found by an independent pathologist in a 2-year study of carcinogenicity in rats Dietary concentration (opm)

Sex

0 38 150 625

Male

0 38 150 625

Female

No.animals examineei

120

60 60 60 120

60 60 60

Hepatic lesions Adenoma

Carcinoma

Any tumour Eosinophilic hepatocytes

Clear cells

0 0 1 0

1 1 2 3

1 1 3 3

16 6 14 12

12 2 1 4

1 0 0

0 0 0 0

1 0 0 1

16 6 12 26

4 2 0 9

1

From Colley et al. (1982a,b), Gopinath (1987), Mallyon (1988b) and Malarkey (1993b). The incidences of liver tumours in concurrent control CD rats in the same laboratory were 2/50, 1/100, 0/55, 0/50 and 0/60 adenomas in males and females; 2/50, 0/100, 1/55, 0/50 and 1/60 adenocarcinomas in males and 1/50, 0/100, 0/55, 1/50 and 1/60 adenocarcinomas in females.

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showed an increased incidence of foci or areas of altered hepatocytes, mainly of eosinophilic hepatocytes and clear cells, when compared with control females. The NOAEL was 38 ppm, equal to 1.3 mg/kg bw per day, on the basis of hepatic effects (periportal glycogen loss and centrilobular fat deposition) at the next highest dose (Colley et al., 1982a,b; Gopinath, 1987; Mallyon, 1988b; Malrkey, 1993b). Dogs In a study conducted in compliance with the principles of GLP (with QA certification), groups of five male and five female beagle dogs were fed diets containing technical-grade prochloraz (purity, 95.2-97.1%) at a concentration of 30,135 or 600 ppm (increased to 1000 ppm from week 57) for 104 weeks. These concentrations corresponded to mean achieved intakes of 0.90,4.1,18 and 28 mg/kg bw per day for females and 0.94,4.5,18 and 29 mg/kg bw per day for males. Control animals received a diet containing the vehicle (corn oil) at the same concentration as that admixed with the highest concentration of test compound. An additional group of two males and two females given 600 ppm prochloraz were treated for 13 weeks (interim kill group). All animals were observed daily, and body weight and food consumption were recorded weekly. Ophthalmoscopy, electrocardiography and laboratory investigations were carried out before treatment and during weeks 13, 26, 50-51, 78 and 104. Samples were taken for haematology, blood chemistry and urine analysis. All dogs were killed by exsanguination under pentobarbital anaesthesia, and a full post mortem was carried out. Bone marrow was obtained by sternal puncture and examined. Various organs were weighed, and a large number of tissues were selected and (except those from interim kill animals) examined microscopically. Four dogs were killed for humane reasons during the treatment period, but none of the deaths nor the findings post mortem were considered to be related to treatment. There were no overt signs of toxicity and no effect of treatment on body weight or food consumption. Ophthalmoscopy and electrocardiography revealed no effects. The mean serum alkaline phosphatase activity in males and females receiving 600 ppm was higher than that of controls during weeks 13,26 and 50. When the dietary concentration of this group was increased to 1000 ppm in week 57, a marked increase in alkaline phosphatase activity was seen, which persisted for the remainder of the dosing period. The activity was also higher in males at 135 ppm from week 13, but the difference was not as marked as at the higher concentration. Slight increases in cholesterol concentration were observed at the highest dietary concentration. Slight but inconsistent increases in platelet count and blood glucose concentration were also seen in these animals, but the effects could not be definitely related to treatment. Treatment had no effect on urinary end-points or faecal occult blood content. Macroscopic examination post mortem revealed no changes that were considered to be related to treatment. At the interim kill, the liver of one animal was heavier than usual, and the liver of one other animal slightly exceeded the normal upper limit when expressed as a percentage of body weight. The prostate of one dog was lighter than expected. At termination, statistical analysis showed that the mean liver weight of males and females at 600/1000 ppm was significantly greater than the corresponding control mean (increase of 42% in males and 30% in females), and the mean prostate weight of animals at these concentrations was significantly lower than the control mean (decrease of 63%). The histopathological treatment-related changes observed after 104 weeks were minimal swelling and rarefaction of centrilobular hepatocytes with associated low-grade hepatitis in all dogs at the highest dietary concentration, minimal swelling and rarefaction of occasional centrilobular hepatocytes and polymorphic leukocyte infiltration in central areas in one dog at the intermediate concentration, and minimal prostatic atrophy or incomplete acinar development with associated stromal, trabecular or capsular fibrosis in four male dogs at the highest dietary concentration. No evidence of neoplasia was found.

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The NOAEL was 30 ppm, equal to 0.90 mg/kg bw per day, on the basis of effects on the liver in one dog at the next highest dose (Woodhouse et al., 1979; Chesterman et al., 1981; Mallyon, 1988c; Malarkey, 1993c) (d)

Genotoxicity

The mutagenic and genotoxic potential of technical-grade prochloraz was investigated in a battery of tests in vitro and in vivo (Table 7). The results of all tests were negative, with the exception of an assay for sister chromatid exchange in vitro, in which a slight increase in frequency was observed in the presence and absence of metabolic activation at doses in the toxic range. Table 7. Results of studies of the genotoxicity of prochloraz End-point In vitro Reverse mutation

Test object

Concentration

Purity (%)

Results

GLP orQA

Reference

GLP

Wilcox (1978)

McGregor et al. (1983)

S. typhimurium TA98, 62.5, 125, 250, 500, TA100,TA1535, TA1537, 1000 ng/plate in dimethyl TA1538 sulfoxide

100 Negative8'15 and 97.9

Forward mutation

Mouse lymphoma L5 1 78Y 0.5, 1.5,5, 15, 50ng/ml Tk locus 30, 40, 50, 60, 70 jig/ml in methanol

97.8

Negative3'6

QA

Chromosomal aberration

Chinese hamster ovary cells

2.5, 15, 30 ug/ml, 20 h -S9 96.1 7, 35, 50 jig/ml, 2 h +S9, in dimethyl sulfoxide; harvesting 1 8 h later

Negative84

GLP& Allen et al. (1986a) QA

Sister chromatid exchange

Chinese hamster ovary cells

5, 10, 20, 25, 30 ng/ml -S9 96.1 20, 25, 30 jig/ml - S9 7, 20, 28, 35 ng/ml +S9 20, 28, 35 M.g/ml + S9 in dimethyl sulfoxide

Weakly positive6 Weakly positivef Toxic

GLP& Allen & Brooker (1983) QA

Unscheduled DNA synthesis

Human embryonic fibroblasts

10,40,70, 100, 130, 160, 97.8 190, 220 ng/ml in methanol

Negative8'8

QA

In vivo Micronucleus formation

Male and female CD-I mouse bone marrow

Single oral dose, 270, 540, 96.3 1 100 mg/kg bw in 1% w/w methylcellulose. Sampling at 24, 48, 72 h

Negative11

GLP& Allen et al. QAs (1986b)

Micronucleus formation

Male and female CD rat bone marrow

Two intraperitoneal injections of 6.25, 25, 100 mg/kg bw, 24 h apart, in corn oil. Sampling 6 h after second injection

95.7

Negative'

Dominant lethal mutation

Male CD1 mice

0,6,25, 100 mg/kg bw per day in corn oil for 8 weeks

NR

Negative)

McGregor & Riach(1983)

Everest & Cliffe (1980)

GLP& Cozens et al. (1980) QA

S9, exogenous metabolic activation system from 9000 x g fraction of rat liver; NR, not reported Positive control substances were used in all assays and gave the expected results. 8

With and without metabolic activation Antibacterial effects of the test material precluded evaluation of mutagenicity at 1000, 500 and in some cases 250 (J,g/plate. An initial test indicated that prochloraz was toxic to the cells, causing mortality at 158 |o.g/ml. No meaningful results were obtained at 60 and 70 Jig/ml, owing to toxicity. d Reduction of about 50% in mitotic index at highest doses e Dose-related, statistically significant increases in frequency at the highest dose in both studies, at which a reduction of about 40% in mitotic index was observed in a preliminary cytotoxicity test f Dose-related, statistically significant increases in frequency at three highest doses 8 Some evidence of toxicity at 100 ug/ml and marked toxicity at 1000 pig/ml; precipitation at 1000 jig/ml h At 48 and 72 h, some reduction in ratio of polychromatic:normochromatic erythrocytes in animals at 1088 mg/kg bw, indicating bonemarrow cytotoxicity. At this dose, 7/20 males and 7/19 females died. 1 In a previous study, a single intraperitoneal dose of 100 mg/kg bw elicited overt signs of toxicity which disappeared within 24 h; at 200 mg/kg bw, the effects persisted but no deaths occurred. The LDjo was 400-800 mg/kg bw. J Mating performance of treated males and subsequent pregnancy rates in untreated females were unaffected by treatment. The mean numbers of implantations, viable young, embryonic deaths and post-implantation losses were unaffected by treatment. At terminal autopsy of males, no obvious treatment-related macroscopic changes were seen, and the mean weights of the testes of treated males compared favourably with that of controls. b

c

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(e)

Reproductive toxicity (i)

Multigeneration studies

Rats The effect of prochloraz (purity, 96.2-97%) on reproductive function in two generations (two litters) of CrL:COBS CD (SD) rats was investigated in a study conducted in compliance with the principles of GLP (QA certification). The test compound was mixed into the diet at a concentration of 0, 38, 150 or 625 ppm, corresponding to mean achieved intakes of 3.1, 13 and 57 mg/kgbwperdayforF 0 malesand3.5,14and58mg/kgbwperdayforF 0 females;3.7,16and 70 mg/kg bw per day for FI males and 4.5,18 and 81 mg/kg bw per day for Fj females. The parent animals were exposed to the fungicide for 9 weeks before mating, and representative offspring were retained to form a second generation. The initial animals were then re-mated with different males and females, and their offspring were discarded when they were about 3 weeks of age, after macroscopic examination/?*^ mortem. Animals forming the second generation were mated about 8 weeks after selection and were also re-mated with different male and female pairings. At autopsy, selected organs from 10 male and 10 female Fj a weanlings, F2a weanlings and Fia adults in each group were weighed, and tissues from the 10 male and 10 female F la adults per group and from five male and five female Fi a and F2a weanlings per group were examined histologically. F0 males at the highest dietary concentration showed a prolongation of aggressive behaviour after re-housing after both matings; this effect was not seen among FI males. A slight prolongation was also seen in FO males at 150 ppm, after the first mating only. The clinical signs seen in FO females at the highest dietary concentration in late gestation and/or the perinatal period were hunched posture, walking on tiptoes, piloerection and pallor. About one-third of the animals were affected in each mating, although not necessarily the same individual each time. These effects were noted less frequently in the FI generation, the females being least affected at the second mating. Food intake before mating was not consistently related to dose but was generally lower than that of controls. Decreased mean body weight was noted in parental males and females of both generations at 625 ppm. At this dose, there was evidence of extended gestation (> 22 days) and parturition in some females, leading to a small number of deaths associated with dystocia in both generations; there were, however, no effects on pregnancy rates. There were no obvious effects on male mating performance. A few females at both matings of the F0 animals and at the first mating of the F la animals lost their litters in the immediate perinatal period. The mean litter size at birth was smaller in both generations at the highest dietary concentration. The mean percentage of pup loss at birth was greater than the control value for both matings of the F0 generation and for the first mating of the FI generation; however, none of the differences from control values was statistically significant. There was also a higher pup mortality rate at birth. At 625 ppm, impaired growth of the offspring to weaning was noted. The incidence of structural anomalies recorded at terminal macroscopic examination of the remaining F la and F2a pups and all F lb and F2b offspring provided no indication of any adverse effect of treatment. Increased mean liver weights were recorded in weanling (14%) and adult Fi a (13%) males at 625 ppm and in p2a weanling females at all three dietary concentrations (9, 9 and 14%, respectively at the low, intermediate and high concentrations). At the highest concentration, the mean thymus weight was lower than the control value in Fi a weanlings of both sexes, and the mean brain weight of Fj female weanlings was also lower than the control value. None of the histopathological changes in the tissues examined was considered to be attributable to treatment. Reproductive performance was affected only at the highest dietary concentration, which was toxic to the parent animals. The NOAEL was 3 8 ppm, equal to 3.1 mg/kg bw per day (Cozens etal., 1982).

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(ii)

Developmental toxicity

Rats In a study conducted in compliance with the principles of GLP (QA audit of final report), groups of 20 mated Charles River CD rats were given technical-grade prochloraz (purity, 96.2%) orally at a dose of 6,25 or 100 mg/kg bw per day on days l-20post coitum, day 1 post coitum being the day on which sperm were detected in a vaginal smear; 31 rats given the vehicle (10% aqueous acacia solution) served as controls. The doses used were determined in a preliminary study in which pregnant rats given the highest dose of 100 mg/kg bw per day showed increased salivation and liver enlargement. Body weight, food consumption and overt signs of toxicity were recorded throughout the study. The dams were killed on day 21 post coitum and necropsied. Their reproductive organs were removed and examined, corpora lutea were counted, and the placenta was weighed. Fetuses were weighed, dissected and examined; half were then decapitated and the heads subsequently sliced and examined. The carcasses of all fetuses were processed and the skeletons stained and examined. Maternal toxicity was seen at 100 mg/kg bw per day, manifested as increased salivation, reduced food consumption, lower body-weight gain (12%) and liver enlargement. At 25 mg/kg bw per day, increased salivation and a slightly higher liver weight were also observed. Dams gained marginally less body weight at both 6 (decrease of 6%) and 25 mg/kg bw per day (decrease of 5%). There were no macroscopic or microscopic findings attributable to treatment. The number of corpora lutea was similar in all groups. The highest dose appeared to be embryotoxic, as the litter size, implantation index and viability index were slightly reduced and the incidence of dead fetuses was marginally increased. In addition, the mean fetal weight was lower and the degree of calcification of sternebrae and vertebral arches was retarded. The mean weight of placentae from treated dams was higher than that of controls, and the difference was dose-related. There was no evidence of a teratogenic response. The NOAEL for maternal toxicity was 6 mg/kg bw per day, as the dose of 25 mg/kg bw per day was slightly toxic to the dams; the NOAEL for developmental toxicity was 25 mg/kg bw per day, on the basis of toxicity to embryos at 100 mg/kg bw per day (Beswick, 1980; Jackson, 1988b). Rabbits In a dose range-finding study of embryotoxicity and teratogenicity, groups of five mated female chinchilla rabbits were given technical-grade prochloraz (purity, 95.2%) by gavage on days 6-18post coitum at a dose of 0,25, 50,100 or 200 mg/kg bw per day. The vehicle, distilled water with 8% carboxymethlcellulose, was given to controls. On day 28 post coitum, all females were killed by cervical dislocation, and the fetuses were removed surgically. Does receiving 200 mg/kg bw per day lost weight on days 7-13 post coitum (days 2-7 of treatment). Thereafter, slight body-weight gain was noted but the rate was lower than that in the control group. The mean food consumption of does in this group during treatment was distinctly reduced, and this was confirmed by the compensatory increase in food consumption after treatment. The mean liver weights (absolute and relative) were dose-dependently increased (absolute, 5.5%, 10%, 22% and 36%, respectively, and relative, 8.1%, 17%, 24% and 42%, respectively). No treatment-related pathological changes were noted in the liver. The small number of females per group, the wide variations within and between the groups and the insufficient number of pregnant does (two) at the highest dose obviated a definitive conclusion, but no dose-related differences were seen in reproductive parameters. No malformed or anomalous fetuses were found in any group (Becker et al., 1989a).

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In another dose range-finding study of embryotoxicity and teratogenicity, groups of six mated female chinchilla rabbits were given technical-grade prochloraz (purity, 95.2%) by gavage on days 6-18post coitum at a dose of 0,200 or 250 mg/kg bw per day. The vehicle, distilled water with 1% carboxymethylcellulose, was given to controls. On day 28post coitum, all females were killed by cervical dislocation, and the fetuses were removed surgically. Four of the six does at 250 mg/kg bw per day died between days 10 and 13 post coitum. All four lost weight from the start of dosing. Somnolence, ataxia and dyspnoea were observed in one doe, and sedation, catalepsy and dyspnoea were seen in a second before death. At necropsy, macroscopic changes were observed in the livers of two does at the highest dose. The liverbody weight ratios varied from 4.4% to 7.0% (control mean, 2.2%), and histological examination of the livers showed moderate to severe increases in hepatocellular lipid in three of four animals and severe fatty changes with minimal-moderate multifocal necrosis in two of four animals. None of the does at 250 mg/kg bw per day had viable young at termination of the study. Of the two surviving does, one did not become pregnant, and the second lost its litter. Of the does at 200 mg/kg bw per day, three had viable young, two aborted and one did not become pregnant. The overall pregnancy rates (including animals that died) were 100% in controls, 83% at 200 mg/kg bw per day and 33% at 250 mg/kg bw per day. Animals at 200 mg/kg bw per day showed a mean weight loss during the first 5 days of dosing; thereafter, some recovered and there was a slight compensatory increase in weight gain after treatment, in comparison with concurrent control values. At this dose, food and water consumption was reduced throughout the dosing period. No macroscopic changes were observed in any doe surviving to termination on day 28 post coitum. The mean liver weights of all does surviving to termination were increased by 37% and 34% at 200 and 250 mg/kg bw per day, respectively, in comparison with concurrent control values, and the mean relative liver weights were increased by 53% and 46% at the two doses, respectively. No treatment-related histological changes were observed in the livers of does surviving to termination. In the three does at 200 mg/kg bw per day with viable young, there was no evidence of an adverse effect of treatment on reproductive or fetal parameters. These results indicate a marked maternally toxic effect, which, in some animals, either prevented implantation or caused early post-implantation loss, so that at termination on day 28 post coitum there was no evidence of implantation and the does were categorized as not pregnant. In other animals, pregnancy was maintained slightly longer, but, because of the stress to the maternal system, total resorption or abortion occurred. In those does that maintained pregnancy to term, there did not appear to be an obvious adverse effect on the fetuses. On the basis of the results of these two studies, a dose < 200 mg/kg bw per day was considered the appropriate highest dose for the main study (Becker et al., 1989b). In a study conducted in compliance with the principles of GLP (QA certification), groups of 16 mated female chinchilla rabbits were given technical-grade prochloraz (purity, 95.2%) at a dose of 10,40 or 160 mg/kg bw per day on days 6-18post coitum by gavage. A group of 17 mated rabbits receiving the vehicle alone (1% carboxymethylcellulose in distilled water) served as controls. The does were killed on day 28 post coitum, and the fetuses were removed surgically. Both does and fetuses were examined. Does at 160 mg/kg bw per day showed significantly reduced food consumption during the dosing period and reduced water consumption during the first 5 days. Weight gain was retarded during the first 5 days of dosing, but the differences from control values did not attain statistical significance. The liver weights (absolute and relative) were significantly increased. There was a slightly increased incidence of non-pregndnt animals and animals with total litter loss; these effects were considered treatment-related in view of the results obtained at higher doses in the dose range-finding studies. Does with viable young at termination had a significantly increased incidence of fetal resorption. None of the other reproductive parameters and none of the fetal PROCHLORAZ 141-182 JMPR 2001

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parameters appeared to be adversely affected by treatment. The NOAEL for both maternal and fetal toxicity was 40 mg/kg bw per day on the basis of maternal toxicity and embryotoxicity at 160 mg/kg bw per day (Becker et al., 1988). In a study previously evaluated by the Meeting (Annex 1, reference 41), groups of 15 New Zealand white rabbits were given prochloraz at a dose of 0,3,12 or 48 mg/kg bw per day by gavage on days 1-28 of gestation. Maternal toxicity was observed at the highest dose, as evidenced by a significant increase in liver weight, with liver discolouration. No evidence of embryotoxicity, fetotoxicity or teratogenicity was found (Palmer et al., 1980). (f)

Special studies (i)

Neurotoxicity

Rats Groups of five male and five female Boots-Wistar rats were given prochloraz (purity, 97.9%) at a single oral dose of 100 mg/kg bw, and control groups received the vehicle (10% aqueous acacia solution). A blood sample was obtained from the tail vein of each rat before dosing, and a further sample was collected by heart puncture immediately after the rats were killed 15 min, 90 min or 6 h after dosing. The samples were analysed for erythrocyte and plasma cholinesterase activity. Prochloraz did not affect cholinesterase activity in either sex at any interval after treatment (Smithson, 1979). Dogs A single oral dose of 20 mg/kg bw of prochloraz (purity, 97.9%) was given by gavage to three pairs of one male and one female beagle dogs; another pair was given an equivalent volume of the vehicle (10% aqueous acacia solution) and served as controls. A blood sample was taken from each dog immediately before and 0.75, 1.5, 3, 6 and 24 h after dosing and analysed for erythrocyte and plasma cholinesterase activity. There was no consistent difference in cholinesterase activity between treated and control animals (Morgan & Stobart, 1979). (H)

Mechanistic studies

Prochloraz (technical-grade, purity, > 95%; and analytical grade, purity, 99.5%) was investigated for effects on the initiation and promotion stages of hepatocarcinogenesis and for its ability to inhibit gap-junctional intercellular communication in the scrape-loading dye-transfer assay in IAR 20 rat liver epithelial cells. In the test for tumour promotion, female Sprague-Dawley rats were initiated with Af-nitrosodiethylamine (30 mg/kg, intraperitoneally) 24 h after partial hepatectomy to maximize any interaction between proliferation and the effects of prochloraz; 2 weeks later, they were given prochloraz on 5 days a week by gavage at 30 or 150 mg/kg bw per day for 10 weeks. Groups of uninitiated and/or non-hepatectomized rats were included. The control group was given the vehicle (corn oil) only. A positive control group was given phenobarbital (500 ppm) in drinking-water for 10 weeks, starting 2 weeks after initiation. In the test for tumour initiation, animals were given prochloraz at 150 mg/kg bw per day by gavage for 12 days, and a control group was given the vehicle according to the same schedule. On day 8 of treatment, all the animals were partially hepatectomized. After 2 weeks' recovery, promotion was started by giving all groups phenobarbital (500 ppm) in drinking-water for 10 weeks. The rats were killed 1 week after the promotion period. Altered hepatic foci were counted by quantitative stereology in liver sections stained for g-glutamyltranspeptidase and glutathione-S-transferase placental form (GST-P).

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Prochloraz inhibited cell-cell communication in the test system used. It had no effect on the initiation of g-glutamyltranspeptidase-positive foci, but significant increases in the percentage of liver tissue occupied by these foci and in the number of GST-P-positive altered hepatic foci per cm3 were recorded at the lower dose. The absence of effects on plasma transaminase activity and on body-weight gain in the prochloraz-treated animals suggests that the doses used in the study were not overtly hepatotoxic. A significantly increased relative liver weight was observed in rats given prochloraz at 150 mg/kg bw per day. The data suggest that prochloraz acts as a weak promoter of hepatocarcinogenesis but does not initiate the process (Kato et al., 1998). Eight pesticides, including prochloraz, were tested in a bioassay based on the induction of preneoplastic lesions in the liver. Rats were divided into three groups of 15 or 16 animals. Group 1 was given a single intraperitoneal injection of 7V-nitrosodiethylamine dissolved in 0.9% saline at 200 mg/kg bw to initiate hepatocarcinogenesis; after 2 weeks on a basal diet, they received a diet containing prochloraz (purity, 94.8%) at a concentration of 625 ppm for 6 weeks, being subjected to partial hepatectomy at week 3. Group 2 was given 7V-nitrosodiethylamine and subjected to partial hepatectomy in the same way but received the basal diet throughout the test period. Group 3 received only 0.9% saline and was then given a diet containing prochloraz from week 2, with partial hepatectomy at week 3. The experiment was terminated at week 8. Tumourpromoting potential was assessed by comparing the number and area (> 0.2 mm) of GST-Ppositive foci in the liver with those in controls given 7V-nitrosodiethylamine alone. Statistical analysis was performed with Student t test. The results were scored on the basis of the difference between the first two groups: a result was considered positive when there was an increase in both the number and area of foci atp < 0.05; a borderline result was an increase in either the number or the area of foci at/? < 0.05; and a negative result was no or a non-significant increase in either parameter. No treatment-related deaths were seen. Body-weight gain at week 8 was impaired in Group 1 (- 12 g; not reported for Group 3). A slight but statistically significant increase (p < 0.01) in relative liver weight was noted, a slight not statistically significant increase in the number of GSTP-positive foci per unit area of liver was found in sections from animals fed prochloraz (8.0 ± 3.3 foci/cm2 in Group 1,6.5 ± 2.2 foci/cm2 in Group 2), and a slight increase (p < 0.05) was found in the total area of GST-P-positive foci (0.71 ± 0.35 mm2/cm2 in Group 1, 0.44 ± 0.22 mm2/cm2 in Group 2). In Group 3, no GST-P-positive foci > 0.2 mm in diameter were seen. Thus, prochloraz is not an inducer of hepatocarcinogenesis (Cabral et al., 1991). (Hi)

Studies on metabolites of prochloraz

A comparison of the acute toxicity of prochloraz and its three main metabolites in plants is shown in Table 8. Groups of five male rats were given M1 or M2 (maj or plant metabolites) at a single oral dose of 3200 mg/kg bw or prochloraz at 1600 mg/kg bw. After dosing with prochloraz or M1, the overt signs of toxicity were similar and were seen within 1 h. Rats in both groups showed signs of central nervous system depression, adopted a hunched posture, had slowed respiration, piloerection and diarrhoea, were cool and limp to touch, tremorous, ataxic and had increased nasal exudate and salivation; in addition, rats given prochloraz had increased lachrymation. Animals had generally recovered within 7 days of dosing with Ml and within 9 days of dosing with prochloraz. Four rats given prochloraz died within 2 days, and one rat given M1 died on the day after dosing. At autopsy, they were found to have gastrointestinal irritation. After dosing with M2, one rat was inactive, adopted a hunched posture and had a wasted appearance, piloerection, staining around the eyes, a nasal exudate and urine staining around the genital region; these signs were seen 2-8 days after dosing. None of the rats died after treatment (Carter & Smithson, 1979a).

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Table 8. Studies of the acute toxicity of metabolites ofprochloraz administered orally Species

Strain

Sex

Vehicle

LD 50 (mg/kg bw) GLP orQA

Reference

N-Propyl-N-2-(2,4,6-trichlorophenoxy)ethylurea (Ml in Figure 2), major plant metabolite Rats Boots-Wistar Male 8% aqueous acacia solution > 3200

Carter & Smithson (1979a)

Dogs

Beagle

Male and female

Gelatin capsules

Watson et al. (1980a)

Dogs

Beagle

Male and female

Gelatin capsules

> 150 NOAEL: 50

Watson et al. (1980b)

N-Formyl-N'-propyl-N'-2-(2,4,6-trichlorophenoxy)ethylurea (M2 in Figure 2), major plant metabolite Rats Boots-Wistar Male 8% aqueous acacia solution >3200

Carter & Smithson (1979a)

Dogs

Beagle

Male and female

Gelatin capsules

> 1000

Watson et al. (1980a)

Dogs

Beagle

Male and female

Gelatin capsules

NOAEL: 250

Watson et al. (1980b)

2,4,6-Trichlorophenol, plant metabolite Rats Boots-Wistar Male

0.4% aqueous cellosize solution

>3200d

Dog

Gelatin capsule

NOAEL: 250

Beagle

Male and female

2-(2,4,6-Trichlorophenoxy)ethanol, rodent metabolite (metabolite 2 in Figure 1) Rats Boots-Wistar Male 0.4% aqueous cellosize solution 800-1600

GLP

Carter & Smithson (1979b) Watson et al. (1980b) Carter et al. (1979a)

Pairs of male and female dogs received Ml at single oral doses (in gelatin capsules) of 50 (day 1) and 150 (day 36) mg/kg bw, M2 at 500 (day 1) and 1000 (day 36) mg/kg bw, or imidazole at 50 (day 1), 50 (day 36) and 10 mg (day 50) mg/kg bw. The control group (one male and one female) received empty gelatin capsules. No effect was seen in dogs given Ml at 50 mg/kg bw or in males at 150 mg/kg bw, but the female at the latter dose appeared subdued and had slightly increased serum alkaline phosphatase activity the following day. M2 at 500 mg/kg elicited neutrophilia and increased serum alkaline phosphatase activity in the male, and M2 at 1000 mg/kg bw caused marginal increases in serum alkaline phosphatase activity and plasma glucose concentration in the female. The female treated with imidazole on day 1 vomited and retched within 1.5 h of dosing, but no toxic effect was seen in the male. On day 36, the male retched but no effect was seen in the female. On day 50, no toxic effect was elicited in either animal (Watson etal., 1980a). Pairs of male and female dogs were given a single oral dose of 250 mg/kg bw ofprochloraz, Ml, M2, imidazole or 2,4,6-trichlorophenol on day 1 of the study, while two males and two females received empty gelatin capsules and served as controls. On day 1, emesis and diarrhoea were observed in the pairs given prochloraz or Ml, and both dogs given imidazole showed emesis and increased salivation and appeared subdued. On day 2, the male given prochloraz had lost weight, and this dog and the female given Ml had slightly increased serum alkaline phosphatase activity; these parameters were normal by day 5. No effect of treatment with M2 was detected. The toxic signs elicited by imidazole appeared to be different from those induced by prochloraz and were further investigated. The pairs given prochloraz or imidazole were given a second oral dose of the same test article on day 29. The female given prochloraz and both dogs given imidazole salivated during administration of the dose and subsequently vomited. In addition, the female given imidazole had excessive diarrhoea, increased salivation and appeared subdued. On day 30, the dogs given imidazole had increased salivation, and the female had a very slightly increased blood urea nitrogen concentration and increased serum alkaline phosphatase activity. These biochemical parameters were normal on day 36, but the female again showed increased salivation when examined clinically. In order to investigate the different responses to doses of imidazole, one of the pairs of control dogs was given a single oral dose of imidazole at 250 mg/kg bw on day 43, and the other pair received empty gelatin capsules. Emesis and increased salivation were observed

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on day 43 in the naive pair given imidazole, but no other effect of treatment was detected. These signs appear to be the only consistent toxic effect of a single dose of imidazole. To investigate whether the instances of increased salivation seen in the first pair during administration of the dose and clinical examination were a conditioned response, these dogs were given a second dose consisting of empty capsules. No effect was elicited. No effect of treatment was seen at autopsy or histopathologically. No effect of treatment with 2,4,6-trichlorophenol was detected. Thus, M2 and 2,4,6-trichlorophenol were less acutely toxic than prochloraz; Ml elicited a toxic response at the same dose, but imidazole appeared to be slightly more toxic (Watson et al., 1980b). Groups of four or five male rats were given 2,4,6-trichlorophenol at a single oral dose of 3200 mg/kg bw. Ten controls received the vehicle. After treatment, a clear nasal exudate was observed in three rats, and diarrhoea and increased salivation were each seen in one animal; these signs occurred on the day of dosing. The rats had recovered by the following day, except for one rat that had a wasted appearance on day 6 (Carter & Smithson, 1979b). Groups of five male rats were given 2-(2,4,6-trichlorophenoxy)ethanol (metabolite 2 in Figure 1) at a single oral dose of 800, 1600 or 3200 mg/kg bw, and a group of controls received the vehicle. Overt signs of toxicity were seen within 30 min of dosing. Rats at all doses showed evidence of central nervous system depression, were tremorous and had piloerection, increased nasal exudate and urine staining around the genital region. In addition, some rats at 1600 or 3200 mg/kg bw were ataxic, convulsive and cool to touch, and some given 1600 mg/kg bw showed increased salivation and closed eyes. All rats given 3200 mg/kg bw and four given 1600 mg/kg bw died within 4 days of dosing, and one control lost condition and died 13 days after dosing. The survivors in the treated groups generally recovered within 7 days, although some remained in poor condition throughout the study. Autopsy of the decedents revealed macroscopic and microscopic evidence of gastrointestinal irritation. None of the findings was significant (Carter et al., 1979a). The mutagenic potential of metabolites of technical-grade prochloraz was assessed in assays for reverse mutation in S. typhimurium in the presence and absence of metabolic activation (Table 9). None of the compounds was mutagenic in this test.

3.

Observations in humans

Prochloraz was first synthesized in 1974 and has been produced on a commercial scale since 1980. Few adverse human effects shown to be due to prochloraz have been reported among Table 9. Results of assays for reverse mutation with metabolites and impurities of prochloraz Metabolite or impurity

Test object

Concentration

Purity Results (%)

W-Propyl-W-2-(2,4,6-trichlorophenoxy)ethylurea (Ml), major plant metabolite

5. typhimurium TA98, TA100,TA1535, TA1537, TA1538

16,31,62, 125, 250 ng/plate in dimethyl sulfoxide

NR

Negative"-11

Everest & Varley (1979)

7V-Formyl-jV'-propyl-,/V'-2-(2,4,6- S. typhimurium TA98, trichlorophenoxy)ethylurea (M2), TA100, TA1535, major plant metabolite TA1537, TA1538

62, 125, 250, 500, lOOOng/platein dimethyl sulfoxide

NR

Negativea,c

Everest & Varley (1979)

2-(2,4,6-Trichlorophenoxy)ethanol, rodent metabolite

S. typhimurium TA98, 5, 15,50, 150,500, 96% TA100,TA1535, 1500, 5000 fAg/plate TA1537;£. co/iCM881 in dimethyl sulfoxide WP2 uvr resistant and CM 891 WP2wvrA

Negativea

GLP Reference orQA

GLP Jones & Gant ( 1992a) &QA

S9, exogenous metabolic activation system from 9000 x g fraction of rat liver; NR, not reported Positive control substances were used in all assays and gave the expected results. • With and without metabolic activation b No evidence of mutagenic activity when assayed up to a concentration of 125 )il/plate; at higher concentrations, antibacterial activity precluded assessment of mutagenicity. c 1000 u,g/plate, maximum solubility

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persons involved in the synthesis, formulation and use (as recommended) of prochloraz and its formulations. The adverse effects comprised two cases of ocular irritation due to splashing of formulation concentrate, one case of cracked and blistered lips after blowing out a blocked spray nozzle, one case of skin rash in a person with multiple allergies who was accidentally soaked in spray and two cases of face and eye irritation after prolonged use of a prochloraz formulation (Davies, 1991).

Comments After oral administration to rats, prochloraz was rapidly and completely excreted in urine and faeces. There was a noticeable sex difference, faecal excretion predominating in females. After administration of a single oral dose of 5 mg/kg bw [14C]prochloraz to male and female rats with cannulated bile ducts, the radiolabel was recovered quantitatively, with no apparent sex difference. Prochloraz was well absorbed, a mean of 74% of the dose being recovered in the bile, urine, cage washings and carcass. Biliary excretion was the major route of elimination. After an oral dose of 5 mg/kg bw, the tissue concentrations were very low; only the liver contained > 0.1 mg/kg 96 h after dosing. By 96 h after a dose of 100 mg/kg bw, the concentrations in liver, kidneys, blood and plasma of animals of each sex and in the lungs and adrenals of females were > 1 mg/kg. The main metabolic pathway at both doses involved cleavage of the imidazole ring and initial loss of small fragments, to give Ml and M2, which, together with a considerable quantity of unchanged prochloraz, were the main compounds found in the faeces. Further metabolism yielded the phenoxyethylurea, which was excreted mainly in the faeces or further metabolized to the phenoxyethanol and then to the acid. These latter compounds were excreted mainly in the urine in free or conjugated forms, and trichlorophenoxyacetic acid was the main metabolite in urine. A small amount of this acid may be further metabolized to trichlorophenol, which was also excreted in the urine. A minor metabolic pathway involves aromatic hydroxylation. Prochloraz has low acute toxicity. The LD50 in rats treated orally was 1600-2400 mg/kg bw, and the main toxic effects were reversible central nervous system depression and gastrointestinal irritation. WHO (1999) has classified prochloraz as 'slightly hazardous'. The LDso after dermal application was > 2100 mg/kg bw in rats and > 3000 mg/kg bw in rabbits, and the LC50 in rats exposed by inhalation for 4 h was > 2.2 mg/1 of air, the highest achievable concentration. The compound was not irritating to the skin of rabbits after a 4-h exposure, was not irritating to the eyes of rabbits and did not sensitize the guinea-pig skin in a Magnusson and Kligman maximization test. In short-term studies in mice, rats and dogs, the liver was the principal target organ. Prochloraz is a potent inducer of the hepatic microsomal mixed-function oxidase system of rats and mice after oral administration. The spectrum of induction was similar to that caused by phenobarbital, with increased content and activity of cytochrome P450 enzymes. In a 14-day range-finding study in dogs given 40 mg/kg bw per day, serum alkaline phosphatase activity increased progressively from day 3 throughout treatment. The NOAEL for the increase in alkaline phosphatase activity at day 3 was 10 mg/kg bw per day. In 13-week studies, liver weights were increased in all three species; this response was considered to reflect induction of the hepatic mixed-function oxidase system. In mice and rats, hepatocyte enlargement was observed, with periportal fat infiltration and glycogen loss in mice. No histopathological changes were observed in the liver in dogs. In all studies, the effect was dose-related and showed partial reversal after a 4-week recovery period. Dogs also had decreased weights of the prostate and testis. The NOAELs were 6 mg/kg bw per day in mice and 2.3 mg/kg bw per day in dogs, but no NOAEL could be identified in rats, as at the lowest dose, 6 mg/kg bw per day, increased liver weights and occasional signs of intoxication (increased salivation, diarrhoea) were observed.

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In long-term studies in mice and rats and in a 2-year study in dogs, the liver was again the principal target organ. The NOAELs were 1.3 mg/kg bw per day in rats and 0.9 mg/kg bw per day in dogs; no carcinogenic effect was observed in rats. In the study in mice, an increased incidence of liver adenomas and carcinomas was found in both males and females at concentrations > 325 ppm. No significant difference from controls was found in the number of liver tumours in animals of either sex at 78 ppm, equal to 7.5 mg/kg bw per day, which was therefore the NOAEL. Prochloraz was hepatocarcinogenic in mice. A comprehensive range of studies of genotoxicity gave consistently negative results, except for a weakly positive response in a test for sister chromatid exchange in Chinese hamster ovary cells in vitro in both the presence and the absence of an exogenous metabolic activation system. The Meeting concluded that prochloraz is unlikely to be genotoxic. Investigation of the effects of prochloraz on the initiation and promotion stages of hepatocarcinogenesis in rats suggested that the substance acts as a weak tumour promoter but does not initiate the process. It was a weak, rodent-specific hepatocarcinogen, with a mode of action similar to that of phenobarbital. The Meeting concluded that the increased incidence of tumours observed in the liver was a threshold phenomenon that was species-specific, and that prochloraz was therefore unlikely to pose a carcinogenic risk to humans. In a two-generation study of reproductive toxicity in rats, reproductive performance was affected only at a concentration of 625 ppm in the diet, as indicated by total litter loss in a few females, reduced mean litter size at birth, higher pup mortality rates at birth and impaired growth of the offspring; furthermore, parental toxicity was observed. The NOAEL for parental and offspring toxicity was 38 ppm, equal to 3.1 mg/kg bw per day. Prochloraz was not teratogenic in either rats or rabbits. In a study of developmental toxicity in rats, a dose of 100 mg/kg bw per day was toxic in dams, embryos and fetuses. The NOAELs were 6 mg/kg bw per day for maternal toxicity and 25 mg/kg bw per day for developmental toxicity. In a study of developmental toxicity in rabbits, both maternal toxicity and embryotoxicity were seen at 160 mg/kg bw per day; the NOAEL for maternal and fetal toxicity was 40 mg/kg bw per day. Prochloraz did not affect plasma or erythrocyte cholinesterase activity in rats or dogs. In a comparative study of the acute toxicity of prochloraz and the plant metabolites Ml and M2 in rats treated orally, the signs of toxicity were qualitatively similar, but prochloraz was more acutely toxic than either of the metabolites. Trichlorophenol was also less acutely toxic than prochloraz. In dogs, M2 and trichlorophenol were less acutely toxic than prochloraz, and Ml elicited a similar toxic response at the same dose. Neither Ml nor M2 induced reverse mutation in S. typhimurium. Since the initial synthesis of prochloraz in 1974 and its commercial introduction in 1980, only a few cases of skin and eye irritation have been reported in humans heavily exposed to products containing prochloraz.

Toxicological evaluation The Meeting concluded that the existing database was adequate to characterize the potential hazards of prochloraz to fetuses, infants and children. The Meeting confirmed the ADI of 0-0.01 mg/kg bw established in 1983, on the basis of a NOAEL for effects on the liver of 0.9 mg/kg bw per day in a 2-year study in dogs, a NOAEL of 1.3 mg/kg bw per day in a 2-year study in rats and a safety factor of 100. The Meeting established an acute reference dose of 0.1 mg/kg bw, on the basis of a NOAEL of 10 mg/kg bw per day for effects on the liver at day 3 (increased serum alkaline phosphatase activity) in a 14-day study in dogs, and a safety factor of 100.

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Levels relevant to risk assessment Species

Study

Effect

NOAEL

LOAEL

Mouse

2-year study of toxicity and carcinogenicity3

Toxicity

78 ppm, equal to 7.5 mg/kg bw per day 78 ppm, equal to 7.5 mg/kg bw per day

325 ppm, equal to 33 mg/kg bw per day 325 ppm, equal to 33 mg/kg bw per day

38 ppm, equal to 1.3 mg/kg bw per day Carcinogenicity 625 ppm, equal to 28 mg/kg bw per dayc Parental toxicity 38 ppm, equal to 3 . 1 mg/kg bw per day Offspring toxicity 38 ppm, equal to 3.7 mg/kg bw per day 6 mg/kg bw per day Maternal toxicity Embryo- and fetotoxicity 25 mg/kg bw per day

150 ppm, equal to 5. 1 mg/kg bw per day

Carcinogenicity

Rat

2-year study of toxicity and carcinogenicity3

Multigeneration reproductive toxicity8 Developmental toxicityb

Toxicity

Rabbit

Developmental toxicityb

Dog

1 4-day study of toxicityb>d Toxicity 2-year study of toxicity3 Toxicity

3 b c d 6

Maternal toxicity 40 mg/kg bw per day Embryo- and fetotoxicity 40 mg/kg bw per day 10 mg/kg bw per day6 30 ppm, equal to 0.90 mg/kg bw per day

1 50 ppm, equal to 13 mg/kg bw per day 150 ppm, equal to 16 mg/kg bw per day 25 mg/kg bw per day 100 mg/kg bw per day 160 mg/kg bw per day 160 mg/kg bw per day 40 mg/kg bw per day6 135 ppm, equal to 4. 1 mg/kg bw per day

Diet Gavage Highest dose tested This study was used to establish the acute reference dose. Based on an increase in alkaline phosphatase activity at day 3

Estimate of acceptable daily intake for humans 0-0.01 mg/kg bw Estimate of acute reference dose 0.1 mg/kg bw Studies that would provide information useful for continued evaluation of the compound Further observations in humans Summary of critical end-points for prochloraz Absorption, distribution, excretion and metabolism in mammals Rate and extent of oral absorption Rapidly and well absorbed; mean of 74% at low dose (bile duct-cannulated rats) Dermal absorption Poor; < 2% in pigs Distribution Rapid and extensive. At high dose, liver, kidneys, blood, and plasma of males and females and lungs and adrenals of females had concentrations > 1 mg/kg 96 h after dosing. Potential for accumulation None Rate and extent of excretion Rapid and complete, significant sex difference, faecal excretion predominating in females (70% vs 59% in males at low dose). Urinary excretion: 65% in males, 41% in females at high dose.

PROCHLORAZ 141-182 JMPR 2001

176 Major pathway: cleavage of imidazole ring and initial loss of small fragments. Further metabolism yields the urea, which is excreted in faeces or further metabolized to the phenoxyethanol and then to the acid, which is excreted in urine in free and conjugated forms. A small amount may be further metabolized to the trichlorophenol.

Metabolism in animals

Minor metabolic pathway involves aromatic hydroxylation. lexicologically significant compounds

Prochloraz

Acute toxicity Rat, LD 50 , dermal Rat, LC50, inhalation Rabbit, skin irritation Rabbit, eye irritation Guinea-pig, skin sensitization (test method) Short-term studies of toxicity Target/critical effect

Lowest relevant oral NOAEL

Lowest relevant dermal NOAEL Lowest relevant inhalation NOAEL Long-term studies of toxicity and carcinogenicity Target/critical effect Lowest relevant NOAEL Carcinogenicity

Rat, LD5o, oral 1600 mg/kg bw > 2100 mg/kg bw >2.2mg/lofair(4h) Not irritating (4 h) Not irritating Not sensitizing (Magnusson and Kligman)

Liver: increased weight (rats, mice, dogs), hepatocyte enlargement (rats, mice), periportal fat infiltration and glycogen loss (mice) Prostate and testis: decreased weight (dogs) 10 mg/kg bw per day (dogs, 14 days, NOAEL based on increase in alkaline phosphatase activity at day 3) 2.3 mg/kg bw per day (dogs, 90 days) No data No data

Liver (mice, rats, dogs), prostate (dogs) 0.90 mg/kg bw per day (dogs), 1.3 mg/kg bw per day (rats) Not carcinogenic in rats Increased incidence of liver adenomas and carcinomas in mice No genotoxic potential

Genotoxicity Reproductive toxicity Reproduction target/critical effect Lowest relevant reproductive NOAEL Developmental target/critical effect Lowest relevant developmental NOAEL Developmental toxicity: 25 mg/kg bw per day (rats)

Decreased litter size, increased pup mortality rate, and impaired growth of pups at parentally toxic dose 3.1 mg/kg bw per day Embryo and fetotoxicity at maternally toxic dose Maternal toxicity: 6 mg/kg bw per day

Neurotoxicity/delayedneurotoxicity

No concern from other studies

Mechanistic studies

Potent inducer of hepatic microsomal monooxygenase system in rats and mice after oral administration, with spectrum of induction similar to that caused by phenobarbital Weak tumour promoter but not an initiator

Medical data

A few cases of skin and eye irritation after heavy exposure to products containing prochloraz

Summary

Value

Study

Safety factor

ADI Acute RfD

0-0.01 mg/kg bw 0.1 mg/kg bw

2 years, dogs and rats, toxicity 14 days, dogs, toxicity

100 100

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177

References Allen, J.A. & Brooker, P.C. (1983) Technical prochloraz: Frequency of sister chromatid exchange in Chinese hamster ovary cells cultured in vitro. Unpublished report No. TOX/86/173-135 from Huntingdon Research Centre & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Allen, J.A., Brooker, P.C., Birt, D.M. & McCaffrey, K. J. (1986a) Technical prochloraz: Metaphase chromosome analysis of CHO cells cultured in vitro. Unpublished report No. TOX/86/173-136 from Huntingdon Research Centre & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Allen, J.A., Proudlock, R. J. & Pugh, L.C. (1986b) Technical prochloraz: Mouse micronucleus test. Unpublished report No. TOX/86/173-132 from Huntingdon Research Centre & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Becker, H., Mueller, E., Vogel, W., Vogel, O. & Terrier, C. (1988) Embryotoxicity study (including teratogenicity) with prochloraz technical in the rabbit. Unpublished report No. PF-86.814 from Schering & RCC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Becker, H., Muller, E. & Vogel, W. (1989a) First dose range-finding embryotoxicity study (including teratogenicity) with prochloraz technical in the rabbit. Unpublished report No. RCC 067511 from Schering & RCC—Addendum 1 to Becker H., Muller E. & Vogel W. (1989), unpublished report No. PF-86.814. Submitted to WHO by Aventis CropScience SA, Lyon, France. Becker, H., Muller, E. & Vogel, W. (1989b) Second dose range-finding embryotoxicity study (including teratogenicity) with prochloraz technical in the rabbit. Unpublished report No. RCC 074891 from Schering & RCC—Addendum 2 to Becker H., Muller E. & Vogel W. (1989), unpublished report No. PF-86.814. Submitted to WHO by Aventis CropScience SA, Lyon, France. Beswick, A.M. (1980) The teratogenicity study of technical prochloraz in male and female rats. Unpublished report No. TX 80024 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Boardman, L.E. (1979) Plasma and tissue distribution studies in the rat following single and repeated oral doses of (3H)-BTS 40542. Unpublished report No. AX 79004 from Corning Hazleton& Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Cabral, R., Hoshiya, T., Hakoi, K., Hasegawa, R., Fukushima, S. & Nobuyuki, I. (1991) A rapid in vivo bioassay for the carcinogenicity of pesticides. Tumori,17, 185-188. Campbell, J.K. (1983) Residues of prochloraz in milk and tissues of a lacting goat fed straw containing residues of radiactive prochloraz. Unpublished report No. METAB/83/8 from FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Campbell, J.K. & Needham, D. (1980) Residues in milk and tissues of a goat dosed orally with 14C-BTS 44596 (major plant metabolite of prochloraz). Unpublished report No. AX 80033 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Campbell, J.K. & Needham, D. (1981) The quantitative excretion of BTS 9608 in the urine of rats after oral administration of prochloraz, BTS 46828 or BTS 44596. Unpublished report No. METAB/81/29 from FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Carter, O.A. & Smithson, A. (1979a) Acute oral toxicity of the metabolites BTS 44595 and BTS 44596 to male Boots Wistar rats. Unpublished report No. TX 79031 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Carter, O.A. & Smithson, A. (1979b) Acute oral toxicity to male Boots Wistar rats of the impurities BTS 42825 and BTS 45186. Unpublished report No. TX 79008 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Carter, O.A. & Smithson, A. (1979c) BTS 40542 impurity BTS 43026 acute oral toxicity to male Boots Wistar rats. Unpublished report No. TX 79022 (2nd edition) from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Carter, O. A., Lancaster, M.C. & Smithson, A. (1979a) Acute oral toxicity in male Wistar rats of the impurity BTS 3037. Unpublished report No. TX 79040 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Carter, O.A., Lancaster, M.C. & Smithson, A. (1979b) Acute oral toxicity in male Boots Wistar rats of the impurity BTS 39883. Unpublished report No. TX 79039 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Carter, O.A., Lancaster, M.C. & Smithson, A. (1979c) BTS 40542 impurity BTS 42140 acute oral toxicity study in male rats. Unpublished report No. TX 79038 (2nd edition) from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France.

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Challis, I.R. & Campbell, J.M. (1983) The effect of prochloraz on the hepatic mixed function oxidase system of the male mouse after oral administration. Unpublished report No. METAB/83/6 from FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Challis, I.R. & Greedy, C.L. (1988) The excretion and distribution of tissue residues in rats dosed orally with prochloraz at 5 mg/kg bodyweight. Unpublished report No. ENVIR/88/39 from Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Challis, I.R. & Greedy, C.L. (1989) The metabolism of prochloraz in the rat following oral dosing at 5 and 100 mg/kg bodyweight. Unpublished report No. ENVIR/88/42 from Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Chesterman, H., Perkin, C.J., Heywood, R., Street, A.E., Prentice, D.E., Woodhouse, R.N. & Majeed, S.K. (1981) Two-year toxicity study in beagle dogs of technical BTS 40542—Final report—Repeated dietary administration for 104 weeks. Unpublished report No. TOX/83/173-2 from Huntingdon Research Centre & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Colley, J., Wood, J.D., Heywood, R., Street, A.E., Prentice, D.E., Gibson, W.A. & Almond, R.H. (1982a) BTS 40542 (prochloraz) chronic toxicity and carcinogenicity study in rats by dietary administration—104 weeks (final report). Unpublished report No. TOX/82/173-8 from Huntingdon Research Centre & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Colley, J., Wood, J.D., Heywood, R., Street, A.E., Cherry, C.P., Gibson, W.A. & Almond, R.H. (1982b) BTS 40542 chronic toxicity and carcinogenicity study in rats by dietary administration (addendum 1 to final unpublished report No. TOX/82/173-8). Unpublished report No. TOX/82/173-20 from Huntingdon Research Centre & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Colley, J., Nunn, G., Heywood, R., Prentice, D.E., Buckley, P., Offer, J.M., Gibson, W.A., Almond, R.H. & Chauter, D.O. (1983) Prochloraz (BTS 40542) tumorigenicity study in mice by dietary administration (final report). Unpublished report No. TOX/83/173-23 from Huntingdon Research Centre & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Colley, J., Gopinath, C. & Offer, J.M. (1988) BTS 40542 tumorigenicity study in mice by dietary administration. Unpublished report No TOX/83/173-23 from Huntingdon Research Centre & Schering, Addendum 1 to Colley J., Gopinath C. & Offer J.M. (1988), unpublished report No. TOX/83/173-23. Submitted to WHO by Aventis CropScience SA, Lyon, France. Cozens, D.D., Reid, Y.J., Woodhouse, R.N., Almond, R.H., Anderson, J. & Ball, S.I. (1980) Dominant lethal gene assay of BTS 40542 (prochloraz technical) in the male mouse. Unpublished report No. TX 80077 from Huntingdon Research Centre & Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Cozens, D.D., Bottomley, A.M., Smith, J.A., Offer, J.M., Greyson, R.L., Gibson, W.A., Almond, R.H., Anderson, J. & Ball, I.S. (1982) The effect of BTS 40542 (prochloraz) on reproductive function of multiple generations in the rat. Unpublished report No. TOX/82/173 from Huntingdon Research Centre & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Cuthbert, J.A. & D'Arcy-Burt, K.J. (1984a) Technical prochloraz: Primary skin irritancy study in rabbits. Unpublished report No. TOX/84/173-124 from Inveresk Research International & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Cuthbert, J.A. & D'Arcy-Burt, K.J. (1984b) Technical prochloraz: Primary eye irritancy study in rabbits. Unpublished report No. TOX/84/173-125 from Inveresk Research International & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Davies, W.W. (1991) Medical statement on the human exposure to prochloraz. Schering (Unpublished, 3rd edition). Submitted to WHO by Aventis CropScience SA, Lyon, France. Dawson, J.R. (1989) The excretion and distribution of tissue residues of prochloraz in rats dosed orally with prochloraz at 100 mg/kg. Unpublished report No. ENVIR/88/51 from Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. D'Souza, G.A. (1995) Prochloraz (14C)-prochloraz BTS 40542 a biliary cannulation study in rats following a single oral dose of 5 mg/kg bodyweight. Unpublished report No. 194/122 from Corning Hazleton. Submitted to WHO by Aventis CropScience SA, Lyon, France. Everest, R.P & Cliffe, S. (1980) BTS 40542 (technical) Micronucleus assay in male and female CD rats of prochloraz. Unpublished report No. TX 80003 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Everest, R.P. & Varley, R. (1979) The in vitro bacterial mutagenicity testing of the plant metabolites BTS 44595 and BTS 44596. Unpublished report No. TX 79123 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France.

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Gale, E.P. (1980) 90 day oral toxicity study with prochloraz technical to male and female CD 1 mice. Unpublished report No. TX 80040 from Corning Hazleton & Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Gopinath, C. (1987) BTS 40542 chronic toxicity and carcinogenicity study in rats by dietary administration, histopathological reexamination of H & E stained sections of liver from all main group rats. Unpublished report No. TOX/82/173-8 from Huntingdon Research Centre & Schering, Addendum 2 to Colley J. et al. (1982), unpublished report No. TOX/82/173-8. Submitted to WHO by Aventis CropScience SA, Lyon, France. Hamilton, D.Y. (1978) The distribution of radiolabelled residues in the tissues of the pig following a single dermal application of BTS 40542. Unpublished report No. AX 78007 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Hounsell, I. A. & Ogle, A. (1987) Technical prochloraz: Acute dermal toxicity in the rat. Unpublished report No. TOX/86/173-142 from Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jackson, M. (1988a) BTS 40542: 13 week oral toxicity study with a 4 week off dose period. Unpublished report No. TOX/83/173-75 from Schering, Addendum 2 to Lancaster M.C. & Shaw J.W. (1979) unpublished report No. TX 79028. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jackson, C.M. (1988b) The teratogenicity study of technical prochloraz in male and female rats. Unpublished reportNo. TOX/83/173-97 from Schering, Addendum 1 toBeswick A.M. (1980), unpublished report No. TX 80024. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jackson, C.M. (1989) BTS 40542: 13 week oral toxicity study in dogs with a four week off dose period. Unpublished report from Schering, Addendum 5 to Lancaster, M.C., Morgan, H.E. & Stobart, J.E. (1979) unpublished report No. TX 79010. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jackson, G.C. & Hardy, C.J. (1987) Technical prochloraz: acute inhalation toxicity (4-hour exposure) in rats. Unpublished report No. TOX/87/173-167 from Huntingdon Research Centre & Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jones, E. & Gant, R.A. (1991) SN 604904: Bacterial mutation assay. Unpublished report No. TOX/91/173-267 from Huntingdon Research Centre & Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jones, E. & Gant, R.A. (1992a) BTS 3037: Bacterial mutation assay. Unpublished reportNo. TOX/92/173-279 from Huntingdon Research Centre & Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jones, E. & Gant, R.A. (1992b) BTS 39883: Bacterial mutation assay. Unpublished report No. TOX/92/173-278 from Huntingdon Research Centre & Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jones, E. & Wilson, L.A. (1988a) Analytical BTS 43298: Ames bacterial mutagenicity test. Unpublished report No. TOX/88/173-191 from Huntingdon Research Centre & Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jones, E. & Wilson, L.A. (1988b) Analytical BTS 42140: Ames bacterial mutagenicity test. Unpublished report No. TOX/88/173-193 from Huntingdon Research Centre & Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jones, E. & Wilson, L.A. (1988c) Analytical BTS 43026: Ames bacterial mutagenicity test. Unpublished report No. TOX/88/173-192 from Huntingdon Research Centre & Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Jones, E. & Wilson, L.A. (1988d) Analytical BTS 40348: Ames bacterial mutagenicity test. Unpublished report No. TOX/88/173-196 from Huntingdon Research Centre & Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Kato, Y., Flodstrom, S. & Warngard, L. (1998) Initiation and promotion of altered hepatic foci in female rats and inhibition of cell-cell communication by the imidazole fungicide prochloraz. Chemosphere, 37, 393-403. Keene, A.T. (1988a) BTS 40542:13 week oral toxicity in the mouse (macroscopic and microscopic observations). Unpublished report No. TOX/82/173-9 from Schering, Addendum 3 to Gale, E.P. (1980) unpublished report No. TX 80040. Submitted to WHO by Aventis CropScience SA, Lyon, France. Keene, A.T. (1988b) 90 day oral toxicity study with prochloraz technical in male and female beagle dogs 4 week off dose period (summary tables and statistical analysis). Unpublished report No. TOX/83/173-74 from Schering, Addendum 1 to Lancaster, M.C., Morgan, H.E. & Stobart, J.E. (1979) unpublished report No. TX 79010. Submitted to WHO by Aventis CropScience SA, Lyon, France.

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Kynoch, S.R., Lloyd, O.K. & Mallard, J.R. (1979) Acute dermal toxicity of BTS 40542 (prochloraz) to male and female rabbits. Unpublished report No. TX 79076 from Huntingdon Research Centre & Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Laignelet, L., Riviere, J.-L. & Lhuguenot, J.-C. (1992) Metabolism of an imidazole fungicide (prochloraz) in the rat after oral administration. Food Chem. Toxicol., 30, 575-583. Lancaster, M.C. (1980) 13-week oral toxicity study with prochloraz technical (BX 9/DM 2723) in male and female dogs with a four week off dose period, histopathological examination of the remaining tissues. Unpublished report No. TX 80034 from Boots, Supplement 1 to Lancaster, M.C., Morgan, H.E. & Stobart, J.E. (1979) unpublished report No. TX 79010. Submitted to WHO by Aventis CropScience S A, Lyon, France. Lancaster, M.C. (1982) BTS 40542: 13 week oral toxicity in the mouse: Histopathological examination. 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(1983) Technical prochloraz assessment of mutagenic potential in the mouse lymphoma mutation assay. Unpublished report No. TOX/83/173-22 from Inveresk Research International & FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. PROCHLORAZ 141-182 JMPR 2001

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Morgan, H.E. & Stobart, J.E. (1979) The effect of a single oral dose of prochloraz technical on the cholinesterase activity in Beagle dogs. Unpublished report No. TX 79029 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Morgan, H.E., Patton, D.S.G., Shepherd, G.M. & Stobart, J.E. (1977) Acute oral toxicity of prochloraz to female baboon. Unpublished report No. TX 78060 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Morgan, H.E., Patton, D.S.G., Shepherd, G.M. & Stobart, J.E. (1978) Acute oral toxicity of BTS 40542 to male and female beagle dogs. Unpublished report No. TX 78049 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Morgan, H.E., Lancaster, M.C., Patton, D.S.G. & Stobart, J.E. (1979) 14 day cumulative oral toxicity study with prochloraz technical in male and female beagle dogs. Unpublished report No. TX 79030 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Needham, D. (1980) The effect of dog gastric juice or plasma on prochloraz. Unpublished report No. AX 80011 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Needham, D. (1981) The excretion of (14C)-phenyl labelled BTS 44596 by male and female rats after a single oral dose. Unpublished report No. METAB/81/10 from FBC. Submitted to WHO by Aventis CropScience S A, Lyon, France. Needham, D. (1982a) The excretion and tissue residues of (14C)-prochloraz in male and female mice following a single oral dose of 100 mg/kg. Unpublished report No. METAB/82/32 from FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Needham, D. (1982b) The metabolism of prochloraz in the rat after oral administration. Unpublished report No. METAB/82/31 from FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Needham, D. (1983a) The effect of prochloraz on the hepatic mixed function oxidase system of the mouse when administered at 80,325 and 1300 mg/kg diet for up to 14 weeks. Unpublished report No. METAB/83/7 from FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Needham, D. (1983b) The effect of prochloraz on the hepatic mixed-function oxidase system of the male rat after oral administration. Unpublished report No. METAB/83/5 from FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Needham, D. (1997) The metabolism of prochloraz in the rat following oral dosing at 5 and 100 mg/kg bodyweight. Unpublished report No. ENVIR/88/42-Amendment to report from Challis, I.R. & Greedy, C.L. (1989) from AgrEvo. Submitted to WHO by Aventis CropScience SA, Lyon, France. Needham, D. & Campbell J.K. (1982) The excretion and tissue residues of (14C)-prochloraz in male and female dogs following a single oral dose of 18 mg/kg. Unpublished report No. METAB/82/30 from FBC. Submitted to WHO by Aventis CropScience SA, Lyon, France. Needham, D. & Challis, I.R. (1991) The metabolism and excretion of prochloraz, an imidazole-based fungicide, in the rat. Xenobiotica, 21, 1473-1482. Needham, D., Greedy, C.L. & Dawson, J.R. (1992) The profile of rat liver enzyme induction produced by prochloraz and its major metabolites. Xenobiotica, 22, 283-291. O'Donovan, M.R. & Smithson, A. (1978) Acute oral toxicity to male and female Boots Wistar rats of BTS 40348 (stages Ib intermediate). Unpublished report No. TX 78114 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Offer, J.M., Gopinath, C., Colley, J.C. & Cannon, M. W. J. (1992) Photomicrographic addendum to histopathology report BTS 145 BTS 40542 tumorigenicity study in mice by dietary administration. Unpublished report No. TOX/83/173-23 from Huntingdon Research Centre & Schering, Addendum 4 to Colley, J., Gopinath, C. & Offer, J.M. (1988), unpublished report No. TOX/83/173-23. Submitted to WHO by Aventis CropScience SA, Lyon, France. Palmer, A.K., Bottomley, A.M. & Billington, R. (1980) The effect of technical prochloraz on pregnancy of the New Zealand white rabbit. Unpublished report No. TX 80083 from Huntingdon Research Centre & Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Peto, R., Pike, M.C., Day, N.E., Gray, R.G., Lee, P.N., Parish, S., Peto, J., Richards, S. & Wahrendorf, J. (1980) Guidelines for simple sensitive significance tests for carcinogenic effects in long-term animal experiments. In: IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Suppl. 2, Longterm and Short-term Screening Assays for Carcinogens: A Critical Appraisal, Lyon, lARCPress, pp 311-346. Phillips, M.W.A. & Swalwell, L.M. (1989) The residues of prochloraz in the edible tissues of a cow following oral administration of prochloraz for 3 days at 1.5 mg prochloraz/kg bodyweight/day. Unpublished report No. ENVIR/89/24 from Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France.

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Reynolds, C.M.M. (1995) Prochloraz clearance of a single oral dose from rat tissue. Unpublished report No. TOX/94/173-321 from AgrEvo. Submitted to WHO by Aventis CropScience SA, Lyon, France. Reynolds, C.M.M. (1996) Prochloraz clearance of a single oral dose from rat tissue. Unpublished report No. TOX/94/173-321- 1st addendum from AgrEvo. Submitted to WHO by Aventis CropScience SA, Lyon, France. Riviere, J.-L. (1983) Prochloraz, a potent inducer of the microsomal cytochrome P450 system. Pestic. Biochem. Physiol., 19, 44-52. Sharp, D.W. (1982) Summary of prochloraz 2-year tumorigenicity study in mice. From FBC (unpublished). Submitted to WHO by Aventis CropScience SA, Lyon, France. Shaw, J.W. (1979) The delayed dermal sensitisation study of BTS 40542 in the female guinea pig. Unpublished report No. TX 79058 from Boots & Quintiles Toxicol. Submitted to WHO by Aventis CropScience SA, Lyon, France. Shaw, J.W. & Carter, O.A. (1976) Acute oral toxicity to male CD 1 mice of BTS 40542 prochloraz. Unpublished report No. TX 76093 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Shaw, J.W., Lancaster, M.C. & Smithson, A. (1979a) Acute oral toxicity study in Boots Wistar and CFY rats with BTS 40542 unformulated material. Unpublished report No. TX 79051 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Shaw, J.W., Crowley, J., Wilcox, K. & Lancaster, M.C. (1979b) BTS 40542: 13 week oral toxicity study in rats with a four week off dose period, quantitative assessment of liver cell size. Unpublished report No. TX 79128 from Boots, Supplement I to Lancaster, M.C. & Shaw, J.W. (1979) unpublished report No. TX 79028. Submitted to WHO by Aventis CropScience SA, Lyon, France. Shepherd, G.M., Smithson, A. & Stobart, J.E. (1996) Prochloraz technical impurities BTS 40348, BTS 41995, BTS 43298 Acute oral toxicity to male Boots Wistar rats. Unpublished report No. TX 78093 (3rd edition, original report 1978) from AgrEvo. Submitted to WHO by Aventis CropScience SA, Lyon, France. Smithson, A. (1979) BTS 40542 (prochloraz technical): the effect of a single dose on cholinesterase activity in male and female Boots Wistar rats. Unpublished report No. TX 79086 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Smithson, A. & Lancaster M.C. (1980) Acute intraperitoneal toxicity of BTS 40542 prochloraz to male CD rats. Unpublished report No. TX 80004 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Wason, S.M. (1991) SN 604904 (ROO1041): Rat acute oral toxicity study. Unpublished report No. TOX/91 /173265 from Schering. Submitted to WHO by Aventis CropScience SA, Lyon, France. Watson, J.E., Lancaster, M.C. & Robinson, A.J. (1980a) Acute oral toxicity in male and female beagle dogs of the plant metabolites BTS 19036, BTS 44595 and BTS 44596. Unpublished report No. TX 80021 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Watson, J.E., Lancaster, M.C. & Robinson, A.J., (1980b) Acute oral toxicity in male and female beagle dogs of the plant metabolites BTS 19036, BTS 44595, BTS 44596 and BTS 45186. Unpublished report No. TX 80010 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. WHO (1999) Recommended Classification of Pesticides by Hazard and Guidelines to Classification 1998-1999 (WHO/PCS/98.21/Rev. 1), Geneva, International Programme on Chemical Safety.Wilcox, P. (1978) BTS 40542 In vitro bacterial mutagenicity testing of pure and technical prochloraz. Unpublished report No. TX 78002 from Boots. Submitted to WHO by Aventis CropScience SA, Lyon, France. Woodhouse, R.N., Almond, R.H. & Ball, M.G. (1979) Validation of the method of analysis and determination of homogenicity and stability of BTS 40542 in rodent and dog diets. Unpublished report No. TX 79059 from Boots, Addendum 1 to Chesterman, H. et al. (1981), unpublished report No. TOX/83/173-2. Submitted to WHO by Aventis CropScience SA, Lyon, France.

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SPINOSAD First draft prepared by A. Bartholomaeus Chemicals and Non-prescription Medicines Branch Therapeutic Goods Administration, Canberra ACT, Australia

Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution and excretion Biotransformation Toxicological studies Acute toxicity Lethal dose Ocular and dermal irritation and dermal sensitization Short-term studies of toxicity Long-term studies of toxicity and carcinogenicity Genotoxicity Reproductive toxicity Multigeneration studies Developmental toxicity Special studies Neurotoxicity Mechanisms of lysosomal vacuolation Studies on metabolites Comments Toxicological evaluation References

183 184 184 184 187 187 187 187 187 189 207 213 213 213 215 217 217 218 219 219 222 224

Explanation Spinosad is the ISO approved name for a mixture of compounds formed as a fermentation product of the soil organism Saccharopolyspora spinosa. The mixture comprises approximately 10 related chemicals, with proteinaceous, carbohydrate and inorganic salt compounds derived from the fermentation process. Two closely related compounds, spinosyn A and spinosyn D, in a ratio of approximately 6:1 or 7:1, represent about 88% of the composition of spinosad and are responsible for most of its insecticidal activity. Spinosyn A and spinosyn D differ only in respect to substitution of a hydrogen by a methyl group at a position that is not metabolically labile. The remainder of spinosad is made up of a number of closely related spinosyns, which differ in the location of other minor substitutions at various sites around the molecule (Figure 1). Spinosad, an insecticide which acts by causing rapid excitation of the insect nervous system, is a new compound and has not previously be,en evaluated by JMPR.

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Figure 1. Structure ofspinosyn A and D

Evaluation for acceptable daily intake 1.

Biochemical aspects (a)

Absorption, distribution and excretion

Groups of five Fischer 344 rats of each sex were given a single dose of [14C]spinosyn A (purity, > 96%) by gavage as a suspension in aqueous 0.5% methylcellulose, at a dose of 10 or 100 mg/kg bw. Other groups were given 14 daily doses of unlabelled spinosyn A by gavage at 10 mg/kg bw, followed on day 15 by a single dose of [14C]spinosyn A at 10 mg/kg bw. All animals were killed 7 days after the last dose. The plasma concentration of radiolabel was followed for 72 h in two groups of three rats of each sex given a single dose of [14C] spinosyn A by gavage at 10 or 100 mg/kg bw. The time to the maximal plasma concentration (Cmax) was 1 h in both males and females at the lower dose and 6 h in males and 12 h in females at the higher dose. The time to half the Cmax was 6 h in males and 12 h in females at the lower dose and 12 h in males and 24 h in females at the higher dose. The distribution of radiolabel in tissues was therefore assessed in groups of three animals, at 6 and 12 h in males and 2 and 24 h in females at 100 mg/kg bw, and at 1 and 6 h in males and 1 and 12 h in females at 10 mg/kg bw. Radiolabel was determined in the adrenals, blood, bone, brain, heart, duodenum, gastrointestinal tract (including contents), gonads, kidneys, liver, lung, mesenteric lymph nodes, perirenal fat, skeletal muscle, skin, spleen, thymus and thyroid and in the carcass. Urine, faeces and expired CC>2 were also analysed for radiolabel. Tissues from animals given repeated doses were sampled 1 h after dosing and again at 6 h in males and 12 h in females. Biliary excretion through bile-duct cannulae was determined in three rats of each sex for 24 h after a single dose of [14C] spinosyn A by gavage at 10 or 100 mg/kg bw. The animals were killed 24 h after dosing, and radiolabel was determined in blood, skin and carcass and in collected urine, faeces and CC>2. The study complied with the requirements of GLP and OECD guideline 417. More than 90% of the radiolabelled dose was recovered in all groups. In animals killed 7 days after dosing, faecal excretion accounted for 85-88% of the administered dose in males and 81-82% in females, mostly within the first 24 h, while 6-10% of the dose was excreted in the urine. The radiolabel in tissues and the carcass accounted for < 3% of the dose. Faecal elimination was biphasic, the half-lives for the initial and terminal phases for males being 9 and 28 h at the lower dose, 14 and 25 h at the higher dose and 9 and 31 h after repeated doses, and those for females being 11 and 35 h at the lower dose, 29 and 42 h at the higher dose and 10 and 44 h after repeated doses.

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All tissues sampled at the Cmax and one-half the Cmax contained measurable radiolabel, but there was at least a 10-fold reduction in concentration between the sacrifice at one-half the Cmax and the final sacrifice for all groups. An increase in concentration in some tissues between these sacrifice times indicated that the distribution in tissues was incomplete. At the lower dose, the highest tissue concentrations (0.3-0.6 ng/g, expressed as equivalents) after 168 h were found in the kidneys, liver (males only), lymph nodes and fat; at the higher dose, the highest concentrations (7-13 ng/g in males and 0.8-41 jig/g in females, as equivalents) after 168 h were found in the kidneys, lymph nodes, fat and thyroid. In animals given repeated doses, 0.2-0.3 |iig/g remained in fat, kidney and lymph nodes (females only) after 168 h. Approximately equal amounts of radiolabel were recovered in the bile of male and female rats, with 41% (higher dose) and 44% (lower dose) in males and 41% (higher dose) and 38% (lower dose) in females. Male and female rats excreted similar amounts in the faeces (20-23%). The radiolabel recovered in bile, faeces, urine, exhaled CO2, skin and remaining carcass accounted for > 90% of that administered in both sexes at both the lower and higher dose. On the basis of the radiolabel detected in urine and bile, 70-80% of both doses was absorbed in both sexes. Given that no bile would be eliminated in the faeces of these animals, the approximately 20% of the dose recovered from faeces represents unabsorbed spinosyn A. The overall elimination rates were rapid, as 77-91% and 57-83% of the radiolabel in males and females, respectively, was recovered in excreta within 24 h and < 3% of the dose was found in tissues and carcass by 168 h after dosing. A proposed metabolic pathway for spinosyn A and D is provided in Figure 2 (Domoradzki et al., 1995). Figure 2. Proposed metabolic pathway for spinosyn A and D

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The absorption, distribution and elimination of radiolabel were studied in groups of three female Fischer 344 rats given [14C]spinosyn A (purity, 97%) at 10 mg/kg bw per day as a suspension in aqueous Methocel for 3 or 7 days and killed 1 or 7 days or 1, 7,14 or 21 days after the last dose. The study was conducted in accordance with the requirements of GLP and OECD guideline 417. The mean total recovery of radiolabel was 87-93%. Most of the recovered radiolabel was excreted in urine (4-6%) and faeces (74-87%) during the first 24 h after administration. The concentrations of residues in tissues declined rapidly on cessation of dosing and were generally below the limit of detection 21 days after the end of the 7-day dosing period. After both the 3-day and the 7-day dosing periods, the total concentration of tissue residues declined to < 0.5% of the administered dose within 24 h. Although the concentration in thyroid 24 h after cessation of dosing for 7 days (2 (ig/g of tissue expressed as equivalents) was not as high as in many other tissues, it declined more slowly, remaining at approximately 0.5 (iig/g of tissue at 7 days and 0.25 |ig/g of tissue at 14 days after cessation of dosing, while the concentrations in other tissues were < 0.1 pig/g of tissue 14 days at this time (Thalaker, 1996). Groups of five Fischer 344 rats of each sex were given a single dose of [14C]spinosyn D (purity, 95.6%) at 100 mg/kg bw by gavage as an aqueous suspension in 0.5% methylcellulose ether, and the concentration of radiolabel was determined in faeces and urine collected for 7 days, expired CO: collected for 72 h and in the adrenals, bone, brain, duodenum, fat, gastrointestinal tract (plus contents), gonads, heart, kidneys, liver, lungs, skeletal muscle, spleen, skin, thymus, thyroid, mesenteric lymph nodes, blood and carcass at terminal sacrifice 168 h after dosing. The study was conducted in accordance with the requirements of GLP and OECD Guideline 417. More than 90% of the administered dose was recovered. Faecal excretion accounted for 84% of the administered dose in males and 92% in females, with 68-73% recovered within the first 24 h. Urinary excretion accounted for 4.9% of the dose in males and 2.8% in females. Spinosyn D was eliminated via the faeces and urine in a biphasic manner, the mean half-lives being 6 h for the initial and 30 h for the terminal phases of faecal excretion and 5 and 33 h for urinary excretion. Less than 0.05% of the radiolabel was recovered as exhaled CO2. At terminal sacrifice, the radiolabel in tissues and carcass accounted for < 1 % of the dose in both males and females, and the final cage wash accounted for < 3% of the dose. The highest concentrations of radiolabel were detected in fat, liver, kidneys and mesenteric lymph nodes (Mendrala et al., 1995a). Three male Fischer 344 rats were given a single dose of [14C]spinosyn D (purity, 95.6%) by gavage in an aqueous suspension in 0.5% methylcellulose ether at approximately 100 mg/kg bw, and bile (from a bile-duct cannula), faeces, urine and CO: were collected for 24 h. The rats were then killed, and the concentration of radiolabel remaining in the tissues and carcass was determined. The study was conducted in accordance with the requirements of GLP and OECD Guideline 417. No overt signs of toxicity were reported. About 95% of the radiolabel was recovered. The proportion of the administered dose recovered in excreta was 22-55% in faeces, 28-40% in bile, 3% in urine and < 1% in expired CO: or in the cage wash. At sacrifice, the tissues and carcass contained about 21 % of the radiolabelled dose. Given that no bile would be eliminated in the faeces of these animals, the authors argued that the approximately 34% of radiolabel excreted in faeces represented unabsorbed spinosyn D. Oral absorption was estimated to account for > 70% of the administered dose (Mendrala et al., 1995b). [14C]Spinosyn A in dipropylene glycol was applied at a dose of 50 mg/kg bw (10 mg/cm2) to the shaved skin of Fischer 344 rats under an occlusive dressing for 24 h, and the animals were killed. The study was conducted in accordance with the requirements of GLP and the Pesticide Assessment Guidelines of the USA's Environmental Protection Agency (Section 85-1). Approximately 94-95% of the administered dose was recovered, but < 1% was absorbed, as

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determined from recovery in urine, faeces, carcass, tissues and expired air. A second group was similarly treated, but, after 24 h of treatment, the administration site was washed and then reoccluded with fresh bandaging for 120 h, at which time the animals were killed. About 2% of the applied dose was absorbed, and 93-94% was recovered (Domoradzki & Shabrang, 1996). (b)

Biotransformation

In the study of Domoradzki et al. (1995), described above, conjugation with glutathione, either directly or after O or N demethylation, was identified as the major path of metabolism. Glutathione and cysteine conjugates of spinosyn A and of O-demethylated spinosyn A were tentatively identified as metabolites in faeces, as well as unconjugated O-demethylated spinosyn A. The biliary and urinary metabolites were tentatively identified as glutathione conjugates of spinosyn A and of O-demethylated spinosyn A. The metabolites identified in liver were the glutathione conjugates of spinosyn A and of O-demethylated spinosyn A. Unconjugated O- and 7V-demethylated spinosyn A were identified in the liver, lung, kidney, thyroid and plasma. In the study of Mendrala et al. (1995a), described above, about half of the administered dose was rapidly eliminated as unchanged spinosyn D and its cysteine conjugate in the faeces of both male and female rats. About 3% of the dose recovered in faeces was identified as an Ndemethylated metabolite of spinosyn D and its glutathione conjugate. A cysteine conjugate of spinosyn D was tentatively identified as the main faecal metabolite, accounting for 9-12% of the radiolabelled dose. The authors proposed that the cysteine conjugate was formed from the metabolism of glutathione conjugates by gut microflora, which is a reasonable hypothesis. The glutathione conjugates of spinosyn D and of JV-demethylated spinosyn D were tentatively identified in urine and faecal specimens from both sexes. A number of minor metabolites were isolated from urine and faeces but were not identified as there was insufficient material (< 3% of the dose). In the study of Mendrala et al. (1995b), described above, metabolites were isolated and identified from pooled bile samples collected at intervals of 2-4 h and 6-8 h. The metabolism of spinosyn D followed essentially the same pathway as that of spinosyn A (Figure 2). Conjugation with glutathione, secretion into the bile and excretion in the faeces were identified as the main routes of metabolism and elimination. Less than 0.03% of the dose was eliminated as unchanged spinosyn D. The main biliary metabolite of spinosyn D was tentatively identified as its glutathione conjugate. Other metabolites identified were the glutathione conjugates of 0- and TV-demethylated spinosyn D. Several minor metabolites were isolated in the bile. 2.

Toxicological studies (a)

Acute toxicity (i)

Lethal dose

The acute toxicity of spinosad is summarized in Table 1. (ii)

Ocular and dermal irritation and dermal sensitization

In a study conducted in accordance with GLP requirements and a modification of OECD guideline 404 (higher dose and duration of exposure), groups of five New Zealand white rabbits of each sex received an application of spinosad (purity, 88%) at 5000 mg/kg bw on intact skin under a semi-occlusive dressing for 24 h. After removal of the dressing and any residual test compound, the application site was assessed for signs of irritation at 1 h and then daily for 14 days.

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Purity

% A/% D (%)

Route

Vehicle

LD5o/LC50 (mg/kg

Reference bw or mg/1 air)

Mouse

CD-I

87.9

NR

Oral

0.5% aqueous methyl cellulose

>5000

Gilbert etal. (1994)

Mouse

CD-I

88.0

NR

Oral

0.5% aqueous methyl cellulosev

Males, 6100 Females, 7100

Gilbert &Yano( 1996)

Rat

Fischer 344

87.9

NR

Oral

0.5% aqueous methyl cellulose

Females, > 5000 Males, < 5000

Gilbertetal. (1994)a

Rat

Fischer 344

88.0

NR

Oral

0.5% aqueous methyl cellulose

Males, > 7500 Females, 5300

Gilbert & Yano ( 1996)

Rat Rat Rabbit

Fischer 344 78.2 Fischer 344 88.0 New Zealand 87.9 white

NR

10% aqueous acacia Oral Inhalation8 Water Dermal

> 2000 (no deaths) >5.2 > 2000 (no deaths)

Wright etal. (1992a) Wolff etal. (1992) Gilbert (1994a)

Rabbit

New Zealand 88.2 white

NR

Dermal

> 5000 (no deaths)

Gilbert (1994b)

Rabbit

New Zealand 87.9 white

NR

Dermal

>5000 (no deaths)

Laska etal. (1992)

Species Spinosad

Water

Spinosyn A and D

Rat

Fischer 344

96.3

46.1/50.2

Oral

0.5% aqueous methyl cellulose

Males, 4400 Females, > 5000

Stebbins & Brooks (1999a)

Rabbits

New Zealand 96.3 white

46.1/50.2

Dermal

0.5% aqueous methyl cellulose

>5000

Stebbins & Brooks 1999b)

All studies complied with the requirements of GLP and the respective OECD or USA Environmental Protection Agency guidelines. NR, not reported a In a previous study (details not provided), there were no deaths at 2000 mg/kg bw. In this study, four of five males died. By combining the results of this study with those of Wright et al. (1992a), Dow calculate an LD50 of 3700 mg/kg bw. b Median equivalent aerodynamic diameter, 2.96 urn, with a geometric standard deviation of 2.67 Jim

A gross pathological examination was conducted on all animals. There were no deaths, clinical signs of toxicity or dermal irritation during the study, and all animals gained weight normally. There were no gross pathological lesions attributed to treatment with spinosad (Laska et al., 1992). In a study conducted in accordance with GLP requirements and OECD guideline 404, spinosad (purity, 87.9%) was applied to the intact skin of New Zealand white rabbits at a dose of 500 mg moistened with water under a semi-occlusive dressing for 4 h. No dermal irritation was seen (Gilbert, 1994b). No deaths, clinical signs of toxicity or dermal irritation and no gross pathological lesions attributed to treatment were seen in groups of five New Zealand white rabbits of each sex that received an application of spinosad (purity, 88%) at 5000 mg/kg bw on intact skin under a semiocclusive dressing for 24 h and followed up for 14 days. The study complied with GLP and was conducted in accordance with OECD guideline 404 (Laska et al., 1992). In groups of five New Zealand white rabbits of each sex that received a dermal application of a mixture of 46.1% spinosyn A and 50.2% spinosyn D at 5000 mg/kg bw on intact skin under a semi-occlusive dressing for 4 h and followed up for 72 h, there were no deaths, clinical signs of toxicity or dermal irritation and no gross pathological lesions attributed to treatment. The study complied with GLP and was conducted in accordance with OECD guideline 404 (Stebbins & Brooks, 1999c).

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Administration of 100 mg of spinosad (purity, 87.9%) into one eye of each of six New Zealand white rabbits produced conjunctival redness (average Draize score, 1.7) and chemosis (average score, 1.3) in all treated eyes within 1 h, which resolved in all but one animal (redness with a score of 1) by 24 h and in the remaining animal by 48 h. Spinosad is a slight eye irritant. The study complied with GLP and was conducted in accordance with OECD guideline 405 (Gilbert 1994c). Administration of 100 mg of a 50:50 mixture of 46.1% spinosyn A and 50.2% spinosyn D to one eye of each of three New Zealand white rabbits produced slight conjunctival redness (average Draize score, 1), slight chemosis (average score, 1) and slight discharge (average score, 1.3) in all treated eyes at 1 h, which resolved in all but one animal (redness and chemosis with scores of 1) by 24 h and in the remaining animal by 48 h. The test substance was a slight eye irritant. The study complied with GLP and was conducted in accordance with OECD guideline 405 (Stebbins & Brooks, 1999d). No evidence of delayed contact hypersensitivity was seen in Hartley albino guinea-pigs treated with with spinosad (purity, 87.9%) by the Buehler method, with induction and challenge doses of 400 mg. The sensitivity of the method was confirmed in a positive control group. The study complied with GLP and was conducted in accordance with OECD guideline 406 (Gilbert, 1994d). In a study of maximization with spinosad (purity, 88%) in female Hartley guinea-pigs, 0.1 ml of 0.5% spinosad and 0.1 ml of a 1% emulsion of spinosad in Freund complete adjuvant were injected subcutaneously at two sites on opposite sides of a shaved area of the scapula region on test day 1 for induction. Sodium lauryl sulfate (10% in vaseline) was applied to the test area on day 6, and, on day 7,0.2 ml of spinosad was applied to the test area and covered with an occlusive dressing for 48 h. After 3 weeks, 0.1 ml of spinosad was applied to a shaved area of the abdominal lateral region and maintained under an occlusive dressing for 24 h. There was no evidence of skin sensitization. A concurrent positive control group treated with dinitrochlorobenzene gave appropriate responses. The study complied with GLP and was conducted in accordance with OECD guideline 406 (Shibata, 1996). In a study of skin sensitization with the Buehler method in male Hartley guinea-pigs, a 50:50 mixture of 46.1 % spinosyn A and 50.2% spinosyn D was applied at a dose of 0.4 g moistened with 0.5% methylcellulose solution three times 1 week apart for induction. The animals were challenged with the same preparation. There was no evidence of skin sensitization. The study complied with GLP and was conducted in accordance with OECD guideline 406 (Stebbins & Brooks, 1999e). (b)

Short-term studies oftoxicity Mice

In a study conducted in accordance with the principles of GLP and OECD guideline 408, groups of 10 CD-I mice of each sex were given diets containing spinosad (purity, 77.6%) at a concentration of 0, 50, 150, 450 or 1200 ppm for 3 months, equal to 0, 6, 18, 57 and 110 mg/kg bw per day for males and 0,8,23,72 and 140 mg/kg bw per day for females. Toxicity was assessed by determining clinical signs at least daily, body weight at least weekly, haematological endpoints (clotting parameters, erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, mean haemoglobin concentration, mean corpuscular volume and

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mean corpuscular haemoglobin concentration) and clinical chemical parameters (alanine and aspartate aminotransferase activity and albumin, globulin, total protein, bilirubin, cholesterol, triglyceride, glucose, creatinine and urea nitrogen concetrations) before sacrifice, gross and histopathological appearance (adrenals, heart, prostate, aorta, Harderian gland, lachrymal gland, bone, kidneys, skin, bone marrow, brain, liver, lungs, spleen, lymph nodes, epididymides, muscle, gonads, eyes, thymus, peripheral nerve, thyroid, gall-bladder, trachea, urinary bladder, uterus, femur and joint, pituitary, tongue, parathyroid, mammary glands and spinal cord) and the weights of the kidneys, liver, heart, spleen, ovaries, testes and brain. Samples of liver, kidney and lung from three animals of each sex per group were examined ultrastructurally. Three males and two females at 1200 ppm died before day 44 due to hepatic necrosis, and the remaining animals at this dose lost approximately 25% of their initial body weight, were cachetic and had mild to moderate microcytic, hypochromic anaemia and marked neutrophilic leukocytosis. Their neutrophils had cytoplasmic basophilia and nuclear hypersegmentation, indicating degeneration and prolonged circulation. Increased alkaline phosphatase activity (by three times that of controls in males and 2.5 times in females), alanine aminotransferase activity (by 15 times in males and 11 times in females), aspartate aminotransferase activity (by eight times in males and five times in females) and globulin concentration (by 12% in males and 41% in females) were seen, with lower albumin concentrations (by 30% in the two sexes). Males had decreased glucose (by 50%), bilirubin (by 33%), cholesterol (by 25%) and triglyceride concentrations (by 55%), and females had decreased glucose (by 60%) and bilirubin concentrations (by 35%). The groups at 1200 ppm were discontinued on day 44, and all animals were necropsied. The clinical signs in animals at 50 and 150 ppm were similar to those seen in controls, but at higher concentrations rough or oily coats, thinness, rapid respiration, hypoactivity, hypothermia, ventral and perineal soiling, alopecia and swollen tail were observed commonly. Transient but statistically significant reductions in body weight and body-weight gain occurred in males at 450 ppm, the terminal body weight being 92% that of controls. Also at this dietary concentration, statistically significant decreases were seen in haemoglobin (-22%), packed cell volume (-11%), albumin concentration (-10%) and lymphocyte count (-3 3 %) and increases in alkaline phosphatase activity (+36%) in males; a statistically significant increase in neutrophil count (+72%) in females; and a statistically significant increase in aspartate aminotransferase activity (twice control value in both sexes) and statistically significant decreases in mean corpuscular volume (-8% in males, -10% in females) and mean corpuscular haemoglobin (-10% in males, -11% in females). Significantly increased absolute and relative weights of the liver were seen in both sexes (0.04% in controls, 0.05% at 450 ppm) and relative weight of the spleen in females at 450 ppm (23 mg/10 g in controls, 37 mg/10 g at 450 ppm). Treatment-related gross alterations were seen only in animals at 1200 ppm and consisted of altered appearance of the liver (eight males, nine females, described only as 'whole tissue alteration') and kidneys (six males, three females, 'whole tissue alteration'), adhesions (two males, five females) and lesions of the liver (nine mice of each sex), enlarged spleen (four males, eight females) and enlarged lymph nodes (10 mice of each sex). The term 'whole tissue alteration' was used to group a number of gross pathological observations affecting an entire tissue, including alterations in colour, texture and appearance. Gross, microscopic and ultrastructural examination revealed extensive effects in a wide range of tissues, most notably vacuolation. Histological alterations observed only at 1200 ppm consisted of: acute necrotizing and chronic inflammation of the renal capsule, acute capsular inflammation and severe multifocal necrosis of the liver, granular vascular leukocytosis and vacuolation of cardiac myocytes, intramural histiocytes and macrophages, acute diffuse interstitial inflammation of the lung, acute diffuse inflammation of the spleen, acute inflammation and severe necrosis of the lymph node, atrophy of the thymus, lymphoid vacuolation of the ileum and vacuolation of the pituitary. The incidences of other histopathological lesions occurring at concentrations other than that terminated prematurely are summarized in Table 2. Electron

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191 Table 2. Histological alterations in mice given spinosad in the diet for 90 days Lesion and intensity

(males/females)

Concentration in diet (ppm)

0

50

150

450

1200

Kidney Multifocal cortical tubular cyst: Slight to moderate Cortical multifocal tubular regeneration: Slight to mbderate Cortical tubular vacuolation: Minimal to marked

0/0 0/0 0/0

0/0 0/0 0/0

0/0 0/0 1/0

0/1 7/1

4/0 2/0

10/2

10/10

Liver Centrilobular cytomegaly: Slight Focal/multifocal granuloma: Minimal to slight Acute multifocal inflammation: Slight to moderate Centrilobular hepatocellular vacuolation: Minimal to slight Diffuse hepatocellular vacuolation: Minimal to slight Moderate to marked Vacuolation, Kupffer cell: Minimal.

0/0 0/0 0/0 0/0 0/0 0/0 0/0

0/0 0/0 0/0 0/0 0/0 0/0 0/0

1/0 0/0 0/0 0/0 0/1 0/0 0/0

6/0 0/4 0/1 8/6 0/3 0/0 0/4

2/0 0/0

Lung Multifocal alveolar macrophages: Minimal to moderate

0/0

0/0

0/0

10/8

10/9

Spleen Lymphoid vacuolar change: Minimal to slight Lymphoid vacuolar change: Moderate Haematopoiesis: Moderate Multifocal lymphocytic necrosis: Slight

0/0 0/0 0/0 0/0

0/0 0/0 0/0 0/0

4/0 0/0 0/0 0/0

8/7 1/0 2/0

4/4 6/5 9/8 6/5

Lymph node Lymphoid vacuolar change: Slight to moderate Histiocytosis: Slight to moderate Multifocal lymphocytic necrosis: Slight to moderate

0/0 0/0 0/0

0/0 0/0 0/0

0/2 1/0 0/2

111

7/6 7/7

5/6 8/6 3/5

Thymus Lymphoid vacuolar change: Slight to moderate Multifocal lymphocytic necrosis: Slight to moderate

0/0 0/0

0/0 0/0

0/0 0/0

3/5 3/5

0/0 0/0

Pancreas Diffuse acinar atrophy: Slight Diffuse acinar vacuolation: Slight to moderate

0/0 0/0

0/0 0/0

0/0 0/0

2/0 10/7

5/0 9/5

Tongue Chronic multifocal inflammation: Slight Multifocal regeneration, muscular layer: Minimal to slight Multifocal vacuolation: Slight

0/0 0/0 0/0

0/0 0/0 0/0

0/0 0/0 0/0

2/2 2/4 1/2

3/5 1/1 6/6

Stomach Multifocal glandular dilation: Slight to minimal Multifocal glandular dilation: Moderate to marked Histiocytosis: Minimal to moderate Mucosal hyaline droplets: Slight to moderate Acute diffuse mucosal inflammation: Minimal to moderate Chronic diffuse mucosal inflammation: Slight Multifocal mucosal mineralisation:Minimal to slight Multifocal mucosal necrosis: Minimal to slight

2/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0

3/1 0/0 0/0 0/0 0/0 0/0 0/0 0/0

5/3 0/0 0/0 0/0 0/0 0/0 4/3 1/0

3/4 7/5 4/2 5/4 4/4 5/3 6/8 4/6

0/0 7/6 4/1 7/6 1/2 6/8 3/4

Ovary Vacuolation: Moderate to marked

0

0

1

9

9

Oviduct Mucosal vacuolation: Slight to marked

0

0

0

7

8

Uterus Submucosal histiocytosis: Minimal to moderate Mucosal vacuolation: Slight to marked

0 0

0 0

0 0

5 9

7 8

Cervix Vacuolation: Slight to marked

0

0

0

4

5

Vagina Vacuolation: Slight to marked

0

0

0

3

7

Epididymis Mucosal vacuolation: Slight to moderate

0

0

0

1

10

Skeletal muscle Multifocal degeneration: Slight Multifocal regeneration: Slight

0/0 0/0

0/0 0/0

0/0 0/0

3/1 3/3

5/4 5/3

Bone marrow Granulocytic hypercellularity Multifocal necrosis: Minimal to slight

0/0 0/0

0/0 0/0

0/0 0/0

0/0 0/1

6/5

Adrenal Chronic capsular inflammation: Moderate to marked Vacuolation, zona reticularis: Slight

0/0 0/0

0/0 0/0

0/0 0/0

0/0 2/0

2/2 8/0

10/7

9/10

0/0 0/0 10/10

0/0

10/10

10/10

From Grothe et al. (1992a)

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microscopy of tissues from three animals of each sex per group revealed an increased intensity (but not incidence) of cytoplasmic lamellar inclusion bodies, from minimal in controls, to moderate in the liver and lungs and moderate-severe in the kidneys of mice at 450 ppm. Lymphoid-cell vacuolar changes (vacuolation), necrosis and/or histiocytosis were reported in the spleen and lymph nodes from mice at 150 ppm, in the thymus at 450 ppm and in ileal Peyer patches at 1200 ppm; the changes in the spleen and lymph nodes were associated with enlargement at 1200 ppm. In the kidneys, cortical tubule cytoplasmic vacuoles seen at 150 ppm were associated with clusters of regenerating cortical cells at doses > 450 ppm, reflecting vacuolar degeneration. Acute or chronic renal capsule inflammation was seen at 1200 ppm. The hepatic effects consisted of hepatocellular vacuolation in females at doses > 150 ppm and in males at 1200 ppm, centrilobular cytomegaly in males at doses > 150 ppm, inflammatory changes in females at > 450 ppm and in males at 1200 ppm, granulomatous changes in females at 450 ppm and severe multifocal necrosis (foci of coagulation necrosis and fibrosis surrounded by thick bands of nuclear debris and necrotic inflammatory cells) at 1200 ppm. The inflammatory and necrotic zones were surrounded by areas of degenerative, enlarged, finely vacuolated hepatocytes. In areas that were not necrotic there were multifocal areas of acute inflammation. Other findings attributed to treatment included increased incidences over those in control animals of lesions affecting the heart (vacuolation and granulocytic vascular leukocytosis), lung (inflammatory changes including necrosis), spleen (inflammatory changes), lymph nodes (inflammation and severe necrosis), bone marrow (granulocytic hypercellularity), pituitary gland (vacuolation), adrenal gland (inflammation) and cervix (histiocytosis) at 1200 ppm; lesions in the spleen (haematopoiesis and necrosis), lung (intra-alveolar macrophages), thymus (necrosis), pancreas (acinar atrophy, vacuolation), tongue (inflammatory changes, regeneration of muscle layer, vacuolation), stomach (moderate-to-marked glandular dilatation, histiocytosis, mucosal hyaline droplets and inflammatory changes), oviduct (mucosal inflammation), uterus (histiocytosis and inflammatory changes), cervix (vacuolation), vagina and epididymides (vacuolation), skeletal muscle (degenerative changes), bone marrow (necrosis) and adrenal gland (vacuolation) at doses > 450 ppm; lesions in lymph nodes (lymphocyte necrosis) and stomach (mucosal mineralization and necrosis) at doses > 150 ppm; and lesions in the ovary (vacuolation) at doses > 50 ppm. The histopathological finding of vacuolation corresponded to ultrastructural evidence of cytoplasmic lamellar inclusion bodies. The vacuolar changes observed were considered by the authors to be consistent with phospholipidosis, a condition resulting from accumulation of polar lipids in lysosomes. The NOAEL was 50 ppm, equal to 6 mg/kg bw per day, on the basis of multiple histological alterations at 150 ppm (Grothe et al., 1992a). Rats In a study conducted in accordance with GLP requirements and OECD guideline 407, groups of five male Fischer 344 rats were given diets containing spinosad (purity, 88%; 76.1% spinosyn A, 11.9% spinosyn D), spinosyn A alone (purity, 96.2%) or spinosyn D alone (purity, 93.0%) at a dietary concentration of 0 (10 animals), 1000 or 3000 ppm for 28 days, equal to 86 and 220 mg/kg bw per day of spinosad, 86 and 220 mg/kg bw per day of spinosyn A and 86 and 250 mg/kg bw per day of spinosyn D. The animals were evaluated for clinical chemical parameters (albumin, alanine aminotransferase, aspartate aminotransferase, 5 '-nucleotidase, Ca, Cl, Na, K, creatinine, globulin, sorbitol dehydrogenase, total protein, blood urea nitrogen), haematological end-points (erythrocyte, differential and total leukocyte and platelets counts, erythrocyte volume fraction, haemoglobin, mean corpuscular haemoglobin, erythrocyte sedimentation rate, mean corpuscular volume, mean corpuscular haemoglobin concentration), urinary parameters (appearance, specific gravity, glucose, ketones, bacteria, occult blood, pH, protein, urobilirubin,

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bilirubin, epithelial cells, urate crystals, erythrocytes, leukocytes), organ weights (adrenals, brain, gonads, heart, kidneys, liver, lungs, pituitary, pancreas, prostate, spleen, thyroid, thymus, uterus) and histological appearance of the adrenals, heart, prostate, aorta, ileum, rectum, blood smear, jejunum, salivary gland, bone, kidneys, skin, bone marrow, lachrymal gland, spinal cord, caecum, lung, spleen, colon, duodenum, mammary gland, stomach, epididymides, testes, eyes, skeletal muscle, thymus, peripheral nerve, thyroid, gall-bladder, oesophagus, trachea, Harderian glands, urinary bladder, pancreas, femur and joint, pituitary, tongue, parathyroid, larynx, nasal tissues and any gross lesions. There were no deaths and no treatment-related clinical signs or ophthalmic effects. The body weight and body-weight gain of rats at 3000 ppm of each compound were lower than those of controls, attaining statistical significance for spinosad (19% and 34% lower, respectively) and spinosyn A (17% and 32%) but not spinosyn D (5.5% and 10%). Reduced weight gains were associated with lower food consumption (32% and 29% lower than controls for spinosad and spinosyn A, respectively), with a reduction also seen for groups at 1000 ppm of spinosad (10%). The significant haematological changes (p < 0.05) that were seen included reduced erythrocyte count (approximately 25% below control value), haemoglobin (-50%), erythrocyte volume fraction (-50%), mean corpuscular volume (-30%) and mean corpuscular haemoglobin (-30%) and increased platelet count (approximately twice control value) in groups at 3000 ppm of spinosad or spinosyn A. Morphological examinations revealed increased incidences of erythrocyte polychromasia and hypochromasia and misshapen, enlarged platelets in rats at 3000 ppm of spinosad and spinosyn A. Significant clinical chemical changes (p < 0.05) occurred at 3000 ppm and consisted of decreased total protein (-11%) and globulin (-14%) concentrations after intake of spinosad; decreased albumin concentration (-5%), decreased alkaline phosphatase activity (-30%) and increased cholesterol concentration (1.7-2 times control value) after intake of spinosad or spinosyn A; increased aspartate aminotransferase activity (2-2.5 times control) after intake of spinosad and spinosyn D (and a non-significant increase of 80% with spinosyn A); and increased K (11%) after intake of spinosyn A. Urinary parameters were unaffected. The weight of the kidney relative to that of brain was significantly decreased by 10% in rats given spinosad at 3000 ppm, and the spleen weight was significantly increased by 20% with spinosyn A. The absolute spleen weights were also significantly increased in groups given spinosyn A or spinosyn D at 3000 ppm. Gross pathological examination revealed oedema of the glandular gastric mucosa and haemolysed blood in the lumen of the stomach in all rats given spinosad or spinosyn A at 3000 ppm. The histopathological changes (Table 3) consisted of increased incidences and/or severity of vacuolar changes in the epithelial cells of the thyroid and tubular epithelial cells in the kidneys (all compounds > 1000 ppm) and in the lymph nodes and thymus (spinosad and spinosyn D), epididymides (spinosad and spinosyn A) and jejunum and seminal vesicles (spinosad) at 3000 ppm. Other effects seen at 3000 ppm were epithelial cell aggregation in skeletal muscle (spinosyn A and spinosyn D), increased extramedullary haematopoiesis in spleen and bone marrow, increased mitotic figures in the glandular mucosa of the stomach (associated with regenerative changes) and alveolar histiocytosis (spinosad and spinosyn A). All compounds at the dietary concentration of 3000 ppm reduced the occurrence of protein droplets in the renal tubule epithelium and degeneration or regeneration in the glandular stomach. A NOAEL could not be identified as histological alterations were seen at all dietary concentrations (McGuirk et al, 1994). In a study conducted in accordance with GLP, groups of 10 male Fischer 344 rats were given diets containing spinosad (purity, 88%; 76.1% spinosyn A, 11.9% spinosyn D) at a concentration of 0,250, 1000 or 1500 ppm for 4 weeks. Groups of 10 rats were killed at 2 and 4 weeks, and, to assess the reversibility of any effects, a further four groups of animals at 0, 1000 and 1500 ppm

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Table 3. Pathological findings attributable to treatment in rats given spinosad, spinosyn A or spinosyn D in the diet for 28 days Lesion

Concentration in diet (ppm) Spinosyn A

Spinosyn D

3000

1000

3000

1000

3000

5

5

5

5

5

5

0 0 0

0 0 0

5 5 5

0 0 0

5 5 5

0 0 0

0 0 4

Bone marrow Haematopoiesis

0

0

5

0

5

0

0

Epididymides Vacuolation of epithelial cells

0

0

4

0

4

0

0

Jejunum Vacuolation

0

0

2

0

1

0

1

Kidney Vacuolation Decreased protein droplets, tubules

2 0

5 0

5 4

5 0

5 5

5 0

5 3

Lung Alveolar histiocytosis

0

0

5

0

5

0

1

Mesenteric lymph nodes Vacuolation

2

1

5

3

5

1

3

Skeletal muscle Aggregation of epithelial cells

0

0

1

0

3

0

3

Spleen Extramedullary haematopoiesis

0

0

4

0

4

0

0

Seminal vesicles Vacuolation, epithelial cells

0

0

2

0

0

0

0

Thymus Vacuolation, macrophages

0

0

2

0

0

0

2

Thyroid Vacuolation of epithelial cells: Slight to moderate

0

5

5

5

5

4

4

Control

Spinosad

0

1000

No. of animals

10

Stomach Mucosal glandular oedema Haemolysed blood Degeneration or regeneration of glandular mucosa: Slight to moderate

From McGuirk et al. (1994)

were killed after an additional 2,4, 8 or 22 weeks on a normal diet. The actual intake of spinosad was calculated to be 21,82 and 120 mg/kg bw per day at the respective dietary concentrations. The experimental parameters determined were clinical signs, body weights, food consumption, serum phospholipid and cholesterol concentrations, gross lesions, kidney and thyroid weights and histological appearance of the epididymides, jejunum, kidneys, liver, lung, mediastinal and mesenteric lymph nodes, seminal vesicles, spleen, thymus and thyroid glands. Only animals at 0 and 1000 ppm that were allowed to recover were examined histologically, as the authors argued that the extent of vacuolation observed at 1000 ppm was sufficient to demonstrate the rate and extent of recovery. The body-weight gain of animals at 1000 and 1500 ppm was 9-10% lower than that of controls, and their feed consumption was approximately 6% lower. A slight but significant increase in serum cholesterol concentration was observed in males at 1500 ppm at week 2; as this change was not found at week 4, it was discounted as toxicologically irrelevant. The weights of the kidney and thyroid relative to body weight were significantly increased in rats at 1500 ppm, by approximately 6 and 24%, respectively. Slight to very slight vacuolation of the thyroid (follicular epithelial cells) and kidney (tubule epithelial cells) was seen at 1000 and 1500 ppm and of the thymus (macrophages) and spleen (macrophages) at 1500 ppm after 2 weeks of treatment. At completion of the 4-week treatment, vacuolation was confined predominantly to the kidney and thyroid in rats at 1000 and 1500 ppm and to the thymus in those at 1500 ppm, isolated individuals

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at 1500 ppm showing vacuolation also in the spleen, mesenteric lymph node and liver. During the recovery phase, the vacuolation of the kidney resolved rapidly, with no effects observed after 2 weeks on a normal diet. The thyroid vacuolation resolved more slowly and was still observed in all but one or two animals at 1000 ppm after 2,4 and 8 weeks on a normal diet; however, this lesion was not detectable 22 weeks after cessation of treatment. The NOAEL was 250 ppm, equal to 21 mg/kg bw per day, on the basis of vacuolation in the kidney and thyroid at 1000 ppm (Yano &Liberacki, 1999a). In a study conducted in accordance with GLP requirements and OECD guideline 408, groups of 10 male and 10 female Fischer 344 rats were given diets containing spinosad (purity, 77.6%; ratio of spinsoynA:spinosynD, approximately 5:1) at a concentration of 0,500,1000,2000 or 4000 ppm for 3 months, equal to 0,34,69,130 and 270 mg/kg bw per day for males and 0,39, 78, 150 and 310 mg/kg bw per day for females. The experimental parameters determined were clinical signs, body weight, food consumption, ophthalmic end-points, haematological parameters (clotting parameters, reticulocyte, erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin, mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration), clinical chemical end-points (albumin, globulin, total protein, bilirubin, cholesterol, triglyceride, glucose, electrolyte, creatinine and urea nitrogen concentrations and the activities of alanine and aspartate aminotransferases, creatine phosphokinase and Y-glutamyl transpeptidase) and urinary parameters (appearance, specific gravity, glucose, ketones, bacteria, occult blood, pH, protein, bilirubin and leukocytes), gross and microscopic appearance (adrenal, heart, prostate, aorta, small and large intestine, salivary gland, Harderian gland, sternum, bone, kidney, skin, bone marrow, brain, liver, lung, spleen, lymph node, stomach, epididymus, muscle, gonad, eye, thymus,' peripheral nerve, thyroid, oesophagus, trachea, urinary bladder, pancreas, uterus, prostate, femur and joint, pituitary, tongue and parathyroid) and organ weights. Owing to the deaths of five males and five females at 4000 ppm, this group was discontinued on day 44, and all animals were necropsied. The cause of death was substantial, treatment-related weight loss, the weights of males being 41 % those of controls and those of females 56% of controls by week 6. The clinical signs seen in most or all animals at this dietary concentration were laboured breathing, thinness, piloerection and 'distension of the penis', with chromorhinorrhoea and hypothermia in one female and two males. Significantly lower body weights were reported in animals at 2000 ppm, from week 11 in males (12% below control) and females (13%) from week 2. At 4000 ppm, the haematological changes seen consisted of regenerative changes in erythrocytes (anisocytosis, polychromasia and erythroblastosis). At 2000 ppm, significant reductions were seen in the erythrocyte count (-11 %, males only), haemoglobin (-40% in males, -6% in females), packed cell volume (-33%, males only), mean corpuscular volume (-35% in males, -10% in females) and mean corpuscular haemoglobin (-33% in males, -4% in females). At 4000 ppm, reticulocyte counts were increased (by 10% in males, 46% in females) and leukocyte counts were increased non-significantly in males (+26%) and significantly in females (+34%). Prothrombin time was slightly but significantly decreased in males at dietary concentrations > 500 ppm (15.6s in controls, 14.3 s at 500 ppm, 14.3 s at 1000 ppm, 13.6 s at 2000 ppm). Examination of the data for individual animals revealed consistent values in each group, indicating that the effect was not due to outliers in either the control or treated groups. The changes in mean values for clinical chemical parameters in animals at 4000 ppm were increased serum inorganic phosphorus (+19% in males, +60% in females), blood urea nitrogen (+70% in males, +80% in females) and cholesterol concentration (+60%, males only), alanine aminotransferase activity (1.5 and 4 times control value in males and females, respectively), aspartate aminotransferase activity (3 and 4 times control), alkaline phosphatase activity (2 and 3 times control), y-ghitamyl transferase activity (2 and 3 times control) and creatine phosphokinase activity (1.2 and 3 times control) and

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reduced triglyceride concentration (-35%, males only). Significant differences from control values at other dietary concentrations were increased inorganic phosphorus (+10% in males, +35% in females) and aspartate aminotransferase activity (3 and 3 times control) at 2000 ppm, increased cholesterol concentrations at > 1000 ppm (+15% in males, +26% in females at 1000 ppm; +37% in males, +31 % in females at 2000 ppm), increased alanine aminotransferase activity in males (+34% at 1000 ppm, +50% at 2000 ppm) and increased blood urea nitrogen in females (+67% at 1000 ppm, +61 % at 2000 ppm), and increased activities of alkaline phosphatase (+67%), alanine aminotransferase (+20%) and g-glutamyl transpeptidase (+27%) in females at 2000 ppm. Urine analysis revealed a slight but significant decrease in mean pH in females at doses > 1000 ppm (8.2 in controls, 8.0 at 500 ppm, 6.6 at 1000 ppm, 5.4 at 2000 ppm) and in males at 2000 ppm (8.2, 8,2, 7.8, 6.4, respectively). Significant changes in the absolute and relative (to body weight) weights of organs were reported as follows: increased adrenal and thyroid weights and decreased uterine weights at 2000 ppm, increased liver weights in females at > 500 ppm and in males at > 1000 ppm, increased heart and spleen weights in females at > 1000 ppm and in both sexes at 2000 ppm, and increased kidney weights at > 1000 ppm (Table 4). The Meeting considered that the increased absolute and relative thyroid weights at 1000 ppm, although not statistically significant, were both treatmentrelated and toxicologically significant, as a clear dose-response relationship was observed, the thyroid is a known target organ of spinosad, and histological effects were observed in this organ at the same dose. Although the weights of a number of other organs relative to body weight were increased at 2000 ppm, the Meeting considered the effects to be secondary to lower terminal body weights reported at that concentration. Gross, histopathological and ultrastructural examination revealed extensive effects in a wide range of tissues, most notably vacuolation. Findings observed only or predominantly in nine or 10 animals at 4000 ppm were distension of the caecum (in nine males and 10 females), small testes (in four males), 'distension of the penis' (seven animals), minimal to moderate chronic multifocal inflammatory necrosis of the liver (four males, three females), multifocal hepatocellular vacuolation of the liver (10 of each sex), acute multifocal inflammation of the lung (eight males, seven females), lymphoid vacuolar changes in the spleen (10 males, nine females), necrosis of the lymph node (three males, one female), atrophy of the thymus (six of each sex), lymphoid vacuolar changes in the thymus (five of each sex), diffuse acinar vacuolation of the pancreas (10 males, nine females), atrophy of the pancreas (two males, three females), diffuse mucosal fibrosis (seven males, two females), glandular mineralization of the stomach (nine males, seven females), glandular epithelial vacuolation of the stomach (nine males, seven females), mucosal vacuolation of the oviduct (nine animals), mucosal vacuolation of the vagina (three animals), moderate to marked hypospermatogenesis (10 animals), mucosal vacuolation of the epididymides (10 animals) and bone-marrow hypocellularity (10 males, eight females). The incidences of gross pathological and histopathological findings at other dietary concentrations are summarized in Table 5. Histopathological changes attributed to treatment were lymphoid-cell vacuolar changes and/or histiocytosis in the spleen, thymus, lymph nodes and ileal Peyer patches at dietary concentrations > 1000 ppm; splenic enlargement was seen at 2000 ppm. The lymphoid vacuolar changes were characterized by the presence of large vacuoles in the cytoplasm of lymphocytes or lymphoblasts, while histiocytosis was characterized by collections of histiocyte-macrophagetype cells with faintly vacuolar cytoplasm in the sinusoids of lymphoid tissues. In kidneys, cytoplasmic vacuoles were seen in the cortical tubules at concentrations > 2000 ppm, accompanied by necrotic changes; together, these lesions were reported to be consistent with vacuolar degeneration. Hepatocellular vacuolation and granulomatous inflammatory changes (the inflammatory response was considered secondary to vacuolar changes in Kupffer cells) were reported in females at concentrations > 1000 ppm and in males at 2000 ppm. The changes in animals at 4000 ppm were more pronounced and associated with necrosis. Other findings

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Table 4. Changes in absolute (g) and relative (mg/100 g) organ weights in rats given diets containing spinosadfor 90 days Dietary concentra- Body Kidney tion (ppm) weight (g) Absolute Relative M

F

M

F

M

F

Heart

Liver Absolute Relative M

F

M

F

Spleen

Uterus

Thyroid and parathyroids Adrenals

Absolute Relative

Absolute Relative

Absolute

Relative

Absolute

M

F

M

M

F

F

Relative M

F

M F

M

F

Absolute Relative M

F

M

F

0

330 200

1.9 1.2 560 610

8.5 4.7 2.6 2.4

880

590

260 300

640

440 190 220

670

340

20 14

5.8 7.0

48

57

14

29

500

360 200

2.1 1.3* 580 630

9.5 5.1* 2.6 2.5*

940

650*

260 320

670

450 180 220

510

250

19 15

5.3 7.4

50

60

14

29

1000

340 200

2.1* 1.4* 610*710*

9.7* 5.5* 2.8* 2.8*

930

670*

270 340*

680

560*200 280*

520

270

25 17

7.1

8.7

53

62

15

32

2000

290* 170* 2.1 1.4* 710*830

9.1 6.0* 3.1* 3.5*

1000* 700*

350*400*

1200* 840*400*480*

460*

270

42* 26* 14* 15*

From Grothe et al. (1992b) */><0.05

58* 69* 20* 40*

198

Table 5. Gross and histopathological lesions in groups of 9-10 rats given spinosad in the diet for 90 days Tissue and lesion

Gross examination Kidney Whole tissue alteration Liver Hepato-diaphragmatic nodule. Whole tissue alteration Lung Whole brown, firm, mottled Lymph node Enlarged Spleen Enlarged Stomach Lesion Testes Enlarged Thyroid Whole tissue alteration Histopathological examination Kidney Cortical tubule necrosis: Minimal to slight Multifocal cortical tubular vacuolation: Slight to moderate Liver Multifocal granuloma: Minimal to moderate Multifocal Kupffer cell vacuolation: Minimal to moderate Heart Progressive cardiomyopathy: Slight Multifocal vacuolation: Minimal to slight Lung Alveolar macrophages: Minimal to slight Spleen Haematopoiesis: Slight to moderate Histiocytosis: Minimal to marked Lymph node Lymphoid cell vacuolar change: Slight to moderate Histiocytosis: minimal to marked Thymus Histiocytosis: Slight to moderate Pancreas Diffuse acinar atrophy: Slight Stomach Glandular dilatation: Minimal to slight Focal hyperkeratosis Ileum Histiocytosis of Peyer patches: Moderate Uterus Mucosal vacuolation: Minimal to moderate Adrenal Cortical vacuolation: Slight Thyroid Acute multifocal inflammation: Minimal to slight Diffuse follicular epithelial vacuolation: Minimal to marked Skeletal muscle Multifocal degeneration: Minimal to moderate Multifocal regeneration: Minimal to moderate

(males/females)

Dietary concentration (ppm)

0

500

1000

2000

4000

0/0

0/0

0/0

2/4

1/0

0/0 0/0

0/0 0/0

0/0 0/0

1/2 0/9

0/0

0/0

0/0

0/0

0/9

10/9

0/0

0/0

0/7

10/10

0/0

0/0

0/0

0/0

10/10

0/0

0/0

0/0

0/0

10/10

0/0

0

0

0

8

0

0/0

0/0

0/0

10/10

0/0

0/0 0/0

0/0 0/0

0/0 0/0

0/2

6/2

10/10

10/10

0/0 0/0

0/0 0/0

0/9 0/9

8/10 8/10

10/10 10/10

0/0 0/0

0/0 0/0

1/1 0/1

1/3 0/1

0/0 2/1

0/1

0/0

0/0

3/10

10/10

0/0 2/1

0/0 3/0

0/0 9/8

10/9 10/10

0/0 0/0

0/0 2/5

0/0 7/6

0/0 9/9

0/3 10/10

9/9 9/9

0/0

0/0

0/0

3/1

4/2

0/0

0/0

0/1

0/1

2/3

3/2 0/0

4/2 0/0

5/5 0/0

8/4 7/7

9/7 0/0

0/0

0/0

0/0

1/1

1/0

0

0

2

9

10

9/0

9/0

7/1

3/4

10/8

0/0 0/0

0/0 6/0

0/0

5/2

0/0

10/9

10/10

10/10

0/0 0/0

0/0 1/0

1/7 0/5

10/10 10/10

9/10

10/9

5/9

From Grothe et al. (1992b) The term 'whole tissue alteration' was used by the pathologist to group a variety of macroscopic findings affecting an entire tissue, such as discolouration, with or without mottling, and varying degrees of alteration to the texture of the tissue.

attributed to treatment included an increase in the severity of vacuolation of cardiac myocytes and cardiomyopathy at > 1000 ppm, myopathy of skeletal muscle (characterized by multifocal fibrils undergoing degeneration or regeneration) in females at > 1000 ppm and in males at 2000 ppm, adrenocortical vacuolation in females at > 1000 ppm, intra-alveolar macrophages in females at > 2000 ppm and in males at 4000 ppm, vacuolation of pancreatic acinar cells at 4000 ppm, and focal hyperkeratosis in the stomach associated with linear papillary or nodular proliferation of the gastric ridge at 2000 ppm. Other findings in the stomach were increased incidences of vacuolation in the epithelium of the gastric mucosa, mucosal fibrosis and glandular mineralization, and an increased severity of gastric glandular dilatation at 4000 ppm. The increased incidence of excessive fluid in the caecum and caecomegaly reported at dietary concentrations > 500 ppm was

SPINOSAD 183-227 JMPR 2001

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considered secondary to a detrimental effect on gut flora that caused osmotic disturbances; however, no histopathological findings were reported in the caecum. An increased incidence of vacuolar changes was also reported in the uterus at dietary concentrations > 1000 ppm, in the oviduct and vagina at 4000 ppm and in the thyroid follicular epithelia at concentrations > 500 ppm. Gross examination of the thyroids showed tham to be enlarged and yellow. Hypospermatogenesis (characterized by the presence of immature forms in the tubules due to maturation arrest) and epididymal vacuolation were reported at 4000 ppm. The histopathological finding of vacuolation corresponded to ultrastructural evidence of cytoplasmic lamellar inclusion bodies (Table 6). Although some pathological findings seen at concentrations < 2000 ppm, were not reported at 4000 ppm, the reduced duration of intake at the latter concentration or autolysis may have been responsible. A NOAEL could not be identified as vacuolatory changes, liver weight changes and decreased prothrombin times were seen at dietary concentrations > 500 ppm. The study authors suggested that the changes found may have been the result of prolonged inhibition of phospholipid catabolism and possibly protein synthesis. They also argued that, given the severity and type of lesions occurring at 500 ppm, the affects were probably reversible (Grothe et al., 1992b). In a study conducted in accordance with GLP requirements and OECD guideline 408, groups of 10 male and 10 female Fischer 344 rats were given diets containing spinosad (purity, 96.3%; ratio of spinosyn A to spinosyn D, 1:1) at a concentration of 0,120, 600 or 1000 ppm for 13 weeks, corresponding to actual achieved intakes of 0,7.7,39 and 65 mg/kg bw per day for males and 0, 9.2, 47 and 80 mg/kg bw per day for females. The following parameters were measured: clinical signs at least daily, ophthalmic end-points before the study and at necropsy, body weights and food consumption at least weekly, haematological parameters (erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin), clinical chemical end-points (albumin, globulin, total protein, bilirubin, cholesterol, triglyceride, glucose, electrolyte, creatinine and urea nitrogen concentrations and alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and creatine phosphokinase activities), urine analysis (appearance, specific gravity, glucose, ketones, occult blood, pH, protein, bilirubin), organ weights (brain, liver, kidneys, heart, adrenals, gonads, spleen, thyroid, parathyroid) and gross and histopathology of the adrenals, heart, prostate, aorta, small and large intestine, salivary gland, Harderian gland, sternum, lachrymal gland, bone, kidneys, skin, bone marrow, brain, liver, lungs, spleen, lymph nodes, stomach, epididymides, muscle, gonads, eyes, thymus, peripheral nerve, thyroid, oesophagus, trachea, urinary bladder, pancreas, uterus, femur and joint, pituitary, tongue, oral and nasal tissues, parathyroid, vagina, cervix and mammary gland. A slight, dose-related, statistically significant reduction in platelet count was seen in both sexes at 600 and 1000 ppm (males, 630,610, 580, 560 x 103/mm3; females, 700, 650, 600,590 x 103/mm3). This effect was discounted by the study authors on the basis that the values for males Table 6. Numbers of animals with cytoplasmic lamellar inclusion bodies in groups of three male and threefemale rats given spinosad in the dietfor 90 days Tissue

Degree

Control

2000 ppm

4000 ppm

Liver

Minimal Slight Minimal Moderate to marked Minimal Slight to marked Minimal Slight to moderate Minimal Severe

4 0 4 0 4 0 6 0 2 0

0 6 0 6 0 6 0 6 0 6

0 6 0 6 NE

Kidney Lung Spleen Thyroid

NE NE

From Grothe et al. (1992b). NE, not examined

SPINOSAD 183-227 JMPR 2001

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were within the range of other controls in the laboratory (550-680) and those for females were only slightly outside this range (630-900), despite the attainment of statistical significance and the clear trend in both sexes. Other 90-day studies with this strain of rat have shown slight to marked increases in platelet counts at dietary concentrations at or above the highest used in this study, which is consistent with the authors' conclusion. At 1000 ppm, slight, statistically significant increases in total protein (< 8%) and albumin (< 6%) concentrations were seen in both sexes, and alanine and aspartate aminotransferase activities were significantly increased in males (by 5060%) and non-significantly in females (by 50%). In both sexes at 1000 ppm, the absolute and relative thyroid weights were significantly increased (+20% above control value). In females at 600 and 1000 ppm, significant increases were found in the weights of the liver (+8%, +19%), spleen (+10%, +43%), heart (+6%,+11 %) and kidney (+8%,+12%), and females at 1000 ppm also had signicantly increased adrenal weights (+16%). Animals of each sex at 600 and 1000 ppm had watery caecal contents. Treatment-related histological effects were confined to animals at the two higher dietary concentrations and involved the thyroid, spleen, lymph nodes, liver (females), kidneys (males) and stomach (females) (Table 7). The changes in organ weights in females were considered by the authors to be unrelated to treatment on the basis that the values were within the range for relevant controls in other studies in the laboratory. However, the effects are consistent with those seen in other 90-day studies with spinosad in this strain of rat, they correlated in most cases with histological findings in the same organs, were statistically significant and were not due to outliers in the data for individual animals. The Meeting therefore concluded that they were treatmentrelated. The epithelial cells lining the thyroid follicles were particularly sensitive, with vacuolation seen in all animals at 600 and 1000 ppm. In the spleen, lymph nodes, liver and kidneys, aggregation of reticuloendothelial cells was observed. In the stomach and liver of females at concentrations > 600 ppm, individual cell necrosis was also seen. The NOAEL was 120 ppm, equal to 7.7 mg/kg bw per day (Yano & Liberacki, 1999b). In a study conducted in accordance with GLP requirements and OECD guideline 408, groups of 10 Fischer 344 rats of each sex were given diets containing spinosad (purity, 88%) at a nominal concentration of 0,30,60,120 or 600 ppm for 13 weeks. Two recovery groups of 10 rats of each sex at 0 or 600 ppm were returned to a normal diet for 4 weeks before sacrifice. The Table 7. Incidences of histopathological lesions in rats given diets containing spinosad for 90 days Lesion

Dietary concentration (ppm) Males

Thyroid Follicular epithelial cell vacuolation: Very slight Follicular epithelial cell vacuolation: Slight Spleen Reticuloendothelial cell aggregation: Very slight Reticuloendothelial cell aggregation: Slight Lymph nodes Aggregates of reticuloendothelial cells: Very slight to slight Mediastinal Mesenteric Submandibular Liver Mononuclear-cell aggregation: Very slight Reticuloendothelial cell aggregation: Very slight to slight Individual cell necrosis: Very slight Kidneys Mononuclear cell aggregation: Very slight Stomach Necrosis, individual cells, glandular mucosa From Yano & Liberacki (1999b); -, no animals examined

SPINOSAD 183-227 JMPR 2001

Females

0

120

600

1000

0

120

600

1000

0/10

0/10

2/10 8/10

0/10

0

9/10 1/10

0/10

0

0

0

8/10 2/19

1/10 9/10

3/10

4/10

5/10

1/10

6/10

0

0

2/10 4/10

0/10

0

0

0

0

3/10 7/10

0/10 2/10 0/10

0/10

_2/10

0/10 3/10

0/10 10/10

0/10 0/10

0/10 0/10

2/10 8/10

8/10 10/10

1/8

0/7

0/8

2/9

6/8

1/10 0/10 2/10

0/10 0/10 0/10

0/10 0/10 0/10

5/10 1/10 0/10

3/10 2/10 0/10

2/10 1/10 0/10

0/10 9/10 8/10

0/10 9/10

0/10

0/10

0/10

3/10

1/10

-

-

2/10

-

-

-

-

0/10

0/10

0/10

6/10

-

0

201

approximate actual achieved doses of spinosad were 0,2.2,4.3, 8.6 and 43 mg/kg bw per day for males and 0, 2.6, 5.2, 10 and 52 mg/kg bw per day for females. The experimental parameters determined included clinical signs, body weight, food consumption, ophthalmic parameters, haematological end-points (erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin, erythrocyte sedimentation rate), clinical chemical parameters (albumin, globulin, total protein, bilirubin, cholesterol, triglyceride, glucose, thyroxine, electrolyte, creatinine and urea nitrogen concentrations and alanine and aspartate aminotransferase and creatine phosphokinase activities), urine analysis (appearance, specific gravity, glucose, ketones, bacteria, occult blood, pH, protein, bilirubin), gross and histopathological appearance of animals at 0 and 600 ppm (adrenals, heart, prostate, aorta, small and large intestine, salivary gland, bone, kidneys, skin, bone marrow, spinal cord, brain, liver, lungs, spleen, mammary gland, stomach, epididymides, muscle, gonads, eyes, thymus, eyes, peripheral nerve, thyroid, oesophagus, trachea, Harderian glands, urinary bladder, pancreas, uterus, femur and joint, pituitary, vagina, tongue, parathyroid and cervix), histopathological appearance of organs of animals at other doses (lungs, liver, kidneys, thyroid, spleen, thymus and lymph nodes) and the weights of the adrenals, brain, gonads, heart, kidneys, liver, spleen and thyroid. No haematological orurological parameters were assessed at the end of the recovery period, but creatinine was measured in males and total protein, albumin, globulin and cholesterol in females, and tissues from the thyroid gland and adjacent tissues (oesophagus, larynx, parathyroid gland and trachea) of animals that had been allowed to recover were subjected to histopathological examinations. The absolute and relative weights of the heart and liver of males at 600 ppm were slightly increased, by 10 and 6%, respectively, and all but the increased absolute liver weight were statistically significant. The absolute weights of the heart and spleen were significantly increased in females by 9% each. In the absence of histological findings in these organs, the authors considered them to be unrelated to treatment. The altered liver, heart and spleen weights were, however, consistent with effects seen in these organs in other studies, and, while the magnitude of the changes and the lack of clinical or histological correlates suggests they are likely to be of minimal toxicological significance, a relationship with treatment cannot be discounted. Organ weights were unaffected after the recovery period. Histopathological findings were restricted to slight vacuolation and enlargement of the epithelial cells lining the thyroid follicles of males at 600 ppm and decreased staining intensity seen occasionally in thyroid follicular cell colloid. The severity, but not the incidence, of epithelial cell vacuolation was partially reversible during the 4week recovery period. Serum thyroxine concentrations were not significantly affected by treatment, although the value for males at 600 ppm was 10% lower than that of controls (4.1 jig/dL in controls and 4.1, 4.2, 4.3 and 3.6 M-g/dL at 30, 60, 120 and 600 ppm, respectively), and an examination of data for individual animals did not reveal the presence of outliers to which the apparent decline might be attributed. Multiple renal adenomas and a focus of hyperplasia of the tubule epithelium were noted in one male rat at 600 ppm, and, although these types of lesions are unusual in rats of this strain and age, similar renal tumours have been reported in a female control in the same laboratory. As similar lesions were not observed in other 90-day studies with spinosad in this strain of rat at higher dietary concentrations, the observation was considered by the Meeting to be unrelated to treatment. The NOAEL was 120 ppm, equal to 8.6 mg/kg bw per day (Yano & Bond, 1994). In a study conducted in accordance with GLP requirements and OECD guideline 412, groups of 10 male and 10 female Fischer 344 rats were exposed (nose only) to an atmosphere containing spinosad (purity, 88%; 76.1% spinosyn A and 11.9% spinosyn D) at a concentration of 0,0.3,1.4 or 9.5 mg/m3 for 6 h/day, 5 days per week, for 14 days. Half the animals of each group were killed at the end of the exposure period, and the remainder were maintained without treatment for a further 15 days to evaluate recovery. The mass median aerodynamic diameter of the particles

SPINOSAD 183-227 JMPR 2001

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was 1-1.6 jiim (geometric standard deviation, 2.4-4.2). The effects of spinosad were assessed by evaluating clinical signs at least daily, body weight and food consumption weekly, haematological end-points (erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin, cell morphology, clotting time), clinical chemical parameters (alkaline phosphatase, alanine and aspartate aminotransferase and creatine phosphokinase activities, urea nitrogen, creatinine, albumin, globulin, glucose, bilirubin, cholesterol, triglyceride and electrolyte concentrations) and gross appearance. Urinary parameters (appearance, specific gravity, pH, bilirubin, glucose, ketones, proteins, blood, urobilinogen) and the weights and histopathological appearance of organs from controls and those at the high concentration (adrenals, heart, prostate, aorta, small and large intestine, salivary gland, lachrymal gland, Harderian gland, bone, kidneys, skin, bone marrow, brain, liver, lungs, spleen, lymph nodes, stomach, epididymides, muscle, gonads, eyes and optic nerve, thymus, peripheral nerve, thyroid, oesophagus, trachea, urinary bladder, pancreas, prostate, cervix, vagina, spinal cord, mammary glands, oral and nasal tissues, uterus, femur and joint, pituitary, tongue and parathyroid) were measured only for the main group at the end of exposure. No effects were observed in any group, either at the end of treatment or after the recovery period. In the absence of any effects, the NOAEL was 9.5 mg/m3, the highest concentration tested (Yano & McGuirk, 1999). Rabbits In a study conducted in accordance with GLP requirements and FIFRA guideline 82-2, spinosad (purity, 88%) was evaluated for its potential to induce dermal irritation and systemic toxicity in New Zealand white rabbits after repeated dermal exposure. Groups of four male and six female rabbits received spinosad at a dose of 0 or 1000 mg/kg bw on the intact skin under a semi-occlusive dressing for 6 h/day for 21 days. The animals were examined for clinical signs, body weight, food consumption, ophthalmic end-points, clinical chemical end-points (alanine and aspartate aminotransferase activities and albumin, 5'-nucleotidase, Ca, Cl, Na, K, P, creatinine, globulin, sorbitol dehydrogenase, total protein, blood urea nitrogen concentrations), haematological parameters (erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin, mean corpuscular haemoglobin, eyrthrocyte sedimentation rate, mean corpuscular volume, mean corpuscular haemoglobin concentration), urinary end-points (appearance, specific gravity, glucose, ketones, bacteria, occult blood, pH, protein, urobilirubin, bilirubin, epithelial cells, urate crystals, erythrocytes, leukocytes), organ weights (adrenals, gonads, heart, kidneys, liver, lungs, pituitary, pancreas, prostate, thyroid, thymus, uterus) and histological appearance of the adrenals, heart, prostate, aorta, ileum, rectum, blood smear, jejunum, salivary gland, bone, kidneys, skin, bone marrow, spinal cord, liver, caecum, lungs, colon, lymph nodes, duodenum, mammary gland, stomach, skeletal muscle, testes, eyes, thymus, peripheral nerve, thyroid, oesophagus, trachea, ovaries, urinary bladder, pancreas, uterus, femur and joint, pituitary, tongue and parathyroid. There were no effects on any parameter examined. The NOAEL was 1000 mg/kg bw per day (Wright et al., 1992b). In a study complying with GLP standards and OECD guideline 410, spinosad (purity, 88%; 76.1% spinosyn A and 11.9% spinosyn D) was applied to the intact skin of groups of five New Zealand white rabbits of each sex at a dose of 0, 100, 500 or 1000 mg/kg bw for 6 h/day under a semi-occlusive dressing 15 times over 21 days. The doses were selected on the basis of the results of a preliminary study in which no clinical signs of toxicity or of dermal irritation were seen in male rabbits exposed dermally to spinosad at 500 or 1000 mg/kg bw for 6 h/day for 4 consecutive days. The experimental parameters determined were clinical signs, food and water consumption, ophthalmic parameters, irritation (weekly and at necropsy), clinical chemical end-points (alkaline phosphatase and alanine and aspartate aminotransferase activities, albumin, bilirubin, 5'-

SPINOSAD 183-227 JMPR 2001

203

nucleotidase, Ca, Cl, creatinine, globulin, glucose, K, sorbitol dehydrogenase, protein, Na and urea nitrogen concentrations) and haematological parameters (erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin). Histopathological examinations were conducted on untreated and treated skin from all animals and on the liver, kidney and stomach from rabbits at 0 or 1000 mg/kg bw per day. Liver, kidney and testes were weighed. Treatment had no effect on any parameter tested. The NOAEL was 1000 mg/kg bw per day (Vedula & Yano, 1994). Dogs In a study complying with GLP standards, pairs of one male and one female beagles were given diets containing spinosad (purity, 88%) at a concentration of 0,200,2000 or 4000 ppm for 4 weeks, equal to 6.5, 62 and 120 mg/kg bw per day for males and 6.8, 54 and 92 mg/kg bw per day for females. The following experimental parameters were determined: clinical signs, body weight, food consumption, ophthalmic parameters, haematological end-points (erythrocyte, leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin, mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration, eyrthrocyte sedimentation rate), clinical chemical end-points (albumin, globulin, total protein, bilirubin, cholesterol, triglyceride, glucose, electrolyte, creatinine and urea nitrogen concentrations and the activities of alanine and aspartate aminotransferase, creatine phosphokinase and y-ghitamyl transpeptidase), urinary end-points (appearance, specific gravity, glucose, ketones, occult blood, pH, protein, bilirubin, leukocytes), organ weights (adrenals, brain, gonads, heart, kidneys, liver, lungs, pituitary, pancreas, prostate, spleen, thyroid with parathyroids) and histological appearance of the adrenals, heart, prostate, aorta, small and large intestine, salivary gland, bone, kidneys, skin, bone marrow, lachrymal gland, spinal cord, brain, liver, lungs, spleen, lymph nodes, sternum, mammary gland, stomach, epididymides, muscle, gonads, thymus, eye with optic nerve, peripheral nerve, thyroid, gall-bladder, oesophagus, trachea, urinary bladder, pancreas, uterus, femur and joint, pituitary, tongue, parathyroid, penis, nasopharynx, buccal cavity, diaphragm and oviducts. Histopathological examinations were conducted on tissues from control animals and those at 4000 ppm and on the following tissues from animals at 200 and 2000 ppm: brain (pons), pituitary, thyroids, parathyroids, adrenals, faucial tonsils, spleen, lymph nodes (cervical), liver, pancreas, intestinal tract and all gross lesions. The results could not be analysed statistically, as the groups were too small. Both animals at 4000 ppm were killed in extremis on day 23 owing to body-weight losses of 0.5 kg in the male and 1 kg in the female, accompanied by reduced food consumption and, in the male, hindlimb weakness. The clinical signs before sacrifice were loose stools with blood and/ or mucus and vomiting. Loose or watery stools were also reported for the male at 2000 ppm, and the female at this concentration lost 0.6 kg, which was associated with a 40-60% reduction in food consumption. Leukocyte counts were increased by approximately 50% over initial values in both animals at 4000 ppm, but, at 2000 ppm, the leukocyte and platelet counts were decreased in the female by 50% and 70%, respectively. Increased activities of alkaline phosphatase, aspartate aminotransferase (by 2-4 times the control value) and alanine aminotransferase (3-4 times control) were seen in animals at dietary concentrations > 2000 ppm, and the concentration of inorganic phosphorus was decreased (by > 60%) in the female at 2000 ppm and in the male at 4000 ppm (by 30%). A 30-40% decrease in the albumin:globulin ratio was seen secondary to increased globulin concentrations in females at dietary concentrations > 2000 ppm and in the male at 4000 ppm. Increased triglyceride concentrations was seen in males > 2000 ppm and in the female at 4000 ppm, increased total cholesterol at 4000 ppm and increased potassium in males > 2000 ppm and in the female at 4000 ppm. Urine analysis revealed occult blood in the male and a reduced pH in the female at 4000 ppm.

SPINOSAD 183-227 JMPR 2001

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As this was a dose range-finding study, the organs of the animals at 4000 ppm which died prematurely were not weighed. In males, thyroid weights were increased by 30% at 200 ppm and by 100% at 2000 ppm and pancreas weights by 50% at 2000 ppm. Liver weights were increased by 25 and 40% and kidney weights by 30% and 40% in the male and female at 2000 ppm, respectively. The weight of the spleen of the female at 2000 ppm was increased threefold. Although the relative adrenal weights were increased in females at 200 and 2000 ppm by 40% and 85%, the weights in males at these concentrations were decreased by 25% and 42%. Gross examination revealed pale livers and red spots in the colon or ileo-caecal region of the intestine in males at concentrations > 2000 ppm and in the female at 4000 ppm, and white foamy fluid in the stomach of the female at 4000 ppm. In both the male and the female at 4000 ppm, histopathological examination revealed cytoplasmic vacuolation or vacuolated cell aggregation in the brain, spinal cord, pituitary, thymus, thyroid, parathyroid, adrenal, faucial tonsil, spleen, bone, bone marrow, lymph nodes, salivary glands, liver, pancreas, gastrointestinal tract, nasal cavity and larynx. Other changes at 4000 ppm were foamy-cell aggregation in the lung, arteritis in the brain, heart, gall-bladder, nasal cavity, lung, kidney, and epididymides of the male dog and lung and urinary bladder of the female, inflammatory cell infiltration in the liver, microgranulomas in the spleen, mucosal atrophy in the stomach, focal haemorrhage in the intestinal mucosa of the colon or caecum, bone-marrow necrosis, lymph node adenitis, acinar-cell atrophy of the pancreas, rhinitis, tracheitis in the male and thymic atrophy in the female. At 2000 ppm, cytoplasmic vacuolation or vacuolated (foamy) cell aggregation was reported in the liver, spleen, brain, thyroid, faucial tonsil, lymph nodes, pancreas, ileum, caecum, colon, rectum and lung; inflammatory cell infiltration in the liver in both sexes; microgranulomas in the spleen, focal haemorrhage in the caecum and haematoma and endocarditis in the heart of the male and congestion and microgranulomas in the spleen and acinar atrophy of the pancreas in the female. At 200 ppm, microgranulomas of the liver, focal haemorrhage in the caecum and arteritis in the lung of the male, and microgranuloma in the spleen and arteritis in the lung of the female were seen. Although the authors of the study considered that the effects observed at 200 ppm were not treatment-related, the Meeting considered that no NOAEL could be identified, as the histological lesions and effects on organ weights seen at 200 ppm were consistent with those seen at higher concentrations and as only one animal of each sex received each concentration (Enomoto, 1992). In a study conducted in accordance with GLP requirements and OECD guideline 409, groups of four beagles of each sex were given diets containing spinosad (purity, 88%) at a nominal concentration of 0,150,300 or 900 (females)/1350 (males) ppm for 13 weeks, equal to 0,4.9,9.7 and 33 mg/kg bw per day for males and 0, 5.4, 10 and 30 mg/kg bw per day for females. The experimental parameters determined were: clinical signs, body weight, food consumption, ophthalmic parameters, haematological end-points (reticulocyte, erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin, mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration, eyrthrocyte sedimentation rate), clinical chemical parameters (albumin, globulin, total protein, bilirubin, cholesterol, triglyceride, glucose, electrolyte, creatinine and urea nitrogen concentrations and alanine aminotransferase, aspartate aminotransferase, creatine phosphokinase and y-glutamyl transpeptidase activities) and urinary end-points (appearance, specific gravity, glucose, ketones, bacteria, occult blood, pH, protein, bilirubin, leukocytes), gross and histological appearance (adrenals, heart, aorta, prostate, small and large intestine, salivary gland, sternum, bone, kidneys, skin, bone marrow, liver, lungs, spleen, lymph nodes, stomach, epididymides, muscle, gonads, eyes, thymus, peripheral nerve, thyroid, parathyroid, gall-bladder, oesophagus, trachea, urinary bladder, pancreas, uterus, femur and joint, pituitary, tongue and parathyroid) and the weights of

SPINOSAD 183-227 JMPR 2001

205

the adrenals, brain, gonads, heart, kidneys, liver, pituitary, pancreas, spleen and thyroids with parathyroids. The highest concentration was reduced to 900 ppm for males from day 38 as one male in this group had to be killed in extremis at week 5 after showing unsteadiness, decreased activity and body-weight loss Two males (including the one that died) at 900/1350 ppm had periocular sebum and watery black stools. Loose stools were common in females at 900 ppm. At week 13, the mean body weights of males and females at the highest concentration were 82 and 88% of control values, respectively, and their mean food consumption was 84 and 89% of control values. In males at 9007 1350ppm, significant (p < 0.05) reductions in erythrocyte volume fraction (by 27%), haemoglobin (27%), erythrocyte count (21-24%) and reticulocyte count (85%, week 13 only) were observed at weeks 7 and 13. Although the leukocyte counts were approximately 60 and 80% of control values at weeks 7 and 13 and the reticulocyte count was 38% of the control values at week 7, the differences did not attain statistical significance. In females, significantly lower values were obtained for mean erythrocyte volume fraction (11 % lower than control), haemoglobin (14%) and mean corpuscular haemoglobin (5%) at week 13 and for leukocyte count (38%), lymphocyte count (43%) and platelet count (3 8%) at week 7. Although not statistically significant, the leukocyte and lymphocyte counts at week 13 for animals at 300 and 900 ppm were 66 and 76% and 62 and 63% of control values, respectively. These haematological findings, in conjunction with bone-marrow necrosis and hypocellularity seen histologically are consistent with a diagnosis of the early stages of aplastic anaemia. In males at 900/1350 ppm, significantly lower serum albumin concentration (22% below control) and albumin:globulin ratio (45%), and increased serum globulin (140% of control value), total cholesterol (130%) and triglyceride (133%) concentrations (week 13 only) were observed in weeks 7 and 13. In females, significantly increased aspartate aminotransferase (335%) and alanine aminotransferase activities (726%), total cholesterol (149%), triglyceride (176%) and serum globulin concentrations (131%) and decreased phosphorus concentration (18%) and albumin:globulin ratio (38%) were observed in week 7, and increased aspartate aminotransferase activity (506%) and decreased serum albumin concentration (22%) and albumin:globulin ratio (37%) at week 13. Although there was also a 10-fold increase in the mean alanine aminotransferase activity over the control value in females at 900 ppm at week 13, this was attributable to an extreme value in an individual animal. Females at 900 ppm had a significant reduction in urinary pH at week 13. The aspartate aminotransferase activity in males at 900/1350 ppm was elevated non-significantly in weeks 7 and 13 (260 and 191% of control), as were the values for alanine aminotransferase activity (221 and 149%). Table 8 shows the alterations in organ weights. The relative (to body weight) weight of the spleen (+72%) was increased in females and that of the thyroid (+80%) in males, and the weights of the liver (+60% in males, +36% in females) and kidney (+45% in males, +26% in females) were increased significantly at the highest dietary concentration. The weight of the pancreas was increased and that of the ovary decreased in females at > 150 ppm; however, there was considerable variation in individual values within each group, which greatly outweighed the variation in the mean values between groups. Furthermore, a dose-response relationship was not Table 8. Changes in weights of organs relative to body weight in dogs given spinosad in the diet for 90 days Dietary concentration (ppm)

0 150 300 900

Body weight (kg)

Kidney

Liver

M

F

M

F

M

F

10 10 10 8.2

9.3 8.7 8.9 8.2

0.40 0.40 0.43 0.58

0.39 0.42 0.41 0.49

2.5 2.5 2.6 4.0

2.6 2.6 2.7 3.5

Spleen

Thyroid

Ovaries

M

F

M

F

-

0.22 0.24 0.24 0.38

0.0070 0.0084 0.0086 0.013*

0.0077 0.012* 0.0091 0.010*

0.020 0.015 0.016 0.012

Pancreas

M

F

0.14 0.20 0.18 0.25*

0.17 0.22 0.20 0.27

From Harada (1994); -, no effect */?<0.05

SPINOSAD 183-227 JMPR 2001

206

seen at 150 or 300 ppm, and no histological alterations were observed in either organ at 150 ppm. The Meeting therefore considered that the apparent effect on the pancreas and ovaries at 150 and 300 ppm was not treatment-related. Gross examination of animals at 900/1350 ppm revealed increased incidences of whitish (granular) areas of the gastric mucosa, distension of the stomach with food and pale livers in both sexes, emaciation, thymic atrophy, pancreatic oedema, lymph node enlargement, brown-yellow spots on the lungs, red spots on the gastric mucosa and thyroid and periocular sebum in males, and hepatic and splenic enlargement and hepatic pallor in females. Histopathological examination (Table 9) revealed increased incidences of vacuolation of cells of the spleen, lymph node, faucial tonsil, ileum, caecum, colon, rectum and pancreas, spermatid giant cells, foamy cell aggregation in the lung, and atrophy of the gastric mucosa in animals at dietary concentrations > 300 ppm. At the highest concentration, there were increased incidences of focal necrosis and cellular depletion in sternal and femoral bone marrow, increased haematopoiesis in the femoral bone marrow and spleen in females, cellular vacuolation in the liver, testis, pituitary, thyroid and parathyroid, brain and spinal cord, atrophy of the splenic white pulp, Table 9. Numbers of dogs with histopathological lesionsin dogs after receiveing diets containing spinosad for 90 days (males/females) Tissue and lesion

Thymus Atrophy Bone marrow Sternal: Focal necrosis/cellular depletion Femoral: Focal necrosis/cellular depletion Femoral: Increased haematopoiesis Spleen Vacuolated cell aggregation in white pulp Atrophic white pulp Lymph nodes Cervical: Vacuolated cell aggregation in lymph follicles Mesenteric: Vacuolated cell aggregation in lymph follicles Faucial tonsil: Vacuolated cell aggregation in lymph follicles Lung Foamy cell aggregation Stomach Atrophic mucosa Ileum Vacuolated cell aggregation in lymph follicles Caecum Vacuolated cell aggregation in lymph follicles Colon Vacuolated cell aggregation in lymph follicles Rectum Vacuolated cell aggregation in lymph follicles Liver Vacuolated hepatocytes Kupffer cell proliferation Pancreas Vacuolated acinar cells Testes Giant spermatid cells Arteritis Vacuolated seminiferous epithelial cells Decreased spermatogenesis Epididymis Arteritis Thyroid Vacuolated C-cells Parathyroid Vacuolated glandular cells Brain Cerebrum: Arteritis in meninx Cerebellum: Vacuolated nerve cells Pons: Vacuolated nerve cells Spinal cord Cervical: Vacuolated nerve cells Thoracic: Vacuolated nerve cells Thoracic: Arteritis in nerve root/meninx Lumbar: Vacuolated nerve cells Eye Arteritis in optic nerve

From Harada( 1994)

SPINOSAD 183-227 JMPR 2001

Dietary concentration (ppm) 0

150

300

900/1350

0/0

0/0

0/1

2/0

0/0 0/0 0/0

0/0 0/0 0/0

0/0 0/0 0/0

1/2 1/3 0/1

0/0 0/0

0/0 0/0

0/0

1/1

4/4 4/1

0/0 0/0 0/0

0/0 0/0 0/0

0/2 1/2 2/3

4/4 4/4 4/4

0/0

0/0

0/1

4/4

0/0

0/0

0/2

4/4

0/0

0/0

1/3

4/4

0/0

0/0

0/2

3/4

2/1

4/4

0/0 0/0

0/0

2/0

3/4

0/0 0/0

0/0 0/0

0/0 0/0

3/1 3/3

0/0

0/0

2/0

4/3

0 0 0 0

0 0 0 0

0 0 0

1

2 2 3 1

0

0

0

2

0/0

0/0

0/0

1/3

0/0

0/0

0/0

4/4

0/0 0/0 0/0

0/0 0/0 0/0

0/0 0/0 0/0

1/0 0/1 3/1

0/0 0/0 0/0 0/0

0/0 0/0 0/0 0/0

0/0 0/0 0/0 0/0

4/0 2/2 2/0 3/3

0/0

0/0

0/0

2/0

207

acinar cells of the salivary gland and thymus, Kupffer cell proliferation, and arteritis in the cerebrum, lung, epididymis, testes, spinal cord and optic nerve. The NOAEL was 150 ppm, equal to 4.9 mg/kg bw per day, on the basis of multiple histological alterations at higher concentrations (Harada, 1994). In a study conducted in accordance with GLP requirements and OECD guideline 452, groups of four beagle dogs of each sex were given diets containing spinosad (purity, 87.2%) for 12 months. The dietary concentration was 0,50,100 or 300 ppm in weeks 1-13, at which time the amount of feed was decreased from 300 g/dog per day to 250 g/dog per day in order to 'prevent the dogs from becoming obese'; subsequently, the concentration of spinosad in the diet was increased to 0,60,120 and 360 ppm from weeks 14-52 to compensate for the reduced food intake. The experimental parameters determined were deaths, clinical signs, body weight, food consumption, ophthalmic end-points, neurological assessment ('functional observational battery') at week 49 or 50, haematological end-points (erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin, mean corpuscular haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin concentration, eyrthrocyte sedimentation rate), clinical chemical parameters (albumin, globulin, total protein, bilirubin, cholesterol, triglyceride, glucose, electrolyte, creatinine and urea nitrogen concentrations and the activities of alanine and aspartate aminotransferase, creatine phosphokinase and g-glutamyl transpeptidase), urinary parameters (appearance, specific gravity, glucose, ketones, bacteria, occult blood, pH, protein, bilirubin, leukocytes), organ weights (adrenals, brain, gonads, heart, kidneys, liver, lungs, pituitary, pancreas, prostate, spleen, thyroid, thymus, uterus) and gross and histopathological appearance (adrenals, heart, prostate, aorta, small and large intestine, salivary gland, bone, kidneys, skin, bone marrow, brain, liver, lungs, spleen, lymph nodes, stomach, epididymides, muscle, gonads, eyes, thymus, peripheral nerve, thyroid, gall-bladder, oesophagus, trachea, urinary bladder, pancreas, uterus, femur and joint, pituitary, tongue and parathyroid). The approximate, average achieved doses were 1.4,2.7 and 8.5 mg/kg bw per day for males and 1.3, 2.7 and 8.2 mg/kg bw per day for females. Effects were seen at 300/360 ppm only. Slight but significantly increased aspartate aminotransferase activity (control, 34 U/l; 300/360 ppm, 50 U/l) and triglyceride concentration (41 and 54 mg/dl) were observed in males in week 26 only. As the values for these two parameters were higher than the control values for each dog, the finding is unlikely to be incidental. Alanine aminotransferase activity was increased nonsignificantly in males at week 26 (44 U/l for controls, 113 U/l at 300/360 ppm) and week 52 (40 and 83 U/l), but the increase was confined to three of the four animals, the fourth animal having normal values at both week 26 and 52. The relative thyroid weight was slightly increased in males (12%) and significantly in females (55%). Vacuolated cell aggregates were seen in the spleen, mesenteric and cervical (females only) lymph nodes and faucial tonsil in both sexes and in the ileum, caecum, colon and rectum in males. Two males also showed vacuolation of glandular cells of the parathyroid gland. The NOAEL was 100 ppm, equal to 2.7 mg/kg bw per day, on the basis of the occurrence of tissue vacuolation and alterations in clinical chemistry at 300/360 ppm (Harada, 1995). (c)

Long-term studies oftoxicity and carcinogenicity Mice

In a study conducted in accordance with GLP requirements and OECD guideline 451, groups of 70 CD-I mice of each sex received diets containing spinosad (purity, 88%; 76.1% spinosyn A, 11.9% spinosyn D) at a concentration of 0, 25, 80 or 360 ppm for up to 18 months, equal to 0,3.4,11 and 51 mg/kg bw per day for males and 0,4.3,14 and 67 mg/kg bw per day for females. Ten animals of each sex per group were killed at 3 and 12 months. Females at 360 ppm

SPINOSAD 183-227 JMPR 2001

208

were killed on day 455 because they had markedly reduced body-weight gain, and excessive deaths occurred. The tissues from this group were not examined, as the authors argued that the maximum tolerated dose had been exceeded and the results for these animals would be of questionable value. Toxicity was assessed by determining clinical signs at least daily, body weight and food consumption at regular intervals, ophthalmic end-points before treatment and at sacrifice, haematological end-points (erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin) and clinical chemical parameters (albumin, globulin, total protein, bilirubin, cholesterol, triglyceride, glucose, electrolyte, creatinine and urea nitrogen concentrations and alanine and aspartate aminotransferase and alkaline phosphatase activities) before each kill, gross appearance at each kill, histological changes in the adrenals, heart, prostate, aorta, small and large intestine, salivary gland, Harderian gland, sternum, lachrymal gland, bone, kidneys, skin, bone marrow, brain, liver, lungs, spleen, lymph nodes, stomach, epididymides, muscle, gonads, eyes, thymus, peripheral nerve, thyroid, gall-bladder, oesophagus, trachea, urinary bladder, pancreas, uterus, pituitary, tongue, parathyroid, oral and nasal tissues, vagina and cervix from controls and groups at the highest concentration at each interim sacrifice (subsets of other groups examined), and at 18 months in controls, females at 80 ppm and males at 360 ppm. The brain, heart, liver, kidneys, testes and spleen were weighed. Treatment-related effects were found only at 360 ppm. The mortality rates were substantially increased by the end of the study, being 24% for male and 18% for female controls, 35% for males and 26% for females at 25 ppm, 29% for males and 13% for females at 80 ppm and 44% for males and 60% for females at 360 ppm, exceeding the acceptable limits for a maximum tolerated dose. Body-weight gain was 10-15% less than that of controls, resulting in significantly lower terminal weights, and food intake was slightly reduced in females. Clinical signs of toxicity (dermatitis of the ear, lachrymation, thin appearance, perineal soiling and roughened coat) were observed. Slight, transient anaemia consisting of significantly decreased erythrocyte volume fraction (10-12% below control value) and haemoglobin (by 8-16%) was seen in both sexes at 3 months and in males and to a lesser extent in females at 12 months, but was not apparent in males at 18 months (females not examined). A slight but significant decrease in albumin concentration (10% below control) and total protein concentration (6-11%) was observed in both sexes at 12 months and to a similar extent in males at 18 months. At 12 months, the phosphate (27%) and sodium (8%) concentrations in females were significantly elevated. An increase in the incidence and severity of hypochromasia and a slightly but significantly decreased calcium concentration (5%) were seen in males at 18 months. Liver weights were increased in both sexes from 3 months, the relative weights (to body weight) being 20 and 27% greater than those of controls for males and females at 12 months, respectively, and the spleen weights were increased in both sexes at 3 months (76% greater than controls for males, 47% for females) and 12 months (110% for males, 30% for females), the relative weights generally being significantly different from those of controla. Gross examination revealed increased incidences of decreased fat and thickening of the glandular portion of the stomach in females at 12 months. At terminal sacrifice, increased incidences of chronic active inflammation of the pinna and a decreased incidence of distension of the ovarian bursa were found in females and an increased incidence of perineal soiling, decreased fat and thickening of the gastric glandular mucosa in both sexes. Histopathological examination revealed vacuolation in a number of tissues at all scheduled sacrifices (3, 12 and 18 months); the tissues affected were the cervix, epididymides, mesenteric lymph nodes, ovaries, pancreas, parathyroid, uterus and vagina at 3 months; epididymides, mesenteric lymph nodes, ovaries, pancreas and parathyroid at 12 months; and epididymides, pancreas and parathyroid at 18 months. Other effects were degeneration or regeneration of the kidneys and increased extramedullary haematopoiesis in the spleen at 3 months; aggregates of alveolar macrophages in the lungs, histiocytosis in the mesenteric lymph nodes, myopathy of skeletal muscle and tongue and inflammation, hyperplasia or hyperkeratosis of the gastric mucosa

SPINOSAD 183-227 JMPR 2001

209

Table 10. Gross and histopathological findings in mice given spinosad in the diet for 18 months Time and finding

Dietary concentration (ppm)

25

0 M 12 months (10 animals) Epididymides Epithelial cell vacuolation: Very slight Epithelial cell vacuolation: Slight Lungs Aggregates of alveolar macrophages Lymph nodes (mesenteric) Sinus histiocytosis Vacuolation, macrophage(s), slight Ovaries Vacuolation: Very slight Vacuolation: Slight Pancreas Vacuolation: Very slight Vacuolation: Slight Parathyroid Vacuolation: Slight Skeletal muscle Myopathy Stomach Hyperplasia, glandular mucosa Chronic inflammation, glandular mucosa Tongue Myopathy: Slight 18 months (50 animals) Chronic active inflammation of pinna Perineal soiling Decreased amount of fat Distended ovarian bursa Diffuse thickening of glandular mucosa of the stomach Epididymides Epithelial cell vacuolation: Very slight Epithelial cell vacuolation: Slight Liver Aggregates of reticuloendothelial cells Lungs Aggregates of alveolar macrophages Lymph node Sinus histiocytosis Pancreas Vacuolation of acini: Very slight Vacuolation of acini: Slight Parathyroid Vacuolation: Slight Skeletal muscle Myopathy Stomach Hyperkeratosis, nonglandular mucosa Hyperplasia, glandular mucosa Hyperplasia, nonglandular mucos: Slight Chronic inflammation, glandular mucosa Tongue Myopathy: Slight

F

10 0

M

360

80 F

M

9 0

M

F

F

0 10

9 0

3

2

4

1

3

1

9

9

1 0

1 0

1 0

0 0

0 0

2 0

8 3

5 3

5 1

2 8

5 0

6 0

7 1

7 2

9 0

9 0

8 0

7 0

1 7

1 8

0

0

0

0

0

0

9

7

0

0

0

0

0

0

1

3

6 2

4 3

5 1

3 4

3 1

4 2

10 10

10 10

0

0

0

0

0

0

3

6

0 1 11

1 1 6 21 2

5 4 8

0 1 11 17 4

1 4 6

1 1 5 27 0

4 8 24

13 7 21 8 32

5

1 44 0

45 1

3

35 2 48*

43 0

41

43

35

39

36

45

26

6

10

6

3

11

8

40*

1

1

0

3

3

1

13*

22 8

16 5

15 5

17 7

22 5

17 9

22 23*

3

0

2

1

5

0

40*

0

0

0

0

0

0

5*

2 26 1 12

6 28 1 18

2 26 1 17

2 25 1 18

0 26 0 12

0* 30 0 15

20* 40* 12* 43*

0

0

0

0

0

0

24*

From Bond et al. (1995a)*Significantly different from control

at 3, 12 and 18 months; and aggregates of reticuloendothelial cells in the liver at 18 months. The key findings are summarized in Table 10. There were no tumours that could be attributed to treatment. The NOAEL was 80 ppm, equal to 11 mg/kg bw per day, on the basis of multiple histological alterations and clinical chemical changes and effects on organ weights at 360 ppm (Bondetal., 1995a). Because of the early deaths of females at 360 ppm in the previous study, an 18-month study was conducted in which groups of 60 CD-I mice of each sex received diets containing spinosad (purity, 88%; 76.1% spinosyn A, 11.9% spinosyn D) at a concentration of 0, 8 or 240 ppm for 18 months, equal to 0,1.1 and 33 mg/kg bw per day for males and 0,1.3 and 42 mg/kg bw per day for females. Ten animals of each sex per group were killed at 12 months for interim investigations.

SPINOSAD 183-227 JMPR 2001

210

Toxicity was assessed by determining clinical signs at least daily, body weight and food consumption at regular intervals, ophthalmic end-points before treatment and at the kills, haematological parameters (erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin) and clinical chemical parameters (albumin, globulin, total protein, bilirubin, cholesterol, triglyceride, glucose, electrolyte, creatinine and urea nitrogen concentrations and alanine and aspartate aminotransferase and alkaline phosphatase activities) before each kill, gross examination at each kill and histopathological examination of females at 0 and 240 ppm only (adrenals, heart, prostate, aorta, small and large intestine, salivary gland, Harderian gland, sternum, lachrymal gland, bone, kidneys, skin, bone marrow, brain, liver, lungs, spleen, lymph nodes, stomach, epididymides, muscle, gonads, eyes, thymus, peripheral nerve, thyroid, gall-bladder, oesophagus, trachea, urinary bladder, pancreas, uterus, pituitary, tongue, parathyroid, oral and nasal tissues, vagina and cervix) and the weights of the brain, heart, liver, kidneys, testes and spleen. The study was conducted in accordance with GLP requirements and OECD guideline 451. Mortality rates were unaffected. Body-weight gain was lower in both sexes at 240 ppm early in the study but was similar to control values thereafter, reflecting acclimatization to the study diet. A statistically significant increase observed in leukocyte count in females at 8 and 240 ppm (approximately twice control values) at 12 months was probably due to a low control value, as the control value at 18 months was similar those of animals at 8 and 240 ppm at both the 12-month interim kill and terminal sacrifice at 18 months. The weight of the liver relative to body weight was significantly increased (by 8-10%) in both sexes at 18 months. Gross examination revealed thickening of the glandular portion of the stomach in both sexes at 240 ppm. Histopathological examination of females at 0 and 240 ppm showed that the alterations at 240 ppm were similar to that observed at 360 ppm in,the previous study, consisting primarily of sinus histiocytosis of the mesenteric lymph nodes, vacuolation of pancreatic acini, vacuolation of the parathyroid, aggregates of alveolar macrophages and alveolar cell hyperplasia in the lungs, skeletal muscle and tongue myopathy and inflammation and hyperplasia or hyperkeratosis of the gastric mucosa (Table 11). Both the nature and incidence of tumours at 240 ppm were similar to those in controls. As limited investigations were carried out on in males and in females at 8 ppm, a NOAEL could not be identified in this study. The results support, however, the NOAEL of 80 ppm (equal to 11 mg/kg bw per day) in the previous study (Bond et al., 1996). Table 11. Histopathological findings at 18 months in control female mice and females given a diet containing spinosad at 240 ppm (n=50)for 18 months Lesion Lung Aggregates of alveolar macrophages Lung Alveolar cell hyperplasia Lymph node Sinus histiocytosis Pancreas Vacuolation of acini Parathyroid Vacuolation, slight Skeletal muscle Myopathy Stomach Hyperkeratosis, nonglandular mucosa Hyperplasia, glandular mucosa Hyperplasia, nonglandular mucosa, Chronic inflammation, glandular mucosa Tongue Myopathy, slight

Control 8

39*

2

6

2

20*

35

41

2

25*

0

22*

7 18 3 14

19* 48* 22* 39*

0

17*

From Bond et al. (1996)* Significantly different from control

SPINOSAD 183-227 JMPR 2001

240 ppm

211

Rats Groups of 65 Fischer 344 rats of each sex received diets containing spinosad (purity, 88%; 76.1 % spinosyn A and 11.9% spinosyn D) at a concentration of 0,50,200,500 or 1000 ppm, equal to 0,2.4,9.5,24 and 49 mg/kg bw per day for males and 0,3,12,30 and 63 mg/kg bw per day for females, for 24 months. Fifty animals of each sex per group were scheduled to receive spinosad for 2 years, 10 of each sex per group were used to assess toxicity and five of each sex per group were used to assess neurotoxicity (see below). Clinical signs of toxicity, body weight and food consumption were assessed at regular intervals during the study, and ophthalmic examinations were performed before treatment and at sacrifice. Haematological parameters (erythrocyte, total and differential leukocyte and platelet counts, erythrocyte volume fraction, haemoglobin), clinical chemical end-points (albumin, globulin, total protein, bilirubin, cholesterol, triglyceride, glucose, electrolyte, creatinine and urea nitrogen concentrations and alkaline phosphatase, alanine and aspartate aminotransferase and creatine phosphokinase activities) and urological parameters (appearance, specific gravity, glucose, ketones, urobilinogen, occult blood, pH, protein, bilirubin, leukocytes) were assessed at 6 and 12 months in 10 animals of each sex per satellite group and at 18 and 24 months in 10 and 20 surviving rats, respectively. Gross examinations were conducted on 10 animals of each sex per satellite group after 12 months, on all decedents and at final sacrifice. Histological examination of all gross lesions, the larynx, nasal and oral tissues, cervix, adrenals, heart, prostate, aorta, small and large intestine, salivary gland, Harderian gland, lachrymal gland, bone, kidneys, skin, bone marrow, brain, liver, lungs, spleen, lymph nodes, stomach, epididymides, muscle, gonads, eyes, thymus, peripheral nerve, thyroid, oesophagus, trachea, urinary bladder, pancreas, uterus, mammary gland, pituitary, tongue and parathyroid was conducted, and the weights of the adrenals, brain, gonads, heart, kidneys, liver, lungs, pituitary, pancreas, prostate, spleen, thyroid, thymus and uterus were determined in 10 rats of each sex per group after 12 months, on all decedents and on surviving control and treated animals. At terminal sacrifice, a limited subset of tissues from animals at 50 and 200 ppm were examined histologically; these comprised the liver, kidney, lungs, thyroid with parathyroids, tissues in which the incidence of alterations at 500 or 1000 ppm suggested a possible treatment-related lesion and tissues with gross lesions. The study complied with the requirements of GLP and OECD guideline 453. Owing to high mortality rates among controls and rats at 1000 ppm (28% male controls, 30% female controls, 88% males and 60% females at 1000 ppm) in weeks 102 and 62, reduced body weight gains (by > 10-15%) at this concentration and clinical signs of overt toxicity (perineal soiling, rapid respiration, thin appearance, decreased body fat reserves), these groups were discontinued on day 714 for males and 611 for females. The mean terminal body weight was approximately 82% those of controls before termination, but food consumption was unaffected. Although a number of statistically significant changes were seen in haematological and clinical chemical parameters at the interim kills, most were minimal and did not persist to final sacrifice and were therefore considered to be transient or incidental. Potentially treatment-related alterations were seen in alkaline phosphatase and aspartate aminotransferase activities and blood urea nitrogen concentration, although in each case the increase was modest and, with the exception of aspartate aminotransferase activity, was not correlated with histological alterations at sacrifice. Alkaline phosphatase activity was significantly increased in males at 500 and 1000 ppm at 18 months (by 24% and 43%, respectively) and in females at 1000 ppm at 6 and 12 months (by 28% and 32%, respectively) and nonsignificantly at 18 months (26%). Aspartate aminotransferase activity was significantly increased in males at 1000 ppm at 12 (35%) and 18 months (42%) and in females at 18 months (54%) and correlated with, but was not necessarily caused by, lesions observed in the heart at this concentration. The blood urea nitrogen concentration was significantly increased in females at 1000 ppm at 6, 12 and 18 months (by 16%, 56% and 38%) and in males at 6 and 18 months (by 12% and 24%). The globulin concentration was slightly but significantly

SPINOSAD 183-227 JMPR 2001

212

increased in males at 500 ppm at 18 and 24 months (by 11 % and 14%), in females at 500 ppm from 6 months onwards (by 9%, 17%, 11 % and 11 %), and in both sexes at 1000 ppm at 12 and 18 months (by 9% and 14% in males, 13% and 8% in females). The values were, however, generally within the range seen in other controls in the laboratory. Statistically significant, treatment-related changes in organ weights were seen at 12 months, consisting of increased absolute and relative weights of the heart (dose-related from 500 ppm in females), kidney, liver (absolute weights significant in females only), spleen and thyroid at 1000 ppm. An increase in absolute and relative ovarian weights at concentrations > 500 ppm was dose-related, and adrenal weights were increased at 1000 ppm in females at 12 months. At 24 months, the absolute and relative thyroid weights were increased in both sexes, and the heart and kidney weights were increased in females at 500 ppm. Multifocal pale areas on the lungs were observed in 1/10 males and 8/10 females at 1000 ppm at 12 months. The histological lesions seen (Table 12) were vacuolation in the kidneys and thyroid, inflammation in the liver, thyroid and prostate, extramedullary haematopoiesis of the spleen and degeneration or regeneration of the glandular mucosa of the stomach. The incidence and/or severity of aggregation of reticuloendothelial cells was increased in the larynx, liver, lymph nodes and spleen. At 500 ppm, there were increased incidences of vacuolation of epithelial cells in the thyroid in both sexes. An increase in the intensity, from very slight to slight or moderate, of reticuloendothelial cell aggregation was observed in the livers of females at 1000 ppm at 12 months, in the mesenteric lymph nodes of both sexes at 12 and 24 months and in the spleen of females at 12 months. The gross findings at termination of groups at 1000 ppm were limited to decreased body fat, increased incidences of perineal soiling, serosanguinous fluid in the thoracic cavity and pale areas on the lungs of a substantial proportion of the animals. A small proportion also had mottled left atria and 21/50 males and 11/50 females had a thrombus in the left atrium. The size of the thyroid gland was increased in four of seven animals. At scheduled sacrifice of the remaining groups, Table 12. Principal histopathological findings in rats given diets containing spinosad for 2 years Lesion

Dietary concentration (ppm)

0

50

M 12 months (n = 10) Kidney Vacuolation of tubules: Slight Larynx Aggregation of reticuloendothelial cells: Slight to very slight Liver Focal or multifocal subacute to chronic inflammation:Very slight Prostate Acute inflammation Spleen Increased extramedullary haematopoiesis Stomach Degeneration or regeneration of glandular mucosa: Very slight Thyroid Vacuolation of epithelial cells Subacute to chronic inflammation 24 months (n = 50) Bone marrow Myeloid hyperplasia Larynx Inflammation: Subacute to chronic Liver Dilatation of sinusoids; focal: Very slight Lung Focal or multifocal subacute to chronic inflammation Thyroid gland Necrosis Vacuolation of epithelial cells Inflammation: Subacute to chronic From Bond etal. (1995b)

SPINOSAD 183-227 JMPR 2001

F

M

500

200 F

M

F

M

1000

F

M

F

0

0

0

0

0

0

0

0

0

9

0

0

0

0

0

0

0

1

2

7

1

1

1

1

0

1

2

3

6

10

4

2

8

6

3

0

0

0

0

0

0

0

0

1

10

0

0

0

0

0

0

0

0

1

9

0 0

0 0

0 0

0 0

0 0

0 0

10 0

10 3

10 10

10 9

1

1

1

2

1

1

1

7

-

6

7

2

13

1

9

5

19

_

1

1

2

2

0

6

0

8

-

14

18

17

16

13

13

16

37

_

0 0 0

1 6 1

0 0 0

0 6 0

0 7 0

0 34 0

0 48 3

16 42 32

— -

213

increased incidences and/or severity were seen of myeloid hyperplasia, inflammation of the larynx, lungs and thyroid, necrosis of the thyroid in females at 500 ppm, slight dilatation of hepatic sinusoids in females and vacuolation of thyroidal epithelial cells in both sexes at concentrations > 200 ppm. There were also increased incidences of altered eosinophilic hepatocellular cells (in 0,4, 7 and 2 males and 0, 6, 9 and 2 females at 0, 50, 200, 500 and 1000 ppm, respectively) and mineralization of pulmonary blood vessels (in 0,11,13,0 and 0 males and 1,12,9,0 and 9 females at 0,50,200,500 and 1000 ppm, respectively); however, the incidences were not dose-related, and the finding is likely to be incidental. The nature, incidence and time to onset of rumours was similar in all groups. The NOAEL was 50 ppm, equal to 2.4 mg/kg bw per day, on the basis of histopathological effects at higher concentrations (Bond et al., 1995b). (d)

Genotoxicity

The results of studies of the genotoxicity of spinosad are summarized in Table 13. (e)

Reproductive toxicity (i)

Multigeneration studies

In a study conducted to GLP standards and OECD guideline 416, groups of 30 SpragueDawley rats of each sex were fed diets containing spinosad (purity, 88%; 76.1% spinosyn A and 11.9% spinosyn D) at concentrations regularly adjusted to deliver an actual dose of 0, 3, 10 or 100 mg/kg bw per day for two generations. After 10 weeks, FO rats were mated to produce Fi a litters, of which 30 rats of each sex per dose were randomly selected to be Fj parental animals. These were mated about 12 weeks after weaning of the last FU litter to produce F2 litters. One week after weaning of F ja litters, F0 parents were again mated to produce F \b litters. All litters were culled to four pups of each sex when possible on day 4postpartum. Clinical signs of toxicity, body weight Table 13. Results of studies of the genotoxicity of spinosad End-point In vitro Reverse mutation Reverse mutation

Test object

Concentration

Purity

Results

Reference

S. typhimurium TA1535, TA1537, TA98, TA100; E. coli WP2uvrA~

310-5000 jig/plate in DMSO

88.0%

Negative ± S9

Garriott et al. (1992a)a

S. typhimurium TA1535, TA98, TA100; E. co/r WP2uvrAS. typhimurium TA1537

100-5000 ng/plate in DMSO

88.0%

Negative ± S9

Lawlor (1996a)b

+/

Negative

50-2500 ng/plate -S9

Forward mutation

L5 1 78Y Tk - mouse lymphoma cells

1-35 ng/ml -S9 15-50ng/ml+S9 in DMSO

88.0%

Negative ± S9

Garriot et al. (1992b)c

Chromosomal aberration

Chinese hamster ovary cells

20-35 ng/ml -S9 1 00-500 fAg/ml+S9 in DMSO

88.0%

Negative ± S9

Garriott et al. (1992c)d

0.5-1000 and 0.01-50 ng/ml, in DMSO

88.0%

Negative ± S9

Garriott et al. (1992d)e

Negative

Garriott et al. (1992e)f

Unscheduled DNA synthesis In vivo Micronucleus formation

Male and female ICR mice

0, 500, 1000, 2000 mg/kg bw 88.0% per day x 2 days, orally in 10% acacia in water

S9, 9000 x g supernatant of Aroclor 1254-induced rat liver; DMSO, dimethyl sulfoxide GLP and QA statements and positive controls included a Test in triplicate. In a preliminary test, concentrations of 1500 and 5000 jig/plate were cytotoxic to TA1537 only. Spinosad promoted the growth of auxotrophs due to trace amounts of histidine and other amino acids as impurities. Absence of revertants was confirmed by replicate plate assay. 6 In a preliminary test, cytotoxicity was seen in TA100 at 3330 and 5000 ng/plate, TA98 at > 2500 ng/plate, TA1535 at 5000 ng/plate, TA1537 > 1250 |j,g/plate. Trace amino acid residues were removed before testing. c Concentration-dependent cytotoxicity seen ± S9, obviating interpretation of result -S9 at > 25 p,g/ml d Conducted in duplicate; cytotoxicity demonstrated in a preliminary study at > 100 |-ig/ml e Conducted in duplicate; cytotoxicity observed at concentrations > 10 ng/ml f Five animal of each sex per dose

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and food consumption were assessed at regular intervals throughout the study. The litter parameters assessed included litter size at birth, numbers of live and dead pups on days 0,1,4,7, 14 and 21, and sex and weight on days 1, 4 (before and after culling), 7, 14 and 21. Gross examinations were conducted on all F0 and FI adults after the last litters were weaned. The liver, kidneys, heart, spleen and thyroid were weighed. Potential target organs and reproductive tissues from all controls and animals at the highest dose were examined histologically. Examination of tissues from rats at the lowest and intermediate doses was restricted to the cervix, coagulating glands, epididymides, heart, kidneys, liver, mesenteric lymph node, ovaries, oviduct, parathyroid, pituitary, prostate, seminal vesicles, spleen, stomach, testes, thyroid, urinary bladder, uterus and vagina. At necropsy, the plasma concentrations of thyroid hormones were determined in 10 FI animals of each sex. At the time of weaning, 10 pups of each sex per dose from the F la , F]b and p2 litters were randomly selected for gross examination; the tissues were not examined histologically. Treatment-related effects on parental animals were observed only at 100 mg/kg bw per day. The clinical signs consisted of increased perineal soiling and vaginal bleeding during lactation of Fi a and p2 pups, and FI females also had an increased incidence of dystocia. The body-weight gain of F0 males was significantly reduced (6%), the body weight of F0 dams during gestation was significantly reduced, and the body-weight gains between parturition and the end of lactation of F la and F lb pups were approximately 16% lower than those of controls. At 100 mg/kg bw per day, the number of pups born alive was significantly reduced by 20-35% in all litters of both parental generations (Fia, Fib and F2 litters), and the pup body weights were 8-12% lower than in controls on days 14 and 21 of lactation (significant only on day 21). Significantly increased relative weights of the heart, kidney, liver, spleen and thyroid were seen in FO parents (in males and females: 22%/ 19%, 13%/19%, 19%/14%, 19%/42%, 128%/26% above control values, respectively) and Fj parents (in males and females: 22%/14%, 15%/13%, 17%/16%, 42%/16%, 71%/30% above control values, respectively) at 100 mg/kg bw per day. Macroscopically, an increased incidence of pale foci was seen in the lungs of F0 adults, two F0 animals had resorbed or dead fetuses, and the incidence of watery caecal contents was increased in FI adults. Histologically, an increase in severity but not incidence was seen for the following findings: degeneration with or without inflammation in the heart, multifocal tubule degeneration or regeneration in the kidneys, sinus histiocytosis of the mesenteric lymph node, and thyroid epithelial-cell vacuolation. Increases in severity and/or incidence was seen for the following effects: inflammation or aggregation of alveolar macrophages in the lungs, sinus histiocytosis in the spleen, inflammation of the prostate, inflammation and necrosis of the thyroid and dilatation of the stomach with or without cellular debris. The clinical observations in Fi a and ¥2 pups were attributable to maternal toxicity and included stomachs void of milk and being cold to touch at doses > 10 mg/kg bw per day in Fla litters and at 100 mg/kg bw per day in F2 litters, thinness or decreased activity in Fj a pups at 100 mg/kg bw per day, and an increased incidence of cannibalization of F2 pups at 100 mg/kg bw per day. The effects in pups of the F la generation at 10 mg/kg bw per day were confined almost entirely to one litter, in which all pups were affected and all died by postnatal day 2. The dam produced a normal litter of healthy pups after the F lb mating. Of the remaining 395 Fla pups at this dose, only one had a stomach devoid of milk and two were cold to touch. No other litter of Fib or F2 pups was affected at this dose. Given the nonspecific nature of these effects and the occurrence only in pups of one dam, the finding is probably incidental. The effects in pups at 100 mg/kg bw per day were likely to be secondary to maternal toxicity rather than a direct effect on the pups. The gross and histopathological findings are summarized in Table 14. Despite the histological alterations in the thyroids of rats at the highest dose, the total thyroxine concentration in the serum was unaffected. No effects were observed in F0 or Fj adults at 3 or 10 mg/kg bw per day. The NOAEL for parental toxicity was 10 mg/kg bw per day on the basis of clinical signs of toxicity, reduced body-weight gain, effects on organ weights and multiple

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Table 14. Gross and histopathological findings in a two-generation study of reproductive toxicity in rats Lesion

Dose (mg/kg bw per day) 0

Gross findings (n= ~ 30) F0: Pale lungs, generalized multifocal F,: Watery contents of caecum F0 histopathological findings (n= 30) Lung Subacute or chronic inflammation, multifocal alveoli or septa: Very slight Subacute or chronic inflammation, multifocal alveoli or septa: Slight Multifocal aggregates of alveolar macrophages: Very slight to slight Multifocal aggregates of alveolar macrophages: Moderate Prostate Chronic active, multifocal inflammation: Very slight Chronic active, multifocal inflammation: Slight Spleen Sinus histiocytosis: Very slight Sinus histiocytosis: Slight Thyroid Chronic active multifocal inflammation of interstitium: Very slight Chronic active multifocal inflammation of interstitium: Slight to mmoderate Focal necrosis: Very slight Multifocal necrosis: Very slight to slight F, histopathological findings Kidneys Multifocal mineralization of tubules: Very slight Multifocal mineralization of tubules: Severe Lung Subacute or chronic inflammation, multifocal alveoli or septa: Very slight Subacute or chronic inflammation, multifocal alveoli or septa: Slight Multifocal aggregates of alveolar macrophages: Very slight Multifocal aggregates of alveolar macrophages: Slight to moderate Prostate Chronic active, multifocal inflammation: Very slight Chronic active, multifocal inflammation: Moderate Spleen Sinus histiocytosis: Very slight Sinus histiocytosis: Slight to moderate Stomach Dilatation of glandular mucosa: Very slight Thyroid Chronic active focal inflammation of interstitium: Very slight Chronic active multifocal inflammation of interstitium: Very slight to slight Multifocal necrosis: Very slight

3

10

100

M

F

M

F

M F

M F

0 2

2 5

0 0

1 1

0 5

1 6

6 8 20 17

4 0 10 0

6 0 6 0

2 0 4 0

3 0 4 0

0 0 8 2

4 0 6 1

7 10 3 2 12 9 12 10

1 0

7 4

1 0

2 0

0 0

0 0

0 0

0 0

0 0

0 0

9 20 21 6

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0

1

0 0 0 0

0 0 0 0

11 16 0 10

5 0

20 0

6 0

19 0

4 0 4 1

5 0 5 0

2 0 3 0

1 0 5 0

3 0

5 17 0 0

10 15 0 2

3 0 2 0

13 16 1 1 12 11 8 7

3 0 5 2

7 3

3 0

2 0

7 2 2 4

0 0

0 0

0 0

0 0

0 0

0 0

13 17 14 5

0

1

0

0

0

1 11

1 0 0

0 0 0

0 0 0

0 0 0

0 0 0

1 1 0 0

0 16 6

5 6 2

From Breslinetal. (1994)

histological alterations at 100 mg/kg bw per day. The NOAEL for reproductive effects was 10 mg/ kg bw per day on the basis of decreased litter size at 100 mg/kg bw per day. The NOAEL for developmental toxicity was 10 mg/kg bw per day on the basis of a reduction in the number of pups per litter and clinical signs (cold to touch and stomachs devoid of milk) at 100 mg/kg bw per day, these effects probably reflecting maternal toxicity (Breslin et al., 1994). (ii)

Developmental toxicity

Rats In a study conducted to GLP standards and OECD guideline 414, groups of 30 pregnant Sprague-Dawley rats were given spinosad (purity, 88%; 76.1% spinosyn A and 11.9% spinosyn D) as a suspension in an aqueous solution of 500 ppm methyl cellulose ether by gavage on days 6-15 of gestation (day 0 designated first day of gestation) at a dose of 0, 10, 50 or 200 mg/kg bw per day. Deaths, clinical signs of toxicity, body weight and food consumption were assessed at regular intervals throughout the study. All surviving rats were killed on day 21 of gestation and examined grossly. The parameters reported were the number and position of fetuses in utero, the numbers of live and dead fetuses, the number and position of resorptions, the number of corpora lutea, fetal sex ratio and body weight and any gross external alterations. Visceral examinations

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were conducted on one-half of the fetuses in each litter, and the other half were subjected to skeletal examinations. The liver, kidney, spleen, thyroid gland, trachea, ovaries, oviducts, uterus, cervix and vagina were taken from two control rats and five at 200 mg/kg bw per day and processed for histological evaluation, and the liver, kidney, spleen, heart and gravid uterus were weighed. Mean maternal body-weight gain was significantly decreased (10%) in dams at 200 mg/kg bw per day during treatment but was similar to that of controls over the entire gestation period owing to a compensatory weight gain on cessation of treatment. Water consumption was increased in dams at 200 mg/kg bw per day from day 16 of gestation. The number of fetuses per litter and the number of viable litters were unaffected. Visceral and skeletal examinations of fetuses revealed a slight, equivocal increase in the fetal and litter incidences of delayed ossification of sternebrae at doses > 50 mg/kg bw per day (with incidences of 7%, 6.8%, 10% and 11 % in fetuses and 33%, 39%, 45% and 56% in litters at 0,10,50 and 200 mg/kg bw per day, respectively). Given the flat dose-response relationship for this observation and the high background incidence, this apparent affect was considered incidental to treatment. Microphthalmia was observed in one fetus at 50 mg/kg bw per day (0.4%) and two at 200 mg/kg bw per day (0.8%), in different litters. The incidence was not statistically significant and was small in terms of the absolute numbers of fetuses affected. Microphthalmia is a relatively rare malformation: the published incidence in other animals from the same source as that used in this study (Charles River Laboratories, USA) during the period in which the study was performed showed only five cases of microphthalmia in 64 789 fetuses, giving an incidence of 0.008% and a maximum incidence of 0.41% (MARTA, 1996). As unilateral developmental effects are also unusual, the apparent increase in the incidence of microphthalmia might be considered incidental. Observations that indicate that unilateral developmental ophthalmic effects do exist and can be triggered by xenobiotic agents include induction of unilateral microphthalmia and anophthalmia in rats with trypan blue (Land et al., 1976) and the existence of a strain of rats with hereditary unilateral microphthalmia (Tokunaga et al., 1987). Conversely, developmental effects such as microphthalmia tend to occur in clusters, and an earlier database of controls (Hood, 1997) showed a maximal incidence of microphthalmia of 1.7%. A clustering of microphthalmia was reported at the Huntingdon Research Centre in the United Kingdom (Palmer, 1977), where only 1:8637 CFY rat fetuses showed microphthalmia between 1973 and 1976, but six to eight fetuses among 600 did so in a subsequent 2-month period. Data supplied by the company indicate that incidences of microphthalmia similar to that observed in this study had occurred in the same laboratory randomly in control and test groups over a number of years. Similarly, data from Huntingdon Life Sciences (1988-93) revealed scattered incidences of microphthalmia or anophthalmia commensurate with those observed in this study. This information provides considerable support for the proposition that the observed incidence of microphthalmia was an incidental cluster effect, independent of treatment. This conclusion is further supported by the absence of microphthalmia in the study of reproductive toxicity and the absence of other developmental effects that would usually be observed concurrently with a teratogenic effect. Consequently, the observation of microphthalmia in this study was considered to be a cluster effect incidental to treatment. Other embryo and fetal parameters evaluated and maternal organ weights were not affected by treatment. The NOAEL for maternal toxicity was 50 mg/kg bw per day on the basis of reduced body-weight gain at 200 mg/kg bw per day. The NOAEL for developmental toxicity was 200 mg/kg bw per day, the highest dose tested, in the absence of effects at this dose (Liberacki et al., 1993). Rabbits In a study conducted to GLP standards and in accordance with OECD guideline 414, groups of 20 pregnant New Zealand white rabbits were given spinosad (purity, 88%; 76.1 % spinosyn A and 11.9% spinosyn D) as a suspension in 500 ppm aqueous methyl cellulose ether by gavage at

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a dose of 0,2.5,10 or 50 mg/kg bw per day on days 7-19 of gestation (day of breeding designated day 0 of gestation). Deaths, clinical signs of toxicity, body weight and food consumption were assessed at regular intervals throughout the study. Gross examinations were conducted on all animals. All does surviving to day 28 of gestation were killed and their fetuses removed surgically. The observations reported were the number and position of fetuses in utero, the numbers of live and dead fetuses, the number and position of resorptions, the number of corpora lutea, fetal sex ratio and body weight and any gross external alterations. In addition, the weights of the liver (with gall-bladder), kidney and gravid uterus were recorded. Faecal output was reduced at 50 mg/kg bw per day. These does also lost weight from the beginning of treatment (-4 g), and their body-weight gain at the end of treatment was 30% lower than that of controls. Two does at the highest dose aborted on days 22 and 27 of gestation and were killed. There were no treatment-related effects on any of the embryo or fetal parameters evaluated. Gross examination of one doe that aborted revealed serosanguinous ascites, atelectasia (left apical lobe), multifocal pale areas in the wall of the gall-bladder, mucoid tracheal exudate, a dark focus in the cortex of one kidney, decreased ingesta in the digestive tract and a haemorrhage in the vaginal wall; the other rabbit that aborted appeared normal. The NOAEL for maternal toxicity was 10 mg/kg bw per day on the basis of reduced body-weight gain and abortion in does at 50 mg/kg bw per day. The NOAEL for embryonal and fetal toxicity was 50 mg/kg bw per day, the highest dose tested, in the absence of effects at this dose (Vedula et al., 1994). (/)

Special studies (i)

Neurotoxicity

In a study conducted in accordance with GLP requirements and FIFRA guideline 81-8, groups of 10 male and 10 female Fischer 344 rats received spinosad (purity, 88.0%; 76.1% spinosyn A and 11.9% spinosyn D) in 500 ppm aqueous methylcellulose at a single dose of 0,200, 630 or 2000 mg/kg bw (active ingredient, doses adjusted for purity) by gavage. A 'functional observational battery' and tests for motor activity, forelimb and hindlimb grip strength and landing foot splay were used to assess neurobehavioural changes in 10 rats of each sex per group before treatment, after 5-6 h and on days 8 and 15 of the study. Body weights were also determined. Gross and neurohistopathological examinations were performed at necropsy on five animals of each sex in the control group and that at the highest dose, on the olfactory lobe, cerebellum, thalamus and hypothalamus, midbrain, pons, cerebellum, medulla oblongata, trigeminal ganglion, pituitary gland, eyes with optic nerves, spinal cord, peripheral nerves, dorsal root ganglia, nasal tissues with olfactory epithelium and skeletal muscles. No deaths occurred during the study. The neurobehavioural and motor activity assays revealed no treatment-related effects. There were no gross pathological or neurohistopathological findings attributable to treatment. The NOAEL was 2000 mg/kg bw per day (Albee et al., 1994). In an evaluation of neurotoxicity conducted concurrently with a 13-week study of toxicity, groups of 10 Fischer 344 rats of each sex were fed diets containing spinosad (purity, 88.0%; 76.1 % spinosyn A and 11.9% spinosyn D) at a concentration of 0,30,60,120 or 600 ppm, 7 days a week for 13 weeks, equal to 0, 2.2, 4.3, 8.6 and 43 mg/kg bw per day for males and 0, 2.6, 5.2, 10 and 52 mg/kg bw per day for females. The study was conducted in accordance with GLP requirements and FIFRA guideline 82-7. A 'functional observational battery' and tests for grip performance, hindlimb landing, foot splay and motor activity were administered to 10 animals of each sex per group before treatment and monthly during dosing. At 13 weeks, the central and peripheral nervous systems of five animals of each sex per group were evaluated. No treatmentrelated effects were observed at any dose. The NOAEL was 600 ppm, equivalent to 43 mg/kg bw per day (Wilmer et al., 1993).

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In the long-term study of toxicity and carcinogenicity of Bond et al. (1995b) in Fischer rats, described above, satellite groups of 10 rats of each sex per group were evaluated for neurotoxic effects of treatment. At 12 months, a 'functional observational battery' consisting of hand holding and open field observations, tests for forelimb and hindlimb grip and landing foot splay and an automated test of motor activity were conducted before treatment and then at 3,6,9 and 12 months. At 12 months, the central and peripheral nervous systems of five animals of each sex in the control group and at the highest dose were evaluated. The only potentially treatment-related effects were an increased incidence of very slight focal hyperplasia of the anterior pituitary (pars distalis) in males (in 0/5 controls and 2/5 at 1000 ppm) and very slight multifocal degeneration of individual fibres of the trigeminal nerve in both sexes (in 1 /5 male controls, 3/5 males at 1000 ppm, 0/5 female controls, 3/5 females at 1000 ppm). Given the low intensity of these findings, the absence of any observed functional deficit in these or the animals in the main study, and the absence of similar alterations in other neural tissue, these observations were considered to be incidental to treatment. The NOAEL for neurotoxicity was 1000 ppm, equal to 49 mg/kg bw per day, the highest dose tested, in the absence of treatment-related effects (Spencer & Yano, 1995). In the 12-month study of toxicity of Harada (1995) in beagle dogs, described above, all animals in each group were evaluated for any neurotoxic effects of treatment after acclimatization. The animals were assessed for general sensory function, rectal temperature, response to auditory and somatosensory stimulation, visual function (including pupillary light reflexes, diameter and equality of pupils), visual, tactile and proprioceptive placing reactions, extensor postural thrust reaction and the presence of spontaneous and positional nystagmus, tonic deviations of the limbs or eyes (strabismus) and impaired righting reactions. In addition, the cranial nerves (except the olfactory and accessory nerves) and reflexes were assessed. No neurological abnormalities were found that could be attributed to treatment. The NOAEL was 300 ppm, equal to 8.2 mg/kg bw per day (Holliday, 1994). (ii)

Mechanisms of lysosomal vacuolation

There is evidence for a common mechanism for the reversible lysosomal vacuolation observed in a range of tissues in mice, rats and dogs. Ultrastructurally, the vacuolated lysosomes have the appearance of lysosomal lamellar bodies. Such histological and ultrastructural changes may arise through processes that prevent degradation of the cell constituents normally processed in lysosomes, by enzyme inhibition or interference in the intra-lysosomal environment required for optimal enzyme activity, or by an excess of substrate (Lullmann-Rauch, 1979; Reasor, 1989). In humans, a range of relatively rare genetic enzyme deficiencies are responsible for this type of lysosomal dysfunction. Mucopolysaccharidoses (e.g. Hunter syndrome, Hurley syndrome) and sphingolipidoses (e.g. Gaucher disease, Niemann-Pick disease), for example, result in accumulation of metabolites in lysosomes due to an excess of substrate resulting from a deficiency in the extralysosomal enzymes that normally act on the respective substance (Robbins & Cotran, 1979). Thus, inhibition of an unidentified enzyme responsible for degradation of a normal metabolic byproduct, with consequent accumulation of that by-product in lysosomes, is a possible explanation for the observed vacuolation. The sponsor presented an alternative mechanism for the widespread vacuolation. They noted that the cytoplasm of the affected cells contained clear vacuoles consisting of variable numbers of secondary lysosomes which contained concentric membrane lamellae. The light microscopic and ultrastructural appearance of these cytoplasmic lamellar bodies was consistent with lesions induced by amphiphilic cationic compounds (Schneider, 1992). The general chemical structure of spinosad—a basic amine group attached over a short side-chain to a hydrophobic moiety—is also consistent with the general structure of amphiphilic cationic compounds. The

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mechanism of the intracellular accumulation of lamellar bodies induced by amphiphilic cationic compounds has been reported (Lullmann-Rauch et al., 1978; Reasor, 1989): The non-protonated compound enters cells by diffusion; once within the cell cytoplasm, the compound can diffuse into lysosomes and become protonated in the acidic environment. The protonated compound then forms a complex with phospholipids (polar lipids) within the lysosome by hydrophobic and electrostatic forces. This lipid complex becomes positively charged and is protected from normal enzymatic attack by phospholipases. Lipid complexes accumulate within the cell due to the diffusion gradient between the plasma and the cytosol and lysosomes, and by the reduced ability to degrade these complexes enzymatically. This accumulation can be reversed by withdrawal of the compound, thus reducing the diffusion gradient and allowing dissociation of the complex between the compound and phospholipids within the cell. The generalized, reversible vacuolation and lysosomal lamellar bodies seen after relatively high doses of spinosad are consistent with the hypothesis suggested by the company. The effect might also be consistent with local enzyme inhibition, either within the lysosomes themselves or more generally in the affected tissues. Given the wide variety of cationic amphiphilic compounds which cause effects similar to those seen with spinosad and the clear association between kinetics and tissue sensitivity, the Meeting considered the hypothesis proposed by Dow AgroSciences to be the more likely explanation. (in)

Studies on metabolites

Spinosyn B and spinosyn K are metabolites of spinosyn A in plants and rats. Spinosyn B is N-demethylated spinosyn A, and spinosyn K is one of the possible 0-demethylated derivatives of spinosyn A. In male and female CD-I mice given spinosyn B (purity, 93.5%) orally in aqueous methyl cellulose, the LD50 was 3200 mg/kg bw. All mice at 5000 mg/kg bw and none at 2000 mg/kg bw died (Gilbert & Stebbins, 1996). In male and female CD-I mice given spinosyn K (purity, 99%) orally in aqueous methyl cellulose, the LD50 was > 5000 mg/kg bw (Gilbert, 1996). Neither spinosyn B (purity, 93.5%) not spinosyn K (purity, 98.7%) induced reverse mutation in Salmonella typhimurium TA1535, TA1537, TA98 or TA100 or Escherichia coli WP2wvrA~ at doses of 0.10-3300 jig/plate in dimethyl sulfoxide for spinosyn B and 0.335000 ng/plate for spinosyn K, in the presence or absence of an exogenous metabolic activation system from rat liver (S9). The tests were conducted in triplicate, and positive controls were included. In preliminary experiments with spinosyn B, cytotoxicity was seen in E. coli WP2wvrA and S. typhimurium TA100 at > 330 ng/plate with S9 and at > 67 ng/plate without S9. In preliminary experiments with spinosyn K, cytotoxicity was seen in TA100 at > 330 p,g/plate with S9 and at > 100 |j,g/plate without S9, and in E. coli WP2wvrA at > 3300 jig/plate with S9 and at > 100 |Hg/plate without S9 (Lawlor, 1996b,c).

Comments The pharmacokinetics and metabolism of the two principal constituents of spinosad, spinosyn A and D, are very similar. Oral administration of spinosyn A or D to rats resulted in rapid but incomplete absorption of > 70% of the dose. Peak blood concentrations of radiolabel were achieved 1 h after administration of 10 mg/kg bw and 2-6 h after administration of 100 mg/kg bw. This delay in achieving peak blood concentrations is likely to reflect saturation of absorption at higher doses. Elimination occurs primarily in the faeces (70-90%) via the bile, and < 10% was recovered from urine. Most of the administered radiolabel was recovered within 24 h. The half-

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times for spmosyn A and D radiolabel were 25-42 h and 29-33 h, respectively. A large proportion of the material excreted in the faeces had been absorbed and eliminated in the bile, primarily as glutathione conjugates of N- and 0-demethylated spinosyns A and D. Excretion as exhaled 14CO2 was negligible. The highest concentrations of tissue residues were identified in fat, liver, kidneys, and lymph nodes. Although the concentrations in the thyroid were not high in comparison with many other tissues shortly after administration of spinosyn A or D, the rate of decline was slow and ultimately resulted in higher concentrations in the thyroid than in other tissues, where the decline was more rapid. Absorbed spinosyn A and D were extensively biotransformed, with glutathione conjugates of N- or 0-demethylated spinosyn A or D as the predominant metabolites. Technical-grade spinosad had little acute toxicity after oral or dermal administration or inhalation; the LD50 values after oral administration were consistently > 2000 mg/kg bw and generally > 5000 mg/kg bw in rats and mice. In one study, however, four of five male rats died after administration of 5000 mg/kg bw by gavage. The LD50 in rabbits treated dermally was > 5 000 mg/kg bw, and the LCso after inhalation in rats was > 5.2 mg/1. Spinosad was not irritating to the skin of rabbits and not sensitizing to the skin of guineapigs. It caused slight eye irritation in rabbits, which resolved within 48 h. An extensive range of effects was observed in both short- and long-term studies with repeated doses, and the effects were similar in mice, rats, and dogs. In short-term studies in mice, rats, and dogs, tissue vacuolation was a consistent observation at the LOAEL. In mice, the overall NOAEL in the 90-day study was 6 mg/kg bw per day, and increased liver weight was also observed at the LOAEL. In rats, the overall NOAEL was 8.6 mg/kg bw per day in three 90-day studies and 21 mg/kg bw per day in two 28-day studies, with increased liver weights again observed at the LOAEL in the 90-day studies. In dogs, the LOAEL in a 28-day study was the lowest dose tested, 6.5 mg/kg bw per day; the NOAEL in a 90-day study was 4.9 mg/kg bw per day; and the NOAEL in a 12-month study was 2.7 mg/kg bw per day. Increased thyroid weights were observed in the 28- and 90-day studies in dogs at doses at and above the LOAEL, in addition to tissue vacuolation. In dogs, however, the lymphatic system was more sensitive to vacuolation than the thyroid, the lymphatic lesions occurring at the LOAEL in both the 90-day and 12-month studies. In long-term studies in mice and rats, tissue vacuolation and other histological alterations were again observed at doses at and above the LOAEL. In mice, the lungs, lymph nodes, stomach, and tongue were the main organs affected at doses above the NOAEL of 11 mg/kg bw per day. The main histological findings were chronic inflammation, hyperplasia, and hyperkeratosis of the stomach, vacuolation of the parathyroid, pancreas, ovaries, and epididymal epithelial cells, and myopathy of the tongue. In rats, the NOAEL in the 2-year study was 2.4 mg/kg bw per day. The primary organ affected at the LOAEL of 9.5 mg/kg bw per day was the thyroid; the lungs, liver, larynx, and bone marrow were affected at higher doses. Vacuolation was limited to the epithelial cells of the thyroid gland, and inflammation was observed in the thyroid, lung, and larynx. Bonemarrow hyperplasia and slight dilatation of liver sinusoids were also observed. Strong similarities in other toxic effects were found between species and in the short- and the long-term studies. At the higher doses used, spinosad was toxic in multiple organs of mice, rats and dogs, resulting in increased serum activity of liver, muscle and cardiac enzymes (alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase and creatininephosphokinase), microcytic hypochromic anaemia and increased spleen, thyroid and liver weights. The histological alterations in a wide range of organs were similar in all species tested, the predominant lesions being cellular vacuolation, inflammatory changes (including necrosis), histiocytosis, regenerative and degenerative changes, increased haematopoiesis and skeletal myopathy. In the long-term study in rats, the thyroid was the most sensitive organ overall, effects occurring at lower doses than in other organs and resolving more slowly after withdrawal of treatment. Vacuolation in the thyroid, the most sensitive toxicological end-point overall, was seen in both short- and long-term studies in rats and was reversible in two studies: a 28-day study in males

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fed diets containing concentrations equal to doses < 120 mg/kg bw per day and a 13-week study in rats of each sex fed diets containing concentrations < 40-50 mg/kg bw per day. Selected tissues from rats and mice in the short-term studies of toxicity were examined by electron microscopy, and the vacuolation was found to be associated with cytoplasmic lamellar inclusion bodies, reflecting a lysosomal storage disorder. While such disorders may arise through a variety of mechanisms which prevent degradation of cell constituents that are usually processed in the lysosomes, spinosad probably acts mainly through a physicochemical mechanism associated with its cationic amphiphilic structure (having both lipophilic and hydrophilic properties in one molecule). A comparison of spinosad, spinosyn A and spinosyn D in a 28-day study in rats treated in the diet revealed notable differences in the toxicological profiles of spinosyn A and D. The toxicological effects of spinosyn A were closely similar to those of spinosad, but spinosyn D failed to produce most of the haematological and clinical chemical alterations seen with spinosad or spinosyn A. Consequently, minor variations in the relative proportions of spinosyn A and D in the technical-grade active ingredient are unlikely to alter its toxicological profile significantly. In long-term studies in mice and rats treated in the diet at doses up to 51 and 49 mg/kg bw per day, respectively, there was no evidence that spinosad is carcinogenic. Spinosad gave negative results in an adequate range of assays for genotoxicity in vivo and in vitro. The Meeting concluded that spinosad is not genotoxic. Given the absence of both genotoxicity in appropriate short-term tests and carcinogenicity in long-term studies in rats and mice, the Meeting concluded that spinosad is unlikely to pose a carcinogenic risk to humans. The reproductive toxicity of spinosad was investigated in a two-generation study in rats. The reproductive effects, a reduced number of pups per litter and clinical alterations in Fi a and p2 pups, reported only at a dietary concentration adjusted to deliver a constant dose of 100 mg/kg bw per day, the highest dose tested, were attributed to nonspecific parental toxicity rather than to a specific toxic effect on the reproductive system. A reduction in the number of pups per litter was observed in each of three generations of pups at 100 mg/kg bw per day. As a similar finding was not observed at 200 mg/kg bw per day in the study of developmental toxicity in rats, the reduction in pup number per litter may reflect preimplantation losses. The NOAEL for reproductive toxicity was 10 mg/kg bw per day. In a study of developmental toxicity in rats, dams were given doses up to 200 mg/kg bw per day. Slightly reduced maternal body-weight gain was observed at the highest dose. Unilateral microphthalmia was found at external examination in two fetuses in separate litters at 200 mg/kg bw per day and in one at 50 mg/kg bw per day. Although this is a rare spontaneous malformation in rats, it was discounted as a cluster effect incidental to treatment, for two reasons. First, a similar incidence of this malformation occurred randomly in control and other groups in studies conducted in the same laboratory with the same strain of rat over a number of years; secondly, it occurred in the absence of the other developmental effects which normally accompany a treatment-related increase in the incidence of malformation. The absence of ocular malformations in the study of reproductive toxicity at doses up to 100 mg/kg bw per day provides further support for this conclusion. On this basis, the NOAEL for maternal toxicity in rats was 50 mg/kg bw per day, and that for developmental toxicity was 200 mg/kg bw per day, the highest dose tested. In a study of developmental toxicity in rabbits, the does were given spinosad on days 7-19 of gestation at doses < 50 mg/kg bw per day, with no evidence of embryo or fetal effects, despite maternal toxicity, consisting of weight loss, abortion, and clinical signs at the highest dose. The NOAEL for maternal toxicity was 10 mg/kg bw per day, and that for embryo and fetal toxicity was 50 mg/ kg bw per day, the highest dose tested. Neurotoxicity was investigated in rats by giving them a single dose of < 2000 mg/kg bw, doses < 43 mg/kg bw per day for 3 months, or doses < 49 mg/kg bw per day for 12 months.

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Comprehensive behavioural and histopathological investigations revealed no evidence of neurotoxicity.

Toxicological evaluation The Meeting concluded that the existing database was adequate to characterize the potential hazards of spinosad to fetuses, infants and children. The most sensitive overall toxicological end-point was thyroid vacuolation in rats treated in the diet in the 2-year study of toxicity and carcinogenicity. The Meeting established an ADI of 0-0.02 mg/kg bw on the basis of the NOAEL of 2.4 mg/kg bw per day in this study and a 100-fold safety factor. Spinosad has little acute toxicity. In studies with repeated doses, no acute toxicological alerts were observed that might indicate the need for establishing an acute reference dose. Levels relevant to risk assessment Species

Study

Effect

NOAEL

LOAEL

Mouse

1 8-month study of toxicity and carcinogenicity3

Toxicity

80 ppm, equal to 1 1 mg/kg bw per day 360 ppm equal to 5 1 mg/kg bw per day15

360 ppm equal to 5 1 mg/kg bw per day

Rat

Rabbit

Dog 3 b c

Carcinogenicity

2-year study of toxicity Toxicity and carcinogenicity3 Carcinogenicity 12-month study of Neurotoxicity neurotoxicity3 Two-generation study Parental and offspring of reproductive toxicity3 toxicity Developmental toxicity0 Maternal toxicity Embryo- and fetotoxicity Developmental toxicity0 Maternal toxicity Embryo- and fetOtoxicity 12-month study of toxicity3

Toxicity

50 ppm, equal to 2.4 mg/kg bw per day 200 ppm equal to 9.5 mg/kg bw per day15 1000 ppm, equal to 49 mg/kg bw per dayb 1 0 mg/kg bw per day

-

200 ppm equal to 9.5 mg/kg bw per day

100 mg/kg bw per day

50 mg/kg bw per day 200 mg/kg bw per dayb

-

10 mg/kg bw per day 50 mg/kg bw per dayb

-

100/1 20 ppm, equal to 2.7 mg/kg bw per day

300 ppm, equal to 8.2 mg/kg bw per day

200 mg/kg bw per day

50 mg/kg bw per day

Diet Highest dose tested Gavage

Estimate of acceptable daily intake for humans 0-0.02 mg/kg bw Estimate of acute reference dose Unnecessary Studies that would provide information useful for continued evaluation of the compound Observations in humans Further investigation of the mechanism of tissue vacuolation

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Relevant end-points for setting guidance values for dietary and non-dietary exposure Absorption, distribution, excretion, and metabolism in mammals Spinosyn A > 70% Rate and extent of oral absorption < 1 % after 24 h in rats Dermal absorption Rapid; highest concentrations of residues in kidney, Distribution lymph nodes, fat, and thyroid at 168 h Rapid, largely complete within 24 h; faeces, > 80%; Rate and extent of excretion urine, 6-10% Limited, but decline in thyroid tissue is slow and Potential for accumulation prolonged Large proportion eliminated unchanged in faeces. Biliary Metabolism in mammals and urinary metabolites primarily glutathione conjugates of spinosyn A and N- and O-demethylated spinosyn A Spinosyn D Rate and extent of oral absorption > 70% Rapid; highest concentrations of residues in kidney, Distribution lymph nodes, fat, and thyroid at 168 h Rate and extent of excretion Rapid, largely complete within 24 h; faeces, > 80%; urine, 6-10% Potential for accumulation Limited, but decline in thyroid tissue is slow and prolonged Large proportion eliminated unchanged in the faeces. Metabolism in mammals Biliary and urinary metabolites primarily glutathione conjugates of spinosyn D and N- and O-demethylated spinosyn D Toxicologically significant compounds Spinosyns A, D Acute toxicity Spinosad LD50, oral

LDso, dermal

LC50, inhalation Dermal irritation Ocular irritation Dermal sensitization Short-term toxicity Target/critical effect Lowest relevant oral NOAEL Lowest relevant dermal NOAEL Lowest relevant inhalational NOAEL Long-term toxicity and carcinogenicity Target/critical effect

Spinosad: > 5000 mg/kg bw, mice and female rats > 2000-< 5000 mg/kg bw, male rats Spinosyn A:D (46:50): males, 4400 mg/kg bw; females, > 5000 mg/kg bw Spinosad: > 5000 mg/kg bw, rabbit Spinosyn A:D (46:50): males and females, > 5000 mg/kg bw > 5.2 mg/1, rats Not irritating, rabbits Slight, rabbits Not sensitizing, guinea-pigs Many tissues, vacuolation; thyroid, increased weight; liver, increased aspartate aminotransferase activity 2.7 mg/kg bw per day > 1000 mg/kg bw per day (rabbits, 21 days) > 9.5 mg/m3 (rats, 14 days)

Lowest relevant NOAEL Carcinogenicity

Mice, rats, and dogs; many tissues, vacuolation and associated alterations in clinical chemical parameters; anaemia 2-year study, rats, 2.4 mg/kg bw per day Not carcinogenic, mice, rats

Genotoxicity

Not genotoxic

Reproductive toxicity Reproductive target/critical effect Lowest relevant reproductive NOAEL Developmental target/critical effect Lowest relevant developmental NOAEL

Reduced number of pups per litter, clinical signs in rat pups secondary to maternal toxicity 10 mg/kg bw per day in a two-generation study in rats No developmental effects in rats or rabbits 50 mg/kg bw per day, rabbits

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No evidence of neurotoxicity in a 12-month study in rats at doses up to 49 mg/kg bw per day

Medical data

No data

Summary

Value

Study

Safety factor

ADI Acute RfD

0-0.02 mg/kg bw Unnecessary

2 years, rats

100

References Albee, R.R., Berdasco, N.M. & Yano, B.L. (1994) XDE-105: Acute neurotoxicity study in Fischer 344 rats. Unpublished reports NosDR-0323-1194-009, DR-0323-1194-009R,DR-0323-1194-009A,DR-0323-1194009B, DR-0323-1194-009C, DR-0323-1194-009D. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Bond, D.M., Stebbins, K.E. & McGuirk, R.J. (1995a) XDE-105: 18-month dietary oncogenicity study in CD1 mice. Unpublished reportNo. DR-0323-1194-006(1). Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Bond, D.M., Yano, B.L., Stebbins, K.E. & McGuirk, R.J. (1995b) DE-105: Two-year chronic toxicity, chronic neurotoxicity and oncogenicity study in Fischer 344 rats. Unpublished reports Nos DR-0323-1194-005 and DR-0323-1194-0051. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom Bond, D.M., Stebbins, K.E. & McGuirk, R.J. (1996) XDE-105: 18-month dietary oncogenicity study in CD-I mice. Unpublished report No. DR-0323 -1194-006(A). Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Breslin, W. J., Quasi, J.F. & Vedula, U. (1994) XDE-105: Two-generation dietary reproduction study in SpragueDawley rats. Unpublished reports Nos DR-0323-1194-008AO, DR-0323-1194-008W2, DR-0323-1194008LO, DR-0323-1194-008P2, DR-0323-1194-008W1, DR-0323-1194-008L2, DR-0323-1194-008L1, DR0323-1 194-008W3 and DR-0323-1194-008. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Domoradzki, J.Y. (1995) XDE-105: Comparison of the Metabolism and Tissue Distribution of 14C-Labelled XDE-105(FactorA)andl4C-LabelledXDE-105 (Factor D)inFischer 344 Rats. Unpublished reportNo T2.2187-19 USA. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom Domoradzki, J.Y. & Shabrang, S.N. (1996) Spinosyn A: Probe study dermal absorption of 14C-labelled spinosyn A in Fischer rats. Unpublished report No. DR-0323-1194-029. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Domoradzki, J.Y., Stewart, H.S., Gilbert, J.R. & Markham, D.A. (1995) XDE-105 (factor A): Metabolism and distribution of 14C labelled XDE-105 (factor A) in Fischer 344 rats. Unpublished report No. DECO-HET-DR0323-1194-012. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Enomoto, A. (1992) XDE-105:4-week dose range finding study in dogs. Unpublished report No. IET 91-0126. from Institute of Environmental Toxicology, Kodaira, Tokyo, Japan. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Garriott, M.L., Rexroat, M.A. & Grothe, D.W. (1992a) The effect of XDE-105 on the induction of reverse mutations in Salmonella typhimurium and Escherichia coli using the Ames test. Unpublished reports Nos 910820AMS3162,910917AMS3162,910924AMS3162. Submitted to WHOby Dow AgroSciences, Letcombe, United Kingdom Gamott, M.L., Michaelis, K.C. & Grothe, D.W. (1992b) The effect of XDE-105 on the induction of forward mutation at the thymidine kinase locus of L5178Y mouse lymphoma cells. Unpublished report No. 910910MLA3162. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Garriott, M.L., Kindig, D.E.F. & Grothe, D.W. (1992c) The effect of XDE-105 on the in vitro induction of chromosome aberrations in Chinese hamster ovary cells. Unpublished reports Nos 910918CAB3162, 911009CAB3162 from Toxicology Research Laboratories, Lilly Research Laboratories. Greenfield, Indianapolis, USA. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Gamott, M.L., Yount, D.J. & Grothe, D.W. (1992d) The effect of XDE-105 on the induction of unscheduled DNA synthesis in primary cultures of adult rat hepatocytes. Unpublished reports Nos 910806UDS3162, 910827UDS3162. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom.

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Garriott, M.L., Brunny, J.D., Kindig, D.E.F. & Grothe, D.W. (1992e) The effect of XDE-105 on the in vivo induction of micronuclei in bone marrow of ICR mice. Unpublished reports Nos 910916MNT3162, 911007MNT3162. Gilbert, K.S (1994a) XDE-105: Acute dermal toxicity study in New Zealand white rabbits. Unpublished report No. DR-0323-1194-017D. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Gilbert, K.S (1994b) XDE-105: Acute dermal toxicity study in New Zealand white rabbits. Unpublished report No. DR-0323-1194-017D1. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Gilbert K.S. (1994c) XDE-105: Primary eye irritation study in New Zealand white rabbits. Unpublished report No. DR-0323-1194-011C. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Gilbert, K.S. (1994d) XDE-105: Dermal sensitization potential in the Hartley albino guinea pig. Unpublished report No. DR-0323-1194-017E. Submitted to WHO by Dew AgroSciences, Letcombe, United Kingdom Gilbert, K.S. (1996) Spinosyn K. Acute oral toxicity study in CD-I mice. Dow Chemical Co., Midland, Michigan, USA, 2 April 1996, DR-0351-5524-001A. Gilbert, K.S. & Stebbins, K.E. (1996) Spinosyn B: 1996 acute oral toxicity study in CD-I. Dow Chemical Co., Midland, Michigan, USA Unpublished report No. DR-0350-1119-001 A. 19 February 1996. Gilbert, K.S. & Yano, B.L. (1996) DE-105: Acute oral toxicity study in Fischer 344 rats and CD-I mice. Dow Chemical Co., Midland, Michigan, USA, 6 March 1996, DR-0323-1194-031. Gilbert, K.S., Johnson, K.A. & Stebbins, K.E. (1994) XDE-105: Acute oral toxicity study in Fischer 344 rats and CD-I mice. Unpublished reports Nos DR-0323-1194-017A, DR-0323-1194-017R, DR-0323-1194-017M. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom Grothe, D.W., Boss, S.M. & Gries, C.L (1992a) A subchronic toxicity study in CD-I mice administered XDE105 in the diet for 3 months. Unpublished report No. MO 1290 from Toxicology Research Laboratories, Lilly Research Laboratories, USA. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Grothe, D.W., Boss, S.M. & Gries, C.L. (1992b) A subchronic toxicity study in Fischer 344 rats administered XDE-105 in the diet for 3 months. Unpublished report No. R20690. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom Harada, T., XDE-105: 13-Week Oral Subchronic Toxicity Study in Dogs, Dow Chemical Japan Ltd., Shinagawa-ku, Tokyo 140, Japan, Unpublished report No IET 91-0079, 01 September 1994 Harada, T. (1995) DE-105: 12-month oral chronic toxicity study in dogs. Unpublished report No. DR-03231194-021 (IET 91 -0080), from Institute of Environmental Toxicology, Tokyo, Japan. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Holliday, T.A. (1994) XDE-105: 12 month oral chronic toxicity study in dogs. Unpublished report No. DECOHET-DR-1194-021 (IET 91 -0080), from Institute of Environmental Toxicology, Tokyo, Japan. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Hood, R.D. (1997) In: Handbook a/Developmental Toxicology, New York: CRC Press, Appendix B, Historical control data. Land, P.W., Policy, E.H. & Kernis, M.M. (1976) Patterns of retinal projections to the lateral geniculate nucleus and superior colliculus of rats with induced unilateral congenital eye defects. Brain Res., 103, 394-399. Laska D.A., Rock G.L. & Grothe D.W. (1992) A 2-week acute dermal irritation and toxicity study in New Zealand white rabbits following a single topical application and 24 hour exposure of XDE-105. Toxicology Research Laboratories, Greenfield, Indianapolis, US A, 23 July 1992.. Unpublished report, Laboratory Project ID.:B06891. Lawlor, T.E. (1996a) Mutagenicity test on XDE-105 in the Salmonella Escherichia coli /mammalian microsome reverse mutation assay preincubation method, with a confirmatory assay. Unpublished report No. 17341-0422RT,DR-0323-1194-032 from Corning Hazleton Inc., Vienna, Virginia, USALawlor, T.E. (1996b) Mutagenicity test on XDE-105 factor B in the Salmonella-Escherichia coli /mammalian microsome reverse mutation assay preincubation method with a confirmatory assay. Unpublished report No. 16974-0-422R from Corning Hazleton Inc., Vienna, Virginia, USA. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Lawlor, T.E. (1996c) Mutagenicity test on XDE-105 factor K in the Salmonella-Escherichia coli /mammalian microsome reverse mutation assay preincubation method with a confirmatory assay. Unpublished report No. 17637-0-422R from Corning Hazleton Inc., Vienna, Virginia, USA. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Liberacki, A.B., Breslin W.J. & Yano, B.L. (1993) XDE-105: Oral gavage teratology study in Sprague-Dawley rats. Unpublished report No. DR-0323-1194-003. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom.

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Liillmamn-Rauch, R. (1979) Drug induced lysosomal storage disorders. In: Dingle, J.T., Jacques, P.J. & Shaw, I.H., eds, Lysosomes in Applied Biology and Therapeutics, Amsterdam: Elsevier North Holland, pp. 49-130. Liillmann, H., Liillmamn-Rauch, R. & Wasserman, O. (1979) Lipidosis induced by lysosomal storage disorders. Biochem. PharmacoL, 27, 1103-1108. MARTA (1996) Historical control data (1992-1994) for developmental and reproductive toxicity studies using the Crl:CD(SD)BR rat. Published and distributed by Charles River Laboratories. McGuirk, R.J., Yano, B.L., Freshour, N.L. & Piasecki, D.A. (1994) XDE-105, factor A and factor D: 28-day dietary toxicity study in Fischer 344 rats Unpublished report No. DR-0323-1194-013. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom Mendrala, A.L., Gilbert, J.R., Stewart, H.S. & Domoradzki, J.Y. (1995a) XDE-105 (factor D): Metabolism and tissue distribution of 14C-labelled XDE-105 (factor D) in Fischer 344 rats. Unpublished report No. DECOHET-DR-0335-0070-00. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Mendrala, A.L., Gilbert, J.R., Stewart, H.S. & Domoradzki, J.Y. (1995b) Bile elimination of XDE-105 (factor D)inFischer 344 rats. Unpublished report No. DECO-HET-DR-03 3 5-0070-002. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Palmer, A.K. (1977) Incidence of sporadic malformations, anomalies and variations in random bred laboratory animals. In: Methods in Prenatal Toxicology: Evaluation of Embryonic Effects in Experimental Animals, Teratology Workshop. April 1977, Berlin, pp. 52-71. Reasor, M. (1989) A review of the biology and toxicological implications of lysosomal lamellar bodies by drugs. Toxicol. App. PharmacoL, 97, 47-56. Robbins, S.L. & Cotran, R.S. (1979) In: Pathologic Basis of Disease, London: W.B Saunders Co., pp. 243-256. Schneider, P. (1992) Drug induced lysosomal disorders in laboratory animals: New substances acting on lysosomes. Arch. Toxicol., 66, 23-33. Shibata, R. (1996) A skin sensitization study of DE-105 in guinea pigs (maximisation test), Bozo Research Centre Inc., Tokyo, Japan. Unpublished report No. B-3106, 19 April 1996. Spencer, P.J. & Yano, B.L. (1995) XDE-105: Chronic neurotoxicity study in Fischer 344 rats. Unpublished report No. DECO-HET-DR-0323-1194-005N. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Stebbins, K.E. & Brooks, K.J. (1999a) Spinosad (spinosyn A & D, 50:50 mixture): Acute oral toxicity study in Fischer 344 rats. Unpublished report No. DR-0360-3530-002, 07 January 1999, Dow AgroSciences, Indianapolis, USA. Stebbins, K.E. & Brooks, K.J. (1999b) Spinosad (spinosyn A & D, 50:50 mixture): Acute dermal toxicity study in New Zealand white rabbits. Unpublished report No. DR-0360-3530-005, 07 January 1999. Dow AgroSciences, Indianapolis, USA. Stebbins, K.E. & Brooks, K.J, (1999c) Spinosad (spinosyn A & D, 50:50 mixture): Acute dermal irritation study in New Zealand white rabbits. Unpublished report No. DR-0360-3530-003, 07 January 1999. Dow AgroSciences, Indianapolis, USA. Stebbins, K.E. & Brooks, K.J. (1999d) Spinosad (spinosyn A & D, 50:50 mixture): Acute eye irritation study in New Zealand white rabbits. Unpublished report dated 07 January 1999. Dow AgroSciences, Indianapolis, USA. Stebbins, K.E. & Brooks, K.J. (1999e) Spinosad (spinosyn A & D, 50:50 mixture): Dermal sensitisationpotential study in Hartley albino guinea pigs. Unpublished report No. DR-360-3530-006, 07 January 1999. Dow AgroSciences, Indianapolis, USA. Thalaker, F.W. (1996) Bioaccumulation of 14C-spinosyn A in female Fischer 344 rats following repeated oral administration of 14C-spinosyn A. Unpublished report No. DR-0323-1194-043. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom Tokunaga, A., Sugita, S. & Otani, K. (1987) Uncrossed retino-geniculate and retino-tectal projections in the hereditary unilaterally microphthalmic rats. Neurosci. Res., 4, 195-210. Vedula, U. & Yano, B.L. (1994) XDE-105: Probe and 21-day repeated dose dermal toxicity study in New Zealand white rabbits. Unpublished report No. DR-0323-1194-018. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Vedula, U., Yano, B.L. & Breslin, W.J. (1994) XDE-105: Oral gavage teratology study in New Zealand white rabbits. Unpublished report No. DR-0323-1194-011. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Wilmer, J.W., Spencer, P.J., Yano, B.L. & Bond, D.M. (1993) XDE-105: 13-week dietary toxicity, 4-week recovery, and 13-week neurotoxicity studies in Fischer 344 rats (neurotoxicity portion). Unpublished report No. DR-0323-1194-001B. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom.

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Wolff, R.K., Allen, D.L., Williams, G.D. & Grothe, D. W. (1992) The acute inhalation toxicity in the Fischer 344 rat of technical XDE-105. Unpublished report No. R33491. Wright, F.L., Keaton, M. J. & Grothe, D. W. (1992a) The acute toxicity of XDE-105 administered orally to Fischer 344 rats. Unpublished report No. R42590 from Toxicology Research Laboratories, Lilly Research Laboratories, USA. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Wright, F.L., Laska, D.A., Poulsen, R.G. & Grothe, D.W. (1992b) A 21-day subchronic dermal toxicity study of XDE-105 in New Zealand white rabbits. Unpublished report No. B08491 from Lilly Research Laboratories, Greenfield, Indianapolis, USA. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Yano, B.L. & Bond, D.M. (1994) XDE-105: 13-week dietary toxicity and 4-week recovery studies in Fischer 344 rats. Unpublished report No. DR-0323-1194-001A. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Yano, B.L. & Liberacki, A.B. (1999a) Spinosad: 4-week dietary toxicity and recovery study in Fischer 344 rats. UnpublishedreportsNos981125S,DR-0323-1194-107. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom Yano, B.L. & Liberacki, A.B. (1999b) Spinosad (50% spinosyn A and 50% spinosyn D): 13-week dietary toxicity study in Fischer rats. Unpublished report No. DR-0360-3530-001 from School of Veterinary Medicine, University of California, Davis, California, USA. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom. Yano, B.L. & McGuirk, R.J. (1999) Spinosad technical (DE-105): 14-day nose only aerosol inhalation toxicity and 2-week recovery studies in Fischer 344 rats. Unpublished report No. 991021. Submitted to WHO by Dow AgroSciences, Letcombe, United Kingdom.

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ANNEX 1 Reports and other documents resulting from previous Joint Meetings of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and WHO Expert Groups on Pesticide Residues 1.

2.

3.

4. 5. 6. 7. 8. 9. 10.

11. 12.

13. 14.

15. 16.

17. 18.

19. 20.

21. 22.

23. 24.

25. 26.

Principles governing consumer safety in relation to pesticide residues. Report of a meeting of a WHO Expert Committee on Pesticide Residues held jointly with the FAO Panel of Experts on the Use of Pesticides in Agriculture. FAO Plant Production and Protection Division Report, No. PL/1961/11; WHO Technical Report Series, No. 240, 1962. Evaluation of the toxicity of pesticide residues in food. Report of a Joint Meeting of the FAO Committee on Pesticides in Agriculture and the WHO Expert Committee on Pesticide Residues. FAO Meeting Report, No. PL/1963/13; WHO/ Food Add./23, 1964. Evaluation of the toxicity of pesticide residues in food. Report of the Second Joint Meeting of the FAO Committee on Pesticides in Agriculture and the WHO Expert Committee on Pesticide Residues. FAO Meeting Report, No. PL/1965/ 10; WHO/Food Add./26.65, 1965. Evaluation of the toxicity of pesticide residues in food. FAO Meeting Report, No. PL/1965/10/1; WHO/Food Add./ 27.65,1965. Evaluation of the hazards to consumers resulting from the use of fumigants in the protection of food. FAO Meeting Report, No. PL/1965/10/2; WHO/Food Add./28.65, 1965. Pesticide residues in food. Joint report of the FAO Working Party on Pesticide Residues and the WHO Expert Committee on Pesticide Residues. FAO Agricultural Studies, No. 73; WHO Technical Report Series, No. 370, 1967. Evaluation of some pesticide residues in food. FAO/PL:CP/15; WHO/Food Add./67.32, 1967. Pesticide residues. Report of the 1967 Joint Meeting of the FAO Working Party and the WHO Expert Committee. FAO Meeting Report, No. PL:1967/M/11; WHO Technical Report Series, No. 391, 1968. 1967 Evaluations of some pesticide residues in food. FAO/PL: 1967/M/l 1/1; WHO/Food Add./68.30, 1968. Pesticide residues in food. Report of the 1968 Joint Meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Committee on Pesticide Residues. FAO Agricultural Studies, No. 78; WHO Technical Report Series, No. 417, 1968. 1968 Evaluations of some pesticide residues in food. FAO/PL: 1968/M/9/1; WHO/Food Add./69.35, 1969. Pesticide residues in food. Report of the 1969 Joint Meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Group on Pesticide Residues. FAO Agricultural Studies, No. 84; WHO Technical Report Series, No. 458, 1970. 1969 Evaluations of some pesticide residues in food. FAO/PL: 1969/M/17/1; WHO/Food Add./70.38, 1970. Pesticide residues in food. Report of the 1970 Joint Meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Committee on Pesticide Residues. FAO Agricultural Studies, No. 87; WHO Technical Report Series, No. 4574, 1971. 1970 Evaluations of some pesticide residues in food. AGP:1970/M/12/1; WHO/Food Add./71.42, 1971. Pesticide residues in food. Report of the 1971 Joint Meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Committee on Pesticide Residues. FAO Agricultural Studies, No. 88; WHO Technical Report Series, No. 502, 1972. 1971 Evaluations of some pesticide residues in food. AGP:1971/M/9/l; WHO Pesticide Residue Series, No. 1, 1972. Pesticide residues in food. Report of the 1972 Joint Meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Committee on Pesticide Residues. FAO Agricultural Studies, No. 90; WHO Technical Report Series, No. 525, 1973. 1972 Evaluations of some pesticide residues in food. AGP: 1972/M/9/1; WHO Pesticide Residue Series, No. 2, 1973. Pesticide residues in food. Report of the 1973 Joint Meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Committee on Pesticide Residues. FAO Agricultural Studies, No. 92; WHO Technical Report Series, No. 545, 1974. 1973 Evaluations of some pesticide residues in food. FAO/AGP/1973/M/9/1; WHO Pesticide Residue Series, No. 3, 1974. Pesticide residues in food. Report of the 1974 Joint Meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Committee on Pesticide Residues. FAO Agricultural Studies, No. 97; WHO Technical Report Series, No. 574, 1975. 1974 Evaluations of some pesticide residues in food. FAO/AGP/1974/M/11; WHO Pesticide Residue Series, No. 4, 1975. Pesticide residues in food. Report of the 1975 Joint Meeting of the FAO Working Party of Experts on Pesticide Residues and the WHO Expert Committee on Pesticide Residues. FAO Plant Production and Protection Series, No. 1; WHO Technical Report Series, No. 592,1976. 1975 Evaluations of some pesticide residues in food. AGP: 1975/M/13; WHO Pesticide Residue Series, No. 5, 1976. Pesticide residues in food. Report of the 1976 Joint Meeting of the FAO Panel of Experts on Pesticide Residues and the Environment and the WHO Expert Group on Pesticide Residues. FAO Food and Nutrition Series, No. 9; FAO Plant Production and Protection Series, No. 8; WHO Technical Report Series, No. 612, 1977.

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230 27. 1976 Evaluations of some pesticide residues in food. AGP: 1976/M/14, 1977. 28. Pesticide residues in food—1977. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues and Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 10 Rev, 1978. 29. Pesticide residues in food: 1977 evaluations. FAO Plant Production and Protection Paper 10 Suppl., 1978. 30. Pesticide residues in food—1978. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues and Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 15,1979. 31. Pesticide residues in food: 1978 evaluations. FAO Plant Production and Protection Paper 15 Suppl., 1979. 32. Pesticide residues in food—1979. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 20, 1980. 33. Pesticide residues in food: 1979 evaluations. FAO Plant Production and Protection Paper 20 Suppl., 1980 34. Pesticide residues in food—1980. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 26, 1981. 35. Pesticide residues in food: 1980 evaluations. FAO Plant Production and Protection Paper 26 Suppl., 1981. 36. Pesticide residues in food—1981. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 37, 1982. 37. Pesticide residues in food: 1981 evaluations. FAO Plant Production and Protection Paper 42, 1982. 38. Pesticide residues in food—1982. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 46, 1982. 39. Pesticide residues in food: 1982 evaluations. FAO Plant Production and Protection Paper 49, 1983. 40. Pesticide residues in food—1983. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 56, 1985. 41. Pesticide residues in food: 1983 evaluations. FAO Plant Production and Protection Paper 61, 1985. 42. Pesticide residues in food—1984. Report of the Joint Meeting on Pesticide Residues. FAO Plant Production and Protection Paper 62, 1985. 43. Pesticide residues in food—1984 evaluations. FAO Plant Production and Protection Paper 67, 1985. 44. Pesticide residues in food—1985. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 68, 1986. 45. Pesticide residues in food—1985 evaluations. Part I. Residues. FAO Plant Production and Protection Paper 72/1,1986. 46. Pesticide residues in food—1985 evaluations. Part II. Toxicology. FAO Plant Production and Protection Paper 72/2, 1986. 47. Pesticide residues in food—1986. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 77, 1986. 48. Pesticide residues in food—1986 evaluations. Part I. Residues. FAO Plant Production and Protection Paper 78,1986. 49. Pesticide residues in food—1986 evaluations. Part II. Toxicology. FAO Plant Production and Protection Paper 78/2, 1987. 50. Pesticide residues in food—1987. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 84, 1987. 51. Pesticide residues in food—1987 evaluations. Part I. Residues. FAO Plant Production and Protection Paper 86/1,1988. 52. Pesticide residues in food—1987 evaluations. Part II. Toxicology. FAO Plant Production and Protection Paper 86/2, 1988. 53. Pesticide residues in food—1988. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 92, 1988. 54. Pesticide residues in food—1988 evaluations. Part I. Residues. FAO Plant Production and Protection Paper 93/1,1988. 55. Pesticide residues in food—1988 evaluations. Part II. Toxicology. FAO Plant Production and Protection Paper 93/2, 1989. 56. Pesticide residues in food—1989. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 99, 1989. 57. Pesticide residues in food—1989 evaluations. Part I. Residues. FAO Plant Production and Protection Paper 100,1990. 58. Pesticide residues in food—1989 evaluations. Part II. Toxicology. FAO Plant Production and Protection Paper 100/2, 1990. 59. Pesticide residues in food—1990. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 102, Rome, 1990.

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231 60. Pesticide residues in food—1990 evaluations. Part I. Residues. FAO Plant Production and Protection Paper 103/1, Rome, 1990. 61. Pesticide residues in food—1990 evaluations. Part II. Toxicology. World Health Organization, WHO/PCS/91.47, Geneva, 1991. 62. Pesticide residues in food— 1991. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 111, Rome, 1991. 63. Pesticide residues in food—1991 evaluations. Part I. Residues. FAO Plant Production and Protection Paper 113/1, Rome, 1991. 64. Pesticide residues in food—1991 evaluations. Part II. Toxicology. World Health Organization, WHO/PCS/92.52, Geneva, 1992. 65. Pesticide residues in food—1992. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 116, Rome, 1993. 66. Pesticide residues in food—1992 evaluations. Part I. Residues. FAO Plant Production and Protection Paper 118, Rome, 1993. 67. Pesticide residues in food—1992 evaluations. Part II. Toxicology. World Health Organization, WHO/PCS/93.34, Geneva, 1993. 68. Pesticide residues in food—1993. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 122, Rome, 1994. 69. Pesticide residues in food—1993 evaluations. Parti. Residues. FAO Plant Production and Protection Paper 124, Rome, 1994. 70. Pesticide residues in food—1993 evaluations. Part II. Toxicology. World Health Organization, WHO/PCS/94.4, Geneva, 1994. 71. Pesticide residues in food—1994. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and a WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 127, Rome, 1995. 72. Pesticide residues in food—1994 evaluations. Part I. Residues. FAO Plant Production and Protection Paper 131/1 and 131/2 (2 volumes), Rome, 1995. 73. Pesticide residues in food—1994 evaluations. Part II. Toxicology. World Health Organization, WHO/PCS/95.2, Geneva, 1995. 74. Pesticide residues in food—1995. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the Core Assessment Group. FAO Plant Production and Protection Paper 133, Rome, 1996. 75. Pesticide residues in food—1995 evaluations. Part I. Residues. FAO Plant Production and Protection Paper 137,1996. 76. Pesticide residues in food—1995 evaluations. Part II. Toxicological and Environmental. World Health Organization, WHO/PCS/96.48, Geneva, 1996. 77. Pesticide residues in food—1996. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group. FAO Plant Production and Protection Paper, 140, 1997. 78. Pesticide residues in food—1996 evaluations. Part I. Residues. FAO Plant Production and Protection Paper, 142,1997. 79. Pesticide residues in food—1996 evaluations. Part II. Toxicological. World Health Organization, WHO/PCS/97.1, Geneva, 1997. 80. Pesticide residues in food—1997. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group. FAO Plant Production and Protection Paper, 145, 1998. 81. Pesticide residues in food—1997 evaluations. Part I. Residues. FAO Plant Production and Protection Paper, 146,1998. 82. Pesticide residues in food—1997 evaluations. Part II. Toxicological and Environmental. World Health Organization, WHO/PCS/98.6, Geneva, 1998. 83. Pesticide residues in food—1998. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group. FAO Plant Production and Protection Paper, 148, 1999. 84. Pesticide residues in food—1998 evaluations. Part I. Residues. FAO Plant Production and Protection Paper, 152/1 and 152/2 (two volumes). 85. Pesticide residues in food—1998 evaluations. Part II. Toxicological and Environmental. World Health Organization, WHO/PCS/99.18, Geneva, 1999. 86. Pesticide residues in food—1999. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group. FAO Plant Production and Protection Paper, 153, 1999. 87. Pesticide residues in food—1999 evaluations. Part I. Residues. FAO Plant Production and Protection Paper, 157,2000. 88. Pesticide residues in food—1999 evaluations. Part II. Toxicological. World Health Organization, WHO/PCS/00.4, Geneva, 2000.

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232

89. Pesticide residues in food—2000. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group. FAO Plant Production and Protection Paper, 163, 2001. 90. Pesticide residues in food—2000 evaluations. Part I. Residues. FAO Plant Production and Protection Paper, 165,2001. 91. Pesticide residues in food—2000 evaluations. Part II. Toxicological. World Health Organization, WHO/PCS/01.3, 2001. 92. Pesticide residues in food—2001. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues in Food and the Environment and the WHO Core Assessment Group. FAO Plant Production and Protection Paper, 167, 2001. 93. Pesticide residues in food—2001 evaluations. Part I. Residues. FAO Plant Production and Protection Paper, in preparation.

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