Toxicological Evaluation of Certain Veterinary Drug Residues in Food

WHO FOOD Toxicological evaluation of ADDITIVES certain veterinary drug SERIES: 49 residues in food Prepared by the fifty-eighth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA)

The summaries and evaluations contained in this book are, in most cases, based on unpublished proprietary data submitted for the purpose of the JECFA assessment.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 JECFA 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.

World Health Organization, Geneva, 2002 IPCS—International Programme on Chemical Safety

This publication is a contribution to the International Programme on Chemical Safety. 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 peerreview 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 (Partici-pating Organizations), following recommendations made by the 1992 United Nations Conference on Environment 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 Organiza-tions, jointly or separately, to achieve the sound management of chemicals in relation to human health and the environment. The summaries and evaluations contained in this book are, in most cases, based on unpublished proprietary data submitted for the purpose of the JMPR assessment.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 Toxicological evaluation of certain veterinary drug residues in food / prepared by the fifty-eighth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA) (WHO food additives series ; 49) 1 .Drug residues - toxicity 2.lvermectin - analogs and derivatives 3.lvermectin - toxicology 4.Tiabendazole - toxicology 5.Cefuroxime toxicology 6. Veterinary drugs - toxicology 7.Food contamination 8.Risk assessment I.Joint FAO/WHO Expert Committee on Food Additives. Meeting (58th: 2002 : Rome, Italy) II.Series ISBN 92 4 166049 X

(NLM classification: WA 712)

CONTENTS Preface

v

Anthelmintic agents Doramectin Tiabenzadole (thiabenzadole) Antimicrobial agents Cefuroxime Annexes Annex 1

Annex 2 Annex 3

Annex 4

Reports and other documents resulting from previous meetings of the Joint FAO/WHO Expert Committee on Food Additives Abbreviations used in the monographs Participants in the fifty-sixth meeting of the Joint FAO/WHO Expert Committee on Food Additives Recommendations on compounds on the agenda and further toxicological studies and information required

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65 75

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PREFACE The monographs contained in this volume were prepared at the fifty-eighth meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), which met at FAO Headquarters in Rome, Italy, 21-27 February 2002. These monographs summarize data on the safety of residues in food of selected veterinary drugs reviewed by the Committee. The fifty-eighth report of JECFA will be published by the World Health Organization in the WHO Technical Report Series. Reports and other documents resulting from previous meetings of JECFA are listed in Annex 1. Abbreviations used in the monographs are listed in Annex 2. The participants in the meeting are listed in Annex 3 of the present publication. JECFA serves as a scientific advisory body to FAO, WHO, their Member States, and the Codex Alimentarius Commission, primarily through the Codex Committee on Food Additives and Contaminants and the Codex Committee on Residues of Veterinary Drugs in Foods, regarding the safety of food additives, residues of veterinary drugs, naturally occurring toxicants, and contaminants in food. Committees accomplish this task by preparing reports of their meetings and publishing specifications or residue monographs and toxicological monographs on substances that they have considered. The monographs contained in this volume are based on working papers that were prepared by working groups before the meeting. A special acknowledgement is given at the beginning of each monograph to those who prepared these working papers. The monographs were edited by E. Heseltine, Lajarthe, 24290 St Leonsur-Vezere, France. The preparation and editing of the monographs included in this volume were made possible through the technical and financial contributions of the Participating Organizations of the International Programme on Chemical Safety (IPCS), which supports the activities of JECFA. 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 organizations participating in the IPCS concerning the legal status of any country, territory, city, or area or its authorities, or 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 those organizations in preference to others of a similar nature that are not mentioned. Any comments or new information on the biological or toxicological properties of the compounds evaluated in this publication should be addressed to: Joint WHO Secretary of the Joint FAO/WHO Expert Committee on Food Additives, International Programme on Chemical Safety, World Health Organization, Avenue Appia, 1211 Geneva 27, Switzerland. -v-

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ANTHELMINTIC AGENTS

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DORAMECTIN First draft prepared by Dr Pamela L Chamberlain Center for Veterinary Medicine Food and Drug Administration, Rockville, Maryland, USA

Explanation Biological data Toxicological studies Special studies on the genetic basis for sensitivity to the toxicity of avermectins Collie dogs Observations in Murray Grey cattle Relative sensitivities of mice, rats, rabbits, dogs and nonhuman primates to the toxicity of avermectins Relative potencies of doramectin, ivermectin and abamectin Observations in humans Polymorphisms in the human MDR-1 gene coding for P-glycoprotein Patients with onchocerciasis treated with ivermectin .... Healthy volunteers treated with ivermectin Comments Evaluation References

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3 4 4 4 4 5 5 6 6 6 7 7 9 10 10

EXPLANATION

Doramectin is a member of the avermectin class of compounds, which includes abamectin and ivermectin. It is a semisynthetic avermectin that has close structural similarity to abamectin and ivermectin. It is used as an endoparasitic agent in nonlactating cattle. Doramectin was previously evaluated by the Committee at its forty-fifth meeting (Annex 1, reference 119), when it established an ADI of 0-0.5 mg/kg bw on the basis of a NOEL of 0.1 mg/kg bw per day for mydriasis in a 3-month study in dogs treated by gavage and using a safety factor of 200. An additional safety factor of 2 was applied because doramectin was not tested in CF-1 mice, which is the test animal most sensitive to the neurotoxic effects of this family of drugs. In 1997, the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) concluded that the sensitivity to avermectins of CF-1 mice was due to a genetic variation that causes reduced expression of P-glycoprotein in the blood-brain barrier (FAO/WHO, 1998). The JMPR further concluded that the results of studies with CF-1 mice were not appropriate for establishing ADIs for avermectins. P-glycoprotein was expressed in the brain and jejunum of all species studied. Pglycoprotein is a cell membrane protein that acts to remove a wide variety of lipophilic -3 -

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compounds from cells, including avermectins. In the capillary endothelium of the central nervous system, it serves as a functional component of the blood-brain barrier. In intestinal epithelium, P-glycoprotein can limit intestinal absorption of a range of compounds. The Committee at its fiftieth meeting (Annex 1, reference 134) accepted the conclusions of the JMPR and considered that it was no longer necessary to apply an additional safety factor of 2 for avermectins and milbemycins that had not been tested in CF-1 mice. Doramectin was re-evaluated by the Committee at its present meeting in order to determine whether removal of the additional safety factor of 2 was appropriate. On the basis of the Committee's decision taken at its fiftieth meeting, the present Committee concluded that use of an additional safety factor of 2 in establishing the ADI for doramectin was no longer necessary. No new data were provided to the Committee. The literature was reviewed for published information on the toxicity of avermectins considered relevant to this evaluation. The Committee reviewed information on the mechanism of the toxicity of ivermectin in a subpopulation of collie dogs and observations of its toxicity in a subpopulation of Murray Grey cattle. The Committee also considered a published review of the relative sensitivities of mice, rats, rabbits, dogs and non-human primates to avermectins. The relative potencies of doramectin, ivermectin and abamectin were also considered. The Committee examined information about variants of the human gene that codes for P-glycoprotein and reviewed observations in humans. 2. 2.1

BIOLOGICAL DATA Texicological

studies

2.1.1 Genetic basis for sensitivity to the toxicity of avermectins (a)

Collie dogs

The genetic basis for the sensitivity of collies to avermectins was studied in 13 clinically normal collies, previously identified as being sensitive or insensitive to ivermectin. Seven animals were identified as sensitive after displaying typical clinical signs of neurotoxicity, including depression, ataxia, mydriasis, salivation or drooling, after receiving a single oral dose of 120 mg/kg bw. The objective of the study was to determine whether altered gene expression of P-glycoprotein or a polymorphism of the canine Mdr\ gene that codes for P-glycoprotein exists in avermectin-sensitive collies. The sensitivity of the CF-1 mouse strain to the neurotoxic effects of avermectin has been traced to a polymorphism of the murine Mdr 1 gene resulting in decreased expression of P-glycoprotein (Umbenhauer et al., 1997). The level of Mdrt expression was similar in sensitive and insensitive collies, as determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Sequence analysis of canine Mdr1 by RT-PCR was conducted on RNA isolated from blood leukocytes obtained from the sensitive and insensitive collies and also from other breeds (one beagle, two golden retrievers and one Staffordshire terrier cross-bred dog). Sequence analysis of clones from three ivermectin-sensitive collies revealed an identical four-base pair deletion in the first 10% of the transcript. This deletion causes a frame-shift mutation resulting in the production of a truncated, non-functional protein. The same four-nucleotide deletion was detected in all samples from ivermectin-sensitive collies, which were also

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5

homozygous for the deletion. Insensitive collies had a heterozygous genotype, with one mutant allele and one wild-type allele. Blood samples from all the other breeds showed homozygosity for the wild-type. The investigators concluded that their study provided evidence that the sensitivity of collies to ivermectin results from a frameshift deletion of four base pairs in the canine Mdrt gene (Mealey et al., 2001). (b)

Observations in Murray Grey cattle

Murray Grey cattle on one farm in the central tablelands of New South Wales, Australia, were reported to be sensitive to the toxicity of avermectin B1. The first cases were noted in October 1985, in 50 Murray Grey heifers aged 18-26 months treated with avermectin B1 at an estimated dose of 175-200 mg/kg bw by injection. Three of the heifers died within 2 days of treatment. Two weeks later, 144 Murray Grey cattle aged 4-18 months and weighing 200450 kg were treated with avermectin B1 at an estimated dose of 120-200 mg/kg bw by injection. The numbers of males and females treated were not stated. Within 48 h of treatment, three steers weighing 400-450 kg developed severe neurological signs, and all three were slaughtered for necropsy. A fourth steer in this group showed mild neurological signs and was slaughtered 19 days after treatment. A field trial was conducted on this farm with 208 Murray Grey cattle, comprising 90 steers that had been treated with avermectin B1 1-2 months earlier and a second group of 118 cattle that had not been treated previously. The animals were weighed and treated with the recommended therapeutic dose (200 mg/kg bw) or injected with the vehicle only. One steer in the group that had not been treated previously developed neurological signs 42 h after treatment and was slaughtered for necropsy. Brain, spinal cord, liver, kidney, lung, heart, spleen, intestines, skeletal muscles, adrenals, lymph nodes and peripheral nerves from the four initial cases, the case found in the field trial and one normal treated animal were examined macroscopically and histologically. No pathological changes were found that would explain the severe neurological syndromes observed. The concentrations of avermectin B1 in plasma and/or serum, liver, brain and spinal cord from the five clinically affected animals and the normal animal were assayed by high-performance liquid chromatography with fluorescence detection. The average concentration of avermectin B1a was 56 mg/ml in brain tissue from affected animals and 4 mg/ml in brain tissue from the normal animal. Two additional field trials were undertaken with Murray Grey cattle in other areas of New South Wales and in Victoria. A total of 83 cattle were treated with at least twice the normal therapeutic dose of avermectin B1. No adverse reactions occurred. The authors stated that no other incidents of toxic effects of avermectins have been reported in Murray Grey cattle. They also noted that the farm on which the adverse reactions were seen had maintained a virtually closed herd for approximately 15 years (Seaman etal., 1987). 2.1.2

Relative sensitivities of mice, rats, rabbits, dogs and non-human primates to the toxicity of avermectins

The relative sensitivities of the central nervous system in mice, rats, rabbits, dogs and non-human primates to avermectins have been reviewed (Lankas & Gordon, 1989). The studies conducted with ivermectin addressed acute toxicity in mice, rats, dogs and rhesus monkeys treated orally; short-term studies of toxicity in

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rats, dogs and rhesus monkeys; and studies of developmental and reproductive toxicity in mice, rats and rabbits. The studies with abamectin given by oral administration comprised long-term studies of toxicity and carcinogenicity in mice and rats, a 1-year study of toxicity in dogs and a study in rhesus monkeys given single doses. The authors concluded that clear species differences exist in the sensitivity of the central nervous system to the toxicity of avermectin, rodents being the most sensitive. A dose of 0.2 mg/kg bw in mice and slightly higher doses in rats resulted in clinical signs of central nervous system toxicity, comprising tremors and ataxia, while these doses caused no adverse effects in rabbits, dogs or rhesus monkeys. The authors described a study of acute toxicity in which groups of two male and two female rhesus monkeys were given abamectin or ivermectin at single oral doses of 0.2, 0.5,1, 2, 8,12 or 24 mg/kg bw. The time between administration of the next higher dose to the same group of monkeys was 2-3 weeks. The authors noted that the minimum single oral dose of ivermectin or abamectin that was toxic (2 mg/kg bw) was approximately 10-fold greater than the human clinical dose of ivermectin. Emesis was the only toxic effect observed in rhesus monkeys after an oral dose of ivermectin of 2 or 8 mg/kg bw; the clinical signs of toxicity observed after a dose of 24 mg/kg bw were emesis, mydriasis and sedation. The authors compared the effect at 8 mg/kg bw with effects seen in a child (age and sex not stated) after the apparently accidental ingestion of approximately 8 mg/kg bw. The toxic effects observed in the child were emesis, mydriasis and sedation. In view of the similarity of the toxic effects observed in rhesus monkeys and the child, the authors proposed that rhesus monkeys are an appropriate model for predicting the acute toxic effects of ivermectin in humans. The NOELfor acute effects after administration of abamectin or ivermectin to rhesus monkeys was 1 mg/kg bw. The review of the developmental and reproductive toxicity of ivermectin included the results of studies conducted in mice, rats and rabbits. The reported NOELs for maternal toxicity in mice, rats and rabbits were 0.1, 5 and 3 mg/kg bw per day, respectively. The reported NOELs for developmental toxicity in mice and rabbits were 0.2 and 1.5 mg/kg bw per day, respectively. A NOEL of 0.2 mg/kg bw per day was reported for neonatal and developmental toxicity in a multigeneration study in rats (Lankas & Gordon, 1989). 2.1.3 Relative potencies of doramectin, ivermectin and abamectin The Committee evaluated the relative potencies of doramectin, ivermectin and abamectin by comparing the NOEL values reported in evaluations by the Committee at its firty-fifth meeting, the JMPR in 1997 and Lankas and Gordon (1989). The studies of reproductive and developmental toxicity in rats and rabbits and the 90day studies of toxicity in dogs, all treated orally, were the only ones in which all three compounds could be compared. On the basis of these data, the Committee concluded that the potency of these compounds was similar. 2.2

Observations in humans

2.2.1 Polymorphism in the human MDR1 gene coding for P-glycoprotein In a study of 461 white volunteers in Germany (294 men and 167 women aged 18-65), DNA samples were analysed for polymorphisms in the MDR1 gene by

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7

polymerase chain reaction-restriction fragment length polymorphism assays. Eleven polymorphisms of MDR1 were identified in this population. The author stated that 16 polymorphisms were identified. The polymorphism identified in exon 26 of the MDR1 gene is associated with a specific alteration in drug transport. Volunteers with this polymorphism showed enhanced uptake of an oral dose of digoxin and a steady-state concentration that was 38% higher than that in volunteers without this polymorphism. The difference was statistically significant. Quantitative immunohistochemistry and western blot analysis were used to analyse the level of expression of P-glycoprotein in the duodenum of the volunteers. A correlation was found between decreased expression of P-glycoprotein and the polymorphism in exon 26. Thus, both the expression level and the functionality of P-glycoprotein are affected by this polymorphism. Controlled clinical trials and in-vitro studies are needed to determine the clinical relevance of other polymorphisms of the MDR1 gene. Data on the distribution of MDR1 polymorphisms in populations of other ethnic origins are not available (Hoffmeyer et al., 2000; Cascorbi et al., 2001). 2.2.2

Patients with onchocerciasis treated with ivermectin

Ivermectin has been administered to several million patients in Africa and Latin America for the treatment of onchocerciasis. A WHO report (WHO, 2001) stated that in 2000 alone, over 23 million people were treated. The reported adverse reactions in treated patients are allergic or inflammatory responses resulting from the killing of microfilarariae, referred to as the 'Mazotti reaction' (Pacque et al., 1989). No cases of acute central nervous system toxicity have been reported after treatment of patients for onchocericasis. When ivermectin was first distributed in 1987 to selected populations for the treatment of onchocerciasis, the drug sponsor contraindicated its use in pregnant women, mothers who were breastfeeding infants under 3 months of age, children under 12 years of age and people with active disease of the central nervous system, such as meningitis or epilepsy. These contraindications were listed as it was not known with certainty whether the blood-brain barrier of neonates and those with central nervous system disease would prevent entry of ivermectin. It was also unknown whether the placenta could prevent transfer of ivermectin to the fetus. The sponsor's evaluation of information that had become available since 1987 caused them to change the original list of contraindications. The new information includes elucidation of the genetic basis for the sensitivity of the CF-1 mouse, identification of P-glycoprotein in the human placenta and in the human fetus from week 28 of gestation (Cordon-Cardo et al., 1989), the finding of no significant risk to the fetuses of pregnant women who had been inadvertently treated with ivermectin (Pacque et al., 1990) and the demonstration that ivermectin is safe for patients with epilepsy (Kipp et al., 1992). As a result, use of ivermectin in pregnant or epileptic patients is now permitted, and treated mothers can breastfeed infants as young as 1 week of age (Brown, 1998). The usual dose administered to humans for the treatment of onchocerciasis is 150 ng/kg bw once every 12 months (Anon, 1999). 2.2.3

Healthy volunteers treated with ivermectin

The pharmacokinetics of orally administered ivermectin was studied in 12 healthy male (race not stated) volunteers aged 18-50 years. A single therapeutic dose of

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12 mg (150-200 fig/kg bw) as a tablet resulted in an average maximal time to peak plasma concentration of 3.6 h, an average maximal plasma concentration of 46 ng/ml and an average area under the plasma concentration-time curve of 880 ng/h per ml. No clinical adverse effects were reported (Edwards et al., 1988).

3.

COMMENTS

The genetic basis for the sensitivity of collie dogs to the neurotoxic effects of ivermectin was studied in four males and three females previously identified as sensitive to ivermectin and in six which showed no increased sensitivity. Sensitive animals were identified as those that exhibited typical clinical signs of toxicity to the central nervous system after receiving ivermectin at an oral dose of 120 jig/kg bw. The levels of P-glycoprotein expression were similar in sensitive and insensitive test animals; however, a specific variant of the gene coding for P-glycoprotein was identified in the sensitive animals that caused production of a severely truncated, non-functional form of P-glycoprotein. The Committee noted that the sensitivity of CF-1 mice to the toxicity of avermectins has also been linked to a variant of the gene responsible for expression of P-glycoprotein. When the levels or functionality of P-glycoprotein are reduced, avermectin compounds may penetrate the bloodbrain barrier and may be more extensively absorbed by the gastrointestinal tract. Sensitivity to the toxicity of avermectin B^ was observed in a herd of Murray Grey cattle in Australia in 1985. Eight of 312 cattle treated with ivermectin at a therapeutic dose of 120-200 ^ig/kg bw by injection showed symptoms of hypersensitivity. The average concentration of avermectin B1a in brain tissue from the affected animals was 56 ng/kg, while that in brain tissue from a normal animal was 4 ng/kg. No adverse reactions occurred in 83 additional Murray Grey cattle from other areas of Australia that were tested for sensitivity to avermectins by treating them with at least twice the normal therapeutic dose of avermectin 61. The Committee evaluated the relative potencies of doramectin, ivermectin and abamectin by comparing the NOEL values reported for reproductive and developmental toxicity in rats and rabbits and in 90-day studies of toxicity in dogs treated orally. These were the only studies with which such a comparison could be made. On the basis of these data, the Committee concluded that the potencies of these compounds are similar. Eleven variants of the human gene coding for P-glycoprotein were identified in a sample population of 461 white volunteers in Germany. One of the variants was correlated with decreased levels of P-glycoprotein expression in the duodenum. Volunteers with this variant gene showed enhanced bioavailability of an oral dose of digoxin, with a steady-state concentration that was 38% higher than in volunteers without the variant gene. The difference was statistically significant. Whether this variant could result in enhanced bioavailability of orally administered avermectins is unknown. Studies of variations in the gene coding for P-glycoprotein in populations of other ethnic groups have not been reported. The Committee noted that, although the effects resulting from variation in the human gene coding for P-glycoprotein are modest, the evidence to date does not exclude the possibility that a subpopulation of humans sensitive to the toxic effects of avermectins may exist.

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Ivermectin has been administered to several million human patients in Africa and Latin America since its introduction in 1987 as the main treatment for onchocerciasis at a recommended dose of 150 ng/kg bw administered once every 12 months. The adverse reactions that have been observed in treated patients have been described as allergic or inflammatory responses resulting from killing of microfilariae, referred to as the 'Mazotti reaction'. No signs of acute central nervous system toxicity have been reported. Ivermectin is now considered safe for use in pregnant women, on the basis of the finding of P-glycoprotein in human placentae and in human fetuses by week 28 of gestation and the absence of adverse effects to the fetus when pregnant women were inadvertently treated with ivermectin. The pharmacokinetics of orally administered ivermectin was studied in 12 healthy male volunteers of unspecified race. A single dose at a therapeutic level of 12 mg (150-200 |ig/kg bw) resulted in an average maximal plasma concentration of 46 ng/ml and an average time to maximum concentration in plasma of 3.6 h. No adverse clinical signs were reported. 4.

EVALUATION

An ADI for doramectin of 0-1 u.g/kg of bw was established on the basis of a NOEL of 0.1 mg/kg bw per day for mydriasis in a 3-month study in dogs treated by gavage, with a safety factor of 100. The Committee noted that removal of the twofold safety factor resulted in an ADI that still provided an adequate margin of safety for all other toxicological end-points of doramectin. The Committee also noted that the resulting ADI for dormectin is 150-200 times lower than the human therapeutic dose of the related compound ivermectin. The Committee took special note of the available information on reduced expression of P-glycoprotein in humans, which results in increased bioavailability of substrates for this transporter. However, the effects on the bioavailability of avermectins and their ability to penetrate the blood-brain barrier are unknown. The Committee recommended that human populations continue to be monitored for possible genetic predisposition to sensitivity to avermectins.

5.

REFERENCES

Anon. (1999) Product information for Stromectol® , ivermectin tablets. In: Physicians' Desk Reference (electronic version), PDR® Electronic Library Medical Economics Company Inc., Montvale, NJ. URL: www.pdrel.com. Brown, K.R. (1998) Changes in the use profile of Mectizan: 1987-1997. Ann. Trop. Med. Parasitol., 92 (Suppl. 1), S61-S64. Cascorbi, I., Gerloff, T, Johne, A., Meisel, C., Hoffmeyer, S., Schwab, M., Schaeffeler, E., Eichelbaum, M., Brinkmann, U. & Roots, I. (2001) Frequency of single nucleotide polymorphisms in the P-glycoprotein drug transporter MDR1 gene in white subjects, din. Pharmacol. Ther., 69, 169-174. Cordon-Cardo, C., O'Brien, J.P., Casals, D., Rittman-Grauer, L., Biedler, J.L., Melamed, M.R. & Bertino, J.R. (1989) Multidrug-resistance gene (P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. Proc. NatlAcad. Sci. USA, 86, 695-698. Edwards, G., Dingsdale, A., Helsby, N., Orme, M.L'E. & Breckenridge, A.M. (1988) The relative systemic availability of ivermectin after administration as capsule, tablet, and oral solution. Eur. J. din. Pharmacol., 35, 681-684.

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FAO/WHO (1998) 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, No. 145; available on the Internet at www.fao.org/ag/agp/agpp/pesticid/jmpr/pm-jmpr.htm. Hoffmeyer, S., Burk, O., von Richter, O., Arnold, H.P., Brockmuller, J., Johne, A., Cascorbi, I., Gerloff, T., Roots, I., Eichelbaum M. & Brinkmann, U. (2000) Functional polymorphisms of the human multidrug-resistance gene: Multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc. Natl Acad. Sci. USA, 97, 3473-3478. Kipp, W., Burnham, G. & Kamugisha, J. (1992) Improvement in seizures after ivermectin. Lancet, ii, 789-790. Lankas, G.R. & Gordon, L.R. (1989) Toxicology. In: Campbell, W.C., ed., Ivermectin and Abamectin, New York: Springer-Verlag, pp. 89-112. Mealey, K.L., Bentjen, S.A., Gay, J.M & Cantor, G.H. (2001) Ivermectin sensitivity in collies is associated with a deletion mutation of the mdrl gene. Pharmacogenetics, 11, 727-733. Pacque, M., Munoz, B., Poetschke, G., Foose, J., Greene, B.M. & Taylor, H.R. (1990) Pregnancy outcome after inadvertent ivermectin treatment during community-based distribution. Lancet, ii, 1486-1489. Pacque, M., Dukuly, Z., Greene, B.M., Munoz, B., Keyvan-Larijani, E., Williams, P.N. & Taylor, H.R. (1989) Community-based treatment of onchocerciasis with ivermectin: Acceptability and early adverse reactions. Bull. World Health Org., 67, 721-730. Seaman, J.T., Eagleson, J.S., Carrigan, M.J. & Webb, R.F. (1987) Avermectin 61 toxicity in a herd of Murray Grey cattle. Aust. Vet. J., 64, 284-285. Umbenhauer, D.R., Lankas, G.R., Pippert, T.R., Wise, L.D., Cartwright, M.E., Hall, S.J. & Beare, C.M (1997) Identification of P-glycoprotein-deficient subpopulation in the CF-1 mouse strain using a restriction fragment length polymorphism. Toxicol. Appl. Pharmacol., 146, 88-94. WHO (2001) Programme for the Prevention of Blindness and Deafness, www.who.int/pbd/ oncho/oncho-brochure.pdf.

TIABENDAZOLE (THIABENDAZOLE) (addendum) First draft prepared by M.E.J. Pronk and G.J. Schefferlie Centre for Substances and Risk Assessment National Institute for Public Health and the Environment Bilthoven, The Netherlands

Explanation Biological data Biochemical aspects: Absorption, distribution and excretion Toxicological studies Acute toxicity Short-term studies of toxicity Reproductive toxicity Multigeneration studies Developmental toxicity Observations in humans Selection of relevant end-points Mechanism of action Clinical signs Toxicity to specific organs Effects on the kidney Effects on the haematopoietic system Developmental toxicity Comments Evaluation References

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11 12 12 12 12 13 14 14 14 16 17 17 18 18 18 21 22 22 24 24

EXPLANATION

Tiabendazole (thiabendazole) is a benzimidazole compound used both as a broad-spectrum anthelmintic in various animal species and for the control of parasitic infestations in humans. It was evaluated by the Committee at its fortieth meeting (Annex 1, reference 104). An ADI of 0-100 u,g/kg bw was established on the basis of reduced body-weight gain in a 2-year study in rats and reduced fetal weight in a study of developmental toxicity in rats, by applying a safety factor of 100 to the NOEL of 10 mg/kg bw per day. At its forty-eighth meeting (Annex 1, reference 128), the Committee reviewed the results of supplementary studies that allowed it to confirm its earlier evaluation. The NOELs in the 12-month study in dogs, the 2-year study of toxicity in rats and the two-generation study of reproductive toxicity in rats were all 10 mg/kg bw per day, identical to the NOEL that had served as the basis for the ADI. The Committee applied a safety factor of 100 and confirmed the ADI of 0-100 u,g/kg bw established at its fortieth meeting. As tiabendazole is also used as a fungicide in plant protection, its toxicity was also evaluated by the 1970 and 1977 Joint FAO/WHO Meeting on Pesticide Residues (JMPR) (FAO/WHO, 1971, 1978). At the 2000 JMPR (FAO/WHO, 2001), at which the residue and analytical aspects of tiabendazole were evaluated, the Meeting -11 -

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12

concluded that the toxicological profile of tiabendazole included effects of concern that might indicate a need for an acute reference dose (RfD). The Meeting recommended that tiabendazole be considered further by JECFA, which had conducted the most recent toxicological assessment of this chemical. The Committee did not receive new data for establishing an acute RfD for tiabendazole. All the data considered had been evaluated and summarized previously by the Committee, at its fortieth and forty-eighth meetings. Those data were reevaluated by the Committee at its present meeting, when it focused on aspects relevant for establishment of an acute RfD. In addition, the Committee consulted the literature for recently published information on the toxicity of tiabendazole and considered those relevant for this evaluation.

2.

BIOLOGICAL DATA

2.1

Biochemical aspects: Absorption, distribution and excretion

After oral administration to mice, rats, dogs and humans, tiabendazole is rapidly absorbed (peak plasma concentrations within 3 h), followed by rapid elimination, which is essentially complete within 1 day for humans and rats and 3 days for dogs and mice. The half-time in humans is < 2 h. In humans, rats and mice, urinary excretion predominated over faecal excretion. In dogs, the amount excreted in faeces was somewhat greater than that in urine (Annex 1, reference 105; Tocco et al., 1966). Given these toxicokinetics, accumulation in the body is not expected. 2.2

Toxicological studies

2.2.1 Acute toxicity The acute toxicity of thiadibendazole is summarized in Table 1. The study by Lankas (1981) was not evaluated previously by the Committee. At doses ranging from 2200 to 11 000 mg/kg bw, the signs noted were decreased activity, bradypnoea, ptosis and loss of righting reflex. In the studies of Robinson (1964), the doses used were not specified. The toxic signs were described as lethargy and stupor. No further details were given. In the study with rabbits treated dermally (Blaszcak & Auletta, 1987), no toxic signs were noted. Table 1. Acute toxicity of thiabendazole Species Sex

Route

LD50 (mg/kg bw)

Mouse

Oral Oral Oral Inhalation3 Oral Dermal

Robinson (1964) 3800 No Robinson (1964) 3300 No Lankas (1981) Yes 4700-5100 3 > 400 mg/m Gurmanetal. (1981) Yes Robinson (1964) 3800 No Blaszcak & Auletta (1987) No >2000

Rat Rat Rat Rabbit Rabbit

Female Male Male and female Male and female Not specified Male and female

GLP, good laboratory oractice; QA, quality assurance Gravimetric concentration; 4 h exposure

a

Reference

GLP/QA

TIABENDAZOLE

13

The rats treated by inhalation had squinted eyes, polypnoea and body-weight loss (Gurman et al., 1981). 2.2.2

Short-term studies of toxicity Rats

In a 13-week study of toxicity reviewed by the Committee at its fortieth meeting (Annex 1, reference 105), groups of 10 male and 10 female Sprague-Dawley Crl:CD BR rats received diets containing tiabendazole at concentrations intended to provide a daily dose of 0, 10, 40, 160 or 320 mg/kg bw. The calculated mean intakes of tiabendazole were 0, 9, 37, 150 and 300 mg/kg bw per day, respectively. Clinical signs were recorded weekly, and haematological end-points were measured in samples taken at weeks 6 and 13. The following observations may be relevant for the acute toxicity of this compound. No effects were seen in controls or in animals at the lowest dose. At 37 mg/kg bw per day, females showed an increased platelet count at both weeks 6 and 13, and both sexes showed an increased incidence of erythroid hyperplasia in the bone marrow. At the two higher doses, the following dose-related changes were seen: an increase in the incidence of alopecia; a decrease in erythrocyte count, haemoglobin concentration and erythrocyte volume fraction at weeks 6 and 13; an increased frequency of abnormal erythrocyte morphology at both times; an increase in platelet count at both times (only at week 13 in males at 150 mg/kg bw per day); and increased incidences of erythroid hyperplasia in bone marrow and haemoglobin pigment in spleen. The Committee at its previous meeting identified a NOEL of 9 mg/kg bw per day on the basis of decreased body-weight gain and food intake in males, increased liver weight and centrilobular hypertrophy, increased thyroid weight, follicular cell hypertrophy and bone-marrow erythroid hyperplasia at doses of 37 mg/kg bw per day and above. The effects on the haematopoietic system at both sampling times (6 and 13 weeks), together with the erythroid hyperplasia in bone marrow, were indicative of anaemia and were considered potentially relevant for acute exposure. The NOEL for these effects was 9 mg/kg bw per day (Myers & Lankas, 1990). Dogs In a 14-week study of toxicity reviewed by the Committee at its fortieth meeting (Annex 1, reference 105), groups of four male and four female beagle dogs received tiabendazole orally in gelatin capsules at a daily dose of 0, 35, 75 or 150 mg/kg bw. Clinical signs were recorded twice daily, and haematological end-points were measured at weeks 4, 8 and 12. The following observations may be relevant for acute toxicity. No changes were seen in controls or those at the lowest dose. Animals at the two higher doses showed dose-related increases in salivation and emesis, the latter effect mainly during the first few weeks of the study. At the highest dose, decreased erythrocyte count, haemoglobin and erythrocyte volume fraction were seen, the effects being more frequent at weeks 4 and 8 than at week 12. The Committee at its previous meeting identified a NOEL of 35 mg/kg bw per day on the basis of histopathological changes in the gall-bladder. This effect was, however, considered not relevant for assessing the effects of acute exposure. The haematological findings at the first two sampling times may be indicative of anaemia

14

TIABENDAZOLE

and were considered potentially relevant for acute exposure. The NOEL for these effects was 75 mg/kg bw per day (Batham & Lankas, 1990). Groups of four male and four female beagle dogs received tiabendazole orally in gelatin capsules at a daily dose of 0,10,40 or 160 mg/kg bw for 1 year in a study of toxicity reviewed by the Committee at its fortieth meeting (Annex 1, reference 105). Clinical signs were recorded daily and haematological end-points were measured in samples taken at weeks 4,12, 26 and 52. The following observations may be relevant to the acute toxicity of this compound. No effects were seen in controls or animals at the lowest dose. Animals at the highest dose showed emesis, especially during the first 3 weeks and in females throughout the first half of the study. Animals at the highest dose showed decreased erythrocyte count, haemoglobin concentration and erythrocyte volume fraction and increased activated partial thromboplastin time and platelet count throughout the study (weeks 4-52). Histological examination showed a dose-related increase in the frequency of distal tubule vacuolation in the kidney of females; a dose-related increase in haemosiderin deposition in the spleen of females, with a very slight, not statistically significant effect in males at all doses with no dose-response relationship; and a dose-related increase in splenic erythropoiesis in animals at the two higher doses. Increased bone-marrow erythropoiesis was seen only in females at the highest dose. The Committee at its previous meeting identified a NOEL of 10 mg/kg bw per day on the basis of (histopathological) changes indicative of haemolytic anaemia. As haematological changes were observed throughout the study, all changes to the haematopoietic system, including the histopathological changes, were considered potentially relevant to acute intake as well. It was noted that the effects in spleen and bone marrow seen after 53 weeks were not found in the 14-week study in dogs (Lankas, 1993). 2.2.3 Reproductive toxicity (a)

Multigeneration studies

Rats In a two-generation study of reproductive toxicity, groups of 33 male and 33 female Sprague-Dawley Crl:CD BR rats received diets containing tiabendazole (purity, > 99%) at concentrations providing a dose of 0, 10, 30 or 90 mg/kg bw per day (Wise & Lankas, 1992). This study was summarized by the Committee at its forty-eighth meeting (Annex 1, reference 129). The only treatment-related findings were effects on food consumption and body-weight gain, for which the Committee previously identified a NOELof 10 mg/kg bw per day. These effects were considered irrelevant for acute exposure. (b)

Developmental toxicity

Mice Three studies of developmental toxicity were undertaken in pregnant JcUCR mice. Tiabendazole (purity, 98.5%) was given orally as a suspension in olive oil by gastric intubation. The animals were killed on day 18 of gestation. These studies were summarized by the Committee at its fortieth meeting (Annex 1, reference 105).

TIABENDAZOLE

15

In the first experiment, mice were given a dose of 0, 700, 1300 or 2400 mg/kg bw on days 7-15 of gestation. Maternal body-weight gain was decreased in a doserelated fashion at all doses, and the mortality rate increased with increasing dose, being 0/39 controls, 0/39 at the lowest dose, 5/39 at the intermediate dose and 24/39 at the highest dose. The weights of the liver, kidney, heart and spleen were increased at all three doses. A dose-related increase in the frequency of resorptions was seen at the two higher doses, and a dose-related decrease in fetal body weight occurred at all doses. In the offspring, the incidence of cleft palate was increased in a dose-related fashion at all doses, and the incidence of fusion of vertebral arches and vertebral bodies was increased in offspring of dams at the two lower doses. In the second experiment, animals were given a dose of 2400 mg/kg bw on a single day between days 6 and 15 of gestation. The maternal mortality rates were 2/7,2/12,1/12,2/11,2/11,6/11,2/11,1/11,4/11 and 6/11 on days 6-15, respectively. The rate of resorptions was increased and the fetal body weight decreased after dosing on any day. Increased frequencies were seen of microencephaly and exencephaly after treatment on day 6, 7 or 8; short or absent tail and anal atresia after dosing on day 9; open eyelids after treatment on day 7,8,10,13 or 14; reduction deformity of the limbs after dosing on day 9,10,11 or 12; cleft palate after dosing on day 8, 9, 10, 11, 12 or 13; fusion of vertebral arches and vertebral bodies after treatment on day 7, 8,9,10 or 13; and fusion of the ribs after dosing on day 7, 8 or 9. In the third experiment, groups of 21-31 mice were given tiabendazole at a single dose of 0, 30, 60, 62, 120, 130, 240, 270, 480, 560, 670, 800, 960, 1200, 1400, 1700, 2000 or 2400 mg/kg bw on day 9 of gestation. Maternal weight gain was decreased at 1200 mg/kg bw, the maternal mortality rate was increased at 1700 mg/kg bw, and the weights of the liver, heart and kidney weight were decreased at 1400 mg/kg bw. The resorption rate was increased at 1700 mg/kg bw, and the fetal body weight was decreased at 60 mg/kg bw. The incidence of reduction deformity of the limbs was increased at 480 mg/kg bw, and that of fusion of vertebral arches and vertebral bodies and of ribs was increased at 240 mg/kg bw (Ogata et al., 1984). In a study of developmental toxicity, groups of 25 pregnant JcklCR mice were given tiabendazole (purity, 99.8%) in olive oil by oral gavage at a dose of 0, 25,100 or 200 mg/kg bw per day on days 6-15 of gestation. The animals were killed on day 18 of gestation. This study was evaluated by the Committee at its forty-eighth meeting (Annex 1, reference 129). Dams at the two higher doses showed doserelated decreases in food consumption and weight gain, and there were dose-related decreases in the number of implantations and fetal body weight at these doses. An increased incidence of delayed ossification was seen at all doses, but this was not dose-related, and the number of affected litters was similar in all groups, including controls. The NOEL was 25 mg/kg bw per day on the basis of the reduced number of implantations at a maternally toxic dose (Nakatsuka et al., 1995). Rats In a study of developmental toxicity, groups of 25 pregnant Sprague-Dawley Crl:CD BR rats received tiabendazole (purity, 98.9%) in 0.5% methylcellulose by gavage at a daily dose of 0,10, 40 or 80 mg/kg bw on days 6-17 of gestation. The animals were killed on day 20 of gestation. This study was evaluated by the

16

TIABENDAZOLE

Committee at its fortieth meeting (Annex 1, reference 105). The food consumption and weight gain of the dams at the two higher doses were decreased in a doserelated manner, and dams at the highest dose showed ptosis and regurgitation. A dose-related decrease in fetal body weight was seen at the two higher doses. The NOEL was 10 mg/kg bw per day (Wise, 1990). Rabbits In a study of developmental toxicity, groups of 18 pregnant New Zealand white rabbits received tiabendazole (purity, 98.9%) in 0.5% methylcellulose by gavage at a dose of 0, 24, 120 or 600 mg/kg bw on days 6-18 of gestation. The animals were killed on day 29 of gestation. This study was evaluated by the Committee at its fortieth meeting (Annex 1, reference 105). Dose-related decreases in food consumption and weight gain were seen at the two higher doses, with a loss of body weight at the highest dose. Four of 18 dams at the highest dose aborted. The mortality rates were 2/18 controls (due to intubation errors), 0/18 at the lower dose, 0/18 at the intermediate dose and 1/18 at the highest dose. A dose-related increase in the rate of early and late resorptions was seen at the two higher doses, and fetuses at these doses had dose-related increased incidences of domed head, hydrocephalus and marked enlargement of the fontanelle. The NOEL was 24 mg/kg bw per day (Hoberman, 1989). In another study of developmental toxicity, evaluated by the Committee at its fortieth meeting (Annex 1, reference 105), groups of 18 pregnant New Zealand white rabbits received tiabendazole (purity, 98.6%) in 0.5% methylcellulose by gavage at a dose of 0, 50, 150 or 600 mg/kg bw per day on days 6-18 of gestation. The animals were killed on day 28 of gestation. Effects were seen only at the highest dose; they comprised decreased maternal food consumption and weight gain, increased rates of early and late resorptions, decreased fetal body weight and increased incidences of variation in lung lobation and incompletely ossified metacarpals. No evidence for compound-related fetal hydrocephaly was found. The NOEL was 150 mg/kg bw per day (Lankas & Wise, 1991/1993). 2.3

Observations in humans

In a study with volunteers, 50 men aged 20-57 years received capsules containing placebo and 50 received 125 mg of tiabendazole twice daily for 24 weeks. Neither the study subjects nor the investigators were aware of who had received placebo. In total, 36 men receiving tiabendazole and 41 receiving placebo completed the study. One man was removed from the study at his own request because of daytime sedation and markedly decreased energy. The other withdrawals were clearly unrelated to the treatment. Weekly interviews were conducted to record side-effects. General physical examinations and laboratory examinations (haematology, measurement of cholesterol, glucose, urea, alkaline phosphatase, thymol turbidity, bilirubin in serum and urine analysis) were carried out before the test and after 4,12 and 24 weeks. The haematological parameters measured were reported only as 'CBC' and haematocrit. Protein-bound iodine in serum and electrocardiographic traces were evaluated only at the beginning and after 24 weeks of the study. This study was previously summarized by the 1977 JMPR (FAO/WHO, 1978), which noted that, under the conditions of the study, tiabendazole was well tolerated,

TIABENDAZOLE

17

and no effect on any of the parameters could be clearly ascribed to treatment. The JMPR identified a NOEL of 3-4 mg/kg bw per day, which was confirmed by the Committee at its fortieth meeting (Annex 1, reference 105). The following observations may be relevant for the acute toxicity of tiabendazole, although the time of onset of clinical signs was not specified. The men reported the following possible side-effects (treated versus placebo): increased appetite (26/50 vs 30/50), flatulence (6/50 vs 3/50), nausea (4/50 vs 2/50), increased urinary frequency (3/50 vs 3/50) and sedation (7/50 vs 5/50) (Colmore, 1965). In a review of studies on the efficacy of tiabendazole against parasites in humans, the standard therapeutic oral dose was 25 mg/kg bw twice daily for 1-4 days, although higher doses were used in some studies. The incidences of minor transient sideeffects were generally 25-30% with the standard dose and higher with higher doses. The effects comprised anorexia, nausea, vomiting and dizziness. Serious side-effects were rare and comprised numbness, collapse, tinnitus, abnormal sensation in the eyes, xanthopsia, enuresis, decreased pulse rate and systolic blood pressure and transient rises in the frequency of cephalin flocculation and in aspartate aminotransferase activity (Campbell & Cuckler, 1969). Side-effects in humans after therapeutic oral doses (not specified) of thiabendazole were reported in another literature review. Common effects were dizziness (the frequency ranging from < 5% to 80%, depending on dosage) and nausea and vomiting (5-15%). Rarely observed side-effects included anorexia, abdominal pain, headache, drowsiness, weariness, heartburn, diarrhoea or constipation, flatulence, blurring of vision, xanthopsia, skin eruption, malodorous urine and vomiting of live Ascaris. The extent to which these frequencies differed from those in untreated subjects is unknown, although in two placebo-controlled studies dizziness was reported to be approximately twice as common in tiabendazole-treated subjects as in placebotreated subjects (Cuckler & Mezey, 1966). In a clinical case report, the following side-effects were reported in 14 of 23 patients with trichinosis who had received tiabendazole orally at a dose of 50 mg/kg bw in two daily doses for 10 days: nausea (11/23), retching (11/23), vomiting (11/23), aversion to tablets (3/23), exanthema (3/23), impotence (2/23), diarrhoea (1/23), liver damage (1/23), fever (1/23) and dizziness (1/23) (Hennekeuser et al., 1969). Again, the incidence of side-effects in untreated patients was not reported, and the extent to which these effects might have been influenced by the underlying condition was not reported.

3.

SELECTION OF RELEVANT END-POINTS

The end-points relevant to acute intake are identified below, on the basis of guidance given by the Commission of the European Union (2001), the 2000 JMPR (FAO/WHO, 2001) and van Raaij (2001). 2.1

Mechanism of action

The mechanism of action of tiabendazole is not clear, but it might involve inhibition of the fumarate reductase system of worms, thereby interfering with their source of

18

TIABENDAZOLE

energy (Parfitt, 1999). As fumarate is a link in the citric acid cycle, which is a common pathway in organisms, this possible mode of action of tiabendazole may also be relevant in assessing the risks of humans after acute exposure. An indication of the toxicity resulting from this mode of action may be found in the mitochondrial dysfunction observed in the renal cortex of mice given a single oral dose of 1000 or 2000 mg/kg bw (see section 3.3.1). 3.2

Clinical signs

The studies of acute toxicity gave oral LD50 values above 2000 mg/kg bw, raising no concern. The clinical signs described in these studies were relatively unspecific (e.g. decreased activity), and the correlation with doses was not reported. It remains unclear whether these signs were substance-specific or related to the dosing procedure. They were therefore not considered in establishment of an acute RfD. In the studies with repeated doses, the only specific clinical sign in rats (13week study) was alopecia, which was observed at the highest dose of 300 mg/kg bw per day, with a NOEL of 150 mg/kg bw per day. However, the onset of this sign was not earlier than after 5 or 6 weeks of treatment, and it is therefore not relevant for acute exposure. It was noted that this effect was also reported in a 28-day study in rats treated orally (summarized by the Committee at its fortieth meeting (Annex 1, reference 105) but not available in the present dossier) at the highest dose of 800 mg/kg bw per day, but not at lower doses up to 200 mg/kg bw per day. In dogs, the only treatment-related clinical signs were salivation and emesis. These effects, particularly emesis, are considered relevant for acute exposure, because vomiting is also a common side-effect in humans. The NOEL for emesis in dogs was 40 mg/kg bw per day. Common side-effects reported in humans after oral doses > 25 mg/kg bw twice daily for 1-10 days comprised anorexia, nausea, vomiting and dizziness (data from 1966-69; no recent data available and often no control data for comparison). NOELs could not be identified. In a placebo-controlled study in volunteers, a dose of 125 mg of tiabendazole twice a day for 24 weeks (equivalent to 3.6 mg/kg bw per day for a 60-kg person) did not cause significant changes in subjective side-effects. 3.3

Toxicity in specific organs

3.3.1 Effects on the kidney No effects on the kidney were found in the studies available to the Committee, after repeated doses in rats or in the studies on reproductive and developmental toxicity in rats and rabbits. In dogs, renal toxicity was observed in the 1-year study (NOEL, 10 mg/kg bw per day) but not in the 14-week study at comparable doses. In contrast, renal toxicity was observed after acute and short-term oral intake of tiabendazole in mice. This effect was described in earlier evaluations by the Committee, as well as in some published articles, but none of these studies was submitted in the dossier. In a short-term study of toxicity, mice were given tiabendazole at a dose of 0, 250 or 500 mg/kg bw per day by gavage for 1, 3, 5 or 7 days. Dose-related renal toxicity was observed, which included tubule dilatation, degenerative desquamation, cell infiltration, fibrosis and regeneration of tubule epithelium. Electron microscopy

TIABENDAZOLE

19

showed evidence of glomerular damage, such as flattening of the foot processes of podocytes and oedematous changes in the mesangium in treated mice. No NOEL could be identified (Tada et al., 1989). Effects on the kidneys were also observed in one 13-week study in rats and some of the long term (2-year) studies in rats that had been evaluated earlier by the JMPR or JECFA but which were not available in the present dossier. In a published report not previously evaluated by the Committee, male Crj:CD-1 (ICR) mice were given tiabendazole (purity, 98.5%) in olive oil as a single dose of 0, 250, 500 or 1000 mg/kg bw by oral gavage, and the kidneys were examined histologically 1,3,5 or 24 h after dosing. The histological findings were desquamation of degenerated cells in proximal tubules and production and release of apical cytoplasmic blebs from epithelial cells into the proximal tubule lumen of treated mice from 1 h onwards. Dilatation of proximal, distal and collecting tubules was apparent in treated mice at 3, 5 and 24 h. Pre-treatment with inducers of the microsomal mono-oxygenase system reduced the renal injury, while pre-treatment with inhibitors of that system enhanced the effects, suggesting that the toxicity was caused by the parent compound rather than by its metabolites. Pre-treatment with inhibitors of organic cation or anion transport indicated that tiabendazole is transported into tubule cells through the organic cation transport system for 1-5 h after dosing. No NOEL could be identified (Tada et al., 1992). Recovery from the nephrotoxicity of tiabendazole was reported in mice after a single dose in a published report that had not previously been evaluated by the Committee. Male and female Crj:CD-1 (ICR) mice were given a single dose of tiabendazole (purity, 98.5%) suspended in olive oil at a dose of 0, 125, 250, 500, 1000 or 2000 mg/kg bw by oral gavage. The animals were necropsied 24 h thereafter. Additional groups of mice were given single oral doses of 1000 or 2000 mg/kg bw and necropsied 1, 2, 3, 5, 7 or 10 days after dosing in order to study recovery from the effects. Further groups of mice were given a single oral dose of 0 or 2000 mg/kg bw and were observed for 10 days to study the effect on the mortality rate. By 10 days after administration of 2000 mg/kg bw, 30% of the females and 60% of the males had died. Mice given 1000 or 2000 mg/kg bw showed higher serum urea nitrogen and aspartate aminotransferase concentrations 24 h after dosing. At the same time, mice given 250 mg/kg bw or more showed dose-dependent renal toxicity. Histological examination showed desquamation of degenerated cells in the proximal tubules and dilatation of proximal, distal and collecting tubules. Necrosis of proximal tubules was observed at doses of 500 mg/kg bw and higher. The changes at 250 mg/kg bw were slight, and the kidneys of mice treated at 125 mg/kg bw were not different from those of control mice. In the recovery experiment, tiabendazole-treated mice had higher urine volumes with higher concentrations of glucose, protein and sodium, in particular during the first few days after administration. The serum urea nitrogen concentrations returned to control levels within 3 and 7 days in mice given 1000 and 2000 mg/kg bw, respectively. The increased aspartate aminotransferase concentration in all treated mice recovered to normal within 3 days. The increased absolute and relative kidney weights had returned to control levels at 5 and 7 days, respectively, but pale foci and rough surface were still observed in the kidneys at the end of the study. The dilatation of tubules observed at both doses did not revert during the 10-day observation period. Mitochondria! swelling was seen in proximal

20

TIABENDAZOLE

tubule epithelium during the first 2 days after treatment. Necrosis of proximal tubules and desquamation of degenerated cells in proximal tubules were not observed after 7 and 10 days of recovery, respectively. Tissue repair processes (cell infiltration, fibrosis, regeneration of tubules) started 3 days after exposure and were still apparent in all animals 10 days after exposure. The Committee concluded that the tiabendazole-induced renal toxicity was most frequent 2 or 3 days after dosing and was followed by regeneration. The NOEL for renal toxicity was 125 mg/kg bw (Tada etal.,1994). In a 13-week study of toxicity, groups of male and female Crj:CD-1 (ICR) mice received a diet containing 0, 0.8 or 1.6% tiabendazole (purity not reported). The report was not included in the dossier and had not previously been evaluated by the Committee. The doses were equivalent to 0, 1200 and 2400 mg/kg bw per day, respectively. Anaemia and liver and kidney damage were the main effects at both doses (Tada et al., 1996). The study was considered to be of less value than others for determination of an acute RfD in view of its long duration and the high doses. Another study of toxicity in mice was reported in a published paper that had not previously been evaluated by the Committee. Male Crj:CD-1 (ICR) mice were given diets containing 0, 0.8, 1.2 or 1.6% tiabendazole (purity, > 99%) for 44 weeks. The doses were equivalent to 0, 1200, 1800 and 2400 mg/kg bw per day, respectively. Neither haematological nor urine analyses were performed. Liver, kidney and gallbladder damage were the main effects at all doses (Tada et al., 2001). The study was considered to be of less value than others for determination of an acute RfD in view of its long duration and the high doses used. The same group of investigators studied the structural alterations to mitochondria in renal proximal tubule cells to determine the mechanism of the acute renal tubule necrosis. The published paper had not previously been evaluated by the Committee. Groups of male CRj:CD-1(ICR) mice were given tiabendazole in olive oil by oral gavage at a single dose of 0, 1000 or 2000 mg/kg bw and were killed 3, 6 or 16 h after dosing. The following end-points were determined: mitochondria! respiration, the tiabendazole concentration in renal cortex and mitochondrial fraction (also at 0.5, 1 and 24 h), histochemical analysis of mitochondrial enzyme (NAD-linked isocitrate dehydrogenase) and the concentration of ATP in the renal cortex. Mitochondrial respiration was statistically significantly decreased in a dose-dependent manner 6 and 16 h after dosing but not 3 h after dosing. When additional groups of mice were treated with tiabendazole at 0, 250 or 500 mg/kg bw and killed 16 h later, no effects on mitochondrial respiration were observed. The concentration of tiabendazole in the renal cortex reached a maximum 1 h after dosing and declined thereafter. The concentration in the mitochondrial fractions followed the same pattern. Effects on the activity of NAD-linked isocitrate dehydrogenase (a marker enzyme of mitochondria) were not observed 3 or 6 h after dosing, whereas significant inhibition of this activity was observed 16 h after administration of 1000 or 2000 mg/kg bw. Dilatation of tubules, observed at all times after administration of 1000 or 2000 mg/kg bw, was observed mainly in the area where inhibition of NAD-linked isocitrate dehydrogenase was found. Necrosis of renal tubules was not observed. The ATP concentration in the renal cortex, determined only 16 h after a single oral dose of 0,

TIABENDAZOLE

21

500, 1000 or 2000 mg/kg bw, was significantly decreased at 1000 and 2000 mg/kg bw but not at the lowest dose (Fujitani et al., 1998). The results of the studies summarized above suggest that, after single doses of tiabendazole to mice, the compound is taken up by tubule cells in the renal cortex through the organic cation transport system and subsequently inhibits mitochondrial respiration, leading to depletion of ATP and ultimately to necrosis of proximal tubule cells. The reduced mitochondrial respiration may be due to inhibition of the fumaratereductase system, which is the putative mechanism of action of tiabendazole (Parfitt, 1999). Tada and colleagues have suggested that cell debris obstructs the tubules, leading to dilatation. This explanation is not entirely consistent, because dilatation was observed at lower doses than necrosis and lasted longer. Dilatation of proximal tubules was observed at single doses of 250 mg/kg bw and higher. The lowest dose of 125 mg/kg bw was therefore the NOEL for acute renal toxicity in mice. 3.3.2 Effects on the haematopoietic system In both rats and dogs, a number of haematological parameters were changed after repeated oral intake of tiabendazole, sometimes with related histopathological changes in the spleen and bone marrow. Although the studies were of short duration (13 weeks in rats and 14 and 53 weeks in dogs), the interim analysis of blood samples (starting at week 4 or 6) showed haematological changes early in the study, which were occasionally more frequent at earlier times than at the end of the study. The related histopathological changes were also observed at doses at which haematological changes were not yet or no longer seen. The haematological and histopathological changes are indicative of anaemia, and, as these changes could have occurred after one or a few doses, they were considered relevant to acute intake. The NOELs for this effect in rats and dogs were 9 and 10 mg/kg bw per day, respectively. In addition to the studies summarized above, the Committee at its fortieth meeting reviewed three other studies of the same or shorter duration in rats treated by gavage. These studies, which were not available at the present meeting, comprised two 4week studies with animals given tiabendazole at doses of 50-1600 mg/kg bw per day and one 13-week study with rats given doses of 25-400 mg/kg bw per day. The 4-week studies also showed haematological changes (NOEL, 100 mg/kg bw per day) and histopathological changes in the spleen and/or bone marrow (LOEL, 50 mg/kg bw per day). In the 13-week study, changes were observed in red blood cell parameters, and histopathological changes were seen in the spleen (NOEL for both effects, 25 mg/kg bw per day). As the studies could not be reviewed by the present Committee, it could not verify whether the bone marrow was affected in the 13-week study. It should be noted that in the study of Colmore (1965) with male volunteers, a dose of 125 mg twice a day for 24 weeks (equivalent to 3.6 mg/kg bw per day for a 60-kg person) did not affect haematological parameters after 4, 12 or 24 weeks of treatment. However, this study has a number of serious shortcomings: the haematological parameters (other than haematocrit and 'CBC') investigated were not specified; only one dose was tested, so that the dose-response relationship

22

TIABENDAZOLE

could not be investigated; and, more importantly, no histopathological examination was carried out, while, in animals, this appeared to be a more sensitive indicator of haematotoxicity than the haematological parameters. 2.4

Developmental toxicity

As short treatments are used in studies of developmental toxicity orteratogenicity and because, in particular, teratogenic effects and resorptions may be induced by a single dose within a certain (sensitive) period, effects on the fetus were considered relevant for setting an acute RfD. With regard to teratogenic effects, domed heads, hydrocephalus and marked enlargement of fontanels were observed in one study in rabbits at doses of 120 mg/kg bw per day and higher. The NOEL was 24 mg/kg bw per day. In another study in rabbits, no such effects were observed at doses up to 600 mg/kg bw per day. The NOEL in this study was 150 mg/kg bw per day, on the basis of an increased incidence of malformations, which was seen relatively often in this species. In mice, the teratogenic effects after a single exposure on day 9 of gestation consisted of deformed limbs at doses of 480 mg/kg bw and higher (NOEL, 26 g/kg bw) and fusion of vertebral arches, bodies and ribs at doses of 240 mg/kg bw and higher (NOEL, 130 mg/kg bw). In rats, no teratogenic effects were observed. Increased resorption rates were observed in mice and rabbits but not in rats. In mice, the NOELs for this effect were 700 mg/kg bw per day when the dams were exposed on days 7-15 of gestation and 1400 mg/kg bw when a single dose was given on day 9 of gestation. In rabbits, the resorption rate was increased at doses of 120 mg/kg bw per day and higher. The NOEL for this effect was 24 mg/kg bw. Another developmental effect that was observed consistently in all the laboratory animal species tested was reduced fetal body weight. This effect is generally regarded as nonspecific and secondary to maternal toxicity, and this would appear to be the case for tiabendazole: decreased fetal body weight occurred at doses at which the dams had reduced food intake and/or reduced weight gain. The reduction in fetal body weight at doses that did not appear to induce concurrent maternal toxicity, as observed in mice given tiabendazole only on day 9 of gestation, might also have been secondary to maternal toxicity, as it is possible that the dams had normal weights after the single dose (day 10 until time of death) but that the fetuses did not recover their normal weights within that period. On the basis of the effects considered relevant for acute intake, the overall NOEL for developmental toxicity, including teratogenicity, was 24 mg/kg bw per day.

4.

COMMENTS

The studies of the acute toxicity of tiabendazole given orally, which gave LD50 values > 2000 mg/kg bw, did not provide any indication of acute effects. The only substance-specific clinical sign relevant for acute exposure in studies with single or repeated doses was emesis in dogs (NOEL, 40 mg/kg bw per day). The common side-effects reported in humans receiving therapeutic doses (> 25 mg/kg bw twice daily for 1-10 days) included anorexia, nausea, vomiting and dizziness. However, these effects were poorly described and did not allow identification of a NOEL. In a study in volunteers, in which controls were given a placebo, a dose of 125 mg of

TIABENDAZOLE

23

tiabendazole twice a day for 24 weeks (equivalent to 3.6 mg/kg bw per day for a 60kg person) did not cause significant changes in subjective side-effects. In the report of its fortieth meeting, the Committee noted renal injury in mice given tiabendazole for 1-7 days. In a number of published papers, the renal toxicity of tiabendazole in mice was investigated after single or repeated oral administration. Although renal toxicity was observed in the studies with repeated doses, these studies were considered of limited value for establishing an acute RfD because of the high doses used (1200,1800 or 2400 mg/kg bw per day in the diet) and their long duration (13-44 weeks). In the studies with single doses, mice received 0, 125, 250, 500, 1000 or 2000 mg/kg bw by gavage. Renal toxicity, mainly in the proximal tubules, was observed at doses of 250 mg/kg bw and higher and consisted of histopathological changes including mitochondrial swelling. The toxic effects were due to the parent compound and were most severe 2-3 days after dosing; after that time, tissue repair processes began. All the effects except tubule dilatation were either fully or partly reversed within 10 days of administration. These studies showed that tiabendazole is taken up by proximal tubule epithelial cells in the renal cortex and ultimately causes necrosis of those cells. The lowest dose of 125 mg/kg bw was the NOEL for acute renal toxicity in mice. Haematotoxicity was observed in studies with repeated oral doses in rats and dogs, lasting 4 and 13 weeks in rats and 14 and 53 weeks in dogs. Analysis of blood samples from week 4 or 6 showed changes indicative of anaemia early in the studies, and these were occasionally seen more often earlier than at the end of the study. Related histopathological changes in the spleen and/or bone marrow were observed at the same and lower doses. As it cannot be excluded that histopathological changes indicative of anaemia could occur after one or a few doses, they were considered relevant for assessing acute exposure. The NOELs in rats and dogs were 9 and 10 mg/kg bw per day, respectively. In a study with volunteers, 50 men received an oral dose of 125 mg of tiabendazole twice a day for 24 weeks (equivalent to 3.6 mg/kg bw per day for a 60-kg person), and 50 other men were given a placebo. Tiabendazole did not affect haematological parameters after 4, 12 or 24 weeks of treatment. However, owing to a number of shortcomings, no NOEL could be identified in this study. In particular, it was not possible to perform histopathological examinations, which in animals appeared to provide more sensitive indicators of haematotoxicity than the haematological parameters. In a study of developmental toxicity in rabbits, changes related to hydrocephalus were observed after oral doses of tiabendazole of 120 mg/kg bw per day and higher (NOEL, 24 mg/kg bw per day). In another study with rabbits, no such effects were observed at oral doses of up to 600 mg/kg bw per day (NOEL, 150 mg/kg bw per day). In mice, teratogenic effects were observed after a single oral dose on day 9 of gestation. They consisted of deformed limbs at doses of 480 mg/kg bw and higher (NOEL, 270 mg/kg bw) and fusion of vertebrae and ribs at 240 mg/kg bw and higher (NOEL, 130 mg/kg bw). Tiabendazole was not teratogenic in rats in doses up to 80 mg/kg bw, the highest tested. Increased resorption rates were observed in mice and rabbits but not in rats. In mice, the NOELs for this effect were an oral dose of 700 mg/kg bw per day when the animals were exposed on days 7-15 of gestation and 1400 mg/kg bw when they were given a single oral dose on day 9 of gestation. Rabbits showed increased

24

TIABENDAZOLE

resorption rates at oral doses of 120 mg/kg bw per day and higher, with a NOEL of 24 mg/kg bw. The overall NOEL for developmental toxicity, including teratogenicity, was 24 mg/kg bw per day.

5.

EVALUATION

Emesis and effects on the kidney, haematopoietic system and development were considered relevant end-points for establishing an acute RfD. The most sensitive effect was haematotoxicity, specifically histopathological changes in the spleen and bone marrow indicative of anaemia, for which almost identical NOELs were found in rats (9 mg/kg bw per day) and dogs (10 mg/kg bw per day). Using these NOELs and a safety factor of 100, the Committee established an acute RfD of 100 u,g/kg bw, the same value as the ADI. In view of the lack of appropriate data for this effect after single doses, the acute RfD is based on data from studies of repeated administration and hence may be conservative. The results of a study designed specifically to generate data after a single dose might allow refinement of the estimated acute RfD.

6.

REFERENCES

Batham, P. & Lankas, G.R. (1990) Thiabendazole—Fourteen-week oral toxicity study in the beagle dog. Unpublished report No. TT #89-9010/84021 from Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania, USA, and Bio-Research Laboratories, Ltd, Senneville, Quebec, Canada. Submitted to WHO by Syngenta, Basel, Switzerland. Blaszcak, D.L. &Auletta, C.S. (1987) Acute dermal toxicity study in rabbits. Unpublished report No. 4004-86 from Bio/dynamics, Inc., East Millstone, New Jersey, USA. Submitted to WHO by Syngenta, Basel, Switzerland. Campbell, W.C. & Cuckler, A.C. (1969) Thiabendazole in the treatment and control of parasitic infections in man. Texas Rep. Biol. Med., 27 (Suppl. 2), 665-692. Colmore, J.P. (1965) Chronic toxicity study of thiabendazole in volunteers. Unpublished report No. NDA 16-096 from University of Oklahoma, Medical Centre Oklahoma City, Oklahoma, USA. Submitted to WHO by Syngenta, Basel, Switzerland. Commission of the European Union (2001) Guidance for the Setting of an Acute Reference Dose (ARfD). Draft guidance document 7199/VI/99 rev. 5 of 05/07/2001, Brussels. Cuckler, A.C. & Mezey, K.C. (1966) The therapeutic efficacy of thiabendazole for helminthic infections in man. Arzneimittelforschung, 16, 411-428. FAO/WHO (1971) 1970 Evaluations of some pesticide residues in food. AGP: 1970/M/12/1. WHO/Food Add/71.42, Rome. FAO/WHO (1978J Pesticide residues in food—1977. Report of the Joint Meeting of the FAO Panel of Experts on Pesticide Residues and the Environment and the WHO Expert Group on Pesticide Residues. FAO Plant Production and Protection Paper 10 Rev., Rome. FAO/WHO (2001) 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 on Pesticide Residues. FAO Plant Production and Protection Paper 163, Rome. Fujitani, T, Tada, Y. & Yoneyama, M. (1999) Effects of thiabendazole (TBZ) on mitochondrial function in renal cortex of ICR mice. Food Chem. Toxicol., 37,145-152. Gurman, J.L., Hardy, R.J., Voelker, R.W. & Davidson, J.L. (1981) Acute inhalation toxicity study in rats. Unpublished report No. 284-129 from Hazleton Laboratories America, Inc., Vienna, Virginia, USA. Submitted to WHO by Syngenta, Basel, Switzerland.

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25

Hennekeuser, H.H., Pabst, K., Poeplau, W. & Gerok, W. (1969) Thiabendazole for the treatment of trichinosis in humans. Texas Rep. Biol. /Wed, 27 (Suppl. 2), 581-596. Hoberman, A.M. (1989) Thiabendazole—Oral developmental toxicity study in rabbits. Unpublished report No. 013-029 (TT #89-9005) from Argus Research Laboratories, Inc., Horsham, Pennsylvania, USA. Submitted to WHO by Syngenta, Basel, Switzerland. Lankas, G.R. (1981) Thiabendazole veterinary (lot ERM-211)—Acute oral toxicity study in rats. Unpublished report No. TT #81-2691 from Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by Syngenta, Basel, Switzerland. Lankas, G.R. (1993) Thiabendazole—Fifty-three-week oral toxicity study in dogs. Unpublished report No. TT #91-068-0 from Merck Research Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by Syngenta, Basel, Switzerland. Lankas, G.L. & Wise, L.D. (1991/1993) Thiabendazole—Oral developmental toxicity studyRabbits. Unpublished report No. TT #90-734-0 from Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by Syngenta, Basel, Switzerland. Myers, B.A. & Lankas, G.R. (1990) Thiabendazole—A 14-week dietary toxicity study in rats. Unpublished report No. TT #90-9002/284-169 from Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania, USA, and Hazleton Laboratories America, Inc., Rockville, Maryland, USA. Submitted to WHO by Syngenta, Basel, Switzerland. Nakatsuka, T, Matsumoto, H. & Ikemoto, F. (1995) Thiabendazole—Oral developmental toxicity study in mice. Unpublished report No. TT #94-9818 from Banyu Pharmaceutical Co., Ltd, Saitama-ken, Japan. Submitted to WHO by Syngenta, Basel, Switzerland. Ogata, A., Ando, H., Kubo, Y. & Hiraga, K. (1984)Teratogenicity of thiabendazole in ICR mice. Food Chem. Toxicol., 22, 509-520. Parfitt, K., ed. (1999) Martindaie. The Complete Drug Reference, 32nd Ed., London: Pharmaceutical Press. van Raaij, M.T.M. (2001) Guidance document for setting an acute reference dose in Dutch national pesticide evaluations. RIVM report 620555 002. National Institute of Public Health and the Environment (RIVM), Bilthoven. Robinson, H.J. (1964) Thiabendazole (preclinical evaluation). Unpublished report from Merck Sharp & Dohme Research Laboratories, Rahway, New Yersey, USA. Submitted to WHO by Syngenta, Basel, Switzerland, [only the part related to acute oral toxicity was provided] Tada, Y., Yoneyama, M., Kabashima, J., Fujitana, T. & Nakano, M. (1989) Effects of thiabendazole on the kidneys of ICR mice. Food Chem. Toxicol., 27, 307-315. Tada, Y, Fujitani, T. & Yoneyama, M. (1992) Acute renal toxicity of thiabendazole (TBZ) in ICR mice. Food Chem. Toxicol., 30,1021-1030. Tada, Y, Fujitani, T. & Yoneyama, M. (1994) Thiabendazole (TBZ) nephrotoxicity and recovery in ICR adult mice. Toxicology, 94, 41-55. Tada, Y, Fujitani, T. & Yoneyama, M. (1996) Subchronic toxicity of thiabendazole (TBZ) in ICR mice. Food Chem. Toxicol., 34, 709-716. Tada, Y., Fujitani, T, Yano, N., Yuzawa, K., Nagasawa, A. & Yoneyama, M. (2001) Thiabendazole induces urinary tract toxicity in male ICR mice. Toxicology, 162, 1-10. Tocco, D.J., Rosenblum, C., Martin, C.M. & Robinson, H.J. (1966) Absorption, metabolism, and excretion of thiabendazole in man and laboratory animals. Toxicol. Appl. Pharmacol., 9, 31-39. Wise, L.D. (1990) Thiabendazole—Oral developmental toxicity study in rats. Unpublished report No. TT #90-713-0 from Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by Syngenta, Basel, Switzerland. Wise, L.D. & Lankas, G.R. (1992) Thiabendazole - Two-generation dietary study in rats. Unpublished report No. TT #90-733-0 from Merck Research Laboratories, West Point, Pennsylvania, USA. Submitted to WHO by Syngenta, Basel, Switzerland.

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ANTIMICROBIAL AGENT

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CEFUROXIME First draft prepared by Gladwin Roberts1 & Carl Cerniglia2 1 Chemical Products Assessment Section, Therapeutic Goods Administration, Department of Health and Aged Care, Canberra, Australia; and 2 National Center for Toxicological Research, Food and Drug Administration, Arkansas, USA

Explanation Biological data Biochemical aspects Absorption, distribution and excretion Biotransformation Toxicological studies Acute toxicity Short-term studies of toxicity Genotoxicity Reproductive toxicity Multigeneration studies Developmental toxicity Peri- and post-natal toxicity Special studies Renal toxicity Hepatic toxicity Microbiological effects Observations in humans Comments Evaluation References

1.

29 30 30 30 32 32 32 33 38 40 40 41 42 43 43 44 45 50 50 58 58

EXPLANATION

Cefuroxime is a cephalosporin antibacterial agent with activity against a range of gram-positive and gram-negative bacteria. The chemical name of cefuroxime sodium is sodium (Z)-(6f?,7R)-3-(carbamoyloxymethyl)-7-[2-(2-furyl)-2-(methoxyimino)acetamide]-8-oxo-5-thia-1-a2abicyclo[4.2.0]oct-2-ene-2-carboxylate. Its structure is shown in Figure 1. The sodium salt is used in veterinary medicine for the treatment of mastitis and is available in two formulations, both applied by intramammary infusion: one for the treatment of clinical mastititis in lactating cattle and the second for the treatment of sub-clinical mastitis in dry cows and to prevent new infections during the dry period. Cefuroxime is also used in human medicine, as eitherthe sodium salt or as the 1 -acetoxyethyl ester (known as cefuroxime axetil). Cefuroxime has not previously been evaluated by the Committee.

-29 -

30

CEFUROXIME

Figure 1. Structure of cefuroxime sodium

2.

BIOLOGICAL DATA

2.1

Biochemical aspects

2.1.1 Absorption, distribution and excretion Hats Groups of three Wistar rats were given [14C]cefuroxime sodium as a single intravenous or intramuscular injection of 25 mg/kg bw or as an intravenous infusion of 50 mg/kg bw over 40 min. The blood concentrations of radiolabel were highest within 30 min of the end of administration and then rapidly declined, with initial halftimes of 0.5-1 h. By 24 h, the blood concentrations were < 10% of the respective peak. It was distributed widely, kidney and liver having higher concentrations of radiolabel than blood; particularly low concentrations were detected in the central nervous system, muscle and fat. Dosing of female rats on day 18 of gestation resulted in very low concentrations of radiolabel in fetal tissue and amniotic fluid. Relatively high concentrations were detected in the milk of lactating rats, and the peak represented 18% of blood concentrations after 1 h. About 80% of a parenteral dose was excreted in urine, and the remainder was excreted in bile, predominantly during the first 24 h. A portion of the radiolabel in bile was subject to enterohepatic recirculation. Daily intramuscular injections for up to 14 days did not affect the kinetics in blood or excretion in urine (Nanbo et al., 1979; Okumura et al., 1979). In anaesthetized rats given a single intravenous injection of 20 mg/kg bw of cefuroxime, the bioavailability in brain was 4.2% that in plasma, thus confirming the low penetration of the blood-brain barrier (Tsai et al., 1999). The absorption of cefuroxime axetil in the small intestine of anaesthetized rats was investigated in situ by perfusion at a concentration of 12,59,120 or 200 nmol/l. Absorption across the intestinal mucosa appeared to occur as a result of an active transport mechanism. Some drug was hydrolysed in the lumen of the intestine, the proportion increasing from 16 to 25% with increasing concentration (Ruiz-Balaguer, etal., 1997). In seven rats given a single dose of 2 mg of cefuroxime axetil by gavage, the peak plasma concentration of cefuroxime was found at about 30 min. About 25-

CEFUROXIME

31

30% of the dose was bioavailable, and a similar amount was considered to have been immediately excreted in the bile. The relatively low bioavailability appears to be due to hydrolysis in the intestine, which reduces the amount of the ester available for absorption, and hydrolysis in blood, which facilitates rapid excretion of cefuroxime in the urine (Ruiz-Carretero et al., 2000). Rabbits Groups of four rabbits were given single intravenous or intramuscular injections or twice daily intramuscular injections of cefuroxime at a dose of 25 mg/kg bw for 15 days. The peak blood concentrations were reached within 15 min after each administration and then rapidly declined. About 75-80% of the drug was recovered in urine within 24 h. No cefuroxime was detected in organs 24 h after the final dose (Okumura et al., 1979). Dogs Three male beagle dogs were given a single intravenous injection of 25 mg/kg bw [14C]cefuroxime sodium. The concentration of radiolabel in blood peaked immediately after dosing and then decreased, with an initial half-time of 0.7 h. About 90% of the dose was excreted in urine; recovery of about 8% in faeces suggested limited excretion in bile (Nanbo et al., 1979). In beagle dogs given an intramuscular dose of 25 mg/kg bw cefuroxime, 4% of the dose was detected in bile; the concentrations were maximal 1-2 h after administration and gradually decreased thereafter up to 24 h (Okumura et al., 1979). Plasma was taken from one male and one female beagle dog given cefuroxime axetil by gavage at a dose of 100, 400 or 1600 mg/kg bw per day. Samples were withdrawn after treatment for 1, 36, 169 and 188 days. Peak concentrations of cefuroxime in plasma were found within 2 h of administration, and the bioavailability was claimed to be proportional to the dose. Unchanged cefuroxime axetil was also detected in the plasma of the female given 400 mg/kg bw per day and in both animals at 1600 mg/kg bw per day, attaining a highest concentration representing about 10% of the peak. De-esterification led to formation of acetaldehyde and acetic acid (Spurling et al., 1986). Humans Cefuroxime sodium was poorly absorbed through the gastrointestinal tract. After oral administration of 1000 mg to two volunteers, only 1% of the administered dose was recovered in urine, and the plasma concentrations were stated to be only just measurable. When cefuroxime sodium was injected, at least 95% of the dose was recovered in the urine, mainly in unchanged form (Foord, 1976). Cefuroxime is widely distributed in the body, entering body fluids and tissues when given at therapeutic concentrations. It crossed the placenta and has been detected in breast milk (Parfitt, 1999). The concentrations of cefuroxime in cerebrospinal fluid were about 10% of those in plasma (Mandell & Petri, 1996). Injection of 750 mg of cefuroxime to women during pregnancy, labour or caesarean

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section resulted in significant concentrations in the amniotic fluid and umbilical blood vessels and in plasma from the neonates (Craft et al., 1981; Phillipson & Stiernstedt, 1982). Moderate amounts of cefuroxime axetil were absorbed after five oral doses of 125,250,500 x 2 and 1000 mg to 12 male volunteers. The bioavailability was about 36% in fasted individuals and 50% after food intake. A linear relationship was found between the dose in fed subjects and both the area under the plasma concentrationtime curve and the peak plasma concentration. The peak plasma concentration was 43% greater in fed subjects than in fasted subjects (Finn et al., 1987). Cefuroxime axetil was hydrolysed in the intestinal mucosa and blood to yield cefuroxime; the resulting concentrations in plasma were variable (Parfitt, 1999). The plasma halftime was 1-2 h, the maximum plasma concentration after oral administration was 6.3 mg/l, and the degree of protein binding was 33-50% (Kalman & Barriere, 1990). Cefuroxime was excreted primarily in urine (Williams & Harding, 1984), and secretion by the renal tubules was responsible for 43-54% of the renal elimination (Foord, 1976). The kinetics was unaffected by repeated oral dosing for 7 days (Sommers et al., 1984). 2.1.2

Biotransformation Rats and dogs

Up to 97% of administered [14C]cefuroxime sodium remained unchanged in the plasma and urine of rats and dogs treated parenterally. However, an unidentified metabolite was detected in the bile of rats, which accounted for about 27% of the material present, the remainder being unchanged parent drug (Nanbo et al., 1979). In another study, in rats given cefuroxime by intramuscular injection, thin-layer chromatography showed that the only compound present in urine, bile and plasma was the parent. The metabolites produced in rats and dogs were not identified (Okumura et al., 1979). Humans Cefuroxime axetil mixed with human blood in vitro was rapidly de-esterified to cefuroxime, with a half-time of 3.5 min. Similarly, in 12 male volunteers given a single oral dose of 1.5 g of cefuroxime axetil, hydrolysis of the ester was virtually complete, and cefuroxime was the only compound detected in blood (Harding et al., 1984). In 11 children given a single oral dose of 10, 15 or 20 mg/kg bw cefuroxime axetil, the unchanged ester was found in the urine of only four subjects and accounted for < 0.1% of the administered dose (Powell et al., 1991). 2.2

Toxicological studies

2.2.1 Acute toxicity The studies of acute toxicity did not comply with good laboratory practice (GLP). As shown in Table 1, cefuroxime was of low acute toxicity in mice and rats after oral or parenteral administration and was slightly toxic in rabbits when administered parenterally.

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33

Table 1. Results of studies of the acute toxicity of cefuroxime sodium in male and female rodents Species

Route

LD (mg/kg bw)

Reference

Mouse Rat Mouse Rat Mouse

Oral Oral Intraperitoneal Intraperitoneal Subcutaneous

> 10 000 > 10000 > 10 000

Rat

Subcutaneous

> 10 000

Mouse

Intravenous

10400

Rat

Intravenous

>8000

Rabbit Mouse Rat Rabbit

Intravenous Intramuscular Intramuscular Intramuscular

> 1 500 >2000 >2000 >300

Tamuraetal. (1979) Tamuraetal. (1979) Tamuraetal. (1979) Tamuraetal. (1979) Capel-Edwards et al. (1979); Tamuraetal. (1979) Capel-Edwards et al. (1979); Tamuraetal. (1979) Capel-Edwards et al. (1979); Tamuraetal. (1979) Capel-Edwards et al. (1979); Tamuraetal. (1979) Tamuraetal. (1979) Tamuraetal. (1979) Tamuraetal. (1979) Tamuraetal. (1979)

~ 10000 > 10 000

The only adverse effect seen after oral administration was diarrhoea. In animals that died after parenteral injection, swelling of the caecum was the main effect seen at autopsy, and destruction of cortical renal tubules was observed only in rats (Tamura etal., 1979). Two cats and four dogs were given a single intramuscular injection of 2 g/kg bw cefuroxime sodium as a 40% solution in water. No signs of toxicity were seen, and the only treatment-related effect was discomfort at the injection site, which persisted for a few minutes after injection. Cynomolgus monkeys given 1.5-2.0 g/kg bw intramuscularly showed slight loss of body weight, and some had diarrhoea (Glaxo Laboratories Ltd, 1986; Capel-Edwards et al., 1979). 2.2.2 Short-term studies of toxicity Rats A series of studies that did not comply with GLP were conducted in rats given cefuroxime sodium parenterally. Groups of five male and five female CD rats received cefuroxime sodium in 0.9% saline solution intravenously at a dose of 0, 50,100, 200 or 400 mg/kg bw per day for 29 (males) or 30 (females) days. Treatment had no effects on body weight, general condition, haematological, blood biochemical or urinary end-points, organ weights or lesions. As only a summary of this study was available, the absence of findings could not be confirmed independently (Glaxo Laboratories Ltd, 1986). Groups of eight male and eight female Charles River CD rats were given cefuroxime sodium in 0.9% saline solution by subcutaneous injection at a dose of 0,

34

CEFUROXIME

100, 200, 400 or 800 mg/kg bw per day for 33 (males) or 34 (females) days. There were no toxic signs, no effects on body-weight gain and no changes in urinary parameters. Inflammation and ulceration at the injection site were noted in animals at the highest dose. Haemoglobin concentration and erythrocyte volume fraction were reduced in some animals given 200 mg/kg bw per day or more, although the effect was significant only at the highest dose. The serum potassium concentration was increased in females at 100 and 800 mg/kg bw per day. Lymphatic dilatation and lymphocyte infiltration were seen in the colonic mucosa in some animals at 800 mg/kg bw per day. There were no significant effects at 100 mg/kg bw per day. As only a summary of this study was available, the absence of findings cannot be confirmed independently (Glaxo Laboratories Ltd, 1986). Groups of 18 male and 18 female SD-JCL rats were given cefuroxime in distilled water by subcutaneous injection at a dose of 200, 500 or 1500 mg/kg bw per day on 6 days per week for 35 days. A similar group of controls was given saline. An additional group of six males and six females given the highest dose was retained for a 5week recovery period after the end of treatment. No deaths or signs of overt toxicity were noted, and there were no effects on body-weight gain, water intake or urinary end-points. A significant fall in erythrocyte count was seen in males at the highest dose and in females at all doses. Significant decreases in haemoglobin concentration and erythrocyte volume fraction were noted in animals of each sex given the highest dose. An increase in leukocyte numbers occurred in males a 500 or 1500 mg/kg bw per day. Increased serum alanine aminotransferase activity was seen in animals of each sex at 1500 mg/kg bw per day and increased alkaline phosphatase activity in males at 500 and 1500 mg/kg bw per day. Bilirubin concentrations were decreased in all groups of treated males and in females at 500 and 1500 mg/kg bw per day. A significant increase in blood glucose concentration was seen in males at the two higher doses, while significant decreases were seen in all groups of treated females. The albumin concentration was decreased and the potassium concentrations were increased in animals at 1500 mg/kg bw per day. At this dose, males showed decreased liver weights. The weight of the kidney was increased in males, and caecal swelling was seen in animals of each sex at the two higher doses of cefuroxime. All the changes were fully or partially reversed on cessation of dosing. No compound- related effects were found on histological examination. A NOEL was not identified (Ito et al., 1979a). Groups of 18 male and 18 female SD-JCL rats were given cefuroxime in distilled water by intraperitoneal injection at a dose of 100, 300 or 1000 mg/kg bw per day on 6 days per week for 35 days. A similar group of controls was given saline. An additional group of six males and six females at the highest dose was allowed a 5-week recovery period after the end of treatment. No deaths or signs of overt toxicity were noted, and there were no effects on body-weight gain or urinary end-points. Females at 1000 mg/kg bw per day had slightly increased water intake. Erythrocyte counts were significantly decreased in males at the highest dose, and increased values were found in females at 300 and 1000 mg/kg bw per day. The haemoglobin concentration and erythrocyte volume fraction were not affected. Blood glucose concentrations were decreased in males and blood urea nitrogen concentrations

CEFUROXIME

35

were decreased in females at 300 and 1000 mg/kg bw per day but with no doseresponse relationship. The potassium concentration was decreased in females at 300 and 1000 mg/kg bw per day and increased in males at 100 and 1000 mg/kg bw per day. Caecal swelling was noted in animals at 300 and 1000 mg/kg bw per day. There were no compound-related effects on organ weights or on histological appearance. All changes were reversed on cessation of dosing. There were no significant effects at 100 mg/kg bw per day (Ito et al., 1979a). Groups of seven male and seven female CD rats were given cefuroxime sodium in 0.9% saline by subcutaneous injection at a dose of 0,100, 300 or 900 mg/kg bw per day for 91 days. The groups given the control and highest dose contained an additional seven rats of each sex which were allowed a 21-day recovery period at the end of dosing. There were no effects on general condition, blood biochemistry or body weight. The groups given 300 and 900 mg/kg bw per day showed reduced erythrocyte count, haemoglobin concentration and erythrocyte volume fraction. Reticulocyte counts were slightly increased in male rats that had reduced erythrocyte counts. Total leukocyte counts were increased in females at the two higher doses, but the differential counts were unaffected. Prothrombin times were slightly increased in animals of each sex given the highest dose and in males at 300 mg/kg bw per day. Urine volume, urinary electrolyte excretion and water intake were increased and specific gravity decreased in animals at 900 mg/kg bw per day. The spleen weight was increased in females at 900 mg/kg bw per day, but the only pathological findings were inflammation, haemorrhage and haemosiderin deposition at the injection sites in rats at 300 and 900 mg/kg bw per day. The changes were reversed at the end of the recovery period. There were no significant effects at 100 mg/kg bw per day. As only a summary of this study was available, the findings cannot be confirmed independently (Glaxo Laboratories Ltd, 1986). Groups of 10 male and 10 female CD rats were given cefuroxime sodium in 0.9% saline by subcutaneous injection at a dose of 0, 50,150 or 450 mg/kg bw per day for 26 weeks. Body-weight gain and general condition were unaffected, except for localized swelling at the injection site. There were slight reductions in erythrocyte volume fraction, haemoglobin concentration and erythrocyte counts, and increases in reticulocyte counts in animals at 450 mg/kg bw per day. The serum protein concentration was slightly decreased and sodium and potassium excretion was increased in animals at 450 mg/kg bw per day. The only pathological changes were those associated with physical damage and inflammatory reactions at the injection site. There were no significant effects at 150 mg/kg bw per day. As only a summary of this study was available, the findings cannot be confirmed independently (Glaxo Laboratories Ltd, 1986). Groups of 26 male and 26 female SD-JCL rats were given cefuroxime in distilled water by subcutaneous injection at a dose of 100, 250 or 750 mg/kg bw per day on 6 days per week for up to 26 weeks. Simitar groups of controls were given saline. Animals were killed after 3 months (eight rats of each sex) and 6 months (10 rats of each sex) of treatment, and after a 3-month recovery period (eight rats of each sex). There were no overt signs of toxicity and no significant effects on urinary end-points. Body-weight gain was slightly depressed in males at 750 mg/kg bw per day despite

36

CEFUROXIME

a small increase in food intake. Erythrocyte count, haemoglobin concentration and erythrocyte volume fraction were lower in females at all doses. Significant decreases were seen in serum aspartate aminotransferase activity in males at all doses and in females at 750 mg/kg bw per day and in bilirubin concentration in males at 250 mg/kg bw per day and in males and females at 750 mg/kg bw per day. Slight decreases in cholesterol, total protein and albumin concentration were seen in rats at the highest dose. Kidney weights were increased in the groups given 250 or 750 mg/kg bw per day, but the effect was not associated with pathological alterations. Caecal swelling was seen in animals of each sex at 250 and 750 mg/kg bw per day after 3 and 6 months of treatment. None of these changes was seen at the end of the recovery period. A NOEL was not identified (Ito et al., 1979b). Groups of 26 male and 26 female SD-JCL rats were given cefuroxime in distilled water by intraperitoneal injection at a dose of 50, 125 or 375 mg/kg bw per day on 6 days per week for 26 weeks. A similar group of controls was given saline. Animals were killed after 3 months (eight rats of each sex) and 6 months (10 rats of each sex) of treatment and after a 3-month recovery period (eight rats of each sex). There were no overt signs of toxicity and no deaths that could be attributed to treatment. Body-weight gain was unaffected; however, feed conversion was reduced at 3 months in animals at 125 or 375 mg/kg bw per day, gradually returning to control levels thereafter. There were no effects on urinary parameters. Leukocyte counts were decreased in females at 125 and 375 mg/kg bw per day; at the highest dose, this effect was associated with an increase in the ratio of neutrophils to lymphocytes. A decreased total protein concentration in males at the highest dose was the only consistent biochemical alteration. Caecal swelling was seen in animals of each sex at 375 mg/kg bw per day at 3 months only, but no histopathological findings were attributable to administration of the compound. The leukocyte counts remained low during the recovery period, whereas all other changes were reversed to control levels. No effects were seen at 50 mg/kg bw per day (Ito et al., 1979b). Dogs A series of studies that did not conform to GLP were conducted, in which dogs were treated with cefuroxime sodium parenterally and with cefuroxime axetil by oral administration. Groups of two male and two female beagle dogs were given cefuroxime sodium in 0.9% saline by intramuscular injection at a dose of 0, 60,180 or 540 mg/kg bw per day for 10 or 11 days. All the dogs gained weight and remained in good health. Haematological, blood biochemical and urinary end-points were unremarkable. The weights of the liver and kidney relative to body weight were increased at 540 mg/kg bw per day, but the lesions were limited to irritation at the injection site. As only a summary of this study was available, the findings cannot be confirmed independently (Glaxo Laboratories Ltd, 1986). Groups of three male and three female beagle dogs were given cefuroxime sodium in saline by subcutaneous injection at a dose of 0, 50 or 500 mg/kg bw per day on 6 days per week for 5 weeks. One animal of each sex per group was allowed a 4-week recovery period at the end of treatment. No overt toxicity was seen, but

CEFUROXIME

37

body-weight loss was seen in male dogs at 500 mg/kg bw per day. Haematological and urinary analyses revealed no significant findings. The bilirubin concentration was decreased in both groups of treated males, but the difference was seen before the beginning of dosing. Organ weights and gross appearance were unaffected. At the end of the recovery period, the end-points in control and treated animals were similar. No effects were seen at 50 mg/kg bw per day (Ito et al., 1979c). Groups of three male and three female beagle dogs were given cefuroxime sodium in saline by intravenous injection at a dose of 0,25 or 250 mg/kg bw per day on 6 days per week for 5 weeks. One animal of each sex per group was allowed a 4-week recovery period at the end of treatment. Body-weight loss was observed in males at the higher dose. There were no signs of overt toxicity and no effects on haematological, blood biochemical or urinary end-points, organ weights or histopathological appearance. At the end of the recovery period, all parameters were similar in control and treated animals. No effects were seen at 25 mg/kg bw per day (Ito et al., 1979c). Cefuroxime axetil was administered as an aqueous suspension to groups of four male and four female beagle dogs twice daily by gavage for 27 weeks. The equivalent doses of cefuroxime were 0, 100, 400 and 1600 mg/kg bw per day. Additional groups of two males and two females given 0 or 400 mg/kg bw per day were allowed a 3-week recovery period. Two dogs were killed during the study because of intercurrent disease (polyarteritis). The only signs seen in the remaining animals were occasional vomiting and salivation at the highest dose. Body-weight gain was slightly retarded in the group at this dose. Ophthalmic, electrocardiographic and urinary parameters were unaltered. At the highest dose, erythrocyte count, haemoglobin concentration and erythrocyte volume fraction were lower and the reticulocyte number was higher than in controls. Prolonged prothrombin and activated partial thromboplastin times were observed in males at 1600 mg/kg bw per day, and the concentration of coagulation factor VII was reduced in this group. Plasma total protein, albumin and cholesterol concentrations were reduced and that of triglycerides increased at 1600 mg/kg bw per day. The weight of the kidneys of males at this dose was increased. No differences were seen between groups after the recovery period. None of the macroscopic or microscopic findings was attributable to treatment. The NOEL was 400 mg/kg bw per day (Spurling et al., 1986). Groups of three male and three female beagle dogs were given cefuroxime sodium by subcutaneous or intramuscular injection at a dose of 0, 50, 150 or 450 mg/kg bw per day for 6 months. The route of administration was varied during the study to minimize the irritating effects of the injections. The compound was initially given in saline, but distilled water was used as the vehicle from day 25. Controls and dogs at 50 mg/kg bw per day were given intramuscular injections into the hind legs, while animals at the higher doses received subcutaneous injections. After 12 weeks of treatment, one animal at the highest dose developed a Heinz body haemolytic anaemia and was killed after a further 2 weeks. Otherwise, the condition and body-weight gain of dogs was generally good throughout the study. Discomfort arose at the site of injection, particularly after the subcutaneous doses. The mean corpuscular haemoglobin concentrations were slightly reduced in dogs

38

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at 150 and 450 mg/kg bw per day. Serum iron values were consistently reduced (significant after 8 weeks), and serum iron binding capacity was increased in animals at the highest dose throughout the study. Analyses of blood biochemistry, urine and organ weights showed no compound-related effects. The only pathological changes were inflammatory reactions at the injection sites in some treated dogs and changes to the bone marrow and reticuloendothelial system consistent with severe haemolytic anaemia in the animal killed at week 14. No effects were seen at 50 mg/kg bw per day. As only a summary of this study was available, the findings could not be confirmed independently (Glaxo Laboratories Ltd, 1986). Monkeys In a study that did not comply with GLP, groups of two male and two female cynomolgus monkeys were given cefuroxime sodium in 0.9% saline by intramuscular injection at a dose of 0,150 or 450 mg/kg bw per day for 28 days. No overt signs of toxicity were seen, although some animals in each treated group produced softer faeces than normal for a few days at the beginning of treatment. Animals at 450 mg/kg bw per day showed transient reductions in erythrocyte count and haemoglobin concentration (significant at day 7), with moderate leukocytosis (neutrophilia and eosinophilia) in males only. These changes had largely reversed by the end of the study. Blood biochemistry and urinary parameters showed no alterations, and there were no compound-related effects on organ weights. Histological examination revealed only inflammatory reactions at injection sites. As only a summary of this study was available, the findings cannot be confirmed independently. A NOEL was not identified (Glaxo Laboratories Ltd, 1986). 2.2.3 Genotoxicity A battery of tests that complied with appropriate standards was conducted to address the genotoxicity of cefuroxime sodium. The results are summarized in Table 2. As expected, cefuroxime was toxic to the bacterial strains used in the mutagenicity assays. Consequently, only relatively low concentrations of cefuroxime sodium could be tested in these studies. The selected concentrations were based on the results of tests for cytotoxicity. Negative results were obtained, except in the test for induction of chromosomal aberrations in cultured human peripheral blood lymphocytes obtained from one woman and one man. In this test, a positive result was seen in the absence of the metabolizing system. Prolonged exposure, for 20-44 h, was required to obtain a positive effect, and no chromosomal damage was observed after exposure for 3 h. The aberrations were predominantly chromatid deletions, with very few chromosome rearrangements. Although the aberration frequencies were markedly higher than in controls, the magnitude of the response was less than proportional to the concentration of cefuroxime. This may have been due to inhibition of mitosis, which increased from 14% to 54% as the concentration was increased. Nevertheless, these positive results obtained in vitro were not confirmed in a test for micronucleus formation in vivo in mice receiving cefuroxime sodium at intraperitoneal doses of up to 10 000 mg/kg bw. Similar clastogenic effects were seen in cultured human lymphocytes treated with two other cephalosporins, cephalonium (Marshall, R., 1995) and cephalexin

39

CEFUROXIME

Table 2. Results of tests for genotoxicity with cefuroxime sodium End-point

Test object

Concentration

Result

Reference

Reverse mutation3

S. typhimuriuml'M535, TA1537 ± S9 TA98 ± S9 TA100±S9

0.0013-0.8 ng/ml

Negative

Ballantyne (1996)

0.0013-1.0 jig/ml 0.031 -0.5 jig/ml

Negative Negative

Reverse mutation3

Reverse mutation"

E. coli strains WP2 0.0013-0.8ng/ml pKM101±S9 WP2 uvrA pKM 101 ± S9 0.00026-0.16 pig/ml

Negative

S. typhimurium TA98, 0.05-2.0 |j.g/ml TA100, TA1535, TA1537, TA15381S9

Negative

Tweats(1977)

100-5000 ng/m I

Negative

Tweats(1977)

Thymidine kinase (tk) 140-4500 fig/ml locus in mouse lymphoma L5178Y cells ±S9

Negative

Fellows (1995)

Positive

Marshall, A. (1996)

Gene Saccharomyces conversion0 cerevisiae JD1± S9 Forward mutationd

Chromoso- Human peripheral blood mal damage lymphocytes in culture in vitrcf

Chromoso- Micronucleus formation mal damage in bone marrow from in vivd CR/H female mice

750-1500 ng/ml, 20 h exposure -S9 2200-4500 jig/ml, 3 h exposure + S9 480 u,g/ml, 44 h exposure -S9 4500 u,g/ml, 3 h exposure -S9

Ballantyne (1996)

Negative

Negative Positive Negative

Negative Two intraperitoneal doses of 100,1000 or 10000 mg/kg bw, 24 h apart

Tweats et al. (1980)

S9, 9000 x g supernatant of rat liver used for metabolic activation Positive controls were 2-nitrofluorene for TA98; sodium azide for TA100 and TA1535; ICR191 forTAI 537 and /V-methyl-A/"-nitro-/V-nitrosoguanidine (MNNG) for £ co//in the absence of S9; 2-aminoanthracene for all S. typhimurium and E. coli strains in the presence of S9. b Positive controls were 2-acetamidofluorene for TA98, methyl methanesulfonate for TA100, MNNG for TA1535, 9-aminoacridine for TA1537 and hycanthone methanesulfonate for TA1538 in the presence and absence of S9. 0 Positive controls were hycanthone methanesulfonate in the presence and absence of S9. d Positive controls were 4-nitroquinoline 1 -oxide in the absence of S9 and benzo[a]pyrene in the presence of S9. e Positive controls were 4-nitroquinoline 1 -oxide in the absence of S9 and cyclophosphamide in the presence of S9. f Positive control was adryamicin by intraperitoneal injection. 3

40

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(Riley, 1996). Another cephalosporin, ceftiofur sodium, did not induce reverse mutation in bacteria, mutations in mammalian cells in vitro or micronuclei in vivo. However, chromosomal damage was seen in Chinese hamster ovary cells after exposure in vitro for 44 h, but not for shorter times (Aaron et al., 1995a). These data on ceftiofur were reviewed by the Committee at its forth-fifth meeting and were considered to be of no biological significance (Annex I, reference 120). Further investigations suggested that the clastogenic effect of ceftiofur is associated with inhibition of cell division (Aaron et al., 1995b). 2.2.4 Reproductive toxicity A series of studies that did not conform to GLP were conducted with parenteral administration of cefuroxime sodium. (a)

Multigeneration studies Mice

Groups of 12 male and 30 female CRN mice were given cefuroxime sodium in distilled water by subcutaneous injection at a dose of 800,1600 or 3200 mg/kg bw per day. Controls were given 0.9% saline. After 60 days, each male was caged with two or three female mice at the same dose When pregnancy was detected, the female mice were re-housed. When most of the female mice were pregnant, the males were killed. The females were divided into two approximately equal groups per dose: one group was killed on day 18 of gestation, and the females in the other group were allowed to deliver and rear their litters. The latter group received their final dose on day 18 of gestation. Three weeks after the birth of the F-i pups, the F0 dams and all but one male and one female from each litter were killed. When the FI pups were 4 weeks old, one male from one litter was selected and re-housed with one female from another litter to give rise to the F2 generation. The FT dams and the F2 pups were killed on postnatal day 21. The only treatment-related sign during the study was subcutaneous swelling at the site of injection. There were no treatment-related effects on body-weight gain, conception rate, numbers of implantation sites or resorptions, numbers of live fetuses, litter weights or the proportions of male mice in either generation. There were no fetal abnormalities, and the body-weight gain and physical development of the pups was unaffected. No reproductive effects were observed at any dose (Griffiths, 1975a). Rats Groups of 20 male and 20 female Sprague-Dawley rats were given cefuroxime sodium in physiological saline by subcutaneous injection at a dose of 0,200,400 or 800 mg/kg bw per day. Males were treated from 60 days before and during mating, while females were treated from 14 days before mating until day 7 of gestation. The dams were killed on day 20 of gestation. Pain during drug administration was noted in animals given the highest dose. Males at doses of 400 mg/kg bw per day and above showed slight increases in food intake, and water intake was increased in all treated groups. Body-weight gain was suppressed in treated females during the latter part of gestation. There were no differences in conception rates or in the numbers of corpora lutea, implantations, resorptions or live fetuses. Fetal body

CEFUROXIME

41

weights and the numbers of malformed fetuses were not influenced by treatment. Neither fertility nor reproduction was affected at any dose (Otaka et al., 1979). (b)

Developmental toxicity Mice

Groups of 10-17 pregnant female CRH mice were given subcutaneous injections of cefuroxime sodium in 0.9% saline solution at a dose of 0, 800, 1600, 3200 or 6400 mg/kg bw per day on days 6-15 of gestation and killed on day 18 of gestation. The animals remained in good condition, and there were no effects on body-weight gain or the numbers of implantation sites, resorptions or live fetuses. Litter weights and the sex ratio of fetuses were unaffected, and no fetal abnormalities were noted. Fetal development was not affected at any dose (Troughton & Bartholomew, 1975a). Rats Groups of 30 pregnant Sprgaue-Dawley rats were given cefuroxime sodium in physiological saline by subcutaneous injection at a dose of 0, 200, 400, 800 or 1600 mg/kg bw per day on days 7-17 of gestation. Twenty females in each group were killed on day 20 of gestation, and the remaining 10 were allowed to deliver and rear their pups for 21 days. Dams in the latter groups were killed on postnatal day 21, and the Fi offspring matured up to 10 weeks of age. One male and two female FT rats per dam in each treatment group were mated; one of the two females was killed on day 20 of gestation, and the other was allowed to deliver and rear the F2 pups for 3 weeks. Pain, bleeding and haematomas at the injection site were common at the highest dose. Dams in all groups showed slight depression in body-weight gain and increased water intake and caecal weight. In the phase at which teratogenesis was examined, lower body weights were seen in FT fetuses at 800 and 1600 mg/kg bw per day, but this effect was not seen in full-term pups. No effects were found on the numbers of implantations, resorptions or live fetuses, sex ratios, the incidences of fetal abnormalities or postnatal development or reproductive capacity in either generation. There were no effects on fetuses at 400 mg/kg bw per day (Otaka etal., 1979). Groups of 30 pregnant Sprgaue-Dawley rats were given cefuroxime sodium in physiological saline by intravenous injection at a dose of 0, 200, 400 or 800 mg/kg bw per day on days 7-17 of gestation. Twenty females in each group were killed on day 20 of gestation, and the remaining 10 were allowed to deliver and rear their pups for 21 days. Dams in the latter groups were killed on postnatal day 21, and the Fi offspring matured up to 10 weeks of age. One male and two female FI rats per dam in each treatment group were mated; one of the two females was killed on day 20 of gestation, and the other was allowed to deliver and rear the F2 pups for 3 weeks. Twitching after injection was noted at doses of 400 mg/kg bw per day and above. Slight depression in body-weight gain was seen in dams at the highest dose and increased water intake and caecal weight in all treated groups. No effects were found on the numbers of implantations, resorptions or live fetuses, sex ratios, the incidences of fetal abnormalities or postnatal development or reproductive capacity in either generation. There were no effects on fetuses at any dose (Otaka et al., 1979).

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Rabbits Groups of 5-11 pregnant Dutch rabbits were given cefuroxime sodium in 0.9% saline by intramuscular injection at a dose of 0, 50, 100, 200 or 400 mg/kg bw per day on days 6-18 of gestation and were killed on day 29 of gestation. Four does given 400 mg/kg bw per day, one given 200 mg/kg bw per day and one given 100 mg/kg bw per day died during the study. The rabbits developed diarrhoea before death, probably due to disturbances of the gut flora. Surviving does showed no effects on body-weight gain, and there were no effects on the numbers of implantation sites, resorptions or live fetuses. The incidence of fetal abnormalities was similar in all groups. Fetal development was not affected at any dose (Troughton & Bartholomew, 1975b). Groups of 10 pregnant New Zealand white rabbits were given cefuroxime sodium in physiological saline by intravenous or subcutaneous injection at a dose of 0, 25, 50,100 or 150 mg/kg bw per day on days 6-18 of gestation and were killed on day 28 of gestation. No deaths occurred during the study, and the body weights were similar to those of controls. Caecal weight was increased in does at the highest dose. No differences were seen in the numbers of implants, resorptions or live fetuses, in the sex ratio, in fetal weights or in the incidences of fetal abnormalities in any group. Fetal development was not affected at any dose (Furuhashi et al., 1979). (c)

Peri- and postnatal toxicity Mice

Groups of 12-15 pregnant CRH strain mice were given cefuroxime sodium in 0.9% saline by subcutaneous injection at a dose of 0, 800,1600 or 3200 mg/kg bw per day from day 16 of gestation to 23 days after parturition. The dams and pups were killed on postnatal day 23. The severity of local tissue damage at the injection site was dose-dependent. There were no effects on maternal body weight, length of gestation or sex ratio. The litter sizes were reduced at 1600 and 3200 mg/kg bw per day but with no dose-response relationship. Postnatal development of pups and the proportion of pups successfully weaned were unaffected. There were no effects on reproductive capacity at 800 mg/kg bw per day (Griffiths, 1975b).

Flats Groups of 20 pregnant Sprague-Dawley rats were given cefuroxime sodium in physiological saline by subcutaneous injection at a dose of 0,200,400 or 800 mg/kg bw per day from day 17 of gestation to day 20 after parturition. The dams were killed on postnatal day 21. One male and two female Fi offspring per dam in each group were mated at 10 weeks of age; one of the two females was killed on day 20 of gestation, and the other was allowed to deliver and rear the F2 pups for 3 weeks. Body-weight gain and food and water intakes were increased in F0 dams in all groups during lactation. No differences in gestation or parturition or in the numbers of implantations or live pups, sex ratios or body weights of offspring were found in any generation. Caecal weights at the time of weaning were increased in male and female FI pups of dams at 400 and 800 mg/kg bw per day. Pre- and postnatal

CEFUROXIME

43

development, fertility and reproductive capacity were unaffected at any dose (Otaka etal., 1979). Rabbits Groups of 10-16 pregnant Dutch rabbits were given cefuroxime sodium in 0.9% saline by intramuscular injection at a dose of 0, 50, 100 or 200 mg/kg bw per day from day 19 of gestation to 6 weeks after parturition. Surviving does and offspring were killed on postnatal days 42-44; however, several deaths occurred before parturition: one at the lowest dose, two at the intermediate dose and six at the highest dose. The deaths were attributed to perturbation of the maternal gut flora and secondary toxaemic effects on the liver. Another three does given 200 mg/kg bw per day either aborted or lost their entire litters soon after parturition. There were no effects on maternal body weight, length of gestation, sex ratio or litter size. The weights of pups of does at the highest dose were consistently low at parturition and throughout lactation, but their postnatal development and survival were unaffected. There were no effects on offspring of does at 100 mg/kg bw per day (Griffiths, 1975c). 2.2.5 Special studies (a)

Renal toxicity

Studies on nephrotoxic potential that did not comply with GLP were carried out in rats, rabbits and dogs. Rats Groups of 10 male and 10 female Sprague-Dawley JCL rats were given cefuroxime sodium in distilled water as a single injection, either subcutaneously at 0,1000 or 3000 mg/kg bw or as intravenously at 0,500 or 1500 mg/kg bw. It was not clear when the tests were conducted or when the animals were killed, but they were probably killed 24 h after treatment. The specific gravity and sodium and potassium content of urine were increased in females at 3000 mg/kg bw, and the urinary sodium concentration was decreased in males at this dose. Decreased blood concentrations of creatinine were seen in females, of potassium in males and of uric acid in males and females, all at 3000 mg/kg bw. The blood concentrations of magnesium in females at 1500 mg/kg bw and of urea nitrogen in both sexes at 500 and 1500 mg/kg bw were increased. Renal function tests and histopathological examination of a range of tissues revealed no treatment-related changes (Ito et al., 1979d). Groups of 10 male and 10 female Sprague-Dawley JCL rats were given cefuroxime sodium in distilled water by subcutaneous injection at a dose of 0, 800 or 1600 mg/kg bw per day or by intravenous injection at 0,400 or 800 mg/kg bw per day for 1 week. The results of urine analysis were unremarkable. At 1600 mg/kg bw per day given subcutaneously, the blood concentrations of glucose, protein and albumin were decreased in males, those of creatinine and uric acid were decreased in females, and that of sodium was increased in females. The blood urea nitrogen concentration was increased at both intravenous doses, and the blood concentrations of uric acid and sodium were increased at 800 mg/kg bw per day. Renal function tests and histopathological examination of a range of tissues revealed no treatmentrelated changes (Ito et al., 1979d).

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Rabbits Groups of five male and five female New Zealand white rabbits were given cefuroxime sodium in distilled water as a single intramuscular injection of 0,100 or 300 mg/kg bw. It was not clear when the tests were conducted or when the animals were killed, but they were probably killed 24 h after treatment. The urinary chloride concentration was increased in females at both doses. Tests for blood biochemistry and renal function and histopathological examination of a range of tissues revealed no treatment-related changes (Ito et al., 1979d). Groups of five male and five female New Zealand white rabbits were given cefuroxime sodium in distilled water by intramuscular injection at a dose of 0 or 100 mg/kg bw per day for 1 or 4 weeks. After 4 weeks of dosing, increased urine volume associated with decreased sodium, potassium and chloride concentrations were observed in treated females. Increased creatinine and phenolsulfonphthalein clearance were seen in females treated for 1 week but not in those treated for 4 weeks. Blood biochemical and histopathological examination of a range of tissues revealed no treatment-related changes (Ito et al., 1979d). Dogs Groups of three male and three female beagle dogs were given cefuroxime sodium in distilled water as a single intravenous injection of 0 or 1000 mg/kg bw. Urinary and blood analyses gave unremarkable results. Phenolsulfonphthalein clearance was increased in treated females. Histopathological examination of a range of tissues revealed no treatment-related changes (Ito et al., 1979d). Groups of three male and three female beagle dogs were given cefuroxime sodium in distilled water by intravenous injection at a dose of 0, 250 or 500 mg/kg bw per day for 1 week. No treatment-related changes were found in the results of urinary analysis or blood biochemistry, tests for renal function or histopathological examination of a range of tissues (Ito et al., 1979d). (b)

Hepatic toxicity Dogs

In a study that did not comply with GLP, two male beagle dogs were given cefuroxime sodium in saline by subcutaneous injection at a dose of 0, 100 or 300 mg/kg bw per day for 10 days. One dog was given a single daily dose, while the other was given three divided doses, 8 h apart. There were no effects on body weight or general condition. Blood samples were taken 1, 4 and 8 h after injection on the first and eighth days and 24 h after the initial dose on the first, second and tenth days. The activities of alkaline phosphatase, alanine and aspartate aminotransferases, sorbitol dehydrogenase and glutamate dehydrogenase were not affected at any time. No histopathological changes were observed in the liver. As only a summary of this study was available, the findings cannot be confirmed independently (Glaxo Laboratories Ltd, 1986).

CEFUROXIME

(c)

45

Microbiological effects

Several studies were available on the effects of orally administered cefuroxime on the faecal bacterial flora of humans. These studies were carried out according to appropriate standards for study protocol and conduct. Six healthy male volunteers aged 22-40 (weighing 70-86 kg) were given 10 oral doses of 60 mg of cefuroxime axetil, 8 h apart, for a total dose of about 8.5 mg/kg bw. Bowel function was recorded, and faecal samples were collected for microbiological analysis 2 days before and during treatment. Three of the six volunteers had diarrhoea during the dosing period which lasted for 2 days. Another had mild abdominal discomfort for 1 day. A decline in the number of Escherichia coli and conforms (>2 log) was seen in the faeces of the three men who developed diarrhoea. Similarly, there was a> 2 log-fold decrease in the number of Streptococcus faecalis in two men with diarrhoea. S. faecalis could not be detected in the third. The count of Candida spp. increased from 103to 109in two men with diarrhoea and to 105 in the other. It had been shown previously that oral doses of cefuroxime and other cephalosporins can increase the proportion of yeast (Candida albicans) in the gastrointestinal flora (Thomakos et al., 1998). The number of Bacteroides spp. declined from 1010 to 103and those of Peptostreptococcus and Peptococcusspp. decreased from 1010to < 103 in two of the men with diarrhoea and to 5 x 106 in the third. After cessation of dosing, the microbial counts returned to pre-treatment levels. There were no effects on Clostridium spp. (Wise et al., 1984). Oral administration of cefuroxime axetil at a dose of 250 mg twice daily for 4.5 days to 10 healthy volunteers (five men, five women; mean age, 35 years) caused gastrointestinal disturbances. One of the subjects developed a feeling of nausea, and five reported a bloated feeling; seven reported soft faeces, and two detected a change in odour. Diarrhoea developed in four volunteers, and six reported an increased number of defaecations during a day. One woman developed vaginitis. Bowel function returned to normal within 5 days of completing treatment. Decreased total numbers of anaerobes and total aerobes were found in several individuals. The number of Enterobacteriaceae was reduced or even nil in six persons. Those of Streptococcus and Candida spp. were increased. Pseudomonas aeruginosa appeared in the faeces of four persons during the treatment period. Clostridium difficile and C. perfringenswere not detected in any faecal samples, and no clostridial toxins were found. In three persons in whom Enterobacteriaceae were eliminated, recolonization with the sensitive species E. coli, Citrobacter freundii or Klebsiella ozaenae occurred. The populations of faecal flora returned to pre-treatment proportions about 7 days after cessation of treatment (Leigh et al., 1990). Treatment of eight patients for acute exacerbation of bronchitis with cefuroxime axetil at a dose of 250 mg (~ 4.1 mg/kg bw) twice a day for 10 .days resulted in reduced numbers of Staphylococci, Enterobacteriaceae and clostridial populations. These returned to pre-treatment levels 14 days after cessation of treatment. C. difficile appeared in three patients (Novelli et al., 1995). In another study, in which 15 recipients of liver transplants were given 6.3 g of cefuroxime (100 mg/kg bw),

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0.6 g of tobramycin and 0.5 g of nystatin three times daily for 19-21 days to suppress the gut flora, only a few samples contained C. difficile. The diarrhoea induced by the treatment was self-limiting (Hove et al., 1996). Minimum inhibitory concentrations (MICs) have been reported for a range of bacterial species. Cefuroxime was included for comparison in a multicentre trial of agents against more than 42 000 gram-positive and gram-negative organisms. This survey from 79 medical centres and community hospitals in the USA made it possible to establish MIC5o and MIC90 values for Proteus mirabilis, P. vulgaris, Escherichia coll and Enterococcus faecalis (Murray et al., 1994). These data and data for the same organisms reported by Marshall, A. (1995) are shown in Table 3. The results are based on an inoculum density of 107 CFU/ml. The two reports show closely similar values. MIC values for cefuroxime (sodium) against 100 bacterial strains, comprising 10 isolates of 10 genera derived from human gut isolates, were determined by an agar dilution method (Table 4). The bacteria tested are usually considered the most relevant and representative of human gut flora. Of the 10 groups tested, five genera (Enterococcus spp., Lactobacillus spp., Bifidobacterium spp., Peptostreptococcus spp. and Clostridium spp.) showed a strong inoculum effect (more than two antibiotic dilutions) between the undiluted culture and a 10~2 dilution, and two genera (Bacteroides spp. and Bifidobacterium spp.) showed an inoculum effect between the 10~2 and 1O"4 dilutions. The information available indicates that the lowest relevant MICso, at the highest inoculum level of 109 CFU/ml, was 8 ng/ml for Bifidobacterium spp. (Marshall, R., 1995). In a limited study of the susceptibility of 124 bacteria of aerobic origin isolated from patients with spontaneous bacterial peritonitis, the MIC50 and MIC90 values for E. coll were both 4 ng/ml, and the MIC50for Enterococcus spp. was > 16 pig/ml (Saderetal.,1995). In a study specifically designed to determine the effects of inoculum size on MIC values, a clear inoculum effect was seen in several bacteria. Many cephalosporin antibiotics have an inoculum effect, which may be related to drug deactivation or metabolism (Goldstein et al., 1991). The significance of this observation is twofold.

Table 3. Minimum inhibitory concentrations (MICs) of clinical isolates in two studies Organism

Escherichia coli Proteus mirabilis Proteus vulgaris Enterococcus faecalis

Murray et al. (1994)

Marshall, A. (1995)

MIC50

MIC90

MIC50

MIC90

4 <2 >64 32

8(10942) 4 (3 822) > 64 (341) 32 (2 624)

4 1 256 8

4(10) 2(7) 256 (3) 256(10)

Numbers in parentheses are numbers of isolates studied.

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47

Table 4. Minimum inhibitory concentrations (MICs) of cefuroxime against predominant intestinal microflora at three inoculum levels Genus

Escherichia coli Proteus spp. Enterococcus spp. Lactobacillus spp. Fusobacterium spp. Bacteroides spp. Bifidobacterium spp. Peptostreptococcus spp. Clostridium spp. Eubacterium spp.

MIC50 (fig/ml)

Nominal 109 CPU

Nominal 107 CPU

Nominal 105 CPU

8 8 256 128 128 >256 8 128 256 32

4 2 8 4 0.5 256 1

2 1 4 1

4 32 32

1 2 16

0.062 8

0.25

First, at high inocula, the activity of cefuroxime is greatly reduced; therefore, use of MICs for concentrations of organisms lower than those found in vivo in the gastrointestinal tract will result in overestimates of the potential of cefuroxime to affect the microflora adversely. Secondly, the data show potential p-lactamase production, which in vivo would lead to inactivation of cefuroxime. This conclusion is supported by the work of Barry et al. (1977). The effects of co-culture on the MIC and minimum bactericidal concentration of cefuroxime against 10 combinations of bacteria from the human gastrointestinal microflora were determined by agar dilution and by broth microdilution. The results (Table 5) showed that the activity of cefuroxime was significantly reduced, by a factor of up to 20, by co-culture as compared with monoculture. The relatively sensitive Fusobacterium mortiferum strain became less susceptible when tested in co-culture with a more resistant, p-lactamase-producing Proteus vulgaris strain. This was not observed in all cases. The results also showed that the gut microrganisms can protect themselves against the antimicrobial effects of cefuroxime, thus affording protection of the overall gut ecosystem (Pridmore, 1996). Indeed, significant (3lactamase activity has been detected in human faeces (Nord et al., 1989). Particular organisms are known to produce the enzyme, and some, like Bacteroides fragilis, have strains that produce high concentrations (Cornick et al., 1990). In a study of the p-lactamase activity in bacteria used to determine the MICs of cefuroxime, 21 of the 100 strains representative of the human gut flora had intrinsic P-lactamase activity, and a further eight isolates produced p-lactamase in the presence of cefuroxime (Marshall, A., 1996). Cefuroxime is unaffected by many of the common p-lactamases and is effective against bacterial strains that are resistant to other p-lactam antibiotics. Cefuroxime is stable to many bacterial p-lactamases, especially plasmid-mediated enzymes that are commonly found in Enterobacteriaceae (Medical Economics Co., 2002). A model was used to assess the effects of cefuroxime on bacteria representative of the human gut flora in vitro under conditions that mimicked those in the human

CEFUROXIME

48

Table 5. Effect of co-culture of intestinal microflora on the microbiological activity of cefuroxime Organism

MBC in co-culture Individual broth Individual MIC (jig/ml) MBC (jig/ml) (ng/ml)

Bifidobacterium adolescentis Bacteroides distasonis

0.25 >256

0.25 >256

Proteus vulgaris Fusobacterium mortiferum

>256 0.25

>256 0.25

>256

Enterococcus faecalis Bifidobacterium spp.

>256

>256 256

>256 >256

Enterococcus faecalis Bifidobacterium spp.

>256

>256 32

>256

16

Proteus mirabilis Clostridium perfringens

16 4

32 64

16 16

Bacteroides distasonis Peptostreptococcus spp.

32 1

32 2

2 2

Proteus vulgaris Bifidobacterium spp.

>256

>256 2

>256

Bacteroides uniformis Peptostreptococcus magnus

>256

>256 1

>256

Proteus mirabilis Fusobacterium spp. Peptostreptococcus asaccharolyticus Fusobacterium spp.

4

1 1

0.125

>256 4

64

2 2

32 0.016

128 0.016

128 0.031

0.25 0.016

0.25 0.016

0.25 0.031

MBC, minimum bactericidal concentration

gut. Two concentrations were used: one was based on the maximum concentration of cefuroxime residues expected to be found in the human gut, and the second was comparable to the MICs for cefuroxime against the respective human gut isolates. The effects of cefuroxime were investigated against five groups of bacteria (Clostridium sporogenes, Cl. difficile, Peptostreptococcus magnus, P. anaerobius, and Bacteroides spp.) found in the human gut. The drug was added to sterile anaerobic cooked-meat medium supplemented with pepsin and incubated anaerobically at pH 2 and 37 °C for 1 h. After adjustment to pH 7 and the addition of physiological levels of bile salts and pancreatin, the tester strain was added, and the culture was incubated anaerobically for 18 h. The results are given in Table 6. Eight of the 10 strains tested showed no pronounced change or increase in viable count. The results obtained for 10 bacterial strains in this investigation suggest

CEFUROXIME

49

Table 6. Effects of cefuroxime on human intestinal microflora in vitro Bacterial strain

Clostridium sporogenes

Clostridium difficile

Cefuroximej Inoculum Total viable count (cfu/ml) Cefuroxime • MIC (mg/ml) concentration density After 18 h (mg/ml) (cfu/ml) On inoculation incubation

0 2 4

5.0 x10 6 8.0 xlO 8 4.8 x10 6 5.1 x10 6 1.1 x10 6 1.6x10 6 1.3x10 6

9.8 x10 8 8.1 x 107 6.3 x10 6 6.4 x10 8 1.0x10 7 6.3 x 1 06

2

0 2 4

1.6X10

Peptostreptococcus magnus

0 2

3.2 X10 8 3.9 x10 6 3.7 x10 8

3.7 x10 6 6.9 x10 7

0.5

Peptostreptococcus anaerobius

0 2

4.8 xlO 8 6.0 x10 6 5.1 x10 7

2.5 x10 8 5.6 x 1 07

1

8

256

Bacteroides thetaiotaomicron

0 2 256

1.0 X10

1.2x10 6 6.0 x10 5 6.5 x10 5

7.6 x10 8 9.6 x10 7 2.8 x10 7

256

Bacteroides spp.

0 2 256

7.2 x10 5 1.5 X10 8 6.0 x10 5 7.2 x10 5

8.0 x10 8 4.5 x10 8 <2.5x105

2

3.5 xlO

8

2.2 x10 6 1.7x10 6 2.1 x10 6

6.3 x10 7 1.0x10 7 <2.5x10 5

4

4.0 X10

8

3.4 x10 6 2.7 x10 6 2.2 x10 6

2.6 X10 8 2.1 x 108 8.3 x10 7

256

Lactobacillusspp.*

Lactobacillus spp.

Escherichia coll a

Escherichia coli

a

0 2 128 0 2 128 0 2 8 0 2 8

8

9.2 x10 6 9.3 xlO 8 9.8 x 106 8.7 x10 7

1.1 x10 9 2.9 x 108 8.7 x10 8

2

1.0x10 7 7.7 x10 6 1.0x10 7

1.2x10 9 3.9 x10 8 1.9x10 8

4

1.1 X10

9

Duplicate names indicate different strains/isolates.

that cefuroxime had no detectable effects at the concentrations used, which were equal to or greater than those likely to be achieved in the human gut in vivo. Reduced numbers of one strain of Bacteroides spp. and one strain of Lactobacillus spp. were found, but in both cases only after exposure to very high concentrations (> 128 u,g/ml) chosen as the geometric mean MIC against these organisms (Pridmore, 1995).

50

2.3

CEFUROXIME

Observations in humans

Published reviews of adverse effects in patients treated with cefuroxime sodium and cefuroxime axetil at therapeutic doses indicate that these drugs are generally well tolerated by adults and children. Gastrointestinal disturbances such as stomatitis, nausea and diarrhoea, particularly after oral dosing, occur frequently and are due to the effects of the drug on the colonic microflora. Administration by intramuscular injection commonly causes irritation, thrombophlebitis and pain at the injection site. Hypersensitivity reactions are relatively uncommon, and central nervous system toxicity is rare. Increased liver enzyme activity and blood urea nitrogen concentration and anaemia and other haematological abnormalities (i.e. eosinophilia, leukopenia, neutropenia and positive Coombs test [antibodies to red blood cells}) have been observed occasionally. These changes have been mild, asymptomatic and transient and are regarded as clinically inconsequential (Smith & Le Frock, 1983; Perry & Brogden, 1996). Pseudomembranous colitis is infrequent and occurs at a low incidence (Parfitt, 1999). High doses of cefuroxime were associated with the development of reversible encephalopathy characterized by dulled sensibility or stupor, myoclonic jerks and asterixis (negative myoclonus) in four patients. Three of the patients were over 60 years of age and had renal failure, and the condition arose after two to three intravenous injections of 750 mg. The other patient had been given 125 mg of cefuroxime axetil orally three times a day for 2 years and had then received a dose of 6000 mg for 4 days. In each case, the condition resolved on cessation of treatment (Herishanu et al., 1998). A study was conducted in 106 women who received cefuroxime during the first trimester of pregnancy. The women were enrolled prospectively and paired for age, smoking habits and alcohol consumption. The daily dose of cefuroxime was 5001000 mg, and the duration of treatment was 5-10 days. The risk for spontaneous abortion, fetal distress or malformations in neonates was not increased. The rate of induced abortions was significantly higher in the treated group, but the number of live births, the birth weights and postnatal motor development were similar in the two groups (Berkovitch et al., 2000). The case records of 78 women aged 19-38 years who had been treated with cefuroxime during pregnancy were analysed retrospectively. Treatment had been given during the first trimester to 13 women, during the second trimester to 19 women and during the third trimester to 46 women. The physical and mental development of the children born to these mothers was evaluated for 18 months after birth. No abnormalities were detected (Manka et al., 2000).

3.

COMMENTS

The Committee considered the results of studies on pharmacokinetics and metabolism, acute and short-term toxicity, genotoxicity, fertility, reproductive and developmental toxicity, toxicity to the kidney and liver, microbiological safety and studies in humans. Many of the older studies were available as summary reports

CEFUROXIME

51

only and thus could not be assessed, nor was it possible to determine whether the studies had been carried out according to appropriate standards for study protocol and conduct. The newer studies on genotoxicity and microbiological activity were carried out according to appropriate standards. Toxicological data Cefuroxime, when administered as cefuroxime sodium, was poorly absorbed from the gastrointestinal tract of humans after oral administration. When injected into rats and humans, cefuroxime was widely distributed throughout the body. Metabolism was negligible in rats, dogs and humans; however, the Committee noted that significant transformation occurred after intramammary treatment of cows with cefuroxime sodium. Elimination occurred rapidly, largely in the urine, in rats, rabbits, dogs and humans and also in food-producing animals. A small proportion was excreted in the bile of rats and dogs. More absorption occurred after oral administration of cefuroxime axetil in rats, dogs and humans. The ester was rapidly hydrolysed in the intestinal mucosa and blood to yield cefuroxime, acetaldehyde and acetic acid. Thus, the spectrum of toxicity of cefuroxime axetil in vivo would be the same as that of cefuroxime. In a study summarized in the residue monograph (FAO, 2002), which was conducted according to good laboratory practice (GLP), eight lactating dairy cows (weighing 402-599 kg) were given [14C]cefuroxime sodium by intramammary infusion after each of three successive milkings at a nominal dose of 250 mg per quarter per treatment, for a total dose of 3000 mg. Approximately 95% of the dose administered to two of the cows held in metabolic cages was accounted for, with 78% in milk, 10% in urine, 4.6% in faeces and 2.4% in the cage wash. The concentration of cefuroxime equivalent (0.10 mg/kg) in blood peaked 24 h after treatment and declined steadily to 0.01 mg/kg at 196 h. The concentrations were higher in the plasma fraction (0.13 mg/kg at 24 h) than in serum. The elimination appeared to follow a biphasic model, suggesting distribution in a body compartment other than blood or plasma. Elimination was primarily in milk (75-82%) and urine (6-14%). Parent compound accounted for about 20% of the residue in urine, while the remaining residues were in three unidentified polar fractions separated by liquid chromatography. Parent compound also accounted for about 20% of the total residues in milk during and immediately after treatment, declining to about 6% several days after treatment and later to < 2%. The Committee considered that factors other than metabolism, such as compound instability, might have accounted for much of this depletion from milk; however, no data were available to confirm this hypothesis. At slaughter 7 days after the third dosing, no antimicrobiologically active residues were extracted from the kidney, the edible tissue with the highest concentration of radiolabelled residues. About 60% of the radiolabel was extractable from kidney, but very little co-eluted in chromatographic analysis with the parent compound. Cefuroxime sodium was of low acute toxicity when given orally, and it was not lethal at single oral doses of up to 10 000 mg/kg bw in mice and rats. The main adverse effect seen was diarrhoea. Parenteral administration by the subcutaneous, intramuscular, intraperitoneal or intravenous route of single doses of up to 2000 mg/kg bw was also well tolerated in mice, rats, cats, dogs and cynomolgus monkeys. Rabbits were the most sensitive to the acute toxicity of cefuroxime.

52

CEFUROXIME

In short-term studies of toxicity, rats were given parenteral doses of cefuroxime sodium for periods of 29-91 days (50-1500 mg/kg bw per day) and 26 weeks (50750 mg/kg bw per day). Reductions in erythrocyte count, haemoglobin concentration and erythrocyte volume fraction were observed consistently at doses of 1001500 mg/kg bw per day, and decreased serum protein concentrations were detected in some studies in animals given doses of 375-1500 mg/kg bw per day. The only pathological change, apart from inflammation and haemorrhage at the injection site, was swelling of the caecum at doses of 250-1500 mg/kg bw per day. No effects were seen at 50 mg/kg bw per day for 26 weeks. Dogs were given cefuroxime sodium parenterally for periods of 10-35 days (50540 mg/kg bw per day) and 26 weeks (50-450 mg/kg bw per day). Body-weight loss was observed at 250 and 500 mg/kg bw per day for 35 days, but body weight was not affected at any dose during the 26-week study. Haemoglobin concentrations were reduced at 150 and 450 mg/kg bw per day for 26 weeks, and lower serum iron concentrations were found at 450 mg/kg bw per day. One animal given 450 mg/kg bw per day developed severe haemolytic anaemia after 12 weeks. The pathological changes in other dogs were restricted to inflammatory reactions at the injection site. No effects were found at a dose of 50 mg/kg bw per day for 26 weeks. Dogs were treated orally with aqueous solutions of cefuroxime axetil, providing a dose of 0, 100, 400 or 1600 mg/kg bw per day as cefuroxime, for 27 weeks. Treatment-related effects were seen only at 1600 mg/kg bw per day. Body-weight gain was retarded. Erythrocyte count, haemoglobin concentration and erythrocyte volume fraction were reduced, and the reticulocyte count was increased. Clotting times were prolonged, the concentrations of coagulation factor VII and plasma total protein, albumin and cholesterol were reduced, and the concentration of triglycerides was increased. The weight of the kidneys was increased in males, but there was no histological change attributable to cefuroxime. The NOEL was 400 mg/kg bw per day. Cynomolgus monkeys were given cefuroxime sodium by intramuscular injection at a dose of 0,150 or 450 mg/kg bw per day for 28 days. Soft faeces were reported in some animals in each treatment group, but no further details were available. At the highest dose, the erythrocyte count and haemoglobin concentration were reduced during the initial stages of the study. Inflammation at the injection site was the sole pathological finding. Assays covering an adequate range of genotoxic end-points were conducted with cefuroxime sodium. The results were negative, with the exception of an assay for chromosomal aberration in vitro in the absence of metabolic activation, but effects were seen only after prolonged exposure. The Committee concluded that this result, when taken in conjunction with the negative results in a test for micronucleus formation in the bone marrow of mice treated intraperitoneally, was not of biological significance. It concluded that cefuroxime does not pose a genotoxic hazard. No long-term studies of toxicity were carried out with cefuroxime. The drug has no significant genotoxic activity and is not chemically related to known carcinogens. Furthermore, cefuroxime was poorly absorbed from the gastrointestinal tract. No neoplastic or preneoplastic lesions were observed in 26-week studies in rats and dogs given repeated parenteral doses. Under the circumstances, the Committee concluded that studies of carcinogenicity were unnecessary. Multigeneration studies of reproductive toxicity and peri- and postnatal toxicity were carried out in mice (800-3200 mg/kg bw per day) and rats (200-800 mg/kg bw

CEFUROXIME

53

per day) treated subcutaneously. Fertility and reproduction in parental animals and postnatal development of the offspring were unaffected at any dose. Developmental toxicity was investigated in mice (800-6400 mg/kg bw per day) and rats (2001600 mg/kg bw per day) treated subcutaneously. There was no effect on fetal development. Rabbits received cefuroxime at intramuscular doses of 50-400 mg/kg bw per day during gestation and/or until 6 weeks after parturition. A dose-related increase in the mortality rate of dams was observed at all doses, with diarrhoea occurring before death. This effect was probably due to disturbances of the gut flora, a recognized phenomenon in rabbits given antimicrobial agents. There were no effects on fetal development or postnatal growth and survival at any dose. Special studies designed to identify potential renal toxicity were undertaken in rats (given single doses of up to 3000 mg/kg bw and up to 1800 mg/kg bw per day for 1 week), rabbits (given single doses of up to 300 mg/kg bw and 100 mg/kg bw per day for 4 weeks) and dogs (given single doses of 1000 mg/kg bw and up to 500 mg/kg bw per day for 1 week). There was no evidence of renal toxicity. In a special study to investigate hepatic toxicity in dogs, the concentrations of a range of serum liver enzymes were not affected by doses of cefuroxime of 100 and 300 mg/kg bw per day for 10 days. Humans treated with cefuroxime sodium or cefuroxime axetil have generally shown few toxic side-effects. Gastrointestinal disturbances are frequent, particularly after oral dosing, and irritation and pain commonly occur at sites of injection. Hypersensitivity reactions are relatively uncommon, and central nervous system toxicity is rare. Increased serum concentrations of liver enzymes and blood urea nitrogen and anaemia and other haematological abnormalities are observed occasionally. These changes are mild and are generally regarded as inconsequential. In elderly patients with renal failure, which impedes the elimination of cefuroxime, higher systemic concentrations have been associated with reversible encephalopathy. Data from use during pregnancy, although limited, did not show any adverse effects on the developing fetus or the neonate. The most relevant study for determining a toxicological NOEL is the 27-week study in dogs given cefuroxime axetil orally. Owing to the rapid conversion of the axetil ester to cefuroxime, the effects observed are likely to be due to the latter. Studies in which cefuroxime sodium was given by parenteral injection are less relevant for assessing the acceptable intake of cefuroxime in food. Hence, the NOEL for toxicity was 400 mg/kg bw per day. A safety factor of 100 was considered appropriate in view of the existing database for a compound with a long history of use. Therefore, an ADI of 0-4 mg/kg bw could be established on the basis of the toxicological data. Microbiological data Oral administration of cefuroxime axetil at therapeutic doses of 2.5-8 mg/kg bw per day to human patients or volunteers resulted in gastrointestinal disturbances. The signs and symptoms included soft faeces, nausea, a bloated feeling and diarrhoea. Further investigation showed that these overt signs were associated with decreased numbers of bacteria or, in extreme cases, elimination of certain colonic flora. In a few subjects, there was evidence of overgrowth of pathogenic bacteria or yeasts, suggesting disruption of the protective barrier effect in the colon. The

54

CEFUROXIME

populations of colonic flora returned to normal within 7-14 days after cessation of treatment. Cefuroxime has been tested for its inhibitory activity against microorganisms representative of the human colonic microflora. The most sensitive species were Bifidobacterium spp., with an MICso value of 8 mg/ml at an inoculum density of 109 CFU/ml. It has been shown that the activity of cefuroxime is reduced when large inocula are used or when two or more organisms are combined in co-culture, possibly due to the production of p-lactamase. In an in-vitro model of the gut that mimics the conditions found in the human colon, cefuroxime at very high concentrations inhibited the growth of two of 10 co-culture combinations of bacteria from the human gastrointestinal tract. These results indicate that the intestinal microflora can protect themselves from the antimicrobial effects of cefuroxime, thus affording protection to the overall gut ecosystem. A decision-tree for evaluating the potential effect of veterinary drug residues on human intestinal microflora was developed by the Committee at its fifty-second meeting (Annex 1, reference 140) and is reproduced in Figure 1. At its present meeting, the Committee used the decision-tree to answer the following questions in its assessment of cefuroxime: 1. Does the ingested residue have antimicrobial properties? Yes. The parent compound is present as a residue and has been shown to have antimicrobial activity. Significant transformation occurred after intramammary treatment of cows with [14C]cefuroxime sodium, but the degradation products were not identified and their antimicrobial activity is unknown. 2. Does the drug residue enter the lower bowel by any route? Yes. Cefuroxime sodium is poorly absorbed in mammals, including humans. As approximately 1% of an oral dose of cefuroxime was absorbed systemically in humans, most of an oral dose enters the intestines. 3. Is the ingested residue transformed irreversibly to inactive metabolites by chemical transformation, metabolism mediated by the host or intestinal microflora in the bowel and/or by binding to intestinal contents? No specific information was available on the metabolism of cefuroxime by intestinal microflora. Cefuroxime undergoes negligible metabolism in rats, dogs and humans; however, intestinal microflora have the potential to deactivate cefuroxime, as they have P-lactamase enzymes. As there was no direct evidence that cefuroxime is metabolized to inactive metabolites by the intestinal microflora, it was assumed that microbiological activity is retained in the human gastrointestinal tract. 4. Do data on the effects of the drug on the colonic microflora provide a basis to conclude that the ADI derived from toxicological data is sufficiently low to protect the intestinal microflora? No. A number of studies, including those to establish MIC50 values, experiments with co-cultures, investigations of p-lactamase activity and pH effects and studies with volunteers, have indicated that cefuroxime may have adverse effects on the intestinal microflora.

CEFUROXIME

55

5. Do clinical data from the therapeutic use of the class of drugs in humans or data from in-vitro or in-vivo model systems indicate that effects could occur in the gastrointestinal flora? Yes. Gastrointestinal effects are the most commonly reported adverse reactions to therapeutic use of cefuroxime in humans; they include diarrhoea, nausea, vomiting and abdominal pain. 6. Determine the most sensitive adverse effect of the drug on the human intestinal microflora. The available data indicate that disruption of the colonization barrier is the main concern with cefuroxime, rather than emergence of resistance. In clinical practice, cefuroxime is used to treat patients with acute sinusitis, otitis media, chronic bronchitis and skin and urinary-tract infections. Cefuroxime is active against many typical aerobic gram-positive and gram-negative bacteria associated with these infections. There is always concern about decreased susceptibility to antimicrobial agents with increasing resistance; however, cefuroxime is not indicated for use against the predominant anaerobic bacteria commonly associated with the human gastrointestinal tract, and the antimicrobial resistance of such organisms has not been reported. 7. If disruption of the colonization barrier is the concern, determine the MIC of the drug against 100 strains of predominant intestinal flora and take the geometric mean MIC of the most sensitive genus or genera to derive an ADI using the formula for estimating an ADI. Other model systems may be used to establish an NOEC to derive an ADI. An evaluation of the MIC50 values for relevant gastrointestinal microflora provides a figure of 8 ng/ml for Bifidobacterium spp. This value can be used to calculate a microbiological ADI, as follows: Upper limit of ADI

=

MIC50 x MCC

FA x SF x BW where: MIC50 = minimum inhibitory concentration for 50% of strains of the most sensitive relevant organism. The MICso for the most sensitive relevant genus of the gut flora was 8 ng/ml (8 |ig/g) for Bifidobacterium spp. MCC = mass of colonic contents; a value of 220 g, determined by the Committee at its forty-seventh meeting (Annex 1, reference 125), was used in the calculation. FA = available fraction of the dose: the microbiologically active residue is cefuroxime. As only about 1 % of an oral dose of cefuroxime was systemically absorbed in humans, the fraction of the dose available to the gastrointestinal microflora is 100%. Thus, the value in the equation is unity (1). SF = safety factor: the magnitude of the safety factor depends on the quality and quantity of the microbiological data available. A value of 1 is appropriate when extensive relevant microbiological data are available, as is the case in the current assessment. Thus, the safety factor should be unity (1).

Figure 1. Decision-tree for determining the potential adverse effects of residues of veterinary antimicrobial drugs on the human Intestinal microflora Assess the effects of veterinary drug residues, including metabolites, on the human intestinal microflora. Yes

No

Does the ingested residue have antimicrobial properties (recommendation 1(a))?

Does the drug residue enter the lower bowel (e.g. with the food Yes bolus, by biliary circulation, and/or by mucosal secretion) (recommendation 1(b))?

Conclude that the drug residue will not affect the intestinal microflora and use toxicological data to derive the ADI (recommendation 1(b)).

Yes

No

Conclude that the drug residue will not affect the intestinal microflora and use toxicological data to derive the ADI (recommendation 1(a)).

Is the ingested residue transformed irreversibly to inactive metabolites by chemical Yes transformation, metabolism mediated by the host or by the intestinal microflora in the bowel, and/or by binding to intestinal contents (recommendations 1(c) and 1(d)).

Do data on the effects of the drug on the colonic microflora provide a basis to conclude that the ADI derived from toxicological data is sufficiently low to protect the intestinal microflora (recommendation 1(e))7

Conclude that the drug residue will not affect the intestinal microflora and use toxicological data to derive the ADI (recommendation 1(e)).

No

No

Yes

Determine the most sensitive adverse effect(s) of the drug on the human intestinal microflora, including selection of drug-resistant populations, disruption of the barrier to colonization or changes in the metabolic activity of the microflora in the gastrointestinal tract that have been specifically linked to adverse effects on human health.

Conclude that the drug residue will not affect the intestinal microflora and use toxicological data to derive the ADI (recommendations 1(c) and 1(d}).

Do clinical data from therapeutic use of the class of drugs in humans or No data from in vitro or in vivo model systems indicate that effects could occur in the gastrointestinal microflora (recommendation 1(f))? Conclude that the drug residue will not affect the gastrointestinal microflora and use toxicological data to derive the ADI.

If emergence of antimicrobial resistance is the concern, conduct tests in vitro (continuous culture of faecal inocula) or in vivo (rodents inoculated with human flora); challenge the model system with an antibioticresistant species and determine the concentration of the drug that does not select for resistance or the antibiotic-resistant strain when compared with absence of the drug. Use the dose of the drug that has no effect to derive an ADI (recommendation 2(d)).

If disruption of the colonization barrier is the concern, determine the MIC of the drug against 100 strains of the predominant intestinal flora, and take the geometric mean MIC of the most sensitive genus or genera to derive an ADI using the equation" developed at the forty-seventhmeeting of the Committee (Annex 1, reference 125). Other model systems may be used to establish a no-observed-effect concentration (NOEC) to derive an ADI (recommendation 2(b)). A more realistic ADI can be derived by conducting tests in vitro (continuous culture of faecal inocula) or in vivo (rodents inoculated with human flora). Challenge the model systems with appropriate species (e.g. Clostridium difficile. Salmonella spp., Enterococcus spp., Escherichis coli) and determine the concentration of the drug that does not alter the shedding characteristics of the organisms when compared with absence of the drug. Use the dose of the drug that has no effect to derive an ADI (recommendation 2(c)).

If the concern is change in a specific enzymatic activity that is directly linked to adverse effects on human health, conduct tests in vitro (continuous culture of faecal inocula) or in vivo (rodents inoculated with human flora) to determine the concentration of the drug that does not alter that specific enzymatic activity when compared with absence of the drug. Use the dose of the drug that has no effect to derive an ADI (recommendation 2(e)).

• The equation Is as follows: Upper limit of ADI (ug/kg of body weight) =

MIC (pg/g) x Mass of colonic contents (g) Fraction of oral dose bioavailable x Safety factor x Weight of human (kg)

where: MIC50 = Minimum concentration of an antimicrobial drug that completely inhibits the growth of 50% of the cultures of a particular microorganism, as judged by the naked eye, after a given period of incubation. For the purpose of the evaluation, the MIC50 value is the mean MIC50 for the strain(s) of the relevant species tested. Alternatively, the lowest MICSO value for the most sensitive species can be used. Although MIC50 values are usually expressed in micrograms per millilitre, they are expressed as micrograms per gram in this equation so that the ADI will be in micrograms per kilogram. When the MIC50 value is converted to these units, it is assumed that the density of the experimental medium is 1 g/ml. A value of 220 g is used for the mass of the colonic contents, and a value of 60 kg is used for the weight of an adult. The safety factor used to take account of uncertainty about the amount and relevance of data available for review may range from 1 to 10. A value of 1 is used when extensive relevant microbiological data are provided.

58

CEFUROXIME

BW = body weight: a value of 60 kg has been adopted for an adult. Hence,

8 ng/g x 220 g Upper limit of ADI =

1 x 1 x 60 kg Upper limit of ADI =

4.

29 ng/kg bw

EVALUATION

The Committee noted that there is significant transformation of cefuroxime in cattle, which does not occur in laboratory animals or humans. The toxicological activity of the majority of the residue has not been characterized. Therefore, the Committee established a temporary ADI of 0-30 mg/kg bw on the basis of the MIC5o for Bifidobacterium spp., pending the results of studies to (1) identify the residues in milk and clarify whether the residues other than parent compound are due primarily to metabolism or to non-metabolic decomposition of parent cefuroxime; and (2) characterize the toxicological significance of non-parent residues in milk. This information should be provided for evaluation in 2004. The ADI is based on a microbiological end-point and is significantly lower than it would be if it were based on a toxicological end-point. An extra safety factor was not applied to account for the fact that the ADI is temporary, because the concern is the possible toxicity of the transformation products rather then their antimicrobial activity.

5.

REFERENCES

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Pridmore, A. (1995) Studies on the survival of selected human gut flora following incubation with cefuroxime in a simple in vitro gut model. Unpublished report No. DWS/026/95 from Don Whitley Scientific, West Yorkshire, United Kingdom. Submitted to WHO by ScheringPlough Animal Health, Middlesex, United Kingdom. Pridmore A (1996) Determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of cefuroxime against selected co-culture combinations of bacterial gut isolates. Unpublished report No. DWS/008/96 from Don Whitley Scientific, West Yorkshire, United Kingdom. Submitted to WHO by Schering-Plough Animal Health, Middlesex, United Kingdom. Riley, S. (1996) Cephalexin: Induction of chromosome aberrations in cultured human peripheral blood lymphocytes. Unpublished report No. 808/57-1052 from Corning Hazleton, Harrogate, United Kingdom. Submitted to WHO by Schering-Plough Animal Health, Middlesex, United Kingdom. Ruiz-Balaguer, N., Nacher, A., Casabo, V.G. & Merino, M. (1997) Non-linear intestinal absorption kinetics of cefuroxime axetil in rats. Antimicrob. Agents Chemother., 41, 445-448. Ruiz-Carretero, P., Nacher, A., Merino-Sanjuan, M. & Casabo, V.G. (2000) Pharmacokinetics and absolute bioavailability of oral cefuroxime axetil in the rat. Int. J. Pharm., 202, 89-96. Sader, H.S., Runyon, B.A., Erwin, M.E.& Jones, R.N. (1995) Antimicrobial activity of 11 newer and investigational drugs tested against aerobic isolates from spontaneous bacterial peritonitis. Diagn. Microbiol. Infect. Dis., 21,105-110. Smith, B.R. & LeFrock, J.L. (1983) Cefuroxime: Antimicrobial activity, pharmacology, and clinical efficacy. Ther. Drug Monit.,5, 149-160. Sommers, O.K., Van Wyk, M., Williams, P.E.O. & Harding, S.M. (1984) Pharmacokinetics and tolerance of cefuroxime axetil in volunteers during repeat dosing. Antimicrob. Agents Chemother., 25, 344-347. Spurling, N.W., Harcourt, R.A. & Hyde, J.J. (1986) An evaluation of the safety of cefuroxime axetil during six months oral administration to beagle dogs. J. Toxicol. Sci., 11, 237-277. Tamura, J., Sato, N., Moriwaki, T., Nagasawa, R., Aoishi, M., Okamura, C., Sutoh, M., Tatami, A. & Ezaki, H. (1979) Study of the safety of cefuroxime (1). Acute toxicity in mice, rats and rabbits. Chemotherapy (Tokyo), 27(Suppl. 6), 124-129. Thomakos, N., Maraki, S., Liakakos, T., Macheras, A., Kanavaki, S., Marinis, E., Sehas, M., Margioris, A.N. & Samonis, G. (1998) Effect of cefamandole, cefuroxime and cefoxitin on yeast fecal flora of surgical patients. Chemotherapy, 44, 324-327. Troughton, H.J. & Bartholomew, J.E. (1975a) Teratogenicity of cephalosporin 640/359: Subcutaneous administration to female CRH mice on days 6 to 15 of pregnancy. Unpublished report from Glaxo Research, United Kingdom. Submitted to WHO by Schering-Plough Animal Health, Middlesex, United Kingdom. Troughton, H.J. & Bartholomew, J.E. (1975b) Teratogenicity of cephalosporin 640/359: Subcutaneous administration to female Dutch rabbits on days 6 to 18 of pregnancy. Unpublished report from Glaxo Research, United Kingdom. Submitted to WHO by ScheringPlough Animal Health, Middlesex, United Kingdom. Tsai.T.H., Cheng, F.C., Chen, K.C., Chen, Y.F. & Chen, C.F. (1999) Simultaneous measurement of cefuroxime in rat blood and brain by microdialysis and microbore liquid chromatography. Applications to pharmacokinetics. J. Chromatogr. B Biomed. Sci. Appl., 735, 25-31. Tweats, DJ. (1977) Mutagenicity screen. Cefuroxime sodium salt II/6 (TH4/01) batch number BRC 1212 (milled). Unpublished report No. 77ZIN23 from Glaxo Research Ltd, United Kingdom. Submitted to WHO by Schering-Plough Animal Health, Middlesex, United Kingdom. Tweats, D.J., Halliday, I.J. & Tiley, M.J. (1980) Mutagenicity screen. Micronucleus test: Cefuroxime sodium II/6 milled. Unpublished report No. 80TA4 from Glaxo Research Ltd, United Kingdom. Submitted to WHO by Schering-Plough Animal Health, Middlesex, United Kingdom. Williams, P.E.O. & Harding, S.M. (1984) The absolute bioavailability of oral cefuroxime axetil in male and female volunteers after fasting and after food. J. Antimicrob. Chemother., 13,

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ANNEXES

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ANNEX 1 REPORTS AND OTHER DOCUMENTS RESULTING FROM PREVIOUS MEETINGS OF THE JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

General principles governing the use of food additives (First report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 15,1957; WHO Technical Report Series, No. 129,1957 (out of print). Procedures for the testing of intentional food additives to establish their safety for use (Second report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 17,1958; WHO Technical Report Series, No. 144, 1958 (out of print). Specifications for identity and purity of food additives (antimicrobial preservatives and antioxidants) (Third report of the Joint FAO/WHO Expert Committee on Food Additives). These specifications were subsequently revised and published as Specifications for identity and purity of food additives, Vol. I. Antimicrobial preservatives and antioxidants, Rome, Food and Agriculture Organization of the United Nations, 1962 (out of print). Specifications for identity and purity of food additives (food colours) (Fourth report of the Joint FAO/WHO Expert Committee on Food Additives). These specifications were subsequently revised and published as Specifications for identity and purity of food additives, Vol. II. Food colours, Rome, Food and Agriculture Organization of the United Nations, 1963 (out of print). Evaluation of the carcinogenic hazards of food additives (Fifth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 29,1961; WHO Technical Report Series, No. 220,1961 (out of print). Evaluation of the toxicity of a number of antimicrobials and antioxidants (Sixth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 31,1962; WHO Technical Report Series, No. 228, 1962 (out of print). Specifications for the identity and purity of food additives and their toxicological evaluation: emulsifiers, stabilizers, bleaching and maturing agents (Seventh report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 35,1964; WHO Technical Report Series, No. 281, 1964 (out of print). Specifications for the identity and purity of food additives and their toxicological evaluation: food colours and some antimicrobials and antioxidants (Eighth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 38,1965; WHO Technical Report Series, No. 309, 1965 (out of print). Specifications for identity and purity and toxicological evaluation of some antimicrobials and antioxidants. FAO Nutrition Meetings Report Series, No. 38A, 1965; WHO/Food Add/24.65 (out of print). Specifications for identity and purity and toxicological evaluation of food colours. FAO Nutrition Meetings Report Series, No. 38B, 1966; WHO/Food Add/66.25.

-65-

ANNEX 1 11.

12.

13.

14.

15.

16.

17.

18. 19.

20.

21.

22.

23.

24.

66

Specifications for the identity and purity of food additives and their toxicological evaluation: some antimicrobials, antioxidants, emulsifiers, stabilizers, flour treatment agents, acids, and bases (Ninth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 40,1966; WHO Technical Report Series, No. 339,1966 (out of print). Toxicological evaluation of some antimicrobials, antioxidants, emulsifiers, stabilizers, flour treatment agents, acids, and bases. FAO Nutrition Meetings Report Series, No. 40A, B, C; WHO/Food Add/67.29. Specifications for the identity and purity of food additives and their toxicological evaluation: some emulsifiers and stabilizers and certain other substances (Tenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 43,1967; WHO Technical Report Series, No. 373, 1967. Specifications for the identity and purity of food additives and their toxicologica! evaluation: some flavouring substances and non nutritive sweetening agents (Eleventh report of the Joint FAO/WHO Expert Committee on Food Additives). "FAO Nutrition Meetings Series, No. 44, 1968; WHO Technical Report Series, No. 383, 1968. Toxicological evaluation of some flavouring substances and non nutritive sweetening agents. FAO Nutrition Meetings Report Series, No. 44A, 1968; WHO/Food Add/68.33. Specifications and criteria for identity and purity of some flavouring substances and non-nutritive sweetening agents. FAO Nutrition Meetings Report Series, No. 44B, 1969; WHO/Food Add/69.31. Specifications for the identity and purity of food additives and their toxicological evaluation: some antibiotics (Twelfth report of the Joint FAO/ WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 45, 1969; WHO Technical Report Series, No. 430, 1969. Specifications for the identity and purity of some antibiotics. FAO Nutrition Meetings Series, No. 45A, 1969; WHO/Food Add/69.34. Specifications for the identity and purity of food additives and their toxicological evaluation: some food colours, emulsifiers, stabilizers, anticaking agents, and certain other substances (Thirteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 46, 1970; WHO Technical Report Series, No. 445, 1970. Toxicological evaluation of some food colours, emulsifiers, stabilizers, anticaking agents, and certain other substances. FAO Nutrition Meetings Report Series, No. 46A, 1970; WHO/Food Add/70.36. Specifications for the identity and purity of some food colours, emulsifiers, stabilizers, anticaking agents, and certain other food additives. FAO Nutrition Meetings Report Series, No. 46B, 1970; WHO/Food Add/70.37. Evaluation of food additives: specifications for the identity and purity of food additives and their toxicological evaluation: some extraction solvents and certain other substances; and a review of the technological efficacy of some antimicrobial agents. (Fourteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 48, 1971; WHO Technical Report Series, No. 462, 1971. Toxicological evaluation of some extraction solvents and certain other substances. FAO Nutrition Meetings Report Series, No. 48A, 1971; WHO/Food Add/70.39. Specifications for the identity and purity of some extraction solvents and certain other substances. FAO Nutrition Meetings Report Series, No. 48B, 1971; WHO/Food Add/70.40.

ANNEX 1 25. 26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

67

A review of the technological efficacy of some antimicrobial agents. FAO Nutrition Meetings Report Series, No. 48C, 1971; WHO/Food Add/70.41. Evaluation of food additives: some enzymes, modified starches, and certain other substances: Toxicological evaluations and specifications and a review of the technological efficacy of some antioxidants (Fifteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 50,1972; WHO Technical Report Series, No. 488, 1972. Toxicological evaluation of some enzymes, modified starches, and certain other substances. FAO Nutrition Meetings Report Series, No. 50A, 1972; WHO Food Additives Series, No. 1, 1972. Specifications for the identity and purity of some enzymes and certain other substances. FAO Nutrition Meetings Report Series, No. 50B, 1972; WHO Food Additives Series, No. 2, 1972. A review of the technological efficacy of some antioxidants and synergists. FAO Nutrition Meetings Report Series, No. 50C, 1972; WHO Food Additives Series, No. 3, 1972. Evaluation of certain food additives and the contaminants mercury, lead, and cadmium (Sixteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 51,1972; WHO Technical Report Series, No. 505, 1972, and corrigendum. Evaluation of mercury, lead, cadmium and the food additives amaranth, diethylpyrocarbamate, and octyl gallate. FAO Nutrition Meetings Report Series, No. 51 A, 1972; WHO Food Additives Series, No. 4, 1972. Toxicological evaluation of certain food additives with a review of general principles and of specifications (Seventeenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 53, 1974; WHO Technical Report Series, No. 539, 1974, and corrigendum (out of print). Toxicological evaluation of some food additives including anticaking agents, antimicrobials, antioxidants, emulsifiers, and thickening agents. FAO Nutrition Meetings Report Series, No. 53A, 1974; WHO Food Additives Series, No. 5, 1974. Specifications for identity and purity of thickening agents, anticaking agents, antimicrobials, antioxidants and emulsifiers. FAO Food and Nutrition Paper, No. 4,1978. Evaluation of certain food additives (Eighteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 54, 1974; WHO Technical Report Series, No. 557, 1974, and corrigendum. Toxicological evaluation of some food colours, enzymes, flavour enhancers, thickening agents, and certain other food additives. FAO Nutrition Meetings Report Series, No. 54A, 1975; WHO Food Additives Series, No. 6, 1975. Specifications for the identity and purity of some food colours, enhancers, thickening agents, and certain food additives. FAO Nutrition Meetings Report Series, No. 54B, 1975; WHO Food Additives Series, No. 7, 1975. Evaluation of certain food additives: some food colours, thickening agents, smoke condensates, and certain other substances. (Nineteenth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No. 55, 1975; WHO Technical Report Series, No. 576, 1975. Toxicological evaluation of some food colours, thickening agents, and certain other substances. FAO Nutrition Meetings Report Series, No. 55A, 1975; WHO Food Additives Series, No. 8, 1975.

ANNEX 1 40.

41.

42. 43. 44.

45. 46.

47.

48. 49. 50.

51. 52. 53.

54. 55.

56.

57. 58.

59.

68

Specifications for the identity and purity of certain food additives. FAO Nutrition Meetings Report Series, No. 55B, 1976; WHO Food Additives Series, No. 9, 1976. Evaluation of certain food additives (Twentieth report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Food and Nutrition Meetings Series, No. 1, 1976; WHO Technical Report Series, No. 599, 1976. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 10, 1976. Specifications for the identity and purity of some food additives. FAO Food and Nutrition Series, No. 1B, 1977; WHO Food Additives Series, No. 11, 1977. Evaluation of certain food additives (Twenty-first report of the Joint FAO/ WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 617, 1978. Summary of toxicological data of certain food additives. WHO Food Additives Series, No. 12, 1977. Specifications for identity and purity of some food additives, including antioxidant, food colours, thickeners, and others. FAO Nutrition Meetings Report Series, No. 57, 1977. Evaluation of certain food additives and contaminants (Twenty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 631, 1978. Summary of toxicological data of certain food additives and contaminants. WHO Food Additives Series, No. 13, 1978. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 7,1978. Evaluation of certain food additives (Twenty-third report of the Joint FAO/ WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 648, 1980, and corrigenda. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 14, 1980. Specifications for identity and purity of food colours, flavouring agents, and other food additives. FAO Food and Nutrition Paper, No. 12, 1979. Evaluation of certain food additives (Twenty-fourth report of the Joint FAO/ WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 653, 1980. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 15, 1980. Specifications for identity and purity of food additives (sweetening agents, emulsifying agents, and other food additives). FAO Food and Nutrition Paper, No. 17, 1980. Evaluation of certain food additives (Twenty-fifth report of the Joint FAO/ WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 669, 1981. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 16, 1981. Specifications for identity and purity of food additives (carrier solvents, emulsifiers and stabilizers, enzyme preparations, flavouring agents, food colours, sweetening agents, and other food additives). FAO Food and Nutrition Paper, No. 19, 1981. Evaluation of certain food additives and contaminants (Twenty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 683, 1982.

ANNEX 1 60. 61. 62.

63. 64. 65.

66.

67. 68. 69. 70.

71. 72. 73.

74. 75. 76.

77.

78. 79. 80.

69

Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 17, 1982. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 25,1982. Evaluation of certain food additives and contaminants (Twenty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 696, 1983, and corrigenda. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 18, 1983. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 28, 1983. Guide to specifications General notices, general methods, identification tests, test solutions, and other reference materials. FAO Food and Nutrition Paper, No. 5, Rev. 1, 1983. Evaluation of certain food additives and contaminants (Twenty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 710, 1984, and corrigendum. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 19, 1984. Specifications for the identity and purity of food colours. FAO Food and Nutrition Paper, No. 31/1, 1984. Specifications for the identity and purity of food additives. FAO Food and Nutrition Paper, No. 31/2, 1984. Evaluation of certain food additives and contaminants (Twenty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 733, 1986, and corrigendum. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 34, 1986. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 20. Cambridge University Press, 1987. Evaluation of certain food additives and contaminants (Thirtieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 751, 1987. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 21. Cambridge University Press, 1987. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 37, 1986. Principles for the safety assessment of food additives and contaminants in food. WHO Environmental Health Criteria, No. 70. Geneva, World Health Organization, 1987 (out of print). The full text is available electronically at www.who.int/pcs. Evaluation of certain food additives and contaminants (Thirty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 759, 1987 and corrigendum. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 22. Cambridge University Press, 1988. Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 38, 1988. Evaluation of certain veterinary drug residues in food (Thirty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 763, 1988.

ANNEX 1 81.

70

Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 23. Cambridge University Press, 1988. 82. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41, 1988. 83. Evaluation of certain food additives and contaminants (Thirty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 776, 1989. 84. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 24. Cambridge University Press, 1989. 85. Evaluation of certain veterinary drug residues in food (Thirty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 788,1989. 86. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 25, 1990. 87. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/2, 1990. 88. Evaluation of certain food additives and contaminants (Thirty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 789, 1990, and corrigenda. 89. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 26, 1990. 90. Specifications for identity and purity of certain food additives. FAO Food and Nutrition Paper, No. 49, 1990. 91. Evaluation of certain veterinary drug residues in food (Thirty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 799, 1990. 92. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 27, 1991. 93. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/3, 1991. 94. Evaluation of certain food additives and contaminants (Thirty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 806,1991, and corrigenda. 95. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 28, 1991. 96. Compendium of Food Additive Specifications. Joint FAO/WHO Expert Committee on Food Additives (JECFA). Combined specifications from 1st through the 37th Meetings, 1956-1990. FAO, 1992 (2 volumes). 97. Evaluation of certain veterinary drug residues in food (Thirty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 815, 1991. 98. Toxicological evaluation of certain veterinary residues in food. WHO Food Additives Series, No. 29, 1991. 99. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/4, 1991. 100. Guide to specifications—General notices, general analytical techniques, identification tests, test solutions, and other reference materials. FAO Food and Nutrition Paper, No. 5, Ref. 2, 1991. 101. Evaluation of certain food additives and naturally occurring toxicants (Thirty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series No. 828, 1992. 102. Toxicological evaluation of certain food additives and naturally occurring toxicants. WHO Food Additive Series, No. 30,1993.

ANNEX 1

71

103. Compendium of food additive specifications: Addendum 1. FAO Food and Nutrition Paper, No. 52, 1992. 104. Evaluation of certain veterinary drug residues in food (Fortieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 832, 1993. 105. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 31, 1993. 106. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/5,1993. 107. Evaluation of certain food additives and contaminants (Forty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 837, 1993. 108. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 32, 1993. 109. Compendium of food additive specifications: Addendum 2. FAO Food and Nutrition Paper, No. 52, Add. 2, 1993. 110. Evaluation of certain veterinary drug residues in food (Forty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 851,1995. 111. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 33,1994. 112. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/6,1994. 113. Evaluation of certain veterinary drug residues in food (Forty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 855,1995, and corrigendum. 114. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 34, 1995. 115. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/7, 1995. 116. Evaluation of certain food additives and contaminants (Forty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 859, 1995. 117. Toxicological evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 35, 1996. 118. Compendium of food additive specifications: Addendum 3. FAO Food and Nutrition Paper, No. 52, Add. 3, 1995. 119. Evaluation of certain veterinary drug residues in food (Forty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 864, 1996. 120. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 36, 1996. 121. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/8, 1996. 122. Evaluation of certain food additives and contaminants (Forty-sixth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 868,1997. 123. Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 37, 1996. 124. Compendium of food additive specifications, addendum 4. FAO Food and Nutrition Paper, No. 52, Add. 4, 1996.

ANNEX 1

72

125. Evaluation of certain veterinary drug residues in food (Forty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 876, 1998. 126. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 38, 1996. 127. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/9, 1997. 128. Evaluation of certain veterinary drug residues in food (Forty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 879, 1998. 129. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 39, 1997. 130. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/10, 1998. 131. Evaluation of certain food additives and contaminants (Forty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 884, 1999. 132. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 40, 1998. 133. Compendium of food additive specifications: Addendum 5. FAO Food and Nutrition Paper, No. 52, Add. 5, 1997. 134. Evaluation of certain veterinary drug residues in food (Fiftieth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 888, 1999. 135. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 41, 1998. 136. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/11, 1999. 137. Evaluation of certain food additives (Fifty-first report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 891, 2000. 138. Safety evaluation of certain food additives. WHO Food Additives Series, No. 42, 1999. 139. Compendium of food additive specifications, addendum 6. FAO Food and Nutrition Paper, No. 52, Add. 6, 1998. 140. Evaluation of certain veterinary drug residues in food (Fifty-second report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 893, in press. 141. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 43, 2000 142. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/12, 2000. 143. Evaluation of certain food additives and contaminants (Fifty-third report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, in press. 144. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 44, 2000. 145. Compendium of food additive specifications, addendum 7. FAO Food and Nutrition Paper, No. 52, Add. 7, 1999. 146. Evaluation of certain veterinary drug residues in food (Fifty-fourth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, in press.

ANNEX 1

73

147. Toxicological evaluation of certain veterinary drug residues in food. WHO Food Additives Series, No. 45, 2000. 148. Residues of some veterinary drugs in animals and foods. FAO Food and Nutrition Paper, No. 41/13, 2000. 149. Evaluation of certain food additives and contaminants (Fifty-fifth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series No. 901,2001. 150. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 46, 2001. 151. Compendium of food additive specifications: Addendum 8. FAO Food and Nutrition Paper, No. 52, Add. 8, 2000. 152. Evaluation of certain mycotoxins in food (Fifty-sixth report of the Joint FAO/ WHO Expert Committee on Food Additives). WHO Technical Report Series No. 906, 2002. 153. Safety evaluation of certain mycotoxins in food. WHO Food Additives Series, No. 47/FAO Food and Nutrition Paper 74, 2001. 154. Evaluation of certain food additives and contaminants (Fifty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, in press. 155. Safety evaluation of certain food additives and contaminants. WHO Food Additives Series, No. 48, in press. 156. Compendium of food additive specifications: Addendum 9. FAO Food and Nutrition Paper, No. 52, Add. 9, 2001. 157. Evaluation of certain veterinary drug residues in food (Fifty-eighth report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, in preparation.

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ANNEX 2 ABBREVIATIONS USED IN THE MONOGRAPHS

ADI ATP bw CPU Fo

F1 F2 FA FAO GLP IPCS JECFA

LD50 LOEL MBC MCC MIC MNNG NAD NOEC NOEL QA RfD RT-PCS S9 SF WHO w/v

acceptable daily intake adenosine triphosphate body weight colony-forming units parental generation first filial generation second filial generation available fraction of the dose Food and Agricultural Organization of the United Nations good laboratory practice International Programme on Chemical Safety Joint FAO/WHO Expert Committee on Food Additives median lethal dose lowest-observed-effect level minimum bactericidal concentration mass of colonic contents minimum inhibitory concentration /V-methyl- N -nitro-A/-nitrosoguanidine nicotinamide adenine dinucleotide no-observed-effect concentration no-observed-effect level quality assurance reference dose reverse transcriptase polymerase chain reaction 9000 x g microsomal frcation of rat liver safety factor World Health Organization weight per volume

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ANNEX 3 Joint FAO/WHO Expert Committee on Food Additives Rome, 21-27 February 2002

Members Professor A. Anadon, Department of Toxicology & Pharmacology, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, Madrid, Spain Dr D.Arnold, Acting Director, Federal Institute for Health Protection of Consumers and Veterinary Medicine, Berlin, Germany Dr J. Boisseau, Director, National Agency for Veterinary Medicinal Products, French Food Safety Agency, Fougeres, France (Chairman) Professor A.R. Boobis, Section on Clinical Pharmacology, Division of Medicine, Imperial College School of Medicine, Hammersmith Campus, London, England Dr R. Ellis, Food and Safety Inspection Service, United States Department of Agriculture, Washington DC, USA Dr K. Greenlees, Toxicology Team, Office of New Animal Drug Evaluation, Center for Veterinary Medicine, Food and Drug Administration, Rockville, Maryland, USA (Rapporteur) Dr J.D. MacNeil, Center for Veterinary Drug Residues, Saskatoon Laboratory, Canadian Food Inspection Agency, Saskatoon, Saskatchewan, Canada (Rapporteur) Dr J.G. McLean, Camberwell, Victoria, Australia (Vice-Chairman) Dr E.S. Mitema, Department of Public Health, Pharmacology and Toxicology, Faculty of Veterinary Medicine, College of Agriculture and Veterinary Sciences, University of Nairobi, Kabete, Kenya Professor J. Palermo-Neto, Department of Pathology, Faculty of Veterinary Medicine, University of Sao Paulo, Sao Paulo, Brazil Dr J.L. Rojas Martmez, Chief, Toxicology Section, National Laboratory of Veterinary Services, Ministry of Agriculture and Animal Husbandry, Heredia, Costa Rica

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ANNEX 3

78

Dr S. Soback, Head, National Residue Control Laboratory, Kimron Veterinary Institute, Beit Dagan, Israel Dr R.W. Stephany, Head, Laboratory for Residue Analysis, National Institute of Public Health and the Environment, Bilthoven, Netherlands Secretariat Dr C.E. Cerniglia, Director, Division of Microbiology, National Center for Toxicological Research, Food and Drug Administration, Jefferson, Arkansas, USA (WHO Temporary Adviser) Dr P. Chamberlain, Division of Human Food Safety, Office of New Animal Drug Evaluation, Centerfor Veterinary Medicine, Food and Drug Administration, Rockville, Maryland, USA (WHO Temporary Adviser) Dr A. Fernandez Suarez, National Institute of Agricultural Technology, Food Technology Institute, Food Protection Division, Buenos Aires, Argentina (FAO Consultant) Dr L.G. Friedlander, Off ice of New Animal Drug Evaluation, Centerfor Veterinary Medicine, Division of Human Food Safety, Residue Chemistry Team, Food and Drug Administration, Rockville, Maryland, USA(FAOConsultant) Dr J.L. Herrman, Scientist, International Programme on Chemical Safety, WHO, Geneva, Switzerland (Joint Secretary) Mrs E. Heseltine, Communication in Science, Lajarthe, 24290 St Leon-surVezere, France (Editor) Mr M. Luetzow, Food Quality and Strandards Service, Food and Nutrition Division, Food and Agricultural Organization of the United Nations, Rome, Italy (Joint Secretary) Dr K. Mitsumori, Laboratory of Veterinary Pathology, School of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan (WHO Temporary Adviser) Dr S.W. Page, International Programme on Chemical Safety, World Health Organization, Geneva, Switzerland (WHO Staff) Mrs M.E.J. Pronk, Center for Substances and Risk Assessment, National Institute of Public Health and the Environment, Bilthoven, Netherlands (WHO Temporary Adviser)

ANNEX 3

79

Dr P.T. Reeves, National Registration Authority for Agricultural and Veterinary Chemicals, Kingston, Australian Capital Territory, Australia (FAO Consultant) Mr D. Renshaw, Food Standards Agency, London, England (WHO Temporary Adviser) DrG. Roberts, Manager, Chemical Products Assessment Section, Therapeutic Goods Administration, Commonwealth Department of Health and Aged Care, Woden, Australian Capital Territory, Australia (WHO Temporary Adviser) Dr F. Shojaee AliAbadi, Director, Food Quality Control Laboratory, Khatam Co., Tehran, Iran (FAO Consultant) Dr S. Sundlof, Director, Center for Veterinary Medicine, Food and Drug Administration, Rockville, Maryland, USA (Chairman, Codex Committee on Residues of Veterinary Drugs in Foods) Professor G.E. Swan, Professor of Pharmacology and Toxicology and Head of Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Pretoria, South Africa (FAO Consultant) ProfessorF.R. Ungemach, Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Germany (WHO Temporary Adviser)

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ANNEX 4 RECOMMENDATIONS ON COMPOUNDS ON THE AGENDA AND FURTHER INFORMATION REQUIRED Anthelmintic agents Doramectin Acceptable daily intake: Residues:

0-1 mg/kg bw The MRLs that were recommended at the forty-fifth and fifty-second meetings (WHO TRS 864, 1996, and WHO TRS 893, 2000, respectively) were not reconsidered and were maintained.

Ivermectin Acceptable daily intake:

Residue definition:

0-1 mg/kg bw (established at the fortieth meeting of the Committee (WHO TRS 832, 1993)) Ivermectin B1a

Recommended maximum residue limits (MRLs)3 Species

Milk

Cattle

10

a

The MRLs that were recommended at the thirty-sixth and fortieth meetings of the Committee (WHO TRS 799 (1990) and 832 (1993), respectively) were not reconsidered and were maintained.

Tiabendazole (thiabendazole) Acceptable daily intake:

Acute reference dose: Residues:

0-100 jig/kg bw (established at the fortieth meeting of the Committee (WHO TRS 832, 1993)) 100 mg/kg bw The MRLs that were recommended at the fortieth meeting (WHO TRS 832, 1993) were not reconsidered and were maintained.

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ANNEX 4

82

Antimicrobial agents Cefuroxime Acceptable daily intake: Residue definition:

0-30 |tig/kg bw (temporary)a Cefuroxime

Recommended maximum residue limits (MRLs) Species

Milk (HP/kg) 50b

Cattle a

b

Results of studies to (1) identify the residues in milk and clarify whether the residues other than parent compound are due primarily to metabolism or to non-metabolic decomposition of parent cefuroxime; and (2) characterize the toxicological significance of non-parent residue in milk are required for evaluation in 2004. The recommended MRL is temporary because the ADI is temporary.

Dihydrostreptomycin and streptomycin Acceptable daily intake:

Residue definition:

0-50 ng/kg bw (group ADI for dihydrostreptomycin and streptomycin established at the forty-eighth meeting of the Committee (WHO TRS 879, 1998)) Sum of the concentrations of dihydrostreptomycin and streptomycin

Recommended maximum residue limits

Cattle Sheep a

(

200 200

The MRLs that were recommended at the fifty-second meeting of the Committee (WHO TRS 893, 2000) were not reconsidered and were maintained.

Lincomycin Acceptable daily intake:

Residue definition:

0-30 mg/kg bw (established at the fiftyfourth meeting of the Committee (WHO TRS 900, 2001)) Lincomycin

M

83

ANNEX 4

Recommended maximum residue limits (MRLs)8 Species

Cattle Pigs Chickens a

b

Muscle

Liver

Milk

(mg/kg)

Kidney (mg/kg)

Fat

(ng/kg)

(mg/kg)

(mg/kg)

200 200

500 500

1500 500

100 100

150

The temporary MRLs for muscle, liver, kidney and fat of cattle and sheep that were recommended at the fifty-fourth meeting of the Committee (WHO TRS 900, 2001) were not extended. The MRLs for fat that were recommended at the fifty-fourth meeting of the Committee were maintained. A separate MRL of 300 mg/kg for skin with adhering fat in pigs was recommended in order to reflect the high concentrations found in the skin of pigs. For consistency, an MRL of 300 mg/kg for skin with adhering fat in chickens was also recommended.

Neomycin Acceptable daily intake: 0-60 mg/kg bw (established at the forty-seventh meeting of the Committee (WHO TRS 876,1998)) Residues: Following a request by the Codex Committee on Residues of Veterinary Drugs in Foods at its Twelfth Session (ALINORM 01/31, paragraph 90) the Expert Committee considered information on the registration of injectable neomycin products as well as how they were used with respect to Good Practice in the Use of Veterinary Drugs. In addition, a proposal from the sponsor to increase the MRL in milk was considered. The Committee noted that use of neomycin for injectable administration had been withdrawn in some countries and was under review in others. The Committee also considered information on the toxicity of neomycin in calves; however, it concluded that toxicity in target animals represents an animal welfare issue that falls outside its mandate. The Committee concluded that there was insufficient justification for changing the MRLs and maintained the MRLs that were recommended at its forty-third, fortyseventh, and fifty-second meetings (WHO TRS 855, 1995; WHO TRS 876,1998; and WHO TRS 893, 2000, respectively). The Committee recommended that these MRLs be reviewed when the toxicity of neomycin is re-evaluated in response to the request of the Codex Committee on Residues of Veterinary Drugs in Foods at its Thirteenth Session (ALINORM 03/31, paragraph 18).

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84

Oxytetracycline Acceptable daily intake:

Residue definition:

0-30 mg/kg bw (group ADI for tetracycline, oxytetracycline and chlortetracycline; established at the fiftieth meeting of the Committee (WHO TRS 888, 1999)) Oxytetracycline, expressed as parent drug

Recommended maximum residue limits (MRLs)a Species

Muscle

(mg/kg) Fish a

200

The MRLs that were recommended in cattle, pigs, sheep, poultry, and giant tiger prawn (Penaeus monodon) at the fiftieth meeting of the Committee (WHO TRS 888, 1999) were not reconsidered and were maintained. The MRL applies only to oxytetracycline.

Thiamphenicol Acceptable daily intake:

Residues:

0-5 mg/kg bw (established at the fiftysecond meeting of the Committee (WHO TRS 893, 2000)) The temporary MRLs in muscle, liver, kidney and fat of pigs and muscle of fish were not extended because the information requested at the fifty-second meeting (WHO TRS 893, 2000) was not provided.

Insecticides Cyhalothrin Acceptable daily intake: Residue definition:

0-2 mg/kg bw (temporary)a Cyhalothrin

Recommended maximum residue limits (MRLs)b Species

Muscle (mg/kg)

Cattle Pigs Sheep a

b

20 20 20

Liver (mg/kg)

Kidney (mg/kg)

Fat

Milk

(mg/kg)

(mg/kg)

20 20 20

20 20 20

400 400 400

30

Results of appropriate studies to establish a no-observed-effect level (NOEL) for neurobehavioral effects in laboratory animals are required for evaluation in 2004. The recommended MRLs are temporary because the ADI is temporary. In addition, results of the validation of the analytical method for sheep liver to demonstrate a limit of quantification of 10 mg/kg are required for evaluation in 2004.

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85

Cypermethrin Acceptable daily intake:

0-50 mg/kg bw (established at the fortyseventh meeting of the Committee (WHO TRS876, 1998)) Cypermethrin

Residue definition:

Recommended maximum residue limits (MRLs)a Species Sheep a

Muscle

Liver

Kidney

(mg/kg)

(mg/kg)

(mg/kg)

20

20

20

Fat 200

The ADI established at the forty-seventh meeting (WHOTRS 876, 1998) was for a 45:55 cis:trans mixture. Information provided to the Committee at the present meeting was for an 80:20 cis.trans mixture for topical use. Because the cis isomer is more toxic than the trans isomer, the Committee compared the theoretical maximum daily intake of the 80:20 cis:trans mixture with the ADI for a-cypermethrin, which consists only of cis isomers.

a-Cypermethrin Acceptable daily intake:

Residue definition:

0-20 mg/kg bw (established at the fortyseventh meeting of the Committee (WHO TRS876, 1998)) a-Cypermethrin

Recommended maximum residue limits (MRLs) Species

Cattle Sheep

Muscle

Liver (mg/kg)

Kidney

Fat

Milk

(mg/kg)

(mg/kg)

(mg/kg)

(mg/kg)

100 100

100 100

100 100

1000 1000

100

Phoxim Acceptable daily intake:

Residue definition:

0-4 mg/kg bw (established at the fiftysecond meeting of the Committee (WHO TRS 893, 2000)) Phoxim

ANNEX 4

86

Recommended maximum residue limits (MRLs) Species

Cattlea Pigs Goats Sheep

Muscle (mg/kg)

Liver (mg/kg)

Kidney

Fat

Milk

(mg/kg)

(mg/kg)

(mg/kg)

50

50 50 50 50

50 50 50 50

400 400 400 400

10

50 50 50

Temporary MRLs, pending the receipt of information on a GLP-compliant study on residue depletion in cattle, which is required for evaluation in 2004

Production aid Melengestrol acetate Acceptable daily intake:

Residue definition:

0-0.03 fig/kg bw (established at the fiftyfourth meeting of the Committee (WHO TRS 900, 2001)) Melengestrol acetate

Recommended maximum residue limits (MRLs) Species

Liver (MO/kg)

Fat (ng/kg)

Cattle

2

5