WHO FOOD Toxicological evaluation of ADDITIVES certain veterinary drug residues in food · 2019-05-23 · WHO FOOD ADDITIVES SERIES: 51 Toxicological evaluation of certain veterinary

WHO FOODADDITIVESSERIES: 51

Toxicological evaluation ofcertain veterinary drugresidues in food

Prepared by the sixtieth meetingof the Joint FAO/WHO ExpertCommittee on Food Additives(JECFA)

IPCS—International Programme on Chemical Safety

World Health Organization, Geneva, 2003

The summaries and evaluations contained in this book are, in most cases,based on unpublished proprietary data submitted for the purpose of theJECFA assessment.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

WHO Library Cataloguing-in-Publication Data

Toxicological evaluation of certain veterinary drug residues in food /prepared by the sixtieth meeting of the Joint FAO/WHO Expert Com-mittee on Food Additives (JEFCA).

(WHO food additives series ; 51)

1. Neomycin - toxicity 2.Quinolizines - toxicity 3.Trichlorfon - toxic-ity 4.Carbadox - toxicity 5.Drug residues - toxicity 6.Veterinarydrugs - adverse 7.Food contamination 8.Risk assessment I.JointFAO/WHO Expert Committee on Food Additives. Meeting (60th :2003 : Geneva, Switzerland) II.Series

ISBN 92 4 166051 X (NLM classification: WA 712)ISSN 0300-0923

© World Health Organization 2003

All rights reserved. Publications of the World Health Organization can be ob-tained from Marketing and Dissemination, World Health Organization, 20 Av-enue Appia, 1211 Geneva 27, Switzerland (tel: +41 22 791 2476; fax: +41 22 7914857; email: [email protected]). Requests for permission to reproduce ortranslate WHO publications – whether for sale or for noncommercial distribu-tion – should be addressed to Publications, at the above address (fax: +41 22791 4806; email: [email protected]).

The designations employed and the presentation of the material in this publica-tion do not imply the expression of any opinion whatsoever on the part of theWorld Health Organization concerning the legal status of any country, territory,city or area or of its authorities, or concerning the delimitation of its frontiers orboundaries. Dotted lines on maps represent approximate border lines forwhich there may not yet be full agreement.

The mention of specific companies or of certain manufacturers’ products doesnot imply that they are endorsed or recommended by the World HealthOrganization in preference to others of a similar nature that are not mentioned.Errors and omissions excepted, the names of proprietary products aredistinguished by initial capital letters.

The World Health Organization does not warrant that the information containedin this publication is complete and correct and shall not be liable for any dam-ages incurred as a result of its use.

This publication contains the collective views of an international group of anddoes not necessarily represent the decisions or the stated policy of the WorldHealth Organization.

CONTENTS

Preface......................................................................................................... v

Antimicrobial agentsNeomycin ............................................................................................... 3Flumequine ............................................................................................ 25

InsecticideTrichlorfon (metrifonate) ........................................................................ 31

Production aidCarbadox ................................................................................................ 49

AnnexesAnnex 1 Reports and other documents resulting from

previous meetings of the Joint FAO/WHOExpert Committee on Food Additives ............................... 61

Annex 2 Abbreviations used in the monographs ........................... 71Annex 3 Participants in the sixtieth meeting of the

Joint FAO/WHO Expert Committee onFood Additives .................................................................. 73

Annex 4 Recommendations on compounds on the agendaand further toxicological studies and informationrequired ............................................................................ 75

This publication is a contribution to the International Programme onChemical Safety.

The International Programme on Chemical Safety (IPCS), establishedin 1980, is a joint venture of the United Nations Environment Programme(UNEP), the International Labour Organisation (ILO), and the WorldHealth Organization (WHO). The overall objectives of the IPCS are toestablish the scientific basis for assessing the risk to human health andthe environment from exposure to chemicals, through international peer-review processes, as a prerequisite for the promotion of chemical safety,and to provide technical assistance in strengthening national capacitiesfor the sound management of chemicals.

The Inter-Organization Programme for the Sound Management ofChemicals (IOMC) was established in 1995 by UNEP, ILO, the Foodand Agriculture Organization of the United Nations, WHO, the UnitedNations Industrial Development Organization, and the Organisation forEconomic Co-operation and Development (Participating Organizations),following recommendations made by the 1992 United Nations Confe-rence on Environment and Development to strengthen cooperation andincrease coordination in the field of chemical safety. The purpose of theIOMC is to promote coordination of the policies and activities pursuedby the Participating Organizations, jointly or separately, to achieve thesound management of chemicals in relation to human health and theenvironment.

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

v

PREFACE

The monographs contained in this volume were prepared at the sixtieth meetingof the Joint FAO/WHO Expert Committee on Food Additives (JECFA), which metat WHOHeadquarters in Geneva, Switzerland, 6–12 February 2003. Thesemonographs summarize data on the safety of residues in food of selected veterinarydrugs reviewed by the Committee.

The sixtieth report of JECFA will be published by the World Health Organizationin the WHO Technical Report Series. Reports and other documents resulting fromprevious meetings of JECFA are listed in Annex 1. Abbreviations used in themonographs are listed in Annex 2. The participants in the meeting are listed inAnnex 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 Committeeon Food Additives and Contaminants and the Codex Committee on Residues ofVeterinary Drugs in Foods, regarding the safety of food additives, residues ofveterinary drugs, naturally occurring toxicants, and contaminants in food. Commit-tees accomplish this task by preparing reports of their meetings and publishingspecifications or residue monographs and toxicological monographs on substancesthat they have considered.

The monographs contained in this volume are based on working papers thatwere prepared by working groups before the meeting. A special acknowledgementis given at the beginning of each monograph to those who prepared these workingpapers. The monographs were edited by E. Heseltine, Lajarthe, 24290 St Léon-sur-Vézère, France.

The preparation and editing of the monographs included in this volume weremade possible through the technical and financial contributions of the ParticipatingOrganizations of the International Programme on Chemical Safety (IPCS), whichsupports the activities of JECFA.

The designations employed and the presentation of the material in thispublication do not imply the expression of any opinion whatsoever on the part ofthe organizations participating in the IPCS concerning the legal status of anycountry, territory, city, or area or its authorities, or concerning the delimitation of itsfrontiers or boundaries. The mention of specific companies or of certain manu-facturers’ products does not imply that they are endorsed or recommended bythose organizations in preference to others of a similar nature that are notmentioned.

Any comments or new information on the biological or toxicological propertiesof the compounds evaluated in this publication should be addressed to: Joint WHOSecretary of the Joint FAO/WHO Expert Committee on Food Additives, InternationalProgramme on Chemical Safety, World Health Organization, Avenue Appia, 1211Geneva 27, Switzerland.

– v –

1NEOMYCIN

ANTIMICROBIAL AGENTS

2 NEOMYCIN

3NEOMYCIN

NEOMYCIN (addendum)

First draft prepared by

Mr Derek RenshawFood Standards Agency, London, England

Dr Carl CernigliaDivision of Microbiology, National Center for Toxicological Research, Food

and Drug Administration, Jefferson, Arkansas, USAand

Professor Kunitoshi MitsumoriLaboratory of Veterinary Pathology, School of Veterinary Medicine, Faculty

of Agriculture, Tokyo University of Agriculture and Technology, Tokyo,Japan

Explanation ................................................................................. 3Biological data ............................................................................. 4

Identity and use .................................................................... 4Mechanism of action on bacteria ......................................... 4Microbiological safety ........................................................... 5

Insensitivity of intestinal microflora ................................ 5Acquired resistance ....................................................... 6Decision tree .................................................................. 6

Ototoxicity ............................................................................. 13Effects on hearing .......................................................... 13Mitochondrial DNA mutation .......................................... 14Mechanism of ototoxicity ............................................... 17

Comments ................................................................................. 18Evaluation ................................................................................. 19References ................................................................................. 20

1. EXPLANATION

The Committee considered neomycin at its forty-third, forty-seventh, fifty-secondand fifty-eighth meetings (Annex 1, references 113, 125, 140 and 157). At its forty-third meeting, the Committee established a temporary ADI of 0–30 µg/kg bw on thebasis of a NOEL of 6 mg/kg bw per day for ototoxicity in a 90-day study in guinea-pigs and a safety factor of 200. The ADI was made temporary in view of deficienciesin the genotoxicity data. Studies of mutagenicity were requested for evaluation in1996.

At its forty-seventh meeting (Annex 1, reference 125), the Committee considerednew data on genotoxicity for neomycin. On concluding that neomycin was notgenotoxic, it established an ADI of 0–60 µg/kg bw on the basis of the NOEL of6 mg/kg bw per day for ototoxicity in the 90-day study in guinea-pigs and a safetyfactor of 100.

– 3 –

4 NEOMYCIN

Following a request by the Codex Committee on Residues of Veterinary Drugsin Foods at its Twelfth Session (Codex Alimentarius Commission, 2000), theCommittee at its fifty-eighth meeting considered information on registration ofinjectable neomycin products and on their use with respect to good practice in theuse of veterinary drugs. The Committee also considered data on the toxicity ofneomycin in calves, but it concluded that the information was relevant only to thewelfare of the target animals and therefore fell outside its mandate.

The Codex Committee on Residues of Veterinary Drugs in Foods at its ThirteenthSession (Codex Alimentarius Commission, 2001) requested the Committee toevaluate new data on the safety of neomycin. Two submissions were made, oneaddressing the microbiological aspects of the safety of neomycin to consumers andthe other addressing the evidence for a link between the presence of a specificmutation to mitochondrial DNA in humans and increased susceptibility to amino-glycoside-induced ototoxicity.

The ADIs that could be derived from studies in bacteria in vitro, from studies ofhuman flora-associated animals in vivo and from studies in humans were evaluated.

2. BIOLOGICAL DATA

2.1 Identity and use

Neomycin is used to treat superficial infections in humans and is given orally tocattle, sheep, pigs, goats and poultry for bacterial gastrointestinal infections and byintramammary administration to treat mastitis. It is an aminoglycoside and is activeagainst bacteria that grow aerobically. It is considered to be inactive againstanaerobes, and its activity against facultative bacteria in vitro is lower in anaerobicenvironments than in air (Annex 1, reference 113).

Neomycin is produced by Streptomyces fradiae. Preparations are complexesconsisting of neomycin A, neomycin B and neomycin C, generally containing morethan 90% neomycin B, the remainder being mainly neomycin C. Neomycins B andC both contain three amino sugars attached by glycosidic linkage to the centralhexose. Neomycin A, more appropriately referred to as neamine, is a hydrolysisproduct of either neomycin B or neomycin C and usually comprises less than 1% ofthe mixture. Neomycin A has a bicyclic ring system with four amino groups. NeomycinB has a total of six amino groups and consists of neamine and neobiosamine B, adisaccharide of D-ribose and neosamine B. Neomycin C is a stereoisomer andconsists of neamine and neobiosamine C, a disaccharide from D-ribose andneosamine C (stereoisomer of neosamine B). When tested by antibiotic dilutiontechniques against aerobic and facultative bacteria, the activity of neomycin B isgenerally greater than that of neomycin C, which is greater than that of neamine.

2.2 Mechanism of action on bacteria

The bacteriocidal effects of aminoglycosides are brought about by disruption ofcellular transport mechanisms as a result of the formation of abnormal cell membranechannels by abnormal proteins (Prescott et al., 2000). Neomycin reacts with 30Sribosomal subunits of prokaryotic cells by electrostatic attraction. This blocks theformation of a complex of mRNA with formethionine and tRNA and induces misreading

5NEOMYCIN

of the genetic code on the mRNA template (Brown, 1988). The result is a change inthe conformation of the ribosomal binding protein, with concomitant errors in readingthe code of the mRNA (Mingeot-Leclercq et al. 1999), and a non-functional proteinis produced (Brown, 1988).

As it is a polycation, neomycin binds to negatively charged bacterial surfaceanions such as the lipopolysaccharide of gram-negative bacteria, teichoic acids ofgram-positive bacteria and polar portions of phospholipid in both types of bacteria.Active transport is required for neomycin to traverse the membrane so that it canreach the ribosomal target site. Such transport mechanisms are lacking in a numberof the anaerobes studied to date, and for this reason they are generally not sensitiveto the aminoglycosides. Aminoglycosides are moved across the cytoplasmicmembrane by the membrane potential after non-specific association with a transporterin the cytoplasmic membranes. Ribosomes and nucleic acids act as binding sinksfor transported aminoglycosides and contribute significantly to total cell uptake. Inthe presence of sufficient concentrations of neomycin, transport may cause somerelease of cell components, including potassium, amino acids and nucleotides, fromexposed bacterial cells by damaging the cell wall.

Aminoglycosides have additional effects on microorganisms, including inter-ference with the cellular electron transfer system, induction of RNA breakdown,disruption of polysomes into inactive monosomes, inhibition of translation, blockingof initiation of DNA replication, effects on DNA metabolism and damage to cellmembranes (Brown, 1988; Prescott et al., 2000). At least some of these effects arelikely to be due to the mistranslation of mRNA.

2.3 Microbiological safety

2.3.1 Insensitivity of intestinal microflora

As noted above, the transport of aminoglycosides into facultative anaerobes isan energy-dependent system requiring electron transport (Bryan & Kwan, 1981).Under anaerobic conditions, the membrane potential of facultative bacteria isdiminished, and transport of aminoglycosides is markedly impaired. Facultativebacteria that are sensitive to neomycin under aerobic conditions are much lesssusceptible under anaerobic conditions owing to the lack of an effective proton motiveforce for transport in these organisms.

Although aminoglycosides bind in vitro to both the cell surfaces and the ribosomesof Bacteroides and Clostridia, these and other obligate anaerobes are naturallyinsensitive to aminoglycosides. Their insensitivity is not related to the production ofinactivating enzymes nor to mutations that reduce ribosomal affinity. In fact, proteinsynthesis by Bacteroides fragilis and Clostridium perfringens in vitro is inhibited byaminoglycosides, to which they are otherwise insensitive. Rather, the insensitivityof anaerobes appears to be due to insufficient transmembrane driving force or theamount of cell membrane transporter (Bryan et al., 1979; Bryan & Kwan, 1981;Rasmussen & Tally, 1993). Thus, the insensitivity results from an inability of thecells to accumulate sufficient intracellular concentrations of neomycin for it to beinhibitory.

6 NEOMYCIN

2.3.2 Acquired resistance

There are three categories of resistance to aminoglycosides: that due tospontaneous mutation (rare, occurring with a probability of 10–8 to 10–10 in a cellpopulation (Moellering 1983)); that due to altered cell permeability to the aminoglyco-side; and that due to inheritance of plasmid-encoded resistance factors which specifyinactivating enzymes. The latter is more commonly reported for clinically relevantspecies. Most of the acquired resistance to aminoglycosides in aerobic bacteria isdue to the acquisition of inactivating enzymes which modify the drug bound to thetransporter, preventing ribosomal binding. The plasmid resistance factors (R factors)against neomycin encode phosphotransferases, acetyltransferases and nucleotidyl-transferases. These enzymes are in certain cases active against more than oneaminoglycoside but do not necessarily cause complete inactivation. Thus, bacteriathat inherit plasmids which encode these inactivating enzymes may become resistantor less sensitive to other aminoglycosides (Lechevalier, 1975; Davies, 1986;Moellering, 1983).

2.3.3 Decision tree

At its fifty-second meeting (Annex 1, reference 141), the Committee developeda decision tree to address the potential adverse impacts of antimicrobial residueson human intestinal microflora (Figure 1). At its present meeting, the Committeeused this decision tree to answer the following questions in its assessment ofneomycin.

1. ‘Does the ingested residue have antimicrobial properties?’

Yes, but the spectrum of activity against the major groups of intestinal bacteria islimited, as shown in the table of minimum inhibitory concentrations (MICs) for relevantintestinal bacteria tested under standard conditions specified by the NationalCommittee of Clinical Laboratory Standards (USA; 1993) (see Table 1). Gastro-intestinal anaerobes are insensitive to neomycin. In a survey of gastrointestinalbacteria tested at standard and high inoculum densities, the lowest MIC50 valueswere recorded for the most sensitive relevant genera, Eubacterium (8 µg/ml) andLactobacillus (64 µg/ml), respectively. The potency of neomycin against Escherichiacoli in vitro decreased as the inoculum density increased or the oxygen availabilitydecreased. The MIC50 for E. coli at a high inoculum density was 64 µg/ml underaerobic test conditions and 128 µg/ml under anaerobic conditions; the latter valuewas higher than those of other intestinal organisms. The MIC50 of 64 µg/ml wasused by the Committee in its calculations, as Lactobacillus was the most sensitiverelevant strain at high inoculum density.

2. ‘Does the drug residue enter the lower bowel by any route?’

Yes. Most of an oral dose is excreted in faeces, without metabolism. Neomycinis poorly absorbed from the intestinal tract of humans and animals and from cows’udders. The amount of orally administered neomycin recovered in urine was lessthan 10%. In healthy humans given a single oral dose of neomycin sulfate at

1000 mg per person, the proportion of neomycin absorbed from the gastrointestinal

7NEOMYCIN

Assess the effects of veterinary drug residues including metabolites,on the microflora of the human gastroinestinal tract

Does the ingested residue haveantimicrobial properties? Section 1a

Does the drug residue enter the lowerbowel by any route (e.g., with the foodbolus, by biliary circulation, and/or by

mucosal secretion)? Section 1b

Conclude that the drug residuewill not affect the intestinal

microflora and use toxicologicaldata to derive the ADI

Conclude that the drug resi-due will not affect the intesti-nal microflora and use toxico-logical data to derive the ADI

Is the ingested residue transformed irrevers-ibly to inactive metabolites by chemical trans-

formation, host metabolism or intestinalmicroflora metabolism in the bowel and/or bybinding to intestinal contents? Section 1b–d

Do a literature survey and other submitteddata on the effects of the veterinary drug

on the colonic microflora provide a basis toconclude that the ADI derived from toxico-logical data is sufficiently low to protect the

intestinal microflora? Section 1e





Do data derived from therapeutic use ofthe drug class in humans or in modelsystems in vitro or in vivo indicate that

effects could occur in the gastrointestinalmicroflora? Section 1f

Determine the most sensitive adverse effect(s) of thedrug on human intestinal microflora. Adverse effects

such as selection of drug-resistant populations,disruption of the colonization barrier or changes in the

metabolic activity of intestinal microflora that have beenlinked to specifically to adverse effects on human health

should be considered.

Conclude that the drugresidue will not affect the

intestinal microflora and usetoxicological data to derive

the ADI

NoYes

NoYes

No

No

Yes

Yes

Yes No

Figure 1. Decision tree for determining adverse microbiological effects of residuesof antimicrobial drug in food-producing animals

tract was < 10% on the basis of blood and urine analysis (Poth et al., 1950; Kunin etal., 1960). Very young calves were found to absorb slightly more (Aschbacher &Feil, 1994). Neither neomycin B nor neomycin C was preferentially absorbed indogs given either molecule orally (Freyburger & Johnson, 1956).

8 NEOMYCIN

It is generally accepted that most of the neomycin that is ingested is excreted.As neomycin is a polycation, much of the ingested material is apparently adsorbedto faecal contents. Therefore, a conservative microbiological safety assessmentwould assume that 100% neomycin is excreted chemically unaltered in faeces.

Table 1. Minimum inhibitory concentrations (MICs) of neomycin against majorbacterial groups tested under the conditions of the National Committee ofClinical Laboratory Standards of the USAa

Bacterial species Strains MIC50 (µg/ml)b

and genusLowc Highd

Supplemented Wilkins– Supplemented Wilkins–blood medium Chalgren- blood medium Chalgren-

glucose glucosemedium medium

Bacteroides 15e,f > 128 > 128 > 128 > 128Bifidobacterium 12e 16 128Clostridium 11e,5f > 128 128 > 128 > 128Enterococcus 10e,2f > 128 128 > 128 > 128Escherichia coli (aerobic)g 13e 16 64Escherichia coli (anaerobic)g 13e > 128 > 128Eubacterium 9e 8 > 128Fusobacterium 5e,3f 16 32 > 128 128Lactobacillus 15e,2f >128 32 > 128 64Peptostreptoccoccus/ 16e,14f >128 32 > 128 128 Peptococcus

a Corrected version of the original tables that appeared in reference 141 in Annex 1b MIC50 values are for all strains included in the assay, tested by the standard guideline of

the National Committee of Clinical Laboratory Standards for determining MICs ofanaerobes by the agar dilution technique.

c The cell concentration of low-density inocula was 1 ∞ 108 cells/ml. The amount of inoculumper spot on agar medium was approximately 2 ∞ 105 cells, assuming 0.002 ml of cultureper spot.

d The cell concentration of high-density inocula was 1 ∞ 1010 cells/ml. The amount ofinoculum per spot on agar medium was approximately 2 ∞ 107 cells, assuming 0.002 ml ofculture per spot.

e Number of tested strains of which the MIC values for Wilkins–Chalgren-glucose mediumwere used to calculate summary values.

f Number of tested strains of which the MIC values for supplemented blood medium wereused to calculate summary values.

g The same set of 13 strains was tested aerobically and anaerobically with the same inoculaand lots of media. The inocula used to determine the aerobic MIC were incubated at 37 °Caerobically.

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3. ‘Is the ingested residue transformed irreversibly to inactive metabolites by chemicaltransformation, metabolism mediated by the host or intestinal microflora in the bowelor binding to intestinal contents?’

Neomycin undergoes negligible biotransformation after parenteral administrationand binds avidly to intestinal and faecal contents. It is not clear what percentage ofan oral dose is inactivated or whether the inactivation is dose-dependent. Althoughstudies have been conducted of binding and inactivation of neomycin in faecalsuspensions in vitro, the solids and soluble components of faecal specimens arecomplex and heterogeneous, varying substantially among individuals. Thus, thesestudies cannot be considered exhaustive for defining the kinetics or the physico-chemical mechanisms of binding or inactivation of neomycin. Nevertheless, theyprovide useful information for assessing the effects and safety of residues of neomycinin milk and tissues.

In an early study of binding of aminoglycosides to dog faeces, neomycin wasincubated in a dilute suspension of faeces, and the amount of neomycin bound tofaecal solids was calculated as the difference between the amount added to thesuspension and the amount measured (by microbiological assay) in the supernatantafter the mixture had been incubated and centrifuged. Initially, 75% of the neomycinwas calculated to have been pelleted with faecal solids in the incubation mixture.After two washings of the pelleted solids and acid extraction (method unspecified),47% of the added neomycin was calculated to be retained with the faecal solids.The same experiment was conducted with other aminoglycosides, includinggentamycin, kanamycin and paromomycin, with similar results (Wagman et al., 1974).

Neomycin was incubated at various concnetrations with similarly dilutesuspensions of faecal specimens obtained from nine healthy volunteers. As in theprevious study, ‘binding’ of neomycin to faeces was calculated from the amount ofadded activity that remained in the supernatant solution after removal of faecal solidsby centrifugation of the incubation mixture. The percentage of concentrations ofneomycin up to 500 µg/ml that was bound was calculated to be 83–98%. The amountbound depended on the amount of neomycin added per gram of diluted faeces. Theauthors reported that no destruction or inactivation of neomycin occurred duringincubation in the supernatant solutions, but data were not provided (Hazenberg etal., 1984). The same test system was used to compare the binding of variousaminoglycosides and antibiotics used in selective decontamination of the bowel.Neomycin and polymyxin B showed substantially higher percentages of binding perunit weight of antibiotic than did tobramycin, gentamycin and cephradine (Hazenberget al., 1983a).

Neomycin at various concentrations was incubated with suspensions of individualfaecal specimens collected from eight healthy volunteers. The suspensions containedhigher concentrations of faecal solids than in the studies described above, and twoincubation times (0 and 24 h) were studied in duplicate. As in the other studies, themicrobiological activity remaining in the supernatants was used to calculate the ‘percent inactivation’ relative to the amount of added neomycin. The microbiologicalactivity in the supernatants of pelleted faecal solids depended on the concentration

10 NEOMYCIN

of added neomycin, and the percentage of added neomycin that was ‘inactivated’decreased with increasing concentrations of neomycin. Nearly 100% binding wasseen in incubation mixtures in which the ratio of neomycin to faecal wet weight was≤ 5 mg to 1 g. This ratio was consistent with the observations of Hazenberg et al.(1984) and Wagman et al. (1974). The authors concluded that the ‘inactivation’occurred during the time it took to mix the suspension and harvest the supernatantsolution (roughly 50 min), because no difference in the per cent inactivation wasseen at 0 and 24 h (Veringa & van der Waaij, 1984).

In all four studies summarized above, a microbiological assay was used to assessthe concentration of neomycin. The recovery of neomycin from the test system wasnot reported. The only amounts of neomycin measured were the concentrationsremaining in supernatant solutions after centrifugation of faecal solids. Thus,estimates of ‘per cent binding’ or ‘per cent inactivation’ reflect microbiological activityexpressed as a percentage of the total neomycin activity added to the test system.The net decrease in the detectable microbiological activity of soluble neomycin couldhave been due to a number of competing reactions, including: inactivation by ionicbinding to insoluble components of the slurry (including bacterial cells); inactivationby enzymatic modification (phosphorylation, acetylation); artefactual physical trappingof neomycin by faecal solids during centrifugation; active transport and uptake ofneomycin by sensitive bacterial cells; and inactivation by binding to solublecomponents of the slurry. Whatever the cause, the detectable microbiological activitywas reduced substantially within a short time in these dilute faecal incubations. Thisdecrease in soluble microbiological activity, by whatever mechanism, was saturable.Thus, the faecal slurries had a finite capacity to inactivate neomycin. Overall, theresults indicate that neomycin is partially or fully inactivated at the levels found asresidues. The data are, however, difficult to verify, as discussed above, and the nextquestion in the decision tree should be addressed.

4. ‘Do data on the effects of the drug on the colonic microflora provide a basis toconclude that the ADI derived from toxicological data is sufficiently low to protectthe intestinal microflora?’

Yes. Orally administered neomycin is used as adjunct therapy (at a dose of4–8 g/day) for hepatic coma and for ‘preparation’ or ‘selective decontamination’ offacultative anaerobes in the bowel for surgery (9–21 g with purgatives over 3 days).A reversible intestinal malabsorption syndrome with loss of digestive enzymes andflattening of intestinal villi has been described after oral dosing with neomycin atž 3 g/day for more than several days (Jacobson et al., 1960; Faloon et al., 1962).Malabsorption of fats, protein, cholesterol, carotenes, mono- and disaccharides,vitamin B12, sodium, calcium and iron have been reported, due in part to directinhibition of absorption after interaction of neomycin with the nutrient. Neomycin isthought to disrupt micelle formation of bile acids and neutral sterols, thus leading totheir increased excretion in faeces (Sirtori et al., 1991). In vitro, addition of neomycinto human bile and bile salt solutions resulted in precipitates of bile salts and bilirubin,indicating that there may be a relationship between binding in vitro and themalabsorption of fat observed when neomycin is given orally (Faloon et al., 1962).Prepared solutions of sodium glycocholate and sodium taurocholate are precipitatedby the addition of neomycin, but the precipitation is reversed as the pH is raised,

11NEOMYCIN

with maximum precipitation at - pH 6.0. The extent to which this occurs after ingestionof the concentrations at which residues occur has not been determined. The dosesat which malabsorption is observed are more than 1000 times higher than thetoxicological ADI (0–60 µg/kg bw), and the calculated microbiological ADI is basedon microbiological data.

In a study of the effects of antibiotics on human gut flora, changes in facultativebacteria, yeast and Clostridia were monitored in patients in hospital for non-gastrointestinal diseases. The bacterial populations in faecal specimens collectedbefore, during and after treatment with neomycin were enumerated. Four patientsreceived neomycin orally at 4 g/day for 7 days, and 10 patients received 2 g/day for6 days. The populations of all the bacterial groups that were monitored decreasedduring treatment. The group most severely affected was enterococci, followed by E.coli non-lactose fermenters, lactobacilli, Clostridia and Proteus species. The numbersof yeasts and Klebsiella-like organisms increased, as did those of antibiotic-resistantpopulations (Daikos et al., 1968). In a study in which facultative bacteria and yeastsin faecal specimens from patients given neomycin orally were cultured, 2 g/day ofneomycin reduced the numbers of facultative bacteria (Poth et al., 1950, 1951). Inpatients treated with neomycin orally at a dose of 3 or 5–6 g/day, although the totalbacterial counts remained high (1010–1011 cells/g faeces), the number of enterococcifell by two to three orders of magnitude, and the decreases in the numbers of coliformsand Clostridia were even greater. The populations of bifidobacteria and Bacteroideswere more constant (Finegold et al., 1965, 1983).

Neomycin was administered to healthy volunteers at a dose of 1 g three timesdaily for 5 days, and fresh stool samples were collected before, during and aftertreatment and monitored for facultative bacteria and strict anaerobes. The mean E.coli counts were not affected by treatment, but two of the eight volunteers showedsubstantial differences in counts. It was concluded that this regimen had little effecton the counts of aerobic bacteria in the colon (Arabi et al., 1979).

In all the studies summarized above, oral doses 2000 mg were given per adult.The responses varied, but the effects on facultative bacteria reflected a generaltrend. The concentration of neomycin residues remaining after veterinary useafter,3.6 mg, is > 500 times lower than the doses used in these studies. As the numbersof anaerobic bacteria are not detectably affected at therapeutic doses, there is littlereason to expect that the populations in the lower bowel would be affected by dailyingestion of 3.6 mg.

The absence of any change in the population of anaerobic bacteria in humansgiven neomycin at doses 2000 µg is consistent with recent findings in HFA micegiven neomycin, in which no changes in the populations of subpopulations of E.coli, ‘neomycin-resistant E. coli’, enterococci, ‘neomycin-resistant enterococci’ orlactobacilli were found at the highest concentration of neomycin added to drinking-water (20 µg/ml) for 6 weeks (Kotarski, 2002). Assuming that a 40-g mouse ingests5 ml of neomycin-containing water per day, the amount of neomycin ingested wouldbe (20 µg/ml ∞ 5 ml/day per 40-g mouse), or 150 mg/60 kg bw. Thus, the toxicologicalADI of 0–3.6 mg/60-kg person (equal to 0–60 µg/kg bw) is sufficient to protect againstchanges in bacterial composition, including emergence of resistance.

12 NEOMYCIN

The toxicological ADI of 0–60 µg/kg bw is further justified by the finding ofHazenberg et al. (1983b) that no changes in bacterial subgroups were induced inHFA mice given neomycin at 1000 µg/ml in water for 35 days, but changes wereinduced with 2000 µg/ml. The NOEL for changes in bacterial subpopulations in thisstudy was thus 7500 mg/60-kg person. Although the study was not designed todetermine a microbiological ADI, it nevertheless provides information suggestingthat the NOEL for changes in a bacterial population is much higher than the currenttoxicological ADI. The available data (Table 2) thus support the toxicological ADI of0–60 µg/kg bw (equal to 0–3.6 mg/60-kg person), which would protect the humangastrointestinal flora. The current toxicological ADI for neomycin should thereforenot be changed, consistent with the positive answer to question 4 and recommen-dation 1(e) of the guidance for the decision tree (Annex 1, reference 147).

Table 2. ADIs that could be derived from various studies on neomycin

Type of study No-effect observations ADI (mg/60-kg Referenceequivalent bwper day)a

MIC test for > 100 MIC50 of most sensitive ADI = (64 µg/ml ∞ 200 g) / Van Saene etstrains of intestinal relevant genus, Lacto- (1.0 ∞ 1.0 ∞ 60-kg person) al. (1985);bacteria (high bacillus, under conditions = 14 mg/kg bw Annex 1,inoculum density) of high inoculum density = 840 mg/60-kg person reference 114

= 64 µg/ml. Inactivation incolon due to binding nottaken into account, asminimum bactericidalconcentrations of sensi-tive aerobic facultativebacteria increased 30–640-fold when faeceswere added to test broth.

HFA mice dosed No changes in E. coli, ADI = 125 mg/kg equi- Hazenburgorally in drinking- gram-negative anaerobes, valent bw x 60 kg et al. (1981)water total number of resistant 7500

anaerobes at 125 mg/kgbw per day

Humans No changes in numbers 3000 Finegold et al.of Bacteroides, Clostridium, (1965)neomycin-resistant E. coliat oral dose of 3 g/day for5 days

Adapted from Cerniglia & Kotarski (1999)a ADIs are deriuved only from comparisons of MICs with data derived in vivo, to indicate themagnitude of the differences. Additional, relevant, unpublished information that may beavailable from registration files should be taken into account in making final decisions andestablishing an appropriate ADI.

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2.4 Ototoxicity

2.4.1 Effects on hearing

High systemic doses of aminoglycosides can be ototoxic and nephrotoxic inhumans and other mammals. In some countries, neomycin is not allowed forparenteral use as a veterinary drug because of concern about toxicity to targetanimals. In those countries that permit this use, it is generally restricted for treatmentof serious gram-negative infections that are resistant to less toxic medications.

Deafness occurred in one of two calves treated with neomycin at an intramusculardose of 2.2 mg/kg bw twice daily for 13 days and in one of two calves given 4.5 mg/kgbw intramuscularly twice daily for 12 days. Nephrotoxicity was seen in all fourneomycin-treated calves. No adverse effects were seen in two control calves givenbenzylpenicillin at an intramuscular dose of 3600 U/kg bw twice daily for 7 days(Crowell et al., 1981).

Neomycin is considered to be the most toxic of the authorized aminoglycosidedrugs (Riviere et al., 1991). Assessment of the damage to cultures of hair cells fromthe outer cochlea of neonate mice showed neomycin to be the most potent of severalaminoglycoside and aminocyclitol drugs investigated. The order of potency wasneomycin > gentamycin > dihydrostreptomycin > amikacin > neamine >spectinomycin (Kotecha & Richardson, 1994).

Aminoglycoside-induced ototoxicity may affect one or both ears (Lerner et al.,1998). Ototoxicity is more likely to occur after parenteral use than after oraladministration. A review of studies of ototoxicity in humans (Govaerts et al., 1990)showed wide variation in estimates of the incidence of ototoxicity in patients treatedwith aminoglycosides, ranging from 0% to 63% for cochlear toxicity and 0% to 72%for vestibular toxicity. In a prospective study of 32 patients given long-term treatmentwith neomycin (neomycin sulfate at 500 mg per person every 6 h for 3 months),6.7% of the patients developed cochlear toxicity, as determined by high-frequencyaudiometry (Rappaport et al., 1986). The risk of ototoxicity after orally administeredneomycin is increased for patients with impaired renal function or gastrointestinalinflammation (Kavanagh & McCabe, 1983). Loss of hearing in the high frequencyregion was experienced by 9 of 17 children (53%) aged 2–7 years with gastroenteritiswho had been given neomycin orally at doses of 50–100 mg/kg bw per day for6–9 days (Zelenka et al., 1966).

High concentrations of aminoglycosides in plasma can result in transfer to theperilymph and endolymph of the inner ear. As diffusion back into the bloodstream isslow, the drug can accumulate in the perilymph and endolymph. The accumulationis dose-dependent but saturable, and the aminoglycoside can stay in the perilymphfor prolonged periods. The half-life of aminoglycosides in perilymph is 10–15 timeslonger than that in serum (Lortholary et al., 1995; Chambers & Sande, 1996)

Aminoglycosides can damage the vestibular and cochlear sensory hair cells ofthe vestibular epithelium and the organ of Corti, which can result in impairment ofauricular function, including loss of balance and irreversible deafness. The sensory

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cells do not regenerate, and retrograde degeneration of the auditory nerve (eighthcranial nerve) follows. The nerve cells become affected only when the hair cells aremissing. Aminoglycoside-induced cochlear toxicity is one of the commonest causesof acquired deafness in humans but is less common in neonates and children thanin adults (Matz, 1993; Chambers & Sande, 1996; Lerner et al., 1998).

Although all aminoglycosides can affect both cochlear and vestibular function,some preferential toxicity is evident. In the case of neomycin, it is principally auditoryfunction that is affected. Tinnitus is often the first symptom of effects on the cochlea,and there may be auditory impairment if exposure to the drug is not discontinuedwithin a few days. Perception of high-frequency sounds is lost first; if exposure tothe drug continues, perception of lower frequencies is lost progressively (Langman,1993; Chambers & Sande, 1996)

Although auditory (cochlear) toxicity is more common, neomycin can also causevestibular toxicity. The symptoms of severe vestibular toxicity include nausea,vomiting, vertigo, nystagmus and difficulty with gait (Lerner et al., 1998).

The Committee assessed data on the ototoxicity of neomycin at its forty-thirdmeeting (Annex 1, reference 114). Several studies of the effects of neomycin onguinea-pigs (Riskaer et al., 1956; Brummett et al., 1985) and on cats (Hawkins,1952; Hawkins et al., 1953) were considered. In addition human case studies weretaken into account (Waisbren & Spink, 1950; Lindsay et al., 1960; Halpern & Heller,1961; King, 1962; Fields, 1964; Greenberg & Momary, 1965). The Committee couldnot identify a NOEL from the studies in cats but identified a NOEL of 6 mg/kg bw perday for ototoxicity in guinea-pigs given neomycin orally. The Committee at its forty-seventh meeting established an ADI of 0–60 µg/kg bw by applying a safety factor of100 to the NOEL of 6 mg/kg bw per day from the study on guinea-pigs (Annex 1,reference 126).

2.4.2 Mitochondrial DNA mutation

Two groups of patients with aminoglycoside-induced ototoxicity have beenidentified: one in which the effect was the result of prolonged or high-dose exposureto the drug and a second that in which signs appeared after minimal exposure,suggesting an idiosyncratic susceptibility (Bacino et al., 1995). Clusters of susceptibleindividuals exist in certain families, suggesting that genetic factors might play a role.Families with raised susceptibility to the drug were reported in the literature as earlyas 1957 (Horiguchi & Moriyama, 1957). After a maternal inheritance pattern ofaminoglycoside-associated deafness was identified in several Japanese (Higashi,1989) and Chinese (Hu et al., 1991) families and in a large Arab–Israeli family (Jaberet al., 1992), the hypothesis of mitochondrial inheritance of the susceptibility wasraised. The observations that every known mitochondrial genetic disease hassensorineural hearing loss or deafness as a common phenotype and thataminoglycoside-poisoned hair cells show mitochondrial dysmorphology areconsistent with this hypothesis (Hutchin et al., 1993).

As the bacteriocidal activity of aminoglycosides was known to involve interactionwith ribosomal RNA in bacteria and as mitochondrial ribosomes are structurally

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more similar to bacterial ribosomes than to mammalian cytosolic ribosomes, it wassuspected that susceptibility to aminoglycoside-induced ototoxicity might be due toa mutation of mitochondrial DNA at a site corresponding to the production of ribosomalRNA. This mutation was sought in a large Arab–Israeli family in which 55 membershad maternally inherited sensorineural deafness, which could be traced back throughfive generations to one common female ancestor (Prezant et al., 1993). The mutationwas thought to be homoplasmic, as family members had either severe hearing lossor normal hearing, with no intermediate impairment. Formal segregation analysispredicted that the simultaneous inheritance of a mutation to mitochondrial DNA andan autosomal recessive mutation caused the disease phenotype. The entiremitochondrial DNA from the blood of two deaf patients from the Arab–Israeli familywas sequenced, as was DNA corresponding to the mitochondrial 12S and 16S rRNAgenes (between nucleotides 648–1601 and 1671–3229, respectively) from twoChinese patients who were members of other families with maternally inheritedaminoglycoside-induced deafness. The results were compared with the sequencesfor 278 control individuals, consisting of 35 Arab–Israelis and whites, Asians andblacks in equal proportions. The investigation of the Arab–Israeli family also includedsequencing of mitochondrial DNA from one non-deaf family member and a non-deaf, unrelated Arab, and all the sequences were compared with the published‘Cambridge sequence’ of the 16 569-base pair human mitochondrial genome(Anderson et al., 1981). Several rare sequence variations (mutations) were detectedin mitochondrial DNA from each of the four deaf individuals. Only one mutation wascommon to all three families: an adenine to guanine (A to G) homoplasmic pointmutation at nucleotide 1555 (A1555G mutation), which is in a region of the 12SrRNA gene that codes for a highly conserved domain of the small rRNA. This mutationwas not found in any of the 278 controls, whereas the other mutations were eachfound in a few control individuals. Aminoglycosides are known to bind to this regionof the 12S rRNA gene in bacteria (Moazed & Noller, 1987; Gravel et al., 1987).Furthermore, the A1555G mutation was the only one of the identified mutations thataffected a sequence of bases that was evolutionarily conserved. The sequence hasbeen identified in mice, rats, cattle and humans. It codes for a domain of the smallrRNA that is essentially the same in bacteria, plants, invertebrates and mammals.

The homoplasmic A1555G mutation was detected in mitochondrial DNA fromthe blood of seven of 41 (17%) unrelated individuals in the USA of diverse ethnicorigins who had hearing loss that had developed after they had received amino-glycosides (Fischel-Ghodsian et al., 1997). The ethnic groups of the persons withthe mutation included whites, Hispanics and Asians. Four of the seven individualswith the mutation had a family history of aminoglycoside-induced ototoxicity. In threeof those with the mutation, the onset of deafness had occurred a number of years(17 years in one case) after exposure to aminoglycosides.

Mitochondrial DNA from blood and hair of members of two Japanese and threeChinese families (the subjects of previous reports by Higashi, 1989 and by Hu et al.,1991) with maternally inherited hypersensitivity to aminoglycosides showed the sameA1555G mutation in all the deaf individuals investigated. The mutation was not foundin 414 unaffected people, including 274 Asians, who were studied as controls. Themutation was also detected in four of 74 people in a Chinese hospital who had gonedeaf after treatment with aminoglycosides (Hutchin et al., 1993).

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Three Mongolian families with maternal inheritance of susceptibility tostreptomycin-induced ototoxicity were identified at a school for the deaf. Bloodsamples from five deaf members of these three families (three from one family andone from each of the other families) were screened for A633G and A1555G mutationsof the 12S r RNA gene and the A1736G mutation of the 16S rRNA gene. Controlblood samples from 400 Mongolians with normal hearing was also screened. TheA1555G mutation was detected in four of the five deaf subjects, including the threefrom the same family, but in none of the 400 control individuals. The other mutationsinvestigated were present in four deaf subjects (including the three members of thesame family and the person without the A1555G mutation), but were also present inthe control population at an individual prevalence of approximately 5% (Pandya etal., 1997).

The A1555G mutation to the 12S rRNA gene in mitochondrial DNA was alsofound in 12 families in a village in Zaire where 53 of the 348 inhabitants were deaf.All of the families were said to have a common female ancestor who had lived about150 years previously. Verbal tradition indicated that many members of the familyhad suddenly become profoundly deaf in 1954, and the high incidence of deafnesshad continued to the present. It seems unlikely that exposure to aminoglycosidesplayed a part in this incident, as only one of the affected individuals had ever beentreated with an aminoglycoside (Matthijs et al., 1996).

The A1555G mutation has thus been detected in the mitochondria of familieswith high rates of deafness in China (Hutchin et al., 1993; Prezant et al., 1993),Israel (Prezant, et al., 1993), Japan (Hutchin et al., 1993), Mongolia (Pandya et al.,1997), the USA (Fischel-Ghodsian et al., 1997) and Zaire (Matthijs et al., 1996). Insome but not all cases the deafness was associated with prior exposure toaminoglycosides.

The A1555G mutation was detected in 10 of 319 unrelated Japanese out-patientswith sensorineural hearing loss; 21 of the patients had received aminoglycosides byinjection. Hearing loss was also found in 14 of 140 patients with cochlear implants;22 of these patients had received aminoglycosides. In both series, the frequency ofhearing loss was higher in the patients with a history of aminoglycoside use (33% inthe out-patients and 59% in those with cochlear implants) (Usami et al., 2000).

A Japanese family with maternally inherited sensorineural hearing loss wasreported, which had no history of aminoglycoside injection. There was no otherknown etiology for the deafness in this family. The A1555G mutation was detectedin mitochondrial DNA from blood from all three family members with hearing losswho were investigated (Iwasaki et al., 2000).

The A1555G mutation was detected in mitochondrial DNA from leukocytes offour of 46 deaf–mute Japanese persons; the A3243G mutation was not detected inany of them. Two of the 46 deaf people investigated reported on questionnaires thatthey had received streptomycin injections, and both of these people had the A1555Gmutation. The A1555G mutation was not detected in blood samples from 27 peoplewith normal hearing or 110 patients with adult-onset sensorineural hearing loss.

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The mutation therefore appeared to be a contributing factor to prelingual deafnessbut not to late-onset sensorineural deafness (Oshima et al., 2001).

Seventy Spanish families with sensorineural deafness, comprising 36 congenitalcases and 34 of late onset, were investigated for the presence of the A1555G mutationin mitochondrial DNA and compared with 200 control subjects. The mutation wasfound in the 19 of the 70 families with maternally inherited sensorineural deafnessbut in none of the controls. In 12 of the 19 affected families, all the members with themutation who had received aminoglycosides became deaf, representing 30% of thedeaf people in these families. None of the deaf people in the seven other affectedfamilies had been treated with aminoglycosides. Overall, 18% of the people withboth the mutation and deafness had been treated with aminoglycosides. The medianage at onset of deafness was significantly lower (p < 0.001) in people with themutation who were treated with aminoglycosides (5.6 years) than in those with themutation who did not receive aminoglycosides (41 years). The probability that aperson with the mutation would develop deafness by the age of 30 years wasestimated to be 96% if they were treated with an aminoglycoside and 40% if theywere untreated (Estivill et al., 1997).

Mitochondrial DNA from two Italian sisters and three of their children who haddeveloped profound high-frequency hearing loss after receiving aminoglycosidesdid not have the A1555G mutation. Nevertheless, sequencing of the 12S rRNA geneshowed that they all had a thymidine deletion around nucleotide position 961 (Casanoet al., 1999).

An investigation was conducted of the 12S rRNA gene in peripheral blood from35 Chinese students in a school for the deaf, none of whom had the A1555G mutation.Comparison of the base sequences with the standard ‘Cambridge’ mitochondrialsequence and with results for 100 control subjects with normal hearing showed thatonly three of the patients had mutations to this gene (T1243C, T1520C and961∆T+Cn) that were not also found in controls. The mutations were not found inareas of the gene known to be critical for aminoglycoside binding in the bacterialhomologue; however, one of them (T1520C) was in the mitochondrial D-loop, whichis also the site of the A1555G mutation found in other studies. The authors suggestedthat the effect of these mutations might be similar to that of the A1555G mutation:altering the three-dimensional structure of the gene to increase susceptibility toaminoglycoside-induced ototoxicity. One of the three unique mutations detectedwas an insertion at nucleotide 961 of multiple sequences coding for cytosine(961∆T+Cn). The authors claimed that such heteroplasmic mutations to mitochondrialDNA are usually restricted to only a few tissues, suggesting the possibility that somedeaf people with no unique mutations to 12S rRNA in their blood cells might havesignificant mutations in sensory cells in their ears that contributed to their deafness(Bacino et al., 1995).

2.4.3 Mechanism of ototoxicity

Prezant et al. (1993) postulated that the mutation at position 1555 on mitochondrialDNA might change the three-dimensional structure of the small rRNA in such a wayas to bring about greater binding of aminoglycosides and consequently increased

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toxicity. Hutchin et al. (1993) suggested that the mutation causes increased bindingof aminoglycosides at the ribosome, resulting in inhibition or mis-translation of proteinsynthesis. As mitochondrial polypeptides comprise part of several enzyme complexes(oxidative phosphorylation complexes I, III, IV and V) involved in ATP production,reduced production of a crucial protein could result in reduced ATP production. LowATP levels in the cochlea might lead to an imbalance of ion concentrations in thestria vascularis, endolymph or hair cells, which could cause accumulation of Ca2+ inthe hair cells, leading to cell death.

In line with this proposed mode of action, it was observed that the rate ofmitochondrial protein synthesis was decreased by about 30% in the presence of2 mg/ml paramycin in lymphoblastoid cell lines from carriers of the A1555G mutation(from the Arab–Israeli family previously investigated), as compared with cell linesfrom control individuals (Guan et al., 2000). In contrast, comparison of the proteinproduction by lymphoblastoid cell lines derived from deaf and non-deaf members ofa family in Zaire who had the mutant (A1555G) mitochondrial DNA showed thatexposure to 0.1 mg/ml of neomycin, gentamycin or streptomycin had no effect onthe amount of protein translation in cells of either deaf or non-deaf individuals (Matthijset al., 1996).

In preliminary studies, Southern blot analysis of DNA extracted from bloodsamples from the Arab–Israeli family previously described revealed no grossdeletions, insertions or rearrangements of mitochondrial DNA (Jaber et al., 1992).Biochemical studies of the effects of aminoglycosides on oxidative phosphorylationcomplexes I, III, IV and V, which include all 13 mitochondrially encoded polypeptides,in lymphoblastoid cell lines derived from members of the family showed thatmitochondrial protein synthesis was generally normal, but oxidative phosphorylationcomplex V showed more activity in cells from deaf family members than in thosefrom their non-deaf siblings, and the activity of complex III was greater in cells fromhearing and deaf family members than in control cells from unrelated Arabs (Prezantet al., 1992). Prezant et al. (1993) suggested that the development of deafness inthis family might require the presence of both the mitochondrial A1555G mutation(causing increased activity of complex III) and homozygosity for an autosomalrecessive mutation (causing increased activity of complex V), non-deaf familymembers being heterozygous for the recessive mutation. This hypothesis for themechanism of deafness differs from that proposed by Prezant et al. (1992), as theincreased activities of complexes III and V would be expected to result in increasedATP production rather than the hypothesized reduction. It is possible that thebiochemical effects underlying deafness are expressed in the ear but not inlymphoblastoid cell lines.

3. COMMENTS

The ADIs that could be derived from studies in bacteria in vitro, from studies ofhuman flora-associated animals in vivo and from studies in humans were evaluated.The available relevant microbiological data indicated that the toxicological ADI of0–60 µg/kg bw would protect the gastrointestinal flora of humans. The lowest ADIthat could be set on the basis of the results of all the available microbiological studies

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would be 0–14 mg/kg bw (840 mg per person). This value is higher than the existingADI of 0–60 µg/kg bw (3.6 mg per person), which was set on the basis of toxicologicaldata. Intake of residues at levels that would expose consumers to up to 60 mg/kgbw (3.6 mg per person) would not be expected to have adverse effects on the gutmicroflora. Consequently, there is no need to change the current ADI on the basis ofthe microbiological data.

The Committee affirmed that recent papers indicate that there is a causalrelationship between the presence of a point mutation in which the adenine at the1555 position of the 12S rRNA gene on mitochondrial DNA is changed to a guanine(A1555G mutation) and the development of deafness. Hearing loss can be due toboth genetic and environmental factors. Systemic exposure to a large dose ofaminoglycosides can bring about deafness, and genetic factors may make somepeople more susceptible to the ototoxic effects of aminoglycosides than others.Although people with the A1555G mutation appeared to be more susceptible toaminoglycoside-induced ototoxicity, it was not clear whether any of them had receivedneomycin. Nevertheless, it was considered prudent to assume that the effectsobserved were relevant to all aminoglycosides, including neomycin.

Some families with the A1555G mutation appeared to be at increased risk ofhearing loss even in the apparent absence of exposure to aminoglycosides. Thus,people with this mutation may be more susceptible to ototoxicity caused by a varietyof environmental factors, one of which is exposure to aminoglycosides. The mutationis inherited from the mother and has been demonstrated in various ethnic groups,including Chinese, Japanese, Mongolian, Spanish and Arab–Israeli, and in individualsin the USA of diverse origin. Only a few families and individuals with the A1555Gmutation have been identified worldwide.

The Committee recognized that people with the A1555G mutation might besusceptible to aminoglycoside-induced ototoxicity and might become deaf afterreceiving therapeutic doses of aminoglycosides. Since the dose and route ofadministration of aminoglycosides were not given in the reports of the studies inhumans, a dose–response relationship could not be established for an increasedrisk of ototoxicity after administration of aminoglycosides to people with the A1555Gmutation. No quantitative data were available for identifying a NOEL for the ototoxicityof neomycin or any other aminoglycoside in people with the A1555G mutation.

4. EVALUATION

The Committee noted that the current ADI for neomycin of 0–60 µg/kg bw hadbeen set by applying a safety factor of 100 to the NOEL of 6 mg/kg bw per day forototoxicity in a 90-day study in guinea-pigs. This safety factor comprises a 10-foldfactor to compensate for extrapolation of results from guinea-pigs to humans andanother 10-fold factor to account for interindividual variation within the humanpopulation.

The Committee was aware that systemic exposure to large doses of amino-glycosides in excess of the recommended therapeutic doses could result in deafnessin any person, irrespective of the presence of the mitochondrial DNA mutation.Nevertheless, deafness has been reported in people with the A1555G mutationgiven therapeutic doses of aminoglycosides. The recommended oral therapeuticdose of neomycin for adults is about 12 000 mg per person per day. The Committee

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noted that this dose is more than 3000 times greater than the current ADI for neomycinof 0–60 µg/kg bw (3.6 mg per person). This ADI is adequate to assure the health ofall consumers, including those with the A1555G mutation.

The Committee concluded that there was no need to alter the ADI of neomycinto account for the possible susceptibility of the subpopulation with the A1555Gmutation or to account for the microbiological properties of neomycin.

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Higashi, K. (1989) Unique inheritance of streptomycin-induced deafness. Clin. Genet., 35,433–436.

Horiguchi, S. & Moriyama, S. (1957),Familial occurrence of hearing disturbances due to theuse of streptomycin. Otolaryngology (Tokyo), 29, 13–16.

Hu, D.N., Qui, W.Q., Wu, B.T., Fang, L.Z., Zhou, F., Gu, Y.P., Zhang, Q.H., Yan, J.H., Ding,Y.Q. & Wong, H. (1991) Genetic aspects of antibiotic induced deafness: Mitochondrialinheritance. J. Med. Genet., 28, 79–83.

22 NEOMYCIN

Hutchin, T., Haworth, I., Higashi, K., Fischel-Ghodsian, N., Stoneking, M., Saha, N., Arnos, C.& Cortopassi, G., (1993) A molecular basis for human hypersensitivity to aminoglycosideantibiotics. Nucleic Acids Res., 21, 4174–4179.

Iwasaki, S., Tamagawa, Y., Ocho, S. & Hoshino, T. (2000) Hereditary sensorineural hearingloss of unknown cause involving mitochondrial DNA 1555 mutation. Otorhinolaryngology,62, 100–103.

Jaber, L., Shohat, M., Bu, X., Fischel-Ghodsian, N., Yang, H.-Y., Wang, S.-J. & Rotter, J.I.(1992) Sensoneural deafness inherited in a tissue-specific mitochondrial disorder. J. Med.Genet., 29, 86–90.

Jacobson, E.D., Chodos, R.B. & Faloon, W.W. (1960) An experimental malabsorption syndromeinduced by neomycin. Am. J. Med., 28, 524–533.

Kavanagh, K.T. & McCabe, B.F. (1983) Ototoxicity of oral neomycin and vancomycin.Laryngoscope, 93, 649-653.

King, J.T. (1962) Severe deafness in an infant following oral administration of neomycin. J.Med. Assoc. Georgia, 51, 530–531.

Kotarski, S.F. (2002) Review of exploratory studies using neomycin to develop model testsystems to examine the effects of antimicrobials on the human gut flora. Report No. ER-0802-7922-2002-001. Submitted to WHO by Pharmacia-Upjohn.

Kotecha, B. & Richardson, G.P. (1994) Ototoxicity in vitro: Effects of neomycin, gentamycin,dihydrostreptomycin, amikacin, spectinomycin, neamine, spermine and poly-L-lysine.Hearing Res., 73, 173–184.

Kunin, C.M., Chalmers, T.C. & Leevy, C.M. (1960) Absorption of orally administered neomycinand kanamycin. New Engl. J. Med., 262, 380–385.

Langman, A.W. (1993) Neomycin ototoxicity. Otolaryngology, 110, 441–444.Lechevalier, H.A. (1975) The 25 years of neomycin. CRC Crit. Rev. Microbiol., 3, 359–397.Lerner, S.A., Gaynes, R.P. & Nordstrom-Lerner, L. (1998) Aminoglycosides. In: Gorbach, S.L.,

Bartlett, J.G. & Blacklow, N.R., eds, Infectious Diseases, 2nd Ed., Philadelphia: W.B.Saunders Co., pp. 775–790.

Lindsay, J.R., Proctor, L.R. & Work, W.P. (1960) Histopathological inner ear changes in deafnessdue to neomycin in a human. Laryngoscope, 70, 382–392.

Lortholary, O., Tod, M., Cohen, Y. & Petitjean, O. (1995) Aminoglycosides. Med. Clin. N. Am.,79, 761–787.

Matthijs, G., Claes, S., Longo-Mbenza, B. & Cassiman, J.-J. (1996) Non-syndromic deafnessassociated with a mutation and a polymorphism in the mitochondrial 12S ribosomal RNAgene in a large Zairean pedigree. Eur. J. Hum. Genet., 4, 46–51.

Matz, G.J. (1993) Aminoglycoside cochleal ototoxicity. Ototoxicity, 26, 705–712.Mingeot-Leclercq, M.-P., Glupczynski, Y. & Tulkens, P. (1999) Aminoglycosides: Activity and

resistance. Antmicrob. Agents Chemother., 43, 727–737.Moazed, D. & Noller, H.F. (1987) Interaction of antibiotics with functional sites in 16S ribosomal

RNA. Nature, 327, 389–394.Moellering, R.C., Jr (1983) In vitro antibacterial activity of the aminoglycoside antibiotics. Rev.

Infect. Dis., 5 (Suppl. 2), S212–S232.National Committee for Clinical Laboratory Standards (1993) Methods for Antimicrobial

Susceptibility Testing, 3rd Ed., Villanova, Pennsylvania, p. 23.Oshima, T., Kudo, T. & Ikeda, K. (2001) Point mutation of the mitochondrial genome in Japanese

deaf-mutism. Otorhinolaryngology, 63, 329–332.Pandya, A., Xia, X., Radnaabazar, J., Batsuuri, J., Dangaansuren, B., Fischel-Ghodsian, N. &

Namce, W.E. (1997) Mutation in the mitochondrial 12S rRNA gene in two families fromMongolia with matrilineal aminoglycoside ototoxicity. J. Med. Genet., 34, 169–172.

Poth, E.J., Fromm, S.M., Wise, R.I. & Hsiang, C.M. (1950) Neomycin, a new intestinal antiseptic.Texas Rep. Biol. Med., 8, 353–360.

Poth, E.J., Martin, R.G., Fromm, S.M., Wise, R.I. & Hsiang, C.M. (1951) A critical analysis ofneomycin as an intestinal antiseptic. Texas Rep. Biol. Med., 8, 631–644.

23NEOMYCIN

Prescott, J.F., Baggot, J.D. & Walker, R.D. (2000) Antimicrobial Therapy in Veterinary Medicine,3rd Ed., Ames: Iowa State University Press, pp. 191–228.

Prezant, T.R., Shohat, M., Jaber, L., Pressman, S. & Fischel-Ghodsian, N. (1992) Biochemicalcharacterisation of a pedigree with mitochondrially inherited deafness. Am. J. Med. Genet.,44, 465–472.

Prezant, T.R., Agapian, J.V., Bohlman, M.C., Bu, X., Oztas, S., Qui, W.-Q., Arnos, K., Cortopassi,G.A., Jaber, L., Rotter, J.I., Shohat, M. & Fischel-Ghodsian, N. (1993) Mitochondrial ribosomalRNA mutation associated with both antibiotic-induced deafness and non-syndromicdeafness. Nature Genet., 4, 289–293.

Rappaport, B.Z., Fausti, S.A., Schechter, M.A. & Frey, R.H. (1986) A prospective study ofhigh-frequency auditory function in patients receiving oral neomycin. Scand. Audiol., 15,67–71.

Rasmussen, B.A. & Tally, F.P. (1993) Antimicrobial resistance in Bacteroides. Clin. Lif. Dis., 16(Suppl. 4), S390–S400.

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van Saene, J.J., van Saene, J.J., Stoutenbeek, C.P. & Lerk, C.F. (1985) Influence of feces onthe activity of antimicrobial agents used for decontamination of the alimentary canal. Scand.J. Infect. Dis., 17, 295–300.

Sirtori, C.R., Manzoni, C. & Lovati, M.R. (1991) Mechanisms of lipid-lowering agents. Cardiology,78, 226–235.

Usami, S.-I., Abe, S., Akita, J., Namba, A., Shinkawa, H., Ishii, M., Iwasaki, S., Hoshino, T., Ito,J., Doi, K., Kubo, T., Nakagawa, T., Komiyama, S., Tono, T. & Komune, S. (2000) Prevalenceof mitochondrial gene mutations among hearing impaired patients. J. Med. Genet., 37, 38–40.

Veringa, E.M. & van der Waaij, D. (1984) Biological inactivation by faeces of antimicrobialdrugs applicable in selective decontamination of the digestive tract. J. Antimicrob.Chemother., 14, 605–612.

Wagman, G.H., Bailey, J.V. & Weinstein, M.J. (1974) Binding of aminoglycosides to feces.Antimicrob. Agents Chemother., 6, 415–417.

Waisbren, B.A. & Spink, W.W. (1950) A clinical appraisal of neomycin. Ann. Intern. Med., 33,1099–1119.

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25FLUMEQUINE

– 25 –

FLUMEQUINE (addendum)


Dr L. Ritter

Department of Environmental Biology, University of Guelph, Guelph,Ontario, Canada

Professor A.R. Boobis

Section on Clinical Pharmacology, Faculty of Medicine, Imperial College,London, England

Dr K. Greenlees

Office of New Animal Drug Evaluation, Center for Veterinary Medicine, Foodand Drug Administration, Rockville, Maryland, USA

and

Dr K. Mitsumori

Laboratory of Veterinary Pathology, School of Veterinary Medicine, Facultyof Agriculture, Tokyo University of Agriculture and Technology, Tokyo,

Japan


Hepatotoxicity ....................................................................... 26Mechanism of tumorigenicity in mice ................................... 26


1. EXPLANATION

Flumequine is a fluoroquinolone compound with antimicrobial activity againstgram-negative organisms and is used in the treatment of enteric infections in foodanimals. It also has limited use in humans for the treatment of urinary-tract infections.Flumequine was evaluated by the Committee at its forty-second, forty-eighth andfifty-fourth meetings (Annex 1, references 110, 128 and 146). At its forty-eighthmeeting, the Committee established an ADI of 0–30 µg/kg bw based on a toxicologicalend-point (hepatotoxiciy in male CD-1 mice in a 13-week study).

At its forty-eighth meeting, the Committee evaluated information related interalia to a NOEL for hepatotoxicity and the mechanism of tumour induction. The presentCommittee, at the request of the Codex Committee on Residues of Veterinary Drugsin Foods at its Thirteenth Session (Codex Alimentarius Commission, 2001), evaluatednew studies that had been carried out to elucidate further the mechanism offlumequine-induced hepatocarcinogenicity in mice.

26 FLUMEQUINE

2. BIOLOGICAL DATA

2.1 Hepatotoxicity

In short-term and long-term studies of toxicity that had been evaluated by theCommittee at its forty-second meeting, oral administration of flumequine causeddose-related hepatotoxic effects in rats and mice. Hypertrophy, degenerative changesand focal necrosis of hepatocytes were observed in rats at 400 and 800 mg/kg bwper day in a 2-year study, and in CD-1/ICR mice at 400 and 800 mg/kg bw per dayin an 18-month study. The prevalence of hepatotoxic lesions increased with durationof treatment. At its forty-eighth meeting, the Committee noted that male mice werethe most sensitive to flumequine-induced liver damage.

2.2 Mechanism of tumorigenicity in mice

The results of long-term studies that had been evaluated previously by theCommittee showed no carcinogenic effects in rats, but a dose-related increase inthe incidence of liver tumours was observed in CD-1 mice at doses > 100 mg/kg bwper day. The tumour incidence paralleled hepatotoxic changes and was significantlyhigher in male than in female mice. As flumequine was inactive in a range of testsfor genotoxicity, including assays for gene mutation in bacteria and mammaliancells in vitro and for chromosomal aberrations in vivo and in vitro, the mechanism oftumorigenicity was unclear. At its forty-eighth meeting, the Committee reviewed theavailable toxicological database in order to determine if the hepatocarcinogenicityof flumequine resulted from a genotoxic or a non-genotoxic mechanism. Genotoxiccarcinogens act directly on DNA in the target tissue, inducing DNA or chromosomaldamage such as strand breaks or mutations, which can typically be assessed inassays for genotoxicity and short-term assays in rodents. Non-genotoxic carcinogensdo not have this activity, and non-genotoxic carcinogenicity can result from inductionof cytotoxicity and cell proliferation, which probably cause tumorigenicity in targetorgans by sustained mitogenic stimulation. Non-genotoxic tumorigenesis in rodentliver can arise through several mechanisms, including hepatotoxicity. Flumequineproduced consistently negative results when evaluated in various assays forgenotoxicity in vitro and in vivo.

Flumequine was hepatotoxic, causing hepatocellular degeneration and focalnecrosis in male and female mice. The dose-related severity of the lesions paralleledthe incidence of liver tumours. The occurrence of foci of altered hepatocytes is animportant intermediary step in hepatotoxicity-induced liver tumorigenicity.

The Committee at its forty-eighth meeting concluded that the liver tumorigenicityobserved in mice exposed to flumequine was the result of a non-genotoxicmechanism, secondary to hepatotoxicity-induced necrosis–regeneration cycles. TheCommittee noted that, as the tumorigenicity was secondary to hepatotoxicity, theNOEL for both hepatotoxicity and carcinogenicity was 25 mg/kg bw per day. TheCommittee also noted that the NOEL for hepatotoxicity was determined from a 13-week study and extrapolated to the dose required for tumour formation observed atthe end of the 18-month study in mice.

The Committee at its present meeting evaluated new information on themechanism of action of flumequine-induced mouse liver tumorigenicity. Administration

27FLUMEQUINE

of flumequine in the diet of CD-1 mice at a concentration of 4000 ppm for 30 weeks(equivalent to the lowest dose in the 18-month study of carcinogenicity in mice) orafter a single intraperitoneal injection of N-nitrosodiethylamine induced basophilicliver foci in males. Flumequine also increased the number of 8-hydroxydeoxy-guanosine adducts in liver. These responses are consistent with oxidative DNAdamage and can be associated with carcinogenicity (Yoshida et al., 1999).

In another study, heterozygous p53-deficient mice (which have increasedsensitivity to genotoxic carcinogens) that received a diet containing 4000 ppm offlumequine for 26 weeks developed basophilic liver foci at a time when there was noevidence of necrosis. The absence of cell death at the dose tested (which did notcause necrosis) showed that this would not confound interpretation of the significanceof the altered liver foci (Takizawa et al., 2001).

In a 13-week study of two-stage hepatocarcinogenicity in mice, administrationof a diet containing flumequine at a concentration of 4000 ppm induced altered liverfoci in mice subsequently exposed to a mixed promoting regimen of D-galactosamineand phenobarbital, indicating that flumequine acted as a short-term initiator. A lowerdose of flumequine (500 mg/kg bw) caused DNA strand breaks in a ‘comet’ assay ata time when liver damage was not evident. The absence of liver damage at thedose tested showed that this would not confound interpretation of the comet assay.The liver was more sensitive if it was undergoing cell proliferation due to regenerationor juvenile growth. Similarly, other tissues such as stomach, colon and urinary bladdershowed more DNA breaks in response to various doses of flumequine, with dose-dependent DNA damage in these organs in adult mice 3 h, but not 24 h, aftertreatment. The results of these studies suggest that flumequine has initiating potentialand that the hepatocarcinogenicity in mice might involve DNA strand breakage(Kashida et al., 2002).

Quinolones like flumequine exert their antibacterial activity by inhibiting bacterialtopoisomerase II (DNA gyrase). Although there are some structural similaritiesbetween bacterial and mammalian topoisomerases, they differ substantially in overallstructure. Fluoroquinolones in general have a much lower affinity for mammaliantopoisomerases than for bacterial enzymes. Information on inhibition of mammaliantopoisomerases by flumequine was not available to the Committee at its forty-eighthmeeting.

Flumequine at doses that inhibit mammalian topoisomerase II might induce DNAdamage, and the damage might be involved in mutagenic or other genotoxic stepsin carcinogenicity. The Committee noted that there was inadequate evidence toconfirm that the observed carcinogenicity of flumequine was secondary to inhibitionof topoisomerase II or that this hypothesized mechanism is the sole basis for thetumour induction observed in the lifetime study of carcinogenicity in mice.

3. COMMENTS

The Committee at its forty-second and forty-eighth meetings evaluated thetumorigenicity of flumequine. At its forty-second meeting, the Committee noted that

28 FLUMEQUINE

there was evidence of compound-related hepatotumorigenic effects in male mice.As flumequine was inactive in a range of tests for genotoxicity, the mechanism oftumorigenesis was unclear. The Committee at its forty-eighth meeting consideredthat hepatocellular necrosis and regeneration subsequent to hepatotoxicity was therelevant mechanism for the induction of liver tumours by flumequine.

Flumequine has generally been considered to be a non-genotoxic carcinogenwith only promoting activity. The Committee at its present meeting reviewed newstudies on flumequine-induced tumorigenicity that were not available at the forty-eighth meeting, in which the mechanism of action in male mice was furtherinvestigated. Although the results of ‘comet’ assays indicated that flumequine causeddouble-strand DNA breaks, the Committee noted the limitations of this assay andconsidered that those results alone could not fully substantiate a genotoxicmechanism for the observed hepatocarcinogenicity of flumequine.

4. EVALUATION

The Committee concluded that the new data raised further questions about themechanism by which flumequine increases the incidence of liver tumours in malemice. The Committee evaluated evidence that supported the involvement of bothgenotoxic and non-genotoxic mechanisms. It noted that flumequine was not genotoxicin a comprehensive battery of assays in vitro and in vivo; however, in the absence ofnecrosis, it induced basophilic foci and DNA strand breaks in a ‘comet’ assay. TheCommittee therefore could not dismiss the possibility that flumequine induces tumoursin mouse liver by a mechanism that includes genotoxic effects. It was, however,unable to identify the genotoxic effects involved in liver tumour formation or a thresholdfor those effects.

The Committee concluded that it could not support an ADI and withdrew the ADIthat it had established at its forty-eighth meeting. Before establishment of an ADIcan be considered, the Committee would wish to receive additional data on thegenotoxic effects involved in tumour formation.

5. REFERENCES

Codex Alimentarius Commission (2001) Report of the Thirteenth Session of the CodexCommittee on Residues of Veterinary Drugs in Foods, Charleston, SC, USA, 4–7 December200, Rome: Food and Agriculture Organization of the United Nations (unpublished documentALINORM 03/31).

Kashida, Y., Sasaki, Y.F., Ohsawa, K., Yokohama, N., Takahashi, A., Watanabe, T. & Mitsumori,K. (2002) Mechanistic study on flumequine hepatocarcinogenicity focusing on DNA damagein mice. Toxicol. Sci., 69, 317–321.

Takizawa, T., Mitsumori, K., Takagi, H., Onodera, H., Yasuhara, K., Tamura, T. & Hirose, M.(2001) Modifying effects of flumequine on dimethynotrosamine-inducedhepatocarcinogenesis in heterozygous p53 deficient CBA mice. J. Toxicol. Pathol., 14,135–143.

Yoshida, M., Miyajima, K., Shiraki, K., Ando, J., Kudoh, K., Nakae, D., Takahashi, M. & Maekawa,A. (1999) Hepatotoxicity and consequently increased cell proliferation are associated withflumequine hepatocarcinogenesis in mice. Cancer Lett., 141, 99–107.

29TRICHLORFON

INSECTICIDE

30 TRICHLORFON

31TRICHLORFON

TRICHLORFON (addendum)


Dr Pamela L. ChamberlainCenter for Veterinary Medicine, Food and Drug Administration, Rockville,

Maryland, USA


Biochemical aspects ............................................................. 31Absorption, distribution and excretion ........................... 31

Toxicological studies ............................................................. 34Genotoxicity ................................................................... 34Reproducitive toxicity ..................................................... 34

Multigeneration studies ........................................... 34Developmental toxicity ............................................ 36Special studies on toxicity to mammalian germ cells 38

Special studies: Neurotoxicity ........................................ 38Observations in humans ....................................................... 39


1. EXPLANATION

Trichlorfon (metrifonate) was evaluated by the Committee at its fifty-fourth meeting(Annex 1, reference 147), when it established an ADI of 0–20 µg/kg bw on the basisof a NOEL of 0.2 mg/kg bw per day for inhibition of erythrocyte acetylcholinesteraseactivity in humans treated orally, applying a safety factor of 10.

A re-evaluation of the ADI for trichlorfon was requested on the basis of theavailability of new data that were not reviewed by the previous Committee. TheCommittee at its present meeting considered the results of additional studies on thepharmacokinetics of trichlorfon and on genotoxicity, reproductive toxicity,developmental toxicity, toxicity to mammalian germ cells and studies in humans.

2. BIOLOGICAL DATA

2.1 Biochemical aspects

2.1.1 Absorption, distribution, and excretion

The pharmacokinetics of trichlorfon and its metabolite dichlorvos was studied inhealthy human volunteers and patients with various levels of renal impairment definedby creatinine clearance. The study was conducted in accordance with EuropeanCommunity Good Clinical Practice Guidelines, the Declaration of Helsinki (1989)and the German Drug Laws. The 24 participants (15 men and 9 women aged 45–75years and weighing 45–94 kg) were divided into four groups according to their

– 31 –

32 TRICHLORFON

creatinine clearance rates. Healthy volunteers (group A) had a creatinine clearance> 90 ml/min per 1.73 m2; individuals with renal impairment were assigned to groupswith creatinine clearance = 60–90 ml/min per 1.73 m2 (group B), 30–60 ml/min per1.73 m2 (group C) or < 30 ml/min per 1.73 m2 (group D). There were four men andtwo women in groups A, C and D and three men and three women in group B.Treatment consisted of a single 50-mg tablet of trichlorfon administered orally. Clinicalevaluations were performed 0 (before dosing),1, 2, 3, 4, 12 and 24 h afteradministration. Clinical laboratory end-points were evaluated before dosing andduring the final examination. A 12-lead electrocardiogram (ECG) was recorded beforedosing and 1, 4 and 24 h afterwards. Blood samples for measurements ofpharmacokinetics were collected 0, 10, 20, 30, 45 and 60 min and 1.5, 2, 3, 4, 6, 8,10, 12, 16 and 24 h after dosing. Plasma and erythrocyte cholinesterase activitywas measured before dosing and 1, 24 and 168 h afterwards.

The adverse clinical effects observed included diarrhoea, headache, transientincreases in amylase and lipase activity and weakness. No treatment-related effectswere found in clinical laboratory and ECG end-points. The differences in thepharmacokinetics of trichlorfon and dichlorvos in the various groups were notstatistically significant. The geometric mean half-life for trichlorfon in healthyvolunteers was 3 ± 1 h, whereas those in groups B, C and D were 3 ± 1 h, 3 ± 1 hand 2 ± 1 h, respectively. The renal clearance of trichlorfon and dichlorvos decreasedin proportion to the severity of renal impairment; however, as renal excretion ofunchanged drug or dichlorvos contributed only 1–2% of the total dose to the overallclearance of trichlorfon, the systemic concentrations of trichlorfon and its activemetabolite did not affect renal function. No significant inhibition of erythrocytecholinesterase was found in healthy or renally impaired participants. The investigatorsexplained that trichlorfon causes a gradual increase in inhibition of erythrocytecholinesterase activity, steady-state inhibition being reached within a few weeks atthe daily doses used in the treatment of Alzheimer disease. The authors did notspecify the doses used in this indication, but the dosage regimens used in reportedclinical trials consist of a loading dose of 0.5, 0.9 or 2 mg/kg bw per day for 2 weeks(Cummings et al., 1998), followed by a maintenance dose of 0.2, 0.3 or 0.65 mg/kgbw per day for 10 weeks. In the present study, plasma cholinesterase activity wasinhibited by 60–80% 1 h after dosing, with greater inhibition seen in healthy volunteers.The investigators concluded that renal function had no significant effect on thepharmacokinetics of trichlorfon and that doses need not be adjusted for patientswith renal impairment (Dingemanse et al., 1999; Heinig & Dietrich, 1999).

The pharmacokinetics of trichlorfon was studied in healthy volunteers given thedrug alone or in combination with a magnesium- and aluminum hydroxide-containingantacid, cimetidine or ranitidine, in two studies conducted in accordance with goodclinical practice guidelines and the 1975 Declaration of Helsinki and its revisions. Inthe first study, 12 female and six male volunteers aged 55–75 years and of normalbody weight were given three single 50-mg oral doses of trichlorfon 1week apart.Six participants were dosed orally 5 min later with 10 ml of a magnesium- andaluminum hydroxide-containing suspension. Another six participants were dosed90 min later with 10 ml of the same suspension. The remaining six received noadditional treatment. Blood samples for assay of trichlorfon, dichlorvos andcholinesterase activity were collected before dosing and 0.5, 1, 1.5, 2, 3, 6 and 8 hafter dosing. An additional sample was collected at 24 h for determination of

33TRICHLORFON

cholinesterase activity. In the second study, six female and 10 male volunteers aged45–58 years and of normal body weight were given each of the following treatmentsseparated by a 1-week washout period: cimetidine at 400 mg in a tablet twice dailyfor 4 consecutive days, with 400 mg of cimetidine and a 50-mg trichlorfon tabletadministered on day 5; ranitidine at 150 mg in a tablet twice daily for 4 consecutivedays, with 150 mg of rantidine and a 50-mg trichlorfon tablet administered on day 5;or a single oral dose of 50 mg trichlorfon. Blood samples for assay of trichlorfon anddichlorvos were collected before administration and 10, 20, 30, 45 min and 1, 1.5, 2,3, 4, 6, 8, 10 and 12 h afterwards. Blood samples for determination of cholinesteraseactivity were collected before dosing and 1, 2 and 24 h after dosing on day 5 of eachperiod and in the follow-up phase of the study 7 and 14 days after administration oftrichlorfon. Plasma samples for assay of cholinesterase activity were collected ondays 1, 4 and 5 before dosing and on day 5, 24 h after dosing, in each period and 7and 14 days after administration of trichlorfon in the last study period. In both studies,all volunteers were given a complete medical examination including medical history,physical examination, chest X-ray, laboratory investigations, vital signs and an ECG,before entering and after completion of the study.

Nausea (first study) and headache (second study) were the only adverse eventsconsidered possibly to be related to treatment. There were no clinically relevantchanges in ECG or clinical laboratory end-points. The pharmacokinetics of trichlorfonand dichlorvos were not significantly affected by concomitant administration of theantacid preparation or by intake of cimetidine or ranitidine. The authors explainedthat the terminal elimination half-lives of trichlorfon and dichlorvos could not bedetermined in the first study because many samples contained insufficient concentra-tions of these compounds during the elimination phase. The geometric meanelimination half-life of trichlorfon in the second study was 2–3 h. In the first study,erythrocyte cholinesterase activity was only slightly inhibited and was notdistinguishable from baseline values. The authors explained that multiple dosesmust be administered to attain significant inhibition of this enzyme and, eventually,therapeutically relevant, steady-state inhibition. In the second study, the authorsreported that the cholinesterase activity in plasma was constant on days 1, 4 and 5of the study with the first two treatments, but the level of inhibition was not given.After the third treatment period, the geometric mean inhibition of plasmacholinesterase activity 24 h after dosing was 35% that of the baseline value on day5. The authors concluded that concomitant administration of a magnesium- andaluminum hydroxide-containing antacid, cimetidine or ranitidine, had no effect onthe pharmacokinetics of trichlorfon or dichlorvos in a healthy volunteer population(Heinig et al., 1999).

The effects of food and time of administration on the pharmacokinetics oftrichlorfon were studied in healthy volunteers. The studies were conducted accordingto good clinical practice guidelines and the 1975 Declaration of Helsinki and itsrevisions. In the first study, 12 male and two female participants aged 50–68 yearsand of normal body weight were given two single oral doses of 50 mg of trichlorfon1 week apart. After an overnight fast (10 h), the volunteers received the followingtreatments, in random order: a 50-mg tablet of trichlorfon orally or a 50-mg tabletwithin 5 min of eating a breakfast consisting of 22 g of protein, 76 g of fat, 54 g ofcarbohydrate and 1015 total calories. Blood samples for assay of trichlorfon anddichlorvos were collected before administration and 10, 20, 30 and 45 min and 1,

34 TRICHLORFON

1.5, 2, 3, 4, 6, 8 and 10 h after dosing. Blood samples for determination of erythrocyteand plasma cholinesterase activity were collected before administration and 1, 2, 3,4, 6, 8, 10 and 24 h after dosing. In the second study, 12 healthy male volunteersaged 24–45 years and of normal body weight were given the following treatments,separated by a 1-week washout period: a tablet containing 80 mg of trichlorfon at8:00 after an overnight fast (10 h), with a meal 4 h after dosing; a tablet containing80 mg of trichlorfon at 19:00, after a meal at 12:00; and a tablet containing 80 mg oftrichlorfon at 22:00 after a meal at 18:00. Blood samples were collected for assay oftrichlorfon and dichlorvos before administration and 10, 20, 30 and 45 min and 1,1.5, 2, 3, 4, 6, 8 and 12 h after dosing. Blood samples for determination of erythrocyteand plasma cholinesterase activity were collected before administration and 0.5, 1,2, 3, 4, 6, 8 and 12 h after dosing.

Nausea and diarrhoea (first study) and headache (second study) were the onlyadverse events considered possibly to be related to treatment. The bioavailability oftrichlorfon and dichlorvos after breakfast was approximately 95% of that after fasting.The maximum concentrations of trichlorfon and dichlorvos were reduced toapproximately 56% with food, and the time to maximal plasma concentration wasincreased. The terminal elimination half-lives of trichlorfon or dichlorvos were notaffected; the geometric mean elimination half-life of trichlorfon in the first and secondstudy was 2 ± 1 h. In the first study, erythrocyte cholinesterase activity was notsignificantly changed from the values before administration . Plasma cholinesteraseactivity was almost completely inhibited 1 and 3 h after administration. In the secondstudy, inhibition of erythrocyte cholinesterase activity was low and erratic, with highstandard deviations, after all treatments. The mean plasma cholinesterase activitywas inhibited maximally (ž 95%) 1 and 3 h after treatment in the first and secondtreatment phases and 3 h after treatment in the third treatment phase. The enzymewas still measurably inhibited (by approximately 50%) 24 h after dosing. The authorsconcluded that intake of food and time of administration had no relevant effect onthe pharmacokinetics of trichlorfon or dichlorvos or the pharmacodynamics oferythrocyte or plasma cholinesterase activity (Heinig & Sachse, 1999).

2.2 Toxicological studies

2.2.1 Genotoxicity

The only information on the genotoxicity of trichlorfon that was not reviewed bythe Committee at its fifty-fourth meeting was the results of two tests for sisterchromatid exchange reported in the same paper. In a test conducted in humanlymphocytes in vitro, trichlorfon at a concentration of 10, 20, 30, 40, 50 or 60 µg/mldid not induce sister chromatid exchange; however, when mice were given trichlorfonat a dose of 30, 60 or 120 mg/kg bw, sister chromatid exchange was observed inbone-marrow cells (Madrigal-Bujaidar et al., 1993).

2.2.2 Reproductive toxicity

(a) Multigeneration studies

Groups of 40 male and 40 female Sprague-Dawley rats received diets containingtrichlorfon (purity, 98%) at a concentration of 150, 500 or 1750 mg/kg, equivalent to7.5, 25 and 88 mg/kg bw per day, for two generations, with one mating per generation.

35TRICHLORFON

The parameters evaluated in adults and pups included clinical observations, bodyweight, food consumption and gross and tissue lesions. In addition, plasma anderythrocyte cholinesterase activities were determined in 10 F0 and F1 animals ofeach sex per dose after 8 weeks of treatment, during the premating phase andagain at termination. Plasma, erythrocyte and brain cholinesterase activities weredetermined in F1 and F2 generation pups at the time of culling (day 4 post partum)and at weaning (day 21 post partum). One pup of each sex per litter was chosenrandomly from each litter and killed on lactation day 4 and another pair on lactationday 21, until 10 pups of each sex per dose had been obtained. This study wasconducted in accordance with the United States Environmental Protection AgencyPesticide Assessment Guidelines and Health Effect Guidelines and the OECDguidelines for testing of chemicals.

No remarkable clinical signs were observed in adults or pups of either generation.Significantly decreased body weights were observed in males and females at thehighest dietary concentration during the premating phase. No meaningful effectswere observed on food consumption by either sex. In the gestational phase of thesecond generation, the body weights of animals at the highest dose were significantlydecreased on days 0 and 6 of gestation. The body weights of animals at the highestdose were also decreased during lactation, but the difference was not statisticallysignificant. Treatment had no effect on food consumption during gestation in eithergeneration. Food consumption during the lactation phase was significantly reducedin F0 dams at the highest dose during week 2 of lactation. In animals of the secondgeneration at the highest dose, food consumption during lactation was significantlyreduced in weeks 1, 2 and 3.

Dams in the first generation at the highest dose had a significantly reducedlactation index (live pups per litter on lactation day 21/live pups per litter on lactationday 4 after culling ∞ 100). In the second generation, significant decreases in thebirth index (pups born per litter/implantation sites per litter) and mean litter sizewere observed at the lowest and highest doses, but there was no dose–responserelationship. Treatment-related decreases in pup body weight were observed inboth generations at the highest dose. Gross and histopathological findings wereunremarkable in adults and pups of both generations.

Significant decreases in plasma cholinesterase activity were observed in F0generation adult females at the two higher doses and in males at the highest dose.Erythrocyte cholinesterase activity was significantly reduced in adult F0 males andfemales at the highest dose after 8 weeks of dietary intake of the test compound. Inthe F1 generation, plasma cholinesterase activity was significantly reduced in adultfemales at the two higher doses and in males at the highest dose. Erythrocytecholinesterase activity was significantly reduced in adult males and females at thetwo higher doses. At termination of the F0 generation, plasma cholinesterase activitywas significantly reduced in females at all doses, erythrocyte cholinesterase activitywas significantly reduced in males and females at the two higher doses, and braincholinesterase activity was significantly reduced in females at all doses and in malesat the highest dose. At termination of the F1 generation, plasma cholinesterase activitywas significantly decreased in females at the highest dose, erythrocyte cholinesteraseactivity was significantly decreased in females at all doses and in males at thehighest dose, and brain cholinesterase activity was significantly decreased in femalesat all doses and in males at the highest dose. Assessment of neonatal cholinesterase

36 TRICHLORFON

activity on lactation day 4 showed significantly decreased erythrocyte cholinesteraseactivity in F1 males at the two higher doses. On lactation day 21, significant decreasesin plasma and brain cholinesterase activity were observed in males and females atthe highest dose. In the F2 generation, no significant decreases in cholinesteraseactivity were observed in males or females on lactation day 4. On lactation day 21,significant decreases in plasma cholinesterase activity were observed in males andfemales at the highest dose, and significant decreases in brain cholinesterase activitywere observed in males at the two higher doses and in females at the highest dose.The treatment-related effects observed at the lowest dose in this study includedsignificantly decreased plasma cholinesterase and brain cholinesterase activities inF0 adult females at term and significantly decreased erythrocyte and braincholinesterase activities in F1 adult females at term. A NOEL could not be identifiedin this study (Astroff et al., 1998).

(b) Developmental toxicity

The sensitive period and the doses of trichlorfon required to produce brainhypoplasia in offspring were examined in pregnant white guinea-pigs. The numberof animals in each treatment group was not stated. Trichlorfon was administeredeither by stomach tube or by subcutaneous injection. Three groups received an oraldose of 25, 50 or 100 mg/kg bw on days 42, 43 and 44 of gestation. One groupreceived oral doses of 200, 100 and 200 mg/kg bw on days 42, 43 and 44 of gestation,respectively. One group received oral doses of 150, 150, 100 and 150 mg/kg bw ondays 40, 41, 42 and 43 of gestation, respectively. One group received a single oraldose of 165 mg/kg bw on day 44 of gestation. Only one group was treatedsubcutaneously, with a dose of 125 mg/kg bw. An untreated control group wasincluded. The pups were decapitated within 24 h after natural birth or immediatelyafter surgical removal around day 64, and various brain sections were separatedout and weighed.

In an earlier study, a significant reduction in brain weight was observed in offspringborn to dams treated orally with trichlorfon at a dose of 125 mg/kg bw on days 42,43 and 44 of gestation (Mehl et al., 1994). In the more recent study, no reduction inthe weight of the cerebellum or other brain structures was found in offspring ofdams at 25 or 50 mg/kg bw , but significant weight reductions were observed in theoffspring of dams given 100 mg/kg bw. A dose–response related increase in brainweight reduction was observed in a comparison of the groups given 100 mg/kg bw,200–100–200 mg/kg bw and 150–150–100–150 mg/kg bw. Clinical signs of toxicitywere observed in dams treated with 100, 150 or 200 mg/kg bw. The cerebellumappeared to be most sensitive brain structure to exposure on days 42–44, thediencephalon and medulla oblongata to treatment on days 45–50 and the colliculusto treatment on days 51–53 of gestation. The NOEL was 50 mg/kg bw (Hjelde et al.,1998).

The brain hypoplasia seen after exposure to trichlorfon has been attributed toDNA alkylation damage and inhibition of DNA alkyltransferase repair (Badawi, 1998;Mehl et al., 2000).

In a study of cytogenetic and developmental effects on pre-implantation, mid-gestation and near-term mouse embryos treated with trichlorfon during the zygote

37TRICHLORFON

stage, female mice were given an intraperitoneal dose of 100 mg/kg bw (18 animals)or 200 mg/kg bw (21 animals) 6 h after presumed conception. A control group of 18pregnant animals received distilled water. Developmental outcomes andmicronucleus formation during the pre-implantation phase were assessed on day 3of gestation; and developmental and aneugenic outcomes at mid-gestation wereassessed on day 9. On day 17 of gestation, the embryos were removed, sexed,weighed and examined for external malformations.

No signs of toxicity related to treatment with trichlorfon were observed. The meannumber of cells in embryos in both treated groups was significantly lower than in thecontrol group, and the mean number of micronuclei was significantly increased inboth treated groups compared with the control group. The number of live embryosper dam was significantly lower in the group given 100 mg/kg bw than in controls.The mean number of somites in the treated groups was significantly lower than inthe control group. A significant increase in mosaic aneuploidy, including monosomicor trisomic cell lines, was associated with treatment. The percentage of dead orresorbed embryos tended to be higher in treated than in the control group, but thedifferences were not significant. There was no increase in the incidence of externalmalformations in the treated groups, and the body weights of male and female fetusesexposed to trichlorfon were comparable to those of controls. The authors concludedthat exposure to trichlorfon around the time of fertilization induces a high frequencyof micronuclei, aneuploidy and developmental retardation in embryos from the pre-implantation to the mid-gestation stage. Thereafter, embryos with micronuclei orchromosomal damage appeared to develop and could appear normal by near term(Tian et al., 2000).

A study of the effects of trichlorfon on spindle morphology and chromosomalsegregation was conducted in fertilized mouse embryos in vitro. Epididymal spermobtained from male mice was capacitated and added to 385 oocytes collected fromsuperovulated female mice, and the mixture was incubated for 2 h. By that time,most of the fertilized eggs had reached anaphase II of the second maturation division.Some fertilized embryos were incubated further to the first mitotic division.Chromosomal analysis was performed on one-cell embryos arrested in metaphase.To study the effect of trichlorfon on fertilization, chromosomal segregation andspindles at anaphase II, 73 oocytes were incubated in 50 µg/ml of trichlorfon for 1 hbefore fertilization, then transferred to a chemical-free medium and fertilized. Inaddition, 300 oocytes were exposed for a total of 3 h: 1 h before fertilization andduring the 2-h fertilization. The treated embryos were then processed for chromo-somal analysis or immunofluorescence. To study the effects of trichlorfon on spindlesand chromosomes of maturing oocytes, the oocytes were incubated for 8 or 16 h inthe presence or absence of 50 µg/ml trichlorfon. By that time, most of the oocyteshad emitted a first polar body and were arrested at metaphase II.

Trichlorfon had no significant effect on the rate of fertilization, nor did it affect theseparation of chromatids or the distribution of chromosomes at anaphase II. Withregard to effects on maturing oocytes, aberrant spindles were observed in a largepercentage of oocytes after 8 h of exposure to trichlorfon. During the advancedstages of meiosis I, chromosomes appeared to be incapable of proper alignment atthe equator. Polar body formation occurred at about the same time in trichlorfon-treated oocytes and controls. After 16 h, most of the treated and control oocytes had

38 TRICHLORFON

reached metaphase II. The spindles were highly aberrant in a large percentage ofmetaphase II stages, and chromosomes were frequently unaligned and located atdifferent distances from the poles. The authors considered that the effects observedon spindle formation and chromosomal alignment placed oocytes at high risk forerrors in chromosomal segregation and might make them more prone tonondisjunction, predisposing the fertilized egg and embryo to trisomy (Yin et al.,1998).

(c) Toxicity to mammalian germ cells

A study of aneuploidy induction in male mouse germ cells was conducted in vivoin groups of five male F1 mice given a single intraperitoneal injection of trichlorfon at200, 300 or 400 mg/kg bw. The control group was given the solvent, dimethylsulfoxide. The mice were killed 22 days after treatment, the time it normally takesfor mouse spermatocytes to develop into mature sperm, and sperm were collectedfrom the cauda epididymis. As the same investigators had established that trichlorfonhas no effect on the duration of meiosis in male mouse germ cells, this samplingschedule was appropriate. A method involving multicolour fluorescence hybridizationin situ with chromosome-specific DNA probes was used to identify gain or loss ofindividual chromosomes during meiosis. Approximately 5000 sperm cells were scoredfrom each group. A significant, dose-related increase in the percentage of disomic(X-X-8, Y-Y-8, X-Y-8, X-8-8 or Y-8-8 phenotype) cells was observed at all dosesover that in controls. Trichlorfon also caused spindle disturbances in Chinese hamsterV79 cells in vitro. The authors noted that aneuploidy can result from non-disjunctionor chromosome loss during meiosis. Chromosomal non-disjunction can result frommalfunctioning of the processes involved in chromosomal segregation. The integrityof the spindle apparatus is considered to be of central importance in chromosomalsegregation. The authors concluded that trichlorfon could be regarded as a germ-cell aneugen in vivo (Sun et al., 2000; Schmid et al., 2001).

2.2.3 Special studies: Neurotoxicity

The maximal effects of trichlorfon on soluble neuropathy target esterase (NTE)and the regional distributions of NTE and acetylcholinesterase were studied in agroup of 16 adult hens aged 20–24 months. The hens were given a single intravenousdose of 200 mg/kg bw. To prevent acute death, each was given a subcutaneousinjection of atropine sulfate at 20 mg/kg bw, 10 min before and 15 min after dosingwith trichlorfon. Clinical signs of delayed neuropathy were studied in four of thehens for 28 days. The remaining 12 were used to measure the activities of NTE andacetylcholinesterase in three regions of the brain (cerebrum, midbrain andcerebellum) and three regions of the spinal cord (cervical, thoracic and lumbar)after 6, 24 and 48 h, with four hens per time. A group of six hens served as untreatedcontrols.

No signs of delayed neuropathy were observed in the treated hens. NTE activityin all the regions measured was inhibited by 14–45% 6 h after dosing, 6–15% 24 hafter dosing and 5–10% 48 h after dosing. Peak inhibition thus occurred at 6 h in allregions and ranged from 15% to 44%. The greatest inhibition was found in themidbrain (44%) and thoracic cord (36%). The average inhibition of NTE during the6–48-h period after dosing varied from 10% in the cerebellum to 23% in the midbrain(Tian et al., 1998).

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2.3 Observations in humans

A cluster of congenital abnormalities was identified in a Hungarian village in1989–90. Of 15 live births, 11 (73%) were affected by abnormalities, six were twinbirths, and four of the 11 had trisomy 21 (Down syndrome). Two of the cases ofDown syndrome also had endocardial cushion defect. Other abnormalities observedwere a ventricular septal defect with pulmonary atresia, an inguinal hernia, stenosisof the left bronchus, anal atresia, choanal atresia, cleft lip and Robin sequence. Of61 children born in this village between 1980 and 1988, only three had hadmalformations. The incidence of Down syndrome in the reported cluster wasapproximately 200 times greater than that in the general Hungarian population. Acase–control study revealed that the mothers of all the infants with abnormalitiesreported having eaten ‘contaminated’ fish during the index pregnancy. It was foundthat several ponds around the village used for fish farming had been treated with a40% trichlorfon formulation at a level of 500 mg/l. The composition of the formulationwas not described. The average concentrations of trichlorfon in 12 carp, 12 amurand 10 European wels collected from treated ponds, frozen and analysed 10 dayslater were 0.15, 0.13 and 0.26 mg/kg, respectively. Because trichlorfon rapidlydegrades in biological systems, the initial trichlorfon content of the fish was estimatedto have been as high as 100 mg/kg. The estimated daily fish consumption was250 g, resulting in a daily ‘worst-case’ estimated intake of 25 mg per person or400 µg/kg bw. The authors pointed out that the non-specificity of the observed adverseoutcomes argued against a single cause. Nevertheless, because mutagenic andteratogenic effects of trichlorfon have been observed in vitro, in vivo and in studiesin experimental animals, the authors suggested that reproductive hazards associatedwith exposure to high doses of trichlorfon should be further explored (Czeizel et al.,1993).

Chromosomal effects in lymphocytes were studied in 31 people who hadattempted suicide by self-poisoning with trichlorfon. The actual or estimated doseswere not stated. Three blood samples were collected 3–6, 30 and 180 days afterthe poisoning incidents. The controls were patients who had undergone surgery fora hernia or appendicitis in the surgical department of the same hospital, and theauthors reported that the rate of chromosomal breakage in these controlscorresponded to that in the general population.

Aneuploidy was found in 14% of 500 lymphocytes from the first blood sample,26% of 400 cells from the second sample and 16% of 480 cells from the third sampletaken from the victims, with 4% found in controls (Czeizel, 1994).

The results of a double-blind, placebo-controlled, single-centre study of the safety,tolerability and pharmacokinetics of trichlorfon in patients with Alzheimer diseasewere used to calculate the NOEL for inhibition of erythrocyte cholinesterase activity.A total of 27 patients were given an oral loading dose of trichlorfon by capsulecontaining 1.5, 2.5, 4 or 4 mg/kg bw per day for 6 days, followed by a daily oralmaintenance dose of 0.25, 0.4, 0.65 or 1 mg/kg bw for 21 days. The mean inhibitionof erythrocyte cholinesterase activity at the end of the treatment was 14%, 35%,66%, 77% and 82% with the placebo and the four treatments, respectively. A linearextrapolation of the data on inhibition of erythrocyte cholinesterase activity resultedin an estimated NOEL of 0.1–0.2 mg/kg bw (Bieber et al., 1996).

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3. COMMENTS

The pharmacokinetics of trichlorfon was studied in 24 volunteers with renaldisease who had various levels of impairment of renal clearance, in 34 healthyvolunteers who also received magnesium- and aluminium hydroxide-containingantacids or H2 receptor antagonists (cimetidine or ranitidine) and before and after ameal in 26 healthy volunteers. Trichlorfon was administered as a single oral dose of50 mg to the volunteers with renal disease, as three oral doses of 50 mg given1 week apart to the volunteers also receiving antacids and as a single oral dose of50 or 80 mg to the volunteers before or after a meal. The pharmacokinetics oftrichlorfon was not significantly altered in any of these studies. The reportedelimination half-life was approximately 2 h, similar to the value found by the Committeeat its fifty-fourth meeting. In all studies, trichlorfon caused significant reductions inplasma cholinesterase activity, while erythrocyte cholinesterase activity was relativelyunaffected. The authors concluded that multiple doses were required to attainsignificant inhibition of erythrocyte cholinesterase activity and a steady state oftherapeutically relevant inhibition. The data available to the Committee wereinsufficient to determine whether significant species differences exist with regard tothe pharmacokinetics of trichlorfon. The Committee noted that higher NOELs wereobserved in studies in which trichlorfon was administered in feed rather than bydirect oral administration in tablets or by gavage. Therefore, differences inpharmacokinetics may result from differences in the bioavailability of the dosageform administered.

Trichlorfon has been tested in a large number of studies for genotoxicity coveringa wide range of end-points, with considerable variation in the results for most end-points. Both positive and negative results were obtained in tests for bacterial mutationsand for gene mutation in mammalian cells in vitro, but the results of studies of effectson chromosomes in mammalian cells in vitro (chromosomal aberrations or sisterchromatid exchanges) were uniformly positive. Mostly negative results were foundin assays in mammals in vivo assessed by the Committee at its fifty-fourth meeting,comprising tests for somatic cell mutations in bone marrow (sister chromatidexchange, negative result in a single study), micronucleus formation (negative resultsin five of six studies) and chromosomal aberrations (negative results in three of fivestudies). Mostly negative results were also found in assays for germ cell mutagenicityin vivo evaluated by the Committee at its fifty-fourth meeting, comprising dominantlethal mutations (negative results in six of nine studies) and chromosomal aberrationsin spermatogonia or spermatocytes (negative results in three of four studies). TheCommittee at its present meeting received further data on mutagenicity, comprisingpositive results in studies of sister chromatid exchange in vivo but not in vitro.Trichlorfon was a germ cell aneugen in laboratory animals in vivo. There was alsolimited evidence from observations in poisoned humans that trichlorfon causedaneuploidy and chromosome damage in lymphocytes. A study involving pregnantwomen suggested that exposure to uncertain concentrations of residues of trichlorfonin fish may have caused trisomy 21 (Down syndrome) in their offspring as a result ofgerm cell aneugenicity. The Committee at its fifty-fourth meeting noted that bioassaysfor carcinogenicity in rats and mice gave negative results and identified a NOEL fordevelopmental toxicity. The Committee at its present meeting concluded that theweight of the evidence from the assays for mutagenicity in vivo indicated that

41TRICHLORFON

trichlorfon residues in animal-derived foods would not present a carcinogenic hazardto consumers.

In a two-generation study of reproductive toxicity, trichlorfon was administeredin the diet to groups of rats at concentrations providing a dose equivalent to 0, 7.5,25 or 88 mg/kg bw per day. The parameters evaluated in adults and pups includedclinical end-points, body weight, food consumption and gross and histologicalappearance. The body weights of F0 males and females at the highest dose weresignificantly decreased during the pre-mating phase, although feed consumptionappeared to be unaffected by treatment. The body weights of F1 dams weredecreased during gestation and lactation. The feed consumption of pups in the firstand second generation at the highest dose was decreased during the lactation phase.The lactation index (live pups per litter on lactation day 21/live pups per litter onlactation day 4 after culling ∞ 100) was significantly decreased in the first generationat the highest dose. In the second generation, significant decreases in birth index(pups born per litter/implantation sites per litter) and mean litter size were observedat the lowest and highest doses, but there was no dose–response relationship. Thebody weight of pups at the highest dose was decreased in both generations on day21. No abnormalities were observed at gross and histological examination of adultsand pups of either generation. At termination, significant decreases in plasma andbrain cholinesterase activity were reported in F0 females at all doses. In addition,erythrocyte cholinesterase activity was significantly decreased in females and malesat the two higher doses. Brain cholinesterase activity was significantly decreased inF0 males at the highest dose. At termination of the F1 generation, plasmacholinesterase activity was significantly decreased in adult males and females atthe two higher doses. Erythrocyte cholinesterase activity was significantly decreasedin females at all doses and in males at the highest dose. Neonatal erythrocytecholinesterase activity was significantly decreased in F1 males at the two higherdoses on day 4 of lactation. On day 21 of lactation, significant decreases in brainand plasma cholinesterase activity were observed in male and female pups at thehighest dose. In the F2 generation, significant decreases in plasma cholinesteraseactivity were observed in male and female pups, and significant decreases werefound in brain cholinesterase activity in females at the highest dose and in males atthe two higher doses on day 21 of lactation. A NOEL could not be identified in thisstudy, as significantly decreased plasma and brain cholinesterase activities wereseen at term in adult F0 females at the lowest dose and significantly decreasederythrocyte and brain cholinesterase activities in F1 adult females at the lowest dose.The Committee noted that the LOEL in this study (7.5 mg/kg bw per day) was lowerthan the NOEL of 30 mg/kg bw per day identified in a three-generation study ofreproductive toxicity in rats by the Committee at its fifty-fourth meeting. It also noted,however, that cholinesterase inhibition was not evaluated in that study. Had thatbeen done, it is reasonable to assume that the NOEL for that study would havebeen lower than 30 mg/kg bw per day. This assumption is supported by the NOEL of5 mg/kg bw per day for inhibition of erythrocyte cholinesterase activity identified bythe Committee at its fifty-fourth meeting in a 16-week study of toxicity in rats treatedorally. On the basis of these considerations and the fact that inhibition ofcholinesterase activity was the most sensitive effect in offspring in the present study,with an NOEL of 7.5 kg/kg bw per day, the Committee concluded that the reproductivetoxicity of trichlorfon in rats had been adequately assessed.

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In a study of developmental toxicity in guinea-pigs, designed to evaluate brainhypoplasia in offspring exposed in utero, trichlorfon was administered either bystomach tube or by subcutaneous injection. Oral doses ranging from 25 to 200 mg/kgbw per day were administered to groups of rats on days 40–44 of gestation in variousregimens. Clinical signs of toxicity typical for this substance, including hypersalivationand hindlimb weakness, were observed in dams given trichlorfon at 100, 150 or200 mg/kg bw. The NOEL for brain hypoplasia in offspring was 50 mg/kg bw.

In a study of the cytogenetic and developmental effects of trichlorfon on pre-implantation, mid-gestation and near-term mouse embryos and fetuses in vivo, groupsof mice were given an intraperitoneal injection of 100 or 200 mg/kg bw 6 h afterpresumed conception. Developmental outcomes and micronucleus formation duringthe pre-implantation phase were assessed on day 3 of gestation, and developmentaland aneugenic outcomes during the mid-gestation period were assessed on day 9of gestation. On day 17 of gestation, the embryos were removed, sexed, weighedand examined for external malformations. No clinical signs of toxicity were observedin the treated mice. The mean number of cells in embryos in both treated groupswas significantly lower than that in the control group, and the mean number ofmicronuclei was significantly increased in both treated groups compared with controls.The number of live embryos per dam was significantly lower among those given100 mg/kg bw than in controls. The mean number of somites in the trichlorfon-treated groups was significantly lower than in controls. Significantly more fetuseswith abnormal numbers of chromosomes were found to be associated with trichlorfontreatment. The incidence of external malformations and the body weights of maleand female fetuses in the trichlorfon-treated groups were comparable to those ofcontrols. This study provides evidence that exposure to trichlorfon around the timeof fertilization could result in induction of micronuclei, aneuploidy and developmentalretardation in embryos from pre-implantation to mid-gestation. Embryos withmicronuclei or aneuploidy may no longer show abnormalities at term. The Committeenoted that these effects resulted from intraperitoneal injection of doses 5000 to10 000 times greater than the current ADI established for orally administeredtrichlorfon by the Committee at its fifty-fourth meeting.

The effects of trichlorfon on fertilization, spindle morphology and chromosomalsegregation were studied in mouse oocytes exposed in vitro to a concentration of50 µg/ml. Aberrant spindles were observed in maturing oocytes after 8 h of exposure,and the chromosomes appeared to be incapable of proper alignment at the equator.After 16 h, most of the treated oocytes had highly aberrant spindles, and thechromosomes were frequently unaligned and located at different distances from thepoles. Such effects on spindles and chromosome alignment could have aneugeniceffects on fertilized eggs and embryos. The Committee noted that effects on spindlesleading to aneuploidy have thresholds. The Committee also noted that, assuming100% bioavailability, systemic exposure to trichlorfon resulting from consumption ofthe entire ADI would be orders of magnitude lower than the dose used in this study.Exposure to trichlorfon at the ADI would therefore pose a negligible risk to humanoocytes.

Aneuploidy induction was studied in sperm cells collected from groups of malemice 22 days after a single intraperitoneal injection of trichlorfon at a dose of 200,300 or 400 mg/kg bw. A significant, dose-related increase in the percentage of spermcells with an extra chromosome was observed at all doses. On the basis of this

43TRICHLORFON

experiment, the Committee concluded that trichlorfon is a male mouse germ-cellaneugen in vivo. The Committee noted that these effects resulted from intraperitonealinjection of doses several orders of magnitude higher than the current ADI fortrichlorfon established by the Committee at its fifty-fourth meeting.

The maximal effects of trichlorfon on soluble neuropathy target esterase activityand regional distribution of neuropathy target esterase and acetylcholinesterasewere studied in brain and spinal cord of hens given trichlorfon at a single intravenousinjection of 200 mg/kg bw. Peak inhibition of neuropathy target esterase activityoccurred 6 h after dosing and ranged from 15 to 44%. No signs of delayed neuropathywere found during the 28-day observation period in four treated hens. Havingreviewed several contemporary studies, the Committee at its fifty-fourth meetingalso concluded that trichlorfon did not cause delayed neuropathy in hens.

A cluster of congenital anomalies was identified in a Hungarian village between1989 and 1990. Of 15 live births, 11 (73%) were affected by abnormalities, and sixwere twin births. Four of the 11 affected infants had trisomy 21 (Down syndrome).Nine different physical abnormalities were observed. The mothers of all the affectedinfants reported having eaten fish during pregnancy. Several ponds around the villageused for fish farming had been treated with a trichlorfon formulation at a level of500 mg/l. The average concentrations of trichlorfon measured or estimated in thetypes of fish consumed ranged from 0.15 to 100 mg/kg. The exposure of fathers totrichlorfon was not evaluated in this study. This is the only known report of reproductiveeffects in humans possibly associated with oral exposure to trichlorfon, despite itswidespread use as an anthelmintic.

The Committee acknowledged that the results of this study were not conclusiveand provided limited evidence of a possible association between birth defects inhumans and oral intake of trichlorfon in food. In addition, the published report didnot include confirmation of the magnitude or frequency of intake or even whetherintake had occurred. The Committee at its fifty-fourth meeting evaluated studies ofdevelopmental toxicity with trichlorfon conducted in four animal species. In thesestudies, teratogenic effects were seen only at very high, maternally toxic doses. Inaddition, as multigeneration studies of reproductive toxicity did not provide evidenceof paternally transmitted teratogenicity, the Committee considered that the effect onexposed males had been assessed. The Committee at its present meeting reviewedthe assessment by the Joint Meeting on Pesticide Residues in 1993 (WHO, 1994)of dichlorvos, the major metabolite of trichlorfon. That Meeting concluded thatdichlorfos was not teratogenic in mice, rats or rabbits, even at doses that were toxicto maternal animals. In addition, at 12 mg/kg bw per day, dichlorvos had noreproductive effects in rats in a three-generation study. On the basis of theseconsiderations, the Committee concluded that the information from the study inhumans would not significantly affect its risk assessment of trichlorfon.

Chromosomal effects were studied in the lymphocytes of 31 people who hadattempted suicide by taking unknown doses of trichlorfon. There appeared to be anincrease in per cent aneuploidy in blood samples collected 3–6, 30 and 180 daysafter the incidents. An increase was also found in the rate of chromatid andchromosome-type aberrations. The Committee concluded that the intake that hadresulted in these effects far exceeded the ADI established for trichlorfon by theCommittee at its fifty-fourth meeting.

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4. EVALUATION

The additional information reviewed by the Committee for this re-evaluation oftrichlorfon included further information on its pharmacokinetics and genotoxic,reproductive and developmental toxicity. In addition, a NOEL for teratogenicity inguinea-pigs was established. The information did not provide evidence that any ofthese effects was more sensitive than inhibition of acetylcholinesterase activity, andthe Committee concluded that inhibition of acetylcholinesterase activity is the mostrelevant end-point for establishing an ADI for trichlorfon.

The Committee at its fifty-fourth meeting established the ADI for trichlorfon onthe basis of a NOEL of 0.2 mg/kg bw per day in a study of volunteers with Alzheimerdisease treated with trichlorfon. In this study, the volunteers were given a loadingdose of 0.5 mg/kg trichlorfon daily for 2 weeks, followed by a maintenance dose of0.2 mg/kg per day for 8 weeks. The Committee at its fifty-fourth meeting concludedthat the maintenance dose had not significantly enhanced the inhibition of erythrocytecholinesterase activity established in patients by the loading dose. Therefore, itconcluded that the maintenance dose was the NOEL in this study. Because theNOEL was derived from a study in humans, a safety factor of 10 was applied to theNOEL to derive the ADI.

The present Committee re-examined the basis on which it had established theADI for trichlorfon at its fifty-fourth meeting and concluded that the dose it hadidentified then as the NOEL was nevertheless effective in maintaining the steady-state level of inhibition of erythrocyte cholinesterase activity and was therefore moreappropriately considered a LOEL. The Committee concluded, however, that an ADIcould be derived from this study by applying an additional factor of 10 to this LOEL.This conclusion is supported by supplemental information that included a lineardose extrapolation of data from a study of 27 volunteers with Alzheimer disease,who were treated with loading doses of 1.5–4 mg/kg bw per day for 6 days, followedby maintenance doses of 0.25–1 mg/kg bw per day for 15 days. The linearextrapolation resulted in an estimated NOEL for inhibition of cholinesterase activityin the range 0.1–0.2 mg/kg per day. This provides further support that the NOEL forinhibition of erythrocyte cholinesterase activity in humans is very close to 0.2 mg/kgbw per day. Furthermore, the Committee recalled that a clear NOEL of 0.2 mg/kgper day for inhibition of erythrocyte cholinesterase activity was established in a 10-year study of toxicity in monkeys treated orally, which it had evaluated at its fifty-fourth meeting. If that study had been selected as the basis for setting the ADI, asafety factor of 100 would have been applied, resulting in an ADI of 0–2 µg/kg bw.The present Committee thus amended the ADI for trichlorfon from 0–20 µg/kg bw to0–2 µg/kg bw on the basis of the LOEL of 0.2 mg/kg bw per day for inhibition oferythrocyte acetylcholinesterase activity in humans treated orally and a 100-foldsafety factor.

5. REFERENCES

Astroff, B.A., Freshwater, K.J. & Eigenberg, D.A. (1998) Comparative organophosphate-inducedeffects observed in adult and neonatal Sprague-Dawley rats during the conduct ofmultigeneration toxicity studies. Reprod. Toxicol., 8, 619–645.

Badawi, A.F. (1998) O6-Methylguanine and O6-methylguanine-DNA methyltransferase activityin tissues of BDF-1 mice treated with antiparasitic drugs. Toxicol. Lett., 94, 199–208.

45TRICHLORFON

Bieber, F., Orazem, J. & Mas, J. (1996) A double-blind, placebo-controlled, randomized, single-center study of the safety, tolerability and pharmacokinetics of metrifonate (BAY a 9826) inpatients with probable Alzheimer’s disease. Unpublished report No. MMRR-1340 from Bayer,Wuppertal-Elberfeld, Germany. Submitted to WHO by Bayer Animal Health, Germany.

Cummings, J.L., Cyrus, P.A., Bieber, F., Mas, J., Orazem, J. & Gulanski, B. (1998) Metrifonatetreatment of the cognitive deficits of Alzheimer’s disease. Metrifonate study group. Neurology,51, 1214–1221.

Czeizel, A.E. (1994) Phenotypic and cytogenetic studies in self-poisoned patients. Mutat. Res.,313, 175–181.

Czeizel, A.E., Elek, C., Gundy, S., Metneki, J., Nemes, E., Reis, A., Sperling, K., Timar, L.,Tusnady, G. & Viragh, Z. (1993) Environmental trichlorfon and cluster of congenitalabnormalities. Lancet, 341, 539–542.

Dingemanse, J., Halabi, A., Kleinbloesem, C.H., Heinig, R. & Blume, H. (1999) Pharmacokineticsand pharmacodynamics of the acetylcholinesterase inhibitor metrifonate in patients withrenal impairment. Ther. Drug Monit., 21, 310–316.

Heinig, R. & Dietrich, H. (1999) Pharmacokinetics of metrifonate and its metabolite dichlorvosin healthy volunteers and patients with renal impairment. Clin. Drug Invest., 18, 35–46.

Heinig, R. & Sachse, R. (1999) The effect of food and time of administration on thepharmacokinetic and pharmacodynamic profile of metrifonate. Int. J. Clin. Pharmacol. Ther.,37, 456–464.

Heinig, R., Boettcher, Z., Herman-Gnjidic, Z. & Pierce, C.H. (1999) Effects of a magnesium/aluminum hydroxide-containing antacid, cimetidine or ranitidine on the pharmacokineticsof metrifonate and its metabolite DDVP. Clin. Drug Invest., 17, 67–77.

Hjelde, T., Mehl, A., Schanke, T.M. & Fonnum, F. (1998) Teratogenic effects of trichlorfon(metrifonate) on the guinea-pig brain. Determination of the effective dose and the sensitiveperiod. Neurochem. Int., 32, 469–477.

Madrigal-Bujaidar, E., Cadena, R.S., Trujillo-Valdez, V.M. & Cassani, M. (1993) Sister-chromatidexchange frequencies induced by metrifonate in mammalian in vivo and in vitro systems.Mutat. Res., 300, 135–140.

Mehl, A., Schanke, T.M., Johnsen, B.A. & Fonnum, F. (1994) The effect of trichlorfon and otherorganophosphates on prenatal brain development in the guinea pig. Neurochem. Res., 19,569–574.

Mehl, A., Rolseth, V., Gordon, S., Bjoraas, M., Seeberg, E. & Fonnum, F. (2000) Brain hypoplasiacaused by exposure to trichlorfon and dichloros during development can be ascribed toDNA alkylation damage and inhibition of DNA alkyltransferase repair. NeuroToxicology, 21,165–174.

Schmid, T.E., Attia, S., Baumgartner, A., Nuesse, M. & Adler, I.D. (2001) Effect of chemicals onthe duration of male meiosis in mice detected with laser scanning cytometry. Mutagenesis,16, 339–343.

Sun, F.Y., Schmid, T.E., Schmid, E., Baumgartner, A. & Adler, I.-D. (2000) Trichlorfon inducesspindle disturbances in V79 cells and aneuploidy in male mouse germ cells. Mutagenesis,15, 17–24.

Tian, Y., Xie, X., Piao, F. & Yamauchi, T. (1998) Delayed neuropathy and inhibition of solubleneuropathy target esterase following the administration of organophosphorus compoundsto hens. Tohoku J. Exp. Med., 185, 161–171.

Tian, Y., Ishikawa, H. & Yamauchi, T. (2000) Analysis of cytogenetic and developmental effectson pre-implantation, mid gestation and near-term mouse embryos after treatment withtrichlorfon during zygote stage. Mutat. Res., 471, 37–44.

WHO (1994) Pesticide residues in food—1993 evaluations. Part II. Toxicology, WHO/PCS/94.4, Geneva.

Yin, H., Cukurcam, S., Betzendahl, I., Adler, I.-D. & Eichenlaub-Ritter, U (1998) Trichlorfonexposure, spindle aberrations and nondisjunction in mammalian oocytes. Chromosoma,107, 514–522.

47CARBADOX

PRODUCTION AID

48 CARBADOX

49CARBADOX

– 49 –

CARBADOX (addendum)


Professor Fritz R. Ungemach

Institute of Pharmacology, Pharmacy and Toxicology, Veterinary Faculty,University of Leipzig, Leipzig, Germany


Reproductive toxicity ............................................................ 50Genotoxicity and carcinogenicity .......................................... 51


1. EXPLANATION

The quinoxaline-1,4-dioxide compound carbadox (methyl-3-[quinoxalinyl-methylene]carbazate-N1,N4-dioxide) is used in pigs as a growth-promoting agentfor the improvement of weight gain and feed efficiency and as an antibacterial drugfor prevention and control of dysentery and bacterial enteritis in pigs.

Carbadox was evaluated by the Committee at its thirty-sixth meeting (Annex 1,reference 92). At that time, the Committee was not able to establish an ADI becausecarbadox and some of its metabolites (desoxycarbadox and hydrazine) were foundto be genotoxic and carcinogenic in rodents. The final metabolite, quinoxaline-2-carboxylic acid (QCA), was not found to be carcinogenic or mutagenic in animals.Studies of residues showed rapid depletion of the parent substance and its genotoxicmetabolites in liver and muscle within 72 h, to concentrations of < 2 µg/kg, within thelimit of detection of the analytical method available at that time (MacIntosh et al.,1985). QCA was the most persistent metabolite and was the only residue detectablein edible tissues of pigs 72 h after dosing. After 28 days’ withdrawal, its concentrationwas < 30 µg/kg in liver and 5 µg/kg in muscle, representing the limits of quantificationof the analytical method used at that time, which was based on extraction by alkalinehydrolysis. Uncertainty remained about the nature of the bound residues in liver;however, the Committee concluded that the bound residues in liver 28 days aftertreatment would not represent a risk for consumers. On the basis of data from studieson the toxicity of QCA, on the metabolism and depletion of carbadox and on thenature of the compounds released from the bound residues, the Committeeconcluded that residues resulting from use of carbadox in pigs were acceptableprovided that the recommended maximum residue limits of 0.03 mg/kg in liver and0.005 mg/kg in muscle, based on the levels of and expressed as QCA, were notexceeded. It concluded that use of carbadox according to good practice in veterinarymedicine (no use in finisher pigs and a withdrawal period of at least 28 days) doesnot represent a dietary hazard to human health.

Carbadox was banned for use in food-producing animals in the European Unionin 1999 because of its genotoxic and carcinogenic properties and for the safety of

50 CARBADOX

workers. Health Canada issued an order to stop the sale of carbadox in 2001. Reportsof misuse and cross-contamination, combined with a better analytical capacity todetect desoxycarbadox, showed that this carcinogenic residue was present in tissuesand rendered products obtained from leftover tissue of pigs that had not beenadequately withdrawn. These resulted in serious concern about the safety of thisdrug (Vilim & Lambert, 2001).

At the request of the Canadian Government, the Codex Committee on VeterinaryDrug Residues in Foods placed carbadox on the list of priorities for the sixtiethmeeting of the Expert Committee and asked it to review all relevant data on thetoxicology and residues of carbadox and its metabolites in porcine tissues, includinganalytical methods for their detection that had been generated since the previousevaluation by JECFA.

2. BIOLOGICAL DATA

2.1 Reproductive toxicity

At its previous evaluation, the Committee considered studies on the reproductivetoxicity of carbadox and its persistent end-metabolite QCA. In a three-generationstudy, with two litters per generation, Charles River C-D rats were given dietscontaining carbadox at concentrations providing a dose of 0, 1 or 2.5 mg/kg bw perday. No evidence of reproductive toxicity was found in the first two generations.Occasional differences from control group parameters were sporadic and considerednot to be related to treatment. It was concluded that carbadox had no effect onfertility, lactation or the neonate at doses up to 2.5 mg/kg bw per day. The potentialreproductive toxicity of QCA was studied in rats and rabbits. In a three-generationstudy of reproductive toxicity in rats given diets containing QCA at concentrationsproviding doses up to 100 mg/kg bw per day, no treatment-related effects wereobserved. In studies of developmental toxicity in New Zealand white rabbits givenan oral dose of 0, 25, 50 or 100 mg/kg bw per day on days 7–18 of gestation, therewas no evidence of maternal toxicity, embryotoxicity or teratogenicity. The NOELwas 100 mg/kg bw per day.

A study of the teratogenicity of carbadox in rats has been reported since theCommittee’s previous evaluation. No statement was included about the complianceof the study with good laboratory practice. The protocol and conduct of the studywere compliant with recognized testing guidelines for developmental toxicity, exceptthat there were only 10 animals per group and treatment was not begun before day8 of gestation. An aqueous solution of carbadox was administered by oral gavageonce daily to groups of 10 pregnant Sprague-Dawley rats at a dose of 0, 10, 25, 50or 100 mg/kg bw on days 8–15 of gestation. The dams were observed daily forbody-weight gain. The animals were killed on day 21 of gestation, the fetuses wereremoved surgically, and the numbers of implants, resorptions and live pups werecounted. The fetuses were weighed and examined for sex ratio and for external,skeletal and internal malformations. The viability of the pups and the number ofcorpora lutea were not reported.

Maternal body-weight gain was significantly reduced in a dose-related mannerat all doses, but none of the treated dams died. After discontinuation of treatment,

51CARBADOX

the body-weight gain recovered on days 15–21, except for animals at the highestdose. At doses of up to 50 mg/kg bw per day, the number of live pups was nodifferent from that in the control group, with a mean of 13.5 fetuses per litter.Embryolethality was seen at the highest dose, the number of live fetuses beingreduced to 2.4 per litter; the percentage of late resorptions was 82%, with completeresorptions in five dams, compared with 3.4% in the control group. No treatment-related effects on early resorptions were reported. A dose-related reduction in fetalbody weight was seen, which was statistically significant at doses ž 25 mg/kg bwper day. At the highest dose, carbadox induced a significantly increased incidenceof malformations: in 24 fetuses examined for external malformations, short tail wasrecorded in 11, kinky tail in five, brachygnathia in five, ectrodactyly in three, club footin one and generalized oedema in one. The skeletal malformations observed weremainly fused vertebrae, found in 8 of 17 fetuses. Internal examination revealedhydrocephaly in two of seven fetuses. The fetal sex ratio was affected at 100 mg/kgbw per day, but not statistically significantly so.

The author concluded that maternally toxic doses of carbadox caused embryo-toxicity and embryolethality. Carbadox was also considered to be teratogenic. Theoccurrence of malformations and severe maternal toxicity at the highest dose testedsuggested that adverse effects on maternal animals and a direct action on theconceptus contributed to fetal malformations and embryo and fetal deaths. The NOELwas 10 mg/kg bw per day for embryotoxicity and 50 mg/kg bw per day forteratogenicity. No NOEL for maternal toxicity could be identified (Yoshimura, 2002).

The NOEL of 10 mg/kg bw per day is well above the NOEL of 2.5 mg/kg bw perday found in studies of reproductive toxicity in rats and also exceeds the lowestdose of 1 or 5 mg/kg bw per day that had adverse effects in rats in long-term studiesof toxicity evaluated previously by the Committee (Annex 1, references 86 and 92).QCA, the predominant residue after carbadox treatment, showed no adverse effectsin studies of reproductive toxicity at doses - 100 mg/kg bw per day and wasconsidered not to be teratogenic. Therefore, the results of this new study do notaffect the previous evaluation of the Committee with regard to reproductive anddevelopmental toxicity and its overall conclusion on risk characterization of carbadox.The main findings of studies of reproductive toxicity with carbadox are summarizedin Table 1.

2.2 Genotoxicity and carcinogenicity

In its previous evaluation, the Committee considered the results of a range oftests for genotoxicity in bacteria and mammalian cells with carbadox and itsmetabolites desoxycarbadox, methyl carbazate and its possible hydrolysis producthydrazine, and QCA. Positive results were obtained with carbadox in 14 of 15 tests.Desoxycarbadox gave negative results in many test systems, but positive findingswere reported for cell transformation in mouse cells, for chromosomal aberration inrat bone marrow and for reverse mutation in Salmonella typhimurium with microsomesfrom rats treated with polychlorinated biphenyls. Hydrazine gave positive results inthree mutagenicity test systems. Methyl carabazate gave negative results in threeand equivocal results in one test system. The mutagenic potential of QCA was testedin only two assays, for reverse mutation in bacteria and for chromosomal aberrations

52 CARBADOXTa

ble

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f rep

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53CARBADOX

in human lymphocytes, with negative results. The results of the mutagenicity testsindicate that carbadox, desoxycarbadox and hydrazine have genotoxic potential,while the genotoxic potency of desoxycarbadox appears to be lower than that of theparent substance carbadox.

The Committee also previously reviewed several long-term studies in whichcarbadox and its metabolites were administered in the diet to rodents. The mainfindings are summarized in Table 2.

In rats, carbadox was reported to cause a dose-dependent increase in theincidence of benign and malignant hepatic tumours and of mammary tumours atdoses ž 1 mg/kg bw per day. Doses > 25 mg/kg bw per day were toxic, andadministration could not be continued. The results of all the studies demonstratedthe carcinogenic potential of carbadox, even thought there were relatively few animalsper dose. Studies in monkeys and studies of ‘relay toxicity’ (in which laboratoryanimals are fed meat from farm animals treated with the agent) in rats and dogswere not adequate to assess carcinogenicity. In two studies with long-term administra-tion of desoxycarbadox to rats, increased incidences of tumours were recorded atall doses. Desoxycarbadox was not only a potent hepatocarcinogen but increasedtumour incidence in a dose-related manner at other sites, including the mammarygland and skin. Hydrazine was shown to be carcinogenic in mice and rats but not inhamsters. No treatment-related increase in tumour incidence was recorded in ratstreated with methyl carbazate at doses - 10 mg/kg bw per day for 21 or 23 monthsor in rats or mice given a diet containing QCA at doses - 100 mg/kg bw per day.

The results of the studies with methyl carbazate and the persistent end-metaboliteQCA thus provided no evidence of genotoxic or carcinogenic potential. The NOELof QCA was 50 mg/kg bw per day. The Committee concluded that carbadox,desoxycarbadox and hydrazine are genotoxic carcinogens. The Committee alsonoted that the tumorigenic potential of desoxycarbadox was apparently greater thanthat of the parent compound and that it therefore probably makes a significantcontribution to the tumorigenic activity of carbadox in rats. The results of studies ofthe metabolism of carbadox suggested, however, that desoxycarbadox is a relativelyshort-lived intermediate between carbadox and QCA. No NOEL could be identifiedfor carbadox, desoxycarbadox or hydrazine in long-term studies in rodents treatedin the diet. Because of the genotoxic and carcinogenic nature of carbadox,desoxycarbadox and its possible metabolite hydrazine, the Committee was not ableto establish an ADI or to determine a safe concentration of total residues in theabsence of any threshold.

An evaluation of carbadox residues was completed in 1998 which resulted incodification of a revised tolerance for residues of carbadox and its metabolites ofcarcinogenic concern in edible tissues and their risk to the consumer (Food andDrug Administration, 1998). That document referred to the review of the Committeeat its thirty-sixth meeting and stated that the end-points of toxicological concern forcarbadox and its metabolites were their genotoxicity and carcinogenicity. The studiesof genotoxicity, the long-term studies of toxicity and carcinogenicity summarized inTable 2 and a study of toxicity in monkeys given repeated doses were reviewed.

54 CARBADOXTa

ble

2. T

umou

r inc

iden

ces

obse

rved

in lo

ng-te

rm s

tudi

es o

f tox

icity

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mitt

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ex 1

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2)

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no.

of

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our i

ncid

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kg b

wpe

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amm

ary

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55CARBADOXTa

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sex

per

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24 m

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s 0

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24 m

onth

s 5

75%

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12–2

4 m

onth

s10

95%

28%

5%10

%12

mon

ths

2510

0% 2

7%47

%18

%

Rat

10 m

onth

s 0

14 o

f eac

h se

x pe

r dos

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mon

ths

2510

0% h

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ocel

lula

rca

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, Bal

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fem

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46 w

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50Lu

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mou

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0%

Mou

se, S

wis

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wee

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b/Se

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21 o

f eac

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mal

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90%

fem

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56 CARBADOXTa

ble

2 (c

ontd

)

Spec

ies,

no.

of

Leng

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Tum

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ne (c

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, CBA

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days

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of e

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sex

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% /

3.4%

per d

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/ 0%

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% /

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/ 66

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% /

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%

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ster

, old

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mg/

anim

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g/ra

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%27

%

57CARBADOX

From the results of these studies, ‘no residue’ levels were determined from S0 valuesfor the carcinogenic compounds carbadox, desoxycarbadox and hydrazine. The S0values were transformed to Sm values to correct for human food intake. ‘No residue’of a compound is considered to remain in edible tissue when the residue ofcarcinogenic concern in the total diet does not exceed the S0. The S0 is defined asthe concentration of total residue of carcinogenic concern of the test compound inthe total diet of test animals that corresponds to a maximum lifetime risk of cancer inthe test animals of 1 in 1 million. The S0 values were calculated for carbadox andeach of its carcinogenic metabolites from a low-dose linear statistical model, as106 ng/kg for carbadox, 61 ng/kg for desoxycarbadox and 11 ppb (µg/kg) forhydrazine. The lowest value of 61 ng/kg obtained for desoxycarbadox was designatedas the S0 of carbadox residues. The Sm is the permitted concentration of residues ofcarcinogenic concern in a specific edible product (Food and Drug Administration,1998).

The S0 values were calculated with 99% confidence limits by use of the one-hitlinear extrapolation model (for review, see Calabrese, 1983) on the basis of hepatictumours in male and female rats combined. The result obtained with the model wasmore conservative than that found with the Mantel Bryan extrapolation (for review,see Calabrese, 1983), anticipating an S0 of 680 ng/kg with 99% confidence limitsand a 10–6 risk for desoxycarbadox (L. Friedlander, personal communication, 2003).As further details of the assignment of S0 values (e.g. studies included) were notavailable, the results of the extrapolations could not be confirmed independently.

Because the total human diet is not derived solely from food-producing animals,a correction for food intake is made to determine the concentrations of residues ofcarcinogenic concern that should be permitted in edible animal tissue. Given a totaldaily diet of 1500 g for humans, up to 500 g are assumed to be due to the consumptionof meat, with 300 g comprised of muscle, 100 g comprised of liver, 50 g comprisedof kidney and 50 g comprised of fat. Thus, for each edible tissue the Sm is calculatedon the basis of the lowest S0 for desoxycarbadox, as shown in Table 3.

At the time of the review, no regulatory analytical method was available to monitorcarbadox residues of carcinogenic concern at the level of the S0 and Sm in muscleand liver of pigs. The results of a study of carbadox residues in porcine tissue

Table 3. Consumption figures and calculated Sm values for total carbadox-derivedresidues of carcinogenic concern in edible pig tissues

Tissue Consumption (g) Fraction of total diet S0 Sm(ng/kg) (ng/kg)

Muscle 300 1/5 61 305Liver 100 1/15 61 915Kidney 50 1/30 61 1830Fat 50 1/30 61 1830

S0, concentration of total residue of carcinogenic concern of the test compound in the totaldiet of test animals that corresponds to a maximum lifetime risk of cancer in the test animalsof 1 in 1 million; Sm, permitted concentration of residues of carcinogenic concern in aspecific edible product (Food and Drug Administration, 1998)

58 CARBADOX

subjected to digestion with either pepsin or pancreatin and with a more sensitivedetection method indicated the presence of desoxycarbadox at concentrations thatsubstantially exceeded the Sm value in liver throughout the experimental period of15 days after withdrawal. When the concentration of the marker residue, QCA, hadfallen below the MRL of 30 µg/kg, the concentration of residues of desoxycarbadoxin liver still exceeded the established Sm by a factor of 4–5 (Heird, 2002/2003).Therefore the MRL of 30 µg/kg for liver recommended by the Committee at its thirty-sixth meeting is inadequate to monitor the absence of residues of carbadox ofcarcinogenic concern.

The Committee has not to date used the approach of extrapolating irreversibleeffects of chemicals at low doses for which thresholds have not been identified to a‘virtually safe dose’ by using mathematical models, such as the one-hit linear modelor the Mantel Bryan model, in evaluating residues of veterinary drugs in food. Themodels used for extrapolating to a ‘virtually safe dose’ have been criticized on severalbases, such as lack of validity, the absence of consistent low-dose linearity inexperimental systems, arbitrary selection of the slope in the Mantel Bryan probitanalysis, the considerable diversity of predictions in the very low dose range withvariations of several orders of magnitude, insufficient recognition of the effects ofpharmacokinetics, and lack of consideration of individual variation in susceptibilitywithin the human population. Although such estimates of risk have substantialinherent uncertainties, the one-hit low-dose extrapolation model is assumed to be aconservative approach, as it is least likely to result in underestimation of carcinogenicrisks in humans and has been reported to show the closest fit to human responserates derived from epidemiological studies (Calabrese, 1983).

3. COMMENTS

In a study of developmental toxicity, carbadox was administered orally to pregnantrats at a dose of 0, 10, 25, 50 or 100 mg/kg bw per day on days 8–15 of gestation.None of the treated dams died. Maternal body-weight gain was significantlydecreased in a dose-related manner at all doses; weight gain recovered aftercessation of treatment, except in animals at the highest dose. The animals werekilled on day 21 of gestation, and their fetuses were removed surgically. Carbadoxwas embryotoxic and fetotoxic, as indicated by a dose-related reduction in fetalbody weight, which was statistically significant at doses of 25 mg/kg bw per day andhigher. The number of live fetuses and the resorption rate in dams at doses of50 mg/kg bw per day or less were not different from those of the control group.Embryolethality occurred at the highest dose, the percentage of late resorptionsbeing 82% and the number of live pups being reduced by more than 80% whencompared with controls. At the highest dose, carbadox induced external, skeletaland internal malformations at rates of 47%, 45% and 28%, respectively. Theabnormalities recorded were short tail, kinky tail, brachygnathia, ectrodactyly, clubfoot, generalized oedema, fused vertebrae and hydrocephaly. Carbadox wasconsidered to be embryotoxic and fetotoxic as a consequence of its strong maternaltoxicity and to be teratogenic in rats. The NOEL was 10 mg/kg bw per day forembryotoxicity and 50 mg/kg bw per day for teratogenicity. An NOEL for maternaltoxicity could not be identified. The Committee noted that the NOELs for

59CARBADOX

developmental toxicity in this study were well above the NOEL of 2.5 mg/kg bw perday found in the studies of reproductive toxicity in rats previously evaluated by theCommittee.

No new experimental data on the metabolism of carbadox or on the genotoxicityor carcinogenicity of carbadox and its metabolites were provided. The Committeewas aware of an evaluation based on linear extrapolation to estimate a ‘virtuallysafe dose’ for carbadox and its metabolites of carcinogenic concern. This estimatewas made on the basis of the incidence of hepatic tumours in rats in long-termstudies of carcinogenicity previously evaluated by the Committee. The evaluationresulted in codification of tolerance for residues of carbadox and the metabolites ofcarcinogenic concern in edible tissues and their risks to the consumer. The Committeedid not consider this approach appropriate owing to the substantial inherentuncertainties involved.

The results of a recent study of residues of carbadox in pigs indicated longerpersistence of substantial amounts of the carcinogenic metabolite desoxycarbadoxin liver at the time when the concentration of the marker residue, QCA, fell belowthe MRL of 0.03 mg/kg and to the end of the experiment 15 days after withdrawal.

4. EVALUATION

Carbadox was reviewed by the present Committee primarily on the basis of newinformation on residue levels, which indicated that the metabolite desoxycarbadoxwas present in edible tissues even at the end of the 15-day experimental withdrawalperiod. The Committee confirmed that the information previously submitted indicatedthat both carbadox and desoxycarbadox should be regarded as carcinogens thatact by a genotoxic mechanism. Although the Committee was aware that linearextrapolation has been used to estimate a ‘virtually safe dose’ of carbadox, theCommittee concluded that it was not possible to identify a dose of carbadox thatposes an acceptable risk to consumers. The Committee therefore did not establishan ADI for carbadox.

5. REFERENCES

Calabrese, E.J., ed. (1983) Principles of Animal Extrapolation, New York: John Wiley & Sons,pp. 555–574.

Food and Drug Administration (1998b) Mecadox 10 type A medicated article (carbadox).Freedom of information summary, supplement to NADA No. 041-061, Washington DC.

Heird, C.E. (2002/2003) Concentration and depletion of carbadox, deoxycarbadox andquinoxaline-2-carboxylic acid (QCA) in tissue and rendered material of growing swine afterconsumption of carbadox at 50 g/ton of feed. Unpublished report from Southwest Bio-Labs, Inc. Submitted to WHO by Phibro Animal Health, Fairfield, New Jersey, USA.

MacIntosh, A., Lauriault, G. & Neville, G.A. (1985) Liquid chromatographic monitoring of thedepletion of carbadox and its metabolite desoxycarbadox in swine tissues. J. Assoc. Off.Anal. Chem., 68, 665–671.

Vilim, A. & Lambert, G. (2001) Health risk assessment: Carbadox in swine. Human SafetyDivision, Bureau of Veterinary Drugs, Health Products and Food Branch, Ottawa: HealthCanada.

Yoshimura, H. (2002) Teratogenic assessment of carbadox in rats. Toxicol. Lett., 129, 115–118.

lxiANNEX 1

REPORTS AND OTHER DOCUMENTS RESULTING FROM PREVIOUSMEETINGS OF THE JOINT FAO/WHO EXPERT COMMITTEE ON FOOD

ADDITIVES

1. General principles governing the use of food additives (First report of the JointFAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings ReportSeries, No. 15, 1957; WHO Technical Report Series, No. 129, 1957 (out of print).

2. Procedures for the testing of intentional food additives to establish their safety foruse (Second report of the Joint FAO/WHO Expert Committee on Food Additives).FAO Nutrition Meetings Report Series, No. 17, 1958; WHO Technical ReportSeries, No. 144, 1958 (out of print).

3. Specifications for identity and purity of food additives (antimicrobial preservativesand antioxidants) (Third report of the Joint FAO/WHO Expert Committee on FoodAdditives). These specifications were subsequently revised and published asSpecifications for identity and purity of food additives, Vol. I. Antimicrobialpreservatives and antioxidants, Rome, Food and Agriculture Organization of theUnited Nations, 1962 (out of print).

4. Specifications for identity and purity of food additives (food colours) (Fourth reportof the Joint FAO/WHO Expert Committee on Food Additives). These specificationswere subsequently revised and pyÇlmìhed a}tspecifications for identity and purityof food additives, Vol. II. Food colours, Rome, Food and Agriculture Organizationof the United Nations, 1963 (out of print).

5. Evaluation of the carcinogenic hazards of food additives (Fifth report of the JointFAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings ReportSeries, No. 29, 1961; WHO Technical Report Series, No. 220, 1961 (out of print).

6. Evaluation of the toxicity of a number of antimicrobials and antioxidants (Sixthreport of the Joint FAO/WHO Expert Committee on Food Additives). FAO NutritionMeetings Report Series, No. 31, 1962; WHO Technical Report Series, No. 228,1962 (out of print).

7. Specifications for the identity and purity of food additives and their toxicologicalevaluation: emulsifiers, stabilizers, bleaching and maturing agents (Seventh reportof the Joint FAO/WHO Expert Committee on Food Additives). FAO NutritionMeetings Series, No. 35, 1964; WHO Technical Report Series, No. 281, 1964(out of print).

8. Specifications for the identity and purity of food additives and their toxicologicalevaluation: food colours and some antimicrobials and antioxidants (Eighth reportof the Joint FAO/WHO Expert Committee on Food Additives). FAO NutritionMeetings Series, No. 38, 1965; WHO Technical Report Series, No. 309, 1965(out of print).

9. Specifications for identity and purity and toxicological evaluation of someantimicrobials and antioxidants. FAO Nutrition Meetings Report Series, No. 38A,1965; WHO/Food Add/24.65 (out of print).

10. Specifications for identity and purity and toxicological evaluation of food colours.FAO Nutrition Meetings Report Series, No. 38B, 1966; WHO/Food Add/66.25.

11. Specifications for the identity and purity of food additives and their toxicologicalevaluation: some antimicrobials, antioxidants, emulsifiers, stabilizers, flourtreatment agents, acids, and bases (Ninth report of the Joint FAO/WHO ExpertCommittee on Food Additives). FAO Nutrition Meetings Series, No. 40, 1966;WHO Technical Report Series, No. 339, 1966 (out of print).

– 61 –

lxii12. Toxicological evaluation of some antimicrobials, antioxidants, emulsifiers,

stabilizers, flour treatment agents, acids, and bases. FAO Nutrition MeetingsReport Series, No. 40A, B, C; WHO/Food Add/67.29.

13. Specifications for the identity and purity of food additives and their toxicologicalevaluation: some emulsifiers and stabilizers and certain other substances (Tenthreport of the Joint FAO/WHO Expert Committee on Food Additives). FAO NutritionMeetings Series, No. 43, 1967; WHO Technical Report Series, No. 373, 1967.

14. Specifications for the identity and purity of food additives and their toxicologicalevaluation: 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.

15. Toxicological evaluation of some flavouring substances and non nutritivesweetening agents. FAO Nutrition Meetings Report Series, No. 44A, 1968; WHO/Food Add/68.33.

16. Specifications and criteria for identity and purity of some flavouring substancesand non-nutritive sweetening agents. FAO Nutrition Meetings Report Series, No.44B, 1969; WHO/Food Add/69.31.

17. Specifications for the identity and purity of food additives and their toxicologicalevaluation: some antibiotics (Twelfth report of the Joint FAO/WHO ExpertCommittee on Food Additives). FAO Nutrition Meetings Series, No. 45, 1969;WHO Technical Report Series, No. 430, 1969.

18. Specifications for the identity and purity of some antibiotics. FAO Nutrition MeetingsSeries, No. 45A, 1969; WHO/Food Add/69.34.

19. Specifications for the identity and purity of food additives and their toxicologicalevaluation: some food colours, emulsifiers, stabilizers, anticaking agents, andcertain other substances (Thirteenth report of the Joint FAO/WHO ExpertCommittee on Food Additives). FAO Nutrition Meetings Series, No. 46, 1970;WHO Technical Report Series, No. 445, 1970.

20. Toxicological evaluation of some food colours, emulsifiers, stabilizers, anticakingagents, and certain other substances. FAO Nutrition Meetings Report Series,No. 46A, 1970; WHO/Food Add/70.36.

21. Specifications for the identity and purity of some food colours, emulsifiers,stabilizers, anticaking agents, and certain other food additives. FAO NutritionMeetings Report Series, No. 46B, 1970; WHO/Food Add/70.37.

22. Evaluation of food additives: specifications for the identity and purity of foodadditives and their toxicological evaluation: some extraction solvents and certainother substances; and a review of the technological efficacy of some antimicrobialagents. (Fourteenth report of the Joint FAO/WHO Expert Committee on FoodAdditives). FAO Nutrition Meetings Series, No. 48, 1971; WHO Technical ReportSeries, No. 462, 1971.

23. Toxicological evaluation of some extraction solvents and certain other substances.FAO Nutrition Meetings Report Series, No. 48A, 1971; WHO/Food Add/70.39.

24. Specifications for the identity and purity of some extraction solvents and certainother substances. FAO Nutrition Meetings Report Series, No. 48B, 1971; WHO/Food Add/70.40.

25. A review of the technological efficacy of some antimicrobial agents. FAO NutritionMeetings Report Series, No. 48C, 1971; WHO/Food Add/70.41.

26. Evaluation of food additives: some enzymes, modified starches, and certain othersubstances: Toxicological evaluations and specifications and a review of thetechnological efficacy of some antioxidants (Fifteenth report of the Joint FAO/

lxiiiWHO Expert Committee on Food Additives). FAO Nutrition Meetings Series, No.50, 1972; WHO Technical Report Series, No. 488, 1972.

27. Toxicological evaluation of some enzymes, modified starches, and certain othersubstances. FAO Nutrition Meetings Report Series, No. 50A, 1972; WHO FoodAdditives Series, No. 1, 1972.

28. Specifications for the identity and purity of some enzymes and certain othersubstances. FAO Nutrition Meetings Report Series, No. 50B, 1972; WHO FoodAdditives Series, No. 2, 1972.

29. A review of the technological efficacy of some antioxidants and synergists. FAONutrition Meetings Report Series, No. 50C, 1972; WHO Food Additives Series,No. 3, 1972.

30. Evaluation of certain food additives and the contaminants mercury, lead, andcadmium (Sixteenth report of the Joint FAO/WHO Expert Committee on FoodAdditives). FAO Nutrition Meetings Series, No. 51, 1972; WHO Technical ReportSeries, No. 505, 1972, and corrigendum.

31. Evaluation of mercury, lead, cadmium and the food additives amaranth,diethylpyrocarbamate, and octyl gallate. FAO Nutrition Meetings Report Series,No. 51A, 1972; WHO Food Additives Series, No. 4, 1972.

32. Toxicological evaluation of certain food additives with a review of general principlesand of specifications (Seventeenth report of the Joint FAO/WHO Expert Committeeon Food Additives). FAO Nutrition Meetings Series, No. 53, 1974; WHO TechnicalReport Series, No. 539, 1974, and corrigendum (out of print).

33. Toxicological evaluation of some food additives including anticaking agents,antimicrobials, antioxidants, emulsifiers, and thickening agents. FAO NutritionMeetings Report Series, No. 53A, 1974; WHO Food Additives Series, No. 5,1974.

34. Specifications for identity and purity of thickening agents, anticaking agents,antimicrobials, antioxidants and emulsifiers. FAO Food and Nutrition Paper, No.4, 1978.

35. Evaluation of certain food additives (Eighteenth report of the Joint FAO/WHOExpert Committee on Food Additives). FAO Nutrition Meetings Series, No. 54,1974; WHO Technical Report Series, No. 557, 1974, and corrigendum.

36. Toxicological evaluation of some food colours, enzymes, flavour enhancers,thickening agents, and certain other food additives. FAO Nutrition Meetings ReportSeries, No. 54A, 1975; WHO Food Additives Series, No. 6, 1975.

37. Specifications for the identity and purity of some food colours, enhancers,thickening agents, and certain food additives. FAO Nutrition Meetings ReportSeries, No. 54B, 1975; WHO Food Additives Series, No. 7, 1975.

38. Evaluation of certain food additives: some food colours, thickening agents, smokecondensates, 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.

39. Toxicological evaluation of some food colours, thickening agents, and certainother substances. FAO Nutrition Meetings Report Series, No. 55A, 1975; WHOFood Additives Series, No. 8, 1975.

40. Specifications for the identity and purity of certain food additives. FAO NutritionMeetings Report Series, No. 55B, 1976; WHO Food Additives Series, No. 9,1976.

41. Evaluation of certain food additives (Twentieth report of the Joint FAO/WHO ExpertCommittee on Food Additives). FAO Food and Nutrition Meetings Series, No. 1,1976; WHO Technical Report Series, No. 599, 1976.

lxiv42. Toxicological evaluation of certain food additives. WHO Food Additives Series,

No. 10, 1976.43. Specifications for the identity and purity of some food additives. FAO Food and

Nutrition Series, No. 1B, 1977; WHO Food Additives Series, No. 11, 1977.44. 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.

45. Summary of toxicological data of certain food additives. WHO Food AdditivesSeries, No. 12, 1977.

46. Specifications for identity and purity of some food additives, including antioxidant,food colours, thickeners, and others. FAO Nutrition Meetings Report Series, No.57, 1977.

47. Evaluation of certain food additives and contaminants (Twenty-second report ofthe Joint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 631, 1978.

48. Summary of toxicological data of certain food additives and contaminants. WHOFood Additives Series, No. 13, 1978.

49. Specifications for the identity and purity of certain food additives. FAO Food andNutrition Paper, No. 7, 1978.

50. Evaluation of certain food additives (Twenty-third report of the Joint FAO/WHOExpert Committee on Food Additives). WHO Technical Report Series, No. 648,1980, and corrigenda.

51. Toxicological evaluation of certain food additives. WHO Food Additives Series,No. 14, 1980.

52. Specifications for identity and purity of food colours, flavouring agents, and otherfood additives. FAO Food and Nutrition Paper, No. 12, 1979.

53. Evaluation of certain food additives (Twenty-fourth report of the Joint FAO/WHOExpert Committee on Food Additives). WHO Technical Report Series, No. 653,1980.


55. Specifications for identity and purity of food additives (sweetening agents,emulsifying agents, and other food additives). FAO Food and Nutrition Paper,No. 17, 1980.

56. Evaluation of certain food additives (Twenty-fifth report of the Joint FAO/WHOExpert Committee on Food Additives). WHO Technical Report Series, No. 669,1981.


58. Specifications for identity and purity of food additives (carrier solvents, emulsifiersand stabilizers, enzyme preparations, flavouring agents, food colours, sweeteningagents, and other food additives). FAO Food and Nutrition Paper, No. 19, 1981.

59. Evaluation of certain food additives and contaminants (Twenty-sixth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 683, 1982.



62. Evaluation of certain food additives and contaminants (Twenty-seventh report ofthe Joint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 696, 1983, and corrigenda.

lxv63. Toxicological evaluation of certain food additives and contaminants. WHO Food

Additives Series, No. 18, 1983.64. Specifications for the identity and purity of certain food additives. FAO Food and

Nutrition Paper, No. 28, 1983.65. 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.

66. Evaluation of certain food additives and contaminants (Twenty-eighth report ofthe Joint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 710, 1984, and corrigendum.

67. Toxicological evaluation of certain food additives and contaminants. WHO FoodAdditives Series, No. 19, 1984.

68. Specifications for the identity and purity of food colours. FAO Food and NutritionPaper, No. 31/1, 1984.

69. Specifications for the identity and purity of food additives. FAO Food and NutritionPaper, No. 31/2, 1984.

70. Evaluation of certain food additives and contaminants (Twenty-ninth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 733, 1986, and corrigendum.


72. Toxicological evaluation of certain food additives and contaminants. WHO FoodAdditives Series, No. 20. Cambridge University Press, 1987.

73. Evaluation of certain food additives and contaminants (Thirtieth report of the JointFAO/WHO Expert Committee on Food Additives). WHO Technical Report Series,No. 751, 1987.



76. 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.

77. Evaluation of certain food additives and contaminants (Thirty-first report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 759, 1987 and corrigendum.

78. Toxicological evaluation of certain food additives. WHO Food Additives Series,No. 22. Cambridge University Press, 1988.


80. Evaluation of certain veterinary drug residues in food (Thirty-second report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 763, 1988.

81. Toxicological evaluation of certain veterinary drug residues in food. WHO FoodAdditives Series, No. 23. Cambridge University Press, 1988.

82. Residues of some veterinary drugs in animals and foods. FAO Food and NutritionPaper, No. 41, 1988.

83. Evaluation of certain food additives and contaminants (Thirty-third report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 776, 1989.


lxvi85. Evaluation of certain veterinary drug residues in food (Thirty-fourth report of the

Joint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 788, 1989.

86. Toxicological evaluation of certain veterinary drug residues in food. WHO FoodAdditives Series, No. 25, 1990.

87. Residues of some veterinary drugs in animals and foods. FAO Food and NutritionPaper, No. 41/2, 1990.

88. Evaluation of certain food additives and contaminants (Thirty-fifth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 789, 1990, and corrigenda.


90. Specifications for identity and purity of certain food additives. FAO Food andNutrition Paper, No. 49, 1990.

91. Evaluation of certain veterinary drug residues in food (Thirty-sixth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 799, 1990.



94. Evaluation of certain food additives and contaminants (Thirty-seventh report ofthe Joint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 806, 1991, and corrigenda.


96. Compendium of food additive specifications (Joint FAO/WHO Expert Committeeon Food Additives (JECFA)). Combined specifications from 1st through the 37thmeetings, 1956–1990. Rome, Food and Agricultural Organization of the UnitedNations, 1992 (2 volumes).

97. Evaluation of certain veterinary drug residues in food (Thirty-eighth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 815, 1991.

98. Toxicological evaluation of certain veterinary residues in food. WHO Food AdditivesSeries, No. 29, 1991.


100. Guide to specifications—General notices, general analytical techniques,identification tests, test solutions, and other reference materials. FAO Food andNutrition Paper, No. 5, Ref. 2, 1991.

101. Evaluation of certain food additives and naturally occurring toxicants (Thirty-ninthreport of the Joint FAO/WHO Expert Committee on Food Additives). WHOTechnical Report Series No. 828, 1992.

102. Toxicological evaluation of certain food additives and naturally occurring toxicants.WHO Food Additive Series, No. 30, 1993.

103. Compendium of food additive specifications: addendum 1. FAO Food and NutritionPaper, No. 52, 1992.

104. Evaluation of certain veterinary drug residues in food (Fortieth report of the JointFAO/WHO Expert Committee on Food Additives). WHO Technical Report Series,No. 832, 1993.


lxvii106. Residues of some veterinary drugs in animals and food. FAO Food and Nutrition

Paper, No. 41/5, 1993.107. Evaluation of certain food additives and contaminants (Forty-first report of the



109. Compendium of food additive specifications: addendum 2. FAO Food and NutritionPaper, No. 52, Add. 2, 1993.

110. Evaluation of certain veterinary drug residues in food (Forty-second report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 851, 1995.



113. Evaluation of certain veterinary drug residues in food (Forty-third report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 855, 1995, and corrigendum.



116. Evaluation of certain food additives and contaminants (Forty-fourth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 859, 1995.



119. Evaluation of certain veterinary drug residues in food (Forty-fifth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 864, 1996.



122. Evaluation of certain food additives and contaminants (Forty-sixth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 868, 1997.


124. Compendium of food additive specifications, addendum 4. FAO Food and NutritionPaper, No. 52, Add. 4, 1996.

125. Evaluation of certain veterinary drug residues in food (Forty-seventh report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 876, 1998.



lxviii128. Evaluation of certain veterinary drug residues in food (Forty-eighth report of the




131. Evaluation of certain food additives and contaminants (Forty-ninth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 884, 1999.

132. Safety evaluation of certain food additives and contaminants. WHO Food AdditivesSeries, No. 40, 1998.


134. Evaluation of certain veterinary drug residues in food (Fiftieth report of the JointFAO/WHO Expert Committee on Food Additives). WHO Technical Report Series,No. 888, 1999.



137. Evaluation of certain food additives (Fifty-first report of the Joint FAO/WHO ExpertCommittee on Food Additives). WHO Technical Report Series, No. 891, 2000.

138. Safety evaluation of certain food additives. WHO Food Additives Series, No. 42,1999.


140. Evaluation of certain veterinary drug residues in food (Fifty-second report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 893, 2000.

141. Toxicological evaluation of certain veterinary drug residues in food. WHO FoodAdditives Series, No. 43, 2000


143. Evaluation of certain food additives and contaminants (Fifty-third report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 896, 2000



146. Evaluation of certain veterinary drug residues in food (Fifty-fourth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 900, 2001



149. Evaluation of certain food additives and contaminants (Fifty-fifth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries No. 901, 2001.

lxix150. 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 theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 909, 2002.



157. Evaluation of certain veterinary drug residues in food (Fifty-eighth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 911, 2002.



160. Evaluation of certain food additives and contaminants (Fifty-ninth report of theJoint FAO/WHO Expert Committee on Food Additives). WHO Technical ReportSeries, No. 913, 2002.


162. Evaluation of certain veterinary drug residues in food (Sixtieth report of the JointFAO/WHO Expert Committee on Food Additives). WHO Technical Report Series,No. 918, 2003.



ANNEX 2 73

ANNEX 2

ABBREVIATIONS USED IN THE MONOGRAPHS

ADI acceptable daily intakebw body weightECG electrocardiogramF0 parental generationF1 first filial generationF2 second filial generationFAO Food and Agricultural Organization of the

United NationsGLP good laboratory practiceIPCS International Programme on Chemical SafetyJECFA Joint FAO/WHO Expert Committee on Food

AdditivesLOEL lowest-observed-effect levelMIC minimum inhibitory concentrationMRL maximum residue limitNOEL no-observed-effect levelNTE neuropathy target esteraseQCA quinoxaline-2-carboxylic acidWHO World Health Organization

– 71 –

ANNEX 3 73

– 73 –

ANNEX 3

JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES

Geneva, 6–12 February 2003

Members

Dr D. Arnold, Consultant, Berlin, Germany (Vice-Chairman)

Professor A.R. Boobis, Section on Clinical Pharmacology, Division of Medicine,Faculty of Medicine, Imperial College, London, England

Dr R. Ellis, Senior Regulatory Scientist, Division of Human Food Safety, Officeof New Animal Drug Evaluation, Center for Veterinary Medicine, Food andDrug Administration, Rockville, MD, USA (FAO Rapporteur)

Dr K. Greenlees, Toxicologist, Division of Human Food Safety, Office of NewAnimal Drug Evaluation, Center for Veterinary Medicine, Food and DrugAdministration, Rockville, MD, USA (WHO Rapporteur)

Dr L.D.B. Kinabo, Sokoine University of Agriculture, Morogoro, Chuo Kikua,United Republic of Tanzania

Dr J. MacNeil, Head, Centre for Veterinary Drug Residues, Canadian FoodInspection Agency, Saskatoon Laboratory, Saskatoon, Saskatchewan,Canada

Professor J.G. McLean, Camberwell, Victoria, Australia (Chairman)

Dr K. Mitsumori, Professor, Laboratory of Veterinary Pathology, School ofVeterinary Medicine, Faculty of Agriculture, Tokyo University of Agricultureand Technology, Tokyo, Japan

Dr G. Moulin, Agence Française de Sécurité Sanitaire des Aliments, Fougères,France1

Dr J.L. Rojas Martínez, Ministerio de Agricultura y Ganadería, LaboratorioNacional de Servicios Veterinarios, Barreal de Heredia, Heredia, CostaRica

Dr S. Soback, Head, National Residue Control Laboratory and DepartmentFood Hygiene, Kimron Veterinary Institute, Beit Dagan, Israel

Secretariat

Dr C.E. Cerniglia, Division of Microbiology, National Center for ToxicologicalResearch, Food and Drug Administration, Jefferson, AR, USA (WHOTemporary Adviser)

1 Dr G. Moulin was unable to attend the meeting but was involved in the discussionsbefore and during the meeting.

74 ANNEX 3

Dr P. Chamberlain, Toxicologist, Division of Epidemiology, Office of Surveillanceand Compliance, Center for Veterinary Medicine, Food and DrugAdministration, Rockville, MD, USA (WHO Temporary Adviser)

Dr M. Ema, Division of Risk Assessment, Biological, Safety Research Centre,National Institute of Health Sciences, Tokyo, Japan (WHO TemporaryAdviser)

Dr A. Fernández Suárez, National Institute of Agricultural Technology, FoodTechnology Institute, Food Protection Division, Buenos Aires, Argentina(FAO Consultant)

Dr L.G. Friedlander, Physiologist, Division of Human Food Safety, Office ofNew Animal Drug Evaluation, Center for Veterinary Medicine, Food andDrug Administration, Rockville, MD, USA (FAO Consultant)

Dr M. Luetzow, Food Quality and Standards Service, Food and NutritionDivision, Food and Agriculture Organization of the United Nations, Rome,Italy (FAO Joint Secretary)

Dr S.W. Page, International Programme on Chemical Safety, World HealthOrganization, Geneva, Switzerland (Acting WHO Joint Secretary)

Dr P.T. Reeves, National Registration Authority for Agricultural and VeterinaryChemicals, Kingston, ACT, Australia (FAO Consultant)

Mr D. Renshaw, Food Standards Agency, London, England (WHO TemporaryAdviser)

Dr L. Ritter, Department of Environmental Biology, University of Guelph,Guelph, Ontario, Canada (WHO Temporary Adviser)

Dr S. Sundlof, Center for Veterinary Medicine, Food and Drug Administration,Rockville, MD, USA (Chairman, Codex Committee on Residues of VeterinaryDrug Residues in Foods)

Professor G.E. Swan, Professor of Pharmacology and Toxicology and Head ofDepartment of Paraclinical Sciences, Faculty of Veterinary Science,University of Pretoria, Pretoria, South Africa (FAO Consultant)

Professor F.R. Ungemach, Institute of Pharmacology, Faculty of VeterinaryMedicine, University of Leipzig, Leipzig, Germany (WHO Temporary Adviser)

ANNEX 4 75

– 75 –

ANNEX 4

RECOMMENDATIONS ON COMPOUNDS ON THE AGENDA ANDFURTHER INFORMATION REQUIRED

Antimicrobial agents

Neomycin

Acceptable daily intake: The ADI of 0–60 µg/kg bw (established at theforty-seventh meeting of the Committee (WHOTRS 876, 1998)) was maintained.

Residue definition: Neomycin

Recommended maximum residue limits (MRLs)a

Species Liver Kidney Milk(µg/kg) (µg/kg) (µg/kg)

Cattle 500 10 000 1500

a The MRLs of 500 µg/kg for cattle muscle and fat and all other MRLs recommended atthe forty-seventh meeting of the Committee (WHO TRS 876, 1998) were maintained.

Flumequine

Acceptable daily intake: The ADI established at the forty-eighth meetingof the Committee (WHO TRS 879, 1998) waswithdrawn.

Residue definition: The MRLs for cattle, pigs, sheep, chickensand trout established at previous meetings(WHO TRS 879, 1998; WHO TRS 900, 2001)were withdrawn.

Antiprotozoal agent

Imidocarb

Acceptable daily intake: 0–10 mg/kg bw (established at the fiftiethmeeting of the Committee (WHO TRS 888,1999))

Residue definition: Imidocarb free base

76 ANNEX 4

Recommended maximum residue limits (MRLs)

Species Fat Kidney Liver Milk Muscle(µg/kg) (µg/kg) (µg/kg) (µg/kg) (µg/kg)

Cattle 50 2000 1500 50 300

Insecticides

Deltamethrin

Intake considerations: The Joint FAO/WHO Expert Meeting onPesticide Residues performed a dietary riskassessment and estimated that the theoreticalintake of deltamethrin residues from pesticideuse would account for 25% of the ADI,equivalent to 150 µg (FAO Plant Productionand Protection Paper No. 172, 2002). Thesum of the theoretical concentrations ofdeltamethrin residues from use as a veterinarydrug and as a pesticide use would be no morethan 415 µg, equivalent to 68% of the ADI.

Residue definition: The Committee affirmed that the MRLsrecommended at the fifty-second meeting(WHO TRS 893, 2000) were compatible withthe ADI.

Dicyclanil

Acceptable daily intake: 0–0.007 mg/kg bw (established at the fifty-fourth meeting of the Committee (WHO TRS900, 2001))

Residue definition: Dicyclanil

Recommended maximum residue limits (MRLs)

Species Muscle Liver Kidney Fat(µg/kg) (µg/kg) (µg/kg) (µg/kg)

Sheep 150 125 125 200

ANNEX 4 77

Trichlorfon (metrifonate)

Acceptable daily intake: The Committee amended the ADI fortrichlorfon from 0–20 µg/kg to 0–2 µg/kg bw

Residue definition: The Committee confirmed the MRL for cows’milk and the guidance levels for muscle, liver,kidney and fat of cattle recommended at thefifty-fourth meeting (WHO TRS 900, 2001).

Production aid

Carbadox

Acceptable daily intake: The Committee confirmed the opinion,expressed at its thirty-sixth meeting (WHOTRS 799 1990), that an ADI could not beestablished.

Residue definition: The Committee decided to withdraw the MRLsof carbadox recommended at the thirty-sixthmeeting (WHO TRS 799 1990).

Documents

WHO FOOD Toxicological evaluation of ADDITIVES certain veterinary drug residues in food · 2019-05-23 · WHO FOOD ADDITIVES SERIES: 51 Toxicological evaluation of certain veterinary