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1、WHO FOOD ADDITIVES SERIES:89Prepared by the ninety-eighth meeting of the Joint FAO/WHO Expert Committee on Food AdditivesToxicological evaluation of certain veterinary drug residuesin foodWHO FOOD ADDITIVES SERIES:89Prepared by the ninety-eighth meeting of the Joint FAO/WHO Expert Committee on Food

2、AdditivesToxicological evaluation of certain veterinary drug residuesin foodThe summaries and evaluations contained in this book are,in most cases,based on unpublished proprietary data submitted for the purpose of the JECFA assessment.A registration authority should not grant a registration on the b

3、asis of an evaluation unless it has first received authorization for such use from the owner who submitted the data for JECFA review or has received the data on which the summaries are based,either from the owner of the data or from a second party that has obtained permission from the owner of the d

4、ata for this purpose.World Health Organization,Geneva,2025Toxicological evaluation of certain veterinary drug residues in food:prepared by the ninety-eighth meeting of the Joint FAO/WHO Expert Committee on Food Additives(JECFA)(WHO Food Additives Series,No.89)ISBN(WHO)978-92-4-010461-7(electronic ve

5、rsion)ISBN(WHO)978-92-4-010462-4(print version)ISBN(FAO)978-92-5-139512-7ISSN 0300-0923 World Health Organization and Food and Agriculture Organization of the United Nations,2025Some rights reserved.This work is available under the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 IGO licenc

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10、o.int/amc/en/mediation/rules.Suggested citation.Toxicological evaluation of certain veterinary drug residues in food:prepared by the ninety-eighth meeting of the Joint FAO/WHO Expert Committee on Food Additives(JECFA).Geneva:World Health Organization and Food and Agriculture Organization of the Unit

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18、ies of WHO or FAO.iiiCONTENTSPreface vResidues of specific veterinary drugs 1Clopidol 3Fumagillin dicyclohexylamine 23Imidacloprid 63Annex 1Reports and other documents resulting from previous meetingsof the Joint FAO/WHO Expert Committee on Food Additives 74Annex 2Abbreviations used in the monograph

19、s 88Annex 3Participants in the ninety-eighth meeting of theJoint FAO/WHO Expert Committee on Food Additives 90Annex 4Recommendations on substances on the agenda 92vPREFACEThe monographs contained in this volume were prepared at the ninety-eighth meeting of the Joint FAO/WHO Expert Committee on Food

20、Additives(JECFA),which met at the headquarters of the Food and Agriculture Organization of the United Nations(FAO)in Rome,Italy,on 2029 February 2024.These monographs summarize the data on the safety of residues in food of selected veterinary drugs reviewed by the Committee.The ninety-eighth report

21、of the meeting has been published by WHO as WHO Technical Report No.1055.Reports and other documents resulting from previous meetings of JECFA are listed in Annex 1.The participants in the meeting are listed in Annex 3 of the present publication,and the conclusions of the Committee are provided in A

22、nnex 4.JECFA serves as a scientific advisory body to FAO,WHO,their Member States and the Codex Alimentarius Commission,primarily through the Codex Committee on Food Additives,the Codex Committee on Contaminants in Food and the Codex Committee on Residues of Veterinary Drugs in Foods,regarding the sa

23、fety of food additives,residues of veterinary drugs,naturally occurring toxicants and contaminants in food.Committees accomplish this task by preparing reports of their meetings and publishing specifications or residue monographs and toxicological monographs,such as those contained in this volume,on

24、 substances that they have considered.The toxicological monographs contained in this volume are based on working papers that were prepared by WHO experts.A special acknowledgement is given at the beginning of each monograph to those who prepared the working papers.The monographs were edited by Ms El

25、isabeth Heseltine,France.Many unpublished proprietary reports are submitted to the Committee by various producers of the veterinary drugs under review,and,in many cases,they represent the only data available on the substances.The WHO experts based the working papers they wrote on all the data that w

26、ere submitted,and all these reports were available to the Committee when it made its evaluations.All experts who participated in the ninety-eighth meeting completed declaration of interest forms.No conflicts of interest were identified.Any comments or new information on the biological or toxicologic

27、al properties of the compounds evaluated in this publication should be addressed to:WHO Secretary of the Joint FAO/WHO Expert Committee on Food Additives,Department of Nutrition and Food Safety,World Health Organization 20 Avenue Appia,1211 Geneva 27 SwitzerlandRESIDUES OF VETERINARY DRUGS3ClopidolC

28、lopidolFirst draft prepared bySilvana Lima Grniak 1,Silvia Pieiro 2 and Alan Boobis 31 School of Veterinary Medicine,University of So Paulo,So Paulo,Brazil2 Center for Veterinary Medicine,US Food and Drug Administration,Rockville(MD),United States of America(USA)3 National Heart and Lung Institute,I

29、mperial College London,London,United Kingdom of Great Britain and Northern Ireland(United Kingdom)1.Explanation 32.Biological data 42.1 Biochemical aspects 42.1.1 Absorption,distribution and excretion 42.2 Toxicological studies 52.2.1 Acute toxicity 52.2.2 Short-term studies of toxicity 62.2.3 Long-

30、term studies of toxicity and carcinogenicity 82.2.4 Genotoxicity 92.2.5 Reproductive and developmental toxicity 112.3 Microbiological effects 142.4 Observations in humans 153.Comments 163.1 Biochemical data 163.2 Toxicological data 163.3 Microbiological data 184.Evaluation 185.References 201.Explana

31、tionClopidol(International Union of Pure and Applied Chemists IUPAC name,3,5-dichloro-2,6-dimethyl-1H-pyridin-4-one;Chemical Abstracts Services CAS number 2971-90-6)is a pyridone derivative that is structurally related to quinolones(see Fig.1).Clopidol is a coccidiostat and is approved for use in br

32、oiler chickens and replacement layers(pullets)to prevent coccidiosis caused by various Eimeria species.(13).It exerts its anti-protozoal effect by inhibiting mitochondrial respiration for energy production in the early life-cycle stages of the parasite(sporozoites and tropozoites)(4).The available p

33、roducts are not recommended for feeding to laying hens or to pullets after 16 weeks of age.The approved rate of inclusion of clopidol in chicken feed is 80250 mg/kg feed,fed continuously as the sole ration.The 4Toxicological evaluation of certain veterinary drug residues in food Ninety-eighth JECFAW

34、HO Food Additives Series,No.89,2025withdrawal period for approved clopidol products is 07 days.Clopidol is not currently registered for use as a pesticide.Clopidol has not previously been evaluated by the Committee.The Committee evaluated clopidol at the request of the Codex Committee on Residues of

35、 Veterinary Drugs in Foods(CCRVDF)at its twenty-sixth Session(5)in order to establish relevant health-based guidance values and to recommend maximum residue limits(MRLs)in chicken liver,kidney,muscle and skin/fat.Figure 1Chemical structure of clopidolClCH3OHNClH3CThe Committee reviewed data provided

36、 by the sponsor.Additionally,the AGRIS,CAB Abstracts,CAS Embase,PubMed,Scopus and WOS databases were searched with the terms“clopidol”and“tox”.Seventeen articles of potential relevance were identified.Summary information on toxicology was also available in reviews by the American Conference of Gover

37、nmental Industrial Hygienists(ACGIH)(6)and the Health Council of the Netherlands Committee on Updating of Occupational Exposure Limits(7).For evaluation of the impact of residues of clopidol on the human intestinal microbiome,a search of literature in the public domain was conducted in a library cat

38、alogue that contains 228 databases,including PubMed and Scopus.Information relevant to microbiological assessment was sought by using combinations of the terms“clopidol”,“microbiome”,“microbiota”,“bacteria”,“gut”,“gastrointestinal”,“intestinal”and“antimicrobial resistance”.The search did not identif

39、y any reference relevant to an impact of clopidol residues on the human intestinal microbiome.2.Biological data2.1 Biochemical aspects2.1.1 Absorption,distribution,and excretionIn a study conducted before good laboratory practice(GLP)guidelines were adopted,50 mg/kg bw of 36Cl-labelled clopidol(puri

40、ty not specified)were administered once in an oral capsule to 12 rats(sex or strain not specified).Groups of two animals were terminated at intervals of 4,24,48,72,168 and 264 hours after the rats had received the capsules,and radioactivity was determined in organs,plasma and excreta.About 10%of the

41、 radioactivity was still in the 5Clopidolstomach 4 hours later.The urine and faeces excreted during the first 4hours after dosing did not contain any significant amount of radioactivity.Apart from the stomach,appreciable quantities of radioactivity were found,in decreasing order,in the plasma,kidney

42、s,whole blood,liver,lungs and heart.Twenty-four hours after dosing,6065%and 3540%of the radioactivity was found in the urine and faeces,respectively.After 168hours,100%of the radioactivity had been excreted in urine and faeces.The biological half-life of disappearance from rat tissues was in the ord

43、er of 10hours.There did not appear to be any significant accumulation of radioactivity in any of the tissues(8).In another study conducted before GLP guidelines were adopted,80 New Zealand white rabbits(sex not specified)received by oral gavage,once a day for up to 5 days,16 mg/kg bw of 14Cclopidol

44、suspension(purity not specified)in 1%carboxymethylcellulose.One animal was killed at intervals of 0,24,48,96,112,128,160 and 224 hours after the first dose,and radioactivity was determined in organs and excreta.14CClopidol was rapidly absorbed,and 90%or more was excreted via the urine within 24 hour

45、s after the final dose.No accumulation of radioactivity occurred in tissues,and no radioactivity was detected in expired air.Three major radioactive components were detected in rabbit urine,two of which were identified:the first was unaltered clopidol,accounting for 47%of the total urinary radioacti

46、vity,and the second was 3,5-dichloro-2-hydroxymethyl-6-methylpyridin-4-ol,which accounted for 32%.The third substance,which contributed an average of 20%to urinary radioactivity,was considered most likely to be the O-glucuronide conjugate of the hydroxylated metabolite(9).2.2 Toxicological studies2.

47、2.1 Acute toxicityMost data on the acute toxicity of clopidol were available only in summary form.A study conducted in male rats(strain,age and experimental procedures not specified),reported an oral LD50 of 18 000 mg/kg bw for clopidol(purity not specified)(10,abstract only).In a study in rats,rabb

48、its and guinea-pigs(strain,sex,age and experimental procedure not specified)cited by the ACGIH(6),the LD50 of clopidol was reported to be 8000 mg/kg bw.In an unpublished GLP-compliant study(11),the acute oral toxicity of clopidol(99.12%purity)was assessed in Sprague Dawley rats.Clopidol was administ

49、ered by gavage at a single oral dose in an aqueous suspension to fasted rats(five of each sex per dose)at a dose of 0,500,1000 or 2000 mg/kg bw.There were no deaths.Light-brown stools were observed on day 2 in all animals 6Toxicological evaluation of certain veterinary drug residues in food Ninety-e

50、ighth JECFAWHO Food Additives Series,No.89,2025at 2000 mg/kg bw,and a soiled perineal region was observed in one male at 2000 mg/kg bw on day 2.A tendency to decreased body weight was observed in both sexes at 2000 mg/kg bw on day 2,and a decrease in weight gain was observed in males at this dose.No

51、 significant gross findings related to clopidol were found at necropsy.The authors concluded that the LD50 for a single oral dose of clopidol is 2000 mg/kg bw for both sexes.The summary by the ACGHI(6)included a study(12)in which it was found that clopidol in its dry form caused minor irritation to

52、the eyes of rabbits but was virtually non-irritating to both intact and abraded rabbit skin.When applied as a 10%alcohol-based suspension,it was marginally more irritating.No additional data were provided on its potential to cause irritation or to induce skin sensitization.2.2.2 Short-term studies o

53、f toxicityIn an unpublished,4-week,GLP-compliant toxicity study(13),clopidol(99.12%purity)was administered to Sprague Dawley rats(five of each sex per dose)by gavage at a dose of 0,30,100,300 or 1000 mg/kg bw per day.General health,mortality and moribundity were checked twice daily.Body weights and

54、food intake were recorded weekly.Urine and blood samples were collected during the final week of observation,when ophthalmological examinations were conducted.At termination,necropsy,organ weight and histopathological examinations were performed.There were no deaths during the study.Light-brown stoo

55、ls were observed in both sexes at 1000 mg/kg bw per day.Significant decreases in body weight were found in both sexes at 300 and 1000 mg/kg bw per day,in a dose-dependent manner.The mean body weight on day 28 was reduced by 10.9%and 20.1%in males and 8.0%and 11.6%in females at 300 and 1000 mg/kg bw

56、per day,respectively,as compared with the vehicle control group.Significant decreases in food consumption were observed in males from 100 mg/kg bw per day and in females at 300 and 1000 mg/kg bw per day;however,the changes were within the historical control range of the test laboratory.Significant,d

57、ose-related increases in water consumption were observed in males in all treated groups,and a similar tendency was observed in females at doses of clopidol 100 mg/kg bw per day.No ophthalmological abnormalities were observed.Significant decreases in urine specific gravity were observed in males trea

58、ted at 300 and 1000 mg/kg bw per day.Decreases in red blood cells,haemoglobin and haematocrit and increases in the distribution width of red cells and haemoglobin were observed in both sexes at 1000 mg/kg bw per day.The changes in haemoglobin distribution width in males and in haemoglobin and red ce

59、ll distribution width in females were statistically significant.The changes were,however,all within the historical control range of 7Clopidolthe laboratory.Significant increases in the albumin:globulin ratio were observed in both sexes at 1000 mg/kg bw per day,which were attributed to a decrease in

60、globulin.Significant,dose-related decreases in total bilirubin were observed in males at 300 and 1000 mg/kg bw per day,with a similar tendency in females at those doses.Blood urea nitrogen was significantly increased in males at 1000 mg/kg bw per day and creatinine in males at 100,300 and 1000 mg/kg

61、 bw per day,the changes nevertheless being within the laboratory historical control range,as was a significantly increased relative weight of liver in females at 1000 mg/kg bw per day.There were no clopidol-related gross findings at necropsy.The no-observed-adverse-effect level(NOAEL)was 100 mg/kg b

62、w per day,based on decreased body weights of animals of each sex at 300 mg/kg bw per day.In an unpublished,GLP-compliant,13-week toxicity study(14),Sprague Dawley rats(10 of each sex per dose)were given clopidol(purity,99.12%)by oral gavage at a dose of 0,20,60 or 180 mg/kg bw per day.Animals were o

63、bserved daily for clinical signs of toxicity,and body weight and food consumption were recorded weekly.Urine and blood samples were collected during the final week of observation,when ophthalmological examinations were also conducted.At termination,necropsy,organ weight and histopathological examina

64、tions were performed.Ten additional rats(five male and five female)were assigned to the vehicle control and the groups at 180 mg/kg bw per day to evaluate recovery during a 4-week recovery period after the end of administration.There were no deaths.Light-brown stools were observed in four of 15 fema

65、les at 180 mg/kg bw per day during the administration period.Significant decreases in body weights were observed in both sexes at 180 mg/kg bw per day,with a tendency to recuperate during the recovery period.Significant decreases in food consumption were observed in both sexes at 60 and 180 mg/kg bw

66、 per day;however,the changes were observed only in the early stage of the administration period(males:days 17,females:days 121).Significant increases in water consumption were observed in males at 60 and 180 mg/kg bw per day and in females at 20 and 180 mg/kg bw per day,which persisted in both sexes

67、 at 180 mg/kg bw per day during the 4-week recovery period.The changes were,however,within the laboratory historical control range.Ophthalmological examination revealed no abnormalities.Males at 180 mg/kg bw per day showed minor,non-significant increases in the level of ketone bodies,in specific gra

68、vity and in the level of protein in the urine.These changes were not considered toxicologically significant and returned to normal during the 4-week recovery period.Significant decreases were observed in haemoglobin in males at 180 mg/kg bw per day and in mean corpuscular haemoglobin concentration i

69、n males at 60 and 180 mg/kg bw per day.A significant increase in neutrophil count and a significant shortening of activated partial thromboplastin time were observed in females at 180 mg/kg bw per day.These changes were within the laboratory historical control range,and 8Toxicological evaluation of

70、certain veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives Series,No.89,2025the animals recovered fully during the recovery period.Significant increases in the albumin:globulin ratio were observed in males at 20 and 180 mg/kg bw per day and in females at 60 and 180 mg/kg bw per d

71、ay.Significant increases in creatinine were observed in males at 60 and 180 mg/kg bw per day,and these changes were outside the historical control range of the laboratory.Significant decreases in total bilirubin were observed in males at 180 mg/kg bw per day;however,the levels returned to normal dur

72、ing the 4-week recovery period.Discolouration,reduced size,rough surface and hydronephrosis were observed in the kidney of one male at 180 mg/kg bw per day.The incidence and severity of chronic progressive nephropathy increased in males at this dose as compared with the vehicle control group,and the

73、se did not recover during the 4-week recovery period.The authors concluded that oral administration of clopidol to Sprague Dawley rats for 13 weeks increased the occurrence of chronic progressive nephropathy,accompanied by a significant increase in creatinine levels in males receiving 180 mg/kg bw p

74、er day.The increase in chronic progressive nephropathy could be considered toxicologically significant because of its higher incidence and severity than in the vehicle control group,and the condition did not improve during the subsequent 4-week recovery period.It should be noted,however,that chronic

75、 progressive nephropathy occurs naturally in rats,males being markedly more susceptible in terms of onset,frequency and progression of severity.According to Hard et al.(15),this condition is not relevant to human health.No toxicologically significant changes were observed in females.The NOAEL was 60

76、 mg/kg bw per day,based on significant decreases in body weight in both sexes at 180 mg/kg bw per day.2.2.3 Long-term studies of toxicity and carcinogenicityLimited information was available from summaries(16,17)of two unpublished studies on the long-term toxicity of clopidol.In an unpublished study

77、(18)conducted before GLP guidelines were adopted,clopidol(purity not specified)was administered in the diet of rats(40 of each sex per dose;strain and age not specified)at a concentration of 0,30,100 or 300 mg/kg feed(equivalent to 0,1.5,5.0 and 15 mg/kg bw per day)for 24 months.The maximum dose tes

78、ted was below that which had caused adverse effects in shorter term studies.No information was available on the periods or methods for evaluating the animals(i.e.when body weight and food consumption were recorded,clinical evaluation,urine and blood sample collection and gross and microscopic examin

79、ations of major organs and tumour incidence).The results showed no alteration in any parameter evaluated(growth,appearance,behaviour,haematology,clinical chemistry,histopathology or tumour incidence).No more details were provided.9ClopidolIn another unpublished study(19)conducted before GLP guidelin

80、es were adopted,clopidol(purity not specified)was administered in the diet of beagle dogs(four of each sex per dose;age not specified)at a concentration of 0,20,60 or 200 mg/kg feed(equivalent to 0,0.5,1.5 and 5 mg/kg bw per day)for 24 months.The periods and the methods for evaluating the animals we

81、re not specified(i.e.periods when the body weight and food consumption were recorded,clinical evaluation conducted,urine and blood samples collected and histopathological study performed).The results showed no alteration in any of the parameters evaluated.No more details were provided.2.2.4 Genotoxi

82、cityOnly one publication that addressed the genotoxicity of clopidol was identified in the literature(20).The publication is in Chinese,with a brief abstract in English,which summarizes several studies,the details of which are unclear.Eighteen hours after female Kumming mice,presumed to be pregnant,

83、were given a gavage dose of 160 mg/kg bw of clopidol,there was a notable increase in the incidence of micronucleated polychromatic erythrocytes in fetal livers,at a frequency of 6.5%as compared with 2.6%in a negative control group.In what appeared to be a separate study,administration of a lower dos

84、e of clopidol of 50 mg/kg bw resulted in a higher rate of polyploids(3%)in bone marrow cells in comparison with the rate seen in the control group,which was 0.4%.There was no increase in the number of cells with chromosomal aberrations.In a separate study,male mice received an intraperitoneal inject

85、ion of 0,6.25,25 or 50 mg/kg bw of clopidol.At 50 mg/kg bw there was a statistically significant increase in sperm abnormalities(5.34%)in comparison with the negative control(3.28%).In a separate study,mice were given clopidol by intraperitoneal injection at a dose of 10,30 or 100 mg/kg bw clopidol.

86、The number of sister chromatid exchanges per bone-marrow cell was statistically significantly increased,to 4.27,5.23 and 5.96,respectively,as compared with the control rate of 2.88 exchanges per cell.In a dominant lethal assay,male Kumming mice dosed daily by gavage with 0,6.25,25 or 100 mg/kg bw of

87、 clopidol for 5 consecutive days were mated serially with six groups of females over 6 weeks.In the high-dose group of mated females,the average number of living embryos per pregnant female was significantly lower(10.7)than in the control group(average,12.2)mated 3 weeks after administration but not

88、 mated at any other time.Additionally,the number of dead embryos in this group was significantly increased to 1.22 per female,as compared with 0.44 in the control group,when mated 3 weeks post-administration but not at other times.The mutagenic index for this group was increased when they were mated

89、 26 weeks after administration and was 10Toxicological evaluation of certain veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives Series,No.89,2025statistically significant at several time points,increasing to 10.1,as compared with 3.4 in the control group,when mated after 3 weeks.

90、The sponsor submitted three new studies of genotoxicity:two in vitro(bacterial reverse mutation and chromosome aberration assays)and one in vivo(bone marrow micronucleus assay).(a)In vitroIn an unpublished GLP-compliant study(21),clopidol was evaluated for its potential to induce reverse mutation in

91、 four histidine auxotroph strains of Salmonella typhimurium,TA100,TA1535,TA98 and TA1537,and a tryptophan auxotroph strain of Escherichia coli,WP2 uvrA,both in the presence and the absence of an exogenous metabolic activation system,which was the cofactor-supplemented post-mitochondrial supernatant

92、fraction(S9)of liver homogenate from rats pretreated with phenobarbital and 5,6-benzoflavone.Clopidol(99.88%purity)was suspended in dimethyl sulfoxide and added at 0,50,150,500,1500 or 5000 g/plate to the bacteria with or without metabolic activation.Appropriate positive controls were used for each

93、test condition(with metabolic activation:2-aminoanthracene for all bacterial strains other than TA98,benzoapyrene for TA98;without metabolic activation:sodium azide for TA1535 and TA100,2-nitrofluorene for TA98,4-nitroquinoline-1-oxide for WP2 uvrA,acridine mutagen ICR 191 for TA1537),all of which p

94、erformed satisfactorily.Clopidol was not mutagenic to bacteria.In a second unpublished GLP-compliant in vitro study(22),clopidol was evaluated for its potential to induce structural and numerical chromosome aberrations in Chinese hamster lung cells(CHL/IU,#CRL-1935)in the presence and absence of an

95、exogenous metabolic activation system,comprising cofactor-supplemented post-mitochondrial supernatant fraction(S9)of liver homogenate from rats pretreated with phenobarbital and 5,6-benzoflavone.Clopidol(99.88%purity)was suspended in dimethyl sulfoxide.No change in the frequency of chromosomal aberr

96、ations was found after 6 hours of treatment with metabolic activation at 75,150 or 300 g/mL or without metabolic activation,after 6 or 24 hours of treatment at 75,150 or 300 g/mL,as compared with vehicle control.Benzoapyrene and 4-nitroquinoline-1-oxide were used as positive controls,which performed

97、 as expected.The results indicated that clopidol was not clastogenic in mammalian cells.(b)In vivoIn an unpublished GLP-compliant in-vivo bone marrow micronucleus test(23),groups of six male Sprague Dawley rats were treated by oral gavage,once daily for 2 consecutive days,with clopidol(99.88%purity)

98、at 500,1000 or 2000 mg/kg bw per day.A concurrent negative control group received the vehicle 11Clopidolonly(water),while a positive control group received a single dose of 40 mg/kg bw cyclophosphamide monohydrate 24 hours before termination.Bone marrow smears were collected from six rats in each do

99、se group 24 hours after dosing for evaluation of micronuclei and cytotoxicity.No statistically significant or dose-related increase over the negative control was observed in the frequency of micronucleated polychromatic erythrocytes at any dose of clopidol.There was no statistically significant diff

100、erence in the ratio of polychromatic erythrocytes at any dose of clopidol when compared with that in the negative control.A clear increase in the number of micronucleated polychromatic erythrocytes was observed in the positive control group.Clopidol did not induce micronuclei in male Sprague Dawley

101、rat bone marrow cells under the experimental conditions of this study.2.2.5 Reproductive and developmental toxicity(a)Reproductive toxicityNo studies on the effect of clopidol on reproductive performance in animal species were found in the literature,nor were any provided by the sponsor.A brief summ

102、ary of one study,without details,was provided by ACGIH(6).Groups of 22 male and 38 female rats(controls comprised 66 males and 264 females)were given clopidol in their diet at 0,30,100 or 300 mg/kg feed(equivalent to 0,1.5,5 and 15 mg/kg bw per day)for three generations,each generation producing two

103、 litters.No impairment of reproduction or fertility was seen in any generation,and animals showed normal growth and survival.(b)Developmental toxicity In the multi-generation study of Morello et al.(undated),described by ACIGH(6),a third litter was produced from the third generation,and the fetuses

104、were examined before delivery.There was no evidence of any substance-related structural alterations.In a publication in Chinese,with a brief abstract in English(24),it was reported that pregnant Wistar rats(1112 animals per group)were given oral doses of clopidol(98%)at 0,4,20,100 or 200 mg/kg bw pe

105、r day(mode of administration not specified but presumably by gavage)on days 615 of gestation.In animals at the highest dose,a reduction in maternal body weight was observed.At the two highest doses(100 and 200 mg/kg bw per day),a notable decrease in pregnancy rates(p 0.01)was observed.As the animals

106、 were already pregnant at the time of treatment,however,this observation suggests that the decrease in pregnancy rates may have been due to pregnancy loss.This could have a consequence of the loss of weight in the dams.At 200 mg/kg bw per day,adverse fetal outcomes were noted,including reduced fetal

107、 body weight and length(p 0.01).An increase in delayed sternum ossification was also observed in a large number of high-dose fetuses but this was not statistically significant.One fetus in the high-dose group had exencephaly and had a short tail(unclear whether this was the same fetus).Short tail wa

108、s also seen in one control fetus.The authors concluded that these malformations had occurred naturally(only very limited details available).At a dose of 100 mg/kg bw per day,a decrease in the average weight of the litters was observed.In an unpublished,GLP-compliant,dose-range finding study of prena

109、tal developmental toxicity submitted by the sponsor(25),pregnant Sprague Dawley rats(four or five per dose)were treated once daily by gavage with 0,10,30,100 or 300 mg/kg bw per day of clopidol(99.88%purity)in water on days 519 of gestation.All females were examined daily for clinical signs from the

110、 start of dosing,and body weight and food consumption were recorded at regular intervals.The animals were killed on gestation day 20;organ weights and necropsy findings were recorded and caesarean section performed.The fetuses were examined for body weight,placental weight and external abnormalities

111、.At the highest dose of 300 mg/kg bw per day,a significant decrease in gestational body weight and in weight gain were observed in the dams,accompanied by a significant decrease in food consumption.A decrease in gravid uterine weight was also observed at this dose.Enlarged adrenal gland,small thymus

112、 or loss of fur were observed,but the findings were considered not to be related to administration of clopidol,as the incidence was not dose-related.Significant decreases in litter size were observed due to the significant increases in post-implantation loss and also in fetal body weight and in plac

113、enta weight.External malformations were observed in one fetus(i.e.protruding eye,exencephaly,small and bent tail and subcutaneous oedema)at 300 mg/kg bw per day.The NOAEL in this study was 100 mg/kg bw per day for maternal effects and for effects on embryo-fetal development,seen as decreases in litt

114、er size due to post-implantation loss,decreases in fetal body weight and placenta weight,and external malformations in one fetus at 300 mg/kg bw per day.In the main study of developmental toxicity provided by the sponsor(26),clopidol(99.88%purity)was administered in water by oral gavage on days 620

115、of gestation to groups of 25 pregnant Sprague Dawley rats at a dose of 0,40,100 or 250 mg/kg bw per day.All animals were observed daily for mortality and moribundity,and clinical observations,body weights and food consumption were recorded at appropriate intervals.On day 21 of gestation,the animals

116、were killed,necropsy was performed,and the internal organs were examined for macroscopic abnormalities.The uteri,placentae and ovaries were examined,and the numbers of fetuses,early and late resorptions,total implantations and corpora lutea were recorded.The fetuses were weighed,sexed and examined f

117、or external malformations and developmental variations.13ClopidolNo effects of clopidol were observed on clinical signs,abortions or survival at any dose.Significant reductions in the body weight of pregnant animals were observed on days 821,and reductions in overall weight gain were noted at doses

118、of 100 mg/kg bw per day and above.Additionally,significant decreases in corrected body weight were observed in all treated groups,with a clear doseresponse relationship.A marked decrease in food consumption was observed at doses of 100 and 250 mg/kg bw per day,specifically on days 57 of gestation.At

119、 250 mg/kg bw per day,a significant reduction in the weight of gravid uteri was observed.No abnormalities related to the test substance were detected at necropsy.In caesarean section examinations,complete litter resorption was observed in 5 of 21animals at 250 mg/kg bw per day.There was also a signi

120、ficant increase in the number of early resorptions.These changes contributed to a notable increase in post-implantation loss,resulting in a reduction in the overall size of the litters.Significant dose-related decreases in the body weights of live fetuses were observed in all clopidol-treated groups

121、.Significant reductions in placental weights were seen at doses of 100 or 250 mg/kg bw per day,with a clear doseresponse relationship.At 250 mg/kg bw per day,external malformations comprising cleft palate,exencephaly and gastroschisis were observed in one fetus each from a separate litter,resulting

122、in a significant increase in fetal incidence.No external variations were noted.A significant increase in the incidence of fetal malformations was seen at doses of 100 and 250 mg/kg bw per day,including absent or hemicentric thoracic centrum,and in litter incidences at 250 mg/kg bw per day.At 250 mg/

123、kg bw per day,other malformations,such as absent ribs,fused ribs,malpositioned sternebrae and absent lumbar vertebrae,were observed in one fetus.A hemicentric lumbar centrum and a fused lumbar centrum were observed in one fetus each at this dose.Significant increases in the fetal incidences of skele

124、tal variations were seen at doses of 100 and 250 mg/kg bw per day.Litter incidences were increased significantly only at 250 mg/kg bw per day.The variations included supernumerary sites of the lumbar rib(14th rib),asymmetric sternebrae,asymmetric ossification of sternebrae and thoracic centrum,unila

125、teral ossification of sternebrae and fused sternebrae.Additionally,the fetal and litter incidence of retardation was significantly increased at doses of 100 and 250 mg/kg bw per day,characterized by bipartite or dumbbell ossification of sternebrae and thoracic and lumbar centrum,and unossified thora

126、cic centrum.A doseresponse relationship was also observed in the reduction of ossification centres in the cervical vertebrae and sacral and caudal vertebrae in all groups treated with clopidol.The number of ossification centres in the metatarsus and in the first and second phalanges of hindlimbs was

127、 significantly decreased at 100 and 250 mg/kg bw per day,and decreases in ossification centres in the sternebrae and in the first and second phalanges of the forelimbs were seen at 250 mg/kg bw per day.14Toxicological evaluation of certain veterinary drug residues in food Ninety-eighth JECFAWHO Food

128、 Additives Series,No.89,2025Malformations such as absent renal papillae were observed in two fetuses from different litters at doses of 100 and 250 mg/kg bw per day,and an absent lung lobe was noted at 250 mg/kg bw per day.No variations were observed in these cases.No NOAEL could be identified in th

129、is study.The lowest-observed-adverse-effect level(LOAEL)for maternal toxicity was 40 mg/kg bw per day,based on a significant decrease in food consumption at this dose.Similarly,the LOAEL for developmental effects was 40 mg/kg bw per day,based on reductions in fetal weight and in the number of skelet

130、al ossification centres.Very limited information was available on the developmental toxicity of clopidol in rabbits.In a study by Woodhouse et al.(1965),described only in a summary without details(6),rabbits were given clopidol orally at a dose of 0,1.5,5 or 15 mg/kg bw per day on days 816 of gestat

131、ion for two matings.It is assumed that after the first mating litters were delivered naturally and were observed for an unspecified period.Clopidol had no effect on fertility,gestation,viability,lactation or growth of the pups.Offspring from the second mating were examined before natural delivery on

132、 gestation day 30.No effects attributable to clopidol were found.A study on the developmental toxicity of clopidol in rabbits was identified in the published literature(27).However,the study was considered uninformative,as it involved co-administration with methyl benzoquate,and there was a high inc

133、idence of infestation of the dams with coccidiosis and pneumonia.2.3 Microbiological effectsThe impact of clopidol residues on the human intestinal microbiome was evaluated in a decision-tree approach adopted by the Committee at its sixty-sixth meeting(28),which complies with guideline GL36(R2)of th

134、e International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products(VICH)(29).The approach entails answering three questions to determine whether establishment of a microbiological acceptable daily intake(mADI)is necessary.Determine,first,whether

135、the drug residues and/or its metabolites are microbiologically active against representative human intestinal microbiota;secondly,whether the drug residues enter the human colon;and,thirdly,whether residues that enter the human colon remain microbiologically active.If the answer to any of these ques

136、tions is“No”,there is no need to calculate an mADI,and the assessment need not be completed.If an mADI is needed,two end-points of concern for human health should be considered:disruption of the colonization barrier of the human intestinal microbiome and increases in the populations of resistant bac

137、teria in the human intestinal microbiome.In an unpublished minimum inhibitory concentration(MIC)study(30),reported to be GLP-compliant,representative bacterial strains from the human intestinal microbiome were tested against clopidol to determine 15Clopidolwhether its residues directly impact the hu

138、man intestinal microbiome.A total of 600 isolates,comprising 10 predominant groups of bacteria from the human intestinal microbiome,were isolated from faeces freshly obtained from five healthy women and one healthy man.The following taxonomic groups were tested:Bacteroides spp.,Bifidobacterium spp.,

139、Clostridium spp.,Eubacterium spp.,Fusobacterium spp.,Peptostreptococcus/Peptococcus,Enterococcus spp.,Bacteroides fragilis,Escherichia coli and Lactobacillus spp.The MIC50 and MIC90 were determined by reference methods provided by the Clinical and Laboratory Standards Institute(CLSI;31,32,33).The va

140、lues for both MIC50 and MIC90 for 60 isolates of each group were 500 g/mL,showing that clopidol had no measurable activity against the tested isolates.The quality control strains used in this study were:E.coli ATCC 25922 and Enterococcus faecalis ATCC 29212 as reference for non-fastidious bacteria,a

141、nd Bacteroides fragilis KCTC 5013,Bacteroides thetaiotaomicron KCTC 5015,Clostridium difficile ATCC 700057 and Eggerthella lenta ATCC 43055,as reference for anaerobes.Quality control testing was conducted according to CLSI recommendations,and all MIC results were in the range of the approved CLSI qu

142、ality control values for marbofloxacin,ceftiofur and doxycycline.As clopidol had no measurable antibacterial activity against the representative bacterial strains of the human intestinal microbiome tested in this study,the answer to the first question of the assessment of whether clopidol residues a

143、re microbiologically active against representative human intestinal microbiota is“No”.Thus,neither a microbiological ADI nor,by extension,a microbiological acute reference dose(mARfD)is necessary.2.4 Observations in humansThe sponsor did not submit any data and there was only limited information els

144、ewhere on the effects of clopidol in humans.The only mention was found in a review of clinical testing of new antimalarial compounds(34),which included a brief section on clopidol.Clopidol was administered to healthy volunteers(number not specified)at various dosages(neither dosage nor mode of admin

145、istration specified).Total doses of about 7.8 g(approximately 110 mg/kg bw in a 70 kg person)given over 310 days caused transitory,spontaneous,reversible paresthaesia.Some volunteers also developed ephemeral ptosis,diplopia,changes in visual accommodation,ataxia,deep tendon reflex hyperactivity or c

146、louding of consciousness.A total dose of 6 g(approximately 85 mg/kg bw in a 70 kg person)or less administered for 3 days to volunteers infected with various strains of Plasmodium falciparum did not cause neurological abnormalities.16Toxicological evaluation of certain veterinary drug residues in foo

147、d Ninety-eighth JECFAWHO Food Additives Series,No.89,20253.Comments3.1 Biochemical data The pharmacokinetics of clopidol was studied in rats and rabbits,and metabolism was studied in rabbits.No in-vitro study of the metabolism of clopidol was found in the open literature or provided by the sponsor.I

148、n a published study,after 36Clclopidol was administered orally to rats,most of the radioactivity was found in plasma,kidneys,blood,liver,lungs and heart.By 24 hours after dosing,6065%of the radioactivity was detected in urine and 3540%in faeces.The biological half-life of the radioactive material in

149、 rat tissues was estimated to be about 10 hours,with no marked accumulation in any tissues(22).In another published study,rabbits received oral doses of 14Cclopidol daily for up to 5 days.The result indicated rapid absorption and excretion of 14Cclopidol,with over 90%of the dose excreted in urine wi

150、thin 24 hours after the final dose.No accumulation of radioactivity was seen in the tissues,and no radioactivity was detected in expired air after repeated daily doses.Analysis of urine revealed three major radioactive components.The first was identified as unchanged clopidol and the second as 3,5-d

151、ichloro-2-hydroxymethyl-6-methylpyridin-4-ol,constituting approximately 47%and 32%,respectively,of the total urinary radioactivity.The third component,which accounted for an average of 20%of urinary radioactivity,was considered most likely to be the O-glucuronide conjugate of the hydroxylated metabo

152、lite(23).3.2 Toxicological data In rats,the oral median lethal dose(LD50)of clopidol was 2000 mg/kg bw(11).In an unpublished study that complied with good laboratory practice(GLP),(13)rats were given clopidol by gavage for 13 weeks at a dose of 0,20,60 or 180 mg/kg bw per day.The no-observed-adverse

153、-effect level(NOAEL)was 60 mg/kg bw per day,as decreases in body weight gain were seen at 180 mg/kg bw per day.Two unpublished non-GLP-compliant studies(18,19)of the long-term toxicity of clopidol were identified in minimal detail.Clopidol was administered in the feed to rats for 24 months at a conc

154、entration of 0,30,100 or 300 mg/kg feed(equivalent to 0,1.5,5.0 and 15 mg/kg bw per day).No alterations were found in haematology,clinical chemistry,histopathology or tumour incidence.Clopidol was administered in the diet of dogs at a concentration of 0,20,60 or 200 mg/kg feed(equivalent to 0,0.5,1.

155、5 and 5 mg/kg bw per day)for 24 months.No alterations were found in haematology,clinical chemistry or histopathology.17ClopidolThree GLP-compliant studies of the genotoxicity of clopidol were submitted by the sponsor.Negative results were found for clopidol in an Ames test(21),an in-vitro chromosoma

156、l aberration test(22)and a micronucleus study in rats in vivo(23).The Committee concluded that clopidol is unlikely to be genotoxic.In repeat-dose studies of up to 90 days duration in rats,clopidol caused no preneoplastic changes relevant to humans,and it was not genotoxic in a range of well conduct

157、ed studies.The limited information available indicated that it was not carcinogenic after chronic administration to rats.The Committee concluded that clopidol is unlikely to pose a carcinogenic risk at the levels present in the diet from its use as a veterinary drug.No reliable studies on the impact

158、 of clopidol on reproductive performance were provided by the sponsor or found in the published literature.A brief summary of one study,without details,was available from ACGIH(6).Rats were given clopidol in their diet at 0,30,100 or 300 mg/kg feed(equivalent to 0,1.5,5 and 15 mg/kg bw per day)for t

159、hree generations,each generation producing two litters.Neither reproduction nor fertility was impaired in any generation,and the animals showed normal growth and survival.In an unpublished GLP-compliant study of developmental toxicity(25),rats were given clopidol by gavage at 0,40,100 or 250 mg/kg b

160、w per day on days 620 of gestation.The lowest-observed-adverse-effect level(LOAEL)for both maternal and developmental toxicity was 40 mg/kg bw per day,the lowest dose tested,which caused a decrease in maternal food consumption and body weight gain and decreased fetal body weight.The Committee conclu

161、ded that clopidol is not teratogenic in rats.In view of the absence of an effect on reproduction(in a study reported in limited detail),the absence of genotoxicity in well conducted studies and the absence of any other indication of an effect on reproductive organs in repeat-dose studies,the Committ

162、ee concluded that residues of clopidol in the diet from its use as a veterinary drug are unlikely to be a risk to reproduction or to offspring.18Toxicological evaluation of certain veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives Series,No.89,20253.3 Microbiological data The im

163、pact of clopidol residues on the human intestinal microbiome was evaluated in a decision-tree approach,which complies with VICH guideline GL36(R2)of the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products(VICH)(29),adopted by the Com

164、mittee at its sixty-sixth meeting(28).The approach entails answering three questions to determine whether establishment of a microbiological acceptable daily intake(mADI)is needed.Determine,first,whether the drug residues and/or its metabolites are microbiologically active against representative hum

165、an intestinal microbiota;secondly,whether the drug residues enter the human colon;and,thirdly,whether residues that enter the human colon remain microbiologically active.If the answer to any of these questions is“No”,there is no need to calculate an mADI,and the assessment need not be completed.If a

166、n mADI is needed,two end-points of concern for human health should be considered:disruption of the colonization barrier of the human intestinal microbiome and increases in the populations of resistant bacteria in the human intestinal microbiome.In a minimum inhibitory concentration(MIC)study,submitt

167、ed by the sponsor and reported to be GLP-compliant(30),a group of bacterial strains representative of the human intestinal microbiome were tested with clopidol to determine whether its residues directly impact the microbiome.Clopidol had no measurable activity against the isolates(MIC 500 g/mL).The

168、answer to the first question of the assessment whether clopidol residues are microbiologically active against representative human intestinal microbiota is“No”.Therefore,neither an mADI nor,by extension,a microbiological acute reference dose(mARfD)is necessary.4.EvaluationThe Committee established a

169、n ADI for clopidol of 00.04 mg/kg bw based on a LOAEL of 40 mg/kg bw per day for decreased maternal body weight gain and fetal body weight in a developmental toxicity study in rats.An uncertainty factor of 1000 was applied,which comprises 100 for interspecies and intraspecies differences and additio

170、nal factors of 2 to account for the use of a marginal LOAEL and 5 for database uncertainty.The Committee concluded that,in view of the low acute oral toxicity of clopidol and the absence of developmental toxicity or any other toxicological effect likely to be elicited by a single dose,it was unneces

171、sary to establish an ARfD for clopidol.19ClopidolSummary and conclusions Table 1Studies relevant to risk assessmentSpecies/study type(route of administration)Doses(mg/kg bw per day)Critical end-pointNOAEL(mg/kg bw per day)LOAEL(mg/kg bw per day)RatShort-term(13-week)study(gavage)0 20,60 and 180 Redu

172、ction in body weight 60 180 Developmental toxicity study(gavage)0,40,100 and 250 Maternal toxicity:reduced body weight gain and decreased food consumption40 a,bDevelopmental toxicity:reduced fetal weight and decreased number of ossification centres40 a,bLOAEL:Lowest-observed-adverse-effect level;NOA

173、EL:No-observed-adverse-effect level;a Lowest dose tested;b Pivotal study for derivation of the ADI:26 Bae JS.A 13-week repeated-dose oral toxicity study of clopidol followed by a 4-week recovery study in Sprague Dawley rats.Gyeonggi-do:Nonclinical Research Institute,CorestemChemon Inc;2023.Microbiol

174、ogical effectsClopidol had no measurable activity against the tested isolates from the human intestinal microbiome(MIC 500 g/mL).The Committee concluded that no mADI or mARfD was needed.ADI The Committee established an ADI for clopidol of 00.04 mg/kg bw,based on a LOAEL of 40 mg/kg bw per day for de

175、creased maternal body weight gain and fetal body weight in a developmental toxicity study in rats.An uncertainty factor of 1000 was applied,which comprises 100 for interspecies and intraspecies differences and additional factors of 2 to account for the use of a marginal LOAEL and 5 for database unce

176、rtainty.ARfDThe Committee concluded that it was unnecessary to establish an ARfD for clopidol.Residue definition The MR for clopidol in chicken liver,kidney,muscle and skin/fat is clopidol.20Toxicological evaluation of certain veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives Se

177、ries,No.89,2025Estimated dietary exposure For clopidol included at 250 mg/kg feed at 24-hour withdrawal and the most conservative ratio of marker residues to total residues(MR:TR)considered of 0.5,the global estimate of chronic dietary exposure(GECDE)values for adults and the elderly,children and ad

178、olescents and infants and toddlers were 32.9,33.5 and 28.6g/kg bw per day,respectively(82%,84%and 71%,respectively,of the upper bound of the ADI of 40 g/kg bw).MRLs The Committee recommended MRLs of 10 400 g/kg(liver),8800 g/kg(kidney),4100 g/kg(muscle)and 2600 g/kg(skin/fat)in chickens.5.References

179、1.Ryley J.Studies on the mode of action of quinolone and pyridone coccidiostats.J.Parasitol.,1967;53:11511160.2.Reid WM,Taylor EM,Johnson J.A technique for demonstration of coccidiostatic activity of anticoccidial agents.Trans.Am.Microscope Soc.,1969;88:148159.3.Kant V,Singh P,Verma PK,Bais I,Parmar

180、 MS,Gopal A et al.Anticoccidial drugs used in the poultry:an overview.Sci Int.2013;1(7):2615.doi:10.5567/sciintl.2013.261.265.4.Fry M,Williams RB.Effects of decoquinate and clopidol on electron transport in mitochondria of Eimeria tenella(Apicomplexa:coccidia).Biochem Pharmacol.1984;33(2):229240.doi

181、:10.1016/0006-2952(84)90480-5.5.Report of the 26th session of the Codex Committee on Residues of Veterinary Drugs in Foods,1317 February 2023,Portland,Oregon,USA(REP23/RVDF26).Codex Alimentarius Commission,Rome;2023.Available at:https:/www.fao.org/fao-who-codexalimentarius/meetings/detail/en/?meetin

182、g=CCRVDF&session=266.Clopidol.In:Threshold Limit Values(TLVs)and Biological Exposure Indices(BEIs).:American Conference of Governmental Industrial Hygienists,Cincinnati,OH,USA;2013.ISBN 978-1607260592.7.Clopidol;health-based reassessment of administrative occupational exposure limits(2000/15OSH/079)

183、.Health Council of the Netherlands,Committee on Updating of Occupational Exposure Limits;2003.The Hague,Kingdom of the Netherlands.8.Smith GN,Watson BL.Metabolism of 36Cl-clopidol(3,5-dichloro-2,6-dimethyl-4-pyridinol)in rats.Poultry Sci.1969;48:437443.9.Cameron BD,Chasseaud LF,Hawkins DR.Metabolic

184、fate of clopidol after repeated oral administration to rabbits.J.Agric.Food Chem.1975;23:269274.doi:10.1021/jf60198a052.10.Plisek K,Dvorak M,Sevcik B,et al.Examination of toxic effects of metichlorpindol(in Czech).Vet.Spofa.1970;12:11125.(Chem.Abstracts 74:138780p).21Clopidol11.Bae JS.A single-dose

185、oral toxicity study of clopidol in Sprague-Dawley rats.Nonclinical Research Institute,CORESTEMCHEMON Inc.,Gyeonggi-do,Republic of Korea;2023.12.Olson KJ.Toxicological properties of 3,5-dichloro-2-6-dimethyl-4-pyrindol.Dow Chemical Company,Biochemical Research Laboratory,Midland,MI,USA;1963.13.Bae JS

186、.A 4-week repeated-dose oral toxicity DRF study of clopidol in Sprague-Dawley rats.Nonclinical Research Institute,CORESTEMCHEMON Inc.,Gyeonggi-do,Republic of Korea;2023.14.Bae JS.A 13-week repeated-dose oral toxicity study of clopidol followed by a 4-week recovery study in Sprague-Dawley rats.Gyeong

187、gi-do:Nonclinical Research Institute,CORESTEMCHEMON Inc;2023.15.Hard GC,Banton MI,Bretzlaff RS,Dekant W,Fowles JR,Mallett AK et al.Consideration of rat chronic progressive nephropathy in regulatory evaluations for carcinogenicity.Toxicol.Sci.2013;32(2):268275.doi:10.1093/toxsci/kfs305.16.Dow Chemica

188、l Company.Communication.Documentation(1973).In:Threshold limit values and biological exposure indices,5th edition.American Conference of Governmental Industrial Hygienists,Cincinnati OH,USA;2013.17.PubChem compound summary for CID 18087,clopidol.National Library of Medicine,National Center for Biote

189、chnology Information,Bethesda,MD,USA;2004 Available at:https:/pubchem.ncbi.nlm.nih.gov/compound/Clopidol.18.McColister DD,Brown MT,Sadek E.Report of a 2-year dietary feeding study of meticlorpindol in rats.Dow Chemical Company,Biochemical Research Laboratory,Midland,MI,USA;1963.19.McColister DD,Brow

190、n MT,Sadek E.Report of a 2-year dietary feeding study of meticlorpindol in beagle dogs.Dow Chemical Company,Biochemical Research Laboratory,Midland,MI,USA;1963.20.Bao H,Jiang Z,Gong G.Mutagenicity of clopidol(in Chinese).Nanjing Nongye Daxue Xuebao,1992;15:8995(Chem.Abstracts.118:116247v).21.Pak BS.

191、Bacterial reverse mutation assay with clopidol.Nonclinical Research Institute,CORESTEMCHEMON Inc,Gyeonggi-do,Republic of Korea;2023.22.Pak BS.In vitro chromosome aberration test in Chinese hamster lung cells with clopidol.Nonclinical Research Institute,CORESTEMCHEMON Inc.,Gyeonggi-do,Republic of Kor

192、ea;2023.23.Kim MS.Evaluation of clopidol in male Sprague-Dawley rats bone marrow micronucleus assay(oral gavage study).Nonclinical Research Institute,CORESTEMCHEMON Inc.,Gyeonggi-do,Republic of Korea;2023.24.Jiang Z,Bao H,Shi X.Teratogenic effect of clopidol in rats(in Chinese).Zhongguo Shouyi Xueba

193、o.1999;19:390392.25.Bae JS.An embryo-fetal development DRF study of clopidol by oral administration in Sprague-Dawley rats.Nonclinical Research Institute,CORESTEMCHEMON Inc.,Gyeonggi-do,Republic of Korea;2023.26.Bae JS.An embryo-fetal development study of clopidol by oral administration in Sprague-D

194、awley rats.Nonclinical Research Institute,CORESTEMCHEMON Inc.,Gyeonggi-do,Republic of Korea;2023.27.Varewyck H,Peeters JE,Halen P,Lampo P,Bouquet Y.Influence of the anticoccidials clopidol/methylbenzoquate and robenidine on fertility and progeny of female rabbits.Br.Vet.J.1984;140(2):202206.22Toxico

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196、a,Switzerland;2006.(https:/www.who.int/publications/i/item/9789241599397).29.GL36(R2)(Safety microbiological ADI).Studies to evaluate the safety of residues of veterinary drugs in human food:general approach to establish a microbiological ADI.Revision 2.International Cooperation on Harmonisation of

197、Technical Requirements for Registration of Veterinary Medicinal Products(VICH),Brussels,Belgium;2019.Available at:https:/vichsec.org/en/guidelines/pharmaceuticals/pharma-safety/antimicrobial-safety.html).30.Jeong SH.Microbiological impact of clopidol on human normal gut microbiota.Hoseo Biomedical R

198、esearch Center Hoseo University,Republic of Korea;2024.31.Clinical and Laboratory Standards Institute(CLSI),2018.Methods for antimicrobial susceptibility testing of anaerobic bacteria.Approved standard Ml1-A9.Vol.38,Number 19.32.Clinical and Laboratory Standards Institute(CLSI),2018.Performance stan

199、dards for antimicrobial disk and dilution susceptibility test for bacteria isolated from animals.Approved standard VET08-4th ed.Vol.38,Number 14.33.Clinical and Laboratory Standards Institute(CLSI),2021.Performance standards for antimicrobial susceptibility testing.Approved standard M100-Ed31.Vol.41

200、,Number 3.34.Canfield CJ,Rozman RS.Clinical testing of new antimalarial compounds.Bull.World Health Organ.1974;50(34):203212.PCMID:PMC2481191.23Fumagillin dicyclohexylamineFumagillin dicyclohexylamineFirst draft prepared byMayumi Ishizuka1,Silvia Pieiro2,Alan Boobis3 and Angelo Moretto 1 Faculty of

201、Veterinary Medicine,Hokkaido University,Sapporo,Japan2 Center for Veterinary Medicine,US Food and Drug Administration,Rockville(MD),United States of America3 National Heart and Lung Institute,Imperial College London,London,United Kingdom4 Department of Cardiac Thoracic Vascular Sciences and Public H

202、ealth,University of Padova,and University Hospital,Padua,Italy1.Explanation 242.Biological data 252.1 Biochemical aspects 252.1.1 Absorption,distribution and excretion 252.1.2 Biotransformation 272.1.3 Effects on enzymes and other biochemical parameters 282.2 Toxicological studies 282.2.1 Acute toxi

203、city;lethal doses 282.2.2 Short-term toxicity 282.2.3 Long-term toxicity 352.2.4 Genotoxicity 362.2.5 Reproductive and developmental toxicity 422.2.6 Special studies 482.3 Microbiological effects 492.4 Observations in humans 503.Comments 513.1 Biochemical data 513.2 Toxicological data 523.3 Microbio

204、logical data 543.4 Observations in humans 543.5 Metabolites or degradation products of fumagillin 554.Evaluation 555.References 5824Toxicological evaluation of certain veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives Series,No.89,20251.ExplanationFumagillin(IUPAC name:(2E,4E,6E

205、,8E)-10-(3R,4S,5S,6R)-5-methoxy-4-(2R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl-1-oxaspiro2.5octan-6-yloxy-10-oxodeca-2,4,6,8-tetraenoic acid;Chemical Abstracts Services No.23110-15-8)is a mycotoxin used as an antimicrobial agent for the treatment of microsporidian infections in honeybees and in v

206、arious fish species.Fumagillin is poorly soluble in water and undergoes rapid ultraviolet and thermal degradation.Therefore,commercial formulations used in veterinary medicine contain fumagillin as the dicyclohexylamine(DCH,IUPAC name N-cyclohexylcyclohexanamine;Chemical Abstracts Services No.101-83

207、-7)salt in a 1:1 stoichiometric ratio,to increase its stability and water solubility(Fig.1).Figure 1Chemical structures of fumagillin and of DCHNHSalt formed by loss and gain ofH ion at*and#respectively Fumagillin(MW 458.55)Dicyclohexylamine(MW 181.32)*#OOHOOCOOOH3CCH3CH3H3CThe mode of action of fum

208、agillin is based on inhibition of type-2methionine aminopeptidase(MetAP-2)activity by formation of a covalent bond with the histidine moiety of the enzyme(1).MetAP-2 is a cytosolic enzyme,which removes the initial methionine from the amino terminus of newly synthesized proteins for subsequent post-t

209、ranslational modifications,affecting the function of many proteins.Fumagillin DCH is used as a veterinary drug in feed for fish and for honeybees or by immersion bath treatment for fish.The dosage used in fish is 1550 mg of fumagillin base per kg bw in feed for 30 consecutive days or 60 mg fumagilli

210、n base per litre of water in an immersion bath for 5 consecutive days.According to good veterinary practice,the withdrawal period for use in fish is 28 days for both treatment regimens(water temperature not specified).The inclusion rate for use in bees is 2025 mg fumagillin base per litre of sugar s

211、yrup.Bees should be treated in the autumn after honey supers(boxes placed on beehives for bees to store honey)have been removed or in spring at least 4 weeks before the start of honey flow.25Fumagillin dicyclohexylamineFumagillin has been used in human medicine for certain infectious diseases and fo

212、r cancer(25).Fumagillin DCH is not currently registered for use as a pesticide.Fumagillin DCH has not previously been evaluated by the Committee.The Committee evaluated fumagillin DCH at the present meeting at the request of the Codex Committee on Residues of Veterinary Drugs in Foods at its Twenty-

213、sixth Session(6)with a view to establishing relevant health-based guidance values and recommending MRLs for fish and for honey.In veterinary medicine,fumagillin is administered only as the DCH salt;however,because the fumagillin DCH salt dissociates into the two moieties and consumers would be expos

214、ed to the residues of both,the Committee evaluated both fumagillin and DCH.2.Biological dataThe Committee reviewed data provided by the sponsor and conducted a search of the scientific literature in the following publicly accessible databases,with the search terms(fumagillin OR dicyclohexylamine)AND

215、“toxicity”:Web of Science,PubMed and Scopus.In Google Scholar,the Committee searched for papers containing the terms:“fumagillin”,“dicyclohexylamine and toxicity”,“metabolism”,“metabolites”,“in vivo”,“rats”,“mice”and“human”.The Committee also searched references in publications of the European Chemi

216、cals Agency(ECHA)and the International Agency for Research on Cancer(IARC).In total,33 papers on fumagillin and 13 on DCH relevant to the assessment were identified.For evaluation of the impact of residues on the human intestinal microbiome,a search for literature in the public domain was conducted

217、in a library catalogue that covers 228 databases,such as PubMed and Scopus.The search was conducted with combinations of the terms“fumagillin”,“DCH”,“dicyclohexylamine”,“microbiome”,“microbiota”,“bacteria”,“gut”,“gastrointestinal”,“intestinal”and“antimicrobial resistance”.No literature relevant to t

218、he impact of fumagillin residues on the human intestinal microbiome was identified.2.1 Biochemical aspects2.1.1 Absorption,distribution and excretion(a)FumagillinNo data on the metabolism or kinetics of fumagillin in mammalian species were submitted by the sponsor or available in the publicly availa

219、ble literature.When fumagillin DCH was administered at 30 or 60 mg/kg bw to trout 26Toxicological evaluation of certain veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives Series,No.89,2025through an indwelling catheter placed in the dorsal aorta,the plasma clearance fit a two-com

220、partment model,with a rapid phase half-life of 20 min but a prolonged phase half-life of 5.4 days(7).(b)Dicyclohexylamine (DCH)No data on the metabolism or kinetics of DCH in mammalian species were submitted by the sponsor.Rabbits(number not reported)received a single dose of 85 mg/kg bw DCH by gava

221、ge,and absorption from the gastrointestinal tract was observed.No DCH was detected in the blood,but more than 1 mg/mL was detected in the urine 130 min after administration.The animal(sic)died 160 min after dosing(Filov,1968,cited in 8).Absorption of DCH in situ was studied in isolated flow-through

222、loops of the small intestine of male rabbits(strain not reported)and Wistar rats(9).Rabbits were given 150 mL of a 30 mg/kg solution of DCH in pH 7.4 isotonic phosphate buffer,and rats received 10 mL of a 0.5 mg/mL solution in pH 6.0 McIlvaine buffer.The absorption rate constants were 0.44/hour in r

223、abbits and 0.33/hour in rats,indicating relatively rapid intestinal absorption of DCH in both species.Groups of three to five male rabbits(strain not stated,2.5 kg)and four to five male Wistar rats(200250 g)received a single dose of DCH in McIlvaine buffer,pH 6.0,by gavage at 50 mg/rabbit and 5 mg/r

224、at,and urine and faeces were collected at 24 and 48 hours.Additional animals were treated intravenously with DCH in McIlvaine buffer,pH 6.0,at a dose of 10 mg/rabbit and 1 mg/rat.Urine and faeces were collected at 24 and 48 hours.Urinary and faecal excretion of unchanged DCH over 2 days was low in b

225、oth species after both oral(0.08%and 0.30%of the administered dose in rabbits and 5.88%and 0.25%in rats,respectively)and intravenous(4.55%and 0.47%in rabbits and 14.84%and 0.59%in rats,respectively)administration.Excretion was greater during the first 24 hours than in the subsequent 24 hours.These d

226、ata suggest that the substance is rapidly and extensively absorbed and metabolized(Table 1).Table 1Urinary and faecal excretion of unchanged DCH over 48 hours after a single gavage or intravenous doseMatrixRabbit (percentage excreted)Rat (percentage excreted)OralIntravenousOralIntravenousUrine0.080.

227、034.551.125.881.3214.841.65Faeces0.300.120.470.210.250.110.590.17 Source:Suenaga et al.(9)Each value represents the mean standard deviation for three or five rabbits and for four or five rats;The oral dose of DCH was 50 mg/rabbit and 5 mg/rat;and the intravenous dose of DCH was 10 mg/animal for rabb

228、its and 1 mg/animal for rats27Fumagillin dicyclohexylamine2.1.2 BiotransformationNo data on the biotransformation of fumagillin or DCH in mammalian species in vivo were submitted by the sponsor or available in the publicly available literature.(a)FumagillinNo relevant data were submitted.(b)Dicycloh

229、exylamine (DCH)Rat liver homogenate was incubated with 0.04 mg/mL DCH in physiological saline for 1 hour,and rabbit liver slices were incubated with 0.08 mg/mL DCH in physiological saline for up to 18 hours.No change in DCH concentration was seen.In addition,rat and human blood was incubated with 0.

230、16 mg/mL and 1 mg/mL DCH in physiological saline,respectively,for 24 hours,with no change in DCH concentration(Filov,1968,cited in 8).In another study(9),liver post-mitochondrial supernatant from one rabbit(presumed to be male)and several rats(presumed to be male Wistar)was incubated with DCH at 1 m

231、g/mL(unclear whether this was the final concentration)in the presence of an NADPH regenerating system for 3 hours in an aerobic or anaerobic atmosphere.Unchanged DCH was found at 1,2 and 3 hours.DCH was rapidly and extensively metabolized by rabbit liver under aerobic but not under anaerobic,conditi

232、ons.Metabolism was much slower in rat liver and was apparent only under aerobic conditions(Fig.2).The metabolites were not identified.Figure 2Metabolism of DCH in 10 000 g supernatant fraction of rabbit and rat liverUnder aerobic conditionUnder anaerobic conditionIncubation time(hours)Remaining DCH(

233、percentage of dose)100500RabbitRatRabbitRat1 2 3 1 2 3 Incubation time(hours)100500Remaining DCH(percentage of dose)1 2 3 1 2 3 Each value represents the mean standard deviation of two or three experiments;Source:Suenaga et al.(9)28Toxicological evaluation of certain veterinary drug residues in food

234、 Ninety-eighth JECFAWHO Food Additives Series,No.89,20252.1.3 Effects on enzymes and other biochemical parametersNo data were submitted by the sponsor or identified in the literature search.2.2 Toxicological studies2.2.1 Acute toxicity;lethal doses(a)FumagillinThe oral LD50 of fumagillin in mice was

235、 reported to be 2000 mg/kg bw(10).(b)Dicyclohexylamine (DCH)A single oral gavage dose of DCH at 0.160.30 mL/kg bw administered to a group of rats(number not reported)caused tonic convulsions,sedation and deterioration of the general condition in all animals;doses 0.18 mL/kg bw caused death within 3

236、hours to up to 2 days.The LD50 was 0.22 mL/kg bw,equivalent to 200 mg/kg bw(8).In another study,158316 mg/kg bw DCH were administered once to five rats,which were observed for 14 days.Decreased appetite and activity,rapid increase in weakness,collapse and death were observed at doses 200 mg/kg bw,wi

237、th an LD50 of 240 mg/kg bw.Gross autopsy of the dead animals revealed haemorrhagic areas in the lungs,discolouration of the liver and acute gastroenteritis;the internal organs of survivors were normal(8).2.2.2 Short-term toxicity(a)FumagillinIn an unpublished study that conformed to GLP(11),fumagill

238、in DCH salt at a dose of 0,0.5,2,8 or 32 mg/kg bw per day(equivalent to doses of fumagillin of 0,0.36,1.44,5.76 and 23 mg/kg bw per day,respectively)was administered in 0.5%methyl cellulose in distilled water by gavage to Sprague Dawley rats(five males and five females per dose)for 2 weeks.Assessmen

239、t of toxicity was based on clinical signs,deaths,body weights,food and water consumption,ophthalmological examination,urinalysis,haematological testing,clinical biochemistry testing,organ weights and necropsy findings.There were no deaths and no clinical signs related to the test article.Significant

240、,doseresponse related decreases in body weights and weight gain were observed for both sexes at the two highest doses.Mean body weight had decreased by 9.3%and 17.4%in males and by 10.7%and 16.8%in females at 5.76 and 23 mg/kg bw per day,respectively,by day 14 as compared with the vehicle control gr

241、oup.In the groups given 5.76 and 23 mg/kg bw per day,body weight gain was significantly lower in males(24.7%and 46.2%)and in females(41.3%and 65.1%at the two doses).Significant decreases in food and water 29Fumagillin dicyclohexylamineconsumption were observed on day 13 in males at 23 mg/kg bw per d

242、ay.The clinical chemistry changes were slight and not dose-dependent,with the exception of alkaline phosphatase(ALP),which decreased by up to 33%at the highest dose.As there were no histopathological changes in the liver or in other serum liver enzymes,the changes in ALP were considered to be of no

243、toxicological significance.Significant decreases in the absolute weight of the spleen were found in both sexes at doses 5.76 mg/kg bw per day.Significant decreases in the absolute weight of the pituitary gland were observed in males at doses 5.76 mg/kg bw per day,with a non-significant decrease in f

244、emales at the highest dose.Non-significant decreases in the absolute weights of the prostate gland and of both ovaries were observed at 23 mg/kg bw per day.No treatment-related changes were found at necropsy.The NOAEL in this study was 1.44 mg/kg bw per day,based on significant decreases in body wei

245、ght gain in both sexes at 5.76 mg/kg bw per day In another unpublished GLP-compliant study(12),fumagillin DCH salt at a dose of 0,2.4,6 or 15 mg/kg bw per day(equivalent to fumagillin at 0,1.73,4.32 and 10.8 mg/kg bw per day,respectively)was administered by oral gavage in 0.5%methyl cellulose in wat

246、er to groups of 10 male and 10 female Sprague Dawley rats for 13 weeks.Toxicity was assessed from clinical signs,deaths,body weights,food and water consumption,ophthalmological examination,urinalysis,haematological tests,clinical biochemistry tests,organ weights and findings at necropsy.There were n

247、o deaths.Slightly soft stools and overgrown teeth were observed in a few animals of each sex at 10.8 mg/kg bw per day,which were outside the range of historical control data.Dose-related decreases in body weights and weight gain were observed in both sexes in all treated groups.Mean absolute body we

248、ights were significantly lowered on day 91,by 11.7%,11.6%and 23.1%in males and by 7.5%,12.2%and 15.5%in females at 1.73,4.32 and 10.8 mg/kg bw per day,respectively,as compared with the vehicle control group.Food consumption was significantly reduced in all treated groups,but water consumption was un

249、changed.Changes were observed in a number of haematological and clinical biochemistry parameters at doses 1.73 mg/kg bw per day,which were considered not toxicologically relevant.Significant decreases in the absolute,but not the relative,weights of some organs were found in both males and females(Ta

250、ble 2).A significant increase in the relative weight of the liver was observed only in females at 10.8 mg/kg bw per day.Necropsy of teeth from animals of each sex at 10.8 mg/kg bw per day showed thick periodontal membrane,fracture,hyperplasia of the gingival epithelium or hyperplasia of cementoblast

251、s.The incidence of fatty change in 30Toxicological evaluation of certain veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives Series,No.89,2025liver in females at doses 4.32 mg/kg bw per day was significantly higher than in the vehicle controls.Degeneration or atrophy of the testis

252、,reduced sperm and cell debris in the lumen of the epididymis,and atrophy in the seminal vesicle and coagulating gland were observed in males at 10.8 mg/kg bw per day.The incidence of lesions in the epididymis was significantly increased over that in the vehicle control group.The NOAEL for fumagilli

253、n was 1.73 mg/kg bw per day,based on decreased body weight gain at 4.32 mg/kg bw per day,with changes in organ weights and liver fat.Table 2Absolute and relative organ weights of Sprague Dawley rats treated with fumagillin DCHDose(mg/kg bw per day)Weight:absolute01.734.3210.8 relative to body weight

254、MalesTerminal body weight(g)Mean522.03462.75*459.63*397.99*Standard deviation17.9138.8346.7026.12Adrenal gland,left Absolute weight(g)Mean0.02740.02680.02750.0271Standard deviation0.00260.00380.00690.0027Adrenal gland,left Relative weight(%)Mean0.00530.00580.00590.0068*Standard deviation0.00060.0007

255、0.00100.0008Adrenal gland,right Absolute weight(g)Mean0.02570.02610.02750.0267Standard deviation0.00340.00350.00650.0021Adrenal gland,right Relative weight(%)Mean0.00490.00560.0059*0.0067*Standard deviation0.00070.00050.00080.0007Pituitary gland,Absolute weight(g)Mean0.01400.01320.01330.0120*Standar

256、d deviation0.00170.00120.00090.0014Pituitary gland,Relative weight(%)Mean0.00270.00290.00290.0030Standard deviation0.00030.00030.00030.0003Thymus,Absolute weight(g)Mean0.36110.39190.39330.3452Standard deviation0.05710.07500.07310.0996Thymus,Relative weight(%)Mean0.06950.08460.08610.0868Standard devi

257、ation0.01280.01390.01740.0242Prostate gland,Absolute weight(g)Mean0.77340.71800.5860*0.5290*Standard deviation0.15530.1422 0.13420.1001Prostate gland,Relative weight(%)Mean0.14860.15640.12810.1323Standard deviation0.03160.03470.03140.0195Testis,left Absolute weight(g)Mean2.15362.02972.23021.9131Stan

258、dard deviation0.22080.25220.13320.3230Testis,left Relative weight(%)Mean0.41270.43870.4897*0.4797*Standard deviation0.04200.04450.05850.0687Testis,right Absolute weight(g)Mean2.13762.06232.17701.8874Standard deviation0.22240.26920.09840.298931Fumagillin dicyclohexylamineTestis,right Relative weight(

259、%)Mean0.41010.44580.4782*0.4735*Standard deviation0.04650.04930.05490.0643Epididymis,left Absolute weight(g)Mean0.76310.71650.71020.5370*Standard deviation0.04630.13220.06760.0804Epididymis,left Relative weight(%)Mean0.14630.15440.15620.1349Standard deviation0.00940.02330.02400.0177Epididymis,right

260、Absolute weight(g)Mean0.76530.72100.72630.5410*Standard deviation0.06350.13010.05360.0830Epididymis,right Relative weight(%)Mean0.14690.15540.15990.1358Standard deviation0.01380.02360.02340.0173Spleen,Absolute weight(g)Mean0.96070.7913*0.87700.8015*Standard deviation0.13730.07000.07510.0735Spleen,Re

261、lative weight(%)Mean0.18450.17160.17160.2017Standard deviation0.02910.01640.01640.0180Kidney,left Absolute weight(g)Mean1.54291.4065*1.3807*1.3462*Standard deviation0.12120.08910.15660.0903Kidney,left Relative weight(%)Mean0.29580.30580.30040.3386*Standard deviation0.02380.03230.01580.0169Kidney,rig

262、ht Absolute weight(g)Mean1.54641.4193*1.3945 1.3351*Standard deviation0.08180.0938*0.15740.0836Kidney,right Relative weight(%)Mean0.29640.30810.30360.3358*Standard deviation0.01560.02730.02090.0139Heart,Absolute weight(g)Mean1.54101.46801.47331.2979*Standard deviation0.11160.07790.12250.1012Heart,Re

263、lative weight(%)Mean0.29530.3186*0.3216*0.3262*Standard deviation0.02040.02340.02100.0151Lung,Absolute weight(g)Mean1.89241.7335*1.6951*1.6224*Standard deviation0.09260.12460.10450.1451Lung,Relative weight(%)Mean0.36280.37600.37090.4077*Standard deviation0.01880.03000.02980.0268Brain,Absolute weight

264、(g)Mean2.20722.21392.23312.1453Standard deviation0.10080.08580.16160.0854Brain,Relative weight(%)Mean0.42310.4808*0.4887*0.5409*Standard deviation0.02120.03520.04490.0377Liver,Absolute weight(g)Mean14.517112.0953*12.8262*11.3587*Standard deviation0.95470.89581.38331.0789Liver,Relative weight(%)Mean2

265、.78182.6170*2.78952.8531Standard deviation0.17240.09570.06450.1808Thyroid with parathyroid gland,left Absolute weight(g)Mean0.01060.01010.01080.0089Standard deviation0.00140.00150.00290.0014Thyroid with parathyroid gland,left Relative weight(%)Mean0.00200.00220.00240.0022Standard deviation0.00030.00

266、040.00070.0004Thyroid with parathyroid gland,right Absolute weight(g)Mean0.01170.01000.01070.0088*Standard deviation0.00110.00130.00260.0016Thyroid with parathyroid gland,right Relative weight(%)Mean0.00220.00220.00240.0022Standard deviation0.00020.00040.00070.000432Toxicological evaluation of certa

267、in veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives Series,No.89,2025FemalesTerminal body weight(g)Mean274.16251.93*240.14*230.40*Standard deviation22.3517.8913.2016.35Ovary,left Absolute weight(g)Mean0.04680.04140.0347*0.0344*Standard deviation0.00960.00500.00380.0034Ovary,lef

268、t Relative weight(%)Mean0.01720.01650.01440.0150Standard deviation0.00380.00230.00100.0017Ovary,right Absolute weight(g)Mean0.04970.0417*0.0370*0.0356*Standard deviation0.00770.00630.00740.0064Ovary,right Relative weight(%)Mean0.01830.01660.01540.0156Standard deviation0.00350.00260.00290.0030Adrenal

269、 gland,left Absolute weight(g)Mean0.01560.03300.0321*0.0318*Standard deviation0.00300.00330.00500.0022Adrenal gland,left Relative weight(%)Mean0.01320.01320.01340.0139Standard deviation0.00140.00170.00190.0016Adrenal gland,right Absolute weight(g)Mean0.03470.03170.03160.0306Standard deviation0.00410

270、.00360.00470.0020Adrenal gland,right Relative weight(%)Mean0.01270.01260.01320.0133Standard deviation0.00120.00140.00180.0012Pituitary gland,Absolute weight(g)Mean0.01680.01670.0147*0.0141*Standard deviation0.00150.00110.00190.0024Pituitary gland,Relative weight(%)Mean0.00620.00660.00610.0061Standar

271、d deviation0.00080.00060.00100.0011Thymus,Absolute weight(g)Mean0.25710.29910.29630.2746Standard deviation0.04200.06260.06630.0752Thymus,Relative weight(%)Mean0.09410.11850.12310.1203Standard deviation0.01610.02260.02430.0381Uterus and cervix,Absolute weight(g)Mean0.73091.00940.71380.8348Standard de

272、viation0.19620.41040.16110.2913Uterus and cervix,Relative weight(%)Mean0.26630.4044*0.29670.3582Standard deviation0.06810.16520.06080.1079Spleen,Absolute weight(g)Mean0.60570.56800.5317*0.4968*Standard deviation0.03810.05320.07480.0614Spleen,Relative weight(%)Mean0.22220.22570.22180.2159Standard dev

273、iation0.02320.01740.03100.0242Kidney,left Absolute weight(g)Mean0.83470.78580.75910.7819Standard deviation0.08190.05350.06000.0426Kidney,left Relative weight(%)Mean0.30440.31250.31660.3400*Standard deviation0.01700.01810.02560.0176Kidney,right Absolute weight(g)Mean0.83750.78790.77370.7981Standard d

274、eviation0.08190.06000.04420.0379Kidney,right Relative weight(%)Mean0.30560.31320.32280.3477*Standard deviation0.02030.01910.02000.0260Heart,Absolute weight(g)Mean0.92750.89140.87590.8573*Standard deviation0.06220.05510.03030.042633Fumagillin dicyclohexylamineHeart,Relative weight(%)Mean0.33890.35450

275、.3656*0.3737*Standard deviation0.01650.01900.02130.0323Lung,Absolute weight(g)Mean1.38901.2487*1.1973*1.2344*Standard deviation0.10530.14690.11330.1041Lung,Relative weight(%)Mean0.50760.49560.49860.5366Standard deviation0.02930.04630.03930.0397Brain,Absolute weight(g)Mean2.04642.04111.95041.9745Stan

276、dard deviation0.13410.10050.06690.0688Brain,Relative weight(%)Mean0.74840.8126*0.8145*0.8611*Standard deviation0.04520.05090.05400.0714Liver,Absolute weight(g)Mean6.90257.04606.64057.0663Standard deviation1.06210.40500.37530.5116Liver,Relative weight(%)Mean2.52372.80592.77043.0733*Standard deviation

277、0.38540.20500.17990.2139Thyroid with parathyroid gland,left Absolute weight(g)Mean0.00820.00770.0068*0.0069Standard deviation0.00120.00150.00090.0010Thyroid with parathyroid gland,left Relative weight(%)Mean0.00300.00310.00280.0030Standard deviation0.00050.00060.00040.0004Thyroid with parathyroid gl

278、and,right Absolute weight(g)Mean0.00830.00860.0068*0.0070Standard deviation0.00180.00110.00100.0012Thyroid with parathyroid gland,right Relative weight(%)Mean0.00310.00340.00280.0030Standard deviation0.00070.00050.00040.0005Statistically significant at:*p=0.05;*p=0.01;*p=0.001(b)Dicyclohexylamine (D

279、CH)In a study reported in Japanese with an abstract and summary tables in English(8,13),male and female CD rats were treated orally according to the Japanese guidelines for testing the toxicity of chemicals at 0,20,70 or 200 mg/kg bw per day DCH in corn oil for 28 days.Additional groups of rats in t

280、he control and highest dose groups were kept for a 14-day recovery period.Eight of 13 rats of each sex showed clinical signs and died after receiving the fourth administration of the highest dose.Myocardial degeneration was observed in one male that died,but no histopathological changes that would e

281、xplain the cause of death were observed in the other rats that died.Clinical signs such as salivation and convulsions were observed in male and female rats treated at doses 70 mg/kg bw per day;however,the signs disappeared during the recovery period.Body weight and food intake were significantly red

282、uced in male and female rats that received 200 mg/kg bw per day.The body weights at the end of treatment were 285 g in male and 184 g in female treated rats and 355 g and 232 g in male and female controls,respectively.Weight loss persisted until the end of the recovery period(male/female:333 g/236 g

283、 vs 399.9 g/266 g in controls),but food intake had recovered by the end of the study.No statistical analysis was performed on the results of urinalysis,haematology,clinical chemistry and 34Toxicological evaluation of certain veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives Seri

284、es,No.89,2025organ weights at 200 mg/kg bw per day.Increased leukocyte counts were seen in females at the highest dose at the end of treatment(11 300/mm vs 4000/mm in controls).Clinical chemistry results at the end of treatment showed increased inorganic phosphorus and calcium concentrations in rats

285、 treated at 200 mg/kg bw per day in both males and females.Values in treated and control rats respectively for phosphorus were:males 9.2/9.0 mg/dL,females 10.5/6.2 mg/dL.For calcium the values for treated and control rats were:males 9.4/9.0 mg/dL,females 10.7/8.8 mg/dL).Urinalysis revealed no signif

286、icant findings.Adrenal weights were elevated in male and female animals at 200 mg/kg bw per day(absolute/relative,treated rats vs controls:males:62.5 mg/0.220 mg/g vs 47.3 mg/0.134 mg/g;females:88.2 mg/0.478 mg/g vs 61.0 mg/0.263 mg/g),ovarian weights were significantly reduced in female rats at 70

287、mg/kg bw per day(79.6 mg/0.357 mg/g)or 200 mg/kg bw per day(59.9 mg/0.325 mg/g)as compared with controls(98.2 mg/0.425 mg/g).No histopathological correlation was observed.The NOAEL in this 28-day repeated-dose study of DCH in rats was 20 mg/kg bw per day based on clinical signs and a significant dec

288、rease in ovarian weight,without histopathological changes,at 70 mg/kg bw per day.In a GLP-compliant study reported by the European Chemicals Agency(14),Wistar rats(78 weeks of age)were given DCH in olive oil by gavage at 0,10,30 or 90 mg/kg bw per day for 90 days.Additional satellite groups were use

289、d to assess recovery,but details were not available.DCH markedly changed the clinical condition of the animals,inducing mainly convulsions and pronounced salivation in both sexes at the highest dose.At 30 mg/kg bw per day,these signs were seen only sporadically,and only salivation was observed in ma

290、les at the lowest dose.All clinical signs disappeared after administration ceased.Haematological analysis showed decreased activated partial thromboplastin time at the highest and intermediate doses and decreased platelet counts,but only at the lowest dose in males;in females,haematocrit values incr

291、eased in all treatment groups,while activated prothrombin time decreased,and total red blood cell count and haemoglobin values increased at the highest dose.These changes were reversible,although the activated prothrombin time in females remained decreased after the recovery period.In biochemical te

292、sts,the following changes were detected:in males,sodium ion concentrations decreased at all doses,while there was a delayed increase in sodium ion value and an increase in total protein values at the lowest and intermediate doses.At the highest dose,there was a delayed decrease in total protein valu

293、es,an increase in bilirubin values,a decrease in aspartate transaminase(AST)and increases in total protein,albumin and sodium ion values in females.The absolute and relative weights of the thymus decreased in males at the highest dose,and the absolute and relative weights of the adrenals increased a

294、t the highest dose in both sexes.Males at the intermediate and highest doses 35Fumagillin dicyclohexylamineshowed a statistically significant increase in relative liver weights.Urine specific gravity increased in a dose-dependent manner in animals of each sex at the intermediate and highest doses.Ch

295、anges in urine specific gravity were reversible and were considered to be of no toxicological significance.Histopathology of the liver in males at the mid and high doses revealed an increased incidence of vacuolation of fatty droplets.These reversible changes were considered to reflect an adaptive r

296、esponse.Histopathology also revealed oedema in the submucosa of the stomach of males at all doses.The microscopic changes observed in the stomach were probably due to contact with the test substance,but they were not considered toxicologically significant.No histopathological changes were observed i

297、n the adrenals.The NOAEL was 10 mg/kg bw per day based on haematological and clinical chemistry changes at 30 mg/kg bw per day.2.2.3 Long-term toxicity(a)FumagillinNo data were submitted.(b)Dicyclohexylamine (DCH)Studies by Pliss(15),evaluated in volume 22 of the IARC Monographs on the Carcinogenic

298、Risk of Chemicals to Humans(16),on the long-term toxicity of DCH administered orally,subcutaneously and/or intramuscularly were described with minimal detail and were not useful for risk assessment.They are described here for completeness.MouseDCH(chemically pure)in a 2.6%solution in sunflower oil w

299、as administered daily by subcutaneous injection to groups of 22 male and 35 female strain D mice(CC57 white crossed with C57 black)at a dose of 0.05 mL for 1112.5 months(total dose,60.179.3 mg).Four of the 15 mice(sex unspecified)that survived for 12 months developed sarcomas at the site of injectio

300、n(15).(The IARC Working Group noted the absence of solvent-treated controls.)DCH nitrite(chemically pure)was administered daily by subcutaneous injection to groups of 31 male and 23 female strain 0 mice at a dose of 0.1 mL of a 1%aqueous solution for 1213 months.Tumours were observed in five of the

301、23 mice(sex unspecified)that survived for 12 months,comprising two hepatocellular adenomas,one papillary cystadenoma of the lung,one papillary adenoma of the lung and one cavernous haemangioma of the liver(15).(The IARC Working Group noted the absence of solvent-treated controls.)36Toxicological eva

302、luation of certain veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives Series,No.89,2025RatDCH nitrite was administered orally to 17 male and 13 female rats(strain unspecified),daily,at a dose of 1 mL in a 3%aqueous solution on 6 days a week for 12 months(total dose,9180 mg).After

303、 17 months,one rat had a mesenteric sarcoma(15).(The IARC Working Group noted the lack of concurrent controls.)DCH nitrite was administered subcutaneously to 34 male and 22 female rats(strain unspecified)at 0.5 mL of a 2%aqueous solution weekly.Seven of 31 rats(sex unspecified)that lived 12 months d

304、eveloped tumours at various sites(15).(The IARC Working Group noted the inadequacy of the reporting of data and the lack of appropriate controls.)Groups of 25 male and 25 female rats(strain unspecified)were given multiple subcutaneous injections of 30 mg DCH for 8 weeks,when necrosis occurred at the

305、 injection site.Administration was therefore continued by feeding the rats six times a week for 52 weeks with a diet containing DCH in 0.5 mL of a 5%solution of sunflower oil(total dose,8875 mg).One hepatic neoplasm was found after 84 weeks and one sarcoma of the omentum after 90 weeks(15).(The IARC

306、 Working Group noted the inadequacy of the reporting of data and the lack of appropriate controls.)2.2.4 Genotoxicity(a)FumagillinIn vitroIn an unpublished study conducted according to GLP(17),fumagillin DCH was evaluated for its potential to induce reverse mutation in four histidine auxotroph strai

307、ns of Salmonella typhimurium,TA100,TA1535,TA98 and TA1537,and a tryptophan auxotroph strain of Escherichia coli,WP2 uvrA,both in the presence and absence of an exogenous metabolic activation system(hepatic S9 from Aroclor 1254-treated male Sprague Dawley rats)at 0,50,150,500,1500 and 2000 g/plate.Th

308、e test article was suspended in dimethyl sulfoxide.No increase in the number of revertants per plate was observed in any of the test strains at any dose.No cytotoxicity was observed,in the presence or absence of a metabolic activation system.The mean number of revertants in the positive control for

309、each test strain was clearly higher than that in the negative control.The results indicate that fumagillin DCH is not mutagenic in bacteria.In a study that did not comply with GLP,the clastogenicity of fumagillin DCH was tested at concentrations of 0.34,0.68,1.02,3.07 and 9.20 g/mL of fumagillin DCH

310、 dissolved in 1:1 water:sugar syrup and incubated with cultured peripheral blood lymphocytes isolated from five male subjects.The mitotic index,proliferative index and nuclear division index were calculated to assess cytotoxicity(18).37Fumagillin dicyclohexylamineAll concentrations of fumagillin DCH

311、 tested significantly increased the frequency of sister chromatid exchange per cell;however,the increase was more than twice that of the negative control only at the highest concentration,at which there was 50%reduction in mitotic index.Clear clastogenicity was therefore observed only at the highest

312、 concentration(9.20 g/mL).Significant increases over the negative control in the frequency of structural chromosomal aberration were observed at the three higher concentrations.Furthermore,the three highest test concentrations increased micronucleus formation and significantly decreased the mitotic,

313、proliferative and nuclear division indexes as compared with the negative control.The results indicate that fumagillin has cytotoxic and clastogenic effects on cultured human lymphocytes.The Committee considered these conclusions unreliable,primarily because the biological significance of the observe

314、d increases cannot be evaluated in the absence of historical control data for negative controls.In addition,the conclusions were weakened by several methodological shortcomings:the absence of a clear rationale for selection of dose levels;misuse of cytochalasin B in the micronucleus test and of 5-br

315、omo-2-uridine(5-BrdU)in the sister chromatid exchange test;parallel use of five different donors,with pooling of data in the absence of an appropriate statistical analysis;and treatments performed only in the absence of S9 metabolic activation.The rationale for selection of solvent and consequent id

316、entification of the highest dose for treatment was questionable because of the limited solubility of the test compound in an aqueous medium.The in vitro clastogenic potential of fumagillin DCH in human lymphocytes therefore cannot be supported.In an unpublished,GLP-compliant study(19),fumagillin DCH

317、 was evaluated for its potential to induce structural and numerical chromosomal aberrations in Chinese hamster lung cells in the presence or absence of an exogenous metabolic activation system(hepatic S9 from Aroclor 1254-treated male SD rats).The test article was suspended in dimethyl sulfoxide.The

318、 frequency of metaphases with structural chromosomal aberrations was increased in a dose-dependent manner,although the increase was statistically significantly only at 600 g/mL after 6 hours of treatment with metabolic activation,and the maximum increment was above the historical control range.Never

319、theless,cytotoxicity(relative increase in cell count,43%)was also observed at the same dose.Without metabolic activation,6-hour treatment at 80,160,300 or 320 g/mL or 24 hour treatment at 37.5,75,130 or 150 g/mL fumagillin DCH did not induce any damage in chromosomes.The study author concluded that

320、fumagillin DCH has no clastogenic activity.The Committee agreed with this conclusion and noted that,at the next lower dose(550 g/mL),although the frequency of aberrant cells was also outside 38Toxicological evaluation of certain veterinary drug residues in food Ninety-eighth JECFAWHO Food Additives

321、Series,No.89,2025the range of historical control data,it was not statistically significantly different from the concurrent negative control.There was,however,substantial cytotoxicity(50%reduction in the relative increase in cell count).In vivoIn a study that did not comply with GLP,fumagillin DCH in

322、 50%sugar solution was administered to white mice by gavage at a dose of 10,15 or 20 mg/kg bw per day for 7 days.Fumagillin at 15 and 20 mg/kg bw induced a significant increase in the frequency of micronuclei(24.60 2.37 per 1000 cells and 53.00 4.59,per 1000 cells respectively)as compared with the n

323、egative control(8.20 1.39 per 1000 cells)(21).This study could not be evaluated properly due to the limited reporting,which obviated an adequate assessment of the method used and the results.In a study that did not comply with GLP,groups of 6-week-old BALB/c mice were treated orally with fumagillin

324、DCH in 1:1 watersugar syrup at a dose of 25,50 or 75 mg/kg bw at 24-hour intervals for 7 days(18).Significant reductions in the mitotic index were seen at all three doses(3.47 0.04%,3.17 0.01%and 2.27 0.02%,respectively)as compared with the negative control(6.00 0.01%).Fumagillin DCH significantly i

325、ncreased the frequency of micronuclei(4.98 0.35%,8.45 0.57%and 12.02 0.37%,respectively)over that in the negative control(1.04 0.28%).The frequency of numerical chromosomal aberrations(aneuploidy and ploidy)and structural chromosomal aberrations was significantly increased at the highest dose as com

326、pared with negative controls.These results suggest that fumagillin is genotoxic(clastogenic)in vivo.The Committee concluded that the study is not suitable for risk assessment because of major methodological shortcomings.The biological significance of the results cannot be assessed in the absence of

327、historical negative control values.The rationale for selection of doses is questionable,as they were based on the therapeutic dose range of fumagillin used in beekeeping and not on appropriate toxicity or cytotoxicity parameters.The time at which bone-marrow cells were assessed after the last admini

328、stration was not indicated.Justification was not provided for use of animals of each sex in the micronucleus test and of only male animals in the chromosomal aberration test.Colchicine,which should be used to provide evidence of,and accumulate,metaphases,apparently was not administered.The results w

329、ere expressed as the total number of aberrations per experiment rather than as the number of cells with aberrations per animal.Cytogenetic analysis was performed with the G-banding technique for identifying specific chromosomal aberration(e.g.translocations,insertions),which is not appropriate for a

330、ssessing general clastogenic effects.The induction of more of the relatively rare Robertsonian translocations than of the 39Fumagillin dicyclohexylaminemore common chromosomal aberrations,such as breaks,appears to be unlikely.The number of cells scored in the micronucleus test was insufficient,and l

331、imited reporting of data obviates a reliable evaluation of the results.In a study that did not comply with GLP,6-month-old male BALB/c mice were treated with fumagillin DCH in watersugar syrup at a dose of 5,10 or 20 mg/kg bw by gastric intubation,once daily for 7 days.Watersugar syrup was used as a

332、 negative control and cyclophosphamide(15 mg/kg bw)as a positive control(23).The frequency of numerical chromosomal aberrations(aneuploidy and ploidy)was significantly increased at both 10 and 20 mg/kg bw per day.Structural chromosomal aberrations were significantly more frequent than in negative co

333、ntrols only at the highest dose.The results indicate that fumagillin is genotoxic(clastogenic)in vivo at concentrations of 10 and 20 mg/kg bw per day.The Committee considered that the study was not adequate for appraisal,as the data reported were similar to the those of Stanimirovic et al.(22),at least those for aneuploidies,polyploidies,gaps and acentrics/breaks,despite the different doses of fum