Residue evaluation of certain veterinary drugs: Joint FAO/WHO Expert Committee on Food Additives, ninety-eighth meeting 20–29 February 2024

Joint FAO/WHO Expert Committee on Food Additives Ninety-eighth Meeting Rome, 20–29 February 2024 33 ISSN 1817-7077 FA O J E C FA M o n o g r a p h s RESIDUE EVALUATION OF CERTAIN VETERINARY DRUGS

FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS WORLD HEALTH ORGANIZATION Rome, 2024 Joint FAO/WHO Expert Committee on Food Additives Ninety-eighth Meeting Rome, 20–29 February 2024 RESIDUE EVALUATION OF CERTAIN VETERINARY DRUGS 33 ISSN 1817-7077 FA O J E C FA M o n o g r a p h s Required citation: FAO & WHO. 2024. Residue evaluation of certain veterinary drugs – Joint FAO/WHO Expert Committee on Food Additives, 98th Meeting 20–29 February 2024. Joint FAO/WHO Expert Committee on Food Additives (JECFA) Monographs, No. 33. Rome. https://doi.org/10.4060/cd2487en The designations employed and the presentation of material in this information product do not imply the expression of any opinion whatsoever on the part of the Food and Agriculture Organization of the United Nations (FAO) or World Health Organization (WHO) concerning the legal or development status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. 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Cover photo: ©FAO/Karen Minasyan iii SPECIAL NOTE While the greatest care has been exercised in the preparation of this information, FAO expressly disclaims any liability to users of these procedures for consequential damages of any kind arising out of, or connected with, their use. iv Contents List of participants ......................................................................................................................v Abbreviations ........................................................................................................................... vii Introduction .................................................................................................................................1 Clopidol.......................................................................................................................................6 Fumagillin dicyclohexylamine ..................................................................................................40 Imidacloprid (fin fish) - Addendum ..........................................................................................83 Annex 1 – Summary of recommendations from the 98th JECFA on compounds on the agenda and further information required ..................................................................................89 Annex 2 – Summary of JECFA evaluations oof veterinary drug residues from the 32nd meeting to the present ...............................................................................................................93 Use of JECFA reports and evaluations by registration authorities Most of the evaluations and summaries contained in this publication are based on unpublished proprietary data submitted to JECFA for use when making its assessment. A registration authority should not consider granting a registration based on an evaluation published herein unless it has first received authorization for such use from the owner of the data or any second party that has received permission from the owner for using the data. vList of participants Ninety-eighth Meeting of the Joint FAO/WHO Expert Committee on Food Additives Rome, 20–29 February 2024 Members Professor (Emeritus) Alan R. Boobis National Heart and Lung Institute, Imperial College London, London, United Kingdom of Great Britain and Northern Ireland (Vice-Chairperson) Dr Alan Chicoine Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada (Chairperson) Mr Peter Cressey Senior Scientist, Institute of Environmental Science and Research Limited, Christchurch Science Centre, Christchurch, New Zealand Dr Holly Erdely Residue Chemistry Team, Division of Human Food Safety, Center for Veterinary Medicine, United States Food and Drug Administration, Rockville, Maryland, United States of America (FAO Rapporteur) Professor Silvana Lima Górniak Department of Pathology, School of Veterinary Medicine and Animal Sciences, University of São Paulo, São Paulo, Brazil Professor Angelo Moretto Department of Cardiac, Thoracic, Vascular and Public Health Sciences, University of Padua, Padua, Italy (WHO Rapporteur) Professor Susanne Rath University of Campinas, Department of Analytical Chemistry, São Paulo, Brazil Dr Rainer Reuss Safe Work Australia, Canberra, Australia Experts Dr Anke Finnah German Federal Office of Consumer Protection and Food Safety, Berlin, Germany Mr Samuel Fletcher United Kingdom Veterinary Medicines Directorate, Addlestone, Surrey, United Kingdom of Great Britain and Northern Ireland Dr Amy-Lynn Hall Residue Chemistry Team, Division of Human Food Safety, Center for Veterinary Medicine, Food and Drug Administration, Rockville (MD), United States of America vi Professor Lingli Huang College of Veterinary Medicine, Huazhong Agricultural University, Wuhan City, China Dr Mayumi Ishizuka Laboratory of Toxicology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Japan Dr Anne-Marie Jaques Agency for Veterinary Medicinal Products, National Agency for Food, Environmental and Occupational Health and Safety, Fougères, France Dr Hui-Seung Kang Ministry of Food and Drug Safety, Chungcheongbuk-do, Republic of Korea Dr Cheetham Lawrence Mingle Food and Drugs Authority Ghana, Accra, Ghana Dr Silvia A. Piñeiro Center for Veterinary Medicine, United States Food and Drug Administration, Rockville, Maryland, United States of America Ms Tina Zuidema Wageningen Food Safety Research, Wageningen, Netherlands (Kingdom of the) Secretariat Dr Vittorio Fattori Agrifood Systems and Food Safety Division, Food and Agriculture Organization of the United Nations (FAO), Rome, Italy (FAO JECFA Secretariat) Ms Elisabeth Heseltine France (WHO editor) Ms Ngai Yin Ho Department of Nutrition and Food Safety, World Health Organization (WHO), Geneva, Switzerland (WHO consultant) Dr Markus Lipp Agrifood Systems and Food Safety Division, FAO, Rome, Italy (FAO Secretariat) Mr Soren Madsen Department of Nutrition and Food Safety, WHO, Geneva, Switzerland (WHO JECFA Secretariat) Dr Keya Mukherjee Agrifood Systems and Food Safety Division, FAO, Rome, Italy (FAO Secretariat) Dr Magdalena Niegowska Conforti Agrifood Systems and Food Safety Division, FAO, Rome, Italy (FAO Secretariat) vii Abbreviations ADI acceptable daily intake ARfD acute reference dose bw body weight CAS Chemical Abstracts Services CCRVDF Codex Committee on Residues of Veterinary Drugs in Foods CIFOCOss Chronic Individual Food Consumption database summary statistics CMC carboxymethyl cellulose CV coefficient of variation DAD photodiode array detector DCH dicyclohexylamine DD degree-days DMSO dimethylsulfoxide dSPE dispersive solid phase extraction ELISA enzyme-linked immunosorbent assay ESI+ positive mode electrospray ionization EU European Union FAO Food and Agriculture Organization of the United Nations equiv equivalent GC gas chromatography GCB graphitized carbon black GEADE global estimated acute dietary exposure GECDE global estimated chronic dietary exposure GLP Good Laboratory Practice GVP good veterinary practice HBGV health-based guidance value HILIC hydrophilic interaction liquid chromatography HPLC high-performance liquid chromatography HRP highest reliable percentile INN International Non-Proprietary Names IUPAC International Union of Pure and Applied Chemists JECFA Joint Expert Committee on Food Additives LC-DAD-MS liquid chromatography - diode array detector - mass spectrometry LCGA lateral-flow colloidal gold immunoassay viii LC-HRMS liquid chromatography-high resolution mass spectrometry LC-MS liquid chromatography-mass spectrometry LC-MS/MS liquid chromatography with tandem mass spectrometry LLOQ lower limit of quantification LOD limit of detection LOQ limit of quantification LPS large portion size LSC liquid scintillation counting mADI microbiological acceptable daily intake MAPA Brazilian Ministry of Agriculture, Livestock and Food Supply: Ministério da Agricultura e PecuáriaMetAP-2 type-2 methionine aminopeptidase mARfD microbiological acceptable reference dose MIP-SPCE molecularly imprinted polypyrrole polymer modified screen printed carbon electrode MR marker residue MRL maximum residue limit MRM multiple reaction monitoring MR:TR marker residue to total residue NMR nuclear magnetic resonance OECD Organization for Economic Co-operation and Development ppm parts per million PSA primary secondary amine QuEChERS Quick, Easy, Cheap, Effective, Rugged and Safe RASFF Rapid Alert System for Food and Feed SD standard deviation SPE solid phase extraction SRM selected reaction monitoring TAD total administered dose tADI toxicological acceptable daily intake tARfD toxicological acceptable reference dose TLC thin layer chromatography TR total residue TRA total radioactivity administered average TRR total radioactive residue USA United States of America ix UTL upper tolerance limit UV ultraviolet VICH International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products WHO World Health Organization

FAO JECFA Monograph 33 1 Introduction This volume of FAO JECFA Monographs contains residue evaluation of certain veterinary drugs prepared at the Ninety-eighth Meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA), held from 20 to 29 February 2024. This JECFA meeting was convened specifically to consider residues of veterinary drugs in food-producing animal species. The tasks for the Committee were to further elaborate principles for evaluating the safety of residues of veterinary drugs in food and for establishing acceptable daily intakes (ADIs) and/or acute reference doses (ARfDs), and to recommend maximum residue limits (MRLs) for substances on the agenda when they are administered to food-producing animals in accordance with good veterinary practice in the use of veterinary drugs. The enclosed monographs provided the scientific basis for the recommendations of MRLs. Background In response to the growing use of veterinary medicines in food animal production systems internationally and the potential implications for human health and fair-trading practices, a Joint FAO/WHO Expert Consultation on Residues of Veterinary Drugs was convened in Rome in November 1984 (FAO and WHO, 1985). One of the major recommendations of this consultation was the establishment of the Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF) and the periodic convening of an appropriate expert body to provide independent scientific advice to this Committee and to member countries of FAO and WHO. At its first session, in Washington, DC, in November 1986, the CCRVDF reaffirmed the need for such a scientific body and made several recommendations and suggestions to be considered by JECFA (FAO and WHO, 1986). In response to these recommendations, the 32nd JECFA meeting was devoted entirely to the evaluation of residues of veterinary drugs in food - a new responsibility for the Joint FAO/WHO Expert Committee on Food Additives. Ninety-eighth Meeting of JECFA The present volume contains monographs on the evaluations of residue data of three substances scheduled for evaluation at the Ninety-eighth Meeting of the Committee: clopidol, fumagillin dicyclohexylamine, and imidacloprid. The evaluation of an additional substance, ethoxyquin, was planned, but data was not submitted by the sponsor. An evaluation of ethoxyquin is therefore not presented in this monograph. The monographs are prepared in a uniform format consistent with the data provided and the specific request for risk assessment by CCRVDF. The format includes identity of substance, residues in food and their evaluation, metabolism studies, tissue residue depletion studies, methods of residue analysis, a final appraisal of the study results, and if appropriate, recommendations on MRLs. A summary of the recommendations on compounds on the agenda and further information required is included in Annex 1. In addition, a summary of JECFA evaluations of residues of veterinary drugs in foods from the 32nd meeting to the present Ninety-eighth Meeting can be found in Annex 2. The monographs of this volume must be considered in the context of the full report of the meeting, which will be published in the WHO Technical Report Series. On-line editions of residues of some veterinary drugs in animals and foods are available at https://www. fao.org/food-safety/resources/publications/en/, where JECFA documents can be found listed under categories. The database containing the most recent information on veterinary drugs and their residues in foods as evaluated by JECFA can be found at https://www.fao.org/food/food-safety-quality/scientific- advice/jecfa/jecfa-vetdrugs/en/. FAO JECFA Monograph 332 Contact and feedback More information on the work of the Committee is available from FAO at https://www.fao.org/food- safety/scientific-advice/en/. Please send questions and feedback to jecfa@fao.org. FAO JECFA Monograph 33 3 References and other sources FAO & WHO [Food and Agriculture Organization of the United Nations and World Health Organization]. 1985. Residues of Veterinary Drugs in Foods. Report of a Joint FAO/WHO Consultation, Rome, 29 October–5 November 1984. FAO Food and Nutrition Paper, No. 32. FAO & WHO. 1986. Report of the First Session of the Codex Committee on Residues of Veterinary Drugs in Foods (ALINORM 87/31). Washington, D.C., 27–31 October 1986. FAO & WHO. 2006. Updating the Principles and Methods of Risk Assessment: MRLs for Pesticides and Veterinary Drugs. Report of the FAO/RIVM/WHO Workshop: “Updating the Principles and Methods of Risk Assessment: Maximum Residue Levels (MRLs) for Pesticides and Veterinary Drugs” held in Bilthoven, the Kingdom of the Netherlands, 7–10 November 2005. FAO & WHO. 2012. Joint FAO/WHO Expert Meeting on Dietary Exposure Assessment Methodologies for Residues of Veterinary Drugs. Final report including report of stakeholder meeting. FAO & WHO. 2014. Report of the Twenty-second Session of the Codex Committee on Residues of Veterinary Drugs in Foods, San José, Costa Rica, 27 April–1 May 2015 (REP15/RVDF). JECFA [Joint FAO/WHO Expert Committee on Food Additives]. 1969. Specifications for the Identity and Purity of Food Additives and their Toxicological Evaluation: Some antibiotics (Twelfth Report of the Joint FAO/WHO Expert Committee on Food Additives). FAO Nutrition Meetings Report Series, No. 45; WHO Technical Report Series, No. 430. JECFA. 1982. Evaluation of Certain Food Additives and Contaminants (Twenty-sixth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 683. JECFA. 1983. Evaluation of Certain Food Additives and Contaminants (Twenty-seventh Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 696. JECFA. 1988. Evaluation of Certain Veterinary Drug Residues in Food (Thirty-second Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 763. JECFA. 1989. Evaluation of Certain Veterinary Drug Residues in Food (Thirty-fourth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 788. JECFA. 1990. Evaluation of Certain Veterinary Drug Residues in Food (Thirty-sixth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 799. 4 FAO JECFA Monograph 28 JECFA. 1991. Evaluation of Certain Veterinary Drug Residues in Food (Thirty-eighth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 815. JECFA. 1993. Evaluation of Certain Veterinary Drug Residues in Food (Fortieth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 832. JECFA. 1995. Evaluation of Certain Veterinary Drug Residues in Food (Forty-second Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 851. JECFA. 1995. Evaluation of Certain Veterinary Drug Residues in Food (Forty-third Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 855. JECFA. 1996. Evaluation of Certain Veterinary Drug Residues in Food (Forty-fifth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 864. FAO JECFA Monograph 334 JECFA. 1998. Evaluation of Certain Veterinary Drug Residues in Food (Forty-seventh Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 876. JECFA. 1998. Evaluation of Certain Veterinary Drug Residues in Food (Forty-eighth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 879. JECFA. 1999. Evaluation of Certain Veterinary Drug Residues in Food (Fiftieth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 888. JECFA. 2000. Evaluation of Certain Veterinary Drug Residues in Food (Fifty-second Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 893. JECFA. 2001. Evaluation of Certain Veterinary Drug Residues in Food (Fifty-fourth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 900. JECFA. 2002. Evaluation of Certain Veterinary Drug Residues in Food (Fifty-eighth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 911. JECFA. 2003. Evaluation of Certain Veterinary Drug Residues in Food (Sixtieth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 918. FAO JECFA Monograph 28 5 JECFA. 2004. Evaluation of Certain Veterinary Drug Residues in Food (Sixty-second Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 925. JECFA. 2006. Evaluation of Certain Veterinary Drug Residues in Food (Sixty-sixth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 939. JECFA. 2006. Residue Evaluation of Certain Veterinary Drugs. 66th Meeting 2006, Joint FAO/WHO Expert Committee on Food Additives, FAO JECFA Monographs, No. 2. JECFA. 2009. Evaluation of Certain Veterinary Drug Residues in Food (Seventieth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 954. JECFA. 2009. Residue Evaluation of Certain Veterinary Drugs. 70th Meeting 2008, Joint FAO/WHO Expert Committee on Food Additives, FAO JECFA Monographs, No. 6. JECFA. 2010. Residue Evaluation of Certain Veterinary Drugs. Meeting 2010, Evaluation of data on ractopamine residues in pig tissues. FAO JECFA Monographs, No. 9. JECFA. 2012. Evaluation of Certain Veterinary Drug Residues in Food (Seventy-fifth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 969. JECFA. 2012. Residue Evaluation of Certain Veterinary Drugs. 75th Meeting 2011, Joint FAO/WHO Expert Committee on Food Additives, FAO JECFA Monographs, No. 12. JECFA. 2014. Evaluation of Certain Veterinary Drug Residues in Food (Seventy-eighth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 988. JECFA. 2014. Residue Evaluation of Certain Veterinary Drugs. 78th Meeting 2013, Joint FAO/WHO Expert Committee on Food Additives, FAO JECFA Monographs, No. 15. JECFA. 2016. Evaluation of Certain Veterinary Drug Residues in Food (Eighty-first Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 997. JECFA. 2016. Residue Evaluation of Certain Veterinary Drugs. 81st Meeting 2015, Joint FAO/WHO Expert Committee on Food Additives, FAO JECFA Monographs, No. 18. FAO JECFA Monograph 33 5 JECFA. 2017. Residue Evaluation of Certain Veterinary Drugs. 85th Meeting 2017, Joint FAO/WHO Expert Committee on Food Additives, FAO JECFA Monographs, No. 21. JECFA. 2018. Evaluation of Certain Veterinary Drug Residues in Food (Eighty-fifth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 1008. JECFA. 2020. Residue Evaluation of Certain Veterinary Drugs. 88th Meeting 2019, Joint FAO/WHO Expert Committee on Food Additives, FAO JECFA Monographs, No. 24. JECFA. 2020. Evaluation Certain Veterinary Drug Residues in Food (Eighty-eighth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 1023. JECFA. 2022. Evaluation of Certain Veterinary Drug Residues in Food (Ninety-fourth Report of the Joint FAO/WHO Expert Committee on Food Additives). WHO Technical Report Series, No. 1041. JECFA. 2023. Residue Evaluation of Certain Veterinary Drugs. 94th Meeting 2022 (Virtual), Joint FAO/WHO Expert Committee on Food Additives, FAO JECFA Monographs, No. 28. FAO JECFA Monograph 336 Clopidol First draft prepared by Amy-Lynn Hall, Rockville, United States of America Samuel Fletcher, Norwich, United Kingdom of Great Britain and Northern Ireland Tina Zuidema, Wageningen, Kingdom of the Netherlands Peter Cressey, Christchurch, New Zealand and Susanne Rath, Campinas, Brazil Alan Chicoine, Saskatoon, Canada Rainer Reuss, Barton, Australia Identity International Non-proprietary Names (INN): Clopidol Synonyms: clopindol; 3,5-dichloro-2,6-dimethyl-1H-pyridin-4-ol; meticlorpindol; coyden IUPAC name: 3,5-dichloro-2,6-dimethyl-1H-pyridin-4-one Chemical abstract service No.: 2971-90-6 Structural formula: O H ClCl N Molecular formula: C7H7Cl2NO Molecular weight or molecular weight of the salt form: 192.04 g/mol Other information on identity and properties Appearance: White to light-brown, crystalline solid; powder Melting point: >320 °C Solubility: in water, 10 mg/L Log P: 2.6 (estimated value) FAO JECFA Monograph 33 7 Residues in food and their evaluation Conditions of use Clopidol is a pyridone-derivative structurally related to the quinolones. Pyridones are the carbonyl tautomeric forms of hydroxypyridines. The tautomeric equilibrium of 3,5-dichloro-2,6-dimethyl-1H- pyridin-4-one and 3,5-dichloro-2,6-dimethyl-1H-pyridin-4-ol is a common phenomenon with aromatic compounds (Monastyrskyi, Kyle and Manetsch, 2014). Clopidol is a coccidiostat approved for use as an aid in the prevention of coccidiosis in broiler chickens and replacement layers (pullets) caused by certain Eimeria species. Clopidol inhibits the development of sporozoites and trophozoites of certain Eimeria species (Noack, Chapman and Selzer, 2019). Available products are recommended not to be fed to laying hens or to pullets after 16 weeks of age. Clopidol is also approved as an aid in the prevention of leucocytozoonosis caused by Leucocytozoon smithi in turkeys grown for meat purposes only. In the United States of America, clopidol is approved for use as a Type A medicated article to manufacture a Type C medicated feed in the concentration range of 0.0125 percent w/w (weight by weight) to 0.025 percent w/w clopidol. When feeding 0.025 percent w/w clopidol, the dosage should be reduced to 0.0125 percent w/w or the medication entirely withdrawn five days prior to slaughter. In turkeys, regardless of whether the sole ration of clopidol is fed at 0.0125 or 0.025 percent w/w, clopidol should be withdrawn five days before slaughter. In New Zealand, clopidol is registered for use in broiler chickens at a concentration of 125 mg/kg (0.0125 percent w/w) with a 2-day withdrawal period. In the case of severe infection, the dosage can be increased to 250 mg/kg (0.025 percent w/w). In Canada, clopidol is registered for use in broiler chickens at a rate of 125 mg/kg (0.0125 percent w/w) with a 0-day withdrawal period. In the Republic of Korea, clopidol is registered for use in broiler chickens at a rate of 80 to 250 mg/kg with a withdrawal period of 5 or 7 days, depending on usage. Clopidol is not currently registered for use as a pesticide. Dosage Clopidol is administered in feed at a dose rate of 0.008 percent (80 mg/kg feed) to 0.025 percent (250 mg/kg feed), which can be fed to the birds continuously until slaughter (broilers), or until 16 weeks of age (replacement pullets). Some product labels recommend that the dose should be reduced from 250 mg/kg feed to 125 mg/kg feed five days prior to slaughter for broilers. Pharmacokinetics and metabolism Pharmacokinetics in laboratory animals Literature references for pharmacokinetic and metabolism studies conducted in rats and rabbits were provided by the sponsor and are summarized below. The sponsor did not provide additional pharmacokinetic or metabolism studies in laboratory animals. Twelve white rats were each administered an oral capsule containing 10 mg of [36Cl]-clopidol (approximately 50 mg 36Cl-clopidol/kg bw) under anaesthesia and were euthanized at 4, 24, 48, 72, 168 or 264 h post-dosing (Smith and Watson, 1969). Urine, faeces, blood and tissues (heart, liver, lungs, spleen, kidney, testes, muscle, fat, bone, skin and stomach) were collected at each timepoint. At 4 h post-dosing, urine and faecal samples did not contain a significant amount of radioactivity. Approximately 58 percent of the dose was excreted in the urine, and approximately 42 percent was excreted in the faeces, from 24 to 168 h post-dosing, with the levels recovered dropping quickly from 24 h post-dosing onwards, indicating the rapid elimination of clopidol. Mean distribution of radioactivity in whole blood at FAO JECFA Monograph 338 4 h post-dosing was approximately 18 mg equiv/kg, indicating that the radioactive compound was being circulated throughout the body. At 4 h post-dosing, mean radioactivity was approximately 28 mg equiv/kg in plasma and 8 mg equiv/kg in red blood cells, suggesting that the radioactive compound was not bound to proteins. This was confirmed by the authors by demonstrating that the radioactivity was easily extracted from the plasma and cells with methanol. Radioactivity in the blood rapidly decreased, with concentrations below 1 mg equiv/kg by 24 h and at 0.05 mg equiv/kg at 264 h post-dosing. The limit of quantification (LOQ) of the method used to quantify radioactivity was 0.02 mg equiv/kg. The pharmacokinetics of clopidol were also studied in rabbits. Eight adult New Zealand white rabbits were administered either a single oral dose (16 mg/kg) or multiple (16 mg/kg per day) oral doses up to five days of [14C]-clopidol and slaughtered at various timepoints after dosing (Cameron, Chasseaud and Hawkins, 1975). Urine and faeces were collected from each rabbit at each timepoint. Radioactivity was measured by combustion and liquid scintillation counting (LSC). Results indicated that most of the oral dose of [14C]-clopidol was rapidly absorbed and excreted in the urine, with 99 percent of a single dose and 97 percent of two daily doses being present in the urine 24 h after the last dose. Additional rabbits were slaughtered at different times after receiving the last of five daily doses. In rabbits slaughtered at 16 and 32 h after the last of five daily doses, less than 1 percent of the total dose remained in the total carcass. Concentrations of radioactivity expressed as clopidol equivalents were greater in the plasma than in the tissues. Based on the carcass and plasma levels, the authors calculated the biological half-life of clopidol and its metabolites to be approximately 3 h in rabbits. Pharmacokinetics in food-producing animals Chickens No pharmacokinetics studies in chickens were provided by the sponsor. Metabolism in laboratory animals While no traditional in vivo or in vitro metabolism studies in laboratory species were provided by the sponsor, the literature referenced by the sponsor for rats and rabbits does include metabolism data. Those metabolism data from the above-referenced literature are summarized below. In the Smith and Watson study (1969), tissues from rats administered an oral capsule containing 10 mg of [36Cl]-clopidol (approximately 50 mg [36Cl]-clopidol/kg bw) were collected, including heart, liver, lungs, spleen, kidney, testes, muscle, fat, bone, skin and stomach. Tissues were analysed for radioactivity by a combustion procedure and LSC. Results indicated that, at 4 h post-dosing, approximately 10 percent of the radioactivity was present in the stomach tissues. At 4 h post-dosing, kidney tissues had the highest level of radioactivity (24 mg equiv/kg), followed by liver (~13 mg equiv/kg), suggesting rapid elimination from these tissues. Radioactivity rapidly decreased over time, with concentrations of [36Cl]-clopidol dropping to below 1 mg equiv/kg in all tissues by 48 h (Table 1). The authors plotted the rates of elimination for rat kidney, liver, muscle and fat tissues. and calculated a biological half-life of 10 h for [36Cl]-clopidol. The authors noted that preliminary identification of the radioactive compound in the tissues by paper chromatography indicated the presence of unchanged clopidol only. In the study reported by Cameron, Chasseaud and Hawkins (1975), rabbit liver, kidney, bladder, gastrointestinal tract and muscle tissues were collected and analysed by combustion and liquid scintillation counting. The limits of detection (LOD) for clopidol in the tissues were from 0.01 to 0.04 mg equiv/kg. FAO JECFA Monograph 33 9 Table 1. Distribution of radioactivity in various tissues of rats fed [36Cl]-clopidol Tissue Results in ppm (mg equiv/kg) at timepoint (h) 4 24 48 72 96 168 264 Heart 10.72 1.08 0.05 <0.02 0.12 0.02 0.03 Liver 12.68 1.80 0.08 <0.02 0.07 0.05 <0.02 Lungs 11.01 1.14 0.07 0.03 0.25 <0.02 <0.02 Spleen 6.45 0.54 0.08 <0.02 0.06 <0.02 <0.02 Kidney 24.09 2.58 0.09 0.04 0.14 0.06 <0.02 Testes 6.32 6.17 0.05 0.03 0.30 0.03 <0.02 Muscle 6.03 0.56 <0.02 0.05 0.07 <0.02 <0.02 Fat 1.22 0.17 0.04 <0.02 0.57 <0.02 <0.02 Bone 3.47 0.48 0.04 0.08 0.03 <0.02 0.02 Skin 7.03 0.97 0.48 0.89 0.41 0.42 0.15 Stomach 1 203.59 0.74 0.09 0.22 0.11 0.10 0.05 Whole blood 17.92 1.80 0.10 0.14 0.06 0.10 0.05 Plasma 27.87 - 0.09 0.03 0.08 0.08 <0.02 Red cells 8.13 0.91 0.05 0.04 0.04 0.05 <0.02 Source: adapted from Smith, G. N. & Watson, B. L. 1969. The metabolism of 36Cl-Clopidol (3,5-Dichloro-2,6- Dimethyl-4-Pyridonol) in rats. Poultry Science, 48: 437–443. Figure 1. Proposed metabolic pathway of clopidol (UC3), involving hydroxylation (UC2) and glucuronidation (UC1) OH Cl Cl Cl N UC3 OH OH Cl N C UC2 Cl OH O-gluc Cl N C UC1 Source: Cameron, B. D., Chasseaud, L. F. & Hawkins, D. R. 1975. Metabolic fate of clopidol after repeated oral administration to rabbits. Journal of Agricultural and Food Chemistry, 23: 269–274. FAO JECFA Monograph 3310 Concentrations of radioactivity were low in all tissues, with no accumulation of radioactivity apparent in those animals that had received five daily doses of clopidol. Three major radioactive components in rabbit urine were separated by thin layer chromatography (TLC), and designated as UC1, UC2 and UC3 (Figure 1). UC2, accounting for 32 percent of the urine radioactivity, was characterized by mass spectrometry to be 3,5-dichloro-2-hydroxymethyl-6-methylpyridin-4-ol. UC3, accounting for 47 percent of urinary radioactivity, was determined to be unchanged clopidol. UC1, accounting for 21 percent of the urinary radioactivity, was suggested to be a glucuronide conjugate of UC2. The authors provided the following proposed metabolic pathway of clopidol, involving hydroxylation and then glucuronidation of the resulting alcohol. Metabolism in food-producing animals Chicken A search of the literature conducted by the Committee provided additional information regarding the metabolism of clopidol in the target species, chickens, in addition to the sponsor-provided metabolism and residue kinetics report. In the Smith (1969) study, six-week-old chickens were continuously fed a feed containing 0.0125 percent [36Cl]-clopidol (125 mg/kg feed) for seven days. The specific activity of the compound was 0.154 µCi/g. Chickens were euthanized while still on medicated feed and blood, muscle and liver samples were collected. Blood, muscle and liver samples were subjected to combustion to measure total radioactivity and then isolated and identified by infrared, X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) analyses. The identity was also further verified by paper chromatography and crystallography. Most of the radioactivity was associated with unchanged clopidol and a minor component of radioactivity was determined to be traces of inorganic [36Cl]-chloride. In a sponsor-provided metabolism study (Kim, 2023) reported to be Good Laboratory Practice (GLP)-compliant, twenty 21-day old Arbor Acre broilers weighing approximately 1 kg, and acclimated for seven days, were dosed with a single oral dose of 25 mg [3H]-clopidol/ kg bw, with an aqueous solution of 5 000 mg/L. Broilers were subjected to a 12 h light/12 h dark cycle during the acclimation and testing phases. To make the dosing solution, [3H]-clopidol (0.1 mg; Figure 2) and non-radiolabelled clopidol (749.9 mg) were added to 150 mL of 0.5 percent CMC solution (carboxymethylcellulose sodium salt in sterilized water) and were homogenized. The content and homogeneity of clopidol in the dose solution was confirmed by LSC analysis. The specific activity of [3H]-clopidol was 4.8 Ci/mmol with a purity reported to be 100 percent. Figure 2. Radiolabelled [3H]-clopidol O H ClCl CH3**3HC N Source: adapted from Kim, J-H. 2023. Metabolism and residue kinetics of [3H]Clopidol in Broiler. Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology. Sponsor submitted. FAO JECFA Monograph 33 11 To assess the extent of tritium exchange with water, the radioactivity values of both wet and dry samples were measured. Dry samples were prepared by drying tissues at 50 °C for 24 to 30 h and adding 5 mL of Soluene-350 solution (a tissue solubilizer). Samples were shaken at 180 rpm and 50 ± 1 °C for 24 h until completely dissolved. An aliquot of each sample was taken and mixed with a scintillation cocktail and radioactivity was measured by LSC. The wet samples were solubilized without drying and radioactivity was measured by LSC. The sponsor reported that radioactivity in the samples was very low after day 3 and was not measured further. Only the 6-h and 1-day post-dosing samples were reported. Numerous samples at both timepoints exceeded the VICH-recommended (Veterinary International Committee for Harmonisation) acceptance criterion of <5 percent (from -40.2 to +28.8 percent), suggesting that the tritium label was not stable. Broilers were euthanized using xylazine and succinylcholine, followed by exsanguination at 6 h, and 1, 3, 5 and 10 days post-dosing. Liver, skin and fat in natural proportions (skin/fat), muscle and kidney tissues were collected, ground and homogenized on dry ice, and stored at 20 °C until analysis. The tissue extracts were subjected to radio-HPLC (high performance liquid chromatography) to separate clopidol and its metabolite(s). Kidney samples were extracted twice using a mixture of acetonitrile and water at 1 000 rpm for ten minutes. Samples were centrifuged and all extracts were combined and subjected to clean-up using MgSO4, C18E and graphitized carbon black (GCB). After shaking and centrifugation, the resulting supernatant was evaporated using nitrogen gas, and brought up to 0.9 mL with dimethylsulfoxide (DMSO). The sample was filtered (0.22 µm) prior to analysis and an aliquot of filtrate was mixed with the scintillation cocktail for analysis by radio-HPLC to separate clopidol and its metabolites. Liver, muscle and skin/fat samples were extracted through agitation (1 000 rpm, 10 min), twice, using a mixture of acetonitrile:water (8:2 v/v). After centrifugation, all extracts were combined and subjected to clean-up. Muscle and liver extracts underwent clean-up using MgSO4, C18E and GCB, while skin/fat extracts were cleaned up using MgSO4 and primary secondary amine (PSA). After shaking and centrifugation, the resulting supernatant was evaporated using nitrogen gas and brought up to 1 mL with DMSO. The sample was filtered (0.22 µm) prior to analysis and an aliquot of filtrate was mixed with the scintillation cocktail for radioactivity quantitation by LSC. Simultaneously, analysis by radio-HPLC to separate clopidol and its metabolites was performed. The remaining tissue after extraction was dried at 30–35 °C and thoroughly mixed. The total weight was measured and 0.1 g of dried sample was combusted with cellulose in a sample oxidizer and subjected to LSC analysis, to determine the amount of radioactivity associated with the non-extractable residue. Table 2 illustrates the recovery of [3H]-clopidol in broiler tissues. Total recovery was greater than 90 percent in all tissues. Table 3 shows the distribution of radioactivity, as %TRR (total radioactive residue), in the broiler tissues following oral administration of [3H]-clopidol. Overall, extractability of radioactivity was almost complete, with all tissues at all timepoints (except liver at 6 h) exceeding 90 percent extractability. FAO JECFA Monograph 3312 Table 2. Recovery of [3H]-clopidol in liver, fat, muscle and kidney tissues of broilers Tissue Levela Recoveryb (extract, %) (mean ± SD) CVc (%) Unextractable (%)d (mean ± SD) Total recoverye (%) Liver 1 94.3 ± 0.4 0.4 6.3 ± 0.0 100.6 2 100.0 ± 1.6 1.6 5.8 ± 0.1 105.8 3 101.3 ± 0.8 0.8 5.7 ± 0.4 107.0 Skin/Fat 1 95.6 ± 1.0 1.0 1.7 ± 0.1 97.3 2 98.1 ± 0.1 0.1 1.5 ± 0.0 99.6 3 95.2 ± 1.6 1.6 2.7 ± 0.4 97.9 Muscle 1 97.0 ± 0.1 0.1 11.6 ± 0.1 108.6 2 91.6 ± 0.8 0.8 9.2 ± 0.1 100.87 3 91.7 ± 0.4 0.4 7.5 ± 0.1 99.2 Kidney 1 91.1 ± 0.0 0.0 2.3 ± 0.0 93.4 2 91.4 ± 0.1 0.1 2.4 ± 0.0 93.8 3 92.2 ± 0.7 0.7 2.3 ± 0.1 94.5 Notes: aLevel 1: 10% level of total radioactivity administered, Level 2: 1% level of total radioactivity administered, Level 3: 0.1% level of total radioactivity administered; b(Detected dpm/fortified dpm) × 100; cCoefficient of variation: SD/mean × 100%; dRemaining residue after extraction; eExtract (%) + Unextractable (%); SD: standard deviation Source: adapted from Kim, J-H. 2023. Metabolism and residue kinetics of [3H]Clopidol in Broiler. Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology. Sponsor submitted. Table 3. Distribution of radioactivity, expressed as %TRR (total radioactive residue), in broiler tissues following oral administration of [3H]-clopidol at a dose of 25 mg/kg bw Tissue Time post-dose (days) Fraction %TRR (mean ± SD) Liver 6 h Extract 88.5 ± 2.2 Unextractable 11.6 ± 2.2 Total 100.0 ± 0.0 1 Extract 91.0 ± 2.3 Unextractable 10.8 ± 2.8 Total 100.0 ± 0.0 3 Extract 100.0 ± 0.0 Unextractable - Total 100.0 ± 0.0 5 Extract - Unextractable - Total - 10 Extract - Unextractable - Total - Skin/Fat 6 h Extract 100.0 ± 0.0 Unextractable - Total 100.0 ± 0.0 1 Extract 100.0 ± 0.0 Unextractable - Total 100.0 ± 0.0 FAO JECFA Monograph 33 13 Table 3. Distribution of radioactivity, expressed as %TRR (total radioactive residue), in broiler tissues following oral administration of [3H]-clopidol at a dose of 25 mg/kg bw (cont.) Tissue Time post-dose (days) Fraction %TRR (mean ± SD) Skin/Fat 3 Extract 100.0 ± 0.0 Unextractable - Total 100.0 ± 0.0 5 Extract - Unextractable - Total - 10 Extract - Unextractable - Total - Muscle 6 h Extract 90.2 ± 3.9 Unextractable 9.9 ± 3.9 Total 100.1 ± 0.0 1 Extract 100.0 ± 0.0 Unextractable - Total 100.0 ± 0.0 3 Extract - Unextractable - Total - 5 Extract 100.0 ± 0.0 Unextractable - Total - 10 Extract - Unextractable - Total - Kidney 6 h Extract 97.4 ± 0.7 Unextractable 3.1 ± 0.3 Total 100.0 ± 0.0 1 Extract 100.0 ± 0.0 Unextractable - Total 100.0 ± 0.0 3 Extract 100.0 ± 0.0 Unextractable - Total 100.0 5 Extract - Unextractable - Total - 10 Extract - Unextractable - Total - Source: adapted from Kim, J-H. 2023. Metabolism and residue kinetics of [3H]Clopidol in Broiler. Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology. Sponsor submitted. FAO JECFA Monograph 3314 Radio-HPLC analysis of the tissue extracts showed the presence of two radiolabelled components: unchanged [3H]-clopidol and a metabolite. These two radiolabelled components were isolated by eluting from the HPLC column at the specific retention time and the eluates from each tissue were extracted and identified by liquid chromatography-mass spectrometry (LC-MS). Unchanged [3H]-clopidol was confirmed by comparison with an authentic standard. Through mass measurement and fragmentation patterns, the metabolite was identified as 3,5-dichloro-2-hydroxymethyl-6-methylpyridin-4-ol (exact mass: 206.99 g/mol), a hydroxylated metabolite. Table 4 illustrates the distribution of radioactivity amongst unchanged [3H]-clopidol and the hydroxylated metabolite. [3H]-clopidol comprised more than 80 percent of the TRR in all broiler tissues. The hydroxylated metabolite was found in all tissues, but only at the 6-h timepoint and was never above 10 percent of the TRR. The sponsor did not propose a metabolic pathway for clopidol in chickens. Table 4. Radio-HPLC analysis of tissue extracts, expressed as %TRR (total radioactive residue), following oral administration of [3H]-clopidol at a dose of 25 mg/kg bw Tissue Time post-dose (days) Fraction %TRR (mean ± SD) Liver 6 h Clopidol 84.4 ± 2.5 Hydroxylated metabolite 4.1 ± 0.7 Total fractioned 88.5 ± 2.2 1 Clopidol 91.0 ± 2.3 Hydroxylated metabolite - Total fractioned 91.0 ± 2.3 3 Clopidol 100.0 Hydroxylated metabolite - Total fractioned 100.0 5 Clopidol - Hydroxylated metabolite - Total fractioned - 10 Clopidol - Hydroxylated metabolite - Total fractioned - Skin/Fat 6 h Clopidol 93.1 ± 2.1 Hydroxylated metabolite 7.0 ± 2.1 Total fractioned 100.1 ± 0.0 1 Clopidol 100.0 ± 0.0 Hydroxylated metabolite - Total fractioned 100.0 ± 0.0 3 Clopidol 100.0 ± 0.0 Hydroxylated metabolite - Total fractioned 100.0 ± 0.0 5 Clopidol - Hydroxylated metabolite - Total fractioned - 10 Clopidol - Hydroxylated metabolite - Total fractioned - FAO JECFA Monograph 33 15 Table 4. Radio-HPLC analysis of tissue extracts, expressed as %TRR (total radioactive residue), following oral administration of [3H]-clopidol at a dose of 25 mg/kg bw (cont.) Tissue Time post-dose (days) Fraction %TRR (mean ± SD) Muscle 6 h Clopidol 85.1 ± 4.2 Hydroxylated metabolite 5.1 ± 0.8 Total fractioned 90.2 ± 3.9 1 Clopidol 100.0 ± 0.0 Hydroxylated metabolite - Total fractioned 100.0 ± 0.0 3 Clopidol - Hydroxylated metabolite - Total fractioned - 5 Clopidol - Hydroxylated metabolite - Total fractioned - 10 Clopidol - Hydroxylated metabolite - Total fractioned - Kidney 6 h Clopidol 87.8 ± 3.2 Hydroxylated metabolite 9.7 ± 4.3 Total fractioned 97.5 ± 0.7 1 Clopidol 100.0 ± 0.0 Hydroxylated metabolite - Total fractioned 100.0 ± 0.0 3 Clopidol - Hydroxylated metabolite - Total fractioned - 5 Clopidol - Hydroxylated metabolite - Total fractioned - 10 Clopidol - Hydroxylated metabolite - Total fractioned - Source: adapted from Kim, J-H. 2023. Metabolism and residue kinetics of [3H]Clopidol in Broiler. Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology. Sponsor submitted. Comparative metabolism The sponsor did not provide comparative metabolism studies. The literature provides limited references on the metabolites present in laboratory animals (rats and rabbits), although the data do suggest that clopidol is the only major compound present in rat tissues. In rabbit, the data suggest that clopidol undergoes hydroxylation to 3,5-dichloro-2-hydroxymethyl-6-methylpyridin-4-ol which then undergoes glucuronidation of the resulting alcohol. The literature also suggests rapid absorption and excretion of clopidol. Data provided from the literature for chickens (Smith, 1969) and from the sponsor (Kim, 2023) also suggest the major residue is clopidol which undergoes hydroxylation to 3,5-dichloro-2- hydroxymethyl-6-methylpyridin-4-ol (Kim, 2023), similar to the proposed metabolic pathway in rabbits. FAO JECFA Monograph 3316 Tissue residue depletion studies Radiolabelled residue depletion study Chickens In a sponsor-provided radiolabelled residue depletion study, reported to be GLP-compliant (Kim, 2023), twenty 21-day old Arbor Acre broilers weighing approximately 1 kg, and acclimated for seven days, were dosed with a single oral dose of 25 mg [3H]-clopidol per kg bw. Broilers were subjected to a 12 h light/12 h dark cycle during the acclimation and testing phases. The dose solution (5 000 mg/L) was prepared to allow dosing at ca. 25 mg [3H]-clopidol/kg bw. Radiolabelled [3H]-clopidol (Figure 2; 0.1 mg) and non-radiolabelled clopidol (749.9 mg) were added to 150 mL of 0.5 percent CMC solution (carboxymethylcellulose sodium salt in sterilized water) and were homogenized. The content and homogeneity of clopidol in the dose solution was confirmed by LSC analysis. The specific activity of [3H]-clopidol was 4.8 Ci/mmol with a reported purity of 100 percent. Broilers were euthanized using xylazine and succinylcholine, followed by exsanguination, at 6 h, and 1, 3, 5 and 10 days post-dosing. Liver, skin and fat in natural proportions (skin/fat), muscle and kidney tissues were collected, homogenized on dry ice, and stored at 20 °C until analysis. The sponsor indicated that [3H]-clopidol in tissues was stable for 14 days. Total radioactivity was determined by combustion, followed by LSC, and all radio assays were performed in duplicate. Concentrations of the marker residue, clopidol, were determined by radio-HPLC analysis. Total residues were highest in kidney tissues (29.2 mg equiv/kg), followed closely by liver (28.8 mg equiv/kg), muscle (14.3 mg equiv/kg) and skin/fat (7.3 mg equiv/kg). Total residue concentrations decreased rapidly in all tissues, with TRR concentrations below the LOQ in all tissues except muscle by day 5 post-dosing (Table 5). Clopidol residue concentrations, comprising more than 80 percent of the TRR, depleted most slowly from liver, followed by kidney, muscle and skin/fat. Clopidol residue concentrations were below the LOQ in all tissues except liver, by day 3 post-dosing (Table 6). The data confirm that clopidol is a suitable marker residue. Ratios of marker to total radioactive residues are presented in Table 7, with ratios able to be calculated only at the 6 h and 1 day timepoints because of the rapid elimination of clopidol. The sponsor-reported marker to total ratios remained generally stable amongst the tissues and timepoints. However, because of the uncertainty about the stability of the tritium radiolabel, the Committee considered it appropriate to evaluate dietary exposure with a range of marker residue to total residue (MR:TR) values. Table 5. Mean total radiolabelled residues (mg equiv/kg) in tissues of broilers orally administered 25 mg [3H]-clopidol/kg bw, once Time post-dose (days) Kidney Liver Muscle Skin/Fat 6 h 29.222 28.811 14.334 7.257 1 4.934 4.762 1.333 0.849 3 0.148 0.357 <LOQ 0.087 5 <LOQ <LOQ 0.086 <LOQ 10 <LOQ <LOQ <LOQ <LOQ Notes: LOQ for kidney is 0.00882 mg equiv/kg; LOQ for liver is 0.00151 mg equiv/kg; LOQ for muscle is 0.00042 mg equiv/kg; LOQ for skin/fat is 0.00166 mg equiv/kg Source: adapted from Kim, J-H. 2023. Metabolism and residue kinetics of [3H]Clopidol in Broiler. Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology. Sponsor submitted. FAO JECFA Monograph 33 17 Table 6. Mean clopidol residues (mg equiv/kg) in tissues of broilers orally administered 25 mg [3H]-clopidol/kg bw, once Time post-dose (days) Kidney Liver Muscle Skin/Fat 6 h 25.703 24.288 12.176 6.758 1 4.934 4.330 1.333 0.849 3 <LOQ 0.357 <LOQ <LOQ 5 <LOQ <LOQ <LOQ <LOQ 10 <LOQ <LOQ <LOQ <LOQ Notes: LOQ for kidney is 0.342 mg equiv/kg; LOQ for liver is 0.183 mg equiv/kg; LOQ for muscle is 0.114 mg equiv/kg; LOQ for skin/fat is 0.183 mg equiv/kg Source: adapted from Kim, J-H. 2023. Metabolism and residue kinetics of [3H]Clopidol in Broiler. Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology. Sponsor submitted. Table 7. Marker to total residue ratios (MR:TR) in broiler tissues Time post-dose (days) Kidney Liver Muscle Skin/Fat 6 h 0.88 0.84 0.85 0.93 1 1.0 0.91 1.0 1.0 3 NA 1.0* NA <LOQ 5 NA NA NA <LOQ 10 NA NA NA <LOQ Notes: *MR:TR is based on a liver sample from one animal. All other values were <LOQ. Source: adapted from Kim, J-H. 2023. Metabolism and residue kinetics of [3H]Clopidol in Broiler. Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology. Sponsor submitted. Residue depletion study with non-radiolabelled drug Chicken In a sponsor-provided residue depletion study, reported to be GLP-compliant, sixty-six 21-day-old Ross broilers were fed 125 mg clopidol/kg feed or 250 mg clopidol/kg feed as the sole ration for 14 days. Six chickens per dose per timepoint were euthanized using xylazine and succinylcholine, followed by exsanguination at 1, 3, 5, 7 and 10 days post-dosing. Broilers were subjected to a 20 h light/4 h dark cycle. Six broilers were used as control animals and were euthanized on the first day after the 14-day dosing period. The remaining broilers were split into two treatment groups, with one group receiving 125 mg clopidol/kg feed and the second group receiving 250 mg clopidol/kg feed. At each withdrawal timepoint, muscle, skin and fat in natural proportions (skin/fat), liver and kidney samples were collected from each broiler and stored at -20 °C prior to shipment to the analytical laboratory. Tissue samples were analysed in triplicate by a validated liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method. The tissues showed similar depletion profiles of clopidol, regardless of dose level (125 mg/kg or 250 mg/kg feed). Clopidol concentrations in all tissues were highest at 1-day post- final dosing, with all residues below the LOQ (50 µg/kg) by day 5 (kidney, liver, muscle) and day 7/10 (skin/fat: 125 mg/kg/250 mg/kg feed) (Table 8 and Table 9). FAO JECFA Monograph 3318 Table 8. Clopidol residue concentrations (µg/kg) in tissues of broilers administered 250 mg clopidol/kg feed Slaughter timepoint (days) Animal ID Results (µg/kg) Muscle Liver Kidney Skin/Fat 1 15 <LOQ <LOQ <LOQ 887 38 1 139 2 508 2 716 1 033 39 1 516 4 781 3 803 1 032 40 2 032 3 099 2 622 1 075 41 1 349 4 601 4 029 794.9 42 <LOQ <LOQ <LOQ 494.2 3 55 122 358.2 358.9 199.2 56 <LOQ 111.9 100.9 95.3 57 <LOQ 128.9 68 84.6 58 79.4 201.9 128.1 84.7 59 98 194.6 111.3 150.6 60 309.6 478.3 412.3 173.4 5 13 <LOQ <LOQ <LOQ <LOQ 14 <LOQ <LOQ <LOQ 62.3 16 <LOQ <LOQ <LOQ 48.7 17 <LOQ <LOQ <LOQ 58.4 18 <LOQ <LOQ <LOQ 62.6 37 <LOQ <LOQ <LOQ 93.9 7 31 <LOQ <LOQ <LOQ <LOQ 32 <LOQ <LOQ <LOQ <LOQ 33 <LOQ <LOQ <LOQ <LOQ 34 <LOQ <LOQ <LOQ 54.6 35 <LOQ <LOQ <LOQ <LOQ 36 <LOQ <LOQ <LOQ 68.7 10 19 <LOQ <LOQ <LOQ <LOQ 20 <LOQ <LOQ <LOQ <LOQ 21 <LOQ <LOQ <LOQ <LOQ 22 <LOQ <LOQ <LOQ <LOQ 23 <LOQ <LOQ <LOQ <LOQ 24 <LOQ <LOQ <LOQ <LOQ Note: <LOQ: below the limit of quantitation (50 µg/kg) Source: adapted from Jeong, S., Jong-hwan, K., Jeong-Ran, M., Chang-hun, L. & Min-cheol, S. 2023. A study on residue depletion of clopidol in edible tissues of chickens. Report Number RED22018. Hoseo Biomedical Science Research Center, Hoseo University. Sponsor submitted. FAO JECFA Monograph 33 19 Table 9. Mean (± SD) clopidol residue concentrations (µg/kg) in tissues of broilers administered either 125 mg clopidol/kg feed or 250 mg clopidol/kg feed Dose level (mg/kg in feed) Withdrawal (days) Kidney Liver Muscle Skin/Fat 125 1 3 016 ± 442.1 3 420 ± 732 1 537.3 ± 224 619.5 ± 279.3 3 121.2 ± 68.7 160.1 ± 115.3 128.3 87.8 ± 15.4 5 111.2 <LOQ <LOQ 73.2 ± 31.7 7 <LOQ <LOQ <LOQ <LOQ 10 <LOQ <LOQ <LOQ <LOQ 250 1 3 293 ± 726.9 3 747 ± 1 118.6 1 509 ± 381.3 886.1 ± 219.3 3 196.6 ± 148.7 245.6 ± 143.4 152.3 ± 106.3 131.3 ± 49.8 5 <LOQ <LOQ <LOQ 69.3 ± 16.5 7 <LOQ <LOQ <LOQ 61.7 ± 10.0 10 <LOQ <LOQ <LOQ <LOQ Note: <LOQ: below the limit of quantitation (50 µg/kg) Source: adapted from Jeong, S., Jong-hwan, K., Jeong-Ran, M., Chang-hun, L. & Min-cheol, S. 2023. A study on residue depletion of clopidol in edible tissues of chickens. Report Number RED22018. Hoseo Biomedical Science Research Center, Hoseo University. Sponsor submitted. Review of published literature – residue depletion data Drug residues in the organs of broiler chickens after flock treatment with Rigecoccin R (clopidol) The study conducted by Czegledi-Jankó, Balla and Tóth (1976), was not conducted in line with VICH Guidelines or the principles of GLP. Three hundred broiler chickens (strain Tetra B) were administered clopidol in feed at an inclusion rate of 125 mg/kg feed. After 10 weeks of treatment, 25 birds were slaughtered daily on day 0, 1, 2 and 3 after removal of clopidol from the feed. The same was done after 16 weeks of treatment (Table 10). Samples of liver, kidney, thigh and breast muscle, intestinal contents and blood were taken, homogenized and analysed using gas chromatography (LOD/LOQ not mentioned); the samples were extracted using three different methods, which were roughly equivalent in terms of recovery from spiked samples. The samples that were analysed were homogenates from all 25 birds per timepoint studied; it is not stated whether or how many duplicate samples were analysed. FAO JECFA Monograph 3320 Table 10. Clopidol residues in broiler chickens after long-term preventative treatment with Rigecoccin R at 125 mg/kg dietary dose leveled Duration of treatment (weeks) Slaughter timepoint (days) Concentration of clopidol detected (µg/kg) in tissues Muscle Liver Kidney Blood Intestinal contents 10 0 870 3 600 1 290 1 590 1 260 1 440 1 300 590 720 800 2 270 330 260 320 610 3 100 120 180 200 - 16 0 620 1 370 430 680 2 020 1 310 610 470 360 700 2 380 320 200 280 - 3 70 380 240 120 420 Source: adapted from Czeglédi-Jankó, G., Balla, J., & Tóth, L. 1976. Drug residues in the organs of broiler chickens after flock treatment with Rigecoccin R (clopidol). Acta Veterinaria Academiae Scientiarum Hungaricae, 26(4): 445–53. PMID: 1052659. Tissue residues of clopidol (3,5‐dichloro‐2,6‐dimethyl‐4‐pyridinol) in chickens in relation to withdrawal times The study, conducted by Ekström, Slanina and Dahlström‐King (1984), was not conducted in line with VICH guidance or with GLP. Broilers (males and females; strain Hybro) were fed clopidol at a rate of 125 mg/kg in feed for 34 days, starting when they were one day old. Four birds per day were slaughtered for 10 days after withdrawal of the medicated feed. The birds weighed between 1.2 and 1.4 kg at slaughter. Samples of thigh muscle and liver were taken from each bird, pooled and stored at -20 °C, for a maximum of one month, until analysis (in duplicate) using a gas chromatographic method. The method is described in Ekström and Kuivinen (1984). The results of the study showed residues of about 7 000 µg/kg in liver and 3 000 µg/kg in muscle at zero days (8 h after the last medicated feed was consumed). Once clopidol was no longer being added to the feed, there was a rapid decrease in the tissue concentrations over the first two days. This was followed by a levelling out of the residue concentration in samples of both liver (200–800 µg/kg) and muscle (50 to 180 µg/kg) between days 2 and 10 post treatment. No individual data were provided in this paper, so only the trends can be seen in Figure 3. It was also noted that there was a correlation between the levels found in the liver and muscle of treated birds (Figure 4). The authors note that, at the time of writing, the withdrawal periods authorized for the inclusion rate of 125 g/tonne feed was 0 days in the United States of America, 3 days in Sweden and 7 days in Japan. FAO JECFA Monograph 33 21 Figure 3. Clopidol levels in liver and muscle samples from broiler chickens slaughtered daily after withdrawal of clopidol-containing feed 4 5 6 7 3 1 2 0 pp m C lo pi do l Days 1050 1 2 3 4 6 7 8 9 Liver Muscle Notes: Each point shows the results for pooled tissue samples from four chickens; each sample was analysed in duplicate; ppm = mg/kg; black circles: liver; clear circles: muscle Source: reproduced from Ekström, L. G., Slanina, P. & Dahlström‐King, L. 1984. Tissue residues of clopidol (3,5-dichloro-2,6-dimethyl-4-pyridinol) in chickens in relation to withdrawal times. Food Additives & Contaminants, 1(1): 17–22 Figure 4. Relationship between clopidol levels in liver and muscle samples from broiler chickens slaughtered daily after withdrawal of clopidol-containing feed 1.0 5.0 0.1 pp m C lo pi do l i n liv er ti ss ue ppm Clopidol in muscle tissue 0.1 1.00.03 Note: R2: 0.92 (day 0 values not included) Source: reproduced from Ekström, L. G., Slanina, P. & Dahlström‐King, L. 1984. Tissue residues of clopidol (3,5-dichloro-2,6-dimethyl-4-pyridinol) in chickens in relation to withdrawal times. Food Additives & Contaminants, 1(1): 17–22. FAO JECFA Monograph 3322 Determination of clopidol residues in chicken tissues by liquid chromatography: part II Distribution and depletion of clopidol in chicken tissues The study, conducted by Pang et al. (2001a) was not claimed to be compliant with VICH guidance or with GLP. One hundred chickens (22–42 days old) were administered feed with 125 mg/kg clopidol mixed into it for 12 days. Ten chickens per day were slaughtered randomly, starting 8 h after withdrawal of the clopidol from the feed, until no clopidol was detected in the samples taken from the chickens. Samples of liver (whole), kidney (whole), upper breast (100 g), lower breast (100 g) and leg muscle (100 g) were taken for analysis. Samples were blended on arrival at the laboratory. Samples were analysed using a validated LC-MS method (Pang et al., 2001b) with an LOD of clopidol of 5 µg/kg. Additionally, frozen storage stability was demonstrated for clopidol in chicken muscle samples for three months at -18 °C using incurred samples at three fortification levels: 15, 55 and 1 019 µg/kg. Stability data for other tissues were not reported. Although the feeding conditions were the same, the intake of clopidol for each chicken was different, which may be due to the inhomogeneous mixture of the feed, or the intake and absorption of each chicken, leading to relatively large variability of individual residue levels (Table 11 and Table 12). Table 11. Clopidol residues in chicken samples Timepoint (day) Animal no. Clopidol (µg/kg) Upper breast Lower breast Leg muscle Liver Kidney 1 1 1 500 1 486 1 222 3 956 3 610 2 1 529 1 412 1 382 3 733 4 195 3 2 294 2 147 1 824 5 600 3 610 4 889 861 765 3 289 2 146 5 2 059 2 118 1 910 6 311 5 073 6 1 882 1 971 1 882 4 267 3 512 7 2 176 1 824 2 029 6 133 4 293 8 1 676 1 412 1 147 3 022 1 951 9 2 029 2 000 1 735 5 067 4 780 10 1 382 1 176 1 353 4 622 3 024 Mean 1 742 1 641 1 525 4 600 3 619 SD 430 436 412 1 154 1 031 CV% 24.7 26.6 27 25.1 28.5 2 1 1 429 1 400 1 114 4 000 4 444 2 314 314 257 511 1 067 3 429 443 343 1 447 1 600 4 943 1 057 914 4 085 3 200 5 714 686 657 1 872 2 578 6 1 971 1 914 1 686 3 319 2 043 7 471 486 414 2 043 1 956 8 1 714 1 771 1 486 1 277 4 178 9 800 814 743 1 277 2 311 10 457 514 500 2 936 1 956 Mean 924 940 811 2 277 2 978 SD 585 575 487 1 238 1 640 CV% 63.3 61.1 60 54.4 55.1 FAO JECFA Monograph 33 23 Table 11. Clopidol residues in chicken samples (cont.) Timepoint (day) Animal no. Clopidol (µg/kg) Upper breast Lower breast Leg muscle Liver Kidney 3 1 82 82 85 268 225 2 115 94 88 585 200 3 92 82 82 439 275 4 209* 238* 194* 2 293* 974* 5 76 71 70 171 128 6 106 115 112 683 275 7 108 85 85 610 317 8 82 94 88 439 275 9 65 33* 70 390 226 10 90 100 78 122 341 Mean* 91* 90* 84* 412* 251* SD* 16 13 12 196 64 4 1 26 27 25 107 109 2 20 24 18 74 62 3 22 18 17 85 52 4 8 8 9 38 26 5 18 26 21 126 76 6 22 20 21 56 60 7 68* 58* 66 350* 171 8 33 32 34 170 93 9 29 33 30 84 114 10 26 25 41 136 114 Mean* 23* 24* 28 97* 88 SD* 7* 8* 16 42* 41 5 1 16* 13 17 ** ** 2 8 4 7 ** ** 3 9 10 9 16 14 4 6* 6 9 16 14 5 8 6 9 12 12 6 9 9 13 28 20 7 10 10 10 16 18 8 8 9 9 22 20 9 9 Nd 6 Nd Nd 10 9 7 11 17 16 Mean* 9* 7 11 17 16 SD* 1 4 4 9 8 CV% 8.1 60.6 40.9 51 49.6 FAO JECFA Monograph 3324 Table 11. Clopidol residues in chicken samples (cont.) Timepoint (day) Animal no. Clopidol (µg/kg) Upper breast Lower breast Leg muscle Liver Kidney 6 1 5 5 5 Nd Nd 2 8 7 14 Nd Nd 3 Nd 8 12 10 12 4 7 8 5 8 6 5 5 5 5 10 6 6 7 7 7 Nd Nd 7 7 8 12 24* 22 8 Nd Nd 7 Nd Nd 9 Nd Nd Nd Nd Nd 10 5 Nd 7 10 10 Mean 4 5 7 4* 6 SD 3 3 4 5* 7 CV% 72.8 72.7 56.7 119.5 131.3 7 All samples had undetectable levels (<5 µg/kg) of clopidol from day 7. Notes: *Values were omitted from statistical calculations; using ‘Dixon testing1’, they were considered outliers. **Results not reported Source: adapted from Pang, G. F., Cao, Y. Z., Fan, C. L., Zhang, J. J. & Li, X. M. 2001a. Determination of clopidol residues in chicken tissues by liquid chromatography: part II. Distribution and depletion of clopidol in chicken tissues. Journal of AOAC INTERNATIONAL, 84(5): 1343–1346. PMID: 11601451. Table 12. Clopidol mean concentrations Timepoint (days) Mean clopidol concentration (µg/kg) Upper breast Lower breast Leg muscle Liver Kidney 1 1 742 1 641 1 525 4 600 3 619 2 924 940 811 2 277 2 978 3 91* 90* 84* 412* 251* 4 23* 24* 28 97* 88 5 9* 7 11 17 16 6 4 5 7 4* 6 Notes: *Values were omitted from statistical calculations; using ‘Dixon testing1’, they were considered outliers. Source: adapted from Pang, G. F., Cao, Y. Z., Fan, C. L., Zhang, J. J. & Li, X. M. 2001a. Determination of clopidol residues in chicken tissues by liquid chromatography: part II. Distribution and depletion of clopidol in chicken tissues. Journal of AOAC INTERNATIONAL, 84(5): 1343–1346. PMID: 11601451. FAO JECFA Monograph 33 25 From the above data, the authors calculated the elimination half-lives (t½el) for each tissue (Table 13). Table 13. Elimination half-lives (t½el) for various chicken tissues Tissue Elimination half-life (h) Liver 11.8 Kidney 13.3 Upper breast 13.7 Lower breast 14.3 Leg muscle 15.5 Source: adapted from Pang, G. F., Cao, Y. Z., Fan, C. L., Zhang, J. J. & Li, X. M. 2001a. Determination of clopidol residues in chicken tissues by liquid chromatography: part II. Distribution and depletion of clopidol in chicken tissues. Journal of AOAC INTERNATIONAL, 84(5): 1343–1346. PMID: 11601451. Method for the determination of clopidol in muscle, skin/fat, liver and kidney of chickens (sponsor) In summary, sample preparation involves the addition of 2 mL of water to 2 g of ground and homogenized tissue. This mixture is agitated for 5 min, after which 10 mL of acetonitrile is added. Following agitation, the mixture undergoes centrifugation at 4 ºC and 4 500 g for 10 min. The resulting supernatant is collected and extracted by adding 10 mL of hexane. After agitation and centrifugation at 4 ºC and 4 500 g for 10 min, the upper layer (hexane) is removed. The remaining extract is evaporated and the resulting residue is resuspended in 1 mL of 50 percent aqueous acetonitrile. The mixture is agitated, followed by centrifugation at 4 ℃, 4 500 g for 5 min. The supernatant is filtered (0.2 µm) and subjected to analysis by LC-MS/MS. The separation of clopidol is performed on a Poroshell 120-EC-C18 column (2.1 × 100 mm, 2.7 µm), at 40 ºC, utilizing a mobile phase containing aqueous 0.1 percent v/v formic acid (solvent A) and acetonitrile containing 0.1 percent v/v formic acid (solvent B). The gradient started at 95% solvent A. At 1 min solvent A linear decreased from 95% to 5% in 8 min. At 11 min, the gradient is returned to its initial condition of 95% solvent A within 1 min and the column is allowed to equilibrate for 5 min, resulting in a total run of 17 min. The flow rate is 0.35 mL/min and the injection volume is 1 µL. Detection is carried out by tandem mass spectrometry, or MS/MS, using an API4000 (Sciex) mass spectrometer and the electrospray ionization source operated in positive mode. Quantitation is performed using acquisition of ions in the multiple reaction mode (MRM). The precursor ion for clopidol is m/z 192 and the product ions are m/z 101 and m/z 87.1, with m/z 192 to 101 being the transition for quantitation. The method underwent a validation procedure and the following parameters were determined: specificity, linearity, LOD, LOQ, accuracy and precision (intra- and inter-day). Specificity: In order to determine the specificity, blank samples and samples fortified with clopidol were analysed without addition of clopidol. Specificity was assessed by checking the signal of the blank extracts for interferences at the retention time corresponding to clopidol. Linearity: On three different days, a matrix matched calibration curve was prepared at 0, 0.2, 0.5, 1, 2, 5, 10 and 50 ng/mL by adding clopidol to the extract of blank samples. Calibration curves were constructed by plotting the area versus the added concentration and carrying out least squares linear regression. FAO JECFA Monograph 3326 The linearity was considered acceptable if the linear coefficient of correlation was at least 0.99 and if the maximal relative error for each concentration was <±15 percent. LOD and LOQ: The LOD and LOQ for clopidol were set based on signal-to-noise (S/N) ratios. Accuracy and precision: On one day, three muscle samples were fortified at four levels: 1, 2.5, 5 and 10 µg/g. These samples were used to determine the accuracy and intra-day precision. The same procedure was carried out on three different days to assess the inter-day precision. As part of method validation, the sponsor did not determine the stability of clopidol in chicken tissues. However, the sponsor noted that, as part of the radiolabelled residue depletion study, clopidol was stable in chicken tissues for 14 days. As part of the non-radiolabelled residue depletion study, the sponsor noted that all samples were stored at -20 °C and were analysed within 2–3 days of collection. Therefore, the Committee determined that because chicken tissue samples had been analysed shortly after collection, it was not necessary to conduct further stability testing. The validation parameters are shown in Table 14. Table 14. Validation parameters of the LC-MS/MS method for the determination of clopidol in chicken Validation parameter Fortification level Muscle Skin/Fat Liver Kidney Accuracy (%) 1.0 µg/g 86.57 89.54 88.28 85.46 2.5 µg/g 84.24 86.42 84.89 84.38 5.0 µg/g 86.45 88.41 89.24 86.13 10.0 µg/g 87.05 90.77 84.04 85.16 Inter-day precision (%) 1.0 µg/g 2.5 6.5 12.6 3.8 2.5 µg/g 2.6 6.2 11.0 3.7 5.0 µg/g 2.0 7.9 8.1 3.7 10.0 µg/g 1.8 4.1 6.4 3.1 LOD (µg/kg) 25 25 25 25 LOQ (µg/kg) 50 50 50 50 Range (ng/mL) 0.2–50 0.2–50 0.2–50 0.2–50 Linearity (r) >0.99 >0.99 >0.99 >0.99 Specificity No interference observed No interference observed No interference observed No interference observed Source: adapted from Jeong, S., Jong-hwan, K., Jeong-Ran, M., Chang-hun, L. & Min-cheol, S. 2023. A study on residue depletion of clopidol in edible tissues of chickens. Report Number RED22018. Hoseo Biomedical Science Research Center, Hoseo University. Sponsor submitted. Overall comment on validation of the method According to VICH GL49 (Studies to evaluate the metabolism and residue kinetics of veterinary drugs in food-producing animals: validation of analytical methods used in residue depletion studies), linearity, accuracy, precision, LOD, LOQ, selectivity, stability in matrix, processed sample stability and robustness should be determined. The validation of the analytical method was based on parameters outlined in Codex or OECD guidelines that don’t include stability in matrix, processed sample stability and robustness. The performance characteristics determined meet the acceptance criteria. However, stability in matrix, processed sample stability and robustness were not determined. Limited information on the stability of clopidol in matrices is available in the public literature. FAO JECFA Monograph 33 27 Matus and Boison (2016) developed an LC-MS method for the determination of coccidiostats in poultry liver and as part of the validation study fortified poultry liver tissues were stored at 20 °C and checked weekly for eight weeks. No significant loss in recovery was reported for clopidol, indicating that it is supposedly stable in poultry liver for at least two months when frozen. However, only limited information was provided. Furthermore, multiple RASFF notifications (Rapid Alert System for Food and Feed – European system for rapid sharing of food safety related issues) for clopidol in poultry muscle have been reported (Banach et al., 2017), which further confirm that clopidol residues in matrices are relatively stable since it has been regularly detected and reported. The Committee concluded that, because the sponsor noted the stability of clopidol in chicken tissues to be 14 days as part of the radiolabelled residue depletion study and because samples collected as part of the non-radiolabelled residue depletion study were stored at -20 °C and analysed within 2 to 3 days of collection, further stability testing was not necessary. Methods of analysis for clopidol residues in tissues Analytical methods for the detection of residual clopidol in a variety of matrices using a variety of techniques have been published over the years. In recent years, LC-MS/MS or liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) using positive mode electrospray ionization (ESI+) appears to be the preferred analytical technique (Dasenaki and Thomaidis, 2019; Moloney et al., 2012; Pietruk et al., 2015; Pietruk, Olejnik and Posyniak, 2018; Rusko et al., 2019; Rydchuk et al., 2023; Valese et al., 2017; Wang et al., 2022), although older examples of gas chromatography (GC)-based methods are also available (Ekström and Kuivinen, 1984; Fang et al., 2009; Pang et al., 2001b). Tandem mass spectrometry is the preferred detection technique due to its combination of high selectivity and high sensitivity. All methods reported in the literature focus on the determination of the parent compound, unmetabolized clopidol. Clopidol is often included in multi-residue methods used for the simultaneous detection of multiple coccidiostats or veterinary drugs. Additionally, multiple screening approaches using different techniques have been published (Chao et al., 2020; Jiang et al., 2014; Li et al., 2020; Radi, El-Naggar and Nassef, 2014). Modern confirmatory methods for clopidol generally include reversed phase liquid chromatography for separation (i.e. Barreto et al., 2017; Pietruk, Olejnik and Posyniak, 2018; Rydchuk et al., 2023; Valese et al., 2017), although an example of a hydrophilic interaction liquid chromatography (HILIC) based method is also available (Dasenaki and Thomaidis, 2019). Generally, C18 columns are used in combination with a gradient elution and a mobile phase consisting of water and acetonitrile including formic acid and ammonium acetate or ammonium acetate as modifiers. After separation, detection and quantitation is generally performed using tandem mass spectrometry in multiple reaction monitoring (MRM) mode, monitoring two ion-transitions which use the protonated molecule as the precursor ion at m/z 192, with m/z 101 and m/z 87 as the quantifying and qualifying ions, respectively (Barreto et al., 2017; Dasenaki and Thomaidis, 2019; Rydchuk et al., 2023). Multiple sample preparation approaches, such as liquid-liquid extractions and dispersive solid phase extraction (dSPE), have been presented and they all seem viable based on the method performance characteristics (Dasenaki and Thomaidis, 2019; Rydchuk et al., 2023). Clopidol is often included in official residue control plans, which means that the published analytical methods are subjected to rigorous validation procedures, such as (EU) 2002/657/EC and (EU) 2021/808, which can help verify their analytical performance. Several validated methods for determining clopidol in muscle and organ tissue deemed representative and most relevant have been described below in more detail. FAO JECFA Monograph 3328 Screening methods Chao et al. (2020) developed a lateral-flow colloidal gold immunoassay (LCGA) for the detection of clopidol in poultry muscle. Samples are extracted using methanol, defatted using n-hexane and then reconstituted using a methanol phosphate buffer solution. The method was validated in terms of selectivity, sensitivity, linearity and recovery. Low cross-reactivity with other coccidiostats and an LOD of 0.14 µg/kg in chicken muscle was reported. Radi, El-Naggar and Nassef (2014) developed an electropolymerized molecularly imprinted polypyrrole polymer modified screen printed carbon electrode (MIP-SPCE) for the detection of clopidol in poultry muscle. Samples were hydrolysed using an ethanol:HCl 1 mol/L mixture (1:1, v/v) and the final mixture was neutralized before analysis. The method was validated in terms of recovery and reproducibility. Jiang et al. (2014) developed a one-step enzyme-linked immunosorbent assay (ELISA) for the detection of clopidol in poultry muscle. Samples are extracted using 0.01 mol/L NaOH:acetonitrile (1:10 v/v) and further diluted using 0.01 mol/L phosphate buffer. The method was validated in terms of matrix effects, selectivity and sensitivity. An LOD of 0.3 µg/kg in poultry muscle was reported. Confirmatory methods Rydchuk et al. (2023) developed an LC-MS/MS method for the determination of 14 coccidiostats, including clopidol, in poultry liver and muscle tissues. Both liver and muscle samples are prepared using a combined acetonitrile and 0.2 mol/L phosphate-citrate buffer extraction of the ground and homogenized material. After pH neutralization, a liquid-liquid extraction is performed using ethyl acetate and dichloromethane. Next, the upper layer is evaporated and reconstituted using 0.5 percent ammonium acetate in 40 percent methanol which is then defatted using n-hexane. Finally, the extract is cleaned up further using a microcentrifuge at 21 000 g. Reversed phase liquid chromatography is performed using a Waters™ ACQUITY UPLC BEH С18 analytical column (50 × 2.1 mm, 1.7 μm) using a gradient elution with 0.1 percent formic acid in water as mobile phase A and 0.1 percent formic acid in methanol as mobile phase B. Finally, detection and quantitation are performed using tandem mass spectrometry in MRM mode. Multiple labelled internal standards were included in the method, although it is not specified which one was used to correct for clopidol. The method was validated as a quantitative confirmatory method according to (EU) 2021/808 guidelines, including identification and confirmation criteria, selectivity, linearity, matrix effects, recovery, repeatability and reproducibility. For chicken muscle, an LOD of 0.9 µg/kg and an LOQ of 3.6 µg/kg were reported, and for chicken liver, an LOD of 1.2 µg/kg and LOQ of 4.1 µg/kg were reported. The method has been used by the national reference laboratory of veterinary drug residues controls in Ukraine. Dasenaki and Thomaidis (2019) report a hydrophilic interaction LC-MS/MS based method for the determination of 16 coccidiostats in poultry, bovine, porcine, ovine and rabbit tissues. Homogenized samples are extracted using acetonitrile and dispersive solid phase extraction (dSPE) clean-up is performed using MgSO4 and C18 sorbent. The extract is evaporated and reconstituted using 1 mmol/L ammonium formate with 0.1 percent formic acid in acetonitrile (20:80, v/v) and filtered (0.22 µm). Chromatography is performed using a Waters™ ACQUITY UPLC BEH HILIC (100 × 2.1 mm, 1.7 μm) column under isocratic elution using acetonitrile as mobile phase A and aqueous ammonium formate 1 mmol/L with 0.1 percent formic acid (v/v) as mobile phase B. Detection and quantitation is performed using tandem mass spectrometry in MRM mode. The electrospray ionization source is operated in positive mode (ESI+). The method was fully validated as a quantitative confirmatory method according to (EU) 2002/657/EC in all aforementioned matrices and the validated parameters include identification and confirmation criteria, selectivity, linearity, matrix effects, recovery, repeatability, reproducibility FAO JECFA Monograph 33 29 and sensitivity. Furthermore, proficiency test materials were analysed for further method performance verification. LODs for clopidol between 0.263 and 0.513 µg/kg were reported for the investigated matrices. The method has been used in the Greek National Residue Control Plan. Barreto et al. (2017) developed an LC-MS/MS method for the determination of 14 coccidiostats, including clopidol, in poultry muscle. Homogenized samples were extracted with acetonitrile using an Ultra-Turrax® system for tissue disruption. A low-temperature clean-up was performed and after centrifugation the supernatant was evaporated. Next, the extract was simultaneously reconstituted and defatted using n-hexane and water:acetonitrile (1:1, v/v). Finally, the aqueous phase was used for analysis. Chromatography was performed with a Agilent Poroshell 120 ECC18 column (50 × 3.0 mm, 2.7 µm) using a gradient elution consisting of water as mobile phase A and acetonitrile as mobile phase B, both containing 5 mmol/L formic acid and 5 mmol/L ammonium acetate. Detection and quantitation were performed using a linear ion trap-tandem mass spectrometer in MRM mode. The electrospray ionization source was operated in positive mode (ESI+). Robenidine-d8 was used as an internal standard. The method was validated as a quantitative confirmatory method according to (EU) 2002/657/EC and Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA) guidelines, including linearity, sensitivity, repeatability, reproducibility, recovery, matrix effects and selectivity for poultry muscle. A calculated LOD of 2.5 µg/kg and LOQ of 6.25 µg/kg for poultry muscle were reported. The method has been used in the Brazilian National Residue Control Plan. Appraisal Clopidol is a coccidiostat registered for use in several Member States at inclusion rates of 80–250 mg/kg feed in broiler chickens and pullets up to 16 weeks of age. The withdrawal periods range from 0–7 days. Clopidol is not authorized for use in laying hens. Metabolism Limited literature exists on the pharmacokinetics and metabolism of clopidol in laboratory animals and most studies were conducted over 50 years ago. The literature suggests that, in the laboratory animal (rats and rabbits), clopidol is rapidly absorbed and eliminated. However, no studies were provided for assessing the pharmacokinetics in chickens or for comparing the metabolism of clopidol in laboratory animals to that in chickens. Nevertheless, similarities in the metabolism reported in published papers on laboratory animals and in the sponsor-provided radiolabelled metabolism study in chickens (Kim, 2023), in which clopidol was identified as the major residue in tissues, were noted. In the study in chickens, the metabolite 3,5-dichloro-2-hydroxymethyl-6-methylpyridin-4-ol was identified. However, this metabolite constituted <10 percent of the TRR and was detected only at 6 h post-dose. While the same metabolite was detected in rabbits, it was not reported in rats, the laboratory species used in the toxicology studies used to determine the acceptable daily intake (ADI). Radiolabelled residue depletion Results from the radiolabelled residue depletion study (Kim, 2023) indicate that clopidol rapidly depletes from chicken tissues, with most of the total radioactivity and clopidol residue concentrations below the LOQ by days 5 and 3 post-dose, respectively. Clopidol constituted >80 percent of the TRR, confirming its suitability as the marker residue. MR:TR could be calculated only at 6 h and 1 day after dosing in all tissues, because of the rapid elimination of clopidol. The sponsor reported MR:TRs of 1 in kidney, muscle, and skin/fat and 0.91 in liver, 1 day after dosing. FAO JECFA Monograph 3330 Limitations with the study were noted. Numerous samples at the 6 h and 1 day timepoints exceeded the VICH-recommended acceptance criterion of ≤5 percent (from -40.2 to +28.8 percent) for tritium exchange with water, suggesting that the tritium label was not stable. Because of this uncertainty, the Committee considered it appropriate to use a conservative MR:TR value of 0.5 in assessing dietary exposure. In addition, clopidol is intended as a continuous use product via feed. Radiolabelled clopidol was administered once via an oral aqueous solution. Although VICH GL46 allows for the employment of gavage or bolus dosing for drugs intended for oral administration, it also is recommended that the dose be divided and given in the morning and afternoon to better approximate actual use conditions. In addition, for continuously administered drugs, a steady state should be achieved. The sponsor did not provide data regarding steady state in the edible tissues of chickens. Clopidol is registered for use at an inclusion rate of 80 mg clopidol/kg feed to 250 mg clopidol/kg feed. In the radiolabelled residue depletion study, chickens were dosed based on their bodyweight. However, based on the calculated mg/kg bodyweight doses for both the 125 and 250 mg feed inclusion rates from the non-radiolabelled residue depletion study of 11 and 22 mg/kg bw per day, respectively, the Committee concluded that the use of a 25 mg/kg bodyweight dose rate was acceptable. The Committee noted that animals were euthanized with xylazine and succinylcholine. However, because the LC-MS/MS was operated in selected reaction monitoring (SRM) mode, interference with chemical analysis due to use of chemical euthanasia was not expected. Lastly, the Committee noted that the numerical values for those residue concentrations determined to be below the LOQ were not provided, so the Committee could not verify that the concentrations were indeed below the LOQ. Non-radiolabelled residue depletion A non-radiolabelled clopidol residue depletion study (Jeong et al., 2023) conducted in chickens continuously fed either the approved label dose of 125 or 250 mg clopidol/kg feed for 14 days, further corroborated previous findings that clopidol rapidly depleted from the edible tissues of broilers. Clopidol concentrations depleted most slowly from skin/fat in both treatment groups. By days 7 and 10 after the final dose, clopidol concentrations in chickens treated with either inclusion rate were below the LOQ (50 µg/kg) in all tissues. No explanation was provided for the similar residue concentrations observed despite the difference in inclusion rates. The Committee noted similar limitations with the sponsor-provided non-radiolabelled residue depletion study as were found with the radiolabelled residue depletion study. Specifically, animals were euthanized with xylazine and succinylcholine and numerical values for those residue concentrations below the LOQ were not provided. Non-radiolabelled residue depletion data found in the published literature are all from studies where the dose rate was 125 mg clopidol/kg feed, which is around half the maximum dose authorized in Member States in accordance with Good Veterinary Practice (GVP). In all three studies (Czeglédi-Jankó, Balla and Tóth, 1976; Ekström, Slanina and Dahlström‐King, 1984; Pang et al., 2001a), samples were analysed for parent clopidol only, which is in line with the pharmacokinetic data which demonstrate only limited metabolism. The duration of treatment differed between the studies. In one study (Pang et al., 2001b), birds were treated for 12 days, in the second (Ekström, Slanina and Dahlström‐King, 1984), the birds were treated for 5 weeks (34 days) and in another (Czeglédi-Jankó, Balla and Tóth, 1976), they were treated for either 10 or 16 weeks before withdrawal of the medicated feed and slaughter for sampling. FAO JECFA Monograph 33 31 In all studies, liver and kidney had the highest levels of residues at each timepoint studied, followed by muscle tissues. None of the studies investigated residues in skin or fat, which is a requirement of VICH GL48. From the studies where the data were presented, there is a trend for those birds that were treated for a longer duration to have lower concentrations of clopidol in their tissues (Figure 5). This may be due to larger birds consuming less feed on a w/w basis, and thus less clopidol, or it may be that elimination becomes more efficient when constantly exposed (increased metabolism due to induction of liver enzymes is less likely to be a factor, as there appears to be no reduction in MR:TRR over time (Kim, 2023)). Other sources of difference between the studies include the strain of chicken used, with faster growing birds becoming more predominant over time, and the analytical methodology used, which is not always described in great detail. Figure 5. Comparison of residue levels in chicken livers from published studies 4 000 5 000 3 000 1 000 2 000 0 Co nc en tr at io n (µ g/ kg ) Sample timepoint (days) 41 2 3 CJ-10 CJ-16 Pang-12 0 Notes: CJ-10: residue levels after treatment with 125 mg/kg feed for 10 weeks, as reported in Czeglédi-Jankó, Balla and Tóth (1976); CJ-16: residue levels after treatment with 125 mg/kg feed for 16 weeks, as reported in Czeglédi-Jankó, Balla and Tóth (1976); Pang-12: residue levels after treatment with 125mg/kg feed for 12 days, as reported in Pang et al. (2001a); bw: body weight Source: based on Czeglédi-Jankó, G., Balla, J., & Tóth, L. 1976. Drug residues in the organs of broiler chickens after flock treatment with Rigecoccin R (clopidol). Acta Veterinaria Academiae Scientiarum Hungaricae, 26(4): 445–53; Pang, G. F., Cao, Y. Z., Fan, C. L., Zhang, J. J. & Li, X. M. 2001a. Determination of clopidol residues in chicken tissues by liquid chromatography: part II. Distribution and depletion of clopidol in chicken tissues. Journal of AOAC INTERNATIONAL, 84(5): 1343–1346. The sponsor-provided non-radiolabelled residue depletion study was sufficient to calculate percentile concentrations and corresponding one-sided 95 percent confidence interval over the 95th percentile of residue concentrations (95/95 upper tolerance limit, UTL) in chicken skin/fat at 1-day withdrawal. Because quantifiable residues in chicken kidney, liver and muscle were found at only two sampling timepoints (day 1 and 3), regression analysis could not be used to determine UTLs in those tissues. Therefore, a 95/95 UTL was calculated at a single timepoint (1-day withdrawal) for these tissues, using the results of the 250 mg/kg feed inclusion rate. FAO JECFA Monograph 3332 Analytical methodology The Committee assessed the validation data against the requirements for analytical methods published in Codex Guideline CAC/GL 71-2009. A sponsor-provided validated LC-MS/MS method was considered suitable for routine monitoring of clopidol as the marker residue in chicken liver, kidney, muscle and skin/fat. The LOQ of the method is 50 µg/kg. In addition, public literature provides information on the development of analytical methods, including extraction, clean-up where required, and analysis of clopidol residues in poultry muscle, liver and kidney tissues. The methods described are single analyte methods, or multi-residue methods, which can detect or confirm multiple coccidiostats in tissue samples. These methods can be used as part of a residues control programme. Recent publications present validation data and confirm the reliability of the methods described. Overall, it is considered that there is enough information in the public domain to allow a Member State or regional competent authority to set up a valid method to analyse chicken tissue samples for residues of clopidol. Dietary exposure assessment Chronic dietary exposure assessment When used as a veterinary drug, dietary exposure to clopidol was estimated based on the potential occurrence of clopidol residues in chicken tissues. Residue concentrations were taken from measurements made at 24-h withdrawal (day 1) for an inclusion rate of 250 mg/kg feed. The studies reported residue concentrations in terms of clopidol (the marker residue). The available studies provide residue data for both chicken liver and kidney. However, the available food consumption information on consumption of chicken kidney includes only a single individual. Given that chicken liver is much more commonly consumed and contained higher residue concentrations than chicken kidney, only chicken liver was included in the assessment of chronic dietary exposure to clopidol. Given uncertainty around the MR:TR, sensitivity of dietary exposure estimates to this parameter was assessed by deriving dietary exposure estimates based on three MR:TRs for all tissue types: 1, 0.9 and 0.5. Clopidol residue values used to estimate dietary exposure were derived from the regression analysis of depletion for skin with fat and from the median residue determined at 24-h for chicken muscle and chicken liver. Based on incurred clopidol residues at 24-h withdrawal time in chicken muscle, chicken liver and skin with fat (250 mg/kg feed) and a MR:TR of 1, the global estimates of chronic dietary exposure (GECDE) for the adults and the elderly, children and adolescents, and for infants and toddlers were 16.5, 16.8 and 14.3 μg/kg bw per day, respectively, which represent 41, 42 and 36 percent, respectively, of the upper bound of the ADI of 40 µg/kg bw (Table 15). Based on incurred clopidol residues at 24-h withdrawal time in chicken muscle, chicken liver and skin with fat (250 mg/kg feed) and a MR:TR of 0.9, the GECDE for adults and the elderly, children and adolescents, and for infants and toddlers were 18.3, 18.6 and 15.9 μg/kg bw per day, respectively, which represent 46, 47 and 40 percent, respectively, of the upper bound of the ADI of 40 µg/kg bw. Based on incurred clopidol residues at 24-h withdrawal time in chicken muscle, chicken liver and skin with fat (250 mg/kg feed) and a MR:TR of 0.5, the GECDE for adults and the elderly, children and adolescents, and for infants and toddlers were 32.9, 33.5 and 28.6 μg/kg bw per day, respectively, which represent 82, 84 and 71 percent, respectively, of the upper bound of the ADI of 40 µg/kg bw. FAO JECFA Monograph 33 33 As part of the GECDE methodology, further estimates of chronic dietary exposure were carried out. Instead of using the highest mean and the highest reliable percentile consumption across surveys, the calculations were carried out using the mean and the highest reliable percentile for each individual national survey from available datasets (FAO/WHO Chronic individual food consumption database, CIFOCOss). The highest GECDE for each age class for each country was determined. For the inclusion rate of clopidol at 250 mg/kg feed and the most conservative MR:TR of 0.5, the mean (range) of 35 country-specific estimates for clopidol dietary exposure for adults and the elderly at 24-h withdrawal was 8.5 (1–27.9) µg/kg bw per day, or 21 percent (2–70 percent) of the upper bound of the ADI. The mean (range) of 26 country-specific estimates of clopidol dietary exposure for children and adolescents at 24-h withdrawal was 13.8 (0.6–33) µg/kg bw per day, or 35 percent (1–83 percent) of the upper bound of the ADI. The mean (range) of 18 country-specific estimates of clopidol dietary exposure for infants and toddlers at 24-h withdrawal was 16 (2.7–27.9) µg/kg bw per day or 40 percent (7–70 percent) of the upper bound of the ADI. As no acute reference dose (ARfD) was necessary, acute dietary exposure (global estimate of acute dietary exposure, GEADE) was not assessed for clopidol. Maximum residue limits In recommending maximum residue limits (MRLs) for clopidol in chicken liver, kidney, muscle and skin/fat, the Committee considered the following factors: z The Committee established an ADI of 0–0.04 mg/kg bw for clopidol. z The Committee concluded that establishment of an ARfD for clopidol was unnecessary. z Clopidol is registered for use in several Member States. The withdrawal periods range from 0–7 days for use of clopidol at inclusion rates of 80–250 mg/kg feed in broilers and replacement layers (pullets) up to 16 weeks of age. z Clopidol is not authorized for use in laying hens. z Clopidol is rapidly absorbed and excreted after oral administration. z Clopidol is a suitable marker residue in all edible tissues of chickens. z In the radiolabel study, numerous samples at both times exceeded the VICH-recommended acceptance criterion for exchange of tritium with water, suggesting that the tritium label was unstable. Because of this uncertainty, the Committee considered it appropriate to use a conservative MR:TR value of 0.5 in assessing dietary exposure. z The study of non-radiolabelled residue depletion at the highest inclusion rate (250 mg/kg feed) was sufficient to determine the mean marker residue and 95/95 UTL concentrations in chicken skin/fat at 1-day withdrawal. z Quantifiable residues in chicken kidney, liver and muscle were found at only two sampling times. Therefore, regression analysis could not be used to determine UTLs in those tissues. A 95/95 UTL was calculated at a single timepoint for these tissues, with the results of the 250 mg/kg feed inclusion rate at 1-day withdrawal. z A validated LC-MS/MS method was considered suitable for routine monitoring of clopidol as the marker residue in chicken liver, kidney, muscle and skin/fat. FAO JECFA Monograph 3334 Ta bl e 15 . G lo ba l e st im at e of c hr on ic d ie ta ry e xp os ur e (G E C D E ) fo r cl op id ol in c hi ck en ti ss ue s Ca te go ry Ty pe M ed ia n co nc en tr at io n1 (µ g/ kg ) M ea n co ns um pt io n, wh ol e p op ul at io n2 (g /k g b w pe r d ay ) H RP co ns um pt io n, co ns um er s o nl y3 (g /k g b w pe r d ay ) M R: TR ra tio Ex po su re (µ g/ kg b w pe r d ay ) G EC DE 4 M ea n H RP µg /k g b w pe r d ay % AD I 25 0 m g/ kg cl op id ol : 2 4 h p os t-w ith dr aw al , M R: TR = 1. 0 Ad ul ts an d th e e ld er ly Po ul try m us cle Ch ick en m us cle 1 24 4 1. 25 11 .2 1. 00 1. 6 14 .0 14 .0 Po ul try o ffa l Ch ick en li ve r 2 80 4 0. 88 1. 59 1. 00 2. 5 4. 4 2. 5 Po ul try tr im m ed fa t Ch ick en fa t 72 5 0. 06 0. 35 1. 00 0. 04 0. 25 0. 04 TO TA L 16 .5 41 Ch ild re n an d ad ol es ce nt s Po ul try m us cle Ch ick en m us cle 1 43 0 2. 75 13 .3 1. 00 3. 4 16 .5 16 .5 Po ul try o ffa l C hi ck en o ffa l 3 85 0 0. 06 2. 04 1. 00 0. 17 5. 7 0. 17 Po ul try tr im m ed fa t Ch ick en fa t 96 0 0. 12 0. 65 1. 00 0. 09 0. 47 0. 09 42 TO TA L 16 .8 In fa nt s a nd to dd ler s Po ul try m us cle Ch ick en m us cle 1 43 0 3. 96 11 .2 1. 00 4. 9 14 .0 14 .0 Po ul try o ffa l C hi ck en o ffa l 3 85 0 0. 06 1. 08 1. 00 0. 25 3. 0 0. 25 Po ul try tr im m ed fa t Ch ick en fa t 96 0 0. 10 0. 63 1. 00 0. 07 0. 45 0. 07 TO TA L 14 .3 36 25 0 m g/ kg cl op id ol : 2 4 h p os t-w ith dr aw al , M R: TR = 0. 9 Ad ul ts an d th e e ld er ly Po ul try m us cle Ch ick en m us cle 1 43 0 1. 25 11 .2 0. 90 1. 7 15 .5 15 .5 Po ul try o ffa l Ch ick en li ve r 3 85 0 0. 88 1. 59 0. 90 2. 7 4. 9 2. 7 Po ul try tr im m ed fa t Ch ick en fa t 72 5 0. 06 0. 35 0. 90 0. 05 0. 28 0. 05 TO TA L 18 .3 46 Ch ild re n an d ad ol es ce nt s Po ul try m us cle Ch ick en m us cle 1 43 0 2. 75 13 .3 0. 90 3. 8 18 .3 18 .3 Po ul try o ffa l C hi ck en o ffa l 3 85 0 0. 06 2. 04 0. 90 0. 19 6. 4 0. 19 Po ul try tr im m ed fa t Ch ick en fa t 96 0 0. 12 0. 65 0. 90 0. 10 0. 52 0. 10 TO TA L 18 .6 47 FAO JECFA Monograph 33 35 Ta bl e 15 . G lo ba l e st im at e of c hr on ic d ie ta ry e xp os ur e (G E C D E ) fo r cl op id ol in c hi ck en ti ss ue s ( co nt .) Ca te go ry Ty pe M ed ia n co nc en tr at io n1 (µ g/ kg ) M ea n co ns um pt io n, wh ol e p op ul at io n2 (g /k g b w pe r d ay ) H RP co ns um pt io n, co ns um er s o nl y3 (g /k g b w pe r d ay ) M R: TR ra tio Ex po su re (µ g/ kg b w pe r d ay ) G EC DE 4 M ea n H RP µg /k g b w pe r d ay % AD I In fa nt s a nd to dd ler s Po ul try m us cle Ch ick en m us cle 1 43 0 3. 96 11 .2 0. 90 5. 5 15 .5 15 .5 Po ul try o ffa l C hi ck en o ffa l 3 85 0 0. 06 1. 08 0. 90 0. 27 3. 4 0. 27 Po ul try tr im m ed fa t Ch ick en fa t 96 0 0. 10 0. 63 0. 90 0. 08 0. 51 0. 08 TO TA L 15 .9 40 25 0 m g/ kg cl op id ol : 2 4 h p os t-w ith dr aw al , M R: TR = 0. 5 Ad ul ts an d th e e ld er ly Po ul try m us cle Ch ick en m us cle 1 43 0 1. 25 11 .2 0. 50 3. 1 27 .9 27 .9 Po ul try o ffa l Ch ick en li ve r 3 85 0 0. 88 1. 59 0. 50 4. 9 8. 9 4. 9 Po ul try tr im m ed fa t Ch ick en fa t 72 5 0. 06 0. 35 0. 50 0. 09 0. 51 0. 09 TO TA L 32 .9 82 Ch ild re n an d ad ol es ce nt s Po ul try m us cle Ch ick en m us cle 1 43 0 2. 75 13 .3 0. 50 6. 8 33 .0 33 .0 Po ul try o ffa l C hi ck en o ffa l 3 85 0 0. 06 2. 04 0. 50 0. 34 11 .4 0. 34 Po ul try tr im m ed fa t Ch ick en fa t 96 0 0. 12 0. 65 0. 50 0. 18 0. 94 0. 18 TO TA L 33 .5 84 In fa nt s a nd to dd ler s Po ul try m us cle Ch ick en m us cle 1 43 0 3. 96 11 .2 0. 50 10 .0 27 .9 27 .9 Po ul try o ffa l C hi ck en o ffa l 3 85 0 0. 06 1. 08 0. 50 0. 49 6. 1 0. 49 Po ul try tr im m ed fa t Ch ick en fa t 96 0 0. 10 0. 63 0. 50 0. 14 0. 91 0. 14 TO TA L 28 .6 71 N ot es : M R : m ar ke r re si du e; T R : to ta l re si du e; H R P : hi gh es t re li ab le p er ce nt il e; G E C D E : gl ob al e st im at es o f ch ro ni c di et ar y ex po su re ; 1 M ed ia n co nc en tra tio n of th e m ar ke r re si du e at t he s pe ci fi ed t im es a ft er t he e nd o f tr ea tm en t ex pr es se d as c lo pi do l; 2 H ig he st m ea n co ns um pt io n fi gu re s ba se d on w ho le p op ul at io n co ns id er ed f ro m t he a va il ab le da ta se t; 3 H ig he st r el ia bl e pe rc en ti le f oo d co ns um pt io n fi gu re s ba se d on c on su m er s on ly c on si de re d fr om th e av ai la bl e da ta se t; 4 G EC D E is th e s um o f t he h ig he st ex po su re at th e hi gh es t r el ia bl e pe rc en til e of c on su m pt io n fo r a fo od a nd th e m ea n di et ar y ex po su re s o f t he o th er fo od . So ur ce : A ut ho rs ’ o w n el ab or at io n. FAO JECFA Monograph 3336 The Committee recommended MRLs of 10 400 µg/kg (liver), 8 800 µg/kg (kidney), 4 100 µg/kg (muscle) and 2 600 µg/kg (skin/fat) in chickens. For calculation of the UTL at a single timepoint, the Committee followed the approach described at https://www.itl.nist.gov/div898/handbook/prc/section2/prc263.htm. The general formula for any UTL is: UTL: mean residue concentration + (k × standard deviation) where k is a factor chosen to ensure the specified coverage and confidence (95/95). FAO JECFA Monograph 33 37 References Banach, J. L., van Asselt, E. D., Hoogenboom, R., Razenberg, L., Boon, P. E., van Horne, P., Mengelers, M. J. B. & van der Fels-Klerx, H. J. 2017. Chemical and physical hazards in the Dutch poultry meat chain. RIKILT report, No. 2017.001. https://doi.org/10.18174/401913 Barreto, F., Ribeiro, C., Hoff, R. B. & Costa, T. D. 2017. A simple and high-throughput method for determination and confirmation of 14 coccidiostats in poultry muscle and eggs using liquid chromatography-quadrupole linear ion trap-tandem mass spectrometry (HPLC-QqLIT-MS/MS): Validation according to European Union 2002/657/EC. Talanta, 168: 43–51. https://doi.org/https://doi. org/10.1016/j.talanta.2017.02.007 Cameron, B. D., Chasseaud, L. F. & Hawkins, D. R. 1975. Metabolic fate of clopidol after repeated oral administration to rabbits. Journal of Agricultural and Food Chemistry, 23: 269–274. https://doi. org/10.1021/jf60198a052 Chao, M., Liu, L., Song, S., Wu, X. & Kuang, H. 2020. Development of a gold nanoparticle-based strip assay for detection of clopidol in the chicken. Food and Agricultural Immunology, 31(1): 489–500. https://doi.org/10.1080/09540105.2020.1737655 Czeglédi-Jankó, G., Balla, J. & Tóth, L. 1976. Drug residues in the organs of broiler chickens after flock treatment with Rigecoccin R (clopidol). 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Food Chemistry, 393: 133260. https://doi.org/10.1016/j.foodchem.2022.133260 FAO JECFA Monograph 3340 Fumagillin dicyclohexylamine1 First draft prepared by Anke Finnah, Berlin, Germany Susanne Rath, Campinas, São Paulo, Brazil Anne-Marie Jacques, Fougères, France Peter Cressey, Christchurch, New Zealand and Holly Erdely, Rockville, United States of America Alan Chicoine, Saskatoon, Canada Rainer Reuss, Canberra, Australia Identity International Non-proprietary Names (INN): Fumagillin dicyclohexylamine Synonyms: Fumagillin: Amebacilin, Flisint, Fugillin, BS-16576, Fumagillinum Dicyclohexylamine: N-cyclohexylcyclohexanamine, cyclohexanamine, N-cyclohexyl-DCHA, DCH IUPAC name: Fumagillin: (2E,4E,6E,8E)-10-{[(3R,4S,5S,6R)-5-methoxy-4-[(2R)-2- methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl] oxy}-10-oxodeca-2,4,6,8-tetraenoic acid Dicyclohexylamine: N-cyclohexylcyclohexanamine Chemical abstract service No.: Fumagillin: 23110-15-8 DCH: 101-83-7 Structural formula: Fumagillin (a) as the dicyclohexylamine salt (b) H ba N H O O O O O O Me O Molecular formula: Fumagillin: C26H34O7 DCH: C12H23N Molecular weight: Fumagillin: 458.54 g/mol DCH: 181.32 g/mol 1　Although the request of the CCRVDF was to evaluate fumagillin, the Committee interpreted the request as an evaluation of fumagillin DCH, as this is the form in which the compound is used as a veterinary drug. FAO JECFA Monograph 33 41 Other information on identity and properties Appearance: Fumagillin: light yellow needles, yellowish white powder DCH: colourless or light-yellow liquid Melting point: Fumagillin: 189–194 °C DCH: -0.1 °C Aggregate state at 20 °C: Fumagillin: solid DCH: liquid Solubility: Fumagillin: in water: 3.3 mg/L, in ethanol or DMSO: soluble; DCH: sparingly soluble in water: 0.08 g/100 mL (at 25 ºC), soluble in ethanol, ether and benzene UVmax: Fumagillin: 330–335 nm; DCH: not applicable Log Kow: Fumagillin: 4.79; DCH (neutral form): 4.37 Background Fumagillin is a mycotoxin originally produced by the fungus Aspergillus fumigatus and currently used in a synthesized form. Fumagillin is registered as a veterinary drug in several Member States. It is used as an antimicrobial compound for the treatment of microsporidian infections in various fish species and in honeybees. Fumagillin has been used in human medicine for certain infectious diseases (Maillard et al., 2021; Molina et al., 2002; Guruceaga et al., 2019), and to treat various cancers, by inhibiting the formation of new blood vessels around growing tumours (angiogenesis), thereby limiting their blood supply (Ingber et al., 1990). Fumagillin is poorly soluble in water and undergoes rapid ultraviolet and thermal degradation. Therefore, to increase its stability and water solubility, commercial formulations used in veterinary medicine contain fumagillin as the dicyclohexylamine (DCH) salt in a 1:1 stoichiometric ratio. The mode of action of fumagillin is based on inhibition of type-2 methionine aminopeptidase (MetAP-2) activity via formation of a covalent bond with the histidine moiety (His231) of the enzyme. MetAP-2 is a cytosolic enzyme which removes the initial methionine from the amino terminus of newly synthesized proteins, for subsequent post-translational modifications, which affects the function of many proteins (Arico-Muendel et al., 2009; Guruceaga et al., 2019). Fumagillin DCH has not previously been evaluated by the Committee. The Committee evaluated fumagillin DCH at the present meeting at the request of the twenty-sixth session of the Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF), with a view to recommending maximum residue limits (MRLs) for fish and for honey. As fumagillin is only used in veterinary medicine as the DCH salt, the majority of data evaluated was on fumagillin DCH. However, because the fumagillin DCH salt dissociates into the two moieties and consumers would be exposed to the residues of both, the Committee evaluated fumagillin and DCH. The sponsor provided unpublished proprietary studies as well as data from studies in the published literature to support the assessment. FAO JECFA Monograph 3342 In addition, the Committee conducted a search of peer-reviewed scientific literature in the following publicly accessible databases: Agricola, Web of Science, PubMed, Springer Protocols, Food Science and Technology Abstracts, PhishPharm, CABI VetMed Resource and ZB Med Search Portal. Keywords relevant to the use, metabolism and pharmacokinetics of fumagillin DCH and residue monitoring in fish species and honeybees were used (Table 1). The literature search resulted in 21 articles that were considered relevant for the evaluation. Table 1. Inclusion and exclusion criteria for the literature search Inclusion criteria Exclusion criteria Any article on fumagillin residues or on DCH residues in any fish species or honey Any article on the efficacy of fumagillin Any article on withdrawal periods of fumagillin in any fish species or honey Any article on environmental contamination with fumagillin or DCH Any article on analytical methods for determining fumagillin or DCH residues Any article on resistance in target organisms Any article on degradation products of fumagillin or DCH Articles in languages other than English Any article on kinetics and metabolism of fumagillin or DCH Any article on MRLs of fumagillin Any publication year Residues in food and their evaluation Conditions of use Fumagillin DCH is approved for the treatment of microsporidia infections in various fish species, including carp (Sphaerospora renicola, Myxobolus cyprinid), eels (Pleistophora giardi, Myxobolus giardia), and trout (Sphaerospora sp., Myxobolus cerebralis) (Kano and Fukui, 1982; Molnar, Baska and Szekely, 1987; Rigos et al., 2000). In apiculture, fumagillin is approved for treatment of infections caused by Nosema apis and Nosema ceranae (van den Heever et al., 2016; Huang et al., 2013). In several European countries (the United Kingdom of Great Britian and Northern Ireland, Spain, Belgium, Greece, Hungary, and Romania) exceptional temporary authorization has previously been given to use fumagillin under veterinary supervision to treat nosemosis in positively diagnosed apiaries (Higes et al., 2011). Dosage The dosage used in fish is 15–50 mg of fumagillin base per kg bw in medicated feed for 30 consecutive days, or 60 mg fumagillin base per litre of water in an immersion bath for five consecutive days. According to good veterinary practice (GVP), 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 20–25 mg fumagillin base per litre of sugar, administered once weekly for 6–8 weeks. Bees should be treated in the autumn after honey supers have been removed, or in the spring, with treatment completed 4 weeks before the start of honey flow. Fumagillin DCH is not currently registered for use as a pesticide. FAO JECFA Monograph 33 43 Pharmacokinetics and metabolism Pharmacokinetics and metabolism in laboratory animals No data on the pharmacokinetics and metabolism of fumagillin in laboratory animals were available. To provide further context on the pharmacokinetics and metabolism of the DCH component of fumagillin DCH, the Committee evaluated one published study on metabolism and excretion of DCH (administered as DCH alone) in laboratory animals (Suenaga, Wada and Ichibagase, 1983). In rabbits and rats, absorption rate constants from the small intestines were determined as 0.44 and 0.33 per hour, respectively, after gavage treatment with DCH at doses of 50 mg/rabbit and 5 mg/rat for 23 to 43 days, indicating that intestinal absorption of DCH is rapid in both species. Urinary and faecal excretion of unchanged DCH was low in both species (0.08 percent and 0.30 percent of the administered dose in rabbits and 5.88 percent and 0.25 percent in rats, respectively) over 2 days after administration of DCH, suggesting that the substance is quickly metabolized. In liver supernatant from rabbits and rats in vitro, DCH was rapidly metabolized in rabbits but not in rats under aerobic conditions and was metabolized only slightly under anaerobic conditions in both species. The metabolites were not identified. Pharmacokinetics and metabolism in food-producing animals One published paper on the kinetics of fumagillin in fish (rainbow trout, Oncorhynchus mykiss) was available, and one study of metabolism in rainbow trout was provided by the sponsor. Fumagillin In a study reported in the literature (Laurén et al., 1989), the plasma kinetics of fumagillin was evaluated in rainbow trout (both sexes; bw 100–300 g). The fish were kept at a water temperature of 15 °C, and fumagillin DCH was administered directly into the dorsal aorta at four doses (60, 30, 6 and 3 mg/kg bw). For the two high dosage groups (30 and 60 mg/kg bw), no pharmacokinetics results were reported due to mortality of the test animals (death within 360 minutes). Histological examination revealed extensive toxic alteration in liver and posterior kidneys. At the two lower dosage groups, plasma clearance of fumagillin at both dosage regimens fit a two-compartment model with a rapid alpha phase (estimated half-life, about 20 min) and a prolonged beta phase (5.4 days). As the slopes of the alpha and beta phases were similar at the two doses, the data were combined for calculation of kinetics parameters. The calculated volume of distribution was high (231 ± 64 mL/kg) (Table 2). DCH concentrations were not measured. A study of fumagillin metabolism in rainbow trout (one-year old; n = 50; 100 to 275 g bw), which was reported to be compliant with good laboratory practice (GLP), was provided by the sponsor (Kim, 2023). The test substance was a mixture of tritium-radiolabelled fumagillin and unlabelled fumagillin DCH. The fish received a dose of 50 mg/kg bw fumagillin by gavage. The water temperature was kept at 15 °C. The sponsor reported that fumagillin was randomly labelled with tritium; as such, the precise positions of the tritium labels in the fumagillin molecule were unknown. The extent of exchange of the tritium radiolabel with water was assessed 6 hours after dosing. Most samples exceeded the acceptance criterion of ≤5 percent recommended by the Veterinary International Committee on Harmonization (VICH, 2011) (range, from -22.2 to +15.6 percent), suggesting that the tritium label was unstable. Almost 50 percent of the radioactive residues could not be extracted, although various solvents were tested, including toluene, acetonitrile acidified to pH 3 and acetonitrile adjusted to pH 10. No explanation was provided for the limited extractability of the radioactive residue. FAO JECFA Monograph 3344 Table 2. Plasma pharmacokinetic parameters of fumagillin in rainbow trout after intra-aortal administration Dose 6 mg/kg bw (n = 2) 3 mg/kg bw (n = 2) Weighta (g) 313.6 ± 19.6 C (µg/mL) 53.1, 32.7 21.1, 9.5 A (µg/mL) 46.9, 26.2 19.3, 7.3 αa (min-1) 0.0172 ± 0.0043 T½ α (min) 20.7 ± 4.6 B (µg/mL) 6.2, 6.5 2.2, 1.8 βa (min-1) 0.00004 ± 0.00002 T½ βa (min) 7734 ± 2960 Vdαa (mL/kg) 231 ± 64 Notes: aValues from pooled data, n=4; C: concentration; A, α, B and β: macro-constants of the compartment model; T½ α: half-life of the alpha phase; T½ β: half-life of the beta phase; Vdα: volume of distribution of the alpha phase Source: adapted from Laurén, D. J., Wishkovsky, A., Groff, J. M., Hedrick, R. P. & Hinton, D. E. 1989. Toxicity and pharmacokinetics of the antibiotic fumagillin in yearling rainbow trout (Salmo gairdneri). Toxicology and Applied Pharmacology, 98(3): 444–453. The concentration of fumagillin in fillet ranged from 0.2 to 2.3 mg equiv/kg (0.2–2.1 percent of the total administered dose), whereas the concentrations of unextractable residues ranged from 0.4 to 1.4 mg equiv/kg (0.3–1.7 percent of the total administered dose) throughout the study. Only the parent compound, fumagillin, was identified in fillet. It was therefore proposed that fumagillin was not metabolized. As no radiolabelled DCH was used in this study, no data were available on the metabolism and depletion of DCH in fish. Like other veterinary drugs used in apiculture, fumagillin DCH does not appear to be metabolized in honeybees, and most fumagillin DCH is likely to end up in beeswax and honey. Degradation products The fumagillin portion of fumagillin DCH is subject to degradation under conditions relevant for the treatment of fish and honeybees (Figure 1). It can be degraded by exposure to light, producing biologically active degradation products with activity similar to that of fumagillin (Kochansky and Nasr, 2004), or hydrolysed under basic conditions to produce fumagillol, which has about 10 percent of the biological activity of fumagillin (Gochnauer and Furgala, 1962). Thermal degradation of fumagillin leads to formation of dihydroxyfumagillin, a biologically inactive compound (Kochansky and Nasr, 2004). Based on structural considerations, the Committee concluded that fumagillin degradation products are unlikely to be of greater toxicological concern than the parent compound (see Fumagillin DCH toxicological monograph). The Committee noted that no information on concentrations of fumagillin degradation products in fish tissues or honey was available. Comparative metabolism No data was provided to the Committee, or available from the published literature, to allow comparison of the metabolism between species. FAO JECFA Monograph 33 45 Figure 1. Fumagillin and its degradation products identified in honey O OH Fumagillin UV degradation product Hydrolysis product (fumagillol) Thermal degradation product (dihydroxyfumagillin) O O O O O O OH O O O O HO HO O OH O O O O O OH O O O Sources: van den Heever, J. P., Thompson, T. S., Curtis, J. M. & Pernal, S. F. 2015a. Determination of Dicyclohexylamine and Fumagillin in Honey by LC-MS/MS. Food Analytical Methods, 8(3): 767–777; Nozal, M. A. J., Bernal, J. L., Martín, M. A. T., Bernal, J., Alvaro, A., Martín, R. & Higes, M. 2008. Trace analysis of fumagillin in honey by liquid chromatography-diode array-electrospray ionization mass spectrometry. Journal of Chromatography A, 1190(1-2): S. 224–231. Tissue residue depletion studies One study on the residue depletion of radiolabelled fumagillin in rainbow trout and 12 studies of residue depletion with unlabelled fumagillin DCH in rainbow trout, carp and eels were provided by the sponsor. All the studies were reported to be GLP-compliant. Studies with each fish species given unlabelled fumagillin DCH were conducted at two water temperatures and two administration routes (oral in feed and via immersion bath). One study of residue depletion with non-radiolabelled fumagillin DCH in honeybees was provided by the sponsor. FAO JECFA Monograph 3346 Radiolabelled residue depletion studies Fish In the radiolabelled residue depletion study (Kim, 2023), 50 one-year-old rainbow trout (bw 100–275 g), were treated with a mixture of [3H]-fumagillin and unlabelled fumagillin DCH at a total dose of about 50 mg/kg bw of fumagillin. To achieve the intended dose, a solution of 50 000 mg/L was prepared from 0.02 mg [3H]-fumagillin (as fumagillin base), and 499.98 mg unlabelled fumagillin (present as fumagillin DCH) added to 10 mL of 0.5 percent CMC solution (carboxymethylcellulose sodium salt in sterilized water). The test was performed in a circular tank system at a water temperature of 15 ± 3 °C. For dose administration, test animals were anesthetized by bathing the fish for 2-3 minutes in ethyl 3-aminobenzoate methanesulfonate at a concentration of approximately 70 mg/L. Fish were treated via gavage based on their individual body weights within 3 minutes of being anaesthetised. The fumagillin was randomly labelled with tritium. The extent of exchange of the tritium radiolabel with water was assessed 6 hours after dosing. Numerous samples exceeded the VICH-recommended acceptance criterion (VICH, 2011) of <5 percent (range, from -22.2 to +15.6), indicating that the tritium label was unstable. A dose of 50 mg/kg bw of fumagillin was administered via gavage. Fillet samples from ten fish per timepoint were collected at 6 and 12 hours, and 1, 2, and 7 days after administration. Individual body weights were recorded after removing surface water from the fish and before tissue collection. The tissues were homogenized using a sample mixer and dry ice, after which they were stored at approximately -20 °C until analysis. Storage stability samples were prepared by spiking a known amount (10 percent of total administration dose) of [3H]-fumagillin into untreated samples. These samples were stored under the same conditions as the samples from the dosing group. Validated liquid scintillation counting (LSC) and radio-HPLC (high performance liquid chromatography) methods were used to determine the concentrations of radiolabelled fumagillin. The radiochemical purity of [3H]-fumagillin, determined with a radio-HPLC method, was reported to be 100 percent. To assess the extent of exchange with water, the radioactivity values of both wet and dry samples were measured using LSC. The dry samples were prepared as follows: muscle samples (0.5 g) were dried at 50 °C for approximately 12 hours, and 5 mL of soluene-350 solution (tissue solubilizer) was added. The samples were shaken at 250 rpm and 50 ± 1 °C for approximately 12 hours until they were completely dissolved. Subsequently, an aliquot (1 mL) was mixed with a scintillation cocktail (12 mL), and the radioactivity was measured by LSC. The wet samples collected from the rainbow trout were solubilized without drying and measured using the same method as described above. For sample extraction, 10 g of muscle sample was transferred into a 50 mL tube and extracted up to two times with 80 percent acetonitrile in water first, followed by 0.1 percent formic acid in acetonitrile. The extracts were combined, and the total volume was measured. An aliquot (30 mL) of the extract was evaporated to approximately 0.05 mL using nitrogen gas, and subsequently brought up to 0.3 mL with a mixture of 5 mM ammonium formate and 0.1 percent formic acid in methanol. Afterwards, the solution was centrifuged at 12 000 rpm (4 °C) for 3 minutes, and 0.1 mL of the supernatant was mixed with 4 mL of scintillation cocktail for radioactivity quantification using LSC. Following this, the solution was analysed by radio-HPLC for metabolite characterization. The remaining tissue after extraction was air-dried for an unknown time and thoroughly mixed. The total weight was measured, and an aliquot (ca. 0.1 g) of the dried sample was combusted in a sample oxidizer followed by LSC analysis to determine the amount of radioactivity in the non-extractable residues. Monophase-S was used as 3H liquid scintillator. FAO JECFA Monograph 33 47 The efficiency (recovery) of the radioactivity by the sample oxidizer was determined by combustion of the control tissue sample, spiked with a known amount of radiolabelled fumagillin, to check combustion and trapping efficiencies, which was 93.7–98.8 percent. The recovery test was performed before analysing the samples. The radiochemical purity of [3H]-fumagillin was determined, using a radio-HPLC method, to be 100 percent, indicating the absence of any other radioactivity apart from [3H]-fumagillin. The identification of [3H]-fumagillin was initially confirmed through HPLC using authentic analytical standards (non-radiolabelled fumagillin) by comparing the retention times. Both analytical methods were validated and values for the limit of quantification (LOQ) and the limit of detection (LOD) are provided in Table 3. Linearity was observed between 0.0001 and 0.01 mg/L with a correlation coefficient greater than 0.999. Table 3. Calculation of LOD and LOQ for LSC-analysis and for radio-HPLC analysis LSC analysis Background (dpm)a LOD (dpm) LOQ (dpm) Actual LOD LOQ %TRAb mg/kg %TRA mg/kg Muscle extract 21.5 13.4 58.0 0.000011 0.00010 0.000047 0.00044 Muscle unextractable 79.5 24.8 91.4 0.000020 0.00019 0.000074 0.00069 Radio-HPLC analysis LOQ (dpm)c Actual LOQ %TRA mg/kg Muscle Extract 12 400.00 0.01 0.09 Notes: total radioactivity administered (TRA) average 123 630 000 dpm; specific activity 2 538 GBq/mmol (3.321 × 108 dpm/ug); aMeasured from control sample; bTotal radioactivity administered; cThe lowest dpm detected by radio-HPLC Source: adapted from Kim, J.-H. 2023. Final Report: Metabolism and Residue Kinetics of [3H]-Fumagillin in Rainbow Trout. Republic of Korea, KRICT. The injection precision of the radio-HPLC was assessed by injecting a standard solution containing 10 ng/mL of [3H]-fumagillin six times. The precision, expressed as the coefficient of variation, was determined to be 0.6 percent. The accuracies, represented by the average recoveries across all fortified levels, ranged from 96.9 to 102.3 percent. Precision (CV) was below 2 percent, meeting the acceptance criteria of ≤10 percent. Unextractable residues ranged from 1.9 to 4.4 percent and total recoveries were from 100.8 to 106.7 percent. To evaluate the stability of fumagillin in tissue samples under frozen conditions below -20 °C, a study on storage stability was conducted. The mean storage stability of the tissue samples treated with [3H]-fumagillin was 95.5 ± 1.1 percent for 14 days, showing that [3H]-fumagillin in tissue was stable for at least 14 days. The homogeneity of the dose formulation in 0.5 percent CMC solution was evaluated, yielding a coefficient of variation of 3.9 percent across concentrations at the top, middle and bottom, satisfying the acceptance criteria (≤10 percent). In addition, the concentration met acceptance criteria (100 ± 15 percent of the nominal concentration). FAO JECFA Monograph 3348 To assess the extent of tritium exchange with water, the wet and dry samples of each tissue were analysed by comparing the radioactivity of both samples. Most of the samples showed negative values; however, some fillet tissues exhibited a maximum ratio of 15.6 percent at 6 hours after administration. The amount of fumagillin recovered in the extra ct accounted for 1.1 percent of the total administered dose (TAD) at 6 hours after administration, equivalent to 1.2 mg/kg. Fumagillin levels increased to 2.1 percent of TAD at 12 hours, corresponding to 2.3 mg equiv/kg. However, the percentage levels on days 1, 2, and 7 continuously declined to 1 percent, 0.9 percent, and 0.2 percent of TAD, respectively, corresponding to 1 mg/kg, 0.9 mg/kg, and 0.2 mg/kg, respectively. The unextractable residue of the total radioactive residue (TRR) recovered accounted for 1.2 percent of TAD at 6 hours after administration, corresponding to 1.3 mg/kg. However, by day 7, the TRR decreased to 0.3 percent of TAD, corresponding to 0.4 mg/kg. After administering fumagillin to rainbow trout, the unchanged fumagillin detected by radio-HPLC in the muscle tissues was collected and subjected to analysis using liquid chromatography-mass spectrometry (LC-MS) for identification of fumagillin. The mass spectra indicated the presence of ions at m/z 459 as the protonated molecular ion (M+H+) and m/z 481 as the sodium adduct molecular ion (M+Na+). The identification and confirmation of the unchanged fumagillin were achieved by comparison with an authentic fumagillin standard. The TAD values obtained through additional extraction with toluene, acidic, and basic solutions ranged from 0.006 to 0.008 percent, 0.13 percent, and from 0.07 to 0.09 percent, respectively, corresponding to 0.006–0.007 mg/kg, 0.12–0.14 mg/kg, and 0.07–0.1 mg/kg. These findings indicate that a large percentage of the TRR (from 38 to 63.2 percent) was present in the fraction of the unextractable residues (Table 4). Table 4. Distribution of radioactivity in tissues following oral administration of [3H]-fumagillin to rainbow trout, percent of total radioactive residues and mean values ± SD from 10 fish per time point Time (days) Fraction Mean ± SD %TRR mg equiv/kg 6 h Extract 48.5 ± 6.0 1.2 ± 0.4 Unextractable 51.6 ± 6.0 1.3 ± 0.5 Total 100.0 ± 0.0 2.5 ± 0.9 12 h Extract 62.0 ± 14.0 2.3 ± 1.4 Unextractable 38.0 ± 14.0 1.2 ± 0.5 Total 100.0 ± 0.0 3.5 ± 1.3 1 Extract 41.5 ± 5.1 1.0 ± 0.4 Unextractable 58.5 ± 5.1 1.4 ± 0.5 Total 100.0 ± 0.0 2.4 ± 0.9 2 Extract 36.9 ± 10.7 0.9 ± 0.5 Unextractable 63.2 ± 10.7 1.7 ± 0.9 Total 100.0 ± 0.0 2.6 ± 1.2 7 Extract 55.8 ± 30.9 0.2 ± 0.0 Unextractable 63.2 ± 6.0 0.4 ± 0.1 Total 100.0 ± 0.0 0.5 ± 0.2 Notes: %TRR: percent of the total radioactivity residue; SD: standard deviation; LOQ: <0.09 mg/kg Source: adapted from Kim, J.-H. 2023. Final Report: Metabolism and Residue Kinetics of [3H]-Fumagillin in Rainbow Trout. Republic of Korea, KRICT. FAO JECFA Monograph 33 49 The concentration of fumagillin in rainbow trout fillet increased from 1.2 mg equiv/kg at 6 hours after dosing to 2.3 mg equiv/kg at 12 hours, and then decreased to 0.2 mg equiv/kg on day 7. The radiolabelled residue depletion study indicates that the parent compound, fumagillin, is a suitable marker residue. Residue depletion studies with non-radiolabelled drug Fish The sponsor submitted residue depletion studies reported as GLP-compliant with non-radiolabelled fumagillin in rainbow trout, carp and eels (National Institute of Food and Drug Safety Evaluation (NIFDS), 2015a, 2015b, 2015c). For each species, studies were conducted at two water temperatures and two administration routes (oral via feed and immersion bath). A nominal dose of 50 mg/kg bw for 30 consecutive days was used for oral administration; for the immersion bath, fish were exposed to fumagillin at a concentration of 60 mg/L for 5 consecutive days. The product used in these studies was Fumagil-C, containing 50 g/kg of fumagillin (administered as fumagillin DCH), and 13.6 g/kg of ascorbic acid, in glucose. Medicated feed was prepared by mixing Fumagil-C with feed under light-protected conditions, and fish oil was uniformly sprayed onto the medicated feed to prevent release of the drug into the water. Fumagillin was quantified in the medicated feed and constituted 80–110 percent of the intended concentration. Fumagillin was not quantified in the water of the immersion bath. The feeding rate was 0.5–1.0 percent of the fish body weight, considering the specific appetite levels of the fish species tested. The medicated feed was administered every morning at the same time and the process was considered complete if consumption occurred within 30 minutes of supply. Details such as the exact quantity of feed administered and consumed, and the weight of the fish in each tank, were not provided. In the studies provided, the assessment was solely of depletion of fumagillin. Residues of DCH were not quantified in the sampled tissues. Ten fish were euthanized at each of 1, 3, 7, 14 and 28 days after the last oral dose and 1, 3 and 7 days after exposure in an immersion bath, and samples of fillet (muscle with skin in natural proportions) were collected. Fumagillin was quantified in the samples with a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method separately validated for each species. The LOQ for fumagillin in fish fillet for all species was 5 µg/kg. Rainbow trout The sponsor conducted four studies using non-radiolabelled fumagillin (test product Fumagil-C) in rainbow trout. These included two studies involving the oral administration of fumagillin via medicated feed at two different water temperatures (15 ± 3 ºC and 22 ± 3 ºC), as well as two studies exposing fish to fumagillin in an immersion bath at two different water temperatures (13 ± 3 ºC and 25 ± 3 ºC) (NIFDS, 2015a). Rainbow trout, weighing 720 ± 90 g, were housed in a concrete tank measuring 5 × 5 × 1.5 m for acclimation, administration, and sampling purposes. Prior to the start of the experiment, a 2 to 4-week acclimation period was observed under laboratory conditions with voluntary feeding. At the end of treatment, fillet and mixed organ samples were collected at various timepoints, and the quantification of fumagillin was carried out using a validated LC-MS/MS method. The calibration graph for fumagillin exhibited linearity (r > 0.99) over the concentration range of 5–500 µg/kg, with an LOQ of 5 µg/kg. FAO JECFA Monograph 3350 Oral administration Two separate studies were conducted at two water temperatures (15 and 22 ºC). Ten fish were euthanized at each of 1, 3, 7, 14 and 28 days after the last treatment and fillet and mixed organ samples (liver, kidney, spleen, stomach, and intestine) were collected. The concentrations of fumagillin determined in the fillet samples at 15 °C and 22 °C are shown in Table 5 and Table 6, respectively. Table 5. Concentration of fumagillin in fillet from rainbow trout with time after oral daily dose (medicated feed) of fumagillin (50 mg/kg bw) for 30 days at 15 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 15 15 10 678.38 10 444.31 1 452.67 319.59 3 45 15 10 504.98 10 86.24 1 208.00 381.35 7 105 15 10 75.52 6 <LOQ 205.20 70.62 14 210 15 10 <LOQ 0 <LOQ <LOQ - 28 420 15 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015a. Final Report (A): A Study on Residues Depletion of Fumagillin in Rainbow Trout. Republic of Korea, NIFDS. Table 6. Concentration of fumagillin in fillet from rainbow trout with time after oral daily dose (medicated feed) of fumagillin (50 mg/kg bw) for 30 days at 22 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 22 22 10 1 558.46 10 566.50 3 258.40 915.60 3 66 22 10 496.93 10 58.41 1 113.45 329.76 7 154 22 10 47.14 4 <LOQ 105.2 43.09 14 308 22 10 <LOQ 0 <LOQ <LOQ - 28 616 22 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015a. Final Report (A): A Study on Residues Depletion of Fumagillin in Rainbow Trout. Republic of Korea, NIFDS. In the mixed organs of fish maintained at 15 ºC, the mean (±SD) residue levels of fumagillin (considering only the values above the LOQ) were 1 299.43 ± 694.65 μg/kg on day 1 and 897.36 ± 656.25 μg/kg on day 3. On day 7, fumagillin was detected in 7 out of 10 mixed organ samples with levels of 96.68 ± 104.89 μg/kg. On day 14, fumagillin was quantified in 4 out of 10 samples at a level of 7.72 ± 2.82 μg/kg. After that, no fumagillin was detected in any of the samples. FAO JECFA Monograph 33 51 In the mixed organs of fish maintained at 22 ºC, the mean (±SD) residue levels of fumagillin (considering only the values above the LOQ) were 2 787.44 ± 1 623.04 μg/kg on day 1 and 950.15 ± 660.31 μg/kg on day 3. On day 7, fumagillin was detected in 5 out of 10 samples with levels of 44.75 ± 45.85 μg/kg. On day 14, fumagillin was quantified in 4 out of 10 samples at a level of 11.48 ± 6.55 μg/kg. After that, no fumagillin was detected in any of the samples. Figure 2 shows the concentration of fumagillin in rainbow trout fillet versus degree-days post-dose from the residue depletion studies conducted at two different water temperatures. Figure 2. Residue depletion profile of fumagillin in rainbow trout fillet at two water temperatures after in-feed administration of fumagillin 100 1 000 10 0 Co nc en tr at io n of fu m ag ill in ( µg /k g) 10 000 Degree-days post-dose Water temperature 15 °C 500100 200 300 4000 100 1 000 10 0 Co nc en tr at io n of fu m ag ill in ( µg /k g) 10 000 Degree-days post-dose Water temperature 22 °C 22 66 154 308 616 Source: Authors’ own elaboration, based on data submitted to the Committee. Immersion bath Rainbow trout were exposed to fumagillin in an immersion bath at a concentration of 60 mg/L for 5 days at two water temperatures, 13 ± 3 ºC and 25 ± 3 ºC. Ten fish were euthanized at each of 1, 3 and 7 days after treatment and fillet samples were collected. The concentrations of fumagillin determined for the lower and higher temperatures are shown in Table 7 and Table 8, respectively. Table 7. Concentration of fumagillin in fillet from rainbow trout with time after immersion bath of fumagillin (60 mg/L) for 5 days at 13 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 13 13 10 22.21 9 <LOQ 94.43 27.71 3 39 13 10 <LOQ 0 <LOQ <LOQ - 7 91 13 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015a. Final Report (A): A Study on Residues Depletion of Fumagillin in Rainbow Trout. Republic of Korea, NIFDS. FAO JECFA Monograph 3352 Table 8. Concentration of fumagillin in fillet from rainbow trout with time after immersion bath of fumagillin (60 mg/L) for 5 days at 25 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 25 25 10 50.11 10 11.25 103.43 33.70 3 75 25 10 7 1 <LOQ 7 - 7 175 25 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015a. Final Report (A): A Study on Residues Depletion of Fumagillin in Rainbow Trout. Republic of Korea, NIFDS. Figure 3 shows the concentration of fumagillin in rainbow trout fillet versus degree-days post-dose from the depletion studies at two different water temperatures. Figure 3. Residue depletion profile of fumagillin in rainbow trout fillet at two water temperatures after immersion bath treatment with fumagillin at a dose of 60 mg/L 10 100 0 Co nc en tr at io n of fu m ag ill in ( µg /k g) 1 000 Degree-days post-dose Water temperature 13 °C 10020 40 60 800 Co nc en tr at io n of fu m ag ill in ( µg /k g) Degree-days post-dose Water temperature 25 °C 500 100 150 200 10 100 1 000 0 Source: Authors’ own elaboration, based on data submitted to the Committee. In the published literature, one residue depletion study of fumagillin, as DCH salt, in rainbow trout (>50 g) is described by Guyonnet et al. (1995). Two doses were evaluated; 3 mg/kg bw and 15 mg/kg bw. The fish were held at constant water temperatures (16–17ºC) and a water flow of 150 L/min. Fumagillin was administered as medicated feed (5 g of fumagillin as the DCH salt per 100 g of feed) twice per day for 10 consecutive days. Twelve fish per timepoint were euthanized at 1 hour, and at 4, 10 and 21 days after the end of the treatment. Muscle samples were collected and fumagillin determined by an HPLC-UV method. The LOQ and LOD of the method were 20 µg/kg and 7 µg/kg, respectively. The mean concentrations of fumagillin (± SD) determined at 1 hour post-dose was 30 ± 8 µg/kg and 46 ± 24 µg/kg for the doses of 3 and 15 mg/kg bw, respectively. In the muscle sampled on 4, 10 and 21 days post-dose, no fumagillin was detected. FAO JECFA Monograph 33 53 Carp Four studies were conducted using non-radiolabelled fumagillin (test product Fumagil-C) in Carp (Cyprinus carpio). These comprised two studies involving the oral administration of fumagillin via medicated feed at two water temperatures (13 ± 3 ºC and 25 ± 3 ºC), and two studies exposing fish to fumagillin via immersion bath at two water temperatures (13 ± 3 ºC and 25 ± 3 ºC) (NIFDS, 2015b). The carp (750 ± 50 g bw) were housed in a concrete tank (5 × 5 × 1.5 m) for acclimation, administration, and sampling. Prior to the start of the experiment, they were acclimated under laboratory conditions for 2–4 weeks with voluntary feeding. Fillet and mixed organs were sampled and fumagillin was quantified by a validated LC-MS/MS method. The calibration graph for fumagillin was linear (r > 0.99) in the concentration range of 5 to 500 µg/kg, with an LOQ of 5 µg/kg. Oral administration Carp were treated with fumagillin, once a day, via feed at a nominal dose of 50 mg/kg bw for 30 consecutive days. Two studies were carried out at different water temperatures (13 and 25 ºC). Ten fish were euthanized at each of 1, 3, 7, 14 and 28 days after the treatment and fillet and mixed organ samples (liver, kidney, spleen, stomach, and intestine) were collected. Fumagillin was quantified by a validated LC-MS/MS method. The concentrations of fumagillin determined in the samples analysed in the two studies are shown in Table 9 and Table 10, respectively. Table 9. Concentrations of fumagillin in carp fillet with time after oral daily dose (medicated feed) of fumagillin (50 mg/kg bw) for 30 days at 13 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 13 13 10 1 224.13 10 534.28 2 566.93 765.80 3 39 13 10 404.50 10 79.31 885.45 272.76 7 91 13 10 37.26 6 <LOQ 87.33 28.15 14 182 13 10 8.5 1 <LOQ 8.5 - 28 364 13 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015b. Final Report (B): A Study on Residues Depletion of Fumagillin in Carp. Republic of Korea, NIFDS. FAO JECFA Monograph 3354 Table 10. Concentrations of fumagillin in carp fillet with time after oral daily dose (medicated feed) of fumagillin (50 mg/kg bw) for 30 days at 25 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 25 25 10 2 256.13 10 867.45 5 234.27 1 732.10 3 75 25 10 658.01 10 205.35 1 282.00 356.94 7 175 25 10 59.02 8 <LOQ 122.34 38.45 14 350 25 10 <LOQ 0 <LOQ <LOQ - 28 700 25 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015b. Final Report (B): A Study on Residues Depletion of Fumagillin in Carp. Republic of Korea, NIFDS. Figure 4 shows the concentrations of fumagillin in carp fillet versus days post-dose from the depletion studies at two different water temperatures. Figure 4. Residue depletion profile of fumagillin in carp fillet at two water temperatures after oral administration of fumagillin 100 1 000 10 0 Co nc en tr at io n of fu m ag ill in ( µg /k g) 10 000 Degree-days post-dose Water temperature 13 °C 100 200 300 4000 100 1 000 10 0 Co nc en tr at io n of fu m ag ill in ( µg /k g) 10 000 Degree-days post-dose Water temperature 25 °C 200 400 600 8000 Source: Authors’ own elaboration, based on data submitted to the Committee. In the mixed organs of carp maintained at 13 ºC, the mean (± SD) residue levels of fumagillin (considering only the values above the LOQ) were 1 894.68 ± 880.31 μg/kg on day 1 and 724.06 ± 488.24 μg/kg on day 3. On day 7, fumagillin was quantified in 7 out of 10 samples with levels of 76.65 ± 89.81 μg/kg. On day 14, fumagillin was quantified in 4 out of 10 samples at a level of 10.77 ± 7.84 μg/kg. After that, no fumagillin was detected in any of the samples. In the mixed organs of carp maintained at 25 ºC, the mean (± SD) residue levels of fumagillin (considering only the values above the LOQ) were 2 926.57 ± 1 785.48 μg/kg on day 1 and 1 171.26 ± 635.35 μg/kg on day 3. On day 7, fumagillin was quantified in 8 out of 10 samples with levels of 130.20 ± 82.68 μg/kg. On day 14, fumagillin was quantified in 2 out of 10 samples at a level of 6.78 ± 0.61 μg/kg. After that, no fumagillin was detected in any of the samples. FAO JECFA Monograph 33 55 Immersion bath Carp were exposed to fumagillin via an immersion bath at a concentration of 60 mg/L for 5 days at two water temperatures, 13 ± 3 ºC and 25 ± 3 ºC. Ten fish were sampled at each of 1, 3 and 7 days after the treatment and fillets were collected. Fumagillin was quantified by a validated LC-MS/MS method. The concentrations of fumagillin determined in the fillet samples analysed in the two studies at 13 and 25 ºC are shown in Table 11 and Table 12, respectively. Table 11. Concentrations of fumagillin in carp fillet with time after immersion bath of fumagillin (60 mg/L) for 5 days at 13 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 13 13 10 26.48 10 6.71 81.31 24.44 3 39 13 10 17.22 4 6.60 28.50 9.75 7 91 13 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015b. Final Report (B): A Study on Residues Depletion of Fumagillin in Carp. Republic of Korea, NIFDS. Table 12. Concentrations of fumagillin in carp fillet with time after immersion bath of fumagillin (60 mg/L) for 5 days at 25 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 25 25 10 34.39 7 <LOQ 67.21 22.70 3 75 25 10 11.2 1 11.20 11.20 - 7 175 25 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015b. Final Report (B): A Study on Residues Depletion of Fumagillin in Carp. Republic of Korea, NIFDS. Figure 5 shows the concentrations of fumagillin in carp fillet versus degree-days post-dose from the depletion studies at two different water temperatures (treatment immersion bath). In the mixed organs of carp maintained at 13 ºC, the mean (± SD) residue levels of fumagillin (considering only the values above the LOQ) were 33.30 ± 30.72 μg/kg on day 1 and 14.24 ± 12.36 μg/kg on day 3. On day 7 and day 14, fumagillin was not detected in any of the samples analysed. In the mixed organs of carp maintained at 25 ºC, the mean (± SD) residue levels of fumagillin (considering only the values above the LOQ) were 40.30 ± 27.08 μg/kg on day 1 and 9.67 ± 3.11 μg/kg on day 3. On day 7 and day 14, fumagillin was not detected in any of the samples analysed. FAO JECFA Monograph 3356 Figure 5. Residue depletion profile of fumagillin in carp fillet at two water temperatures after immersion bath with fumagillin at a dose of 60 mg/L Co nc en tr at io n of fu m ag ill in ( µg /k g) Degree-days post-dose Water temperature 13 °C 500 100 150 200 10 100 1 000 0 Co nc en tr at io n of fu m ag ill in ( µg /k g) Degree-days post-dose Water temperature 25 °C 100 200 300 4000 10 100 1 000 0 Source: Authors’ own elaboration, based on data submitted to the Committee. Eels Four studies were conducted using non-radiolabelled fumagillin (test product Fumagil-C) in eels (Anguilla japonica). These comprised two studies involving the oral administration of fumagillin in medicated feed at different water temperatures (20 ± 3 ºC and 28 ± 3 ºC), and two studies exposing fish to fumagillin in an immersion bath at different water temperatures (20 ± 3 ºC and 28 ± 3 ºC) (NIFDS, 2015c). The eels (240 ± 55 g bw) were housed in a concrete tank (3 × 1 × 0.9 m) for acclimation, administration and sampling. Prior to the start of the experiment, they were acclimated under laboratory conditions for 2–4 weeks with voluntary feeding. Fillet and mixed organs were sampled and fumagillin quantified by a validated LC-MS/MS method. The calibration graph for fumagillin was linear (r >0.99) in the concentration range of 5 to 500 µg/kg, with an LOQ of 5 µg/kg. Oral administration Eels were treated with fumagillin, once a day, via feed, at a nominal dose of 50 mg/kg bw for 30 consecutive days. Two studies were carried out at different water temperatures (20 ºC and 28 ºC). Ten eels were euthanized at each of 1, 3, 7, 14 and 28 days after the treatment and fillet and mixed organ samples (liver, kidney, spleen, stomach, and intestine) were collected. Fumagillin was quantified by a validated LC-MS/MS method. The concentrations of fumagillin determined in the samples analysed in the two studies are shown in Table 13 and Table 14, respectively. FAO JECFA Monograph 33 57 Table 13. Concentrations of fumagillin in eel fillet with time after oral daily dose (medicated feed) of fumagillin (50 mg/kg bw) for 30 days at 20 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 20 20 10 904.04 10 440.32 2300.23 569.39 3 60 20 10 425.39 10 143.20 1256.29 357.37 7 140 20 10 14.24 2 <LOQ 22.35 11.48 14 280 20 10 20.21 2 <LOQ 23.40 3.40 28 560 20 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015c. Final Report (C): A Study on Residues Depletion of Fumagillin in Eel. Republic of Korea, NIFDS. Table 14. Concentration of fumagillin in eel fillet with time after oral daily dose (medicated feed) of fumagillin (50 mg/kg bw) for 30 days at 28 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 28 28 10 1 714.26 10 555.89 3 257.54 994.01 3 84 28 10 540.80 10 109.44 1 209.65 309.05 7 196 28 10 31.98 5 <LOQ 78.42 33.26 14 392 28 10 18.78 2 <LOQ 26.79 8.02 28 784 28 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015c. Final Report (C): A Study on Residues Depletion of Fumagillin in Eel. Republic of Korea, NIFDS. Figure 6 shows the concentrations of fumagillin in eel fillet versus days post-dose from the depletion studies at two different water temperatures. In the mixed organs of eels maintained at 20 ºC, the mean (± SD) residue levels of fumagillin (considering only the values above the LOQ) were 1 563.49.68 ± 1 232.08 μg/kg on day 1 and 658.44 ± 497.80 μg/kg on day 3. On day 7, fumagillin was detected in 4 out of 10 samples with levels of 96.55 ± 82.72 μg/kg. On day 14, fumagillin was quantified in 3 out of 10 samples at a level of 12.94 ± 7.50 μg/kg. No fumagillin was detected in any of the samples collected at day 28. In the mixed organs of eels maintained at 28 ºC, the mean (± SD) residue levels of fumagillin (considering only the values above the LOQ) of fumagillin were 4 303.64 ± 2 592.14 μg/kg on day 1 and 702.45 ± 577.11 μg/kg on day 3. On day 7, fumagillin was quantified in 3 out of 10 samples with levels of 36.05 ± 39.30 μg/kg. On day 14, fumagillin was quantified in 2 out of 10 samples at a level of 6.44 ± 0.76 μg/kg. No fumagillin was detected in any of the samples collected at day 28. FAO JECFA Monograph 3358 Figure 6. Residue depletion profile of fumagillin in eel fillet at two water temperatures after oral administration of fumagillin 100 1 000 10 0 Co nc en tr at io n of fu m ag ill in ( µg /k g) 10 000 Degree-days post-dose Water temperature 20 °C 600200 4000 Co nc en tr at io n of fu m ag ill in ( µg /k g) Degree-days post-dose Water temperature 28 °C 1 000200 400 600 8000 100 1 000 10 0 10 000 Source: Authors’ own elaboration, based on data submitted to the Committee. Immersion bath Eels were exposed to fumagillin in an immersion bath at a concentration of 60 mg/L for 5 days at two water temperatures: 20 ± 3 ºC and 28 ± 3 ºC. Ten fish were euthanized at each of 1, 3 and 7 days after the treatment and fillets and mixed organ samples were collected. Fumagillin was quantified by a validated LC-MS/MS method. The concentrations of fumagillin determined in the samples analysed in the two studies are shown in Table 15 and Table 16, respectively. Table 15. Concentration of fumagillin in eel fillet with time after immersion bath of fumagillin (60 mg/L) for 5 days at 20 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 20 20 10 41.35 9 <LOQ 85.34 22.57 3 60 20 10 20.38 6 <LOQ 54.2 17.63 7 140 20 10 <LOQ 0 <LOQ <LOQ - 14 280 20 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015c. Final Report (C): A Study on Residues Depletion of Fumagillin in Eel. Republic of Korea, NIFDS. FAO JECFA Monograph 33 59 Table 16. Concentrations of fumagillin in eel fillet with time after immersion bath of fumagillin (60 mg/L) for 5 days at 28 ± 3 ºC Time post-dose (day) Time post-dose (DD) Temp (ºC) Number of fish Concentration of fumagillin (µg/kg) Standard deviationa (µg/kg)Meana n >LOQ Minimum Maximum 1 28 28 10 44.11 9 <LOQ 106.87 37.34 3 84 28 10 15.43 1 <LOQ 15.43 - 7 196 28 10 <LOQ 0 <LOQ <LOQ - 14 392 28 10 <LOQ 0 <LOQ <LOQ - Notes: Data not corrected for recovery; aConsidering only the values >LOQ (5 μg/kg); DD: degree-days Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015c. Final Report (C): A Study on Residues Depletion of Fumagillin in Eel. Republic of Korea, NIFDS. Figure 7 shows the concentrations of fumagillin in eel fillet versus days post-dose from the depletion studies at two different water temperatures (treatment via immersion bath). Figure 7. Residue depletion profile of fumagillin in eel fillet at two water temperatures after immersion bath with fumagillin at a dose of 60 mg/L Co nc en tr at io n of fu m ag ill in ( µg /k g) Degree-days post-dose Water temperature 20 °C 1000 200 300 10 100 1 000 0 Co nc en tr at io n of fu m ag ill in ( µg /k g) Degree-days post-dose Water temperature 28 °C 100 200 300 4000 10 100 1 000 0 Source: Authors’ own elaboration, based on data submitted to the Committee. In the mixed organs of eels maintained at 20 ºC, the mean (± SD) residue levels of fumagillin (considering only the values above the LOQ) of fumagillin were 55.10 ± 27.78 μg/kg (n=9) on day 1, 32.60 ± 39.91 μg/kg (n=6) on day 3 and 5.98 ± 0.05 μg/kg (n=2) on day 7. On day 14, fumagillin was quantified in one out of 10 samples, at a concentration of 6.10 μg/kg. In the mixed organs of eels maintained at 28 ºC, the mean (± SD) residue levels of fumagillin (considering only the values above the LOQ) of fumagillin were 4.52 ± 42.82 μg/kg (n=9) on day 1, 32.81 ± 23.68 μg/kg (n=3) on day 3 and 5.76 ± 0.63 μg/kg (n=3) on day 7. On day 14, fumagillin was not detected in any of the samples. FAO JECFA Monograph 3360 Conclusions Levels of fumagillin were determined at several timepoints after both administration routes and water temperatures in all three fish species. Slightly higher concentrations of fumagillin residues in fillet were generally found in fish euthanized at the higher water temperature in all studies. The Committee noted that the residue concentrations of fumagillin in fish exposed via immersion baths were almost 100 times lower than after oral administration. The Committee also noted that the higher water temperatures used in these studies may not be optimal for all species used. When the Committee combined the data on residue depletion in fish after oral administration of fumagillin DCH (normalized to degree-days), the depletion profiles were similar for the three species (Figure 8). The Committee also noted that no quantifiable fumagillin residues were found at the approved GVP withdrawal period (28 days), including in the most conservative scenario (coldest water temperature 13 °C, corresponding to 364 degree-days). Figure 8. Residue depletion profile combined of fumagillin in rainbow trout, carp and eel fillet data at different water temperatures following oral administration of fumagillin DCH at a dose of 50 mg/kg bw for 30 consecutive days. Regression line (blue), UTL 95/95 regression line (orange), UTL 95/99 regression line (yellow) and UTL 99/99 regression line (grey). Co nc en tr at io n of fu m ag ill in ( µg /k g) 100 000 10 000 1 000 100 10 Time (Degree-days) 25050 100 150 200 0 0 Sources: NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015a. Final Report (A): A Study on Residues Depletion of Fumagillin in Rainbow Trout. Republic of Korea, NIFDS; NIFDS. 2015b. Final Report (B): A Study on Residues Depletion of Fumagillin in Carp. Republic of Korea, NIFDS; NIFDS. 2015c. Final Report (C): A Study on Residues Depletion of Fumagillin in Eel. Republic of Korea, NIFDS. Authors’ own elaboration. FAO JECFA Monograph 33 61 Bees In one study in honeybees, reported to be GLP-compliant, six beehives at each of three apiaries were treated with Fumidil-B (containing 20 g fumagillin per kg product as fumagillin DCH) at a dose of 25 g product dissolved in 20 L of sugar water (corresponding to 25 mg/L fumagillin solution), once a week for 4 or 5 consecutive weeks (Jeong, 2023). Sugar water was provided in a honeybee feeder (0.8 L sugar water containing 20 mg fumagillin per hive), and all was consumed within 1 day of supply. The applied doses were reported to be lower (75.6–98.6 percent) than the intended doses (Table 18). The sponsor explained the lower concentrations were due to storage of the formulation in a transparent container, allowing UV degradation to occur. The treatment duration (4–5 weeks) was shorter than approved according to GVP (6–8 weeks). Table 18. Concentration ranges in sugar water used for treatment and percent of the intended doses of fumagillin Apiary Fumagillin concentration % of intended dose A 23.68–23.88 mg/L 95.07% B 16.52–21.09 mg/L 75.62% C 24.49–24.84 mg/L 98.61% Source: adapted from Jeong, S.-H. 2023. Final Report: A Study on Fumagillin Residues in Honey Samples and Determining Withdrawal Period (Study No. RED22023). Republic of Korea, HBSRC. The treatment was administered in spring, 1 week before onset of honey flow (from 8 March to 9 April). Honey samples were taken starting 1 week after onset of honey flow (from 14 April to 16 May), although GVP requires that the treatment should be finished by 4 weeks before the start of honey flow. The exact location of sampling in the beehives (i.e. whether samples were taken from honey supers or elsewhere) was not reported. Robinie, or ‘Black Locust’ (Robinia pseudoacacia) was identified as the main honey crop for all three apiaries. Information on climate conditions on treatment and sampling days for each apiary was provided, but no further data on agro-ecological conditions for the three apiaries were available. Honey from each beehive was collected before treatment and at various times after the last treatment. In one apiary (Apiary A), four samples were taken on each of days 16, 31, 36, 42 and 45; in the second apiary (Apiary B), only two samples per beehive were taken, on days 24 and 29 after the last treatment; and, in the third apiary (Apiary C), two samples were taken, on days 22 and 44 after the last treatment. Honey samples from all apiaries were analysed for residues of fumagillin, and samples from Apiary A were also analysed for DCH concentrations. No samples of beeswax were taken. No details on sampling (e.g. amount of pooled honey, pH, and moisture content of all pooled honey samples) were provided. An LC-MS/MS method was used for determination of fumagillin concentrations in honey (LOD 2 µg/kg, LOQ 5 µg/kg). Only fumagillin itself was measured (i.e. without degradation products). The accuracy, evaluated on three separate days and at four fortification levels (5, 10, 100 and 500 ng/mL), exhibited a range from 85.8 percent to 115.5 percent, while precision varied from 0.4 to 8.1 percent. For inter-day accuracy and precision, the average accuracy across the three days was between 99.4 percent and 107 percent, with average precision ranging from 3.6 to 13.8 percent. In all three apiaries, the residue concentrations of fumagillin in honey samples decreased with time after treatment (Table 19). In one apiary, fumagillin was detected at concentrations (mean ± SD) of 110.7 ± 97.3 µg/kg, 19.01 ± 10 µg/kg, 5.882 ± 1.2 µg/kg, <LOQ, and <LOQ on days 16, 31, 36, 42, and 46 FAO JECFA Monograph 3362 after treatment, respectively. In the second apiary, fumagillin concentrations of 20.45 ± 17.67 µg/kg and 16.59 µg/kg (only one quantifiable sample) were measured on days 24 and 29 after treatment, respectively. In the third apiary, no fumagillin residues were detected in samples collected on days 22 and 44 after treatment. Table 19. Fumagillin concentrations in honey from three apiaries with samples from six beehives per site, values <LOQ were excluded from calculations of mean values Apiary Treatment duration Day after final treatment Mean ± SD (µg/kg) A 4 weeks 16 110.700 ± 97.300 31 19.010 ± 10.000 36 5.882 ± 1.200 42 <LOQ 45 <LOQ B 5 weeks 24 20.450 ± 17.670 29 16.590 ± 0.000 C 4 weeks 22 N/D 44 N/D Notes: SD: Standard deviation; LOQ: Limit of quantification (5 μg/kg); N/D: not detected Source: adapted from Jeong, S.-H. 2023. Final Report: A Study on Fumagillin Residues in Honey Samples and Determining Withdrawal Period (Study No. RED22023). Republic of Korea, HBSRC. DCH was determined in one apiary only, with an LC-MS/MS method (LOD 12 µg/kg, LOQ 20 µg/kg). The intraday accuracy ranged between 95.4 percent and 106.8 percent and precision between 1.2 percent and 3.5 percent on day 1. On day 2, accuracy ranged between 110.2 percent and 126.9 percent and precision between 1.4 percent and 10.1 percent. On day 3, accuracy ranged between 101.9 percent and 115.0 percent and precision between 0.6 percent and 5.1 percent. In terms of inter-day accuracy and precision, the average accuracy over the 3 days was between 102.5 percent and 116.2 percent, and the average precision was between 5.9 percent and 9.7 percent. Residue levels of DCH in honey samples decreased with time after treatment (Table 20). On days 16, 31, 36 and 42 after the last treatment, DCH was detected at mean concentrations ± SD of 1 698.1 ± 1 160.5 μg/kg, 524.9 ± 261.5 μg/kg, 296.9 ± 106.2 μg/kg and 32.5 ± 3.1 μg/kg, respectively. On day 45, the residue levels of DCH were below the LOQ in all samples. Table 20. DCH concentrations in honey from Apiary A, values <LOQ were excluded from calculations of mean values Day after treatment Mean ± SD 16 1 698.1 ± 1 160.5 31 524.9 ± 261.5 36 296.9 ± 106.2 42 32.5 ± 3.1 45 < LOQ Notes: SD: Standard deviation, LOQ: Limit of quantification (20 µg/kg) Source: Jeong, S.-H. 2023. Final Report: A Study on Fumagillin Residues in Honey Samples and Determining Withdrawal Period (Study No. RED22023). Republic of Korea, HBSRC. FAO JECFA Monograph 33 63 The Committee noted that DCH concentrations in honey were at least an order of magnitude higher than those of fumagillin on each day of sampling. The concentrations of fumagillin and DCH residues differed among the beehives, with a wide range; standard deviations were close to the mean at higher concentrations and in the order of half the mean at lower concentrations. The Committee noted that several recommendations from VICH GL 56 (VICH, 2018) were not met. For example, treatment was not performed according to GVP, only three apiaries were included instead of four, and no information was available on agro-ecological conditions or beekeeping management practices. Validation of the analytical method provided by the sponsor was insufficiently described for fumagillin, and no description of the method and no validation data were available for DCH. Methods of analysis for residues in tissues It is noteworthy that the prevailing focus in the literature on analytical methods primarily revolves around the determination of fumagillin, with limited attention given to the quantification of DCH. Fumagillin Fumagillin, due to its physicochemical properties, has been quantified in fish tissues and honey using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Additionally, older methods employing high performance liquid chromatography (HPLC) with a photodiode array detector (DAD) or ultraviolet (UV) detector have been documented in the literature for the determination of fumagillin, capitalizing on its absorption in the UV region (Fekete et al., 1995; Guyonnet, Richard and Hellings, 1995). Nevertheless, contemporary analytical preferences in food analysis favour mass spectrometry detection due to its selectivity and the attainability of lower limits of quantitation. In general, the electrospray ionization source is operated in the positive mode, resulting in protonated fumagillin as the precursor ion and different mass/charge (m/z) transitions have been monitored, including m/z 459.2→177.0, 459.1→233.3 and/or 459.2→131 (Dmitrovic and Durden, 2013; Thompson, van den Heever and Pernal, 2018; van den Heever et al., 2015a; Lopez et al., 2008). It is important to mention that most of the analytical methods reported in the literature only focus on the determination of fumagillin, without considering the quantitation of DCH. The chromatographic separation of fumagillin is typically conducted in the reverse phase mode using non-polar stationary phases, such as C18 or C8 (Guruceaga et al., 2019). For the mobile phase, acetonitrile has been the most used organic modifier. Nevertheless, in some methods, methanol or a mixture of acetonitrile and methanol has been used (Kanda et al., 2011; van den Heever et al., 2015a). As additives of the mobile phase, formic acid, acetic acid, ammonium formate/formic acid and ammonium formate have all been used (Lopez et al., 2008; Nozal et al., 2008; Higes et al., 2011; Dmitrovic and Durden, 2013; van den Heever et al., 2015a). Exploiting the acidic nature of fumagillin, Guyonnet et al. (1995) developed an ion-pairing method using a mobile phase with a pH of 7.8, where tetrabutylammonium acts as a cation to retain fumagillin onto a C8 column. Finally, an alternative method using a normal-phase liquid chromatographic method was proposed by Fekete et al. (1995) for the determination of fumagillin in fish tissues using a silica gel stationary phase and a hexane-dichloromethane-dioxan-2- propanol-acetic acid as mobile phase. For the sample preparation procedure, fumagillin has been extracted with water or acetonitrile containing formic acid from the food matrix. The Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) approach has also been used. Clean-up of the extracts has been carried out employing solid phase extraction (SPE) using polymeric sorbents and weak anion exchange cartridges (Guyonnet, Richard and Hellings, 1995; Dmitrovic and Durden, 2013; Kanda et al., 2011; van den Heever et al., 2015a; Nozal et al., 2008). FAO JECFA Monograph 3364 Nozal et al. (2008) proposed a method for the determination of residues of fumagillin in honey using liquid chromatography coupled to a diode array detector and mass spectrometer (LC-DAD-MS). The sample preparation procedures involve solid-phase extraction on polymeric cartridges (Strata X-33 µm) to isolate fumagillin from diluted honey. Chromatographic separation of fumagillin was conducted in isocratic mode on a C18 column (150 × 3 mm, 5 μm). The mobile phase consists of a mixture of 20 mM ammonium formate in water and acetonitrile (61:39, v/v) at 35 °C, with a flow rate set at 1 mL/min. As internal standard, roxithromycin was used. Average analyte recoveries, influenced by botanical origin, range from 88 percent to 96 percent. The LOQ of the LC-DAD-MS method varied between 3 and 10 µg/kg. This method has been applied to determine fumagillin residues in honey samples collected from veterinary-treated beehives infected by Nosema ceranae and fed with Fumidil-B at different doses. The authors verified that fumagillin is very unstable under irradiation, and it rapidly degrades into four compounds with the same molecular mass, which must be diastereoisomers of neofumagillin, formed by the cyclization of the chain. These compounds are eluted at higher retention times than fumagillin. Degradation products at lower retention times than fumagillin were also verified in the chromatograms. One suggested compound is dihydroxyfumagillin, formed by hydrolysis of the unstable epoxide. Screening methods In the literature, an enzyme-linked immunosorbent assay (ELISA) method is reported as a screening method for fumagillin in honey, with detection levels of at least 20 µg/kg (Assil and Sporns, 1991). Confirmatory methods Numerous confirmatory techniques employing LC-MS/MS are documented in the literature for the determination of fumagillin alone or in the presence of other residues of veterinary drugs (multiclass methods) in food matrices. Honey Lopez et al. (2008) described a multiclass method including the determination of fumagillin in honey using LC-MS/MS. The separation was achieved using a Phenomenex Polar RP Synergy column (50 × 2 mm, 4 µm) with a guard column of the same stationary phase. The mobile phase was a mixture of water and acetonitrile with 0.1 percent formic acid added under gradient elution. The ionization of fumagillin was conducted by operating the electrospray ionization source in positive mode. For quantitation and identity confirmation, the following transitions were monitored: m/z 459.1→233.3 and m/z 459.1→215.3. Kanda et al. (2011) reported a method of the determination of residues of fumagillin in honey using the QuEChERS approach followed by LC-MS/MS quantitation. The chromatographic separation was conducted in gradient mode on a C8 column (100 × 2 mm, 5 μm) maintained at 40 °C. The mobile phase comprised a mixture of a 2 mM ammonium formate added to 0.01 percent formic acid solution and methanol, with a flow rate set at 0.2 mL/min. Fumagillin extraction was carried out using acetonitrile containing 0.1 percent formic acid and adding the QuEChERS salts (sodium chloride, trisodium citrate dihydrate, and 4 g magnesium sulfate). Clean-up was carried out using solid-phase extraction with an Oasis® mixed-mode weak anion-exchange cartridge. The electrospray ionization source was operated in the negative ion mode. For quantitation and identity confirmation, the following transitions were monitored: fumagillin m/z 456.9→131 (quantitation) and m/z 456.9→102.8 and 456.9→175 (identity confirmation). The limit of quantitation (LOQ) was established at 0.1 μg/kg. The combination of LC-MS/MS with the QuEChERS method demonstrated good potential for the accurate determination of fumagillin residues in honey. FAO JECFA Monograph 33 65 Van den Heever et al. (2015a) detailed a method for the determination of fumagillin, DCH and fumagillin degradation products in honey. Quantitative analysis was conducted using LC-MS/MS with two internal standards, namely DCH-d10 and roxithromycin. For quantitation and identity confirmation, the following transitions were monitored: fumagillin m/z 459.2→177 and 459.2→102.8, DCH m/z 182.0→83 and 182→100, DCH-d10 m/z 192.2→83 and 192.2→100 and roxithromycin m/z 837.4→158. The chromatographic separation was achieved using an Xterra MS-C18 column (4.6 × 100 mm, 3.5 µm) maintained at 30 ºC, with a mobile phase consisting of a mixture of water and methanol, with ammonium formate/formic acid added to both. The sample preparation procedure involved weighing 5 g of honey and adding the internal standards and water (10 mL). After agitation using a vortex and mechanical shaker for 1 hour, the mixtures underwent centrifugation, and the supernatant was subjected to clean up using a reversed polymeric phase in SPE. Following washing and drying of the sorbent, the retained analytes were eluted with acetonitrile containing 5 percent formic acid. Quantitation relied on matrix matched calibration curves within the concentration range of 10 to 500 ng/g. The LOQ of the method was established at 10 ng/g. Method provided by the sponsor for the determination of fumagillin in rainbow trout, carp, and eel tissues The Committee assessed the validation data for determination of fumagillin against the requirements for analytical methods published in the Codex Guideline CAC/GL 71-2009 (FAO and WHO, 2014). Fumagillin was determined in trout, carp and eel fillets with a LC-MS/MS method (NIFDS, 2015a, 2015b, 2015c). In summary, the sample preparation involves the addition of 20 mL of acetonitrile with 0.1 percent formic acid to 5 g of homogenized tissue. Following agitation, the mixture undergoes centrifugation at 4 ºC and 2 600 g for 15 min. The resulting supernatant is collected, and the solvent removed (leaving a residue of 0.5 mL) using a rotary evaporator at 40 ºC. Subsequently, 5 mL of water is added to the residue, and the solution undergoes clean-up using a Strata-X cartridge previously conditioned with methanol and water. The cartridge is washed with 5 mL water: methanol 60:40 v/v. Fumagillin is then eluted with 10 mL of acetonitrile containing 0.1 percent formic acid. The solvent is removed using a rotary evaporator at 40 ºC and the resulting residue is resuspended in 1 mL of methanol containing 0.1 percent formic acid and 10 mM ammonium formate. The mixture is agitated, followed by centrifugation at 4 ℃, 15 000 g for 10 minutes. The supernatant is filtered (0.2 µm) and subjected to analysis by LC-MS/MS. The separation of fumagillin is performed on a Phenomenex Luna C18 column (2 x 100 mm, 3 µm), at 35 ºC, utilizing a mobile phase containing aqueous 0.1 percent v/v formic acid and 10 mM ammonium formate (solvent A), and methanol containing 0.1 percent v/v formic acid and 10 mM ammonium formate (solvent B). The flow rate is set at 0.25 mL/min, employing a gradient elution as follows: from 0 to 2 min, 95:5 v/v A:B; 5 min to 5:95 v/v A:B; 10 min 5:95 v/v A:B; 15 min 95:5 v/v A:B; 20 min 95:5 v/v A:B). The mass spectrometer conditions were electrospray source operating in the positive mode and source temperature of 350 ºC. Quantitation is performed using acquisition of ions in the selected reaction- monitoring mode, using the transition of m/z 459.3→131 for fumagillin. For identity confirmation, two additional transitions for fumagillin are monitored: m/z 459.3→103.1 and m/z 459.3→177.1. Linearity is observed in the range of 5 to 500 µg/kg. The validation parameters are shown in Table 21. FAO JECFA Monograph 3366 Table 21. Validation parameters of the LC-MS/MS method for the determination of fumagillin in fillet of trout, carp and eel Parameter Trout fillet Carp fillet Eels fillet Precision (CV, n=5) 19.2% (5 µg /kg) 16.6% (10 µg /kg) 12.4% (5 µg /kg) 9.4% (10 µg /kg) 13.9% (5 µg /kg) 12.7% (10 µg /kg) Accuracy (n=5) 80.6% (5 µg /kg) 88.3% (10 µg /kg) 79.2% (5 µg /kg) 81.5% (10 µg /kg) 74.1% (5 µg /kg) 79.4% (10 µg /kg) LOQ 5 µg/kg 5 µg/kg 5 µg/kg Analytical range (µg/kg) 5–500 5–500 5–500 Linearity (r) >0.99 >0.99 >0.99 Specificity/selectivity No interference observed No interference observed No interference observed Source: adapted from NIFDS (Korean National Institute of Food Drug Safety Evaluation). 2015a. Final Report (A): A Study on Residues Depletion of Fumagillin in Rainbow Trout. Republic of Korea, NIFDS; NIFDS. 2015b. Final Report (B): A Study on Residues Depletion of Fumagillin in Carp. Republic of Korea, NIFDS; NIFDS. 2015c. Final Report (C): A Study on Residues Depletion of Fumagillin in Eel. Republic of Korea, NIFDS. Method provided by the sponsor for the determination of fumagillin in honey Fumagillin in honey underwent analysis utilizing LC-MS/MS, with quantitation performed using a matrix-matched calibration curve spanning a concentration range of 5 to 250 µg/kg. However, the absence of details regarding the analytical method impedes the verification of data reliability. The accuracy and precision of the method are outlined in Table 22. Table 22. Validation parameters of the LC-MS/MS method for the determination of fumagillin in honey Parameter Honey Precision (CV, n=5) 13.8% (5 µg /kg) 5.1% (10 µg /kg) 4.6% (100 µg/kg) 3.6% (500 µg/kg) Accuracy (n=5) 101.9% (5 µg /kg) 107.0% (10 µg /kg) 100.8% (100 µg/kg) 99.4% (500 µg/kg) LOQ 5 µg/kg Analytical range (µg/kg) 5–250 Linearity (r) >0.99 Specificity/selectivity No interference observed Source: adapted from Jeong, S.-H. 2023. Final Report: A Study on Fumagillin Residues in Honey Samples and Determining Withdrawal Period (Study No. RED22023). Republic of Korea, HBSRC. The stability of fumagillin in honey and fish tissue samples was not adequately demonstrated for normal conditions of laboratory handling or for typical storage conditions. Information in the literature (van den Heever et al., 2015a) indicated that fumagillin is not stable when exposed to UV light or at common temperatures in hives (about +34 °C). FAO JECFA Monograph 33 67 Dicyclohexylamine No methods were submitted by the sponsor for the analysis of DCH in fish tissues or honey. An LC-MS/MS method for analysis of DCH in honey was described in the published literature (van den Heever et al., 2015a), which had an LOQ of 10 µg/kg. In summary, honey samples are diluted with water and cleaned up by solid phase extraction. Chromatographic separation is performed on a C18 column. The electrospray ionization source is operated in the positive ion mode. Quantification is performed by acquisition of ions in the selected reaction-monitoring mode, with the transitions of m/z 182→83 used for quantification and m/z 182→100 used for identity confirmation. The linear range of the matrix-matched calibration curve, with DCH-d10 as internal standard, was 10–500 µg/kg, with a linear correlation coefficient >0.99. Precision and accuracy were evaluated at three concentrations, 10, 100, and 500 µg/kg and on three days. The inter-day precision ranged from 5.9 percent to 9.7 percent and the accuracy from 98.3 percent to 104 percent. The estimated limit of quantitation (LOQ) was 10 µg/kg. Overall comment on validation of the analytical methods Fumagillin The information on the performance of the analytical methods for fumagillin in fish and honey provided by the sponsor consisted only of a summary of validation data, making it difficult to confirm whether the methods adhered to full validation parameters in accordance with Codex Guideline CAC/GL 71-2009 or VICH guidelines. The Committee considered that, while the lack of full validation reports was a source of uncertainty, the methods were suitable for monitoring purposes. Dicyclohexylamine The Committee considered that the method described in the publicly available literature (van den Heever et al., 2015a) is suitable for monitoring DCH residues in honey. Stability of fumagillin Fumagillin is composed of a decatetraenedioic acid linked to a cyclohexane through an ester bond. The cyclohexane is further characterized by the presence of a methoxy group, an epoxide, and an aliphatic chain derived from a terpene, featuring an additional epoxide. The primary functional groups contributing to the instability of the molecule are the epoxides. Numerous published papers in the literature explore the stability of fumagillin, with a particular emphasis on photodegradation and thermal degradation. Fumagillin undergoes degradation in both acidic (1 mol/L HCl) and alkaline (1 mol/L NaOH) conditions. Furthermore, when exposed to standard fluorescent light, approximately 40 percent degradation occurs within a six-hour period (Guruceaga et al., 2019). As outlined by Higes et al. (2011), the efficacy of Fumidil-B is influenced by various factors, encompassing storage, treatment preparation, and the quantity consumed by bees. Notably, exposure to UV radiation, such as sunlight, markedly diminishes the initial concentration of fumagillin within a few hours, while temperature exerts an influential impact on its degradation. To evaluate the stability of fumagillin, the authors conducted assessments in 50 percent sugar syrup at three different concentrations of Fumidil-B (1, 1.5, and 2.5 g in 250 mL of sugar syrup). Stability analyses were performed for each concentration in the absence and presence of UV radiation (6 W short/longwave UV lamp) at temperatures of 4 °C, 22 °C (room temperature), 30 °C (drying oven), and 40 °C (drying oven). Results indicated that fumagillin underwent rapid decomposition under light exposure, with this degradation accelerated by FAO JECFA Monograph 3368 higher temperatures. Specifically, after 70 days, samples exhibited 30 percent decomposition at 4 °C, 60 percent decomposition at 22 °C, and 65 percent decomposition at 30 °C. At 40 °C, under light exposure, fumagillin became undetectable after 20 days. In the absence of light (utilizing amber vials), fumagillin demonstrated greater stability at lower temperatures, with only 12 percent decomposition observed at 4 °C after 70 days. However, exposure to UV irradiation, even with amber vials, resulted in complete decomposition of fumagillin after 40 days, with UV-decomposed fumagillin products detectable for up to 60 days. According to the sponsor, fumagillin prepared in sugar water (25 mg/L) undergoes degradation within a day when exposed to light. However, it maintains stability for up to 7 days when stored under light protection. The stability of fumagillin and DCH in honey under simulated hive and ambient storage conditions, both in the presence and in the absence of light was investigated by van de Heever et al. (2015a). Honey samples (Buram honey Company, Turkey) were weighed (5 g) into either amber coloured or clear 50 mL centrifuge tubes. The samples were fortified at 500 µg/kg with fumagillin (free acid form) and with DCH, followed by shaking for 30 min on a mechanical shaker to homogenize the samples as well as possible. Simulated shelf condition stability samples (n = 93) were weighed into clear 50 mL centrifuge tubes to enable exposure of the honey to ambient fluorescent light. Amber coloured centrifuge tubes were divided into two sets consisting of 93 tubes, with one set being stored in an incubator at 34 °C in darkness, to simulate hive conditions, and the other set kept at 21 °C in the dark, simulating bulk storage conditions in drums. Fumagillin and DCH were quantified by a validated LC-MS/MS method (LOQ of 10 µg/kg). The results are shown in Figure 9. Fumagillin and its UV degradation products exhibit high instability when exposed to fluorescent light. This rapid degradation of both fumagillin and its UV decomposition products contrasts with the comparatively stable behavior observed for DCH under identical conditions. In the absence of UV-decomposed fumagillin standards, the calibration curve of fumagillin was utilized for the quantification of UV-decomposed fumagillin. Furthermore, DCH demonstrates significantly greater stability than fumagillin at hive and room temperatures, particularly in the absence of light. At room temperature upon exposure to light, fumagillin has an estimated half-life of 3 days, compared to that of 829 days for DCH. The authors indicate that it is unlikely that fumagillin or its UV degradation products will be detected in any appreciable amounts in honey destined for human consumption, when the commercial formulations are used according to GVP. Conclusion UV decomposition products of fumagillin retain their biological activity, whereas the thermally degraded fumagillin does not. The hydrolysed product (fumagillol) also retains some biological activity, albeit only about 10 percent of that of fumagillin. Based on this, fumagillin is likely to represent the major residue. However, it may not represent the total activity, since degraded and hydrolysed products still have some biological activity. Exposure to light results in a decrease of fumagillin concentration, but some activity of the degraded fumagillin would remain. After exposure to light the M:T ratio would be <1. FAO JECFA Monograph 33 69 Figure 9. Concentrations of fumagillin, UV-fumagillin and DCH under simulated storage conditions Co nc en tr at io n of fu m ag ill in ( µg /k g) 600 400 200 Days Bulk storage (21 °C in darkness) 19 6 17 5 15 5 12 6 11 298918477716355484542383531282421171410730 0 Fumagillin DCH Fumagillin UV-Fumagillin DCH Co nc en tr at io n of fu m ag ill in ( µg /k g) 600 400 200 Days Shelf conditions (21 °C exposed to light) 19 6 17 5 15 5 12 6 11 298918477716355484542383531282421171410730 0 Fumagillin DCH Co nc en tr at io n of fu m ag ill in ( µg /k g) 600 400 200 Days Hive (34 °C in darkness) 19 6 17 5 15 5 12 6 11 298918477716355484542383531282421171410730 0 Source: adapted from van den Heever, J. P., Thompson, T. S., Curtis, J. M. & Pernal, S. F. 2015a. Determination of Dicyclohexylamine and Fumagillin in Honey by LC-MS/MS. Food Analytical Methods, 8(3): 767–777. FAO JECFA Monograph 3370 Appraisal Introduction Fumagillin (IUPAC name: (2E,4E,6E,8E)-10-{[(3R,4S,5S,6R)-5-methoxy-4-[(2R)-2-methyl-3- (3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro[2.5]octan-6-yl]oxy}-10-oxodeca-2,4,6,8-tetraenoic acid; Chemical Abstract Service No. 23110-15-8) is a mycotoxin used as an antimicrobial agent for the treatment of microsporidian infections in honeybees and in various fish species. Fumagillin is poorly soluble in water and undergoes rapid ultraviolet and thermal degradation. Therefore, to increase its stability and water solubility, commercial formulations used in veterinary medicine contain fumagillin as the dicyclohexylamine (DCH, IUPAC name N-cyclohexylcyclohexanamine; Chemical Abstract Services No. 101-83-7) salt in a 1:1 stoichiometric ratio. Fumagillin DCH is used as a veterinary drug in feed for fish and honeybees or via immersion bath treatment for fish. The mode of action of fumagillin is based on inhibition of type-2 methionine aminopeptidase (MetAP-2) activity via formation of a covalent bond with the histidine moiety of the enzyme. MetAP-2 is a cytosolic enzyme, which removes the initial methionine from the amino terminus of newly synthesized proteins for subsequent post-translational modifications, which affects the function of many proteins. Fumagillin DCH has not previously been evaluated by the Committee. The Committee evaluated fumagillin DCH at the present meeting at the request of the twenty-sixth session of the CCRVDF with a view to recommending maximum residue limits (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 exposed to the residues of both, the Committee evaluated both fumagillin and DCH. The dosage used in fish is 15–50 mg of fumagillin base per kg bw in medicated feed for 30 consecutive days, or 60 mg fumagillin base per litre of water in an immersion bath for 5 consecutive days. The GVP 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 20–25 mg fumagillin base per litre of sugar syrup, administered once weekly for 6–8 weeks. Bees should be treated in the autumn after honey supers have been removed, or in spring, when the treatment should be completed at least 4 weeks before the start of the honey flow. Fumagillin DCH is not currently registered for use as a pesticide. Metabolism A study of fumagillin metabolism in rainbow trout, which was reported to be GLP-compliant, was provided by the sponsor (Kim, 2023). The fish received a dose of 50 mg fumagillin per kg bw (mixture of tritium-radiolabelled fumagillin and unlabelled fumagillin DCH) by gavage. The water temperature was kept at 15 °C. The precise positions of the tritium labels in the fumagillin molecule were unknown. The extent of exchange of the tritium radiolabel with water was assessed 6 hours after dosing. Most samples exceeded the VICH-recommended acceptance criterion (VICH, 2011) of < 5 percent (range, -22.2 to +15.6), suggesting that the tritium label was unstable. Almost 50 percent of the radioactive residues could not be extracted, although various solvents were tested. No explanation was provided for the limited extractability of the radioactive residue. Only the parent compound, fumagillin, was identified in fillet extracts. It was therefore proposed FAO JECFA Monograph 33 71 that fumagillin was not metabolized. As no radiolabelled DCH was used in this study, no data were available on the metabolism and depletion of DCH in fish. Like other veterinary drugs used in apiculture, fumagillin DCH does not appear to be metabolized in honeybees, and most fumagillin DCH probably appears in beeswax and honey. Degradation products The fumagillin portion of fumagillin DCH is subject to degradation under conditions relevant for the treatment of fish and honeybees. It can be degraded by exposure to light, producing biologically active degradation products with activity similar to that of fumagillin (Kochansky and Nasr, 2004), or hydrolysed under basic conditions to produce fumagillol, which has about 10 percent of the biological activity of fumagillin (Gochnauer and Furgala, 1962). Thermal degradation of fumagillin leads to formation of dihydroxyfumagillin, a biologically inactive compound (Kochansky and Nasr, 2004). The Committee concluded that fumagillin degradation products are unlikely to be of greater toxicological concern than the parent. The Committee noted that no information on concentrations of fumagillin degradation products in fish tissues or honey was available. No information on degradation of DCH was available. Residue depletion Radiolabelled residue depletion study Fish In the study of radiolabelled residue depletion (Kim, 2023), 50 one-year-old rainbow trout, bw 100–275 g) were treated with a mixture of [3H]-fumagillin and unlabelled fumagillin DCH at a total dose of about 50 mg of fumagillin per kg bw. To achieve the intended dose, a solution of 50 000 mg/L was prepared from 0.02 mg [3H]-fumagillin (as fumagillin base), and 499.98 mg unlabelled fumagillin (present as fumagillin DCH) were added to 10 mL of 0.5 percent CMC solution (carboxymethylcellulose sodium salt in sterilized water). The fumagillin was randomly labelled with tritium. The extent of exchange of the tritium radiolabel with water was assessed 6 hours after dosing. Numerous samples exceeded the VICH-recommended acceptance criterion of <5 percent (range, from -22.2 to +15.6), indicating that the tritium label was unstable. A dose of 50 mg/kg bw of fumagillin was administered via gavage. Fish were euthanized and fillet samples collected from each of 10 fish at 6 and 12 hours and 1, 2 and 7 days after administration. The tissues were homogenized and stored at about -20 °C until analysis. Validated LSC and radio-HPLC methods were used to determine the concentrations of radiolabelled fumagillin. The radiochemical purity of [3H]-fumagillin, determined with a radio-HPLC method, was reported to be 100 percent. The concentration of fumagillin in fish fillet increased from 1.2 mg equiv/kg at 6 hours after dosing to 2.3 mg equiv/kg at 12 hours and decreased to 0.2 mg equiv/kg on day 7. The radiolabelled residue depletion study indicates that the parent compound, fumagillin, is a suitable marker residue. FAO JECFA Monograph 3372 Non-radiolabelled residue depletion studies Fish Residue depletion studies with unlabelled fumagillin in rainbow trout, carp and eels were assessed (NIFDS, 2015a, 2015b, 2015c). For each species, studies were conducted at two water temperatures and two administration routes (oral and immersion bath). A nominal dose of 50 mg/kg bw for 30 consecutive days was used for oral administration, and for the immersion bath, fish were exposed to fumagillin at a concentration of 60 mg/L for 5 consecutive days. The product used in these studies was Fumagil-C, containing 50 g of fumagillin per kg (administered as fumagillin DCH). Medicated feed was prepared by mixing Fumagil-C with feed under light-protected conditions, and fish oil was uniformly sprayed onto the medicated feed to prevent release of the drug into the water. Fumagillin, quantified in the medicated feed, constituted 80–110 percent of the intended concentration. Fumagillin was not quantified in the immersion bath. In the studies provided, the assessment was solely of depletion of fumagillin. Residues of DCH were not quantified in the sampled tissues. Details such as the exact quantity of feed administered and consumed and the weight of the fish in each tank, were not provided. Ten fish were sampled at 1, 3, 7, 14 and 28 days after the last oral dose and 1, 3 and 7 days after exposure in an immersion bath, and fillet (muscle with skin in natural proportions) was collected. Fumagillin was quantified in the samples with a LC-MS/MS method separately validated for each species. The LOQ for fumagillin in fish fillet for all species was 5 µg/kg. Rainbow trout (Oncorhynchus mykiss): After oral administration, the highest mean ± SD fumagillin concentration in fillet (1 558 ± 916 µg/kg, n=10) was observed on day 1 after dosing at a water temperature of 22 ºC, and the peak individual concentration was 3258 µg/kg. By 14 days after treatment cessation at both temperatures, all fumagillin concentrations were below the LOQ of the method (5 µg/kg). For the immersion bath treatment, fumagillin residues were quantifiable only on day 1 after dosing (mean concentration ± SD of 22 ± 28 µg/kg, n=9) at a water temperature of 13 ± 3 ºC. At 25 ± 3 ºC, the mean concentration ± SD of fumagillin determined on day 1 was 50 ± 34 µg/kg (n=10); on day 3, one of the 10 fish fillets sampled contained a concentration of 7 µg/kg. Carp (Cyprinus carpio): After oral administration, the highest mean concentration of fumagillin in fillet (2 256 ±1 732 µg/kg, n=10) was found on day 1 after dosing at a water temperature of 25 ºC, with an individual peak concentration of 5 234 µg/kg. By 28 days after treatment cessation, all fumagillin concentrations in fillet were below the LOQ of the method (at both temperatures). For the immersion bath treatment, quantifiable fumagillin residues in fillet persisted until day 3 after dosing at both water temperatures. All mean concentrations were < 34 µg/kg; the maximum concentration was 81 µg/kg in a sample collected on day 1 (13 ºC). Eels (Anguilla japonica): After oral administration, the highest mean concentration ± SD of fumagillin in eel fillet (1 714 ± 994 µg/kg, n=10) was found on day 1 after dosing at a water temperature of 28 ºC, with a maximum individual concentration of 3 258 µg/kg. At 14 days and 28 days after treatment cessation, all fumagillin concentrations in fillet were below the LOQ (5 µg/kg) of the method for the lower and higher water temperatures, respectively. In the immersion bath treatment, quantifiable fumagillin residues persisted until day 3 after dosing for both water temperatures. All the mean concentrations were <44 µg/kg; the maximum individual concentration was 107 µg/kg, in a sample collected on day 1 (28 ºC). FAO JECFA Monograph 33 73 Summary of fish non-radiolabelled residue depletion studies Depletion of fumagillin was observed after several withdrawal times after both treatment routes and water temperatures in all three fish species. Slightly higher residue concentrations of fumagillin in fillet were generally found in fish harvested at the higher water temperature in all studies. The Committee noted that the residue concentrations of fumagillin in fish exposed in immersion baths were almost 100 times lower than after oral administration. The Committee also noted that the higher water temperatures used in these studies may not be optimal for all species used. When the Committee combined the data on residue depletion in fish after oral administration of fumagillin DCH (normalized to degree-days), the depletion profiles were similar for the three species. The Committee also noted that no quantifiable fumagillin residues were found at the label withdrawal period (28 days), including in the most conservative scenario (coldest water temperature 13 °C, corresponding to 364 degree-days). Honeybees In one study in honeybees, reported to be GLP-compliant, six beehives each at three apiaries were treated with Fumidil-B (containing 20 g fumagillin per kg product as fumagillin DCH) at a dose of 25 g product dissolved in 20 L of sugar water (corresponding to 25 mg/L fumagillin solution), once a week for 4 or 5 consecutive weeks (Jeong, 2023). Sugar water was provided from a honeybee feeder, and all was consumed within 1 day of supply. The applied dosages were reported to be lower (75.6–98.6 percent) than the intended doses, and the treatment duration (4–5 weeks) was shorter than that according to GVP (6–8 weeks). Treatment was administered in spring, 1 week before onset of honey flow, and honey samples were taken starting 1 week after onset of honey flow (although GVP requires that the treatment should be finished 4 weeks before the start of honey flow). An LC-MS/MS method was used for determination of fumagillin concentrations in honey (LOD 2 µg/kg, LOQ 5 µg/kg). Only fumagillin itself was measured (i.e. without degradation products). In all three apiaries, the residue concentrations of fumagillin in honey samples decreased with time after treatment. In one apiary, fumagillin was detected at concentrations (mean ± SD) of 110.7 ± 97.3 µg/kg, 19.01 ± 10 µg/kg, 5.882 ± 1.2 µg/kg, <LOQ on days 16, 31, 36, 42, and 46 after treatment, respectively. In the second apiary, fumagillin concentrations of 20.45 ± 17.67 µg/kg and 16.59 µg/kg (only one quantifiable sample) were measured on days 24 and 29 after treatment. In the third apiary, no fumagillin residues were detected on days 22 and 44 after treatment. DCH was determined in one apiary only, with a LC-MS/MS method (LOD 12 µg/kg, LOQ 20 µg/kg). Residue levels of DCH in honey samples decreased with time after treatment. On days 16, 31, 36 and 42 after the last treatment, DCH was detected at a mean concentration ± SD of 1 698.1 ± 1 160.5 μg/kg, 524.9 ± 261.5 μg/kg, 296.9 ± 106.2 μg/kg and 32.5 ± 3.1 μg/kg, respectively. On day 45, the residue levels of DCH were below the LOQ in all samples. The Committee noted that DCH concentrations in honey were at least an order of magnitude higher than those of fumagillin. The concentrations of fumagillin and DCH residues differed among the beehives, with a wide range and standard deviations close to the mean at higher concentrations and in the order of half the mean at lower concentrations. The Committee noted that several recommendations from VICH GL 56 (VICH, 2018) were not met. For example, treatment was not performed according to GVP, there were too few study sites, no information was available on agro-ecological conditions or beekeeping management practices, validation of the analytical method provided by the sponsor was insufficiently described for fumagillin, and no description of the method and no validation data were available for DCH. FAO JECFA Monograph 3374 Analytical method Fumagillin. The Committee assessed the validation data for determination of fumagillin against the requirements for analytical methods published in the Codex Guideline CAC/GL 71-2009 (FAO and WHO, 2014). An LC-MS/MS method has been developed and validated for analysis of fumagillin in trout, carp and eels (NIFDS, 2015a, 2015b, 2015c) as well as in honey (Jeong, 2023). The estimated LOQ was 5 µg/kg for fish fillet and honey. The stability of fumagillin in fish tissue and honey samples was not adequately demonstrated under normal conditions of laboratory handling or typical storage conditions. Information in the literature (van den Heever et al., 2015a) indicated that fumagillin is not stable when exposed to UV light or at common temperatures in hives (about +34 °C). The information on the performance of the analytical methods for fumagillin in fish and honey consisted only of a summary of validation data, making it difficult to confirm whether the methods adhered to full validation parameters in accordance with Codex Guideline CAC/GL 71-2009 or VICH guidelines. The Committee considered that, while the lack of full validation reports was a source of uncertainty, the methods were suitable for monitoring purposes. Dicyclohexylamine. No methods were submitted for the analysis of DCH in fish tissues or honey. An LC-MS/MS method for analysis of DCH in honey was described in the publicly available literature (van den Heever et al., 2015a), which had an LOQ of 10 µg/kg. The Committee considered that the method is suitable for monitoring DCH residues in honey. Estimated dietary exposure Chronic dietary exposure assessment Fumagillin Dietary exposure to fumagillin was estimated according to the potential occurrence of fumagillin residues in fish fillet and honey. For fish, residue concentrations were taken from measurements in rainbow trout, carp and eels that received a nominal dose of fumagillin of 50 mg/kg bw per day. The studies reported residue concentrations in terms of fumagillin (the marker residue). Data for the three species were combined, and regression analysis was used to estimate residue concentrations at a withdrawal period of 364 degree-days (28 days at 13 °C). At this withdrawal period, the regression line concentration of fumagillin was < LOQ (5 μg/kg). For estimation of dietary exposure, a fumagillin residue concentration of LOQ/2 (2.5 μg/kg) was applied. While no metabolites of fumagillin in fish were identified, only 37–62 percent of the TRR was recovered from rainbow trout tissue by extraction. Therefore, a marker residue to total residue ratio (MR:TR) of 0.5 was used to estimate chronic dietary exposure. Food consumption information in the FAO/WHO Chronic Individual Food Consumption – Summary Statistics database (CIFOCOss) were combined for all fish types to give total fish consumption. In apiculture, fumagillin is not used during honey flow; therefore, residues of fumagillin should not be present in honey. As no residue data were available from studies conducted according to GVP, the actual fumagillin concentrations in honey are not known. Therefore, for estimation of dietary exposure, the concentration of fumagillin residue in honey was assumed to be at the LOQ (5 μg/kg). FAO JECFA Monograph 33 75 Fumagillin is not metabolized in honey, although substantial degradation may occur. In honey in a hive, the concentration of fumagillin was reported to decrease by 32 percent over 28 days (van den Heever et al., 2015b). Although the toxicological significance of the fumagillin degradation products has not been investigated (see toxicological and microbiological evaluation), the Committee concluded that they are unlikely to have greater toxicological activity than fumagillin. Therefore, a conservative MR:TR of 0.5 was used to estimate dietary exposure. Food consumption information in CIFOCOss were combined for all honey types to give total honey consumption. According to the assumptions described above, the global estimates of chronic dietary exposure (GECDE) for adults and the elderly, children and adolescents, and infants and toddlers were 0.06, 0.10 and 0.11 μg/kg bw per day, respectively, which represent 2 percent, 3 percent and 4 percent of the upper bound of the acceptable daily intake (ADI) of 3 µg/kg bw. Details of the GECDE estimates are included in Table 23. Country-specific estimates of chronic dietary exposure were also determined. Instead of using the highest mean and the highest reliable percentile consumption from all surveys, the calculations were made with the mean and the highest reliable percentile in each national survey from available datasets (CIFOCOss). The highest GECDE for each age class for each country was determined. In accordance with the assumptions described above, the mean (range) of 43 country-specific estimates for fumagillin dietary exposure for adults and the elderly was 0.015 (0.003–0.053) µg/kg bw per day or 0.5 percent (0.1–1.8 percent) of the upper bound of the ADI (3 µg/kg bw). The mean (range) of 32 country-specific estimates of fumagillin dietary exposure for children and adolescents was 0.027 (0.006–0.090) µg/kg bw per day, or 0.9 percent (0.2–3.0 percent) of the upper bound of the ADI. The mean (range) of 23 country-specific estimates of fumagillin dietary exposure for infants and toddlers was 0.037 (0.008–0.097) µg/kg bw per day or 1.3 percent (0.3–3.2 percent) of the upper bound of the ADI. As no acute reference dose (ARfD) was necessary, acute dietary exposure, GEADE was not assessed for fumagillin. Dicyclohexylamine Information was available on residues of DCH in honey; however, the design of the study did not allow assessment of DCH residues when the veterinary drug is used in accordance with GVP. For estimation of dietary exposure, it was assumed that DCH should not be present in honey, and the LOQ (10 μg/kg) was used as an estimate of the median residue concentration after chronic dietary exposure. As DCH is not metabolized in honey and DCH is reasonably stable in honey, an MR:TR of 1 was used to estimate chronic dietary exposure. No information was available on the concentration of DCH residues in fish. In order to provide guidance on a potential target level for DCH in fish (Cfish), the maximum residue concentration consistent with the upper bound of the ADI (20 μg/kg bw) was back-calculated from the following equation: GECDE = (HRPFish × CFish) + (MeanHoney × CHoney) where: z HRPFish is the highest reliable percentile consumption of fish (0.019 kg/kg bw); z CFish is the maximum concentration of DCH in fish (in μg/kg); z meanHoney is the population mean consumption of honey (0.0014 kg/kg bw); and z CHoney is the assigned concentration of DCH in honey (10 μg/kg). Setting the GECDE to the upper bound of the ADI (20 μg/kg bw) results in an approximate value of Cfish of 1 050 μg/kg (rounded to 1 000 μg/kg). The calculation was based on food consumption by infants and toddlers, the age group with the highest food consumption per kg bw. FAO JECFA Monograph 3376 Ta bl e 23 . G lo ba l e st im at e of c hr on ic d ie ta ry e xp os ur e (G E C D E ) fo r fu m ag il li n in fi sh a nd h on ey Ca te go ry Ty pe M ed ia n co nc en tr at io n1 (µ g/ kg ) M ea n co ns um pt io n, wh ol e po pu la tio n2 (g /k g b w pe r da y) H RP co ns um pt io n, co ns um er s on ly 3 (g /k g b w pe r da y) M R: TR ra tio Ex po su re µg /k g b w/ da y G EC DE 4 M ea n H RP µg /k g bw /d ay % AD I Ad ul ts an d th e e ld er ly Fi sh an d se af oo d Fi sh 2. 5 2. 38 10 .6 0. 50 0. 01 2 0. 05 2 0. 05 2 Ho ne y Ho ne y 5 0. 28 0. 95 0. 50 0. 00 3 0. 00 9 0. 00 3 TO TA L 0. 05 5 2 Ch ild re n an d ad ol es ce nt s Fi sh an d se af oo d Fi sh 2. 5 3. 14 18 .0 0. 50 0. 01 6 0. 09 0 0. 09 0 Ho ne y Ho ne y 5 1. 21 1. 48 0. 50 0. 01 2 0. 01 5 0. 01 2 TO TA L 0. 10 2 3 In fa nt s a nd to dd ler s Fi sh an d se af oo d Fi sh 2. 5 2. 69 19 .3 0. 50 0. 01 3 0. 09 7 0. 09 7 Ho ne y Ho ne y 5 1. 41 8. 76 0. 50 0. 01 4 0. 08 8 0. 01 4 TO TA L 0. 11 1 4 N ot es : M R : m ar ke r re si du e, T R : t ot al r es id ue , H R P : h ig he st r el ia bl e pe rc en ti le , G E C D E : g lo ba l e st im at es o f ch ro ni c di et ar y ex po su re 1 F or fi sh , n o re si du es g re at er t ha n th e L O Q w er e de te ct ed a t th e sp ec ifi ed w it hd ra w al t im e an d a va lu e of L O Q /2 w as u se d as t he m ed ia n re si du e co nc en tr at io n. R es id ue s of fu m ag ill in sh ou ld n ot b e p re se nt in h on ey an d th e L O Q o f t he av ai la bl e a na ly tic al m et ho d w as u se d as th e m ed ia n re sid ue co nc en tra tio n, ex pr es se d as fu m ag ill in ; 2 H ig he st m ea n co ns um pt io n fi gu re s ba se d on w ho le p op ul at io n co ns id er ed fr om th e av ai la bl e da ta se t; 3 H ig he st re li ab le p er ce nt il e fo od c on su m pt io n fi gu re s ba se d on c on su m er s on ly c on si de re d fro m th e av ai la bl e da ta se t; 4 G EC D E is th e su m o f t he h ig he st ex po su re a t t he h ig he st re lia bl e pe rc en til e of c on su m pt io n fo r a fo od a nd th e m ea n di et ar y ex po su re s o f t he o th er fo od So ur ce : A ut ho rs ’ o w n el ab or at io n. FAO JECFA Monograph 33 77 Acute dietary exposure assessment The Committee concluded that it was unnecessary to establish an ARfD for fumagillin. The Committee established an ARfD for DCH of 0.7 mg/kg bw. No information was available to derive appropriate residue concentrations of DCH in fish or honey for estimation of acute dietary exposure (GEADE). In the GEADE method, the information on food consumption is on large portion sizes (97.5th percentile consumers only food consumption from single-day food surveys). Large portion sizes are reported for two population groups; children and the general population. For large portion sizes of fish and honey for children and the general population, the maximum DCH residue concentrations that would not result in exceedance of the ARfD are 22 000 and 25 000 μg/kg in fish for children and the general population, respectively, and 130 000 μg/kg in honey for both children and the general population. The details of this approach are outlined below. GEADE = LPS × CFish or Honey where: z LPS is the large portion size (0.0313 kg/kg bw for fish consumption by children, 0.0278 kg/kg bw for fish consumption by the general population and 0.0055 kg/kg bw for honey consumption by children or the general population). z Cfish or honey is the maximum concentration of DCH in fish or honey (μg/kg), consistent with the ARfD. The GEADE was set to the ARfD (700 μg/kg bw) and Cfish or honey was calculated. In the GEADE method, each relevant food is considered individually, as it is assumed that an individual would not be a high consumer of more than one food in a 24-hour period. Maximum residue limits In recommending MRLs for fumagillin DCH in fish and honey, the Committee considered the following factors: z The Committee established an ADI of 0–0.003 mg/kg bw for fumagillin. The Committee established an ADI of 0–0.02 mg/kg bw for DCH. z The Committee concluded that it was unnecessary to establish an ARfD for fumagillin. The Committee established an ARfD of 0.7 mg/kg bw for DCH. z Fumagillin DCH is approved in one Member State for use in fish. For application to fish, fumagillin DCH is administered in feed at a dose of 15–50 mg fumagillin base/kg bw for 30 consecutive days or in immersion baths containing fumagillin base at a concentration of 60 mg/L for 5 consecutive days. A withdrawal period of 28 days is applied for either use in fish (no water temperature specified). z Fumagillin DCH is approved in several Member States for use in honeybees. For application to honeybees, fumagillin DCH is incorporated into a sugar solution. The inclusion rate is 20–25 mg of fumagillin base per litre, administered once weekly for 6–8 weeks. Honeybees should be treated in the autumn after honey supers have been removed or in spring, when treatment should be completed 4 weeks before start of honey flow. z Data from a study with radiolabelled fumagillin were used to assess the depletion of fumagillin in rainbow trout at a water temperature of 15 ºC after a single oral dose. No studies of radiolabelled DCH were available. FAO JECFA Monograph 3378 z Fumagillin was identified as the marker residue in fish fillet and is considered suitable for monitoring residues. As no reliable MR:TR for fumagillin in fish fillet was identified, a conservative MR:TR value of 0.5 was applied in the dietary exposure assessment. z DCH was identified as the marker residue in honey and is considered more suitable for monitoring residues than fumagillin, which is unstable in this matrix. An MR:TR of 1 was used for DCH in the dietary exposure assessment, as it is not metabolized in honey, and DCH is reasonably stable in this matrix. z Data on residue depletion after administration of non-radiolabelled fumagillin DCH were available in rainbow trout, carp and eels. Only the concentrations of fumagillin were measured in tissues and not those of DCH. z Data were available on the concentration in honey of non-radiolabelled fumagillin and for a subset of samples, DCH concentrations were available. z Suitable LC-MS/MS analytical methods are available for the determination of the marker residues (fumagillin in fish and DCH in honey) and may be used for monitoring. z No suitable analytical method is currently available for the determination of DCH in fish. The Committee recommended an MRL in fish fillet of 10 µg/kg, which corresponds to twice the LOQ of the analytical method for the marker residue fumagillin. The Committee recommended that residues of DCH (including any potential metabolites) be monitored when fumagillin DCH preparations are used in fish to ensure that the concentration is < 1 000 µg/kg, which is a target level compatible with the upper bound of the ADI. The Committee noted that a suitable analytical method for the determination of DCH in fish fillet should be developed. The Committee recommended an MRL in honey of 20 µg/kg, which corresponds to twice the LOQ of the analytical method for the marker residue DCH. Data limitations and explanation of the approach taken by the Committee for recommending MRLs Fish Given the data challenges and sources of uncertainty in the fish residue depletion studies provided (no confirmation of the dose administered/consumed, weight of fish not stated, unconsumed feed not measured, treatment water not analysed, DCH residues not determined), the Committee acknowledged that the fumagillin residue concentrations reported in such studies may not accurately reflect fumagillin residue concentrations under GVP. Nevertheless, the Committee noted that no quantifiable fumagillin residue concentrations (LOQ 5 μg/kg) were observed in fish fillet in any study at the sampling time corresponding to GVP (withdrawal period of 28 days, no temperature indicated). At the approved withdrawal period and the lower water temperature (13 ºC, resulting in 364 degree-days), the Committee recommended an MRL for fumagillin in fish fillet of 10 µg/kg (twice the LOQ of the analytical method). The Committee considered it unlikely that fish harvested according to GVP (28 days or 364 degree-days) would contain fumagillin residues in fillet that exceed this value. The Committee noted that the withdrawal period according to GVP was reported as time (4 weeks) and not degree-days, and that other risk management options (such as longer withdrawal periods) could be considered if fumagillin DCH is to be used at water temperatures outside the range of those used in the studies reviewed (13–28 ºC). FAO JECFA Monograph 33 79 No data were provided or were available in the scientific literature on residue concentrations of DCH in fish fillet after administration of fumagillin DCH. No studies were available of use of radiolabelled DCH in fish, and its metabolism remains unknown. Therefore, the Committee was unable to define a marker residue or to assess depletion of DCH residues in fish. The MRLs recommended by the Committee for fumagillin residues in fish are protective of consumers and compatible with GVP. As fumagillin is currently used only in association with DCH in veterinary medicine, however, DCH residues may also be present when fumagillin residues are detected. The Committee used the limited information available to the current meeting to provide guidance on a potential target level for DCH in fish (see exposure section). The Committee recommended that residues of DCH (including any potential metabolites) be monitored when fumagillin DCH preparations are used in fish to ensure that the concentration is < 1 000 µg/kg, a target level compatible with the upper bound of the ADI. The Committee noted that a suitable analytical method for determination of DCH in fish fillet should be developed. Should JECFA receive sufficient data on DCH residues resulting from fumagillin DCH use in fish, the Committee may refine its recommendations. Honeybees The Committee noted that the data on fumagillin DCH residues in honey were not generated according to GVP and are likely to be overestimates of the residue concentrations that might be present if GVP were followed. When fumagillin DCH is used according to GVP, residues of neither fumagillin nor DCH should be present in honey. No data on the concentrations of bioactive degradation products of fumagillin or DCH in honey were provided with the residue studies. In view of the reported instability of fumagillin residues and the relative persistence of DCH in honey, the Committee recommended that DCH be used as the marker residue for this matrix. 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Republic of Korea, NIFDS. NIFDS. 2015b. Final Report (B): A Study on Residue Depletion of Fumagillin in Carp. Republic of Korea, NIFDS. NIFDS. 2015c. Final Report (C): A Study on Residue Depletion of Fumagillin in Eel. Republic of Korea, NIFDS. Nozal, M. A. J., Bernal, J. L., Martín, M. A. T., Bernal, J., Alvaro, A., Martín, R. & Higes, M. 2008. Trace analysis of fumagillin in honey by liquid chromatography-diode array-electrospray ionization mass spectrometry. Journal of Chromatography A, 1190(1-2): 224–231. https://www.doi.org/10.1016/j. chroma.2008.03.019. Rigos, G., Kotzamanis, I., Gialamas, I., Nengas, I. & Alexis, M. 2000. Toxicity and digestibility of fumagillin DCH in gilthead sea bream, Sparus aurata L. Journal of Fish Diseases, 23(2): 161–164. https://www.doi.org/10.1046/j.1365-2761.2000.00211.x FAO JECFA Monograph 3382 Suenaga, A., Wada, T. & Ichibagase, H. 1983. Studies on Synthetic Sweetening Agents. XVIII. Metabolism of Sodium Cyclamate. (7). Dicyclohexylamine, a Metabolite of Sodium Cyclamate in Rabbits and Rats. Chemical and Pharmaceutical Bulletin, 31(6): 2079–2084. https://www.doi. org/10.1248/cpb.31.2079 van den Heever, J. P., Thompson, T. S., Curtis, J. M. & Pernal, S. F. 2015a. Determination of Dicyclohexylamine and Fumagillin in Honey by LC-MS/MS. Food Analytical Methods, 8(3): 767–777. https://www.doi.org/10.1007/s12161-014-9956-x van den Heever, J. P., Thompson, T. S., Curtis, J. M. & Pernal, S. F. 2015b. Stability of dicyclohexylamine and fumagillin in honey. Food Chemistry, 179: 152–158. https://www.doi. org/10.1016/j.foodchem.2015.01.111 van den Heever, J. P., Thompson, T. S., Otto, S. J. G., Curtis, J. M., Ibrahim, A. & Pernal, S. F. 2016. Evaluation of Fumagilin-B® and other potential alternative chemotherapies against Nosema ceranae-infected honeybees (Apis mellifera) in cage trial assays. Apidologie, 47(5): 617–630. https:// www.doi.org/10.1007/s13592-015-0409-3 VICH (Veterinary International Conference on Harmonization). 2011. GL46 (MRK): Studies to Evaluate the Metabolism and Residue Kinetics of Veterinary Drugs in Food-Producing Animals: Metabolism Study to Determine the Quantity and Identify the Nature of Residues. Adopted at Step 7 of the VICH Process by the VICH Steering Committee in February 2011 for implementation in February 2012. https://vichsec.org/en/guidelines/pharmaceuticals/pharma-safety/metabolism-and-residue-kinetics.html VICH. 2018. GL56 (MRK): Studies to Evaluate the Metabolism and Residue Kinetics of Veterinary Drugs in Food-Producing Species: Study Design Recommendations for Residue Studies in Honey for Establishing Maximum Residue Limits and Withdrawal Periods. Adopted at Step 7 of the VICH Process by the VICH Steering Committee in June 2018 for implementation by June 2019. https://vichsec.org/en/ guidelines/pharmaceuticals/pharma-safety/metabolism-and-residue-kinetics.html FAO JECFA Monograph 33 83 Imidacloprid (fin fish) - Addendum First draft prepared by Samuel Fletcher, Norwich, United Kingdom of Great Britain and Northern Ireland Rainer Reuss, Barton, Australia and Susanne Rath, Campinas, São Paulo, Brazil Addendum to the monograph prepared by the 94th Meeting of the Committee and FAO JECFA Monograph 28 Background At its twenty-fifth meeting, the Codex Committee on Residues of Veterinary Drugs in Foods (CCRVDF) requested an evaluation of imidacloprid for use in all fin fish and for the Joint FAO/WHO Expert Committee on Food Additives (JECFA) to recommend maximum residue limits (MRLs) for muscle and fillet (muscle with skin in natural proportions). Imidacloprid (CAS no. 138261-41-3) was previously reviewed by the Committee at its 94th meeting (FAO and WHO, 2023). The Committee derived a toxicological acceptable daily intake (tADI) of 0–0.05 mg/kg bw per day and a toxicological acute reference dose (tARfD) of 0.09 mg/kg bw. Due to the absence of a study to assess the impact of imidacloprid on representative human intestinal microbiota, it was not possible to determine a microbiological acute reference dose (mARfD) or a microbiological acceptable daily intake (mADI), thus the Committee was unable to establish an acute reference dose (ARfD) or an acceptable daily intake (ADI) for imidacloprid. As the Committee could not establish an ADI or an ARfD, MRLs could not be recommended. Current evaluation Imidacloprid was on the agenda for the current meeting to complete the assessment. The previously derived tADI of 0–0.05 mg/kg bw/d and the tARfD of 0.09 mg/kg bw were confirmed at the current meeting as the overall health-based guidance values (HBGVs), as the microbiological data provided indicated that mADI and mARfD were not required for imidacloprid. No additional residue data were submitted for the current meeting. The Committee reviewed the data contained in the monograph prepared by the 94th Meeting of the Committee, which contained the residue data used to generate upper tolerance limits and estimate dietary exposure (FAO and WHO, 2023). For ease of reference, the key information is represented below. FAO JECFA Monograph 3384 Residues in food and their evaluation Conditions of use Imidacloprid is registered as the veterinary medicinal product Ectosan Vet 1 000 mg/g powder for treatment solution for fish (hereafter referred to as Ectosan), which contains 100 percent imidacloprid. This product is currently authorized for use in Norway (MTnr. 20-13358) and is indicated for the treatment of pre-adult and adult salmon lice Lepeophtheirus salmonis infestation in Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss. It is a bath treatment for use in closed containment vessels (well-boats) only, due to environmental concerns. The product has an approved withdrawal period of 98 degree-days for both Atlantic salmon and rainbow trout. Dosage The authorized dosing regimen is 20 mg imidacloprid per litre of sea water for a period of 60 minutes in a well-boat. It has been noted that the duration of immersion in the treatment baths might be extended to up to 6 hours because of the method used to administer the product. Appraisal At the current meeting, JECFA established an ADI of 0–0.05 mg/kg bw/d and an ARfD of 0.09 mg/kg bw, based on the toxicological effects of the drug. Pharmacokinetics The pharmacokinetics in fish appears to be similar in all species for which data are available. Imidacloprid is distributed to all tissues in various proportions and hardly metabolized. The only metabolite seen is 5-hydroxy imidacloprid. The Committee identified imidacloprid as the sole marker residue in Atlantic salmon and rainbow trout fillet and determined that a value of 0.7 was appropriate for the marker residue to total radioactive residue ratio (MR:TRR). Residue Depletion The authorized dosing regimen is 20 mg/L administered in a treatment bath for 60 minutes. However, in field conditions it has been noted that it is not always possible to remove the fish from the treatment baths within a reasonable time, which is why data from studies using increased durations of immersion (up to 360 minutes) were provided. The Committee considered the residue depletion study by Longshaw (2020) to be the pivotal study, as this gave the worst-case results in terms of extent and persistence of imidacloprid residues in Atlantic salmon fillet (Table 1). It was noted that this was because the salmon had been exposed to the treatment solution for longer than the approved duration (6-fold longer). However, it was also noted that this was a practical consideration since the salmon had to be treated in well-boats to prevent exposure of the environment to imidacloprid. As a result, in some cases it can take longer than the approved treatment time to remove the salmon from the treatment bath. It was also noted that this was the study used to set the withdrawal period for the one approved product in a Member State. FAO JECFA Monograph 33 85 Table 1. Mean concentration (± SD) of imidacloprid (µg/kg) in Atlantic salmon fillet 1, 7, 14, 21, 28 and 33 days post exposure Days post treatment (Degree-days) 60 min Mean ± SD 196 min Mean ± SD 360 min Mean ± SD 1 (15.6) 359 ± 63 740 ± 83 1 372 ± 114 7 (108.8) 71 ± 15 160 ± 24 296 ± 56 14 (206.8) 12.73 ± 3.81 27.45 ± 9.87 54.73 ± 15.90 21 (313.2) <LLOQ 6.29 ± 2.28a 9.57 ± 3.70 28 (422.1) <LLOQ <LLOQ 2.68 ± 1.64a 33 (508.6) <LLOQ <LLOQ <LLOQ Notes: LLOQ: lower limit of quantification (4 µg/kg); awhere one or more samples were <LLOQ, the value was taken as 2 µg/kg (1/2 LLOQ) in order to calculate the mean; SD: standard deviation Source: based on Longshaw, M. 2020. Extended ECTOSAN® bath exposure and sampling of Atlantic salmon (Salmo salar) for residue analysis. Report ARD-0014. Ardtoe Marine Laboratory, Ardtoe, Argyll, UK. Sponsor submitted. It should be noted that there are no data available for repeated treatments, but imidacloprid would be expected to accumulate in fish tissues upon repeated treatments that occur too close together. The MRLs recommended for salmon fillet are based on the upper limit of the one-sided 95 percent confidence interval over the 95th percentile of residue concentrations (95/95 upper tolerance limit, or UTL) for the 98 degree-day post-treatment data from the non-radiolabeled residue depletion study in Atlantic salmon. The tolerance limits for imidacloprid in Atlantic salmon fillet are shown in Figure 1. Figure 1. Tolerance limit considerations for imidacloprid in Atlantic salmon fillet Co nc en tr at io n of im id ac lo pr id ( µg /k g) 10 000 1 000 100 10 Time (Degree-days) 35050 100 150 250200 300 0 0 Notes: blue: regression line; orange: 95/95 UTL regression line; yellow: 95/99 UTL regression line; grey: 99/99 UTL regression line Source: based on Longshaw, M. 2020. Extended ECTOSAN® bath exposure and sampling of Atlantic salmon (Salmo salar) for residue analysis. Report ARD-0014. Ardtoe Marine Laboratory, Ardtoe, Argyll, UK. Sponsor submitted. FAO JECFA Monograph 3386 Dietary exposure assessment Dietary exposure from pesticide residues The Committee noted at the 94th meeting of JECFA that dietary exposure to imidacloprid may also occur through its multiple registered uses as a pesticide. A review of previous assessments of pesticide residues showed that dietary exposure from this source is low and most likely not a substantial contributor to overall dietary exposure. Chronic dietary exposure estimates At the 94th JECFA, the global estimate of chronic dietary exposure (GECDE) to imidacloprid residues was estimated based on the potential occurrence of imidacloprid residues in Atlantic salmon muscle and all fin fish meat. However, no ADI was recommended at that meeting. The ADI established at the current meeting is 0–0.05 mg/kg bw. Based on incurred residues in Atlantic salmon (fillet) and a withdrawal period of 98 degree-days, the estimate of chronic dietary exposure for adults and the elderly (1.0 μg/kg bw per day) is 2 percent of the upper limit of the ADI. For children and adolescents estimated exposure (2.7 μg/kg bw per day) is 5 percent of the upper limit of the ADI, and for infants and toddlers, estimated exposure (0.9 μg/kg bw per day) is 2 percent of the upper limit of the ADI. Based on consumption of total fin fish meat, the exposure for adults and the elderly (1.8 μg/kg bw per day) is 4 percent of the upper limit of the ADI. For children and adolescents, exposure (3.8 μg/kg bw per day) is 8 percent of the upper limit of the ADI, and for infants and toddlers, exposure (1.2 μg/kg bw per day) is 2 percent of the upper limit of the ADI. Acute dietary exposure estimates At the 94th JECFA, the global estimate of acute dietary exposure (GEADE) to imidacloprid residues was estimated based on the occurrence of residues in Atlantic salmon muscle and all fin fish meat. However, no ARfD was recommended at that meeting. At the current meeting, the ARfD was established as 0.09 mg/kg bw (90 µg/kg bw). The acute dietary exposure for adults and children (6.2 and 6.6 μg/kg bw, respectively) based on consumption of Atlantic salmon was 7 percent of the ARfD. Acute exposure based on consumption of fin fish (34.1 and 23.8 μg/kg bw) was 38 and 26 percent of the ARfD for adults and children, respectively. Maximum residue limits In recommending MRLs for imidacloprid in fin fish fillet (muscle with skin in natural proportions) and muscle, the Committee considered the following: z The ADI for imidacloprid is 0–0.05 mg/kg bw. z The ARfD for imidacloprid is 0.09 mg/kg bw. z Imidacloprid is used as a pesticide and as a veterinary drug. z Imidacloprid is authorized for use in Atlantic salmon and rainbow trout. The maximum recommended dose is 20 mg/L once, by immersion in a seawater treatment bath for 60 min. The withdrawal period is 98 degree-days for both species. FAO JECFA Monograph 33 87 z Under field conditions, it may not be possible to remove all the fish from the treatment bath immediately after 60 min. Therefore, extended exposure up to 360 min was considered. z Imidacloprid is the marker residue in Atlantic salmon and rainbow trout muscle and fillet. z The ratio of the concentration of marker residue to that of total residue was calculated to be 0.7. z Data on residues in Atlantic salmon and rainbow trout were provided which were derived with a validated analytical method for quantifying imidacloprid in muscle and fillet. z A validated analytical method for determining imidacloprid in Atlantic salmon and rainbow trout muscle and fillet is available and may be used for monitoring purposes. z The MRL recommended for salmon and trout muscle, or fillet, is based on the 95/95 UTL at 98 degree-days from an unlabelled residue depletion study in Atlantic salmon. z The Committee recommended an MRL for Atlantic salmon and rainbow trout fillet (muscle with skin in natural proportions), or muscle, of 600 µg/kg. The Committee recommended that the MRL be extrapolated to all fin fish. When considering the possibility of recommending the extrapolation of MRLs, the Committee referred to the discussion at the CCRVDF on the “Proposed approach for the extrapolation of maximum residue limits of veterinary drugs to one or more species” (FAO and WHO, 2021). Although there are no pharmacokinetic or residue depletion data currently available for species other than Atlantic salmon and rainbow trout, experience shows in general that fish do not metabolize pharmaceutical compounds to a great extent. As such, it is considered to be unlikely that the MR:TRR in non-salmonid fin fish species would be much different from that seen in Atlantic salmon. It should be noted, however, that water temperature influences the extent of absorption, extent of metabolism, and the rate of elimination in fish. The data available do not address rate or extent of metabolism at higher water temperatures. Many other farmed fin fish species are kept at higher temperatures than salmonid species, and so might have different metabolic profiles, including an increased metabolism which would lead to a lower MR:TRR being calculated, leading to an underestimate of human dietary exposure. Nonetheless, the Committee considered that, as the worst-case scenario had been used for estimating the likely dietary exposure, there would be a margin of safety for the proposed MRL that could take into account slight differences in metabolism between salmonids and non-salmonids. FAO JECFA Monograph 3388 References FAO & WHO (Food and Agriculture Organization of the United Nations & World Health Organization). 2021. Report of the 25th Session of the Codex Committee on Residues of Veterinary Drugs in Foods. https://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?lnk=1&url=https%253A%252 F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FMeetings%252FCX-730-25%252FREPOR T%252FFinals%252FREP21_RVDFe.pdf FAO & WHO. 2023. Residue evaluation of certain veterinary drugs – Joint FAO/WHO Expert Committee on Food Additives, 94th Meeting (Virtual) 16–27 May 2022. Joint FAO/WHO Expert Committee on Food Additives (JECFA) Monographs, No. 28. Rome. https://doi.org/10.4060/cc5153en Longshaw, M. 2020. Extended ECTOSAN® bath exposure and sampling of Atlantic salmon (Salmo salar) for residue analysis. Report ARD-0014. Ardtoe Marine Laboratory, Ardtoe, Argyll, UK. Sponsor submitted. FAO JECFA Monograph 33 89 Annex 1 - Summary of recommendations from the 98th JECFA on compounds on the agenda and further information required Clopidol (coccidiostat) Acceptable daily intake The Committee established an ADI for clopidol of 0–0.04 mg/kg bw based on a lowest-observed-adverse-effect level (LOAEL) of 40 mg/kg bw per day for decreased maternal and foetal body weight gain. An uncertainty factor of 1000 was applied, which comprises 100 for inter and intra-species differences and additional factors of 2 to account for the use of a marginal LOAEL and 5 for database uncertainty. Acute reference dose The Committee concluded that it was unnecessary to establish an ARfD for clopidol. Estimated dietary exposure For clopidol included at 250 mg/kg in feed at 24 h withdrawal and the most conservative ratio of marker residues to total residues (MR:TR) considered of 0.5, the global estimate of acute dietary exposure (GECDE) for adults and the elderly, children and adolescents, and infants and toddlers were 32.9, 33.5 and 28.6 μg/kg bw per day, respectively (82, 84 and 71 percent, respectively, of the upper bound of the ADI of 40 µg/kg bw). Residue definition The marker residue for clopidol in chicken liver, kidney, muscle and skin/fat is clopidol. Maximum residue limits The Committee recommended MRLs of 10 400 µg/kg (liver), 8 800 µg/kg (kidney), 4 100 µg/kg (muscle) and 2 600 µg/kg (skin/fat) in chickens. FAO JECFA Monograph 3390 Fumagillin dicyclohexylamine (antimicrobial agent) In veterinary medicine, fumagillin is administered only as the dicyclohexylamine (DCH) salt. As the fumagillin DCH salt dissociates into the two moieties, consumers would be exposed to residues of both. The Committee evaluated both fumagillin and DCH. Acceptable daily intake The Committee established an ADI of 0–0.003 mg/kg bw for fumagillin and an ADI of 0–0.02 mg/kg bw for DCH. Acute reference dose The Committee concluded that it was unnecessary to establish an ARfD for fumagillin. It established an ARfD of 0.7 mg/kg bw for DCH. Estimated dietary exposure For potential fumagillin residues in fish fillet and honey, the GECDE values for adults and the elderly, children and adolescents, and infants and toddlers were 0.06, 0.10 and 0.11 μg/kg bw per day, respectively, which represent 2, 3 and 4 percent of the upper bound of the ADI of 3 µg/kg bw. There was insufficient information to estimate dietary exposure (chronic or acute) to DCH. Residue definition The marker residue for fumagillin DCH in fish fillet is fumagillin. The marker residue for fumagillin DCH in honey is DCH. Maximum residue limits The Committee recommended an MRL in fish fillet of 10 µg/kg for the marker residue fumagillin. The Committee recommended that residues of DCH (including any potential metabolites) be monitored when fumagillin DCH preparations are used in fish to ensure that the concentration is < 1 000 µg/kg, a target level compatible with the upper bound of the ADI. The Committee noted that a suitable analytical method for the determination of DCH in fish fillet should be developed. The Committee recommended an MRL in honey of 20 µg/kg for the marker residue DCH. FAO JECFA Monograph 33 91 Imidacloprid (parasiticide) Imidacloprid (neonicotinoid parasiticide) Imidacloprid exerted very low or no measurable antibacterial activity against the representative bacterial strains of the human intestinal microbiome tested. The Committee concluded that no mADI or mARfD was required Acceptable daily intake The Committee established an ADI of 0–0.05 mg/kg bw, based on a NOAEL of 5.25 mg/kg bw per day for decreased body weight gain in the extended one-generation reproduction study, with application of a safety factor of 100 to allow for interspecies and intraspecies differences. Acute reference dose The Committee established an ARfD of 0.09 mg/kg bw based on a BMDL05 of 9 mg/kg bw for acute neurobehavioural effects in rats and a safety factor of 100 to allow for interspecies and intraspecies differences. Residue definition The marker residue for imidacloprid in fin fish is the parent molecule, imidacloprid. Dietary exposure For Atlantic salmon only, the GECDE was 1.0, 2.7 and 0.9 μg/kg bw per day (2, 5 and 2 percent of the upper bound of the ADI of 50 μg/kg bw) for adults and the elderly, children and adolescents, and toddlers and infants, respectively. For all fin fish, the GECDE was 1.8, 3.8 and 1.2 μg/kg bw per day (4, 8 and 2 percent of the upper bound of the ADI of 50 μg/kg bw) for adults and the elderly, children and adolescents, and toddlers and infants, respectively. The GEADE, based on consumption of Atlantic salmon, was 7 percent of the ARfD for adults and children (6.2 and 6.6 μg/kg bw, respectively); the GEADE for all fin fish was 38 and 26 percent of the ARfD (34.1 and 23.8 μg/kg bw) for adults and children, respectively. Maximum residue limits The Committee recommended an MRL for Atlantic salmon and rainbow trout fillet (muscle with skin in natural proportions) and/or muscle of 600 µg/kg. It further recommended that the MRL be extrapolated to all fin fish. FAO JECFA Monograph 3392 Future work and recommendations Recommendations relating to specific veterinary drugs, including ADIs and proposed MRLs, are given in section 3 of each monograph, which includes recommendations relating to future work by the JECFA Secretariat. Guidance for the Safety Evaluation of Residues of Veterinary Drugs with incomplete data packages The Committee adopted the guidance and welcomes comments from CCRVDF. JECFA Toolbox for Veterinary Drug Residues Risk Assessment The toolbox is expected to be available by the end of 2024 and will be publicly available on the FAO website. FAO JECFA Monograph 33 93 Annex 2 - Summary of JECFA evaluations of veterinary drug residues from the 32nd meeting to the present The following table summarizes the veterinary drug evaluations conducted by JECFA at the 32nd (1987), 34th (1989), 36th (1990), 38th (1991), 40th (1992), 42nd (1994), 43rd (1994), 45th (1995), 48th (1997), 50th (1998), 52nd (1999), 54th (2000), 58th (2002), 60th (2003), 62nd (2004), 66th (2006), 70th (2008), 75th (2011), 78th (2013), 81st (2015), 85th (2017), 88th (2019), 94th (2022), and 98th (2024) meetings. These meetings were devoted exclusively to the evaluation of veterinary drug residues in food. This table must be considered in context with the full reports of these meetings, published as WHO Technical Report Series. Some notes regarding the table: z The “ADI/ArfD” column provides the ADI and, when applicable, the ArfD established by the Committee. When no ARfD is stated, an ArfD has not been established. z The “ADI Status” column refers to the ADI and indicates whether an ADI was established; if a full ADI was given, or if the ADI is temporary (T). z Where an MRL is temporary, it is indicated by “T”. z Where a compound has been evaluated more than once, the data given are for the most recent evaluation, including the 78th meeting of the Committee. Substance ADI/ARfD (µg/kg bw) ADI Status JECFA MRL (µg/kg) Tissue Species Marker residue and other remarks Abamectin ADI: 0-1 (JMPR 1995) Full 47 (1996) 100 50 Liver, Fat Kidney Cattle Avermectin B1a Albendazole ADI: 0-50 Full 34 (1989) 100 5000 Muscle, Fat, Milk Liver, Kidney Cattle, Sheep MRLs analysed as 2-amino- benzimidazole, expressed as albendazole equivalents Amoxicillin ADI: 0-0.7 Full 75 (2011) 50 Muscle, Liver, Kidney, Fat Milk Cattle, Pig, Sheep Cattle, Sheep Amoxicillin ADI: 0-2 Full 85 (2017) 4 50 Fillet, Muscleb Finfisha Ampicillin ADI: 0-3 Full 85 (2017) 50 Muscleb Finfisha Ampicillin Apramycin ADI: 0-30 Full 75 (2011) 5000 Kidney Cattle, Chicken Apramycin Avilamycin ADI: 0-2000 (as avilamycin activity) Full 70 (2008) 200 Muscle, Kidney, Pig, Chicken, Turkey, Rabbit Dichloroisoeverninic acid (DIA), expressed as avilamycin equivalents Skin/Fat Pig, Chicken, Turkey, Rabbit 300 Liver Azaperone ADI: 0-6 Full 52 (1999) 60 Muscle, Fat Pig Sum of azaperone and azaperol 100 Liver, Kidney FAO JECFA Monograph 3394 Substance ADI/ARfD (µg/kg bw) ADI Status JECFA MRL (µg/kg) Tissue Species Marker residue and other remarks Benzylpenicillin ADI: <30µg/ person/day of the penicillin moiety Full 36 (1990) 50 Muscle, Liver, Kidney All species Benzylpenicillin 4 Milk Bovine Somatotropins ADI: Not specified Full 78 (2013) Not specified Muscle, Liver, Kidney, Fat, Milk Cattle Carazolol ADI: 0-0.1 ARfD: 0.1 Full 52 (1999) 5 Muscle, Fat/ Skin The Committee noted that the concentration of carazolol at the injection site may exceed the ADI that is based on the acute pharmacological effect of carazolol. 25 Liver, Kidney Pig Carbadox No ADI or ARfD 60 (2003) No MRL The Committee decided that quinoxaline-2- carboxylic acid is not an appropriate marker residue Ceftiofur ADI: 0-50 Full 48 (1997) 1000 Muscle Cattle, Pig Desfuroylceftiofur 2000 Liver, Fat 6000 Kidney 100 Milk Cattle Cefuroxime No ADI or ARfD 62 (2004) No MRL Chloramphenicol No ADI or ARfD 62 (2004) No MRL Chlorpromazine No ADI or ARfD 38 (1991) No MRL Chlortetracycline, Oxytetracycline, Tetracycline ADI: 0-30 (group ADI) Full 58 (2002) 200 Muscle Cattle, Pig, Sheep, Poultry Parent drugs, either singly or in combination 600 Liver 1200 Kidney 400 Eggs Poultry 100 Milk Cattle, Sheep 200 Muscle Fish, giant prawn Oxytetracycline only Clenbuterol ADI: 0-0.004 Full 47 (1996) 0.2 Muscle, Fat Cattle, Horse Clenbuterol 0.6 Liver, Kidney Cattle, Horse 0.05 Milk Cattle Clopidol ADI: 0–40 No ARfD Full 98 (2024) 10 400 Liver Chickens Clopidol 8800 Kidney 4100 Muscle 2600 Skin/fat Closantel ADI: 0-30 Full 40 (1992) 1000 Muscle, Liver Cattle Closantel 3000 Kidney, Fat 1500 Muscle, Liver Sheep 5000 Kidney 2000 Fat FAO JECFA Monograph 33 95 Substance ADI/ARfD (µg/kg bw) ADI Status JECFA MRL (µg/kg) Tissue Species Marker residue and other remarks Colistin ADI: 0-7 Full 66 (2006) 150 Muscle, Liver, Fat Cattle, Sheep, Goat, Chicken, Turkey, Pig, Residue definition is the sum of Colistin A and colistin B. The MRL includes skin + fat where appropriate (chicken, turkey, pigs). 200 Kidney Rabbit 50 Milk Cattle, Sheep 300 Eggs Chicken Cyfluthrin ADI: 0-20 Full 48 (1997) 20 200 Muscle, Liver, Kidney Cattle Cyfluthrin 40 Fat Milk Cyhalothrin ADI: 0-5 Full 62 (2004) 20 Muscle, Kidney Cattle, Sheep, Pig Cyhalothrin 400 Fat Cattle, Sheep, Pig 20 Liver Cattle, Pig 50 Liver Sheep 30 Milk Cattle, Sheep Cypermethrin α-Cypermethrin ADI: 0-20 (group ADI) Full 62 (2004) 50 1000 100 Muscle, Liver, Kidney Fat Milk Cattle, Sheep Cattle, Sheep Cattle Total of cypermethrin residues (resulting from the use of cypermethrin or α-cypermethrin as veterinary drugs) Danofloxacin ADI: 0-20 Full 48 (1997) 200 Muscle Cattle, Danofloxacin 400 Liver, Kidney Chicken 100 Fat For chicken fat/skin in normal proportions 100 Muscle Pig 50 Liver 200 Kidney 100 Fat Deltamethrin ADI: 0-10 (1982 JMPR) Full 60 (2003) 30 Muscle Cattle, Chicken, Sheep, Salmon Deltamethrin 50 Liver, Kidney Cattle, Sheep, Chicken500 Fat 30 Milk Cattle 30 Eggs Chicken Derquantel ADI: 0-0.3 Full 78 (2013) 0.3 0.4 Muscle Kidney Sheep Derquantel 7 Fat 0.8 Liver Dexamethasone ADI: 0-0.015 Full 70 (2008) 1 Muscle, Kidney Cattle, Pig, Horse Dexamethasone 2 Liver Cattle, Pig, Horse 0.3 Milk Cattle Diclazuril ADI: 0-30 Full 50 (1998) 500 Muscle Sheep, Rabbit, Poultry Diclazuril Poultry skin + fat 3000 Liver 2000 Kidney 1000 Fat FAO JECFA Monograph 3396 Substance ADI/ARfD (µg/kg bw) ADI Status JECFA MRL (µg/kg) Tissue Species Marker residue and other remarks Diflubenzuron No ADI or ARfD 81 (2015) No MRL Diflubenzuron ADI: 0–20 No ARfD 88 (2019) 10 Muscle plus skin Salmon Dihydro- streptomycin Streptomycin ADI: 0-50 (group ADI) Full 58 (2002) 600 Muscle, Liver, Fat Cattle, Pig, Chicken, Sheep Sum of dihydrostreptomycin and streptomycin 1000 Kidney 200 Milk Cattle, Sheep Dimetridazole No ADI or ARfD 34 (1989) No MRL Diminazene ADI: 0-100 Full 42 (1994) 500 12000 Muscle Liver Cattle Diminazene 6000 Kidney 150 Milk Doramectin ADI: 0-1 Full 62 (2004) 10 5 Muscle Muscle Cattle Pigs Doramectin 100 Liver Cattle, Pigs 30 Kidney Cattle, Pigs 150 Fat Cattle, Pigs 15 Milk Cattle Emamectin benzoate ADI: 0–0.5 Full 78 (2013) 100 Muscle Salmon Emamectin B1a Fillet (muscle with skin) Trout Enrofloxacin ADI: 0-2 Full 48 (1997) No MRL Eprinomectin ADI: 0-10 Full 50 (1998) 100 2000 Muscle Liver Cattle Eprinomectin B1a 300 Kidney 250 Fat 20 Milk Erythromycin ADI: 0-0.7 Full 66 (2006) 100 Muscle, Liver, Chicken, Turkey Erythromycin A Kidney, Fat/Skin 50 Eggs Chicken Estradiol-17β ADI: 0-0.05 Full 52 (1999) Not specified Muscle, Liver, Kidney, Fat Cattle Ethion ADI: 0–2 ARfD: 20 88 (2019) No MRL Febantel, Fenbendazole, Oxfendazole ADI: 0-7 (group ADI) Full 50 (1998) 100 Muscle, Kidney, Fat Cattle, Goat, Horses, Sum of febantel, fenbendazole and oxfenbendazole, expressed as oxfendazole sulfone equivalents 500 Liver Pig, Sheep 100 Milk Cattle, Sheep Fenbendazole (see Febantel) Fluazuron ADI: 0-40 Full 48 (1997) 200 500 Muscle Liver, Kidney Cattle Fluazuron 7000 Fat Flubendazole ADI: 0-12 Full 40 (1992) 10 Muscle, Liver Pig Flubendazole 200 Muscle Poultry 500 Liver Poultry 400 Eggs Poultry FAO JECFA Monograph 33 97 Substance ADI/ARfD (µg/kg bw) ADI Status JECFA MRL (µg/kg) Tissue Species Marker residue and other remarks Flumequine ADI: 0-30 Full 66 (2006) 500 Muscle Cattle, Sheep, Pig, Chicken Flumequine 1000 Fat 500 Liver 3000 Kidney Trout The MRLs are temporary for Black Tiger Shrimp and Shrimp. The MRLs for shrimp applies to all fresh water and marine shrimp. 500 Muscle Black Tiger Shrimp 500T Muscle Shrimp Flumethrin ADI: 0-4 Full 85 (2017) 6 Honey Flumethrin (trans-Z1 and trans Z2 diastereomers at a ratio of approximately 60:40). ADI: 0–4 ARfD: 5 Full 88 (2019) No MRL Fosfomycin No ADI ARfD: 80 88 (2019) No MRL Fumagillin dicyclo- hexylamine Fumagillin: ADI: 0–3 No ARfD DCH: ADI: 0–20 ARfD: 700 Full 98 (2024) 10 20 Fillet Fish Honey Fumagillin DCH The Committee recommended that residues of DCH (including any potential metabolites) be monitored when fumagillin DCH preparations are used in fish to ensure that the concentration is <1000 µg/kg, a target level compatible with the upper bound of the ADI. A suitable analytical method for the determination of DCH in fish fillet should be developed. Furazolidone No ADI or ARfD 40 (1992) No MRL Gentamicin ADI: 0-20 Full 50 (1998) 100 Muscle, Fat Cattle, Pig Gentamicin 2000 Liver 5000 Kidney 200 Milk Cattle Gentian violet No ADI or ARfD 78 (2013) No MRL Halquinol ADI: 0–200 ARfD: 300 Full 88 (2019) 40 350 500 9000 Muscle Skin plus fat Liver Kidney Swine Sum of 5-chloroquinolin- 8-ol (5-CL), 5,7-dichloroquinolin-8- ol 5,7-DCL (5,7-DCL) and their glucuronide metabolites: 5-CLG (expressed as 5-CL equivalents) and 5,7- DCLG (expressed as 5,7-DCL equivalents) FAO JECFA Monograph 3398 Substance ADI/ARfD (µg/kg bw) ADI Status JECFA MRL (µg/kg) Tissue Species Marker residue and other remarks Imidacloprid No ADI or ARfD ADI: 0–50 ARfD: 90 Full 94 (2022) 98 (2024) No MRL 600 Fillet (Muscle with skin) and/or Muscle Atlantic salmon and rainbow trout Imidacloprid Imidacloprid ADI based on NOAEL of 5.25 mg/kg bw per day. ARfD based on BMDL05 of 9 mg/kg bw. The Committee recommended that the MRL be extrapolated to all fin fish. Imidocarb ADI: 0-10 Full 60 (2003) 300 Muscle Cattle Imidocarb, free base 1500 Liver 2000 Kidney 50 Fat, Milk Ipronidazole No ADI or ARfD 34 (1989) No MRL Isometamidium ADI: 0-100 Full 40 (1992) 100 Muscle, Fat, Milk Cattle Isometamidium 500 Liver 1000 Kidney Ivermectin ADI: 0–10 ARfD: 200 Full 81 (2015) 30 800 100 400 15 20 10 Muscle Liver Kidney Fat Liver Fat Milk Cattle Pig, Sheep Cattle Ivermectin B1a. The Committee considers that the presence of high concentrations of ivermectin residues at the injection site is product dependent and must be assessed on a case-by-case basis during marketing authorization by comparison of suitable acute dietary exposure estimates with the ARfD. ADI: 0–10 ARfD: 200 Full 88 (2019) 20 15 15 10 Fat Kidney Liver Muscle Sheep, pigs and goats The MR in sheep, pigs and goats in ivermectin B1a (H2B1a, or 22,23- dihydroavermectin B1a) ADI: 0–10 ARfD: 200 Full 94 (2022) 15 30 20 50 Muscle Liver Kidney Fat Pigs 30 60 Muscle Liver Sheep, goats 20 Kidney 100 Fat Lasalocid sodium ADI: 0–5 Full 81 (2015) 400 Muscle Chicken, Turkey, Quail, Pheasant Lasalocid A 1200 Liver 600 Kidney 600 Fat/Skin Levamisole ADI: 0-6 Full 42 (1994) 10 Muscle, Kidney, Fat Cattle, Sheep, Pig, Poultry Levamisole 100 Liver FAO JECFA Monograph 33 99 Substance ADI/ARfD (µg/kg bw) ADI Status JECFA MRL (µg/kg) Tissue Species Marker residue and other remarks Lincomycin ADI: 0-30 Full 62 (2004) 200 Muscle Chicken, Pig Lincomycin A separate MRL of 300 µg/kg for skin with fat adhering fat for pigs was recommended to reflect the concentrations found in skin of pigs. MRL was also extended skin/fat for chicken. 500 Liver Chicken, Pig 1500 500 100 150 Kidney Kidney Fat Milk Pig Chicken Chicken, Pig Cattle Lufenuron ADI:0-20 Full 85 (2017) 1350 Muscleb Finfisha Lufenuron Melengestrol Acetate ADI: 0-0.03 Full 66 (2006) 1 Muscle Cattle Melengestrol acetate 10 Liver 2 Kidney 18 Fat Metronidazole No ADI or ARfD 34 (1989) No MRL Monensin ADI: 0-10 Full 70 (2008) 10 Muscle, Liver, Kidney Chicken, Turkey, Quail Monensin 75 (2011) 10 Muscle, Kidney Cattle, Sheep, Goat 20 Liver Sheep, Goat 100 Liver Cattle Cattle liver MRL revised at 75 JECFA 100 Fat Cattle, Sheep, Goat, Chicken, Turkey, Quail 2 Milk Cattle Monepantel sulfone ADI: 0-20 Full 78 (2013) 500 7000 Muscle Liver Sheep Monepantel sulfone 1700 Kidney 13000 Fat ADI: 0-20 Full 85 (2017) 300 2000 Muscle Liver Cattle Monepantel sulfone 1000 Kidney 7000 Fat Moxidectin ADI: 0-2 Full 50 (1998) 20 Muscle Cattle, Deer Moxidectin The Committee noted very high concentrations and great variation in the residue levels at the injection site in cattle over a 49-day period after dosing. 50 Muscle Sheep 100 Liver Cattle, Deer, Sheep 50 Kidney Cattle, Deer, Sheep 500 Fat Cattle, Deer, Sheep Narasin ADI: 0-5 Full 70 (2008) 15 Muscle, Kidney Chicken, Pig Narasin A Temporary MRLs for cattle, replaced with full MRLs in cattle tissue 75 (2011) 50 Liver, Fat Chicken, Pig 15 Muscle, Kidney Cattle 50 Liver, Fat Cattle Neomycin ADI: 0-60 Full 60 (2003) 500 Muscle, Fat, Liver Cattle, Chicken, Sheep, Turkey Goat, Pig, Duck Neomycin 10000 Kidney 1500 Milk Cattle 500 Eggs Chicken FAO JECFA Monograph 33100 Substance ADI/ARfD (µg/kg bw) ADI Status JECFA MRL (µg/kg) Tissue Species Marker residue and other remarks Nicarbazin ADI: 0-400 Full 50 (1998) 94 (2022) 200 Muscle, Liver, Kidney, Fat/ Skin Chicken (broilers) N,N’-bis (4-nitrophenyl)urea ADI: 0–900 No ARfD 4000 Muscle Chicken 4,4’-dinitrocarbanilide (DNC) 15000 Liver 8000 Kidney 4000 Skin plus fat Nitrofurazone/ Nitrofural No ADI 40 (1992) No MRL Olaquindox No ADI or ARfD 42 (1994) No MRL The Committee recommended no MRLs but noted that 4µg/kg in muscle of pigs of the metabolite MQCA (3-Methylquinoxaline- 2-carboxylic acid) is consistent with Good Veterinary Practice. Oxfendazole (See Febantel) Oxolinic acid No ADI or ARfD 43 (1994) No MRL Oxytetracycline See chlortetracycline Permethrin No ADI or ARfD 54 (2000) No MRL Phoxim ADI: 0-4 Full 62 (2004) 50 Muscle, Liver, Kidney Goat, Pig, Sheep Phoxim 400 Fat Pirlimycin ADI: 0-8 Full 62 (2004) 100 Muscle, Fat Cattle Pirlimycin 1000 Liver 400 Kidney 100 Milk Porcine Somatotropin ADI: Not Specified 52 (1999) Not Specified Muscle, Liver, Kidney, Fat Pig Procaine benzylpenicillin ADI: <30µg/ person/day of the penicillin moiety Full 50 (1998) 50 Muscle, Liver, Kidney All species Benzylpenicillin 4 Milk Progesterone ADI: 0-30 Full 52 (1999) Not Specified Muscle, Liver, Kidney, Fat Cattle Propionyl- promazine No ADI or ARfD 38 (1991) No MRL Ractopamine hydrochloride ADI: 0-1 Full 66 (2006) 10 Muscle, Fat Cattle, Pig Ractopamine 40 Liver 90 Kidney Ronidazole No ADI or ARfD 42 (1994) No MRL Sarafloxacin ADI: 0-0.3 Full 50 (1998) 10 80 20 Muscle Chicken, Turkey Sarafloxacin Liver, Kidney Fat/skin FAO JECFA Monograph 33 101 Substance ADI/ARfD (µg/kg bw) ADI Status JECFA MRL (µg/kg) Tissue Species Marker residue and other remarks Selamectin ADI: 0–10 ARfD: 400 Full 88 (2019) No MRL ADI: 0–50 ARfD: 400 Full 94 (2022) No MRL Spectinomycin ADI: 0-40 Full 50 (1998) 500 Muscle Cattle, Chicken, Pig, Sheep Spectinomycin 2000 Liver, Fat 5000 Kidney Chicken 2000 Eggs Cattle 200 (µg/L) Milk Spiramycin ADI: 0-50 Full 48 (1997) 200 Muscle Cattle, Chicken, Pig For cattle and chicken, MRLs are expressed as the sum of spiramycin and neospiramycin. 600 300 800 Liver Kidney Kidney Cattle, Chicken, Pig Cattle, Pig Chicken 300 Fat Cattle, Chicken, Pig For pigs, the MRLs are expressed as spiramycin equivalents (antimicrobial active residues). 200 (µg/L) Milk Cattle Streptomycin (See dihydro- treptomycin) Sulfadimidine (Sulfamethazine) ADI: 0-50 Full 42 (1994) 100 25 Muscle, Liver, Kidney, Fat Milk Cattle, Sheep, Pig, Poultry Cattle Sulfadimidine Sulfathiazole No ADI or ARfD 34 (1989) No MRL Teflubenzuron ADI: 0–5 Full 81 (2015) 400 Muscle Salmon Teflubenzuron 400 Muscle plus skin in natural proportion Salmon Testosterone ADI: 0-2 Full 52 (1999) Not specified Muscle, Liver, Kidney, Fat Cattle Tetracycline (See chlortetracycline) Thiamphenicol ADI: 0-5 Full 58 (2002) No MRL Tiabendazole (Thiabendazole) ADI: 0-100 Full 58 (2002) 100 100 Muscle, Liver, Kidney, Fat Milk Cattle, Pig, Goat, Sheep Cattle, Goat Sum of tiabendazole + 5-hydroxy tiabendazole Tilmicosin ADI: 0-40 Full 70 (2008) 100 Muscle, Fat Cattle, Pig, Sheep Tilmicosin 1000 Liver Cattle, Sheep 1500 Liver Pig 300 Kidney Cattle, Sheep 1000 Kidney Pig 150 Muscle Chicken 100 Muscle Turkey 2400 Liver Chicken 1400 Liver Turkey 600 Kidney Chicken 1200 Kidney Turkey 250 Skin/Fat Chicken, Turkey FAO JECFA Monograph 33102 Substance ADI/ARfD (µg/kg bw) ADI Status JECFA MRL (µg/kg) Tissue Species Marker residue and other remarks Trenbolone acetate ADI: 0-0.02 Full 34 (1989) 2 Muscle Cattle β Trenbolone for muscle 10 Liver α-Trenbolone for liver Trichlorfon (Metrifonate) ADI: 0-2 Full 66 (2006) 50 Milk Cattle Trichlorfon 50 Muscle, Liver, Kidney, Fat Guidance MRLs at the limit of quantitation of the analytical method for monitoring purposes. No residues should be present in tissues when used with Good Veterinary Practice. Triclabendazole ADI: 0-3 Full 70 (2008) 250 Muscle Cattle Keto-triclabendazole 850 Liver Cattle 400 Kidney Cattle 200 Muscle Sheep 300 Liver Sheep 200 Kidney Sheep 100 Fat Sheep, Cattle Tylosin ADI: 0-30 Full 70 (2008) 100 Muscle, Liver, Kidney Cattle, Pig, Chicken Tylosin A 100 Fat Cattle, Pig 100 Skin/Fat Chicken 100 Milk Cattle 300 Eggs Chicken Xylazine No ADI or ARfD 47 (1996) No MRL FAO JECFA Monograph 33 103 FAO technical papers FAO JECFA MONOGRAPHS Note: JECFA Monographs are available in English only. 1. Combined compendium of food additive specifications, JECFA specifications monographs from the 1st to the 65th meeting. Vol. 1: Food additives A–D; Vol. 2: Food additives E–O; Vol. 3: Food additives P–Z; Vol. 4: Analytical methods, test procedures and laboratory solutions. 2 Residue evaluation of certain veterinary drugs, Joint FAO/WHO Expert Committee on Food Additives, 66th meeting 2006. 3 Compendium of food additive specifications, Joint FAO/WHO Expert Committee on Food Additives, 67th meeting 2006. 4 Compendium of food additive specifications, Joint FAO/WHO Expert Committee on Food Additives, 68th meeting 2007. 5 Compendium of food additive specifications, Joint FAO/WHO Expert Committee on Food Additives, 69th meeting 2008. 6 Residue evaluation of certain veterinary drugs, Joint FAO/WHO Expert Committee on Food Additives, 70th meeting 2008. 7. Compendium of food additive specifications, Joint FAO/WHO Expert Committee on Food Additives, 71st meeting 2009. 8. Safety evaluation of certain contaminants in food, Joint FAO/WHO Expert Committee on Food Additives, 72nd meeting 2010. Joint FAO/WHO publication: WHO Food Additives Series No. 63/FAO JECFA Monographs 8. 9. Residue evaluation of certain veterinary drugs, Joint FAO/WHO Expert Committee on Food Additives, Meeting 2010, Evaluation of data on ractopamine residues in pig tissues. 10. Compendium of food additive specifications, Joint FAO/WHO Expert Committee on Food Additives, 73rd meeting 2010. 11. Compendium of food additive specifications, Joint FAO/WHO Expert Committee on Food Additives, 74th meeting 2011. 12 Residue evaluation of certain veterinary drugs, Joint FAO/WHO Expert Committee on Food Additives, 75th meeting 2011. 13 Compendium of food additive specifications, Joint FAO/WHO Expert Committee on Food Additives, 76th meeting 2012. 14 Compendium of food additive specifications, Joint FAO/WHO Expert Committee on Food Additives, 77th meeting 2013. 15 Residue evaluation of certain veterinary drugs, Joint FAO/WHO Expert Committee on Food Additives, 78th meeting 2014. 16 Compendium of food additive specifications, Joint FAO/WHO Expert Committee on Food Additives, 79th meeting 2014. FAO JECFA Monograph 33104 17. Compendium of food additive specifications, Joint FAO/WHO Expert Committee on Food Additives, 80th meeting 2015. 18. Residue Evaluation of Certain Veterinary Drugs, Joint FAO/WHO Expert Committee on Food Additives, 81st meeting 2015. 19. Compendium of Food Additive Specifications, Joint FAO/WHO Expert Committee on Food Additives, 82nd Meeting 2015. 20. Compendium of Food Additive Specifications, Joint FAO/WHO Expert Committee on Food Additives, 84th Meeting 2017. 21. Residue Evaluation of Certain Veterinary Drugs, Joint FAO/WHO Expert Committee on Food Additives, 85th meeting 2017. 22. Compendium of Food Additive Specifications, Joint FAO/WHO Expert Committee on Food Additives, 86th Meeting 2018. 23 Compendium of Food Additive Specifications, Joint FAO/WHO Expert Committee on Food Additives, 87th Meeting 2019. 24. Residue Evaluation of Certain Veterinary Drugs, Joint FAO/WHO Expert Committee on Food Additives, 88th meeting 2019. 25. Compendium of Food Additive Specifications, Joint FAO/WHO Expert Committee on Food Additives, 89th Meeting 2020. 26. Compendium of Food Additive Specifications, Joint FAO/WHO Expert Committee on Food Additives, 91st meeting 2021. 27. Compendium of Food Additive Specifications, Joint FAO/WHO Expert Committee on Food Additives, 92nd meeting 2021. 28. Residue Evaluation of Certain Veterinary Drugs, Joint FAO/WHO Expert Committee on Food Additives, 94th Meeting 2022 (Virtual). FAO Technical Papers are available through the authorized FAO Sales Agents or directly from Sales and Marketing Group, FAO, Viale delle Terme di Caracalla, 00153 Rome, Italy.

RESIDUE EVALUATION OF CERTAIN VETERINARY DRUGS Joint FAO/WHO Expert Committee on Food Additives Ninety-eighth Meeting 20–29 February 2024 This volume of FAO JECFA Monographs contains residue evaluation of certain veterinary drugs prepared at the Ninety-eighth Meeting of the Joint FAO/ WHO Expert Committee on Food Additives (JECFA), held 20–29 February 2024. This JECFA meeting specifically convened to consider residues of veterinary drugs in food. The Committee elaborated principles for evaluating the safety of residues of veterinary drugs in food, for establishing acceptable daily intakes (ADIs) and acute reference doses (ARfDs) and for recommending maximum residue limits (MRLs) for such residues when the drugs under consideration are administered to food-producing animals in accordance with good practice in the use of veterinary drugs (GVP). The enclosed monographs provide the scientific basis for the recommendations of MRLs, including information on chemical identity and properties of the compounds, pharmacokinetics and metabolism, residue depletion studies and analytical methods validated and used for the detection and quantification of the compounds. This publication and other documents produced by JECFA contain information that is useful to all those who work with or are involved with recommending or controlling maximum residue limits for veterinary drugs in food. CD2487EN/1/10.24 ISBN 978-92-5-139126-6 ISSN 1817-7077 9 7 8 9 2 5 1 3 9 1 2 6 6