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Background to COM review 1. Flunixin in the form of the meglumine salt is a non-steroidal anti-inflammatory (NSAID) drug and a non-narcotic analgesic drug with antipyretic activities. It is used in veterinary medicine (including food-producing animals) but it is not used in human medicine. Flunixin-meglumine dissociates in-vivo to Flunixin and meglumine. In 1997, the European Medicine Evaluation Agency's (EMEA) Committee on Veterinary Medicinal Products (CVMP) considered the safety of Flunixin-meglumine as part of its review of old veterinary medicinal products, operating under Council Regulation 2377/90, which covers the marketing authorisation of pharmacologically active substances used in veterinary medicines. 2. With regard to meglumine, the CVMP considered that it could be regarded as an excipient that had the purpose of increasing the solubility of flunixin. The CVMP concluded that "meglumine used as an excipient at up to 1.5 mg/kg bw does not fall within the scope of Council Regulation (EEC) number 2377/90."1 3. The Food Standards Agency is concerned about the possible mutagenicity of Flunixin-meglumine, meglumine and Flunixin. The Food Standards Agency considered that it would be useful to seek the opinion of the COM. Assessment of Mutagenicity data 4. As a general comment Members noted that relatively high levels of micronuclei had been documented in all of the in-vivo bone-marrow micronuclei studies submitted on these chemicals which had all been undertaken in the early 1980s by one particular contract laboratory. Flunixin 5. The available mutagenicity data were relatively old and of limited value. Negative results were reported in Salmonella typhimurium2 and in an in-vivo micronucleus assay in BS1 mice using intraperitoneal administration of two doses separated by 24 hours (200 mg/kg bw reduced to 150 mg/kg bw) and harvested 6 hours after the last dose.3 The DNA damage assay in Escherichia coli p3478 repair deficient strain showed an increase in DNA damage at one interim concentration, but this finding was not seen in a repeat test.4 A positive result was claimed in a mitotic gene conversion assay in Saccharomyces cerevisiae.5 6. Overall Members agreed that there was limited evidence that Flunixin was mutagenic in-vitro but there was no evidence to suggest that Flunixin had mutagenic potential in-vivo. Members felt the in-vitro mutagenicity data on this compound were inadequate and considered this should be raised with the CVMP. Meglumine 7. Negative results were reported in an old plate incorporation assay in a limited number of Salmonella typhimurium strains6. A positive result had been reported in a bone-marrow micronucleus assay in BSI mice using intraperitoneal administration of 500 or 1000 mg/kg bw suspended in 0.25% methylcellulose (two doses given 24 hours apart and harvest 6 hours after last dose)7. A repeat test using 1000 mg/kg bw was also positive. Members queried how the repeat test could have been undertaken on the same day as the initial test. It was noted that negative results were obtained in a separate in-vivo micronucleus assay using intraperitoneal administration of two doses given 24 hours apart at up to 600 mg/kg bw of meglumine to CD1 mice8. Members noted that a clear positive control response had only been seen in males and not females in this latter assay and that the sampling regimen (24 and 48 hours after last dose) differed from the study in BS1 mice. 8. A number of in-vivo bone marrow micronucleus assays were recently undertaken for the Committee using a variety of single/double intraperitoneal dosing regimens at dose levels of 500 mg/kg bw and 1000 mg/kg bw and sampling for micronuclei at 6 or 24 h after the last dose.9 There was some evidence for an effect 6 h after the second dose in one study using Alpk:ApfCD-1 mice, but no definite conclusions could be reached as the result was not repeatable, there was considerable individual animal variation and the observed effect could have been complicated by toxicity. 9. The Committee agreed that there was a need to further evaluate the potential mutagenicity of meglumine. This is discussed in the COM evaluation section below. Flunixin meglumine 10. Members agreed that the mutagenicity data on Flunixin-meglumine (ie the mixture used in veterinary medicines) was difficult to assess. Negative results were reported in an old plate incorporation assay using a limited number of Salmonella typhimurium strains10. Negative results were also reported in an in-vitro UDS assay using rat hepatocytes11. Positive results were documented in a mitotic gene conversion assay in Saccharomyces cerevisiae12,13. There was also evidence for DNA damage in two separate tests in Escherichia coli p3478 repair deficient strain but not in a third test14,15,16. However there was limited evidence for a mutagenic effect in three separate mouse lymphoma assays17,18,19 and evidence for an equivocal response in a cytogenetics test in Chinese Hamster Ovary cells20. 11. Overall the Committee agreed that there was limited evidence that Flunixin-meglumine was an in-vitro mutagen. Members noted that negative results had been documented in an in-vivo micronucleus assay in BS1mice using intraperitoneal administration of two doses (up to 80 mg/kg bw) separated by 24 h and sampling 6 h after the last dose. Members were concerned with regard to the adequacy of the selection of dose levels21 In addition the inconclusive in-vivo data on meglumine suggest that a definite conclusion regarding Flunixin-meglumine cannot be reached. COM Evaluation 12. The Committee noted the poor quality of mutagenicity data on Flunixin, meglumine and Flunixin-meglumine. Members noted that while negative results obtained in carcinogenicity bioassays with Flunixin-meglumine in the rat and the mouse provided some reassurance with regard to Flunixin, they were not informative with regard to meglumine. 13. The Committee was aware of several in-vivo bone-marrow micronucleus assays using meglumine but noted that the results were inconsistent. Thus there was evidence in two separate studies (in BS1 and Alpk:ApfCD-1 mice) for a mutagenic effect following intraperitoneal dosing of 1000 mg/kg bw following a treatment regime of two doses of meglumine (24 hour apart) with a sampling time of 6 hours after the second dose (ie 30 hours after the first dose).7,9 One of these studies had been undertaken by a Committee member. However this result was not repeated in two recent bone marrow micronucleus assays in mice which had been conducted for the Committee which attempted to repeat these findings using an equivalent treatment regime.9 14. The Committee considered a number of options for further investigating the mutagenicity of meglumine. The Committee agreed the most appropriate way forward would be to request additional in-vitro assays with meglumine to properly assess whether meglumine had mutagenic potential in-vitro before considering the need for any further in-vivo mutagenicity studies with meglumine. Thus data were required from an in-vitro chromosomal aberration assay and a mouse lymphoma assay, which together with the data currently available for a test in bacteria using Salmonella, would complete the in-vitro package to modern standards given in the COM guidance. In this case the weak and inconsistent induction of micronuclei in mice after two treatments with meglumine 24 hours apart with sampling 6 hours after the last treatment would be disregarded as strain-or-system specific effects of no genotoxic relevance if these two additional in-vitro tests were negative. 15. Thus negative data were available for a test in bacteria using Salmonella, but data were required from an in-vitro chromosomal aberration assay in mammalian cells and a mouse lymphoma assay to complete the in-vitro package to modern standards given in the COM guidance.22 If these were negative, there would be no concerns regarding meglumine. Consideration of presentation by Schering-Plough on mutagenicity testing strategy (6 February 2003). 16. Schering-Plough (referred to as the data holder in this statement) suggested that two further in-vivo mutagenicity studies be carried out with meglumine, namely a bone-marrow chromosome aberration assay in rats and a bone-marrow micronucleus test in rats. 17. Members considered the presentation and concluded that provision of two adequately conducted in-vitro tests as outlined in COM guidance with negative results would obviate the need for any further in-vivo testing. The purpose of these assays would be initial screening of meglumine and its metabolites in-vitro. The data holder accepted this proposal. 18. COC Members considered some additional data23 provided by the data holder on the conduct of the carcinogenicity studies in the rat and mouse using Flunixin-meglumine and drew conclusions with regard to the adequacy of these studies for the assessment of Flunixin-meglumine and meglumine (see paragraph 12 above). The COM noted comments from the data holder on the rapid dissociation of Flunixin-meglumine into Flunixin and meglumine and the suggested fast metabolism of meglumine. The COM agreed that it would be useful for the data holder to consider undertaking appropriate ADME studies with meglumine using radiolabelled material. COM conclusion and recommendations 19. The COM concluded:
March 2003 References 1. CVMP, 1999b, "Meglumine - Summary Report", unpublished document number EMEA/MRL/622/99-FINAL (dated July 1999) of Committee on Veterinary Medicinal Products (CVMP), European Medicines Evaluation Agency (EMEA), 7 Westferry Circus, Canary Wharf, London E14 2HB, England. 2. Ames Test - S tuphimurium; flunixin. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref D15342. 3. Micronucleus-flunixin. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref D15343. 4. DNA polymerase deficinet assay; Escherischia coli; flunixin NMG. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref D15344. 5. Mitotic gene conversion; Sachromyces cerevisiae - flunixin. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref A15345. 6. Ames Test - S typhimurium; N-methyl-D-glucamine. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref D15346. 7. Micronucleus-N-methyl-D-glucamine. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref 15347. 8. Mouse micronucleus - meglumine. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref D15348. 9. Anon. Activity of methylglucamine in the mouse bone marrow micronucleus assay. Report submitted to April 2002 COM meeting. 10. Ames Test - S typhimurium; flunixin meglumine. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref 15335. 11. Assessment of genotoxicity in an unscheduled DNA synthesis assay using adult rat hepatocyte primary cultures. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref A18272. 12. Mitotic gene conversion; Sachromyces cerevisiae - flunixin meglumine. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref A15340. 13. Mitotic gene conversion; saccromyces cerevisiae - flunixin meglumine. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref A15341. 14. DNA polymerase deficient assay; Escherischia coli; flunixin meglumine. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. D15338. 15. Evaluation of flunxin meglumine in the bacterial DNA repair test. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. D15339. 16. SCH 14714 NMG (BanamineR) DNA repair test with Escherichia coli. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref A 18270. 17. Evaluation of SCH 14714 NMG for mutagenic potential using L51787 Tk +/- cells. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref D14318. 18. Mouse lymphoma - flunixin meglumine. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref D 15336. 19. Mouse lymphoma mutation assay - Banamine. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref A18271. 20. Chromosome aberration-Chinese hamster. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref A18463. 21. Micronucleus-flunixin meglumine. Flunixin Meglumine MRL Submission Dossier Study Reports, Volume 13. Ref D 15337. 22. Committee on Mutagenicity (2000). Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment. Guidance on a strategy for testing of Chemicals for mutagenicity. 23. Schering-Plough (2003). Additional carcinogenicity data provided to COM in confidence at meeting of 6 February 2003.
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