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Background to COM review 1. Dimetridazole (DMZ) is an antiprotozoal substance that is used to maintain the health of certain farm animals. In the UK it is permitted for use as a feed additive for turkeys and guinea-fowl and as a veterinary medicine for game birds (pheasants and partridges only). The European Commission is currently considering whether to suspend use as a feed additive, but this would not directly affect veterinary medicinal use. 2. In the last few years, DMZ has been assessed by the EU’s Committee on Veterinary Medicinal Products (CVMP)(1,2) and by the EU’s Scientific Committee on Animal Nutrition (SCAN)(2). These two committees disagreed with one another over the interpretation of the mutagenicity data. The CVMP considered that more information was needed before it could conclude on mutagenicity, whereas the SCAN considered that there was sufficient information to allow it to conclude that there was no genotoxic hazard to consumers. The specialist expert advice of COM was sought by the Food Standards Agency (FSA). The FSA is particularly concerned about the safety of consumers of foods derived from animals given DMZ. Assessment of mutagenicity data 3. DMZ was mutagenic in bacteria.(4-6) Studies using nitroreductase proficient and deficient strains of Salmonella typhimurium TA100 showed that this pathway accounted for a proportion but not necessarily all of the mutagenicity seen in bacteria.(7) Positive results were reported in Saccharomyces cerevisiae D4 demonstrating that DMZ was mutagenic in eukaryotic cells.(8) Members noted that there was inconsistency in the interpretation of the data for the in-vitro mutation assays in the mouse lymphoma assay. For the cytogenetics assay in CHO-K1 cells the data were reported as negative but were not traceable. In addition there were very limited details available regarding the in-vitro UDS assay in V79 Chinese hamster lung fibroblasts and no conclusions could be reached on the results of this test. 4. . However sufficient details were available to assess the genotoxicity of DMZ in human lymphocytes using the comet assay.(9) It was established that DMZ was genotoxic under aerobic conditions in the absence of exogenous metabolic activation. The addition of Aroclor 1254 induced rat liver S-9 fraction abolished the genotoxicity of DMZ in this test system. Using the same treatment concentration of DMS, the magnitude of the response (mean tail moment derived from 200 cells) was reduced under anaerobic conditions and abolished by the addition of anti-oxidants (8-hydroxyquinoline, vitamin C, catalase or superoxide dismutase). Thus, DMZ was genotoxic in the in-vitro comet assay in human lymphocytes and that the activity observed in this assay was consistent with oxidative DNA damage. 5. The Committee agreed that DMZ was mutagenic in-vitro. In vivo data Insects 6. Members agreed that no weight could be given to the negative results reported in the sex-linked recessive lethal mutation assay in Drosophila melanogaster.(10) Mammals 7. Negative results were reported in published in-vivo micronucleus assays in the mouse using intraperitoneal and oral routes of administration.(11) However no conclusions could be reached by the Committee in view of the limited details of these tests that were available. 8. The full reports made available to the Committee of the three in-vivo studies form the basis for the Committee conclusions regarding the ability of DMZ to express the mutagenic potential shown in vitro in mammals. 9. Negative results were obtained when DMZ was investigated in an adequately conducted bone marrow micronucleus test in the mouse, DMZ being given orally at 2 daily doses up to 915 mg/kg.(12) Negative results were also obtained in an in- vivo liver UDS assay when DMZ was given orally to male rats at single doses up to 1000 mg/kg but the Committee noted that this study had not been performed to current standards since the analyses were limited to examination of primary hepatocytes at a single harvest time (15 hours).(13) [The OECD guideline (486) adopted in 1997, recommends an early sampling time (at 2-4 hours) as well as a later sampling time (12-16 hours). Compounds such as dimethylnitrosamine would not be detected as positive if only the later sampling time point was used.] Negative results were also obtained when DMZ was examined for its ability to induce mutations in germ cells in a dominant lethal assay in male mice.(14) Animals were given 5 daily doses of up to 1000 mg/kg and then subjected to sequential mating over 8 weekly periods. 10. The Committee agreed that the further data were needed to provide adequate reassurance that the mutagenic activity seen in-vitro was not expressed in-vivo. This would comprise a liver UDS assay in rats conducted in accordance with the OECD guideline; it was however recommended that 4 animals should be analysed at each dose level and time point. Carcinogenicity data 11. The Committee agreed that the available long-term carcinogenicity bioassays in rats had not, from the limited data available, been conducted to contemporary standards.(15) Benign mammary tumours had been documented in two of these studies and some limited evidence was available to support a hormonal mechanism for the induction of these tumours. However, the blood concentration of progesterone was raised in female rats but not in males, whereas benign mammary tumours had been produced in both sexes. Overall, no reassurance regarding the absence of genotoxicity could be derived from the available carcinogenicity bioassays. Conclusion 12. Dimetridazole (DMZ) has mutagenic activity in vitro. The Committee was provided with full reports of in-vivo studies to investigate the ability of DMZ to induce micronuclei in bone marrow of mice, UDS in primary hepatocytes of male rats, and dominant lethal mutations in the germ cells of male mice. In all cases the oral route was used, with dose levels up to about 1 gram/kg per day. Negative results were consistently obtained. However the Committee concluded that the rat liver UDS assay had not been adequately conducted and recommended that a further rat liver UDS assay conducted in accordance with OECD guidelines (but using 4 animals per dose/sampling time point) was required to provide full reassurance that DMZ (or its metabolites) does not have significant mutagenic activity in mammals in vivo.
1. SWP, 1999, "Dimetridazole: review of safety aspects with reference to the SCAN draft Opinion", unpublished working paper of the Safety Working Party of the EU's Committee on Veterinary Medicinal Products (CVMP). 2. EMEA, 1997, "Dimetridazole (1)", "Dimetridazole (2)" & Dimetridazole
(3)", Summary Reports. Available on the web site of the European Agency
for the Evaluation of Medicinal Products (EMEA): 3. SCAN, 2000, "Opinion of the Scientific Committee for Animal Nutrition on the use of dimetridazole in animal feedingstuffs", European Commission, Health & consumer Protection Directorate-General, Directorate C - Scientific Opinions. Available on the internet at: http://euroa.eu.int/comm/food/fs/sc/scan/out51_en.pdf 4. Voogd CE, van der Stel JJ & Jacobs JJJAA, 1974, "The mutagenic action of nitroimidazoles: I. Metronidazole, nimorazole, dimetridazole & ronidazole", Mutat. Res., 26: 483-490. 5. Voogd CE, van der Stel JJ & Jacobs JJJAA, 1979, "the mutagenic action of nitroimidazoles. IV. A comparison of the mutagenic action of several nitroimidazoles an some imidazoles", Mutat. Res., 66: 207-221. 6. Rosenkranz HS, Speck WT & Stambaugh JE, 1976, "Mutagenicity of metronidazole: Stucture-activity relationships", Mutat. Res., 38: 203-206. 7. Lindmark DG & Müller M, 1976, "Antitrichomonad action, mutagenicity and reduction of metronidazole and other nitroimidazoles", Antimicrobial Agents Chemother., 10: 476-482. 8. Voogd CE, van der Stel JJ & Jacobs JJJAA, 1980, "The mutagenic action of quindoxin, carbadox, olaquindox and some other N-oxides on bacteria and yeast", Mutat. Res., 78: 233-242. 9. Ré JL, De Méo MP, Laget M, Guiraud H, Castegnaro M, Vanelle P & Duménil G, 1997, "Evaluation of the mutagenic activity of metronidazole and dimetridazole in human lymphocytes by the comet assay", Mutat. Res., 375: 147-155. 10. Kramers PGN, 1982, "Studies on the induction of sex-linked recessive lethal mutations in Drosophila melanogaster by nitroheterocyclic compounds", Mutat. Res., 101: 209-236. 11. Oud JL, Reutlinger AHH & Branger J, 1979, "An investigation into the cytogenetic damage induced by the coccidiostatic agents amprolium, carbadox, dimetridazole and ronidazole", Mutat. Res., 68: 179-182. 12. Rhone Poulene. Dimetridazole: micronucleus test in the mouse using oral route. Fournier E and Cordier A. Report Ref. ST/CRV/Tox No 11. 3 April 1986. 13. Rhone Poulene. Dimetridazole. In vivo – in vitro DNA repair test in the Fischer 344 rat hepatocyte. Melcion C and Cordier A. Report ref. ST/CRV/Tox 214. 14 March 1988. 14. Rhone Poulene (May and Baker). Dimetridazole. Dominant lethal study in the mouse by the oral route. Dale M. Res. Report No 3041 November 1977. 15. Cohen SM, Erturk F, von Esch AM, Crovetti AJ & Bryon GT, 1973, "Carcinogenicity of 5-nitrofurans, 5-nitroimidazoles, 4-nitrobenzenes and related compounds", J. Natl. Cancer Inst., 51: 403-417.
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