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Introduction Risk assessment of benomyl, carbendazim and thiophanate-methyl 1. Benomyl, carbendazim and thiophanate-methyl belong to the methyl benzimadazole carbamate (MBCs) class of chemicals. The MBC class of chemicals are widely used in approved pesticide products as fungicides and also in veterinary medicines in particular as anithelmintics in both food producing and companion animals. These chemicals act by interfering with microtubule formation during mitosis. The COM has provided advice to the U.K regulatory Authorities namely the Pesticides Safety Directorate (PSD) and the Veterinary Medicines Directorate (VMD) of the Ministry of Agriculture, Fisheries and Food on the most appropriate approach for the risk assessment of MBCs. (1-3) 2. In 1993 the COM agreed that it was reasonable to assume that aneuploidy inducing chemicals (particularly those that function by interfering with the spindle apparatus of cell division) have a threshold of action.(1) The safety evaluation of aneuploidy inducing chemicals (aneugens) acting by inhibition of microtubule formation is based on the identification of a threshold dose below which aneuploidy does not occur. The Committee provided advice on methodology for identifying thresholds in 1993, namely appropriate in-vitro experiments in human lymphocytes using the detection and quantification of non-disjunction, chromosome loss and centromere positive micronuclei using FISH (Fluorescent in-situ hybridisation) analysis of selected chromosomes for centromeric DNA. This advice was used by PSD and VMD when requesting data from approval/licence holders of products containing MBCs. In 1996, the Committee considered the results of experiments undertaken with benomyl and carbendazim and concluded that the studies had been satisfactorily conducted and the data indicated No Observed Effect Levels (NOELs) for these two chemicals.(4,5,6) It was noted that that it would be difficult to define precise thresholds for activity from these data and the mathematical models that had been used for their analysis. Appropriate studies which provided evidence for a threshold effect have also been undertaken with thiophanate-methyl. (7) 3. The U.K Advisory Committee on Pesticides (ACP) considered that the available in-vitro aneuploidy data were consistent with a threshold for MBC-induced aneuploidy. The ACP considered that in-vitro aneuploidy threshold studies should be regarded as providing data that underpinned the regulatory decision rather than providing critical NOELS for direct use in setting Acceptable Daily Intakes (ADIs) and Acceptable Operator Exposure Level (AOEL) values. The conclusions of the U.K review have been passed to the rapporteur for the ongoing EC review (under Directive 91/414/EEC) of the use of MBCs as agricultural pesticides. 4. In the case of consumer safety for veterinary medicines, ADIs and Maximum Residue Limits (MRLs) in edible tissues are set on a substance specific basis by the EU Committee for Veterinary Medicinal Products (CVMP). All currently authorised veterinary medicines have been assessed on the basis of the concept that aneuploidy induced by spindle inhibitors is a threshold effect. Background to current review 5. The Committee was asked by the ACP to consider the conclusions reached by the European Commission's Group of Specialised Experts who were considering the classification and labelling of benomyl, carbendazim and thiophanate-methyl under the Dangerous Substances Directive 67/548/EEC. The Committee considered a draft summary record of the meeting of the Specialised Experts held on the 1-2 September 1999.(8) The Committee was asked by the ACP to advice on the applicability of extrapolating to germ cells, evidence for thresholds for induced aneuploidy obtained in studies on somatic cells, and the relevance of those conclusions for the approach used by PSD to evaluate aneuploidy data in the risk assessment of agricultural pesticides. The Committee has not been asked to comment on the proposals for classification and labelling of these MBCs. Conclusion reached by Specialised Experts 6. The Specialised Experts concluded that "..current knowledge does not allow extrapolation to meiotic cells of the in-vitro finding of a threshold [for induced aneuploidy in somatic cells]. Meiosis I is fundamentally different from mitosis in the structures and processes involved in chromosome segregation. Due to the current lack of knowledge on the interaction of aneugens with these possible targets, the concept of a threshold for induced aneuploidy in germ cells is as yet a hypothetical one." 7. The COM considered that the critical piece of evidence used to reach this conclusion came from the publication by de Stoppelaar et al (1999) which reported diploidy (i.e polyploidy but not aneuploidy) in sperm of rats exposed to carbendazim.(9) de Stoppelaar et al concluded that their findings suggested that diploidy in sperm is induced at a lower dose level than micronuclei in peripheral blood erythrocytes (no micronuclei were seen in this study). The Committee reaffirmed that there was adequate information available on the mechanism of interaction of MBCs with microtubules to assess the effects of these chemicals in both somatic and germ cells. The Committee agreed it therefore important to review the results obtained by de Stoppelaar et al in detail in order to comment on the conclusions reached by the Specialised Experts. COM consideration of de Stoppelaar et al Mutagenesis, 14, 621-631, 1999 8. Groups of 5 Wistar (Unilever) rats aged 13-14 week were given a single oral dose of carbendazim (50, 150, 450 or 800 mg/kg bw) in corn oil. The control group received corn oil only. A further group of 5 rats were given an intraperitoneal dose of 150 mg/kg bw carbendazim in corn oil. The animals were killed at thirty-one or 50 days (at 450 mg/kg bw only) and epididymal sperm isolated. In a second experiment, groups of three rats received a single oral dose of carbendazim (2.5, 5, 10, 20, 30, 40, 50, 100, 150, 450, 800 mg/kg bw) in corn oil. A group of 4 rats received 3 mg/kg bw mitomycin C (intraperitoneal) and the negative control received corn oil only. One day before and 48 hours and 72 hours after treatment peripheral blood samples were collected from the tail vein for assessment of micronucleated erythrocytes. The animals in the second experiment were killed thirty-one days after treatment and epididymal sperm isolated. Fluorescence in situ hybridisation was carried out using DNA probes specific for rat chromosomes 4 and Y (and 19 in the second experiment). Additional analyses were undertaken using some animals from the first experiment for chromosome 4 and 19 to confirm the presence of diploid sperm. Five thousand sperm were scored on two slides per animal (i.e 10,000 sperm per animal). Only one slide was scored for rats treated intraperitioneally or in trials where rats were killed at 50 days after treatment. 9. The main finding of experiment one was a small but dose related increase in the absolute frequency of diploid sperm (0.03% to 0.22%) following oral dosing and analysis of sperm at 31 days after treatment. An increase in sperm classified as diploid was only seen in one of five animals following intraperitoneal treatment with carbendazim at 150 mg/kg bw. No increase in 'diploid' sperm was seen in animals killed 50 days after treatment with an oral dose of 450 mg/kg bw carbendazim. The Committee noted that a smaller increased frequency of diploid sperm was reported in the second experiment in animals given an oral dose of 800 mg/kg bw carbendazim which may have resulted from sub-optimal exposure conditions in the experiment. No micronuclei were induced in peripheral blood erythrocytes following oral treatment of up to 800 mg/kg bw after sampling peripheral blood at 24 or 48 hours post treatment. 10. The Committee noted that the mechanism of action of carbendazim involved interference with the formation of polar microtubules. This effect combined with differences in the type of nuclear organising centres (NOC's) for germ cells in first meiotic division in males (a single pole) and in females (multiple poles) would result in carbendazim inducing polyploidy in sperm and aneuploidy in oocytes. The Committee agreed that the finding of diploid but not aneuploid sperm by de Stoppelaar et al (8) was to be expected. The Committee noted the finding of aneuploid oocytes in hamsters given a single oral dose of 1000 mg/kg bw carbendazim was also an expected finding.(10) The Committee considered that in the case of MBCs such as benomyl, carbendazim and thiophanate-methyl that affect the formation of spindles it was scientifically plausible for such compounds to be aneugenic in both somatic and germ cells. There is currently no evidence to suggest that MBCs are capable of modifying meiosis I specific events such as chromosome pairing. The Committee agreed that it would be expected that MBCs had a threshold of activity in somatic and germ cells. 11. The Committee considered that the results obtained by de Stoppelaar et al did not provide evidence for a lower threshold for aneuploidy in germ cells compared to somatic cells. The Committee felt that the analysis of peripheral blood samples for micronuclei in rats undertaken by de Stoppelaar et al was suboptimal in that a 24 hour sampling time point should have been used as micronucleated erythrocytes may have been efficiently removed by the spleen in rats and as noted in paragraph 9 above exposure conditions used may not have been optimal for the production of micronuclei. The Committee agreed that a more appropriate study in somatic cells for comparison with germ cells in the rat would be an investigation of the dose-response for the formation of micronuclei containing aneuploid chromosomes in polychromatic erythrocytes obtained in bone marrow smears from rats treated using a similar protocol to that used by de Stoppelaar et al. The Committee noted that such data would be informative with regard to any differences between aneuploidy in somatic and germ cells and agreed to review the subject when appropriate studies had been undertaken. Even if it could be established that the effects on sperm occur at lower doses than for somatic cells, this would not invalidate the concept of a threshold effect. Conclusions 12. The Committee agreed the following conclusions i) The aneuploidy induced by methyl benzimadazole carbamates (specifically benomyl, carbendazim and thiophanate-methyl) which act by inhibiting spindle formation is a threshold related effect. There is a sound scientific basis to assume that these chemicals have a threshold of action in both somatic and germ cells. The Committee did not agree with the interpretation reached by the European Commission's Group of Specialised Experts in fields of carcinogenicity, mutagenicity and reprotoxicity at its meeting of the 1-2 September 1999 particularly with regard to the finding by de Stoppelaar et al (8) of diploid sperm in rats. The Committee considered the finding of diploid sperm to be an expected effect of carbendazim on male germ cells undergoing meiosis and entirely consistent with the known effects of this chemical on microtubule formation. ii) The Committee concluded that de Stoppelaar et al (9) had not adequately demonstrated a lower threshold for aneuploidy in male germ cells of the rat compared to somatic cells. The Committee agreed that a more appropriate study in somatic cells for comparison with germ cells in the rat would be an investigation of the dose-response for the formation of micronuclei containing aneuploid chromosomes in polychromatic erythrocytes obtained in bone marrow smears from rats using a similar treatment protocol to that used by de Stoppelaar et al. The Committee agreed to review the subject when appropriate studies had been undertaken. iii) The Committee agreed that the approach used for risk assessment of MBCs by regulatory authorities for pesticides and veterinary medicines and the strategy outlined in the Pesticide Safety Directorate position paper on the role of aneuploidy in the risk assessment of agricultural pesticides were acceptable but would need to be reviewed should a marked difference in sensitivity to aneuploidy induced by these chemicals be reported between germ cells and somatic cells. iv) The Committee agreed that these conclusions were only relevant to aneuploidy inducing chemicals acting by spindle inhibition. The risk assessment (i.e consideration of thresholds in somatic and germ cells) of aneuploidy inducing chemicals acting via other mechanisms needed to be considered on a case by case basis. Secretariat June 2000 References 1. 1993 Annual Report of the Committees on Toxicity, Mutagenicity, Carcinogenicity of Chemicals in Food, Consumer Products and the Environment. 2. 1995 Annual Report of the Committees on Toxicity, Mutagenicity, Carcinogenicity of Chemicals in Food, Consumer Products and the Environment. 3. 1996 Annual Report of the Committees on Toxicity, Mutagenicity, Carcinogenicity of Chemicals in Food, Consumer Products and the Environment. 4. Unpublished report 1996. Carbendazin induction of aneuploidy in cultured peripheral blood lymphocytes. Final Report.* 5. Unpublished report 1996. Benomyl induction of aneuploidy in cultured peripheral blood lymphocytes. Final Report. * 6. Elhajouji A, Van Hummelen P and Kirch-Volders M (1995). Indicators for a threshold of chemically induced aneuploidy in-vitro in human lymphocytes. Environmental and Molecular Mutagenesis, 26, 292-304. 7. Unpublished report 1996. Thiophanate-methyl induction of aneuploidy in cultured peripheral blood lymphocytes. Final Report. 8. European Chemicals Burea (1999). Draft Summary record. Specialised Experts in fields of carcinogenicity, mutagenicity and reprotoxicity meeting of the 1-2 September 1999. ECBI/49/99- Add.1 Rev.2 9. de Stoppelaar JM, van de Kuil T, Bedaf M, Verharen HW, Slob W, Mohn GR, Hoebee B and van Benthem J (1999). Increased frequencies of diploid sperm detected by multicolour FISH after treatment of rats with carbendazim without micronucleus induction in peripheral blood erythrocytes. Mutagenesis, 14, 621-631. 10. Jeffay SC, Libbus BL, Barbee RR and Perreault SD (1996). Acute exposure of female hamsters to carbendazim (MBC) during meiosis results in aneuploid oocytes with subsequent arrest of embryonic cleavage and implantation. Reproductive Toxicology, 10, 183-189. * Subsequently published: Bently K, Kirkland D, Murphy M and Marshall R (2000). Evaluation of thresholds for benomyl and carbendazim-induced aneuploidy in cultured human lymphocytes using flourescein insitu hybridisation. Mutation Research, 464, 41-51.
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