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Introduction 1. The COM have been asked for advice by PSD on a new pesticide active ingredient which is undergoing evaluation through the independent Advisory Committee on Pesticides (ACP). The referral statement is as follows; "PSD have asked for advice from the COM on the most appropriate strategy to be taken with regard to the testing for aneugenicity of ethaboxam in-vivo and the potential for risk of site of contact in-vivo aneugenicity effects and their likely significance for risk assessment. This is not a full referral of the mutagenicity data to the COM." 2. Ethaboxam is a new fungicide and is formulated as a 100 g/L suspension concentrate for control of grapevine downy mildew caused by Plasmopara viticola. The proposed pattern of use is for up to 5 applications per season, with an interval of 7 to 10 days depending on weather conditions and disease pressure. Thus the key sites of contact for evaluation are the upper respiratory tract (in particular the nasal passages) and possible skin contact to concentrate and in-use dilution. 3 The data holder (LG Life Sciences) and representative (Huntingdon Life Sciences) attended the COM meeting of the 12 October 2006 to make a short presentation and to answer COM queries regarding the evaluation of ethaboxam for aneugenicity. COM consideration of areas for discussion 4. The Committee considered the submitted data which included the draft PSD evaluation which presented information on structure, use, ADME studies, general toxicology, mutagenicity, carcinogenicity and reproduction,1,2 and copies of the mutagenicity studies relevant to the evaluation of aneugenicity which had 1,2 been submitted to PSD.3-8 (Some information on a possible approach to the assessment of aneugenicity in-vivo in the gastrointestinal tract and the evaluation of aneugenicity data was also presented to members.9,10) Members derived an evaluation of these studies and considered the areas of mutagenicity evaluation to raise with the data holder and their representative. 5. The areas of discussion related to:
6. The primary objective of the COM was to consider an appropriate testing strategy for aneugenicity which could be recommended to the ACP. Data holder presentation 7. The data holder (LG Life Sciences) and representative (Huntingdon Life Sciences; HLS) were asked to make a short presentation to the COM and to answer members queries. All comments were made by the data holders representative. 8. HLS noted that some of the fungicidal activity of ethaboxam was due to interference with the fungal cytoskeleton possibly by inhibition of tubulin subunits. The negative findings in the Ames and MLA tests indicated no potential for gene mutagenicity. The in-vitro cytogenetics and MN tests in PBLs were considered to be positive in the absence of exogenous metabolic activation. However two negative bone marrow MN assays were available in the rat (oral dosing) and in the mouse (intraperitoneal dosing). In addition following a request from the ACP, a re-analysis of sections from the gastrointestinal tract from the rat 2 year chronic toxicity/carcinogenicity assay had not indicated any effects on mitosis. HLS also noted that no adverse toxicity had been reported in a 28 day dermal toxicity study in the rat. It was also considered that potential site of contact concentrations (skin and via oral ingestion) would be low. COM questions for data holder and representative 9. A summary of the response given by the data holder's representative on the areas for discussion (see paragraphs 5, 7 and 8 above) is given below. 10. HLS noted that the initial strategy had placed a weight of evidence on the mouse lymphoma assay and the available in-vivo MN assays and thus the initial in-vitro study in PBLs had not specifically incorporated an investigation of MN formation. The data holder's representative noted the comments made by the COM and evaluation of the in-vitro chromosomal aberration assay and the mouse lymphoma assay. HLS noted the evaluation of the COM with regard to the in-vitro MN assay in PBLs regarding the assessment of dose response for aneugenicity and clastogenicity and the possible effects of adding exogenous metabolic activation systems. HLS considered that the use of 0.5% carboxymethyl cellulose was a relatively normal approach to oral dosing in in-vivo bone marrow MN assays. The COM considered that oral absorption would have been limited in comparison to that seen in the oral rat kinetics study (up to 60%) using radiolabelled ethaboxam which had used 1% CMC and Tween 80 as a surfactant to aid solubility in the dosing vehicle. HLS noted the interpretation of the in-vivo bone marrow MN assays in rats and mice reached by the COM. 11. The data holder and HLS withdrew from the meeting so that the COM could derive its conclusions. Additional data submitted by industry: LG Life Sciences (1 November 2006) 12. The data holder and representative submitted reasoned arguments concerning the COM conclusions and additional investigations for polyploidy from the in-vitro cytogenetics assay in human peripheral blood lymphocytes.4 13. The COM noted the new data but agreed that it would still be appropriate to investigate the aneugenicity and clastogenicity in-vitro to describe appropriate dose-response data and to determine the NOELs. The COM noted the data holder had agreed to undertake a further in-vivo BM MN assay in mice using intraperitoneal administration. Members agreed that this study should include dose-response, multiple sampling times, and an evaluation of whole chromosomes. The COM considered that the results of the repeat in-vivo BM MN assay in mice would be important for regulatory decision making with regard to ethaboxam. COM consideration and conclusions 14. The COM considered that an appropriate investigation of polyploidy as an indicator for potential aneugenicity had been undertaken in the in-vitro chromosomal aberration study in peripheral blood lymphocytes given the predominant effects of ethaboxam on cell division. The COM agreed that more evaluation of the mouse lymphoma data to predict potential for aneugenicity might have been possible. 15. The COM agreed that a further evaluation for aneugenicity and clastogenicity should be undertaken in-vitro to describe appropriate dose-response data and to determine the NOELs. The COM noted that the potential NOEL for non disjunction might be lower than for chromosome loss or gain and this end point should also be investigated. 16. The COM agreed the NOEL for MN induction in PBLs in-vitro was likely to be lower than the value of 5 µg/ml reported in the existing study in PBLs but probably above the tested concentration of 1.0 µg/ml. 17. The COM considered that it would be difficult to undertake an evaluation of site of contact aneugenicity in-vivo for ethaboxam given the current knowledge of potential approaches. A pragmatic in-vivo testing strategy should include a re-test using intraperitoneal dosing in mice with evaluation of MN in the bone marrow to include dose response, multiple sampling times and an evaluation of whole chromosomes. Members agreed that consideration should also be given to investigating MN formation in other tissues in this study such as the liver, germ cells and splenocytes. Members considered it was important to measure plasma and tissue concentrations of ethaboxam. The COM considered that the results of the repeat in-vivo BM MN assay in mice would be important for regulatory decision making with regard to ethaboxam.
February 2007
References 1. General information on ethaboxam volume 1 submission 91/414/EEC (draft in-confidence PSD evaluation) 2006. 2. Detailed evaluation volume 3 91/414/EEC (draft in confidence PSD) 2006. 3. Mouse Lymphoma Assay. In confidence Huntingdon Life Sciences project LKF 038, 21 December 2001. (in confidence report). 4. In-vitro Chromosome aberration assay in human peripheral blood lymphocytes. Huntingdon life Sciences project LKF/039, 24 October 2001. (in confidence report). 5. In vitro investigation of toxicity using Cytochalasin B in human peripheral blood lymphocytes. Huntingdon Life Sciences project LKF/087, 12 February 2003. 6. In-vitro MN assay in human PBLs. Huntingdon Life Sciences report on LGC-30473. Draft in confidence report July 2006. 7. Rat bone marrow MN assay. Huntingdon Life Sciences project LKF/046, 15 November 2001. (in confidence report). 8. In-vivo mouse micronucleus test. Huntingdon Life Sciences project LKY24, 13 November 1996. (in confidence report). 9. Rosefort C et al (Mutagenesis, 19, 277-284, 2004). 10. Vanhauwaert A et al (Mutagenesis, 16, 39-50, 2001).
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