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Background

1. The COM has 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." 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.

2. The Committee considered the submitted data at the October 2006 meeting which included the draft PSD evaluation which presented information on structure, use, ADME studies, general toxicology, mutagenicity, carcinogenicity and reproduction and copies of the mutagenicity studies relevant to the evaluation of aneugenicity which had been submitted to PSD. (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.) The COM heard a presentation from the data holder (LG Life Sciences) and representative (Huntingdon Life Sciences).

3. The COM agreed that a number of additional studies should be undertaken to further evaluate the potential for in vitro and in vivo aneugenicity as summarised from statement MUT/07/S1 below:

i. 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.

ii. 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.

*= No Observed Effect Level, ** MN = micronucleus, *** = Bone Marrow

Introduction to current review

4. HLS submitted an interim report of the additional studies requested by COM on 16 April 2007. The COM discussed these data at its meeting of the 15 May 2007. HLS made a short presentation of the results of the additional studies during this meeting. Additional data on the results of studies for non disjunction were submitted during the COM meeting of the 15 May 2007.²

COM consideration of areas for discussion

5. The COM considered the new data submitted.^1,2 There were a number of questions relating to the interpretation of the in vitro MN assay with regard to the derivation of NOELs in binucleate and mononucleate cells and the appropriate statistical level of significance to apply. With regard to the repeat intraperitoneal in vivo bone marrow MN assay in mice, members considered that the justification of the top dose needed further explanation as there was evident excessive toxicity and mortalities reported at the top dose of 300 mg/kg bw in the main study. Members also agreed that HLS should be questioned on the selection of tissue levels in the testes as a surrogate for the bone marrow and whether in vitro data on non disjunction were available (subsequently submitted during the COM meeting of 15 May 2007).²

Presentation to COM from HLS

6. HLS reported the data from the repeat in vitro MN assay in peripheral blood lymphocytes (PBLs) using both 24 hour and 48 hour PHA stimulation. FISH analysis using probes for chromosomes 1,8,11,17 and 18 in binucleate cells was undertaken. Overall NOELs after 24 hour and 48 hour PHA stimulation were interpreted to be 7 µg/ml and 4 µg/ml respectively. There was no evidence for non disjunction.

7. With regard to the repeat in-vivo bone marrow MN assay in mice (groups of 7 male mice dosed intraperitoneally twice at 50 mg/kg bw, 150 mg/kg bw or 300 mg/kg bw (24 hours apart) with bone marrow sampling 24 hours and 48 hours after the last dose), a small statistically significant (P<0.01) increase in the group mean incidence of micronucleated polychromatic erythrocytes (MNPCEs) was reported at the 24 hour and 48 hour sampling times after the second intraperitoneal dose of 300 mg/kg bw ethaboxam (in 1% methylcellulose/0.1% Tween 80). The trend analysis was statistically significant at the 48 hour sampling time point. A statistically significant reduction in percentage of polychromatic erythrocytes (PCEs) had been documented at both 150 mg/kg bw and 300 mg/kg bw ethaboxam (P <0.001 or <0.0001) with a statistically significant trend (P<0.001). The increase in MNPCEs at the top dose of 300 mg/kg bw reported at both the 24 hour and 48 hour sampling time points was not considered biologically relevant as it was only seen at the high dose where there was significant systemic toxicity and mortality reported, the individual and group mean data were within the historical control ranges and the low vehicle control MNPCE rate increased the statistical significance.

8. The toxicokinetic analysis for ethaboxam revealed evidence of exposure in the plasma, liver, spleen and testes. Tissue concentrations had not been measured in the bone marrow due to the small amount of tissue. HLS considered that tissue concentrations of ethaboxam were at least equivalent to testes and this was supported by evidence of bone marrow toxicity (reduced PCE frequency). Overall HLS suggested ethaboxam did not induce biologically significant increases in MNPCEs and therefore no subsequent FISH analysis for whole chromosomes had been undertaken.

9. HLS concluded that the request for additional data from COM had been adequately fulfilled. NOELs for binucleate PBLs were interpreted to be 6 µg/ml (24 hour stimulation) 3 µg/ml (48 hour stimulation). There was no evidence for induction of non disjunction. Ethaboxam was not mutagenic in a repeat intraperitoneal bone marrow MN assay in mice with additional toxicokinetic data in directly and indirectly exposed tissues. Overall HLS concluded there was no evidence for genotoxicity in vivo.

COM questions for HLS

10. In response to a question from the COM on the evaluation of NOELs for the in vitro MN assay , HLS reported that the dose spacing in the study was very small and the size of increase in MN frequency at low concentrations was small.

11. In answer to questions on the conduct of the repeat in vivo bone marrow mouse MN assay. HLS considered the process had been adequate and the top does level (300 mg/kg bw) satisfactory. The COM considered the level of toxicity seen at the top dose level was excessive. Members asked for an explanation of the cause of the small increase in MNPCEs seen in the study. HLS noted the response in animals was within the historical control range, and the response had been seen at highly toxic doses at the limit of the MTD and could be interpreted as a toxic-stress related response. Members asked if further investigation using procedures to identify whole chromosomes would be useful. HLS considered there were too few MNPCEs on slides at the top dose level for any meaningful analysis for the presence of whole chromosomes.

12. The COM asked for more explanation regarding the assertion that tissue concentrations in the testes could be regarded as equivalent to bone marrow. HLS considered that testes were representative of peripherally exposed tissue and hence similar to bone marrow. HLS noted that higher tissue levels in liver and spleen might in part represent surface retention of ethaboxam following intraperitoneal dosing. Members asked if tissues had been specially prepared to avoid such contamination by intraperitoneally dosed ethaboxam. HLS reported that standard operating procedures had been used. COM members considered that surface retention of ethaboxam in spleen and liver was unlikely at 24 hours post dose. Overall the time course seen for all tissues and plasma suggested a reduction in tissue concentrations at 6-12 hours post dose, a peak at between 12-24 hours post dose, with a relatively slow elimination (compared to plasma) for liver, spleen and testes. Overall COM members were not convinced that testes could be selected as a representative tissue of bone marrow.

13. The data holder, HLS and JSCI withdrew from the meeting so that COM could derive conclusions.

COM consideration and conclusions

14. The COM considered a number of questions in deriving conclusions:

i. The interpretation of new in vitro MN data in PBLs: The COM agreed that NOELS were slightly lower than the submitted test data reported; approximately 4 µg/ml (24 hour PHA stimulation) and 2 µg/ml (48 hour PHA stimulation).

ii. The need for additional in vitro data on aneuploidy and chromosome disjunction: Members noted no evidence for non disjunction had been reported. The COM noted that ethaboxam treatment resulted in a greater effect in mononucleate compared to binucleate cells. The mechanism of ethaboxam induced effects in vitro was unclear and needed to be resolved.

iii. The interpretation of the new in vivo MN data in mice given ethaboxam via intraperitoneal dosing: The small statistically significant increase in MNPCEs seen in the repeat study at high doses (2 x 300 mg/kg bw in 1% methylcellulose/0.1%Tween 80) resulting in severe toxicity and increased mortality at both the 24 hour and 48 hour post dose bone marrow sampling times is a similar result to the first intraperitoneal study where small statistically significant increase in MN in the bone marrow was reported in mice (following intraperitoneal dosing of 2 x 486 mg/kg bw (in aqueous 1% methylcellulose) with sampling at 48 hours post dose). Severe toxicity and increased mortality were also reported in the first intraperitoneal bone marrow mouse MN assay with ethaboxam. The COM noted the evidence for aneugenicity of ethaboxam in vitro and considered that there was no adequate explanation for the small increases in bone marrow micronuclei seen in the intraperitoneal in vivo tests with ethaboxam. The COM could not exclude a direct aneugenic effect of ethaboxam in these studies. The COM did not agree that the increase in MNPCEs seen in these studies could definitely be related to a toxic-stress response. Thus overall the top dose level in the repeat intraperitoneal bone marrow MN assay of 300 mg/kg bw should be regarded as a LOAEL. The COM agreed that further evaluation of the slides from the repeat bone marrow MN assay at the top dose level with additional procedures to identify whole chromosomes should be undertaken if sufficient micronuclei could be identified.

iv. The need for additional in vivo MN data in other tissues (eg spleen): The COM agreed that further information could be provided for other tissues but this would require additional in vivo studies in mice using intraperitoneal dose levels below 2 x 300 mg/kg bw. There would need to be additional toxicokinetic evaluation undertaken. The COM considered that such a request would need to come from the Advisory Committee on Pesticides (ACP).

v. The use of tissue concentration data for risk assessment: The COM did not agree from the data submitted, that ethaboxam concentrations in testes could be used as a surrogate for bone marrow. The COM noted the absence of bone marrow tissue levels from the repeat bone marrow MN assay in mice. The COM suggested that in this instance an initial risk assessment could be undertaken using the peak plasma levels of ethaboxam at the LOAEL for MN induction in bone marrow reported in the repeat intraperitoneal bone marrow MN assay, although further evaluation of the suitability of this approach for risk assessment of site of contact aneugenicity would need to be considered by the ACP.

October 2007

References

1. Ethaboxam: Further investigation of potential for aneugenicity in vivo and in vitro, Interim assessment. 16 April 2007. Huntingdon Life Sciences.

2. Ethaboxam: Presentation to COM 15 May 2007. Results of studies requested by COM. Interim results of investigations for non disjunction. Huntingdon Life Sciences.

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