Expert 14

As reported in round 1, I consider the mouse bone marrow micronucleus test (Gollapudi, 1985) of limited relevance in the context because of no clear demonstration of target tissue exposure (no reduction of %PCE) and because of a limited statistical sample (1000 PCE/animal) as well. Furthermore, the bone marrow micronucleus test doesn't seem to be an adequate in vivo follow-up assay for IARC carcinogenic haloalkanes and haloakenes which are genotoxic in vitro as shown by Morita et al. (1997), Mutation Research 389,3–122. The clastogenicity issue cannot be ruled out for three main reasons: - The presence of obsolete in vitro clastogenicity assays. - The absence of reliable in vivo assays which are relevant for clastogenicity. - A "positive" outcome for the comet assay in different organs (Sasaki et al., 2000). On this basis I would strongly recommend the performance of a robust in vitro micronucleus test in human lymphocytes (OECD TG 487) in the presence and absence of GSH. A negative outcome in this test would, in my opinion, eventually clarify the issue of clastogenicity of 1,3-D and no further actions would be needed. In addition, this outcome would also cover the aneugenicity issue. If a positive outcome will be obtained then, an in vivo comet assay compliant with the OECD TG 489 would be needed and would be highly relevant for the potential in vivo clastogenicity of haloalkanes and haloakenes. In order to rule out the aneugenic potential a FISH analysis of micronuclei would also be requested in the slides generated in the in vitro micronucleus test.

Expert 5

With the information available I would give it very little weight. First, as already indicated by one reviewer it is not known if reaches the target organ. Second, the bone marrow micronucleus test is not very sensitive for haloalkanes or haloalkenes in comparison to the ames test. Third, little or nothing is known about the metabolism of 1,3-D or its epoxide in bone marrow. To address the concern for the micronucleus test, some information on exposure in bone marrow needs to be available and if possible, on metabolism of 1,3-d and its epoxide in vitro. Also, retesting of the relevant assays for clastogenic effects with purified 1,3-D would be important.

Expert 1

As said previously it is not only that but also the fact that the risk assessment plainly discounts the relevance of the in vivo DNA strand break assays. Believe the cleanest way to resolve this would be a new OECD 474 study
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Expert 14
04/19/2019 05:21

To user 717898 [Expert 1] : The in vivo mammalian alkaline Comet Assay is regulated by OECD TG 489 and currently represents the ultimate in vivo follow up study when positive findings are observed in the in vitro cytogenetic assays (e.g. micronucleus test). Many of these in vitro genotoxins do not reach at a sufficient level the bone marrow (negative findings in the in vivo bone marrow micronucleus test with no clear target tissue exposure). In these cases the in vivo comet assay does represent, at present, the obligate in vivo follow-up study.
Furthermore, I do not consider a good idea to recommend a new OECD 474 study (bone marrow micronucleus test) for the reasons I indicated in round 1 (Result 2.1) where I reported for the bone marrow micronucleus test by Gollapudi (1985) as main shortcoming, the absence of a clear target tissue exposure. This is corroborated by the findings that IARC carcinogens aloalkanes and haloakenes including 1,3-D, are poor or no inducers of micronuclei in the bone marrow in vivo as reported by Morita et al., (1997).

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Expert 1
04/21/2019 20:07

To use 615591: I am not sure why OECD 489 is (or would be) the 'ultimate in vivo follow up study when positive findings are observed in the in vitro cytogenetic assays" - where is this stated in the guideline? I do agree that OECD 474 has more limitations regarding exposure but for BB there is plenty of other in vivo data available that show that there is systemic (and bone marrow) exposure. OECD 474 is a measuring mutations, the Comet assay is an indicator test. Don't get me wrong, I like the Comet assay and it has an important place in the testing strategy but in the given case here I would prefer the in vivo micronucleus test

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Expert 14
04/22/2019 03:23

To user 717898 [Expert 1] : I was of course referring to compounds positive in the in vitro cytogenetic assays (e.g. chromosome aberration assay; micronucleus assay) for which negative findings in the in vivo bone marrow micronucleus test with no clear target tissue exposure are observed. Based also on my direct experience, many of these in vitro genotoxins do not reach at a sufficient level the bone marrow. In these cases, the in vivo comet assay does represent an obligate in vivo follow-up study since alternative methods (e.g. induction of micronuclei in the rat/mouse colon and intestinal epithelium and also in liver of young rats) are not yet validated. Aneugenicity would not be covered in these cases and additional ad hoc in vitro evaluations would clarifying the MoA. I would also note that the Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays is only used as in vivo follow-up study in case of positive outcome for gene mutation in vitro (Ames test, mammalian cell gene mutation assay).

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