Limitations were noted in the study design of the mouse bone marrow micronucleus test (Gallapudi, 1985) by the peer review. Two reviewers considered the evidence for overall clastogenicity to be equivocal, and one judged it as positive, although the test itself was considered to be negative by all three. Two of the reviewers consider this test to be clearly or slightly relevant. One reviewer raised the concern of the limited utility of this test for haloalkanes and haloakenes. Question: Please comment on whether the negative mouse micronucleus assay (inhalation) has any utility as part of the weight of evidence assessment to address the in vivo clastogenic potential of 1,3-D, and comment on what further information you would want to clarify the mouse miconucleus test and to address the concern for potential chromosomal effects?

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).

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

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).