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.
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.
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