Expert 1

Believe the differences in opinion stem from weighing the validity and strength of assays differently. The definition I applied to 'mutagenic' was broader than as in 'gene mutation', including 'chromosome mutations', and this is where a see a certain weakness of the data package. Regarding the last question: Not sure where this is going. The in vivo assays are done in order to provide insights on in vivo genotoxicity potential which seems all relevant for risk assessment. Yes, some of the endpoints looked at may be more relevant than others but in the given context I don't believe one assay trumps it all

Expert 14

Yes, actually the question itself leads to misinterpretations. I considered in question 2.6 the overall conclusion of 1-,3-D in terms of gene mutation induction, bearing also in mind that question 2.7 was dealing with the clastogenic potential of 1,3-D. Referring to the same question, after it was re-phrased, and the EPA framework for weighing evidence for a mutagenic mode of action (MOA) in which the term "mutagenic" indicates the capacity of either the carcinogen or its metabolite to react with or bind to DNA in a manner that causes mutations (gene mutations and structural chromosome aberrations, both in vitro and in vivo) I would judge 1,3-D as equivocal. As reported from my side in round 2, I considered the induction of gene mutation of no concern, since the positive findings in the Ames test and mammalian cell mutation assays (Myhr and Caspary, 1991) were ruled out by the negative findings obtained, mainly by the Big Blue rat study, via oral route (Young 2018). However, the concern for clastogenicity is not ruled out neither in vitro nor in vivo with the available data-set. The quoted mouse bone marrow micronucleus test by Gollapudi (1985) with a negative outcome, cannot be considered reliable in relation to the absence of a clear demonstration of target tissue exposure (e.g. no reduction of %PCE). In addition, its relevance is further limited by additional shortcomings which include an unjustied difference in the percentage of PCE in the negative control group between male and female animals and within female animals at 24- and 48-hour sampling times and by a limited statistical sample (1000 PCE/animal). Furthermore, the bone marrow micronucleus test doesn't seem to represent 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. On the other hand, I would mention the positive results observed in different organs in the alkaline single cell gel electrophoresis (comet) assay after treatment with 1,3-D (Sasaky et al., 2000). In this study 1,3-D, when the substance was injected intraperitoneally to mice, induced marked and statistically significant increases in DNA breakage in the stomach, liver, kidney, blood, lung, brain and bone marrow three hours after treatment. Notably, the effect observed in bone marrow, though significantly different from the untreated control was marginally affected compared to the other organs/tissues. Histopathological observation excluded the presence of signs of necrosis in the organs in which DNA damage was observed. This last observation is further strengthened by the negative findings observed in the same organs at the 24-hour sampling time. The study, even though not OECD compliant, is in my opinion sufficiently reliable and therefore clastogenicity cannot be excluded and cannot be ruled out by the negative findings obtained in the bone marrow micronucleus test (Gollapudi 1985) for the reasons discussed before.

Expert 5

I interpreted mutagenic as the ability to induce mutations in ANY mutational assay (bacterial, cell culture, in vivo). If the question was interpreted as only for an in vivo assay, then I would have indicated "clearly not mutagenic". If the question referred to humans I would have answered most likely not mutagenic, since the in vivo mutagenic assay by Young, was clearly negative, yet 1,3-D showed some activity the Ames test, albeit much less when highly purified and the most recent peer-reviewed article I could find (Eder E, 2006) did not include dose-response data, or statistical significance.
3 votes 3 0 votes
Expert 7
04/17/2019 12:26

As noted, in risk assessment, we are judging whether there is a concern for humans. Thus, the question we are addressing is 1,3-D’s potential to induce mutation in vivo. Given that “metabolic profiles seems to be sex, route and species invariant”, the totality of the in vivo evidence does NOT support a mutagenic concern for humans (nor does DNA reactivity/mutagensis support a cancer concern). And if I am reading the comments correctly, I think there is consensus that the induction of gene mutation in vivo is of no concern. But there is a remaining issue regarding clastogenicity as pointed out in the comments. Also, Although I can not rule this out, I do not think it is likely that 1,3-D would be a direct clastogen (vs secondary consequence) when it does NOT appear to interact with DNA and induce gene mutations (albeit there are exceptions). Also, I do not view this as a major issue in the context of a weight of evidence for carcinogenicity given that results of the relevant chronic bioassays do not provide convincing evidence of carcinogenicity. However, if the sponsor wants to strengthen the conclusions regarding genotoxicity, as pointed out in 6.5, “the cleanest way to resolve this” would be a new study (OECD TG 487 or 474).

Expert 14
04/19/2019 04:51

To user 232578 [Expert 7] : Reactive compounds to DNA can be clastogenic but not necessarily mutagenic (induction of point mutation) because the specific MoA. If a gene is broken down by a clastogenic MoA (e.g. large deletion) this is not detected by a conventional assay for the induction of gene mutation (except by the in vitro mouse lymphoma TK assay).

Expert 7
04/19/2019 05:47

I am not in disagreement with 617591 [Expert 14] . My only point was, in general, effective DNA reactive mutagens typicall y induce point/gene mutations (including deletions) and chromosomal aberrations. But there are exceptions of chemicals that are only clastogenic and do not induce
point mutations. Again, if the sponsor wants to strengthen the conclusions regarding genotoxicity, they need to address clastogenicity.

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