How do you interpret the in vivo genotoxicity studies for 1,3-D summarized in the white paper?

Answer 1: “Big blue assay for Gallapudi…..”: I cannot completely follow the Colleague in this respect. I think that this study, though performed before adoption of the relevant OECD Guideline (TG 488), it essentially adheres to the main requirements of the TG. I only noted (my comments in round 1) that the administration period was 14 days instead of 28 days requested for slow proliferating tissues/organs (e.g. liver). The background of mutation frequencies appears to regular (see also background mutation frequencies in the rat study by Young, 2018). Concerning the route of exposure in the study by Young (2018), I cannot see a real problem, since administration by diet is one of the recommended routes of exposure. In this respect, the only issue I can see, is the use of the maximum tolerated dose (MTD) as the top dose. From the white paper it is not possible to assess this, but I believe that MTD was used as top dose. Overall, the in vivo studies with transgenic animals relevant for induction of point mutations are the most reliable among the studies available. Based on this, I concluded that these results can overrule the concern for point mutation observed in some in vitro studies and in particular in the Ames test (Lawlor 2009) and in mammalian cells (Myhr and Caspary, 1991).

Answer 2: I agre with the Colleague. Concerning the dose-response for the observed resorptions (3 and 4) and also the increase from 1.7% to 7.7% for resorption 3 and its possible statistical significance I would not pay much attention. Statistical significance has not been reported in the white paper and I believe that this is due to the high variability of results obtained among the animals.

Concerning the Gallapudi Big Blue assay, I don't agree with the white paper. The OECD guideline specifies the use of 5 animals to detect a 2 fold change in mutant fraction (However, as 1,3-D is likely weakly mutagenic (if at all)) 5 animals would not be sufficient to detect a 1.5 fold increase . Yet this could be important biologically. Also, since 1,3-D is at best weakly mutagenic, and mutations in the BB assay simply integrate over time, the 28 day treatment period would have doubled the sensitivity.
The Young BB assay was much more thorough, with a larger number animals and a longer exposure period, but the fixation was only several days for the last exposures (but longer for the earlier exposures). The sensitivity of the assay might have been increased with a longer fixation period. Although the OECD guideline states oral exposure is acceptable, it also states that the anticipated route of exposure should e considered when designing the assay; and inhalation exposure seems more relevant for 1,3-D.
Nonetheless, although certain limitations can be pointed out, (others were identified by the above reviewers)I think the weight of evidence is still against in vivo genotoxicity for 1,3-D. The in vivo tests referred to in the question, do not show any clear evidence of genotoxicity, If unlimited resources were available, the sensitivity of the post-labelling assay and the BB assays could be increased, but at biologically relevant exposures it would likely not change risk estimation.

Don't think the acceptance of the Gallapudi 1985 study is all that relevant since there is a recent, guideline compliant TGR study available which is clearly negative. As stated earlier, for this study (Young 2018), the lack of through substantiation of the maximum dose used. Given the votes shown in the above table, it seems like there is good consensus on a lack of mutation by BB but less clarity on the 'clastogenicity' side

Nothing to add