Do you agree with the conclusions of the Zeiger (2005; see page 13) review (please explain your answer)? In your explanation please indicate if the studies conducted since the Zeiger review (e.g., Big Blue rat assay via oral route (Young, 2018) add to or detract from these conclusions.

Paragraph 1: Disagreement. It should be noted that 1,3 D without epichlorohydrin is positive in the Ames test when tested in the absence of metabolic activation and in its presence using solvents different from DMSO (e.g. ethanol) (e.g. Lawlor 2004).

Paragraph 2: The issue that the in vivo genetic damage induced by 1,3 D in rats and mice is limited to non-specific DNA strand breakage and is not of direct DNA interaction is highly speculative and not based on data. In the case of comet assay by Sasaki et al., cytotoxicity in the presence of strong positive results was excluded by histopathological analyses. In addition the positve results in the Ames test in different tester strains support a direct effect on DNA.

Paragraph 3: For the first part of this paragraph , please see my previous comments. For the 32P postlabelling study by Reddy (1997) my view, as also noted previously, is that the study is of questionable relevance because the low dosages of 1,3-D both by inhalation (30 and 60 ppm) and by oral gavage (12.5 and 25 mg/kg/day), compared to the other available in vivo studies (e.g Gollapudi 1998; Young 2018). In addition, the 32P-postlabeling method is less sensitive for the detection of non-aromatic DNA adducts than aromatic adducts because of analytical difficulties in the separation of non-aromatic adducts from unmodified (normal) nucleotides (Phillips et al., 2000). For UDS (Ghia et al., 1993), I would consider the assay not sufficiently sensitive as also proposed by EFSA that does not consider the in vivo unscheduled DNA synthesis (UDS) assay as a suitable follow-up study for a positive results in in vitro gene mutation tests (EFSA Journal 2017, Clarification of some aspects related to genotoxicity assessment).

Paragraph 4. As pointed out in section 2.5 the presence of epichlorohydrin in the in vivo genotoxicity studies could not represent a confounder factor since it has been shown in a number of cytogenetic studies a negative outcome for epichlorohydrin in mice and rat at dose-levels up to 200 mg/kg bw (Rossi et al., 1983; Tsuchimoto and Matter, 1981; Kirkhat, 1981; Salamone et al., 1981; Terada et al., 1992; Asita et al., 1992; Dabney et al., 1979; Sram et al., 1981).

The Big Blue rat assay via oral route (Young, 2018) add useful information and the negative results obtained in this test permit to overrule the positive outcome obtained in the Ames test.

Not fully - as described previously I do not agree with a blank exclusion of the studies done wit 1% epichlorhydrin, nor with the conclusion that the positive results from the Comet assay and alkaline elution can be ignored. To note, this was written in 2005, the Comet assay is now an OECD guidline method.
I do agree, though, with the statment that the data do not support that 1,3-D is a DNA reactive carcinogen.

I generally agree with Zeiger, although I believe that pure 1,3-D has some limited genotoxic potential in vitro. The 32P-labelling assay as cited by Zeiger to deny the presence of DNA adducts is not really sensitive for small adducts, (as would be expected for 1,3-D). This does not mean that DNA adducts are not present. They simply cannot be detected because the limit of the assay. However, DNA breakage observed (e.g. comet assay) could be the result of intermediate steps in the repair of small DNA adducts (excision repair) and not necessarily via a secondary to toxicity. This calls into question the conclusion that "whenever DNA or chromosome breaks is seen in the absence of adducts, a secondary mechanism of action always be considered". However, the Young study offers strong support to the lack of genotoxicity in vivo. I agree with Zeiger that 1,3-D in unlikely to have any genotoxic or carcinogenic effects in humans under realistic exposure conditions.