With the available information from genotoxicity studies, and with the pattern of tumor results from the cancer bioassays, what is your overall conclusion on the clastogenic potential of 1,3-D in humans following in vivo exposure (please explain)?

The bone marrow micronucleus test is likely the most relevant to this question, and it suffers from a number of shortcomings; perhaps the most serious of which is the lack of evidence that 1,3-D or its metabolites reaches the bone marrow. Although there is no unequivocal evidence indicating clastogenic potential, it cannot be ruled out. However, as indicated in question 9.1 the highest levels of exposure in the inhalation studies in mice, that were below the KMD were several orders of magnitude above the worst case scenarios in humans, and the lack of convincing evidence for clastogenic effects at such high doses argues for a very low probability of clastogenicity in humans.

As indicated in the genotoxicity revised question 9.1 and as also answered previously to question 2.10, genotoxicity must be thoroughly and deeply investigated with the appropriate in vitro and in vivo test batteries for hazard identification, regardless of cancer bioassay information/results, since the implication of mutations are not limited to cancer but involve genetic diseases and other somatic illnesses.
Based on the available information from genotoxicity studies, my overall conclusion on the clastogenic mutagenic potential of 1,3-D is that it is "Equivocal". This view is based on the fact that all negative in vitro and in vivo studies relevant for clastogenicity are not fully reliable for different reasons. More specifically, the quoted mouse bone marrow micronucleus test by Gollapudi (1985) is not fully reliable due to several shortcomings which include:
• no clear demonstration of target tissue exposure (in addition no reduction of %PCE);
• an unjustified difference in the percentage of PCE in the negative control group between male and female animals and within female animals at the 24 and 48 hour sampling times;
• a limited statistical sample (1000 PCE/animal).
In addition, it should be noted that the bone marrow micronucleus test is not an adequate in vivo follow-up assay for the 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, the positive results observed in different organs (stomach, liver, kidney, bladder, lung, brain and bone marrow) in terms of DNA breakage in the alkaline single cell gel electrophoresis (Comet assay) following intraperitoneal injection of 1,3-D to mice (Sasaky et al., 1998), is suggestive of a clastogenic potential of this compound. In addition, some of these organs (e.g. liver, kidney, bladder, lung) are potential tumor target organs of 1,3-D. This study, even though not OECD compliant (performed before the issue date of the relevant OECD Guideline 489) is in my opinion sufficiently reliable and therefore clastogenicity of 1.3-D cannot be excluded.
Possible future actions to rule out the potential clastogenic concern, as also highlighted from my side in the previous debate (question 6.5), include the performance of an in vivo comet assay compliant with the OECD TG 489, with the default organs (liver and duodenum) and additional potential tumor target organs e.g. kidney, bladder, lung.

The WoE considering all data indicates a lack of clastogenicity in vivo. The inconsistencies in the dataset with regards to micronucleus data as well as in the DL study prevent me from ticking the 'clearly' box