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Background to COM review 1. Halonitromethanes (HNM's) are a class of compound characterised by
the presence of one or more halogen atoms together with a nitro moiety
on a single central carbon. They differ from the Halomethanes (HM's) by
the presence of the nitro moiety. The HNM's have recently been identified
as disinfection by-products (DBP) in drinking water in the US (Richardson
et al 1999) and had received a high priority ranking for investigation
by the Environmental Protection Agency (Weinberg et al 2002 http://www.epa.gov/athens/publications/EPA_600_R02_068.pdf).
2. Members were told that the only available published data related to the in-vitro studies in the three publications (Plewa et al 2004, Kundu et al 2004a, b). Some preliminary findings had been reported by the EPA to the DH Toxicology Unit regarding in-vivo testing of the HNM's in transgenic medaka fish. Members noted the availability of an NTP negative in-vivo bone marrow mouse micronuclei test with trichloronitromethane (TCNM; chloropicrin), but that there were no appropriate in-vivo mutagenicity data available for other HNMs. Data considered by the COM 3. The COM discussed the in-vitro COMET studies, conducted using CHO cells (Plewa et al 2004). Members noted that HNMs tested were cytotoxic in CHO cells and that apoptosis induction had not been recorded in these studies. In addition none of the trials had included the addition of an exogenous metabolising fraction. However members agreed that HNMs were genotoxic in this test system and that the rank order reported by the study authors was a reasonable guide to relative potency for in-vitro DNA damage. It was noted that the data precluded a determination of absolute potency of HNMs compared to the positive controls used in the study but the data were consistent with HNMs being more potent in the test system than ethyl methanesulphonate. 4. The COM reviewed the available bacterial mutagenicity data obtained from plate incorporation trials using Salmonella typhimurium strains both in the presence and absence of an exogenous metabolising fraction (Kundu et al 2004a). The strains tested included TA 98, TA 100, TA 104 and a strain RSJ100 which expresses rat glutathione transferase theta (GSTT1-1) known to activate halomethanes. Members agreed that the standard plate incorporation approach had been undertaken in this study used two plates per dose point (instead of the normal three) but that reproducible small increases in the number of revertant colonies had been reported. It was noted that HNMs might volatilise and therefore procedures to prevent evaporation might have been more appropriate. In a further study (Kundu et al 2004b) trials had been undertaken using pre-incubation in screw-capped glass vials using Salmonella typhimurium strain TA 100 at 370C for 30 minutes followed by plate incorporation assessment of revertant colonies. Members felt that some residual loss of HNMs could still have occurred and that it would have been appropriate to undertake pre-incubation at a lower temperature of 300C. Overall members considered that the estimates of relative potency for in-vitro mutagenesis in Salmonella should be interpreted with caution. COM evaluation of the data 5. Members considered the potential mechanisms of HNM induced mutagenicity in Salmonella typhimurium strains and agreed this included the potential for both direct acting and metabolically activated mutagenic responses. This might include an oxidative pathway but it was evident that there were possible differences between the nine chemicals in the HNM group. Whether direct activity was due to HNM carbocation formation (loss of the halogen resulting in a reactive, positively charged carbon) and DNA alkylation in an SN1 reaction or alternatively SN2 substitution (in which nucleophilic attack precedes halogen loss), was not clear. Members felt that, unlike the halomethane group of compounds, there was no convincing evidence for a glutathione mediated pathway with HNMs. Members observed that it was difficult to derive clear conclusions regarding mechanism and potency in the bacterial mutagenicity tests, but overall brominated HNMs appeared from the limited data to be more potent in-vitro mutagens than chlorinated HNMs. 6. The COM discussed the potential testing strategy that might be applied to HNMs and agreed that each individual HNM needed to be tested. It was agreed that the standard COM strategy was appropriate in this instance but should be modified so that the in-vivo rat liver UDS assay was the first in-vivo test. Members felt that a liver UDS assay would be more appropriate than the more usual in-vivo micronucleus test because of the likelihood that potential direct acting chemicals or reactive intermediates would not reach the bone marrow intact. Also, due to the presence of potentially direct acting mutagens a site of contact assay was considered appropriate (for example the COMET assay in the stomach). Members felt that an in-vitro liver UDS assay would be a useful pre-screen. COM overall conclusions 7. Overall, in accordance with the COM strategy, HNM's should be regarded as in-vitro mutagens and potential in-vivo mutagens. It was considered that there was a need for appropriate in-vivo testing. The COM suggested:-
If either of these studies yielded positive results, then the chemical
under test should be considered to be an in-vivo mutagen.
June 2005
References Kundu B, Richardson SD, Swartz PD et al (2004a) Mutagenicity in
salmonella of halonitromethanes: a recently recognized class of disinfection
by-products in drinking water. Mut. Res. 562 39-65
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