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In vitro bacterial mutagenicity:

Thein vitro mutagenicity of benzotrichloride has been tested in in vitro bacterial systems based on the Ames test. The reported studies are similar to the OECD guideline 471.

In a study from Yasuo (1978), the authors tested the mutagenic activity of benzotrichloride (CAS n° 98-07-7) with (1) a bacterial reverse mutation test by following a methodology similar to the OECD guideline 471 and (2) with a recombination assay performed according to Kada, Tutikawa and Sadaie (1972) (Mutation Research 16: 165-174).

For the bacterial reverse mutation test following strains were used:Salmonella TA 98, TA 100, TA 1535 and Escherichia coliWP2 try hcr and WP2 try B/r. All Salmonella strains are assumed to have been tested with and without the metabolic activation system (i.e. rat liver S9 mix) and the E. coli strains were tested both with and without the metabolic activation system. For Salmonellastrains TA 98 and TA 1535 reported tested concentrations are 0.5 and 1.0 µmoles/plate, while for SalmonellaTA 100 1.0 and 2.0 µmoles/plate was tested. The concentrations used to test theE. coliWP2 try hcr strain were 0.25, 0.51, 1.02, 1.53, 2.05 and 2.56 µmoles/plate, while for E. coli WP2 B/r try 0.51, 1.02, 2.05, 2.56, 3.07 and 4.09 µmoles/plate of the test substance were tested. However for all tested strains, it does not appear very clear whether more concentrations were tested and if only positive results were reported. The number of revertant colonies were counted and compared to the control to assess the mutagenicity of the test substance.

For the recombination assay the mutagenic potential of the test substance was tested with Bacillus subtilis M45 (rec-) and H17 (rec+) with and without metabolic activation system at concentrations of 1.5, 2.6 and 3.6 µmoles/plate. The assessment of the mutagenicity is based on the inhibition of the growth zone.

Finally for all these experiments the test substance was dissolved in DMSO and solvent controls were performed (study is unclear on this point for some strains) while no positive controls were executed.

Under the test conditions, the mutagenicity of benzotrichloride was clearly positive in the reverse mutation assay with metabolic activation system to Salmonella TA 98, TA 100, TA 1535 and E. coli WP2 try hcr. Slight mutagenic activity was observed for E. coli WP2 try hcr without metabolic activation and the peak value forE. coli WP2 B/r try with metabolic activation was about 3 -fold the control level. Furthermore the mutagenicity of the test substance was also considered positive in the recombination assay for Bacillus subtilis M45 and H17 without metabolic activation.

Thus considering the overall study, benzotrichloride may be considered a potent mutagen with metabolic activation. Therefore, further testing would be required to clarify the situation based on this experiment.

In an other study from Kudoley (1986), the authors tested mutagenic activity of benzotrichloride (CAS n° 98-07-7) following a methodology also similar to the OECD guideline 471. Only, three strains of S. typhimurium(i.e. TA 100, TA 98 and TA 1538) were exposed with metabolic activation to the test substance using the plate incorporation method. The metabolic activation system (liver S9) was derived from arochlor treated rats. Furthermore solvent controls and positive control substances (i.e. 2-acetylaminofluorene and benzo(a)pyrene; both with metabolic activiation and methylnitrosoguanidine without metabolic activation) were included. It is not clear how the authors chose the tested strains.

Under the test conditions, a 2 to 5 fold induction of mutants was observed in comparison to spontaneous background levels after exposure to the test substance. Thus, the in vitro mutagenicity assay exposing S. typhimurium TA 100, TA 98 and TA 1538 to benzotrichloride with metabolic activation revealed that the test substance is mutagenic. This test should then be considered as positive with metabolic activation. Hence, further testing would also be required based on these results.

Both studies follows a methodology similar the OECD guideline 471 based on the Ames test method but the study from Yasuo (1978) contains less deviations to the guideline. Furthermore, the double approach used in the Yasuo study brings more evidences that benzotrichoride is a potent mutagen in in vitro bacterial systems with a metabolic activation. Finally, more details are provided on the criteria for interpretation of results in the study from Yaso (1978). Altogether, these elements were the basis to chose this study as the key study.

So far, based on both results, according to Annex VII further tesing would be required. However, according to the column 2 of the specific rules for adaptation from column 1 of the REACH regulation n° 1907/2006, an in vitro study on cytogenicity in mammalian cells or an in vitro micronucleus study of the annex VIII requirement does not usually need to be conducted if the substance is known to be carcinogenic category 1 or 2 or mutagenic category 1, 2 or 3. In this case, benzotrichloride is classified in the CLP regulation n° 1272/2008 EC as a carcinogen category 1B, hence no study on cytogenicity in mammalian cells or an in vitro micronucleus is required.

Hence, no further testing is required on an in vitro Mammalian system. Besides, as no information on in vivo testing was freely available, then no information will be here further discussed.


Short description of key information:
In vitro bacterial mutagenicity: positive

Endpoint Conclusion:

Justification for classification or non-classification