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In vitro bacterial mutagenicity:

The in vitro mutagenicity of benzotrichloride has been tested in in vitro bacterial systems mainly based on the Ames test. The first two reported studies are somehow similar to the OECD guideline 471 and the last one is based on recognized scientific principles.

In a study from MacMahon (1979), the authors tested the mutagenicity potential of benzoyl chloride (CAS n° 98 -88 -4) with a methodology similar to the OECD guideline 471, with the recommended strains of salmonella typhimurium and E.coli. They conducted this test with a slightly modified protocol proposed by Ames for the plate incorporation. They did a gradient technique for the plate incorporation allowing to test a full range of concentration ranging from 0.1 µg/mL to 1000 µg/mL or to cytotoxic concentrations. They included also in this test four other strains of S.typhimurium to cover all major kind of mutations. All strains were tested with and without metabolic activation and positive and negative controls were included in the experiment.

With this gradient technique, the concentration range over which chemically induced mutant colonies are present is recorded. The minimal inhibitory concentration is observed as a clear zone in the agar gel. This level is a basis for the expert to judge on the mutagenicity potential of the test substance compared with positive controls. It is assumed then than all substances showing no revertants over 1000 µg/mL or to the concentration showing first sign of toxicity are considered as negative in this test. Besides a complementary analysis on the number of revertants may be undergone to confirm the expertise.

According to the authors, this test was negative for benzoyl chloride for all strains. Even if this test system requires an expert judgement, we could assume that this test is enough robust and sensitive to detect mutagens. And since all strains returned negative, benzoyl chloride should not be considered as a potent mutagen in in vitro bacterial systems based on these results.

In an other study from Yasuo (1978), the authors tested the mutagenic activity of benzoyl chloride (CAS n° 98 -88 -4) with (1) a bacterial reverse mutation test in a E. coli strain WP2 hcr and (2) with a recombination assay performed according to Kada, Tutikawa and Sadaie (1972) on Bacillus subtilis strains..

For the bacterial reverse mutation test following strains may have been used:Salmonella TA 98, TA 100, TA 1535, TA 1536, TA 1537, TA 1538 and Escherichia coli WP2 try hcr and WP2 B/r try. As not all results are presented in the study, we can only state that E. coliWP2 try hcr was tested both with and without the metabolic activation system (i.e. rat liver microsome fraction S9) and the concentrations used were 2.1 and 3.6 µmoles/plate. To assess the mutagenicity of the test substance the number of revertant colonies were counted and compared to the control .

For the recombination assay the mutagenic potential of the test substance was tested with Bacillus subtilis M45 (rec-) and H17 (rec+) at concentrations of 0.4 and 0.7 µmoles/plate. The assessment of the mutagenicity for this latter point is based on the inhibition of the growth zone.

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

Thus, under the test conditions, the mutagenicity of benzoyl chloride was negative in the reverse mutation assay with and without metabolic activation system to E. coli WP2 try hcr and also for the other tested strains no induction of mutation was observed. Furthermore, the mutagenicity of the test substance was also negative in the recombination assay for Bacillus subtilis M45 and H17.

Thus considering the overall study, benzoyl chloride should be considered as non mutagenic in in vitro bacterial system. Further testing may be conducted to clarify the situation for in vivo cases since the authors report that these results could be due to the susceptibility of the test substance to hydrolyse in an aqueous system.

In a last study from Kawazoe (1982), the authors tested the mutagenic activity of benzoyl chloride (CAS n° 98-88-4) (more specific antimutagenic effect on UV-induced and X-ray induced mutagenesis) with a bacterial reverse mutation test according to Kawazoe, Kato and Takahashi (1981) [Chem. Pharm. Bull. (Tokyo), 29:2631-2638] with slight modifications described in this study. Two E. coli B (argF-) strains were used, namely H/r30R and Hs30R. Further, few details are given on materials and methods. However, results are sufficiently described. Criteria for interpretation are not clearly reported but dose effect relationship are assessed in the publication, it could then be assumed that the presence of a dose-effect relationship is the criteria for positive results. It is also assumed that more than three concentrations were tested but not reported.

In the test conditions, no induction of the revertants was observed for both strains, even when tested at lethal doses. Hence, this reverse mutation test should be interpretated as negative.

These studies are well documented and are based on good scientific principles. Few details are missing and minor deviations are observed in the study from Kawazoe (1982) and Yasuo (1978) respectively. Besides, the study from MacMahon is very similar to the OECD guideline 471. In addition, further strains have been tested and the incubation method has been improved to give more sensitive results. The interpretation criteria for the study of MacMahon may be questionnable since they are based on an expert judgement. However, the results of these experiments are con sitent and results of the MacMahon study are described with great care and with the more details. Altogether, these elements were the basis for the choice of the MacMahon study as a key study since the approach was the most straight forward and reliable available data.

Furthermore, all results are negative and prove that there is no concern on the mutagenicity potential of benzoyl chloride. As introduced by the study of Yasuo (1978) however, these results may be due to the succeptibility of the test substance to hydrolyse. Hence, as the hydrolysis may induce an inactivity , further in vivo testing is not required at this level but in vivo mutagenicity could be recommended to be completely sure. So far however, further in vitro testing on Mammalian systems as part of the annex VIII is not required since the in vitro bacterial mutagenicity is considered negative.


Short description of key information:
In vitro bacterial mutagenicity

Endpoint Conclusion:

Justification for classification or non-classification