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Bacterial reverse mutation assay

To perform a bacterial reverse mutation assay, ethyl benzoate was dissolved in DMSO. The substance was tested in concentrations of 15 to 5000 µg per plate in the presence and of 5 to 5000 µg per plate in the absence of S9. In the absence of S9-mix the substance was found to be bacteriotoxic towards the strain TA102 at 500 μg/plate, towards the strain TA98 at 1500 μg/plate, and towards the strains TA100, TA1535, and TA1537 at 5000 μg/plate. In the presence of S9-mix ethyl benzoate was bacteriotoxic towards the strain TA102 at 1500 µg/plate and towards the strains TA98, TA100, and TA1535 at 5000 µg/plate. Precipitation of the test compound and the plates was not observed. The test compound failed to induce a significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. In conclusion, these results indicate that ethyl benzoate under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

In vitro gene mutation study in mammalian cells

A study was conducted according to OECD Guideline 476 to assess the potential mutagenicity of the read across substance methyl benzoate on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. Chinese hamster (V79) cells were treated with the test item at up to seven dose levels, in duplicate, together with vehicle (solvent) and positive controls in the presence and absence of an S9 metabolic activation system. Four treatment conditions were used for the test. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2 % final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose range of the test item was selected based on the results of a preliminary cytotoxicity test and were as follows:

4 hour without S9: 85, 170, 340, 680, 906.67, 1133.33, 1360 µg/mL

4 hour with S9 (2 %): 85, 170, 340, 680, 906.67, 1133.33, 1360 µg/mL

24 hour without S9: 85, 170, 340, 680, 906.67, 1133.33, 1360 µg/mL

24 hour with S9 (1 %): 42.5, 85, 170, 340, 680, 1020, 1360 µg/mL

The vehicle (solvent) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system. The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. As a conclusion, the test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

In vitro cytogenicity study in mammalian cells

The test item methyl benzoate (read across), dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleated cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis. The test concentrations were: without S9 mix: 8.8, 15.5, 27.1, 47.3, 82.9, 145, 253.8, 444.1, 777.1, 1360 µg/mL (Exp I and II); with S9 mix: 8.8, 15.5, 27.1, 47.3, 82.9, 145, 253.8, 444.1, 777.1, 1360 µg/mL (Exp I) and 82.9, 145, 253.8, 444.1, 777.1, 1360 µg/mL (Exp II). In Experiment I, no precipitation of the test item in the culture medium was observed. Precipitation occurred microscopically in Experiment II, in the absence of S9 mix, at 777.1 µg/mL and above at the end of treatment. No relevant influence on osmolarity or pH value was observed. Phase separation was observed in Experiment I at 1360.0 µg/mL in the absence and at 444.1 µg/mL and above in the presence of S9 mix and in Experiment II at 777.1 µg/mL and above in the presence of S9 mix. No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis could be observed up to the highest applied concentration. In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item were close to the range of the solvent control values and within the range of the laboratory historical control data. In conclusion, it can be stated that under the experimental conditions reported, the test item Methyl Benzoate did not induce micronuclei in human lymphocytes in vitro when tested up to the highest required concentration.


Justification for selection of genetic toxicity endpoint
There are three in vitro genetic toxicity tests available, a bacterial reverse mutation assay (Ames test), a gene mutation assay in mammalian cells (HPRT) and a chromosome aberration assay.

Short description of key information:
In vitro gene mutation in bacteria:
The test substance was not mutagenic in a bacterial reverse mutation assay (Ames Test).
In vitro gene mutation study in mammalian cells:
The read across substance was not mutagenic in an in vitro mammalian cell gene mutation test (HPRT Test) in V79 cells.
In vitro cytogenicity study in mammalian cells:
The read across substance was not clastogenic and not aneugenic in an in vitro micronucleus test in human lymphocytes.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available cytogenicity and mutagenicity test data did not indicate a mutagenic, clastogenic or aneugenic property of the test substance. On the basis of these data the substance is not considered to be classified for genetic toxicity under Directive 67/548/EEC (DSD) or under Regulation (EC) No 1272/2008 (CLP).

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