Reaction Mass of (1R)-1-[(1S)-3,3-dimethylcyclohexyl]ethyl formate and (1R,2S)-2,6,6-trimethylcycloheptyl formate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is negative in the Ames test with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA, with and without metabolic activation (OECD TG 471)

The substance is negative in the mouse lymphoma assay, with and without metabolic activation (OECD TG 476)

The substance is negative in the micronucleus assay with human lymphocytes, both with and without metabolic activation (OECD TG 487)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

An in vitro mutagenicity test using bacteria was performed according to OECD TG 471, and in compliance with GLP. The ability of the substance to induce mutations was investigated using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and E. coli strain WP2 uvr A using a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). In the absence of the S9, the highest dose tested was 78.1 µg/plate, while in the presence of S9 mix it was 313 µg/plate based on two dose-range finding tests. No precipitation was observed at any dose level with and without S9 mix. The bacterial growth inhibition was observed at > 78.1 μg/plate in all test strains without S9, > 156 μg/plate in TA100, TA1535, TA98 and TA1537 and > 313 μg/plate in WP2uvrA with S9. The numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all test strains, both with and without metabolic activation. Therefore the substance was concluded to give negative results in the Ames test.

Mouse lymphoma assay

In a study, performed according to OECD TG 476 and in compliance with GLP, the substance was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). Four tests were performed. The test substance was diluted in dimethylsulfoxide (DMSO). The highest concentrations of the substance evaluated for mutagenicity were 57 μg/mL and 73 μg/mL in the absence and presence of S9-mix, respectively, based on cytotoxicity. Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and presence of the S9-mix, respectively; DMSO served as negative control in all cases.

In all tests the negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls. Therefore, all tests were considered valid. In both the absence and presence of S9-mix the substance was toxic to the cells resulting in a reduction in initial cell yield and suspension growth. The relative total growth (RTG) at the highest concentration evaluated in the absence of S9-mix (57, 55 and 53 μg/mL) was 0.3%, 19% and 16%, for test 2, 3 and 4, respectively. The relative total growth (RTG) at the highest concentration evaluated in the presence of S9-mix (73 μg/mL) was 7% and 3%, for test 2 and 3, respectively. In all tests evaluated for the mutant frequency in both the absence and presence of S9-mix no increase in mutant frequency (MF) by more than 88 or 126 mutants per 1,000,000 clonable cells, i. e. no equivocal or positive response, compared to the negative control was observed at any dose level. It is therefore concluded that under the conditions used in this study, the substance is not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in both the absence and presence of metabolic activation (S9-mix).

In vitro micronucleus test

In an in vitro mammalian cell micronucleus test, performed according to OECD TG 487 and in compliance with GLP, the substance was examined for its potential to induce micronuclei in cultured binucleated human lymphocytes in both the absence and presence of a metabolic activation system (S9 -mix). Two independent in vitro micronucleus tests were conducted for which blood was obtained from two different donors. DMSO was used as solvent for the test substance. Dose levels ranging from 3.6 to 1841 µg/mL were tested as final concentrations in the culture medium. Cytotoxicity was calculated from the Cytokinesis-Block Proliferation Index (CBPI). In the first test, in the presence and absence of metabolic activation (S9-mix) the treatment/recovery time was 4/20 hours (pulse treatment). In the second test, in the continuous treatment group, the treatment/recovery times were 20/28 hours. In the first test, in both the pulse treatment groups (with and without S9-mix) a dose related cytotoxicity was observed. Three dose levels of the test substance, together with the negative controls and positive controls, were analysed for micronucleus induction in binucleated lymphocytes. The selected dose levels were: 230, 115 and 28.8 μg/mL (pulse treatment group with metabolic activation) and 115, 57.6 and 28.8 μg/mL (pulse treatment group without metabolic activation). In the second test, in the continuous treatment group without metabolic activation, a dose related cytotoxicity was observed. Three dose levels of the test substance (200, 100 and 50 μg/mL), together with the negative controls and positive controls, were analyzed for micronucleus induction in binucleated lymphocytes. In both the first and the second test, the negative controls were comparable with the historical data of the test facility. Treatment with the positive controls Cyclophosphamide, Mitomycin C and Vinblastine sulphate resulted in statistically significant increases in the numbers of binucleated cells containing micronuclei, when compared to the numbers observed in the cultures treated with the solvent control (negative control). This demonstrates the validity of the study. In both the first and second test, in all treatment groups, the test substance did not show a significant increase in the number of binucleated cells containing micronuclei, at any of the concentrations analysed, when compared to the numbers found in the concurrent negative controls. From the results obtained in two independent in vitro micronucleus tests it is concluded that, under the conditions used in this study, the substance was not clastogenic and/or aneugenic to cultured human lymphocytes.

Justification for classification or non-classification

Based on the negative results in three in vitro genotoxicity tests, classification of the substance for genotoxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its amendments.