Reaction mass of 3,5,6,6-tetramethyl-4-methyleneheptan-2-one and (E)-3,4,5,6,6-pentamethylhept-3-en-2-one

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 471)

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

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

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 and in case of TA100 and TA1535 in the presence of S9, the highest dose tested was 78.1 µg/plate, while for TA98, TA1537 and WP2 uvr A 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. Bacterial growth inhibition was observed at 78.1 μg/plate in all test strains without S9 mix, at 78.1 μg/plate in TA100 and TA1535 with S9 mix, at 313 μg/plate in WP2 uvrA with S9 mix and 156 μg/plate or higher in TA98 and TA1537 with S9 mix. The numbers of revertant colonies in the substance treatment groups were less than two times that observed in each negative control in all test strains and therefore, the mutagenicity of the substance was judged negative. As such, it is concluded that the substance has no ability to induce mutations under the present test conditions.

 

Mouse lymphoma assay

The ability of the substance to induce gene mutations in mammalian cells was studied in the OECD TG 476 mouse lymphoma assay both with and without metabolic activation which was conducted in compliance with GLP. The cells were treated for 4 hours with concentrations from 10 to 85 μg/mL without and from 20 to 275 μg/mL substance with metabolic activation. Cultures were selected for cloning for mutant selection based on their relative suspension growth (from 20 to 60 μg/mL without S9 and from 20 to 125 μg/mL with S9). The relative total growth for the cloned cultures ranged from 17% to 97% for cultures treated without activation and from 8% to 101% for cultures treated with activation. The results were negative. Also negative results were observed in the confirmatory repeat using 24 hours exposure in a concentration range from 1.0 to 75 μg/mL (concentrations selected for cloning were in a range of 25 to 70 μg/mL). The relative total growth for the cloned cultures ranged from 7% to 77%. Under the test condition, the substance was considered to give negative results in the mouse lymphoma assay.

In vitro micronucleus test

The ability of the substance to induce micronuclei in cultured human lymphocytes was tested in an OECD TG 487 study and in compliance with GLP. Three treatment schedules were examined: pulse treatment (4 hours) with and without metabolic activation (S9), and continuous treatment (20 hours) without metabolic activation. The concentrations analysed were: 20, 60 and 120 μg/mL (4 hr +S9); 15.6, 31.1 and 62.5 μg/mL (4 hr -S9); 20, 60 and 100 μg/mL (20 hr -S9). Validity of the test was confirmed by the negative and positive control results. Dose-related cytotoxicity occurred under all three treatment conditions. None of the treatments induced a substance-related increase in micronuclei in binucleate lymphocytes, indicating that 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, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.