A mixture of: 4-(2,2,3-trimethylcyclopent-3-en-1-yl)-1-methyl-2-oxabicyclo[2.2.2]octane; 1-(2,2,3-trimethylcyclopent-3-en-1-yl)-5-methyl-6-oxabicyclo[3.2.1]octane; spiro[cyclohex-3-en-1-yl-[(4,5,6,6a-tetrahydro-3,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]furan]; spiro[cyclohex-3-en-1-yl-[4,5,6,6a-tetrahydro-4,6',6',6'a-tetramethyl)-1,3'(3'aH)-[2H]cyclopenta[b]]furan]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is negative in the Ames test (OECD TG 471).

The substance is negative in the Chromosome Aberration test (OECD TG 473).

The substance is negative in the Mouse Lymphoma Assay (OECD TG 490).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The bacterial reverse mutation (Ames) study

The substance is tested in the Ames test (OECD TG 471), using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and E. coli strain WP2uvrA. In the range finding test (plate incorporation), the substance was tested up to a concentration of 5000 µg/plate. The substance was toxic towards all tester strains in both 5000 µg/plate and 500 µg/plate. In the main test, the substance was tested up to a concentration of 500 µg/plate in two mutation experiments (plate incorporation). In the first experiment, toxicity was observed in all tester strains at a concentration of500µg/plate. In strains TA100 and TA1538, toxic responses were also observed at 125µg/plate. No growth in bacterial background lawns and/or substantial decreases in revertant colony frequency were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation. Based on this, the substance is not mutagenic in the Ames test.

In vitro cytogenicity

The substance is tested in the Mammalian Chromosome Aberration test (OECD TG 473). The substance was evaluated for chromosome aberrations in two experiments and an additional confirmatory test. In the first experiment, the substance was tested in absence of S9 -mix for 18 and 32 hours and in the presence of S9 -mix for 3 hours with 15 and 29 hours of fixation time at concentrations up to 500 μg/mL. In the second experiment the substance was tested in the presence of S9 -mix for an exposure time of 3 hours with 15 and 29 hours fixation time at concentrations up to 500 μg/mL. An additional confirmatory test was performed in absence of S9 -mix with a harvest time of 18 hours and concentrations up to 75 μg/mL. Cytotoxicity was observed in all treatments and based on these data, the concentrations for metaphase analysis were determined. No treatment related increases in aberrant cells were observed, neither did the substance induce an increase in the numbers of polyploid cells, either in the absence or presence of metabolic activation. Both positive control compounds, ethyl methanesulphonate and cyclophosphamide, caused large statistically significant increases in the proportion of aberrant cells showing the sensitivity of the test system. Based on this, the substance is not cytogenic in the Mammalian Chromosome Aberration test.

Mouse lymphoma assay test

The substance is tested in the Mouse Lymphoma Assay (OECD TG 490). Mouse lymphoma L5178Y cells cultured in vitro were exposed to the substance in duplicate, both with and without metabolic activation (S9 -mix). The substance was tested in eight dose levels in the first experiment: 1 to 40 µg/mL for the 4-hour –S9 exposure and 3 to 200 µg/mL in the 4-hour +S9 exposure and in the second experiment: 3 to 45 µg/mL for the 24-hour –S9 exposure and 10 to 350μg/mL for the 3 -hour +S9 exposure. The substance was tested beyond the limit of the solubility. In both experiments no significant increase in the mutation frequency at the TK locus was observed after treatment with the substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls. Based on this, the substance is not mutagenic in the Mouse Lymphoma Assay.

Justification for classification or non-classification

Based on the results of the genemutations in bacterial cells (Ames test), cytogenicity information (Chromosome Aberration test) and the genemutations in mammalian cells (Mouse Lymphoma Assay), the substance is not genotoxic and therefore does not have to be classified for genotoxicity in accordance with EU CLP (EC no. 1272/2008 and its amendments).