3-ethoxy-1,1,5-trimethylcyclohexane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

- In vitro Micronucleus Test (OECD 487, K, rel.1): non clastogenic and non aneugenic up to cytotoxic concentations

- In vitro Gene Mutation Assay at the hprt locus (OECD 476, K, rel.1): not mutagenic at the hprt locus of L5178Y mouse lymphoma cells up to toxic, or high toxic concentrations

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A Bacterial Reverse mutation Assay (Ames test) was performed in 1978 with several deviations (Gloxhuber, 1978).

The quality of this study was not considered to be sufficient so an other Ames Test was performed according to the OECD guideline No.471 and in compliance with GLP (Sokolowski, 2015). No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies

The substance is therefore considered as non-mutagenic according to the Ames test.

Moreover, an in vitro Micronucleus Test was performed according to the OECD Guideline No. 487 and in compliance with GLP (Morris, 2016). In this test, cultured peripheral human lymphocytes were exposed to test item in the presence and absence of a metabolic activation system. Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the absence and presence of a standard metabolizing system (S9 at a 2% final concentration) and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 The dose selection for the main test was based on the toxicity seen in the preliminary toxicity test and near optimum toxicity was achieved in the three exposure groups of the main experiment. The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, in the 4-hour exposure groups in the absence and presence of S9. In the 24-hour exposure group the test item did induce some small but statistically significant increases in the frequency of binucleate cells with micronuclei at 54 and 72 µg/mL. However, since these increases did not exceed the upper limit of the historical control range for a vehicle control, and they were set against a low vehicle control value they were considered to be of no toxicological significance.

The substance is therefore considered to be non-clastogenic and non-aneugenic to human lymphocytesin vitro.

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP (Lloyd, 2016), L5178Y tk^+/- mouse lymphoma cells were exposed to test substance in the presence and absence of a metabolic activation system. Negative, vehicle and positive control groups were also included in each mutagenicity test. Metabolic activation system used in this test was 2 % S9 mix (final concentration). S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254. 

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 31.25 to 1000 µg/mL (limited by solubility of the test substance formulation in culture medium). Steep concentration-related toxicity was observed and the highest concentrations to give>10% relative survival (RS) were 31.25 µg/mL in the absence of S-9 and 62.5 µg/mL in the presence of S-9, which gave 72% and 68% RS, respectively.

In the Mutation Experiment twelve concentrations, ranging from 5 to 100 µg/mL in the absence of S-9 and from 10 to 200 µg/mL in the presence of S-9, were tested.Seven days after treatment, the highest concentration analysed to determine viability and 6TG resistance in the absence of S-9 was 40 µg/mL, which gave 3% RS. Steep concentration-related toxicity was observed between 30 and 40 µg/mL in the absence of S-9, giving 85% and 3% RS, respectively, therefore both concentrations were analysed. The highest concentration analysed in the presence of S-9 was 110 µg/mL,which gave 13% RS.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore, the study was accepted as valid.

 When tested up to toxic, or highly toxic concentrations in the presence or absence of S-9, respectively, no statistically significant increases in mutant frequency, compared to the vehicle control MF, were observed following treatment with the test substance at any concentration analysed and there were no statistically significant linear trends. Treatment up to a highly toxic concentration (40 µg/mL, giving 3% RS) in the absence of S-9 did not affect the interpretation of the data, which were clearly negative. 

Under the test conditions, test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic, or highly toxic concentrations for 3 h in the presence or absence, respectively, of a rat liver metabolic activation system (S-9).

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 and to the GHS.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.