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in vitro:

Gene mutation in bacteria

The potential mutagenicity of 4-tert-butylcyclohexanol was investigated in two bacterial reverse mutation assays (Ames test).

One bacterial gene mutation assay (Ames test) with the test substance was conducted in compliance with OECD Guideline 471 and under GLP conditions (2012-0124-DGM). Based on the results of a preliminary cytotoxicity test Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with the test substance at concentrations of 1.0, 3.16, 10.0, 31.6, 100 and 316 µg/plate using the plate incorporation assay and pre-incubation method (20 min pre-incubation time), respectively. Both experiments were performed in the absence and presence of a liver microsomal activation system (S9 mix). Cytotoxicity was observed in all S. typhimurium strains at the highest concentration (316 µg/plate) with and without metabolic activation. The induced number of revertants per plate was comparable to the vehicle control for all strains tested with and without metabolic activation. The positive controls included showed the expected results. Thus, under the experimental conditions reported, the test substance did not induce mutations in the absence and presence of metabolic activation in the selected strains of S. typhimurium.

In a further Ames test investigating the gene mutation potential of 4-tert-butylcyclohexanol similar to OECD Guideline 471, the S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were exposed to concentrations of 10-5000 µg/plate in the presence and absence of metabolic activation system (S9 mix) using the plate incorporation assay and the preincubation method, respectively (88-0660-DKM). Cytotoxicity occurred starting at concentrations of 250 and 500 µg/plate, respectively. Based on the results of the experiment, the test substance was considered to be non-mutagenic in the selected strains of S. typhimurium with and without S9 mix.

In summary, 4-tert-butylcyclohexanol was non-mutagenic in bacteria in the presence and absence of metabolic activation.

Cytogenicity in mammalian cells

An in vitro mammalian chromosome aberration test was performed with 4-tert-butylcyclohexanol in Chinese hamster lung fibroblasts (V79) according to OECD Guideline 473 (adopted in 1983) and in compliance with GLP (97-0366-DGM). The test substance concentrations for chromosome analysis were selected in a preliminary cytotoxicity study using ten test concentrations in the range of 2.5 to 2000 µg/mL. The preliminary experiments revealed a systematic influence of the test substance which led to a reduction in the mitotic index.

In the main test, concentrations of 10, 60 and 100 µg/mL were used to analyse chromosomal aberrations after exposure to the test substance for 18 h in the presence of metabolic activation. Test concentrations of 50, 250 and 500 µg/mL as well as 20, 100 and 200 µg/mL were selected for analysis of chromosome aberrations after test substance exposure for 3 h in the presence of metabolic activation. Cells were harvested 18 h after start of exposure. Moreover, tests with a harvest time of 28 h were performed, in which cells were exposed to 60 µg/mL for 28 h in the absence of metabolic activation and to 200 µg/mL for 3 h in the presence of metabolic activation, respectively. All mutant frequencies were within the range of historical control data. A statistically significant increase in the frequency of cells with chromosomal aberrations was observed in the experiment with a sampling time of 28 h at 200 µg/mL in the presence of metabolic activation. As this chromosomal aberration frequency of 3.8% does not exceed the normal range of the test system (< 5%) and no dose-dependent abnormalities in the other experiments could be detected, this statistical significance was due to the low concurrent negative control (0%) and not a consequence of a clastogenic activity of the test substance. The frequency of polyploid cells in both parts of the experiment was within the expected range (< 10%). The positive controls showed the expected increase in the rate of chromosome aberrations, thus indicating the sensitivity of the assay.

It is concluded that 4-tert-butylcyclohexanol did not induce biological significant increases in the chromosomal aberration frequency in cultured Chinese hamster lung fibroblasts (V79) with and without metabolic activation and is therefore considered not to be clastogenic.

Gene mutatation in mammalian cells

4-tert-Butylcyclohexanol was tested for its potential to induce mutation at the thymidine kinase (TK) locus in mouse lymphoma L5178Y cells according to OECD Guideline 476 under GLP conditions (2012-0126-DGM). In a preliminary experiment with and without metabolic activation (S9 mix) pronounced to complete cytotoxicity in form of decreased survival was noted starting at a concentration of 250 µg/mL and test item precipitation was observed starting at 1000 µg/mL. In the main test (mutagenicity test) L5178Y mouse lymphoma cells were treated with test substance concentrations of 15.63, 31.3, 62.5, 125 and 250 µg/mL both in the absence and presence of metabolic activation. The exposure duration was 3 h and 24 h in experiments without S9 mix and 3 h in two independent experiments with S9 mix. The treatment of cells in all experiments was followed by an expression period of 2 days and a selection period of 11-14 days in the presence of trifluorothymidine.

The mutation frequency of the cultures treated with the test substance in the presence and in the absence of S9 mix was within the range of the negative control values and the normal range of 50 to 170 mutants per 1E+06 viable cells, whereas the positive controls caused a pronounced increase in the mutation frequency. Cytotoxicity was noted at the top concentration of 250 µg/ mL in the absence and presence of metabolic activation. No change was noted in the ratio of small to large mutant colonies.

Thus, based on the test result 4-tert-butylcyclohexanol was not mutagenic in the mouse lymphoma forward mutation assay and did not exhibit clastogenic potential at the concentration-range investigated.

In conclusion, 4-tert-butylcyclohexanol is considered not to cause genetic damage, since all in vitro genetic toxicity studies revealed negative results.

in vivo:

No data on in vivo genetic toxicity is available.


Justification for selection of genetic toxicity endpoint
No study was selected, since all available in vitro genetic toxicity studies were negative.

Short description of key information:
In vitro:
- Gene mutation in bacteria (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 (OECD 471)
- Cytogenicity in mammalian cells (Mammalian chromosome aberration assay): negative with and without metabolic activation in Chinese hamster lung fibroblasts (V79) (OECD 473)
- Gene mutation in mammalian cells (Mammalian cell gene mutation test, TK): negative with and without metabolic activation in mouse lymphoma L5178Y cells (OECD 476)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of 4-tert-butylcyclohexanol do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.