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Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol was tested in the standard Ames Test according to OECD TG 471 and GLP in bacteria strains TA 1535, TA 1537, TA 98 and TA 100 ofS. thyphimurium. The test substance was tested both in the presence and absence of metabolic activation in concentrations up to 5000 µg/plate. No mutagenic or bacteriotoxic effect was noted.

Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol was also negative in another standard Ames Test conducted under GLP and according to OECD TG 471 withS. thyphimuriumTA 1535, TA 1537, TA 98 and TA 100 in the presence and absence of metabolic activation. Test substance concentrations were up to 5000 µg/plate without reaching cytotoxicity. No mutagenic effect was noted.

Chromosomal Abberation Tests:

Human lymphocytes were treated with Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol at four dose levels, in duplicate, together with negative and positive controls according to OECD TG 473 and GLP. Two treatment conditions were used, i. e. 24 hours exposure both with and without the addition of an induced rat liver homogenate metabolising system. The dose range was selected from a series of eight dose levels and was 312.5 to 2500 µg/ml for both treatment conditions. All negative (solvent) controls gave frequencies of aberrations within the range expected for normal human lymphocytes. All the positive control treatments gave highly significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol demonstrated no significant increases in the frequency of aberrations in either of the treatment conditions. Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol was shown to be non-clastogenic to human lymphocytes in vitro.

In another chromosomal abberation test according to OECD TG 473 and GLP, human lymphocyte cell cultures were exposed to Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol at different concentrations ranging from 0 to 6970 µg/ml depending on the exposure time (19 h or 43h) and the use of metabolic activation. In this study the top dose without S9 -mix was 1743 µg/ml, which closely resembles the limit of 10 mM. No genotoxic effects were seen under these conditions. The top dose with S9-mix was 6970 µg/ml, which is 40 mM and four times as much as the upper limit of the guideline. No genotoxic effects were seen in concentrations up to 3485 µg/ml, which is about 20 mM and twice as much as the adviced upper limit. Concentrations of >= 4000 µg/ml (>= 23 mM) lead to weak clastogenic effects. These very high concentrations do not correspond to the current guideline and understanding of culture conditions. Therefore, the weak clastogenic results at very high concentrations are considered to be due to unphysiological conditions. The test substance itself is regarded as being not genotoxic.

Mammalian Cell Gene Mutation Test:

In a cell gene mutation assay in accordance with OECD TG 476 and GLP, the potential of Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus using the cell line V79 was investigated. The treatment period was 4h in the first experiment with and without metabolic activation. In the second experiment, the treatment period was 24h without metabolic activation and 4h in the presence of metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. Cytotoxic effects were observed in the absence of metabolic activation, whereas with metabolic activation no cytotoxicity was seen up to the highest test concentration of 10 mM.

In conclusion it can be stated that under the experimental conditions Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol did not induce mutations in the hypoxanthine-guanine phosphoribosyl transferase locus assay using the V79 cell line with or without metabolic activation.

Mammalian Erythrocyte Micronucleus Test:

Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol was tested in a micronucleus test in accordance with OECD TG 474 and GLP in CD-1 mice (5 animals/sex/dose), dosed once by gavage with 150, 300 and 600 mg/kg bw. Bone marrow cells were harvested 24 hours (negative control and all dose levels) and 48 hours post-treatment (for negative control and high dose). The vehicle used was aqueous methylcellulose (1%).

There were no signs of toxicity during the study. Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol did not cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes at the 24 hour sampling time. A statistically significant decrease in the ratio of polychromatic to normochromatic erythrocytes was obtained at the 48 hour sampling time for animals treated with the high dose level of the test substance (600 mg/kg bw) compared with the concurrent vehicle control group. However, this decrease at the 48 hour sampling time was notregarded as biological relevant, because the values were in the range of historical control data. No significant increase in the frequency of micronucleated erythrocytes in bone marrow up to the dose of 600 mg/kg bw after treatment with Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol was observed. In conclusion Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol was found to be not clastogenic or aneugenic in this test.

Short description of key information:
Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol has no genotoxic potential, since the test item was found to be negative in all tests including the Ames Test, the Chromosomal Abberation Test in vitro, the Mammalian Cell Gene Mutation Test in vitro and the Erythrocyte Micronucleus Test in vivo.

Endpoint Conclusion:

Justification for classification or non-classification

Based on the negative in vitro results supported by negative in vivo testing, Tetrahydro-4-methyl-2-(2-methylpropyl) -2H-pyran-4-ol has not to be classified and labeled as mutagenic according to Directive 67/548/EEC and Regulation 1272/2008/EC.