Phenol, 2,4-bis[(dodecylthio)methyl]-6-methyl-

Endpoint summary

Additional information

in vitro:

- Gene mutation in bacteria:

The test substance was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2uvrA at concentrations ranging from 33 to 5000 µg per plate (CIBA 703300, 2001). The tests were conducted, using the pre-incubation and plate incorporation method, on agar plates in the presence and absence of an Phenobarbital induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity. Positive control compounds demonstrated the sensitivity of the assay and the metabolising potential of the S9 mix.

The results obtained in both experiments were similar. No mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition. No precipitation was observed at any concentration. In the plate incorporation test, no toxic effects were observed with and without metabolic activation. In pre-incubation test, a minor toxic effect was observed in the presence of metabolic activation in strain TA 98 at 5000 µg/plate. No toxic effects were observed in the remaining strains.

- Chromosomal aberration in mammalian cells:

The test item, dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments (CIBA 745900, 2002). In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 µg/ml) was chosen with respect to the current OECD Guideline 473. No toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

- Gene mutation in mammalian cells (read-across to the structural analogon 4,6-bis(octylthiomethyl)-o-cresol)

A data matrix including detailed read-across justification is attached to the Chemical safety report.

The read-across substance differs in the length of the alkyl chain at the thio ether. Specifically, it is a C8 chain in comparison to a C12 chain in the target substance. Due to the shorter chain length, the molecule is smaller and predicted to be of slightly better solubility in water. Therefore, it is considered to be of better systemic availability which might result in a slightly higher toxicity compared to the target substance.

As both C8 and C12 chains are metabolized via beta oxidation, both substances are predicted to have the same metabolites. Overall read-across is justified.

The test item was tested in a gene mutation study in V79 cells according to OECD guideline 476 and GLP requirements (Ciba Geigy 1991). The system allows the detection of base-pair substitutions, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested by the appearance of cells resistant to 6-thioguanine (6-TG) and can be (quantified by comparison of the numbers of 6-TG-resistant colonies in the treated and control cultures. To ensure that any mutagenic effect of metabolites of the test substance found in mammals is also detected, a parallel series of experiments is performed, in which its metabolic turnover is simulated in vitro by the addition of an activation mixture containing rat-liver microsomes and co-factors to the cell cultures.

The test item was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The cells were treated in the experiments with microsomal activation for 5 hours and in the experiment without microsomal activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment).

Mutagenicity test with microsomal activation

The original experiment was performed at the following concentrations: 25, 50, 100, 200, 300, 400 and 500 µg/mL. Because the intended toxicity was not obtained in the original experiment, in the confirmatory experiment the concentrations applied were increased to 50, 100, 200, 400, 600, 800 and 1000 µg/mL. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant deviations of the mutant frequencies as determined by the screening with 6-TG.

Mutagenicity test without microsomal activation

Both, the original and the confirmatory experiment were performed at concentrations of 50, 100, 200, 400, 600, 800 and 1000 µg/mL. In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no significant deviations of the mutant frequencies as determined by the screening with 6-TG.

In both investigations with and without microsomal activation, a mutant factor greater than 3.0 together with a difference in the treated and untreated dishes of at least 20 clones per 10e6 cells plated was not detected and there was no indication of a concentration mutant-frequency relation in any experiment.

in vivo:

Cytogenicity in vivo (read-across to the structural analogon 4,6-bis(octylthiomethyl)-o-cresol):

In a GLP conform Micronucleus Test according to OECD guideline 474, the test item was administered by gavage to Chinese hamsters (Ciba-Geigy Ltd. 861249, 1987). In this experiment the animals were treated once with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. The experiment was performed to evaluate any mutagenic effect on polychromatic erythrocytes in bone marrow cells in vivo.

The bone marrow smears from the animals treated with the dose of 5000 mg/kg showed no statistically significant increase (p >0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 1.73% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.16%) treated with the vehicle (arachis oil) alone.

It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item.

Short description of key information:
in vitro: Ames Test: negative; CIBA 703300, 2001, OECD Guideline Study
Chromosome abberation assay: negative; CIBA 745900, 2002, OECD Guideline Study
Since there is no data on gene mutation in vitro for the substance (EC 438-600-3) available, a read-across to the structural analogon 4,6-bis(octylthiomethyl)-o-cresol has been conducted:
Gene mutation in mammalian cells, HPRT assay, V79 cells, with and without metabolic activation: negative (GLP, OECD 476; Ciba-Geigy 894550, 1990)

in vivo: A read-across to 4,6-bis(octylthiomethyl)-o-cresol was also conducted regarding genetic toxicity in vivo:
Micronucleus test, Chinese hamster: negative (GLP, OECD 474; Ciba-Geigy 861249, 1987)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

There is no indication given to classify the test substance for its mutagenic potential according to requirements of the EU Annex VI of directive 67/548/EEC and EC/1272/2008 as amended for the second time in Directive EC 286/2011 as well as to UN-GHS, respectively.