Amides, tall-oil fatty, N,N-di-Me

Endpoint summary

Additional information

Ames test

The potential for DMATO to induce gene mutations in the bacterial reverse mutation assay was evaluated in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and in Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without metabolic activation (S9 mix). The following concentrations were tested in Experiment I (plate incorporation test): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate. The following concentrations were tested in Experiment II (pre-incubation test): 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate. Appropriate negative, solvent and positive controls were tested. All test solution concentrations and controls were tested in triplicate. The plates incubated with the test material showed normal background growth up to 5000 µg/plate with and without metabolic activation in both experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. Minor toxic effects were observed only in the presence of metabolic activation in strains TA 1535 and TA 1537 in Experiment I at 5000 µg/plate and in Experiment II in strain TA 1535 at 1000 and 2500 µg/plate. No substantial increase in revertant colony numbers of any of the five tester strains was observed at any dose level, in the presence or absence of metabolic activation. There was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The test material did not induce gene mutations by base pair changes or frameshifts under the experimental conditions. DMATO is therefore considered to be non-mutagenic in the bacteria reverse mutation assay

Mouse lymphoma assay

The genotoxic potential of DMATO was evaluated in a mammalian cell gene mutation assay, according to OECD 476 and GLP. The assay determined whether the test substance induces mutations at the mouse lymphoma thymidine kinase locus using the L5178Y cell line. The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation (S9 mix) and a treatment period of 4 h. In the second experiment the cells were treated with the test item for 4 hours with and for 24 hours without metabolic activation. The highest concentration of the pre-experiment (4200 μg/mL) was chosen with regard to the solubility properties of the test item in DMSO and aqueous medium. The concentration range of the main experiments was limited by the cytotoxicity of the test item. Test concentrations chosen for mutation rate analysis were as follows: Experiment 1 without S9 mix: 1.9, 3.8, 7.5, 15.0 and 30.0 µg/mL; with S9 mix: 7.5, 15.0, 30.0, 45.0 and 60.0 µg/mL.

Experiment 2 without S9 mix: 1.9, 3.8, 7.5, 15.0 and 30.0 µg/mL; with S9 mix: 3.8, 7.5, 15.0, 30.0 and 60.0 µg/mL.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were valid. It is concluded that under the conditions of the study, DMATO did not induce mutations at the thymidine kinase locus in L5178Y cells in the presence and absence of metabolic activation. DMATO is therefore considered to be non-mutagenic in this assay.

Chromosomal aberration assay

The test item DMATO, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in three independent experiments. In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment IIA, in the absence of S9 mix, where only 50 metaphases were evaluated. The highest applied concentration in this study (5000.0 µg/mL of the test item) was chosen with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473.

In the absence of S9 mix, cytotoxicity was observed at the highest evaluated concentrations. In the presence of S9 mix, concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. No clastogenicity was observed at the concentrations evaluated in the presence of S9 mix. In Experiment IIA in the absence of S9 mix one statistically significant increase (3.5 % aberrant cells, excluding gaps) was observed after continuous treatment with 49.0 µg/mL being slightly above the range of the historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps). No dose-dependency was observed. In Experiment IIB this finding could not be confirmed. However, one statistically significant increase was observed after treatment with 40.0 µg/mL (2.0 % aberrant cells, excluding gaps). This value is within the range of the historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and therefore not biologically relevant. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, DMATO is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest evaluable concentrations.

 

Justification for selection of genetic toxicity endpoint
The in vitro battery of three genotoxicity studies are presented as weight of evidence approach. each of the three studies are reliable and valid as key studies but no individual key study is sufficient to address the genetic toxicity endpoint but the three key studies together provide sufficient weight of evidence to establish no adverse genotoxic response.

Short description of key information:
Studies of reverse mutation in bacteria (Ames test), forward mutation in mammalian cells (mouse lymphoma assay) and clastogenicity in mammalian cells are available for the submission substance.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification for genotoxicity is not triggered by the negative results of the available in vitro studies.