2-Propenenitrile, reaction products with 3-amino-1,5,5-trimethylcyclohexanemethanamine

Endpoint summary

Additional information

A bacterial reverse mutation assay was performed to test the potential of test substance to cause gene mutation as per OECD Guideline 471, EPA OPPTS 870.6100 and EU Method B.13/14, in compliance with GLP. The study included a preliminary compatibility test, a preliminary concentration range finding test, an initial mutation test and a confirmatory mutation test. In the preliminary concentration range finding test as well as in the initial mutation test, the plate incorporation method was used. In the confirmatory mutation test, the pre-incubation method was used. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli WP2 uvrA in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/b-naphthoflavone-induced rats. Based on the results of the preliminary experiment, the examined test concentrations in the main tests were 5,000, 1,581, 500, 158.1, 50, 15.81 and 5 μg/plate with and without metabolic activation. The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test substance treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. In the initial mutation test, the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 1,581μg/plate concentration with metabolic activation (the mutation factor value was 1.57). However, there was no clear dose response, the observed mutation factor value was well below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range. Furthermore, higher number of revertant colonies compared to the distilled water control were detected for untreated control (MF: 1.57) also in this strain with metabolic activation, indicating a higher than usual variability in this case. In the confirmatory mutation test, the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 1,581 μg/plate concentration with metabolic activation. The observed mutation factor value was 1.52. However, no clear dose-dependent relationship was observed, and the observed mutation factor value was well below the biologically relevant threshold value. The number of revertant colonies was within the historical control range. Inhibitory, cytotoxic effect of the test substance was seen in the confirmatory mutation test in all examined bacterial strains at 5,000μg/plate concentration with and without metabolic activation. The evaluation criteria was satisfied for the study. No mutagenic effect of test substance was observed either in the presence or absence of metabolic activation system under the conditions of this bacterial reverse mutation assay (Hargitai J, 2014).

Anin vitromammalian cell assay was performed in CHO K1 Chinese hamster ovary cells at the hprt locus to test the potential of test substance to cause gene mutation as per OECD Guideline 476 and EU Method B.17, in compliance with GLP. Treatments were carried out for 5 h with and without metabolic activation (±S9-mix) and for 24 h without metabolic activation (-S9-mix). Treatment concentrations up to 2,200 μg/mL for the mutation assay were selected for the main tests based on the results of a preliminary toxicity test. In the main assays, a measurement of the survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of the 7 d expression period following the treatment) and mutagenicity (colony forming ability at the end of the 7 d expression period following the treatment, in the presence of 6-Thioguanine as a selective agent) were determined. In assay 1 with and without metabolic activation and assay 2 without metabolic activation, no statistically significant increase in the mutant frequency was observed in presence of cytotoxicity. There was no dose response to the treatment. In Assay 2, in the presence of S9-mix (5-h treatment), excessive cytotoxicity of the test substance was observed at 2,200, 2,000 and 1,800 μg/mL concentrations. Slight, statistically significant increase was observed in this experiment at 500 μg/mL concentration, although the observed mutation frequency was within the historical control range and there was no dose response to the treatment (a trend analysis showed no effect of treatment). Furthermore, the increase was not significant when compared to the untreated control sample. Thus, it was considered as being within the normal biological variability of the test system. The evaluation criteria were satisfied for the study. No mutagenic effect of test substance was observed either in the presence or absence of metabolic activation system under the conditions of this HPRT assay (Hargitai J, 2015).

A study was conducted to evaluate the potential of the test substance to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-, as per OECD Guideline 487, in compliance with GLP. Based on the results of a preliminary experiment, the main assay was conducted in the form of two experiments. Experiment 1 consisted of concentrations of 31, 63, 125, 250, 375, 500 and 1,000 μg/mL for 3 h treatment and 24 h recovery without S9 mix, and concentrations of 31, 63, 125, 250, 375, 500 and 1,000 μg/mL for the 3 h treatment and 24 h recovery with S9 mix. Experiment 2 consisted of concentrations of 15.6, 31.3, 62.5, 125, 250, 375 and 500 μg/mL for the 24 h treatment and 20 h recovery, without S9 mix and concentrations of 62.5, 125, 250, 500, 750, 1,000 and 2,000 μg/mL for the 3 h treatment and 24 h recovery with S-9 mix. Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (i. e., population doubling) of cells which is based on the starting cell count and the final cell count at the time of harvesting. For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test substance, for the vehicle and the positive controls, in 1,000 mononucleated cells per culture (total of 2,000 mononucleated cells per dose). Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture. No precipitate was observed in the culture medium at the end of the treatment periods in any of the experiments. No significant increase in the frequency of micronucleated cells was noted in either experiment. All acceptance criteria were met. The study was therefore considered to be valid. The test substance did not induce any chromosome damage or damage to the cell division apparatus in cultured L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system (Sarlang S, 2015).

Justification for selection of genetic toxicity endpoint
Guideline-compliant study conducted according to GLP

Short description of key information:
The test substance was not mutagenic or clastogenic in bacterial and mammalian cell lines.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of in vitro genotoxicity testing, the substance does not require classification for this endpoint according to the EU CLP criteria (EC 1272/2008).