Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames (OECD 471): positive with S. typhimurium TA 100 and TA 1535 without metabolic activation, negative with TA 98, TA 102 and TA 1537 with and without metabolic activation (1999); positive with S. typhimurium TA 1535 without metabolic activation, negative with TA 98, TA 100, TA 102 and TA 1537 with and without metabolic activation (2001)

HPRT (OECD 476): negative in V79 cells with and without metabolic activation

Micronucleus test (OECD 487): negative in cultured human lymphocytes with and without metabolic activation

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2001). In two independent experiments, the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were exposed to the test substance using the plate incorporation method. Based on the results of a pre-experiment, test substance concentrations of 15 to 5000 µg/plate were selected for the incubation with and without metabolic activation in the first experiment. In the second experiment, concentrations of 50 to 5000 µg/plate were selected for the incubation with and without metabolic activation. No precipitation occurred up to the highest investigated dose. In the absence and presence of metabolic activation the test substance was bacteriotoxic towards the strain TA102 at 1500 and 5000 µg/plate, respectively. In the concentration range investigated the test substance induced a significant and dose related increase in the mutation frequency of the tester strain TA1535 in the absence of metabolic activation. The results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system. Under the conditions of this experiment, the test substance was mutagenic in the S. typhimurium strain TA1535 without metabolic activation.

A second bacterial gene mutation assay was performed with the test substance according to OECD Guideline 471 and in compliance with GLP (1999). In two independent experiments, the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were exposed to the test substance using the plate incorporation method. Based on the results of a pre-experiment, test substance concentrations of 50 to 5000 µg/plate were selected for the incubation with and without metabolic activation in the first experiment. In the second experiment, concentrations of 15 to 5000 µg/plate were selected for the incubation with and without metabolic activation. No precipitation occurred up to the highest investigated dose. The test substance was bacteriotoxic towards the strains TA98, TA102 and TA1537 at 5000 µg/plate without metabolic activation and towards the strain TA1537 at 1500 µg/plate and towards the strains TA98, TA100 and TA102 at 5000 µg/plate with metabolic activation. In the concentration range investigated the test substance induced a dose related increase in the mutation frequency of the tester strains TA100 and TA1535 without metabolic activation. The increase in the mutation frequency of strain TA1535 reached a significant level in both experiments. However, with TA100 significant values were measured only in one experiment. The test substance did not induce a significant increase in the mutation frequency of any of the tester strains with metabolic activation. The results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system. Under the conditions of this experiment, the test substance was mutagenic in the S. typhimurium strains TA100 and TA1535 without metabolic activation.

Increases in mutation frequencies of strain TA 1535 in the first study (2001) and of strain TA 100 in the second study (1999) are low (less than doubled) compared to the control and results of TA 100 were not reproducible in the second experiment. Since statistical significance should not be the only determining factor for interpretation of results and reproducibility within experiment is not given, positive results for strains TA 1535 and TA 100 of both studies (2001 and 1999) are considered to have no biological relevance.

Gene mutation in mammalian cells

The mutagenic activity of the test substance was evaluated in an in vitro mammalian cell gene mutation test according to OECD Guideline 476 and in compliance with GLP (2002). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. Based on the results of a preliminary test, cells were exposed for 3.5 h with and without metabolic activation up to concentrations of 10 mM in Experiment 1. In a second independent experiment, cells were exposed for 3.5 h with and 24 h without metabolic activation up to concentrations of 10 mM. Cytotoxicity was determined by measuring the relative colony forming ability and/or relative total growth. Cytotoxic effects were observed at 10 mM following 24 h treatment without metabolic activation and beginning at 3 mM and higher following 3.5 h treatment with metabolic activation. No significant increase in the number of mutant colonies was observed up to the maximum concentration of 10 mM in the presence and absence of metabolic activation. Appropriate reference mutagens, used as positive controls, confirmed the efficacy of the test system as well as the full activity of the metabolizing system. In conclusion, the test substance did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

 

Cytogenicity in mammalian cells

The potential of the test substance to induce mirconuclei was investigated in an in vitro mammalian cell micronucleus test in cultured human lymphocytes performed according to OECD Guideline 487 and GLP (2015). A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cells were incubated for 4 h with and without metabolic activation with test substance concentrations ranging from 10.8 to 1662 µg/mL (approx. 10 mM). Since the cultures fulfilled the requirements for cytogenetic evaluation, the preliminary test was designated Experiment 1. In Experiment 2, cells were incubated for 20 h without metabolic activation up to 1662 µg/mL. The cells were prepared 40 h after start of treatment. In each experimental group two parallel cultures were analysed and at least 1000 binucleate cells per culture were evaluated for cytogenetic damage. No precipitation of the test substance in the culture medium was observed. Phase separation was observed in Experiment 1 and 2 in the absence of metabolic activation at 1662 µg/mL and in Experiment 1 in the presence of metabolic activation at 949.7 µg/mL and above at the end of treatment. No relevant influence of the test substance on osmolarity or pH value was observed. In Experiment 1 in the absence and presence of metabolic activation, no cytotoxicity was observed up to the highest applied concentration. In Experiment 2 in the absence of metabolic activation, no clear cytotoxicity occurred up to the highest applied concentration. However, cytostasis of 47.9% was observed at the highest applied and evaluated concentration. No relevant increase in the number of micronucleated cells was observed after treatment with the test substance in both experiments with and without metabolic activation. However, in Experiment 2 one single statistical significant increase in micronucleate cells was observed at the highest evaluated concentration of 1662 µg/mL. Since the value was within the range of the historical control the finding was considered as biologically irrelevant. Mitomycin C, demecolcin and cyclophosphamide were used as positive controls and induced statistically significant increases in cells with micronuclei. In conclusion, the test substance did not induce micronuclei in the in vitro micronucleus test in human lymphocytes under the experimental conditions reported. Therefore, the test substance is considered to be non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to phase separating concentrations.


Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.