Reaction mass of (2R*,4R*,4aR*,9bS*)-2,4-dimethyl-4,4a,5,9b-tetrahydroindeno[1,2-d][1,3]dioxine and (2R*,4R*,4aS*,9bR*)-2,4-dimethyl-4,4a,5,9b-tetrahydroindeno[1,2-d][1,3]dioxine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

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

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

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 TA 98, TA 100, TA 102, TA 1535 and TA 1537 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 metabolic activation and of 5 to 5000 µg/plate for the incubation 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. In the absence of metabolic activation the test substance was bacteriotoxic towards the strains TA102 at 1500 µg/plate and towards the strains TA1537 and TA100 at 5000 µg/plate. In the presence of metabolic activation the test substance was bacteriotoxic towards the strains TA1535, TA1537, TA100, and TA102 at 5000 µg/plate. In the concentration range investigated, the test substance did not induce a significant increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. The negative and positive control data were between the minimum and maximum value of the historical control data of the laboratory. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains in the presence and absence of metabolic activation.

 

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 (2015). The test substance was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. Based on the results of a pre-test, cells were exposed for 4 and 24 h to the test substance up to concentrations of 1020 µg/mL in the absence and presence of metabolic activation. No or moderate cytotoxic effects indicated by a relative cloning efficiency or cell density below 50% occurred in the first experiment at the highest analyzable concentration of 510 µg/mL with and without metabolic activation. Although the next higher concentration was closely spaced, exceedingly severe cytotoxic effects did not leave enough viable cells for mutation frequency analysis at 765 µg/mL and above. In the second experiment relevant cytotoxicity was noted at the maximum analyzable concentration of 500 µg/mL with and without metabolic activation. Again, exceedingly severe cytotoxicity occurred at the next higher concentration of 575 µg/mL with and 750µg/L without metabolic activation. No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration of 510 µg/mL. The mutant frequency generally did not exceed the historical range of solvent controls. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation 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 (2014). The test substance was dissolved in DMSO and cytotoxicity of the test substance was investigated in a preliminary test. Cells were incubated for 4 h with and without metabolic activation up to 2043 µg/mL (approx. 10 mM). Since the cultures fulfilled the requirements for cytogenetic evaluation with S9 mix, this preliminary test was designated Experiment IA. The experimental part without metabolic activation was repeated with a top dose of 2000 µg/mL and narrow concentration spacing (Experiment IB). Additionally, two further independent experiments (Experiment IIA and IIB) were performed. In Experiment IIA, cells were incubated for 4 h with S9 mix and 20 h without S9 mix and in Experiment IIB, cells were incubated for 20 h without S9 mix. 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. In the absence and presence of S9 mix, concentrations showing clear cytotoxic effects were not evaluable for cytogenetic damage, except for Experiment IIB in the absence of S9 mix, where clear cytotoxicity was observed at the highest evaluated concentration (600 µg/mL). No relevant increase in the number of micronucleated cells was observed after treatment with the test substance. However, in Experiment IB in the absence of S9 mix increases above the range of the historical solvent control values were observed after treatment with 300 and 600 µg/mL. The values were not statistically significant and no dose-dependency was observed. In Experiment IIA and IIB in the absence of S9 mix single statistically significant increases within the range of the historical solvent control values were observed after treatment with 257.2 and 600 µg/mL, respectively. The findings were regarded 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 cytotoxic or the highest evaluable concentration.

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, as amended for the eighteenth time in Regulation (EU) 2022/692, and are therefore conclusive but not sufficient for classification.