Indeno[4,3a-b]furan, decahydro-2,2,7,7,8,9,9-heptamethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames,+/- S9, negative, Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2 uvrA), (OECD TG 471, GLP).

- In vitro chromosome aberration study, +/- S9, negative, Chinese Hamster Lung (CHL) cells, (OECD TG 473, GLP).

- In vitro gene mutation assay, +/- S9, negative, mouse lymphoma L5178Y cells TK+/-, (OECD TG 490, GLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- In vivo mammalian erythrocyte micronucleus test, negative, micronucleated polychromatic erythrocytes in mice, (OECD TG 474, GLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames - OECD TG 471

A Bacterial Reverse Mutation Test (Ames) was performed in accordance with OECD TG 471 and under GLP conditions to determine the mutagenic potential of the test substance. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9-mix). The dose range of the main test was determined in a preliminary toxicity assay using TA100 and WP2uvrA- and was determined to be 50 to 5000 µg/plate. Both main tests were performed at 50, 150, 500, 1500, and 5000 µg/plate. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test substance caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test substance was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test substance precipitate was observed on the plates at any of the doses tested in either the presence or absence of the S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test substance, either with or without metabolic activation. Based on the observed results, the test substance was considered to be non-mutagenic under the conditions of this test.

 

In vitro chromosome aberrations - OECD TG 473

An in vitro Chromosome Aberration study was conducted in accordance with OECD TG 473 and under GLP conditions. Duplicate cultures of Chinese Hamster Lung (CHL) cells were treated with the test substance at several dose levels, together with vehicle and positive controls. Four treatment regimens were used: a 6(18)-hours exposure, both with and without the addition of an induced rat liver homogenate metabolising system (5 % S9-mix) in Experiment 1; Experiment 2 included a 24-hourcontinuous exposure in the absence of S9 -mix and a repeat of the 6(18)-hours exposure with a 1 % final concentration of S9 -mix. The dose levels used were selected on the basis of molecular weight, solubility and the results of a preliminary toxicity test. The dose range for the Experiment 1 6(18)-hour exposures, both with and without the S9 -mix, was 0.63 to 80 µg/mL, 0.63 to 10 µg/mL for the 24-hour exposure group and 2.5 to 60 µg/mL for the with S9 -mix (1%) exposure group. Results showed that the vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for the CHL cell line. Furthermore, all of the positive control materials induced highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any significant increases in the frequency of cells with aberrations in any of the exposure groups. The dose levels of the test substance were shown to be toxic to CHL cells in vitro and optimal levels of toxicity were achieved in all exposure groups. The test substance did not induce any biological significant, dose-related increases in the frequency of cells with structural or numerical chromosome aberrations either in the presence or absence of the S-9 mix or after various exposure times. The test substance can be considered non-clastogenic to CHL cells in vitro, under the conditions of this test.

 

In vitro gene mutations in mammalian cells - OECD TG 490

An in vitro gene mutation study in mammalian cells was performed according to OECD TG 490 and in compliance with GLP to evaluate the mutagenic potential of the test substance by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions. The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. The study procedures described in this report were based on the most recent OECD guideline. In the first experiment, the test substance was tested up to concentrations of 30 and 100μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 10 and 17 % in the absence and presence of S9-mix, respectively. In the second experiment, the test substance was tested up to concentrations of 40 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 11 %. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95 % control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95 % control limits of the distribution of the historical positive control database. It was therefore, concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, the test substance did not induce a biologically relevant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the test substance did not induce a biologically relevant increase in the mutation frequency. The test substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

 

In vivo Mammalian Erythrocyte Micronucleus test - OECD TG 474

An in vivo Mammalian Erythrocyte Micronucleus test was performed according to OECD TG 474 and under GLP conditions. The study was performed to assess the potential of the test substance to produce damage to chromosomes or aneuploidy when administered to mice. A range-finding test was performed to find suitable dose levels of the test substance, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test substance toxicity between the sexes; therefore, the main test was performed using only male mice. With no evidence of any toxicity with the test substance via either route of administration the micronucleus test was conducted using the intraperitoneal route to maximise exposure in groups of 7 mice (males) at the maximum recommended dose (2000 mg/kg) only. Animals were killed 24 or 48 hours later, the bone marrow was extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test substance dose groups when compared to their concurrent control groups. However, in both instances marked reductions were observed and this was taken to indicate that systemic absorption had occurred. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test substance when compared to the concurrent vehicle control groups. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. Based on the results of the test, the test substance was considered to be non-genotoxic under the conditions of the test.

Justification for classification or non-classification

Based on the results of the gene mutations in bacterial cells (Ames test, OECD TG 471), in vitro chromosome aberration test (OECD 473), in vitro gene mutations in mammalian cells (OECD TG 490) and the in vivo mammalian erythrocyte micronucleus test (OECD TG 474)  the substance is not genotoxic and therefore, is not classified for genotoxicity in accordance with EU CLP (EC no. 1272/2008 and its amendments).