5H-Cyclopenta[h]quinazoline, 6,7,8,9-tetrahydro-7,7,8,9,9-pentamethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- The substance is negative in the Ames test (OECD TG 471)

- The substance did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments (according to OECD TG 473). The substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.

- The substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic according to OECD TG 490.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test (OECD TG 471): The in vitro gene mutation study in bacteria was conducted according to OECD TG 471 and GLP principles. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the substance, using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day (pre-incubation method) using a similar dose range to the range-finding test, fresh cultures of the bacterial strains and fresh substance formulations. Additional dose levels and an expanded dose range were selected in order to achieve both four non-toxic dose levels and the toxic limit of the substance. Results: The vehicle (dimethyl formamide) 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the range-finding test (plate incorporation methodology), the substance caused a substantial reduction in the frequency of revertant colonies of bacterial strains TA1535, TA98 and TA1537 (presence and absence of S9-mix) and WP2uvrA (absence of S9-mix only), initially from 500 µg/plate. These responses were not accompanied by any visible reduction in the growth of the bacterial background lawns. No toxicity was noted for bacterial strains TA100 (absence and presence of S9-mix) or WP2uvrA (presence of S9-mix). In the main test (pre-incubation methodology), the substance caused both a substantial reduction in the frequency of revertant colonies and/or a visible reduction in the growth of the bacterial background lawns of all of the tester strains except WP2uvrA (presence of S9-mix). Weakened bacterial background lawns were initially noted at 50 µg/plate for TA1535 and TA1537 (absence of S9-mix). The sensitivity of the bacterial tester strains to the toxicity of the substance varied between strain type, exposures with or without S9-mix and experimental methodology. A substance precipitate (oily in appearance) was observed at 5000 µg/plate. This observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the substance, either with or without metabolic activation or exposure method. Conclusion: The substance was considered to be non-mutagenic under the conditions of this test.

Chromosomal aberration (OECD TG 473): This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al., 1990). This test is conducted according to OECD TG 473 and GLP principles. Duplicate cultures of human lymphocytes, treated with the substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; i.e. in Experiment 1, 4-hours in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2 % final concentration with cell harvest after a 20-hour expression period and a 4-hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4-hours exposure with addition of S9 was repeated (using a 1 % final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows: The final concentration of the substance was 4, 8, 16, 24, 32, 48, 64 µg/mL for a 4-hour treatment and 20-hour recovery period (without S9). The final concentration of the substance was 8, 16, 32, 48, 64, 80 µg/mL for a 4-hour treatment and 20-hour recovery period (with S9 2%). The final concentration of the substance was 4, 8, 16, 24, 32, 48 µg/mL for a 24-hour treatment without S9. The final concentration of the substance 4, 8, 16, 32, 40, 48, 56, 64 µg/mL for a 4-hour treatment and 20-hour recovery period (with S9 1%). Results showed that all vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The substance did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50 % mitotic inhibition or greater. Conclusion: The substance was considered to be non-clastogenic to human lymphocytes in vitro.

Gene mutations in mammalian cells (OECD TG 490): The study was conducted according to a method that was designed to assess the potential mutagenicity of the substance on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. This study was performed according to OECD TG 90 and GLP principles. Two main mutagenicity tests were performed. In these main tests, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the substance at eight dose levels in duplicate, together with solvent (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9). The dose range of the substance used in the second main test was selected following the results of a preliminary toxicity test and the first experiment where optimum toxicity was not achieved. The dose levels plated for cloning efficiency and expression of mutant colonies were as follow for the exposure groups: 4, 8, 16, 20, 24, 28, 30 µg/mL (without S9) and 4, 8, 16, 32, 36, 40, 44 (with S9 2%). Results: The maximum dose level used was limited by a substance-induced toxicity. No precipitate of the substance was observed at any of the dose levels in either the absence or presence of metabolic activation, at the end of the exposure period. The solvent control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The substance did not induce any increases in the mutant frequency at any of the dose levels in the main test that exceeded the Global Evaluation Factor (GEF), using a dose range that achieved optimum levels of toxicity in both of the exposure groups, and at least four analysable dose levels in each exposure group. In conclusion, the substance did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay. ·    

Justification for classification or non-classification

Based on the results of the genemutations in bacterial cells (Ames test, OECD TG 471), gene mutations in mammalian cells (OECD TG 473 and OECD TG 490) the substance is not genotoxic and therefore does not have to be classified for genotoxicity in accordance with EU CLP (EC no. 1272/2008 and its amendments).