2-(2H-benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, AMES: negative
OECD 473, CA: negative
OECD 476, HPRT : negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro studies

AMES:

In a reverse gene mutation assay conducted according to OECD guideline 471, the bacteria strains (TA 100, TA 1535, TA 1537 and TA 98) of S. typhimurium and E. coli WP2 uvrA were exposed to the 2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol (>99% pure) in DMSO at concentrations of 625, 1250, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation in a plate incorporation test.

The test substance was tested up to precipitating and cytotoxic (TA 98, 5000 µg/plate) concentrations. Only one experiment with duplicates was performed. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in this test.

Chromosomal Aberration:

An in vitro chromosomal aberration test according to OECD guideline 473 and GLP was conducted with 2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol in Chinese hamster lung fibroblasts (V79 cells).

Cells were incubated for 4 and 18 hours with 12.5, 25 and 50 µg/mL test substance in the absence and presence of metabolic activation. Precipitation was observed from 25 µg/mL on and higher. The cells were analyzed after 18 hours (about 1.5-fold of the normal cell cycle time) and 28 hours (more than 1.5-fold of the normal cell cycle time) incubation time, covering the intervals at which maximum aberration frequencies were expected.

Ethylmethanesulfonate and cyclophosphamide were used as positive controls without and with metabolic activation, respectively and were found to induce chromosomal aberrations as expected.

According to the results of the present in vitro cytogenetic study, the test substance 2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol did not lead to a relevant increase in the number of structural chromosomal aberrations including and excluding gaps either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other selecting different exposure times (4 and 18 hours) and sampling times (18 and 28 hours). The types and frequencies of structural chromosome aberrations were close to or within the range of the concurrent negative and vehicle control values at both sampling times and clearly within in the range of the historical negative control data. Additionally, no increase in the number of cells containing numerical chromosomal aberrations was observed in the absence and the presence of metabolic activation.

Thus, under the experimental conditions chosen in this study the test substance is not a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.

  

HPRT Test:

An in vitro cell gene mutation test according to OECD guideline 476 and GLP was conducted with 2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol using Chinese hamster ovary cells (CHO).

Cells were incubated for 4 hours with metabolic activation and for 4 and 24 hours without metabolic activation. In the presence of S9 mix no cytotoxicity was observed but precipitation was visible starting at 50 µg/mL test substance and above. Without S9 mix strong decreases in the number of colonies could be seen in Experiment I starting at 3.13 µg/mL after 4 hours and at 100 µg/mL after 24 hours exposure in Experiment II. Only cultures at concentrations below were chosen for evaluation. Precipitation did not occur in Experiment I without metabolic activation but was seen at 12.5 µg/mL and above in Experiment II.

In both experiments, in the absence of metabolic activation, at least the highest concentrations tested for gene mutations were clearly cytotoxic. In contrast, in the presence of metabolic activation, no cytotoxicity was observed up to the highest concentration evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two independent experiments.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, Ethylmethanesulfonate and 7,12-Dimethylbenzanthracene, led to the expected increase in the frequencies of forward mutations.

Thus, under the experimental conditions of this study, the test substance 2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and presence of metabolic activation.

In conclusion, 2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol was found not to be mutagenic in vitro in a bacterial reverse mutation assay and in a chromosomal aberration and a gene mutation test in mammalian cell culture.

Justification for classification or non-classification

Based on the available in vitro data and according to CLP Regulation (EU) 1272/2008 and Directive 67/548/EEC, respectively,

2-(2H-Benzotriazol-2-yl)-4-(1,1,3,3-tetramethylbutyl)phenol should not be classified for mutagenicity.