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In-vitro study: Bacterial systems

The test substance sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate was tested in the Salmonella typhimurium reverse mutation assay according to OECD Guideline 471 and EU method B.14. Five strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) were exposed to the test substance at concentrations ranging from 10 - 5000 µg/plate with or/and without S9 mix. Toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in some test groups with and without metabolic activation at the highest investigated concentration. Up to the highest investigated concentration, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings. The positive and solvent controls were valid. Under the conditions of this assay, the test substance revealed no indication for a mutagenic effect in both the presence and absence of S9 mix, in all five strains of Salmonella typhimurium used.

In-vitro study: Mammalian cell gene mutation test

An in vitro mammalian cell assay was performed according to OECD Guideline 476. The test article sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate was assessed for its potential to induce point mutations at the HGPRT locus using V79 cells of the Chinese hamster in vitro. The study was performed in four independent experiments, using identical procedures, each with and without liver microsomal activation. The test article was tested with the following concentrations:

Exp. I and II with and without S9 mix; 10.0; 30.0; 60.0 and 100.0 µg/mL; Exp. III with and without S9 mix; 80.0 µg/mL; Exp. IV with and without S9 mix: 45.0 and 70.0 µg/mL.

According to the pre-experiment for toxicity the concentration ranges were selected to yield concentration related toxic effects. The highest concentration caused a low level of survival (13.2 % without metabolic activation and 22. 7 % with metabolic activation) and the survival at the lowest concentration was approximate in the range of the negative control. Up to the highest investigated dose no relevant increase in mutant colony numbers was obtained in two independent experiments . Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test article did not induce point mutations at the HGPRT locus in V79 cells.

In-vitro study: Chromosome aberration test

An in vitro chromosome aberration test was performed according to OECD Guideline 473 and EU method B.10. The test article sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese Hamster in vitro. Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment with the test article. The treatment interval was 4 h. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following dose levels were evaluated: without S9 mix: 7 h: 110 µg/mL; 18 h: 8; 80; 110 µg/mL; 28 h: 110.00 µg/mL. With S9 mix: 7 h: 130 µg/mL; 18 h: 10; 50; 120 µg/mL; 28 h: 130 µg/mL. Treatment of the cells with 110 µg/mL (without S9 mix) and 130 µg/mL (with S9 mix) reduced clearly the plating efficiency of the V79 cells. Also the mitotic index was reduced with the highest concentration at any fixation interval in the presence and absence of S9 mix. There were significant enhancements of cells with structural aberrations after treatment with the test article at any fixation interval with metabolic activation by S9 mix. Without metabolic activation only at fixation interval 18 h a significant enhancement of the aberration rate could be observed.

Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate was concluded to be positive in the presence and absence of metabolic activation in the V79 cytogenetic assay.

In-vivo study: Mouse micronucleus assay

The test article sodium 3 -(2H-benzotriazol-2 -yl)-5 -sec-butyl-4 -hydroxybenzenesulfonate was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The assay was performed according to OECD Guideline 474 and EU method B.12. The test article was suspended in 0.5 % Methocel solution. This suspending agent was used as negative control. The volume administered orally was 20 ml/kg bw 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 3000 mg/kg bw. In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

In-vivo study: Unscheduled DNA Synthesis

The test article sodium 3 -(2H-benzotriazol-2 -yl)-5 -sec-butyl-4 -hydroxybenzenesulfonate was assessed in the in vivo UDS assay for its potential to induce DNA repair (UDS) in the hepatocytes of rats. The assay was performed according to OECD Guideline 486 and EU method B.39. The test article was formulated in 1% carboxymethylcellulose suspension (1% CMC). 1% CMC was used as negative control. The volume administered orally was 10 mL/kg bw. After a treatment period of 12 - 14 hours the animals were narcotized and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to 3HTdR which is incorporated if UDS occurs. The test article was tested at the following dose levels: 100; 330; and 1000 mg/kg bw. For each dose level, including the controls, hepatocytes from three treated animals were assessed for the occurrence of UDS. Up to the highest dose level tested no animal expressed toxic reactions. No dose level of the test article revealed UDS induction in the hepatocytes of the treated animals as compared to the current negative controls. Under the conditions of this UDS test, sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate did not induce DNA-damage leading to repair synthesis in the hepatocytes of the treated rats.


Short description of key information:
Sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzene did not reveal any indication for mutagenicity in the Ames test or in a gene mutation test in cultured mammalian cells (HGPRT assay). In an in vitro chromosomal aberration test in cultured V79 cells, positive effects were noted in the presence and absence of metabolic activation. However, in an in vivo mouse micronucleus assay the test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice. In addition in an UDS assay in vivo the test article did not induce DNA-damage leading to repair synthesis in the hepatocytes of treated rats. In conclusion, the test substance is not considered to be mutagenic in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available results of the three different in vitro and two vivo genetic toxicity studies, sodium 3-(2H-benzotriazol-2-yl)-5-sec-butyl-4-hydroxybenzenesulfonate has not to be classified and labelled as genotoxic according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).