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Key value for chemical safety assessment

Additional information

Ames

The aim of the study was to investigate the potential of the test item to induce gene mutations in S. typhimurium with and without metabolic activation. The study was performed according to OECD 471, adopted in 1983. The test item was dissolved in DMSO. The dose range was determined in a preliminary toxicity assay using 50 to 5000 µg/ plate. Cytotoxicity was excluded according to normal growth. For the main test, concentrations ranging from 4 µg to 1000 µg/ plate using the direct plate incorporation method with and without external metabolisation (S9- mix) were used. The bacterial strains S. typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included and remained within the historical range. An independent repetition of the experiment was performed. No toxicity of the test substance to the bacteria was observed up to 1000 µg/plate. A precipitate was visible when the test substance was mixed with the agar at the 1000 and 333 µg/ plate samples. The precipitate was still visible when the colonies were counted and impeded the counting of the 1000 µg/ plate samples. In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results. Under the selected test conditions the test item is considered to be non-mutagenic in the Ames test neither with nor without metabolic activation.

Chromosomal aberration

The aim of the study was to investigate the DNA damaging potential of the test item with and without metabolic activation using isolated human lymphocytes. The study was performed according to OECD 473, adopted in 1987. Four experiments were performed: two of them without and two with metabolic activation (microsomes of Aroclor induced rats). After 48 hours of incubation the test item was added. In both experiments with metabolic activation and in one experiment without metabolic activation system the test item was washed out after a 3 hour exposure time and the cultures were cultivated for another 17 hours. In one experiment without metabolic activation the cultures were treated with the test item for 20 hours. In all experiments Colcemid was added 2 hours before the end of the cultivation period, and then the cells were fixed and slides prepared for evaluation. The test substance was suspended in DMSO and added to the medium as duplicate for each concentration of 0.6, 1.78, 5 and 15 µg/mL. 1 % DMSO (v/v) served as vehicle control and MMS and Cyclophosphamide served as positive controls. No cytotoxicity was observed for the test item. No statistically significant differences in the number of metaphases with numerical aberrations were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls with or without metabolic activation. No statistically significant increases in the number of metaphases with structural aberrations or with gaps were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls with or without metabolic activation. The positive control substances caused significantly higher numbers of metaphases with structural aberrations than found in the negative controls, with or without metabolic activation. No relevant evidence of induction of structural chromosomal aberrations for the test item was observed in cultured human lymphocytes, neither with nor without metabolic activation. Therefore the test item is considered to have no DNA damaging potential.

HPRT

The test item, CCPIB was tested in a Mammalian Gene Mutation Test in CHO-K1 cells according to OECD guideline 476 and EU Method B.17. The test item was dissolved in acetone and the following concentrations were selected on the basis of cytotoxicity investigations and the solubility of the test item made in a preliminary study (without and with metabolic activation using S9 mix). In the performed Main Mutation Assays the concentration levels were chosen mainly based on the solubility of test item. For Experiment 1 and Experiment 2, the test item concentrations used in the presence and in the absence of S9 were in the no precipitating range, except the concentration of 200 μg/mL, where precipitation in the medium was observed during the treatment period. Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:

Experiment 1, 5-hour treatment period without and with S9 mix: 25, 50, 75, 100, 125, 150 and 200 μg/mL

Experiment 2, 20-hour treatment period without S9 mix: 25, 50, 75, 100, 125, 150 and 200 μg/mL

Experiment 2, 5-hour treatment period with S9 mix: 25, 50, 75, 100, 125, 150 and 200 μg/mL

At concentrations of 200 μg/mL precipitation in the medium was observed during the treatment period.

Duration of phenotypic expression was 8 days following exposure. The duration of selection period was 8 days, too.

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no biologically significant differences between treatment and control groups and no dose-response relationships were noted.

In Experiment 2, at a concentration of 200 μg/mL, the average mutant frequency (8.42) obtained from cells in duplicate cultures (7.92 and 8.91) was slightly above the historical control value (8.00), when tested without S9 mix over a prolonged treatment period (20 hours). This slight increase at the precipitation causing concentration was not statistically significant and thus not considered to be biologically relevant.

The 5-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.

As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose-response relationships were noted.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures. CCPIB tested both without and with metabolic activation (S9 mix), did not induce relevant increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells. Thus, CCPIB was not mutagenic under the conditions of this study.


Justification for selection of genetic toxicity endpoint
GLP and guideline compliant

Short description of key information:
The test item was investigated for its genotoxic potential in three in vitro assays, namely Ames, CA and HPRT. In these tests no mutagenic or DNA-damaging potential was observed with or without metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data on genotoxicity the test item ist not subjected to classification and labelling according to Regulation 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).