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Point mutation in bacteria

In a GLP-compliant study following OECD guideline 471, S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 were treated with 33,3 – 5000 µg/plate test substance by the pre-incubation method, (CCR, 1993). The assay was performed in two independent experiments using identical procedures, both with and without liver microsomal activation. In experiment I, a clear dose-dependent increase in revertant colonies was observed in the strains TA 1537 and TA 1538 from 33.3 µg to 333.3 µg/plate and in strain TA 98 from 33.3 µg to 100 µg/plate all in the presence of metabolic activation. At higher concentrations the induced revertant rate decreased due to overlapping toxic effects of the test article. In experiment II, a clear dose-dependency was observed in strain TA 1538 from 100 µg to 1000 µg/plate and in strain TA 98 from 33.3 µg to 100 µg/plate in the presence of metabolic activation. Likewise, the induced revertant rate decreased due to overlapping toxic effects. The obtained effect in strain TA 1537 in the presence of S9 mix was less distinctive and a clear dose-dependency could not be observed in experiment II. However, together with the clear dose dependent effect in experiment I and the relatively high value at 100 µg/plate, a mutagenic response could not be excluded but the evaluation criteria for a mutagenic effect (reproducibility) was not fulfilled. In conclusion, under the experimental conditions reported, the test article induced point mutations by frame shifts in the genome of the strains TA 1538, and TA 98 in the presence of metabolic activation.

Similar results were obtained in a second study performed on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100 (CCR, 1988). The assay was performed once, in the presence and absence of a liver microsomal activation system with the following concentrations: 10, 100, 333.3, 1000 and 5000 µg/plate. A significant dose-dependent increase in revertant colony numbers was obtained in the Salmonella typhimurium strain 1538 up to a concentration of 1000 µg/plate in the presence of metabolic activation. At 5000 µg/plate the number of revertants was reduced thus indicating the beginning of toxicity. This mutagenic response was confirmed in an independent repeat experiment performed with this strain only. In all other tested groups no relevant increases in the number of revertant colonies were observed. In conclusion, under the experimental conditions reported, the test article induced point mutations by frame shifts in the genome of strain TA 1538.

In a third study (GLP, OECD 471), the test article was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 (CCR, 1990). The assay was performed in two independent experiments, both with and without liver microsomal activation. The test article was tested at the following concentrations: 10, 100, 333.3, 1000 and 5000 µg/plate. Up to the highest investigated dose, no significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control. The presence of liver microsomal activation did not influence these findings. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies. In conclusion, under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frame shifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Similar results were obtained in a fourth GLP-compliant study (CCR, 1988), where the test article was assessed by a single plate incorporation test using Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The test article was tested at concentrations ranging from 10 to 5000 µg/plate. Up to the highest investigated dose, no significant 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. In conclusion, under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frame shifts in the genome of the strains used.

Chromosome Aberration

The test article was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation by S9 mix (RCC 1993). Preparation of chromosomes was done 7 h (0.1 mg/ml), 18 h (0.01, 0.03 and 0.1 mg/ml) and 28 h (0.5 mg/ml without and 0.1 mg/ml with S9 mix) after start of the treatment with the test article. The treatment interval was 4 h. Both, in the absence and presence of S9 mix the test article did not increase biologically relevantly the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (0.0 % - 5.5 %) were in or near to the range of the control values; 1.0 % - 3.5 %. In one experiment (28 h, without S9 mix), a relative high spontaneous aberration rate (4.5 %) were found with 2.0 % cells carrying exchanges (data not reported). To overcome possible incalculable effects, several experimental points (one control and 6 treatment groups) at this interval were repeated in an independent experiment. In this experiment a slight increase in the aberration rate was found as compared to the corresponding control. However, this increase was observed only in one of the two slides. In addition, the increase (5.5 %) was near to the control rate (3.5 %) and not statistically significant. Therefore, this effect was not regarded as biologically relevant. No relevant deviation from the control data was found after treatment with the test article regarding polyploid metaphases. The positive controls showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that in the described study and under the experimental conditions reported, the test article did not induce structural chromosome aberrations in the V79 Chinese hamster cell line.

Point mutation in mammalian cells

In a mammalian gene mutation test (BASF, 2011) according to OECD guideline 476 and in compliance with GLP, the test article was tested for mutagenic effects at the HPRT locus in Chinese hamster ovary (CHO) cells in vitro. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. Analyzed concentrations were 12.5; 25.0; 50.0; 100.0 and 200.0 µg/ml with S9 mix and 50.0; 100.0; 200.0; 400.0 and 800.0 µg/ml without S9 mix. In the second experiment, the cells were exposed to the test substance for 4 hours with S9 mix (12.5; 25.0; 50.0; 100.0 and 200.0 μg/mL) and for 24 hours without S9 mix (100.0; 200.0; 400.0; 600.0 and 800.0 μg/mL). In this study, in both experiments in the absence and the presence of metabolic activation the highest concentrations tested for gene mutations were clearly cytotoxic. The test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in both experiments performed independently of each other. The mutant frequencies at any concentration were nearby the range of the concurrent negative control values and within the range of our historical negative control data. The increase in the frequencies of mutant colonies induced by the positive control substances EMS and MCA clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The positive control values as well as the vehicle control values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Overall conclusion

The potential of the test article to induce genomic point mutations was first tested in four independent Ames tests with several strains of S. typhimurium. Two were clearly negative, whereas the two other experiments had positive findings in the strains TA 1538 (both tests) and TA 98 (one test). Due to the ambiguous nature of these results no clear conclusion regarding point mutagenicity can be drawn. To further describe the mutagenic potential of the test article and to clarify whether classification regarding genotoxicity is required, two additional mutagenicity tests have been performed. In a HPRT test conducted with mammalian cells it was clearly demonstrated that the test substance does not cause point mutations. Furthermore, in a chromosomal aberration no potential of the test substance to cause chromosomal damage was evident. Therefore, based on the available data, the test substance is regarded as not mutagenic and not clastogenic and does not need to be classified. This is in line with “ECHA guidance on information requirements and chemical safety assessment Chapter R7a, Figure R.7.7 -1”, which states that no further genotoxicity studies are required for a substance which was tested positive in an Ames test when results from an HPRT test and from a clastogenicity test are negative. Consequently no classification is warranted.


Short description of key information:
- Ames tests (CCR, 1988a, 1988b, 1990 and 1993): Ambiguous (two plate incorporation tests were negative, one plate incorporation and one pre-incubation test were positive (OECD 471).
- chromosome aberration in vitro (CCR, 1990): negative (OECD 473)
- HPRT test (BASF, 2011): negative (OECD476)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the data, classification for genetic toxicity is not warranted under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for genetic toxicity is not warranted under Regulation (EC) No.1272/2008.