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Key value for chemical safety assessment

Additional information

The key study for mutagenicity in bacteria was performed according to OECD guideline 471 and according to GLP (Synthesia 2009b). Four Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA strain were used. The test substance was suspended in water and assayed in doses of50-5000mg which were applied

to plates in volume of 0.1mL. Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance C.I. Pigment Red 177 was non-mutagenic for all the used bacterial strains with as well as without metabolic activation.

 Another Ames test was performed with only four strains and without an independent repeat experiment (Ciba-Geigy Ltd 1979). Its procedure was comparable to a guideline study with acceptable restrictions. The plate incorporation protocol was applied and the maximal evaluated concentration was 2000 µg/plate in the absence of cytotoxicity or precipitation. Liver homogenate from arochlor induced rats was used for metabolic activation. No increase in revertant frequency was induced by treatment with the pigment.

The assessment of mutagenicity in mammalian cells in vitro was performed by two GLP-compliant studies for unscheduled DNA synthesis (Ciba-Geigy Ltd. 1986a, b). The experiment without metabolic activation was performed with a human fibroblast cell line (CRL 1121), and the experiment with metabolic activation was performed with primary hepatocytes from arochlor 1254 induced rats. Both studies together fulfill the requirements of OECD testing guideline 482. In the studies, the optional inhibition of semi-conservative DNA replication was not included. However, discrimination between cells in S-phase and repairing cells was done.

The test concentrations that could be applied were limited by the solubility of the pigment: In rat hepatocytes, 0.4, 2, 10 and 50 µg/ml were applied using DMSO as vehicle. In human fibroblasts, 0.8 , 4, 20 and 100 µg/ml were scored. 2-Acetylaminof luorene at 0.2 mg/L was used as positive control for the study with metabolic activation and (4-nitroquinoline- N-oxide at 5µM was used for the study. Incubation with the pigment did not induce an increase in unscheduled DNA synthesis.

Pigment Red 177was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the absence and the presence

of a metabolizing system (BASF 2012c). The study followed OECD testing guideline 473 and was performed under GLP.

According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were evaluated (higher concentrations were tested but not evaluated due to strong precipitation):

1st Experiment: 4-hour exposure, 18-hour sampling time, without and with S9 mix: 0;5; 10; 20μg/mL. 

2nd Experiment: 18-hour exposure, 18-hour sampling time, without S9 mix

0; 2.5; 5; 10;μg/mL; 18-hour exposure, 28-hour sampling time, without S9 mix 0;10; 20μg/mL; 4-hour exposure, 28-hour sampling time, with S9 mix; 0;5; 10; 20μg/mL

A sample of 100 metaphases for each culture was analyzed for chromosomal aberrations, except for the positive control cultures where only 50 metaphases were scored due to clearly increased aberration rates.

The negative controls gave frequencies of aberrations within the range expected for the V79 cell line. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations.

In the main experiments no clear cytotoxicity was observed after 4 or 18 hours exposure period at any experimental condition, except in the 1st Experiment in the presence of metabolic activation where all test groups scored for chromosomal damage showed a mitotic rate of about 50% of control. However, strong test substance precipitation in culture medium was observed in all experimental parts. Thus, dose selection for cytogenetic evaluation was based on the occurrence of precipitates in the test groups. No increase in the frequency of cells containing structural or numerical chromosome aberrations was demonstrated and the pigment was found to be non clastogenic in vitro.

Justification for selection of genetic toxicity endpoint
random; more than one study needed for this endpoint.

Short description of key information:
Pigment Red 177 is not mutagenic in the Ames test (Synthesia 2009b, Ciba-Geigy Ltd 1979) and in the test for unscheduled DNA synthesis in primary hepatocytes and in a human fibroblast cell line (Ciba-Geigy Ltd 1986a, b). It did not cause chromosome aberrations in mammalian cells in vitro as tested in a GLP-compliant study following OECD guideline 473 (BASF 2012c). The more recent Ames test was performed under GLP and following OECD guideline 471. The UDS tests were performed under GLP and together fulfill the requirements of OECD guideline 482.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).