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in vitro

The test substance was tested for its mutagenic potential at doses of up to 5000 μg/plate in Salmonella typhimurium strains TA1535, TA100, TA1537, TA98 and TA 102 with and without metabolic activation according to OECD guideline 471 (CIBA GEIGY 1986). GLP accordance was not stated in the report. The study was performed as independent standard plate test. S-9 mix of Aroclor 1254 -induced rat livers served as metabolic activation system.

The test article was suspended in dimethylsulfoxide and precipitated in soft agar from the lowest concentration of 20 µg/plate used in the assay.

An increase in the number of his+revertants was not observed and a negative result was suggested. Cytotoxic effects were not observed.

The positive control substances (2-aminoanthracene for the strains TA 98, TA 100, TA 1537 and Cyclophosphamide for the strains TA 1535 with metabolic activation; Daunorubicin-HCl for the strain TA 98, 4-nitroquinoline-N-oxide for the strain TA 100, mitomycin C for the strain TA 102, sodium azide for the strain TA1535, 9-aminoacridine for the strain TA 1537 without metabolic activation; all substances dissolved in DMSO produced valid responses.

Thus, under the experimental conditions chosen here, the test substance was assessed to possess no mutagenic effects in Salmonella typhimurium strains in vitro.

 

In vivo

The test substance was tested for chromosomal damage (clastogenicity) in Chinese hamsters using the micronucleus test method in a study according to OECD guideline 474 (CIBA GEIGY 1986). For this purpose, the test substance, dissolved in 0.5 % aqueous solution of sodium carboxymethylcellulose, was administered once per oral gavage to male and female animals at a dose levels of 5000 mg/kg body weight in a volume of 20 ml/kg body weight.

Animals treated with the vehicle (0.5% CMC) alone served as negative control. The positive control chemical, cyclophosphamide for clastogenic effects led to the expected increase in the rate of micronucleated polychromatic erythrocytes.

 

After administration of the test item, the animals were sacrificed and the bone marrow of the two femora was prepared 16, 24 and 48h after application. After staining of the preparations, 1000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei.

 

According to the results of the present study, the administrations of the test compound did not lead to any increase in the number of micronucleated polychromatic erythrocytes. The rate of micronuclei (0.18 %) was nearly the range as that of the concurrent negative control (0.13 %). No toxic effects were observed as determined from the ratio of polychromatic to normochromatic erythrocytes.

 

Thus, under the experimental conditions chosen here, the substance does not have any chromosome-damaging (clastogenic) effects in bone marrow cells in vivo.

No further experimental data regarding mutagenicity in vitro is considered necessary as the pigment is insoluble both in fat and water and shows strong precipitation in culture medium. Most importantly, the toxicokinetic study using 14C-labelled pigment showed that the pigment is not taken up after ingestion. Mutagenic impurities are more sensitively picked up in the Ames test because bacteria tolerate precipitates much better than cultivated cells and higher doses can be applied. Overall, the available information on genotoxicity is considered adequate for hazard assessment.


Short description of key information:
The pigment is not mutagenic in bacteria as shown in the Ames test with S. typhimurium TA1535, TA100, TA1537, TA98 and TA 102 with and without metabolic activation at doses of up to 5000 µg/plate (OECD 471; Ciba-Geigy 1986a). It was not genotoxic in the micronucleus test in Chinese hamster after gavage application of 5000 mg/kg bw (OECD 474, Ciba-Geigy 1986b).

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 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.

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