A mixture of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium ((1-(4(or 5)-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-pentylphenylazo)-2-naphtholato))chromate(1-)

Endpoint summary

Additional information

In vitro

Ames test:

The mutagenicity of the test substance was evaluated in the standard Ames test according to OECD TG 471 and GLP in the bacteria strains TA 1535, TA 1537, TA 98 and TA 100 of S. thyphimurium. The test substance was tested both in the presence and absence of metabolic activation (S9 mix from induced rat liver) at concentrations of 100, 333, 1000, 3330 and 5000 µg/plate. A preliminary toxicity test was carried out in the tester strain TA100 with concentrations ranging from 1 to 5000 µg/plate, from which it was concluded that 5000mg/plate could be used as the highest concentration. 0.1 ml DMSO/plate was used as solvent control. Three Petri dishes were prepared per strain per concentration and in order to confirm the results, the experiments were repeated. In the mutagenicity assay the test substance induced a 5 to 45 fold increase in the number of revertant (His+) colonies in the tester strains TA1537, TA98 and TA100 both in the absence and presence of S9-mix in two independent experiments. Tester strain TA1535 was considered negative in both experiments. The positive controls gave the expected results. Based on the results of this study it was concluded that the test substance can be considered as mutagenic in the Ames test.

Chromosome aberration test:

In a mammalian cell cytogenetics assay according to OECD TG 473 and GLP, the test substance formulated in acetone was assessed for its potential to induce structural chromosome aberrations in V79 cells up to cytotoxic and/or precipitating concentrations in the absence and the presence of metabolic activation by S9 mix. Two independent experiments were performed, each set up with two parallel cultures, in which the first experiment included an exposure time of 18 hrs and the second a exposure and/or harvest time of 28 hrs. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations. The concentrations of the test substance in both experiments ranged from 0 to 350 µg/mL depending on the exposure time and the use of metabolic activation. The concentrations used were chosen according to an initial range–finding cytotoxicity test. The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive controls, i.e. cyclophosphamide and ethylmethanesulfonate, led to the expected increase in the number of cells containing structural chromosomal aberrations. The test substance did not cause any increase in the number of structurally aberrant metaphases incl. and excl. gaps in both experiments, with and without a metabolizing system. In addition no increase in the frequency of cells containing numerical aberrations was noted.  In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce structural chromosome abberations in V79 cells when tested up to cytotoxic and/or precipitating concentrations.

Mammalian Cell Gene Mutation test:

In a cell gene mutation assay in accordance with OECD TG 476 and under GLP, the potential of the test substance to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus using the cell line V79 was investigated. The treatment period was 4h in two independent experiments (each with duplicate cultures) at concentrations of 6; 10; 15; and 20 µg/ml with metabolic activation and concentrations of 6; 10; 30; 60 and 100 µg/ml without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive andvalid. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. Cytotoxic effects were observed in the absence of metabolic activation (relative plating efficiency of 84.8%, 80.5% and 49.1% at 10, 15 and 20 µg/ml, respectively), whereas with metabolic activation no cytotoxicity was seen up to the highest test concentration of 100 µg/ml.Higher concentrations precipitated in the culture medium. In conclusion it can be stated that under the experimental conditions the test substance did not induce mutations in the HPRT-locus with or without metabolic activation.

In vivo

Micronucleus test:

In a micronucleus test in accordance with OECD TG 474 and GLP in Swiss mice (5 animals/sex/ dose) the test substance was dosed once by gavage at 5000 mg/kg bw. The dose was selected based on the results in a preliminary toxicty study. The dosing volume was 10 ml/kg bw and corn oil was used as vehicle. Bone marrow cells were harvested 24, 48 and 72 hours (negative control and all dose levels) and 48 hours post-treatment (for positive control). There were no signs of toxicity during the study. The test substance did not cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes at the 24, 48 and 72 hour sampling time. No significant increase in the frequency of micronucleated erythrocytes in bone marrow at a dose of 5000 mg/kg bw after treatment with the test substance was observed. The positive control cyclophosphamide induced the appropriate response. In conclusion the test substance was found to be not clastogenic or aneugenic in this test.

Short description of key information:
The test substance was found to be positive in the standard Ames test (OECD 471, GLP). No genotoxic potential was found in the Chromosomal Abberation Test in vitro (OECD 473, GLP), in the Mammalian Cell Gene Mutation Test in vitro (OECD 476, GLP) and in the Erythrocyte Micronucleus Test in vivo (OECD 474, GLP).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was found to be positive in the standard Ames test. However, no genotoxic potential was found in the Chromosomal Abberation Test in vitro, in the Mammalian Cell Gene Mutation Test in vitro and in the Erythrocyte Micronucleus Test in vivo. Based on these results, the test substance does not need to be classified and labelled for mutagenicity according to Directive 67/548/EEC and Regulation 1272/2008/EC.