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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance did not induce any genotoxic effects in the studies conducted

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance did not induce genotoxic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The test item was tested in the standard plate incorporation test in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at concentrations from 0.16 to 100 µg/plate both in the presence and absence of metabolic activation. No cytotoxicity was observed at any concentration. At concentrations of 20 µg/plate and above, precipitation of the test stubstance was observed. An increase in the number of revertant colonies could not be observed up to the highest dose of 100 µg/plate. Vat Blue 20 is thus not mutagenic in the Ames test under the experimental conditions chosen.

The structural analogue was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item suspensions/solutions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed. Based on solubility features, the test item was assayed, in the absence and presence of S9 metabolism, at a maximum dose level of 100 µg/mL and at a wide range of lower dose levels: 50.0, 25.0, 12.5, 6.25, 3.13, 1.56, 0.781 and 0.391 µg/mL. By the end of treatment, precipitation of the test item was noted at the two highest dose levels. No toxicity was observed at any concentration tested, in the absence or presence of S9 metabolic activation. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Main Assay I (-/+S9): 50.0, 25.0, 12.5, 6.25 and 3.13 μg/mL

Main Assay II (-S9): 50.0, 31.3, 19.6, 12.2 and 7.64 μg/mL

Main Assay II (+S9): 80.0, 50.0, 31.3, 19.6 and 12.2 μg/mL

Selection of dose levels used in Main Assay I was performed taking into account precipitation observed in the preliminary cytotoxicity assay. The dose range used in Main Assay II was modified to focus on the highest concentrations that could be tested. No relevant increases in mutant numbers or relevant five-fold increases in mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments, indicating the correct functioning of the assay system. It is concluded that Vat Green 1 does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

The investigation for possible cytogenetic effects of the structural analogue was included into a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity screening test in rats, as the test item is not soluble in water or organic solvents.

In this test, no relevant differences in clinical signs were observed between male and female animals from the main and recovery groups. Based on these results, the genotoxicity assessment was performed including male animals only. The presence of a dark precipitate in many urine samples collected from treated animals was considered a proof of absorption.

Following treatment with the test item, no relevant increase in the number of micronucleated PCEs over the concurrent negative control was observed at any dose level. The incidences of micronucleated PCEs were comparable to historical control data for negative control animals. A marked increase in the frequency of micronucleated PCEs was observed in the positive control group.

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

The incidence of micronucleated PCEs of the negative control group fell within the historical control range (95% confidence limit). Statistically significant increases in the incidence of micronucleated PCEs over the negative control values were seen in the positive control group. The induced response was compatible with the historical control range, demonstrating the laboratory proficiency in the conduct of the test. Five animals per group were available for micronucleus slide analysis. Based on the stated criteria, the assay was therefore accepted as valid.

No significant within-group heterogeneity was observed; hence thec² test was used to compare treated groups with the control group. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated PCEs over the negative control value was observed at any dose level. No significant dose-effect relationship was found after a trend test evaluation.

 

Dose level

Incidence in micronucleated PCEs

PCE/s(PCEs+NCEs)

[mg/kg/day]

Mean

SE

Range

[%] over the mean control value

0.00

0.6

0.1

0.5 - 0.8

100

62.5

0.6

0.1

0.5 - 0.8

100

250

0.6

0.1

0.3 - 1.0

96

1000

0.7

0.1

0.3 - 1.0

99

Mitomycin-C
2.00 mg/kg

9.3

0.7

7.0 - 11.0

91

Conclusion

On the basis of the results obtained, it is concluded that the test substance does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

Justification for classification or non-classification