Phenol, 2,4-dinitro-, sulfurized, leuco derivatives

Endpoint summary

Additional information

An aqueous Leuco Sulfur Black 1 formulation containing less than typical sulfur dye concentration (30.2% instead of 52.75 %) was tested in the bacterial reverse mutation assay with Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 using the plate incorporation method at five dose levels, in triplicate, both with and without a metabolic activation system (S9 mix from induced rat liver). Two independent experiments were conducted. The assay was performed according to OECD test guideline No. 471 and GLP. The dose range was 312.5 to 5000 µg/plate. Distilled water was used as solvent.

The concurrent positive controls gave increases in revertants within the expected ranges.

The test item did not cause toxicity. Precipitations occurred at and above 625 µg test item/plate. No significant increase in the number of revertants was recorded for any of the bacterial strains with any dose of the test item either with or without metabolic activation.

In this assay Leuco Sulfur Black 1 precipitated at and above test item concentrations of 625 µg/plate, demonstrating that the highest possible Leuco Sulfur Black 1 concentrations which can be studied in the Ames test were achieved. Thus, the assay is fully valid for the assessment of the mutagenic potential of Leuco Sulfur Black 1.

 

Leuco Sulfur Black 1 was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster according to OECD test guideline No. 476 and GLP. The study was performed in two independent experiments. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

Precipitation of the test item was observed at 125 µg/mL and above with and at 250 µg/mL and above without metabolic activation.

Relevant cytotoxic effects defined as a relative cloning efficiency I below 50% in both parallel cultures occurred at 500 µg/mL in both experiments without metabolic activation. In the presence of metabolic activation cytotoxic effects as described above were noted at 125 µg/mL and above in the first experiment.

The positive controls ethylmethanesulfonate and DMBA showed a distinct increase in induced mutant colonies.

No relevant and reproducible increase in mutant colony numbers/10E6cells was observed in the main experiments up to the maximum concentration. All mutant frequencies remained well within the historical range of solvent controls. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In conclusion, Leuco Sulfur Black was not mutagenic in theHPRTassay with and without metabolic activation.

 

Leuco Sulfur Black 1, suspended in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments according to OECD test guideline No. 473 and GLP.

In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations. Cells were exposed to the test item for 4 hours with and without S9 in experiment I and again for 4 hours with S9 and continuously for 18 hours in experiment II. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The chosen treatment concentrations were between 2.3 and 300 µg/mL (with and without S9). The evaluated concentrations were 19.5, 39.1 and 78.1 µg/mL in experiment I and 37.5, 75 and 150 µg/mL in experiment II.

In the absence and presence of S9 mix no cytotoxicity was observed up to the highest evaluable concentration. Due to strong test item precipitation concentrations higher than those examined could not be evaluated for cytogenetic damage.

No clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. However, in experiment II in the presence of S9 mix a single statistically significant increase in the number of aberrant cells excluding gaps (3.0 %) was observed at the highest evaluable concentration (150 µg/mL). This value was clearly in the laboratory’s historical solvent control data range (0.0 – 4.0 % aberrant cells excluding gaps) and is therefore regarded as biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, the test item was not clastogenic in this in vitro chromosome aberration assay.

Short description of key information:
Leuco Sulfur Black 1 was neither mutagenic in the Ames test nor in the HPRT assay on V79 cells and was non-clastogenic in the in vitro chromosome aberration assay using V79 cells. All assays were performed with and without metabolic activation system.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data demonstrate that Leuco Sulfur Black 1 has no genotoxic potential. Thus, it is not subject to classification and labelling according to Directive 67/548/EECand Regulation 1272/2008/EC.