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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
see confidential details on test material
see confidential details on test material


Species / strain
Species / strain / cell type:
other: TA1535, TA1537, TA1538, TA98, TA100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix derived form Aroclor 1254 induced rat livers
Test concentrations with justification for top dose:
0, 8, 40, 200, 1000 and 5000 µg/plate (experiment I, with and without S9 mix)
0, 312.5, 625, 1250, 2500 and 5000 µg/plate (experiment II, with and without S9 mix)
Vehicle / solvent:
sterile distilled water
Untreated negative controls:
Negative solvent / vehicle controls:
distilled water
True negative controls:
Positive controls:
Positive control substance:
other: -S9 mix: ENNG (TA100, TA1535), 9-AA (TA1537), 4-NOPD (TA1538), 4-NQO ( TA98); +S9 mix: 2-AA (TA1535), BP (all other strains)
Details on test system and experimental conditions:
Five concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method.
0.1 mL of the diluted test item or negative control solution was added to 2.0 mL of trace supplemented top agar at 45°C in sterile tubes. A 0.1 mL aliquot of bacterial suspension and 0.5 mL of the S9 liver microsome mix or buffer was added. The mix was overlaid onto sterile plates of Vogel-Brunner agar. After approximately 48 hours at 37°C the plates were scored for revertant colonies and thinning of background lawn
Evaluation criteria:
For a substance considered to be positive, it should have induced a dose-related and statistically significant increase in the mutation rate in one or more strains. To be considered negative the numbr of revertatns compared to spontaneous revertants should be less than twofold at each dose level.
All data are statistically analysed using the methods recommended by UKEMS (Kirkland DJ (ed) Statistical evaluation of mutagenicity test data. Reprot - part III (1989)- Cambridge university press)

Results and discussion

Test results
Species / strain:
other: TA1535, TA1537, TA1538, TA98, TA100
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
No toxicity was exhibited. Precipitation was observed with doses at and above 625 µg/plate. this did not interfere with the scoring of revertant colonies. No significant increases in the numbers of revertant colonies were recorded for any of the strains of Salmonelly used at any dose level either with or without S9.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion

Executive summary:

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.