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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 weeks
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
yes
Remarks:
Cloning efficiency value (125%) outside the range and the value for mutation frequency was within 95% control limits of historical solvent control database, deviation had no effect on the results of the first mutation experiment in the absence of S9-mix.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99 %
Specific details on test material used for the study:
Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99%.

Method

Target gene:
Mutations in L5178Y mouse lymphoma cells, specifically deficiencies in thymidine kinase (TK) levels due to the forward mutation of TK+/- to TK-/-
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homoegenate) prepared from male Sprague Dawley rats, dosed with phenobarbital (80mg/kg bodye weight) and ß-napyhoflavone (100mg/kg body weight)
Test concentrations with justification for top dose:
Based on the results of the dose-range finding test the following concentrations were selected for the first mutagenicity tests I the absence and presence of S9-mix: 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/ml of exposure medium. For the second mutagenicity test concentrations of 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5 and 25 µg/ml were used. These concentrations were chosen because the test item was deemed non-toxic and difficult to dissolve in aqueous solutions. The highest concentration was determined by solubility in the culture medium, the highest test item concentration showed slight precipitate in the exposure medium.
Vehicle / solvent:
Ethanol
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate

Results and discussion

Test results
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
In conclusion, Disperse Blue 359 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the mutagenic potential of Disperse Blue 359 by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

In the first experiment, Disperse Blue 359 was tested up to concentrations of 25 µg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. In the second experiment, Disperse Blue 359 was again tested up to concentrations of 25 µg/ml in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. Disperse Blue 359 precipitated in the culture medium at test item concentrations of 12.5 and 25 µg/ml after the three hour treatment and at 25 µg/ml and after the 24 hour treatment.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Disperse Blue 359 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, Disperse Blue 359 did not induce a significant increase in the mutation frequency.

In conclusion, Disperse Blue 359 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.