Glycine, N-[5-(acetylamino)-4-[2-(2-bromo-4,6-dinitrophenyl)diazenyl]-2-methoxyphenyl]-N-(2-methoxy-2-oxoethyl)-, methyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 December 2014 to 16 January 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliance with GLP and testing guidelines, coherence among data, results and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disperse Blue ANT

IUPAC Name:

Disperse Blue ANT

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Test item: Disperse Blue ANT

Method

Target gene:

The test item Disperse Blue ANT was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate from induced rat (rat mixed induction)

Test concentrations with justification for top dose:

In Main Assay I: 5000, 2500, 1250, 625 and 313 µg/plate.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Toxicity and Main Assay I were performed using the plate incorporation method.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

Doubling rate (Chu et al 1981)
Regression line

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item induced dose related and statistically significant increases based on a “doubling rate” in all tester strains, in the absence and presence of S9 metabolism with the exception of TA1535 and WP2 uvrA.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

See Final Report

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item, Disperse Blue ANT, induces reverse mutation by base substitutions or frameshifts in bacteria in the absence and presence of S9 metabolism in the Salmonella strains TA98, TA100 and TA1537, under the reported experimental conditions.

Executive summary:

The test item Disperse Blue ANT was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in dimethylsulfoxide (DMSO).

Toxicity test: The test item Disperse Blue ANT was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations. Due to the dark color of plates the background lawn was not analysable at the two highest dose levels. Large increases in revertant numbers were observed both in the absence and presence of S9 metabolic activation with all tester strains with the exception of TA1535 and WP2 uvrA without S9 metabolism and WP2 uvrA with S9 metabolism. Due to a toxic effect, induced mutant colonies were markedly reduced at the highest dose level with TA98 and TA 100 tester strain in the presence of S9 metabolism.

Main Assay I: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed by using the following dose levels: 5000, 2500, 1250, 625 and 313 µg/plate. Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations. At these dose levels the background lawn was not analysable due to the dark color of plates. The test item induced large increases in the number of revertant colonies using the plate incorporation method in all tester strains in the absence and presence of S9 metabolism with the exception of TA1535 and WP2 uvrA. Since a clear positive response was observed, no further experiment was undertaken.

Conclusion: It is concluded that the test item, Disperse Blue ANT, induces reverse mutation by base substitutions or frameshifts in bacteria in the absence and presence of S9 metabolism in the Salmonella strains TA98, TA100 and TA1537, under the reported experimental conditions.