alkoxy substituted aryl diazo heterocyclic aryl nitrile

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 27 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Test material form:

solid: particulate/powder

Details on test material:

Test item: Disperse Yellow DYLA 1306

Method

Target gene:

The test item Disperse Yellow DYLA 1306 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:

According to solubility, in Main Assay I: 248, 124, 62.0, 31.0 and 15.5 µg/plate.
Main Assay II: 248, 165, 110, 73.3, 48.9 and 32.6 μg/plate (TA100 +S9)

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle 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.
A second separate experiment were performed on the TA100 tester strain in order to confirm the positive results obtained in the first experiment.

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

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Species / strain:

S. typhimurium TA 98

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

Test results 4

Species / strain:

E. coli WP2 uvr A

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

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

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

Test results 6

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

in the Main Assay 1, test result was questionable, in Main Assay II, there was a weak positive result

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

Additional information on results:

The test item induced increases in the number of revertant colonies, which were at least two-fold greater than the control value at the highest dose levels with TA100 tester strain in the presence of S9 metabolic activation.

Remarks on result:

not determinable because of methodological limitations

Remarks:

as the substance is a nitro-compound, the bacterial nitroreductase causes a bacteria-specific positive effect

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item Disperse Yellow DYLA 1306 might probably induce reverse mutation in Salmonella thyphimurium TA100 tester strain in the presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item Disperse Yellow DYLA 1306 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: Based on results obtained in a preliminary solubility trial, the test item Disperse Yellow DYLA 1306 was assayed in the toxicity test at a maximum concentration of 248 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 78.4, 24.8, 7.84 and 2.48 µg/plate. Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations. No toxicity was observed with any tester strain in the absence or presence of S9 metabolism. Increases in revertant numbers were observed in the presence of S9 metabolism with TA100 tester strain.

Main Assay: On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 248, 124, 62.0, 31.0 and 15.5 μg/plate.

Precipitation of the test item was observed at the end of the incubation period at the two highest concentrations both in the absence and presence of S9 metabolic activation.

No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. The test item induced increases in the number of revertant colonies in the presence of S9 metabolism with TA100 tester strain reaching a two-fold increase at the highest concentration tested, which showed precipitation of the test substance, compared to the vehicle control, but not compared to the untreated control (MF 1.8 -fold).

An additional confirmatory experiment (Main Assay II) was performed in which TA100 tester strain was treated in the presence of S9 metabolism at the dose levels of 248, 165, 110, 73.3, 48.9 and 32.6 μg/plate. Dose related increases in revertant numbers were observed. These increases were greater than twice the control values at the two highest dose levels, which were the dose levels with precipitation, thus indicating a possible positive response with TA100 tester strain in the presence of S9 metabolism.