Benzyl 4-[(1-butyl-5-cyano-1,6-dihydro-2-hydroxy-4-methyl-6-oxopyridin-3-yl)azo]benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

solid: particulate/powder

Method

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:

rat-liver post mitochondrial supernatant fraction

Test concentrations with justification for top dose:

313, 625, 1250,2500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

other: 2-aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: each test item concentration, the negative and positive and ontrols were tested in triplicates. 2 experiments were run.

CITOTOXICITY
Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation with the concentrations of 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. Precipitation of the test item was observed at concentrations of 185.2 to 5000 µg/plate.
From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.
In the first mutagenicity test performed with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with test item at any concentrations.
In the second mutagenicity test carried out with and without metabolic activation, again treatment of the tester strains with the test item did not lead to an increase in the number of revertant colonies at any concentrations.
In both mutagenicity tests normal background growth was observed with all strains at all concentrations. The number of revertant colonies was not reduced. Precipitation of the test item on the surface of the agar plates was observed at concentrations of 625 to 5000 µg/plate.
In the experiments negative (solvent) and positive control treatments were included for all strains. The mean numbers of revertant colonies on negative control plates were found to be within acceptable ranges. The positive controls induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain functioning and the activity of the S9-mix.

Applicant's summary and conclusion

Conclusions:

The test item is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item was tested for mutagenic effects in vitro in histidine-requ¡ring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. The following strains were used: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coliWP2 uvrA.

The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. Each test item concentration, the negative and positive and ontrols were tested in triplicates. The test item was dissolved in dimethylsulfoxide (DMSO) and tested at five concentrations: 313, 625, 1250,2500 and 5000 µg/plate.

In order to confirm the results, the experiments were repeated with and without metabolic activation at the same concentrations used in the first experiment. The test with metabolic activation was carried out as pre-incubation assay. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

Previously, a pre-experiment for toxicity (range finding test) was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiment was performed with and without metabolic activation with the concentrations of 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 µg/plate.

Normal background growth was observed with both strains. The number of revertant colonies was not reduced at any concentrations tested. Precipitation of the test item was observed at concentrations of 185.2 to 5000 µg/plate.

From the results obtained, the highest concentration suitable for the first mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.

In the first mutagenicity test performed with and without metabolic activation, no substantial increase in the number of revertant colonies was observed after treatment with test item at any concentrations.

In the second mutagenicity test carried out with and without metabolic activation, again treatment of the tester strains with the test item did not lead to an increase in the number of revertant colonies at any concentrations.

In both mutagenicity tests normal background growth was observed with all strains at all concentrations. The number of revertant colonies was not reduced. Precipitation of the test item on the surface of the agar plates was observed at concentrations of 625 to 5000 µg/plate.

In the experiments negative (solvent) and positive control treatments were included for all strains. The mean numbers of revertant colonies on negative control plates were found to be within acceptable ranges. The positive controls induced appropriate increases in the number of revertant colonies in all experiments, thus demonstrating the correct strain functioning and the activity of the S9-mix.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base-pair changes or frameshifts in the genome of the strains used.

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.