1-ethyl-1,2-dihydro-6-hydroxy-4-methyl-5-[(2-nitrophenyl)azo]-2-oxonicotinonitrile

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 to 22 March 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Principles of method if other than guideline:

In addition to the standard plate incorporation test (Ames Test) a modified protocol using preincubation with hamster S9 supplemented with flavine mononucleotide was used. This protocol has been proposed by Prival for assessing the mutagenic activity of azo dyes.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

425-310-7

EC Name:

-

Cas Number:

77889-90-8

Molecular formula:

C14H10ClN5O4

IUPAC Name:

5-[2-(4-chloro-2-nitrophenyl)diazen-1-yl]-6-hydroxy-1,4-dimethyl-2-oxo-1,2-dihydropyridine-3-carbonitrile

Test material form:

solid: particulate/powder

Method

Target gene:

Reverse mutation to histidine prototrophy using histidine auxotrophic mutants.

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-Mix from rat and hmster liver

Test concentrations with justification for top dose:

with and without metabolic activation: 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance is not water soluble

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

a: untreated control b: solvent controls (0 ug/plate)

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

other: N-Methyl-N-nitro-N-nitrosoguanidine (MNNG); 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Incubation period: 48 h at approx. 37 °C


NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators.
Thinning of the bacterial lawn was evaluated microscopically.

Evaluation criteria:

Criteria for a positive response

A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

If the test substance does not produce reproducible increases of at least 2 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this system.
The test results must be reproducible.

Results and discussion

Test results

Test results 1

Key result

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

Key result

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

Key result

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

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

Any other information on results incl. tables

Sterility checks and control plates

Sterility of S9-mix and the test compound were indicated by the absence of contamination in the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies.

Solubility

Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 2500 µg/plate and lower doses. Visible precipitation of the test compound on the plates was observed at 500 µg/plate and above.

Toxicity

The highest concentration was 50 mg/ml of the test compound in DMSO, which provides a final concentration of 5000 µg/plate. Further dilutions of 2500, 500, 100, 20 and 4 µg/plate were used in both experiments.

The test compound proved to be not toxic to the bacterial strains.

Applicant's summary and conclusion

Conclusions:

The test substance is not mutagenic in the standard plate test (Ames test) and in the preincubation method according to Prival.

Executive summary:

The test substance was tested for mutagenicity in the Ames and Prival test with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and, only in the Ames test, with the strain Escherichia coli WP2 uvrA.

Two independent mutagenicity studies were conducted in the standard plate test (Ames test) and in a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolising system derived from rat liver homogenate or hamster liver homogenate. For both studies, the compound was suspended in DMSO, and each bacterial strain was exposed to 6 dose levels. Doses for both studies ranged from 4 to 5000 µ/plate.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

No cytotoxicity was not observed either in the absence or in the presence of metabolic activation.

In the absence of the metabolic activation system the test compound did not result in relevant increases in the number of revertants in any of the bacterial strains in the plate incorporation test. Also in the presence of rat liver activation system (10 % (v/v)), treatment of the cells with the test substancedid not result in relevant increases in the number of revertant colonies.

In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Privalthe test substancedid not result in relevant increases in the number of revertant colonies with any of the tester strains.

Summarising, it can be stated thatthe test substanceis not mutagenic in the standard plate test (Ames test) and in the preincubation method according to Prival.