Trisodium 7-[[4-chloro-6-[ethyl[3-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methoxy-2-sulphonatophenyl)azo]naphthalene-2-sulphonate

Genetic toxicity: in vitro

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Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From November 12, 1999 to December 04, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

red powder

Specific details on test material used for the study:

Purity: 52%
Concentration of stock solution in deionized water: 50 mg/mL

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: deficient in lipopolysaccharide layer and in DNA excision repair system

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: defective in the uvrA system of DNA repair

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction (rat liver homogenate)

Test concentrations with justification for top dose:

Plate incorporation test: 1. 0, 50, 160, 500, 1600 and 5000 µg/plate
Repeat plate incorporation test: 2. 0, 50, 160, 500, 1600, 2500 and 5000 µg/plate

Vehicle / solvent:

Deionized water

Controls

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Remarks:

deionized water

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 1-methyl-3-nitro-1-nitrosoguanidine and 2-aminoanthracene

Details on test system and experimental conditions:

To 2 mL of molten top agar in a sterile test-tube, 0.1 mL of the tester strain culture, graded quantities of the test substance in 0.1 mL solution and, for the S-9 series, 0.5 mL of S-9 Mix were added. The contents of the test tube were rapidly mixed and poured onto the surface of previously prepared minimal agar plates with Vogel-Bonner E mixture. The plate were incubated upside down at 37°C for 2 d, after which the number of revertants colonies appearing was counted.

In addition, sterility of the test medium, solubility of the test substance and toxicity to the test strains were evaluated.

Evaluation criteria:

Doubling of the spontaneous mutation rate (control) and dose-related increase in the mean number of revertants

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

in both tests

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All positive controls produced significant increases in the number of revertant colonies.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was mutagenic to Salmonella typhimurium with and without metabolic activation (Ames test).

Executive summary:

A study was conducted to determine the mutagenic potential of the substance according to OECD Guideline 471, EU Method B13/14 and US EPA OPPTS 870.5100, in compliance with GLP. Four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli strain WP2uvrA were exposed to the test substance at concentrations of 0 to 5000 µg/plate with or without metabolic activation (S-9 Mix) for 48 h. Negative and positive controls were valid. The test substance induced mutagenic activity in the strain of Salmonella typhimurium TA 1535 both under the presence as well as absence of metabolic activation. The number of revertants was greater than twice the number of spontaneous mutations. Mutagenicity was negative in all other strains tested. Under the study conditions, the substance was considered to be mutagenic with and without metabolic activation (Stammberger, 1999).