C.I. Reactive Yellow 161

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:

1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only hamster S9, only Salmonella strains

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

400-640-4

EC Name:

-

Cas Number:

198153-89-8

Molecular formula:

C32ClH25N10Na2O9S3

IUPAC Name:

disodium 2-(4-{[4-({3-[2-(5-carbamoyl-1-ethyl-2-hydroxy-4-methyl-6-oxo-1,6-dihydropyridin-3-yl)diazen-1-yl]-4-sulfonatophenyl}amino)-6-chloro-1,3,5-triazin-2-yl]amino}phenyl)-6-methyl-1,3-benzothiazole-7-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

Reactive Yellow 161

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Hamster liver S9

Test concentrations with justification for top dose:

10.0; 100.0; 333.3; 1000.0; and 5000 μg/plate
based on a toxicity test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and relative non-toxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

other: 4-NOPD

Details on test system and experimental conditions:

Pre-incubation test
For each strain and dose level, including the controls, a minimum of three plates was used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl Test solution at each dose level,solvent control, negative control, or reference mutagen solution (positive control),
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
100 µl Bacteria suspension (cf. test system, pre-culture of the strains),
After pre-incubation 2.0 ml of molten 45 °C overlay agar was added to each tube. The mixture was poured an minimal agar plates.
After solidification the plates were incubated upside down for 72 hours at 37° C in the dark.

Evaluation criteria:

A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of FAT 40'162/B to induce gene mutations according to the pre-incubation test for azo dyes using the Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was te-sted in triplicate. The test article was tested at the following concentrations:

10.0; 100.0; 333.3; 1000.0; and 5000.0 µg/plate.

Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred in the test groups with and without metabolic activation at the highest investigated dose.

The plates incubated with the test article showed normal Background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Up to the highest investigated dose, no significant and repro-ducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

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

Therefore, FAT 40'162/B is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.