tetrasodium 4-{4-[(9,10-dioxo-4-{[4-(2-sulfophenoxy)phenyl]amino}-9,10-dihydroanthracen-1-yl)amino]phenoxy}benzene-1-sulfonate 4-{4-[(9,10-dioxo-4-{[4-(4-sulfophenoxy)phenyl]amino}-9,10-dihydroanthracen-1-yl)amino]phenoxy}benzene-1-sulfonate

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:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No bacterial tester strain to detect cross-linking mutagens included (e.g. E.coli wp2/uvra)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OFFICIAL JOURNAL OF THE EUROPEAN COMMUNITIES, No. L 383 A, Volume 35, 160-162, Annex to Commission Directive 92/69/EEC of July 31, 1992: Salmonella typhimurium - Reverse Mutation Assay, December 29, 1992.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Disodium 2-{4-[(9,10-dioxo-4-{[4-(4-sulfonatophenoxy)phenyl]amino}-9,10-dihydroanthracen-1-yl)amino]phenoxy}benzenesulfonate and Disodium 4,4'-[(9,10-dioxo-9,10-dihydroanthracene-1,4-diyl)bis(iminobenzene-4,1-diyloxy)]dibenzenesulfonate

EC Number:

941-784-2

Molecular formula:

C38H26N2O10S2.2Na

IUPAC Name:

Reaction mass of Disodium 2-{4-[(9,10-dioxo-4-{[4-(4-sulfonatophenoxy)phenyl]amino}-9,10-dihydroanthracen-1-yl)amino]phenoxy}benzenesulfonate and Disodium 4,4'-[(9,10-dioxo-9,10-dihydroanthracene-1,4-diyl)bis(iminobenzene-4,1-diyloxy)]dibenzenesulfonate

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): FAT 21030/D (Irganol Grün BLS roh trocken).
- Batch N°: 16.36.
- Purity: 67 %.
- Appearance: Greenblack fragments
- Expiry date: December 31, 1998.
- Storage: Room temperature.

Specific details on test material used for the study:

Test material: FAT 21030/D (Irganol Grün BLS roh trocken)
Batch No.: 16.36
Purity: 67 %
Appearance: Green black fragments.
Expiry date: December 31, 1998
Storage: Room temperature

Method

Target gene:

Salmonella typhimurium histidine auxotrophs

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction: Rat-liver post mitochondrial supernatant.

Test concentrations with justification for top dose:

Concentrations tested in the range finding test: 20.58, 61.73, 185.19, 555.56, 1666.67 and 5000 µg/plate

Concentration range in the mutagenicity test:
- Original experiment: 92.13, 276.40, 829.19, 2487.56 and 7462.69 µg/plate
-Confirmatory experiment: 92.13, 276.40, 829.19, 2487.56 and 7462.69 µg/plate

In preliminary test conducted at test concentration of 5000 µg/plate, normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 7462.7 µg/plate with and without metabolic activation. (The purity of the tested batch is 67%, hence 7462.7 µg/plate correspond to 5000 µg/plate of pure substance).

Vehicle / solvent:

Bidistilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cyclophosphamide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation);
Agar plate incorporation : 0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl and was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water.

Evaluation criteria:

A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response
The test substance will be considered positive in the test system if the following condition is met:
- At least a reproducible meaningful increase of the mean number of revertant per plate above that of the negative control at any concentration for one or more of the strains tested. Generally a concentration-related effect should be demonstrable.

Statistics:

A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.

Results and discussion

Test results

Species / strain:

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

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

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Range finding test

Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 7462.7 ug/plate with and without metabolic activation. (The purity of the tested batch is 67 %, hence 7462.7 µg/plate correspond to 5000 µg/plate of pure substance).

Mutagenicity test, original experiment

In the experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with FAT 21030/D (Irganol Grün BLS roh trocken) did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

Mutagenicity test, confirmatory experiment

In the experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with FAT 21030/D (Irganol Grün BLS roh trocken) no increase in the incidence of histidine-prototrophic mutants was observed in comparison with the negative control.

In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria.

There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

Applicant's summary and conclusion

Conclusions:

FAT 21030/D and its metabolites did not induce gene mutations in the strains Salmonella typhimurium used (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100).

Executive summary:

The purpose of this study is to evaluate the test compound for mutagenic activity in bacterial test systems in the presence and absence of a rat liver metabolic activation system. This experiment was performed in compliance with GLP and according to the OECD Guideline 471 (Bacterial Reverse Mutation Assay).

FAT 21030/D, purity 67%, batch no. 16.36, was tested for mutagenic effects in vitro in histidinerequiring strains of Salmonella typhimurium. The following strains of Salmonella typhimurium were used: TA 98, TA 100, TA 1535 and TA 1537. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was dissolved in bidistilled water and tested at five concentrations in the range of 92.1 to 7462.7 µg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 92.1 to 7462.7 µg/plate. (The purity of the tested batch is 67%, hence 7462.7 µg/plate correspond to 5000 µg/plate of pure substance.) 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.

In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 21030/D led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. Hence, it was concluded that FAT 21030/D and its metabolites did not induce gene mutations in the strains Salmonella typhimurium used (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100).