1,1'-diethyl[3,3'-bianthra[1,9-cd]pyrazole]-6,6'(1H,1'H)-dione

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:

From ferbruay 22 to March 13, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 31, 1992

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 13, 1987

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Vat Red 013

IUPAC Name:

Vat Red 013

Test material form:

solid

Method

Target gene:

Histidine locus

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:

Rat liver S9 fraction

Test concentrations with justification for top dose:

4.12, 12.35, 37.04, 111.11, 333.33 and 1000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle:acetone was found to be the best suited vehicle yielding an applicable suspension of 10 mg/ml.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Solvent alone

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

Solvent alone

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in 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 20.0 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.

NUMBER OF REPLICATIONS: 3 per test substance concentrations and per control

DURATION
The plates were inverted and incubated for about 48 hours at 37 ± 1.5 °C in darkness

EVALUATION
The plates were evaluated by counting the number of colonies and determining the background lav/n

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:

other: TA 98, TA 100, TA 1535 and 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

Any other information on results incl. tables

Range finding test

Six concentrations of the test substance ranging from 4.1 to 1000.0 ug/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. 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 1000.0 ug/plate with and without metabolic activation.

Mutagenicity test

In the original experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with the test substance did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the

negative control.

In the confirmatory experiments performed with and without metabolic activation, again after treatment of strains TA 98, TA 100, TA 1535 and TA 1537 with the test substance 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.

Applicant's summary and conclusion

Conclusions:

Not mutagenic

Executive summary:

Method

The test substance, was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium, according to the OECD guideline 471. 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 aceton and tested at five concentrations in the range of 4.12 to 1000.0 ug/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 the same concentrations. 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.

Results

In both experiments, performed with and without metabolic activation, none of the tested concentrations of the test substace led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.