Anthra[9,1,2-cde]benzo[rst]pentaphene-5,10-dione, amino-, reaction products with 1-amino-9,10-anthracenedione and tetrabromo-8,16-pyranthrenedione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Ames test

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Anthra[9,1,2-cde]benzo[rst]pentaphene-5,10-dione, amino-, reaction products with 1-amino-9,10-anthracenedione and tetrabromo-8,16-pyranthrenedione

EC Number:

215-520-5

EC Name:

Anthra[9,1,2-cde]benzo[rst]pentaphene-5,10-dione, amino-, reaction products with 1-amino-9,10-anthracenedione and tetrabromo-8,16-pyranthrenedione

Cas Number:

1328-25-2

Molecular formula:

C126H58N4O10

IUPAC Name:

anthra[9,1,2-cde]benzo[rst]pentaphene-5,10-dione, amino-, reaction products with 1-amino-9,10-anthracenedione and tetrabromo-8,16-pyranthrenedione

Test material form:

solid: particulate/powder

Details on test material:

Vat Black 9

Method

Target gene:

histidine

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:

S-9 fraction (rat liver homogenate)

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500 and 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitroso-guanidine, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Standard plate test
Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation) or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

Preincubation test
The experimental procedure is based on the method described by Yahagi et al. (5) and Matsushima et al. (6).
0.1 ml test solution, 0.1 ml bacterial suspension and
0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.

After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Evaluation criteria:

Positive effects:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Positive and negative controls were valid

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was considered not to be mutagenic in Salmonella typhimurium TA98, TA100, TA1535, and TA1537.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471. Four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) were exposed to the test substance at concentrations of 20 to 5000.0 µg/plate with or without metabolic activation (S-9 Mix) for 48 h.

A weakly, bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed only using TA 100 in the standard plate test without S-9 mix at doses > 500 µg/plate. Incomplete solubility of test substance in DMSO was observed at from about 2500 µg/plate onward.

Negative and positive controls were valid. The test substance with and without metabolic activation did not induce mutagenic activity in bacteria. Under the study conditions, the test substance was considered not to be mutagenic.