Disodium 1-amino-9,10-dihydro-9,10-dioxo-4-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]amino]anthracene-2-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

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:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 1-amino-9,10-dihydro-9,10-dioxo-4-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]amino]anthracene-2-sulphonate

EC Number:

240-267-2

EC Name:

Disodium 1-amino-9,10-dihydro-9,10-dioxo-4-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]amino]anthracene-2-sulphonate

Cas Number:

16102-99-1

Molecular formula:

C22H18N2O11S3.2Na C22H16N2Na2O11S3

IUPAC Name:

disodium 1-amino-9,10-dioxo-4-({4-[2-(sulfooxy)ethanesulfonyl]phenyl}amino)-9,10-dihydroanthracene-2-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

Name Reaktiv-Brillantblau P
Other names : F - 64 357

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from rat liver

Test concentrations with justification for top dose:

The first experiment was performed with all tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an
appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity.
Thinning of the bacterial lawn was controlled microscopically.
In combination with the second experiment, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly
mixed with 0.1 ml of 10-6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control
is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).


The test compound was tested at doses of 4 to 10000 µg/plate in the first experiment and proved to be not toxic to the bacteria.

For mutagenicity testing in the second experiment, 5000 pg/plate was chosen as the highest dose.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: N-Methyl-N-nitro-N-nitrosoguanidine; 2-Aminoanthracene

Details on test system and experimental conditions:

Top agar is prepared for the Salmonella strains by mlxlng 100 ml agar (0.6 % agar, 0.5 %NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. With E. coli histidine is replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients are added (in order) to 2 ml of molten top agar at 45°C:
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution
0.5 ml S9 Mix (if required) or buffer
After mixing, the liquid is poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for 48 to 72 hour at 37°C in the dark, colonies (his+ revertants) are counted.

Positive controls
Positive control plates were included for each strain. The following substances were used as positive controls.
a) without metabolic activation:
Na-azide: TA 100, TA 1535;
9-Aminoacridine: TA 1537
2-Nitrofluorene: TA 98, TA 1538
N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG): WP2uvrA

b) with metabolic activation:
Benzo[a]pyrene: TA 98, TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA
2-Aminoanthracene: TA 98, TA 100, TA 1535, TA 1537, TA 1538, WP2uvrA

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

up to 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Remarks:

up to 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or presence of S-9 Mix. No dose dependent effect was obtained.

Applicant's summary and conclusion

Conclusions:

Reactive Blue 19:1 is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Executive summary:

Reactive Blue 19:1 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 µg/plate to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains. On the basis of the preliminary test results the top dose level did not exceed 5000 µg/plate.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Reactive Blue 19:1 did not result in relevant increases in the number of revertant colonies.

Summarizing, it can be stated that Reactive Blue 19:1 is not mutagenic in these bacterial test systems neither with nor without exogenous metabolic activation at the dose levels investigated.