Reaction mass of disodium 2-({4-methyl-2-[(4-{[5-methyl-1-(2-sulfonatophenyl)hexan-3-yl]amino}-9,10-dioxo-9,10-dihydroanthracen-1-yl)methyl]pentyl}amino)benzene-1-sulfonate,disodium 4-{5-methyl-3-[(4-{[5-methyl-1-(4-sulfonatophenyl)hexan-3-yl]amino}-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]hexyl}benzene-1-sulfonate anddisodium 4-{5-methyl-3-[(4-{[5-methyl-1-(2-sulfonatophenyl)hexan-3-yl]amino}-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]hexyl}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:

June 16th, 1998 to June 25th, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guideline For Testing Of Chemicals, 471 Bacterial Reverse Mutation Test Adopted: July 21st 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69, L 383 A, Annex B 14
EEC Directive 92/69, L 383 A, Annex B 13

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: U.S. EPA: 798.5265 The Salmonella typhimurium reverse mutation assay

Version / remarks:

U.S. EPA: 798.5265 The Salmonella typhimurium reverse mutation assay Fed. Reg. 50, Subpart F, September 1985

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: U.S. EPA: 798.5100 The Escherichia coli WP2 and WP2 uvrA reverse mutation assay

Version / remarks:

U.S. EPA: 798.5100 The Escherichia coli WP2 and WP2 uvrA reverse mutation assay Fed. Reg. 50, Subpart F, September 1985

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino[3-isopropylpropane-1,3-diyl]]]bis(benzenesulphonate)

EC Number:

276-822-0

EC Name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino[3-isopropylpropane-1,3-diyl]]]bis(benzenesulphonate)

Cas Number:

72749-90-7

Molecular formula:

C40H46N2O8S2.2Na

IUPAC Name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino[3-isopropylpropane-1,3-diyl]]]bis(benzenesulphonate)

Test material form:

solid: particulate/powder

Details on test material:

Acid Blue 221

Method

Target gene:

histidine and tryptophan

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

first experiment withand without metabolic activation: 50, 160, 500, 1600, 5000 μg/plate
second experiment withand without metabolic activation: 16, 50, 160, 500, 1600, 5000 μg/plate

Vehicle / solvent:

Formulation of test compound
suspended in double-distilled water at appropriate concentrations immediately before use.

Formulation of reference compounds
sodium-azide dissolved in double-distilled water final concentration: 1.0 μg/plate for strain TA 100 and TA 1535
9-aminoacridine dissolved in DMSO final concentration: 50.0 μg/plate for strain TA 1537
2-nitrofluorene dissolved in DMSO final concentration: 2.5 μg/plate for strain TA 98
MNNG dissolved in DMSO final concentration: 4 μg/plate for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations:
0.5 μg/plate for strain TA 98 and TA 100
1.0 μg/plate for strain TA 1535 and TA 1537
30.0 μg/plate for strain WP2uvrA

The frozen stock solutions of each compound were diluted progressively up to the final concentration on the day of treatment.

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

other: 1-methyl-3-nitro-1-nitrosoguanidine (MNNG); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

Assay procedure
The first mutation test was performed in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance.
Positive and negative controls as well as solvent controls were included in each test.
Triplicate plates were used.
The first mutation experiment also assessed the toxicity of the test substance by evaluation of the bacterial lawn in order to select a suitable range of dose levels for a second mutation test. The highest concentration was usually 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 μg/plate.
Further dilutions of 1600, 500, 160 and 50 μg/plate were used.
A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
If the number of evaluable concentrations was less than 5, a repeat mutation experiment on the basis of toxicity results in the first test was performed.
If negative or equivocal results obtained, a second mutation experiment was performed on the basis of toxicity results in the first test as a pre-incubation test.
For mutagenicity testing top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 ml of a 0.5 mM histidinebiotin solution. With E. coli histidine was replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution (dissolved in double-distilled water)
Only for the second mutagenicity test this top-agar liquid was pre-incubated for approximately 20 minutes.
After mixing, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2% (w/v) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (his+ and trp+ revertants) were counted with an automatic colony counter (Artec counter Model 880). The counter was calibrated for each test by comparison of manual count data of three control plates with automatic data of the colony counter.
A correction factor was determined to compensate for differences between manual and automatic count. This correction factor was used to automatically adjust the observed number of colonies on each plate to more accurately reflect the actual number of colonies present.

Rationale for test conditions:

In accordance with test guidelines.

Evaluation criteria:

Criteria for a valid assay
The assay is considered valid if the following criteria are met:
-the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
-the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range

Criteria for a positive response
A test compound is classified as mutagenic if it has either of the following effects:
a) it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial backgrounc;tlawn
b) it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

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 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

Key result

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 3

Key result

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 4

Key result

Species / strain:

S. typhimurium 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

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

Sterility checks and control plates
Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.

Solubility and toxicity
The test compound was suspenden in double-distilled water and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 μg/plate. Further dilutions of 1600, 500, 160, 50 and 16 μg/plate were used.
The test compound did not precipitate on the plates up to the highest investigated dose of 5000 μg/plate.
The test compound proved to be not toxic to the bacterial strains in the mutagenicity experiments.

Mutagenicity
In both independent mutation tests the number of colonies per plate with each strain as well as mean values of 3 plates are given.
The test compound did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or in the presence of S9-mix in either mutation test. No dose-dependent effect was obtained.
All positive controls produced significant increases in the number of revertant colonies.
Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.

Applicant's summary and conclusion

Conclusions:

The results lead to the conclusion that Acid Blue 221 is not mutagenic in these bacterial test systems either in the absence or in the presence of an exogenous metabolizing system.
The substance is therefore not classified in accordance with CLP criteria.

Executive summary:

Acis Blue 221 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

 

Two independent mutagenicity studies were conducted, each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate.

For both studies, the compound was dissolved in double-distilled water, and each bacterial strain was exposed to 5 dose levels in the first and 6 dose levels in the second experiment. Concentrations for both studies ranged from 50 to 5000 μg/plate.

 

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All the positive control compounds showed the expected increase in the number of revertant colonies.

 

Toxicity: In both mutagenicity experiments toxicity was not observed either with or without metabolic activation.

 

Mutagenicity: In the absence and in the presence of the metabolic activation system, Acid Blue 221 did not result in relevant increases in the number of revertants in any of the bacterial strains.

 

Summarizing, it can be stated that Acid Blue 221 was not mutagenic in this bacterial mutation test at any dose level in either the absence or in the presence of an exogenous metabolic activation.