Sodium 4-[[3-(acetylamino)phenyl]amino]-1-amino-9,10-dihydro-9,10-dioxoanthracene-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:

2000-09-26 - 2000-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-[[3-(acetylamino)phenyl]amino]-1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

EC Number:

274-675-7

EC Name:

Sodium 4-[[3-(acetylamino)phenyl]amino]-1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Cas Number:

70571-81-2

Molecular formula:

C22H17N3O6S.Na

IUPAC Name:

sodium 4-[(3-acetamidophenyl)amino]-1-amino-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

The test compound was suspended in deionized 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 and 50 µg/plate were used in the first plate incorporation test.
In the second experiment with the strain TA 98 dilutions of 5000, 1600, 500, 160, 50, 16, 5 and 1.6 µg/plate were chosen.
Visible precipitation of the test compound on the plates was observed at 1600 µg/plate and above.
Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 1600 µg/plate and lower concentrations.
In the first plate incorporation test toxicity was observed at a concentration of 1600 µg/plate in the absence of metabolic activation with the tester strain TA 100.

Vehicle / solvent:

Solvent: deionised water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

Evaluation criteria:

Assay considered valid if:
-Solvent control data are within the laboratory´s normal control range for the spontaneous mutant frequency
-Positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory´s normal range

Test compound is classified as mutagenic if has either following effects:
-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 background lawn
-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 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

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

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

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

Test results 4

Species / strain:

S. typhimurium TA 102

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 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

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 6

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

ambiguous

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: -
- Effects of osmolality: -
- Evaporation from medium: -
- Water solubility: > 10 g/L
- Precipitation: yes; from 1600 µg/plate onwards
- Definition of acceptable cells for analysis: -
- Other confounding effects:


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

Remarks on result:

other: no dose-dependency observed

Applicant's summary and conclusion

Conclusions:

In the presence of metabolic activation, treatment of the bacterial strain with Acid Blue 324 resulted in increases in the number of revertant colonies with the strain TA 98 without showing any dose-dependency; hence not meeting the criteria for a positive result.

Executive summary:

Acid Blue 324 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 and TA 102 of Salmonella typhimurium. This mutagenicity study was conducted (plate incorporation test), in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. Additionally a repeat of the plate incorporation test was performed with the strain TA 98 in the presence of S9-mix.

For both studies, the compound was suspended in deionized water, and each bacterial strain was exposed to 5 dose levels, in the repetition with the strain TA 98 to 8 dose levels.

Visible precipitation of the test compound on the plates was observed at 1600 µg/plate and above. The concentrations for the first plate incorporation test were 50, 160, 500, 1600 and 5000 µg/plate. Because of the increased mutation frequencies (2.1 to 5.2 -fold without dose-dependency) observed with the strain TA 98, dose levels from 1.6 to 5000 µg/plate were chosen for the second plate incorporation test with TA98 in the presence of the metabolizing system.

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.

In the first plate incorporation test toxicity was observed at a concentration of 1600 µg/plate in the absence of metabolic activation with the tester strain TA 100.

In the presence of metabolic activation, treatment of the bacterial strain with Acid Blue 324 resulted in increases in the number of revertant colonies with the strain TA 98 without showing any dose-dependency; hence not meeting the criteria for a positive result.