Salts of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthenium chloride and 4-{[1-hydroxy-6-({[5-hydroxy-6-(phenyldiazo)-7-sulfonaphthalen-2-yl]carbamoyl}amino)-3-sulfonaphthalen-2-yl]diazo}benzoic acid (2:1)

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:

from 2008-05-14 to 2008-07-03

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

not applicable

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with

Metabolic activation system:

2-Aminoanthracene, 2AA

Test concentrations with justification for top dose:

The examined concentration levels were different at each examined bacterium strain. The maximum test concentration was 5000 or 2000 μg/plate in case of Salmonella typhimurium strains (depending on the test method and on the presence or absence of metabolic activation) and 5000 μg/plate in case of Escherichia coli WP2 uvrA:
- Initial Mutation Test (Plate Incorporation Test): 2000; 632.5; 200; 63.25; 20; 6.325 and 2 µg/plate (S. typhimurium strains) and 5000; 1581.1; 500; 158.1; 50; 15.81 and 5 μg/plate (E. coli WP2 uvrA).
- Complementary Plate Incorporation Test in S.typhimurium TA 100: 2; 0.633; 0.2 μg/plate (–S9 Mix); 5000 and 2000 μg/plate (+S9 Mix); in TA 1535: 5000 and 2000 μg/plate (±S9 Mix) and in TA 1537: 2 and 0.633 μg/plate (±S9 Mix).
- Confirmatory Mutation Test (Pre-Incubation Test) the examined concentration levels were in case of S.typhimurium TA 98 and TA 1535: 2000; 632.5; 200; 63.25; 20; 6.325; 2 and 0.633 μg/plate; in case of TA 100 and TA 1537: 200; 63.25; 20; 6.325; 2; 0.633 and 0.2 μg/plate. In case of E. coli WP2 uvrA the 5000; 1581.1; 500; 158.1; 50; 15.81 and 5 μg/plate concentration levels were tested.
- Complementary Pre-Incubation Test the examined concentration levels were in case of S.typhimurium TA 98 and TA 1535: 5000 and 2000 μg/plate (±S9 Mix) and in TA 100 and TA 1537: 2000; 632.5 and 200 μg/plate (±S9 Mix).

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Details on test system and experimental conditions:

see attachment for details

Evaluation criteria:

The colony numbers for control, positive control and the test plates were determined, the mean values and appropriate standard deviations were calculated. The mutation factor (MF) was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (exact, not rounded values were used for this calculation).

Statistics:

not applicable

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and 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:

not applicable

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

F 213 Red is considered as non mutagenic.

Executive summary:

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, F 213 Red is considered non-mutagenic in this bacterial reverse mutation assay.