Registration Dossier

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:
2008-04-21 to 2008-08-19
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: Reaction product of Acid Yellow 42, Acid Black 2 and Basic Violet 1
- Lot/batch No.: 2008041541
- Expiration date of the lot/batch: 2010-01-31
- Stability under test conditions: see expiry date
- Storage condition of test material: store cool and dry
- Analytical purity: 100 % (96.8 %, UVCB substance)

Method

Target gene:
Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of F 46 Black in independent experiments, in plate incorporation tests and in pre-incubation tests.
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the results of a preliminary toxicity test concentrations were chosen for the plate incorporation tests (initial and complementary) and pre-incubation tests (initial and complementary).
In the initial mutation test (plate incorporation test) the examined concentration levels were: 60; 24; 9.6; 3.84; 1.536; 0.614 and 0.246 μg/plate (Salmonella typhimurium strains) and 500; 158.1; 50; 15.81; 5; 1.581 and 0.5 μg/plate (Escherichia coli WP2 uvrA). In the complementary plate incorporation test only the Escherichia coli WP2 uvrA was examined at the concentration levels of 5000 and 1581.1 μg/plate (±S9 Mix).
In the confirmatory mutation pre-incubation test the examined concentration levels were in case of Salmonella typhimurium strains: 24; 9.6; 3.84; 1.536; 0.614; 0.246 and 0.098 μg/plate. In case of Escherichia coli WP2 uvrA the 1581.1; 500; 158.1; 50; 15.81; 5 and 1.581 μg/plate concentration levels were tested. In the complementary pre-Incubation test the examined concentration levels were in case of in case of Salmonella typhimurium TA 98 and TA 100: 375; 150; 60 and 24 μg/plate (+S9 Mix), in case of Salmonella typhimurium TA 1535: 150; 60; 24; 0.098 and 0.031 μg/plate (+S9 Mix), the concentration levels of 0.098 and 0.031 μg/plate in absence of metabolic activation (–S9 Mix) were also tested, in case of Salmonella typhimurium TA 1537: 150; 60 and 24 μg/plate (+S9 Mix).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, distilled water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
S. typhimurium: TA 98
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine, 4-NOPD (or NPD)
Remarks:
4 μg/plate, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
S. typhimurium: TA 100; TA 1535
Positive control substance:
sodium azide
Remarks:
2 µg/plate, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
S. typhimurium: TA 1537
Positive control substance:
9-aminoacridine
Remarks:
50 µg7plate, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
E. coli WP2uvrA
Positive control substance:
methylmethanesulfonate
Remarks:
2 µL/plate, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
S. typhimurium: TA 100; TA 98; TA1535; TA 1537 and E. coli WP2uvrA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
2 μg/plate for S. typhimurium strains; 50 μg/plate for E. coli WP2uvrA; With metabolic activation [10 % (v/v) rat liver]
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
S. typhimurium: TA 100; TA1535; TA 1537 and E. coli WP2uvrA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
2 μg/plate for S. typhimurium strains; 50 μg/plate for E. coli WP2uvrA; With metabolic activation [30 % (v/v) hamster liver]
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Remarks:
S. typhimurium: TA 98
Positive control substance:
congo red
Remarks:
250 μg/plate; With metabolic activation [30 % (v/v) hamster liver]
Evaluation criteria:
A test item is considered mutagenic if: – a dose–related increase in the number of revertants occur and/or
– a reproducible biologically relevant positive response for at least one of the dose groups occurs in at
least one strain with or without metabolic activation.
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.


Statistics:
a statistical evaluation of the results is not regarded necessary

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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 46 Black was considered non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

Based on the results obtained in the present Ames test according to EU method B.13/14 and OECD guideline 471 the test item F 46 Black was not considered mutagenic.