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Toxicological information

Genetic toxicity: in vitro

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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:
2003-06-06 to 2003-06-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented, scientifically sound study conducted according to GLP and OECD guideline 471 (Bacterial Reverse Mutation Assay).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

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)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tungsten Oxide Blue
- Molecular formula (if other than submission substance): WO
- Substance type: Active
- Physical state: Blue to black powder
- Analytical purity: 99.9 %
- Purity test date: 2003-04-15
- Stability under test conditions: 5 years
- Storage condition of test material: Ambient temperature
- Solubility in water: 118 mg/L at 20 degrees C

Method

Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Additional strain / cell type characteristics:
other: See additional information on methods below
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
- 62, 185, 556, 1667 and 5000 ug/plate
-The test substance solution of the highest concentration group was prepared by suspending 500 mg of the test substance in 10 mL of 0.5 % carboxymethyl cellulose. The suspensions for the lower concentrations were prepared by subsequent dilution of one volume of the higher concentrated solution with two volumes of 0.5 % carboxymethyl cellulose. The test substance was suspended and diluted immediately before the start of the Ames test. The time between the preparation and the administration to the last petri dish was less than 3 hours.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 0.5 % carboxymethyl cellulose
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Remarks:
N/A
Positive controls:
yes
Remarks:
N/A
Positive control substance:
other: 2-Aminoanthracene- used for tester strains TA98 (1 ug/plate), TA100 (2 ug/plate) and TA1535 ( 2 ug/plate)
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation Migrated to IUCLID6: used for tester strain TA97a (10 ug/plate)
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxy-anthraquinone- used for tester strain TA102 (50 ug/plate)
Remarks:
with metabolic activation
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation Migrated to IUCLID6: used for tester strain TA98 (2 ug/plate)
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation Migrated to IUCLID6: used for tester strains TA100 (2 ug/plate) and TA1535 (1 ug/plate)
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine- used for tester strain TA97a (10 ug/plate)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
Vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % carboxymethyl cellulose
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: t-Butyl-hydroperoxide- used for tester strain TA102 (50 ug/plate)
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION
-In agar (plate incorporation).

DURATION
- Exposure duration: 2 days
- Selection time (if incubation with a selection agent): 2 days

SELECTION AGENT (mutation assays)
-Histidine

NUMBER OF REPLICATIONS
-3 replications were used for the test substance and positive control, and 6 replications were used for the solvent control.

NUMBER OF CELLS EVALUATED
-The number of viable cells in the overnight-cultures was in the range of 2 x E8 cells/mL.

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn.


OTHER: The actual batch of the strains was tested for ampicillin resistance (TA102: ampicillin/tetracycline resistance), UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances in September 2000. The bacteria were store frozen since that time.
Evaluation criteria:
Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result were:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
-For the strains with a low spontaneous revertant rate (TA98 and TA1535): 2 1/2 fold of the amount of the spontaneous revertants.
-For the strains with a high spontaneous revertant rate (TA97a, TA100 and TA1020: 1 2/3 fold of the amount of the spontaneous revertants.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: 118 mg/L at 20 degrees C
- Precipitation: Although the test substance was not soluble, the particles were so fine that they were not visible in any of the concentration groups.

RANGE-FINDING/SCREENING STUDIES
The test substance was evaluated in a preliminary toxicity test using the Plate Incorporation Assay method. The test substance was tested at concentrations ranging from 21-5000 ug/plate in tester strain TA100 in the absence of metabolic activation. The test substance was not toxic up to 5000 ug/plate. It was therefore decided to use 5000 ug/plate as the highest concentration for the Ames Assay, which was the limit concentration according to the guidelines. Each of the other 4 concentrations were 1/3 of the preceding one.

COMPARISON WITH HISTORICAL CONTROL DATA
Solvent control values were within historical range.

OTHER
All positive control substances increased the mutation frequency to more than the threshold values stated above. As 2-aminoanthracene, 1,8-dihydroxy-anthraquinone and 7,12-dimethyl-benz[a]anthracene require metabolic activation for mutagenicity, the result of these substances demonstrated the efficiency of the metabolizing system.
There were no substantial increases in the number of mutants in any of the tested bacterial strains at any of the tested concentrations of the test substance. The addition of S9 metabolic activation did not change these results.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The results were verified by a second independent experiment.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this assay, Tungsten Oxide Blue was non-mutagenic in the Ames test with Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the presence and absence of metabolic activation up to 5000 ug/plate, which was the limit concentration for this substance.