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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:
July 26 - October 01, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: Testing Methods Concerning New Chemical Substances
Qualifier:
according to
Guideline:
other: Notice on Standards Issued by Ministry of Labor Based on provisions of Article 57-3, Paragraph 1 of Industrial Safety and Health Act
Qualifier:
according to
Guideline:
other: Partial Revision of Notice on Standards Issued by Ministry of Labor Based on Provisions of Article 57-3, Paragraph 1 of Industrial Safety and Health Act
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): E-BW102
- Physical state: Reddish brown powder
- Stability under test conditions: Stability of the test substance was confirmed by the data obtained from the sponsor
- Stability in water: Stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Preliminary test: TA100, TA1535, TA98, TA1537 and WP2uvrA:
Without and with S9-mix: 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate

Main study 1 and 2: TA100, TA1535, TA98, TA1537 and WP2uvrA:
Without and with S9-mix: 313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterilized distilled water
- Justification for choice of solvent/vehicle: In the solvent selection test, the test substance was dissolved at 50 mg/mL in distilled water. Increases in temperature, discoloration and foaming were not observed when the test substance was mixed with distilled water.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9

Migrated to IUCLID6: 0.5 µg/plate in distilled water for TA1535
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9

Migrated to IUCLID6: 80 µg/plate in DMSO for TA1537
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-(2-Fury)-3-(5-nitro-2-furyl)acrylamide, 0.01 µg/plate in DMSO for TA100 and WP2 uvrA and 0.1 µg/plate in DMSO for TA98
Remarks:
without S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 0.5 µg/plate in DMSO for TA98, 1.0 µg/plate in DMSO for TA100, 2.0 µg/plate in DMSO for TA1535 and TA1537, and 10 µg/plate in DMSO for WP2 uvrA
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Preliminary test: 1 plate/dose (2 plates/dose for the negative and positive controls)
- Main test 1 and 2: 3 plates/dose

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
The test substance was judged to have mutagenicity (positive) when the test substance induced a dose-dependent increase in the number of the revertant colonies (mean) to a level equal to or greater than 2-fold of the negative (solvent) control value (mean value) in any one of the tester strains with or without S9 mix, and when the dose-dependent increase was reproducible. Other results were judged to be negative. When the test substance was positive, mutation activity (number of revertant colonies/mg) was calculated.
Statistics:
No statistical analysis was performed.

Results and discussion

Test resultsopen allclose all
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:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No mutagenicity was observed up to and including the top dose of 5000 µg/plate

Any other information on results incl. tables

Results and tables, see the attached document "Tables bacterial reverse Mutation Test".

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Bacterial reverse mutation test with E-BW102 performed according to OECD 471 Guideline and GLP principles.

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that E-BW102 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.