Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
yes
Remarks:
2AA used as sole positive control substance with S9-mix
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
July 23, 1999
Qualifier:
according to
Guideline:
other: Guidelines stipulated by the Japanese Ministry of Labor and Japanese Ministry of International Trade and Industry
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Lot/batch No.: 004716.H9
- Physical state: yellow powder
- Stability under test conditions: stable (at least 96 h in DMSO)
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
his and trp operon
Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
the S9-mix was prepared from uninduced male Golden Syrian Hamster liver
Test concentrations with justification for top dose:
- Combined range finding/First experiment: 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
- Second experiment: 3, 10, 33, 100 and 333 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Remarks:
the Ames test is regularly conducted in the testing facility
Positive controls:
yes
Positive control substance:
other: Without S9-mix: 2 µg SA for TA1535, 10 µg NF for TA1537 and 4 µg DM for TA98 in saline, or 650 µg MMS for TA100 and 1 µg 4-NQO for WP2uvrA in DMSO. With S9-mix: 1, 2.5 or 5 µg 2AA, respectively for TA1535 and TA98, TA1537 or WP2uvrA and TA100 in DMSO
Remarks:
SA (sodium azide), NF (2-nitrofluorene), DM (daunomycine), MMS (methylmethanesulfonate), 4-NQO (4-nitroquinoline N-oxide) and 2AA (2-aminoanthracene)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation (in triplicates)

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 hours

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 were evaluated
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria: (1) the negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain. (2) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance. (3) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

- A test substance is considered negative (not mutagenic) in the test if: (a) the total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation. (b) The negative response should be reproducible in at least one independently repeated experiment.
- A test substance is considered positive (mutagenic) in the test if: (a) it induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant. (b) The positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No reduction of the bacterial background lawn and no decrease in the number of revertants was observed
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Precipitation on the plates was obsenved at the start and at the end of the incubation period at concentrations of 333 µg/plate and upwards. In the second experiment, the test substance precipitated in the top agar concentration of 333 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
Performed

Any other information on results incl. tables

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. The strain-specific positive control values were within the laboratory background historical control data ranges, except for TA1535 in the absence of S9-mix (first experiment). However, since the response of the mean number of revertant colonies was more than 10-fold compared to the solvent control value and the mean number of revertant colonies of TA1535 was just outside the limit of the range, the validity of the test was considered not to be affected.

Applicant's summary and conclusion

Conclusions:
negative