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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:
15 May 2001 to 08 June 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
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: T-7479
- Physical state: Clear, colorless liquid
- Analytical purity: Not indicated but treated as 100% pure
- Expiration date of the lot/batch: 23 March 2002
- Stability under test conditions: Ethanol: Not indicated
- Storage condition of test material: At room temperature in the dark.

Method

Species / strain
Species / strain / cell type:
bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100. WP2uvrA also used
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
First Experiment 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate.
Vehicle / solvent:
-ethanol
- best solubility characteristics
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, daunomycine, methylmethanesulfonate, 4-nitroquinoline N-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION:

Combined Range Finding/First Experiment

Selection of an adequate range of doses was based on a range finding test in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Seven concentrations of the test substance, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate were tested in triplicate.

Second Experiment

Five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.

The highest concentration of Test Substance used in the subsequent mutation assay was the recommended 5 mg/plate.

Experimental procedure

The test substance was tested both in the absence and presence of S9-mix in each strain, in two independent experiments.

Top agar in top agar tubes was molten and heated to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 ± 1 °C for 48 h. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli) were counted.
Evaluation criteria:
No formal hypothesis testing was done.
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 at least a 2-fold, dose related increase in the number of revertants with respect to the number 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
Species / strain:
other: 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
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
None
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 Criteria used for interpretation of results: expert judgment

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The test substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA in two independent experiments.

The test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. The test substance did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at all concentrations tested and no biologically relevant decrease in the number of revertants was observed.

The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

The test substance did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

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