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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: diluted in acetone

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Five concentration of the test material (50,150,500,1500 and 5000 µg) were assayed in triplicate against each testster strain, using the direct plate incorporation method.
Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
The test material was accurately weighed and appximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. Formulated concentratoins were adjusted to exclude the stated water/impurity content (30.3%) of the test material. Prior to use, the solvent was dried using molecular sieves. Vehicle and positive controls were used in parallel with the test material.
Evaluation criteria:
The test materila may be considered positive in this test system if the following critiera are met:
The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

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:
not determined
Vehicle controls validity:
valid
Untreated negative 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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material caused no visible reduction in the growth of the bacterial bacground lawn at any dose level. The test material was, therefore , tested up to the maximum recommended dose level of 5000 µg/plate. A oily precipitate was observed at and above 1500 µg/plate, this did not prevent the scoring of revertant colonies.
No significant increases in the grequencey of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
All of the positive control chemicals used in the test induced marked increases in the frequeency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

See attached tables 2 -5 as follows:

Table 2. Test results: Experiment 1 - Without Metabolic Activation.

Table 3. Test results: Experiment 1 - With Metabolic Activation.

Table 4. Test results: Experiment 2 - Without Metabolic Activation.

Table 5. Test results: Experiment 2 - With Metabolic Activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

No significant increases in the frequencey of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.
The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The method was designed to meet the requirenments of the OECD guidance for:

Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission

Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98, TA 100 and Escherichia coli strain WP2uvrK were treated with solutions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ~tgIplate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results. The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ~.tg/plate. An oily precipitate was observed at and above 1500 j.tg/plate, this did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion. The test material was considered to be non-mutagenic under the conditions of this test.