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)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (rat)
Test concentrations with justification for top dose:
25-500 µg/plate for TA 98
50-1000 µg/plate for TA 1535, TA 1537, TA 100 and TA 102
Vehicle / solvent:
DMSO
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98, without metabolic activation
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 and TA 1535, without metabolic activation
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
TA 102, without metabolic activation
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537, without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-Aminofluorene, 2-Aminoanthracene
Remarks:
all strains, with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1st main experiment: plate incorporation, 2nd main experiment: preincubation

DURATION
- 1st main experiment: 72 hours incubation
- 2nd main experiment: 30 min preincubation, 72 hours incubation

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
Criteria for determination of a valid test:
- In the solvent control, each tester strain culture must exhibit a characteristic mean number
of spontaneous revertants.
- To ensure that appropriate numbers of bacteria are plated, overnight culture titers must be
in excess of 108 bacteria/mL.
- The mean of each positive control must exhibit a significant increase in the number of
revertants over the mean value of the respective vehicle control. In case of no significant
increase in the number of revertants by addition of 2-Aminofluorene, a parallel significant
increase by addition of 2-Aminoanthracene will be regarded as sufficient and vice versa.
- Normally, at least four non-toxic dose levels are required to evaluate the assay data.
Exceptions from this requirement, however, may be justified.

Criteria for evaluation of test results:
For a test compound to be considered positive, it must (in two independent experiments)
cause at least a doubling in the mean revertants per plate of at least one tester strain. This
increase must be accompanied by a dose response towards increasing concentrations of the
test article. A test article that does not meet these criteria will be called non-mutagenic in
bacteria. Single increases in revertant frequencies, which are not dose-related and not
reproducible in two independent tests are considered non-relevant. If however these
increases do occur in both tests, this will be taken as an indication of a mutagenic effect.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In a pre-experiment, the toxicity of the test item was determined with strains TA 98 and TA
100. Reduced background lawn was observed in the following cases:
- 500 - 5000 µg/plate; TA 98; without S9 mix.
- 5000 µg/plate; TA 98; with S9 mix.

Precipitation of the test item was observed in the following cases:
- 1600 - 5000 µg/plate; TA 98; with and without S9 mix.
- 1600 - 5000 µg/plate; TA 100; with and without S9 mix

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 without metabolic activation
negative with metabolic activation

Under the conditions of this study, the test item did not induce gene mutations by base pair changes or frame-shifts in the genome of the tester strains used. Thus, the test item is considered non-mutagenic in this bacterial reverse mutation assay.