Registration Dossier

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
Remarks:
GLP

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:
50 - 5000 µg/plate
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Tester strain TA 98 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Tester strains TA 100 and TA 1535 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Tester strain TA 102 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Tester strain TA 1537 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminofluorene and 2-Aminoanthracene
Remarks:
Tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 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

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The following criteria must be met for the mutagenicity assay to be considered valid:
- 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 10E+08 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.

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 item. A test item 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
Key result
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
Remarks:
5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the plate incorporation test reduced background lawn was observed in the following cases:
5000 µg/plate; TA 98, TA 1535 and TA 1537; without S9 mix.
In the preincubation test reduced background lawn was detected in the following case:
5000 µg/plate; TA 1537 ; without S9 mix.
Additionally, precipitation was observed in the preincubation test in the following cases:
5000 µg/plate; TA 98, TA 100, TA 102, TA 1535 and TA 1537 ; with S9 mix
5000 µg/plate; TA 98, TA 100, TA 102, TA 1535 and TA 1537 ; without S9 mix
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative without metabolic activation
negative with metabolic activation

Under conditions tested, 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.