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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:
2012-07-11 until 2012-07-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
, adopted 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Method

Species / strain
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of Phenobarbital/ß-naphtoflavone induced rats.
Test concentrations with justification for top dose:
0; 33; 100; 333; 1 000; 2 750 and 5 500 μg/plate
Vehicle / solvent:
The test substance was dissolved in DMSO.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
With S9: 2-aminoanthracene (all strains), Without S9: N-methyl-N'-nitro-N-nitrosoguanidine (TA 1535, TA 100); 4-nitro-o-phenylenediamine (TA98); 9-aminoacridine (TA 1537); 4-nitroquinoline-N-oxide (E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (experiment I) preincubation test (experiment II)
DURATION: Plate incorporation: 48 hours at 37°C, pre-incubation for 20 min at 37°C followed by a 48 - 72 h incubation of the prepared plates
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY:
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer (the titer is determined only in the experimental parts with S9 mix both for the
negative controls (vehicle only) and for the two highest doses in all experiments).
Evaluation criteria:
Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the
historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in
the number of revertant colonies within the range of the historical positive control data or
above.
- Fresh bacterial culture containing approximately 109 cells per mL were used. For approval
the titer of viable bacteria was ≥ 108 colonies per mL.

Assessment criteria
The test substance is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about
doubling of the spontaneous mutation rate in at least one tester strain either without
S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control
range under all experimental conditions in at least two experiments carried out
independently of each other.
Statistics:
not applicable

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5500 µg/plate in the standard plate test with S9; occasionally in the pre-incubation assay at test concentrations from 2750 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants, slight reduction in the titer) was observed in the standard plate test only with metabolic activation at 5500 μg/plate using the salmonella strains.
In the preincubation assay bacteriotoxicity (slight decrease in the number of his+ revertants, slight reduction in the titer) was occasionally observed depending on the strain and test conditions from 2750 μg/plate onward using the salmonella strains.
Test substance precipitation was found from 2750 μg/plate onward 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

Conclusively, the test item is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.