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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:
23 Apr - 24 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline 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:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Freie und Hansestadt Hamburg, Behörde für Soziales, Familie, Gesundheit und Verbraucherschutz, Hamburg, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon
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:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
1.0, 3.16, 10.0, 31.6, 100, and 316 µg/plate with and without metabolic activation
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
methylmethanesulfonate
other: 2-amino anthracene
Details on test system and experimental conditions:
Experiment 1:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 - 72 h

Experiment 2:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

Experiment 1+2:
NUMBER OF REPLICATIONS: 3 plates per condition per experiment

DETERMINATION OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn, reduction in the number of spontaneous revertants, degree of survival of the treated cultures
Evaluation criteria:
The test item is considered positive in the Ames test if
- at one or more concentrations the number of revertants is reproducibly increased (in comparison to the solvent control; 2-fold in TA 98, TA 100, and TA 102), 3-fold in TA 1535 and TA 1537) in at least one strain with or without metabolic activation.
- a concentration-related increase of the revertants is observed.
- positive results have to be reporducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.
Statistics:
Mann and Whitney test (p <= 0.05)
Spearman's rank correlation coefficient

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
Remarks:
at 316 µg/plate (highest concentration) in both experiments in all strains with and without S9 mix
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The test substance was examined in a preliminary cytotoxicity test (plate incorporation test with and without S9 mix) in TA 100 using ten concentrations ranging from 0.316 to 5000 µg/plate. Cytotoxicity was noted at concentrations of 316 µg/plate and higher, thus, 316 µg/plate was chosen as the highest concentration in the main test.

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In the plate incorporation assay with metablic activation, a more than 3-fold increase in the number of revertants was observed for TA 1537 at a concentration of 3.16 µg/plate (7.3 ± 2.3 revertants) compared to the vehicle control (2.0 ± 1.0 revertants). However, this increase was not dose-dependent and was not reproducible using the preincubation method. Furthermore, compared to the mean number of revertants of the historical control data (7.8 ± 1.9 revertants) for strain TA 1537 in the presence of metabolic activation, the increase in the number of revertants for the test substance was no longer evident.

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

In the reverse mutation assay in bacteria the test substance did not induce biologically significant increases in the number of revertants und is thus considered non-mutagenic in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 under the test conditions chosen.