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
Study period:
2013
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Principles of method if other than guideline:
/
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
yellow, clear viscous oil

Method

Target gene:
TA98: his D 3052
TA100: his G 46
TA102: his G428
TA1535: his G46
TA1537: his C3076
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
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 3160 and 5000 μg V125226 per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-amino-anthracene
Details on test system and experimental conditions:
Plates: 3 per concentration and experiment
Experiments: 2 independent experiments, each with and without metabolic activation
Evaluation criteria:
In our laboratory, a test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY, see section 6, reference 3., compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100 and TA102 and 3-fold of the solvent control for TA1535 and TA1537 in both independent experiments. The Mann and Whitney test (p ≤ 0.05, see section 6, reference 3.) may be used to determine statistical significance.
- in addition, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient, see section 6, reference 3.) is observed;
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

The range of spontaneous reversion frequencies per plate is based on Kirkland9
TA98:
20 -
60
TA100:
100 -
200
TA102:
240 -
320
TA1535:
10 -
35
TA1537:
3 -
20
The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment.
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:
see 'Evaluation criteria'

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:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Main study Six concentrations ranging from 31.6 to 5000 μg V125226/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

Test item precipitation was noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at the top concentration of 5000 μg V125226/plate in all test strains. In addition, cytotoxicity was noted in the plate incorporation test without and with metabolic activation at the top concentration of 5000 μg V125226/plate in test strains TA98 and TA100 or TA100, respectively, and in the preincubation test without and with metabolic activation in test strain TA1537. The reduction of the number of revertants by more than 50% in test strain TA1537 in the preincubation test without metabolic activation at 31.6 μg/plate is considered to be caused by the high variation in individual counts and not due to cytotoxicity.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for V125226, tested up to a concentration of 5000 μg/plate, that led to test item precipitation and, in addition, to cytotoxicity in some strains, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test). The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

Under the present test conditions v125226 tested up to a concentration of 5000 ug/plate, that les to test item precipitation and/or cytotoxicity, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.