Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jul 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine gene locus; tryptophan gene locus
Species / strainopen allclose all
Species / strain:
other: TA 1535, TA 100, TA 1537, TA 1538 and TA 98
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male Sprague-Dawley rat liver S9 mix
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced male Sprague-Dawley rat liver S9 mix
Test concentrations with justification for top dose:
0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 mg/plate
The highest dose tested should cause toxicity (thinning of the background lawn of bacteria) or should correspond to the substance's precipitation range (visible precipitates on the plates) but should not exceed 5 mg/plate or 5 μL/plate.







Vehicle:
DMSO
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
benzo(a)pyrene
cyclophosphamide
ethylmethanesulphonate
other: 4-Nitro-o-phenylenediamine, N-Methyl-N’-nitro-N-nitrosoguanidine, Anthracene-2-amine
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ca. 72 hours (S. typhimurium) and ca. 48 hours (E. coli)

- all plates were prepared in triplicates
Evaluation criteria:
A positive response was considered if the number of revertants of the compound groups compared to the number of revertants of the negative group was reproducibly higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.
Statistics:
The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA1535, TA100, TA1537, TA1538 and TA98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
Growth inhibition of the background lawn was observed in all tester strains from 1.0 mg per plate onwards without metabolic activation.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Species / strain:
other: TA1535, TA100, TA1538 and TA98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Growth inhibition of the background lawn was observed from 2.5 mg per plate onwards.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Growth inhibition of the background lawn was observed in all tester strains from 1.0 mg per plate onwards without metabolic activation.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
Precipitates in the agar were found from 1.0 mg/plate onwards.

Any other information on results incl. tables

All S. typhimurium strains used (TA1535, TA100, TA1537, TA1538 and TA98) showed increased reversion to prototrophy when incubated with the test substance at doses between 0.1 and 5.0 mg/plate in the absence of metabolic activation. The experiments with S9 mix gave exclusively negative results.

The Escherichia coli strain used (WP2uvrA) did not show increased reversion to prototrophy with the test substance at any doses tested in the absence or in the presence of S9 mix.

Applicant's summary and conclusion

Executive summary:

In conclusion it can be stated that under the experimental conditions reported, the test substance induced gene mutations by base-pair changes or frame-shifts in the genome of the S. typhimurium strains used, when tested up to highest recommended dose level of 5.0 mg/plate in the absence of S9 mix. Therefore, the test substance has to be considered as directly mutagenic in the Ames test.