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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 20th to July 5th, 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
May 26th, 1983
Deviations:
yes
Remarks:
Salmonella Typhimurium Reverse Mutation Assay Modified Version for Azo-Dyes
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Test I (plate incorporation test): rat liver S9 mix
Test II (pre-incubation test): hamster liver S9 mix
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 μg/plate
Concentration range in the main test (without metabolic activation): 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 μg/plate
Vehicle / solvent:
Bi-distilled water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation - rat liver S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation - rat liver S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
congo red
other: 2-aminoanthracene
Remarks:
with metabolic activation - hamster liver S9 mix
Details on test system and experimental conditions:
According to the results of the pre-experiment, the concentrations applied in the main experiment were chosen. The maximum concentration was 5000.0 μg/plate. According to the dose selection criteria the test article was tested at the following concentrations: 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 μg/plate. For each strain and dose level, including the controls, three plates were used. The following materials were mixed in a test tube and poured into the selective agar plates:
- 100 μl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control).
- 500 μl S9 mix (for the test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation).
- 100 μl bacteria suspension.
Rationale for test conditions:
A pre-experiment on toxicity was run on TA 98 and TA 100. The plates incubated with the test article showed normal background growth up to 5000.0 μg/plate with and without S9 mix in all strains used.
Evaluation criteria:
A test article is considered mutagenic if in strains TA 98 and TA 100 the number of reversions is at least twice higher and in strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Remarks:
exp. I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
exp. I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
exp. I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
exp. I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No toxic effects, evident as a reduction in the number of revertants, occured in the test groups with and without metabolic activation up to the highest investigated dose. An isolated and moderate reduction of revertant colonies occured in the strain TA 98 in the absence of metabolic activation at 2500 μg/plate in the second experiment. This effect was considered as a fluctuation of the frequency of spontaneous reversions rather than a true toxic effect. The reduction was not observed at any other concentration of the test article and did not occur in the first experiment.
The plates incubated with the test article showed normal background growths up to 5000.0 μg/plate with and without S9 mix in all strains used.
No substantial increase in revertant colony numbers of any of the four tester strains were observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagen were used as positive controls. They showed a distinct increase in induced revertant colonies.

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.