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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:
08 Sep 1995 to 26 Sep 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
1987
Qualifier:
according to
Guideline:
other: 84/449/EEC B.14
Version / remarks:
1992
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
MHW Japan, 1986
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Target gene:
his- (S. typhimurium), trp- (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S-9 mix
Test concentrations with justification for top dose:
Preliminary Cytotoxicity Assay (with and without metabolic activation): 20.58, 61.73, 185.19, 555.56, 1666.67, 5000.00 µg/plate
Bacterial Assay (with and without metabolic activation): 5000, 2500, 1250, 625 and 312.5 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
mitomycin C
other: 2-Aminoanthracene (2-AA); 4-Nitroquinoline (4-NQ)
Details on test system and experimental conditions:
PRELIMINARY CYTOTOXICITY ASSAY: A range finding test was carried out with strains S. typhimurium TA 100 and E. coli WP2 uvrA with and without metabolic activation at six concentrations (20.58 to 5000.0 µg/plate) of the test substance and one negative control. The plates were inverted and incubated for about 48 hours at 37±1.5°C in darkness. After incubation they were evaluated by counting the colonies and determining the background lawn. One plate per concentration and negative control was used.

METHOD OF APPLICATION: in agar (plate incorporation);

PROTOCOL:
- Bacterial cultures were prepared from frozen stocks and grown in liquid nutrient broth medium by incubating overnight for 8 hours. 0.1 mL of the overnight cultures were mixed with 2 mL of top agar, either 0.5 ML of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 mL of the activation mixture (experiments with activation) and 0.1 mL of a solution of the test substance, the positive control or the solvent and poured on minimal agar in Petri dishes. Each Petri dish contained about 20.0 mL of minimal agar. The top agar was composed of 0.6% agar and 0.6% NaCl. In the experiment with Salmonella the top agar was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+)biotin dissolved in water. In the experiment with E. coli it was supplemented with 10% of 0.5 mM L-tryptophan dissolved in water.
- The test substance (purity 98.6%) in DMSO was tested on five histidine-auxotrophic strains of Salmonella typhimurium and one tryptophan-auxotrophic strain of Escherichia coli, by plate incorporation at five concentrations (312.5 to 5000.0 µg/plate). Two independent assays were performed, both experiments with and without S9 metabolic activation. After preparation, the plates were inverted and incubated for about 48 hours at 37±1.5°C and evaluated by colony counting and determining the background lawn. Concurrent strain-specific mutagens were applied to all strains as positive controls in both experiments.
- For all strains triplicate plates were used for all test substance and positive control treatments. For solvent controls 5 plates were used.

Evaluation criteria:
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met:
- At least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains: TA98, TA1535, TA1537, E. coli WP2 uvrA.
- A reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least factor of 1.5 for strains TA100 or TA102
- Generally a concentration-related effect should be demonstrable.
Statistics:
None.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA102, TA1535 and TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY CYTOGENICITY ASSAY: Normal background growth was observed with both strains tested. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and 5000.0 µg/plate without metabolic activation.

MUTAGENICITY ASSAY: In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced with increasing concentration. Therefore, the test substance exerted no toxic effect on the growth of the bacteria. The positive controls produced a marked increase of the number of revertant colonies.

Applicant's summary and conclusion

Conclusions:
In this GLP compliant study, performed according to OECD TG 471, the test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli
Executive summary:

In a reverse gene mutation assay in bacteria, the test substance (purity 98.6%) in dimethylsulphoxide (DMSO) was tested on five histidine-auxotrophic strains (TA98, TA100, TA102, TA1535 and TA1537) of Salmonella typhimurium and on the tryptophan-auxotrophic strain WP2uvrA of Escherichia coli, by plate incorporation, at concentrations of 0 (solvent control), 312.5, 625, 1250, 2500 and 5000 µg/plate. Two independent assays were performed, both experiments with and without S9 metabolic activation (S9 fraction from Arochlor induced rat liver). After preparation, the plates were inverted and incubated for about 48 hours at 37±1.5°C and evaluated by colony counting and determining the background lawn. Concurrent strain-specific mutagens were applied to all strains as positive controls in both experiments. In the range finding test normal background growth occurred with both strains, with or without metabolic activation and no appreciable cytotoxicity was observed at 5000 µg/plate, the highest concentration evaluated. The test substance did not increase the number of revertants in any of the strains used, with or without metabolic activation, in either experiment when compared to the vehicle controls. There was no evidence of toxicity to the bacteria at any concentration, in either experiment. The positive controls produced a marked increase of the number of revertant colonies. The test substance and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used in the study.