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Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to an internationally accepted technical guideline and in compliance with GLP in a contract research organization.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
of 1997
Occasional incubation temperature from 34.2°C to 39.9°C. In view of good growth and comparable results in all tests, study results were considered to be unaffected by this.
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material



Target gene:
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA102, TA1535, TA1537
Additional strain / cell type characteristics:
other: essential amino acid requiring strains
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver microsomal fraction (S9 mix), supplied by Molecular Toxicology, Inc., Boone, North Carolina, USA
Test concentrations with justification for top dose:
Preliminary toxicity test (only with TA100): 5.0, 12.5, 25, 50, 125, 250, 500, 1250, and 5000 μg/plate
Main Tests (Experiments 1 and 2, with all strains): 0.50, 5.0, 50, 500, and 5000 μg/plate

In addition, in several instances, 0.05 and 0.005 μg/plate levels were also assayed, but being all negative for mutagenicity these results in general were not reported. Only for the independent repeat experiment (Experiment 2) for TA98 (-S9) the results of the 0.05 µg/plate level were reported, because in this experiment no data were available for the 0.5 µg/plate concentration, as no background lawn was observed.
Vehicle / solvent:
Justification for choice of solvent/vehicle: Outcome of solubility trials conducted at the testing laboratory.
Untreated negative controls:
testing for spontaneous reversion and in some assays with TA100, TA102 and TA1535 mainly (-S9) water controls included
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene and mitomycin C.
Details on test system and experimental conditions:
Two independent assays (Plate Incorporation tests) were conducted (Experiments 1 and 2), each without and with metabolic activation (-/+ S9 mix)

METHOD OF APPLICATION: in agar (plate incorporation)

Preliminary toxicity test: Water and vehicle controls: all plating performed in duplicate; Test concentrations: all plating performed in triplicate.
Experiments 1 and 2: All test substance concentrations, vehicle controls and positive controls were plated in triplicate. On very few occasions single plates were not considered due to reduced background lawn.

In the main tests (Experiments 1 and 2) moderate precipitate was evident at 5000 µg/plate in all but one assays. Only in Experiment 2 with TA1535 (-S9 mix) precipitate was not recorded at 5000 µg/plate. In the preliminary toxicity test, precipitate was moderate in degree at 2500 µg/plate and moderat to heavy in degree at 5000 µg/plate.

The following positive controls were used to check mutability of the bacteria and activity of the S9 mix:

Without metabolic activation (-S9 mix):
Sodium azide: 1.5 μg/plate: - strains: TA 1535, TA 100
9-Aminoacridine: 75 μg/plate: - strain: TA 1537
2-Nitrofluorene: 2.0 μg/plate: - strain: TA 98
Mitomycin C: 0.5 μg/plate: - strain: TA 102

With metabolic activation (+S9 mix):
2-Aminoanthracene: 1.0 μg/plate: - strains: TA 98, TA 100, TA 102, TA 1535, TA 1537

Incubation time: 48 h at approximately 37 +/- 2 degrees. Occasional incubation temperature from 34.2°C to 39.9°C. In view of good growth and comparable results in all tests, study results were considered to be unaffected by this.

Cytotoxicity was tested in the preliminary assay with a single strain, TA100, in the presence and absence of metabolic activation.

Genetic markers, such as histidine/biotin requirement, crystal violet sensitivity, ampicillin resistance, ultra violet sensitivity, tetracycline resistance and spontaneous reversion rates of the cultures to histidine independence have been checked in the testing laboratory. Each strain demonstrated the strain genotypes expected. In addition cell titres of each strain were measured at use.

Evaluation criteria:
A concentration was considered toxic if one or both of the following criteria were met:
1) a reduction greater than 50% in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction hasd to be accompanied by an abrupt dose-dependent drop on the revertant count.
2) A reduction observed in the background lawn.

The test substance was considered positive in the assay when:
1) statistically significant dose-related increase in the mean number of revertants for at least one tester strain, over a minimum of two test concentrations. Data sets for strains TA98, TA100 and TA102 were considered positive if the mean number of revertants at the peak of the dose-response was two times (or greater) than the mean solvent control values. Data sets for strains TA1535 and TA1537 were considered positive if the mean number of revertants at the peak of the dose-response was three times (or greater) than the mean solvent control values.
Not specified.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 102, TA 1535 and TA 1537
Metabolic activation:
with and without
in both experiments (Experiment 1 and 2)
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
In all but one incidence, precipitate moderate in degree at 5000 µg/plate
Vehicle controls validity:
Untreated negative controls validity:
not applicable
Positive controls validity:
Additional information on results:
There was no evidence of reproducible cytotoxicity at any of the concentrations tested.

Positive controls were considered to be valid as in general their revertant colony number values were more than threefold higher than those of concurrent vehicle controls.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'. Remarks: in both experiments (Experiment 1 and 2)

Applicant's summary and conclusion

Interpretation of results (migrated information):
negative without and with metabolic activation (S9 mix)

In both main experiments, the test material was found to be non-mutagenic for all of the tested Salmonella strains, TA98, TA100, TA102, TA1535 and TA1537, both without and with metabolic activation.