Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
of 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: essential amino acid requiring strains
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix (supplied by MOL. TOX Molecular Toxicology).
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
other: essential amino acid requiring strain
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix (supplied by MOL. TOX Molecular Toxicology).
Test concentrations with justification for top dose:
Preliminary screening test (only with TA100 & WP2 uvrA): 5.0, 1.0, 0.5, 0.1 and 0.05 mg/plate
Main Tests (Experiments 1 and 2, with all strains): 5.0, 1.0, 0.5, 0.1 and 0.05 mg/plate
Vehicle:
DMSO
Controlsopen allclose all
Negative controls:
yes
Remarks:
testing for spontaneous reversion
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 2-nitrofluorene, 2-aminoanthracene and 9-aminoacridine
Remarks:
for Salmonella strains
Negative controls:
yes
Remarks:
testing for spontaneous reversion
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene and methyl methanesulfonate
Remarks:
for Escherichia coli
Details on test system and 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)

NUMBER OF REPLICATIONS: All plating performed in triplicate

Evaluation of toxicity was based on reversion frequency, viability and integrity of the background lawn.

Precipitate was not evident at any tested concentration of WS400517.

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: 5 μg/plate: - strains: TA 1535, TA 100
9-Aminoacridine: 100 μg/plate: - strain: TA 1537
2-Nitrofluorene: 5 μg/plate: - strain: TA 98
Methyl methanesulfonate: 0.001 mL/plate: - strain: WP2 uvrA

With metabolic activation (+S9 mix):
2-Aminoanthracene: 1.0 μg/plate: - strains: TA 98, TA 100, TA 1535, TA 1537
10 μg/plate: - strain: WP2 uvrA

Genetic markers, such as histidine/biotin requirement, crystal violet sensitivity, ampicillin resistance, ultra violet sensitivity, tetracycline resistance for Salmonella strains and tryptophan requirement for growth and sensitivity to Mitomycin C for E. coli, and spontaneous reversion rates have been routinely checked in the testing laboratory.
Evaluation criteria:
The test material was considered to exhibit mutagenic activity in this assay if the following criteria were met:
1) there was a statistically significant difference (at p < = 0.05) in the number of revertants between the solvent control and the test material, as determined by One Way Analysis of Variance (Anova).
2) In response to a test material concentration, strain TA98 or TA100 responded with a mean reversion frequency twofold or more greater than that of the corresponding solvent control plates, or strains TA1535, TA1537 or WP2 uvrA responded with a mean reversion frequency threefold or more greater than that of the corresponding solvent control plates. In addition, the response must be dose-dependent or increasing concentrations of the test material must show increasing mean reversion frequencies. In evaluating the results, the magnitude of any increase in reversion frequency and the degree of associated toxicity (if any) is given due consideration.

Accordingly, the response of a test material was considered to be negative if (1) the Anova did not disclose a statistically significant difference (at p = 0.05) from the corresponding solvent control, (2) the increases (if any) in mean reversion frequency for TA98 or TA100 were < twofold and that (if any) for TA1535, TA1537 or WP2 uvrA were < threefold that of the corresponding solvent control plates and (3) there was no evidence of a dose-dependent response.

A response was considered equivocal if it did not fulfil the criteria of either a negative or a positive response and/or the Study Director did not consider the response to be either positive or negative.
Statistics:
Numbers of revertant conlonies were statistically analysed using One Way Analysis of Variance to determine statistically significant differences of test concentrations or positive controls from vehicle controls at p<=0.05.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
of 5000 µg/plate, without precipitate
Vehicle controls valid:
other: See "Additional information on results"
Negative controls valid:
not applicable
Positive controls valid:
other: See "Additional information on results"
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: in both experiments (Experiment 1 and 2)
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in both experiments (Experiment 1 and 2)
Cytotoxicity:
no, but tested up to limit concentrations
Remarks:
of 5000 µg/plate, without precipitate
Vehicle controls valid:
other: See "Additional information on results"
Negative controls valid:
not applicable
Positive controls valid:
other: See "Additional information on results"
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: in both experiments (Experiment 1 and 2)
Additional information on results:
The historical reference ranges for vehicle and positive controls were not presented in the study report and therefore the validity of the controls could not be assessed. However, the presented revertant colony number values of concurrent negative controls were similarly low to those of the test material treated groups, whilst those of positive controls were substantially higher (statistically significant, p<0.05), consistent with what would be expected from valid negative and positive controls. In addition, the numbers of spontaneous revertants/plate were similarly low to those of the corresponding vehicle controls.

Applicant's summary and conclusion

Conclusions:
negative without and with metabolic activation (S9 mix)

In both main experiments, the test material was found to be non-mutagenic and non-cytotoxic at 5000 µg/plate and below this level for all of the tested Salmonella and E. coli strains, both with and without metabolic activation.