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Toxicological information

Genetic toxicity: in vitro

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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
Study period:
December 01, 2009 - December 24, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD guideline and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
other: The standard that the Minister of Labor establishes based on the Industrial Safety and Health Law Article 57-2 Paragraph 1 (Notification No, 77 of JMOL on September 1, 1988 and Notification No. 67 of JMOL on June 2, 1997)
Qualifier:
according to
Guideline:
other: Notification of testing methods relating to the new chemical substances (Notification No. 1121002 of JMHLW, No. 2 of JMETI, No. 031121002 of JMOE on November 21, 2003)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): X-22-1622
- Appearance at ordinary temperature: colourless transparent liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Experiment 1 (Preliminary test): TA98, TA100, TA1535, TA1537 and WP2uvrA:
Without and with S9-mix: 5, 20, 78, 313, 1250 and 5000 µg/plate
Main study: TA98, TA100, TA1535, TA1537 and WP2uvrA:
Without and with S9-mix: 313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:
- Solvent used: Acetone
- Justification for choice of solvent: the test substance is insoluble in water and dimethylsulfoxide, whereas the test substance is soluble in acetone with a solubility of 100 g/L or more. And the test substance is judged to be stable in acetone.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3 (5nitro-2-furyl)acrylamide in DMSO for TA98 (0.1 μg/plate), TA100 and WP2uvrA (0.01 μg/plate)
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 for TA1537 (in DMSO 80 μg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 for TA1535 (in distilled water 0.5 μg/plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for TA1535 (2.0 μg/plate) and WP2uvrA (10.0 μg/plate)
Remarks:
with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 for TA98, TA100 and TA1537 (in DMSO 5.0 μg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in duplicate in each strain. According to the OECD guideline the use of duplicate plating is acceptable when scientifically justified, as no toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate.
Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered positive:
- a clear dose-related increase in the number of the revertant colonies,
- and more than two-fold increase in the number of the revertant colonies compared with the negative control in two indepently repeated experiments.
Statistics:
Not performed.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed at dose levels of 625, 1250, 2500 and 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The mutagenic activity of the substance X-22-1622 was evaluated according to OECD 471 guideline and GLP principles. It is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The mutagenic activity of the substance X-22-1622 in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay was evaluated according to OECD 471 guideline and GLP principles. The test was performed in 2 independent preincubation assays, both in the absence and presence of S9-mix.

Precipitation was observed from 625 µg/plate up to the top dose level of 5000 µg/plate and no toxicity was observed. Adequate negative and positive controls were included.

The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in a repeated experiment for all strains. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.