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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:
October 01 - 10, 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Directive 92/69/EEC, B.14 "Ames test" and B.13
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Details on test material:
- Name of test material (as cited in study report): Nikkol VC-IP
- Substance type: Colourless to light yellow transparent liquid
- Physical state: Liquid
- Stability under test conditions: stable
- Storage condition of test material: In refrigerator in the dark

Method

Target gene:
Histidine gene in S. typhimurium
Tryptophan gene in E. coli
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:
Aroclor 1254 induced rat liver S9-homogenate
Test concentrations with justification for top dose:
Preliminary test: 3-10-33-100-333-1000-3330-5000 μg/plate
First and second test: 10-33-100-333-1000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: the test substance was dissolved in ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix

Migrated to IUCLID6: 1 μg/plate in saline for TA1535
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix

Migrated to IUCLID6: 60 μg/plate in milli-Q water for TA1537
Positive control substance:
other: daunomycine, 4 μg/plate in DMSO for TA98
Remarks:
without S9 mix
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix

Migrated to IUCLID6: 650 μg/plate in DMSO for TA100
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix

Migrated to IUCLID6: 10 μg/plate in DMSO for WP2 uvr A
Positive control substance:
other: 2-aminoanthracene in DMSO (5 and/or 10%) for all tester strains
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

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:
- Other: precipitation of test substance
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or
without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in
any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

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
Untreated negative 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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: slight precipitation was observed at doses of 1000 μg/plate and higher.
RANGE-FINDING/SCREENING STUDIES:
No toxicity and mutagenicity was observed up to concentrations 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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in
two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory background historical
ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that the NIKKOL VC-IP is not mutagenic in the Salmonella typhimurium reverse mutation assay
and in the Escherichia coli reverse mutation assay.