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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: yellowish solid
- Analytical purity: > 95%, detals given in substance characterization
- Lot/batch No.: 0004416917
- Expiration date of the lot/batch: May 26, 2013
- Stability under test conditions: stable
- Storage condition of test material: room temp, protected from light

Method

Target gene:
hprt
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/b-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
0.13 0.25 0.5 1.0 2.0 3.0 mg/L (experiment 1; 4h, without S9)
0.94 1.9 3.8 7.5 15.0 30.0 mg/L (experiment 1; 4h, with S9)
46.9 93.8 187.5 375.0 750.0 1500.0 mg/L (experiment 2; 24h, without S9)
0.94 1.9 3.8 7.5 15.0 30.0 mg/L (experiment 1; 4h, with S9)
Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9:150 μg/mL ethyl methanesulfonate, with S9: 1.1 μg/mL 7,12-dimethylbenz(a)anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium):3-4 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days

SELECTION AGENT (mutation assays): 6-thioguanine


NUMBER OF REPLICATIONS: Population doubling time 12 - 16h

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-muta¬genic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concen¬trations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 23.4 µg/mL and above in the absence of metabolic activation and at 187.5 µg/mL with metabolic activation following 4 hours treatment. After 24 hours treatment without metabolic activation cytotoxic effects occurred at 93.8 and 187.5 µg/mL and at the maximum concentration of 3000 µg/mL.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 23.4 µg/mL and above in the presence and absence of metabolic activation following 4 hours treatment and at 750.0 µg/mL and above without metabolic activation following 24 hours treatment.
There was no relevant shift of pH and osmolarity of the medium even in the stock solution of the test item.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The test material is not geno-toxic under the conditions of the test.
Executive summary:

In this guideline (OECD 476) study conducted with GLP certification, the test material was considered to be non-genotoxic. The HPRT test was conducted in Chinese hamster lung fibroblasts cells with and without metabolic activation. Cytotoxicity was clearly observed in the 1st Experiment in the presence of metabolic activation at the two highest concentrations tested. Test concentrations were tested up to 3000 µg/l.