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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
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:
other: Guideline study, GLP

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:
- Name of test material (as cited in study report): Anisaldehyde
- Physical state: Liquid, yellowish, clear
- Analytical purity: 99.3 area-%
- Lot/batch No.: AP11-0143
- Expiration date of the lot/batch: August 08, 2012
- Storage condition of test material: Room temperature, under nitrogen

Method

Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 µg/ml) and amphotericin B (1 %).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
85 - 1360 µg/ml
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9: ethylmethane sulfonate 0.150 mg/mL = 1.2 mM; with S9: 7,12-dimethylbenz(a)anthracene 1.1 µg/mL = 4.3 µM
Details on test system and experimental conditions:
DURATION
- Preincubation period: 24 hours
- Exposure duration: 4, 24 hours
- Expression time (cells in growth medium): approx. 10 days
- Selection time (if incubation with a selection agent): 8 days

SELECTION AGENT (mutation assays): 6-thioguanine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Mutagenic, if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations
in the experiment, or 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 corresponding 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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 10.6 to 1360 µg/mL (->10 mM) to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 1360 µg/mL with and without metabolic activation following 4 and 24 hours treatment.

COMPARISON WITH HISTORICAL CONTROL DATA: Solvent and positive control were within the historical control data range

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects were observed in the first experiment at 340 µg/mL and above in culture II in the absence of metabolic activation.
In the presence of metabolic activation cytotoxic effects were noted at 1020 µg/mL in culture II and at 1360 µg/mL in both parallel cultures.
In the second experiment cytotoxic effects occurred at 340 and 425 µg/mL in culture I and at 680 µg/mL and above in both cultures without metabolic activation (24 hours treatment).
With metabolic activation relevant cytotoxicity was noted at 1020 and 1360 µg/mL in both parallel cultures.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. The mutation frequency remained well within the historical range of solvent controls.

Applicant's summary and conclusion