Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-10-09 to 2012-11-06
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:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian gene mutation assay in vitro

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DEBR 012553

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster ovary cells
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham´s F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix prepared from incuded rat liver
Test concentrations with justification for top dose:
Experiment I (without S9-mix, 5 hours treatment):
53.8, 107.5, 215, 430 and 860 µg/mL
Experiment I (with S9-mix, 5-hours treatment):
53.8, 107.5, 215, 430 and 860 µg/mL
Experiment II (without S9-mix, 20 hour treatment):
53.8, 107.5, 215, 430, 860 µg/mL
Experiment II (with S9-mix, 5-hour treatment):
53.8, 107.5, 215, 430, 860 µg/mL

The highest test concentration of 860 µg/mL corresponds to 10 mM.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ham´s F12 medium
- Justification for choice of solvent/vehicle: suitable solvent to dissolve test item
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DOSE SELECTION EXPERIMENT
In a pre-test on cytotoxicty no cytotoxicity was observed up to concentrations of 5000 µg/mL in the presence or absence of S9. As the highest test concentration according to guideline is 10 mM or 5000 µg/mL, whichever is obtained first, 10 mM (corresponding to 860 µg/mL) was selected as the highest test concentration for the main experiment.

MAIN EXPERIMENT

DURATION
- Exposure duration: 5 hours (with and without S9) and 20 hours (without S9)
- Expression time (cells in growth medium): 8 days

SELECTION and FIXATION
At the end of the expression period the cultures from each of the dose levels were resided at 2x105 cells per 100-mm dish (five dishes) in selection medium. After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 500000

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
The test item would have been considered to be mutagenic in this assay if all the following criteria were met:
• The assay is valid.
• The mutant frequency at one or more doses is significantly greater than that of the relevant control.
• Increase of the mutant frequency is reproducible.
• There is a clear dose-response relationship.
The test item would have been considered to have shown no mutagenic activity if no increases were observed which met the criteria listed above.

ASSAY ACCEPTANCE CRITERIA
• The mutant frequency in the negative (solvent) control cultures is within the range (min-max) of historical laboratory control data.
• The positive control chemicals induce a statistically significant and biologically relevant increase in mutant frequency.
• The cloning efficiency of the negative controls is between the range of 60% to 140% on Day 1 and 70% to 130% on Day 8.

Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
• Mutant frequency between the negative (solvent) and the test item or positive control item treated groups.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY
Comparable toxicity was observed in treatment groups when compared to the negative (solvent) controls, both in the absence and in the presence of the metabolic activation, confirming the response seen in the dose selection cytotoxicity assays, that the Crotonic acid is a non-cytotoxic item.

MUTATION DATA
In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistical differences between treatment and control groups and no dose-response relationships noted. In Experiment 2, the mutant frequencies of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.

POSITIVE CONTROL/NEGATIVE CONTROL
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures. The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory. In the Experiment 1 the mutation frequencies of EMS were higher (1374.67 and 1421.62 ) than the historical control (1204.29) value, but this deviation did not influence the quality or integrity of the study.

PH AND OSMOLALITY
The pH and osmolality of control and treatment solutions did not show any alterations compared to the concurrent control groups in Experiments 1 and Experiment 2

Applicant's summary and conclusion

Conclusions:
It is concluded that the test item, Crotonic acid, was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells under the conditions of the present study.