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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: according guideline and GLP, well documented, read across substance

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham`s 12, supp. with 1% Pen/Strep, 1% amphotericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from phenobarbital and β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
1st Experiment
without S9 mix (4-hour exposure period)
0; 2.5; 5.0; 10.0; 20.0; 40.0; 1 300.0; 2 600.0; 5 200.0 μg/mL
with S9 mix (4-hour exposure period)
0; 2.5; 5.0; 10.0; 20.0; 40.0 μg/mL


2nd Experiment
without S9 mix (4-hour exposure period)
0; 250.0.; 500.0; 1 000.0; 2 000.0; 3 000.0; 4 000.0; 5 200.0 μg/mL
with S9 mix (4-hour exposure period)
0; 3.1; 6.3; 12.5; 25.0; 50.0 μg/mL

3rd Experiment
without S9 mix (24-hour exposure period)
0; 3.1; 6.3; 12.5; 25.0; 125.0; 250.0; 500.0 μg/mL
Vehicle / solvent:
Using deionized water as solvent a homogeneous suspension of the test substance was obtained. Therefore, culture medium (Ham's F12) was selected as vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
wihtout metabolic activation
Positive controls:
yes
Positive control substance:
other: methylcholanthrene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24h
- Time schedule: Day 1 seeding, Day 2 Test substance incubation (20 – 24 hours after seeding); exposure period (4-hour and 24-hour); washing of the cultures (4-hour exposure); 1st cytotoxicity determination (cloning efficiency 1: survival), Day 3 washing (24h exposure), Day 5 1st passage, DAy 7-9 2nd passage of the treated cells with seeding in the selection medium ("TG" medium); 2nd cytotoxicity determination (cloning efficiency 2: viability)
- Fixation time (start of exposure up to fixation or harvest of cells): from Day 16 drying, fixation, staining and counting of the selected colonies

NUMBER OF REPLICATIONS: 3 independent experiments

NUMBER OF CELLS EVALUATED: all

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

- Check of pH, osmolarity, solubility and cell morphology
Evaluation criteria:
Cytotoxicity determined and expressed as absolute and relative cloning efficiency
Mutant frequency calculated and expressed uncorrected and corrected frequency

The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0 – 15.95 mutants per 10^6 clonable cells
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.
Statistics:
An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative/vehicle control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in all experiments in the absence of S9 mix at least in the highest applied concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Osmolarity and pH values were not influenced by test substance treatment
- Effects of osmolality: Osmolarity and pH values were not influenced by test substance treatment
- Precipitation: The test substance was poorly soluble either in all tested vehicles or in culture medium. Therefore, in the pretest using culture medium as most suitable vehicle precipitation occurred from the lowest applied concentration onward.

RANGE-FINDING/SCREENING STUDIES: pre-test for dose selection

COMPARISON WITH HISTORICAL CONTROL DATA: yes

Applicant's summary and conclusion

Conclusions:
The test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.