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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
Study period:
April 17, 2012 to September 03, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(adopted July 21, 1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
dated May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): N-Ethylpiperidine
- Physical state: Liquid, colorless to yellow
- Analytical purity: 97.1 g/100 g determined by 1H-NMR-analysis
- Lot/batch No.: 000STD77L0
- Test substance No.: S1325711
- Stability in solvent: Not indicated
- Storage condition of test material: Room temperature
- On the day of the experiment (immediately before treatment), the test item was dissolved in deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v).
- The osmolarity and pH-value were determined in the solvent control and in the highest concentration of the pre-experiment without metabolic activation: solvent control: Osmolarity mOsm: 378 / pH-value: 7.39; N-Ethylpiperidine (1140 µg/mL): Osmolarity mOsm 333 / pH-value: 7.95

Method

Target gene:
HPRT locus

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, neomycin (5 μg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of 8 - 12 weeks old male Wistar rats [Hsd Cpb: WU], treated with 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days.
Test concentrations with justification for top dose:
Range finding pre-experiment: 8.9 to 1140 μg/mL (≈10 mM), in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
Main experiment: 35.6; 71.3; 142.5; 285; 570; 1140 μg/mL
1st Experiment: with and without S9 mix (4-hour exposure period):
2nd Experiment: without S9 mix (24-hour exposure period); with S9 mix (4-hour exposure period)
The cultures at the lowest concentration in the presence and absence of metabolic activation (experiment I and II) were not continued since a minimum of only four analysable concentrations is required by the guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(water)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
0.150 mg/mL (1.2 mM). Dilutions of the stock solution were prepared on the day of the experiment and used immediately.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(water)
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
1.1 µg/mL (4.3 µM)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration:
1st experiment: 4 h exposure with and without S9 mix.
2nd experiment: 4 h exposure with S9 mix and 24 h without S9 mix.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)

NUMBER OF REPLICATIONS: duplicates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency;

Evaluation criteria:
A test item was classified as positive if it induced 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 was considered non-mutagenic in this system.
A positive response was described as follows:
- A test item was classified as mutagenic if it reproducibly induced a mutation frequency that was three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item was classified as mutagenic if there was a reproducible concentration-related increase of the mutation frequency. Such evaluation was considered also in the case that a threefold increase of the mutant frequency was not observed.
- However, in a case by case evaluation this decision depend on the level of the corresponding solvent control data. If there was 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 was discussed. The variability of the mutation rates of solvent controls within all experiments of the 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 was judged as significant whenever the p-value (probability value) was 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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: 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. No precipitation occurred up to the highest concentration with and without metabolic activation following 4 and 24 hours treatment.

RANGE-FINDING/SCREENING STUDIES:
- Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. The individual concentrations were spaced by a factor of 2

COMPARISON WITH HISTORICAL CONTROL DATA:
- In both experiments of the study (with and without S9 mix) the range of the solvent controls was from 8.5 up to 54.6 mutants per 10E6 cells; the range of the groups treated with the test item was from 4.5 up to 56.8 mutants per 10E6 cells. In experiment I with metabolic activation the number of mutants in the solvent control in culture I exceeded the upper limit of the historical control data. The data were regarded as acceptable however since the data of the mutant frequency in culture II and the mean value of both cultures were within the historical range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- A cytotoxic effect indicated by a relative cloning efficiency I below 50% in both parallel cultures was solely observed in the second experiment without metabolic activation at the maximum concentration of 1140 μg/mL equal to approximately 10 mM.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

- No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. The threshold of three times the mutation frequency of the corresponding solvent control was exceeded in the first culture of the second experiment without metabolic activation at 1140.0 μg/mL. This isolated increase was judged as biologically irrelevant however, as it was not reproduced in the parallel culture under identical experimental conditions at almost an identical level of cytotoxicity. Cytotoxicity was severe at this experimental point with a relative cloning efficiency I of 16.1% indicating an irreproducible cytotoxic artefact.

- A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A p-value below 0.05 was solely detected in culture I of the second experiment without metabolic activation. This trend however, was judged as biologically irrelevant as it was not reproduced in culture II and based on a cytotoxic artefact as described above.

- In the first culture of experiment I the cloning efficiency II (viability) of the solvent control with metabolic activation fell just short of the acceptance criteria (40% versus 50%). The data were regarded as acceptable however, as the cloning efficiency II of the parallel culture was fully acceptable (60%).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative