Registration Dossier

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-06-20 to 2007-02-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) and EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test) without deviation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
: The testing facility reported the following deviation to the protocol: the concentration of the positive control used in the absence of metabolic activation was updated. The deviation had no detrimental impact on the outcome of the study.
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
: The testing facility reported the following deviation to the protocol: the concentration of the positive control used in the absence of metabolic activation was updated. The deviation had no detrimental impact on the outcome of the study.
Principles of method if other than guideline:
not applicable
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): (+-)-Trans-3-methyl-1-[(4-methylphenyl)sulfonyl]-4-phenyl-4-piperidinecarbonitrile (T000268)
- Molecular formula (if other than submission substance): not applicable
- Molecular weight (if other than submission substance): not applicable
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: no data
- Physical state: beige solid
- Analytical purity: 100 %
- Impurities: not applicable
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: batch BEA351
- Expiration date of the lot/batch: 2007-05-31
- Stability under test conditions: no data
- Storage condition of test material: room temperature
- Other: no data

Method

Target gene:
not applicable
Species / strain
Species / strain:
lymphocytes: human
Details on mammalian cell lines (if applicable):
not applicable
Additional strain characteristics:
other: See "Any other information on materials and methods incl. tables"
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/beta-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
- Experiment 1 (with and without S9): 11.8, 20.6, 36.0, 63.0, 110.3, 193.0, 337.7, 591.0, 1034.3 and 1810.0 ug/mL (20.6, 36.0 and 63.0 ug/mL were selected for metaphase analysis)
- Experiment 2 (without S9): 11.8, 20.6, 36.0, 63.0, 110.3, 193.0, 337.7, 591.0, 1034.3 and 1810.0 ug/mL (36.0, 63.0 and 110.3 ug/mL were selected for metaphase analysis)
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol (the final concentration of ethanol in the culture medium was 0.5 % (v/v))
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubility properties and its relative non-toxicity to the cell cultures.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
: ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation

Migrated to IUCLID6: (EMS) at 495-550 ug/mL
Negative controls:
no
Solvent controls:
yes
Remarks:
: ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation

Migrated to IUCLID6: (CPA) at 30.0 ug/mL
Details on test system and conditions:
METHOD OF APPLICATION: in medium
- In Experiment 1 (4-hour pulse treatment), about 80 hrs after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm^2 cell culture flasks. The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test substance. For the treatment with metabolic activation solvent and positive controls were performed. After 4 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test substance was discarded and the cells were re-suspended in “saline G". The washing procedure was repeated once.
- In Experiment 2 (22-hour continuous treatment), about 80 hours after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm^2 cell culture flasks. The culture medium was replaced with complete medium (with 10 % FCS) containing the test substance without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed. All cultures were incubated at 37 deg C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

DURATION
- Preincubation period: not applicable
- Exposure duration: 4 hours (Experiment 1) and 22 hours (Experiment 2)
- Expression time: ~15 hours (Experiment 1) and 0 hours (Experiment 2)
- Selection time: not applicable
- Fixation time: 22 hours

SELECTION AGENT:
- not applicable

SPINDLE INHIBITOR:
- Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL).

STAIN:
- The cells used for evaluation of cytogenetic damage were stained with Giemsa or according to the Fluorescent plus Giemsa technique.

NUMBER OF REPLICATIONS:
2

NUMBER OF CELLS EVALUATED:
- One hundred well-spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with 46 +/- 1 centromere regions were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: One thousand cells per culture were counted for determination of mitotic index.

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.
- Determination of endoreplication: yes
- Other: no data

OTHER:
- Following cell harvest by centrifugation, the cultures were harvested by centrifugation 22 hours after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37 deg C for 20 to 25 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide.
- The slides were evaluated using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates.
- Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 ug/mL) to reassure the replication time of the cultured lymphocytes for each experiment.
Evaluation criteria:
- The test substance was classified as non-mutagenic if: 1) the number of induced structural chromosome aberrations in all evaluated dose groups was in the range of the historical control data (0.0-4.0 % aberrant cells, exclusive gaps); and 2) no significant increase of the number of structural chromosome aberrations was observed.
- The test substance was classified as mutagenic if: 1) the number of induced structural chromosome aberrations was not in the range of the historical control data (0.0-4.0 % aberrant cells, exclusive gaps); and 2) either a concentration related or a significant increase of the number of structural chromosome aberrations was observed.
- If the above mentioned criteria for the test substance were not clearly met, the classification with regard to the historical data and the biological relevance was discussed and/or a confirmatory experiment was performed.
- The test substance could be classified as aneugenic if the number of induced numerical aberrations was not in the range of the historical control data (0.0-1.5 % polyploid cells).
Statistics:
- Statistical significance was confirmed by means of the Fisher's exact test (p<0.05); however, both biological and statistical significance was considered together.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to precipitating concentrations
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In Experiment 1, no relevant influence on pH was observed (solvent control pH 7.6 versus pH 7.7 at 1810 ug/mL).
- Effects of osmolality: In Experiment 1, no relevant influence of osmolarity was observed (solvent control 440 mOsm versus 356 mOsm at 1810 ug/mL).
- Evaporation from medium: no data
- Water solubility: <0.010 g/L
- Precipitation: In Experiment 1, precipitation of the test substance in the culture medium was observed at 63 ug/mL and above in the absence and presence of S9 mix. In Experiment 2, in the absence of S9 mix, precipitation was observed at 110.3 ug/mL and above.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment 1. See relevant sections for results.

COMPARISON WITH HISTORICAL CONTROL DATA:
- In both experiments, in the absence and presence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test substance (0.0-2.5 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (1.0-2.5 % aberrant cells, exclusive gaps) and clearly within the range of the historical control data; 0.0-4.0 % aberrant cells, exclusive gaps.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In Experiments 1 and 2, in the absence and presence of S9 mix, no cytotoxicity was observed after treatment up to the highest applied concentration being far in the range of test substance precipitation.

Any other information on results incl. tables

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test substance (0.0-0.2 %) as compared to the solvent control values (0.0-0.2 %).

The positive controls showed distinct increases in cells with structural chromosome aberrations.

The proliferation index of the lymphocytes in solvent control cultures in the 22 hour preparation interval with and without S9 mix (4 hour treatment; 1.02 and 1.51, respectively), in the 22 hours preparation interval without S9 mix (continuous treatment; 1.75), was checked by analyzing the proportion of mitotic cells in the 1st, 2nd, and 3rd metaphase (M1, M1 +, M2 and M3) indicating that the lymphocytes divided about a 1.5 times within the early preparation interval. This was also proven by the occurrence of sufficient numbers of mitotic cells and by a clear clastogenicity observed after treatment with the positive control substances.

Applicant's summary and conclusion

Conclusions:
The test substance was evaluated for induction of chromosome aberrations in human lymphocytes in the presence and absence of S9 metabolic activation. Under the conditions of the study, it was concluded that the test substance was negative for induction of chromosome aberrations in the presence and absence of metabolic activation.
Executive summary:

not applicable