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Toxicological information

Genetic toxicity: in vitro

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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant near-guideline study, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

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): 2, 4, 6-trimethylbenzaldehyde
- Storage condition of test material: cool and dark place, in a nitrogen environment.
- Physical state: Colorless to pale yellow, transparent liquid

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other: Chinese Hamster Lung (CHL/IU)
Details on mammalian cell type (if applicable):
- Type and identity of media: Neonatal calf serum (Lot No. NBQ07, Mitsubishi Chemical Corporation) was added to Eagle MEM medium (Lot No. 421001, Nissui Pharmaceutical Co., Ltd.) at 10 v/v%.
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and 5,6-benzoflavone induced SD rats rat liver S9-mix
Test concentrations with justification for top dose:
Chromosomal aberration study: 0, 40, 80, 160 µg/mL in the absence of S9 mix and 0, 70, 140, 280 µg/mL in the presence of S9 mix.
Chromosomal aberration study (confirmatory study): 0, 80, 110, 140 µg/mL in the absence of S9 mix and 0, 140, 200, 260 µg/mL in the presence of S9 mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: This solvent was selected because the test substance was found to be dissolved at 150 mg/mL or more in DMSO.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the precence of metabolic activation
Details on test system and experimental conditions:
CELL GROWTH INHIBITION TEST: A preliminary toxicity test was performed on cell cultures using a 6-hour exposure period (both with and without metabolic activation) followed by an 18-hour recovery period in treatment-free media. The dose range used was 20 to 360 µg/mL.

CHROMOSOMAL ABERRATION STUDY:
EXPERIMENT 1
- METHOD OF APPLICATION: in medium
- DURATION: A chromosomal aberration study was conducted in the same way as the cell growth inhibition study.
- DOSE LEVELS SELECTED FOR METAPHASE ANALYSIS: Chromosomal aberration study: 0, 40, 80, 160 µg/mL in the absence of S9 mix and 0, 70, 140, 280 µg/mL in the presence of S9 mix; Chromosomal aberration study (confirmatory study): 0, 80, 110, 140 µg/mL in the absence of S9 mix and 0, 140, 200, 260 µg/mL in the presence of S9 mix

SPINDLE INHIBITOR: Colcemid

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: cell growth inhibition study: 50 metaphase cells; chromosomal aberration study: 100 cells per dish.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

DETERMINATION OF CYTOTOXICITY
- Cell growth rate
Evaluation criteria:
The study result was regarded as positive if incidence of cells with structural or numerical aberrations increased 10% or more and increases in the incidence were dose-dependent or if 5% of higher incidence was shown to be reproducible. Otherwise, the result was regarded as negative. If the result was judged to be positive, the D20 value (concentration at which 20% or more cells show abnormality) was determined.
Statistics:
Cell growth rates were calculated as percentages of the cell count in the negative control group, and concentrations at which 50% inhibition is achieve were calculated.

Results and discussion

Test results
Species / strain:
mammalian cell line, other: Chinese Hamster Lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: The 50% inhibition concentration was about 140 µg/mL in the absence of S9 mix and about 250 µg/mL in the presence of S9 mix. Evidence of apparent inhibition of cell growth with cell growth rates lower than 50% was obtained at 160 µg/mL in the absence of S9 mix and 280 µg/mL in the presence of S9 mix, both the highest doses selected in the chromosomal aberration study. In addition, structural aberrations slightly increased in the absence of S9 mix.

CHROMOSOMAL ABERRATION STUDY
In the absence of S9-mix, the cell growth rate was 90.7%, 78.5% and 7.8%, respectively, at 40, 80 and 160 µg/mL. The incidence of structural aberrations was 0.5%, 0.5% and 15.5%, respectively, at 40, 80 and 160 µg/mL compared to 0% in the negative control group and 53.5% in the MMC (positive control) group. Incidence of numerical aberrations was less than 5.0% in all groups. The D20 value was calculated to be 0.23 mg/mL (S=0.016) in terms of structural aberrations. In the presence of S9-mix, the cell growth rate was 101.2%, 93.7% and 16.9%, respectively, at 70, 140 and 280 µg/mL.
The incidence of structural aberrations was 1.0%, 0% and 22.5%, respectively, at 40, 80 and 160 µg/mL compared to 0% in the negative control group and 32.0% in the MMC (positive control) group. The incidence of numerical aberrations was less than 5.0% in all groups. The D20 value was calculated to be 0.30 mg/mL (S=0.021) in terms of structural aberrations.

CONFIRMATORY STUDY
In the absence of S9-mix, the cell growth rate was 76.7%, 71.4% and 12.1%, respectively, at 80, 110 and 140 µg/mL. The incidence of structural aberrations was 1.0%, 0% and 19.0%, respectively, at 80, 110 and 140 µg/mL compared to 1.0% in the negative control group and 56.0% in the MMC (positive control) group. The incidence was less than 5.0% in all groups. The D20 value was calculated to be 0.21 mg/mL (S=0.019) in terms of structural aberrations. In the presence of S9-mix, the cell growth rate was 91.9%, 84.1% and 16.2%,respectively, at 140, 200 and 260 µg/mL. The incidence of structural aberrations was 2.5%, 5.0% and 23.5%, respectively, at 140, 200 and 260 µg/mL compared to 0% in the negative control group and 60.0% in the CPA (positive control) group. Incidence of numerical aberrations was 2.0%, 5.0% and 0%, respectively, at 140, 200 and 260 µg/mL compared to 0.5% in the negative control group. The D20 value was calculated to be 0.28 mg/mL (S=0.021) in terms of structural aberrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

The test substance is considered to be clastogenic under the conditions of this test.
Executive summary:

In a GLP-compliant chromosome aberration test, Chinese Hamster Lung (CHL/IU) cells were exposed to the test substance with and without metabolic activation (S9 -mix).The study consisted of a cell growth inhibition test and two separate experiments. Except in the cell growth inhibition test, duplicate cultures of Chinese Hamster Lung (CHL) cells that had been treated with the test material in either the presence or absence of metabolic activation, were evaluated for induction of chromosome aberrations. Duplicate vehicle and positive controls were included in parallel in both experiments. In both experiments, the exposures were 6(18)-hour both with and without metabolic activation. The dose levels used were selected on the results of the cell growth inhibition test. The dose levels in experiment 1 were 0, 40, 80, 160 µg/mL in the absence of S9 mix and 0, 70, 140, 280 µg/mL in the presence of S9 mix. The dose levels in experiment 2 were 0, 80, 110, 140 µg/mL in the absence of S9 mix and 0, 140, 200, 260 µg/mL in the presence of S9 mix. Experiments were considered to have been conducted properly because there were no significant differences in the incidence of aberrant cells between two dishes used; incidence of aberrant cells was lower than 5% in the negative control group; and incidence of cells with aberrations other than gaps was 20% or higher in the positive control group. In the first experiment, chromosomal aberrations increased at the highest dose in both the presence and absence of S9 mix, but increases were not dose-dependent. In the second experiment, structural aberrations increased in both the presence and absence of S9 mix, showing reproducibility of the finding obtained in the first study. In addition, increases in structural aberrations were dose-dependent in the presence of S9 mix.In conclusion, the test material was considered to be clastogenic to CHL cells in vitro.