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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 16, 2008 to February 6, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF
Version / remarks:
No 12 Nousan No 8147
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Cas Number:
915972-17-7
Molecular formula:
C33H39NO9
IUPAC Name:
[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate
Test material form:
solid
Details on test material:
- Analytical purity: >94 %
Specific details on test material used for the study:
Batch no. 080722
Description: Pale yellow green powder

Method

Species / strain
Species / strain / cell type:
mammalian cell line, other: Chinese hamster Lung (CHL) cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: CHL cells established from the lung of Chinese hamster

MEDIA USED
- Type and identity of media: MEM (Gibco BRL), supplemented with 10% inactivated newborn calf serum, 100 IU/ml Penicillin, 100 μg/ml streptomycin and 2 mM L-Glutamine.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes, Stock confirmed to the Mycoplasma-free
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix induced by phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Preliminary toxicity assay: 19.5 to 5000 μg/mL
Assay: 1st experiment (6 hr exposure): concentrations of 78.1, 156, 313 or 625 μg/mL without and with metabolic activation
2nd experiment (24 hr exposure): 30.9, 46.3, 69.4, 104 or 156 μg/mL without metabolic activation
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: Due to the limited solubility of the test substance in water, dimethylsulfoxide (DMSO) was selected as the vehicle, and has been demonstrated to be suitable in the in vitro cytogenetic test.
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:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 6 hours (short term treatment, absence and presence of a metabolic activation system) or 24 and 48 hours (continuous treatment, absence of a metabolic activation system)

SPINDLE INHIBITOR: 0.2 μg/mL colcemid was added 2 hours before cell harvest

STAIN: 2% Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slides were prepared by dropping the harvest cultures on clean glass slides. The slides were stained with 2% Giemsa.

NUMBER OF CELLS EVALUATED: 100 cells were examined per replicate culture (200 per dose)

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE:
Slides were coded prior to analysis. 100 cells were examined per replicate culture (200 per dose) and were scored for structural aberrations and for numerical aberrations (polyploidy).

DETERMINATION OF CYTOTOXICITY
- Method: Previously performed HPRT study

OTHER EXAMINATIONS:
- Determination of polyploidy: Only polyploid cells having 3 or more copies of haploid number of chromosomes were scored as a numerical chromosome aberration cell. Since the CHL cells have 25 chromosomes in the modal number, the cell with 37 or more chromosomes was recorded as a polyploid cell.
- Determination of endoreplication: Endoreduplication was classified as polyploidy.
Evaluation criteria:
The test material was considered positive if reproducible and significant Increases in the frequencies of structurally (excluding gaps) or numerically aberrant metaphases were observed with a dose-related response.
Statistics:
The number of structurally aberrant metaphases and the number of polyploid metaphases at each concentration were statistically compared with those of corresponding solvent controls using a chi-square test.

Results and discussion

Test results
Key result
Species / strain:
mammalian cell line, other: Chinese hamster Lung (CHL) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
continuous treatment: 104 and 156 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was observed at 313 and 625 μg/mL

RANGE-FINDING/SCREENING STUDIES:
Nine doses of the test material ranging from 19.5 to 5000 μg/mL in the absence and presence of S9-mix were evaluated in the preliminary growth inhibition test. Precipitation was observed at 313 μg/mL and above both in the absence and presence of a metabolic activation system, Over 50 % cell growth inhibition was not observed in the short-term test but was observed in the continuous test at the concentrations of 156 μg/mL and above.

HISTORICAL CONTROL DATA
- Positive historical control data: Valid
- Negative (vehicle) historical control data: Valid

Applicant's summary and conclusion