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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
Study period:
2003-01-07 to 2003-03-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant Guideline Study (OECD 473)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Human venous blood from healthy, adult donors was drawn into sterile, heparinized “vacutainers”. Whole blood cultures were initiated in 15 mL centrifuge tubes by adding 0.6 mL of fresh heparinized blood into a sufficient volume of culture medium so that the final volume was 10 mL in the assay without metabolic activation after the addition of the test article in its chosen vehicle or was 10 mL in the assay with metabolic activation after the addition of the test article in its chosen vehicle and the S9 mix.
Metabolic activation:
with and without
Metabolic activation system:
S9 from rat liver
Test concentrations with justification for top dose:
1st experiment:
test concentrations: 3.39 - 500 µg/mL with and without metabolic activation
evaluated concentrations: 20.2, 41.2, 58.8, 84.0 µg/mL without metabolic activation and 3.39, 6.92, 14.1, 20.2 pg/mL with metabolic activation

2nd experiment:
test concentrations: 1.25 - 75 µg/mL without metabolic activation and 5 - 75 µg/mL with metabolic activation
evaluated concentrations: 20.0, 25.0, 37.5, 75.0 µg/mL with and without metabolic activation
Vehicle / solvent:
Ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 1st exp.: 3h (with and without S9 mix), 2nd exp.: 3 h (with S9 mix) and 22 h (without S9 mix)
- Expression time (cells in growth medium): 22 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF EXPERIMENTS AND REPLICATIONS: 2 independent experiments, duplicate cultures

NUMBER OF CELLS EVALUATED: One hundred cells, if possible, from each duplicate culture from four concentrations of the test article, the negative and vehicle controls, and one dose level from the positive control cultures were analyzed for the different types of chromosomal aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Percent polyploidy and endoreduplication were also analyzed by evaluating at least 100 metaphases per culture, if available.
Evaluation criteria:
Evaluation of a Positive Response.
A test article was considered positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when p <= 0.01) in the number of cells with chromosomal aberrations was observed at one or more concentrations. The linear trend test evaluated the dose responsiveness. If a significant increase was seen at one or more concentrations, a dose-response should be observed.
Evaluation of a Negative Response.
A test article was considered negative for inducing chromosomal aberrations if no significant increase was observed in the number of cells with chromosomal aberrations at any of the concentrations.
Equivocal Evaluation.
Although most assays give clearly positive or negative results, in rare cases the data set would preclude making a definitive judgment about the activity of the test article. Results might remain equivocal or questionable regardless of the number of times the assay is repeated.
Statistics:
Statistical analysis employed a Cochran-Armitage test for linear trend and Fisher’s Exact Test (Thakur et al., 1985).

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and solubility:
1st exp.: In the assay with and without metabolic activation, a precipitate was observed after dosing at 58.8 µg/mL and higher; slight precipitate was observed after dosing at 14.1 up to 41.2 µg/mL.
2nd exp.: In the assay without metabolic activation, a slight precipitate was observed after dosing at 75.0 µg/mL. A precipitate was observed on the slides prepared from the cultures treated with 37.5, 50.0, and 75.0 µg/mL. In the assay with metabolic activation, a slight precipitate was observed after dosing at 75.0 µg/mL. A precipitate was observed on the slides prepared from the cultures treated with 50.0 and 75.0 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The chromosomal aberration rates ater treatment with the test item were within the historical contral data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Test 1: Due to excessive toxicity, only dead cells were present on the slides prepared from the cultures treated with 500 µg/mL. Reductions of 19%, 21%, 14%, 0%, 21%, 14%, 14%, and 51% were observed in the mitotic indices of the cultures treated with 6.92, 9.89, 14.1, 20.2, 28.8, 41.2, 58.8, and 84.0 µg/mL, respectively, as compared with the vehicle control cultures.
Test 2: Reductions of 8%, 33%, 28%, and 49% were observed in the mitotic indices of the cultures treated with 25.0, 37.5, 50.0, and 75.0 µg/mL, respectively, as compared with the vehicle control cultures.

OTHER:
The test article did not induce polyploidy or endoreduplication.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion