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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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 26 July 1994 to 07 October 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Longer exposure than the one indicated in the current version of the OECD Guideline. 200 cells in metaphase analysed (300 recommended)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
other: Chinese lung (CHL) cell line
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: A cell line of fibroblast from Chinese hamster lung was obtained from the Division of Genetic and Mutagenesis, National Institute of Hygienic Sciences.
- Suitability of cells: in accordance with OECD guideline 473

MEDIA USED
Type and identity of media including CO2 concentration if applicable:
10% calf serum (inactivated by heating at 56°C for 30 minutes, lot number: 48N1141, Gibco Laboratories Co. Inc.) medium was prepared routinely from minimum essential medium of Eagle (powder form, lot number: 74K4338, Gibco Laboratories Co. Inc.).

Atmospheric condition: 5% carbon dioxide and 95% air in the humid conditions at 37°C using an automatic carbon dioxide gas cell incubator (Napco Model 6200).
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Top dose examined in the study was 5000 μg/ml. Cell growth inhibition test was performed as preliminary study (dose-finding test) using 6 dose levels (156.25, 312.5, 625, 1250, 2500, 5000 μg/ml). The cell growth rate of each exposed group was compared with that of the control group which was regarded as 100%. Because decrease of cell growth rate from 156.25 to 5000 μg/ml was not significant, 5000 μg/ml was selected as the top dose of chromosomal aberration test.
Vehicle / solvent:
Distilled water was used as vehicle for test substance.
Dimethylsulfoxide was used as vehicle for positive control.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Culture medium:
10% calf serum (inactivated by heating at 56°C for 30 minutes, lot number: 48N1141, Gibco Laboratories Co. Inc.) medium was prepared routinely from minimum essential medium of Eagle (powder form, lot number: 74K4338, Gibco Laboratories Co. Inc.).

DURATION
- Preincubation period: 5ml of medium containing 4x10^3 cells/ml was added to petri dish for 3-days incubation.
- Exposure duration: 6 hours (metabolic activation method), 24 or 48 hours (direct method)
- Expression time (cells in growth medium): 18 hours (metabolic activation method)
- Fixation time (start of exposure up to fixation or harvest of cells): not provided

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Two hours before preparing the specimens, colcemid was added to all dishes to reach a final concentration of 0.2 μg/ml. After the incubation, 2 ml 0.2% trypsin was added to dissociate the layered cells. Cells were transferred into a centrifuge tube containing 5 ml of fresh medium followed by 5-minutes centrifugation at 1000 rpm. After removal of supernatant, cells were treated with 4 ml of 75 mM potassium chloride at 37°C for 15 minutes. Furthermore, they were fixed with 1 ml cold fixative, which consisted of methanol and acetic acid in 3:1 (v/v) ratio prepared immediately before use. After the repeating of the orientation i.e., centrifugation, supernatant discard and the fixation with 4 ml of cold fixative, the cool suspension was adjusted by the fixative properly, and was dripped on two fixed spots on each slide. Slide was stained for 15 minutes with 1.4% Giemsa, which was diluted with sorensen buffer solution (pH 6.8).

NUMBER OF CELLS EVALUATED: 200 cells per group

OTHER EXAMINATIONS:
- Determination of polyploidy: The aberration of chromosome was divided into morphological and numerical. Structural chromosomal aberrations were “gaps” (of the chromatid and chromosomal types), “breaks or exchanges of the chromatid type” (of dicentric and ring chromosome, etc.) and others (fragmentation etc.). As for numerical aberration, only polyploid cells were recorded. Gap was subjected to an unstained site of chromatid where was wider than the width of chromatid and aligned on its longitudinal axis. Break would be justified when such unstained site was extremely large through it aligned on its longitudinal axis. Each aberrant category was scored, considering cells with one of these aberrations as aberrant cells.
Evaluation criteria:
In the case where statistical analysis revealed significant increase in frequencies, dose-dependence and/or the reproducibility of the test, the test substance was considered to induce chromosomal aberrations.
Statistics:
An x-square test was used to analyse frequencies of the structural aberrations including gap and the polyploid cells. When the consequence was significant, an x-square test and Fisher’s direct probability method (2x2) were applied to comparison of negative control group with each exposed group.

Results and discussion

Test results
Key result
Species / strain:
other: Chinese lung (CHL) cell line
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Test substance was considered to have a negative effect on chromosomal aberration of CHL cells under the provided conditions.
Executive summary:

The in vitro cytogenicity in mammalian cells of the test substance was determined during a GLP study performed according to a method similar to OECD Guideline for Testing of Chemicals 473 using Chinese lung (CHL) cell line.

Cell growth inhibition was examined using 6 dose levels (156.25, 312.5, 625, 1,250, 2,500, 5,000 μg/ml) in 24- and 48-hour treatment of direct method and in metabolic activation method. Based on the results, concentrations of 1,250, 2,500, and 5,000 μg/ml for the direct and metabolic activation methods.

With regard to direct method, the frequency of structural aberrant was below 1.5% in negative control group and 2.5% in exposed groups, whereas the frequency of structural aberrant was 97.5% in the positive control groups. As for metabolic activation test, the frequency of structural aberrant cells of negative control group in the absence and in the presence of S9mix was 1.0 and 1.5% respectively, while that of dosed groups showed 1.5% (with S9mix) and 1.0% (without S9mix). In contrast, positive control group showed significantly high frequency of structural aberrant cells (69.0%), compared with positive control and dosed groups.  

The test item is considered to be non-mutagenic under the conditions of the test.