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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results. This study has been performed according to OECD 473 guidelines and GLP principles. Reliability given as 2 due to results being obtained on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Notification on Partial Revision of Testing Methods Relating to New Chemical Substances (Notification No 700 of Kanpogyo, No 1039 of Yakuhatsu and No 1014 of 61 Kikyoku, December 5 1986
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
Type:
Constituent
Test material form:
other: Solid

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
other: Chinese Hamster Lung Fibroblasts (Clone No 11)
Details on mammalian cell type (if applicable):
Chinese Hamster Lung (chl) cells were supplied by National Insitute of Hygienic Sciences on September 28 19988. The modal number of chromosomes is 25 per cell. The time required for doubling the cell number is about 15 hours. CHL cells preserved by freezing were defrosted and cultured. Three days after incubation, a subculture was made, and the cells in logarithmic growth phase were used for the test. The passage number of cells used in the chromosomal aberration test was 26 for 24 and 48-h treatment by the direct method, and 26 by the metabolic activation method.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5, 6 benzoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test (Cell Growth Inhibition Test, cell division inhibition):
The dose range of test item used was 0, 100, 200, 400, 600, 800, 1000, 1200, 1400 and 1600 µg/ml.

24-hour tretament without metablic activation: 0, 150, 300 and 600 µg/ml
48-hour treatment withou metabolic activation: 0, 100, 200 and 400 µg/ml

With metabolic activation: 0, 200, 400 and 800 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: pure water

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:
Used in the absence of S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Used in the presence of S9
Details on test system and experimental conditions:
Cell culture
5ml of 10% Newborn Calf Serum/Minimum Essential Medium (NCS/MEM) containing 15000 or 5000 cells/ml ws plated in Petri dish and cultured for 2 or 3 days. Monolayer cells in logarithmic growh phase were exposed to the test item at 2 or 3 days after culturing under the following conditions
Temperature: 37 ± 0.5°C
Humidity : almost 100%
Atmosphere: air containing 5% CO2

Test substance and positive control solutions
Test solutions: The test substance was dissolved in pure water. The solution was diluted with pure water to make the required concentrations and added to the medium at the rate of 1%. Test solutions were prepared immediately before the start of each test and used within 2 hours.

Positive control solutions: Positive control substances were dissolved in pure water and diluted with pure water to the required concentration. Positive control solutions were prepared immediately before the start of each test and used witin 2 hours.


Cell Growth Inhibition Test : Cell cultures were exposed to the test material for 24 or 48 hours. Test with metabolic activation were treated for 6 hours. At the end of the treatment period S9 Mix / MEM and test material was removed and the dishes rinsed. 5ml 10% NCS/MEM was then added to the dishes and the cell in cubated for 18 hours. At the end of incubation, the medium was removed and the cells detached from each dish with 2ml of trypsin and counted using a Microcell Counter. Two dishes were used and counted for each concentration.


Cell Division inhibition test: Cell culture and treatment were as for the cell growth inhibition test. Two hours prior to the end of incubation, colcemid was added to the medium. Chromosome preparations were made and observed microscopically for the incidence of mitotic cells.


CHROMOSOMAL ABERRATION TEST :
Test substance - the test was carried out at 3 dose levels prepared by diluting the maximum test concentration. The treatment regimes were-
24 hour treatment by the direct method at 150, 300 and 600 μg/l.
48 hour treatment by the direct method at 100, 200 and 400 μg/l.
6 hour treatment by the meatbolic activation method at 200, 400 and 800 μg/l.

Positive control substances - The following test methods and concentrations were used
Mitomycin by the direct method 0.05μg/l.
Cyclophosphamide by the metabolic activation method 10μg/l.

Direct method
Cell cultures were exposed to the test material for 24 or 48 hours. Two hours prior to the end of incubation colcemid was added to the medium after which chromosome preparations were made
A vehicle (pure water) control and Mitomycin positive control groups were maintained under identical conditions but not exposed to the test item.

Metabolic activation method
Cell cultres treated with S9 Mix/MEM were exposed to the test material for 6 hours after whch the S9 Mix/MEM including the test material was removed and the dishes rinsed. 5ml of 10% NCS/MEM was then added to the dishes and the cells were incubated for 18 hours. Two hours prior to the end of incubation colcemid was added. Chromosome preparations were made at the end of incubation.

A vehicle control (pure water) and Cyclophosphamide positive control groups were maintained under identical conditions but not exposed to the test item.
A culture without S9 Mix was treated with test substance at the same concentrations and treatment times to clarify the effect of metabolic activation.

NUMBER OF REPLICATIONS: Two


NUMBER OF CELLS EVALUATED:
100/culture, 200/ test concentration

OBSERVATION OF CHROMOSOMAL ABERRATIONS:
The number of cells with numerical aberrations (polyploid, endoreduplication) and with chromatid-type or chromosome-type structural aberrations ( gap, break, exchange etc) were checked by observing 100 well-spread metaphases from each culture. The incidence of cells with numerical and / or structural aberrations was obtained by observing 200 metaphases for each dose. The structural aberration incidence was calculated with and without gaps.
A gap was only scored when a clear discontinuity (larger than a chromatid width) was evident and when the distal part of the chromatid or chronosome showed no dislocation. Microscope slides were coded and scored blind.

Evaluation criteria:
The results were assessed as follows. The incidence of cells (mean value for two flasks) with aberrations including gaps was
less than 5% - Negative (-)
5% or more but less tha 10% - Suspect Positive (±)
10% or more but less than 20% - Positive (+)
20% or more but less than 50% - Positive (++)
50% or more - Positive (+++)

The test substance is judged to be positive for induction of chromosomla aberration when the incidence of 10% or more was dose related or reproducible. Reexamination was performed whenever the result was suspect positive, or positive at only one dose level.

Results and discussion

Test results
Species / strain:
other: Chinese Hamster Lung Fibroblast (clone No 11)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The values for the two dishes were not markedly different, the incidence of cells with aberrration did ot exceed 5% in the negative (vehicle) control groups, the incidence of cells with structural aberrations excluding gap was 10% or more in the positive control groups and there was no contamination with microorganisms in the cultures. The test resuts were therefore considered to be valid.

Inhibition test on cell growth and cell division
Cell growth inhibition see Table 1 and Fig 1 in attached background information
The growth rate in the vehicle control was considered to be 100%. 50% growth inhibition concentrations of the test substance were about 400 μg/ml for 24 hour treatment and about 120 μg/ml for 48 hour treatment by the direct method, and about 1000μg/ml by the metabolic activation method.
The cell division inhibition test was carried out at several concentrations close to the LC50 to determine the possible dose level in the chromosomal aberration test. Mitotic metaphases of chromosomes sufficient to assess chromosomal aberration were observed at doses of 400 μg/ml or less for 24 hour treatment, 800 μg/ml or less for 48 hour treatment by the metabolic activation method and 600 μg/ml or less by the metabolic activation method. Therefore 600 μg/ml for 24 hour treatment and 400μg/ml for 48 hour treatment by the direct method, and 800 μg/ml by the metabolic activation method were used as the maximum concentrations in the chromosomal aberration test. Additionally, two lower concentrations with geometric progression of 2 were used in each treatment.

Chromosomal aberration test
Direct method (see table 2 and Fig 2 in attached background information.)
-24 hour treatment, The incidence of cells with structural chromosomal aberrations including gaps were
150μg/ml = 5.0% , suspect positive
300μg/ml = 13.0% , positive
600μg/ml = 39.5%, positive
The incidence of structural aberration was dose related
The incicence in the vehicle control was 1.5% and within normal range,
The incidence in the positive control group treated with Mitomycin was 55%, indicating induction of chromosomal aberration.
The incidence of polyploid cells was negative in each treatment group.
-48 hour treatment, The incidence of cells with structural chromosomal aberrations including gaps were
100μg/ml = 2.50% , negative
200μg/ml = 7.5% , suspect positive
400μg/ml = 59.5%, positive
The incicence in the vehicle control was 1.0% and within normal range,
The incidence in the positive control group treated with Mitomycin was 90.5%, indicating induction of chromosomal aberration.
The incidence of polyploid cells was negative in each treatment group.

Metabolic activation method (Table 3 and Fig 3)
With S9 Mix -The incidence of cells with structural chromosomal aberrations including gaps were
200μg/ml = 4.5% , negative
400μg/ml = 26.0% ,positive
800μg/ml = 94.0%, positive
The incidence of structural aberration was dose related
The incicence in the vehicle control was 3.0% and within normal range,
The incidence in the positive control group treated with Cyclophophamide was 67.5%, indicating induction of chromosomal aberration.
The incidence of polyploid cells was negative in each treatment group.
Without S9 Mix -The incidence of cells with structural chromosomal aberrations including gaps were
200μg/ml = 4.5% , negative
400μg/ml = 2.0% , negative
800μg/ml = 1.5.0%, negative
The incidence of structural aberration was dose related
The incicence in the vehicle control was 2.5% and within normal range,
The incidence in the positive control group treated with Cyclophophamide was 3.0% and within normal range.
The incidence of polyploid cells was negative in each treatment group.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
The test item induced structural chromosomal aberrations dose dependently within the dose range 150 - 600 μg/ml in the 24 hour treatment and within the dose range 200 - 400 μg/ml in the 48 hour treatment by the direct method and within the dose range 400 - 800 μg/ml by the metabolic activation method.
Executive summary:

Introduction. 

The effect of the test item on chromosomal aberration induction was investigated using Chines hamster lung fibroblasts (CHL cells). The method was conducted in accordance with the Notification on Partial Revision of Testing Methods Relating to New Chemical Substances (Notification No 700 of Kanpogyo, No 1039 of Yakuhatsu and No 1014 of 61 Kikyoku, December 5 1986, complaint with OECD guidelines.

Methods. 

Chromosomal aberration tests were carried out at doses of 150, 300 and 600 μg/ml for 24 hour treatment, at 100, 200 and 400 μ

g/ml for 48 hour treatment by the direct method (without metabolic activation), and at 200, 400 and 800 μg/ml by the metabolic activation method. Positive control Mitomycin was used at 0.05 μg/ml for 24 and 48 hour treatment by the direct method, and Cyclophophamide was used at 10 μg/ml by the metabolic activation method.

Results.

The test item induced structural chromosomal aberrations dose dependently within the dose range 150 - 600 μg/ml in the 24 hour treatment and within the dose range 200 - 400 μg/ml in the 48 hour treatment by the direct method and within the dose range 400 - 800 μg/ml by the metabolic activation method.

The incidence of polyploid ccells was negative in each treatment group.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the normal range.

The positive control groups induced chromosomal aberrations.

Conclusion.

The test item was considered to be clastogenic to CHL cells in vitro