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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:
key study
Study period:
1975
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
publication
Title:
CHROMOSOME TESTS WITH 134 COMPOUNDS ON CHINESE HAMSTER CELLS IN VITRO - A SCREENING FOR CHEMICAL CARCINOGENS
Author:
Ishidate, M. et al.
Year:
1977
Bibliographic source:
Mutation Research, 48 337-354

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not applicable
GLP compliance:
no
Type of assay:
other: in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung fibroblast cell line (CHL)
Details on mammalian cell type (if applicable):
- originally established from the lung of a young adult by Dr. T. Utakoji, Cancer Institute, Tokyo
- karyotype consists of 25 chromosomes
- maintained by 5-day passages
- grown in a monolayer in petri dishes with Eagle's MEM (GIBCO F-11) supplemented with 10 % calf serum
- doubling time was estimated as 18.2 h at their exponential growth at 37 °C in a 5 % CO2 atmosphere
Metabolic activation:
without
Test concentrations with justification for top dose:
0.0313 , 0.0625 or 0.125 mg/mL
Vehicle / solvent:
- physiological saline
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
solvent: physiological saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 h
- Fixation time (start of exposure up harvest of cells): 24 and 48 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg/mL; added to the medium 2 h before harvest)
STAIN (for cytogenetic assays): 1% Giemsa's buffered solution (pH 6.8)

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED: the number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases

DETERMINATION OF CYTOTOXICITY
- only examinded in a pre-test (growth inhibition test)
- the dose inducing a 50% growth inhibition was taken as the highest dose in the main test

Growth inhibition tests carried out before the chromosome tests were started:
The 50% growth inhibition dose was estimated as follows:
Several different doses of each agent were separately added to the 3-day-old cultures (about 6 x 10E3 cells/3-cm dishes). The doses were prepared by a factor of 2 from the maximal dose, estimated from the data on LD50, which appeared in references for the test item. The cells in a monolayer were washed, fixed with 10% formalin solution, and then stained with 0.1% crystal violet solution for 3 min. After washing and drying each dish was placed under a photodensitometer to measure the color absorption values from which relative cell densities on the dishes were easily calculated. The color absorption values obtained reflected well the actual number of cells that survived at the bottom of each petri dish.


OTHER EXAMINATIONS:
- Determination of polyploidy: yes

OTHER:
- types of aberration were classified into 5 groups: chromatid gaps, chromatid breaks, chrorrmatid or chromosomal translocation, ring formation and fragmentation or pulverization
Evaluation criteria:
CHL cells commonly have less than 3.0% cells with chromosomal aberrations. Therefore, the final judgement given to all experimental groups was as follows: Negative if less than 4.9% of the aberration was detected – even when doses of the agent were elevated to sub-lethal amounts, where almost no mitosis was observed; suspicious if between 5.0 and 9.9%, and positive if between 10.0 and 19.9% (+), 20.0 and 49.9% (++) and more than 50.0% (+++). When no reasonable dose response was obtained, experiments with different doses were carried out to confirm its reproducibility.
Statistics:
no data

Results and discussion

Test results
Key result
Species / strain:
mammalian cell line, other: Chinese hamster fibroblast cell line (CHL)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
94% cells with abberations
Cytotoxicity / choice of top concentrations:
other: cytotoxicity can be assumed as the dose where 50% growth inhibition occured in the pre-test was used as the highest dose
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- no data

COMPARISON WITH HISTORICAL CONTROL DATA:

- CHL cells commonly have less than 3.0% cells with chromosomal aberrations. This value was taken for judgement.

Any other information on results incl. tables

A D20 value (the dose (mg/mL) at which chromosomal aberrations were detected in 20% of metaphases) of 0.0451 mg/mL was obtained.

Results chromosome test (only 24 values are presented):

Dose [mg/mL]

No of Metaphases

Polyploid cells [%]

Cells with structural chromosome aberrations [%], 24 h

 

 

24 h

48 h

G

B

T

R

F

Total

 

 

 

 

 

 

 

 

 

 

 

None

100

1

-

1

0

0

0

0

1

Solv

 

-

-

-

-

-

-

-

-

0.0313

100

0

1

1

3

2

0

0

6

0.0625

100

1

0

2

7

16

0

0

22

0.125

100

1

T

0

46

91

0

0

94

G: chromatid gaps

B: chromatid or chromosomal breaks

T: translocation

R: ringformation

F: fragmentation

Applicant's summary and conclusion