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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:
no data
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

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:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Methacrylonitrile
EC Number:
204-817-5
EC Name:
Methacrylonitrile
Cas Number:
126-98-7
Molecular formula:
C4H5N
IUPAC Name:
methacrylonitrile
Details on test material:
- Name of test material (as cited in study report): 2-methyl-2-propenenitrile (MAN)
- Stability under test conditions: The lot used for the study has been confirmed to be stable during the study
- Storage condition of test material: Shield from the light at cool temperature

Method

Target gene:
No data
Species / strain
Species / strain / cell type:
other: Chinese hamster lung(CHL/IU) cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's MEM media (Nissui Seiyaku, Japan) with 10% of calf serum (Cansera International, lot number of 2608311).
Additional strain / cell type characteristics:
other: No more than 10 th-subculture after thawing of the purchased culture (4th of subculture).
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-Benzoflavone induced rat liver, S9
Test concentrations with justification for top dose:
- Zero, 0.17, 0.34, 0.67 mg/mL (short-term treatment for 6 hours) in the absence of metabolic activation
- Zero, 0.17, 0.34, 0.67 mg/mL (continuous treatment for 24 hours) in the absence of metabolic activation
- Zero, 0.068, 0.14, 0.27, 0.54 mg/mL short-term treatment for 6 hours) in the presence of metabolic activation
Vehicle / solvent:
Distilled water (lot number K8H73, Ohtsuka Seiyaku Kojo, Japan).
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium.


DURATION
- Exposure duration:
Without S9 mix: 6 or 24 hours
With S9 mix: 6 hours.
- Incubation time: (If the exposure duration is 6 hours) 18 hours in culture in treatment-free media.

STAIN (for cytogenetic assays): 3% Giemsa solution (diluted with 1/15 M of phosphate buffer solution, pH of 6.8).

NUMBER OF REPLICATIONS: 2.


DETERMINATION OF CYTOTOXICITY
- Method: cell growth rate, mitotic index. Cell growth rate of less than 20% were excluded from further chromosome aberration analysis. Mitotic index of 0.5% and above were subjected for the determination of cytotoxicity of the test substance.
Cytotoxicity 20% and above


OTHER EXAMINATIONS:
- Determination of polyploidy: Yea.
- Other: Observation of morphologic abnormalities.


Evaluation criteria:
Biological evaluation of significance and dose-dependent analysis.
Statistics:
Fisher's exact test and Cochran-Armitage test.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: Chinese hamster lung(CHL/IU) cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
20 % at 0.54 mg/mL
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
other: Chinese hamster lung(CHL/IU) cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
87.5 % at 0.67 mg/mL (4 hours), 109 % at 0.67 mg/mL (24 hours)
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
Cells with structural aberrations were increased dose dependently after short term treatment with metabolic activation (frequency: 7.5-62.0 %). Polyploidy did not show a dose-dependent increase but was significantly induced at 0.068 mg/mL and 0.14 mg/mL during short term treatment with metabolic activation.
Remarks on result:
other: strain/cell type: Chinese hamster lung(CHL/IU) cells
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Chromosome analysis in short-term treatment with S9 mix 

 

Concentration of methacrylonitrile (mg/mL)

Concentration of CPA μg/mL

Zero

0.068

0.14

0.27

0.54

5

Number of cells analysed

200

200

200

200

Tox

200

TAG

 

5

 

19*

 

31*

 

129*

Tox

127*

 

2.5 %

9.5 %

15.5 %

64.5 %

-

63.5 %

TA

 

4

 

15*

 

30*

 

124*

 

Tox

124*

 

2.0 %

7.5 %

15.0 %

62.0 %

-

62.0 %

Polyploid (%)

 

0.50

3.13*

1.88*

0.14

Tox

0.13

Cell number (%)

100.0

80.3

80.7

67.5

20.2

-

 

Mitotic Index (%)

-

-

-

1.6

Tox

-

TAG = total number of cells

TA = total number of cells with aberration except gap

* indicates p < 0.05

Tox = not examined because of cytotoxicity

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation

Chromosome analysis showed that the test material induced morphologic abnormalities and polyploidy after 6 h exposure in the presence of metabolic activation.