Methacrylonitrile

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Methacrylonitrile

EC Number:

204-817-5

EC Name:

Methacrylonitrile

Cas Number:

126-98-7

Molecular formula:

C4H5N

IUPAC Name:

2-methylprop-2-enenitrile

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).

Controls

Controls 1

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

Controls 2

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 results

Test results 1

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

Test results 2

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.