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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:
15 April 2002-10 July 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
The study has been performed according to METI guidelines and according to GLP principles. The METI guidelines for the Chromosome Aberration test are equivalent to the OECD guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
The requirements of the Japanese New Chemical Substance Law (METI) to assess the potential chromosomal mutagenecity of a test material are equivalent to the OECD guideline 473 requirements.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Vinylene Carbonate
- Physical state: clear colourless liquid
- Analytical purity: 99.9%
- Purity test date: 7 January 2002
- Lot/batch No.: 011220
- Storage condition of test material: approx. 4 ºC in the dark, under nitrogen

Method

Target gene:
chromosomes in metaphase
Species / strain
Species / strain / cell type:
other: Chinese Hamster Lung
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's Minimal Essential Medium (MEM) with HEPES buffer and Earle's Salts and supplemented in-house with 10% foetal bovine serum and antibiotics.
- Culture conditions: at 37 ºC with 5% CO2 in air
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix, induced by phenobarbitone and beta-naphthoflavone
Test concentrations with justification for top dose:
Preliminary cell growth inhibition test: dose levels 0, 3.36, 6.72, 13.44, 26.88, 53.75, 107.5, 215, 430, 860 µg/ml
Chromosome aberration test -S9 mix: 26.88, 53.75 and 107.5 µg/ml
Chromosome aberration test +S9 mix: 26.88, 40.32 and 80.63 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Minimal Essential Media (MEM)
- Justification for choice of solvent/vehicle: soluble at 10 mM concentration
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
MEM
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.1 µg/ml -S9 mix
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
5.0 µg/ml +S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 hrs (-/+ S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.1 µg/ml)
FIXING SOLUTION: methanol:glacial acetic acid (3:1 v/v)
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: all treatments were performed in duplicate.


NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: a total of 1000 cells were counted and the number of cells in metaphase recorded and expressed as the mitotix index and as a percentage of the vehicle control value.


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: Precipitation


OTHER:
Evaluation criteria:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 23 to 27 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage recommended in the 1983 UKEMS guidelines for mutagenicity testing.
Aberrations recorded by the slide scorer were checked by a senior cytogeneticist. Cells with 38 or more chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported. Endoreduplicated cells are recorded and are included in the polyploid cell total number. If there was a dose-related increase in endoreduplicated cells then they are reported separately.
The % of cells showing structural chromosome aberrations (breaks and exchanges) was calculated and reported. the number of gap-type aberrations was recorded.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results
Species / strain:
other: Chinese Hamster Lung
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility:
- Precipitation: No precipitate of test material was observed at any dose level.
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES:


COMPARISON WITH HISTORICAL CONTROL DATA:


ADDITIONAL INFORMATION ON CYTOTOXICITY:

Any other information on results incl. tables

The test material induced statistically significant increases in the frequency of cells with aberrations both in the presence and absence of metabolic activation. There were significant increases at the maximum dose levels selected for metaphase analysis, which were 107.5 µg/ml for the absence of S9 and 80.63 µg/ml for the presence of S9.

The test material induced statistically significant increases in the number of polyploid cells at an intermediate dose level 53.75 µg/ml in the absence of S9 only. Polyploidy was not observed at 107.5 µg/ml and this was considered to be the result of the highly clastogenic and toxic response at this dose level, slowing down the rate of mitosis and causing cell-cycle synchronisation. This suggestion is supported by the mitotic index data.

There was a small statistically significant increase in polyploid cells in the presence of S9 at 40.32 µg/ml.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
positive

The test material induced a statistically significant increase in the frequency of cells with chromosome aberrations, both in the presence and absence of a liver enzyme metabolising system.
The test material induced a statistical increase in polyploidy in the absence and presence of S9.
The test material was therefore considered to be clastogenic to CHL cells in vitro.
Executive summary:

Vinylene carbonate was studied for chromosomal aberration potential in vitro in Chinese Hamster Lung cells in a test equivalent to OECD 473. Reliable positive and negative controls were included.

The test material induced a statistically significant increase in the frequency of cells with aberrations both in the absence and the presence of metabolic activation. In additions, Vinylene carbonate induced a srtatistically increased incidence in polyploidy cells, both in the absence and in the presence of metabolic activation. Based on these observations Vinylene carbonate is considered clastogenic in vitro.