Fluoroethylene

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Deviations:

no

Remarks:

The study was conducted according to the guideline in effect at the time of study conduct.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Fluoroethylene

EC Number:

200-832-6

EC Name:

Fluoroethylene

Cas Number:

75-02-5

Molecular formula:

C2H3F

IUPAC Name:

fluoroethene

Details on test material:

- Purity: 99.99%

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham’s F12 medium without hyposanthine (F12 w/o hx) containing 5% dialyzed heat-inactivated foetal bovine serum without antibiotics. In cytotoxicity and chromosome aberration experiments, penicillin (50 units/mL) and streptomycin (50 micrograms/mL) were included in the culture medium.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

other: BH4 clone of the CHO-K1 cell line

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Chromosome aberration assay (without activation): 0, 10, 40, 70, and 100% (target concentrations); 0.0, 8.1, 42.9, 72.3, and 104.1% (analytical concentrations)
Chromosome aberration assay (with activation): 0, 10, 25, 50, and 75% (target concentrations); 0.0, 8.3, 25.9, 49.6, and 75.1% (analytical concentrations)
Confirmatory chromosome aberration assay (without activation): 0, 10, 40, 70, and 100% (target concentrations); 0.0, 8.8, 46.5, 77.8, and 111.4% (analytical concentrations)
Confirmatory chromosome aberration assay (with activation): 0, 10, 25, 50, and 75% (target concentrations); 0.0, 12.3, 35.4, 63.3, and 91.3% (analytical concentrations)

Controls

Untreated negative controls:

yes

Remarks:

Nitrogen

Positive controls:

yes

Positive control substance:

other: For trials without activation: 6.44 mM methane sulfonic acid, ethyl ester (ethylmethane sulfonate, EMS); phosphate buffered saline was used as the solvent. For trials with activation: approximately 2.5% vinyl chloride (VC) mixed with air.

Details on test system and experimental conditions:

METHOD OF APPLICATION: The day following culture initiation, the medium was replaced with treatment medium. Specially designed 1.65 L glass chambers were used to expose the cell cultures. A separate chamber was used for each test level. Tissue culture flasks (4/test level) were uncapped and placed into the chambers immediately prior to the exposure period. The cells were incubated with the test material for 5 hours without activation or 2 hours with activation. During the treatment period, the flasks were incubated on a rocker panel to maximize exposure. After incubation, the flasks were flushed once with air, the treatment medium was removed, and the flasks were rinsed with fresh culture medium and incubation continued for 24-26 hours. Colcemid® was added to the medium approximately 2-3 hours before the end of this incubation period to arrest metaphase cells. Cell from pairs of like-treated flasks were collected and pooled in centrifuge tubes to form two replicate samples for harvest. The cells were then incubated for 15 minutes in a hypotonic solution of sodium citrate and fixed in two changes of 3:1 mixture of methanol and glacial acetic acid. Centrifugation was required between each of these steps and after the final fixation step. Cells were resuspended in fixative and one slide per culture prepared by applying an aliquot of the fixed cells onto microscope slides and air-drying them. The slides were stained by Giemsa and permanently mounted.

DURATION
- Exposure to the test substance began the day following cell plating in culture medium.
- Cells were incubated with the test substance for 4 hours (-S9 and +S9) and 20 hours (-S9).
- After incubation, the flasks were flushed once with air, the treatment medium removed, and the flasks rinsed with fresh culture medium. The incubation was continued for 24-26 hours. Colcemid® was added to the medium approximately 2-3 hours before the end of this incubation period.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid®

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 independent trials

NUMBER OF CELLS EVALUATED: At least 100 metaphases per concentration level (50 from each duplicate culture).

DETERMINATION OF CYTOTOXICITY
- Method: A cytotoxicity assessment was conducted and fifty to 100 metaphase cells per test concentration were scanned and scored. Cell cycle delay was judged fo be present where marked increases in first metaphase cells were seen in the relative distribution of first metaphase, first-to-second metaphase, and second metaphase cells, as compared to the control. The cells were distinguished by their characteristic chromosome staining, resulting from differential uptake of 5-bromodeoxyuridine.

OTHER: A pair of flow meters was used to generate the appropriate test substance-nitrogen mixture. Atmospheric samples were drawn from each exposure chamber with a glass syringe and analyzed with a gas chromatograph equipped with a flame ionization detector.

Evaluation criteria:

Assessment of a positive response was based on:
• A statistically significant increase (p < 0.05) in the percentage of cells with chromosomal aberrations was seen in one or more treatment groups relative to the negative control response.
• The observed increased frequencies were accompanied by a concentration-related increase.
• These effects must be reproducible (i.e., evident in both trials under activated or nonactivated conditions).

The test substance was judged negative if:
• There was neither a statistically positive response at a minimum of one test level nor a significant dose-related increase in chromosomal aberrations.

Results which do not meet these criteria for positive or negative assessments were evaluated on a case-by-case basis.

Statistics:

For each trial the following were evaluated statistically: the number of structural aberrations, expressed on a per-cell basis; the percent abnormal cells; and the percent cells with more than one aberration. Chromatic and isochromatid gaps were recorded, but excluded from the calculations.

A Mann-Whitney U Test comparing each treatment level with the negative control was used to evaluate the data for aberrations per cell. The percent abnormal cells and percent cells with more than one aberration were evaluated using a Fisher Exact Test to compare each treatment level with the negative control. Statistical significance was judged at the 5% level.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Non-activated, cytotoxicity studies showed a significant cell cycle delay only at the relatively high concentration of 96.3%. With activation, moderate cell cycle delay at 52.1%, and severe cell cycle delay at 61.3% or more was observed. See Table 1 for complete results.

Trials Without Activation:
The initial evaluation of 100 cells per treatment level resulted in a statistically significant increase in the percent abnormal cells at only the highest test concentration in nonactivated Trial I; no significant treatment effects were detected in nonactivated Trial II (see Table 2). In an effort to clarify the discrepant findings in the nonactivated trials, an additional 100 cells per trial were scored from the negative control and the highest test substance concentration, where possible. (The data in the tables represent the total of all cells scored.) This analysis also yielded statistically significant increases at the highest test concentration in the first trial; aberrations per cell and percent abnormal cells were elevated. However, nonactivated Trial II remained negative. Since the actual test concentrations in both nonactivated trials were comparable, a combined analysis of percent abnormal cells for both trials was done. This showed a statistically significant (p= 0.04) increase at the highest test concentration. However, the aberration frequencies observed at this level fall in the range of historical control data for the assay. For this reason, and since the effect observed in nonactivated Trial I was not reproduced in Trial II, the test substance is considered equivocal without activation.

Trial With Activation:
The evaluation of 100 cells per test level showed statistically significant increases in aberrations per cell and percent abnormal cells at the three lowest test concentrations (see Table 3). In activated Trial II, the initial evaluation of 100 cells per treatment level showed no significant increases in aberration indices at any test concentration. To clarify the apparent discrepancy with activated Trial I, 100 additional cells per treatment level were scored from Trial II. This analysis (see Table 3) showed significant increases in percent cells with more than one aberration at 35.4 and 63.3% test substance atmospheres; increases in the other two aberration indices were not statistically significant. However, the occurrence at two treatment levels of several cells with more than one aberration was judged to represent confirmation of the positive findings in activated Trial I. Thus, the test substance is considered positive with activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation
ambiguous without metabolic activation

The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). The test substance exhibited clastogenic activity in CHO cells with S-9 activation. Without activation, the findings were equivocal. Under the conditions of this assay, the test substance is positive.

Executive summary:

The test substance was evaluated for clastogenic (chromosome-damaging) activity in Chinese hamster ovary (CHO) cells in vitro following 2-hour treatment with metabolic (S9) activation and 5-hour treatment without metabolic (S9) activation. Two independent trials were conducted. The maximum target concentration tested was 100%. Metaphase cells were evaluated and aberrations per cell, percent abnormal cells, and percent cells with > 1 aberration reported. Under nonactivated conditions, cytotoxicity studies showed significant cell cycle delay only at a test substance concentration of 96.3% (in nitrogen). With activation, moderate cell cycle delay was observed at 52.1% and severe cell cycle delay was evident at a test substance concentration of ≥ 61.3%. In the chromosome aberration studies, equivocal results were obtained following 5-hour nonactivated treatments. After 2-hour treatments with S-9, significant chromosome aberration induction was seen at test substance concentrations ranging from 8.3-63.3%. Under the conditions of this assay, the test substance is positive.