4-fluoro-1,3-dioxolan-2-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 8 December 2003 to 30 March 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP cmpliance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

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:

other: Korea National lnstitute of Environmental Research, Test Guidelines of Chemicals, Notification 1998-41, 23 December 1998

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Chromosome aberration (clastogenic potential)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

483-360-5

EC Name:

-

Cas Number:

114435-02-8

Molecular formula:

C3H3FO3

IUPAC Name:

4-fluoro-1,3-dioxolan-2-one

Test material form:

other: Solid with a low melting point (i.e. liquefied solid)

Details on test material:

Name of the test susbtance: Monofluoroethylene carbonate

Method

Species / strain

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster Lung cells

Metabolic activation:

with and without

Metabolic activation system:

Mammalian liver post-mitochondrial fraction (S9) obtained from Molecular Toxicology lncorporated and prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Dose-levels were:
- 5, 10, 20, 50, 100, 200, 500,1000, 2000 and 5000 µg/mL for the first experiment with and without S9,
- 30, 40, 50, 60, 70 and 80 µg/mL for the confirmation experiment with and without S9.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

CELLS:
The cells were a line of Chinese Hamster Lung (CHL) cells, obtained from American Type Culture Collection (ATCC). The CHL cell line was selected because it is recommended for the chromosome aberration test by other various regulatory authorities and background data are available. The modal chromosome number is 25 and the average generation time is 15-17 hours.

CULTURE ESTABLISHMENT
CHL cells in logarithmic growth were trypsinized and subcultured at low density into tissue culture flasks. After 2 or 3 days incubation at 37 °C in an atmosphere of 5% (v/v) C02 100% humidity, cultured cells at suitable confluence were selected for treatment.

CULTURE TREATMENT CONDITIONS AND HARVEST TIME
The test system was suitably labeled (using a color-coded procedure) to clearly identify the study number, test article, positive and negative control groups, absence or presence of metabolic activation system, dose levels and treatment conditions. ln each experiment duplicated cultures were employed for test article treatment, also negative (vehicle) and positive control articles were included in duplicate.
A series of more than six dose levels of test article spaced at two-fold intervals were employed in chromosome aberration experiment both in the absence and in the presence of metabolic activation system. Duplicate cultures were treated with the test article, the negative (vehicle) and positive controls both in the absence and in the presence of metabolic activation system. The final concentration of 10% S9 mix in the test system was 10% (v/v). Cultures treated in the absence of S9 mix were received an equal volume of 0.2 M Phosphate buffer. See "Any other information on materials and methods incl. tables" for more details.

PREPARATION OF METAPHASE SPREADS
Six harvesting dose levels were selected from a series of more than six dose levels of test article treatment. Approximately 2 hours prior to harvest, colcemid® was added to give a final concentration of approximately 0.2 μg/mL to arrest dividing cells in metaphase. The monolayer of these cultures were then harvested. The suspension from each flask was transferred to a plastic centrifuge tube and the cells were pelleted by centrifuging at 100 x g for 5 minutes. The supernatant was removed and cells were resuspended in 5 mL (hypotonic) of 0.075 M KCI at 37°C for 15 minutes to allow cell swelling to occur. 1 mL of fresh, ice-cold fixer solution was added for pre-fixation. The cells were pelleted by centrifuging at 200 x g for 5 minutes. Cells were resuspended in 5 mL of fresh, ice-cold fixer solution and the fixative was changed by centrifugation at 200 x g for 5 minutes. This procedure was repeated several times until the supernatants become clean. Cells were kept in fixative in the refrigerator before slides are made, but slides were not be made on the day of harvest in order to ensure that cells were adequately fixed. Cells were pelleted and resuspended in a minimal amount of fresh fixative so as to give a milky suspension. Two drops of suspension were transferred on to clean microscope slides. After the slides had dried the cells were stained for 10 minutes in 3% (v/v) Giemsa in pH 6.8 Sörenson buffer. The slides were then rinsed and dried.

SELECTION OF DOSES FOR CHROMOSOME ANALYSIS
At least 1000 cells were scored from slides prepared from each culture to assess the mitotic indices. The mitotic index (Ml) of every slide scored was taken from the number of mitotic cells seen in a total of at least 1000 cells.
ln the absence and in the presence of metabolic activation system, metaphases were not observed at above 100 μg/mL dose level, so 50 μg/mL dose level was selected for highest dose level.
ln the absence and in the presence of metabolic activation system of confirmation experiment, about 50% reduction in Ml was observed at 70 μg/mL dose level, so 70 µg/ml dose level was selected for highest dose level.
This was the maximum dose and next two lower doses were taken for scoring.

SCORING OF CHROMOSOME ABERRATIONS
Slides from the test article treatments and controls were coded using randomly generated letters by a person not involved with slide scoring. Labels bearing only the study reference number and the assigned code were used to cover treatment details on the slides. 100 metaphases from each coded slide were analyzed for chromosome aberrations. Only cells with the modal number of chromosome 25±2 were considered acceptable for analysis. Cells with greater numbers of chromosomes observed during this evaluation were noted and recorded separately. Observations were recorded on raw data sheets by following criteria with the microscope stage coordinates of any aberrant cell.
- Structural aberration:
* Gap (g)
* Chromatid break ( chtb)
* Chromatid exchange (chte)
* Chromosome break (chrb)
* Chromosome exchange (chre)
* Other
- Numerical aberration:
* Hyperploid
* Polyploid
* Endoreduplication (end)

Evaluation criteria:

TREATMENT OF DATA
After completion of microscopic analysis, data were decoded. The aberrant cells in each culture were categorized as follows:
1) Cells with structural aberrations including gaps
2) Cells with structural aberrations excluding gaps
3) Cells with numerical aberrations
4) Cells with numerical aberrations excluding hyperploid

ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria are met:
1) The proportion of cells with structural aberrations (excluding gaps) in negative control cultures falls within the normal range
2) The proportion of cells with numerical aberrations in negative control cultures falls within the normal range
3) The positive control chemicals induce statistically significant increase in the proportion of cells with structural aberrations
4) At least 160 cells are analyzable at each dose level.

EVALUATION CRITERIA
The test article was considered as positive in this assay if:
1) The assay is valid (see acceptance criteria)
2) Statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at one or more dose level
3) 2) exceed the normal range
4) If the result of 2) is reproducible, the test article will be considered as positive on structural aberrations
5) Statistically significant increase in the proportion of cells with numerical aberrations (excluding hyperploid) occurs at one or more dose level
6) 5) exceed the normal range
7) If the result of 5) is reproducible, test article will be considered as positive on numerical aberrations

Statistics:

Normally, the proportion of cells in category 2) and 4) (see "TREATMENT OF DATA") for each treatment condition were compared with the proportion in concurrent negative controls by using Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant. A further statistical test (for linear trend) used to evaluate possible dose-response relationships.

Results and discussion

Test results

Key result

Species / strain:

mammalian cell line, other: Chinese Hamster Lung

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ln the chromosome aberration experiment, the dose levels for chromosome analysis were selected 10, 20, 50 μg/mL dose levels in the absence and in the presence of metabolic activation system (-S9 mix 6+18, +S9 mix, 6+18).
Significant increases in the proportion of cells with structural aberrations were observed in cultures at 50 μg/mL dose level in the absence of metabolic activation system (p ≤ 0.05). Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels.
Significant increases in the proportion of cells with structural aberrations were observed in cultures at 50 μg/mL dose level in the presence of metabolic activation system (p ≤ 0.05). Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels.
Since a positive result was obtained, confirmation experiment was performed under the same treatment condition with modified dose levels. Treatment was applied for 6 hours followed by 18 hours recovery period prior to harvest both in the absence and in the presence of metabolic activation system. The confirmation experiment dose levels for chromosome analysis were selected 50, 60, 70 μg/mL dose levels both in the absence and in the presence of metabolic activation system.
Significant increases in the proportion of cells with structural aberrations were observed in cultures at all dose levels in the absence of metabolic activation system (p ≤ 0.05). Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels.
Significant increases in the proportion of cells with structural aberrations were observed in cultures at all dose levels in the presence of metabolic activation system (p ≤ 0.05). Significant increases in the proportion of cells with numerical aberrations were not observed in cultures at all dose levels.

Negative (vehicle) and positive control treatments were included in each treatment condition. The proportion of cells with structural aberrations on the negative control treatments fell within acceptable ranges, while the positive control treatments induced clear increases in the proportion of cells with structural aberrations.

Any other information on results incl. tables

The following table summarizes the mitotic indices for all treatment conditions and selecting dose levels:

Experiment	Treatment condition	Treatment concentration (µg/mL)	Mitotic index (%)
			A	B
Chromosome aberration experiment	- S9 6 + 18	0 5 10 20 50 100 200	8.6 8.5 8.2 6.1 5.3 0.0 0.0	8.3 8.1 8.4 5.9 5.7 0.0 0.0	C NC C C C NC NC
	+ S9 6 + 18	0 5 10 20 50 100 200	8.7 8.5 8.4 6.2 5.7 0.0 0.0	9.0 8.5 8.0 6.9 5.5 0.2 0.0	C NC C C C NC NC
Confirmation experiment	- S9 6 + 18	0 30 40 50 60 70 80	8.9 6.1 5.5 5.8 5.1 3.6 1.8	8.7 6.3 6.0 4.5 4.8 4.5 2.6	C NC NC C C C NC
	+ S9 6 + 18	0 30 40 50 60 70 80	8.8 6.3 5.0 5.8 6.2 3.8 2.0	9.2 6.0 5.1 5.2 5.7 4.9 2.8	C NC NC C C C NC

Table 1: Summary of numbers and types of structural aberrations, - S9 mix, 6 hour treatment 18 hour recovery (6 +18)

Treatment (µg/mL)		Replicate	Cells counted	Cells with strutural aberrations
				chtb	chte	chrb	chre	g	Other	Total(+g)	Total(-g)
- S9 6 + 18	Dimethylsulfoxide	A B	100 100	1 1	0 0	0 0	0 0	2 1	0 0	3 2	1 1
		Total	200	2	0	0	0	3	0	5	2
	10	A B	100 100	0 0	0 0	0 2	0 0	1 2	0 0	1 4	0 2
		Total	200	0	0	2	0	3	0	5	2
	20	A B	100 100	0 2	0 3	0 0	0 0	0 2	0 0	0 7	0 5
		Total	200	2	3	0	0	2	0	7	5
	50	A B	100 100	3 14	12 22	0 2	0 0	3 6	0 0	15 30	14 28
		Total	200	17	34	2	0	9	0	45	42
	MMC 0.1 µg/mL	A B	100 100	5 10	9 11	0 0	0 0	3 2	0 0	14 19	11 17
		Total	200	15	20	0	0	5	0	33	28

Table 2: Summary of numbers and types of structural aberrations, + S9 mix, 6 hour treatment 18 hour recovery (6 +18)

Treatment (µg/mL)		Replicate	Cells counted	Cells with strutural aberrations
				chtb	chte	chrb	chre	g	Other	Total(+g)	Total(-g)
+ S9 6 + 18	Dimethylsulfoxide	A B	100 100	0 2	0 0	0 0	0 0	2 2	0 0	2 4	0 2
		Total	200	2	0	0	0	4	0	6	2
	10	A B	100 100	0 2	0 0	0 0	0 0	1 1	0 0	1 3	0 2
		Total	200	2	0	0	0	2	0	4	2
	20	A B	100 100	0 4	0 2	0 2	0 0	2 1	0 0	2 8	0 7
		Total	200	4	2	2	0	3	0	10	7
	50	A B	100 100	5 3	10 5	0 2	0 0	7 4	0 0	20 12	16 10
		Total	200	8	15	2	0	11	0	32	26
	B(a)P 5 µg/mL	A B	100 100	1 9	15 15	0 0	0 0	2 3	0 0	17 20	15 19
		Total	200	10	30	0	0	5	0	37	34

Table 3: Summary of numbers and types of structural aberrations, - S9 mix, 6 hour treatment 18 hour recovery (6 +18), confirmation experiment

Treatment (µg/mL)		Replicate	Cells counted	Cells with strutural aberrations
				chtb	chte	chrb	chre	g	Other	Total(+g)	Total(-g)
- S9 6 + 18	Dimethylsulfoxide	A B	100 100	0 0	0 0	0 0	0 0	0 1	0 0	0 1	0 0
		Total	200	0	0	0	0	1	0	1	0
	50	A B	100 100	7 14	18 21	0 1	0 0	3 2	0 0	25 31	23 30
		Total	200	21	39	1	0	5	0	56	53
	60	A B	100 100	34 21	32 36	3 2	0 0	7 2	0 0	61 47	59 47
		Total	200	55	68	5	0	9	0	108	106
	70	A B	100 100	35 65	25 67	1 7	0 0	6 12	0 0	59 83	58 83
		Total	200	100	92	8	0	18	0	142	141
	MMC 0.1 µg/mL	A B	100 100	10 3	5 12	0 0	0 0	2 1	0 0	17 15	15 14
		Total	200	13	17	0	0	3	0	32	29

Table 4: Summary of numbers and types of structural aberrations, + S9 mix, 6 hour treatment 18 hour recovery (6 +18), confirmation experiment

Treatment (µg/mL)		Replicate	Cells counted	Cells with strutural aberrations
				chtb	chte	chrb	chre	g	Other	Total(+g)	Total(-g)
+ S9 6 + 18	Dimethylsulfoxide	A B	100 100	0 0	0 0	0 0	0 0	2 1	0 0	2 1	0 0
		Total	200	0	0	0	0	3	0	3	0
	50	A B	100 100	12 9	26 24	2 2	0 0	6 3	0 0	41 27	38 26
		Total	200	21	50	4	0	9	0	68	64
	60	A B	100 100	2 3	6 12	1 2	0 0	4 3	0 0	11 15	9 15
		Total	200	5	18	3	0	7	0	26	24
	70	A B	100 100	25 52	39 53	2 7	0 0	9 10	0 0	63 68	58 68
		Total	200	77	92	9	0	19	0	131	126
	B(a)P 5 µg/mL	A B	100 100	6 10	22 19	0 0	0 0	2 1	0 0	27 21	26 20
		Total	200	16	41	0	0	3	0	48	46

Applicant's summary and conclusion

Conclusions:

Based on the results that the treatment of Monofluoroethylene carbonate induced increases in the proportion of cells with structural aberrations both in the absence and in the presence of metabolic activation system.
lt is concluded that Monofluoroethylene carbonate exhibit clastogenic activity in cultured Chinese Hamster Lung cells when tested under the conditions employed for this test.