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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 6 July 2007 to 19 October 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliance

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(19 December 2006)

Type of assay:

mammalian erythrocyte micronucleus test

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

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

This strain was used because it is routinely used at the testing facility for this type of studies.

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: young adults
- Weight at study initiation: 27.4 to 28.0 g
- Assigned to test groups randomly: yes. Computer randomization
- Fasting period before study: no
- Housing: The mice were housed with five per cage, under conventional conditions in one room, in sterilised macrolon cages (type III), fitted with a grid cover of stainless steel with a bedding of wood shavings (Lignocel) and shreds of paper as environmental enrichment (Enviro-dri).
- Diet: ad libitum. The animals received a commercial rodent diet (Rat and Mouse No. 3 Breeding Diet, RM3); Batch 5650 (expiry date 8 October 2007); Supplier: SDS Special Diets Services, Witham, England.
- Water: ad libitum. Each cage was supplied with domestic mains tap-water suitable for human consumption. The tap-water was supplied by N.V. Hydron Midden-Nederland and given in polypropylene bottles.
- Acclimation period: yes, 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 40-70%
- Air changes: 10 air changes per hour
- Photoperiod: 12 hours light and 12 hours dark

IN-LIFE DATES: From: 4 July 2007 To: 12 July 2007

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil

Details on exposure:

On day 0 and day 1 (prior to dosing), the test substance was freshly dissolved in corn oil, at concentrations of 10, 5 and 2.5 mg/mL. Prior to each dosing, the animals were weighed and the given dosing volume was 10 mL/kg-bw.

Frequency of treatment:

The test substance was dosed twice intraperitoneally, on two successive days, with an interval of ca. 24 hours.

Post exposure period:

24 hours after treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Negative control (corn oil)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Treated group

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Treated group

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Treated group

Doses / concentrations 5

Dose / conc.:

0.75 mg/kg bw/day (nominal)

Remarks:

Mitomycin C (positive control)

No. of animals per sex per dose:

5 females per groups except the group at the dose-level 100 mg/kg bw/day: 7 females (two extra mouse were treated to replace mortality)

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
The mice were treated once intraperitoneally (0.075 mg/mL; 10 mL/kg) with the mutagen mitomycin C.

Examinations

Tissues and cell types examined:

From each mouse, the bone marrow cells of both femurs were immediately collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grünwald Giemsa solution. The other fixed but unstained smear was kept in reserve and discarded after analysis of the stained smear.

Details of tissue and slide preparation:

The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines, taking care that areas selected for evaluation were evenly distributed over the whole smear.The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of rnicronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

Evaluation criteria:

The study was considered valid because the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and the negative controls were within the historical range.
A test substance is considered to cause chromosomal damage and/or damage to the mitotic apparatus, if the mean number of MPE/2000 PE is statistically significantly higher, when compared to the mean number of the vehicle controls.
A test substance is considered to be negative in the micronucleus test if it produces no positive response at the used dose level.
Both statistical significance and biological relevance are considered together in the evaluation.

Statistics:

The statistical procedures used in the evaluation of data are generally as follows: Data were analysed by Analysis of Variance (ANOVA); if necessary, data were analysed after square root transformation (sqrt(x+ 1)) to 'normalise' the counts (Lovell et al. 1989). If the ANOVA yields significant results, pairwise comparisons between treated and control groups were made.

All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of Califomia Press, Berkeley, 1992).

Results and discussion

Test results

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Cytotoxic to the bone marrow cells

Additional information on results:

STATISTICAL ANALYSIS OF THE TEST RESULTS
* Group 4 (100 mg/kg bw/day) for PE: Statistical analysis (two-way Anova t-test and Dunnett test) of the results indicated there was a marginal statistically significantly (*p<0.05) decrease in the mean number of PE, when compared to the mean number of PE found in the negative control group (group 1; corn oil). This indicates that the test substance reached the bone marrow and induced cytotoxicity to the bone marrow cells. Furthermore, the trend analysis showed a significant linear decrease in the mean numbers of PE.

* Groups 2, 3 and 4 (25, 50 and 100 mg/kg bw/day, respectively) for MPE: Statistical analysis (two-way Anova) of the results indicated there were no statistically significant differences in the mean numbers of MPE, at any of the dose levels used, when compared to the mean number of MPE found in the negative control group (group 1; corn oil). This indicates that treatment with Monofluoroethylene carbonate, up to 100 mg/kg bw/day, did not result in genotoxicity to bone marrow cells.

* Positive control for MPE: Statistical analysis with both Anova (t-test) and the Kruskal-Wallis test indicated there was a statistically significant (***p<0.001) increase in the number of MPE, when compared to the mean number of MPE found in the negative control group (groupl; corn oil). This indicates that the positive control substance reached the bone marrow and induced genotoxicity to the bone marrow cells. The latter results demonstrate the validity of the test system.

CLINICAL SIGNS
* Group 4 (100 mg/kg bw/day) - 4 hours after the first administration: All animals (including reserve animals) showed blepharospasm (bilateral) and lethargy. All animals of treatment group 2 (25 mg/kg bw) and treatment group 3 (50 mg/kg bw), did not show any clinical signs.

* Group 4 (100 mg/kg bw/day) - 4 hours after the second administration: One reserve animal and two animals (31 and 33) were found dead. Two another animals (35 and 39) showed blepharospasm (bilateral), piloerection, hunched posture and lethargy. Another animal (37) showed no clinical signs.

* Groups 2 and 3 (25 and 50 mg/kg bw/day, respectively) - 4 hours after the second administration: All animals showed piloerection.

* Group 3 (50 mg/kg bw/day) - prior to sacrifice: One animal (27) showed blepharospasm (bilateral), piloerection, hunched posture and lethargy.

Any other information on results incl. tables

Table 1: Group numbers (mean ± S.D.) of MPE per 2000 PE, 24 hours after the final administration of Monofluoroethylene carbonate 

Group number:		1: Negative control (corn oil)	Monofluoroethylene carbonate (dose-level/day)			5: Positive control mitomycin C (0.75 mg/kg)
Sex	N		2: 25 mg/kg	3: 50 mg/kg	4: 100 mg/kg
Female	5 4^1)	2.0 ± 1.2	2.2 ± 1.3	2.0 ± 1.6	2.8 ± 0.5	34.6 ± 10.1***

¹⁾ As a result of treatment with the test substance, three out of seven mice died prior to scheduled sacrifice.

N: number of animals per treatment group

*** p<0.001 (statistical analysis: Anova t-test, confirmed by Krushal-Wallis test)

 

Table 2: Group numbers (mean ± S.D.) of PE per 2000 E, 24 hours after the final administration of Monofluoroethylene carbonate

Group number:		1: Negative control (corn oil)	Monofluoroethylene carbonate (dose-level/day)			5: Positive control mitomycin C (0.75 mg/kg)
Sex	N		2: 25 mg/kg	3: 50 mg/kg	4: 100 mg/kg
Female	5 4^1)	107.8 ± 6.83	112.6 ± 14.3	98.6 ± 13.9	84.3 ± 8.1*	91.4 ± 20.5

¹⁾ As a result of treatment with the test substance, three out of seven mice died prior to scheduled sacrifice.

N: number of animals per treatment group

* p<0.05 (statistical analysis: Anova t-test, confirmed by Krushal-Wallis test)

 

Table 3: Individual data of the microscopic evaluation of the bone marrow smears

Group	Mouse	SD	bwd(0)	bwd(1)	PE	NE	MPE	MNE
1 1 1 1 1	1 3 5 7 9	24 24 24 24 24	27.3 28.1 26.3 27.4 29.0	27.7 27.9 27.0 28.1 28.0	109 96 112 113 109	91 104 88 87 91	2 1 4 1 2	0 0 1 0 0
2 2 2 2 2	11 13 15 17 19	24 24 24 24 24	27.1 28.9 27.0 25.1 28.9	26.0 29.6 27.4 26.3 29.1	113 128 111 121 90	87 72 89 79 110	1 4 1 3 2	0 0 0 0 0
3 3 3 3 3	21 23 25 27 29	24 24 24 24 24	30.0 29.7 25.4 28.9 26.1	30.1 29.6 26.8 28.1 26.1	110 106 110 82 85	90 94 90 118 115	3 1 4 0 2	0 0 0 0 0
4 4 4 4 4 4 4	31* 33* R2016 R2017* 35 37 39	-- -- 24 -- 24 24 24	26.3 28.4 31.3 31.7 26.2 28.1 28.4	26.2 27.1 29.3 30.0 26.1 25.8 27.9	-- -- 83 -- 79 96 79	-- -- 117 -- 121 104 121	-- -- 3 -- 3 3 2	-- -- 0 -- 1 0 0
5 5 5 5 5	41 43 45 47 49	24 24 24 24 24	26.0 27.1 28.9 28.3 29.8	25.7 27.3 29.5 28.8 29.4	103 91 88 115 60	97 109 112 85 140	29 40 49 23 32	1 0 0 0 0

Group:

1 = Negative control (corn oil)

2 = Monofluoroethylene carbonate (25 mg/kg bw/day)

3 = Monofluoroethylene carbonate (50 mg/kg bw/day)

4 = Monofluoroethylene carbonate (100 mg/kg bw/day)

5 = Mitomycin C (positive control; 0.75 mg/kg bw)

 

Mouse: mouse number

SD: scheduled dead after all treatments (h)

bwd(0): body weight (g) prior to the first administration

bwd(1): body weight (g) prior to the second administration

PE: number of PE scored per 200 E scored

NE: number of NE scored per 200 E scored

MPE: number of MPE scored per 2000 PE scored

MNE: number of MNE scored per number of NE scored

* As a result of treatment with the test substance, this animal died prior to scheduled sacrifice.

Applicant's summary and conclusion

Conclusions:

Under the conditions used in this study it is concluded that, the test substance Monofluoroethylene carbonate was cytotoxic to the bone marrow cells but did not show any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in female mice, treated intraperitoneally with Monofluoroethylene carbonate, up to 100 mg/kg bw.

Executive summary:

The test substance Monofluoroethylene carbonate was examined for its mutagenic potential in a bone marrow micronucleus test, performed with female mice. The dose levels administered (100, 50 and 25 mg/kg bw/day) were based on treatment related toxicity, observed in a micronucleus test performed with male mice and the same dose levels (information provided by the sponsor).

Animals were treated twice intraperitoneally, on two successive days with an interval of 24 hours, with three graded dose levels of the test substance Monofluoroethylene carbonate. The high dose level group (4), consisted of 7 animals, and each animal received a dose of 100 mg/kg bw/day. The mid dose level group (3), consisted of 5 animals, and each animal received a dose of 50 mg/kg bw/day. The low dose level group (2), consisted of 5 animals, and each animal received a dose of 25 mg/kg bw/day. The vehicle control group (1) consisted of 5 animals, and each animal was dosed in a similar way with the vehicle corn oil. A positive control group (5) consisted of 5 animals, and each animal was given a single intraperitoneal dose of mitomycin C at 0.75 mg/kg bw. At 24 hours after all treatments, all surviving mice were euthanized, bone marrow cells were collected and pooled from both femurs and processed into smears for microscopic examination.

The number of polychromatic erythrocytes (PE) per 200 erythrocytes (E) and the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each mouse.

The group mean number of polychromatic erythrocytes (PE) per 200 erythrocytes (E) was statistically significantly lower in the animals treated with the highest dose level (100 mg/kg bw/day) of Monofluoroethylene carbonate, compared to the mean number found in the vehicle control animals, treated with corn oil. This result is a confirmation that Monofluoroethylene carbonate reached the target cells in the bone marrow and induced cytotoxicity to the bone marrow cells.

The mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in the animals, treated with dose levels up to 100 mg/kg bw/day, were not statistically significantly higher than the mean number found in the vehicle control animals, treated with corn oil. Therefore, Monofluoroethylene carbonate is not genotoxic to bone marrow cells. For the mice of the positive control group, the mean number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly higher than the mean number found in the vehicle control mice. The latter results demonstrate the validity of the test system.

Under the conditions used in this study it is concluded that Monofluoroethylene carbonate was cytotoxic to the bone marrow cells, but did not show any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in female mice, treated intraperitoneally with Monofluoroethylene carbonate, up to 100 mg/kg bw.