[No public or meaningful name is available]

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:

11/Jan/18 - 27/Mar/18

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether

EC Number:

609-858-6

Cas Number:

406-78-0

Molecular formula:

C4H3F7O

IUPAC Name:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether

Test material form:

liquid

Details on test material:

Batch number: 51806180
Expiry: 29 November 2018
Purity: 99.99% (Area %)

Specific details on test material used for the study:

Name: 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether
Other name: HFE-347-pc-f (Product name: ASAHIKLIN AE-3000)
CAS number: 406-78-0
Fomula: CF3CH2OCF2CF2H
Molecular weight: 200.07
Purity: 99.993%
Lot number: 51712010

The test substance was put into an airtight container and stored at room temperature in the test substance storage room (permissible range from 10

Test animals

Species:

mouse

Details on species / strain selection:

Crl:CD1(ICR), SPF

Sex:

male

Details on test animals or test system and environmental conditions:

27 male mice were purchased for the study, which were 6 weeks old when purchased. At administration, the mice were 7 weeks old and were within ±20% of the mean body weight (29.7-36.6g). Animals were observed at receipt and healthy mice were quarantined and acclimatised for 6 days. The clinical signs were observed daily during the quarantine and acclimatisation periods. Body weights were measured at animal receipt and completion of quarantine. After quarantine and acclimatisation, healthy mice were allocated by a body weight-stratified random sampling method on the day before administration. Mice were allocated to 5 groups at 5 animals per group for administration. Remaining animals after allocation (2) were excluded from the study.

At animal receipt, the animal number was assigned arbitrarily and 5 or less animals per cage were accommodated in order of the numerical order. Four animals of the smaller number in each cage were given the marking from 1 to 4 on their tails, using oil-based ink. The fifth animal was not marked. After the allocation, a new animal number was assigned. Two to three animals were housed per cage (1 to 2 cages per dose). The animals were given the marking from 1 to 5 on their back using water-based ink. Different colours were used for each dose.

The temperature and relative humidity used for the study were 22.9-23.9°C and 48.7-60% respectively. The air change rate was 10-15 times per hour and there was a 12h on/ 12h off light cycle. The mice were kept in polycarbonate cages with a flat bottom (210W x 320D x 130H mm), at a rearing density of 5 animals/ less per cage before the allocation and 3 animals/ less per cage after the allocation. Cages, cage lids, bedding, enrichment and water bottles were changed at completion of quarantine. Cages and bedding were changed when animals were transferred for a dissection. For their diet, animals were given free access to diets of autoclaved MF pellets and the water from Hita City water supply was chlorinated at about 3-5ppm, by adding sodium hypochlorite (Pure lox). The animals were given free access to the water. Contaminants in the drinking water were analysed twice per year and before the animal receipt, it was confirmed that the latest analytical data met the criteria of the ordinance.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Olive oil

Details on exposure:

Test substance group and negative control group: Administration was repeated twice within a 24h interval by the oral gavage, which is generally used for the micronucleus test. The test substance solutions were dispensed using a 1mL disposable syringe with a disposable stomach probe. Administration was carried out at 10mL/kg based on the body weight on the administration day

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Twice (24 hour interval)

Post exposure period:

1 hour

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5 males

Positive control(s):

Mitomycin C (MMC) was selected as the positive control substance and was stored in the test substance room at room temperature.
Distilled water for injection was added to a vial containing 2 mg of MMC. MMC was dissolved to prepare 10mL of 0.2mg/mL MMC solution. The solution was used within 4 hours after preparation. On the second administration day of the test substance, an intraperitoneal administration of the positive control substance was carried out one time at 10mL/kg, based on the body weight on the administration day, using a 1mL disposable syringe with a disposable needle.

Examinations

Tissues and cell types examined:

Bone marrow cells from the femur

Details of tissue and slide preparation:

The animals were euthanised by a cervical dislocation. The femur was removed, and bone marrow cells were collected with approx. 0.8mL of heat-inactivated foetal bovine serum into a centrifuge tube and the tube was centrifuged at 1000rpm for 5 minutes. A small amount of the cell suspension was smeared onto a glass slide. The smears were completely air-dried and fixed with methanol. Then, the specimens were stained with 3vol% Giemsa solution prepared with 1/15 mol/L phosphate buffer solution (pH 6.8) and treated with 0.004% w/v% citrate aqueous solution.
The specimens of the negative and positive control groups and 3 doses in the test substance group were observed. Slide numbers were allocated randomly to specimens to be observed. The specimens were observed microscopically (x1000) in a blinded manner.

Evaluation criteria:

Frequency of micronuclei:
4000 polychromatic erythrocytes (PCE) per animal (2000 PCE per specimen) were observed and the frequency of micronucleated polychromatic erythrocytes (MNPCE) (MNPCE/PCE) was calculated.

Growth inhibition of bone marrow cells:
A total of 500 erythrocytes (TE) per animal (250 TE per specimen) were observed and the ration of PCE to TE (PCE/TE) was calculated.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

The means of the MNPCE/PCE in bone marrow cells in mice treated at each dose level were within the range of historical data for the controls.

Toxicity:

yes

Remarks:

Evidence of toxicity was seen in mice dosed with either 1000 or 2000 mg/kg.

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Vehicle controls used as negative controls.

Positive controls validity:

valid

Remarks on result:

other: No remarks

Additional information on results:

Clinical signs: No animals died in any groups. In the test substance group, the toxicity symptom was observed at 1000 and 2000 mg/kg/day. No abnormalities were observed in the negative or positive control groups.

Body weight: No significant difference was noted in body weight when compared with the negative control group in all doses of the test substance, until the day after the second administration.

MNPCE/PCE and PCE/TE: The means of the MNPCE/PCE in the negative and positive control groups were 0.120% and 6.850% respectively. The means of the MNPCE/PCE at 500, 1000 and 2000mg/kg/day of the test substance group were 0.050%, 0.085% and 0.060% respectively. At the positive control groups, statistically significant differences were observed at the 1% level. At all doses in the test substance group, MNPCE/PCEs were within the range of the historical data of the negative control. The means of the PCE/TE of the negative and positive control groups were 50.0% and 45.4% respectively. The means of the PCE/TE at 500, 1000 and 2000 mg/kg/day of the test substance groups were 48.3%, 42,2% and 47.7% respectively.

Any other information on results incl. tables

The means of the MNPCE/ PCE in the negative and positive control groups were within the range of the historical data in the testing facility. The mean of MNPCE/ PCE in the positive control was showed a statistically significant increase at both sides of the 1% level. In the test substance group, the PCE/TE was 20% of more compared with that of the negative control group at all observation doses. Therefore, it was confirmed that the test was conducted appropriately.

The means of the MNPCE/ PCEs of the test substance group at all observation doses were within the range of the historical data of the negative control. Therefore, the results were judged to be negative.

In the PCE/TE, since no statistically significant differences were observed in any doses of the test substance group compared to the negative control group, the exposure of the test substance to bone marrow cells was not proved. In the micronucleus test, the toxicity symptom was observed at 1000 and 2000 mg/kg/day. Therefore, it was judged that the potential to induce the micronuclei was evaluated adequately by the results of this study.

Applicant's summary and conclusion

Conclusions:

It was concluded that HFE-347pc-f did not induce the micronuclei under the present test conditions

Executive summary:

1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether was administered to 7-week old Crl:CD1(ICR) male mice twice, at a 24 hour interval by oral gavage, in order to investigate the ability of HFE-347pc-f to induce micronuclei. The LD50 of the test substance was 2000mg/kg/day or more in the result of the acute oral toxicity test in male and female rats, from the information provided by the sponsor. Therefore, in the micronucleus test, the highest dose selected was 2000mg/kg/day, with two lower doses of 1000 and 500mg/kg/day set based on a geometric ratio of 2. Since it was found that there was no difference of the LD50 between males and females, only male mice were used for the micronucleus test.

The vehicle, olive oil was administered at 10mL/kg twice, at a 24 -hour interval by oral gavage, as a negative control. Mitomycin C was administered intraperitoneally at 2mg/kg/day once as the positive control.

In the micronucleus test, no animals died at any dose until 24 hours after the second administration. Therefore, doses of observation of specimens were selected as 500, 1000 and 2000mg/kg/day. The frequencies of the micronucleated polychromatic erythrocytes to polychromatic erythrocytes (MNPCE/PCE) in bone marrow cells and the ratio of PCE to total erythrocytes (PCE/TE) were examined. The means of MNPCE/PCEs of the test substance group at all observation doses were within the range of the historical data of the negative control. Therefore, the results were judged to be negative.

In the PCE/TE, since no statistically significant differences were observed in any doses of the test substance group compared to the negative control group, the exposure of the test substance to bone marrow cells was not proved. In the micronucleus test, the toxicity symptom was observed at 1000 and 2000mg/kg/day. Therefore, it was judged that the potential to induce the micronuclei was evaluated adequately by the results of this study.

Consequently, it was concluded that 1,1,2,2-tetrafluoroethyl-2,2,2-trifluoroethyl ether did not induce the micronuclei in mice bone marrow cells under the present test conditions.