2,2-Difluoroethyl Acetate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

This genotoxicity evaluation was combined with a 28-day repeated-dose toxicity study by gavage. Only the information relevant for the micronucleus evaluation were described here (for the ‘classical’ repeated-dose toxicity study, see IU section 7.5.1).

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 29 AUGUST 2014 TO 5 MAY 2015.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Remarks:

The present micronucleus evaluation was done during a 28-day repeated dose toxicity study by oral route.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2,2-difluoroethyl acetate

EC Number:

801-773-4

Cas Number:

1550-44-3

Molecular formula:

C4H6F2O2

IUPAC Name:

2,2-difluoroethyl acetate

Test material form:

liquid

Remarks:

Clear and colorless.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD).

Details on species / strain selection:

Rats have historically been used in safety evaluation studies for oral toxicity testing. The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at the testing facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina, U.S.A.
- Females: nulliparous and non pregnant.
- Age at study initiation: 7 weeks old.
- Weight at study initiation: the males mean body weight varied between 224.9 and 228.4 g and the females mean body weight varied between 171.6 and 176.7 g within all dose groups. The weight variation of selected animals did not exceed ± 20% of the mean weight for each sex.
- Assigned to test groups randomly: yes, the animals were distributed by computerized, stratified randomization into study groups.
- Fasting period before study: not specified.
- Housing: animals were housed in groups in solid-bottom caging with bedding and appropriate species-specific enrichment.
- Diet: all animals were fed PMI® Nutrition International LLC Certified Rodent LabDiet® 5002 ad libitum.
- Water: all animals were provided tap water ad libitum.
- Acclimation period: 6 days. The animals were released from quarantine based on normal observations for body weights and clinical signs.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC.
- Humidity (%): 30-70%.
- Photoperiod: Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle.

IN-LIFE DATES: From 16 September 2014 to 14 October 2014.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Solvent used: corn oil.
- Justification for choice of solvent: the chosen solvent was among those recommended in the guideline and it was shown through sampling and analysis that the test material at 10 to 100 mg/mL in the vehicle and stored at room temperature for up to approximately 15 days was stable.
- Concentration of test material in vehicle: 0, 10, 30 and 100 mg test material/mL of vehicle.
- Amount of vehicle (gavage): 10 mL/kg bw.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved in corn oil. Neither the amount nor nature of any contaminants in the vehicle was expected to affect the integrity or validity of this study. Dose formulations were prepared without a correction for the sponsor reported purity. Dosing formulations of the test material were prepared and used within the established range of stability and stored at room temperature until used.

Duration of treatment / exposure:

29 days.

Frequency of treatment:

Animals were dosed daily at approximately the same time (± 2 hours) by intragastric intubation at a dose volume of 10 mL/kg body weight for 29 days. The amount of test material each animal received was based on the most recently collected body weight and the formulation concentration. Control animals were dosed with corn oil at a volume of 10 mL/kg of body weight.

Post exposure period:

Post exposure period is only relevant for the repeated-dose toxicity study described in details under IUCLID section 7.5.1. For micronucleus evaluation, assessments were performed on Day 4 and on the day of sacrifice (Day 30) and a post exposure period is therefore not applicable.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle alone.

No. of animals per sex per dose:

- 10 animals/sex/dose in the control and 1000 mg/kg/day test groups,
- 5 animals/sex/dose in the 100 and 300 mg/kg/day test groups.

Control animals:

yes, concurrent vehicle

Positive control(s):

No positive control was used in this specific study. The study report presents the historical control data (see in section "Any other information on results incl. tables") including positive control data, demonstrating proficiency of the testing facility in the conduct of the test, as requested by the guideline.

Examinations

Tissues and cell types examined:

The frequency of micronuclei was analysed in the peripheral blood by analysing reticulocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
Samples from each exposure level and the negative control were analyzed at both collection time points.

TREATMENT AND SAMPLING TIMES
Peripheral blood samples were collected by sublingual bleeding from 5 animals per sex per group. There were 2 blood collections, a bleed on day 4 and on the day of sacrifice prior to the scheduled sacrifice.

DETAILS OF SLIDE PREPARATION
Approximately 60-120 μL of blood was collected from each animal directly into a labelled microcentrifuge tube containing 350 μL of anticoagulant/diluent (anticoagulant) found in the In Vivo MicroFlow Plus® Rat Micronucleus assay kit (Litron Laboratories, Rochester, New York). The tubes were capped and inverted several times to mix the blood with anticoagulant. The blood/anticoagulant mixture was stored at room temperature for up to 6 hours or refrigerated for up to 24 hours before fixing. The blood samples were fixed in duplicate (approximately 180 μL of blood/anticoagulant mixture each) in 2 mL ultra-cold methanol and stored below -75ºC until processed.

METHOD OF ANALYSIS:
The micronucleus evaluation was conducted by flow cytometry. Whenever feasible, at least 20000 reticulocytes were analyzed per blood sample. The samples were analyzed and evaluated using the In Vivo MicroFlow Plus® Rat Micronucleus assay kit.

Evaluation criteria:

- Toxicity was evaluated by the frequency of immature erythrocytes (%RETs) among the total erythrocytes (RETs plus NCEs).
- The frequency of micronucleated reticulocytes (%MN-RETs) was used as a measure of induction of aneugenic or clastogenic alterations by the test material.
Where:
RET: reticulocyte
NCE: normochromatic erythrocytes
MN-RET: micronucleated reticulocyte

Statistics:

Micronucleus data was evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. For each treatment group, the mean and standard deviation of % RETs and % MN-RETs was calculated. Data was transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation was appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the nontransformed proportion. All micronucleus data analyses was one-tailed and conducted at a significance level of 5%. See also Table 1 in "Any other information on materials and methods incl. tables".

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Remarks:

The vehicle (negative) control group exhibited a response consistent with the %MN-RETs historical control data.

Negative controls validity:

valid

Remarks:

The negative control was the vehicle administered alone, see above.

Positive controls validity:

not applicable

Additional information on results:

- Induction of micronuclei: No statistically significant or biologically relevant increases in the micronucleated reticulocyte frequency (% MN-RET) were observed in any evaluated test substance treated group of male or female animals at either time point.
- Appropriateness of dose levels and route: A reduction in %RETs among the total erythrocytes was observed indicating that the test substance reached the target cells. In male rats statistically significant decreases were observed at 300 and 1000 mg/kg bw/day at day 4. In female rats, although not statistically significant, a dose related decrease in %RETs was also observed at day 4. However, no decrease in %RET was observed at day 30 in either male or female animals.

Any other information on results incl. tables

Dose formulation analysis:

The formulation analyses showed that the test material was at the targeted concentrations and stable when stored at room temperature for up to 15 days. See more details in the IUCLID section 7.5.1.

 

Historical control data:

Table 2: Historical Control Data

Negative control animals^a,b

Parameter	% RET- Males	% RET - Females	% MN-RET- Males	% MN-RET - Females
Mean	1.64	1.41	0.11	0.11
Standard deviation	0.90	0.67	0.05	0.05
Range	0.59 – 5.55	0.34 – 4.14	0.03 – 0.31	0.02 – 0.37

Positive control animals^a,c

Parameter	% RET- Males	% RET - Females	% MN-RET- Males	% MN-RET - Females
Mean	1.26	0.83	1.03	1.03
Standard deviation	0.87	0.52	0.34	0.55
Range	0.25 – 3.96	0.07 – 2.38	0.27 – 1.85	0.31 – 3.12

^a Historical control data was compiled from studies conducted at DuPont Haskell Global Centers since 2009.

^b Negative controls include all vehicles, routes of administration and sample times.

^c The positive control is cyclophosphamide, administered by oral gavage. Blood was collected 48 hours after a single administration.

 

Tabulated results:

Table 3: Micronucleus Evaluation for Male Rats

Dose group	Group 1 0 mg/kg bw/day	Group 2 100 mg/kg bw/day	Group 3 300 mg/kg bw/day	Group 4 1000 mg/kg bw/day
RET % :                                                             Mean                                                                           SD (^a)
Day 4	4.79	4.02	3.84*	2.79*
	0.63 (5)	0.87 (5)	0.23 (5)	0.48 (5)
Day 30	1.61	1.30	1.47	1.53
	0.29 (5)	0.37 (5)	0.40 (5)	0.88 (5)
MN-RET % :                                                       Mean                                                                            SD (^a)
Day 4	0.11	0.08	0.08	0.09
	0.03 (5)	0.03 (5)	0.03 (5)	0.04 (5)
Day 30	0.07	0.08	0.07	0.07
	0.02 (5)	0.05 (5)	0.02 (5)	0.03 (5)

(^a) Standard deviation (number of values included in calculation).

* Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test.

 

Table 4: Micronucleus Evaluation for Female Rats

Dose group	Group 1 0 mg/kg bw/day	Group 2 100 mg/kg bw/day	Group 3 300 mg/kg bw/day	Group 4 1000 mg/kg bw/day
RET % :                                                              Mean                                                                            SD (^a)
Day 4	2.00	2.17	1.76	1.52
	0.26 (5)	0.57 (5)	0.60 (5)	0.38 (5)
Day 30	0.97	0.94	0.72	0.90
	0.23 (5)	0.30 (5)	0.24 (5)	0.14 (5)
MN-RET % :                                                         Mean                                                                              SD (^a)
Day 4	0.08	0.09	0.07	0.10
	0.04 (5)	0.04 (5)	0.04 (5)	0.03 (5)
Day 30	0.06	0.07	0.07	0.05
	0.02 (5)	0.03 (5)	0.03 (5)	0.03 (5)

(^a) Standard deviation (Number of values included in calculation).

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, 2,2-Difluoroethyl acetate showed no evidence of in vivo cytogenicity with no statistically significant or biologically relevant increase in micronucleated reticulocytes in rats.

Executive summary:

The cytogenic potential of 2,2-Difluoroethyl acetate was investigated in the context of a 28-day repeated-dose toxicity study by gavage. This study was performed according to OECD test guidelines 407 (for the repeated-dose toxicity study) and 474 (for the micronucleus evaluation) under GLP compliance.

Four groups of male and female Crl:CD(SD) rats were administered 0, 100, 300, and 1000 mg/kg bw/day test material by gavage for 29 days consecutively (10 animals per sex in the control and 1000 mg/kg/day test groups and 5 animals per sex in the 100 and 300 mg/kg/day test groups).

Peripheral blood samples were collected from 5 animals per sex on days 4 and 30 for micronucleus evaluation by flow cytometry. The frequency of micronucleated reticulocytes (%MN RETs) was used as a measure of induction of aneugenic or clastogenic alterations by the test material. Samples from the negative control and each exposure level were analysed at both collection time points.

No statistically significant or biologically relevant increases in the micronucleated reticulocyte (RET) frequency were observed in bone marrow in any test material treated group of male or female rats at either time point. The negative control group exhibited %MN-RETs values consistent with the historical control data.

Under the conditions of this study, 2,2-Difluoroethyl acetate showed no evidence of in vivo genotoxicity with no biologically relevant increase in micronucleated reticulocytes.