2,2-Difluoroethyl Acetate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 JUNE 2013 to 11 NOVEMBER 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

2009

Deviations:

yes

Remarks:

Some temperature and relative humidity deviations occurred but they did not adversely affect the results or interpretation of this study.

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

no

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) rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: male and female rats were obtained from Charles River Laboratories International, Inc., Raleigh, North Carolina.
- Females nulliparous and non-pregnant: yes.
- Age at study initiation: approximately 8 weeks old.
- Weight at study initiation: male rats weighed between 250 and 292 grams and female rats weighed between 185 and 221 grams at the time of exposure. The weight variation in animals used on an exposure did not exceed ± 20% of the mean weight of each sex.
- Fasting period before study: none.
- Housing: except during exposure, animals were housed individually in solid bottom caging with Enrich-O-Cob for bedding and enrichment.
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except during exposure.
- Water: tap water ad libitum except during exposure.
- Acclimation period: 6-day quarantine. During this period, animals were weighed and observed for clinical signs of disease. Animals that were released from quarantine, had been gaining weight at a normal rate and had no overt signs of disease.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25°C. The chamber temperatures were briefly 1.0ºC higher than the upper target range during the 350 ppm exposure. The chamber temperature was within the animal room temperature specifications.
- Humidity (%): 58 - 99%. The humidity exceeded the upper limit in all exposures however the high humidity did not persist throughout the entire exposures.
The chamber temperature and relative humidity deviations did not adversely affect the results or interpretation of this study.
- Air changes (per hr): 10 L/minute, which produced approximately 32 air changes per hour. The chamber oxygen concentrations ranged from 20.6 to 20.9%.
- Photoperiod: animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle.

IN-LIFE DATES: From 8 July 2013 to 14 August 2013.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus and exposure chamber volume: The exposure chamber was constructed of glass (cylindrical) with a nominal internal volume of 19L.
- Method of holding animals in test chamber: Before the exposures, animals were placed in stainless steel, wire-mesh modules (sexes separate) and then exposed, whole-body, inside the 19-L glass exposure chamber. The modules were placed on a stainless steel stand inside the exposure chamber so that the animals were elevated from the bottom of the exposure chamber.
- Air generation: Houseline generation (and chamber supply) air, was metered to the round-bottom flask with a Brooks model 5850E mass flow controller and carried the vapor and air mixture into a glass transfer tube that led to the exposure chamber.
- Treatment of exhaust air: The test atmosphere was exhausted through a dry-ice cold trap followed by an MSA charcoal/HEPA filter cartridge prior to discharge into the fume hood.

TEST ATMOSPHERE
- Brief description of method and equipment used: Chamber atmospheres were generated by flash evaporation of H-30299 in air. The test substance was metered into a heated round-bottom, flash evaporation flask with a Harvard Apparatus model 22 Syringe Infusion Pump. The round-bottom flask was heated to 175°C to vaporize the test substance. Houseline generation (and chamber supply) air, was metered to the round-bottom flask with a Brooks model 5850E mass flow controller and carried the vapor and air mixture into a glass transfer tube that led to the exposure chamber. Chamber concentrations of the test substance were controlled by varying the test substance feed rate to the round-bottom flask. The infusion pump and the heating mantle were monitored and controlled by the Camile Inhalation Toxicology Automated Data System (CITADS).
- Samples taken from breathing zone: yes. Prior to the start of the exposure phase, the distribution of the test substance atmosphere within the exposure chamber was determined to ensure an even atmosphere delivery to the animals. Chamber samples were collected from 2 separate locations in the exposure chamber and at the chamber reference port.
- Time needed for equilibrium of exposure concentration before animal exposure: Approximately 20 minutes are needed to reach equilibrium.

VEHICLE
None used.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Using Gas Chromatography with Flame Ionization Detection (GC-IFD). For further details, see in the field "Any other information on materials and methods incl. tables".

Duration of exposure:

4 h

Remarks on duration:

Animals were exposed to the test substance during both the time it took for the chamber to reach concentration (approximately 20 min) and the time it took for the test substance to be purged from the chamber.

Concentrations:

The tested concentrations were 350 ± 31, 1500 ± 310 and 5600 ± 820 ppm (equivalent to 1.77 ± 0.16, 7.60 ± 1.57, 28.36 ± 4.15 mg/L*, respectively).

* Conversion from ppm to mg/L:
Test concentrations (mg/m3) = Test concentrations (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
Test concentrations (ppm) = 350 ± 31 or 1500 ± 310 or 5600 ± 820
Molecular weight (g/mol) = 124.09
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Test concentrations (mg/m3) = [350 ± 31 or 1500 ± 310 or 5600 ± 820] x 124.09 / 24.5
Test concentrations (mg/m3) = 1773 ± 157 or 7597 ± 1570 or 28363 ± 4153
Test concentrations (mg/L) = 1.77 ± 0.16 or 7.60 ± 1.57 or 28.36 ± 4.15

No. of animals per sex per dose:

5 males and 5 females per dose group.

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days.
- Frequency of observations and weighing: animals were observed for mortality and response to alerting stimuli during exposure and immediately after they were removed from the exposure chamber. During the 14-day recovery period, all rats were observed each day for mortality and were weighed and observed for clinical signs of toxicity.
- Necropsy of survivors performed: on the last day of the recovery period, all rats were sacrificed by isoflurane anesthesia, exsanguinated, and necropsied. All animals were given a complete gross pathology examination of their internal organs including observation of the nasal passages.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5 600 ppm

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: equivalent to > 28 mg/L

Mortality:

All animals survived the exposures and subsequent 14-day recovery period.

Clinical signs:

other: See in the field "Any other information on results incl. tables".

Body weight:

In general, the severity and duration of body weight losses observed during the recovery periods were concentration related:
1) 350 ppm: on the day after the exposure, 7 of 10 rats lost between 1.1 and 4.8% of their initial body weight followed by normal weight gain throughout the remainder of the recovery period.
2) 1500 ppm : nine of 10 rats lost between 0.71 and 13% of their initial body weight on the day following the exposure. Minor weight losses ranging from 0.19 to 2.6% were observed in some rats from test day 3 until test day 7, followed by normal weight gain throughout the second week of the recovery period.
3) 5600 ppm: all rats lost between 8.4 and 15% of their initial body weight on the day after the exposure. Weight losses from 0.74 to 6.1% were observed in half of the rats on post-exposure day 2, followed by normal weight gain throughout the remainder of the recovery period.

Gross pathology:

No gross lesions were observed in any rats at necropsy.

Any other information on results incl. tables

Chamber Distribution of Vapor:

 

Chamber samples were collected from 2 separate locations in the exposure chamber and at the chamber reference port. Indeed the male rats were placed near the bottom of the chamber, and the female rats were placed above them near the middle of the chamber during exposures; therefore the samples for determination of chamber homogeneity were collected from these 2 locations. The samples were averaged, and the individual samples were compared to the overall average. All samples taken from where the animals were exposed were less than 1.1% different from the overall mean value determined; therefore, the vapor atmosphere was considered homogeneously distributed and the use of the reference port for air sampling was considered adequate.

 

Mean exposure concentrations:

 

The mean chamber test material vapor concentrations were 350 ± 31, 1500 ± 310 and 5600 ± 820 ppm for the exposures, see Table 1 below. The chamber atmospheres were considered adequate for inhalation testing in rats.

 

Table 1: Chamber concentrations of test material

	ppm			mg/L
n	Mean	S.D.	Range	Mean	S.D.	Range
16	350	31	290 – 400	1.8	0.16	1.4 – 2.0
16	1500	310	1000 – 1900	7.6	1.6	5.3 – 9.6
16	5600	820	4100 - 6600	28	4.2	21 - 33

N: number of samples

S.D. standard Deviation

 

Clinical Observations:

 

During exposure:

All rats displayed normal alerting response during all 3 exposures. 

There were no clinical signs of toxicity observed in any rats in the 350 ppm exposure group during the exposure. 

Rats in the 1500 exposure group displayed decreased activity throughout the exposures.

Rats in the 5600 ppm exposure group displayed decreased activity throughout the exposures. In addition, rats in this group displayed labored breathing and/or gasping during the exposure.

 

Post exposure:

There were no clinical signs of toxicity observed in any rats in the 350 ppm exposure throughout the recovery period. 

On the first day of the two-week recovery period, one male rat in the 1500 ppm exposure group displayed lethargy, hunched posture, and had a red stained head. There were no other biologically significant clinical signs of toxicity observed in any rats in the 1500 ppm exposure group throughout the 14-day recovery period.

Clinical signs of toxicity observed in all rats after the 5600 ppm exposure were ataxia, labored breathing, and lethargy; other clinical signs observed in some of the 5600 ppm rats after the exposure included hunched posture, clear discharge from the mouth and red stained fur on the face or head. On the day following the 5600 ppm exposure, clinical signs observed in 5 of 10 rats included hunched posture, lethargy, labored breathing, and red stained fur on the face or head. There were no abnormal clinical signs of toxicity observed in any rats from the 5600 ppm group from post-exposure day 2 throughout the remainder of the recovery period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the 4-hour LC50 of 2,2-Difluoroethyl acetate (administered as vapor) was greater than 5600 ppm (i.e. greater than 28 mg/L) in male and female rats.

Executive summary:

The acute inhalation toxicity of 2,2-Difluoroethyl acetate was investigated in a study performed according to OECD test guideline 403 under GLP compliance.

Three groups of five male and five female Crl:CD(SD) albino rats were exposed whole-body for a single 4-hour exposure to three concentrations of 350 ± 31, 1500 ± 310 and 5600 ± 820 ppm (equivalent to 1.77 ± 0.16, 7.60 ± 1.57, 28.36 ± 4.15 mg/L, respectively). 2,2-Difluoroethyl acetate was generated as a vapour. During a 14-day recovery period, all animals were weighed and observed for mortality and clinical signs of toxicity. A gross pathological examination was performed on all animals at the scheduled necropsy.

There were no deaths during this study.

There were no clinical signs of toxicity observed in any rats in the 350 ppm exposure group during the exposure or the recovery period. All rats displayed normal alerting response during exposure to the three concentrations; however, rats in the 1500 and 5600 ppm exposure group displayed decreased activity throughout the exposure. In addition, rats in the 5600 ppm exposure group displayed labored breathing and/or gasping during the exposure. On the first day of the two-week recovery period, one male rat in the 1500 ppm exposure group displayed lethargy, hunched posture, and had a red stained head. There were no other biologically significant clinical signs of toxicity observed in any rats in the 1500 ppm exposure group throughout the 14-day recovery period. Clinical signs of toxicity observed in all rats after the 5600 ppm exposure were ataxia, labored breathing, and lethargy; other clinical signs observed in some of the 5600 ppm rats after the exposure included hunched posture, clear discharge from the mouth and red stained fur on the face or head. On the day following the 5600 ppm exposure, clinical signs observed in 5 of 10 rats included hunched posture, lethargy, labored breathing, and red stained fur on the face or head. There were no abnormal clinical signs of toxicity observed in any rats from the 5600 ppm group from post-exposure day 2 throughout the remainder of the recovery period.

On the day after the exposure, 7 of 10 rats in the 350 ppm exposure group lost between 1.1 and 4.8% of their initial body weight followed by normal weight gain throughout the remainder of the recovery period. Nine of 10 rats in the 1500 ppm exposure group lost between 0.71 and 13% of their initial body weight on the day following the exposure. Minor weight losses ranging from 0.19 to 2.6% were observed in some rats in the 1500 ppm group from test day 3 until test day 7, followed by normal weight gain throughout the second week of the recovery period. All rats in the 5600 ppm group lost between 8.4 and 15% of their initial body weight on the day after the exposure. Weight losses from 0.74 to 6.1% were observed in half of the 5600 ppm rats on post-exposure day 2, followed by normal weight gain throughout the remainder of the recovery period.

There were no gross lesions observed in any rats in this study.

Under the conditions of this study, the acute inhalation 4-hour LC₅₀ value of 2,2-Difluoroethyl acetate was determined to be greater than 5600 ppm (equivalent to greated than 28.36 mg/L) in male and female rats.