(E)-1,2-difluoroethylene

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Initiation of study: March 18th 2019. Necropsy: April 12th 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

traditional method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(E)-1,2-difluoroethylene

EC Number:

216-629-0

EC Name:

(E)-1,2-difluoroethylene

Cas Number:

1630-78-0

Molecular formula:

C2H2F2

IUPAC Name:

(E)-1,2-difluoroethene

Test material form:

gas

Specific details on test material used for the study:

Test substance: E-1,2-difluoroethylene (abbreviation: TKNl)
Appearance: Colorless gas
Lot No.: N1181201
Purity: 99.9%
Supplier: Daikin Industries, Ltd.
Storage conditions: Room temperature (actual range: 19.0°C-22.7°C, acceptable range: 10°C-30°C), no condition to light, airtight container.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age at receipt: 7 weeks old
Age at exposure: 8 weeks old

Quarantine and acclimatization
Quarantine period was set for 5 days from animal receipt. The following examinations were conducted during the quarantine period :
· Clinical observation: Once a day
· Body weight measurement: The day of animal receipt and the day of end of quarantine Acclimatization period was set from the day of animal receipt to the day before exposure. The animals were daily observed during the acclimatization period. It was confirmed that there were no abnormalities in clinical sign or in increasing body weights during the
quarantine/acclimatization period.

Body weight range at dosing
Males: 281.8-337.4 g, females: 181.3-225.4 g
It was confirmed that the body weight of the animals used in this study was within ±20% of the mean body weight of males and females in each group, moreover, it was confirmed that the body weight was within ±20% of the mean weight for each sex of previously exposed at same age.

Allocation to group
Animal selection
All males and females were selected for the animal assignment based on the results of examinations during quarantine and acclimatization periods.
Animal assignment
The animals were allocated to study group using the stratified-by-weight randomization method based on the body weight on the allocation day. This study used 18 males and 18 females.

Acclimatization to restraint tube
The animals held in a restraint tube for nose-only inhalation exposure (Muenster Ltd.) to acclimatize it for 30 minutes after the animal allocation.

Animal identification
Each animal was identified by the following methods:
Before assignment: Marking the last 1 or 2 digit of animal number on tail by a permanent marker
After assignment: Back subcutaneous implant of a microchip in which an animal number was registered; a microchip reader (IPT-300 and DAS- 5002: Bio Medic Data Systems, Inc.)
The animal numbers before animal allocation were 29001-29021 in males and 69001- 69021 in females.

Handling of surplus animals
Surplus animals that were not used for the study were euthanized 6 days after the experimental starting date, by exsanguination via the abdominal aorta under anesthesia by intraperitoneal injection ofthiopental sodium (Ravonal: Nipro ES Pharma Co., Ltd.).

Animal housing
Housing rooms
Rat/Mouse/Guinea pigs/Rabbits housing room (Rooms 7118 and 7119)

Housing environmental
Temperature
Actual range: 21.1°C-23.2°C, acceptable range: 19.0°C-25.0°C

Relative humidity
Actual range: 36.1%-63.6%, acceptable range: 35.0%-75.0%

Ventilation
6 to 20 times/hr, all fresh air
The ventilation frequency is periodically measured twice a year. It was confirmed to be within the acceptable range as described in the standard operating procedure (SOP) of the test facility.

Lighting
12 hr/day, 7:00 to 19:00

Housing materials
Cages
Polycarbonate cages (W 220 x D 380 x H 183 mm: Tokiwa Kagaku Kikai Co., Ltd.) Sterilization before use: Autoclave sterilization
Frequency of replacement: The day of animal allocation, thereafter, once a week

Feeders
Stainless steel feeders for pellet food (Tokiwa Kagaku Kikai Co., Ltd.) Sterilization before use: Autoclave sterilization
Frequency of replacement: At the time of a cage replacement

Watering bottles
Polycarbonate watering bottles (700 mL; Tokiwa Kagaku Kikai Co., Ltd.) Sterilization before use: Autoclave sterilization
Frequency of replacement: At the time of a cage replacement

Racks
Stainless steel racks (Tokiwa Kagaku Kikai Co., Ltd.)
Disinfection before use: Wiping with sodium hypochlorite solution Frequency of disinfection: At the time of a cage replacement

Enrichment
In order to improve animal welfare, the following enrichment products were used.
Items: Gnawing material and nest material
(Diamond Twists: Envigo and Paper Clean: Japan SLC, Inc.) Analysis results of contaminants such as residual pesticides were obtained from the supplier of enrichment, and the contaminant level was confirmed to meet the criteria specified in the SOP of the test facility.

Number of animals per cage
3 animals (same sex) /cage

Bedding Type
Bedding for laboratory animals (Beta Chip: Charles River Laboratories Japan, Inc.) Sterilization before use: Autoclave sterilization
Frequency of replacement: At the time of a cage replacement

Contamination check
The analysis results of contaminants such as residual pesticides were obtained from the supplier of bedding, and the contaminant level met the criteria specified in the SOP of the
test facility.

Food
Pellet food for experimental animals (MF: Oriental Yeast Co., Ltd., lot No. 181123)

Feeding
Provided ad libitum, except that no food was provided during inhalation exposure. Food was changed at the same time as the feeder replacement.

Contamination check
The analysis results were obtained from the food supplier, and the levels of contaminants such as residual pesticides in used lots were confirmed to meet the criteria specified in the SOP of the test facility.

Drinking Water
Tap water filtered through a 5-µm pore size filter followed by irradiation with ultraviolet light

Water supply method
Provided ad libitum, except that no water was provided during inhalation exposure. Water was changed at the same time as the water bottle replacement.

Analyses
Water quality was periodically (twice a year) analyzed by an external agency (Ibaraki Pharmaceutical Association Inspection Center). The values obtained from the analysis were confirmed to meet the criteria specified in the SOP of the test facility.

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

The rats held in the restraint tubes were connected to the inhalation chamber at 5 minutes or more (actual time: 5--6 minutes) had elapsed from the start of the test atmosphere generation, which was sufficient time for concentration equilibrium, to start exposure. The time to concentration equilibrium (t95) was approximately 30 seconds that was calculated according to the OECD guideline with an inner volume of the chamber (approx. 2.5 L) and air flow ratio oftest atmosphere supply (16 Umin.).

In consideration of the time required to the concentration equilibrium in the air over time, the time point to start of exposure was defined at 5 minutes or more after the start of the test atmosphere generation. The rats were dismounted from the chamber to terminate exposure 4 hours after the start of exposure.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

The test substance in the test atmosphere was quantified by a gas chromatography (hereinafter abbreviated as "GC"), then, the exposure concentration was calculated. See attached background material for full details on GC analytical method.

Duration of exposure:

4 h

Concentrations:

Target concentration were 5000 ppm, 20000 ppm, and 100000 ppm.
Actual exposure concentrations were 5160 ppm, 20940 ppm, and 105660 ppm.

No. of animals per sex per dose:

6 males and 6 females per dose group.

Control animals:

no

Details on study design:

Observation and measurement
The parameters listing in the following sections were examined. The day of exposure was designated as day 1.

Clinical signs
Daily observation was conducted from the day of exposure to the day of necropsy with the following frequency.
Exposure day: Fourth a day (pre-exposure, immediately after the end of exposure, and 1
and 2 hours after the end of exposure)
Other day: Once a day

Body weight
Body weight was measured with an electrical balance (PB-3002S: Mettler Toledo International Inc.) on the following schedule.
Measurement day: Before exposure on day 1, day 2, day 4, day 8, and day 15

Pathological examination
Necropsy
After the observation on day 15, all animals were subjected to necropsy after the euthanization by exsanguination from the abdominal aorta after anesthesia by intraperitoneal injection ofthiopental sodium (Ravonal: Nipro ES Pharma Co., Ltd.).

Histopathological examination
Any tissues or organs were not collected, preserved, or subjected to histopathological examination, since there were no gross abnormalities.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 100 000 ppm

Based on:

test mat.

Exp. duration:

4 h

Mortality:

None

Clinical signs:

other: No abnormalities were observed in any males or females in each group throughout the observation period

Body weight:

In the 5000 ppm group, body weight loss was observed in 4 females on day 2.

In the 20000 ppm group, body weight loss was observed in 3 males and 1 female on day 2, and in 2 females on day 4.

In the 100000 ppm group, body weight loss was observed in 3 males and 5 females on day 2, and in 1 female on day 4.

Gross pathology:

There were no gross abnormalities in any males or females of each group

Any other information on results incl. tables

Inhalation exposure

 Exposure concentration

Actual exposure concentrations were 5160 ppm, 20940 ppm, and 105660 ppm to the target concentrations of 5000 ppm, 20000 ppm, and 100000 ppm.

The concentration homogeneity was confirmed, since the coefficient  variation between 3 positions was 0.7%, 1.0% and 0.6% to the target concentrations of 5000 ppm, 20000 ppm, and 100000 ppm; these were within the criterion of the homogeneity, which was to be 15% or below.

The temporal concentration stability was confirmed, since the relative errors were -0.8%- 1.6%, -1.0%-1.7%, and -1.7%-2.1%, and the coefficient variations of the exposure concentration were 1.3%, 1.4%, and 1.9% to the target concentrations of 5000 ppm,

20000 ppm, and 100000 ppm; these were within the criteria of the stability, which were to be within ±10% on the relative error and 10% or below on the coefficient variation.

Nominal concentrations were 5220 ppm, 21910 ppm, and 108150 ppm to the target concentrations of 5000 ppm, 20000 ppm, and 100000 ppm.

 Air flow rate of chamber

Air flow rate of test atmosphere supply was 16.0 L/min, and exhaust flow rate was 13.8 L/min respectively. It was confirmed that they were intended ranges.

Environmental condition

Temperature was 20.9°C-23.8°C, relative humidity was 12.4%-14.7%. Temperature was within the acceptable range of animal housing. Since pressurized air used for the test atmosphere preparation was dry, relative humidity was low. However, it was reported that the low relative humidity did not have an influence on the results of inhalation toxicity study. Therefore, it was concluded that the test results were not affected.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

50% lethal concentration of the test substance is more than 100000 ppm under the condition of this study

Executive summary:

OECD Guideline 403 (Acute Inhalation Toxicity).

Test organism: Rat (strain: Crj:CD(SD) ).

Target concentration were 5000 ppm, 20000 ppm, and 100000 ppm

Actual exposure concentrations were: 5160 ppm, 20940 ppm, and 105660 ppm

Exposure Duration: 4 hours (nose only)

No mortalities. No clinical changes in any males or females of each group.

No gross abnormalities were observed at necropsy in any males or females after the observation period.

Conclusion: 50% lethal concentration of the test substance is more than 100000 ppm under the condition of this study.