2-methylenepropane-1,3-diyl diacetate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting, 2019-08-23
Experimental Completion, 2019-09-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2-methylenepropane-1,3-diyl diacetate

EC Number:

223-225-8

EC Name:

2-methylenepropane-1,3-diyl diacetate

Cas Number:

3775-29-9

Molecular formula:

C8H12O4

IUPAC Name:

2-[(acetyloxy)methyl]prop-2-en-1-yl acetate

Specific details on test material used for the study:

Identification: MPDAc
Chemical name: 2-methylenepropane-1,3-diyl diacetate
CAS No.: 3775-29-9
Test Item Number: TY19174CH
Molecular Weight: 172. 18
Appearance: Colorless and transparent liquid
Purity: 99.5%
Solubility: 28.9 g/L in water
Storage Conditions: Keep container sealed, Store in a cool area away from incompatible substances
Expiry Date: 2020-08-30

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number and Sex Total: 12, 6 males and 6 females (females were virginal). 6 (3 males and 3 females) were used in the study, an additional 6 (3 males and 3 females') spare animals were transferred into training colony in Testing Facility.
Age and Body Weight: Animals were 8~ 10 weeks of age and body weights ranged from 217.44 g to 222.71 g (females) and 228.02 g to 272.54 g (males).
Source: Zhejiang Vital River Laboratory Animal Technology Co., Ltd.
Animal receipt date: 2019-08,23

Animal Identification
Individual animals were identified with a unique identification number by tail marking. Cage cards
were labeled for each cage,

Receipt and Acclimation
All animals were examined after receipt by the veterinary staff and were quarantined/acclimated for
5 days.

Animal Housing and Environment
Animals were housed in Room 306, The Animal Usage Certificate Number is SYXK 2016-0166, Animals were group housed (up to 3 same sex animals together) in solid bottom cages, with corn bedding, No other species were housed in the same room. The room was controlled and
monitored for humidity (targeted range 40% to 70%) and temperature (targeted range 20 °C to 26 °C)
with more than 15 air changes/hour. The room was on a 12-hour light/dark cycle.

Environmental Enrichment
The animals were provided with manipulatives for environmental enrichment.

Feed and Water
Animals were supplied with rodent feed (lot number 201906;20) front a contract vendor (Jiangsu
Synergetic Pharmaceutical Bioengineering Co., Ltd, ad libitum. Nutritional components and environmental contaminants in the diet were analyzed routinely by the vendor and an independent laboratory, respectively. The feed analysis results met the requirement. Animals were provided reverse-osmosis purified and chlorinated water ad libitum by water bottles. The animal drinking water was analyzed for contaminants by an independent laboratory. The feed and water analysis results can meet the requirement of standard.

Animal Care and Use Committee
IACUC applications relating to the protocol involving the care or use of animals on this study were
reviewed and approved by EnHealth Institutional Animal Care and Use Committee (IACUC) prior to
the initiation of such procedures. IACUC members monitored the study for animal welfare issues.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.98 - <= 3.36 µm

Geometric standard deviation (GSD):

>= 2.31 - <= 2.45

Details on inhalation exposure:

Rats were exposed to the test item aerosol for 4 h. Diet and water were not available during the
exposure period. Actual concentration and pa1iicle size distribution were measured. The exposing day was designated as D1 .
An exposure concentration of 5.00 mg/L were used. The duration of observation continued to D15 if the mortality was less than 3.

Route: Nose-only Dynamic Inhalation.
Method: Rats were fasten in. restraining tubes respectively and exposed to a stable aerosol of test item, which generated by Animal Nose-Only Inhalation system, and the air exchanged approximately 12-15 times per hour.
Frequency: Once daily.

Particle Size Distribution
The particle size distribution were measured for I min with 28.3 L/min airflow rate using cascade
impactor and were measured 4 times (approximately every 1 h) during the exposure period.

Exposure Chamber Conditions
The oxygen concentration, carbon dioxide concentration, temperature and relative humidity were
monitored and recorded by inhalation system during the 4 h exposure period. Oxygen concentration should be at least 19% and carbon dioxide concentration should not exceed 1%. The chamber temperature should be maintained at 22~26 °C and relative humidity was 34.399% - 56.6463 %. However during the study the actual relative humidity of about 0.5h during the 4h of exposure period was 34.399%-39.854%, which was less than 40%. Due to the small deviation of relative humidity, and timely connected the diluent gas to a humidifier to improve the relative humidity in the exposure chamber, the actual concentration of the test item was still acceptable. It was considered that this protocol deviation had no effect on the reliability and integrity of the result.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Exposure concentration: nominal 5 mg/L, measured average concentration 5.250 ± 0.411 mg/L

The aerosol of test item were sampled for 3 min with 2 L/min airflow rate using glass fiber
membranes. The glass fiber membranes were weighed before and after sampling. Aetna!
concentration was calculated by the equation below and the CEL-712 mass concentration monitor
was calibrated. The actual concentration were measured and recorded by CEL-712 mass
concentration monitor, the value should be within ±10% of 5.0 mg/L (4.5-5.5 mg/L).
Equation, mass of membranes after sampling - mass of membranes before sampling/ (airflow rate x
time).

No. of animals per sex per dose:

3 males and 3 females

Control animals:

no

Details on study design:

Duration of Observation, 14 days.

Clinical Observations
Detailed Observations: Detailed observations were conducted once on D1, 4 h during the exposure period, after the exposure (D1), and once daily on D2, D3, DS, and Dl4.
Cage side Observations, Exception of the day of detailed observations, cageside observations were
conducted once daily for all animals.
Body Weights
All survival animals were weighed once on D1, D1 (before dosing), D2, D4, D8 and Dl5.
Pathology
Necropsy and gross observations were conducted for survival animals on D15. No tissues were
collected due to no gross pathological changes were found.

Results and discussion

Effect levels

Sex:

female

Dose descriptor:

LC50

Effect level:

> 5.25 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

1 female animal was found dead at D2.

Clinical signs:

lethargy (hypoactivity)

Remarks:

Please see other findings

Body weight:

During study period, decreased of the mean body weight in surviving animals was observed (female:
6.66%; male: 8.00%) when compared to the mean body weight on D1, however, the mean body·
weight increased on D4 and recovered to normal on D15.

Gross pathology:

No macroscopic obse1vatio11s ·were obse1ved in all animals at necropsy. No histapathological
examination was conducted.

Other findings:

No clinical signs were observed of all animals during the 4 h exposure period; After the exposure, all animals showed arched back, decreased activity and disheveled fur, 2 of them (1F01, 1M02) even showed abnormal respiratory sounds.
1 female animal (1F01) was found dead at D2. All signs were recovered on D2 of the rest surviving animals except 1 male rat (1MO1). Signs of arched back and disheveled fur of this male rat were also recovered on D2 but decreased activity persisted until D3.
No other clinical signs were observed in the rest observation time point.

Applicant's summary and conclusion

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

Based on the results that 1 female rat was found dead in this study, the median lethal concentration
(LC50) was considered to be greater than 5.250 mg/L for rats when administered MPDAc by inhalation.
According to the criteria for toxicity assessment list in Section 13, the acute inhalation toxicity of
MPDAc was considered to be Category 5 of GHS (Globally Harmonized System of Classification and.
Labelling of Chemicals), and the LC50 cut-off value was considered to be greater than 5~12.5 mg/L.

Executive summary:

Objective: The purpose of this study was to determine the toxicity and dose-response relationship of MPDAc when administered by Inhalation to rats, which could be provided on the classification of chemical which cause acute toxicity, administration of chemical labels, justification of dosage selection for other toxicological studies, and protection of chetnical manufacture and application.
Methods: According to The Guidelines for the Testing of Chemicals-Health Effects (the second version) (China Environment Publishing Group, Sep, 2013); 436 Acute Inhalation Toxicity-Acute Toxic Class Method, a concentration of 5.00 mg/L was used in this study. Total 6 rats (3 males and 3 femailes) were exposed to
the test item aerosol for 4 h using HRH-MNE3026 animals nose-only inhalation exposure system. The actual concentration were measured and recorded by CEL-712 mass concenh·ation monitor (which was calibrated by gravimetric filter analysis), and the particle size distribution were measured either to determined MMAD (mass median aerodynamic diameter) and GSD (geometric standard deviation). The exposure day was designated as DI.

Detailed observations were conducted once dming the 4 h exposure period, and on D-1 (the day before exposure), D2, D3, D8, and D14. Exception on the day of detailed observations, cage side observations were conducted once daily for all animals. Toxicity signs and viability were recorded in details. All survival animals were weighed once on D-1, D1 (before exposure), D2, D4, D8, and D15. Necropsy
and gross observations were conducted for dead animals or survival animals on D15.
Results: During the 4 h exposure period, the actual concentration of the test item aerosol was 5.250 ± 0.411 mg/L, the MMAD was 2.98~3.36 μm and the GSD was 2.31~2.45. After the exposure, 1 female animal was found dead at D2; All animals showed arched back, decreased activity and disheveled fur, 2 (1 males and 1 female) of them. even showed abnormal respiratory sounds. All signs were recovered untH D3. No other clinical signs were observed in the rest observation time point.
Decreased of mean body weight in smviving animals was observed on D2 when compared to mean body weight on D1, however, the mean body weight increased on D4 and recovered to normal on D15.
No macroscopic observations were obse1ved in all animals at necropsy.
Conclusions, Based on the results that I female rat was found dead in this study, the median lethal concentration (LC50) was considered to be greater than 5.250 mg/L for rats when administered MPDAc by inhalation.
According to appendix 3d of The Guidelines for the Testing of Chemicals-Health Effects (the second version) (China Envirinment Publishing Group, Sep, 2013): 436 Acute Inhalation Toxicity-Acute Toxic Class Method, the acute inhalation toxicity of MPDAc was considered to be Category 5 of GHS, and the LC50 cut-off value was considered to be greater than 5~12.5 mg/L.