2-methylenepropane-1,3-diyl diacetate

Endpoint summary

Description of key information

Acute Oral Toxicity: The acute median lethal oral dose (LD50) to rats of 2-methylene-1-3-propanediol diacetate was demonstrated to be between 300 and 2000 mg/kg bodyweight.

Acute Toxicity Inhalation: 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.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 04 December 2013 and 27 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to OECD TG 423 in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

17 December 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

EPA 712-C-02-190. 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 12 Nousan No. 8147, 2000

Version / remarks:

JMAFF 12 Nousan No. 8147, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Specific details on test material used for the study:

Identification: 2-methylene-1-3-propanedioldiacetate
Chemical name: MPD (2-Methylene- 1,3- propanediol)
Intended use: Food contact material
Appearance: Colourless liquid
Storage conditions: Ambient temperature, in the dark Supplier:
Sponsor Batchnumber: 201210
Expiry date: December 2017
Purity: 99%
Samplereceipt: 31 January 2013

The Sponsor was responsible for the characterisation of the test substance and the documentation of the methods of synthesis, fabrication or derivation and stability

Species:

rat

Strain:

other: Crl:CD (SD) albino rats

Sex:

female

Details on test animals or test system and environmental conditions:

Animal supply, acclimatisation and allocation Healthy nulliparous and non-pregnant female Crl:CD (SD) albino rats were obtained from Charles River (UK) Ltd. The animals were allocated without conscious bias to cages within the treatment groups. They were housed in groups of three rats of the same sex. Each animal was assigned an alpha-numeric code and identified uniquely within the study by tail marking. Each cage label was colour-coded and was identified uniquely with the study number, dose level and animal mark. The animals were allowed to acclimatise to the conditions described below for at least 5days before treatment. For those animals selected for this study, their bodyweights were in the range 214to 252g and they were approximately eight to twelve weeks of age prior to dosing (Day 1).

Animal housing, diet and water supply Animals were housed inside a barriered rodent facility. The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms.
The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were set to maintain the range of 19 to 23”C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Environmental parameters are archived with the departmental raw data.
Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily.
Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the public supply fail. The cages were solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved softwood bark-free fibre bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals.
The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet), except for overnight prior to and approximately four hours after dosing. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
Each cage of animals was provided with an Aspen chew block for environmental enrichment. Chew blocks were provided throughout the study and were replaced when necessary. Each cage of animals was provided with a plastic shelter for environmental enrichment, which was replaced at the same time as the cages.
Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinised and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier. Certificates of analysis were received routinely from the supplier of the chew blocks. Since the results of these various analyses did not provide evidence of contamination that might have prejudiced the study, they are not presented.
No other specific contaminants that were likely to have been present in the diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test substance was formulated at concentrationsof 30 and 200 mg/mL in the vehicle and administered at a volume of 10 mL/kg bodyweight.
The test substance formulationswere prepared on the day of dosing.
The appropriate dose volume of the test substance was administered to each rat by oral gavage using a plastic syringe and plastic catheter. A record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly. Formulations were stirred before and throughout the dosing procedure.

Doses:

Formulation The test substance was formulated at concentrationsof 30 and 200 mg/mL in the vehicle and administered at a volume of 10 mL/kg bodyweight. The test substance formulationswere prepared on the day of dosing. The absorption of the test substance was not determined. Determination of the homogeneity, stability and purity of the test substance or test substance formulations were not undertaken as part of this study. Detailed records of test substance usage were maintained. The amount of test substance necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

The appropriate dose volume of the test substance was administered to each rat by oral gavage using a plastic syringe and plastic catheter. A record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly. Formulations were stirred before and throughout the dosing procedure.

Serial observations
Mortality Cages of rats were checked at least twice daily for any mortalities.
Clinical observations Animals were observed soon after dosing and at frequent intervals for the remainder of Day1. On subsequent days, surviving animals wereobserved once in the morning and again at the end of the experimental day(with the exception of Day 15 - morning only). The nature and severity, where appropriate, of the clinical signs and the time were recorded at each observation. All surviving animalswere observed for 14 days after dosing.
Bodyweight The weight of each rat was recorded on Days1 (prior to dosing), 8 and 15 or at death. Individual weekly bodyweight changes and group mean bodyweights were calculated.

2.5 Necropsy and macropathology
Method of kill All surviving animalswere humanely killed on Day 15 by carbon dioxide asphyxiation.
Macroscopic pathology All animals were subject to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities. The macroscopic appearance of all examined organs was recorded.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - < 2 000 mg/kg bw

Based on:

test mat.

Mortality:

Animals dosed at 300 mg/kg
Two animals(Nos. A1 and A4) dosed at 300 mg/kg were found dead on the morning of Day2. Clinical signs prior to death comprised of loose faeces, piloerection, elevated gait (Nos. A1 and A4), underactivity ( No. A1) and hunched posture (No. A4). These signs were seen from approximately one hour after dosing. Macroscopic examination ofthe animals revealed asmall caecumand spleen, congestion of the subcutaneous tissue (characterised by darkened tissues), yellow fluid content in the small and large intestines, pallor of the lungs, spleen, kidneys and small and large intestine (No. A1 and A4).

Animals dosed at 2000 mg/kg All three females dosed at 2000 mg/kg died on Day 1, two were found dead approximately 3.5 hours following dosing and the remaining animal (No. A7 was killed for welfare reasons due to poor condition). Clinical signs prior to death comprised of underactivity, piloerection, irregular breathing, blue skin colouring to the extremities (Nos. A7, A8 and A9), hunched posture (Nos. A7 and A8), reduced body tone (Nos. A8 and A9) elevated gait, unsteady posture and shallow breathing (No. A7). These signs were seenfrom approximately 30 minutes post dosing. Macroscopic examination of the animals revealedcongestion of the subcutaneous tissue, heart, lungs, liver and spleen (characterised by darkened tissues/organs), clear yellow fluid in the stomach (Nos. A7, A8 and A9), congestion of the kidneys (A8), pallor of the kidneys (Nos. A7 and A9), yellow fluid content in the small and large intestine (No. A9) and a small caecum (Nos. A7 and A8).

Clinical signs:

other: Animals dosed at 300 mg/kg Clinical signs of reaction to treatment for surviving animals dosed at 300 mg/kg comprised of loose faeces (No. A3 and A6), underactivity (Nos. A2, A3 and A6), piloerection (No. A2, A5 and A6), unsteady gait (No. A5), hunched p

Gross pathology:

For surviving animals at 300 mg/kg, macroscopic examination at study termination on Day15 revealed pallor of the kidneyin one female (No. A5). No abnormalities were revealed in the remaining surviving animalsat the macroscopic examination.

Conclusions:

The acute median lethal oral dose (LD50) to rats of 2-methylene-1-3-propanediol diacetate was demonstrated to be between 300 and 2000 mg/kg bodyweight.

Executive summary:

The study was performed to assess the acute oral toxicity of 2-methylene-1-3-propanediol diacetate, a food contact material, to the rat.

Two groups of three fasted female rats received a single oral gavage dose of thetest substance, formulated in corn oil, at a dose level of 300 mg/kg bodyweight.  As results at this dose level indicated the acute (median) lethal oral dose of the test substance to be greater than 300 mg/kg bodyweight, in compliance with the study guidelines a further group of three fasted females were similarly dosed at 2000mg/kg bodyweightto complete the study.

Results

All three animals dosed at 2000 mg/kg died on Day 1 and two animalsdosed at 300 mg/kg were found dead on Day 2.  Clinical signs observed prior to death in one or more animal comprised loose faeces, piloerection, underactivity, hunched posture, irregular breathing, blue skin colouring to the extremities, reduced body tone, elevated gait, unsteady posture and shallow breathing. Macroscopic examination of the animals revealed a small caecum and spleen, congestion of the subcutaneous tissue, heart, lungs, liver and spleen, yellow fluid content in the stomach or small / large intestines, pallor of the lungs, spleen, kidneys and small and large intestine and small caecum.

Clinical signs of reaction to treatment for surviving animals dosed at 300 mg/kg comprised loose faeces, underactivity, piloerection, unsteady gait, hunched posture and yellow staining around the peri genital region. These signs were first noted approximately one hour after dosing.  Recovery of surviving animals, as judged by external appearance and behaviour, was complete by Day 5 for all except one animal who continued to have yellow staining around the peri genital region until termination on Day 15.

All surviving animals at 300 mg/kg were considered to have achieved satisfactory bodyweight gains throughout the study.

Macroscopic examination at study termination for surviving animals at 300 mg/kg on Day 15 revealed pallor of the kidney in one female.  No abnormalities were observed in any other surviving animal at the macroscopic examination.

Conclusion

The acute median lethal oral dose (LD50) to rats of 2-methylene-1-3-propanedioldiacetate was demonstrated to be between 300 and 2000 mg/kg bodyweight.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

500 mg/kg bw

Quality of whole database:

Reliability 1 is assigned because the study conducted according to OECD TG 423 in compliance with GLP, without deviations that influence the quality of the results.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

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

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

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

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

> 5.25 mg/L air

Physical form:

inhalation: aerosol

Quality of whole database:

Reliability 1 is assigned because the study conducted according to OECD TG 423 in compliance with GLP, without deviations that influence the quality of the results.

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An acute oral toxicity study has been conducted according to EC Guideline B.1 tris and OECD Guideline 423. Six rats received single dose of 300 mg/kg and further tree rats received dose of 2000 mg/kg. The dose was administered by oral gavage; animals were observed for up to 15 days post dose.

Two animals receiving 300 mg/kg and all animals receiving 2000 mg/kg were found dead or were killed due to poor clinical condition. On this basis it is concluded that the acute (median) lethal oral dose (LD₅₀) was between 300 and 2000 mg/kg. Furthermore, clinical signs observed in the animals receiving the 300 mg/kg dose indicated some toxic effect however these were largely seen to recover by Day 5 of observation.

LD₅₀ cut-off value of 500 mg/kg calculated in accordance with EU test method B.1 tris, Appendix 1C.

Justification for classification or non-classification

As mentioned above the LD₅₀was in the range of 300-2000 mg/kg. In accordance with Annex I, Section 3.1 of the CLP Regulation classification for oral toxicity Category 4 shall be applied.