2-methylenepropane-1,3-diyl diacetate

Acute Toxicity: oral

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

17 December 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

EPA 712-C-02-190. 2002

Deviations:

no

Test guideline 4

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

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: 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

Test animals

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.

Administration / exposure

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.

Results and discussion

Effect levels

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.

Applicant's summary and conclusion

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.