Diallyl hexahydrophthalate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 November 2016 (reception of animals) to 13 December 2016 (experimental completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Relative humidity values (min 28-29 %) which lie outside the expected range (30-70%) where recorded twice during the study. The vehicle, PEG400, was not confirmed by the Sponsor before experimental start. These had no effect on study integrity/validity.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Relative humidity values (min 28-29 %) which lie outside the expected range (30-70%) where recorded twice during the study. The vehicle, PEG400, was not confirmed by the Sponsor before experimental start. These has no effect on study integrity/validity.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

yes

Remarks:

Relative humidity values (min 28-29 %) which lie outside the expected range (30-70%) where recorded twice during the study. The vehicle, PEG400, was not confirmed by the Sponsor before experimental start. These has no effect on study integrity/validity.

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:

Diallyl hexahydrophthalate

EC Number:

237-580-1

EC Name:

Diallyl hexahydrophthalate

Cas Number:

13846-31-6

Molecular formula:

C14H20O4

IUPAC Name:

1,2-bis(prop-2-en-1-yl) cyclohexane-1,2-dicarboxylate

Test material form:

liquid

Remarks:

Colourless

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical name: 1,2-Cyclohexanedicarboxylic acid
- Test item name: MDAC
- Other name: Diallyl hexahydrophthalate
- CAS number: 13846-31-6
- Description Colourless liquid
- Source and lot No.of test material: Supplied by the Sponsor (Osaka Soda Co., Ltd.,Japan), Lot No. 40201
- Expiration date of the lot/batch: 26 January 2019
- Purity test date: 99.2%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25 °C, below 70% Relative humidity), protected from light.
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: Frestly formulated at 200 mg/mL in PEG400 on the day of use, assumed stable for the duration of the test.
- Reactivity of the test substance with the solvent/vehicle: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: Formulated at 200 mg/mL in PEG400 on day of administration.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI Wistar rats

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D- 97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks old
- Animal identification: Animals were individually identified using numbers written on the tail with an indelible marker pen. The numbers were allocated based on CiToxLAB Hungary Ltd.' s Master File, for each animal allocated to the treatment groups. Cages were identified by cards with information about study code, sex, dose group, cage number and individual animal numbers.
- Weight at study initiation: 220 - 232 g
- Fasting period before study: The night before treatment animals were fasted. The food (not water) was withheld during an overnight period. The test item
was administered by oral gavage in the morning and food returned 3 hours after treatment.
- Housing: 3 animals / cage. Cage Type II. polypropylene/polycarbonate. Lignocel 3/4-S Hygienic Animal Bedding (J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany). For nesting, Arbocel (natural) crinklets (J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) were available to animals during the study. Animals were housed by group to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.
- Diet (e.g. ad libitum): ad libitum from 3 hours after treatment with test item. Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice –
breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (batch number: 141 8884, expiry date: 31 January 2017).
- Water (e.g. ad libitum): ad libitum. Water quality control analysis is performed every three months and microbiological assessment performed monthly. Results are retained in the archives at CiToxLAB Hungary Ltd.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 - 23.7 °C
- Humidity (%): 28 - 60%
The temperature and relative humidity were recorded twice daily during the acclimatisation period and throughout the study.
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light (6 a.m to 6 p.m)

IN-LIFE DATES: From: 03 November 2016 to 13 December 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

VEHICLE
- Vehicle name: Poly(ethylene glycol) 400 (PEG 400). Manufactured by Sigma Aldrich
- Concentration in vehicle: Freshly formulated at a concentration of 200 mg/mL in the vehicle, PEG 400. Formulations were magnetically stirred continuously up to the end of dose administration procedures.
- Justification for choice of vehicle: The toxicological characteristics of PEG 400 are well known and it is routinley used as a vehicle within this test system.
- Lot no. BCBQ0052V
- Expiry date: 31 December 2016
- Storege: Room temperature

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg (200 mg/mL x 10 mL/kg)


CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: The initial dose level was selected by the Study Director to be that which is most likely to produce mortality in some of the dosed animals. In the lack of any preliminary toxicological information, 2000 mg/kg bw was selected as the starting dose.

Doses:

Two groups of three female Crl:WI rats were treated with MDAC at a dose level of 2000 mg/kg bw (Group 1 and Group 2). Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw. As one animal was found dead with no further mortality observed, a confirmatory group (Group 2) was treated at the same dose level. One animal was found dead in the confirmatory group; therefore no further testing was required according to OECD 423 and Commission Regulation (EC) NO 440/2008 of 30 May 2008, B.1.Tris.

No. of animals per sex per dose:

3 female rats per dose (6 animals used in total)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations were performed on all animals at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weight was recorded day before treatment (Day -1), day of treatment (Day 0) and weekly thereafter.In the case of found dead animals, a final
body weight measurement was performed right after the recognition of death.
- Necropsy of survivors performed: yes
- Other examinations performed:
Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
All animals were subject to necropsy and macroscopic examination. After examination of the external appearance, the cranial, thoracic and the abdominal cavities were opened and the organs and the tissues were observed.

Clinical signs, body weight, body weight gain and gross macroscopic data were all tabulated and reported.

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

>= 2 000 mg/kg bw

Based on:

test mat.

Mortality:

MDAC caused mortality in 2/6 animals (one for each treatment group) at a dose level of 2000 mg/kg bw. Animal number 3614 died on Day 1 and animal number 3615 died on Day 0.

Clinical signs:

Found dead animals showed decreased activity (slight to moderate), hunched back, and in one animal piloerection up to the time of death.
Surviving animals showed decreased activity in 2/4 animals up to Day 2, hunched back in 3/4 animals, up to Day 4, and piloerection in 3/4 animals up to Day 7.

Body weight:

Minimal body weight gains and slightly reduced body weights in 2/4 animals were observed in the surviving MDAC treated animals during the study. These changes were considered possible test-item related effects.

Gross pathology:

In found dead animals, diffuse/focal dark/red discoloration of the non-glandular/glandular mucosa, were considered to be test item-related. Diffuse dark/red discoloration of the non-collapsed lungs found, was regarded as agonal or post mortem.
In surviving animals, there was no evidence of macroscopic changes in animals dosed at 2000 mg/kg bw.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The acute oral LD50 value of MDAC was found to be equal or above 2000 mg/kg bw in female Crl:WI Wistar rats. According the CLP criteria, MDAC is not classified for acute oral exposure.