Diallyl hexahydrophthalate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 June 2018 to 03 April 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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

These deviations are considered to have no impact on the outcome of the study or on interpretation of the results.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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
- Source and lot/batch No.of test material: Osaka Soda; Bx 40201
- Expiration date of the lot/batch: 26 January 2019
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature, protected from light
- Stability under test conditions: no data
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for Dose Range Finding study (study code: 17/277-220PE [4]).

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Commercial laboratory animal supplier.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 13 weeks
- Weight at study initiation: Males: 429 – 494 g, females: 255 – 313 g
- Fasting period before study:
- Housing: Type II polycarbonate cage
- Diet: ssniff® SM R/M "Autoclavable complete diet for rats and mice ad libitum
- Water: ad libitum
- Acclimation period: 12 days

DETAILS OF FOOD AND WATER QUALITY: 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: 883 29966, expiry date: 31 October 2018 and batch number: 840 33675, expiry date: 31 January 2019), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 – 25.0 °C (target range 22 ± 3 °C)
- Humidity (%): 34 – 69 % (target range 30-70 %)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 03 July 2018 To: 07 August 2018

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The dosing solutions were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight.

Vehicle:

polyethylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Formulations were prepared up to 4 days before use and were kept at room temperature until use.
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on the results of a short solubility test performed at the Test Facility, PEG 400 was selected as vehicle for this study in agreement with the Sponsor, based on the formulation and analytical trials. The same vehicle was used in the Dose Range Finding (DRF) study [4].
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): A0378559
- Purity:

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Mid dose

Doses / concentrations 4

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Study Director in consultation with the Sponsor based on available acute oral toxicity data (LD50 between 300 and 2000 mg/kg bw in rats [3]) and information from a Dose Range Finding study in the rat (Citoxlab study code 17/277-220PE [4]) where test item related mortality was seen at 1000 mg/kg bw and slight toxicity was seen at 300 mg/kg bw/day.
- Rationale for animal assignment (if not random):
- Fasting period before blood sampling for clinical biochemistry: yes
- Rationale for selecting satellite groups: n/a
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
- Other:

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Not specified
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (pentobarbital) d
- Animals fasted: Yes
- How many animals: 5 male, 5 female
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Other examinations:

The following retained organs were also processed and microscopically examined at request of the Sponsor:

• liver and lymphoid tissues (BALT, mandibular and mesenteric lymph nodes, spleen, thymus, Peyer’s patch) of all male and female animals in all dose groups,

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow).

Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item related clinical signs were observed in the Low and Mid dose groups during the study. The incidental piloerection in one Mid dose male (#3004) on Day 17 was considered as not test item-related.

In the found dead (#4006) or pre-terminally euthanized (#4002, 4007, 4009) High dose males, liquid faeces (4/4), hunched back (4/4), piloerection (4/4), slight or moderate decreased activity (3/4), red discharge at the nose or snout (3/4), noisy respiration (1/4) and red faeces (1/4) were observed. Similar findings were seen in one animal (#4003) from Day 5, but it completely recovered from Day 20. Besides these, hunched back, piloerection or noisy respiration were occasionally seen in the other surviving animals for 1-2 days. Three High dose males were completely symptom-free during the study.

In the found dead (#4503, 4504, 4505, 4512) High dose females, slightly decreased activity (1/4), hunched back (1/4) and piloerection (3/4) were seen before death. One animal (#4504) was found dead without any previous clinical symptoms. In the surviving animals, piloerection (7/8), hunched back (4/8), soft or liquid faeces (2/8) or slightly decreased activity (1/4) were recorded occasionally for 1-2 days. One High dose female (#4510) was completely symptom-free during the study.

Mortality:

mortality observed, treatment-related

Description (incidence):

One High dose male (#4006) was found dead on Day 17 and three High dose males (#4002, 4007, 4009) were pre-terminally euthanized between Day 14 and 20. This was around the mating period, therefore three animals had to be replaced with other males to mate the females and one male was preterminally euthanized after successful mating.

In the High dose female group, four animals (#4503, 4504, 4505, 4512) were found dead around the end of the gestation period or in the beginning of lactation period:

• 4503: Animal was found dead on PPD3 (Day 42). (The single live born pup also died.)
• 4504: Animal was found dead on GD22 (Day 39). Pregnancy was confirmed at necropsy.
• 4505: Animal was found dead on GD21 (Day 38). Pregnancy was confirmed at necropsy.
• 4512: Animal was found dead on GD22 (Day 40). Pregnancy was confirmed at necropsy.

The hepatic effects were considered as cause of death of these animals. There was no mortality in the Control, Low and Mid dose groups during the study.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly higher aspartate aminotransferase activity (AST/GOT) (p<0.01) and alanine aminotransferase activity (ALT/GPT) (p<0.05) were measured in the male High dose group, reflecting hepatic damage seen at histology. Several other parameters linked to liver or kidney function also changed in individual male High dose animals, but without attaining statistical significance in the High dose group mean values, most probably because of the high individual variabilities in the data (total bilirubin, urea, ALKP, GGT, etc.). The High dose females had normal values. No test item-related findings were seen in the Low or Mid dose groups.

Statistical significances recorded in the bile acid and potassium concentrations were considered to be incidental, there was no clear relationship with dose and all recorded values were within the historical control ranges. These differences were considered not to reflect an effect of the test item.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, increased liver and kidney organ weights were recorded in the male and female High dose groups, the liver of the Mid dose males and the kidney of the Mid dose females were also slightly increased. The livers were discoloured and/or enlarged in the High dose group. At histopathology, massive hepatocellular necrosis, multifocal bile duct hyperplasia and periportal fibrosis were seen in the High dose livers. Other histological changes seen only in early death animals were considered to be secondary to liver necrosis. There was no test item-related macroscopic or microscopic finding in the Low and Mid dose groups, the small differences in organ weights in these groups were not considered to reflect any adverse changes.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

kidney

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

The NOAEL for Systemic toxicity for the adults was considered to be 100 mg/kg bw/day.