Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis[4-(1,1-dimethylethyl)phenyl]-2,5-dihydro-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-12-21 - 1994-07-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study (OECD TG 407) performed under GLP with deficiencies to current guidelines: Wet weighing of coagulating glands, epididymides, prostate and seminal vesicles was not performed. No FOB is part of the study (a subchronic study is not available). However, wet weights of thyroid and ovaries were determined and histopathological endpoints for the detection of endocrine effects were included in this reliable study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted on 12th May 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Chemical Substance Law (1987) according to the notification of December 9, 1986 by EA, Environmental Agency (No. 700); MHW, Ministry of Health and Welfare (No. 1039) and MITI, Ministry of International Trade and Industry (No. 1014).

Deviations:

not specified

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

416-250-2

EC Name:

-

Cas Number:

84632-59-7

Molecular formula:

C26 H28 N2 O2

IUPAC Name:

3,6-bis(4-tert-butylphenyl)-1H,2H,4H,5H-pyrrolo[3,4-c]pyrrole-1,4-dione

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd, Füllinsdorf, Switzerland
- Age at study initiation: ca. 6 weeks old
- Body weight range at acclimatization: Males: 142.5 – 157.4 g, females: 115.3 – 129.1 g
- Housing: Individually in Makrolon type-3 cages with autoclaved standard softwood bedding
- Diet: Pelleted standard Kliba 343 rat maintenance diet (Klingentalmuehle AG, Kaiseraugst, Switzerland), ad libitum
- Water: Community tap-water from Füllinsdorf, ad libitum
- Acclimation period: 6 days under laboratory conditions after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 10 - 15 changes/hour
- Photoperiod: 12 hours fluorescent light/12 hours dark, music during the light period

IN-LIFE DATES:
- From: 1993-12-29 To: 1994-02-09

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures (w/v) were prepared using a homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle: standard vehicle for studies of this type
- Concentration in vehicle: 5, 20 or 100 mg/mL
- Amount of vehicle: dose volume of 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose formulations of the pretest and of the test were analysed to check homogeneity, concentration and stability.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

- 5 animals/sex/dose level (main study)
- additional 5 animals/sex at 0 and 1000 mg/kg bw/d (recovery assessment)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based upon the results of a non-GLP 5-day dose range-finding study (RCC Study Number 360641) in which the test item was administered orally by gavage to rats.
- Post exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily
- Mortality/Viability: Once daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- On day 1 of the pretest, on days 1, 8, 15, 22 and 28 of the treatment period and on days 1, 8 and 14 of the recovery period.
- Terminal body weights were recorded at necropsy.

FOOD CONSUMPTION: Yes
- Once during the acclimatization period and weekly thereafter.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: at 4 weeks (all animals) and at 6 weeks (all recovery animals)
- Heine BETA 200 Ophthalmoscope (Eisenhut Vet. AG, 4123 Allschwil, Switzerland)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 weeks and 6 weeks
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, for approximately 18 hours before blood sampling
- How many animals: all animals
- Parameters examined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Reticulocyte count, Reticulocyte fluorescence ratios, Nucleated erythrocytes (normoblasts), Heinz bodies, Methemoglobin, Total leukocyte count, Differential leukocyte count, Red blood cell morphology, Thromboplastin time (=prothrombin time), Activated partial thromboplastin time.
- Other: Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a microhematocrit glass capillary tube.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks and 6 weeks
- Metabolism cages used for collection of urine: yes
- Parameters examined: Glucose, Urea, Creatinine, Uric acid, Bilirubin (total), Cholesterol (total), Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl transferase, Calcium, Phosphorus, Sodium, Potassium, Chloride, Albumin, Protein (total), Globulin, Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks and 6 weeks
- Animals fasted: Yes, urine was collected during the 18-hour fasting period into a specimen vial
- Parameters examined: Volume (18-hour), Specific gravity, Osmolality, Color, Appearance, pH, Protein, Glucose, Ketone, Bilirubin, Blood, Nitrite, Urobilinogen, Urine sediment.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, after 4 weeks and after 6 weeks
All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone (NARCOREN) and killed by exsanguination.

The following organ weights were recorded on the scheduled dates of necropsy: adrenals, brain, heart, kidneys, liver, ovaries, pituitary, spleen, testes, thyroid including parathyroid gland. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.

All organ and tissue samples, as defined under Histopathology, were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY: Yes
Slides of all organs and tissues marked with an asteriks which were collected at scheduled sacrifice from the animals of control and high-dose groups were examined; samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution: adrenals*, aorta, bone (sternum, femur), bone marrow (sternum, femur), brain, cecum, colon, duodenum, epididymides, esophagus, eyes with optic nerve, harderian gland, femur including joint, heart*, ileum, jejunum, kidneys*, larynx, lacrimal gland, exorbital, liver*, lung infused with formalin*, lymph nodes, mandibular, mesenteric, mammary gland area, nasal cavity, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary gland, (mandibular, sublingual), seminal vesicles, sciatic nerve, skeletal muscle, skin, spinal cord, (cervical, midthoracic, lumbar), spleen*, stomach*, testes*, thymus, thyroid incl. parathyroid gland, tongue, trachea, urinary bladder infused with formalin, uterus; vagina and gross lesions.

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights and their all ratios and clinical laboratory data:
When the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex. The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. The Fisher's exact test was applied to the ophthalmoscopy data.

Results and discussion

Results of examinations

Details on results:

Mortality/Viability and Clinical signs:
No unscheduled deaths or clinical signs of reaction to treatment were noted in all groups. At 1000 mg/kg bw/d, red faeces were noted in all animals. This finding was caused due to coloration by the test item.

Body weights:
In all dose groups (50, 200, 1000 mg/kg bw/d), the body weights of the animals were not affected by the treatment with the test item compared with controls.

Food consumption:
Up to and including the highest dose group of 1000 mg/kg bw/d, no adverse effects on the food consumption of the animals were noted.

Haematology / Clinical biochemistry /Urinalysis:
The assessment of hematological, clinical biochemical and urinalysis data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free recovery period.

GROSS PATHOLOGY / HISTOPATHOLOGY:
- Organ weights: No test item related changes in organ weights were noted.
- Macroscopic and microscopic findings:
At macroscopic- as well as at histopathologic examination, there were no test item abnormal findings of toxicological relevance. At terminal necropsy of the animals sacrificed at the end of the treatment period, the red discoloration of the gastrointestinal tract was considered to be due to passive coloration by the test item. This finding was confirmed at microscopic examination of the gastrointestinal tract which revealed the presence of colored particles of the test item.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test-item related adverse effects up to and including the high dose level of 1000 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

ANALYSIS OF DOSE FORMULATIONS

 

Acclimatization/Pretest:

The mean concentrations of the homogeneity samples taken during acclimatization/pretest were found to be 103.0 %, 92.6 % and 100.8 % of the nominal concentrations for dose groups 2 (5 mg/mL). 3 (20 mg/mL) and 4 (100 mg/mL), respectively. The homogeneity varied in the range from -7 % to +5 % of the mean concentrations.

Administration/Test:

The mean concentrations of the homogeneity samples taken during administration/treatment were found to be 102.5 %, 102.8 % and 105.5 % of the nominal concentrations for dose groups 2 (5 mg/mL). 3 (20 mg/mL) and 4 (100 mg/mL), respectively. The homogeneity varied in the range from -7 % to +10 % of the mean concentrations.

The test item was found to be stable in corn oil at room temperature for at least two hours.