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

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:

1995-02-01 to 1995-02-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study. Not allrecommended organs were weighed and not all tissues/organs examined histopathologically. No FOR was performed (no 90-day study available.)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

92/69/EEC, 31-Jul-1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted May 12, 1981

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Revised Japanese Chemical Substance Law (1987) according to the notification of Dec. 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:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd., Wolferstrasse 4, 4414 Fuellinsdorf/Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 137.4 - 152.8 g, Females: 113.3 - 126.7 g
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages with autoclaved standard softwood bedding ('Lignocel', Schill AG, CH-4132 Muttenz).
- Diet: Pelleted standard Kliba no. 343, Batch nos. 82/94 and 84/94 rat maintenance diet ('Kliba', Klingentalmuehle AG, CH-4303 Kaiseraugst) ad libitum.
- Water: Community tap-water from Fullinsdorf was available ad libitum.
- Acclimation period: From 25-JAN-1995 to 31-JAN-1995 under laboratory conditions, after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 46-66
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1995-01-25 To: 1995-03-15

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was weighed into a glass beaker on a tared Mettler PM 480 balance and the vehicle was added. The mixture (w/v) was prepared using a homogenizer. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer.
- Rate of preparation: Daily prior to each application

VEHICLE
- Justification for use and choice of vehicle: Standard vehicle for studies of this type, recommended in the guidelines.
- Amount of vehicle: 10 mL/kg bw per treatment day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability of the test article/vehicle mixtures were determined in samples taken during acclimatization and during week 3 of the treatment. The analyses were performed according to a photometrical method.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days per week for a total of 28 days.

Doses / concentrations

Remarks:

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

No. of animals per sex per dose:

Dose groups:
5 males and 5 females in each group.
Recovery groups:
5 males and 5 females (0 and 1000 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on acute oral test
- Post-exposure recovery period in satellite groups: yes (0 and 1000 mg/kg bw/day), 14 days

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Observations for mortality/viability and clinical signs of toxicity were recorded once daily.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weights were recorded weekly and twice during week 4 of treatment and week 2 of the recovery period.

FOOD CONSUMPTION:
The food consumption was recorded once during the acclimatization period and weekly thereafter.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
at 4 weeks (on all animals) and at 6 weeks (on all recovery animals)
- Examinations:
A description of any abnormality was recorded. 10 - 90 minutes after the application of a mydriatic solution (Dispersa AG, CH-8442 Hettlingen) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimmed light using a Heine BETA 200 Ophthalmoscope (Eisenhut Vet. AG, CH-4123 Allschwil).

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
at 4 weeks: all animals
at 6 weeks: all recovery animals
- Anaesthetic used for blood collection: Yes (light ether anesthesia)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked:
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 cell morphology
Coagulation: Thromboplastin time
Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
at 4 weeks: all animals
at 6 weeks: all recovery animals
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked:
Glucose
Urea
Creatinine
Uric acid
Bilirubin, total
Cholesterol, total
Triglycerides
Phospholipids
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Creatine kinase
Alkaline phosphatase
Gamma-glutamyltransferase
Calcium
Phosphorus
Sodium
Potassium
Chloride
Albumin
Protein, total
Globulin
Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected during the 18-hours fasting period into a specimen vial.
- Metabolism cages used for collection of urine: yes (model K. Ehret & Co., Emmendingen, Germany)
- Animals fasted: Yes
- Parameters checked:
Volume (18-hour)
Specific gravity
Osmolairity
Color
Appearance
pH
Protein
Glucose
Ketone
Bilirubin
Blood
Urobilinogen
Urine Sediment

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

NECROPSY
after 4 weeks - all animals
after 6 weeks - recovery group
All animals were weighed and necropsied and descriptions of all macroscopic abnormalities were recorded. At the end of the treatment or recovery period the animals were anesthetized by intraperitoneal injection of NARCOREN (Rhone Merieux GmbH, D-88471 Laupheim) at a dose of 2.0 mL/kg bw (equivalent to 320 mg sodium pentobarbitone/kg body weight), weighed and sacrificed by exsanguination.

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Tissues collected: Adrenals; aorta; bone (sternum, femur); bone marrow (sternum, femur); brain; cecum; colon; duodenum; epididymides; esophagus; eyes with optic nerve and Harderian gland; femur including joint; heart; ileum; jejunum; kidneys; larynx; lacrimal gland, exorbital; liver; lung; 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.

Tissues examined: Slides of adrenals, heart, kidneys, liver, lungs, spleen, stomach and testes collected at terminal sacrifice from the animals of the control and high-dose groups were examined by a pathologist. The same applied to the testes from males of 50 and 200 mg/kg bw/day treatment groups and to all gross lesions from all animals.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
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 the organ to brain weight ratios were determined.

Other examinations:

none

Statistics:

The following statistical methods were used to analyze the body weights, food consumption, organ weights and 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:

CLINICAL SIGNS AND MORTALITY
All animals survived their assigned study period.
No clinical signs of toxicity were observed in any group. The only finding of note was a discoloration of the feces which occurred in animals of all treated groups during the treatment period.

BODY WEIGHT AND WEIGHT GAIN
There was no effect on body weight in any group. The body weight gain was similar in all groups and no statistically significant differences occurred during the study.

FOOD CONSUMPTION
The ranges for individual food consumption were similar in treated and control rats. There were no treatment-related significant differences in mean absolute and relative food consumption noted for any treated group when compared with the vehicle control group.

OPHTHALMOSCOPIC EXAMINATION
There were no treatment-related ophthalmoscopic findings noted for any group.

HAEMATOLOGY
The assessment of hematology data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free period.

CLINICAL CHEMISTRY
The assessment of clinical biochemistry data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free period.

URINALYSIS
The assessment of urinalysis data indicated no changes of toxicological significance at termination of the treatment nor at the end of the treatment-free period.

ORGAN WEIGHTS
No treatment-related changes of organ weights, organ to body weight and organ to brain weight ratios were observed during the study.

GROSS PATHOLOGY
The reddish discoloration of the mucosa of various segments of the gastrointestinal tract of animals primarily of group 3 (200 mg/kg bw/day) and 4 (1000 mg/kg bw/day) was considered to be due to deposits of the test article.

HISTOPATHOLOGY: NON-NEOPLASTIC
Minimal to slight seminiferous tubular atrophy was noted in the testes of three rats of the high dose group (1000 mg/kg bw/day) at the end of the main study period. This finding was no longer in evidence following the recovery period.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

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/day

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

ANALYSIS OF DOSE FORMULATIONS:

Adequate homogenicity of the test item in the dose formulations and adequate accuracy of preparations were confirmed by the analytical results.