Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1987

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:

May 12, 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 19, 1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

401-540-3

EC Name:

-

Cas Number:

84632-65-5

Molecular formula:

C18H10Cl2N2O2

IUPAC Name:

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

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM Kleintierfarm Madoerin AG, Fuellinsdorf/Switzerland
- Age at study initiation: 7-9 meeks
- Weight at study initiation: males: 147 - 173 g, females: 157 - 181 g
- Fasting period before study:
- Housing: Individually
- Diet (e.g. ad libitum): Pelleted standard Kliba no. 343 Batch 44/86 rat maintenance diet ('Kliba', Klingentalmuehle AG, Kaiseraugst, Switzerland), at
libitum.
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was weighed into a glass beaker on a tared Mettler PK 300 balance and the vehicle, polyethylene glycole (PEG 400), was added. The mixture was prepared daily prior to administration using a homogenizer and kept stable during application with a magnetic stirrer.
Concentrations, homogeneity, and stability were checked by RCC analytical laboratories for all preparations.

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg body weight

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based upon the results in acute toxicity studies, which did not indicate any toxicity at limit test doses.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for mortality were recorded twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Signs of toxicity were assessed twice daily. A description of any abnormalities were recorded and the subsequent progress was monitored.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded weekly during the acclimation and treatment period using an on-line
electronic recording system.

FOOD CONSUMPTION: Yes
- The food consumption was recorded once during the acclimation period and weekly thereafter using an online electronic recording system.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed at termination of treatment.
Ten minutes after the application of a mydriatic solution (Dispersa AG, Winterthur / Switzerland) the cornea, lens, anterior chamber, vitreous body and ocular fundus of both eyes were examined under dimed light using a Heine Miroflex 2 Ophthalmoscope (Eisenhut Vet. AG, Allschwil / Switzerland)
- Dose groups that were examined: Ophthalmoscopic examinations were performed on all animals.

HAEMATOLOGY: Yes, from the retro-orbital plexus
- Time schedule for collection of blood: after 4 weeks
- Anaesthetic used for blood collection: Yes, under light ether anesthesia
- Animals fasted: Yes, the animals were fasted for 18 hours before blood sampling but water was provided.
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 4 weeks
- Animals fasted: Yes, the animals were fasted for 18 hours before blood sampling but water was provided.
- How many animals: all animals
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: after 4 weeks, over an 18-hour period into a specimen
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals were necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed
by experienced prosectors supervised by a pathologist. All animals surviving to the end of the observation period and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination after 4 weeks.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution.
Adrenal glands, Heart, Kidneys, Liver, Spleen, Gross lesions.
Where there was a compound related alteration in an organ, this organ was histologically examined in all animals of all dose groups.

Organ weights: The following organ weights were taken from all animals necropsied at termination of treatment: Adrenal glands, Kidneys,
Liver, Testes.

HISTOPATHOLOGY: Yes
Slides of all tissues collected at terminal sacrifice from the animals of the control and high-dose groups as well as all animals which died spontaneously were examined by a pathologist. Treatment-related morphologic changes in the organs of any highdose animal required histological evaluation of the same organs in lower dose groups until a no-effect-level was determined. All abnormalities were described and included in the report.

HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology were processed, embedded and cut at a thickness of 2-4 micrometers and stained with hematoxylin and eosin. Special stains were used at the discretion of the pathologist.

Other examinations:

No other examinations were performed.

Statistics:

The following statistical methods were used to analyze the body weights, food consumption and organ weights:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- If 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.
- The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
- Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
- Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Starting with day 21 until termination of treatment all rats of the high dose group (1000 mg/kg bw) showed red discolored extremities. In addition red
discolored feces were observed in the same animals betmeen day 18 and termination of the test. No other symptoms related to test article treatment were observed.

Treatment-unrelated signs and symptoms:
Prior to death, one female animal of the group with 300 mg/kg showed moderate sedation (days 17-19), rales (days 18-19) and moderate ruffled fur (days 17-19).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Due to a possible intubation error, two female animals, one of the control group and one of the group 300 mg/kg died spontaneously at day 17 respectively 20 of test.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

The assessment of hematological data indicated no changes of toxicological significance at termination of the treatment.

Treatment-unrelated: All statistical differences in the results of the hematology parameters were considered to be incidental and of normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The assessment of biochemical data indicated no changes of toxicological significance at termination of the treatment.

Treatment-unrelated: All statistical differences in the results of the clinical biochemistryparameters were considered to be incidental and of normal biological variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The assessment of urinalysis data indicated no changes of toxicological significance at termination of the treatment.

Treatment-unrelated: All statistical differences in the results of the urinalysis parameters were considered to be incidental and of normal biological variation.

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

HISTOPATHOLOGY:
- No treatment related microscopic findings were recorded. From the tissues examined no cause of death could be established for the two rats which died. The various spontaneous microscopic findings recorded are within the normal range observed in this age and strain of rat. They can be attributed to subclinical infections, spontaneous congenital abnormalities or physiological status.
The nephroblastoma which mas recorded in one male of the 300 mg/kg body weight group is a seldom encountered malignant kidney tumor which occurs spontaneously with a Iow incidence in many rat strains and is not related to treatment.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

System:

other: none

Applicant's summary and conclusion

Conclusions:

Based upon the results obtained in the study, the NOAEL for the 28-day repeated dose oral toxicity test of the substance was considered to be higher than 1000 mg/kg/day for male and female rats when administered orally by gavage.

Executive summary:

In a study according to OECD Test Guideline 407 under GLP conditions, Wistar rats were treated with three doses of the substance (100, 300 and 1000 mg/kg bw). The dose were applied by oral (gavage) daily for four weeks. Besides some clinical findings caused by the colour of the substance, no treatment-related effects were observed. Therefore, the NOAEL was considered to be higher than 1000 mg/kg/day for male and female rats.