Reaction mass of 3,6-Bis(3-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione, 3-(3-Chlorophenyl)-6-(4-chlorophenyl)-2,5-dihydro- pyrrolo[3,4-c]pyrrole-1,4-dione and 3,6-Bis(4-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione

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:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Chemical Substances Control Law 1987, Notification of Nov. 21 2003 by MHLW (No. 1121002), METI (No. 2) and ME (No. 031121002).

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

- Description: Orange-red powder
- Test substance storage: At room temperature in the dark
- Stability under storage conditions: Stable
- Expiry date: 15 August 2007

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Males: Control (Group 1): 177 ± 8.5g, 50 mg/kg (Group 2): 176 ± 7.0g, 150 mg/kg (Group 3): 179 ± 7.5g, 1000 mg/kg (Group 4): 177 ± 7.3g; Females: Control (Group 1): 151 ± 3.4g, 50 mg/kg (Group 2): 152 ± 3.0g, 150 mg/kg (Group 3): 151 ± 2.4g, 1000 mg/kg (Group 4): 150 ± 7.7g
- Housing: Group housing of 5 animals per sex in Macrolon plastic cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in MIll type; height 15 cm.) with sterilised sawdust as bedding material (Woody-Clean type 3/4, Tecnilab-BMI BV, Someren, The Netherlands) and paper as cage-enrichment (Enviro-dri, Tecnilab-BMI BV, Someren, The Netherlands). No cage-enrichment was provided during overnight activity monitoring.
- Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF-1, Lage, Germany).
- Water: Free access to tap water.
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 - 22.6
- Humidity (%): 35 - 94
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 20% (w/w) Ethylacetate in propylene glycol (specific density 1,009 g/mL)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for density of the vehicle. Storage conditions: At ambient temperature.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at NOTOX and on information from the sponsor.
- Amount of vehicle: 5 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Samples of formulations prepared after the in-life phase were analysed to check stability over 5 hours, homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations). These formulations were prepared similarly as those used during the in-life phase. The analytical method used was based on the results of a separate project for the development and validation of the analytical method (NOTOX project 449358).
- Results: Test substance formulations in 20% (w/w) ethylacetate in propylene glycol were noted as stable for at least 5 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 98 - 106% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 150, 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5/sex/dose/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of a 5-day dose range finding study
- Post-exposure recovery period in satellite groups: 14 days (control and 1000 mg/kg bw group)

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Mortality / Viability: At least twice daily.
DETAILED CLINICAL OBSERVATIONS: At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and on a weekly basis thereafter, this was also performed outside the home cage in a standard arena.
FUNCTIONAL OBSERVATIONS: During week 4 of treatment, the following tests were performed on all animals: hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP), motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
BODY WEIGHT: Treatment period: on days 1, 8, 15, 22 and 28, Recovery period: on days 1, 8 and 14.
FOOD CONSUMPTION: weekly
CLINICAL LABORATORY INVESTIGATIONS: Blood samples were collected from all animals under iso-flurane anaesthesia (Abbott Laboratories Ltd., Zwolle, The Netherlands) between 7.00 and 10.30 a.m, immediately prior to scheduled post mortem examination at the end of the treatment and recovery period. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One, Bad Haller, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (1.0 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). The following parameters were measured in blood samples: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time, Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate.

Sacrifice and pathology:

NECROPSY: All animals surviving to the end of the observation period were deeply anaesthetised using isoflurane vapour (Abbott Laboratories Ltd., Zwolle, The Netherlands) and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution (Klinipath, Duiven, The Netherlands): Adrenal glands; Aorta; Brain [cerebellum, mid-brain, cortex]; Caecum; Cervix; (Clitoral gland); Colon; Duodenum; Epididymides; (Eyes with optic nerve [if detectable] and Harderian gland); (Female mammary gland area); (Femur including joint); Heart; Ileum; Jejunum; Kidneys; (Larynx); (Lacrimal gland, exorbital); Liver; Lung, infused with formalin; Lymph nodes - mandibular, mesenteric; (Nasopharynx); Oesophagus; Ovaries; Pancreas; Peyer's patches [jejunum, ileum] if detectable; Pituitary gland; (Preputial gland); Prostate gland; Rectum; (Salivary glands - mandibular, sublingual); Sciatic nerve; (Seminal vesicles); (Skeletal muscle); (Skin); Spinal cord -cervical, midthoracic, lumbar; Spleen; Sternum with bone marrow; Stomach; Testes; Thymus; Thyroid including parathyroid [if detectable]; (Tongue); Trachea; Urinary bladder; Uterus; Vagina; All gross lesions. Tissues mentioned within brackets were not examined microscopically as there were no signs of toxicity or target organ involvement.
ORGAN WEIGHTS: The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, Epididymides, Kidneys, Ovaries, Testes, Brain, Heart, Liver, Spleen, Thymus
HISTOPATHOLOGY: All organ and tissue samples, as defined under Histopathology, were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Statistics:

The following statistical methods were used to analyse the data:
- 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 of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. No statistical analysis was performed on histopathology findings. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. 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

Details on results:

MORTALITY: No mortality occurred during the study period.
CLINICAL SIGNS: Red discolouration of faeces was observed in all group 2 (50 mg/kg bw) animals during week 4, and in all group 3 (150 mg/kg bw) and 4 (1000 mg/kg bw) animals from week 1 onwards, and had resolved at the end of recovery. Red staining of fur observed in high dose animals was related to exposure to the test substance and had disappeared at the end of recovery. These findings were considered toxicologically irrelevant. In control females hunched posture, breathing difficulties (i.e. laboured respiration, gasping and rales) and piloerection were observed. Rales were also observed in one group 4 male in week 4. These findings were considered to be related to technical handling procedures and were therefore considered to be of no toxicological importance. Other findings that were noted included alopecia (several body parts), chromodacryorrhoea, scabs and wounds (periorbital region and the neck) and salivation. These findings are commonly noted in rats of this age and strain and housed and treated under the conditions in this study and considered of no toxicological significance.
FUNCTIONAL OBSERVATIONS: NEUROBEHAVIOUR, OPHTHALMOSCOPIC EXAMINATION: Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment.
BODY WEIGHT AND WEIGHT GAIN: Body weights and body weight gain of treated animals remained in the same range as controls over the 4-week study period.
FOOD CONSUMPTION : Food consumption before or after allowance for body weight was similar between treated and control animals.
HAEMATOLOGY: Haematological parameters of treated rats were not affected by treatment. Minor statistically significant differences in prothrombin time (PT) values arising between controls and females receiving 150 mg/kg/day at the end of treatment, but not at the end of recovery, were considered to have arisen by chance and not to represent a change of biological significance.
CLINICAL BIOCHEMISTRY: The following statistically significant changes at end of treatment distinguished treated animals from controls: - decreased total bilirubin, urea and creatinine levels at the end of treatment in males at 1000 mg/kg/day; - decreased total bilirubin and inorganic phosphates males, and increased alanine aminotransferase (ALAT) levels in females at 150 mg/kg/day; - decreased inorganic phosphates in males at 50 mg/kg/day. In the absence of any corroborative findings in other parameters (macroscopy, microscopy, haematology), the magnitude of these observed effects and the fact that they had resolved by the end of recovery, they were considered of not toxicological relevance. In females at 1000 mg/kg/day, a slight but statistically significant increase in potassium level was found at the end of recovery. Based on the magnitude of the effect, the absence of this effect at the end of treatment and the variation within the group this finding was considered to be toxicologically irrelevant.
ORGAN WEIGHTS: Absolute and relative organ weights of treated animals were considered to be similar to those of control animals. Statistically significant changes in relative kidney weights of males dosed at 150 mg/kg/day were considered not to be a sign of toxicity based on the magnitude of the effect, absence of this effect in animals treated at 1000 mg/kg/day and absence of corroborative findings in macroscopy and microscopy.
GROSS PATHOLOGY: Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. Reddish contents of the gastrointestinal tract were considered to be related to the test substance colour, but to be of no toxicological importance. Incidental findings among control and treated animals at end of treatment and at end of recovery included pelvic dilation and foci in kidneys, foci in epididymides, smaller seminal vesicles, enlarged spleen, red discolouration of the thymus, enlarged mandibular lymph nodes, diaphragmatic hernia of the liver and cysts and fluid in the uterus. These findings are occasionally seen among rats used in these types of studies. In the absence of correlated microscopic findings these were considered changes of no toxicological significance.
HISTOPATHOLOGY: There were no microscopic findings recorded which could be attributed to treatment with the test substance. All microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

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 adverse effect at the limit dose

Target system / organ toxicity

Critical effects observed:

no