Benzonitrile, 3,3'-(2,3,5,6-tetrahydro-3,6-dioxopyrrolo[3,4-c]pyrrole-1,4-diyl)bis-

Endpoint summary

Description of key information

The test item was administered orally by gavage to groups of 10 male and 10 female Sprague-Dawley rats at dose levels of 0 mg/kg body weight/day (vehicle CMC), 15 mg/kg bw/d, 150 mg/kg bw/d and 1000 mg/kg bw/d for 28 days (OECD guideline 407, EU method B.7, GLP conditions). Clinical examinations, clinical pathology, histopathology and gross pathology did not reveal any finding in treated animals. The no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in both sexes.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-02-23 to 1993-08-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant Guideline study (OECD Guideline 407 adopted 1981). Main deviations from OECD 407 adopted 2008: no FOB or neurobehavioural examinations, less organs weighed, few organs histopathologically examined.

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted Sept. 19, 1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent, England
- Age at study initiation: 28 ± 1 days (at delivery)
- Weight at study initiation: 64 to 91 g
- Fasting period before study: no
- Housing: in groups of five according to sex in metal cages with wire mesh floors
- Diet: Special Diet Services Rat and Mouse Maintenance Diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 22°C
- Humidity: 32-62%
- Air changes: approximately 19 per hour
- Photoperiod: 12 hours artificial light (0700 - 1900 hours) in each 24-hour period

IN-LIFE DATES: From: 1993-03-09 To: 1993-04-01 or 1993-04-15 (recovery groups)

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
A 10% w/v suspension of test item was prepared freshly each day with 1 % w/v aqueous methylcellulose until a smooth paste was formed.

VEHICLE
- Concentration in vehicle: 1 %
- Amount of vehicle: 10 mL / kg bw / day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical results indicate that the doses were accurately formulated during the toxicity study. The results also confirm that the formulations were homogeneous and stable from the time of preparation to completion of dosing.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily, seven days per week

Remarks:

Doses / Concentrations:
0, 15, 150, 1000 mg / kg bw / day
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females (plus 5 males and 5 females per satellite group: vehicle and high dose)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: rat acute toxicity data and a 7-day preliminary oral toxicity study [HRC Report No.: CBG 583/930621]
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice a day
- Observations: checked for signs of ill health, behavioural changes or toxicosis.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to dosing on Day 1 (Week 0) and subsequentiy at weekly intervals

FOOD CONSUMPTION:
- Food consumption for each group of animals determined and mean daily diet consumption calculated as g food/kg body weight/week: Yes (from group mean)

WATER CONSUMPTION: No (visual appraisal only)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
I. on day 30 (all surviving non-recovery group animals)
II. on day 44 (all surviving animals of recovery groups)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: I) 40, i.e surviving animals except recovery groups; II) all surviving animals of recovery groups
- Parameters examined: packed cell volume, haemoglobin, red blood cell count, MCHC, MCV, platelet count, total white cell count, differential white blood cell count: neutrophils, lymphocytes, eosinophils, basophils, monocytes. Morphological abnormalities: poly- / hypochromasia, anisocytosis, Rouleaux formation.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see HAEMATOLOGY
- Animals fasted: Yes
- How many animals: see HAEMATOLOGY
- Parameters examined: glucose, total proteins, albumin, globulin, total globulin minus albumin, albumin/globulin ratio, urea nitrogen, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, cholesterol, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: night before blood collection
- Metabolism cages used for collection of urine: not specified
- Animals fasted: Yes
- Parameters examined: volume, pH, specific gravity, proteins, total reducing substances, glucose, ketones, bile pigments, urobilinogen, haem pigments, epithelial cells, polymorphonuclear and mononuclear leucocytes, erythrocytes, organisms, renal tubule casts, sperm, orther abnormal constituents

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (all animals)
adrenals, aorta, brain (medullary, cerebellar, and cortical sections), caecum, colon, duodenum, eyes (Davidson's fluid as fixative), femur (for bone and marrow sections) with joint, Harderian gland, head (to preserve nasal cavity, paranasal sinuses, oral cavity, nasopharynx, middle ear, teeth, lachrymal gland and Zymbal's gland), heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes (cervical and, mesenteric), mammary glands, oesophagus, ovaries, pancreas, pharynx, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical level), spleen, stomach, testes (including epididymis), thymus (where present), thyroid (with parathyroid), tongue, trachea, urinary bladder, uterus, vagina, any macroscopically abnormal tissue

ORGAN WEIGHT (all animals):
adrenals, brain, kidneys, liver, spleen, ovaries, testes (with epididymides)

HISTOPATHOLOGY: Yes
- preserved: all listed under GROSS PATHOLOGY
- Histopathologically examined (vehicle and high dose group, except satellite groups):
adrenals, heart, kidneys, liver, spleen, testes (including epididymis), abnormal tissue

Statistics:

Significant differences between control animals and those treated with the test substance were expressed at the 5% (* P<0.05) or 1% (** P<0.01) level.

Bodyweight gains, organ weight and clinical pathology data: Fisher's exact test followed by Mantel's test for a trend in proportions, otherwise Bartlett's test for heterogeneity of variance between treatments. Logarithmic transformation if significant heterogeneity was found at the 1 % level. If distribution homoscedastic, ANOVA followed by Williams' test for dose-related response. If distribution heteroscedastic, Kruskal-Wallis analysis followed by Shirley's test.

Covariate analysis: if data correlations at the 10% level of significance, data adjustments (with final bodyweight as covariate).

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment-related clinical signs were noted and there were no mortalities.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant differences in bodyweight or bodyweight gains between treated and control rats.

FOOD CONSUMPTION
There was no obvious difference in food consumption between treated and control rats.

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
There were no differences between treated and control rats that were considered to be test item related.

CLINICAL CHEMISTRY
There were no differences between treated and control rats that were considered to be test item related.

URINALYSIS
There were no differences between treated and control rats that were considered to be test item related.

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
There were no differences between treated and control rats that were considered to be test item related.

GROSS PATHOLOGY
No tteatment-related changes were observed in the terminal or recovery groups.

HISTOPATHOLOGY
No treatment-related changes were observed in the tissues examined at termination. The minor/incidental changes observed are considered to be of no toxicological importance.

HISTORICAL CONTROL DATA: not specified

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Lack of test item related toxic effects at all dosages applied, incl. limit dose.

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Procedure and observations

The test item was administered orally by gavage to groups of 10 male and 10 female Sprague-Dawley rats at dose levels of 0 mg/kg body weight/day (vehicle CMC), 15 mg/kg bw/d, 150 mg/kg bw/d and 1000 mg/kg bw/d for 28 days. A detailed clinical observation was performed in all animals. Body weights and food consumption were determined in F0 animals. Clinicochemical and hematological examinations performed in all animals towards the end of the administration period. All animals were assessed by gross pathology; weights of selected organs were recorded and a histopathological examination was performed.

Clinical examinations, clinical pathology, histopathology and gross pathology did not reveal any finding in treated animals.

Discussion

Thus, under the conditions of this four-week toxicity study the oral administration by gavage to male and female rats did not reveal signs of toxicity. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000 mg/kg bw/d in both sexes.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.