3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea

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:

14 June 1990 - 12 July 1990

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

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:

12 May 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea

EC Number:

406-370-3

EC Name:

3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea

Cas Number:

58890-25-8

Molecular formula:

C27 H36 N4 O2

IUPAC Name:

1-cyclohexyl-3-[4-({4-[(cyclohexylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Test material form:

solid

Details on test material:

- Name of test material (as cited in study report): MDI/CHA
- Physical state: white yellowish solid
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males: 179 to 198 g; females: 120 to 156 g
- Fasting period before study: Overnight
- Housing: Animals were housed 5 to a cage (same sex) in stainless steel suspended cages with wire mesh floors.
- Diet: Free access to standard pelleted laboratory animal diet (Kliba, Klingentalmuehle AG, 4303, Kaiseraugst, Switzerland).
- Water: Free access to tap water.
- Acclimation period: Sixteen days (7 days pre- and 10 days post randomisation).

DETAILS OF FOOD AND WATER QUALITY:
Diet: Each batch was analysed for contaminants and results were examined and archived.
Water: Results of chemical and contaminants analyses were examined and archived quarterly.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 25
- Humidity (%): 50 - 74
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 June 1990 To: 12 July 1990

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Frequency: Once daily, approximately the same time each day, 7 days per week.
Dose volume: 5 mL/kg body weight

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was weighed into a glass flask on an analytical balance and the vehicle (w/w) added.
The test article formulation was daily prepared immediately prior to dosing. Homogeneity during
treatment was maintained using a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Due to analytical limitations, only samples of the high dose concentrations prepared after completion of the treatment period were analysed to check accuracy of preparations. Formulation procedures used were identical to those used during the study.

Duration of treatment / exposure:

28 days.

Frequency of treatment:

Once daily, approximately the same time each day, 7 days per week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

200 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:

Five

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Based on data from previous studies in rats.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily. Severity of observations were graded.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly and on the day preceding termination, prior to overnight fasting.

FOOD CONSUMPTION:
- Weekly

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Both eyes were examined following instillation of atropine sulphate solution before commencement of treatment and during the last week of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately prior to post mortem examination, between 8.00 and 10.00 a.m.
- Anaesthetic used for blood collection: Yes (ether) / No / Not specified
- Animals fasted: Yes, Yes, overnight before blood sampling, but water was provided.
- How many animals: all rats/sex/group
- Parameters checked: According to test guidelines.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Immediately prior to post mortem examination, between 8.00 and 10.00 a.m.
- Animals fasted: Yes, overnight before blood sampling, but water was provided.
- How many animals: all rats/sex/group
- Parameters checked: According to test guidelines.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

NECROPSY:
All animals surviving to the end of the observation period (day 29) were deeply anaesthetised by in traperitoneal injection of sodium pentobarbital and subsequently exsanguinated. All necropsies were performed by experienced prosectors and descriptions of all macroscopic abnormalities recorded.

ORGAN WEIGHTS:
The following organ weights (and terminal body weight) were recorded at termination: Adrenal glands, Heart, Kidneys, Liver, Spleen and Testes.

HISTOTECHNOLOGY:
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin. All slides for histopathological examination were dispatched to RCC (UK) Ltd., Hereford, England.

HISTOPATHOLOGY:
Slides of adrenals, heart, kidneys, liver and spleen, collected at termination from all animals of the
control and high dose group as well as from all gross lesions of all animals were examined by a
pathologist.

Statistics:

The following statistical methods were used to analyse the body weight, organ weights and clinical laboratory data:
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 of 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. All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
The exact Fisher-test was applied to the ophthalmoscopic examination data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One male receiving 200 mg/kg/day showed chromodacryorrhoea over the study period and rales on one occosion during week 3. In the absence of corroborative findings in the opposite sex and in the absence of a treatment related distribution these findings were considered not to represent a sign of toxicity. Alopecia was noted over 10 days or more among 3/5 females receiving 1000 mg/kg/day. However, as alopecia is commonly noted among rats of this age and strain, it was considered not to have arisen as an effect of treatment.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Minor statistically significant changes noted between control and treated animals in inorganic phosphate and calcium values, were considered to have occurred fortuitously and not to be of toxicological significance.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weights and relative organ weights of treated animals were indistinguishable from those of control animals. Statistically significantly decreased relative spleen weights in comparison with control weights among females receiving 1000 mg/kg/day were within normal biological variation and considered not to be of toxicological significance.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

Analysis of dose preparations: The accuracy of preparation testing revealed that the highest nominal
concentration analyzed was in agreement with the concentrations prepared.

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No treatment-related changes observed.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with the substance, dosed via oral gavage at dose levels 50, 200 or 1000 mg /kg bw/day resulted in a NOEL for males and females of 1000 mg/kg bw/day based on absence of adverse effects at 1000 mg/kg bw/day, the highest dose tested.

Executive summary:

In this subacute 28-day toxicity study performed according to OECD 407 guideline and GLP principles, MDI/CHA was administered daily by gavage to SPF-bred Wistar rats to 0, 50, 200 and 1000 mg/kg bw/day.
There were no changes in clinical appearance, body weights, food consumption, ophthalmoscopic examination, clinical laboratory investigations, macroscopic examination, organ weights and microscopic examination that were considered to be an effect of treatment. Based on the absence of
adverse effects at 1000 mg/kg bw/day, the NOEL for males and females in this study is at least 1000 mg/kg bw/day, the highest dose tested.