A mixture of: 3,3'-dicyclohexyl-1,1'-methylenebis(4,1-phenylene)diurea; 3-cyclohexyl-1-(4-(4-(3-octadecylureido)benzyl)phenyl)urea; 3,3'-dioctadecyl-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:

06 July 1990 - 03 August 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

1‐cyclohexyl‐3‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

EC Number:

604-940-8

Cas Number:

154099-21-5

Molecular formula:

C39 H62 N4 O2

IUPAC Name:

1‐cyclohexyl‐3‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Constituent 2

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:

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

Constituent 3

Reference substance name:

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

EC Number:

406-690-3

EC Name:

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

Cas Number:

43136-14-7

Molecular formula:

C51 H88 N4 O2

IUPAC Name:

3‐octadecyl‐1‐[4‐({4‐[(octadecylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Test material form:

solid

Details on test material:

Appearance: White yellowish solid
Storage conditions: At room temperature in the dark

The data on the individual constituents CHA/MDI/CHA (EC 406-370-3) and ODA/MDI/ODA (EC 406-690-3) for physicochemical, environmental fate, ecotoxicological and mammalian toxicological endpoints are very similar to the data available for the different compositions of the multi-constituent or represent the outer boundaries of the data on the multi-constituent. Hence, they confirm that the different compositions are expected to have similar properties for physicochemical, environmental fate, ecotoxicological and mammalian toxicological endpoints.

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
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: males : 151 to 199 g; females: 123 to 163 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: Seventeen 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): 17.5 - 23
- Humidity (%): 50 - 74
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 July 1990 to 02 August 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)

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)
- Animals fasted: 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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental findings noted among treated males included transient hunched posture accompanied by rough coat and alopecia, temporarily accompanied by scabs, in the neck region. Incidental findings noted among treated females included rales and intermittently chromodacryorrhoea.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Minor statistically significant difference arising between controls and animals receiving 1000 mg/kg/day were essentially similar as controls and considered to have arisen by chance and not to represent a change of biological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted between control and treated rats that could be related to treatment with the substance. Statistically significant changes noted between control and treated animals for sodium values (males treated at 1000 mg/kg bw/day), creatinine values (males treated at 200 mg/kg bw/day) and total protein values (females treated at 200 mg/kg bw/day), remained in the range normally expected for rats of this age and strain and were considered to be the result of normal biological variation and not of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

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 detected.

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/ODA 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.