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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 July 1990 - 07 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
12 May 1981
Deviations:
no
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

Constituent 1
Reference substance name:
A mixture (1:2:1) of: bis(N-cyclohexyl-N'-phenyleneureido)methylene; bis(N-octadecyl-N'-phenyleneureido)methylene; bis(N-dicyclohexyl-N'-phenyleneureido)methylene
EC Number:
406-550-1
EC Name:
A mixture (1:2:1) of: bis(N-cyclohexyl-N'-phenyleneureido)methylene; bis(N-octadecyl-N'-phenyleneureido)methylene; bis(N-dicyclohexyl-N'-phenyleneureido)methylene
IUPAC Name:
Reaction mass (1:2:1) of bis(N-cyclohexyl-N'-phenyleneureido)methylene and bis(N-octadecyl-N'-phenyleneureido)methylene and bis(N-dicyclohexyl-N'-phenyleneureido)methylene
Test material form:
solid: particulate/powder
Details on test material:
Description: White yellowish solid
Test substance storage: At room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species: Wistar rat, outbred, SPF quality.
Rationale: Recognised by international guidelines as a recommended test system (e.g. EPA, DECO, EEC).
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 : 149 to 181 g; females: 107 to 143 g
- Fasting period before study: No data.
- 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: Fourteen days (7 days pre- and 7 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): 21 ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 July 1990 to 06 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.
Actual concentrations of preparations were in agreement with the treatment levels as per protocol.
Duration of treatment / exposure:
28 days.
Frequency of treatment:
Once daily, approximately the same time each day, 7 days per week.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
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: A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day) to provide a basis for selection of dose levels for the 28-day study.
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 intraperitoneal 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):
Alopecia (which is commonly noted in rats of this age and strain) was noted in 1 control male, 2 males receiving 200 mg/kg/day, 1 female receiving 50 mg/kg/day and 3 females receiving 1000 mg/kg/day. With the exception of 1 female receiving 1000 mg/kg/day (animal 37), which showed alopecia generally over the whole study period, alopecia was a transient or incidental phenomenon.
Hunched posture was noted in 1 male receiving 1000 mg/kg/day on day 5 and in 1 female receiving 1000 mg/kg/day on days 5 to B. Given the incidental nature, this finding was considered not to be of toxicological significance.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the 4 week study period. Compared to control males, low body weights and body weight gain was noted among males receiving 200 mg/kg/day over the study period. However, in the absence of a treatmentrelated distribution, these differences were considered to have occurred fortuitously and not to be of toxicological significance.
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
Description (incidence and severity):
Determination of the red cell distribution width, white blood cell counts and differential white blood cell counts of a few control males were not measured due to an error in calibration. This, resulted in a lack of confidence in the white blood cell value for control males. However, comparison of white blood cell values between the treated groups and comparison of other haematology parameters between treated and control animals showed, in all cases, values in the range normally expected for rats of this age and strain. Statistically significantly decreased mean corpuscular haemoglobin among males receiving 1000 mg/kg/day was essentially similar to the control value and not of toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no differences noted between control and treated rats that could be related to treatment with the substance. Statistically significantly increased alanine aminotransferase values in treated females when compared to control females were considered the consequence of control values at the lower limits for this age and strain of rats and therefore not to be of toxicological significance. Other minor statistically significant changes noted between control and treated males were considered to be the result of normal biological variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Organ weights and relative organ weights of treated animals were indistinguishable from those of control animals. Heart weights, which were noted to be statistically significant different from control weights among males receiving 200 mg/kg/day and after allowance for body weight among females receiving 200 or 1000 mg/kg/day were considered to have occurred by chance and 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:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Microscopic examination of adrenals, heart, kidneys, liver and spleen exhibited no alterations that were not commonly seen in rats of this age and strain. All changes were within normal biological variation and not of toxicological significance.
Details on results:
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.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related changes detected.
Key result
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 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 NOAEL 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/DCHA 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 NOAEL for males and females in this study is at least 1000 mg/kg bw/day, the highest dose tested.