N,N''-(methylenedi-4,1-phenylene)bis[N'-cyclohexylurea];N-(4-[[4-[[(phenylamino)carbonyl]amino]phenylmethyl]phenyl]-N'-cyclohexylurea;reaction mass of: N,N''-(methylenedi-4,1-phenylene)bis[N'-phenylurea]

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:

October-November 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study has been performed in 1994 according to OECD and/or EC guidelines and according to GLP principles. Consequently, data requirements according to updated guidelines are not included.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

KY-RB

IUPAC Name:

KY-RB

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): KY-RB
- Physical state: powder (white)
- Analytical purity: 93%
- 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
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 135-154 grams (males), 105-124 grams (females)
- Fasting period before study: no
- Housing: group housing of 5 animals/sex/cage in stainless steel suspended cages with wire mesh floors
- Diet: free access to standard pelleted laboratory animal diet (Kliba 343, Switzerland)
- Water: free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1 November 1994 To: 28 November 1994

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared daily immediately prior to dosing. Adjustment was made for specific gravity of the vehicle.


VEHICLE
- Justification for use and choice of vehicle (if other than water): substance is stable in vehicle for at least 4 hours
- Concentration in vehicle: 0, 10, 40 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For analysis of formulations, samples of week 2 formulations were analysed to check stability, homogeneity (highest and lowest concentration) and accuracy of preparations (all concentrations).

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on absence of toxicity in a 5-day dose range finding study (3 rats/sex/dose group at dose levels of 50, 200 and 1000 mg/kg bw/day, NOTOX Project number 131366).

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

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

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: week 4
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (o/n)
- How many animals: all
- Parameters checked according to Guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to post mortem examination
- Animals fasted: Yes (o/n)
- How many animals: all
- Parameters checked according to Guideline.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (according to Guideline)
HISTOPATHOLOGY: Yes (according to Guideline)

Statistics:

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 for each sex.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow normal distribution.
The exact Fisher-test was applied to the ophthalmoscopic examination data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

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):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: no mortality was observed; no treatment related clinical signs were observed. Incidental observations of no toxicologal significance included salivation, rales, a kink in the tail , alopecia and scabs

BODY WEIGHT AND WEIGHT GAIN: no differences between controls and treated animals over the study period

FOOD CONSUMPTION: no differences in food consumption before or after allowance for body weight between treated and control animals

OPHTHALMOSCOPIC EXAMINATION: no treatment related adverse effects

HAEMATOLOGY: no treatment related adverse effects; observed decrease in PT and PTT in treated females is the result of a slightly higher control value, and considered of no toxicological relevance.

CLINICAL CHEMISTRY: no treatment related adverse effects; observed statistically significant values are considered to result from slightly higher control values or to have occurred by chance.

ORGAN WEIGHTS: no treatment related difference between treated and control animals

GROSS PATHOLOGY: no treatment related observations; the reddened thymus observed in one treated male is within the normal range of spontaneous findings for rats of this strain and age.

HISTOPATHOLOGY: NON-NEOPLASTIC: no treatment related microscopic findings; the small number of changes observed were within the range commonly seen for rats of this strain and age

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 substance related adverse effects observed up to and including 1000 mg/kg bw/day, the highest dose tested.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analysis of dose preparations:

Test substance formulations in propylene glycol were stable for at least 4 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 90% to 102% of nominal, which is considered to represent an acceptable level of accuracy for formulations of this type.

Applicant's summary and conclusion

Conclusions:

In an oral 28-day repeated dose toxicity study with rats, the NOAEL for KY-RB is ≥ 1000 mg/kg bw/day, based on the absence of substance related toxicologically relevant effects up to and including the highest dose tested.

Executive summary:

Sprague-Dawley rats were administered KY-RB daily by gavage at dose levels of 0, 50, 200 or 1000 mg/kg bw/day for 4 weeks according to OECD 407. Formulation analysis confirmed that formulations were prepaed accurately and homogenously, and were stable under the conditions used in the study.

No substance related toxicologically relevant effects were observed up to and including the highest dose tested. Based on the absence of adverse effects, the NOAEL for KY-RB in this study is ≥1000 mg/kg bw/day.