N,N''-(methylenedi-4,1-phenylene)bis[N'-octyl]urea

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:

sept 2002-febr 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Neurological investigations were based on OECD 424 and EPA 799, 9620-62-158

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-060-3

EC Name:

-

Cas Number:

122886-55-9

Molecular formula:

Hill formula: C31H48N4O2 CAS formula: C31H48N4O2

IUPAC Name:

N,N’’-(methylenedi-4,1-phenylene)bis(N’-octylurea)

Constituent 2

Reference substance name:

N-{4-[(4-{[(octadecylamino)carbonyl]amino}phenyl)methyl]phenyl}-N’-octylurea

Cas Number:

117328-86-6

Molecular formula:

Hill formula: C41H68N4O2 CAS formula: C41H68N4O2

IUPAC Name:

N-{4-[(4-{[(octadecylamino)carbonyl]amino}phenyl)methyl]phenyl}-N’-octylurea

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:

Hill formula: C51H88N4O2 CAS formula: C51H88N4O2

IUPAC Name:

N,N’’-(methylenedi-4,1-phenylene)bis(N’-octadecylurea)

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): KY-EU
- Appearance: white powder
- Storage condition of test material: in darkness at room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France
- Age at study initiation:6 weeks
- Weight at study initiation: males: 199 g (range 164-214 g); females: 166 g ( range 156-175 g)
- Fasting period before study: no
- Housing: individually in suspended wire-mesh cages.
- Diet: ad libitum A04 C pelleted maintenance diet, distributed weekly
- Water: free access to filtered tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 November 2002 To: 11 December 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/w) aqueous methylcellulose solution

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was ground to a fine powder, using a mortar and pestle, and then mixed with the required quantity of vehicle in order to achieve the concentrations of 30, 90 and 200 mg/mL. The test item dosage forms (suspension in vehicle) were prepared on a weekly basis and were stored at 4°C prior to use.

VEHICLE
- Lot/batch no.: 81K0299

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogenity of the dosage forms was tested before the treatment period (lowest concentration and highest concentration) and analyzed by HPLC with UV detection. Each dosage form prepared for homogenity was analysed for stability after 0, 4, 5 and 7 days storage at 4°C (HPLC with UV detection). The concentration of samples taken from each dosage form (including the control) prepared for use in weeks 1 and 4 was determined.

Duration of treatment / exposure:

29 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150, 450, 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: The dose levels were selected on the basis of the results of a 7-day range-finding toxicity study by oral route performed in the same species (CIT/Study No. 24561 TSR), in which no signs of toxicity were observed at the dose levels of 150, 450 and 1000 mg/kg bw/day. Consequently, the same dose-levels were used for the main study.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the beginning of the treatment period and once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: at least once before allocation of the animals to groups, on the first day of treatment, and then once a week until the end of the study.

FOOD CONSUMPTION:
- recorded once a week, recorded over 7-day period during the study. Food consumption was calculated per animal and per day.

WATER CONSUMPTION: not determined

OPHTHALMOSCOPIC EXAMINATION: not determined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period.
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: Yes, for at least 14 hours
- How many animals: all
- Parameters checked: according to guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period.
- Animals fasted: Yes, for at least 14 hours
- How many animals: all
- Parameters checked: according to guideline

URINALYSIS: not detremined

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: at the end of the treatment period.
- Dose groups that were examined: all
- Battery of functions tested: FOB

ORGAN WEIGHTS
- organ weight at the end of the treatment (aorta, brain, epididimydes, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroids with parathyroids)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, according to guideline
HISTOPATHOLOGY: Yes, according to guideline

Statistics:

Statistical analysis was performed on body weight, food consumption, hematology, blood chemistry and organ weight data (number of animals/sex >3 in each group). Normality of data distribution was tested with Kolmogorov-Lilliefors test, followed by Bartlett test, Fisher test, Dunn test, Student test, Dunett test or Mann-Whitney/Wilcoxon test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

slighly lower body weight gain observed in high dose males

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

changes in plasma Na and Cl (males) and K (females)

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

slightly increased kidney weight in high dose males.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

BODY WEIGHT AND WEIGHT GAIN: body weight of high dose males were slightly lower at days 22 and 29 of the treatment period, and slightly lower bodyweight gain (13%) in high dose males.

CLINICAL CHEMISTRY: increased levels of sodium and chloride in mid and high dose males, and increased levels of potassium in high dose females. No historical control data were included. Relative kidney weight was slightly increased (11%) in high dose males. No historical control data were included. Based on the present available information, the effects on plasma ions are considered toxicologically relevant in the high dose group.

ORGAN WEIGHT: Relative kidney weight was slightly increased (11%) in high dose males.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Higher levels of plasma Na and Cl and increased relative kidney weight in high dose males. In high dose animals slightly lower body weight gain was observed in males, whereas females showed higher plasma potassium levels.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In an oral 28-day repeated dose toxicity study with rats, at 1000 mg/kg bw/day, males showed slightly lower body weight gain, slightly increased plasma levels of sodium and chloride, and higher relative kidney weight. High dose females showed slightly increased levels of plasma potassium. Based on the effects observed in high dose animals, the NOAEL for systemic effects in this study is set at 450 mg/kg bw/day.

Executive summary:

KY-EU was given by oral administration (gavage) to male and female rats at dose levels of 0, 150, 450 and 1000 mg/kg bw/day for 29 days. The study was performed according to EU guidelines for repeated dose toxicity testing. In high dose males a slightly lower body weight gain was observed, together with slightly increased levels of plasma sodium and chloride and increased relative kidney weight. In high dose females, plasma levels of potassium were slightly increased. Based on the effects observed in high dose animals, the NOAEL for systemic effects in this repeated dose toxicity study is set at 450 mg/kg bw/day.