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

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 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 424 (Neurotoxicity Study in Rodents)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA, Guideline 799, 9620-62-158, 1997

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:

C31H48N4O2

IUPAC Name:

3-octyl-1-[4-({4-[(octylcarbamoyl)amino]phenyl}methyl)phenyl]urea

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): KY-UN
- Stability under test conditions: not stated
- Storage condition of test material: at room temperature and protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genet-Saint-Isle, France
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: 196 g to 227 g (males) and 172 g to 191 g (females)
- Fasting period before study: no
- Housing: individually housed in suspended wire-mesh cages, with autoclaved sawdust in metal tray under each cage
- Diet: ad libitum A04 C pelleted maintenance diet, batch No. 30905 (SAFE, Villemoisson, Epinay-sur-Orge, France)
- Water: ad libitum tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 October 2003 To: 21 November 2003

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose solution

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle.
The test item was ground to fine powder using a mortar and pestle, suspended in the vehicle to concentrations of 30, 90 and 200 mg/mL and homogenized by magnetic stirrer.
The test item dosage forms were prepared on a weekly basis and stored at +4 °C and protected from light prior to use.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg/day
- Batch no. (if required): 112K0988

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity:
Before the treatment period, two dosage forms were prepared, under conditions representative of those of the study: a dosage form at 1 mg/mL and a dosage form at 200 mg/mL. From each dosage form, duplicate samples were taken at three different levels of the container (top, middle, bottom) and analyzed for concentration of the test item to evaluate the homogeneity on the day of preparation.

Stability:
Each dosage form prepared for homogeneity analysis was sampled after 0 (just after preparation), 4 and 9 days' storage at +4°C. Each sample taken was analyzed as soon as possible after sampling. In each case, the mean result of the homogeneity analysis was taken as the initial value for the stability test.

Concentration:
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. 26491 TSR). In this study, there were no mortality, clinical signs or effects on body weight and food consumption at any dose-level (150, 450 and 1000 mg/kg/day). No treatment-related necropsy findings were noted.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: 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: 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 AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: part of the FOB
- Dose groups that were examined: all

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

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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment period
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity (see also under 'other examinations'

Additional: rectal temperature (Day 26)

Sacrifice and pathology:

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

Other examinations:

All animals were evaluated once at the end of the treatment period, including a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity.
The animals were randomized. All animals were observed in the cage, in the hand and in the standard arena.
The following parameters were assessed and graded:
"touch escape" or ease of removal from the cage, in the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis), in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.

Then, the following parameter measurements, reflexes and responses were recorded:
touch response,
forelimb grip strength,
pupillary reflex,
visual stimulus response,
auditory startle reflex,
tail pinch response,
righting reflex,
landing foot splay,
rectal temperature.
Finally, motor activity of all animals was measured once by automated infra-red sensor equipment (not validated) over a 15-minute period.

Statistics:

see attached figure

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):

mid and high dose females: lower mean bw gain first week and higher mean bw gain third week

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

lower mean food consumption first week in 450 and 1000 mg/kg bw/day groups.

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, treatment-related

Description (incidence and severity):

Higher cholesterol level was noted in females given 450 and 1000 mg/kg/day, together with higher levels of triglycerides in these groups

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

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; chromodacryorrhea was observed in 1/5 and 3/5 males given 450 and 1000 mg/kg/day, respectively, during the second part of the treatment period. Although this sign only appeared in intermediate- and high-dose groups, it was seen in some ·individuals, not associated with other clinical signs and it is a clinical observation commonly reported in untreated laboratory rats; consequently, it is considered not test substance related.

BODY WEIGHT AND WEIGHT GAIN: overall mean body weight gain was similar in control and treated animals; however, lower mean body weight gain was noted in females given 450 and 1000 mg/kg/day during the first week of treatment (12 and 8 grams in 450 and 1000 groups, respectively, vs 23 grams in controls) whereas moderately higher mean body weight gain was recorded during the third week of treatment in the same female groups (20 and 25 grams, respectively in 450 and 1000 groups, respectively, vs 9 grams in controls). These effects on body weight gain are considered test substance related. However, as the observed effect on body weight gain was transient and final mean body weight gain was not affected compared to controls, this effect is considered not toxicologically relevant.

FOOD CONSUMPTION: statistically significant slightly lower mean food consumption was noted in females given 1000 mg/kg/day during the first week of treatment (15.5 vs. 19.9 g/animal/day in controls). Although these effects on food consumption are considered test substance related, this effect was restricted to 1 week in the dosing period and overall mean food consumption was similar in control and treated animals, and the lower mean food consumption had no effect on overall mean body weigh gain.

HAEMATOLOGY: no test substance related differences between control and treated animals. The observed higher mean prothrombine time in males given 150 mg/kg bw/day is considered not toxicologal relevant, as this effect was not observed at higher dose levels.

CLINICAL CHEMISTRY: higher mean cholesterol level in females given 450 and 1000 mglkg/day (2. 7 and 2.5 mmol/L,
respectively, vs. 1.9 mmol/L in controls, together with higher levels of triglycerides (0.62 vs 0.38 mmol/L in dosed groups versus controls); As these efffects were not observed in males, are not statistically significant at the high dose-level, and absence of a dose-relationship, these effects are considered not toxicologically relevant. The observed slightly lower levels of protein in high dose males and calcium in mid dose females are considered not toxicologically relevant.

NEUROBEHAVIOUR: FOB: There were no treatment-related changes in autonomic, physiological or neurotoxicological parameters. Motor activity was unaffected by treatment with the test item.

ORGAN WEIGHTS: no treatment related effects on organ weights

GROSS PATHOLOGY: no treatment related necropsy findings were observed

HISTOPATHOLOGY: NON-NEOPLASTIC no test substance related changes have been observed

Additonal: no effect on rectal temperature (Day 26)

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No toxicologically relevant adverse effects up to and including the highest dose tested.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

homogeneity:

mean values were between 0 and -4% of nominal values, with a coefficicent variation (100 x SD/mean) of 4 and 10%

stability:

deviation on day 9 from initial value on day 0 was 5 to 9%

deviations from nominal values in administered dosage forms were 1 to 4% in week 1 and 0 to 6% in week 4

Accuracy was 97 to 102% (with precision, CV% of 5 -10%)

Applicant's summary and conclusion

Conclusions:

In a 28-day repeated dose oral toxicity study with rats, KY-UN did not result in toxicologically relevant adverse effects. Based on these observations, the NOAEL for KY-UN is 1000 mg/kg bw/day.

Executive summary:

Sprague-Dawley rats were administered KY-UN daily by gavage at dose levels of 0, 150, 450 or 1000 mg/kg bw/day for 4 weeks according to EC guideline B7 with neurotoxicological investigation according to OECD 424. Formulation analysis confirmed that formulations were prepaed accurately and homogenously, and were stable under the conditions used in the study.

Transient effects on food consumption and on mean body weight gain was observed in mid and high dose females, but had no effect on overall mean food consumtion and on overall mean body weight gain. Slightly higher mean cholesterol level in females given 450 and 1000 mglkg/day and slightly higher levels of triglycerides were not observed in males. In the absence of clear statistical significancy and absence of a dose-relationship, these effects are considered not toxicologically relevant.

Based on the absence of toxicologically relevant adverse effects in this study, the NOAEL for KY-UN is 1000 mg/kg bw/day.