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

Endpoint summary

Description of key information

Based on the results of a sub-acute study (oral; 28 days exposure) performed according to OECD/EC guidelines and GLP principles, a No Observed Adverse Effect Level (NOAEL) for KY-EU of 450 mg/kg was established. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

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

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.

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.

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.

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.

Critical effects observed:

not specified

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February 2017 - 26 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

September 21, 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: National Institute of Environmental Research (NIER) Notification 2016-27

Version / remarks:

December 19, 2016

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Remarks:

SFP

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio Co, Ltd. 699-13, Mokdong-ri, Buk-myeon, Gapyeong-gun, Gyeonggi-do, Korea
- At initiation of dosing males and females were 5 weeks old, males weighed between 112.7 and 130.1 g and females weighed between 93.9 and 107.6 g.
- Fasting period before study: no
- Housing: Stainless steel wire cage, 3-6 animals per cage.
- Diet: Pellet diet for rat [Rodent Diet 20 5053 (Labdiet, USA)] given ad libitum
- Water: R/O water ad libitum
- Acclimation period: All animals were monitored for healthy condition, acclimated and quarantined by observation weight and clinical sign for 8 - 9 days and healthy animals were selected for this study.

DETAILS OF FOOD AND WATER QUALITY:
The absence of contamination in feed was confirmed with periodical analysis results report of manufacture. The water was periodically analyzed in accordance with SOP of Korea Testing & Research Institute.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 23
- Humidity (%): 49.8 to 61.9
- Air changes (per hr): 10 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 16 February 2017 To: 23 June 2017

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Methylcellulose was prepared by distilled water(water for injection) in 0.5 % concentration. After weighing KY-EU, it was suspended by using vehicle (20 mg/mL, 40 mg/mL, 80 mg/mL).
It was not indicated in the study report if formulations were prepared daily/weekly/other.
No information was provided in the study report if adjustment was made for specific gravity of the vehicle. In additon, no information was avialable on if correction was made for the purity/composition of the test item.
The administration volume was calculated (mL/kg) based on the body weight measured before starting of administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of prepared test suspensions was confirmed to be stable by the stability test (KTR Study number : TBK-001208-2016). Concentration of doses at the first preparation was measured, and it was confirmed by sampling from the top, middle and bottom from each dosing solution. Homogeneity was assessed on coefficient of variation(C.V (%)) of dosing solutions (20, 40 and 80 mg/mL) was 19.70 %, 39.04 % and 76.50 % and percents (%) were 98.50 %, 97.60 % and 95.63 % and percent of nominal concentration (%). C.V (%) of dosing solutions (20, 40 and 80 mg/mL) was 19.61 %, 40.12 % and 78.25 % and percents (%) were 98.05 %, 100.30 % and 97.81 %, respectively. These met the acceptance criteria (C.V(%) ≤20.0 %), content (%) : 100 ± 20 % of nominal concentration.

Duration of treatment / exposure:

90 days

Frequency of treatment:

The test substance was administered once/day, 7 days/week

Dose / conc.:

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

Remarks:

It is not clear if a correction was made for specific gravity of the vehicle and purity of the substance

Dose / conc.:

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

Remarks:

It is not clear if a correction was made for specific gravity of the vehicle and purity of the substance

Dose / conc.:

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

Remarks:

It is not clear if a correction was made for specific gravity of the vehicle and purity of the substance

No. of animals per sex per dose:

10 animals/sex/dose for treatment groups
5 animals/sex for control and high dose recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the results of 4 week toxicity study (Study facility : CIT BP 563-27005 Evreux-France, Study No. 24562 TSR), the high dose was 800 mg/kg and other dose was as follows; 400 mg/kg (middle dose), 200 mg/kg (low dose). Also, additional recovery animals in the vehicle and the high dose group was included for observation of reversibility or persistence of any toxic effects after treatment period.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule:

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes / No / Not specified
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / Not specified
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / Not specified
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure, and once a week there after

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: once weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: once weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Twice: before first exposure and last week before necropsy
- Dose groups that were examined: control and high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (16-24 hours)
- How many animals: all
- Parameters checked: in accordance with OECD 408 (1998)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes (16-24 hours)
- How many animals: all
- Parameters checked: in accordance with OECD 408 (1998)

URINALYSIS: Yes
- Time schedule for collection of urine: last week before necropsy
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked: Chemistry (pH, Specific Gravity, Protein, Ketone body, Bilirubin, urobilinogen, Nitrite, Blood, Leukocyte), Volume , Urinary sediments and color.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 12-13
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / functional observations (grip strength / motor activity)

IMMUNOLOGY: No
- Time schedule for examinations: -
- How many animals: -
- Dose groups that were examined: -
- Parameters checked: -

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, according to Guidelines
HISTOPATHOLOGY: Yes, according to Guoidelines

Statistics:

The body weight, food consumption, water consumption, grip strength, motor activity, organ weight and hematological/biochemical data were analyzed using SPSS (Ver 19.0) program. After homogeneity of variances using Levene's test, one way ANOVA analysis was performed to evaluate the significant differences. If the variance was homogeneous and the significance of difference was confirmed, Scheffe's multiple comparison test was performed as a post-hoc test. If the variance was not homogeneous, the data was analyzed by procedure of Dunnett's T3 test. The recovery group was analyzed using T test.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal of the high dose group was found dead. Besides, no compound related mortality was observed at any dose in male and female groups.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males, a significant decrease (p <0.05) was observed in week 2, 4 and 9 in the low dose group and in week 4 in the middle dose group. In females, a significant decrease (p <0.05) was observed in week 3 in the low dose group. In the male recovery group, a significant decrease (p <0.05) was observed in week 17 in the high dose group. In absence of a dose response, the finidngs were considered non-treatment related.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

In low dose males, a significant decrease was observed in week 1. In high dose females, a significant decrease was observed in week 12 and 13. In absence of a dose response, the findings were considered non-treatment related.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

In the high dose male recovery group, basophils(%) was significantly increased (p <0.05). As no change in basophils was seen in any of the treatment groups, this finding was not considered toxicologically relevant.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the high dose male recovery group, basophils(%) was significantly increased (p <0.05). As no change in basophils was seen in any of the treatment groups, this finding was not considered toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In high dose males and low dose females, blood urea nitrogen was significantly increased (p <0.05). In the male high dose recovery group, total protein, triglyceride and calcium were significantly decreased (p <0.05).

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no statistical differences in both sexes.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In the male high dose recovery group, heart weight was significantly increased. However, in absence of any findings in other groups and in absence of histopathological changes, these findings were considered not treatment related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Canibalism was observed in one dead animal. No further macroscopic findings were reported.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Inflammatory cell foci in liver, tubular basophilia in kidney, cortical vacuolation in adrenal gland and interstitial inflammatory cell infiltration in prostate gland were observed in male high dose group.
Tubular basophilia and cortical scar in kidney, hemangiectasis in adrenal gland and cyst in ovary were observed in female high dose group. Inflammatory cell foci in liver, tubular basophilia in kidney and cortical vacuolation in adrenal gland were observed in male high dose recovery group.
The inflammatory cell foci observed in liver in high dose and recovery group of both sexes was consdiered to be equivalent to background incidences observed in repeated dose toxicity studies; the grade of lesions was minimal and the frequency for the lesions was only minimal different from control. Also the tubular basophilia and cortical scar observed in kidney, cortical vacuolation and hemangiectasis observed in adrenal gland, interstitial inflammatory cell infiltration observed in the prostate gland and cyst in ovary were also considered equivalent to background lesions in repeated dose toxicity studies, based on grade and frequency of observations. The neprophathy observed in 1 male of the high dose group is known as protein-overload nephropathy or chronic progressive nephrosis and as degenerative lesions common on older rats. The lesion observed is considered to be at an initial stage and is considered to be incidental.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

In absence of observation of adverse effects in a 90-day oral toxicity study in rats, the NOAEL of KY-EU was set at >= 800 mg/kg bw/day.

Executive summary:

Males and female SD rats were given KY-EU via gavage for 90 days at dose levels of 0, 200, 400 or 800 mg/kg bw/day.

Formulation analysis showed that the formulations were prepared accurately and homogeneously.

No treatment related effecs on mortality, clinical signs, body weight, food consumption, water consumption, functional observation battery, urine analysis, ophthalmoscopy, hematology, clinical biochemistry, marcoscopy, organ weights and histopathology were observed.

In absence of adverse findings the NOAEL was set at >= 800 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

450 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was performed according to OECD/EC guidelines and GLP principles with klimisch code 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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.

Since the effects were not found to be significantly toxic, the test substance is not classified according to Regulation (EC) No 1272/2008.

Males and female SD rats were given KY-EU via gavage for 90 days at dose levels of 0, 200, 400 or 800 mg/kg bw/day. Formulation analysis showed that the formulations were prepared accurately and homogeneously. No treatment related effecs on mortality, clinical signs, body weight, food consumption, water consumption, functional observation battery, urine analysis, ophthalmoscopy, hematology, clinical biochemistry, marcoscopy, organ weights and histopathology were observed. In absence of adverse findings the NOAEL was set at >= 800 mg/kg bw/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The lowest NOAEL was selected as the most relevant endpoint.

For a future update an assessment should be made in order to set an overall NOAEL based on all data available. As a worst case assumption the lowest NOAEL was selected and the starting point for the derivation of the DNELs.

Justification for classification or non-classification

Based on the available data, KY-EU is not classified for repeated oral dose toxicity according to Regulation (EC) No 1272/2008.