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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 12, 2012 ~ April 04, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was performed according to OECD guidelines and GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test) (1995)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-760-8

EC Name:

-

Cas Number:

122886-55-9

Molecular formula:

C31H48N4O2 (component: N,N’’-(methylenedi-4,1-phenylene)bis(N’-octylurea)) C41H68N4O2 (component: N-(4-((4-(((octadecylamino)carbonyl)amino)phenyl)methyl)phenyl)-N’-octylurea) C51H88N4O2 (component: N,N’’-(methylenedi-4,1-phenylene)bis(N’-octadecylurea))

IUPAC Name:

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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): KY-EU
- Physical state: White powder

Test animals

Species:

rat

Strain:

other: Crl:CD(SD) rats, Specific Pathogen Free (SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ORIENT BIO Co., Ltd. (143-1, Sangdaewon-Dong, Jungwon-Gu, Seongnam-Si, Gyeonggi-Do, Korea)
- Age at study initiation: male 9 weeks , female 9 weeks
- Weight at study initiation: Males: 326.9 - 394.0 g; Females: 208.5 - 266.7 g
- Fasting period before study: no data available
- Housing: Administration : Two animals per cage were housed in stainless steel cages; Mating period : One female and one male were housed in stainless steel cages; Gestation and lactation period : Maternal rats and fetuses were housed individually in polycarbonate cage
- Diet (e.g. ad libitum): free access to pellet diet for rat (Cargill Agri Purina, Inc., 56-4, Soryong-dong, Gunsan-si, Jeonbuk, Korea)
- Water (e.g. ad libitum): free access to filtered and UV irradiated water
- Acclimation period: 19 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-22.7
- Humidity (%): 49.0-54.8
- Air changes (per hr): 10 ∼ 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11-12-2012 To: 23-01-2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

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

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was administered as a suspension in the vehicle. The test substance was grounded to a fine powder, mixed with the vehicle to achieve concentrations of 50, 100 and 200 mg/mL. This was prepared on a weekly basis and stored at 4 degrees Celsius, as this was confirmed to be a stable solution. Dosing volume was 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity was evaluated for the formulation of the first day and fifth week before administration. Sample solutions were prepared by sampling from upper, middle and bottom in bottle with KY-EU formulation. Homogeneity was assessed by coefficient of variation (C.V (%)) and content (%). Homogeneity and content (%) of KY-EU in 0.5 % methylcellulose were analyzed in accordance with validated analytical method (KTR/Study No. : KG-2012-374).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: One male and one female were cohoused in stainless steel cages.
- M/F ratio per cage: 1/1 during mating
- Mating period: 25-DEC-2012 to 08-JAN-2013
- Proof of pregnancy: females were examined for the presence of a vaginal plug or sperm, when found this was referred to as day 0 of pregnancy

Duration of treatment / exposure:

Male rats were exposed at each dose levels for 2 weeks prior to mating, during mating period and 2 weeks post mating period (at least 28 or more days).
Female rats were exposed at each dose levels from 2 weeks prior to mating, to day 3 of lactation including the mating and gestation period.

Frequency of treatment:

Once daily, 7 days a week

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

The dose-levels were selected on the basis of the results of the pilot study by 5-week oral route (gavage) in Sprague-Dawley rats (KTR/Study No. : KG-2012-465). KY-EU was given orally by gavage to rats at 500 and 1000 mg/kg bw/day. There was a significant body weight loss in the 1000 mg/kg B.W. group in female rats. Therefore, 1000 mg/kg bw/day was used as the high dose and doses of 250 and 500 mg/kg bw/day were selected as the medium and low dose, respectively, using a scaling factor of 2.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: checked once a day during the non-treatment period and twice a day during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on animal arrival and allocation. Males and females were measured on the first day of dosing, weekly after and at termination. During the gestation and lactation periods, females were measured as follows:
1) During the gestation periods : Days 0, 3, 6, 9, 12, 15, 18 and 20 of gestation
2) During the lactation periods : Days 0 and 4 of lactation

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Rats were supplied with a certain amount of diet on days of body weight measurement and food consumption was recorded for the animals on the following days. During the mating periods, food consumption was not measured. In addition, on day 4 of lactation a diet was supplied on day 3 of lactation and food consumption was recorded on the following day.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: as it is an OECD421 study, females and pups were sacrificed at day 4 of lactation
- Organs examined: Absolute organ weight were measured and their relative organ weight (organ-to-body weight ratios) were calculated from the terminal body weight for the following organs of all live animals when they were sacrificed. 10 % neutral buffered formalin was used for fixation and preservation, except testes, epididymides and eyeball. Bouin's fixative was used for testes and epididymides and Davidson's solution was used for eyeball.
- Histopathology examination: Gross finding organ, testes, epididymides, ovaries and uterus were embedded in paraffin, sectioned, stained with hematoxylin and eosin (H&E) and examined microscopically in the vehicle control and 1000 mg/kg bw/day groups.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Other: A vaginal smear was taken daily for each female during the pre-mating period (14 days) and regularity and length of the cycle was examined.

Fetal examinations:

Bodyweight of neonates were determined at day 0 and day 4 after parturition
- External examinations: Yes

Statistics:

Program : SPSS ver.19 (SPSS Korea Data Solution Co., Ltd.)
Quantitative continuous data (such as bw, bw gain, food consumption and organ weight): One-way analysis of variance (ANOVA), when statistical significant data were analyzed by the multiple comparison procedure of Dunnett or Scheffe.
Other data (number of corpora lutea, total implantations, live and dead fetuses and fetal alterations): Kruskal-Wallis non parametric ANOVA, followed by the Mann-Whitney U-test where appropriate

Indices:

Mating index (%) = (No. of animals with successful copulation/No. of mated animals) × 100
Fertility index (%) = (No. of impregnating animals/No. of animals with successful copulation) x 100
Pregnancy index (%) = (No. of pregnant animals/No. of animals with successful copulation) x 100
Delivery index (%) = (No. of dams with live newborns/No. of pregnant dams) x 100
Sex ratio = No. of live male pups/No. of live female pups
Viability index (%) = (No. of live pups on day 4 of lactation/No. of neonates at birth) x 100
Pre-implantation loss % = ((No. of corporalutea - No. of implantation)/No. of corpora lutea) x 100
Post-implantation loss % = ((No. of implantation - litter size) / No. of implantation) x 100

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Salivation was observed in 1000 mg/kg bw/day group in 1 male and 2 females. This finding was not considered to be related to the KY-EU treatment, because the incidence occurred at a very low frequency, temporarily and restrictively. Statistical changes in bodyweight were noted on the third week (decrease) and fifth week (increase) in 1000 mg/kg bw/day male group compared with the vehicle control group. Food consumption was statistically decreased in 250 mg/kg bw/day group on the first week compared with the vehicle control group. Because the occurrences were not in time- and dose- dependent manners, body weigh gain changes and food consumption were not considered to be toxicologically relevant. No treatment-related changes were observed in absolute and relative organ weights. At histopathological examination, minimal focal interstitial inflammatory cell infiltration in kidney and mammary adenocarcinoma were observed in 1000 mg/kg bw/day male group and 500 mg/kg bw/day female group, respectively. Since the occurrence were common lesions in rats, these were considered to be incidental and not toxicologically relevant. In male neonates, body weight was significantly decreased in 500 mg/kg bw/day group on day 4 of lactation compared with the vehicle control group. The change was not considered due to the KY-EU treatment, since the occurrence was not in a dose dependent manner.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No treatment-related effect was seen in all treatment groups in the following parameters examined: gestation length, the number of corpora lutea, implantation sites, delivery index, live and dead pups, live and death rate to implantation, sex ratio, viability index, body weight of pups in both sexes on day 0 of lactation and pre- and post-implantation losses. By observation of live pups at birth, there were no externally malformed pups in any groups. At necropsy of pups, no gross finding was observed in any groups. Consequently, it was considered that no adverse effects of the test substance on reproduction and development in rats were observed.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Analytical verification:

C.V (%) for the formulations of the first day were 4.1 %, 2.4 % and 1.4 % for G2, G3 and G4. And contents (%) were 91.3 %, 92.7 % and 105.8 % for G2, G3 and G4,

respectively. These results met the acceptance criteria (C.V (%) ≤ 5.0 %, Content (%): 100 ± 10 %). C.V (%) for the formulations of the fifth week were 4.0 %, 1.3 % and 3.6 % for G2, G3 and G4, respectively. And contents (%) were 96.0 %, 101.1 % and 101.0 % for G2, G3 and G4, respectively. These results met the acceptance criteria (C.V (%) ≤ 5.0 %, Content (%): 100 ± 10 %).

Homogeneity and content (%) of KY-EU in 0.5 % methylcellulose were analyzed in accordance with validated analytical method (KTR/Study No. : KG-2012-374). KY-EU formulations for the first day and fifth week were considered homogeneous and accurately prepared.

Applicant's summary and conclusion

Conclusions:

Based on the results of an OECD421 study, it was determined that the repeated oral dose of KY-EU to parental animals resulted in no adverse effects on all parameters examined up to and including 1000 mg/kg bw/day. Therefore, the NOAEL of the test substance is considered to be above 1000 mg/kg bw/day in both sexes for general toxicity, reproductive capability and F1 neonates.

Executive summary:

A Reproduction and Developmental Toxicity Screening Test with KY-EU via gavage in male and female rats was performed according to OECD 421 guideline and GLP principles. No treatment-related changes were observed in body weight, absolute and relative organ weight of parental rats. There were no treatment-related changes in estrus cycle, fertility and pregnant indices among the groups.

There were no treatment-related changes in the number of corpora lutea and implantations, pre-implantation loss, post-implantation loss, litter size at birth, neonatal death, sex ratio of neonates, viability index, body weight of neonates on day 0 and 4 of lactation or gross external findings at any of the doses tested. Based on the results, it was determined that the repeated oral dose of KY-EU to parental animals resulted in no adverse effects on all parameters examined at 1000 mg/kg bw/day. Therefore, the No Observed Adverse Effect Level (NOAEL) of the test substance is considered to be above 1000 mg/kg bw/day in both sexes for general toxicity, reproductive capability and F1 neonates.