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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

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 priciples.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(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)

Sex:

male/female

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): min 21.5 ℃, max 22.7 ℃
- Humidity (%): min 49.0 %, max 54.8 %
- Air changes (per hr): 10 ∼ 15) changes/hr
- 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:

The test substance, prepared by the test substance preparation method, was orally administered once a day, 7 days a week.

Details on mating procedure:

Normally, 1:1 (one male to one female) mating was used in this study. Each morning the females were examined for the presence of a sperm or a vaginal plug. Day 0 of pregnancy was defined as the day which a vaginal plug or a sperm was found. Pregnancy was determined with implantation marks on the uterus at necropsy was performed.

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 (%).
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.

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 dayly, 7 days a week

Details on study schedule:

Male rats were administered by oral gavage 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 administered by oral gavage at each dose levels from 2 weeks prior to mating, to day 3 of lactation including the mating and gestation period.

Doses / concentrations

Remarks:

Doses / Concentrations:
250, 500 and 1000 mg/kg b.w.
Basis:
nominal conc.

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 B.W.. There was a significant body weight loss in the 1000 mg/kg B.W. group in female rats. Therefore, 1000 mg/kg B.W. was used as the high dose and doses of 250 and 500 mg/kg B.W. were selected as the medium and low dose, respectively, using a scaling factor of 2.

Positive control:

no

Examinations

Parental animals: Observations and examinations:

- Clinical signs including mortality, moribundity, general appearances, deaths and behavior changes were checked once a day during the non-treatment period and twice a day during the treatment period and recorded with date, time of finding and duration.
- Body weight and body weight gain:
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 follow:
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: 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.
- 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 B.W. groups.

Oestrous cyclicity (parental animals):

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.

Litter observations:

litter size, body weight on PND 0 and 4, sex ratio, mortality

Postmortem examinations (parental animals):

Necropsy finding, organ weight, histopathological findings

Postmortem examinations (offspring):

Necropsy finding

Statistics:

Program : SPSS ver.19 (SPSS Korea Data Solution Co., Ltd.)
One-way analysis of variance (ANOVA) with Dunnett or Scheffe nonparametric ANOVA with Mann-Whitney U-test
P < 0.05 or 0.01

Reproductive indices:

pregnancy period, No. of corpora lutea, No. of implantation, pre- and post- implantation loss, fertility, pregnancy and delivery indices, mating index, sex ratio, viability

Offspring viability indices:

live pups on PND 0 and 4, dead pups on PND 0, viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

In clinical signs, salivation was observed in 1000 mg/kg B.W. 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. In body weight gain, the statistical changes were noted on the third week (decrease) and fifth week (increase) in 1000 mg/kg B.W. male group compared with the vehicle control group. In food consumption, a statistic decrease was noted in 250 mg/kg B.W. group on the first week compared with the vehicle control group. Because the occurrences were not in time- and dose- dependent manners, body weight gain changes and food consumption were not considered due to the KY-EU treatment. No treatment-related changes were observed in body weight, absolute and relative organ weight. At histopathological examination, minimal focal interstitial inflammatory cell infiltration in kidney and mammary adenocarcinoma were observed in 1000 mg/kg B.W. male group and 500 mg/kg B.W. female group, respectively. Since the occurrence were common lesions in rats, these were considered to be incidental.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

In male neonates, body weight was significantly decreased in 500 mg/kg B.W. 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 dose dependent manners.
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.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

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 B.W .. Therefore, No Observed Adverse Effect Level (NOAEL) of the test substance is considered to be above 1000 mg/kg B.W . in both sexes for general toxicity, reproductive capability and F1 neonates.

Executive summary:

A Reproduction and Developmental Toxicity Screening Test with KY-EU 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 finding 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 B.W .. Therefore, No Observed Adverse Effect Level (NOAEL) of the test substance is considered to be above 1000 mg/kg B.W . in both sexes for general toxicity, reproductive capability and F1 neonates.