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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(1995)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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.

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

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.

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

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

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.

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

Reproductive effects observed:

not specified

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.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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, the NOAEL for parental and reproduction toxicity was determined to be at least 1000 mg/kg bw/day.

Short description of key information:
An OECD 421 study was performed under GLP. Based on the absence of adverse effects, the NOAEL for parental and reproduction toxicity was determined to be at least 1000 mg/kg bw/day.

The extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity.

Effects on developmental toxicity

Description of key information

An OECD 414 study was performed under GLP. Based on the absence of adverse effects, the NOAEL for developmental toxicity was determined to be at least 2000 mg/kg bw/day.

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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.

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

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.

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

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.

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

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-17 untill 2018-03-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:(SD), SPF

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ORIENT BIO Co. Ltd., Mokdong-ri, Buk-myeon, Gapyeong-gun, Gyeonggi-do, Korea
- Number of animals: 96 (males) and 96 (females)
- A range of age and body weight at the time of receipt: 9 weeks old, 277.2 g – 324.7 g (male), 7 weeks old, 203.4 g – 253.1 g (female)
- A range of age and body weight at the time of administration: 10 weeks old, 217.8 g – 280.6 g (female)
- Number of animals (administration): 92 females
- Acclimatisation: All animals were monitored for health condition, acclimated and quarantined by body weight and clinical sign for 6 days and healthy animal was selected for this study.
- Fasting: Quarantine, acclimatisation and after group assignment: stainless stell wire cage, 1 male and 1 female. During gestation: polysulfone cage, 1 female.
- Diet: Free access to pelleted rodent diet (Rodent Diet 20 5053 (Labdiet USA).
- Water: Free access to R/O water

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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 to 22.5
- Humidity (%): 47.6 to 54.1
- Air changes (per hr): at least 10-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5%(w/v) metylcellulose

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a suspension in the vehicle : the test substance was ground to fine powder, using a mortar and pestle and then mixed with the required quantity of vehicle in order to achieve the concentrations of 50, 100 and 200 mg/mL. The test substance was prepared daily based on the analytical method validation (KTR Study No.: TBK-001208-2016, Analytical method validation of the KY-EU in dosing formulation by using HPLC).
No information was provided whether or not adjustment was made for specific gravity of the vehicle. In addition, no information was provided in the study rport on whether or not correction was made for the purity/composition of the test item.
Animals were given a dose volume of 10 ml/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed before treatment (1st dosing) based on the analytical method validation (KTR Study No.: TBK-001208-2016, Analytical method validation of the KY-EU in dosing formulation by using HPLC). The results were as follows and met the acceptance criteria (precision: less than 10 %, accuracy: 80 – 120 %).
0 mg/mL: -
50 mg/mL: 0.52% homogeniticy, 101.58% concentration.
100 mg/mL: 0.29% homogencity, 102.63% concentration.
200 mg/mL: 0.16% homogenicity, 101.17% concentration.

Details on mating procedure:

1:1 (one male to one female).
Each morning, the females were examined for the presence of sperm in vagina or vaginal plug (once/day). Day 0 of gestation was defined as the day on which mating evidence was confirmed.

Duration of treatment / exposure:

The substance was administrated once/day, from implantation to one day prior to the day of caesarean section (GD 5 – GD 20).

Frequency of treatment:

Daily

Duration of test:

See duration of treatment

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

23

Control animals:

yes, concurrent vehicle

Details on study design:

As a result of dose range finding study (KTR Study No.: TBK-001206-2016, dose: 0, 500, 1000, 2000 mg/kg), the treatment-related effect of test substance administration and mortality had not been observed in high dose (2000 mg/kg).

The administration volume was calculated to 10 mL/kg based on most recently measured body weight.

Maternal examinations:

Throughout the test period, general clinical observations, signs of toxicity including mortality and prolonged parturition (dams) were made at once a day for acclimation period and twice a day for a treatment period (before/after administration).
Body weight:
- males: at receipt
- females: at receipt, on GD0, GD5, GD8, 11, 14, 17 and 20, and on the day of scheduled sacrifice GD21.

Food consumption per cage was measured. A weighed amount of feed was supplied to each cage and the remaining feed was measured the next day.

On GD 21 (caesarean section), after blood collection under anesthesia with isoflurane (Forane, JW Pharmaceutical, Korea) the animals were sacrificed by exsanguination form the aorta. The external surface, all orifices, cranial cavity, thoracic (abdominal) organs and their contents were macroscopically checked.

After gross necropsy, absolute and relative (organ-to-body weight ratio) weights of next organs were measured in all animals. For bilateral organs (*), the weight of the left and right organ was measured respectively and combined weight was calculated:
Liver, Spleen, Heart, Kidney*, Lung, Adrenal gland*, Uterus, Ovary*, Thymus, Brain, Pituitary gland.

Ovaries and uterine content:

Gravid uteri including the cervix was weighed. Also, number of corpora lutea and implantation was examined for pregnant animals. The uteri that appear non-gravid was examined by using ammonium sulphide staining.
Implantation ratio (%) = (No. of implantation / No. of corpora lutea) × 100
Pre-implantation loss (%) = [(No. of corpora lutea - No. of implantation) / No. of corpora lutea] × 100
Post-implantation loss (%) = [(No. of implantation – litter size) / No. of implantation] × 100

Fetal examinations:

Fetuses was examined as follows.
Sex (and ratio), body weight (by sexes and with sexes combined, placenta weight and external observations for all fetuses (alterations)
sex ratio = [(liver fetuses of male/live fetuses of total) ×100]
No. of fetal deaths and viable fetuses

Approximately one-half of live fetuses (odd numbered) from each litter was prepared and was fixed in 95 % ethanol. And then fetuses was stained alizarin red S and alcian blue. After examination, fetuses was preserved in 100 % glycerin.

The remaining live fetuses (even numbered) was fixed in bouin's solution (fixed preparation) and was examined.

The internal organs were fixed with 10 % neutral buffered formalin individually except eyes (Davidson's fixative solution) and the following tissues was preserved. Besides, histopathologiacl examination was carried out on the kidneys which necropsy findings were observed.
Liver, Kidney, Adrenal gland, Heart, Lung with bronchus, Brain, Pituitary gland, Spleen, Ovary (uterus), Skeletal muscle (sciatic nerve), Vagina, Tongue, Trachea, Esophagus, Thyroid gland, Parathyroid gland, Thymus, Stomach, Intestine (small/large intestine), Urinary bladder, Skin, Salivary galnds, Pancreas, Eye, Sternum, Bone, Spinal cord, lymph node.

Statistics:

Data collected during the study was presented as mean ± S.D. and was analyzed by SPSS program (Ver 19.0). The Levene's test was performed for homogeneity of variances and then One way ANOVA analysis was performed to evaluate the significant differences between groups. In the One way ANOVA analysis, there were no significant difference and additional post-hoc analysis was not be performed. The confidence interval was 95 % level. Besides, frequency data was examined by using the Kruskal-Walis test and body weight and placental weight of fetus was analyzed using the ANCOVA (analysis of covariance).

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed during the study period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No treatment-related moribund (or dead) animals were observed during the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In dams, there were no significant difference in body weight (gain) for gestation period (including treatment period) and at three-day intervals.
Also, no significant differences was observed in corrected terminal body weight and net body weight change after measurement of gravid uterus weight at caesarean section.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In dams, there were no significant difference for gestation period (including treatment period).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males of the high dose group, the absolute lung weight was significantly increased but there were no significant difference in relative weight at 2000 mg/kg. No further changes were observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

On the 21th day of pregnancy, the post-mortem macroscopic finding showed that kidney discolorations were observed in 2 animals at 500 mg/kg. However, there were no necropsy findings at 0, 1000, 2000 mg/kg.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Uterine gravid weight: no treatment-related findings

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal body weight changes:

no effects observed

Description (incidence and severity):

In fetus on the caesarean section, there were no significant difference in body weight (by sexes and with sexes combined) at any dose group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In skeletal alterations, number of ossification was on a similar level with the control group.
Also, observed alterations (retardations, variations, malformations) were lacked doserelationship and most of findings were observed in control group, too. Therefore, it was not considered to be treatment-related alterations.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In soft tissue (visceral) alterations, observed alterations (variations, malformations) were lacked dose-relationship and most of findings were observed in control group, too. Therefore, it was not considered to be treatment-related alterations.

Other effects:

no effects observed

Description (incidence and severity):

Placental weight of live fetuses: no effects observed.
Implantation loss: no effects observed.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Dose level (mg/kg bw/day)	0	00	1000	2000
Pregnant/total dams	21	22	21	20
Implantation ratio (%)	96.9	98.6	96.4	100
No of corpora lutea	14.4	14.3	14.6	13.7
Pre-implantation loss (percent)	3.1	1.4	3.6	0.0
Post-implantation loss (percent)	2.3	3.4	5.8	2.3
Body weight on day 21 (g)	417.5	423.0	419.7	427.1
Body weight gain day 5-20 (g)	168.3	174.1	171.7	168.1
Gravid uterine weight (g)	101.04	100.03	102.18	99.72
Mean live offspring (number)	289	300	285	266
Live offspring (percent)	13.8	13.6	13.6	13.3
Mean fetal/pup body weight males (g)	5.75	5.79	5.87	5.88
Mean fetal body weight females	5.47	5.44	5.50	5.48
Mean fetal body weight (sexes combined)	5.60	5.62	5.66	5.69
Malformations (including runts) number and percent of fetuses per litter	No overall figures presented No treatment related observations
Variations (% per litter) -external -soft tissue -skeletal	No overall figures presented No treatment related observations

 

Dose level (mg/kg bw/day)	0	00	1000	2000
Pregnant/total dams	21	22	21	20
Implantation ratio (%)	96.9	98.6	96.4	100
No of corpora lutea	14.4	14.3	14.6	13.7
Pre-implantation loss (percent)	3.1	1.4	3.6	0.0
Post-implantation loss (percent)	2.3	3.4	5.8	2.3
Body weight on day 21 (g)	417.5	423.0	419.7	427.1
Body weight gain day 5-20 (g)	168.3	174.1	171.7	168.1
Gravid uterine weight (g)	101.04	100.03	102.18	99.72
Mean live offspring (number)	289	300	285	266
Live offspring (percent)
Mean fetal/pup body weight males (g)	5.75	5.79	5.87	5.88
Mean fetal body weight females	5.47	5.44	5.50	5.48
Mean fetal body weight (sexes combined)	5.60	5.62	5.66	5.69
Malformations (including runts) number and percent of fetuses per litter	No overall figures presented No treatment related observations
Variations (% per litter) -external -soft tissue -skeletal	No overall figures presented No treatment related observations

 

Conclusions:

In conclusion, the NOAEL (no observed adverse effect level) for Prenatal Developmental Toxicity Study of KY-EU was considered to be 2000 mg/kg based on no
treatment-related effect at 2000 mg/kg in dams and fetuses.

Executive summary:

In a teratogenicity study in rats, pregnant females were given (day 5 post mating till the day prior to scheduled caesarean section (the 20th day of preganacy) oral doses of 500, 1000 or 2000 mg/kg bw/day.

No treatment-related moribund (or dead) animals and clinical signs were observed at all treatment group during the study period.

No changes in body weight (gain) was observed at any dose level.

In fetus on the caesarean section, there were no significant difference in body weight (by sexes and with sexes combined) at any dose group.

In food consumption of dams, there were no significant changes at 500, 1000, 2000 mg/kg.

At necropsy on the 21th day of pregnancy, there were no treatment-related findings at 0, 1000, 2000 mg/kg. Besides, kidney discoloration observed in 2 animals at 500 mg/kg confimed to be focal nephropathy and no abnormal findings. This focal nephropathy was known as protein-overload nephropahty or chronic progressive nephrosis and as degernerative lesion common in older rats. The lesion observed in this study was not considered to be treatment-related lesion considering the frequency of lesion (1 case) and degree (moderate).

In organ weight, there were no significant difference in dams at 500, 1000, 2000 mg/kg. In lung at 2000 mg/kg, absolute weight significantly increased was not considered to be treatment-related effect considering no significant difference of relative weight and small difference as compared with control.

In caesarean section, there were no significant difference in weight of gravid uterus and placental weight at 500, 1000, 2000 mg/kg. In fetuses, there were no observations in external malformations and deaths for fetuses and, the number of live fetuses of each sex in litters (sex ratio) showed no significant difference at any dose group. Also, in dams with implants, there were no significant difference in number of corpora lutea, implantation sites, resorptions and in implantation ratio, pre/post-implantation loss at 500, 1000, 2000 mg/kg. These no significant changes for developmental endpoints (copora lutea, implantations, implantation loss, sex ratio) was on a similar level with previous study (Study No. KG-2012-373‘Reproduction/Developmental toxicity

screening test’, dose level – 0, 250, 500, 1000 mg/kg). In soft tissue (visceral) alterations, observed alterations (variations, malformations) were

lacked dose-relationship and most of findings were observed in control group, too. Therefore, it was not considered to be treatment-related alterations.

In skeletal alterations, number of ossification was on a similar level with the control group. Also, observed alterations (retardations, variations, malformations) were lacked doserelationship and most of findings were observed in control group, too. Therefore, it was not considered to be treatment-related alterations.

In conclusion, the NOAEL (no observed adverse effect level) for Prenatal Developmental Toxicity Study of KY-EU was considered to be 2000 mg/kg based on no treatment-related effect at 2000 mg/kg in dams and fetuses.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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, the NOAEL for maternal and developmental toxicity was determined to be at least 1000 mg/kg bw/day.

In a teratogenicity study in rats with KY-EU, pregnant females were given (day 5 post mating till the day prior to scheduled caesarean section (the 20th day of preganacy) oral doses of 500, 1000 or 2000 mg/kg bw/day. The NOAEL (no observed adverse effect level) for Prenatal Developmental Toxicity Study of KY-EU was considered to be 2000 mg/kg based on no treatment-related effect at 2000 mg/kg in dams and fetuses.

Justification for classification or non-classification

Based on the available information, the substance does not need to be classified for reproduction and developmental toxicity in accordance with the CLP Regulation.