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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-17 to 2017-12-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-07-17 to 2017-12-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29th July, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control data base. In addition, the rat is a required species in the regulatory guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 10 weeks, females: 10 weeks
- Weight at study initiation: females at start of administration: 232.9–284.8 g, males at start of administration 352.5–406.0 g
- Housing: Stainless wire mesh cage, 260W×350D×210H (mm), Polycarbonate cage, 260W×420D×180H (mm)
Two animals (during the quarantine-acclimation period); One animal (during the dosing period)
One male and one female (during the mating period)
One female and neonates (during the lactation period)
- Diet: ad libitum, Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C)
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of feed analysis was provided by the manufacturer, Envigo RMS, Inc. The results of feed analysis met the allowable standard of this facility.
Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year by the Research Institute of Health & Environment, Chungbuk (184, Osong saengmyeong 1 (il)-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea) according to the Regulation of Quality Criteria for Potable Water and Test (Ministry of Environment Ordinance No. 684, Revision Dec. 30, 2016). The results of water analysis met the allowable standard of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 -23.9
- Humidity (%): 45.6-58.4
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed using an electronic balance (CP423S, ENTRIS423i-1S, Sartorius, Germany) and placed in a container. The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer, and then the vehicle was gradually added to yield the desired concentrations. The dosing formulation was stored in a refrigerator (4.0–5.6°C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was dissolved in it.
- Lot/batch no.: MKBZ9899V, MKCC0462
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
As a result of homogeneity and stability analyses conducted in the Biotoxtech Study No.: B16852, the 0.5 and 500 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 hours at room temperature and for 7 days under refrigeration.
Analyses of the dosing formulations were conducted using a Gas Chromatography (GC-2010 series, Shimadzu Corp., Japan). Analyses of the dosing formulations were conducted based on the method used in Biotoxtech Study No.B16852 and samples were taken three times from the middle layer of each dosing formulation prior to dosing and analyzed for verification of dose concentration. The results of dose concentration analyses were determined to be in the range of 106.40–110.83%. These results were within the acceptable limits (±15% of nominal values).
Duration of treatment / exposure:
Males of the main group were dosed once daily for a total of 49 days (for 2 weeks prior to mating, during 2 weeks of mating and 21 days of post-mating). Also, males and females of the recovery group were dosed once daily for 49 days. Females of the main group were dosed once daily for 2 weeks prior to mating until Postpartum Day 13. Also, females showing no evidence of parturition signs were dosed until Gestation Day 25.
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 (+ 6 animals in the control and high dose for recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previously conducted 2-week repeated oral dose range finding study (Biotoxtech Study No.: B16853), all five males and all five females were found moribund or dead at 1,000 mg/day bw/day. Decreases in body weight and/or food consumption were shown in males at 100 mg/kg bw/day and in both sexes at 300 mg/kg bw/day. Increases of the absolute and relative liver weights (up to ca. 50%) were noted in both sexes at 300 mg/kg bw/day. An enlargement of the liver was observed in one male at 300 mg/kg bw/day. Therefore, the high dose level was selected at 200 mg/kg bw/day. Then, the mid and low dose levels were selected at 75 and 25 mg/kg bw/day, respectively.
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: All animals were observed for general condition and clinical signs at least once daily throughout the study. All animals were observed for moribundity and mortality twice daily. Females were also observed for signs of abortion and premature birth.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed prior to dosing and once weekly for the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of males of the main groups and animals of both sexes of the recovery groups were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and recovery periods, the day prior to necropsy and on the day of necropsy (body weights after fasting). Body weights of females of the main groups were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, the day prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption of males of the main groups and animals of both sexes of the recovery groups was recorded just prior to dosing on Day 0 (one day before the first dosing), once a week during the dosing and recovery periods (except during mating) and the day prior to necropsy. Food consumption of females of the main group was recorded just prior to dosing on Day 0 (one day before the first dosing), once a week throughout the dosing period (except during mating), on Gestation Days 0, 6, 13 and 19, on Postpartum Days 0, 3 and 12 and the day prior to necropsy. Residual feed was recorded on the next day. Individual food consumption was calculated by subtracting the amount of residual feed from the given quantity.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (appr. 18 h)
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (appr. 18 h)
- How many animals: all animals
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
Six males per group were randomly selected from the main group animals in addition to all male recovery animals for urinalysis a few days before necropsy. Fresh (3-hour) urine and 24-hour urine samples were collected from selected animals and analyzed. Animals were fasted during the fresh urine collection, but were allowed free access to drinking water. Parameters listed in table 3 were examined.

OTHER: Sensory function and motor activity:
Six males and six females per group were randomly selected from the main groups in addition to all recovery animals and evaluated for the following examinations a few days before necropsy. The tests on the dams of the main group were carried out on lactation days 6–11. Parameters examined were: Pinna reflex, auditory reflex, corneal reflex, pupillary reflex, grip strength, motor activity.

Thyroid hormone analysis
Blood samples were taken based on the following schedule:
- from at least two pups (preferably one male and one female) per litter on PND 4 and on PND 13, each (When the number of pups allowed, from pups available above the culling target of 8 pups/litter from at least two pups per litter on both PNDs 4 and 13 otherwise. When only one pup was available above the culling target, only one pup was used for the determination of PND 4. When the litter size was below the culling target, blood collection was not carried out on PND 4. On PND 13, blood samples of all male and female pups were taken and pooled according to sex for analysis.)
- from all adult males and dams at termination
Animals were fasted for more than 18 hours before necropsy (except pups) and anesthetized with isoflurane. Blood samples were collected from the heart (PND 4) or abdominal aorta in a vacutainer. Blood samples of male and female pups were pooled by litter. Blood samples were centrifuged at 3,000 rpm for 10 minutes to obtain serum. Serum samples from the pups on PND 13 and all adult males were analyzed.
Thyroid hormones (T4 and TSH) were preferably measured in “total”.
Oestrous cyclicity (parental animals):
The estrous cycle examination for females of the main group was conducted from dosing initiation day to the confirmed copulation day or necropsy day. Smears of vaginal mucosa were prepared daily in the morning. Prepared smears of the vaginal mucosa were stained with Diff Quick stain (Hemacolor® for microscopy, Merck, Germany). Stained vaginal mucosa smears were examined using a light microscopy. The estrous cycle is divided into four stages; proestrus, estrus, metestrus and diestrus. One estrous cycle was defined as the period between the day of estrus and the day prior to next estrus. Estrous cycle was calculated from the dosing initiation day to the day before mating initiation. Regularity of the estrous cycle were examined from the dosing initiation day to the day of copulation. Vaginal smears were examined on the day of necropsy to determine the stage of the estrous cycle and used for correlation with histopathology of the female reproductive organs.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined:
live birth index and survival rate in pups, external examination and determination of sex ratio of pups, body weights, anogenital distance, nipple retention and thyroid hormone analysis (blood collection on post-natal day 4)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
Males of the main group were sacrificed on Day 50, and females of the main group were sacrificed on Postpartum Day 14. All animals of the recovery group were sacrificed two weeks after the final dosing. Non-delivered females were sacrificed on Gestation Day 26. Dams, whose pups were all dead, were sacrificed as soon as possible. All surviving animals were sacrificed by exsanguination from the abdominal aorta under isoflurane (Hana Pharm. Co., Ltd., Republic of Korea, Lot No.: 7007) anesthesia.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Tables 4 and 5 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
Dead pups and pups sacrificed on PND 13 or shortly thereafter were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which should be examined for signs of altered development.
The thyroids from all male pups and all female pups per litter sacrificed on PND 13 were preserved in 10% neutral buffered formalin.
Statistics:
The statistical analysis of this study was conducted using the SAS program (SAS® 9.3, SAS Institute Inc., U.S.A.).
Reproductive indices:
mating index, male fertility index, female fertility index, gestation index
Offspring viability indices:
live birth index, viability index (on postnatal day 0 and 4)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed in males and females at 200 mg/kg bw/day in the main and recovery groups, however, it was considered to have little toxicological significance since it was caused by physicochemical characteristics of the test item.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the main group, a significant decrease in body weights was noted in pregnant females at 200 mg/kg/day compared to controls from gestation day (GD) 7 to postpartum day (PPD) 13 (body weights on PPD 13; -10.6% vs. controls). In the recovery group, a decreasing tendency of body weights was noted in females at 200 mg/kg bw/day (body weights on Day 49; -8.6% vs. controls) although there was no statistical difference. This decrease or decreasing tendency of body weights was considered to be a test substance-related adverse effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the main group, a significant decrease in food consumption was noted in females at 200 mg/kg bw/day on GD 7 (-16.4% vs. controls) and PPD 13 (-15.8% vs controls). It was considered to be a test substance-related adverse effect since it was related to the decrease of body weight gain.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
In animals of both sexes in the main and recovery group, there were no test substance related effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex tests compared to the control group. Furthermore there was no test substance related effect on grip strength when compared to the control group. In the main group, there was a statistically significant decrease in ambulatory counts and/or vertical count in both sexes at 200 mg/kg bw/day and in females at 25 mg/kg bw/day. However, the statistical significance had little toxicological meaning because it was a temporary change or it showed no dose-dependency. In the recovery group, there was a statistically significant decrease in ambulatory count in females at 200 mg/kg bw/day. However, it was considered not to be a test substance related adverse effect since the values were similar levels to the control group of the main group.
Immunological findings:
effects observed, non-treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related changes were observed in the liver.
Centrilobular hepatocellular hypertrophy was observed at minimal to slight severity in males at 75 mg/kg bw/day and in males and females at 200 mg/kg bw/day in the main groups. At the end of the recovery period, this finding was not observed in all males and females at 200 mg/kg bw/day in the recovery group. Centrilobular hepatocellular hypertrophy was completely recovered in all animals at 200 mg/kg bw/day after recovery period. It was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature in the absence of associated inflammation or necrosis, and it was completely recovered in all animals after the recovery period in this study.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
An increase in post-implantation loss late and decreases in live birth index and viability index of pups on Postnatal Day (PND) 4 were noted in two dams whose pups were all dead at 200 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: reduced food consumption and body weight
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not specified
Description (incidence and severity):
An increase in post-implantation loss late and a decrease in live birth index and viability index of pups on Postnatal Day (PND) 4 were noted in two dams whose pups were all dead at 200 mg/kg bw/day. However, in the low dose group (25 mg/kg bw/day), there was also one female whose pups were all dead. Therefore, there is no dose response relationship regarding the incidence of litter losses (0/12 in control, 1/12 in the low dose group, 0/11 in the mid dose group, 2/11 in the high dose group). Unfortunately, the highest incidence of 2/11 dams with litter loss was in the highest dose group. No historicol control data of the laboratory were provided for reproductive and developmental effects. However, in the Historical Control Data for Development and Reproductive Toxicity Studies using the Sprague-Dawley (CrI:CD®BR) Rat Compiled by MARTA, 1993, there is evidence that amongst about 3500 females out of 209 studies the incidence of females with dead pups was 0.41 with a minimum of 0 up to 2.2. Therefore, it can finally not be concluded with certainty, whether the occurrence of 2/11 dams with dead pups is really substance-related or probably incidential in nature.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in body weights was noted in male pups and female pups at 200 mg/kg bw/day on PNDs 0 (-12% M/F vs controls), 4 (18/14% M/F vs controls) and/or 13 (26/25% M/F vs controls). This change was considered to be a test substance-related adverse effect.
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Domed head was observed in one pup (0.64%) of one dam (Animal ID: 2310) at 75 mg/kg/day on PND 0. However, this change was considered to be incidental and not related to the test substance.
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
No test substance-related adverse effects were noted in the results of the mean litter size, external examination of pups and sex ratio of pups and viability index of pups on PND 0. On PNDs 0, 4 and/or 13, a decrease in body weights of pups was noted in male and female pups at 200 mg/kg bw/day. No test substance-related effects were noted in the results of anogenital distance (AGD) index of pups, nipple retention of male pups and T4 of pups. The test item had no endocrine disrupting potential in the pups under the condition of this study. With regard to the possible questionable effect of 2/11 dams with complete litter losses and its consequences for decreases in live birth and viability incidences and also the decreased pup body weights (Pubs of dam 2402: 38%; pubs of dam 2404: 24%) it should be noted that these effects were only observed together with overt signs of systemic toxicity in the maternal animals in the form of reduced body weights and food consumption as well as liver effects at the maternal toxic dose of 200 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Conclusions:
The NOAEL for reproductive toxicity was considered to be 200 mg/kg bw/day for males and 75 mg/kg bw/day for females. The NOAEL for developmental toxicity was considered to be 75 mg/kg bw/day for pups.
Executive summary:

The purpose of the combined repeated dose oral gavage toxicity study with the reproduction/developmental toxicity screening was to determine the No Observed Adverse Effect Level (NOAEL) of the test item for both the general systemic and reproduction/developmental toxicity including gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal development in addition to the neurotoxic potential and endocrine disrupting potential of the test substance when administered orally to male and female rats at dose levels of 0 (control), 25, 75 and 200 mg/kg bw/day. Males of the main group were dosed once daily for a total of 49 days (prior to mating for 2 weeks, during 2 weeks of mating and 21 days of post-mating), and females of the main group were dosed once daily for 2 weeks prior to mating, throughout gestation and for 13 days after delivery. Also, males and females of the recovery group were dosed for 49 days.

All animals of the main and recovery groups survived the duration of the study. Salivation was observed in males and females at 200 mg/kg bw/day in the main and recovery groups, however, it was considered to have little toxicological significance since it was caused by physicochemical characteristics of the test item. Dystocia and delayed delivery were observed in one female at 25 mg/kg bw/day. It was considered to be incidental since there was no dose-dependent relationship. In the detailed clinical signs, salivation was observed in some females at 200 mg/kg bw/day in the main and recovery groups. A decrease in body weight gains was noted at 200 mg/kg bw/day in females in the main and recovery groups. A decrease in food consumption was noted in females at 200 mg/kg bw/day in the main group. No test substance-related adverse effects were noted in the results of sensory function, motor activity, urinalysis, hematology, clinical chemistry and thyroid hormone analysis in adult animals of both sexes in the test substance-dosed groups. In the main group, increases in the absolute and/or relative organ weights of the liver were noted in both sexes at 75 and/or 200 mg/kg bw/day. Hepatocellular hypertrophy was observed in males at 75 mg/kg bw/day and in both sexes at 200 mg/kg bw/day. It was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature.

An increase in post-implantation loss late and a decreases in live birth index and viability index of pups on Postnatal Day (PND) 4 were noted in two dams whose pups were all dead at 200 mg/kg bw/day. However, in the low dose group (25 mg/kg bw/day), there was also one female whose pups were all dead. Therefore, there is no dose response relationship regarding the incidence of litter losses (0/12 in control, 1/12 in the low dose group, 0/11 in the mid dose group, 2/11 in the high dose group). Unfortunately, the highest incidence of 2/11 dams with litter loss was in the highest dose group. No historicol control data of the laboratory were provided for reproductive and developmental effects. However, in the Historical Control Data for Development and Reproductive Toxicity Studies using the Sprague-Dawley (CrI:CD®BR) Rat Compiled by MARTA, 1993, there is evidence that amongst about 3500 females out of 209 studies the incidence of females with dead pups was 0.41 with a minimum of 0 up to 2.2. Therefore, it can finally not be concluded with certainty, whether the occurrence of 2/11 dams with dead pups is really substance-related or probably incidential in nature.

No test substance-related adverse effects were noted in the results of the estrous cycle, mating period, mating index, gestation period, male and female fertility indexes, gestation index, mean litter size, external examination of pups and sex ratio of pups and viability index of pups on PND 0. On PNDs 0, 4 and/or 13, a decrease in body weights of pups was noted in male pups and female pups at 200 mg/kg bw/day. No test substance-related effects were noted in the results of anogenital distance (AGD) index of pups, nipple retention of male pups and T4 of pups. The test item had no endocrine disrupting potential in the pups under the condition of this study.

With regard to the possible questionable effect of 2/11 dams with complete litter losses and its consequences for decreases in live birth and viability incidences and also the decreased pup body weights it should be noted that these effects were only observed together with overt signs of systemic toxicity in the maternal animals in the form of reduced body weights and food consumption as well as liver effects at the maternal toxic dose of 200 mg/kg bw/day. Taken together, the results of the study indicated that there is no selective or specific reproductive or developmental toxic effect, which may require respective classification and labeling.

Based on these result, the NOAEL of the test item for systemic toxicity was considered to be 200 mg/kg bw/day for males and 75 mg/kg/day for females. The NOAEL for reproductive toxicity was considered to be 200 mg/kg bw/day for males and 75 mg/kg bw/day for females. The NOAEL for developmental toxicity was considered to be 75 mg/kg bw/day for pups.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29th July, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control data base. In addition, the rat is a required species in the regulatory guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 10 weeks, females: 10 weeks
- Weight at study initiation: females at start of administration: 232.9–284.8 g, males at start of administration 352.5–406.0 g
- Housing: Stainless wire mesh cage, 260W×350D×210H (mm), Polycarbonate cage, 260W×420D×180H (mm)
Two animals (during the quarantine-acclimation period); One animal (during the dosing period)
One male and one female (during the mating period)
One female and neonates (during the lactation period)
- Diet: ad libitum, Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C)
- Water: ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of feed analysis was provided by the manufacturer, Envigo RMS, Inc. The results of feed analysis met the allowable standard of this facility.
Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year by the Research Institute of Health & Environment, Chungbuk (184, Osong saengmyeong 1 (il)-ro, Osong-eup, Heungdeok-gu, Cheongju-si, Chungcheongbuk-do, Republic of Korea) according to the Regulation of Quality Criteria for Potable Water and Test (Ministry of Environment Ordinance No. 684, Revision Dec. 30, 2016). The results of water analysis met the allowable standard of this facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 -23.9
- Humidity (%): 45.6-58.4
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed using an electronic balance (CP423S, ENTRIS423i-1S, Sartorius, Germany) and placed in a container. The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer, and then the vehicle was gradually added to yield the desired concentrations. The dosing formulation was stored in a refrigerator (4.0–5.6°C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was dissolved in it.
- Lot/batch no.: MKBZ9899V, MKCC0462
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
As a result of homogeneity and stability analyses conducted in the Biotoxtech Study No.: B16852, the 0.5 and 500 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 hours at room temperature and for 7 days under refrigeration.
Analyses of the dosing formulations were conducted using a Gas Chromatography (GC-2010 series, Shimadzu Corp., Japan). Analyses of the dosing formulations were conducted based on the method used in Biotoxtech Study No.B16852 and samples were taken three times from the middle layer of each dosing formulation prior to dosing and analyzed for verification of dose concentration. The results of dose concentration analyses were determined to be in the range of 106.40–110.83%. These results were within the acceptable limits (±15% of nominal values).
Duration of treatment / exposure:
Males of the main group were dosed once daily for a total of 49 days (for 2 weeks prior to mating, during 2 weeks of mating and 21 days of post-mating). Also, males and females of the recovery group were dosed once daily for 49 days. Females of the main group were dosed once daily for 2 weeks prior to mating until Postpartum Day 13. Also, females showing no evidence of parturition signs were dosed until Gestation Day 25.
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a previously conducted 2-week repeated oral dose range finding study (Biotoxtech Study No.: B16853), all five males and all five females were found moribund or dead at 1,000 mg/day/day. Decreases in body weight and/or food consumption were shown in males at 100 mg/kg/day and in both sexes at 300 mg/kg/day. Increases of the absolute and relative liver weights (up to ca. 50%) were noted in both sexes at 300 mg/kg/day. An enlargement of the liver was observed in one male at 300 mg/kg/day. Therefore, the high dose level was selected at 200 mg/kg/day. Then, the mid and low dose levels were selected at 75 and 25 mg/kg/day, respectively.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: All animals were observed for general condition and clinical signs at least once daily throughout the study. All animals were observed for moribundity and mortality twice daily. Females were also observed for signs of abortion and premature birth.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed prior to dosing and once weekly for the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of males of the main groups and animals of both sexes of the recovery groups were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and recovery periods, the day prior to necropsy and on the day of necropsy (body weights after fasting). Body weights of females of the main groups were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, the day prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption of males of the main groups and animals of both sexes of the recovery groups was recorded just prior to dosing on Day 0 (one day before the first dosing), once a week during the dosing and recovery periods (except during mating) and the day prior to necropsy. Food consumption of females of the main group was recorded just prior to dosing on Day 0 (one day before the first dosing), once a week throughout the dosing period (except during mating), on Gestation Days 0, 6, 13 and 19, on Postpartum Days 0, 3 and 12 and the day prior to necropsy. Residual feed was recorded on the next day. Individual food consumption was calculated by subtracting the amount of residual feed from the given quantity.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (appr. 18 h)
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (appr. 18 h)
- How many animals: all animals
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
Six males per group were randomly selected from the main group animals in addition to all male recovery animals for urinalysis a few days before necropsy. Fresh (3-hour) urine and 24-hour urine samples were collected from selected animals and analyzed. Animals were fasted during the fresh urine collection, but were allowed free access to drinking water. Parameters listed in table 3 were examined.

OTHER: Sensory function and motor activity:
Six males and six females per group were randomly selected from the main groups in addition to all recovery animals and evaluated for the following examinations a few days before necropsy. The tests on the dams of the main group were carried out on lactation days 6–11. Parameters examined were: Pinna reflex, auditory reflex, corneal reflex, pupillary reflex, grip strength, motor activity.

Thyroid hormone analysis
Blood samples were taken based on the following schedule:
- from at least two pups (preferably one male and one female) per litter on PND 4 and on PND 13, each (When the number of pups allowed, from pups available above the culling target of 8 pups/litter from at least two pups per litter on both PNDs 4 and 13 otherwise. When only one pup was available above the culling target, only one pup was used for the determination of PND 4. When the litter size was below the culling target, blood collection was not carried out on PND 4. On PND 13, blood samples of all male and female pups were taken and pooled according to sex for analysis.)
- from all adult males and dams at termination
Animals were fasted for more than 18 hours before necropsy (except pups) and anesthetized with isoflurane. Blood samples were collected from the heart (PND 4) or abdominal aorta in a vacutainer. Blood samples of male and female pups were pooled by litter. Blood samples were centrifuged at 3,000 rpm for 10 minutes to obtain serum. Serum samples from the pups on PND 13 and all adult males were analyzed.
Thyroid hormones (T4 and TSH) were preferably measured in “total”.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4)

HISTOPATHOLOGY: Yes (see table 5 )
Statistics:
The statistical analysis of this study was conducted using the SAS program (SAS® 9.3, SAS Institute Inc., U.S.A.).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was observed in males and females at 200 mg/kg bw/day in the main and recovery groups, however, it was considered to have little toxicological significance since it was caused by physicochemical characteristics.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the main group, a significant decrease in body weights was noted in pregnant females at 200 mg/kg/day compared to controls from gestation day (GD) 7 to postpartum day (PPD) 13 (body weights on PPD 13; -10.6% vs. controls). In the recovery group, a decreasing tendency of body weights was noted in females at 200 mg/kg/day (body weights on Day 49; -8.6% vs. controls) although there was no statistical difference. This decrease or decreasing tendency of body weights was considered to be a test substance-related adverse effect.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the main group, a significant decrease in food consumption was noted in females at 200 mg/kg/day on GD 7 (-16.4% vs. controls) and PPD 13 (-15.8% vs controls). It was considered to be a test substance-related adverse effect since it was related to the decrease of body weight gain.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
In animals of both sexes in the main and recovery group, there were no test substancerelated effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex tests compared to the control group.
In animals of both sexes in the main and recovery groups, there was no test substancerelated effect on grip strength when compared to the control group. In the main group, there was a statistically significant decrease in ambulatory counts and/or vertical count in both sexes at 200 mg/kg/day and in females at 25 mg/kg/day. However, the statistical significance had little toxicological meaning because it was a temporary change or it showed no dose-dependency. In the recovery group, there was a statistically significant decrease in ambulatory count in females at 200 mg/kg/day. However, it was considered not to be a test substancerelated adverse effect since the values were similar levels to the control group of the main group.
Immunological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related adverse effects in total thyroxine (total T4) and thyroid-stimulating hormone (TSH) levels in adult males of the main and recovery groups and F1 male pups and female pups on PND 13 at 25, 75 and 200 mg/kg/day. Other statistical significances were considered not to be test substance-related changes because of small differences and the values were within the range of historical reference data.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the main group, the absolute and/or relative organ weights of the liver were significantly increased in both sexes at 75 and/or 200 mg/kg/day.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic examination at necropsy did not reveal treatment-related changes. At the end of the recovery period, macroscopic examination at necropsy did not reveal test substance-related changes. In addition, among three dams whose pups were all dead, only one dam showed dilatation with gas in the stomach, enlargement of the adrenal glands, and small thymus and spleen. The others showed no gross findings. Also, two non-delivered females were revealed as non-pregnant females but no findings were noted. The other macroscopic findings were considered to be incidental and not related to the test substance.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related changes were observed in the liver.
Centrilobular hepatocellular hypertrophy was observed at minimal to slight severity in males at 75 mg/kg bw/day and in males and females at 200 mg/kg bw/day in the main groups. At the end of the recovery period, this finding was not observed in all males and females at 200 mg/kg bw/day in the recovery group. Centrilobular hepatocellular hypertrophy was completely recovered in all animals at 200 mg/kg bw/day after recovery period. It was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature in the absence of associated inflammation or necrosis, and it was completely recovered in all animals after the recovery period in this study.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: reduced food consumption and body weight

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The NOAEL of the test item for systemic toxicity was considered to be 200 mg/kg bw/day for males and 75 mg/kg bw/day for females.
Executive summary:

The purpose of the combined repeated dose oral gavage toxicity study with the reproduction/developmental toxicity screening was to determine the No Observed Adverse Effect Level (NOAEL) of the test item for both the general systemic and reproduction/developmental toxicity when administered orally to male and female rats at dose levels of 0 (control), 25, 75 and 200 mg/kg bw/day. Males of the main group were dosed once daily for a total of 49 days (prior to mating for 2 weeks, during 2 weeks of mating and 21 days of post-mating), and females of the main group were dosed once daily for 2 weeks prior to mating, throughout gestation and for 13 days after delivery. Also, males and females of the recovery group were dosed for 49 days. All animals of the main and recovery groups survived the duration of the study. Salivation was observed in males and females at 200 mg/kg bw/day in the main and recovery groups, however, it was considered to have little toxicological significance since it was caused by physicochemical characteristics. Dystocia and delayed delivery were observed in one female at 25 mg/kg bw/day. It was considered to be incidental since there was no dose-dependent relationship. In the detailed clinical signs, salivation was observed in some females at 200 mg/kg bw/day in the main and recovery groups. A decrease in body weight gains was noted at 200 mg/kg bw/day in females in the main and recovery groups. A decrease in food consumption was noted in females at 200 mg/kg bw/day in the main group. No test substance-related adverse effects were noted in the results of sensory function, motor activity, urinalysis, hematology, clinical chemistry and thyroid hormone analysis in adult animals of both sexes in the test substance-dosed groups. In the main group, increases in the absolute and/or relative organ weights of the liver were noted in both sexes at 75 and/or 200 mg/kg bw/day. Hepatocellular hypertrophy was observed in males at 75 mg/kg bw/day and in both sexes at 200 mg/kg bw/day. It was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature.

Based on these result, the NOAEL of the test item for systemic toxicity was considered to be 200 mg/kg bw/day for males and 75 mg/kg bw/day for females.