Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-01-05 to 2017-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

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:
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
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Orient Bio Inc. 322, Galmachi-ro, Jungwon-gu, Seongnam-si, Gyeonggi-do 13201, Republic of Korea
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 7 weeks (Males) and 9 weeks (Females)
- Weight at study initiation: 349.4-425.3 g at the initiation of dosing (Males), 231.8-282.9 g at the initiation of dosing (Females)
- Fasting period before study: none
- Housing: during acclimation, pre-treatment, treatment and post-mating in stainless-steel cage (1 or 2 animals), during mating one male and one female animal in stainless-steel cage, during gestation (1 female) and lactation (1 female with pups) in poly sulfone cages
- Diet: Lab Diet® #5053 PMI Nutrition International, USA, ad libitum
- Water: ad libitum filtered, ultraviolet light-irradiated municipal tap water
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
Microbial monitoring for diet was performed at KIT and a certificate of analysis for the diet was provided by the supplier

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was weighed and suspended in corn oil with a magnetic stirrer for about 5 min to prepare the target concentration. The high dose formulation was prepared first and then the lower dose formulations were prepared by diluting the higher dose formulation with corn oil.

VEHICLE
- Justification for use and choice of vehicle: Corn oil was considered non-toxic with this dose volume (2 mL/kg), and it has been used in previous studies because of the solubility of the test item with this vehicle.

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: singly in poly sulfone cages (260W×420L×180 mm)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of dose formulations were conducted according to a validated GC method (KIT Study No.: G316052) in the test site. Samples for dose formulation analysis were transferred at room temperature. Results of dose formulations were 107.4, 102.3 and 95.6% at each dose levels of 50, 200 and 800 mg/kg. They were acceptable as the mean concentration was within ±15% of the nominal concentration.
Duration of treatment / exposure:
Dosing of the males has begun 14 days prior to mating and was continued till the day prior to sacrifice (at least 28 days). Dosing of the females has begun 14 days prior to mating and was continued till lactation day (LD) 13. Animals in recovery group were not mated and were assigned to a 2-weeks recovery period after the completion of administration.
Frequency of treatment:
once daily
Details on study schedule:
- Age at mating of the mated animals in the study: Age at mating of the mated animals in the study: 12 - 14 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
No. of animals per sex per dose:
vehicle control: 18
50 and 500 mg/kg bw dose groups: 12
800 mg/kg bw dose group: 18
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose-range finding study
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week
Detailed clinical observations were made in all animals individually for abnormalities. Signs noted were included (but are not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern), changes in gait, posture, clonic and tonic movements, stereotypical and bizarre behavior, and difficult and prolonged parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, food consumption was observed once a week

ORGAN WEIGHTS:
Organs weighed at terminal and recovery sacrifice: see table 3 in the field 'any other information on material and methods incl. tables'


THYROID HORMONE ANALYSIS:
Blood samples were taken from all adult animals at termination from the caudal vena cava. Thyroid hormone (T4 and TSH) concentrations in the serum samples of the males were determined.
Estrous cyclicity (parental animals):
A vaginal smear was taken daily for each female from the beginning of the 14 days prior to mating with continued monitoring into the mating period until there was evidence of mating. Furthermore, regularity and length of the estrus cycle during the treatment period until mating was examined.
In addition, vaginal smear of sacrificed females were taken at termination to examine the stage of the estrus cycle and allow correlation with histopathology of female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in male parental generations:
testis weight, epididymis weight, Seminal vesicles (with coagulation gland) weight, prostate weight, adrenal gland weight
The reproductive organs collected from the main group were further processed to sildes, stained with H&E, and examined microscopically.

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 in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were euthanized on the day after final dosing using isoflurane, and necropsied.
- Female animals: All surviving main group females on LD 14 were euthanized using isoflurane.

All surviving recovery group males and females were euthanized at least 14 days after the first scheduled sacrifice of males and females using isoflurane and necropsied.
Non-parturition females were euthanized after GD 27 using CO2, and macroscopic findings, tissue preservation, pregnancy confirmation and microscopic findings were conducted.

All scheduled sacrificed surviving main and recovery group animals were fasted more than 16 hours (overnight) prior to scheduled sacrifice.

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 and 2 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
All pups on PND 13 were euthanized using CO2.

GROSS NECROPSY
- Gross necropsy consisted of external examinations for abnormalities.

Blood samples were collected on PND 4 from culled pups due to cardiac puncture and on PND 13 from caudal vena cava (remaining pups) for thyroid hormone analysis.
Statistics:
Mean values and standard deviations were calculated in the final report. Statistical analyses for comparisons of the various dose groups with the vehicle control group were conducted using Pristima System or Statistical Analysis Systems. Data was considered to be significant when p<0.05 or p<0.01.
Multiple comparison tests for different dose groups were conducted. Variance of homogeneity was examined using the Bartlett’s Test. Homogeneous data was analyzed using the Analysis of Variance (ANOVA) and the significance of inter-group differences were analyzed using Dunnett’s Test. Heterogeneous data was analyzed using Kruskal-Wallis Test and the significance of inter-group differences between the control and treated groups were assessed using Dunn’s Rank Sum Test.
For comparing control group and recovery group, the data was analyzed for homogeneity for variance using F-test. Homogeneous data was analyzed using T-test and the significant difference between control and recovery group was assessed using Dunnett’s Test. Heterogeneous data was analyzed using Kruskal-Wallis Test and significant difference between control and recovery group was assessed using Dunn’s Rank Sum Test.
One-way analysis of covariance (ANCOVA) was used to analyze pup body weight. The litter size was used as the covariate. Litter data was statistically evaluated using the statistical unit as a litter.
Data presented as frequencies was analyzed by χ2-test followed by the Fisher's exact test where necessary.
Reproductive indices:
please refer to table 3
Offspring viability indices:
please refer to table 3

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In males at 200 and 800 mg/kg bw, salivation was observed in 12 and 18 animals, respectively. In females at 200 and 800 mg/kg bw, salivation was observed in 3 and 18 animals, respectively. It was considered test item-related but not toxicologically relevant since it was considered to be attributed to the palatability of the test item.
Other clinical signs were observed in this study but were not considered test item-related since these findings were observed with low frequency or occurred sporadically and did not show a dose-response relationship.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test item-related decrease in body weight gain (76% and 69% of control, respectively) was observed in males and females at 800 mg/kg bw during the treatment days 1 to 50.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related changes in food consumption were observed in both sexes during the study.
Statistically significant changes in food consumption was found for a few time periods during the study, but were scattered over time and sex and thus were not considered test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant decrease of platelet counts (PLT, down to 80% of control) was observed in both sexes at 800 mg/kg bw. Also, decrease but not statistically significant of absolute and relative reticulocyte counts (RETA and RET%, 87% and 90% controls, respectively) in males and significantly decrease of absolute and relative reticulocyte counts (83% and 83% controls, respectively) in females were observed at 800 mg/kg bw. Increase but not statistically significant of absolute monocyte counts (MONA, 1.50-fold over control) and a significant increase of relative monocyte counts (MON%, 1.56-fold over control) were observed in males at 800 mg/kg bw. And significant decreases of absolute and relative eosinophil counts (EOSA and EOS%, 43% and 50% of controls, respectively) were observed in males at 800 mg/kg bw. These changes were not considered adverse as there were no histopathological correlates and reversed after recovery period.
Other statistically significant changes were not considered test item-related, because these changes were minimal, lacked a dose-relationship, were not correlated with microscopic changes and/or were observed only in recovery group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase of alanine aminotransferase (ALT, 1.59-fold over control) was observed in males at 800 mg/kg bw and significant increase of total bilirubin (TBIL, 1.41-fold over control) was observed in females at 800 mg/kg bw. These changes were not considered adverse as there were no histopathological correlates and reversed after recovery period.
Other statistically significant changes were not considered test item-related, because these changes were minimal, lacked a dose-relationship, were not correlated with microscopic changes and/or were observed only in the recovery group.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A statistically significant increase in relative liver weight (1.11-fold over control) in males and absolute and relative liver weights (up to 1.14-folds over controls) in females were observed at 800 mg/kgbw. Significantly decreased absolute and relative adrenal glands weights (down to 74% of controls) in males and decrease but not significant of absolute and relative adrenal glands weights (down to 84% of controls) in females were observed. Significantly decreased absolute prostate and seminal vesicles weight (with coagulation gland, 83% of control) in males and increased absolute and relative heart weights (up to 1.11-fold over controls) in females were observed at 800 mg/kg bw. These changes were not considered adverse as there were no histopathological correlates and reversed after the recovery period.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic findings observed during this study were considered to be incidental or spontaneous and of a nature that is commonly seen in SD rats at low incidence or severity.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic findings (e.g. slight dilatation or mineralisation of renal tubules) observed during this study were considered to be incidental or spontaneous since they were infrequent, of low severity and similarly distributed among control and test item-treated groups.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test item-related changes in thyroid hormone (T4) were observed in adult males.

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Decreased regularity of estrus cycle (75%) was observed in females at 800 mg/kg bw.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
An increased number of dead or cannibalized pups were found and a statistically significant increase in gestation period and perinatal death was observed at 800 mg/kg bw. A statistically significant decrease in live litter size and viability was observed at 800 mg/kg bw.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
200 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
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive function (estrous cycle)
reproductive performance
Key result
Dose descriptor:
LOAEL
Effect level:
800 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
Dose descriptor:
LOAEL
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive function (estrous cycle)
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
800 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
An increased number of dead or cannibalized pups were found and a statistically significant increase in perinatal death was observed at 800 mg/kg bw. A statistically significant decrease in live litter size and viability was observed at 800 mg/kg bw.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In male pups at 800 mg/kg bw, a statistically significant decrease in covariate adjusted body weight was observed (88% of control) on PND 0 and significant decrease in body weight was observed (91% of control) on PND 13. In female pups at 800 mg/kg bw, a statistically significant decrease in covariate adjusted body weight was observed (83% and 90% of control on PND 0 and PND 13, respectively).
Food consumption and compound intake (if feeding study):
not examined
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:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
No test item-related changes in thyroid hormone (T4) were observed in pups during the study.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
200 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
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
800 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

Applicant's summary and conclusion

Conclusions:
Adverse effects regarding reproduction/development were observed at 800 mg/kg bw in combination with systemic adverse effects.
Executive summary:

The test item was administered by oral gavage to Sprague-Dawley rats ( at least12 animals per sex per group) at dose levels of 0, 50, 200 and 800 mg/kg with a dose volume of 2 mL/kg in corn oil as vehicle. Males and females were dosed for two weeks prior to mating and continued through the day before sacrifice in males (at least 50 days), and continued through the lactation day (LD) 13 in females. Additional animals in the recovery group at 0 and 800 mg/kg (6 animals per sex per group) were also administered but not mated, and then assigned to 2 weeks of recovery period after the completion of administration.

No deaths or moribund animals occurred in any group throughout the study. One dead fetus was observed in the uterus o one female. It was considered to have toxicological relevance since test item-related systemic (decreased body weight gain) and reproduction/developmental (increased perinatal death and decreased live litter size) adverse effects were observed at 800 mg/kg bwduring the study.

No test item-related change was observed in food consumption, functional behavior examination, motor activity examination, macroscopic and microscopic findings.

A significant decrease in body weight gain was observed in males and females at 800 mg/kg bw. Changes were noted in hematology, clinical chemistry and organ weights, but these were not considered adverse since they were not correlated with microscopic findings and reversed after recovery period.

In reproductive/developmental observations, at 800 mg/kg bw, a significant increase in gestation period and perinatal death and decrease in live litter size and viability index were observed. A significant decrease in body weight of male and female pups was observed on PND 0 (down to 83% of control) and 13 (90% of control).

In conclusion, systemic adverse effects including decreased body weight gain were observed at 800 mg/kg bw. Regarding reproduction/developmental effects, decreases in regularity of estrus cycle, live litter size, viability index and body weights in F1 pups were observed at 800 mg/kg. Increases in gestation period and perinatal death were also observed at 800 mg/kg bw. Therefore, the No-Observed-Adverse-Effect Level (NOAEL) for general toxicity and reproduction/developmental toxicity was considered to be 200 mg/kg bw /day, respectively.