Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 November 2019 - 03 June 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name: Rikabinol HB
- Lot number: 2862
- Purity: 95.5%
- Description: Flake
- Storage conditions: Room temperature, protected from light, in air tight container
- Water content: ≤0.11%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 11 to 12 weeks of age
- Weight at study initiation: males - 383 g to 451 g (mean: 416 g); females - 199 g to 267 g (mean: 241 g)
- Housing: Plastic solid-floored cages (W440 × D275 × H180 mm) with bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23°C ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10 to 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours per day (from 07:00 to 19:00)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For each dose concentration, the requisite amount of the test article was weighed and suspended with an appropriate volume of the vehicle using a agate mortar. The suspension was transferred to a measuring cylinder and the vehicle was added to make the 20, 60 and 200 mg/mL test article formulations (low, middle and high-dose formulations). The test article formulations were dispensed into lots for one day’s use in brown glass bottles for dosing.
Sampling for confirming the concentration and homogeneity was carried out from a beaker before dispensing while stirring with a magnetic stirrer

- Frequency of Preparation: At maximum, a 6-day quantity of the test article formulation was prepared at one time and used within 6 days after preparation.


VEHICLE
- Concentration in vehicle: 20, 60 or 200 mg/mL (corresponding to 100, 300 and 1000 mg/mL dose levels)
- Amount of vehicle: 5 mL/kg

- Storage condition: 1 to 10°C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability:
It has been verified by the testing facility that the dose formulations of Rikabinol HB at 5.00 and 200 mg/mL (vehicle: corn oil) were stable and homogeneous at room temperature (1°C to 30°C) for 24 hours after storage or in a refrigerator (1°C to 10°C) for 7 days in a brown glass bottle.

- Dose Analysis:
The concentration and homogeneity of Rikabinol HB in test article formulations were analyzed by the gas chromatography (GC) method at the tested facility. Analysis showed that conentrationof test item in test solutions ranged from 101.0% to 107.0% of nominal concentration and the coefficient of variation was not more than 4.9%. All results for concentration and homogeneity met the acceptable range [concentration: proportion to the nominal concentration is within 100% ± 10% (evaluation by the mean value), homogeneity: CV (coefficient of variation) is 10% or less].


-The analytical method is shown briefly in the following:
GC system
GC: 6890N (Agilent Technologies, Inc.)
Injector: 7683 (Agilent Technologies, Inc.)
Data processor: MSD Chem Station (Agilent Technologies, Inc.)

Measurement conditions
Column: DB-5ms (0.53 mm I.D.×30 m, film thickness 1.5 μm, Agilent Technologies Inc.)
Carrier gas: Helium
Flow mode: Constant flow mode
Flow rate: 5 mL/min
Injection port: Split injection port
Split ratio: 5:1
Injention port temperature: 260°C
Detection: Flame ionization detector (FID)
Detection tempurature: 320°C
Flow rate of hydrogen: 30 mL/min
Flow rate of air: 360 mL/min
Flow rate of make up gas (Nitrogen): 5 mL/min
Temperature of column oven: 240°C (Hold 10 min) → 300°C (30°C/min, Hold 3 min)
Injection volume: 1 μL
Details on mating procedure:
After selection of animals (healthy animals which showed no abnormalities in clinical signs or body weight gain during the quarantine and acclimation period), female rats at 10-11 weeks of age were housed together overnight with male rats at 11-12 weeks of age on a one-to-one basis. When a vaginal plug was observed in the vagina or sperm was present in the vaginal smear on the following morning, the females were regarded as copulated successfully. That day was designated as GD 0.

Number of animals per cage:
On the starting day of mating and after: single housing (1 male/cage) or group housing (2 to 3 females/cage)
During mating: pair housing (a pair of 1 male and 1 female/cage)
Successfully copulated female: single housing (1 animal/cage)
Duration of treatment / exposure:
Duration of dosing was from GD 5 to GD 19
Frequency of treatment:
Once daily
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Concentration - 0 mg/mL; Dose Volume - 5 mL/kg
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Concentration - 20 mg/mL; Dose Volume - 5 mL/kg
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Concentration - 60 mg/mL; Dose Volume - 5 mL/kg
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Concentration - 200 mg/mL; Dose Volume - 5 mL/kg
No. of animals per sex per dose:
Each group consisted of 22 copulated females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were set in reference to the results of the “Rikabinol HB: Combined repeat dose toxicity study and reproductive/developmental toxicity screening study in sprague-dawley rat (Study No. MOG0017, dose levels: 100, 300 and 1000 mg/kg, 10 animals/group/sex, Huntingdon Life Sciences). From these results, a dose level of 1000 mg/kg, which corresponds to the recommended highest dose in the Toxicity Test Guideline, was selected as the high dose like the Combined repeat dose toxicity study and reproductive/developmental toxicity screening study. Dose levels of 300 and 100 mg/kg, which were divided with the common ratio of approximately 3, were selected as the middle and low dose, respectively.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
All animals were observed 3 times for clinical signs including mortality, any abnormality in the external appearance, excretions, nutritional condition, posture and behavior (excitement, convulsions, sedation, abnormal gait) — before dosing, immediately after dosing and 1 to 3 hours after dosing — on each day of dosing and once (in the morning) each on the other days. On GDs 5, 6, 12 and 19, each animal was put on the hand for periodical observation

BODY WEIGHT:
All animals were weighed on GDs 0, 5, 7, 10, 12, 15, 17 and 20, and body weight gain during the dosing period (GD 5 to GD 20) was calculated. Body weights were measured between 08:32 and 10:46 (before dosing during the dosing period).

FOOD CONSUMPTION:
For all animals, cumulative food consumption was measured on GDs 0-5, 5-7, 7-10, 10-12, 12-15, 15-17 and 17-20, and the one-day food consumption calculated for each animal. Total food consumption during the gestation period (GD 0 to GD 20) was calculated. Both the amount of feed remaining or supplied were measured between 08:33 and 11:49 (before dosing during the dosing period).

ORGAN WEIGHT:
For all animals, the thyroid with parathyroid and uterus (including the conceptus, if found) were weighed. The relative organ weight per 100 g body weight was calculated from these absolute organ weights and animal’s body weight at necropsy (body weight corrected by subtracting the pregnant uterus weight). The relative weight of the uterus was not calculated. For the thyroid with parathyroid, the right and left organs were weighed separately, but evaluation was conducted on the total of both sides.

HISTOPATHOLOGY:
The Thyroid and Parathyroid of all animals were fixed and preserved in phosphate buffered 10% formalin. The thyroids (bilateral) and parathydoid (unilateral) of all dams in the high dose group and control group were examined microscopically. Microscopy for the middle and low dose groups was not performed as changed related to test article dosing were not suspected in the high dose group.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination:
For pregnant females, the ovary and uterus were excised. For the ovaries, the number of corpora lutea was counted. For the uterus, the uterine wall was cut and the numbers of live fetuses and resorptions as well as their classification (implantation sites, resorbed embryos, placental remnant, early macerated fetuses, late macerated fetuses and dead fetuses) were counted and recorded. The sum of the numbers of live fetuses and resorptions was indicated as the number of implantations. Placentae of live fetuses were observed macroscopically for gross anomalies and then weighed individually. For 1 female in the 100 mg/kg group and 1 female in the 300 mg/kg group without macroscopic implantations, the uterus was subjected to clarification by 2% NaOH solution to examine whether implantation sites were present or absent. Since there were no implantation sites, these animals were judged to be non-pregnant and all the data of these animals were excluded from the evaluation.
Fetal examinations:
- External examinations:
All live fetuses were examined for the presence or absence of external anomalies including those in the oral cavity. The distance from the anus to the genital papilla was measured to judge their sex, and body weight was measured for each fetus. For the live fetuses, anogenital distance (AGD, unit: 0.1 mm) was measured using a slide gage.

- Visceral Examination
For all groups including the vehicle control group, approximately half the live fetuses in each litter were fixed in Bouin’s solution. The internal organs were observed for the presence/absence of visceral anomalies or variations by Wilson’s free hand razor method for the head, and by Nishimura’s micro-dissection method for the thoracic and abdominal regions, and gender was confirmed by internal reproductive organs. In addition, a photograph was taken at the time of observation for anomalies or variations that could not be confirmed in the specimen at a later date (saved in the test materials but was not evaluated). After the examination, all visceral specimens were preserved in Bouin’s solution.

- Skeletal examinations:
The remaining half of the live fetuses in all groups including the vehicle control group were fixed in 70% alcohol and Alizarin-red S stained clear skeletal specimens were prepared by the modified Dawson’s method after the gender was confirmed by internal reproductive organs. The fetuses were observed under a stereomicroscope for the presence/absence of skeletal anomalies or variations. For the progress of ossification, the number of ossified sternebrae, metacarpal bones, metatarsal bones, and sacral and caudal vertebrae were counted. After the examination, all skeletal specimens were preserved in 100% glycerin.
Statistics:
- Wilcoxon’s rank-sum test (levels of significance: 0.05 and 0.01, two-tailed) - the indices of pre-implantation loss, implantation, post-implantation loss (for total and each class), external anomalies, placentas with gross anomalies, visceral anomalies, visceral variations, skeletal anomalies and skeletal variations.

- Fisher’s exact test - the sex rate of live fetuses and the incidence of dams bearing fetuses with external anomalies, placentas with gross anomalies, skeletal anomalies/variations or visceral anomalies/variations

The analyses were performed using an integrated statistical package, SAS Release 9.1.3 (SAS Institute Inc.).
Indices:
- Pre-implantation loss index (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] × 100
- Implantation index (%) = (Number of implantations / Number of corpora lutea) × 100
- Post-implantation loss index (%) = (Number of resorptions / Number of implantations) × 100
- Index of external anomalies (%) = (Number of live fetuses with external anomalies / Number of live fetuses examined) × 100
- Index of placentas with gross anomalies (%) = (Number of placentas with gross anomalies / Number of placenta) × 100
- Index of visceral anomalies (%) = (Number of fetuses with visceral anomalies / Number of fetuses examined) × 100
- Index of visceral variations (%) = (Number of fetuses with visceral variations / Number of fetuses examined) × 100
- Index of skeletal anomalies (%) = (Number of fetuses with skeletal anomalies / Number of fetuses examined) × 100
- Index of skeletal variations (%) = (Number of fetuses with skeletal variations / Number of fetuses examined) × 100
- Sex rate of live fetuses (%) = (Number of live male fetuses / Number of all live fetuses) × 100
- Index of occurrence of dams having fetuses with external anomalies (%) = (Number of dams bearing fetuses with external anomalies / Number of dams) × 100
- Index of occurrence of dams bearing placentas with gross anomalies (%) = (Number of dams bearing placentas with gross anomalies / Number of dams) × 100
- Index of occurrence of dams with fetuses having visceral anomalies or variations (%) = (Number of dams with fetuses having visceral anomalies or variations / Number of dams) × 100
- Index of occurrence of dams with fetuses having skeletal anomalies or variations (%) = (Number of dams with fetuses having skeletal anomalies or variations / Number of dams) × 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No deaths/abortions occurred in any group and no abnormal clinical signs were observed throughout the gestation period.
Mortality:
no mortality observed
Description (incidence):
No deaths occurred in any group and no abnormal clinical signs were observed throughout the gestation period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, a statistically significant low value compared to the vehicle control group was recorded in body weight on GDs 10, 12, 15, 17 and 20, and cumulative body weight gain during the dosing period (GD 5 to 20).
At 300 mg/kg, a statistically significant low value compared to the vehicle control group was recorded in cumulative body weight gain during the dosing period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, statistically significant low values compared to the vehicle control group were recorded in mean daily food consumption during each period of GD 5-7, 7-10, 10-12, 12-15 and 15-17, and in total food consumption for the total gestation period (GD 0 to 20).
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:
effects observed, treatment-related
Description (incidence and severity):
Serum Hormone (T3, T4 and TSH) Concentration:
At 1000 mg/kg, there was a statistically significant high value in TSH compared to the vehicle control group, but no significant difference in T3 and T4.
At 100 and 300 mg/kg, there was no statistically significant difference in TSH, T3 and T4 compared to the vehicle control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the test article dose groups, no statistically significant difference was observed in the thyroid gland weight and the pregnant uterine weight compared to the vehicle control group.
At 1000 mg/kg, a statistically significant low value was recorded in the corrected body weight [the value obtained by subtracting the weight of the pregnant uterus from the body weight on the day of cesarean section (gestation day 20) of the dam] compared to the vehicle control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross lesions were observed in the external appearance, and in the main organs/tissues in the thoracic or abdominal cavity in any animal.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No histological findings, which were considered to be the effects of the test article dosing, were observed in the thyroid gland and parathyroid gland.
Histopathological findings: neoplastic:
not examined
Details on results:
Cesarean Section Data on Dams
The number of pregnant rats were 22, 21, 21 and 22 at 0 (vehicle control), 100, 300 and 1000 mg/kg, respectively.
No statistically significant differences from the vehicle control group were noted in the number of corpora lutea, number of implantations, pre-implantation loss, or implantation index at the terminal phase of the gestation in any test article dose group.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions occurred in any group and no abnormal clinical signs were observed throughout the gestation period.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No effect of the test article dosing were observed post-implantation loss index.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No effect of the test article dosing were observed in the number of resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No effect of the test article dosing were observed in the number of resorptions.
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
The number of pregnant rats were 22, 21, 21 and 22 at 0 (vehicle control), 100, 300 and 1000 mg/kg, respectively. No deaths/abortions occurred in any group and no abnormal clinical signs were observed through the dosing period. At 1000 mg/kg, food consumption decreased after the start of dosing and body weight gain was suppressed during the dosing period, and lower value was recorded in the corrected body weight on GD 20. At 300 mg/kg, body weight gain was lower during the dosing period. These changes were considered test article-related. At 1000 mg/kg, higher TSH values were observed compared to the control group. However, no abnormality was observed in T3 and T4, and there was no effect of the test article dosing on the histopathology of thyroid and parathyroid. Therefore, it was considered to be an incidental change. No gross abnormalities were observed at necropsy in any group, and no changes related to the test article dosing were observed in the weight of thyroid and parathyroid.
There were no differences between the vehicle control group and each dosing group for the number of corpora lutea, number of implantations, pre-implantation loss index and implantation index at the end of pregnancy, and no test article-related effects were noted.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, a statistically significant low value was recorded in the body weight of live fetuses compared to the vehicle control group.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg, the number of live female fetuses was significantly lower than that of the vehicle control group, but it was considered to be incidental because the change was not dose-related.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No effect of the test article dosing were observed on the sex ratio.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A complex anomaly of the thread-like tail and anal atresia was found in 1 fetus at 1000 mg/kg. However, it was considered spontaneous, as it was only expressed in 1 fetus and its incidence was within the historical control data at the test facility (Min.-Max.: 0.00-0.37%, 28 studies, 2014-2019). In addition, 1 fetus at 100 mg/kg had anasarca, and 1 fetus at 300 mg/kg had proboscis, all of which were considered to be incidental because of dose-independent changes.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant differences were noted in the incidences of dams having anomalous fetuses and incidences of fetuses with any anomaly between the vehicle control group and each dosing group.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related effects on the visceral morphology of live fetuses.
Although a statistically significant differences were noted compared to the vehicle control group, it was considered to be incidental because of dose-independent change.
Details on embryotoxic / teratogenic effects:
There were no differences between the vehicle control group and each dosing group for the number of resorptions, post-implantation loss index, the number, sex ratio and AGD of live fetuses or the placenta weights, and no test article-related effects were noted. At 1000 mg/kg, low body weight of live fetuses were noted, and it was considered test article-related.
No macroscopic anomaly considered test article-related was found in the external appearance of live fetuses or the placenta. No treatment-related effects were observed on visceral or skeletal morphology of live fetuses. At 1000 mg/kg, the number of ossified sacral-caudal vertebra were low value in the progress of ossification, which was considered to be a possible change due to ossification delay accompanying development suppression shown by low body weight.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, suppression of body weight gain accompanied by a decrease in food consumption was noted during the dosing period in dams at 1000 mg/kg, and suppression of body weight gain was noted in dams at 300 mg/kg. There were no effects on reproductive function in pregnant dams in any dose group. Low body weight in live fetuses and low value of the number of sacral-caudal vertebrae were noted at 1000 mg/kg. However, there were no effects on the external appearance, visceral or skeletal morphology of the fetuses at this dose levels. Therefore, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 100 mg/kg, and the NOAEL for the embryo-fetal development was 300 mg/kg.