Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

NOAEL = 300 mg/kg/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 August 2012 to 21 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen as the test species because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available in this laboratory.
Rats were obtained from a reputable commercial supplier.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Commercial laboratory animal supplier
- Age at study initiation: (P) 70 days;
- Weight at study initiation: (P) Males: 328 to 388 g; Females: 225 to 268 g;
- Fasting period before study:
- Housing: The gridded cages used during pairing were suspended over trays covered with absorbent paper which was changed daily. For cages with solid floors, softwood based bark-free fibre was used as bedding and was sterilised by autoclaving and changed at least twice each week. Cages, cage-trays, food hoppers and water bottles were changed at appropriate intervals.
- Diet: The animals were allowed free access to a standard rodent diet (SDS VRF1 Certified diet)
- Water: Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes: Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod: Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance, Rikabinol HB, was prepared for administration as a series of graded concentrations in the vehicle. The required amount of the test material was weighed out,
transferred to a mortar and ground to a fine powder. Some vehicle was added and mixed using a pestle to form a paste. Further amounts of vehicle were gradually added and mixed to
produce a smooth, pourable suspension.
The suspension was poured into a measuring cylinder, which was wetted with vehicle and the mortar was thoroughly rinsed with vehicle which was also added to the cylinder. The suspension was then made up to the required volume with vehicle, transferred to a beaker and mixed using a high shear homogeniser until homogenous. The suspension was transferred
into containers, via syringe, whilst magnetically stirred. The remaining concentrations were formulated in ascending order using the same method.

DIET PREPARATION
- Rate of preparation of diet (frequency): All formulations were prepared weekly
- Storage temperature of food: 2-8°C

VEHICLE
- Justification for use and choice of vehicle: Corn oil is a widely accepted vehicle
- Amount of vehicle (if gavage): 5 ml/kg
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
- Further matings after two unsuccessful attempts: no; After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.
- After successful mating each pregnant female was caged (how): no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix. Specimen formulations (typically 400 mL) were prepared at concentrations of 5 and 200 mg/mL and equally split between four amber glass screw-capped bottles. The stability was assessed following storage at ambient temperature (nominally 21°C) for 0, 1 and 2 hours and 1 day (Bottle 1), and refrigeration (nominally 2 to 8 °C) for 1 day, 8 days and 15 days (Bottles 2, 3 and 4). Prior to initial sampling on each day, the formulation was mixed by 20-fold inversion and magnetically stirred for a minimum of five minutes. The homogeneity and stability of formulations were confirmed as part of this study after 24 hours storage at ambient temperature and after 15 days during refrigerated storage.
Samples of each formulation prepared for administration on the first and last occasions were analysed for achieved concentration of the test substance.

Analytical column: DB5ms, 30 m x 0.53 mm id., film thickness 1.5 μm
Injector temperature: 300°C
Injector mode: Splitless
Injection volume: 1 μL
Column temperature: Initial: 100°C for 1 min
Rate 1: 10°C/min to 260 °C
Rate 2: 30°C/min
Final: 300°C for 2 min
Carrier gas: Helium, 10 mL/min
Detector: Flame ionisation
Detector temperature: 360°C
Detector gas: Hydrogen, 30 mL/min
Air, 360 mL/min
Nitrogen (make-up gas), 5 mL/min
Approximate retention time: 14.8, 15.0 & 15.1 minutes (Rikabinol HB peaks)
16.4 minutes (internal standard)
Duration of treatment / exposure:
The test substance was administered to F0 males for two weeks before pairing up to necropsy after a minimum of five weeks and to F0 females for two weeks before pairing, throughout pairing and gestation, up to Day 6 of lactation.
Animals of the F1 generation were not dosed.
Frequency of treatment:
All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time each day, seven days per week.
Details on study schedule:
- F1 parental animals not mated
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study (0, 100, 300 and 1000 mg/kg/day) were selected in conjunction with the Sponsor based on findings from a 7-day preliminary repeat dose study with this compound performed at these laboratories (Huntingdon Life Sciences Report Number: MOG0005).
- Rationale for animal assignment: The test substance was administered to F0 males for two weeks before pairing and to F0 females for two weeks before pairing.
Positive control:
No positive control was used.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment, weekly from Week 2 for all F0 animals and on Days 0, 6, 13 and 20 of gestation and Days 1 and 6 of lactation for F0 females, detailed observations were recorded at the following times in relation to dose administration:
Immediately before dosing; Between one and two hours after completion of dosing; As late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each F0 animal was recorded during acclimatisation, on the day that treatment commenced (Week 0), weekly thereafter and before necropsy.
The weight of each F0 female was also recorded on Days 0, 6, 13 and 20 after mating and on Days 1, 4 and 7 of lactation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 2 before pairing before dosing
- Anaesthetic used for blood collection: Yes - isoflurane
- Animals fasted: Yes
- How many animals: first five F0 males and females per group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:Week 2 before pairing before dosing
- Animals fasted: Yes
- How many animals:first five F0 males and females per group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the five lowest numbered lactating females in each group at Days 4-6 of lactation.
Oestrous cyclicity (parental animals):
For 15 days before pairing, daily vaginal smears were taken from all females, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle. After pairing with the male, smearing was continued using pipette lavage, until evidence of mating was observed.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight, sperm count in testes,
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities,

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities;
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals; F0 males were killed after Week 5 investigations were completed.
- Maternal animals: All surviving animals. F0 females were killed on Day 7 of lactation.
GROSS NECROPSY
- All F0 adult animals were subject to a detailed necropsy.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.

Adrenals, Prostate, Brain, Seminal vesicles with coagulating glands, Epididymides (L&R), Spleen, Heart, Testes (L&R), Kidneys (L&R), Thymus, Liver, Uterus including cervix and oviducts, Ovaries (L&R). L&R Bilateral organs weighed individually
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F1 offspring were sacrificed at 7 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: For offspring surviving to scheduled termination, a careful external examination was performed for gross abnormalities and externally normal offspring were discarded without internal examination. Externally abnormal offspring were internally examined and any abnormal tissues were retained in an appropriate fixative.


Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including pre-coital interval and mating performance the similarity of the data was such that analyses were not considered to be necessary. All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Reproductive indices:
No data
Offspring viability indices:
No data
Clinical signs:
no effects observed
Description (incidence and severity):
Overall Rikabinol HB was well-tolerated at all doses with no adverse effect on general condition (including the sensory reactivity and grip strength tests) or food consumption. There was low bodyweight gain during treatment for males receiving 1000 mg/kg/day and for females receiving 1000 mg/kg/day during the first and last week of gestation. There was also an effect on motor activity in males at 1000 mg/kg/day with high scores throughout the majority of the one-hour recording period for both rearing and ambulatory activity. There were no observations of overactive behaviour at the assessments of clinical signs during the study.
Mortality:
no mortality observed
Description (incidence):
There were no deaths and detailed physical and arena observations revealed no signs that were attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall bodyweight gain for males receiving 1000 mg/kg/day was significantly low when compared with Controls (Weeks 0-5; p<0.05). Bodyweight gain for males receiving 100 or 300 mg/kg/day was essentially similar to the Controls.
Bodyweight gains for females before pairing did not show any adverse effects of treatment.
During Days 0-6 of gestation, however, females receiving 1000 mg/kg/day showed slightly but significantly high weight gain (p<0.05), whilst during Days 13-20 of gestation weight gain was significantly low when compared with Controls (p<0.01). Overall bodyweight gain during gestation (Days 0-20), did not show any adverse effect of treatment at dose levels up to 1000 mg/kg/day.
Bodyweight gain for females during lactation was unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption for males during treatment and for females before pairing, during gestation and lactation did not show any clear effects of treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
During Week 2 of treatment plasma creatinine levels for both males and females at 1000 mg/kg/day were significantly high when compared with the Controls (p<0.05 and p<0.01 respectively). Males receiving 300 or 1000 mg/kg/day and females receiving
1000 mg/kg/day had significantly high total protein when compared with Controls. In addition males receiving 300 or 1000 mg/kg/day had significantly low glucose levels and males at all dose levels had low phosphorous levels, although a dose response was not apparent. Females receiving 1000 mg/kg/day also showed high cholesterol and triglyceride levels and high potassium and calcium plasma concentrations, when compared with Controls and females receiving 300 or 1000 mg/kg/day showed elevated alanine amino-transferase activities. The Albumin/Globulin ratio for females at 1000 mg/kg/day was also significantly low when compared with Controls.
Several findings in the blood plasma may be secondary to the effect on the liver, namely increased cholesterol and triglyceride levels, with elevated alanine amino-transferase activities in females, and increased total protein in both sexes; all of which may be associated with altered liver metabolism (see effects noted below). Also, as calcium is bound in the plasma to albumin, the increased calcium concentrations seen in the females may be associated with the increase in total protein.
Plasma creatinine levels were high for both males and females however in the absence of any effect on kidney weight and any macroscopic or microscopic findings in the kidney, the aetiology of this finding remains unclear.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Sensory reactivity observations and grip strength values were considered to be unaffected by treatment.
There was an effect on motor activity in males at 1000 mg/kg/day with high scores throughout the majority of the one-hour recording period for both rearing and ambulatory activity. There were no observations of overactive behaviour at the assessments of clinical signs during the study.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with Rikabinol HB were seen in the liver and thyroid.
Minimal centrilobular hepatocyte hypertrophy was seen at 1000 mg/kg/day in both sexes.
The liver was identified as the main target organ, with treatment at 1000 mg/kg/day resulting in centrilobular hepatocyte hypertrophy for both males and females. This microscopic change in the liver correlated with increased organ weight in males and females at 1000 mg/kg/day. Hepatocytic hypertrophy is encountered commonly in rodents following exposure to high levels of a xenobiotic and is normally attributed to induction of hepatocellular enzymes and therefore indicative of metabolic adaptation and it is considered that this is the most likely explanation in this study.
Increased incidence of minimal follicular cell hypertrophy of the thyroid was present at 1000 mg/kg/day in males and females and was characterised by colloid depletion, obliteration of follicular space or partial collapse of follicles lined by hypertrophic thyroid follicular epithelium. At this dose level, the morphological changes seen in the thyroid were considered to be a secondary response to liver enzyme induction and correlated with the significant increase in the liver organ weight.
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
During the two week treatment period before pairing the majority of females showed regular 4/5 day oestrous cycles.
Two Control females, one female receiving 300 mg/kg/day and one female receiving 1000 mg/kg/day were acyclic, with at least ten days without oestrus, and one female at 1000 mg/kg/day showed an irregular cycle (at least one cycle of two, three or six to ten days). The group distribution for these abnormal cycles did not show any relationship to treatment with Rikabinol HB.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm counts were not affected by Rikabinol HB.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating performance and fertility, as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment.
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The test substance was administered by oral gavage and no probems have been noted.

OTHER FINDINGS (PARENTAL ANIMALS)
During Week 2 of treatment plasma creatinine levels for both males and females at 1000 mg/kg/day were significantly high when compared with the Controls (p<0.05 and p<0.01 respectively). Males receiving 300 or 1000 mg/kg/day and females receiving 1000 mg/kg/day had significantly high total protein when compared with Controls. In addition males receiving 300 or 1000 mg/kg/day had significantly low glucose levels and males at all dose levels had low phosphorous levels, although a dose response was not apparent. Females receiving 1000 mg/kg/day also showed high cholesterol and triglyceride levels and high potassium and calcium plasma concentrations, when compared with Controls and females receiving 300 or 1000 mg/kg/day showed elevated alanine amino-transferase activities. The Albumin/Globulin ratio for females at 1000 mg/kg/day was also significantly low when compared with Controls.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Changes related to treatment with Rikabinol HB were seen in the liver and thyroid.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
Offspring survival was unaffected by parental treatment.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic examination of offspring that died before scheduled termination on Day 7 of age did not reveal any abnormality that could be related to parental treatment. Examination of offspring on Day 7 of age did not reveal any abnormality.

OTHER FINDINGS (OFFSPRING)
Offspring bodyweight on Day 1 of age was unaffected by treatment. However for offspring derived from females receiving 1000 mg/kg/day the subsequent gain was high when compared with Controls, attaining statistical significance for Days 1 to 4 for both male and female offspring (p<0.05). Overall the mean weight gain for male and female offspring Days 1 to 7 of age was 118% of Controls; this difference attained statistical significance for female offspring (p<0.05).
Offspring bodyweight gain at 100 or 300 mg/kg/day was unaffected by parental treatment.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
ca. 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: survival and development up to Day 7 of age not affected by treatment up to 1000 mg/kg/day
Key result
Reproductive effects observed:
no
Conclusions:
It is concluded that oral administration of Rikabinol HB to Sprague-Dawley rats at doses of 100, 300 and 1000 mg/kg/day caused mild toxicity at the highest dose, evident as reduced weight gain, increased motor activity for males at 1000 mg/kg/day, and with evidence for adaptive change in the liver and thyroid for both males and females at 1000 mg/kg/day. There was no adverse effect of treatment on mating performance, fertility and offspring survival and development up to Day7 of age, however at 1000 mg/kg/day, litter size was low from implantation.

Therefore for general toxicity and reproductive function the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted (Huntingdon Life Sciences, 2013, Study MOG0017) to assess the general toxicity and reproductive toxicity of HBPA in the rat. The study was conducted in accordance with OECD 422 guideline and in compliance with GLP.

Based on findings from a 7-day preliminary repeat dose study, three groups each comprising ten male and ten female Sprague-Dawley [Crl:CD(SD)] rats received oral admiration of HBPA at doses of 100, 300 or 1000 mg/kg/day formulated in corn oil. The F0 males were treated for two weeks before pairing up to necropsy after a minimum of five weeks and the F0 females were treated for two weeks before pairing until Day 6 of lactation. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose throughout the same period. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.

During the two-week treatment period before pairing the majority of females showed regular 4/5 day oestrous cycles. Pre-coital interval, mating performance and fertility, as assessed by percentage mating, conception rate and fertility index, gestation length and index were unaffected by treatment. At 1000 mg/kg/day the number of uterine implantation sites, the total number of offspring on Day 1 of age and the number of live offspring from Day 1 to Day 7 of age were significantly lower than Controls Offspring survival and sex ratio were unaffected by parental treatment. Offspring bodyweight on Day 1 of age was unaffected by treatment. However, for offspring derived from females receiving 1000 mg/kg/day the subsequent gain was high when compared with Controls. Macroscopic examination of offspring that died before scheduled termination or as scheduled on Day 7 of age did not reveal any abnormalities that could be related to parental treatment.

It is concluded that oral administration of HBPA to Sprague-Dawley rats at doses of 100, 300 and 1000 mg/kg/day there was no adverse effect of treatment on mating performance, fertility and offspring survival and development up to Day7 of age, however at 1000 mg/kg/day, litter size was low from implantation. Therefore for reproductive function the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day.

Effects on developmental toxicity

Description of key information

NOAEL = 300 mg/kg/ day

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
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%
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)
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
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.
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
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.
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.
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
Key result
Abnormalities:
no effects observed
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.
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
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
no
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.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

The prenatal developmental toxicity study (BoZo Research, 2020, Study R-1246) was performed to assess the effects of HBPA on pregnant animals, embryo development after implantation and fetal growth. The study was conducted in accordance with OECD 414 and in compliance with GLP.

 

HBPA, suspended in the corn oil, was administered orally by gavage at the dose levels of 100, 300 and 1000 mg/kg to Sprague-Dawley strain SPF pregnant rats [Crl:CD (SD)] once a day from gestation day (GD) 5 to GD 19, which corresponds to the day of implantation to the day before cesarean section. The animals in the control group received the corn oil, the vehicle, alone in the same manner.

 

The number of pregnant rats were 22, 21, 21 and 22 at 0 (vehicle control), 100, 300 and 1000 mg/kg, respectively. There were no deaths/ abortions nor effects from dosing of the test article on clinical signs or necropsy in any dose group. There was no effect of the test article dosing on the serum hormone (T3, T4 and TSH) concentration, the histopathology and weight of thyroid and parathyroid. At 300 and 1000 mg/kg, suppression of body weight gain was noted during the dosing period.

 

There were no test article-related effects in the number of corpora lutea, number of implantations, pre-implantation loss index or implantation index in the last stage of the gestation period in any dose group. At 1000 mg/kg, low fetal body weight was recorded and the number of sacral-caudal vertebrae (progress of ossification) were low value. There were no remarkable differences between the vehicle control group and these dose groups in the number of resorptions, post-implantation loss index, the number, sex ratio and AGD of live fetuses or the placenta weights. There were no anomalies in the external appearance, placenta, visceral or skeletal morphology of live fetuses.

 

The NOAEL for maternal toxicity was 100 mg/kg based on the suppression of body weight gain during the dosing period in dams at 300 mg/kg or more, although there were no effects on reproductive function in pregnant dams in any dose group. The NOAEL for the embryo-fetal development was 300 mg/kg based on the low body weight in live fetuses at 1000 mg/kg.

Justification for classification or non-classification

The study concludes that the NOAEL for reproductive function for HBPA is 300 mg/kg bw/day. There was no adverse effect of treatment on mating performance, fertility and offspring survival and development up to Day7 of age. The NOAEL for the embryo-fetal development was 300 mg/kg based on the low body weight in live fetuses at 1000 mg/kg. On this evidence this substance will not be classified as toxic to reproduction.