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Description of key information

NOAEL: 300 mg/kg/day

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 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
Specific details on test material used for the study:
Substance name: HBPA
CAS number: 80-04-6
Action: An industrial chemical
Description: White flakes
Storage conditions: At ambient temperature and protected from light
Supplier: Sponsor

Batch number: 7095

Expiry date: 31 December 2012

Purity: 95.6%
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.

IN-LIFE DATES: From: 10 September 2012 To: 28 October 2012
Route of administration:
oral: gavage
Details on route of administration:
Animals received the test material or vehicle control formulations orally at a volume-dosage of 5 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.
Vehicle:
corn oil
Details on oral 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 (if other than water): Corn oil chosen for consistency with preliminary study
- Amount of vehicle (if gavage): 5 mL/kg
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.
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 . The low dose of 100 mg/kg/day was selected with the aim to establish a no observed effect level (NOEL) and the intermediate and high dose levels were selected with the aim to establish a no observed adverse effect level (NOAEL) at between 300 and 1000 mg/kg/day.
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: n/a
- Post-exposure recovery period in satellite groups: n/a
- Section schedule rationale (if not random): n/a
Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes all FO adult animals
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic,
abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded. External and cut surfaces of the organs and tissues were examined as appropriate. Any abnormality in
the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.
For F0 females, the number of implantation sites in each uterine horn was counted. 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.


HISTOPATHOLOGY: Yes For the first five males and females in Groups 1 and 4 at scheduled termination all tissues were subject to histological processing. In addition the liver and thyroid for the first five male and first five female animals in Groups 2 and 3 at scheduled termination were subject to histological processing.

Those tissues subject to histological processing included the following regions:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and pons
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Liver - section from two lobes
Lungs - section from two major lobes, to include bronchi
Spinal cord - transverse and longitudinal section at the cervical level
Sternum - included bone marrow
Stomach - included keratinised, glandular and antrum in sections
Uterus - uterine body with cervix section
For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required for microscopic pathology.



Statistics:
Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented in the report 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.
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological investigations during Week 2 of treatment did not show any differences that could be attributed to administration with Rikabinol HB.
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.
Urinalysis findings:
not examined
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day organ weight analysis for males after 5 weeks of treatment and for females on Day 7 of lactation showed significantly high adjusted liver weights when compared with the Controls (p<0.01).
Organ weights for both males and females at 100 or 300 mg/kg/day were unaffected by treatment with Rikabinol HB.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The incidence and distribution of all findings were consistent with the common background seen in Sprague-Dawley rats of this age at these laboratories.
Neuropathological findings:
not examined
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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
behaviour (functional findings)
Key result
Critical effects observed:
no

Summary of treatment related findings in the liver for animals killed at scheduled termination

Group/sex                                               1M         2M         3M         4M       1F          2F          3F          4F

Dose (mg/kg/day)                                     0           100        300       1000     0           100        300       1000

Hepatocyte Hypertrophy,
Centrilobular

Minimal        0             0            0            4             0            0            0             3

Total         0             0             0            4             0             0           0          3

Number of tissues examined                     5             5            5            5           5            5            5            5

Summary of treatment related findings in the thyroid for animals killed at scheduled termination

Group/sex                                               1M         2M         3M         4M   1F          2F          3F          4F

Dose (mg/kg/day)                                     0           100        300       1000       0           100        300       1000

Follicular Cell Hypertrophy

Minimal         1             1            2            5            0            0            0             3

Total         1             1             2            5           0           0          0          3

Number of tissues examined                     5             5            5            5            5            5            5              5

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.
Executive summary:

Therefore for general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day.

Endpoint conclusion
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

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
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 males were treated for two weeks before pairing up to necropsy after a minimum of five weeks and the 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.

Overall HBPA 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.

The liver was identified as the main target organ, with treatment at 1000 mg/kg/day resulting in centrilobular hepatocyte hypertrophy for males and females. This microscopic change in the liver correlated with increased organ weight in animals 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.

In the thyroid, increased incidence of follicular cell hypertrophy was seen in both sexes receiving 1000 mg/kg/day. At this dose, 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. Such changes are often encountered in rodents where there is hepatic enzyme induction and results from an acceleration of the hepatic de-iodination of thyroxine (T4) to tri-iodothyronine (T3), affecting the feed-back control of thyroid metabolism. As a consequence, there is a compensatory increase of thyrotrophin-releasing hormone (TRH) from the hypothalamus and a consequential increase of thyroid stimulating hormone (TSH) production from basophilic cells of the pars distalis of the pituitary. The consequence of the increased thyroid stimulating hormone is a continuous stimulation of a normal thyroid gland, leading to thyroid hypertrophy/hyperplasia. These thyroid-related findings are not considered of any toxicological significance to man since, in man, the hormone-binding profiles differ to that of rodents, and the rodent thyroid is many times more responsive to hormonal imbalance than the human thyroid.

Several findings in the blood plasma may be secondary to the effect on the liver, i.e. 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. 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.

It is concluded that oral administration of HBPA 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. Therefore for general toxicity the no-observed-adverse-effect level (NOAEL) was considered to be 300 mg/kg/day.

Justification for classification or non-classification

The study concludes that the NOAEL for general toxicity is 300 mg/kg bw/day. For classification at Category 2, as a repeat-dose toxicant, the substance must show evidence of toxicity between 10 and 100 mg/kg bw/day. Therefore HBPA is not classified as toxic to health by prolonged exposure by the oral route under the CLP Regulation (Commission Regulation 1272/2008).