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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
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. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Rikabinol HB
IUPAC Name:
Rikabinol HB
Constituent 2
Chemical structure
Reference substance name:
4,4'-isopropylidenedicyclohexanol
EC Number:
201-244-2
EC Name:
4,4'-isopropylidenedicyclohexanol
Cas Number:
80-04-6
Molecular formula:
C15H28O2
IUPAC Name:
4,4'-isopropylidenedicyclohexanol
Test material form:
solid: flakes
Details on test material:
- Name of test material (as cited in study report): Rikabinol HB
- Substance type: An industrial chemical
- Physical state: White flakes
- Analytical purity: 95.6%
- Lot/batch No.: 7095
- Expiration date of the lot/batch:
- Storage condition of test material: At ambient temperature and protected from light
- Other: Date of receipt: 3 May 2012

Test animals

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 or test system 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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

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.

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

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.