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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 June 2016 till 20 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Distilled Farnesane w/no BHT Lot#: 15P0112023100001
- Expiration date of the lot/batch: 03 March 2017
- Purity test date: 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark
- Stability under test conditions: formulations stable for at least twenty-one days
- Solubility and stability of the test substance in the solvent/vehicle: Formulations were prepared fortnightly and stored at approximately 4 °C in the dark



Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Envigo RMS (UK) Limited, Oxon, UK
- Females: nulliparous and non-pregnant: yes
- Age at study initiation:approximately six to eight weeks old
- Weight at study initiation:males weighed 220 to 258g, the females weighed 165 to 192g
- Fasting period before study: no
- Housing:solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:eight days

DETAILS OF FOOD AND WATER QUALITY: pelleted diet (Rodent 2014C eklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.), water was
supplied from polycarbonate bottles attached to the cage. The diet, drinking water bedding and environmental enrichment were
considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): give twelve hours continuous light and twelve hours darkness.

IN-LIFE DATES: from 03 June 2016 (first day of treatment) and 02 September 2016 (final day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered d by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an
identical manner with Arachis oil BP.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Amount of vehicle (if gavage): 4 mL/kg
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken and analyzed for concentration of Farnesane at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Annex 2. The results indicate that the prepared formulations were within ± 9% of the nominal concentration.
Duration of treatment / exposure:
ninety consecutive days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
2.5 mg/mL
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
25 mg/mL
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
250 mg/mL
No. of animals per sex per dose:
10 males per dose
10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were based on the results of previous toxicity work which included a 14 Day Repeated Dose Range Finding Toxicity Study in the rat (Envigo Study No. 41500675).

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes post dosing and one and hour after dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION : Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: The eyes of all control and treated animals were examined pre-treatment and all control and
high dose animals before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.

HAEMATOLOGY: Yes
Hematological and blood chemical investigations were performed on all surviving animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.

BLOOD CHEMISTRY: Yes
Blood chemistry was evaluated for all animals at the end of the study.


URINALYSIS: Yes
Urinalytical investigations were performed on all animals from each test and control group during Week 12 of the study. Urine samples were collected by housing the animals overnight in metabolism cages under normal hydration but without access to food.

HISTOPATHOLOGY:Yes
Samples of the tissues were removed from all animals and preserved in buffered 10% formalin. All tissues from control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.

Since there were indications of thyroid (both sexes) and spleen (females only) changes,examination was subsequently extended to include similarly prepared sections of the spleen (females only) and thyroids (both sexes) from animals in the low and intermediate groups. Also, Immunohistochemical staining of the kidneys for α-2u-globulin was performed (from additional sections from both kidneys) for males from the control and high dose groups.
Sacrifice and pathology:
On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Other examinations:
Functional Observations:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment:
Detailed individual clinical observations were performed for each animal using a purpose built arena. This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests:
- Motor Activity
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail
1979).
- Forelimb/Hindlimb Grip Strength
An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity:
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
Statistics:
Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry,
Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights. Data were analyzed using the decision tree from the Provantis TM Tables and Statistics Module

Results and discussion

Results of examinations

Clinical signs:
not specified
Description (incidence and severity):
Increased salivation was evident in animals of either sex treated with 1000 mg/kg bw/day from Day 43 (females) and Day 44 (males) onwards. Increased salivation was also evident in animals of either sex treated with 100 mg/kg bw/day albeit to a lesser extent and towards the latter stages of the study. No such effects were detected in animals of either sex treated with 10 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no treatment-related deaths.

One female treated with 1000 mg/kg bw/day was killed in extremis on Day 90. This female started to convulse during warming prior to blood sampling. In the absence of any similar clinical signs evident in the remaining 1000 mg/kg bw/day animals, this death was considered to be incidental.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No overall adverse effect on body weight development was evident.
Food efficiency:
no effects observed
Description (incidence and severity):
No overall adverse effect on food consumption was evident.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmic examination of the eyes from rats treated with 1000 mg/kg bw/day did not indicate any effect of treatment.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters measured.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters measured.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in functional performance considered to be related to treatment at 10, 100 or 1000 mg/kg bw/day.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant effects were detected in the organ weights measured.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic findings detected in surviving adult animals.

One control female, one male ands two females treated with 10 mg/kg bw/day and one male and one female treated with 100 mg/kg bw/day had reddened lungs at necropsy. One male treated with 1000 mg/kg bw/day had a small left seminal vesicle. In the absence of a true dose related response or any histopathological correlates, the intergroup differences were considered not to be of toxicological significance.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid Glands: Minimal follicular cell hypertrophy was present in three control males, one male treated with 10 mg/kg bw/day, three males and two females treated with 100 mg/kg bw/day and six males and three females treated with 1000 mg/kg bw/day. These changes showed a minor increase in animals treated with the test item; however it is considered that there was a degree of individual variation and that the incidence reflects this rather than an effect of treatment.

Spleen: Increased hematopoiesis was present (minimal) in one control female, four females treated with 10 mg/kg bw/day, two females treated with 100 mg/kg bw/day and five females treated with 1000 mg/kg bw/day. It was also present in one control male. Upon evaluation of the spleen from all females there was considerable variation in incidence of this low grade change. As this did not demonstrate any dose-related trend and was apparent in one sex only, this was considered to be the result of individual variation and of no toxicological significance.
Other effects:
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes in functional performance considered to be related to treatment at 10, 100 or 1000 mg/kg bw/day.

There were no inter-group differences considered to be treatment-related during the Behavioral Assessments.

Sensory reactivity scores across all test item-treated dose groups were similar to controls.

Full details of immunohistochemical staining reagents and equipment used are included in the raw data/study records.
Details on results:
The oral administration of Farnesane to rats by gavage, at dose levels of 10, 100 and 1000 mg/kg bw/day, did not result in any significant adverse toxicological effects. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: systemic toxicity

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral administration of Farnesane to rats by gavage, at dose levels of 10, 100 and 1000 mg/kg bw/day, did not result in any significant adverse toxicological effects.
The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.
Executive summary:

The study was designed to investigate the systemic toxicity of the test item, by repeated oral administration to the Wistar Han™:

RccHan™: WIST strain rat for a period of ninety consecutive days at dose levels of 10, 100 and 1000 mg/kg bw/day.  A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). The dose levels were based on the results of previous toxicity work which included a 14 Day Repeated Dose Range Finding Toxicity Study in the rat (Envigo Study No. 41500675). All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

The oral administration of Farnesane to rats by gavage, at dose levels of 10, 100 and 1000 mg/kg bw/day, did not result in any significant adverse toxicological effects. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.