Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Dose levels were selected based on the results of the 14-day preliminary study performed in these laboratories with this compound (Envigo Study Number TMR0053).
Principles of method if other than guideline:
None
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Solid
Details on test material:
None

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test system
The rat (virgin) was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Han Wistar [RccHan™;WIST] strain was used because of the historical control data available at this laboratory.

Animal supply, acclimatisation and allocation
Strain/Species: RccHan™; WIST rat.
Supplier: Harlan (UK) Ltd.
Number of animals: 44 males and 44 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatisation: Five days before commencement of treatment.

Age of animals at the start of the study
Males: 69 to 75 days old
Females: 62 to 68 days old

Weight range of animals at the start of the study
Males: 270 to 310 g
Females: 161 to 198 g

Allocation and identification
Allocation: On arrival and non-selective allocation to cages.
On Day 1 of study all animals were weighed and body weights were reviewed before dosing commenced by Study Management. Body weight of animals did not exceed ±20% of the mean for each sex. Four males were swapped between groups in order to ensure this was achieved.
Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Identification of cages: Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal housing, diet and water supply
Environmental control
Rodent facility: Full barrier - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 19-23ºC and 40-70%. There were no deviations from these ranges.
Lighting: Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal accommodation
Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatisation, gestation, littering and lactation periods.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution: The cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups.
Bedding : Solid bottom cages contained softwood based bark-free fibre bedding, which was changed at appropriate intervals each week.
Number of animals per cage
Pre-pairing: up to five animals
Pairing: one male and one female
Males after mating: up to five animals
Gestation: one female
Lactation: one female + litter

Environmental enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.

Diet supply
Diet: SDS VRF1 Certified pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted (removed overnight before blood sampling for haematology and blood chemistry investigations).

Water supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Formulation
Correction factor: A correction factor of 1.335 was used when calculating quantities of test substance used during dose preparation (including purity and ratio salt/base).
Vehicle: Purified water.
Method of preparation: Sufficient vehicle was added to the test material to obtain a solution and it was mixed by hand using a spatula. It was mixed using a magnetic stirrer to dissolve the test material. It was made up to volume with the remaining vehicle and mixed with a magnetic stirrer.
A series of solutions at the required concentrations were prepared by dilution of individual weighings of the test substance.
Frequency of preparation: Weekly.
Storage of formulation: Ambient (up to two days) or refrigerated (15 days).
Test substance accounting: Detailed records of compound usage were maintained. The amount of test substance necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Administration
Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg.
Volume dose: 10 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as the treated groups.
Frequency: Once daily, at approximately the same time each day.
Sequence: Groups dosed in ascending order.
Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Storage of formulation: The stability was confirmed following storage at ambient temperature (nominally 21°C) for up to two days, and refrigeration (nominally 2-8 °C) for up to 15 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation analysis
Stability and homogeneity: The stability of a solution of the test substance in the vehicle at concentrations of 10 mg/mL and 100 mg/mL was demonstrated over a period of up to 15 days at 2 8ºC and two days at 21ºC.
Achieved concentration: Samples of each formulation prepared for administration in the first and last weeks of treatment were analysed for achieved concentration of the test substance.

Analytical procedure
Apparatus and instrumentation
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Balances fitted with printers: Capability of weighing to 5 or 6 decimal places
General laboratory apparatus and glassware.

Reagents
Control vehicle: Purified water
Acetonitrile (ACN): Gradient HPLC grade
N,N-Dimethylformamide (DMF): HPLC grade
Tetra-n-Butylammonium bromide: Reagent plus, >99%
Propyl-4-hydroxy-benzoate: >99%
Water: Reverse osmosis
Diluent: DMF/water 20/80 v/v

Preparation of standards
A primary standard solution (1000 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of FAT 40061-F TE in diluent (50 mL). A secondary standard solution (250 μg/mL) was prepared by diluting primary standard solution in diluent (20 mL).
Solutions for instrument calibration were prepared by appropriate dilution of the secondary standard solution using diluent containing 1 mL of stock internal standard solution and contained FAT 40061-F TE at nominal concentrations of 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL and 25 μg/mL.
Calibration solutions were injected onto the HPLC, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the chromatographic section.
An internal standard (100 μg/mL) was prepared by dissolving (ca. 10 mg) propyl-4-hydroxybenzoate in diluent (100 mL).

Sample process
A representative sample of test formulation (1 mL, accurately weighed) was diluted using diluent to provide a solution containing FAT 40061-F TE at an expected concentration within the range 10 μg/mL to 20 μg/mL.
The concentration of FAT 40061-F TE in the final solution was quantified by HPLC using UV detection as detailed in the chromatographic section.

Typical chromatographic conditions
Column: Hypersil BDS, C18, 5 μm, 4.6 × 250 mm
Column temperature: 40°C
Sample temperature: 10°C
Mobile Phase A: Tetra-n-butylammonium bromide (2 g/L) in ACN/water 35/65 v/v
Mobile Phase B: Tetra-n-butylammonium bromide (2 g/L) in ACN/water 90/10 v/v

Linear Gradient:
Time (min.) % A % B
0 100 0
1.0 100 0
24.0 80 20
34.0 20 80
35.0 0 100
40.0 0 100
43.0 100 0
53.0 100 0

Flow rate: 1.2 mL/min
Detector wavelength: UV, 254 nm
Injection volume: 10 μL
Run time: 53 minutes
Approximate retention time: FAT40061-F TE: 23.2 minutes
Internal standard: 10.1 minutes

Calculation
The peak area response ratio (test item/internal standard) for FAT 40061-F TE in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration.
The concentration of FAT 40061-F TE was determined using the following equation:

Analysed concentration (mg/mL) = ((Y - I) / S) x (V / 1000)

Where
Y = Peak area response ratio for FAT 40061-F TE in test chromatogram
I = Intercept derived from linear regression of calibration data
S = Slope derived from linear regression of calibration data
V = Dilution factor of sample

Validation of the analytical procedure
The analytical procedure was validated by determining the following parameters:
The specificity of the chromatographic analysis in control sample chromatograms.
The limit of detection and quantification was estimated by examination of control vehicle chromatograms in order to calculate a test substance concentration based on a peak height response equivalent to three times and ten times baseline noise respectively.
The linearity of detector response over the calibration standard concentration range.
The repeatability of the lowest and highest concentration calibration standards.
The method accuracy and precision, by determining six procedural recoveries at nominal concentrations of 10 mg/mL and 100 mg/mL during the method validation.
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (purified water) with known amounts of FAT 40061-F TE. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

Homogeneity and stability in purified water formulations
The homogeneity and stability of FAT 40061-F TE in purified water formulations was assessed at nominal concentrations of 10 mg/mL and 100 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (100 mL) were equally sub-divided (2 × 50 mL) into two amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

Ambient temperature storage (nominally +21ºC)
On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion (representing 0 hour) and 2 hours, duplicate samples (nominally 1 mL) were removed for analysis from the middle of the formulation.
The remainder of the bottle was stored at ambient temperature and after 2 days storage the contents were remixed and sampled as detailed above.

Refrigerated storage (nominally +4ºC)
The remaining bottle was refrigerated on receipt and on Day 2 and Day 15, the bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion and single samples (nominally 1 mL) were removed for analysis from the middle of the formulation.

Concentration in test formulations
For the First and Last week of treatment, freshly prepared test formulations were sampled (4 × 1 mL, 2 × 3 mL for controls accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate samples were analysed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved. For the first week the contingency samples were analysed due to an analytical error for the original analysis.

Results
Method validation
The analytical procedure was successfully validated for FAT 40061-F TE in purified water with respect to the specificity of chromatographic analysis, limit of detection and quantification, the linearity of detector response, repeatability, method accuracy and precision. Results are summarised below:

The specificity of the HPLC assay was demonstrated by the absence of a peak at the characteristic retention time for FAT 40061-F TE in the control sample
chromatogram.
The limit of detection and quantification was estimated as 0.0148 μg/mL and 0.0494 μg/mL respectively.
Linearity was confirmed over the nominal concentration range 5 μg/mL to 25 μg/mL with a coefficient of determination >0.999;
The repeatability was <1% for six replicate injections of standard solutions containing FAT 40061-F TE at nominal concentrations of 5 μg/mL and 25 μg/mL
;
Method accuracy and precision were confirmed:
a mean procedural recovery value of 101.3% (CV=0.49%, n=6) was obtained for 10 mg/mL and 101.3% (CV=0.37%, n=6) was obtained for 100 mg/mL.

Formulation trial
The homogeneity and stability of FAT 40061-F TE in purified water formulations was assessed with respect to the level of concentration at nominal concentrations of 10 mg/mL and 100 mg/mL.
Stability was confirmed during distribution between the bottles and on re-suspension following storage at ambient temperature for 2 days and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the duplicate samples remained within 9% of the initial time zero value and the difference from mean was less than 5%.

Concentration in dose formulations
The mean concentrations of FAT 40061-F TE in test formulations analysed during the study and the deviation of the mean result from the nominal value are detailed. The mean concentrations were within applied limits +10%/-15%, confirming the accuracy of formulation. Difference from mean and coefficient of variation values were within 1% confirming precise analysis.

Conclusion
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The stability was confirmed for FAT 40061-F TE in purified water formulations at nominal concentrations of 10 mg/mL and 100 mg/mL at ambient temperature storage for 2 days and refrigerated storage for up to 15 days.
The mean concentrations of FAT 40061-F TE in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. Difference from mean and coefficient of variation values were within 1% confirming precise analysis.
Duration of treatment / exposure:
Males: Two weeks pre-pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 6 of lactation.
Animals of the F1 generation were not dosed.
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 330, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 per sex per dose
Control animals:
yes
Details on study design:
Treatment groups and doses
Dose levels were selected based on the results of the 14-day preliminary study performed in these laboratories with this compound (Envigo Study Number TMR0053).
In the 2-week preliminary study (Envigo Study Number TMR0053), repeated doses at 250, 500 and 1000 mg/kg/day were well tolerated for 14 days. Based on these results 1000 mg/kg/day was considered to be an appropriate high dose. This and the low and intermediate doses of 100 and 330 mg/kg/day were selected to investigate a dose response and with reference to the Globally Harmonized System of Classification and Labelling of Chemicals (2013).
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Serial observations
Clinical and behavioural observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

Signs associated with dosing
Daily during Week 1 of treatment and once each week thereafter and for females on Days 0, 6, 13 and 20 after mating and Day 1 and 6 of lactation, detailed observations were recorded at the following times in relation to dose administration:
One to two hours after completion of dosing
As late as possible in the working day.

Detailed physical examination and arena observations
Before treatment commenced and during each week of treatment and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”.
After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered 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. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were moved into individual cages prior to transport to the testing room, the length of separation of dam from litter was minimised. The cage labels on these individual cages showed only the study and animal number. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores probe/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
During Week 5 of treatment for males and at Days 4-6 of lactation for females, the motor activity of five lowest numbered surviving males and the five lowest numbered lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.5), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body weight
The weight of animals was recorded as follows:
F0 males:
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.

F0 females:
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing.
Days 0, 6, 13 and 20 after mating.
Day 1, 4, and 7 of lactation.
On the day of necropsy.

Food consumption
The weight of food supplied, that remaining and an estimate of any spilled was recorded as follows:
F0 animals: Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
Days 0-5, 6-12 and 13-19 after mating
Days 1-3 and 4-6 of lactation
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Haematology, peripheral blood
Blood samples were collected after overnight withdrawal of food and prior to dosing at the following occasion:
Week 2 prior to pairing: The five lowest numbered surviving males and females per group

Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyser:
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
Morphology:
Anisocytosis
Microcytosis
Macrocytosis
Hypochromasia
Hyperchromasia

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyser. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyser and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood chemistry
At the same time and using the same animals as for peripheral haematology, further blood samples were collected after overnight withdrawal of food and prior to dosing at the following occasion:
Occasion: Animals

Week 2 prior to pairing: The five lowest numbered surviving males and females per group.

Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyser in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acid (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb) See deviations to protocol.

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.


Vaginal smears
Wet smears : After pairing until mating, using pipette lavage.

Mating
Pairing commenced: After a minimum of two weeks of treatment.
Male/female ratio: 1:1 from within the same treatment groups.
Duration of pairing: Up to two weeks.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation: When positive evidence of mating was detected.
Male/female separation: Day when mating evidence was detected.
Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Parturition observations and gestation length
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Records made during littering phase
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
Sex ratio of each litter: Recorded on Days 1, 4 and 7 of age.
Individual offspring body weights: Days 1, 4 and 7 of age.
Sacrifice and pathology:
Terminal procedures
All adult animals were subject to a detailed necropsy. 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. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Time of necropsy
F0 males: After Week 5 investigations completed.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 7 of lactation.
F1 offspring: Day 7 of age.


Females
The following were recorded:
Each uterine horn: Number of implantation sites.

Offspring
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination: Subject to a complete macroscopic examination.

Organ weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: Initially in modified Davidson’s fluid.
For all animals examined, samples of any abnormal tissues were retained. In those cases where a lesion was not clearly delineated, contiguous tissue was fixed with the grossly affected region.

Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List: The five lowest surviving F0 males and females in Groups 1 and 4 at scheduled termination.
Abnormalities only: All F0 animals.
Kidneys and duodenum: All available animals.
Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light microscopy
Tissues preserved for examination were examined as follows:

Five lowest numbered F0 surviving males and females in Groups 1 and 4.
All specified
Five lowest numbered F0 surviving males and females Groups 2 and 3.
Kidney and duodenum.
All F0 adult animals: Abnormalities only.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Other examinations:
None
Statistics:
None

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Adult animal responses
Signs and mortality
There were no signs seen in association with dosing FAT 40061/F TE at 100, 330 or 1000 mg/kg/day.
Clinical signs seen in all treated groups include yellow staining of various parts of the body, which is considered a consequence of the administration of FAT 40061/F TE (a yellow dye).
Yellow bedding and/or yellow faeces were observed in the cages of treated animals receiving 330 or 1000 mg/kg/day.

Sensory reactivity and grip strength
Sensory reactivity and grip strength were unaffected by treatment.
Motor activity
There were no clear effects on either rearing (high beam) or ambulatory (low beam) motor activity.
Group mean low beam activity scores for males receiving FAT 40061/F TE at 330 or 1000 mg/kg/day were statistically significantly high at the first 6-minute interval compared with Controls. Total scores are also slightly high when compared with Controls, although statistical significance was not attained. In addition, high beam total scores for males receiving FAT 40061/F TE at 330 or 1000 mg/kg/day were marginally low compared with Controls. The slight inter-group differences can be attributed to natural variation.

Body weight
The body weight and body weight change of males receiving FAT 40061/F TE at 100, 330 or 1000 mg/kg/day has been similar to that of the Control during Weeks 0 – 5 of treatment.
The body weight and body weight change of females receiving FAT 40061/F TE at 100, 330 or 1000 mg/kg/day during the first two weeks of treatment prior to pairing was slightly higher than that of the Control, and a dose response was apparent. Weight gains during Week 2 were similar to Controls.
The body weight and body weight change of females receiving FAT 40061/F TE at 100, 330 or 1000 mg/kg/day up to Day 20 of gestation and during Days 1-7 of lactation was similar to that of the Control.

Food consumption
There was no effect of treatment on the food consumption of animals receiving FAT 40061/F TE at 100, 330 or 1000 mg/kg/day during Weeks 1 and 2 of treatment, or on the food consumption of males receiving FAT 40061/F TE at 100, 330 or 1000 mg/kg/day during Weeks 4 and 5 of treatment, or on the food consumption of females receiving FAT 40061-F TE at 100, 330 or 1000 mg/kg/day throughout gestation and until Day 7 of lactation.

Haematology
Haematology investigations in Week 2 of treatment revealed the following:
Haematocrit was statistically significantly low (p<0.05) for males receiving FAT 40061/F TE at 1000 mg/kg/day, when compared with the Control.

White blood cell counts were statistically significantly low (p<0.01) for males receiving FAT 40061/F TE at 1000 mg/kg/day, when compared with the Control.

Lymphocyte counts were statistically significantly low (p<0.05) for males receiving FAT 40061/F TE at 330 mg/kg/day, and statistically significantly low (p<0.01) for males receiving FAT 40061/F TE at 1000 mg/kg/day, when compared with the Control.

Monocyte counts were statistically significantly low (p<0.01) for females receiving FAT 40061/F TE at 100, 330 and 1000 mg/kg/day, when compared with the Control.

These findings occurred in one sex only, and were considered of uncertain significance to treatment.

Blood chemistry
Biochemistry investigations in Week 2 of treatment revealed the following:
Alanine aminotransferase was statistically significantly low (p<0.05) for males receiving FAT 40061/F TE at 1000 mg/kg/day, when compared with the Control.

Chloride concentration was statistically significantly high (p<0.01) for males and females receiving FAT 40061/F TE at 330 or 1000 mg/kg/day, when compared with the Control.
Total protein and albumin were statistically significantly high (p<0.05) for females receiving FAT 40061/F TE at 330 or 1000 mg/kg/day when compared with the Control.

These findings were considered of uncertain significance to treatment.

Pre-coital interval, mating performance, fertility and gestation length
Pre-coital interval, fertility and mating performance were unaffected by treatment with FAT 40061/F TE.
In all treated groups there was a reduction in the number of females with a 22 day gestation length and a higher number of females with a 23 day gestation length compared with the Controls and differences attained statistical significance at 330 or 1000 mg/kg/day. In addition, when compared with contemporaneous control data (CCD), an increased proportion of females with a 23 day gestation length has been observed. Therefore it is considered that this slight shift does not represent an effect of treatment with FAT 40061/F TE.

Organ weights
Organ weights for males killed after 5 weeks of treatment and for females killed on Day 7 of lactation showed no adverse effects of treatment.
The adjusted kidney weights for females killed on Day 7 of lactation were statistically significantly higher than in Controls, however, as no dose response was apparent, this difference is considered to be of no biological significance.
The ovarian weights for females killed on Day 7 of lactation were essentially similar amongst the groups, showing no adverse effects of treatment. The mean weight of the uterus/cervix/oviducts of females receiving 1000 mg/kg/day was statistically significantly lower than in Controls.

Macropathology
The macroscopic examination of males performed after five weeks of treatment revealed no test substance related lesions. The findings of abnormal colour are considered a consequence of administration of a yellow dye.
The macroscopic examination of females performed on Day 7 of lactation revealed no test substance related lesions.
The yellow colouration seen in a variety of tissues and in the gastrointestinal tract contents was considered to be due to the coloured nature of the compound and not representative of any pathological change.

Histopathology
Males killed after 5 weeks of treatment and females killed on Day 7 of lactation
Treatment related findings
Changes related to treatment with FAT 40061/F TE were seen in the kidneys and the duodenum.


F1 litter responses
Offspring clinical signs
The distribution of signs at physical examination showed no relationship to parental treatment.

Litter size, sex ratio and survival indices
One female in Group 2 (2F 55) which received FAT 40061/F TE at 100 mg/kg/day and one female in Group 4 (4F 48) which received FAT 40061/F TE at 100 mg/kg/day failed to litter and was killed on Day 25 after mating. 2F 55 was found to have four implantations at macroscopic examination, and was considered to have been pregnant. 4F 48 was found to have no implantations at macroscopic examination, and was considered not to have been pregnant. All other females were pregnant and all litters were raised to Day 7 of age.
There was no effect of treatment with FAT 40061/F TE on the number of implantations. Litter size, offspring survival and sex ratio were considered unaffected by parental treatment. The viability index (which reflects survival of the offspring between Days 1 and 7 of age) appears slightly low for females in Group 4 (1000 mg/kg/day), but this is largely due to the death of two pups in one litter (4F 44), and is considered incidental and not related to treatment with FAT 40061/F TE.

Offspring body weight
There was no effect of treatment on the mean body weights of male and female offspring at Day 1 of age.
Offspring body weight on Day 1 of age and the subsequent body weight gain up to Day 7 of age was unaffected by parental treatment. The minor differences seen are considered a consequence of natural variation between litters, and not a consequence of parental treatment with FAT 40061/F TE.

Offspring macropathology
Macroscopic examination of offspring that either died prematurely or at scheduled termination on Day 7 of age did not reveal any findings that could be attributed to treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No evidence for general systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Formulation analysis

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision. The stability was confirmed for FAT 40061 /F TE in purified water formulations at nominal concentrations of 10 mg/mL and 100 mg/mL at ambient temperature storage for 2 days and refrigerated storage for up to 15 days. The mean concentrations of FAT 40061/F TE in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. Difference from mean and coefficient of variation values were within 1% confirming precise analysis.

Signs and mortality

Incidence of yellow bedding

 

 

1M

2M

3M

4M

Week 1

0/2

0/2

0/2

2/2

Week 2

0/2

0/2

0/2

2/2

Week 3

0/2

0/2

0/2

2/2

Week 4

0/2

0/2

2/2

2/2

Week 5

0/2

0/2

2/2

2/2

Week 6

0/2

0/2

2/2

2/2

 

 

 

 

 

 

1F

2F

3F

4F

Week 1

0/2

0/2

0/2

2/2

Week 2

0/2

0/2

0/2

2/2

Gestation

 

 

 

 

Day 6

0/10

0/10

10/10

10/10

Day 13

0/10

0/10

10/10

10/10

Day 20

0/10

0/10

10/10

10/10

Lactation

 

 

 

 

Day 1

0/7@

0/4@

4/4@

6/6@

Day 4

0/10

0/9

10/10

9/9

Day 7

0/10

0/9

10/10

9/9

 

 

 

 

 

@ Not all cages recorded in error

Incidence of yellow faeces

 

1M

2M

3M

4M

Week 1

0/2

0/2

0/2

0/2

Week 2

0/2

0/2

0/2

0/2

Week 3

0/2

0/2

0/2

0/2

Week 4

0/2

0/2

0/2

0/2

Week 5

0/2

0/2

0/2

0/2

Week 6

0/2

0/2

0/2

0/2

 

 

 

 

 

 

1F

2F

3F

4F

Week 1

0/2

0/2

0/2

0/2

Week 2

0/2

0/2

0/2

0/2

Gestation

 

 

 

 

Day 6

0/10

0/10

0/10

0/10

Day 13

0/10

0/10

0/10

0/10

Day 20

0/10

0/10

0/10

0/10

Lactation

 

 

 

 

Day 1

0/7@

0/4@

0/4@

0/6@

Day 4

0/10

0/9

1/10

2/9

Day 7

0/10

0/9

0/10

8/9

 

 

 

 

 

@ Not all cages recorded in error

Histopathology

Treatment related findings

Kidneys

Vacuolation of the cortical tubules with granular deposits was seen in females and a few males given 1000 mg/kg/day and in one female given 330 mg/kg/day.

Summary of treatment related findings in the kidneys for males killed after 5 weeks of treatment and females killed on Day 7 of lactation

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

330

1000

0

100

330

1000

 

 

 

 

 

 

 

 

 

Vacuolation/granular deposits, cortical tubules

 

 

 

 

 

 

 

 

Minimal

Slight

Moderate

0

0

0

0

0

0

0

0

0

4

0

0

0

0

0

0

0

0

1

0

0

0

1

8

Total

0

0

0

4

0

0

1

9

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

5

5

8

5

4

5

9

 

 

 

 

 

 

 

 

 

 

Duodenum

Vacuolated cells in the lamina propria were seen in a few males and females given 1000 mg/kg/day and in one male given 100 mg/kg/day.

Summary of treatment related findings in the duodenum for males killed after 5 weeks of treatment and females killed on Day 7 of lactation

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

100

330

1000

0

100

330

1000

 

 

 

 

 

 

 

 

 

Vacuolated cells in the lamina propria

 

 

 

 

 

 

 

 

Minimal

Slight

0

0

1

0

0

0

2

0

0

0

0

0

0

0

1

1

Total

0

1

0

2

0

0

0

2

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

5

5

5

5

4

5

6

 

 

 

 

 

 

 

 

 

 

In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

 

Staging of the oestrous cycle was difficult in most animals as they had not returned to normal cycling following pregnancy but there were no differences between the groups.

 

Incidental findings

All other histological changes were considered to be unrelated to treatment.

Applicant's summary and conclusion

Conclusions:
In the absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development, the no observed adverse effect level was 1000 mg/kg/day.
Executive summary:

The objective of this study was the assessment of general systemic toxic potential of FAT 40061/F TE in rats, including a screen for reproductive/development effects, with administration of the test substance by the oral gavage administration for at least four weeks.

Three groups comprising of ten male and ten female rats received FAT 40061/F TE at doses of 100, 330 or 1000 mg/kg/day by oral gavage administration. Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 6 of lactation. Females were killed on Day 7 of lactation. The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, purified water at the same volume dose as the treated groups. 

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. 

The clinical condition, litter size and survival, sex ratio, body weight and macropathology for all offspring were also assessed. 

Results

The mean concentrations of FAT 40061/F TE in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation. Difference from mean and coefficient of variation values were within 1% confirming precise analysis.

Administration of FAT 40061/F TE at dose levels of 100, 330 and 1000 mg/kg/day was well tolerated with no effects that could be attributed to treatment on clinical condition, survival, food consumption, sensory reaction, grip strength, motor activity or organ weights. In addition there were no effects on pre coital interval, mating performance, fertility, gestation length, offspring survival or development. There were no adverse effects of treatment on haematology/blood chemistry parameters.

During the first week of treatment, mean body weight gain of females receiving 100, 330 or 1000 mg/kg/day was statistically significantly higher than in Controls and a dose response was apparent; there was no effect of treatment on body weight gain of males.

Macroscopic examination of the F0 parent males and females revealed yellow colouration in a variety of tissues and in the gastrointestinal tract contents; this was considered to be due to the coloured nature of the compound and not representative of any pathological change. Histopathological examination of a full range of tissues from these animals revealed vacuolation of the renal cortical tubules with granular deposits in females and a few males given 1000 mg/kg/day and in one female given 330 mg/kg/day and, vacuolated cells in the lamina propria of the duodenum were seen in a few males and females given 1000 mg/kg/day and in one male given 100 mg/kg/day. Neither of these changes was considered to represent an adverse effect of treatment.

Conclusion

It was therefore that in the absence of any evidence for general systemic toxicity or effects on reproductive performance/offspring development that the no observed adverse effect level was 1000 mg/kg/day.