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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:
Experimental start date: 8 December 2021
Final report date: 11 January 2023 (see attached justification)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
EC Number:
212-985-6
EC Name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
Cas Number:
901-44-0
Molecular formula:
C19H24O4
IUPAC Name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
Test material form:
solid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen as the test species because it is accepted 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 at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Strain/Species Crl:CD(SD) rat.

Supplier Charles River (UK) Ltd.

Number 54 males and 54 females.

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization 13 days before commencement of treatment.

Age at start of treatment 42 to 48 days.

Weight range at the start of treatment
Males: 203 to 286 g
Females: 145 to 213 g

Allocation and Identification
Allocation Randomly allocated on arrival.

Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.

Identification of animals
Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.

Identification of cages
Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
On Day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ¿20% of the mean for the appropriate sex. No replacements were necessary.

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize 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 20-24¿C 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 Polycarbonate body with a stainless-steel mesh lid, changed at appropriate intervals.

Cage distribution Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.

Number of animals per cage Up to 4 of the same sex, unless reduced by mortality.

Bedding Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen gnawing material (soft white untreated wood blocks/ balls), Diamond Twists (paper product) and a plastic shelter Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet Teklad 2014C diet.

Availability Non-restricted (removed overnight before blood sampling for hematology or blood chemistry and during the period of urine collection).

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

Availability Non-restricted (except during the period of urine collection).

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the wood based bedding, Aspen gnawing material and Diamond Twists.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.

Procedure A 5% sugar solution (glucose or sucrose) was used to lubricate the cannula immediately before administration.
Reward Each animal was given a piece of natural popcorn (cooked within the facility daily) after the dosing procedure (on return to the home cage). Popcorn was also offered during pre-treatment period from Day -3 but not during the recovery period.
Vehicle:
methylcellulose
Remarks:
0.5% (w/v) methylcellulose 4000 cps aqueous solution
Details on oral exposure:
Method of preparation
Starting with the control group, a sufficient amount of vehicle was dispensed into the appropriate containers prior to starting the test formulations.

Under yellow light the required amount of test item was weighed, ground in a mortar using a pestle and mixed with some vehicle to form a paste. The suspension was homogenized by manual stirring before the final volume was made up with the vehicle. The suspension was transferred into a beaker and mixed using a high shear homogenizer at 11000 rpm for 10 minutes. It was then stirred for a minimum of 15 minutes using a magnetic stirrer.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.

Frequency of preparation
Groups 1 to 3: weekly throughout.
Group 4: Weekly initially and then daily from Day 23.

On Day 3 it was not possible to dose the Group 4 females as the dose could not be drawn up the catheter. The dose was re-suspended in pharmacy and also fresh dose was prepared but these could still not be drawn up the catheter (the females have a narrower gauge catheter due to their smaller bodyweight). Following discussion with the Sponsor it was decided to increase the dose volume to 10 mL/kg from Day 4 and subsequently reduce all the dose concentration by 50%. The Group 4 females were not dosed on Day 3 (see Section 4).

On Day 22 it was again not possible to dose the Group 4 males and females as the dose could not be drawn up the catheter. The dose was re-suspended and re-tested in pharmacy but it could still not be drawn up the catheter. Following discussions with the Sponsor it was decided to prepare and test a smaller volume of dose at 100 mg/mL (a daily volume rather than that required for a week of dosing). This could be drawn up the catheter easily. Consequently, the Group 4 dose was prepared daily from Day 23 (see Section 4).

Expected appearance White / off-white suspension

Storage of formulation Refrigerated (2 to 8°C), unless prepared in the morning before dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Achieved concentration Samples of each formulation prepared for administration in Weeks 1, 4, 8 and 12 of treatment were analyzed for achieved concentration of the test item.

The analytical method involved extraction and dilution of samples in ACN/Water, 60/40 v/v, followed by reverse phase high performance liquid chromatographic analysis with ultra violet detection at 230 nm. Sample concentrations were determined with reference to external standards prepared in the concentration range of 1 µg/mL to 20 µg/mL.
Duration of treatment / exposure:
Minimum period 13 weeks followed by a 4-week recovery period
All surviving Main study animals were treated throughout the necropsy period. Cessation of treatment for the Recovery study animals started on the first day of the necropsy of animals allocated to the Main study. Serial observations were recorded at appropriate intervals.
Frequency of treatment:
Once daily at approximately the same time each day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle - Days 1 to 3: Volume dose 5 mL/kg
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Days 1 to 3: Volume dose 5 mL/kg
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Days 1 to 3: Volume dose 5 mL/kg
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Days 1 to 3: Volume dose 5 mL/kg
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle - From day 4: Volume dose 10 mL/kg
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
From day 4: Volume dose 10 mL/kg
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
From day 4: Volume dose 10 mL/kg
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
From day 4: Volume dose 10 mL/kg
No. of animals per sex per dose:
Main study
10 males / 10 females per treatment group

Recovery study
Control: 5 males / 5 females
1000 mg/kg dose group: 5 males / 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection
The dose levels selected for this study were based on the results of an OECD 422 combined toxicity and reproductive screening study conducted on 2,2-isopropylidenebis(p-phenyleneoxy)diethanol in Sprague-Dawley rats at doses of 100, 300 and 1000 mg/kg bw/day in 0.5% (w/v) methylcellulose 4000 cps aqueous solution.

At 1000 mg/kg bw/day, two females were prematurely sacrificed on Day 6 of treatment, or Day 2 post-partum, following poor clinical condition (piloerection, emaciated appearance, hypoactivity, dyspnea, etc.). As only one animal had to be sacrificed on the first week of exposure at this dose level, this was likely to be incidental. The female sacrificed on Day 2 post-partum (after approximately 6 weeks of treatment) did not have remarkable signs until the day of sacrifice, so it is considered that the timing after gestation likely impacted on its clinical condition. Piloerection and round back were observed in several animals of both sexes, considered to be test item-related. A statistically significant reduction in bodyweight gain was observed in male animals on the first week of treatment. Thereafter, bodyweight gain was similar to control animals. The statistically significantly lower bodyweight reported at termination of male animals after five weeks of treatment (down to -10% versus controls at termination) was attributable to the transient reduction in bodyweight gain observed on the first week of exposure. In females, mean bodyweight was statistically significantly lower during gestation and lactation (down to -12% versus controls at the beginning of lactation). Effects on bodyweight and bodyweight gain in both males and females were not considered to be adverse as they were of slight amplitude.

At 300 mg/kg bw/day, no treatment-related mortality was observed. Ptyalism (salivation) was noted in about half of the animals and two females had piloerection plus round back for one of them. Mean bodyweight gains of males were statistically significantly lower in the first week of treatment and/or over the entire treatment period. These effects were considered to be of limited toxicological significance; mean bodyweights were not toxicologically affected. There were no toxicologically significant effects in mean bodyweight or mean bodyweight gain in females.

According to the OECD Testing Guideline 408, the highest dose level should be chosen with the aim to induce toxicity but not death or severe suffering. Considering that the most significant effect attributable to treatment at 1000 mg/kg bw/day during the OECD 422 was a transient reduction in bodyweight gain during the first week of treatment, this dose-level was not expected to induce death or severe suffering during the OECD 408 and was therefore selected as the high dose-level. Doses of 500 and 250 mg/kg bw/day were selected for the intermediate and low dose levels, respectively.

Examinations

Observations and examinations performed and frequency:
Serial Observations

Clinical and Behavioral Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. 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 acclimatization and recovery periods, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end of each week) and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:

• Pre-dose observation.
• 1 to 2 hours after completion of dosing all groups.
• As late as possible in the working day.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment and recovery, 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 who was unaware of the experimental group identities.

After removal from the home cage, animals were assessed for physical condition and behavior 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 behavior.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory Reactivity Observations and Grip Strength
Sensory reactivity and grip strength assessments were performed on the Main and Recovery study animals (before dosing) during Week 12 of treatment. In addition, all Recovery study animals were assessed during Week 4 of recovery. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. 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 on each day of testing.

The following measurements, reflexes and responses were recorded:

Approach response

A blunt probe was brought towards the head until it was close to the nose (but not touching the whiskers). The reaction was recorded as:

1 No reaction or ignores/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 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 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 12 of treatment (before dosing) the motor activity of all Main and Recovery study animals was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Labcorp. In addition, all Recovery study animals were tested during Week 4 of recovery.

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 necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

Mortality
A viability check was performed near the start and end of each working day. A complete necropsy was performed in all cases.

Body Weight
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

Water Consumption
During Week 11 of treatment and Week 3 of recovery, water consumption was recorded by weight (over a 3-day period on each occasion) for each cage of animals, using water bottles fitted with sipper tubes.

Ophthalmic Examination
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows:

Occasion Animals
Pretreatment All Main and Recovery study animals, including spares
Week 12 All Main study animals of Groups 1 and 4

Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

As there were no treatment-related changes seen in Week 12, the examination was not extended to include Main study animals of Groups 2 and 3 or Recovery study animals during Week 12, or to the Recovery study animals at the end of the recovery period.

Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food at the following occasions:

Occasion Animals
Week 13 All Main study animals
At necropsy (after 13 weeks of treatment) All Main study animals (EDTA sample only) #
Week R4 All Recovery study animals
At necropsy (after 4 weeks of recovery) All Recovery study males (EDTA sample only) #

# Due to the number of clotted EDTA samples at the Week 13 and R4 sampling occasions, additional EDTA samples were taken from the specified animals at necropsy (without overnight deprivation of food).

Sampling in Week 13 and Week R4 was performed on the morning after overnight collection of urine. Animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.

Animals were held under light general anesthesia 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 analyzer:

• Hematocrit (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Absolute reticulocyte count (Retic)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)

* Derived values calculated in ClinAxys.

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. A manual count of the differential white blood cell parameters was performed where necessary.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer 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
Blood samples were collected after overnight withdrawal of food at the following occasions:

Occasion Animals
Week 13 All Main study animals
Week R4 All Recovery study animals

Sampling was performed on the morning after overnight collection of urine. Animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures.

Animals were held under light general anesthesia 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 Cobas 6000 Analyzer in respect of:

• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Total bilirubin (Bili)
• Bile acids (Bi Ac)
• Urea (numerically equivalent to blood urea nitrogen)
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• Cholesterol - high density lipoprotein (HDL)
• Cholesterol - low density lipoprotein (LDL)
• Sodium (Na)
• Potassium (K)
• Total protein (Total Prot)
• Albumin (Alb)

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

Urinalysis
Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight at the following occasions:

Occasion Animals
Week 13 All Main study animals
Week R4 All Recovery study animals

The individual samples were examined for the following characteristics:
Using manual methods:

• Clarity and Color (App) - by visual assessment
• Volume (Vol) - using a measuring cylinder
• pH - using a pH meter
• Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips interpreted using the Clinitek¿500 instrument:
• Bile pigments (Bili)
• Blood pigments (UBld)

Using a Roche Cobas 6000 analyzer:
• Protein - total (T-Prot) and concentration (Prot)
• Glucose - total (T-Gluc) and concentration (U-Gluc)

Estrous cycles
Wet smears Wet smears were taken from the vagina of all Main and Recovery study females using pipette lavage for the 4 consecutive days before scheduled termination. The last smear was taken on the morning of necropsy.

Smears were assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus) at termination and were used to assist in the histological evaluation of estrogen sensitive tissues.

Thyroid Hormone Analysis
Blood samples were collected at necropsy as follows:

Occasion Animals
Week 14 All Main study animals
Week R5 All Recovery study animals

Parameters Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)

Sequence of blood sampling on each occasion In order to minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.

Conditions No overnight deprivation of food.
Anesthetic Isoflurane. The animals did not recover from the anesthesia.
Blood sample site Sublingual vein.
Anticoagulant None.
Blood volume 2.0 mL.
Blood tubes Grenier Minicollect tubes with clotting activator.

Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.

Centrifugation conditions At 2000g for 10 minutes at 4°C.
Aliquot volumes Aliquot 1 (T3 and T4): 0.2 mL of serum
Aliquot 2 (TSH): 0.2 mL of serum
Aliquot 3 (for additional hormones): all remaining serum

Final storage conditions Deep frozen (-60 to -80°C) pending analysis.

Analysis The method of analysis and results are presented in Attachment 14.3 (for T3 and T4) and Attachment 14.4 (for TSH).

As the TSH concentrations were unaffected after 13 weeks of treatment, the serum samples taken from the Recovery study animals after 4 weeks of recovery were not analyzed and subsequently discarded following issue of the final report. To be updated if required following results of T3 and T4 analysis.

The samples for additional hormones were not analyzed and were subsequently discarded following issue of the final report.
Sacrifice and pathology:
Necropsy
All Main and Recovery study 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. The retained tissues were checked before disposal of the carcass.

Schedule Main study animals were killed following 13 weeks of treatment.
Recovery study animals were killed following 13 weeks of treatment and 4 weeks of recovery.
Sequence To allow satisfactory inter-group comparison.

The organs weighed,
Tissue and regions examined

Abnormalities
Adrenals
Aorta
Brain (cerebellum, cerebrum, midbrain)
Bone marrow smear
Cecum
Coagulating glands
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femur (femorotibial joint)
Head
Heart (including auricular and ventricular regions)
Ileum
Jejunum
Kidneys (routine and special staining )
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes - mesenteric
- left axillary
Ovaries
Pancreas
Peyer’s patches
Pituitary
Prostate
Rectum
Salivary glands - submandibular
- sublingual
- parotid
Sciatic nerves
Seminal vesicles
Skeletal muscle
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum (and bone marrow)
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina

Bone Marrow
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods, and from animals killed prematurely during the study.
Fixation Smears were air dried and subsequently fixed in methanol.
Retention No examinations were performed, however, the smears were retained for possible future examination.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:

Testes In modified Davidson’s fluid.
Eyes In Davidson’s fluid.
Bone marrow smears Methanol.

Sperm Analysis
Analysis was performed on all Main study males killed after 13 weeks of treatment and on all Recovery study males killed after 4 weeks of recovery.

Immediately following death, the left vas deferens, left epididymis and left testis were removed from each animal. The epididymis and testis were weighed before processing. The following tests were performed:

Sperm motility
A sample of sperm was expressed from the vas deferens into pre-warmed (37¿C) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 ¿m depth cannula by capillary action and at least 200 sperm per animal analyzed using the Hamilton Thorne IVOS Computer Assisted Sperm Analyzer (CASA).

Sperm morphology
A 200 L micro L aliquot of the sperm/medium mixture (described above) was diluted with 800 micro-L of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared for each male. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male.

Sperm count
The left cauda epididymis of each male was weighed, the tunica was removed, then homogenized for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Homogenization-resistant spermatids count
After removal of the tunica, the left testis was homogenized for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenization-resistant spermatid count using CASA.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal 4 to 5 µm thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full List All animals killed or dying prematurely.

All Main study animals of Groups 1 and 4 killed after 13 weeks of treatment.

Kidneys, liver and abnormalities All Main study animals of Groups 2 and 3 killed after 13 weeks of treatment

All Recovery study animals killed after 4 weeks of recovery.

Routine staining 4-5µm sections were stained with hematoxylin and eosin.

Additional processing and special staining - kidneys (males only) Additional 4-5 µm sections were prepared from the kidneys of all Main study males in Groups 1 and 4 (including early decedent males) and stained by immunohistochemistry for alpha-2u-globulin using a validated immunohistochemistry method.

Light Microscopy
Tissues preserved for examination were examined as follows:

Category Animals Tissues
Premature deaths All animals from all groups. All specified in Section 3.7.2.
Scheduled kill All Main study animals of Groups 1 and 4. All specified in Section 3.7.2.
All Main study animals of Groups 2 and 3. Kidney, liver and abnormalities
All Recovery study animals

A detailed qualitative examination of the right testis 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 into the lumen. Any cell- or stage-specificity of testicular findings was noted.

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.
Statistics:
Please refer to the section "Any other information on materials and methods"

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The general appearance and behavior of the animals were generally unaffected by treatment.
Two females receiving 1000 mg/kg/day (Nos. 115 and 118) had tremors or convulsive episodes lasting up to 45 seconds from Week 12. Female No. 118 was subsequently killed for welfare reasons due to its clinical condition following a 3rd episode. The convulsions were considered to have been triggered by being handled.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were five premature deaths during the treatment period and one during the recovery period but none of these deaths were considered to be associated with treatment.

Male No. 26 (Group 1, Control, Main study) was found dead after dosing in Week 5. There were no signs seen before death and no findings at necropsy. Microscopic findings comprised dilatation of the renal pelvis and of the medullary tubules in the kidney. The major factor contributing to death was undetermined.
Male No. 3 (Group 2, 250 mg/kg/day, Main study) was found dead in Week 2. There were no signs seen before death. Necropsy revealed a distended stomach and small epididymides. Microscopic findings comprised moderate reduced sperm in the cauda of the epididymis. The major factor contributing to death was undetermined.
Male No. 20 (Group 4, 1000 mg/kg/day, Main study) was found dead before dosing in Week 8. There were no signs seen before death. Necropsy revealed a perforated esophagus, clear fluid in the thoracic cavity, adhesions in the thoracic cavity involving multiple organs, a distended stomach and edema of the thymus. Microscopic findings comprised neutrophilic inflammation of the esophagus, fibrinous inflammation of the epicardium in the heart, neutrophilic inflammation of the pleura in the lungs and of the serosa in the thoracic cavity. In addition, interstitial edema of the thymus and decreased cellularity of the white pulp in the spleen were noted. This death was a result of the dosing procedure and not related to treatment with the test item.
Male No. 46 (Group 4, 1000 mg/kg/day, Recovery study) was killed for welfare reasons after dosing in Week 7 due to a marked breathing impairment. Necropsy revealed a perforated esophagus, pale and dark contents in the abdominal cavity and edema of the thymus. Microscopic findings comprised submucosal inflammation of the esophagus, fibrinous inflammation of the epicardium in the heart, neutrophilic inflammation of the pleura and increased alveolar macrophages in the lungs. In addition, increased apoptosis of the lymphocytes in the cortex of the thymus were noted. This death was a result of the dosing procedure and not related to treatment with the test item.
Female No. 118 (Group 4, 1000 mg/kg/day, Main study) was killed for welfare reasons in Week 12 due to a poor clinical condition. It experienced tremors / convulsions lasting up to 45 seconds on three separate occasions whilst being handled for procedures. There were no findings at necropsy. Microscopic findings comprised cortico-medullary mineralization in the kidney. The major factor contributing to death was considered as neurological clinical signs.
Female No. 150 (Group 4, 1000 mg/kg/day, Recovery study) was found dead in Week 3 of the recovery period. There were no signs seen before death. Necropsy revealed an enlarged and ruptured spleen (with red fluid in the abdominal cavity), enlarged lymph nodes (inguinal, mesenteric, pancreatic and renal), masses in the kidneys, masses in the ovaries and masses in the cranium with a dark and thickened meninges. Microscopic findings comprised a lymphoma extending to the bone marrow of the sternum and femur and the femoro-tibial joint, brain, kidney, liver, lungs, ovaries, pituitary, mandibular salivary gland, skin and subcutis, spleen, thymus, uterus, bone of the cranium, and, inguinal, left axillary, pancreatic, renal, and mesenteric lymph nodes. The major factor contributing to death was considered as lymphoma.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The overall bodyweight gain (Weeks 0 to 13) was low, when compared to the controls, for all groups of treated males with a clear dose-response although statistical significance was only achieved for males receiving 500 or 1000 mg/kg/day.

The overall bodyweight gain (Weeks 0 to 13) was slightly low, when compared to the controls, for all groups of treated females with no clear dose response and statistical significance was only achieved for females receiving 1000 mg/kg/day.

After 4 weeks of recovery, the bodyweight gains (Week R0 to R4) for males and females which previously received 1000 mg/kg/day were slightly higher than those of the controls (attaining statistical significance in males only) indicating partial recovery.

Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The overall food consumption (Weeks 1 to 13) was slightly low, when compared to the controls, for all groups of treated males and females, although there was no dose response in females.

After 4 weeks of recovery, the overall food consumption (Week R1 to R4) for males and females which previously received 1000 mg/kg/day had improved but was still slightly lower than those of the controls indicating partial recovery.

Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
The water consumption in Week 11 was high, when compared to the controls, for males and females receiving 500 or 1000 mg/kg/day, although statistical significance was only achieved at 1000 mg/kg/day.

In Week 3 of recovery, the water consumption for males and females which previously received 1000 mg/kg/day was similar to those of controls indicating full recovery.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related ophthalmoscopic findings in Week 12.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The haematological examination during Week 13 revealed low hematocrit, hemoglobin concentration and red blood cell counts, when compared to the controls, for females receiving 500 or 1000 mg/kg/day; hematocrit, hemoglobin concentration and red blood cell counts were also slightly low for females receiving 250 mg/kg/day but statistical significance was only attained for hemoglobin concentration. These inter-group differences were no longer apparent at the end of the recovery period in females which previously received 1000 mg/kg/day, indicating that full recovery had occurred.
Platelet counts were low, when compared to the controls, for males receiving 1000 mg/kg/day and for all groups of treated females although there was no dose response in females. These inter-group differences were no longer apparent at the end of the recovery period in males and females which previously received 1000 mg/kg/day, indicating that full recovery had occurred.
Prothrombin times were slightly increased, when compared to the controls, for all groups of treated females although there was no clear dose response. This inter-group difference was no longer apparent at the end of the recovery period in females which previously received 1000 mg/kg/day, indicating that full recovery had occurred.
Activated partial thromboplastin times were slightly reduced for all groups of treated females although statistical significance was only achieved at 1000 mg/kg/day; this change was still apparent at the end of the recovery period in females which previously received 1000 mg/kg/day.
All other inter-group differences from controls were minor, confined to one sex or were without dose-relationship and were therefore considered to represent normal biological variation. These included the slightly low reticulocyte count for males receiving 500 or 1000 mg/kg/day as 5/9 control values were higher than the historical control range (98 percentile range; 0.099 to 0.229 x1012/L; n=54) and there was no similar change in females.
Due to a small number of clotted EDTA samples at the Week 13 sampling occasion, additional EDTA samples were taken in the morning before necropsy. These repeat results generally confirm the original results although low hemoglobin concentration and red blood cell counts were also noted for males receiving 1000 mg/kg/day. Due to the majority of EDTA samples being clotted for males at the Recovery Week 4 sampling occasion, additional EDTA samples were taken at necropsy.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma during Week 13 revealed high urea concentrations, when compared to the controls, for males and females receiving 1000 mg/kg/day and females receiving 500 mg/kg/day. Creatinine concentration was also high for males and females receiving 1000 mg/kg/day, although statistical significance was only achieved in females. These inter-group differences were still apparent at the end of the recovery period for males which previously received 1000 mg/kg/day, although were no longer apparent for females which previously received 1000 mg/kg/day, indicating that full recovery had occurred in females only.
Cholesterol, and high- and low-density lipoprotein concentrations were high, when compared to the controls, for all treated groups of males and females with a clear dose response. These inter-group differences were still apparent at the end of the recovery period for males which received 1000 mg/kg/day, although statistical significance was only achieved for low-density lipoprotein. These changes were, however, no longer apparent at the end of the recovery period for females which previously received 1000 mg/kg/day, indicating that full recovery had occurred in females only.
Sodium concentrations were slightly low for females receiving 1000 mg/kg/day. This inter-group difference was no longer apparent at the end of the recovery period, indicating that full recovery had occurred.
Albumin concentrations were slightly low for females receiving 500 or 1000 mg/kg/day, resulting in an associated reduction in A/G ratio. This inter-group difference was no longer apparent at the end of the recovery period in females which previously received 1000 mg/kg/day, indicating that full recovery had occurred.
All other inter-group differences from controls, including those that attained statistical significance, were minor, occurred in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation. These included the slightly low potassium concentration for males receiving 1000 mg/kg/day as all individual values in this group were within the historical control range (98 percentile range; 4.0 to 5.4 mmol/L; n=56), apart from a low value for male No. 11 (3.90 mmol/L); potassium concentration was also slightly high for females receiving 500 mg/kg/day but there was no similar change at 250 or 1000 mg/kg/day. Bile acid concentrations were also slightly high for males receiving 1000 mg/kg/day but this difference was associated with two high values for male Nos. 12 and 13 (199.5 and 106.9 µmol/L) in this group which were outside the historical control range (98 percentile range; 4.0 to 97.4 µmol/L; n=56) and there was no similar change in females. These variations were therefore not attributed to treatment.
Endocrine findings:
effects observed, treatment-related
Description (incidence and severity):
The results of the serum triiodothyronine (T3) and thyroxine (T4) concentration analysis were not available at the time of writing this report.

The serum thyroid stimulating hormone (TSH) concentrations were significantly high, when compared to the controls, for males and female which received 1000 mg/kg/day.

The significance of the high TSH and whether the analysis should be extended to include samples from the recovery animals will be evaluated once the results of the T3 and T4 analysis are available.

Please refer to "Any other information on results" for a summary of results after 13 weeks of treatment
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The analysis of urine during Week 13 revealed high volumes, when compared to the controls, for males and females receiving 1000 mg/kg/day and females receiving 500 mg/kg/day, and low specific gravity for females receiving 1000 mg/kg/day.

Urinary pH was low for males and females receiving 500 or 1000 mg/kg/day.

Protein concentrations were low for males and females receiving 1000 mg/kg/day and glucose concentrations were low for males and females receiving 1000 mg/kg/day and females receiving 500 mg/kg/day, but these were considered to be associated with the high urine volumes.

All the above inter-group differences were no longer apparent at the end of the recovery period in males and females which previously received 1000 mg/kg/day, indicating that full recovery had occurred.

All other inter-group differences from controls, including those that attained statistical significance, were minor, occurred in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Sensory Reactivity and Grip Strength
There were no treatment-related findings at the sensory reactivity and grip strength assessments in Week 12.

Hindlimb grip strength was statistically significantly low, when compared to the controls, for females receiving 1000 mg/kg/day. The extent of the difference from controls was, however, small, no associated change to forelimb grip strength (which is a more sensitive marker for any effect on grip strength), all individual values in this group were within the historical control range (0.40 to 0.63 kg; mean 0.49 kg; n=204 from 20 studies) and there was no similar trend in males. Consequently, this was attributed to normal biological variation. This inter-group difference, although no longer statistically significant, was still apparent at the end of the recovery period in females which previously received 1000 mg/kg/day.

Motor Activity
There was no effect of treatment on motor activity in Week 12.

The total low beam scores (cage floor activity) were slightly but statistically significantly reduced, when compared to the controls, for females receiving 1000 mg/kg/day; statistical significance was also achieved for reduced low beam scores at 6 and 12 minutes. There was however no dose relationship, no change to high beam scores (rearing activity) and no similar trend in males. For the individual total values, 4/15 control females had low beam scores that were above the historical control range at these laboratories (low beam range 474.1 to 1332.6 (mean 954.8); n=155 from 15 studies), compared to only 1/10 for the low dose, 1/10 for the intermediate group and 0/15 for the high dose. Consequently, this inter-group difference in female low beam scores in Week 12 was considered to be due to higher than expected control values indicating biological variation not to treatment. This inter-group difference was still apparent at the end of the recovery period in females which previously received 1000 mg/kg/day.

A few other inter-group differences during the one hour assessment period in Week 12 attained statistical significance (decreased high beam scores at 12 minutes for males receiving 1000 mg/kg/day and decreased low beam scores at 6 minutes for females receiving 500 mg/kg/day). Statistical significance was not attained for the total scores and, consequently, these isolated statistically significant differences from controls were attributed to normal biological variation.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
After 13 weeks of treatment, test item-related higher than controls adjusted mean liver weights were seen in males and females given 250, 500 or 1000 mg/kg/day and correlated with hepatocellular centrilobular hypertrophy at microscopic examination.

There were no test-item related changes in liver weight at the end of the recovery phase.
All other differences in organ weights, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; lack of a dose relationship or correlative findings; presence only in absolute and/or adjusted mean weights; and/or the magnitude was considered small.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed after 13 weeks of treatment and after 4 weeks of recovery revealed no test item-related findings.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The microscopic examination performed after 13 weeks of treatment revealed test item-related findings in the kidneys and liver. Trackdown results awaited

Focal basophilia of the proximal tubules was observed in the kidney of both sexes given 1000 mg/kg/day. In addition, focal dilatation of the proximal tubules and interstitial infiltrates of inflammatory cells were observed in females given 1000 mg/kg/day. These findings correlated with high volumes of urine, for males and females receiving 1000 mg/kg/day, and low specific gravity for females receiving 1000 mg/kg/day. In addition, low urinary pH and high urea concentrations were observed for males and females receiving 1000 mg/kg/day. These findings are indicative of compromised urinary function and are regarded as adverse in this study.

Centrilobular hepatocellular hypertrophy was observed in the liver of both sexes given 1000 mg/kg/day and correlated with increased liver weights. In addition, increased high- and low-density lipoprotein concentrations were observed for all treated groups of males and females, when compared to the controls, and were still apparent at the end of the recovery period for males which received 1000 mg/kg/day. These findings, collectively, are indicative of a compromised hepatic function and are regarded as adverse in this study.

All other microscopic findings after 13 weeks of treatment were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item related.

The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present. The estrus cycle evaluated based on vaginal histological stage was within normal limits.

There were no differences in the immunostaining for the presence of alpha-2u-globulin accumulation in the kidneys of males.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
Estrous Cycles
There was no effect of treatment on estrous cycle at the end of the treatment or recovery periods.


Sperm Analysis
The analysis of sperm motility, motion and morphology performed after 13 weeks of treatment or after 4 weeks of recovery revealed no test item-related findings.

All inter-group differences from controls seen after 13 weeks were minor or were consequences of control mean values being outside of historical control ranges and were therefore considered to represent normal biological variation.

Effect levels

Key result
Dose descriptor:
NOAEL
Remarks on result:
other: Not yet confirmed as report in draft stage

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The conclusion will be added upon completion of the examinations currently conducted on the test item.