CAGE SIDE OBSERVATIONS: Yes

- Time schedule: At least twice daily for mortality/viability (once daily on weekends or public

holidays).

DETAILED CLINICAL OBSERVATIONS: Yes

- Time schedule: Observed clinical signs were recorded at least once daily for all animals during

the study. Detailed physical examinations were performed during the acclimatization phase and

at least weekly during the treatment periods. The nature, pnset, severity, reversibility and duration

of clinical signs were recorded. Cages and cage-trays were inspected daily for evidence of illhealth

such as blood or loose feces.

BODY WEIGHT: Yes

- Time schedule for examinations: Each animal was weighed three times during the

acclimatization period, on the 􀂠rst day of test substance administration, then at least weekly

throughout the treatment period. Additionally, diet fasted animals were weighed before necropsy.

FOOD CONSUMPTION: yes

- Time schedule for examinations: The weight of food supplied and of that remaining at the end

of the food consumption period was recorded weekly for all animals during the treatment period.

From these records the mean daily consumption was calculated. Food spillage was also noted.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes

- Time schedule for examinations: During the acclimatization period, all animals were subjected

to an ophthalmic examination. After instillation of an atropinic agent (Mydriaticum, Merck Sharp

and Dohme) each eye was examined by means of an indirect ophthalmoscope. During Week 14

of the treatment period, all surviving animals from all groups were re-examined.

HAEMATOLOGY: Yes

- Time schedule for collection of blood: at the end of treatment

- Anaesthetic used for blood collection: Yes (iso􀂡urane)

- Animals fasted: Yes, overnight

- How many animals: all surviving animals

- The following parameters were examined: Red blood cell count (1), hemoglobin concentration

(2), hematocrit (3), mean corpuscular volume (4), mean corpuscular hemoglobin (5), mean

5/10

corpuscular hemoglobin concentration (6), reticulocyte count (7), white blood cell count and

differential count evaluation (8) and platelet count (9) were assayed using a Advia 120 (Bayer

Diagnostics, Puteaux, France). A blood smear was prepared and stained with Wright stain. It was

examined when the results of

Advia 120 determinations were abnormal. Prothrombin time (10) was assayed on an ACL 3000

(Instrumentation Laboratory, Paris, France).

CLINICAL CHEMISTRY: Yes

- Time schedule for collection of blood: at the end of treatment

- Animals fasted: yes, overnight

- How many animals: all surviving animals

- The following parameters were examined: Total bilirubin (11), glucose (12), urea (13), creatinine

(14), total cholesterol (17), triglycerides (18), chloride (23), sodium (23), potassium (23), calcium

(24) and inorganic phosphorus (25)

concentrations, and aspartate aminotransferase (19), alanine aminotransferase (20), alkaline

phosphatase (21) and gamma-glutamyltransferase (22) activities were assayed on plasma

samples, total protein (15) and albumin (16) concentrations were assayed on serum samples

using an Advia 1650 (Bayer Diagnostics, Puteaux, France). Globulin concentrations and

albumin/globulin ratio values were calculated.

URINALYSIS: Yes

- Time schedule for collection of urine: on study Days 87 or 88 in the morning, overnight urine

samples were collected.

- Metabolism cages used for collection of urine: Not speci􀂠ed

- Animals fasted: Yes

- How many animals: all surviving animals

- The following parameters were examined: general appearance, volume, pH, urinary refractive

index. Glucose, bilirubin, ketone bodies, occult blood, protein and urobilinogen were assayed

using a Clinitek 200+ and Multistix dipsticks (Bayer Diagnostics, Puteaux, France). Microscopic

examination of the urinary sediment was performed after centrifugation of the urine. The

presence of red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals was

graded.

NEUROBEHAVIOURAL EXAMINATION: Yes

- Time schedule for examinations: week 11-12 of treatment

- Dose groups that were examined: all surviving animals

- Battery of functions tested: Motor activity (recording period 90 minutes with data being

collected at 15-minute intervals), sensory reactivity (pupillary re􀂡ex, surface righting re􀂡ex,

corneal re􀂡ex, 􀂡exor re􀂡ex, auditory startle response, tail pinch response), Open 􀂠eld observation

(changes in gait, posture, as well as presence of clonic or tonic movements, stereotypic

behaviour, bizarre behaviour e.g. self-mutilation, walking backward), grip strength (fore- and hindlimb,

recorded as mean of three measurements per animal) .

IMMUNOLOGY: No

Sacri􀂠ce and pathology: GROSS PATHOLOGY: Yes

The following organ weights and terminal body weight were recorded from all animals on the

scheduled day of necropsy:

Adrenal gland, brain, epididymis, heart, kidney, liver, ovary, pituitary gland, prostate gland, spleen,

testis, thymus, thyroid gland (with parathyroid gland) and uterus (including cervix). Paired organs

were weighed together.

HISTOPATHOLOGY: Yes. The samples of following tissues and organs were collected:

Adrenal gland

Aorta

Articular surface (femoro-tibial joint)

Bone (sternum)

Bone marrow (sternum)

Brain

Epididymis

Esophagus

Exorbital (lachrymal) gland

Eye and optic nerve

Harderian gland

Heart

Intestine (duodenum, jejunum, ileum,

cecum, colon, rectum)

Kidney

6/10

Larynx/Pharynx

Liver

Lung

Lymph nodes (submaxillary, mesenteric)

Mammary gland

Nasal cavities

Ovary

Pancreas

Pituitary gland

Prostate gland

Sciatic nerve

Seminal vesicle

Skeletal muscle

Skin

Spinal cord (cervical, thoracic, lumbar)

Spleen

Stomach

Submaxillary (salivary) gland

Testis

Thymus

Thyroid gland (with parathyroid gland)

Tongue

Trachea

Urinary bladder

Uterus (including cervix)

Vagina

All of the above mentioned samples (except exorbital lachrymal gland, larynx/pharynx and nasal

cavities) were embedded in parafin wax. Histological sections, stained with hematoxylin and

eosin, were prepared from all organs and tissue samples from all the animals in the control and

high dose groups, and from the animal (QT3M0972) found dead on Day 15.

Additionally, sections from liver, kidney, lung, thyroid gland, from potential target organs (heart)

and from significant gross findings observed at necropsy were prepared for all the animals in all

intermediate dose groups.

Female Wistar Rj:WI (IOPS HAN) Rat