"Details on Exposure:

PREPARATION OF DOSING SOLUTIONS: The test was administered as a solution in the

vehicle.

* Preparation procedure: According to CiToxLAB France/Study No. 43498 VAS describing the

preparation procedure for a range of concentrations covering the lowest and highest used in

this study: Add the required amount of test item in some amount of vehicle and mix. Then

add the vehicle until the final volume and mix. Check the pH (pH paper) of each test item

dose formulation at the first preparation in the study.

Frequency of preparation: The formulations were prepared for use every 2 to 12 days [based

on test item dose formulation stability (CiToxLAB France/Study No. 43498 VAS) and vehicle

expiry]. The formulations were stored and delivered at room temperature.

VEHICLE

- Justification for use and choice of vehicle (if other than water):

- Concentration in vehicle: 0, 100, 300 and 1000 mg/kg bw/day

- Amount of vehicle (if gavage): 10 mL/kg/day

- Lot/batch no. (if required):

* sodium phosphate monobasic monohydrate: BCBK7914V and BCBR2016V

* NaOH 10N: 1494258

- Preparation: The vehicle was prepared using:

* sodium phosphate monobasic monohydrate (Sigma-Aldrich ref. 71504)

* NaOH 10N

* drinking water treated by reverse osmosis using ELIX 5 (Millipore SA)

- Storage conditions: After preparation, the required amount of vehicle was stored in the

refrigerator set at +5°C. In the afternoon of the day before control and test item dose

formulations preparation, it was placed at room temperature under magnetic stirring.

Details on analytical verification of doses or concentrations:

Ion Chromatography (IC) with conductivity detection was used.

Determination of test item concentrations in dose formulations: Once in Weeks 1 and 4. A

sample was taken from control and test item dose formulations and analyzed using the

validated method.

Acceptance criterion: measured concentration = nominal concentration ± 10%.

The validation of the analytical method was conducted in CiToxLAB France (CiToxLAB

France/Study No. 43498 VAS) prior to dose formulation analysis and precise details

concerning the checked parameters, acceptance criteria and obtained results were

documented in the corresponding validation report.

Duration of treatment / exposure:

The dosage forms were administered daily according to the following schedule:

# males:

- 2 weeks before pairing,

- during the pairing period,

- until euthanasia on Day 32.

# females:

- 2 weeks before mating,

- during the mating period,

- during gestation,

- during lactation until day 4 post-partum inclusive,

- until euthanasia for females with no delivery.

Day 1 corresponds to the first day of the treatment period.

Frequency of treatment: Once a day, at approximately the same time.

- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, on

the basis of the results of:

* a Japanese 4-week toxicity study (Hita Research Laboratories/Test Code No.: B11-0254;

1994) where six male and six female Sprague-Dawley rats per group received the test item

in formulation in water by gavage at 0, 8, 40, 200 or 1000 mg/kg/day (dose-volume = 10

mL/kg).

During the treatment period, no premature deaths occurred. There were no effects on

general health or food intake. A trend towards absence of body weight gain was observed at

1000 mg/kg/day in the last week of treatment in males. Reductions in several red blood cell

parameters (males) and increase in platelet count (females) were seen at 1000 mg/kg/day.

A reduction in total protein was observed in females at 1000 mg/kg group, as well as an

increase in urea nitrogen and creatinine in one female. An increase in ketone bodies and

glucose was observed in urines of one female at 1000 mg/kg. There were no effects on

organ weight. At necropsy, boundary ridge protrusion in the forestomach (plus reddening of

the mucosa in the glandular stomach in one case) was observed in males treated at 1000

mg/kg, and boundary ridge protrusion in the forestomach, roughening and blackening of the

glandular stomach mucosa (one to two females), and fading of the kidney (one female)

were observed in the females. At microscopy, squamous epithelium hyperplasia in the

forestomach was observed in one male at 200 mg/kg/day and all males at 1000 mg/kg/day.

A reduction in glandular stomach parietal cells, surface epithelial cell hyperplasia,

submucosal bleeding, and loss of villi in the duodenum were also observed in the 1000

mg/kg/day males. In females of this group, squamous epithelium hyperplasia in the

forestomach, a reduction in glandular stomach parietal cells, surface epithelial cell

hyperplasia, loss of villi in the duodenum, renal tubular necrosis, and/or urinary casts were

noted in one to three females each time.

After 2 weeks of recovery, changes due to the test item were only observed in the males at

1000 mg/kg/day (reduction in mean corpuscular haemoglobin, squamous epithelium

hyperplasia in the forestomach - lower occurrence and extent compared to the end of

treatment, reduction in relative liver weight, peripheral hepatocyte enlargement of the liver),

* a 7-day toxicity study (2016; CiToxLAB France/Study No. 43737 TSR) where _ve Sprague-

Dawley males received the test item in formulation in pH 7.2 sodium phosphate 0.5M buffer

by gavage at 1000 mg/kg/day (dose-volume = 10 mL/kg). There were no premature deaths

or relevant clinical signs. All males gained weight and the mean food consumption was

correct. There were no necropsy findings.

As the study duration of the present study was also 4 weeks in males (likely 5-6 weeks for

most females), and as the vehicle slightly buffered the formulations compared to the _rst

study, 1000 mg/kg/day was selected as the high dose-level. The low-dose and mid-dose

were selected using a ratio representing approximately a 3-fold interval (i.e. 100 and 300

mg/kg/day).

- Administration: As the test item is corrosive, care was taken to correctly wipe the gavage

tube before administering the animals in order to remove all the dose formulation at the

exterior surface of the tube.

The control and test item dose formulations were stirred just before administration. The

formulations were maintained under continuous magnetic stirring and at room temperature

throughout the dosing procedure."

P0 Male SD (Rjhan: Sd) Rats