Reaction mass of trisodium 2-(hydroxyphosphinato)succinate and pentasodium 1-(hydroxyphosphinato)butane-1,2,3,4-tetracarboxylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2013-12-20 to 2014-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, OECD 408 compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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 (incl. QA statement)

Remarks:

2013-01-10

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: tetrasodium-phosphonoethane-1,2-dicarboxylate; hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate

EC Number:

410-800-5

EC Name:

A mixture of: tetrasodium-phosphonoethane-1,2-dicarboxylate; hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate

Cas Number:

143239-08-1

Molecular formula:

Constituent 1 (tetrasodium phosphonoethane-1,2-dicarboxylate): Hill formula: C4H3Na4O7P CAS formula: C4 H7 O7 P .4Na Constituent 2 (hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate): Hill formula: C8H5Na6O11P CAS formula: C8 H11 O7 P .6Na

IUPAC Name:

Reaction mass of tetrasodium phosphonoethane-1,2-dicarboxylate and hexasodium-phosphonobutane-1,2,3,4-tetracarboxylate

Test material form:

other: liquid

Specific details on test material used for the study:

Purity of the test material is based on the phosphonate composition, with no direct measure of % sodium. ITC 288 is not the "Reaction mass of tetrasodium-phosphonoethane-1,2-dicarboxylate and hexasodium-phosphonobutane-1,2,3,4--tetracarboxylate" (EC 410-800-5) rather the substance "Reaction mass of trisodium-phosphonoethane-1,2-dicarboxylate and pentasodium-phosphonobutane-1,2,3,4--tetracarboxylate" (EC 701-079-0) as demonstrated by the manufacturing specs reported in Section 1.2, legal entity composition.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, le Genest-Saint-Isle, France.
- Age at study initiation: on the first day of treatment, the animals were approximately 6 weeks old.
- Weight at study initiation: The males had a mean body weight of 273 g (range: 255 g to 300 g) and the females had a mean body weight of 182 g (range: 159 g to 221 g).
- Housing: The animals were housed in pairs, by sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2045 cm²) containing autoclaved sawdust (SICSA, Alfortville, France) Nylabone or cocoon was given as enrichment.
The cages were placed in numerical order on the racks. Every 5 to 9 weeks, all the racks were moved clockwise around the room, rack by rack. In this way, for each group, identical exposure to environmental conditions was achieved.
- Diet (e.g. ad libitum): All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch Nos. 1010248 and 6730746 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter).
- Acclimation period: the animals were acclimated to the study conditions for a period of 14 days before the beginning of the treatment period. A larger number of animals than necessary were allocated to the study and acclimated, in order to permit the selection and/or replacement of individuals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: sodium phosphate buffer 0.5M pH 7.2 prepared using: . sodium hydroxide 1mol/L . sodium phosphate monobasic monohydrate, drinking water treated by reverse osmosis using ELIX

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was prepared daily, by mixing the required quantity of vehicle with the required amount of test item. Formulations were transferred to the study room at room temperature and protected from light.
The pH was measured for each formulation on the first day of preparation

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chemical analysis of the dose formulation was performed by ion chromatography with conductivity detection analytical method.

Before the start of treatment the suitability of the dose formulation process was confirmed. The stability was also determined on a range of dose formulations prepared at levels which cover the lowest and highest concentrations proposed for use in this study.

The concentration of the test item in samples of each control and test item dose formulation prepared for use on the first day and last week (Week 13) of treatment of the study was determined. Because of the daily formulation preparation, these analyses were not performed prior to administration of the dose formulations to the animals.

Duration of treatment / exposure:

The dose formulations were administered for a period of 13 weeks.
Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg
Basis:
other: actual ingested, active ingredient

No. of animals per sex per dose:

10 males and 10 females per sex and dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous 4-week toxicity study. In this study, four groups of 5 male and 5 female Sprague-Dawley rats were given 0, 150, 400 or 1000 mg/kg/day (corresponding to 132, 351 and 877 mg/kg/day of active ingredient, respectively) of the test item in distilled water at a constant dose-volume of 5 mL/kg/day by gavage for a period of 28 days.
The No Observed Effect Level was 400 mg/kg/day. At 1000 mg/kg/day, females showed piloerection, ptyalism and fur staining in the last week of treatment and both sexes had increased mean plasma alkaline phosphatase level. There were no target organs.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment period, including weekends and public holidays.
Each animal was observed once a day, at approximately the same time (see § Study plan adherence), for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study.
Observations included (but were not limited to) changes in the skin, fur, eyes and mucous membranes, occurence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling, as well as the presence of
clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) and bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

Functional Observation Battery (FOB)
Each animal was evaluated once in Week 12.
This evaluation included a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and motor activity.
The animals were randomized and all animals were observed in the cage, in the hand and in the standard arena.

The following parameters were assessed and graded:
. in the cage: "touch escape"
. in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
. in the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypic behavior, breathing, ataxia and hypotonia.

Reactivity to manipulation and different stimuli
The following measurements, reflexes and responses were recorded:
. touch response,
. forelimb grip strength,
. pupillary reflex,
. visual stimulus response,
. auditory startle reflex,
. tail pinch response,
. righting reflex,
. landing foot splay,
. rectal temperature (at the end of the observation).

Motor activity
For each animal, motor activity was measured by automated infra-red sensor equipment over a 60-minute period.

BODY WEIGHT: Yes
The body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment and then at least weekly until the end of the study.

FOOD CONSUMPTION: Yes
The quantity of food consumed by the animals in each cage was recorded at least once a week, over a 7-day period, during the study.
Food consumption was calculated per animal and per day..

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examinations were performed on all animals, before the beginning of the treatment period and on control and high-dose animals during Week 13. The pupils of the animals were dilated with tropicamide (Mydriaticum®, Laboratoires Théa, Clermont-Ferrand, France). After assessment of the corneal reflex (at instillation of the tropicamide), the appendages, optic media and fundus were examined by indirect ophthalmoscopy.

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes : the animals were deprived of food for an overnight period of at least 14 hours.
- How many animals: All
- Parameters checked in table 1 were examined.


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity:

OTHER:
Monitoring of estrous cycle
The estrous cycle stages were determined daily during Weeks 12 and 13 from a fresh vaginal lavage (stained with methylene blue), daily for 14 consecutive days at the end of the treatment period.

MALES: seminology
+ Epididymal sperm
The left epididymis was removed, weighed and sperm from the cauda was sampled for motility and morphology investigations. Animals were then s acrificed. The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at −20°C pending further investigation

+ Sperm motility
The sperm was evaluated on a slide, after appropriate dilution. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification. Results are expressed as the proportion of motile and non-motile spermatozoa.

+Sperm morphology
The morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatozoa per slide. Results are expressed as the proportion of spermatozoa in each of the following categories:
. normal,
. normally shaped head separated from flagellum,
. abnormal head separated from flagellum,
. abnormal head with normal flagellum,
. abnormal head with abnormal flagellum,
. normally shaped head with abnormal flagellum.

+ Sperm count
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results are expressed as the number of spermatozoa per cauda and per gram of cauda.

+Testicular sperm
The left testis was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogenization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber.
Results are expressed as the number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (See table2)

Sacrifice
On completion of the treatment period, after at least 14 hours fasting, all animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.

Organ weights
The body weight of each animal was recorded before sacrifice at the end of the treatment period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

Preservation of tissues
For all study animals, the tissues specified in the Tissue Procedures Table were preserved in 10% buffered formalin (except for the eyes and optic nerves and Harderian glands, and the right testis and epididymidis which were fixed in Modified Davidson's Fixative).
A bone marrow smear for the determination of the bone marrow differential cell count was prepared from the femur of each animal sacrificed on completion of the treatment period.

Preparation of histological slides
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert, et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except for the right testis and epididymidis which were stained with hematoxylin/PAS).
The tissue processing was performed at CiToxLAB France.

HISTOPATHOLOGY: Yes (see table 2)
A microscopic examination was performed on all tissues listed in the Tissue Procedure Table for animals of the control and high-dose groups (groups 1 and 4).
In addition testicular staging was performed for control and high-dose males (groups 1 and 4). A detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.
Based on the microscopic results of the high-dose group, the mandibular lymph nodes from the low- and intermediate-dose groups were examined.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
ITC288-related clinical observations were noted at 1000 mg/kg/day only, and consisted of ptyalism (18/20) and abnormal growth of teeth in most males (7/10) and a few females (3/10). Ptyalism was observed mostly starting in Weeks 6-7, although one animal exhibited this observation as soon as Day 13, while abnormal growth of teeth was noted during the last 3 weeks of the study. Two animals at 1000 mg/kg/day (male C23195 and female C23264) were observed with hunched posture and loud breathing during the last two weeks of the study (from day 83), along with thin appearance and a swollen mouth (recorded as increase in size) for animal C23195 and piloerection for animal C23264.

Male C23177 (100 mg/kg/day) was isolated from its cage mates from Day 29 to Day 65, in view of the poor condition due to a scab in the dorsal region (Days 10 to 55). Scabs were also noted in animals from all groups, including controls, and as such, were not considered ITC288-related.

Other clinical signs such as chromodacryorrhea and/or thinning of hair were observed in animals from all groups, and are commonly seen in laboratory Sprague-Dawley rats.

BODY WEIGHT AND WEIGHT GAIN:
There was a dose-related decrease in body weights and body weight gains in males at all doses which reached statistical significance at 1000 mg/kg/day. This observation started during the first week of the study and persisted until the last week of the study. At 1000 mg/kg/day, this resulted in a mean difference
in body weight of -17% and in a decreased overall body weight gain of -30% when compared to controls.

There were no statistically significant ITC288-related changes in body weight and body weight gains in females. There was a trend to reduced body weights and body weight gains at 100 and 1000 mg/kg/day, but in view of the lack of dose-relationship, it was not considered to be toxicologically significant.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There was a trend to reduced food consumption in males at all doses, which reached statistical significance at 1000 mg/kg/day starting in Week 2 until the end of the study. This correlated with reduced body weights and body weight gains.
There was a trend to reduced food consumption in females at 100 and 1000 mg/kg/day, correlating with reduced body weights and body weight gains. In view of the lack of dose-relationship, it was not considered to be toxicologically significant.

OPHTHALMOSCOPIC EXAMINATION
There were no changes in ophthalmology during the study.

HAEMATOLOGY
Changes in hematology parameters were observed in males at 1000 mg/kg/day, and were limited to increased absolute number of white blood cells and neutrophils.
Other changes in hematology values were observed without statistically significance and considered incidental since they reflected the normal inter-animal variation in this species.

CLINICAL CHEMISTRY
There were no ITC288-related changes in biochemistry parameters.
Changes in biochemistry values, including some that resulted in statistically significant differences between groups, were considered incidental since they reflected the normal inter-animal variation in this species.

NEUROBEHAVIOUR
Evaluation of functional observation battery did not elicit any relevant difference between control and treated animals at any dose-level.

ORGAN WEIGHTS
Lower mean body weight was noted in males treated at 1000 mg/kg/day (-17%; p<0.01). This difference was considered to be related to test item administration.

No test item-related organ weight differences were observed.

The few organ weight changes were considered to be of minor magnitude, not dose-related and/or the contribution of lower body weight in males at 1000 mg/kg/day. In the absence of associated microscopic findings, these differences were considered not to be related to test item administration.
This included the lower absolute and relative thymus weights in females at 300 and 1000 mg/kg/day, with no microscopic test item-related correlates at 1000 mg/kg/day. At this dose-level, the decrease in mean absolute and relative-to-body thymus weights was considered to be related to the contribution of a single animal (low absolute and relative weights for C23264, with microscopic moderate lymphoid atrophy).

GROSS PATHOLOGY
At 1000 mg/kg/day, abnormal growth or irregular colour of lower incisor teeth was observed in some males and in one female. These findings correlated with ameloblast lesions seen microscopically and were related to test item, although seen also grossly in one control female.

The other gross findings were seen with similar incidence in both control and test item-treated animals, were seen only at minor incidence in the low or intermediate dose-group and/or correlated with common background findings. Thus they were considered not to be related to test item administration

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related findings were observed in the teeth (alteration of the ameloblasts) and in the lymph nodes (presence of macrophages) from males and females treated at 1000 mg/kg/day.

. teeth
In the lower incisor teeth sampled because of macroscopic findings, there were focal, multifocal or diffuse, minimal to marked disorganization and atrophy of the ameloblasts (inner enamel epithelium). This was associated with minimal to moderate degeneration/necrosis of these cells. Morphologically, the ameloblasts were irregular (either high, cuboidal or flat) and formed folds or pseudo-cysts containing cell debris in their lumen. The adjacent enamel was irregular, containing empty spaces ("holes"). Neither the other components of the incisor tooth (odontoblasts, dentin, cementum, pulp…), nor the molar teeth seen on the section were affected by any microscopic changes.
This lesion was seen at 1000 mg/kg/day in 4/10 males and 1/10 females. The macroscopic lesion of the control female was not associated with any microscopic alterations. However, it is noteworthy that the ameloblasts were not seen in the section although two slides were processed.
In view of the severity of these findings (up to marked), of the presence of degeneration/necrosis of ameloblasts, of the enamel alteration with the consecutive gross lesions and finally of the lower body weights in males at 1000 mg/kg/day which may be related to the teeth changes, these lesions were considered to be adverse in rats. It is noteworthy that this species, like other rodents, has rapidly and continously growing incisor teeth during adulthood, unlike adult humans.
No examination can be performed in other animals including those treated at 100 or 300 mg/kg/day since the teeth without macroscopic abnormalities were not sampled.

. mesenteric lymph node
In the mesenteric lymph nodes from 9/10 males and 10/10 females treated at 1000 mg/kg/day, minimal to moderate infiltrate of macrophages was seen. It was also noted in 1/10 males and 10/10 females treated at 300 mg/kg/day at minimal severity. This was considered not to be adverse in view
of the low magnitude of this change and of the absence of degenerative alteration of these lymph nodes.
The other microscopic findings were considered to be unrelated to test item since they were seen with similar incidence in control animals, at minimal severity and incidence were not dose-related and/or corresponded to background findings seen occasionally in the untreated rats of these strain and age.
A detailed examination of the testes was performed, based on a thorough understanding of tubule development through the different stages of the spermatogenic cycle. No test item-related findings were observed.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS

Monitoring of estrus cycles
There was a trend to decreased number and increased length of estrus cycle at 100 and 1000 mg/kg/day.
In view of the lack of dose-relationship, and the absence of histopathological correlates, it was not considered to be toxicologically significant.

Seminology
Epididymal sperm count, motility and morphology were similar between groups. No relevant differences were observed between control and test item treated animals.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 300 other: mg/kg bw

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: At 1000 mg/kg/bw in males reduced body weight and increase absolute number of white blood cells and in both gender at 1000 mg/kg/bw change in teeth.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Incidence of test item-related gross lesions

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	100	300	1000	0	10	300	1000
Number of animals per group	10	10	10	10	10	10	10	10
Teeth: abnormal growth	0	0	0	3	1	0	0	0
Teeth: irregular color	0	0	0	1	0	0	0	1

bold: related to test item treatment.

 

Incidence of test item-related microscopic lesions

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	100	300	1000	0	10	300	1000
Number of animals per group	10	10	10	10	10	10	10	10
Number of rats with an incisor tooth gross anomaly submitted at microscopic examination	0	0	0	4	1	0	0	1
Teeth: disorganization/atrophy; ameloblasts	0	0	0	4	0	0	0	1
Grade 1	0	0	0	1	0	0	0	0
Grade 2	0	0	0	0	0	0	0	0
Grade 3	0	0	0	2	0	0	0	1
Grade 4	0	0	0	1	0	0	0	0
Teeth: degeneration/necrosis; ameloblasts	0	0	0	4	0	0	0	1
Grade 1	0	0	0	3	0	0	0	1
Grade 2	0	0	0	0	0	0	0	0
Grade 3	0	0	0	1	0	0	0	0
Number of mesenteric lymph nodes at microscopic examination	10	10	10	10	10	10	10	10
Mesenteric lymph node; infiltrate; macrophages	0	0	1	9	0	0	1	10
Grade 1	0	0	1	4	0	0	1	6
Grade 2	0	0	0	4	0	0	0	4
Grade 3	0	0	0	1	0	0	0	0

bold: related to test item treatment.

Applicant's summary and conclusion

Conclusions:

In conclusion, the administration of ITC288 at 100, 300 and 1000 mg/kg/day for 13 weeks to male and female Sprague-Dawley rats was well tolerated and resulted in no mortalities. Adverse changes were observed at 1000 mg/kg/day only and consisted in decreased body weights and body weight gains associated with reduced food consumption in males only. In both gender, there were increased absolute number of white blood cells and neutrophils, and gross and microscopic findings in teeth. As such, the Non Observable Adverse Effect Level (NOAEL) was 300 mg/kg/day.

Executive summary:

Objectives

The objective of this study was to evaluate the potential toxicity of the test item, ITC288, following daily oral administration (gavage) to rats for 13 weeks during an OECD 408 compliant study.

Methods

Four groups of ten male and ten female Sprague-Dawley rats were orally (gavage) administered the vehicle (sodium phosphate buffer 0.5M pH7.2 in drinking water) or the test item, ITC288 (batch No. B288P22E1) at dose-levels of 100, 300 and 1000 mg/kg/day (active ingredient), under the constant dosage volume of 5 mL/kg, daily for 13 weeks.

Evaluations of animals included a daily observations, weekly detailed clinical observation, weekly body weight and food consumption recording, ophthalmological examinations (pre-dose and in Week 13), monitoring of estrous cycle (Weeks 12 and 13), hematology and blood biochemistry (Week 13).

At necropsy, spermogram, organ weight, macro- and microscopic examinations were performed.

Results

• There was no ITC288 in control dose formulation. All ITC288 formulations were within 10% of the expected concentrations in Weeks 1 and 13. Formulations ranged from 96.7% to 101.1% of expected concentrations.
• There were no mortalities during the study. ITC288-related clinical observations were noted at 1000 mg/kg/day only, and consisted of ptyalism (18/20) and abnormal growth of teeth in most males (7/10) and a few females (3/10).
• There were no test item related changes during the functional observation battery.
• There was a dose-related decrease in body weights and body weight gains in males at all doses which reached statistical significance at 1000 mg/kg/day.
• There was a trend to reduced food consumption in males at all doses correlating with reduced body weights and body weight gains in males only.
• There were no changes in ophthalmology during the study.
• There were no changes in estrus cycles that considered to be related to treatment.
• Changes in hematology parameters were observed in males at 1000 mg/kg/day, and were limited to increased absolute number of white blood cells and neutrophils.
• There were no ITC288-related changes in biochemistry parameters.
• Seminology was not considered affected by the treatment with ITC288 at any dose-level.

At necropsy, test item-related lower mean body weights were noted in males treated at 1000 mg/kg/day.

Macroscopic test item-related abnormal growth or irregular color of lower incisor teeth was observed in some males and females treated at 1000 mg/kg/day. These findings correlated with adverse ameloblast disorganization and atrophy associated with degeneration/necrosis of these cells. The adjacent enamel was altered.

In the mesenteric lymph nodes from males and females treated at 300 or 1000 mg/kg/day, non adverse infiltrate of macrophages was seen.

Conclusion

In conclusion, the administration of ITC288 at 100, 300 and 1000 mg/kg/day (active ingredient) for 13 weeks to male and female Sprague-Dawley rats was well tolerated and resulted in no mortalities. Adverse changes were observed at 1000 mg/kg/day only and consisted in decreased body weights and body weight gains associated with reduced food consumption in males only. In both gender, there were increased absolute number of white blood cells and neutrophils, and gross and microscopic findings in teeth. As such, the Non Observable Adverse Effect Level (NOAEL) was 300 mg/kg/day.