Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 Feb 2019 to 23 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)
EC Number:
950-860-4
Molecular formula:
C15H20N4O2
IUPAC Name:
(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)
Test material form:
solid: bulk
Details on test material:
White Crystalline Solid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Receipt
On 13 Feb 2019, Crl: WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. The animals were 9-10 weeks old at initiation of dosing. Males weighed between 259 and 281 g and females between 180 and 198 g. A health inspection was performed before the initiation of dosing.

Justification for Test System and Number of Animals
The rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives. At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist. The study plan was reviewed and agreed by the Animal Welfare Body of Charles River Laboratories Den Bosch B.V. within the framework of Appendix 1 of project license AVD2360020172866 approved by the Central Authority for Scientific Procedures on Animals (CCD) as required by the Dutch Act on Animal Experimentation (December 2014).

Animal Identification
At study assignment, each animal was identified using a chip that was implanted shortly after arrival at the Test Facility.

Environmental Acclimation
The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days before the commencement of dosing.

Selection, Assignment, Replacement, and Disposition of Animals
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals in poor health or at extremes of body weight range were not assigned to groups.

Husbandry
Housing
On arrival and following randomization, animals were group housed (up to 3 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon type IV, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities. The room in which the animals were kept was documented in the study records. Each cage was clearly labeled with a color-coded cage card indicating test facility study no., group, animal numbers, and sex.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 20 to 21°C with an actual daily mean relative humidity of 41 to 51%. A 12-hour light/12-hour dark cycle was maintained (except during designated procedures). Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water was freely available to each animal via water bottles. Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.It was considered that there are no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Administration of Test Materials
The test item and vehicle were administered to the appropriate animals by once daily oral gavage for 7 days. Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube. The first day of dosing was designated as Day 1. The dosing formulations were stirred continuously during dose administration. A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Vehicle:
propylene glycol
Details on oral exposure:
Justification of Route and Dose Levels
The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item. The dose levels were selected based on results of an acute oral toxicity study with oral exposure of PF-07094402 in rats (Test Facility Study No. 20171458), and in an attempt to produce graded responses to the test item. At 2000 mg/kg, one animal was found dead and on the first three days hunched posture, uncoordinated movements, piloerection, salivation, chromodacryorrhoea of the snout and/or lethargy were observed. These signs were not observed from Day 4 onwards. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level was expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Duration of treatment / exposure:
7 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3 males and 3 females
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Vehicle
Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. These trials were not performed as part of this study were not used for dosing. Raw Data of these trials will be retained by the Test Facility.

Test Item Characterization
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item.

Reserve Samples
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility. The sample will be destroyed after the expiry date.

Test Item Inventory and Disposition
Records of the receipt, distribution, and storage of test item were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

Dose Formulation and Analysis
Preparation of Test Item
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a suspension and dosed within 4 hours after adding the vehicle to the test item.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.

Sample Collection and Analysis
Analysis of test item in vehicle for concentration, stability, homogeneity was not performed, however, to limit the impact, the test item preparation was performed with approved procedures and documented in detail. Formulations were visually inspected for homogeneity prior to use and all formulations were used within 4 hours after adding vehicle to the test item. This GLP exception was therefore considered as being minor with no impact on the outcomes and the integrity and the achievement of the objective of the study.
Positive control:
not specified

Examinations

Observations and examinations performed and frequency:
Mortality/Moribundity Checks
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings. Animals showing pain, distress or discomfort which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail, see 9.1.

Clinical Observations
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the Dosing Period. These observations were performed immediately after dosing up to 30 minutes after dosing. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body Weights
Animals were weighed individually on Day 1, 4 and 7, prior to dosing. A fasted weight was recorded on the day of necropsy.

Food Consumption
Food consumption was quantitatively measured on Days 1-4 and Days 4-7.

Water Consumption
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Laboratory Evaluations - Clinical Pathology
Sample Collection
Blood sampling for clinical pathology was performed as part of the necropsy procedure immediately prior to sacrifice when the animal was deeply anaesthetized (for animals surviving to planned necropsy and moribund animals). Blood was collected between 7.00 and 10.30 a.m. from the retro-orbital sinus under anesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands). After collection all samples were transferred the appropriate laboratory for analysis. Animals were fasted (overnight with a maximum of 24 hours) before blood sampling, but water will be available. Samples will be collected according to the following table.

Hematology
Blood samples at a target volume of 0.5 mL were collected into tubes containing K3-EDTA as anticoagulant. Samples were analyzed for the parameters specified in the following table.

Hematology Parameters
White blood cells (WBC) Red Blood Cell Distribution Width (RDW)
Neutrophil (absolute) Hemoglobin
Lymphocyte (absolute) Hematocrit
Monocyte (absolute) Mean corpuscular volume (MCV)
Eosinophil (absolute) Mean corpuscular hemoglobin (MCH)
Basophil (absolute) Mean corpuscular hemoglobin concentration (MCHC)
Red blood cells Platelet
Reticulocyte (absolute)
A blood smear was prepared from each hematology sample. Blood smears were labeled,
stained, and stored.

Coagulation
Blood samples at a target volume of 0.45 mL were collected into tubes containing citrate as anticoagulant. Samples were processed for plasma, and plasma was analyzed for the parameters listed in the following table.

Coagulation Parameters
Prothrombin Time (PT) Activated Partial Thromboplastin Time (APTT)

Clinical Chemistry
Blood samples at a target volume of 0.5 mL were collected into tubes containing Li-heparin as anticoagulant. Samples were processed for plasma, and were analyzed for the parameters specified in the following table. Serum samples at a target volume of 0.25 mL were collected in tubes without anticoagulant. Blood samples were processed for serum (bile acids), which was analyzed for the parameters specified in the following table.

Clinical Chemistry Parameters
Alanine aminotransferase (ALAT) Glucose
Aspartate aminotransferase (ASAT) Cholesterol
Alkaline Phosphatase (ALP) Triglycerides
Total protein Sodium
Albumin Potassium
Bile Acids Chloride
Total Bilirubin Calcium
Urea Inorganic Phosphate (Inorg. Phos)
Creatinine

Necropsy
All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably qualified person, was available.

Organ Weights
The organs identified in the following table were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for the animals euthanized preterminally. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.

Organs Weighed at Necropsy
Brain
Epididymisa
Gland, adrenala
Heart
Kidneya
Liver
Ovarya
Spleen
Testisa
Thymus
Uterus
a Paired organ weight.

Tissue Collection and Preservation
Representative samples of the tissues identified in the following table were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated.

Tissue Collection and Preservation
Animal identification
Brain [cerebellum, mid-brain, cortex] (8 levels)
Cervix
Epididymisa
Gland, adrenal
Gross lesions/masses
Heart
Kidney
Liver
Ovary
Spleen
Stomach
Testisa
Thymus
Uterus
Vagina
a Preserved in Modified Davidson’s fixative prepared at Charles River Den Bosch using Formaldehyde 24%, Ethanol, Acetic acid - glacial (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA). Tissues were transferred to formalin after fixation for approximately 3 days.

Histology
Tissues identified in the table above (except animal identification) were embedded in paraffin (Klinipath, Duiven, The Netherlands), sectioned, mounted on glass slides, and stained with hematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Histopathology
All tissues as defined under Histology (section 4.10.6) were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology. Target tissues identified by the study pathologist during microscopic evaluation were communicated to the Study Director; tissues were evaluated and reported. A peer review on the histopathology data was performed by a second pathologist.
Sacrifice and pathology:
Unscheduled Deaths
For humane reasons, two females at 1000 mg/kg/day were deeply anaesthetized using isoflurane and subsequently exsanguinated. These animals underwent necropsy, and specified tissues were retained, but not weighed.

Scheduled Euthanasia
Animals surviving until scheduled euthanasia had a terminal body weight recorded, were deeply anaesthetized using isoflurane and blood was sampled from the retro-orbital sinus (for clinical pathology). The animals were subsequently exsanguinated and subjected to a full post mortem examination. Animals were fasted (overnight with a maximum of 24 hours) before their scheduled necropsy. Water was available.
Statistics:
Descriptive statistics number, mean and standard deviation will be reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs were observed at 150 and 300 mg/kg/day.
At 1000 mg/kg/day, hunched posture was observed in all males and the surviving female starting on Day 6.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females dosed at 1000 mg/kg/day were euthanized prior to scheduled euthanasia on Days 4 (No. 24) and 7 (No. 23). In these animals, hunched posture, piloerection, eyes partly closed, thin appearance, shallow breathing, decreased activity and/or red discoloured urine were observed. Body weight loss was noted for both females (20% for No. 24 on Day 4, and 18% for No. 23 on Day 7). Histopathological changes in the kidney noted in these animals were similar to those observed in the surviving animals at 1000 mg/kg/day (see also 9.10) and these were considered to be the cause of moribundity.
Additionally, dark red fluid in the urinary bladder was noted in Female No. 24, microscopically correlating to multifocal mucosal haemorrhage, multifocal mixed cell infiltrates, multifocal urothelial hyperplasia and minimal multifocal urothelial degeneration. In Female No. 23, papillary necrosis and mild urothelial hyperplasia were noted additionally. Other histopathological findings in the thymus, spleen and stomach were interpreted to be likely secondary to stress.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights of treated animals at 150 and 300 mg/kg/day remained in the same range as controls over the study period.
A test item related lower body weight gain was observed in males and females at 300 mg/kg/day.
At 1000 mg/kg/day, body weight loss was observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight at 150 and 300 mg/kg/day was similar to the control level over the study period.
At 1000 mg/kg/day, a lower food consumption compared to control animals was observed in both males (0.82x and 0.41x of controls over Days 1-4 and Days 4-7, respectively) and females (0.30x and 0.38x of controls over Days 1-4 and Days 4-7, respectively).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in hematological parameters.
While lower reticulocyte numbers were observed in males at 1000 mg/kg/day, the alterations were considered to be unrelated to administration of the test item because numbers were within the range observed in male rats of this age and strain. It should be noted that no blood sample could be collected from the surviving female at 1000 mg/kg/day, hence hematology parameters were only evaluated up to 300 mg/kg/day in females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical biochemistry parameters in females and in males at 150 and 300 mg/kg/day.
At 1000 mg/kg/day, an increase in urea (2.23x of controls), creatinine (2.66x), glucose (1.47x) and cholesterol (1.63x) levels was observed in males. It should be noted that no blood sample could be collected from the surviving female at 1000 mg/kg/day, hence clinical chemistry parameters were only evaluated up to 300 mg/kg/day in females.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Two females dosed at 1000 mg/kg/day were euthanized prior to scheduled euthanasia on Days 4 (No. 24) and 7 (No. 23). In these animals, hunched posture, piloerection, eyes partly closed, thin appearance, shallow breathing, decreased activity and/or red discoloured urine were observed.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related higher kidney weights and lower thymus weights were noted in terminally euthanized males and female dosed at 1000 mg/kg/day
Higher kidney weights correlated with macroscopic enlargement in males and microscopically, with the presence of crystals and tubular and/or pelvic dilatation in males and females.
All other organ weight differences observed were considered incidental and unrelated to the administration of PF-07094402.
Description (incidence and severity):
Test item-related findings were observed in the urinary tract of males and females dosed at 1000 mg/kg/day.
Test item-related findings included enlargement on the kidney, in 3/3 males, pale foci in the kidney in 1/3 males and 1/3 females, dilatation of the renal pelvis in 1/3 males, ureter dilatation in 1/3 males, and dark red fluid in the urinary bladder of 1/3 females. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings after treatment with PF-07094402 were noted in the kidney, thymus, liver, spleen, ureter, and urinary bladder of the 1000 mg/kg/day group males and females and are summarized below.
Finding relating to the urinary system were similar in preterminal animals

The main histologic findings were noted in the kidneys in terminal and preterminally euthanized animals. Findings included the presence of crystals, up to marked degree, within the lumen of the tubules (predominantly located in the medulla, but also scattered throughout the cortex) and in the renal pelvis, often accompanied by hemorrhage.

The crystals were pale eosinophilic on H&E staining and predominantly starburst shaped, often forming aggregates when present in the renal pelvis. Additionally there was degeneration/regeneration of the tubular epithelium, also up to marked degree, with the same pattern of distribution and often concurrently with the presence of the crystals, tubular dilatation up to mild degree (likely secondary to obstruction by crystals), mild urothelial hyperplasia of the renal pelvis, mixed cell inflammation up to moderate degree, and in males only, minimal fibroplasia. Microscopic kidney changes unique to preterminally euthanized females consisted of mild unilateral papillary necrosis (Animal No. 23).

Additional findings noted in only one male (Animal No. 11) included mild pelvic dilatation (with a large crystal aggregate), mild vascular necrosis and thrombosis in the kidney, and moderate vascular/perivascular inflammation in the liver (portal arterioles). The ureters wer also examined from this animal due to the presence of macroscopic dilatation and this correlated with mild dilatation microscopically, with concurrent moderate mixed cell inflammation.

The urinary bladder was examined microscopically from 2 females due to the macroscopic finding of abnormal dark red content: one terminal female (Animal No. 22, no microscopic correlate) and one preterminal female (Animal No. 24). For Animal No. 24 this generally correlated with mild multifocal mucosal hemorrhage. Additionally mild multifocal urothelial hyperplasia, minimal urothelial degeneration, and minimal multifocal mixed cell infiltrates were noted. These findings were most likely secondary to the presence of crystals in the urine.

Minimal histologic changes in the thymus of terminally euthanized animals included decreased lymphoid cellularity or single cell lymphoid necrosis correlated with lower thymus weights and were interpreted to be secondary to stress. In the spleen, extramedullary hematopoiesis was noted in all groups at comparable incidence and severity (minimal to mild) except the high dose group (1000 mg/kg/day), where none was noted in terminally euthanized animals.
The remaining histologic changes were considered to be incidental findings and were within the range of background pathology enc untered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
mortality

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, administration of PF-07094402 by once daily oral gavage for 7 days was well tolerated in rats at dose levels up to 300 mg/kg/day. Treatment at 1000 mg/kg/day resulted in preterminal deaths in 2/3 females. Test item-related adverse morphologic alterations at 1000 mg/kg/day consisted of crystal deposition in the renal tubules/pelvis (up to marked degree) with secondary effects (tubular degeneration/regeneration, dilatation, inflammation, and urothelial hyperplasia in males and females and fibroplasia in males), dark red content of the urinary bladder, enlarged and/or pale foci in the kidneys, renal pelvic dilatation, ureter dilatation, and higher kidney weights. Furthermore, adverse body weight loss with lower food consumption were observed at 1000 mg/kg/day.
Based on the results presented in this report a No Observed Adverse Effect Level (NOAEL) for PF-07094402 of 300 mg/kg was established.
Executive summary:

Wistar Han rats were treated with PF-07094402 for 7 consecutive days by daily oral gavage at dose levels of 150, 300 and 1000 mg/kg/day.

Two out of three females dosed at 1000 mg/kg/day were euthanized prior to scheduled euthanasia. In these animals, hunched posture, piloerection, eyes partly closed, thinness,

shallow breathing, decreased activity and/or discoloured red urine were observed, as well as severe body weight loss. Histopathological changes in the kidney (crystals in the tubules (cortex and medulla), renal pelvis of the kidney, tubular degeneration/regeneration, mixed cell inflammation, tubular dilatation, and/or urothelial hyperplasia) were considered to be the cause of moribundity.

At 300 mg/kg/day, a test item related slightly lower body weight gain was observed in males and females, which was considered non-adverse at this severity and incidence.

In the surviving animals at 1000 mg/kg/day, hunched posture was observed.

In addition, a test item-related slight body weight loss was observed, which correlated to markedly lower food consumption observed in these animals and were considered to be

adverse. At clinical chemistry, a test item-related increase in glucose, cholesterol, urea and creatinine levels was observed in males at 1000 mg/kg/day (no data available for females at 1000

mg/kg/day). During histopathological examination, adverse test item-related microscopic findings were noted in the in the kidney, ureter and liver, and non-adverse findings were noted in the

thymus and spleen at 1000 mg/kg/day.

The increased urea and creatinine levels, observed at clinical chemistry, correlated with higher kidney weights, macroscopic enlargement and pale foci of the kidney and microscopically with the presence of crystals within the lumen of the tubules (medulla and cortex) and tubular dilation in the kidneys of males and females at 1000 mg/kg/day.

Additional effects observed in the kidneys included degeneration/regeneration of the tubular epithelium, inflammation and urothelial hyperplasia, fibroplasia, pelvic dilatation and/or mild vascular necrosis and thrombosis.

Histologic changes in the urinary bladder (examined for two females at 1000 mg/kg/day only) included mild multifocal mucosal hemorrhage, mild multifocal urothelial hyperplasia, minimal urothelial degeneration, and minimal multifocal mixed cell infiltrates which correlated with the macroscopic finding of abnormal dark red content in these two females.

These findings were most likely secondary to the presence of crystals in the urine. Minimal histologic changes in the thymus included decreased lymphoid cellularity or single cell lymphoid necrosis, correlating with lower thymus weights and were interpreted to be secondary to stress and therefore non-adverse.

Histologic changes in the spleen included the absence of extramedullary hematopoiesis which was considered test item related but non-adverse at this incidence and severity.

No test item-related changes were noted in males and females treated at 150 or 300 mg/kg/day, and in males at 1000 mg/kg/day for any of the remaining parameters investigated in this study (i.e. hematology and coagulation).