"TEST ANIMALS: Healthy male and female Crl: CD® (SD) IBS BR Stock out-bred albino rats were received from Charles River Laboratories, Inc., for use on the study. The animals were individually housed in screen-bottom stainless steel cages. Animal identification consisted of uniquely numbered ear tags and color-coded cage cards. On Study Day 1, the animals were approximately seven weeks old. The males weighed 196 to 223 grams, and the females weighed 156 to 183 grams. Teklad Certified Rodent Diet #8728 and filtered tap water were provided ad libitum. The feed and water were routinely analyzed for contaminants. There were no known contaminants in the feed or water that would be expected to affect the results of the study. The results of these analyses are on file at Primedica Redfield. Environmental controls were set to maintain temperatures of 18° to 26°C (64° to 79°F) with a relative humidity of 30% to 70%. These parameters were recorded at least once daily. A 12:12 hour light:dark cycle was maintained in the animal room.

GROUP DESIGNATION AND TREATMENT: The animals were acclimated for seven days prior to Study Day 1 and were examined by the Staff Veterinarian prior to being released for use on the study. Twenty males and twenty females were randomly assigned to groups by a computer generated weight-ordered distribution such that individual body weights did not exceed ±20% of the mean weight for each sex. Doses were administered via oral gavage for ten consecutive days. The first day of dosing was designated as Study Day 1. Individual dose volumes were calculated based on the most recently collected body weight for each animal.

JUSTIFICATION FOR SPECIES SELECTION, ROUTE OF ADMINISTRATION, AND DOSE LEVEL:

Rats are an animal model for acute toxicity studies of this type. In the assessment and evaluation of the toxic characteristics of a substance, determination of acute oral toxicity is the usual initial step. Dose levels were chosen by the Sponsor.

CLINICAL OBSERVATIONS: Observations for mortality and moribundity were recorded twice daily (a.m. and p.m.). Clinical observations were recorded once daily.

BODY WEIGHTS: Body weights were recorded on Study Days 1, 7, 10, and 11. The Study Day 11 body weights were only used for relative organ weight calculations.

FEED CONSUMPTION: Feed consumption was measured on Study Days 1, 7, and 10.

HEMATOLOGY: Blood samples for hematological analysis were obtained via cardiac puncture prior to necropsy. The animals were anesthetized with carbon dioxide for the collection. Feed was withheld overnight prior to collection. The blood samples were evaluated for the following parameters: total and relative differential leukocyte counts, erythrocyte counts, hemoglobin concentration, hematocrit value, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet counts, prothrombin time, and activated partial thromboplastin time. Specific methods for hematology analyses are located in Appendix 1.

CLINICAL CHEMISTRY: Blood samples were obtained via cardiac puncture prior to necropsy. The animals were anesthetized with carbon dioxide for the collection. Feed was withheld overnight prior to collection. The samples were processed and evaluated for the following parameters: total protein, albumin, globulin, albumin/globulin ratio, glucose, cholesterol, triglycerides, total bilirubin, urea nitrogen, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, calcium, phosphorus, sodium, potassium, and chloride. Specific methods for clinical chemistry analyses are located in Appendix 1.

NECROPSY AND ORGAN WEIGHTS: On Study Day 11, the animals were humanely euthanized via anesthesia with carbon dioxide followed by exsanguination. All animals were submitted for a complete necropsy examination. A complete necropsy was defined as examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. Organ weights were recorded for the following organs prior to fixation: adrenal glands, brain, heart, kidneys, liver, ovaries, spleen, testes, and thymus. Paired organs were weighed together. The following organs and tissues were examined in situ, dissected free, and fixed in 10% neutral buffered formalin: adrenal glands, aorta, bone marrow (sternum), brain (brain stem, cerebellum, cerebrum), cervix, epididymides, esophagus, eyes (with optic nerve), femur (with articular surface), gross lesions, Harderian gland, heart, large intestine (cecum, colon, rectum), small intestine (duodenum, ileum, jejunum), kidneys, lacrimal gland, liver, lungs (with bronchi), lymph nodes (mandibular, mesenteric), mammary gland, ovaries, oviducts, pancreas, pituitary gland, prostate gland, salivary gland (mandibular), sciatic nerve, seminal vesicles, skeletal muscle, skin (abdominal), spinal cord (cervical, thoracic, lumbar), spleen, stomach (forestomach, glandular), testes, thymus, thyroid/parathyroid, tongue, trachea, uterus, urinary bladder. Zymbal's gland, and vagina.

HISTOPATHOLOGY: Fixed tissues were trimmed, embedded, sectioned, and stained with hematoxylin and eosin. The slides were delivered to Toxicology Pathology Associates for microscopic evaluation by Charles Frith, DVM., Ph.D. Histopathology was performed on all tissues from the vehicle control and high-dose group animals and gross lesions from all animals.

STATISTICS: Means and standard deviations were calculated for all quantitative data. Treated groups of the sanie sex were compared to Group 1 at common time points using an ANOVA with a post hoc Dunnett's t Test (Dunnett, C.W., J. Amer. Statis. Assoc. , 50:1096-1121 , 1955). A twosided a of 0.05 or 0.01 was used to determine if statistically significant differences existed between the vehicle control and the treated groups. If no significant differences existed at the 0.05 level, then no comparison was necessary at the 0.01 level."

Blood Urea Nitrogen (BUN)

Endpoint Details

Dataset

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