ACUTE INTRAVENOUS TOXICITY STUDY OF M1 IN SPRAGUE DAWLEY RATS

Transcription

1 Unaudited Draft Report October 30, 2006 ACUTE INTRAVENOUS TOXICITY STUDY OF M1 IN SPRAGUE DAWLEY RATS Author: Hanna Hongchin Ng, Ph.D., D.A.B.T (Study Director) Testing Facility: SRI International Toxicology Laboratory 333 Ravenswood Avenue Menlo Park, CA SRI Study No.: M SRI Project No.: P Study Initiation: Experimental Work Performed: Start 07/26/2006 Finish 10/09/2006 Study Completion: TBD Sponsor: National Institute of Mental Health, NIH, DHHS NCS Building, Rm (MSC 9641) 6001 Executive Blvd. Bethesda, Maryland Sponsor's Representatives: Jamie Driscoll, Program Analyst, NIMH Robert Innis, Ph.D., NIH

2 APPROVAL SIGNATURES Written by: Hanna Hongchin Ng, Ph.D., D.A.B.T. Study Director Date Approved by: Jon Mirsalis, Ph.D., D.A.B.T. Principal Investigator Date SRI International Biosciences Division 333 Ravenswood Avenue Menlo Park, CA

3 TABLE OF CONTENTS APPROVAL SIGNATURES...2 SUMMARY...5 SRI QUALITY ASSURANCE STATEMENT...6 QCL QUALITY ASSURANCE STATEMENT...7 BIOPATHOLOGY SCIENCES QUALITY ASSURANCE STATEMENT...9 KEY PERSONNEL...10 I. PURPOSE OF STUDY...11 II. OBJECTIVE OF STUDY...11 III. MATERIALS AND METHOD...11 A. Experimental Design B. Test and Control Articles C. Test System D. In-Life Evaluations E. Necropsy F. Histopathologic Examination G. Evaluation of Data: H. Control of Bias I. Good Laboratory Practices Compliance J. Retention of Records and Study Samples IV. RESULTS...21 A. Mortality/Morbidity and clinical Observations B. Body Weight C. Food Consumption D. Clinical Pathology E. Necropsy F. Organ Weight G. Histopathology V. DISCUSSIONS AND CONCLUSION

4 TABLE OF CONTENTS (CONCLUDED) TABLES 1. Mortality/Morbidity and Clinical Observations Body Weights Summary Food Consumption Summary Hematology Summary Clinical Chemistry Summary Organ Weight Summary Histopathology Findings Summary...51 APPENDICES A. PROTOCOL... A-1 B. ANALYTICAL CHEMISTRY...B-1 B-1. CERTIFICATES OF ANALYSIS...B-2 B-2. TEST ARTICLE PURITY, FORMULATION CONCENTRATION, HOMOGENEITY AND STABILITY ANALYSES...B-7 C. CLINICAL OBSERVATION FREQUENCY BY ANIMAL...C-1 D. INDIVIDUAL ANIMAL BODY WEIGHT... D-1 E. INDIVIDUAL ANIMAL FOOD CONSUMPTION...E-1 F. INDIVIDUAL ANIMAL CLINICAL PATHOLOGY...F-1 G. INDIVIDUAL ANIMAL GROSS NECROPSY FINDINGS... G-1 H. INDIVIDUAL ANIMAL ORGAN WEIGHTS... H-1 I. HISTOPATHOLOGY...I-1 I-1. PATHOLOGY NARRATIVE...I-2 I-2. HISTOPATHOLOGY SUMMARY...I-4 I-3. INDIVIDUAL ANIMAL HISTOPATHOLOGY...I-17 4

5 SUMMARY The objective of this study is to determine potential toxic effects, and to identify potential target organs of toxicity for the toxicity endpoints examined following a single intravenous (iv) bolus administration of M1. Male and female Sprague-Dawley rats (10/sex/group) were given a single iv dose of M1 at 88.1 µg/kg (528.6 µg/m 2, 100x human dose) on Day 1. A control group (10/sex) was given a single iv dose of vehicle (4.8% ethanol (v/v) and 2.8% (v/v) Cremophor EL in sterile saline) at an equivalent volume on Day 1. Animals were sacrificed on Days 3 and 15 (interim and terminal necropsy, respectively). The following parameters were evaluated: mortality/morbidity, clinical observations, body weights, food consumption, clinical pathology (hematology and serum chemistry), organ weights, necropsy macroscopic observation and microscopic histopathology. All animals survived until their scheduled necropsy. No drug-related effects were observed for clinical observations, body weights, food consumption, clinical pathology, organ weights, macroscopic or histopathologic evaluations. Slight, sporadic changes in clinical pathology parameters did not correlate with histopathologic or other toxicologic parameters, and are considered to be of minimal toxicologic significance. In conclusion, a single intravenous administration of M1 to male and female Sprague- Dawley rats at 88.1 µg/kg (100x human dose) did not produce overt biologically or toxicologically significant adverse effects. The maximum tolerated dose (MTD) is therefore considered to be greater than 88.1 µg/kg (528.6 µg/m 2 ) and the no observed adverse effect level (NOAEL) is considered to be at least 88.1 µg/kg (528.6 µg/m 2 ). 5

6 SRI QUALITY ASSURANCE STATEMENT 6

7 QCL QUALITY ASSURANCE STATEMENT 7

8 QCL QUALITY ASSURANCE STATEMENT 8

9 BIOPATHOLOGY SCIENCES QUALITY ASSURANCE STATEMENT 9

10 KEY PERSONNEL Name David G. Fairchild, D.V.M., M.S., D.A.C.V.P. Jane Han, Ph.D. Hanna Hongchin Ng, Ph.D., D.A.B.T. Kenneth López, D.V.M., M.P.H., D.A.C.L.A.M. Jon Mirsalis, Ph.D., D.A.B.T Janice Nimer/Sherrod Smith Sandra Phillips Henry Sellenthin, M.S. Christina Tang, C.L.S., M.T. Elaine Tran, B.S. Veterinary Pathologist Title Manager, Analytical Chemistry Study Director Attending Veterinarian Principal Investigator Study Monitors Director, Toxicology Support Services Managing Director, BioPathology Sciences Managing Director, Quality Clinical Laboratory, Inc. Supervisor, Dose Preparation 10

11 I. PURPOSE OF STUDY The purpose of this study is to support an application for research or marketing purposes. This study, therefore, was performed in accordance with the U.S. FDA "Good Laboratory Practice for Nonclinical Laboratory Studies," as described in 21 CFR Part 58, Section I. II. OBJECTIVE OF STUDY The objective of this study is to determine potential toxic effects, and to identify potential target organs of toxicity for the toxicity endpoints examined following a single intravenous bolus administration of M1. Information from this study may be used to determine the suitability of the proposed human dose. III. MATERIALS AND METHOD A. Experimental Design Species/Strain/Sex: Rat; Sprague-Dawley; female and male Number of Animals and Dose Levels: Dose Level # Animals Sacrificed Group Target Dose* µg/m 2 µg/kg Dose Conc. Dose Vol. Day 3** Day 15 (µg/ml) (ml/kg) Interim Terminal 1 0 (vehicle) M/5 F 5 M/5 F 2 100x Human Dose M/5 F 5 M/5 F * Maximum human dose is 10 µg per 70 kg person. 10 µg/70 kg = µg/kg x 37 = 5.286µg/m 2. Scaling for the rat gives µg/m 2 / 6 = µg/kg as an equivalent human dose. 100 X = 88.1 µg/kg. ** Day 1 is the day of dose administration Route of administration and justification: Intravenous (iv) via the tail vein. This is the route intended for human clinical trials; therefore iv injection was selected to model the intended route of human administration. Frequency: Single dose on Day 1 Necropsy: Days 3 and 15 11

12 B. Test and Control Articles Test Article: M1 (also known as SP-203) Molecular Weight: 260g/mole Manufacturer: National Institute of Mental Health, NIH (Bethesda, MD) Supplier: National Institute of Mental Health, NIH (Bethesda, MD) Lot No.: Physical Description: Off white powder Storage Conditions: C Characterization of Test Article: Control Article/Vehicle: Ethanol Manufacturer: Lot No.: Physical Description: Storage Conditions: Cremophor EL Manufacturer/Supplier: Batch No.: Physical Description: Storage Conditions: Responsibility of the Sponsor. A Certificate of Analysis (C of A) with information of identity, and purity was provided by the Sponsor with the drug. The C of A was not conducted in accordance with the U.S. FDA GLP compliance. The C of A does not include information of stability of the test article; however, SRI performed chromatographic purity of the test article by measuring both before and after several days of test article in the formulation. Purity was calculated by normalized peak area percentage and purity results were used for evaluation of the test article stability. The same HPLC method was used in concentration and purity analyses of the dose formulations. 4.8% ethanol (v/v) and 2.8% (v/v) Cremophor EL in sterile saline AAPER (Shelbyville, KY) DH1136 Clear colorless liquid C Sigma-Aldrich (St. Louis, MO) 085K0104 Clear light yellow viscous liquid C 12

13 Sterile Saline for Injection, USP Manufacturer: Baxter Healthcare Corp. (Deerfield, IL) Supplier: VWR (Brisbane, CA) Lot No.: C Physical Description: Clear colorless liquid Storage Conditions: C Characterization of Control Article/Vehicle: Preparation of Dose Formulations: Information on the identity, purity, and stability was obtained by recording all of the pertinent information provided on the container labels or from the Certificate of Analysis (C of A) included in Appendix B-1. Dose solutions was prepared by dissolving the appropriate amount of test article in the vehicle to achieve the target concentration using a sterile stir bar for 15 minutes initially and an additional 20 minutes after Cremophor EL was added to the mixture. This produced a clear, colorless thick solution with particles remaining, which was then sonicated for 60 seconds. After sonication, the test article was completely dissolved and the resulting solution was clear and colorless. The initial ph for dosing formulation was determined to be ph The dose solution was aliquoted to volume with sterile saline and then mixed for another 15 minutes. The final ph of the solution was Dose formulations were kept at - 76 to -81 C and protected from light until the day of use. Formulation was brought to room temperature prior to administration to the animals. Characterization of Dose Formulations: Determination of formulation stability and homogeneity was conducted by SRI, concurrently with the study (see Appendix A). Dose formulations were used within the window of stability determined in the stability study. Results of the dose verification are included in Appendix B-2. Dose formulation concentrations were determined using an analytical method developed for this compound and described in Appendix A. The analytical methods are provided in Appendix B-2. 13

14 Test Article Handling: Disposition: Method for Assuring Correct Dose: C. Test System Species: Strain: Supplier: Gender: Age at Initiation: Weight at Initiation: Number of Animals: Animal Care: Quarantine/Acclimation: Housing: At the minimum, the test article and dose formulation were handled with the use of eye protection, latex gloves, and a protective smock or laboratory coat. Unused bulk test article will be returned to the Supplier following the end of the study or saved for subsequent studies at SRI. Residual dose solution samples were stored frozen at -70 ±10 C freezer and will be discarded 30 days after the Final Report is issued. The test article was weighed with calibrated balances. The preparation and administration of each formulation was properly documented and records were maintained showing the amount administered to each animal. Rat Sprague-Dawley Charles River Laboratories (Hollister, CA) Male and female Male: 7 Weeks; Female: 8 Weeks Male: 180g-213 g; Female: 162g-198 g 40 assigned to test General procedures for animal care and housing was in accordance with the National Research Council (NRC) Guide for the Care and Use of Laboratory Animals (1996) and the Animal Welfare Standards incorporated in 9 CFR Part 3, Rats were quarantined for three days. The general appearance of the animals was evaluated by the attending veterinarian, who documented that the animals were healthy before releasing them from quarantine. 5 per cage 14

15 Cages: Light Cycle: Polycarbonate hanging cages with Sanichip bedding. 12 hr light/12 hr dark Temperature: 67 F to 71 F Humidity: 41-64% Food: Water: Purina Certified Rodent Chow, #5002, (Richmond, IN) ad libitum. No contaminants that could interfere with and affect the results of the study are expected to be present in the food. Copies of lotspecific feed analysis reports were included in the study record. Water (purified, reverse osmosis) was provided ad libitum. No contaminants that could interfere with and affect the results of the study were present in the water, based on previous analysis reports. Copies of annual analysis reports are maintained at SRI for reference. Assignment of Animals to Study: Day: Randomization: Identification: Test System Justification: Welfare of the Animals: 1 day before initiation of dose administration Randomly assigned to treatment groups by a computerized body weight stratification procedure. No animals were excluded from the selection process (LABCAT In-Life V6.2). Animals individually identified by ear punch. This is an accepted species to support studies of compounds used or intended for use in humans. Every effort was made to minimize, if not eliminate, pain and suffering in all animals in this study. There were no moribund animals in the study. D. In-Life Evaluations Mortality/Morbidity: Clinical Observation: Once daily. Once daily. 15

16 Body Weights: On Day 1 prior to dose administration and Day 3 and 15 prior to necropsy. Food Consumption: Twice weekly. Quantitatively measured for each cage over a 24 hr-period on Days 1-2, 5-6, 9-10, and Data per cage was collected manually and entered directly into LABCAT In-Life data collection system (Version 6.2). Clinical Pathology Evaluations: Method of Collection: From the retro orbital sinus under CO 2 :O 2 (60:40) anesthesia. Frequency: On Days 3 and 15 Hematology samples were collected using EDTA as the anticoagulant. No anticoagulant was used for serum chemistry samples. Clinical laboratory: Hematology Parameters: Serum Chemistry Parameters: After collection at SRI, the clinical pathology samples were sent to Quality Clinical Labs, Inc (Mountain View, CA). RBC count (RBC) Hematocrit (HCT) Hemoglobin (HGB) Mean Corpuscular Volume (MCV) Mean Corpuscular Hemoglobin (MCH) Mean Corpuscular Hemoglobin Concentration (MCC) WBC count (WBC) WBC differential counts [absolute neutrophil count (ANS), lymphocyte (ALY), monocyte (AMO), eosinophil (AEO), and basophil (ABA)] Platelet count (PLC) Reticulocyte count (RET) Alanine aminotransferase (ALT) Albumin (ALB) Alkaline phosphatase (ALP) Aspartate aminotransferase (AST) Bilirubin [total (TBI) Blood urea nitrogen (BUN) 16

17 E. Necropsy Calcium (CAL) Chloride (CHL) Cholesterol (CHO) Creatinine (CRE) Creatinine phosphokinase (CPK) Gamma glutamyl transferase (GGT) Globulin (GLO) Glucose (GLU) Phosphorus (PHO) Potassium (POT) Protein, total (TPR) Sodium (SOD) Triglycerides (TRI) Albumin/globulin ratio (AGR) Interval: Euthanasia Observations: Tissues Retained: Animals were sacrificed on Days 3 and 15 (interim and terminal necropsy). Approximately 150 mg/kg sodium pentobarbital administered IP. External examination of all body orifices and surfaces and an examination of all cranial, thoracic, and abdominal organs. Gross pathology findings were recorded. Fixed in phosphate-buffered 10% formalin: Adrenal glands Aorta Bone marrow (histology, sternum) Bone marrow smear (cytology, sternum) Bone, femur Brain (fore-, mid-, and hindbrain) Cecum Cervix Colon Duodenum Epididymides Esophagus Eyes, with optic nerve Gross lesions (including tissue masses and abnormal regional lymph nodes) Heart Ileum 17

18 Injection site (tail) Jejunum Kidneys Liver Lungs with bronchi Lymph nodes, mandibular, and mesenteric) Mammary tissue (including nipple and surrounding tissue) Ovaries Pancreas Prostate gland Pituitary gland Rectum Salivary gland Sciatic nerve Seminal vesicle Skeletal muscle Skin, untreated (abdominal; taken with mammary gland) Spinal cord (thoracolumbar junction; retained within spinal column) Spleen Stomach Testes Thymus Thyroid and parathyroid Tongue Trachea Urinary bladder Uterus Vagina Tissues Weighed: Organ weights recorded for all surviving animals at scheduled sacrifice. Paired organs were combined and weighed as a pair except for kidneys Adrenal glands Brain Heart Kidneys Liver Spleen Thymus Testes Ovaries 18

19 F. Histopathologic Examination Groups Evaluated: Tissues Evaluated: Performed by: Method: All groups. Listed above (Section E). Sections of the tissues from the animals were embedded in paraffin, cut approximately 5 microns thick, and stained with hematoxylin and eosin. David G. Fairchild, D.V.M., M.S., D.A.C.V.P. Each lesion listed and coded by the most specific topographic and morphologic diagnoses, severity, and distribution using Systematized Nomenclature of Medicine (SNOMED) codes as a guide. A fourstep grading system (minimal, mild, moderate, and marked) was used to define gradable lesions for comparison between treated and control groups. Records of gross findings for a specimen from postmortem observations were available to the pathologist when examining that specimen microscopically. G. Evaluation of Data: Toxicology Parameters: Statistical Test: Mean and standard deviation were calculated for body weight, clinical pathology, food consumption and organ weight data at each evaluation interval. One-way ANOVA analysis was performed on body weights and food consumption (LABCAT module In-Life v. 6.2), clinical pathology(labcat module HE v. 4.42), and organ weight data (LABCAT module OW v3.24 ). If appropriate a post-hoc analysis (Dunnett s t-test) was done. Criteria for Null Hypothesis Rejection: p < 0.05 H. Control of Bias When evaluating the animals, the technical staff was aware of the each animal s treatment history. However, based on the relatively objective endpoints to be examined, bias is not expected to influence the results of the study. 19

20 I. Good Laboratory Practices Compliance This study is intended to be submitted to and reviewed by the U.S. Food and Drug Administration (FDA) or an equivalent regulatory agency and, therefore, was performed in accordance with the U.S. FDA "Good Laboratory Practice for Nonclinical Laboratory Studies," as described in 21 CFR Part 58, with the following exceptions. Animal water and food analysis was not performed under GLP compliance by the vendors. Characterization of the test article is the responsibility of the Sponsor. The Sponsor submitted a Certificate of Analysis (C of A) that includes information on chemical identity and purity. The C of A for the test article was not conducted in accordance with the U.S. FDA GLP compliance. The C of A does not include information of stability of the test article; however, SRI performed chromatographic purity of the test article by measuring both before and after several days of test article in the formulation. Purity was calculated by normalized peak area percentage and purity results were used for evaluation of the test article stability. The report is included in the final report. Various pre-study activities (e.g. quarantine of animals, pre-study body weights, and randomization) were performed prior to the approval of the protocol. These activities were conducted according to the testing facility Standard Operating Procedures (SOPs), but were conducted before the protocol was signed and therefore may be considered not to be conducted within full FDA GLP compliance. J. Retention of Records and Study Samples The original protocol and amendments, original final report, and all raw data, supporting documents, and records specific to this study will be retained and stored in the Records Center at SRI International, 333 Ravenswood Avenue, Menlo Park, CA All pathology materials (slides, blocks, and wet tissue specimens) will be archived at a commercial archiving facility, Pacific Storage (Sacramento, CA). All pathology materials (slides, blocks, and wet tissue specimens) will be archived at a commercial archiving facility, Pacific Storage (Sacramento, CA). All materials will be maintained for a period of 5 years. At the end of the retention period, the Sponsor will be contacted regarding further disposition of these materials. The Sponsor will be responsible for the retention of records that are derived from portions of the study conducted by the Sponsor. 20

21 IV. RESULTS A. Mortality/Morbidity and clinical Observations Mortality/morbidity and clinical observations are summarized in Table 1. Individual animal clinical observations are presented in Appendix C. All animals survived until their scheduled necropsy. All animals appeared normal throughout the study. B. Body Weight Body weights are summarized in Table 2. Individual animal body weights are presented in Appendix D. No changes in body weight were observed in the treated group compared with the control group. C. Food Consumption Food consumption data are summarized in Table 3. Individual animal data are presented in Appendix E. No statistically significant changes in food consumption were observed in the treated group compared with the control group. Food consumption was lower on the last evaluation day (Day 13-14) for both males and females, but this difference was not statistically significant, and coming at the end of the study, two weeks after initial treatment, is not considered to be toxicologically significant or test article related. D. Clinical Pathology Hematology and clinical chemistry results are summarized in Tables 4 and 5, respectively. Individual animal clinical pathology data are presented in Appendix F. A slight increase of red blood cell count (RBC) and white blood cell count (WBC) and a slight decrease in the reticulocyte count (RET) in the males of the treated group were observed on Day 3. No differences in the hematology parameters were seen in the males of the treated group compared with the controls on Day 15 terminal sacrifice. In females, eosinophil count (AEO) in the treated group was increased on Day 15. A slight increase in calcium (CAL) was seen in the males and females of the treated group on Day 3. Phosphorus (PHO) was slightly increased in the treated groups compared with the controls on Days 3 and 15. These changes in clinical pathology are slight, fall within normal ranges for the rat, and are not correlated with any microscopic or other toxicologic findings. They may represent spontaneous variations unrelated to the treatment with the test article, and therefore, they are considered to be of minimal toxicological significance. 21

22 E. Necropsy Individual animal gross necropsy findings are presented in Appendix G. A number of grossly observable abnormalities were noted in the vehicle control group and test article-treated groups in the following tissues: cecum, lung, thymus, kidney, adrenal gland, liver, spleen, lymph node, salivary gland and uterus. The necropsy findings were sporadic and were not correlated with any test article related findings in the microscopic evaluation; therefore they are considered to be unrelated to treatment with M1. F. Organ Weight Organ weights are summarized in Table 6. Individual animal data are presented in Appendix I. A slight, but statistically significant, increase in the spleen to brain weight ratio was seen in the test article treated females compared with the controls at the interim sacrifice. This change is very slight and is considered to be a sporadic variation. No other changes in organ weight were observed in the treated group as compared with the control group. G. Histopathology Histopathology findings are summarized in Table 7. Individual animal histopathology data are presented in Appendix J. None of the changes presented in the pathology tables were distributed in a manner suggesting a causal relationship of administration of either the vehicle or the 88.1 µg/kg compound formulations. Rather, the changes were considered related to spontaneous disease or conditions and not to the administration of the compound formulations. V. DISCUSSIONS AND CONCLUSION All animals survived until their scheduled necropsy. No drug-related effects were observed for clinical observations, body weights, food consumption, clinical pathology, organ weights, macroscopic or histopathologic evaluations. Slight, sporadic changes in clinical pathology parameters did not correlate with histopathologic or other toxicologic parameters, and are considered to be of minimal toxicologic significance. In conclusion, a single intravenous administration of M1 to male and female Sprague-Dawley rats at 88.1 µg/kg (100x human dose) did not produce overt biologically or toxicologically significant adverse effects. The maximum tolerated dose (MTD) is therefore considered to be greater than 88.1 µg/kg (528.6 µg/m 2 ) and the no observed adverse effect level (NOAEL) is considered to be at least 88.1 µg/kg (528.6 µg/m 2 ). 22

23 Table 1 MORTALITY/MORBIDITY AND CLINICAL OBSERVATIONS Observations & Details Vehicle (µg/kg) Sex: Male M µg/kg IV No. of Animals: Appears Normal Mortality Interim Sacrifice 5 5 Terminal Sacrifice 5 5 Observations & Details Vehicle (µg/kg) Sex: Female M µg/kg IV No. of Animals: Appears Normal Mortality Interim Sacrifice 5 5 Terminal Sacrifice 5 5 No. - Number of animals exhibiting one or more occurrence of the observation. 23

24 Table 2 BODY WEIGHTS SUMMARY (GRAMS) Observations & Details Sex: Vehicle (µg/kg) Male M µg/kg IV Period Day 1 Mean S.D N Day 3 Mean S.D Day 15 Mean S.D Sex: Female Observations & Details Vehicle (µg/kg) M µg/kg IV Period Day 1 Mean S.D. 8 8 N Day 3 Mean S.D. 7 4 Day 15 Mean S.D S.D. - Standard Deviation 24

25 Table 3 FOOD CONSUMPTION SUMMARY (GRAMS) Sex: Male Observations & Details Vehicle (µg/kg) M µg/kg IV Period Day 1-2 Mean S.D. 1 1 N Day 5-6 Mean S.D. 0 0 Day 9-10 Mean S.D. 0 0 Day Mean S.D. 0 0 S.D. - Standard Deviation 25

26 Table 3 (concluded) FOOD CONSUMPTION SUMMARY (GRAMS) Sex: Female Observations & Details Vehicle (µg/kg) M µg/kg IV Period Day 1-2 Mean S.D. 2 0 N Day 5-6 Mean S.D. 0 0 Day 9-10 Mean S.D. 0 0 Day Mean S.D. 0 0 S.D. - Standard Deviation 26

27 Table 4 CLINICAL PATHOLOGY SUMMARY HEMATOLOGY PERIOD: Day 3, SEX: MALE TEST(s): RBC HCT HGB MCV MCH UNITS: x10 6 /mm 3 % g/dl fl pg MEAN SD MEAN 6.32** SD TEST(s): MCC WBC ANS ALY AMO UNITS: g/dl x10 3 /mm 3 x1000/ul x1000/ul x1000/ul MEAN SD MEAN * SD TEST(s): AEO ABA PLC RET UNITS: x1000/ul x1000/ul x10 3 /mm 3 % RBC MEAN SD MEAN * SD WBC corrected for NRBC > 0 *-Significant Difference from Control p<0.05 **-Significant Difference from Control p<

28 Table 4 (continued) CLINICAL PATHOLOGY SUMMARY-HEMATOLOGY PERIOD: Day 15, SEX: MALE TEST(s): RBC HCT HGB MCV MCH UNITS: x10 6 /mm 3 % g/dl fl pg MEAN SD MEAN SD TEST(s): MCC WBC ANS ALY AMO UNITS: g/dl x10 3 /mm 3 x1000/ul x1000/ul x1000/ul MEAN SD MEAN SD TEST(s): AEO ABA PLC RET UNITS: x1000/ul x1000/ul x10 3 /mm 3 % RBC MEAN SD MEAN SD WBC corrected for NRBC > 0 28

29 Table 4 (continued) CLINICAL PATHOLOGY SUMMARY-HEMATOLOGY PERIOD: Day 3, SEX: FEMALE TEST(s): RBC HCT HGB MCV MCH UNITS: x10 6 /mm 3 % g/dl fl pg MEAN SD MEAN SD TEST(s): MCC WBC ANS ALY AMO UNITS: g/dl x10 3 /mm 3 x1000/ul x1000/ul x1000/ul MEAN SD MEAN SD TEST(s): AEO ABA PLC RET UNITS: x1000/ul x1000/ul x10 3 /mm 3 % RBC MEAN SD MEAN SD WBC corrected for NRBC > 0 29

30 Table 4 (concluded) CLINICAL PATHOLOGY SUMMARY-HEMATOLOGY PERIOD: Day 15, SEX: FEMALE TEST(s): RBC HCT HGB MCV MCH UNITS: x10 6 /mm 3 % g/dl fl pg MEAN SD MEAN SD TEST(s): MCC WBC ANS ALY AMO UNITS: g/dl x10 3 /mm 3 x1000/ul x1000/ul x1000/ul MEAN SD MEAN SD TEST(s): AEO ABA PLC RET UNITS: x1000/ul x1000/ul x10 3 /mm 3 % RBC MEAN SD MEAN 0.23* SD WBC corrected for NRBC > 0 *-Significant Difference from Control p<

31 Table 5 CLINICAL PATHOLOGY SUMMARY CHEMISTRY PERIOD: Day 3, SEX: MALE TEST(s): ALT ALB ALP AST TBI UNITS: IU/L g/dl IU/L IU/L mg/dl MEAN SD MEAN SD TEST(s): BUN CAL CHL CHO CRE UNITS: mg/dl mg/dl meq/l mg/dl mg/dl MEAN SD MEAN * SD *-Significant Difference from Control p<

32 Table 5 (continued) CLINICAL PATHOLOGY SUMMARY-CHEMISTRY PERIOD: Day 3, SEX: MALE TEST(s): CPK GGT GLO GLU PHO UNITS: IU/L IU/L g/dl mg/dl mg/dl MEAN 224 NA SD 34.3 NA N MEAN 224 NA * SD 45.5 NA N TEST(s): POT TPR SOD TRI AGR UNITS: meq/l g/dl meq/l mg/dl na MEAN SD MEAN SD NA-Not Applicable; Individual Values < 3 *-Significant Difference from Control p<

33 Table 5 (continued) CLINICAL PATHOLOGY SUMMARY-CHEMISTRY PERIOD: Day 15, SEX: MALE TEST(s): ALT ALB ALP AST TBI UNITS: IU/L g/dl IU/L IU/L mg/dl MEAN SD MEAN SD TEST(s): BUN CAL CHL CHO CRE UNITS: mg/dl mg/dl meq/l mg/dl mg/dl MEAN SD MEAN SD

34 Table 5 (continued) CLINICAL PATHOLOGY SUMMARY-CHEMISTRY PERIOD: Day 15, SEX: MALE TEST(s): CPK GGT GLO GLU PHO UNITS: IU/L IU/L g/dl mg/dl mg/dl MEAN 220 NA SD 33.6 NA N MEAN 237 NA SD 60.3 NA N TEST(s): POT TPR SOD TRI AGR UNITS: meq/l g/dl meq/l mg/dl na MEAN SD MEAN SD NA-Not Applicable; Individual Values < 3 34

35 Table 5 (continued) CLINICAL PATHOLOGY SUMMARY-CHEMISTRY PERIOD: Day 3, SEX: FEMALE TEST(s): ALT ALB ALP AST TBI UNITS: IU/L g/dl IU/L IU/L mg/dl MEAN SD MEAN SD TEST(s): BUN CAL CHL CHO CRE UNITS: mg/dl mg/dl meq/l mg/dl mg/dl MEAN SD MEAN * SD *-Significant Difference from Control p<

36 Table 5 (continued) CLINICAL PATHOLOGY SUMMARY-CHEMISTRY PERIOD: Day 3, SEX: FEMALE TEST(s): CPK GGT GLO GLU PHO UNITS: IU/L IU/L g/dl mg/dl mg/dl MEAN 197 NA SD 67.2 NA N MEAN 198 NA SD 74.8 NA N TEST(s): POT TPR SOD TRI AGR UNITS: meq/l g/dl meq/l mg/dl na MEAN SD MEAN SD NA-Not Applicable; Individual Values < 3 36

37 Table 5 (continued) CLINICAL PATHOLOGY SUMMARY-CHEMISTRY PERIOD: Day 15, SEX: FEMALE TEST(s): ALT ALB ALP AST TBI UNITS: IU/L g/dl IU/L IU/L mg/dl MEAN SD MEAN SD TEST(s): BUN CAL CHL CHO CRE UNITS: mg/dl mg/dl meq/l mg/dl mg/dl MEAN SD MEAN SD

38 Table 5 (concluded) CLINICAL PATHOLOGY SUMMARY-CHEMISTRY PERIOD: Day 15, SEX: FEMALE TEST(s): CPK GGT GLO GLU PHO UNITS: IU/L IU/L g/dl mg/dl mg/dl MEAN 146 NA SD 37.0 NA N MEAN 149 NA * SD 41.5 NA N TEST(s): POT TPR SOD TRI AGR UNITS: meq/l g/dl meq/l mg/dl na MEAN SD MEAN SD NA-Not Applicable; Individual Values < 3 *-Significant Difference from Control p<

39 (1)-Vehicle Control 0 µg/kg IV (2)-M µg/kg IV Table 6 ORGAN WEIGHT SUMMARY (ABSOLUTE) SEX: MALE, Interim Sacrifice (1) (2) GROUP: 1-M 2-M BODY WEIGHT (G)(ABSOLUTE) MEAN SD Adrenal Glands (G)(ABSOLUTE) MEAN SD Brain (G)(ABSOLUTE) MEAN SD Heart (G)(ABSOLUTE) MEAN SD Kidney (left) (G)(ABSOLUTE) MEAN SD Kidney (right) (G)(ABSOLUTE) MEAN SD Liver (G)(ABSOLUTE) MEAN SD Spleen (G)(ABSOLUTE) MEAN SD Testes (G)(ABSOLUTE) MEAN SD Thymus (G)(ABSOLUTE) MEAN SD

40 (1)-Vehicle Control 0 µg/kg IV (2)-M µg/kg IV Table 6 (continued) ORGAN WEIGHT SUMMARY (% BODY WEIGHT) SEX: MALE, Interim Sacrifice (1) (2) GROUP: 1-M 2-M Adrenal Glands (% BODY WEIGHT) MEAN SD Brain (% BODY WEIGHT) MEAN SD Heart (% BODY WEIGHT) MEAN SD Kidney (left)(% BODY WEIGHT) MEAN SD Kidney (right)(% BODY WEIGHT) MEAN SD Liver (% BODY WEIGHT) MEAN SD Spleen (% BODY WEIGHT) MEAN SD Testes (% BODY WEIGHT) MEAN SD Thymus (% BODY WEIGHT) MEAN SD

41 (1)-Vehicle Control 0 µg/kg IV (2)-M µg/kg IV Table 6 (continued) ORGAN WEIGHT SUMMARY (% BRAIN WEIGHT) SEX: MALE, Interim Sacrifice (1) (2) GROUP: 1-M 2-M Adrenal Glands (% BRAIN WEIGHT) MEAN SD Heart (% BRAIN WEIGHT) MEAN SD Kidney (left)(% BRAIN WEIGHT) MEAN SD Kidney (right)(% BRAIN WEIGHT) MEAN SD Liver (% BRAIN WEIGHT) MEAN SD Spleen (% BRAIN WEIGHT) MEAN SD Testes (% BRAIN WEIGHT) MEAN SD Thymus (% BRAIN WEIGHT) MEAN SD

42 (3)-Vehicle Control 0 µg/kg IV (4)-M µg/kg IV Table 6 (continued) ORGAN WEIGHT SUMMARY (ABSOLUTE) SEX: FEMALE, Interim Sacrifice (3) (4) GROUP: 1-F 2-F BODY WEIGHT (G)(ABSOLUTE) MEAN SD Adrenal Glands (G)(ABSOLUTE) MEAN SD Brain (G)(ABSOLUTE) MEAN SD Heart (G)(ABSOLUTE) MEAN SD Kidney (left) (G)(ABSOLUTE) MEAN SD Kidney (right) (G)(ABSOLUTE) MEAN SD Liver (G)(ABSOLUTE) MEAN SD Ovaries (G)(ABSOLUTE) MEAN SD Spleen (G)(ABSOLUTE) MEAN SD Thymus (G)(ABSOLUTE) MEAN SD

43 (3)-Vehicle Control 0 µg/kg IV (4)-M µg/kg IV Table 6 (continued) ORGAN WEIGHT SUMMARY (% BODY WEIGHT) SEX: FEMALE, Interim Sacrifice (3) (4) GROUP: 1-F 2-F Adrenal Glands (% BODY WEIGHT) MEAN SD Brain (% BODY WEIGHT) MEAN SD Heart (% BODY WEIGHT) MEAN SD Kidney (left)(% BODY WEIGHT) MEAN SD Kidney (right)(% BODY WEIGHT) MEAN SD Liver (% BODY WEIGHT) MEAN SD Ovaries (% BODY WEIGHT) MEAN SD Spleen (% BODY WEIGHT) MEAN SD Thymus (% BODY WEIGHT) MEAN SD

44 Table 6 (continued) ORGAN WEIGHT SUMMARY (% BRAIN WEIGHT) (3)-Vehicle Control 0 µg/kg IV (4)-M µg/kg IV * - Significant difference P<.05 SEX: FEMALE, Interim Sacrifice (3) (4) GROUP: 1-F 2-F Adrenal Glands (% BRAIN WEIGHT) MEAN SD Heart (% BRAIN WEIGHT) MEAN SD Kidney (left)(% BRAIN WEIGHT) MEAN SD Kidney (right)(% BRAIN WEIGHT) MEAN SD Liver (% BRAIN WEIGHT) MEAN SD Ovaries (% BRAIN WEIGHT) MEAN SD Spleen (% BRAIN WEIGHT) MEAN * SD Thymus (% BRAIN WEIGHT) MEAN SD

45 (1)-Vehicle Control 0 µg/kg IV (2)-M µg/kg IV Table 6 (continued) ORGAN WEIGHT SUMMARY (ABSOLUTE) SEX: MALE, Terminal Sacrifice (1) (2) GROUP: 1-M 2-M BODY WEIGHT (G)(ABSOLUTE) MEAN SD Adrenal Glands (G)(ABSOLUTE) MEAN SD Brain (G)(ABSOLUTE) MEAN SD Heart (G)(ABSOLUTE) MEAN SD Kidney (left) (G)(ABSOLUTE) MEAN SD Kidney (right) (G)(ABSOLUTE) MEAN SD Liver (G)(ABSOLUTE) MEAN SD Spleen (G)(ABSOLUTE) MEAN SD Testes (G)(ABSOLUTE) MEAN SD Thymus (G)(ABSOLUTE) MEAN SD

46 (1)-Vehicle Control 0 µg/kg IV (2)-M µg/kg IV Table 6 (continued) ORGAN WEIGHT SUMMARY (% BODY WEIGHT) SEX: MALE, Terminal Sacrifice (1) (2) GROUP: 1-M 2-M Adrenal Glands (% BODY WEIGHT) MEAN SD Brain (% BODY WEIGHT) MEAN SD Heart (% BODY WEIGHT) MEAN SD Kidney (left)(% BODY WEIGHT) MEAN SD Kidney (right)(% BODY WEIGHT) MEAN SD Liver (% BODY WEIGHT) MEAN SD Spleen (% BODY WEIGHT) MEAN SD Testes (% BODY WEIGHT) MEAN SD Thymus (% BODY WEIGHT) MEAN SD

47 (1)-Vehicle Control 0 µg/kg IV (2)-M µg/kg IV Table 6 (continued) ORGAN WEIGHT SUMMARY (% BRAIN WEIGHT) SEX: MALE, Terminal Sacrifice (1) (2) GROUP: 1-M 2-M Adrenal Glands (% BRAIN WEIGHT) MEAN SD Heart (% BRAIN WEIGHT) MEAN SD Kidney (left)(% BRAIN WEIGHT) MEAN SD Kidney (right)(% BRAIN WEIGHT) MEAN SD Liver (% BRAIN WEIGHT) MEAN SD Spleen (% BRAIN WEIGHT) MEAN SD Testes (% BRAIN WEIGHT) MEAN SD Thymus (% BRAIN WEIGHT) MEAN SD

48 (3)-Vehicle Control 0 µg/kg IV (4)-M µg/kg IV Table 6 (continued) ORGAN WEIGHT SUMMARY (ABSOLUTE) SEX: FEMALE, Terminal Sacrifice (3) (4) GROUP: 1-F 2-F BODY WEIGHT (G)(ABSOLUTE) MEAN SD Adrenal Glands (G)(ABSOLUTE) MEAN SD Brain (G)(ABSOLUTE) MEAN SD Heart (G)(ABSOLUTE) MEAN SD Kidney (left) (G)(ABSOLUTE) MEAN SD Kidney (right) (G)(ABSOLUTE) MEAN SD Liver (G)(ABSOLUTE) MEAN SD Ovaries (G)(ABSOLUTE) MEAN SD Spleen (G)(ABSOLUTE) MEAN SD Thymus (G)(ABSOLUTE) MEAN SD

49 (3)-Vehicle Control 0 µg/kg IV (4)-M µg/kg IV Table 6 (continued) ORGAN WEIGHT SUMMARY (% BODY WEIGHT) SEX: FEMALE, Terminal Sacrifice (3) (4) GROUP: 1-F 2-F Adrenal Glands (% BODY WEIGHT) MEAN SD Brain (% BODY WEIGHT) MEAN SD Heart (% BODY WEIGHT) MEAN SD Kidney (left)(% BODY WEIGHT) MEAN SD Kidney (right)(% BODY WEIGHT) MEAN SD Liver (% BODY WEIGHT) MEAN SD Ovaries (% BODY WEIGHT) MEAN SD Spleen (% BODY WEIGHT) MEAN SD Thymus (% BODY WEIGHT) MEAN SD

50 (3)-Vehicle Control 0 µg/kg IV (4)-M µg/kg IV Table 6 (concluded) ORGAN WEIGHT SUMMARY (% BRAIN WEIGHT) SEX: FEMALE, Terminal Sacrifice ( 3) ( 4) GROUP: 1-F 2-F Adrenal Glands (% BRAIN WEIGHT) MEAN SD Heart (% BRAIN WEIGHT) MEAN SD Kidney (left)(% BRAIN WEIGHT) MEAN SD Kidney (right)(% BRAIN WEIGHT) MEAN SD Liver (% BRAIN WEIGHT) MEAN SD Ovaries (% BRAIN WEIGHT) MEAN SD Spleen (% BRAIN WEIGHT) MEAN SD Thymus (% BRAIN WEIGHT) MEAN SD

51 Table 7 HISTOPATHOLOGY SUMMARY FATE: Interim sacrifice PERIOD: DAY 3, SEX: MALE GROUP: 1M 2M (1) (2) NUMBER OF ANIMALS: 5 5 # % # % CECUM 5 5 AUTOLYSIS ILEUM 5 5 AUTOLYSIS EPIDIDYMIDES 5 5 HYPOSPERMIA SPERM PRECURSORS EYES, WITH OPTIC NERVE 5 5 RETINAL ROSETTE HEART 5 5 HEMORRHAGE KIDNEYS 5 5 REGENERATION INFLAMMATION, CHRONIC LIVER 5 5 INFLAMMATION LUNGS, WITH BRONCHI 5 5 HEMORRHAGE LYMPH NODE, MESENTERIC 5 5 HEMORRHAGE LYMPH NODE, MANDIBULAR 5 5 HEMORRHAGE PLASMACYTOSIS PROSTATE GLAND 5 5 PERIURETHRAL INFLAMMATION Incidence Calculated by No. of Tissues Scored (1) - VEHICLE (2) MCG/KG M1 # = # of tissues evaluated. 51

52 Table 7 (continued) HISTOPATHOLOGY SUMMARY FATE: Interim sacrifice PERIOD: DAY 3, SEX: MALE GROUP: 1M 2M (1) (2) NUMBER OF ANIMALS: 5 5 # % # % SPLEE PROLIFERATION, EXTRAMED HEMATOPOIESIS TESTES 5 5 ATROPHY THYROID GLAND 5 5 ULTIMOBRANCHIAL CYST BONE, SPINAL COLUM PERIOSTEAL MARROW EXTRUSION Incidence Calculated by No. of Tissues Scored (1) - VEHICLE (2) MCG/KG M1 # = # of tissues evaluated. 52

53 Table 7 (continued) HISTOPATHOLOGY SUMMARY FATE: Interim sacrifice PERIOD: DAY 3, SEX: FEMALE GROUP: 1F 2F (1) (2) NUMBER OF ANIMALS: 5 5 # % # % ADRENAL GLANDS 5 5 HEMORRHAGE BRAI HEMORRHAGE CHOROID PLEXUS, FETAL REST CECUM 5 5 AUTOLYSIS ILEUM 5 5 AUTOLYSIS EYES, WITH OPTIC NERVE 4 5 RETINAL ROSETTE HEMORRHAGE HEART 5 5 HEMORRHAGE KIDNEYS 5 5 MINERAL, MICROSCOPIC OBSERVATION REGENERATION INFLAMMATION, CHRONIC LIVER 5 5 INFLAMMATION LUNGS, WITH BRONCHI 5 5 HEMORRHAGE LYMPH NODE, MANDIBULAR 5 5 HEMORRHAGE PLASMACYTOSIS SPLEE CONGESTION THYROID GLAND 5 5 ULTIMOBRANCHIAL CYST UTERUS 5 5 CONGESTION Incidence Calculated by No. of Tissues Scored (1) - VEHICLE (2) MCG/KG M1 53

54 # = # of tissues evaluated. Table 7 (continued) HISTOPATHOLOGY SUMMARY FATE: Terminal sacrifice PERIOD: DAY 3, SEX: MALE GROUP: 1M 2M (1) (2) NUMBER OF ANIMALS: 5 5 # % # % CECUM 5 5 AUTOLYSIS EPIDIDYMIDES 5 5 HYPOSPERMIA SPERM PRECURSORS HEART 5 5 HEMORRHAGE LIVER 5 5 INFLAMMATION LUNGS, WITH BRONCHI 5 5 EDEMA HEMORRHAGE LYMPH NODE, MESENTERIC 5 5 HEMORRHAGE LYMPH NODE, MANDIBULAR 4 5 HEMORRHAGE HYPERPLASIA, LYMPHOID PLASMACYTOSIS SPLEE CONGESTION TESTES 5 5 ATROPHY THYMUS 5 5 HEMORRHAGE Incidence Calculated by No. of Tissues Scored (1) VEHICLE (2) MCG/KG M1 # = # of tissues evaluated. 54

55 Table 7 (concluded) HISTOPATHOLOGY SUMMARY FATE: Terminal sacrifice PERIOD: DAY 3, SEX: FEMALE GROUP: 1F 2F (1) (2) NUMBER OF ANIMALS: 5 5 # % # % BRAI HEMORRHAGE JEJUNUM 5 5 AUTOLYSIS ILEUM 5 5 AUTOLYSIS HEART 5 5 HEMORRHAGE KIDNEYS 5 5 MINERAL, MICROSCOPIC OBSERVATION REGENERATION LIVER 5 5 INFLAMMATION LUNGS, WITH BRONCHI 5 5 HEMORRHAGE LYMPH NODE, MANDIBULAR 5 5 HEMORRHAGE PLASMACYTOSIS SPLEE CONGESTION Incidence Calculated by No. of Tissues Scored (1) - VEHICLE (2) MCG/KG M1 # = # of tissues evaluated. 55