Toxicology Research Laboratory • ^^ The University of Illinois UlV at Chicago Department of Pharmacology (M/C 868) 1940 W. Taylor St. Chicago, Illinois 60612-7353 20100915156 I Contract No DAMD17-92-C-2001 I Task Order No. UIC-7E 107 TJICmU. Study No. ll £ Title Page Volume 1 of 2 Revised Draft Report for Task Order No. UIC-7E THIRTEEN WEEK ORAL TOXICITY STUDY OF WR242511 IN RATS Sponsor: US Army Medical Materiel Development Activity Test Article. WR242511 Tartrate Contract No.: DAMD17-92-C-2001 Studv Director Barry S. Levine, D.Sc, D.A.B.T. In-Life Phase Completed On January 14, 1994 Performing Laboratory TOXICOLOGY RESEARCH LABORATORY (TRL) University of Illinois at Chicago (UIC) Department of Pharmacology 1940 W. Taylor St. Chicago, IL 60612-7353 The views, opinions, and/or findings contained in this report are those of the authors) and should not be construed as an official Department of the Army position, policy, or decision, unless so designated by other documentation. Page 1 UNCLASSIFIED fiffifrjg CURiTY CLASSIFICATION QP •HIS PAGE Form Approved REPORT DOCUMENTATION PAGE OMB No. 0704-0188 1a. REPORT SECURITY CLASSIFICATION 1b. RESTRICTIVE MARKINGS 3. DISTRIBUTION/AVAILABILITY OF REPORT 2a. SECURITY CLASSIFICATION AUTHORITY Unclassified 2D. DECLASSIFICATION/DOWNGRADING SCHEDULE Unlimited 5. MONITORING ORGANIZATION REPORT NUMBER(S) 4. PERFORMING ORGANIZATION REPORT NUMBER(S) UIC-7E (UIC/TRL Study No. 107) 6b. OFFICE SYMBOL 7a. NAME OF MONITORING ORGANIZATION 6a. NAME OF PERFORMING ORGANIZATION (If applicable) Toxicology Research laboratory U.S. Army Medical Research Acquisition Activity University of Illinois at Chicago 7b. ADDRESS (City. State, and ZIP Code) 6c ADDRESS (City. State, and ZIP Code) ATTN: SGPJJL-RMA-RCD Department of Pharmacology (M/C 868) Fort Detrick 1940 W. Taylor Street Frederick, to 21702 Chicago, 1L 60612-7353 9. PROCUREMENT INSTRUMENT IDENTIFICATION NUMBER 8b. OFFICE SYMBOL Ba. NAME Of FUNDING/SPONSORING (if applicable) ORGANIZATION U.g, ^ y §dlcal DAMD17-92-C-2O01 Materiel Development Activity SGRD-UMP 10. SOURCE OF FUNDING NUMBERS 8c ADDRESS (Gty, State, and ZIP Code) TASK WORK UNIT PROGRAM PROJECT Fort Detrick ELEMENT NO. NO. ACCESSION NO. NO. Frederick, MD 21702-5009 QC 073 30463807 63807A 11. TITLE (Include Security Classification) Thirteen Week Oral Toxicity Study of WR242511 in Rats 12. PERSONAL AUTHOR(S) Wheeler, Clvde W. and Toralinson, M. J. (PAD Levine. Barrv 5, 15. PAGE COUNT 13b. TIME COVERED 14. DATE OF REPORT (Year, Montt). Day) 13a. TYPE OF REPORT 543 FROM1?n/Q? TO Studv 16. SUPPLEMENTARY NOTATION 17. 18. SUBJECT TERMS (Continue on reverse if necessary and identify by block number) COSATI CODES SUB-GROUP FIELD GROUP WR242511 Tartrate Toxicity Rats 19. A3STRACT (Continue on reverse if necessary and identify by block number) This study evaluated the toxicity of WR242511 Tartrate in rats following thirteen weeks of daily oral (gavage) administration. Dose levels studied were 0 (vehicle control), 0.5, 1.5 and 4.5 mg base/kg/day. The primary treatment-related toxic effects of WR242511 were seen in the liver, lungs and RBCs. Males appeared more sensitive than females to the hepatotoxic effects of WR242511 administration. Microscopic liver lesions (hepatocyte degeneration and necrosis), and elevations in serum ALT and/or SDH levels were observed in mid and high dose males. Increased triglyceride and cholesterol levels in high dose females, and increased cholesterol levels in high dose males also suggested potential hepatocellular toxicity. Increases in total bile acids and alkaline phosphatase levels suggested hepatobiliary changes in high dose animals. Pulmonary microscopic lesions (alveolar histiocytosis) were observed in all WR242511-treated groups. These dose-related effects (hepatocyte degeneration and necrosis, and alveolar histiocytosis) probably contributed to the early deaths of seven out often high dose males. Treatment-related mild anemia was observed in mid dose and high dose animals. The lesser methemoglobinemic response seen in high dose males than in high dose females may have been secondary to the greater hepatotoxic effect in males, resulting in a reduction in the production of a direct methemoglobin-forming metabolite. Hemosiderosis in the spleen of high dose females was probably secondary to mild hemolytic anemia. Significant methemoglobin production was also observed in mid and high dose animals. Thymic lymphocyte depletion in high dose males was apparently secondary to stress produced by test article administration, but possibly could also be a direct treatment-related effect. Mild leukocytosis possibly secondary to stress and consisting of increased number of lymphocytes, neutrophils, monocytes. and/or eosinophils was seen in high dose animals and mid dose males. Thrombocytopenia was observed in all WR242511-treated groups. Because alveolar histiocytosis. thrombocytopenia. and hematology changes were seen at the low dose level, a no- adverse effect level of WR242511 could not be determined. ABSTRACT SECURITY CLASSIFICATION 20. DISTRIBUTION.-AVAILABILITY OF ABSTRACT Urclassified • UNCLASSIFIED/UNLIMITED QJ SAME AS RP~ Q OTIC USE3S 22e. OFFICE SYMBOL 22a. NAME CF RESPONSIBLE INOIVIOUAL 23. TELEPHONE (Include Area Code) N/A 3arrv S. Levine (312) 996-5543 SSCURI-" CLASSIFICATION OF THIS °AGE Previous editions are obsolete. DO Form 1473, JUN 86 UNCLASSIFIED Contract No.: DAMD17-92-C-2001 Task Order No.: UIC-7E UIC/TRL Study No.: 107 B ft IF IT STATEMENT OF COMPLIANCE To the best of my knowledge, Study No. 107 entitled "Thirteen Week Oral Toxicity Study of WR242511 in Rats" was conducted in compliance with the Good Laboratory Practices regulations as published in 21 CFR 58, 40 CFR 160 and 40 CFR 792 in all material aspects. The protocol for this study was approved by the UIC Animal Care Committee. Signature Study Director Barry S. Levine, D.Sc, D.A.B.T. Date Pace 2 QUALITY ASSURANCE STATEMENT STUDY TITLE: THIRTEEN WEEK ORAL TOXICITY STUDY OF WR2 42 511 IN RATS STUDY NUMBER: 107 STUDY DIRECTOR: BARRY S. LEVINE INITIATION DATE: 12/3/92 This study has been divided into a series of phases. Using a random sampling approach/ Quality Assurance personnel monitor each of these phases over a series of studies. Procedures, equipment, documentation, etc., are examined in order to assure that the study is performed in accordance with the Good Laboratory Practice regulations of the Food and Drug Administration and the Environmental Protection Agency to assure that the study is conducted according to the protocol. The following are the inspection dates, phases inspected, and report dates of QA inspections of the study. INSPECT ON 12/7/92, TO STUDY DIR 12/7/92, TO MGMT 12/7/92 PHASES: PROTOCOL REVIEW INSPECT ON 10/13/93, TO STUDY DIR 10/14/93, TO MGMT 10/19/93 PHASES: OPHTHALMOLOGIC EXAMINATION INSPECT ON 10/14/93, TO STUDY DIR 10/14/93, TO MGMT 10/19/93 PHASES: FOOD CONSUMPTION, BODY WEIGHT, CLINICAL OBSERVATION AND DOSING INSPECT ON 3/10-11/94, TO STUDY DIR 3/14/94, TO MGMT 3/23/94 PHASES: ANALYTICAL LABORATORY RAW DATA AND DRAFT REPORT INSPECT ON 3/30-4/1/94, TO STUDY DIR 4/1/94, TO MGMT 4/6/94 PHASES: RAW DATA INSPECT ON 5/3-5/94, TO STUDY DIR 5/5/94, TO MGMT 5/6/94 PHASES: DRAFT PATHOLOGY REPORT INSPECT ON 5/18-20/94, TO STUDY DIR 5/20/94, TO MGMT 5/24/94 PHASES: DRAFT REPORT INSPECT ON 9/30/94, TO STUDY DIR 9/30/94, TO MGMT 9/30/94 PHASES: SECOND DRAFT REPORT AdafajKjAjwA ?/Wf</ 6K&k.. QUALITY ASSURANCE DATE Page 3 Contract No.: DAMD17-92-C-2001 Task Order No.: UIC-7E UIC/TRL Study No.: 107 BEfi IF IT Signature Page THIRTEEN WEEK ORAL TOXICITY STUDY OF WR242511 IN RATS Test Article.: WR242511 Tartrate Sponsor: US Army Medical Materiel Development Activity Fort Detrick Frederick, MD 21702-5009 Sponsor Representative: George J. Schieferstein, Ph.D. Testing Facility: TOXICOLOGY RESEARCH LABORATORY (TRL) University of Illinois at Chicago (UIC) Department of Pharmacology 1940 W. Taylor St. Chicago, IL 60612-7353 Barry S. Levine, D.Sc, D.A.B.T. Date Studv Director Clyde W. Wheeler, PhD. Date Toxicologist Study Initiation: December 3, 1992 Dosing Initiation: October 14, 1993 In-Life Completion: January 14, 1994 Page 4 Contract No.: DAMD17-92-C-2001 Task Order No.: UIC-7E UIC/TRL Study No.: 107 71 BfiffH TABLE OF CONTENTS VOLUME 1 TITLE PAGE 1 STATEMENT OF COMPLIANCE 2 QUALITY ASSURANCE STATEMENT 3 SIGNATURE PAGE 4 TABLE OF CONTENTS 5 1. SUMMARY 7 2. INTRODUCTION 7 3. MATERIALS AND METHODS 7 3.1 Test Article 7 3.2 Animals 8 3.3 Experimental Design 8 3.4 Statistical Analyses 11 4 RESULTS 12 4.1 Dosage Formulation Analyses 12 4.2 Mortality and Clinical Signs/Observations 12 4.3 Body Weight 13 4.4 Food Consumption 13 4.5 Clinical Pathology 14 4.6 Ophthalmology 15 4.7 Organ Weights 15 4.8 Pathology 16 5. DISCUSSION/CONCLUSION 17 6. PERSONNEL 19 7. ARCfflVES 19 LIST OF TABLES 1 Summary of Toxic Responses 20 2 Dosage Formulation Analyses 21 3 Summary of Clinical Signs 22 4 Summary of Body Weights 23 5 Summary of Weight Gains 25 Page 5 Contract No. DAMD17-92-C-2001 UIC-7E Task Order No. 107 UICATRL Study No. (saiFir TABLE OF CONTENTS (contd.) r LIST OF TABLES (contd.) LU 6 Summary of Daily Food Consumption 27 7 Summary of Clinical Chemistry Tests 29 8 Summary of Hematology Tests 65 9 Organ Weight Summary (% Brain Weight) 103 10 Summary of Gross and Microscopic Lesions 105 FIGURES 1 Summary of Male Body Weights 106 2 Summary of Female Body Weights 107 APPENDICES 1 Analytical Chemistry Report 1-1 2 Clinical Pathology Methodology 2-1 3 Individual Observations (Clinical Signs) 3-1 VOLUME 2 APPENDICES (contd.) 4 Individual Body Weights and Body Weight Gains 4-1 5 Individual Food Consumption Data 5-1 6 Individual Clinical Chemistry Data 6-1 Individual Hematology Data 7-1 8 Ophthalmology Report 8-1 9 Individual Organ Weights 9-1 10 Pathology Report 10-1 11 Protocol and Protocol Amendments 11-1 12 Study Deviations 12-1 Page 6 Contract No. DAMD17-92-C-2001 Task Order No. UIC-7E 107 UIC/TRL Study No. ffifflRf! 1. SUMMARY This study evaluated the toxicity of WR242511 Tartrate in rats following thirteen weeks of daily oral (gavage) administration. Dose levels studied were 0 (vehicle control), 0.5, 1.5 and 4.5 mg base/kg/day. The primary treatment-related toxic effects of WR242511 were seen in the liver, lungs and RBCs. Males appeared more sensitive than females to the hepatotoxic effects ofWR242511 administration. Microscopic liver lesions (hepatocyte degeneration and necrosis), and elevations in serum ALT and/or SDH levels were observed in mid and high dose males. Increased triglyceride and cholesterol levels in high dose females, and increased cholesterol levels in high dose males also suggested potential hepatocellular toxicity. Increases in total bile acids and alkaline phosphatase levels suggested hepatobiliary changes in high dose animals. Pulmonary microscopic lesions (alveolar histiocytosis) were observed in all WR242511-treated groups. These dose-related effects (hepatocyte degeneration and necrosis, and alveolar histiocytosis) probably contributed to the early deaths of seven out of ten high dose males. Treatment-related mild anemia was observed in mid dose and high dose animals. Hemosiderosis in the spleen of high dose females was probably secondary to mild hemolytic anemia. Significant methemoglobin production was also observed in mid and high dose animals. The lesser methemoglobinemic response seen in high dose males compared to high dose females may have been secondary to the greater hepatotoxic effect in males, resulting in a reduction in the production of a direct methemoglobin-forming metabolite. Thymic lymphocyte depletion in high dose males was apparently secondary to stress produced by test article administration, but possibly could also be a direct treatment-related effect. Mild leukocytosis possibly secondary to stress and consisting of increased number of lymphocytes, neutrophils, monocytes, and/or eosinophils was seen in high dose animals and mid dose males. Thrombocytopenia was observed in all WR242511-treated groups. Because alveolar histiocytosis, thrombocytopenia, and hematology changes were seen at the low dose level, a no-adverse effect level of WR242511 could not be determined. INTRODUCTION This study was conducted to determine the specific target organ toxicity, dose-response relationships and determination of a no-adverse effect level of WR242511 tartrate in rats following thirteen weeks of daily oral administration. The study was conducted in accordance with the specifications of the Sponsor. The rats used in the study are a standard and accepted rodent species for regulatory toxicology studies, and was specified by the Sponsor. Oral administration is the intended clinical route and was also specified by the Sponsor. All methods and procedures were conducted in accordance with the Quality Assurance Programs of the Toxicology Research Laboratory, University of Illinois at Chicago and Pathology Associates, Inc., designed to conform with FDA Good Laboratory Practices Regulations. No unforeseen circumstances affected the integrity of the study. Dosing was initiated on October 14, 1993 and the in-life portion was terminated on January 14, 1994. MATERIALS AND METHODS 3.1 Test Article WR242511 Tartrate (Lot No. DJD-08-235. Batch No. BM05816), a yellow powder, was received on December 15. 1992 and June 16. 1992 from Hemer & Co., and was assigned an in-house chemical number (1720614). The chemical name of the test article is 8-[(4-Amino-l-methylbutyl)amino]5-(l-hexyloxy)-6-methoxy-4-methylquinoline DL Tartrate and the mole fraction of the base is 0.71. It was stored at -15 to -20°C and ambient humidity, and protected from light in an amber bottle. Paae 7 B&H DAMD17-92-C-2001 illl \*A f&\ l? U Contract No. Task Order No. UIC-7E UIC/TRL Study No. 107 The Analytical Chemistry Report is contained in Appendix 1. The test article was initially identified by GC-MS and the purity was determined to be 99.51% + 0.02%. The purity was re-determined following the completion of the in-life portion of the study. At that time, the purity was 99.59% + 0.02%. Thus, the test article was stable under storage conditions. »&* 3.2 Animals Fifty male and female CD® Virus Antibody Free (VAF) rats were obtained from Charles River Breeding Laboratories (Kingston, NY) on October 6, 1993. The animals were approximately 6 weeks old (date of birth August 30, 1993) upon arrival at the UIC AAALAC-accredited animal facility. Each animal was given a study-unique quarantine/pretest number following placement in cages. Animals were singly housed in polycarbonate cages with Anderson bed-o-cob® bedding (Heinold, Kankakee, IL) in a temperature (65-78°F) and humidity (30-70%) controlled room with a 14 hour light/10 2 hour dark cycle. The cage size, 840 cm area and 20 cm height, was adequate to house rats at the upper weight range as described in the Guide for the Care and Use of Laboratory Animals, DHHS (NIH) No. 86.23. All animals were routinely transferred to clean cages with fresh bedding weekly. Certified Rodent Chow No. 5002 (PMI Feeds Inc., St. Louis, MO) was provided ad libitum from arrival until termination, except during an approximate 16-20 hour fast prior to blood collection for clinical pathology and/or necropsy. Tap water from an automatic watering system in which the room distribution lines were flushed daily was provided ad libitum. The water was not treated with additional chlorine or HC1. There were no known contaminants in the feed or water which were expected to influence the study. The results of the bimonthly comprehensive chemical analyses of Chicago water performed by the City of Chicago are documented in files maintained by Quality Assurance. 3.3 Experimental Design All animals were examined daily during the eight day quarantine/pretest period, and were approved for use by the Clinical Veterinarian prior to being placed on test. Near the end of the quarantine/pretest period, 40 animals of each sex were randomized by sex into the groups shown in the following table using a computer-generated randomization program, stratified on the basis of body weight. Number Number Treatment Dose Level of Males of Females (ma base/ka/dav) Group 10 10 0 1 10 10 0.5 : 10 10 1.5 3 10 10 4 4.5 Page 8