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1) OECD 423-OPPTS 870.1100, Acute oral toxicity (rat), 132-002 Acute Oral Toxicity Study of T-7485 Administered to Spraque-Dawley Rats Study Number: 132-002 ACUTE ORAL TOXICITY STUDY OF T-7485 ADMINISTERED TO SPRAGUE-DAWLEY RATS STUDY NUMBER: 132-002 SANITIZED SPONSOR 3M Corporate Toxicology 3M Center, Building 220-2E-02 P.O.Box 33220 St. Paul, MN 55133-3320 DEC 0 9 2003 SPONSOR'S REPRESENTATIVE Ph.D., DABT, CIH Senior Laboratory Manager Telephone: FAX: TESTING FACILITY Primedica Redfield Mail: P.O.Box 308 Deliveries: 100 East Boone St. Redfield, AR 72132 Telephone: 501-397-2540 FAX: 501-397-2002 STUDY DATES Study Initiation: May 22, 2000 Animal Phase Initiation: May 25, 2000 Animal Phase Completion: June 8, 2000 Study Completion: October 10, 2000 Page 1 Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 GLP COMPLIANCE STATEMENT Study Number: 132-002 I certify that this study was performed in compliance with the United States Food and Drug Administration (FDA) Good Laboratory Practice Regulations (21 CFR Part 58) and OECD Regulations [C(81)30(final)] and that the report accurately reflects the raw data. e\. w Jofin Seng, Ph.D. Date Stbdy Director Primedica Redfield Page 2 Acute Oral Toxicity Study of T-7485 Administered to Sprasue-Dawley Rats Study Number 132-002 QUALITY ASSURANCE STATEMENT Study Number: 132-002 This study has been inspected and audited by the Quality Assurance Unit (QAU) as required by the Good Laboratory Practice (GLP) regulations promulgated by the U.S. Food and Drug Administration and OECD Regulations. The following is a record of the dates that auditdinspectionswere performedand reportedby the QAU. DATEOF AUDIT/INSPECTION 05/19/00 npE OF AUD'TANsPECTloN Protocol I DATESREPORTETDO STUDY DIRECTOARND MANAGEMENT 0511 9/00 05/22/00 Randomization 05/22/00 05/25/00 Dose Administration 05/25/00 06/02/00 Amendment #1 06/02/00 0612 1 /00 Formulations Raw Data, Individual Animal Necropsy Records, Histopathology,and Gross Pathology 06/29/00 06/28/00 Raw Data 06/29/00 06/28/00-06/29/00 Tables 06/29/00 0711 1/00 Draft Report 0711 1/00 1011 0100 Final Report 1011 0100 The report accurately reflects the original data. APPROVED BY: \Jd &b Val Gartner, B.A. Date Quality Assurance Auditor Primedica Redfield Page 3 Acute Oral Toxicity Study of T-7485 Administeredto Sprague-Dawley Rats Study Number: 132-002 TITLE ACUTE ORAL TOXICITY STUDY OF T-7485 ADMINISTERED TO SPRAGUE-DAWLEY RATS APPROVED BY: Study Director Prirnedica Redfield Page 4 Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 TABLE OF CONTENTS Page Cover Page....................................................................................................................................... 1 GLP Compliance Statement ............................................................................................................. 2 Quality Assurance Statement ........................................................................................................... 3 Title and Signature Page .................................................................................................................. 4 Table of Contents ............................................................................................................................. 5 Study Abstract .................................................................................................................................. 6 Objective........................................................................................................................................... 7 Materials and Methods ..................................................................................................................... 7 . Archival Statement............................................................................................................................ Results............................................................................... .............................................................. 8 9 Conclusion ........................................................................................................................................ 9 Tables ............................................................................................................................................. 10 Table 1 .Summary of Clinical Observations............................................................................ 11 Table 2 - Summary of Body Weights ....................................................................................... 14 Table 3 - Summary of Body Weight Changes.......................................................................... 17 Appendices ..................................................................................................................................... 20 Appendix 1 - Individual Necropsy Observations ...................................................................... 21 Appendix 2 - Individual Clinical Observations.......................................................................... 23 Appendix 3 - Individual Body Weights ..................................................................................... 28 Appendix 4 - Certificate of Analysis ......................................................................................... 31 - Appendix 5 Protocol and Amendment ................................................................................... 33 Appendix 6 - Protocol Dev.iat.ions............................................................................................. 42 Appendix 7 - Key Personnel..................................................................................................... 44 Page 5 Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 ACUTE ORAL TOXICITY STUDY OF T-7485 ADMINISTERED TO SPRAGUE-DAWLEY RATS STUDY ABSTRACT The objective of this study was to determine the acute effect(s) of a single oral gavage administrationof test material to Sprague-Dawley rats with a fourteen-day recovery. Healthy male and female Crl:CD@(SDI)GS BR stock albino rats were received from Charles River Laboratories, Inc. for use on the study. The animals were individually housed in stainless steel cages. Animal identificationconsisted of uniquely numbered ear tags and cage cards. On Study Day 1, the animals were approximately nine weeks old. The males weighed 257 to 280 grams and the females weighed 170 to 194 grams. The animals were acclimated for a minimum of seven days prior to Study Day 1 and were examined by the Staff Veterinarian prior to being released for use on the study. Fifteen males and fifteen females were randomly assigned to groups such that individual body weights did not exceed +20% of the mean weight for each sex. The study design was as follows: Group Number 1 2 3 Group Designation High-dose Low-dose Mid-dose Dosage Level (mg/kg) 2000 500 1000 Dosage Concentration (mg/mL) 200 50 100 Dosage Volume (mUkg) 10 IO 10 Number of Animals Males Females 5 5 5 5 5 5 A single dose was administered via oral gavage to fasted animals on Study Day 1. The dose volume was 10 mUkg. Individual dose volumes were calculated using fasted body weights recorded on Study Day 1. The animals in Group 1 were treated with 2000 mglkg of test material on Study Day 1 and then held for a 14-day recovery period. Becauseno deaths occurred, the study was terminated. Groups 2 and 3 were not dosed. Observationsfor mortality and moribundity were recorded twice daily (a.m. and p.m.) with no incidence of either occurring throughout the study. Clinical observations were recorded predose and approximately hourly for four hours postdose on the day of dosing then daily thereafter for at least 14 days. Clinical observations included male localized alopecia, urine-stained abdominal fur, and one female had liquid feces at three and four hours postdose. The listed clinical observations did not appear to be related to the test material given that the three observations were unrelated and each occurred in different animals. Body weights were recorded pretest and on Study Days 1, 8, and 15. Between Study Days 1 and 15, mean body weight increased in males (114.0 f 15.9 grams) and females (49.4 f 17.4 grams). During the study, there was no record of body weight loss for either males or females suggesting no adverse effect of a single dose of 2000 mg/kg T-7485 on body weights or body weight changes. On Study Day 15, all animals (non-fasted)were humanely euthanized via carbon dioxide asphyxiation followed by exsanguination and submitted for a complete necropsy examination. Necropsy examination failed to reveal any adverse findings with the exception of a single male and a single female with dilatation of the right kidney. Dilatation of the kidney is usually induced by hydronephritis of the kidney and this condition is not uncommon in rats. Therefore, these findings are not considered related to the test material. In conclusion, a single oral (gavage) administrationof 2000 mglkg T-7485 failed to induce adverse clinical observations, mortality, moribundity, changes in body weights or body weight changes, or gross findings in rats. Page 6 Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 OBJECTIVE The objective of this study was to determine the acute effect(s) of a single oral gavage administration of test material to Sprague-Dawley rats with a fourteen-day recovery. MATERIALS AND METHODS TEST AND CONTROL MATERIALS: The test material and vehicle were identified as follows: Test Material: Lot Number: Date Received: Physical Description: Storage: Amount Received: T-7485 (Potassium Perfluorobutane Sulfonate, PFBS) 2 April 6, 2000 White Powder Room Temperature, Protected from light 500 grams Vehicle: Supplier: Lot Number: Carboxymethylcellulose(medium viscosity) Sigma Chemical Co. 69H0028 The Sponsor assumed responsibilityfor characterization(identity, purity, and stability) determinationsof the test material. The test material was inventoriedwhen received at Primedica Redfield, and a record of all test material usage was maintained. The Certificate of Analysis for the test material is in Appendix 4. Doses were prepared by Primedica Redfield on the day of use. The vehicle was prepared to a concentration of 1% by mixing 10 grams of powdered medium viscosity carboxymethylcellulose with deionized water using a Waring blender. Additional deionized water was added to yield 1000 mL of prepared vehicle. The vehicle was stored refrigerated when not in use. The test material was dissolved in the prepared vehicle to the appropriate concentration and stirred on a magnetic stir plate. A 2 mL sample of the formulated test material was retained and frozen at approximately -20C. TEST ANIMALS: Healthy male and female Crl:CD@(SDI)GS BR stock albino rats were received from Charles River Laboratories, Inc. for use on the study. The animals were individually housed in stainless steel cages. Animal identification consisted of uniquely numbered ear tags and cage cards. On Study Day 1, the animals were approximately nine weeks old. The males weighed 257 to 280 grams and the females weighed 170 to 194 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. Environmentalcontrols were set to maintaintemperaturesof 18" to 26C (64" to 79F) with a relative humidity of 30% to 70%. These parameters were recorded at least once daily. A 12:12 hour 1ight:dark cycle and ten or greater air changes per hour were maintained in the animal room. GROUP DESIGNATION AND TREATMENT: The animals were acclimated for a minimum of seven days prior to Study Day 1 and were examined by the Staff Veterinarian prior to being released for use on the study. Fifteen males and fifteen females were randomly assigned to groups such that individual body weights did not exceed GO% of the mean weight for each sex. Page 7 Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 The study design was as follows: Group Number 1 2 3 Group Designation High-dose Low-dose Mid-dose Dosage Level (mg/kg) 2000 500 1000 Dosage Concentration (mg/mL) 200 50 100 Dosage Volume (mUkg) 10 10 10 Number of Animals Males Females 5 5 5 5 5 5 A single dose was administeredvia oral gavage to fasted animals on Study Day 1. The dose volume was 10 mUkg. Individual dose volumes were calculated using fasted body weights recorded on Study Day 1. The animals in Group 1 were treated with 2000 mg/kg of test material on Study Day 1 and then held for a 14-day recovery period. Because no deaths occurred, the study was terminated. Groups 2 and 3 were not dosed. JUSTIFICATION FOR SPECIES SELECTION, ROUTE OF ADMINISTRATION, AND DOSE LEVEL: Rats are an animal model for acute toxicity studies of this type. The number of animals assigned to the study represented the minimum required to meet the objectives of the study and the OECD guidelines (#Mol). In the assessment and evaluation of the toxic characteristicsof a substance, determination of acute oral toxicity is usually an initial step. Dose levels were chosen in accordance with OECD guideline Mol. CLINICAL OBSERVATIONS: Observationsfor mortality and moribundity were recorded twice daily (a.m. and p.m.). Clinical observationswere recorded predose and approximately hourly for four hours postdose on the day of dosing then daily thereafter for at least 14 days. BODY WEIGHTS: Body weights were recorded pretest and on Study Days 1, 8, and 15. The pretest body weights are not included in this report but are located in the raw data. NECROPSY: On Study Day 15, all animals (non-fasted) were humanely euthanized via carbon dioxide asphyxiation followed by exsanguination and 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. Gross lesions were preserved in neutral buffered formalin. All other tissues were discarded. HISTOPATHOLOGY: No histopathologicalexaminationswere performed. STATISTICS: Means and standard deviations were calculated for all quantitative data. ARCHIVAL STATEMENT All original data and the original final report will be retained at Primedica Redfield's archives, located at 100 East Boone Street, Redfield, Arkansas, for a period of five years after issuance of the final report. Wet tissues, slides, and blocks (if generated)will be retained at Primedica Redfield for one year. After this time, the Sponsor will be contacted for disposition instructions. Page 8 Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 RESULTS CLINICAL OBSERVATIONS: Observations for mortality and moribundity were recorded twice daily (a.m. and p.m.) with no incidence of either occurring throughout the study. Clinical observations were recorded predose and approximately hourly for four hours postdose on the day of dosing then daily thereafter for at least 14 days. Clinical observations included male localized alopecia, urine-stained abdominal fur, and one female had liquid feces at three and four hours postdose. The listed clinical observations did not appear to be related to the test material given that the three observations were unrelated and each occurred in different animals. The summary of clinical observations is in Table 1. The individual clinical observations are in Appendix 2. BODY WEIGHTS AND BODY WEIGHT CHANGES: Body weights were recorded pretest and on Study Days 1, 8, and 15. The pretest body weights are not included in this report but are located in the raw data. The summaries of body weights and body weight changes are in Tables 2 and 3. The individual body weights are located in Appendix 3. Between Study Days 1 and 15, mean body weight increased in males (114.0k 15.9 grams) and females (49.4 f 17.4 grams). During the study, there was no record of body weight loss for either males or females suggesting no adverse effect of a single dose of 2000 mg/kg T-7485 on body weights or body weight changes. NECROPSY: On Study Day 15, all animals (non-fasted) were humanely euthanized via carbon dioxide asphyxiation followed by exsanguination and submitted for a complete necropsy examination. The individual necropsy observations are located in Appendix 1. Necropsy examination failed to reveal any adverse findings with the exception of a single male and a single female with dilatation of the right kidney. Dilatation of the kidney is usually induced by hydronephritis of the kidney and this condition is not uncommon in rats. Therefore, these findings are not considered related to the test material. CONCLUSION In conclusion, a single oral (gavage) administration of 2000 mglkg T-7485failed to induce adverse clinical observations, mortality, moribundity, body weights or body weight changes, or gross findings in rats. Page 9 Acute Oral Toxicity Study of T-7485 Administeredto Sprague-Dawley Rats Study Number: 132-002 TABLES Page 10 Acute Oral Toxicity Study of T-7485 Administeredto Sprague-Dawley Rats Study Number: 132-002 TABLE 1 SUMMARY OF CLINICAL OBSERVATIONS Page 11 W (u (0 (D B(D Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 TABLE 2 SUMMARY OF BODY WEIGHTS Page 14 9cp (D 9(D (D Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 TABLE 3 SUMMARY OF BODY WEIGHT CHANGES Page 17 9(0 rD BODY UEIGHT CHANGE ( G I SD 1 - 8 MEAN3S.D. . +32.2 f 6.6 SD 8 - 15 MEANfS .O . i17.2 f 12.1 S_D _1 -_15___M_EAN_fS.O_. ___+49_.6 _f 1_7.4 _____--------------------..--.-.-------------------------- SD = STUDY DAY Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 APPENDICES Page 20 Acute Oral Toxiclty Study of T-7485 Administeredto Sprague-Dawley Rats Study Number: 132-002 APPENDIX 1 INDIVIDUAL NECROPSY OBSERVATIONS Page 21 Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 APPENDIX I INDIVIDUAL NECROPSY OBSERVATIONS GROUP 1 - 2000 n GlKG - Animal #6209 Animal #6211 Animal #6213 Animal #6215 Animal #6217 Animal #6210 Animal #6212 Animal #6214 Animal #6216 Animal #6218 - Male Male Male Male Male Female Female Female Female Female No adverse findings. No adverse findings. No adverse findings. No adverse findings. Kidneys, Dilatation, Right, Pelvis. No adverse findings. No adverse findings. No adverse findings. No adverse findings. Kidneys, Dilatation, Right, Pelvis. Page 22 Acute Oral Toxiclty Study of T-7485 Administeredto Sprague-Dawley Rats Study Number: 132-002 APPENDIX 2 INDIVIDUAL CLINICAL OBSERVATIONS Page 23 D a, ln (D lu P EE ont rc es emt ots ot ttt etn I ;i -u a, [mo i Acute Oral Toxicity Study of T-7485Administered to Sprague-Dawley Rats Study Number: 132-002 APPENDIX 3 INDIVIDUAL BODY WEIGHTS Page 28 i i B i i 0 0 Acute Oral Toxicity Study of T-7485 Administeredto Sprague-Dawley Rats Study Number: 132-002 APPENDIX 4 CERTIFICATE OF ANALYSIS Page 31 Acute Oral Toxiclty Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 Certificate Of Analysis CJ?9SO3K+ (PFBS), Lot 2 March 10,2000 C+FsSO3X+ 99.82 % I 0.04 % Additionally, the isorna distributionof the sample wap determinedh g '%-"h4Rtechniques and found to contain the following weight pcrccnt composition: I I ~-1 97.86 % 1.96 % L Page 32 Acute Oral Toxic@ Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 APPENDIX 5 PROTOCOL AND AMENDMENT Page 33 Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 Primedica Redfield Protocol RO~ONCw nOb~c 132-002 ACUTE ORAL TOXICITY STUDY OF T-7485 ADMINISTERED TO SPRAGUE-DAWLEYRATS PROTOCOL NO.: 132002 OBJECTWE: Determine the acute effect(s) of a single oral gavage administrationof test material to Sprague-Dawley rats with a fourteen-day recovery. LOCATION OF STUDY AND CONDITIONS OF TESTING: It is the intention of 3M Pharmaceuticals. through the conduct of this study. to generate animd safety data that may be submitted to regulatory authorities. Primedica Redfield, 100 East Boone Skeet Redfield, Arkansas, 72132, is accredited by AAAIAC and licensed by the United States Department of Agriculture to conduct research in laboratory animals. All the conditions of testing will conform to the Animal Welfare Act (CFR 9) and its amendments. Primedica Redfieldwill follow all requirementsspecified in thi approved protocd and all applicable governmental regulations regarding Good Laboratory Pmdices as well as P r i d i Redfield's Standard Operating Procedures. Changes in the protocol may be made by consultation with and approval from 3M Pharmaceuticals followed by written verification of the change. 3M Pharmaceuticals reserves the right to inspect facilities and procedures used for this study by means of announced or unannounced site visits. Primedii Redfieldwill n o m 3M Pharmaceuticalsprwnptty.by telephone prior to release of any data for review SPONSOR 3M 3M Center, Building 220-2E-02 P.O. Box 33220 St Paul, MN 55133-3320 SPONSOR'S REPRESENTATIVE: , Ph.D.. DABT, CIH Senior Laboratory Manaaer Telephone: FAX: TESTING FACILTTY: Primedica Redfield Mail: P.O. Box 308 Deliveries: 100 East Boone St Redfield, AR 72132 Telephone: 501-397-2540 FAX: 501-397-2002 PERSONNEL: Study Director: John Seng, Ph.D. Principle Investigator: Karen Tranter. E A ,IATG Veterinarian: Allan Manus, D.V.M.. M.Sc.,ACLAM Head Technician: Jennifer Dedman. B.S. Pathologist TBD PROPOSED STUDY DATES: Experimental Start Date: May 25,2000 ExperimentalTermination Date: June 8,2000 Draft Report Date: July 14,2000 Page I of 7 5/23/00 Page 34 ~~~ ~ ~ Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number 132-002 Primedica Redfield Protocol Protocol Number: 132- 1. REGULATORCYOMPLIANCEAND QUALITYASSURANCE This study will be conducted in accordance with the following Good Laboratory Practice RegulationdStandarddGuidelines: 21 CFR58 El C(81)30 (Final) (OECD) The Quality Assurance Unit in accordance with Primedica Redfields Standard Operating Procedures(SOPS),will audit the protocd, study conduct, and the final report. 2. lNSmuTlONAL ANIMAL CARE AND USE COMWTTEE REVIEW The protocol will be reviewed and approved by Primedica Redfields InstitutionalAnimal Care and Use Committee (IACUC) for compliance with regulations prior tu study initiation. In the opinion of the Sponsor, indicatedby the signature on this protocol, the study does not unnecessarily duplicate any previous work 3. MATERIALS TESTMATERIAL lDENnFlC.4 TION T-7485 (Potassiumpemuorobutanesulfonate, PFBS) The lot number will be listed in the raw data and final repon P ~ v s r u sDESCRlPnON White powder VEHICLE 1%carboxymethylcellulose(aqueous, medium viscosity) ~DENnFlGATION The lot number will be listed in the raw data and final report PHYSICADLESCRIPTION White powder STORAGE CONDITIONS The bulk test material will be stored in its original container and will be stored at room temperature. A N 4 L Y l l C X CHEMISTRY The Sponsor assumes responsibility for characterization (identity, punty, and stability) of the bulk test material (including under test conditions). Information on the composition and method of synthesis of the bulk test material will be held by the Sponsor. The doses, unless otherwise stated, will be calculated assuming the test material to be 100% pure. DOSE PREPARAnONANO c0"TRATlON Doses will be prepared by Primedica Redfield on the day of use. The carboxymethykellu~will be prepared at 1%in deionized water and may be prepared prior to Study Day 1. Page 2 of 7 5/22/00 Page 35 Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 Primedica Redfield Protocol Protocol Numbw: 132-002 INVENTORY AmrmcsL SAMPLES The test material container(s)will be inventoried when receivedat the testing laboratory, and a record of all test material use will be maintained. The technician dosing the animals will: 1) Recordthe weight avolume of the test material container immediately before and after dosing; 2) Compute and record the amount used and compare it to thetheoretical amount immediately after dosing; 3) Keep the formulated test material use log in the animal study books at all times; 4) Assure the dose used is within *lo% of theoretical. Samples of the formulated test materials will be retained and frrnen at approximately -20C for possible analysis. If no analysis is pwkmned. the samples will be discarded upon finalbabon of the study repat. or as authomed by the Sponsor. TESTMATE~WRAELTENTION Unused test material may be returned to the Sponsor or designee at the termination of the study, or retained for use on future studies. lbe Sponsor will be notitied in advance of shipping, and a t r a n s W letter will accompany the shipment The material will be packed in a suiebk container to maintain the conditions specified by the Sponsor dung transit plus an adequate margin of safely for any transit delays. SAFEWPRECACmONS General safely precautionsas required by Prirnedika Redfield'spolicies and procedures will be followed. The Sponsor's Representativewill be notified of any personnel exposures requiring a physician's examination or care. 4. ANIMALS Species: SbainlSource: Age at Initiation: Number and Sex: Identification: Rat Crl:CP(SD) BR VATIplus out-bred dbino rats, Charles River Laboratories, Inc. Six to eight weeks 15 male and 15 female (female nullipamus and non pregnant) Uniquely numbered ear tag and cage cad ANIMAL HUSBANDRY HOUSING The animals will be individually housed in stainless steel cages. The cages conform to standards set forth in the Guide for the Care and Use of Laboratorv Animals, National Academy Press, Washington, DE.. 1996. FEED Teklad Certified Rodent Diet #8728 will be provided ad libitum. This diet is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Results of the manufacturer's an- are on file at Primedica Redfield. Page 3 of 7 m2/00 Page 36 Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 Primedica Redfield Protocol Procod Number: l324W2 Group Group Number Designation 1 Highdose 2 Lwdose 3 Middose Dosage Level (mgncg) 2000 500 1000 Dosage Concentration (mglmL) 200 50 100 D-ge Vdumo (mLkg) 10 10 10 Number of Animals Males Females 5 5 5 5 5 5 Page 4 of 7 Page 37 5/22/00 ~- ~~ ~ Acute Oral Toxicity Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 Primedica Redfield Protocol Protocol N&r: 132-oOt FREQUENCAYND WRATION OF ADMlNlSTRATlON A single dose will be administered via oral gavage to fasted animals (feed withheld over-night). The dose volume will be 10 mL/kg. The first day of dosing on the study will be Study Day 1. The animals in G m p 1will be treated with 2000 mgkg of test material on Study Day 1with a 14-day recovery period. If deaths m r , then rats will be dosed at loo0 and 500 rng/kg of test material with a 14day recovery. If no deaths occur, the study will be terminated. Individual dose volumes will be calculated using the fasted body weights collected on the day of dosing. JUS77FlCA77ONFOR ROUTE OFADMlNlSTRn77ONAND DOSEk V E L In the assessment and evaluation of the toxic characteristics of a substance, determination of acute oral toxicity is usually an initial step. Dose levels were chosen by the OECD guideline W01. AMEMoRTEM OESERVAnONS CLINICAL OBSERVATK~S Observations will be performed and recorded twice daily ( a m and p.m.) for moribundity and mortality. Clinical observations will be recorded at least once each day. Observations will be recorded predose and approximately hourly for four hours postdose on the day of dosing then daily thereafter for at least 14 days. Observations should indude changes in the skin and fur, eyes, and mutous membranes, and also respiratory. circulatory. autonomic and central nervous system, and m t o m o t o r activity and behavior pattern. Particular attention should be directed to observation of tremors, convulsions. salivation. diarrhea (liquid feces), lethargy, and coma. BODY WIGHTS Body weights will be recorded pretest, on Study Day 1, approximately weekly, and at termination. POSTMORTEM OESERVATlOFJS MORIBUND ANIMLS AND ANIWLS FOUND DEAO Animals unlikely to sunrive until the next scheduled observation will be weighed. euthanized. and necropsied. Animals found dead will be weighed and necropsied. In either case, a complete necropsy will be performed as detailed below. All animals surviving to the end of the study will be humanely euthanized via carbon dioxide asphyxiation. Euthanasia will be performedin accordance with accepted American Veterinary Medical Association ( A M ) guidelines [J. Amer. Vet. Med. Assoc.,20229-249, 19931. Page 5 of 7 5/22/00 Page 38 Acute Oral Toxicity Study of T-7485Administered to Sprague-Dawley Rats Study Number: 132-002 Primedica Redfield Protocol Pmocol Number: 132402 NECROPSY Starting on Study Day 15, the animals will be sacrificed rotating sequentially through groups for each sex. All animals that die during the study, or are killed at study termination, will be subjected to a complete necropsy examination. A complete necropsy is defined as examination of the external surface of the body. all orifices, and the cranial, thwacic, and abdominal cavities and their contents. Gross lesions will be preserved in a suitable fixative for possible histopathology; all other tissues will be discarded. Histopathologywill be provided at additionalexpense to the Sponsor. 6. SrAnsncAl ANALYSIS Means and standard deviations will be calculated for all quantitative data. 7. REPORT An audited draft report will be sent to the Sponsor. Revisions to the initial draft report will be provided to the Sponsor by mail or fax transmission as printed copies of the corrected pages only. Additional revisions or. complete copies of the revised draft reports will be provided at additional expense to the Sponsor. Three copies (one bound, two unbound) of the approved final report will be submitted to the Sponsor approximately two weeks after approval of the draft report. The final report will indude all elements requirdrecommended by the regulations and guidelines. The report will indude, but is not limited to,those items listed below: Descriptive text of the study objective Summary Test material identification (Certificate of Analysis, stability data, and analytical analysis if any are performed) Methods Results and conclusions Body weights Body weight changes Necropsy repoct A copy of the protocol, all protocol amendments, and all protocol deviations that may affect the integrity of the study 8. MAINTENANCOEF R AW DATAAND RECOROS Original data or copies thereof, will be available at Primedica Redfield to facilitate auditing the study during its progress and before acceptance of the final report M e n the final report is completed, all original paper data and the original final report will be retained in the archives of the laboratory for at least fnre years. Wet tissues, slides. and blocks (if generated) will be retained at Primedica Redfield for one year. After one year the storage of the wet tissues, slides. and blocks will be negotiated with the Sponsor. After five years the storage of the paper data will be negotiated with the Sponsor. The Sponsor will be notified prior to disposal of any original study data. Page 6 of 7 WOO Page 39 Acute Oral ToxicQ Study of T-7485 Administered to Sprague-Dawley Rats ~ ~~ Study Number: 132-002 Primedica Redfield Protocol Proiocol Number: 132-oOr 9. Q U A UA~SSURANCE RNlEw This is to ceftify that this protocol has been reviewed by Quality Assurance. Ppn Ftan Pattillo, M.S., RQAP-GLP Manager of Quality Assurance Primedica Redfield 70. APPROVALS s-aa-= Date r f q z Zd,2@ Ph.0, DABT. CIH Date Senior Laboratory Manager 3M ' Cr- // G&do&& Director u Primedica Redfield Page 7 qf 7 Page 40 5mw Acute Oral Toxicrty Study of T-7485 Administered to Sprague-Dawley Rats Study Number: 132-002 Primedica Redfield Protocol Amendment ~~ ~~~ STUDY NUMBER: 132-002 AMENDMENT: 1 Protocol Number: 132-002 - STUDY TITLE: DATE ISSUED: ACUTE ORAL TOXICITY STUDY OF T-7485 ADMINISTERED TO SPRAGUEDAWLEY FIATS June 9,ZOOO AMENDMENT I1 ITEM 1: CHANGED FROM: CHANGED TO: REASON: Protocol 3M Pharmaceuticals 3M Corporate Toxicology Sponsor requested clarification. ITEM 2. CHANGED FROM: CHANGED TO: REASON: Page 1, SPONSOR'S REPRESENTATIVE Telephone: Telephone: Correction d telephone number. ITEM 3 CHANGED FROM: CHANGED TO: REASON: Page 6, Section 5, Necropsy Starting on Study Day 15. the animals will be sacrificed rotating sequentially through groups for each sex. Starting on Study Day 15, the animals (non-fasted) will be sacrificed rotating sequentially through groups for each sex. Fasting of the animals will not be necessary. APPROVALS: , Ph.D., DABT, CIH / ' Senior Laboratory Manager 3 M Corporate Toxicology Dat'e Study Director Primedica Redfield Date 1 Page 41 Primedica Redfield Protocol Amendment Protocol Number: 132-002 STUDY NUMBER: 132-002 -a AMENDMENT: 2 STUDY TITLE: ACUTE ORAL TOXICITY STUDY OF T-7485 ADMINISTERED TO SPRAGUE- DAWLEY RATS DATE ISSUED: October 18, 2000 AMENDMENT # 2 ITEM 7: CHANGED FROM: CHANGED TO: REASON: Page 6, Section 7, Report Three copies (one bound, two unbound) ofthe approved final report will be submitted to the Sponsor approximately two weeks after approval of the draft report. Three copies (two bound, one unbound) of the approved final report will be submitted to the Sponsor approximately two weeks after approval of the draft report. Sponsor request. APPROVALS: Ph.D., DABT, CIH Senior Laboratory Manager 3M Corporate Toxicology Date Study Director Primedica Redfield f Acute Oral Toxicity Study of T-7485 Administeredto Sprague-Dawley Rats Study Number: 132-002 APPENDIX 6 PROTOCOL DEVIATIONS Page 42 Acute Oral Toxicity Study of T-7485 Administeredto Sprague-Dawley Rats Study Number: 132-002 PROTOCOL DEVIATIONS There were no protocol deviations that adversely affected the integrity of the study. Page 43 Acute Oral Toxicty Study of T-7485 Administeredto Sprague-Dawley Rats Study Number: 132-002 APPENDIX 7 KEY PERSONNEL Page 44 Acute Oral ToxicitV Study of T-7485 Administered to Spraque-Dawley Rats Study Number: 132-002 KEY PERSONNEL Study Number: 132-002 Study Director: ............................................... Study Coordinator:......................................... Head Technician:........................................... Veterinarian: .................................................. Quality Assurance:......................................... Report Coordination: ..................................... Report Preparation: ....................................... John Seng, Ph.D. Karen Tranter, B.A., LATG Jennifer Dedman, B.S. Allan Manus, D.V.M., M.Sc., ACLAM Val Gartner, B.A. Amy Babb, B.S. Tarnmy Peckham, Ad. Asst. Tammie Hughey, Ad. Asst. Page 45