Document 3QZD5bQ4LyVLozmDmNO1xYLK0

C O V A -ffC "^ the development services company C ovan e L a b o ra to rie s Inc. P.O. B o x 7545 M ad ison , W isco n sin 53707-7545 P a cka g e s: 3301 K in sm a n B ou leva M ad ison , W isco n sin 53704 Tel: 6 0 8 /2 4 1 -4 4 7 1 Fax: 608/241-7227 THE AM ERICAS Sponsor: 3M St. Paul. Minnesota FINAL REPORT Study Title: 5-Dailv Dose Dermal Absorption/Toxicity Study of T-6684 in Rabbits Author: F. Bud W. McDonald Study Completion Date: December 31, 1997 Testing Facility: Covance Laboratories Inc. 3301 Kinsman Boulevard Madison. Wisconsin 53704 Testing Facility Project Identification: Covance 6329-200 EUROPE Page 1 of 52 ASIA/PACIFIC AFRICA QUALITY ASSURANCE STATEMENT Covance 6329-200 This report has been reviewed by the Quality Assurance Unit of Covance Laboratories Inc., in accordance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations, 21 CFR 58. The following inspections were conducted and findings reported to the Study Director and management. Written status reports of inspections and findings are issued to Covance management according to standard operating procedures. Inspection Dates From To 04/15/97 05/05/97 05/21/97 07/14/97 04/15/97 05/05/97 05/21/97 07/15/97 Phase Protocol Review Dose Administration Protocol Amendment Data/Report Review Date Reported to Study Director and Management 04/15/97 05/05/97 05/21/97 07/15/97 ReDprreesseennfative,. Quality Assurance Unit /Z S /9 7 Date STUDY IDENTIFICATION Covance 6329-200 5-Daily Dose Dermal Absorption/Toxicity Study of T-6684 in Rabbits Test Material Sponsor Sponsor's Representative Study Director Testing Facility Study Timetable Study Initiation Date Experimental (In-life) Start Date In-life End Date Experimental Termination Date Study Completion Date T-6684 3M Toxicology Services Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Roger G. Perkins. PhD, DABT 3M Toxicology Services Medical Department 3M Center. Bldg. 220-2E-02 P.O. Box 33220 St. Paul. MN 55133-3220 (612)733-3222 F. Bud W. McDonald Covance Laboratories Inc. P.O. Box 7545 Madison, WI 53707-7545 608.242.7901 Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, WI 53704 April 18, 1997 May 5, 1997 June 2, 1997 June 2, 1997 December 31,1997 Acute Studies Steven M. Glaza Manager F. Bud W. McDonald Study Director Jeffrey B. Hicks In-life Supervisor Rose M. Bridge Administrative Supervisor Toxicology Support Kathy Myers Manager Calvin L. Horton Supervisor Quality Assurance Nancy M. Centanni Manager KEY PERSONNEL Covance 6329-200 Laboratory Animal Medicine Donna J. Clemons, DVM Diplomate, ACLAM Supervisor Anatomical Pathology Thomas E. Palmer, PhD Anatomical Pathologist Deborah L. Pirkel Laurie Schuller Supervisors Necropsy CONTENTS Covance 6329-200 QUALITY ASSURANCE STATEMENT..............................................................................2 STUDY IDENTIFICATION....................................................................................................3 KEY PERSONNEL.................................................................................................................. 4 SUMMARY............................................................................................................................... 7 OBJECTIVE.............................................................................................................................. 9 REGULATORY COMPLIANCE........................................................................................... 9 TEST AND CONTROL MATERIALS...................................................................................9 Identification..........................................................................................................................9 Purity and Stability..............................................................................................................10 Storage and Retention......................................................................................................... 10 Safety Precautions................................................................................................................10 TEST SYSTEM ....................................................................................................................... 10 Test Animal.......................................................................................................................... 10 H o u s in g ................................................................................................................................. 10 Animal Diet.......................................................................................................................... 11 Selection of Test Animals................................................................................................... 11 Study Design........................................................................................................................ 11 Justification for Species Selection..................................................................................... 11 PROCEDURES....................................................................................................................... 12 Preparation of Exposure Area.............................................................................................12 Dose Administration............................................................................................................1Reason for Route of Administration.................................................................................. 13 Observations of A nim als.................................................................................................... 13 Blood Sample Collections/Shipment................................................................................. 13 Pathology............................................................................................................................. 14 Shipment of Bile and Tissues.............................................................................................14 Statistical Analyses............................................................................................................. 14 Location of Raw Data. Records, and Final Report........................................................... 14 R E S U L T S ..................... Body W eights........... Clinical Observations Dermal Irritation...... Pathology................... DISCUSSION SIGNATURE.............................................................. REFERENCE....................................................... TABLE 1 Individual and Mean Bodv Weights (g)......... 2 Individual Clinical Signs....................................... 3 Individual Dermal Irritation Scores - .............................. Group 1 (Control) - Dimethyl Sulfoxide (0.0 mg/kg/day) 4 Individual Dermal Irritation Scores - ............................... Group 2 - T-6684 (3.0 mg/kg/day)..................................... APPENDIX A ........................................................... Individual Animal Pathology D ata..................... APPENDIX B ...................................... Protocol Deviation.......................... Protocol TP6798............................. Amendment No. 1 To The Protocol Covance 6329-200 .... 15 ....15 ....15 ....15 ....16 ..... 16 SUMMARY Covance 6329-200 This study was done to assess the systemic absorption/toxicity and relative skin irritancy of T-6684 when applied to the skin of rabbits for five consecutive davs. The study was conducted using three male and three female acclimated rabbits of the Hra:(~NZW)SPF strain for each treatment group. Group 1 (Control) 0 Control/ Dose Level Test Material (mg/kg/day) Dimethyl Sulfoxide T-6684 o 3.0a Number of Animals Males Females 33 3 a Administered at a dose volume of 0.1 mL/kg of body weight. The back of each rabbit was clipped free of hair and a single dose of the respective control or test material was administered to the skin of the rabbits for 5 consecutive days. The treatment sites remained intact. The area of application was covered with a gauze patch secured with paper tape around all edges and overwrapped with Saran Wrap and Elastoplast tape to provide an occlusive dressing for each approximate 23-hour exposure period. Clinical observations were conducted predose on Day 1 only and at approximately 1. 2.5. and 4 hours after each control or test material administration on Days 1 through 5. Additional clinical observations were conducted daily thereafter through Day 29. Mortality checks were conducted twice daily on Days 2 through 29 (a.m. mortality check only on Day 29). Body weights were determined on Day -5 for randomization purposes, before the first control or test material administration (Day 1), and at the scheduled sacrifice interval (Day 29). The initial dermal irritation reading was made before the first control or test material administration (recorded as the Day 1 reading). Subsequent readings o f dermal irritation were made after each patch removal on Days 2 through 6 and on Day 13. A blood sample (approximately 4 mL) was collected from each animal on 7 Covance 6329-200 Days -3, 8, 15, and 23. In addition, at the time of necropsy (Day 29), approximately 20 to 40 mL of blood was obtained from each control animal and approximately 20 mL of blood was obtained from each Group 2 animal. All samples were centrifuged and separate samples of serum and cellular fractions were obtained and sent frozen on dry ice to the Sponsor. On Day 29, the animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, exsanguinated and necropsied. The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site, and both kidneys (collected as one sample) were collected from all animals, weighed (volume only determined for bile), and sent frozen to the Sponsor. Application of T-6684 did not result in any test material-related changes in body weight gain or macroscopic findings at necropsy. All animals appeared normal throughout the study with the exception of one Group 1 animal and two Group 2 animals which had decreased feed consumption lor one to three days within the first nine days of the study. Slight dermal irritation was observed in one control animal while slight to moderate dermal irritation was observed in the other five control animals treated with dimethyl sulloxide (DMSO). In the animals treated with T-6684. slight dermal irritation was observed in one animal while slight to moderate dermal irritation was observed in the other five animals. No macroscopic lesions were seen in any of the animals at necropsy. OBJECTIVE Covance 6329-200 The objective o f this study was to assess the systemic absorption/toxicity and relative skin irritancy of test materials when applied to the skin of rabbits for five consecutive days. REGULATORY COMPLIANCE This study was conducted in accordance with the United States Food and Drug Administration Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58, with the exception that analysis of the test material mixtures for concentration, homogeneity/solubility, and stability was not conducted. All procedures used in this study were in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study did not unnecessarily duplicate any previous work. All procedural times presented in this report fall within the acceptable ranges as specified in the Covance Laboratories Inc. (Covance) Standard Operating Procedures (SOPs). TEST AND CONTROL MATERIALS Identification The materials were identified and described as follows: Identification Physical Description T-6684 (test) off-white liquid DMSO (control) Clear colorless liquid Dimethyl sulfoxide was manufactured by Mallinckrodt Chemical. 9 Covance 6329-200 Purity and Stability The Sponsor assumes responsibility for test material purity and stability determinations (including under test conditions). Analysis of the test material mixtures for concentration, homogeneity/solubility, and stability was not conducted. The purity and stability of the control material were considered to be adequate for the purposes of this study. Storage and Retention The control and test materials were stored at room temperature. A reserve sample of each the control and test materials were taken and stored in a freezer set to maintain a temperature of -20C 10C. The control material reserve sample will be retained at Covance for one year. The test material reserve sample was sent to the Sponsor. Any unused test material will be returned to the Sponsor. Any remaining control material may be used for other testing and will not be discarded after issuance of the final report. Safety' Precautions The control and test material handling procedures were according to Covance SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPF strain were received from Covance Research Products Inc., Kalamazoo, Michigan on April 16. 1997. Housing After receipt, the animals were acclimated for a period of at least 7 days. During acclimation and throughout the study, the animals were individually housed in suspended stainless steel cages. Environmental controls for the animal room were set to maintain a temperature of 16 to 22C, a relative humidity of 50% 20%, and a 12-hour light/12-hour to 1 Covance 6329-200 dark lighting cycle. In cases where variations from the required temperature and humidity conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad libitum and a measured amount of Laboratory Rabbit Diet HF #5326. PMI Feeds, Inc. The feed is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Samples of the water are periodically analyzed. There were no known contaminants in the feed or water at levels that would be expected to interfere with or affect the results of the study. Selection of Test Animals The animals were identified by animal number and corresponding ear tag and were placed into study groups using a stratified body weight randomization program. The randomization body weights were obtained on Day -5. Study Design Animals weighing from 2.067 to 2,500 g and approximately 15 weeks of age at initiation of treatment were placed into the following study groups: Group 1 (Control) 2 Control/ Test Material Dimethyl Sulfoxide T-6684 Dose Level Dose Volume Number of Animals (mg/kg/day) (mL/kg/day) Males Females 0.0 0.1 *> 3.0 0.1 J Justification for Species Selection Historically, the New Zealand White albino rabbit has been the animal of choice because o f the large amount of background information on this species. PROCEDURES Covance 6329-200 Preparation of Exposure Area On the day before the first control or test material application, the back and. if necessary (to obtain unblemished skin), the flanks of each rabbit was clipped free of hair with an electric clipper. The clipped area made up approximately 20% of the total body surface area. The test sites (intact skin) were inspected for interfering lesions, irritation, or defects that would preclude the use of any of the animals. The animals were clipped as needed throughout the study (Days 3, 6. 13 and 29). Dose Administration All animals received five consecutive days of administration of the respective control or test material. The first day of treatment was designated as Day 1. Group 1. An individual dose was calculated and measured based on each animal's body weight on the first day of treatment. The control material (dimethyl sulfoxide) was applied to the test site at a dose volume of 0.1 mL/kg in a thin and uniform layer. Group 2. An individual dose of the test material mixture (T-6684 and dimethyl sulfoxide) at a concentration of 30 mg/mL was calculated and measured based on each animal's body weight on the first day of treatment. The test material mixture was applied to the test site at a dose volume of 0.1 mL/kg of body weight in a thin and uniform layer. Each area of application in Groups 1 and 2 was covered with a 4-plv 5-cm x 5-cm gauze patch. Each gauze patch was secured with paper tape around all edges and overwrapped with Saran Wrap and Elastoplast tape to provide an occlusive dressing. Collars were used to restrain the animals during each approximate 23-hour exposure period. One Group 2 male was found without its collar on at the a.m. mortality check on Day 3. The collar was placed back on the animal at this time (see protocol deviations page). Approximately 23 hours after each control or test material application, the restraining collars and patches were removed and any residual material removed from the application sites using tap water and disposable paper towels. Reason for Route of Administration The dermal route is a potential route of exposure in humans. Covance 6329-200 Observations of Animals Clinical observations were conducted predose on Day 1 only and at approximately 1. 2.5, and 4 hours after each control or test material application on Days 1 through 5. Additional clinical observations were conducted daily thereafter through Day 29. Mortality checks were conducted twice daily on Days 2 through 29 (a.m. mortality check only on Day 29). Body weiehts were determined on Day -5 for randomization purposes, before the first control or test material administration (Day 1), and at the scheduled sacrifice interval (Day 29). The initial dermal irritation reading was made before the first control or test material application according to the Draize1technique (recorded as the Day 1 reading). Subsequent readings of dermal irritation were made within 30 to 37 minutes after each patch removal (Days 2 through 6) and on Day 13. Blood Sample Collections/Shipment A blood sample (approximately 4 mL) was collected from a marginal ear vein of each animal on Days -3. 8, 15. and 23. In addition, at the time of necropsy (Day 29), approximately 20 to 40 mL of blood was obtained from each control animal and approximately 20 mL of blood was obtained from each Group 2 animal via the posterior vena cava. All samples were stored at room temperature until centrifuged. After centrifugation, separate samples of serum and cellular fractions were obtained and stored in a freezer set to maintain a temperature of -20C 10C until shipped to the Sponsor. The serum and cellular fraction samples obtained on Days -3. 8. and 15 were shipped frozen (on dry ice) to the Sponsor (James D. Johnson. 3M E.T. & S. Bldg. 2-3E-09, 9j 5 Bush Avenue. St. Paul, MN. 55106) on Day 16. The serum and cellular fraction samples obtained on Days 23 and 29 were shipped to the Sponsor the day after the experimental (in-life) date of termination in the same manner as the samples obtained prior to Day 23. The Sponsor is responsible for the retention and disposition of the samples. Covance 13 Covance 6329-200 does not accept any responsibility for the analysis of the samples collected in this study nor are these results presented in this report. Pathology At termination of the in-life experimental phase (Day 29), animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, exsanguinated, and necropsied in random order. The sites of control or test material application were washed with warm tap water before the necropsy procedure. All animals were subjected to an abbreviated jross necropsy examination and any abnormalities were recorded. The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site, and both kidneys (collected as one sample) were collected from all animals, weighed (volume only determined for bile), and placed in a freezer set to maintain a temperature of-20C ICC. After necropsy, the animals were discarded. Shipment of Bile and Tissues One week after in-life experimental termination, the tissues (whole livers, dermal application sites, and kidneys) and bile were sent frozen (on dry ice) to the Sponsor (James D. Johnson. 3M E.T. & S. Bldg. 2-3E-09. 935 Bush Avenue. St. Paul, MN. 55106). along with documentation of their corresponding weights or volumes. The Sponsor is responsible for the retention and disposition of the samples. Covance does not accept any responsibility for the analysis of the samples collected in this study nor are these results presented in this report. Statistical Analyses No statistical analyses were required by the protocol. Location of Raw Data, Records, and Final Report The raw data, records, and an original signed copy of the final report will be retained in the archives of Covance in accordance with Covance SOP. RESULTS Covance 6329-200 Body Weights Individual and mean body weights are in Table 1. All animals, with the exception of one control animal, exhibited a weight loss from randomization to initiation (in-life) which can be attributed to the acclimation of the animals to restraining collars conducted for 3 days (approximately 21-23 hours/day) before the initial control and test material administration. All animals gained weight from Day 1 to Day 29. Clinical Observations Individual clinical signs are in Table 2. All animals appeared normal throughout the study with the exception of one Group 1 animal and two Group 2 animals which had decreased feed consumption for one to three days within the first nine days of the study. Dermal Irritation Individual dermal irritation scores are in Tables 3 and 4. Slight erythema, edema and fissuring reactions were observed in one control animal while slight to moderate erythema and edema and slight desquamation reactions were observed in the other five control animals treated with dimethyl sulfoxide. Of these five control animals, two animals also exhibited slight coriaceousness reactions. In the animals treated with T-6684. one exhibited slight erythema, edema and fissuring reactions, one exhibited slight erythema and slight to moderate edema reactions, one exhibited slight to moderate erythema and slight edema and fissuring reactions, one exhibited slight to moderate erythema and slight fissuring reactions, one exhibited slight to moderate erythema and edema and slight desquamation and coriaceousness reactions, and one exhibited slight to moderate erythema and edema and slight desquamation, coriaceousness, and fissunng reactions. 15 Covance 6329-200 Pathology Individual animal pathology data (tissue weights, bile volumes, and gross pathology observations) are presented in Appendix A. No macroscopic lesions were observed in the animals at necropsy. DISCUSSION The acute systemic absorption/toxicity and relative skin irritancy of T-6684 was evaluated in male and female albino rabbits when administered dermally for five consecutive days. Application of this material did not result in any test material-related changes in body weight gain or macroscopic findings at necropsy. All animals appeared normal throughout the study with the exception of one Group 1 animal and two Group 2 animals which had decreased feed consumption for one to three days within the first nine days o f the study. Slight dermal irritation was observed in one control animal while slight to moderate dermal irritation was observed in the other five control animals treated with dimethyl sulfoxide. In the animals treated with T-6684. slight dermal irritation was observed in one animal while slight to moderate dermal irritation was observed in the other five animals. f. 0, F. Bud W. McDonald Study Director Acute Studies SIGNATURE Covance 6329-200 ___ IZ.Iilh'7 Date REFERENCE 1. Draize. J. H.. "Acute Dermal Toxicity (Single Exposure)," In: Appraisal o f the Safety' o f Chemicals in Foods, Drugs and Cosmetics - Dermal Toxicity, Association of Food and Drug Officials of the U.S.. pp. 54-56 (1959). Table 1 Individual and Mean Body Weights (g) Covance 6329-200 Males Animal Random Number -ization Day 1 29 Females Animal Random Day Number -ization 1 29 Group 1 (Control) Dimethyl Sulfoxide (0.0 mg/kg/day) F63011 F63012 F63013 2,315 2.430 2.447 2.277 2,529 2,500 2,766 2.388 2,708 F63017 F63018 F63019 2.424 2.380 2.263 2,333 2.755 2,277 2.783 2.244 2.619 Mean 2.397 2.388 2.668 Mean 2.356 2.285 2.719 Group 2 - T-6684 (3.0 mg/kg/day) F63014 F63015 F63016 2.404 2.485 2,447 Mean 2,445 2.402 2 722 2,444 2.754 2.390 2.805 2.412 2.760 F63020 F63021 F63022 2.262 2.438 2.395 Mean 2.365 2.067 2.506 2.429 2.804 2,321 2.735 o nj2 2.682 18 Tabic 2 Individual Clinical Signs Covance 6329-200 Animal Number Sex Observation `Day 1 Hour 1 2.5 4 Day 2 Hour 1 2.5 4 Day 3 Hour 1 2.5 4 Day 4 Hour 1 2.5 4 Day 5 Hour i 2.5 4 67 Day(s| 8 9 10-15 16-29 Croup 1 (Contraili - Dimethyl Sulfoxide (0.0 mg/kg/day) F6301I M Appeared normal y / y / yy - - - Decreased food - // - consumption F630I2 M Appeared normal / y / / / / y / / / y y / / / / / / / F630I3 M Appeared normal / / / y / / y y / / / / y / - / F630I7 F Appeared normal / / yy yy / / / / / y / F63018 F Appeared normal y / / y y / y y y / y y / 1-63019 F Appeared normal y / / / y y / yy y/ / / y / y / Group 2 - T-6684 (3.0 mg/kg/day) 1-63014 M Appeared normal / y / / / / y / yy y/ / / / y FA3015 M Appeared normal / y / / / / / y / - - - y y / y y / Decreased food / yy consumption FA30I6 M Appeared normal / y / / / / y / y y y y y y y y y / / - / F63020 F Appeared normal y / y / - - - y y y- F6302I Decreased food consumption F Appeared normal / y / yy - - - --y - y / yy y y y / F63022 F Appeared normal / y / / / y / y y y / y y / y - y y / / / a Each animal appeared normal prior to control or test material administration / Condition existed. - Condition not evident. 19 Covance 6329-200 Table 3 Individual Dermal Irritation Scores Group 1 (Control) - Dimethyl Sulfoxide (0.0 mg/kg/day) Dermal Reaction Ervthema Edema Atonia Desquamation Coriaceousness Fissuring Ervthema Edema Atonia Desquamation Coriaceousness Fissuring Ervthema Edema Atonia Desquamation Coriaceousness Fissuring Males Study Day 1 2 3 4 5 6 13 Animal No. F63011 0 0 0 12 2 2 0 0 0 17 2 7 0000000 000000 1 0000000 0000000 Animai No. F63012 0 111111 0 0 0 00 11 0000000 0000000 0000000 10 0 0 0 0 0 Animal No. F63013 0 1 1 1 12 1 0 0 0 12 7 1 0000000 10 0 0 0 0 0 00 0 0 0 0 0 0 00 0000 17 Females Study Day 345 6 13 Animai No. F63017 00 i 222 1 0 0 0 12 2 1 0000000 000000 1 000 0 100 0000000 Animai No. F63018 0 1 1 117 7 0 1 1 17 i 00 0 0 0 0 0 10 0 0 0 0 0 00000 i 0 0 0000 00 Animai No. F63019 0 1 1 17 7 7 0 1 1 112 2 00 0 0 0 0 0 10 0 0 0 0 0 0 0 00 00 0 000 0 000 20 *!* '*.**!'mn<* Table 4 Individual Dermal Irritation Scores Group 2 - T-6684 (3.0 mg/kg/day) Covance 6329-200 Dermal Reaction Males Study Day________ 1 2 3 4 5 6 13 Animal No . F63014 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 00 11111 0 0 1 12 2 0 0000000 0000000 0000000 0000000 Animal No. F63015 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 1111I 1 00 11I 11 0000000 0000000 0000000 000000 1 Animal No. F63016 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0 1 1 1 12 1 0000 111 0000000 000000 1 0000000 0000000 Females Study Day 1 2 3 4 5 6 13 Animal No . F63020 00 10 21 0 0 0 1? 1 0000000 000000 1 0 0 00 0 10 0 0 0 0 0 10 Animal No. F63021 0 12o 2 n 0 111 1 0000000 000000 1 00000 i0 0000000 Animal No. F63022 00 111 1 0 0 11 1n 1 0000000 0000000 0000000 000000 1 21 APPENDIX A Individual Animal Pathology Data Covance 6329-200 Appendix A Individual Animal Pathology Data Covance 6329-200 PAGE: 1 ANIMAL NUMBER: F63011 SEX: MALE DOSE GROUP: 1 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 06/02/97 STUDY DAY OF DEATH: 29 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: NOT ENTERED DATE AND TIME OF NECROPSY: 06/02/97 8:59 PROSECTOR: DAVID SCHUETTE RECORDER: KEVIN BILLINGS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. TOM PALMER WEIGHER: JOHN S HALFORD ORGAN NAME ABSOLUTE ORGAN WEIGHT (GRAMS) ORGAN WEIGHT RELATIVE TO BODY WEIGHT (t) ORGAN TO BRAIN WEIGHT RATIO ORGAN STATUS DERMAL APP. SITE (IS) KIDNEY (KD) LIVER lLI) BILE (VOLUME) (BI) 1.2216 15.3938 67.8896 0.5000 WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN* ORGAN NAME *** G R O S S PATHOLOGY OBSERVATIONS *** SEVERITY, KEYWORD(S) OR PHRASE FREE-TEXT COMMENTS AND NOTES GENERAL COMMENT (GC) -NO MACROSCOPIC LESIONS IO -ABBR NEC PERFORM. PAGE : 2 ANIMAL NUMBER: F63012 SEX: MALE DOSE GROUP: 1 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 06/02/97 STUDY DAY OF DEATH: 29 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT- NOT ENTERED DATE AND TIME OF NECROPSY: 06/02/97 8:35 PROSECTOR: DAVID SCHUETTE RECORDER: KEVIN BILLINGS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. TOM PALMER WEIGHER: JOHN S HALFORD ORGAN NAME DERMAL APP. SITE (IS) KIDNEY (KD) LIVER (LI) BILE (VOLUME) (BI) ABSOLUTE ORGAN WEIGHT (GRAMS) 1.1614 16.1562 61.6643 0.5000 ORGAN WEIGHT RELATIVE TO BODY WEIGHT (4) ORGAN TO BRAIN WEIGHT RATIO ORGAN STATUS WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME * * * GROSS PATHOLOGY OBSERVATIONS * ** SEVERITY, KEYWORD(S) OR PHRASE FREE-TEXT COMMENTS AND NOTES GENERAL COMMENT (GC) ...................... -NO MACROSCOPIC LESIONS i -------------- ---------------------A-B-B-R---N-E-C---P-E-R--F-O-R-M.------- I Appendix A Individual Animal Pathology Data Covance 6329-200 PAGE : 3 ANIMAL NUMBER: F63013 SEX: MALE DOSE GROUP: 1 SACRIFICE STATUS- DATE OF DEATH: 06/02/97 STUDY DAY OF DEATH: 29 STUDY WEEK OF DEATH- 5 DATE AND TIME OF NECROPSY: 06/02/97 9 05 PROSECTOR: JILL PAUS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. TOM PALMER SCHEDULED, TERMINAL SACRIFICE TERMINAL BODY WEIGHT: NOT ENTERED RECORDER: KEVIN BILLINGS WEIGHER: JOHN S. HALFORD ORGAN NAME ABSOLUTE ORGAN WEIGHT (GRAMSI DERMAL APP. SITE (IS) 0 7738 KIDNEY (KD) 15 1059 LIVER (LI) 73 3509 BILE (VOLUME) (BI) 0 6000 -------------------------------------- ORGAN WEIGHT RELATIVE TO BODY WEIGHT (I) ORGAN TO BRAIN WEIGHT RATIO -------- ORGAN STATUS ----------WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME *** G R O S S PATHOLOGY OBSERVATIONS *.* SEVERITY, KEYWORD(S) OR PHRASE FREE-TEXT COMMENTS AND NOTES GENERAL COMMENT (GC) -NO MACROSCOPIC LESIONS UiIO - ABBR NEC PERFORM. Wi`.i I:.!W. ;rM*| ?;4#WH.'ji#*|ljjHlHUtlIt.t^l'iii Appendix A Individual Animal Pathology Data Covance 6329-200 PAGE: 4 ANIMAL NUMBER: F63014 SEX: MALE DATE OF DEATH: 06/02/97 STUDY DAY OF DATE AND TIME OF NECROPSY: 06/02/97 8:17 POST-FIX WEIGHER: NOT AVAILABLE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DEATH: 29 STUDY WEEK OF DEATH: 1 TERMINAL BODY WEIGHT: NOT ENTERED PROSECTOR: JILL PAUS RECORDER: KEVIN BILLINGS PATHOLOGIST: DR. TOM PALMER WEIGHER: JOHN S. HALFORD ORGAN NAME ABSOLUTE ORGAN WEIGHT (GRAMS) ORGAN WEIGHT RELATIVE TO BODY WEIGHT (\) ORGAN TO BRAIN WEIGHT RATIO ORGAN STATUS DERMAL APP. SITE (IS) KIDNEY (KD) LIVER (LI) BILE (VOLUME) (BI) 0.4833 12 .5487 68.0339 0.9000 ---------------------........ -..... --------------........ WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME * * * GROSS PATHOLOGY OBSERVATIONS *** SEVERITY, KEYWORD(S) OR PHRASE FREE-TEXT COMMENTS AND NOTES GENERAL COMMENT (GC) ...................... -NO MACROSCOPIC LESIONS -ABBR NEC PERFORM. Corning Hazleton Inc. Madison, Wisconsin USA Appendix A Individual Animal Pathology Data Covance 6329-200 PAGE: 5 ANIMAL NUMBER: F63015 SEX: MALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 06/02/97 STUDY DAY OF DEATH: 29 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT* NOT ENTERED DATE AND TIME OF NECROPSY: 06/02/97 8:25 PROSECTOR: DAVID SCHUETTE RECORDER- KEVIN BILLINGS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. TOM PALMER WEIGHER: JOHN S. HALFORD ORGAN NAME DERMAL APP. SITE (IS) KIDNEY (KD) LIVER (LI) BILE (VOLUME) (BI) ABSOLUTE ORGAN WEIGHT (GRAMS) 1.0044 15.9025 74 .7248 1 .9000 ORGAN WEIGHT RELATIVE TO BODY WEIGHT (%) ORGAN TO BRAIN WEIGHT RATIO SOTRAGTAUNS WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME * * * GROSS PATHOLOGY OBSERVATIONS *** SEVERITY, KEYWORD(S) OR PHRASE FREE-TEXT COMMENTS AND NOTES t GENERAL COMMENT (GC) ...................... -NO MACROSCOPIC LESIONS -ABBR NEC PERFORM. ff Corning Hazleton Inc. Madison, Wisconsin USA Appendix A Individual Animal Pathology Data Covance 6329-200 PAGE : 6 ANIMAL NUMBER: F63016 SEX: MALE DATE OF DEATH: 06/02/97 STUDY DAY OF DATE AND TIME OF NECROPSY: 06/02/97 856 POST-FIX WEIGHER: NOT AVAILABLE DOSE GROUP: 2 SACRIFICE STATUS: DEATH: 29 STUDY WEEK OF DEATH: 5 PROSECTOR: JILL PAUS PATHOLOGIST: DR. TOM PALMER SCHEDULED, TERMINAL SACRIFICE TERMINAL BODY WEIGHT: NOT ENTERED RECORDER: KEVIN BILLINGS WEIGHER: JOHN S. HALFORD ORGAN NAME ABSOLUTE ORGAN WEIGHT (GRAMS) DERMAL APP. SITE (IS) KIDNEY (KD) LIVER (LI) BILE (VOLUME) (BI) --------------------------------- 0 6073 14 9890 61 8841 n'finnn ORGAN WEIGHT RELATIVE TO BODY WEIGHT (V) ORGAN TO BRAIN WEIGHT RATIO ....... . -------- ORGAN STATUS ----------WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME * * * GROSS PATHOLOGY OBS ERV ATI ON S *** SEVERITY, KEYWORD(S) OR PHRASE FREE TEXT COMMENTS AND NOTES Koo) GENERAL COMMENT (GC) -NO MACROSCOPIC LESIONS -ABBR NEC PERFORM. **<**, rff****^!*. .if *'y <\ Appendix A Individual Animal Pathology Data Covance 6329-200 Corning Hazleton Inc. Madison, Wisconsin USA PAGE : 7 kvske:si"vsi TMTMs "POST-FIX WEIGHER: ORGAN NAME s,,Fs:n, T, sr : 2,S' := ABSOLUTE ORGAN WEIGHT (GRAMS) weekofsTM" TMTM5 SCHEDULED, TERMINAL SACRIFICE TERMINAL BODY WEIGHT: NOT ENTERED RECORDER KEVIN BILLINGS " j TM ............... WEIGHER: JOHN S . HALFORD ORGAN WEIGHT RELATIVE TO BODY WEIGHT (V) ORGAN TO BRAIN WEIGHT RATIO ORGAN STATUS DERMAL APP. SITE (IS) KIDNEY (KD) LIVER (LI) BILE (VOLUME) (BI) 0 9965 14 5047 55 4966 1.4000 WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME GROSS P A T H O SEVERITY, KEYWORD(S) OR PHRASE OGY O B S E R V A T I O N S FREE-TEXT COMMENTS AND NOTES Id GENERAL COMMENT (GC) -NO MACROSCOPIC LESIONS - ABBR NEC PERFORM. F ,.! j. Jxff..'*'' Corning Hazleton Inc. Madison, Wisconsin USA Appendix A Individual Animal Pathology Data Covance 6329-200 ANIMAL NUMBER: F63018 SEX: FEMALE DOSE GROUP 1 SACRIFICE STATUS- DATE OF DEATH: 06/02/97 STUDY DAY OF DEATH: 29 STUDY WEEK OF DEATH: 5 DATE AND TIME OF NECROPSY: 06/02/97 8:14 PROSECTOR: DAVID SCHUETTE POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. TOM PALMER SCHEDULED, TERMINAL SACRIFICE TERMINAL BODY WEIGHT: NOT ENTERED RECORDER: KEVIN BILLINGS WEIGHER: JOHN S. HALFORD ORGAN NAME DERMAL APP. SITE (IS) KIDNEY (KD) LIVER (LI) BILE (VOLUME) (BI) ABSOLUTE ORGAN WEIGHT (GRAMS) 0.0229 13 .4273 63 .0821 1 ,6000 ORGAN WEIGHT RELATIVE TO BODY WEIGHT (\) ORGAN TO BRAIN WEIGHT RATIO ORGAN STATUS WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME * * * GROSS PATHOLOGY OBSERVATIONS SEVERITY, KEYWORD(S) OR PHRASE FREE-TEXT COMMENTS AND NOTES GENERAL COMMENT (GC) ...................... UOJ -NO MACROSCOPIC LESIONS -ABBR NEC PERFORM. Corning Hazleton Inc. Madison, Wisconsin USA Appendix A Individual Animal Pathology Data Covance 6329-200 ANIMAL NUMBER: F63019 SEX; FEMALE DOSE GROUP: 1 SACRIFICE STATUS: DATE OF DEATH: 06/02/97 STUDY DAY OF DEATH: 29 STUDY WEEK OF DEATH: 5 DATE AND TIME OF NECROPSY: 06/02/97 0:23 PROSECTOR: JILL PAUS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. TOM PALMER SCHEDULED, TERMINAL SACRIFICE TERMINAL BODY WEIGHT: NOT ENTERED RECORDER: KEVIN BILLINGS WEIGHER: JOHN S. HALFORD ORGAN NAME ABSOLUTE ORGAN WEIGHT (GRAMS) ORGAN WEIGHT RELATIVE TO BODY WEIGHT (%) ORGAN TO BRAIN WEIGHT RATIO ORGAN STATUS DERMAL APP. SITE (IS) KIDNEY (KD) LIVER (LI) BILE (VOLUME) (BI) 0.6150 14 .0486 51.5804 0.6000 WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME * * * GROSS P A T H O L OGY OBSERVATIONS SEVERITY, KEYWORD(S) OR PHRASE FREE-TEXT COMMENTS AND NOTES GENERAL COMMENT (GC) -NO MACROSCOPIC LESIONS - ABBR NEC PERFORM. Corning Hazleton Inc. Madison, Wisconsin USA Appendix A Individual Animal Pathology Data Covance 6329-200 PAGE: 10 ANIMAL NUMBER: F63020 SEX: FEMALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 06/02/97 STUDY DAY OF DEATH: 29 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: NOT ENTERED DATE AND TIME OF NECROPSY: 06/02/97 8:49 PROSECTOR: JILL PAUS RECORDER: KEVIN BILLINGS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. TOM PALMER WEIGHER: JOHN S. HALFORD ORGAN NAME ABSOLUTE ORGAN WEIGHT (GRAMS) ORGAN WEIGHT RELATIVE TO BODY WEIGHT (\) ORGAN TO BRAIN WEIGHT RATIO ORGAN STATUS DERMAL APP. SITE (IS) KIDNEY (KD) LIVER (LI) BILE (VOLUME) (BI) 0.6370 13 .4440 52.3 928 0.8000 ----------------..... .......- --------------........ -------- WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME * * * GROSS PATHOLOGY OBSERVATIONS SEVERITY, KEYWORD(S) OR PHRASE FREE-TEXT COMMENTS AND NOTES GENERAL COMMENT (GC) to -NO MACROSCOPIC LESIONS - ABBR NEC PERFORM. Coining Hazleton Inc. Madison, Wisconsin USA Appendix A Individuai Animal Pathology Data Covance 6329-200 PAGE: 11 ANIMAL NUMBER: F63021 SEX: FEMALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 06/02/97 STUDY DAY OF DEATH: 29 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT: NOT ENTERED DATE AND TIME OF NECROPSY: 06/02/97 8:37 PROSECTOR: JILL PAUS RECORDER: KEVIN BILLINGS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. TOM PALMER WEIGHER: JOHN S. HALFORD ORGAN NAME ABSOLUTE ORGAN WEIGHT (GRAMS) ORGAN WEIGHT RELATIVE TO BODY WEIGHT (I) ORGAN TO BRAIN WEIGHT RATIO ORGAN STATUS DERMAL APP. SITE (IS) KIDNEY (KD) LIVER (LI) BILE (VOLUME) (BI) 0.8344 17.0195 53.0880 1.5000 ....... - -------- WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME * * * GROSS PATHOLOGY OBSERVATIONS *** SEVERITY, KEYWORD(S) OR PHRASE FREE-TEXT COMMENTS AND NOTES GENERAL COMMENT (GC) ...................... -NO MACROSCOPIC LESIONS -ABBR NEC PERFORM. n Appendix A Individual Animal Pathology Data Covance 6329-200 PAGE: 12 ANIMAL NUMBER: F63022 SEX: FEMALE DOSE GROUP: 2 SACRIFICE STATUS: SCHEDULED, TERMINAL SACRIFICE DATE OF DEATH: 06/02/97 STUDY DAY OF DEATH: 29 STUDY WEEK OF DEATH: 5 TERMINAL BODY WEIGHT- NOT ENTERED DATE AND TIME OF NECROPSY: 06/02/97 9:07 PROSECTOR: DAVID SCHUETTE RECORDER: KEVIN BILLINGS POST-FIX WEIGHER: NOT AVAILABLE PATHOLOGIST: DR. TOM PALMER WEIGHER: JOHN S HALFORD ORGAN NAME DERMAL APP. SITE (IS) KIDNEY (KD) LIVER (LI) BILE (VOLUME) (BI) ABSOLUTE ORGAN WEIGHT (GRAMS) 1.0944 14.2647 60.3910 1.6000 ORGAN WEIGHT RELATIVE TO BODY WEIGHT (%) ORGAN TO BRAIN WEIGHT RATIO ORGAN STATUS WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN WEIGHT TAKEN ORGAN NAME * * * GROSS PATHOLOGY OBSERVATIONS *** SEVERITY, KEYWORD(S) OR PHRASE FREE-TEXT COMMENTS AND NOTES GENERAL COMMENT (GC) ...................... -NO MACROSCOPIC LESIONS -ABBR NEC PERFORM. APPENDIX B Protocol Deviation Protocol TP6798 Amendment No. 1 To The Protocol Covance 6329-200 35 Protocol Deviation Covance 6329-200 Protocol Actual Procedure Page 7, 7. Experimental Design, B. Dose Administration, (3) Dose Administration, Ninth Sentence: The rabbits will be collared during each approximate 23-hour application period. Animal No. F63016 (Group 2 Male) was found without its collar on at the a.m. mortality check on Day 3. The collar was placed back on the animal at this time. The patch appeared to have remained in place on the application site This deviation is not considered to have had an adverse effect on the outcome of the studv. Covance 6329-200 C O V A ^TC ^ r n ( O lV lL O N fN r SM viC C S C O -M nt C o va n e la b o ra to rie s Ine. PO. Box 7549 M a d iso n . W isco nsin 53707*7545 P ackages: 3301 K in sm a n B ouiavard M a d iso n . W isco nsin 53704 Tal: 5 0 8 /2 4 1 *4471 Pax: 608/241*722 7 Sponsor: 3M Si. Paul. Minnesota PROTOCOL TP6798 Study Title: 5-Daily Dose Dermal Absorpiion/Toxicity Study of T-6684 in Rabbits Date: April 18. 1997 Testing Facility: Covance Laboratories Inc. 3301 Kinsman Boulevard Madison. Wisconsin 53704 Testing Facility Project Identification: Covance 6329-200 TMC AMERICAS cuiiope ASIA/PACIFIC AFRICA Covance 6329-200 Covance 6329-200 TP6798 Page 2 STUDY IDENTIFICATION 5-Dailv Dose Dermal Absorption/Toxicity Study ofT-6684 in Rabbits Covance No. 6329-200 Test Material T-6684 Sponsor 3M Toxicology Services Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative Roger G. Perkins, PhD, DABT 3M Toxicology Services Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul. MN 55133-3220 (612) 733-3222 Study Director F. Bud W. McDonald Covance Laboratories Inc. P.O. Box 7545 Madison, W1 53707-7545 608.242.7901 Testing Facility Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, WI 53704 Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date Week o f May 5, 1997 Week o f June 2, 1997 Week of July 14, 1997 38 r1 'i ) Covance 6329-200 Covance 6329-200 TP6798 Page 3 1. Study 5-Daily Dose Dermal AbsorptionTToxicity Study in Rabbits 2. Purpose To assess the systemic absorption and toxicity and relative skin irritancy of a test material when applied to the skin of rabbits for five consecutive days 3. Regulatory Compliance This study will be conducted in accordance with the following Good Laboratory Practice Regulauons/Standards/Guidelines with the exception that analysis of the test material mixtures for concentration, solubility, homogeneity, and stability will not be conducted: [ ] Conduct as a Nonregulated Study [X] 21 CFR 58 (FDA) [ ] 40 CFR 160 (EPA-FIFRA) [ ] 40 CFR 792 (EPA-TSCA) [ ] C (81)30 (Final) (OECD) [ j 59 NohSan No. 3850 (Japanese MAFF) [ j Notification Nos. 313 and 870 (Japanese MOHW) All procedures in this protocol are in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. 4. Quality Assurance The protocol, study conduct, and the final report will be audited by the Quality Assurance Unit in accordance with Covance Laboratories Inc. (Covance) Standard Operating Procedures (SOPs) and policies. 5. Test M aterial A. Identification T-6684 B. Physical Description Off-white liquid 39 J Covance 6329-200 Covance 6329-200 TP6798 Page 4 C. Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under test conditions). Samples of test material/vehicle mixture(s) for concentration, homogcneity/solubility, and stability analyses will not be taken. D. Storage Room temperature E. Reserve Samples Reserve sample(s) of each batch/lot of test material will be taken for this study. The test material reserve sample(s) will be stored at Covance in a freezer set to maintain a temperature of-20C z 10C until returned to the Sponsor alter completion of the in-life phase of the study. F. Retention Anv unused test material will be returned to the Sponsor. G. Safety Precautions As required by Covance SOPs and policies 6. Control M aterial A. Identification Dimethyl Sulfoxide (DMSO) B. Physical Description Clear, colorless liquid C. Purity and Stability To be documented by Covance (information from the supplier) D. Storage Conditions Room temperature 40 r : Covance 6329-200 Covance 6329-200 TP6798 Page 5 E. Reserve Samples Reserve sample(s) of each batch/lot of control material will be taken. The control material reserve sample(s) will be stored at Covance in a freezer set to maintain a temperature of-20C 10C. F. Retention Any remaining control material may be used for other testing and will not be discarded after issuance of the final report. G. Safety Precautions As required by Covance SOPs and policies Experimental Design A. Animals (1) Species Rabbit (2) Strain/Sourcc Hra:(NZ\V)SPF/Covance Research Products Inc. (3) Age at Initiation Adult (4) Weight at Initiation 2.0 to 3.0 kg (5) N um ber and Sex 6 males and 6 females (6) Identification Individual numbered ear tag (7) Husbandry (a) Housing individually, in suspended stainless steel cages i 41 Covance 6329-200 ii i ii i Covance 6329-200 TP6798 Page 6 (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds, Inc.). The food is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. (c) W ater Ad libitum from an automatic system. Samples of the water are periodically analyzed for specified microorganisms and environmental contaminants. (d) Contaminants There are no known contaminants in the food or water that would interfere with this study. (c) Environment Environmental controls for the animal room will be set to maintain a temperature of 19'C to 23 C. a relative humidity of 50% ~20%. and a 12-hour light/12-hour dark cycle. The dark cycle may be interrupted due to in-life procedures. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to Covance SOPs. An adequate number of extra animals will be purchased so that no animal in obviously poor health is placed on test. The animals will be placed into study groups using a stratified body weight randomization program within nine days of study initiation. (9) Justification for Species Selection Historically, the New Zealand White albino rabbit has been the animal of choice because of the large amount of background information on this species. 42 y Covance 6329-200 Covance 6329-200 TP6798 Page 7 B. Dose Administration (1) Test Groups Group Control/ Test Material (Control) DMSO T-6684 Dose Level (mc/kp/davl 0.0* 3.0* Number of Animals Males Esim ilo 33 33 * To be administered at a dose volume of 0.1 mL/kg of body weight (2) Preparation of Exposure Area On the day before the initial test and control material application, the back and, if necessary (to obtain unblemished skin), the flanks of each rabbit will be clipped free of hair with an electric clipper. The shaved area will constitute approximately 20% of the total body surface area. The treatment sites (intact skin) will be inspected for interfering lesions, irritation, or defects that would preclude the use of any o f the animals. The animals will be clipped as needed throughout the study. (3) Dose Administration All animals will receive five consecutive days of administration of the respective control or lest material applied to the same respective application site each day. The test material/vehicle mixture will be prepared fresh each day of administration. The first day of treatment will be designated as Day 1. The control material will be applied undiluted at a dose volume of 0.1 mL/kg. The Group 2 test material will be diluted with DMSO and applied at a dose volume of 0.1 mL/kg. The doses for the animals in Groups 1 and 2 will be based on the animal's body weight taken on Day 1 before the first administration and applied to the area of exposure in a thin and uniform layer. The area of application (Groups 1 and 2) will be completely covered with a 4-ply 5.0-cm x 5.0-cm gauze patch. All patches will be secured with paper tape around all edges and overwTapped with Saran Wrap* and Elastoplast' tape to provide an occlusive dressing. The rabbits will be collared during each approximate 23-hour application period. The prepared test mixtures will be stored at room temperature until administration. fi:. ; T]3 sr Covance 6329-200 Covance 6329-200 TP6798 Page 8 (4) Reason for Route of Administration The dermal route is a potential route of exposure in humans. (5) Removal of Test Material Approximately 23 hours after each test or control material application, the patches and collars will be removed and the residual material will be removed using water or an appropriate substance, if necessary. The sites of test and control material application will be washed with warm tap water on Day 29 prior to the tissue collection procedure. C. Observation of Animals (1) Clinical Observations Before control or test material administration, at approximately 1,2.5, and 4 hours after each administration of control and test material on Days 1-5 for clinical signs, daily thereafter for clinical signs, and twice daily (a.m. and p.m.) for mortality starting on Day 2 and continuing to Day 29 (a.m. mortality only on Day 29). Observations may be extended when directed by the study director. (2) Reading of Dermal Irritation Before the initial control or test material administration, the dermal irritation reading will be made and recorded as the Day 1 reading (Attachment 1). Additional dermal irritation readings will be made within 30 to 60 minutes after each patch removal (Days 2-6) and on Study Day 13. Individual dermal irritation records will be maintained for each animal. (3) Body Weights For randomization, before the initial test or control material application (Day 1), on Day 29, or at death (when survival exceeds I day) (4) Blood Sample Collections (a) Frequency Predose (anytime from up to four days before the initial dose administration to Day 1), on Days 8, 15, 22, and at experimental termination (Day 29) 44 Covance 6329-200 Covance 6329-200 TP6798 Page 9 (b) Method of Collection/Numbcr of Animals Blood samples (approximately 4 mL each) will be collected from a marginal car vein of all animals at the intervals predose through Day 22. From the posterior vena cava, approximately 20 mL of blood will be obtained from each animal sacrificed in a moribund condition (if possible), approximately 20 to 40 mL of blood will be obtained from each control animal sacrificed on Day 29 (the maximum volume possible will be obtained), and approximately 20 mL of blood will be obtained from each Group 2 animal sacrificed on Day 29. The blood samples will be stored at room temperature and then centrifuged, and the separate serum and cellular fractions stored in a freezer set to maintain -20C i10C. The serum and cellular fractions obtained through Day 15 will be sent frozen on dry ice to the Sponsor one to two weeks prior to in-life termination. The serum and cellular fractions obtained after Day 15 will be sent frozen on dry ice to the Sponsor within one week after in-life termination. The Sponsor is responsible for the retention and disposition of the samples. The serum and cellular fraction samples will be shipped to: James D. Johnson 3M E.T. & S Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James D. Johnson or his alternate will be notified regarding the shipment of the samples. D. Pathology (1) Unscheduled Sacrifices and Deaths Any animal dying during the study or sacrificed in a moribund condition will be subjected to an abbreviated gross necropsy examination and all abnormalities will be recorded. Animals in a moribund condition will be 45 Covance 6329-200 Covance 6329-200 TP6798 Page 10 anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. The whole liver, bile (all available), an approximate 1-cm x 1-cm section of the dermal application site and both kidneys (collected as one sample) will be collected from all animals dying during the study or sacrificed in a moribund condition, weighed (volume only determined for bile), and placed into a freezer set to maintain a temperature of-20C 1 10C. After necropsy, the animals will be discarded. (2) Scheduled Sacrifice On Day 29, the animals will be anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, exsanguinated, and subjected to an abbreviated gross necropsy examination. The animals will be necropsied in random order (via computer-generated random numbers) and all abnormalities will be recorded. The whole liver, bile (all available), an approximate 1-cm x I-cm section of the dermal application site, and both kidneys (collected as one sample) will be collected, weighed (volume only determined for bile), and placed into a freezer set to maintain a temperature of-20C e 10C. After necropsy, the animals will be discarded. (3) Tissue Sample Shipment The samples (liver, bile, dermal application site, and kidneys) collected at the scheduled sacrifice will be sent frozen on dry ice to the Sponsor within one week after collection. Samples collected at unscheduled sacrifices and deaths (if applicable) will be sent with and in the same manner as the samples from the scheduled sacrifice. The samples and their corresponding weights or volumes will be shipped to the person listed in Section 7.C.(4).(b). The Sponsor is responsible for the retention and disposition of the samples. E. Statistical Analyses No statistical analyses are required. 46 Covance 6329-200 Covance 6329-200 TP6798 Page 11 8. Report A final report including those items listed below will be submitted. Description of the test and control materials Description of the test system Procedures Dates of experimental initiation and termination Tabulation of mortality data by sex and dose level Description of any toxic effects/dermal irritation Tabulation of mean body weights by sex and dose level Gross pathology findings Gross pathology report (if requested by the Study Director) Individual animal tissue weights and bile volumes 9. Location of Raw Data, Records, Reserve Samplc(s), and Final Report Original data, or copies thereof, will be available at Covance to facilitate auditing the study during its progress and before acceptance of the final report. When the final report is completed, control material reserve sample(s). all original paper data, including those items listed below will be retained in the archives of Covance for a period of one year following signing of the final report. One year after signing of the final report, all of the aforementioned materials will be sent to the Sponsor and a return fee will be charged. The Sponsor may elect to have the materials retained in the Covance Archives for an additional period of time and Covance will charge a storage fee. If the Sponsor chooses to have Covance dispose of the materials, a disposal fee will be charged. Protocol and protocol amendments Dose preparation records In-life records Body weights Randomization data Dose administration Observations Anatomical pathology records Sample collection records Shipping records Study correspondence Final report (original signed copy) Covance 6329-200 Covance 6329-200 TP6798 Page 12 The following supporting records will be retained at Covance but will not be archived with the study data. Animal receipt/acclimation records Water analysis records Animal room temperature and humidity records Refrigerator and freezer temperature records Instrument calibration and maintenance records 48 Covance 6329-200 PROTOCOL APPROVAL Covance 6329-200 TP6798 Page 13 R o g ers. Perkins, PhD, DABT Sponsor's Representative 3M F. Bud W. McDonald Study Director Acute Studies Covance Laboratories Inc. Representative 2dfii^vr> Quality Assurance Unit Covance Laboratories Inc. Date H-IX-n Date y -//- Y7 Date 49 Covance 6329-200 Attachment 1 Scoring Scale for Acute Dermal Reactions Covance 6329-200 TP6798 Page 14 Erythema 0 - None 1 - Slight 2 - Moderate 3 - Severe Edema 0 - None 1 - Slight (barely perceptible to well defined by definite raising) 2 - Moderate (raised approximately 1 mm) 3 - Severe (raised more than 1 mm) A to n ia 0 - None 1 - Slight (slight impairment of elasticity) 2 - Moderate (slow return to normal) 3 - Marked (no elasticity) D esq uam ation 0 - None 1 - Slight (slight scaling) 2 - Moderate (scales and flakes) 3 - Marked (pronounced flaking with denuded areas) Coriaccousness 0 - None 1 - Slight (decrease in pliability) 2 - Moderate (leathery texture) 3 - Marked (tough and brittle) Fissuring 0 - None 1 - Slight (definite cracks in epidermis) 2 - Moderate (cracks in dermis) 3 - Marked (cracks with bleeding) 50 Covance 6329-200 c o v a ^Tc ^ tm| OCVClOy(M T SERVICES COm rant AMENDMENT NO. 1 TO THE PROTOCOL C ovane* la b o ra to rie s In c PO. Bos 754$ M adison. W isco n sin 53707-7545 P ackages: 3 3 0 ! K insm an B oulevard M adison, W isco n sin 53704 Tel: 6 0 S /2 4 1 -4 4 7 1 Fax: S O t/2 4 1-7227 PROTOCOL TP6798 5-Daily Dose Dermal Absorption/Toxicity Study of T-6684 in Rabbits Covance 6329-200 Sponsor Testing Facility 3M Toxicology Services Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, WI 53704 Sponsor's Representative Roger G. Perkins. PhD, DABT Study Director F. Bud \V. McDonald This amendment modifies the following portions of the protocol: Effective May 20, 1997 The observance of Memorial Day coincides with Day 22 of the study. To avoid having to conduct the Day 22 bleeds on a holiday, modify the following two sections of the protocol to move the Day 22 bleeds to Day 23. 1. Page 8, 7. Experimental Design, C. Observation of Animals, (4) Blood Sample Collections, (a) Frequency. Modify this section with the following underlined change: Predose (anytime from up to four days before the initial dose administration to Day 1), on Days 8, 15, 22, and at experimental termination (Day 29) THE AMERICAS EUROPE ASIA/PACIFIC AFRICA j j I { j ' 51 Covance 6329-200 Amendment No. Covance 6329-200 TP6798 Page 2 2. Page 9, 7. Experimental Design, C. Observation of Animals, (4) Blood Sample Collections, (b) Method of Collection/Number of Animals. Modify the first paragraph in this section with the following underlined change: Blood samples (approximately 4 mL each) will be collected from a marginal ear vein of all animals at the intervals predose through Day 22. PROTOCOL AMENDMENT APPROVAL Sponsor's Representative 3M fu -eJ J cm F. Bud W. McDonald0 Study Director Acute Studies Covance Laboratories Inc. Representative Quality Assurance Unit Covance Laboratories Inc. <T S-ZI-Ql Date Date 52 C iiv.uk'c is an in d e p e n d e n t, pubhclv held com pany operating in over 15 countries and over 30 offices worldwide, with headquarters in Princeton, New Jersey. USA. Covance is the m ark e tin g nam e tor Covanee Inc. and its subsidiaries around the world, the principal ones ot which are listed on this patje. T h e use or "Covance in this report refers to one or more ot these and other subsidiaries. THE AM ERICAS Pr,nceton 1 800.773.001 1 EUROPE Brussels +32.2.773 29.10 A S IA /P A C IF IC S.nqaccre -6 5 7747233 COVANCE LABO RATO RIES Madison. Wl USA 388 541-LABS 5227 Wnna. VA. USA 300-742-8378 Harrogate. UK 01 1 44 1423 50001 1 Munster. Germany 011 49 251 97980 C O V / V N 'C ^ THE DEVELOPM ENT SERVICES COMPANY Shaping Solutions