Document J3N7JRea9MVrGJr82y60zQYev

C orning H azleton liw. P.O. Box 7545 Madison, Wl 53707-7545 Deliveries: 3301 Kinsman Blvd. Madison, WI 33704 608.241.4471 608.241.7227 Fax Sponsor: 3M St. Paul. Minnesota C O R N IN G Hazleton FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6684 in Rabbits Author: F. Bud W. McDonald Study Completion Date: December 31. 1997 Performing Laboratory': Coming Hazleton Inc. 3301 Kinsman Boulevard Madison. Wisconsin 53704 Laboratory Project Identification: CHW 6329-199 Page 1 of 36 Corning Pharmaceutical Services QUALITY ASSURANCE STATEMENT CHW 6329-199 This report has been reviewed by the Quality Assurance Unit o f Coming Hazleton 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. Inspection Dates F ro m To Phase Date Reported to Study Director Date Reported to Management 12/24/96 01/28/97 02/17/97 03/27/97 12/24/96 01/28/97 02/17/97 03/31/97 Protocol Review Necropsy Protocol Amendment Data/Report Review 12/24/96 01/28/97 02/17/97 03/31/97 12/24/96 01/28/97 02/17/97 03/31/97 / jlsJ s ______________ Representative, Quality Assurance Unit /J?-3 / 9 Date 2 STUDY IDENTIFICATION CHW 6329-199 Single-Dose Intravenous Pharmacokinetic Study o f T-6684 in Rabbits Test Material T-6684 Sponsor 3M Toxicology Service 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 Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612)733-3222 Study Director Study Location Study Timetable Study Initiation Date Experimental (In-life) Start Date In-life End Date Experimental Termination Date Study Completion Date F. Bud W. McDonald Coming Hazleton Inc. P.O. Box 7545 Madison, WI 53707-7545 (608)242-7901 Coming Hazleton Inc. 3301 Kinsman Boulevard Madison, WI 53704 January 3, 1997 January 14, 1997 February 11, 1997 February 11, 1997 December 31, 1997 3 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 KEY PERSONNEL Quality Assurance Nancy M. Centanni Manager CHW 6329-199 Laboratory Animal Medicine Donna J. Clemons, DVM Diplomate, ACLAM Supervisor Anatomical Pathology Deborah L. Pirkel Laurie J. Schuller Supervisors Necropsy 4 CONTENTS QUALITY ASSURANCE STATEMENT STUDY IDENTIFICATION..................... KEY PERSONNEL..................................... S U M M A R Y ................................................. O B JE C T IV E .............................. REGULATORY COMPLIANCE TEST AND CONTROL MATERIALS Identification...................................... Purity and Stability............................ Storage and Retention........................ Safety Precautions.............................. TEST SYSTEM ............................... Test Animal.................................. H o u s in g ......................................... Animal Diet.................................. Selection o f Test Animals............ Study Design................................. Justification for Species Selection P R O C E D U R E S ................................................... Dose Preparation and Administration................... Reason for Route of Administration..................... Observations of Anim als....................................... Blood Sample Collections/Shipment.................... P a th o lo g y ............................................ Shipment o f Bile and Tissues......................... Statistical Analyses............................................ Location o f Raw Data, Records, and Final Report 5 CHW 6329-199 .......2 ..... 3 ..... 4 ..... 7 .... 8 ..... 9 ..... 9 ..... 9 .....9 ..10 ........10 .......10 .......10 .......10 .......11 ...... 11 ...... 11 ..... 12 .... 12 ..... 12 ..... 12 ..... 12 ..... 13 .....13 ....14 ....14 CHW 6329-199 R E S U L T S ................................................................................................................................. 14 Body W eights...................................................................................................................... 14 Clinical Observations...........................................................................................................14 Pathology..............................................................................................................................14 DISCUSSION.......................................................................................................................... 14 SIGNATURE........................................................................................................................... 15 TABLE 1 Individual Body Weights (g ).........................................................................................16 2 Individual Clinical Signs............................................................................................... 18 3 Individual Animal Tissue Weights and Bile Volumes............................................... 20 APPENDIX.............................................................................................................................. 21 Protocol Deviations............................................................................................................. 22 Protocol TP6797..................................................................................................................23 Protocol Amendment No. 1 ................................................................................................35 6 SUMMARY CHW 6329-199 This study was done to assess the level o f systemic exposure of T-6684 when administered by a single intravenous injection to rabbits. The study was conducted using four male and four female acclimated rabbits o f the Hra:(NZW)SPF strain for each treatment group as follows: Group 1 (Control) 2 3 4 5 Control/Test Material DMSO T-6684 T-6684 T-6684 T-6684 Dose Level (mg/kg)a 0.0 1.0 3.0 10.0 30.0 Number of Animalsb Male Female 44 44 44 44 44 DMSO - Dimethyl Sulfoxide a Administered at a dose volume of 0.5 mL/kg. b Two animals/sex/dose level were sacrificed on Day 15. The remaining animals (two animals/sex/dose level) were sacrificed on Day 29. The animals received a single intravenous injection of the control or test material at the indicated dose level into the marginal ear vein of the right ear. The test material was mixed with Dimethyl Sulfoxide (DMSO) to a specific concentration for each dose level in Groups 2-5. The respective control material and test mixtures were administered at a dose volume o f 0.5 mL/kg of body weight. Two animals/sex/dose level were sacrificed on Day 15 and the remaining animals (two animals/sex/dose level) were sacrificed on Day 29. Clinical observations were conducted predose and at approximately 0.5, 2.0, and 4.0 hours after intravenous injection. Additional clinical observations and twice a day 7 CHW 6329-199 mortality checks were conducted daily thereafter until the scheduled sacrifice interval (a.m. mortality check only on days o f scheduled sacrifice). Body weights were determined on Day -7 for randomization purposes, before test or control material administration (Day 1), and at the scheduled sacrifice interval (Day 15 or Day 29). A blood sample (approximately 4-mL) was collected from each animal on the day before control or test material administration. Blood samples (approximately 4-mL) were also collected from the animals at 4-, 8-, 12-, 24-, and 48-hours post-injection, and on Day 8, with the exception of one Group 1 male at the 8-hour collection interval from which only 1.7 mL of blood was collected. An approximate 4-mL blood sample was also collected on Days 15 and 22 from each animal scheduled for sacrifice on Day 29. In addition, at the time of the scheduled sacrifice (Day 15 or Day 29), approximately 20 to 40 mL of blood was obtained from each control animal and approximately 20 mL o f blood was obtained from each Group 2-5 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 15 or 29, the animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected, weighed (volume only determined for bile), and sent frozen to the Sponsor. After being intravenously injected with T-6684 or the DMSO control, all animals appeared clinically normal and gained weight during the study, with the exception o f one Group 3 female (3.0 mg/kg) which exhibited an insignificant loss in weight at its termination interval (Day 15). OBJECTIVE The objective of this study was to assess the level of systemic exposure to the test material, T-6684, when administered as a single intravenous injection to rabbits. 8 REGULATORY COMPLIANCE CHW 6329-199 This study was conducted in accordance with the U.S. Food and Drug Administration's Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58, with the exception that analysis of the test 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. TEST AND CONTROL MATERIALS Identification The test material was identified as T-6684 and described as an off-white liquid. The control material was Dimethyl Sulfoxide, (Sigma Chemical Company, Lot No. 64H1007; Expiration March 19, 1997), and was described as a clear, colorless liquid. 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. Reserve samples of the test and control materials were taken and stored in a freezer set to maintain a temperature o f -20C 10C. The control material reserve sample will be retained at CHW for 1 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. 9 CHW 6329-199 Safety Precautions The test and control material handling procedures were according to CHW SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPF strain were received from HRP, Inc., Kalamazoo, Michigan on December 11, 1996 and maintained at the CHW facility at 3301 Kinsman Boulevard, Madison, Wisconsin. 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 in temperature- and humidity- controlled quarters. The only exception to this was one Group 1 male which was found outside its cage at the time of the a.m. mortality check on Day 2 of the study. Environmental controls for the animal room were set to maintain a temperature of 19 to 23C, a relative humidity of 50% 20%, and a 12-hour light/12-hour dark lighting cycle. The dark cycle was interrupted to conduct in-life procedures. In cases where variations from these 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. 10 ___________________________ CHW 6329-199 Selection of Test Animals The animals were identified by animal number and corresponding ear tag and were placed into study groups based on a stratified body weight randomization program. The randomization body weights were determined on Day -7. Study Design Animals weighing from 2,372 to 2,800 g and approximately 17 weeks o f age at initiation of treatment were placed into the following study groups: Group 1 (Control) 2 3 4 5 Control/Test Material DMSO T-6684 T-6684 T-6684 T-6684 Dose Level (mg/kg)a 0.0 1.0 3.0 10.0 30.0 Number of Animalsb 1 Male Female 44 44 44 44 44 a Administered at a dose volume of 0.5 mL/kg. b Two animals/sex/dose level were sacrificed on Day 15. The remaining animals (two animals/sex/dose level) were sacrificed on Day 29. 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. 11 PROCEDURES CHW 6329-199 Dose Preparation and Administration The test material was diluted with DMSO to achieve a specific concentration for each dose level in Groups 2 to 5. An individual dose of the control material or respective test mixture was calculated for each animal based on its body weight on the day of treatment (Day 1). The control material or respective test mixture was administered by intravenous injection into the marginal ear vein of the right ear over a period of 31 to 53 seconds. The prepared test mixtures were stored at room temperature until administered. After administration, any remaining test mixtures were discarded. Reason for Route of Administration Intravenous injection is an acceptable route to assess systemic exposure. Observations of Animals Clinical observations were conducted predose and at approximately 0.5, 2.0, and 4.0 hours after intravenous injection. Additional clinical observations and twice a day mortality checks were conducted daily thereafter until the scheduled sacrifice interval (a.m. mortality check only for animals sacrificed on Days 15 or 29). Body weights were determined on Day -7 for randomization purposes, before test or control material administration (Day 1), and at the scheduled sacrifice interval (Day 15 or Day 29). Blood Sample Collections/Shipment A blood sample (approximately 4-mL) was collected from a marginal ear vein (left ear) o f each animal on Day -4. Blood samples (approximately 4-mL each) were then collected from a marginal ear vein of each animal at 4-, 8-, 12-, 24-, and 48-hours post-injection, and on Day 8, with the exception of a Group 1 male from which a 1.7-mL blood sample was collected at the 8-hour interval. An approximate 4-mL blood sample was also collected on Days 15 and 22 from a marginal ear vein (either ear) of each animal 12 CHW 6329-199 scheduled for sacrifice on Day 29. In addition, at the time o f the scheduled sacrifice (Day 15 or Day 29) and via the posterior vena cava, approximately 20 to 40 mL o f blood was obtained from each control animal and approximately 20 mL o f blood was obtained from each animal in Groups 2-5. All samples were stored at room temperature until centrifuged. After centrifugation, separate samples o f serum and cellular fractions were obtained. The serum and cellular fraction samples obtained pre-injection through Day 15 were stored in a freezer set to maintain a temperature of-20C 10C until shipped 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) on Day 22. The serum and cellular fraction samples obtained on Days 22 and 29 were stored at CHW and shipped to the Sponsor the day after experimental (in-life) termination in the same manner as the samples obtained prior to Day 22. Pathology On Day 15, the first two animals/sex assigned to each dose level (based on the group assignment randomization) were anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected, weighed (volume only determined for bile), and immediately placed in a freezer set to maintain a temperature of -20C 10C. After tissue/bile collection, the animals were discarded. The remaining two animals/sex/dose level were anesthetized, bled, and exsanguinated on Day 29 in the same manner as the animals sacrificed on Day 15. Shipment of Bile and Tissues The tissues (whole livers and kidneys) and bile collected on Days 15 and 29, along with documentation of their corresponding weights or volumes, 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) on Day 21 and six days after in-life termination, respectively. The Sponsor is responsible for the retention and disposition of the samples. CHW does not accept any responsibility for the analysis of the samples collected in this study nor are these results presented in this report. 13 Statistical Analyses No statistical analyses were required by the protocol. CHW 6329-199 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 CHW in accordance with CHW SOP. RESULTS Body Weights Individual body weights are in Table 1. All animals gained weight during the study, with the exception of one Group 3 female (3.0 mg/kg) which exhibited an insignificant loss in weight (18 g) at its termination interval (Day 15). Clinical Observations Individual clinical signs are in Table 2. All animals appeared normal throughout the study. Pathology Individual animal tissue weights and bile volumes are in Table 3. The animals were not examined grossly, although tissues were saved. DISCUSSION The level of systemic exposure of T-6684 was evaluated in male and female albino rabbits when administered as a single intravenous injection at levels o f 1.0, 3.0, 10.0, and 30.0 mg/kg. All animals appeared clinically normal and gained weight during the study, with the exception of one Group 3 female (3.0 mg/kg) which exhibited an insignificant loss in weight at its termination interval (Day 15). 14 SIGNATURE CHW 6329-199 O.nvfboncJ F. Bud W. McDonald Study Director Acute Studies lWs//?7 Date ' ' 15 CHW 6329-199 Table 1 Individual Body W eights (g) Animal Randomization Initial Sex Number (Day -7) (Day 1) Terminal (Day 15) (Day 29) M ale Female F61522 F61523 F61524 F6I525 F61542 F61543 F61544 F61545 G roup 1 (C ontrol) - DM SO (0.0 mg/kg) 2,604 2,677 2,458 2,422 2,625 2,782 2,492 2,507 2,749 2,900 - 2,293 2,549 2,449 2,641 2,372 2,644 2,525 2,731 2,481 2,788 - * * 2,941 3,018 * * 2,991 3,174 Male Female F61526 F61527 F61528 F61529 F61546 F61547 F61548 F61549 G roup 2 - T-6684 (1.0 mg/kg) 2,598 2,437 2,527 2,410 2,688 2,556 2,665 2,551 2,537 2,544 2,501 2,592 2,691 2,676 2,610 2,719 2,877 2,664 - 2,891 2,829 - * * 3,029 2,875 * * 2,850 3,036 G roup 3 - T-6684 (3.0 mg/kg) M ale Female F61530 F61531 F61532 F61533 F6I550 F61551 F61552 F61553 2,585 2,547 2.599 2,627 2,472 2,447 2,416 2,450 2,691 2,555 2,713 2,754 2,425 2,591 2,487 2,527 - Not required. * A nim al sacrificed on Day 15. 2,694 2,718 - 2,669 2,573 - ' * * 3,181 2,979 * * 2,832 2,880 16 CHW 6329-199 Table 1 (Continued) Individual Body W eights (g) Animal Randomization Initial Sex Number (Day -7) (Day 1) T erm in al (D ay 15) (Day 29) M ale Female F61534 F61535 F61536 F61537 F61554 F61555 F6I556 F61557 G roup 4 - T-6684 (10.0 m g/kg) 2,593 2,419 2,413 2,394 2,800 2,576 2,487 2,493 2,381 2,555 2,493 2,481 2,486 2,612 2,544 2,539 2,953 2,708 - 2,574 2,803 - * * 2,818 2,811 * * 2,871 3,002 M ale Female F61538 F61539 F61540 F61541 F61558 F61559 F61560 F61561 G roup 5 - T-6684 (30.0 mg/kg) 2,436 2,409 2,646 2,594 2,569 2,500 2,610 2,619 2,637 2,427 2,498 2,571 2,637 2,492 2,656 2,610 - Not required. * A nim al sacrificed on Day 15. 2,746 2,620 - 2,771 2,643 - ' * * 2,969 2,860 * 3,093 2,924 17 CHW 6329-199 Table 2 Individual Clinical Signs Animal Sex Number Observation Hour (Day 1)'1 0.5 2.0 4.0 2-8 Male Female Group 1 (Control) - DMSO (0.0 mg/kg) F61522 Appeared Normal / / y F61523 Appeared Normal / / y F61524 Appeared Normal / / y F6I525 Appeared Normal / / y F61542 Appeared Normal / / y F61543 Appeared Normal / / y F61544 Appeared Normal / / y F61545 Appeared Normal / / y y y y y y y y y Male Female Group 2 - T-6684 (1.0 mg/kg) F61526 Appeared Normal / / y F61527 Appeared Normal / / y F 6 1528 Appeared Normal / / y F61529 Appeared Normal / / y F61546 Appeared Normal / / y F61547 Appeared Normal / / y F61548 Appeared Normal / / y F61549 Appeared Normal / / y y y y y y y y y Male Female Group 3 - T-6684 (3.0 mg/kg) F61530 Appeared Normal / / y F61531 Appeared Normal / / y F61532 Appeared Normal / / y F61533 Appeared Normal / / y F61550 Appeared Normal / / y F61551 Appeared Normal / / y F61552 Appeared Normal / / y F61553 Appeared Normal / / y y y y y y y y y a Each anim al appeared normal prior to control or test material adm inistration. * Anim al sacrificed on Day 15. / Condition existed. Days 9-15 16-29 y* y yy yy y* y* yy yy y y* yy yy y* y* yy yy y* y* yy yy y* y* yy yy 18 Table 2 (Continued) Individual Clinical Signs Animal Sex Number Observation H our (Day 1)`1 0.5 2.0 4.0 G roup 4 - T-6684 (10.0 mg/kg) Male F61534 Appeared Normal / / F61535 Appeared Normal / / y F61536 Appeared Normal / / y F61537 Appeared Normal / / y Female F61554 Appeared Normal / / y F61555 Appeared Normal / y F61556 Appeared Normal / / y F61557 Appeared Normal / / y G ro u p 5 - T-6684 (30.0 m g/kg) Male F61538 Appeared Normal / y F61539 Appeared Normal / / y F61540 Appeared Normal / / y F61541 Appeared Normal / / y Female F61558 Appeared Normal / / y F61559 Appeared Normal / / y F61560 Appeared Normal / / y F61561 Appeared Normal / / y a Each animal appeared normal prior to test material administration. * Animal sacrificed on Day 15. / Condition existed. CHW 6329-199 Days 2-8 9-15 16-29 yy yy * yy y yyy yy yy * * yyy yyy yy yy * yyy yyy yy yy * * yyy yyy 19 C o r n i n g H a zleton Inc. Madison, Wisconsin USA TABLE 3 Individual Animal Tissue Weights and Bile Volumes TABLE INCLUDES: S E X - A L L ;G R O U P - A L L ;W E EKS-ALL D E A T H - A L L ;SUBSET-ALL Sex Dose Animal Group Number Kidney (g) Liver (g) Bile (mL) Sex Dose Animal Group Number Kidney (g) Liver (g) Bile (mL) 1 F61522* 15.4076 1 F61523 14.8 7 9 1 1 F61524b 15.2507 1 F61525D 16.3977 2 F6 15 26* 17 2412 2 F61527 13.9506 2 F 6 152 0b 14 . 6 6 3 2 2 F 6 1 5 2 9 0 13 . 4 7 3 9 3 F61530 1 2.9111 3 F61531 1 5.9940 3 F61S32b 15.4256 3 F 6 1 5 3 3 13 . 7 7 9 6 4 F61534 1 6 .0 1 3 5 4 F61535 1 9 .3 3 1 8 4 F61S36b 14.5952 4 F61537b 16.5857 5 F61S38 1 6 .1 9 4 0 5 F61539 1b .5 6 J J 5 F61540b 15.9951 5 F61541b 17.9059 a S a cr i f i c e d on D ay IS. b S a cr i f i c e d on D ay 29. Group 1 (Control) 85.4694 97.1462 95.4341 92.9237 0.1000 1 . 3000 0.3000 0.4000 -_ E i m ethvl S u lfo x id e F 1 F61S42* F 1 F6 5 4 3? F 1 F61544? F 1 F61545 10 - g K>q/hg) 14.5255 14.4908 13 .4878 15.1213 Groun - T6684 (l.Q mq/kq) 92 .1133 97.4926 81.6418 78 . 2645 0.5000 1.3000 1.4000 0.5000 F 2 F61546 F 2 F61547 F 2 F61548p F 2 F615490 12.0996 13 . 6 3 4 1 14.2870 14.2304 70.4048 71.5495 79.8862 65.6058 r o u p _2_ - T S 6 8 4 ( 3 . 0 m a / k a ) 0.7000 0.6000 0.4000 1.7000 F 3 F615S0 F 3 F61551 F 3 F61552p F 3 F615530 13.2410 14.1205 14.5109 15.2481 Group - T6684 (10.0 mg/ko) 77.8536 109.4233 71.0696 81.7135 0 . 3000 1.8000 0.6000 0.5000 F 4 F61554* F 4 F61555* F 4 F61556b F 4 F61557b 13.3187 13 . 9257 14 .1 23 2 11.7310 group - T6684 (30.0 ma/ka) 91.2777 77.5408 87.0437 80.6534 0.9000 0.2000 0.9000 0.8000 F 5 F61558* F 5 F61SS9 F 5 F61560b F 5 F615610 16.4529 13.8245 13.2574 13.8642 70.3337 83.8694 80.7606 91.5821 67.3471 80.3894 70.6502 83.7193 90.1444 65.4078 64.3275 70.2567 74.8795 79.1617 59.5147 76.3246 72.4410 70.6216 93.3025 62.2193 1.1000 1.3000 0 50nn 0.9000 1.3000 0.6000 1.0000 0.8000 1.2000 1.1000 0.9000 0.5000 1 nnnn 0.9000 n 4nnn 0.8000 0.0000 0 finnn 0.5000 1.3000 CHW 6329-199 APPENDIX Protocol Deviations Protocol TP6797 Protocol Amendment No. 1 CHW 6329-199 21 Protocol Deviations CHW 6329-199 Protocol Page 5, 7. Experimental Design, A. Animals, (7) Husbandry, (a) Housing. Individually, in suspended stainless steel cages. Page 8, 7. Experimental Design, D. Observation of Animals, (3) Blood Sample Collections, (b) Method of Collection/Number of Animals, first paragraph, first sentence. Blood samples (approximately 4 mL)... and from the marginal ear vein (left ear) at 4-hours postdose through Day 8. Page 9, 7. Experimental Design, E. Termination (3) Sample Collection. The whole liver, bile, and both kidneys (collected as one sample) from each animal will be collected, weighed (volume only determined for bile), and immediately placed into a freezer set to maintain a temperature of -20C 10C. Actual Procedure One Group 1 male was found outside its cage at the time of the a.m. check on Day 2. Blood samples were collected at the following intervals (postdose) from the right marginal ear vein of the following animals (animal number/group/sex): 12- and 24-hour: F61524 (1M), F61557 (4F). 48-hour and Day 8: F61524 (1M), F61549 (2F), F61555 (4F), F61557 (4F). Only one kidney for Animal No. F61531 (Group 3M) was weighed at the time of tissue collection. The other kidney was weighed the next day. These deviations are not considered to have had an adverse effect on the outcome of the study. 22 Corning Hazleton Inc. P.O. Box 7545 Mad.wm, WJ 53707-7545 Deliveries: 3 3 0/ Kinsman Bird. Madison, W l 53704 6 0 8 .2 4 1 .4 4 7 1 60 8.241.7227 Fax Sponsor 3M St. Paul, Minnesota CHW 6329-199 C O R N IN G Hazleton PROTOCOL TP6797 Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6684 in Rabbits Date: January 3, 1997 Performing Laboratory: Coming Hazleton Inc. 3301 Kinsman Boulevard Madison. Wisconsin 53704 Laboratory Project Identification: CIIW 0329-199 l miint; I'lunnatcuiu jl Vivii\ 23 CHW 6329-199 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study ofT-6684 in Rabbits CHW 6329-199 TP6797 Page 2 CHW No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6329-199 T-6684 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 Roger G. Perkins, PhD, DABT 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-3222 F. Bud W. McDonald Coming Hazleton Inc. P.O. Box 7545 Madison, WI 53707-7545 (608)242-7901 Coming Hazleton Inc. 3301 Kinsman Boulevard Madison, Wl 53704 Week of January 13, 1997 Week of February 10, 1997 Week of March 24. 1997 24 CHW 6329-199 CHW 6329-199 TP6797 Pige 3 1. Study Single-Dose Intravenous Pharmacokinetic Study in Rabbits 2. Purpose To assess the level of systemic exposure when the test material is administered as a single intravenous injection to rabbits 3. Regulatory Compliance This study will be conducted in accordance with the following Good Laboratory Practice Regulations/Standards/Guidelines with the exception that analysis o f 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(8I)30 (Final) (OECD) ( ] 59 NohSan No. 3850 (Japanese MAFF) [ ] 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 the Wisconsin facility of Coming Hazleton Inc. (CHW) Standard Operating Procedures (SOPs) and policies. 5. Test Material A. Identification T -6684 U. Physical Description (To be documented in the raw data) C. Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under test conditions). Samples of test material/vehiclc mixture(s) 25 CHW 6329-199 CHW 6329-199 TP6797 Page 4 for concentration, homogeneity/solubility, and stability analyses will not be taken before administration unless requested otherwise by the Sponsor. These samples (if taken) will be sent to the Sponsor after experimental termination. D. Storage Room temperature El Reserve Samples Reserve sample(s) of each batch/lot o f test material will be taken for this study. The test material reserve sample(s) will be stored at CHW in a freezer set to maintain a temperature of -20C 10C and then returned to the Sponsor after completion of the in-life phase o f the study. F. Retention Any unused test material will be returned to the Sponsor after completion of all related in-life testing. G. Safety Precautions As required by CHW SOPs and policies 6. C ontrol Material A. Identification Dimethyl Sulfoxide (DMSO); Lot number, source, and expiration date to be recorded in the raw data and included in the final report. B. Physical Description (To be documented in the raw data) C. Purity and Stability To be documented by CHW (information from the supplier) D. Storage Room temperature 26 CHW 6329-199 CHW 6329-199 TP6797 Page J E. Reserve Samples Reserve sample(s) of each batch/lot of control material will be taken for this study. The control material reserve sample(s) will be stored at CHW in a freezer set to maintain a temperature o f -20C 10C. F. Retention Any remaining control material may be used for other testing and will not be discarded after issuance o f the final report G. Safety Precautions required by CHW SOPs and policies Experimental Design A. Animals (1) Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP, Inc. (3) Age at Initiation Adult (4) Weight at Initiation 2.5 to 3.5 kg (5) Number and Sex 20 males and 20 females (6) Identification Individual numbered ear lag (7) Husbandry (a) Housing Individually, in suspended stainless steel cages 27 CHW 6329-199 CHW 6329-199 TP6797 Page 6 (b) Food A measured amount o f 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 A d libitum from an automatic system. Samples of the water are analyzed for total dissolved solids, specified microbiological content, selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the food or water that would interfere with this study. (e) E nvironm ent Environmental controls for the animal room will be set to maintain a temperature o f 19C to 23C, a relative humidity o f 50% 20%, and a 12-hour light/12-hour dark cycle. The dark cycle may be interrupted due to in-life procedures. (f) A cclim ation At least 7 days (8) Selection of Test Animals Based on health and body weight according to CHW 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. 28 CHW 6329-199 B. Dose Administration (1) Test Groups CHW 6329-199 TP6797 Page? Group 1 (Control) 2 3 4 5 Control/Test Material Sterile Water T-6684 T-6684 T-6684 T-6684 Dose Level (mg/kg)* 0.0 1.0 3.0 10.0 30.0 Number o f Animals* Male Female 44 44 44 44 44 a Administered at a dose volume o f 0.5 mL/kg. b Two animals/sex/dose level will be sacrificed on Day 15. The remaining animals (two animals/sex/dose level) will be sacrificed on Day 29. C. Dosing Procedures (1) Dosing Route Intravenous injection into the right marginal ear vein over approximately 30 to 60 seconds. (2) Reason for Dosing Route Intravenous injection is an acceptable route to assess systemic exposure. (3) Dosing Duration Single dose (4) Dose Preparation The day of treatment will be designated as Day 1. Individual doses will be calculated based on the animal's body weight taken on Day 1. The Group 1 animals will be treated with DMSO at a dose volume of 0.5 mL/kg, The test material will be diluted with DMSO to achieve a specific concentration for each dose level in Groups 2-5 and administered 29 CHW 6329-199 CHW 6329-199 TP6797 Page* at a dose volume of 0.5 mL/kg. The prepared test mixtures will be stored at room temperature until administered. D. Observation of Animals (1) Clinical Observations Before test or control material administration, at approximately 0.5, 2.0, and 4.0 hours post-injection (Day 1) for clinical signs, daily thereafter for clinical signs, and twice daily (a.m. and p.m.) for mortality until the scheduled sacrifice interval (Day 15 or Day 29). The animals sacrificed on Days 15 or 29 will be observed only once for mortality on those respective days. Observations may be extended when directed by the study director. (2) Body Weights For randomization, before test or control material injection (Day 1), at the scheduled sacrifice interval (Day 15 or Day 29), or at unscheduled death and sacrifices (when survival exceeds 1 day) (3) Blood Sample Collections (a) Frequency Pre-injection (anytime from up to four days before test material administration to Day 1), at approximately 4-, 8-, 12-, 24-, and 48hours post-injection, on Days 8, 15, 22, and at the scheduled sacrifice interval (Day 15 or Day 29) (b) Method of Collection/Number of Animals Blood samples (approximately 4 mL) will be collected from the marginal ear vein (either ear) of all animals on the day before test or control material injection, and from the marginal ear vein (left ear) at 4-hours postdose through Day 8. On Days 15 and 22. additional blood samples (approximately 4 mL) will be collected from the marginal ear vein (left ear if possible) of the animals scheduled for sacrifice on Day 29. 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 Days 15 or 29 (the maximum volume possible will be obtained), and approximately 30 CHW 6329-199 CHW 6329-199 TP6797 Page 9 20 mL of blood will be obtained from each Group 2-5 animal sacrificed on Days 15 or 29. The samples will be stored at room temperature and then centrifuged, and the separate serum and cellular fractions stored in a freezer set to maintain a temperature o f -20C 10C. 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. E. Termination (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 anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. Tissues, as described in section 7.E.(3) Sample Collection, will be collected from any animal dying during the study or sacrificed in a moribund condition. After necropsy, the animals will be discarded. (2) Scheduled Sacrifices On Day 15. the first two animais/sex assigned to each dose level (based on the group assignment randomization) will be anesthetized with sodium pentobarbital (via injection in the marginal car vein), bled via the vena cava, and exsanguinated. The remaining two animals/sex/dosc 31 CHW 6329-199 CHW 6329-199 TP6797 Page 10 level will be anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, and exsanguinated on Day 29. An abbreviated gross necropsy examination will not be done, however, tissues [as described in section 7.E.(3) Sample Collection] will be collected. (3) Sample Collection The whole liver, bile, and both kidneys (collected as one sample) from each animal will be collected, weighed (volume only determined for bile), and immediately placed into a freezer set to maintain a temperature o f -20C 10C. After sample collection, the animals will be discarded. The samples (liver, bile, and kidneys) will be sent frozen on dry ice to the Sponsor within one week after collection. The samples and their corresponding weights or volumes will be shipped to the person listed in Section 7.D.(3)(b). The Sponsor is responsible for the retention and disposition o f the samples. F. Statistical Analyses No statistical analyses are required. 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 Description of any toxic effects Gross pathology findings (if applicable) Gross pathology report (if applicable and requested by the study director) Individual animal tissue weights and bile volumes 32 CHW 6329-199 CHW 6329-199 TP6797 P ig e ll 9. Location of Raw Data, Reserve Sample(s), Records, and Final Report Original data, or copies thereof, will be available at CHW to facilitate auditing the study during its progress and before acceptance o f 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 CHW for a period of one year following signing o f the final report One year after signing of the final report, all o f 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 CHW Archives for an additional period of time and CHW will charge a storage fee. If the Sponsor chooses to have CHW dispose of the materials, a disposal fee will be charged. Protocol and protocol amendments Dose preparation records In-life records Body weights Dose administration Observations Sample collection records Shipping records Pathology Records Study correspondence Final report (original signed copy) The following supporting records will be retained at CHW but will not be archived with the study data. Animal reccipt/acclimation records Water analysis records Animal room temperature and humidity records Refrigerator and freezer temperature records Instrument calibration and maintenance records 33 CHW 6329-199 PROTOCOL APPROVAL CHW 6329-199 TP6797 Page 12 Roger G. Qrkins, PhD, DABT Sponsor's Representative 3M Date l). F. Bud W. McDonald Study Director Acute Studies Coming Hazleton Inc. RepreseptaUve Quality Assurance Unit Coming Hazleton Inc. _________ /- 3 - <y7 Date 377 Date 34 CHW 6329-199 C O V M C E ^ AMENDMENT NO. 1 TO THE PROTOCOL Covanca U tooratsriM in c P.O. B o x 7545 M a to fl, W ia e M iin 53707-7545 PachaoM i 3301 K in m a n B a u lm rd M a d ita n , W iaeonain 53704 Tal: 03/341*4471 Fax: <05/241 -7237 PROTOCOL TP6797 Single-Dose Intravenous Pharmacokinetic Study o f T-6684 in Rabbits Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 CHW 6329-199 Testing Facility Coming Hazleton Inc. 3301 Kinsman Boulevard Madison, WI 53704 Sponsor's Representative Roger G. Perkins, PhD, DABT Study Director F. Bud W. McDonald This amendment modifies the following portions of the protocol: Effective January 3,1997 1. Page 7, 7. Experimental Design, B. Dose Administration, (I) Test Groups. To correctly identify the control material to be used in this study, which was listed incorrectly in the table at the time the protocol was issued by the study director, replace sterile w ater with DMSO. TNC AM M IC A S EUROPE ASIA/PACIFIC AFRICA 35 CHW 6329-199 Amendment No. 1 CHW 6329-199 TP6797 Page 2 Effective January 14,1997 2. Page 5 ,7 . Experimental Design, A. Animals, (4) Weight at Initiation. To accommodate the use o f lighter animals available for use in the study, modify the body weight range with the following underlined change: U l to 3.5 kg PROTOCOL AMENDMENT APPROVAL Roger G. P e tr is , PhD, DABT Sponsor Representative 3M fig fe9 ~ ) Date i). F. Bud W. McDonald Study Director Acute Studies Coming Hazleton Inc. t. Representative Quality Assurance Unit Coming Hazleton Inc. t iSFa&Q'? Date iteti m i Date 36