Document 71JqnrMdqpxgo4rm3dw1J0JYj

C-.orningHazleton laic. P.O. Box 754.i ,Madison,Wi 53707-7545 Delit,eries3:301 Kinsman Blvd. M,zdison,W'l @5-3/'04 608.241.4471 608.241.7227 Fax Sponsor: 3M St.Paul.Minnesota CORNINGHazleton FINAL REPORT Study Title: Single-DoseIntravenousPhannacokineticStudy of T-6684 inRabbits Author: F.Bud W. McDonald Study Completion Date: December ')1.1997 Performing Laboratory: Coming HazletonInc. ')-')KOilnsman Boulevard Madison.Wisconsin 53704 Laboratory ProjectIdentification: CHW 6329-199 Page Iof')6 (@orningllharmaceuricalServices QUALITY ASSURANCE STATEMENT CHW 6329-199 This report has been reviewed by the Quality Assurance Unit of Coming Hazleton Inc.,in accordance with theFood and Drug Administration(FDA) Good Laboratory Practice Regulations,21 CFR 58. The followinginspectionswere conducted and findings reportedto the Study Directorand management. InspectionDates - From To Phase Date Reported to Study Directo 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 ProtocolReview Necropsy ProtocolAmendment 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 Re'prese;dativ'Qeu,a'lity Assurance Unit Date 17 STUDY IDENTIFICATION CHW 6329-199 Single-DoseIntravenousPharmacokineticStudy ofT-6684 inRabbits Test Material T-6684 Sponsor 3M ToxicologyService Medical Department 3M Center,Bldg.220-2E-02 P.O. Box 33220 St.Paul,NIN 55133-3220 Sponsor's Representative Roger G. Perkins,PhD, DABT 3M ToxicologyService Medical Department 3M Center,Bldg. 220-2E-02 P.O. Box 33220 St.Paul,N4N 55133-3220 (612)733-3222 Study Director Study Location Study Timetable Study InitiatioDnate Experimental(In-lifSet)artDate In-*liEfned Date ExperimentalTerminationDate Study Completion Date F.Bud W. McDonald Coming HazletonInc. P.O. Box 7545 Madison,W] 5)707-7545 (608)242-7901 Coming HazletonInc. 3301 Kinsman Boulevard Madison,WI 53704 January3, 1997 January 14, 1997 February 11, 1997 February 11,1997 December 31, 1997 3 Acute Studies StevenM. Glaza Manager F.Bud W. McDonald Study Director JeffreyB. Hicks In-lifSeupervisor Rose M. Bridge AdministrativeSupervisor ToxicologySupport Kathy Myers Manager CalvinL. Horton Supervisor KEY PERSONNEL QualityAssurance Nancy M. Centanni Manager CHW 6329-199 LaboratoryAnimal Medicine Donna J.Clemons,DVM Diplomate,ACLAM Supervisor Anatomical Pathology Deborah L.Pirkel LaurieJ.Schuller Supervisors Necropsy 4 CONTENTS CHW 6329-199 QUALITY ASSURANCE STATEMENT I STUDY IDENTIFICATION 3 KEY PERSONNEL 4 SUMMARY 7 OBJECTIVE 8 REGULATORY COMPLIANCE 9 TEST AND CONTROL MATERIALS Identification 9 9 Purityand Stabilit.y........................................................................ 9 Storageand Retention.......................................................9................ SafetyPrecautions..........................................................10................ TEST SYSTEM .............................................................................. 10 TestAnimal ................................................................................ Housing 10 ................................................................................... 10 Animal Diet................................................................................ Selectionof TestAnimals 10 II Study Design ............................................................................... II JustificatiofnorSpeciesSelection............................................I.I................. PROCEDURES ............................................................................... 12 Dose Preparationand Administratio.n.........................................12................. Reason forRoute of Administratio.n..........................................12................. ObservationsofAnimals ....................................................1.2................. Blood Sample Collections/Shipmen.t.........................................1.2................. Pathology ................................................................................... 13 Shipment of Bilcand Tissues................................................1.3................. StatisticaAlnalyses........................................................................... 14 Location of Raw Data,Records,and FinalReport ...............................1.4................. 5 CHW 6329-199 RESULTS ..................................................................1.4................. Body Weights .............................................................1.4................ ClinicalObservations.......................................................1.4................. Pathology.................................................................14................. DISCUSSION ...............................................................1.4................. SIGNATURE ................................................................1.5................ TABLE I IndividuaBlody Weights(g)..............................................1.6................. 2 IndividuaCllinicaSligns.................................................1.8................. IndividualAnimal TissueWeights and BileVolumes .........................2.0................. APPENDIX .................................................................2.1................. ProtocolDeviations........................................................I.-.)................ ProtocolTP6797 ..........................................................2.'.1................. ProtocolAmendment No. I .................................................'.).5................ SUNUMLARY CHW 6329-199 This studywas done to assessthelevelof systemicexposure of T-6684 when administeredby a singleintravenousinjectiotno rabbits. The studywas conducted usingfourmale and fourfemale acclimatedrabbitsof the Hra.(NZW)SPF strainforeach treatmentgroup asfollows: Group I (Contro 2 3 4 5 ControVrest 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 4 4 4 4 4 4 4 4 4 4 DMSO - Dimethyl Sulfoxide a Administeredata dose volume of 0.5mL/kg. b Two animals/sex/dosleevelwere sacrificeodn Day 15. The remaining animals(two animals/sex/dosleevel)were sacrificeodn Day 29. The animalsreceiveda singleintravenousinjectioonf thecontrolor testmaterialatthe indicateddose levelintothemarginalearveinoftherightear.The testmaterialwas mixed with Dimethyl Sulfoxide(DMSO) toa specificconcentratiofnoreach dose level inGroups 2-5. The respectivceontrolmaterialand testmixtureswere administeredata dose volume of 0.5 mL/kg of body weight.Two animals/sex/dosleevelwere sacrificed on Day 15 and theremaininganimals(two animals/sex/dosleevel)were sacrificeodn Day 29. Clinicalobservationswere conductedpredoseand atapproximately0.5,2.0,and 4.0 hours afterintravenousinjectionA.dditionalclinicaolbservationsand twicea day 7 CHW 6329-199 mortalicthyeckwsereconducteddailtyhereafutnetritlheschedulseadcrifincteerval (a.mm.ortalicthyeckonlyondaysofschedulseadcrificBeo)d.yweightwsere determinedon Day -7 forrandomizationpurposes,beforetestor controlmaterial administrati(oDnay 1),and atthescheduledsacrifiicneterva(lDay 15 orDay 29). A blood sample(approximatel4y-mL) was collectefdrom eachanimalon theday before controlortestmaterialadministrationB.lood samples(approximatel4y-mL) were also collectefdrom theanimalsat4-,8-,12-,24-,and 48-hourspost-injectioann,d on Day 8, withtheexceptionof one Group I male atthe8-hourcollectioinntervaflrom which only 1.7mL of bloodwas collectedA.n approximate4-mL bloodsample was alsocollected on Days 15 and 22 from each animalscheduledforsacrificoen Day 29. Inadditiona,t thetimeofthescheduledsacrific(eDay 15 orDay 29),approximately20 to40 niL of bloodwas obtainedfrom each controlanimaland approximately20 mL ofbloodwas obtainedfrom each Group 2-5animal.Allsampleswere centrifugeadnd separate samplesof serum and cellulafrractionwsere obtainedand sentfrozenon dry icetothe Sponsor.On Day 15 or 29,theanimalswere anesthetizewdith sodium pentobarbital, bledviatheposteriorvena cava,and exsanguinated.An abbreviatedgrossnecropsy examinationwas notdone,however,tissuewsere collectedT.he whole liverb,ile,and bothkidneysfrom eachanimalwere collectedw,eighed (volumeonlydeterminedfor bile)a,nd sentfrozentotheSponsor. Afterbeingintravenousliynj*ectweidthT-6684 ortheDMSO controla,llanimals appearedclinicallnyormal and gainedweightduringthestudy,with theexceptionof one Group 3 female(3.0mg/kg) which exhibiteadn insignificalnotssinweightatits terminatioinnterva(lDay 15). OBJECTIVE The objectiveof thisstudywas toassessthelevelofsystemicexposuretothetest materialT,-6684,when administeredasa singleintravenouisnjectiotno rabbits. 8 REGULATORY COWLLANCE CHW 6329-199 Thisstudywas conducteidnaccordancweiththeU.S.FoodandDrugAdministration's Good LaboratoryPracticeRegulationsforNonclinicalLaboratoryStudies,21 CFR 58, with theexceptionthatanalysisofthetestmixturesforconcentration, homogeneity/solubilitayn,d stabilitwyas notconducted.All proceduresused inthis studywere incompliancewith theAnimal WelfareAct Regulations.Inthe opinionof the Sponsor and studydirectort,hestudydid not unnecessarildyuplicateany previous work. TEST AND CONTROL MATERIALS Identification The testmaterialwas identifieadsT-6684 and describedas an off-whiteliquid.The controlmaterialwas Dimethyl Sulfoxide,(Sigma Chemical Company, Lot No. 64H 1007; ExpirationMarch 19,1997),and was describedas a clear,colorlessliquid. Purity and Stability The Sponsor assumes responsibilitfyortestmaterialpurityand stabilitdyeterminations (includingunder testconditions)A.nalysisof thetestmaterialmixturesfor concentrationh,omogeneity/solubilitayn,d stabilitwyas not conducted.The purityand stabilitoyf thecontrolmaterialwere consideredtobe adequateforthepurposes of this study. Storage and Retention The controland testmaterialswere storedatroom temperature.Reserve samples of the testand controlmaterialswere takenand storedina freezersetto maintaina temperature of -20'C IO*C. The controlmaterialreservesample willbe retainedatCHW forI year. The testmaterialreservesample was senttotheSponsor. Any unused testmaterialwill be returnedtothe Sponsor. Any remainingcontrolmaterialmay be used forothertesting and willnot be discardedafterissuanceof thefinalreport. 9 CHW 6329-199 SafetyPrecautions The testand controlmaterialhandlingprocedureswere accordingtoCHW policies. SOPs and TEST SYSTEM Test Animal Adult albinorabbitsoftheHra:(NZW)SPF strainwere receivedfrom HRP, Inc., Kalamazoo, Michigan on December 11,1996 and maintainedatthe CHW facilitayt3301 Kinsman Boulevard,Madison, Wisconsin. Housing Afterreceiptt,heanimalswere acclimatedfora periodof atleast7 days. During acclimationand throughoutthestudy,theanimalswere individuallhyoused in suspended stainlessteelcages intemperature-and humidity-controlledquarters.The only exceptiontothiswas one Group I male which was found outsideitscage atthetime of the a.m.mortalitycheck on Day 2 of thestudy.Environmentalcontrolsforthe animal room were settomaintaina temperatureof 19' to23'C, a relativheumidityof 50% 20%, and a 12-hourlight/12-houdrark lightincgycle.The dark cyclewas interruptetdo conduct in-lifperocedures.Incaseswhere van'ationfsrom theseconditionsexisted,they were documented and consideredto have had no adverseeffecton the studyoutcome. Animal Diet The animals were providedaccessto waterad libitumand a measured amount of LaboratoryRabbitDiet HF #5326, PMI Feeds,Inc.The feedisroutinelaynalyzedby the manufacturerfornutritionaclomponents and environmentalcontaminants.Samples of the water areperiodicallaynalyzed.There were no known contaminantsinthefeedor water atlevelsthatwould be expectedtointerferweithor affectthe resultosf thestudy. l'O CHW 6329-199 Selection of Test Animals ne animals were identifiedby animal number and corresponding ear tag and were placed intostudy groups based on a stratifiebdody 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 of age at initiation of treatmentwere placed intothe followingstudy groups: Group I (Control) Control/Test Material DMSO Dose Level (mg/kg)a 0.0 2 T-6684 1.0 3 T-6684 3.0 4 T-6684 10.0 5 T-6684 30.0 b Number of Animals Male Female 4 4 4 4 4 4 4 4 4 4 a Administered at a dose volume of 0.5 mL/kg. b Two animals/sex/dose level were sacrificedon Day 15. The remaining animals (two animaWsex/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. PROCEDURES CHW 6329-199 Dose Preparationand Administration The testmaterialwas dilutewdithDMSO to achievea specificoncentratiofnoreach dose levelinGroups 2 to5. An individuadlose of thecontrolmaterialor respectivetest mixturewas calculatedforeachanimal based on itsbody weight on the day oftreatment (Day 1).The controlmaterialorrespectivteestmixturewas administeredby intravenous injectioinntothemarginalearveinof therightearovera periodof31 to5' seconds The preparedtestmixtureswere storedatroom temperatureuntiladministered.After administrationa,ny remainingtestmixtureswere discarded. Reason for Route ofAdministration Intravenousinjectioinsan acceptableroutetoassesssystemicexposure. Observations ofAnimals Clinicalobser-vationwsere conducted predose and atapproximately0.5,2.0,and 4.0 hoursafterintravenousinjectionA.dditionalclinicaolbservationsand twice a day mortalitychecks were conducteddailythereafteurntilthescheduledsacrificienterval (a.m.mortalitycheck only foranimalssacrificeodn Days 15 or 29). Body weightswere determinedon Day -7 forrandomizationpurposes,beforetestor controlmaterialadministratio(nDay 1),and atthescheduledsacrificienterva(lDay 15 or Day 29). Blood Sample Collections/Shipment A bloodsample (approximatel4y-mL) was collectefdrom a marginalearvein(leftear) of each animal on Day -4. Blood samples (approximately4-mL each)were thencollected from a marginalearveinof eacharu'malat4-,8-,12-,24-,and 48-hours post-injection, and on Day 8,with the exceptionof a Group I male from which a 1.7-mL blood sample was collectedatthe8-hourintervalA.n approximate4-mL blood sample was also collecteodn Days 15 and 22 from a marginalearvein(eitheerar)of each animal 12 CHW 6329-199 schedulefdorsacrifiocneDay 29.Inadditioant,thetimeoftheschedulesdacrifice (Day 15 or Day 29) and viatheposteriovrena cava,approximately20 to 40 mL ofblood was obtainedfrom each controlanimal and approximately20 mL of blood was obtained from each animal inGroups 2-5.All samples were storedatroom temperatureuntil centrifuged.Aftercentrifugatiosne,paratesamples ofserum and cellularfractionwsere obtained.The serum and cellulafrractionsamplesobtainedpre-injectiotnhroughDay 15 were storedina freezersettomaintaina temperatureof -20*C IOIC untilshipped frozen(on dry ice)tothe 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 cellulafrractionsamples obtainedon Days 22 and 29 were storedatCHW and shipped to the Sponsor the day after experimental(in-lifet)erminationinthesame manner asthesamples obtainedpriorto Day 22. Pathology On Day 15,thefirstwo animals/sexassignedtoeachdose level(based on thegroup assignmentrandomization)were anesthetizewdithsodium pentobarbita(lviainjectioinn themarginal earvein),bledviatheposteriorvena cava,and exsanguinated.An abbreviatedgrossnecropsy examinationwas notdone,however, tissueswere collected. The whole liverb,ile,and bothkidneysfrom eachanimal were collectedw,eighed (volume only determinedforbile)a,nd immediatelyplacedina freezersettomaintaina temperatureof-20'C 10'C. Aftertissue/bicloellectiotnh,eanimalswere discarded. The remaining two animals/sex/doselevelwere anesthetizedb,led,and exsanguinatedon Day 29 in theswne manner as theanimals sacrificeodn Day 15. Shipment of Bileand Tissues The tissues(whole liversand kidneys)and bilecollectedon Days 15 and 29,along with documentationof theircorrespondingweightsorvolumes,were sentfrozen(on dry ice) totheSponsor (James D. Johnson,3M E.T.& S,Bldg.2-3E-09,935 Bush Avenue, St. Paul,MN, 55106) on Day 21 and sixdaysafterin-lifteerminationr,espectivelyT.he Sponsor isresponsiblefortheretentioannd dispositioonf thesamples. CHW does not acceptany responsibilitfyortheanalysisof thesamplescollectedinthisstudynor are theseresultspresentedin thisreport. 13 StatisticaAlnalyses No statisticaanlalyseswere requiredby theprotocol. CHW 6329-199 Location ofRaw Data, Records, and FinalReport The raw data,recordsa,nd an originaslignedcopy ofthefinalreportwillbe retainedin thearchivesofCHW inaccordancewithCHW SOP. RESULTS Body Weights Individualbody weightsareinTable 1. All animalsgainedweight duringthe study,with theexceptionof one Group 3 female(3.0mg/kg) which exhibitedan insignificanltossin weight(18g)atitsterminatioinnterva(lDay 15). ClinicalObservations IndividualclinicaslignsareinTable 2. All animalsappearednormal throughoutthe study. Pathology Individualanimal tissueweightsand bilevolumes areinTable 3. The animals were not examined grossly,althoughtissueswere saved. DISCUSSION The levelof systemicexposureof T-6684 was evaluatedinmale and female albino rabbitswhen administeredasa singleintravenousinjectioantlevelsof 1.0,3.0,10.0,and 30.0mg/kg. All animalsappearedclinicallnyormal and gainedweight duringthestudy, withtheexceptionofone Group 3 female(3.0mg/kg) which exhibitedan insignificant lossinweightatitsterminatioinnterva(lDay 15). 14 -F34@ t,,), T.Bud W. McDonald Studv Director Acute Studies SIGNATURE CHW 6329-199 /-@- Date t 15 CHW 6329-199 TableI IndividuaBlody Weights(g) Animal -Randomization Initial Sex Number (Day-7) (Day 1) Terminal (Day 15) (Day 29) Male Female F61522 F61523 F61524 F61525 F61542 F61543 F61544 F61545 Male Female F61526 F61527 F61528 F61529 F61546 F61547 F61548 F61549 Group 1 (Control-)DMSO (0.0mg/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 - Group 2 - T-6684 (1.0mg/kg) 2,598 2,437 2,527 2,410 2,688 2,556 2,665 2,551 2,877 2,664 - 2,537 2,544 2,501 2,592 2,691 2,676 2,610 2,719 2,891 2,829 - - Group 3 -T-6684 (3.0mg/kg) 2,941 3,018 2,991 3,174 3,029 2,875 2,850 3,036 Male Female F61530 F61531 F61532 F61533 F61550 F61551 F6]552 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 2,694 2,718 - 2,669 2,573 - 3,181 2,979 2,832 2,880 Not required. Anixnal sacrificeodn Day 15. 16 CHW 6329-199 Table I (Continued) IndividualBody Weights (g) Animal Randomization Initial Sex Number (Day-7) (Day 1) Terminal (Day 15) (Day 29) Group 4 -T-6684 (10.0mg/kg) Male Female F61534 F61535 F6]536 F61537 F61554 F61555 F61556 F61557 Maie Female F61538 F61539 F6]540 F61541 F61558 F61559 F61560 F61561 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 Group 5 - T-6684 (30.0mg/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 2,953 2,708 - 2,574 2,803 - 2,746 2,620 - 2,771 2,643 - 2,818 2,811 2,871 3,002 2,969 2,860 3,093 2,924 Not required. Animal sacrificeodn Day 15. 17 CHW 6329-199 Table 2 IndividualCliniC21 Signs Animal Sex Number Observation Hour (Day 1)" 0.5 2.0 4.0 Days 2-8 9-15 16-29 Group I (Control)- DMSO (0.0mg/kg) Maie Female F61522 F61523 F61524 F61525 F61542 F61543 F6]544 F61545 Appeared Normal Appeared Normal Appeared Normal Appeared Normal Appeared Normal Appeared Normal Appeared Normal Appeared Normal Group 2 - T-6684 (1.0mg/kg) Male Female F61526 F61527 F61528 F61529 F61546 F6]547 F61548 F61549 Appeared Normal Appeared Normal Appeared Normal Appeared Normal Appeared Normal Appeared Normal Appeared Normal Appeared Normal Group 3 - T-6684 (3.0mg/kg) Male F61530 Appeared Normal F61531 Appeared Normal F61532 Appeared Normal F61533 Appeared Normal Female F61550 F6]551 F61552 F61553 Appeared Normal Appeared Normal Appeared Normal Appeared Normal a Each animal appeared normal priortocontrolor testmaterialadministration. Animal sacrificeodn Day 15. Condition existed. 18 Table2 (Continued) IndividuaCllinicaSligns CHW 6329-199 Animal Scx Number Observation Hour (Day I). 0.5 2.0 4.0 Days 2-8 9-15 16-29 Group 4 -T-6684 (10.0mg/kg) Male F61534 Appeared Normal F61535 Appeared Normal F61536 Appeared Normal F61537 Appeared Normal Female F61554 F61555 F61556 F61557 Appeared Normal Appeared Normal Appeared Normal Appeared Normal Group 5 - T-6684 (30.0mglkg) Male F61538 Appeared Normal F61539 Appeared Normal F61540 Appeared Normal F61541 Appeared Normal Female F61558 F61559 F61560 F61561 Appeared Normal Appeared Normal Appeared Normal Appeared Normal a Each animal appeared normal priorto testmaterialadministration. Animal sacrificeodn Day 15. Condition existed. 19 CHW 6329-199 (U E 0 > CU Ed U.tn vi 4J 0 1m4 r_ 0 0 IV ux :i IM CU > E -0 E a0t D 0 03 m Ch r,@w m @ cac* -%0 r@ ,m 0 140 mmm InW0 10 -P In w In In Lwo -InwIwn wwww !WO @OMO "@mLn 0* 0' v- m 0 0'0' m %0 In 000- -W"M mmU, v -"0 00 %D co r- m %o w wwln @@ @w Ir 0 0 Ln -W 0 m %D %D r- a In(h In LAr, r-w C4 w@mw r- In r-" 0 M In M 0w w C4 0 C4 104, 14* %a r-w I 'o4.0.0 ty a0 4.0.0 w @:m --4 In @ r4 m In Irn m 11 'Lwn CY In In m In In tn In w 10 w %D w w -6 4,0.0 w r- In inIn Ln E In In In wwww mmo@ IInn LLnn w %o 0 ww 0000 ID tuw w w w 1. 0000 0 0000 c; 0* 0* cl a aooo a 00 C->00-000000 00@o cn 0. cn im m 0 ca E.3 mv E U@ It z x x4 r4 w w @3 cn i'Z im 41 11 m :3 00 64 x 0) tn Inr- Inr4 ol v w r-Ch0 r, 0 r-Ina, momm - InID@ C4 m ko v r4 r@ w n clir4 Ln LA In w w %a 'M m m In rI4n w %D r4 C4 TEEM o@wo ID @oww @ V InM H m C4@ m (71-W r- %0 ch 0 @W"@ I @m @ 0@ mM MInD n wmvw C, - -In mIn @; %0 w w , In w r- IIn mIn -tn w to 10 xxxx xxrx @v .10 t0 OM-M " - @ In m w a,0 -,n m a, w r: In tn w w %a L. 00 vv In tn In In r@ x x x ww uu cmn m 'a cn .0 20 APPENDIX ProtocolDeviations ProtocolTP6797 ProtocolAmendment No. 1 CHW 6329-199 21 ProtocolDeviations CHW 6329-199 Protocol Page 5,7.Experimental Design,A. Animals, (7)Husbandry, (a) Housing. Individuallyi,nsuspended stainlessteelcages. Page 8, 7.Experimental Design, D. Observation of Animals, (3)Blood Sample Collections,(b) Method of Collection/Number of Animals, first paragraph, firstsentence. Blood samples (approximately4 mL)...and from themarginalearvein(lefetar)at 4-hours postdosethrough Day 8. Page9,7.ExperimentaDlesignE,. Termination (3)Sample Collection. The whole liverb,ile,and both kidneys (collecteadsone sample) from each animalwillbe collected, weighed (volume onlydeterminedfor bile)a,nd immediatelyplacedintoa freezersetto maintaina temperature of -20'C 10'C. Actual Procedure One Group I male was found outside itscage atthetime of thea.m.check on Day 2. Blood samples were collectedatthe followingintervals(postdose)from the rightmarginal ear vein of the followinganimals (animal number/group/sex): 12-and 24-hour:F61524 (IM), F61557 (4F). 48-hourand Day 8: F61524 (IM), F61549 (2F),F61555 (4F),F61557 (4F). Only one kidney forAnimal No. F61531 (Group 3M) was weighed at the timeof tissuecollectionT.he otherkidney was weighed thenext day. These deviationsarenotconsideredtohave had an adverseeffecton theoutcome of the study. 22 L.(irninHgazleton Inc. P.O.B" 7345 Mad.um, W) 53707-7545 Del,twors: 3301 Kins-w. Bit@J. Madison, Wi S3704 608.241.4471 61)9.241.7227 Fax Sponsor. 3M St.Paul,Nfinnesota CHW 6329-199 CORNING Hazleton PROTOCOL TP6797 Study Title: Single-DoscIntravenouPsharmacokincticStudy of T-6684 inRabbits Date: January3, 1997 Performing Laboratory: Coming liazictoInnc. 3301 Kinsman Boulcvard Madison, Wisconsin 53704 l.al)iirato1r'-r%(.ijelcdtcntificati(in: ('IIW 0329-199 23 CI4W 6329-199 STUDY IDENTIFICATION Single-DoseIntravenousPharmacokincticStudy ofT-6694 in Rabbits CHW 6329-1" TP6797 Page2 CHWNO. Test Material Sponsor Sponsoi's Representative Study Director Study Location Proposcd Study Timetable Experimental StartDate Experimental Termination Date Draft Repori 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 Depariment 3M Center,Bldg. 220-2E-02 P.O. Box 33220 St.Paul,MN 55133-3220 (612) 733-3222 F. Bud W. McDonald Coming HazJeton Inc. P.O. Box 7545 Madison, Wl 53707-7545 (608) 242-7901 Coming Hazleton Inc. 3301 Kinsman Boulevard Madison, Wi 53704 Week Week Week ofjanuary 13, 1997 of Febniary 10, 1997 of March 24. 1997 24 CHW 6329-199 CHW 6329@l" TP6797 Page 3 1 . Study Single-Dose Intravenous Pharmacokinctic Study in Rabbits 2. Purpose To assess the levelof systemic exposure when the testmaterialisadministered as a singleintravenous injectionto rabbits 3. Regulatory Compliance This study willbe conducted in accordance with the followingGood Laboratory PracticeRegulations/Standards/Guidelineswith the exception thatanalysisof the testmaterialmixtures forconcentration,solubilityh,omogeneity, and stabilitwyill not be conducted. [ ] Conduct as a Nonregulated Study [X] 21 CFR 58 (FDA) 40 CFR 160 (EPA-FIFRA) 40 CFR 792 (EPA-TSCA) C(g 1)30(Final)(OECD) 59 NohSan No. 3850 (JapaneseMAFF) NotificationNos. 313 and 970 (Japanese MOHW) All procedures in thisprotocolare in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study directort,he study does not unnecessarilyduplicateany previous work. 4. Quality Assurance The protocol,study conduct, and the ftnalreportwillbe audited by the Quality Assurance Unit in accordance with the Wisconsin facilitoyfcoming Hazleton Inc. (CHW) Standard Operating Procedures (SOPS) and policies. 5. Test Material A. Identification T-6684 B. Physical Description (To be documented in the raw data) C. Purity and Stability The Sponsor assumes responsibilityforpurityand stabilitdyeterminations (includingunder testconditions).Samples of testmaterial/vehiclmeixture(s) 25 CHW 6329-199 CHW 6329-199 TP6797 Page 4 for concentration,homogeneity/solubility,and stabilitaynalyses will not be taken before administrationunless requested otherwise by the Sponsor. These samples (iftaken)will be sentto the Sponsor afterexperimentaltermination. D. Storage Room temperature F- Reserve Samples Reserve sample(s) of each batch/lotof testmaterial willbe taken forthisstudy. The testmaterialreservesample(s) will be storedat CHW in a freezersetto maintain a temperature of -20*C IVC and then returned to the Sponsor after completion of the in-lifpehase of the study. F. Retention Any unused testmaterialwillbe returned to the Sponsor aftercompletion of all relatedin-lifetesting. G. Safety Precautions As required by CHW SOPs and policies 6. Control M2teri2i A. Identification Dimethyl Sulfoxide (DMSO); Lot number, source,and expirationdate to be recorded in the raw data and included in the finalreport. B. Ph3'SiC21Description (To be documented in the raw data) C. Purity and Stability To be documented by CHW (informationfrom the supplier) D. Stor2ge Room temperature 26 CHW 6329-199 CHW6329-1" M797 Page 5 E. Reserve Samples Reserve sample(s)of each batch/lotof controlmaterialwfli be taken for this study. The controlmaterialreservesample(s) willbe storedat CHW maintain a temperature of -20*C IOOC. in a freezerset to F. Retention Any remaining controlmaterialmay be used forothertestingand willnot be discardedafterissuance of the finalreporl G. Safety Precautions requiredby CHW SOPs and policies 7. 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 3.5kg (5) Numbcr and Sex 20 maics and 20 females (6) ldcntification Individualnumbered ear tag (7) Husbandry (a) Housing Individually,in suspended stainlesssteelcages 27 CHW 6329-199 CHW 6329-199 T?6797 Page 6 (b) Food A measured amount of LaboratoryRabbitDietHF #5326 (PNU Feeds,lnc.).The food isroutinelaynalyzedby themanufacturer fornutritionaclomponents and environmentalcontaminants. (c) Water Ad libitumfroman automaticsystem.Samplesofthewaterare analyzedfortotaldissolvedsolidss,pecifiemdicmbiological content,selectedelements.heavy metals,organophosphates,and chlorinatehdydrocarbons. (d) Cont2Minants Thereare no known contaminantsinthefoodorwater thatwould interferweiththisstudy. (c) Environment Envirorunentaclontrolsfortheanimalroom willbe setto maintain a temperatureof 19*C to23*C, a relativheumidityof 50*/ot20%, and a 12-hourlight/12-houdrarkcycle.The darkcyclemay be interrupteddue toin-lifperocedures. (f)Acclimation At least7 days (8) SelectionofTest Animals Based on healthand body weight accordingtoCHW SOPS. An adequate number ofextraanimalswillbe purchasedso thatno animalinobviously poor healthisplacedon test.The animalswillbe placedintostudy groupsusinga stratifibeoddy weightrandomizationprogram withinnine days ofstudyinitiation. (9) JUStirIC2tiOfnorSpeciesSelection Historicalltyh,eNew Zealand White albinorabbithasbeentheanimalof choicebecauseof thelargeamount of backgroundinformationon this species. 28 CHW 6329-199 B. Dose Administration (1) Test Groups CHW6329-1" 'rP6797 Page 7 Group I (Control) Control/Test Material Sterile Water 2 T-6684 3 T-6684 4 T-6684 5 T-6684 Dose Level (mg/kg)* Number of Animal Male Female 0.0 4 4 1.0 4 4 3.0 4 4 10.0 4 4 30.0 4 4 a Administered at a dose volume of 0.5 mL/kg. b Two animalstsex/doselevelwillbe sacrificcdon Day 15. The remaining animals (two animalsisex/dosclevel)will be sacfificedon Day 29. C. Dosing Procedures (1) Dosing Route Intravenousinjectionintothe fightmarginal car vein over approximately 30 to 60 seconds. (2) Reason for Dosing Route lntravcnous injectionisan acceptable route to assesssystemic cxposure. (3) Dosing Dur2tion Single dose (4) DoscPrep2r2tion The day of treatment willbe designated as Day 1. Individualdoses will he calculatedbased on the animal'sbody weight taken on Day 1. The Ciroup I animals willbe treatedwith DMSO ata dose volume of 0.5 mLAg. The tcstmaterialwillbe dilutcdwith DMSO toachieve a spt:cificconcentration foreach dose levelinGroups 2-5 and administered 29 CHW 6329-199 CHW6329-1" TP6797 Page 8 ata dose volume of 0.5 mL/kg. The prepared testmixtures willbe stored atroom temperature untiladministered. D. Observation of Animals (1) Clinical Obser-vations Before testor controlmaterialadministration,atapproximately 0.5,2.0, and 4.0 hours post-injectio(nDay 1) forclinicalsigns,dailythereafterfor clinicalsigns,and twice daily(am. and p.m.) formortalityuntilthe scheduled sacrificeinterval(Day 15 or Day 29). The animals sacrificed on Days 15 or 29 willbe observed only once for mortalityon those respectivedays. Observations may be extended when directedby the study director. (2) Body Weights For randomization,before testor controlmaterialinjection(Day 1).at the scheduled sacrificeinterval(Day 15 or Day 29),or atunscheduled death and sacrifice(swhen survivalexceeds I day) (3) Blood Sample Collections (2) Frequency Pre-injection(anytime from up to four days before testmaterial administrationto Day 1),atapproximately 4-,8-.12-,24-, and 48hours post-injectiono,n Days 8, 15, 22, and at the scheduled sacrificeinterval(Day 15 or Day 29) (b) Method of Collection/Number of Animals Blood samples (approximately4 mL) willbe collectedfrom the marginal ear vein (eitherear)of allanimals on the day before test or controlmaterialinjectiona,nd from the marginal ear veiii(left ear)at 4-hours postdose through Day 8. On Days 15 and 22. additionalblood samples (approximately 4 mL) willbe collected from themarginal ear vein (leftcar ifpossible)of the animals scheduled forsacrificeon Day 29. From (lieposteriorvena r-ava,approximately 20 mL of blood will be obtained from each animal sacrificedin a mofibund condition (ifpossible)a.pproximately 20 to 40 mL of blood will be obtained fronieach controlanimal sacrificedon Days 15 or 29 (the maximum volume possiblewillbe obtained),and approximately 30 CffW 6329-199 CHW 6329-199 TP67gr7 Page 9 20 mL ofbloodwillbeobtainedfrom eachGroup 2-5animal sacrificeodn Days 15 or 29. The sampleswillbe storedatroom temperatureand then centrifugeadn,d theseparatseerumand cellulafrractionsstoredin a free= settomaintaina temperaturoef-200C IOOC. The senan and cellulafractionosbtainedthroughDay IS wfllbe sentfro=n on dryicetotheSponsorone totwo weeks priortoin-life terminationT.he senimand cellulafrractionosbtainedafterDay 15willbesentfrozenondryicetotheSponsorwithinone week afterin-lifterminationT.he Sponsorisresponsiblfeorthe retentioannd dispositionf thesamples. The senunand cellulafrractiosnampleswillbe shippedto: James D. Johnson 3M E.T.& S Bldg.2-3E-09 935 Bush Avenue St.Paul,N4N 55106 James D. Johnsonorhisalternatweillbenotifierdegardingthe shipmentofthesamples. E. Termination (1) UnscheduledSacrificeasnd Deaths Any animaldyingduringthestudyorsacrificeidna moribundcondition willbe subjectetdoan abbreviategdrossnecropsyexaminationand all abnormalitiewsillberecorded.Animalsina moribundconditiownillbe anesthetizewdithsodiumpcniobarbit(avliainjectioinntiimcarginalear vein)b,ledviathevenacava,andexsanguinatedT.issues,asdescribed insectio7n.E.(3S)ampleCollectiowni,llbe collectefdromany animal dyingduringthestudyorsacrificienda moribundconditionA.fter necropsy,theanimalswillbe discarded. (2) ScheduledSacririces On Day 15,thefirsttwo animals/seaxssignedtoeachdoselevel(based on thegroupassignmenrtandomizationw)illbe ancsthetizcwdith sodium pcntobarbit(avliainjectiionthemarginalcarvcin)b,ledviathe venacava,and exsanguinatedT.he remainingtwo animaWsex/dosc 31 CHW 6329-199 CHW6329-1" TP6797 Page 10 levelwillbe anesthetizedwith sodium pentobarbital(viainjectionin the marginal ear vein),bled via the vena cava, and exsanguinated on Day 29. An abbreviated grossnecropsy examination willnot be done, however, tissues[asdescribed in section7.E.(3)Sample Collection)will be collected. (3) S2mple Collection T'hewhole liver,bile,and both kidneys (collectedas one sample) from each animal will be collected,weighed (voltuneonly determined for bile),and immediately placed intoa five= setto maintain a temperature of-20*C 10*C. Aftersample collectiont,he animals willbe discarded. The samples (liver,bile,and kidneys)will be sentfrozen on dry iceto the Sponsor within one week aftercollection.T"nesamples and their corresponding weights or volumes willbe shipped to the person listedin Section 7.D.(3)(b).The Sponsor isresponsibleforthe retentionand dispositionofthe samples. F. StatisticaAlnalysts No statisticaalnalyses are required. 8. Report A finalreportincludingthose items listedbelow willbe submitted. Description of the testand control materials Description of the testsystem Procedures Dates ofcxpcrimental initiatioannd termination Description of any toxiceffects Gross pathology findings(ifapplicable) Gross pathology report(ifapplicableand requestedby the study director) Individualanimal tissueweights and bilevolumes 32 CHW 6329-199 CHW 6329-1" TP6797 Page 11 9. LOC2tion of R2w D2t2, Reserve Sample(s)R,ecordsa,nd Fin2l Report Original data, or copies thereof, will be available at CHW to facili= auditing the study during itsprogress and before acceptance of the finalreport. When the fmal reportiscompleted, controlmaterialreservesmple(s), alloriginalpaper data, including thoseitems listedbelow will be retainedin the archives of CHW for a period of one year followingsigriingof the fmal report.One year aftersigning of the finalreport,allofthe aforementioned materialswillbe sent to the Sponsor and a return feewillbe charged. The Sponsor may electto have the materials retainedin the CHW Archives foran additionalperiod oftime and CHW will charge a storage fee. IftheSponsor chooses to have CHW disposeofthe materials,a disposal fee willbe charged. Protocol and protocolamendments Dose preparationrecords In-liferecords Body weights Dose administration Observations Sample collectionrecords Shipping records Pathology Records Study correspondence Finalreport(originalsigned copy) The followingsupportingrecords willbe retainedat CHW with the studydata. but willnot be archived Animal reccipt/acclimatiornecords Water analysisrecords Aiiimal room temperature and humidity records Refrigeratorand freezertemperature records liistrumentcalibrationand maintenance records 33 CHW 6329-199 PROTOCOL APPROVAL CHW 6329-199 TNM Page 12 Roger jrkinsP,hD,D T Sponsor's Rcpmscntative 3M &117 Dft F. Bud W. McDonald Study Director Acute Studies Coming HazletIonc. ZE4 4@4 Represen(ative Quality Assurance Unit Coming Hazleton Inc. Date 1.3-'77 Date 34 CHW 6329-199 C 0 V Alg-C-E--"' THR CKVGLOMEXT SINVICIES COMP@ AMENDMENT NO. I TO THE PROTOCOL PROTOCOL 7?6797 Comnce Laboratorieskw- P.O. Sol 7545 Mgdhm% POCkSo"; Madison. WiSC"Sin 53707-7S4S 3301 Kinsfftan 13"ovard Wisconsin 53704 701:609/241-4471 Fax: 404/241-7=7 Single-DOseIntravenousPhumacokinefic Study of T-6694 inRabbits CFFW 6329-199 Sponsor 3M ToxicologyService Medical Department 3M Center,Bldg.220-2E-02 P.O.Box 33220 St.Paul,MN 55133-3220 TestingFacility Coming HazletonInc. 3301 Kinsman Boulevard Madison,WI 53704 Sponsor's Representative Roger G. Perkins,PhD, DABT Study Director F.Bud W. McDonald This amendment modifiesthefollowingportionosf theprotocol: EffectiveJanuary 3,1997 1 Page 7,7.Experiment2iDesign,B. Dose Administr2tion(,1)Test Groups. To correctlyidentiftyhecontrolmaterialtobe usedinthisstudy,which was listed incorrectliynthe tableatthetimetheprotocolwas issuedby thestudydirector, replacestcrilWeater %%ithDMSO. THE AMERICA$ EU*OPE ASWP=FPC 35 AFITICA CHW 6329-199 Ammdmcnt No. I CHW 6329-199 TP6797 Page 2 EffectiveJ2UU2ry 14,1"7 2. Page 5,7.Experiment2i Design, A. Animah, (4)Weight at Initiation.To accommodate theuse of lightearnimalsavailableforuse inthestudy,modify the body weight rangewiththe followingunderlinedchange: 2j to3.5kg PROTOCOL AMENDMENT APPROVAL @L, Roger G.Pix@)is,PhD, DABT Sponsor Representative 3M C26 rc--g5:7 Date F.Bud W. McDonald Study Director Acute Studies Coming HazletonInc. 19FE8q-7 Date Reoresentative QualityAssurance Unit Coming HazJetonInc. lr(Ff-@/??7 Date 36