Document YG75Q3mv54NDBmgjqGJvoepBO

CLIENT PRIVATE PROTOCOL TITLE: Range findingCytotoxicitAyssay of T-6292,T-6293,T-6294. T-6295 inRat Hepatocytes SRI Study No. BOIO-95 RESEARCH CLIENT 3M Medical Departinent Medicine,HealthPhysics,IndustriaHlygiene,Toxicology 3M Center Building220-2E-02 St.Paul,MN 55144-1000 Study Monitor: StevenC. Gordon, Ph.D.,D.A.B.T. TESTING LABORATORY SRI International ToxicologyLaboratory 333 Ravenswood Avenue Menlo Park,CA 94025 Study Director: CarolE. Green,Ph.D.,D.A.B.T. Telephone:(415)859-4083 FAX: (415)859-2889 APPROVALS: @@@ @? Research Client'sAuthorizedRepresentative -F SRI Studybirector v. May 3, 1995 Date SY Date CLEENT PRIVATE STUDY OBJECTIVE The objectiveof thisstudy istoevaluatethecytotoxicitoyf 4 differentestarticlesas measured by MTT conversion. The ECio and ECIO valueswillbe estimatedfor each test articleand thesedatawillbe used toselecta suitableconcentrationof each testarticlefor subsequentin vitrometabolism studies. Il. APPROACH Hepatocyteswillbe isolatedfrom adultmale Sprague-Dawley rats.The cellswillbe allowed to attachinmonolayer culturein thewellsof a 96 weR plate.The ceus willthen be exposed to eithertestarticles,olvent,or positivecontrolin4 replicatceultures.In this experiment,thecompounds willbe testedin 6 differenctoncentrationsata singletime point (24 hours). -Theconcentrationfsoreach testarticlaeredescribedbelow intheExperimental Design. M. TEST ARTICLE A. Test ArticleIdentification Name: T-6292 Lot No.: To be providedby ResearchClient Molecular weight: 571.2 Name: T-6293 Lot No.: To be providedby Research Client Molecular weight:658.28 Name: T-6294 Lot No.: To be providedby ResearchClient Molecular weight:527.2 2 CLEENT PRRVATE Name: T-6295 Lot No.: To be providedby ResearchClient Molecular weight:538.1 B. Purityand Stability Documentationof theidentitys,trengthp,urity,and stabiliotfy thetestarticlewsillbe the responsibiliotfytheResearchClient.The positivceontrola,cetaminophen,and the solvent control,DMSO, willbe obtainedfrom Sigma ChemicalCo. (St.Louis)and their documentationofthe identitaynd purityofthesechemicalswillbe used. C. Handling and Storage On receiptt,hetestarticlewsillbe placedina secondarycontainer(lightprooiff required)and storedas recommended by theResearchClient.A MaterialSafetyData Sheet (MSDS) or othercomparabledocument specitdngany hazardsand identifyinfgirstaidand clean-upproceduresin caseof an accidentaslpillsha.Ube providedby theResearch Clientand shallaccompany thetestarticlesT.he testarticlewsillbe logged inand storedinBuildingL, Room LB286. D. Dispositioonf theTestArticle Any remainingunused portionofthetestarticlewsillbe returnedtotheResearch Clientaftercompletionof thestudy. IV. TEST SYSTEM A. Background and Justificatiofnor Selectionof the Test System The liveristhemajor siteofmetabolismofmost organicchemicals,both endogenous and foreign.Species-relateddifferenceisnthemetabolismof xenobioticasrewellknown and have been documented to be correlatetdothe toxiceffectsof chemicalson certainspecies (Caldwell,1980;Calabrese,1983).The proposedcolorimetriccytotoxicitayssayhas been used todeterminemammalian cellsurvivaland proliferatio(nNlosrnann,1983;Otoguro etal., 1991). This assaydetectslivingb,ut notdead cells.Because thesignalgeneratedis 3 CLEENT PRIVATE dependenton the degreeofactivaitoonf thecellsc,ytotoxicitpyr,oliferatioann,d activation can allbe measured. This assaydependson thefactthatMTT (which isyellowin aqueous solutionatneutralpH) ismetabolizedby mitochondriaof livingcellstoa blueformazandye. When used as a cytotoxicitayssay,spectrophotometriqcuantitatioonf theformazan generated by ceus aftertheirincubationwiththetestarticlweillgivean indicatioonfthenumber and metabolicactivitoyf the survivingcells. B. Test Species Rat. Adult male Sprague-Dawleyratswillbe used forthepreparatioonfhepatocytes. They willbe purchasedfrom CharlesRiverLaboratoriesand willweigh approximately200250 g atthetime of purchase. The ratstobe used forthisstudywillbe housedinBuildingL, where thecellisolation willbe performed. Ratswillbe takenfrom theshippingcontainersw,eighed,examined for generalhealth,and placedthreeper cage in 22 x 12'h x 8-inchsuspendedpolycarbonatecages labeledwith theirquarantinenumber assignments.Animals willbe quarantinedinthe same room inwhich theywillbe housed. Rats willbe fed (ad libitumP)urinaCertifieRdodent Chow (#5002)and thefeedsupplierwillprovideSRI withan analyticarleportidentifyintghe diettobe freeof contaminantsforeach lotoffeedreceived.A copy ofeach analyticarleport willbe availablaes partofthestudyrecord.Ratswillreceivepurified(deionizedand UVtreatedd)rinkingwaterad libitum.Temperaturewillbe maintainedat72 40F and relative humiditymaintainedbetween 35 and 65 %. A lightcycleof 12 hours lightand 12 hours dark willbe maintained,withlightstartinagt06:00. The laboratoryspecieswillbe quarantinedforatleast3 days beforeuse in an experiment.The LaboratoryAnimal MedicineDepartmentatSRI willissuedocumentationto the Study Directoron thehealthof animalsduringthequarantineperiod.Beforeisolatioonf hepatocytes,theratswillbe anesthetizewdith sodium pentobarbita(l65 mg/kg). V. EXPEREWENTAL DESIGN A. Hepatocyte Isolation Rat hepatocyteswillbe isolatebdy eitherwhole liveror biopsyperfusionasconvenient (Greenetal.,1983). In brieft,hewhole liveror wedges oftheorganwillbe perfusedfirst with a Ca'2_freebuffer,followedby a buffercontainingcollagenase.Hepatocyteswillbe combed freefrom thedigestedtissueand purifiedby simpledifferenticaelntrifugation. 4 CLIENT PRIVATE Isolatedhepatocyteswillbe platedonto collagen-coatecdulturedishes(35 diameter)ina modifiedWaymouth's 752/1culturemedium (CMHla) thatcontains11.2Ag/ml alanine,12.8 Ag/nilserine,24.0 Aglml asparagine,84.0 Ag/nilgentamicinsulfate0,.168 Aglml aminolevuliniaccid,5.0pig/rnolleicacid,5.0lAg/mllinoleiaccid,1.0,ug/mlD,Ltocopherol2,88 ng/ml testosterone2,72 ng/ml estradiol3,93 ng/mi dexamethasone,7.9 jig/mi thyroxin,30 ng/ml glucagon,0.02U/ml insulin0,.1% ITS (CollaborativResearch,Inc.; finalconcentrations5:Aglml transfenin5, lAg/mlinsulina,nd 5 ng/ml selenium)0,.2 mM Lascorbicacid2-phosphate,and 0.2% BSA. The initiaplatingmedium willcontain10% fetal bovineserum. After2 to3 hr ofincubationat37"C inan atmosphereof 95% air:5%C02, themedium willbe aspiratetdoremove nonviable,unattachedcellsand replacedwith CMHla containingthe testarticle. B. -Range Finding CytotoidcityExperhnent (MCIT Assay) 1. Preparationand Administrationof Test Articlesand Controls. Concurrent solventcontrolswillbe examined intheexperiment.Solventcontrolsampleswillconsistof culturemedium and DMSO atthe same concentratiounsed to dissolvethetestarticles. DMSO willbe used so thatthefinalconcentratiodnoes not exceed 10 lallml(1%). Acetaminophen (10mK willbe thepositivecontroland willbe preparedasa stocksolution inculturemedium. Immediatelybeforetheexperiment,an aliquotofeachtestarticl(esolidor pure liquid) willbe dilutedinDMSO toform a concentratesdtocksolution.Serialdilutionwsillbe made from theseinitiasltocks. 2. Test ArticleConcentrations.Six testarticlceoncentrationwsillbe studied: 0.01,0.05,0.10,0.50, 1.0,5.0,10.0mm. 3. Incubationof Hepatocyteswith Test Article.Four replicatmeonolayer culturesof isolaterdathepatocytewsillbe incubatedat37*C for24 hours with thetestarticle or control.A negativecontrol(nocellsw)illalsobe measuredfordetemiinatioonf background staining. 4. NM Assay. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-tdeitprhaeznoyll* bromide] assaywillbe performedasdescribedby Scudieroetal.(1988)asmodifiedby this laborator(yOtoguroetal.,1990).U= (Sigma)willbe dissolveadt5 mg/ml incalciumand magnesium freephosphatebufferedsaline(CMF-PBS) and sterile-filtewrietdha 0.2 Am filter unit.The MTT solutionwillbe diluted1:5inCNMla, and 150 Al willbe added toeach of the24 wellsinthedish.Aftera 2 hour incubationat37*C, theincubatiomnedium willbe 5 CLIENT PRIVATE aspirateadnd 450 glof DMSO willbe addedto solubilizteheMTT-formazan product.After mixing,theabsorbanceat 540 mn willbe measured. 5. Data Collection.The netchange (%) relativteothe solventcontrolwillbe plottedagainstthechemicalconcentration.The EC50 valueswillbe determinedby a computer statisticparlogram designedforsimultaneousfittinogf familiesof sigmoidaldose response curvesusingthefour-parametelrogistiecquation(De Lean etal.,1988). VI. REPORTS A summary reportcontainintghe resultosfthisexperimentwillbe issuedtothe Study Monitor. The testarticlceoncentrationfsorthemetabolismstudywillbe selectebdased on theseresults; IX. RECORDS TO BE MAINTAINED The laboratorynotebooks,allraw data,relevantcommunications,and theoriginalcopy of theFinalReportwillbe transferretdotheSRI Records Center,BuildingB, aftercompletion of thestudy. These materialswillbe maintainedfor 10 years;thentheResearchClientwillbe contactedconcerningfuturedispositioonf therecords. X. REFERENCES Calabrese,E. J. John Wiley & Sons,New York, pp.203-282,1983. Caldwell,J. InEnzymaticBasisofDetoxicationV,ol.I. WilliamB. Jakoby (Ed.),Academic Press,New York, pp. 85-114,1980. DeLean, A.,P.J.Munson, and D. Rodbard. 1978. Am. J PhysioL235,E97-ElO2. Green,C. E.,J.E. Dabbs, and C. A. Tyson. Anal.Biochem. 129,269-276,1983. Mosman, T. J.Immunological.Meth. 65,55-63,1983. Otoguro,K., K. Komiyama, S. Omura, and C.A. Tyson. ATLA 19,356-360(1991). 6 415B592BB9 r)c,2;e;.V395 12:28PM SRI TOX LAB FAX 4 415 859 2Se9 NO.939 P.i FACSEMME lItANSNHSSION Message to: Dr. SteveGordon Company: 3M FAX Number: 612/733-1773 Date: October26, 1995 RE-. Cytotoxicitayssay Number ofPages: 5 From: CarolGrem DirectPhone: (415)8994M SRI Toximlogy Laboratory 333 Ravenswood Avenue Menlo Plark,C)A. 94025 FAX (415)859-2899 (4LS)326-SS12(alt) CC-. V. Weizer C. Tyson Enclosedarecopiesoftbadatafrom theMTT assay.You willalsofindso= notes preparedby thetecbniciaonn thesolubilitoyf thecompounds intheme&um. After reviewingtheraw data(absorbanrevalues)forT-6292,I fo=d thatthecontrols forthatplatewere low. As a reolt,Ithinktheassayforthatchemicalmay need to be repeated.Based on the appearanr-oefft cellstreatedwith T-6292, severetioxicity occurredbetween 0.1 and 0.5 mM. Pleasereview theremlm and uR me so we can discussthisftnt=. OCT.26.i9% 12:29PM SRI TOX LAB FAX 415 859 2889 Project7585 Rat MTT Assay NO.939 P.2 Project75e5 MTT As!R usingRat hepatocytes I I I Hapatocytes Isolatedon 10/16/95. Cellse)Cposedtotestaruciesfor24 hours. MTT assay performed on 10/17/95. At the 24 hour time pointthe media containingtestardelewas aspiraw from the plon and replacedwithMTT assay soluton.1 KITT Assay soluton was incubatedon thecellsfor2 hr I MTT assay solutiownas mmoved after2 hrand the cellswere lysedvvithDMSO - releasingintothe DMSO the Formazen Blue Dye. I I I 7he plateswere read on a MolecularDevices lcneficPlatereaderat540nm. I . Data calcu ons were performed on the S a x o;pme 9 pyogra fbrthe p6te reader. T-6295 (mM) il;@6-vir 0.01 0.05, 0.1 0.5 1.o 6.0 10.0, T-6294 (MM) 0.011_ 0.05 0.1 0.5 1.0 5.0, 10.0 % ofcontrolcalls 127.3 127.3 113.8 8.318 8.798 6.13 14.68 SD 19.6824.42 18.92 3.492 .5.443 5.184 17-89 % ofcontrolcalls 104.7 92.73 48.43 5.366 4.983 6.54 .807 SD 8.301 15.2731 2.411 1.689 3.894 0.919 0.883 Page 1 OCT.26.1995 12:29pm SRI TOX LAB FAX 415 859 2W9 Projed 7565 Rat MTT Assay T-6293 (MM) 0.01 0.05 0.1 0.5 l.o. 6.01 io-oj_ % ofcontrolcells 107.4 95-22 103.5 1C)9.8 121.6 36.71 2.969 SD 23.81 19.58 17.53 24.2 32-13 14.49 1.6861- T-6292 (MM) 0.011 0.051 0.11 0.51 1.0 5.0 10.0 % ofcontroloefls i4-9.-8 128.1 40A 4.r@% 25-99 17.31 227.1 13.59 SD 62.49 36.64 52.7 17.9 4,426 60.39 4.414 APAP (MM) Positivecontrol % ofcontroloells- I 36.'461 SD 8.027 NO. 93c3 P. 3 Page 2 OCT. 29. 19% 12:29PIl SRI TOX LPIB FAX 0 415 859 2BB9 NO. 939 P. 4 160- 140 -Izo10080 - co 0 CL 60 40200 0.001 Cytotoxicityto Rat Hepatocytes T-6295 T-6294 -'T-6293 0.01 0.1 1 10 100 Concentrati(omnM) OCT.26.1995 12:30PM SRI TOX LAB FAX # 41S eM-2BE39 NO.939 P. S Notesotisolubilftoyfcompound duringl@M wq=iment ofOr-t16-17. T-6292 Visiblyprecipitateodut at10,5,and I mM concentmtions T-6293 Not allsolubiuzedat 10,S,and I MM con=Uadons intheDMSO added CMHI a and afteragitadonappemd tohave gone intosoludon. T-6294 Visiblyprecipitateodutat10 and 5 mM concat&ations.No visiblpemipiblteat 1 mM concentration. T-6295 Visiblyprecipitateodutatthe 10 mM conceutmdoll No visiblperecipitataetthe 5 and ImM concentmdon, Notes on appearanceofcallsaftermcubationwithtestarticlefsbr4roxim8ftly 22 houcs. T-6292 Pows 3-5 some cellsspreadolitsome cellsroundc& Rows 6-9very few cellsspreadoutmost rounded. Rc)w 9 crystalpsresmt darkcoloredmedi& 0.6%)o.or,o. t ftK T-6293 Rows 3-6no visiblaebno=alities 0.c I Rows 7-10 have a lotof debris,ther,eUsarerounded orunattachc& i-o T-o C..* %Q.0 T-6294 Rows 3-5no visibleabnormalities. 0. 0 1 - 0 .1 Rows 6-9ceusnotspreadoli4munded, 0,3,. T-6295 APAP Rows 3-5 cellsmosdy spreadoutwith some rounded. Rows 6-7cellsnot spreadoutwithsome debris. Rows 8-9a lotof debris,theoellswen rounde& -Coo ot,p, r-chP,-^ rows 2-3 no visibleabnormalities. Rows 4-6 few to no spreadout calls.Aff@r APPearmee of T629:5after2 hour incubationwithl@M Wells 8-9 had a heavy yellow pxecipitate. Well 7 had a medium yellowprecipitate. Well 6 bad a lightyellow p=ipitate. Wells 1-5 no visibleprecipitate. solution Tlm otber Plateshad no remarkable abuormalities. For plateT-6293 one fow of wellswas repeated. When read by plate'readearzd by visible observafionof colorchange mws 8 aad 9 werr.5.0 mM concmtration. Tids corresponds with an interruptiodnudng the applicationof testarticleasnd time I noticedthe e=. vlw 10/24/95 DE'7.iz.IC395 12:59pm SRI TOX LAB FPX 0 415 859 2BB9 NO. E934 P.1 SRI INTERNATIONAL Toxicology Laboratory 333 Ravenswood Avenue, Bldg 205 Menlo Park,CA 94025-3493 Fax No. (415)859-2889 Altemate Fax No. (415)859-3342 FacsimileTransmittal From: ral DirectPhone: %C Message To: Company: K sjrcwq- Fax Number GI-L I . Date: December 12, 1995 This Message Transmitted from: Fax No. (416)869-2889 3 Number ofPages: A includingthistransmittaslheet MESSAGE: -i@-0, TL--VT ( ryN JA Pleasecaltlhesenderair-a*Ifybuhaveanyprob4cmvathth ptionofthismessage. DEC. 12. 1995 12: 59PM SRI TOX LAB FAX 9 415 859 998i) Project7665 Rat MTT Aawy Project7565 MTT Assay-usingRat hapatocytes I I I HepatocytesIsolateodn 12/M5. 1 Cellsexposed totestarficlefsbr24 hours. M7T assay performed on 1217/95. At the24 hour tme pointthemedia containintgestarficlweas aspiraw from the plateand replacedwfthMTT assay soludon.1 MTT Away solutiownas tnrubaftdon thecallsfor2 hr I MTT assay solutiownas removed aftr 2 hrand thecellswere lysedwithDMSO rewsing IntotheDMSO theFormazan BlueDye. I I I The pwtes were read on a MolecularDevi= YJneboPlatereaderat54onm. Data calculatonwse perfbrmedon theS ax ope na progmm forthep reader. TZi293 (mM) 0.05 0.1 0.51 1.0 6.0 10.0 % of controloelle 84.01 104.7 108.2 45.28 8.739 55.98 97.4 SD 4.495 23.85 14.-95 15.69 1.4401 16.62 10.36 T-6292 '(MM) FC--199 Yoofcontrolcefle so 0.01 0.051 0.1 0.5 1.0 5.0 10.0 106.1 24.86 110.0 41.77 sq.. 33.77 7.882 2.914 EC@p 9.208 2.199 24.44 9.603, 93.92 23.96 O-ot#5'@-ft e) 13'7 Ot APAP Positivecontrol 10 % ofcontrolcells 1 32.341 SD 12.80 Pagel DEC.i2.1995 - l'OOPM- SRI TOX LAB FAX 415 BS9 2989 . NO. E04 P.;i N'D"P0-"ssolub'UtOyfr-OMPO=ddurinMgrr mpaiment ofDw.6-7 T-6292 Compound was notallsolubffizeadt10,5,lmd ImM concentadons. Ihe I MM co=entmdoia appearedto be themost solubirzedintheDNSO and CMRL a and afteragitadonappearedtohave gone intosolutiam. T-6293 Compound was notallsolubui=d at10,5,and Imm i:oncenftadons'.Me I mM concentmtionappemd tobe theinostsolubm=d intbeDMO and CMH Ia md aft NOtafionappeawd tohave gone intosoludm sonic4@ 10,5 and ImM concenbmgonsfor10 mimitesaftearddidwof CMHla toincrem solubft. Notes on appearanceofcellsafterincubationwithteg atfloicfsorqppzoxb@y 22 houm. T-6292 Rows 8-9 Some cellsspreadout sam C*M munded. V-ui-blcerystalosf compound- (5,10 n2K Rows 6-7 Few tono ceu-cellattachmentsmost allcellsappear vL4ble Crystals ofcompound. (1.0,0.5mM) - Row 5 Some flattenecdellswith cell-ceauuwbment (0.1m&j@ Row 4 Mo= cell-ccauttaehment.Few tono crystalsS.ome rouadedcells.(0.05 Row 3 Cellsslightlbyetterthanrow 4. (0.01m&o Row 2 A coupleofroundedceusodmvvfm goo& (Normal macha) T-6293 APAP Row 9 CoM visiblmeany roundedwithsome spreadhavingcell-ceajnlwhment (10.0MNP Rows 6-8 CeUs notvisibldeue tograinydebris.(5.0,1.0,0.5mAi) Row 5 Very grainycellsrounded.(0.1 n2K Row 4 Some cellsareclumped togoffiagrowing on top ofeachother,gminy debrispresent(0.05mNI) Row 3 Good withgminy appearm=. (0.01mK Row 2 A couple of rdunded cellsotbarwisegood. (Nonnal media) rows2-5no visiblaebnormaudes.(Normalnumfla) Rows 6-9few tono spreadoutcells.I(OmM APAP) vlw 1217/95 160 140 120 r0: 100 - 80 41 c so - 40 - 20 0 0.01 IV ,%A 160 - 140 2 120 100 so so 40 20 0 0.01 E@14 FC-95 (glass) ECIO @ 0.14 mmMM EC, = 0 2,5MM ECC5'0 = 00.26 mM 0.1 1 10 Concentration (mM) 160 140 - 120" 00 100' 80 c 6() 40 % r: 20 ECIO @ 1.83mM (glass),0.15mM (sil.-poly) ECSO c 3.97 mM (glass)0,.44 mM (sH.-poly) 0.01 0.1 Concentralon (mM) FX-12 (glass) EC,, = 0.05mM ECSO = 0.09 mM AAAL- 0.1 1 10 Concentration (mM) 160 - 140 120 100 its 0 8wo 60 40 20 0 0I.L01 @@EC_ ECIO x 0.06 m EC50 = 000.'11.044 m 0.1 1 Concentration (mM) jpM.16.i996 41 6*1 SRI TO>< LAB FPX 415 859 2BB9 NO. 2M P. I FACSE%= TRANSAUSSION Nressageto: Dr. SteveGordon Company: 3M FAX Number: 612/733-1773 Date.-Jamiary 16, 1996 RE: Rat bepatocytestudy Nmnbex ofPages-. 3 ]From CarolGrftu -Dwed Phone.-(415)859-4083 SRI nternatio1w To3deoloa Laboratoz7 333 RavmswoRd Aveilue Mago Park, CA 94025 FAX (419)89.2889 (4M326-Ml2(ah) CC: V. Weizer C. Tyson F=Iosed am copws offt datafrom theLDH assaythatwe performedon rat bepaxytes =ubated withft testarticleIsbBowing ttipcrotocolforthemetilboli= study.We used 100 izM ofeach as discussa No signif=m toxicitiysevident 4@@hich &d inmen LDII relme. I suggesthatwe sendtheodw s=plra from6292,6293,and 6295 to ABS foranalysisD.o you agme? We can discusstheseresultsafteryou have a chance toreview thenl Iwould also Uke todiscusstheprojectbudgetbecauseft LDH assaywas notincludedinft originaplrotoroland we now may need torepentdc in=bationfor6294 metaboliam I willtalktoyou soon abouttheseissm. BOII-96 Rat I LDH 1112196 Ille/gsi IVLW LDH a&saylbr75651801 I-Rl lnctibatlopnerfbrmedon 1111/98 SampJes stdred0-5*C untilanalysis LDH spoked on Gameni on 1112/96 Cob In35 mm chshwere scrapedintd1.0 rTd ofPBS afterwashing withPOS Cob were d&ftdt"ed Wih 1.0% bttdnX-100 InPBS 1.0rw I I I I I Cal earnpks wqre kMwS"eo _ "ce- 2X = 100 pi cd nfix+ 100 14 PBSI 6294 c + d, 6293 c. ax n 100 @l cell mix + 200 tA PBS nouna 6292 c + d, 6293 d, 6295 r.+ d I- - I Mecb saffon were aspMW I I --F -- kdo microfuge tuba and stxed urAM an*sis at O-WC Nommi cab wwo trcdbaw wfth 2 ffd of rnedlum Testoffidewserekmbobd withI rd ofrneftm Mediasampleswerebtw dkdw as necxmnry 2X = 100 iAmodkim + 100 PIPBS 16294c+d remainingsinIIPIW notdilutednoTnuM cds were In2 M ofnwdia so 2X Caluculagons: FinalLOH C = dlutonC X LDH/Cell FinalLDH M = dliudwM X LDWnmdia T otal= FinalLDH C + FinalLDH M % releasedintdmadla = FinalLDH M ITotal (expressed as %) 4e As I SAMPLE normal a normal a normal b normal b LDH/CELL dilutionC fmalLOH 384 6 360 61 438 466 6 C 2304 2160 2628 2736 LDHIMEDIA 207 211 242 248 DlufionM rinaLlDH 2 2 2 2 M TotW % releasedInto 414 2718 422 2582 484 3112 496 3232 Average Page 1 SAMPLE 6292 c 6202 c 62 6292 d 62 66[2292 d 0293 c 6293 c 6293 d 6293 d emc 6294 c 6294 d 18294 d 6295 c W95 c 6295 d 6295 d 0011-96RatILDH 1/12196 LDHICELL dlulon C finaLlDH C LDH/MEDIA Dludon M finaLlDH M Total releasedIntom 779 a 4874 Soo 1 808 5656 1 745 6 4470 sis 1 815 5285 1 749 a 4494 717 1 717 6211, 1 7601 a 4600 732 1 732 5232 1 Average 1 909 4 3636 969 41 3876 027 el 3762 649 a 3894 484 1 509 1 552 1 560 1 484 4120 1 609 4385 1 652 4314 1 660 4454 1 Average I 712 4 2848 722 41 2M 750 4 3040 763 4 3052 722 2 704 2 5w 2 544 2 1444 4292 3 1408 4296 1084 4104 2 ioaa 4140 Average 451 6 2705 459 6 2754 709 6 4254 653 6 3918 1 547 1 574 1 286 1 284 1 647 3253 1 674 3328 1 286 4640, 284 4202 Average 1 Page 2