Document Er9QX8DLOnpXmLeKLvneJ1B4

CLIENT PRIVATE AR21G-0 162. PROTOCOL TITLE: bMeytRaabtolainsdmoHfumT-a6n29H2e,paTt-o6c2y9t3e,sT-6294, and T-6295 SRI Study No. BO11-95 RESEARCH CLIENT 3M Medical Department Toxicology Services 3M Center SBtu.ilPdaiunlg, 2M2N0-25E5-10233-3220 CECE y aau 10 1995 Qo 20 : Study Monitor: Steven C. Gordon, Ph.D., D.AB.T. TESTING LABORATORY SRI International Toxicology Laboratory 333 Ravenswood Avenue Menlo Park, CA 94025 Study Director: Carol E. Green, Ph.D., D.AB.T. Telephone: (415) 859-4083 FAX: (415) 859-2889 APPROVALS: EY,7 (Cod Research Client's Authorized Representative Gach Study Director of21fps-- Date ger ate QualitAy ALsLsu.ra.nc.e (Qoedd vay, 95 2/"Da1te 04048 1 CLIENT PRIVATE I. STUDY OBJECTIVE `The objective of this study is to determine the metabolismofthe test articles using isolated hepatocytes system will be used prepared from to compare the rat and human liver. The data metabolismofthe test articles generated in the two by this in species. vitro allowedHetpoaattotcayctheisn wmiolnlobleayiseorlactueldtufrreoamndratthaenndwhilulmbaenilnicvuerbastpeedciwmietnhss.uiTthabelececllosncweinltlrbateions soefletchteedtetsitmceh-epmoiinctasl.s.ThAleipqruoottesoinfctohnetecnutltaunrde 7m-eedtihuoxmyccoonutmaairniinngO-cedlelestwhiylllasbeearcetimvoivtye,daat the cytochrome P-450-associated activity, will be determined in cultures from cach preparation. I. STATEMENT OF PURPOSE `The purpose of this study is to provide data that can be used to support applications for arensdeasrucbhmiotrtemdarpkuertsiunagntpetromsietcstifoonrsp4r0o6d,uc4t0s8,re4g0u9l,ate5d02b,y5t0h3e,F5o0o5d, a5n06d,D5r0u7g, A5d1m0i,ni5s1t2r-a5t1i6o,n 5or18S-e5c2t0i,on7s0365o1ro8r013,54o-r3o6t0hFeorftaphpeliPcuabblleicseHcetailotnhsoSfetrhvieceFeAdcetr.alTFhoiosds,tuDdryuwgialnbdeCcoosnmdeutcitceAdct according to SRI Standard Operating Procedures (SOP's) as well as in compliance with the Food and Drug Administration Laboratory Studies. 21 CFR Part 58 Good Laboratory Practice for Nonclinical IL TEST ARTICLE A. Test Article Identification Name: T-6292 Lot No.: Tp oberovbyi Resd earce hCld ient Molecular weight: 571.2 2 04049 : CLIENT PRIVATE - Name: T-6293 Lot No.: Tp oberovbyi Resed arce hCld ient Molecular weight: 685.28 Name: T-6294 Lot No.: Tp oberovbyi Resed arce hClid ent Molecular weight: 527.2 Name: T-6295 Lot No.: Tp oberovbyi Resd earce hClid ent Molecular weight: 538.1 B. Purity and Stability Documentationofthe identity, strength, purity, and stabilityofthe test articles will be the responsibility of the Research Client. Documentation on the identity and purity of the solvent control, DMSO, will be obtained from the supplier of the compound. C. Handling and Storage On receipt, the test articles will be placed in a secondary container (lightproof) and stored as recommended by the Research Client. A Material Safety Data Sheet (MSDS) or other comparable document specifying any hazards and identifying first aid and clean-up procedures in case of an accidental spill shall be provided by the Research Client and shall accompany the test articles. The test articles will be logged in and stored in Building L, Room LB286. 3 04050 CLIENT PRIVATE D. Disposition of the Test Article Client Any after remaining unused completion of the portion study. of the test articles will be remrned to the Research IV. TEST SYSTEM A. Background and Justification for Selectionofthe Test System `The liver is the major site of metabolism of most organic chemicals, both endogenous ahanvdefboreeeingnd.ocSupmeecinetse-dretloatbeed cdoirfrfeelraetnecdestointhtehetomxeitcaebfoflecitssmooffcxheenmoibciaoltsicosnarceerwtealiln skpneociwens and (Caldwell, 1980; Calabrese, 1983). The recent availability of human tissues for research, particularly high-quality human liver specimens, has contributed greatly to the knowledge base on human xenobiotic metabolism capabilites. In addition, these tissues can be used for in vitro investigations on the metabolism of test chemicals early in the development of new products to more reliably predict the metabolic fateofthe chemical in humans. B. Test Species 1. Rat. Adult Sprague-Dawley rats (3 males and 3 females) will be used for the preparation of hepatocytes. They will be purchased from Charles River Laboratories and will weigh approximately 200-250 g and will be at least 2 months old at the time of receipt. `The rats to be used for this study will be housed in Building L, where the cell isolation will be performed. Rats will be taken from the shipping containers, weighed, examined for general health, and placed three per cage in 22 x 12% x 8-inch suspended polycarbonate cages labeled with their quarantine number assignmens. Animals will be quarantined in the same room in which they will be housed. Rats will be fed (ad libitum) Purina Certified Rodent Chow (#5002) and the feed supplier will provide SRI with an analytical report identifying the dict to be free of contaminants for each lot of feed received. A copyofeach analytical report will be available as partofthe study record. Rats will receive purified (deionized and UVtreated) drinking water ad libitum. Temperature will be maintained at 72 + 4F and relative humidity maintained between 35 and 65%. A light cycle of 12 hours light and 12 hours dark will be maintained, with light starting at 06:00. `The laboratory species will be quarantined for at least 3 days before use in an experiment. The Laboratory Animal Medicine Department at SRI will issue documentation to 4 04051 CLIENT PRIVATE the Study Director on the hepatocytes, the rats will health of animals during the be anesthetized with sodium quarantine period. Before pentobarbital (65 mg/kg). isolation of organ p2r.ocureHmeunmtaonr.ganHizuamtaionnslifvreromspbercaiimne-dnesadwilhlubmeanacoqrugiarneddtonhorrosu.ghLciovoeprerfartoimontwwiothmales and two females will be used. Tissues will be processed as for transplant. The organs will be pTehrefutsiessdueisn twihlelotpheernatbiengshriopopmedwtioththiecel-acboolrdatoorrgyanfoprreceslelrviastoilaotniosnolbuytitohneamnodsptaecxkpeeddiiennitce. method. Hepatocytes will be isolated either by the Human Cell Culture Center (Folkston, GA) SRI . International or they will be obtained from C. Test System Identification Each liver human), followed specimen will be identified by a number indicating the by a letter designating particular specimen. the species (R = rat; H = D. Specimen Characteristics For the metabolism studies, six rats will be used (three males and three females) and. four human specimens (two males and two females). For human specimens, information on age, sex, race, and cause of death will be provided to the Research Client. Other pertinent information that is on drug exposure, smoking, and medical history may be available. E. Special Considerations Related to Human Tissues those spCeocmipmelnestethsaetrtoelsotgnyegteasttiivneg twoilhlepbaetiptiesrfBoramneddConvieraucshesh,uhmuamnandoinmomrusnpoevciirmuesn(aAnIdDS)o,nly and syphilis will be accepted for use on this project. a positive reaction to cytomegalovirus (CMV). Manyofthe donors, however, will have and `Whatever the technicians will results take the of serology testing, all necessary precautions human tissue in its use for will be treated as infectious this project, including wearing proper attire while preparing hepatocytes or subcellular fractions and performing incubations. `The tissues will be handled in a hood to protect the technician from aerosols that may form dcounrtiancgtewxiptehrihmuemntaanltpirsosuceedwuirllesbeanddistionfkeecetpedprweiptahrastoidoinusmstpeerricleh.lorGaltaes,stwhaernewtahsathecdomaensd into autoclaved. Disposable materials will be incinerated. 5 04052 CLIENT PRIVATE V. EXPERIMENTAL DESIGN A. Hepatocyte Isolation convenieRnatt.heHpautmoacyntehsepwaitlolcbyeteissowlialtledbebypreeiptahreerdwhboyltehelipveerrfoursiboinoposfybpieorpfsuyssieocntiaosns (Strom et al., the 1981; organ Green will be et al., 1986; Allen perfused first with and Green, a Ca*free 1991). buffer, In brief, followed the by whole liver or wedges a buffer containing of collagenase. Hepatocytes differential centrifugation. will be combed free from the digested tissue and purified by simple. diameteIrs)oliantaedmhoedpiaftioecdytWeasywmiolultbhe'psla7t5e2d/1onctuoltcuorlelamgeedn-icuomat(edCMcHul1taur)etdhiasthceosn(t3a5inms m11.2 ig/ml agl/anmilnea,mi1n2o.l8euvgul/imnlicsearciinde,, 52.40.0ugu/gm/lmlolaesipcaarcaigdi,ne5,.084u.g0/mulg/lmilnolgeeinctaacmiidc,in1.s0ulufgat/em,l0D.1,6L8tocopherol, 288 ng/m testosterone, 272 ng/ml estradiol, 393 ng/ml dexamethasone, 7.9 ug/ml tfihnyarloxcionnc,e3nt0rantgi/omnsl:gluScuaggo/nm,l 0t.ra0n2sfUe/rrmiln,in5suulgi/n,ml0.i1ns%uliInT,Sa(nCdoll5abnogr/amtlivseelReensieuamr)c,h,0.I2ncm.;M Lbasocvoirnbeicsearcuimd.2-Aphfosrph2atteo,3ahnrd o0f.i2n%cuBbaStAi.onTahte37iniCtiailnpalnataitnmgomsephdeiruemowfil9l5c%onatiarin:5f%etCalO, the medium will be aspirated to remove nonviable, unattached cells and replaced with CMH1a containing the test article. B. Metabolism Experiments 1. prepared as Preparation stock solutions and Administration of in DMSO, maintaining Test Article. The test articles will be the DMSO concentration at 0.1% or less. dTihsecyuswsiilolnsbewidtihluttheed SwtiutdhycuMlotnuirteomreadnidubmastoedgiovne tahefinraelsuclotsnocfetnthreatiraonngdee-tfienrdmiinngedcybtyotoxicity eexxppeerriimmeenntt.aSntdockkepstoliunttihoensoreffrtihgeertaetsotraorrtioclnesicweilulntbiel pusree.parAleidqiumomtsedoifattehlesyepsrtioocrktosotlhuetions will be saved for later analysis by Advanced Bioanalytical Services (Ithaca, NY) to verify the concentrations and homogeneityofthe stock solutions. 2. cultured on Preparation of Conditioned Medium. collagen-coated culture dishes for 0 and 6 Rat hepatocytes will hr. At the appropriate be isolated time point, and the hepatocytes will be scraped into the culture medium. The cells and medium will be aspirated `awnildl itmhmeendbieatceelnytrmiifuxgeedd waittahpapnroexqiumaaltevloylu1m2e00ofXicgefcoorld5 mmeitnhuatneosl.andThseubcsoenqduietnitolnyedfrmoezednifuomr 6 04053 CLIENT PRIVATE shipment to the Research Client. point will be prepared. Approximately 500 mi of conditioned medium at each time 3. duplicate with Incubation of Hepatocytes with Test Article. the test chemical in CMH1a. Samples wil be Cultures will be incubated taken at 2 time-points (0 and in 6 hhre)p.atTochyetefsolilnomweindgiacownittrhoolsutwitlelstaalrstoicblee (i4ncsuebtastoefdqaunaddrsuapmlpilceatsetiankceunbaatti0onas)n;d 62)hr:inc1u)bation `media containing test articles but no cells. The atiached cells wil be scraped into the culture ``vmoelduimuem,ofThiceen-ctohledcmeelltshaannodlmtoedsituopmtwhiellrebaectaisopni.raTtehdeasnadmpilmemsedwiialtletlhyenadbdeecdetnotrainfuegqeudalat high speed in a table top centrifuge for 5 minutes and subsequently frozen for shipment. determi4n.ed byParostpeeicntrAospshaoyt.omeTthreicpprrootecienducroen,teCnotoomfatshseiehebplauteocaystseaycu(lBtruardefsowridl,l 1b9e76). 5. Cytochrome P450 Activity. As a control to indicate the presence of bceytdoectherrommienePd45in0eaascshocpiraetpeadraacttiiovnitoyf, h7e-peattohcoyxtyecso.umTarhienceOl-ldseewtihllylbaetiionncu(bEaCteOdDw)itachti1v0i0tyuwMill 7T-ethoxycoumarin for 1 hr. The culture medium will be assayed for hydroxycoumarin production using the fluorometric method of Greenlee and Poland (1978). ECOD activity will be calculated as the amount of hydroxycoumarin produced/hr/mg protein. 6. Advanced Analysisof Metabolism. Samples will be shipped to Bioanalytical Services, Inc. for analysis of metabolism. Dr. Jack Henion at 7. Data Collection. The protein assay and ECOD assay will be performed using a spectrophotometer and captured electronically, fluorometer, respectively. depending on equipment The values availability. will be recorded manually or Data from both assays will be rsepceocridmeednsanadremKaenpitpuilnaatecdomopnuateMricdraotsaobfatsEexacnedl asrpereaaldssohereetc.orIdnefdoirnmaatilaobnoorantohruymnaontelbioveork. Inc.) `The results of will be returned the metabolite analysis (performed by to SRI and the data will be calculated Advanced Bioanalytical Services, as the nmol of metabolite/mg protein. These data will be included in the final report. The results of the analysis of the liver tissue by Mr. James Johnson at 3M Environmental and Pollution Control wil also be included in the final maintained report. The raw data generated on the metabolite analysis will be kept by Advanced Bioanalytical Services. The raw data on levels of the test and articles in the human liver samples will be kept and maintained by 3M. 7 04054 or CLIENT PRIVATE D. Sample Shipping Addresses `The incubation samples, samples of frozen human and rat liver tissue (approximately 1:g), FBS, BSA, L-ascorbic acid 2-phosphate, and media (conditioned and fresh) will be sent Dr. Jack Henion, Ph.D. A15dvCaantcheedrwBoiooadnaRloyatidcal Services, Inc. Ithaca, NY 14850 A sampleofeach human liver tissue (approximately 1 g) will be sent to: James D. Johnson 3M Environmental Engineering and Pollution Control 9Bu3i5ldBiunsgh2A-v3eE-n0u9e PO Box 33331 St. Paul, MN 55133-3331 `The data on protein content, ECOD, reports, and excess test article wil be seat to: Dr. Steven C. Gordon 3M Medical Department, P. 0. Box 33220 Toxicology Services St. Paul, MN 55133-3220 VI. CONTROL OF BIAS `Human tissues will be used as they become available. VIL STATISTICAL EVALUATION OF DATA Mean and standard deviations will be determined for the replicate incubations for each test article. 8 04055 CLIENT PRIVATE VIIL REPORTS Final Draft Report reports will be wsiulblmibteteidsstuoedthperiRoersteoarscuhbmCilsiesnito.nofthe Final Report. Two copiesofthe IX. RECORDS TO BE MAINTAINED of the `The Final laboratory notebooks, all raw Report will be transferred to tdhaetaS,RrIelReevcaonrtdcsomCmeuntneirc,atBiuoinlsd,inagnBd,thafetoerricgoimnapllectoipoyn ofcotnhtaectsetdudcyo.ncTehrenisnegmaftueturriealdsiswpiolslibteiomnoaifntthaeinreedcofrodrs.10 years; then the Research Client will be X. GOOD LABORATORY PRACTICES (GLP) AND QUALITY ASSURANCE The `This study will be conducted in duties of SRI's Quality Assurance accordance with Unit (QAU) will Good Laboratory Practice Regulations. include inspecting the calculations, inspecting reviewing laboratory work, the Final Report. cAertQiufayliintgypArsospuerraindceentiSftiactaetmioenntanwidllnoatcecboomopkaennytrtyheofFisnaamlplReesp,oratnd of the study. LaboratAofrtyerorthtehestSupdoynhsaosr--bweielnlinbietisautebdm,itmtoeddifiincawtriiotnisngoftothteheprotohteorcopalr--tbyy. either the Testing All agreed-upon `modifications `modifications will and be the in the form reasons for of Protocol Amendments, the modifications and will that will state be signed and the specific dated by the pSepronfsoorrm'esdRienpraecsceonrtdaatnicveewaintdhtShReITeSsttainndgarLdabOopreartaotriyn'gsPSrtoucdeyduDrierse.ctor. All procedures will be area ofTnhoencSotmupdlyiaDnicreecstiogrniwfiilclanbtelymaafdfeectaswatrhee roefsualntysoGfLthPensotnucdoym,ptlhieanScpeondsiorerctwliyl.lIbfetnhoitsified immediately. QAU. This will be documented and the agreed upon outcome will be forwarded to the hours fAorutqhuoarliitzyedasdseusriagnncateepsuorfptohseesSapnodnsmoarymcaoypyinaspneyctsttuhdiys-srteuladtyedduorriingginraelgdualtaar.working 9 04056 Yoel CLIENT PRIVATE | XI. REFERENCES Allen, K. L. and C. E. Green. In: Methods in Toxicology, Volume 1, In Vitro Biological Systems, PartA. C. A. Tyson and J. Frazier (eds) Academic Press Inc., pp. 262-270, Bradford, oh. M. Anal. Biochem. 72, 248-254, 1976. Calabrese, E. J. John Wiley & Sons, New York, pp. 203-282, 1983. Caldwell, J. Press, INneEwnzYyomrakt,icppB.as8i5s-o1f14D,et1o9x8i0c.ation, Vol. I. William B. Jakoby (Ed.), Academic DeLean, A. P. J. Munson, and D. Rodbard. 1978. Am. J. Physiol. 235, E97-E102. Green, C. E., J. E. Dabbs, and C. A. Tyson. Aual. Biochem. 129, 269-276, 1983. Green, od . E. LeValley, and C. A. Tyson. J. Pharmacol. Exp. Ther. 237,931-936, Greenlee, W. F.andA. Poland. Jour. Pharmacol. Exp. Ther. 205, 596-605, 1978. Strom,ISr.onCs.,,J.R.R.L.MJciLratil,n,R.anSd. GJ.oneMsi,chDa.loLp.ouNloovsi.ckiJ., NMa.l.R.CaRnosceenrbIenrsgt., 6A8., N7o7v1o-7t7n8y,, 1G9.82. 10 04057