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. : I CLIENT PRIVATE 5] 16 | PROTOCOL TITLE: TR-a6n2g9e5fiinndRiantg HCeyptoattooxciyctietsy Assay of T-6292, T-6293, T-6294. SRI Study No. B010-95 RESEARCH CLIENT 3M Medical Department Medicine, Health Physics, Industrial Hygiene, Toxicology 3M Center Building 220-2E-02 St. Paul, MN 55144-1000 Study Monitor: Steven C. Gordon, Ph.D., D.A.B.T. `TESTING LABORATORY SRI International `Toxicology Laboratory 333 Ravenswood Avenue Menlo Park, CA 94025 Study Director: Carol E. Green, Ph.D., D.A.B.T. Telephone: (415) 859-4083 FAX: (415) 859-2889 APPROVALS: % oo)Codon Research Client's Authorized Representative oferfos-- Date (dol Jb wants SRI Study Director Izier Date 04030 : Lo CLIENT PRIVATE L STUDY OBJECTIVE `The objective of this study is to evaluate the cytotoxicity o4f different test articles as measured by MTT conversion. The ECyoand EC,, values will be estimated for each test article and these data will be used to select a suitable concentration of each test article for subsequent in vitro metabolism studies. IL APPROACH Hepatocytes will be isolated from adult male Sprague-Dawley rats. The cells will be aelxlpoowseedd ttoo aetittahcehr itensmtoanrtoilcaley,esrolcvuelnttu,reoirnptohseitwievlelcsoonftraol9i6n w4elrlepplliactaet.e Tcuhletucreelsl.s wIinllthtihsen be experiment,thecompoundswillbetestedin 6 different concentrationsata singletimepoint (24 hours). The concentrations for each test article are described below in theExperimental Design. IL TEST ARTICLE A. Test Article Identification Name: T-6292 Lot No.:TpoberovbyiResedarcehCld ient Molecular weight: 571.2 Name: T-6293 Lot No.:TpoberovbyiResd earcehClident Molecular weight: 658.28 Name: T-6294 Lot No.:TpaberovbyiResd earcehClident Molecular weight: 527.2 2 04031 CLIENT PRIVATE Name: T-6295 Lot No.:TpoberovbyiResd earcehCld ient Molecular weight: 538.1 B. Purity and Stability the `Documentationof responsibilityofthe the ideaity, strength, purity, Research Client. The positive and stability of the test articles will be control, acetaminophen, and the solvent control, DMSO, will be obtained from Sigma Chemical Co. (St. Louis) and their documentationofthe idenantdpiurtityyofthese chemicals wi bel usel d. C. Handling and Storage requiredO)narnedcesitpotr,edthaestersetcaormtimcelensdweidllbbyetphleaRceesdeianrcahsCelcioenndt.aryAcoMnattaeirniearl (SlaifgehttyprDoaotfaifSheet (MSDS) or other comparable document specifying any hazards and identifying first aid and cshlaelaln-aucpcoprmopcaendyurtehse tiensctaasretoicflaesn. aTchceidteensttalarstpiiclllesshwaillllbbee plroogvgieddedinbayntdhestRoersedeairncBhuCillideinntgaLn,d Room LB286. D. Disposition of the Test Article Client aAfnteyr croemmpalientiinognuonfustehed sptourdtyi.onofthe test articles will be returned to the Research IV. TEST SYSTEM A. Background and Justification for Selection of the Test System `The liver is the major site of metabolism of most organic chemicals, both endogenous and foreign. Species-related differences in the metabolism of xenobiotics are well known and `(hCaavledwbeeleln, d1o9c8u0m;eCnatleadbrteosbe,e c1o9r8r3e)l.ateTdhetoptrhoepotosxeidc ceoflfoercitsmeotfricchecmyitoctaolxsicointycearstsaaiyn hsapsecbieesen used to determine mammalian cell survival and proliferation (Mosmann, 1983; Otoguro et al., 1991). This assay detects living, but not dead cells. Because the signal generated is 3 C0403 | CLIENT PRIVATE dependent on the degree of activaiton of the cells, cytotoxicity, proliferation, and activation can all be measured. This assay depends on the fact that MTT (which is yellow in aqueous solution at neutral pH) is metabolized by mitochondria of living cells to a blue formazandye. `When used as a cytotoxicity assay, spectrophotometric quantitation of the formazan generated `bmyetcaeblolsliacftaecrtitvhietiyor fintchuebastuirovniwviintgh ctehlelst.est article will give an indicationofthe number and B. `Test Species Rat. Adult male Sprague-Dawley rats will be used for the preparation of hepatocytes. `They 250 g waitllthbeetpiumrecohfapsuerdcfhrasoem.Charles River Laboratories and will weigh approximately 200- will be `The rats to performed. be used for this study will be Rats will be taken from the housed in Building L, where the cell isolation shipping containers, weighed, examined for general health, and placed three per cage in 22 x 12% x 8-inch suspended polycarbonate cages lraoboemleidnwwihtihcthhetihreqyuawirlalntbienehonuusmebd.erRaastssigwnimlelnbtes.feAdn(iamdallisbiwtiulml) bPeurqiunaaraCnetritniefdieidnRtohdeesnatme Chow (#5002) and the feed supplier will provide SRI with an analytical report identifying the diet to be free of contaminants for each lot of feed received. A copy of each analytical report will be available as part ofthe study record. Rats will receive purified (deionized and UV- treated) 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. experim`eTnhte.laTbhoreatLoarbyorsapteocireysAwniillmable MqeudairacnitnieneDdepfaorrtamtelnetastat3SdRaIyswiblelfiosrseueusdeociunmaenntation to the Study Director on the health of animals during the quarantine period. Before isolation of hepatocytes, the rats will be anesthetized with sodium pentobarbital (65 mg/kg). V. EXPERIMENTAL DESIGN A. Hepatocyte Isolation Rat hepatocytes will be isolated by either whole liver or biopsy perfusion as convenient. (Green et al., 1983). In brief, the whole liver or wedgesofthe organ will be perfused first cwiotmhbeadCaf*r2e-effrereombutfhfeerd,igfeosltleodwteidssbuye aanbdufpfuerrifcioendtabiynisnigmpcloleldaigfefnearseen.tiaHlecpeanttorcifyutgeastiwoinl.l be 4 04033 - CLIENT PRIVATE diameteIrs)oliant2edmhoedpiaftioecdytWeasywmiolultbhe'spla7t5e2d/1onctuoltcuorlelamgeedn-icuomat(edCMcHul1taur)e dishes (35 mm that contains 11.2 ug/ml agl/anmilnea,mi1n2o.l8egvu/limnicsearciinde,, 52.40.0pgu/gm/lmlolaesipcaarcaigdi,ne5,.084g./0 umg/lmilnolgeeinctaacmiidc,in1.s0ulufgat/em,l0.D1,6L8- tthoycroopxhienr,ol3,02n8g8/nmgi/gmlluctaegstoons,te0r.o0n2e,U2/7m2l nign/sumlline,st0r.ad1i%ol,IT3S93(Cnogl/lamblordaetxiavmeeRtehsaesaornceh,, 7n.c9.p;g/ml final concentrations: 5 ig/ml transferrin, 5 ascorbic acid 2-phosphate, and 0.2% BSA. g/ml insulin, and The initial plating 5 ng/ml selenium), medium will contain 0.2 mM L10% fetal bovine serum. After 2 to 3 hrofincubation at 37C in an atmosphereof 95% air:5% CO, the medium will be aspirated containing the test article. to remove nonviable, unattached cells and replaced with CMH1a B. Range Finding Cytotoxicity Experiment (MTT Assay) solvent1c.ontrolPsrewiplalrabteieoxnaaminndeAddimninthiesterxapteiroinmeonft.TesStolAvretnitclceosntarnold sCaomnptlreoslsw.illCoconncsuirsrteonft culture medium and DMSO at the same concentration used to dissolve the test articles. DMSO will be used so that the final concentration does not exceed 10 ul/ml (1%). Acetaminophen (10 in culture medium. mM) will be the positive control and will be prepared as a stock solution `will be Immediately before the experiment, an aliquot diluted in DMSO to form a concentrated stock of each test article (solid or pure liquid) solution. Serial dilutions will be made from these initial stocks. 2. Test Article Concentrations. 0.01, 0.05, 0.10, 0.50, 1.0, 5.0, 10.0 mM. Six test article concentrations will be studied: 3. Incubation of Hepatocytes with Test Article. Four replicate monolayer cultures of or control. isolated rat A negative hepatocytes control (no will be incubated cells) will also be at 37C for 24 hours with the test measured for determination of article background staining. 4. bromide] MIT assay will Abesspaeyr.foTrhmeedMaTs Tdes[c3r-i(b4e,5d-bdyimeStchuydlitehrioazeotla-l2.-y(11)9-828,)5-adsipmhoedniyfliteedtrbayzothliisum laboratory (Otoguro et al., 1990). MTT (Sigma) will be dissolved at 5 mg/ml in calcium and `magnesium free phosphate buffered saline (CMF-PBS) and sterile-filtered with 2 0.2 um filter unit. The MTT solution will be diluted 1:5 in CMH1a, and 150 ul will be added to each of the 24 wells in the dish. After a 2 hour incubation at 37C, the incubation medium will be 5 04034 : CLIENT PRIVATE aspirated and 450 ul of DMSO will be added to solubilize the MTT-formazan product. After `mixing, the absorbance at 540 nm will be measured. plotted5a.gainstDtahteacChoelmlieccatliocno.nceTnhteranteitonc.haTnhgee E(C%s),rvealaltuievsewtiolthbeesdoelvteenrtmicnoendtrboly wailclombpeuter statistical program designed for curves using the four-parameter simultaneous fiting of families of logistic equation (De Lean et al., sigmoidal 1988). dose response VI. REPORTS Asummaryreport containing theresultsofthis experimentwillbe issued tothe Study Monitor. The test article concentrations for the metabolism study will be selected based on. these results: IX. RECORDS TO BE MAINTAINED `The laboratory notebooks, all raw data, relevant communications, and the original copy ofthe Final Report will be transferred to the SRI Records Center, Building B, after completion of the study. These materials wil be maintained for 10 years; then the Research Client will be contacted concerning future dispositionofthe records. X. REFERENCES Calabrese, E. J. John Wiley & Sons, New York, pp. 203-262, 1983. Caldwell, J. In Enzymatic BasisofDetoxication, Vol. 1. William B. Jakoby (Ed.), Academic Press, New York, pp. 85-114, 1980. DeLean, A., P. J. Munson, and D. Rodbard. 1978. Am. J. Physiol. 235, E9T-E102. 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, . Omura, and C.A. Tyson. ATLA 19, 356-360 (1991). 6 04035 03.26.1995 12:2S0RE TOX LAB FRX # 415 859 2809 M.59 Pu | FACSIMILE TRANSMISSION Message to: Dr.SteveGordon Company: 3M FAX Number: 612/733-1773 Date: October26, 1995 RE: Cytotoxicityassay Number of Pages: 5 From:Carol Green SDRirIeIcnttPerhnoantei:on(a41l5) 859-4083 `Toxicology Laboratory 333 Ravenswood Avenue MenloPark, CA94025 FAX (415)859-2889 (415)326-5512(alt) CC: CV.. TWyesizoenr `EnclosedarecopiesofthedatafromtheMTTassay. `Youwillalsofindsomenotes preparedbythetechnicianonthe solubility ofthecompoundsinthemedium. After reviewingtherawdata (absorbance values)for T-6292, Ifoundthatthecontrols forthat plate werelow. As aresult, 1thinktheassayforthat chemicalmayneedto `berepeated. Basedonthe appearanceof the cellstreatedwith T-6292, severetoxicity `occurredbetween0,1and 0.5 mM. Pleasereviewtheresultsandcallme sowecan discussthisfurther. Cant 04036 OCT.26.1995 12izM SRI TOK LAB FAX + 415 E59 2669 wes ez Project 7586 RatMTTAssay AeCoPl peexipernodroea ms Tploesroar r. --rE--T ----r T T] -- -- [A16o24nthh orpattmoapndornepielmeadwiicteTodTryasceyTsoton]asr1s. MTT Assay `solution as incubatedonthecellsfor 2 hr [TT [[MTTheTTpaellssaastsaienysgwseolrunettiroohnaewdaDosnSraeOMmtooohvceeudlFaaroftrDeermv2iahcrgesaaKnunidentehDetyec.ePl| lastawerreeaa|fddwSi4et0hD7rMSO|| Dr r [ ata calculationswerey perform+ edontheSoftt e maex kopeerantinngpp+ orvogfe ororratPhmaepeats sTeatder:| ts I Bn m-- fC o m osw o o r or remr m i --y ------------| i e oT w s mT --------ss hs--o----------------| r s ooT osv or e --ha--r -------- Er r s merT r] fooot1 ocrT ----r ore smre es e +o . m i --e --| oosr Ts we ---- stam----------------] [ E-- Eo r es ----r oms---- rr . ' 04037 Page OCT.26.1995 12:29M SRI TOX LAB FAX # 415 659 2089 wom a Project 7565 Rat MTTAssay f [2e%d Teeger --1TTT 1T ] | [r onoe T ofemo1 naorr esiT eW s meo t ] Fo oeoss T Tw m roso eers smaa ] soloooa Tv ms ie mtae ae l-- o rr r r --r Tr rtr [F [ 0T p---- T [ 1or| 1eg o-- T --T|fs1 1 p -- eeoos Ta a mm ses ] ioso seeeesrmTry g ----] ----] rm was -- [osoor a rser s rqa se 1| [fPr oAT Pos1ten | r | ------] C ( mPf FeweseE| sw ragez . 04038 OCT.26.1995 12:29PM SRI TOK LAB FAX # 415 B59 2089 No.5 Pua i 160 140 = g 120 5 100 i.5 2 80 - 2 Ew 20 0 0.001 Cytotoxicity to RatHepatocytes --&-- T6205 --m-- T6204 --A- T6203 \ Xa - \ A\ | \ \ \\ '\ \ \ { W \ ~. \ = 0.01 01 1 10 100 Concentration (mM) . 04039 OCT.26.1995 12:30PM SRI TOX LAB FAX # 415 8592669 ro. ps `NotesonsolubilityofcompoundduringMYTexperiment ofOct16.17, | T-6292 T6293 T6294 Visiblyprecipitatedout at 10, 5,and 1 mMconcentrations CNoMtHa1llasaonludbaifltiezreadgaitta1t0i,o5n,aapnpeda|rmedMtocohnacveentgroanteiionntsoisnotlhuetiDoMn.SO added Vi1msiMolcyonpcernteractiiopn.oiuttaa1t0aendd 5mM concentrations. Novisibleprecipitateat T6295 VSiasnidbly1pmreMcciopnicteanttedroauttiaont.the 10mMconcentration. Novisibleprecipitateatthe Notesonappearanceofcell afterincubationwithtest articlesforapproximately22hours, T6292 RRoowwss36--59vseormyefceewllcselslpsresapdroeaudtosuotmmeocsetllrsoruonudnedde.d. 0-04,0.0F, 0,\ ah 6.5,1.0.5.03 0 mb Row 9crystalspresentdarkcoloredmedia. T6293 RRoovwiss73--160nohvaivseiablleotsobfndoembarliisf,tiehsecell0s.6ar1e r=ounde0d,ormunpattached. 1-6 ) T.0,6:516,. " T-6294 RRoowwss63--95cneollvsinsiobtlseparbenaodromualti,trieosu.nded.6.00.15 = . 04 ch 16 mn T-6295 RRoowwss3-6-S7cceellllssnmootsstplryesapdreoaudtowuitthwistohmseodmeberriosu.n~ded~. Oe 5.5~ - 0s mn .q wo Rows8:9 alotofdebris,thecellswererounded. gu + (man APAP Z0ws 2-3 10 visible abnormalities. Cavheaty Rows 4-6few to no spreadout cells. Ate `AWpeplelsar8a-n9cheofaaTh6e2ad9v5y ayfetlel2rowhporuerciipnitcautbea.tionwithMTTsolution Well `Well 7had 6had aalimgehdtiyeulmyleowllproewcpirpeictiaptiet.ate, `Wells 1-5zovisibleprecipitate. `The other Plateshad no remarkable aboormalities. 3 ForplateT-6293 one rowofwellswasrepeated. When ead by plate readeraad byvisible ionIbtsweerrrv1ua0pt2ti4iono/n9o5dfurcionlgotrhcehaapnpgleicraotwison8oafnteds9twaretricele5s.a0nmdMticmoencIennottriacteidotnh.e`eTrhriosrc.orrespondswithan - ' 04040 DE".12.1995 12:5G9RY Tox LAB FAX # 415 E59 2899 0.64 Pui } .1 TSoRxiIcolIogNyTLaEboRrNatAorTy IONAL Facsimile Transmittal 3M3e3nRlaovPeanrks,wCooAd94A0v2e5nu-e3,43B3ldg 206 From: Coral Green FAalxterNnoa.te(4F1a5x) N8o5.9-(241858)9858-3342 DirectPhone: tw. # 9- 4OP8 ------------------------------ Message To: Rv, Stew Gordan ! Company: JM FaxNumber, 12 [43-1333 Date: December 12, 1895 This Message Transmitted from: Fax No. (416) 866.2869 Numberof Pages: 4 including this transmittal sheet MESSAGE Endoten are He dda From Be copes NTT aspen| De bebiwe Thad Ho tevesa in Hopi & SE nb vm bee "= dhe boo aaditiy" .of de dik aidey ax Ator ChuTaions jainin Br. Q He Grposd Suc porn adhilinwg,e fede pena gud hE fn 1,5, (mt, Sebi CeTosion wt dil Kin of, [mh (36mag bie aqpuima, Sem, biMa hanscoiiting lose ay GpdT, W. d Ul Ao. Ce e } Pleasecallthesenderdi ect ifyouhaveany problemwith"The reception of te`message. 04041 DEC.12.1995 12:59PM SRI TOX LAB FAX # 415 659 2889 No.4 pz Project 7665 RatMTTAssay Pywre --T-- CiolssxpoanE to etarL ses or28E ormsmE -------- ------ ------ --] hoI24hou timepolitthemed Gorantest rideWas -- BSpFaiEd | [MTT ly solutionwas incubatedonthecellsfor 2 hr. 1 --1 MTTraeslesaasyingsoilnuttoiotnhweaDsMrSeOmotvheedFaofrtmeraz2ahnrBalnudetDhyeec.ell|s were sed with DI | SO | | [D[aThteacplaatlecs wuelraetriweoeanrdseonpearfoMrmeodleculoanrtDheeviScoefsiKmianxetoipoerPaltaitnegrepardoegrraamtf5o4r0hmem.pi|s ead: [T o F rem Perr--F e --or-- emT --T f-- oe |-------------- Ee e E w I 7w e +w -- -- o sooss T Ta m w a s o o --s ------ r + oo T1 erar --os] -------- [ee Pep --------| [ E s oF o e pJ o e m T n dmmme --e -- os Te 13 pe -- ' "C= O0.65 eOn att C i T---- [ w ew ee w ; Paget 002042 DEC.12.1995 1:00PM SRI TOX LAB FAX # 415 659 2689 LE Notesonsolubilityofcompoundduring MTT experimentofDec.6.7 T:6292 CaCofnotcemerpnatgoriuatntatidiowonanaasppnppeoeaatrraeleddtstooolbuhboaitvlhiezegemodonaset1itn0sto,losu5,obliaulntidizoe1ndmi,nMthceonDcMenStOraatinodnsC,MThHe1g1msaM. T6293 aCCfootnemcrepangotirutanattdiiowonanaasppnppoeetaarraeleddtstoooluhbbaeitvlheizegemodouasstitn1s0to,lo5us,boilalunitzdieod1n1i.nMthceonDcMeSpOtiaonndsC.MThHe11pmagM. . concentrationsfor 10mitesaferadditionofCMHlSaotoinncrie1a0cs,es5aoglautbdi1leistypd. y Notesonappearanceofcellsaferincubation with tetartilesfor approximately22 hous. T6292 Rcoowmpsoun9d.S(o5m,e1c0emllMss)pread out some celsrounded.Visiblecrystals of Roofwcosm6p-o7unFde.wt(1o.n0,o0c.e5lrl-aceMl)lattachmentsmos llclsappearVisiblecrystals RRooww$4MSoormeecfellalt-ceealeldactetlalcshwmietnht.ceFlle-wcetlol2at0tcarcyhsmtenatl..(S0o,m1meMro)undedcell, (0.05 RRmoMow)w32 CAeclolussplelofirobuegntdteehrdtchetallnslrootwhy4er,wi(s0e.0g1omodM.)(Normalmedia) T6293 APAP RowCell (100mM) visiblemanyrounded with som spreadhaving cell-cell attachment. RRooviws56.V8erCygreainnolytcvelilslissblreoduunedetdo.gr(a0i.1nmydMbr)is. (5.0, 1.0,0.5mM) deRbroiws4prSesoemaetc.el(l0s.a0r5emcMlu)mpedtogethergrowingontopofeachother,grainy RRooww32 GAocoodupwlietohfgrraoiunnydeedpcpeelalrsanocteh.erw(0i.s0e1gmooMd).(Normalmedia) Ro0wwss26--591f0ovwitsoib1l0esapbruoeramdaoluitticeesl,ls(.N(o1rm0amlMmAePdiAaF)) viw 12/795 : 04043 | i i]. "13 i i i= 1d ie ( - mse 1] E eErmsaamgmzeie Bh" E = i EI i en. 6.19% 6:1EHGT RE Tox Lap FAX 41s eso z0e9 Yozm py FACSIMILE TRANSMISSION | Messageto: Cony pany: Dr. Steve Gordon 3M FAX Number: 612/733-1773 Date: Jamuary 16, 1996 RE: Rathepatocytestudy From: Carol Green Direct Phone; (415) 859-4083 SRI International Toxicology Laboratory 333Ravenswood Averme Menlo Park, CA 94025 FAX (415)859-2889 (415)326-5512(alt) NumobfPeagers: 3 CC: V. Weizer C. Tyson IEEISINSeSI ssensuresahsasassns eRORs seranaRtne ERnepcaltonsceydtaesreinccoupbiaetseodfwtihtehdtahoatfesrtoamrttihceleLDfoHllaoswsianygtthhatepwreotpoecroflormedon rat Ae study. WwieuGsaedA100t2M op f eacn has discussseed,,NNoosigTnsiufgigceasntttthoxaitfcwioteryshiesenemdvetishdseebnoottlhiesrm samples from 6292,6293,and6295toABS for analysis, Doyouagree? lWikee ctoandidsicsucussstshtehepsreorjeescutlbtusdafgteetrbyeocauuhsaevtehea LchDaHncaesstoaryevwiaeswnotthienmc,lu1dweoduldalso Twiglilntaallpkrtootoycooulasnodonwaebosuotwtmheasyeinsesueedst,orepeatthe inthe incubation for6294mets. cosoas 801Rat11L.OH912098 ype r ew r 717r 71 TT | 2 fsa perometon1 | [Swe sowoscus: oT o mer nonervom O [-- oTpg1 [| Co vere handysed-- wih10 -- Won KA--RPSTom-- -- -- ---- -- -- ~ ||[| -- [C 1 o 1 sby| ] ] [Xyicumes ogres] emerge |||||] . i [| esen-- dover m m-- ird es--og -- woms-- oedntme--drdewowe--s05S|| |r | | r | [Toostmiceinwnespeesmtivamn1anicifornmasdn| |7 | ] | I[oC puT L rs[ Tsim1 p [ eet-- es -- f | --p -- ] -- ] -- -- PPol JT T oma | ee een r ---- F --r --| FR [E Fin coTit=sT ton xD S-- i-- g ---- s -- -- -- ] -- E zEo r f r re-- m -- EJoTm If ssam |--f-- swernla-- cionefe-- mur {-- Huy EeEDs |Pm R-- E e ON-- fe e --e -- e g frosts [ e sae eame lseal a wasw ss el| eed ow BEE f= E page soonrtLo ss. . CL 0c Croo Ce mmre gg : [Jc fleorewdee|e Tree-- e--ae m-- a ---- s d me e ms Ta--vaw ens] C--O f CCrT -------------- ---- --b -- i --i m -- eu ofE e e = F -- ---- pe|| om| " Boe J[ r oss [ ma E a a e ee m m e e r d o ----te iesa e e e s o m A ] , EEE ews eafem| 2 gfe Fr ee a e m( ea i] n a, E:o t+1] B Po r io r -- ---- b ] ----me --h pein ff C e re r f r] eee ; rrr E Paez