Document 067MkzJ0ZnYpY7OBZkopZ3xom

@HAZLETON reTy"N \ 016% ) J Z NS i ANALYSIS OF T-5794 IN A CELL PROLIFERATION ASSAY IN RAT LIVER CELLS AR26-0302 [EINAL REPORT AUTHOR Maria A. Cifone, Ph.D. PERFORMINGLABORATORY Hazl9e2t0o0n LWeaesshbiunrggtoPni,keInc. Vienna, Virginia 22182 LABORATORYPROJECT ID HWA Study No.: 154-207 SUBMITTTEDO 3M Corporation Building 220-2E-02 3 M Center St. Paul, MN 55144-1000 SCTUODYMPLETIDOATNE January 26, 1994 154-207 1 of 23 605101  WASHINGTON - QUALITY ASSURANCE STATEMENT PROJECT TITLE: Analysis of T-5794 in a Cell Proliferation Assay in Rat Liver Cells PROJECT NO.: 20991 PROTOCOL NO.: 493 HWA STUDY NO.: 154-207 EDITION NO.: 1, Modified for 3M Corporation Quality Assurance inspections of the study and review of the final report of the above referenced project were conducted according to the Standard Operating Procedures of the Quality Assurance Unit and according to the gFeinnedrianlgs refqroumiretmheentisnsopfectthieonsappanrdoprfiianatle Good Laboratory Practice regulations. report review were reported to management and to the study director on the following dates: Inspect Find Necropsy /Tissue Preservation/8-20-93 Draft Report Review/ 11-29,30-93,12-1,6,7-93 Final report review/ 1-10,11-94 8-20-93 12-7-93 1-11-94 M.J. Robertson B. Mullett B. Mullett 154-207 fLedaTity WAsasulranlce Un itNe ERelease 2 005102  WASHINGTON - COMPLIANCE AND CERTIFICATION STATEMENT TPhreacdteiscceriRbeegdulasttiuodnyswaass csoetndufcotretdh iinn tchoemplCioadenceofwiFtehdertahle GRoeogdulaLtaiboonrsato(r21y CFR 5t8h,e 4a0forCeFmRen79t2i,oneadndre40gulCaFtRio1n6s0).or tThheeresiwgenreed pnorotsoicgonliftihcaatntwoduelvdiataifofnesctfrtohme dianttaegrhiatvye boefentherevsiteuwdcydorbythteheiSnttuedrypreDitraetcitoonr,ofwhtohecetersttifireessultthsa.t thTehe raw eCovnaclluuastiioonn woifthtihne ttheestcoanrtteixctleofasthperessetnutdeyddehseirgeninanrdeperveaselnutastioann carpiptreorpirai.ate AgeInerraatweddataas, adorceusmuelnttaotfiotnh,isresctourddys,wilplrotboecoalrsc,hivsepdecibmyenHsazlaendtonfinfaolr areppoerrtisod Aoffteatr tlheeastoneoneyeyaerarperfiooldl,owitnhge ssupbomnissosriomnayoefletchte ftoinahlavereptohretsetomattheeriaslpsonsor. rtiemteainoerdseinntthteoastosrtaogreagefacfialciitliietsy doefsiHganzalteetdonbyfortheanspaodndsiotri.onal period of SUBMITTED BY: Adee Horn AAnsdsroecaiatL.e FSacmi,entB.iSs.t Study Director: fe Te 26,1994 J 1 foxes istriddayDiA.reCcitfoorne, PH.D. Genetic and Cellular Toxicology a,tudy Cafpl2e6ti.o7n Date 7 154-207 3 05103  WASHINGTON - TABLE OF CONTENTS PAGE NUMBER ABSTRACT oo ve vinnie enna. 6 Io SPONSOR. oveveee eee T TL AM.ATERIAGLenTetEiScTsEDAs.sa.yoNoo. ovv iv ene ene e anne. 1 CB.. PIhdyesnitciaflicaDteisocnription D. Date Received FT IV. PROTOCOL NUMBER . ooo vvvveve ie enneennnnns 7 Vo AS.TUDY sDtAuTdEyS i. niotiaotiton Daotetunitaene eee T CB.. EExxppeerriimmeennttaall TSetramritnaDtaitoen Date VI. AS.UPERVISStOuRdYy DPiErReScOtNoNrEL . . . oo vevvvv evn nvennns 1 B. Associate Scientist VIL OBJECTIVE oo voeveoe een eeene eee 1 VIIL DEFINITION ooo veoie aise enn. 8 IX. AM.ATERIAILnSdic. at. or. Coeiols veeeteinnnaennenen. 8 B. Osmotic Pumps and Label for Cell Proliferation Analysis C. Control Articles X. AE.XPERIMDEoNsTingDESPIrGoNced. ur.eo.oo ovvoveneonennene.. 10 CB.. DIomspelanRtaantgieo-nFinodfinOgsmoSttiucdyPumps ED.. TIinsmsuunechiCsotlolcehcetmiiocnalandStaPirneipnagration F. Assessment of Cell Proliferation Rates 154-207 4 005104  WASHINGTON - TABLE OF CONTENTS (CONTINUED) XI. ASSAY EVALUATION CRITERIA . . . . . . . +. cov oven. 11 XII. INTERPRETATION OF RESULTS . . . . . vv vv vv vv vv vv www 12 A. Dose Rangefinding Study B. General Observations c. Summary of Labeled Cell Counts for the Liver VIL. REFERENCES . . oo ooeev eeee ennn nn ae o. XVIII. EXPERIMENTAL DATA TABLES . . . . . . . cv vv vv vv vv v nw 16 AppendiAx Histopathology Report . . . . . .. . ............ 21 154-207 5 005105 @ HAZLETON WASHINGTON ABSTRACT The purpose of this study was to determine the hepatotoxicity of T-5794 by measuring cell proliferation (CP) assayed as S-phase induction in rat liver cells after in vivo treatment. A dose rangefinding study with T-5794 was performed prior to initiation of the cell proliferation assay. In the dose rangefinding assay, T-5794 was administered to rats at doses of approximately 500, 1000, and 2000 mg/kg body weight. A11 rats remained healthy and appeared normal throughout the four-day observation period. Based on the lack of observable toxicity in the dose range-finding assay, a high dose of 3000 mg/kg was chosen for the assay. The cell proliferation assay was initiated at dose levels of 500, 1000, 2000, and 3000 mg/kg. Following a single oral dose of the test material for the cell proliferation assay, five animals per condition were labeled with BrdU for 72 hours using ALZET osmotic pumps. Two animals at 3000 mg/kg and one animal at 2000 mg/kg were found dead during the labeling period. Evidence for histomorphological alterations were observed at 2000 mg/kg and 3000 mg/kg as well as in the DMN positive control animals and in some animals treated with 5710.1. For the cell proliferation assay, sections from the left lateral, median and right lateral lobes of the livers as well as samples from the duodenum were fixed in 10% neutral Formalin, embedded in paraffin, and processed for immunohistochemistry. Each slide was prepared with sections from both liver and duodenum. The duodenum (a rapidly proliferating organ) was used as an internal control for delivery of label and immunohistochemical staining. The percentage of nuclei incorporating label in the liver was determined microscopically. Only hepatocyte nuclei were enumerated. Significant increases in cell proliferation were observed following treatment with the test material at the two lowest dose levels. However, the increases were small (0.05>p>0.01) and higher dose levels where toxicity was observed and cell proliferative likely to occur, were not significantly elevated. Therefore, no dose-related increase in cell proliferation was induced. T-5794 was therefore considered negative for the induction of cell proliferation in rat liver cells. 154-207 6 005106 ? WASHINGTON - a CAenlallyPsriosliofferTa-t5i7o9n4 Aisnsay in Rat Liver Cells I. SPONSOR: 3H Corporation II. MATERIAL TESTED: A. Genetics Assay No.: 154-207 B. Identification: T-5794 (L-13097) C. Physical Description: light amber solid D. Date Received: July 19, 1993 III. TYPE OF ASSAYS: Analysis of Cell Proliferation in Rat Liver Cells Iv. PROTOCOL NUMBER: 493, Edition 1, Modified for 3H Corporation V. STUDY DATES: A. Study Initiation Date: July 29, 1993 B. Experimental Start Date: August 2, 1993 C. Experimental Termination Date: September 29, 1993 VI. SUPERVISORY PERSONNEL: A. Study Director: Maria A. Cifone, Ph. D. B. Associate Scientist: Andrea L. Ham, B.S. VIL. OBJECTIVE: TT-h5e79o4bjebcytimveeasuorfintghiscelalssapyrowlaisfertaotimoenasu(rCeP)hempeaastuorteodxicaistyS-pchaausseed by induction in rat liver cells after in vivo treatment. rCeelp]licpartoiloinferiantiroant lmievaesrureudsintgheanfraicmtuinoonhiosftoccehlelnsicuanldertgeocihnngiquceell(1,2) to Adentiencatlsbrweormeodegoixveynuriadisniengl(eBrdoUr)al idnocsoerpoorfattehde tdeusrtinmgatDeNrAiaslyntahnedsitsh.e Tivers were isolated following administration of BrdU for 72 hours in 154-207 7 0605107 @ HAZLETON - WASHINGTON - vQiuvanotwiiftichatainonALZoEfTcelolssmottihactpuhnapveimipnlcaonrtpeodratseudbcDuNtAaneporuescluyr.sors over the 72-hour period indicates increased cell proliferation in the liver (3). VIII. DEFINITION: Hanepaitnoctraexaisceanitnsceslulchprasolicfaerrboantiotnetrtoachrleoprliadcee annedcrdoitniictrtoitsosluueen(e2,4i)n.duce TOhtehseer pchreomliicfaelrsatimnagy cienldluscemaSy-phbaesdeetiencttehde daubrsienngceSo-fphahseepataontaolxyisicsi.ty. It pisrocneosts apbpuatretnhterehowarecelnlumeprrooulsifmeeracthiaonnismmsaywhaiccth incanthebecaarfcfeicntoegdenidcuring rtheeplipcraotbiaobnili(t5y-8)o.f sCphoenmtianceaolulsy miuntdautciedonscelals wperlollifaseraitnicorneamsaeythiencrease pUrnosbcahbeidluilteyd coefllconpvreorltiifnegratuinornepmaiaryedalsDoNAplaadyducatsrolientoinmutthaetieoxnpsa.nsion of ptrreannesofpolransetdic_clopnoepuolfaticoenlsls.leadSionmge otof tthheeseemeerxgaemnpcleesofacta fbyullaynongneonnogteonxoitcoxmiecchcaarnciisnmogaenndsitasiwseltlheoasregteicnaoltloyxicpocsasricblienogteonsdetuescitng this technique. IX. MATERIALS: A. Indicator Cells oYlodungat adtuhelt tmianeleofratdsosionfg,thweerSeprapguurec-hDasaewdleyfrsotmraHianr,an10S-p1r2awgeueeks Dsatwrlaeiyn,'waFsredseerliecckt,ed MDto (mCarxii:CmDizBeR)g.enetTihcis hehteaelrtohgyeneriatnydomandbreadssure ahcocuessesd atcocoardcionmgontososutracned.ardAnoipmearlastinsgchepdruolceeddurfeosr atnhdiswesrteudyfewdere APnuirmianlasCweerrteifiqeudaranRtoidneendt Cfohrowat(Floeramsutla750d0a2y)s apnrdiowratteorraanddolimbitum. tahsesigdnomseentrantoge-sftiundydinggrouapsssayandandidbeyntiifmipclaatnitoanblweith an ear tag for Amniicmraolisdewnetirfeicaanteisotnhedteivziecdeprfiororthteo cseulrlgerpyrolfoirferparteipoanrataisosany.of cell ceuxlstaunrgeusi,natuesdingduraibonugtth6e0 mhga/rkvgestsodpiruocmedpuernet.obarbital, and were F2o5r5-3t3h4e gcreallns.proAlpipfreroaxtiimoantelayssa2y4, horuartss waefrteerusdeodsintgh,atthreangaendimaflrsom wIenrc.e) anienshtahleattiizonedanuseisntgheHseitaofaanndeeone(mAeLtZhEoTxyflpuurmapnep,erPiantimmaanl-Howoarse, ahosuerpsticTaatlelry, inasneirntaelds wseurbecuatnaensetohuestliyze(ddorwsiatlh CsOu,rfparcieo)r. toServeemnotvya-ltwo of the livers and duodenum (control organ). 154-207 8 05108  WASHINGTON : B. Osmotic Pumps and Label for Cell Proliferation Analysis AwLeZrEeTuseods.motAicsipnugmlpes l(oAtLZA(#C0o4r22p0o7r)atiwoans,usPeadlotAhlrsoou,ghoCuA)t,'thMeodes]tud2yH.LI TThhee ppuummppshwaesrea p20r0e0-fui]llecdapwaictihtyBrwdiUthata paumcponcreatnetraotfio1n0 uo1f/hour. 20 mg/ml. C. Control Articles 1. Vehicle control Agavvaegheicl(eP.0c.o)ntrwoi]thcothnesisvtehiincglef,iveOKCrat(shiwgahsvdiossceodsitbyy oral cTiasrsbuoexsyneftrhoynlcveelhliuclloesec,ont9r0o0l4-3a2n-i4m,alsSigwneareLostubj#ec1t2e1dF06t4o4)t.he sanaimmealmsa.nipuTlhaetidoonssingusevdolufnoer tofhethteissvueehsicldeericvoendtroflromanitmraelasted did not exceed 10 ml/kg. 2. Positive control article Trhaet phoespiattiovceytecsontirnolvivcoo.mpouTnhde pisosiktniovwen tcoontirnodlu,ce S-phase in dLoitm#eth29yFl0n6i7t9r)oswaamsinedos(eDdHN,atC1A5S.06m2g-/7k5g-.9, SFiivgenarCahtsemiwcearle Co., treated P.0.. 3. 3H Corporation cell proliferation control aTwnoimaaldsditfioornaelachcocnotnrtorloslwesruebstiannccleu.ded57c1o0n.s1iswtaisngionfclufdievde as daosneedgawtiitvhe caopnptrrooxlimaftoerlcyel8l10pmrgo/lkigferoafti5o7n1.0.1.Anim5a7l1s1.1werheas pbereonlifsehroawntiotno baessapyo.sitiAvneimailnsawesriengdloeseddosewitchellapproximately 1000 mg/kg of 5711.1. D. Test Article Fthoer ttheestpraerptaircaletiwoansofsutshpeenddeodsinign 0s.o5l%utiCoHCnsatofcotnhceenttersattiaorntsicloef, 5v0o.l0u,mes100f,or20t0heantdest300armtgi/cmlle pdirdiornottoexdcoseiendg.10 Tmhle/kgm.aximum dosing 154-207 9 05109  WASHINGTON - X. EXPERIMENT DESIGN: A. Dosing Procedure tFhievecerlaltspwreorleifetrraetaitoend bayssaoyralangdavtahgreeewirtathsthpeertgersotuparwteirceletrfeoarted wbeyreoraclalcgualvaatgeedfoorn tthhee dboasseisraofnget-hefimnodsitngrestcuednyt." aniDmeallivweeriyghvtoluamneds tshuespteanrsgieotnsdoasdem.inisTtheermeadxidmiudmnovtoluemxeceeodf t1h0emlt/eksgt. artFriecslhe parneypatreasttiiongnspuorfpotsees.t arCtoincfliermaitniovnehiocfletheandconccoenntrtorlastiownereofustehde for taessstaymawtaesrinaolt udnedteerrmicnoendditiinoncsonjoufncptrieopnarwaittihonthainsd dsotsuidyn.g of the B. Dose Range-Finding Study Tghreoupdso,seearcahngger-ofiunpdicnognsissttuidnygwaosf ptherrefeormmaeldeusriatnsg. thrTe-e579t4reawtasment Sroaltusbiweirzeeddosiend0w.i5t%hcaarbsoinxgylmeethtyrlecaetlmleuntlosoef sT-o5l7u9t4ionby(oCrHaCl).gavTahgee (o0r)toaxtic500s,ign1s000a,ndanmdort2a0l00itmyg/wekrgebopdeyrfwoerimgehdt,forDafioulry doabysse.rvatAitons tihnehaleandtioonf,thfeollfoowuerddabyys,penaleltraatniiomnalsofwetrhee tehuotrhaaxn.ized using CO, Cc. Inplantation of Osmotic Pumps wFeorrethperelceolaldedprwoiltihfer2a0t0i0o4n1asosfayB,rdAULZaEtTa Mcoodneclent2rHaL1tioonsmootfic pumps t2o0 msgt/amnld.ardThperocaendiumraelss waenrdeonaenesptuhmeptipzeerdanuismianlg wMaestofaasneepticacaclolryding aifntseerrteddosisnugb.cutTahneeouisnlcyisi(odnorswaals csluorfsaecde)witahppwroouxnidmatcellipys24andhoutrhse waenriemalnso cmloinniitcoarledsiugnntisl oftheinfteicmteioonf. sacTrheifiocsemottiocenpsuumrpes wtehraet there implanted three days prior to sacrifice. D. Tissue Collection and Preparation aEancahlysainsi.mal Thweasthaonreasctihceticzaevdityprwiaosr otopenreemdovaanld tohfeorTgiavnesr froermoved daunoddeFniuxme,d iantniesusturealwitbhuffheirgehdceflolrmatluirnn.overA,crwoasssalsseoctiroenmovoefd from teahcaht alanbiemlalwaasndadfimxiendi.sterTehde dcuoordreencutmlywatso eiancchludaendimaals. anFoirndtihcaetor Tliavteerr,al,5 pmuedpiaarnaffainnd erimgbhetddeadntesreictoironlsobewse.re tSaikmeinlarflryomprtheeparleedft sections of the duodenum were also made and a section of the 154-207 10 CLglio ? WASHINGTON - dacucoodredniunngwatso msotuanntdeadrdonproecacehdurselisdef.or Selxiadmeisnatwieorne baylsoa ppraetphaorleodgist to determine if any abnormalities were present. E. Innunohistochemical Staining TShleidessidoefstwheerelivdeerpawrearfefinsitzaeidnedandforrehdyedtreartmeidnatpiroinorotfocesltlaining. pthreoliBfieorgaetniexonprasimmaeraysuarneddsebyconidnacroyrpaonrattiiboondieosf wBirtdhU pientrooxDiNdAaseu-sing ctoentjruaghaytderdochsltorreipdteavi(dDiAnB)ancdhroamo3g,e3n-dianadminhoebmeantzoixdyilnien counterstain. Paanraallylseils sbylidaespawtehroleogsitsati.ned with hematoxylin and eosin for F. Assessment of Cell Proliferation Rates TthheatsethcetioTnabeolf wtahse dpuroodpeenrulny waadsminmiisctreorsecdopitcoaltlhye eanxiammailn.ed Otnoceensure Teaxbaenlineddelifvorerylowbauslarcondfiifrfmeerde,ncessl.idesInfrtohmisthsetuddyi,ffelraebnetlinlgobweasswere swiitmhilasrecatmioonngs tfhreonlotbhees,ieftCelllatecroaulntilnobgesw.as tThheerepfeorrceenpteargfeoromfed nmuicctreotscoipniccoarlployr.atin~TgheTaabreelasintothbee 1ciovuenrtewdaswerdeeterramnidnoemdly generated b1y0 cxom1p0utseqru.areAs w1a.s0 musmedsqutaoredefiinndeexetdheoccuoluanrtinggridaredai.videAdt lienatsot 2an0d006nufcileelidswepreer esexcatmiionne.d per animal with a minimum of 3 sections Acnoyntanuicnlienig tanhyatbwreorwen bclhureomwoegreenicconhuseidweerreed ucnolnasbiedleerdedanldabealneydnuclei uennulmeesrsataedc.learFiealrdtsifatchtatwacsontpareisneendt.areaOsnlyofhenpeactroosciystewneurceleniotwere included in the evaluation. The slides were coded for (blind) evaluation as to treatment group. S-phase nuclei labeling indices for each animal were calculated as follows: Labeled S-phase nuclei (LI)=nh too.taoe lflnp oa.bea olfedt hepaotocc ytey s nct ouucnle teeid X 100 XI. ASSAY EVALUATION CRITERIA Acamlecaunlataendd sfotrandeaarcdh dterveiaattmieonnt gfroorutpheusipnegrcetnhetagiendiovfidSu-aplhasaenimcaelllsmeawnere Sus-ipnhgaseonvea-lwuaeys.analSytsaitsistoifcavlariaannaclyesitsechonfiqluaebseli(n1g2).indeCxontwraosl peverrfsoursmed treatment group comparisons were done with Dunnet's t-test (13, 14)and 154-207 1 ood &@ WHAASZHLINEGTTOONN : control versus positive and 3M Corporation control group comparisons were done using the Student's t-test. In the case of variance heterogeneity, rank transformations of the data were performed prior to analysis of variance and Dunnet's t-test. An S-phase percentage in a dose group that deviates from the S-phase percentage in the concurrent control group at a significance level of p<0.05 was considered significantly different than the control group. The same procedures were used to analyze terminal body and liver weights. XII. INTERPRETATION OF RESULTS A. Dose Rangefinding Study Three groups, each containing three male rats were dosed P.0. with a single dose of T-5794. The dose levels were approximately 500, 1000, and 2000 mg/kg body weight. For four consecutive days, the rats were observed for mortality and toxic signs. All rats remained healthy and appeared normal throughout the four-day observation period. Since, no overt toxicity was observed, it was decided that the cell proliferation assay would be initiated with four treatments (500, 1000, 2000 and 3000 mg/kg). B. General Observations Treatment-related histomorphologic alterations were observed in some animals which received T-5794 at levels of 2000 and 3000 mg/kg, 5710.1 at a level of 810 mg/kg, and DMN at 15 mg/kg. Affected animals had evidence of hepatocellular injury (hepatotoxicity) and this was characterized microscopically by scattered foci or centrilobular areas of necrosis which in some animals extended to portal areas, or necrosis of individual hepatocytes, chronic-active inflammation and, in some animals, vacuolization. Animals which received T-5794 at 2000 mg/kg had minimal to slight changes whereas at the 3000 mg/kg level these changes were all slight. Details of the histopathology are in Appendix A. Two animals dosed with 3000 mg/kg and one animal dosed with 2000 mg/kg of T-5794 were found dead prior to termination of the BrdU labeling period. The presence of dead animals at 2000 mg/kg in the definitive assay and not in the dose range-finding study may be explained by the additional trama from the pump implantation that occurred in the cell proliferation assay. Cells stained with the brown DAB chromogen were observed in the duodenum from all of the animals used in the study. The presence of label in all the animals indicated proper delivery of the BrdU label and acceptable immunohistochemical staining. 154-207 OOSIAR 12 @ HWAASZHLINEGTTOONN : . The livers of the treated animals did not show a dose-related increase in mean weight compared to control animals. The mean Tiver weight of the positive control was also not significantly elevated even though large increases in DNA synthesis (and subsequent cell proliferation) were induced. However, the 5510.1 treated animals had liver weights that were significantly lower than the control animals. Mean terminal body weights of treatments at 1000, 2000, and 3000 mg/kg and the DMN positive control mean animal weights also were significantly less than control values confirming the presence of toxicity. C. Summary of Labeled Cell Counts for the Liver A summary of the labeled cell counts for each group is shown in Table 1." Individual animal counts are shown in Table 2. The mean labeling index (LI) for each group is presented in the third column in Table 1. There was no apparent preferential labeling in any of the lobes and the label was random within the lobes. The mean background labeling index (Group 1) was 1.61 which indicates that less than 2% of the nuclei had undergone DNA synthesis during the 72-hour labeling period. Significant increases in the labeling index were induced by T-5794 in the dosed Groups 2 and 3 (500 and 1000 mg/kg). The labeling indices at 500 and 1000 mg/kg were 4.55% and 4.43%, respectively. These represent 2.8-fold increases over background (ps0.05). "While these increases are considered significant, higher dose levels (2000 and 3000 mg/kg) did not show increases in cell proliferation. Toxicity was observed in animals at 2000 mg/kg and 3000 mg/kg as represented by animal death and histopathological changes in the liver. Since the increases in cell proliferation that were observed were not dose-related and not associated with the toxic doses, T-5794 was not considered positive for cell proliferation. The mean labeling index of the DMN positive control animals was 44.95 which is significantly elevated (p < 0.01). The 3M control compounds behaved as expected, with 5710.1 being negative for cell proliferation and 5711.1 inducing a positive response (p<0.01). XVI. CONCLUSIONS The test material, T-5794, did not induce significant dose-related changes in the number S-phase cells following a single oral dose of T-5794. The animals were labeled for 72 hours and increases were observed at the two lowest dose levels, but higher doses were negative. T-5794 was therefore evaluated as negative in the induction of cell | proliferation in rat liver cells. 154-207 . 13 065113 2 WASHINGTON - XVII. REFERENCES 1. D(e1F9a8z7i).o, AT.n,muLneoahriys,tocJh.Ae.n,icaHledldeeyt,ectD.iWo.n aofndprTaotltiefresraaltli,ngM.cHe.iNl.s in vivo." J. Histochem. Cytochem. 35, 571-577. 2. L5a-nbireorm,odeT.oLx.y,uriBderigneer,andE.KH.-,thaynndidEianceho,in Pr.oId.en(t19h8e9p)a.toCcoemlplaurliasron of proliferation studies. Toxicologist 9, 64. 3. BMiurtstaelriwso,rthJ,., B.PEr.o,bstA,shbGy.,, Ja.n,d GB.erWmiuldleiza,ns:E'., ACaspcrioatnooc,ol D.a,nd guide for the in vivo rat hepatocyte DNA-repair assay. Mutation Res., 189:123-133, 1987. 4. MDiNrAsaslyinst,hesJi.sC. ianndratButhteeprawtoorctyht,esB.fEo.l:lowiInngduictniovnivooftruenastcmheendtulweidth dinitrotoluene. Carcinogenesis, 3:241-245, 1982. 5. RMaerlsamtaino,nshDi.Sp.,ofCahtetplaetyi,c pR.eCr.o,xisCoomnweayp,rolJ.iGf.e,ratainodnPoapnpd, rJe.pAl.ica(t1i9v8e8). DprNoAlisfyenrtahteosriss dtio(2t-heethhyelphaetxoycla)rpchitnhoagleantiecitayndof[4t-hcehlpoerroo-x6i-s(o2m,e3xCayn1c1edrinoR)es-.2-p4y8,rim6i7d3i9n-y6l7t44h.io] acetic acid (Wy-14,643) in rats. 6. Ccarnacdedro.ck,In:V.M".liv(e1r976C)e.il CCealnlcerp"r,olCiafmeerraotni,onH.aMn.d,exLpienrsiemlle,ntaCl.A.livaendr WAanrswtiecrkd,an.G.P., Elsevier, North Folland Biomedical Press, 7. RCoelquumibraenmoe,ntA.o,f cRealjlalapkrsonlii,ferS.a,tioanndfoSarrmtah,e Di.nSi.tRi.ati(o1n981o)f. liver cReasr.ci4n1o,gen2e0s7i9s-20a8s3.assayed by three different procedures. Cancer 8. oGlfinToisv,erA.rDe.g,eneBrutacthieorn, onN.Lt.umoRr.', foanrdmaAtuibo,n Ji.nC.rat(s195f1e)d. The effect 4-dianinobenzene. J. Exp. Med. 933, 313-324. 154-207 1 005114 : WASHINGTON - 9. DWeisnteirn,,B.HJe.tra(w1-9H7i1)l.i,StNaetwisYotrikc,alznPdriEndciitpiloens,ipnp.Ex1p4e8r-i2n2e0n.tal 10. CDuommmpeatrti,ngC.sWe.ver(a1l955t)r.eatmAenmtusltwiiptlhe acocmopnatrrioslo.n Jp.roAcne.durSetatf.orAssoc. 50, 1096-1121. 11. cDounntnreotlt., BCiMo.met(r1i9c6s4).20,Ne4w82-t4a9b1l.es for multiple comparisons with a 12. Hsatmu,dyA.Tivaenrd eCifffoencet,s Mi.nAd.uce(d199b1y).a sUisnegleofdoceslel ofprDoWlNi.ferEantviiornonmteontal 2nd Molecular Mutagenesis 17(19), 16. 156-207 15 005115 @ HWAASZHLINEGTTOONN : XVIII. EXPERIMENTAL DATA TABLES 154-207 005116 16  WASHINGTON Client: 3M Corporation Client Code: T-5794 . Table 1 Cell Proliferation Summary HUA Assay No.: 154-207 Trial Initiation Date: August 16, 1993 Group/Sex?| Dose Level| (mg/kg) Labeling Index | (%) Liver Weight (grans) | VTeerimgihntal(grBaomdsy) TM [4 161: 0.71 | 12.99% 1.12 322.5 15.6 on 500 455+ 1.23% | 13.62% 1.24 309.6 + 20.9 Mm 1000 4.43% 2.83%| 14.07 2 1.04 307.0 + 38.5 we 2000 271% 1.09 | 12.01% 1.84 251.0 30.10% snd 3000 119% 072 |13.07+1.28 | 263.7% 21.8% Mf 810 1.80 + 1.22 | 12.26 + 0.45%| 255.2 + 14.5% TM 1000 6.62% 1.24%1) 15.02% 2.03 | 311.6 + 35.3 ah 15 44.95% 8.75%%1( 12.10 0.84 | 306.6 + 23.7% ""FPievreceanntiagmealsofpelrabgelreodupheupnlaetsoscytiendniuccalteeid per total number of hepatocytes counted "(FaoturTeaansitma2l0s00p)er group "sTVherheiecleanicmoantlrsolp,er1g0romulp/kg of CHC "`55771110..11 CCoonnttrrooll,, 8110000nmgg//kkgg "Positive control, 15 mg/kg of DMN *** SSiiggnniiffiiccaanntt aatt 0p.'0s10<.01p < 0.05 +4 DIenccrreeaassee iinn tthhee mmeeaann 154-207 17 095117 ? WASHINGTON - . Animal Number 8B4411117710 BB4411117732 B41174 GGrroouupp MSeDan N B4B411118801 B4B411118832 B41184 GGrroouupp MSeDan N B84411119901 4B411119932 B41194 GGrroouupp MSeDan N Group /Sex JJ]] JJi ] TM i2] |on Mn 33MM 33MM MH Table 2 CeIlnldivPirdoulailferAnaitmiaoln ADastsaay LMaebaenling Index(%) 12..9005 21.1343 0.62 10.671 5 63..4587 33..7866 5.10 41.2535 5 13..9557 49..1290 3.24 24..8433 5 TBeordymiWneailght (a) 330134..00 338324..00 319.4 13252..56 5 239476..00 239099..00 297.0 23009..96 5 326620..00 331146..00 283.0 33087..50 5 LivTeerrmiWneiaglht (a) 1112..4967 1134..2596 12.69 121.1929 5 1152..7866 1133..2517 12.1 131..6224 5 1133..1388 1155..2147 13.40 141..0074 5 154-207 18 05118 2 WASHINGTON - Animal Number 84B411220010 B4B411220034 GGrroouupp MSeDan N BB4A11221120 B41213 GGrroouupp SMeDan N 8B4411222210 BB4411222232 B41224 GGrroouupp MSeDan N 884411222256 BB4411222278 841229 GGrroouupp SMDean N Group Sex aa a" SHH oH 6oHH ooHH oH TMTM TMTM TM Table 6 (Con't) CeIlnldivPirdoulailfeArnaitmiaoln ADastsaay Mean Labeling Index(8) 24.3338 2.1.1995 1.2.0791 4 02..9050 0.62 10..1792 3 30..8015 03.1547 1.43 1.18202 5 58..5677 65..1900 6.86 61..6224 5 Terminal Body Weight (a) 226638..00 222276..00 23501..10 4 228638..00 240.0 22613..87 3 22493..00 228505..00 249.0 124857.52 5 332155.000 253306..00 332.0 315..36 5 Terminal Liver Weight (a) 1133..2478 131..7498 121..0814 4 1124..7458 11.99 131..0278 3 1122..1045 1132..0225 11.85 102..4256 5 1155..2811 1117..6027 15:37 125..0032 5 154-207 19 005113 t WASHINGTON Animal Mumber B84411223310 BB4411223323 B41234 GGrroouupp MSeDan Group /Sex aa aa El Table 6 (Con't) CeIlnldivPirdoulailfeArnaitmiaoln ADsastaay LMaebaenling Index(X) 4567..4388 4344..8169 32.86 448..9755 TBeordymiWneailght (a) 239359..00 238213..00 295.0 30263..67 LiTveerrmiWneailght (a) 1112..4766 113.411 12.02 102..8140 154-207 20 005120 @ HAZLETON : } WASHINGTON Appendix A Histopathology Report 154-207 0605121 21 ShazusToOyN : - Pathology Report Analysiisn RofatCeLlilverProClelilfseration Project No. 154-207 Test Material - T-5794 GeneralProtocol The objective of this study was to detect hepatotoxicity caused by the test material by measuring cell proliferation as S-phase induction induced in rat Tiver cells after in vivo treatment. The pathology portion of this study consisted of microscopic examination of duplicate sections, stained by HE methods to detect any treatment-related histomorphologic alterations. Male rats were divided into 8 groups and assigned to study as follows: Group 1 animals received carboxymethylcellulose; Group 2 received T-5794 at 500 mg/kg; Group 3 received T-5794 at 1000 mg/kg; Group 4 received T-5794 at 2000 mg/kg; Group 5 received T-5794 at 3000 ng/kg; Group 6 received 5710.1 at 810 mg/kg; Group 7 received 5711.1 at 1000 ng/kg and Group 8 received DN at 15 mg/kg. The Tatter three groups were common to a companion study. The following is a sumary of histomorphologic findings. Resanu dDilscutssison Treatnent-related histonorphologic alterations were observed in some animals which received T-5794 at levels of 2000 and 3000 mg/kg, 5710.1 at a Tevel of 810 mg/kg, and DMN at 15 mg/kg. Affected animals had evidence of hepatocellular injury (hepatotoxicity) and this was characterized microscopically by scattered foci or centrilobular areas of necrosis which in some animals extended to portal areas, or necrosis of individual hepatocytes, chronic-active inflammation and, in some animals, vacuolization. Animals which received T-5794 at 2000 mg/kg had minimal to slight changes whereas at the 3000 mg/kg level these changes were all slight. Three of five animals which received 5710.1, a positive control, at a level of 810 mg/kg demonstrated evidence of slight to moderate hepatocellular injury. Five animals receiving TS711.1 at a dose of 1000 mg/kg had no evidence of hepatocellular injury. All animals which received DMN at a level of 15 mg/kg 154-207 2 005122 2 WASHINGTON -a- 154-207 showed evidence of minimal to moderate hepatocellular injury. The Individual HistopathologyFindings table details all lesions, severity grades and distribution in individual animals from all groups. Conclusions Hepatocellular injury occurred in groups of animals receiving T-5794 at Tevels of 2000 and 3000 mg/kg, 5710.1 at a level of 810 mg/kg and DHN at 15 mg/kg. Pathologist: Bru MN? DBioprlgoemaHt.e,UllAamnedr,icaDn.V.CHo.i.lege of Veterinary DepPaartthmoelnogtisotfs Pathology (-2a$-49y Date -- 154-207.94 154-207 - 2 005123 @ HAZLETON J WA SRAKER 0 N HAUDADENSDTUSUTDNOOY.PROTOCOL FO. 493 5a Ja LewaA&/| DAROSCAEELNLGPREOLIFFEIRANTSIDTOUNIADYSNSFAOGYR hup.liaotf %sq Rico= ORAL GAVAGE ADMINISTRATION Mitbbyn fo.fox. HGaozoldetLoanboWraasthoirnygtPorna,ctiIcnce. (G(LiPA))Rewgiulllatcioonndsu.ct Tthhiiss psrcoutdoycoiln, cocmrpiltiiacnaclephwaisteh(s) Aosfsutrhaencveorkininacpcroorgdraenscse awnidthfiSnOaPls raetpoHratzlweitloln bWaeshsiunbgjteocnt, toInca.uditThbiys Qsutauldiyty WIT be conducted by HiA at 9200 Leesburg Pike, Vienna, Virginia 22182. PART 1. SPONSOR INFORMATION AND APPROVALS I. sPousoRIDENTIFICATION Company Name: 3 Corporation Address: Building 220-26-021 3H Center, St. Paul MN 55144-1000 Ir. TESTARTICLE IDENTIFIGATION: T5794 anoT-5195 INI. TESTARTICLEANALYSIS DCeetreirsmciicnsatiaosndeoffintheed itnestthearGtLiPcleregsutlaabtiilointsy aofndFDtAhe(2t1estCFRarc5i6c.1l0e5)c,haErPaAcTrSeCsApon(s40ibCiFlRi.cy792o.f10t5h)e, SpaonndsoErP.A-FIFRA (40 CFR 160.105) is the IV. OTIFOIFRECGULAATORTY SIUBMOISSNION I5n8.1o0r;derEPAt-oTScCoAm,ply40wiCcFhR U7.9S2..10f;edeEPrAa-lFLrFeRgAu,la4t0ionCFRc.od1e6s0.1(0F)DA,and21cCeFrRtain Pfaorreeigonf sagesntcuideys,iscotnosubeltisnugbaliacbtoedrattooritehse maguesntcyb.e nHotAifmiaeidntiafinasllaor Riansdtiecratescvhheiduclhe agoefncsyt,udiLefs awnyh,ichBigfhatllreucnedievre rethgeularteosruyltsrevoifewt.hisPlsteuadsye: -- 5 tntecernines 20 ma 0 meactsca C--0 eeacrima -- marr -- J wow puJlo)mo = "2Jomm BEA. 9 Toe? 005124 WASHINGTON Aon To FROTOGOL 493 Vv. STUDYDATES - seoposed Experimental. Start Dever Proposed Experimental Termination Dace: VI. AROP FSR TUO DYPV ROA TOCL OL seuty Director: Tari A Cfo Fo Sponsor's Authorized Repeosencacive: Choglibiea SO Date: Nulia20,4993 75 ret 005125 2 WASHINGTON ADDENDUM TO PROTOCOL 493 DARPO_SCAREELNOLLGIEFFEIRNASDTTUIDAIYSNOSFAONGYR ORAL GAVAGE ADMINISTRATION 1. Purpose 2. HWA Study Coordinator 3. Experimental Design A. Animals A) species (2) strain/Source (3) Age at Initiation @) Nusber/sex (5) Number/Group (6) Identification (7) Husbandry (a) Housing Dperfoilnieferaatdoisoen rsatnugdeiesfortocbeell conducted in rats. Andrea Ham, B.5.. Rat YDoauwlnegyasdtulrtaimnalpeusrcohfasethdefSropnraHgaurel-an SSpprraagguuee--DDaawwlleeyy,(SIDnOc.BR)(HoSrD:CHhaarrllaens R(iCvre1r:CLoabBoRr.)atories, Inc. 10 - 12 weeks 9 males 3 males per group Ear tags AfLoLlloavpepdl.icabAlneimHalAs SwOiPlsl wbiellisboelated bpeyrscexa.ge Adnuirmianlgsqwuiarlalntbieneh,ousbeudt wtiwlol bienihtioautsieodn.singSlaynitparriyorcatogesexwpielrlimbeent uasneidm.alsPweirtshoninnelthheanadnliimnagl fthaecility . wpirloltebcetivreequgiarremdenttso waenadreqsuuiiptmaebnlte. 7/93 307 005126  WASHINGTON ) Food () Water (@) Environment () Quarantine (8) Randomization (9) Justification B. Study Design ADDENDUM TO PROTOCOL 493 Purina Certified ad Liblcun. Feed RiosdeanntalGyzheodwby500t2h,e msapneucfiafciteudrehreavfyormectoanlcse,ntraaftlaitoonxsino,f corhglaonroipnhaotsepdhahtyedsr,ocaarnbdonssp,ecified nfuitlreiewnitcsh. theThimsanuifnafcotrumraetri.on is on aTanpalwyazteedr,biaadnnLuiabllloyuno.n aThe vater is mriectrrooosrpgeacntiisvnes,baspeisstifcoirdessp,echiefaiveyd metals, alkalinity, and halogens. mEavienrytaianttetmepmtpewrialtlurebse wmaidtehinto 57258F1456.F wAit1h2-haourrelaltiigvhet/h1u2m-ihdoiutry of dark cycle will be maintained. aAcLLlaenaismtalssevewinlldaybse aqfutaerranrteicneeidptfor wfirlolm trheeleassuepplailelr.aniAmalvseteprriinoraritoan the assay initiation. Unussibnegrsc.ompAuntiemra-lgsenewrialtledberuannidqoumely girdoeunptsifviieldlbbyeeairdetnatgi.fieTdrbeyatmceangte Tabel. Rcaetlsl hpriosltiofreircaatliloynhasvteudibeese.n used for Tthhereestutdryeawtimlelntbegrocuopnsd.ucteEdachusionfgthe three groups will consist of 3 male zats. 7/93 wot 7 005127 2 WASHINGTON ADDENDUM TO PROTOCOL 493 GC. Group Designation and Treatment Regimens Number of Rats Duration GroupNo, Male Route (Dave) 1 3 0 " 2 3 20 " 3 3 20 " D. E. F. 7/93 Dosing Procedures (1) Dose Volume (2) Dose Levels (3) Duration of Treatment Dosing Formulations (1) Preparation of Dosing Formulations (2) Storage of Dosing Formulations Animal Observations (1) Clinical Examinations Tohrealroguatveageof (aPdO)m.iniTshterattieosnt will be material will solubilized in one of the following solvents: water, 0.5% aqueous carboxymethylcellulose solution, or corn oil. ANlolt atonimeaxlcseewdil1l0 bmel/kdgosbeoddbyasweeidghutp.on individual body weights. Assigned by Protocol Amendment Adlalysanaifmtaelrs rweiclelivibengeauthsainngilzeedd4ose. Dosing formulation will be prepared just prior to dosing. Solutions will be prepared at daomsbiinegnt (t0e-2mpheoruartsu)r.es and held until aDnadilmyorotbaselrivtaytifoonrs,thefordurtaotxiiconsiogfns the study. 50f 7 005128 @HWAASZHLIENGTTOONN t RecaondrRepdorscs @ oconds Maintained AsoEmN To FROTOGOL 433 orton Tesesmanion of the shores TTrEaptonsutseidotno sseecriemeaitiss.iahsedRateasim Tolerated Dose (MID). Doses will ALL av data, documentation, pre April 005129  WASHINGTON ADDENDUM To PROTOCOL 493 + aTybpeero.f assay and protocol o Dcaotmepsletoifons.tudy iniciation and + Hveatlhuoadts.ion cricerta. ++ CTonnccelrupsrieocnasc.ion of results. + TCeasbtularresufolrtms. presented in + References. J. ANIMALCARE ANDUSESTATEMENT mIenthtohdesopsrienioapnproofprtihaeteS,tudtyheDisrteucdtyord,oesnomsotltdeumpaltiicvaetetesnsytinpgrevious Tpoprrkopwriitahceh.isThmaicserpiraolt,ecealndWitlhle bmeambreervieawneddsbpyecitehse HsIelAeTcAtCeUdCarfeor SoErpilnttaanaclse.iIhE nroetguliantocroymplgiuaindceel,ineasmocdoinfciecranitnigontwhiellcabree and use BTeequsiernetd.to Atnhye MchiaAnTgAeCsUCofforrevitshieoinrsreovfietwh.is approved protocol will K. CHAORNREG VIE SIOS NS Aidnoiycnumcehtnahtniegsde,sproostriagcnreeeldv.ibsyioTnhcseheSopSfotnutsdhoyisrDviairplepclrtoobvree,dnopdarttoietfdoi,ceodlaonfwdilmalaniybnetcahiannegde of Tevision 9 Tee 7 005130 3M Internal Correspondence cc: To: From: Subject: Date: R. G. Perkin-s 220-2E-10 B. C. Copley - 53-35-02 R. D. Howells -53-35-02 Key to FC Alcohol Tox Samples September 14, 1995 T5877 TS710 T5711 He TS794 T5795 T5878 `Wide Range EtFOSE Analytical Request 41220 FM 3924 Lot 547 Retain from 2 year feeding study ~~ L-13203 `Narrow Range EtFOSE Lot 884 "Typical Raw Material for FC-807 Wide Range MeFOSE Lot 555 Typical Raw Material for FX-845 Narrow Range MeFOSE Notebook 97900-107-2 Lab Prepared Sample Wide Range EFOSE Lab Prepared Sample `Wide Range MeFOSE Lab Prepared from `Washed POSF Analytical Request 41220 L-10059 Analytical Request 41220 L-1276 Analytical Request 41343 L-13097 Analytical Request 41343 L-13098 Analytical Request 42607 IEDR Attachments 005131 REQ 41220 R. Howells - in [eroe [pm | GC/MS analyses of these samples were accomplished using a 25 m X 0.32 mm HP-1 GC column to introduce the samples into the Finnigan SSQ-70 mass spectrometer. The sample components were ionized ruastienogfc1h0emdiecgarleeisonpiezratmiionnutwei.thTmheetrheasnuletsaosftthheerseeagaennatlygasse.s cTohmebiGnCedcowlituhmnthweiarscoorprereastpeodnfdrionmg4GC0/tFoI3D0a0rCeaata percents show the following: Component LD. NR N-EFOSE Lot |W.R.N-EFOSE Lot 547 erossess comsonemy |oms ons | lomsonaolcoum s oonmeon| lopsonmoopne c[nuno a n| lcmsonaoleouns o[n imeor| lolpmoonen s sonma lloows nsee[s oiwussor|| llomsoonernssonaocloomnwsson|oeowsmn| |cosonmonoimroc[ooonenon ons 005132 Component ID. L1276 = N-McFOSE Lot N-Methyl-carboxamides (Rr-C(O)N( 146% C2FsSOIN(Me)CH2CH20H 024% -- J CAF9SO2N(Me)CH2CH20H CsF11S02N(Me)CH2CH20H CeF13S02N(Me)CH2CH20H CsF17S02N(Me)CHaCHICI CrF15S02N(Me)CH2CH20H N-MeFOSE_ CsFi7SO2N(Me)CH2CH20H CoF19S0IN(Me)CH2CH20H cosonuas CoF17S02N(Me)(CH2CH20) Liss| lee| lion | [ins | loose | lose | 169% 83.88% 087% 034% Further work has been done byGC on these samples which involves derivatizationofthe alcohols with trifluoroacetic anhydride (TFAA) and again with BSA (to give the trimethylsilyl ethers). This work was designed to investigate the potential problems that could be overlooked by any one method of analysis. Preliminary results show that analyzing the EtFOSE underivatized could hidea significant amount of N-E(FOS Amide under the C-3 alcohol peak in wide range material. However, analyzing the same material that has been dbeyritvhaetdiezreidvawtiitzheTd FC-A8Aalschoohwols.thTahteanBySEAtFdOeSriEv-acthilvoerhiadsentohtatbeisenpreevsaelnutiatnetdhyeets,abmuptlesiimsilnaorwprcoobmlpelemtsealryemasked expected because of the numberofdifferent components in the sample. The same sortof problems will most likely exist with MeFOSE and wil be even more complicated in the analysis of MeFOSEA. 712093 005133 REQ 41343 J. Grant (GC/MS analyses of these samples were accomplished using a 25 m X 0.32 mm HP-1 GC column to introduce the samples into the Finnigan SSQ-70 mass spectrometer. The sample components were ionized using chemical ionization with methane as the reagent gas. The GC column was operated from 40 to 300 C at a rate of10 degrees per minute. The resultsofthese analyses combined with their corresponding GC/FID area. percents show the following: `Component LD. NccrssAoongicon N-Ethyl-carboxamides (Re-C(O)N( J I---- N-EtFOS Amide( CsF17SO2N( C4F9SO2N(EY) OH CsFuSO2N(Et)CH2CH20H I J---- CeF13SO2N(EY)CH2CH20H `W.R. N-EtFOSE Precut 97900-112-1 `W.R. N-EtFFOSE `main cut 97900-112-2 [izes 68.34% Jone127% [[ oe oe| Loe ome | lJ i04n1%e o leee| | os lows| 0.62% 042% 148% 2.16% 20.16% [ex lume| a 60.86 % Component LD. N.R. N-MeFOSE B.P. 132 97900-107-2 005134 lorsonomenloeenom| lommono Jee e s| oesignJoi ma s| lopsonan Amide 97900-111 Jee | Sm =| |nc orossm me sonle owsn| lorsonen lee |] that include hydrides, chlorine in the backbone, and trace unidentified high boilers 005135 To: From: Subject: Date: 1. Muggli 53-65-02 R.M. Payfer 2362811 (612)733-4212 SA&C Analytical Request No. 42607 Dec. 21, 1993 iGnCt/roMdSucaentahleyssiasmopfltehiisntsoatmhpeleFiwnanisgaanccSoSmQp-li7s0hemdasussisnpgeact2ro5mmeteXr0..3T2hme msaHmPp-l1e cGoCmpcoonleunmtnstwoere fironoimz4ed0 u1s0i3n0g0chCemaitcaalraitoeniozfat1i0ondewgirtheemsetpehranmeinaustet.heGrCeaagneanltysgiass.witThheflGamCe cioonliuzmatniownadsetoepcetriaotnewdas amlassosdosnpee,cawnordkt.heTahreearepseuirtcsenoft tvhaelsueesanfarloymstehsis(wwohirckhwaerreenaoptpnleiceedstsoartihleypqeuaanktiitdaenttiivtei)ess hfroowmtthhee following: Mol. Weight |Component LD. 13202 N-MeFOSE N-Methyl-carboxamides CyFis-C(OIN(Me)H| 0.06% |sr curusomuer lose|n I-- loss|n IP -- C4FsSO2N(Me)CH2CH2OH Lose| |so |oCroFu1s3o80m2uNm(Moer)CeHcCrH2oOnH 338% N-MeFOSE _CoF17S0zN(Me)CH2CHZOH |sis Josusommaoncnon |er |cirusomuacricsuon Loss| | sn |rucsonmcrcon [ess | CeF16SF5-S02N(Me)CH2CH20H 0.29% 005136 [on |CaF17S02N(Me)(CHzCH20)2H 005137