Document jmZpjObr8Q4Drk1QJL9rbBE2O

2 HWAASSHHIENGSTOAN 154-209 ANALYSIS OF T-5878 IN A CIENLLRAPTROLLIIVFEERRACTEILOLNS ASSAY EINAL REPORT. ATOR Maria A. Cifone, Ph.D. PERFORMING LABORATORY Viema, Virginia 22182 Hazleton Washington, Inc. 9200 Leesburg Pike ORATORY HWA Study No.: 154-209 MITTED T 3M Corporation Building 220-2E-02 3M Center St. Paul, MN 55144-1000 STUDY COMPLETION DATE November 1, 1994 7 5 ' 005138 1 of 34  WASHINGTON QUALITY ASSURANCE STATEMENT PROJECT TITLE: Analysis of T-5878 in a Cell Proliferation Assay in Rat Liver Cells PROJECT NO.: 20991 PROTOCOL NO.: 493 HWA STUDY NO.: 154-209 EDITION NO.: 1, Modified for 3M Corporation Quality Assurance inspection(s) 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 general requirements of the appropriate Good Laboratory Practiceregulations. Findings from the inspections and final report review were reported to management and to the study director on the following dates: Inspection/Date Dosing and pump implantation (surgery)/ 2-7-94 Draft report review/ 7-12,13,14-94 Final report review/ 11-1-94 FindingsReported 2-8-94 7-14-94 11-1-94 Auditor B. Mullett B. Mullett B. Mullett FwquateilAsPsuraancie lUnt DfateioReleased 154-209 2 005139 2 WASHINGTON COMPLIANCE AND CERTIFICATION STATEMENT The described study was Practice Regulations as conducted set forth in in compliance with the the Code of Federal Good Laboratory Regulations (21 CFR 58, the 40 CFR 792, and 40 CFR 160). aforementioned regulations or There were the signed no significant deviations from protocol that would affect the integrity of the study or the interpretation of the test results. The raw data have been reviewed by the evaluation of the test article Study Director, who as presented herein certifies that the represents anappropriate conclusion within the context of the study design and evaluation criteria. Al raw data, documentation, records, protocols, specimens and final gFeanetratleedastas oanereyseuarltfoolflotwhiisngstsuudbymiwsislilonbeofarthcehivfeindalbyrHeapzolrtetotno ftohre reports aspponesroiro.d After the one year period, the sponsor may elect to have thesematerials retained in the storage facilities of Hazleton for an additional period of time or sent to a storage facility designated by the sponsor. SUBMITTED BY: fondo Z Horn Andrea L. Ham, M.S. Associate Scientist /i)94 Date Study Director: (7l? on Maria A. Cifone, PH. D. Study Director Genetic and Cellular Toxicology ltd Study Completion Date 154-209 3 005140 ? WASHINGTON TABLE OF CONTENTS PAGE NUMBER ABSTRACT + +e eee eieiine eee oeenes 6 To SPONSOR . ov vowovww eee T II. MATERIAL TESTED . . vows 1 A. B. GIedneenttiicfsicAastsiaoyn No. Cc. D. PDhaytseicRaelceDievsecdription III. TYOFAPSSAEYS v voovvooooenennnnnnns 1 IV. PROTOCOL NUMBER . ov www vvvvvwwoonnnnnnon 7 Ve STUDYDATES . oo ooo eeannneenneeeeeneeeeee 1 BA.. ESxtpuedryimIennittailatiSotnartDatDeate C. Experimental Termination Date VI. SUPERVISORYPERSONNEL . oo vv vv vee vee eencooone 7 AB.. ASstsuodcyiaDtierecStcoirentist VIL OBJECTIVE ove veeeeeeennneeeeeeeeeeees 1 VIIL DEFINITION + oe eee eeeneeeeeeeeeeeeoees 8 IX. MATERIALS . + oo ooeo noo eoem aam n mn 8 A. B. OIsnmdoitciactorPumCpesllsand Label for Cell Proliferation Analysis C. D. CToensttroAlrtiAcrlteicles X. AE.XPERIMDEoNsTinDgESPIrGoNced ureooo vvvvve eevee eooonoees 9 B. Cc. TIimspsluaentaCtoilolnectoifoOnsmaontdicPrePupmaprsation D. E. AsIsmemusnsomheinsttoocfhemCielclal PrSotaliinfienrgation 154-209 4 005141 ? WASHINGTON TABLE OF CONTENTS (CONTINUED) XI. ASSAY EVALUATION CRITERIA . + + + ev vv vv vv veo o nnn 11 XII. AI.NTERGPeRnEeTraAlTIOOObNFserRvEaStUioLnTs.S oo oo cov vn ooo eee eee 12 B. Summary of Labeled Cell Counts for the Liver XIII. CONCLUSIONS + + + vooeoo meee 13 XIV. REFERENCES veo ovvo oom onm nnm nnnm .. 14 XV. EXPERIMENTAL DATA TABLE . oo ov ooo oom mon nooo ee 15 AAPPPPEENNDDIIXX A B IInnddiivviidduuaall AAnniimmaall BanoddySlainddeLiLvaebreliWnegighItnsdicaensd Li. ve.r.t.o .... 17 APPENDIX C BHoidsytoWpeaitghhotlogRyatiRoesport. . . .. o = v oo ooo.vv.eoeonononooo oo. 24 29 APPENDIX D Statistical Analysis of Labeling Indices . ......... 32 154-209 5 005142 ? WASHINGTON ABSTRACT mTheeasupruirnpgosecelolf pthrioslifsetruadtyiwoans (tCoP)deatsesraymeidneastheS-phheapsaetotionxdiuccittiyonofinT-r5at878livbeyr C2e0l00l,s aanfdter30i0n0 mvgi/vkog.treaDtimmeentth.ylniTtherosdaomsiensech(oDsMeN)n aftor15theng/sktgudywasweriencl50u0d,ed1a0s00,a positive control. aIdnmitnheistceelrledpranodliffeirvaetiaonnimaalsssayp,eracosnidngilteionorawleredosleaboefledthweitthestBrmdaUtefroiral72was ohobusresrveudsinagt A50L0Z,ET100o0smoatnidc20p0um0psm.g/kgNobuhtistionmcorrepahsoeldogmiictaolsesalwteerreatioobnsservweedreat d30i0m0ethmgy/Ikngi.trosTarniantemen(tD-MrN)elaptoesditicvheangceonstrwoelreanailmsaolso.bserved in the dFioflfleorweinncgesdetienrmtihneatliaobnelitnhgat itnhdeircees,werseecntoiontsreaftrmomentth-erellaetfetdlaltoebruallarlobe of `itmhmeulniovheirsst,och.aesmiwsetllry.as Esaamcphlesslidferownastheprdeupoadreendumw,ithwerseecptirooncsessferdomfobroth liver {anndterdnuaoldencuonn.trolTheforduoddeelniuvmery(aofraplaibdelly apnrdoliifmemruantoihnigstoocrghaenn)icawlasstuasiendinags. anThe mpiecrrcoesnctoapgiecaolflyn.ucleOinlyinchoerppaotroacyttiengnulcalbeeil wienretheenulmievreartewda.s deThteermcionnterdol animals rhaadngeadlafbreolmin6g.73intdoex7o.8f3.1.46Wheanndcotmrpeaatreidnganviemhailcslehaadndltarbeealtiendg girnoduipcsesantdhatthere w(e0r.e01<spi<g0n.i0f5i)canatndiancrpeoassietsiveintrtehendlwaabselionbgserivnedde.x a~tThe200a0vearnadge30v0a0lumegs/ksghowed hientcerreoagseesneoiunscovlalriapnrcoeliwfheernaticoonmpvareirnegalvseohicolbeseranvdedtrineattehde gproosuiptsi.ve cSoingtnrioflicant animals. pTr-o5l8i7f8erwaatsiotnherienforraet cloinvseridecrelelds.positive for the induction of cell 154-209 6 005143 ? WASHINGTON a CAenlallyPsriosliofferTa-t5i8o7n8 Aisnsay in Rat Liver Cells I. SPONSOR: 3M Corporation II. MATERIAL TESTED: A. Genetics Assay No.: 154-209 B. Identification: T-5878 C. Physical Description: waxy cream colored solid D. Date Received: January 19, 1994 III. TYPE OF ASSAYS: Analysis of Cell Proliferation in Rat Liver Cells Iv. PROTOCOL NUMBER: 493, Edition 3, Modified for 3 Corporation V. STUDY DATES: A. Study Initiation Date: January 10, 1994 B. Experimental Start Date: February 7, 1994 C. Experimental Termination Date: May 26, 1994 VI. SUPERVISORY PERSONNEL: A. Study Director: Maria A. Cifone, Ph.D. B. Associate Scientist: Andrea Ham, M.S. VII. OBJECTIVE: TTh-e587ob8jebyctimveeasuorfintghiscelalssapyrowlaisfertaotmieoansu(rCeP) hmeepaastuorteodxicasityS-pchaausseed by induction induced in rat liver cells after in vivo treatment. 154-209 7 005144 @ HWAASZHLIENGTTOONN - Cell proliferation measured the fraction of cells undergoing cell replication in rat liver using an immunohistochemical technique (1,2) to detect bromodeoxyuridine (BrdU) incorporated during DNA synthesis. Animals were given a single oral dose of the test material and the Tivers were isolated following administration of BrdU for 72 hours in vivo with an ALZET osmotic pump implanted subcutaneously. Quantification 72-hour period of cells that have incorporated DNA precursors over indicates increased cell proliferation in the liver the (3). VIII. DEFINITION: Hepatotoxicants such as carbon tetrachloride and dinitrotoluene induce an increase in cell proliferation to replace necrotic tissue (2,4). These proliferating cells may be detected during S-phase analysis. Other chemicals may induce S-phase in the absence of hepatotoxicity. It is not apparent how cell proliferation may act in the carcinogenic process but there are numerous mechanisms which can be affected during replication (5-8). Chemically induced cell proliferation may increase the probability of spontaneous mutations as well as increase the probability of converting unrepaired DNA adducts into mutations. Unscheduled cell proliferation may also play a role in the expansion of preneoplastic_populations leading to the emergence of a fully transformed clone of cells. Some of these examples act by a nongenotoxic mechanism and it is theoretically possible to detect nongenotoxic carcinogens as well as genotoxic carcinogens using this technique. IX. MATERIALS: A. Indicator Cells Young adult male rats of the Sprague-Dawley strain, 10-12 weeks old at the time of dosing, were purchased from Charles River Laboratories, Raleigh, NC (Cr1:CDBR). This healthy random bred strain was selected to maximize genetic heterogeneity and assure access to a common source. Animals scheduled for this study were housed according to standard operating procedures and were fed Purina Certified Rodent Chow (Formula 5002) and water ad libitum. Animals were quarantined a minimum of 7 days prior to random assignment to study groups and identification by ear tag for the dose rangefinding assay and by implantable microidentification device for the cell proliferation assay. The cell proliferation assay was initiated with rats that ranged from 289 to 355 grams. Within 2 hours after dosing, the animals were anesthetized using Metofane (methoxyflurane, Pitman-Moore, Inc.) inhalation anesthesia and one ALZET pump per animal was aseptically inserted subcutaneously (dorsal surface). Seventy-two hours later, animals were anesthetized with CO, prior to removal of the Tivers and duodenum. 154-209 8 005145 ? WASHINGTON B. Osmotic Pumps and Label for Cell Proliferation Analysis wAeLrZeETuseods.motiAcsipnugmlpes T(oAtLZA(C04o2r3p0o1r)atiwoans, usPeadlotAhrlosuog,hoCuAt)',thMoedes]tud2yH.LI TThhee ppuummppshwaserea 2p0r0e0-fui]llecdapawciitthyBwridUthata apumcponcreatnetraotfio1n0 uo1f/hour. 20 mg/m. C. Control Articles 1. Vehicle control gAavvaegheicl(eP.0c.o)ntrwoilthcotnhseisvteihnigclef,ive0.5ra%tshiwghasvdiossceodsitbyy oral C90a0r4b-o3x2y-m4e)t.hylcTeilssluuelsosefro(mCHCv)ehi(cSliegmac,ontLrootl 12a1nFi-m0a5l4s4;werCeAS # dSeurbijveecdtedfrfoom tthreeatseadmeamnainmialpsu.latiTohnesdoussiendgfovrolutmhee otifsstuhees vehicle control animals did not exceed 10 ml/kg. 2. Positive control article Trhaet phoespiattiovceytecsontirnolvivcoo.mpouTnhde pisosiktnoiwvne tcoontirnodlu,ce S-phase in dLiomte#th8y2Hl0n3i6t5r)osawmaisnedos(eDdMN,atCA1S5#.062m-g7/k5g-.9, SFiigvnearaCthsemiwcearle Co., treated P.0.. D. Test Article Ftohre ttheestpraertpiacrlaetiownasofsutshpeenddeodsinign CsHoClutaitoncsoncofenttrheatitoensst oarfti5c0l,e, V1o00l,ume2s00foarndth3e00tnegs/tmlarptriciloer dtoiddonsoitnge.xceeTdhe10mamxli/mkugm.dosing X. EXPERIMENT DESIGN: A. Dosfng Procedure Ffiovrethreatscelplerpcroonldiifteiroantiownereasstarye.atedDelbyiveorrayl voglauvmaegse wwietrhe T-5878 tcaaadrlmgcieuntliasttdeoesdree.don diTtdhhee nmobataxsiiesmxucmeoefdvotlh1eu0mmmelo/soktfg.trheeceFnrtteesshtaniapmrratelipcawlreeaitgishoutnsspeannodsfiottnhesest Carotnifcilrematiinonvehoifcltehewecroenceunsetdratfioornanoyf ttheestitnegstpumraptoesrei.al under dceotnedrimtiinoends oinf pcroenjpuanrcattiioonn awnidthdotshiisngsotfudyt.he assay was not 154-209 9 005146 @ HWAASZHLIENGTTOONN - B. Cc. D. E. 154-209 Implantation of Osmotic Pumps For the cell proliferation assay, ALZET Model 2ML1 osmotic pumps (Lot #042301) were preloaded with 2000 ul of BrdU at a concentration of 20 mg/ml. The animals were anesthetized using Metofane and one pump per animal was aseptically inserted subcutaneously (dorsal surface) approximately 2 hours after dosing. The incision was closed with wound clips and the animals monitored until the time of sacrifice to ensure that there were no clinical signs of infection. The osmotic pumps were implanted three days prior to sacrifice. Tissue Collection and Preparation Each animal was anesthetized prior to removal of organs for analysis. The thoracic cavity was opened and the liver removed and fixed in neutral buffered formalin. A cross section of duodenum, a tissue with high cell turnover, was also removed from each animal and fixed. The duodenum was included as an indicator that label was administered correctly to each animal. For the livers from high dose (Group 5) animals, 5 u paraffin embedded sections were taken from the left lateral, median and right anterior lobes. Once it was determined that no treatment-related Tobular differences were present, slides from the left lateral Tobe were prepared from each animal. Sections of the duodenum were also made and a section of the duodenum was mounted on each slide containing a liver section. Slides were also prepared according to standard procedures for examination by a pathologist to determine if any abnormalities were present. Immunohistochemical Staining The slides were deparaffinized and rehydrated prior to staining. The slides were stained for determination of cell proliferation as measured by incorporation of BrdU into DNA using Biogenix antibodies with peroxidase-conjugated streptavidin and a 3,3-diaminobenzidine tetrahydrochloride (DAB) chromogen and hematoxylin counterstain. Assessment of Cell Proliferation The section of the duodenum was microscopically examined to ensure that the label was properly administered to the animal. Once label delivery was confirmed, slides from the different lobes of the high dose animals were examined for lobular differences. Labeling was similar among the lobes therefore cell counting was performed with sections from the left lateral lobe from all animals. The percentage of nuclei incorporating label in the liver was determined microscopically. The areas to be counted were randomly generated by computer. A 1.0 mm square indexed 10 005147 ? WASHINGTON occouulnatringgriadread,ividAetdleianstto 210000x n1u0clseqiuawreersewaesxamuisnededtopedrefainniemalthweith a minimum of 3 sections and 6 fields per section. Acnoyntanuicnlienig tahnaytbwreorwen bclhureomowgeerenicconhuseidewreerde ucnolnasbiedleerdedanldabealneydnuclei uennluemsesrataedc.learFiealrdtsifatchtatwacsontpareisneendt.areaOsnlyofhenpeactroosciystewenruceleniotwere eivnaclluuadtedioninasthetoevtarleuaattmieonnt.groTuhpe. slides were coded for (blind) Sf-oplhlaowsse:nuclei labeling indices for each animal were calculated as Labeled S-phase nuclei (LI)=ntoo.talofnol.abeolfedhephaetpoactyotceystecnouucnlteeid X 100 XI. ASSAY EVALUATION CRITERIA Tchoeuntperdopowretreionasnaolfyzetdhebynumrbeepreatoefdcmeelalssurleasbelaendalytsoistheofnuvmabreirancoef c(eAlNlOsVA) tsepchhenriiqcuietsy ttoestdetweasrmianlesoanuytilsliizdeed, toandtesrtelavtaerdianicnetehroamcotgieonneietfyf.ects. The Acadldciutlaitoendaltoltyhc.oendauvcetragoene-vwaalyueANfOrVoAm, thDeunntehtrtee'sslti-dteesst,ofTeearcphsa-anJiomnaklheewraes dtaetsat., anSdeerAepgpreesnsdiioxnC tefsotrsstfaortisttriecnadl usainnaglysbiosthoufntlraabneslfiongrmeidndiacneds.ranked Fteorrmitnhael tebromdiynawleigwhhtoleratbioodsy,weaigmhetasn, anTdivesrtawnediagrhdtsd,eviaantdiolnivewrereto cS-aplhcausleatevdalufeosr.eacShtattirsetaitcmaelntagnraoluypsisusoinfgltahbeeliinngdiviinddueaxl waasnimpaelrfmoeramned ugsrionugp conoem-pwaaryisoannsalywseirse odfonevawriitahnceDuntneecth'nsiqute-st.estCoanntdroclontvreor]susvertsruesatment pcaosseitiovfevagrroiuapncecomhpeatreirsoognesneiwteyr,e droannek utsrianngsfotrhmeatSitoundsento'fsthte-tedsatt.a weIrnethe performed prior to analysis of variance and Dunnet's t-test. Atolabboedlyinwgeigihndtexr,atitoermiinnaal dobsoedygwreoiugphtt,hatterdmeivniaalteslivferromwetiheghtvalaunedsliivner tchoensicdoenrceudrresnitgnicfonitcraonltlygroduipffeartenatstihgannifithceancceontlreovlel groofupp.s0.05 was 154-209 1 005148 @ HWAASZHLIENGTTOONN - XII. INTERPRETATION OF RESULTS A. General Observations A11 animals survived treatment. No treatment-related h20i0s0tommogr/pkhgolbougticianlcreaalsteedratmiiotnosseswerweereobsoebrsveerdvedat at5003,00010m0g0/kagn.d Treatment-related changes were also observed in the dimethylnitrosamine (DMN) positive control animals. Details of the histopathology are in Appendix A. 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 Tabel and acceptable immunohistochemical staining. There was no apparent preferential labeling in any of the lobes and the label was random within the lobes. Two of the dose groups (Groups 3 and 5) had mean liver weights that were significantly elevated (p<0.01) above the Group 1 control liver weights but the increases were not dose-related. The mean liver weight of the positive control was not significantly elevated even though large increases in DNA synthesis (and subsequent cell proliferation) were induced. The 500 mg/kg (Group 2) animals also had a mean terminal body weight that was less than the Group 1 control value (0.01<p<0.05). No dose-related trend in body weights or liver weights was observed. However, when the liver to body weight ratios were determined, there were significant increases in the liver to terminal body weight ratios with p<0.01 Groups 3 through 5. B. 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 Appendix A. The mean labeling index (LI) for each group is presented in the third column in Table 1. The mean background labeling index (Group 1) was 1.46 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-5878 in dosed Groups 4 and 5 (2000 mg/kg and 3000 mg/kg; 0.01 < p < 0.05). The labeling indices at 400 and 800 mg/kg were 6.92% and 7.83% respectively which represent 4.7- to 5.4-fold increases over background." Large increases were observed at 500 mg/kg and 1000 mg/kg, but the heterogeneous nature of the response resulted in a lack of significance. There was also indication of a 154-209 12 005149 2 WASHINGTON ssitgantiifstiiccaanlt panoasliytsiivse. trTenhde dmueeantolabterleiantgmenitn.dexSoefe AtphpeenDdMiNxpDosiftoirve c(opn<tro0l.01a)n.imals was 34.96 which is significantly elevated TrheelsaetedresiunlctrseasdeesmonisntrtahteeLIthaitn Tth-e587l8iveirnduicnedmalseignriatfsicaanfttedrosaesLairnggeleionrcarleadseosseinatthceoncleanbterliantgionisndeoxf w2e0r0e0 malgs/okgobansderv3e0d00imng/tkhge. DtMrNeatpeodsitpiovseiticvoentrcoolntraonlimaalnsi.malsThewasmea3n4.9l6abe(lpin<g0.i0n1d)e.x in the DHN- XIII. CONCLUSIONS TSh-ephatseestcmealtlesrifaoll,lowT-i5n8g78a, siinngdluecedorasligndiofsiecaonft20c0h0angmegs/kginantdhe30n0u0mbemrg/kg. Tthreendaniimnaltshewemreean lalbaebleeldingforin7d2exhowuasrs obansdervaedsiginniftihecanttreadtoesde-rgerlouaptse.d Ts-y5n8t7h8eswiassitnherratefolrieveervacleulalst.ed as active in the induction of DNA 154-209 13 005150 ? WASHINGTON XIV. REFERENCES 1. DeFazio, A., Leary, J.A., Hedley, D.W. and Tattersall, M.H.N. (1987). Immunohistochemical detection of proliferating cells in vivo. J. Histochem. Cytochem. 35, 571-577. 2. Lanier, T.L., Berger, E.K., and Eacho, P.I. (1989). Comparison of S-bromodeoxyuridine and 'H-thymidine in rodent hepatocellular proliferation studies. Toxicologist 9, 64. 3. Butterworth, B.E., Ashby, J., Bermudez, E., Casciano, D., Mirsalis, J., Probst, G., and G. Williams: A protocol and guide for the in vivo rat hepatocyte DNA-repair assay. Mutation Res., 189:123-133, 1987. 4. Mirsalis, J.C. and Butterworth, B.E.: Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene. Carcinogenesis, 3:241-245, 1982. 5. Marsman, D.S., Cattley, R.C., Conway, J.G., and Popp, J.A. (1988). Relationship of hepatic peroxisome proliferation and replicative DNA synthesis to the hepatocarcinogenicity of the peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643) in rats. Cancer Res. 48, 6739-6744. 6. Craddock, V.M. (1976). Cell proliferation and experimental liver cancer. In: "liver Cell Cancer", Cameron, H.M., Linsell, C.A. and Warwick, G.P., Elsevier, North Holland Biomedical Press, Amsterdam. 7. Columbano, A., Rajalaksmi, S., and Sarma, D.S.R. (1981). Requirement of cell proliferation for the initiation of liver carcinogenesis as assayed by three different procedures. Cancer Res. 41, 2079-2083. 8. Glinos, A.D., Butcher, N.L. R., and Aub, J.C. (1951). The effect of liver regeneration on tumor formation in rats fed 4-diaminobenzene. J. Exp. Med. 933, 313-324. 9. Ham, A. and Cifone, M.A. (1991). Use of cell proliferation to study liver effects induced by a single dose of DMN. Environmental and Molecular Mutagenesis 17(19), 16. 154-209 1 005151 @ HWAASZHLIENGTTOONN ' ; XV. EXPERIMENTAL DATA TABLE 154-209 15 005152 2 WASHINGTON Client: 3M Corporation Client Code: T-5678 Table 1 Cell Proliferation Sumary HHA Assay No.: 154-209 Trial Initiation Date: February 7, 1994 Group/Sexta Page,| Labbeleling, Indexb | Liv(egrranWse)ight | TWeerimgihntal(grBaomdsy)|| LWeiivgehrt/Bo(d%)y (mg/kg) " 0c | 1.46% 1.23 [12.92+1.06 [333.5%14.3 |3.67%0.20 Mm 500 | 6.91 7.04 [14.56% 0.75 [291.2 % 21.0%] 5.01 0.25 a [1000 | 6.73% 5.1 [16.09 + 0.58%1(307.8 + 11.3 | 5.23 0.09%1 a | 2000 | 6.92% 3.49%1[18.15 1.15 [309.6 + 16.8 | 4.57 0.32% SM [3000 | 7.83% 5.04%1[15.80 1.96%41|356.3 22.5 | 4.44 x 0.29% md 150| 38.96 + 14.86 (13.13 0.73 [318.4% 7.7 | 4.12+0.19 3bFpievrecenatnaigmealsofpelrabgerleoduphepatocyte nuclei per total number of hepatocytes counted (atVTeehaisctle200c0o)ntrol, Carboxymethylcellulose positive control, 15 mg/kg of DMN ++* SSiiggnniiffiiccaanntt aatt 0p.<010<.01p 5 0.05 +4 DIenccrreeaassee iinn tthhee mmeeaann 154-209 16 005153 @ HWAASZHLIENGTTOONN - APPENDIX A Individual Animal and Slide Labeling Indices 154-209 17 005154  WASHINGTON Slide #AnimalID 16 47773 17 47773 18 47773 Group 1M 1M nm L# abeled 34 10 9 C# ounted %Labeled 700 700 700 4.86 1.43 1.29 Mean SD 2.52 2.02 19 47774 mM 20 47774 Mm 21 47774 mM 5 700 7 700 4 700 Mean SD 0.71 1.00 0.57 0.76 0.22 22 47775 mM 23 47775 mm 24 47775 pL 2 700 5 700 2 700 Mean SD 0.29 0.71 0.29 0.43 0.25 25 47776 m 26 47776 mM 21 47776 TM 11 700 3 700 8 700 Mean SD 1.57 0.43 1.14 1.05 0.58 28 47771 TM 29 47771 TM 30 47777 TM 16 700 18 700 19 700 Mean SD 2.29 2.57 2.711 2.52 0.22 GROUP MEAN 1.46 GROUP SD 1.23 154-209 18 005155  WASHINGTON Slide# AnimalID 46 47 47778 47778 I] 47778 Group #Labeled 2MMu 115294 Mn 140 #Counted %Labeled 70000 2127..7711 700 20.00 MesaOn 202..1540 5409 4477777799 MH 2470 7000 62.7816 51 47779 Mu 39 700 5.57 Mea0n 51..0958 552 4477778800 MMnn 54 47780 Ed 2315 770000 53..0000 13 700 1.86 Me0an 31..2599 5565 4477778811 M1u] 1198 770000 22..7517 57 47781 Mu 23 700 3.29 MBeEa)n 02..8368 5598 4747778822 22MM 60 47782 M 3222 770000 34..5174 14 700 2.00 MesaDn 31.2294 GGRROOUUPP SMEDAN 76..0941 : 154-209 19 005156 SLATE Slide# Anima1l0 6621 4477778833 6 47783 Group #Labeled 3MMn 1120 MH 22 #Counted %Labeled 770000 1I.n43 700 34 Mesapn 02..9120 6654 4477778844 3MMm 66 47784 MH 998 770000 1144..1040 125 0 17.86 Hea0n 125..1393 6678 4477778855 MMn 69 47785 Mn 4358 770000 65..4433 38 700 5.43 MesaDn 05..5786 770 4477778866 33MM 72 47786 M a578 70000 68..7219 63 700 9.00 MesaDn 81.070 7734 4747778877 3MMH 75 47787 3M 122 770000 31.77 1 700 2.00 Me0an 12..0488 GROUP MEAN GROUP SD 56..1713 154-209 20 005157 ? WASHINGTON Slide # Animal 31 32 4477778888 3 47788 ID Group #Labeled #Counted %Labeled aN aM n 73 700 700 1100..1443 aN 55 700 7.86 Mean 9.48 sD 1.41 3 35 4477778899 aaMM 36 47789 aN 2338 770000 35..2493 49 700 7.00 Mean 5.24 sD 1.86 37 38 4477779900 aM aM 39 47790 aM 61 89 700 700 8.71 12.71 9% 700 13.57 Mean 11.67 sD 2.59 40 41 4477779911 aM aM 42 47791 aM 21 33 700 700 3.00 4.71 23 700 3.29 Mean 3.67 sD 0.92 43 44 4477779922 aaMM 45 47792 aM 2318 770000 44..0403 37 700 5.29 Mean 4.57 sD 0.65 GGRROOUUPP MSEDAN 63..9429 154-209 21 005158 ? WASHINGTON Slid#e Aninal 10 1 47793 2 47793 3 47793 Group #Labeled #Counted % Labeled 5M 84 5M 66 SM mn 715 11.75 700 9.43 700 10.14 Mean SD 10.44 1.19 4 47794 5M 5 47794 5M 6 47794 5M 24 700 32 700 17 700 Mean SD 3.43 4.57 2.43 3.48 1.07 7 47795 5M 8 47795 5M 9 47795 5M 66 700 60 700 67 700 Mean SD 9.43 8.57 9.57 9.19 0.54 10 47796 SM 11 47796 SM 12 47796 SM 109 700 15.57 112 700 16.00 85 700 12.14 Mean SD 14.57 2.11 13 47797 5M 14 47797 5M 15 47797 5M 8 700 10 700 13 700 Mean SD GROUP MEAN GROUP SD 1.14 1.43 1.86 1.48 0.36 7.83 5.04 154-209 22 005159 ? WASHINGTON Slide # AnimalID 76 7 47758 47758 78 47758 Group #Labeled o oM 260 364 6M 435 #Counted %Labeled 700 700 37.14 52.00 700 62.14 Mean 50.43 sD 12.57 79 80 47759 47759 6M 6M 265 251 81 47759 El 171 700 700 37.86 35.86 700 24.43 Mean 32.71 sD 7.24 82 83 47760 47760 6M 6M 8 47760 6M 155 26 700 700 22.14 35.14 369 700 52.71 MesaDn 3165..6374 85 8 47761 47761 6M 6H 81 47761 6M 270 292 700 700 38.57 4171 285 700 40.71 Mean 40.33 SD 1.61 8889 4477776622 aoMM 15645 90 47762 o 89 770000 7.71 23.57 700 12.71 Mean 14.67 sD 8.11 GGRROOUUPP MSEDAN 3144..9866 154-209 2 005160 ? WASHINGTON APPENDIX B IndividuBaoldyAnWiemiaglhtBoRdaytioasnd Liver Weights and Liver to 154-209 % 005161 2 WASHINGTON STUDY WKBER: 154209 ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS DRAFTS ABSOLUTE ORGAN VEIGHTS (3) "ORAFT* SEX GDRoOsUeP NANNGAELR ORGAN ABBREVIATION: LI - LIVER 8T0E0RYMIENIAGLHT (9) LI KWo o1 ssmm 3 wo wnars KKool1 ssomnes ause 1n2aa Ko1 sm Fle ne NBER IN GRwowwp:: STANDARD OEY: s5s 15 a WI 1s WWoozz ssoenmse saemoo sswo NWoozzosmein 0 30 Wiess Woz sim 260 ls WER TH GReOwUPs: al5e s se STANDARD DEV: 20 ors WMooz3 msmem aszeos l18e3o1 WWo o3 mmeemess emaoo sdoses Wo mw wo ls NOUBER IN GWReO:: STANDARD DEY: ar5e 165.09 ns osm 154-209 25 005162 e WASHINGTON STUDY nse: 154209 ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS SORFTS ABSOLUTE GRGAN WEIGHTS (3) *ORAFT* CRA ABBREVIATION: LT - LIVER SEX00oSEw ANWINBALE T8E0R0MI1NAVLT (9) UI WNBoo4ioosammm msmoo ao 1i2sm sm ENLG omemmm swmeo isnm reSnTANDnARoDWwDoE:Vw we5ss e5Lss wKooss smmwe NWoOsE osoemse WOE sem NBER IH GRoOUnP: swowo bv: mwoesnse mwo0s Iusse ao wn we5s sa5 225 196 WNo oe smmmm 9 so 12s MNoose No emnmmmaeo sqweooo ideeses MuksScTRANIONRDGeoDwEwV:: as5e Bs a 7 on 154-209 2% 005163 ? WASHINGTON STuor Nuva: 154209 AVALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS SORAFTS ORGAN-TO-TERKINAL BODY WEIGHT RATICS (X) "ORAFT ORGAN ABRREVIATION: LI = LIVER SEGXowOOSNENERANIBOLOT TWTERM(I0N)AL LI WWo orseem waab sdooww WNNooo1aa emmemmmsme ssmmeioeo vseon ssw NNER IN GRWOeWa:: ss5s 5see STANONRD DEV: ws ose WNWooozF: smoommmy omawseeo saeos san WWooz: smoomm saeedo asoms NNER IH GRWOwUP:: l5z 5som STANDARD OEY: wo oa WWoo ssmmea aaezso ssamm WWoo33 memmess aweo ssaaem Kos mw mo san NBER IN GeROw: so5s 552% STAOARD DEV: ws oo 154-209 005164 27  WASHINGTON STUDY NMBER: 154209 ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS SORAFTS ORGAN-TO-TERMINAL 6GOY VEIGHT RATIOS (X) *DRAFT ORGAN ABBREVIATION: LI - LIVER SEX OSEGOANIMNAALBER TERNI8M0A0L1 WT (9) LI NNooa4 mwem assoo aadsma WWoo4& ssmmee 2w0o sdoame Wo4 sms woo ans NOMBER IN GRWOeUaP:: STANOARD OEY: n5e 68 A5 S os HKooss smmmss sasmso edaesw KKooss esammess 3ao0s 4an4es Kos mms amo 400 NBER IN GRMOUAP: 3453 5La STANDARD DEV: 2s om HKoos6 wsmss waeo a3m1e0 KKoo66 sseenw e30o0 4s2o9n Ko 6 sz eo 439 NNGER IN GRWOUoP:: ie5d 5daze STANDARD DEV: 77 oa 154-209 005165 2 @ HWAASZHLIENGTTOONN - APPENDIX C Histopathology Report 154-209 005166 29 SIAZLEIN Pathology Report Analysis of Cell Proliferation in Rat Liver Cells Project No. 154-209 eneral Protocol Thirty, young adult, male Sprague-Dawley rats were placed in six groups of five rats/group. Group 1 served as the vehicle control. Groups 2, 3, 4, and 5 served as the low-, low-mid-, high-mid-, and high-dose groups, respectively, receiving 500, 1000, 2000, and 3000 mg/kg of the test material, T-5878, via oral gavage. Group 6 served as the positive control, receiving 15 mg/kg of dimethyInitrosamine (DMN) via oral gavage. After dosing, an ALZET Model 2ML1 osmotic pump containing 20 mg/mL of bromodeoxyuridine (BrdU) was implanted subcutaneously in each rat while it was under Metofane anesthesia. Seventy-two hours after pump implantation, all rats were anesthetized, exsanguinated, and necropsied. Liver, duodenum, and all gross lesions from each rat were placed in 10% neutral-buffered formalin and processed as per HWA SOPs. These tissues from all rats were evaluated microscopically by a boardcertified veterinary pathologist. Histopathology Treatment-related change in the liver consisted of minimally to slightly increased mitoses in Group 5 (3000 mg/kg) rats. Group 6 (DMN treated) rats had varying severities of centrilobular necrosis, hepatocellular hypertrophy, chronic inflammation, peliosis, and increased mitoses. Various spontaneous disease lesions and incidental findings, including chronic inflammation of the spleen capsule in a Group 3 rat and renal pelvis dilatation in three rats, are unrelated to treatment. The chronic active inflammation with acanthosis of the nonglandular stomach of a Group 2 rat is probably an effect of gavage trauma. 154-209 005167 30 @ HAZLETON Sumary The test material, T-5878, when administered to male Sprague-Dawley rats in single oral gavage doses of 500, 1000, 2000, and 3000 mg/kg, produced increased mitoses in the liver of rats dosed at 3000 mg/kg. No treatmentrelated histomorphologic changes were noted in the liver of rats dosed at 500, 1000, and 2000 mg/kg. Pathologist: Samuel V. Machotka, D.V.M., D.A.B.T., Date Diplomate, American College of Veterinary Pathologists Department of Pathology 154-209 005186! 5 31 @ HWAASZHLIENGTTOONN - APPENDIX D Statistical Analysis of Labeling Indices 154-209 005169 2 2 WASHINGTON Methods: The proportions of the number of cells labeled to the number of cells counted were analyzed by repeated measures analysis of variance (ANOVA) techniques to determine any significant dose, slide, and related interaction effects. The sphericity test was also utilized to test variance homogeneity. The model used was: : proportion = pu + dose + slide + slidexdose + ~. Additionally, since the study did not show any variation, the average value from the 3 slides calculated to conduct one-way ANOVA, Dunnett's test [1], and regression tests for trend using data. significant between-slide of each animal was then t-tests, Terpstra-Jonckheere both untransformed and ranked Results: Since the sphericity test rejected variance homogeneity in comparing vehicle vs positive control (p = .0227), the Greenhouse-Geisser probabilities were used for this significance evaluation. There was no significant heterogeneity in comparing vehicle vs treated groups (p = .7922). As Text Table 1 indicates, there is no significant finding in comparing vehicle with treated groups. Within- and between-group slide-to-slide variations were not significant in either case. Only the positive control showed highly significant elevation in labelling over vehicle control. The data based on the average values showed homogeneous variance (p = .1449) in comparing vehicle vs treated groups for the untransformed data. However, closer examination of the individual values in each group along with the means and standard deviations (Text Table 2) indicates that the analyses based on the rank-transformed data may be necessary. As Text Tables 2 and 3 indicate, there is significant difference between vehicle and treated groups in transformed data (p = .0314). Dose 2000 and 3000 showed significant increase in labelling over control. Furthermore, there was a significant positive trend as indicated in Text Table 4 using Terpstra-Jonckheere test (p = .0049) and regression of rank-transformed data (p = .0090) even though the regression based on the untransformed data did not show any significance. There was no significant lack of fit for both regressions (p = 0.252 and 0.473). The following notations are used to denote direction and statistical significance: * = significant at p < 0.05 ** = significant at p < 0.01 t = effect in the positive direction Text Table 1 - Univariate ANOVA Vehicle vs Positive ControlVehicle vs Treated Groups Dose p 0.0005 **0.3029 Slide p0.27920.9965 PS0l.i2d0e5x3D0o,se22 mmmmeeee---------------------------- 154-209 005170 n ? WASHINGTON Text Table 2 - The Descriptive Statistics Dose (ng/kg) Mean untrans$f0ormed VVeehhiiccllee vs Posi1t.i4v56e0Control0.9958 Positive 34.9620 13.1132 VVeehhiiccllee vs Trea1t.e4d56G0ro|ups 0.9958 5100000 69.67300 57..34845059 32000000 679.286300 35..43624200 Median 36.16.0750000 31.20050000 55..27460000 9.1900 RMaenank-Transfor0med 83..00000000 11..55841111 144..02000000 63..50597444 1174..00000000 38..82713508 15.8000 7.7910 Text Table 3 - One-Nay ANOVA and Dunnett's T-Test VUenhtircalnesvfosrPnoedsitivTerCaonnstfroormled UntranVsefhoircnleed vsTreTartaensdfoGrrnoedups Treatment 0005 ** VVeehhiiccllee vvss 1500000 VVeehhiiccllee vvss 32000000 L000 #3022 0314 * J00881276 1+ "l00135115 #*11 Text Table 4 - Test for Trend for Vehicle vs Treated Groups TReergprsetsrsiao-nJonocfkhUenetrreanTsefsotrned Data 00490 +#1 Regression of Rank Transforned Data 20090 #4 DiscussTiheon:results of the present study based on the rank-transformed data indicate that there was significant increase in cell proliferation at Dose 2000 and 3000 compared to control due to treatment by the chemical. There was also indication of a significant positive trend due to treatnent. References: [1] Ajit K. Thakur, A Fortran Program to Perforn the Nonparametric Terpstra- Jonckheere Test, Computer Programs in Biomedicine 18: 235-240, 1984. [2] SAS (Statistical Analysis System), SAS Institute, Cary, NC, 1991. 154-209 005171 3 WASHINGTON Ha StudyNo. _[SY80F Protocol No. 493, Edicion 3 ANALYSTS OF CELL PROLIFERATION IN RAT LIVER CELLS aHnadzleFtDAonGoWaosdhiLnagbtoorna,torIync.Pra(cHtWAi)cewi(lGlL?)coRnedguucltattihoinss.stuTdhyisinprcootmopcloila,ncecriwtitihcalEPA bpyhasQeu(asl)ityofAstsheurawnocrek iinn apcrcoogrrdeasnsceanwditthheSOfPisnalatrHeapzolrettwoinllWasbheinsgutbojne,ctIntco. audit T2h2i18s2.study will be conducted by HWA at 9200 Leesburg Pike, Vienna, Virginia PART 1. SPONSOR INFORMATION AND APPROVALS I. SPONSORIDENTIFICATION Company Name: _3 Corporation Address: Building220-2802.3 Center.St.Paul,MN55144:1000 II. TESTARTICLEIDENTIFICATION: TEE2 -5878 shy. III. TESTARTICLEAVALYSIS cDheatrearcatfenraicsitoincsofasthedeftiesntedaritnictlhee GsLt?abirleigtuylatainodnstheofteFsDtA a(r21tiCcFlRe t5h8e.10r5e)s,ponEsPiAb-iTlSiCtAy(o40f CcFheR S7p9o2n.s1o0r5.), and EPA-FIFRA (40 CFR 160.105) is 1V. NOTIOFFIREGUCLATAORYTSUIBMIOSSINON 5I8n.1o0r;derEPAt-oTScCoAm,pl4y0wiCtFhR U7.9S2..10f;edeErPaAl-FIrFeRgAu,la4t0ionCFRcod1e6s0.1(0F)DA,and21cCeFrRtain Pfaorrteiogfn aagsetnucideys,isctoonsbueltisnugbmiltatbeodrattooritehse amguesntcyb.e nHoUtAifimeadintiafinasllaor Biansdciecratescwhheidcuhleagoefncsyt,udiiefs anwyh,ichBigfhatllreucnedievre rtehgeularteosrulytsrevoifewt.his Plsteuadsye: OJ vesecernines BI mon CJ sontsen CJ sonora Owe CJ wow C3 oe CJ ome 10/93 Page 1 005172 @ HAZLEETTON : V. STUDY DATES VI. APPOFRSTO UDYVPROATOCLOL pesca to. 453, Eakin 3 - 10/93 Page 2 005173 WASHINGTON Protocol No. 493, Edition 3 PART 2. STUDY PROTOCOL ANALYSIS OF CELL PROLIFERATION IN RAT LIVER CELLS I. OBJECTIVE The objective of this assay is to detect hepatotoxicity caused by the tiensdtucmtaitoenriianldubcyedmeiansurraitnglicveerllceplrlosliaffetreartioinn v(iCPv)o mteraesatumreendt.as S-phase Cuenldlerpgrooilnigfecerlaltiorneplisicadteisoingneidn rtaotmelaisvuerreustihnegfarnactiimomnunoofhicsetlolcshemical technique (1,2). Animals are given a single oral dose of the chemical and the livers are isolated following administration of bromodeoxyuridine (BrdU) for 72 pump implanted subcutaneously. QhuoaurnstifinicvaitvioonwiotfhcaenllAsLZtEhTat hoasvmeotic incorporated DNA precursors over the 72-hour period has been shown to be useful for the evaluation of chemicals that may cause increased cell proliferation in the liver (3). II. DEFINITION Haenpaitnoctroexaisceanitnsceslulchparsolicfaerbroantiotnetrtoachrleoprliadcee annedcrodtiincitrtoitssouleuen(e2,4i)n.duce These proliferating cells may be detected during S-phase analysis. Other compounds may induce S-phase synthesis in the absence of hepato- toxicity. It is not apparent how cell proliferation acts in the carcinogenic process, but there are numerous processes that can be parfofleicfteerdatdiuorninmgayrepilniccreaatsieonth(e5-8p)r.obaCbihleimtiycalolfyspionndtuacneedoucsellmutations as wmeultlatiaosnsinpcrrieoarsetothea probability of repair process. converting DNA adducts into Unscheduled cell proliferation may also play emergence aofroalefulilny thteraenxspfaonrsmieodn cloofnepreomfcocpelllass.tic Scoemlelsofletahdesiengetxoamptlhees act by a nongenotoxic mechanism. It is therefore possible to detect nongenotoxic carcinogens as well as genotoxic carcinogens using this technique. III. MATERIALS A. Animals Young adult ac the time moafledosriantgs, ofwiltlhe beSppruargcueh-aDsaevdlefyromstrHaairnl,an 1S0p-1r2aguweeekDsawloelyd, Tne. Inc. ((CHrS1D::CSDprBaRg)u.e-DaTwhiiseyh(eSaDlt)hByR)raonrdomChabrrleedsstRriavienr hLaasborbaeteonriseesl,ected to maximize genetic heterogeneity and at the same time assure access to a common source. 10/93 Page 3 005174 WASHINGTON Protocol No. 493, Edition 3 TdhueresaniamnadlswilwlillbebefehdouPsuerdinaacGcoerrdtiinfgiedto RsotdaenndtardChoowper(aftoirnmgulparo5c0e0-2) adThaneydswrpaarttiesorrtaotdobeliubsuiesteuadnn.dforwiTlhtleheybwaesislralaynbdewoimlqllyuabraeasnstiaignnneeesddthetaotmiizsnetidudmbyuemfgororofeupsse.ven surgery. TPhietnaann-iHmoaolrse,wilIlnc.b)e ainnehsatlhateitoinzedaneusstihnegsiMaetoafnadneone (pmuestphopxeyfrluarnainmea,l wSielvlenbtey-tawsoepthoiucrasllylatienrs,eratneidmaslusbcwuitlalnsobueslaynes(tdhoerstailzedsuwrifatche)C.O prior to removal of the livers and duodenum (control organ). 3. OsmoticPumpsandLabelforGellProliferationAnalysis AWiLlZlETbeosusmeodt.ic pAumspisngl(eALZLAotCowripllorabteiouns,edPatlhoroAulgtheo,utCAt)h,e Mstouddeyl. 2MTLh1e Ar(LaBZtreEauT)ofMato1d0ealulc/2ohMnrLc.1entorTsahmteoitoipncumpposfumpw2i0hllmags/baeal.2010l0leudlwictahpacbirtoymowdietohxyuaripduimnpe c. Control Arcicles 1. Vehiclecontrol Afovrehciecllleprnoelgiafteirvaeticoonn.trolThceyonwsiilsltibneg ocfreaatmeidniwmiucmh othfefvievehicrlaets moerthsoodlsven(tususaellleycteodralforgavtahgee)teusstedmatfeorriatlh.e tTehset msaatmeeridaolsing ptorsesaitbmleentsdowsiilnlg bveoluenmepsloyfoerd ofroarlthgeavavgeehicvliellconnottroelx.ceWehderaebout 10 ml/kg body weight. 2. Positiveconcrolarcicle Tihneraptosihteipvaetoccyotnetsrolinarvitviec.lesThuesedposairteivkenowcnontcroolindfuorcecSe-lplhase pberoltirfeeartaetdiobny wpielrlosb.e 15 mg/kg of DMN. At Least five race will D. TescArticle Utnelsetsesd saspecsuipfpileidedb.y cAhenyspoopnesroart,iontshepetrefstormaertdicolnetwhielltenstormaarltliyclebe bsyuchtheasspgorninsdoirngp,rioerxtrtaocttihoen,inoiftiastoilovnento-fextcehstainngge. muAsLtLbeopesrpaetciiofnised performed on the test article will be described in the final report. 10/93 Page & 005175 WASHINGTON Protocol No. 493, Edition 3 IV. EXPERIMENTALDESIGN A. DosingProcedure Aseplreecltiinotnnafroyr ttheesttweisltlabreticpleerfournmleedsstoa dveetheircmlien/esolvveehnitclei/ssoslpveecnitfied mbeythtyhleceSlplounlsoosre., Mcaatrebroixaylmsethwyhliccehllmualyosbee, sceolrencteoidlionrcluadneothwaetrer, Sthuetcatbelset vaerhtiiccllee/sionlvveonltu.mesRatthsatwiwlilllbenottreeaxtceededbyaboourtal1g0avmalg/ekgwibtohdy wSepiognhsto.r. AlDtIeNrnwaitlel rbeoutdeisssooflveedxpoisnursetemraiylebederieoqnuiezsetdedwatbeyr.theFresh pSroelpvaenrtatioornsvehoifcltehewitlelstbeartuisceldefoarndapnyositteisvteingconputrrpoolsse.in tShteability boef tthhee rteesstpomnastiebriilailtyunodfertheconSpdointsioorn.s of preparation and dosing will B. DoseSelection Ubenle1ssg/ksgpecoirfhiaeldfotthheerwLiDse,, wthhiechheivgehresits dloesses.selFeocutreddowsielsl wuislulalbley sferloencteeadchusdionsge alpepvreloxiamnadtecloyntrtowlo-fgrooludpdwiillultiboen sutseepds.to Fainvaelyzaenimcaellsl proliferation at 72-hours. C. ImplaonftOsamottiicPuompns ABLrZdlETacMoadeclonc2eHLn1troastmiootnicofpu2m0psmg/waill.l beFoplrleowlionagdeddowsiicnhg w2i0t0h0 utlheoftest m(amteetrhioaxly,flutrhaenea,nimPiaclasanw-iMlolorbee, anIencs.t)hetiinzheadlautsiionngaMneetsotfheasniea and one pSuumrpfacpee)r. aniThmealiwniclilsiboen awislelptbiecalcllyoseidnsweirtthedwosuunbdcutclainpesousalnyd t(hdeorsal aarneimanloscmlionniictaolredsigunnstilofthienfetcitmieono.f sTahcerifoiscmeotitco peunmspusrewitlhlatbechkeerpet in the rats for three days prior to sacrifice. D. TC issoue llecandtPreiparoatinon Eanaaclhysainsi.malThweillthboeracaincestchaevtiitzyedwilplriober otopenreemdovaanld othfeorlgiavnesr froermoved adunoddefniuxme,d aintniesusuteralwibthufhfiegrhedceflolrmatluirnn.overA, crwoislsl salescotiboen orfemoved 10/93 Page 5 005176 @HAZLETON WASHINGTON Protocol No. 493, Edition 3 E. ImmunchistochemicalStaining F. Assoe fCes llPs rolim fere ation nRat tes 005177 WASHINGTON Protocol No. 493, Edition 3 V. DATAPRESENTATION Tfhoer efaicnhaltrienpepooritntw,illforincthleudenegtahteivfeolcloonwtirnogl,inpfoosrimtaitvieoncoinntrtoalb,ulaarndfeoarcmh analyzed treacment! + Tfhoer ceaaclhculaanitueadl3San-aplhyazseed+fosrtanSd-pahradsed.eviation among the three slides VI. ASSAYEVALUATIONGRITERIA bAemecaanlcualndatesdtanfdoarrdeacdhevtiraetaitomnenftorgrtoheuppeurscienngtatghee oifndiSv-ipdhuaasle caenlilmsalwimlelan Sp-eprhfaosremedvaluuseisn.g onSet-awtaiystiacnaallysainsalyosfivsaroifanlcaebeltiencghniinqdueexs w(i9l).l beControl V(e1r0,s1u1s).treIantmetnhet cgraosuepofcovmaprairainscoenshewtielrlogbeenediotnye,wirtahnkDutnrnaents'fsormta-ttieosnt of tGhretesdca.ta wSitluldenbet'spertf-otremsetdwiplrliorbetuoseadnalfyosriscoomfpavrairsioanncoef tahned pDousninteitv'es cgroonutprolthvaetrsduesviattheesvefhriocmletheconSt-rpohla.se ApnercSe-npthaagsee pienrctehnetacgoencuirnraentdose SCoingtnrioflicagnrtoluyp adtiffaerseingtniftihcaannctehelecvoenltroolf pgrso0u.p0.5 will be considered VIL. REFERENCES 1. D(e1F9a8z7i)o., AI.m,muLneoahriys,toJc.hAe.n,icaHledldeeyt,ectD.iUo.n oafndpTraotltiefresraalcli,ngM.cHe.lNl. in vivo. J. Histochea. Cycochea. 35, 571-577. 2. Lbarnotmeord,oxTy.Lu.r,idiBneergearn,d E*.HK-.thyaaniddiEnaechoi,n rPo.dI.ent(1h9e8p9a)toCcoemlplaurliasron of 5proliferation studies. Toxicologist 9, 64. 3. 4. 5. 10/93 3B.u,ctePrrvoobrseth,, G.B,.E.a,ndAsGh.byW,illJi.,amsB:ermuAdepzr,ocoE.c,olCaasncdiagnuoi,deD.f,orMitrhsealiins, Vivo rac hepatocyte DNA repair assay. Mutation Research, 189:123133, 1987. MSiyrnstahleissi,s Ji.nC.ratsnhdepBauttotecrywtoersthf,olBl.oEw.i:ngIinnduvcitvieontorfeautmnesncthewdiutlhed DNA dinitrotoluene. Carcinogenesis, 3:241-245, 1982. MRaerlsamtaino,nshDi.Sp.,ofCahtetplaetyi,c Rp.eCr.o,xisCoomnewayp,rolJ.iCf.eraatnidonPopapn,d Jr.eAp.lic(a1t98i8v)e. DNA spyrnotlhiefseirsatotros tdhei(h2e-peatthoyclahrecxiyln)opghecnhiacliattyeofandthe(4p-ecrholxoirsoo-m6e-(2,3yRe1s.1di4n8o,)-627-3p9y-r6i76m4i.dinylthioacetic acid (y-16,643) in rats. Cancer Page 7 005178 WASHINGTON Protocol No. 493, Edition 3 6. cCarnacdedro.ck, `IVn.iM."Li(v1e9r76)C.ellCeClalnceprr"o,lifCaemreartoino,nH.aMn.d, eLxipnesreilmle,nCc.aAl. lainvder Varwick,G.P., Elsevier, North Holland Biomedical Press, Amsterdas. 7. oCoflucseblalnop,roAl.i,ferRaajtailoanksfaoir, tSh.e ainnditiSaartmiao,n oD.fS.lRi.ver(1c96a1r)c.inogReenqsusiirsemeasnt assayed by three different procedures. Cancer Res. 41, 2079-2083. 8. GLliivneors,reAg.eDn.e,ratBiutocnheorn, tNu.mLo.rR.foramnadtAiuobn, iJn.Cr.ats(1f95e1d)4-Tdhiesmienfofbeecntzeonfe. 3. Exp. Med. 933, 313-326. 9. MWicnGerra,w-HBi.lJ.l, (N1e97w1)Y.orSkt,at2insdtEidciatlioPnr,inpcpi.pl1e49s-i2n20E. xperineDnesciagnl, 10. sDuenvneertatl, tCr.eHa.tme(n1t9s55)w.ithAamcuolnttirpolle. cJo.mpAamr.isoSntatp.rocAessdoucr.e 5f0o,r 1c0o9m6p-aring 121. 11. Dcuonnntertotl,. BC.iWo.met(r1i9c64s).20, N4e6w2-t4a9b1l.es for multiple comparisons with a VIII. REPORTFORMAT The final report will provide the following information. + TSepsotnsmoarteirdieanltifiidceantciiofnl.cacion and Assay Number. A physical dienscclruidpecdioinn tohfisthesectteisotn.material and dace of receipt will be + + DTyaptees ooffassstauydy anidniptrioattoicoonl anndusbceorm.pletion. NIanmteesrporfetaSttiuodny oDifrercetsourl,ts.Senor Technician, Scientist HCoinsctloursiicoanls.control data for negative and positive control cultures. + TSeisgtnatruerseusltsofprSetsuednyteSdupeirnvitsaobrulaarndfoSrtmusd.y Director. + MEevtahloudast.ion criteria ++ QRueafleirteynceAsssurance statement. 10/93 Page 005179 WASHINGTON Protocol No. 493, Edition 3 IX. CHAORNREGVIESIOSNS Asniygnecdhabngyestheorstruedvyisidoinrsectoofr,thidsateadp,proavneddmparionttoacionledwiwlilchbethidsocupmreonttoecdo,l The sponsor will be notified of any change or revisions. X. RETCOBOEMR AIND TAIS NED Aglelnerraatweddataas, adorceusmuelnttaotfiotnh,isresctourddys,wiplrlotboecoalrsc,hivaendd ifinnatlhersetpoorratgse ffiancaillitrieepsortof tHoaztlheetosnponfsoorr.at least After one the oyneearyefaorllpoewriinogd,sutbhmeissspioonnsoorf the may felaeccitlittioeshavoef Htahezleatfoonrefmoerntiaonneadddmiattieorniaallsperrieotdainoefd tiimne tohre storage sent to a storage facility designated by the sponsor. 10/93 Page 9 005180 3M Internal Correspondence cc: To: From: Subject: Date: RG. Perkins - 220-2E-10 B.C. Copley - 53-38-02 R. D. Howells - 53-35-02 Key to FC Alcohol Tox Samples September 14, 1995 T5877 T5710 T5711 T5794 T5795 F878 `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 Analytical Request 41220 L-10059 `Wide Range MeFOSE Lot 555 Typical Raw Material for FX-845 Narrow Range MeFOSE NLoatbePbroeopkar9e7d90S0a-m1p0l7e-2 Analytical Request 41220 L-1276 Analytical Request 41343 L-13097 `Wide Range EtFOSE Notebook 97900-112-2 Lab Prepared Sample Wide Range MeFOSE Lab Prepared from `Washed POSF Analytical Request 41343 L-13098 Analytical Request 42607 PHI Rtlo Attachments 005181 REQ 41220 ' R. Howells : introduc`eGCth/eMsSaamnpalleysseisntooftthheeFsiensnaimgpalneSsSwQe-r7e0amcacsomspslpieschterdomuestienrg. 2T2h5e msamXpl0e.3c2ommpmonHePn-t1s GweCrecoilonuimznedto using chemical ionization with methane as the reagent gas. The GC column was operated from 40 to 300 C at a rateof10 degrees per minute. The results of these analyses combined with their corresponding GC/FID area percents show the following: |nI oomcommioe econ {oma en | `Component LD. N.R. N-EtFOSE Lot `W.R. N-EtFOSE Lot 547 lmsoneelm ousenopo s n| lcrsonmeosmx en weon| lcmsoneuooum s elnimo e n| lcomsonm{eonm s a|s amson| lcrmsonenoewsm| aoms| ane wns 005182 I lies| `Component 1D. 11276 - N-McFOSE Lot ceusona C2FsSON(Me)CH2CH20H l 024% o| CsF7S02N(Me)CH2CH20H 115% cCAFr9SuO2sN(Me)CH2CcHrc2r0iHon cisonomciczon CEF17SOIN(Me)CH2CHaCL CrF15502N(Me)CH2CH20H N-MeFOSE_ CsF17S0zN(Me)CHzCH20H CoF15S02N(Me)CH2CH20H -- CsF17502N(Me)(CHzCH20)2H OtherHigh Boilers 162% [as| loose| 013% 169% 83.88% 087% 034% 155% Further work has been done byGCon these samples which involves derivatization of the 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 EFOSE underivatized could hide a significant amount of N-E(FOS Amide under the C-3 alcoholpeak in wide range material. However, analyzing the same material that has been derivatized with TFAA shows that any EGFOSE-chloride that is present in the sample is now completely masked by the derivatized C-8 alcohol. The BSA derivative has not been evaluated yet, but similar problems are expected because of the number of different components in the sample. The same sort of problems will most likely exist with MeFOSE andwillbe even more complicated in the analysis of MeFOSEA. 712193 005183 REQ 41343 J. Grant introducGeCt/heMSsaamnpalleysseisnotfotthheesFeinsnaimgpalneSsSwQe-r7e0amcacosmspslpieschterdomuesteirn.agT2h5e msamXpl0e.3c2ommpmoHnPen-t1s GweCrecoilonuimznedto urastienogfc1h0edmeigcraelesionpiezratmiionnutwei.thTmheetrheasnueltsasotfhethreesaegaennatlygasse.s cTohmebiGnCedcowiltuhmnthweiarscooprerreastpeodndfirnogmG4C0/tFoI3D 0arC0eaata percents show the following: Component LD. cvsonn N-Ethyl-carboxamides (R-C(OIN(EY) crsoneicoon arson Nc-oE(uFsOSoAnmeidne ( CSF17SON(EY) opsonEacmasor C4F9SOIN(ENCH2CHI0H ersongcrcrion CsF13SON(ENCHZCH20H -- CiF1sSON(ENCH2CH20H WR N-EFOSE |WR N-EFOSE Precut main cut 97900-1121 97900-1122 ll 6i8.m34%eow 127% s| foe low | le [ome | ll i04o1%n eeee|| lise Joss | L[ a062e% oo 042% n| 2016% 60.86% Le 352% f2u.5m5%e | `Component ID. NR N-McFOSE BP. 132 97900-1072 005184 [-------- PE-- FS loonLoms e | omsomen Amide 97900-111 lee | omsonen [see | lE omsomen EE |le.e || that include hydrides, chlorine in the backbone, and unidentified high boilers 005185 To: From: Subject: Date: 1. Muggli 5365-02 R.M. Payfer 236-2B-11 (6127834212 SAZC Analytical Request No. 42607 Dec. 21, 1993 GinCt/roMdSucaentahleyssiasmopfltehiisntsoatmhpeleFiwnanisgaanccSoSmQpl-i7s0hemdasussinsgpeact2r5ommetXer0..3T2hme msaHmPp-l1e GC column to components were ionized using chemical ionization with methane as the reagent gas. The GC column was operated falrsoomd4o0net,o 3a0n0dCtheataareraatpeeorfce1n0tdveaglrueeessfrpeorm mtihinsutweo.rkGwCeraenaalpypsliisedwittohtfhleampeeaikoniidzeanttiiotniedsetfercotmiotnhewas mass spec work. The resuits of these analyses (which are not necessarily quantitative) show the following: |os loeomwle omww| |e Jorcsommaomo[onson| [eos Jourusommoon[oonzos n| 005186 [ onrunsoms CeF17802N(Me)(CHzCH20)2H 005187