Document G5md4DxgXz27nRKwmK5d1v42q

AR 226 110/ `TOXIKON FINAL REPORT: 01-7019-G1 I CHO/HGPRT FORWARD MUTATION ASSAY - ISO (T-6889.7) DeviAuStadhhour, PhD. | EiMnaarlRcehp2o8r,t2D0a0t2e: l . MANAGEMENT OFTHE STUDY. Performing Laboratory Toxikon Corporation B1e5dfWoirgdg,inMsAAv0e1n7u3e0 `Sponsor: 3M 3M Center 220-2E-02 Saint Paul, MN 55144 ~ =g S gs =3 =I z3 we OF 3 _ -- J CONTAIN NO CR! 000003 `"CTHoOxi/kHonGCPoRrpTorFaotriwoanrdMutation Assay ISO(T-6889.7) EeE ProjeccR t NiumE berr: 0m1-I70Im19I-GB1msmmieeeeseteoerreemsmrae Title Page TABLEOFCONTENTS . TaofbColntenets Study Summary Quality AssuranceStatement Study Director Signature and Verification Dates 10 Purpose 20. References 30 Compliance 40 50 IIddeennttiiffiiccaattiioonnoofftthheeTTeessttaSnydstCeomntrol Aticles 60 7.0 EJxupsertiimfenitcaoal tfDTieesosintgnSysantdReoumteof Administration 80 Dosage 90 EvaluationCriteria 100 Results 110 Conclusion 120 Records 13.0 Confidentiality Agreement Table Table: TableI: TableIV: TableV: Table VE TableVII: PaPraraalllleellCCyltoontionxgicEiftfyiAcisesnacyy (Non-Activated) PHaGraPlRleTlCMlonuingtEfaficAigsesncaeyy((nANcotnei-vAatcsetdi)viatesd) HGPRT MutagAsesany(eActsiviatesd) Summary Summary ooffMMuuttaannttCCoolloonnyyFFrreeqquueennccyyiinn HGPRT Assay HGPRTAssay (Non-Activated) (Activated) Page20f18 . 000004 Toxikon Corporation CHO/HGPRT Forward MutationAssay -ISO(T-6889.7) Project Number: 01-7019-G1 STUDY SUMMARY . `The test article, Ammonium Perfluorooctanoate (FL-143), did not induce mutations attheHGPRT locusasevidencedby theabsenceof astatisticallysignificantincreaseinthe numofbmuetarnt colonies at the highest analyzable test concentration as compared to the negative controls in the presence or absenceofan exogenous mammalian metabolic activation system. The statistically significant increase in the numberofmutant coloniesinpositive controls in both the activated and `non-activatedassays vertihfepiroepedr functioningofthetestsystem. Therefore, basedonthe evaluation criteria of the protocol, the test article is considered non-mutagenic, under the experimental conditions employed. Page3 of 18 0001005 TCoHsOik/oBnGCPorRpTorFaotriwoanrd Mutation Assay- SO (T-6889.7) RPre oject Numbeerr: 01.7019-G1eee QUALITY ASSURANCE STATEMENT . `This study forthin 21 was conducted CFR, Part 58. in compliance with U.S. Food and Drug Administration regulations set TexheemspetctfiroonmsotfhtishGeoordegLualbaotiroantsonroytPrpaecrtifcoermSetdabteymoenrt,uinndcelrutdheedchdairraeccttieorniozaftTioonxainkdosntCaorbpiorloatifittohnye, testarticleand its mixturewithcarriers,21 CFR,Parts 58.and1580.1153. r`eTphoerQtueadltiottyheAsSstuurdaynDcierUecntiotrcaonnddtuocTtoexdiiknosnp'escMtiaonnasgeomnetnhte.followingdates. Thefindings were INSPECTIONS DATE OF INSPECTION DATE REPORTED _ DATE REPORTED STUDY DIRECTOR MANAGEMENT ------------------ DOSING 01/02/02 ou02/02 01/02/02 CELL COUNTING 02/07/02 02/07/02 0207/02 RAW DATA 0328/02 03/28/02 03/28/02 FINALREPORT 03/28/02 --_---- 03728002 03/28/02 3 z Mansi Dsai,B.Sc. Quality Assurance Ddaee d fay Paged of 18 000006 ZT`ProCoxe HjieOkc/otnBNGCue oPmrRbpTeorrF:aoot0ri1wo-an7t r0d19M-u.Gt1ation Assay - ISO (T-6889.7) STUDY DIRECTOR SIGNATURE AND VERIFICATION DATES . `rTehqisusirteumdenytmseofttwhiethGtohoedtLeacbhonriactaolrryePqruaicrteicmeenRtesgoulfattihoensp,ro2t1ocCoElR.,ThPearstt5u8d,ywailtshotmheeetxweimtpthitohnes `noted in the QA Statement. Protocol Number: StudyDirector: 3MC/VITRO/003-01/000 Devaki Sadhu, Ph.D. Company: Toxikon Corporation Signature: / (I la Date: 03)28f, ox Study Supervisor: Leigh Waugh-Cohen, B.A. VERIFICATIONDATES: `The study dates were as follows: ProtocolEffectiveDate: `Test Article Receipt: Project Log Date: Technical Initiation: `Technical Completion: Final Report Date: 12/12/01 12/12/01 12/12/01 02/05/02 02/20/02 03/28/02 Page Sof 18 000007 Toxikon Corporation `CPrHoOje/cHtNGuEmRbTerF:o0r1w.a7r0d19M-uGt1ation Assay-ISO (T-6889.7) 10 PURPOSE i`tTsheabCilHiOty/tHoGPinRdTuceFofrowrawradrdMumtuattaitoinonAsssaatytehvealhuyaptoexdantthheimnuet-aggueanniincepoptheonstpihaloroibafostyelsttarratniscfleervaisae (HGPRT) locus. The CHO assay system utilized toxic that are presumed deficient in the purine salvage enzyme purine analogs HGPRT. to select for resistant cells 20 REFERENCES `Thetestwasconductedbased uponthefollowingreferences: 21 BainodlRogeipcraolduEcvtailvueatTiooxniociftMye,dAiNcaSlVDAeAvMicIeIsS--OPa1r0t939:3-T3e:st1s99fo3r. Genotoxicity, Carcinogenicity 22 OECD Guidelines for the testing of Chemicals, "Genetic Toxicology:InvittaMammalian Cell Gene Mutation Tes," Test Guideline # 476, current version. 23 Hsie, AW. Casciano, D.A, Couch, DB., Krahn, DF., ONeill, J.P,, Whitefield, B.L. "EPA's Gene Tox Program," Mutation Research, 86: 193-214 (1981). 30 COMPLIANCE The present study conformed to requirementsincludedthe current all applicable laws and FDA, 21 CFR,Part 58 regulations. Specific regulatory - GoodLaboratoryPractice for `Nonclinical Laboratory Studies. 4.0 IDENTIFICATION OF THE TEST AND CONTROL ARTICLES iTnhfeormfaotliloonwidnogesinnoftorampaptliyo.nThweasSposnusppolriweadsbryestphoensiSbploefnosroarllwthesetraervteircleacphpalriaccatbeler.izaCtoinofniddeanttaiaals specified in the GLP regulations. 4.1 Test Article: Ammonium Perfluorooctanoate (FL-143) CAS/Code #: 3825-26-1/ (T-6889.7) LovBatch#: Lot 332 Physical State: Solid Color: Whi/Crtysteal Density: Not Supplied by Sponsor (NS) pH:4-7 Stability: Stable: Solubility: >1g/mL Storage Conditions: Room Temperature, -20C or 4C Safety Precautions: Standard Toxikon Laboratory Safety Precautions Page6 of 18 000008 `CTHoxOikHaGn CPoRrpTorFaotriwoanrd Mutation Assay- ISO (1-6889.7) Project Number: 01.7013-G1 42 Control Articles- (Toxikon Supplied) 4Q2C.1InNveegnattiovr#ey:CoLnPtRr-ol01A-rt1i1c-le00N1a-mCeC: Ham's F-12 Complete Mediuni . Physical State: Liquid Color: Colorless SSatfoertaygePrCeocnaduittiioonnss::SRtaonodmarTdeLmapbeorraattuorrey Safety Precautions 4Q2C.2InPvoesnittoirvye C#:onCtSrCo-l0Ar1t-i0c3l-e0N0a4m-CeC(Non-Activated Assay): Ethylmethanesulfoxide (EMS) Physical State: Liquid Color: Clear SSatfoertaygePrCeocnaduittiioonnss::S4tan2dCard Laboratory Safety Precautions 422.3 Positive Control Article Name (Activated QC Invento#r:y CSC-97-09-013-CC Assay): Dimethylbenzanthracene (DMBA) Physical State: Liquid Color: Clear SSatfoertaygePrCeocnaduittiioonnss::S4t1a2ndaCrd Laboratory Safety Precautions 50 IDENTIFICATION OF THE TEST SYSTEM CChuilnteurseeCHoalmlesctteiornO,vMaarnyas(sCaHsO,)Vcierlgilnsiuat.ilTihzeedcienlltshwiesraesdsearyiwveerdfeorbotmaainneodfrvom ta bhieopArsmyeoriifcaacanhiTnynepsee `hamster. 60 JUSTIFICATIONOFTEST SYSTEM AND ROUTE OF ADMINISTRATION 6a.t1thTehihsyapsosxaayntehvianleu-agtueadnithnee mpuhtoaspgheonriicbpoostye!nttiraalnsofefarastees(t HarGtPicRleT)vialoicuss.abiTlhiitsy tloociunsduisceremsuptoantsiiobnlse fhoyrpopxarntohidneuaoncdftHguGiaPnoRiTnne,farnomentzhyecmueltthuarteimsepdiurmienionsrndoerermtaonlscyentltlhsesainzde aDNAl.Icenllnlsoortmoaswlaclevlaslgs,e `HwGhePnRaTtoexnizcysmuebsatlaonncgewciatlhlehdy6p-otxhainotghuianneinaenidsgiunacnliundeeadnidnitnhecgororwptohrmaeitdneitudomD,NiAt ,is tshaelrveabgyedcabuysitnhge cell death. exposed to Exponentially growing cells, a various concentratioonfs sensitive the test to the toxic effects material.Ifthe test of 6-thioguanine, were materialis potentially ``bmuetcaogmeeniicn,canpoarbmlaelocfeulltsili(zwihnigchhypcoaxnanutthiliinzee,hgyupaonxiannet,hionre,6-tghuiaongiunaeniannedf6r-otmhitohgeuacnuilnteu)remmuetadtieumt.o `DHNowAevesyrn,tmheustiasntiscemlalsderetpaoisnstihbelierabbiyliatlytteomagtreoswynatshweetlicl apsatnhowramyasl.cTelalkseinn ctuolgteutrheerm,etdhieusemfbiencdaiunsges i6n-dtihciaotgeuatnhiantet.heThbearseifsorfoer, sceellelcstitohnatofgHroGwPRtoTfmourtmanctolsoinsietshe ilnactkhoefaprneysenabcieliotyft6o-tuthiiloigzueatnhienetoaxriec `assumed to have undergone mutation either spontaneousolry by exposure to the test material. Page 7of 18 000009 TCoHsOik/aHnGCPorRpTorFaotriwoanrd Mutation Assay - ISO (T-6889.7) Project Number: 01-7019-G1 r`aThnegeCoHfOc/heHmGiPcaRlTclAassess.hassbeaenusyed extensiveilny the detection ofmutagenic activity ofawide . 6S:p2oTnshoert'esstsaprectiifcilceatwiaosn.aTdhmiisniwsatsertehdeionpvtiitmraol, trohurtoeoufgaahdmsiolnviesnttractoimopnataivabilleabwliethitnthheitetsetstsyssytsteemr,n.per 70 EXPERIMENTAL DESIGN 71 CellLine nTehceeCsHsOat-royKs1elceecltltlhienecewlalscusletluercetsetdofror iets htidhgehbucalcokncgirngoeuanbidliftryeaqnudenrcaypiodf doubling HGPRT" time. It was mutants. not 72 Maintenance ofCHO Cells TCohmeplCeHteOmceedllisuwmecroensgirstoewdnofinHacmo'msplFe-t1e2 Hmaemd'isumF-c1o2ntmaeindiinugm1,0b%ufffetearlebdovwiinthese10rummM(FBHSE)P,E1S-.2 `semrMumL--fgrleuetacmoimnpel,et1e00-muendiitusm/.mLTpheneicicellllisn,waenrde1i0n0cu-bga/temdL asttr3e7pt1omCyc,in5.I1nc%omCpOlje,teamneddsiautmurawtaesd humidity. 73 Forward Mutation Assay 7.3.1 MetabolicActivation System (S9): `1T2h5e4.S9ThmeicSr9osroatmallivefrrahcotmioongewnaastperwepaasrepdurfcrhoamseSdprfargoumeMoDlaewcluelyarrTatoxliivceorlsoignyduIcnecd(w1i5t7hIAnrdousctlroiral iPsaorckitrdircivceo,faBcotoonres,anNdCi2nc8o6m0p7l)etaendmestdoiruedm awta-s80p+r5ep'aCreudntijlusutseb.efAorecoemxbpionsautrieonanodf Sus9edfraacstitohne, NmeAtaDbPol(icdisacotdiivuamtisoanl)syatstaemf.inaTlhceonccoefnatcrtaotriomnioxftu4r.5e mcgon/smisLteadndof2.i4smocgi/trmaLte, (retsrpiescotdiivuemly.salTth)eaSn9d fraction wasaddedto cach flask at aconcentratoif2o0n pL/mL. 7A.3p2arallPealraclyletlotCoyxtioctiotxyicaistsyayAswsaasy performed along with the Mutation assay. The cultures were treatedexactlyas the mutation plates, fixed, stained, and colonies counted. incubated fo9r days followingtheremovofathle test article, 73.3 PreparationofTest Cultures: A10pcpellr/doishxai2nd4mhtroiauprltiscpaerteiolparrtyaolleexlpcoystuorteo,xidcuiptlyipclaatteesw10e0rmemspelaeteadstwe2e0rd0ecseeleldse/d6a0mt ma ddeinsshi.ty of 1 x 734 Exposure Periods: a`cTthievcaetlelds waessraey.exApfotseerd etoxptohseurtees,t acretlilcslewfeorre17whaosuhresdintwthiecenowni-tachtiPvBatSedaansdsasyuapnpdle5mheonutresdinwitthhe complete medium. Page of 18 000010 ``TCoHxiOk/oHnGCPorRpTorFaotriwoanrd Mutation As- SsO(T-a6889y.7) BProG jectNumber: 01-7019-G1ieee 73.5 Phenotype Expression: Approximate4l8y hours following termination ofnon-activatedexposureperiodand approximately , 64 %; hours platedat 1 fxol1l0owcienlgltsepremrina1t0i0omnomfadcitisvha.tTehdeexcpeolslusrweepreeripoads,sceedllsewveerrye4t8r-y7ps2ihnoizuerds, ctoounmtaeidn,taainnd exponential growth during phenotypic expression for 6 days. 7.3.6 Selective Growth: Fmoultlaonwticnegllst.heThpehesneolteycptiivce emxepdreisusmioinncpelruidoedd,hcyeplolxsawntehrieneg-rforween Hianms'esleFc-t1iv2ewmiethdi1u0m%tfoetsaellbeoctvifnoer dsueprluimc,atep)enwiceirleliuns-esdtrefpotromeyaccihn t(esPt/Sc)onadintdion1.0 CeplMl d6e-ntshiitoyguwaansin2e.xT1e0n/1d0is0hemsm(fpievterifdoismh. cTahceh cultureswereincubatedfor9daystoallowcolonies to develop. 7Co3nc7urrPeanrtallyl,elthCelocnlionnigEnfgfeifcfiiecniceync(yPwCaE)s:determined by plating cells in selective medium without c6e-ltlhsi/o6g0umanmindei.shS.iTxhdeischuelstuwreersewesreeetdehdenfionrceuabcahtecdofnocren9tdraatyisonto (atlhlroewe cforloomneyafcohrmdautpiloicna.te) at 200- 73.8 Termination: A`twitthhGeieenmdsoaf. tOhnelyinccoulboantiiesonwpietrhi5o0d,orpmloatrees wceelrlesrwienrseecdowuintthed.PBS, fixedin methanolandstained 74 Control Articles 74.1 Positive Control Article: : The positive control for the non-activated system was Ethylmethanesulfoxide (EMS). For the `activated system, Dimethylbenzanthracene (DMBA) was used asthepositive control. H7a.4m2's NFe-g1a2tiCvoempColnettreolMAerdtiiculem: served as the negative control article for the non-activated and activated assays. 80 DOSAGE 8.1 Thetestarticlewasdissolivntehde culture mediumat aconcentrationof 5 mg/mLasper OECD guidelines. 1:128, 1:256 and 1:512 dilutionsof 5 mg/mL concentrationofthetest article 82 Dose Selection 82.1 A cytotoxicity preliminary dose-range finding was performed using several dilutionsofthe 5 mg/mLtest article and 1:128, 1:256 and 1:512 dilutions were selected for testing. Thepositive control for the non-activated assay, EMS, was used at a concentrationof 244 ng/mL.Thepositive controlfor the non-activated assay, DMBA, was used at a final concentration of9 pg/mL. Page 9of 18 000012 `CTHoxOi/koHnGCPoRrpTorFaotriwoanrd MutationAssay -1S0 (I-6389.) * Ee PrEojer ct Nume ber:r 01-70e19-Ge 1 em r seeeeee . i ee oi rmsscm es 9.0 EVALUATION CRITERIA 9.1 Evaluation of Test Results The resusofthe CHO/HGPRT Locus Mutation Assay were evaluated based on the number of TG-resistantmutantsper 1 x 10 survivingcells. Thesignificanceofthe testresults were determined by using the statistical program, Tallarida, R.S. and R.B. Murray's Pharmacological Calculations Procedure, ANOVA (analysisofvariance) and Newman- Keuls Test for confirmation. `fThrisesqtautiesotnfictcahlyemetetshtaordtdicelteecrommipnaerdeidfttohtehreenweagsatiavseicgonnitfrioclaanrtt(ipcle<. 0.05)increaseinthemutation `eTxhheitbeistteadraticrleeprwoadsuccoinbsleidaenrdedstattoihsativcealclayussiegndifaipcoasntitiinvcerreeasspeoinnstehienntuhmebaessraoyfimfutthaenttesstpaerrti1clxeh1a0d cells over its concurrent negative control article. 10.0 RESULTS ApreliminaryRangeFindingAssaywasconductedtodeterminethedilutionsofthe testarticleto be usedinthe Forward Mutation Assay. Exposure todilutionsofthe test article resulted in tchyetosteoxfiicndiitnygrs,antghirnege Assay. fdriolumti5o0ns%rcaenlglisnugrvfirvoalm a1t:11:2182t8o a1n:d58120%wgerreeatuetirlaitzed1:i2n56thaenFdo1r:w5a1r2d. BMuatsaetdioonn `The parallel cytotoxicity assay indicated that the positive control article exhibited a cell survival `percentage of 10% and 45% in the non-activated and activated assays respectively (Table I). The dilutionsofthe test article extract, 1:128, 1:256 and 1:512 exhibited cell survival values of 105, 99 and 102 respectiveilnythe non-activated assay and 94, 104 and 118% respectiveilny the activated assay. The negative control activated assays respectively. article exhibited values of 79 and 110% in the non-activated and Fi`nThdeintegstAasrstaicyleinwansont-eascttedivaattetdhaestsharye.e dNiolnuetioofnst(h1e:t12h8r,ee1d:i2l5u6t,io1n:s5t1e2s)teesdtsahbloiwsehded ianstatthiestRiacnalgley significantincreaseinthe numberofmutants per 1 x 10survivingcells as compatroethde `pocsoirtrievsepocnodnitnrgolnsegdaitdivsehcoowntarosltsatiisntbicoatlhlty hseignnoinf-iaccanttivianctreedasaendinactthievantuemdbaesrsaoyfs m(uTtaabnletss Ip-eVrII1).x T1h0e surviving cells as compared tothe correspondingnegativecontrols. 11.0 CONCLUSION `lTohceusteasst aervtiicdlee,ncAemdbmyotnhieumabPseernfcleuoofroaoctsatnaotiastteic(aFllLy-1s4i3g)ni,fdiicdanntoitncirnedauscee imnuttahteinonusmbaterthoefHmGuPtaRnTt colonies at the highest analyzable test concentration as compared to the negative controls in the psirgensiefnicceantorinacbrseeanseceinoftahennuemxbogeernoofumsumtaanmtmacloiloanniemseitnapboosliitcivaectciovnattrioolnssiynstbeomt.h tTheheactsitavtaitsetdicaalnldy `non-activated assays verified the proper functioningof the test system. `Therefore, based on the Page 100f 18 000012 `CTHoxOik/oHn CGoRrpTorFaotriwoanrd Mutation Assay -1SO (T-6889.7) Project Number: 01-7019-G1 eevxapleuraitmieonntalcrcitoenrdiiatioofnstehmeplporyoetdoc.ol, the test article is considered non-mutagenic, under the 120 RECORDS 12.1 Originalrawdata is archivedat Toxikon Corporation. 122 Acopyof the final report and any amendments is archived at Toxikon Corporation. 123 The originalfinalreportand forwarded to the Sponsor. acopy ofanyprotocol amendmentsordeviations is 124 sAplelcuifniucsateidont.estarticlesshallberetumedtothe Sponsobyr Toxiperkspoonnsor,'s 12.5 aFiuntahlorriezpaotritosnw/ialplprnoovtablefrroempTroxoikdoune.xcceepdt infull,without the written 130 CONFIDENTIALITY AGREEMENT Statementsofconfidentialweirtey as agreed upon prior to study initiation. Page 11 of 18 000013 E aB EtRes re ore e `Table I: Parallel Cytotoxicity Assay Il I ial Extract (Mutagen Assay) Activatio per Plate . urvival* Qusie 1Activated [232 240 2371 236 |118] * Percentage Survival= Average colonies per plate / numberof cells `plated (200 cells) x 100 % Page 120f18 000014 sian [res i a fame Tome me ild l f a ai rere ww [wwwwe] / 000015 - RO em eran [orem Fr T e eee | somes| -- ( ---- E I-- ld ----d ------ | f-- e-- e[ ----E ----e ---- -- e f|m-- m ree [ ---- ----E ---- -- e ---- fa --mee e || ES e ----| Ere SE eee 000016 : fo---- ieeeoem di HGPRT MUTAGENEASsIsaSy EpE p mEso .l y|peoplnilapS ldollEhalfbellaooll]llao]] Epp E omems:r |opoids[Li eeLaloeeL lfE[fm aLLoaEofalLlaE La aiAa feol ] EE E p E o p|obtlaeao em r bS afaLulaab]at lspi]afaa]s) 000017 | HoPRTMiUTAyGENEASSIASY [ bm pe me ) -- | bormeaen:|ute| | sn|oai o | i |T |ee ||stne| a] FI oreemmseemsspmor oe|| ovon||m oooo||on|| cooo[|o | 0||| oooo ||] oco|e|[20]] Ei tomyt:e:|oio|eie|eoo|on| ein|ase|i on|oiot|oih|eooe| 000018 mt nrFgrntincurs mr || ep a i m m , cs i -- ------ ------ -- -- sen | 000019 = --_---- SUMOFMMUTAANrTCROaLeOrYNvYePnsEaryQUI EGRNRECASYAY ee oE e remr fom p e e o r ee e ] aw [Ermer {er ww [a T [Emrame fepm ea ] ow] -- 000020 :: DEC-12-20H0E1D 12:32 PH TOKIKON Fat No. 7812711133 bn i `TOXIKON TEST PROTOCOL FDA GLP GUIDELINES CHO/HGPRT FORWA(TR-D688M9U.7T)ATION ASSAY - ISO TOXIKONPROTOCOLNUMBER: 3MC/VITRO/003-01/000 PROTOCOLDATE: 12/03/01 EFFECTIVEDATE: 12/12/01 I COMPLIANCE. 21 CFR, Part 58 Good Laboratory Practice for Nonclinical Laboratory Studies MANAGQEFM THE ESTN UDT Y. `Performing Laboratory: - `Toxikon Corporation 15 Wiggins Avenue Bedford, MA 01730 Sponsor: 3M Company 3M Center BCuoirlpdoirnagte02T2o0x-i0c2o-lEo-g0y2 St. Paul, MN 55144 000021 _ DEC-12-2001 WED 12:3 PM TOXIKON CAE Dotmeeerbne Fae rsyi CBO/HGPRT ForwardMutationAssay -ISO (1-6389.7) FAX NO. 7812711133 Je Tn Devaki Sadhu, Ph.D. `Toxikon Corporation 15 Wiggins Avenue Bedford, MA 01730 "PROTOCOL ACCEPTANCE fine Date nc Quality Assur "Toxikon Ct 15 Wiggins Avenue Bedford, MA 01730 Ble Date ' Lda[llBea" `Sponsor's Representative 3M Company 3CoMrpCoernatteerToxicology Building 0220-02-E-02 St. Paul, MN 55144 _n/i3/s) Date P. 03/12 Page 2of 11 090022 __ DEC-12-2001 WED 12:3PH2 TOKIKON FAX NO. 7812711133 P. 04/12 TCoxHiOkoHnGCPoRrpTorFatoirownardMutationAssay - ISO (T-6899.7) F Effer Dactet:1i2o 1v20e1 me e-- r ---------- tall -- eniese-- ereeeeee-- eset TABLE OF CONTENTS Title Page Protocol Acceptance TableofContents 10 Purpose 20 References 30 Compliance 40 IdentificationofTest and Control Articles 50 Identificationof Test System 60 Justificationofthe Test SysantdReoumte of Administration 70 Experimental Design - 80 Dosage 9.0 EvaluationCriteria 100. Records 110 ConfidentialityAgreement 120 Protocol Amendments/Deviations Page3of 11 000023 DEC-12-2001 VED 12:32 PH TORIKON Toulon Corporation FFCrrHooOstco/acHllGNDPaatRbeTseFr1o3:r0Sw3aM0r1GdVMuItTatRioOnAA2ss0a1y0-010SO (T-6889.7) Erte Dae 121200 FAK NO. 7812711133 P0512 1.0 PURPOSE . . The CHO/HGPRT Forward Mutation Assay (with Confirmation) evaluates the mutagenic ppohtoesnpthioarlio boastyf elsttraanrstifcelreavsiea(tHsGaPbRiTl)ittlooycuisn.duTcheefCorHwOardasmsuatyastyiosntseamtuttihleizheys ptooxxainc tphuirnien-egaunaanlionges to select for resistant cellsthatare presumed deficient in the purine salvageenzymeHGPRT. The gene mutations are induced at the HGPRT locus in cultured Chinese hamster ovary (CHO) cells. 2.0 REFERENCES `The test will be conducted based upon the following references: 2.1 Biological EvaluationofMedical Devices-Part 3: Tests for Genotoxicity, Carcinogenicity and Reproductive Toxicity, ANSVAAMI/ISO 10993-3: 1993. 22 OECD Guidelines for the testing ofChemicals, "Genetic Toxicology: In.vitra Mammalian Cell Gene Mutation Test," Test Guideline # 476, current version. 23 Hsie, AW, Casciano, D.A., Couch, D.B,, Krahn, D.F., ONeill, J.P., Whitefield, B.L. "EPA's Gene Tox Program," Mutation Research, 86: 193-214 (1981). 24 Extraction procedures,ifapplicable, will be based upon the standard titled Biological `EvaluationofMedical Devices-Part 12: Sample Preparation and Reference Materials, EN/ISO 10993-12 (1997). > 3.0 COMPLIANCE The study will conform to all applicable laws and regulations. Specific regulatory requirements includethe current Good Laboratory Practicefor Nonclinical Laboratory Studies, FDA, 21 CFR, Part 58. 4.0 IDENTIFICATION OF THE TEST AND CONTROL ARTICLES Thefollowing information will be supplied by the Sponsorona test requisition form or other correspondence wherever applicable; it does not apply to confidential information. The Sponsor `will be responsible foralltest article characterization data as specified in the GLP regulations. `Test and control articles (exclusiveofextracts)that are mixed with carriersrequireverification of croentcuernnterdattiootnh,ehSopomnosgorenfoeriacthnadyrascttaebirliiztayt.iSonaamnpdlevseoriffitceasttioann,dwchoenrtreovleraratpipclleicmaibxlte.ures will be Paged ofl 000024 _ DEC-12-2001 WED 12:32 PH TOKIKON FAK BO. 7812711133 + TCoHxiOkBonGCPoRrpTorFaotriwonardMutationAssay - SO (T-6889.7) P`Prroottooccooll NDuameb:e1r2:0I3M0C1/VITRO/002-01/000 Effective Date: 121201 41 Test Article: To Be Determined (TBD) CLAoSB/aCtocdhe##:: Physical State: DCeonlsoirt:y: PStEa:bility: ESxolpuibrialtitiyo:n Date: Storage Conditions: Safety Precautions: 4.2 Control Articles (Toxikon Supplied): T4.o2x.i1kNoengaQtCiv#e CTonBtDrol Article Name: TBD Physical State: Liquid CSotloorrag:eC:olRoorloesms.Temperature Safety Precautions: Standard Laboratory Safety Precautions 4.2.2 Positive Control Articles: 4.22.1 Positive Control Article Name: TBD `TPohxysiikcoalnSQtaCte#: TBD Color: Storage: Safety Precautions: Known Mutagen. Appropriate Laboratory Safety `Precautions 4.2.2.2 Positive Control Article Name: TBD P`hTyosxiickaolnSQtaCte#: TBD Calor: SSatfoertaygeP:recautions: Known Mutagen. Appropriate Laboratory Safety `Precautions P. 08/12 . PageSof 11 000025 DEC-12--2012001 VED 12:3 33 PH TOKIKON FAK HO. 7812711133 P. 01/12 TCoHxiOkHonGCPoRrpTorFaotriownardMutationAssay - 1S (T-6889.7) `PPrroottaocceoll NDautmeb:e1r2:/I03M8C1/VITRO/002-01/000 Effective Dates 12/1201 -50 IDENTIFICATION OF THE TEST SYSTEM . CCuhlitnuersee HCoalmlsectteironO,vaRorcykv(iCllHeO,)Mcaerlylls auntidl.izTehdeinctelhlissaasresadyearirveedobftraoimnaendforvoamritahneAbimopesyroifacTycahpinenese hamster. 6.0 JUSTIFICATION OF TEST SYSTEM AND ROUTE OF ADMINISTRATION 6a.t1thTehihsypaosxsaanytehvianleu-agtueasntihneempuhtoasgpehnoircibpoostyelnttiraalnsoffearatseest(aHrGtiPclReTv)ialtocsusa.bilTihtiys1l0oicnudsuicsermeusptoantsiiobnlse fhoyrpopxraondtuhcitnieonanodfHgGuaPnRiTne,farnomentzheymceultthuarteimsepdreisuemntininorndoerrmatlo cseylnltshaesnidzealDlNoAws.ceInllntoormsaallvcaeglels, `twhheeHnGaPtRoxTicesnuzbystmaenacelocnagllweidt6h-htyhpiooxguaanntihnienie ainndclguudaendininetahnedgrioncwotrhpomreadtiedumi,ntoitDisNsAa,lvtahgeerdebbyy ecaxupsoisnegdcteollsdeevaetrha.l Ecxopnocneennttriaatliloynsgorfotwhientgescetlmla,tesreinasli,tiIveftthoettheesttomxaitcereifaflecitssopoft6e-ntthiailolgyumauntiangee,nairce,: ninocrampaablceeolflsut(iwlhiizcihngcahnypuotxialinztehihnyep,oxgaunatnhiinnee,, ogrua6n-itnheioagnudan6i-ntehfiorgoumantihneec)umltuutraetmeetdoibuemc.oHmoewever, `smyunttahensticselilssmraedtaeinpothsesiirblaebbiyliatlotyergnraotewsaysnwteheltliacspantohrwmaaylsc.elTlaskienncutlotguertehemre, dtiheusme bfeicndaiunsges DinNdiAcate tthhaitogtuhaenibnaesi.sTfhoerresfeolrecet,icoenlosftHhaGtPgRrTowmutotafnotrsmiscotlhoenileasckoinfthaenpyreabsielintcyetoofu6t-itlihzieotghueantioxnieca6r-e aTshseuCmeHdOt/oHhaGvPeRuTndAesrsgaoynheasmubteaetniounseeditehxetresnpsoivnetlaynicnoutshleydoertbeycteioxnpoofsmuutrtaeogetnhiecteascttimvaitteyroiafla. wide rangeofchemical classes. 6te.s2tTshysetetme.st Tarhteiscleewarielltbheeaodnmliynriosutteerseodfiandmviitnrios,tdriarteicotnlyavoaritlahbrleoiungathhissotlevsetnstycsotmepma.tible with the 7.0 EXPERIMENTAL DESIGN 7.1 Cell Line `The CHO-K1 cell linc is selectedforits high cloning ability and rapid doubling time. To reduce tbheesneelgeacttiedveagcaoinntsrtolthairstipchleenforteyqpueeanncydorfeHtGumPeRd Ttomnuotramnatlsgtrooawstlhomweadsipuomssfiobrlet,hrceelelcourlmtuorreescdaayns before use. 7`T.h2e CMaHiOntecenlalnscweiollfbCeHgrOoCwenlilnscomplete Ham's F-12 medium, buffered with 10mMHEPES. uCnoimtpsl/meltepemniecdililiunm, awinldl c1o0n0siusgt/moif 1st0r%epfteotmaylcbionv.iInnecsoemrpulmet(eFmBSe)d,iu1-m2ims MseLr-ugml-ufrteaemicnoem,pl1e0t0e `medium. The cells willbeincubated at 371C, 51% CO, and saturated humidity. Page Gof 11 000026 DEC-12-2001 HED 12:33 PH TORIKON FAK KO. 7812711133 P. 08/12 TCeHstOkManGCPoRrpTorFaotriwonard Mutation Assay - 150 (T-6889.7) PPrroottooccooll NDautmeb:e1r2:0S3M0C1IVITRO/02.01000 Effective Date: 121201 7.3 The Cytotoxicity Cytotoxicity Assay Assay (f necessary)isconducted without metabolic activation, prior to the . Mutation Assay. The highest concentration is based on maximum solubility,10 mg/m, or `sSupbosntsaonrcespceocnicfeincattriaotniso.n,Linqeugiadtitveestacrotnitcrloelswainldlbsoelvteensttecdonattro1l0(0iuflaipmpll-i0c.a0b1leu)li/sml.teEstaecdhitnetsrtiplicate. `CTuhletcuurletsuraeresairnieteixatpeodsiend60tomthmedtiessthaersatitcl2e0a0ndcecloln/tprloaltse afpoprr1o5x-i1m7ahtoeulrys2.4Ahtoutrhseeprnidoortfothdeosing. exposureperiod,dishesare rinsed with mediumor Phosphate Buffered Saline (PBS) and ipenrciuobda,tetdhfeodriasnhesadadrietifoinxaeld 6i-n9medthaayntosol,alsltaoiwncedolwiotnhiGteiosemdsevaealnopd. cAotlotnhieesencdouonftthede.. iRenlcautbiavteion survival is obtained by comparing the numberofsurviving colonies for cach dose to thatofthe `negative or solvent control. 7.4 Forward Mutation Assay 7.4.1 Metabolic Activation System ($9) The $9 microsomal fraction is prepared from Sprague Dawley rat livers induced with Aroclor 1254. The SO rat liver isocitric cofactors and hioncmoomgpelneatteemiesdsituormedisatpr-e8p0a:r:e5djCuusnttiplriuosre.toAexcpoomsbuirneatainodn ofS9 useda fraction, sthe metabolic activation system. The cofactor mixture consistsofsocitrate (trisodium salt) and NADP (disodium salt) at afinal concentration of4.5 mg/mland 2.4 mg/m, respectively. The S9 fraction isaddedto eachflaskor directlyto the cofactor mixture at a concentrationof 20 ul/ml. 7.4.2 Parallel Cytotoxicity Assay Aexapcatrlayllaels ctyhteomtuoxtiactiitoynapslsaatyesi,s ipnecrufboartmeeddfaorlo6n-g9wdiatyhstfhoelMluowtiantgiothnerasesmayo.vaTlhoefctuhlteurteesstaarretitcrlee,ated fixed, stained, and colonies counted. 7.4.3 PreparationofTest Cultures Approximate2l4y hours prior to exposure, duplicate 100 mm mutation plaaretseeedesd at a density of5 x 10 celis/dishandtriplicate parallel cytotoxicity plates are seeded at 200 cells/60 mm dish. 7.4.4 Exposure Periods Tchheemcieclalslsw)ilinl tbheeenxopno-saectditvoatthedetaessstayaratnicdle4-f6orho1u5r-s17inhtohuersac(t4i-v6atheodurasssfaoyr. hAifgthelryerxepaocstuivree, cells are washed at leastoncewith mediumor PBS and supplemented with complete medium. Pawgaee7of oft 1 000027 DEC-12-2001WED 12:33 PH TORIKON FAK NO. 7812711133 P. 09/12 T`oCslRkOonGCPoRrpTorFaotriwoanrdMutationAsay- ISO (T6899) PPrroottooccooll NDuameb:e1r2:0S3M0C1IVITRO/02-0L/000 Effective Date: 1212001 ee 7.4.5 Phenotype Expression ' Approximately 24-48 hours following terminationofexposure period, cells are trypsinized, ``macintoaiunaennxdpotpnleaenttedidaal,tgr1oxwt1h0dcuerlilnsgppehren1o0t0ympimcdeixsphr.esTshieocneflolrs aarpeprpoaxsismeadteevleyr7y-498d-ay7s2.hours to 7.46 Selective Growth Following the phenotypic expression period, cells are grownin selective medium to select for `femtuatlabntovcielnles.sTerhuem,sepleencitciviellmien-dsituremptionmcylcuidnes(Ph/ySp)oxaanndth1i0ne12-f6r-etehiHogAuMan'iSne.F-1A2twoitatlhof1t0e%nddiisalhyezsed (fivefrom each duplicate)areused foreachtest condition. Cell density is 2 x 10 / 100 mm petri dish. The cultures are incubated for 6-9 days to allow coloniteos develop. C7to.hn4i.co7ugrPuraaernnailtlnleyel,.CtlShoienxcidlniogsnhEiefnsfgiaecrifeefnsicecyeid(eePndicCfsyEo)dreetaecrhmicnoendcebnytrpaltatiionng(ctehlrleseifnrsoemleectaicvhe dmuepldiicuatme)wiatth2o0ut0 cells/60 mm dish. The cultaurertheesn incubated for 6-9daysto allow colony formation. 7.4.8 Termination At the end ofthe incubation period, plara e rt insee dwis th PBS, fixed in methanol and `stained with Giemsa. Only colonies with 50 or more cells are counted. 7.5 Control Articles 7.5.1 Positive Control Article - `NTihteropqousiintoivleinceo-n1t-rooxlifdoer(t4hNeQn)o.nF-oarcttihveataecdtisvyasttedemsyiss. tEetmh,yl9m,e1t0,h-anDeismueltfhoyxli-d1e,2(-EbMeSnz)anotrh4r-acene (DMBA) or Dimethylnitrosamine (DMN) can be used as the positive control. 7.5.2 Negative Control Article `The appropriate solvent, extractant or cell culture medium will serve as the negative control article. 7.6 Confirmatory Assay TCohnefriersmualttsoorfytAhsesaFyo.rTwhaerdCoMnuftiartmiaotnorAyssAasysawiyllwiblle bceonpfeirrfmoerdmtehdraosugdhesacnriibneddepinenSdeecnttions 7.0 and 8.0. Page 8 of 1 000028 DEC-12-2001 WED 12:34 PH TOKIKON FAK KO. 7812711133 P. 10/12 CTHoOxt/kHonGPCoRrTporFaotriwoanrdMutatinAssay - 150 (6885.7) PPrroottooccooll DNautmeb:e1r2:0S3M0C1IVITRO/0Z-0L/000 EffecetiveeDate: 121201 8.0DOSAGE . 8.1 PreparationofTest Anicle 8T.h1e.1teMsetdairctaiclleDewivlilcbees:extractedatratios specified by EN/ISO 10993-12. Extraction vehicles may be oneofthe following media: serum-free medium, complete medium, or0.9% USP Sodium Chloride for Injection, USP (NaCl). Extraction conditions will beas specified by Sponsor: (pleasecheckdesired condition) 71022C1ffo2orr2o24nehohuorCusr @ 5307112CCffoorr 7722 hhoouurrss ` 371C for 24 hours per Sponsors directions (____"C for hours). `wEixtthraScotsdipruempaCrheldorwiidtehmwieldibeumdiwliultledbowittehst2eXd amte1d0i0u%m n(ndeatt)esctoendceatnt5ra0t%ioenx.trEaxcttraccotnscepnrterpaatrieodn (considered neat). Modifications to test article preparation will beas specified by the Sponsor. 8.1.2 Solid Solid/Liquid Test Articles test articles wil be dissolved or suspendedin avebicle appropriateforthe test system or as scpoencciefniterdabtiyotnhseoSrpaosnssopre.ciLfiiqeudibdytetshetaSrptoinclseosrw.ill be administered as received at predetermined 8.2 Dose Selection 8`.m2a.x1imTuhnen csoolnucbeinltircayotifotnhsefoterstthaertMiuctleatoiroSnpAosnssaoyr aspreecsiefilceacttieodnsb.aIsfendeocnestsoaxriyc,itay Ciyntfootromxaitciiotny, Assay wciolnlcbenetpreatrifoonrmwehdictho cdaeutseersmialneotwoxilceivteyl.oIffstuorxviicviatly (isapepvriodxeiam,attehleydo1s0e36s)sahonudlfdoiunrclloudweerthdeosheisg,hest i`nmcalxuidminugmcoonnecednotsreawtiitohnwniollapbpearbeanstedcyotnottohxeicliitmyi.toIff1so0laupbiplairteynotrcy1t0otmogxi/cmi,tywihsiocbhseevrevredi,s ltohweer (inocrraesmesnptesc.ifIiendtbhye tahbeseSnpcoensooftro)x.icSiutbys,elqiuqeuindttedsotseasrtwicillesdweiclrlebaeseitensatpedpraot x1i0m0atule/mhall-f0-.l0o1g ulm. Dose selection modificatiwoinls be as specifiedby the Sponsor. o8f.2m.e2dMiecdailcdaelvidceeviecxetreaxcttrsaacrteswniolt baenaalnyazleydzueadlaetssonotehceornwciesnetrreaqtuiiorneod,nliyn (wnheiacthcexatsraectt)h.eDilutions justificationshallbe indicated in the final report. An initial Cytotoxicity Assayisnot performed for device extracts. Page9 of 11 000029 _ DEC-12-2001 VED 12:34 PH TORIKON FAR KO. 7812711133 PRI Toxikon Corporation P'rCoHtoOc/oHlGNPuRmbTeFro:rSwMarCdVMIuTtRatOiMonO2A0s10s-0aI0SyO (T-6889.7) PrEfofteocctoivleDaDattee::1122/0132/0011 9.0 EVALUATION CRITERIA The resultsofthe CHO/HGPRT Locus Mutation Assay will be evaluated on the basisofthe b`neudmebteerromfTinGe-drebsyiscittahnetr omunteoanfttshpeefro1llxow1i0ngsmuertvhivoidnsg: cells. The significanceof the test results will 9.1 The test resultsareanaulsinygasztateistidcal program such as Tallarida, R.S, and RB. KMuerurlasyT'essPthfaorrmcaocnofliorgmiactailonCoalfcpualiartiwoinssePcroomcpeadruirsoen,sA. NTOhiVsAsta(tainsatlicyaslismeoftvhaordiadnectee)rmainndesNiefwthmearne-is a significant (p <0.05)increasein the mutation frequencyof the test article compared to the inelglatbievaencaolnytzreodlbayrttihclee.mTehtehordeosuflLtsinoebatraiRneegdrfeosrsithoenumsuitnagtiaonprforegqruaemncsyucaht atshe"vLairnieoaursRdeogsreeslseivoelns PI"robgyrRaJm.s,TSaplrlairnigdear-aVnedrlRagB,. NMeurwraYyo,rk(,Ma1n9u86a,lopfpP1h0a-r13m)a.coTlhoigsiwciClaldceutleartmiionnesifwtihtehrCeoimspauptoseirtive. dose response. s9t:a2tTishtiectaellsytsairgtniicfliecainstcpoonssiitdievreeddotsoehraevsepocnasueseodr aatploesaisttivoenreetsepsotnasretiicfltehdeotseestsharotwicsleasrheopwrosduacible tstoattihseticcaolrlryessipgonnifdiicnagntneignatcivreeicoanntsthreeolnaurtmibcleer.ofmutants per 1x 10 surviving -cells as compared `9T.h3eCcoonnffiirrmmaattiioonn Aassssaayywill validate the reproducibility ofthe mutagenesis assay. 10.0 RECORDS 10.1 Original raw data will be archived at Toxikon Corporation. 10.2 Acopyof thefinal report and any amendments will be archived at Toxikon Corporation. 10.3 Theoriginal final report and acopyofany protocol amendments or deviations will be forwarded to the Sponsor. 104 Allunused testarticlewillbehandledasspecifiedinthe Test Requisition Form. Otherwise, all remaining test article will be discarded. 11.0 CONFIDENTIALITY AGREEMENT Statementsofconfidentiality may be agreed upon prior to study initiation. Page 10 of 11 000030 DEC-12-20W0E1D 12:34 PH TOKIKON FAK No. 7812711133 P2712 ``TCxHiOk/onHGCoPrRpTorFaotriwonardMutationAssy- ISO (16883. PPrroottooccoollDNautmb:e1r2:0S3M0C1/VITRO/O201/000 ET ffecT tive DatE e: 121201 E eRe aT SS ieermereaeeres 12.0 PROTOCOL AMENDMENTS/DEVIATIONS . Awrliltcinhga,ngseisgnteodtbhyetahpeprSotvueddy pDirroetcotcoorl,adnadtetdh,earnedasmoaninftoaritnheedcwhiatnhgtehsewpirllotboecodl.ocNumoepnrtoetdocionl amendments will be made without written approval in the form ofa Sponsor Communication Log bteitmweeoefntthheechSapnognes.or and the Study Directorwhichwillbe generated asclosely as possible to the Page 110f 11 000021