Document DG82reXM49Edo4VnKqZnKQpwQ

AR226-0132 G-s Final Report . Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures With PFOS -- comersTo . 01887 COVANCE.> FINAL REPORT "UNSCHEDULED DNA SYNTHESIS IN RAT LIVER PRIMARY CELL CULTURES `WITH PFOS AUTHOR Maria A. Cifone, PhD PERFORMING LABORATORY Covance Laboratories Inc. 9200 Leesburg Pike Vienna, VA 22182 LABORATORY PROJECT IDENTIFICATION Covance Study No.: 20784-0447 Sponsor Study No.: T6295.19 SUBMITTED TO 3M Corporate Toxicology 3M Center Building 220-2E-02 St. Paul, MN 55144-1000 STUDY COMPLETION DATE `November 9, 1999 ) Page 1 0f20 01888 Covance 20784-0447 QUALITY ASSURANCE STATEMENT Unscheduled DNA Synthesis In Rat Liver Primary Cell Cultures With PFOS `The report has been reviewed by the Quality Assurance UnitofCovance Laboratories Inc., in ParcoctoercdtainocneAwgietnhctyhe(EGPoAo-dTLSaCbAo)r,atToirtyleP4ra0cotfictehreeUgu.lSa.tiCoondsaeofseFtedfeorrtahliRnetghuelaEtnivoinrsoPnamretnt79a2l, and wreiptohratendytaopptlhiecSatbluedyamDeirnedcmteonrtasn.d Tsthuedyfodlilroewctionrgmiannsapegcetmieonnts.weWrreictotnednuscatteudsarnedpotrhtesfoifndings pirnoscpeedcutrieosn.s andfindingsare issued to Covance management according to standard operating Inspection Dates Phase DatDeisreRcetpoorratnedd StotuSdtyu~dy~ Auditor Director Management 0831/99 WeighingofTest Article 08/31/99 P. Caceres 11299 DrafPtrRoetpoocorlt aRnedview 17299 P. Caceres 11/09/99 Final Report Review 11/0999 C. Smith Reprsbentaatitve, QuT ality Ase surance Unit Da1te0/5/58 2 01889 Covance 20784-0-447 STUDY COMPLIANCE AND CERTIFICATION The described study was conducted in compliance with the Good Laboratory Practice regulations as set forth in the Environmental Protection Agency (EPA-TSCA), Title 40ofthe U.S. Code of Federal Regulations Part 792, and with any applicable amendments. There were no significant deviations from the aforementioned regulations or the signed protocol that would affect the integrityofthe study or the interpretationofthe test results. The raw data have been reviewed by the Study Director, who certifies that the evaluationofthe test article as presented hercin represents an appropriate conclusion within the contextof the study design and evaluation criteria. Al test and control results in thisreportare supported by an experimental data record and this record has been reviewed by the Study Director. Study Director: ia A. Cifone, PhD, Genetic and Cellular Foxicology 2-99 Study Completion Date 91890 3 TABLE OF CONTENTS Covance 20784-0:447 STSUpoDnYsoIrNFORMATION css TAessstayArItnicfloermation SSutpuedryvDiastoersy Personnel OBJECTIVE... TEST SYSTEM RATIONALE csc MATTesEtRSIyAstLeSm AND METHODS ccs] Control Articles EXDPosEiRnIgMPrEoNcTedAuLreDESIGN ccs UAsDsSayAAscscaeyptance Criteria `Assay Evaluation Criteria CONCLUSION css 13 RECORDS TO BE MAINTAINED wns 14 DATA TABLES wns 13 HISTORICAL CONTROL DATA nnd | 4 01891 Covance 20784-0447 ABSTRACT The test `primary article, PFOS, was tested cell cultures. PFOS was in the in insoluble vitro assay for unscheduled DNA synthesis in water and Williams Medium E. In in rat liver dthiemebtohtytlosmuloffoxtihdeef(ubDeMaStOS)0,1 tmhge/temsLt.artTihcleepfroercimpeidtaateswusapsenasbisoenntwiatth40p1recmigp/itmaLt.e tDhatMsSetOtlwedasto ctehsotsaerntiaclsetphreevpeahrieclde.in DInMtSheOasasnadyddoessecrdiibnetdoimnetdhiias.reIpnormte,driatuhme,paatsoucsypteensswieornewietxhpofsineed to the, precipitate was observed at 4010 pg/mL `tBoapsdeodsoenfosroltuhbeiliintyviitnrDo MUDSSOaasnsdaym.edFiifutmee,naccoonncceennttrraattiioonnsooff4P0F0O0Spwge/rmel awpapsliseedl,erctaendgiansgtfherom t0r.e2a5t0edppgl/atmeLs ttoo 4th0e00cepllg/demnLs.itCyoyfttothoexiccointtyrowlaspldateetserampipnreodxibmyatceolmypa2r3ihnogurthseacfetlelr dineintsiiattyioofntohfe treatment. The test article was excessively cytotoxic at and above 50.0 pg/ml, weakly cytotoxic 2125.0 pg/ml, and noncytotoxic at and below 10.0 pg/ml. The cellular morphology was suitable offotrhaenadlyossieslaetvealnsdsbheolwoewd2a5n.0elpegv/amtli.onNion meevaidnennecte nfuocrlaenaringcrraeiansse(iNnNUGD)Sorwainstohbesenruvmedb.erNoofne nnuecglaetiivceonitnatihneining 5viotrromaosrseayneftorgruanisncs.hedTuhleedteDstNarAticslyen,thPeFsOiSs,inwraast ltihveerrefporriemaervyalcuealtlecdulatsures. 01892 + - Covance20784:0447 STUDY INFORMATION Sponsor 3M Corporate Toxicology Test Article Sponsor's Identification: PFOS (FC-95), Lot 217 Date Received: August 19, 1999 Physical Description: White crystalline powder Storage Conditions: Ambient temperature Assay Information Type of Assay: Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures Protocol No.: 447 Edition 17 Covance Study No.: 20784 3M Study No.: T6295.19 Study Dates Initiation Date: August 20, 1999 - Experimental Start Date: September 1, 1999 Experimental Termination Date: September 22, 1999 Supervisory Personnel Study Director: Maria A. Cifone, PhD Laboratory Supervisor: Agnes Nourbakhsh, BS OBJECTIVE `The objective of this in vitro assay was to evaluate the ability of the test article, PFOS, ora metabolite, to induce DNA damage in rat primary hepatocytes by measuring unscheduled DNA synthesis (UDS). `TEST SYSTEM RATIONALE This assay aims to establish whether the test article or its metabolites interacts with rat liver primary cell (hepatocyte) cultures to induce DNA damage followed by DNA repair measured as UDS. Hepatocytes were isolated from liver and exposed in vitro to the test article and tritiated thymidine CH-TdR). need for the addition oPfraimmaircyrohseopmataolcyatcteisvhataivoen ssuyfsftiecmie.ntAmnetaaubtoolriacdiaocgtrivaipthyitcoteelcihmniinqautee the described by Williams (1977) was used to measure incorporation of *H-TdR into DNA. Because only a small percentageof the cells enter S-phase (replicative DNA synthesis) during the DexNpoAsudraempaegreiocda,utsheedibnycotrrpeoartamteinotnowfit>hHt-hTedtRestinatrotiDclNe.ATihseuesxeidstaesnacemaenadsudreegorfetehoefrDepNaAir of 01893 - Covamce207840447 `damage is inferred from an increase in nuclear grain counts in treated hepatocytes compared to nuclear grain counts from untreated hepatocytes. The typesofdetectable DNA damage are unspecified but must be recognizable by the cellular repair system and result in the incorporation ofnucleic acid bases including *H-TdR into the DNA. MATERIALS AND METHODS Test System "The indicator cells for this assay were hepatocytes obtained from a single adult male, Fischer 344 rat, weighing 214.0 grams. The animal was purchased from Charles River Laboratories, Raleigh, NC, and was housed in a shipping crate and received food supplied for shipping by the vendor and water ad libitum. The rat was used for the surgical procedure within 2 daysofreceipt, and iwnajsecatnieosntahnetdiezxedsabnegfuoirneastuerdgdeurryiwnigtthhaebporuotce6d0urmeg./kg sodium pentobarbital by intraperitoneal The Cell cCeollllsewcetrioenoabntdaiCnueldtubryepienrftuhseisoencotfitohneonlitvheer UinDsSituaswsiayt)h. a Mcoolnloalgaeynaesrecsuolltuutrieosn,weWreMEC (see established on plastic coverslips in culture dishes and were used the same day for initiationofthe UDS assay. Media and Cell Culture Conditions. Williams' Medium E supplemented with 2 mM cLu-lgtluurteammiendei,u1m0,0apngd/wmalsstmroedpitfoimeydcifnorsculafcahtes,peacnidfi1c5r0eqpugi/rmelmegnetn.taTmhiceihne(paWtoMcEyt)eswwaesrteheobbtaasiened `by perfusionoflivers in situ with HBSS/EGTA followed by WMEC:Hanks' balanced salts (CaTM-and Mg"*-free) containing 0.5 mM ethyleneglycol-bis(p-aminoethyl ether)-N, N-tetraacetic acid and 50 mM HEPES buffer at pH 7.2 (HBSS/EGTA), and WMEI containing 50-100 units/mlof collagenase and 50 mM HEPES buffer at pH 7.2 (WMEC). The cultures were established in WMEI supplemented with 10% fetal bovine serum (WME+). All cell cultures were maintained as monolayers in a humidified incubator at 35 to 37.5 C in an `atmosphere of4 10 6% CO in air. After the established period, the culture labeling was initiated using WMEI containing 10 pCi/ml *H-TdR at 40 to 60 Ci/mMole (WME-treat). Control Articles Vehicle Control Article. The vehicle control cultures received a concentrationofthe vehicle equivalent to the vehicle in the test article-treated cultures and were subjected to allofthe experimental manipulation that the test article-treated cultures received. Dimethylsulfoxide (DMSO; CAS 67-68-5, Lot #128H3460, Sigma Chemical Co.) ata concentration of 1% was used for all trials. Positive Control Article. The positive control compound is known to induce UDS in rat hepatocyte primary cell cultures. 2-Acetylaminofluorene (2-AAF, CAS# 53-96-3, Lot#57H0293, Sigma Chemical Co.) was dissolved in DMSO and diluted 1:100 into WME-treat foar final concentrationof0.10 pg/ml (4.48 x 107 M). 2 0189% Covance 20784-0-447 EXPERIMENTAL DESIGN Dosing Procedure A preliminary solubility test was performed with the test article, PFOS, in water, Williams' Media E and DMSO. The test article was insoluble in all vehicles but formed an acceptable suspension in DMSO at 401 mg/mL. DMSO was therefore chosen as the vehicle. `When dosed into media for mammalian cells, the test article formed a suspension/translucent liquid with very fine particlesofprecipitate at 4010 pg/mL. Based on this information, a concentrationof4000 g/m was selected as the top dose. A fresh preparationoftest article in the vehicle was used for the assay. A suspension/solution of test article in DMSO was serially diluted with DMSO to obtain the final desired concentrations oftest article. DMSO stocks were dosed 1:100 into media to createthe desired dosing. concentrations. `The sponsor is responsible for the determination and documentationofthe analytical purity and compositionofthe test article and the stability and strengthofthe dosing solutions. UDS Assay Dose Determination. A concurrent cytotoxicity assessment was used to select appropriate doses for the particular, fresh primary cultureof hepatocytes used in this study. A range of fificen concenirations from 0.25 pg/ml to 4000 pg/ml was applied to the cells. A viable cell count, based on trypan blue dye exclusion, was then obtained approximately 23 hours after initiation of the treatments. This information was used to choose six concentrations for analysisofnuclear labeling starting with the highest dose that resulted in a sufficient number of surviving cells with intact morphologies and proceeding to successively lower doses. Cell Collection and Culture. This assay was based on the procedures described by Williams (1980), and Butterworth et al. (1987). The hepatocytes were obtained by perfusionofliver in situ for four minutes with HBSS/EGTA then for 11 minutes with WMEC. The hepatocytes were obtained by mechanical dispersionofexcised liver tissue in a culture dish containing WMEC. The suspended tissue and cells were then allowed to settle to remove cell clumps and rdeebsruisspeprnidoerdtioncWollMecEti.on.AfTtheer ocbotlaleicntiendgcaelvliasbulsepecenlslicoonuwnta,sacesnertireisfoufge3d5anmdmthceulctelulrepedlilsehtes (at least 5 per dose level) were inoculated with approximately 0.5 x 10 viable cells in 3 ml of WME: per dish. Culture dishes used for the UDS analysis contained round plastic coverslips, `while dishes used to assess attachment efficiency and toxicity had no coverslips. Tino2ehstuambildiisfhitehdeactelmloscuplhteurreesc,ocnetlalisnwienrge4atlol6ow%edCaOn.atUtnaacthtmaecnhtepderceiloldsowef2rehotuhresn arte3m5ovteod37a.n5dC the cultures were refed with WMEL The UDS assay was initiated 1.4 hours later by replacing the media in the culture dishes with 2.5 ml WME-treat containing the test article at the desired 04395 3 Covance 20784-0-447 concentration. Each treatment, including the positive and vehicle controls, was performed on at least five cultures, twoof which were used for cytotoxicity measurements. Termination and Cytotoxicity Measurement. After an exposure periodof 19.6 to 20 hours, the UDS assay was terminatedby washing the cell monolayers twice with WMEL The cultures used for the cytotoxicity measurements were refed with WMEI and retumed to the incubator. At approximately 23 hours afte the initiationofthe treatments, cytotoxicity was assessed as cellular `morphology and cell survival relative to the vehicle control. Viable cell counts were obtained using trypan blue dye exclusion. `The triplicate labeled cell cultures for UDS analysis were refed with WMEI containing 1 mM thymidine and incubated for 30 minutes. The nuclei in the cells were swollen by addition of 1% sodium citrate to the coverslips (containing the cell monolayers) for 12 minutes, and then the cells were fixed with cold acetic acid:ethanol (1:3) and dried overnight. The coverslips were `mounted on glass slides, dipped in a solutionofKodak NTB2 emulsion and deionized water, and dried. The coated slides were stored for 6 days at 2 to 8C in light-tight boxes containing packets ofDrierite. The emulsions were then developed in Kodak D19, fixed, and stained using Williams' modified hematoxylin and eosin procedure. Slide Analysis. The cells were examined microscopically at approximately 1500X `magnification under oil immersion and the field was displayed on the video screenofan ``anuutmobmeartoifcgcroauintnesr.inUthDrSeewnaucslmeaera-ssuirzeeddcbyytocpoluanstmiincg naruecalseaardjgarcaeinnts taondeascuhbtnruacclteiunsg (tcheytaovpelraasgmeic count). This value is referred to as the net nuclear grain count. The coverslips were coded to prevent bias in grain counting. `The net nuclear grain count was determined for fifty randomly selected cells on cach coverslip. Only nuclei with normal morphologies were scored, and any occasional nuclei blackened by grains too numerous to count were excluded as cells in which replicative DNA synthesis (Sphase) occurred rather than repair synthesis. Normally, 150cellsper dose level were read (SO from eachofthe triplicate coverslips). The complete number of 150 cells per dose may not be available due to toxicity or qualityofpreparation. Grain count data obtained for a given treatment is acceptable as partofthe evaluationif obtained from at least two replicate cultures and at least 100 cells per dose. The mean net nuclear grain count is determined from the triplicate coverslips for each treatment condition. Assay Acceptance Criteria An assay normally is considered acceptable for evaluation of the test results only ifall of the csrtiutdeyridailriesctteodrbeexleorcwisaerse ssactiiesnftiiefdi.c jTuhdigsmleinsttinignmmaodyinfoytinegncthoempcraistseriaallotrecsonssitiudaetriionnsg,etvheunststhtehat might affect assay reliability and acceptance. Cell Culture Conditions. normally exceeds 70%. A The viabiolfitthye hepatocytes variety of factors can affect cell collected yield and from the viability, perfusion so values process below 01896 9 - Covance20784.0-447 70% are not uncommon nor necessarily detrimental. A lower limit for acceptability is set at 50% in order to avoid the possible use of a damaged, unrepresentative sampleofcells. `The viabilityofthe monolayer cell cultures used for the assay treatments must be 70% or greater. `Normally, the viabilityofattached cells is about 90%. `The numberof viable cells in the vehicle contro] cultures should remain reasonably stable over the experimental time period because rapidly declining (dying) cultures may not respond in a representative manner to the test article treatments. Therefore, the average number ofviable cells in the vehicle control cultures must not be less than 50%ofthe cell number at the beginning of the treatment period. Acceptable Controls. The average net nuclear labeling in the vehicle control cultures mustbe in the rangeof 5.00 to 1.00. In addition, no more than 10%of the cells should contain five or more net nuclear grains. Failure to meet eitherofthese criteria will invalidate the assay. `The positive control is used to demonstrate that the cell population employed was responsive and the methodology was adequate for the detectionofUDS. For test articles causing weak or no uDseSd taoctiinvdiitcy,atteheUDavSer(asgceerAessspaoynsEevatloutahteioponsCirtiitveericaonstercotliotnroefatmtheinstsrempourst)t.exFcoereda ebsottharctriictleeria clearly causing dose-related UDS activity, an assay is acceptable in the absence oaf positive. control lost for technical reasons. UAcDcSepatnaabllyseiHsibguth iDtoissen.ecIetsissanroyttonescheosswatrhyattoainnicnlcurdeeatsoexiinc cdoonsceesnt(rleastsiotnharnes9ul5t%s isnurevxicveasls)iivnethe toxicity as measured by either survivals below 50% or observationofpoor cellular morphology. `Hcioguhnltsyotroxpiocodrocseelsluclaanr mreosruplthoilnorgeyduacneddmiancyorlpeoardattoioanrotfif*aHct-s TdudrRinrgesgurlatiinnganianlydseicsr.eaTsheed hgirgahinest analyzed dose must approach excessive toxicity, or result in test article insolubility, or reach the highest applicable dose of mg/ml. Acceptable Number of Doses. A minimumofsix dose levelsmustbe analyzed for nuclear grain counts. Repeat trials need only augment the numberofanalyzed dose levels in the first trial to achieve a totalofsix different concentrations. Grain count data obtainedfor a given treatment is acceptable as partofthe evaluation if obtained from at least two replicate cultures and at least 100 cellsperdose. Assay Evaluation Criteria aSretviecrlealascraictteirivaehianvtehebeUeDnSesatsasbalyi.shTehdewhciricthe,riiaffmoert,apwoisliltpirvoevriedsepoanbsaesiasrefobraesveadluoantiaosntaotifastitceaslt analysisof Covance historical data and calculationofthe required minimum increase in nuclear Iabeling using an approach described by Casciano and Gaylor (1983). 01897 -10- -_-- CovanceQ0784047 The test article is considered active in the UDS assay at applied concentrations that cause: An increase in the mean net nuclear grain count to at least five grains per nucleus above the concurrent vehicle control value, and/or; Anincrease in the number of nuclei withfiveormore net grains such that the percentage of these nuclei in test cultures is 10% above the percentage observed in vehicle control cultures. Generally,ifthe first condition is satisfied, the second often will be met. However, satisfaction ofonly the second condition can also indicate UDS activity. When net nuclear labeling is e`lmeivnaitmeudma,btohveeotvheeravlelhpicaltetecroonftrtohlelarbeeslpionngsbeutisdcooenssindoetrreedaicnh tthhee esvtaaltiusattiicoanl.ly DsiigfnfiefriecnatntDNA- damaging agents can give a varietyof nuclear labeling patterns, and weak agents may strongly affect only a minority ofthe cells. Therefore, bothofthe above conditionsareconsidered in an evaluation. A dose-related increase in UDS for at least two consecutive applied concentrations is also desirable to evaluate a test article as active in this assay. In some cases, UDS can increase with dose and then decrease to near-zero with successively higher doses.Ifthis behavior is associated `with increased toxicity, the test article can be evaluated as active.Ifan isolated increase occurs for a treatment far removed from the toxic doses, the UDS is considered spurious. `The test article is considered negativeifnoneofthe above criteria are met. `When, inthe judgment ofthe study director, results are neither clearly positive nor clearly negative, the presenceof a dose response, the frequency distributionofcellular responses, and the reproducibility of data among slides is considered. `oTrhceaprocsiintoigveenciocntrirsokl ansuscolceiaarteldabweiltihngthise nUoDt Stoabcetiuvsietydofasthaeretfeestreanrctieclpeo.inUt DtoSeseltiicmiatteed mbuytategsetnic. agents in this assay is probably more dependent on the type of DNA damage inflicted and the. available repair mechanisms than on the potencyofthe test agent as a mutagen or carcinogen. Some forms of DNA damage are repaired without the incorporation ofnew nucleic acids. Thus, the positive controls are used to demonstrate that the cell population employed was responsive: and the methodology was adequate for the detectionofUDS. Em 01398 Covance 20784-0-447 RESULTS A preliminary solubility test was performed and the test article, PFOS, was insoluble in water and Williams' Media E. In DMSO, the test article formed a translucent light-yellow suspension `with a few undissolved piecesoftest article on the bottomofthe vial at a concentration of 501 mg/mL. At 401 mg/mL, a translucent light-yellow suspension with a small amountof foam on top was observed. When dosed into media for mammalian cells, the test article formed a suspension/translucent liquid with very fine particlesofprecipitate at 4010 pg/mL. At concentrations from 253 mg/mL to 15.6 mg/mL in DMSO, PFOS formed a translucent off-white to white homogenous suspension with a small amount of foam on top. When dosed into media, 312 pg/mL formed a transparent media-colored solution. Based on this information, a concentrationof4000 pg/mL was selected as the top dose. In the UDS assay, the test article formed a translucent light-yellow homogeneous suspension with a small amount of foam on top in DMSO at 400 mg/mL. The stock became atransparent colorless solution at 50.0 mg/mL. Tn media prior to treatment, a precipitate was present from 4000pg/mLto 1000 g/mL. Fifteen concentrationsofPFOSwereapplied, ranging from 4000 pg/mL to 0.250 pg/mL. The test article was excessively cytotoxic at and above 50.0 cpegl/lmulLa,r mweoarkplhyolcoytgoytwoxaisc sauti2t5ab.l0epfgo/rmaLn,alaysnidsnaotnacnydtobteolxiocw a2t5a.n0dpgb/emlLo.w 10.0 pg/mL. The `The hepatocytes were collected at a calculated viability of 92.3% based on trypan blue exclusion. `The treatments were initiated within 3 hours with cell monolayers that were 96.7% viable, with an attachment efficiencyof94.5%. After an additional 22.7 hours in culture (which encompassed the 19.6-hour treatment period), the average viable cell count in the control cultures was 86.0%ofthe viable count at the beginningof the treatments. This stability in cell number and the normal morphological appearanceofthe cells indicated that the hepatocyte cultures were in good metabolic condition for the UDS assay. `The vehicle control values were a mean net nuclear grain countof-4.74 and a mean percent nuclei containing five or more net grainsof2.67%. These values were usedto determine the `minimum criteria for a positive UDS response. Foar treatment to be considered positive, there: `must be an increase in the mean net nuclear grain count to at least five grains per nucleus above the concurrent vehicle control value, and/or an increase in the numberof nuclei with five or more net grains such that the percentageofthese nuclei in test cultures is 10% above the percentage observed in vehicle control cultures. The criteria for a positive response in this assay were a `mean net nuclear grain count exceeding 0.26 and /or at least 12.67%ofthe nuclei containing five or more net grains. Noneofthe treatments with the test article samples caused a positive response in either the mean nuclear grain count or in the nuclei containing five or more net grains. 01899 1. Covance 20784-0-447 `The 2-AAF treatment induced large increases in nuclear labeling that exceeded both criteria used to indicate UDS. This positive response confirmed that the hepatocyte cultures were. metabolically active during the UDS assay. Heavily-labeled nuclei that were blackened with numerous grains represent cells undergoing DNA replication (S-phase) as opposed to DNA repair. The number present in this study was low and did not interfere with the assay. CONCLUSION The test article did not induce unscheduled DNA synthesis, as measured by mean net nuclear grain counts or the percentage ofnuclei containing five or more net grains. Doses were chosen for each assay based on cell counts determined at 20-24 hours after dosing and the analyzed treatments covered 2 good rangeofcytotoxicity for evaluation ofUDS. The test article, PFOS, `was therefore evaluated as negative in the in vitro assay for unscheduled DNA synthesiins rat liver primary cell cultures. 013800 a1. Covance 20784-0447 RECORDS TO BE MAINTAINED Al raw data, documentation, records, protocol and the final report generated as a resultofthis study will be archived in the storage facilities of Covance-Vienna for at least one year following submissionofthe final report to the Sponsor. After the 1-year period, the Sponsor may elect to have the aforementioned materials retained in the storage facilities of Covance-Vienna for an additional period of time, or sent to a storage facility designated by the Sponsor. REFERENCES Butterworth, B.E., J. Asby, E. Bermudez, D. Casciano, J. Marsalis, G. Probst and G. Williams (1987) Aprotocol and guide for te in vitro rat hepatocyte DNA-repair assay, MutationResearch, 189, 113-121. Casciano, D.A. and D.W. Gaylor (1983) Statistical criteria for evalutating chemicals as positive or negative in the hepatocytes DNA repair assay, Mutation Research, 122:81-86. Williams, GM. (1977) Detectionofchemical carcinogens by unscheduled DNA synthesis in rat iver primary cell culture, Cancer Research, 37:1845-1851. Williams, GM. (1980) The detectionofchemical mutagens-carcinogens by DNA repair and `mutagenesis in liver cultures, in: F. de Serres and A. Holaender (Eds.), Chemical Mutagens, Plenum Press, N.Y, Vol. 6, pp. 61-79. 01901 "14 Covance 20784-0-447 DATA TABLES 01302 -15- Covance 20784-0447 TABLE 1. SUMMARY OF DATA FROM THE RAT HEPATOCYTE UDS ASSAY TEST ARTICLE: PFOS COVANCE STUDY NO.: 20784-0-447 TRIAL INITIATION DATE: 1-September-99 Test Condition Concentration MeanNet ~~ %Cells ~~ Mean Nuclear wiz5 Cyto. Grains Mean Grains MNG) (1) NG) (3) % Survival A233 hours @ Vehicle Control - DMSO 1.00 % 474 267 1903 1000 Positive Control 2-AAF 0.100 pg/mL 1935 96.00 1617 885 `Test Material 250 pgml 281 100 pgmL 37 500 pg/ml 451 250 pgml 317 100 pgmL 4.04 0500 pg/mL 487 667 2081 65.2 6.00 261 1033 333 1877 96.8 533 158 1093 067 17.39 96.3 133 1929 1022 (1) Averageof net nuclear grain counts on triplicate coverslips (150 total cells). Net `muclear grains = Nuclear grain count - Average cytoplasmic grain count. (2) Average percentageofcells withgreaterthan or equal to $ net nuclear grains on triplicate coverslips (150 total cells). (3) Averageof cytoplasmic grain counts on triplicate coverslips (150 total cells). (4) Survival = Numberofviable cells per unit area relative to the solvent control. 2-AAF = 2-acetylaminofluorene DMSO = Dimethyl sulfoxide 01903 16. Covance 20784-0-447 TABLE 2. INDIVIDUAL SLIDE DATA ASSAY NO. 20784-0-447 TRIAL INITIATION DATE: 09/01/99 Slide Code Dose | MeanNet | Standard | Mean | %Cellsw/=5 (ug/ml) | Nuclear | Deviation | Cytoplasmic| Net Nuclear Grains Grains Grains 8 [ve a3 | 1 ies6 | 2.00 6 [ ve [ aie [1 174 | 4.00 4 | ve [| se [1 2280 | 2.00 DoseAverage | | 474 | 082 | 1903 | 2.67 Cells in S-Phase 0/1500 screened for S-Phase = 0.00% 9 [ pc [ 2360 [ -- [ 1806 [ 9800 7 [| pc | 2066| -- [ 1458 [10000 2 [ pc [ B78 |1158| 9000 DoscAverage | | 1935 | 504 | 1617 | 9600 Cells in S-Phase 7/1500 screened for S-Phase =0.47% 5 [250 T 4[T1588 [800 3 [250 | 33 [1 2164 | 800 1 [250 | 36 [1 2400 | 400 DoscAverage | | 281 | 121 | 2081 | 667 Cell in S-Phase 4/1500 screened for S-Phase = 0.27% 18 [too [ 330 [TT 213 4.00 17 [100 [27[7T 2m | 6.00 16 [100 1sp [1"237 | 8.00 DoscAvenge | | 371 | 125 | 2261| 6.00 Cells in S-Phase 5/1500 screened for S-Phase = 0.33% 15 [ 500 [ soaTT 2006 ] 4.00 14 [ 500 [ 40a| 1 204| 400 13 [ 500 | 26 [1 13% | 2.00 DoseAverage | | 451 | 168 | 1877 | 3.33 Cells in S-Phase 2/1500 screened for S-Phase = 0.13% 01994 -17- Covance 20784-0-447 TABLE 2. INDIVIDUAL SLIDE DATA (Continued) ASSAY NO. 20784-0447 TRIAL INITIATION DATE: 09/01/99 Slide Code | Dose | MeanNet| Standard Mean % Cells w/= 5 (ng/mL) | NGurcalienasr | Deviation | CyGtroapilnass.mic NetGrNauicnlsear 21 [25[366 [1 1392 | 4.00 20 [25[30 [T1748| 2.00 DoscAv1erage 1|7250 | asa| [ie 317 | 144 | 1584 | [1000 533 Cells in -Phase 4/1500 screened for S-Phase = 0.27% 24 Tro [a3 TT 1608 J 0.00 25 2 ||w10o0 [ [ 39 | a [116 w 5 56|| 20..0000 DoscAverage | | 404 | 031| 17.39 | 067 Cells in S-Phase 0/1500 screened for S-Phase = 0.00% 12 Toso[41a TT 1908 | 2.00 110 [[oosso0| [ 346 | 702 | -- | ise 1 228 | | 2.00 0.00 DoscAverage | | 487 | 189 | 1929 | 133 Cell in -Phase 1/1500 screened for S-Phase = 0.07% Total Cells in S-Phase 23/12000 screened for S-Phase = 0.19% VC = Vehicle Control PC = Positive Control 01995 -18- HISTORICAL CONTROL DATA HISTORICAL VEHICLE CONTROLS IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY Covance 20784-0-447 Study Number 1 2 3 4 5 6 7 8 9 10 11 2 13 14 15 16 17 18 19 20 Average Range: Low High UDS Grains/ ~~% ofNuclei with>5 Nucleus + SD* Net Nuclear Grains * 353 268 037 074 074 124 "141 036 0.47 087 099 0.16 "145 080 073 -136 278 "1.83 267 463 143 = 128 [ 27 61 80 60 73 13 80 53 53 93 93 73 61 60 33 07 07 40 33 5.06 + 291 353 00 074 93 Average Cyto Grains 15.44 1617 878 1059 1328 1747 1687 14.54 1030 2157 14.69 1073 14.62 12.95 1863 11.29 11.50 1049 1649 20.05 1432 3.54 829 21.57 Numberofdata points is 20. Data included from 5/90 to 1/99 *Averageofnet nuclear grain counts triplicate or duplicate coverslips (150 cells analyzed) Average values for triplicate or duplicate coverslips (150 cells) analyzed SD = Standard deviation . "10 01906 Covance20784-0-447 HISTORICAL CONTROL DATA (continued) "HISTORICAL POSITIVE CONTROLS IN VITRO UNSCHEDULED DNA SYNTHESIS ASSAY Study Number 1 2 3 4 5 6 7 8 9 10 1 12 3 14 15 16 17 18 19 20 Average Range: Low High UDSGrains/ ~~ %of Nuclei with = 5 Nucleus +SD* Net Nuclear Grains 29.45 100.0 15.53 920 735 633 1235 887 1418 973 14.45 927 16.09 100.0 9.51 787 13.17 90.7 16.92 973 17.38 867 4.46 46.0 15.59 993 1069 8438 13.79 927 14.66 893 1433 913 1463 96.7 1681 98.7 17.51 913 1445 + 489 89.19 = 1336 Average Cyto Grains 25.71 11.52 12.53 1131 1625 16.05 15.73 15.98 1427 1051 15.68 10.89 10.73 11.68 14.09 17.85 12.2 11.50 1049 1649 1407 & 365 446 46.00 1049 2945 100.00 2571 Numberofdata points is 20. Data included from 5/90 to 1/99 `The positive control used is 10 pg/ml 2-AAF *Averageofnetnuclear grain counts triplicate or duplicate coverslips (150 cells) analyzed Average values for triploriducplaictatee coverslips (150 cells) analyzed SD = Standard deviation 01907 20 COVANCE. STUDY TITLE: AMENDMENT TO THE STUDY PROTOCOL Page of1 Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures PROTOCOL NO.: 447 Edition 17 COVANCE STUDY NO: 20784-0-447 `Amendment #1. 1 Section 4.2.3 Termination and Cytotoxicity Measurement `The third sentence in paragraph 3 is changed from "The cells will be fixed with cold acetic acid:ethanol (1:3) and dried for at least 24 hours." to "The cells will be fixed with cold acetic acid:ethanol (1:3) and dried at least overnight." Reason: Overnight allows for sufficient drying. 2 Section 4.2.3 Termination and Cytotoxicity Measurement `The third sentence in paragraph 4 is changed from "The emulsion-coated slides will be stored for 7 to 10 days at 2 to 8C in light-tight boxes containing a desiccant." to "The emulsion-coated slides will be stored for 6 10 9 days at 2 to 8C in light-tight boxes. containing a desiccant." Reason: Previous studies indicated that a shorter exposure period was needed. 3. t`Thheeprreoftoecroeln:ce to Mirsalis, Tyson and Butterworth, 1982 is deleted from Section 4.2.2 of Reason: The reference refers to procedures for an in vivo/in vitro UDS assay. Study Director: Mipre (1 C "GMeanpetaicA.anCidfCoenlleu,laPy K].Toxicology g.i-9 Date 01903 COVANCE.> ee AMENDMENT TO THE STUDY PROTOCOL Page 1 of1 STUDY TITLE: `Unscheduled DNA Synthesis in Rat Liver Primary Cell Cultures PROTOCOL NO.: 447 Edition 17 CO AEST Ne `COVANCE STUDY NO.: Amendment #2. 20784-0-447 1. Section 1.0 Sponsor Identification The nameofthe sponsor is changed from 3M Corporation to 3M Corporate Toxicology. Resson: Sponsor request. - Study Director: a a ; /, aA. Cifone, PhJ). GGeernleettic aannddCCeellllualrarT'oTxoixiccoolloogyyy -9-99 Date 013909 ' ANCE SovANCE COVANCESTUDY No. JC 7:54 ={ ~ #4 F PROTOCOL NO. 447 EDITION 17 "UNSCHEDULED DNA SYNTHESIS IN RAT LIVER PRIMARY CELL CULTURES Covance Laboratories Inc. (Covance) will conduct this study in compliance with Good Laboratory Practice (GLP) regulations and standards. This protocol, at least one critical phase of the work in progress, and the final report will be subject to audit by Quality Assurance in accordance with Standard Operating Procedures maintained at Covance. The study will be conducted by Covance at 9200 Leesburg Pike, Vienna, Virginia 22182 (Covance-Vienna). PART 1. SPONSOR INFORMATION AND APPROVALS 10 SPONSOR IDENTIFICATION Company Name: Address: 3M Corporation 3M Center `Building 220-2E-02 St. Paul, MN 55144-1000 20 TEST ARTICLE IDENTIFICATION - 00f0c 0000000000 2m Hof Ah Tb29c Ig 30 TEST ARTICLE ANALYSIS "The Sponsor will be responsible for the determination and documentation of the analytical purity and composition of the test article and the stability and strength of the dosing solutions. 40 NOTIFICATION OF REGULATORY SUBMISSION In order to comply with GLP regulations and standards, consulting laboratories must be notified if all or part of a study is intended for regulatory submission. Covance maintains a master schedule of studies which fall under regulatory review. Please indicate which agency, if any, might receive the resultsofthis study: DO Undetermined ~~ OFDA ~~ [MEPA-TSCA ~~ DVEPAFIFRA DO MAFF O MOHW DO oEcD O None 0 Other. -- 1198 Tof13 013910 COVANCETM PROTOCOL NO. 447 EDITION 17 50 STUDY DATES Proposed Experimental Start Date: pliy es(999 Proposed Experimental Termination Date:_Oitotes1599 60 APPROVAL OF STUDY PROTOCOL Study Director: L : 5 Marif A. Cifone, Ph.D. Date: 7) 20/99 Testing Facility Management: Bi C.- fof Brian C. Myhr, Ph.D. Associate Director Sponsor's Authorized Representative: Paes Teor Marvin T. Case, D.V.M., Ph.D. Date: __B]as)g4 Due: Lh Baga Jes 198 . 20f13 91311 COVANCETM PROTOCOL NO. 447 EDITION 17 PART 2. STUDY PROTOCOL UNSCHEDULED DNA SYNTHESIS IN RAT LIVER PRIMARY CELL CULTURES 10 OBJECTIVE `The objective of this in vitro assay i to evaluate the ability oaf test article,or a `metabolite, to induce DNA damage in rat primary hepatocytes by measuring unscheduled DNA synthesis (UDS). 20 TESTSYSTEM RATIONALE `This assay aims 10 establish whether the test articleor its metabolites can interact with rat liver primary cell (hepatocyte) cultures to induce DNA damage followed by DNA repair measured as UDS. Hepatocytes will be isolated from liverand exposed in vitro to the test article and tritiated thymidine CH-TdR). Primary hepatocytes have sufficient metabolic activity to eliminate the need for the addition ofamicrosomal activation system. An autoradiographic technique described by Williams (1977) is used to measure: incorporation of *H-TdR into DNA. Because only asmall percentage of the cells will enter S-phase (replicative DNA synthesis) during the exposure period, the incorporation of *H-TAR into DNA can be used as a measure of the repair of DNA damage caused by treatment with the test article. The existence and degree of DNA damage will be inferred from an increase in nuclear grain counts in treated hepatocytes compared to nuclear grain `counts from untreated hepatocytes. The types of detectable DNA damage are unspecified but must be recognizable by the cellular repair system and result in the incorporation of nucleic acid bases including *H-TdR into the DNA. 30 MATERIALS 31 Test System `The indicator cells for this assay will be hepatocytes obtained from a single adult, `male, Fischer 344 rat (weighing 1500 300 g), which will be purchased from the Charles River Breeding Laboratories, Incorporated or another qualified dealer. Animals will be received by animal care personnel, examined, and housed according to applicable Covance SOPs. Food and water will be supplied ad libitum. The animals will be used for the surgical procedure within two days of receipt. Animals will be anesthetizedprior to surgery to obtain the hepatocytes (sodium pentobarbital at about 60mg/kg by intraperitoneal injection) and exsan`guinated during the procedure. 1198 3013 01912 COVAANNCEC.E PROTOCOL NO. 447 EDITION 17 3.2 33 1/98 `The cells will be obtained by perfusion of the liver in situ with a collagenase solution, WMEC, (see Section 4.2, UDS ASSAY). Monolayer cultures will be established on plastic coverslips in culture dishes and used the same day for initiation of the UDS assay. 3.1.1 Media and Cell Culture Conditions Williams' Medium E supplemented with 2 mM L-glutamine, 100 g/ml streptomycin sulfate, and 150 pg/ml gentamicin (WMEI) is the base culture medium, and is modified for each specific requirement. The `hepatocytes will be obtained by perfusion of livers in situ with HBSS/EGTA followed by WMEC: Hanks' balanced salts (Ca++and Meg-++-free) containing 0.5 mM ethyleneglycol-bis(B-aminoethyl ether)-N, N-tetra-acetic acid and 50 mM HEPES buffer at pH 7.2 (HBSS/EGTA), and WMEI containing 50 to 100 units/ml of collagenase (WMEC). The cultures are established in WMEI supplemented with 10% fetal bovine serum (WME+). All cell cultures will be maintained as monolayers ina humidified incubator at 35 to 37.5C in an atmosphereof 4 to 6% CO, in air. After the establishment period the culture labeling is initiated using. 'WMEI containing 10 pCi/ml *H-TdR at 40 to 60 Ci/mMole (WME-treat). `When water is the vehicle of choice, the concentration of *H-TdR will be adjusted so that the final concentrationof label in the medium (aftear 1:10 dilutionofthe water stocks) will be about 10 pCi/ml. Test Article Solid or liquid test articles are suitable for this assay. The test article is identified in Part 1 of this protocol. Storage conditions will be as specifiedbythe Sponsor. Control Articles 33.1 Vehicle Control Article The vehicle control article will be the vehicle selectedforthe test article. `The vehicle may be specified by the Sponsorr selected by the process described in Section 4.1, Test Article Handling. The vehicle willbe used in the vehicle control cultures at a concentration equivalent to the vehicle in the test article-treated cultures. The vehicle control cultures will be handled identically to the test article treated cultures. An untreated (negative) control, consisting of assay procedures performed on cells `exposed to WME-treat only, will be included when acetone is used as the vehicle. 4of 13 01313 COVANCETM PROTOCOL NO. 447 EDITION 17 332 Positive Control Article The positive control article, 2-acetylaminofluorene (2-AAF; CAS # 53-963), is known to induce UDS in rat hepatocyte primary cell cultures. The 2-AAF will be dissolved in DMSO and diluted 1:100 into WME-treat foar final concentration of 0.10 ug/ml (4.48 x 10M). 40 EXPERIMENTAL DESIGN 41 Test Article Handling If solubility information is not provided by the Sponsor, a preliminary solubility test will be carried out with serum-free culture medium or sterile deionized water, dimethyl sulfoxide (DMSO; CAS #67-68-5), ethanol (CAS #64-17-5), or acetone b(eCAdiSss#o6l7v-e6d4-a1t)t.hIefhtihgehesvtehdiecslieroefdcchooniccenetirsactuilotnurien mWMeEd-iturme,atth.e tSetsotcakrtsioclleutwiiolnls in waterwill be diluted 1:10, in DMSO or ethanol will be diluted 1:100, and in acetone will be diluted 1:200 in WME-treat. The vehicle selected will be the one which gives the best solubility and dispersion characteristics after dilution in culture medium. In some cases, test articles are apparently insoluble in vehicles thatare compatible with tissue culture. In such cases, the vehicle chosen and the highest dose tested will be that in whichan evenly dispersed suspension can be prepared. 42 UDS Assay 42.1 Dose Determination and Culture Conditions The maximum dose will be determined on acase by case basis taking into `account both solubility and any relevant cytotoxicity information available: on the test article. The highest dose tested will be 5 mg/ml, unless higher doses are specified by the Sponsor. Relatively insoluble test articles will be tested up to approximately 2 times the solubility limit,if insoluble article does not interfere with the enumeration of nuclear grains. `The top dose tested for liquid test articles will be 5 mg/ml, which may be determined by weight or calculated from the specific gravity. Solubility testing will be the same as described above. 1/98 . Sof13 01914 ANCE> COVANCE. PROTOCOL NO. 447 EDITION 17 1f marked pH changes are noticed during the solubility test, neutralization, with HCI or NaOH, will be performed to maintain a normal culture pH range (6.8 to 7.4) only after consultation with the Sponsor. A concurrent cytotoxicity assessment is an integral part of the UDS assay used to select appropriate doses for a particular, fresh primary culture of hepatocytes. A range of 15 concentrations will usually be applied to the cells. Fewer concentrations may be initiated when toxicity information is available. A viable cell count (trypan blue dye exclusion) will then be obtained 20 to 24 hours after initiation of the treatments. At least six concentrations willbechosen for analysis of nuclear labeling, starting with the highest dose that results ina sufficient number of surviving cells with intact morphologies and proceeding to successively lower doses. Repeat trials may contain as few as 5 dose levels at the discretion of thestudy director. The test article solution will be prepared immediately before use. `The maximum final concentrations of vehicle in WME-treat will beupto 10% water, 1% DMSO or ethanol,or 0.5% acetone. 4.22 Cell Collection and Culture `This assay is based on the procedures described by Williams, (1980), Mirsalis, Tysonand Butterworth, (1982) and Butterworth er al, (1987). `The hepatocytes will be obtainedbyperfusion of livers in sifu with HBSS/EGTAfor abou4t minutes followed by WMEC for about 11 minutes. Depending on the condition of the liver, this timemaybe altered by +2 minutes. The hepatocytes will be obtained by mechanical dispersionofexcised liver tissue in a culture dish containing WMEC. The suspended tissue and cells will then be allowed to settle to remove cell clumps and debris prior to collection. The collected cell suspension will be centrifuged and the cell pellet resuspended in WME+. After obtaining a viable cell count, aseriesof culture dishes (at least 5perdose level) will `be inoculated with approximately 0.5 x 10 viable cells in 3 ml of WME+. Culture dishes that will be used for UDS analysis will contain round plastic coverslips while dishes used to assess attachment efficiency and toxicity will have no coverslips. An attachment period of 1.5 to 2 hours at 35 to 37.5C in a humidified atmosphere containing 4 to 6% CO, will be usedtoestablish the cell cultures. Unattached cells will then be removed and the cultures will be. refed with WMEL 1/98 60f13 01915 COVANCETM PROTOCOL NO. 447 EDITION 17 `iTnhteheUDcuSltuarsesadyiswhiells bweitihni2t.i5atmeld wWiMtEh-intrtehraete choonutrasinbiyngretphleactiesntgatrhteicmleedatia tpheerfdoesrimreeddwciotnhceanttlreaatsitonf.iveEcauclhtucroens.troTlhraened otefstthaersteicrleeptlriceaattemecnutltwuirlelsbweill be used for UDS analysis and two replicates will be used for the cytotoxicity measurements. Dishes will be labeled per Covance SOP. 4.2.3 Termination and Cytotoxicity Measurement After an exposure period of 18 to 20 hours, the treatments and labeling cwuillltubreesteursmeidnfaotredtbheycwytaosthoixnicgittyhemceealslumroenmoelnatyserwsilwibteh rmeefdeiduwmi.thTWheME and retuned to the incubator. AL20 to 24 hours after the initiation of the treatments, cytotoxicity will be. acosnstersosle.d aVsiacbelleluclealrlmcoorupnhtsolwoilglybaendobcteallinseudrvuisvianlgrterlyaptiavnebtloutehdeyev.ehicle exclusion. `The triplicate labeled cell cultures for the UDS assay will be refed with 'WMEI containing 1mMthymidine. The nuclei will be swollen by addition of 1% sodium citrate to the coverslips (containing the cell monolayers) for 8 to 12 minutes. The cells will be fixed with cold acetic acid:ethanol (1:3) and dried for at least 24 hours. The coverslips will be mounted on glass slides. `The cell survival and morphology obtained from the cytotoxicity cultures `will be use to chose doses levels for autoradiography. Slides will be dipped in an emulsion of Kodak NTB2 and water, and dried. The emulsion-coated slides will be stored for 7 to 10 days at 2 to 8C in lighttight boxes containing a desiccant. The emulsions will be developed with Kodak D19 developer and Kodak Rapid Fixer, then stained with a modified hematoxylin and eosin procedure. 424 Slide Analysis `The cells willbeexamined microscopically at approximately 1500x `magnification under oil immersion and the field displayed on the video screenofan automatic counter. UDS will be measured by counting `nuclear grains and subtracting the average number of grains in three 198 Tof 13 01916 COVANCETM PROTOCOL NO. 447 EDITION 17 nuclear-sized cytoplasmic areas adjacent to each nucleus (cytoplasmic count). This value is referred to as the net nuclear grain count. The coverslips willbe coded to prevent bias in grain counting. `The net nuclear selectedcells on grain each count will coverslip. bOenrloyutniuncelleyi dweittehrnmoirnmeadlfomror5p0horlaongdioemsly will be scored, and any occasional nuclei blackened by grains too numerous to count will be excluded as cells in which replicative DNA sdyonstehelseivselowciclulrrbeedrreaatdhe(r50thfarnormepeaaicrhsoyfntthheesitsr.iplNiocratmealcolvye,rs1l5i0psc)e.llTshpeer `complete number of 150 cells per dose may not be available due to toxicity or quality of preparation. Grain count data obtained for a given treatment is acceptable as part of the evaluationif obtained from at least two replicate cultures and at least 100cellsper dose. The mean net nuclear gwreaaitnmceonutnctownidliltiboen.deOtcecramsiinoendalflryo,matchoevterrisplliipcamteaycovbeerrselciposunftoerdeaatcha later dateorby a different technician. Since a different cell population will cgaelnceurlaaltliyobnoefsctohreedm,etahnefaovrertahegetrciopluinctatfeortr5e0atcmeelnlts.will be used in the 5.0 DATA 51 Data Presentation "vTehheicflienaclornetproor,t pwoisllitiinvcelcuodnetrtohle,faonldloewaicnhg ainnafloyrzmeadtitorneaitnmtenatb:ular form for the The mean net nuclear grain count for triplicate cultures with standard deviation (usually a total of 150 cells). The mean percentageofcellshaving five or more net nuclear grains for triplicate cultures. The mean cytoplasmic grain count for triplicate cultures (usually three areas per cell in a total of 150 cells). + T`phereumnietaanresaurivnivtahle cfourltduurpelircelaatteivceulttourtehse, vwehhiicclheicsotnhteronluvmableure oxf1v0i0ab%l.e cells 1/98 * 8of 13 01917 COVANCETM PROTOCOL NO. 447 EDITION 17 52 198 Information regarding test article solubility in the culture medium, viability of the hepatocyte cultures, and the percent of cells in DNA replicative synthesis (Sphase) will also be provided. Assay Acceptance Criteria, `An assay will be considered acceptable for evaluation of the test resultsonly ifall of the criteria listed below are satisfied. This listing may not encompass all test situations, thus the study director exercises scientific judgment in modifying the criteria or considering events that might affect assay reliability and acceptance. 52.1 Cell Culture Conditions `The viability of the hepatocytes collected from the perfusion process normallyexceeds 70%. A variety of factors can affect cell yield and viability, so values below 70% are not uncommon nor necessarily detrimental. A lower limit for acceptability is set at 50% in order to avoid. the possible useof a damaged, unrepresentative sample of cells. `The viabilityofthe monolayer cell cultures used for the assay treatments `mustbe 70% or greater. Normally, the viabiliotfy attached cells is about 90%. The number of viable cells in the vehicle control cultures should remain reasonably stable over the experimental time period because rapidly declining (dying) cultures may not respond in a representative manner to the test article treatments. Therefore, the average number of viable cells in the vehicle control cultures must not be less than 50%ofthe cell number at the beginning of the treatment period. 522 Acceptable Controls `The average net nuclear labeling in the vehicle control cultures must be in the rangeof-5.00 to 1.00. In addition, no more than 10% of the cells should contain five or more net nuclear grains. Failure to meetcitherof these criteria will invalidate the assay. The positive control is used to demonstrate that the cell population employed was responsive and the methodology was adequate for the detectionofUDS. For test articles causing weakor no UDS activity, the 90f13 . 01313 COVANCETM PROTOCOL NO. 447 EDITION 17 carvietrearigaeurseesdpotnosiend1i0ctahtee UpoDsiSti(vseeceoSnetcrotliotnre5a.3t)m.enFtosrmausttestexarcteiecldebcolteharly caabusseinncgeoafdoaspeo-srietliavteedcoUntDrSolalcotsitviftoyr, taenchansiscaaylwrielalsobnesa.cceptable in the 52.3 Acceptable High Dose UtiDsSnoatnanleycsiesssbaurtyittoisinncelcuedsesatroyxitcodsohsoews (tlheastsatnhainnc9r5ea%sesuinrvciovnacle)nitnratthieon orebssuelrtvsaitnioenxocfespsoiovertcoexlilcuiltayr masormpehaosluorgeyd.bHyiegihtlheyrtsouxrivcivdaolssesbeclaonwre5s0ul%t ionr reduced incorporationof *H-TdR resulting in decreased grain counts or T`phoeorhcieglhleusltaramnaolrypzheodldoogsyeamnudsmtaaypplreoaadcthoeaxrctiefsascitvsedutorxiincgitgyr,aoirn arensaullytsiisn. test article insolubility,orreach the highest applicable dose of5mg/ml. 524 Acceptable Numberof Doses A minimumofsix dose levels will be analyzed for nuclear grain counts. Repeat trials need only augment the number of analyzed dose levels in the first trial to achievae total of six different concentrations. Grain count data obtained foar given treatment is acceptable as part of the evaluation if obtained from at least two replicate cultures and at least 100 cells per dose. 53 ASSAY EVALUATION CRITERIA Several criteria have been established which, if met, will provide a basis for evaluation of atest article as active in the UDS assay. The criteria foar positive response are based on iansctraetaissteicianl nauncalleyasirslaobfeClionvgaunscienghiasntoaripcparlodaactha daensdcrciablecdulbaytiCoansocfitahneoraenqduiGraeydlmoirn(i19m8u3m). `The test article will be considered active in the UDS assay at applied concentrations that cause: An increase in the mean net nuclear grain count to atleast five grains per nucleus above the concurrent vehicle control value, andlor; 19% - 100f13 01319 COVANCETM PROTOCOL NO. 447 EDITION 17 + Anincrease in the number of nuclei with five or more net grains such that the percentage of these nuclei in test cultures is 10% above the percentage observed in vehicle control cultures. Generally,if the first condition is satisfied, the second often will be met. However, satisfactionofonly the second condition can also indicate UDS activity. When net nuclear labeling is elevated above the vehicle control labeling but does not reach the statistically significant minimum, the overall patternofthe response is considered in the evaluation. Different DNA-damaging agents can give a varoi fne uct leayrlabeling pattems, and weak agents may strongly affect onlya minority of the cells. Therefore, bothofthe above conditions will be considered in an evaluation. A dose-related increase in UDS for at least two consecutive applied concentrations is also desirable to evaluate a test article as active in this assay. In some cases, UDS can increase with dose and then decrease to near-zero with successively higher doses. Ifthis behavior is associated with increased toxicity, the test article can be evaluated as active. If an isolated increase occursfor atreatment far removed from the toxic doses, the UDS will be considered spurious. "The test articleisconsidered negative if none of the above criteria are met. `When, inthe judgement of the study director, results are neither clearly positive nor clearly negative, the presence of a dose response, the frequency distributionof cellular responses, and the reproducibility of data among slides is considered. "The positive control nuclear labeling will not be used as a reference point to estimate mutagenicor carcinogenic risk associated with the UDSactivityof the test article. UDS elicited by test agents in this assay is probably more dependent on the typeof DNA damage inflicted and the available repair mechanisms than on the potency ofthe test `agent as a mutagenor carcinogen. Some forms of DNA damage are repaired without the incorporation of new nucleic acids. Thus, the positive controls will be used to demonstrate that the cell population employed was responsive and the methodology was adequate for the detectionofUDS. 1/98 1lof13 01920 COVANCETM PROTOCOL NG. 447 EDITION 17 6.0 REFERENCES Butterworth, B.E., J. Ashby, E. Bermudez, D. Casciano, J. Mirsalis, G. Probst and G. Williams (1987) A protocol and guide for the in vitro rat hepatocyte DNA-repair assay, Mutation Research, 189, 113-121. (Casciano, D.A. and D.W. Gaylor (1983) Statistical criteria for. `evaluating chemicals as positive or negative in the hepatocytes DNA repair assay, Mutation Research, 122:81-86. Williams, G.M. (1977) Detectionof chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell culture, Cancer Research, 37:1845-1851. `Williams, G.M. (1980) The detection of chemical mutagens-carcinogensby DNA repair and mutagenesis in liver cultures, in: F. de Serres and A. Hollaender (Eds.), Chemical Mutagens, Plenum Press, NY, Vol. 6, pp. 61-79. 7.0 REPORT FORMAT Covance employs a standard report format for each assay design. The final report will provide the following information. * Sponsor identification. * Quality Assurance statement. + Statement of GLP compliance. Signatureofstudy director. * Test article identification and Covance Study Number. A physical descriptionof the test article and date of receipt willbe included in this section. * Typeofassay and protocol number. + Dates of study initiation and completion. + Identification of study director and senior technician. * Methods. + Evaluation criteria. * Interpretation of results. + Conclusions. * References. Test results presented in tabular form. * Historical control data. 1/98 : 120f 13 61921 COVANCETM PROTOCOL NO. 447 EDITION 17 30 CHANGES OR REVISIONS Any changes or revisions study director, dated, and of this approved maintained with protocol will this protocol. be documented, signed by the The Sponsor will be notified of any changes or revisions 90 ANIMAL CARE AND USE STATEMENT sIntutdhye doopeisninoont odfuptlhiecSatteudaynyDiprreecvtioor,usnwooarlktewmiatthivtehitsestetsitngarmteictlhe,odasndarteheapnpurmopbreiratae,ndthe fsopreccioemspsleilaenccteedwairteharpepgruolpartioartye.guTihdielsipnreostcooconlcewrinlilnbgetrheevciaerweeadnbdyutsheeoCfoavnaimnacles-.IIAfCnUotC ainppcroomvpelidapnrcoet,ocaomlowdiilflibcaetsieonntwtiollthbeeCroevquainrecde.-JAAnCyUcChfaonrgtehseoirrrreevviiseiwo.ns of this 100 RECORDSTO BE MAINTAINED oAfllthriaswstduatday,wdiollcubmeenatracthiiovne,d irnectohredss,toprraogteocfoalc,ilaitnidestohfe Cfionvaalnrceepo-rVtiegnennaefroartaaetsdleaasrtesounlet tyheearSfpoolnlsoowrinmgasyubelmeicststioonhoavfethtehefainfaolrreempeonrttitoonetdhemSapteornisaolrs.reAtfatienredthien otnheeysetaorrapgeeriod, facilities of Covance-Vienna designated by the Sponsor. for an additional periodoftime or sent to a storage facility 1198 130f13 01922