Document ba6dYJ9mabjp2rVqD2nMn1pe0

A lU U -o ^ o Pathology Associates International A Company of Science Applications International Corporation toEmptarm+OmmdCarmnr Sponsor: St. Paul,3MMinnesota PROTOCOL Study Title: i PerfClueollrPoorocltiafneeraStiuolnfoSntiucdAycwidithPoNta-Esstihuym(lPPFSeOarlfStluA(PoFr3oOMoSc;tTa3n-M7e0su9Tl1-f6.o12n)9ai5mn.1iRd6ao)t,EsatnhdanNo-lE(tNh-yEl tPFeOrfSluEo;r3oMctaTne-6su3l1fo6n.1a1m),ide Date: January 12, 1999 Performing Laboratory GaitherR1s5.bOFu.irWrgs,t.fMiSeacldriyeRnlaconeadsd20878 Laboratory Study Identification: Study Number: (1132-100) PAI Project Number: (Histology number to be assigned by PAI by protocol amendment) 15 Worman's Mill Court, Suite I * Frederick, Maryland 21701 * (301) 663-1644 * (301) 663-8994 FAX 004350 Study Number 1132-100 Page 2 Study Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluoroctanesulfonamide (PFOSA 3M T-7091.1) in Rats Purpose To assess cell proliferation and peroxisome proliferation in rats administered test material in the diet. Sponsor 3M Corporate Toxicology Building 220-2E-02, 3M Center St. Paul, MN 55144-1000 Study Representative Marvin T. Case, D.V.M, Ph.D. 3M Corporate Toxicology Phone No.: 651 733-5180 Fax No.: 651 733-1773 Email: mtcase@mmm.com Alternative Study Representative Andrew M. Seacat, Ph.D. 3M Corporate Toxicology Phone No.: 651 575-3161 Fax No.: 651 733-1773 Email: amseacat@mmm.com Study Location R.O.W. Sciences 15 Firstfield Road Gaithersburg, Maryland 20878 004351 Study Number 113P2a-g1e030 Study Monitor Sandra R. Eldridge, Ph.D. Pathology Associates International Phone No. 301 624-2036 Fax No. 301 663-8994 Email: srepaisaic@aol.com Study Director Gary W. Wolfe, Ph.D., D.A.B.T. R.O.W. Sciences Phone No.: 301 330-3723 Fax. No.: 301 330-3738 Email: gwolfe@lab.row.com Principal Investigator Sandra R. Eldridge, Ph.D. Pathology Associates International Phone No. 301 624-2036 Fax No. 301 663-8994 Email: SREPAISAIC@aol.com Study Pathologist Carolyn Moyer, D.V.M., Diplomate, A.C.V.P. Pathology Associates International Phone No. 301 624-2928 Fax No. 301 663-8994 Proposed Study Timetable In-life Start Date: To be added by protocol amendment; Day 0 In life End Date: To be added by protocol amendment Audited Draft Report Date: To be added by protocol amendment Regulatory Compliance This study will be conducted in the spirit of Good Laboratory Practice (GLP) regulations. 004352 Study Number 113P2a-g1e040 Animal Care and Use Statement All procedures in this protocol are in compliance with the Animal Welfare Act Regulations, 9 CFR 1-4. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. Quality Assurance Not applicable. Test Materials Test Material: N(3cM-oEm)tFpOleSteEd by Identification: N-EtFOSE Lot Number: FM 3929 Purity: 99.2% Stability: > 5 years SCCtohoanrradagicteitoerniss:tics: room temp. waxy solid P(3cMFoOm) Spleted by PFOS 217 99% > 5 years room temp. white powder pPP(atmrFFooOOetbonSSecdAAoamldednetd) by pWWa(tmrooyyetb-one1cd4oam,ld6ed4ne3td) by > 5 years room temp. amber waxy solid Reserve (Archive) Samples A reserve sample (approximately 5 g) of each lot will be taken and stored.at room temperature. These samples will be transferred to the Sponsor after completion of the in-life phase to be retained in accordance with 40 CFR 792.195. Disposition of Test Material After authorization from the Sponsor, any remaining test material will be returned to: FPMB3SMahtu.axoiPrlnCvdNaeiiounonrNl.gp,:CooM2a.r6:2asi50net61e,-n5-2Te1D7Es-o3.o7-Vx30t3i-a.c231Mo,-7355l.7o5M,13g18Py40hC4.eD-n1.t0e0r0 004353 Study Number 113P2a-g1e005 Animals Species: Rat Strain: Crl:CD(SD) IGS BR Source: Charles River Laboratories, Inc., Raleigh, NC Age at Initiation of Treatment: Preferable 6 weeks of age, but not more than 8 weeks of age Weight at Initiation of Treatment: 150 to 300 g Number and Gender: males Identification: unique identification by individual car tags and cage cards 004354 Study Number 113P2a-g1e006 Husbandry Housing: Diet: Single housed in hanging stainless steel wire cages Teklad 7012 Certified Rodent Diet. Fresh food will be provided weekly. Fnmueetetrdaielsins,tsaa.nflaaSltypozexecidnif,ibecydhtlnhoueritnmriaeatnentushfyaadncrtauolycreasrerbfsooarnrcse,oononcrgefnailnteroaapttihoRon.ssOpoh.Wfatse.psSe,ccaiifenindecdsephse.ecaivfyied Water: TbsSpocaetipteclniewfcsia.ectseTm.rh,iceprrwoobavetiesd.reidTsahadenalrilebysizutueltdmsaovtfilaethaaesnsteatwuatonoamtliymasteiecsswpaeraerteyoreninafrgilfseoyrastcteoRmn.Otao.mrWwin.aatnetrs and Contaminants: Tsemlwiuxshiabpttetehseedtscrattithinuaeniclddse(yit2tssostd)eupibaldorfeneytoecdp.nrtro(tpe3irsoa)aelslannsybitdbopi/lnovreereass.tnethrinNeumtcoSaitnnpuleorfaaenonleslifdyomtrhoraeherllsaawfeveteaeecdtdoecnmroatnanaatsdmtieldewrinveiaaraeltenlsesdtras,spsuiaonwfrsfcesielciurlbiledelaainensstoigtntnhottaeehibreniflttyeeteermrisfntsegre Environment: Tbcgoerhetnewatitteneaerurngoaeiu3rts0ecld%yh.taenamAngdep1se72/rh0-aoh%tuuo.rruerwTsleiialgmrlehbptbe/e1erm2taw-tauheironeetunarai6nnd4edadrahk.nudcmy7icd9lietFywwailrilethbmeaomrneailtaiontirtveaedinheudm. iTdeitnyor Acclimation: Atswetansiltrilimtnbogaefldsrdeuowmrsiiinolnlgvgbe.tedhAiafscrnpcoilemmirmiaotlahsdtee.wdsAittlunoldibmtyeh.aeolsfbastcheiarlvitteyadrfeofodrriasgemeansineeidrmaolurhmueanolstfuh7itaadnbadlyessufpoitrraiobterisltitotiyntghfoer Randomization: Uftsiohmsroienueaglodcfchnoraogmntropdeuuoxpmtceeoriez-fgdaeetainocen2hr,aSstteh.eDxed.wwroaiefnlildtghnoheomttmvbnaeeruaimasnttaibwoteinersstioigcwfhattil,htlyeaansdasdniifgitfmhneermaelmnsetneo.taftneoabgcorhdosyuepwxs,euAisgethdtthse Justification: Rreaqtusirweimllebnetsufsoedr aberocaduesnet sopfetchieese.xtensive historical data base, and the FDA 004355 Study Number 113P2a-g1e070 Group Designations, Dietary Levels and Scheduled Sacrifice Time Points Number of Male Rats G(rToiumpeNPuominbt)er (C0opnptrmol) 300N-1E0t0FO3S0Eppm 2P0FpOpSm 1P0F0OpSpAm W10y0-014p,p6m43 oTfoAtanlimNaol.s (48 1hrs) 10 10 10 10 10 10 5 65 (7 d2ays) 10 5 5 5 5 5 5 40 (14 d3ays) 10 5 5 5 5 5 5 40 (1 wk re4covery; 10 55 5 5 5 5 40 (4TwoAtkanlrie5mNcaoolv.seorfy; 10 50 55 5 30 30 30 5 30 5 30 5 25 40 225 Dosing Procedures Method of Administration Dietary. Animals in Groups 1through 3 will receive test diet for 48 hours, 7 days, and 14 days, respectively. Animals in Groups 4 and 5 will receive test diet for 14 days followed by a 1 or 4 week recovery period, respectively. Reason for Dosing Route The potential human exposure is by the oral route. Dose Preparation Before initiation of treatment, dose preparation of each test material will be mixed. A l dose preparations will be stored at room temperature. Dose preparation will be documented and reported. See Attachment I for test diet preparation procedures. 004356 Study Number 113P2a-g1e008 Retention Sample Samples (approximately 100 g) will be taken from the dose preparation and stored at room temperature. Unless used for analyses, these samples will be discarded at least 1month after completion of the in-life phase. Observation of Animals Clinical Observations Each animal will be observed twice daily (a.m. and p.m.) for mortality and moribundity; findings will be recorded as they are observed. Body Weights Prior to treatment (at randomization), weekly for Week 1 through 4 weeks of recovery. Food Consumption Weekly for Week 1through 4 weeks of recovery. Clinical Chemistry Animals will be fasted overnight before animal's scheduled necropsy; blood will be collected from ajugular vein into an EDTA-coated tube. Serum enzyme levels of alanine aminotransferase (ALT), alkaline phosphatase, aspartate aminotransferase (AST), cholesterol and triglycerides will be determined. Termination Unscheduled Sacrifices and Deaths Necropsies will be done. Animals to be sacrificed will be anesthetized with C 02, weighed, and exsanguinated. G04357 Study Number 113P2a-g1e009 Scheduled Sacrifices Interim Sacrifices At 48 hrs, 7 days, and 14 days, animals will be fasted overnight, bled for serum samples, anesthetized with C 02, weighed, and exsanguinated. NOTE: Two serum samples will be needed, (1) a 0.5 ml sample for clinical chemistry and (2) a 1.5 ml sample for compound level analysis. The abdominal cavity of each animal will be opened, the liver will be removed and weighed, and liver samples will be collected. Animals will be discarded after liver collection. Terminal Sacrifices After 1 and 4 weeks of recovery, animals will be fasted overnight, bled for serum samples, anesthetized with C02, weighed, exsanguinated, and necropsied. NOTE: Two serum samples will be needed, (1) a 0.5 ml sample for clinical chemistry and (2) a 1.5 ml sample for compound level analysis. Postmortem Procedures Necropsy The necropsy will include an examination of the external features of the carcass; all external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. Cell Proliferation Tissue Collection and Immunohistochemical Evaluation Representative samples of the left lateral lobe of the liver and any macroscopic lesions of the liver will be collected and preserved in zinc formalin. After fixation, each sample of liver will be delivered to: FSP15raaetnWhddoerolraoircgmRky.,aAnME'lssadsrMoryicdliialgalnteeCd,soPI2uhn1r.tt7De,0r.Sn1uaittieonIal 004358 Study Number 11P3a2g-e10100 Proliferation cell nuclear antigen (PCNA) evaluation will be done on the samples. In addition, liver sections prepared from the same tissue block will be stained with hematoxylin and eosin and examined microscopically. Palmitoyl-CoA Oxidase Tissue Collection and Analyses A sample (approximately 500 mg) of the right lateral lobe of the liver will also be collected from select animals and flash-frozen in liquid nitrogen. See Attachment II for procedure. The liver tissue will be stored in a freezer set to maintain -60 to -80 C until analyzed by Covance for palmitoyl-CoA Oxidase activity. The liver samples to be analyzed will include all study animals, EXCEPT for the Wy-14,643 animals and all animals from the 4-week recovery groups. In addition to this study, samples from a previous 3M study will be analyzed for palmitoyl-CoA Oxidase activity; these samples consist of liver samples from 35 rats and 35 guinea pigs. Tissue Collection for Electron Microscopic Evaluation Sections of liver from all animals will be collected, minced to approximately one millimeter cubes and placed in a fixative appropriate for electron microscopy. The containers and fixative will be provided by PAL Electron microscopy will be performed on one animal per treatment group exhibiting the highest cell proliferative response as well as one control animal at the discretion of the Sponsor, from one time point as well as the 4-week recovery. Thus, EM will be performed on one animal from the control, N-EtFOSE (one dose only to be determined), PFOS, PFOSA, and Wy groups at one of the time points, as well as the 4 week recovery, for a total of 10 animals. Remaining Liver Tissue The remaining liver tissue will be frozen and stored at -60 to -80 C for possible future analysis. Organ Weights At the scheduled sacrifices, the liver will be weighed. Histopathology Liver from each animal that is examined for cell proliferation will be stained with hematoxylin and eosin, and examined microscopically for histopathologic changes. 004359 Study Number 11P3a2g-e10101 Reports One copy of the draft report will be sent to the Sponsor. The report will include the following information: Experimental Design and Methods Results butcpmcdmcbfmeolelloaooiistoaiollnndsdrctlmcradeiiyyrtmprccsoaiocartaawtlwsarosoinollcuttcleeyanyepooicoiillrfsabpgtgy-pieuutaCsihhsrhimcrelceattoaorscstopcAlvolioobohtbaonigsnoatsobseiynexosnuarrgeivnmsrvdreessaavasspettsauistioetiosloitnmnoasnscsnetsnivtsities Record Retention All raw data, documentation, records, protocol, specimens, and final report generated as a result of this study will be archived in the storage facilities of PAI for a period of 1 year following submission of the final report to the Sponsor. One year after submission of the final report, all of the aforementioned materials will be sent to the Sponsor and a return fee will be charged. All raw data stored on magnetic media will be retain by PAI. 04360 Study Number 11P3a2g-e10102 PROTOCOL APPROVAL MS3MtuadrCvyionRrpeCoparraseetse,enTDtoa.Vxtiiv.cMeol.o, gPyh.D. SPPraaitnnhdcorilpaolgRey.InAEvlsedssrotiicdgigaaetteo,srPIhn.tDer.national Date 004361 Study Number 11P3a2g-e10103 Attachment: I Test Diet Preparation Procedures 1) Determine the amount of test diet (feed) that is to be prepared and weigh out that amount of feed. 2) Calculate the amount of test article that is needed to prepare the test diet at the desired concentration. 3) Accurately weigh out the necessary amount of test article. 4) Transfer the weighed test article to a container and add a small volume of acetone to container. Manually mix to dissolve the test material adding acetone as necessary (typical ratio of test material to acetone is 1 g: 15-20 ml acetone). Visually inspect test material/acetone for solubility of test material. 5) Prepare a pre-mix by transferring the dissolved test material into 4 kg of feed in a Hobart mixing bowl. Mix for 10 minutes. Transfer the premix to a larger mixer, add remaining amount of weighed diet, mix for 30 minutes. 004362 Study Number 11P3a2g-e10104 Attachment: II Collection of Tissue Samples for Biochemical and Molecular Analysis Because of the extreme instability of certain enzymes and biomolecules, it is essential that tissues be harvested as soon after death as possible and flash frozen immediately in liquid nitrogen. Failure to follow these procedures may lead to loss of the entire sample and all the energies and resources that were invested into generating the samples. Therefore, make every effort to comply with the following: 1) Harvest the tissue samples as soon as possible after death. Delays may allow for biodegradation and/or inactivation of the desired endpoint. 2) Immediately submerse the tissue sample directly into liquid nitrogen. Dry ice or other alternatives will not suffice. It is important that the tissue be immersed directly in liquid nitrogen Transferring it to a dry vessel (or sample container) suspended in liquid nitrogen will not suffice. The tissue may freeze to the vessel wall and will then be impossible to remove without completely destroying the vessel (or sample container). 3) Be absolutely sure to maintain the tissue frozen. It should be stored in a sealed container at 70 C and shipped or transferred on dry ice. If needed, the frozen sample can be fractured (broken into portions for different applications) by placing in a crucible which contains liquid nitrogen to keep the sample frozen while grinding/fracturing. 004363