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3775) Pathology Associates International 8 -- EE7R 8--4 Co-- mpany of Sci-- ence Applicatio-- ns Intemationa-- l Corporation. -- T 3 -- E. _ Sponsor: 3M St. Paul, Minnesota PROTOCOL Study Title: Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T-6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), and N-Ethyl Perfluoroctanesulfonamide (PFOSA 3M T-7091.1) in Rats Date: January 12, 1999 Performing Laboratory R.O.W. Sciences 15 Firstfield Road Gaithersburg, Maryland 20878 . Laboratory Study Identification: Study Number: (1132-100) PAI Project Number: (Histology number to be assigned by PAI by protocol amendment) SOs aE gtreerie 15 Worman's Mill Court, Suite I * Frederick, Maryland 21701 * (301) 663-1644 * (301) 663-8994 FAX 005969 Study Number 113P2a-g1e020 Study Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EtFOSE; 3M T6316.11), Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; 3M T-6295.16), andN-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, PhD. 3M Corporate Toxicology Phone No.: 651 733-5180 Fax No.: 651 733-1773 Email: micase@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 005970 Study Number 1132-100 Page3 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, DAB.T. ROW. Sciences Phone No.: 301 330-3723 Fax. No.: 301 330-3738 k `Email: gwolfe@lab.row.com Principal Investigator Sandra R. Eldridge, Ph.D. `Pathology Associates Intemational Phone No. 301 624-2036 Fax No. 301 663-8994 Email: SREPAISAIC@aol.com Study Pathologist Carolyn Moyer, D.V.M, Diplomate, AC.V.P. Pathology Associates International : Phone No. 301 624-2928 Fax No. 301 663-8994 Proposed Study Timetable In-lfe 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. 005971 Study Number 1132-100 Page 4 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-EFOS TPEOS ==TPro TW Tien Ii I 3(cmompletedby | (c3oMmpletedby (`tapromobteoecanoldddemdebyn)|| pa(trmooebtnoedcaomldendyebdy | [Fe rree p[reerrp[ ee | CoTnodriatgieons: oom temp. Toor temp: oomtemp: | Reserve (Archive) Samples A reserve sample (approximately 5 g)ofeach lot will be taken and stored.at room temperature. `These samples will be transferrteod the Sponsor after completion of the in-life phase to be retained in accordance with 40CFR 792.195. Disposition of Test Material After authorization from the Sponsor, any remaining test material will be retuned to: Mackin Case, D.V.M,, Ph.D. Corporate Toxicology Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 Phone No.: 651-733-5180 Fax No.: 651-733-1773 005972 Study Number 1132-100 Pages. Animals i [Species -- [Ra - oo -- 71 [romrm------f---- 005973 Study Number 113P2a-g1e060 Husbandry = Single housed inhanging ssiness sel wie cages 1 `Teklad 7012 Certified Rodent Diet. Fresh food will be provided weekly. | Feed is analyzed by the manufacturer for concentrations of specified heavy mnuettrailesn,tsa.flaStpoexciinf,icehdlonurtirniaetnetshyadnraolcyasrebsoanrse,oonrgfailneopathoRs.pOh.aWt.esS,caienndcessp.ecified | Water: `boTtatplewsa.tTerh,epwraotveirdiedsaadnalliybzietudmavtilaeaasnttawuototmatiicpmwearteeyresianrgfosrysctoenmtaomriwnaatnetrs and Scsipeecnicfeics.microbes. The results of these analyses are on file at R.O.W. Contaminants: | Tsuhbestsatnucdeysdpiorteecnttoiraalnldy/porretsheentSpinonasniomrahlafveeedcoannsdidwaetreerd,pionscsliubdleinignttehrefteersitng mliastteerdiainl (S2)eainfd (p3o)saibbolvee.syNctounreaolflytehlesaetecdonmtaatemriinaalnstassawreelreaassotnhaebelyms ewxiptehcttheids tsotubdey.present in animal feed or water at levels sufficient to interfere Environment: Acclimation: | Tbehtewteaernge3te0d%teamnpde7ra0t%u.reTseamrpeebreattwuereena6n4dahnudmi7d9itFywairtehmaonrieltaotrievde humidity c`gornetaitneuroauisrlcyh.anAge1s2/-ohuorurwilllibgehmati/nt1ai2dnae-rdkh.coyuclre will be maintained. Ten or | { AStnairtmaolfsdowsiilnlgb.e Aancicmallismwitalolttbehedeobfasceirlivteydfofroragmeinneriamtuhmeaolfth7adnadyssupirtaiboirlittoytfhoer | tweisltlibneg druermionvgetdhifsrpoermitohde. Astnuidmya.ls that are diseased or unsuitable for testing. Randomization: I I Justification: | Utismiengofcroamnpduotmeirz-agteinoenr,attheed wreaingdhotmvanruimatbieornosfwtihteasasniigmnamlesnotfteoagcrhouspesx,uAstedthe sfohroueladchnogtroeuxpceoefdea2c'hSs.eDx.woiflltnhoetmbeeasntawteiistgihcta,llaynddiftfhereenmte.an body weights Rraetqsuiwrielmlebnetsusfeordabercoaduesnet sopfectihees.extensive historical data base, and the FDA 005974 Study Number 113P2a-g1e0?0 Group Designations, Dietary Levels and Scheduled Sacrifice Time Points f1 "Numberof Male Rats -- = |}--GCrroiumpeNPumobnerg||_ 0Conptrmol)1|3010N-0 EF3OS00 E5pm||2P0FpOpSm||1P0F0OgSAp|l W1y6-6104p,om643||ofTAotnailmNao.ls i (481hrs) 65 diays) 14 d3ays) | (1 wi r4ecover | (4 wk r5ecove Cootal mNo.o |] s 5 ! 5 I 5 55 5 I Tolwu lol l=o|l =] Dosing Procedures Method of Administration Dietary. Animals in Groups | through 3 will receive test diet for48 hours, 7 days, and 14 days, respectively. Animals in Groups 4 and 5 will receive test diet for 14 days followed by a 1or 4 week. recovery period, respectively. Reason for Dosing Route `The potential human exposure isby the oral route. Dose Preparation Before initiation of treatment, dose preparation of each test material will be mixed. Al dose preparations willbe stoatrreoodm temperature. Dose preparation will be documented and reported. See tachmeInotr test diet preparation procedures. 005975 Study Number 113P2a-g1e080 Retention Sample `Samples (approximately 100 g) will be taken from the dose preparation and stored at room temperature. Unlessusedfor analyses, these samples will be discarded at least 1 month after completion of the in-ife phase. Observation of Animals Clinical Observations Eachanimal willbe observed twice daily (a.m.and p.m.)formortality and moribundity; findings will be recordaesd they are observed. Body Weights Prior to treatment (at randomization), weekly for Week 1 through 4 weeksofrecovery. Food Consumption Weekly for Week 1 thro4uwegekhs 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 willbeanesthetized with CO,, weighed, and exsanguinated. 005976 `Study Number 113P2a-g1e00 Scheduled Sacrifices Interim Sacrifices At48 hrs, 7 days, and 14 days, animals will be fasted overnight, bled for serum samples, anesthetized with CO,, weighed, and exsanguinated. NOTE: Two serum samples will be needed, (1) a0.5 ml sample for clinical chemistry and. (2)a 1.5 ml sample for compound level analysis. `Theabdominalcavity ofeachanimalwillbe opened,theliver willberemovedand `weighed, and liver sampleswillbecollected. Animals wil 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 CO,, weighed, exsanguinated, and necropsied. NOTE: Two serum samples will be needed, (1) a0.5 ml sample for clinical chemistry and (2)a 1.5 ml sample for compound level analysis. Postmortem Procedures Necropsy `The necropsy will include an examinationofthe external featuresofthe 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: PSaatnhdorlaogRy. AEslsdorciidagtee,sPIhn.tDe.mational F1r5edWeorrimcka,nM'asrMyillalnCdou2r1t7,0S1uitTe 005977 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 S00 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 uniil 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-weck 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 Sectionsofliver from all animals will be collected, minced to approximately one millimeter cubes and placed in afixative 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-ECFOSE (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 wil be weighed. Histopathology Liver from each animal that is examined for cell proliferation will be stained with hematoxylin and cosin, and examined microscopically for histopathologic changes. a 005978 Study Number 11P3a2g-e10110 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 dose analyses c`lmionritcaallitoybservations bbooddyy wweeiigghhttschanges fteosotdmactoenrsiualmpctoinosnumption : pclailnmiciatlopyalt-hCoolAoogxyirdeassueltasctivities mmiaccrroossccooppiiccoobbsseerrvvaattiioonnss cuelltlrapsrtorluicfteurraatlioonbsaesrsveastsimoennsts. 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, allofthe aforementioned materials will be sent to the Sponsor and a return fee will be charged. All raw data stoon rmagenetdic media will be retain by PAL 005979 Study Number 1132-100 Page 12 PROTOCOL APPROVAL easea, D[VMG, e Study Representative 3M Corporate Toxicology ROW, ay . Stuc i 0. ) fe, PhD, DAP' Sdignces eand dra R._ Eldri2 dge, PRD. Principle Investigator Pathology Associates International 1p ree ' / iz /27 42 27 005980 Study Number 11P3a2g-e10130 Attachment: 1 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)Calculatetheamountoftest articlethatisneeded topreparethetestdietatthe desired concentration. 3) Accurately weigh out the necessary amooufntestt article. 4) Tranthsewfeiegherd testarticleto acontainaenrd add a small volume of acetone to container. Manuallymixtodissolvethetestmaterial addingacetoneas necessary (typical raotfiteost matteo arcetioneaisl1 g: 15ml-ace2ton0e). Visuallyinspecttest material/acetone for solubility oftest 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. 005981 Study Number 11P3a2g-e10104 Attachment: TL 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 inactivationofthe 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 adryvessel (or sample container) suspended in liquid nitrogen will not suffice. The tissuemayfreeze 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 70C and shipped or transferroend 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. 005982