Document qmxRORMRGwm2JG68L30VvvOXk

FEF 2 Pathology Associates International S55E A CCoommpaannyy ooff Science ApplApiplciacatiionosnIsnItnetemrantatiioonaall CoCroprpooration a ro 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 PotassiumSalt(PFOS; 3M T-6295.16), and N-Ethyl Perfluoroctanesulfonamid (PFOSA 3M T-7091.1) in Rats Date: January 12, 1999 Performing Laboratory R.O.W. Sciences Gaither1s5buFirrgs,tfMiaerldylRaonadd20878 Laboratory Study Identification: Study Number: (1132-100) PAI Project Number: (Histology number to be assigned by PAI by protocol amendment) A 15 Worman's Mill Court, Sulte 1 * Frederick, Maryland 21701 + (301) 663-1644 = (301) 663.8994 FAX 000163 Study Number 113P2a-g1e020 Study Cell Proliferation Study with N-Ethyl Perfluorooctanesulfonamido Ethanol (N-EFOSE; 3M T6316.11), Perflucrooctane 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 220-2-02, 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: 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 000164 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., D.AB.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 Intemational 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 addedbyprotocol amendment AuditedDraftReportDate: Tobeaddedbyprotocolamendment Regulatory Compliance "This study will be conducted in the spirit of Good Laboratory Practice (GLP) regulations. 000165 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 TestMaterial |NETOSE (completedby TT | hl | PFOS (completedby. ha PFOSA (tobeaddedby | aPmoenidnment) [Wy Tash] | (tobeaddebdy parmievndment) i p1 r i] orage Toor Ep. oom temp. room temp. Conditions: (Teer [re e [ees"[ees= EE]| 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 completionofthe 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 retuned to: B3MuaMirlvCdioinrnpgoC3ra3as0te.e,2TD5o..xV0i.3c,oMl.3o,gC3Pehn.tD.er St. Paul, Minnesota' 35144-1000 Phone No.: 651-733-5180 Fax No.: 651-733-1773 000166 Study Number 1132-100 Pag5e RpAnimals epee f[eeo reoem s | Re Tr Pla Ves of a Bi Seno | [rer 000167 Study Number 113P2a-g1e00 Husbandry owing:| sinc stinging ns se irc I{ Water: II Teklad 7012 Certified Rodent Diet. Fresh food will be provided weekly. | F`meeetadlsi,s aanflaaltyozxeind,bcyhltohreinmaatneufhaycdtruorcearrbfoonrsc,onocregnatnroapthioonssphoaftespse,ciafnidedspheecaivfyied nutrients. Specified nutrients analyses are on file at R.O.W. Sciences. { { Tap water, provided bottles. `The wateris ad libitum via an analyzed at least automatic two times watering per year system or water for contaminants and | Scsipeecnicfeics.microbes. The resultsofthese ana. lyses are on file at R.O.W. Contaminants: | Tsuhbestsatnucdeysdpiorteecnttoiralalnyd/porrestehnetSipnoannsiomralhafveeedcoannsdidwearteerd,pionscsliubdleinignttehrefteersitng | lmiastteerdiailn (it2s)elafnord (p3o)ssaibbolvee.strNuoctnueraollfytrheelsaetecdonmtaatmeirnainaatlssawreelrleaassotnhaebiltyems || ewxiptehcttheids stotbudeyp.resent in animal feed or waterat levels sufficientot interfere Environment: | bTehtewetaernge3t0ed%tamnpder7a0n%i.reTseamrpebreattwuereena6n4dahnudmidiFtywairethmaonrietloirveed humidity `cgornetaitneruoauisrlcyh.anAges1/2h-ohuorurwillilghbte/1m2a-ihnotuarindeadr.k cycle il be maintained. Ten or Acclimation: | Astnairtmaoflsdowsiilnlgb,eaAcncilmiamlastewdiltlobteheobfasceirlivteydffoorragmenienriamtuhmeaolfth7adnadyssupirtaiboirlittoythfeor wtielsltibneg druermionvgetdhisfrpoermiotdh.e Astnuidmy.als that are diseased or unsuitable for testing. | Randomization: | Utismiengofcroamnpduotmri-zgaetinoenr,attehedwreaingdhotmvanruimatbieornsofwittheasasniigmnamlesnotfteoagcrhouspesx,uAstedhe || i sfhoroueladchnogtroeuxpcoefede+ac2hSs.eDx.woilfltnhoetmbeeasntawteiistgihcta,llaynddiftfhereenmte.an body weights Justification: | Rats will be used because of the extensive historical data base, and the FDA. | 1 `requirements for a rodent species. | 00168 Study Number 113P2a-g1e0?0 Group Designations, Dietary Levels and Scheduled Sacrifice Time Points f = - ~ Number of Male Ra -- || _GCrToiumpeNPuombienr)||(0Conptproml) 1 (48 hrs) |300N1-0E0tFO3S0Eppm | 2P0FpOpSm||10PF0OpSpAm|]1W8y0-104p,p6r43||ofToAtanliNmoa. || 10 5 6| days) ! 1 14 d;ays) ' sls] | [I11 wk r.ecover ' EE : [Em] [_(4 wk recover I~ Tom No.o 0 Twwlw[ ]ow ' |[ ow] Dosing Procedures Method of Administration Dietary. Animals in Groups I through 3 will receive test diet for 48 hours, 7 days, and 14 days, respectively. Animals in Groups 4and 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 preparationof each test material will be mixed. All dose `preparations will be stored at room temperature. Dosepreparation will be documented and reported. See Attachment I for test diet preparation procedures. 00169 Study Number 113P2a-g1e0s0 Retention Sample `Samples (approximately 100 g) will be taken from the dose preparation andstoredat room temperature. Unless used for analyses, these samples willbediscarded at least 1 month after completion of the in-life phase. Observation of Animals Clinical Observations Each animal findings will will be observed twice daily (a.m.and be recorded as they are observed. p.m.) for mortality and`moribundity; Body Weights Prior to treatment (at randomization), weekly for Week 1 through 4 weeks ofrecovery. Food Consumption `Weekly for Week 1 throug4h weeks of recovery. Clinical Chemistry Animals a jugular will be fasted overnight before animal's vein into an EDTA-coated tube. Serum secnhzeydmueleldevneelcsroofpaslya;nbilnoeoadmwiinloltbraencsoflelreacsteed(AfLrTo)m, 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 CO,, weighed, and exsanguinated. 00170 . Study Number 1132-100 Page9 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. `The abdominalcavity ofeachanimal wilbe opened,theliverwillbe removedand `weighed, and liver samples willbecollected. Animals will be discardedafterliver 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, (120).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 lef lateral lobe of the liver and any macroscopic lesions of the liver willbecollected and preserved in zinc formalin. After fixation, each sample of liver will be delivered to: PSaatnhdorlaogRy. AEslsdorciidagtee,sPIhn.tDe.mational F1r5edWeorrimcka,nM'asrMyillalnCdou2r1t7,0S1uite I 000171 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 Asample (approximatelyS00mg)ofthe right lateral lobeofthe liver wil also be collected from select animals and flash-frozen in liquid nitrogen. See Attachment I 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 placedin 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-EGFOSE (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 A the scheduled sacrifices, the liver wil be weighed. Histopathology Liverfromeachanimalthat is examined for cell proliferation will be stained with hematoxylin and eosin, and examined microscopically for histopathologic changes. 000172 Study Number 11P3a2g-e10110 Reports One copy of the draft report will be sent to the Sponsor. The report wil include the following information: Experimental Design and Methods Results dmoorstealaintaylyses clinical observations bbooddyy wweeiigghhttschanges fteosotdmactoenrsiualmpctoinosnumption : cplailnmiciatlopyalt-hCoolAoogxyirdeassueltsactivities `macroscopic observations `ulmtircarsotsrcuocptiurcaolbosbesrevravtaitoinosns cell proliferation assessments 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 submissionofthe 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 retum fee will be charged. All raw data stoonmragenetdic media will be retain by PAL 000173 Study Number 1132-100 Page 12 PROTOCOL APPROVAL arsTC Study Representative 3M Corporate Toxicology Ala 0, ).00, Satrudyy DWci. hWyolfe, PhD, DA. (3 tree Salas Dai ped Principle Investigator Pathology Associates International 4297 000174 Study Number 11P3a2g-e10130 Attachment: 1 Test Diet Preparation Procedures 1) Determine the amount oftestdiet (feed) that is to be prepared and weigh out that amount of feed. 2)Calculatetheamountof testarticlethatis neededtopreparethetestdietatthe desired concentration. 3) Accurately weigh out the necessary amount of test article. 4) Transferthe weighedtestarticleto acontainerand add asmall volumeofacetone to container. Manuallymixtodissolvethetestmaterialaddingacetoneasnecessary (typicalratiooftest material to acetone is 1 g: 15-20 ml acetone). Visually inspect test materialfacetone for solubilityoftest material. : 5) Prepare apre-mibyx transferringthedisstoesltmvateeridalinto 4kgoffeedin a Hobart mixing bowl. Mix for 10 minutes. Transfer the premix to a larger mixer, add remaining `amount of weighed diet,mixfor30 minutes. 000175 Study Number 1132-100 Page 14 Attachment: TI Collection of Tissue Samples for Biochemical and Molecular Analysis Because of the extreme instability of certain enzymes and biomolecules,i 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 resourcesthatwere 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 adry 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 sureto maintain the tissue frozen. It should be stored in a sealed container at 70Candshippedortransferredondryice. Ifneeded,thefrozensamplecanbefractured (broken into portions for different applications) by placing in a crucible which contains liquid nitrogen to keep the sample frozen while grinding/fracturing. 000176 -