Document q33XjEk1e7a9apy3J9ErN9zwq

COVAANNCCEE AR R86 -- 1358 Sponsor: 3M FINAL REPORT Study Title: 4-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys Author: PeteJr. Thomford, PhD Study Completion Date April 9, 2002 Performing Laboratory Coven Lorrie ne 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Laboratory Project Identification: - Covance 6329-222 `Sponsor Project Identification: 3M Study No. T-6295.6 Page 1 0f235 0001.37 - em COMPLIANCE STATEMENT 4-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys All aspectsofthis study were in accordance with the Environmental Protection Agency Good Laboratory Practice (GLP) Standard4s0, CFR 792. However, during the timeframe of March 10, 1998, through April 16, 1998, prestudy procedures such as general husbandry, health screening activities, and testing for assignment to study were conducted. The protocol was finalized on April 17, 1998. While the performance of these prestudy events without a final protocol represents a GLP deviation, they did not affect either the outcome or the interpretationofthe data. ae Study Bffector Covance Laboratories Inc. Andrew M. Seacat, PhD Study Monitor 3M O=Sforacoy Date : 000138 CovanMceT6632209-52262 QUALITY ASSURANCE STATEMENT `This report has been reviebwytehde Quality Assurance Unit of Covance Laboratories Inc. The following inspections were conducted and findings reported to the study director (SD) and associated management. Inspection Dates From To Phase 0421/98 04121/98 Protocol Review 05/20/98 05/20/98 Protocol Amendment Review 05/20/98 05/20/98 Protocol AmendmentReview 0521/98 05/21/98 ClinicalLaboratoryInspection 06/01/98 06/01/98 Postlife 07/08/98 07/13/98 DataReview 08/14/98 08/14/98 Protocol Amendment Review 08/10/98 08/18/98 Report Review 06/04/99 06/04/99 Protocol Amendment Review 03/27/02 03/27/02 ProtocolAmendmentReview 0327/02 03/27/02 Report Review DateReported to Study Director and Study Director Management 0421/98 05/2098 05120098 0521/98 06/01/98 0078//1143//9988 0086//0148//9998 03/27/02 03/27/02 Representative Quality AssuranceUnit Bue 000139 3 CovanIcMe 6T36292-52262 STUDY IDENTIFICATION 4-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys Test Material Sponsor Study Monitor Altemate Study Monitor Study Location Study Director Study Timetable Study Initiation Date In-Life (Experimental) Start Date In-Life Termination Date Experimental Termination Date Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) 3M Toxicology Services Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 Andrew M. Seacat, PhD 3M 651.575.3161 Marvin T. Case, DVM, PhD. 3M 651.733.5180 Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 PeteJr.Thomford, PhD. Covance Laboratories In. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 608.241.7207 April 17, 1998 April 23, 1998 May 22,1998 April ,2002 000140 4 _-- CovanMcTeT-6e6322s99252s62 KEY PERSONNEL Study Director Study Toxicologist Study Coordinator Supervisor, Large Animal Toxicology Supervisor, Dose Formulation Supervisor, Laboratory Animal Medicine Clinical Pathologist Supervisor, Clinical Pathology Anatomical Pathologist Anatomical Pathologist Supervisor, Anatomical Pathology PeteJr.Thomford, PhD Dale Aldridge, BS Patricia K. McKee Pesik, BS, LAT Mecchelle Bordeaux, LAT Dixic Bushee, BS, LATG Donna J. Clemons, DVM, MS Diplomate, ACLAM Robert L. Hall, DVM, PhD Diplomate, ACVP (Clinical Pathology) Ronald Markevitch, BS, MT (ASCP) Dawn G. Goodman, VMD. Diplomate, ACVP Directorof Pathology `Thomas E. Palmer, PhD Kimberly W. Durland, BS, HT 000141 5 Covance 6320-222 CONTENTS Page ABSTRACT costs ST) FURZOSE....cocssmsmessesmessomsmrmmmssmss I) REGULATORY COMPLIANCE.......... ss-------- TEST MATERIAL, VEHICLE, AND SOLVENT........... nmmmnnsrntuntl2 TSUMBIETB vse nner dZ Reserve (ATChive) SAMPIES............oe DISPOSION. rr nsnenenn A rm------ 13 TEST SYSTEM... ens rorsss--_5 TTUAEARDI FCRsImOrNmmmmsms sssesmn-- seS s ener-- o 13 HouandsMaiintnenagnce.......... r------------hyEY JUSHECRLON rs mmm PROCEDURES mmission mms `DGO1SOEUpPDLEePsAIiBHgONna.n.ad.Dt.O.SirErornnrsmrmLsEVeEmI.r.i..n.s.i....ecrorovensrnrnorcrmrinemnrerer ens1 ClMinicealOOtFBSAeQrMh vaUtBiIoo SrHs.T.Ad .t.O..N...w..........ccrverrenem eee-- scs-- sen-- es e-- ner--)1 T RBeOcBtaYlWBoEdIy TGemHpeIraSure.s.r..r...n..n..e...s..e..m..s. esesA esonnonsI oe or 18 BEloromd HPOFTOMISOLNeEveDlDEErMiaAtionN ..... ..... ..... .ooeooooerererrerererereornrerorsorone1}8 CHRCal PAHOIORY...crsrsreemsmssnmsnre AddBiIOtCOiNCCoHiOnN.a...l...ecrrememsrrireoroeo cn 19 nd FPaElmRitOoyS])COiA aOXirdasSepDEgErMMmINsAOmNSs.rgono snrmecas eeeeeor r2d0l) Cell PrONfEration EVBIUAHON .........cvevrenemsesoneseneoresmrensororororon LiverPFOSDOCTIIAON .......veveeecsssensososenroeorerorrr 20 20 a HHSUOPALNOIORY .ossrnssnnonensmsnsososenenenen 1 21 SISA] ABBIYSES recensione 22 RECORDRETENTION c.cccnrsssmensnsnssnsnsnsnssnsesonninnn 22 6 000142 - OOOO `CONTENTS (Continued) RESULTS .ov-csscomummnussnmmmammsinssensss TCroveMe 6Tw129s22 Page I remy Body Weights... BI EE Clinical ODSETVALONS........ooccoosivssrssssesssesessessssesssosesssee FoodConsumption........... Rectal Body Temperatures... wr. ee RRA ot 23 wren ws Blood HormoneAnalyses ........ ------------ Clinical Pathology........c.c..ccuue orssssrerseremssmaion Cell Proliferation EVAIUGtion ...........ceewveeerseeeereeerenn Anatomical PAthOlOGY...........ceewuueeriisserssssersesrsrseesisen smote sr -- ssa orm dS end worms 'CONCLUSIONS..... errs asarate wo mds PATHOLOGYREPORT ccnumunsmmrmmenssrsisminsensmsestsmummniamnsnss IN. COMMENTS ON THE DATA ......ooccoverccmrserne ---- remmromin3l `CODES, ABBREVIATIONS, AND UNITS..... ---- natmmms3S General Codesand Abbreviations ...................... Codes for Clinical Pathology. onsen sss ssrssssesersen Abbreviations and Units for Clinical Hematology .. Abbreviations and Units for Clinical Chemistry ...... ora sro Y srm---- mers srna---- mornin 36 ---------------- CodesforAnatomical Pathology. rr --. etm ore 8 `TABLES 1 Summ ofa Clirnicyal Observations A.M./Weekly.. sr-- nrrrrrrrr1n 2 Sumomfa Clirnicyal Observa3t0Miionuntess Postdose..................... rod 3 SummaryofClinical Observations 60 Minutes POSIAOSE..............o.oowrrrererrrrrn 83 4 SummaryofClinical Observations 90 Minutes POSdOSE...............o.ocer... dd 5 Summary ofClinical Observations Unscheduled............ wnnmrnereS 6 Suom fBodm y Wea ightr Datay (Kg) .......ovceevevrevsrssrsrmrsmesesnon 86. 7 SummaryofRectal Body Temperature Data (C) ............covrmomrrmrrmsneendd 8 SummaryofClinical Hematology Data - Day -7........cccevevrmmmmmmsmsnn S| 9 Summary ofClinical Hematology Data - Day 29............... A 10 SummaryofClinical Chemistry Data - Day -7.............ccccoeemmmmmmmmrssmmmsrerssn 9 11 Summary ofClinical Chemistry Data = Day 2 ......o.ccc.ceermrrmnen 12 SummaryofClinical Chemistry Data - Day 7 ..........o.oooevrorrene 13 SummaryofClinical Chemistry Data - Day 14............o.ooeoerrene wereresymnrnd snd? 7 000143 CovancMeT63e292-52262 CONTENTS (Continued) Page TABLES 14 SummofaClirnicyal Chemistry Data-Day 29... 15 SummaryofClinical Chemistry Data - Day 30 -------- 83 89 16 17 Incidence of Macroscopic IncidenceofMicroscopic ODSETVAIONS OBServations.......... rors swmm---- 91 APPENDS Lupino ws -- Protocol Deviations. Protocol ........ - s---------------------- 95 s------------ ProtocolAmendment No. | mm---------------- Lf Protocol AmendmeNnot. 2... amm------------------_ 1 PrO10CO1 AMENAMENOt.3 css soso 20 Protocol AMERIMENt NO. 4... A Material Safety Dats SHEE ..rnmsnnsnmssemmnsnssssnsiosonn 125 APPENDS Lummis IndividualAnimal Fate Data... IndividualClinicalObservations ........ Individual Rectal Body TemperatureData (C) sss e131 A wsm-- 133 -- 140 APPENDIX 3... Individual Body WeightData (kg) wmmm----------eli ws-- 143 APPENDIX A. ------ Individual Clinical Hematology Data... Individual ClinicalChemistry Data... smn 4S ssn HE ssn 156 APPENDIX 5 sr ------ Individual Animal Pathology Data evr wml 100 rn 191 QualityASSUTAnCE StatAeTmLeYEnItCS.r..r.rorcnrnrnn 204 Summaryand Individual Hormone Analyses Data........... comm ZiS Figure | - Mean Estradiol Data (R/L) - MALES ..........corcrcorcnrnronrn200 Figure 2 - Mean Estradiol Data (pg/mL) - Females... A + Figure 3 -MeanTriiodotDahtay(1gr/doL)n-ine Males.....o.ovcorcormronn 211 Figure 4 - Mean Triiodothyronine Data (ng/dL-) Females... 212 APEENDIL commands DOS CONITMAON ADBIYSIS REPO. 214 COMPOUN SHabity REPOL creer nnn 219 : 000144 CovancIeM 6T36292-25262 CONTENTS (Continued) Page CenificateofAnalysis you QualityAssurance Statem-e3nMt ...... ---------- 21 ss------ 226 APPENDIXS..... Cell Proliferation Report... A-- 227 be -------------------- 5 000145 ABSTRACT CovanMceT663299-52262 `The purpose of this study was to provide data for determining an estimated `maximum-tolerated dose and lower dose levels to be used in a chronic toxicity study and 10 assess the effectofthe test material, Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295), on critical enzyme levels, hormones, and other selected biochemical parameters Male and female cynomolgus monkeys were assigned to three groups (two animals/sex in Group 1, three animals/sex in Group 2, and one animalsex in Group 3). Animals in Groups 2 and 3 received gelatin capsules containing 0.02 and 2.0 PFOS/kgof body weight/day (mg/kg/day), respectively, triturated with lactose (1:999 and 1:9, ww, respectively). Animals in Group 1 received gelatin capsules containing lactose only. The total material dose level for each group was 20.0 mg/kg/day. Food was provided once or twice daily. Water was provided ad libitum. The animals `were observed twice daily (a.m. and p.m.) for mortality and moribundity. At least once daily, animals were examined for abnormalities and signsoftoxicity, and food consumption was assessed qualitatively. Inaddition, animals were observed approximately 30, 60, and 90 minutes postdose for signsofpoor health or abnormal behavior. Body weight data were collected twice weekly beginning 2 weeks before initiationoftreatment, on Day -1, on the fist dayoftreatment, and twice weekly thereafter. Rectal body temperatures were recorded twice weekly beginning 2 weeks before initiationoftreatment. Blood samples for hormone analyses (estradiol, estrone, estriol, thyroid stimulating hormone, triiodothyronine (T3), and thyroxin (T4)] were collected before initiation of treatment and predose on Day 29. Serum was harvested, and the samples were sent to Ani Lytics Inc, for analyses. Blood samples for serum PFOS concentration analyses were collected before initiationoftreatment and predose on Days 2,3 (approximately 24 hoursaftrthe first and second dose, respectively), 7, 14, and 29. Serum was harvested, and samples were sent to the Sponsor for analyses. Blood samples were collected for hematology and clinical chemistry tests before initiation of treatment and on Day 29. In addition, blood for clinical chemistry tests was collected predose on Days 2, 7, and 14. Additional blood was collected at the time of exsanguination, and samples were shipped to the Sponsor for possible future analysis. On Day 30, the animals were anesthetized, weighed, exsanguinated, and necropsied. At necropsy, macroscopic observations were recorded, and selected tissues were collected 10 000146 CovanMceTE632290.5262 and preserved. In addition, a sample of liver was collected from each animal for palmitoyl CoA oxidase activity analyses. Representative samplesofliver. testes, and pancreas were collected from each animal and sent to Pathology Associates International for proliferation cell nuclear antigen evaluation. A sample of liver was collected from ach animal and setontthe Sponsor for PFOS concentration analyses. Microscopic examinations were done on the adrenals, eye, femoral bone marrow, lung, liver. kidney, `pancreas, spleen, testes, and thymus from each animal. All animals survived to the scheduled sacrifice. There were no test material-related effects on clinical observations, body weights, food consumption, rectal body temperatures, clinical pathology, or macroscopic or microscopic findings. There were no apparent test material-related effects on estrone, estriol, thyroid stimulating hormone, or thyroxin. There were no apparent test material-related effects on estradiol or triiodothyronine at 0.02 mg/kg/day. Given the low numberofanimals in this study, itis not possible to determine conclusively whether there was atest material-related effect on estradiol or triiodothyronine. Estradiol levels for the male and female given 2.0 mg/kg/day were numerically lower than thoseofthe control animals on Day 29 and their respective prestudy values. Triiodothyronine levels for the male and female given 2.0 mg/kg/day were numerically lower than thoseofthe control animals on Day 29; however, their prestudy values were also considerably lower than the control values. Triiodothyronine levels for animals given 0 or 0.02 mg/kg/day were numerically higher on Day 29 compared with their prestudy levels while triiodothyronine levels for `animals given 2.0 mg/kg/day were numerically lower compared with their prestudy levels. Cell proliferation, as determined by measuring the proliferating index, was not increased in the liver, testes, or pancreasofmonkeys. In cynomolgus monkeys treated with 0, 0.02, or 2.0 mg PFOS/kg/day orally for at least 4 weeks, there were no apparent test material-relatedeffectson clinical observations, `body weights, food consumption, rectal body temperatures, or clinical or anatomical pathology findings. The one male and one female treated with 2.0 mg PFOS/kg/day appeared to have lower levelsofestradiol on Day 29. 000147 n CovanScMeT6-36292-925262 PURPOSE `The purposeofthis study was to provide data for determining an estimated `maximum-tolerated dose and lower dose levels to be used in a chronic toxicity study and 0 assess the effect of the test material, Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295), on critical enzyme levels, hormones, and other selected biochemical parameters. REGULATORY COMPLIANCE All aspectsofthis study were done in accordance with the Environmental Protection Agency Good Laboratory Practice Standards, 40 CFR 792. TEST MATERIAL, VEHICLE, AND SOLVENT Test Material `The test material, Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295), Lot No. 217, is awhite to off-whitepowderand is 86.9% pure. It was received at Covance on September 4, 1997. The test material was stored at room temperature. Information on synthesis methods, stability, purity, composition, or other characteristics that define the test material is on file with the Sponsor. The CertificateofAnalysis is in Appendix 7. Vehicle `The vehicle was lactose (Spectrum, New Brunswick, New Jersey), Lot No. NNO192 (expiration date February 13, 1999). It was received at Covance on March 30, 1998. The vehicle was stored at room temperature. Information on synthesis methods, purity, composition, or other characteristics that define the vehicle is on file with the manufacturer. 000148 2 CovanMceT632295-2622 Solvent "The solvent was acetone, USP (Spectrum, Gardena, California), Lot No. LH0253 (expiration date June 2000), and is 99.8% pure. It was received at Covance on June 23, 1997. The vehicle was stored at room temperature. Information on synthesis methods, purity, composition, or other characteristics that define the solvent is on file with the manufacturer. Reserve (Archive) Samples Areserve sample (10 g each)ofeach lotofthe test material, vehicle, and each test `materiallactose trituration was taken before initiation of treatment and stored at room temperature. These samples were transferred to the Sponsor after completionofthe in-life phase. Disposition Remaining test material will be retained at Covance for use in possible future studies. TEST SYSTEM Test Animal Young adult to adult cynomolgus monkeys were obtained from Covance Research Products Inc. (Alice, Texas),on March 10, 1998. The animals weighed 2.0 to 2.5 kg at initiation of treatment. Identification Each animal was assigned a permanent number upon arrival and identified with collar tag before initiationoftreatment. All data for an animal are recorded under this number. Acclimation Sevenmalesand seven females were received on March 10, 1998, transferred from the `Covance in-house stock colony on March 16, 1998, and aclimated in Animal Room 2015 for 38 days before initiationoftreatment. In general, animals in this 1B 000149 CovanScMe T6E32209.5262 shipment appeared healthy. During acclimation, the animals were examined for abnormalities indicative of health problems. In addition, three tuberculosis tests. a physical examination, and a fecal flotation for parasites were done on cach animal. `The physical examination done after the animals were received revealed no major abnormalities. Results of the tuberculosis tests were negative for all animals: the results are recorded in the data. The results of the fecal flotation tests indicated the animals were negative for parasite ova. One male and one female were eliminated from study consideration based on clinical pathology findings or abnormal fecal observations during acclimation. There were six males and six females in the randomization for assignment to groups (two animals/sex in Group 1, three animals/sex in Group 2, and one animal/sex in Group 3). Animals not selected for the study were transferred to the Covance stock colony. Housing and Maintenance Animal Room 2015 was used for this study. Environmental controls for the animal room were set to maintain 18 to 29C,a relative humidityof 30 to 70%, and a 12-hour light/12-hour dark cycle. Variations from these conditions are documented in the data and are considered to have hadnoeffect on the outcomeofthe study. "The animals were housed individually in stainless steel cages. Certified primate diet (#8726C, Harlan Teklad) was provided once or twice daily. The lot numbers are recorded in the data. The diet is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Resultsofspecified nutrient and contaminant analyses are on file with Covance-Madison. Fruits or additional supplements were provided, but did not require analysis `Water was provided ad libitum. Samplesofthe water are analyzed for specified `microorganisms and environmental contaminants. The results are on file with Covance-Madison. `There were no known contaminants in the diet or water at levels that would have interfered with this study. 14 000150 CovanMce 6T36299-52262 Justification PFOS is a known hepatic peroxisome proliferator (PP) in the rat. When exposed to PP, `nonhuman primates (such as the cynomolgus monkey) respond similarly to humans (i.e, Tow 10 no hepatic response) and therefore are an appropriate human surrogate species. PROCEDURES "This study was conducted in accordance with the Protocol dated April 17, 1998, and Protocol Amendment Nos. 1 through 4. The protocol, protocol amendments, and protocol deviations are in Appendix 1 Group Designations and Dose Levels Selectionofanimals for the study was based on clinical observations, clinical pathology, and other data as appropriate. Animals were assigned to treatment groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment group. Dose Level Total Material Dose _ Numberof Animals Gr1 o(uCpon.trol) (mg/0kg/day) Level (mg/kg/day) Males Females 200 2 2 2 (Low dose) 0.02 3 (High dose) 20 220000" 31 31 a The control group (Group 1) received an equivalent amount of lactose in gelatin capsules as the total material administered to Group 3. b The low-dose group received the test material triturated with lactose (1:999, ww). The high-dose group received the test material triturated with lactose (1:9, ww). Dose Preparation Gelatin capsules (Torpac, Inc., Fairfield, New Jersey), Size No. 2, Lot No. 122630 (expiration date June 26, 2002), were used for dose administration. The test material riturated with lactose and lactose only (control group) were dispensed into capsules twice `weekly and stored at room temperature. s 000151 Covance 6329222 Tass Vehicle. Lactosewasused as the vehicle in the dose preparations. Dose levels were based on the vehicle as supplied for Group 1. For Group 1 dose preparations, the specified amount of lactose was weighed and transferred into the gelatin capsule. The top and bottom halves of cach capsule were joined, and the capsule was placed into the appropriate container labeled with study number, group number, animal number, and dose level. `Test Material. For Group 2 and 3 dose preparations, the test material was dissolved in acetone and triturated with lactose (1:99 and 1:9, wiw, respectively) once before initiationoftreatment. Acetone (Spectrum, Gardena, California), Lot No. LH0253 (expiration date June 2000) was used to dilute the test material with lactose. Trituration with lactose was necessary to facilitate capsule preparation. For Group 3 dose preparations, the specified amount of test material was weighed, placed into a labeled mixing container, and the appropriate volumeofacetone was added to the container. The preparation was stirred manually until the test material was dissolved. `The specified amount of lactose was weighed, placed into the mixing container, and thoroughly mixed using a spatula. This preparation was exposed to air until the acetone. was evaporated and stirred periodically. Samplesofthe finished preparation fordose analyses were taken directly from the container. The triturated test material was transferred to container and stored at room temperature and protected from light and `moisture. For Group 2 preparations, the required amountofthe Group3 preparation was measured andplaced into a labeled mixing container. The specified amountoflactose was `weighed, placed into the mixing container, and thoroughly mixed using a spatula for `approximately5 minutes. Samplesofthe finished preparation for dose analyses were taken directly from the container. The triturated test material was transferred to a container and stored at room temperature and protected from light and moisture. `The specified amountoftriturated test material was weighed and transferred into the gelatin capsule. The top and bottom halvesof each capsule werejoined, and the capsule was placed into the appropriate container labeled with study number, group number, animal number, and dose level. 16 000152 CovanMceT6632299-52262 Dose Analyses Samples (1g each) were collected from the top, middle, and bottom of the test `materiallactose preparations for the low- and high-dose groups and stored at room temperature. The samples were shipped under ambient conditions to the Sponsor for analysis (see Protocol Deviations for exceptions). Homogeneity samples collected from the middleof the preparations were used for the prestudy stability analysis. One set of samples (1 g each) were taken from the low- and high-dose test material/lactose preparations at the end of the in-ife phase and shipped to the Sponsor for analysis (see Protocol Deviations for exceptions). Resultsofthe homogeneity, stability, and dose confirmation analyses provided by the `Sponsor are in Appendix 7. Method of Administration Gelatin capsules were used for test material preparation administration to compare with data from previous toxicology studies using the oral route, which is the most likely route of exposure. `The dose preparations were administered orally in gelatin capsules once daily (7 days/week) for 29 days (see Protocol Deviations for exceptions). The dosing tubes were flushed with 5mLofreverse osmosis water. Individual doses were calculated based `on the most recently recorded body weights, with the exceptionofbody weight collection days when the previous body weight was used. Animals were dosed at approximately the same time each day. Clinical Observations `The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. Each animal was observed daily, and food consumption was assessed qualitatively; abnormal findings were recorded. Animals were observed approximately 30, 60, and 90 minutes postdose for signsofpoor health or abnormal behavior. Effects were recorded as they were observed. Animals were observed once weekly; abnormal findings or an indicationof normal was recorded. 1" 000153 CovanMceT6632299-52262 Body Weights Individual body weight data were recorded twice weekly beginning 2 weeks before initiationoftreatment (Monday and Thursday), on the fist day of treatment, and twice weekly thereafter. An additional body weight was recorded on Day -1 for the Day 1 dose calculations. Rectal Body Temperatures Rectal body temperatures were taken at approximately 11:00 twiceweeklybeginning 2 weeks before initiationoftreatment [Monday and Thursday (sce Protocol Deviations for exceptions). Animals were not anesthetized for the procedure. Blood Hormone Determination Approximately 5 mLofwhole blood were collected from a femoral vein of each animal before initiationoftreatment (Day -7) and predose on Day 29. Blood was collected between 07:00 and 09:00. Animals were fasted overnight before collections. All samples were collected without anticoagulant, maintained at room temperature, and allowed to clot. Samples were centrifuged within 1 hour after collection, and serum samples were harvested. Serum was divided into two approximately equal aliquots (blood collected on Day 29 only) and stored in a freezer set to maintain -60 to -80C until packed ondry ice and shipped to Ani Lytics Inc., for analyses. The samples were analyzed for estradiol, estrone, estriol, thyroid stimulating hormone, triiodothyronine (T3), and thyroxin (T4) Resultsofthese analyses were provided by Ani Lytics Inc., and are in Appendix 6. Serum PFOS Level Determination Approximately5 (Day -7 only) or2 mLofwhole blood were collected from a femoral veinofcach animal before initiationoftreatment (Day -7) and predose on Days 2,3 (approximately 24 hours after the first and second dose, respectively), 7, 14, and 29. Animals were fasted ovemight before collections. All samples were collected without anticoagulant, maintainedatroom temperature, and allowed to clot. Samples were centrifuged within | hour after collection, and serum was harvested and stored in a freezer set to maintain -60to -80C until packed ondry ice andshipped to the Sponsor for analyses. The samples were analyzed for POS. Resultsofthese analyses will be: reported separately by the Sponsor. " 000154 CovanScMe T6E32290-25262 Clinical Pathology Blood samples were collected from cach animal on Days -7 and 29 for hematology and clinical chemistry tests. In addition, blood for clinical chemistry tests was collected from each animal predose on Days 2, 7, and 14. Animals were fasted ovemight: water was providedad libitum. Blood was collected from the femoral vein. The following were evaluated: Hematology red blood cell (erythrocyte) count hemoglobin differential blood cell count segmented neutrophil count hematocrit `mean corpuscular volume lymphocyte count monocyte count `mean corpuscular hemoglobin eosinophil count `mean corpuscular hemoglobin concentration basophil count platelet count white blood cell (leukocyte) count blood cell morphology reticulocyte count glucose: urea nitrogen creatinine total protein albumin globulin total bilirubin cholesterol triglycerides aspartate aminotransferase alanine aminotransferase: alkaline phosphatase Clinical Chemistry `gamma glutamyl transferase: sorbitol dehydrogenase creatine kinase: calcium inorganic phosphorus sodium potassium chloride sameyrluamsbeile acids lipase pancreatic-specific amylase: Additional Blood Collection `Whole blood (approximately 20 mL) was collected from the vena cava of each animal at the timeofexsanguination (See Protocol Deviations for exceptions). The anticoagulant waspotassium EDTA. Samples were transferred into containers (approximately 5 mL. ach) and stored ina freezersetto maintain -60 to -80C until packed on dry ice and. shipped to the Sponsor possible future analysis. 000155 19 CovanMceT6E3299-52262 Necropsy On Day 30, animals that were fasted overnight were anesthetized with ketamine and xylazine, weighed, bled for required tests, exsanguinated, and necropsied. Animals were necropsied in random order. `The necropsy included a macroscopic examination of the external surfaceof the body; all orifices; the cranial cavity; the brain and spinal cord; the nasal cavity and paranasal sinuses; cervical tissues and organs; and the thoracic, abdominal, and pelvic cavities and viscera. Palmitoyl CoA Oxidase Determinations A sampleofthe right lateral lobe of iver was collected from each animal at scheduled sacrifice. The sample was flash-frozen in liquid nitrogen and stored in a freezer set to maintain -60 to -80C until analyzed for palmitoy] CoA oxidase activity. Cell Proliferation Evaluation Representative samplesofthe liver, testes, and pancreas were collected and preserved in zinc formalin. After fixation, samples for proliferation cell nucear antigen (PCNA) evaluation were embedded in paraffin and shipped to Pathology Associates International for PCNA evaluation. PCNA evaluation (including examination of slides stained with hematoxylin and eosin) was done on the samples. Resultsofthe evaluation provided by Pathology Associates International are in Appendix 8. Liver PFOS Determination A sampleofliverwas collected from each animal at sacrifice, weighed, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C untilpackedon dry ice and shipped with plasma samples to the Sponsor for PFOS analysis. Resultsofthese analyses will be reported separately by the Sponsor. 000156 20 CovanIcMe 6T36292-52262 Tissue Preservation The following tissues (when present) or representative samples were collected and preserved in 10% neutral-buffered formalin, unless otherwise specified,for possible: future microscopic examination: adrenal (2) aorta brain cecum cervix colon duodenum epididymis (2) esophagus eyfeisxa[tpirvees(e2r)v]ed in Davidson's femur with bone marrow (articular surfaceofthe distal end) gallbladder heart ileum jejunum Kidney 2) liver lung lymph node (mesenteric) `mammary gland muscle (thigh) opavnacrrye(a2s) pituitary prostate rectum salivary gland sciatic nerve [mandibular (2)) seminal skin vesicle (2) spinal cord (cervical, thoracic, and lumbar) spleen sternum with bone marrow stomach testis (2) thymus thyroid (2) with parathyroid trachea urinary bladder uvtaegriunsa Histopathology `The adrenals, eye, femoral bone marrow, lung, liver, kidney, pancreas, spleen, testes, and thymus were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and `examined microscopically from each animal (see Protocol Deviations for exceptions). Bone marrow smears from the sternumofeach animal at the scheduled sacrifice were prepared, stained with Wright's stain, and retained for possible examination. 21 000157 CovanMceT6632299-52262 Statistical Analyses Only data collected on or afte the first dayoftreatment were analyzed statistically. Statistical analyses were not done on data collected for Group 3 due to the small number ofanimals. One-way analysisofvariance [ANOVA (Winer, 19712)) was used to analyze initial body weights, rectal body temperature, palmitoyl CoA oxidase activities, and continuous clinical pathology values Levene's test (Levene, 1960) was done to test for variance homogeneity. In the case of heterogeneityof variance atp < 0.05, transformations were used to stabilize the variance. ANOVA was done on the homogeneous or transformed data.Ifthe ANOVA was significant, Dunnett's t-test (Dunnett, 1964) was used for control versus treated group comparisons. `One-way analysisof covariance [ANCOVA (Winer, 1971b)] was used to analyze body weights, with initial body weights as the covariate. Although Levene's test for variance `homogeneity was done (see above), no transformations were used because covariance adjustment removed extraneous heterogeneity. Ifthe ANCOVA was significant, least squares means t-test was used for control versus treated group comparisons. For each sex, Group 2 was compared with Group 1 (Control). Group comparisons were evaluated at the 5.0% two-tailed probability level. RECORD RETENTION All raw data, documentation, records, protocol, and specimens generated as a result of this study will be archived in the storage facilitiesof Covance-Madison for a period of 1 year. One year after the submissionofthe final report, the Sponsor will determine the final dispositionofthe materials. All raw data stored on magnetic media and an original copyofthe final report will be retained by Covance-Madison. 000158 -_ CovawnMcTeTa66322m99-s52262 Within 1 year after submission of the final report, all of the aforementioned materials (as appropriate) from the Sponsor's designees (3M E.T. & S and Ani Lytics Inc.) will be sent to the Sponsor (Andrew Seacat, PhD, 3M). Pathology Associates Intemational (PAI) i data or specimens produced by PAL responsible for the maintenanceof any raw RESULTS Dose Analyses Results of analyses provided by the Sponsor are in Appendix 7. Dose analysis results indicate that the average + standard deviation for the 2.0 mg/kg/day dose level prepared at a 1:9 dilution was 80 + 02%oftarget. The average + standard deviation for the 0.02 mg/kg/day dose level prepared at a 1:99 dilution was 54% + 02% oftarget. The stability samples obtained from the middleofeach mixture on May 22, 1998, were 79% and 71%oftarget for the 2.0 and 0.02 mg/kg/day preparations, respectively. Inherent variability and lackofsufficient sensitivity in the analytical `method for measuring (PFO; T-6295) resulted in stability and routine analysis data that were outside ofthe generally recognized as acceptable standard limits o+f 15% Clinical Observations Clinical observations are summarized in Tables 1 through 5; individualdataare in Appendix 2. Al animals survived to the scheduled sacrifice. There were no testmaterial related clinical observations. Body Weights Body weight data are summarized in Table 6; individual data are in Appendix 3. There were no apparent test material-related effects on body weights. 2 000159 CovanIcMe 6T36292-52262 Food Consumption Food consumption data are summarized in Table | (Summary of Clinical Observations - A M./Weekly); individual data are included in the individual clinical observations in Appendix 2. `There were no test material-related effects on food consumption. Rectal Body Temperatures Rectal body temperature data are summarized in Tables 7; individual data are in Appendix 2. There were no test material-related effects on rectal body temperatures. Blood Hormone Analyses Summary and individual hormone analyses data are in Appendix 6. Estradiol and triiodothyronine analyses data are illustrated in Figures 1 through 4 (Appendix 6). `There were no apparent test material-related effects on estrone, estriol, thyroid stimulating hormone, or thyroxin. There were no apparent test material-elated effects on estradiol or triiodothyronine at 0.02 mg/kg/day. Given the low numberof animals in this study, itis not possible to determine conclusively whether there was a test material-related effect on estradiol or riiodothyronine. Estradiol levels for the male and female given 2.0 mg/kg/day were numerically lower than thoseofthe control animals on Day 29 and their respective prestudy values. Triiodothyronine levels for the male and female given 2.0 mg/kg/day were numerically lower than thoseofthe control animals on Day 29; however, their prestudy values were also considerably lower than the control values. Triiodothyronine levels for animals given 0 or 0.02 mg/kg/day were numerically higher on Day 29 compared with their prestudy levels while triiodothyronine levels for animals given 2.0 mg/kg/day were numerically lower compared with their prestudy levels. 000160 2 CovanMceT663290-52262 Clinical Pathology Hematology and clinical chemistry data are summarized in Tables 8 through 15; individualdataare in Appendix 4. The Pathology Report contains a discussionof the data. Administration of PFOS had no effects on clinical pathology test results. Cel Proliferation Evaluation Resultsof cell proliferation evaluation provided by Pathology Associates Intemational are: in Appendix 8. Cell proliferation, as determined by measuring the proliferating index, was not increased in the liver, testes, or pancreasofmonkeys. Anatomical Pathology Incidences ofmacroscopic and microscopic observations are summarized in Tables 16 and 17. Individual data are in Appendix 5. The Pathology Report contains a discussion ofthe data. `There were no test material-related macroscopic or microscopic changes in the organs and tissues examined. CONCLUSIONS In cynomolgus monkeys treated with 0, 0.02, or 2.0 mg PFOS/kg/day orally for a least 4 weeks, there were no apparent test material-related effects on clinical observations, `body weights, food consumption, rectal body temperatures, of clinical or anatomical `pathology findings. The one male and one female treated with 2.0 mg PFOS/kg/day appeared to have lower levelsofestradiol on Day 29. 000161 2 SIGNATURES Counce 3T2e92m2e2 oma R.a. Patricia K. McKee Pesik, BS, LAT CSotvuadngyeCooLradbionraattoorries Inc 0a Apr|zoz Date L Ls i A Study Direcfor Covance Laboratories Inc. Date 4 000162 26 Covance 6329-222 -_-- MTs REFERENCES Dunnett, C. W., "New Tables for Multiple Comparisons with a Control," Biometrics, 20:482-491 (1964). Levene, H., "Robust Tests for Equality of Variances," ContritobPurobtabiiliotynansd `Statistics, (eds.) L. Olkin et al, Ch. 25, pp. 278-292, Stanford University Press: Stanford, California (1960). `Winer, B. J, "Design and Analysis of Single-Factor Experiments," Statistical Principles in Experimental Design, Second Ed., Ch. 3, pp. 149-260, McGraw-Hill: New York, New York (19712). Winer, B. ., "AnalysisofCovariance," Statistical Principles in Experimental Design, Second Ed, Ch. 10,pp. 752-812, McGraw-Hill: New York, New York (1971b). 000163 27 PATHOLOGY REPORT Covance 6320-222 Teme SUMMARY `The purposeofthis study was to provide data for determining an estimated `maximum-tolerated dose and lower dose levels to be used in a chronic toxicity study and 10 assess the effectof the test material, Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295), on critical enzyme levels, hormones, and other selected biochemical parameters Administration ofPFOS had no effects on clinical pathology test results and caused no test material-related macroscopic or microscopic changes in the organs and tissues examined METHODS `Three groups ofmale and female cynomolgus monkeys were administered the test `material orally via capsule ata dose level of0 (control group; received the vehicle, lactose), 0.02, or 2.0 mg/kgofbody weightday (mg/kg/day) for 29 days. The control `group had two animals/sex, the low-dose group had three animals/sex, and the high-dose `group had one animallsex Blood was collected for hematology and clinical chemistry tests before initiation of treatment (Day -7) and on Day 29. In addition, blood for clinical chemistry tests was collected on Days 2,7, and 14.Theanimals were sacrificed and necropsied on Day 30; `macroscopic observations were recorded, and tissues were preserved in fixative as specified by the protocol. Samplesof liver were collected and frozen for determination of hepatic palmitoyl CoA oxidase activity and for determinationof PFOS content (by the Sponsor). Samplesofliver, testes,andpancreas were collected and preserved in zinc formalin for evaluationofproliferation cell nuclear antigen (PCNA; by Pathology Associates International). Microscopic examinations were done on adrenals, eye, femoral `bone marrow, lung, liver, kidney, pancreas, spleen, testes, and thymus from all animals. Resultsofclinical pathology tests for the control and low-dose groups were compared statistically. = 000164 -_ RESULTS AND DISCUSSION Mortality All animals survived to the scheduled sacrifice. CovanMcTeT6a3229s5-262s2 Clinical Pathology Individual values for hematology and clinical chemistry tests are in Appendix 4. Mean values and the resultsofstatistical comparisons are in Tables through 15. Day -7. Clinical pathology test results indicated no obvious group or individual health abnormalities. Days 2,7, and 14. Administration of te test material had no effects on clinical chemistry test results a these collection intervals. Statistically significant differences for clinical chemistry results between the control and low-dose groups were inconsistent over time and between the sexes. Several of the differences were similar to differences present at Day -7, and none ofthe differences exhibited a strong dose relationship (i.c., the high-dose animals did not exhibit notable differences for the same parameters). Day 29. Administrationofthe test material had no effects on hematologyor clinical chemistry test results at Day 29. Statistically significant differences for red blood cell count, neutrophil count, monocyte `count, and hepatic palmitoy] CoA oxidase were inconsistent between the sexes and exhibitneod dose relationship. Statistically significant differences for triglycerides and alkaline phosphatase were similar to differences observed at Day -7. Anatomical Pathology Anatomical pathology findings for each animal are in Appendix 5. Incidence summaries ofthe macroscopic and microscopic observations are in Tables 16 and 17. 29 000165 [--T--n-- Wseneeks.exaTmhienreed,wearned ntoermeawcerroescompeaijnocdrfiwfcmeinccroesscboopiwcefinndcinogstitahnedoergaarnesdsnadils inthe inciodf neynncdineg,Allmroscopl observationswereconsidered incidental and unrelated to the test material. A Rebet r I Fah, VEL271DAY Diplomate, ACVP Nore Cl) (Clinical Pathology) WDDNairdo: neGommA e MNAN) Decor of Pathlogs NoshAmeria H a OE 04hon Deed P 55 fa p evp n 000166 30 COMMENTS ON THE DATA CovanMce1636295-2622 Various modelsofcalculators, computers, and computer programs were used to analyze data in this study. Because different models roundoffor truncate numbers differently. values in some tables (.g., means, standard deviations, or individual values) may differ slightly from those in other tables, from individually calculated data, or from statistical analysis data. Neither the integrity nor the interpretationof the data was affected by these differences. Some tabular data were compiled using Excel Version 7.0 software, `The summary tables for clinical observations indicate the number of animals for which a condition was observed without regard to the specific nature, severity, reversibility, `number of incidences/animal, or the lengthoftime the condition persisted. Only observations other than normal are indicated on the summary clinical observations. tables. The dayofinitiationoftreatment is "Day 1." 31 000167 -_-- CovanMcTeT66a32299s-522s62 CODES, ABBREVIATIONS, AND UNITS General Codes and Abbreviations Codes for Clinical Pathology Abbreviations and Units for Clinical Hematology Abbreviations and Units for Clinical Chemistry Codes for Anatomical Pathology Note: The following lists ofcodes, abbreviations, and units are used by Covance. Some, but not necessarily all, ofthis information may be needed for this report. 000168 2 WK N Mean; MEAN CAM SD; S.D; STAND DEV; . STANDARD DEV; sd LNA P UNSCHED DISPATCH MIN 0BS CovanIcMe 6T36292.52262 General Codes and Abbreviations Week. Number of measurements in a group. Arithmetic mean, Covariate-adjusted mean Standard deviation. Group 2 mean is significantly different from the mean of the control group (Group 1)atp < 0.05. PNroesveanltue; not applicable; not present Unscheduled. `Observations transferred from the in-life `module of the data collection system to the necropsy module for reference during necropsy. Observations are duplicates of the last in-ife observations. Minute. Observation. Animal Death Codes: T Terminal sacrifice. 000163 3 Ns QS/QNS NR FS sc SH H SL L st 1 NF u DTDOT DB be] TE RE EE SE PC PD Pl PL PA co HB PLASMO NO AGG FR uD NO COAG CovanIcMe 6T36292-52262 Codes for Clinical Pathology GENERAL CODES No sample Quantity not sufficient FNiobrrienpesattra(nsdasmple volume not sufficient for repeat analysis) Sample clotted Slightly hemolyzed Hemolyzed Slightly lipemic Lipemic Slightly icteric Teteric Animal not fasted UAnnsicmhaeldduileedd/omnotreistbund bleed Died during bleeding Technician judgment to repeat test Technical error (instrument or technician error that results in unacceptable spilled, entry data, e.g., of invalid unacceptable data) instrument output, sample sRpeeclolridnignegrreorr,roirn(crorerceocrtdeddatien)correct data, e.g., wrong number, SEnatmrpyliernrgorer(rionrcorrect keyboard entry) Platelets clumped PPllaatteelleettss dineccrreeaasseedd Platelets large CPloaltoerleitnstearpfpeeraesr awditehqutaetste Heinz bodies observed Plasmodium No aggregation Fractious Unable to determine No coagulation 3 000170 -_ CovanMcTeT66e32209m-522e62 Codes for Clinical Pathology (Continued) RESULTS NOT INCLUDED IN STATISTICAL ANALYSES Hemolyzed clinical chemistry or coagulation samples `Samples from animals at unscheduled intervals Prothrombin times (PT)greaterthan 50 seconds Activated partial thromboplastin Bleed times (BLETIME) greater times (PTT) greater than 30 minutes than 10 seconds CODES FOR BLOOD CELL MORPHOLOGY `pTohiekifloolclyotowsiinsg (sPcaOlIeK)w,aspoulsyecdhrtoommaesaisau(rPeOtLhYe)d,eghryepeoocfhraonimsaosciyato(sHisYP(OA)N,ISaOn)d,toxic neutrophils (TOXNEUT): Scale Degree = Normal for the species 1 Slight 2 Moderate 3 Marked 4 Not applicable Presence Not present Rare Few Moderate Many 000171 35 -_-- CovanScMeTT663e2200-s2526s2 Abbreviations and Units for Clinical Hematology Test Red blood cell count Hemoglobin Hematocrit Mean corpuscular volume Mean corpuscular hemoglobin PMleataenlectocropuunstcular hemoglobin concentration Mean platelet volume Reticulocyte count HAbesionlzubtoedryetciocuunlotcyte count Erythrocyte sedimentation rate Prothrombin time Activated partial thromboplastin time `Thrombin time Activated coagulation time Fibrinogen Fibrin/ibrinogen degradation products Platelet aggregation Collagen Adenosine diphosphate. BAllepehdain2g-atnitmieplasmin Methemoglobin MPylealsomiad/heermyotghlrooibdinratio Estimated myeloid/erythroid ratio D`iWfhfietreenbtlioaoldbcleololdcoceulnltcount Nucleated red blood cell count Corrected white blood cell count Segmented neutrophil count Bandneutrophil count Lymphocyte count Monocyte count Eosinophil count AnBiassoopchyitlosciosunt Polychromasia Abbreviation (Units) RBC (E6/UL or X10%uL) HGB (G/DL) HCT (%) MCV (FL) MCH (PG) MCHC (%) PLT (E3/UL or X10%4L) MPV (FL) RETIC (%) RETIC HEINZ (E3/UL (%) or X10%uL) ESR (MM/HR) PT (SEC) PTT (SEC) TT (SEC) ACT (SEC) FBR (MG/DL) FDP (UG/ML) PAGGICOL (%) PAGG/ADP (%) ANTIPLAS (%) BLE TIME (SEC) METHGB (%) PLA HGB (MG/DL) ME RATIO EST MEE RATIO WBC (E3/UL or X10/uL) NRBC (7100 WBC) COR WBC (E3/UL or X10/uL) N-SEG (E3/UL or X10%uL) and % N-BAND (E3/UL or X10*/uL) and % LYMPH (E3/UL or X10%/uL) and % MONO (E3/UL or X10%4L) and % BEOASSION((EE33//UULLoorrXX1100%%uuLL))aanndd%% ANISO (-1,23) POLY (1.2.3) 36 000172 -_-- CovanMcTeT6ee32w99-5s2262 Abbreviations and Units for Clinical Hematology (Continued) Test Poikilocytosis Abbreviation (Units) POKK (1,23) Hypochromasia Howell-Jolly bodies HYPO (123) HIBODY (12,34) Basophilic tipping Toxic neutrophils BASTIP (-12,3) TOXNEUT (123.4) Atypical lymphocytes Aqueous white blood cell count (right eye) ~~ ATYPLYM (1.234) R EYE (WBC/UL) Aqueous white blood cell count (left eye) L EYE (WBC/UL) 000173 37 [-- resis Abbreviations and Units for Clinical Chemistry Test Glucose Urea nitrogen Urea Creatinine Total protein Albumin Globulin Albumin/globulin ratio. Total bilirubin Direct bilirubin Indirect bilirubin Cholesterol Triglyceride Urea nitrogen/creatinine ratio Total lipids Phospholipids High-density lipoprotein cholesterol Low-density lipoprotein cholesterol Uric acid Aspartate aminotransferase Alanine aminotransferase Alkaline phosphatase Gamma glutamyl transferase Sorbitol dehydrogenase Lactate dehydrogenase Creatine kinase Amylase Lipase Palmitoyl CoA oxidase Calcium Ionized calcium Inorganic phosphorus Sodium Potassium Chloride Magnesium Zinc Strontium Tron Abbreviation (Units) GLU (MG/DL) UN (MG/DL) "UREA (MG/DL) CREAT (MG/DL) TPRO (G/DL) ALB (G/DL) GLOB (G/DL) A/G RATIO T BILI (MG/DL) D BILI (MG/DL) 1BILI (MG/DL) `CHOL (MG/DL) TRIG (MG/DL) 'UN/CREAT (RATIO) T LIPIDS (MG/DL) P LIPIDS (MG/DL) HDL (MG/DL) LDL (MG/DL) UA (MG/DL) AST/SGOT (IU/L) ALT/SGPT (IU/L) ALK PHOS (IU/L) GGT (IUL) SDH (IU/L) LDH (IU/L) CK (UL) AMYLASE (TUL) LIPASE (TUL) PCOAO (IU/G) CA (MG/DL) ION CA (MG/DL) 1PHOS (MG/DL) NA (MMOL/L) K (MMOL/L) CL (MMOL/L) MG (MEQ/L or MG/DL) ZN (MG/L or PPM) SR (MG/L or PPM) FE (UG/DL) 000174 Coepaiirn Abbreviations and Units for Clinical Chemistry (Continued) Test Excess iron Total iron binding capacity Unbound iron binding capacity Percent iron saturation Plasma cholinesterase Red blood cell cholinesterase Brain cholinesterase Caudate putamen Hippocampus Frontal cortex Cerebellum Bicarbonate Serum hemoglobin Serumbile acids Fecal bile acids Average fecal weight Fecal bile acids (calculation) Osmolality Electrophoresis Albumin Alpha-1-globulin Alpha-2-globulin Beta globulin Gamma globulin High-density lipoprotein Low-density lipoprotein Very-low-density lipoprotein Insulin Adrenocorticotropic hormone Cortisol Glucagon Triiodothyronine Thyroxine `Pancreatic-specific amylase Creatine kinase isoenzymes BB MB MM `Abbreviation (Units) EX FE (UG/DL) TIBC (UG/DL) UIBC (UG/DL) FE %SAT (%) CHEP (MUML) `CHER (MUML) CHEB (MUML) CAUD PUT (UMOL/G) HIPPOCAM (UMOL/G) F CORTEX (UMOL/G) `CEREBELL (UMOL/G) BICARB (MMOL/L) SER HGB (MG/DL) SBA (UMOL/L or MG/DL) FBA (UG/ML) FCC WGT (G) FBA (MG/Day) (OSMO (MOSM/KG) E ALB (G/DL) EA-1 (G/DL) EA-2(G/DL) E BETA (G/DL) E GAMMA (G/DL) E-HDL (%) E-LDL (%) E-VLDL (%) INSULIN (UUML) ACTH (PG/ML) CORTISOL (UG/ML) GLUCAGON (PG/ML) T3 (NG/DL) T4 (UG/DL) P AMYL (UL) CK-BB (UL) CK-MB (UL) CK-MM (UL) 39 000 ($S Covance 6329-222 -_ MTess Codes for Anatomical Pathology MICROSCOPIC CODES Distribution of Findings Focal Diffuse. Multifocal Grades for Severity or Amount 1 Minimal- the least amountofchange that can be observed with the light microscope 2 Slight - less than average amountof change, but readily discernible as abnormal 3 Moderate- the average amountofchange that is expected for a lesion 4 Moderately severe (marked)- a marked amountof change with possible loss of function of the affected cells or organs 5 Severe - a great amountofchange with probable loss of function of the affected cell or organs and frequently involves large areasof the organ 40 000001176 vo ql | FE IEial 0 en eee oe pe Coa i : : Pogo fy, Progitlragrdint: i Elww o EF C22 8 zo $a Pram 000177 i i Hiei eo oo FifA SLI ige coc cc LEED LL F5 UBEL igL ic coc oc i Belfi oo os oy 28 | i i i DIED En Bor i| os .pigiids8as af gsBEsagt i 8 FF (iE FFE: ! 000178 ii gf i C theHiiEo oD.ol . sieve PBhlionm 2i0 53 | oo {ofOOLF REgRaLRiEn gHBfEe ofinnaen 000179 5 i Eg [7 BEE. 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Sonne me we me i ES i284" mn mm ooemoem meee i #00 ST EE me anne eee PPoOnRi HE PiTRE3 ji oE BClE Co i Ph a A [IE Pogo CE #5 P1000 EE PoE Eb i000 id: 8 SE H- Pgh ogi PE By og fF OFopi,ffTH 000228 o googi 8F lignin iris gp Bipacnpn : i [284mm PoE Ta momen mm mmm momen mm mmo ee eee mn eee am was me ne oo non eee = Bri BEEDEd Eos og og 8: EE Tg 3 oi i OO: 8 BE PEON HOE on Pog iEEE monn Ei i: 3 co iE biol Pod TOE Pol 3 3i i i Po Po Powel Pog EH : Id PPRfE E ogiEfoC FfPF PPoEs RsEig fEOFF HBEE EoO. OEoc TREfgd op 5BES Tg 000229 APPENDIX 1 Protocol Deviations Protocol APmroetnodcomlent No. 1 Protocol Amendment No. 2 Protocol Amendment No. 3 Protocol Amendment No. 4 Material Safety Data Sheet Cove 6T1a29s2s2 000230 94 CovanMceT623299-52262 Protocol Deviations Protocol. Group Designations and Dosage Levels. Dose levels were 0, 0.02, and 2.0 mg/kg/day. The control group (Group 1) was to receiveanequivalent amount of lactose in gelatin capsules as the total material administered to Group 3. The total `material was 20.0 mg/kg/day for each group; however, the low- and high-dose groups were to receive test material triturated with lactose (1:99, w:w, and 1:9, wiw, respectively). Actual Procedure. Because the capsule broke during dose administration, the following animals may not have received the full amountofdose preparation: Animal No. 105350 (Group 1 male) on Day 1; Animal No. 105367 (Group 1 female) on Days 2,7, and 14; Animal No. 105344 (Group 2 male) on Day 7; and Animal No. 105369 (Group 3 female) on Day2. Protocol. Dose Analysis. Stability. "One setofsamples (approximately 1 geach) will be taken from the low- and high-dose test materiallactose preparations at the endofthe inlife phase and analyzed for test material content" Actual Procedure. Two reserve samples (10 g) were shipped to the sponsorforstability analysis. The samples for stability analysis (1 g) were retained as reserve samples, until shipped to the Sponsorn March 19, 2002. Protocol. ObservationofAnimals. Rectal Body Temperatures. "Beginning 2 weeks before initiationoftreatment, rectal body temperatures will be taken at approximately 11:00 on two days/week (Monday and Thursday). Actual Procedure. Rectal body temperatures were recorded between 12:50 and 13:30 on Day -14 and between 08:17 and 08:40 on Day -10. Protocol. Serum PFOS Level Determination. Frequency. "Before initiationoftreatment (Day -7 or -6), and prior to treatment on Days 2 (approximately 24 hours after the first dose), 7, 14, and 29." 9 000231 . CovanMceT6.32290-52262 Protocol Deviations (Continued) Actual Procedure. On Day 2, incorrect blood collection tubes were used for the collectionofblood for clinical chemistry test; therefore, blood collected for serum PFOS level determinations was used for clinical chemistry tests. Remaining serum from clinical chemistry tests was usedastheDay 2 sample for serum PFOS level determinations. In addition, whole blood (approximatel2y mL) was collected from a femoral veinofeach animal before treatment on Day 3 (approximately 24 hours afte the second dose). Protocol. Additional Blood Collection. "At scheduled and unscheduled necropsies, blood (as much as possible, up to 20 mL) will be collected from animals at the time of exsanguination." Actual Procedure. Additional blood was not collected from Animal Nos. 105364 (Grou2p female), 105365 (Group 2 female), and 105369 (Group 3 female). Protocol. Postmortem Procedures. Tissue Preservation. "The following tissues (when present) from each animal will be preserved in 10% neutral-buffered formalin, unless otherwise specified, for possible future microscopic examination: Actual Procedure. Femoral bone marrow from Animal Nos. 105349 and 105350 (Group 1 males), 105344 and 105348 (Group 2 males), 105345 (Group 3 male), 105366 (Group 1 female), and 105364 (Group 2 female) were insufficient; therefore, these tissues were not examined microscopically. This tissue is listed with the appropriate comments in the pathology data sheets for each individual animal. Summary tables do not include it as having been examined. `These deviations are not expected to have affected the resultsofthe study. 000232 9% COVANCE> Sponsor: 3M St. Paul, Minnesota PROTOCOL Study Title: Week Capsule Toxiiy Study with Perfuorooetans Sufoic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys Date: April 17, 1998 Performing Laboratory: Coane Labonte os. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Laboratory Study Identification: Proposal No. 90545 Covance 6329-222 000233 97 Connce 6329-222 ht Study 4-Week Capsule Toxicity Study with Perfluoraoctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys Purpose "To provide data for determining an estimated maximum-tolerated dose and lower dose. levels to be used in a chronic toxicity study and to asesstheeffectofthe test material on erica enzyme levels, hormones, and other elected biochemical parameters Sponsor aM Toxicology Services Building 20-26-02, 3M Center St. Paul, Minnesota 55144-1000 Study Monitor, Andrew M. Seacat, PhD Mm Telephone No.: 612.575.3161 Facsimile No.: 612.733.1773 Alternate Study Monitor Paul Lieder, PD, DABT aM Telephone No.: 612.737.2678 Facsimile No.: 612.733.1733 Study Location Covance Laboratories Inc 3301 Kinsman Boulevard Madison. Wisconsin 53704-2595 Mailing Address: PO Box 7545 Madison, Wisconsin 53707-7545 000234 9% Study Director PeteJr. Thomford, PhD Covance Laboratories Inc. `Telephone No.: 608.241.7207 Facsimile No.: 608.242.2736 Covance632P9a-g2e232 Study Toxicologist Dale Aldridge, BS Covance Laboratories Inc. Proposed Study Timetable In-Life Start Date: April 23, 1998 In-Life End Date: May 22, 1998 Audited Draft Report Date: September 3, 1998 Regulatory Compliance `This study willbe conducted in compliance with the Environmental Protection Agency Good Laboratory Practice Regulations as set forth in Title 40 of the US Code of Federal Regulations, Part 792, issued November 29, 1983 (effective December 29, 1983), and `with any applicable amendments 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 opinionof the Sponsor and study director, the study does not unnecessarily duplicate any previous work. Quality Assurance `The protocol. study conduct. and final report will be audited by the Covance Quality Assurance Unit (QAU). The proliferation cell nuclear antigen evaluation data and report will be audited by the QAU of Pathology Associates International. The dose analysis and plasma and liver PFOS analysis and report will be audited by the QAU of 3M Environmental Laboratories. The blood hormone determinations and report will be: audited by the QAUofAniLytics Inc. 000235 9 _-- `Test Material Covance 6r32Pu9a-eg2e2ss2 Identification Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) Lot Number `The lot number will be maintained in the raw data. Parity Responsibility of the Sponsor Stability Responsibility of the Sponsor Storage Conditions At room temperature Characteristics Information on synthesismethods, composition, or other characteristics that define the test material is on file with the Sponsor. Vehicle Identification Lactose Lot Number `The lot number will be maintained in the raw data. Purity On file with the manufacturer Stability On fie with the manufacturer 000236 100 Cove 6329222 --------eh} Storage Conditions At oom temperature Gelatin Capsules Cbaepssuuplpelsie(dSibzye tNhoe. ma2n)uofbatctauirneerd.frGoemlaTtoirnpacca,psIunlce.s(wFialilrfbieesldt,orNeedwatJerrsoeoym);tleomtpenruamtbuerer.wiAll cdoapyofthe CertificateofAnalysis providedby the manufacturer will be maintained in the Reserve (Archive) Samples A reserve sample.ofeach lot of test material, vehicle, and each test materallactose: riuration (10g each) will be taken and sored at room temperature. These samples will be transferred 10 the Sponsor after completion of the in-lfe phase. DispositionofTest Material `Any remaining test material will be retained at Covance for use in possible future studies Animals Species Cynomolgus monkey (purpose-bred) Source Covance Research Products Inc., Alice, Texas Age at InitiationofTreatment Young adultadu Weight at Initiationof Treatment Approximately 2 0 4 kg. Number and Sex Six males and six females Identification Collar tag 000237 101 -_-- Husbandry Covance 6329-222 Paes Housing Individual; animals will be housed in suspended, stainlesssteelcages. Diet Centified primate diet (#8726C, Harlan Teklad) once or twice daily. The diet is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Resultsof specified nutrient and contaminant analyses are on file at Covance-Madison. Fruit, vegetables, or other supplements may be provided but will not require analysis. Water Ad libitum. Samples of the water arc routinely analyzed for specified microorganisms `and environmental contaminants. The results are on file at Covance-Madison. Contaminants `There are no known contaminants in the diet or water at levels that might interfere. with this study. Environment Environmental controls for the animal room willbe set to maintain 18 t0 29C, a relative humidity of 30 to 70%, and a 12-hour light/12-hour dark cycle. Acclimation Minimum of 4 weeks Randomization Animals will be weighed, stratified by body weight, and allocated to the number of blocks equal to the number of animals to be selected for each group. Animals in each block will then be assigned to groups using computer-gencrated random numbers. 000238 102 _-- Covance 632hP9au-g2ee2?2? Justification PFOS is aknown hepatic peroxisome proliferator (PP) in the rat. When exposed to PP, nonhuman primates (such as the cynomolgus monkey) respond similarly to humans (ie, low 10 no hepatic response) and therefore arc an appropriate human surrogate. species. Group Designations and Dosage Levels _-- Group Dose Level Total Material Dose Number of Animals (mg/kg/day) Level (mgfkg/day) Males Females 1 (control) o 2 (low-dose) 0.02 3 (high-dose) 20 200 2 2 20.0" 3 3 200 1 i a The control group gelatin capsules as (Group 1) will receive an equivalent amount the total material administered to Group 3. of lactose in b. The low-dose (Group 2) will receive the test material triturated with lactose: (1:999, ww). The high-dose (Group 3) will receive the test material trturated with lactose. (1:9, ww). Dosing Procedures Method of Administration Orally by gelatin capsules, daily (7 days/week) for 28 days Reason for Dosing Route `To compare with data from previous toxicology studies using the oral route, which is the most likely route of exposure Dose Preparation Capsules wil be prepared at least twice weekly. The size and number of capsules will depend on the physical characteristics of the test material, the dose level, und the. weight of the monkey. Individual daily doses will be based on the most recent individual body weights, with the exception of body weight collection days when the previous body weight will he used. Dose levels will be based on thevehicleas 000259 103 --_-- Covance 632eP9aa-g2ee2s2 supplied for be triturated Group 1. For the Groups 2 and 3 withlactose (1:99 and 1:9, wiw, dose preparations, respectively) once the test material before initiation will of treatment. Trituration with lactose is necessary to failate capsule preparation. dose preparations willbestored at room temperature until dosed. All Dose Analysis, Dose analyses willbedone by the Sponsor. `Homogeneity b`Soatmtpolmeosf(atphpertoexsitmmaatteelryia1lglaecatcohs)e wpirlelpbareactoilolnescftoerdtfhreomlothwe a(nopd,hmiigdhd-ldeo,saengdroups `and analyzed for test material content. All samples willbe stored at room temperature until analyzed. Stability Hfoormtohgeepnreeisttyudsyamsptalbeislitcyolalnealcytseids.frOomnethseetmoifddslaemopfletshe(apprpepraorxaitmiaotneslywil1lg bceaucshe)d willbe end taken from the lowof the in-lfe phase and andhigh-dose test materialactose analyzed for test material content. preparations at the `Sample Shipping `Samples will be shipped under ambient conditions to Kris J. Hansen, PhD 3MET.&S Bldg. 2-38-09 935 Bush Avenue `StT.elPeapuhlon,eM:in6n1e2s.o7t7a8.$65011086 Facsimile: 612.778.6176 Kris J. Hansen or her aliemate wil be notified regarding the shipment ofthe samples. Analysis of for test will be provided for inclusion material content will in the final report. be done on the samples. Results 000240 104 Observationof Animals Covance 63e2P9wa-g2ee2d2 Clinical Observations Each animal will beobserved twice daily (a.m. and p.m.) for mortality and `moribundity; findings will be recorded as they are observed. Each animal will be observed daily and food consumption will be assessed qualitatively; abnormal findingswillbe recorded. Once weekly, each animal will be: observed; abnormal findings, or an indication that the animal is normal wil be. recorded. During treatment, cach animal willbe observed for signs of poor health or abnormal behavior approximately 30, 60, and 90 minutes posidose. `Additional findings will be recorded as they are observed, Body Weights Beginning two weeks before initiationof treatment, twice weekly (Monday and Thursday), on the first day of treatment, and twice weekly thereafter. An additional body weight will be recorded on Day -1 for the Day | dose calculations. Rectal Body Temperatures Using nonanesthetized animals. Beginning 2 weeks before initiation of treatment, rectal body temperatures will be taken at approximately 11:00 on two days/week (Monday and Thursday). Clinical Pathology Frequency Unscheduled Collections `When possible, blood will be collected for clinical pathology tests from animals sacrificed at unscheduled intervals 000241 105 Cove 6329-222 _-- mu Scheduled Collections Hematology and clinical chemistry will be done once befor initiation oftreatment (Day -7 or 6) and on Day 29. Clinical chemistrywilllso be done prior to the daily dose on Days 2,7, and 14, MethodofCollection AAnntiimcaolasguwlilalntbewifalstbeedpoovtearsnsiighutm;EbDloToAd wfiorllhbeemcaotlolleocgteydtfesrtso.m aNfoemaonrtiaclovaegiunl.ant s used for clinical chemistry tests. Tests Hematology rheedmobgllooobdicnell (erythrocyte) count hmeemaantoccorriptuscular volume: mmeeaann ccoorrppuussccuullaarr hheemmoogglloobbiinn concentration wplhaitteelebtlcoooudntcell (leukoeste) count dbilfoeordntceilallmbolropohdolceolgycount reticulocyte count Clinical Chemistry glucose ucrreeaatinniitnreogen alwbaulmiprnotein goltoablulbiinlirubin digrreeactbeirlitrhuabnin2.i0f mtogt/adlLb)iirubinis nchioglleyscteerriodles aallkaanliinneeapmhionsopthraantsaiseerase gasapmarmtaatgelaumaimnoytlraannssifeerrasssee scroerabtiionle dKeihnaysderogenase calcium isnoodriguamnic phosphorus potassium schelrourmidbeile acids pamayslease pancreaticspecific amylase 000242 106 _-- Blood Hormone Determination Comnec 6529222 Pen Frequency Before initiationof trzatment (Day -7 or -6) and prior to treatment on Day 29; blood willbe collected between 7:00am. and 9:00 am. Numberof Animals An MethodofCollection Animals will be fusted overnight; bloodwilbe collected from a femoral vein. Approximately5 mL of blood willbe collected without anticoagulant and allowed 10 clot for serum samples. `Sample Handling Samples for serum willbe centrifuged within 1 hour ater collection, and serum will be harvested. `Serum will bestored ina freszer se to maintain -60 to -80C until packed on dry ice and shipped to: Dr. Das AniLytics Inc. 2Ga0i0thGeirrsabrudrgSt,reMeatr,ySluaintde 22000877 Telephone No.: 301.921.0168 Facsimile No.. 301.977.0433 AniLytics Inc. will be notified regarding shipmentof samples. Tests Samples will be analyzed by AniLytics In.,for estradiol, estrone, estriol, thyroid stimulating hormone, triiodothyronine (T3), and thyroxin (T4). `Serum PFOS Level Determination Frequency Before initiation of treatment (Day -7 or -6), and prior 1 treatment on Days 2 (approximately 24 hours afer the first dose)7, 14, and 29 000243 107 _-- Number of Animals An Covance 6h3P2aege9-e2n2l22 MethodofCollection Animals wil be fasted overnight; blood (approximately 2 mL) willbe collected from a femoral vein without an anticoagulant `Sample Handling h`aSravmepslteesd.wilSlebreucmenstarmipfluegsedwwililthbiens1tohroeudrinafatfrrceoelzleerscteitont,oamnadinsteariunm-w6i0lltobe-80C. Sample Shipping `Serum samples will be packed on dry ice and shipped to: Kris J. Hansen, PhD MET. &S Bldg. 2.3E.09 935 Bush Avenue St. Paul, Minnesota. 55106 `Telephone: 612.778.6018 Facsimile: 612.778.6176 KriJs.Hansen or her alterate wilbel notified regarding the shipmentof the samples. Analysisofserum for PFOS will be done on the samples. provided for inclusion in the final report Resultswillbe Additional Blood Collection Abet csoclhleedcutleeddfarnodm uannsimcahlesdualtetdhenetcirmoeposifcse,xsbalngouoidna(tai5omn.uchBlaosopdosfsribolme,eaucphtaoni2m0almLwi)llwiblel tmraainnstfaeirnre-d60inttoo c-o8n0taCinuenrtsil(sahpipprpoexdimtaotetlhye S5pmoLnsoearcfho)rapnodsssitvoreefdutinuraefarneaelzyseirs,sct to 000244 108 _-- Termination Cove 6329-222 Pen Unscheduled Sacrifices and Deaths Necropsies wibeldonle. Animal to be sacrificed will be ancsthetized with sodium pentobarbital, weighed, bled for required tests, and exsanuinated `Scheduled Sacrifice Aanfetsetrhaettliezaesdtwitwheseokdsioufmtpreenattombeanrtb,itaalll,suwreviigvhiengd,anbilmedalfsorwilrlbeeqfuiursedtetedstos,vernight, then exsanguinated, and necropsied. Postmortem Procedures Necropsy "The necropsy wil include examination of all orifices cranial cavity texhteebrrnaailnsaurnfdascpeinoafltcheorbdrawiinl;ltbheeeexxatemrinnaeldsuwrhfeanceevoefrtthiesssupeintarlicmomridnganisdpceurtfsourrmfeadces of ctehrovriaccailc,tiasbsduoemsiannadl,onrgdanpselvic cavities and viscera enaxstaelmacalvsituyrfaancde opafrtahneasbaoldsyinuses Palnitoyl CoA Oxidase Determinations A sample of the right lateral lob. of liver wil be collected from cach animal at the sfcrehcezdzurlesedts1a0crmifaiicnet.aiTnh6e0sa0mp8le0wiCll ubneiflaansahl-fyrzoezdenfoirn plailqmuiidtonyiltrCoogeAn,oaxinddassteoarecdtiinv.a Cell Proliferation Evaluation Representative samples of the lve, estes, and pancreas will be collected and preserve in zine formalin. eAfmtbeerdfdiexadtiionn,paarmapflinesanfdorshprioplpiefdertaot:ion cell nuclear antigen (PCNA) evaluation will be 0024s 109 Sandra R. Eldridge, PhD Pathology Associates Interational 15 Worman's Mill Court Sue 1 Frederick, Maryland 21701 Telephone: 301.663.1644 ext. 2201 Facsimile: 301.663.8994 Covnee62P9a.g2e2s2 Pathology Associates International willbe notified regarding shipment of samples. PCNA evaluation will be done on the samples. Resuls will be provided for inclusion in the final report Liver PFOS Determination A sample ofliver (any non-formalin treated liver remaining afer sampling for histopathology) will be collected from each animala the scheduled sacrifice, weighed. fash-frozen in liquid nitrogen, and stored inafreezer set to maintain -60 10 80C until shipped with plasma samples to 3M (Kris Hansen) for analysis, Analysis of iver for PROS willbe done onthesamples. Results willbeprovided for inclusiion the final report Tissue Preservation "The following tissues (when present) from each anianal wil be preserved in 10% neutral-buffered formalin, unless otherwise specified, for possible future: microscopic examination saodrreanal 2) brain cecum cceorlvoinx duodenum eepsiodpihdaygmuiss (2) 12) 0be preserved in feDmauvridwsiotnh'bsonfiexamtiavrer]ow galalrbtliacdudlearr surface of the distal end) pmaensccrnteearsic lymph node piitary prercotsutmae sscailaitviacrynegrlvaend [mandibular (2)) sskeemlientaall vmeussiccllee((2t)high) skin spiannadl lcuomrbdar(c)ervical, thoracic, splecn sternum with bone marrow no 000246 Conece9P:a2e2s2 heart iileejuumnum kleisdinoensy (2) lluinvegr omvaamrmya@r)y gland (females only) stomach ttehsytimsus(2) wthaycrhoeiad (2) with parathyroid uurtienraursy bladder vagina Bone Marrow Smear hFerlodmfortpheosstseirbneumfuotfuraecehxaamniinmaatlioant)unscheduled and scheduled sacrifices (made and Histopathology "aTnhde tahdryemnuaslsf, oeyme,eafcemhoarnailmbalonweilmlabrereomw,beldudnge,dliinvepr,aKridan,ey,sepcatnicorneeads,,sstpaliencend,weistthes, hematoxylin and cosin, nd examined microscopically. Reports A draft report that includes the following information will be prepared and submitted: Experimental Design and Methods Rmeosrualltisty clinical observations bfoooddy cwoenisguhmtsption rceicitiaclalbopdaythtoelmopgeyrraetsuurless phaolrmmiotonyel aCnoalAysoexsi(dparsoevaicdteidvibtyesAniLytics) dose analyses (provided by 3M) pmmilacacrsromosasccaoonppidicclioovbbesrseerPrvFvaaOttiSioonlnsesvels (provided by 3M) cdeolsepraonlailfyesreasti(opnreovvaildueadtiboyns3(Mp)rovided by Pathology Associates International) 000247 nm Covance 6329-222 --_--_-- Pais Statistical Evaluation Levene's test will be done to test for variance homogeneity. In thecaseof heterogofevnareiaincteaytp 5 0.05, transformations will be used to stabilize the variance. Comparison testswill ake variance heterogeneity into consideration. One-way analysis of variance (ANOVA) will be used (ifapplicable) to analyze initial body weights, rectal body temperature, palmitoyl CoA oxidase activities, and `continuousclinical pathology values. If the ANOVA is significant, Dunnett t-test `will be used for control versus treated group comparisons. `One-way analysisofcovariance (ANCOVA) will be used to analyze body weights, with initial body weights as the covariate. If the ANCOVA is significant, covariate-adjusted means will be used for control versus treated group comparisons. `Group comparisons (Group 2 versus Group 1)willbe evaluated at the 5.0% two-tailed probability level. Only data collected on orafterthe irs day of treatment will be analyzed statistically. At the end of1 year after issuance of the audited draft report,ifno requested revisions or instructions to finalize have been communicated by the Sponsor, then the audited draft report will be considered `final and issued as the final report, signed by the studydirector, and submitted t0 the Sponsor. Any modifications or changes to the audited draft report requested 1 year afier issuance willbe performed at additional cost to the Sponsor. Record Retention All raw data, documentation, records, protocol, specimens, and final report generated as a resultofthis study will be archived in the storage facltisofCovance for a period of 1 year following submissionofthe final report t0 the Sponsor. One year after submission of the final report, alof the aforementioned materials will be sent 10 the Sponsor, and a return fee will be charged. The Sponsor may elect to have the materials retained in the Covance archives for an additional period of time. and Covance will charge astorage fee 1 the Sponsor chooses to have Covance disposeof the materials, charged. All raw data stored on magnetic media will be retained adisposal fee by Covance, will be: 000248 12 _-- protocol and protocol amendments dose preparation records in-aalcnicfielmiarmleacrtoeircodensipt arnainmdaolmirzoaotimonmsainenance dcloisniecaaldmoibnsiesrtvraattiioonns bfoooddycwoenisguhtmsption niscaampllpeactohlolleoctgiyonrecords aStnaattisotmiiccalalnaplaytshoelsogy records sttiusduyecsoprerceismpeonnsd(ewnecte and in parafin) bfilnoaoldreapnodrti(souregisniadlessigned copy) "The following supporting records will archived with the study data. be retained at Covance:Madison Cones ne2n2 but will not be fweactderanaanlsylsyissisrerceocrodrsds arenfirmiaglerraotoormaenndvfirreoenzmeernttemrpeecroartdusre records rinosotmrutmeemnptecraaltiubrreatrieoncoarndds mfaoirnttesetnamnacteeriraelcosrtdosrage 000249 13 Come 229: FE PROTOCOL APPROVAL [dus ik Soul StudryewMoMn.iSteoarcat, PhD M SPatikudyDirkiorThepord, Covance Laboratories Inc Fass Bae Da 000250 114 COVANCE.> PROTOCOL AMENDMENT NO. 1 Counce 6329222 Week Cape Toxicity Study with Peruorooetane Suloic Acid - 0 Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys Seon 3M. St. Pal, Mines StudyMonior: AndrewM,Sesca, PHD. Testing Facility: Covance Laboratories Ine. Madison, Wisconsin SuStduydyDDiircecotorr: PPeerteJlr.TThhoomfnorfdo,tPPhD0 "This amendment modifi the folowing porns ofthe protocol: Effective April 17, 1998 1. Pfaignee",thDeodsiungrPoroaceedsumree,nM,etdheoledtofthAedtmeixntinstrhatisocni.oTnoepmore pwrietchissehley following Orally bygeuti capes, diy (7 daysweek for at east 28 days 2 Pamogdeiy11t,heBlsoaodmpHonrdmionnegDmeettehromdi,neadlotn,hSiasmspelneenHcaendalningre,plSaecentwietnhc2teh.e To following See4r1u0mmwaiilltabeidni-v6i0de0d 5n0toCtwuonsipppraocxkiemdatoenlydreyqueacl lanodtshsipapedd s0oredin frecaer 000251 us ree EsTISn=E E aion T S--_-- -- 3. Page 14, Postmortem Procedures, Tissue Preservation. To correct an error in 3perms LsTus? -- # 70> 7 < 000252 116 COVANCE> PROTOCOL AMENDMENT NO. 2 Covance 6329-222 4-WeekCapsule Toxicity Smdy with PerfluorooctaneSulfonicAcid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys Sponsor: 3M, St. Paul, Minnesota Study Monitor: Andrew M. Seacat, PhD SP uiyDe iovor r iToomfot0 Pd "Testing Facility: ~ Covance Laboratories Inc., Madison, Wisconsin `Study Director: Peter J. Thomford, PD "This amendment modifies th following portionsofthe protocal: Effective May 15, 1998 1. Page 13, Termination, Unscheduled Sacrifices and Deaths. To. change the anesthetic to be used before exsanguination because the barbiturate may interfere `with tissue analyses, delete the text in this section and:replace with the following: Necrowipllsbeideonse. Animalstobesacrificedwillbe anesthetiwzietdh ketamine and xylazine, weighed, bled for required tests, and exsanguinated. 2 Page 13, Termination, Scheduled Sacrifice. To change the anesthetic to be used before exsanguination because the barbiturate may interfere with tissue analyses, delete the textin this section and replace with the following: Afterat least 4 weeksoftreatment,allsurvivinganimalswillbefasted overnight, then anesthetized with ketamine and xylazine, weighed, bled for required tests, exsanguinated, and necropsied. 000253 ww 000254 18 re E --R -- _-- s- ee bye? AMENDMENT APPROVAL r r a `Study Monitor rene. `StudyDirector BO plonitor did pod ito a SiPgneedndcompeyn a 2 paso! S2O 7 hrT poor-- 000255 1s PROTOCOL AMENDMENT NO. 3 Covance 6329-222 4-WeePkotCaaspssiuulmeSTalotxi(cPiFtOySS;tuTd-y62w9i5t)h iPnerCfylnuoormooolcgtuanseMSounlfkoenyisc Acid Sponsor: Study Monitor: `Testing Facility: Study Director: 3M, St. Paul, Minnesota Andrew M. Seacat, PhD C_PoevtearnJ.ceThLoambofroartdo,riPehsDInc., Madison, Wisconsin `This amendment modifies the following portions of the protocol. Effective April 17, 1998 1. tPeastgemsat,erVieahlicbleef,orSetomriaxgiengCownidtihttihoensl.actTosoe,inacdldudtehethfeolsloolwvienngt uafsteedr tthoidsissescotlivoen.the Solvent Identification Acetone Lot Numbers The lot numbers will be maintained in the raw data. Purity On fille with the manufacturer Stability On file with the manufacturer Storage Conditions At room temperature: Characteristics dIenffionrematthieosnoolvnenstynitsheosnisfimleetwhitohdst,hecommapnousfiacttiuorne,ro.r other characteristics that 000256 120 ProtocolCAomveanndcmee6n3t29N-o2.223 --- uPaege? 2. oPfaagcee7t,onDeosinintghePprroecpeadruartieosn,oDfotsheePdroseepsa,radteileotne,tSheisntseenntceen5c.e aTnodirnecplluadceetwhiethustehe following. aFcoerttohneeGarnodutpri2tuarnatded3 wdiotshelparcetpoasrea(ti1o:n9s9,9 tahnedte1s:t9,mawt:ewri,arlewsiplelctbievedliys)soonlvceedbienfore: initiationoftreatment. Effective April 17, 1998 and April 26,1999 3. sPapgee1t6c,heRireecfcoorryddRreetteennttiioonn.reqTuoirceormreencttsafnoroAminsisLiyotnicisn Itnhce. parnodtoScoMl EanTd. t&o S I(enftfeercntaitvieonaAlpr(ielff1e7c,ti1ve99A8p)rialnd26t,o1c9l9a9ri)f,yatdhde trheequfiorlelmoweinntg.for Pathology Associates m`Waittehriinal1sy(eaasraappfreorprsiuabtmei)sfsiroonmoAfntiheLyftiincasl Irnecp.oratn,dal3loMfEt.hTe.a&foSrewmielnltbieonseednt to (thPeADS)poisnsroesrp(onAsnidbrleewfoSreatcheatm,aPihnDte,n3aMn)c.eofPaatnhyolroagwy dAastsaocoiratsepsecIinmteernnsatpiroondaluced byPAL Effective December 15, 1998 4. PPaagrea1g3r,aphPo3s,tSmeonrtteenmceP2r.ocTeoduirnecsl,udCeelelvaPlruoaltiifoenraoftsiiodneEsvasltuaaitnieodnw,ith whietmhattohxeyfloilnlaownidnegosin in the PCNA evaluation, delete this sentence and replace cPoCsiNnA) weivlallbueatdioonne(ionncltuhdeinsgamepxlaemsi.nationofslides stained with hematoxylin and 000257 121 ProtocolCAonvaicem6e32N9o.223 -_ Th AMENDMENT APPROVAL AndrUewnMd. iSaccw, 0. ID Lina TMStudy Monitor Study riDeoionr Fi Covance Laboratories 16. Bae /21/99 52 000258 12 COVANCE PROTOCOL AMENDMENT NO. 4 Covance 6329222 WeePkotCaaspssiulmeSTaoxti(ciPtFyOSS;uTd-y6w2i9t5h)PienrCfyuonroomolcgtaunse MSolnfkoenyicsAcid SSptoudoyrMonitor: AWSnMdF.Sareacmaete,PwhrD. STetsutdiyngDiFatceicltiotyr::_ PCeotvear)n.ceLThoamobrd.oPArIDnca.Mtadiosonr,Wiisceonssin This amendment modifies the followingpotionsofth protocol. EffeJuclyt15i, v20e00 1 PSiasegsnueetse1f2n,ocSr2eec.roTmuopmoPeuFfnfOdeSlteLvheeelvsesslpeopDraesrtaaerte'rlsmyid,nedacetiliseoonn,'thSirasmespeplnrteenShcheeiapepnildngre,epPolfaasrceangwerahsptho3oF, following Resultswillbe reporidseparately by he sponsor. 2 ddeePclaigeseiot1nh4i,sLsireveneptroernPtcFetahOenrSedsDreuetlpetlraomcfiewnnaidttliyhosints,eoSfefonlstoeuwneicnseg3f.orTcoomrepfoleucntdtlheevseplosnsseopru'astcly, Results will be reporid spare by he sponsor. 3. oPfagaenal1y5s,isReopfotrtsss,isResourltcso.mpToouendcvtehlesseppoanrsatoerl'ys,ddeecliestieonteoorwepirnhge resus plasm and liver PROS levels (provided by 3M) 000259 123 ProcCAonnesn2e9.e2224 --_-- AMENDMENT APPROVAL hm LrnowgM.hSaeacrat, P2AD. Ey SMtudy Monitor Study Diettor va Covance Laboratories ln. 5 7/3aan 000260 124 Covance 6329-22 umes wAre sneesTuey I[nA car nter -- Sieinad raenseent CSionEpfyoErriIaaaNnttiG,onF1ef9so9e7rr,utehMaei.mnseuCsropotepsaysiwnig3niapnnrgdo/poaernrdldyMoamnwmuiifLoaincLsstiiunnrgginofgo.nCoorpmkrpsoadnuyc.ts 1} hlreio'onrnefaogrroaeraeotsviesonotnnsa1sSocooosptiaeidnedintrFounl.dSwhithaneno changes unless 2) Shiesttrnieorutheed cwoipcyh mhoer hSneveonrciigoinnaofl e1asrnFiansgsla& oprrotoitch.erhcairseean. ThReocaweF:iona pp -- rans Fiuorocnemical surfactant 0SSBENOEEER 0Ua1Pa6a37 Fauntaaa. c0o0:CSsTi1iIsoSn..0i3ie0s5e4n11s aSaB-nGEETS-0e10e4s-5 00s-5e11a25s.o8n08e5-d8 SSsiorsreReastio:eFse:1br0Auu-agr0uys8t109,0,19917958 inenepion rey Ed TOFrTOAToSASmSSIIsMN pPPEEERRRFFFLLLUOUSOMIROOOAAMLLIOONLL SSSUULFFLOOFWTO.ELW.TLE.LL SaTre0ersi.ms2e0er-s3s 3832 aL4be PFooTTAAGSaSiIiMn PPEERFRLFOOORORAMLO,L SSAUFLOWFTOEW.T1E11L. Sdaargaisoiesnes 31 L1g8 i hse oan SOahIrrLooImnNpGmaPevOesrsI::NT1LLLCC.I .. WAMIAA ElRromTion MTEL 11111 SPR aviv M1 BAle beens PpERCeENTnYONTLE:.......c. 8TYei VIPSGEOSTITYS 11MfrD ipe-- h(eEogn ceeAtaaOredGo,ooEfnr:ee Towing ponder. 000261 125 -_ CovanuIceMTT6e6322w902s5262s F6e0b8r:uarFyC-9159,FL1U99O7RAD Brand Fluorochenical Surfactant ace 2 3. FIRE AND EXPLOSION KAZARD DATA Te FFLLAuSBHBPLOIENTL:IMoIT.S |oCoE.L.I. WNoane AFULTAOBIAGBNLIETIOLNINTITESMPEVREALT:U1RE1S.D.. NW/AA EXWTaItNeGrU,ISCHaIrNbGoMnEDdIiAo:xide, Ory chemical, Foun SPPWEeoCasIrAiLtifvuFelIlREprperFsoIstGueHrcTeItNiGvoerPpcRlrOoeCtEshDsiUunRrgEe,S:deimnacnluddibnrgeathheilansgt,Appsaerlaft-ucso,ntabiunnekde,r cost Parnodtpeacnttisv,e cboavnedrsinagroTuonrd earxepso,sedwaiasrtassandofltehgse,hefaadc.e mask, and UNSUeSeUALHazFaIrRdEouAsNDDeEcXoPsLpOoSsIOiNtioHnAZAsReDcSt:ion for products of combustion. REACTIVITY DATA Crm-- STABILITY: Stable INNCoOtMPaApTpIlBiIcLaIbTlYe.- MATERIALS/CONDITIONS TO AVOID: HAZARDOUS POLYMERIZATION: Hazardous polymerization will not occur. HAFCZlaAurRobDroOinUdSeM,oDnEoTCxoOixMdiPecOSIVaanTpdIoOrCNsa,rPbRoOGnDaUsCeDTsiSo:xoirdeP,artOixciudleasteosf.Sulfur, Hydrogen 5. ENVIROWNENTAL TNFORATION or Tr SPCOIboLLsmeproRevusenpdopnorsreec:waautteiornstoraovsoidotdhuesrtisnegc.tioCnAs.UTIOVNaAcuusv,icuuusse WCelteansewreepcionugld abepparnoveidgnimteitoanl csoounrtcaei.ner.CleaSnealupthreesicdounetaiWnietrh.water. Place in an OEC7O0MnMoEtNDErDeleDaIsSePOStAoL:katernays processes that could result ionr aseqwueart.ic cDooncneotntruasteioinnsprgordeuacttesr tohran Bi1an/td1e0ursitoarfli.atlheCoorlsobwcueosssttmieorEncCiSaplroordfuaccLtiCslSiwtiyclolnicneintnthcrelautpdieroensH.Fe.nceIDniocfsipnoeasracalotmsbuisntiabnle alternative: Dispose of naste product in a facility peraitted to 126 000262 Covance 6329-22 -_-- wrews warFyc.9150,F1L9O07OD Brand Fluorschemical Surfactant once 3 5. ENVIRONMENTAL INFORMATION (continued) TTI accept chemical waste. EWSBTRReuOnngMiEaItATQAUSLSotnifOEAiTsAnF:(iLsehpoLaCsSso, suFsartohcehaidruMsirn=neosu(PRioaye,phaPlaeisntipnrosTeelssest)i=e3msmeangil, G$laarinpoeozrbst=)swiasn. i ane. Eso, Gamma MAIR = $0 mark, counrny REVVGooUlcLaALtTeiOslRseY OHFrO'gRasnMiTcEIxCeOomNms:peouSnodlsv:entWsA:.WIA. SGEaiotnnocrwesardeigzauploaasta.si.o"ns 1va6r.y,EncsonnsaulztarcaopcpsliHcaasbtlee Nreogmuairstiaonshomoer a(uhtehnouriist.ies TThoihs,prEoINdEuScSt,cComopblLieAsTEwSi,thWItThSe canhdasiKocraeln.registration requiressots of ecThE KmAaIzAaRnDD:cHuoss:PRESSURE: No REACTIVITY: No ACUTE: Yes GHONIC: vos 6. SUGGESTED FIRST AID or T-- evSeTiaccneotdominea.tra:lGyuclisumsehdieiytees wneidcihcallarSgtetosnmcoiuonnc:s of water for at least 15 swCaTonenntcaoagumimiaantcaerlt:yedlculsohnisnkgi.n wIfithSrlrairtgeetioanmopuanrtseisotferwatcehri.doRespohvyeetotan. Contasinated clotning before ress Hasn wcCTfltainSaloanmosy:mspyrsopstsoncsontoicncuuer,, crheislavea ppawryssoinciotno: fresh air. If 1Osrionnteuo:plasses of water. Gall a physician. 7. enecAUTIonARYINFORMATION +. vorrloreecyreiocno:ntact. Wear vented goggles. Cm 000263 127 CovancMeT.6632299-52262 E8a0b8r:uaFrCy-9159, FL19U9O7RAD Brand Fluorocheaical Surfactant PAGE 4 7. PRECAUTIONARY INFORMATION (continued) Tr SKAINviPdROTsEkCiTnIOcNo:ntact. Wear appropriate gloves when handling this mraetceormieanld.ed:A pabiurtylofrgulbboevre.s naUdsee ofnreonorthseorfeollofowitnhge fmaotlelroiwaiin(gs) are pcoevresroinnagl, pcroovteercatlilosn. itPemrsoteacstinveecesgasrasreynttso p(roetvheenrtthsakninglcoovnetsa)ct:shohueladd pboelymeatdheyloefneeiptohleyrvionfyltihdeenfeollcohwlionrgidseat(eSrairaalnse:x). `REUCsOeMMwEiNtDhEDaVpEpNrToIpLrAiTaItOeN:local exhaust ventilation. Use in a wellveimesinstnositiloantaseddeqbueaarlteoeaw,. ruescPeoraonvaiepdnpedrsodpsrueifxafptioecsiuerrneetspilvrienanitttoisrl.yatiporInofteetcoxthisaouansi.tntavienntilation RE`SAPvIoRiAdTbOrReYatPhRiOnTgECToIfONd:ust. Select one of the following NIOSH approved hFaacelcsfop-rimdraaasntkcoerssuwipbtpahlsieOeddSKonAairraeigrrueblsoaprtinireoantcsoo:rn,cenfhtuarllalft--ifsoaancsekofdGuusscttontaannsddaimmniiassntttsrreeassndppiirriaanttoorr,, ull-face supplied air respirator. PREDoVEnNoTtIONeatO,F AArCCiInDkENoTrALsacIkNeGEwSThIeOnN:using this product. Wash exposed bareefaosretheoartionugg.hly with soap and water. Kash hands after handling and REKCeOeMpMEcNoDnEtDaiSnTeOrRAGdEr:y. Keep container closed when not in use. FINRoEnfAlNaDssEaXbPlLeO.SION AVOIDANCE: OTHNEoRsPoRkEiCnAgU:TISOaNoAkRiYngINwFhOiRMlAeTIuOsNi:ng this product can result in cofontthaeninhaatziaorndouosf dtheecostpoobsaictcioonandpr/oodrucstasckemenantdionleedaditnostehcetifoonrm4atofion This HDS. WAS HAZARD RATINGS: WPEEARLSTOHN:AL2PROFTLEACMTMIAOBNI:LITXY:(Se0s RpErAeCcTaIuVtIiToYn:s, 0section 7.) EXPOSURE LIMITS REOrENT VALE oI TIPE AUTH Skane PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOONNAATTEE...... 00.11 MMGa/iMk PPOOTTAASSSSIIUUMM PPEERRFFLLUUOORROOAALLKKYYLL SSUULLFFOONNAATTEE...... 00.11 MMGG//MM} TTWaA M aM v TTWHAA M SH.YY 128 000264 --- CovManTIceMeT6-3w6292s-0252e62 arFyC.9195,FL1U9O9R7AD Brand Fluorochesical Surfactant Paces EXPOSURE LIMITS (continued) MoREoTENT VAWE UNIT Tyee ue ska POTASSIUM PERFLUGROALKYL SULFOMTE. 0.1 hams haany "tihnecSlKpuIodNitneNgOnTtAmiTuaIclOoNuc:sontareLisibbsrutatenideonsuatnbodstetayhneec,esoevieitrnhadelirlcabteyex.dpaosiwurirbteohrnb'eYy' thuendesrutaSnKsIaNusrefreartsto or, more partieeterly, by direct contact with the substance. Vehicles can alter skin sbsorptin S"OaUR:CE OF9EHXPROeScUoRsEsenLdIeMdITEOxApToAs:ure Guidelines Ta. WEALTH HAZARD OATA Crm evHeilcdonEtaycet:Irritation: pain, and tearing. signs/sysptoss can include redness, swelling, SKIH1Niglndcso/SwsTkyAisCnpT:tIornrsitcaatnioninc(laufdteerrperdonelsosn,gedsweolrlirnegp,eataendd ictocnhtiancgt.): Heaxytebnededabstionreb.ed through the skin and persist In the body for an IOHAaLyATbIeONh:aratul if inhaled. Htaiye.be absorbed by inhalation and persist in the body for an extended Single overexposure, above recossanded guidelines, may cause: ISrorrietnaetsisonof (tuhpepernosreesapnidrattohrryo)a:t,sciogungsh/isnygspatnodssscnaeneziinngc.lude IFISnugAeLsLtOiwoEnD:is not a likely route of exposure to this product. tIhlilsnemssatsearyiarlesult fron a single swallowing of a moderate quantity of May be haratul 5f sxallowed. TaUgteangTeCnIiTcYi:ty assays indicate the product is not mutagenic. 129 000265 8Fe0b5r:uarFyG-9185,FL1U9O8R7AD Brand Fluorochesical Surfactant Covan3cMe 1T229925262 we 8. HEALTH HAZARD DATA (continued) Tr REoTPetRv.OeDltUseG.rTaItVoEg/eDnEiVcE.LOiPnHEtNhTeALratTOaXtINo:ral doses below maternally toxic OTAHERPrHoEdAuLctTHTHoAxZicAiRtDyINSFuOmRsMaArTyIOSNhSeet 1s available. SECTION CHANGE OATES - Cm PRECAUTIONRY INFO. SECTION CHANGED SINCE Aigust 23, 1696 ISSUE Abbreviations: NID | het Determined WA - Not Appiisasie GA - Appreriaiely TSIhREep"LCToiErnDrf,aocrtIsNaCtaLaiUoDonIfNiGnt,henBiUdsTateMhoarteesrLuIieNadIlT.ESDa.f37e40t,yFAAKONEsYStaNISONhPeLNeIAtERDRA(NM4Ta5IR0ER5D)A,NT1YEsXOPbFReElSiSeEvDedOnto "PHhEEeRRtFGhOHeRArMNATNASCShEeILIO9TFHYUpOSrRAo'GdEuFcItOTFNE1ST3SRA1D4FE%G.RfoArUsPeAaRrTpIAiCrsUtLLrAeeRvsipaPorUnRsFiPObuSlrEeposfOeRorCaOdnUedRtSeSEru.mLitOneFisnhgs for UCiasnneira'assfeflmaycettNhEohdSe'ofuSsheuessavnsdoerr sspsppipiklcnioacwtaliieoodnng.eof aGn&idvIencnonpttrrhooaeluscvta,Jrtiseotsusys osfeofsefnamcthtiioscrlhs.tahrtaehtet tRhReriuCsueLrarevapluuraptoesetahned3Hsuplreoadbulcetfotro duesteerrsainseetAheNdAToefrueIst o1f Taiptplifcoarti8on. i0une`'aptrroroovritshd,eesormeiimnsofstoieroanspstoisosonribiialnlitteeyrlaetctithoertnonsisclinecfiotrrhisosniassinfSaorrasaneaertfvieiorcne,staWoyhhiaatvasesscsursetsmoeunlstread. C{{ehnpffraoerrsmaeasmtttiiiootnniooinbnsttahiaesnedHtoOfSriotmsavcaaasniGpsaibtieaetbsasngseisrseacatyolfy10s%cferoboromaco4ny5,.curInr'anatseiassiohnes } 000aO6 130 Covace 32922 -_ Tews APPENDIX 2 Individual Animal Fate Data Individual Clinical Observations Individual Rectal Body Temperature Data (C) 000267 131 HA i e| gsElmnnannnnnnnn | 831asssssasanss LE gp | BREESE iq, : Ee EE Blesseenees L pj EE EB N fiEnE inLg 1 30 EG gi) ziddnaadiag BiiEEmEaaa i HE iRERERREERRRR EOE | os izssssssenin 2%22 ||| wFkaUji[sRBERBE3Ri3Rz3Es3EE3RE3Rs8Ri8Re8Es3R Bl Beers ge igs! I| i BiEssmEEmRiRs EEE i lmmonnnannnnn ! o = 000268 0 L188 LIE PE i fig E PHfIi 2ipzi LEE te : PPippli HERE EN iE 8 THE 8 HEHE Ei 000269 ig P EEiEg sFEfI ERu E IEE 3 ei dik gi: id PEPLEsE [HE PEE Eo: 8: i 8 gifi FER o: gE: 4 BE BoB DE . : 000270 Fo o. oisdd 08 PE oee PEE PRL Li Ei Bigs Hi: ff LEE POE EER ESE og PPOEgLEE2 000271 Ef ig PE LEE er P1220 5] men ems smmasccennass a 2 2 PEd E Poti Ig B5 ri fsi . Tt E os II i i EREi p= LE EH 2 {Eiv - 000272  PL18E5i PEE PE Pad igi EE i i PEE LE EE i: gi i digi DEE Eg Hic8 w8d8112 Af gy li ee eee a0TANR 8 ae 288 ii genad POET HH LE EE 8 000273 ' BEE fyi i i PB cree an cee eee $8 Ei if i i di ~ofRRR 2B ef} .o2NAR 2 LosEs iid i PE id 1EE gid iE PPEEELE [LH PE BEH EE2 . : ; ooozva PE EE LPiEEBi. erent TE eri gE senna fFIoEiEi Eod Peli Pggic Pegi f CE a 000275 Fo. FB oo oR mo: lel Eom: om ow os Pel BOOB Emo: SoBe momo: om mo: ; oH Si iggE iE 23 He 4 od ssf 8 odie nom oR mos z Hie u om ion mo: Pod 5 C PPg2EiiSss TifEieRd :. E g g fP giEggiy Eiede aRzesg:t i Ee en tee ween ma ETRY sEgifE i sds i s 000276 o Ps n PoE Poi Bll mo: oom: Pgs, gE Eiddfiegieres be nba i nT ae emome wen PETA peEi nisi Lg DEE Dsfp reel foe fusBs unss fs 000277 -_-- Covance 6329-222 MTIMeT-%62s95e6 APPENDIX 3 Individual Body Weight Data (kg) 000278 142 Boo.ommomo1omomm oo Peel o3oEono@mommoq Briel ow oImonomom3 os 8 PELs moron om os : i LE rT BE a ane oa am a g Fog Tomes momen Ios : Bigiw om or onmom oz Bo Biplan mo: ou om Pol A fi p3 lom$ imix tgimag me2s PR os 1d leplemommorimIana Pi FF Fd PEs ss Dpgizzn lala igeis DEERE Ef EE Ehoarid go. Bio Ho Po Pog Pogiel Wom od FOR 3 Blige mom onomomm os F000 z ToPlad, WseElAendemEIframeinanded PEs 8 fod [ELE EI I 000280 APPENDIX 4 Individual Clinical Hematology Data Individual Clinical Chemistry Data CovanMceTT623e299s-5226s2 000281 145 5 : FH 8 av eey vg = Pay met ame sgi Blo WmEs ommEa os PFi ooEggiR,E omi:t om: pdpEey TF EER PPB a.m 2 3T2 E Eel; PARRY :FARR :pd OE Fae ama Fo.c lm Bo. Beodmdimiis 8 g 000252 x Cee of see ata i HE ii goo oo Be sme sl s PET . pip EE TE aie een 2 POE i BsE,EFg ae tud bte pBl aoovng aaltan gBso ag mitemd, gdm dae by Eid i i 000283 &385, PHP I PEE gn THEY Rl: 2 Yo Gaia EB ZEEiEE 000284 i gli mein mmat a gl aT ee bu Pa om amd ; B BelLFEEse 2m8e8wE n A piirtih ipd ai pREEE gi 3 3 PILE be sta asen do 2 23S%iaBr gei EYo pggp EdeA n pe id FEoHeB iiRa E FIat emaee eBso, Ho mF emi: ! cap heeaie fea sii fd iT" $f9i0f5e,, PfaHaE. iisd 000255 3 5! se o% 3 La ii Lb Po ed 5f0f . goofrgi mT. ses om a amb 5 ome. od : Es 4; Feo ote Fann ain Fu stg imum ge : 3 male ow ggii f dnarfd as meut m biog 3d i i fide fa id 000286 i i gil , 1 PP sodar:ri r2 Lipid 2 i TeihEy. fo POgErLi:d E o; E 000287 oi as sie gas aie = nz y EL sues ssp pie a sg Tafgr. FE: dE ET 3: JBi EAE T He IEe 40 opis IEIE #0 LEHAN ER ER 3d i i 000288 i fi ii El ser BE Bish seat ese ade hp EE FPlOrgEi gdLpae m eae e a SEOHE RFee iFama i PORE: i i SBguziaFmoartet iPaems aann ibso Tog marta. gid famed id i i 0002.9 i $Ba EEi0s3 f 3 dH CoE RTE PE i i ;ipEEgie 84 |FEIigg sooo 2 SEEEELC C5 HPI hEiRl EE TR 000290 gf ses amok os i ar ee 3 I ET ; B of0 . 0om. om p oi i gg-i RE Rae R SirE pep HET PERE PES oh. anto.aain, & 23% E; pel pees pdedapon PoE EEE IEE Foz fen oaie fap oatae $B5E0B5.3E. F3EBR,RE f3d 000291 8 Fi El ; fB ggeReende Lrifk mr mmmmm aaEtTeosw om , PRBy Ea:FoamCCaie Tune esa Bog Hp iud juga gdm tag dy pd i i 000292 3 HET higE ; 7 E i E T PoRtei: i fEaEgRF EF EE T iR:eg# ie0 Bd 5 Fi. so. 2 P PEoEhL, E 50 oe Trias ie 000293 i : , ead wma giel TTT TETF aNBifE fie wn Ee aun nie 3 EET aPo g. moms es ofa men ofa o pfiEteE Lam eae Fiz iz 2 : PR Ta Tame Ty BOE i. sfc. Ba i CE . . PR : : goi fend Dame abe bs gi i i 000294 i EB ge o sense Lith TTT F lFaF gFyuFplEoEetTe FRoe EEzie SEER i FOos i 2 Pofsa fag Pasa bg BORE ET TTT a EI 4 - i foi REE geasin 2 PSgido s si tebi: d anats bi: s 000295 i : i ui Foe aba Toe tn Ba pPog oaEad:iEp nt 2gee 3ge Cini sift fama fe paddy : 55d Zs TE P FElSat oW l uGeE | mI em ai. e 2 = oPoE h imial ii Boi. tm. 3: PoE BETEL fEsgl fd 000296 i El go : TTC Bl, ii HES mat wmode a ee Be one 0% Fziiagn ig ol Fen ofa : Fone a8n 2 Foo ged Cd 58 CH . Pol : TEs . : 000297 i 3i BeBlH BREET AER ig 8 fod =f Br Demea t femmt es Be g ghee 8 8 Pog jE james OBE BR. sso. F theR FEAT opr beg oaie gd srpgie = g3 em a8i fesneie ig i 000298 I D8 la iona t, 2 18 8H 5 . Fiighgyl 2 IIT eiir.: EEG 3 5 PH atime i WEE i 2B ge % 708 Ts 000293 8 iesl 23% ss: gE 3 o&)l EET mast 5 i I iol BE ona mena : no LI : we wen ene ele ow PEER eee rats iH itm nan Bn LEAge C. E . Td HLH - or 508 i 000300 i i. 8% =" gE gem 3 LI) mE ma os ff. : HUG $3 si 82% aa 288 527 2 ifs os FTir8ike taste i togpE aig PHBE fama] Be eros,ess aig EE : : #1 i 000301 FI IO HTPaLode FEawAono bs Lig gi dm jae is EBiHpifEgyF mba,: EE A A 000302 i* o . eal AR VEC wp En og Ei &gif BIC gma x PdiBEf.i a ue vB aan owe ; BE H2 eBE eiEe a%e mwanPw@e os OEHeosdPes iPoasn ibl Po mgma LLiE deseivf3enasia 3 bg ik i 000303 ff mE mE 3 Sn 8 gE mE i. meat . og F3887gf R3 gfEF of2 L {3 2 F3Pii3 ginlB.e8l3T : reTz: a apt gdos i:: g2 E : EEE RLE : i Poin fed fame fe Hy szedgomaia Pogt hiigfsoromic fi:oams gin Bi: e WHiilsg,p fFaH, iit 000304 i i oi ssa, zamoaee CH seaman t TOE ge gd > iE fo: 5 3 SEER ERR EEE gE PPoi oandineimPeClTs Bim. tm. 1: 000305 gl mE ome os ia HBEEH1! am ede ame nge ii a :ii 33 in wan 28a PTH nim BE 5 gedbeet 58 CH . sz! . Re =~ 8 ME EEE ilan andi, , 3 ]IR =~ < = : i FE 000306 i fol BET mE og HI . . ; Bie BL ME 2s2e8s s82ie osg i058 poe eee ete oo PELE rasta. = i Ehime fama ig Hg regs oe 2d i i 000307 PEHSRE ET is . HE EE 5S magma Poh alae ine i; 000308 33 osm osEe ses ai: #i sal BEET ZEE EAS oz Bee abe en a3e oo FPR ORa Lt en ete a Pola E oma oz PEE Pane Bona d, 8 fai d BE 0 8 CH . . PRL : i RE ERLE RR id i 000309 3 Et E338" Eg Em 3 ; B BgldEe E sm ms mig e A . Po Bpii jmus BEF. vEeEt gumR at + 5.0 fesse fas aia fg 53d i i Sigs EL PEER fd 000310 go Bf ee aimode T Ineaznebn Ioa sopHI fd Ta 2 BEiiisslp,l W oneEyeeIeteL = EEN S i 5 3 agin s gE gs 000311 5 El gel BEER Ema a 8 Li[1 JE 88 Ig! P. oEEa = 5 Pole mE om os Siig H2ER:583EEL1 Fig Ame efa RweAn wha z i oe 2 FER:FREETS E E HEHE 0 gg fi =o - og a mamot aioet on PR :: i fzeeie fessor bsg fd 000312 i i gi BE oma og 5 5.0 BEES EEE En 3 5 PoE ii i 4 - I r $2E,3E01 L seae T Fagzrg in Bg = OEE IRR ose ge i Sol . a5 go z 285 on, Popa ste Pann aie Bs gid i 000313 ome mes H 0d T ii a geti, HE i JE$r0E8gli ibBusTatefamatd:eg F PiOEi Rhr LTth ay a8 o: g= oThEiTam fae anid Bc. tl. oo . 000314 gl RT TEER oo Ei ia RBE E mE enade o FEiEaEla WIEa N EPR i =2 Flj rg tiii e n oflag m ti s TOE Fume amaze fa EEE Cd & . . 1 matt amass |e FR 3 i Ere gtBangfag . 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E 8800E 8 4 i Bias ld TIE iy {EER Ei iC**g33i Bi adil CCehlt EE oy 1g oEEREE 8. 1 SeEggEtER fesR EE a Efid (5EEEER EEE gpos BlEs8 Beal [|PEP gEEHsEREl Pee | ioEEE8 BEGG 335328 35%8542.%3.07 F3E0S2!: HEE Po 3 i 000334 PPPEEEED EaRarpfA RaE EE s Pati EEE. gil B BoB iEnel oc gHL 0 PoeosE88i1igEE i| IiaEzEaEPiGfdEsE; oREgEgE EEEs ECl S iOi BidR Egr GthEigEh DiEghResLE HEARN BL PEE Dom [1 PPaodgi PEE iLiBangki Peni | i EEE | EEE wil (Ee; fn sl efi a Sg,u2ERiEEsBE gz,0. 385,20 EE sh obE bed iEE FHA tH gr, i 000335 PEEL DPEaElRsE GpiLEfEF PR ETT PoC BELL oigE LooeEiE PEE O PB ME E dEEeE 2:PREBe ERgEicESPEEE EE EG3M8E EH Eel Ee ENE cogEEEE BH gEdE 2 fiaEd aPH 2%i; oBt&gfaoEnehEEile PoE FOE EigBEit g FiE fE sisi Pole |CgHhiaEEElE fEE sdEggnh iBo 5HBrREE Eg Pai El 88.5508 2 DLoEEE gp RgsoEaiEgt HBgEl EHEEY TIES gIF pperlOe ani |HE pa pnaEian: ged PPEaEgRaEnDi D| TaTe tli 000336 PRE PgEefi le ES EERE OL gain el {RRERl El go oCEEsn i [ FEREEE EESEBE mE fH BPBoEpille e niies aEEEE 5Bo Ppa ig 5BE5zU|fiRg3C30 fH3iiEo-TiiTsEE CEiE 5m5EE 8h.re8 | = Fig] 8 CE EHEC emmin Ee i g Popa RN PoEan i EZ 1 5 Bie mE vp .EE333 EE! B zi EGERD (afeifEtiaE dfegd PBiieell HmEiSi al ogi E Bgfueed Ld PRE Es E Pal LD E Bega Pad | pla Pgaiii 1 EEEgEF ioEEEED 1&7, Eo i 000337 LEEEE PEL E H AEEeE .tPooi cemEllbEH B EgiEtt dEB s CooEil EcelEe 5 E JPfPErogoOEERiosEmELrY peed g ESuE 8 8 FEGiiomEl dhs PogboiMgEl hkaan EE R BG o E F ECaEE mEi5 e OLE (85.858, 21 [DgaaaElE | Seg Dee DRa I fiedERlEaEi2 ne EBHa Gg ht oe Eine i phpmms Po 000338 _-- CovanuMceTr6322e95-2s622 APPENDIX 6 Quality Assurance Statement Summary and Individual Hormone Analyses Data Figure 1 - Mean Estradiol Data (pg/mL-) Males Figure 2 - Mean Estradiol Data (pg/mL) - Females Figure 3 - Mean Triiodothyronine Data (ng/dL) - Males Figure 4 - Mean Triiodothyronine Data (ng/dL) - Females NOTE: This appendixofthe report contains information supplied by Ani Lytics Inc. and has been reviewed by the Quality Assurance UnitofAni Lytics Inc. 000339 203 ---- [m te--ems CAAYdTdInC?S smoumcmamsimssoen vnmssep sor Eafstubpvhe1n2Teieh3eeolg,yoSr5ee7deopnsnLoi,rnaiStesgdadelE ties,nntetd"be tbBahreatetlsofonwlaiywtadaersswrpreovoeirevtwespdsotfoeaDrrlecocBtmoapreelgiretalesnnncetewoithe sooRoe tay geritlLD LLL Tinie, SSieetietfeecntehd aedlgte same SPONSOR: COVAMCE LABS /4 seonz mires 6365, 73 4, 5h goeseits CH-5% Taadeem STWY: (327-222 ser sowse, | seid: L598 e771 oe 204 000340 Bost m2 pd 7I 88 fr szan ggEge 4 ii RN . Poggi cEFoliEe Ravlfm. i vTe o.m iBso LE BS : : 1% Sf fas ar faasar dg SPoEgg i Er iE i IEeE fling fea ls Togs fama fangs 000341 i5 FEI , oHIEf BAL EEEee sEEd Ppg AbLl i HI 5: S1E1P. T1oE8lEE E f3 G1s3 i rf vTm 3a: amu . a . aI3:2s. Flag Fig : + PPEEE e fom f Btaaamsa dste PPoOaE fi med smed5os TfR idEeaE dEopEa i. 000342 = Bom mms os LE ma ama oo , ER EEE gE 2 prib imal vad bs Fi, fly : 5 if Bil fag sf Ggsaay is @ g8 ERl odd giv Sgmd aed PRED EE E3i BES El. gE gn. id 000343 B8l0 sJ4x3x3a smear dad Yh. 4 ofPhFEonsagdn ofmga PPoEg Ltl :PoBE i, d [.I . SHE EERE RE FlE 1 iE p RB FlI yE I EE : JWU ME : Pip. sdeme PPoE ohplciiedermjfaanmianglee 22 &! 2% $4 8 2= 2% Bb8. 2 000344 r8i 7 | | /ji7 [8 / / y ]/ | 2s 1 /1 =A | / Iz Ea |i I I E 33 i i 2 !| I/ |BE3 iE2 &g : =8 |i // es33 & B5 Vd 073 | / & [Ivo 22 [Ivo | | \/ | |J \ $8838i RINgReTNe 5 (bd) 000345 Fo#1 u5l \ \t o| |//For / /|/ 3\ I @E \ |/ w | / a: |\ /I gE \| issw |\ 2| 5S5 | ! w7 |\ |/ 1:=83 i[282233 [leg /// Eel 23s /1 = 84 i g o& = frp \\ // 9 ! \ /I / | ood 4 Ln 8 8 8 2 2 8 38 8 8 2 (uid) 000346 5 | 3i%t s [8 8% 3= s | sie 1 8@ \\\ 3See2sg8 ||! s =iy \ Bs S \ cow) ;|| 3 \i I 22 \ = \ |1I . .8 \ | 5 :2 \ i s \ i 2 I 3 \\ | 2 \ E \Vo | = \ | \ \| EE EREER EREE EET ~ (Wp/Bu) 000347 o P Q i F RE $ 2 5 5 ~ 3 2 z \ J .a 2 2 5 E32 = | 4{ h\ Eggeys | \\ lEeHsH] || \\ $94] | \\ }/ \ / \ | \\ || \\ || \ j| \ I \ | \ \ |I \ | 5 8 = 9 A |i 1 1 redeem B g882883382888R8 to (pou) 000348 -_-- CovanMceTT6ee322w09.s526s2 APPENDIX 7 Dose Confirmation Analysis Report Compound Stability Report Certificate ofAnalysis Quality of Assurance Statement NOTE: This appendix ofthe report contains information supplied by the Sponsor `and has been reviewed by the Quality Assurance Unitof3M. 000349 23 -_-- Covance 6329222 wmTews DPoersfeluCroonofcitramnaetsiuolnf6Ao3nr2ai9lc-y2As2ci2is,dR(eP3pootMratsMsefidouirmcSatSlauldDtye(ptPittFenO:uSm4)b-eiWnre:CeyTk-nCo6am2po9su5ll.ge6sTMoxoinckietyysStCuodvyawnicteh Author: Andrew M. Seacat Final Report Date: 228/01 IDpnoosstoeadsucicountmfiiosrnamlatt(iKonPFaOnaSl)ystiistwrearteedpeirfloacrtmoesed,oTnhesa2m.p0lemsg/okfg/pdearlyudoorsooclteavneelswulafsonpircepcairedd aPstRaOm1Sp9lelwsacwwteorPseeFO(pSrse:apmlaparlceetdonsauetm,Cbeoarvnsa:ntchCeeL0o.nI#0241-m12g3/0-k9Ag8,/adtaahnyeddoCisLneIiFlteiv1oe2ln0wo6afAst1hperreeiinsppaerfcetdivpaethlay1)d,9e9oT9hewfhsoeew smtiuxdty.ureEiwgehrtesacomlplleecstewdeorne1co3ll.e9ct8ed.anSdamsepnltetso f3rMomEtnhveitroopn,mmeintdadlleLaabn,d Abdodirtieonoaflssch pismaimdpdlleesofwcearchcomlilxetcutreed ona5s-s2e2s-s98c,oamnpdouwnedrstsaebinltittyotnheth3eMdoEsnivnigrvoenhmiecnlteafloLmabthfeor M"eATnhtaehldyootsdicseaulcmoLnmafbaiorrrmyaattoiroynRdeaptoarwte(1r)e, cBorlileefcltye,ddsoesceorcdoinnfgirtmoatimoentwhaosd dpeesrcfroirbmeeddfbuylldyiilnuttihneg tthhee olanc-tposaeidroesaegesnatmptltersab1u0t00y-lfaomlmdowriituhmMhiyldlr-oQgewantseru,fawtheiacchcworedriengthfeontehxetrparcotceeddusrieng r(1e)s.peTchteiveelxyt,ratcotsbrriongmtCheLIPF#O1S20l5evAe1lsainndthCLIliKne1a2r0a6nAg1ewoefrtehethiennsluumcene.1F.o2r00tancdh 12 suasmepdlaes,c(o1npe,ctmiiodndlfeacatnordfoborttcoamlc)u,ltahtinagvtehreagpeerPceFnOtSrescpoivkeerdy.mantalrix ecxatsreasc,tsvaamlpuleeswawsere analyzed vearnunsexturscsted curve sing HPLC-ESMSIMS. RT1e:hs9eudlritelssuutlitosniindliaccattoesdetwhaasth8e0=ave0r.a0g2e%o<fStDhfeorttahrgeet2c.0omngc/enktgr/atdiaony.dTohsee alevveelraprge=epaSreDdfaotra tthhee t0a.r0g2etmc/okncee/ndtaraytidoonse(SleeeveTlapbrleep|aarnedacta3lc1u:l9a9t9iodnisluitnitohn.isplpaectnodsiev)w.as 54.2 0.02% of `weTrhest7a9bi%liatyndsa7m1pl%esoftthhate wtaerrgeetocboanicneendtrratoimontfhertmhiedd2l.e0oafncdatchhe m0i.x0t2umrge/okng/5d-a2y2.d9o8se dleovseilnpgrpeeprairoadt.ions respectively. This the dosepreparations ere stabi for the entire Signature; ini 10 Banca `Andrew M. Seacat Ph.D. 000350 214 _-- CMoTunEce9:5162922 Dos Ansys Report CowvanMTceTe6e329m9522e62 SL tudy wHitahnPseernlu.orJo. ocAtnaanleytsiuclaflonLiacboArcaitdRoerPfyoeRtreaepnsocseri:tufmrSoalmt t(hPeF2OS6)-WieneCkynCaopmslugleusToxicity CMPoeorvnfaklnuecoyerso6oo3cn2tt0ahn-ec2sD2ue2lt,feo6rn3ma2it9ne-a2(t2Pi3Fo,OnoS3)fMtinMheeLdPiciaarlnvDdeeSpesetnsrndruumCmeobneScrane:mnptTlrc-east2.i9eo5Pn.r1o,joefAcntalIydteinciaflcsatuidoyn: FACT TOX030, 3M Laboratory request No. U2275, 214 pp 2000 000351 25 iz ) 38 dw;;io Foyi LT TET] Hie 1| pielJ i i i ii L{iEdan 1 jot i l i HE 233] fr 8 hi iL. BE 3 alll! HH 000352 33 A i8 i BRE iPi Po Phill ! heel 1 bhp oo 2 32 i Pr1 opo IE yb REEL bT) : goal nl 0m AEE ProtE i oE IE bNTE E|p 3 000353 82 8g3id9z3 g . Eley iPolEg 3 i -2 gles: PE Fl8 :?% oz Lobb Bll you Epi HELE iB 000354 _-- cC opreee Cettn kisny , A SOhcSsu ootn Frio Septeber 6.2000 CSovatScMee6T3w6209s.5262 CPSteoutdevyreDiTehLtoamovforro,srTodor,xiPieshsaDal.oegy M$3a0d1dKoinn1sm5a3n7B0i4v Re. CCoomvapnocuend69S2a0t.2i2s(R7.e6p2o9s56), 326-23 (T6295). Peforsacunemionsie per d(Tohyev.ipneAfeu6oC2rr9s.ea2s2ta2ne,cs6ouf3l2Ao5nn.i2ac2l.syibds(pCorotefamsAsi)idnudmtssdiatlM(ePaRfroOrS,h5.eF2Co0.0t09t1Lu.otai 2t1o7nwsdtfrien s`PEcE-oiNmNpMMooRuRnn,dwaaawnssdybeeeildendemgent9noi0tcaa.l4hla9rn%aalhCycesStiFsCito1eFcf7ChASnsi0qsu3le-ostb.Ky2P+1oar7evcsicoodurssmlhcybo,iwiosnnDyonaefcmtLeCsmi/beoMrSpn,1*mH1-9N97M,R, `Additionally, '*F-NMR and 1HNMRconductedon August 24% 2000(3) were: Sai{`bchnoelcmrepvafwiroaernsdrgteosts3thiueegnnid'fhiecFta-nlmtNlMdypRieefacrforarmdepemldeeeustpenridnogohdwne.hDcTceohocmeptmohPbsseFterOsiS1ou%,fi1F9Cs9t.7wiaontLodfoSPivndor2i1con7atsoedsothvast s r-- ACdhenecMr. iSewatTP.hD.Snel Toniesoss Speci 000355 219 -_-- Peter Thomford, Ph.D. Page2 September 6, 2000 CovawnMcTTe -6e6322m99-25s262 References: 1. PDiavyifseironR.MM.arCcerhti9f,i2ca0t0e0OfAnalysis FC-95, Lot217. 3M Specialty Chemicals 2. KFCe.s9t5e,rloTt.21F7luAonraolcyhteimcailcaRleqIuseosmter$3Di0s3t0ri.buStiAo&n CbyAn'aFly-tNicMaRl LSapbe.ctrDoescceopmyb.er 1,1997 3. K"ePsFt-neNrMRT.SpCehcetmriocscaolpyC.haCraocmtpearriziastoinontoofFPC-F9O5S/2(1F7Ca-n9a5l,ysliots2f1r7o)mRbyeq"#H5-30N30M&-R Request No. 61886. 3M SA&C Analytical Lab - 236-2B-11. August 24, 2000. 000356 20 _-- lo CovanMcreT62e3299s5.26s2 CPreonn8etR.0re1es42e0A1s8032 al LFaoxb(8o10r)a2T0t1-o1e2r5i30ecs41,9)a2I0n1n1c5.00 INTERIM CERTIRFevIiCsiAo3TnE OF ANALYSIS Centre Analytica3lMLaPbroordautcotr:iePsFCOOS,ALRoetfe2r1e7nce #: 023-0184 RefPaerrietyn:c#e:86S9D%-018 ESEy IHC) 21.. CMaagneensium 35. PSoodmums! 65. INenkal 7 TooTT,IprTtpyORY "aTocurss) rparey R(eGfweendsCompan POAR Ea [rome| 21 0000001swrreasn +35 614s399wwiannn 0001wm 7._<00000051wwraishm [moran Sari] s NoreDees 033 wets org1anCAorndoes ICT 52.. BFirvoonmdee 45. NNiimes Orga7nicASPcrioidmeieatc 2TTpAea FleP5 erEEBNA Analy |2 Bcyawboogn 35. iNifworgen 5. Pome | 21 |3 5. 521. 000001905wwwiaamnnt| | i5 6 0DDo0ogmmamsn | 7 Sr 21 DOirwmaann 5 0o28lwoamnmns TThoeowreeticlalVVaauhee=oo17%.8% Theoretical abe 0 21 oRaszwasns | 5 Usman TDheeormtnaVleogI- Donee. | 3 5 satms ans common peter: 000357 21 TrCoveanMceTw66322s9925262 A CePrAnoBnetR:er(s1ee4) 231.8032 aeCotegeL,FaaPxAb(o61r4)2aT6031--t1-wo1w2r5e3icoera0sr1i,9)a2aI3bn11ec6a.8r0s INTERIM CERTIRFevIisCioAnT3E OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-0184 Date ofLast Analysis: 08/31/00 Expiration Date: 08/31/06 Storage Conditions: Frozen 10C Re-assessment Date: 08/31/06 F"lPuuorriidte=y, 033%) 01.0590%-%N(MsuRmofimmeputraitlieism,pu1ri9t0i5e%s, 1o.r4ga5n%i+cLsCci/dMiS mimppuruitire0si,38t8.%4i1P%eOsAnoA,r,ganic ToPaulriitmypu=ri1t0y0f%1rom3all.es0ts==781635%.%07% ?Potassium is expected in this salt form and is therefore not considered an impurity. o"bPusreirtvyebdyoDrStCisissagmepnleer.ally not applicable to materialsoflow purity. No endotherm was "iSnuolrfguarniicnatnhieosnammpeltehoadppceoanrdisttioonbse. cTonhveeratneidontoreSsOul,taangdreheesnwceelldewtietchtetdheussiulnfgutrhe doentethremirneasutlitosn, itnhtehSOeleismennotaclonansaildyesriesd, lsehnidmipnugrictoyn.fidence to ti interpretation. Based MHEFABA THrefplaufolrwoosrcoebtutcysreiicdacid NPFFPPAA NPoenmaaffuuoorrooppernotpasnnooiicccaicidd "Theoretical value calulaions based on the empirical formula,CiSOK" (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160), connotea Puge2or2 000358 22 _-- CovanMcrTe-6e6292s.92s5262 e CPer0on8vet:Fre6a1e0r2s31.8O0v0o2 Ste CtFLe.aaPcbA(8oT16Er)A2aOtT5_ovra1miowe1e(ss16n,2)i2a9I5b-ne1co53.r0s0 INTERIM CERTIRFevIiCsiAo3TnE OFANALYSIS `Centre Analytical Laboratories COA Reference #: 023.0184 LOMSPusyProfle: EE -- -- S-- C eooe 1 wm m sa NcaountrdevC:esG,TrsheaesnpdCeac4tridavecluyCr.nv6Tevsh.aeldLuCeiSswkveearlweucitawhlseacseuCl7,caatlevcadulluuasetiewndagustshicenalgCc4tuahleantadevdCerw6aisgntergaensdualrtadvfcearrloaimbgtrehareteisoCunl4t romtheC6and C8standcuarrveds. PrepuedBy: Charilso fom Scientist, Cente Analytical Laboratories Dsioefitor Reviewed By: in Flaherty Daote t Laboratory Manager, Centre Analytical Laborstores conmaisa Pigesofs 000359 23 CovcwSeMi6Tr32e299s.5262 33MMEENVNIRVONIMERNTOANL LMABEORNATTOARYLLABORATORY Note to File Project or Study Number: FACT-TCRO0L Associated Study Number: _L#I E00M -16S82 Thecop dtFrPEOS os 171d 17 (5000912 30018) maybecnn yrsO03 5 ilyvaeons sisWTSGirly),W277 holy, nd 801044. Qlodegradation). ee We orlorky LRiescaoCrdleedmeBny: (sf, 1 o aBsi0330011 Oplos]., Somers . 24 cHactscoyit0sgtod 000360 Cove 6329.22 _-- ress 000361 225 TCiovarnMceTe6392s952262 SponsorProtCocroleNSouFdACNTo-T0C3R3O001L8 QUALITY ASSURANCE STATEMENT reLCvotie1en7w1eS"dwptuhadasysreNesuvwimeebrweerdrb0e2vy3iC0eew1e8nd,tfeeornAtncelondyd,iucc"atCalhacrLeaarcbdtoeriranitgzoarttioieosCneSQaaudaelliyAtvoyfaAPlsyFsdOuerSaal,ncLeLaobUtno2ir1to7raiAnlsld `PSrtaacntdiacredStOapnedraartdis.ng APrlocendduirnegss,wteherSrteupdoyrtPerdottooctohl,SatnuddylDl iapcplticaablnedGtoomoadnaLgaebmoernetr TM lomDeuccted SDuebRyeDpioreeotitod Conve Mangement SoopDsueeRMespsorsiedmieont | ProcolReview 31900 somo amo G2 aLbiice 714m me pending Pa3erpSaasynitdnywdwSdoion 2@m020 aeame nmaanmo 4oRaawDiaRewew M00 sw Ponting FS5.reoSpurnoadia)orn Soluion 71400 000 Pending FrpSuusnidoundSoosen 62T9a.a30m00 3m0000 Pnenadimng 7sRoeponnRevew samo won Pending In8tDeruimRReevpioerRwesvnidew 48900 soo Pending , 27k TQuiaality AssWuirngance Offices Conse Arts Lorri oe, S90 Bw 4E4sOR1GIaNAcL tDOCcUoMnEoNr*T SE pA pe 000362 26 Covance 6329222 - mTewss APPENDIX 8 Cell Proliferation Report NOTE: This appendix ofthe report contains information supplied by Pathology `Associates International and has been reviewed by the Quality Assurance Unit ofPathology Associates Intemational. 000363 27 Covce 632922 -_ MTewse A CoPaomtfhoSlpcoagryAcasesAopcpnicaattyeosnistieermmaattioonnaal Coors EE. a COP motes aL CELLPROLIFERATION REPORT . WEEK CPAOPTSAUSLSEITUOMXIS"CACILOTTVYA(SNPTRCUOESDS,YTTW-U6ID2YT9NH9IUPNMECBYERNROF6M3LO9L.UG13UOSRMOOSNKUOELCYFSOTNIACANCIED PREPARED FOR: BUITLoDxINCGo2L2O0.Ga2Y6.S0E2R,3VMICCEENSTER STPAUL, MN $51441000 PREPAREBYD: PATH1O5LWOGOFYRAESDRSEORCMIMIICALKT,LAEMCSODIUNN2RT1T7E6,R'1SNUAITSTIEO1NAL NOVEMBER 11, 1999 TE emt Go Sa Fr Ve 0GAG 000364 228 Covance 6329-222 3MT-62956 `CoFvianaacleSCtealdlyPNroulmifbeerarti6o3n2R9e-p22o2r.t Page2 CELL PROLIFERATION REPORT 4-WAECEIKDCPAOPTSAUSLSEITUOMXISCAILTTY(SPFTOUSD;YT-W6I29T5H)PIEN RCFYLNUOOMROOLOGCUTSAMNOESNUKLEFYOSNIC 'COVANCE STUDY NUMBER 6329-222 PURPOSE d`Tohseepaunrdpolsoewoefrdtohsesetleuvdeylws1astboeuprsoevdidinedaactharfoonricdettoexricmiitnyisntguadyneasntditmoataessdmeassxtihmueme-ftfeoclteorfattheed testmaterialoncriticalenzymelevels, hormones,andotherselectedbiochemicalparameters. r`Tehpirsesreenptosrtt,hesucbelmlitptreodlifbeyraPtaitonhofliongdyingAsssaoncdiaitnetserpIrnetteamtaitoinofnoarl C(oPvAaYn)cteoStthuedysNtuudmybSepron6s3o2r,5-32M2,3 entitled (PFOS; *4-Week T-6295) iCnapCsyunloemoTolxgiucsitMyoSntkuedyys"w.ithAlPlerafslpueocrtosocotfanteheSutlafsoknsicasAscociidatPeodtawsistihumPASIa'lst `pGoorotidoLnaboofrtahtiosrsytPurdayctwiecree (cGoLnPd)ucRteegduliantcioomnpslsiasnectefowritthhitnhTeitElnev4ir0oonfmetnhteaUl SPrCootedcetioofn FAegdeenracly Regulations, Part 792, issued November 29, 1983 (effective December 29, 1983), and with any applicable amendments. MATERIALS AND METHODS `Tissue Collection for Cel Proliferation ``Tawndooanenaniimmpaaleprlesressxeixncionndtorsoelggrroouupp13,(2t.h0remega/nkigm/adlasyp)ewresreexsiacnrdiofisceegdarfotuepf2ou(r0.w0e2emkgs/okgn/ddiaeyt). A representative sample fixed in zine formalin, of the liver, testes (males only) and processed and embedded to paraffin pancreas block by from each animal was Covance per protocol specifications. block, a slide Tissue blocks were was prepared for then shipped to PAI for H&E evaluation and sectioning and staining. immunohistochemical From each detection of proliferating cell nuclear antigen (PCNA), a markerofcll proliferation. Immunohistochemistry for Cell Proliferation Sections of (Superfrost pPalursa,ffFiins-heemrbeScdideendtitfiiscs,uePsitwtesrbeurcguht,atPA) um to and placed on positively ensure adhesion during charged slides processing for PCNA. Standard SOtpaenrdaatridngimPmruoncoehdiusrteofcohremIimcmaulnomheitshtoodchsewmeircealusSetdaitnoinsgta(inSOtPiss#u7e0s7)f.or PCNA Briefly, (PAI's tissue sections A1723) were incubated with a and reagents required for monoclonal antibody to PCNA (DAKO, the avidin-biotin peroxidase (ABC Kit, lot lot #016, #PK-6100, PAI PAI No. No. Kloc3a2l4i)zemdebtyhotdhefocrhtrhoemdaegteenct3io,n3o'fdtihameinaonbtiegnezni-dainnteib(oDdAyBc;omSpilgemxa. PCNA expression Chemical Co. lot in cells was. #18H8201). `Tissue sections were counterstained with hematoxylin 000365 229 _--- CovManITcMeTe6-3w6292-s9252s62 Cell Proliferation Measurements: CoFinvalSCaiuldnPNrouclmifbeserarti6on32Re9p.o2r2t aged 3`0Th0e0hpeerpcaenttoacgyeotfeipnsrol1i0ffeirealtdisongfcleilvlesr,(aprtolleiafserta5ti0n0gLienydedsi,gPcIe)lwsoasftdheetetremstiense(dmablyesscoonrliyn)g,aatnldeaastt slteaaisnti2n0g0r0uancainndarcceollns osfitohsfestptaeundcdyrteiasssupeetrhaantiwmaasl.notAinnecguabtaitveedcwointthrtohlselpirdiemwaarsyainnticboldyu.idnethde qFuoarlicteyll opfrosltiafienriantgi,onpervoacleusastiinognsa,nsdlisdeecstwioenriengf,irsptotpeenrtuisaledpaattltoemwsmaogfnicfelilcualtaironpr(ol1i0f0e%ra)titoonj,udagned. fohirsltiovmeorr,pahnodlo4g0i0cXchafnorgtese.steCselalndpproalnicfrereaatsi)oan swdaesscthreinbeqduaanbtoivfiee.daHihisghterommagonirfipcwahtaiosonflu(or2t0gh0eyXr eavsasleusasteeddbfyorccvealllupartoilinfgertathieonH. &E slide prepared from the same tissue block for cach animal Statistical Analysis Due to the smal sample size, statistical analysis was not performed. RESULTS Cell Proliferation iInndtihveidluivaelr,atneismtaesl acenldl pparnoclirfeearsatoifoncdoanttraoalraendprteessetmnatteedriinalStercetaitoendIIan(iTmaabllsew1a).s sCiemlillaprroliferation Histopathology wSeictthihonesmaftrooxmyltihne asnadmecotsiisnsu(eHbAlEo)cksforusheidstfoopratphroelpoagriactieovnaolufaPtiCoNnA-tsotfsaciinleitdatselitdheesiwnteerreprsettaaitinoend ofthe immunostained slides. Individual animalfindings are presented in Appendix 1. sSeicntgiloenssoecftvioenrsaonfdplaivnecr,repaasncfrreoasm,6aynoduntgesatdesultfrfoemmal6eymoounnkgeyasdurletprmeasleentimnognckoentyrsol,anldow-sdionsgel:e B(0a.c0h2 tmigs/skuge/sdeacyt)i,onawndashisgtha-idneodsew(i2t.h0hmegm/aktgo/xdyalyi)n tarnedatemoesnitn.grTohuepspwuerproeseevaolfutahtiesdehviasltuoaltoigoincawllays. talotedreotrercmoinnfeouwnhdetthheerspoercinfioctitmyoropfhtohleoPgiCcNAchastnagiensingweorbeseorcvceudrriinntgheisnetahneismealtsi.ssues which may t`eTshteicruelsaurlttsissshuoeswefdrotmhatmanloe smiognnikfeicyasntocrhtahnegelsivewreraendobpsaenrcvreedasintiesistuheesr ftrhoemlifveem,alpeanmcorenakse,yso,r `which wouldalter the interpretationofthe PCNA staining in this study. 000366 230 -_-- `Covance6329-222 M3MTT-e62s9566 DISCUSSION `FoaClSCteulo dlyPNrum omlbiefra e63r2aR9tee -pi2oo2r2nt. Paget `Imnotnhkeepyrse,saenndttsthuedyl,ivceerlalpnrdolpiafnecrraetiaosnowfafsemmeaalseumroendkweiythsifnrtohmecloivnetrr,toels(te0smagn/dkgp/adnacyr)e,alsoowfdmoaslee a(n0e.0s2timmga/ktge/ddmaayx)i,maunnd-thoilgehrdaotesdedo(2s.e0amngd/kgl/owdearyd)ogsreoluepvsealfstoerbfeouusrewdeienksaochnrsotnuidcyttoxoidceitteysrtmuidnye saenledcttoedasbsieolsosgtihcaelepfafreacmetotefrst,hetotiensctlumdaetceerilalporonlicfreirtaitciaolne.nzyme levels,hormones, and other ``Tphaenrceredaisd,ansodteatpepremairnetodbbyepracoleilfleprraotliinfgeirnatdiivceersewshpiocnhsewteortehsiemtielsatrmaitnaelrliaalniimnatlhse. liver, testesor SUMMARY nIonttihnecprreeasseendtisntuthdeyl,ivceerl,ltepstresoorpalnciaresafdseotefermrmoinnkaeedybts.yimeaosunrin,g the proliferating index, was 000367 231 Covance 6329-222 _---- MT6%56 CoFraicseSCtauldlyrNoulmfbeeriro6n32R9e.2g2t2 ILTABLE 000368 232 Cone 12922 -_ Tews TABLCEoT.vCaEnL cPeROsUPoEoRA,IOeNsR9V2O2R2IEYS [2-002monaryGowcose| ioasir|--Oave Oran Sinamay |aTiosse ota sox si] tros |sare] [[22-000%2mmaaricoorGGoowwddeessee |FTiioossssessr||--0--0e05%t00a%--]|----isain--m--||| = 000367 Covance 329-22 -_-- wrews ComFei sSatyPmroet5eR5ip8n APPENDIX 000370 234 - TCRovanTEcea6e3m29.e22 [I -- Individual AnimeawleHekiCaspsutleoToxpFiicniatdyitnSgtsuhd-yMoalle Moongkeyys FNurmber Ter Tom == [105345 |Nosignificant ndings|Nosignet dings|Nosgn:Figs| Individual Animal Bstopathology Findings - Female Monkeys 4-week Capsule Toxicity Study HNuimbler [105367 Toe |Nosignificantfindings [Nosgnificantfindings] [l05370 [No igifcant ndings |Nosignificantfindings] 00031 235 - Comme 629.22 wmTewme ILSIGNATURE PAGE WekCale ToitSadywithPeorsocianeSlo Ac PotasiumSalt(FOS; "T-629i5n)CynomolgusMonkeys (CovanceStudyNumber6329-222) swine f---- (SieJ. BVeSPDp: Eo742-99 PAP Publis: E4T. SN7,ra fh = ii 0005.2 236 Covance 6329222 IM T-62956 CoFviannacleCSltuldPyrNoulmifbeerra6on3R2e9po2r2t2 IV. QUALITY ASSURANCESTATEMENT VOUS 3 237 CovanMceT639295-2622 PathAomloorgyyASsosaocimarteesrIentaercnaamtieornal EA :--. Cell Proliferation Report WeePkotCaaspssiuulmeSaTlotri(cPiFySOt;udTy-6W2i95t)hiPneCrfyinuoomrooolcgtaasnMeoSunlkfeonyiscAcid CoranceStyNmber: 6329.22 QUALITY ASSURANCE STATEMENT TArhiimsecoeelmpUrntbifpteyr(atQeihAoeUnU)dpSsrtj.eecgEtibeassmbbyeGniisdPpnrctteLocnadiboaonredaAgdnePirecd;in(bcSysC(lAGheL)P.)PrAeIhgQeiuaolnaistly recoo aaf nbenepposct nsciesprroenceooftreeporretcedodbeyddhuea.GATLh:eFlowingabe 3 InmDeasiteaiton Pome mstst MaDantsesFcinmdeinnlgSRaedpoyrPtaebdotoosPiAsLs aGs2u7s05MR1058 TSSuipympeoDrLiaanbageslDniodncgumeation oss oonoss 0W8i72s70s508.1098 FDirsCCellllPPrakitraoinonRReeppoorrtt woess xr Som BB aie M Qual Arcs Spins uh Die m Teoar Co an rac Hand 1701 GO El GOGLE ud 74 28