Document NRvExx84Xr5e0d9v6VzjnLVb

RECEIVED OPPT MCIC AR 226 - 06IS REGEN ore . W00AUS -3 PH 2: 38 andes i Final Report - 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats PREPATMRED FOR COVANCE STUDY NUMBER: 000003 COVANCETM - Spo3nsMor. St. Paul, Minnesota FINAL REPORT Study Title: . 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats Author. Peter J. Thomford, PhD - Study Completion Date: June 30, 2000 . Performing Laboratory: Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 - Laboratory Study Identification: Covance 6329-225 Sponsor Study Identification: Study No. 3M T-6314.1 Page 1 0f 560 - 000004 Covance 6329-225 -_ wre - `QUALITY ASSURANCE STATEMENT `This report, with the exception of Appendix 7, hasbeen reviewed by the Quality Assurance Unitof Covance Laboratories Inc., in accordance with the Food andDrug - AAdpmpiennidsitxra7tiiosnge(nFeDrAa)teGdoboydanLdabiosrtahteorreyspPornascitbiicleitRyeogfutlhateioSnpso,ns2o1rC. FTRhe58.folTlhoweidnagta in imnasnpaegcteimoennstwere conducted and findings reported to the study director and study director Tspection Date Reported to = Dates Study Director and From To Phase Study Director Management 08/31/98 08/31/98 Protocol Review 08/31/98 08/31/98 08/31/98 Material Dispensation 08/31/98 04/12/99 04/29/99 Report Review 04/29/99 = 04/02/99 04/07/99 Data Review 05/04/99 06/02/99 06/02/99 Protocol Amendment Review 06/02/99 04/19/00 04/28/00 04/28/00 04/20/00 04/28/00 04/28/00 Report Review Protocol Amendment Review Protocol Amendment Review 04/20/00 04/28/00 04/28/00 - CRepe resenr tativi e fo thstn Quality Assurance Unit Dat So, sore 2 000005 _-- STUDY IDENTIFICATION Covance 6329-225 MTeM 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats - `Test Material N-Methyl Perfluorooctanesulfonamido Ethanol (MeFOSE, T-6314) Sponsor - M Toxicology Services `Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 Study Monitor Andrew M. Seacat, PhD 3M Toxicology Services 651.575.3161 Alternate Study Monitor A Marvin T. Case, DVM, PhD 3M Toxicology Services 651.733.5180 Study Location Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2505 - Study Director Peter J. Thomford, PhD Covance Laboratories Inc. P.O. Box 7545 Madison, Wisconsin 53707-7545 608.241.7207 Study Timetable Study Initiation Date In-Life Start Date In-Life End Date Study Completion Date August 27, 1998 September 1, 1998 December 2, 1998 June 30,2000 3 000006 CovanIcMeT6-36239-124215 KEY PERSONNEL Study Director Peter J. Thomford, PhD . Study Toxicologist `Thomas E. Ryan, BS Study Coordinator Nii Van Bruce-Konuah Supervisor, SmallAnimalToxicology Nathan E. Snortum, BA, BT, ALAT . Supervisor, Dose Formulation Dixie Bushee, BS, LATG Supervisor, Chemistry/Tox Support Brian Schoenike, BS Supervisor, Laboratory Animal Medicine Donna J. Clemons, DVM Diplomate, ACLAM - Clinical Pathologist Robert L. Hall, DVM, PhD. Diplomate, ACVP (Clinical Pathology) Supervisor, Clinical Pathology Ronald Markevitch, BS, MT (ASCP) . Anatomical Pathologist Sandra R. Eldridge, PhD Pathology Associates Intemational Anatomical Pathologist Johnnie J. Eighmy, DVM, MS Diplomate, ACVP Diplomate, ABT - Supervisor, Anatomical Pathology Deborah Pirkel, ALAT = 4 000007 CONTENTS CovanIcMeT6-36239-124215 Page - EL R---- REGULATORY COMPLIANCE... || J SS -------- ANIMALS AND HUSBANDRY ...c.crmssssssssssnnns 12. J Group Designations and DOSE LEVELS... a 13 ---- | FT ----| FE OPHAIMOIORY ------| rrr | ~ SCeHruCm PPerAflRuOorIooOcGtaYnet Sulfonic Acid Lr evel (PROS) Di ECrMnations ................. 11 18 OIG Cell WEIGHS... Proliferation Tissue Collection and Immunohistochemical EValuation............ 19 19 - Palmitoyl-CoA Oxidase LIVE PROSARISES Tissue Collectionand ARGIYSeS. rns rrr 19 19 FT ------------ J ------ mmm------0 - RECORD RETENTION ..coccrrmmmmmmmmmmsssssssssssses21 - 5 000008 CONTENTS(Continued) CovancMeT6-36239124.215 Page Clinical OBSEVations And SUNVVAL ovr OPBURITIOIORY rrr A 23 2 ---- _ E CHC]PALBOIe ORY rrr 2 CSeelrluPmroPleirfferlautoiroonoTcitsasnueeSuCloflolreictciAocniadndLeIvmemlu(nPoFhiOsSt)oDcehteemrimcianlatEVilouna.ti.o.n............................2255 AJ RQIOC PALROIOBY wrens 25 CONCLUSIONS cnt:26 SIGNATURES cernssss: 21 OPHTHALMOLOGY REPORT crn 29 PATHOLOGY REPORT: 30 COMONM THEEDATNAc TS ss31 CODES, ABBREVIATIONS, AND UNITS... Ee 39 ------ CodesfOr Clinical PAROIORY rrr 81 - `AAbbbbrreevviiaattiioonnssaannddUUnniittssffoorr CClliinniiccaall CHEHMEAMOIISOTRYY.v .. ce 46 `Abbreviationsand Usfor ClinicalUMAIYSIS Je rcs 88 ------ TAB1LREeSsults of HomoAgnaleysnes(ePPiI) ty ....ccrinsnsns 31 2 3 RReessuullttssoOffSDtOabSiElitPyreApBaIraYtSieosn A(RPAlPY)Ses crn (ppm)... 32 35 4 5 S Summu aryoOm ffCOlpihnm tichaalla OtiDc OSr BCSIEy VIAVOANLSIO.N.S........v.rrvcrvrrvrnrcssr58s6 . 6 7 Summaryof Summaryof Body Body Weight Weight DCahtaang(e 8Dat)(.g).. 39 cscs 61 8 Sum ofFm oodCaONSrUMPy UON Da (g) rrr 63 3 6 000009 CovanIcMeT6-362391-242.51 CONTENTS (Continued) Page TABLES ~ 9 SummaryofTest Material Consumption Data (Ig/kg/day) ........vwe:65 10 Summ ofa Clirnicyal Hematology Data- Week S..........ocvwuvsssusssisssssssssssnns 67 11 SummofaClirnicyal Hematology Data- Week 14.........occouverrrrrsnnsssssssisssiiens 1 12 Summary ofClinical Chemistry Data - Week 5.........ccocumummsrsmmmsmssssssssssssns 15 13 Summary ofClinical Chemistry Data - Week 14........cccovvrsvsssnsssnssssssssnsrsnnns 81 N 14 Summary ofClinical Urinalysis Data-Week 14 ......oocvvvrsvsssnssssisssssssssrerens 87 15 SummaryofOrgan Weight Data - Week 5 SACHfiCe .....o..vvvcrrrcrrrrcersisssssesrnes 89. 16 Summary of Organ Weight Data - Week 14 SQCTHICe rrr: 91 17 18 InciofdMaecrn OSCcOPe IC Incidence of MicroSCOPIC OBSEIVALONS OBSEIVaLiONS ......vccvvevrresessssssssnssssssssssssesssssssssssens ......vvrverrerrrrmrrrrssssssssiss 109 112 19 Incidence of Severity of Selected Microscopic OBSErVALONS.........rwwrwrns 121 Protocol Amendment NO. 1 ..cc....ceorsuusrrsssmssssssssssssssmsssssssssssssssssssssssssssssssssssess 144 - Protocol Amendment NO. 2 .....c.cewusuessssssssssssssssssssssssssssssssssssssssssssssssssssssssses 146 Individual Animal Fate Data...........oouusssssmsssssssmssssssssssssssssssssssssssssssssssssssssssses 152 _ `Individual Clinical ODSEIVAONS........cccccvversssssssssssssssssssssssssssssssssasssssssssssessasessss 157 Individual Ophthalmic OBSEIVAHONS......oceversssrsssssssssssssssssssssssesssssssssssssssssss 113 Individual Body Weight Data (8) .c.vewerersrisrrrsmmrmrmsssssssssess 190 Individual Food CONSUMPLON Data (8) v.vvvereevemmvsmsmsssnsssrrrrners 202 Individual Test Material Consumption Data (Mg/Kg/day).........c.cewwrsmsmsessssssssssenses 213 IndividualClinical HematoDlatoagy ......swesmsmsmsinsrssrssssrrmssse2rs4s - Individual Clinical ChEMISTY Data .........ccoocvverssssssssssssssssmsssssssssssssssssssssssssssssess 248 Individual Clinical UHRAlYSis DAta............ccceverssssssrsssssssssssssssssssssssssssssssssasssssss 216 FS I -- : APPENDI7.X 546 Cell POHICTAON REPOM cesveerressceersrrsssssssmssssssssssssssssssssssssssssmeersssssseessssasessasssse SAT - 7 000010 ABSTRACT CovanMce 6T329-]225 "The purposeofthis study was to assess the toxicity ofthe test material, ~ 'N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) when administered in the diet to rats for at least 13 weeks. Four groups of Crl:CD(SD) IGS BR rats were exposed to N-MeFOSE in the diet for at least 13 weeks. Each group contained 20 males and 20 females. The dietary concentrations were 0 (control group), 3, 30, and 100 ppm. Food was provided ad libitum, except when animals were fasted. Water was provided ad libitum. Theanimalswere observed twice daily (a.m. and p.m.) for mortality and - `moribundity. At least once weekly, each animal was removed from its cage and examined for abnormalities and signs oftoxicity. Body weight data were collected on the first day oftreatment and weekly thereafter. Food consumption data were collected weekly. Ophthalmic examinations were done before initiation of treatment and during Week 14. - Blood samples were collected for hematologyand clinical chemistry tests during Weeks 5 and 14. Urine samples for urinalysis tests were collected during Week 14. During `Week5s and 14, blood was collected from five animals/sex/group for serum perfluorooctane sulfonic acid (PFOS) analyses. At the Week 5 sacrifice, samples of the ~ liver were collected from five animals/sex/group for palmitoyl-CoA oxidase activity and liver PFOS analysis. Animals sacrificed at Week 5 were not necropsied. During Week 14, theremaininganimals were anesthetized, weighed, exsanguinated, and necropsicd. At necropsy, macroscopic observations were recorded, selected organs were weighed, and selected tissues were collected and preserved. Samples ofliver were collectedfromall - `surviving animals for palmitoyl-CoA oxidase activity and liver PFOS analysis. Samples of the liver were collected from five animals/sex/group for proliferation cell nuclear antigen (PCNA) evaluation and microscopic examination. Microscopic examinations were done. on tissues from each animal in the control and high-dose groups (terminal sacrifice only) - and from the low-dose animal that was sacrificed at an unscheduled interval. Microscopic examinations were also done on the liver and stomach from each terminal sacrifice animal iin the mid-dose group, and on the liver from each terminal sacrifice animal in the low-dose: group. : 000011 CovanMceT633219-42125 One female given 3 ppm N-MeFOSE was sacrificed on Day 59 due 10 severe laceration of the tail. There were no macroscopic or microscopic changes to indicate that its moribund condition was test material-related. All otheranimals survived 10 the scheduled sacrifice. No clinical observations were noted that were considered to represent primary effects of the test material. Statistically significantly lower mean body weights were noted beginning at Week 4 and continuing through the endofstudy for animals given 100 ppm. Statistically significantly lower mean body weights were noted beginning at Week 7 and continuing through the end of study for males given 30 ppm. Body weight changes were - statistically significantly lower in a sporadic manner for animals given 30 or 100 ppm. `Overall body weight changes (Weeks 1-14) were statistically significantly lowerforall `groups given N-MeFOSE. Although statistically significant only at Week 13, food consumption was in general lower for males given 100 ppm. For females given 100 ppm, - statistically lower food consumption was apparent at Weeks 3, 4, 6,7, 11, and 12. Dietary administration of N-MeFOSE was associated with lower hematocrit for males and females fed 100 ppm;higherurea nitrogen for males and females fed 100 ppm; higher - albumin for males fed 100 ppm: lower globulin for males fed 30 or 100 ppm; lower cholesterol and triglycerides for males fed 30 or 100 ppm and females fed 100 ppm; higher alanineaminotransferase for males fed 100 ppm; and higher hepatic palmitoyl CoA oxidase for males and females fed 100 ppm. The effects were relatively mild with the exception of those on cholesterol and triglycerides for the high-dose males; those effects - were considered to be of moderate severity. All of the test material-related clinical pathology findings were more prominent at Week 14 than at Week 5. Statistically significant and test material-related changes in terminal body weights and liver - weights were seen in males and/or females in the two highest dose groups. Statistically significant decreases were seen in the terminal body weights of males fed 30ppmand in both males and females fed 100 ppm of N-MeFOSE. Statistically significant increases were seen in the mean liver-to-body weight percentage and mean liver-to-brain weight - ratio in males fed 30 ppm, in mean absolute liver weight and mean liver-to-body weight percentage in fomales fed 30 ppm, and in al liver weight parameters in males and females fed 100 ppm. 9 000012 CovanMceT631294-2125 Testmaterialrelated macroscopic findings were noted ata low incidence in theliverand. `glandular stomachofmales and femalesin the 100 ppm dose group only. Liver findings included organs that were large and diffusely dark. In the glandular stomach, dark foci or . areas weresometimesseen in the gastric mucosa. Test material-related microscopic findings were seen in the liver ofanimals fed 30 and 100 ppm of N-MeFOSEand in the glandular stomach of males and females in the 100 ppm group only. No test material-related changes were seen in animals fed 3 ppm of - N-MeFOSE. In the liver, one or more test material-related findings affected all animals fed 100 ppm and included slight-to-moderate centrilobular or diffuse hepatocellular hypertrophy, minimal-to-slight coagulative necrosis, minimal hepatocellular pigment, and `minimal-to-slight centrilobular to midzonal or diffuse hepatocellular vacuolation. In - animals fed 30 ppm, centrilobular hepatocellular hypertrophy was also seen, although the incidence and severity was decreased at this dose. In addition, a low incidenceofminimal hepatocellular vacuolation occurred in 30 ppm dose group males, and the liver oaf single female fed 30 ppm hadminimalcoagulative necrosis. A possible test material-related - change in the glandular stomachofanimals fed 100 ppm was arelatively low incidence of `mucosal erosions. The erosions wereofminimal to slight severity and involved a very small portionofthe mucosal surface in all cases. . Based on anatomic pathology, clinical pathology, and body weight effects at 30 and 100 ppm the no-observable-adverse-effect level was determined to be less than 30 ppm when N-MeFOSE is administered at concentrations of0, 3, 30 and 100 ppm to Cr:CD(SD) IGS BR rats for at least 13 weeks through dietary administration. Based on the body weight effects at 3, 30, and 100 pp, the no-observable-effect level was determined to be less than 3ppm when N-MeFOSE is administered at concentrations of 0,3,30 and 100 ppm to Crl:CD (SD) IGS BR rats for at least 13 weeks through dietary administration. In the absenceofcorrelative changes in anatomic pathology and clinical pathology the body weights effects at 3 ppm were not considered to be biologically - adverse. - 10 000013 CovanIcMe 6T36291-42215 PURPOSE The purposeofthis study was to assess the toxicity of the test material, N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) when administered inthediet toratsforatleast 13 weeks. REGULATORY COMPLIANCE All aspectsofthis study were in accordance with theFoodand Drug Administration Good Laboratory Practice Regulations as set forth in Title 21 of the US Code of Federal Regulations, Part 58, issuedDecember22, 1978 (effective June 20, 1979), and with any - applicable amendments. TEST MATERIAL Test Material "The test material, N-Methyl Perfluorooctanesulfonamido Ethanol (MeFOSE, T-6314), Lot No. F-11364, isanoff white powder. It was received at Covance on August 3, 1995. `The test material was stored at room temperature. Information on synthesis methods, stability, purity, composition, or other characteristics that define the test materialison file with the Sponsor. Reserve (Archive) Sample A reserve sample (approximatel5y g) of the test material was taken and stored at room temperature. This sample will be transferred to the Sponsor after authorization from the Sponsor. - Disposition Remaining test material will be returned afer authorization from the Sponsor. . 1 000014 Covance 6329-225 e--e----------------eeereeeereerreeReEeL ANIMALS AND HUSBANDRY Test Animal N Male and female Crl:CD(SD) IGS BR rats were obtainedfromthe Portage, Michigan, facilityofCharles River Laboratories, Inc., on August 17, 1998. The animals were 39 to 45 days old at initiation of treatment. The males weighed from 184 to 236 g, and the females weighed from 140 to 179 g at initiation of treatment. Identification Each animal was assigned a temporary number upon arrival. Before initiation of treatment, a microchip identification device was implanted into each animal. After - randomization for placement on test, eachanimalwas assigned a permanent number, and the microchip was coded with that number. All data for an animal are recorded under these numbers. Acclimation Ninety-two males and 92 females were received on August 17, 1998, and acclimated in Animal Room 348 for 15 days before initiationoftreatment. In general, animals in this . shipment appeared healthy. During acclimation, the animals were examined for abnormalities indicative of health problems, an ophthalmic examination was done, and body weights were recorded for all animals at randomization. - `Housing and Maintenance Animal Room 348 was used for this study. Environmental controls for the animal room were set to maintain 18 to 26C, a relative humidityof30 to 70%, and a 12-hour light/12-hour dark cycle. "The animals were housed individually (except for the first 7 days of acclimation when animals were group-housed) in stainless steel, screen-bottom cages. 12 000015 CovanMcTe 66332104-2.215 Certified rodent diet #5002 meal, (PMI Nutrition International) was provided (see Protocol Deviations for exceptaidloibnitsum), except when animals were fasted. The diet is routinely analyzed by the manufacturer for nutritional components and - environmental contaminants. The results are on file with Covance-Madison. Waterwas providedadlibitum. Samples of the water are routinely analyzed for specified microorganisms and environmental contaminants. The results are on file with - Covance-Madison. "There were no known contaminants inthe diet or water at levels that would have interfered with this study. Animals not selected for the study (12 males and 12 females) were removed from the Study room and used for training procedures. Justification Historically rats have been used in safety evaluation studies and are recommended by appropriate regulatory agencies. PROCEDURES "This study was conducted in accordance with the Protocol dated August 27, 1998, and - Protocol Amendment Nos. 1, 2, and 3. The protocol, protocol amendments, and protocol deviations are in Appendix 1. - Group Designations and Dose Levels The animals were examined by a laboratory animal veterinarian on August 31, 1998, and found to be suitable for study consideration. Selection of animals for the study was based on clinical observations, body weights, ophthalmic examinations, 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. At the timeofrandomization, the weight variationofthe animalsforeach sex used did not 13 000016 Covance 6329-225 eeeeererm------------eeSXe EEe exceed 2 standard deviations of the mean weight. Group mean body weights were analyzed using Levene's test for homogeneityofvariance at the 5.0% probability level and found to be homogeneous. Animals were assigned to the study according to the following ~ design: Group NuMamlbeerofAnFiemmaalles Dietary Levels. (ppm MeFOSE)* 1 (Control 20 20 0 - 2 (Low) 20 20 3 3 (Mid 20 20 30 4 (High) 20 20 100 a Dose levels were expressed as ppmof McFOSE. b The control animals received the basal diet only. Dose Preparation Dietary concentrations were based on the test mateasrsuipplaield. Before initiation of _ treatment, diets of 1,3, 30, 100, and 500 ppm concentrations were mixed. Diets were prepared for stability analyses at the 1-ppm and 500-ppm concentrations and for homogeneity analyses at the 3, 30, and 100 ppm concentrations. Diets were prepared once every 4 weeks for use in the in-life phase. - PreparationofDiet for Group 1. The appropriate amount of diet was weighed into a labeledcontainerand acetone was added at the same concentration as the high-dose concentration. `The combination of the diet and acetone was then mixed for 15 minutes. - PreparationofDiets for Group2s through 3. Each dose level was prepared independently. Aspecified amount of diet was weighed into a labeled container and a specified amount of diet for Group4 was weighed for each diet. A trench was formed in the feed in the mixing bowl and the amount of diet for Group 4 was transferred to the . trench. The contents of the mixing bowl were thoroughly mixed for 15 minutes. Preparation of Diet for Group 4. A specified amountofdiet was weighed into a labeled container. The required amountoftest material was weighed and transferred into a Iabeled container. Approximately 5 mL of acetone was added to the container and mixed manually. Incrementsof approximately 5 mL acetone were added as necessary until the test material had dissolved. To prepare a premix, the diet was transferred into a labeled 14 000017 CovanMceT6633291-42215 Hobart mixing bowl. The test material and acetone were added to the mixing bowl, overlaid witah portionofdiet from the mixing bowl, and thoroughly mixed. A portion of diet from the mixing bowl was transferred 10 a second mixing bowl, mixed manually to ~ recover residual test material, and returned t0 the first mixing bowl. The contentsofthe mixing bowl were thoroughly mixed. `Samples for dose analyses were taken directly from the mixing bowl. - The prepared test diets were stored at room temperature in covered containers until dispensed into feeding jars. - Retention Samples During the in-life phase, samples (approximately 100 g) were taken from cach dose: preparation for dose analyses and stored at room temperature. These samples were discarded on May 11, 1999 (see Protocol Deviations for exceptions). Method of Administration Dietary admixture was used because the potential routeofexposure in humans is oral `The dose preparations were administered ad libitum for at least 13 weeks, unless otherwise specified. - Dose Analyses Analyses for the concentration of test material in the dose preparations were done by `Covance using an analytical method, MP-M324-MA, supplied by the Sponsor and validated by Covance. Homogeneity was determined for the 3-ppm, 30-ppm, and 100-ppm dose preparations once pretest. One sample (approximately 100 g) each from the top, middle, and bottom of the dose preparations mixed for homogeneity analyses were collected, divided into . three subsamples for extraction and analysis, and analyzed for test material content. All samples were stored at room temperature until analyzed within 7 days of mixing. - 1s 000018 CovanMcTe.6632391-42215 Four samples (approximately 100 g each) were taken from the 1-ppm dose level concentrationofdiet preparations mixed pretest. One sample was analyzed on the day of `mixing and used as the baseline value. One sample was stored at room temperature for at least 19 days, then analyzed.Athird sample was stored at room temperature for at least 32 days, then analyzed. The remaining sample was stored in a freezer set to maintain -10 to -30C for 8 weeks, then analyzed. In addition, two samples (approximately 100 g each) were taken from the S00-ppm dose preparation mixed pretest. One sample was analyzed on the day of mixing. The second sample was stored at room temperature after - at least 32 days, then analyzed. Two additional samples (approximately 100 g cach) were takenfromthe 3-ppm dose preparation mixed for Weeks9 through 12. One sample was stored at room temperature for at least 19 days,thenanalyzed. The second sample was. stored at room temperature for at least 32 days, then analyzed. The sample collected for dose confirmation analysis was used as the baseline sample. During the in-ife phase, samples (approximately 100 g) from all dose preparations were collected and analyzed for dose confirmation. All samples were stored at room ; temperature until analyzed. Clinical Observations _ `The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity (see Protocol Deviations for exceptions). Signs of poor health or abnormal behavior were recorded as they were observed. At least once weekly, each animal was removed from its cage and examined. Any unusual or abnormal findings were recorded. Body Weights Individual body weight data were recorded on the first dayoftreatment, and weekly thereafier. Food Consumption Individual food consumption data were recorded weekly during treatment. Food consumption was not measured during Week $ for animals scheduled for sacrifice during Week S. 16 000019 CovanIcMeT6-32391-24215 Ophthalmology `Ophthalmic examinations were done before initiationoftreatment and during Weck 14. The pupils were dilated with 0.5% Mydriacyl, and the eyes were examined by a . veterinarian with an indirect ophthalmoscope. Clinical Pathology Blood samples were collected from 10 animals/sex/group during Weeks and 14. Urine - samples were collected from 10 animals/sex/group during Week 14. Animals were fasted `overnight, (see Protocol Deviations for exception) and urine was collected chilled (approximately 16 hours before blood sampling); water was providedad libitum. Blood was collected froam jugular vein. PotassiumEDTA was usedas the anticoagulant for 2 hematology tests. No anticoagulant was used for the clinical chemistry samples. Animals were bled in random order. The following were evaluated: Hematology - red blood cell erythrocyte) count hemoglobin white blood cell (leukocyte) count differential blood cell count hematocrit segmented neutrophil count `mean corpuscular volume lymphocyte count mean corpuscular hemoglobin `monocyte count - mean corpuscular hemoglobin concentration eosinophil count basophil count platelet count blood cell morphology Reticulocyte count smears were made and held for possible future examination. Clinical Chemistry glucose aspartate aminotransferase urea nitrogen alanine aminotransferase . creatinine `gamma glotamyltransferase total protein albumin calcium inorganic phosphorus globulin sodium total bilirubin potassium cholesterol chloride - triglycerides > 17 000020 CovanMcTe-6633291-42.215 Urinalysis volume bilirubin specific gravity blood - prpoHtein `umriocbriolsicnoopgiecnexamination of sediment gkleutcoonsees. appearance - Serum Perfluorooctane Sulfonic Acid Level (PFOS) Determination During Weeks 5 and 14, five animals/sex/group were fasted overnight, (see Protocol Deviations for exception) anesthetized with carbon dioxide, and blood (approximately 4 mL) was collected from ajugular vein. All samples were collected without - anticoagulant, allowed to clot at room temperature, and centrifuged. Serum was harvested and stored in a freezer set to maintain -60 to -80C until packed on dry ice and shipped to the Sponsor for analyses. The samples were analyzed for PFOS. Results of analyses wil be reported separately by the Sponsor. Necropsy A necropsy was doneonthe Group 2 female that was sacrificed atan unscheduled interval. During Week 5, five animals/sex/group were fasted overnight (see Protocol Deviations for exception), anesthetized with carbon dioxide, bled for serum PFOS samples, weighed, and exsanguinated. These animals were discarded without necropsy. `The liver was removed from these animals, weighed, and collected for palmitoyl CoA oxidase and PFOS analysis. During Week 14, the remaining animals were fasted overnight, anesthetized with carbon dioxide, bled for clinical pathology tests (10 animals/sex/group with the exception of Group 2 female) and serum PFOS samples (five animals/sex/group), weighed, exsanguinated, and necropsied. Liver samples were collected for palmitoyl CoA oxidase, PCNA, and PFOS analysis. Animals were = necropsied in random order. `The necropsy included a macroscopic examinationofthe external featuresofthe carcass; al external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. - 18 000021 CovanMce 6T3621924215 Organ Weights After 13 weeksoftreatment, the following organs (when present) were weighed; paired organs were weighed together: adrenal (2) brain Kidney (2) . olivvearry @ spleen testis (2) thymus thyroid (2) with parathyroid Organ-to-body weight percentages and organ-to-brain weight ratios were calculated. - Cell Proliferation Tissue Collection and Immunohistochemical Evaluation At the terminal sacrifice, representative samples of the left lateral lobe of the liver were collected from five animals/sex/group and preserved in zinc formalin. After fixation, `samples were embedded in paraffin and shipped to Pathology Associates International - (Frederick, Maryland) for proliferation cell nuclear antigen (PCNA) evaluation. In addition, liver sections were stained with hematoxylin and eosin and examined `microscopically. Resultsofthe evaluation areinAppendix 7. Palmitoyl-CoA Oxidase Tissue Collection and Analyses At the scheduled sacrifices, the right lateral lobe of the liver was collected from each `animal, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C ~ until analyzed by Covance for palmitoyl CoA oxidase activity. Liver PFOS Analyses At the scheduled sacrifices, a portion of the liver from each animal was flash-frozen in - liquidnitrogenand stored in a freezer set to maintain -60 to -80C until packed on dry ice and shipped to the Sponsor for PFOS and metabolites analyses. Results of these analyses willbe reported separately by the Sponsor. - 000022 _-- Conwmeer53e29225 `Tissue Preservation The following tissues (when present) from the animal that was sacrificed at an unscheduled interval, or from each animal sacrificed at theterminalsacrifice were ~ collected and preserved in 10% neutral-buffered formalin, unless otherwise`specified: ahdirsesnal 2) . bcroamian cervix opvaancrrye@as) pTioturittasry rectum colon salivary gland [mandibular (2)] duodenum sciatic nerve. `epididymis (2) . esophagus sskeemlientaall vmeussiccllee ((2t)high) eye (2) skin femur with bone marrow (articular spinal cord (cervical, mid-thoracic, and surface of the distal end) lumbar) Harderian gland spleen py esis @) heart sternum with bone marrow - ileurn with Peyer's patch (lymphoid stomach jejunum kidney (2) thymus thyroid (2) with parathyroid lesions trachea. - liver lung with mainstem bronchi uurtienraursy bladder l`myammpmhanroydegsla(nmdesse(nfteemrailcesaonndlym)andibular) vZaygmibnaal's gland Histopathology Tissues (as appropriate) were embedded in paraffin, sectioned, stained with hematoxylin and eosin,and examined microscopically from each animal inthe control and high-dose - `groups and from the low-dose animal that was sacrificed at an unscheduled interval. Due to microscopic findings noted in the high-dose group, the liver and stomachfromanimals iin the mid-dose group, and the liver from animals in the low-dose group were also. `embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined - microscopically. : 000023 CovanMceT-638291-42215 Macroscopic lesions from animals in the low- and mid-dose groups were embedded in `paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. Bone marrow smears from the femur ofeach animal were prepared at the scheduled sacrifice after 13 weeksoftreatment, stained with Wright's stain, and retained for possible examination. Statistical Analyses Levene's test (Levene, 1960) was done to test for variance homogeneity. In the case of heterogeneity of variance at p < 0.05, transformations were used to stabilize the variance. Comparison tests took variance heterogeneity into consideration. `One-way analysisofvariance [ANOVA (Winer, 1971a)] was used to analyze body weights, body weight changes, food consumption, continuous clinical pathology values; organ weights, organ-to-body weight percentages, and organ-to-brain weight ratios. If 3 the ANOVA was significant, Dunnett's t-test (Dunnett, 1964) was used for control versus. treated group comparisons. Group comparisons (Groups 2 throug4h versus Group 1) were evaluated at the . 5.0%, two-tailed probability level. Only data collected on or afte the first day of treatment was analyzed statistically. RECORD RETENTION Al raw data, documentation, records, protocol, and specimens generated as a result of this study will be archived in the storage facilities ofCovance-Madison for a period of at least 1 year. Atleast 1 year after the submissionofthe final report, the Sponsor will - determine the final dispositionofthe materials. All rawdata stored on magnetic media, the protocol and protocol amendments, study correspondence, and an original copy of the: final report will be retained by Covance-Madison. - PCNA evaluation data, paraffin blocks, and tissue slides will be retained by Pathology Associates International. - 21 000024 CovanMceT683219-42215 Liver and serum samples sent to theSponsor and analysis data generated from these samples will be retained by the Sponsor. - RESULTS Dose Analyses Results of the homogeneity, stability, and dose preparation analyses are in Tables 1 - through 3. Mean values of the homogeneity analyses ranged from 74.0-78.0%, 84.0-96.7%, and 77.5-84.2% of the theoretical concentrations for the diets containing 3, 30, and - 100 ppm N-MeFOSE, respectively. These resalts indicate that the mixing procedure produced a homogeneous distribution of the test materialin the dose preparations; although variability generally appeared slightly greater at the 3 and 30-ppm dietary concentrations. Resultsofstability analysesofsamples stored for 19 and 32 days at room temperature and 8 weeks under frozen conditions indicated that the mean concentrations ranged from 54.4-62.5%ofthe initial concentrations of 1 ppm for diet mixed pretest. Samples stored RN for 32 days at room temperature indicated that themeanconcentration was 98.4% of the initial concentrationsof S00 ppm. Results of stability analysesofsamples stored for 19 and 32 days at room temperature indicated that the mean concentrations ranged from 93.2:94.0% of the inital concentrations of 1 ppm for diet mixed at Week 4. Samples Stored for 19 days at room temperature indicated that the mean concentration was 89.0% - ofthe initial concentrations of 3 ppm. `The mean concentrationsofthe dose preparation analyses for all levels ranged from 63.7 to 110.0% of the theoretical concentrations. Inherentvariabilityand lack of Sufficient sensitivity at low levels in the analytical method for measuring N-McFOSE resulted in homogeneity, stability and routine analysis data that were in many cases outside. ofthe standard limitsof +/- 15%. These results will be reevaluated in conjunction with the analytical results from the blood and liver level determinations when they are provided by . the sponsor. . 2 000025 CovanMceT63e294-2125 Clinical Observations and Survival Clinical observations are summarized in Table 4;individualdata are in Appendix 2. Individual animal fate data are also in Appendix 2. One female given 3 ppm N-McFOSE was sacrificed on Day 59 due to severe laceration of the tail. There were no macroscopic or microscopic changes to indicate that its moribund condition was test material-related. All other animals survived to the scheduled sacrifice. : No clinical observations were noted that were considered effectsofthe test material. `Ophthalmology - Ophthalmic observations are summarized in Table 5; individual data are in Appendix 2. `The Ophthalmology Report contains a discussionofthe data. Animals selected for the study had no lesions at the prestudy examination. No test - `material-related ophthalmic observations were noted at the Weck 14 examination. Body Weights ~ Body weight and body weight change data are summarized in Tables 6 and 7; individual body weight data are in Appendix 3. Testmaterialrelated lower body weights were noted for males given 30 and 100 ppmand 3 for males and females given 100 ppm. Statistically significantly lower mean body weights were noted beginning at Week 4 and continuing through the endofstudy for males and females given 100 ppm. Statistically significantly lower mean body weights were noted beginning at Week 7 and continuing through the endofstudy for males given 30 ppm. Body weight changes were statistically significantly lower in a sporadic manner for males given 30 ppmand for animals of both sexes given 100 ppm. Overall body weight changes (Weeks 1-14) were statistically significantly lower for all groups given N-MeFOSE. Food Consumption Food consumption data are summarizedin Table 8; individual data are in Appendix 4. - 23 000026 Covance 329225 -_ uTem Although statistically significant only at Week 13 for males given 100 ppm, food consumption was lower for males given 30 and 100 ppm when compared to those of controls for most ofthe study. Females given 100 ppm had statistically significantly lower food consumption than thoseofcontrols at Weeks 3, 4,6, 7, 11, and 12. The overall `mean food consumption for females fed the 100-pp concentration was significantly lower by 17.5% when compared with thatofthe controls. `Test Test Material Consumption material consumption data are summarizedin Table 9; individual data are in Appendix 4. - Animals were fed diets containing 3, 30, or 100ppm N-MeFOSE, T-6314. The mean amountsof test material consumedby the animals in these groups were 0.148 to 0.304 mg/kg/day; 1.390 to 3.094 mg/kg/day; and 5.054 to 10.291 mg/kg/day for males, respectively. Values for females were 0.184 10 0.319 mg/kg/day; 2.204 to 3.395; - and 6.494 to 10.378, respectively. The overall mean test material consumption for males feddiets containing 3, 30, or 100 ppm N-MeFOSE, T-6314 were 0.203, 2.041, and 6.844 mg/kg/day, respectively. The overall mean test material consumption for females fed diets containing 3, 30, or 100 ppm N-MeFOSE, T-6314 were 0.240, 2.588, . and 7.702, respectively. Clinical Pathology Hematology, clinical chemistry, and urinalysis data are summarized in Tables 10 through 14; individual data are in Appendix 5. The Pathology Report contains a discussionofthe data Dietary administration of N-MeFOSE was associated with lower hematocrit for males and - females fed 100 ppm; higher urea nitrogen for males and females fed 100 ppm; higher albumin for males fed 100 ppm; lower globulin for males fed 30 or 100 ppm: lower cholesterol and triglycerides for males fed 30 or 100 ppmand females fed 100 ppm; higher alanine aminotransferase for males fed 100 ppm; and higher hepatic palmitoyl CoA - oxidase for males and females fed 100 ppm. The effects were relatively mild with the exception of those on cholesterol and triglycerides for the high-dose males; those effects : = 000027 CovanIcMeT6-362391.42.215 were considered to be of moderate severity. Allofthe test material-related clinical pathology findings were more prominent at Week 14 than at Week 5. : Serum Perfluorooctane Sulfonic Acid Level (PFOS) Determination Resultsofserum PFOS level determination will be reported separately by the Sponsor. : Cell Proliferation Tissue Collection and Immunohistochemical Evaluation Results ofcell proliferation and immunohistochemical evaluation provided by Pathology Associates Intemational are in Appendix 7. Liver PFOS Determination Resultsofliver PFOS level determination will be reported separately by the Sponsor. Anatomic Pathology Terminal body weights, absolute organ weights, organ-to-body weight percentages, and organ-to-brain weight ratios are summarizedin Tables 15 and 16; incidences of _ `macroscopic and microscopic observations are summarized in Tables 17 and 18; the incidence of severity of microscopic observations is summarized in Table 19. Individual dataare in Appendix 6. The Pathology Report contains a discussion of the data. Statistically significant and test material-related changes in terminal body weights and liver - `weights were seen in males and/or females in the two highest dose groups. Statistically significant decreases were seen in the terminal body weightsofmales fed 30 ppm and in both males and females fed 100 ppm of N-MeFOSE. Statistically significant increases were seen in the mean liver-to-body weight percentage and mean live-to-brain weight ratio in males fed 30 ppm, in mean absolute liver weight and mean liver-to-body weight percentage in females fed 30 ppm. and in al liver weight parameters in males and females fed 100 ppm. - 2 000028 CovanMcTe-6362391-24215 Testmaterialrelated macroscopic findings were noted atalow incidence in the liver and `glandular stomachofmales and femalesin the 100 ppm dose group only. Liver findings included organs that were large and diffusely dark. In the glandular stomach, dark foci or ~ areas were sometimes seen in the gastric mucosa. Test material-related microscopic findings were seen in the liver ofanimals fed 30 and 100 ppm of N-McFOSE and in the glandular stomachofmales and females in the 100ppmgroup only. No test material-related changes were seen in animals fed 3 ppm of - N-MeFOSE. In the liver, one or more test material-related findings affected all animals fed 100 ppm and included slight-to-moderate centrilobular or diffuse hepatocellular hypertrophy, minimal-to-slight coagulative necrosis, minimal hepatocellular pigment, and minimal-to-slight centrilobular to midzonal or diffuse hepatocellular vacuolation. In - animals fed 30 ppm, centrilobular hepatocellular hypertrophy was also seen, although the incidence and severity was decreased atthisdose. In addition, a low incidenceofminimal hepatocellular vacuolation occurred in 30 ppm dose group males, and the liver ofa single female fed 30ppmhad minimal coagulative necrosis. A possible test material-related - change in the glandular stomachofanimals fed 100ppm was a relatively low incidence of mucosal erosions. The erosions wereofminimal to slightseverityand involved a very small portion of the mucosal surfaceina cases. CONCLUSIONS Based on anatomic pathology, clinical pathology, and body weight effects at 30 and 100ppmthe no-observable-adverse-ffect level was determined to be less than 30 ppm - `when N-MeFOSE is administered at concentrations of 0, 3, 30 and 100 ppm to Crl:CD%(SD) IGS BR rats for at least 13 weeks through dietary administration. Based on the body weight effects at 3, 30, and 100 ppm, the no-observable-cffect level was determined to be less than 3 ppm when N-MeFOSE is administered at concentrations of - 0,3,30 and 100 ppm to Crl:CD*(SD) IGS BR rats for at least 13 weeks through dietary administration. In the absence of correlative changes in anatomic pathology and clinical pathology the body weighs effects at 3 ppm were not considered to be biologically adverse. - 000029 CovanMcTe .6632391-24215 SIGNATURES Posen : Niian Bruce-Konuah Study Coordinator . Covance Laboratories Inc. _ Peter. 2 4 / Study Director Covance Laboratories Inc. 29 June 2 ovo Date Dat 2 AL . 27 000030 Covance 6329-225 _-- Mpa REFERENCES Dunnett, C. W., "New Tables for Multiple Comparisons with a Control," Biometrics, 20:482-491 (1964). Levene, H., "Robust Tests for Equalityof Variances," ContritboPurobtabiiloitynansd `CSatlaitifsotrincsi,a ((e1d9s6.0)).1. Olkin et al., Ch. 25, pp. 278-292, Stanford University Press: Stanford, . Winer, B. 1, "Design and Analysis of Single-Factor Experiments,"Statistical Principlesin ExperimentalDesign, Second Ed, Ch. 3, pp. 149-260, McGraw-Hill: New York, New York (19712). : 000031 CovanMcTe-6633219-42.215 - OPHTHALMOLOGY REPORT Ophthalmic examinations were done before initiation of treatment and during Week 14. : Animals selectedforthe study had no lesions at the prestudy examination. No test materiab-related ophthalmic observations were noted at the Week 14 examination. Donna J. Clspons, DVM, MS . iplomate, AFLAM 14, 200 . 2 000032 PATHOLOGY REPORT CovanMceT683219-42215 SUMMARY "The purposeof this study was to assess the toxicityofthe test material, N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE), when administered in the diet to rats for at least 13 weeks. The test materialwas administered at dose levels of 3, 30, and - 100 ppm. Dietary administrationofN-MeFOSE was associated with lower hematocrit for males and femalesfed100ppm;higherurea nitrogenformalesandfemalesfed 100 ppm;higher ~ albumin for males fed 100 ppm: lower globulin for males fed 30 or 100 ppm; lower cholestearnodl triglycerides for males fed 30 or 100ppmand females fed 100 ppm; higher alanine aminotransferase for males fed 100 ppm: and higher hepatic palmitoyl CoA oxidase for males and females fed 100 ppm. The effects were relatively mild with the - exceptionof those on cholesterol and triglycerides for the males fed 100 ppm; those effects were considered to be of moderate severity. Allofthe test material-related clinical `pathology findings were more prominent at Week 14 than at Week 5. . Statistically significant and test material-related changes in terminal body weights and liver weights were seen in males and/or females in the two highest dose groups. Statistically significant decreases were seen in the terminal body weights of males fed 30ppmand in bothmalesand females fed 100 ppm. Statistically significant increases were seen in the mean liver-to-body weight percentage and mean liver-to-brain weight ratio in males fed - 30 ppm, females in mean absolute liver weight and fed 30 ppm, and in al liver weight mean liver-to-body weight percentage in parameters in males and females fed 100 ppm. "Testmaterialrelated macroscopic findings were noted ata low incidence in the liver and - `glandular stomach ofmalesand females in the 100 ppm dose group only. Liver findings included organs that were large and diffusely dark. In the glandular stomach, dark foci or areas were sometimes seen in the gastric mucosa. - "Testmaterialrelated microscopic findings were seen in the liverofanimals fed 30 and 100 ppm of N-McFOSE and in the glandular stomach of males and females in the 100 ppm group only. No test material-rlated changes were seen in animals fed 3 ppm of - 30 000033 Covance 6329-225 - wre N-MeFOSE. In the liver, one or more test material-related findings affected all animals fed 100 ppm, and included slight-to-moderate centrilobular or diffuse hepatocellular hypertrophy, minimal-to-slight coagulative necrosis, minimal hepatocellular pigment, and . `minimal-to-slight centrilobular to midzonal or diffuse hepatocellular vacuolation. In animals fed 30 ppm, centrilobular hepatocellular hypertrophy was also seen, although the: incidence and severity was decreased at this dose. In addition, a low incidenceofminimal hepatocellular vacuolation occurred in 30 ppm dose group males, and the liver of a single female fed 30ppmhad minimal coagulative necrosis. A possible test material-related - changeinthe glandular stomach ofanimals fed 100 ppm was a relatively low incidence of mucosal erosions. The erosions wereofminimal to slight severity and involved a very small portion of the mucosal surface in all cases. METHODS Four groupsof Cr:CD(SD) IGS BR rats (20 animals/sex/group) were fed diets 2 containing the test material at adose level ofO (control groups; fed basal diet only), 3, 30, or 300 ppm. During Week 5, five animals/sex/group were sacrificed to obtain liver samples for determination of hepatic palmitoyl CoA oxidase activity (by Covance) and analysis for PFOS and metabolites (by 3M). One female fed 3 ppm was sacrificed and . necropsied during Wec9k becauseofpoor health (unscheduled sacrifice). The remaining animals were sacrificed and necropsied during Week 14 (terminal sacrifice). Blood was collected for hematology and clinical chemistry tests during Weeks 5 and 14. Urine was collected for urinalysis tests during Week 14. At necropsy, macroscopic . observations were recorded, organ weights were obtained (terminal sacrifice only), and tissues were placed in fixative as specified by the protocol. In addition, samples of liver were collected at the terminal sacrifice for evaluationofproliferation cell nuclear antigen (PCNA; by Pathology Associates International; five animals/sex/group), and both - scheduled sacrifices for determination of hepatic palmitoyl CoA oxidase activity (by Covance) and analysis for PFOS and metabolites (by 3M). Microscopic examinations `were done on protocol-required tissues from all control and high-dose animals sacrificed during Week 14 and the 3 ppm dose group female sacrificed in a moribund condition - during Week 9 (unscheduled sacrifice). Tn addition, livers, stomachs, and macroscopic - 31 000034 CovanMcTe66332194-2.215 lesions were examined from all terminal sacrifice animals fed 30 ppm, and macroscopic lesions and livers only were examinedfromanimals fed 3 ppm. . Statistically significant differences cited in the Results and Discussion section are based on comparisons between the control and treated groups. RESULTS AND DISCUSSION Mortality One female fed 3 ppm (Animal No. C96016) was sacrificed during Week 9 (Day 59) becauseofpoor health. All other animals survived to the respective scheduled sacrifice. Clinical Pathology Weeks 5 and 14. There were several statistically significant or otherwise notable differences for clinical pathology results between control and treated animals. Differences considered to be effectsofthe test material were lower hematocrit for males and females. fed 100 ppm: higher urea nitrogen for males and females fed 100 ppm; higher albumin for `males fed 100 ppm, lower globulin for males fed 30 or 100 ppm; lower cholesterol and ~ triglycerides formalesfed 30or 100 ppmandfemales fed 100 ppm;higheralanine aminotransferase for males fed 100 ppm; and higher hepatic palmitoyl CoA oxidase for males and females fed 100 ppm. The effects were relatively mild with the exception of those on cholesterol and triglycerides for the males fed 100 ppm; those effects were considered to be of moderate severity. All of the test material-related clinical pathology - findings were more prominent at Week 14 than at Week 5. `The mild effects on hematocrit and globulin were consistent with animals failing to gain weight appropriately. Although mildly higher urea nitrogen and albumin suggested a - relative dehydration for animals given 100 ppm, there were no effects on urine volume or urine specific gravity consistent with dehydration. The mild effect on alanine aminotransferase was indicative of the hepatocellular degeneration, and mildly higher hepatic palmitoyl CoA oxidase was indicative of peroxisomal proliferation. The effects on - cholesterol and triglycerides were the most prominent findings, and suggested lipid metabolism by the liver was altered. : = 000035 CovanIcMeT6-362391-42.215 Of uncertain relationship to administration of the test material was statistically lower mean corpuscular volume for females fed 100 ppm. The difference for mean corpuscular Volume became smaller over time (i, from Week 5 to Weck 14). Other statistically | significant differences were considered incidental and unrelated to administrationofthe test material. Anatomic Pathology - Unscheduled Deaths. The one female fed 3 ppm that was sacrificed in a moribund condition on Day 59 (Animal No. C96016) had no macroscopic or microscopic changes to indicate that its moribund condition was test material-related. `Terminal Sacrifice Terminal Body Weights and Organ Weights. Test material-related changes were sen in the terminal bodyweightsandin absolute and relative liver weights in animals fed - 30 and 100ppm N-MeFOSE. Mean terminal body weights were statistically significantly decreased in males and females that were fed 100 ppm of N-MeFOSE and in males only fed 30 ppm. The decreased mean . body weights at both dose levels were considered to be test material-related. Statistically significantand test material-related increases were seen in the mean liver-to-body weight percentage and the mean iver-to-brain weight ratio in males fed 30 ppm. inmeanabsolute liver weight and mean liver-to-body weight percentage in Tfehmealleivserfewdei3g0hptpimn,craenasdeisnaclorlrleilvaetrewdemiagchrtospcaorpaimceatlelryswiintmhallaersgeanlidvefresmainle3soffed1510m0alpepsmi.n the 100 ppm dose group and microscopically with the presence of hepatocellular hypertrophyinall males and females in the high-dose group. . 3 000036 CovanIcMeT6-362391-42.215 Additional statistically significant absolute or relative organ weight changes affected several other organs in both sexes and in multiple dose groups. Because there were no `macroscopic or microscopic findings that correlated with the weight changes, the ) remaining organ weight variations were considered to be within the rangeofnormal biologic variation or were attributed to the lower body weightsin treated versus control animals. Macroscopic Findings. Testmaterialrelated macroscopic findings were noted ata low - incidence in the liver and glandular stomachofmales and females fed 100 ppm N-MeFOSE. Livers that were large were seen in 3 of 15 males and in 0 females fed 100ppm,anddiffuselydarkliversoccurredin 2 of 15malesand 1of 15femalfeesd 100 ppm. No liver changes occurred in animals fed the control material or in animals fed - lower doses of N-MeFOSE. The large and diffusely dark livers correlated microscopically with hepatocellular hypertrophy intheaffected animals. In the glandular stomachs, dark fociorareasoccurredat an incidence of0 of 15, 1 of 15, 0 f 15, and 2 of 15 in males, and at an incideonfce0 of 15,0 of 14, 0 of 15, and 3 of 15 in females in the control, - 3 ppm. 30 ppm, and 100 ppm groups respectively. The dark foci or areas generally correlated microscopically with smal erosions in the glandular mucosa. Diffuse reddening and mottling of the thymus and mandibular lymph nodesaswell as red . foci or areas in the thymus were relatively common findings and frequently correlated microscopically with hemorrhage in the affected organs. Because these findings did not occur in a dose-relatedmannerand because rats in multiple dose groups, including control males, were sometimes affected, the findings were considered to be agonal changes that were not associated with test material administration. All remaining macroscopic findings - `were considered to be spontaneous and incidental and not related to test material administration. Microscopic Findings. Test material-related microscopic findings were seen in the liver - ofanimals fed 30and 100 ppm of N-MeFOSE and in the glandular stomach of males and females in the 100ppmgroup only. There were no test material-related findings in the liversofanimals fed 3 ppm. 3 000037 CovanMcTe-6362391-24215 Testmaterialrelated findings in the liver included hepatocellular hypertrophy, coagulative necrosis, hepatocellular pigment, and hepatocellular vacuolation. The incidence and severity of these findings tended to be greater in 100 ppm animals. The incidence of - `pertinent liver findings is presentedin Text Table 1. Text Table 1 Incidence of Selected Microscopic Findings in the Liver GrowNamberSex `Number Examined IM 15 IM 15 3M 1s 4M__IF 15 15 OF 14 3F 15 4F_ 15 Hypertrophy, hepatocellular, diffe 0 0 0 0 0 0 0 1 - Hypertrophy, hepatocellular, Centrilobular 0 0 14 15 0 0 7 14 Necrosis, coagulative Pigment, hepatocellular 0003 o002 00 1 0 0007 - Vacuolation, hepatocellular, centrilobular tomidzonal 0 0 2 7 0002 Vacuolation, hepatocellular, diffuse 0 0 2 2 0 0 0 0 `As noted in the above table, hepatocellular hypertrophy generally affected centrilobular areasofthe liver in animals fed 30 and 100 ppm, but the iver was more diffusely hypertrophied with abundant in one female fed 100 ppm. The hypertrophic hepatocytes were enlarged cytoplasm that ofien had a granular to globular eosinophilic appearance. In Someanimals in the high-dose group (two males and seven females), the cytoplasm of hepatocytes also contained minimal amounts ofa brown granular pigment at the periphery - of the cell. In addition, liver cells frequently were vacuolated (see Text Table 1), particularly in males given 100 ppm, with the vacuolation generally affecting hepatocytes in centrilobular and midzonal areas. Diffuse hepatocellular vacuolationwasalso seen in two males in each of the 30 and 100 ppm dose groups, but in no other animals. Lastly, . minimal to slight coagulative necrosis seen in3 of 15 males fed 100 ppm and 1 of 15 females fed 30 ppm was likely related to dietary administration of the test material since no control or 3 ppm dose group animals were similarly affected. . A possible test material-relted change in the glandular stomach was the presence of `mucosal erosions. The erosions wereofminimal to slight severity and involved a very smal portionofthe mucosal surface in all cases. "The erosions were seen with an . 35 000038 -_-- TCovwanMcmTe-66r33219e-42.2m15 ainncdid3eoncfe1o5 fi0noffem1a5l,es0oinf1th5e,caonndtro2lo,f3015ippnmm,alaensd,a10n0daptpamngirnocuipdsenrceespoecft0iovefl1y5.,T0hoef15, iprnetsheengcaestorficermouscioonssamaitcnreocsrcooppsiyc.allOyngeenmearallelfyecdor3replpamteadlswoithhatdhea dslairgkhtfgocaisotrriacreeraossisoenen : that was collected as a gross lesion at necropsy. Since no erosions were seninany of the animals' stomachs examined from animals fed considered unlikely to be test material-related. 30 ppm, the lesion in the 3 ppm male was - cAlhlanrgeemsaiannidngtympiiccarloosfcorpaitcs ofifndthiinsgasgweeraendcosntrsaiidne.red to be spontaneous and incidental (Ztae UH DRiopbleormtaLt.e,HaAllC,VDPVM, PhD - (Clinical Pathology) . ie J. es Eigyfny, DVMIMS iplomate, ACVP Diplomate, ABT une. /9, 2000 te Datbe /19 foo 36 000039 EE CovanIcMe|T6632391T-42Y215} (COMMENTS ON THE DATA Vdaatraioiunsthmiosdsetludsoy.fcaBleccualuasteordsi,ffceoremnptutmeordse,lsanrdoucnodmopfuftoerr ptrruongcraatmesnwuemrbeerussedidffteoreanntally,yze - svlailguhetslyifnrsoommtehtoasbeleisn o(ct.hge.,r tmaebalness,, fsrtoanmdianrddivdiedvuiaaltliyoncsa,lcourlaitneddivdiadtuaa,lovralfureosm) mstaatyisdtiifcfaelr `analysis data. differences. Neither the integrity nor the interpretation of the datawasaffected by these - `reTfhleecntusmtbheernoufmabneirmoaflasnliimstaeldsinastshieghneeaddtiongeaocfhtghre osuupmamtatrhye tsatbalrteosfftohr eclisntiucdayl.observations `cTohnedistuimonmawraystoabbsleerfvoerdclwiintihcaolutobrseegravradtitoontshiensdpieccaitfeicthneatnuurmeb,esrevoefraitnyi,mraelvserfsiobrilwihtiy,ch a - `numberofincidences/animal, or the lengthoftime the condition persisted. Only observations tables. other than normal are indicated on the summary clinical observations . `Ecaocmhmeannitma"lAnwiimtahlohbassernvoatsiiognnsifriecacnotrdfienddiansgs""Noirndmiacla"tetdhoronutghheouitndtihveidsutauldcylihnaiscalthe observations tables. `The specific details for comments indicated with a "C" can be found in at the the individual clinical observations endofeach group for each sex. tables that are `The start day of initiation ofa study week of treatment is "Day (c.g. a body weight 1, Week recorded 1." on Body weight data are Day 1is considered a entered Week 1 at the body N wweeiigghhtt,cahabnogdey dwaetiaghatrerceaclocrudleadteodnfDroamyt8heisfcirosntsdiadyeorfetdhaeWseteukdy2wbeoedkytwoetihghetf)i.rst Bdoadyyof atrhee finodlilcoawtiendgisnttuhdeytwaebleeks (wei.tg.h,tWheedeakyIbevialnugetshaerfeircsatldcauylaotfedthferofomlDloawyinIgtwhereokugh 7) and (e.g., Week 1 values are indicated as "Day 8."). Weekly food consumption and test `tmhaetefroilallocwoinngsusmtputdiyowneaerke (cca.lgc.u,lWateeedkfr1ovmatlhueesfiarrstedcaalycouflattheedsftruodmy Dwaeyek1 ttohtrhoeugfhirs7t).day of s`iTghneivfiaclaunetsffiogrurseusmtmhaanryias napdprionpdriivaitduealfotrestthemadtaetraiadluecotnosluimmpittaitoinonasroefrtehpeordtaetdawciotlhlemctoiroen and reporting software. 37 000040 ) - 00000000000CoS0 unMctee6e20 a9.wl25 `COMMENTS ON THE DATA (Continued) Food consumption values are reported in whole numbers; however, PTS carries food consumption values to one place to the right of the decimal for food efficiency and test - `material consumption calculations. The differences in values generated do not influence the interpretationofthe calculation. cTohnescumopmtmieonntw"aSsPnIoLtLrEeDc"orodnedindduievitdouaslpifloloagdecdounrsiunmgptthieoinntdeartvaalt.ables indicates that food `The comment "NOT TAKEN" on individual food consumption data tables indicates the animal died before the endofthe food consumption interval. Differences in the population size (N) on the summary tablesforclinical and anatomic - pathology are explained on the individual data tablesorthe codes sheets. "The calculation for individual test material `consumption is: `Test material consumption = - Coin en Sy DietConcenWtreatnioonfxFoeodrs yy Wg GD Fi where: Daily Body Weight Gai=n - BLiy stBodyWWeeilghoD f eraFF itBoyWeoigyhtW ol ter s__ and: Day Factor = (Food Consumption Start Day-oDfaFirysBtodyWeight of trv + 22 Compton ie and: Food ConsumopotidConnnIpntieorvnaElnd=Day.FokCrm Doy s38 000041 CODES, ABBREVIATIONS, AND UNITS General Codes and Abbreviations Codes for Clinical Pathology Abbreviations and Units for Clinical Hematology Abbreviations and Units for Clinical Chemistry Abbreviations and Units for Clinical Urinalysis Codes for Anatomic Pathology CovanIcMeT6-362391-42.215 Note: The following listsof codes, abbreviations, and units are used by Covance. Some, but not necessarily al,of this information may be : needed for this report. - 39 000042 CovanMceT633219-42215 General Codes and Abbreviations WK N - Mean; MEAN SD; $.D.; STAND DEV; . STANDARD DEV; sd NA P c - UNSCHED DISPATCH TBW # co Week. Number of measurements in a group. Arithmetic mean. Standard deviation. Group mean s significantly different from the mean of atps 0.05. the control group (Group 1) No value; Present. not applicable; not present. Comment found at the endofeach group for each sex. Unscheduled. Observations transferred from the in-life module of the data collection system to the necropsy module for reference during ntheecrloapstsyi.n-lOifbesoebrsveartviaotnisonasr.e duplicates of Terminal body weight. Number. Clinical observation. Animal Death Codes: 1 T M Interim sacrifice 1. `Terminal sacrifice. Sacrificed at an unscheduled interval. 40 000043 Codes for Clinical Pathology Covance 6329-225 Tear GENERAL CODES - Ns No sample QS/QNS Quantity not sufficient NR FS No repeat (sample volume not sufficient for repeat analysis) Fibrin strands sc Sample clotted - SH Slightly hemolyzed H Hemolyzed SL Slightly lipemic L Lipemic - st 1 Slightly icteric Ieteric NF Animal not fasted u Unscheduled/moribund bleed DT/DOT Animal died on test DB Died during bleeding . i] Technician judgment to repeat test TE Technical error (instrument or technician error that results in unacceptable data, .g., unacceptable instrument output, sample RE spilled, entryofinvalid data) Recording error (recorded incorrect data, e.g. wrong number, | spelling error, incorrect date) EE Entry error (incorrect keyboard entry) SE Sampling error PC Platelets clumped PD Platelets decreased . PI Platelets increased PL Platelets large PA Platelets appear adequate co HB Color interferes with test Heinz bodies observed PLASMO Plasmodium - NO AGG No aggregation FR Fractious UTD Unable to determine NO COAG No coagulation - 4 000044 -_-- CovanMcTeT-663e3219u-42.2n15 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) greater than 50 seconds Activated partial thromboplastin Bleed times (BLETIME) greater times (PTT) greater than 30 minutes than 110seconds CODES FOR BLOOD CELL MORPHOLOGY `pTohiekifloolclyotwoisinsg (sPcaOlIeK)w,aspoulsyecdhrtoommaesaisau(rPeOtLhYe)d,eghryepeoocfharnoimsaosciyato(sHiYsP(OA)N,ISoOr)b,asophilic stippling (BASTIP) or the (TOXNEUT), or atypical presence of lymphocytes Howell-Joll bodies (ATYPLYM): (HJBODY), toxic neutrophils Scale Degree Presence - - Normal for the species Not present 1 Slight Rare 2 Moderate Few 3 Marked Moderate 4 Not applicable Many URINE APPEARANCE The Camy | Miscellaneoms - A Pale B Straw E Amber 1 Black F Bown P Blue/green 7 Clear K Hazy M Debris 0 Feces C Yellow G Red Q Blue SDy Dark yellow HHGwGreeenn ROOmrangge L Cloudy -- - a2 000045 Codes for Clinical Pathology (Continued) CovanIcMe 6T36293-12215 _-- URINE CHEMISTRY MULTISTIX STRIP - Urine Glucose Urine Ketone Urine Blood = Negative = Negative ~ Negative + 100 mg/dL. ++ 250 mg/dL. + SmgdL ++ 15 mg/dL + Small ++ Moderate +++ 500 mg/dL ++++ 1,000 mg/dL +++ 40 mg/dL +++ 80 mg/dL +++ Large +++ 22,000 mg/dL +4+++ 160 mg/dL Urine Urobilinogen Urine Bilirubin - = 02mgdl = Negative + ImgdL + Small ++ 2mg/dL +++ dmg/dL +++ 8mgldL ++ Moderate +++ Large - (1 mg = approximately 1 Ehrlich unit) URINE SEDIMENT - Cells, Crystals, Casts, and Comments Bacteria A Amorphous urates Q Sperm 0 Not present B Amorphous phosphates R Fecal contamination 1 Few C Uric acid S$ Pinworm ova found 2 Moderate D Triple phosphates T Pinworm larvae found 3 Many - E Calcium oxalate U Parasite ova found GF CaGrlacniuulmarcacrabstosnate H Hyaline casts 0 Not present 1 Cellular casts 11-5 per field - J Waxy casts 2 6-10 perfield K Unknown crystal 3 11-20 per field P_ Mucous threads 4 >20 per field - a3 000046 CovanMcTe66331249.-2125 Abbreviations and Units for Clinical Hematology Test Abbreviation (Units) - HReemdogbllooobdincell count RHBGCB ((EG6//DULL) or X10%/uL) Hematocrit Mean corpuscular volume HCT (%) MCV (FL) Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration MCH (PG) MCHC (%) - PMleataenleptlcatoeulnett volume PLT (E3/UL or X10/uL) MPV (FL) Reticulocyte count RETIC (%) AHbesionlzubtoedryetciocuulnotcyte count RHEETIINCZ ((E%3)/UL or X10%uL) : Erythrocyte sedimentation rate Prothrombin time PETSR(S(EMCM)/HR) TAhctriovmabtiend ptairmte.ial thromboplastin time PTT (SEC) TT (SEC) - Activated coagulation time Fibrinogen ACT (SEC) FBR (MG/DL) Fibrin/fibrinogen degradation products Platelet aggregation FDP (UG/ML) Collagen Adenosine diphosphate: PPAAGGGG//CAODLP ((%%)) . Alpha 2-antiplasmin Bleeding time ABNLTEITPILMAES ((S%E)C) Methemoglobin Plasma hemoglobin METHGB (%) PLA HGB (MG/DL) Myeloid/erythroid ratio Estimated myeloid/erythroid ratio M/E RATIO EST ME RATIO - D`iWfhfietreenbtlioaoldbcleololdcoceulnltcount WBC (E3/UL or X10%uL) Nucleated red blood cell count NRBC (/100 WBC) Corrected white blood cell count COR WBC (E3/UL or X10%uL) - Segmented neutrophil count Band neutrophil count N-SEG (E3/UL or X10%4L) and% N-BAND (E3/UL or X10*/4L) and % Lymphocyte count LYMPH (E3/UL or X10*/uL) and % Monocyte count MONO (E3/UL or X10%4L) and % Eosinophil count EOSIN (E3/UL or X10*/uL) and % AnBiassoocphyitlosciosunt BASO (E3/UL or X10%4L) and % ANISO (1,23) Polychromasia POLY (123) - a 000047 CovanMceTs63321926215 Abbreviations and Units for Clinical Hematology (Continued) Test Poikilocytosis - Hypochromasia Howell-Jolly bodies Basophilic stippling. Abbreviation (Units) POIK (-,1,2,3) HYPO (-,1,2.3) HIBODY (-,1,2,3,4) BASTIP (-,1,2,3) `Toxic neutrophils TOXNEUT (-,1,2,3,4) - Atypical lymphocytes Aqueous white blood cell count (right eye) ATYPLYM (-,1,2,3.4) REYE (WBC/UL) Aqueous white blood cell count (left eye) LEYE (WBC/UL) - # 000048 .- 0000 [Srpe--l 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 | AmCryelaatisnee kinase Lipase Palmitoyl CoA oxidase Calcium Ionized calcium Inorganic phosphorus - Sodium Potassium Chloride `Magnesium Zinc - Strontium Iron 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) IBILI (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 (IU/L) SDH (IU/L) LDH (IU/L) ACMKY(LIUA/LS)E (IU/L) LIPASE (IU/L) PCOAO (1U/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) " 000029 Cone 6329.25 _ wre 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 Serum bile acids Fecal bile acids Average fecal weight * Fecal bile acids (calculation) Osmolality Electrophoresis Albumin Alpha-1-globulin ~ Alpha-2-globulin Beta globulin GHiagmh-mdaengsliotbyulliipnoprotein Low-density lipoprotein | Very-low-density lipoprotein Insulin Adrenocorticotropic hormone Cortisol Glucagon "Triiodothyronine - `Thyroxine Creatine kinase isoenzymes BB MB MM `Abbreviation (Units) EX FE (UG/DL) TIBC (UG/DL) UIBC (UG/DL) FE %SAT (%) CHEP (MU/ML) CHER (MU/ML) CHEB (MU/ML) 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) E A-1 (G/DL) EA-2 (G/DL) E BETA (G/DL) E GAMMA E-HDL (%) (G/DL) ELDL (%) E-VLDL (%) INSULIN (UU/ML) ACTH (PG/ML) `CORTISOL (UG/ML) GLUCAGON (PG/ML) T3 (NG/DL) T4 (UG/DL) CK-BB (UL) CK-MB (U/L) CK-MM (U/L) : a 000050 CovanIcMeT6-36239-124215 Abbreviations and Units for Clinical Urinalysis Test Urine volume Abbreviation (Units) U VOL (ML) - 8 hour urine volume Specific gravity 8 HR VOL (ML) SPGR Urine osmolality Quantitative urinary/cerebrospinal U OSMO (MOSM/KG) QUAN PRO (MG/DL) 2 fluid protein Urine protein excretion PRO EXC (MG) Urine chemistry Multistix strip Urine pH UPH Urine protein Urine glucose UPRO (MG/DL) UGLY - Urine ketones, UKET Urine bilirubin Urine blood UBILI UBLOOD Urine urobilinogen Urine reducing substances UROBILI URESUB . Microscopic examinationofurine sediment Red blood cells per high-power field `White blood cells per high-power field RBC (PER HPF) WBC (PER HPF) Epithelial cells per high-power field Bacteria per high-power field EPITH (PER HPF) BACT (PER HPP) . Casts per low-power field CASTS (PER LPF) Crystals per low-power field CRYSTALS (PER LPF or Urine appearance PER LPF2) URINE APP! or URINE APP Comments COMMENTS, Miscellaneous Codes and Abbreviations for Clinical Pathology Fecal occult blood - Fecal parasite detection Hemolytic potential Osmolality Not applicable Not applicable: Not applicable 0SMO (MOSM/KG) - 43 000051 CovanMcTe 663321942215 Codes for Anatomic Pathology Code Definition - ANIMAL DEATH CODES 1 Interim sacrifices 1 T `Terminal sacrifice M Sacrificed at an unscheduled interval MACROSCOPIC CODES - EX NOT TAKEN Indicates that organ weight is excluded from calculations. Organ weight not taken; explanation given in necropsy notes. MISSING Organ missing or lost UNSUITABLE Organ technically unsuitable for weighing AUTOLYTIC Organ autolyzed and could not be weighed EXCLUDE Weight was taken, but was excluded from all calculations MICROSCOPIC CODES Codes Prefacing Neoplastic Findings | B- Primary, benign neoplasm M- Primary, malignant neoplasm LN- MLeotcaasltlaytiincvanseiovpelnaesomplasm X- Other neoplasm : Code Definition Distribution of Findings Focal Diffuse - Multifocal . a9 000052 Codes for Anatomic Pathology (Continued) Covance 6329-225 wre Grades for Severity or Amount - 1 Minimal - the least light microscope: amountof change that can be observed with the 2 Salsiaghbtno-rlmeasslthan average amountof change, but readily discernible 3 Moderate - the average amountof change thatis expected for a - 4 lesion Moderately severe (marked) - a marked amountofchange with 5 pSoesvseirbele- laosgsroeaftfuanmcotuinotnooffcthhaenagfefewctietdh cperlolbsaobrleorlgoasnssof function of the affected organ cell or organs and frequently involves large areasofthe Other Microscopic Codes TL Total P Finding present - Finding not present - MN Mean `TISSUE ABBREVIATIONS . Abbreviation IN Definition Lymph node cL Gland STOMACH, GL STOMACH, NONGL Glandular stomach Nonglandular SALIV GL, MANDIB LN, ANT MES/PANC Mandibular salivary gland Anterior mesenteric/pancreatic lymph node: AUDITORY SEB GL LACRIMAL GLAND, EX Auditory sebaceous gland Exorbital lacrimal gland HEMATO NEOPLASIA Hematopoietic neoplasia - LACRIMAL GL, INT CAVITY, ABDOM Internal lacrimal gland Abdominal cavity SALIV GL,PAROTID LN, TRACHEOBRON Parotid salivary gland Tracheobronchial lymph node: . 50 000053 CovanMcTe-6633219-42.215 Table 1 Resultsof Homogeneity Analyses (ppm) Mixed 8/20/98 13-WEEK DIETARY TOXICIETYTHSATNUODLY (WNI-TMHeFNO-SME,ETT6H3Y1L4)PIENRRFALTUSOROOCTANESULFONAMIDO T6314 (ppm) - Samples Location Replicate 3 30 100 Top 21 22.419 323778 7796.54 E Me3an 22.2094047" 252.960067) 7761..751.95) Middle 1 2.24 25.2 75.0 2 227 253 79.7 . Me3an 22.2124(740) 2255.21(840) 889L.45(814) Bottom 1 2.03 237 83.2 23 31.094 2214.6 8817..68 Mean 234780) 269897) 842(842) a Each value in parenthesis is the percent of theoretical. = 51 000054 - Covance 6329-225 OO ure Table 2 ResultsofStability Analyses (ppm) Mixed 8/20/98 13-WEEK DIETARY TOXICITEYTHSATNUODLY(WNI-TMHeFNO-SME,ETTH63Y1L4)PIENRRFALTUSOROOCTANESULFONAMIDO. . Storage Conditions "Replicate 1 T6314 (ppm) 500 Initial 1188 415 2 0954 468 Mean 1.42(142) 442(88.4) 19 day, room temperature 1 073 - 2 081 - Mean 0772(112) - - 32 day, [I 444 room temperature 2 425 Mean - 435 (87.0) - 32day, 1 0709 - room temperature' 2 0.850 - Mean 0780 (78.0) - 8 week, 1 07m - - frozen 2 0995 - Mean 0.887887) - a Each value in the parenthesis is the percentof theoretical. b Reinjected - 52 000055 _-- CovwanMcreT663e3291.42215 `Table 2 (Continued) Resultsof Stability Analyses (ppm) Mixed 9/24/98 13-WEEK DIETARY TOXICIETYTHSATNUODLY(WNI-TMHeFNO-SME,ETT6H3Y1L4)PIENRRFALTUSO.ROOCTANESULFONAMIDO. T6314 (ppm) - Storage Conditions Replicate 500 T Initial 1- 0.788 2 0.656 - Mean - 0722 (72.2 19 day, room temperature 1 - 0.707 _ 2 0.651 Mean 0.679.679) 32 days, room temperature 1 - 2 Mean 0.688 0.657 0.673 (673) a Each value in the parenthesis is the percentoftheoretical - 33 000056 A -_-- `Table 2 (Continued) CovwanMcTTe66e33219M4-2.2n15 ResultsofStability Analyses (ppm) Mixed 10/22/98 13-WEEK DIETARY TOXICITEYTHSATNUODLY(WNI-TMHCFNO-SME,ETT6H3Y1L4)PIENRRFALTUSOROOCTANESULFONAMIDO. T6314 (ppm) - Storage Conditions Replicate 3 Initial 1 224 - 2 249 Mean 2.37 (79.00 19 day, room temperature 118 2 2.33 Mean 2.11(703) a Each value in the parenthesis is the percent of theoretical. - 54 000057 - _-- Covance 6329-225 wre Table 3 Results of Dose Preparation Analyses (ppm) - PERFLUORO13O-CWTEAENKEDSIUELTFAROYNATOMXIIDCOITEYTHSATNUODLY(WNI-TMHEFNO-SMEE,TTH-6Y3L14) IN RATS. Week Replicate 03 T-6314 (ppm) ~ 30 100 14 1a 216 2 a 21 297 270 873 852 Mean - 214713) 284(947) 863(363) 14 21 .- 1.98 im - - . - Mean - 191(7) - - 14 1. 2a - - 2. 2m - - Mean - 238(193) - - 58 1a ass 279 893 - 2a Mean a 30s 3300110) 270 275017) 83 858(858) onz 21aa 22 249 276 268 89.7 872 Mean a 237(790) 272(90.7) 885(885) 1304 1a 25 262 98.1 2a Mean a 2m 247(823) 264 263(817) 98.7 98.4(98.4) - a b BEealchowvatlhueefimn tpaorfenqtuhaenstiistaistitohne(p<e0r.c5enptpomf)t.heoretical Retest. d Retention. : 5s 000058 232 223 56 ;i 532 52 phioii mer tc: onions 32 2 2 P 22223 phim oe 22s 22 SION ARP 3H22 2 a ums of soy ian cur ou a) way 5 wom, ow ww us, 283 32a 2333 3 | Bod, ld ld dB H323 38 " eeprom wens in it ri 22s 22 o 322 32& % wes of. MOF OW OR OT afi 8322 3e3S o wires wife A AL RB. BEL GBR ve en eo men om 233 R3 o Ee a BOT OR OWT a 2Hgs & mos ofa EFL ANREP. EL GB ve teen eo mem mao 233 3 & o wees ofa SOTO OW OT ao 332 a33 7" ee of BOAR EP. EEL GBR 0 en oo oan we Bg2 a$ n wiles of BVO RT wd 32 33 > n srs ate Nia AWE BP BL oP ve teen eo mem mao 233 2 = u 223 3 > 7s Samar of Chaichua 333 3 < 13 i Eat www Be ] 332 332 n BOR Ge eo Mh M0 Me Ch She 233 322 " 3g2 8$ oe . . ' . . 3333 8 233sgg z n 33HHg & | i nn ESR IT ERE 233 282 | Some oe ge vat tra 3g 3g3 u 2gg8 & 8s 232 828 5 3g33 8 w 222 832 a 223 28 282 &323 0 222 82 ot mn of rg is ua SESE a i x: nl 2223 a 2 22233 * 2) g22 9% 2223 3 El 332 33 * 333 23 7 232 22 5 2233 82 % 2233 a2 10 2323 101 Tm TR STRUT am ot i ld 2322 & 0 2323 2& 103 Sem of re tr cn SE 2322 2 = 104 232s 8 10s g333 3 10s maryof orp maneoes EE emone BD OBEE URE TEN seen HD ENR DEB SEM 2322 B = 107 2322 B 108 3222 B 109 2322 B& 10 2222 iB m 22 22a & n 2223 B* I oe om 2233 +<3 i me to 0 mom 1 oo a ne ct of mers cron 5 PR 23 a2 = ns octsmn of wiconspte coersions ER 323 a2 ne 2222 8S wm mansion 20 0 0 100s WG080I ooo Np pO 13 33 3 a 1 3 3 2332 88 ns 2333 8 no 223 88 1 ciomc of svi of Setceed Hirssrpc chsration RE: 23g3 8 > 2 333 58 = 233s 38 APPENDIX 1 CovanMcTe -636201-42215 Protocol Deviations . Protocol Protocol Amendment No. 1 Protocol Amendment No. 2 Protocol Amendment No. 3 - 124 coo127 Protocol Deviations CovanMceT-638291-42215 Protocol. Housing. Diet. "Certified Rodent Diet #5002, meal (PMI Nutrition _ International)adlibitum, unless otherwise specified." Actual Procedure. The animals were given Teklad 8728C pelleted food from August 17, 1998 to August 23, 1998. Protocol. Dose Preparation. Retention Samples. "During the in-life phase, samples (approximately 100 g) wil be taken from each dose preparation sampled for dose analyses and stored at room temperature. Unless used for analyses, these samples will be discarded - approximately 1 month after completionofthe in-life phase." Actual Procedure. Retention samples were discarded after approximately 1 month of the completionof the in-life phase. Protocol. Observation of Animals. Clinical Observations. "Each animal will be observed twice daily (a.m. and p.m.) for mortality and moribundity, recording findings astheyare: observed." Actual Procedure. On Days 16 through 30, the a.m. and p.m. mortality and moribundity checks were not documented. Protocol. Termination. Scheduled Sacrifices. "After at least 4 weeks of treatment, five animals/sex/group will be fasted overnight, bled for serum samples, anesthetized with carbon dioxide, weighed, and exsanguinated." Actual Procedure. Fasting of animals at the Week 5 scheduled sacrifice was not documented. - 125 000128 Covance 6329-225 - O0 OO 0 mre Protocol Deviations (Continued) Protocol. Experimental Design. Postmortem Procedures. "The following tissues (when present) or representative samples will be collected from each animal and preserved in 10% phosphate-buffered formalin, unless otherwise specified." Actual Procedure. Some tissues, requiredby the protocol, were not available for histopathologic examination. Missing tissues are listed with appropriate comments in the pathology data sheets for individual animals. Summary tables do not include them as having been examined. - Thesedeviations are not expected to have affected the resultsofthe study. 126 000129 . COVANCE Sponsor: s 3M St. Paul, Minnesota PROTOCOL Study Title: 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido - Ethanol (N-MeFOSE, T-6314) in Rats : Date: August 27, 1998 Performing Laboratory: Covance Laboratories Inc. 3301 Kinsman Boulevard _ Madison, Wisconsin 53704-2595 Laboratory Study Identification: - Proposal No. 90545D Covance 6329-225 Sponsor Project Identification: 3M T-6314.1 - 127 000130 Covance 6329-225 Page2 . Study 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MEFOSE, T-6314) in Rats : Purpose Toassessthe toxicity of the test material when administered in the diet to rats for at least 13 weeks - Sponsor 3M Toxicology Services Building 220-2E-02, 3M Center - St. Paul, Minnesota 55144-1000 Study Monitor Andrew M. Seacat, PhD . 3M Toxicology Services Telephone No.: 651.575.3161 Facsimile No.: 651.733.1773 ~ Alternate Study Monitor Marvin T. Case, DVM, PhD 3M Toxicology Services Telephone No.: 651.733.5180 Facsimile No.: 651.733.1773 Study Location Covance Laboratories Inc. 3301 Kinsman Boulevard - Madison, Wisconsin 53704-2595 Mailing Address: PO Box 7545 Madison, Wisconsin 53707-7545 - 128 000131 --_-- Study Director Peter J. Thomford, PhD Covance Laboratories Inc. ~ Telephone No.: 608.241.7207 Facsimile No.: 608.242.2736 Toxicologist `Thomas E. Ryan, BS Covance Laboratories Inc. Covance 6320:225 Pues Proposed Study Timetable In-Life Start Date: September 1, 1998 - In-Life End Date: December 3, 1998 Audited Draft Report Date: May 26, 1998 Regulatory Compliance - `This study will be conducted in compliance with the Food and Drug Administration Good Laboratory Practice Regulations as set forthinTitle 21 of the US Code of Federal Regulations, Part 58, issued December 22, 1978 (effective June 20, 1979), and with any applicable amendments. Animal Care and Use Statement All procedures in this protocol areincompliance with the Animal Welfare Act Regulations, 9 CFR 1-4. In the not unnecessarily duplicate any opinion previous of the work. Sponsor and study director, the study does `Quality Assurance The protocol, study conduct, and final report will be audited by the Covance Quality Assurance Unit (QAU). The proliferation cell nuclear antigen (PCNA) evaluation, data, - and report will be auditedby the QAU of Pathology Associates International. Liver and `serum analyses, data, and report will be audited by the QAU of 3M Environmental `Technology and Safety Services. 129 0001.32 . `Test Material EE Covance 63P2a9ge2s5 ~ NId-eMnettihfyiclaPtieornfluorooctanesulfonamido Ethanol (MeFOS, T-6314) "LTohte Number lot numbers will be maintainedin the raw data. : Purity Responsibilityofthe Sponsor Stability - Responsibility of the Sponsor Storage Conditions At room temperature Characteristics Information on synthesis methods, composition, or other characteristics that define the test material is on file with the Sponsor. : Reserve (Archive) Samples A reserve sample (approximately 5 g)ofeach lot will be taken and stored at room temperature. These samples wil be transferred to the Sponsor after completionof the in-life phase. Dispositionof Test Material After authorizationfromthe Sponsor, any remaining test material will be returned to: - Andrew M. Seacat, PhD 3M TBuoixlidcionlgo2g2y0S-e2rEv-i0c2e,s 3M Center St. Paul, Minnesota 55144-1000 - Telephone No.: 651.575.3161 Facsimile No.: 651.733.1773 - 130 0001.33 Animals Covance 6320-225 Pages `Species . Rat Strain Cr:CD*(SD) IGS BR - Source Charles River Laboratories, Inc., Portage, Michigan Age at Initiationof Treatment - Preferably less than 6 weeksofage but not more than weeks of age Weight at Initiationof Treatment 1000300 g Number and Sex 80 males and 80 females Identification Implantable microchip identification device Husbandry - Housing Individual (may be group-housed during acclimation). Animals will be housed in suspended, stainless steel cages. - Diet Certified Rodent Diet #5002, meal (PMI Nutrition International) ad libitum, unless `otherwise specified. The diet is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Specified nutrient and contaminant A analyses are on file at Covance-Madison. - 131 000134 A -_-- Covance 63P2P9ua-ge2e2s5s Water Ad libitum. Samplesofthe water are routinely analyzed for specified microorganisms and environmental contaminants. The resultsareon 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 will be set to maintain 18 t0 26C, a relative humidity of 30 to 70%, and a 12-hour light/12-hour dark cycle. The light/dark eyele may be interrupted to accommodate in-life procedures. Acclimation Atleast 1 week 3 Randomization Selectionofanimals for the study will be basedonclinical observations, ophthalmic. `examinations, and otherdataasappropriate. Animals wil be assigned to treatment `groups using a computerized blocking procedure designed to achieve body weight ~ balance with respect to treatment groups. At the timeofrandomization, the weight variationofthe animalsofeachsex used will not exceed +2 standard deviationsofthe mean weight, and the mean body weight for each groupofeach sex will not be statistically different at the 5.0% probability level. - Justification Rats historically have been used in safety evaluation studies and are recommended by `appropriate regulatory agencies. . 132 000135 . _-- Covance 6P32a9g2e2r57 Group Designations and Dietary Levels Group MNaulmeberofAnFimeamlasle Dictar-- y Levels (ppm MeFOS)* - 1 (Control 20 20 0 32 ((MLiodw)) 4 (High) 2200 2 220 20 330 100 a Dose levels are expressed as ppm of MeFOS. b The control animals will receive the basal dietonly. Dosing Procedures = Method of Administration Dietary, 7 days/weekforat least 13 weeks. Treatment will continue through the day before necropsy. Reason for Dosing Route `The potential routeof exposure in humans is oral. Dose Preparation All dose preparations will be mixed according to the study-specific mixing procedure - developed by Covance. Dose concentrations will be based on the MeFOS content as supplied. All dose preparations wil be stored at room temperature. - `Baenfdo5r0e0inpiptimawtiiolnolfbtermeiaxtemde.nt, dose preparations of 1 ppm, 3 ppm, 30 ppm, 100 ppm Dose preparations will be mixed at least once every 4 weeks during the in-life phase. Retention Samples During the in-life phase, samples (approximately 100 g) will be taken from each dose preparation sampled for dose analyses and stored at room temperature. Unless used for analyses, these samples will be discarded approximately 1 month after `completionofthe in-life phase. - 133 000136 Covance 6329-225 Pages Dose Analyses By Covance, using a method supplied by the Sponsor and validated by Covance _ Homogeneity Homogeneity will be determined for 3-ppm, 30-ppm, and 100-ppm dose preparations once pretest. One sample (approximately 100 g) eachfromthe top, middle, and bottom ofthe dose preparations mixed for homogeneity analyses will be collected, divided into three subsamples for extractionand analysis, and analyzed for test material content. All samples will be stored at room temperature until analyzed within 7 days after mixing. "Homogeneity analysis will be repeatedibf atch size changes by more than 30%. Stability - Four samples (approximately 100 g each) will be takenfromthe 1-ppm dose level concentrationofdiet preparations mixed pretest. One sample will be analyzed on the dayofmixing and used as the baseline value. One sample will be stored at room . temperature for at least 19 days, then analyzed. A third sample will be stored at room temperature after atleast 32 days, then analyzed. The remaining sample will be stored inafreseettozmaeintrain -10 to -3fo0r8weeCks,thenanalyzed. In addition, two samples (approximately 100g each) will be taken from the 500-ppm dose preparation mixed pretest. One sample will be analyzed on the day of mixing. `The second sample will be wil be stored at room temperatureafter at least 32 days, then analyzed. Dose Confirmation - During the in-life phase, samples (approximately 100 g) from all dose preparations will be analyzed. Al samples will be stored at room temperature until analyzed. : 1 0001.37 i _-- Observationof Animals CovanceP 632P9ae -g2e2s5 Clinical Observations } Each animal will be observed twice daily (a.m. and p.m.) for mortality and moribundity, recording findings as theyare observed. At least once weekly, each animal will be: observed (cage will be opened, and the animal will be removed); abnormal findings or an indicationofnormal will be recorded. Additional findings will be recorded as they are observed. Body Weights Eachanimal wil be weighed at least once prior to treatment, on the first day of treatment, and weekly thereafter. Food Consumption Food consumptionwil be recorded weekly during treatment. Food consumption will not be measured during Week S for animals scheduled for sacrifice during Week 5. Ophthalmic Examinations Ophthalmic examinations will be done for eachanimal before initiation of treatment and beforethe Weck 14 scheduledsacrifice.Theeyesofeachanimalwillbe examinbeyda veterinarian using an indirect ophthalmoscope. A mydriatic agent wil be instilled into the eyespriorto examination. Clinical Pathology - Frequency Hematology and clinical chemistry during Weeks5 and 14; urinalysis during Week 14 Number of Animals - `Ten animalgsex/group (the same animals will be used at each interval,ifpossible) MethodofCollection Animals will be fasted overnight; blood will be collected froam jugular vein. The - anticoagulant will be potassium EDTA for hematology tests. Urine will be collected chilled overnight (approximately 16 hours). 135 000138 Covance 63P2a9g-e22150 Tests Hematology - red blood cell (erythrocyte) count hemoglobin platelet count white blood cel leukocyte) count hematocrit mean corpuscular volume bdilfofoerdencteilallmbolropohdolceolglycount mean corpuscular hemoglobin `mean corpuscular hemoglobin concentration reticulocyte examined) smear (made, but not Clinical Chemistry - glucose urea nitrogen creatinine total protein albumin . gchloolbeusltienrol triglycerides total bilirubin alanine aminotransferase `gamma glutamyltransferase aspartate aminotransferase: calcium isnoodriguamnic phosphorus potassium chioride Urinalysis appearance volume specific gravity - pH protein urobilinogen glucose ketones bilirubin blood microscopic examinationofsediment Serum Perfluorooctane Sulfonic Acid (PFOS) Analyses Frequency and NumberofAnimals Five animals/sex/group after at least 4 and 13 weeks of treatment. Samples after 4 weeks of treatment wil be collected from animals selected for the interim sacrifice. - 136 0001.39 _-- Covancee63P2ea9g-e2n2115 MethodofCollection Animals will be fasted overnight; blood (approximately 4 mL)willbe collected . from a jugular vein. Samples wil be collected without anticoagulant, Sample Handling Blood samples will be allowed to clot at room temperature and centrifuged. Serum samples will be harvested and stored in a freezer set to maintain - 60 10 -80C. Samples will be packed on dry ice and shipped to: Kris J. Hansen, PhD = 3M Environmental Technology and Safety Services 935 Bush Avenue Building 2-38-09 St. Paul, Minnesota 55133-3331 Telephone No.: 651.778.6018 Facsimile No.: 651.778.6176 Serum samples will be analyzed for PFOS and metabolitesbythe Sponsor. Results will be reported separately by the Sponsor. Termination Unscheduled Sacrifices and Deaths Necropsies willbe done. Animals 10 be sacrificed will be anesthetized with carbon dioxide, weighed, and exsanguinated. Scheduled Sacrifices After at least 4 weeks of treatment, five animals/sex/group will be fasted overnight, bled for serum samples, anesthetized with carbon dioxide, weighed, and exsanguinated. The abdominal cavityofeach animal will be opened, the liver will be removed and weighed, and liver samples will be collected for palimitoyl CoA and PFOS analysis. Animals will be discarded after liver collection. - 137 0001.40 . -_-- Covance 6329-225 Page 12 After at least 13 weeksoftreatment, all surviving animals will be fasted overnight, bled for serum samples (five animals/sex/group) and clinical pathology tests (10/sex/group), anesthetized with carbon dioxide, weighed, exsanguinated, and necropsied. Liver sampleswillbe collected for palimitoyl CoA, PCNA, and PFOS analysis. Postmortem Procedures Necropsy `The necropsy willincludean examinationof the external featuresofthe carcass; all external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. - Organ Weights At the scheduled sacrifice after 13 weeksoftreatment, the following organs (when present) will be weighed; paired organs will be weighed together: adrenal (2) brain kidney (2) liver ovary (2) spleen ttehsytimsu(s2) thyroid (2) with parathyroid Organ-to-body weight percentages and organ-to-brain weight ratios will be calculated. Bone Marrow Smear _ From the femurofeach animal at the scheduled sacrifice after 13 weeksoftreatment only; made but not examined Cell Proliferation Tissue Collection and Immunohistochemical Evaluation - At the scheduled sacrifice afer 13 weeksoftreatment, representative samplesofleft lateral lobe of the liver from cach animal will be collected and preserved in zinc formalin. - After fixation, each sample of liver will be embedded in paraffin, and the paraffin blocks will be shipped to: . 138 0001.41 Covance 63P2a9g-e22153 Sandra R. Eldridge, PhD Pathology Associates International - 15 Worman's Mill Court, Suite I Frederick, Maryland 21701 Telephone No.: 301.663.1644, ext. 2201 Facsimile No: 301.663.8994 Proliferation cell nuclear antigen (PCNA) evaluation will be done on the samples. Results will be provided for inclusion in thefinalreport. Palmitoyl-CoA Oxidase Tissue Collection and Analyses At the scheduled sacrifices, the right lateral lobeofthe liver will be collected from each - animal and flash-frozen in liquid nitrogen. The liver tissue will be stored in a freezer set to maintain -60 to -80C until analyzed by Covance for palmitoyl-CoA oxidase activity. Liver PFOS Analysis A At scheduled sacrifices, the remaining portion of the liver from cach animal after other required collections will be stored in a freezer set to maintain -60 to -80C. Samples will be packed on dry ice and shipped to Kris J. Hansen, PhD, 3M Environmental `Technoloangdy Safety Services. Liver samples will be analyzed for PFOS and `metabolites by the Sponsor. Results will be reported separately by the Sponsor. Tissue Preservation The following tissues (when present) from each animal that is found dead, sacrificed at an unscheduled interval, or sacrificed at the scheduled sacrifice after 13 weeks of - treatment will be preserved inl0% neutral-buffered formalin: 2 139 0003.42 Covne 63T2a9e215d aaodrrteanal (2) poavnacrrye(a2s) R c`ecbcreaurinmn colon `depuioddiedynmuims (2) . esophagus eye (2) piLHtruoiAstaatrsy salivary gland [mandibular (2)] ssceimaitniaclnevresviecle (2) skeletal muscle (high) skin femur with bone marrow (articular surface `spinal cord (cervical, mid-thoracic, and Haorfdetrhieaonfgtlhaenddistal end) spllueemnbar) ~ heart sternum with bone marrow ileum with Peyer's patch (lymphoid stomach aggregate) jejunum ttehsytimsu(s2) kidney (2) thyroid (2) with parathyroid lesions. trachea lilvunegr with mainstem bronchi uurtienraursy bladder Iymph nodes (mesenteric and mandibular) `mammary glands (females only) vagina Zymbal's gland Histopathology `Tissues (as appropriate) from each animal in the control and high-dose groups and each asencitmiaolnetdh,atsdtiaeisneodrwiistshachreimfaitceodxyaltiannaunndsccohseidnu,leadndinetxearvmailnweidllmbicereosmcboepdicdaeldlyi.nparaffin, pMaarcafrfoisnc,ospeicctiloenseido,nsstfarionmedawniitmhalhseimnattohxeylloiwn-aannddemosiidn-,daonsde egrxoaumpisnewdilmlibceroesmcobpeidcdaleldy.in Suspected target organs noted at the high dose will be examined microscopically from - each animal (at the Sponsor's request and added by amendment). Reports _ One copyofthe draft report will be sent to the Sponsor. The report will include the following information: . a 000143 Covance 63P2a9g-e22155 Experimental Design and Methods Results dose analyses. - `mortality cbloidniycawleoibgshetrsvations body weight changes food consumption - test material consumption `ophthalmic findings clinical pathology results palmitoyl CoA oxidase activities `macroscopic observations - `microscopic observations cell proliferation assessments (provided by the Sponsor's designee) Statistical Evaluation Levene's test will be done to testforvariance homogeneity. In the case of`heterogeneity - of variance at p < 0.05, transformations will be used to stabilize the variance. Comparison tests will take variance heterogeneity into consideration. One-way analysisofvariance (ANOVA) will be used (ifapplicable) to analyze body weights, body weight changes, food consumption, continuous clinical pathology values, and organ weight data. If the ANOVA is significant, Dunnett's t-test will be used for control versus treated group comparisons. ~ Ifthe ANOVA shows significance for body weights at Week 1, one-way analysis of covariance (ANCOVA) will be used to analyze body weights, with initial bodyweightsas the covariate. Ifthe ANCOVA is significant, covariate-adjusted means will be used for control versus treated group comparisons. - Group comparisons (Groups 2 through 4 versus Group 1) will be evaluated at the 5.0%, two-tailed probability level. Only data collected on or after the first day of treatment will beanalyzed statistically. . 141 000144 Covance 32P9ag.e22156 At the end of 1 year after issuance of the audited draft report,ifno requested revisions or instructions to finalize have been communicatedbythe Sponsor, then the audited draft report will be considered `final' and issued as the final report, signed by the study director, . and submitted to the Sponsor. Any modificationsor changes to the audited draft report requested 1 year after issuance `will be performed at additional cost to the Sponsor. "Two copiesofthe signed final report (one unbound and one bound) will be sent to the client. Record Retention - All raw data, documentation, records, protocol, specimens, and final report generated as a result of this study will be archived in the `storage facilities ofCovance-Madison for a period of 1 year following submissionofthe final report to the Sponsor. One year after `submission ofthe final report, the aforementioned materials will be sent to the Sponsor, . and a return fee will be charged; all raw data stored on magnetic media, protocol, study `correspondence, and the original final report will be retained by Covance. The Sponsor `may elect to have the materials retained in the Covance archives for an additional period of . time, and Covance will charge a storage fee.Ifthe Sponsor chooses to have Covance disposeof the materials, a disposal fee will be charged. PCNA antigen evaluation data will be retained by Pathology Associates International. - Liverand Sponsor. serum samples sent to the Sponsor and analysis data will be retained by the - 142 0001.25 Covance 63P2a9g-e21275 . PROTOCOL APPROVAL L`oAddrohw Ma. Soeacsat, PWhDM.Sain?" SMtudy Monitor yStuidypDirto7 r 7 Covance Laboratories Inc. Dae730/ pr i 13 000146 COVANCE PROTOCOL AMENDMENT NO. 1 Covance 6329-225 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats Sponsor: 3M, St. Paul, Minnesota Study Monitor: ~~ Andrew M. Seacat, PhD `Testing Facility: Covance Laboratories Inc., Madison, Wisconsin . SStHudYyDDirEecEtor:: DPReteLr J. TThRomRfBorRdY,RPIhDD eee `Thisamendment modifies the following portion of the protocol: : Effective August 27, 1998 1. Page 3, Proposed Study Timetable. To correct the audited draft report date, delete the text in this section and replace with thefollowing: : In-Life Start Date: September 1, 1998 IAnu-dLiitfeedEDnrdafDtatRee:poDretcDeamteb:erM3a, y19296,8 1999 - Effective October 1, 1998 2. IPmamguen1o2h,iPsotsotcmhoermticeamlPErvoacleudautrieosn,,CPelalraPrgorlaipfher1a.tiTono Tissue reflect Collection and the decision to ~ perform proliferation cell nuclear antigen evaluation for five animals/sex/group only, delete this paragraph and replace with the following: Athte tlheefslcahteerdaullleodbesoacfrtifhiecelaifvteerrf1r3omwefievkesoanfimtarlesa/tsmeexn/tg,rroeupprewsilelntbaeticvoellseacmtpeldeasnodf preserved in zinc formalin. - 144 0001477 - _-- ProtocoCloAvmaenncdem6e3n2tN9o2.251 Tw Page? AMENDMENT APPROVAL ST lu dil: 2 Coad / It/s [32 . AStnuddfyeMwoMn.itoSeracat, PhD Date M - PET ThomgBed, PhD SCtouvdayncDeigLiaboratories Inc. Date [0 fief [-- | 145 000148 - COVANCE. - PROTOCOL AMENDMENT NO. 2 Covance 6329-225 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats Sponsor: 3M, St. Paul, Minnesota Study Monitor: ~~ Andrew M. Seacat, PhD Testing Facility: ~ Covance Laboratories Inc., Madison, Wisconsin - Study Director: PeteJr.Thomford, PhD `This amendment modifies the following portionofthe protocol: Effective October 21, 1998 R 1 [Page 8, Dose Preparation, Dose Analysis, Stability. To reflect the decision to `perform additional stability analysis, add the following to this section: `Two additional samples (approximately 100 g each) will be taken from the 3-ppm dose preparation mixed for Week 9 through 12. One sample will be stored at _ room temperature for at least 19 days, then analyzed. The second sample will be stored at room temperature for at least 32 days, then analyzed. The sample collected for dose confirmation analysis will be used as the baseline sample. 16 000149 - _-- Protocol CAomveanndcmee6n3t29N-o2.252 Pye 2 Effective December 4, 1998 - 2. Page 13, Postmortem Procedures,LiverPFOS Analysis, Sentence 1. To clarify collectionofliver sections for PFOS analysis, delete this sentence and replace with the following: oAtthesrchreedquulierdedsaccorlilfeicctesi,ontshewilrlembaeifnliansghp-forrotzieonnionfltihqeuildinvietrrforgoemneaancdh astnoirmeadlianfater : freezerset tomain-6t0toa-8i0nC. Effective December 15, 1998 - 3. Page 13, Postmortem Procedures, Cell Proliferation Tissue Collection and Immunohistochemical Evaluation, Paragraph 3. To include examination of liver sections will be stained with hematoxylin and eosin as part of the cell proliferation evaluation, delete this paragraph and replace with the following: Proliferation cell nuclear antigen (PCNA) evaluation will be done on the samples. In addition,liversections will be stained with hematoxylin and eosin and . examined microscopically. Results will be provided for inclusion in the final report. Effective January 25, 1999 - 4. Page 14, Postmortem Procedures, Histopathology, Paragraph 3. Liver and stomach were identified as target organs in animals in the high-dose group and will be examined in animals from the mid-dose group. Therefore delete this paragraph and replace with the following: - Liver and stomach from cach animal in the mid-dose group will be embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. . 147 000150 - -_-- ProtocolCAomvaenncdeme63n2t9N-2o2:52 ewe33 Effective February 8, 1999 - 5. wPaasgeid1e4n,tiPfioesdtamsotratregemtPorrogcaendiunreasni,mHailsstionptahtehmoilodg-yd,osPeagrraogurpapahnd3.wilLliver and be examined in animals from the and replace with the following: low-dose group. Therefore delee this paragraph Lainviemralainndtshteolmoawc-hdofsreomgrcoaucph awinlilmableienmtbheeddmeidd-idnopsaeragffrionu,psaecntdiolniveedr,fsrtoaimnecdacwhith hematoxylin and cosin, and examined microscopically. AMENDMENT APPROVAL 77 7 AStnuddryeMwoMni.toSeracat, PhD - aM /7 Date T - Pet fT. Study Thofogbra, Dirdatr PhD, Covance Laboratorie Inc. Bate 4 148 ee 000151 - COVANCE> PROTOCOL AMENDMENT NO. 3 Covance 6329-225 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats Sponsor: 3M, St. Paul, Minnesota Se nSSTee Study Monitor: AndrewM. Seacat, PhD Testing Facility: ~~ Covance Laboratories Inc., Madison, Wisconsin - Study Director: PeteJr.Thomford, PhD `This amendment modifies the following portionofthe protocol: - Effective August 28, 1998 2 Page 4. To include the vehicle used to dissolve the test `material before mixing with the diet, add the following section. - Vehicle Identification Acetone . Lot Numbers `The lot numbers will be maintained in the raw data. Purity On file with the manufacturer Stability On file with the manufacturer - 149 000152  Protocol CAommeencdammNaeorn2st= 3 _(]) - PmPaege2? ASttorroaogem Ctoenmpdeirtaitounrse = Characteristics dIenffionrematthieovnehoinclseynitshoensifsilmeewtihtohdst,hecmoamnpuofsaicttiuorne,r.or other characteristics that AMENDMENT APPROVAL (hichso TY Bos =(777 . Andrew M. Scacat, PhD ~ Study Monitor 3M fo Ba PelhT. PhD Study Direbtor Covance Laboratories Inc. Day 2 QD To Co VAT ras ect Gn Or sr - 150 -- 000153 APPENDIX 2 CovanMcTe-6633219-42.215 Individual Animal Fate Data Individual Clinical Observations. - Individual Ophthalmic Observations - 151 000154 2332 a o 12 - 33 Sha RE 7 DEER RNASE am Bi 22 & 153 333 a5 150 E33: &5 155 2332 2 156 32 2 3 157 me 4s mbR esweeso sion rons rr 32 2 22 158 23 2a 0 2233 223 161 wr eld 2333 5 @ 23 253 @ | oo 13 se mt srs CS 15 ANDO NAS BO SIGIFICANE PINGINGS 33 233 164 33 g 2 Ps 2323 32 166 | ais 3 o-------- -- 2232 3 0 2323 H 168 - EE 333 a 8 ps SR 2323 J m os 52H 233 a a nm os 5 SE 22332 3 m 2233 33 4 23 22 3 1s g333 3 ve | pod nsw we 2333 g m 233 82 1 3 2 8 i I. ERE 233 &22 180 323 222 181 333 &k = tions cogints ometicns 2233 &2 1 2233 2 18 2283 2> 185 23 &22 186 : & w 32 2 2 158 0 rem Covance 6329-225 APPENDIX 3 Individual Body Weight Data (g) ~ 189 000192 32 a222 190 Sotvisnt soy ian sen (1 Sem B mW OW OB a we ow ow om 233 22? 01 os SEH 2232 a2 192 Lp -- 32 2 &2 193 ivi soy ian sen (1 2323 32 194 . 2mm ery sonore ey win wm, EH nos. 6 2233 23 195 Seivioat soy ian en a1 323 28 3 107 is SRE 233 8H2 198 ivan boty san ues a1 232 8F3E] 199 . tun sty ion aca a1 EE 232 82a 20 APPENDIX 4 CovanMce 6r329i.25 Individual Food Consumption Data (g) Individual Test Material Consumption Data (mg/kg/day) : 1 000204 332$ & m rvs roocommntion sta. (4) IN ails HH ewe ow aw swe om om 333 &2 0 223 233 mn PO 32323 208 233 323 26 - EH 223 32 s El - ER 223 3B8 28 | gE on EE 53332 38233 & 20 i33s2 |2 wm ie eh 332 32 LJ an | gH HB 8 TH TH THE ETTEE | od #8 #8 # # # 8 8 # 32 HW BB OH OH OH HB H OH HB HB H 25 m 232 332 J En iis Tt tas meses ts in FH OH OH wd wid mdi md wid 0H 3233H & as 33 333 " iis vn tempaves 3333 8 mn rivioul Tes waterio Cansngon saa (sya) 23383 B 2a vin Tu tia, Comin a (aan) 232 3B = 29 ie EB 222 388 2 ivan st cera Comme nes eg aldor) 32238 om | ns ot | niin et cris Gmeoti s itt 333E2 a m APPENDIX Individual Clinical Hematology Data Individual Clinical Chemistry Data Individual Clinical Urinalysis Data Covance 6329-225 IMT-63141 . 23 6n022e mE WF W OW OW dd 223 33 2 sepeix 5 wa a BOT WOR OR fa Congy OE 233 @38 2s WE GT WR ATR WP BL ND. Ue we mm me 23H3 8 => 26 opens 3 Conny BRE Ban of. SPL AL BP. BULB, ve tem ee mem me 333 88 m 2333 B us Aopendin Comes SB 3333 3 2 sopentix 3 wan ae STOW WOR cosEB 323 328 20 Coo4 wnte 00 oisg oma 33 332 = Bes of. EFL SUL BR. BL BP. ve eee -- 332 a m mE oT ST ETL RR. SEL BR. VU mee ee wen me 233 323 i J -- [---- 323 J35 2 Grown 4 sossevels 135 os tistm esros 323 323 25 BE BONER a 333 333 26 wa of. WOT OW OWT de 32233 3 El BE ofl A AVR EP. BEL BG. NO tee ee omen we Grou: 1 Dove Laval0: `Dosagevats pomHeros. rT 3233 3iB EY [-- cos OTL BE Th SMR BO. EPL Bm wo om wm 3333 3 m 3223 o 2x0 Aopenti 3 cons BE 322 3 3 21 wan ae M0 TOW OWT de Gro 3 toe tats 3 Pp 332 3 a %2 is gs Ea BT FRY | SEH om omg Eo WB 333g 3 Ra of BFL APR MW. BL BEL. vee ow orem we 3233 ~ 24 BE ofa ST AL BW. BNL WR CU ee ee wom mo :52 :3 us 3222 26 sensin s 5B 22 238 uw 332 33H us 2233 83 2 223 3&# 20 223 333 21 vis os 5 | gf 4 fH OH oH 3 B 8 323 3 a 252 = i BReoo Ms w0% owTR Cwh owB ow B B0d3 233 3P] & 2 Aopen 3 a -- aegon Comey PRE gm oF FB IB oH BoB 2 Hs wt wT Wow Ww wt 33 33 ast 22 333 % 25 3323 3 256 Sependix 5 cones REE 3g233 2 232 23 258 223 383 2 p-- comngs 358 ] Bou oF ion oR oH o Ed 352 3Z " | 323 3a3 21 222 3a3 x2 pent 5 Coop 3 Sontele 30 ooiees eammeot gs SRE 233 33I ES) opentix 5 coms 23 222 433 4 Gre 4 sos evel 109 oan tts emwar 322 &33 265 seoenaix 3 [-- ons tie7mmerce cos PE 822 32@ 26 Geo 3 msetevli 30 osstnt,sm art 22 33 3 27 3 23 N 28 33332 & x 333 33 m cr a ts 0 waes ome ee 3>3 33 m 32322 a m CT ee i3:: 3223 3 Ed 3233 > zs 2223 76 3332 g m Aependin 3 corss 3 rope 2 soeteats 3 ons ie rmmerce 222 23 El ee se or smn an wm,rca 322 33 ED 23 332& 20 Groups 3 Dose Levels 30 oosage Unit: pomNeroS. or Gre4 on ert 10 an ie enmr 3223 2 = 223 3a2 x ties em gt 3233 82 w 223 32NS EY ieET Pps ow 2> 3&z as seveniin CT REE 23333 & EY 3322 8 El APPENDIX 6 Individual Anatomic Pathology Data e[e--n--r"e - 8 0nnz91 | IN sos AAS on :25i 2 2 232 2 20 3232 2 1 2322 32& 0 3 H32 ] Een 35 3 - 23 23 322 323 26 232 a23 7 322 28= 2 223 328 2 23 H3 a 0 >333 2 sot 32233 a 0 22z23 303 23 232 ~ 0 33 32 & 20s 3332 3 06 23 32D S 307 33 323 B 08 223 32 2 22 3B2 310 22 322 an 22 322 @ 312 asl GE Sa l TOh Ty oootLE olerp, 32225 an 23 32 5~ 3 Sroka Aton setenv re 32 323 7) as H 233 316 22 382 an 222 38B a1 23 33 8 319 33: 5& EY PearaL RRSEL Mr TRG 1H mars his: 5 322 283 EY Soivioul ruten colony uta RE 32 332 a | EERIE BH we BR ry ER SO 333 32 m 233 283 En 32 3 8& ns Sotividans matemic Pchaiony aca ERE 33 3 2 2 | " ste creme soc srr am wen not. 2 88 8 8 Ed 22a g 28 a82S8 2 a&S8 & 30 ggs8 2 = g 8 & m 28 8 & EY 88 a&3 En 82 &88 3s aii sme en a So 888 8 ky 336 28 &g m = fa Lo == Fite gg o2B 5 | gf, = = = IF g 8 3g8 22 20 2gg 2= a aa88 " EC 9S8 a2> ws 388 84 4 22S8s @ 35 8S 82 346 m---------- eit 2g&8 347 8ggc g ww 288a 39 gg8 8 0 gg g ssi 28 &aG Eo) aa28 3 2gg 89 354 222 8a 56 gsg: g - ag88 " EY S8 888 3% S88 8& 30 gg g2 0 838a a 0 g88 8 ES 3g28 04 ge 8& 26 222 aa 36 o38e 83 361 28 e H 368 288 ]< 3 28 a & 30 S2s S3as om 228 &o3u mn g3 43 In 2g3 4J ED 288& CE as 88a 6 EE a, ss 0 2S8 8g " RE it mn 38 a2QB ES 23 828 nm 228 82& 0 | Ee oe. a g gg2 a ei, a = 28g i8 a iL 2g 8&& wn 38 8@$ am POUT Ra 289 12 ass 82 82$ ES ror TE ET ware 223 a@3 Ww 283 8 a 288 88 $22 8 TM | EE RC s8g& g " 2g2 8 8 8g2 " g 84 0 soncirssem semen, mor: 107 222 822 35 22 83 3% 3g 2 El g 22= 08 erinEA SLETH ewe i} 28g 2g 3 | 23 22a an 28: 32= on 28 2& wn roE r ES RT TD ars 22 23 5 an sm ey ne er es 10 8 22 ws ym me rn J ggs 2$ as rvs dtentc mseieny uta 288 2gg 05 22 28 a0 | SEE BH oe I SE BEER se 2<gg22 ws 2Ss 2B 8 a 2g2 23 a0 2g2 B an 282 2& an 3322S @ an e8sa au aa33 @ as 2233S B a6 gg28 an 2g =8R as 2&S =88 a 88 h8 C 2gg 8 - a rset ens, to xs EE 288 8 a oso ston stacy aca re g8 Ee gg2 5 2228 as  i a i = 2g2 38 a i5g ba a gg2 - eis emt eon we CRgg 8 wn pd an dat Ee BER g 52 88. 2 a o g & Po divin smi Polen aa EH | rr EE SLE TR ewe noes 10 og.h os  g8a8 7a2 a6 | gg 2 @ o8k% 2: as SER INTL TI SOTALS Re, sa oo. <&32 I2 EE g . gEEsXi: 23 2 wn <a 2 4& &= "a 38g 52 w 3 2 * as 22= < wo r 233 2 8 " g g2 "w 223a23 ast 23 &22 as2 22aaS - a 222g < as SS8 i a 455 g2g< & ass 233S 22 a7 28S 22& ass 822 22 a5 233 2? & 2) S8s 222 61 22 2?& 0 <gz 22c 0 88 2 ao <gg >2 65 gg a& 6 28 a 3 a6 i Ave Sho as BAAR ER TT 38 25H 68 ereEL 88 23 0 3sSS a & an g2 2 = an o 8 1 g&x < n gsg s a <82= 3 an A LT IU ATI 0 J fe, sur, vo ge= # ars 8 2 3 476 cg & im ettt hsi g2 2[3 an GRR 5 oR EE AY Th TR - eSs 82a a cg&E & TM s< <> = Ee g8 7 ie w g & aw e& " = a 88 222 ass 88c 2a& a6 22 =G aw 2332 2 g8s 88 5 83 2f8o] 0 8 22 2 33S a3a 0 22 &&& 0 882 & o 288e & as gg && 49 S93s a8 < a 22 ae8 a cgg a$& a 82 a&g 500 tvs Aten ston ven PSE 288 aSE son 88 i&g] so g&&ec @ 03 3 88 ~ 04 232 ae8 05 323 a 3 506 8ES a sor 222 &2B 08 34 aa EY 82aa sto 38aa > su rare SE TT amare 22 &2 sn 3egg aa & sn 82 a2 si 282 a5 sts ett em eins so ggg 2 sis 38 82 si 22 8=& sis ]| = EE +H3i iJg mio BEEEE 88 a9  &g FE <gg && 21 288 a8a = iris dteni colon ata re gg< && = | EERE SE Ce eT. <gg &&Ss EN comms Tio NRwT Rae Sh g& &8" 25 2S2 a82 526 S2 &&2 2 cgS &&8 2 222 a& 0 3g82 2 m 2SS aa 532 222 a&8 ES 322 && 34 frie es Pp KG opXY 22 &&g 55 22 a&2 ES 288 a2 s El 8g& 8 EY 282aa B 59 Loteiamt stmt sasstoy sa SEB 238 a2 EY - og| a = sat 28 a= u se 88& 2 sn coms Tica ThE as Sh 2e &g = su cg a25 sis -_-- CovmanMcTTe-66e33219u-42.215 APPENDIX 7 Cell Proliferation Report Note: This appendixofthe report contains information supplied by Pathology Associates International and has been reviewed by the Quality Assurance Unit of Pathology Associates International. - 546 000543 ~7 CovanMce 63T29.25 a comppaannyPyaotfhSoclioegnycAesAspopcliicaatteisonIsntinetrenrantaitoinoanlal Cora poratio = -- .. FINAL CELL PROLIFERATION REPORT 13-WEEK DIETARY TOXICITY STUDY WITH N-METHYL PERFLUOROOCTANESULFONAMIDO ETHANOL (N-MeFOSE, T-6314) IN RATS 'COVANCE STUDY NUMBER 6329-225 PREPARED FOR: ~ 3M RN PREPARED BY: TOXICOLOGY SERVICES BUILDING 220-2E-02, 3M CENTER ST. PAUL, MN 55144-1000 PATHOLOGY ASSOCIATES INTERNATIONAL 15 WORMAN'S MILL COURT, SUITE I FREDERICK, MD 21701 MAY 12, 2000 TSWormeMIrsCour,Sut T +Fredericki.artandT1701 (0) 65.7694 Gar} eeFa - 547 000550 CFoivnaanMCcTeel.l663P32r19ol-4i2.f2e15ration Report Covance Study Number 632Pa9g5e25 TABLE OF CONTENTS - L `CELL PROLIFERATION REPORT IL TABLES III. SIGNATURE PAGE IV. QUALITY ASSURANCE STATEMENT Vv. APPENDIX I - 34 000553 - CovanIcMeT6-362391-42.215 CovaFinncael CSteuldyPrNoluimfberearti6on32R9e-p3o2r5t Page3 CELL PROLIFERATION REPORT PERFLUOR1O3O-CWTEEAKNEDSIUETLAFROYNATMOXIIDCOITEYTHSATUNDOYL CCOVANCE STUDY NUMBER W(NI-TMHeFNO-SME,ETT-H6Y31L4) 6329-225 IN RATS PURPOSE The diet 1p0uraptossfeorofatthleeassttu1d3ywweaeskso. assess the toxicityofthe test material when administered in the . Chrneiipstreiseredenpto1srt3t,hWeseucbeelmlkitptDreoildeitfbaeryryaPtTiaootxnhiofcliiontdgyiynSgAtssusadonycdiwaiitntethserNIp-nrteMeterattnhiaotynilofnPoarelrCf(olPvuAaoInr)ceto StthuedystNuudymbSepron6s3o9r,6.33M5,5 - $sQrtUaucMdtyeicFweeOr(SeGELc,Po)nTd-Ru6ec3gt1ue4lda)tiiInonncRsoamatpssl"is.atnAcfloelrtwhaistipnhecTttihsteloeFfo2to1hdeoatnasdksDraussgocAidamtiendoioswtcirttaahtniPeosAnuTlG'foonpgao.mrLitadibooonrEoarpteo1sr.y) 58, issued December 22, 1978 (effective June 20, 197f),thaenUdSwiCtohdaenyofapFpeldiecraablleReagmuelantdioancst,eFay MATERIALS AND METHODS - Tissue Collection for Cell Proliferation hFeipvaetoacneilmlaullasr pperorlisfeexratpieorn.gArouspecitniongroofupthse 1letfhlraotuergahl 4lobweeoretfhsacrliifviecrefdrodumrciangchwoeef$k 13 rats for por - cPgLerelaoleuenpuactl(woegaarnsrosua.ppnsrteiTpg1iae-srn4se)u(dePwfCbaoNlrsoAcH)kf,&sixaEweemdraeveaansldhuaitppiproeondceoasnsdPedAiImtmofuonrpoashreiacsftftiioonncihnbeglmoaicnckdalsbtydaeitnCieoncgtv.iaonnFcreoofmppereroalciphfreobrltaootccikno.gl rkerofcell proliferation. Tmmunobistochemistry for Cell Proliferation SS(SeOucptNeirAofnisoosSfttpanaPldruaasrf,dfiFinims-mheuemrnbheSidcsdietendotcitfhiiescsm,uiePcsiatwliesrbmeuertgchhu,todaPstA5w)eurtmeo eannsdurpelacaeddheosniopnosdiutriivenlgy used to stain tissues for pcrhoacregsesdinsglifdeosr PC - pSaenOrtPtivbiofddoayrsteiom(PmAuEnBNoChAiKs(ittDo,cAhlKoeOtm,i#sPtlKort-y6#)10.0160,,BrPiPeAAfIIlyN,Noo.t.iAsK1s7u2523s4e))ctaimnoedntsrheowadegernfetorsirtnhecquubiarteedd fwoirtthheaavmiNdoiAnnc.(apSriAaacpno HaeIn8aiHb8ie2d0zy1)es.omdplTeixbs.sy.uetPhsCeecNtciAhornesoxmpwaregeresensicoo3nu3ni'tndericesaltmlaisinnioenbaelwnliztpihdaisnees of the (DAB; celel dceytcelceti(oGny,of,thGe, aantnigden): Sigma Chemical `Coy lon - pwraismairnyclaundteidbodiny,the staining run and consisted odf studhyhteimsastuoextyhlaitn.wasAnnoetgaitnicvuebactoendtrwoi]thsisnhee . 549 } 000552 . Covance 6329-225 Final CMeTl.l6P3ro1l4if.e1ration Report `Covance Study Number 632P9a-g2e2s5 Cell Proliferation Measurements 3 For cell quality proliferation of staining, evaluations, slides were first perused at low processing and sectioning, pattem of cell magnification labeling (c.g. (100X) to judge centrilobular or panlobular, extrahepatocellular proliferation, endothelium), and histomorphologic changes. such as Kupffer cells, bile duct epithelium, Cell proliferation was then quantified at higher `imnadgenxi,fiLcIa)tiwoans(d2e0t0eXr)m.ineTdhebypesrccoernitnaggaet olfeahstep3a0t0oc0ytheespaitnocSytepshaisne1o0frtahnedocemlllycsyeclleect(eldabfeileilndgs - per animal. Histomorphology was further animal evaluated for cell proliferation. assessed by evaluating the serial H&E slide for each Statistical Analysis - TLIhebeSttwuedeennt'csontteroslt a(ntdwot-rseiadtemde,nutngerqouuaplsvaursiianngceM)icwraossoufsteEdxctoeltevsterfsoironsta5t.i0s.ticAalPsivganilfuiecaonfceleisns than 0.05 wasjudged to be statistically significant. RESULTS - Cell Proliferation ITnhdeivpiedrucalenatnaigemaolfapnrdoligfreoruaptimngeahenpacteollcyptreosl,ifaesradteiotnerdmaitnaedarebyprtehseenltaebedliinngSiencdteixon(LITI)(, Twabalse n1o)t. - significantly ats. The LI wianscrseiagsneidfiicnanttrleyatleedssgtrhoaunpcsonctormoplsariendfetmoatlheatraotsffcroontmrgolrsouipnsc3itahnedr male 4. or female Histopathology - wSeictthiohnesmaftrooxmyltihne asnadmecotsiisnsu(eHb&lEoc)ksfoursheidstfoopratphroelpoagriactieovnaloufatPiCoNnA-tostfaaciinleitdatselitdheesiwnteerreprsettaaitnieodn of the immunostained slides. Appendix I Individual animal findings and group summaries are presented in - The results showed interpretationofthe no changes in the liver tissue PCNA staining in this study. of male and female rats that would alter the ) 350 000553 - SUMMARY CovanMcTe66332104-.2215 CovFainncael SCetluldyPrNoulimfberearti6on32R6e.p5o2r5t Pages In the present study, cell proliferation was measured within the liver of male and female rats wferoemkscoonntrsotlud(y0.ppNmo),stlatoiwstdicoaslely(s3igpnpimf)ic,anmtidincdroesaese(s30inpcpemll)paronldifheirgahtiodno,seas(d1e0t0eprmpimn)edafbtyert1h3e - PCNA labeling index (LI) were observed in male or female rats after perfluorooctanesulfonamido ethanol. Although cell proliferation was 13 weeks ofN-methyl statistical controls in mid dose (30 ppm) and high dose (100 ppm) females, the decrease ly was less than not dose- r`eTlhaetreedf,orea,ndthitshedeicnrdeiavsieduwaalsannoitmcaolnsviadleureesdwteorbee w`biitohliongictahlelyrraenlgeevaontb.served in control animals. - = 000554 . CovanIcMe6T3-26931-42.215 CovFaincnea CSetluldyPrNoulimfberearti6on32R5e-p3o3r5t Pages IL TABLES Legend + SEM = Standard Error of the Mean = 552 00055.5 i CCoovnanTece66S31t29u0-y212N5o. 6320225 Table II-1. Cell Proliferation in Rat Liver . ro - DoseGroup 1 (Control 0 pom) |_Sex Animal | | Number | Laboling index (oy WMT__coCsosoeoes | 0023478 ((CCoonnotrr00ppom - MMMTTTCoCCsooosssootoose || g00'21se25s73] ean 0a - -- [2(ooww 3pom ppm) |[W w s|eocmesee za ||osoos0.r1e50 - E 22 (LowC=T 33opbpwomm) -- --( WM o -- cCeosssar|-- r [0022243|| 5E (i= 5E d--0--ppm) Sss0pom FUE M A CeseJp |0o1t62 | Coe| moliesa| - 3E (Vid -30 pp t = {MT TT Ces050 o0.d065e|| Mean oer : L[e 4oiHgignh f110000po pppmm)) toT)oiTe|"GGs sasessoamone rsTT0pao2o1o9a n0||| [C4r(Figh 00 om) ---- caso0|s 0.008 TT e Sew ro The Pagel 553 000554 _ CovanScMeT6.362391-242.51 Table 1-1. Gell Profferaton in Rat Liver Covance Study No. 6326-225 - ro Dose Group Sex NAunmimbaelr|| iLnadbeexlin(%g) - |[T1(((CCCooonnntttrrroolol--000pppoppmmm))) FFFT_ o| sCCoos5o0se8s5 e| 0.001z17s6083 1 (Control-0ppm) 1(Control 0ppm) |FFTT__c|e_ec|ooos002er105er| - TTTWem oas TTT sem Too| [[22(CCooww=3ppoomm)) CF eToeonr [ooaesd] [[-222((CCoo3owww=p33 pppoomm))m)~T T~ TE T FFT[TI CCco6o0se06oe||o[so0002l.22852o35 - TwSEMee0n108s [S50(M Wiiag 33300oppppppmm)))~~ |TF"FFT|| ToCGsoes0otzzsss|to00o00o68i38m|| . [TC3300pdpm) T -- F 1 Co | s oo5eTs 2 -| 4-- re Fgh 00 ppm) CTT FT T|_CsoM 0ease |aoo 0.on 09m%o| - J44 J (rigHh -i11000g0pppphmm)) bi1g00phom) -- 1"F'F T"TCCooeeo0ads6| |FT Coeoar | 0oa.r12|8 o1s|2 [-4(igh -100pom)-- |I F _CoeMoesasn [00.11122 8| -- sen oom - Page 2 554 000557 - CovanMcTe66332194-.2215 CovFainncael SCteuldyPrNoluimfberearti6o3n2R0e.p2o2r5t Page? - IIL SIGNATURE PAGE Submitted by: Project Manager: Sandra R. Eldridge, Ph.D. SA5200 Date Project Pathologist: ~ Ly ey Carolyn Moy, D.V.M,, Diplomate, A.C.VF. Date 5/r5/ oe - 555 000555 - CovanIcMeT6-362391-42.215 `CovFainncael CSetluldyPrNoluimfberearti6on32R9e-p2o2r5t Pages - IV. QUALITY ASSURANCE STATEMENT - 336 000559 - EH PatAChoolomogfySpcAiesncaseAocpnciicsaytotsensteImanttnaerCnoaptainonal CovanIceM6T3269.42125 EEEo .= Cell Proliferation Report - Perfluoro1c3ta-nWeeseuklfDoineatmairdyoToExtihcaintoylS(tuNd-yMewFitOhSEN-,MTe-t6h3y1l4) in Rats Covance Study Number: 6329-225 - QUALITY ASSURANCE STATEMENT . pATrhsiossmuurlcaegnlaclteepdUrnobiltyife(trhQaetAiUUo.)nS.apsrForojeeoqcdutiarhneadds bDbyreuetghneAdiGmnoisonpeidcsttLeradabtoiaronandto(raFyuDdAPir)ta.ecdtiTcbheye(tcGheLelPl)PprAroIelgiufQleuaratalitioitnosny ifenpsopretctiisoansn/aaucdciutrsatpeerreffolremcetidonaonfdtrheeporretceodrdbeydtdhaetaQ.AUT.he following table is a record offoe InDsapetcetioofn Phase Inspected MDaantaegFeimnednitn/gPsrRoejpeocrtteMdantoagPeArI - 1024/2033//9080 MiSctrudoytDoimay and Supporting Documentation 12723198 04/03/00 0045//0132//0000 FDirnaaflt CCeellll PPrroolliiffeerraattiioonn RReeppoorrtt 0054//1023//0000 Xo Karen g E. A Butler 1 Quality Assurance Officer D5at/e 2foo ~ 15 Worman's Mil Cou, Sue | + Frederick, 557 Maryland 21701 (307) 663-1684 + 301) 353-6900 000569 - CovanIcMeT6-362391-42.215 CovFainncael SCetluldyPrNoluimfberearti6on32R9e-p2o2r5t Pages APPENDIX I - 558 000561 - : Covance 6329-225 Individual Animal Findings COVAINMTC-E63#1643.219-225 Perfluoroo1c3t-aWneeseuklDfioentaamriydoToExtihcaitnyolSt(uNd-yMweiFtOhSNE-,MeTt-h6y3l14) in Rats - Tissue: Liver --OMeANNUEIMMRBAEL_R _ SEX DOSE GROUP HISTOLOGIC FINDINGS OOOO" co6027 F 3 C9026 F 3 No Significant Findings No Significant Findings cos021 F 3 cos23 F 3 No Significant Findings No Significant Findings ~ 96038 F 3 No Significant Findings Cosas. M 3 No Significant Findings coos M 3 NoSignificant Findings cosoaz M 3 No Significant Findings - Cosoar M 3 Cossg TMM 3 No Significant Findings No Significant Findings Cos96s. M a No Significant Findings C9967 M 4 No Significant Findings Coses M 4 Coss M 4 No Significant Findings No Significant Findings cose? M 4 No Significant Findings Cos048 F 4 No Significant Findings co604s F a - co60s6 F a No Significant Findings No Significant Findings Cos04s F 4 No Significant Findings co604s F a No Significant Findings Cos9s. F ' Cos997 F 1 No Significant Findings No Significant Findings cososs F 1 No Significant Findings cose? F 1 No Significant Findings 96000 F 1 Cos909 mM 1 No Significant Findings No Significant Findings coso0z mM 1 No Significant Findings cosos M ' No Significant Findings - C9595 mM 1 cos906 mM 1 No Significant Findings No Significant Findings = 5% 0006562 . ndividust AnisFindings CoCvoaunNcCeE5T#2e93.i2n25 5 Peruor1o3a:WceaekncDinalroynTaEonticieldnyoS(ayMewPitOhSXE-,MeTt4h5y1!4 in Rats Thue: Liver oar sex bose Group HISTOLOGICFINDINGS . e oss wm e 2 e NoSinifcntrFiendeinsgse own wu 2 comm 2 NoSinifcnFindings NoSignificantFindings - osm 2 om m 2 No Significant Findings No Signin Findings PE 2 coos F 2 No Significant Findings No ignfan Findings . oon oF 2 cows F 2 No ignifcntFindings No igifcant Findings con 2 oa F 2 NoSignifcnt Findings NoSinicn Findings - oon F 2 ows F 2 NoSinifcnt Findings NoSinicnt Findings cows F 2 NoSignificant Findings 360 000563