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. oo R260 : TpH! - COVANCE Draft Report 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats J covers nase 0045477 COVANCE> Sponsor: 3M St. Paul, Minnesota AUDITED DRAFT May 25, 1999 FINAL REPORT Study Title: 13-Week Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314)inRats Author: Peter J. Thomford, PhD Study Completion Date: To be determined 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 of 551 004548 -_-- .00 TCove anIcMeT-662n 391.42.215 = QUALITY ASSURANCE STATEMENT ~ 004549 CovanMcTe-6632391-42215 STUDY IDENTIFICATION 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 Study Location Study Director Study Timetable Study Initiation Date: In-Life Start Date: In-Life End Date Study Completion Date Marvin T. Case, DVM, PhD 3M Toxicology Services 651.733.5180 Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 PeterJ. Thomford, PhD Covance Laboratories Inc. P.O. Box 7545 Madison, Wisconsin 53707-7545 608.241.7207 August 27, 1998 September 1, 1998 December 2, 1998 To be determined ~~ 004550 Covance 6329-225 re r------ ee SCALE], . KEY PERSONNEL Study Director Study Toxicologist Study Coordinator Supervisor, Small Animal Toxicology Supervisor, Dose Formulation Supervisor, Chemistry/Tox Support Supervisor, Laboratory Animal Medicine Clinical Pathologist Supervisor, Clinical Pathology Anatomical Pathologist Anatomical Pathologist Supervisor, Anatomical Pathology Peter J. Thomford, PhD `Thomas E. Ryan, BS Nii Van Bruce-Konuah Nathan E. Snortum, BA, BT, ALAT Dixie Bushee, BS, LATG Brian Schoenike, BS Donna J. Clemons, DVM Diplomate, ACLAM Robert L. Hall, DVM, PhD Diplomate, ACVP (Clinical Pathology) Ronald Markevitch, BS, MT (ASCP) Sandra R. Eldridge, PhD Pathology Associates Intemational Johnnie J. Eighmy, DVM, MS Diplomate, ACVP Diplomate, ABT Deborah Pirkel, ALAT - 004551 CONTENTS CovanScMeT6-362391-42.215 Page REGULATORY COMPLIANCE..c.cccrcnssnrsssnnsnnsssessmnnee 11 DEPOSIHON.rrenesmsesmensensmseeorenoeeseoeoeeoeoee 11 ANIMALS AND HUSBANDRY... 12. HOUSING ad MAINENANCE.....c.ccoserverernsnsenessssessssssssessrsosesesossesssomnn 12. PROCEDURES... 13 Group Designations and DOSE LEVES........vevevrererersssessssssssssrerosemrssos7n 13. . RELENHION SAMPIES...vcrrvevvnsnsssnsssnsrsrssseseesesesssesessessse serene 1 DE T Clinioal OBYEIVEIONS ..coucreresrssmmersrsssesssmmmmmmmmmmmmmeenssssssisscssisssssseoresmsonsesnens 56 OClPAHINCEAI]MOPAIHOOZIYO.R.Y...c.rrecrrevrrrvreorrevrrevrreerneennenssvsteesnonnssevssenssessesssessseemnsesnsssssssssssssssssssssssssssssssssesosesmsnesesn1e1n11 Serum Perfluorooctane Sulfonic Acid Level (PFOS) Determination................c.us. 18. NOEFCGTAONPWSEYIcGoHvSrs..scscsccscercrscnremrssntssvssesssrssnmssssssssmseremnmssnsseissssmeessesssmsssetss esssssssses mseossssssns snrmssnss 18 19) Cell Proliferation Tissue Collection and Immunohistochemical Evaluation .................. 19 PLIaVlEmritPoRyOlS-CAoRAIOYxSEiSd.a.s.e.cT.icscsvuverCvoslolreecstrinosnnsasnsdenANianliyssess.s.s.e..s.s.s..s.s..s.e.s..s.sccsceeseeseeesssssossssscsssosssssesrosances 19 19. HISIOPRHOIOBY .orseeeceurssscerssrssaesmmssssasmecssssssssmesssssasssssssssssssssesssssssssssssssssesessasse2s0ss 5 004552 CONTENTS (Continued) CovanMceT6632198-2125 - Page Clinical ObSETvations and SUIVIVAL.............suusuussssssssssssssssssssssssssmssssssmsssssssssssssssssesses 22. OPMBAINIOIORY .ccnrsssscermssssseensssssersmsssssesessesasmsesmssssssssssssssesssmssmseesssssss 3 BOAY WEIBRIS....orcrererrerssssesenssnssessessesssssesssssssssessssssssssss sss ssssssseseesos 23 "Test Material CONSUMPUON. eer24 CCl PANOIOZY...vorerercrcrersnrsesosinnsesesnsesessssssssssssssesessesersssssso2n8s. SCeelrluPmrPoelriffelruaotrioonocTtiasnseueSuCloflolneicctiAocniadnLdeIvmemlu(nPoFhiOsSt)oDcehteemrimcianlatEiVoln.u.a.t.i.o.n.........u...s..s.e.e.w.e.e2.e24s5. Liver PFOS DELEIMUNALON .....v..c.trvrs11111101101118181808851808158088188088210830018 0s es ennasenasens2n9s ADSIOMIC PANOIOBY ..cornresesnssssssessssssseseesssssssessrmssssssmssssrsssssssssessassssssssssssssssssens 23 REFERENCES wmmmmmmmmsmssmmmmmmmmmsssnmoand OPHTHALMOLOGY REPORT... 29 COMMENTS ON THE DATA .crrrsrnnnssssssessseso3s1 CODES, ABBREVIATIONS, AND UNITS... 40 General Codes and ADDIEVIBIONS ............c.ccvvcvvvvvnverrenssrsnssssssssssssssssssssssssssssssssssssssss4nn1s `ACbobdreesvifaotriColnisniacnadl PUantithsOfIoOrZYCl.i..n.i..c.a.l...H..E..M.cAsuOusIusOsuZmsYmsss remeron ssssssmsssssssssssmssssmssmssssssssmssssassassssasses 852. `Abbreviations and Units for Clinical CREMISILY ..............csuusummsssmssmsssssssssssssssssssasssssasses &7 ACbObereSviFaOtriAonRs OanIdCUnPitAsNOfoIrOCRlYine ical URNAlYSiS....r .....ccceesssssuseee srsssssssssssssssss ssssssassssses 4S9O TABLE 1 Results of Homogeneity Analyses (PPM)..........ceveevsessssssssesssssssssssssssssssssessssssess 53 2 ResultsofStability Analyses (PPM).......couuuurevsesssssssssssssssssssssssssssssssssssssssssssnss 4 3 Results of DOSe Preparation AnalySes (DPM).........coeuerssessrsssesnsesinsnsns ST 4 Summary Of Clinical OBSEIVALONS .............coussussrrsesssssssssssssssssssssssssssssssssssse3ss8s 5 Summary ofOphthalmic OBSEIVAtIONS ................csussussussussssssssssssesssssssssssssassassass8as0s 6 Summary of Body Weight Data (8)..........ccuusmurersssssssssssssssssssssssssssssssssssssssss6s1s 7 Summary of Body Weight Change Data (8) ...........cuusuuemsssssssssssssssssssesssssssss6es3s 8 Summary ofFood CONSUMPLION Data ()....c......covcsessssueersssmssssssssssssssssssassssss6es3s 9 Summary of Test Material Consumption Data (Mg/Kg/day)........oevr-rrrrree: 67 * 004553 ConMeTg821942215 CONTENTS (Continued) `TABLE (Continued) Page 10 11 SummaryofClinical SummaryofClinical Hematology Hematology Data - Wek Dat-a Week 514.... ......vvnrcersrr61n39 12 13 Summary of Clinical SummaryofClinical Chemistry Chemistry Data- Week Data - Week 5........ovmreoseecee TT 14....ooermoemern 83 14 15 Summary of Summaryof COlrigniacnalWeUFiRgAhItYSDIa5taD-AWteae.k.5 ...e. .eem. vro. ssmomesenreere9819 16 17 Summary Incidence oOffMOarCgraonsCWOepiigchtOBDSaEtIaV-AWLOeNeSkv 14.e ...r ...s ..ori rsre nen11913 18. 19 Incidence Incidence oOff SMeIvCerrOiStCyOoPfIcSeOleBcSteEd IMIVCATOLSICOOpINcSO.B.SE.I.VL.IvONrSreerrvsrssseen 114 123 APPPrOEINOCDOI!XDEL VIRHONS s ess ses r} 19261 ------ PrOLOCON PrOKOCON AMENAMENE ATENAMENE NO. NO. 2L ..rve rorsr rssmo nsssi ises 146 148 PrO10CO] AMENMENt NO.3. 11 SRIPndEivNiDduIaXl ADRuIsALsmFautesmDAsAmc mmmmsmmr manmone smsssss sssssss msnnnn 119536 IInnddiivviidduuaall OClpinhicaall iOcBOSBCSIEVNAVHAOONNSS.........r.r.rcrcrvirrssssosisnseesnnosnonns 111559 APPENDIX3... 191 Individual Body Weight Data (8)... 192 APPENDIX 4... 203 Individual Food CONSUMPHON DAA ()rrr 204 Individual Test Material COnSUmption Data (Mg/kg/day) reves 215 AEFINDIRS, spss 38 IInnddiivviidduuaall CClliinniiccaall HCHEEMMAIOSITOYEDYADA a..t..a....r.rr.rissssrsssnsi2ss22650 Individual Clinical Urinalysis Data... 218 APInPdEivNiDduIaXl A6DB.tOcMincsPAsthsOIsOEYsDsaas.s..ssmsmssssssnsssssns239910 APCPelElNPDroIliXfeTraotironrTrisescuerCrolrlsecmtimonsamnmdsIsmmsunsoshissstoscshesmsicsamlsEVsUsatsioin.n.s..s..s.s..n..o.s. 5n344s98 BE -------------------------------------------- x 004554 CovanMceT6E3219-42125 ABSTRACT `The purposeofthis study was to assess the toxicity of the test material, NN-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) when administered in the diet to rats for at least 13 weeks. Four groupsofCrl:CD"(SD) IGS BR rats were exposed to N-MeFOSE in the dietforat least 13 weeks. concentrations w Eac ere h 0 group (contr contained ol group), 20 3, males and 30, and 10 20 0p females. pm. The dietary Food was provided ad libitum, except whenanimalswere fasted. Water was provided adlibitum. The animals were 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 of toxicity. Body weight data were collected on the first day of treatment and weekly thereafter. Food consumption data were collected weekly. Ophthalmic examinations were done before initiation oftreatment and during Week 14. Blood samples were collected for hematology and clinical chemistry tests during Weeks 5 and 14. Urine samples for urinalysis tests were collected during Week 14. During Weeks 5 and 14, blood was collected from five animals/sex/group for serum perfluorooctane sulfonic acid (PFOS) analyses. At the Week 5 sacrifice, samplesofthe 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, the remaining animals were anesthetized, weighed, exsanguinated, and necropsied. At necropsy, macroscopic observations were recorded, selected organs were weighed, and selected tissues were collected and preserved. Samples of liver were collectedfrom all 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 in the mid-dose group, and on the liver from each terminal sacrifice animal in the low-dose group. x 004555 CovanMcTe.6632391-42215 One female given 3ppm N-MeFOSE 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 testmaterialrelated. All other animals survived to the scheduled sacrifice. No clinical observations were noted that were considered to represent primary effects of the test material. Statistically significantly lowermeanbody weights were noted beginning at Week 4 and continuing through the end of study 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 significantlylowerin a sporadic manner for animals given 30 or 100 ppm. Overallbody 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 lowerformales given 100 ppm. For females given 100 ppm, statistically lower food consumption was apparent at Weeks 3, 4,6, 7, 11, and 12. Dietary administrationof 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 were considered to be of moderate severity. All of the test material-elated 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 weightsofmales fed 30 ppmand 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 iver-to-body weight percentage in females fed 30 ppm, and in al liver weight parameters in males and females fed 100 ppm. Testmaterial related macroscopic findings were noted at a low incidence in the liver and `glandular stomachofmales and 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 { 004556 - areas were sometimes seen in the gastric mucosa. Covance 6329-225 000 rem `Test material-related microscopic findings wereseenin the liverofanimals fed 30and 100 ppmofN-MeFOSE and in the glandular stomachofmalesand females in the 100 ppm group only. No test material-related changes were seen in animals fed 3 ppm of N-McFOSE. 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 hepatocellularpigment,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 decreasaetd this dose. In addition, a low incidenceofminimal hepatocellular vacuolation occurred in 30 ppm dose group males, and the liver ofa single: female fed 30 ppm had minimal coagulative necrosis. A possible test material-related change in the glandular stomachofanimals fed 100 ppm was a relatively low incidence of `mucosal erosions. The erosions were ofminimal to slight severity and involved a very small portion of the mucosal surface in all cases. Based on the anatomic and clinical pathology results at 30 and 100 ppm and body weight effects at 3, 30, and 100 ppm, the no-observable-adverse-effect level was determined to be less than 3 ppm when N-MeFOSE is administered at concentrationsof0, 3, 30and 100 ppm to Cri:CD*(SD) IGS BR rats for at least 13 weeks through dietary administration. EY 004557 _-- CovwanMcTre.663e2391-42215 PURPOSE `The purposeofthis study was to assess the toxicity of the test material, N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) when administered in the diet to rats for at least 13 weeks. REGULATORY COMPLIANCE All aspectsofthis study were in accordance with the Food and Drug Administration Good Laboratory Practice Regulations as set forthinTitle 21ofthe US CodeofFederal Regulations, Part 58, issued December 22, 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. To be supplied by Sponsor,isan off 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 material i on file with the Sponsor. Reserve (Archive) Sample A reserve sample (approximately 5 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 retuned after authorization from the Sponsor. Nn 004558 _-- CovManScMTeT6-36e2391-4m2.215 ANIMALS AND HUSBANDRY Test Animal Male and female Crl:CD'(SD) IGS BR rats were obtained from the Portage, Michigan, facility ofCharles River Laboratories, Inc., on August 17, 1998. The animals were 39 to 45 days old at initoifatrteaitmoennt. The males weighed from 184 to 236 8 and the females weighed from 140 to 17g9 at initiationoftreatment. 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, each animal was assigned a permanent number, and the microchip these numbers. was coded with that number. All data for an animal are recorded under Acclimation Ninety-two males and 92 females were received on August 17, 1998, and acclimated in Animal Room 348 for 15 days before initiation of treatment. In general, animals in this shipment appeared healthy. During acclimation, the animals were examined for abnormalities indicativeofhealth 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 humidity of30 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. w 004559 - O00 `CovwanMcreT631e294-2125 Certified rodent diet #5002 meal, (PMI Nutrition International) was provided (see Protocol Deviations for exceptions) ad libitum, 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. `Water was provided ad libitum. 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 in the 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 and 2. 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. Selectionofanimals 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 time of randomization, the weight variation of the animals for each sex used did not exceed +2 standard deviations of the mean weight. Group mean body weights were analyzed using Levene's test for homogeneity of variance at the 5.0% probability level and LS 004560 CounMcTe.66239124215 ~ found to be homogeneous. Animals were assigned to the study according to the following design: Group --NMuamleberofAnFiemmalasle 1 (Control) 20 20 2 (Low) 20 2 3 (Mid) 20 2 4 (High) 2 2 a Dose levels were expressed as ppm of MeFOSE. b Thecontrol animals received the basal diet only. pDipemtaMreyFLOevSeEl)s* 0 3 30 100 Dose Preparation Dietary concentrations were based on the test material as supplied. 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-ppmand 500-ppm concentrations and for homogeneity analyses at the 3, 30, and 100ppm concentrations. Diets were prepared once every 4 weeks for use in the in-life phase. Preparation of Diet for Group 1. The appropriate amountofdiet was weighedinto a labeled container and 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. Preparation of Diets for Groups 2 through 3. Each dose level was prepared independently. A specified amountofdiet was weighed into a labeled container and a specified amountofdiet for Group 4 was weighed for each diet. A trench was formed in the feed in the mixing bowl and the amountofdiet for Grou4p 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 labeled container. Approximately 5 mLofacetone was added to the container and mixed `manually. Increments of approximately $ mL acetone were added as necessary until the test material had dissolved. To prepare a premix, the diet was transferred into a labeled Hobart" mixing bowl. The test material and acetone were added to the mixing bowl, TM 004561 CovnIcMeT6-36230124015 `overlaid with a portionofdiet from the mixing bowl, and thoroughly mixed. A portion of diet from the mixing bowl was transferred to a second mixing bowl, mixed manually to recover residual test material, and returned to 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 feedingjars. Retention Samples During the in-life phase, samples (approximately 100 g) were taken from each 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 routeof exposure in humans is oral. `The dose preparations were administered ad libitur 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 daysofmixing. 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 " 004562 CovanMcTe 6329.225 `mixing and used as the baseline value. One sample was stored at room temperaturefor at least 19 days, then analyzed. A third sample was stored at room temperature for at least 32 days, then analyzed. The remaining sample was storedin afreezer set to maintain 10 to -30C for 8 weeks, then analyzed. In addition, two samples (approximately 10g0 each) were taken from the 500-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 each) were takenfromthe 3-ppm dose preparation mixed for Weeks 9 through 12. One sample was stored at room temperaturefor at least 19 days, then analyzed. 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-life phase, samples (approximately 100 g) fromalldose 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.) formortalityand moribundity (see Protocol Deviations for exceptions). Signsofpoor 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 day of treatment, and weekly thereafter. 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 5. ha 004563 CovanMcTe-6632391-24215 - Ophthalmology Ophthalmic examinations were done before initiationoftreatment and during Week 14. The pupils were dilated with 0.5% Mydriacyl", and the eyes were examinedby a `veterinarian with an indirect ophthalmoscope. Clinical Pathology Blood samples were collected from 10 animals/sex/group during Weeks 5 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 wasprovidedad libitum. Blood `was collected from a jugular vein. Potassium EDTA was used as the anticoagulant for hematology tests. No anticoagulant was usedfor the clinical chemistry samples. Animals were bled in random order. The following were evaluated: `Hematology red blood cell (erythrocyte) count `hemoglobin hematocrit `mean corpuscular volume `mean corpuscular hemoglobin `mean corpuscular hemoglobin concentration platelet count `white blood cell (leukocyte) count differential blood cell count `segmented neutrophil count lymphocyte count `monocyte count eosinophil count basophil count blood cell morphology Reticulocyte count smears were made and held for possible future examination. glucose urea nitrogen creatinine total protein albumin globulin total bilirubin cholesterol triglycerides Clinical Chemistry aspartate aminotransferase. alanine aminotransferase gamma glutamyltransferase calcium inorganic phosphorus. sodium `potassium chloride wx 004564 volume. specific gravity pH protein glucose ketones CovanIcMeT6-62391242.15 Urinalysis bilirubin blood urobilinogen `microscopic examinationofsediment appearance Serum Perfluorooctane Sulfonic Acid Level (PFOS) Determination During Weeks 5 and 14, five animals/sex/group were fasted ovemight, (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 ondryice and shipped to the Sponsor for analyses. The samples were analyzed for PROS. Results of analyses will be reported separately by the Sponsor. Necropsy A necropsy was done on the Group 2 female that was sacrificed at an unscheduled interval. During Week 5, five animals/sex/group were fasted overnight (se 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, theremaininganimals 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 examination of the external features of the carcass; all external body orifices; the abdominal, thoracic, and cranial cavities; organs; and tissues. TM 004565 -_-- CoMmeMsTT.e6en3o1mn41s Organ Weights After 13 weeksoftreatment, the following organs (when present) were weighed; paired organs were weighed together: adrenal (2) brain Kidney (2) liver ovary (2) spleen testis (2) thymus. thyroid (2) with parathyroid Organ-to-body weight percentages and organ-to-brain weight ratios were calculated. ACetltlhPertoelrimfienraaltisoancrTifiiscse,uereCporlelseecnttaitoinvaensdamIpmlmesunoofhtihsetloefcthleamteircalalloEbevoafltuhateiolinver 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. Tn addition, liver sections were stained with hematoxylin and eosin and examined `microscopically. Resultsofthe evaluation will be in Appendix 7. Palmitoyl-CoA Oxidase Tissue Collection and Analyses At the scheduled sacrifices, the right lateral lobeofthe 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 liquid nitrogen and 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 will be reported separately by the Sponsor. 19 004566 -_-- CovawnMcTer-663e2391-42215 Tissue Preservation `The following tissues (when present)fromthe animal that was sacrificed at an `unscheduled interval, or from each animal sacrificed at the terminal sacrifice were collected and preserved in 10% neutral-buffered formalin, unless otherwise specified: adrenal (2) aorta brain cecum cervix colon duodenum epididymis (2) esophagus eye (2) sufrefmaucrewith bone marrow (articular of the distal end) Harderian gland heart ileum with Peyer's patch (lymphoid aggregate) jejunum Kidney (2) lesions liver lung with mainstem bronchi lymph nodes (mesenteric and mandibular) `mammary glands (females only) ovary 2) pancreas pituitary prostate rectum salivary gland [mandibular (2)] sciatic nerve seminal vesicle (2) skeletal muscle (thigh) skin spinal cord (cervical, mid-thoracic, and lumbar) spleen sternum with bone marrow stomach testis (2) thymus thyroid (2) with parathyroid trachea urinary bladder uterus vagina Zymbal's gland Histopathology `Tissues (as appropriate) were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically from each animal in the 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, theliverand stomach from animals in the mid-dose group, and the liver romanimals in the low-dose group were also embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. . 20 004567 _-- CovawnIcMreT663e329124215 pMaarcarffoisnc,ospeicctiloenseido,nssftraionmeadnwiimtahlhseimnattohxeylloiwn-aannddemosiidn-,daonsde gerxoaumpisnewdermeicreomsbceopdidceadlliyn. sBaocrniefimcearafrtoerw 1s3mewaeresfksroofmttrheeatfmeemnutr, ostfaeiancedh waintihmaWlrwiegrhetprsetpana,readnadtrtehteaisncehdedfourlepdossible examination. Statistical Analyses hLeetveernoege'neteistty(oLfevveanreia,nc1e96a0t)p wsas0.d0o5,netrtaontsefsotrmfaortivoanrsiawnecreehuosmeodgetnoeisttayb.iliIzne tthheecvaasreiaonfce, `Comparison tests took variance heterogeneity into consideration. `weOingeh-twsa,ybaondalyyswiesigohftvacrhiaanngcees,(fAoNodOVcoAns(uWmipnteiro,n,19c7o1nat)i]nwuoauss ucsleindictaol apnaatlhyozleogbyodvyalues; othregaAnNwOeiVghAtsw,aosrgsaignn-itfioc-abnot,dyDuwneniegthtt'spetr-cteesntta(gDeusn,neatntd, o1r9g6a4n)-twoa-sbruasiendwefoirghctonrtartoilosv.erIsfus treated group comparisons. 5G.r0o%u,ptcwoom-tpaairliesdopnrsob(aGbrioliutpysl2evtelh.roOunglhy4dvaetrascuosllGercoteudpo1n) owrerafeteervatlhueaftiersdtadtatyheof treatment was analyzed statistically. RECORD RETENTION Athlilsrsatwuddyatwai,lldboecuamrecnhtiavteidoinn,trheecsotrodsr,agperoftacoicloilti,esaonfdCsopveacnicmee-nsMagdeinesroantefdoaars paerreisoudlot foaft least I year. Atleast 1 year afer determine the final disposition of the the submission of materials. All the raw final data report, stored the on Sponsor magnetic will media, the protocol and protocol amendments, study correspondence, final report will be retained by Covance-Madison. and an original copy of the PCNA evaluation data, paraffin Associates International. blocks, and tissue slides will be retained by Pathology LsiavmeprleasndwisllerbuemrestaamipnleedsbsyentthetoSptohnesSopr.onsorand analysis data generated from these 2 004568 -_-- CovManITcMeTe633u1294a2.215 RESULTS Dose Analyses Results through of 3. the homogeneity, stability, and dose preparation analyses are in Tables 1 7M7e.a5n-8v4a.l2u%eos fofthtehethheoomroegtiecnaelictoynacneanltyrsaetsiornasnfgoerd tfhre odmie7t4s.c0o-n7t8a.i0n%i,ng834,.300-,96a.n7d%,10a0ndppm N-MFOSE, respectively. These results indicate that the mixing procedure produced a homogeneous distribution of the test material in the dose preparations; although variability generally appeared slightly greater at the 3 and 30-ppm dietary concentrations. 8Rewseuletkssofunsdtaebrilfirtoyzaennacloynsdeisotfiosnasmipnldeicsatsetdortehdatfotrhe19meaannd c3o2ncdeanytsraattiroonsomratnegmepderfartoumre and f5o4r.43-26d2a.y5s% aotfrtohoe mintiteimalpecroantcuenrteriantdiiocnasteod f1thaptptmhefomredainetcmoincxeendtrpartetieosnt.waSsa9mp8l.e4s%ostfotrheed initial concentrationsof 500 ppm. and 32 days at room temperature Resultsof stability analysesofsamples indicated that the mean concentrations stored ranged for 19 from s9t3o.r2e-d9f4o.r0%1o9fdtahyes aitnitrioalocmontceemnpterraattiuornesiondfi1captpedmtfhoartdtiheet mmeixaendcaotncWeenterkat4i.oSnawmapsle8s9.0% ofthe initial concentrations of3 ppm. `The mean concentrations of the dose preparation analysesforall levels ranged from 63.7 10 110.0% ofthe theoretical concentrations. Inherent variability and lackofsufficient 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 outsideofthe 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. CClliinniiccaalloObbsesrevravtaitoinsonasreasnudmSmuarrviizveadlin Table 4; individual data are in Appendix 2. Individual animal fate data are also in Appendix 2. Othneetafile.maTlheegrievewner3epnpommaNc-rMosecFoOpiScEowramsicsraocrsicfoipciedc ocnhaDnagyes59todiuneditcaotseetvhearteiltsacmeorraitibounnodf condition was test material-related. All other animals survived to the scheduled sacrifice. 2 004569 _-- CovManMcTeT.663e2391-M42215 No clinical observations were noted that were considered effectsofthe test material. OOpphhtthhaallmmicoloobgseyrvations are summarized in Table S; individualdataare in Appendix 2. `The Ophthalmology Report contains a discussionof the data. Animals selected for thestudyhad no lesions at the prestudy examination. No test `material-related ophthalmic observations were noted at the Week 14 examination. Body Weights Body body weight weight and data body weight change are in Appendix 3. data are summarized in Tables 6and 7; individual Testmaterialrelated lower body weights were noted for males given 3 and 30ppmand for males and females given 100 ppm. Statistically significantly lower mean body weights `were noted beginning at Weck 4 and continuing through the endof study for males and fbeemgailensnigianvtgenWe1e00kp7pma.nd Sctoanttiistniucianlglytshirgoniufgihcatnhtelyelnodwoefrsmteuadyn fboordmyalweesigghitvsenwe3r0epnpomt.ed Body weight changes were statistically significantly lower in a sporadic manner for males given 30 ppm and for animals of both sexes given 100 ppm. Overall body weight changes (Weeks 1-14) were statistically significantly lowerforall groups given N-MeFOSE. Food Food cCoonnssuummptpitoinondata are summarized in Table 8; individual data are in Appendix 4. 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 cfoonotdroclosnsfourmpmtoisotnotfhtahnetshtousdeyo. fFceomnatlreolssgaitveWnee1k0s0 p3,p4m, h6,ad7,st1a1t,isatincdall1y2.sigTnhieficoavnetrlaylllower `mean food consumption for females fed the 100-ppm concentration was significantly lower by 17.5% when compared with that of the controls. 23 004570 CovanMcTe-6632391-42215 `Test Material Consumption `Test material consumption data are summarized in Table 9; individual data are in Appendix 4. Animals were fed dicts containing 3, 30, or 100 ppm N-MeFOSE, T-6314. The mean amountsoftest material consumed by 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 to 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 fed diets 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 summarizedin Tables 10 through 14; individual data are in Appendix 5. The Pathology Report contains a discussionofthe data. Dietary administration ofN-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 exceptionof 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. SReesrulutmsoPfesreflruuomroPoFctOaSneleSvuellfdoentiecrmAicniadtiLoenvweilll(bPeFrOeSp)orDteetdesrempianraatteiloynby the Sponsor. x 004571 - @ @@O@O@O@O@O@0@0@0@0@0@0@ CovamnMcmeT6e3s219-42125 Cell Proliferation Tissue Collection and Immunohistochemical Evaluation Resultsofcell proliferation and immunohistochemical evaluation provided by Pathology Associates International will be in Appendix 7. Resultsofthe cell proliferation evaluation will be provided by Pathology Associates International. 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 ofseverityofmicroscopic observationsis summarizedin Table 19. Individual data are in Appendix 6. The Pathology Report contada isciusnsis on 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 weights ofmales fed 30 ppm and in both males and females fed 100 ppmof 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 females fed 30 ppm, and in al liver weight parameters in males and females. fed 100 ppm. `Test material-related macroscopic findings were noted at a low incidence in the liver and glandular stomach of males and 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. Test material-related microscopic findings were seen in the liver of animals fed 30 and 100 ppmofN-MeFOSE and 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 bd 004572 CovanIcMeT6-362391-42.215 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 liverofasingle female fed 30 ppm had minimal coagulative necrosis. A possible test material-related change in the glandular stomachofanimals fed 100 ppm was a relatively low incidence of mucosal erosions. The erosions wereofminimal to slight severity and involved avery `small portionof the mucosal surfaceinall cases. CONCLUSIONS Based on the anatomic andclinicalpathology results at 30 and 100 ppmand body weight effects at 3, 30, and 100 ppm, the no-observable-adverse-effect level was determined to be ess than 3 ppm when N-MeFOSE is administered at concentrationsof0, 3, 30 and 100 ppm to Crl:CD*(SD) IGS BR rats for at least 13 weeks through dietary administration. 2% 004573 SIGNATURES CovanMcTe -636291-42215 - Nii van Bruce-Konuah Date Study Coordinator Covance Laboratories Inc. Peter J. Thomford, PhD Date Study Director Covance Laboratories Inc. 2 004574 CovanMcTe-66293.124215 REFERENCES Dunnett, C. W., "New Tables for Multiple Comparisons with a Control," Biometrics, 20:482-491 (1964). Levene, H., "Robust Tests for Equality of Variances," ContritobPruobtabiiliotynansd Statistics, (eds.) I. Olkin et al, Ch. 25, pp. 278-292, Stanford University Press: Stanford, California (1960). `Winer, B. J., "Design and AnalysisofSingle-Factor Experiments," StatisticalPrinciplesin `Experimental Design, Second Ed., Ch. 3, pp. 149-260, McGraw-Hill: New York, New York (1971). 28 004575 -_-- CovManIcMTeT6-36e321942M.215 OPHTHALMOLOGY REPORT Ophthalmic examinations were done before initiation of treatment and during Week 14. `Amnaitmerailasl-sreelleactetdedofpohrtthhaelsmtiucdoybhseardvantoiolnessiwoenrseatnottheedpratestthuedWyeexeakmi1n4ateixoanm.inaNtoiotne.st Donna J. Clemons, DVM, MS Diplomate, ACLAM Date 004576 2 PATHOLOGY REPORT MTL Covance 6329-225 - SUMMARY `The purposeofthis study was to assess the toxicity of the test material, N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE), when administered in the diet to rats for at least 13 weeks. The test material was administered at dose levelsof3, 30, and 100 ppm. Dietary administrationof N-MeFOSE was associated with lower hematocrit for males and femalesfed 100 ppm;higherureanitrogenformalesandfemales fed 100ppm;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 cholesteranodl triglycerides for the males fed 100 ppm; those. effects were considered to be of moderate severity. Allof 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 30 ppm and in both males and 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, in mean absolute liver weight and mean-liver-to-body weight percentage in females fed 30 ppm, and in all liver weight parameters inmalesand females fed 100 ppm. "Test material-related macroscopic findings were noted at a low incidence in the liver and `glandular stomach of males and 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. "Test material-related microscopic findings were seen in the liver ofanimals fed 30 and 100 ppm of N-MeFOSE and 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 ppmof N-MeFOSE. In the liver, one or more test material-related findings affected all animals H 004577 CovanIcMeT6-362391-24215 - 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 hadminimal coagulative necrosis. A possible test material-related change in the glandular stomachofanimals fed 100 ppm wasa relatively low incidence of `mucosal erosions. The erosions wereofminimal to slightseverity and involved a very `small portion of the mucosal surface in all cases. METHODS Four groupsofCrl:CD (SD) IGS BR rats (20 animals/sex/group) were fed diets containing the test material at a dose level of0 (control group; fed basal diet only), 3, 30, or 300 ppm. During Week 5, five animals/sex/group were sacrificed to obtain liver samples for determination ofhepatic palmitoyl CoA oxidase activity (by Covance) and analysis for PFOS and metabolites (by 3M). One female fed 3 ppm was sacrificed and necropsied during Week 9 because of poor health (unscheduled sacrifice). The remaining. `animals were sacrificed and necropsied during Week 14 (terminal sacrifice). Blood was collected for hematologyand clinical chemistry tests during Weeks S 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 evaluationof proliferation 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). In addition, livers, stomachs, and macroscopic lesions were examined from all terminal sacrifice animals fed 30 ppm, and macroscopic lesions and livers only were examined from animals fed 3 ppm. a 004578 _-- CovanMMcTeT6633e2194-u2.215 Statistically significant differences cited in the Results and Discussion section are based on comparisons between thecontroland treated groups. RESULTS AND DISCUSSION Mortality One female fed 3 ppm (Animal No. C96016) was sacrificed during Week 9 (Day 59) becauseofpoor health. Al other animals survived to the respective scheduled sacrifice. Clinical Pathology `Weeks and 14. There were several statistically significant or otherwise notable differences for clinical pathology results between control and treated animals. Differences cfoedns1i0d0erpepdmt;ohbiegheefrfeuctrseoafntihterotgeesnt fmoartemrailaelswaenrdefleomwaelreshefmeadto1c0r0iptpfmor;mhailgehseraanldbfuemmianlefosr `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;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 cfionndsiindgesrweedrteo mboeorefmpordoemrinaetnetsaetveWrietey.k A14ltohfanthaettWesetemkat5e.rial-related clinical pathology 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 dehydrationforanimals 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 hcheoplaetsitcerpoallmaintdoytlriCgolyAceroixdiedsasweerweasthiendmiocsattipveroomfipneernotxifisnodmiangls,praolnidfesruagtigoens.teTdhleipeifdfects on `metabolism by the liver was altered. Ocforpuunsccerutlaairnvroellautmieonfsohripfetmoaaldemsinfiedst1ra0t0iopnpmo.f tThehetedstifmfaetreernicaelfwoarsmestaantisctoircpalulsycluloawrer mean volume became smaller over time (i... from Week to Week 14). Other statistically 004579 -_-- CovanwMcTeM.6T63239e1-24u215 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. Testmaterial related changes were seen in the terminal body weights and in absolute and relative liver weights in animals fed 30 and 100 ppm N-MeFOSE. Mean terminal body weights were statistically significantly decreased in males and females that were fed 100ppmofN-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 significant and test material-related increases were seen in the mean liver-to-body weight percentage and the mean liver-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 all liver weight parameters in males and females fed 100 ppm. The liver weight increases correlated macroscopically with large livers in 3 of 1 males in the 100 ppm dose group and microscopically with the presenceof hepatocellular hypertrophy in all males and females in the high-dose group. 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 range of normal biologic variation or were attributed to the lower body weights in treated versus control animals. Macroscopic Findings. Test material-related macroscopic findings were noted at a low incidence in the liver and glandular stomach of males and females fed 100 ppm 004580 : _-- CovanMcTTe.683e2391-42n215 N-MFOSE. Livers that were large were seen in 3 of 15 males and in 0 females fed 110000 ppppmm., aNnod dliifvefruscehlyandgaerskolcicveurrsroecdciunrraendimianl2s foefd1t5hemacloenstraonldma1teorfia1l5orfiemnalaensimfaelds fed TwiotwherhedpoasteoscoelfluNl-arMehyFpOeSrEtr.ophTyheinltahrgeeafafnedctdeidffaunsiemlayldsa.rkInlitvheersgcloarnrdeullaatresdtmoimcarcohssc,opdiacraklly afoncdioartaarneiansciodcecnucreroefd 0atoafn1i5,nc0idoefnc1e4,o00ofoff151,5,an1do3fo1f5,105 oinff1e5m,aalneds i2notfhe15coinntrmoall,es, 3 ppm. 30 ppm. and 100 ppm groups respectively. The dark fociorareasgenerally correlated microscopicallywith small erosions in the glandular mucosa. Dfoicfifuosrearreedadseinnitnhgeatnhdymmoutstlwienrgeofrtehlaetivtehlyymcuosmamnodnmafnidnidibnuglsaranldymfrpehquneondtelsyacsorwreelllataesdred microscopically with hemorrhage in the affected organs. Because these findings did not ``moaclceusr,iwnearedosseo-mreetliamteeds mafafnencteerd,antdhebfeicnaduisnegsrawtesriencmounlstiidpleereddostoe bgeroaugposn,alincchlaudnignegsctohnattrol `wweerree ncoontsaisdseorceidatteodbweitshpotnetstanmeatoeursiaalndadimnicniidsetnrtaatliaonn.d nAollt rreelmaatiendintgo mteasctrmoastceorpiailc findings administration. Microscopic Findings. Testmaterialrelated microscopic findings were seen in the liver ofanimals fed 30 and 100 ppm of N-MeFOaSnEd in the glandular stomachofmalesand females in the 100 ppm group only. There were no test material-related findings in the liversofanimals fed 3 ppm. `Test material-related findings in the liver included hepatocellular hypertrophy, coagulative nseecvreorsiitsyo,fhtehpeatsoeceflilnudlianrgspitgemnednetd,taonbdehgerpeaattoecrelilnul1a0r0vpacpumolaantiimoanl.s.ThTeheinicnicdiednecneceanodf pertinent liver findings is presentedin Text Table 1. 004581 EN -_ CovaMnIcMeTT63e3219u-4221r5 Text Table 1 Incidence of Selected Microscopic Findings in the Liver Group Number/Sex. Number Examined IM 2M 3M 4M IS 15 15 15 IF 2F 3F 4F 1s 14 15 15 Hypertrophy, hepatocellular, diffise Hypertrophy, hepatocellular, 0 0 0 0 Centrilobular Necrosis, coagulative 0 0 14 15 0003 Pigment, hepatocellular Vacuolation, hepatocellular, 0002 centrilobular to midzonal Vacuolation, hepatocellular, diffe 0 0 0 0 2 2 7 2 _-- 0001 0 0 7 14 00 1 0 oo 0 0 7 9g 9 o 2 go go go o 00 aArseansotoefdthien ltihveerabinovaenitmaablles,fheedpa3t0ocaenldlu1l0ar0 hpyppme,rtbruotpthhyegleinveerrawllaysamfoferceteddifcfeunsterliylobular whiytpheratbruonpdhainetd icnytoonpelfaesmmatlheatfeodft1e0n0hpapdma.grTahneulhayrpteortgrloopbhuilcarheepoasitnoocpyhtielsicwearppeeaernalnacreg.ed Tn hseopmaetoacnyitmeaslsalisnotchoenthiagihn-eddomsiengirmaolupam(otuwnotmsaloefas abnrdoswenvegrnafneumlaalresp)i,gmtehentcyattotphleapsemriopfhery opfartthiecuclealrll.y Iinn maaddlietsiogni,veinve1r0ce0llpspmf,rewqiutehnttlhyewvearceuovlaactuioolnagteender(aslcelyTaefxftecTtaibnglehe1p),atocytes itnwcoenmtarlielsobiunlacracahnodfmtihdezo3n0alanadrea1s0.0 pDipfmfudsoesheepgartooucpesl,lubluatr ivnacnuooloatthieornanwiamsalasl.soLsaesetlny,in `1m5infiemmaalletso sfleidgh3t0cpoapgmuwlaatsivelinkeelcyrorseilsatseeden0 idni3etoafry1a5dmmianliesstrfaetdio1n00ofptphme taenstd m1aotefrial since no control or 3 ppm dose group animals were similarly affected. Amucpoosssailbleerotseisotnmsa.teTrihael-erreolsaitoendscwhearnegeofinmtihneimgalalntdoullairghsttosmevaecrhitwyaasndthienpvroelsveendcae voefry sinmcaildlepnocretioon0ofoffth1e5,m0ucoofs1a5l,saunrdfa2ceofin1a5llicnamsaesl.es,Thaenderaotsainonisncwiedreencseeeonf0wiotfh 1a5n, 0 of 15, aprneds3enocfeo1fS eirnofseimoanlsesmiicnrotshceocpoinctarlolly, g3e0neprpaml,lyacnodrr1e0la0tepdpwmigthrotuhpesdraerskpefcotciiveolry.arcTahseseen he 004582 NTE `Covance 6329-225 - in the gastric mucosa at necropsy. One male fed 3 ppmalso hadaslight gastric erosion that was collected as a gross lesion at necropsy. Since no erosions were seen in anyof the animals' stomachs examinedfromanimals fed 30 ppm, the lesionin the 3 ppm male was considered unlikely to be test material-related. All remaining microscopic findings were considered to be spontaneous and incidental changes and typical of ratsofthis age and strain. Robert L. Hall, DVM, PhD Date Diplomate, ACVP (Clinical Pathology) Johnnie J. Eighmy, DVM, MS Date Diplomate, ACVP Diplomate, ABT a 004583 -_ CovwanMcTTe 66e2391w.422r15 COMMENTS ON THE DATA dVaatraioiunsthmisodsetludsyo.fcaBleccualuasteordsi,ffceormenptutmeordse,lsanoducnodmopfuftoerr tprruongcraatmesnuwmebreerussdeidffteoreanntally,yze svlailguhetslyinrsoommethotasbeleisn o(ct.hge.r, tmaebalness,,rstoamndianrddivdiedvuiaaltliyoncsa,lcoulaitneddivdiadtuaa,lovralfureosm)msataytidsitficfaelr analysis data. differences Neither the integrity nor th interpretationofthe data was affectedbythese r"eTfhleecntusmtbheernoufmabneirmoaflsanliismtaeldsianssthiegnheedadtiongeoacfhthgerosuupmamtatrhye ttaabrltesofftohreclisntiucdayl.observations `cTohnedistuimonmawrays toabbsleerfvoerdclwiintihcaolutobrseegravradtitoonsthiensdpieccaitfeicthneatnuurmeb,esrevoerfiatnyi,mraelvserfsoirbwihlyi,ch a numberofincidences/animal, or the length of time the condition persisted. Only observations tables. other than normal are indicated on the summary clinical observations cEaocmhmeannitm"alAnwiimtahlohbassernvoatsiiognnsifriceacnotrdfienddiansg"s"Noirnmdiacla"tetdhoronutghheouitndtihveidsutauldcylihnaiscalthe observations tables. iTnhdeicsapteecdifwiictdhetaai"lCs"fcorancobmemfeonutnsd aint tthhe iennddiovfideuaalchclignricoaulpofbosrercvaacthiosnesx.tables that are The start dayofinitiationoftreatment is "Day ofa study week (e.5.,a body weight 1, Week recorded 1." on Body Day 1 weight data are is considered a entered Wek I at the body wweeiigghhtt,cahabnogdey dwaetiagharterceaclocrudleatdeodnrDoamy t8heisficrostnsdiadyeroefdtaheWseteudky2wbeoedky(owetihgehtf)i.rst Bdoadyyof atrhee ifnodlilcoawtiendgisnttuhdeytwaebleeks (wei.tgh.tWheeedakyIbevianlguetshearfeircsatldcauylaotfetdhfrofmolDloawyin1gtwhereokug(hc.7g).,and cWoeneskump1 tviaoluneasraercealincduilcaatteeddraosm"Dtahy f8i.r"s)t.daWyeeokfltyhefsotouddcyowneseukmpttoiothneafnidrsttedsatymaotfetrhieal - following study week (e.g., Week | values are calculated from Day 1 through 7). - 004584 -_-- CouMnMcTeT6633e2194u2.215 ~ COMMENTS ON THE DATA (Continued) `The values for summary and individual test material consumption are reported with more significant figures than is appropriate for the data due to limitationsofthe data collection and reporting software. Food consumption values are reported in whole numbers; however, PTS carries food `consumption values to one place to the rightofthe decimal for food efficiency and test `material consumption calculations. The differences in values generated do not influence the interpretationofthe calculation. `The comment "SPILLED" on individual food consumption data tables indicates that food consumption was not recorded due to spillage during the interval. `The comment "NOT TAKEN" on individual food consumption data tables indicates the animal died before the end ofthe food consumption interval. Differences in the population size (N) on the summary tables for clinical and anatomic pathology are explained on the individual data tables or the codes sheets. 38 004585 - COMMENTS ON THE DATA (Continued) Teer Covance 6329-225 - "The calculation for individual test material consumption is: `Test material consumption = Emm BarDoesbcy Weg FestCtsosomyiBn37VgCF a i where: Daily Body Weight Gain = lLLDBtWWeeiigh tm DFoo eiytWSeygWamor and: Day Factor = FoodConsumpion StDay Dayo istBody Weight vat F020Coosplonler. and: Food ConsumFpotnitoCnonInmtpetrvEoa=ndlDyFoodCompSounDy ~35- 00455 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 CounMceT.63821924215 Note: The following lstsofcodes, abbreviations, and units are used by Covance. Some, but notnecessarilyall, ofthis information may be needed for this report. "0 004587 WK N Mean; MEAN SD; $.D.; STAND DEV; hd STANDARD DEV; sd =NA P c UNSCHED DISPATCH TBW # co CovanMcTe -636219-42215 General Codes and Abbreviations Week. Numberofmeasurements in a group. Arithmetic mean. Standard deviation. Group mean is significantly different from the meanofthe control group (Group 1) atp< 005. No value; not applicable; not present. 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 necropsy. Observations are duplicates of the last in-life observations. `Terminal body weight. Number. Clinical observation. Animal Death Codes: 1 T M Interim sacrifice 1. Terminal sacrifice. Sacrificed at an unscheduled interval. - 004588 Ns QS/QNS NR FS sc SH H SL L st 1 NF u DT/DOT DB bo] TE RE EE SE PC PD Pl PL PA co HB PLASMO NO AGG FR uD NO COAG CovanIcMeT6-362391-42.215 Codes for Clinical Pathology GENERAL CODES No sample Quantity not sufficient No repeat (sample volume not sufficient for repeat analysis) Fibrin strands Sample clotted, Slightly hemolyzed Hemolyzed Slightly lipemic Lipemic Slightly icteric Ieteric Animal not fasted Unscheduled/moribund bleed Animal died on test Died during bleeding `Technicianjudgment to repeat test Technical error (instrument or technician error that results in unacceptable data, e.g., unacceptable instrument output, sample spilled, entry of invalid data) Recording error (recorded incorrect data, .g., wrong number, spelling error, incorrect date) Entry error (incorrect keyboard entry) Sampling error Platelets clumped Platelets decreased Platelets increased Platelets large: Platelets appear adequate Color interferes with test Heinz bodies observed Plasmodium No aggregation Fractious Unable to determine No coagulation <2 004589 CovanIcMeT6-3629124215 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 times (PTT)greaterthan 110 seconds Bleed times (BLETIME)greaterthan 30 minutes CODES FOR BLOODCELLMORPHOLOGY `The following scale was used to measure the degreeofanisocytosis (ANISO), poikilocytosis (POIK), polychromasia (POLY), hypochromasia (HYPO), or basophilic stippling (BASTIP) or the presence ofHowell-Jolly bodies (HJBODY), toxic neutrophils. (TOXNEUT), or atypical lymphocytes (ATYPLYM): Scale Degree - Normal for the species 1 Slight 2 Moderate 3 Marked 4 Not applicable Presence Not present Rare Few Moderate Many A Pale B Straw C Yellow D Dark yellow URINE APPEARANCE Color E Amber F Bown G Red H Green 1 Black P Blue/green Q Blue R Orange Clarity J Clear K Hazy L Cloudy Miscellaneous M Debris O Feces < 0045: 90 --- CounMcMeTT8631s2492.215 Codes for Clinical Pathology (Continued) URINE CHEMISTRY MULTISTIX" STRIP __ UrineGlucoss _UrneKetone____UrineBlood ~ Negative + 100mg/dL = Negative + Smgldl Negative + Small ++ 250mg/dL +++ 500mg/dL ++ 1S mg/dL ++ 40 mg/dL ++ Moderate ++ Large +++ 1,000mg/dL Hert 22000mgldll +++ 80mg/dL +++ 160mgd Urine Urobilinogen -- + 02 mg/dL mgd ++ 2mgldL +++ 4mg/dl + 8mg/dL (1 mg = approximately I Ehrlich unit) Urine Bilirubin = Negative + Small ++ Moderate +++ Large URINE SEDIMENT Celis, Crystals, Casts, and Comments A Amorphousurates B_ Amorphous phosphates Q Sperm R Fecalcontamination C Uricacid D Triple phosphates S Pinwovoafroumnd T Pinworm larvae found E Calciumoxalate F Calciumcarbonate U Paraovsaifotuned G Granularcasts H_Hyaline casts 0 Not present I Cellular casts J Waxycasts, 115 per field 2 6-10perfield K Unknown crystal P_ Mucousthreads 3 11-20perfield 34 X>p20eprerffieeldd Bacteria 0 Not present 1 Few 2 Moderate 3 Many i 004591 CovanMceT63249-1225 Abbreviations and Units for Clinical Hematology "Test Red blood cell count Hemoglobin Hematocrit Mean corpuscular volume MMeeaann ccoorrppuussccuullaarr hheemmoogglloobbiinn concentration PMleataenleptlcatoeulnett volume Reticulocyte count AHbesionlzubtoedryetciocuunltocyte count Erythrocyte sedimentation rate. Prothrombin time Activated partial thromboplastin time `AcTthirvoamtbeidn ctoiamgeulation time Fibrinogen Fibrin/fibrinogen degradation products PClaotlelleatgaegngregation Adenosine diphosphate Alpha 2-antiplasmin MeBlteheedmionggltoimbein Plasma hemoglobin Myeloid/erythroid ratio Estimated myeloid/erythroid ratio `White blood cell count Differential blood cell count Nucleated red blood cell count Corrected white blood cell count Segmented neutrophil count Band neutrophil count Lymphocyte count Monocyte count Eosinophil count Basophil count Anisocytosis Polychromasia Abbreviation (Units) RBC (E6/UL or X10%uL) HGB (G/DL) HCT (%) MCV (FL) MMCCHHC(P(G%)) PLT (E3/UL or X10%4L) MPV (FL) RETIC (%) HREETIINCZ ((E%3)/UL or X10%uL) ESR (MM/HR) PT (SEC) PTT (SEC) ATTCT(S(ESCE)C) FBR (MG/DL) FDP (UG/ML) PAGG/COL (%) PAGG/ADP (%) ANTIPLAS (%) MBELETHTIGMBE((%S)EC) PLA HGB (MG/DL) M/E RATIO [EST M/E RATIO WBC (E3/ULor X10%4L) NRBC(/100 WBC) (COR WBC (E3/UL or X10%/uL) N-SEG (E3/UL or X10%uL) and % N-BAND (E3/UL or X10%uL) and % LYMPH (E3/UL or X10%uL) and % MONO (E3/UL or X10%/uL) and % EOSIN (E3/UL or X10%/4L) and % BASO (E3/UL or X10"4L) and % ANISO (-,1,2.3) POLY (1,23) & 004592 MTG Covance 6329-225 - Abbreviations and Units for Clinical Hematology (Continued) Test Poikilocytosis Hypochromasia Howell-Jolly bodies Basophilic stippling "Toxic neutrophils Atypical lymphocytes. `Aqueous white blood cell `Aqueous white blood cell count count (right eye) (left eye) Abbreviation (Units) POIK (-1,2.3) HYPO (-,1,2.3) HIJBODY (-,1,2,3,4) BASTIP (-,1,2,3) `TOXNEUT (-,1,2,3,4) ATYPLYM (-,123,4) REYE (WBC/UL) LEYE (WBC/UL) oe 004593 CovanMcTe-6633219-42.215 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 glutamy transferase Sorbitol dehydrogenase Lactate dehydrogenase Creatine kinase: Amylase Lipase Palmitoyl CoA oxidase: Calcium Tonized calcium Inorganic phosphorus Sodium Potassium Chloride Magnesium Zinc Strontium Tron Abbreviation (Units) GLU (MG/DL) UN (MG/DL) UREA (MG/DL) CTRPERAOT((G/MDGL/)DL) ALB (GIDL) GLOB (G/DL) A/GRATIO T BILI (MG/DL) D BILI (MG/DL) 1BILI (MG/DL) CHOL (MG/DL) TRIG (MG/DL) UN/CREAT (RATIO) TLIPIDS (MG/DL) P LIPIDS (MG/DL) HDL (MG/DL) LDL (MG/DL) UA (MG/DL) ASTISGOT (UL) ALT/SGPT (IU/L) ALK PHOS (IU/L) GGT (IU/L) SDH (IU/L) LDH (ULL) CK (IU/L) AMYLASE (IU/L) LIPASE (IU/L) PCOAO (IU/G) CIOAN(CMAG/(DMLG)/DL) 1PHOS (MG/DL) NA (MMOL/L) K (MMOL/L) CL (MMOL/L) MG (MEQ/L or MG/DL) ZN (MGIL or PPM) SR (MG/L or PPM) FE (UG/DL) Et 004594 CovanIcMeT6633219-42.215 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 `Gamma globulin High-density lipoprotein 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) FCEHE%PSA(MTU(M%)L) CHER (MUML) CHEB (MUML) CAUD PUT (UMOL/G) HFICPOPROTCEAXM((UUMMOOLL//GG)) 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) SMO (MOSM/KG) EEAA-L1B(G(/GD/LD)L) EA2 (GDL) E BETA (G/DL) E GAMMA (G/DL) E-HDL (%) EE--LVDLLDL(%()%) INSULIN (UUML) ACTH (PG/ML) CORTISOL (UG/ML) GLUCAGON (PG/ML) T3 (NG/DL) T4 (UG/DL) CK-BB (UL) CK-MB (ULL) CK-MM (ULL) 5 004595 Abbreviations and Units for Clinical Urinalysis CovanScMeT6-362391-42.215 TUersitne volume. 8 hour urine volume. Specific gravity Urine osmolality Quauntiidtaptriovteeiunrinary/cerebrospinal Urine protein excretion Urine chemistry Multistix" strip Urine pH Urine protein Urine glucose Urine ketones Urine bilirubin UUrriinnee burlooboidlinogen Urine reducing substances Microscopic examination of urine sediment Red blood cells per high-power field `White blood cells per high-power field Epithelial cells per high-power field Bacteria per high-power field Casts per low-power field Crystals per low-power field Urine appearance Comments AUbbVrOeLvi(atMiLo)n (Units) 8HR VOL (ML) SP GR U OSMO (MOSM/KG) QUAN PRO (MG/DL) PRO EXC (MG) UPH U PRO (MG/DL) UGLY UKET U BILI UURBOLBIOLOID URE SUB RBC (PER HPF) WBC (PER HPF) EPITH (PER HPF) BACT (PER HPF) CASTS (PER LPF) CRYSTALS (PER LPFI or PER LPF2) URINE APP1 or URINE APP2 `COMMENTS Miscellaneous Codes and Abbreviations for Clinical Pathology Fecal occult blood Fecal parasite detection Hemolytic potential Osmolality Not applicable Not applicable Not applicable `OSMO (MOSM/KG) hy 004596 Code T1 M 000000 Codes for Anatomic Pathology Definition ANIMAL DEATH CODES ITnetremriinmalsascarcirfiifciecse 1 Sacrificed at an unscheduled interval CovwanMcreT6-e312w94221r5 ENXOTTAKEN MIUSNSSUIINTGABLE EXAUCTLOULDYTEIC MACROSCOPIC CODES OInrdgiacnatweseitghhatt onrogtatnakweeni;ghextpilsaenaxtciloundegdifvreonmicnanleccurloaptsiyonnsotes OOrrggaann mteicshsniincgalolry luonsstuitable for weighing `OWregiagnhtawutaoslytzaekdena,nbdutcowualsd enxoctlbuedewdeifgrhoemdal calculations MICROSCOPIC CODES BC-odes Prefacing NeoplParsimtaircyF,ibnedniinggnsneoplasm MN- MPertiamsatrayt,imcanleiognpalnatsmneoplasm r x LOotchaelrlyneionpvalsaisvme neoplasm ~o 004. 597 CovanMcTe-6633219-42.215 Codes for Anatomic Pathology (Continued) Code Definition Distribution of Findings Focal Diffuse Mukifocal Grades for Severity or Amount 1 Minimal - the least amount of change that can be observed with the light microscope 2 Slight-lessthanaverage amountofchange, butreadilydiscernible as abnormal 3 Moderate- the average amountof change that is expected for a lesion 4 Moderately severe (marked) - a marked amountof change with possible lossoffunction of the affected cells or organs 5 Severe -a great amount of change with probable loss of function of the affected cel or organs and frequently involves large areasofthe organ Other Microscopic Codes TL Total P Finding present - Finding not present MN Mean < 004s98 CovanIcMeT6-36239-124215 Codes for Anatomic Pathology (Continued) TISSUE ABBREVIATIONS Abbreviation IN GL STOMACH, GL STOMACH, NONGL SALIV GL, MANDIB LN, ANT MES/PANC AUDITORY SEB GL LACRIMAL GLAND, EX HEMATO NEOPLASIA LACRIMAL GL, INT CAVITY, ABDOM SALIV GL,PAROTID LN, TRACHEOBRON Definition Lymph node Gland Glandular stomach Nonglandular Mandibular salivary gland AAnutdeirtioorrymseesbeancteeroiucs/pgalnacnrdeatic lymph node `Exorbital lacrimal gland Hematopoietic neoplasia Internal lacrimal gland Abdominal cavity Parotid salivary gland Tracheobronchial lymph node - 004599 NTI Covance 6329-225 - Table 1 Results of Homogeneity Analyses (ppm) Mixed 8/20/98 13:WEEK DIETARY TOXICIETYTHSATNUODLY (WNI-TMHFNO-SEM,ETT6H3Y1L4)PIENRRFALTUSOROOCTANESULFONAMIDO Samples Location _ Replicate 3 T6314 (ppm) 30 100 Top 1 2.19 337 79.4 2 243 278 76.5 3 2.09 256 76.7 Mean 224747" 2909.7) T71.5(715) Middle 1 2 3 `Mean 2.24 227 2.14 2.22(74.0) 252 253 25.1 25.2(84.0) 75.0 79.7 89.5 81.4(81.4) Bottom 1 2 3 Mean 2.03 3.04 1.94 2.34 (78.0) 237 27.6 29.4 269(89.7) a Each value in parenthesis is the percentoftheoretical. 832 81.6 87.8 84.2(84.2) x 004600 CommMeT-06.3212451 Table 2 ResultsofStability Analyses (ppm) Mixed 8/20/98 13-WEEK DIETARY TOXICITEYTHSATNUODLY(WNI-TMHeFNO-SME,ETT6H3Y1L4)PIENRRFALTUS.OROOCTANESULFONAMIDO T6314 (ppm) Storage Conditions Replicate 1 500 Initial 1188 415 2 0954 468 Mean 142(142) 442884)" 19 day, room temperature 1 0733 - 2 08 - Mean 0772772) - 32day, room temperature. 1 2 Mean - 444 425 435.(81.0) 32day, 10709 - room temperature' 2 0.850 - Mean 0780(78.0) - 8 week, frozen 10m - 2 0995 - Mean 0.887(887) - a Each value in the parenthesis is the percent of theoretical. b Reinjected 004601 ES CoumeMeTs6s3291:42.215 Table 2 Results of Stability Analyses (ppm) Mixed 9/24/98 13-WEEK DIETARY TOXICIETYTHSATNUODLY (WNI-TMHeFNO-SME,ETT6H3Y1L4)PIENRRFALTUSOROOCTANESULFONAMIDO T6314 (ppm) Storage Conditions Replicate 500 T Initial 1- 0.788 2- 0656 Mean - 0722(12.2* 19 day, room temperature 1 - 2Mean - 0.707 0.651 0.679 (61.9) 32 days, room temperawre 1 - 2Mean - 0.688 0.657 0.673 (67.3) `a Each value in the parenthesis is the percentof theoretical. = 004602 ComMnTc-e6e3o1n4s1 Table 2 Resultsof Stability Analyses (ppm) Mixed 10/22/98 13-WEEK DIETARY TOXICIETYTHSATNUODLY(WNI-TMHcFNO-SME,ETT6H3Y1L4)PIENRRFALTUSOROOCTANESULFONAMIDO T6314 (ppm) Storage Conditions Replicate 3 Initial 1 22 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. x 004603 CovanMceT6E3219-42125 `Table 3 ResultsofDose Preparation Analyses (ppm) 13-WEEK TOXICITY STUDY WITH N-METHYL PERFLUOROOCTANESULFONAMIDO [ETHANOL (N-MEFOSE, T-6314) IN RATS Week Replicate 0 3 T-6314 (ppm) 30 100 1-4 1 a 2.16 29.7 87.3 2 a Mean = 212 21.0 85.2 214(713) 284(947) 863 (863) 14 1-1 - - 2 - 1.84 - - Mean - 191(63.7) - - 1-4 1 - 248 - - Me2an -- 222388193) -- - - 58 1a 355 279 83 2 a 305 270 823 Mean a 330(110) 27.5(91.7) 85.8(85.8) 92 1 a 224 27.6 89.7 Me2an aa 223479(190) 2267..82(907) 8878.52(88.5) 13/14 1 a 251 26.2 98.1 Me2an aa 224472(823) 262.643(877) 9988..74(98.4) a Below the limit of quantitation (<0.5 ppm). b Each value in parenthesis is the percent of theoretical. d RReetteesntt.ion. ~ 0046 04 28 a2 voi t 82 3 8 Co i 2 232 3 w vot ] snr pe Che EE od BE a Ea t 2g 28 61 " }i svt sop er LE ! . 23 Bol fd dH, dd dh 3 8 a Co i Soma ot et ie oe saw www www a t 823: 5 a co i mr 6 set isSe an 0 wea w wom om wow ww i RN Bel, of dl EE, dh dB E823 B " Co i Coe mwa mw oma t 228 52 65 ' ii 23SS 23 & & cof| Bary of Tas Materia onmmption Buc rararden o =u wmoome ome one Gw 32S a2 @ vo omy et min meetin is mir uw 13 13 1 3 a us 22283s a - Co i ; res ule SnRTT OORW wOw RTe wma . OB Sh 4h Ts wn om, : 323S 5E Co i os wif EF SUL OE. REL BR US we wm em wen 23= 22 3 3 oi WE of BOTW OR Rm Lam : 23g %2 n ` i: ] re me ly sw nas we lla EAL RO SDR Mm se mm ee BOR Bote op oa os w 3am 8os a } i 228gsg & " ! ii EI Sy ts wa, nous ofl ofl. BOF RW wm ome nolid t 239 S8] " Co i wile uf BF SUL NW. BEL GDR en om wen wen ' 2S2s 828 " Co i Se se yc re ms i : BOR OP OW Ow som H$s 8 75 ` ii mrs oe EF. AL BW. SSL GBR eee jem sem wea . 2S2 88 7 Co i 55 " i e 32 & n Co i 83 2 8 Co i Boge booth BB : 82g< o oy oI i 2$ 88 a Coi =o i aS 8<&@ Co our Gow ome nme we am tmu om mu BOW ON FORO OW ROBB gig od; BoB 2% RW ` $. gz " Cy EOE OR BOE OP OB WB :: 5$s< - oy} Boob bh Oh WR ; 28<3g " co } | [. . oy i 28 g2 wn Co i rotor 30 2S aga F] Co s 2 8g vo { Fogg 3 ` g82288 0 vo mary of crn teh wen ES 32 2& eso HD MEE 3 | or ot i3 i 2 23a& a vod: ecm CB3 CHE SER IB aww ED OSEN DHE DR , S2 >2 9 vodi i 32 322 9% Codi ' o32S >a8 5 cod ` 232 [a43 9% vo i 232 >2 = od i' 2S a&> [| 2S >32 9 vo i i 2S 2> 34 100 IR i 2S >23 01 "3 2S _> & 102 vo i Summary of Organ Weight Data. pl o meer CHV OIES HEU ews Bota far tae aa2 103, ' {i i 2$s "&2 0 Co 23S a82 105 IE| cy ot orp wane ca 2Ss &a3 106 vod S2s a2a 107 Coi t} g2e 2 a 108 Co S2 *>a 0 vod { 2SgS a& N no Co i : aes 2 & Crees 2S< a2 n odi gem, 23 238 TRE BB BBB BRR , 5 0 0 8 1s os ! ms Co i incidence of Microscopic Observations reer rm o $ 22 EEE EEE ' ESRB PPE ROL SY ne coli 2 cESHRSEMNLRRaT,cBos mmrooosno 282 fii see us Cd Sntdence of Mcrocepis hearasions ra CHER ee Pedi Ett i ee ER 8SBBE OL3B 32$ 8& 16 Co 2$I laemma 22 CEESe LEe B e LSE wn Co i 2S mon a2a TAME HsDiRs OaBeB RE eos us Co cide of Merseepic aseraticns re CBN (ve erren wees ws sno Lon ` EpEE 232 2a no vodi EnrAIo ES Fn asc FORE, TEASED TH mare men? CRE, masmormic eoGL 08) on 2 [-- 2&3 sett it 8 8 M3 4 oe . ea 4 0 wa 120 Co 2S3$ 333 2 vod ote of ire comets rei a pe oT 2$. Copmomse 2&2 ERSTE ED UR 8338 | RE $3 3 38 3 8 3 P ter tr eee e Co i toe so es rns ins =O are, snc, camicsian SE , Piiid iii ! 3$ 33 13 vo i | Sncgence of sve of selected Wcrescpic omarion ma 2S N3 En iod g2 3 nN ns Co APPENDIX 1 Protocol Deviations Protocol Protocol Amendment No. 1 Protocol Amendment No. 2 Protocol Amendment No. 3 Covance 6329-25 IMT-6314.1 le N 004673 Protocol Deviations CoveMrT.6832391042215 Protocol. Housing. Diet. "Certified Rodent Diet #5002, meal (PMI Nutrition International) ad libitum, unless otherwise specified." Actual Procedure. The animals were given Tekiad 8726C pelleted food from August 17, 1998 to August 23, 1998. Protocol. Dose Preparation. 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." Actual Procedure. Retention samples were discarded after approximately 1 monthofthe completionofthe 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 findingsasthey are 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 weeksoftreatment, five animals/sex/group will be fasted overnight, bled for serum samples, anesthetized with carbon dioxide, weighed, and exsanguinated." Actual Procedure. Fastingofanimals at the Week 5 scheduled sacrifice was not documented. 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." TZ 004674 Covance 6329225 IMT-6314.1 Actual Procedureex.amiSnoatmieont.issMuiesss,irnegqutiirsesduebsyartehelipsrtoetdowciolt,h wapeprreopnroitataevaciloabmlme efonrtisn the histopathologic pathology data sheets for individual animals. Summary tables do not include them as having been examined. These deviations are not expected to have affected the resultsofthe study. 78 004675 CCOOVVAANRCEEL.> Sponsor: 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 9 004676 `Covance 632P9a-g2e225 - Study 13-Week Dietary Toxicity Study with N-Methyl PerfluorooctanesulfonamidoEthanol (N-MCFOSE, T-6314) in Rats Purpose To assess the toxicity ofthe test material when administered in the diet t0 ratsforat 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 C3o3v01anKcienLsambaonraBtoourlieevsaIrndc. Madison, Wisconsin 53704-2595 Mailing Address: PO Box 7545 Madison, Wisconsin 53707-7545 130 004677 Covmce 632025 Page3 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. `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 LRaegbuolraattioornys,PrPaacrttic5e8,ReigsusuleadtiDoencsaesmsbeetr f2o2r,th19in78Ti(telfef2ec1toifvtehJueneUS20C,o1d9e7o9)f,Feadnedrwailth any applicable amendments. Animal Care and Use Statement Al procedures in this protocol arein compliance with the Animal Welfare Act Regulations, 9 CFR 1-4. In the opinionofthe Sponsor and study director, the study does not unnecessarily duplicate any previous work. Quality Assurance The protocol, study conduct, } and final report wil be audited by the Covance Quality aAsnsdurreapnocret Uwinliltb(eQaAuUd)i.tedTbhye tphreolQifAerUatioofnPcaetllhonluocglyeaArsasnotciigaetnes(IPnCtNerAn)atieovnaallu.atiLoinv,erdaatnad. Serumanalyses, data, and report will be audited by the QAU of 3M Environmental Technology and Safety Services. oy 004678 Covmee 63290225 Pages Test Material Identification N-Methyl Perfluorooctanesulfonamido Ethanol (MeFOS, T-6314) Lot Number The lot numberswillbe maintainedin the raw data. Purity Responsibilityofthe Sponsor Stability Responsibilityofthe 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 Areserve sample (approximately 5 g) of each lot will be taken and stored at room temperature. These samples will be transferred to the Sponsor afer completion of the inlife phase. | DispositionofTest Material After authorization from the Sponsor, any remaining test material will be returned to: Andrew M. Seacat, PhD 3ToMxicology Services Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 TFeaclseipmhiolneeNoN.o:.:665511..753735..13717631 nr 004679 Covance 632P9a-g2e2s5 - `Animals Species Rat Strain Crl:CD'(SD) IGS BR Source Charles River Laboratories, Inc., Portage, Michigan Age at Initiation of Treatment Preferably less tha6n weeksof age but not more than 8 weeksofage Weight at Initiation of Treatment 10010300 g Number 80 males and and Sex 80 females Identification Implantable microchip identification device: Husbandry : Housing Individual (mabye 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 analyses arc on file at Covance-Madison. Ty 004680 Covance 6329225 Page Water Adlibitum. Samplesofthe water are routinely analyzed for specified microorganisms `and environmental contaminants. Theresultsare on file at Covance-Madison. TChoenrteamariennaontksnown 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 0 26C, a relative humidityof 30 to 70%, and a 12-hour ight/12-hour dark cycle. The light/dark cycle may be interrupted to accommodate in-life procedures. Acclimation Atleast 1 week Randomization Selectionofanimals for the studywillbe based on clinical observations, ophthalmic examinations, and other data as appropriate. Animals will be assigned to treatment groups using a computerized blocking procedure designed to achieve body weight balance with respect to treatment groups. At the time of randomization, the weight Variation of the animalsofeach sex used will not exceed #2 standard deviations of the `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. . Bo 004681 Comme 632P0ag2e25 Group Designations and Dietary Levels Group NMuamlbeerofAnimFaelmsale Dietary Levels (ppm MeFOS)* 1 (Control) 20 2(Low) 2 20 20 0 3 3 (Mid) 4 (High) 20 20 20 20 30 100 "b2 TDhoesceolnetvreolslaanriemeaxlpsrweislsledecaesipveptmhoefbaMseaFlOdiSe.tonly. Dosing Procedures Method of Administration Dietary, 7 days/week for at least 13 weeks. Treatment will continue through the day before necropsy. Reason for Dosing Route The potential route of 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 will be stored at room temperature. Before initation of treatment, dose prepasations of1 ppm. 3 ppm. 30 ppm, 100 ppm and 500 ppm wil be mixed. Dose preparations will be mixed at least once every 4 weeks during the in-life phase. Retention During the Samples 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 willbe discarded approximately | month after completion of the inclife phase. } TM5 004682 Comme 32925 Pages DBoysCeovAananlcyes,eussing a method supplied by the Sponsor and validatedbyCovance Homogeneity `Homogeneitywillbe determinedfor 3-ppm, 30-ppm, and 100-ppm dose preparations once pretest. One sample (approximately 100 g) eachfromthe top, middle, and bottom of the dose preparations mixed for homogeneity analyses will be collected, divided into three subsamples for extraction and analysis, and analyzed for test material content. All sampleswillbe stored at room temperature until analyzed within 7 days after mixing. `Homogeneity analysis will be repeatedifbatch size changes by more than 30%. Stability Four samples (approximately 100 g each) will be takenfromthe1-ppmdose level concentrationofdiet preparations mixed pretest. One sample will be analyzed on the dayofmixing and used as the baseline value. One samplewillbe stored at room temperature for at least 19 days, then analyzed. A third sample will be stored at room temperature after at least 32 days,thenanalyzed. The remaining sample will be stored ina freezer set to maintain -10 to -30C for 8 weeks, then analyzed. In addition, two samples (approximately 100 g each) will be taken from the 500-ppm dose preparation mixed pretest. One sample will be analyzed on the dayofmixing. The second sample will be will be stored at room temperature after at least 32 days, then analyzed. . Dose Confirmation During the in-life phase, samples (approximately 100 g)fromall dose preparations will be analyzed. Al samples will be stored at room temperature until analyzed. 004683 Be Observation of Animals `Covance 6329-225 - we Clinical Observations `Each animal will be observed twice daily (a.m. and p.m.) for mortalityand moribundity, recording observed (fcaigenwdialilsnbtehgeosypeanreed,obasnedrvtehde.anAitmalleaswitlol nbceerweemeokvleyd,);eaacbhnaonrimmaallfwiinldlinbges or an indication of normal will be recorded. Additional findings will be recorded as they are observed. Body Weights `Eachanimalwillbe weighed at least once prior to treatment,`onthe first day of treatment, and weekly thereafter. Food Consumption Food consumption will be recorded weekly during treatment. Food consumption will not be measured during Week $ for animals scheduled for sacrifice during Week 5. Ophthalmic Examinations Ophthalmic examinations will be done for eachanimal before initiationoftreatment and `before the Week 14 scheduled sacrifice. The eyes ofeachanimalwill be examined by a Veterinarian using an indirect ophthalmoscope. A mydriatic agent wil be instilled into the eyes prior to examination. Clinical Pathology . Frequency Hematology and clinical chemistry during Weeks 5 and 14; urinalysis during Week 14 Number of Animals Ten animals/sex/group (the same animals will be used at each interval,ifpossible) Method of Collection Aanntiimcaolasguwlialnltbweilflasbteedpoovtearsnsiiguhmt;EbDlTooAd fwoirllhbeemactoollleocgtyedtef sts.r aUrjo iungeum lwairllvbeien.colTlheceted. chilled overnight (approximately 16 hours). TH 004684 Covance 6320225 Page 10 Tests Hematology red blood cell (erythrocyte) count hemoglobin hematocrit `mean corpuscular volume `mean corpuscular hemoglobin `mean corpuscular hemoglobin concentration platelet count white blood cell (leukocyte) count differential blood cell count blood cell morphology reticulocyte smear (made, but not examined) glucose urea nitrogen creatinine total protein albumin globulin cholesterol iglycerides total bilirubin Clinical Chemistry alanine aminotransferase `gamma glutamyhransferase aspartate aminotransferase calcium inorganic phosphorus. sodium potassium chloride Urinalysis appearance volume specific gravity oH protein : urobilinogen glucose ketones bilirubin bmliocordoscopic examinationofsediment Serum Perfluorooctane Sulfonic Acid (PFOS) Analyses Frequency and Number of Animals Five animals/sex/group after atleast 4 and 13 weeks of treatment. Samples after 4 weeks of treatment will be collected from animals selected for the interim sacrifice. ~35 004685 `Covance 63P2a9g-e22151 - Method of Collection Animals will be fasted overnight; blood (approximately 4 mL) will be collected from a jugular vein. Samples will be collected without anticoagulant. `Sample Handling Blood samples will be allowed to clot at room temperature and centrifuged. Serum samples willbe harvested and 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 Technology and Safety Services 935 Bush Avenue Building 2-3E-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 will be done. Animals to be sacrificed will be anesthetized with carbon dioxide, weighed, and exsanguinated. Scheduled Sacrifices After at least 4 weeksof treatment, five animals/sex/group will be fasted overnight, bled for serum samples, anesthetized with carbon dioxide, weighed, and exsanguinated. The abdominal cavity of each 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. 004686 wy Counce 32Pa9g2e2152 Afer atleast 13 weeks of treatment, all surviving animals will be fasted overnight, bled faonresstehreutimzesdamwpiltehsc(afrivbeonandiimoaxlisd/es,ewxe/igrgohuepd), aenxdsacnlgiuniicnaaltpeda,thaonldognyectreostpssi(e1d0./sLexi/vgerroup), samples willbecollected for palimitoyl CoA, PCNA, and PFOS analysis. Postmortem Procedures Necropsy The necropsy wil include an examinationofthe external features ofthe carcass; all externalbody orifice; the abdominal, thoracic, and cranial cavities; organs; and tissues. Organ Weights At the scheduled sacrifice afer 13 weeksoftreatment, the following organs (when present) will be weighed; paired organs wil be weighed together: adrenal (2) bKriadineny (2) liver ovary @) spleen ttehsytmsu(s2) thyroid (2) with parathyroid Organ-to-body weight percentages and organ-to-brain weight ratios will be calculated. Bone From Marrow Smear the femurofeach animal at the scheduled sacrifice after 13 weeks of treatment ; only; made but not examined CAetllthPersoclihfeedrualteidonsaTcriisfsiuceeaCfotlelre1c3tiwoeneaknsdofImtmreuantomheinstt,orcehpreemsiecntaaltiEvveaslaumaptlieosnof left Jateral lobe of the iver from each animal will be collected and preserved in zinc formalin. After fixation, each sample of iver will be embedded in paraffin, and the paraffin blocks will be shipped to 140 004687 Covance 63P2a9g-e22135 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 the final report. Palmitoyl-CoA Oxidase Tissue Collection and Analyses At the scheduled sacrifices, the right lateral lobeoftheliverwil be collectedfromeach `animal and flash-frozen in liquid nitrogen. The liver tissu will be stored ina freezer set to maintain -60 to -80C until analyzed by Covance for palmitoyl-CoA oxidase activity. Liver PFOS Analysis At scheduled sacrifices, the remaining portion ofthe liver from eachanimalafer 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 Technology and 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: 004688 141 Covance 63Pa2g9e22154 adrenal 2) aorta brain cecum cervix colon duodenum epididymis (2) esophagus efyeemur with bone marrow (articular surface oftheofthedistal end) Harderian gland heialerutm with Peyer's patch (lymphoid aggregate) jejunum Kidney (2) lesions lilvuenrg with mainstem bronchi lmyammpmhanroydegsla(nmdesse(nfteemrailcesanondlym)andibular) ovary @ pancreas pituitary prostate sraelcitvuarmy gland [mandibular (2)] sciatic nerve. seminal vesicle (2) skeletal muscle (thigh) sspkiinnal cord (cervical, mid-thoracic, and lumbar) ssptleerennum with bone marrow stomach testis (2) tthhyyrmouis.d (2) with parathyroid trachea urinary bladder uterus vagina Zymbal's gland TaHinisissmtuaoelpsta(htaahstoadlipoepgsryooprriiastsea)crfirfiocmedeaatchananuinmsaclhiendutlheecdoninttreorlvaalnwdilhibgeh-edmobseedgdreoduipns paanrdafefainc,h sectioned, stained with hematoxylin and eosin, and examined microscopically. : Mpaarcarffoisnc,ospeicctiloenseido,nssftraoinmeadnwiimtahlsheimnattohxeylloiwn-aannddemoisidn-,doasned gerxoaumpisnewdillmibceroesmcobpeidcdaleldy.in Suspected target organs noted at the high dose will be examined microscopically fom cach 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: 142 404689 Covance 63P2a9g-e22155 Experimental Design and Methods Results dose analyses `mortality clinical observations body weights 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 caseofheterogeneity ofvariance atp 0.05, transformations will be used to stabilize the variance. Comparison tests will take variance heterogeneity into consideration. Owenieg-hwtsa,ybaondalyyswiesigohftvacrhiaanngcees,(AfoNoOdVcAo)nswuimlpltbieonu,secdon(tifinapupoluiscacblilnei)catlopaantahloylzoegbyovdaylues, and organ weight data. Ifthe ANOVA is significant, Dunnett's t-test will be used for control versus treated group comparisons. : IcfotvhaeriAanNcOe V(AANsChOoVwsA)sigwinlilfibceanucseefdortoboandaylywzeeigbhotdsyatweWieghetks,1w,iotnhei-nwitaiyalabnoadlyysiwseiogfhts as the covariate. Ifthe ANCOVAissignificant, 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 be analyzed statistically. 004690 143 . ComesPag2e 1s6 Aitnstthreucetinodnsotf1o fyienaalrizaefthearviessbueaenncecoofmtmhueniacuadtiteeddbdyratfhterSeppoornts,oirf,ntoherneqtuheesatueddirteevdisdiroafnts or report will be considered final and issued as the fina report, signedby the study director, and submitted to the Sponsor. `Anymodifications or changes to the audited draft report requested 1 year afer issuance will be performed at additional cost to the Sponsor. Two copiesofthe signedfinalreport (one unboundand one bound) wil be sent to the client. RAlelcroarwddRaettae,ndtoicounmentation, records, protocol, specimens, and final report generated as a preesruilotodfotfhi1 syesatrudfyolwliolwlibnegasrucbhmiivsesdioinntohfetshteoriangael rfaecpiolirttietsootfheCoSvpaonnscoer-.MaOdniesoyneafroraafter asunbdmaisrseitounronffteheewiflilnablercehpaorrtg,edt;healalfroarwemdeanttaisotnoerdedmaotnermiaalgsnewtililcbmeedsieant, tporotthoecoSlp,onsstourd,y mcoaryreeslpeoctndteonchea,veatnhdetmhaetoerriigailnarleitnaailnerdeipnortthewiClolbveanrceetaairncehdibveysCfoovraanncea.ddiTthieonSalpopnersioord of time, and Covance will charge a storage fee. If the Sponsor chooses to have Covance disposeof the materials, a disposal fee will be charged. PCNA antigen evaluation data willbe retained by Pathology Associates International. Liver and serum samples sent to the Sponsor and analysis data will be retained by the . Sponsor. a, 004691 PROTOCOL APPROVAL amon TT een Los 01.Sais?" w M. Seacat, PhD Study Monitor 3M 7/31/43 Date. PStiuedry J.% rd, PAD : Covance Laboratories Inc. Date 5 004692 SC COVANCE PROTOCOL AMENDMENT NO. 1 Covance 6329-225 13-Week Dietary ToExtihcaintoylS(tNud-yMewiFtOhSNE-,MTe-t6h3y1l4P)eirnflRuaotrsooctanesulfonamido -Sponsor: 3M, St. Paul, Minnesota Study Monitor: Andrew M. Seacat, PhD Testing Facility: Covance Laboratories Inc., Madison, Wisconsin Study Director: _ Peter J. Thomford, PhD This amendment modifies the following portionofthe protocol: Effective August 27, 1998 I. dPealgeete3,thPertoepxtosiendthSistusedcytiToinmaentdabrleep.lacTeowicotrhretchte tfohlelaouwdiintg:ed draft report date, IInn--LLiiffee EStnadrtDDaattee:: DSeecpetmebmebrer3,1,19199898 `Audited Draft Report Date: May 26, 1999 Effective October 1, 1998 : 2. PIamgmeun1o2h,iPsotsotcmhoermtiecmalP rEovcaeludautrieosn,,CePlalraPrgorlaipfhera1.tiTonoTriesfslueec Collection and the decision 0 ponelryf,odremleptroelithfiesraptairoangcreallphnuacnldearrepalnatciegewnitehvatlhueatfioolnlofwoirngf:ive animals/sex/group Athtetlheetslcahteedraullleodbseaocrfiftihceelaifveerrf1r3omwefeivkesoanfimtarlesa/tsmeexn/t,grroeupprewsilelntbaeticvoellseacmtpeldeasnodf preserved in zinc formalin. 004693 +6 PracCAomvaennced2m9eFN:nugo2te51 AMENDMENT APPROVAL / s" 7 w M. Seacat, PhD `Study Monitor 3M 1/5 [92 Date Per]. Study Dij Covance rd, PhD or Laboratories Inc. Date ;a 004694 147 COVANCE. PROTOCOL AMENDMENT NO. 2 Covance 6329-225 ree EO foe omer eet 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: Peter J. Thomford, PhD "This amendment modifies the following portionoftheprotocol: Effective October 21, 1998 1 Page 8, Dose Preparation, Dose Analysis, Stability. To reflect the decision to perform additional stability analysis, add the following to this section: Two dose additional samples preparation mixed (approximately 100 for Week 9 through g each) will be taken 12. One sample will from the be stored 3-ppm 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. .0463s 148 Protocol CAomveanndcmee6n3t29N-o2.225 Page2 Effective December 4, 1998 2. Page 13, Postmortem Procedures, Liver PFOS Analysis, Sentence 1. To clarify collection of liver sections for PFOS analysis, delete this sentence and replace with the following: A`otthsecrhreedquulierdedsaccorlilfeicctesi,ontshewirllembaeinfilnasghp-forrotzieonnoifntlhiqeuildinviertrforgoemn aenadchsatnoirmeadlianfater freezer set to maintain -60 to -80C. 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 partofthe 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, liver sections 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.Liverand . stomach were identified as target organsinanimals 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. 004696 149 ProtocolCAomvaenncdeme6n3t29N2o225 --_-- wm Effective February 8, 1999 5. Page14, Postmortem Procedures, Histopathology, Paragraph 3.Liverand `was identified as target organinanimalsinthemid-dose group and will be examinedinanimalsfrom the low-dose group. Therefore delete this paragraph and replace with the following: Liverandstomach fromeachanimainthemid-dosegroupandliverfromeach animalin the low-dose groupwillbe embeddedin paraffin,sectioned, stainedwith hematoxylin and eosin, and examined microscopically. AMENDMENT APPROVAL /7 | Andrew M. Seacat, PhD `Study Monitor 3M Q) uit 3//1049 Dae . Peed 7.Thefsrd,PhD, Study Dirdofor Covance Laboratories Inc. 2% 004697 150 COVANCE . PROTOCOL AMENDMENT NO. 3 DR AFT Covance 6329-225 ee 13-Week Dietary Toxicity Study with N-MethylPerfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats `Sponsor: 3M, St. Paul, Minnesota `Study Monitor: Testing Facility: Andrew M. Seacat, PhD Covance Laboratories Inc., Madison, Wisconsin Study Director: Peter. J. Thomford, PhD This amendment modifies the following portionofthe protocol: Effective August 28, 1998 1 Page 4. To include. the vehicle used to dissolve the test material `before mixing `with the diet, add the following section. Vehicle Acetone Identification Lot Numbers . The lot numbers will be maintained in the raw data. Purity On file with the manufacturer Stability On file with the manufacturer = 004698 ProtocolCAmoenmdmnenst Nmo.s3 Page2 Storage Conditions `At room temperature. ICnhfaorramcatteiroinstoincssynthesis methods, composition, or other characteristics that define thevehicleis on filewiththe manufacturer. AMENDMENT APPROVAL -- Andrew M. Seacat, PhD ee Study Monitor 3M -- Peter J. Thomford, PRDee Study Director Covance Laboratories Inc. A Date -- eDateesseee-- 004699 152 APPENDIX 2 Individual Animal Fate Data Individual Clinical Observations `Individual Ophthalmic Observations Tere Covance 6329-225 - 00470; 153 0 ai |B booms ommammn aR RO , 23 goo EE IEE 833g 54 Co i WTM 323g ggaiiE B iBOBD E DGEERE EBEEAERRROMHRE ROR d2 38 * : g SHE OE bose IER mEBmEaml mHmE 8Rodg !\ 23 Shooi BER BEIER WR 3 2 835 156 ; gE Dom i oa mmawn wd BOR \ $82S SEEM | EER BAERS OEE OR J 32 5 ; BE seam a , 533<3 = Co ; po wr Ss z$g g " _j sxe em rc res , < 335 @ ; PUSS ------ | 2S &3gSy 161 od ! =Sc 8 i a nn g 53a i ropendix 2 nn eaec rt 8233 2 10 wr \ Co i en 52 ! 165 ` i n GGounme x1a wwioaessourcmar neess g2 6a3 166 5 et !' oi i3 mr x 10 Tiecrn ns r- .` 8 a33 1 vodi ov rivs | 8 &35 @ vy| seven 2 - 23 a3 > 169 Co i EE e" ` 33:.s : Te nn : 355 & m Cy| opnr x ai tm srins }i 8 535 m . |i mmm . $g< g Co i tam wor wns ` 28e R8B ns vo i en SE Tn mmm , <gg33 i ett os, sin {i $ 3 8 16 Co i ey an ammooss aas wwso csucurme rpors i} <8 2 m odi { 28 & us vo : mo mime s 2 m9 : {; s8 J33 0 Co i mavens cg sion SEE u i 8 833 w Co | | i} gg 88 w Co | ves von wb a wo ssc PoE 32 3@8 13 5 SE i Co 1 . 23 83S 18 Co i arte i ns snr rss i 82 388 1s Co i i 32s 38 156 Co i ereom JR ------ i aren wn aso suspic: Ps i 232 58 1" Co i | 8 3& a 1 co i . vs telat opiate chserntions SHE arn Jp ---- \ 2S 3a 159 od . i { 32 Sa3g 190 odi APPENDIX 3 Individual Body Weight Data (g) CovanMceT66321942215 "+4 004738 sein mr 1 gmow om Em EL. wm ow \ gmooB BE oWmoooo# HB @ : Jg22 8 wn Co i wow 0B o o BoB OB 8 8 t 82g2 HE OB OE OH OH HB 4 ow we we ow 33 193 . i|. ] nt a ts Eo 28253B BoB ow BOE OB 8 8 OH i 54 Co i cri gmo@ oo # BH OB om om om om ox i S$ Eg8Bw o 3og sE O80BB 88 OoOBwB mOmF oodoOmB BBoOBB oO&B BBoH oOBBOE 2OOBB oOoHm BO& OB# oOOmBE O## oOO#BB 0OHE OO#BE OOBH 19s Co | tansNwvpenndiix s3 3s 1 wr mE om oBo o% 8B B38 | g3S Bog8gwm EE OB oBmOB BOOEBE OB OBBE BOOBE a ow om ow ow 3& 196 Co . i | rn # BF o op oo og 8B oR BOR t 3g233 { coon ans 0 e B3c oogomggw & #B## OE0BOHE OBE OOBB BOOOBER 98 Co i 2 gz2 I2 BEE 32 ii 2ec ggmM 2BOB8 BoE& %# 2 og o o# o@ # co | Individual Body Might Data (9) = EE Z B { J3Sg2s 8gomSamcor: PEAxLEE 2-3 re BoBmH 3 OBOBB OBOOHB 0 vo { ett ea ah ue 0 B i E#2 RBEE B&E O3B fi 23ec 8 8 E#E B#E BBEB B# 3& 20 ` i t ] sO OB OB OB , i 228 3e3 n Co i APPENDIX 4 CounIcMeT3-62391242.51 Individual Food Consumption Data (g) Individual Test Material Consumption (mg/kg/day) 6 604750 8 8 WOE EF ov o@ op BE oou ! 3$o 8MW8 i#t wBoBo OBE OiB i#n8 #BE iE 8EE 8B a Cy} ] " acona i | ornSEE SEY ET me Wm oH 2 OB OE 8 8% 3 o3 8 3 ! g BF # B EB BB BR OB = d432 aBou@Es kOoi HF HiBhEOBOEBdE OOHBHe oe ow ow ome ow om 0s Cdi g gm oo# O#H JEMe oB& BOB O# # OH OB }4 g25s $B BE OE OE OE OB OB OB OE OE OB Ha 26 Co | | 2 2 FI -- FB E BE BH . Bm I FE ZI 3 2 0 os G2 t CREse 2BYJ2 WW=AYTEWT8ie 2 oE i Y%J WLEEF EE EAE X+ TE2R O3BB I a E TE TYTS TF RYT CE Ee Co i ggEEoEOEeS Wolee MWOoB Womee HHOOEHS oOHBOEOT"E OoHB }4 SggS O@EEm a BE oH OBETE E @ HiTH se ow ewe ow sue oe 0 Co i HOE = J FB FE BE XZ OE B OB g g E2 OEEF OOH OOEB OH O2H OBB OE o3e oBfL oBe 38 209 . ii cour: wae 2-3 pe i A 9 SE ~N BEE Bl is in ; ggE EoH B#8O# it ggo 4OB g838#88 88 #B B 8 B# m Co : SgHi vOiEe wE ie Bede ;| 3=32 odcasoer oHt OB OB 8 mn Co : | 2 5 Ef . = 8 EB OB i $ g3 y# Co Et al a , Si2Ss 223 2 Codi Stl Sos neo goo HOH OH TETE TELE TERE ` 22 EH BH OH OH OH OH OH OH OH OBE OH 38 us Co 1 ie en 888 ki bE 3 33 io a be i" ii { TS o83:S amt EBEEEE IIIT 5 a 3 TI TTAB borINN horIME orTEN MrTM MFTB Cy i 2 ei ot is covooio cs tn sd i om Wd BOTH OH Bon oH i 382332 m Co | Sa i oaus pu mmm omwmk 00 gmk edn ogni 5 k 323o Hi eH ed HOH OH OH oe & a HOH oo Co } ! S2 323 2 Co | . I S-------------- go TE TE TH { 32Zgs #3 "TMH OgH H & Co i ei et rad Soames Sth marr g ae HnonoH ooB# 4 | soe 5 EBHEEEEBNE EDREE 83 PY vo i . 5 2a ton Ss crt cement. rds Sf edd Ee SE mame mm mie t : o a oH HTH g 233 m Co i gow ETE Ste 3 i 3 f o Sai & i i 33 i. cy} teks es sri ost va. ta it g i3 2 Co i APPENDIX 5 Individual Clinical Hematology Data Individual Clinical Chemistry Data Individual Clinical Urinalysis Data CovanScMeT6.36239-124215 - RS 004772 mE aM BOTROT BOoWomom om oH HE t o WH 2$ J3 Ey Codi mE BTR RRM ogOood BEOEB B BE BBoBmaSB t: 23gg 33 = Co 1 J og 40 BE. NAT WP. HR BD 0 Sm me ue #@w odd rir Err gE 32 FBS Nob EE bb oo a3d om Co i ate RO. ASL MR. AW WE, YU then ee mee sen wou 40H 1 03 4 11 | a6 ne Pou i 1 3 i # i i : F gg 83 29 . ii Bi oi t 28 335 20 Co i | nt 5 a oi: iF: i g2 3J = Co | gE oB OM OOM OBB : $s 33 [I | mE oan BTR RN S ga HpB onoBoBo# BomD oommoom@ '! 2-8 i EI g23 m Co ' ito FUTSTes To a ocTmessroanizo. RE of. SFL AOL BR. EDM HR, US tem oo wen we Group3: DoseLevels `Dosage UnitspomNarOS. or ` goon n d1B8 44434d7s rborn r ' g g53 24 ' i4 HE We BAR FR. BEL MD. te me ma se EgoEo H EEFHEE4EEE4 E8 4 FB Fb ii 2sg &8 2s Co i BM: oo: i | 32gs &2 EY Coi ooA : 2A i t! g a 2 = Coi ooenain 5 co EE BE WW. WOT WOW OR de 23 aa 28 vo i | eis Con tes mE a BFR RTM Ei om oB om om om oR ' SEH OBB BoM om oB 2 2 OWN TERE && Co 1 | BE. NE SR. EEL ve ee me me we E gm EHoE E HoEd dEdE IFoF Fb |i S me gn oe fh 4 A 4 mm 1. a, o 33 0 Co i | oi ee EE MSR MM BU Mt ue em we w#rooHo8 Hod oH0H o3f i3 33 FOF8 1O40}i i ;' g Em EL ROW OM o4or oH o bb 8gad oFn BR12 1Ow 3OB8R 4OR o1F o2R 2BF" os b1oe 241 . ii | 1 gZz32 [------ sevunsin 3 A-- Group: 1 Dose Levels 0 Dosage bast: ppmNaros a2 BE t | wn ivod BOB HH HB NE . PTR oW $2g g 244 . ii | Mm wf. STWR Me Wom oH BBB oR : 2 28 us Co 1 | . Ban af. NO. AOL BR. EN ER, eS tees wo sem wo 823 32 6 Co 1 SE M0 TL ND. WL MR me em woe se goon 4 dow og bo i -i g2 32 2 + BF FEB Moa w Co i pendix 3 co ERE tow ties om ts nn | EEE A 4 go: oor ! 22$ &3 248 ` ii otis ha 1-74 | crs nts0 on ntssot 28 &23 uw Co i ! ris es E soo oF Fo oH oH OB O41 3 ' go 2 FEg8 # EE4 of HAou 4 fF & g 250 . ii Seo BF fo oH oB 4 3% 3 ' g 2 Be Ge bh BM Bb fe 83 21 . i: | 05 fom oH BE OH oB FF IH BoB WOE : < Bf dol ROW s2 = Co rv gEmoo o4 8 Ffo oommooHuoom@ ouoO omoH ;t 22< =I Ed T d 8g2 23 ' |: Bf4 Th Hu 11 | ; F FREE TFB oF g = Cy | || ggos o8F 8Bf4 #H3 oHoH #OHB 1o3 %#4 %i || s Boiob ROW OR oF bob 52 8 25 . |i [---- ge Bo0% 8 BoB mon om | 2$ Ra wr Gi TE The RT 8S&$ 256 [A i || @ 4 3 LE BoBouoB 8g$< 2 Co } = ! gSc$ F PB & 258 .: v | 'i 2S &32S 2 vod v =n g2$ hd 1a w Cy; i{ 2S &$2 2% Co i | | | ]|| s gEmo08 gE FBFd oo@BoH8 ooHH 8 dH son 11p0% i dF ok ot ress an ne rn gSoooom oopF 43 oo#H HBooHB 11 00%f 88 ;| 82$sg 83 0 Co 1 | Bh Me phe wb BB | gm o@ 4 oH HoH 408 of 2&2$s s 2 ; 23< 2B2 26 { - i wd 2S 22 8 265 to pana 3 Cosh PRY sm woop 4 o wom 30% i i 2g$s & x6 ; ggwaooo@om oo% f4 iHoHoH # OH 33 08% 3 :| 2&2$s Bd i ga ooF of om oBo@ HOB } 282gs a a : gem om op o}om oH oB o#o : 2&$s 2> ; "3 t! 8Ss$ 2 m } =3 }L )! z2g 2 o m H` ii ti $s 2& m ; <S228 m Coi: 22S 882 m odi rt P& $Ss 88& ns ng !| i " Ie i @c 88 6 i & i 2g 28 m : Group: 1 boseLevel: 8 Dosage nit: pom Heros. ` go iE i { 22g =o aE 9 &8 n i tes cs nie eert om 2 : ECE : 888-g 3 5 a sample vas overflowing at the tise of collection. 2 vd t rnin 5 songh S00501 gor cL i ; 22g5 88 ET 'i @ oor ` g$c i 81 =S 8@2 i JE A g2g. 283 i tos gm HoH t $S 28 Ea ; punt 5 gm aim 2&gs & us ro i i amin meg 3 L 2E rd 't 82sg 88 26 i oss s EE gi ; gee o oo: 11 i 2gz 2&@ = Co i " ORE gm os of fp 1 ff OF GP ` | g2$ &2 ' :i as ne2s ] @: ppb F Ee | gat of 1 1 Fb RG ' @ or or 7 Ff FE t igs & eee ComnSIcMeTL6-6332L1942.,51 - APPENDIX 6 Individual Animal Pathology Data 0 004837 ir em ! | SE re 8B @ 2291 ` i Soh Sek)Bh nt Ca LA ME lice (hf Het re: rioat m ous V 2$ og32 m ; 2eS 3~& m Cd GE SnRe The A En el A &2g< 8& Co earn A TE ER RS FEE a aes nee Ce28 &2 | 5 TA i 2<S 6@a 26 { << & 7 i EEE BEE REERR , TH FOLIOS TISSUES MEREMENC Ee, To (6m) ETE (0), sar a8), F 383 = i ] Ceg 5& rd rm. { TEE mere 2 Co SIESE dnel,l Be TT BR STU | sg2223 00 Co i 2$ 52& 01 Co i EnES zg.gg wn Cy 1 23 I iencaerasncntisrtratntntreiesane 3a@s 0 Co i eee ATS880OU0R ia), SRSARYSaco tn,Sonia Ves Uo. 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TELL cmeena, cn 2am ii agg TRREESelbemameemey 28 2 Co i erreer ag 322s I er eemsememssssirsn ii El ai i tepons, corso woos i ag2g 2g 54 .: ' EE, BE looS Ay ae rn 1 Bnd i gg2 83 545 ` i I ekRSehr, NEPATOCHLLLLAR,CONTASLOMLAR, SLIGHT IRCER oie coBIT: cue THROIO/TAAATITRONS NOT FOND TH EE,LLXhemMCKIAL PRESEN ' &&3 822? st6 i3 BE a CE LR a , g2& 23 547 ! 5v -_ CovManMcTeT.638e219-42W215 - APPENDIX 7 Cell Proliferation Tissue Collection and Immunohistochemical Evaluation NOTE: This appendixofthe report contains information supbpyPlatihoelodgy Associates International and has been reviewed by the Quality Assurance Unit of Pathology Associates International. * 605095 MTL Covance 6329-225 - Cell Proliferation Tissue Collection and Immunohistochemical Evaluation (to be provided by Pathology Associates International) si9 605096 APPENDIX 8 Client-Supplied Items CovanIcMeT6-36321942.215 Note: This appendix contains information supplied by the Sponsorand has been reviewed by the Quality Assurance Unit of [insert client name. 550 6050: 97 Client-Supplied Items CovanMceT6.36294215 605098 551 Conance is an independent, publicly held company F operating in over 15 countries and over 0 offices. worldwide, with headquarters in Princeton, New Joris, USA aCnodssinscesubissitdhiearmiaerskaertoiunngdntahmeewofrolrdC,otvhaencpreinIcnicpal o"nCeosroanfcweh"icinhtahries lriesptoerdtorneftehriss0pagoen.eTohremoursee ooff these and othe subsidiaries. TPHrEnceAeMnERICAS 18007730011 eBousnsoesse w522m930 ASosgwappaucniric esa `Covance LasoRaroRiEs . M8a8s5o4n11WL4U8S(A5227) Vae0rrnia2V6A37U0SA HO1a1 a4g4 ,142U3KSc00n1 M01i1na49.28G1om5a7r9y80 605099 THE DEVELOPMENT SERVICES COMPANY Shaping Solutions