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. AR&26_06/6 Fi. nEagl ogRepo. rt ~ 4-Week Range-Finding Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats = PREPARED FOR: - 000564 COVANCETM Sponsor: . IM St. Paul, Minnesota - FINAL REPORT Study Title: Per4f-lWuoercokocRtaanngees-uFlifnodnianmgidDoieEttahraynoTlox(iNci-tMyeSFtOuSdEy,wiTt-h6N3-14M)etihnyRlats Author: : Peter J. Thomford, PhD Study Completion Date: July 5,200 - Performing Laboratory: Covance Laboratories Inc. _ Madi3s3o0n1, KWiisncsomnasninBou5l3e7v0a4r-d2595 Laboratory Study Identification: Covance 6329-224 - Sponsor Project Identification IMT-63142 - Page 1 of 373 000565 _-- CovaMnMcTer6633e291-m42224 QUALITY ASSURANCE STATEMENT `ATshsisurraenpocret,UnwiittohftChoeveaxcnecpetiLoanboorfatAoprpieensdIinxc.7i, nhaascbceoerndarnecveiweiwtehd tbhyetFhoeoQduaalnidtyDrug - AAdpmpiennidsitxra7tiwoans(gFeDnAe)raGteodobdyaLanbdoriasttohreyrPersapcotnsiicbeilRietgyuolfatPiaontsh,ol2o1gyCFAsRso5c8.iatTehse data in International. study director The and following inspections were study director management conducted and findings reported to the - Tnspection Date Reported to Dates From To Study Director and Phase Study Director Management 06/04/98 06/04/98 Protocol Review 07/13/98 07/13/98 Postlife 06/04/98 07/13/98 - 09/16/98 09/25/98 Data Review 10/01/98 10/09/98 Data Review 09/25/98 10/09/98 10/19/98 10/22/98 Report Review 06/02/99 06/02/99 Protocol Amendment Review 10/22/98 06/02/99 05/02/00 05/03/00 Report Review 05/03/00 - Representa 8 Quality Assurance Unit ODaSte July 2000 - 2 000556 - STUDY IDENTIFICATION wCmovarnMcTe 6Te3629u1-422224 4-Week Range-Finding Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats Test Material N-Methyl Perfluorooctanesulfonamido Ethanol (MeFOSE, T-6314) Sponsor ~ 3M Toxicology Services Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 RB Study Monitor Andrew M. Seacat, PhD 3M Toxicology Services 651.575.3161 Alternate Study Monitor ~ Study Location - Study Director MarviTn. Case, DVM, PhD 3M Toxicology Services 651.733.5180 Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Peter J. Thomford, PhD Covance Laboratories Inc. P.O. Box 7545 Madison, Wisconsin 53707-7545 608.241.7207 Study Timetable Study Initiation Date June 8, 1998 In-Life Start Date In-Life End Date June 12, 1998 July 13, 1998, Study Completion Date July 5, 2000 - 3 000567 Covance 6329-224 -_ wTeM2 KEY PERSONNEL 4-Week Range-Finding Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats Study Director Peter J. Thomford, PhD Study Toxicologist Study Coordinator `Thomas E. Ryan, BS Nii van Bruce-Konuah Supervisor, Small Animal Toxicology Nathan E. Snortum, BA, BT, ALAT Supervisor, Dose Formulation Supervisor, Chemistry/Tox Support Dixie Bushee, BS, LATG Brian Schoenike, BS Supervisor, Laboratory Animal Medicine Donna J. Clemons, DVM Diplomate, ACLAM Clinical Pathologist Robert L. Hall, DVM, PhD Diplomate, ACVP (Clinical Pathology) `Supervisor, Clinical Pathology Ronald Markevitch, BS, MT (ASCP) Anatomical Pathologist Richard Hamilton Bruner, DVM Diplomate, ACVP Pathology Associates International - Supervisor, Anatomical Pathology Deborah Pirkel, ALAT 4 000568 - CONTENTS Covance 6329224 wre Page . BUREORE mmm i REGULATORY COMPLIANCE... 10 TEST MATERIAL ANDVEHICLE vc. 10 BTOSrMEEHELc.crssmssmsmassmsssm-- sssnmsomsonemeemnmemermere 10 RESEIVE (ATCHIVE) SAUDIES resrmremememeeemne 11 Di smmmsmmmmmmmm--------k POOCEDURES commis] DGOrSoEupPDIeEsiPgAnTatBiOonNs v and Dietary LEVEL ..s .......rrmrnrmrs emnrnnrnnmrnne 1133 _ RECTION FE SAIMPIES rvs -- 1 CHICA OBSEIVAONS rrr mssemsemremsmemenrennemne 16 BFoOdGy CWOMESUIIIPHGONHr S..e . re 16 16 . SerumPerfluorooctane SulAcfidLoevenl(iPROcS)Determination. ..................16 CHCA PANOIORYrns 16 ORBAN WEIGHS... 18 Cell Proliferation Tissue Collection and Immunohistochemical Evaluation............. 18 e Palmitoy}-CoA Oxidase Tissue COLEaS nCdAtBIiYSoESn.....eerovemrenrre 18 ES SHRUSCAIARISES crores20 RECORD RETENTION .......c.c.. mm----------0 . 5 000569 CONTENTS (Continued) CovancMeT63e2042224 Page Clinical OBSErVatiaonsdSUNVIVALreser 21 Body Weightsand Body Weight CHANGES... mre 22 Test MaterialCORSUTON....c..cceeserene 23 _ Serum Perfluorooctane Sulfonic Acid Level (PFOS) Determination...................23 ClRICALPANOIORY vores23 LiCevlelrPrPoFliOfSeraDtitonCTrisMsuae Ctolileoctnio.n.an.dcTm.mucnvohsisvtoschseremiscaslsEVeAIe Iation n...o .....n ..2.24. ADBIOMCELPADOIORY reese24 - CONCLUSIONS... 25 SIGNATURES .c.conmssmsmsmsmssssssssssssssmsmsmsnsnmos 36 - PATHOLOGY REPORT... 28 COMMENTS ON THE DATA wees 33 CODES, ABBREVIATIONS, AND UNITS... 35 - `CGoedneersaflOCr oCldiensiacanldPAADtBhIEOVIIOOBNSY..........c.v.eeronernsrnesrsrnsnnmensnmmem3snn631 ``AAbbbbrreevviiaattiioonnss aanndd UUnniittss oorrCClliinniiccaallHCREETSBTO.I.O.Z..Y....e .enr rrrr rrro orer mmemse4402n CAbObEreSvifaOtriAonRsBOaMndICUAnLitPsAoLNrOIClOiRnYical Ur ENAIYSISr .........r ..rorcorrnrnroremr44e5n4 TABLES 1 Results ofHOMOeneity ANlYSes (PI)... 2Results Of SLabiltyABBIYSES (DP) rere 1 49 - 3 4 RSeusmulmtasrOyfoDfoCslienPicarl OeDSpTaVAADrGUIOYaNSEStS..i..o.n..v(PvPMv).r.r. rmemrsreemmrmrrnerns 5S|3 5 6 SSuumoomffmBBoom addyyWrWea EiIgyghhr ttCDhaatyang(e 8Da)ta.(.) . omroemme8 n 54 7 8 SSuummmarmyooaffTrFeosy0tdMCatOerNialSCUonMsuDPmApItAiO(o8Nn)Da.ta..(.m.gr/.krgr/rdamyr)m.r.e.memmrore 6662 - 9 10 SSuummmmaarryyooffCClliinniiccaallCHeHmaEtoMlogIySDaTtd.Y..D.AR. .....cocercosscmonoamrs o nerrso6782 11 SummofaClirnicyal Urinalysis Data... sorrnreror1r6 - 6 00057ry0 CovancMeT6.32891-24224 CONTENTS (Continued) Page TABLES 12 SummaryofAbsoluteOrgan Weight Data (8)... 18 13 Suom fOrgm an-1a 0-BodryWeiyghtPETCERAgS ........rvrrrrrrrrnr8n1 14 SumofmOrgaan-tr0-Bryai Weight RAOS........rrrenrnrrrsrrn8o4 15 Incidence of MACrOSCOPIC OBSEIVIIONSv.81 16 InciofdMIeCrnoSCcOpeic OBSEIVIIONSverses89 _ 17 Incidence of Severityof Selected Microscopic OSCIVAtIONS.........vrern9.1 APPENDUL LussmmmimmsmmmmmnssssnsmmnsnmmmmnmnS8 FE -- PROIOCOL....rrsrsisssssssissnss 100 APPENDIX 2...coscnrnssissssnssssnssnsssnss 123 I0ividualANIMA Fate Da... 128 IndividualClinical OBSEIVALONS rset121 BOPENDIN Susmssmmssmmsmmmmmmmmmmnsn 193 - Individual Body Weight Data(g)verre 138 KEPENDUR Assmann 138 Individual Food CONSUMPUION Daa (@)rss 139 Individual Test Material Consumption Data (ME/KE/day)............rovrrern 143 APPENDIX 5. 146 Individual Clinical Hematology Data... 141 Individual ClinicalCHEMISTYDA... 159 Individual Clinical Urinalysis Data... 161 = AEP Cosmin 59 Individual AbsoluteOrgan Weight Data (8) reser 180 Individual Organ-10-Body Weight PETCERAS.....r.rrrrrrner 192 IndividualOrgan-1W0e-igBhtrRAatIiOSn ocr 204 IndiARivmaliPadthOuIOaY l Dat. errno 216 APPENDIX 7... Coll PrORRTAION REPO... 359 360 - 7 00057vt1 Covance 6329-224 - OO OO OO wTeM2 ABSTRACT `The purpose of this 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 4 weeks. Six groups of Crl:CD(SD) IGS BR rats were exposed to N-MeFOSE in the diet for at least 4 weeks. Each group contained six males and six females. The dose levels were 0 (controlgroup), 1, 10, 30, 100,and 500ppm. Food was provided ad libitum, except when animals were fasted. Water was provided adlibitum. The animals were observed twice daily (a.m. and p.m.) for mortality and : moribundity. At least once each week, each animal was removed from its cage and `examined for abnormalities and signs of toxicity. Body weights and food consumption data were collected twice weekly for at least 4 weeks. Blood and urine samples were collected for hematology, clinical chemistry, and urinalysis tests from all surviving animals - afte4r weeksoftreatment.Afterat least 4 weeksoftreatment, blood was also collected fromall surviving animals for serum perfluorooctane sulfonic acid (PFOS) analyses. On Day 32, the 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 iver were collectedfromall surviving animals for palmitoyl-CoA oxidase activity (byCovance) and proliferation cell nuclear antigen (PCNA) evaluation and liver PFOS analysis (by the Sponsor). The animal that died on test was also necropsied, but organ weights were not recorded. Microscopic ~ examinations were done on tissues fromeachanimal One animal given 500 ppm N-MeFOSE died on Day25. All other animals survived to the scheduled sacrifice. No clinical observations were noted that were considered to represent primary effects of the test material. Lower body weight, body weight gains, and food - consumption in animals given 500 ppm were test material-related. Dietary administration of N-MeFOSE ata dose levelof500 ppm for approximately 4 weeks was associated with several effects on clinical pathology test results including - moderately lower red blood cell count, hemoglobin, and hematocrit; lower absolute osinophil count; mildly higher urea nitrogen; moderately higher albumin and mildly to . 8 000572 CovanMcTe 8633291-42224 moderately lower globulin; markedly lower cholesterol; mildly to moderately higher total bilirubin, aspartate aminotransferase, and alanine aminotransferase; andmoderatelyhigher hepatic palmitoyl-CoA oxidase. Of uncertain relationship to administration of N-MeFOSE . were lowerurine pHformalesfed 500 ppmand smalldifferencesforanimalsfed 100ppm including higher urea nitrogen, alburnin, and alanine aminotransferase (males only) and lower cholesterol.Ofthe differences for animals fed 100 ppm, only the difference for alanine aminotransferase was statistically significant, - Mean absolute and relative (to brainandbody) liver weightsfor all males and females given 100 and 500 ppm N-MeFOSE were significantly increased over control values, and these variations were regarded as treatment-related. All additional mean organ weights, `which varied significantly from control values, were considered to be within the range or - normal biologic variation, or were manifestations of light weight loss associated with physiologic stress. Treatment-related microscopic findings were restricted to the liver and consisted of `minimal to severe centrilobular hepatocellular hypertrophy. Hypertrophic changes were slightly more severe in males; and at the high-dose level, liver cell swelling in several males was associated with acute necrosis. Necrosis was attributed to restricted blood supply ischemia) from swollen liver cells rather than direct hepatocellular damage. Additional microscopic findings were consistent with common, spontaneous alterations in laboratory rats or changes associated with stress, nutritional deprivation,or tissue processing artifact. Based on the resultsofthis study, dietary administration of N-MeFOSE to Crl:CDSD) 1GS BR rats for at least 4 weeks resulted in adverse effects at a dose levelof500 ppm. `The no-observable-adverse-effect level was determined to be 100 ppm. - 9 at 000573 PURPOSE CovanMcTe 329-224 "The purpose ofthis study was to assess the toxicity of the test material, i} N-Methyl Perfluorooctanesulfonamido Ethanol (N-McFOSE, T-6314) when administered inthediet to ats for atleast 4 weeks. REGULATORY COMPLIANCE "The studywasconducted in compliance with the Food and Drug Administration Good Laboratory Practice Regulations as set forthinTile 21 of the US CodeofFederal Regulations, Part 58, issued December 22, 1978 (effective June 20, 1979), and with any - applicable amendments. TEST MATERIAL AND VEHICLE Test Material The test material, N-Methyl Perfluorooctanesulforamido Ethanol (MeFOSE, T-6314), Lot No. F-11364, is anoffwhite powder. It was received at Covance on August 3, 1995. - Information on synthesis methods, stability, purity, composition, or other characteristics that defin the test materia s on ile with the Sponsor "The test material was stored at room temperature. Vehicle "The vehicle was acetone (Spectrum Quality Products Inc., Gardena, California), Lot - No. LH0253 (expiration date: June 2000). It was received at Covance on June 23, 1997. "The vehicle was stored at room temperature. - Information on synthesis methods, stability, puriy, composition, or other characteristics that define the acetone is on fle with the manufacturer. R 10 000574 CovanMcTe 6635291-42224 Reserve (Archive) Samples Areserve sample {approximately 5 g)ofthe test material wastakenand stored at room the Sponsor. temperature. These samples will be transferred to the Sponsor after authorization from Disposition `Remaining test material was retained for future use (see Protocol Deviations page for exception). TEST SYSTEM `Test Animal Male and female Crl:CD(SD) IGS BR rats were obtained from the Raleigh, North Carolina, facility of Charles River Laboratories, Inc., on June 2, 1998. Theanimalswere - 31 to 37 days old at initiation of treatment. The males weighed from 144 to 194 g, and the females weighed from 114 to 158 g at initiationoftreatment. - EIdaecnhtiafniicmaatliownas 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 R the microchip was coded with the permanent number. Alldata foran animal are recorded under these numbers. Acclimation Forty-one males and 41 females were received on June 2, 1998, and acclimated in Animal Room 349 for 10 days before initiation of treatment. In general, animals in this shipment appeared healthy. During acclimation, the animals were examined for abnormalities indicative ofhealth problems, and body weights were recordedfor all animals at - randomization. - u 00057v5e CovanMce 6T36291-42226 Housing and Maintenance Animal Room 349 was used for this study. Environmental controls for the animal room were set to maintain 18 to 26C, a relative humidity of 30 to 70%, and a N 12-hour light/12-hour dark cycle. Variations from these conditionsare documented in the data and are considered to have had no effect on the outcome of the study. `The animals were housed individually (except for the first 7 days of acclimation when animals were group-housed) in stainless steel, screen-bottom cages. Certified rodent diet #5002 meal, (PMI Nutrition International) was provided ad libitum, except whenanimals were fasted. The diet is routinely analyzedbythe manufacturer for nutritional components and environmental contaminants. The results are on file with - Covance-Madison. `Water was providedadlibitum. Samples of thewaterare 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 (fivemalesand five females) were removed from the `studyroomand used for training procedures. : Justification Rats historically 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 June 8, 1998, and - Protocol Amendment No. 1. The protocol, protocol amendment, and protocol deviations are in Appendix 1. - 12 0005.76 CovanMcTe.6362391-24224 `The animals were examinedby a laboratory animal veterinarian on June 10, 1998, and found to be suitable for study consideration. Selection ofanimals for the study was based on clinical observations, body weights, 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 42 standard deviationsofthe mean weight. Group mean body weights were analyzed `using Levene's test for homogeneity of variance at the 5.0% probability level and found to `behomogeneous. Animals were assigned to the study according to the following design: Group Designations and Dietary Levels Group M`aNluember of AFneimmaallse (DpieptmarMyeLFeOvSe)ls* _ 1 (Control) 2 (Low) 6 6 6 6 0 1 3 (Low-Mid) 6 6 10 4 (Mid) 6 6 30 5 (Mid-High) 6 6 100 6 (High) 6 6 500 ~ a Dose levels were expressed as ppm of MeFOS. b The control animals received thebasaldiet only. Dose Preparation - Dietary concentrations were based on the test material as supplied. Diets were prepared pretest for homogeneity, and twice for use in the in-life phase. PreparationofDiet for Group 1. The appropriate amountofdiet was weighed into a - labeled container, and acetone was added at the same concentratiaosn the high-dose concentration. The combinationofthe diet and acetone was then mixed for 15 minutes. PreparationofDiet for Group 6. Each dose level was prepared independently. A ~ specified amountofdiet was weighed into a labeled Hobart mixing bowl. The required amount of test material was weighed and transferred into a labeled container. Approximately 5 mL of acetone was added to the container and mixed manually. - 13 000577 CovanIcMeT6-36293-124224 Increments of approximately 5mLacetone 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 bow, overlaid with _ a portionofdiet from the mixing bow, 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 contents of the mixing bowl were: thoroughly mixed. PreparationofDietsfor Groups 2 through 5. Each dose level was prepared independently. A specified amountofdiet was weighed into a labeled Hobart mixing `bowl and a specified amountofdietforGroup 6 was weighed for each diet. A pocket was formed in the feed in the mixing bowl and the amountofdit for Group 6 was transferred - to the pocket. The contentsofthe mixing bowl were thoroughly mixed for 10 minutes. `Samples for doseanalyseswere taken directly from the mixing bowl. - `The prepared test diets were stored at room temperature in covered containers until dispensed into feeding jars . Retention Samples Samples (approximately 100 g) were taken from each dose preparation sampled for dose: analyses and stored at room temperature. These samples were discarded on December 9, 1998 (see Protocol Deviations page for exception). 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 Sponsorand - validated by Covance. Homogeneity was determined for all test material dose preparations once pretest. One sample cach (approximately 100 g) from the top, middle, and bottom of the dose - preparations mixed for homogeneity analyses was collected, divided into three subsamples for extraction and analyzed for test material content. All samples were stored at room - 14 . 000578 CovanMceT6832i042224 temperature until analyzed. In addition, one sample each (approximately 100 g) from the top, middie, and bottomofthe 1-ppm dose preparation mixed for the in-life portionofthe study was collected, divided into three subsamples for extraction and analysis, and analyzed for test material content. To evaluate the stability of the test material in the diet, four setsofsamples (approximately 100 g each) were taken from the highest test material concentration used for the study. One set was analyzed on the day of mixing; asecond sample of the - high-dose preparation was stored at room temperature for at least 19 days, then analyzed. "The third sample of the high-dose preparation was stored at room temperature for 32 days, then analyzed. Another sampleofthe high-dose preparation was stored at room temperaturefor62 days, then analyzed. The remaining sample of the high-dose - preparation was stored in a freezer set to maintain -10 to -30C for 8 weeks, then analyzed. In addition, three samples (approximately 100 g each) were taken from the Tow-dose level preparation mixed for the in-life portionofthe study and analyzed. Homogeneity samples collected from the middle of the low-dose preparation were " analyzed on the dayofmixing andusedas the baseline value. Samples (approximately 100 g)fromal dose preparations were analyzed. The homogeneity sample collected from the middle of the low-dose level preparation mixed for Weeks 1 through 4 was used for dose confirmation. All samples were stored at room temperature until analyzed. Method of Administration - Dietary admixture was used because the potential route of exposure in humansisoral. `The dose preparations were administered ad libitum for at least 4 weeks, unless otherwise specified. : " 000579 CovanMcTe 6633291-42224 Clinical Observations `The animals were observed twice daily (a.m. and p.m.) for mortality and moribundity. Signsofpoor health or abnormal behavior were recorded as they were observed. At least _ once each week, each animal was removed from ts cage and examined. Any unusual or abnormal findings were recorded. Body Weights Individual body weight data were recorded on the first dayoftreatment and twice weekly thereafter. - Food Consumption Individual food consumption data were recorded twice weekly during treatment. - Serum Perfluorooctane Sulfonic Acid Level (PFOS) Determination After at leas4t weeks of treatment, animals were fasted overnight, anesthetized with carbon dioxide, and blood (as mich as possible) was collected via cardiac puncture from all surviving animals. All samples were collected without anticoagulant, allowed to clot, . at room temperature and centrifuged. Serum was harvested and stored in a freezer set to `maintain -60 to -80C until packed on dry ice and shipped to the Sponsor for analyses. `The samples were analyzed for PROS. Results ofanalyses wil be reported separateblyy the Sponsor. Clinical Pathology After at least 4 weeks of treatment, blood and urine samples were collected from cach animal. Animals were fasted overnight, and urine was collected chilled (approximately 16 hours before blood sampling); water was provided ad libitum. The animals were anesthetized with carbon dioxide; blood was collected via cardiac puncture. Potassiom EDTA was used as anticoagulant for hematology tests. Animals were bled in random order. The following were evaluated. - 16 000550 Covance 6329-224. _-- Mees Hematology red blood cell (erythrocyte) count white blood cell (leukocyte) count hemoglobin differential blood cell count hematocrit segmented neutrophil count 3 mean corpuscular volume lymphocyte count mean corpuscular hemoglobin monocyte count mean corpuscular hemoglobin concentration eosinophil count platelet count basophil count - Reticulocyte count smears were made and held for possible future examination. Clinical Chemistry glucose alanine aminotransferase - urea nitrogen `gamma glutamyltransferase creatinine aspartate aminotransferase. total protein calcium albumin inorganic phosphorus globulin sodium - cholesterol potassium total bilirubin chloride Urinalysis appearance glucose - volume ketones. specific gravity bilirubin PH blood protein microscopic examination of urobilinogen sediment `The remainingurine (upto 10mL)foreachaniwamssatorledin afreseettozmaientarin ~1010 -30C. Samples were packed on dry ice and shipped to the Sponsor. Urine samples will be stored by the Sponsor for possible future analysis. Necropsy A necropsy was done on the animal that was found dead. After at least 4 weeks of . treatment, all surviving animals were fasted overnight, anesthetized with carbon dioxide, bled for clinical pathology tests and serum PFOS samples, weighed, exsanguinated, and necropsied. Animals were necropsied in random order. - " 0005521 CovanMcTe-663239124224 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. Organ Weights At the scheduled sacrifice, the following organs (when present) were weighed; paired organs were weighed together (see Protocol Deviations for exception): : adrenal (2) ovary (2) brain spleen epididymis (2) testis (2) Kidney (2) thymus ~ liver thyroid (2) with parathyroid Organ-to-body weight percentages and organ-to-brain weight ratios were calculated. - Cell Proliferation Tissue Collection and Immunohistochemical Evaluation At the scheduled sacrifice, representative samples of left lateral lobe of the liver were collected fromeach animal and preserved in zinc formalin. Afr fixation, samples were `embedded in paraffin and shipped to Pathology Associates International for proliferation - cell nuclear antigen (PCNA) evaluation. Resultsofthe evaluation are in Appendix 7. Palmitoyl-CoA Oxidase Tissue Collection and Analyses _ At the scheduled sacrifice, a sample (approximately 500 mg)ofthe right lateral lobe of liver was collected from each animal, flash-frozen in liquid nitrogen, and stored in a freezer set to maintain -60 to -80C until analyzed for palmitoyl-CoA oxidase activity. - Liver PFOS Analyses A portion ofthe liver was collected from each animal at the scheduled sacrifice 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. Resultsofthese analyses will be reported - separately by the Sponsor. - I 0005352 CovanMcTe -636239-124242. `Tissue Preservation `The following tissues (when presen) from each animal were collected and preserved in 10% neutral-buffered formalin: adrenal (2) ovary (2) aorta pancreas brain pituitary cecum prostate . cervix rectum colon salivary gland mandibular (2)] duodenum sciatic nerve esophagus seminal vesicles epididymis (2) skeletal muscle (thigh) - eye (2) skin femur with bone marrow (articular spinal cord (cervical, mid-thoracic, surfaceof the distal end) and lumbar) Harderian gland spleen heart sternum with bone marrow . ileum with Peyer's patch (lymphoid stomach aggregate) testis [(2) preserved in Bouin's fixative] jejunum thymus. kidney (2) thyroid (2) with parathyroid lesions. trachea liver urinary bladder - lung with mainstem bronchi uterus lymph nodes (mesenteric and vagina mandibular) Zymbal's gland mammary glands (females only) : Tissues were shipped to Pathology Associates International for processing. The adrenals, brain, eyes, kidneys, liver, mesenteric lymph node, pancreas, spleen, testes, and ovaries from each animal were embeddedinparaffin, sectioned, and stained with hematoxylin and eosin (see Protocol Deviations). The tissue slides were examined microscopically by - Dr. Richard H. Bruner, DVM, DAVCP of Pathology Associates International. Bone marrow smearsfromthe femur of each animala the scheduled sacrifice were prepared, stained with Wright's stain, and retained for possible examination. 2 19 000583 CovanMcTe -6362391-24224 Statistical Analyses Levene' test was done to test for variance homogeneity. In the caseofheterogeneity of variance atp s 0.05, transformations were used to stabilize the variance. Comparison - tests took variance heterogeneity into consideration. One-way analysisofvariance (ANOVA) was used (if applicable) to analyze body weights, body weight changes, food consumption, continuous clinical pathology values, and organ ~ weight data. If the ANOVA was significant, Dunnett's t-test was used for control versus treated group comparisons. Group comparisons (Groups 2 through 6 versus Group 1) were evaluated at the 5.0%, two-tailed probability level. Only data collected on or after the first day of : treatment was analyzed statistically. RECORD RETENTION All raw data, documentation, records, protocol, and specimens generated as aresult of this study willbearchived in the storage facilitesofCovance-Madison fora period of 1 year. One year after the submissionofthe final report, the Sponsor will determine the - final disposition of the materials. All raw data stored on magnetic media, the protocol and protocol amendments, study correspondence, andanoriginal copy of the final report will be retained by Covance-Madison. ~ PCNA evaluation data, paraffin blocks, and tissue slides will be retained by Pathology Associates International. Liver, urine, and serum samples sent to the Sponsorand analysis data will be retained by the Sponsor. - 20 000584 - RESULTS Covance 6329-224 wTeM2 Dose Analyses Results of the homogeneity, stability, and dose preparation analyses are in Tables 1 through 3. Mean values of the homogeneity analyses ranged from 102-268%, 95.9-102%, 93.7:97.0%, 94.4-95.8%, and 94.0-101%ofthe theoretical concentrations for the diets containing 1, 10, 30, 100, and the analysesofdiets mixed on 500 ppm June 11, N-MeFOSE, 1998, for the respectively. Becausetheresults diet containing | ppm were high of (118%, 268%, and 129%), they were reanalyzed. The resultsofthereanalyses were 116%, 114%, and 102%. These results indicate that the mixing procedure produced a - homogeneous distribution of the test material in the dose preparations. Resultsofstability analyses of samples stored for 19, 32, and 62 days at room temperature and 8 weeks under frozen conditions, indicated that the mean concentrations were - 190.2%, 83.4%, 70.6% and 97.2%, respectively,ofthe theoretical concentrations of 500 ppm. Resultsof stability analyses of samplesfromthe 1-ppm theoretical concentration, indicated that the mean concentrations were 132% and 72.6% for the diet preparations mixed pretest and the in-life portion of the study, respectively. Therefore, the dose preparations were stable for 62 days at room temperature storage and weeks under frozen conditions for mean concentration of 500 and 1 ppm, respectively. `The mean concentrations of the dose preparation analyses for all levels ranged from 63.3% 10 114%of the theoretical concentrations. These data indicate that the levels of . N-methyl perfluorooctanesulfonamido ethanol (N-MeFOSE, T-6314) in the dose preparations were acceptable only for dose levels of 10, 30 100, and 500 ppm. - Clinical Observations and Survival Clinical observations are summarized in Table 4; individual data are in Appendix 2. Individual animal fate data are also in Appendix 2. . 2 00535 CovanMceT663321924224 One animal given 500 ppm N-McFOSE died on Day 25. This animal exhibited severe hepatocellular hypertrophy and moderately-severe livernecrosisat microscopic exam. All other animals survived to the scheduled sacrifice. : No clinical observations were noted that were considered effectsofthe test material. Body Weights and Body Weight Changes Body weight and body weight change data are summarized in Tables 5 and 6; individual data are in Appendix 3. Although not statistically significant, animals given 1, 10, 30, or 100ppm N-MeFOSE had - lower mean body weights than those of controls for mostofthe study. Test material related lower body weight and body weight gains were noted for animals given 500 ppm. For males and females given 500 ppm, statistically lower body weights were apparent beginning on Days 8 and 11, respectively, and continuing through the end of study. Mean - body weight gains for animals given 500 ppm were lower than thoseofcontrol animals throughout the study. _ FFoooodd cCoonnssuummptpitoinondata are summarized in Table 7; individual data are in Appendix 4. Statistically test material related decreases in food consumption were noted in males given 500 ppm throughout the study and in females given 500 ppm beginning on Day4 and continuing throughout the study. The overall mean food consumption for mals fed the 100- or 500-ppm concentration was significantly reduced compared with thatofthe controls, and the overall mean food consumption for females fed the 00-ppm concentration was significantly reduced compared with that of the controls. There were no significant food consumption differences noted in animals given 1, 10 or 30 ppm N-MeFOSE. z2 00055"6 CovanMcTe-663239124224 `Test Material Consumption Test material consumption data are summarizedinTable 8. Individualdataare in Appendix 4. Animals were fed diets containing 1, 10, 30, 100, or 500 ppm. N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314). The mean amounts oftest material consumed by the animals in these groups were 0.087 to 0.140 mg/kg/day; 0.816 to 1.325 mg/kg/day; 2.432 to 4.030 mg/kg/day; 7.768 10 - 13.192 mg/kg/day; and 35.168 to 62.612 mg/kg/day for males, respectively. Values for females were 0.096 to 0.147 mg/kg/day; 0.848 to 1.399 mg/kg/day; 2.612 to 4.124 mg/kg/day; 8.485 to 13.802; and 38.527 10 62.217, respectively. The overall test `material consumption ranged from 0.105 to 44.590 mg/kg/day for themales and = 0.111 to 45.050 mg/kg/day for the females. Serum Perfluorooctane Sulfonic Acid Level (PFOS) Determination - Results of serum PFOS level determination will be reported separately by the Sponsor. Clinical Pathology ~ Hematology, coagulation, clinical chemistry and urinalysis data are summarized in Tables 9 through 11; individual data are in Appendix 5. The Pathology Report contains a discussion of the data. Dietary administration of N-McFOSE at a dose levelof500 ppm for approximately - 4 weeks was associated with several effects on clinical pathology test results including `moderately lowerred blood cell count, hemoglobin, and hematocrit; lower absolute: eosinophil count; mildly higher urea nitrogen; moderately higher albumin and mildly to `moderately lower globulin; markedly lower cholesterol; `mildly to moderately higher total `bilirubin, aspartate aminotransferase, and alanine aminotransferase; and moderately higher hepatic palmitoyl-CoA oxidase. Ofuncertain relationship to administration of N-MeFOSE were lower urine pH for males fed 500 ppm and small differences for animals fed 100 ppm including higher urea nitrogen, albumin, and alanine aminotransferase (males only) and - lower cholesterol. Of the differences for animals fed 100 ppm, only the difference for alanine aminotransferase was statistically significant. 000587 CovanMcTe 6.38291-42224 Cell Proliferation Tissue Collection and Immunohistochemical Evaluation Resultsofcell proliferation and immunohistochemical evaluation provided by Pathology `Associates International are 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. = TAenramtionmailcbaoldPyawtehioglhotgs,y absolute organ weights, organ-to-body weight percentages, and organ-to-brain weight ratios are summarized in Tables 12 through 14; incidences of `macroscopic and microscopic observations are summarizedin Tables 15 and 16; the - incidence of severity of microscopic observations is summarized in Table 17. Individual data are in Appendix 6. The Pathology Report contains a discussionofthe data. Mean absolute and relative (to brain and body) liver weightsfor all males and females . given 100 and 500 ppm N-MeFOSE were significantly increased over control values, and these variations were regarded as treatment-related. All additional mean organ weights, which varied significantly from control values, were considered to be within the range or `normal biologic variation, or were manifestations of slight weight loss associated with physiologic stress. Treatment-related microscopic findings were restricted to theliverand consisted of `minimal to severe centrilobular hepatocellular hypertrophy. Hypertrophic changes were slightly more severe in males; and at the high-dose level, liver cell swelling in several males - was associated with acute necrosis. Necrosis was attributed to restricted blood supply (ischemia) from swollen iver cells rather than direct hepatocellular damage. Additional microscopic findings were consistent with common, spontaneous alterations in laboratory rats or changes associated with stress, nutritional deprivation, or tissue processing artifact - u < 000588 CovanMcTe 6-3B291-24242. CONCLUSIONS Based on the resultsofthis study, dietary administrationofN-MeFOSE, T-6314 to . Crl:CDSD) IGS BR rats for at least 4 weeks resultedinadverse effects at adose level of 500 ppm. The no-obscrvable-adverse-effect level was determined to be 100 ppm. . 2 000539 Covance 6329-224 ee ree - SIGNATURES 1 4: Nii van Bruce-Konuah ~ SCtouvdayncCeooLradbionraattoorries Inc. July 5, 2000 Date - Study Director Covance Laboratories Inc. 26 - 000539 CovanMceT.63B291-24242. 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 `CSatlaitifsotrincis,a ((e1d9s6.0))I.. Olkin et al., Ch. 25, pp. 278-292, Stanford University Press: Stanford, - ``EWxipneerr,imBe.ntJ.a,l"DDeessiiggnn, aSnedcoAnnadlEyds.i,soCfh.Si3n,gplpe.-F1a4c9t-o2r6E0x,peMrciGmreanwt-sH,i"lSlta:tiNsteicwalYPorrikn,ciplesin New York (19712). - `SWeicnoern,dBE.d.J,., C"hA.na1l0y,sipsp.of75C2o-v8a1r2ia,nMcceG,"rSatwa-tHiistlilc:alPNreinwciYpolerski,nNEexwpYeorrikm(e1n97Dt1eabs)li.gn, . 27 000531 PATHOLOGY REPORT CovanMcTe 6-36291-42224 . SUMMARY `The purposeofthis study was to assess the toxicityofthe test material, N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) when administered in the diet to rats for at least 4 weeks. The test material was administered at dose levels of 1, 10, 30, - 100, and 500 ppm. Dietary administration of N-MeFOSE at a dose level of 500 ppm for approximately 4 weeks was associated with several effects on clinical pathology test results including - `moderately lower red blood cell count, hemoglobin, and hematocrit; lower absolute eosinophil count; milly higher urea nitrogen; moderately higher albumin and mildly to `moderately lower globulin; markedly lower cholesterol; mildly to moderately higher total bilirubin, aspartate aminotransferase, and alanine aminotransferase; and moderately higher - hepatic palmitoyl-CoA oxidase. Of uncertain relationship to administration of N-MeFOSE were lower urine pH for males fed 500ppm and small differencesforanimals fed 100 ppm including higher urea nitrogen, albumin, and alanine aminotransferase (males only) and lower cholesterol. Ofthe differences for animals fed 100 ppm,onlythe difference for _ alanine aminotransferase was statistically significant. "Treatment-related microscopic findings were restricted 10 the liver and consisted of `minimal to severe centrilobular hepatocellular hypertrophy. Hypertrophic changes were slightly more severeinmales; and at the high-dose level, liver cell swelling in several males - was associated with acute necrosis. Necrosis was attributed to restricted blood supply (ischemia) from swollen liver cells rather than direct hepatocellular damage. Additional `microscopic findings were consistent with common, spontaneous alterations in laboratory rats or changes associated with stress, nutritional deprivation, or tissue processing artifact. 2 000532 CovanMceT683219-42224 METHODS A Six the groups of Crl:CD(SD) IGS BR rats (six animals/sex/group) were fed test material at a dose levelof O (control groups fed basal diet only), 1, diets containing 10, 30, 100, or 500 ppm. Before the schedule sacrifice and necropsy during Week 5, blood and urine were collected for hematology, clinical chemistry, and urinalysis tests. At necropsy, macroscopic ofbixsaetrivvaetaisonsspewceifrieedrebcyortdheedp,rootrogcaonl.weIinghatdsdiwteiorne, osbatmapilneesd,oafnldivteirswseuersewceorlelepcltaedcefdorin evaluation ofhepatocellular proliferation (by Pathology Associates International), determination of hepatic palmitoyl-CoA oxidase activity (by Covance), and analysis for & 'PFOS and metabolites (by the Sponsor). The animal that died on test was also necropsied, but organweightsand liver samples for the special analyses were not obtained. Microscopic examinations were done on the following tissues from cach animal: adrenals, brain, eyes, kidneys, liver, mesenteric lymph node, pancreas, spleen, testes, - ovaries, and lesions. `Statistically significant differences cited in the Results and Discussion section are based on comparisons between thecontroland treated groups. RESULTS AND DISCUSSION Mortality Animal No, C94523 given 500 ppm N-MeFOSE died on Day 25. The remaining animals survived to the scheduled sacrifice. - Clinical Pathology Individual values for hematology, clinical chemistry, urinalysis, and hepatic palmitoyl-CoA oxidase are in Appendix 5. Mean values for each group and the results of statistical `comparisons are in Table9s through 11. . 2 000533 CovanMceT6-36291-42228 Week 5. There were several statistically significaonrt otherwise notable differences for clinical pathology results between control and treated animals; only oneofthe statistically significant differences affected animals fed less than 500 ppm. Differences considered to . beeffects of the test material were lower red blood cell count, hemoglobin, and hematocrit, lower absolute eosinophil count; higher urea nitrogen; higher albumin and lower globulin; lower cholesterol; higher total bilirubin, aspartate aminotransferase, and alanine aminotransferase; and higher hepatic palmitoyl-CoA oxidase. With the exception of lower globulin andhigheraspartate aminotransferase for the females, these differences wweerreestlaotwisetricuarlilnyesipgHniffiocramnat.lesOffeudnc5e0r0tapipnmrealnadtisonmsahilpdtioffaedrmeinnciesstfroartiaonnimofalNs-fMeed F1O00SEppm including higher urea nitrogen, albumin, and alanine aminotransferase (males only) and lower cholesterol. Ofthe differences for animals fed 100 ppm,onlythe minor difference = for alanine aminotransferase was statistically significant. At dose levels below 100 ppm, clinical pathology results were unaffected by treatment. The effects on the erythrocyte parameters (moderately decreased), eosinophil count - (decreased), and globulin (mildly to moderately decreased) were consistentwithanimals in relatively poor health, failing to gain weight appropriately. Mildlyhigherurea nitrogen and moderately higher albumin were most consistent with mild dehydration and indicated the effects on other parameters (e.g., the erythrocyte parameters) might have been greater if the animals were normovolemic. The mild to moderate effects on alanine aminotransferase, aspartate aminotransferase, and total bilirubin were indicativeofthe hepatocellular degeneration. The effect on cholesterol was marked, but the mechanism for change was not apparent. Moderately higher hepatic palmitoyl-CoA oxidase was | indicativeofperoxisomal proliferation. Anatomical Pathology - Individual anatomical pathology data are in Appendix 6. Individual absolute organ weights, organ-to-body weight percentages, and organ-to-brain weight ratios are also in Appendix 6. Mean values for absolute and relative organ weights for eachgroupare in Tables 12 through 14. Incidence summaries of the macroscopic and microscopic observations are in Tables 15 and 16; the incidence of severity of selected microscopic - observations is summarized in Table 17. 000534 CovanMceT663321924224 Unscheduled Death. Animal No, C94523 given 500 ppm N-MeFOSE died on Day 25. Microscopically, this animal exhibited severe hepatocellular hypertrophy and `moderately-severe liver necrosis. `Terminal Sacrifice Organ Weights. Mean absolute and relative (to brain and body) liver weights for all `males and females given 100 and 500ppm N-McFOSE were significantly increased over `comnetarnoolrvgaalnuewse,iaghntds,thwehseicvharviaartiieodnsswigenrieficraengtalrydferdoamsctornetartomlenvta-lrueelsa,twede.reAlclonasdiddietiroendalto be within the range or normal biologic variation, or were manifestations of slight weight loss associated with physiologic stress. Macroscopic Observations. Treatment-related, gross changes were restricted 0 the liver and stomach of males given 500 ppm N-MeFOSE. Animal Nos. C94521 and C94522 displayed enlarged livers, and in Animal No. C94522, hepatic enlargement - `was associated with discoloration (diffusely brown with light areas). Microscopically, liver changes were attributed to hepatocellular hypertrophy and necrosis. In addition to liver changes, dark or red mucosal areas were present in the glandular stomach ofAnimal Nos. 94521and C94526 given 500 ppm N-MeFOSE and in the glandular stomach of one control male (Animal No. C94493). Gastricareaswere compatible with early "stress" `erosions or ulcers. Microscopic Observations. Treatment-related microscopic changes were restricted to the liver of both sexes and consisted of hypertrophy (swelling)ofliver cells in centrilobular regions ofthe hepatic lobules. Occasionally, hypertrophic liver cells were associated with acute hepatic necrosis. Hepatocellular hypertrophy was not observed in controls or in animals given 1 or 10 ppm N-MeFOSE. At higher dose levels, hypertrophic changes were slightly more prevalent and severe in males, and at dose levels of 30, 100, and - 500 ppm N-MeFOSE, all males displayed minimal to slight, moderate to moderately-severe, and moderately-severe to severe liver cell hypertrophy, respectively. Liver cell hypertrophy was not observed in females given 30 ppm N-MeFOSE; however, at dose levels of 100 and 500 ppm N-MeFOSE, hepatocellular hypertrophy in females - ranged from slight to moderate and from moderate to moderately-severe, respectively. Hepatocellular hypertrophy in both sexes was compatible with the proliferation of 31 000595 Come su9221 cytoplasmic smooth endoplasmic reticulum. Although at lower doses hypertrophy was restricted to centrilobular regions,atthehigh-dose level, hypertrophic hepatocytes often extended into panlobular regions. Inadditiontohypertrophicchanges,moderate livercellnecrosiswas observedin two `males given 500ppm. Necwr asao ttrs ibui teds tolocalischemiaassocwiithalitverecedll swelling and occlusionofregional blood supplies. Minimal hepatic necrosis in oneofsix `males given 10 or 100 ppmand in oneofsix females given 30 ppm was consistent with - commonhepaticalterations inlaboratoryrats. All othermicroscopicfindingswere consistent with common spontancous alterations in laboratory rats, including changes related to stress, nutritional status or tissue processing artifact. [aX BH fsa - Robert L. Hall, DVM, PhD ite, Diplomate, ACVP (Clinical Pathology) Richard H. Bruner, DVM Diplomate, ACVP Pathology Associates International 32 - 000596 CovanMcTe 6.38291-42224 (COMMENTS ON THE DATA `dVaatraiionusthmiosdsetludsyo.fBcaelccauulasteordsi,ffceormenptutmeordse,lsanrdoucnodmpouftfeorr ptrruongcraatmesnwuemrbeerussedidffteoreanntallyy,ze - vslailguhetslyinfrsoommethtoasbeleisn (oct.hge.,r mtaebalness,, fsrtoanmdianrddivdiedvuiaaltliyoncsa,lcourlaitneddivdiadtuaa,lovralfureos)m mstaatyisdtiifcfaelr analysis data. Neither the integrity nor the interpretation of thedatawas affectedbythese differences. - rTefhleenctusmtbheernoufamnbiemroalfsalniismteadlisnastsheighneedatodeoiafcnthhggerosuupmamtatrhye tsatbarltesfofortchleisntiucdayl.observations cTohnedistuimonmawraystoabbsleerfvoerdclwiintihcaolutobrseegravradtitoonstihnedsipceactieftichenatnuurmeb,esrevoefraintyi,mraelvserfsiobrilwihtiy,ch a - number of incidences/animal, or the length of time the condition persisted. Only observations other than normal are indicated on the summary clinical observations tables. . cEaocmhmeannitm"alAnwiimtahlobhassernvoatsiiognnsifriecacnotrdfienddiansg"s"Noirnmdiacla"tetdhroonutghheouitndtihveidsutauldcylihnaiscatlhe observations tables. ~ Tinhdeicsapteecdifwiictdhetaai"lCs"focrancboemmfeonutnsd ianttthhee iennddivoifdueaalcchlignricoaulpobfosrerevaacthiosnexs.tables that are Tsthaertdoafyaosftiundiytiwateieonko(fc.tgr.e,aatmbeondtyiswe"iDgahyt 1r,eWcoeredked1o."n DBaoydy1 wiesicgohntsiddaetraeadreaeWnteeerked1 abtotdhye wweeiigghhtt, cahabnogdey dwaetiaghatrerceaclocrudleadteodnfDroamy t8heisfcirosntsdiadyeroefdtaheWseteukdy2wbeoedkytwoetihgehtf)r.st Bdaoydyof - the are following indicated study in the week tables (c.g., with Week 1 values are calculated from Day 1 through the day being the first dayofthe following week 7) and (mea.tge.riWaleceokns1uvmapltuieosnaraereincdailccautleadteadsf"rDoamyth8"e).firsWtedeakyloyftfohoedsctoundsyuwmepetikotnoatnhde tfeirsstt day of the following study week (c.g., Week 1 values are calculated from Day 1 through 7). `siTghneivfiaclaunetsffiogrurseusmtmhaarnyisaanpdprionpdriivaitduealfotrestthemadtaetraiadluecotnosluimmpittaitoinonasreofretphoerdtaetdawciotlhlemcotrieo:n `and reporting software. - 3 000597 CovanMceT6633291-42224 COMMENTS ON THE DATA (Continued) Diet test material concentrations provided by the data collection system (PTS) are calculated to the nearest 0.1 ppm. These values are used for calculationof test material - consumption. For diet preparation purposes, the values are rounded to the appropriate accuracy for the balance used. Food consumption values are reported in whole numbers; however, PTS carries food consumption values to one place to the rightofthe decimal for 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 endofthe food consumption interval. Differences in the population size (N) on the summary tables for clinical and anatomical ~ pathology are explained on the individual data tables or the codes sheets. `The calculation for individual test material consumption is: Test material consumption = * Dic Concenaionsod Compton Foot Comampen Fes Body Weigh of ea (DuylBodyWeighGuey Foo] where: Daily Body Weight Gain = - TyoLLttBBooddyyWWeeiigghhttooffrearall.FDiaytfBodFyWeBigyhtWofeieghlof Er and: Day Factor = (Fond Consmpic StDy. DyfFi Body Weof rga C22onsrptiern and: Food ConsumFpotoidCoonnsuImnptteirovnalEn=d Day. ood Consumption Sat Day On pages 4 and 7ofthe protocol, the test material is indicated as MeFOS, T-6314 instead - of MeFOSE, T-6314. . H . 000523 CovanMceT633219-42224 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 Anatomical Pathology Note: The following listsof codes, abbreviations, and units are used by Covance. Some, but not necessarily al,ofthis information may be - needed for this report. - 3s 000539 CovanMcTe -6362391-24224 General Codes and Abbreviations WK N - Mean; MEAN SD; $.D.; STAND DEV; STANDARD DEV; sd * - = NA P c . UNSCHED DISPATCH TBW # co WNeuemkb.er ofmeasurements in a group. Arithmetic mean. Standard deviation. Grtohuepmemaenaonfsthseignciofnitcraonltlgyrdoiufpfe(reGnrtofurpom1) Noatvpal<ue0;.0n5o.t applicable; not present. CProemsemnet.nt found at the endofcach group for each sex. OUbnssecrhveadtuiloends.transferred from the in-lfe: `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. 36 0C0Es59 Cones 629224 ---- wm Codes for Clinical Pathology `GENERAL CODES - NS No sample QS/QNS NR Quantity not sufficient No repeat (sample volume not sufficient for repeat analysis) FS sc `Fibrin strands `Sample clotted - SH Slightly hemolyzed H SL Hemolyzed Slightly lipemic L Lipemic st Slightly icteric - 1 Icteric NF Animal not fasted u Unscheduled/moribund bleed DT/DOT `Animal died on test DB . El TE RE ~ EE SE Died during bleeding Technician judgment to repeat test "uTneacchcneipctaalbelrerodrat(ai,ns.tgr.u,meunntacocreptteachbnlieciiannsterrurmoerntthoatutrpeustu,ltssaimnple spilled, entry of invalid data) Recording error (recorded incorrect data, .g., wrong number, spelling error, incorrect date) Entry error (incorrect keyboard entry) `Sampling error PC Platelets clumped PD Platelets decreased PI - PL PA co Platelets increased Platelets large Platelets appear adequate Color interferes with test HB Heinz bodies observed PLASMO Plasmodium - NO AGG No aggregation FR Fractious UTD NO COAG Unable to determine No coagulation : 7 000661 CovanMcTe -6362391-24224 Codes for Clinical Pathology (Continued) RESULTS NOT INCLUDED IN STATISTICAL ANALYSES - Hemolyzed clinical chemistry or coagulation samples `Samples from animals at unscheduled intervals PArcotitvhartoemdbipanrttiiamletsh(rPoTm)bogprleaastteirntthiamnes50(PsTeTco)ngdrseaterthan 110 seconds Bleed times (BLETIME) greater than 30 minutes CODES FOR BLOOD CELL MORPHOLOGY - `pTohiekifloolclyotwoisnigs (scPaOlIeKw),aspoulsyecdhtroommaesaisau(rPeOtLhYe)d,eghryepeoocfharnoimsaosciyato(sHisYP(OA)N,ISoOr)b,asophilic stippling (BASTIP) or the presence of Howell-Jolly bodies (HJBODY), toxic neutrophils (TOXNEUT), or atypical lymphocytes (ATYPLYM): J Scale Degree --Pres-- ence . - Normal for the species. 1 Slight Not present Rare 2 Moderate 3 Marked Few Moderate 4 Not applicable Many URINE APPEARANCE - Color Clarity Miscellaneous - A Pale B Straw E Amber 1 Black 7 Clear F Brown P Blue/green K Hazy M Debris 0 Feces C Yellow G Red Q Blue L Cloudy D Dark yellow H Green R_ Orange , 38 060602 CovanIcMeT6-36293-124224 Codes for Clinical Pathology (Continued) URINE CHEMISTRY MULTISTIX STRIP - - Urine Glucose Urine Ketone Urine Blood ~ Negative + 100 mgldL ~ Negative + 5mgldl Negative + Small ++ 250 mg/dL +++ 500 mg/dl ++ 15 mgldL +++ 40 mg/dL ++ Moderate +++ Large - +++ 1,000 mg/dL abr 22000mgdl +++ 80 mg/dL Here 160 mgldl Urine Urobilinogen Urine Bilirubin . ~ 02mgldl + Imgdl ~ Negative + Small ++ 2mgldL + 4mg/dL ++ Moderate +++ Large +++ 8mgldl - (1 mg = approximately 1 Ehrlich unit) -- URINE SEDIMENT - Cells, Crystals, Casts, and Comments Bacteria A Amorphous urates B Amorphous phosphates Q Sperm R Fecal contamination 0 Not present 1 Few C Uric acid D Triple phosphates S Pinworm ova found T Pinwormlarvae found 2 Moderate 3 Many - E Calcium oxalate U Parasite ova found F Calcium carbonate G Granular casts H Hyaline casts I Cellular casts 0 Not present 11-5 per field - J Waxy casts K_ Unknown crystal 2 6-10 per field 3 1120 per field P_ Mucous threads 420 per field 3 000603 CovanMcTe 6633291-42224 Abbreviations and Units for Clinical Hematology Test Abbreviation (Units) Red blood cell count - Hemoglobin RBC (E6/UL or X10%4L) HGB (G/DL) Hematocrit Mean corpuscular volume HCT (%) MCV (FL) Mean corpuscular hemoglobin Mean corpuscular hemoglobin concentration MCH (PG) MCHC (%) - MPleataenleptlactoeulnett volume PLT (E3/UL or X10%4L) MPV (FL) Reticulocyte count Absolute reticulocyte count RETIC (%) RETIC (E3/UL or X10%4L) . Heinz body count Erythrocyte sedimentation rate HEINZ (%) ESR (MM/HR) PArcotitvhartoemdbipanrttiiamlethromboplastin time PT (SEC) PTT (SEC) `Thrombin time TT (SEC) Activated coagulation time Fibrinogen FABCRT ((MSEGC/)DL) Fibrin/fibrinogen degradation products FDP (UG/ML) Platelet aggregation Collagen PAGG/COL (%) Adenosine diphosphate | Alpha 2-antiplasmin PAGG/ADP (%) ANTIPLAS (%) Bleeding time Methemoglobin BLE TIME (SEC) METHGB (%) Plasma hemoglobin Myeloidlerythroid ratio PLA HGB (MG/DL) ME RATIO Estimated myeloid/erythroid ratio EST MEE RATIO - `DWifhfietreenbtlioaoldbcleololdcoceulnltcount WBC (E3/UL or X10%4L) Nucleated red blood cell count NRBC (/100 WBC) Corrected white blood cell count `Segmented neutrophil count COR WBC (E3/UL or X10%uL) N-SEG (E3/UL or X10%uL) and % = Band neutrophil count N-BAND (E3/UL or X10%uL) and % Lymphocyte count LYMPH (E3/UL or X10*/4L) and % Monocyte count MONO (E3/UL or X10%uL) and % `Eosinophil count EOSIN (E3/UL or X10/4L) and % Basophil count - Anisocytosis BASO (E3/UL or X10%4L) and % ANISO (-,1,2.3) Polychromasia POLY (123) 2 40 0C004 CovanMcTe 6-36291-24242. Abbreviations and Units for Clinical Hematology (Continued) Test Abbreviation (Units) Poikilocytosis 3 Hypochromasia POIK (12.3) HYPO (12.3) Howell-Jolly bodies Basophilic stippling. HIBODY (12.3.4) BASTIP (12.3) "Toxic neutrophils Atypical lymphocytes TAOTXYNPELUYTM ((112..23..344)) - `Aqueous white blood cell count (right eye) Aqueous white blood cell count (left eye) REYE (WBC/UL) LEYE (WBC/UL) a 000605 CovanIcMeT6-3289124224 Abbreviations and Units for Clinical Chemistry Test - GlUurecaosneitrogen Urea Creatinine Total protein Albumin - Globulin Albumin/globulin ratio Total bilirubin Direct bilirubin Indirect bilirubin . ChTroilgelsytceerriodle Urea nitrogen/creatinine ratio Total lipids PHhiogshp-hdoelnispiitdyslipoprotein cholesterol Low-density lipoprotein cholesterol AUsrpiacratcaitde aminotransferase Alanine aminotransferase . GAlakamlminaegplhuotsaphmayttarsaensferase 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) TCRPERAOT((GM/GDL/)DL) ALB (G/DL) GLOB (G/DL) A/G RATIO TBILI (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) AST/SGOT (IU/L) ALT/SGPT (IUL) GALGKT P(IHUO/LS)(IU/L) SDH (IU/L) LDH (IU/L) CK (UL) LAIMPYALSAES(EIU/(LI)U/L) PCOAO (IU/G) CA (MG/DL) PIOHNOCSA((MMGG//DDLL)) NA (MMOL/L) CKL(M(MMOMLO/LL/)L) MG (MEQ/L or MG/DL) ZN (MG/L or PPM) SFER ((UMGG//DLLo)r PPM) - 2 000696 CovanMceT632E9-2L24 Abbreviations and Units for Clinical Chemistry (Continued) Test . TEoxtcaelssiriornonbinding capacity PUenrbcoeuntndiriornonsabtiunrdaitniogncapacity RPleadsbmlaocohdolcienlelsctheroalsien:esterase - BraCianudchaotleinpeusttearmaesne Hippocampus Frontal cortex Cerebellum . BiScearrubmonhaetmeoglobin Serum bile acids FAevcearlabgielefeaccaildsweight . Fecal bile acids (calculation) OsElmeocltarloipthoyresis AAllpbhuam-1i-nglobulin Alpha-2-globulin - GBeatmamgalobgulloibnulin HLiogwh--ddeennssiittyy lliippoopprrootteeiinn Very-low-density lipoprotein InAsdurleinnocorticotropic hormone: Cortisol GTlruiicoadgoothnyronine - TChryeraotxiineneKinase isoenzymes. BB MB MM Abbreviation (Units) ETIXBFCE((UUGG//DDLL)) IBC (UG/DL) FE %SAT (%) CCHHEEPR ((MMUU/MMLL)) CCHAEUBD (PMUUT/M(LU)MOL/G) HFICPOPROTCEAXM((UUMMOOLL//GG)) CEREBELL (UMOL/G) SBIECRAHRGBB((MMMGO/LD/LL)) SBA (UMOLIL or MG/DL) FBA (UG/ML) FFCBCA W(MGGT/D(aGy)) SMO (MOSM/KG) EALB (G/DL) EA (GDL) EA2(G/DL) EE GBEATMAM(AG/(DGL/)DL) E-HDL (%) E-LDL (%) E-VLDL (%) AINCSTUHLI(NPG(MULU)ML) CGOLRUTCIASGOOLN(U(GP/GM/LM)L) T3 (NG/DL) T4 (UG/DL) CK-BB (UL) CCKK--MMBM((UULLL)) - " 000667 Covance 6329-224 - MTeM2 Abbreviations and Units for Clinical Urinalysis TUersitne volume AUbbVrOevLi(atMiLo)n (Units) - 8 hour urine volume Specific gravity 8 HR VOL (ML) SPGR QUurainnteitoastmiovlealuirtiynary/cerebrospinal U OSMO (MOSM/KG) QUAN PRO (MG/DL) fluid protein - Urine protein excretion PRO EXC (MG) Urine chemistry Multistix strip Urine pH UPH Urine protein Urine glucose UPRO (MG/DL) UGLY - Urine ketones Urine bilirubin UKET U BILE Urine blood Urine urobilinogen UBLOOD UROBILI Urine reducing substances URESUB - MicRreodscbolpoiocdecxeallmsinpaetrihoingohf-uproiwneer sfeiedlidment RBC (PER HPP) White blood cells per high-power field Epithelial cells per high-power field WBC (PER HPF) EPITH (PER HPF) ~ Bacteria per high-power field Casts per low-power field BACT (PER HPF) CASTS (PER LPF) Crystals per low-power field CRYSTALS (PER LPF1 or PER LPF2) Urine appearance Comments 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 0SMO (MOSM/KG) - "4 000603 CovanMcTe -663239124224 Codes for Anatomical Pathology Code Definition - ANIMAL DEATH CODES T "Terminal sacrifice D Found dead MACROSCOPIC CODES EX NOT TAKEN Indicates that organ weight is excluded from calculations Organ weight not taken; explanation given in necropsy notes - MISSING `Organ missing or lost "UNSUITABLE AUTOLYTIC Organ technically unsuitable for weighing Organ autolyzed and could not be weighed EXCLUDE `Weight was taken, but was excluded from all calculations MICROSCOPIC CODES Codes B- Prefacing NeoplParsimtaircyF,ibnedniinggnsneoplasm ~ M- Primary, malignant neoplasm N- Metastatic neoplasm a Locally invasive neoplasm X- Other neoplasm ~ Code Definition DistributionofFindings Focal Diffuse Multifocal - 000609 CovanIcMeT6-36293-124224 Codes for Anatomical Pathology (Continued) Grades 1 for Severity or Amount `Minimal - the least amountof change that can be observed with the - 2 light microscope Slight - less than average amount of change, but readily discemible as abnormal 3 Moderate - the average amount of change that is expected foar 4 lesion Moderately severe (marked) - a marked amountof change with possible loss of function of the affected cells or organs 5 tSheevearfefe-ctaedgrceealtl oarmoourngatnosfacnhdafnrgeequweintthlyprionbvaobllveeslolsasrgoefafruenacstoifotnhoef organ - Other Microscopic Codes Total P Finding present - Finding not present MN Mean TISSUE ABBREVIATIONS Abbreviation Definition LN Lymph node GL STOMACH, GL Gland Glandular stomach STOMACH, NONGL Nonglandular SALIV GL. MANDIB Mandibular salivary gland - LN, ANT MES/PANC AUDITORY SEB GL Anterior mesenteric/pancreatic lymph node Auditory sebaceous gland LACRIMAL GLAND, EX Exorbital lacrimal gland HEMATO NEOPLASIA Hematopoietic neoplasia LACRIMAL GL, INT CAVITY, ABDOM Internal lacrimal gland Abdominal cavity - SALIV GL,PAROTID Parotid salivary gland LN. TRACHEOBRON CATHETER EXIT Tracheobronchial lymph Catheterization sie: exit node site from the body CATHETER ENTRANC CATHETER EXIT Catheterization site: entrance site into the vessel Catheterization site: tissues (vascular or - extravascular) associated with the catheter near its tp | % 000610 Coners 9e2a4 Tae1 ResultsofHMoinmedo$1g9A9en8alnysecs(tppym) PWeeeukoRarngoeFiondicngDiiEaatrhaynnoTlcos(ioN-MleSFtoOudSynE,wTa6i3Nm1-4tMi)eithdhRaots -- Ta py SampleLocation _Reptete [0 2 ww Top 2 oiumr FH wSean 7m1 52 wy m2ss om a4 am Midd MoTnis1s101 2 a3 9G5u9659 ws w28e1037) ms 9M5Aa G) se s4u5106) a Boom MoFn r132032) ow 9S8n6686 m21y070) woe 267 e95y8058 \a sSm5A0N am Me2Fm n1d4m3040) wSei 10200) aow0 83050) o2s2 MAG) aas T0040 Eachvl pretest sh porenof hers 2g 82 a CovanMceT6-3219-42224. Table 1 (Continued) Results ofHomogeneity Analyses (ppm) Mixed 6/11/98 Per4f-lWueoreokocRtaanngees-uFlifnodnianmgidDoieEttahraynoTlox(iNci-tMyeSFtOuSdEy,wiTt-h6N3-1M4)etihnyRlats -- eeeeee -- T-631r 4 (ppmn ) - `Sample Location _ Replicate 1 Top 110 2 12 3123 2 Mean LI8(118) Top" 21 11213s 3110 Mean 116(116) 2 Middie 1291 2 140 3 37m Mean 2.68 (268) Middle" 1 Ls - 21 316 Mean 114 (114) Bottom 120 2145 . 3121 Mean 129(129) Bottom" 1 1.01 2 098 3106 _ Mean 1.02(102) "a Each value in parenthesis is the percent of theoretical. b Reassay. . 8 000612 CovanTMceTg63321924224 Table 2 Results ofMSitaxbeildit5y/A1n9a9l8yses (pp) - Per4f-lWuoereokocRtaanngees-uFlifnodnianmgidDoieEttahraynoTlox(icNi-tMyeSFtOuSdEy,wiTt-h63N1-4M)etihnyRlats Se ------T T6314 (E ppm) . Storage Conditions _ Replicate 1 500 Initial 2113163s si 8 312s 522 - Mean 1.32(132) 505(101) rAtoloemasttem1p9erdaatyusr,e, 2J- a45a7a Mean - 451902) rAtoolemasttem3p2erdaaytsu,re 21 -- 441240 Mean - 417 (83.4) 8frwoezeekns, 21- 04 an Mean - 486972) 6t2emdpayesr,atruoroem 2r-- 03024 Mean - 353 (106) e3 ETach value in parentheses is the percentE oftheoe retical a 000613 Covanrcei329a:2n24 `Table 2 (Continued) Results of Stability Analyses (ppm) Mixed 6/30/98 4-Week Range-Finding Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-63] 14) in Rats T-6314 (ppm) - Storage Conditions _ Replicate 1 Initial 1 2 Mean 0.777 0.674 0.726 (72.6)" "a Each value in parenthesis is the percent of theoretical. - 0 000614 Commre a02i24 Tabl3e Results ofDose Preparation Analyses (ppm) Per&f-lWuoereokoRcatanngeesuFlifnodriangmiDdiotEathanoTols(icNk-tMycSFtOuSdEy,wiTth-N6-Me3tih1nyRl4.at)s Weck Replicate 0 T 0 TE Gp0) Ws Le 2[EbTl 10s0e x276 1wa4s M3en.. a2m68s eT Cs 9-21021) 2-7@57) I-LTOLT) 4-86672) : - - 23 - o11m6 -- -: -- -- 4 MaTn b oLmISG 10- 0 2 boon 927 ! 22033 omn9 4asm Restsfo tMhee1pnpm.levelsw0e7re60de2r6iv)edfrom96th1e0s6am)plec2o1ll3e0cLte0d)rom5th0em0i5dd)leof4t8h56d1o0s)e Bperlepoawrtahtieolnismfiorohfomqougeanenittyiat(n<aa0l.ytsipispomn). | 4Eraecahsayv.a inparenthesis thepercent oftheoretical. g3 8a |& a Ta3(bContlinueed) ResoafDloss Preparation Ansys (pm) P4e-WreekfRaungoe FrindoingoDicEeftaatrnyoeTlo(srNi-aiMyClSFtOuoSdEyn,wTi-at6h3Nm1-4M)ieithndyRlaots Wek Repliene 01 TeEe pm 0% iw 50 21Cosoets Mean OSB@ C- omme2 - BEOED FFEE - - F Each aloe arenes thepercentoS f heretical. 4 easy rre em e88 &2 2 g2 25 amo wt 2g 82Eg st mma of ty eta cuts 0) | &82 23 s 55 SE SI ETR H Whee . 22g 2g 5% [I -- 222 88 = 5 omar o mw Ca Sc gg8 5 mary of 20 wane coro at 0) 22 88< 9 maryo sub seta: Coa ots (4) 28ga3 mneyo aay vei Chae ts 1 g2 38 |& o ar pw en, rere onc mnrte 83 &8 * @ S28g ggs 8 " 2g 88 Cl 3S 8&& & mary of Ten Maaciah Cmroption ac waaroe) 2g E&8 @ res oe OT OW ORT wl 2g &8 s Es oe NTR AR EW. SL MR oe em om mem omer &g &8 ke Be oF BOT WORT aM gge g k0 Ee af ETL STR ER. EL MN, ve em ee em ne z<5 ] 3a n 82 8 & n 22 8&3 32 8& " 2e2 &&@ 7s g8 :2 [3 g2: R2 7 Samy of Abate oon wah vc 9) 228s 2 388 2z ams o on se el 283 2 = 80 232 2&G ul come Gy wi ects | VEEL fe due Smroome osm ow 2gg 2 & mary of capac ig Secesages 88 8w3= om ot co i cen A ET TI Wes - WE fm dm dm dm de dm fm dw dem dhe dwder TTT i g83 2B " 88& 5 282 a&s 8 ms ps ee Sr [= || waremiesmt -- 2g8 2 k EEE EEEEE RE 8 2gg a& A TT TL es 2888 a 8 | ERE aa 222 a2a 0 22 8aG ot ET Tk fp Ee sgg8 2 @ 2 22&& ~ 9 | n0n yen esc eT 2g2 8 8 " 28 288 % 232 8&e % Eiiiiiliiiiid ggg8 22 a Covance 6329-224 Te . APPENDIX 1 Protocol Deviations Protocol - Protocol Amendment No. 1 } 98 020662 CovanMcTe -6362391-24224 Protocol Deviations Protocol. Test material. Disposition of Test Material. After authorizationfromthe `Sponsor,anyremaining test material will be returned to the Sponsor. Actual Procedure. Remaining test material was retained for future use. - Protocol. Dosing Procedures. Retention Samples. "Samples (approximately100g) will be taken from cach dose preparation sampled for dose analyses and stored at room temperature. Unless used for analyses, these samples will be discarded approximately 1 month after completionof the in-life phase." Actual Procedure. Retention samples were not discarded approximately 1 month afier completionof the in-life phase. Protocol. Termination. Organ Weights. At the scheduled sacrifice, thyroid (2) with parathyroid was tobeweighed. _ Actual Procedure. One thyroid with parathyroid was not weighed for Animal No. C9459. Protocol. Experimental Design. Postmortem Procedures. "The following tissues (when present) will be collected from each animal and preserved in 10% phosphate-buffered formalin." Gross lesions were not to be processed and examined `microscopically. - Actual Procedure. Some tissues, required by the protocol, were not available for histopathologic examination. Missing tissues are listed with appropriate comments in the pathology data sheets for individual animals. Summary tables do not include them as having been examined. Gross lesions were processed and examined microscopically. These deviations are not expected to have affected the results of the study. . % 000663 Protocol COVANCE Sponsor: 3M St. Paul, Minnesota. PROTOCOL Study Title: - 4-Week Range-Finding Dietary Toxicity Study with N-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats . Date: June 8, 1998 Performing Laboratory: `Covance Laboratories Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704-2595 Laboratory Study Identification: - Proposal No. 90545C Covance 6329-224 TLa4,2 100 000664 - Conce 6329-226 --_--_---s Study 4-Week Range-Finding Dietary Toxicity Study with N-Methyl . Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats Purpose To assess the toxicity ofthe test material when administered in the diet to rats for at least 4 weeks Sponsor TM `Toxicology Services - `Building 220-2E-02, 3M Center St. Paul, Minnesota 55144-1000 Study Monitor Andrew M. Seacat, PhD - 3M Toxicology Services Telephone No.: 612.575.3161 Facsimile No.: 612.733.1773 - Alternate Study Monitor Marvin T. Case, DVM, PhD 3M Toxicology Services Telephone No.: 612.733.5180 Facsimile No.: 612.733.1773 Study Location `Covance Laboratories Inc. 3301 Kinsman Boulevard - Madison, Wisconsin 53704-2595 Mailing Address: PO Box 7545 Madison, Wisconsin 53707-7545 . 101 000665 - --_--m Study Director Peter J. Thomford, PhD Covance Laboratories Inc. - `Telephone No.: 608.241.7207 Facsimile No.: 608.242.2736 Covance 6329-224 Pep Toxicologist - `Thomas E. Ryan, BS Covance Laboratories Inc. Proposed Study Timetable Tn-Life Start Date: June 12, 1998 - Io-ELndiDfatee: July 13. 1998 Audited DraftReportDate: October 18, 1998 Regulatory Compliance 5 "Thisstudywillbe conductedincompliancewiththe FoodandDrugAdministrationGood Laboratory Practice Regulations assetforth inTike 21 of the US Code of Federal Regulations, Part 58, issued December 22, 1978 (effective June 20, 1979), and with any applicable amendments - Animal Care and Use Statement Allproceduresin thisprotocolareincompliancewiththeAnimalWelfareAct nRoetguulnanteicoensss,ar9iClyFdRup1l-i4c.atIenatnhyeporpeivniioounsofwtorhke.Sponsor and study director, the study does Quality Assurance "The protocol, study conduct, and final report will be audited by the Covance Quality Assurance Unit (QAU). The proliferation cell nuclear antigen evaluation, dats, and report will be audited by the QAUof Pathology Associates International. Liver and serum = analyses, data, andreportwill be audited by the QA of 3M Environmental Technology and Safety Services. 2 102 000666 Test Material Covance 632P9a-g2e2s4 . Identification N-Methyl Perfluorooctanesulfonamido Ethanol (MeFOS, T6314) Lot Number `The lot numberswillbe maintained in the raw data. Purity Responsibility of the Sponsor _ Stability Responsibility of the Sponsor Storage Conditions At room temperature Characteristics Information on synthesis methods, composition, or other characteristics that define the. test materialison file with the Sponsor. - Reserve (Archive) Samples A reserve sample (approximately 5 g) of each lot will be taken and stored at room temperature. These samples will be transferred to the Sponsor after completionofthe in-life phase. Disposition of Test Material After authorization from the Sponsor, any remaining test material willbereturned to Andrew M. Seacat, PhD = 3M Toxicology Services Building 220-2E-02. 3M Center St. Paul, Minnesota 55144-1000 Telephone No.: 612.575.3161 Facsimile No.: 612.733.1773 - 103 000667 Avimals Covance 632P9a-g2e2s4 ~ Species Rat Strain Crk:CD'(SD) IGS BR Source Charles River Laboratories, Inc., Raleigh, North Carolina. . Age at Initiation of Treatment Preferablylessthan 6 weeksof age,butnotmorethan 8weeksofage Weight at InitiationofTreatment 10010300 Numberand Sex 36 males and 36 females ~ Identification Implantable microchip identification device Husbandry - Housing Individual (may be group-housed during acclimation). Animals will be housed in suspended, stainless steel cages. . Diet Certified Rodent Diet #5002, meal (PMI Nutrition International) ad libitum, unless otherwise specified. The diet is routinely analyzedbythe manufacturer for nutritional components and environmental contaminants. Specified nutrient and contaminant analyses are on fil at Covance-Madison. - ws 000663 - Covance 632P9ag2e26 Water Ad libirum. Samples of the water are routinely analyzed for specified microorganisms. `and environmental contaminants. The results areonfile at Covance-Madison. Contaminants `Thereare no knowncontaminantsinthedietor wateratlevelsthat might interfere with this study. Environment Environmental controlsforthe animalroomwillbe set to maintain 18 to 26C. a relative humidityof 30 to 70%, and a 12-hour light/12-hour dark cycle. - Acclimation Atleast 1 week Randomization Selectionofanimals for the study will be based on body weights, clinical observations, andotherdataas 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 timeofrandomization, the weight variationofthe `animalsof eachsexusedwillot exceed 2 standard deviationsofthe mean weight, - `and the mean body weight for each groupofeach sex will not be statistically different at the 5.0% probability level, Justification - Rats historically havebeenused in safety evaluation studies and are recommended by appropriate regulatory agencies. _ 105 000663 . a--------------------------C-- onse--e 529-- .226 `Group Designations and Dietary Levels Grow Male Female GpmMeFOS) `Number of Animals Dietary Levels = Group Male Female (ppm MeFOS)* 1 (Control) 6 6 [) 2 (Low) 6 6 [] 3 (Low-Mid) 6 6 10 4 (Mid) 6 6 30 - 5 (Mid-High) 6 6 100 6u (HDigoh)wlwh mope6 dwpm 6MOS, 500 b The control animals will receive the basal diet only. - Dosing Procedures `DiMeettahryo.d7ofdAadymsiiwneiesktrfaotriaonleat weeks. Treatment will continue through the day before necropsy Reason for Dosing Route `The potential routeofexposure in humansisoral. - Dose Preparation AdlelvdeolsoepeprdebpyarCaotviaonncsew.illDboesemicxoendceanctcroartdioinnsg twioltlhbeesbtuadsye-dsopencithfeicMmCixFiOnSg pcrooncteednutraes supplied. All dose preparations willb stored at oom temperature. - Retention Samples `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 sampleswillbe discarded approximately | monthafter completionofthe in-life phase. - 106 000670 Covance 6329-226 - _-- ews Dose Analyses By Covuasinngacmeeth,odsuppliedbythe SponsorandvalidatedbyCovance - `Homogeneity `Homogenweiilltbyedeterminedforalltreated doselevelpreparationsoncepretest. Onesampiecach (approximately 100 g)fromthe top, middle, and bototfothme dose: `preparationsmixed for homogeneity analyses will be collected, divided into three: - subsafomrepxtlracetison and analysis,andanalyzedfor testmaterial content. All `sampleswillbestoredat roomtemperatureuntilanalyzed.Inaddiotniesoamnpl,e `each (approximately 100 g) from the top, middle, and bottomofthe 1-ppm dose: `preparation mixed for the in-lfe portion of the study willbecollected, divided into three subsamples for extractionandanalysis, andanalyzedfor test material content. - Homogeneity analysiswillbe repeatedifbatch size chanbygmeorse than 30%. Stability Four setsof samples (approximately 100 g each) will be taken from the low- and - high-dose level concentrationsofdie preparationsmixedpretest. Homogeneity `samplescollected frtheomidmdleofthepretest dose preparations will be analyzed on theday ofmixingandusedasthebaseline value. Theremaining samplesfrom low- dose level preparationmixedpretestwillbe discarded. One sampleofthe high-dose. preparation will bestoredatroomtemperatureforat least 19 days, then analyzed. - "The third sampleofthe high-dose preparationwillbe stored at room temperature after at least 32 days, then analyzed. Theremaining sampleofthe high-dose preparation wibesltorledin afreseettozmaeintrain -10 to 30Cfor 8weeks,thenanalyzed. - In addition, three samples (approximately 100 geach) will be takenfromthe low-dose: level preparationmixedfor the in-lfe portionofthe stanudandalyyzed according to the schedule used for the samplesofpre-test high-dose preparation. Homogeneity samples collectedfromthe middleofthe low-dose level preparation will be analyzed on the dayofmixing and used as thebaselinevalue. Dose Confirmation `Samples (approximately 100 g)fromalldose preparations will be analyzed. The homogeneity sample collected from the middle of the low-dose level preparation - mixedforthein-ifeportionofthe studywillbeusedfordoseconfirmation. All `samples will be stored at room temperature until analyzed. 107 - 000671 Cones 6329-226 FE ----. :4 Observation of Animals - Clinical Observations Each animal will be observed twice daily (a.m. and p.m.) for mortality and `moribundity,recording findings asthey are observed. At least onceweekly, cach `animalwill be observed(cagewill be opened,andtheanimalwillberemoved); - abnormalfindings or an indicationofnormalwillbe recorded. Additional findings will `be recorded as they are observed. ~ Body Weights `Each animal willbeweighedatleast once priortotreatment, onthe firstdayof treatment, and twice weekly thereafter. Food Consumption Food consumption will be recorded for each animal twice weekly during treatment. Clinical Pathology - FAfrteeqruaetnlceyas 4 weeksoftreatment AlNumber of Animals - Method of Collection Animalswillbe fasted overnight: blood willbe collected from a jugular vein. The anticoagulant will be potassium EDTA for hematology tests. Urine will be collected. chilled overnight (approximately 16 hours). 108 000672 Tests Hematology Covance 63P2a9g-e22140 - hermedogbllooobdicnell (erythrocyte) count platelet count white blood cel leukocyte) count he`mmaeatnoccroriptuscular volume dbilfofoerdencteilallmbolropohdocleolglycount - ``mmeeaann ccoorrppuussccuullaarr hheemmoogglloobbiinn concentration rereixcaumlioncyetde)smear (made, but not Clinical Chemistry - glucose urea nitrogen galaamnimnae agmhiintoatmryarnasnfsefrearsaese: ctroetaatlipnrinoetein acsaplacrituamte aminotransferase albumin globalin inorganic phosphorus sodium - cholesterol total bilirubin pcholtoarsisdieum Urinalysis - aVpopelaurmaence. kgleutcoonsees specific gravity bilirubin prpoHtein mbilcoroodscopic examination of sediment urobilinogen "Theremaiurinnei(unp gto 10mL)for achanimalwill stored i afreezerset (0maintain -1010-30C. Samples willbepacked on dry ceandshipped to: - K3rMisEJn.vHiarnosnemne,atPahlDTechnology and Safety Services 935 Bush Avenue BStu.ilPdaiunl,g 2M-i3n5n-e0so9ta 55133-3331 TFeaclseipmhiolneeNoN.o:.: 661122..777788..66107168 - 109 000673 . P----L--x SP -- SerumPerfuroocaneSuan Acid(PFOS) Analyses - wei sat west rst erates Frequencyand NumberofAnimals delle rz we ned of last Methodof Collection nent of Go. - jAungiumlaalrsvewiinl. bSeafmapslteesdwoivlelrbniegchotl;lebcltoeoddw(istphporuotxainmtaitceolayg2umlaLnt).will be collected from a Fo1 Peslytis BSiamopolsestHianodsiwnigtb wed lotsoer ndcng, Sn Samepleos wwiilllbhhparvteeodns6d7 esrseedd fpeeto ro itn 00 S5K5Mi5EBnJo.vHanAsemn,ePDTechnology ad Sty Services . TSBotaePlgaihnnotgneM3N5io0sm56e125757153630315851 Face No, 6137786176 wSilelrsruenppolrets wsiel s abnytaefoSlrPpRoyOnS.sdrol byth Sponsor, Ress Termination . NeUcntsocphseideuslewdiSlahcrdiofniec,eAanndiDelathbse sre willbanswhithecoen EI AScehedaulcedsSacwriefisce of eaten,slsuing imal will ed ovisbed fo incl ptolgy ts nd BOS sample, then sncsheted with cen dowide, - `weighed, exsanguinated, and necropsied. io - - 000674 . Postmortem Procedures Covance 63P2a9g-e22182 Necropsy - "The necropsy will include an examinationof the external featuresofthe carcass; all external body orifices: the abdominal, thoracic, and cranial cavities; organs; and tissues. : OArtgtahensWceheidguhltesd sacrifice, the following organs (when present) will be weighed; paired. organs will be weighed together: adrenal 2) ovary (2) - brain spleen epididymis (2) testis (2) lkiivdenrey (2) tthhyyrmouisd (2) with parathyroid - `Organ-to-body weight percentages and organ-to-brain weight ratios will be calculated. Bone Marrow Smear From the femur of each animal at the scheduled sacrifice only; made but not examined Cell Proliferation Tissue Collection and Immunohistochemical Evaluation At the scheduled sacrifice, representative samples of left lateral lobe ofthe liver from. each animal willbecollected and preserved in zinc formalin. - After fixation, each sample of liver wil be embedded in paraffin, and the paraffin blocks willbe shipped to: `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 - IH 000675 ~ Covance 63P2a9g-e22183 Proliferation cell nuclear antigen (PCNA) evaluation will be done on the samples. Results will be provided for inclusion inthe final report. Palmitoyl-CoA Oxidase Tissue Collection and Analyses At thescheduledsacrifice,asample (approximately S00 mg)ofthe right lateral lobe of the liverwillalsobecollectedfromeach animal and flash-frozen in liquid nitrogen. "Thelivertissuewillbestoredin afreezersettomaintain -60to -80Cuntilanalyzed. > by Covance for palmitoyl-CoA oxidase activity. Liver PFOS Analysis At scheduled sacrifice,a portionofthe liverfromeach animal will be stored in a freezerset to maintain -60 to -80C. Samples will be packedondry ice and shipped to Kris J. Hansen, PhD, 3M Environmental Technology and Safety Services. Liver samples will beanalyzedfor PFOS and metabolites by the Sponsor. Results will be: reported separately by the Sponsor. - Tissue Preservation Thefollowingtissues (whenpresent)fromeachanimalwillbepreservedin 10% neutral-buffered formalin: - aaodrrteanal (2) jkeijdunneyum(2) brain lesions cecum liver cervix lung with mainstem bronchi colon lymph nodes (mesenteric and - duodenum esophagus mandibular) `mammary glands (females only) epididymis (2)) ovary (2) efyeemu(r2)with bone marrow (articular ppiatnucirteaarys - surface of the distal end) Harderian gland prostate rectum heart ileum with Peyer's patch (lymphoid sscailaitviacrynegrlvaend [mandibular (2)] aggregate) `seminal vesicles _ n 009676 : [--Fa-- se 14 sskkielnetal muscle (thigh) tthhyyrmouisd (2) with parathyroid - spiannadl cuomrbderc)ervical mid-boracke, turriancahreyabladder spslteeremnum with bone marrow vutaegriunsa stteosmtasc[h(2) preserved in Bouin's fixative] Zymbal's gland - H"Tihsetaodpraetnhalosl,ogbyain, eyes, kidneys. iver, mesenteric ymph node, pancreas, spleen. testes,andovaries fromeachanimalwillbeembeddedinparaffin, sectioned, and tained with hematoylin and coin. - "Tissue slides willbe shipped to: RPiacthhaorldogHy.ABsrsuonceiri,eDs.IVn.tMer.naDtiAoVnaClP - 6O2h1i7o OCpeenrtarteiPoanrsk Drive WTeelsetpChhoenseteNro,.:Oh5i1o3.4757096.99600 Facsimile No. $13.779.9603 - "Tissue sideswillbe examined microscopicbaylDlry.Brunerand theresultsfromhis evaluation willbe included inthefinal report. After completion of the examination, slides will be retuned toCovance:Madison. . Reports One copy ofthedraft reportwillbe sent to the Sponsor. The report will nclude the following information: Experimental Design and Methods Rdeossuelatnsalyses mcloirnaiclailtoybservations . body weights 1s 000677 ConesasFza9:t2s26 body weight changes f{eosotdmcatoenrsiuamlpctoinosnumption - clinical pathology results organ weights organ-to-body weight percentages oparlgmaint-otyo]-bCraoiAn woexiigdahsteraatcitosives ~ `mmiaccrroossccooppiiccoobbsseerrvvaattiioonnss cell proliferation assessments (providedby the Sponsor's designee) Statistical Evaluation Levene's test will bedone to test for variance homogeneity. In the caseof = hvaertiearnocgee.neCitoymopfavraisroinatenastctswepil<lt0a.k05e,vtarrainasnfcoerhmeatteiroongsewnieliltbyenuosecdotnosisdtearbaitliizoen.the One-way analysis of variance (ANOVA) willbeused(ifapplicable) to analyze body - weights, body weight changes,foodconsumption, continuous clinical pathology uvaslueesf,orancodnotrroglanvewresiugshrtedtatead. gIrftohuep cAoNmpOaVriAsoinsss.ignificant, Dunnett's t-testwillbe If the ANOVA shows significance for body weights at Week 1, one-way analysis of = covariance (ANCOVA) will be used to analyze body weights, with initial body weights asthecovariate. Ifthe ANCOVAissignificant, covariate-adjusted means willbe used for control versus treated group comparisons. - Group comparisons (Groups 2 throug6h versus Group 1) will beevaluated at the \5e.c0a%t,mtewnot-twaiilllebdeparnobaalbyizleidtysalteiveslt.iOcnallydata collectoendorafter thefirstdayof At the end of 1yearafter issuanceoftheauditeddraft report,if no requested revisions or and submited to the Sponsor. - instructions to finalize have been communicated by the Sponsor, then the audited draft report will be considered final'and issuedasthe final report, signedby the study director, . Any modifications orchanges 0theauditeddraftreport requested 1yearaeissuance `will be performed at additional cost to the Sponsor. : He 000673 . _-- Covance 6329-224 he "Two copiesofthe signed final report (one unbound and one bound) will be sent to the client. - Record Retention All raw data, documentation, records, protocol, specimens, and final report generated as a resultofthis studywillbearchivedinthestoragefacilities ofCovance-Madison for a periodof 1yearfollowing submissionofthe final report to the Sponsor.All raw data - stored on magnetic media, the protocol and protocol amendments, study correspondence, `andtheoriginal report will be retbayiConvaencde. One year after submission of the final report, alolf the aforementionedmaterialswill be sen to the Sponsor, and a return fee will be charged. The Sponsor may elect to have the materials retainedin the Covance archives ~ foranadditional periodof time,and Covancewillchaastrorag gefe ee.Ifthe Sponsor chooses to have Covance dispose ofthe materials, a disposal fee will be charged. PCNAevaluationdata,paraffinblocks,andtissueslideswillberetainedby Pathology Associates International. Liver, urine,andserumsamples sent to the Sponsor and analysis data will be retained by `the Sponsor. ~ 115 000679 . cmavermazt EEE EE --1 PROTOCOL APPROVAL Luda)M Seas 12/33 _ -`ASntdudryeMwonM.itSoeracat, PhD Date - rl, ford, PI Study `tor Date / ; Covance Laboratories Inc. 0 - 000630 Protocol Amendment No. 1 COVANCE.> PROTOCOL AMENDMENT NO. 1 Covance 6329-224 4-Weck Range-Finding Dietary Toxiiy Study withN-Methyl = Perfluorooctanesulfonamido Ethanol (N-MeFOSE, T-6314) in Rats Toomer ML SPmMEmees Study Monitor: AndrewM. Seaca, PAD - Testing Facility: Study Director, _ Covance Laboratories Ic. Madison, Wisconsin PeteJr. Thomford, PID "This amendment modifi the following porotf thieporotnocosl - Effective June 8, 1998 1 Pwiatghteh.e dTioetn,cdldudethtehfeovlelboiwcilnegusseectdioond.issolveth testmaterialbeforemixing Vehicle Identification Acetone "LTohte Nltumubmebresr willbemsintined i herawdata. POunriftiyl with the manufacturer `OSntabfilleitwyiththe manufscurer SAttorroaogme tCeomnpdeirtaitounrse wm 000651 . --_-- Protocol CAomveanndcmee6n3t2N9o..261 Page? Characteristics _ thInaftodremfaitnieotnhoenveshyinctlheesiissomneftlheodwsi,thctohmpeomsaintuifoanc,tourreort.her characteristics Effective June 19, 1998 2. Page,ClinicalPathology, Methodof CollectionToindtihactaadiftfereent - metofcholloectidon i required to provide additional blood, delete the text in this section and replace with the following: Animals will be fasted overnight and anesthetized with carbon dioxide; blood will ~ be collectedviacardiac puncture. The anticoagulant will be potassium EDTA for hematology tests. Urine will be collected chilled overnight (approximately 16 hours). 3. Page 11,SerumPerflooSurlfooniocAccitd(aPnFOeS)Analyses, Methodof - Collection. To indicate thatadifferent methodofcollection is required to provide additional blood for PFOS analyses, delete the text inthis sectionandreplace with the following: `Animalswillbe fasted overnightandanesthetized with carbon dioxide; blood (as `much as possible) wilbecollected via cardiac puncture. Samples will be collected without anticoagulant. 4. Page 11, Termination,Scheduled Sacrifice. To indicate achangeinnecropsy - procedures required for collection of additional blood for PFOS analysis, delete the. text in this sectionandreplacewiththe following: After at least 4 weofetrekatmesnt, allsurvivinganimals will be fasted overnight, ~ then anesthetized with carbon dioxide, bled for clinical pathology tests and PFOS. samples, weighed, exsanguinated, and necropsied. A ns 000652 . ProtocoCloAvmaenncdeme6a3t29N-o2.24| ---------------------- Pg Effective July 13, 1998 - 5. Page3, Quality Assurance. To indicate that quality assurance for tissue processing wil be done by Pathology Associates Intemational, delete the text inthis section and replace with the following: "The protocol, study conduct, and final report wil be audited by the Covance - Quality Assurance Unit (QAU). Tissue processing will be audited by the QAU of Pathology Associates International, Ohio Operations. The proliferation cell `muclearantigen evaluation,data,andreportwillbeauditedbythe QAUof Pathology Associates International, Frederick, Maryland. Liver and serum - analyses,data,andreportwil beauditedbythe QAUof 3MEnvironmental Technology and Safety Services. 6. Page12,PostmortemProcedures,Histopathology. Torefthledeecisciontto ship tissues to Pathology Associates Intemational for processing, delete the text in - this section and replacewiththe following: Tissuewill be shippedforprocessitnog: Ms. Sheree Lovelace: Pathology Associates International Ohio Operations, 6217 Centre Park Drive: West Chester, Ohio 45069 The adrenals, brain, eyes, kidneys, liver, mesenteric lymph node, pancreas, spleen. testes,andovaries from each animalwillbe embedded in paraffin, sectioned, and stained with hematoxylin and eosin. - Tissue slideswillbe examined microscopbiycRiaclhalryd H. Bruner, D.V.M. DAVCP of Pathology Associates International and the results from his evaluation will be incladed in the final report. After completionofthe examination, slides and tissues will be retuned to Covance-Madison. - 1 000683 - Protocol CAomveanndcmee6n3t29N.o2.241 Pages Effective July 20, 1998 7. Page2,Study MonitorandAlternate StudyMonitor.Toindithcecahatngee intheareacode,delete thetextinthesesectionandrepwithltheafollcowieng: SAtnuddryewMoMn.itSeoarcat, PhD - `MTelephone No.: 651.575.3161 Facsimile No.: 651.733.1773 - AMlatrervniaTnt.e SCatsued,yDMVoMni,tPorhD T3eMleTpohxoinceolNoo.g:y S6e5r1vi.c7e3s3.5180 Facsimile No.. 651.733.1773 8 Page,DispositionofTestMaterial.To indicatethechangeinthearea. ode, delete the textinthese sectionandreplace with the following: A0f:ter authorization from the Sponsor, any remaining test material will be returned - A3nMdrew M. Seacat, PhD TBuoixlidcionlgog2y20S-e2rEv-i0c2es, 3M Center St. Paul, Minnesota. 55144-1000 . TFaeclseipmhiolneeNoN.o:.: 665511..753735..13713631 9. Page 10, Clinical Pathology, Tests, Urinalysis, Paragraph 2. To indicate the change in theareacode, deletethetextinthis paragraphandreplace with the - following: `Theremaiurninei(unptgo 10mL)foreachanimalwillstoredinafreezerset to `maintain +10 t0 -30C. `Samples willbe packed on dry ice and shipped to: 120 000684 Brotocol CAomveanndcmee6n3t29N-o2.24| --_---- Paes Kris J. Hansen, PhD 3M Environmental Tectmology and Safety Services. . 935 Bush Avene: Building 2-38-09 St. Paul, Minnesota 55133-3331 Telephone No.: 651.778.6018 Facsimile No.: 651.278.6176 - 10. Page 11, SerumPerfluorooctaneSulfonicAcid (PFOS)Analyses, Sample Handling, Paragraph 1. To indicate the change in theareacode,deletethe text inthis paragraph and replace with the following: 2 Bloodsampleswilbeallowedtoclotat room temperatureandcentrifuged. `Serumsampleswillbeharvestedandscoredin afreezersettomaintain 60t0 -80C. Sampleswillbepackedondryiceandshippedto: Kas J. Hansen, PhD - 3M Environmental Technology and Safety Services. 935 Bush Avenue Building 2-36-09 STte.lePapuhlo,nMeiNmon.e.so6t5a1.5757183.36-0313831 - Facsimile No.: 651.778.6176 Effective December 15, 1998 11. Page13,PostmortemProcedures,CellProliferationTissueCollectionand - ImmunohistochemiealEvaluation, 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 wil be done onthe samples. - In addition, liversectionswillbe stainedwith hematoxylin and eosin and. examined microscopically. Resultswillbe provided for inclusion in the final report. 121 000655 --_--_--mmm ProvenCAovnanacet632a9-.2241 Paes AMENDMENTAPPROVAL AndrewM.Seacat, PhD `StudyMonitor - Mm me [07 nn[977 Date - ~ Jl % C4 Study Dis CovanceLaboratoriesInc. _ 122 000685 CovanTce6633291-224 | APPENDIX 2 Individual Animal Fate Data Individual Clinical Observations - 123 000687 | SELB | BEE EEE no: | gg 2& 2 | oh (BEM! BEE ERES id | 28 2 ns 2g3 &8S 126 -- OU 75 wo amo ssmarions riers g8 22 iY 5 SE g88 2& = 228 8&& 3 | mn 3 emer misrr g3g g8 o 8283 a 1 a ERE 223&8 & mn CovanMcTe-663321942224 . APPENDIX 3 Individual Body Weight Data (g) 133 00069 a som ggo 8g 1 = 5 28 S88 8 15 teint vty tan outs a) 2g2 3: 136 sects 3 2S B32R 137 CovanMcTe-663239124224 : APPENDIX 4 Individual Food Consumption Data (g) Individual Test Material Consumption Data (mg/kg/day) . 138 000702 88 d2 282 33= 140 gg &3 ww 2 2 3 142 ives ene cea Conmticn oe (n/n) 33 3S 3 . ERE g < 2@ 1 5 IE 23 :3 us CovanMcTe 6-3B291-42224 APPENDIX Individual Clinical Hematology Data Individual Clinical Chemistry Data - Individual Clinical Urinalysis Data - ke 000710 pw Me BOF RW adie 22s oi w THTOE Mh th Fide Mh fe 3oB 17 msmys oe S po NR ORT a g3g 2 1 EAIET IE mE Th BTR AVE Se ER NRO WOM 8g33 8 ww pe ET ST AVE Be BR MRT ess 354 mmm 3=g3 1% ps cones OR 2g8 a5 151 Aopenti 5 come 2 22 32 > | 192  8325 1 pm BOF RRR ade 38 3N @ 154 pms me. XSS ATR Rw BR BR YU Sm Mr em Mm E] 2 MH 155 ri neg 35 Em Gh WR WR Me SN UT em om me gg i8 56 pendix $ coPoI s fri :::: ssc 3 : : : gm cc: i 8g 83 i 23 El 3 158 es cs 3g 8 15 sepsis covosy 84 g2 ot 160 2gg a & 161 823 By 162 Cg = [AEE TE g83 0 ha ess 23 f be bok g833 1 | gaw'ooi@n B% fomoemoonma o#1 2Bodom 1 | gs = Be hl Mh RR hd 33g 2 165 ee [-- | gmooo@ FF oEOEOER BOA 2 = we, oy wy ws we, ea ow 28g: 2 166 gg3 F do + i as eto a on con a ecin 3 = 167 8 + uote va contin a th so of cts 2 & 168 enti 3 cogs oe 88 3& & 1 Aooensix 5 comes PEE gg 3&2 10 got of bf EB 8 s8g3 a& m S2s @3 m o + tasasteesting a g33 8 m p-- conesy os + amiewanvnoni a5 he soe of coblction. 3e3 &8 m p-- coos 2 222 38I) 1s es cy 21 sgg5 : ns 8g 32 m ropentix 5 covesPRE ] orb bod E <:3 APPENDIX 6 Individual Absolute Organ Weight Data (g) Individual Organ-to-Body Weight Percentages Individual Organ-to-Brain Weight Ratios Individual Animal Pathology Data CovanIcMeT6-36293-124224 - 179 000743 S2 3 = 150 Siva ose oem igh ata 0) 88 ao3n 181 <g 3S2 a te on i a 1 | Tam Ea g2$ 3 = 22 3>> 15 2g2S 15 om vnseen reas orn sr eryoem oa? 2 2 g 186 2g Sa ww | CUE Te TTTE :: 3 - I i 2 Sa 190 28 2# | 91 toi Geman eh. ecestazen 8 a3g 192 & ions rp by Sia: Prcstns g2g <a 193 8g 3g8 194 otekantorgan o-soy Miah. prcuanes 82 3a 195 Sin rp pty an rcs 22 S & 196 8 Soak Canons wep. pecans 88 3 g 197 Snviot cepa-c-y i ecenten g g && 198 gg 8 Ld 199 ein cv tos ih rss ge 10 niin Spann wah. Prceans 22g &a3 01 nt rnc oy is g2 8g$ m I ais z2: ws | PPoEgEREBDOE B R OME E ON mT m EuaE g E OE ow g 2 22 204 Piogm omiOME OE OE om im 2gg28 2 25 GOEL am 22 g 206 ettcooetn Mk cen . 28 207 Stunt Opa co-bein igh acion onc ORI EER 88 3 5 08 me avr, sera somes STEAE met. ge 3 & 2 se pn ye recorme re ma gg 33 0 gg 33 an ] 5gg$3 " [Em -- g < 3 2m g e a 2g8 3 2] as g2 3z8 26 i e33s iB a 2g g i 218 gsg i 9 8 32s EY Sg2 Z& 2 2S 3 g m Ce82 3 ~ m -2] z3@ 2 2gS 32 2s Te 3ggs g 2g 3< m eg g : EY <gs g w 22g 3 $ 20 or g2 | &g3 = gc & psi ee pe gm SEERIS, core cr Mn EA 0) gg2 3 EA se es rm se sn ta a gE a hE 8 SEE 2g 2 &g 4 || 2 g g ns Srviaun anand Phan saa g282& 2 28 2g2 El 82 2 2 &8g & TM Soividnt nnn cons ows 2e -2& 2x0 gg8 8 nn tei ees tons a 228 Z & nm S3s 2J uw g83 & we Sm pre J c &gg us 3g c&S@ 26 3g22 E El s82 a5 ue S222 29 gS2 22 a 20 g2gg @ 21 28 &I x BEST Till SY or cere 5 MUR nyoMGRITECE, TS SED, Tong aceiricy 22 3 < Ee) 28 2B 25 g2e 2G 255 2g2 2&S 26 282 g = 27 2g &8 5 22@8 2 222@8 = 20 S8 28& 21 222&8 & %2 g2 &&< El 282@3 260 82 28 25 32g: " ntviont dant actors cs EDFr", FE fare os g2 g El | Coven 6329-224 eg< & = , 8e 2& 2% SEEURSEEB aB en,E BRU PEme mBe,EaEn ser || 2gg& g m gg gz m 22 2 & m 28 && m 223 28@ m 232 @&8 ES | BEERMT com, BSgy PE Gmppg o 2g3 5@ ES 28& g m 28 @= & m 32@ 8 EE 222s z = 20 &232 a 3 228 2 > ES | = Eo Ei =u EE &g82 2 m 2e& 2=% En 2gg z3 ns 83 & * 26 222& " wm 222 2&8 288 228 I&g] x gg w&&= 2% iasgg 38 a& = <S2@ 4 El g2@& 204 g22a 25 aii sm oes ee 3 333 2% 282 @82 El 82 2a8 28 82 285 2 g82 2 300 8 g on EE ERBn pn omen Z288& 0 g8g 3g 0 222 22 2 04 2g2 288? 305 28 23 306 288 223 07 oink nine colony ues 222 I] 08 | . 2gZ2 & Bei, mesnan 309 gg &3 310 | Sm erin aSec y we an 2 2 g 2a3 sit oo . . , ' , , ' . . 82& a EH | ce ng en GREENBRAE SE | Fn Tan a Sine 2gg 23 an &g8 31 82 @ 3 as 2g2 @2g 36 ] om 2 ggg an 82 2Z&z 2g3 2 as gg&2  g& z " EY 383 m 2g2g m Bp ESRRa Sr W 0 IE BN NEpelinTREElo (I r RY 32 &% Ex 28 22 Ed | BERRA BREE SOS RR ggg 2 1] 226 S222 Rr 2 288 zg EY - FEET Ted . rats, casonte, vpn. 2S8 && 29 g2 2g 0 238S 2g& ] EE ETE ERETRE <g3: 288& $ EY 2&2 x@ EX 22 32&S as 282 ?g& 36 eg8 32 Rr El Snivians inns rleny es e2 28e? 3m g88 & 88 g 0 22 &2e? 1 33?2 a ES 223 < Ee) 2g988 2 34 8g8 g ss 38 22? 36 | e 8@2 = Ed g82 8 ws 32g 22? 319 g 32 0 3gg 2 sn 28 22 32 gg 28 ES gg 22B? 354 8S 3 5& 355 LB IT IRETmLTm a mmm--"-- 822 &g 356 2g @8 = 37 g82 as Covance 6329-224 - OO OO O wTeu2 APPENDIX 7 Cell Proliferation Report Note: This appendixofthe report contains information supplied by Pathology Associates International and has been reviewed by the Quality Assurance Unit of Pathology Associates International. 2 359 000823 [t-- oi Cell Proliferation Report LcBEaETr)L)pAAcCaompora oyfmSeosSsfropetSieonrcshammohesnterer CCeoorpornsnineP= eeote noyAn coutrrouotoBoNaroRT EEXANCETIANGCETSATRooYyNTFOXVEICMAITLYGSSTeiUVDAYsLOWSIETFH MoEsTNIEAT . rexzasa von: a rSoEmcToavioad-nyn Ssimtvsicooens - J-- PATHA OP LOGYASE So OCIATESbSoIaNoi TmERSNAoTItONAL - avin ET - 360 000924 CovancMeT63291.42224 CoFmiancSaolyPNroetn5eR2ei5pe2tn4t TAOFBCOL NTENETS L CELLPROLIVERATIONREPORT IL. TABLES IM. SIGNATURE PAGE IV. QUALITYASSURANCESTATEMENT V. APPENDIX - 361 000925 Covance 6329-224 --------------------------------------E--A--L FilCllProkifiaRtepioornt CELLPROLIFERATIONREPORT = hed - PEWEREKFRLANGUE-OFIRNDIONGODICETTARAYETNTOXHEIACNISOTLYU(SNTL-UMDeFFYOWOSIE,TNHT-AN63-1MM4)EIITNHRDYALTOS COSV TUDA YNUN MBERC 6329E -224 PURPOSE `dTiheetptuoruptosfeoorfattlheeasstta4dwyeweakss1.0 assessthetoxicityofthetestmaterialwhenadministeredinthe ``Trheipesersespcoirstt,hseucbemliltptreodlibfyizPaatitohnfoilnodgiynAgsssaoncdiiantteesrIpnrteetmaatitoinofnoalrC(oPvAaYn)cteo StthaedsytNoudmySbpeorn6s3o2r9,-I2M2,4 - ePcteerd fi"4n-WoecrkoRemcgEtothaaFninonlde(iNns-gMsEDFtiOScfEt,owyTu-6s31Tr4o)xIaicniiRtyadtSso"w.mAdlylawisthpoeNft-chMeettatssyksl DsrseuogciAsdtmeidnwiistthatPiAoIn'sGpooortdiLoanboofratthoirsysPtruadcyticwser(eGcLoPn)dRuecgtueladtiinocnsosmspsleitnfcoretwhiitnhTtihtele2Fo1oodfatnhde UaSndCwoidtehaonfyFsepdpslriaclsRbelgeuslmaetnidomnse,ntPsac.t 58,issuedDecember22, 1978(effective June20, 1979), MATEARNDIMAETLHODSS TiasneCollfoer CecllPtroliifeoratnion - S`ihexptaonciemlianllsaprperrolsicfxerpaetiron. oAospecitniognroofwtpheel|ftthrloatuergahllo6bweeorfetshaeclriivfeircfedrodmueraicnghowfee6krat4afpoerr. sspeexcpifeircpatoiwonps.(Tgirsoeuupssb1l-o6c)kwsawesrfeisxkeidpapneddptroocPeAsIsfeodrtsoepcatriaofnfiinngbalnodcsktabiyniCnogv.aFnrcoempeearcphrobtlooccokl, c2eslllimduecwicaasrspnrioipgaerno(dPfCoNrAH)A,E&mcavrakieursotfiocnelalnpdroilmimfeurnaotkioins.tochemica)detection ofproliferating - IommrwackistfoorcCehllePmroilisfetrartiyon (SSeucpteirofnosostfpPlaursa,ffFiias-bcerrbSocdideeadttiises,ocPsiwtsebruerCgaht,aPtA)5 jtuoennsduprleaacdehdeosnipoonsdiutirvienlgypcrhoacregsesdisnigfdeosr SPOCPNAf.orSitmamnudnaosbdiisiocmhemimsrym ). Broicflbmy,etithiossdussesweecrtteiuosnoesdwtecorestih anicnubteiastseudmeswiftoihrPaCcmoNnAoa(clPoAln'asl p`aemrtuixbioddyasteo(PACBNCAE(lDitAeKKOi,t,ltot#0#1P6K,-6P1A0T0N,o.PAA1L7N2o3.) aKnd32re4a)gmenettshreoqdufiroerdtfhoertdehteeacvtiidoinno-bfitothien aanmctilgMe)n-wsaatsilboocdaylcizoemdpblyext.hPecChNroAmeaxgperne3s,s3i*odniaimnicneolbseinnzaidlilnpeha(sDeAsBo;fStihge cmeallCchyecalnieca(GlyC,o5.,lGot; w#aIsSHiEn2c0l1u)d.edTiinstshueessteacitniionngsrwuenraencdcoonsuisntetdoefswrtiutsdhyhateimsiasutneoxtyehlaitdnw.asAnneogtaitnicvuebcaotnedtrwoiltshltihdee: - primaryamtibody. - 362 000926 Covance 6329-224 ----------------------erLSe TAEZ, C FinalSo CatllyProm blirfere a2Rte5pi5eo3rnt4 CellProliferation Messurements - Fqouarlciteyllopfrsotfafienritngi,onpeeovcaeluaastiinogns,asdidseecstwieornoinfgi,nptpaetreurseodfaclelolwlmaabgelniinfgic(a6ti5o,nc(e1n0t0rXi)lotbouljaurdoger `epeninobeldar, oexattraadbhehipsettoeonccoxllpihooeilropgrioelcicfhe)arnatg,ieosn.,CseulclhparKupoffler wciaelslfts,hbeeinlqeruadnuatcitfiteepdiaitthehoilgihunemr, `imnadgnei,fLicIa)twioans(d2e0t0eXr)m.inTehdbeypesrococrcitaagagetolefahsetp3a0to0c0yhteepsaitnocytpehsaisneo10ftrahnedcoemlllycsyeclleect(eldabeellindgs. - pmeirnsamlimoalv. iasioftuosocureplhlaoplrootlgiyfweereatsidfounr.therassessedbyevaluatingtheserialHAE slideforcach `StatisticsAnalysis: - PTIhebeSttwudoesnatc'osnttreoalta(ntdwot-rseisdtemde,autngerqouuaplsvuasriianngcMei)cwraossoufsteEdxtcoetlevsetrfsoirosnt5a.t0is.ticAals2ivgniafilcoafnuc0e.ie0n5 `was judgedtobestatistically sigaificat. RESULTS CeltProliferation I2n)d.iTvihdeupaelracneinmtaalgmeodfgperooekfpmraetimncgehlelppertoofsiyfteecstaisodndeattearmairnepeedbsycathe ilanbSeelcitngiionndIe(xT(aLbIl)edsid1 annodt differsignificantlybetweencontrolandtrestedgroupsofmale ar femalerat. - Imnizgmraolmepwe6rraelreepsa,rtiheedrfeowratshinsogrPoCuNp.Aslidepresentfor animalC94523;therefore,fiveoutofsix Histopathology `iSetchthioenmsaftrooxmytlhinemsadmeeotsiisnm(HbAlEoc)kfsourschidsftoopraptrheoplaorgaitcicovnalousftPiConNA-o sftcaiilnteadtselitdheeiswnteerrepsrettaaitnieodn - oAfptpheenidmixm.mnostaislnideeds. Individual animal findingsandgroup summariesarepresented in TihernesntitseshoorfwtehpdenPorCcNheaAnsgitessiiaainntghtienltiihviesrosttiusndmys.eoTfhmealliveearsnfdofemmaolneermaisdt-hhaitgwhoduolsdeaalntderttwhoe - hciognhcdomoisteamntailnefrlaatmsm,aatnordytwreoshpiognshed.ovTeh.fiesmnaelcerroastisscwaacshcnoonttaaicnceodmfpoacnailendecbryosiassweictohnoduatrya regenerativecffoctasdeterminedby aaincreasedPCNALIL - 363 0003827 Covance6329-224 -- ---------------- eSl ------ LR4, SUMMARY CoFvaoCSetludPyNreombbateiro3n2R8e.p2e4r.t ges - Ifrnotmhceopnrtersolet(0sptapdmy),,cleolwlpdroowiofe(r1aptoiomn)w,alsomwe-arisdurdoesdew(i1th0ipnptmh)e,limviedrodofsmea(l3e0 apnedn)f,emmiadl-ehriagths dose (100ppas),wad highdose (500ppm)groupsater 4weeksonstody. Nostistically osbisgneirfviecadnitnimncarloeaosersfienmacleel aprtoslaiffteerrat4iwoen,ek8ssodefteNr-cmzeitnhedyblpyetrhfelPuCoNrAoolcabtealniengsiunldfeoxne(stLehDna)niwodelor.e - 364 000328 CovancMeT633219-42224 CFinal Co SetllyProlv ifoersatSioe n334 Page ~ ILTABLES Legend SEM =StaEnrrodrofatherMedan - 365 000929 CovneMeT3e291.24224 - Tal 1.Co roto Li ofl Aa4Woks ANCoOvFaOnBceEStudyNo.6230-224 ee TW Coma 1oasew | Tw osm we | Tw cowee een | Tw cee | oars | ase em | Ween |o3sow | - EieemGon |W sem |osew| |cessr |oaarw | WTcosss oomaw | Tw cee 1oraem | Tw cesm ose | - 3-10ppm(owt)|W |Cossad |osrre| a -- Tw cesses |oie | Tw oes |oosew | eae ~ fee] bos "as00%| -- arLm: Tw cows |_oosew | x z CE | Tw cesie |ozs| - ee LET E CRG) een Toten| fem Tosa soe] -- Tw Tw ces 0.337% Cous1T_T0263|% -- -------- - Tw oseao |oosew| 7 Mean |0.284| % - a --aaCeeomteen ES -- Sor -- a-- (NP:sidanot or Coa8as Co4528 | 0.197% TI fo - 366 000550 eb ctes tressJE sr [T cesF tas|oosaw| [C ceF sae oak| [ITT cess |F oses% | TF ceaT sst onsen | [T ceF ass| o.im%| [Me | on asi%| - sem ooemw | [E-ippmowy | F Coasss | 0.136% | [T ceF ase| 0.38% | [C ceF ss oanix | [TT CeasssF | 040i | [F ceaT sst |ost | - ==a = And [3-70ppm (Lowi) | TF ------1 Fo T4538 0.180% Cousai |0.080_%| Coasaz |0.110%| a ' ee Es ae [TF |cessar|019%| a= [_F ceas_ es |_ 012| %| = [T |cesF ses[otei% | [F cesI sso |ooso%| 71 1 [5-700ppem(ui-viigh) | [_o3se| % [ce |a oos so%e| 1 [oo82%| = 1 [oosi%| a F 1 Coasse 0.055% [TeT an | naam| a [6-500ppm(High) | 1 F CuassT F Co4s88 0.193% 0.027% } ee rs a - T [ F TcCooF uuseso ||o0i.1e93%_| - 367 000831 -- CoSv tadya Numbn er6c 329e -24 Pag7e wmsarman - Submitted by: mie adllG) . srw Project Pathologist: { z 4 Doe Carolyn Moye, DVM., ACVP. T7135 o0~ Date - 368 000932 Covance 6329-224 IMT-63142 Cov`aFniczaelCSetuldlyPNruomlbiefre6r3aR2et9p-io2or2nt4 Piped - IV. QUALITYASSURANCESTATEMENT - 369 000933 EER retclonfAsscscoicieastienseImnatteirnoantsi!onal Covance 6T32e9s22i4 SREY CellProliferation Report 4-WeekRange-FindingDietary ToxicityStudywith N-Methyl PerfluoroctEtahannoel (sN-uMeiFOfSEo, Tn-6a31m 4)iinRdatos. - `CovanceStodyNumber: 6329-224 QUALITYASSURANCESTATEMENT - Theceallpyroali)no epEoek tbnybBreeiE mpetcnoy aodnBsoiFmodeTbsye(hoOie1l7e PpAT Qiiusy Ee ot eta:Th iow i oh . Dest [or---- LwuSmieicztison tiSMnPooheamtDyIsnsemeemstmedenicn waonvevn Managsment/ProjectManager . fogA PC ESai oper iSiene 2ehaR Quality AsseanceOfficer se 3 k ST ea WT ROT - 370 . 000834 CovanMcTe -6632391-24224 CoFvianncaelSCteuldlyPNruomtbiefre6r32aR6et-pi2oo2rn4t Pages APPENDIXI - an 000835 CovanMceT6632391-24224 TnDoCl tte oe 4-WIonduikiRdesalAnmimalgEDleieart-yaTeFmzieekitcySpFvtihanddeidynwlgisttoihngLNi-oivMeecrtg:kyl PerfieorosctEtahaenosl(sN-iMeiFOvSEm,Ta63m14s)iInRdaets AvimalNumber_ Sex Doos Group Htslogic Findings own 0M Ten (Coto) ToSigal Findings coun Couns M M 11--00ppppmm ((CCoonnttrrooll)) NNooSSiiggapiiffiiccaannttFFiinnddionggss Coun 1-0ppm: (Control) NoSigificantFindings - Cours ous M M 11--00ppppm ((CCoonnttrrooll)) NNooSSiiggnaiiffiiccaannttFFiinnddiinnggss Coun M 21ppm (Low) NoSignificantFindings CCoouunr MMu 2211ppppmm ((LLooww)) NNoo SSiigggiiffiiccaanntt FFiinnddiinnggss se M coun M 215m Low) 21pen (Low) NoSigificast Findings NoSignificantFindings coum M 2-1pen (Low) coum Mo 10pmGowmit) NoSignificant Findings No SigaificantFindings Coun M 30pmLownid) Couss Mo 30pmLowmd) NoSignificantFindings NoSignificantFindings - couse cous Mo Mo 33100pmmmLGoowwaniidd)) NYoo SSiiggnaiifiiccaanntFFionddiinnggss Cousts Mo Cour 10pmGowmid) 4-30ppm (i) No SigpificantFindings NoSigpificantFindings so M 430pp (i) cost M 430ppm(id) NoSignificantFindings NoSigaificant Findings - ccoaostzs MM 4-3300ppuenn((5i8d)) MNooSSiigpnsiiffiiccaannttFFiinnddiinnggss ccuosuss MMo S140-030ppmp((h4idg)h) YNooSSiiggnpiiffiiccaacnktFFiinnddiinnggss - couse coenr M Mo S100pmMthigh) 5100ppm (Mig) No SigificntFindings MoSignificantFindings ccoonuss M M5 51 1pppmm0 0 (QiMdi-h0 0 hiigghh)) NoFSiogcniafliNcaenctroFsiinsdings us Mo 5100pm (Midhigh) cous M 508ppm(High) No SignificantFindings Yo SignificantFindings . ccooeus " 66550000pppa((iigghh)) No SFiognciafliNceacnrtoFsiinsdings Coun M 6500ppm igh) Focal Necrosis ccoouuses MM 550000ppppm(Giisghh)) NNooSSiiggaaiiffiicceannttFFiinnddiinnggss . an 000836 Covance 6329224 _OOOOO0O@O@O@O@w0 re@w FdrC t ol Po toy Yto Gte RIopnt IndividualAnimalHistomerpFihndoinglsongLiivecr: 4-WeskRangDie eta- ryToF xicii ty Sn tudyd witi hN-n Metg hyl PerfisoresctEtahannoel(sN-uMelFOfSEo,Tn-6s314m) iin Rdates - _AsteeiNewher Sex DowGrosp [HeepcPadep CscozewTns FFFF 111-000ppppmmm CC(Coounmtmroo)l)) N"NoNoSoSSiiiggginaiifffiiccceasnntttFFFiiinnndddiiinnngggsss - Coo4su20 oumz FFF F 11-00pppmm (CCoonturo)l) 1dpm Cound NNNoooSSiSiiggginaififcciacaannnttFFFiiinnndddiiinnngggsss ccouem EF 2211pppnm QGoow)) NoNSoiSgiuginfiificcaannttFFiinnddiinnggss Coas3s. * 2-1ppm (Low) `NoSignificantFindings Cos F 2-1 pp (Low) Cous31 2-1pp (Low) NNooSSiiggnniiffiiccaannttFFiinnddiinnggss. CSoesas FPF H2o-1ppmpomw(mLoiwd) ) See FF 310pmmmd NNNoooSSSiiiggngaiiififccoaamnntttFFFiiinnndddiiinnngggsss - ocuwan FFEP 33i100ppmm(oowvmad)) NNooSSiiggnaiifficcmannttFFiinnddiinnggss ousme FFFF 3i100ppmm(Coowwmds)) NNooSSiipgiafiiccaunntFFiinnddiinnggss oowwss FF 4+3300ppppmm((6i)) NNooSSiiggniifficcaannttFFiinnddiinnggss cen FE +30ppm0450) No SigifcatFindings = Coesas F 4-30ppm (Mid) No Significant Findings C549 F 4-30ppm (Mid) NoSignificant Findings ed F 4-30ppem(Mid) NoSignificantFindings Coss F 5-100ppem(Mid-high) No SignificantFindings Css? - ouusse PPFF Ss1l0o0opppmmO(Meid)i NNooSSiiggniiffiiccaannttFFiinnddiinnggss Css Coss. Couss7 Coss - cum F 6S0pmCie) FocalNecrosis 94568 9461 F 5-100ppm(Mid-high) F 5-100ppem (Mad-high) F 5-100ppea(Mid-high) F 6-500ppm(Fligh) F 6-500ppm(High) F 6-500ppm (High) F 6-500ppm(High) No Significant Findings No SignificantFindings NoSignificantFindings NoSignificantFindings NoSignificant Findings FocalNecrosis NoSignificant Findings Co4s62 F 6-500ppm(High) `No SignificantFindings - an 0009.57