Document reV97O7VJDyZX1OY517a68GQE

SDRIPTSTH-6295.8, T-6316.4, T-6868.2, T0711 T-7132.1 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY `Title: Comparative Molecular Biology of Perfluorooctanesulfonate (PFOS, T-6295), N-cthyl perfluorooctanesulfonamido ethanol (N-E{FOSE, T-6316), N-Ethyl perfluorooctanesulfonamide (N-ECFOSA, T-6868), Perfluorooctanesulfonamido acetate (FOSAA, T-7071), and/or Perfluorooctanesulfonamide (FOSA, T-7132) of in Rats and Guinea Pigs following Oral dosing. Final Report Date: May 25, 2004 Study Numbers. T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 Strategic Toxicology Study Number: DT-15-B Sponsor: Study Location(s): Study Director: Study Toxicologist: In-Life Start Date In-Life End Date In-Life Start Date In-Life End Date In-Life Start Date In-Life End Date 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul MN 55133-3220 1. 3M Strategic Altemative Toxicology Laboratory 3M Center, Building 270-SB-181 Saint Paul, MN 55133-3220 2. UniversityofMinnesota, Duluth Dept. ofBiochemistry and Molecular Biology Schoolof Medicine 10 University Drive Duluth, MN 55812-2496 Andrew M. Seacat Ph.D, DABT Toxicology Specialist 3M Medical Dept. Corporate Toxicology and Regulatory Services Deanna Luebker M.S Senior Toxicologist 3M Medical Dept. Protocol: Protocol: Amendment #1: Amendment #1: Amendment #2: Amendment #2: 11/16/1998 1120/1998 03/01/1999 03/05/1999 02/19/2001 02/23/2001 1 SDRTPITST-B6295.8, T-6316.4, 6868.2, T-7071.1, T-7132.1 `SumTmaorayfCbonltenets Aes} ADmTeInSdBmePnrtotNoucomlbePrroc|ePdruorecse.dures. rm mo] AITOSTEMOu 0 co et mms ----------0 Results -- LBiIvReOrGFRlu)orPocheRmicMal Conrceontmraetions rmsensesessmsmrseismarermmmae momma 12 ClinicalChemisty. mm CPoanlclmuistiooyn]s.COAOxidase (PCONO)BCVY....crerrrensenr serene 1136 SHNMR PIGS rims 1) F "TablIe.o Average CumulativeDose. m-- 19 "Table 2. SumofBmodyaweigrhtsynd BW CHANGES... 20 "`TTaabbllee 43..PSeurmcmeanrtyIoniftiaOlrBgoadnwyeWiegihgthsta,nCdOOMrgIaNnG0Body WDA.eight RatO. cv 22 ""TTaabbll56ee.. LLiivveerr-PaenrdflKuiodrnoseuyl-ftoonaBmoiddeysWaenidgMhettRaabtoiliotse,VCaolmubeisnferdoDmaRtast,sand Guinca Pigs. Analyses it performed atthe UniveOrFRsOCiHOtSIyE ...rvvvsvsvsvesssssee J "ATnaabllyes7e.sSpuemrmfaorrmyeodfLatitvheer3PMerEfnluvoirroosnumlefnotnaalmiLdaeb.s and Metabolite Values from Rats and Guinea Pigs.2% "TableHepatic Palmitoyl CoAOxidaseACV, COmbInGdDA ......vvrvvrverrrronrrn 27 `A`pApppeennddiixx 21.-DAneavlyitaictail10oLtinhveserSPraomtpocloelL..a.b Identification...._.__._______________._...22 ``AAppppeendnidxi43x.. BCoudmuylWateiivgehDtoIsenInddiivivdaunaidlSdaundmumSaaurmlymaDrayta.d.ata... A 31 ``AAppppeendnidxi65x.OLrigvaenr wFeliugohrtosc,hOermigcaanltDoaBtaW. rIatniosd. IindiavvniidduSdaulmuamnaadrlSyumdmataar.y..d.a.t.........__.... W45 `A. Ratand GuineaPig LiverFCconcentrati(oAnlslunaireutggs). Analyasttehse Universiotfy B. Ratan Guinca Pig. Liver FCPercoefDnoste Evaluations. Analysesat the Universiotfy CR . oRatLtiversPFOSXmAni alayi tsthee3sM Environmental Lab. ona5l0 (DA.ll unRitastaarnedggGu.inTehaePi3gMLiEvnevrirFoCnPmeenrtcaelntLaobfDoonlsyeEavnaalluyazteidornast.lAinvearlyssaempsleast)t.he 3M environmenta5l 4 Lab. 55 AppenE.diTxec7h:niCclailniRceaploCrhte:miLisvterry.Fluorocarbon Metabo-lUniivteresistyofRocheste--r. ------l5 IndivanidSdumumaarlyClinical CRETISTY DOR... creer 65 AB.. SStaattiisstticcssoonnCClliinniiccaall CChehmiestrmisfifoorrsrGauesi,nmseaalPegasn,dmafleemaalnedvafleumeaslecvoambliuneesd.c..ombined. 10708 Appendix 8. HepaticPalmitoy] Co-A 0X38 ACAI o.oo 135 A`pApppeennddiixx 91.0: EFfifneacltRoefpaocruttfeoFrCFaOdSmAin(isTt-raTtIio3n02so.n catalase and acyICoA oxidase expression..... 113493 `AppAenpdipx1e121:,nEFndidnpaioliRxnetpoCrortrfeolratFiOonSAtoAL(iIv-e70r7PL.F1O)S.._________________ 1i6s2) `AB.. CCoorrrrooeeffeeffllffeeccaattssbbttyydiiidifffoofeerrennennttttrreeaattmmeennttggrroouuppssto0 liivveerr PPFROOSSiinn GuineaPrais.g._,_.m..a__l.e_.s..a.n..d...f..e.m1a&l2e COMBI... 185 2 SDRIPSTHT6295, T6316.4,T-6868., T-107L1, T-7132. Summary Adult male and female Sprague-Dawley rats and Harlan guinea pigs received oral gavage dosesofvehicle control (2% Tween 0), 40 mg/kg/day (mkd) perfluorooctanesaulfonate (PFOS, T-6295), 40 mkd or 160 mkd N-Ethyl perfluorooctanesulfonamido ethanol (NE{FOSE, T-6316), or40 mg/kg/day N-Ethyl perfluorooctanesulfonamide (N-E(FOSA, T6868) for four days and were humanely sacrificed on the fifth day. Male SpragueDawley rats received oral gavage dosesof vehicle control (propylene glycol), 160 mkd perfluorooctanesulfonamidoacetate (FOSAA, M556, T-7071),or40 mkd perfluorooctansulfonamide (FOSA, T-7132) for four days and were humanely sacrificed on the fifth day. Al animals survived. The percent initial body weights were significantly decreased in rats by 40 mkd PFOS and 160 mkd N-E(FOSE significantly reduced body weight compared to controls to 85% and 87% inital body weight, at average liver PFOS concentrations of> 600 ug/g. 40 mkd FOSA and 160 mkd FOSAA significantly reduced body weight to 93% and 95 % of initial body weight, respectively, at average liver PFOS concentrations of 140 ug/g and 200 g/g, respectively. Neither 40 mkd NEtFOSEof 40 mkd N-EtFOSA had a significant effect on body weight in ats. In the rat, liver to body weight ratios were significantly increasbeyd 40 mkd PFOS at average liver rPaFtsOfSrcoomncalelntortahteriodnossoe gf>ro6u0p0s,ubg/ugt.thIenscercehaasendgelsivwererteo bnootdysiwgeniifgihctanrtatcioosmpwaerreedftoountdhein control. "The guinea pig percent body weight was significantly decreased by 160 mkd N-E(FOSE and40mkd PFOS 10 91% and 93% initial body weight, at average liver PFOS concentrationsof419 g/g and 171 g/g, respectively. Guinea pig liver to body weight ratios were unchanged. Guinea pig kidney to body ratios were significantly increased by treatment in all the dose groups measured, i. 40 mkd N-EFOSE, N-EFOSA, or PFOS. In rats, cholesterol (CHOL), riglycerides (TRIG), alkaline phosphatase (ALKP), and aspartate aminotransferase (AST) were significantly lowered in rats treated with40 mkd PFOS and 160mkd N-EGFOSE. Significant decreases in serum potassium (K") occurred in rats treated with 40 mkd PFOS,40 mkd N-E(FOSE and 40 mkd N-E(FOSA. In rats, 40 mkd PFOS caused asignificant increase in hepatic palmitoyl CoA oxidase (PCoAO) activity, but 160 N-EXFOSE did not. Exposureof ats to 160 mkd N-E-FOSE caused a doublingofthe specific activity of lauryl CoA oxidase (LCoAO) and a 2-fold increase in the concentration ofmRNA encoding for PCAO in rat liver, but catalase activity and catalase mRNA were unchanged. Exposure of rats to 40 mkd PFOS caused a 2-fold increase in LCoAO activity and a 3 to 6-fold increase in PCOAO mRNA expression in rats. Treatmentwith40 mkd FOSA or 160 mkd FOSAA caused a significant increase in hepatic cytochrome P430 content and Acyl CoA oxidase activity in male rats. PFOS was apparentlay more potent peroxisome proliferator than N-EFOSE. PFOS significantly induced PCOAO activity whereas N-EWFOSE increased, but did not significantly induce PCOAO activity, even though both treatments achieved similarly high liver PFOS concentrationsofgreater than 600 g/Kg. PCoAO activity was not 3 DSRTPITST-B6295.8, T-6316.4, 68682, T-1071.1, T-7132.1 determined for the 40 mkd FOSA and 160 mkd FOSAA treated male rats, but a 2-fold induction of acyl CoA oxidase activity occurred at relatively lower average liver PFOS concentrations of 193pig/gand 140 ug/g , respectively, with an equivalent or greater fraction of the total liver fluorochemical was contributed by the parent compound or the metabolite FOSA from the FOSAA treated rats. These data suggest that FOSA may be an equally potent peroxisome proliferator to PFOS, but not the N-acetyl metabolites of N-Et FOSE. In the guinea pig, potassium (K*) values were significantly reduced by 40 mkd PFOS and N-EtFOSA. Neither 40 mkd PFOS or N-EtFOSA, nor 40 mkd or 160 mkd N-EWFOSE. caused peroxisome proliferation in either gender ofguinea pigs. `These results showed that PFOS, N-EtFOSE, FOSA and FOSAA all caused indications ofperoxisome proliferation in rats, but not in guinea pigs. Theseresults are in concordance with effectsofclassical peroxisome proliferators, in that the response is specific to certain species 5 SDRTPITST-B6295.8, T6316,T-6868.2, T0711, T-7132. Introduction: `The objectiveofthis study was to investigate and compare the molecular mechanisms of peroxisome proliferation in rats and guinea pigs. Three compounds derived from perfluorooctane sulfonate and known to cause peroxisome proliferation in the rat were initially tested, perfluorooctanesaulfonate (PFOS), N-Ethyl perfluorooctanesulfonamide (N-EtFOSA) and N-Ethy! perfluorooctanesulfonamido ethanol (N-EtFOSE). By amendment, two other compounds, perfluorooctanesulfonamido acetate (FOSAA) and perfluorooctanesulfonamide (FOSA) were added to the study. The ultimate metabolite of N-EFOSA, N-E(FOSE, FOSAA and FOSA was presumed to be PFOS, however there is some debate about that. The hypothesis that these fluorochemicals would induce peroxisome proliferation in the rat, but not the guinea pig, was based on several lines of evidence indicating that guinea pigs and primates are resistant 0 peroxisome proliferation. However, the molecular and biochemical mechanisms that differentiate the response of these species to peroxisome proliferators for the perfluorosulfonamiwdaess unclear. The specific aimsofthis study were to: 1. To elucidate the molecular response in both rats and guinea pigs by measuring the induction of mRNAofgenes that are associated with peroxisome proliferation. 2. To measuring the hepatic activityofperoxisomal enzyme systems and fatty acid binding proteins. 3. To perform standard toxicity testsof serum clinical chemistry and to examine the liver for histological changes which may indicate an explanation for the species differences seen in response to these compounds. 4. To correlate any observed alterations of the above functions to liver and serum levels ofperfluorosulfonamides and their metabolites. `This study was part ofa seriesofinvestigations designed to understand the `molecularand biochemical mechanisms for the effectsofthese compounds observed invivo. This study was carried-outi collaboration with other investigators. Dr. Ken `Wallace, Universityof Minnesota Duluth, who has been engaged in studies designed to understand the effectsofthese perfluorosulfonamides on mitochondrial bioenergetics, `performed molecular and biochemical analysesofthe inductionofgenes associated with peroxisomal proliferation and/or cell replication. Dr. Kris Hansen and Lisa A. Stevenson ofthe 3M Environmental Technology and Safety Services lab performed quantitative serum and liver perfluorosulfonamide metabolite analyses. Dr. Lin Xu performed quantitative liver perfluorosulfonamide metabolite analyses on some samples in Dr. Marion Anders lab at the Universityof Rochester Study Timelines: `This study (DT15-B) was conducted in three parts, the protocol and protocol amendment numbers 1 and 2. The protocol had an in-life start date of 11/16/98 and an in-lfe end date of 11/20/98. Amendment number 1 had an in-life start date of 3/1/99 and an in-life end dateof 3/5/99. Amendment number 2 had an in-life start dateof2/19/01 and an inlife end date of 223/01. Amendment #3 was procedural only. s SbRTPITSTH-62958, T-6316.4, 68682, T0711, T-7132.1 Regulatory Compliance: "This was an exploratory study and thus classified as non-GLP as explained in TOX SOP 0950, Strategic Toxicology Lab GLP Program Procedure. Test Material: "The sponsor provided samplesofall luorochemicals to the investigators. Analytical `documentationofthe stating material was the responsibilityofthe sponsor. A chemical `composition specification sheet was kept on file. Compoundswere stable at room temperature. Test material was stored tightly sealed at room temperature. "The T-numbers, chemical names, abbreviations used for these samples, and the chemical structuresofeach ofthe compounds that were tested in this study are given below. The currently accepted abbreviations for each compound are in bold 1. Vehicle control: 2% Tween 80, or propylene glycol. 2. T-6295: Perfluorooctane sulfonic acid, potassium salt (perfluorooctanesulfonate), POS, FC-95, Formula: C4fsS0s- K', MW = 538.1 mole). 3. T6316: Necthylperfluorooctane sulfonamido ethanol, (narrow Range N- Ethyl Perfluorooctanesulfonamido ethyl alcohol), N-EFOSE, Formula: C,fsSO:N(C:HICH,CHOH, MW = 571.06). EtFOSE, FC-10, 4. T-6868: Neethyl perfluorooctane sulfonamide, (perfluorooctane sulfonyl ethylamide), PFOSA (as sated in the protocol, but not used for ths compound in this report), N-EtFOSA, PFOSEA (abbreviation used by the 3M Environmental lab), FX-12, Formula CifSO:NHCHi, MW = 527.2). 5. T7071: Perfluorooctanesulfonamido acetate, FOSAA, MSS6, (Formula iFSONHCH,CO0, MW -- 556 g/mole) 6. T-7132 Perfluorooctanesulfonamide , FOSA, PFOSA (abbreviation used by the 3M Environmental Lab), Formula CsF17SO;NHs, FOSA, MW = 499.06 gmole). 7. Wyeth-14643 (WY, MW =323.79 g/mol) was obtained from Chemsyn Science Laboratories, Lexena, KS. Wyeth-14643 (WY), was added to DTISA as positive control dose group for hepatic peroxisome proliferation. 6 DSRTPITST-B6295.8, T6316, T-6868.2, T107L1, T-7132.1 Methods `The methods and dose groups for eachpart ofthis study are summarized below. Detailed methods are given in the protocol and amendments. Deviations to the protocol are listed. in Appendix | DTIS-B Protocol Procedures "Theprotocolhad an in-lfe start date of 11/16/98 and an in-life end date of 11/2098. Under the protocol, eight male and eight female Sprague Dawley Rats, 10-12 weeks old weighing approximately 250-300 grams at the timeofinitiation were obtained from Harlan Laboratories, Inc. Eightmaleand eight female Hartley Guinea Pigs 10-12 weeks old and weighing approximately 600 to 750 grams at the timeofinitiation were obtained from Harlan Laboratories, Inc. Each dose group contained 2 animals/sex/species. The dose groups were vehicle control (2% Tween 80), 40 mg/kg/day N-Et FOSE, 40 mg/kg/day N-EFOSA, and 40 mg/kg/day PFOS. A 20 mg/mL suspensionofcach test compound was prepared in 2% Tween 80 in a glass tissue grinder. The animals received four consecutive daily doses. The dayofthe first dose was designated day zero, thus the doses were administered on days 0,1,2 and 3ofthe study. Rats receive the test compound suspended in 2% Tween 80 or the vehicle control by oral gavage at a volume of2 ml/kg body weight. The guinea pigs received their daily oral dose volume of2 mk by droplet in the back of the mouth. The animals were humanely sacrificed on day4 Amendment Number 1 Procedures Amendment number 1 had an in-life start date of 3/1/99 and an in-life end date of 3/5/99. The purpose of amendment 1 wastoadd groups ofboth male and female rats and guinea pigs treated with vehicle control, N-E(FOSE (T-6316) or PFOS (T6295). The histological and clinical chemistry resultsofthe treatment groups under the protocol were not remarkably different than in the controls, and the tissues for northern blot analysis were delayed during shipping and were degraded. Therefore, amendment 1 was designed to replace the specimens that were lost and elevate the dose of N-E(FOSE administered to achieve a more effective level, `Twelve rats and twelve guinea pigs total (6 male, 6 female/species) were used under the protocol amendment #1. Each dose group contained 2 animals/sex'species. `The dose groups were vehicle control (2% Tween 80), 160 mg/kg/day N-Et FOSE and 40 mg/kg/day PFOS. For N-EtFOSE, a suspension of 80 mg/ml N-E(FOSE in 2% Tween 80 was prepared and a volume of2 ml/kg was administeredbyoral gavage to the rats and by droplet in the back of the mouth to the guinea pigs on days 0 through day 3 ofthe study. `This dose was comparable to the cumulative doseofN-ECFOSE that induced peroxisomal PCOAO activity in the rat in a 4 week feeding study with 300 ppm N-EWFOSE (Ref. 3M Medical Dept. T-6316.1), and was less thanhalfofthe LDS0. 7 SDRTPITST-B6295.8, T-6316.4, T-6868.2, T0711, T-7132.1 For PFOS, a suspension of20 mg/m of PFOS in 2% Tween 80 was prepared, anda volume of2 mU/kg was administered by oral gavage to the rats and by droplet in the back. of the mouth to the guinea pigs on day zero through day 3of the study. The cumulative dose of PFOS delivered under protocol amendment #1 was ~160 mg/kg, as wasused in the protocol. The cumulative dose was below the LDS0 for PFOS and well above the threshold for inducing peroxisome proliferation in the rat Amendment Number 2 Procedures "The purposeofamendment 2 was to add groups male ras treated with vehicle control, perfluorooctanesulfonamidoacetate (FOSAA) or perfluorooctanesulfonamide (FOSA) at doses equivalent to the doses of N-EFOSEand PFOS, respectively, in protocol amendment #1 in order to compare the effects and metabolite profilesofthese compounds in ats at equivalent doses. Amendment numbe2r had an in-life start date of 2/19/01 and an in-life end date of2/23/01. Nine male rats were used under the protocol amendment #2. Each dose group contained 3 male Sprague Dawley Rats, 10-12 weeks old weighing approximately 250-300 grams at the timeof initiation were obtained from Harlan Laboratories, Inc. The dose groups were vehicle control (propylene glycol), 160 mg/kg/day FOSAA, and 40 mg/kg/day FOSA. The vehicle control was delivered by oral `gavage ata volumeof S mUkg body weight on days zero through da3yofthe study. A larger volume of 5 ml/g was used than in protocol amendment #1 because only rats were dosed by oral gavage, whereas the guinea pigs cannot be dosed by oral gavage. For FOSAA, a suspension of 32 mg/ml FOSAA in propylene glycol was prepared and a volume ofS ml/kg was administered by oral gavageto the rats on days 0-3. This dose achieved a cumulative dose of 640 mg/kg FOSAA. For FOSA, adoseof40 mg/kg body weight was administered via gavage to rats on day 210 through day 3of the study. A suspension ofmg/ml ofFOSA in propylene glycol was prepared, and a volume of5 ml/kg was administered by oral gavage to the ats. This dose achieved a cumulative dose of 160 mg/kg aftr four daysofdosing. The dose of FOSA was the same as the dose of PFOS administered under amendment #1 ofthis apdromtioncioslt.eTrehdeuLnDdeSr0hfiosr pFrOotSoAcoilswnaost kbneolwonw,thbeutLtDhSeOcufmourlPatFiOvSeodfo2s5e1ofmFgO/kSgAfor POS. in com oil, as a pointofreference. Specimen Handling: Liver and sera were collected and frozen rapidly after euthanasia according to the details described in the protocol and amendments. A one to two gram aliquotofthe liver samples shipped in dry ice to the analytical laboratories listed in the protocol and amendments. The identificationofeach liver sample sent to each lab is listed in Appendix 2. s DSRIPITST-H6295.8, T-6316.4, 6868.2, T-707L1, T-71321 Analytical methods Certain liver samples were sent for analysis by previously published methods for P450 content, Lauro] CoA oxidase activity (Poosch and Yamazaki 1986) and protein content (Bradford 1976) in the laboratory of Ken Wallace Dept of Biochemistry and Molecular Biology at the University ofMN by the following methods: Enzyme Sample Preparation ~ The enzyme fraction consisted of the 6,000 g. supermatantof a 10% (wt/vol) homogenate of 0.5-1.0 g frozen liver tissue in 300 mM mannitol-10 mM HEPES-1 mM EGTA (pH 7.2). Protein concentration was estimated according to the method of Bradford using commercial bovine serum albumin as standard. L-Cod Oidase Assay ~ The equivalent of ca., 5 pg/ml tissue homogenate was suspended in 60 mM KH:PO-0.02 % Triton X100 (pH 7.4) containing 1 mM phydroxyphenylacetate (PHPA), 4 units/ml peroxidase, 20 uM FAD, and 60 uM lauryl-CoA (LCOA). The reactions were allowed to incubate at 37C for 30 min in a shaking water bath and terminated by adding 3 volumes of 2 mM KCN in 100 mM sodium carbonate (pH 10.5). The concentration of HO; generated during the reaction was estimated from the fluorescence of PHAP as measured with an excitation wavelength of 317 nm and emission at 405 nm. The fluorescence was calibrated with commercial HO; and the results are expressed as nmol peroxide generated/min/mg mitochondrial protein. Protein was `quantitated by the Bradford method. Catalase Assay - The activity of catalase was estimated by a modification of the original method published by Claiborne and Fridovich (J. Biol. Chem. 254, 424552,1979), which is based on the direct measurement of H0, disappearance as quantified spectrophotometrically at 240 nm. In this procedure, the tissue sample: was diluted in 50 mM potassium phosphate (pH 7.0). The medium was warmed 10 27C and the reaction initiated by adding 10.3 mM H0x. The progress of the reaction was monitored at 240 mm for min. Catalase activity was estimated pfrrootmeinth(eTaibnilteial1).liOnenaer urnaitte o(facEt*i=vi4t3y.i6s dmefMincemd TMas)thaatndameoxupnrteosfseednzasymueniwthsi/cmhg catalyzes the decomposition of| mole of HO; per min. Northern Blot Analyses - Quantitation of mRNA for both acylCoA oxidase (ACOAO) and catalase were performed by Northern blot analysis of quick frozen liver samples from treated rats and guinea pigs. Approximately | goffrozen liver was powderized in liquid nitrogen using a mortar/pestle. Total RNA was. recovered using the PERFECT RNATM isolation kit and the concentration quantified spectrophotometrically at 260nm. The RNA was electrophoresed on a 1% agarose gel, blot transferred t0 a cellulose membrane and hybridized to the cforrormespproinmdeirnsgtoracnad,om3l5y0 [b*a?sPe] lsaebqeuleendceoolifgtohnueclreeostpiedcetsivtehartatwelirveerPgCenRe.ampmlRifNieAd 9 DSRTPITSTB.62958, T-6316.4, 68682, T-1071L 7132.1 band density was quantified autoradiographically using phospho-imaging software. Certain Liver samples were sent for analysis for the parent compound (s) and the `metabolites by the 3M Environmental Technology and Safety Services using published `methods (Hansen et al. 2001). The detailsofthe methods used and the resultsofthe analyses that were completed were issued in an analytical report from the 3M Environmental Lab on May 8, 2003 (3M Study No. FACT-TOX ~107, 3M Laboratory LIMS No.E01-0129). The resultsof analyses that were completed are included in this report and integrated with the body weight and liver weight data. The abbreviations that the 3M Environmental lab used to identify compounds were different than the ones listed above, and are found associated with the primary data tables from the lab. The lab analyzed foar metabolite, perfluorooctanesulfonamidofethylacetate (PFOSAA), that was never dosed. The abbreviations used for cach analyte that the lab used as standards and analyzed for in liver samples are given below, followed by the chemical formula and the abbreviation used in this report. PFOS= PFOSA = PFOSAA = EIFOSE= MsS6= PFOSEA= Perfluorooctanesulfonamide (Formula C4F;,S0;-, PFOS) Perfluorooctanesulfonamide (Formula CsFi7SO:NH;, FOSA) Perfluorooctanesulfonamidoethyl)acetate. (This compound was also referredtoas Perfluorooctanesulfonamidoacetate in the raw data tables of FACT TOX 107). (Formula CyF17SO:N(C:HsJCH,COO', N-EtFOSAA), narrow Range N-Ethyl Perfluorooctanesulfonamido ethyl alcohol (Formula CiFy:SO:N(C:H,CHCHOH, N-EtFOSE) Perfluorooctanesulfonamidoacetate (Formula C.F,SO:NHCH,CO0" FOSAA) Perfuorooctane sulfonyl ethylamide (Formula C.F,SONHCH, ,NE(FOSA) Certain other liver samples were analyzed for parent compounds and metabolitesof the fluorocarbons by LC-MS/MS by Dr. Lin Xu in the laboratory of M. W. Anders DepartmentofPharmacology and Physiology, University of Rochester using previously published methods (Hansen et al. 2001). The results are reported in this report and integrated with the body weight and liver weight data. Certain other liver samples were sent to Covance in Madison WI and were analyzed for palmitoyl Co-A Oxidase activity as an indicatorofperoxisome proliferation using a validated methodbasedon published methods (Lazarow 1981). The principleof the assay is that in the presenceof palmitoyl-Co-A, the third step of the b-oxidation spiral involves the reduction of NAD to NADH, which can be measured spectrophotometrically a1:340 nm. The results were reported the 3M on October 22, 2002 ina letter report from Covance and the data is included in this report. No formal report was written by. Covance. 10 DSRTPITST-B6295.8, T-6316.4, 68682, T-7071.1, 7132.1 Results Biological Parameters All animals survived to the end of the study. No gross observations were noted during the in-life phaseor at necropsy. `The cumulative doses ranged from approximately 33 to 53 mg fluorochemical in rats, and from approximately 67 to 123 mg fluorochemical in guinea pigs receiving 40 mg/kg/day. `The 160 mg/Kg/day dose group animals received proportionally higher cumulative doses (Table 1). The body weights ofthe guinea pigs that were dosed at different times were really different, which accounted for the wide range in cumulative dose in the guinea pigs. Individual and summary cumulative dose data are shown in Appendix 3. Average body weights decreased significantly over the dosing period for female rats given 40 mkd PFOS and 160 mkd N-EtFOSE (Table 2). Average body weights decreased significantly over the dosing period for male rats given 40 mkd PFOS or FOSA and 160 mkd N-EFOSE or FOSAA (M556). Individual and summary body weight data are shown in Appendix 4. `The male rats given N-E{FOSE at 40 mkd had significantly increased liver weights (Table 3). Male rats given PFOS at 40 mkd had a significant increase inliverto body weight ratios. Male rats treated with PFOS, N-EtFOSE and N-EFOSAat40 mkd all had significantly lowered kidney weights and Kidney to body weight ratios. Female kidney `weight and kidney 10 body weight ratios were not significantly different from control values. Kidney weight data was not obtained for the other dose groups. Individual and `summary body weight data are shown in Appendix Male and female percent of initial body weights on day four were combined for each dose group and analyzed together. The combined rat percent body weight was significantly decreased by 40 mkd PFOS, FOSA, and FOSAA,andby 160 mkd NEtFOSE (Table 4). The combined guinea pig percent body weight was significantly decreased by PFOS, N-E{FOSA and N-EFOSA at 40 mkd, and by N-E(FOSE at 160 mkd. `Organ weight ratios (liver to body weight and kidney to bodyweight where available) were combined for males and females independently from each dose group in all parts ofthe study and analyzed together. Combining the relative organ weights was done to increase theN for each dose group for analysis and isjustified because the organ `weights have been normalizedbybody weight. PFOSat40 mkd significantly increased liver weight to body weight ratios in the rat combined data (Table 5). The combined male and female guinea pig liver to body weight ratios were not significantly different from the control group values for anyofthe treatments given. Significantly increased kidney to body weight ratios were found in guinea pigs treated with NEFOSE, N-E(FOSA, or PFOS at 40 md. nu SDRTPITST-B6295.8, T-6316.4, T-68682, T-10711 T-7132.1 Liver Fluorochemical Concentrations "The liver fluorochemical concentrations in rats and guinea pig livers are summarized from the analyses performed at the Universityof Rochester (Table 6), and at the 3M Environmental ab (Table 7). The individual and summary liver fluorochemical concentrations data are presented in Appendix 6. The 3M Environmental lab analyzed liver samples from the PFOS 40 md and the N-EtFOSE 160 mkd rat dose groups and did not measure fluorochemical levels in the guinea pig livers. Aliquots ofthe livers that were analyzed by the 3M Environmental lab were also analyzed by Drag Andres lab at the UniversityofRochester. The individual rat liver data showed that one lotofthe samples analyzed at Rochester had a high background of FOSA in the control group. The Rochester analyses tended to have higher PFOS and FOSA determinations than the 3M Environmental ab, however the values for PFOS are listed as 35% accuracy, for FOSAA + 50% accuracy, and for FOSA, FOSAA, EtFOSE and N-EFOSA the values are. listed as qualitative only in the final report (FACT TOX 170). Therefore, the differences in the measurement of PFOS and FOSA fall within the experimental error inherent with the methods used. "The percentofthe dose that was in the liver was calculated for each dose group from the amountofthe total liver PFOS containing species, derived from the sum of the. concentrationsofall luorochemical species detected in the liver and designated TLPFOSX, and for the amountof PFOS itselfin the liver. The amount (mg)ofall fluorochemical species detected in the liver was derived from the TLPFOSX times the liver weight. The percent of fluorochemical dosed present in the liver was derived for both the TL PFOSX and for PFOS itself for the values derived from both the University of Rochester lab and the 3M Environmental lab (Appendix 6, B and D). In rats dosed with PFOS, the percent of PFOS dosed present in the liver as PFOS ranged from about 17 1023 percent in both male and female rats, andwas consistent between labs. In guinea pigs dosed with PFOS, the percentage of the total amount of PFOS dosed that was present in theliverswas far less than in the rat, between 3% and 5%. Guinea pig livers were only analyzed at the University of Rochester so a comparison between labs for the fluorochemical content in the livers ofguinea pigs cannot be determined. However, it can be seen from the data presented in Tables B and D in Appendix 6 that the TL PFOSX values derived at the UniversityofRochester and at the 3M Environmental for rats treated with 160 mg/kg/day N-EXFOSE are about the same at cach lab. "The UniversityofRochester analyzed both rat and guinea pig livers from animals treated with either 40 mkd PFOS, N-EtFOSE or N-E(FOSA, or 160 mkd N-E(FOSE. The TLPFOSX in guinea pigs treated with 160 mkd N-ECFOSE was lower than in the rat, particularly in the males guinea pigs. The percentofthe dose that was present in the liver as PFOSX was approximately 2-fold the percentofthe dose present in the liver as PFOS itself, in both guinea pigs and in most rats treated with N-EtFOSE and FOSA. However, the female rats treated with 160 mkd N-EWFOSE had greater than 70%ofthe TLPFOSX as PFOS in thei livers. Conversely, the percentageofthe dose that was present as PFOS in the liver of rats treated with 160 mkd FOSAA had high levels of FOSAAitselfand FOSA, with low levels of PFOS, n SDRTPITTS.H6295.5, T6316.4, T68652, F-70711, T7132 Clinical Chemistry Serum clinical chemistries for rats and guinea pigs are presented in Appendix 7. Clinical chemistry values were combined from males and females in each species and statistics were performed. In rats, cholesterol (CHOL), triglycerides (TRIG), alkaline phosphatase (ALKP), and aspartate aminotransferase (AST) were significantly lowered in rats treated with 40 mkd iPnFrOatSs atnredat1e6d0wimtkhd4N0-mEk{dFOPSFEO.S,S4ig0nimfikcdanNt-dEec(rFeOasSeEsaninds4e0rummkpdotNa-sEsWiuFmOS(KA',)woictchurthreed `most significant decreases occurring inthe 40 mkd PFOS dose group. Albumin (Alb) and total protein (TP) were significantly increased by 40 mkd PFOS, and creatinine (CREAT) was significantly increased in the 40 mkd PFOS and 40 mkd N-E(FOSA dose `groups. These changes in clinical chemistry are consistent with previous studies with PFOS and N-EtFOSE. None of theotherclinical chemistry parameters were significantly different from control values in rats. In the guinea pigs combined male and female clinical chemistry analysis, there were no significant changes in cholesterol, triglycerides, alkaline phosphatase, or aspartate aminotransferase. Albumin, total protein and creatinine were not significantly changed. Potassium (K') values for male and female the guinea pigs combined were significantly reduced by 40 mkd PFOS and 40 mkd N-E(FOSA, similartothe rat. Alanine aminotransferase (ALT) was significantly increased by treatmentofguinea pigs with 40 `mkd PFOS.. None of the other clinical chemistry parameters were significantly different from control values in ats Palmitoyl CoA oxidase (PCoAO) activity Hepatic palmitoyl CoA oxidase (PCoAO) activity data for males and females from cach dose group was combined and analyzed together. PFOS at 40 mg/kg/day for four days caused a significant increase in hepatic PCOAO activity in rats (Table 8). N-EWFOSE at c1h6a0nmgkedocfcuorr4reddayinstdhiedgnuoitnseiagpniigf.icIanndtilvyiidnucarleaasnedhseupamtmiacrPyCPOCAoOAiOn vraatsl.uesNoforsimganlifeiscaanntd females separately are shown in Appendix . Peroxisomal enzyme activity and gene expression The effects of four days of oral dosing of 160 mg/kg/day N-EFOSE or 40 mg/kg/day PFOS for four on catalase and acylCoA oxidase gene expression and enzyme activity in liver tissue from exposed rats and guinea pigs are presented in Appendix 9. These data were presented as a poster at the 2001 Society of Toxicology meeting (Wallace ef al. 2001). Acute exposure of rats to 160 mkd N-Et-FOSE caused a doubling of the specific activity of LCOAO and a 2-fold increase in the concentration of mRNA encoding for 5 DSRTPITSTB.62958, T-6316.4, T:68682, T1071, T7132. PeoAO in liver from both male and female rats (Appendix 9). Catalase activity and catalase mRNA were unchanged by exposure to 160 mkd N-Et-FOSE in livers from both sexes. Exposure of ats 10 40 mkd PFOS caused a 2-fold increase in LCoAO activity for both sexes, and possibly a slight increase in catalase activity in liver from male, but not female, rats. Acute exposure of rats to 40 mkd PFOS caused a 3- to 6-fold increase in PCA mRNA expression that was more pronounced in female compared to male rats. Exposure of guinea pigs to N-Et-FOSE and PFOS did not stimulate LCoAO activity or catalase activity in either sex. The guinea pig mRNA encoding for PCOAO was undetectable, even following exposure to N-Et-FOSE or PFOS (Appendix 9). Treatmentofrats with 40 mkd FOSA caused significant increases in hepatic cytochrome P450 content and Acyl CoA oxidase activity (Appendix 10). Treatmentofrats with 160 mkd FOSAA caused significant increases in hepatic cytochrome P450 content and Acyl CoA oxidase activity (Appendix 11). The induced the expressionoftheseof these proteins in liverofrats indicated that FOSA and FOSAA were peroxisome proliferators in rat. For the 40 mkd FOSA and 160 mkd FOSAA treated male rats, a 2-fold induction ofacyl CoA oxidase activity occurred at average liver PFOS concentrations of 193 ug/g. and 140 ug/g, respectively. These liver PFOS concentrations were lower than the liver concentrations in the PFOS treatment group. In FOSA treated rats, the parent compound, FOSA, contributed an equivalentorgreater fractionofthe total liver fluorochemical as did the FOSA metabolite PFOS and cach of these fluorochemical species represented approximately 0.3%ofthe dose in the liver. In contrast, the FOSA-glucuronide present in FOSA treated rats contributed only a small fractionofthe TLPFOSX (Appendix 10). In FOSAA treated rats, the metabolite FOSA, and the parent compound, FOSAA, cdoindttrhiebumteetdabaonli2tteoP3FOtiSm,esa,nrdesrpeepcrteisveenltye,dtahepparmooxuinmattoeftlyh0.t1o4ta%l alnivder0.fl2u4o%roocfhetmheicdaolsethiann the liver, respectively (Appendix 11). Taken together, these data suggest that FOSA may be an equally potent peroxisome proliferator to PFOS in rats, and that the N-acetyl metabolites of N-Et FOSE (NE{FOSAA, and FOSAA) are weaker peroxisome proliferators than either POS or PFOSA. The compounds FOSA and FOSAA, were not tested in guinea pigs. Discussion "The analytical data showed that FOSA was consistently identified as a metabolite of PFOS, whether PFOS was administered directly or formed as a metabolite, but the source. ofthe amino group is not readily apparent. FOSA was present in someofthe control ssuabmmpiltetsesduabtmdiitftfeedrefnotrtainmaelsy.sTehseatforgmiavteinontiomfe,PbFuOtSnAotfirnoomthPeFrOgSroturpesaotfedcoanntirmoallsswamapsles determined in all ofthe liversofall PFOS treated animals that were analyzed at the Universityof Rochester, and at the 3M environmental lab for the same samples, which 1 SDRIPTSTH-62955, T-6316.4, T6868, T707L1,T7132. had no background PFOSofPFOSA in the concurrent control from cither lab. Clarification of the formationof the FOSA following treatment with PFOS should be: obtained in further studies. Whatever its routeofformation, FOSA was metabolized to FOSA N-glucuronide. Nco-uElWdFaOrSisEe aflrcoomhotlhegiNv-edseertihseyltaotaiornaongfeoN-fEmaFjOoSrEaanldcomhionlo,ramnedtaNb-oEli(tFesO.SAFAOScoEuladlcaorhiosle by the oxidation ofthe alcohol to the carboxylic acid. Glucuronidation of the parent NEtFOSE alcohol would give the observed N-EtFOSE alcohol glucuronides. FOSAA could be formed by the N-deethylationof N-EIFOSAA or by the oxidationofFOSE alcohol, or both. FOSA could be formed by the N-decthylation of N-EtFOSA or by the removalofthe carboxymethyl group of FOSAA as glyoxylate. FOSA N-glucuronide mFaOySAbeAfwoorumledd bgiyvethFeOglSuAcuorronliodsasotfiotnhoefgFlOycSiAn.e mLooisestoyftwohuelcdagribvoexyPmFeOtShydlirgercotluyp. from "cTohnececnotrrraetliaotniosnwoerfae faenawolyfztehdebmyodsotsseiggnriofuipcainnt rtaotxsicandengdupionienatspitog.s (liAvpeprePnFdiOxS 12). "PTFheOSdeactre4a0semkindtahendpeNr-cEenWtFiOnSitEialatbo16d0ymwekightto wa5s%maonsdt8s7tr%onignliytiaelffbeocdtyedwienigrhatts,bayt average liver PFOS concentrationsof > 600 ug/g. The percent initial bodyweightwas also decreased in rats by FOSA at 40 mkd and FOSAA (M556) at 160 mkd to 93% and 95% of initial body weight, respectively, at average liver PFOS concentrations of approximately 140 t0 200 g/g. A greater fractionofthe total fluorochemicalin the liver oFfOtShAe.FSOiSmiAlaArltyr,eattheedFaOniSmAaltsrewaatsedparneismenatlasshathdeaphairegnhtlcivoemrpcoonucnedntorrataisotnhoefmtehteabolite unmetabolized parent compound. Thus these perfluorosulfonamide fluorochemical species likely contributed to the body weight effec to a greater degree than the metabolite PFOS in the FOSAA and FOSA treatment groups. "bTohdeyrwaenikgohrtdeirnorafttshweaesffPecFtOofStahte4d0ifmfkerden>t FtrOeSatAmeantt4g0rmoukpds >onNi-nEc(reFaOsSesElaitve1r6w0emigkhdt >to FOSAA at 160 mkd occurring at average liver PFOS concentrations o>f 600 pig/g for tPrFeOatSedaanndimNa-lEst,FrOesSpEe,ctiavpeplry.xima~Atlelldyo1s9e3 gurgo/ugpsfohraPdFiOnScrAe,asaenddli1v4e0r tuog/bgodfoyrwFeOigShAtAratios, but only the liver to body weight ratiosofthe PFOS treated rats were significantly increased compared to control values by Dunnett's t-test ~ Correlationofthe clinical chemistry endpointsofpotassium, cholesterol and triglyceridteos liver PFOS concentrations in rats are not shown due to the limited numberofdata points for cach determination. `The decrease in the percent initial body weight was most strongly effected in guinea pigs by N-EFOSE at 160 mkd and PFOS at 40 mkd andto 91% and 93% initial body weight, ataverage liver PFOS concentrations of 419 and 171 g/g, respectively. A greater fraction of the total fluorochemical in the liverofthe N-E{FOSE treated guinea pigs was present as the metabolites FOSAA and N-EtFOSAA than occurred in rats. Thus, these: 1s SDRIPTSTH-6295.8, 6316.4,T-6868.2, T0711 T-7132.1 perfluorooctanesulfonamide fluorochemical species likely contributed to the body weight effect in guinea pigs. These data suggest that rats metabolize N-EtFOSE to PFOS more quickly than guinea pigs, perhaps due to the inducibilityofcytochrome P430s in rats, as were shown in this report for rats, but was not determined for guinea pigs `The percent initial body weight was also decreased in guinea pigs to 94% initial body weight by N-Et FOSA at 40 mkd and growth was held to 99% initial body weight by 40 `mkd N-E{FOSE, at average liver PFOS concentrationsofapproximately 100 ug/g in the N-EIFOSA treatment group and 66 g/g in the N-EXFOSE treatment group, respectively. Opingls.y HPoFwOeSvearn,dtFheO4S0AmwkedreNa-nEa(lFyzOeSdEftorreiantmtheentligvreoruopftghueinNe-aEpWigFOliSvAerstrheaadtead lgaurigneea fractionofthe total fluorochemical in the liver present as the metabolites N-E(FOSAA and FOSAA which when combined, were equal to or greater than the concentration of PFOS in the liver and thus may have made a significant contribution to the observed effects on body weight. The liver weight to body weight ratios in guinea pigs, in contrast to rat, were all rdeacnrkeoarsdeedr,oafltthhoeudgehcnreoansoefd tlhiveesretcohabnogdeyswweeirgehtsiegfnfiefcitcianntgluyidniefafpeirgenstwtahsan Nco-nEtr(oFl.OSTAheat 40 mkd > N-EXFOSEat40 mkd > PFOS at 40 mkd > N-E(FOSE at 160 mkd. This order is roughly the inverse orderof the decreased body weight effect in guinea pigs for cach of these dose groups. Given the lack of the liver responses in guinea pigsofeither htheaptaatnomIengtraalpyeroirtoinnedaulctiinojenoctfiopneroofx1i0s0ommeg/prKogliPfFerOaStincgaeusnezdymdeeastchoiunplgeuidnweiatphitghsebfuacntot in ats (See DTIS A), suggests that the peroxisome proliferation in the rat sa protective mechanism. Correlation of the clinical chemistry endpoints in guinea pigs showed that the rank order oPfFtOheS eaftf4ec0tomfktdhe> dNi-ffEeWreFnOtStAreaattm4e0ntmkgdr,ouopcscuornridnegcraetaasveedrasgeerulmivperotPaFsOsiSumcolnecveenltsrwataisons of 148 ug/g and 100ug/g, respectively. In contrast, treatmentof guinea pigs with 40 mkd N-EXFOSE had no effect on the Serum potassium levels at average liver POS. concentrationsofapproximately 45 g/g in female and 88 g/g in male guinea pigs. dCihfofleersetnetrtohlaanncdonttrriogllyvcaelruiedse,s caonndcesnhtorwateidonnso ipnargtuiicnuelaarptirgesndwebreetwneotensidgonsiefigcranotulpys or strong correlation to liver PFOS concentrations. Conclusions All treatments caused increased liver to body weight ratios in rats but not guinea pigs, with the increase caused by PFOS being the greatest among all the fluorochemicals tested. The evidence revealed the classical signs of peroxisome proliferation in rats, but not guinca pigs, caused by these acute exposures. These data provide strong evidence that: 1) N-E-FOSE and PFOS stimulate both the transcriptional and translational expression of acylCoA oxidase in ats in vivo, and 2) there is a marked difference in the 16 bSRTPITST-B6295.8, T6316, T-6868.2, T0711, T-7132.1 response of rats and guinea pigs 10 in vivo exposure to these two fluorochemicals. These results are very consistent with the suggestion that these fluorochemical compounds are: "peroxisome proliferators" in rats and, much like what has been demonstrated for the classical "peroxisome proliferator" chemicals, guinea pigs are resistant to ths effect of fluorochemical exposures. n pSPiTthT6295, T:6316.4, 68082, T0711, 171321 Signature Page Prepared by: _ nhs . SAtnuddryeDwirMe.ctSoeracat, Ph.D, DAB. S/25/0y Dae MN SIRPSTTB-6295.8, T-6316.4, T-68682, T0711, T7132. Summary Tables Variable = `Table 1. Average CumulativeDose Cumulative Dose_ng) soerectes 005E_GRO rsex u cont NMeaon a0.0 a0.0 N-BtROSMORKA sNXo 02 0 02.0 e5an 938..96 10.202.5 N-ELFOSEIGOMKA NeX an 2163.4 2163.8 N-EeROSEAORKA NX 42 3 a2 s Mean 10.062.2 14.70.6 erOSd0mkd N Meana66.7 475.0 Mss6-leomkd sNo M3i2s.4sing a3 le MseDan MMiissssiinngg 11.656.1 cont dNeana0.0 70.0 FOSMOmkg sNo i0.s0sing 03.0 Mseban MMiissssiinngg 41.03.0 N-Btrosadomkd NMXean 236.6 251.2 N-ELROSELGOMKA sNXo 12 b 20.5 Mseoan 12.284.3 16.739.5 NELFOSEAOMKA MNean 239.1 252.6 Prosdonkd sNo a1.4 04.1 Mseoan 33.33.2 43.62.8 1 SDTIRSB PTTT-631-6.4,6T-62868.92,T75071..1, 8T-71,32.1 `Ta2.bSumlmaery ofBody weights and BWchan eFs w SPECIES DOSE_GRO BWDO BWDA(g) BW.DO IEWDD BWDA(g) BW_DO oe cont Ns( a a 4 4 4 Mseoan 243541 2a243 1607 26196 254003 6110 E-FOSA4OmKS No 2 2 2 2 2 2 Mean 61187 25587 915 71 0 7426 91 E-FOSE160mkd N2 2 2 2 2 2 Mseoan 2653 2a33 0a2 5255 12033 13) -EXFOSEOmkd Nz 2 2 2 2 : Mseoan 0635 2CI 0 37036 7313 0100 Prosdomkd MNeana425 39a 8 9 03 haz 401 MS56-160mkd sNo M2i1s3sing M1i9s5sing M2issing 2378 23 3 83 Mean Missing Missing Missing 258 ogg! gg! cont SND aMissing Miissing Miissing 87 21s 19 Mseoan 2704 7am 2103 1297 12897 2107 FOSAdOmkg Vean MMiissssiinngg MMiissssiinngg MMiissssiinngg 2353 33s! 3gg - SND zMissing M2issing M2issing 26 216 2a EXFOSAGOmKd Mean 214 208 97 303 310 102 N= v so 28 32 22 I2 21 21 EXFOSE160mkd mean 201 st ert 286 os! est N- sNozs 12 32 82 229 28 EtFOSE40mkd Mean 228 20 101 32 36 100 Prostomkd sND 46 31 hl4 11 1a 01 Mean 205 se 290 ot grt sp 1a 15 4 12 15 3 Significantlydifferentfromcontrol valuesbyDuets Lest. 0 DSRTPITSTB-62958, T-6316.4, 6868.2, T0711, T7132.1 `Ta3.bSumlmaery ofOrgan weights and Organto BodyWeightRatios. A RLF M SsPECIE DGORSPE LV WLw WB KW KnWoBW LW LWWB KW WwKWB ap Nt Na Mem 196 4 0043 42 3 20006 4 197 40032 9 46 02006 NEFOSA NsD 277 0E00s 201 0001 2 101 2 0003 2 09 2 0001 2 soma sMDem 11488 0o0o2 4055 00000018 22667 0037 005 55 01 0008 0000 N16E0FmOkdSE N 2 2 0 0 220 0 MSeDan 9053 00000430 MMiissiinngg MMiissiinngg 0816 00000317 MMiissiinngg MMiissssiinngg NAoEmFkOdSE N 2 2 2 z 222 2 Mean 208 sD 1s 003 s2 om 00 0008 000 264 18 003 ool Ss 01 0007 000 APoOmSkd ON 4 4 2 2 42 2 SMDem 61398 00003053 4011 000070 91564 00003084 0S1O 00000018 3 1M60SmSkd N Missing Mising Missing Mising 3 3 0 0 SMeDan MMiissssiinngg MMiissiinngg MMiissssiinngg MMiissssiinngg 01008 00000434 MMiissssiinngg MMiissssiinngg CONT N Mema81 4008 219 0z000 7 121 700a 229 02 09 FOSA SND 0Mi9sing M0i0s0s3ing M0i1ssing 0M0i0s0ig 314 30005 000 00000 omg SMeDan MMiissssiinngg MMiisssiinngg MMiisssiinngg MMiisssiinngg 01107 00000436 MMiissssiinngg MMiissssiinngg ANoEmFkdOSA N 2 2 2 2 222 2 Men 80 sD 04 00% 18 oom 02 0008 0001 141 0046 25 03 0001 01 ooos' 000 N16B0FmOkdSE N 2 2 0 0 220 0 MSeDan 7014 0000402 MiMsissiinngg MMiissiinngg 11231 00004080 MMiissssiinngg MMiissssiinngg oNmBkFdOSE N 2 2 2 2 222 2 Mem 99 0043 20 000 1sol 0047 27 gos! PROS OsND 401 400 z02 omkd 20001 $ 12 a00s 021 02 00 Mem 82 oo! 17 000 132 go! 22 os! SD__07___oo0 01 000 12 000+ 00 000 Significantlydiffrent from control values by Dunnett's vest 2 DSRTPITST-B6295.8, T-6316.4, T-6868.2, T-107L1, T7132.1 oSFPECTES DOS`ETaGbRlOe. 4. PercentInitial B5od4y DWei0ght, Cont T MeanF108 N-EFOSMOmKE N so d6 Mean gt N-EFOSEIGOmKd MsNeoan g14at N-ECFOSE4OMKE sMNoean 24gt PrOS4Omkd sNo os1 Mean gf so 5 Rr cont Neanou106 N-EtFOSEAORKE sNo 34 Mseoan 1201 N-EtFOSAMOmKE eN an 4100 Msse-l60mkd sMMNoean 33gg! FOSA4OmKg Nso o39 ean gt N-EtFOSEL6OmKA sNeoan g44st PFOS4Omkd s Neo ansgg! sp 3 "Significantlydiffrent from control values by Dunnett' test Combined Data zn fSiTt sTissTosaTo TA Table 5. Liver- and Kidney- to Body Weight Ratios, Combined Data. Yow Bou bone woes gm `Significantly different from control values by Dunnett's t-test x S DTIR SB PTT-T 6316-4,T6 -68682.2, 9 T-10751.1,8 T713,2. `Table 6. Liver Perfluorosulfonamides and Metabolite Values from Rats and Guinea Pigs. Analyses performed at the University of Rochester. (SAPlECitsSaErXe DOSE FOS FOSA_ FOSAR FIFOSAR FOSE ELFOSE WETFOSE FOSA ES ore Roc _RoC _R ROC MRCoC ROC _gluc. _gluc wf om Mea_n TNA N7A NCA R]WA N[ M Wmo wn osNO 2MA N2A NoA oNA o Moow oWm N2m EdtornoksA Mseoan 13004..6 0ol.s6 NwAm WoAm MMOoMm oomm 00.:20 N EtFoSN E 2 2 2 2 22 2 2 T6omkd Mean 350.2 16.2 217.9 388.2 28.7 1.9 6.1 0.3 NNso 32391s 72.00 120300 s2 ed 20.8 02 7 235 0B 1 E0tmFkOdSE Mseoan 4445.37 73.s1 3e0z.4 2100.55.0 31..42 00.19 10.22 00.03 p0rmoksd NW 3 3 oo 00 wsepan 114100.085 2347..35 Wwwm WmAA mMomMMom Nn Mo cor oMNean 10.1 0NA NoA oNA m0 ow0 o0m n0m sonO N2A N2A MoWoA M ooN W0m N2A E0tnFodsA sMoean 2963.03 00.25 wWmA mWAA Mmoomw oomm 00..10 EtFosE on 2 2 2 2 22 2 2 T6omkd Moan 488.6 8.2 129.4 261.6 16.0 2.3 2.0 0.5 NWsp 529.8 42l6 s26.4 2113.8 820.3 20.2 21.1 02 0 E0tmFkodsE vsepan 8884.04 04.39 288..62 51.46.0 31.65 1o.s3 0o.n4 00.04 p0rmoksa nN 4 a oo oo Msepan 11934.85 4313..71 NNAA WmAA mMoowM oomm nNmA RF car XMNean 01.0 24.4 WoA oWA mooom o0m N0A sono W2M 204 m2 om2 M2 ow2 o2m # ETt6FoomskEd sMoean 839.16.9 1184.1 s6.78.5 23031d.5 4274.62 61.s4 00.12 00.:20 pros N 2 27 0 0 00 0 0 u SDRTPITSHT6295, T-63164, T-6868.2, T-10711, T-1132.1 Toned vean 866.4 18.3 NA WA mow om n M wsse Nsp03130 23.2 swmo M ON0 om o NA o L60mkd ean 140.3 316.8 $55.2 WA mow om wa cor NsXp 28s. s 11411o11308 W0w NLNO)N 0 NA o Mseoan 00.00 11039.s2 M NA WNA mMOoNw oomm NNAA r0omskag N03 30 oo o 3 espan 12963..84 1167.72.9 NNA AMWA mMooww oomm 00.14 EtFosE oN 2 2 2 2 22 2 2 T6omkd vean 1124.3 18.5 94.3 294.6 12.1 3.8 0.3 0.1 pros WsNp 2166. 29.5 0237 nsl8 o 2.5 o1.2 00.0 0o.0 0nd vean 969.9 17.8 NA mA mom om A sp 88.2 19:5 WA wa NNW NA 2 SDTIRSB PTT-6T316.-4, 6686822,T9-70751.1,T8713,2. `Table 7. Summary of Liver Perfluorosulfonamides and Metabolite Values from Rats and Guinea Pigs. Analyses performed at the 3M Environmental Lab. STPEsEC SEX TaOrBeE CEO3S F3OMS ECFOSM T ECFOST E 3RM WECROSA_3M TF ww FT 7=u 7EN TO Mseoan N0A.151 00.00 00.02 MNAM N 0.1 mNM N EcrosN e 2 22 2 21 L6omkd Mean 604,000 109.0 244.5 302.5 118.5 0.9 pros sWNo 52.657 1271.3 220.7 601.2 213.4 N0a a0mka Mean 803.500 0.1 0. WA 0.4 mA car Nso 2ers 200 200 w0m 010 0ma Mseoan 00.12938 00.00 00.12 NwAm 0N.A1 mWWA NEtroosen 2 22 2 22 160mkd Mean 908.000 89.0 317.5 147.0 169.5 0.4 pros sWwD 02 .510 92.5 127.7 402.4 210.6 00.1 a0mkd Mean 800.000 0.1 0.3 NA 0.1 ma sb 16.57 00 00 wa 00 wa 2 SDRIPTSTH.62958, T6316.4, T68682, T707LL T7132. `Table Hepatic Palmitoyl CoA Oxidase Activity, Combined Data S2PECIES C0o%r GRO CI]FORT Nseoan 20.8 N-EtFOSMMOmKA N eand2 N-BtFOSElOmkd sNo 13.0 esoan 03.6 NeEtEOSEAOmKd N Meand2 erose0nkd x 07.6 dseoan 31.6 con exan76 N-EROSEIGORKE sNo d3.2 dseoan 113.6 erosd0nkd v eansgt so 5. Significant diferent from control values by Dunner' List. zn SDRIPTSTH.62958, T-6316.4, T8682, T-7071.1, T7132.1 Appendix 1. Deviations to the Protocol `The 3M Medical Department identification number for N-Ethyl perfluorooctanesulfonamide, T-6868, was not listed in the protocol. `The protocol used abbreviations for N-Ethyl perfluorooctanesulfonamide (T-6868) of PFOSA and FX-12. Protocol amendment numbe2r lists the abbreviations for perfluorooctanesulfonamide (T-7132a)s FOSA, PFOSA and FOSAmide. The abbreviations used in this report for N-Ethyl perfluorooctanesulfonamide was N-E(FOSA and for perfluorooctanesulfonamide was FOSA. "The protocol stated that the animals would be dosed on days "zero through 4of the study". That was atypographical error, as evidenced by the fact that even the example calculation in the protocol was for a 4-day dosing period, nota 5-day dosing period. The actual dosing period was for days zero through threeofthe study, and the animals were sacrificed n day fourofthe study. Animals were not weighed in most cases on day oneofthe study, and in some cases on day threeofthe study. In those instances, the previous days' body weights were used for determinationofdosing volume. Protocol amendment #2 stated the Dr. M. Wempe at the University of Rochester would `perform the metabolite analysisof the liver samples from animals treated with T-7071.1, T-7132.1. Due to personnel changes, Dr. Xin Lu at the University of Rochester `performed these metabolite analyses instead. Kidney weight was not obtained during necropsy under amendment number 2 on 3/5/99. Clinical chemistry was not performed on all serum samples. Histological evaluation ofliver, kidney and tests were not performed. Serum `perfluorosulfonamides and metabolite values were not obtained. Induction of mRNA for the following genes associated with peroxisomal proliferation andor cell replication were not analyzed for in liver: Peroxisome Proliferation Activating Receptor (PPAR), Liver fatty acid binding protein (L-FABP) and Proliferating Cell Nuclear Antigen (PCNA). = Appendix 2 -- Analytical Liver Sample Lab Identification. (Study / [Record [Dose group ne |Necropsy|Specimen|Amt [Amtsent [Date [Type sentto [to [Amt [Amt sent to|sentto Fo [er |o| to [rofo] (1t11o65s9e8 SlOeRRoDaO0sH3Ev [Ccoonntt |HTiiu20s8leivrer || 00b3o2l8-1Tag |~TTog [uNaA || [im [pre[ep [ene[ep pa|S|p washer |oiA s |e | pez [[Spe2eeefJooeo || veEwssser | | oSRA9iTss [[TToe JW | | [v[e feeyp apre | |repupee]]] : e [[ opr d BE0TMp | eewl|ip ppee]] 2L sf Ev mp--|vr_s_ --orpr | | [8G0148 I"Nr- ou Top 11/20/98 [RTE [OR hee eee ad TM peg [7 feEP eo e e pe (3/41994020 Imkd [21 fo [eR Re, | wm | oso oe| freoeent(on He[[rFovTeN[toi,e|[ SS ||h ooSsse [[TTeOr FTTe I |ORO0464|M [Cont |_3/5/9liver | 0.76]-1g |OR00460F [Cont | 3/5/99liver | 07471~1g ro seas |-1g |-1g | |-1g|~1g | 18 | I cl WAAil aes mee Pa i P poppe |e ee|a oa me| [F5 fpom oor||v vm |e wo ea] a pe 0|Wwiwe |iwe A li| Ei:od 0Wesl oA re cl | I lL c WcAECAA f[rfpoomm prrooss|Sswemie] oosree ve oe]| Br OO ccGc Wcoe CI a Ee aE nt[2 TRo07a3M [Cont|2r23i0tliver INA |=Tg |NA_|=Tg | pss Eom [_8[1R00749|M_|FOSAA| 2/23/01fliver \Veh ~ |___[1RO0TSOM |FOSAA| 2;23/01fiver NA |~1g NA |=1g INA |~1g | NA |-1g | |6[1R00747|M [FOSA|2/23/01fiver NA |-1g INA |~1g | . DSRTPITSTB-62958, T-6316.4, T6868, T-7071.1, T7132.1 Appendix 3. Cumulative Dose Individual and Summary data. oF Toon 88Gc0oTrAiE8Se 00 99660000005512 Mxean 40.0 ES 00 N-ECFOSMORkd 880G1O4L49808 91601 xMean 298.6 5 pt) N-BCFOSE160RKd 99600000005543 116660 xdean 2163.4 sp a3 N-BCFOSEAOmkd 880G0o11448878 110032 xMean 2102.2 so 0.6 PrOSdOmKd 88G6o011449912 9954 99660000005556 3389 xMean 166.7 5 32.4 u con s8G0011447787 0 6960000004456 xean 40.0 5 0.0 N-ELFOSMOmkd s8cGoOrLi4eEz1 112233 xean 2122.5 5 0.0 N-ECFOSEL6ORKA 99660000004478 116671 xMean 2163.8 0 a5 N-ECFOSEAOmKd 88060011447890 111250 3 DSRTPITST-B6295.8, T-6316.4, 768682, T-1071.1, T-7132.1 SPECIES SEX DOSE_GRO NTM Mseoan PrOS4OmKd 88G6o0l144s813 99660000004590 NMean so cont 8BRR0O4400440L 99880000447609 NMsean N-ELFOSMOmkd 88RR0O4S004445 Mean sp N-ELFOSEI6Omkd 9ORR0O00447TL2 MNean sp N-ELFOSEAOmkd 88RR0O44004432 Ndean 0 PFOS4OmKd 88RR0O24004467 99R80000447743 Ndsean M wSSe-lcomkd SPECIES SEX DcOoSntE'GRO 118R0O00774498 N1800750 Msepan be1s00782 11880000774483 88R0042003332 9800463 C2umulativebose_ (7g) 0117.6 111057 3399 I75.0 als 0 0 40.0 00 3386 236.6 15 112262 2124.3 2.9 4308 239.1 14 3385 3219 333.2 3.3 116656 1568 116.65.1 Cu0mulative Dose_(ng) 00 00 0 2 SRPTTT-631-6.4,6T-62868.92, T-51071..1, 8T-71,32.1 DTISB NSRG0R6E 02 MseDan 00.00 FOSA4OmKg 11R0000774456 4309 N1R00747 431 Msepan 4305.0 N-ELFOSAdOmkd 8BRR0O44003376 5511 Mean 251.2 so 0.5 N-ELFOSEIGOmkd 99RROO0D4AEGES 118745 Mean 2179.5 sp 6.3 N-ELFOSEAOmkd 8BRR0O44003345 5533 NMean 252.6 so 0.1 PEOS40mKd 88RR0014003385 4S9O 99RR000044667E 4453 Nvean h4)6.8 sp 3.3 3 DSRTPITST-B6295.8, T-63164, T-6868.2, T-7071.1, T-7132.1 EY EE gma TE a RE, ITI 3 we " 2 " <) o| 28 gg gg 2g FlEddg.92 # S.88 f.4f d.d84 dddd8 2 | 25 wn 2a = of FEERR.ER 8.54 8 R.RG 8 83 333. 3 | lowe i _ _ an El a [2388 $3 83 gS $388 :: 2: =z L|le i 32 BR < ih i fh g g g? a & 2Ez3 Ex A x8 & -- g o b 2co3n2e2.0800 37 54.393. 48%ddad, 8954.a2,7 YdiTsl.44), = of 9.9% 4 4.0% 8 a. o8s 32 l= ey 3 . oho ney 2 qa 48 4d, 3. < BEd Bd B ELE f dos R BEES CEH Es o i EER8. 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Technical Report: Liver Fluorocarbon Metabolites -- University of Rochester. Title: Summaryof Quantitative Analysis of Fluorocarbon Metabolites in Rat Liver Samples Lin Xu and M. W. Anders "The paren"tThaisndim.ebtrabioflsituemsmoaftrhyeofutohreocaanaslbyosnissowefrievedretsearmmpilneesdfiromivreartssgaimvpelnesabryaLnC-oMffSlAuMoSro,caTrhboen, resus are presented below: control rt"sT,hrferogmroruatpssgoifivevne4r0smagm/pklge/sdwaeyreFOanSaAlyozerda:lyT,haendfifsrtogmroautps(gGivreonup1610) mcogn/skigs/teddaoyfFliOvSerAsAfom ((TMaSbSl6e)1)o.ralTlyh.e dTahtea scohnocwenttartatPioFnOsSofpwaarsetnhtecmoamjoproumnetdasboalnidtethfeorunmdetiabtohleitleisveirnsfovferrastswegirveemneFaOsuSrAeGd oFfOaStAs gNi-vgelnucFuOroSnAidAe(vMa3s6i)de:ntPifFiOedSaasnadmFiOnSoAr.metIatbioslintoet.ewTorwtohymethtaatbotlhietceosnwtreorleaindiemnatlifsi(edIRiOn0t7h42v,ers F1iRs0c0h7e4r33,44anadts1RmOa0i7n4ta3i)nceodnitaitnheedUnsiigvneirfsiictaynotfcRonoccehnetsrtaetiroVnisovafrFiOumSAd.id nAotpasrahlolweldsenteacltyasbsloefvers from concentrations of FOSA. fiom cont"rTohlerstcsc,onfdrogmroausp (gGirveonup402)mogf/rkagt/sdwaaysPtFrOeSadanailnlyN,ofvreommberrat,s g19i9v8e.n 4G0romugp/k2g/cdoanytaNin-eEdFlOivSeErs a`cloombpoolunrdasla,ndaniddernoifmcdrtmsetgaibveonli4t0emagreksghdoawny iNn-TEaIbFlOeS2.A oTrhalelyd.atTahsehocwotnhactelnivterrs afortfopimaorarntstsgiven sPiFvOenS Nc-onEtAaFiOnSdEpaalrceonhtoclocmopntoauinndedPsFeOveSr;alFmOeStaAbowlaitsesa.lsoThfeomuanjdoirn mtehetsaebosalmiptleesw.erTehNe-fEiIvFeOsSfAoAm, aPtFsOS, aanndd FFOOSSAAAN-(lMuScSu6r)o.niTdhe.e mPiFnOorSmweatsabtohleitmeasjwoerrmeetFaObSaAl,icFiOnSlEivaelscofhraolm, rNa"tsEgFiOveSnENa-lEc(oFhoOlSAgh;ucFuOroSnAide, aconndtrFoOlSrAtsNd-igdluncotarcoonnitdaeinwdeertecitdaebnlteifcioendcaesntmriantoironmsetoafpbaolrietnets.coImnpcoonutnrdasstoomGertaobuopli1t,ets.he ivrs rom ats, from"Trhiesthgiirvdegnr4o0upm(gGkrgodupay3)PwFaOsSteorsatleldy,iannMdarfcrho,m r1a9t9s9.givGernou1p603mc/okntea/idnaedylNi-veErAsFfOrSoEm acolnctorhooll oarnadllyo.wTchoencconnicretnitroantsioofnFs OofStAhewcehreemiicdeanltiafnieddmietlaivbeorlsifcoamrersahsogwinveinnPTFabOlSe.3.InAtshewivtehrGsroofrupas2,gFivOeSn Ath-eEmIiFnOoSrEmeatlcaobhoolli,te\s-iEdIenFtOifSiAedA,wePrFeOFSO,SnAd, FFOOSSEAAalc(oMhSolS,6)V-wEeIreFOiSdeEntailfcioehdoalsgmlaujcourromneitdaeb,oalnidcF,OaSnAd Nslucuronide. Similar to Group I, control animals showed detectable hepatic concentrationsofFOSA. DSicshceumses)i,onb:utThdeonseotdaatlaloawloiwnfceornecnlceussiaobnosuatbhoeuetxhaectvreorutaesomeftmestbaobloiliuteeoffortmhaetifolnu.orFoOcaSrAbownasstudied (sc mcoentsaibsotleinttel,ybiudtentthiefsieoduracseaofmetthaeboalmiitneoofgrPoFuOpSi, wnhoetrheeardiPlyFapOpawreanst.admWihnaitsetveererdidsirocuetyeoofrffoorrmamteidoans,a FOSA was metabolized to FOSA N-ghucuronide aise fromNt-hEFNO-SdEcEthayllcaohtoilongoifves-EisUeF0O.S2Eraanlcgoehoolf,maajndorNa-nEdtmFiOnSoArAmectoaubloldiatreiss.e bFyOtShEe oaxlicdoahtoilocnooufltdhe aNl-kEoAhoFlOtSoEtahleccoahrobloxgylulciucraocniidd.es.GluFcuOrSonAiAdactoiuolndofbethfeorpmarecdnbtyN-thEeFNO-dSeEctahlycloahtoilonwoofulNd-EgiCvFeOtSheAAoobfsberyvedthe orxeimdoavtailonooftfhFeOcSaErbaolxcyomoelt,hyolrgbrotohu.p oFfOFSOAScAouAldasbgelyfooxrymlaetde.byFthOeSNA-dNe-cgtlhuylcautrioonniodfe mN-aEyIbFeOfSoAromrebdybythe the glucuronidation ofFOSA. 5 SDRTPITST-E6295.8, T-6316.4, T-68682, 1071.1, T-7132.1 sive FOS"ATheorfltososfofFtOheSAglAycaipnpeemaoriserteylawtoivuelldy sgtirvaeigPhFtfOorSwadridr.ectlLyo.ssofthe carboxymethyl group would g :2 3 : 2g 2% 1333333333 PoE P| RITE p2o |T: _T i 355% 855585 i f 333: PL E5 z ; g 23 Boat Ea 2 ddd3 EEEEEEEE g5525982 [HHH 5i i ATT 3 [a2 EEEgEgEEEEdg 3 EEEEEE] 2 FL| eT 38 PE j lil igLH E n| eikl | aE F FAT TT i EL <tet=te] = , [Hfo0 RRR EREER EeEY #1] EEELEIn 3 1818] 1 8) 82 2[ 2 : H [ diE E H e 3 EERE EEe5 EET EYY g 5 [9999995 TIES 3g $i |8%iz z SE {ET g pk FETs EH | 3. 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Bsz3s :2z 3.8z 52 Ber 53823 58,08 8. dnd 2 E% EB 2saag5. s.3 dv3e F 2% 3Rz Fe Tozs 5.3% 28 Bx. Biz3a%a 22y .8. 22 es Brzzirozgo.z B Eiss 3.3: 3 8.8 5 F8 3.335 2.33 gE eg 222220 823 .38 02 cf2.2%azs33%2. .8388: sg2.28a .833 3 3.882 2.33 2.3% 2.33 Tfy 382 .33 333 3.38 82 3.5% Fez. Ffa B85 x 2.8g 38: 3.3% Brrr.dz23ss.diz oz oz ds 3.0 F2%Raasa2 2. 2.3 32%2 a.83 2file i5i5i33lg, 5 L 2 fais EE i. ads = = 4 Bb IH 23%32.8F% essed Za eldan $32: 3 5 2523.88 3333.3835 5.38% sagws og 2528 .Fy se28. 3x8 2.8% soyr oz og sng. mmr ily 3.3% gor of 2zgz fs sas ds sic sass og 8g 3 3.5 5238.58.92 3.83% s.mz2 See .37 sere? 3s radar 2.8% gf ss fe .38 saz dae aire 32x pn sry r 23 rams die adi sdrs gf saza. 88 gaan dla nda sags og 282.33 memefnz: endna giz: oo seg? 3% mons.dda on Bis goss gf s2g2.8% maa.88a2 3.38% goa oo $923.83 sem. 080 3.38: 2373 4 sezr. dd ss dlo 2.38% gaexa og fis. fms diz 288s sz og LRLLa. Hd Lol Bad sxfa df zras.d uw 2d vg gE e 4 ry $338 FE] # g g 58 a 5 iE n AF e angi os w2eE dR ou sad sa ald fg z hE 8 359.8% 3203.38 3 3.3%: 2.3% 4 LEneagds senda oz owdrz sds hE Emme E onzan.@ fos 3.35 1.3 Rd \Erzssr ds WRon EB WF Re WEY os EERE $zz.E.f, REE.E 2 e553 5 ENE hE3aasr dn eres di ow size 2.824 2#2E.9.9. 2 ELE.3 BR E.Eon meez.@ 2 3 sad Tz sad nog seef. Re 3.8 Ze gad 1 of REIN EERE saad os size 3.36 hd 2s 228.2 1 WEF noag Rd WE 279s. % =ran.d i oz ogres. loz smnn.38 3 sal aoe e833 23% ald ono 3.88 2.38 hE 8339.38 2333.38 1 3.3383 3.3% 18% zosr.3f sedi oz 2EEx sayz EEEEITRENEHTRE IENEAE PANEE WE 2s oaggd d sexs.3 3 re 2fas 2.33 4 sez 8533 ss 3 Ee i Hig. fia. 1 La, i is sd 2# 5 g 7 4 WH 4 g iE 2d Zw aeg.3 2 $38 z5s2.8% sd: sess do| g Z 88 zae.8d : 2.-2: zesg."a| 2 - 2 B.E.%. 5 gRs.5.5 3 2.85 8 sag.d 5 | o 3d2s3 =ret%ad2z| 92 85% eng E- g F= 2.42 samedi] . ~ g8 sls 5.80 rxmaarncggpndga|l 322 2.28 seedy] =3v 3.37 38.3% 8 2..28% 2aezd2z| =5 22..38%2 g3adsspeddys)l dg . E 28 3 3 2ag.d on | 8 25 2 a Lis i HF 5| 8 2 gl 1 g Sg igEz Ez TprIiLs LER A. Statistics on Clinical Chemistries for rats, male and female values combined. (CHOL mid. By Dose Group (mg/Ko/day) OSunmemwaaryyAnofovFiat RRsqquuaarree Adj RMoooatnMoefaRneSsqpuonasree.Eror Observations (or Sum Wats) 00760311768112 2105824217431 2 SMooudrecle DF AnalysSiusmooffVSaqriuaanrcees 4 530.1636 Mean1S5qu2a5r4e F10R0a0t1i3o [Er3or 271 6415925520378550 W11733296 P0r0o0v0o2 CLOevNeTl Means for OnewNauymAbneorva Mean 8 sem StEsrrmoer NNEEFFOOSSEATO6m0mKkYa 41 w500o000 15083411 NPFEOFSOAS0EmdkOdmid 45 TS01o0 s47s59 StdErrorusMeseaanpsooalneddSetsdtiDmeavtiaetoifonse.rror variance CLeOvNelT NEFOSAOmKY Nombe8r meMreasn 4 sooo 7S6U0D8e2v SW Er2M6o8a8n8 sem 27386 NNEEFFOOSSEE146O0mmkkda PFOSAOmKY 41 5T030000000 116s? ssa277 5 501000 16249 72670 DCiOfMNeTan(l-Moan] NEFOSAtOmId CONT ~MeNanEsFCOoSmpAaOrmiskoins 208070500 03.80705000 NEFOSE1A8Om7k0d 80000 PFO3S4I7O7M5K0I 30000 N-EIFOSE4f8607m5k0d 31.0000 n TTIISLLER NPFEOFSOAS0EmdKOmd 1a17857500 NEFOSET60mkd Sa8750 308..09000000 310000 203000000 230000 Apa `Comp0a0r5isons for each pair using Students t 210t080 ACOSNOTILSD NEFOSAOmK 11C2O2N8T6 NEFOSM"O9m8k7i% N-EFOSEA8O7k3S8 987% 15.8755 T8755 PFNO-ESFAOOSmEKdYomkd 2"1108775386 NEFOSET60mid 10618 1758873525 1578873525 5.8987 21013 Positive values showpais`ofCmoemapnasrtihastwoiatnehsasicgoinfrclanutsyindgifeDruenntn.ett Method 274]180 ACbOs(NDTAILSD 14C5O8N8T8 NNEEFFOOSSAEdOOmmidd "51.399962288 PFNEOFSOASoEm16K0mka 138.01247152 20209000000 0.1000 PFO2S1I9O7m5k8d 5788339922 214440s6e4 Cholesterol in rats male and female combdiPionseeirdteinfvoter.vDaTlSue,s sSihgorwepdauictsioofnbmye4a0nsmkthdaPaFoIOsSigannfdca1n6t0y mid N-EIFOSE. 202..10000000 00000 N-EIFOSE1116006m1k8d 25.81008173 2311.47958029 n 0 bis 1 Campi. DosoGroup (myn . R : y A : ON Zn s IY Vv AO W Vbh)IAN CONT wn Meroswoms brosios eT b wa TrAi0s7LIFR ``OSnuemwmaayryAonfoFviat RRsSqquuaarreo Ad RMoooatnMoefaRnesSpqounasree Eror Observations (or Sum Was) 0.012081195672 0.4119887258 16 Source DF AnalySsuionsfofVaSrqiaanrcees MeanSquare FRao EMrodoerl 23 0281205500000000 0027S50T00 P1r.o56v7o7 Coal 15 29300000 019533 02484 Love! `Means for OnevNaoymbAenrova Mean NECFONOTSAtOmId 44 i12i1s5s0 PFNOESFO4S0Emdiodmkd 44 112828000 S0u20E0r4o1r 000222000088444111 Std Error usMeasanapsoaonldedSetsdtDimoavtieaotofnesrrorvariance. LCeOvNeTl Nombe4r 12M1e5a0n 05Su6De7v NNEEFFOOSSEdAOOmMkdd 44 1t1sies 0o.s1e8r9e2v0i7 PFOSAOmKd 4 22000 0216025 StE0r2M1e2a3n 000208436055 0.10801 PDiF-OMSeAa0nmflH-dMeanl] ~~ NCEOFNOTSAdOMY NEFOSEdOmKS PFOSMIeOaMnKsdComparisCoOnNsT 0000000050 00..005000000000 005277s5 0082258 N-EFO0S2A7W5O0m0K0S 00.202050000000 03 Alphas PAFbOSS(AD0ImLkSdD cNEoFnOtSAtOmId NEFOSEOmkd Compfo0ae0a5crhpaiirussingoStundenstst 2.17t 882 PFO0S8IO4m2A7d 05C9O5N2T7 NEFO"S0M3O7m0k2i1 o03s10e27 00.6442502277 "os4a2s02277 oor2r ora02r 034827 Positivevaluess`hCowompapiasroifsmwoeianhnsasctohnattraolreussiinggniDcuannnteytfteMreetnhto.d 268dl204 APsFODSIALOSmDd 07C4O4N8T7 CNOENFTOSAtOMK "00.7596498577 NEFOSEdOmkd 0.26957 N-EFO0S5E7o5m0k0d0 000053020050000000000 NEIFOS"E0A0O7m0K2S7 ""001324052277 "osisar dPioesrieinvte,values show pais of means tha ae signifcanty PHOS mg/dL By Dose Group (mg/kg/day) TorAs071R ~ A=p IAN yr ne A -- Ry "a YN IN 8g aos TW; \V 53 ~---- i wo Vv Co werosaions VY prose teSe A [aNEFOSEAmI w00es t OSnouwamyAmorfoaFriat RRSsqquuaarreeAd Fool Mean SquaroErr o03s0i0e0r3t9 osorrt MeObasnerovfaRtieosnpso(nrsSeumWots) arizTss NSooudrec!e OF AnaSulmofoyVfasSrqiiuaansrcees 3 sarees MeanPSqeuwse FR3a7ti8o ErCoorwl i2s 197498674357050 017990873 PorodvooF cLooventt Mefora OnewNnauymAbnesorva Mean Su Eror Tm oss NNEEFFOOSSAEtOOmmkdd PrOSiOmd 330 Si 3 wm oousss osu Lovet 516 EroruMseeasnaspoaonldeSdedstDiamvataetoofnasorvariance Nombor Mean SuDev SWErMem cNoEnFtOSAtOna NEFOSEOmka G 3 mshsoo i 00 orozmsw oss 00153470080 032404 PROSiOmd + sto zen agar DCiOcNMTe'anf Meant] MCoOaNnTsComNpaErFiOsSonEsAOmkd 000000 072500 N-EFOSA1I7O2m5k0d0 PFO1S.i72O5m0d0 NENFEOFSOSAEidOOmmad PrOSiOmd 07722550000 72500 100000000000 So0000 0T0o0o oroooo 00000 000000 Aptos Comp00orsaeacrhpair ussingoStundensts ADS 278: 02 CONT "NEFOSEAmks N-EFOSAIOm PFOSiOmkd Con 136081 asst 035508 035500 7s TbIi0s7LIFR NNEEFFOOSSAEOMMMKGd PFOSAOmKd 008.435459010 035509 1".03306099911 "036091 0330690911 103366009011 3e91 136091 MaleandfePmoasilteiveatvsacloumesbsihnoewdCpaaisanodfPmheoasncslitnhiacaarcohesmiignsitfriiceasn.tNlyodisfiegrcenhta.ngefromconirol. 7% Tbiim Tomgt. Dose Grp iit ET SzS 4 Ov -<T: a ( s l Oa k C) omnia econo. NEIFOSAMOTNIEIFOSEAOmks |ESAidMwPtOsFt =Duar "WithContre = TTIILSLER `SOunmemvaayryAonfoFvita ReRqSquuiarreoAdj RMoooatnoMfeRaensSpqounasree Eror `Observations (orSum Wats) 0038757521041 00036508439313 2% MSoodurecle CEroroar l OF Analys'iSsoufmoVfarSiqaunacees MeanSquare FRaio 149 000089615785303030 0o0ot0e4s2s0 Psraomvso 2 017833333 ogorrsa 00102 Lovel MeansforOnewNauymAbneorva Mean StdEror NCEOFNOTSAtOMKY 48 002377550000 0000247557 NNEEFFOOSSEEdO1m60kmd 43 0o4r0s000000 00003041725 PFOSAOmkd 5 040000 003108 Std Error us`eMseaanspoaonldedStedstDiemvaitaeotifoensrorvariance. CLoOvNelT Numbe8r 037M5e0a0n 00S3U6D3e5v NENEFFOOSSAEd6O0mmS 43 00247005000000 00..005000000000 NPEFFOOSSAE0dmOKmkd 45 004227000000 00.019058754435 PFDiOMSeaAnO(ml-dMeani] CNOENFTOSE60mkd NNEEFFOOSSAEOdmOkmdd Aphas PAFbSO(SOAIOLmSdD NCEOFNOTSE160mkd NNEEFFOOSSEAdLOOmmiidd PFOSAOmkd `MeansNCEoFmOpSaEri1s6o0nmskd 0000000020 00000200000000 00..0134255 000112255 0145 018 003C2O5N0T0 00.001020500000 0011112258 Compar0i0s5onfo each pai using Student' t 209+301 PFOS0A.0O9m1A9d9 N-EFOSE01S0O8mk2d 00C5O0N8T2 "00.0085604222 00018870s7 000078251927 00004477443333 00001133991155 00002233443344 Positive values show parso`CfommepaarnissotnhsatwiatrhoasicgonntircoalnutsyindgifeDruennnte,tt's Method 270dl438 PAFbOSSAIOLmSKDd 00C7O4N8T4 CNOENFT.OSEIGOkd 00..010437936 NNEEFFOOSSAEtsOOmmKidd 0"0..0000226588 SEM00M1e2a5n0 00.0020500000 000044788970 NEFOS0.M1O4m50k0d0 00..111225050000 00000000000000 NEFOS00M4O7m4k3I3 00001230941354 00.1100228844 N-EIFO0S.E1d4O5m0k0d0 00..112152050000 00.000000000000 N-EIFO0S0E4A7O4m3A3d 00001233041354 "00.1100228848 7% TTIIOSLIFR aPioseirtoinvte.valuesshowpaisof meansthatare significant AbioforrvatTBeIL,maleandfemalcombineddata.no igchanges. " ToTiLs R Amg By Dose . A | VY] [4 afA _A : EISEN ~~ SOV NW . e4re Eo DoseGroup (mg/day) 5 ) oN, a ans. 0 TTIISLLER ``OSnuemwmaayryAonfoFviat RRSSqquuaarree Ad RoMooatnMoefaRneSsqpuoanrseeEror `Observations (or Sum Wats) o0.s59086807772 307381251673244% Sour OFAnalySsuionsfoVfaSriqaancees MeanSquare F Rao ENrooder! 184 216704312451030 o00e0s0i6o2s5 Preeosvsoz Cowl 2 44373913 020700 00018 Level Means for OnevaNauymAbneorva Mean Su Eror CNOENFTOSALOmId 48 s37s00s0 0o11s15e9 NN-EEFFOOSSEE1s6o0mmikds 24 33s2sm0m o02s231e9 PFOStOmd 5 4300 014116 Std Error usMeesaanspoaonldedStesdtDiomvatieaotfonesror variance LeCvOeNlT Numbe8r 36M1e8a0n 03S8W7D0e6v8 NENFEOFSOES1A6d0Ommkda 42 33720800000 00231126123228 PFNOESFAOOSmEkddomid 53 3435670s000 00128580009070 Sid0E.r1M3e6a8n5 00.118501010 00.0172580804 DiMean(l-Meanl] ~~ PFOSAOmkdMeansNCEomFpOaSriAsOonmsk.d PFOSAOmkd 000000 066000 07C4O7N5T0 NEWFOS0EA7O8m5k0d0 CNoENFTOSAOmK NEFOSEdomk "006764070500 "078500 000080705000 00.0080705000 012500 005750 00.0132755000 0.00000 NEFOSEI60mi "1.11000 045000 036250 0.32500 Apha= PAFbOsS(4DoImLkSdD NCEOFNOTSAdOmKd NENEFFOOSSEE1O6m0imkdd Compar0i0so5ns for each pair using Students t 210t091 PFO0S4A1O9A3I9 NEWF0O2S1M5O1T6K8 0.3C60O4N6T4 00326195416646 00341688598 00331381868 0033480511658 0012304238 003166815784 NEWOS0E3A4O0m1k6d6 0.03.63845389 "002.44562889 Positive values show pais`CoofmmpearainssontshwaitahesaicgoninftircoalnutslyindgifDeurennnte.tt Method 272]184 AbPsFO(SDAAOImLKSdD 025C7O7N3T5 NCEOFNOTSALOMKY 0"0..4432895805 NENFEOFSOES4EOImSkOdm "00.4381865608 N-EFOSE11.6101m0k0d0 00.3465205000 003020500000 N-EFOS0E156505m1K9d5 061127248 ""00281351228 st TorsTL 178PoaDfsooisoeeGtvraoluuesshowpisofmeansthtar snfcanty 1s ECSl 1..9 I ES YN AY X eo \ om weroReroomsecrisoetmeoisss TSaetrsi osoGroup(ng) 00s at o0n0sa | | OnSeuwmaymaAorrforFyia RRSRoqsouqtuaHararoaenASaoErr o0s03m66a01s20z5526 OMbesearnvoaftiRoensspo(onrSsaumWeis) S8sT=e VSoodaa OFs ArSuiomfoVSfasarSciuhasnmicnees Mem"Szmomee aFxRaeo CErrnoor w 2 T2a35a0r25e00 oOTe pooovmr cLoovnett Means forOnNyomAbrerve aMseamn SoumEreorr NNEEFFOOSsEAoTSmOia NEFOSEiomia i2 sGea7so0o ooorzarsoscaess ProSiomd 5 Tew omer Lovet StdErorusMes apeoNoaulnmeabaSdeearsDtienmvaitaetosifMonaesrwrvariaSncue.ey SuErNem NCeortosnoma NEFOSE Soma T4 asestsoeo0 2 sao oomaseann Oawass o03o27n4 00 PrNoEFsOdSmEidaoma i5 eTmeso oooorm oOoissee PFDiO-SVeIaOrmf}dMearl] PFOSIOnkdMeansCNoEmFpaOrSiAsoMnOsM 000000 075000 NEFOSaEaAeO0m 13C7O7N5T0 NENEFFOOSSEAdDomNkd Con "0o7s9e0s0000 Rims 220070500000 33750 0co0r0s0e 00236725600 026250 000000 Nerosetsoma Rp ss000 D570 0120 NEFOSE1f4G4O0m0k0 008678500000 000301020500 Torsao Apts [aos \ ---- APNrseosriiossuoma PROo3SarUamenksmSs NE2F1Op0S0M0rO1m]kI "am NEFOSGSeaEiArsT o"asCOreNmT NNoiEnFroossctiioommaa GOooossonistems prSSiiaamr DSSiaieamsr Sooamsaemsn Poste valuesstowproCofmpmaerainonhsatwiarnsciogfnatnotoy diDrot.ms ethos AForsoOSIt2oL7rS5oullDses osecsosnsts SNNoEenrroosstvioonmaa Dtoaisseeezs Nirostooma adanse = GLUmgGE rBeyeDnoseru R -- Ps A "ADAL ASTY NEFOSES"taG6noaorrmsedea 2peiZsacsaeoen AY ~~ i v[7 x ---- CONT NEFOSKfTroEsIeFiOeSaeEaOsmsky . -- S9ohrPeOt PooComo 1 Duets Rss [ Som-- at RareAc oGsriozszszer GNRhooamnnaoiefaaRnnesSsqo(uaxnrSeuEmrirg) SGoosrseooe=sry NsTowo!e 0" AriomfaVSarouiiasngcmres Men"sUwrwso Forwaes is Grisso moss peer oa 2 Sse sms owe Loovvert Means for OnenNaoyrAonwoT rva mMean SuCEiroer = TILLER pris NENFEOFSOAStEO6m0mIkdd PFN-OESFOOmSEk4domid 42 w2e1s00 40341m3 54 uZmar7Tes0 327024m% StdError usMeesaanpsooalneddeStsdtiDmeavitaetoifonesrorvariance LeCvoenlt Numbe8r 20M3e5a0n aSruDeesv NNE-IEFFOOSSEA6O0mmiidt NEFOSEdOmid 42 s211o00 149s9s461%6 4 mars asses PFOSAOmKd 5 M760 286671 Sur3M1e0a7n3 33S.a0r0t0 22882106 PFfMOoSaAnO(KMYean] CNEOFNOTSAMOmIA NNEEFFOOSSEEs6O0mmikdd PFOSOmidMeansNCEoFmpOaSrMisOoTnKsY CONT 250.010000 205010000 s2a9100000 5542.815000 136600 3297070%0 80708000 180 82500 Alphas (C0o05mfopr aachpraiiusisngSotudnentsst AbsOILSD 210t091 PFOSIOmkd NEFOSAAOMK CONT PNFEOFSOASOAmLKOIm CONT 5018.06571 60088767-18as9a8r8r "i895 "49477 sa076 NNEEFFOOSSEE1s6O0mmikdd 221031870 20056177 198782173 Positive values show pars`oCfommpeaarnissotnhsatwirteh asicgonntircoalnutsyindgifDeurnennte,tt's Method 272]184 PFAOsS(AOOImLiSdD 40C5O5N0T7 CNOENFTOSAdOmd T3.e01r4m1 NENFEOFSOESdEOTmOiMdG 42897251610 NEWOSE6A2O8k5d0 387775500 730705000 NEIFOS2EA1O1M7I 5"592782677 50281%7 Poasfiotriovnetv.alues showpais of means that are significant N-EFOSET13680.m6K0d0 18121550000 703070500 N-EFOSET2G9O3m8K0d 180851137 2387128%2 8 Tbirs oi Abmgt 84D as: CASN = 2 VY] :1" ESEa TTNN I A >A aA EJ | \/ fa---- heroine or iva -- we = 5 TTIIOS7LIFR OSnuemwmayarAonyfovFiat RsRqquuaarree Ad) RMoaoatnMoefaRnesSpqounasree Eror Observations (or Sum Wets) o0s5a0s6e0r7r2 03371552613744 zn MSoodurecle ErCoora OF Analys'iSsoufnVoafrSiqaunacees MeanSquare FRaio 184 2167498312451030 006018063255 P6r6o3b8o2F 2 44373913 0200 00018 Lovel Means for OnewNauymAbneorva Mean StErmor NCEOFNOTSAtOmKY 48 3a70s00s0 0o111s175E NNEEFFOOSSEE146O0mmkkda 42 332s00 0o2t2e3r1e9 PFOSAOmKd 5 4300 014118 StdErrorusMesoaanpsooalneddSetsdtiDmeavtiaetoifonso.orvariance CLeOvNelT Number8 36M1e2a5n0 038S7W0D6e8v NNEEIFFOOSSEA1O6m0Kmdkd 42 337000000 00321162212382 PFNOESFEOOSmEKddomkd 45 433500000 00218508000907 SWE0.1M3e6a8n5 00..1155801010 00.0172580804 DisMean(lMean(] ~ PFOSAOmkdMeansNCEomFpOaSriMsOonmsK.S CONT NEWFOSEAOmkd NPEFFOSO4S0AmLkdOmY 00.8060000000 006060000000 007048775500 00.7182550000 NCEOFNOTSE4Omkd 07784570500 001028570500 000030705000 000030705000 NEFOSE160mHa "11000 045000 036250 032500 Alphas PAFbOs(SD4I0fmLcSdD NECOFNOTSAdOmId NNEEFFOOSSEE146O0mmdkd `Compar0i0s5ons for each pair using Students t 2.10!091 PFO0S4A1O9mA3d9 NEFO0S2M1O5T1K6Y5 0.3C60O4N6T4 00326105418646 00341688508 003331188568 00538450116985 001324038 001366187548 NEIFOS0E3A4O0m1k6d6 0."03.63845389 "002.44562889 Positive values show pais`CoofmmpearainssonthsawtitrhesaicgonnitrcoalnutsyinigfDeruennnt.ett Method 272]184 APbFsO(SADOLmSKdD 025C7O7N3T5 CNO-NEFTOSALOmKd 00..4432895505 NENFEOFSOES4EOTmGkOdM 00.438165608 N-EFOSE11.6101m0K0d0 003465205000 00.3020500000 N-EIFOS0E5156501m9kd5 00.1162147248 ""00284351228 6 TorOsLER TPBPoestDriovoseavaluesshowpisofmeans that aesigan 2. LNa A n ESAS pA ~~ AY wo WV "oDrossGroNuEpF(OSmAcaMOarMrBoIFsOmSEaOmnAg SSI0uN0sPeOtF WoDesureSaosmel | Rsqure OSneuvmamy aAorrfoFryia ose RNeRaoaontmMeosfaARnegsSpoanoseErr 0r03060i265s6 Obsarations (orSumWots) = VSoodrac DF An'SsoufmVoyafrSiaqsnucees " pra MeTmZe F9Raaio ErCoosa 2" 723288a25m00 G0as p00o0r0 cLoovett Maans forOnNomAbaerrn| Mean Tesi SourErzoor NNEEFrOoSsEaToomoimias NEFOSEdOma 23 i oBaa0o0 eT roazcsseest ores FRoSimia 5 Tei orerer Love S10 ErousWeasaraponolsedSeisatDiomavtieaoofnasrrvarias Nomber Mem Sev SiEnNoan cNoEnFtosnoma NEFOSEroom 42 aossom Ooamre sam Oomwe ootimon 02000 NPErFoOSSiEoimoamua 5i sTmeno ooaosims oOisoesmt POrfMOeSai(mliMaeanl] PROSHOnKSMeansCNoEmpFaOriSsMonOsm 200000 075000 NEFOSsEMemisd 11C3O7N5T0 NNEEFrOoSESdAOmmkad ont D7e9s00t0 zr 000070500000 570 o00u0r00s0 222% 00230337500 0oooa0 NErosEtsoma iio pr D0 om NEFOSETTaSdO0m0d0 006557050000 00030103050 w fwo o ap com-pleit2 ingutes nNt Seirsea ems rdGSoamiyms |NN FO2clammme NErOnmvRerNaonmmomSns |SoomG2ueO0nnN uBmoeseroma SSiEs ddaTm dmoym oamaen AT t Sa t i iila"l _-- EE n amt e Sie . iA y ---- - A | A ON | L"E A D AAN1 hm ) || ` IZ VI Av ROR | | ROJ oni NEFOSITNrEIFOSEAomia .E0hPa om Pi con _ ioSresneposos _5--5 RaSeLySmreeee EgLs pr re oOyRIETErToEEmn wsTreEgRs atEa Tw 5 mum hm oe oih A iYoOnE VYOm EFOTSa =oTsE = Si . TLItLLER NNEEFFOOSSAEL1O6m0Kmdka PFNOESFAOOSmEkddomid 42 amtaosm0 330143m 54 2umTew 320r4e%m StdError us`eMseaapnosoalneddSetsdtiDmoavtieaotfonesrorvariance LeCvOeNlT Numbe8r 20M3e5a0n0 eSeUDrev NENFEFOOSSEA6d0Ommkdd 42 a211s0000 416969649106 PFNOESF4OS0Emdiodmid 45 aum7a7es00 s28e66a7n0 SW Er3M1o0a7n3 33S.a0r0t0 212881260 DitMeanll-Mean[] ~~ PFOSAOmkdMeansNCEomWpOaSriAsMonOsM.K CONT PFNOESFAOOSmAkOdmK 250.010000 20510000 2598010000 CNO-ENFTOSEdomkd 6524815000 2F9705000 80705000 NEFOSET60mi 136.600 150 62500 Alphas PAFbOsS(ADOImLSD CNOENFTOSALOmid NNEEFFOOSSEE1640ommkdd Co0m05 pfo aeacrhpaiirussingoStundenstst 2.10t091 PFOS5A1O.m0k51d NEFOSMO0T%K6Y7 1C8O9N5T8 "5108..98578 0"48941777 dsgaa0r7r6 221031870 20056177 198182113 Positive values show pars`oCfommepaarnissotnhsatwairthesaicgonnitrcoalnutsyindgifDeurnennte.tt's Method 27d2l18 PAFbOsSDAI0LmSKD 40.c5o5n0t7 NCEOFNOTSAdOmId T27301111 NNEEFFOOSSEEIsGoOmmikdd 2489.2715619 NEIFOSE6A2O8m5k0d 387775500 730705000 NEIFOSE2A3O1m1k7d "592782677 5E0z817 aPiotseiretnito.values show pars of means that ae sigifcanty N-EFFOSE1T3660.m6K0d0 18112.550000 703070500 N-EFOSE126003m8A0 180851137 -3z87128%2 8 Torrs on BUNmg/dL.ByDoseGroup (mg/kg/day) - A A7a 0, Aa, e | N J w=N ETOv I) te crfniri dA oo ooyn 0 TpIri0s7LIFR ``OSnuemwmaayryAonfoFviat RRsSqquuaarree Ag RoMooatnMoefaRneSsqpuoanrseeEror Observations (or Sum Was) 00.218140800228 330118317156 % Source OF AnalysSiusnofoVfaSriqaancees MeanSquare FRao EMroodrel 184 207740557050000 11068403784 P1r7o1v0o8r Coal 2 28162500 122046 0.1892 Level Means for OnowNauymAbneorva Mean Su Eror CNOENFTOSAOmid 84 11867050000 116657016 NNEEFFOOSSEE1d6o0mmkkdd 43 2te070s00 1196055096 PFOSHOmKd 5 188000 14763 1d Error usMeesaanspoaonldedSetdsDtoavitatoofnesrror variance LCovOeN!T Number8 18M7e5a0n0 44S9u6D0e3v NNEEFFOOSSAEL6O0mmikdd 43 2160000000 226444897459 PFNOESFAOOSmEkddomid 35 1t8e80r0s0 22126177935 St 1.M5e8a9n6 11.52227457 01.91609857 DiNfE=FMOoaSnE 6M0emakna] PFCOOSNtTOmkd NENFEOFOSSAELdOommKKYd NEFOSEI0MMeaKnSs CompaPrFiOsSoInOsm.kd CONT 302000000000 302000000000 030255000000 352255000000 020055000000 200000000000 600000 280000 275000 Alphas ANb-sE(FDOISLES1D60mHa PFCOONSTA0mK NNEEFFOOSSEA4LOOmMkAd Compf0o0aseacrhpaii ussingoStundenstst 200t01 NEFOSE5I604M1K3d9 PFO1S8IOaMSksS 14C2O7N60T Ga824i56810 433E6e0E8s0 33848s8i8e9 00077272040 2t5gs8asss 124283110044 Positive values show pais`CoofmmpearainssontshwaiatrhesaigconnitfrcoalnutsyidnigfeDruenntn.ett Mothod 270]438 AbNsE(FDOiSLEStD6Omk 27C9O3N9T2 PCFOONSTA0mHd 5a0s39s44 NENFEFOOSSEA4LOOmMkKdd 42761669933 NEFOSE5A2I5M0k0d0 220050000000 007050000000 NEFOS"E00A2I7m0k4 "225238140848 "pa1r38y59 N-EFOS6A.M0O0m0i0t0 228705000000 007050000000 N-EFOS0A7M2O2m9k6d 1".r8a3s4t8084 p48r85e50 91 r5 o LL PEEp n t ssnstom as ta ety i AD A I) gmigo"o LO ol-- ) :: 3 ~ - Wi CONT N-EFOSAORMIEIOSEdOmi 00hPOF N -- J| usoGroup (oR 005 008 | WgeeoSnreesv u_z SEeSLyrrven = oe% h ofaer gor%PVEA SmmNimtgImms cMeTrnismmess arCemme Ta BO EEZO5G E19y ---I -- TE=T" M=wEw ygomm SAEiESaSn, P 1 PuE 0m Ib0ez E-o SM E a sI Gtin] Se [ ShEEePss-- 1 to{ pE2mE2 FE2nEEemn oa=anmm E2ROrSeattiinron EroasonnsConprSEtaRoomrYs werosti5nom%tis | NEFOSEaNInaOynn PRPmOro=Sve in 22 i am om a Aeron, npgn na=8 = = Iw:s 2 5 ow amenasmap:gennonss s5risnt.he | werogiognelsy II"TaEmll ewmeIa rEo| sesoifsamenss Soegong Ssos ine T BunN = = i=B mona de A o2i: vans we00isr.e, ay = formere, r= WLswnypnmiey | mo H L " i eA, a i2200 | Sed o VEFVRT & CO Te: l = || cnaGrp (rom) 00s oss - Sil w rrn lwsz LC -- 0 LL 2 sWowr 7iEe ErToEEnD aoTemE one # i EZ BEE #% Soit EAWR ESET 2s5r sutnn Tpr0i7s11 FR NNEEFFOOSSAEt1O6m0KmSia PNEFFOOSSAE0dmOKmYKd 43 e7ss2ss0e0 659918sm 54 67612205000 5s3e5e6m5 StdErrorusMesoaanpsooalneddSstsdtiDmeavtieaotofnse.rrorvariance LCoOveNlT Numbe8r soMerasn 1S7W0Demv NNEEFFOOSSEA1O6m0KmkAd 43 7ca2s0e0 7409471350 PNFOESFAOOSmEKddOmid 45 6T62i00s0 80o4st3 SWE6M2e6a0n5 3203837303 230517526 DCifO=NMToan-Moan(] NNE-FEFOOSSEAdOOmmKd NEPFFOOSSAEOmTK6d0mK CONT MeanNsEFCoOmSpaMrOimsoKnSs. 13080705000 103.80705000 "21972875500 49..00050000 25.4583 11.5833 NEFOSE1A7O8m7k5d0 4000000000 5705560303 PFO2S9IO2m5k0d 9500550000 205003030 Alphas CAbOsN(TDILSD NNEEFFOOSSEAdtOOmMKKdd PFNOEFSOASoEmTK6d0mKd Compa0r0issonsforeachpair using Studentts 208t301 12C5O3N4T7 NEFOSA"M1O.M47K6I8 NEFOSEA2m5k2d3 1245726328 1"17377226688 1r37e26s8 sseas86s2 7T7s67e1 CnTseers PFOSIeO3M5K2S i7t76e7r1 5s7ss7eds Positive valuesshow pais`CoofmmpearainssontshwaiahreasicgonnifrclanutsyinfgerDeunntn.ett Method 270id438 CAOsN(TOILSD 16.C1O9N61T NNEEFFOOSSEAdsoOmmiIdd 599661111 PFNEOFSOASOEm16d0mkd 4354258878 Poesrietnitve,valuesshowpaisofmeansthat aesignifcanty ALT UL By Dose Group (mgolday) N-EFOSE2158.04m5k8d3 17155883333 0205030303 N-EIFOSET8E408m6K2d 17155663%8 ""210547670418 % Tbrrs on | O Ha RBO eSAm SS . v | |l DoseGre an roerko)se ciamamon oSuss TDeares oss Rqure OSnionvaanAracFaa ogrerss [NRooonno oiaiaRnesSpoanrs Err S0Ta3araseesmornstd Gosanston (orSm ig) oe Soduen OF% AnSuomfaovTlarSrasaneecawys MenMSayleer FRato ECrhroa 3ws mTesriems wperoroene cLoovnet Means for OnNaovAraoooT yva nMean Ssiurno NENNFEErFoFoSosEsneiioosnmokmsaa 5i GaGoawwm asswasnmn Prosiomia i Sam doe Lovet StEroruWseo poNasanemdbs5atDroavtoeesaroncvariaSncsev SETom oKNEEFnroossatiosnokmaa SSo oo@mGameener mrrammw sasoauams NEPFroosSiEioma IS BGeenm ptowmn dosem NDEirfotsetsMEomsa]l NEFOSEIueGInoMsGCamPaErOaoSmsmked rCaONeT NEFOSEAOsomed CPNroeoFnsoisoemsaoia DeSeemme ggcmosmso daoasoooo pTGroo]oo NErosaioma Sater ase iors S50 Alphas Compf0o0ra5eacrh1}aisisngoSonnss NEFOSTSaoMr300I80m70 TSSo0or0oo0o 0 wi ww=oan. | woo0A s _PliFnoynI gona sGeeeri Bom EE am Bolml E$E9 SBE EaE o=m itt frI eerliietsnoms idl mMoWieeem, Abs(Dif}-LSD omCONT iSEz Hom BB TagT stasS Z 2h= etn A : = F||EgN dTMwd A = V ON VA / =\TEZ=NNY [& Des E feTrsAG WA Pe [."% i" fSoe,Tmmar `DoseGroup (myKg/day) os o jsao ET re id Lt 2 gB 5=r T12I SEBZ umErmIsE ueEgnwu== tu WI WP yu Si Bu a Fsims 1 !(208 32% 9g8 Serum ian = " TtIdO7LLFR PFOSAOmKG 5 47600 StdErroruseMsoaanpsooalneddeStsdtiDmeavtiaetoifonos.ror variance CLOovNelT Number8 siMemasn 3S042Do8v NNEEFFOOSSEA1d6O0MmKkYa 43 7w3e0s0 178756012s NPFEOFSOAS0EmdKOdmKd 15 maT2eso000 36 3964 10705 SWEM1M0e7a1n2 74.745815 12698832 DCfOMNoTan( Moan] NENFEFOOSSEA4MOOmmKIdd PFNEOFSOAS0Em1K60mka CONT MeNaEnFsOCSomEpdaOrmiskodns 90.01020500 90.01020500 143778775500 38478550000 587083 ose3s NEFOSAaMOrmkod 0807050000 205890%00 PFO4S3I7O7m5k0i 235498050000 104.90308030 Apa ACbOSN(TDILSD NNEEFFOOSSAEiOOmmiIdd PFNOEFSOASOEmIi6dOmks `C00o5 mfopreaachpririusisngSotudnentsst 209!01 25C0O5N35T NEFOS2E1A55D9A1d N-EIFO"S1A2M8O0T9K1Y "22185080911 S8s64831100 352846381100 1245.27080568 111.30183752 72578385 PFO1S5I2Om0k0d6 7170132782 231166950946 Positive values show pars`oCfommpoaarinssonthsawtiahresaicgonnitrcoalnutsyindigfDeruennnte,tt's Method 270dl438 ACbOsN(TDFLSD 32C3O7N1T6 NNEEFFOOSSEAdiOOmMdd 32015722200 PFNOESFAOoSmEk1d60mH 16488675770 Poasfifteirvonetv.aluesshow paisofmeanstha aresignificantly `SiCgomdbeicnreedasaetsdiantAaLfKoPr,maAlSeTs aannddfTeRmIalGesb.y 40 kd PFOS and 160 mika N-EIFOSE in ats. N-EIFOSE5168.07m0k8d3 0985383333 140.09030303 N.EIFOSE2146708m5k8d 12153613355 2d1o6s5i2926 07 -- 2 = ATES d1a.,80 a A\ fo N 112A 60 AA SUANYN " INV A OTT = cori ar4ora BW TpIri0s7LIFR OSnuemwmaayryAnoof viat RRsSqquuaarree Ad) RMoooatnMoefaRneSsqpuoanrseeEror Observations or Sum Wis) 00.311520232879 53001..96806817 5 Source DFAnalysSiusmofofVaSrqiuanacees MeanSquare FRato ENordoer! 4 6360947044.592508 3T762001833 P2r0o4b2F0 Cot 2 o7379.478 wes os Love! Means for OnewNaoymAbneorva Mean StEror NCEOFNOTSAtOmKS 48 a20s3500 3210546%6 NEN-FEOFOSSEEd1O6m0Kmd 42 228170800 4330143% PFOSAOmK 5 ren 21219 1dErrorusMeseaanpsooalneddStesdtDimeavtieaotifoensrorvariance CLeOvNelT Number8 20M3e5a0n 7S848D7o0v NN-EEFIOFSOASEL1O6m0Emdkd 42 s211000 416963649106 PFN-OESIAFOOSmEkdomid 54 gTmae7sn0 s286e67s1 SWE3M1e0a7n3 3935.7010 22882106 PFDOitSMAeOanm(kld-Meanl] ~ CNOENFTOSAOmK NNEEFFOOSSEE1d6o0mmkkdd PFOSAOmkd MeansNCEomFpaOrSisAoOnsm.k 250.010000 20510000 5542180500 729705000 138.600 10 5C8O.1N0T0 NEIFOSEA6O2m8k5d0 209000000 387775500 682755000 730705000 Aphas APsFO(SDALOSMYD CNOENFTOSAOmKd NN-EEFFOOSSEEId6O0mmKidd Compar0i0so5ns for each pair using Students t 2.10t001 PFOS"8O1M0K5S1 N-EIFOSSA0OM8K7S 1C8.O9N5T8 ""1680.896878 409641777 984401776 2320131870 Se05e1r7 e1o8r8a1rs NEWFOS2E3O1m1k7d "S5o2r6271 97028137 Posto values show pairs`oCfommpeaarnissotnhsatwiatrheasicgonntircoalnutsyindgifeDruennnte,tt's Method 272]184 PFAOSS(ADOLmSKD) 40c5o5n0t7 NECFONOTSAIOMKd 73206174111 NNEEFFOOSSEE4OTmSKOdm s482.2751619 N-EFOSE1T36606m0K0d 18112.55000 70700%0 NEFOSET62O9m3K8d0 180851137 "152782.13582 9 Toros on = bo pd sm] =ra fn L0HPpBorsyieBedevtalevesshowposofmean hotar sigfcanty LAAAN A ACS : &, 2) = Sa oecr iy Roue ERE e Rqure -- fi-- i] ozsstss RRNeooaamrnoefRAogsdsaoanrssErr SSeiasvseeornys Gosenaton or Smvig) = oShue or% AnaisomfoVlyiasrmsnsmceees Me"sleos Foreas ETrra i2s dSioersemss taomsnei moewre LSoovent VansforOnevNaoyArnio"va I ean SuEror NNNeEErrrooosssaetirisooonmnkskaa IDF omomwH mreure Frosionia HEA Lovet SiErruWseesaarpNoa oomleodasrn sDioavtdofVeernrvaraSiOev SuEMom KNEFnrorossmeonsoimaa TPuoTammmo msmeeown Uo"mo0 PNeEsrtFoasnEiaoms F PoI m ] ee aErHes Ooneste) cGoonot Mo"nFRsOTSCiooimmpsiasrisonsNEFSOSEoiOsMd NEFOSEJIaSnOeMsds NNEreiorrsooissoEeniiaoonmaa aTsmooeeosss 100omso PRiois]] Hoo3rs] NErosaioms rr tire sao NEFOSpZAisOernoS0 foSi0o0e0 10 poe ns sSwrnrreomoup"nonwearossnigtmy weroregmy aSPio,ee.wn,,e eSTEmmTMo OTEEhmmmam neseMGSaaRmm esosM2mUkwR ei fr Trt etn wiod1)n am Pew ImEE " sarrEepEtobe meenous82m ets = LD ipHriSn V A\WCIAN L(O=YYLOx Y)) wewrooroM4EoUnU8mmHyR 'E Gg re er Wercieeroseioms | GAPE Teo RJ FEr sLrmo - s=m EETmm =% tm By=o%oFoEeEEEusmemEElnmh wrwaEeeRmg aoocmseo oimoe eiraA monTam ue TprIiOsLIFR NENEFFOOSSAEL1O60mmIkda PFNOESIAFOOSmEk4dOmid 24 usstso0m o1g3776 45 1t9a2e0 0og8T1e1r3 StdErroruse`MseaanpsoaonldedSetsdtDimoavtieaotfonersror variance LCoOveNlT Number8 1eM9e3a7n5 16S8U5D0e2v NNEEFFOOSSEA1t6O0MmKkYa 42 1u5s1s00000 035831568530 NPFEOFSO4S0EomdkOdmkd 45 1ue760 215a16m88 SWE0M5e9a5n7 0245008020 e1.r436e1 NDEiFfMOoSanA(MlMOeKaAn] NCEOFNOTSEdOmd NEPFFOOSSAE0Tm6K0mK NEFOSAOMMeKaGns ComparCiOsNonTs 60200500000 010602050000 4J705000000 01.7172550000 550000 387500 NEFOSE147O5m0k0d0 001020050000 6758000000 PFOS3I4O0m0K0d0 11..7875500000 201000000000 Alphas ANbEsF(ODSIALdSODmId CNOENFTOSEdOmid PFNOESFAOS0EmTk6d0mkd `Com0fo0pr5eaachpririusisng Sotudnentsst 210091 NEFOS2M8O0M4K3d1 08C8O1N8T5 NEFOS1E.1A4m4k3d1 oBasiesss 22308416885 22380413815 e19s55t20 00563598046 C0o20s5t2e0 PFOSoIsOsmikzd 015089854779 12.s3e2e4r8s0 Positive values show pairs`CoofmmpearainssonthsawtitarhesaigcnoinftircoalntusidnigfeDruenntn.ett Method 272]184 NAEbsF(ODSAALtSODmd 1.C62O2N9T7 NCEOFNOTSEdOmkd 2S62513478 PFNOESFAOoSmEKdISOM 2038117333 Paiofseirienvte.values show paisofmeans that are signfcanty Ks mmol By Dose Group (mgKgiday) N-EIFOSE515G0O0m0id0 337857500000 20.1000000000 N-EIFOSE1.19G5O5m2d0 006230550260 p1.3r24i60 102 TorsLR v . e > s i 5 LA, AN 7 > . on werotamaroVweI]To NEFOSCIoRROSS `DoseGroup (mgKaiday) SUITS! had ir, To Sowaty oss Rss SOneovamyAaorfonrFia o7asrss RRVoeaoaatnMroolaRnAegSsapaornaErr QQ$as2aa0ae0os6ai0sz0 Ossarvatons (orSum Wot) 7 VSoodcaa DFi AnSuamofoTVfiaSruivaasnrcsees Meomses i FRaio CErloow 2 NTmeoans oarmers p<roooork Loovnet Means for nevNaomAben7ra cMaoaon SuoEormr NNNEEEFFFOOoSSsEIEmooommmaakd 32DOm33R000 oaoismme Prosimia 53% ose Lov S40ErroruseesaarpsoNoaolnmebdSesratOaoitaoofMnaestaonrvariaSnicdeDev SiEnMoan cNNEoErrroossenacnoimaa 47 aeShmaoono oi00as10re0 30"720s00t%0 PErorsoiosmiioa n i 8S0m0 oomseer 0oi2r6 COfoentloan Meant) 00C0O0N0T0 MeaNnEsFCOoSmpEaI1rO3i0sm0oin0ds0 NEFOSTE0M0m0i0 NEFO2S2M75O0M0 NNENEFEFrOOoSSsEAEimsmioaama F2123a7030o0000 2D2a30r00s00000000 0308007000000000 a0o0so00oo0 PrOSimia 30 200000 Hoo S060 Aphas Co00smpforasacrh|pii ssingoSudnentss PFO33S50I0O60m00k00d cr17eo0s0e0o0 0 0bis16 ffNefiosscsiooma PrCeONT NEFO2S"1ED0Io0G0sOsMy NEFOSEsHomisd 003% Seas 5 NEFOSSTMieoenemd PNNrioirsroiosastmsiiioaommaa. Za1a1si6rs%e SSpoeiams prSheaenres SGSlioesee Posto vues showposCofmamns hptwairnoaassioratnoadoioft. nsesthod cRoosin21e51d|s 4c5o5r NNNeWErorososesnicieioommmaaa 3o5o3se8eo8n3s Prsiomia So PGorseteenvalvesshowposofmeanthatar sian Ci- mmoliL ByDoseGroup (mg/kg/day) PFOOoSiosmkoed SSokoiemay [fob [ =f |= MTAeA LAYF ETN | a C) / TE comns cara on ow Rare OSnovoa Ayra 02ers RReoaasnre roSpqauanrseyErr opToirraen tontoSum ig) ] oosuen OFi AnSuosmfVoySalSrhsmcweoes Messuam FoRwaino HCohra 5B sSloomse sSeoems moeeer Lovur Ma forOnnTAs orTaoswsen Souwmny KNNEEErFroooSsstEaosiooommniaa 3Pollikeoem ei1mmEm 104 TTIIOSLIFR PROSAOmd 5 10200 1000 5Error us`eMseaapnosoalneddeStdsDovtiaotifonemsrroravartianece LCoOveN!T Numbe8r 00M6e2a5n 26S6U9D2e7v NENFEFOOSSAEd1O6M0KmkSa 42 11004805000 0100900s0 PFNOESF4OS0Em4KOdmKd 54 11002102050 216828019164 SWE0r8M4e3m7 0099507040 o13o15r0 DNi-fEMFoOaSnE(1M60eman] PFNOESFAOS0AmLKOIMIA NECOFNOTSEOmid NEFOSEI6M0eakn]s CompaNrEisIoFnOsSMOTKY 005.000000000 005000000000 220705000000 125205000000 337500 28750 PFO2S0I0O0m0k0d 010.500000000 017357050000 Apha= AbNsE(FDOISLES6D0mkd NPFEOFSOASOAmLiOdmAd NCOENFTOSE4omk `Comf0opr0seaachpririusisng Sotudnentsst 210!001 NEFOSEI46703T9K9S9 NEFOS3M6O0T4K0d6 PF1O9S6M5m7k6d "13.8908459766 3D3esrte6k8 12607997688 "13375429269 io0e6s8 s24e29r68 Positive values show pars`oCfommepaarnissotnhsatvairthesaicgonnitrcoalnutsyindigfeDruenntn.ett Method 27d2l18 AbNsE(FOOISLESTD6Om 1.C47o9n8t2 PFNOESFAOSOAmdKO)MKd "2a1s2s5e8z6 NCOENFTOSEdOmid 30173054882 NEFOS2E7I5m0k0d0 202755000000 008020500000 NEFO1S3E54m9d8 Za02106688 S232s7t8648 aPiosfiotrinvetvalues showpaisof means tha are significantly `GREA mad. By Dose Group (mo/Ko/day) 33C7O5N0T0 218377550000 00.5020500000 03C7O2N2T0 0i.30e277240 223271706040 105 bri w Jw a AAE, AA, S YD A Se A S <y > WW " pre ---- be DossGu(oneirroscimross Sooen ! o00es L_--inT iini _ Sa--n---"in a oe% wyFT 0iYtaT rmEVT euirn =t=em w i ZB EE -3nimr OnYfLe1tw5 intm YO=mz eEaen, iI oBinR= ai. TTTEaEFsoo Jtiyer onetuom SSa oEuie 1 E 1mg 2F ia EaEHrEs = EaRCpese vS2dJ rsS arEBiynRE vea wpom Swoeeefs ouoen oiee 27Bh =TnR = I= E 4&R 7 a n oz J Goeodsee cwi:gbgots #5 TbiIsLER APsFODSIALOSmDd NNEEFFOOSSEA1O6m0Kmkdd NCEoFNOTSE4omkd PFO0SI0O0m2kd2 0000005439834 00009350007659 NEI2F10O0S9M1OTKS ""00..1040459%3 "00005340031 0033776 N-EFOS0E.10G0O5m3k6s4 "00..1045849071 ""00.0162970717 N.EFOS0E0A3O0m0k6d0 00.0132003041 0o10z5 Positivevaluesshowpars`oCfommpeaarinssotnhsawiathesaigcnointfrioclanutsyidnigfeDruennnte.tt's Method 272]180 PAFbOsS(4D0LmkSdD 008C0O8N7T5 NNEEFFOOSSMEO1M6KOSm 000.0160821648 NCEOFNOTSEdOmd ""000.60801695 Poasiifteriovnet.valuesshowpaisofmeansthataresignificant Cdeicnrieaalscehinempoitsatsssifuomr ably r4a0im(kadlPeFsOaSn,dNf-eEmaIlFeOsScEoamnbdinNe-dEInFDOSTA1,58w,itShhtohwesmossigtnisfiigcnainfticant cIhnacnrgeaesseodcciunrtrhien4g0imnithde4P0FOmSkanPdF4O0SmdoisoN-gEroWuFpO.SACrdeoatsienignreo(uCpsR.EAT) was signicanty 009C5O0N7T5 000.3036777776 000047142s2 107 am B. Statistics on Clinical Chemistries for Guinea Pigs, male and female values combined. w ERA - A / p E01 a A w AErAi ~NG7 NN ol i RY NY . No= - > -- CONT NEFOSAonyd Prosios ~ EMhPar ---- on N-EIFOSEOmId Wotdeer: Nor / Berens " TTIIOS7LIFR OSneuwmamyaAroryfovFait RRsSqquuiarreoAd RMoeoatnMoefaRnesSpqounasree ror `Observations(orSum Wats) 0o360e357e825 a02a3a37635T 19 Source DF AnalysSiusomfoVfaSrqiuaanrcees MeanSquare Rat EMrodoerl 155 387138.8180426 15149519028 P2r.o8w1F7 Coal 18 11761947 esses 01327 Level Means for OnewNauymbAenrova Mean CNEOFNOTSAdOmKd 64 4T0i078s30 PFNOESFOASOEmdiodmkd i5 m549a00s0 Su9E5ro%r etewe 10449 1d Error usMeesanapsoaonldedSetsdtiDmoavtieaotofnesrrorvariance. CLOovNelT Numbe6r 400M8e3a3n 19S947Do8v5 NENEFFOOSSEAddOOmmdY 44 7m1m7s0 2307453404 PFOSAOmKd 5 549000 207497 StEr8M1e5n6 1115281775 9280 DiNfE=FMOoaSrAOMmeKaGn] PCFOONSTAOmid NEFOSEdOmkd NEFMOeaSnAsICOomMpKarisonPsFOSOmkd CONT N-EIFOSEAOmd 160.08050000 10608050000 311468816677 230753278500 3r1a6i6s70 1240815607 5070008030 5070008030 Alphas `Comfopr eaach0pr0asiiusisng Sotudnentsst 213t144 ANbEsF(OOSLAdSODmId NEFOS5MO2M1k7d PFO1S6I8O5M7K6d 0C4O7N9T8 CPOFNOSTAOmKd J0e47s9s8 sitsaoan0 12583730974 NEFOSEdOmKS 21608 2888 26.4382 N-EIFOSE2d1o6m0i3d 218248382882 382147 Posiive valuesshCoomwpparaiissonosf wmietahnasctonhtaroaleussiinggniDfucnannettyt'dsifMeeretnhto.d 262]898 AbNSE(FDOISOALdSODMKd 7C9O8N3T8 PCFOONST40mkd 23534768474 NEFOSEMm 339421 Poasiifotriovnte,valuesshowpaisofmeans thaaresignificant Cholesterol, Male andfomal guinea pig combined. 109 TbiIsLLER 10 --1 -- RTH = x AVA ally sq N A A T N ASi \ 3 \/ V \ w]\/ 7 d | CONT sy N-EIFOSAWOmKE Prosi NEFOSEOm |EMER PF Spt Wahsata) a TTII0S7LIFR OSnuemwamyaArnoyofvFait RsRqquuaarree Adj RMoeoatnMofeRaensSpqounasree Error Observations (or Sum Wets) 0709.1258019 080122112765 1 SMooudrecle CErlroor w OF Analys'iSsoufmVoairSiqaunacees MeanSquare FRaio 23 4033575500000000 00316225901070 P0r3o4v4o4r 15 47300000 oats 07838 Level Mans forOnowNauymAbnerova Mean StErmor CNOENFTOSAtOmKd 44 n12e2r0s0 o03o0nm PFNO-ESFAOOSmEk4odmid 44 1u24s00 0o301w21 19Error usMeesaanpsooalneddSetdsDevtiatoiifonesmrroravartianece LoCvOeN!T Numbe4r 1M9ea0n 080S5W7D0e0v NN-EEFFOOSSEASOOMmKdd 44 112224000000 o0s4e9e6r6e8d5 PFOSAOmK 4 ris oastaar StE0r.M40e2a8n5 002294485393 022087 NDEifF=OMoSaEnfd-OMmeiadn] PNFEOFSOAS0AmKddOmd CONT NEFMOeSaEnAsICMomKpdarisonsNEFOSAMOmki 0.00000020 00020000000000 ""00422785 00202755 PF0O2S7I5O0M0K0S 00007050000000 015 042C5O0N0T0 00.212550000000 0000000 Apha= Comparisons for eac0h0p5ai using Students t 217t882 ANb-sE(FOOISLES4ODmKd NEFOS"E045O2m8K13d NEFOS0A7M2O8M1k3S PFO0S6A5Om3K1d3 05C0O3N1T3 PNEFFOOSSAAOdmOKMKd ""00752381133 009881133 00582381i3 007770831133 CONT 050313 70313 ores 0213 Positive valuesshCoomwpparaiissoonfsmietahnasctohnattoaleussiignngiDfucnannettytdsifMeeretnhto.d 26d8l20 AbNSEF(ODSIEAdSODmKs 07C1O7N8T8 PFNEOFSOAS0AmdKOMKd 00.9919728888 CoNT Exe Podsiifteirevnetv.aluesshowpaisofmeans thatarosignificant PHOS mg/dL By Dose Group (mo/Ko/day) nm . x To LATE os A a0fmt CA NA2 (( VV Vv eons N-EIFOSAOMWIEFOSEIPTrEose |E`aStcuhdePnatsrt DuWmeo ttsn Dose Group(mgKgiday) s--m--iid 00s. 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Apa PAbFsODSIAHOLmSdD CNOENFTOSEOmid NEFOSAOMKA ~~ PFOSMIeOamnKsdCompariNsEoFnsO.SEAOmki 0000.00407050. 00000407050000 005016078 00000142157 Compfo0ae0a5crhpaiirussingoStundenstst 213t144 PFO0S.I1O4M3k91S NEFOS0E1A0O5m1d4 "00180851124 f0a16r09 "0.00264 ones 0.0C51O6N6T7 00.000004010607 000833 00C8O8N1T2 00.1134123771 013855 N-EFOS0A0L6O0m0k0d0 00000182353030 0000000 NEWFO0S.A0d0O2m6K4 000...1113648088045 Posie valuess`hCoomwpapraiissoonsfwmiehanasctohnattraoleussiignngDifucnannettydtifMeertehnto.d 262I]898 PAbFsO(SDAIOLmSdD 01C1O8N2T8 CNOENIFTOSE4Omid 01o62T074 NEFOSAdOMK 0.17283 aPioesriteinvte.values show pairs of means tha ae sigifcanty Alb mgd. 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J----I--Tewn SSuItE NHNErFooSsaost,oionnss iPoEEBmEm Aaamme FLoow Err aas araiiadsovoirfmoercvaScbw SErtem Toe mw See 0 TorsTLFR NNEEFFOOSSEAOImOkmd ProSiomd 43 5 smmoorss mreineds rome CDOiNMTea'nfMean() MCoOaNnTsComPpRaOriSsMoDnMsK ooo 326067 NEFOSSAOiTeKS PNNrEEOFFSOOiSoSEmAiiiaoOtmmkdd SSaiaoeww 2z0102r0%0 200.2050000 "1000 Apho= Comp0f0orsaeacrhpiiussingoStnudss AsO CONT 213|104 `PROSAmks NEFOSMOmks CPNOONESTFH,OSMOM aL2i8mn5ae2 a6s4ond7msee0 "2s08s.53a79738120 NEvOSEma ror aera per PosiivevalvessChowopairsmofmwpeiahnasactoanttrraoleuissiinggnDsfucnanontey dniMfeeresmnto. ASOIL26S2ol0D08 cont coPrnO'SOmt 9"1207%% NNEEFFOOSSEiAOOmmd 4467.65228888 t55s50 stor NEIFOSSsE4A1O6T7 2T7505000 00000 NEFOS2E1d0O7m3i2d S"2s07e50 03631 = ASTUnPossBoiyDotionvs:eevaGlrueosshowpairsofmeansthataresignificantly wo - 3 A AlN PA AOA e D S N 1 DeN7Y Y \ [== sy wr os V Dose Group(mOKoN/EdIEyF)OSEAOTA Sy o% Gigy 005 1s TbIi0s7LIFR OSneuwmamyaAronyfovFiat RRsSqquuaarreoAd) RoMooatnMoefaRneSsqpuoanrseeError `Observations (or SumWats) 00..01803003537 s2016.81422418 1 NSooduer!ce ErCoorl DF AnalysSiusomfoVfaSrigaanrcees MeanSqure FRatio 13s 6660953.73621393 242612027 P0r4o5b2F9 18 73001053 asset 07191 Level MeansforOnewNauymAbnerova Mean StEror CNEOFNOTSALOmId 46 aate0r fs0se6a2 PFNOESFAOOSmEkddomid 45 sstass00 ossuer StdError usMeesanapsooalndedSetdsDevtiatoiifonrsmoravartianece LCoOveNlT Numbe6r atMeearn 10S144D7e2v NENFEFOOSSEAdtOOmMkKdd 44 msss0000 471728128 PFOSAOmK 5 57400 150100 SWE M4e1a8n3 230868019 6713 PDFfO:SMAeaOnmfKl-AMeanl] NCEOFNOTSE4OmKd NEFOSAOMK PFOMSeIaOnMKsSCompariNs-oEnFsO.SEAOmki CONT 10.09000000 019000000 1913233383 1Ci1s21353003 192335503 1090106070 Apha= Comparisonsfo00e5ach pai using Students t 213t144 PAFbSO(SOAIoLmSkD PFO2S8I4O7M7K0S N-EWOS2E8d3Om0k4d 16C.0O3N1T3 CNEOFNOTSEdomkd "218630038143 S19i7s30k8 1259.79300888 NEFOSALOmIA Tossa Z0se82 2r.1475 Posiive valuess`hCoowmpapraiissoonfsmweitahnasctohnattraoleussiinngifDcuannnteytditfMereetnhto.d 2621898 APsFODSIAOLmSidD 23c9o5n6t CNOENFTOSEdOmkd 23260581830 NEFOSAdOMK 33.9319 N.EFO1S3A.O15m0d0 1011029051006007 N.EFOS"1A7O.m05K4I4 270.5487852 Biase aPiofsoireinvte.values show pais of means tha ae significant ALT UL By Dose Group (ngiKglday) 126 TorsLR SYS TOC) " 2 No NT w / x Non werovime rooms TERE N-EFOSEOmMg `DoseGroup(maKoiday) isuney ow ee `ouwiagy os Rsaure OSnoemvmaayryAorroFria oars RoReoSaqtunaMroafaARnefsSpaonrseErr 0S833t60a80i33r81s7 Ohsanation (orSumWots) fH oSdasc!o DF AnSusomfoVyafrSiasancmes 3 "s512001 Mewmaes FsRaaieo EnCelrot % 1s16i37z80e5 SWissss prooomet Lovet Means for OnewNaoyrAencrva Moan SuEror NNcEEoFFnOtOSSMEOOmmd Ti ammmo aoemm i km 3mm ProSiomd 5 wow zens Low 514 EroruseasaanpNooaomnlbdeedSreisatDiomvaitateooMrfesaa.tnorvariSa@ncDesv SidEntoan CNoEnrtosnoma NEFoSEdmia Ti ammame ian asneem ere "Toeaattes san ProSiomia 5 sooo 430 Tozas OPrfOMSeiaonmdMoni] PROMSeI0aO0nM0Ks0SC0o|mparNiEsoFnsOSaArOsK eCOrNT NEWOSE1A35O0m0d0 NNeeCorrnootssneisaommaa T28157055000700 sB007a00w0 io`ssotemor 30p70r500e00 Alphas PrAOS5io0m 0 Compor0a.e02a51cr3hp:14ai4r ussingoStundenstst PrOSSeitmoS NEFOZSMOwmd aCvONrT MEFOSEaddmmisd ww Tbisoi fnoeonrsocsisoommaa 2p433007i9281 SPSarews adasmam SsTeomee Poin au Csohnovwepnaess ofmwainnssshotntrewSyinDaonmesdoiad pProst2tdn1isdoan 1co5nt NSNeoWrroossemuommaa ipSezmAaen LonPuniGBnirognwsvarionuhowosofmons hrsiicanly |Is = PLA AOA 22 A Al ~ AN Ty hr coop) -- Voss proses | BAR N-EFOSEdOMKS Susarg ooWRT OSes: ro f fieory oor RjokdmaiSdar Err ehSoeir Oetmion t Sum Wot) jSoaoco Of5ArumoofaVloaSamnssmcenoss MemSSwyme Fortea HToorm $B odwwes agweer moesre Loovn Mearsor nNeoAonynoTmMean SuSEirsr PRNroEFsriooossmtsiioaonmaa P P oTmmo< sBime - 4ErortaesapnNocmtetSntstorottean"oNfaoanvariSeuDsoy Sun omn or Tore was Tm 2s wHeNrrEorososisontnoisnoinaa D oMmmmeeo mwseumns Gweumm NDeOorosrtEeiinma] PrOSsSioomana sM,ERGgEeS] 350m Soon NEFOSfpMrOoim rc_ceONT SNeorno =om] pPi d - Soom ] asses Avtar Compor00aasch\rpauisinsgSdontns jNfseOrneoiwsiDma eroisoionnisa2sNerosi tima men eed WeFofsowiivmra Scomur SNoenrosma a aon ade T edEaEms p Co noe om 5nohft]ooe mmame--ssows ffNoresrOeohsLtnSiDona TPceeor SNoenrossioms oTirmeems = w] A asmtSory ase ro (git Positivevalues showpairsofmeansthataresignificantly lNye" .A iHxR oS = CS - Adel >- -- ~~ [ C= o|| [ry tl `Dose Group(moKoiday) os oss 2 TTIIOSLIFR SOunmemwaaryyAonfovFiat RResqquuaarree Ad RMoeoatnMofeRaensSpqounasree Error Observations (or Sum W's) 00779531297249 113.87.24728171169 Source OF AnalysSiusomfoVfaSrgiaanrcees MeanSguare FRato MEordroerl 135 74148748832353 S2702885267 1P9r.1o8w6F6 Co 18 21663158 20351 <0001 Love! Means forOnevNaoymAbneorva Mean Su Eror CNOENFOTSAtOmId 46 114715803 00780653440 NPEFFOOSSA0Emdkodmid 45 11430400000 00787623743 1d Error u`sMeesaanspoaonldedStedstDiomvaitaeotofnrsorvariance LCovOeN!T Numbe6r aMteasn 1S7u2D2o0v0 NNEEFFOOSSEAdtOOmmkKdd 44 1030870500 114510402010 PFOS40Omkd 5 13400 207384 SWE070M8e1a7n 007705701010 082738 DCifO=NMTean-Moanf] NN-EEFFOOSSAELdoOmmkKd PFOStOmKd CMOoNaTns~ComNpEarFiOsoSnEsAMkd 108030303030 01.0803033030 570483333333 356205000000 NEFOS5MO0m8K%S 03020500000 235000 PRO7SI4O3MK%! 253650000000 000000 Alphas `Comparisons0fo0r5each pir using Students t 213t144 AsCO(NDTLSD 2.C1O26N%T "NEFOS0E5A4I3m9k5I NEFOS2M7O0m8k0S5 NNEEFFOOSSEAdOOmMKKG 025740630955. 2o6i04s18 2o6s0i4s1i8 PFOSHOmKd 520325 3.12045 D154 PFO5SI2O0mK5d 03.112209864 232025 Positive values`sChoompwapraiissonosfwmietahnascotnhtaroalruosisinggnDifuicnannteltytdiMfeertehnto.d 262dl898 ACsONLT SD 26C2o2n6t5 NENFEOFSOASdEOdmmKkdd 12.1059181837 PFOSA0mKd esz6s Paiotseirienvte.values show pair of means that are significant CH mmol By Dose Group (mg/day) 130 ois = '|. VIu YN Y - S 1 ) M CONT NEFOSAMOmks PrOSIN | EXPO J ow N-EFOSE4OmAS Suse owWrSoneel vou rsare OSnovya Arsora ososste RroeeoaniSosro SSoaranas Cotonou Ws) % sowre or} stiomthsom MomRsuMre FESrauio T fo R EBEwS SRkTsEer owt MeorCaaTryAersal an er NPEKrFosooissoEnniosinsmta iPoomneEemm utmn S40 utee aapmreedsxfsaotveicifrarovtrs SNErroosseaimomad DSoOa eRs diamsee boemme Prosionss PoE Eu DbNosor)scoiorma) SoosaouessTCoEmSprSSSiooemnmimea prOCjsiaoomnemkss NEFosa$M3o0nm0s0 BNosetorn o diree STomme Sdiees oasmo Ait [ER--oss -- \ -- Jr8 25c0o%r 2"3N3EosScoiroensa pros"iOomok,a NErosSMosms a 0 bis 1 PnVreoovsioisemumaa oSGgorooes sISesmns 32i20hm80 S2Fi0io0rns Posie ves shrowpmassofmweasho 2ataadsv sigaae mnedsornetn AfrsoiOseLSuDma a3c3b7oeinrs f irtom SSoireas CREAmgPtoGsriBteiyevenovsaleuGersosuhpowpa(irnsolfmeansthatare significantly " IA ANA\ osm YIYN <INS. -- No - omc tgs CONT NEFOSAdmkl PrOSiN NEFOSEOmKS oeEMPOM w--_-- on WarSoveel Sars Asa OS nevayT om ozosens fRrRooennde AbdSuanrdErr SSieaseiiToaenn Cotman Sums) % Hsoura orSsnahgimSvooalStoagmrmmemsee MeCmoSanmso oFoRami HSo hah OOmme Sdhoammes meet SLoovn Moons fo noeorAoa"Tov owMeosm SDueereir PNNreEoFrsooisstsioomma Is oSGalememe oooooooms Lov Si ror gTe apNeoolmS"iansGoaovoaoaonrn variance SuDoy Sram our T owe omens Ogos 1 spp r[CopLins 5i n wonuomnl ai=dzm n Rotts en I orSschorreTsTamye ewZmaeruom T 5 e =x == |2 e Ju RE ao: w E oN gonnee .s, vePU rTo E romnasy werwdeerna [bE osoany ue i OE IEE T a opr cvo i5e en s Thsatetrs, fA Tir otee o2n i Li " erie a oig Positivevaluesshowpairsofmeansthataresignificantly x F I TN A lLVaAEaR( T - Vv \ J CONT NEFOSMOmMd PrOSis N-EIFOSEAOmk = | EAChPAr Stenrs al Duoty - TIAS07LIFR OSnuemwmayarAonyfovFiat RsReqquuaarree Ad) RMoeoatnMoefaRnesSpqounasree Eror Observations (or Sum Wets) 00..10340289935 31428322916065 19 Source OF AnalysSiusomfoVfaSrqiuaanrcees MeanSquare FRaio MEordroerl 153 182179460558735 2o1i3s1s12 P0r7o5v0o Coal 18 20897.158 este 05375 Level Means for OnewNauymbAenrova Mean CNEOFNOTSAtOmId 64 r1amtse0 NPFEOFSOAS0EmOKm)kd i5 a105000 Sw14E2r1o9r r7a4t1ss 15576 1dErrorusMeesaanpsoaonldedSeisdtiDmevaitaetoiofnse.rorvariance CLoOvNe!T Numbe6r uMmealn aSaUsDuerv NENF-OEFSOESdAdoOmmiIdd 44 12338520%0 42450348638 PFOSAOmkd 5 10500 17282 SWEm7M.e4a1n0 2T2609223 774 DiNfEMFeOaSnA(i-OMMeKadn] CN-OENFTOSE4Omkd PFOSA0mK NEFMOeSaAnsMOCoMmKpadrisons. CONT 1000308030 100.30303030 JWs0205000 243.91166677 NEFOS1E6A2m5k0d0 a0o0l0e0r0 18.2500 PFO3S3I5O0m0A0d 128a2e5e0r0 00000 Alphas `Comparisons foreach0p0asiusingStudents t 213t144 ANbEsF(ODSIAfLdSODMKd NEFOS2MO4m9K%d 37C5O0N6T7 NEFOS7E42O4m3k8d NCEOFNOTSEdOmkd S2rsaesT 44238060130 S2340908 PFOSAOmkd Jes000 217883 Sis80 PFO1S6I.O3m0K0d0 23115758080 469518 Posiive values sChomopwapraiissoonsfmweitahnastchonatraolreusiignngfDiucnannettyt'dsifMeertehntod 262ol898 ANbsE(FDOISLASODmkd 48C.7O7N2T7 CNOENFTOSEdmkd "5542.81686903 PFOSAOmid oz aPiotsrieinvte.values show pairs ofmeansthat are significant Above i for the male and female uineapigscombined clinical chemistrystatistics. 134 TTIIOS7LIFR Appendix 8. Hepatic Palmitoyl Co-A oxidase activity Go F cont 50G104TA8865 21..0000 99660000005512 33..0000 vean 32.250 sp 0.957 N-ECFOSAMOmKd 886G0O1144380B 31..0000 vean 22.000 so aie N-ECFOSEL6O0mkd 996G0000005543 33..0000 Mxean 23.000 so 0.000 N-ECFOSEAOmkd 880G101448837 22..0000 Mean 22.000 so 0.000 rOS40mKd 88660011448512 9G60000005565 NMean sD 2.00 M3.i0s0sing 53.00 32.l606872 cour 88660011447787 11..0000 99660000004456 22..0000 vean 31.500 sp 0571 N-ELFOSMOmkd 8s6c0o114d8s12 11..0000 vean 21.000 so 0.000 N-ECFOSEI60mkd 996G0000004487 MNean so M2i.s00sing 12.000 Missing N-ECFOSEAOmKd 886G0O1L448709 11.:0000 135 TpIri0s7LIFR SPECIES SEX DOSEGRO I Mseoan PrOS4Omkd 8sc6o01144s8e3 99660000005409 Mean sp Rr cow 88RR0O44004410 99RR0000446790 Mseoan N-ELFOSAMOmkd 8BRROOAA004445 MNean so N-ECFOSEL6Omkd 9SRR0O00447721 Mean so N-ECFOSEAOmkd 88RROO44004432 Mean so PrOS4Omkd 88RR0044004476 SSRR0000447734 Mean so M uSSe-lsOmkd SPECIES SEX DcOoSnEtGRO 11RR0O0O774489 N1R00750 Mseoan TM1R00742 11RR0000774434 88SRRR000440004336233 T2 ORO. 10..000000 12..0000 33..0000 32.250 0.957 M4i.s00sing 67..0000 35.667 1828 MMiissssiinngg M0issing Missing 58..0000 25.500 2.121 MMiissssiinngg M0issing Missing MMiissssiinngg 1108..0000 214.000 5.657 MMiissssiinngg M0issing MMiissssiinngg MPiCsOsRiDn_gU_ MMiissssiinngg 049..:000000 136 TIO7LIFR TIS FOSA4OmKg N-ELFOSAdOmkd N-ECFOSEL60mkd N-ELFOSEAOmkd PFOS4OmKd NSRG0AET MseDan 118R0000774465 N1R00747 Mseoan BBRROO44003376 xMean so 99RR0O0446656 Mean so BBRR0O44003345 Mean sp 88RR0044003389 99RR0000446678 Nvean sD 94 .00 54.1530509 MMiissssiinngg M0issing MMiissssiinngg MMiissssiinngg oMissing Missing 2109..0000 %15.500 0.707 MMiissssiinngg M0issing Missing M1i1s.s00ing 2210..0000 317.333 5.508 137 TTIISLL FR 138 Tr0i7s11 FR Appendix 9. Effect of acute FC administration on catalase and acylCoA oxidase expression The following report summarizes the effects of in vivo administration of N-EFOSE or PFOS on catalase and acylCoA oxidase gene expression and enzyme activity in liver tissue from exposed rats and guinea pigs, and was presented as a poster at the 2001 SwoecrieetdyeroifveTdoxfircoomloagpyprmoexeitmiantgel(yWa1llgacoef effroazle.n2l0i0v1e)r. frToimsseuaecshaonfaltywzoedmianlethaisndsttuwdoy female rats and guinea pigs that received either 40 mg/kg/day PFOS or 160 mg/kg/day 2- (N-cthylperfluorooctanesulfonamido)-cthyl alcohol (N-EtFOSE) orally for 4 days, or an equivalent volume of 2% tween 80 (vehicle control) from March 1, 1999 to March 4, 1999. All Portions of animals were the respective killed livers on March were quick $, one frozen day after the last consecutive dose. in liquid nitrogen and shipped on dry ice to Duluth for analysis. Eonfazym1e0%Sa(mwptl/veolP)rehpoamroatgieonna--teoThfe0.e5n-z1y.0megfrfarcotzieonn lciovnesristitsesdueofinth3e060,0m00Mgmsaunpneirtnoalt-a1n0t tmheMmeHtEhPoEdSo-f1BrmadMfoErdGTusAin(gpcHom7m.2e)r.ciaPrlotbeoivninceonsceernutmraatliobnumwiansasessttiamnadtaerdd.according to L-Cod Oxidase Assay ~ The equivalent of ca., 5 pg/ml tissue homogenate was shuysdproexnydpehdeniynla6c0etamteM(PKHHP:AP)O,L-04.0un2it%s/mlTrpietroonxiXda1s0e0, 2(0pHuM7.4F)ADc,ontaanidni6n0g u1MmlaMurypl-- CoA (LCoA). water bath and The reactions were allowed to incubate terminated by adding 3 volumes of 2 at 37C for mM KCN 30 in min 100 in a mM shaking sodium carbonate (pH 10.5). The concentration of H0; generated during the reaction was estimated from the fluorescence of PHAP as measured with an excitation wavelength of 317 and nm the and emission at 405 nm. results are expressed as The nmol fpleuroorexsicdeencgeenwearsatceadl/imbriant/emdgwmiitthocchoomnmderricailalprHot0ei;n (Table 1). Protein was quantitated by the Bradford method. Catalase Assay - The activityofcatalase was estimated by a modification of the original method published by Claibome and Fridovich (J. Biol. Chem. 254, 4245-52, 1979), which is based on the direct measurement of H;O; disappearance as quantified smpMectprootpahostsoimuemtrpihcoaslplhyaatte2(4p0Hn7m..0). InTthheismperdocieudmurwea,sthweatrimsseude stoam2p7leCwaansddtihleutreedacinti5o0n initiated by adding 10.3 mM H:0x. The progress of the reaction was monitored at 240 nm for 5 min. Catalase activity was estimated from the initial linear rate ( E***=43.6 mM" a`meomu)ntaonfd eexnpzryemsesewdhaischunciattsa/lmygzepsrottheeidne(cToambploesi1t).ioOnnoef u1nuitmooflaecotfivHitOy;is pdeerfimnien.d as that 139 TorIsLLER Northern Blot Analyses - Quantitation of mRNA for both acylCoA oxidase (ACoAO) and catalase were performed by Northern blot analysisofquick frozen liver samples from treated rats and guinea pigs. Approximately 1 g of frozen liver was powderized in liquid nisiotlraotgieonn uksiitnganadmotrhtearc/opnecsetnlet.ratTiootnalquRanNtAifiweadssrpeeccotrvoeprheodtoumseitnrgictahlelPy EaRt F2E6C0nTm.RNTAhTMe RNA was electrophoresed on a 1% agarose gel, blot transferred to a cellulose membrane and hybridized to the corresponding randomly [**P] labeled oligonucleotides that were PCR amplified from primers to ca., 350 base sequence of the respective rat liver gene. mRNA band density was quantified autoradiographically using. phospho-imaging software. RESULTS: Rats -- Both male and female rats express LCoAO and catalase activities, and there is no substantial difference in enzyme activities between the sexes. The same is true for the constitutive expression of mRNA for both enzymes, with the exception that unexposed malerats do not express the message for PCOAO. Acute exposure 10 N-EL-FOSE causes a doubling of the specific activity of LCoAO in liver from both male and female rats, but catalase activity is unchanged. Associated with this is a proportionate 2-fold increase in the concentration of mRNA encoding for PCOAO, but no change in catalase mRNA, in livers from both sexes. Again, there is no remarkable sex difference in response to N-Et-FOSE exposure, at-least with this limited numberofanimals tested (n=2). Exposure of rats to PFOS elicits a similar doubling of LCAO activity for both sexes, and possibly a slight increase in catalase activity in liver from male rats. Hepatic catalase activity in female rats does not appear to be affected by PFOS exposure. Unlike N-EtFOSE, which caused a doubling of mRNA band intensity for pCoAO, acute exposure to PFOS caused a 3-6 fold increase in PCOAO mRNA expression. This enhanced expression of message was more pronounced in female compared to male rats, and in both sexes resulted in no greater LCOAO enzyme activity than was observed athalf the mRNA level for rats exposetdo N-Et-FOSE. Guinea Pigs - The response 10 guinea pigs to exposure to N-EtFOSE and PFOS was dramatically different from that observed for rats. The constitutive activity of LCoAO in unexposed guinea pigs of either sex was very low (near LOD values) whereas catalase activity was 1.5-3 times higher. However, the activityofneither enzyme was stimulated following acute exposure to N-E-FOSE or PFOS in vivo. Perhaps the most dramatic difference between species is the fact that guinea pig mRNA encoding for PCOAO was undetectable, even following exposure to N-Et-FOSE or PFOS. Part of the explanation for this observation may be that we were using hybridization probes developed against 140 TbIr0i7L1 FR the sequence for rat known at this time. liver ACOAO because the It may be that the guinea sequence of the pig sequence is guinea pig enzyme is sufficiently different not that trahte sPeCquRenpcreo.ducAtltlahcokusghcomthpilsemmeanytariintdyeefdorbeadaenquaartteifahcytb,ritdhiezaatbisoenntcoe tohfe cpornosbteituftoirvetlhye expressed consistent message and the inability to stimulate expression of PCoAO with the low and uninducible enzyme activity in guinca pigs. mRNA are CONCLUSIONS: stimulate both the These data transcriptional parnodvitrdaenssltartoinognaelviedxepnrecsestihoanto: f1a)cNy-lECto-AFOoSxiEdaasned PFOS in rats in vivo, and 2) vivo exposure there is 10 these a marked difference two fluorochemicals. in the response These results of rats and guinea pigs to in are very consistent with the suggestion and, much that like these what fluorochemical compounds are "peroxisome proliferators" in rats has been demonstrated for the classical "peroxisome proliferator" cnohtemgiicvaelns,tgheuidnaetaa pfiogrs oarrgearneswiesitagnhttstootrhinsecefrfoepcstyoffifnlduionrgoscahnemdicthaulsecxpaonsnuortesc.omWmeenwterone washseotchieartedexwpiotshurpeertooxicsiothmeer pargoleinfteractaiuosne.d Hhoewpeavteorm,egbaalsyedoronotohuerrdaitnad,icwateiosnussptehcatt tahrate the evidence will reveal the classical signs `guinea pigs, caused by these acute exposures. of peroxisome proliferation in ras, but not 141 TTHIOSTLIFR SummaryofScreen forHepaticPerorisomeProliferation Avimal sSpeociexSe Treatment BWiody wLiivePrrLoivweirncPon4t0ent OxLiaduaysleCoA acCtailviatsye uRgNAW pmCRoNAAO CmaRtaNlaAse ( ( (mee) (umolimg)A(ctaimvoiltmyin/mg) (Uniting re Contro ol Control protein) >) GROG Ra M Veh OROOMGH Ra M Veh RODE) Ra F Veh ROTO Ra F Veh 298 12 ISLS 0086 313 ISI 1456 0.067 210 76 1883 01s 201 70 153 0055 4 209 20m 1267 53 1585 2650d 753 92 102 Ll 219 2047 338 100 9s 100 1085 9RODM6S Ra M NFOES.E 22 13 ISLS 0331 ROOMS Ra M NE. FOSE 231 ILI 1349 0284 9ROMTL Rat F NE. FOSE 174 75 1658 004 9ROMT2 Ra F NE. FOSE 17S 73 2158 0188 2295 3s 20 1401 nr om3 26 2m os 78 1861 193 2 178 "2 me 1m 30 wu ORODM6T Rai M PROS ORODMGS Rai M PROS 257 119 1154 29 125 13 0423 0241 ROTI Ra F PROS ROOT Ra FE PFOS 175 83 141 0077 1S3 73 2386 0099 By 2m 39 2 2%69 208 1372041 198 99 1556 2 a 1ss1 277 157 60 1804 44 1473 9G000IS 9G0046 9GO0SI 9G00S2 Gpig M Veh Gpig M Veh Gpig F Veh Gpig F Veh 296 113 1752 0123 298 119 227 0089 274 119 1803 0188 289 142 221 007 9G00047 9G00M4S 9600053 9600s Gpig M NFOES-E Gpig M FNOES.E Gpig F NFOES.E Gpig F NFOES.E 20 87 2127- 2 226 85 2691 0013 230 96 1764 0126 236 89 283 0195 9G00049 Gpig M PROS 215 72 1532- @ 9G000SO Gpig M PFOS 9GO000SS Gpig F PROS 240 10 221 221 84 1278 0074 0031 9GO000SS G)piignsuffFicienPtFsOaSmple fo2a3n7alys7is4 1716 0016 (0d) notdetectable L347 Lond 133907 Lend 103 97 12 5506 Lesnd 09 ass 23nd 91 109 M3. ow 07 393 17m 92 07 4168 177m 105 1s aml 128m 104 as. ow - 09 4608 12700 09 6129 65nd 97 103 14 S16 12 9% uw oTr0i7s11 FR Appendix 10: Final Report for FOSA (T-7132.1). Tile: Comparative Molecular Biology of Perfluorooctanesulfonamide (FOSA, T- 7132) in Rats following four consecutive daysof dosing. Final Report February 18, 2004 Study Number: DT1S Protocol Amendment Number: 2. 3M Medical Department Study Number: T-7132.1 `Study Director: Andrew Seacat Ph.D. Analytical laboratories: 1. Kendall B. Wallace, Ph.D. DABT. Professor, DeptofBiochemistry and Molecular Biology Schoolof Medicine 10 University Drive Duluth, MN 55812-2496 2 DrXinLu Department of Pharmacology and Physiology 601 Elmwood Ave, box 711 Rochester, New York 14642 Study Protocol Tide: Comparative Molecular Biology of Peroxisome Proliferation in Rats and Guinea Pigs Study Initiated: In-Life Start Date: November 16, 1998 In-Life End Date: December 21, 1998 143 TrIi0s7L1FR Purpose: (`Tvheehipculrepcoosnetorfotl)hiosr 4s0tumdgy/Wkags/tdoaytrpeeartftlhuroereoomcatlaenersautlsftorneaamtieddew:itCh8eFi1th7eSrOn2oNcHo2m.po(uFnOdSA, cPeFrOtaSiAn,toFxOicSoAlmogiidceal, eTf-f7ec1t3s2,)s.uTchheassthuedpyatwiacspedreosixginseodmetopreovlailfueartateitonh,eomftethaibsocloismmpoaundnd and to compare these effects to other compounds dosed at the same concentration. Methods Test Materials: P1e2r0f0l6u7o-r1o0o8c,taGn.esMuolofroenam1i0d/2e8(/0F0O,STA-,71P3F2O)SwAa,sFiOnvSeAsmtiigdatee,d.C8TFh1e7vSeOh2icNlHe2coNntBro#l was propylene glycol. Dose Groups: `vTohlrueememaolfe5rmatls/kregcbeoivdeydwperiogphytl.ene glycol as the vehicle control by oral gavage at a `oTnheretehrmoaulgehrfaotsurreofctchiveedsatuddyo,seanodf4w0ermegeuFtOhaSnAi/zeKdgobnoddayywfeiivgeh.t,Avsiausopreanlsgiaovnaogef8onmgd/amyls o`TfhiFsOdSosAinign prergoipmyelnetneagclhyiceovledwaascuprmeuplaarteidv,eadnodsea ovfol1u6m0emog/f$kgmalf/tkegr wfoausrasdumcicneissstievreedd.ays ofdosing. This of this protocol dose was the sameasthedoofsPFeOS administered and is comparableto effective dose levels for hepatic under amendment peroxisome | bpruotltihfeercatuimounlaintirvaetsodofsePFofOSFOfSouAndadimnitnhiestleirteerdatuunred.eTrhtehisLDprSo0tocfoorlFisObSeAloiws ntohte kLnDoSw0n,for PFOS 251 mg/kg for PFOS in com oil, as apointofreference. MethodofSpecimen Collection: i`nTthoe~li1vergwpaiescresemaonvdefdlaasshr-afpriodzleyn daisrpeocstlsyibilnelaifqtueirdenuitthranoagseina.inTthareedlipvoerlsypwreorpeyldeinveided (Nalgenc) containers. The containers were moved to dry ice and weighed afer all liquid nitrogen had evaporated. The tissue was stored at -700 C and shipped on dry ice. Sera: Upto 10 mlofblood was collectedfromeach animal into glass serum tubes. Following clotting, thebloodwascentrifuged for 10 minutes at 110x0 g at 4 C. the sera was transferred to new tubes and be centrifuged again for 10 minutes at 1100x g at 4 C. 1" Tb0r7i01 FR `Two aliquots ofthe serum sample (~ 0.75 mi) were saved for possible metabolite analysis. Specimen Handling: A 1-2 gram aliquotofthe liver samples that were flash frozen in liquid nitrogen were: shipped in dry ice 10 the analytical laboratories listed according to the livers sample identification chart below to: Kendall B.Wallace, Ph.D. DAB.T. Professor, Deptof Biochemistry and Molecular Biology School of Medicine UniversityofMinnesota 10 University Drive Duluth, MN 55812-2496 Liver samples were analyzed by previously published methods for P450 content, Lauroyl CoA oxidase activity (Poosch and Yamazaki 1986) and protein content (Bradford 1976) in the laboratory of Ken Wallace Dept ofBiochemistry and Molecular Biology at the University of MN. Another 1-2 gram aliquotofthe liver sample was sent to: Dr. Michael Wempe Laboratory of M. W. Anders DepartmentofPharmacology and Physiology 601 Elmwood Ave, box 711 Rochester, New York 14642 Dr. Lin Xu performed the analysisofthese samples and provided abrief summaryof the analysisofliver samples from rats given a rangeoffluorocarbons. The parent and metabolitesofthefluorocarbonswere determined in liver samples by LC-MS/MS using previously published methods (Hansen ef al. 2001). LiverSample Identification Chartfor T-7132.1 [Sample#[Animal # [Species[Sex [DoseGroup | T_TTT [3 [iRo074z | Rat |WJoomol | [210743[Rat [M Joontol | [3 [TRO07&4|Rat |WJoorkal | [4 [iR00745| Rat |W_|40 mgkgday FOSA | [5 ]iR00746 | Rat |M40mokgiday FOSA | 1s TorTsL FR [& TiR00747|RatM]J40mgikgiday FOSA 1s TrIi0s7L1 FR Results and Discussion cAovnetrraolggerbooudpyowneidgahytssofofutrhaendFOfiSvAe t(rTeaabtlmeen1)t. grLoivueprwweerieghstisgnwifeirceanntolty sliogwneifritchaanntltyhe different between the treated and control groups, but the liver weight asa body weight was significantly increased in the FOSA treatment group percentage of TlihveerrsesaumlptlseosfiPn4di5c0atceodnttehnatt (FFOiSguArei1n)duacneddAtchyelexCporeAssoixoindoafsethaecstieviptryot(eFiingsuraend2)FfOorStAheis therefore a hepatic peroxisome proliferator in rats. `(TThaeblceon2c)e.ntTrhaetidoantsoafsthhoew ptahratenPtFcOoSmpwoausntdheanmdajmoertambeotlaibtoelsitien lfiovuenrds wienrtehemleiavserusreodf rats given FOSA and Approximately 0.3 FOSA N-glucuronide was identifiedas a minor percentofthe cumulative dose was present in metabolite. the liver as either the p3)a.reTnhtecloomwpopuenrdce,ntFaOgSeoA,foFrOaSsAthienmtehteabliovleirtaenPdFtOhSe,aoppnaerdenaty laofwercothnevelrasstiodnosoef(FTOabSlAe to ePtFaOl.S2h0a0s3)alwshoibcehenobnsoetrevdedintihna-tvFitOroSAmiwcraosscoomnavleratneddltiovePr FslOiSce imne-vtiatbroolbiysmlisvteurdsileisce(sXuat a low rate, but not by microsomes or cytosol . stiginsifnioctaentwocrotnhcyentthraattithoenscoonftrFoOlSaAn.imaFlusrt(h1eRr0m0or7e4,2,no1RP0F0O7S43o,raFnOdSIAR0N0-7g4l4u)cucroonntiadiened owfelrievefrosufndroimn Ftihescchoenrtr3o4l4sraamtpslmeasinwtiatihnahediginhthbeacUknigvreorusnidtoyfofFORoScAh.esAterpaVriavllaerliaunmaldyisdis not show detectable concentrationsof FOSA. These results suggest that the control liver osfamFpOlSesA,foarsthnios msettuadybowleirsemctoonPtaFmOiSnaotredFOatSsAomN-egplouicnutrotniimdeeexh-avdiovcocbuyrrterdacienqtuhaentciotnitersol samples 7 Tr0i7sLLFR Signatures: Report prepared by, Andrew M. Seacat, PhD, DABT Date Study Director Reviewed by, Daniel C. Hakes Date Sponsor representative us Tb0r7i11 FR References: `Bqruaadnfiiotride,soMf.prMo.te(i1n97w6i)l.izAinrgaptihde aprnidncsiepnlseiotifvpermoetetihno-ddyfeorbtihnediqnuga.ntAintaaltiBonioofcmhiecmr7o2g,r2a4m854. sHpaencsifeinc,,Kq.uaJn,tiCtalteimveenc,haL.racA.t, erEilzlaetfisoonno,foMr.gEa.n,iacndflJuoohrnoscohne,miHc.alOs.i(n2b0i0o1l)o.giCcoamlpmoautrnidc-es. Environmental Science and Technology 38, 766-110. Lazarow, P. B. (1981). Assayofperoxisomal beta-oxidationoffatty acids. Methods Enzymol 72, 315.9. `PoCoosAcohx,idM.aseS.,acatnivditYyaumsaiznagkai,laR.urKo.yl(-1C9o8A6-).baDseetderfmluionraotmieotnroifcpaesrsoayx.isBoimoaclhifmatBtyioapcyhly-s Acta 884, 585-93. Wallace, K. B., Lucbker, D. J. Butenhoff, J. L., and Seacat, A. M. (2001). pPeerrfolxuiosroomoectparnoelisfuelrfaotnorasteinanradts2,-b(uNt-entohtylgpueirnfelauopirgoso.ctTaonxeiscuollfoognisatmi6d0o,)-3c4t8hyA!bsatlrcaochtolIDa:re 1657. XN-uE,tLh.y,lS-eNa-c(a2t-,hAy.drMo.x,yeBtuhtyelnpheorfffl,uJo.rLo.o,catnadneAsnudlefrosn,amMi.deW.(N(-2E00(3F)O.SBEi)otbryanrastfolrivmeartion of microsomes, cytosol and slices. In Toxicological Sciences, Suppl. Vol. 72, p. 314. 149 Tor0is71E1R Tables `Table 1 BiologicalParameters _ [D(TCo1m5p,arAamteinvedmMoelnetc#ul2arBiologyofPerfucroocianestfonamide (FOISA, 1-7132) in R[ ats [ folow] ing fourconsecutidvaeysofdosing, [DT15 Body Weight (BW),LiverWeight (LW),Thyroid Weight (TW) | --T [Date 2rtoi01 [Y272t0] DoseGroup [dBaWyt ([dBaWy2 l[dBaWy3 |[dBaWy4 d[BaWy [LW (g) [WLWeIB ([0T)W Ro eo | a0 0 wm rorsx [oe oaml | 269 278] ms mm ow s37%r) s | a 3[1RO07A con 5 280) 7070 KX a|i - ? [ose] wr 266] 271 275] 281] 1075 = 071 C0] 11]13 15 12 0.06]0.14%]0.14] I TRO074[40mgkgiday TI 5 [rosa [ [a|an] om 4ri[or|s ROTH 40 iy|202 6 IFosA = Tos avis 7R074" 4[0Fomsghkgiday 25 25 T0584] 4 2%[1084 9 |"283 285 252 242 10.76) CE [8100 qo] 41] 16] 0.09 0.34% 0.09] PualveTTest | | 021 043 0.06] 002 001 044] 001 0.32 fi"To-mTceostnt(ounlpaired, on tiled, equal variance). A P-value of 0.05wasconsidered significantly diffrent 150 TorAs7LIR `Table 2 Liver Fluorocarbon Concentration Hepatic ConcetaionofFuorocaboannsd Flurocarbon Metabolites iLiovfReatsrGivsen FOSA (40 pi | Avimal iversn pe pom | roopsm)a|| roGmsa| glronide [1 [wove]woot|ota0| oof isu od om [5 [woos]wooo| wr| od oma od ow [CoTwovss|wos | oawr| 163d 1957 aml oad [5[woos]w rosa |omos| 21a was aml 0x9 eimone| bow Toso| wd wd snl oud mg/kg/day) Note: nm. =notmeasured. 151 i SipT it--------T-- TI-- O `Table 3 Percent Fluorocarbon Dose in Liver [Percent Dose in oe Liver TS [35%1 PrT ee _ S[50a05T 0 aoT hn oST ke [01] (mg) |doseTM ((ppm) |(mg) loaner (ppm) |(mg) gd f [ Bar ir omo [ o --9 do m m E=E; ] [p Teee dpde or1 T 4[E T== 1] lavg |] | TT Twos | | so1 TT Tea | [let1 t |8[1R00746|FOSA|660.2]660.2|214.3] 2.32] 0.38 1741] 19] 029] |___6[1R00747 [FOSA| 614.3] 614.3 2028] 2.16] 0.35]1639] 17] 0.8 avg|T [49343 2T 08] 032 177.90 1.91 _030 soTT| 1 T2681 020] 00s 1624] 0.18] 0.2 Tb0r7i11 FR Figures Figure 1 Cytochrome P450 content in Liver HE Total CYTP450 Content in RaFtOLSiAver Following Treatment With AEvrargoorstrreespresen ne f on) foe 5t ooo oun Control SE rosa Yt 153 TorAsLI ER Figure 2: Lauroyl CoA oxidase activity in liver LCoA-Oxidase Activity in Rat Liver Homogenate Following TreatWimtheFOnStA pa Fro Ec mme * f= in$Emw Fm Sew II =aw! 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