Document nmMRBqQQVO7DvZ62mgRJ3Za38

A226r/qo3 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 3M MEDICAL DEPARTMENT, CORPORATE TOXICOLOGY Title: Comparative Molecular Biology of Perfluorooctanesulfonate (PFOS, T-6295), N-ethyl perfluorooctanesulfonamido ethanol (N-EtFOSE, T-6316), N-Ethyl perfluorooctanesulfonamide (N-EtFOSA, 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: 3M Specialty Chemicals Division 3M Center, Building 236 Saint Paul MN 55133-3220 Study Locations): 1. 3M Strategic Alternative Toxicology Laboratory 3M Center, Building 270-SB-181 Saint Paul, MN 55133-3220 2. University of Minnesota, Duluth Dept, of Biochemistry and Molecular Biology School of Medicine 10 University Drive Duluth, MN 55812-2496 Study Director: Andrew M. Seacat Ph.D., DABT Toxicology Specialist 3M Medical Dept. Corporate Toxicology and Regulatory Services Study Toxicologist: Deanna Luebker M.S Senior Toxicologist 3M Medical Dept. In-Life Start Date In-Life End Date In-Life Start Date In-Life End Date In-Life Start Date In-Life End Date Protocol: Protocol: Amendment #1: Amendment #1: Amendment #2: Amendment #2: 11/16/1998 11/20/1998 03/01/1999 03/05/1999 02/19/2001 02/23/2001 1 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Table of Contents Sum m ary.................................................................................................................................................................... 3 M ethods...................................................................................................................................................................... 7 DT15-B Protocol Procedures...............................................................................................................................7 Amendment Number 1 Procedures.....................................................................................................................7 Amendment Number 2 Procedures.....................................................................................................................8 Analytical methods................................................................................................................................................ 9 R esu lts.......................................................................................................................................................................11 Biological Parameters.......................................................................................................................................... 11 Liver Fluorochemical Concentrations...............................................................................................................12 Clinical Chemistry....................................... 13 Palmitoyl CoA oxidase (PCoAO) activity........................................................................................................ 13 Conclusions.............................................................................................................................................................. 16 Signature P age........................................................................................................................................................ 18 Summary Tables..................................................................................................................................................... 19 Table 1. Average Cumulative D ose................................................................................................................... 19 Table 2. Summary o f Body weights and BW changes................................................................................. 20 Table 3. Summary o f Organ weights and Organ to Body Weight Ratios...................................................21 Table 4. Percent Initial Body Weight, Combined Data.................................................................................22 Table 5. Liver- and Kidney- to Body Weight Ratios, Combined Data.....................................................23 Table 6. Liver Perfluorosulfonamides and Metabolite Values from Rats and Guinea Pigs. Analyses performed at the University o f Rochester.........................................................................................................24 Table 7. Summary o f Liver Perfluorosulfonamides and Metabolite Values from Rats and Guinea Pigs. Analyses performed at the 3M Environmental Lab........................................................................................ 26 Table 8 Hepatic Palmitoyl CoA Oxidase Activity, Combined D ata............................................................27 Appendix 1. Deviations to the Protocol............................................................................................................ 28 Appendix 2 - Analytical Liver Sample Lab Identification...........................................................................29 Appendix 3. Cumulative Dose Individual and Summary data...................................................................31 Appendix 4. Body Weight Individual and Summary Data..........................................................................35 Appendix 5. Organ weights, Organ to BW ratios. Individual and Summary data...............................40 Appendix 6. Liver Fluorochemical Data. Individual and Summary data.............................................45 A. Rat and Guinea Pig Liver FC concentrations (All units are pg/g). Analyses at the University o f Rochester...............................................................................................................................................................45 B. Rat and Guinea Pig Liver FC Percent o f Dose Evaluations. Analyses at the University of Rochester............................................................................................................................................................... 50 C. Rat Liver PFOSX Analyses at the 3M Environmental Lab.............................................................54 (All units are pg/g. The 3M Environmental Lab only analyzed rat liver samples).................................. 54 D. Rat and Guinea Pig Liver FC Percent o f Dose Evaluations. Analyses at the 3M environmental Lab. 55 E. Technical Report: Liver Fluorocarbon Metabolites - University of Rochester....................59 Appendix 7: Clinical Chemistry.........................................................................................................................65 Individual and Summary Clinical Chemistry Data.........................................................................................65 A. Statistics on Clinical Chemistries for rats, male and female values combined..................................... 71 B. Statistics on Clinical Chemistries for Guinea Pigs, male and female values combined.................... 108 Appendix 8. Hepatic Palmitoyl Co-A oxidase activity................................................................................ 135 Appendix 9. Effect o f acute FC administration on catalase and acylCoA oxidase expression......139 Appendix 10: Final Report for FOSA (T-7132.1)......................................................................................... 143 Appendix 11: Final Report for FOSAA (T-7071.1).................................................................................. 162 Appendix 12, Endpoint Correlation to Liver PFOS.................................................................................. 182 A. Correlation o f effects by different treatment groups to liver PFOS in rats.......................................182 B. Correlation o f effects by different treatment groups to liver PFOS in Guinea Pigs, males and female combined............................................................................................................................................................. 185 2 S0 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Summary Adult male and female Sprague-Dawley rats and Harlan guinea pigs received oral gavage doses o f vehicle control (2% Tween 80), 40 mg/kg/day (mkd) perfluorooctanesulfonate (PFOS, T-6295), 40 mkd or 160 mkd N-Ethyl perfluorooctanesulfonamido ethanol (NEtFOSE, T-6316), or 40 mg/kg/day N-Ethyl perfluorooctanesulfonamide (N-EtFOSA, T6868) for four days and were humanely sacrificed on the fifth day. Male SpragueDawley rats received oral gavage doses o f vehicle control (propylene glycol), 160 mkd perfluorooctanesulfonamidoacetate (FOSAA, M556, T-7071), or 40 mkd perfluorooctansulfonamide (FOSA, T-7132) for four days and were humanely sacrificed on the fifth day. All animals survived. The percent initial body weights were significantly decreased in rats by 40 mkd PFOS and 160 mkd N-EtFOSE significantly reduced body weight compared to controls to 85% and 87% initial body weight, at average liver PFOS concentrations o f > 600 pg/g. 40 mkd FOSA and 160 mkd FOSAA significantly reduced body weight to 93% and 95 % o f initial body weight, respectively, at average liver PFOS concentrations of 140 pg/g and 200 pg/g, respectively. Neither 40 mkd NEtFOSE o f 40 mkd N-EtFOSA had a significant effect on body weight in rats. In the rat, liver to body weight ratios were significantly increased by 40 mkd PFOS at average liver PFOS concentrations of > 600 pg/g. Increased liver to body weight ratios were found in rats from all other dose groups, but these changes were not significant compared to the control. The guinea pig percent body weight was significantly decreased by 160 mkd N-EtFOSE and 40 mkd PFOS to 91% and 93% initial body weight, at average liver PFOS concentrations o f 419 pg/g and 171 pg/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.e. 40 mkd N-EtFOSE, N-EtFOSA, or PFOS. In rats, cholesterol (CHOL), triglycerides (TRIG), alkaline phosphatase (ALKP), and aspartate aminotransferase (AST) were significantly lowered in rats treated with 40 mkd PFOS and 160 mkd N-EtFOSE. Significant decreases in serum potassium (K+) occurred in rats treated with 40 mkd PFOS, 40 mkd N-EtFOSE and 40 mkd N-EtFOSA. In rats, 40 mkd PFOS caused a significant increase in hepatic palmitoyl CoA oxidase (PCoAO) activity, but 160 N-EtFOSE did not. Exposure o f rats to 160 mkd N-Et-FOSE caused a doubling of the specific activity o f lauryl CoA oxidase (LCoAO) and a 2-fold increase in the concentration o f mRNA encoding for PCoAO in rat liver, but catalase activity and catalase mRNA were unchanged. Exposure o f 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. Treatment with 40 mkd FOSA or 160 mkd FOSAA caused a significant increase in hepatic cytochrome P450 content and Acyl CoA oxidase activity in male rats. PFOS was apparently a more potent peroxisome proliferator than N-EtFOSE. PFOS significantly induced PCoAO activity whereas N-EtFOSE increased, but did not significantly induce PCoAO activity, even though both treatments achieved similarly high liver PFOS concentrations o f greater than 600 pg/Kg. PCoAO activity was not 3 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B determined for the 40 mkd FOSA and 160 mkd FOSAA treated male rats, but a 2-fold induction o f acyl CoA oxidase activity occurred at relatively lower average liver PFOS concentrations o f 193 pg/g and 140 p g /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-EtFOSE. 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-EtFOSE caused peroxisome proliferation in either gender o f guinea pigs. These results showed that PFOS, N-EtFOSE, FOSA and FOSAA all caused indications o f peroxisome proliferation in rats, but not in guinea pigs. These results are in concordance with effects o f classical peroxisome proliferators, in that the response is specific to certain species. 4 sz SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Introduction: The objective o f this study was to investigate and compare the molecular mechanisms o f 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, perfluorooctanesulfonate (PFOS), N-Ethyl perfluorooctanesulfonamide (N-EtFOSA) and N-Ethyl 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-EtFOSA, N-EtFOSE, 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 o f evidence indicating that guinea pigs and primates are resistant to peroxisome proliferation. However, the molecular and biochemical mechanisms that differentiate the response o f these species to peroxisome proliferators for the perfluorosulfonamides was unclear. The specific aims of this study were to: 1. To elucidate the molecular response in both rats and guinea pigs by measuring the induction of mRNA o f genes that are associated with peroxisome proliferation. 2. To measuring the hepatic activity o f peroxisomal enzyme systems and fatty acid binding proteins. 3. To perform standard toxicity tests o f 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 o f the above functions to liver and serum levels of perfluorosulfonamides and their metabolites. This study was part o f a series o f investigations designed to understand the molecular and biochemical mechanisms for the effects o f these compounds observed invivo. This study was carried-out in collaboration with other investigators. Dr. Ken Wallace, University o f Minnesota Duluth, who has been engaged in studies designed to understand the effects o f these perfluorosulfonamides on mitochondrial bioenergetics, performed molecular and biochemical analyses of the induction o f genes associated with peroxisomal proliferation and/or cell replication. Dr. Kris Hansen and Lisa A. Stevenson o f the 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 University of 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 o f 11/16/98 and an in-life end date o f 11/20/98. Amendment number 1 had an in-life start date o f 3/1/99 and an in-life end date of 3/5/99. Amendment number 2 had an in-life start date of 2/19/01 and an in life end date of 2/23/01. Amendment #3 was procedural only. 5 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 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 samples of all fluorochemicals to the investigators. Analytical documentation o f the starting material was the responsibility of the sponsor. A chemical composition specification sheet was kept on file. Compounds were 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 structures o f each o f the 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), PFOS, FC-95, Formula: C8Fi7S0 3- K+, MW = 538.1 g/mole). 3. T-6316: N-ethylperfluorooctane sulfonamido ethanol, (narrow Range N- Ethyl Perfluorooctanesulfonamido ethyl alcohol), N-EtFOSE, EtFOSE, FC-10, Formula: C8F17S 0 2N(C2H5)CH2CH20 H , MW = 571.06). 4. T-6868: N-ethyl perfluorooctane sulfonamide, (perfluorooctane sulfonyl ethylamide), PFOSA (as stated in the protocol, but not used for this compound in this report), N-EtFOSA, PFOSEA (abbreviation used by the 3M Environmental lab), FX-12, Formula C8F17S 0 2NHC2Hs, MW = 527.2). 5. T-7071: Perfluorooctanesulfonamido acetate, FOSAA, M556, (Formula C8F17S 0 2NHCH2C 0 0 -, MW - 556 g/mole) 6. T-7132: Perfluorooctanesulfonamide, FOSA, PFOSA (abbreviation used by the 3M Environmental Lab), Formula C8Fi7S0 2NH2, FOSA, MW = 499.06 g/mole). 7. Wyeth-14643 (WY, MW = 323.79 g/mol) was obtained from Chemsyn Science Laboratories, Lexena, KS. Wyeth-14643 (WY), was added to DT15A as positive control dose group for hepatic peroxisome proliferation. 6 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Methods The methods and dose groups for each part of this study are summarized below. Detailed methods are given in the protocol and amendments. Deviations to the protocol are listed in Appendix 1. DT15-B Protocol Procedures The protocol had an in-life start date o f 11/16/98 and an in-life end date o f 11/20/98. Under the protocol, eight male and eight female Sprague Dawley Rats, 10-12 weeks old weighing approximately 250-300 grams at the time o f initiation were obtained from Harlan Laboratories, Inc. Eight male and eight female Hartley Guinea Pigs 10-12 weeks old and weighing approximately 600 to 750 grams at the time of initiation 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-EtFOSA, and 40 mg/kg/day PFOS. A 20 mg/mL suspension o f each test compound was prepared in 2% Tween 80 in a glass tissue grinder. The animals received four consecutive daily doses. The day o f the first dose was designated day zero, thus the doses were administered on days 0,1,2 and 3 o f the study. Rats received the test compound suspended in 2% Tween 80 or the vehicle control by oral gavage at a volume of 2 ml/kg body weight. The guinea pigs received their daily oral dose volume of 2 ml/kg by droplet in the back of the mouth. The animals were humanely sacrificed on day 4 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 o f amendment 1 was to add groups o f both male and female rats and guinea pigs treated with vehicle control, N-EtFOSE (T-6316) or PFOS (T-6295). The histological and clinical chemistry results o f the 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 o f N-EtFOSE 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-EtFOSE in 2% Tween 80 was prepared and a volume of 2 ml/kg was administered by oral gavage to the rats and by droplet in the back o f the mouth to the guinea pigs on days 0 through day 3 o f the study. This dose was comparable to the cumulative dose o f N-EtFOSE that induced peroxisomal PCoAO activity in the rat in a 4 week feeding study with 300 ppm N-EtFOSE (Ref. 3M Medical Dept. T-6316.1), and was less than half o f the LD50. 7 SS SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B For PFOS, a suspension of 20 mg/ml of PFOS in 2% Tween 80 was prepared, and a volume o f 2 ml/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 3 o f the study. The cumulative dose o f PFOS delivered under protocol amendment #1 was -1 6 0 mg/kg, as was used in the protocol. The cumulative dose was below the LD50 for PFOS and well above the threshold for inducing peroxisome proliferation in the rat. Amendment Number 2 Procedures The purpose o f amendment 2 was to add groups male rats treated with vehicle control, perfluorooctanesulfonamidoacetate (FOSAA) or perfluorooctanesulfonamide (FOSA) at doses equivalent to the doses of N-EtFOSE and PFOS, respectively, in protocol amendment #1 in order to compare the effects and metabolite profiles o f these compounds in rats at equivalent doses. Amendment number 2 had an in-life start date o f 2/19/01 and an in-life end date of 2/23/01. 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 time o f 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 at a volume o f 5 ml/kg body weight on days zero through day 3 of the study. A larger volume o f 5 mL/Kg 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 o f 5 ml/kg was administered by oral gavage to the rats on days 0-3. This dose achieved a cumulative dose of 640 mg/kg FOSAA. For FOSA, a dose of 40 mg/kg body weight was administered via gavage to rats on day zero through day 3 o f the study. A suspension o f 8 mg/ml of FOSA in propylene glycol was prepared, and a volume o f 5 ml/kg was administered by oral gavage to the rats. This dose achieved a cumulative dose o f 160 mg/kg after four days o f dosing. The dose o f FOSA was the same as the dose o f PFOS administered under amendment #1 o f this protocol. The LD50 for FOSA is not known, but the cumulative dose o f FOSA administered under this protocol was below the LD50 for PFOS o f 251 mg/kg for PFOS in com oil, as a point o f reference. 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 aliquot o f the liver samples shipped in dry ice to the analytical laboratories listed in the protocol and amendments. The identification o f each liver sample sent to each lab is listed in Appendix 2. 8 ss SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Analytical methods Certain liver samples were sent for analysis by previously published methods for P450 content, Lauroyl CoA oxidase activity (Poosch and Yamazaki 1986) and protein content (Bradford 1976) in the laboratory o f Ken Wallace Dept o f Biochemistry and Molecular Biology at the University o f MN by the following methods: Enzyme Sample Preparation - The enzyme fraction consisted o f the 6,000 g supernatant o f a 10% (wt/vol) homogenate o f 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-CoA Oxidase Assay - The equivalent o f ca., 5 pg/ml tissue homogenate was suspended in 60 mM KH2PO4-O.O2 % Triton X I00 (pH 7.4) containing 1 mM p hydroxyphenylacetate (PHPA), 4 units/ml peroxidase, 20 pM FAD, and 60 pM 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 o f 2 mM KCN in 100 mM sodium carbonate (pH 10.5). The concentration o f H2O2 generated during the reaction was estimated from the fluorescence o f PHAP as measured with an excitation wavelength of 317 nm and emission at 405 nm. The fluorescence was calibrated with commercial H 2O2 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 o f the original method published by Claiborne and Fridovich (J. Biol. Chem. 254, 424552, 1979), which is based on the direct measurement o f H2O2 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 to 27C and the reaction initiated by adding 10.3 mM H2O2. The progress o f the reaction was monitored at 240 nm for 5 min. Catalase activity was estimated from the initial linear rate ( E240=43.6 mM '1cm '1) and expressed as units/mg protein (Table 1). One unit o f activity is defined as that amount o f enzyme which catalyzes the decomposition o f 1 pmole o f H2O2per min. Northern B lot Analyses - Quantitation o f 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 1 g o f frozen 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 to a cellulose membrane and hybridized to the corresponding randomly [32P] labeled oligonucleotides that were PCR amplified from primers to ca., 350 base sequence o f the respective rat liver gene. mRNA 9 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 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 details o f the methods used and the results o f the analyses that were completed were issued in an analytical report from the 3M Environmental Lab on May 8,2003 (3M Study No. FACT-TOX -1 0 7 ,3M Laboratory LIMS No.E01-0129). The results of 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 for a metabolite, perfluorooctanesulfonamido(ethyl)acetate (PFOSAA), that was never dosed. The abbreviations used for each 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 = EtFOSE = M556 = PFOSEA = Perfluorooctanesulfonamide (Formula C8FI7S 0 3-, PFOS) Perfluorooctanesulfonamide (Formula C8F17SO2NH2, FOSA) Perfluorooctanesulfonamido(ethyl)acetate. (This compound was also referred to as Perfluorooctanesulfonamidoacetate in the raw data tables o f FACT TOX 107). (Formula C8F17S0 2N(C2H5)CH2C0 0 - , N-EtFOSAA). narrow Range N-Ethyl Perfluorooctanesulfonamido ethyl alcohol (Formula C8F17S 0 2N(C2H5)CH2CH20 H , N-EtFOSE) Perfluorooctanesulfonamidoacetate (Formula CsF17S 0 2NHCH2C 0 0 ' FOSAA) Perfuorooctane sulfonyl ethylamide (Formula C8F17S 0 2NHC2H5 , NEtFOSA) Certain other liver samples were analyzed for parent compounds and metabolites of the fluorocarbons by LC-MS/MS by Dr. Lin Xu in the laboratory o f M. W. Anders Department of Pharmacology and Physiology, University o f 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 indicator of peroxisome proliferation using a validated method based on published methods (Lazarow 1981). The principle o f the assay is that in the presence o f palmitoyl-Co-A, the third step o f the b-oxidation spiral involves the reduction of NAD to NADH, which can be measured spectrophotometrically at 340 nm. The results were reported the 3M on October 22,2002 in a letter report from Covance and the data is included in this report. No formal report was written by Covance. 10 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Results Biological Parameters All animals survived to the end o f the study. No gross observations were noted during the in-life phase or 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 o f the guinea pigs that were dosed at different times were greatly 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-EtFOSE or FOSAA (M556). Individual and summary body weight data are shown in Appendix 4. The male rats given N-EtFOSE at 40 mkd had significantly increased liver weights (Table 3). Male rats given PFOS at 40 mkd had a significant increase in liver to body weight ratios. Male rats treated with PFOS, N-EtFOSE and N-EtFOSA at 40 mkd all had significantly lowered kidney weights and kidney to body weight ratios. Female kidney weight and kidney to 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 5. 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, and by 160 mkd NEtFOSE (Table 4). The combined guinea pig percent body weight was significantly decreased by PFOS, N-EtFOSA and N-EtFOSA at 40 mkd, and by N-EtFOSE 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 o f the study and analyzed together. Combining the relative organ weights was done to increase the N for each dose group for analysis and is justified because the organ weights have been normalized by body weight. PFOS at 40 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 any of the treatments given. Significantly increased kidney to body weight ratios were found in guinea pigs treated with NEtFOSE, N-EtFOSA, or PFOS at 40 mkd. 11 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Liver Fluorochemical Concentrations The liver fluorochemical concentrations in rats and guinea pig livers are summarized from the analyses performed at the University o f Rochester (Table 6), and at the 3M Environmental lab (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 mkd and the N-EtFOSE 160 mkd rat dose groups and did not measure fluorochemical levels in the guinea pig livers. Aliquots of the livers that were analyzed by the 3M Environmental lab were also analyzed by Drag Andres lab at the University of Rochester. The individual rat liver data showed that one lot of the 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 lab, however the values for PFOS are listed as 35% accuracy, for FOSAA 50% accuracy, and for FOSA, FOSAA, EtFOSE and N-EtFOSA the values are listed as qualitative only in the final report (FACT TOX 170). Therefore, the differences in the measurement o f PFOS and FOSA fall within the experimental error inherent with the methods used. The percent o f the dose that was in the liver was calculated for each dose group from the amount of the total liver PFOS containing species, derived from the sum o f the concentrations o f all fluorochemical species detected in the liver and designated TLPFOSX, and for the amount o f PFOS itself in the liver. The amount (mg) o f all 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 o f 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 to 23 percent in both male and female rats, and was consistent between labs. In guinea pigs dosed with PFOS, the percentage o f the total amount o f PFOS dosed that was present in the livers was far less than in the rat, between 3% and 5%. Guinea pig livers were only analyzed at the University o f Rochester so a comparison between labs for the fluorochemical content in the livers o f guinea 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 University of Rochester and at the 3M Environmental for rats treated with 160 mg/kg/day N-EtFOSE are about the same at each lab. The University of Rochester analyzed both rat and guinea pig livers from animals treated with either 40 mkd PFOS, N-EtFOSE or N-EtFOSA, or 160 mkd N-EtFOSE. The TLPFOSX in guinea pigs treated with 160 mkd N-EtFOSE was lower than in the rat, particularly in the males guinea pigs. The percent o f the dose that was present in the liver as PFOSX was approximately 2-fold the percent o f the 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-EtFOSE had greater than 70% o f the TLPFOSX as PFOS in their livers. Conversely, the percentage o f the dose that was present as PFOS in the liver o f rats treated with 160 mkd FOSAA had high levels o f FOSAA itself and FOSA, with low levels o f PFOS. 12 CO SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 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 PFOS and 160 mkd N-EtFOSE. Significant decreases in serum potassium (K7) occurred in rats treated with 40 mkd PFOS, 40 mkd N-EtFOSE and 40 mkd N-EtFOSA, with the most significant decreases occurring in the 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-EtFOSA dose groups. These changes in clinical chemistry are consistent with previous studies with PFOS and N-EtFOSE. None o f the other clinical 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-EtFOSA, similar to the rat. Alanine aminotransferase (ALT) was significantly increased by treatment o f guinea pigs with 40 mkd PFO S.. None of the other clinical chemistry parameters were significantly different from control values in rats. Palmitoyl CoA oxidase (PCoAO) activity Hepatic palmitoyl CoA oxidase (PCoAO) activity data for males and females from each 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-EtFOSE at 160 mkd for 4 days did not significantly increase hepatic PCoAO in rats. No significant change occurred in the guinea pig. Individual and summary PCoAO values for males and females separately are shown in Appendix 8. Peroxisomal enzyme activity and gene expression The effects o f four days o f oral dosing o f 160 mg/kg/day N-EtFOSE 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 o f Toxicology meeting (Wallace et al. 2001). Acute exposure o f rats to 160 mkd N-Et-FOSE caused a doubling o f the specific activity of LCoAO and a 2-fold increase in the concentration of mRNA encoding for 13 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B PcoAO 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 o f rats to 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 o f rats to 40 mkd PFOS caused a 3- to 6-fold increase in PCoAO 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). Treatment of rats with 40 mkd FOSA caused significant increases in hepatic cytochrome P450 content and Acyl CoA oxidase activity (Appendix 10). Treatment o f rats with 160 mkd FOSAA caused significant increases in hepatic cytochrome P450 content and Acyl CoA oxidase activity (Appendix 11). The induced the expression o f these o f these proteins in liver o f rats indicated that FOSA and FOSAA were peroxisome proliferators in rats. For the 40 mkd FOSA and 160 mkd FOSAA treated male rats, a 2-fold induction of acyl CoA oxidase activity occurred at average liver PFOS concentrations o f 193 pg/g and 140 pg/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 equivalent or greater fraction o f the total liver fluorochemical as did the FOSA metabolite PFOS and each of these fluorochemical species represented approximately 0.3% of the dose in the liver. In contrast, the FOSA-glucuronide present in FOSA treated rats contributed only a small fraction o f the TLPFOSX (Appendix 10). In FOSAA treated rats, the metabolite FOSA, and the parent compound, FOSAA, contributed an 2 to 3 times, respectively, the amount o f the total liver fluorochemical than did the metabolite PFOS, and represented approximately 0.14% and 0.24% of the dose in 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 o f N-Et FOSE (NEtFOSAA, and FOSAA) are weaker peroxisome proliferators than either PFOS 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 of the amino group is not readily apparent. FOSA was present in some o f the control samples submitted for analyses at a given time, but not in other groups o f control samples submitted at different times. The formation o f PFOSA from PFOS treated animals was determined in all o f the livers o f all PFOS treated animals that were analyzed at the University o f Rochester, and at the 3M environmental lab for the same samples, which 14 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B had no background PFOS o f PFOSA in the concurrent control from either lab. Clarification o f the formation o f the FOSA following treatment with PFOS should be obtained in further studies. Whatever its route of formation, FOSA was metabolized to FOSA iV-glucuronide. A-EtFOSE alcohol gives rise to a range of major and minor metabolites. FOSE alcohol could arise from the TV-deethylation o f A-EtFOSE alcohol, and A-EtFOSAA could arise by the oxidation o f the alcohol to the carboxylic acid. Glucuronidation o f the parent NEtFOSE alcohol would give the observed A-EtFOSE alcohol glucuronides. FOSAA could be formed by the A-deethylation o f A-EtFOSAA or by the oxidation o f FOSE alcohol, or both. FOSA could be formed by the A-deethylation of A-EtFOSA or by the removal o f the carboxymethyl group o f FOSAA as glyoxylate. FOSA A-glucuronide may be formed by the glucuronidation of FOSA. Loss of the carboxymethyl group from FOSAA would give FOSA or loss o f the glycine moiety would give PFOS directly. The correlation o f a few o f the most significant toxic endpoints to liver PFOS concentrations were analyzed by dose group in rats and guinea pigs (Appendix 12). The decrease in the percent initial body weight was most strongly effected in rats by PFOS at 40 mkd and N-EtFOSE at 160 mkd to 85% and 87% initial body weight, at average liver PFOS concentrations o f > 600 pg/g. The percent initial body weight was also decreased in rats by FOSA at 40 mkd and FOSAA (M556) at 160 mkd to 93% and 95 % o f initial body w eight, respectively, at average liver PFOS concentrations o f approximately 140 to 200 pg/g. A greater fraction o f the total fluorochemical in the liver o f the FOSAA treated animals was present as the parent compound or as the metabolite FOSA. Similarly, the FOSA treated animals had a high liver concentration o f the unmetabolized parent compound. Thus these perfluorosulfonamide fluorochemical species likely contributed to the body weight effect to a greater degree than the metabolite PFOS in the FOSAA and FOSA treatment groups. The rank order o f the effect o f the different treatment groups on increases liver weight to body weight in rats was PFOS at 40 mkd > FOSA at 40 mkd > N-EtFOSE at 160 mkd > FOSAA at 160 mkd occurring at average liver PFOS concentrations o f > 600 pg/g for PFOS and N-EtFOSE, apprximatelyl93 pg/g for PFOSA, and 140 pg/g for FOSAA treated animals, respectively. All dose groups had increased liver to body weight ratios, but only the liver to body weight ratios of the PFOS treated rats were significantly increased compared to control values by Dunnett's t-test Correlation o f the clinical chemistry endpoints of potassium, cholesterol and triglycerides to liver PFOS concentrations in rats are not shown due to the limited number o f data points for each determination. The decrease in the percent initial body weight was most strongly effected in guinea pigs by N-EtFOSE at 160 mkd and PFOS at 40 mkd and to 91% and 93% initial body weight, at average liver PFOS concentrations o f 419 and 171 pg/g, respectively. A greater fraction o f the total fluorochemical in the liver o f the N-EtFOSE treated guinea pigs was present as the metabolites FOSAA and N-EtFOSAA than occurred in rats. Thus, these 15 ca SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 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 inducibility o f cytochrome P450s 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-EtFOSE, at average liver PFOS concentrations o f approximately 100 pg/g in the N-EtFOSA treatment group and 66 pg/g in the N-EtFOSE treatment group, respectively. Only PFOS and FOSA were analyzed for in the liver o f the N-EtFOSA treated guinea pigs. However, the 40 mkd N-EtFOSE treatment group guinea pig livers had a large fraction o f the total fluorochemical in the liver present as the metabolites N-EtFOSAA 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 rats, were all decreased, although non of these changes were significantly different than control. The rank order o f the decreased liver to body weight effect in guinea pigs was N-EtFOSA at 40 mkd > N-EtFOSE at 40 mkd > PFOS at 40 mkd > N-EtFOSE at 160 mkd. This order is roughly the inverse order o f the decreased body weight effect in guinea pigs for each of these dose groups. Given the lack of the liver responses in guinea pigs o f either hepatomegaly or induction o f peroxisome proliferating enzymes coupled with the fact that an Intraperitoneal injection o f 100 mg/Kg PFOS caused death in guinea pigs bu not in rats (See DTI 5 A), suggests that the peroxisome proliferation in the rat is a protective mechanism. Correlation of the clinical chemistry endpoints in guinea pigs showed that the rank order o f the effect o f the different treatment groups on decreased serum potassium levels was PFOS at 40 mkd > N-EtFOSA at 40 mkd, occurring at average liver PFOS concentrations o f 148 pg/g and lOOpg/g, respectively. In contrast, treatment o f guinea pigs with 40 mkd N-EtFOSE had no effect on the serum potassium levels at average liver PFOS concentrations o f approximately 45 jug/g in female and 88 pg/g in male guinea pigs. Cholesterol and triglycerides concentrations in guinea pigs were not significantly different than control values, and showed no particular trend between dose groups 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 o f peroxisome proliferation in rats, but not guinea pigs, caused by these acute exposures. These data provide strong evidence that: 1) N-Et-FOSE and PFOS stimulate both the transcriptional and translational expression o f acylCoA oxidase in rats in vivo, and 2) there is a marked difference in the 16 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B response o f rats and guinea pigs to 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 this effect o f fluorochemical exposures. 17 5 SRPT T-6295.8, T-6316.4,1-0*68.2, T-77J. i , T-7132.1 DT15-B Signature Page Prepared by: <Z<Z 8 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Summary Tables ______________________ Table 1. Average Cumulative Dose Variable = Cumulative Dose (mg)______________________________ DOSE GRO CONT N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE4 Omkd PFOS40mkd M556-160mkd CONT FOSA40mkg N-EtFOSA4 Omkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS40mkd N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD SEX F 4 0.0 0.0 2 98.6 3.9 2 163.4 4.3 2 102.2 0.6 4 66.7 32.4 Missing Missing Missing 4 0.0 0.0 Missing Missing Missing 2 36.6 1.5 2 124.3 2.9 2 39.1 1.4 4 33.2 3.7 M 4 0.0 0.0 2 122.5 0.0 2 163.8 4.5 2 117.6 4.0 4 75.0 41.8 3 166.1 1.5 7 0.0 0.0 3 40.0 1.3 2 51.2 0.5 2 179.5 6.3 2 52.6 0.1 4 46.8 3.2 19 7 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Table 2. Summary of Body weights and BW changes SEX FM DOSE GRO BWDO BWD4(g) %BW DO IBWD0 GP CONT (g) (g) N4 444 Mean 451 474 107 469 SD 234 223 6 246 N- N2 222 EtFOSA40mkd Mean 617 587 95 770 SD 18 25 1 1 N- N2 222 EtFOSE160mkd Mean 253 233 92 255 SD 6 449 N- N2 222 EtFOSE40rakd Mean 635 629 99 736 SD 0 2 0 30 PFOS40mkd N4 444 Mean 425 398 94 480 SD 213 195 2 278 M556-160mkd N Missing Missing Missing 3 Mean Missing Missing Missing 258 CONT FOSA40mkg SD N Mean SD N Mean Missing 4 204 7 Missing Missing Missing 4 211 7 Missing Missing Missing 4 103 2 Missing Missing 8 7 277 19 3 253 NEtFOSA40mkd NEtFOSE160rakd NEtFOSE40mkd PFOS40mkd SD Missing N2 Mean 214 SD 8 N2 Mean 201 SD 5 N2 Mean SD N Mean 228 6 4 205 Missing 2 208 4 2 175f 1 2 229 3 4 l 77t SD 14 19 IcSTignificantly different from control values by Dunnett's t-test Missing 2 97 2 2 87f 3 2 101 4 4 861" 4 6 2 303 4 2 286 8 2 312 1 4 290 12 BWD4(g) %BW 44 503 110 240 6 22 726 94 41 22 233 92 10 1 22 734 31 4 429 235 3 246f 18 7 297 18 3 235+ 16 2 100 0 4 91 8 3 96t 9 7 107 2 3 93f 4 2 310 102 11 22 252f 29 2 316 1 4 2511" 15 88^ 8 2 101 0 4 87* 3 20 4* SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Table 3. Summary of Organ weights and Organ to Body Weight Ratios. SEX FM DOSE LW LW/B KW KW/BW LW LW/B KW GRP (g) W (g) ratio ratio (g) W (g) CONT N4 4 2 2 44 2 Mean 19.6 SD 7.7 0.043 0.005 4.3 0.1 0.006 0.001 19.7 0.039 4.6 10.1 0.003 0.9 N-EtFOSA N 2 2 2 2 22 2 40mkd N-EtFOSE Mean SD N 18.8 1.4 2 0.032 0.001 2 4.5 0.5 0 0.008 0.001 0 26.7 0.037 5.5 2.6 0.003 0.1 22 0 160mkd N-EtFOSE 40mkd Mean SD N 9.3 0.5 2 0.040 0.003 2 Missing Missing 2 Missing Missing 2 8.6 0.1 2 0.037 0.001 2 Missing Missing 2 PFOS Mean SD N 20.8 1.5 4 0.033 0.002 4 5.2 0.0 2 0.008 0.000 2 26.4 0.036 5.5 1.8 0.001 0.1 44 2 40mkd Mean 13.8 0.035 4.1 0.007 16.4 0.038 5.0 SD 6.9 0.003 0.1 0.000 9.5 0.004 0.1 M556- N Missing Missing Missing Missing 3 3 0 160mkd CONT Mean SD N Missing Missing 4 Missing Missing 4 Missing Missing 2 Missing 10.8 0.044 Missing Missing 0.0 0.003 Missing 2 77 2 FOSA 40mkg Mean SD N 8.1 0.9 Missing 0.038 0.003 Missing 1.9 0.1 Missing 0.009 12.1 0.041 2.9 0.000 1.4 0.003 0.0 Missing 3 3 0 N-EtFOSA 40mkd Mean SD N Missing Missing 2 Missing Missing 2 Missing Missing 2 Missing 10.7 0.046 Missing Missing 0.1 0.003 Missing 2 22 2 Mean 8.0 0.038 1.8 0.008 14.1 0.046 2.5 N-EtFOSE 160mkd SD N 0.4 2 0.002 2 0.2 0 0.001 0 0.3 0.001 0.1 22 0 N-EtFOSE Mean SD N 7.4 0.1 2 0.042 0.001 2 Missing Missing 2 Missing 12.1 0.048 Missing Missing 1.3 0.000 Missing 2 22 2 40mkd Mean 9.9 0.043 2.0 0.009 15.0f 0.047 2.7 PFOS 40mkd SD 0.1 N4 0.000 4 0.2 2 0.001 2 1.2 0.004 0.1 44 2 Mean 8.2 0.047' 1.7 0.009 13.2 0.052t 2.2 SD 0.7 0.001 0.1 0.000 1.2 0.004 0.0 ^Significantly different from control values by Dunnett's t-test O ooof-- 0o04--* KW/B W 2 0.006 0.001 2 0.008 0.000 0 Missing Missing 2 0.007 0.000 2 0.008 0.001 0 Missing Missing 2 0.009 0.000 0 Missing Missing 2 0.000 0 Missing Missing 2 0.008T 0.000 2 0.000 21 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B _____________ Table 4. Percent Initial Body Weight, Combined Data SPECIES DOSE GRO T%BW DO CONT N8 Mean 108 SD 6 N-EtFOSA4 Omkd N 4 Mean 95t SD 1 N-EtFOSEl60mkd N 4 Mean 92f SD 2 N-EtFOSE4 Omkd N 4 Mean 99f SD 1 PFOS40mkd N8 Mean 93f SD 5 CONT N 11 Mean 106 SD 3 N-EtFOSE40mkd N 4 Mean 101 SD 2 N-EtFOSA40mkd N 4 Mean 100 SD 3 M556-160mkd N 3 Mean 96* SD 9 FOSA40mkg N3 Mean 93f SD 4 N-EtFOSEl60mkd N 4 Mean 87f SD 5 PFOS40mkd N8 Mean 861- + _ ..' ' ' ~ SD 3 Significantly different from control values by Dunnett's t-test 22 TO SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Table 5. Liver- and Kidney- to Body Weight Ratios, Combined Data. SPECIES DOSE_GRO LW/BW KW/BW ratio ratio GP CONT N8 4 Mean 0.041 0.006 SD 0.004 0.001 N-EtFOSA40mkd N 4 4 Mean 0.034 0.008t SD 0.003 0.000 N-EtFOSE160mkd N 4 0 Mean 0.038 Missing SD 0.002 Missing N-EtFOSE40mkd N 4 4 Mean 0.034 0.008t SD 0.002 0.000 PFOS40mkd N8 4 Mean 0.036 0.008f SD 0.004 0.000 R M556-160mkd N 3 0 Mean 0.044 Missing SD 0.003 Missing CONT N 11 4 Mean 0.040 0.009 SD 0.003 0.000 FOSA40mkg N3 0 Mean 0.046 Missing SD 0.003 Missing N-EtFOSA40mkd N 4 4 Mean 0.042 0.008 SD 0.004 0.001 N-EtFOSE160mkd N 4 0 Mean 0.045 Missing SD 0.003 Missing N-EtFOSE4 Omkd N 4 4 Mean 0.045 0.008 SD 0.003 0.001 PFOS40mkd N8 4 Mean 0.050f 0.009 SD 0.004 0.000 ^Significantly different from control values by Dunnett's t-test 23 7/ SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Table 6. Liver Perfluorosulfonamides and Metabolite Values from Rats and Guinea Pigs. Analyses performed at the University of Rochester. (All units are ug/g)_________________________________________________________________________________________ SPEC SEX DOSE PFOS FOSA FOSAA ETFOSAA FOSE EtFOSE NETFOSE FOSA IES _GRP __ROC __ROC __R _ROC _ALC _ROC _gluc_ _gluc ROC CONT N 0 000 00 0 0 Mean NA NA NA NA NA NA NA NA SD NA NA NA NA NA NA NA NA N- N 2 200 00 0 2 EtFOSA 40mkd Mean 104.6 0.6 NA NA NA NA NA 0.2 SD 30.1 0.8 NA NA NA NA NA 0.0 N- N 2 222 22 2 2 EtFOSE 160mkd NEtFOSE 40mkd PFOS 40mkd CONT NEtFOSA 40mkd NEtFOSE 160mkd NEtFOSE 40mkd PFOS 40mkd CONT NEtFOSE Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N 350.2 339.9 2 44.7 45.3 3 140.8 110.5 1 0.1 NA 2 96.3 23.0 2 488.6 59.8 2 88.4 84.0 4 194.5 131.8 1 0.0 NA 2 16.2 7.0 2 7.1 3.9 3 24.3 37.5 0 NA NA 2 0.5 0.2 2 8.2 4.6 2 4.9 0.3 4 41.7 33.1 2 4.4 0.4 2 217.9 388.2 103.0 98.1 22 30.4 6.2 0 NA NA 0 NA NA 0 105.0 27.5 0 NA NA 0 NA NA 0 NA NA NA NA 22 129.4 261.6 56.4 113.8 22 28.2 8.6 0 NA NA 0 NA NA 2 54.0 1.6 0 NA NA 0 NA NA 2 28.7 1.9 0.8 0.7 22 3.2 0.9 1.4 0.1 00 NA NA NA NA 00 NA NA NA NA 00 NA NA NA NA 22 16.0 2.3 8.3 0.2 22 3.5 1.3 1.6 0.5 00 NA NA NA NA 00 NA NA NA NA 22 6.1 3.5 2 1.2 0.2 0 NA NA 0 NA NA 0 NA NA 2 2.0 1.1 2 0.4 0.1 0 NA NA 0 NA NA 2 0.3 0.1 2 0.3 0.0 0 NA NA 0 NA NA 2 0.1 0.0 2 0.5 0.0 2 0.4 0.0 0 NA NA 0 NA NA 2 160mkd PFOS Mean SD N 891.9 37.6 2 18.1 1.1 2 67.5 5.5 0 201.9 33.4 0 47.2 6.4 24.6 1.5 00 0.2 0.1 0 0.2 0.0 0 24 ? SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 40mkd M556 Mean 866.4 SD 143.0 N3 18.3 4.2 3 NA NA 3 NA NA 0 160mkd CONT FOSA 40mkg NEtFOSE 160mkd PFOS 40mkd Mean SD N Mean SD N Mean SD N Mean SD N Mean SD 140.3 85.4 2 0.0 0.0 3 193.4 26.8 2 1124.3 166.1 2 969.9 88.2 316.8 114.1 5 109.2 113.6 3 177.9 16.2 2 18.5 9.5 2 17.8 19.9 555.2 113.8 0 NA NA 0 NA NA 2 94.3 23.7 0 NA NA NA NA 0 NA NA 0 NA NA 2 294.6 115.8 0 NA NA NA NA NA NA 00 NA NA NA NA 00 NA NA NA NA 00 NA NA NA NA 22 12.1 3.8 2.5 1.2 00 NA NA NA NA NA NA 0 NA NA 0 NA NA 0 NA NA 2 0.3 0.0 0 NA NA NA NA 0 NA NA 0 NA NA 3 0.4 0.1 2 0.1 0.0 0 NA NA 25 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Table 7. Summary of Liver Perfluorosulfonamides and Metabolite Values from Rats and Guinea Pigs. Analyses performed at the 3M Environmental Lab. SPEC SEX DOSE PFOS FOSA N- N- FOSAA N- IES GRP 3M 3M EtFOSAA EtFOSE 3M EtFOSA 3M -3M 3M R F CONT N 1 32 0 10 Mean 0.151 0.0 0.2 NA 0.1 NA SD NA 0.0 0.0 NA NA NA N- N 2 22 2 21 EtFOSE 160mkd Mean 604.000 109.0 244.5 302.5 118.5 0.9 SD 5.657 11.3 24.7 67.2 13.4 NA PFOS N 2 22 0 20 40mkd Mean 803.500 0.1 0.1 NA 0.1 NA SD 67.175 0.0 0.0 NA 0.0 NA M CONT N 2 22 0 10 Mean 0.298 0.0 0.2 NA 0.1 NA SD 0.136 0.0 0.1 NA NA NA N- N 2 22 2 22 EtFOSE 160mkd Mean 908.000 89.0 317.5 147.0 169.5 0.4 SD 90.510 9.9 17.7 42.4 10.6 0.1 PFOS N 2 22 0 20 40mkd Mean 800.000 0.1 0.3 NA 0.1 NA SD 16.971 0.0 0.0 NA 0.0 NA 26 7V SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Table 8 Hepatic Palmitoyl CoA Oxidase Activity, Combined Data SPECIES DOSE GRO CONT N-EtFOSA4 Omkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS40mkd CONT N-EtFOSE160mkd PFOS40mkd N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD PCOAO U 8 2 0.8 4 2 1.0 3 3 0.6 4 2 0.6 7 3 1.6 7 6 3.2 4 13 7.6 5 16^ 5.1 t Significantly different from control values by Dunnett's t-test g 27 75 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 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 number 2 lists the abbreviations for perfluorooctanesulfonamide (T-7132) as FOSA, PFOSA and FOSAmide. The abbreviations used in this report for N-Ethyl perfluorooctanesulfonamide was N-EtFOSA and for perfluorooctanesulfonamide was FOSA. The protocol stated that the animals would be dosed on days `zero through 4 o f the study". That was a typographical error, as evidenced by the fact that even the example calculation in the protocol was for a 4-day dosing period, not a 5-day dosing period. The actual dosing period was for days zero through three of the study, and the animals were sacrificed n day four o f the study. Animals were not weighed in most cases on day one o f the study, and in some cases on day three o f the study. In those instances, the previous days' body weights were used for determination o f dosing volume. Protocol amendment #2 stated the Dr. M. Wempe at the University o f Rochester would perform the metabolite analysis of the liver samples from animals treated with T-7071.1, T-7132.1. Due to personnel changes, Dr. Xin Lu at the University o f 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 o f liver, kidney and testis were not performed. Serum perfluorosulfonamides and metabolite values were not obtained. Induction o f mRNA for the following genes associated with peroxisomal proliferation and/or 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). 28 76 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Appendix 2 - Analytical Liver Sample Lab Identification. # XStudy/ Tube Animal se Dose Necropsy Specimen Amt Amt sent Amt Amt Record number group Date Type sent to to sent to sent to (g)Covance Rocheste 3M r 1-19-01 Env Univ MN lab (1- Duluth d1o1s/1e6d/98 4PNFF10O1XF-E/O-Sm11t9SE2k/,,.d98. tvc3mNF3PD1woe/O6/F-o4k1henE0OsdS//eti99tcermESno99lde;;lk-84d0022222211524309 18R04032MCont 11/20/98liver 11111111117840362452356897098982888885840788788863888988811RGGGRRGGGGGGGGGGGG000000000000000000044111111111111111040444444444444444463988798788788888133MMMMFMMMMMMFFFFFFF EECECCCCFFFECCPPPNFNNNNNNNOOOttOtotFFFoooooo---FFFF-----eeennnnnnnOOOeSSSSOOOOtttttttttttEEEESSSSSSSAAAA11111111111111111111111111111111113/////////////////22222222222222222/500000000000000000//////////////////999999999999999999988888888888888888lllllllllllllllllliiiiiiiiiiiiiiiiiivvvvvvvvvvvvvvvvvveeeeeeeeeeeeeeeeeerrrrrrrrrrrrrrrrrr 999999RRRRRR000000000000444444766667156490MFMFMF NFCCNNFCFOOOoo-o--eeennnSSSttttttEEE 3333////5555////99999999lllliiiivvvveeeerrrr 3/5/99liver 3/5/99liver 0000....443758201989~~~~1i11g9gg 19-01) (3/8/99) ~g NA1 NA~ 1 g NA~1 9 ~g NA1 0.49~1g ~1g NA 0.544~1g ~1g NA 0.468~19 ~lg NA 0.901~1g ~lg NA 0.638~1g ~1g NA 0.387~1g ~1g NA 0.374~1g ~1g NA 0.817~1g ~1g NA 0.484~1g NA- 1 g 0.786~1g ~1g NA 0.57~1g ~1g NA 0.538~1g ~19 NA 0.483~1g ~1g NA 0.85~1g ~ig NA 1.025~1g ~1g ~19 000...0887.18746867~~~~1111gggg ~~~~1111{g?gg ~~~~ii11gg9g 0.812~1g ~1g ~1g 0.593~1g ~1g ~1g 29 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 2%; 26 2223317890 32 33 34 35 36 37 38 39 40 41 42 V42cFF1Do/0Oe6O1on0h9SmSst/rAme0Aokd1Adlk,d, 1999959499596996992979839990911RRGRGGGGGGGGRRGGRGQ00000000000000000000000000000000000447440000000000040774675456545544545322847MFFMMMMFMFFMFMMFFF CPPPNPPCCFFNFNFPFPPCCNNOOOOOoFFFFFFFFoooo-----eeeeenOOOOOnnOOOnnSSSSSttttttttttSSSEEEEESSSSS. 3/5/99liver 33333/////55555/////9999999999llllliiiiivvvvveeeeerrrrr 3/5/99liver 3/5/99liver 3/5/99liver 3/5/99liver 3/5/99liver 3/5/99liver 3/5/99liver 3/5/99liver 3/5/99liver 3/5/99liver 3/5/99liver 2/23/01liver 4562789311111111RRRRRRRRO000000000000000777777774445444465704893MMMMMMMMCCFFFFFFOOOOOOoonnSSSSSSttAAAAAAAAA 22222222////////2222222233333333////////0000000011111111lllllllliiiiiiiivvvvvvvveeeeeeeerrrrrrrr 0.834~1g ~1g ~1g 000.00..877..8407912363--~~~11111gg9gg ~~~~~11111gg9gg ~~~~~11i11ggggg 0.78~1g ~1g ~1g 0.422~19 ~1g ~1g 0.727~1g ~1g ~1g 0.527~19 ~1g ~1g 0.834~1g ~1g ~1g 0.812~1g ~1g -1g 0.7~1g ~1g -1 g 0.808~1g ~1 g ~1g 0.815~1g ~1g ~1g 0.716~1g ~1g -1g 0.517~1g ~1g ~1g NA NNNNNNNNAAAAAAAA ~1g ~~~~~~~~11i11111g.g99ggag NA NNNNNNNNAAAAAAAA ~~~~~~~~~1111J1111ggggggggg 30 77 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Appendix 3. Cumulative Dose Individuai and Summary data. SPECIES SEX DOSE GR GP F CONT ID 8G01485 8G01486 9G00051 9G00052 N Mean SD Cumulative Dose (mg) 0 0 0 0 4 0.0 0.0 N-EtFOSA4 Omkd 8G01488 8G01490 N Mean SD 96 101 2 98.6 3.9 N-EtFOSEl60mkd 9G00053 9G00054 N Mean SD 166 160 2 163.4 4.3 N-EtFOSE40mkd 8G01487 8G01489 N Mean SD 103 102 2 102.2 0.6 PFOS40mkd 8G01491 8G01492 9G00055 9G00056 N Mean SD 95 94 38 39 4 66.7 32.4 M CONT 8G01477 8G01478 9G00045 9G00046 N Mean SD 0 0 0 0 4 0.0 0.0 N-EtFOSA40mkd 8G01481 8G01482 N Mean SD 123 123 2 122.5 0.0 N-EtFOSE160mkd 9G00047 9G00048 N Mean SD 167 161 2 163.8 4.5 N-EtFOSE40mkd 8G01479 115 8G01480 120 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B SPECIES DOSE_GRO ID N Mean SD Cumulative _Dose _(mg) 2 117.6 4.0 PFOS40mkd 8G01483 8G01484 9G00049 9G00050 N Mean SD 115 107 39 39 4 75.0 41.8 R CONT 8R04040 0 8R04041 0 9R00469 0 9R00470 0 N4 Mean 0.0 SD 0.0 N-EtFOSA4 Omkd N-EtFOSEl60mkd 8R04044 8R04045 N Mean SD 9R00471 9R00472 N Mean SD 38 36 2 36.6 1.5 126 122 2 124.3 2.9 N-EtFOSE40mkd 8R04042 8R04043 N Mean SD 40 38 2 39.1 1.4 PFOS40mkd 8R04046 8R04047 9R00473 9R00474 N Mean SD 38 35 31 29 4 33.2 3.7 SPECIES M556-160mkd DOSE GRO CONT 1R00748 1R00749 1R00750 N Mean SD ID 1R00742 1R00743 1R00744 8R04032 8R04033 9R00463 165 166 168 3 166.1 1.5 Cumulative _Dose_ (mg) 0 0 0 0 0 0 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B FOSA40mkg N-EtF0SA4 Omkd N-EtFOSEl60mkd 9R00464 N Mean SD 1R00745 1R00746 1R00747 N Mean SD 8R04036 8R04037 N Mean SD 9R00465 9R00466 N Mean SD 0 7 0.0 0.0 40 39 41 3 40.0 1.3 51 51 2 51.2 0.5 184 175 2 179.5 6.3 N-EtFOSE40mkd PFOS40mkd 8R04034 8R04035 N Mean SD 8R04038 8R04039 9R00467 9R00468 N Mean SD 53 53 2 52.6 0.1 49 50 45 43 4 46.8 3.2 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Appendix 4. Body Weight Individual and Summary Data. SPECIES SEX GP F DOSE GRO CONT ID 8G01485 8G01486 9G00051 9G00052 N Mean SD NEtFOSA40mkd 8G01488 8G01490 N Mean SD NEtFOSEl60mkd 9G00053 9G00054 N Mean SD NEtFOSE40mkd 8G01487 8G01489 N Mean SD PFOS40mkd 8G01491 8G01492 9G00055 9G00056 N Mean IBWDO G 621 684 243 257 4 451.3 233.9 604 630 2 617.0 18.4 257 249 2 253.0 5.7 635 635 2 635.0 0.0 608 610 234 246 4 424.5 IBWD1 G Missing Missing Missing Missing 0 Missing Missing Missing Missing 0 Missing Missing Missing Missing 0 Missing Missing Missing Missing 0 Missing Missing Missing Missing Missing Missing 0 Missing BWD2 G 632 692 260 272 4 464.0 230.0 604 651 2 627.5 33.2 263 252 2 257.5 7.8 648 641 2 644.5 4.9 591 575 242 244 4 413.0 BWD3 G 642 689 Missing Missing 2 665.5 33.2 583 623 2 603.0 28.3 Missing Missing 0 Missing Missing 648 633 2 640.5 10.6 579 556 Missing Missing 2 567.5 BWD4 G 636 695 274 289 4 473.5 223.1 569 604 2 586.5 24.7 230 236 2 233.0 4.2 630 627 2 628.5 2.1 574 558 221 237 4 397.5 BW GAIN 15 11 31 32 4 22.3 10.8 -35 -26 2 -30.5 6.4 -27 -13 2 -20.0 9.9 -5 -8 2 -6.5 2.1 -34 -52 -13 -9 4 -27.0 %BW DO 102.42 101.61 112.76 112.45 4 107.3 6.1 94.21 95.87 2 95.0 1.2 89.49 94.78 2 92.1 3.7 99.21 98.74 2 99.0 0.3 94.41 91.48 94.44 96.34 4 94.2 35 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B SPECIES SD CONT 8G01477 8G01478 9G00045 9G00046 N Mean SD NEtFOSA40mkd 8G01481 8G01482 N Mean SD NEtFOSE160mkd 9G00047 9G00048 N Mean SD NEtFOSE40mkd DOSE_GRO 8G01479 8G01480 ID N Mean SD PFOS40mkd 8G01483 8G01484 9G00049 9G00050 N Mean SD 213.1 723 637 257 260 4 469.3 245.9 769 771 2 770.0 1.4 261 248 2 254.5 9.2 714 757 IBWDO C 2 735.5 30.4 737 702 244 235 4 479.5 277.5 Missing Missing Missing Missing Missing 0 Missing Missing Missing Missing 0 Missing Missing Missing Missing 0 Missing Missing Missing Missing IBWD1 G 0 Missing Missing Missing Missing Missing Missing 0 Missing Missing 196.4 751 650 281 278 4 490.0 246.5 773 773 2 773.0 0.0 261 254 2 257.5 4.9 720 743 BWD2 G 2 731.5 16.3 727 653 246 248 4 468.5 257.5 16.3 194.8 755 653 Missing Missing 2 704.0 72.1 754 662 296 298 4 502.5 240.2 753 729 748 2 750.5 3.5 723 2 726.0 4.2 Missing 240 Missing 0 Missing Missing 226 2 233.0 9.9 725 712 754 BWD3 G 2 739.5 20.5 756 BWD4 G 2 734.0 31.1 675 621 Missing Missing 2 648.0 38.2 668 594 215 240 4 429.3 235.1 19.9 31 25 39 38 4 33.3 6.551 -40 -48 2 -44.0 5.7 -21 -22 2 -21.5 0.7 -2 -1 BW GAIN 2 -1.5 0.7 -69 -108 -29 5 4 -50.3 49.0 2.0 104.29 103.92 115.18 114.62 4 109.5 6.2 94.80 93.77 2 94.3 0.7 91.95 91.13 2 91.5 0.6 99.72 99.87 %BW DO 2 99.8 0.1 90.64 84.62 88.11 102.13 4 91.4 7.6 36 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B R F CONT 8R04040 8R04041 9R00469 9R00470 N Mean SD 205 213 201 197 4 204.0 6.8 NEtFOSA40mkd 8R04044 8R04045 N Mean SD 220 208 2 214.0 8.5 NEtFOSE160mkd 9R00471 9R00472 N Mean SD 204 197 2 200.5 4.9 NEtFOSE40mkd 8R04042 8R04043 N Mean SD 232 223 2 227.5 6.4 PFOS40mkd 8R04046 8R04047 9R00473 9R00474 N Mean SD 221 208 202 188 4 204.8 13.7 M M556-160mkd 1R00748 254 1R00749 254 1R00750 267 Missing Missing Missing Missing 0 Missing Missing Missing Missing 0 Missing Missing Missing Missing 0 Missing Missing Missing Missing 0 Missing Missing Missing Missing Missing Missing 0 Missing Missing 263 262 270 274 279 202 192 4 236.8 46.1 283 267 2 275.0 11.3 191 185 2 188.0 4.2 303 285 2 294.0 12.7 284 265 191 176 4 229.0 53.5 264 265 267 217 209 Missing Missing 2 213.0 5.7 218 215 210 201 4 211.0 7.4 217 210 205 2 211.0 8.5 205 2 207.5 3.5 Missing 174 Missing 0 Missing Missing 175 2 174.5 0.7 235 227 223 2 229.0 8.5 231 2 229.0 2.8 212 189 Missing Missing 2 200.5 16.3 200 178 175 153 4 176.5 19.2 258 250 263 262 247 227 13 2 9 4 4 7.0 5.0 -10 -3 2 -6.5 4.950 -30 -22 2 -26.0 5.7 -5 8 2 1.5 9.2 -21 -30 -27 -35 4 -28.3 5.9 -4 8 -40 106.34 100.94 104.48 102.03 4 103.4 2.4 95.45 98.56 2 97.0 2.2 85.29 88.83 2 87.1 2.5 97.84 103.59 2 100.7 4.1 90.50 85.58 86.63 81.38 4 86.0 3.7 98.43 103.15 85.02 37 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B SPECIES SEX DOSE_GRO CONT N Mean SD ID 1R00742 1R00743 1R00744 8R04032 8R04033 9R00463 9R00464 N Mean SD 3 258.3 7.5 IBWDO C 250 269 256 297 295 279 294 7 277.1 19.4 FOSA40mkg 1R00745 1R00746 1R00747 N Mean SD 250 249 259 3 252.7 5.5 NEtFOSA40mkd 8R04036 8R04037 N Mean SD 305 300 2 302.5 3.5 NEtFOSE160mkd 9R00465 9R00466 N Mean SD 292 280 2 286.0 8.5 NEtFOSE40mkd 8R04034 8R04035 N 312 311 2 3 265.0 4.4 IBWD1 G 259 278 260 Missing Missing Missing Missing 3 265.7 10.7 3 265.3 1.5 BWD2 G 260 285 267 374 368 286 303 7 306.1 46.5 254 245 265 3 254.7 10.0 250 243 263 3 252.0 10.1 Missing 371 Missing 0 Missing Missing 367 2 369.0 2.8 Missing 283 Missing 0 Missing Missing 267 2 275.0 11.3 Missing 375 Missing 378 02 3 256.0 8.2 BWD3 G 264 292 268 320 305 Missing Missing 5 289.8 23.9 3 246.3 17.8 BWD4 G 272 295 276 321 307 298 313 7 297.4 18.3 239 233 255 3 242.3 11.4 227 225 254 3 235.3 16.2 306 310 304 2 305.0 1.4 309 2 309.5 0.7 Missing 272 Missing 0 Missing Missing 231 2 251.5 29.0 314 316 318 315 22 3 -12.0 25.0 BW GAIN 22 26 20 24 12 19 19 7 20.3 5.0 -23 -24 -5 3 -17.3 10.7 5 9 2 7.0 2.8 -20 -49 2 -34.5 20.5 4 4 2 3 95.5 9.4 %BW DO 108.80 109.67 107.81 108.08 104.07 106.81 106.46 7 107.4 1.8 90.80 90.36 98.07 3 93.1 4.3 101.64 103.00 2 102.3 1.0 93.15 82.50 2 87.8 7.5 101.28 101.29 2 38 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B PFOS40mkd Mean SD 8R04038 8R04039 9R00467 9R00468 N Mean SD 311.5 0.7 295 303 286 275 4 289.8 12.0 Missing Missing Missing Missing Missing Missing 0 Missing Missing 376.5 2.1 353 363 282 262 4 315.0 50.5 316.0 2.8 275 279 Missing Missing 2 277.0 2.8 315.5 0.7 257 261 257 229 4 251.0 14.8 4.0 0.000 -38 -42 -29 -46 4 -38.8 7.3 101.3 0.0 87.12 86.14 89.86 83.27 4 86.6 2.7 39 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Appendix 5. Organ weights, Organ to BW ratios. Individual and Summary data. SPECIES SEX DOSE GRO ID LW G LW BW RA KW KW BW RA GP F CONT 8G01485 24.50 0.04 4.30 0.01 8G01486 27.70 0.04 4.20 0.01 9G00051 11.90 0.04 Missing Missing 9G00052 14.20 0.05 Missing Missing N 44 2 2 Mean 19.575 0.043 4.250 0.006 SD 7.704 0.005 0.071 0.001 NEtFOSA40 mkd 8G01488 8G01490 N Mean SD 17.80 19.80 2 18.800 1.414 0.03 0.03 2 0.032 0.001 4.10 4.80 2 4.450 0.495 0.01 0.01 2 0.008 0.001 NEtF0SE16 Omkd 9G00053 9G00054 N Mean SD 9.60 8.90 2 9.250 0.495 0.04 0.04 2 0.040 0.003 Missing Missing Missing 0 Missing Missing Missing 0 Missing Missing NEtFOSE40 mkd 8G01487 8G01489 N Mean SD 19.70 21.80 2 20.750 1.485 0.03 0.03 2 0.033 0.002 5.20 5.20 2 5.200 0.000 0.01 0.01 2 0.008 0.000 PFOS40mk d 8G01491 8G01492 9G00055 18.50 20.90 8.40 0.03 0.04 0.04 4.20 4.00 Missing 0.01 0.01 Missing SRPT T-6295,8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B *3 SPECIES 9G00056 N Mean SD CONT 8G01477 8G01478 9G00045 9G00046 N Mean SD NEtFOSA40 mkd 8G01481 8G01482 N Mean SD NEtFOSE16 Omkd 9G00047 9G00048 N Mean SD NEtFOSE40 mkd DOSE_GRO 8G01479 8G01480 ID N Mean SD PFOS40mk 8G01483 d 7.40 4 13.800 6.895 32.30 23.30 11.30 11.90 4 19.700 10.052 28.50 24.80 2 26.650 2.616 8.70 8.50 2 8.600 0.141 25.10 27.60 LW G 2 26.350 1.768 27.60 0.03 4 0.035 0.003 0.04 0.04 0.04 0.04 4 0.039 0.003 0.04 0.03 2 0.037 0.003 0.04 0.04 2 0.037 0.001 0.04 0.04 LW BW RA 2 0.036 0.001 0.04 Missing 2 4.100 0.141 Missing 2 0.007 0.000 5.20 3.90 Missing Missing 2 4.550 0.919 0.01 0.01 Missing Missing 2 0.006 0.001 5.40 0.01 5.60 2 5.500 0.141 Missing 0.01 2 0.008 0.000 Missing Missing 0 Missing Missing 5.40 Missing 0 Missing Missing 0.01 5.60 KW 2 5.500 0.141 5.00 0.01 KW BW RA 2 0.007 0.000 0.01 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 8G01484 9G00049 9G00050 N Mean SD 20.60 7.20 10.00 4 16.350 9.463 0.03 0.03 0.04 4 0.038 0.004 R F CONT 8R04040 9.00 0.04 8R04041 8.80 0.04 9R00469 7.60 0.04 9R00470 7.10 0.04 N 44 Mean 8.125 0.038 SD 0.922 0.003 NEtFOSA40 mkd 8R04044 8R04045 N Mean SD 7.70 8.20 2 7.950 0.354 0.04 0.04 2 0.038 0.002 NEtFOSEie Omkd 9R00471 9R00472 N Mean SD 7.50 7.30 2 7.400 0.141 0.04 0.04 2 0.042 0.001 NEtFOSE40 mkd 8R04042 8R04043 N Mean SD 9.80 9.90 2 9.850 0.071 0.04 0.04 2 0.043 0.000 PFOS40mk 8R04046 8.90 0.04 4.90 Missing Missing 2 4.950 0.071 1.90 1.80 Missing Missing 2 1.850 0.071 1.90 0.01 Missing Missing 2 0.008 0.001 0.01 0.01 Missing Missing 2 0.009 0.000 0.01 1.60 2 1.750 0.212 Missing 0.01 2 0.008 0.001 Missing Missing 0 Missing Missing Missing 0 Missing Missing 2.10 0.01 1.80 2 1.950 0.212 1.70 0.01 2 0.009 0.001 0.01 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B SPECIES d 8R04047 9R00473 9R00474 N Mean SD M556160mkd DOSE GRO CONT 1R00748 1R00749 1R00750 N Mean SD ID 1R00742 1R00743 1R00744 8R04032 8R04033 9R00463 9R00464 N Mean SD FOSA40mk g 1R00745 1R00746 1R00747 N Mean SD NEtFOSA40 mkd 8R04036 8R04037 N 8.40 8.30 7.30 4 8.225 0.670 10.74 10.77 10.79 3 10.767 0.025 LW G 10.73 10.82 10.70 14.20 13.30 12.00 13.10 7 12.121 1.434 10.77 10.81 10.64 3 10.740 0.089 14.30 13.90 2 0.05 0.05 0.05 4 0.047 0.001 0.04 0.04 0.05 3 0.044 0.003 LW BW RA 0.04 0.04 0.04 0.04 0.04 0.04 0.04 7 0.041 0.003 0.05 0.05 0.04 3 0.046 0.003 0.05 0.04 2 1.60 Missing Missing 2 1.650 0.071 0.01 Missing Missing 2 0.009 0.000 Missing Missing Missing Missing 0 Missing Missing KW Missing Missing Missing 2.90 2.90 Missing Missing 2 2.900 0.000 Missing Missing 0 Missing Missing KW_BW_RA Missing Missing Missing 0.01 0.01 Missing Missing 2 0.009 0.000 Missing Missing Missing Missing 0 Missing Missing Missing Missing 0 Missing Missing 2.40 0.01 2.50 2 0.01 2 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Mean SD NEtFOSEie Omkd 9R00465 9R00466 N Mean SD NEtFOSE40 mkd 8R04034 8R04035 N Mean SD PFOS40mk d 8R04038 8R04039 9R00467 9R00468 N Mean SD 14.100 0.283 13.00 11.10 2 12.050 1.344 15.80 14.10 2 14.950 1.202 13.50 14.70 11.90 12.50 4 13.150 1.226 0.046 0.001 0.05 0.05 2 0.048 0.000 0.05 0.04 2 0.047 0.004 0.05 0.06 0.05 0.05 4 0.052 0.004 2.450 0.071 Missing 0.008 0.000 Missing Missing 0 Missing Missing 2.70 Missing 0 Missing Missing 0.01 2.60 2 2.650 0.071 2.20 2.20 Missing Missing 2 2.200 0.000 0.01 2 0.008 0.000 0.01 0.01 Missing Missing 2 0.008 0.000 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Appendix 6. Liver Fluorochemical Data. Individual and Summary data. A. Rat and Guinea Pig Liver FC concentrations (All units are pg/g). Analyses at the University of Rochester. SPEC SEX DOSE IES GRP GP F CONT ID 8G01485 8G01486 9G00051 9G00052 N Mean SD PFOS ROC NA NA NA NA 0 NA NA N-EtFOSA 40mkd 8G01488 8G01490 N Mean SD 125.80 83.30 2 104.55 30.05 N-EtFOSE 160mkd 9G00053 9G00054 N Mean SD 590.50 109.80 2 350.15 339.91 N-EtFOSE 40mkd 8G01487 8G01489 N Mean SD 76.70 12.70 2 44.70 45.25 PFOS 40mkd 8G01491 127.50 8G01492 NA FOSA ROC NA NA NA NA 0 NA NA 1.10 0.00 2 0.55 0.78 11.20 21.10 2 16.15 7.00 4.30 9.80 2 7.05 3.89 67.60 NA FOSAA R NA NA NA NA 0 NA NA ETFOSAA ROC NA NA NA NA 0 NA NA FOSE ALC ROC NA NA NA NA 0 NA NA EtFOSE ROC NA NA NA NA 0 NA NA NETFOSE glue NA NA NA NA 0 NA NA FOSA glue NA NA NA NA 0 NA NA NA NA NA NA NA 0.26 NA NA 00 NA NA NA NA NA 0 NA NA NA NA 00 NA NA NA NA 0.23 2 0.25 0.02 290.70 457.60 28.10 1.43 8.57 0.25 145.00 2 217.85 103.03 318.80 2 388.20 98.15 29.20 2 28.65 0.78 2.37 2 1.90 0.66 3.58 2 6.08 3.53 0.35 2 0.30 0.07 26.00 85.50 4.20 0.82 1.02 0.25 34.80 2 30.40 6.22 124.40 2 104.95 27.51 2.20 2 3.20 1.41 0.96 2 0.89 0.10 1.35 2 1.19 0.23 0.29 2 0.27 0.03 NA NA NA NA NA NA NA NA NA NA NA NA SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B M CONT 9G00055 9G00056 N Mean SD 37.50 257.30 3 140.77 110.50 8G01477 8G01478 9G00045 9G00046 N Mean SD NA NA 0.10 NA 1 0.10 NA 3.00 2.20 3 24.27 37.53 NA NA NA NA 0 NA NA NA NA 0 NA NA NA NA NA NA 0 NA NA N-EtFOSA 40mkd 8G01481 8G01482 N Mean SD 112.50 80.00 2 96.25 22.98 N-EtFOSE 160mkd 9G00047 9G00048 N Mean SD 530.80 446.30 2 488.55 59.75 N-EtFOSE 40mkd 8G01479 8G01480 N Mean SD 29.00 147.80 2 88.40 84.00 PFOS 40mkd 8G01483 304.00 8G01484 90.80 9G00049 312.80 0.30 0.60 2 0.45 0.21 11.40 4.90 2 8.15 4.60 5.10 4.70 2 4.90 0.28 69.80 71.00 14.50 NA NA 0 NA NA 169.30 89.50 2 129.40 56.43 34.20 22.10 2 28.15 8.56 NA NA NA NA NA NA NA 00 NA NA NA NA NA NA NA NA NA NA NA NA 00 NA NA NA NA NA NA 0 NA NA 342.10 181.10 2 261.60 113.84 52.90 55.10 2 54.00 1.56 NA NA NA NA NA 0 NA NA 21.80 10.10 2 15.95 8.27 2.40 4.60 2 3.50 1.56 NA NA NA NA NA 0 NA NA NA NA NA NA 0 NA NA NA NA 0 NA NA 2.46 2.13 2 2.30 0.23 0.96 1.67 2 1.32 0.50 NA NA NA NA NA 0 NA NA NA NA NA NA 0 NA NA NA NA 0 NA NA 2.75 1,26 2 2.01 1.05 0.31 0.47 2 0.39 0.11 NA NA NA NA NA 0 NA NA NA NA NA NA 0 NA NA 0.12 0.16 2 0.14 0.03 0.49 0.48 2 0.49 0.01 0.36 0.40 2 0.38 0.03 NA NA NA 46 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 9G00050 N Mean SD 70.50 4 194.53 131.80 11.60 4 41.73 33.14 NA 0 NA NA R F CONT 8R04040 8R04041 9R00469 9R00470 N Mean SD NA NA 0.00 NA 1 0.00 NA NA NA 4.60 4.10 2 4.35 0.35 NA NA NA NA 0 NA NA N-EtFOSA 40mkd 8R04044 8R04045 N Mean SD NA NA 0 NA NA NA NA NA NA 00 NA NA NA NA N-EtFOSE 160rakd 9R00471 9R00472 N Mean SD 865.30 918.50 2 891.90 37.62 17.30 18.80 2 18.05 1.06 63. 60 71. 40 2 67 .50 5.52 N-EtFOSE 40mkd 8R04042 8R04043 N Mean SD NA NA 0 NA NA NA NA NA NA 00 NA NA NA NA PFOS 40mkd 8R04046 NA 8R04047 9R00473 9R00474 N NA 967.50 765.30 2 NA NA 21.20 15.30 2 NA NA NA NA 0 NA 0 NA NA NA NA NA NA 0 NA NA NA NA 0 NA NA 178.20 225.50 2 201.85 33.45 NA NA 0 NA NA NA NA NA NA 0 NA 0 NA NA NA NA NA NA 0 NA NA NA NA 0 NA NA 29.80 64.60 2 47.20 24.61 NA NA 0 NA NA NA NA NA NA 0 NA 0 NA NA NA NA NA NA 0 NA NA NA NA 0 NA NA 7.45 5.34 2 6.40 1.49 NA NA 0 NA NA NA NA NA NA 0 NA 0 NA NA NA NA NA NA 0 NA NA NA NA 0 NA NA 0.11 0.28 2 0.20 0.12 NA NA 0 NA NA NA NA NA NA 0 NA 0 NA NA NA NA NA NA 0 NA NA NA NA 0 NA NA 0.23 0.24 2 0.24 0.01 NA NA 0 NA NA NA NA NA NA 0 47 SEPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Mean SD 866.40 18.25 NA 142.98 4.17 NA NA NA M M556 1R00748 238.40 447.30 614.40 NA -160mkd 1R00749 81.90 235.70 424.00 NA 1R00750 100.70 267.40 627.10 NA N 3 330 Mean 140.33 316.80 555.17 NA SD 85.45 114.12 113.77 NA CONT 1R00742 NA 154.10 NA NA 1R00743 NA 113.20 NA NA 1R00744 NA 273.20 NA NA 8R04032 NA NA NA NA 8R04033 NA NA NA NA 9R00463 0.00 2.60 NA NA 9R00464 0.00 2.80 NA NA N 2 500 Mean 0.00 109.18 NA NA SD 0.00 113.59 NA NA FOSA 40mkg 1R00745 163.20 1R00746 1R00747 N Mean SD 214.30 202.80 3 193.43 26.81 195.70 NA 174.10 163.90 3 177.90 16.24 NA NA 0 NA NA NA NA NA 0 NA NA N-EtFOSA 40mkd 8R04036 8R04037 N Mean SD NA NA 0 NA NA NA NA NA NA NA NA 000 NA NA NA NA NA NA N-EtFOSE 160mkd 9R00465 9R00466 N 1241.70 1006.80 2 25.20 11.70 2 111.00 376. 40 77.50 212. 70 22 NA NA NA NA NA O NA NA NA NA NA NA NA NA NA O NA NA NA NA NA O NA NA NA NA O NA NA 13.90 10.30 2 NA NA NA NA NA O NA NA NA NA NA NA NA NA NA O NA NA NA NA NA O NA NA NA NA O NA NA 2.92 4.66 2 NA NA NA NA NA O NA NA NA NA NA NA NA NA NA O NA NA NA NA NA O NA NA NA NA O NA NA 0.24 0.29 2 NA NA NA NA NA O NA NA NA NA NA NA NA NA NA O NA NA O .44 0.39 0.34 3 0.39 0.05 NA NA O NA NA 0.09 0.08 2 48 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Mean SD 1124.25 18.45 166.10 9.55 N-EtFOSE 40mkd 8R04034 8R04035 N Mean SD NA NA 0 NA NA NA NA 0 NA NA PFOS 40mkd 8R04038 NA NA 8R04039 9R00467 9R00468 N Mean SD NA 907.50 1032.30 2 969.90 88.25 NA 3.70 31.80 2 17.75 19.87 94.25 23.69 NA NA 0 NA NA NA NA NA NA 0 NA NA 294.55 115.75 NA NA 0 NA NA NA NA NA NA 0 NA NA 12.10 2.55 NA NA 0 NA NA NA NA NA NA 0 NA NA 3.79 1.23 NA NA 0 NA NA NA NA NA NA 0 NA NA 0.27 0.04 NA NA 0 NA NA NA NA NA NA 0 NA NA 0.09 0.01 NA NA 0 NA NA NA NA NA NA 0 NA NA SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B B. Rat and Guinea Pig Liver FC Percent of Dose Evaluations. Analyses at the University of Rochester. SPECIES SEX DOSE_GRO GP F CONT ID 8G01485 8G01486 9G00051 9G00052 N Mean SD TL PFOSX (ROC)_ (ug/g)_ Missing Missing Missing Missing 0 Missing Missing TL PFOSX (ROC)_ (mg) Missing Missing Missing Missing 0 Missing Missing % DOSE TL PFOSX (ROC ) (%) Missing Missing Missing Missing 0 Missing Missing N-EtFOSA 40mkd 8G01488 8G01490 N Mean SD Missing 83.5 1 83.5 Missing Missing 1.7 1 1.7 Missing Missing 1.6 1 1.6 Missing N-EtFOSE 160mkd 9G00053 9G00054 N Mean SD 1388.4 630.2 2 1009.3 536.1 13.3 5.6 2 9.5 5.5 8.0 3.5 2 5.8 3.2 N-EtFOSE 40mkd 8G01487 8G01489 N Mean SD 198.8 186.5 2 192.6 8.7 3.9 4.1 2 4.0 0.1 3.8 4.0 2 3.9 0.1 PFOS 40mkd 8G01491 8G01492 9G00055 Missing Missing 40.5 Missing Missing 0.3 Missing Missing 0.9 % DOSE PFOS (ROC) (%) Missing Missing Missing Missing 0 Missing Missing 2.3 1.6 2 2.0 0.5 3.4 0.6 2 2.0 2.0 1.5 0.3 2 0.9 0.8 2.5 Missing 0.8 50 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B M CONT N-EtFOSA 40mkd N-EtFOSE 160mkd N-EtFOSE 40mkd PFOS 40mkd 9G00056 N Mean SD 8G01477 8G01478 9G00045 9G00046 N Mean SD 8G01481 8G01482 N Mean SD 9G00047 9G00048 N Mean SD 8G01479 8G01480 N Mean SD 8G01483 8G01484 9G00049 9G00050 N 259.5 2 150.0 154.9 Missing Missing 0.1 Missing 1 0.1 Missing 112.9 80.8 2 96.8 22.7 1081.1 735.8 2 908.4 244.2 125.2 236.8 2 181.0 78.9 Missing Missing 327.3 82.1 2 1.9 2 1.1 1.1 Missing Missing 0.0 Missing 1 0.0 Missing 3.2 2.0 2 2.6 0.9 9.4 6.3 2 7.8 2.2 3.1 6.5 2 4.8 2.4 Missing Missing 2.4 0.8 2 4.9 2 2.9 2.8 Missing Missing Missing Missing 0 Missing Missing 2.6 1.6 2 2.1 0.7 5.6 3.9 2 4.8 1.2 2.7 5.4 2 4.1 1.9 Missing Missing 6.0 2.1 2 4.9 3 2.7 2.0 Missing Missing Missing Missing 0 Missing Missing 2.6 1.6 2 2.1 0.7 2.8 2.4 2 2.6 0.3 0.6 3.4 2 2.0 1.9 7.3 1.7 5.7 1.8 4 51 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Mean SD R F CONT 8R04040 8R04041 9R00469 9R00470 N Mean SD N-EtFOSE 160mkd 9R00471 9R00472 N Mean SD PFOS 40mkd M M556160mkd CONT 8R04046 8R04047 9R00473 9R00474 N Mean SD 1R00748 1R00749 1R00750 N Mean SD 1R00742 1R00743 1R00744 8R04032 204.7 173.4 Missing Missing 4.6 4.1 2 4.4 0.4 1162.0 1304.7 2 1233.3 100.9 Missing Missing 988.7 780.6 2 884.7 147.1 1300.1 741.6 995.2 3 1012.3 279.6 Missing 113.2 Missing Missing 1.6 1.1 Missing Missing 0.0 0.0 2 0.0 0.0 8.7 9.5 2 9.1 0.6 Missing Missing 8.2 5.7 2 7.0 1.8 14.0 8.0 10.7 3 10.9 3.0 Missing 1.2 Missing Missing 4.1 2.7 Missing Missing Missing Missing 0 Missing Missing 6.9 7.8 2 7.3 0.6 Missing Missing 26.1 19.6 2 22.8 4.6 8.5 4.8 6.4 3 6.6 1.8 Missing Missing Missing Missing 4.2 2.8 Missing Missing Missing Missing 0 Missing Missing 5.1 5.5 2 5.3 0.2 Missing Missing 25.5 19.2 2 22.4 4.5 1.6 0.5 0.6 3 0.9 0.6 Missing Missing Missing Missing 52 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B FOSA 40mkg N-EtFOSE 160mkd PFOS40mkd 8R04033 9R00463 9R00464 N Mean SD 1R00745 1R00746 1R00747 N Mean SD 9R00465 9R00466 N Mean SD 8R04038 8R04039 9R00467 9R00468 N Mean SD Hissing 2.6 2.8 3 39.5 63.8 359.3 388.8 367.0 3 371.7 15.3 1771.5 1324.0 2 1547.7 316.4 Missing Missing 911.2 1064.1 2 987.7 108.1 Missing 0.0 0.0 3 0.4 0.7 3.9 4.2 3.9 3 4.0 0.2 23.0 14.7 2 18.9 5.9 Missing Missing 10.8 13.3 2 12.1 1.7 Missing Missing Missing 0 Missing Missing 9.8 10.8 9.4 3 10.0 0.7 12.5 8.4 2 10.5 2.9 Missing Missing 23.9 31.0 2 27.4 5.0 Missing Missing Missing 0 Missing Missing 4.4 5.9 5.2 3 5.2 0.8 8.8 6.4 2 7.6 1.7 Missing Missing 23.8 30.0 2 26.9 4.4 53 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.I, T-7132.1 DT15-B C. Rat Liver PFOSX Analyses at the 3M Environmental Lab. (All units are |xg/g. The 3M Environmental Lab only analyzed rat liver samples) SPECIES SEX DOSE GRO ID PFOS 3M FOSA 3M N-EtFOSAA-3M N-EtFOSE 3M FOSAA 3M N-EtFOSA 3M R F CONT 8R04040 8R04041 9R00469 9R00470 N Mean SD NA NA NA 0.15 1 0.15 NA NA 0.01 0.03 0.05 3 0.03 0.02 NA NA 0.23 0.21 2 0.22 0.01 NA NA NA NA 0 NA NA NA NA NA 0.07 1 0.07 NA NA NA NA NA 0 NA NA N-EtFOSE160mkd 9R00471 9R00472 N Mean SD 608.00 600.00 2 604.00 5.66 101.00 117.00 2 109.00 11.31 262.00 227.00 2 244.50 24.75 255.00 350.00 2 302.50 67.18 128.00 109.00 2 118.50 13.44 0.89 NA 1 0.89 NA PFOS40mkd M CONT 8R04046 8R04047 9R00473 9R00474 N Mean SD NA NA 851.00 756.00 2 803.50 67.18 1R00742 1R00743 1R00744 8R04032 8R04033 9R00463 9RQ0464 N Mean SD NA NA NA NA NA 0.39 0.20 2 0.30 0.14 NA NA 0.08 0.08 2 0.08 0.00 NA NA NA NA NA 0.05 0.02 2 0.04 0.02 NA NA 0.12 0.16 2 0.14 0.03 NA NA NA NA NA 0.24 0.12 2 0.18 0.08 NA NA NA NA 0 NA NA NA NA NA NA NA NA NA 0 NA NA NA NA 0.04 0.06 2 0.05 0.01 NA NA NA NA NA 0.08 NA 1 0.08 NA NA NA NA NA 0 NA NA NA NA NA NA NA NA NA 0 NA NA SRPT T-6295,8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B N-EtFOSE160mkd 9R00465 9R00466 N Mean SD 844.00 972.00 2 908.00 90.51 96.00 82.00 2 89.00 9.90 330.00 305.00 2 317.50 17.68 PFOS40mkd 8R04038 8R04039 9R00467 9R00468 N Mean SD NA NA 788.00 812.00 2 800.00 16.97 NA NA 0.16 0.12 2 0.14 0.03 NA NA 0.30 0.25 2 0.28 0.03 117.00 177.00 2 147.00 42.43 NA NA NA NA 0 NA NA 177.00 162.00 2 169.50 10.61 NA NA 0.12 0.11 2 0.11 0.01 0.37 0.50 2 0.44 0.09 NA NA NA NA 0 NA NA D. Rat and Guinea Pig Liver FC Percent of Dose Evaluations. Analyses at the 3M environmental Lab. SPECIES SEX DOSE_GRO ID TL PFOSX (3M)_ (ug/g) TL PFOSX (3M)_ (mg) % DOSE TL PFOSX (3M) (%) R F CONT 8R04040 Missing Missing Missing 8R04041 Missing Missing Missing 9R00469 0.26 0.0 Missing 9R00470 0.48 0.0 Missing N2 2 0 Mean 0.4 0.0 Missing SD 0.2 0.0 Missing % DOSE PFOS (3M) (%) Missing Missing Missing Missing 0 Missing Missing N-EtFOSE 160mkd 9R00471 9R00472 N Mean SD 1408.00 1407.00 2 1407.5 0.7 10.6 10.3 2 10.4 0.2 8.4 8.4 2 8.4 0.0 3.6 3.6 2 3.6 0.0 55 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B PFOS 40mkd M556160mkd CONT FOSA 40mkg N-EtFOSE 8R04046 8R04047 9R00473 9R00474 N Mean SD 1R00748 1R00749 1R00750 N Mean SD 1R00742 1R00743 1R00744 8R04032 8R04033 9R00463 9R00464 N Mean SD 1R00745 1R00746 1R00747 N Mean SD 9R00465 Missing Missing 975.00 880.00 2 927.5 67.2 1300.10 741.60 995.20 3 1012.3 279.6 154.10 113.20 273.20 0.00 0.00 0.79 0.35 7 77.4 107.5 358.90 388.40 366.70 3 371.3 15.3 1580.00 M is s in g Missing 8.1 6.4 2 7.3 1.2 14.0 8.0 10.7 3 10.9 3.0 1.7 1.2 2.9 0.0 0.0 0.0 0.0 7 0.8 1.2 3.9 4.2 3.9 3 4.0 0.2 20.5 M is s in g Missing 25.7 22.1 2 23.9 2.6 8.5 4.8 6.4 3 6.6 1.8 Missing Missing Missing Missing Missing Missing Missing 0 Missing Missing 9.8 10.8 9.4 3 10.0 0.7 11.2 M is s in g Missing 22.5 19.0 2 20.7 2.5 Missing Missing Missing 0 Missing Missing Missing Missing Missing Missing Missing Missing Missing 0 Missing Missing Missing Missing Missing 0 Missing Missing 6.0 56 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 160mkd PFOS 40rakd 9R00466 N Mean SD 8R04038 8R04039 9R00467 9R00468 N Mean SD 1675.00 2 1627.5 67.2 Missing Missing 811.00 804.00 2 807.5 4.9 18.6 2 19.6 1.4 Missing Missing 9.7 10.1 2 9.9 0.3 10.6 2 10.9 0.4 Missing Missing 21.2 23.4 2 22.3 1.5 6.2 2 6.1 0.1 Missing Missing 20.6 23.6 2 22.1 2.1 57 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B E. Technical Report: Liver Fluorocarbon Metabolites - University of Rochester. Title: Summary of Quantitative Analysis o f Fluorocarbon Metabolites in Rat Liver Samples Lin Xu and M. W. Anders This is a brief summary o f the analysis o f liver samples from rats given a range o f fluorocarbons. The parent and metabolites o f the fluorocarbons were determined in liver samples by LC-MS/MS. The results are presented below. Three groups o f liver samples were analyzed: The first group (Group 1) consisted o f livers from control rats, from rats given 40 mg/kg/day FOSA orally, and from rats given 160 mg/kg/day FOSAA (M556) orally. The concentrations o f parent compounds and their metabolites in livers were measured (Table 1). The data show that PFOS was the major metabolite found in the livers o f rats given FOSA; FOSA V-glucuronide was identified as a minor metabolite. Two metabolites were identified in the livers o f rats given FOSAA (M556): PFOS and FOSA. It is noteworthy that the control animals (1R00742, 1R00743, and 1R00743) contained significant concentrations o f FOSA. A parallel analysis o f livers from Fischer 344 rats maintained in the University o f Rochester Vivarium did not show detectable concentrations o f FOSA. The second group (Group 2) o f rats was treated in November, 1998. Group 2 contained livers from control rats, from rats given 40 mg/kg/day PFOS orally, from rats given 40 mg/kg/day V-EtFOSE alcohol orally, and from rats given 40 mg/kg/day V-EtFOSA orally. The concentrations o f parent compounds and identified metabolites are shown in Table 2. The data show that livers from rats given PFOS contained parent compound PFOS; FOSA was also found in these samples. The livers from rats given V-EtFOSE alcohol contained several metabolites. The major metabolites were V-EtFOSAA, PFOS, and FOSAA (M556). The minor metabolites were FOSA, FOSE alcohol, V-EtFOSE alcohol glucuronide, and FOSA V-glucuronide. PFOS was the major metabolite in livers from rats given V-EtFOSA; FOSA and FOSA V-glucuronide were identified as minor metabolites. In contrast to Group 1, the livers from control rats did not contain detectable concentrations o f parent compounds or metabolites. The third group (Group 3) was treated in March, 1999. Group 3 contained livers from control rats, from rats given 40 mg/kg/day PFOS orally, and from rats given 160 mg/kg/day V-EtFOSE alcohol orally. The concentrations o f the chemicals and metabolites are shown in Table 3. As with Group 2, PFOS and low concentrations o f FOSA were identified in livers from rats given PFOS. In the livers o f rats given V-EtFOSE alcohol, V-EtFOSAA, PFOS, and FOSAA (M556) were identified as major metabolites, and the minor metabolites identified were FOSA, FOSE alcohol, V-EtFOSE alcohol glucuronide, and FOSA Vglucuronide. Similar to Group 1, control animals showed detectable hepatic concentrations o f FOSA. Discussion: These data allow conclusions about the overall metabolic fate o f the fluorocarbons studied (see Scheme), but do not allow inferences about the exact routes o f metabolite formation. FOSA was consistently identified as a metabolite o f PFOS, whether PFOS was administered directly or formed as a metabolite, but the source o f the amino group is not readily apparent. Whatever its route o f formation, FOSA was metabolized to FOSA V-glucuronide. V-EtFOSE alcohol gives rise to a range o f major and minor metabolites. FOSE alcohol could arise from the V-deethylation o f V-EtFOSE alcohol, and V-EtFOSAA could arise by the oxidation o f the alcohol to the carboxylic acid. Glucuronidation o f the parent V-EtFOSE alcohol would give the observed V-EtFOSE alcohol glucuronides. FOSAA could be formed by the V-deethylation o f V-EtFOSAA or by the oxidation o f FOSE alcohol, or both. FOSA could be formed by the V-deethylation o f V-EtFOSA or by the removal o f the carboxymethyl group o f FOSAA as glyoxylate. FOSA V-glucuronide may be formed by the glucuronidation o f FOSA. 0/ & 59 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B The fate o f FOSAA appears relatively straightforward. Loss o f the carboxymethyl group would give FOSA or loss o f the glycine moiety would give PFOS directly. />7 60 T-7071.1 FR DTI 5 Table 1. Hepatic Concentrations of Fluorocarbons and Fluorocarbon Metabolites in Livers of Rats Given FOSA (40 mg/kg/day) or FOSAA (M556) (160 mg/kg/day) (Group 1) Sample # Animal # 1 1R00742 2 1R00743 3 1R00744 4 1R00745 5 1R00746 6 1R00747 7 1R00748 8 1R00749 9 1R00750 Weight o f liver sample Sex Treatment (g) M Control 0.4600 M Control 0.9236 M Control 1.1287 M FOSA 0.2402 M FOSA 0.7905 M FOSA 0.8305 M FOSAA 0.5520 M FOSAA 0.9373 M FOSAA 0.5320 Note: n.m. = not measured. PFOS (ppm) 0.0 0.0 0.0 163.2 214.3 202.8 238.4 81.9 100.7 FOSA (ppm) FOSAA (ppm) FOSA Nglucuronide (ppm) 154.1 0.0 0.00 113.2 0.0 0.00 273.2 0.0 0.00 195.7 n.m. 0.44 174.1 n.m. 0.39 163.9 n.m. 0.34 447.3 614.4 0.00 235.7 424.0 0.00 267.4 627.1 0.00 T-7071.1 FR DTI 5 Table 2: Hepatic Concentrations of Fluorocarbons and Fluorocarbon Metabolites in Livers from Rats Given PFOS (40 mg/kg/day), /V-EtFOSE alcohol (40 mg/kg/day). or /V-EtFOSA (40 mg/kg/day) (Group 2) Sample # Animal # Sex Treatment 1 8R04032 M Control 2 8R04033 M Control 3 8R04041 F Control 4 8G01477 M Control 5 8G01478 M Control 6 8G01485 F Control 7 8G01486 F Control 8 8G01483 M PFOS 9 8G01484 M PFOS 10 8G01491 F PFOS 11 8G01479 M /V-EtFOSE alcohol 12 8G01480 M /V-EtFOSE alcohol 13 8G01487 F /V-EtFOSE alcohol 14 8G01489 F V-EtFOSE alcohol 15 8G01481 M /V-EtFOSA 16 8G01482 M /V-EtFOSA 17 8G01488 F /V-EtFOSA 18 8G01490 F IV-EtFOSA Note: n.m. = not measured. Weight o f liver samples PFOS (g) (ppm) 0.7516 0.0 1.1 0.0 0.413 0.0 1.081 0.0 0.6009 0.0 1.206 0.0 0.8228 0.0 0.7756 304.0 0.789 90.8 1.1795 127.5 0.7712 29.0 0.7744 147.8 0.7137 76.7 0.4796 12.7 0.707 112.5 0.993 80.0 0.6395 125.8 0.754 83.3 N- N- FOSE EtFOSE FOSA FOSAA EtFOSAA alcohol alcohol (ppm) (ppm) (ppm) (ppm) (ppm) 0.0 0.0 0.0 0.0 n.m. 0.0 0.0 0.0 0.0 n.m. 0.0 0.0 0.0 0.0 n.m. 0.0 0.0 0.0 0.0 n.m. 0.0 0.0 0.0 0.0 n.m. 0.0 0.0 0.0 0.0 n.m. 0.0 0.0 0.0 0.0 n.m. 69.8 n.m. n.m. n.m. n.m. 71.0 n.m. n.m. n.m. n.m. 67.6 n.m. n.m. n.m. n.m. 5.1 34.2 52.9 2.4 0.96 4.7 22.1 55.1 4.6 1.67 4.3 26.0 85.5 4.2 0.82 9.8 34.8 124.4 2.2 0.96 0.3 0.0 0.0 0.0 n.m. 0.6 0.0 0.0 0.0 n.m. 1.1 0.0 0.0 0.0 n.m. 0.0 0.0 0.0 0.0 n.m. /V-EtFOSE alcohol glucuronide (ppm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n.m. n.m. n.m. 0.31 0.47 1.02 1.35 0.00 0.00 0.00 0.00 FOSA Nglucuronide (ppm) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 n,m. n.m. n.m. 0.36 0.40 0.25 0.29 0.12 0.16 0.26 0.23 / 62 T-7071.1 FR DT15 Table 3. Hepatic Concentrations of Fluorocarbons and Fluorocarbon Metabolites in Livers of Rats Given ZV-EtFOSE alcohol (160 mg/kg/day) or PFOS (40 mg/kg/day) (Group 3) Sample # Animal # Sex Treatment 19 9R00463 M Control 20 9R00464 M Control 21 9R00469 F Control 22 9R00470 F Control 23 9R00465 M ZV-EtFOSE alcohol 24 9R00466 M ZV-EtFOSE alcohol 25 9R00471 F ZV-EtFOSE alcohol 26 9R00472 F ZV-EtFOSE alcohol 27 9R00467 M PFOS 28 9R00468 M PFOS 29 9R00473 F PFOS 30 9R00474 F PFOS 31 9G00045 M Control 32 9G00046 M Control 33 9G00051 F Control 34 9G00052 F Control 35 9G00047 M ZV-EtFOSE alcohol 36 9G00048 M ZV-EtFOSE alcohol 37 9G00053 F ZV-EtFOSE alcohol 38 9G00054 F ZV-EtFOSE alcohol 39 9G00049 M PFOS 40 9G00050 M PFOS 41 9G00055 F PFOS 42 9G00056 F PFOS Weight o f PFOS liver (ppm) samples (g) FOSA (ppm) FOSAA ZV- (ppm) EtFOSAA (ppm) FOSE alcohol (ppm) NEtFOSE alcohol (ppm) ZV-EtFOSE alcohol glucuronide (ppm) PFOSA glucuronide (ppm) 0.4459 0.903 0.8444 0.5802 0.1707 0.885 0.304 0.2791 0.5589 0.411 0.6576 0.4673 0.1608 0.3882 0.712 0.59 0.7258 0.713 0.9192 0.44 0.787 0.43 0.9398 0.5605 0.0 0.0 0.0 0.0 1241.7 1006.8 865.3 918.5 907.5 1032.3 967.5 765.3 0.0 0.0 0.0 0.0 530.8 446.3 590.5 109.8 312.8 70.5 37.5 257.3 2.6 n.m. 2.8 n.m. 4.6 n.m. 4.1 n.m. 25.2 111.0 11.7 77.5 17.3 63.6 18.8 71.4 3.7 n.m. 31.8 n.m. 21.2 n.m. 15.3 n.m. 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 11.4 169.3 4.9 89.5 11.2 290.7 21.1 145.0 14.5 n.m. 11.6 n.m. 3.0 n.m. 2.2 n.m. n.m. n.m. n.m. n.m. 376.4 212.7 178.2 225.5 n.m. n.m. n.m. n.m. 0.0 0.0 0.0 0.0 342.1 181.1 457.6 318.8 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. 13.9 10.3 29.8 64.6 n.m. n.m. n.m. n.m. 0.0 0.0 0.0 0.0 21.8 10.1 28.1 29.2 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. 2.92 4.66 7.45 5.34 n.m. n.m. n.m. n.m. n.m. n.m. n.m. n.m. 2.46 2.13 1.43 2.37 n.m. n.m. n.m. n.m. 0.00 0.00 0.00 0.00 0.24 0.29 0.11 0.28 n.m. n.m. n.m. n.m. n.m. 0.00 0.00 0.00 2.75 1.26 8.57 3.58 n.m. n.m. n.m. n.m. 0.00 0.00 0.00 0.00 0.09 0.08 0.23 0.24 n.m. n.m. n.m. n.m. n.m. 0.00 0,00 0.00 0.49 0.48 0.25 0.35 n.m. n.m. n.m. n.m. /M 63 T-7071.1 FR DT15 Note: n.m. = not measured. M etabolism o f Fluorocarbons in Rats ii R -S-N ii ------------------ R -S -N Oii R--S -N H -G lu c u ro n id e Oii FOSA W-glucuronide PFOS 64 T-7071.1 FR DT15 Appendix 7: Clinical Chemistry. Individual and Summary Clinical Chemistry Data. SPEC SEX DOSE GRP ID GP F CONT 8G0148 8G0148 9G0005 9G0005 N Mean SD NEtFOSA 40mkd 8G0148 8 8G0I49 N Mean SD NEtFOSE 40mkd 8G0148 7 8G0148 N Mean SD PFOS 8G0149 40mkd 1 CHOL CA PHOS TOIL ALB TP BUN GLU ALKP AST ALT LDH Na+ K+ Cl- CREA TRIG mg/dL mg/dL mg/dL g/dL g/dL mg/dL mg/dL U/L U/L U/L U/L mmol/L mmol/L mmol/L mg/dl mg/dL mg/dL1 63 13.1 9.0 0.2 2.7 5.6 14 416 156 46 50 257 141 NR 103 0.5 124 62 11.9 9.7 0.1 2.4 5.0 13 202 117 45 46 118 144 12.0 102 0.4 132 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 23 NR NR 0.2 1.9 4.3 14 310 202 37 42 133 141 12.9 104 0.3 78 3 2 2 3 333 3 3 333 3 2 3 33 49.2 12.50 9.35 0.17 2.33 4.97 13.7 309.3 158.3 42.7 46.0 169. 142.0 12.45 103.0 0.40 111.3 23.1 0.85 0.49 0.06 0.40 0.65 0.6 107.0 42.5 4.9 4.0 76.3 1.7 0.64 1.0 0.10 29.1 67 11.6 8.7 0.2 2.6 5.7 17 198 126 38 49 160 138 5.8 100 0.5 107 116 12.6 8.6 0.2 2.9 6.3 13 263 122 38 56 158 138 6.6 2 2 2 2 2 22 2 2 222 2 2 91.5 12.10 8.65 0.20 2.75 6.00 15.0 230.5 124.0 38.0 52.5 159. 138.0 6.20 34.6 0.71 0.07 0.00 0.21 0.42 2.8 46.0 2.8 0.0 4.9 1.4 0.0 0.57 70 11.7 9.5 0.1 2.5 5.5 17 182 121 32 34 117 141 11.0 100 0.5 143 2 22 100.0 0.50 125.0 0.0 0.00 25.5 99 0.6 96 23 12.7 9.2 0.2 2.4 5.0 10 286 125 32 43 166 141 13.2 104 0.4 117 2 2 2 2 2 22 2 2 222 2 2 2 22 46.3 12.20 9.35 0.15 2.45 5.25 13.5 234.0 123.0 32.0 38.5 141. 141.0 12.10 101.5 0.50 106.5 33.6 0.71 0.21 0.07 0.07 0.35 4.9 73.5 2.8 0.0 6.4 34.6 0.0 1.56 3.5 0.14 14.8 75 12.4 10.2 0.1 2.6 5.6 15 326 163 77 60 286 133 6.1 100 0.5 127 65 T-7071.1 FR DT15 CONT 8G0149 2 9G0005 5 9G0005 6 N Mean SD 8G0147 7 8G0147 8 9G0004 5 9G0004 6 N Mean SD 71 NR NR 2 73.0 2.8 23 48 23 NR 3 31.0 14.7 11.9 7.4 0.4 2.6 NR NR NR NR NR NR NR NR 2222 12.15 8.80 0.23 2.60 0.35 1.98 0.25 0.00 11.2 8.3 0.3 2.5 11.7 9.2 0.1 2.4 NR NR 0.1 1.7 NR NR NR NR 2233 11.45 8.75 0.15 2.20 0.35 0.64 0.13 0.44 NEtFOSA 40mkd 8G0148 1 8G0148 2 N Mean SD NEtFOSE 40mkd 8G0I47 9 8G0148 0 N Mean SD 55 49 2 52.0 4.2 23 23 2 22.5 0.0 12.8 9.7 0.1 2.6 11.8 8.9 0.1 2.6 2222 12.30 9.30 0.10 2.60 0.71 0.57 0.00 0.00 12.8 9.0 0.3 2.6 12.4 9.6 0.1 2.3 2222 12.60 9.30 0.18 2.45 0.28 0.42 0.18 0.21 5,6 10 315 110 44 55 344 134 4.1 100 0.5 101 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 22 5.60 12.5 0.00 3.5 2 320.5 7.8 2 136.5 37.5 222 60.5 57.5 315. 0 23.3 3.5 41.0 2 133.5 0.7 2 5.10 1.41 2 22 100.0 0.50 114.0 0.0 0.00 18.4 5.3 15 154 110 64 45 303 144 12.1 103 0.5 168 5.1 16 211 194 49 52 113 140 NR 103 0.4 183 4.0 16 275 329 36 52 141 141 14.2 107 0.3 84 NR NR NR NR NR NR NR NR NR NR NR NR 33 3 3 333 3 2 3 33 4.80 15.7 213.3 211.0 49.7 49.7 185. 141.7 13.15 104.3 0.40 145.0 0.70 0.6 60.5 110.5 14.0 4.0 102. 2.1 6 1.48 2.3 0.10 53.4 5.5 12 485 147 54 51 208 135 7.5 97 0.6 201 5.5 16 194 128 47 57 218 136 6.7 101 0.7 103 22 2 2 222 2 2 2 22 5.50 14.0 339.5 137.5 50.5 54.0 213. 135.5 7.10 99.0 0.65 152.0 0 0.00 2.8 205.8 13.4 4.9 4.2 7.1 0.7 0.57 2.8 0.07 69.3 5.4 13 449 163 117 62 514 138 12.7 98 0.5 154 4.8 16 372 108 41 47 151 140 11.9 103 0.5 126 22 2 2 222 2 2 2 22 5.10 14.5 410.5 135.5 79.0 54.5 332. 139.0 12.30 100.5 0.50 140.0 0.42 2.1 54.4 38.9 53.7 10.6 256. 1.4 7 0.57 3.5 0.00 19.8 66 T-7071.1 FR DTI 5 PFOS 40mkd 8G0148 3 8G0148 4 9G0004 9 9G0005 0 N Mean SD 52 54 NR 23 3 42.8 17.6 12.6 9.1 0.1 3.0 11.6 7.6 0.2 2.9 NR NR NR NR NR NR 0.3 1.8 2233 12.10 8.35 0.20 2.57 0.71 1.06 0.10 0.67 CONT 8R04040 8R04041 9R00469 9R00470 N Mean 96 77 86 81 4 85.0 SD 8.2 NEtFOSA 40mkd 8R04044 8R04045 N Mean 82 88 2 85.0 SD 4.2 NEtFOSE 160mkd 9R00471 9R00472 N Mean SD NR NR 0 NR NR NEtFOSE 40mkd 8R04042 8R04043 70 89 12.9 12.1 NR NR 2 12.50 10.5 10.7 NR NR 2 10.60 0.4 0.3 0.4 0.4 4 0.38 0.57 0.14 0.05 11.8 8.2 0.3 4.3 3.8 3.1 3.6 4 3.70 0.50 3.8 12.2 8.3 0.3 4.1 2222 12.00 8.25 0.30 3.95 0.28 0.07 0.00 0.21 NR NR 0.4 NR NR NR 00 NR NR NR NR 11.3 9.5 NR NR 10 0.40 NR NR NR 0.2 3.5 12.0 10.1 0.3 3.7 6.2 16 6.2 17 NR NR 4.5 16 33 5.63 16.3 0.98 0.6 7.1 15 6.7 13 5.9 20 6.5 19 44 6.55 16.8 0.50 3.3 7.0 14 342 102 49 57 131 133 131 115 47 62 155 138 NR NR NR NR NR NR 248 270 70 66 348 134 3 240.3 105.7 3 162.3 93.5 333 55.3 61.7 211. 5a 12.7 4.5 119. 0 3 135.0 2.6 426 351 188 215 4 295.0 267 214 253 274 4 252.0 112.8 26.8 86 76 374 84 69 343 80 72 416 94 78 307 44 4 86.0 73.8 360. 0 5.9 4.0 46.3 149 149 148 148 4 148.5 0.6 328 283 69 66 139 151 4.1 2.7 NR 6.4 3 4.40 1.87 6.8 5.7 7.2 7.4 4 6.78 0.76 4.5 95 0.8 118 101 0.7 94 NR NR NR 103 0.3 85 3 33 99.7 0.60 99.0 4.2 0.26 17.1 98 0.6 75 98 0.5 57 105 0.4 81 104 0.5 62 4 44 101.3 0.50 68.8 3.8 0.08 11.1 106 0.7 53 7.4 19 22 7.20 16.5 0.28 3.5 NR 24 315 2 321.5 9.2 NR 234 2 258.5 34.6 184 80 70 180 22 2 74.5 68.0 159. JC 7.8 2.8 29.0 60 77 NR 150 2 150.5 0.7 NR 4.6 2 4.55 0.07 NR 102 0.6 70 2 22 104.0 0.65 61.5 2.8 0.07 12.0 NR NR 35 NR NR NR NR NR NR NR NR NR NR NR NR 01 0 1 110 0 0 0 01 NR 24.0 NR 184.0 60.0 77.0 NR NR NR NR NR 35.0 NR NR NR NR NR NR NR NR NR NR NR NR 6.7 14 248 236 71 56 219 147 5.1 105 0.6 40 6.8 18 318 229 77 87 222 147 5.7 100 0.5 105 67 T-7071.1 FR DT15 PFOS 40mkd N Mean 2 79.5 SD 13.4 8R04046 57 8R04047 9R00473 9R00474 N Mean 23 NR NR 2 39.8 SD 24.4 2222 11.65 9.80 0.25 3.60 0.49 0.42 0.07 0.14 11.9 8.1 0.3 4.1 12.4 8.1 0.4 4.6 NR NR NR NR NR NR NR NR 2222 12.15 8.10 0.35 4.35 0.35 0.00 0.07 0.35 CONT 8R04032 8R04033 9R00463 9R00464 N Mean 88 73 92 86 4 84.8 SD 8.2 12.0 11.6 NR NR 2 11.80 11.0 11.1 NR NR 2 11.05 0.4 0.4 0.4 0.4 4 0.40 0.28 0.07 0.00 3.6 3.7 3.7 3.1 4 3.53 0.29 NEtFOSA 40mkd 8R04036 8R04037 N Mean 75 79 2 77.0 SD 2.8 NEtFOSE 160mkd 9R00465 9R00466 N Mean 50 NR 1 50.0 SD NR N- 8R04034 63 11.8 9.4 0.2 3.4 11.9 10.5 0.3 3.5 2222 11.85 9.95 0.25 3.45 0.07 0.78 0.07 0.07 NR NR 0.4 3.1 NR NR 0.4 3.4 0022 NR NR 0.40 3.25 NR NR 0.00 0.21 12.2 11.0 0.4 3-7 22 6.75 16.0 0.07 2.8 7.5 16 8.0 20 NR NR NR NR 22 7.75 18.0 0.35 2.8 6.5 18 6.8 15 6.6 25 6.0 25 44 6.48 20.8 0.34 5.1 6.4 14 2 2 22 2 2 283.0 232.5 74.0 71.5 220. 147.0 5 49.5 4.9 4.2 21.9 2.1 0.0 303 165 58 66 197 147 345 NR NR 2 324.0 29.7 153 NR NR 2 159.0 8.5 66 77 443 NR NR NR NR NR NR 22 2 62.0 71.5 320. 0 5.7 7.8 173. 9 146 NR NR 2 146.5 0.7 376 216 327 249 4 292.0 72.8 239 301 325 308 4 293.3 37.5 80 71 330 87 70 746 72 65 310 130 85 1291 44 4 92.3 72.8 669. 3 25.9 8.6 460. 7 150 150 153 148 4 150.3 2.1 300 341 68 54 179 152 2 5.40 0.42 3.2 3.3 NR NR 2 3.25 0.07 7.5 5.4 5.9 8.5 4 6.83 1.43 4.0 2 22 102.5 0.55 72.5 3.5 0.07 46.0 102 0.6 43 104 0.7 44 NR NR NR NR NR NR 2 22 103.0 0.65 43.5 1.4 0.07 0.7 99 0.6 82 99 0.5 132 101 0.5 105 101 0.4 137 4 44 100.0 0.50 114.0 1.2 0.08 25.5 104 0.6 85 6.6 17 22 6.50 15.5 0.14 2.1 6.0 19 347 2 323.5 33.2 178 335 2 338.0 4.2 238 84 60 178 222 76.0 57.0 178. 5 11.3 4.2 0.7 63 94 227 151 2 151.5 0.7 143 5.0 2 4.50 0.71 6.6 102 0.6 86 2 22 103.0 0.60 85.5 1.4 0.00 0.7 104 0.6 39 6.4 23 22 6.20 21.0 0.28 2.8 7.0 16 244 2 211.0 46.7 330 160 2 199.0 55.2 266 68 89 241 222 65.5 91.5 234. 0 3.5 3.5 9.9 69 67 422 148 2 145.5 3.5 153 4.4 2 5.50 1.56 5.2 104 0.5 24 2 22 104.0 0.55 31.5 0.0 0.07 10.6 99 0.5 114 68 T-7071.1 FR DT15 EtFOSE 40mkd 8R04035 N Mean 70 2 66.5 S D 4.9 PFOS 40mkd 8R04038 63 8R04039 9R00467 9R00468 N Mean 49 59 NR 3 57.0 SD 7.2 11.0 2 11.60 0.85 9.8 2 10.40 0.85 0.2 2 0.30 0.14 12.3 10.6 0.6 12.2 NR NR 2 12.25 9.6 NR NR 2 10.10 0.4 0.4 NR 3 0.47 0.07 0.71 0.12 3.4 6.6 19 2 22 3.55 6.80 17.5 0.21 0.28 2.1 4.6 7.8 21 4.5 7.8 20 4.0 7.1 17 NR NR NR 3 33 4.37 7.57 19.3 0.32 0.40 2.1 243 2 286.5 61.5 365 346 379 NR 3 363.3 16.6 258 2 262.0 5.7 203 160 227 NR 3 196.7 33.9 68 66 272 222 68.5 66.5 347. 0 0.7 0.7 106. 1 71 90 527 77 93 482 59 83 274 NR NR NR 333 69.0 88.7 427. 9.2 5.1 135. 0 150 2 151.5 2.1 148 150 147 NR 3 148.3 1.5 4.8 2 5.00 0.28 3.1 3.0 4.6 NR 3 3.57 0.90 101 0.6 70 2 22 100.0 0.55 92.0 1.4 0.07 31.1 100 0.8 55 103 0.7 54 101 0.6 42 NR NR NR 3 33 101.3 0.70 50.3 1.5 0.10 7.2 1. The LOD for CHOL was 45 mg/dL. When CHOL < LOD, half o f LOD (22.5 mg/dL) was entered to perform statistics * 69 T-7071.1 FR DT15 T-7071.1 FR DTI 5 A. Statistics on Clinical Chemistries for rats, male and female values combined. CHOL mg/dL By Dose Group (mg/Kg/day) Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.701781 0.631612 10.64141 72.52273 22 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 4530.1636 17 1925.0750 21 6455.2386 Mean Square 1132.54 113.24 307.39 F Ratio 10.0013 Prob>F 0.0002 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 1 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 84.8750 81.0000 50.0000 73.0000 50.1000 Std Error 3.762 5.321 10.641 5.321 4.759 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 84.8750 7.6052 N-EtFOSA40mkd N-EtFOSE160mkd 4 81.0000 1 50.0000 5.4772 ? N-EtFOSE40mkd 4 73.0000 11.1654 PFOS40mkd 5 50.1000 16.2496 Std Err Mean 2.6888 2.7386 ? 5.5827 7.2670 Dif=Mean[i]-Mean[j] CONT N-EtFOSA40mkd CONT 0.0000 -3.8750 Means Comparisons N-EtFOSA40mkd 3.8750 0.0000 N-EtFOSE40mkd 11.8750 8.0000 PFOS40mkd 34.7750 30.9000 N-EtFOSE160mkd 34.8750 31.0000 //J 71 T-7071.1 FR DTI 5 N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd -11.8750 -34.7750 -34.8750 -8.0000 -30.9000 -31.0000 0.0000 -22.9000 -23.0000 22.9000 0.0000 -0.1000 23.0000 0.1000 0.0000 Alpha= Abs(Dif)-LSD CONT N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10980 CONT N-EtFOSA40mkd N-EtFOSE40mkd -11.2256 -9.8736 -1.8736 -9.8736 -15.8755 -7.8755 -1.8736 -7.8755 -15.8755 21.9758 15.8392 7.8392 11.0618 5.8987 -2.1013 PFOS40mkd 21.9758 15.8392 7.8392 -14.1994 -24.4942 N-EtFOSE160mkd 11.0618 5.8987 -2.1013 -24.4942 -31.7509 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Idl 2.74189 Abs(Dif)-LSD CONT N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd N-EtFOSE 160mkd CONT -14.5888 -13.9926 -5.9926 18.1412 3.9275 Positive values show pairs of means that are significantly different. Cholesterol in rats male and female combined for DT15. Sig reduction by 40 mkd PFIOS and 160 mkd N-EtFOSE. 72 //? T-7071.1 FR DT15 13.0 12.5 12.0 S 11.5 11.0 Ca mg/dL By Dose Group (mg/Kg/day) N-EtFOSE40mkd Dose Group (mg/Kg/day) Student's t 0.05 Dunnett's 0.05 l 0 T-7071.1 FR DT15 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.28157 0.101962 0.418828 11.975 16 Source Model Error C TotaI Analysis of Variance DF Sum of Squares 3 0.8250000 12 2.1050000 15 2.9300000 Mean Square 0.275000 0.175417 0.195333 I- Ratio 1.5677 Prob>F 0.2484 Means for Oneway Anova Level Number CONT 4 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 4 Mean 12.1500 11.9250 11.6250 12.2000 Std Error 0.20941 0.20941 0.20941 0.20941 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 4 12.1500 0.544671 N-EtFOSA40mkd 4 11.9250 0.189297 N-EtFOSE40mkd 4 11.6250 0.567891 PFOS40mkd 4 12.2000 0.216025 Std Err Mean 0.27234 0.09465 0.28395 0.10801 Dif=Mean[i]-Mean[j]| PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd Means Comparisons PFOS40mkd CONT 0.000000 0.050000 -0.05 0.000000 -0.275 -0.225 -0.575 -0.525 N-EtFOSA40mkd 0.275000 0.225000 0.000000 -0.3 N-EtFOSE40mkd 0.575000 0.525000 0.300000 0.000000 Alpha= Abs(Dif)-LSD PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t + 2.17882 PFOS40mkd CONT -0.64527 -0.59527 -0.59527 -0.64527 -0.37027 -0.42027 -0.07027 -0.12027 N-EtFOSA40mkd -0.37027 -0.42027 -0.64527 -0.34527 N-EtFOSE40mkd -0.07027 -0.12027 -0.34527 -0.64527 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Ml 2.68294 Abs(Dif>LSD CONT PFOS40mkd -0.74457 CONT -0.79457 N-EtFOSA40mkd -0.56957 N-EtFOSE40mkd -0.26957 Positive values show pairs of means that are significantly different. PHOS mg/dL By Dose Group (mg/Kg/day) 74 T-7071.1 FR DT15 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.471871 0.339839 0.889171 9.78125 16 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 8.476875 12 9.487500 15 17.964375 Mean Square 2.82562 0.79063 1.19763 F Ratio 3.5739 Prob>F 0.0470 Means for Oneway Anova Level Number CONT 4 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 4 Mean 10.8250 9.1000 10.1000 9.1000 Std Error 0.44459 0.44459 0.44459 0.44459 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 4 10.8250 0.27538 N-EtFOSA40mkd 4 9.1000 1.08012 N-EtFOSE40mkd 4 10.1000 0.64807 PFOS40mkd 4 9.1000 1.22474 Std Err Mean 0.13769 0.54006 0.32404 0.61237 Dif=Mean[i]-Mean[j] CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd Means Comparisons CONT N-EtFOSE40mkd 0.00000 0.72500 -0.72500 0.00000 -1.72500 -1.00000 -1.72500 -1.00000 N-EtFOSA40mkd 1.72500 1.00000 0.00000 0.00000 PFOS40mkd 1.72500 1.00000 0.00000 0.00000 Alpha= Abs(Dif)-LSD CONT 0.05 Comparisons for each pair using Student's t t 2.17882 CONT N-EtFOSE40mkd N-EtFOSA40mkd -1.36991 -0.64491 0.35509 PFOS40mkd 0.35509 fX . 75 T-7071.1 FR DTI 5 N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd -0.64491 0.35509 0.35509 -1.36991 -0.36991 -0.36991 -0.36991 -1.36991 -1.36991 Positive values show pairs of means that are significantly different. Male and female rats combined Ca and Phos clinical chemistries. No sig change from control. -0.36991 -1.36991 -1.36991 /A 5 76 T-7071.1 FR DT15 TBIL mg/dL By Dose Group (mg/Kg/day) N-EtFOSE160if?fSDS Dose Group (mg/Kg/day) Student's t 0.05 Dunnett's 0.05 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.485514 0.377201 0.069491 0.358333 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 0.08658333 19 0.09175000 23 0.17833333 Mean Square 0.021646 0.004829 0.007754 F Ratio 4.4825 Prob>F 0.0102 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 3 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 0.387500 0.275000 0.400000 0.275000 0.420000 Std Error 0.02457 0.03475 0.04012 0.03475 0.03108 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 0.387500 0.035355 N-EtFOSA40mkd 4 0.275000 0.050000 N-EtFOSE160mkd 3 0.400000 0.000000 N-EtFOSE40mkd 4 0.275000 0.095743 PFOS40mkd 5 0.420000 0.109545 Std Err Mean 0.01250 0.02500 0.00000 0.04787 0.04899 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSE160mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd Means Comparisons PFOS40mkd N-EtFOSE160mkd 0.000000 0.020000 -0.02 0.000000 -0.0325 -0.0125 -0.145 -0.125 -0.145 -0.125 CONT 0.032500 0.012500 0.000000 -0.1125 -0.1125 N-EtFOSA40mkd 0.145000 0.125000 0.112500 0.000000 0.000000 N-EtFOSE40mkd 0.145000 0.125000 0.112500 0.000000 0.000000 Alpha= 0.05 Comparisons for each pair using Student's t Abs(Dif)-LSD PFOS40mkd N-EtFOSE160mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd -0.09199 -0.08622 -0.05042 0.047433 0.047433 2.09301 N-EtFOSE160mkd -0.08622 -0.11875 -0.08597 0.013915 0.013915 CONT 0.05042 -0.08597 -0.07272 0.023434 0.023434 IN-EtFOSA40mkd 0.047433 0.013915 0.023434 -0.10284 -0.10284 N-EtFOSE40mkd 0.047433 0.013915 0.023434 -0.10284 -0.10284 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD CONT PFOS40mkd -0.07464 N-EtFOSE160mkd -0.11473 CONT -0.09396 N-EtFOSA40mkd -0.00258 N-EtFOSE40mkd -0.00258 iZS T-7071.1 FR DT15 Positive values show pairs of means that are significantly different. Above is for rat TBIL, male and femal combined data, no sig changes. I2.& T-7071.1 FR DT15 27 T-7071.1 FR DT15 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.595877 0.506072 0.315634 3.752174 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 2.6441413 18 1.7932500 22 4.4373913 Mean Square 0.661035 0.099625 0.201700 F Ratio 6.6352 Prob>F 0.0018 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 3.61250 3.70000 3.25000 3.57500 4.36000 Std Error 0.11159 0.15782 0.22319 0.15782 0.14116 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 3.61250 0.387068 N-EtFOSA40mkd 4 3.70000 0.316228 N-EtFOSE160mkd 2 3.25000 0.212132 N-EtFOSE40mkd 4 3.57500 0.150000 PFOS40mkd 5 4.36000 0.288097 Std Err Mean 0.13685 0.15811 0.15000 0.07500 0.12884 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.00000 0.66000 -0.66000 0.00000 -0.74750 -0.08750 -0.78500 -0.12500 -1.11000 -0.45000 CONT 0.74750 0.08750 0.00000 -0.03750 -0.36250 N-EtFOSE40mkd 0.78500 0.12500 0.03750 0.00000 -0.32500 N-EtFOSE160mkd 1.11000 0.45000 0.36250 0.32500 0.00000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd CONT -0.41939 0.215166 0.369464 0.215166 -0.4689 -0.31858 0.369464 -0.31858 -0.33156 0.340166 -0.3439 -0.36858 0.555195 -0.12428 -0.16174 N-EtFOSE40mkd 0.340166 -0.3439 -0.36858 -0.4689 -0.24928 N-EtFOSE160mkd 0.555195 -0.12428 -0.16174 -0.24928 -0.66312 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT PFOS40mkd 0.257735 N-EtFOSA40mkd -0.43859 CONT -0.42955 N-EtFOSE40mkd -0.48859 N-EtFOSE160mkd -0.31668 28 81 T-7071.1 FR DT15 Positive values show pairs of means that are significantly different. TP By Dose Group (mg/Kg/day)__________________________ Dose Group (mg/Kgiday) -05 0.05 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.674822 0.60256 0.361958 6.834783 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 4.8939239 18 2.3582500 22 7.2521739 Mean Square 1.22348 0.13101 0.32964 F Ratio 9.3386 Prob>F 0.0003 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 6.51250 6.85000 6.20000 6.77500 7.64000 Std Error 0.12797 0.18098 0.25594 0.18098 0.16187 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 6.51250 0.397986 N-EtFOSA40mkd 4 6.85000 0.443471 N-EtFOSE160mkd 2 6.20000 0.282843 N-EtFOSE40mkd 4 6.77500 0.170783 PFOS40mkd 5 7.64000 0.350714 Std Err Mean 0.14071 0.22174 0.20000 0.08539 0.15684 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.00000 0.79000 -0.79000 0.00000 -0.86500 -0.07500 -1.12750 -0.33750 -1.44000 -0.65000 N-EtFOSE40mkd 0.86500 0.07500 0.00000 -0.26250 -0.57500 CONT 1.12750 0.33750 0.26250 0.00000 -0.31250 N-EtFOSE160mkd 1.44000 0.65000 0.57500 0.31250 0.00000 /a ? 82 T-7071.1 FR DTI 5 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd -0.48095 0.279880 0.354880 0.279880 -0.53771 -0.46271 0.354880 -0.46271 -0.53771 0.693982 -0.12817 -0.20317 0.803769 -0.00856 -0.08356 CONT 0.693982 -0.12817 -0.20317 -0.38022 -0.28868 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT PFOS40mkd 0.565854 N-EtFOSA40mkd -0.2658 N-EtFOSE40mkd -0.3408 CONT -0.4926 N-EtFOSE160mkd -0.46636 Positive values show pairs of means that are significantly different. N-EtFOSE 160mkd 0.803769 -0.00856 -0.08356 -0.28868 -0.76044 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.312227 0.159389 60.99861 301.6087 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 30404.528 18 66974.950 22 97379.478 Mean Square 7601.13 3720.83 4426.34 F Ratio 2.0429 Prob>F 0.1312 Level CONT Means for Oneway Anova Number 8 Mean 293.500 Std Error 21.566 /3 83 T-7071.1 FR DT15 N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS40mkd 4 322.500 2 211.000 4 284.750 5 347.600 30.499 43.133 30.499 27.279 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 293.500 87.8879 N-EtFOSA40mkd 4 322.500 19.9416 N-EtFOSE 160mkd 2 211.000 46.6690 N-EtFOSE40mkd 4 284.750 45.6317 PFOS40mkd 5 347.600 28.6671 Std Err Mean 31.073 9.971 33.000 22.816 12.820 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.000 25.100 -25.100 0.000 -54.100 -29.000 -62.850 -37.750 -136.600 -111.500 CONT 54.100 29.000 0.000 -8.750 -82.500 N-EtFOSE40mkd 62.850 37.750 8.750 0.000 -73.750 N-EtFOSE160mkd 136.600 111.500 82.500 73.750 0.000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd CONT -81.051 -60.867 -18.958 -60.867 -90.617 -49.477 -18.958 -49.477 -64.076 -23.117 -52.867 -69.727 29.380 0.517 -18.813 N-EtFOSE40mkd -23.117 -52.867 -69.727 -90.617 -37.233 N-EtFOSE160mkd 29.380 0.517 -18.813 -37.233 -128.152 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT PFOS40mkd -40.5507 N-EtFOSA40mkd -72.6711 CONT -83.0141 N-EtFOSE40mkd -92.9211 N-EtFOSE160mkd -48.7569 Positive values show pairs of means that are significantly different. 84 /s/ T-7071.1 FR DTI 5 A 3 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.595877 0.506072 0.315634 3.752174 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 2.6441413 18 1.7932500 22 4.4373913 Mean Square 0.661035 0.099625 0.201700 F Ratio 6.6352 Prob>F 0.0018 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 3.61250 3.70000 3.25000 3.57500 4.36000 Std Error 0.11159 0.15782 0.22319 0.15782 0.14116 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 3.61250 0.387068 N-EtFOSA40mkd 4 3.70000 0.316228 N-EtFOSE160mkd 2 3.25000 0.212132 N-EtFOSE40mkd 4 3.57500 0.150000 PFOS40mkd 5 4.36000 0.288097 Std Err Mean 0.13685 0.15811 0.15000 0.07500 0.12884 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.00000 0.66000 -0.66000 0.00000 -0.74750 -0.08750 -0.78500 -0.12500 -1.11000 -0.45000 CONT 0.74750 0.08750 0.00000 -0.03750 -0.36250 N-EtFOSE40mkd 0.78500 0.12500 0.03750 0.00000 -0.32500 N-EtFOSE160mkd 1.11000 0.45000 0.36250 0.32500 0.00000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd CONT -0.41939 0.215166 0.369464 0.215166 -0.4689 -0.31858 0.369464 -0.31858 -0.33156 0.340166 -0.3439 -0.36858 0.555195 -0.12428 -0.16174 N-EtFOSE40mkd 0.340166 -0.3439 -0.36858 -0.4689 -0.24928 N-EtFOSE160mkd 0.555195 -0.12428 -0.16174 -0.24928 -0.66312 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT PFOS40mkd 0.257735 N-EtFOSA40mkd -0.43859 CONT -0.42955 N-EtFOSE40mkd -0.48859 N-EtFOSE160mkd -0.31668 tS3 86 T-7071.1 FR DT15 Positive values show pairs of means that are significantly different. TP By Dose Group (mg/Kg/day)__________________________ Dose Group (mg/Kg/day) 0 05 0.05 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.674822 0.60256 0.361958 6.834783 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 4.8939239 18 2.3582500 22 7.2521739 Mean Square 1.22348 0.13101 0.32964 F Ratio 9.3386 Prob>F 0.0003 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 6.51250 6.85000 6.20000 6.77500 7.64000 Std Error 0.12797 0.18098 0.25594 0.18098 0.16187 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 6.51250 0.397986 N-EtFOSA40mkd 4 6.85000 0.443471 N-EtFOSE160mkd 2 6.20000 0.282843 N-EtFOSE40mkd 4 6.77500 0.170783 PFOS40mkd 5 7.64000 0.350714 Std Err Mean 0.14071 0.22174 0.20000 0.08539 0.15684 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.00000 0.79000 -0.79000 0.00000 -0.86500 -0.07500 -1.12750 -0.33750 -1.44000 -0.65000 N-EtFOSE40mkd 0.86500 0.07500 0.00000 -0.26250 -0.57500 CONT 1.12750 0.33750 0.26250 0.00000 -0.31250 N-EtFOSE 160mkd 1.44000 0.65000 0.57500 0.31250 0.00000 /3 V 87 T-7071.1 FR DT15 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd CONT N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd -0.48095 0.279880 0.354880 0.279880 -0.53771 -0.46271 0.354880 -0.46271 -0.53771 0.693982 -0.12817 -0.20317 0.803769 -0.00856 -0.08356 CONT 0.693982 -0.12817 -0.20317 -0.38022 -0.28868 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT PFOS40mkd 0.565854 N-EtFOSA40mkd -0.2658 N-EtFOSE40mkd -0.3408 CONT -0.4926 N-EtFOSE160mkd -0.46636 Positive values show pairs of means that are significantly different. GLU mg/dL By Dose Group (mg/Kg/day) N-EtFOSE160mkd 0.803769 -0.00856 -0.08356 -0.28868 -0.76044 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.312227 0.159389 60.99861 301.6087 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 30404.528 18 66974.950 22 97379.478 Mean Square 7601.13 3720.83 4426.34 F Ratio 2.0429 Prob>F 0.1312 Level CONT Means for Oneway Anova Number 8 Mean 293.500 Std Error 21.566 88 fSS T-7071.1 FR DTI 5 N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS40mkd 4 322.500 2 211.000 4 284.750 5 347.600 30.499 43.133 30.499 27.279 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 293.500 87.8879 N-EtFOSA40mkd 4 322.500 19.9416 N-EtFOSE160mkd . 2 211.000 46.6690 N-EtFOSE40mkd 4 284.750 45.6317 PFOS40mkd 5 347.600 28.6671 Std Err Mean 31.073 9.971 33.000 22.816 12.820 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.000 25.100 -25.100 0.000 -54.100 -29.000 -62.850 -37.750 -136.600 -111.500 CONT 54.100 29.000 0.000 -8.750 -82.500 N-EtFOSE40mkd 62.850 37.750 8.750 0.000 -73.750 N-EtFOSE160mkd 136.600 111.500 82.500 73.750 0.000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE 160mkd 0.05 Comparisons for each pair using Student's t * PFOS40mkd -81.051 -60.867 -18.958 -23.117 29.380 2.10091 N-EtFOSA40mkd -60.867 -90.617 -49.477 -52.867 0.517 CONT -18.958 -49.477 -64.076 -69.727 -18.813 N-EtFOSE40mkd -23.117 -52.867 -69.727 -90.617 -37.233 N-EtFOSE160mkd 29.380 0.517 -18.813 -37.233 -128.152 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method 2.72184 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd CONT -40.5507 -72.6711 -83.0141 -92.9211 -48.7569 Positive values show pairs of means that are significantly different. 89 T-7071.1 FR DT15 /3 7 T-7071.1 FR DT15 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.264802 0.110024 3.301116 18.375 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 74.57500 19 207.05000 23 281.62500 Mean Square 18.6438 10.8974 12.2446 F Ratio 1.7108 Prob>F 0.1892 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 3 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 18.7500 16.0000 22.0000 16.7500 18.8000 Std Error 1.1671 1.6506 1.9059 1.6506 1.4763 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 18.7500 4.49603 N-EtFOSA40mkd 4 16.0000 2.44949 N-EtFOSE160mkd 3 22.0000 2.64575 N-EtFOSE40mkd 4 16.7500 2.21736 PFOS40mkd 5 18.8000 2.16795 Std Err Mean 1.5896 1.2247 1.5275 1.1087 0.9695 Dif=Mean[i]-Mean[j] N-EtFOSE160mkd PFOS40mkd CONT N-EtFOSE40mkd N-EtFOSA40mkd Means Comparisons N-EtFOSE160mkd PFOS40mkd 0.00000 3.20000 -3.20000 0.00000 -3.25000 -0.05000 -5.25000 -2.05000 -6.00000 -2.80000 CONT 3.25000 0.05000 0.00000 -2.00000 -2.75000 N-EtFOSE40mkd 5.25000 2.05000 2.00000 0.00000 -0.75000 N-EtFOSA40mkd 6.00000 2.80000 2.75000 0.75000 0.00000 Alpha= Abs(Dif)-LSD N-EtFOSE160mkd PFOS40mkd CONT N-EtFOSE40mkd N-EtFOSA40mkd 0.05 Comparisons for each pair using Student's t f1 2.09301 N-EtFOSE160mkd PFOS40mkd CONT -5.64139 -1.84581 -1.42760 -1.84581 -4.36980 -3.88889 -1.42760 -3.88889 -3.45463 -0.02704 -2.58488 -2.23104 0.72296 -1.83488 -1.48104 N-EtFOSE40mkd -0.02704 -2.58488 -2.23104 -4.88559 -4.13559 N-EtFOSA40mkd 0.72296 -1.83488 -1.48104 -4.13559 -4.88559 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD CONT N-EtFOSE160mkd -2.79392 PFOS40mkd -5.03944 CONT -4.46373 N-EtFOSE40mkd -3.46693 N-EtFOSA40mkd -2.71693 /3 * 91 T-7071.1 FR DT15 Positive values show pairs of means that are significantly different. Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.63982 0.563993 36.65097 243.875 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 45338.050 19 25522.575 23 70860.625 Mean Square 11334.5 1343.3 3080.9 F Ratio 8.4379 Prob>F 0.0004 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 3 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 272.625 298.250 194.000 247.250 181.600 Std Error 12.958 18.325 21.160 18.325 16.391 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 272.625 37.3896 N-EtFOSA40mkd 4 298.250 50.1290 N-EtFOSE160mkd 3 194.000 39.9500 N-EtFOSE40mkd 4 247.250 17.5760 PFOS40mkd 5 181.600 31.9343 Std Err Mean 13.219 25.065 23.065 8.788 14.281 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd PFOS40mkd Means Comparisons N-EtFOSA40mkd CONT 0.000 25.625 -25.625 0.000 -51.000 -25.375 -104.250 -78.625 -116.650 -91.025 N-EtFOSE40mkd 51.000 25.375 0.000 -53.250 -65.650 N-EtFOSE160mkd 104.250 78.625 53.250 0.000 -12.400 PFOS40mkd 116.650 91.025 65.650 12.400 0.000 / 3? 92 T-7071.1 FR DT15 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE 160mkd PFOS40mkd 0.05 Comparisons for each pair using Student's t t 2.09301 N-EtFOSA40mkd CONT N-EtFOSE40mkd -54.2427 -21.3506 -3.2427 -21.3506 -38.3554 -21.6006 -3.2427 -21.6006 -54.2427 45.6612 26.6915 -5.3388 65.1908 47.2931 14.1908 N-EtFOSE 160mkd 45.6612 26.6915 -5.3388 -62.6341 -43.6217 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Id| 2.70438 Abs(Dif)-LSD CONT N-EtFOSA40mkd -35.0721 CONT -49.5590 N-EtFOSE40mkd -35.3221 N-EtFOSE160mkd 11.5218 PFOS40mkd 34.5190 Positive values show pairs of means that are significantly different. AST U/L By Dose Group (mg/Kg/day)________________________ PFOS40mkd 65.1908 47.2931 14.1908 -43.6217 -48.5162 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.468919 0.357112 11.9777 75.875 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 2406.7833 19 2725.8417 23 5132.6250 Mean Square 601.696 143.465 223.158 F Ratio 4.1940 Prob>F 0.0134 Level CONT Means for Oneway Anova Number 8 Mean 89.1250 Std Error 4.2348 / Vo 93 T-7071.1 FR DT15 N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS40mkd 4 75.2500 3 63.6667 4 71.2500 5 66.2000 5.9889 6.9153 5.9889 5.3566 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 89.1250 17.7074 N-EtFOSA40mkd 4 75.2500 7.9739 N-EtFOSE160mkd 3 63.6667 4.0415 N-EtFOSE40mkd 4 71.2500 4.0311 PFOS40mkd 5 66.2000 8.0436 Std Err Mean 6.2605 3.9870 2.3333 2.0156 3.5972 Dif=Mean[i]-Mean[j] CONT N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd CONT 0.0000 -13.8750 -17.8750 -22.9250 -25.4583 Means Comparisons N-EtFOSA40mkd 13.8750 0.0000 -4.0000 -9.0500 -11.5833 N-EtFOSE40mkd 17.8750 4.0000 0.0000 -5.0500 -7.5833 PFOS40mkd 22.9250 9.0500 5.0500 0.0000 -2.5333 N-EtFOSE 160mkd 25.4583 11.5833 7.5833 2.5333 0.0000 Alpha= Abs(Dif)-LSD CONT N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.09301 CONT N-EtFOSA40mkd N-EtFOSE40mkd -12.5347 -1.4768 2.5232 -1.4768 -17.7268 -13.7268 2.5232 -13.7268 -17.7268 8.6332 -7.7671 -11.7671 8.4862 -7.5638 -11.5638 PFOS40mkd 8.6332 -7.7671 -11.7671 -15.8553 -15.7748 N-EtFOSE160mkd 8.4862 -7.5638 -11.5638 -15.7748 -20.4691 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD CONT CONT -16.1961 N-EtFOSA40mkd -5.9611 N-EtFOSE40mkd -1.9611 PFOS40mkd 4.4586 N-EtFOSE160mkd 3.5287 Positive values show pairs of means that are significantly different. ALT U/L By Dose Group (mg/Kg/day) /V/ 94 T-7071.1 FR DT15 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.478735 0.368994 9.014511 74.20833 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 1417.9917 19 1543.9667 23 2961.9583 Mean Square 354.498 81.261 128.781 F Ratio 4.3624 Prob>F 0.0114 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 3 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 73.2500 62.5000 86.6667 69.0000 81.8000 Std Error 3.1871 4.5073 5.2045 4.5073 4.0314 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 73.2500 6.2278 N-EtFOSA40mkd 4 62.5000 7.0000 N-EtFOSE 160mkd 3 86.6667 8.7369 N-EtFOSE40mkd 4 69.0000 12.9872 PFOS40mkd 5 81.8000 10.8028 Std Err Mean 2.2019 3.5000 5.0442 6.4936 4.8311 Dif=Mean[i]-Mean[j] N-EtFOSE160mkd PFOS40mkd CONT N-EtFOSE40mkd N-EtFOSA40mkd Means Comparisons N-EtFOSE160mkd PFOS40mkd 0.0000 4.8667 -4.8667 0.0000 -13.4167 -8.5500 -17.6667 -12.8000 -24.1667 -19.3000 CONT 13.4167 8.5500 0.0000 -4.2500 -10.7500 N-EtFOSE40mkd 17.6667 12.8000 4.2500 0.0000 -6.5000 Alpha= 0.05 Comparisons for each pair using Student's t t N-EtFOSA40mkd 24.1667 19.3000 10.7500 6.5000 0.0000 /V<? 95 T-7071.1 FR DT15 Abs(Dif)-LSD N-EtFOSE160mkd PFOS40mkd CONT N-EtFOSE40mkd N-EtFOSA40mkd N-EtFOSE160mkd -15.4052 -8.9122 0.6433 3.2564 9.7564 2.09301 PFOS40mkd -8.9122 -11.9328 -2.2061 0.1433 6.6433 CONT 0.6433 -2.2061 -9.4337 -7.3039 -0.8039 N-EtFOSE40mkd 3.2564 0.1433 -7.3039 -13.3413 -6.8413 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD CONT N-EtFOSE160mkd -3.0877 PFOS40mkd -5.3480 CONT -12.1893 N-EtFOSE40mkd -10.6788 N-EtFOSA40mkd -4.1788 Positive values show pairs of means that are significantly different. N-EtFOSA40mkd 9.7564 6.6433 -0.8039 -6.8413 -13.3413 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.502305 0.397527 23.94016 70.41667 24 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 10990.342 19 10889.492 23 21879.833 Mean Square 2747.59 573.13 951.30 F Ratio 4.7940 Prob>F 0.0076 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 3 N-EtFOSE40mkd 4 Mean 91.3750 73.5000 32.6667 82.2500 Std Error 8.464 11.970 13.822 11.970 /43 96 T-7071.1 FR DTI 5 PFOS40mkd 5 47.6000 10.706 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 91.3750 30.2982 N-EtFOSA40mkd 4 73.5000 15.5027 N-EtFOSE 160mkd 3 32.6667 7.7675 N-EtFOSE40mkd 4 82.2500 33.9644 PFOS40mkd 5 47.6000 6.3482 Std Err Mean 10.712 7.751 4.485 16.982 2.839 Dif=Mean[i]-Mean[j] CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd N-EtFOSE160mkd CONT 0.0000 -9.1250 -17.8750 -43.7750 -58.7083 Means Comparisons N-EtFOSE40mkd 9.1250 0.0000 -8.7500 -34.6500 -49.5833 N-EtFOSA40mkd 17.8750 8.7500 0.0000 -25.9000 -40.8333 PFOS40mkd 43.7750 34.6500 25.9000 0.0000 -14.9333 N-EtFOSE160mkd 58.7083 49.5833 40.8333 14.9333 0.0000 Alpha= Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t t 2.09301 CONT N-EtFOSE40mkd N-EtFOSA40mkd -25.0535 -21.5591 -12.8091 -21.5591 -35.4310 -26.6810 -12.8091 -26.6810 -35.4310 15.2096 1.0372 -7.7128 24.7858 11.3135 2.5635 PFOS40mkd 15.2096 1.0372 -7.7128 -31.6904 -21.6596 N-EtFOSE160mkd 24.7858 11.3135 2.5635 -21.6596 -40.9122 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.70438 Abs(Dif)-LSD CONT CONT -32.3716 N-EtFOSE40mkd -30.5220 N-EtFOSA40mkd -21.7720 PFOS40mkd 6.8657 N-EtFOSEI 60mkd 14.8770 Positive values show pairs of means that are significantly different. Combined rat data for males and females. Sig decreases in ALKP, AST and TRIG by 40 mkd PFOS and 160 mkd N-EtFOSE in rats. /</ 97 T-7071.1 FR DT15 GLU mg/dL By Dose Group (mg/Kg/day) /V5 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.312227 0.159389 60.99861 301.6087 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 30404.528 18 66974.950 22 97379.478 Mean Square 7601.13 3720.83 4426.34 F Ratio 2.0429 Prob>F 0.1312 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 293.500 322.500 211.000 284.750 347.600 Std Error 21.566 30.499 43.133 30.499 27.279 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 293.500 87.8879 N-EtFOSA40mkd 4 322.500 19.9416 N-EtFOSE 160mkd 2 211.000 46.6690 N-EtFOSE40mkd 4 284.750 45.6317 PFOS40mkd 5 347.600 28.6671 Std Err Mean 31.073 9.971 33.000 22.816 12.820 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE160mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.000 25.100 -25.100 0.000 -54.100 -29.000 -62.850 -37.750 -136.600 -111.500 CONT 54.100 29.000 0.000 -8.750 -82.500 N-EtFOSE40mkd 62.850 37.750 8.750 0.000 -73.750 N-EtFOSE160mkd 136.600 111.500 82.500 73.750 0.000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE 160mkd 0.05 Comparisons for each pair using Student's t t 2.10091 PFOS40mkd N-EtFOSA40mkd CONT -81.051 -60.867 -18.958 -60.867 -90.617 -49.477 -18.958 -49.477 -64.076 -23.117 -52.867 -69.727 29.380 0.517 -18.813 N-EtFOSE40mkd -23.117 -52.867 -69.727 -90.617 -37.233 N-EtFOSE160mkd 29.380 0.517 -18.813 -37.233 -128.152 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd N-EtFOSE 160mkd CONT -40.5507 -72.6711 -83.0141 -92.9211 -48.7569 99 T-7071.1 FR DT15 Positive values show pairs of means that are significantly different. RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.295198 0.138575 228.9437 361.6957 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 395163.0 18 943473.8 22 1338636.9 Mean Square 98790.8 52415.2 60847.1 F Ratio 1.8848 Prob>F 0.1569 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE 160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 514.625 169.000 234.000 283.750 384.600 Std Error 80.94 114.47 161.89 114.47 102.39 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 514.625 345.236 N-EtFOSA40mkd 4 169.000 20.017 N-EtFOSE160mkd 2 234.000 9.899 N-EtFOSE40mkd 4 283.750 95.318 PFOS40mkd 5 384.600 141.952 Std Err Mean 122.06 10.01 7.00 47.66 63.48 Dif=Mean[i]-Mean[jJ CONT PFOS40mkd N-EtFOSE40mkd N-EtFOSE 160mkd N-EtFOSA40mkd CONT 0.000 -130.025 -230.875 -280.625 -345.625 Means Comparisons PFOS40mkd N-EtFOSE40mkd 130.025 230.875 0.000 100.850 -100.850 0.000 -150.600 -49.750 -215.600 -114.750 N-EtFOSE 160mkd 280.625 150.600 49.750 0.000 -65.000 N-EtFOSA40mkd 345.625 215.600 114.750 65.000 0.000 /H7 100 T-7071.1 FR DTI 5 Alpha= Abs(Dif)-LSD CONT PFOS40mkd N-EtFOSE40mkd N-EtFOSE160mkd N-EtFOSA40mkd 0.05 Comparisons for each pair using Student's t t 2.10091 CONT PFOS40mkd N-EtFOSE40mkd -240.495 -144.181 -63.670 -144.181 -304.205 -221.808 -63.670 -221.808 -340.111 -99.631 -251.825 -366.799 51.080 -107.058 -225.361 N-EtFOSE160mkd -99.631 -251.825 -366.799 -480.990 -351.549 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT CONT -311.574 PFOS40mkd -225.224 N-EtFOSE40mkd -150.723 N-EtFOSE160mkd -212.016 N-EtFOSA40mkd -35.973 Positive values show pairs of means that are significantly different. Na+ mmol/L By Dose Group (mg/Kg/day) N-EtFOSA40mkd 51.080 -107.058 -225.361 -351.549 -340.111 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.429799 0.303087 1.94829 148.913 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 51.50109 18 68.32500 22 119.82609 Mean Square 12.8753 3.7958 5.4466 F Ratio 3.3919 Prob>F 0.0310 Level CONT Means for Oneway Anova Number 8 Mean 149.375 Std Error 0.6888 101 T-7071.1 FR DT15 N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS40mkd 4 151.000 2 145.500 4 149.250 5 147.600 0.9741 1.3776 0.9741 0.8713 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 149.375 1.68502 N-EtFOSA40mkd 4 151.000 0.81650 N-EtFOSE160mkd 2 145.500 3.53553 N-EtFOSE40mkd 4 149.250 2.87228 PFOS40mkd 5 147.600 1.51658 Std Err Mean 0.5957 0.4082 2.5000 1.4361 0.6782 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd CONT N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd Means Comparisons N-EtFOSA40mkd CONT 0.00000 1.62500 -1.62500 0.00000 -1.75000 -0.12500 -3.40000 -1.77500 -5.50000 -3.87500 N-EtFOSE40mkd 1.75000 0.12500 0.00000 -1.65000 -3.75000 PFOS40mkd 3.40000 1.77500 1.65000 0.00000 -2.10000 N-EtFOSE160mkd 5.50000 3.87500 3.75000 2.10000 0.00000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd CONT N-EtFOSE40mkd PFOS40mkd N-EtFOSE160mkd 0.05 Comparisons for each pair using Student's t f N-EtFOSA40mkd -2.89431 -0.88155 -1.14431 0.65421 1.95520 2.10091 CONT -0.88155 -2.04659 -2.38155 -0.55847 0.63906 N-EtFOSE40mkd -1.14431 -2.38155 -2.89431 -1.09579 0.20520 PFOS40mkd 0.65421 -0.55847 -1.09579 -2.58875 -1.32460 N-EtFOSE 160mkd 1.95520 0.63906 0.20520 -1.32460 -4.09318 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Ml 2.72184 Abs(Dif)-LSD CONT N-EtFOSA40mkd -1.62237 CONT -2.65146 N-EtFOSE40mkd -3.12237 PFOS40mkd -1.24813 N-EtFOSE160mkd -0.31733 Positive values show pairs of means that are significantly different. K+ mmol/L By Dose Group (mg/Kg/day) 102 //? T-7071.1FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.744756 0.688035 0.848512 5.282609 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 37.813543 18 12.959500 22 50.773043 Mean Square 9.45339 0.71997 2.30787 F Ratio 13.1302 Prob>F <.0001 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 6.80000 4.52500 5.50000 5.20000 3.44000 Std Error 0.29999 0.42426 0.59999 0.42426 0.37947 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 6.80000 1.06100 N-EtFOSA40mkd 4 4.52500 0.41130 N-EtFOSE160mkd 2 5.50000 1.55563 N-EtFOSE40mkd 4 5.20000 0.37417 PFOS40mkd 5 3.44000 0.65803 Std Err Mean 0.3751 0.2056 1.1000 0.1871 0.2943 Dif=Mean[i]-Mean[j] CONT N-EtFOSE160mkd N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT 0.00000 -1.30000 -1.60000 -2.27500 -3.36000 Means Comparisons N-EtFOSE160mkd 1.30000 0.00000 -0.30000 -0.97500 -2.06000 N-EtFOSE40mkd 1.60000 0.30000 0.00000 -0.67500 -1.76000 N-EtFOSA40mkd 2.27500 0.97500 0.67500 0.00000 -1.08500 Alpha= 0.05 Comparisons for each pair using Student's t t PFOS40mkd 3.36000 2.06000 1.76000 1.08500 0.00000 l$ 0 103 T-7071.1 FR DT15 Abs(Dif)-LSD CONT N-EtFOSE160mkd N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT -0.89132 -0.10930 0.50836 1.18336 2.34374 2.10091 N-EtFOSE160mkd -0.10930 -1.78265 -1.24382 -0.56882 0.56853 N-EtFOSE40mkd 0.50836 -1.24382 -1.26052 -0.58552 0.56417 N-EtFOSA40mkd 1.18336 -0.56882 -0.58552 -1.26052 -0.11083 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT CONT -1.15476 N-EtFOSE160mkd -0.52583 N-EtFOSE40mkd 0.18572 N-EtFOSA40mkd 0.86072 PFOS40mkd 2.04338 Positive values show pairs of means that are significantly different. PFOS40mkd 2.34374 0.56853 0.56417 -0.11083 -1.12744 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.26878 0.106287 2.256164 101.8261 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 33.67935 18 91.62500 22 125.30435 Mean Square 8.41984 5.09028 5.69565 F Ratio 1.6541 Prob>F 0.2043 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 Mean 100.625 103.500 104.000 101.250 Std Error 0.7977 1.1281 1.5953 1.1281 // 104 T -7071.1 FR DT15 PFOS40mkd 5 102.000 1.0090 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 100.625 2.66927 N-EtFOSA40mkd 4 103.500 1.91485 N-EtFOSE160mkd 2 104.000 0.00000 N-EtFOSE40mkd 4 101.250 2.62996 PFOS40mkd 5 102.000 1.58114 Std Err Mean 0.9437 0.9574 0.0000 1.3150 0.7071 Dif=Meanm-MeanD] N-EtFOSE160mkd N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT Means Comparisons N-EtFOSE160mkd ISi-EtFOSA40mkd 0.00000 0.50000 -0.50000 0.00000 -2.00000 -1.50000 -2.75000 -2.25000 -3.37500 -2.87500 PFOS40mkd 2.00000 1.50000 0.00000 -0.75000 -1.37500 N-EtFOSE40mkd 2.75000 2.25000 0.75000 0.00000 -0.62500 CONT 3.37500 2.87500 1.37500 0.62500 0.00000 Alpha= Abs(Dif)-LSD N-EtFOSE160mkd N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT 0.05 Comparisons for each pair using Student's t t 2.10091 N-EtFOSE160mkd N-EtFOSA40mkd PFOS40mkd -4.73999 -3.60496 -1.96576 -3.60496 -3.35168 -1.67968 -1.96576 -1.67968 -2.99784 -1.35496 -1.10168 -2.42968 -0.37229 -0.02764 -1.32721 N-EtFOSE40mkd -1.35496 -1.10168 -2.42968 -3.35168 -2.27764 CONT -0.37229 -0.02764 -1.32721 -2.27764 -2.37000 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT N-EtFOSE160mkd -1.47982 N-EtFOSA40mkd -0.88552 PFOS40mkd -2.12586 N-EtFOSE40mkd -3.13552 CONT -3.07046 Positive values show pairs of means that are significantly different. CREA mg/dL By Dose Group (mg/Kg/day) iSZ 105 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.557128 0.458712 0.070907 0.573913 23 Source Model Error C Total Analysis of Variance DF Sum of Squares 4 0.11384783 18 0.09050000 22 0.20434783 Mean Square 0.028462 0.005028 0.009289 F Ratio 5.6609 Prob>F 0.0039 Means for Oneway Anova Level Number CONT 8 N-EtFOSA40mkd 4 N-EtFOSE160mkd 2 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 0.500000 0.625000 0.550000 0.550000 0.680000 Std Error 0.02507 0.03545 0.05014 0.03545 0.03171 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 8 0.500000 0.075593 N-EtFOSA40mkd 4 0.625000 0.050000 N-EtFOSE160mkd 2 0.550000 0.070711 N-EtFOSE40mkd 4 0.550000 0.057735 PFOS40mkd 5 0.680000 0.083666 Std Err Mean 0.02673 0.02500 0.05000 0.02887 0.03742 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd CONT Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.000000 0.055000 -0.055 0.000000 -0.13 -0.075 -0.13 -0.075 -0.18 -0.125 N-EtFOSE160mkd 0.130000 0.075000 0.000000 0.000000 -0.05 N-EtFOSE40mkd 0.130000 0.075000 0.000000 0.000000 -0.05 Alpha= 0.05 Comparisons for each pair using Student's t t CONT 0.180000 0.125000 0.050000 0.050000 0.000000 iS 3 106 T-7071.1 FR DTI 5 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd N-EtFOSE 160mkd N-EtFOSE40mkd CONT PFOS40mkd -0.09422 -0.04493 0.005364 0.030069 0.095075 2.10091 N-EtFOSA40mkd -0.04493 -0.10534 -0.05401 -0.03034 0.033776 N-EtFOSE160mkd 0.005364 -0.05401 -0.14897 -0.12901 -0.06777 N-EtFOSE40mkd 0.030069 -0.03034 -0.12901 -0.10534 -0.04122 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.72184 Abs(Dif)-LSD CONT PFOS40mkd 0.069975 N-EtFOSA40mkd 0.006814 N-EtFOSE160mkd -0.10258 N-EtFOSE40mkd -0.06819 CONT -0.0965 Positive values show pairs of means that are significantly different. Clinical chemistries for all rats (males and females combined in DT15B. Shows significant decrease in potassium by 40 mkd PFOS, N-EtFOSE and N-EtFOSA, with the most significant changes occurring in the 40 mkd PFOS dose group. Creatinine (CREAT) was significantly increased in the 40 mkd PFOS and 40 mkd N-EtFOSA dose groups. CONT 0.095075 0.033776 -0.06777 -0.04122 -0.07448 /sV 107 T-7071.1 FR DTI 5 B. Statistics on Clinical Chemistries for Guinea Pigs, male and female values combined. /S5 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or SumWgts) 0.303785 0.164542 23.365 49.44737 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 3573.102 15 8188.846 18 11761.947 Mean Square 1191.03 545.92 653.44 F Ratio 2.1817 Prob>F 0.1327 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 40.0833 71.7500 34.3750 54.9000 Std Error 9.539 11.682 11.682 10.449 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 40.0833 19.9785 N-EtFOSA40mkd 4 71.7500 30.4344 N-EtFOSE40mkd 4 34.3750 23.7500 PFOS40mkd 5 54.9000 20.7497 Std Err Mean 8.156 15.217 11.875 9.280 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd PFOS40mkd CONT N-EtFOSE40mkd Means Comparisons N-EtFOSA40mkd PFOS40mkd 0.0000 16.8500 -16.8500 0.0000 -31.6667 -14.8167 -37.3750 -20.5250 CONT 31.6667 14.8167 0.0000 -5.7083 N-EtFOSE40mkd 37.3750 20.5250 5.7083 0.0000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd PFOS40mkd CONT N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 N-EtFOSA40mkd PFOS40mkd -35.2147 -16.5576 -16.5576 -31.4970 -0.4798 -15.3394 2.1603 -12.8826 CONT -0.4798 -15.3394 -28.7527 -26.4382 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnetfs Method |d| 2.62898 Abs(Dif)-LSD CONT N-EtFOSA40mkd PFOS40mkd CONT -7.9838 -22.3787 -35.4644 N-EtFOSE40mkd -33.9421 N-EtFOSE40mkd 2.1603 -12.8826 -26.4382 -35.2147 Positive values show pairs of means that are significantly different. Cholesterol, Male and female guinea pig combined. 109 T-7071.1 FR DTI 5 /S 7 110 T-7071.1 FR DTI 5 l$ S T-7071.1 FR DT15 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.079281 -0.1509 0.602426 12.175 16 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 0.3750000 12 4.3550000 15 4.7300000 Mean Square 0.125000 0.362917 0.315333 F Ratio 0.3444 Prob>F 0.7938 Means for Oneway Anova Level Number CONT 4 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 4 Mean 11.9750 12.2000 12.4000 12.1250 Std Error 0.30121 0.30121 0.30121 0.30121 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 4 11.9750 0.805709 N-EtFOSA40mkd 4 12.2000 0.588784 N-EtFOSE40mkd 4 12.4000 0.496655 PFOS40mkd 4 12.1250 0.457347 Std Err Mean 0.40285 0.29439 0.24833 0.22867 Dif=Mean[i]-Mean[j] N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT Means Comparisons N-EtFOSE40mkd N-EtFOSA40mkd 0.000000 0.200000 - 0.2 0.000000 -0.275 -0.075 -0.425 -0.225 PFOS40mkd 0.275000 0.075000 0.000000 -0.15 CONT 0.425000 0.225000 0.150000 0.000000 Alpha= Abs(Dif)-LSD N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT 0.05 Comparisons for each pair using Student's t t 2.17882 N-EtFOSE40mkd N-EtFOSA40mkd -0.92813 -0.72813 -0.72813 -0.92813 -0.65313 -0.85313 -0.50313 -0.70313 PFOS40mkd -0.65313 -0.85313 -0.92813 -0.77813 CONT -0.50313 -0.70313 -0.77813 -0.92813 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.68294 Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd -0.71788 -0.91788 -0.99288 CONT -1.14288 Positive values show pairs of means that are significantly different. PHOS mg/dL By Dose Group (mg/Kg/day) iS f __________ 112 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.137384 -0.07827 0.776343 8.98125 16 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 1.1518750 12 7.2325000 15 8.3843750 Mean Square 0.383958 0.602708 0.558958 F Ratio 0.6371 Prob>F 0.6054 Means for Oneway Anova Level Number CONT 4 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 4 Mean 9.05000 8.97500 9.32500 8.57500 Std Error 0.38817 0.38817 0.38817 0.38817 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 4 9.05000 0.58023 N-EtFOSA40mkd 4 8.97500 0.49917 N-EtFOSE40mkd 4 9.32500 0.27538 PFOS40mkd 4 8.57500 1.32256 Std Err Mean 0.29011 0.24958 0.13769 0.66128 Dif=Mean[i]-Mean[i] N-EtFOSE40mkd CONT N-EtFOSA40mkd PFOS40mkd Means Comparisons N-EtFOSE40mkd CONT 0.000000 0.275000 -0.275 0.000000 -0.35 -0.075 -0.75 -0.475 N-EtFOSA40mkd 0.350000 0.075000 0.000000 -0.4 PFOS40mkd 0.750000 0.475000 0.400000 0.000000 Alpha= Abs(Dif)-LSD N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.17882 N-EtFOSE40mkd CONT N-EtFOSA40mkd -1.19608 -0.92108 -0.84608 PFOS40mkd -0.44608 I 113 T-7071.1 FR DT15 CONT N-EtFOSA40mkd PFOS40mkd -0.92108 -0.84608 -0.44608 -1.19608 -1.12108 -0.72108 -1.12108 -1.19608 -0.79608 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Ml 2.68294 Abs(Dif>LSD CONT N-EtFOSE40mkd -1.19782 CONT -1.47282 N-EtFOSA40mkd -1.39782 PFOS40mkd -0.99782 -0.72108 -0.79608 -1.19608 Positive values show pairs of means that are significantly different. the two analyses directly Above are for guinea pig Ca and PHOS, male and female combined. No sig changes occured T-7071.1 FR DT15 \iZ T-7071.1 FR DT15 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.926687 0.909768 1.140108 8.823529 17 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 213.59259 13 16.89800 16 230.49059 Mean Square 71.1975 1.2998 14.4057 F Ratio 54.7738 Prob>F <.0001 Means for Oneway Anova Level Number CONT 4 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 12.8000 6.6500 12.2000 4.6800 Std Error 0.57005 0.57005 0.57005 0.50987 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 4 12.8000 1.01653 N-EtFOSA40mkd 4 6.6500 0.69522 N-EtFOSE40mkd 4 12.2000 0.96264 PFOS40mkd 5 4.6800 1.54661 Std Err Mean 0.50827 0.34761 0.48132 0.69166 Dif=Mean[i]-MeanO] CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd Means Comparisons CONT N-EtFOSE40mkd 0.00000 0.60000 -0.60000 0.00000 -6.15000 -5.55000 -8.12000 -7.52000 N-EtFOSA40mkd 6.15000 5.55000 0.00000 -1.97000 PFOS40mkd 8.12000 7.52000 1.97000 0.00000 Alpha= Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd 0.05 Comparisons for each pair using Student's t t 2.16037 CONT N-EtFOSE40mkd N-EtFOSA40mkd -1.74164 -1.14164 4.40836 -1.14164 -1.74164 3.80836 4.40836 3.80836 -1.74164 6.46773 5.86773 0.31773 PFOS40mkd 6.46773 5.86773 0.31773 -1.55777 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| OfiAQA* Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT -2.13593 -1.53593 4.01407 6.09368 Positive values show pairs of means that are significantly different. Above is for guinea pig combined male and femal potasium levels. Sig reduced by PFOS and N-etFOSA at 40 mg/Kg/day. 116 T-7071.1 FR DT15 TBIL mg/dL By Dose Group (mg/Kg/day) T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.05849 -0.12981 0.106758 0.171053 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 0.01062061 15 0.17095833 18 0.18157895 Mean Square 0.003540 0.011397 0.010088 F Ratio 0.3106 Prob>F 0.8174 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 0.158333 0.150000 0.162500 0.210000 Std Error 0.04358 0.05338 0.05338 0.04774 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 0.158333 0.091742 N-EtFOSA40mkd 4 0.150000 0.057735 N-EtFOSE40mkd 4 0.162500 0.110868 PFOS40mkd 5 0.210000 0.143178 Std Err Mean 0.03745 0.02887 0.05543 0.06403 Dif=Mean[iJ-Mean[j] PFOS40mkd N-EtFOSE40mkd CONT N-EtFOSA40mkd Means Comparisons PFOS40mkd N-EtFOSE40mkd 0.000000 0.047500 -0.0475 0.000000 -0.05167 -0.00417 -0.06 -0.0125 CONT 0.051667 0.004167 0.000000 -0.00833 N-EtFOSA40mkd 0.060000 0.012500 0.008333 0.000000 Alpha= Abs(Dif>-LSD PFOS40mkd N-EtFOS E40mkd CONT N-EtFOSA40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 PFOS40mkd N-EtFOSE40mkd CONT -0.14391 -0.10514 -0.08612 -0.10514 -0.1609 -0.14271 -0.08612 -0.14271 -0.13137 -0.09264 -0.1484 -0.13855 N-EtFOSA40mkd -0.09264 -0.1484 -0.13855 -0.1609 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Idi 2.62898 Abs(Dif)-LSD PFOS40mkd N-EtFOSE40mkd CONT N-EtFOSA40mkd CONT -0.11828 -0.177 -0.16204 -0.17283 Positive values show pairs of means that are significantly different. Alb mg/dL By Dose Group (mg/Kg/day) liS 118 T-7071.1 FR DT15 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.215626 0.058751 0.340474 2.473684 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 0.4780088 15 1.7388333 18 2.2168421 Mean Square 0.159336 0.115922 0.123158 F Ratio 1.3745 Prob>F 0.2887 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 2.26667 2.67500 2.45000 2.58000 Std Error 0.13900 0.17024 0.17024 0.15226 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 2.26667 0.382971 N-EtFOSA40mkd 4 2.67500 0.150000 N-EtFOSE40mkd 4 2.45000 0.129099 PFOS40mkd 5 2.58000 0.471169 Std Err Mean 0.15635 0.07500 0.06455 0.21071 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT Means Comparisons N-EtFOSA40mkd PFOS40mkd 0.000000 0.095000 -0.095 0.000000 -0.225 -0.13 -0.40833 -0.31333 N-EtFOSE40mkd 0.225000 0.130000 0.000000 -0.18333 CONT 0.408333 0.313333 0.183333 0.000000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd -0.51315 -0.39181 -0.28815 CONT -0.0601 119 T-7071.1 FR DT15 PFOS40mkd N-EtFOS E40mkd CONT -0.39181 -0.28815 -0.0601 -0.45897 -0.35681 -0.1261 -0.35681 -0.51315 -0.2851 -0.1261 -0.2851 -0.41898 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT N-EtFOSA40mkd -0.16945 PFOS40mkd -0.22868 N-EtFOSE40mkd -0.39445 CONT -0.51679 Positive values show pairs of means that are significantly different. TP By Dose Group (mg/Kg/day)________________________ 6.5 6.0 5.5 4.5 4.0 N-EtFOSE40mkd Dose Group (mg/Kg/day) Student's t 0.05 Dunnett's 0.05 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.3468 0.21616 0.550989 5.321053 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 2.4177456 15 4.5538333 18 6.9715789 Mean Square 0.805915 0.303589 0.387310 F Ratio 2.6546 Prob>F 0.0863 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 4.88333 5.75000 5.17500 5.62000 Std Error 0.22494 0.27549 0.27549 0.24641 Level CONT Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev 6 4.88333 0.611283 Std Err Mean 0.24956 /7 120 T-7071.1 FR DTI 5 N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd 4 5.75000 0.378594 4 5.17500 0.330404 5 5.62000 0.694262 0.18930 0.16520 0.31048 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT Means Comparisons N-EtFOSA40mkd PFOS40mkd 0.000000 0.130000 -0.13 0.000000 -0.575 -0.445 -0.86667 -0.73667 N-EtFOSE40mkd 0.575000 0.445000 0.000000 -0.29167 CONT 0.866667 0.736667 0.291667 0.000000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT 0.05 Comparisons for each pair using Student's t L 2.13144 N-EtFOSA40mkd PFOS40mkd N-EtFOS E40mkd -0.83043 -0.65781 -0.25543 -0.65781 -0.74276 -0.34281 -0.25543 -0.34281 -0.83043 0.108594 0.025532 -0.46641 CONT 0.108594 0.025532 -0.46641 -0.67804 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif>LSD CONT N-EtFOSA40mkd -0.06836 PFOS40mkd -0.14047 N-EtFOSE40mkd -0.64336 CONT -0.83632 Positive values show pairs of means that are significantly different. 121 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts} 0.018487 -0.17782 2.382342 14.52632 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 1.603509 15 85.133333 18 86.736842 Mean Square 0.53450 5.67556 4.81871 F Ratio 0.0942 Prob>F 0.9621 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 14.6667 14.5000 14.0000 14.8000 Std Error 0.9726 1.1912 1.1912 1.0654 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 14.6667 1.21106 N-EtFOSA40mkd 4 14.5000 2.38048 N-EtFOSE40mkd 4 14.0000 3.16228 PFOS40mkd 5 14.8000 2.77489 Std Err Mean 0.4944 1.1902 1.5811 1.2410 Dif=Mean[i]-Mean[j] PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd Means Comparisons PFOS40mkd CONT 0.000000 0.133333 -0.13333 0.000000 -0.3 -0.16667 -0.8 -0.66667 N-EtFOSA40mkd 0.300000 0.166667 0.000000 -0.5 N-EtFOSE40mkd 0.800000 0.666667 0.500000 0.000000 Alpha= Abs(Dif>LSD PFOS40mkd CONT N-EtFOSA40mkd N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 PFOS40mkd CONT N-EtFOSA40mkd -3.21150 -2.94144 -3.10631 -2.94144 -2.93168 -3.11105 -3.10631 -3.11105 -3.59056 -2.60631 -2.61105 -3.09056 N-EtFOSE40mkd -2.60631 -2.61105 -3.09056 -3.59056 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT PFOS40mkd -3.65919 CONT -3.61602 N-EtFOSA40mkd -3.87617 N-EtFOSE40mkd -3.37617 Positive values show pairs of means that are significantly different. GLU mg/dL By Dose Group (mg/Kg/day) / > 1 122 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.053125 -0.13625 106.4679 282.0526 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 9539.66 15 170031.28 18 179570.95 Mean Square 3179.9 11335.4 9976.2 F Ratio 0.2805 Prob>F 0.8386 Means for Oneway Anova Level Number CONT 6 N-EtF OSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 261.333 285.000 322.250 272.400 Std Error 43.465 53.234 53.234 47.614 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 261.333 93.863 N-EtFOSA40mkd 4 285.000 137.033 N-EtFOSE40mkd 4 322.250 114.782 PFOS40mkd 5 272.400 86.777 Std Err Mean 38.319 68.516 57.391 38.808 Dif=Mean[i]-Mean[j] N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT Means Comparisons N-EtFOSE40mkd N-EtFOSA40mkd 0.0000 37.2500 -37.2500 0.0000 -49.8500 -12.6000 -60.9167 -23.6667 Alpha= 0.05 Comparisons for each pair using Student's t PFOS40mkd 49.8500 12.6000 0.0000 -11.0667 CONT 60.9167 23.6667 11.0667 0.0000 Abs(Dlf)-LSD N-EtFOSE40mkd 2.13144 N-EtFOSE40mkd N-EtFOSA40mkd -160.464 -123.214 PFOS40mkd -102.379 CONT -85.566 / 70 123 T-7071.1 FR DTI 5 N-EtFOSA40mkd PFOS40mkd CONT -123.214 -102.379 -85.566 -160.464 -139.629 -122.816 -139.629 -143.523 -126.346 -122.816 -126.346 -131.018 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT N-EtFOSE40mkd -119.760 N-EtFOSA40mkd -157.010 PFOS40mkd -158.423 CONT -161.602 Positive values show pairs of means that are significantly different. Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.159836 -0.0082 59.956 153.0526 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 10258.114 15 53920.833 18 64178.947 Mean Square 3419.37 3594.72 3565.50 F Ratio 0.9512 Prob>F 0.4409 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 184.667 130.750 129.250 152.000 Std Error 24.477 29.978 29.978 26.813 Level CONT Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev 6 184.667 80.2438 Std Err Mean 32.759 /// 124 T-7071.1 FR DT15 N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd 4 130.750 11.1168 4 129.250 23.6414 5 152.000 70.1391 5.558 11.821 31.367 Dif=Meanti]-Mean[j] CONT PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd Means Comparisons CONT PFOS40mkd 0.0000 32.6667 -32.6667 0.0000 -53.9167 -21.2500 -55.4167 -22.7500 N-EtFOSA40mkd 53.9167 21.2500 0.0000 -1.5000 N-EtFOSE40mkd 55.4167 22.7500 1.5000 0.0000 Alpha= Abs(Dif)-LSD CONT PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 CONT PFOS40mkd N-EtFOSA40mkd -73.7812 -44.7157 -28.5732 -44.7157 -80.8232 -64.4760 -28.5732 -64.4760 -90.3631 -27.0732 -62.9760 -88.8631 N-EtFOSE40mkd -27.0732 -62.9760 -88.8631 -90.3631 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT CONT -91.0039 PFOS40mkd N-EtFOSA40mkd N-EtFOSE40mkd -62.7790 -47.8288 -46.3288 Positive values show pairs of means that are significantly different. /r z 125 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.083057 -0.10033 21.12468 50.68421 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 606.3219 15 6693.7833 18 7300.1053 Mean Square 202.107 446.252 405.561 F Ratio 0.4529 Prob>F 0.7191 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 46.1667 44.2500 55.5000 57.4000 Std Error 8.624 10.562 10.562 9.447 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 46.1667 10.1472 N-EtFOSA40mkd 4 44.2500 7.7621 N-EtFOSE40mkd 4 55.5000 41.2189 PFOS40mkd 5 57.4000 15.0100 Std Err Mean 4.143 3.881 20.609 6.713 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSE40mkd CONT N-EtFOSA40mkd Means Comparisons PFOS40mkd N-EtFOSE40mkd 0.0000 1.9000 -1.9000 0.0000 -11.2333 -9.3333 -13.1500 -11.2500 CONT 11.2333 9.3333 0.0000 -1.9167 N-EtFOSA40mkd 13.1500 11.2500 1.9167 0.0000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSE40mkd CONT N-EtFOSA40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 PFOS40mkd N-EtFOSE40mkd CONT -28.4770 -28.3044 -16.0313 -28.3044 -31.8382 -19.7308 -16.0313 -19.7308 -25.9958 -17.0544 -20.5882 -27.1475 N-EtFOSA40mkd -17.0544 -20.5882 -27.1475 -31.8382 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT PFOS40mkd -22.3956 N-EtFOSE40mkd -26.5153 CONT -32.0640 N-EtFOSA40mkd -33.9319 Positive values show pairs of means that are significantly different. ALT U/L By Dose Group (mg/Kg/day) 173 126 T-7071.1 FR DTI 5 70 65 60 55 3 50 < 45 40 35 30 N -E tF O S E 4 0 m kd Dose Group (mg/Kg/day) Student's t 0.05 Dunnett's 0.05 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.47363 0.368357 6.390531 51.89474 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 551.2061 15 612.5833 18 1163.7895 Mean Square 183.735 40.839 64.655 F Ratio 4.4990 Prob>F 0.0192 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 47.8333 53.2500 46.5000 60.0000 Std Error 2.6089 3.1953 3.1953 2.8579 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 47.8333 4.1191 N-EtFOSA40mkd 4 53.2500 3.8622 N-EtFOSE40mkd 4 46.5000 11.6762 PFOS40mkd 5 60.0000 4.3012 Std Err Mean 1.6816 1.9311 5.8381 1.9235 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd Means Comparisons PFOS40mkd N-EtFOSA40mkd 0.0000 6.7500 -6.7500 0.0000 -12.1667 -5.4167 -13.5000 -6.7500 CONT 12.1667 5.4167 0.0000 -1.3333 N-EtFOSE40mkd 13.5000 6.7500 1.3333 0.0000 Alpha= Abs(Dif)-LSD PFOS40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 PFOS40mkd N-EtFOSA40mkd -8.61470 -2.38727 CONT 3.91872 N-EtFOSE40mkd 4.36273 i7*/ 127 T-7071.1 FR DTI 5 N-EtFOSA40mkd CONT N-EtFOSE40mkd -2.38727 3.91872 4.36273 -9.63153 -3.37568 -2.88153 -3.37568 -7.86411 -7.45901 -2.88153 -7.45901 -9.63153 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD PFOS40mkd N-EtFOSA40mkd CONT N-EtFOSE40mkd CONT 1.99340 -5.42807 -9.69983 -9.51141 Positive values show pairs of means that are significantly different. Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.10172 -0.07794 110.3027 211.6316 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 20666.12 15 182500.30 18 203166.42 Mean Square 6888.7 12166.7 11287.0 F Ratio 0.5662 Prob>F 0.6457 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 177.500 186.000 237.000 252.800 Std Error 45.031 55.151 55.151 49.329 Level CONT Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev 6 177.500 81.343 Std Err Mean 33.208 )7S 128 T-7071.1 FR DT15 N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd 4 186.000 31.454 4 237.000 185.801 5 252.800 103.541 15.727 92.900 46.305 Dif=Mean[i]-Mean[j] PFOS40mkd N-EtFOSE40mkd N-EtFOSA40mkd CONT Means Comparisons PFOS40mkd N-EtFOSE40mkd 0.0000 15.8000 -15.8000 0.0000 -66.8000 -51.0000 -75.3000 -59.5000 N-EtFOSA40mkd 66.8000 51.0000 0.0000 -8.5000 CONT 75.3000 59.5000 8.5000 0.0000 Alpha= Abs(Dif)-LSD PFOS40mkd N-EtFOSE40mkd N-EtFOSA40mkd CONT 0.05 Comparisons for each pair using Student's t t 2.13144 PFOS40mkd N-EtFOSE40mkd N-EtFOSA40mkd -148.693 -141.912 -90.912 -141.912 -166.243 -115.243 -90.912 -115.243 -166.243 -67.062 -92.259 -143.259 CONT -67.062 -92.259 -143.259 -135.737 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT PFOS40mkd -100.294 N-EtFOSE40mkd -127.684 N-EtFOSA40mkd -178.684 CONT -167.422 Positive values show pairs of means that are significantly / 7 129 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.793274 0.751929 1.727876 138.4211 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 171.84825 15 44.78333 18 216.63158 Mean Square 57.2827 2.9856 12.0351 F Ratio 19.1866 Prob>F <.0001 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 141.833 136.750 140.000 134.400 Std Error 0.70540 0.86394 0.86394 0.77273 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 141.833 1.72240 N-EtFOSA40mkd 4 136.750 1.50000 N-EtFOSE40mkd 4 140.000 1.41421 PFOS40mkd 5 134.400 2.07364 Std Err Mean 0.70317 0.75000 0.70711 0.92736 Dif=Mean[i]-Mean[j] CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd Means Comparisons CONT N-EtFOSE40mkd 0.00000 1.83333 -1.83333 0.00000 -5.08333 -3.25000 -7.43333 -5.60000 N-EtFOSA40mkd 5.08333 3.25000 0.00000 -2.35000 PFOS40mkd 7.43333 5.60000 2.35000 0.00000 Alpha= Abs(Dif)-LSD CONT N-EtFOSE40mkd N-EtF OSA40mkd PFOS40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 CONT N-EtFOSE40mkd IN-EtFOSA40mkd -2.12630 -0.54395 2.70605 -0.54395 -2.60418 0.64582 2.70605 0.64582 -2.60418 5.20325 3.12946 -0.12054 PFOS40mkd 5.20325 3.12946 -0.12054 -2.32925 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method Idi 2.62898 Abs(Df)-LSD CONT N-EtFOSE40mkd N-EtFOSA40mkd PFOS40mkd CONT -2.62265 -1.09887 2.15113 4.68268 Positive values show pairs of means that are significantly different. Cl- mmol/L By Dose Group (mg/Kg/day) S7? 130 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.405319 0.286382 2.382342 101.2105 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 58.02456 15 85.13333 18 143.15789 Mean Square 19.3415 5.6756 7.9532 F Ratio 3.4079 Prob>F 0.0452 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 103.667 99.500 101.000 99.800 Std Error 0.9726 1.1912 1.1912 1.0654 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 103.667 1.75119 N-EtFOSA40mkd 4 99.500 1.73205 N-EtFOSE40mkd 4 101.000 2.94392 PFOS40mkd 5 99.800 2.94958 Std Err Mean 0.7149 0.8660 1.4720 1.3191 Dif=Mean[i]-Mean[j] CONT N-EtFOSE40mkd PFOS40mkd N-EtFOSA40mkd Means Comparisons CONT N-EtFOSE40mkd 0.00000 2.66667 -2.66667 0.00000 -3.86667 -1.20000 -4.16667 -1.50000 PFOS40mkd 3.86667 1.20000 0.00000 -0.30000 N-EtFOSA40mkd 4.16667 1.50000 0.30000 0.00000 Alpha= Abs(Dif)-LSD CONT 0.05 Comparisons for each pair using Student's t t 2.13144 CONT N-EtFOSE40mkd PFOS40mkd -2.93168 -0.61105 0.79189 N-EtFOSA40mkd 0.88895 I7 X 131 T-7071.1 FR DT15 N-EtFOSE40mkd PFOS40mkd N-EtFOSA40mkd -0.61105 0.79189 0.88895 -3.59056 -2.20631 -2.09056 -2.20631 -3.21150 -3.10631 -2.09056 -3.10631 -3.59056 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT CONT -3.61602 N-EtFOSE40mkd -1.37617 PFOS40mkd 0.07415 N-EtFOSA40mkd 0.12383 Positive values show pairs of means that are significantly different. CREA mg/dL By Dose Group (mg/Kg/day) Oneway Anova Summary of Fit RSquare RSquareAdj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.295588 0.154706 0.126359 0.5 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 0.10050000 15 0.23950000 18 0.34000000 Mean Square 0.033500 0.015967 0.018889 F Ratio 2.0981 Prob>F 0.1434 Means for Oneway Anova Level Number Mean CONT 6 0.400000 N-EtFOSA40mkd 4 0.575000 N-EtFOSE40mkd 4 0.500000 PFOS40mkd 5 0.560000 Std Error 0.05159 0.06318 0.06318 0.05651 Level CONT Std Error uses a pooled estimate of error variance Means and Std Deviations Number Mean Std Dev 6 0.400000 0.089443 Std Err Mean 0.03651 179 132 T-7071.1 FR DT15 N-EtFOSA40mkd N-EtFOSE40mkd PFOS40mkd 4 0.575000 0.095743 4 0.500000 0.081650 5 0.560000 0.194936 0.04787 0.04082 0.08718 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT Means Comparisons N-EtFOSA40mkd PFOS40mkd 0.000000 0.015000 -0.015 0.000000 -0.075 -0.06 -0.175 -0.16 N-EtFOSE40mkd 0.075000 0.060000 0.000000 -0.1 CONT 0.175000 0.160000 0.100000 0.000000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd CONT 0.05 Comparisons for each pair using Student's t t 2.13144 N-EtFOSA40mkd PFOS40mkd N-EtFOSE40mkd -0.19044 -0.16567 -0.11544 -0.16567 -0.17034 -0.12067 -0.11544 -0.12067 -0.19044 0.001150 -0.00309 -0.07385 CONT 0.001150 -0.00309 -0.07385 -0.1555 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Df)-LSD CONT N-EtFOSA40mkd -0.03943 PFOS40mkd -0.04115 N-EtFOSE40mkd -0.11443 CONT -0.19179 Positive values show pairs of means that are significantly different. TRIG mg/dL By Dose Group (mg/Kg/day) * VA 133 T-7071.1 FR DTI 5 Oneway Anova Summary of Fit RSquare RSquare Adj Root Mean Square Error Mean of Response Observations (or Sum Wgts) 0.130895 -0.04293 34.82966 123.2105 19 Source Model Error C Total Analysis of Variance DF Sum of Squares 3 2740.575 15 18196.583 18 20937.158 Mean Square 913.52 1213.11 1163.18 F Ratio 0.7530 Prob>F 0.5375 Means for Oneway Anova Level Number CONT 6 N-EtFOSA40mkd 4 N-EtFOSE40mkd 4 PFOS40mkd 5 Mean 128.167 138.500 123.250 105.000 Std Error 14.219 17.415 17.415 15.576 Std Error uses a pooled estimate of error variance Means and Std Deviations Level Number Mean Std Dev CONT 6 128.167 42.6447 N-EtFOSA40mkd 4 138.500 45.3836 N-EtFOSE40mkd 4 123.250 24.0468 PFOS40mkd 5 105.000 17.2482 Std Err Mean 17.410 22.692 12.023 7.714 Dif=Mean[i]-Mean[j] N-EtFOSA40mkd CONT N-EtFOSE40mkd PFOS40mkd Means Comparisons N-EtFOSA40mkd CONT 0.0000 10.3333 -10.3333 0.0000 -15.2500 -4.9167 -33.5000 -23.1667 N-EtFOSE40mkd 15.2500 4.9167 0.0000 -18.2500 PFOS40mkd 33.5000 23.1667 18.2500 0.0000 Alpha= Abs(Dif)-LSD N-EtFOSA40mkd CONT N-EtFOSE40mkd PFOS40mkd 0.05 Comparisons for each pair using Student's t t 2.13144 N-EtFOSA40mkd CONT N-EtFOSE40mkd -52.4938 -37.5867 -37.2438 -37.5867 -42.8610 -43.0034 -37.2438 -43.0034 -52.4938 -16.3000 -21.7863 -31.5500 PFOS40mkd -16.3000 -21.7863 -31.5500 -46.9518 Positive values show pairs of means that are significantly different. Comparisons with a control using Dunnett's Method |d| 2.62898 Abs(Dif)-LSD CONT N-EtFOSA40mkd -48.7727 CONT -52.8660 N-EtFOS E40mkd -54.1893 PFOS40mkd -32.2797 Positive values show pairs of means that are significantly different. Above is for the male and female guinea pigs combined clinical chemistry statistics. tV / 134 T-7071.1 FR DTI 5 Appendix 8. Hepatic Palmitoyi Co-A oxidase activity SPECIES SEX DOSE GRO GP F CONT ID 8G01485 8G01486 9G00051 9G00052 N Mean SD PCOAO U 2.00 1.00 3.00 3.00 4 2.250 0.957 N-EtFOSA4 Omkd 8G01488 8G01490 N Mean SD 3.00 1.00 2 2.000 1.414 N-EtFOSE160mkd 9G00053 9G00054 N Mean SD 3.00 3.00 2 3.000 0.000 N-EtFOSE40mkd 8G01487 8G01489 N Mean SD 2.00 2.00 2 2.000 0.000 PFOS40mkd 8G01491 8G01492 9G00055 9G00056 N Mean SD 2.00 Missing 3.00 6.00 3 3.667 2.082 M CONT 8G01477 8G01478 9G00045 9G00046 N Mean SD 1.00 1.00 2.00 2.00 4 1.500 0.577 N-EtFOSA40mkd 8G01481 8G01482 N Mean SD 1.00 1.00 2 1.000 0.000 N-EtFOSEl60mkd 9G00047 9G00048 N Mean SD 2.00 Missing 1 2.000 Missing N-EtFOSE40mkd 8G01479 1.00 8G01480 1.00 T-7071.1 FR DTI 5 SPECIES SEX DOSE GRO ID N Mean SD PCOAO U 2 1.000 0.000 PFOS40mkd 8G01483 8G01484 9G00049 9G00050 N Mean SD 1.00 2.00 3.00 3.00 4 2.250 0.957 R F CONT 8R04040 8R04041 9R00469 9R00470 N Mean SD Missing 4.00 6.00 7.00 3 5.667 1,528 N-EtFOSA4 Omkd 8R04044 8R04045 N Mean SD Missing Missing 0 Missing Missing N-EtFOSEl60mkd 9R00471 9R00472 N Mean SD 5.00 8.00 2 6.500 2.121 N-EtFOSE4Omkd 8R04042 8R04043 N Mean SD Missing Missing 0 Missing Missing PFOS40mkd 8R04046 8R04047 9R00473 9R00474 N Mean SD Missing Missing 10.00 18.00 2 14.000 5.657 M M556-160mkd SPECIES SEX DOSE GRO CONT 1R00748 1R00749 1R00750 N Mean SD ID 1R00742 1R00743 1R00744 8R04032 8R04033 9R00463 Missing Missing Missing 0 Missing Missing PCOAO U Missing Missing Missing 0.00 4.00 9.00 136 T-7071.1 FR DT15 9R00464 N Mean SD 9.00 4 5.500 4.359 FOSA40mkg 1R00745 1R00746 1R00747 N Mean SD Missing Missing Missing 0 Missing Missing N-EtFOSA4 Omkd 8R04036 8R04037 N Mean SD Missing Missing 0 Missing Missing N-EtFOSEl60mkd 9R00465 9R00466 N Mean SD 19.00 20.00 2 19.500 0.707 N-EtFOSE40mkd 8R04034 8R04035 N Mean SD Missing Missing 0 Missing Missing PFOS40mkd 8R04038 8R04039 9R00467 9R00468 N Mean SD Missing 11.00 21.00 20.00 3 17.333 5.508 137 T-7071.1 FR DTI 5 /Y 138 T-7071.1 FR DT15 Appendix 9. Effect o f acute FC administration on catalase and acylCoA oxidase expression The following report summarizes the effects o f in vivo administration o f N-EtFOSE 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 Society o f Toxicology meeting (Wallace et al. 2001). Tissues analyzed in this study were derived from approximately 1 g of frozen liver from each of two male and two female rats and guinea pigs that received either 40 mg/kg/day PFOS or 160 mg/kg/day 2(N-ethylperfluorooctanesulfonamido)-ethyl alcohol (N-EtFOSE) orally for 4 days, or an equivalent volume o f 2% tween 80 (vehicle control) from March 1, 1999 to March 4, 1999. All animals were killed on March 5, one day after the last consecutive dose. Portions o f the respective livers were quick frozen in liquid nitrogen and shipped on dry ice to Duluth for analysis. Enzyme Sample Preparation - The enzyme fraction consisted o f the 6,000 g supernatant of a 10% (wt/vol) homogenate o f 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 o f Bradford using commercial bovine serum albumin as standard. L-CoA Oxidase Assay - The equivalent o f ca., 5 pg/ml tissue homogenate was suspended in 60 mM KH2PO4-O.O2 % Triton X I00 (pH 7.4) containing 1 mM phydroxyphenylacetate (PHPA), 4 units/ml peroxidase, 20 pM FAD, and 60 pM laurylCoA (LCoA). The reactions were allowed to incubate at 37C for 30 min in a shaking water bath and terminated by adding 3 volumes o f 2 mM KCN in 100 mM sodium carbonate (pH 10.5). The concentration o f H20 2 generated during the reaction was estimated from the fluorescence of PHAP as measured with an excitation wavelength of 317 nm and emission at 405 nrn. The fluorescence was calibrated with commercial H20 2 and the results are expressed as nmol peroxide generated/min/mg mitochondrial protein (Table 1). Protein was quantitated by the Bradford method. Catalase Assay - The activity o f catalase was estimated by a modification of the original method published by Claiborne and Fridovich (J. Biol. Chem. 254, 4245-52, 1979), which is based on the direct measurement o f H20 2 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 to 27C and the reaction initiated by adding 10.3 mM H20 2. The progress o f the reaction was monitored at 240 nm for 5 min. Catalase activity was estimated from the initial linear rate ( E240=43.6 mM' 'em '1) and expressed as units/mg protein (Table 1). One unit o f activity is defined as that amount o f enzyme which catalyzes the decomposition o f 1 pmole o f H20 2per min T-7071.1 FR DTI 5 Northern Blot Analyses - Quantitation o f mRNA for both acylCoA oxidase (ACoAO) and catalase were performed by Northern blot analysis o f quick frozen liver samples from treated rats and guinea pigs. Approximately 1 g o f frozen 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 to a cellulose membrane and hybridized to the corresponding randomly [32P] labeled oligonucleotides that were PCR amplified from primers to ca., 350 base sequence o f 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 male rats do not express the message for PCoAO. Acute exposure to N-Et-FOSE causes a doubling o f the specific activity o f 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 o f 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 number o f animals tested (n=2). Exposure of rats to PFOS elicits a similar doubling o f LCoAO 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 o f mRNA band intensity for pCoAO, acute exposure to PFOS caused a 3-6 fold increase in PCoAO mRNA expression. This enhanced expression o f message was more pronounced in female compared to male rats, and in both sexes resulted in no greater LCoAO enzyme activity than was observed at half the mRNA level for rats exposed to N-Et-FOSE. Guinea Pigs - The response to guinea pigs to exposure to N-Et-FOSE and PFOS was dramatically different from that observed for rats. The constitutive activity o f LCoAO in unexposed guinea pigs o f either sex was very low (near LOD values) whereas catalase activity was 1.5-3 times higher. However, the activity o f neither enzyme was stimulated following acute exposure to N-Et-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 l? 7 140 T-7071.1 FR DT15 the sequence for rat liver ACoAO because the sequence o f the guinea pig enzyme is not known at this time. It may be that the guinea pig sequence is sufficiently different that the PCR product lacks complementarity for adequate hybridization to the probe for the rat sequence. Although this may indeed be an artifact, the absence o f constitutively expressed message and the inability to stimulate expression o f PCoAO mRNA are consistent with the low and uninducible enzyme activity in guinea pigs. CONCLUSIONS: These data provide strong evidence that: 1) N-Et-FOSE and PFOS stimulate both the transcriptional and translational expression o f acylCoA oxidase in rats in vivo, and 2) there is a marked difference in the response o f rats and guinea pigs to 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 this effect of fluorochemical exposures. We were not given the data for organ weights or necropsy findings and thus cannot comment on whether exposure to either agent caused hepatomegaly or other indications that are associated with peroxisome proliferation. However, based on our data, we suspect that the evidence will reveal the classical signs o f peroxisome proliferation in rats, but not guinea pigs, caused by these acute exposures. /?? 141 T-7071.1 FR DT15 Summary o f Screen for Hepatic Peroxisome Proliferation Animal Specie Se Treatment Body Liver Liver P 450 LaurylCoA Catalase ug pCoAO Catalase sX wt wt Protein content Oxidase activity RNA/u mRNA mRNA (g) (g) (mg/g) (nmol/mg) Activity 1 (% (% (nmol/min/mg) (Units/mg Control Control protein) )) 9R00463 Rat M Veh 9R00464 Rat M Veh 9R00469 Rat F Veh 9R00470 Rat F Veh 298 12 313 13.1 210 7.6 201 7.1 151.8 145.6 188.3 153.3 0.086 0.067 0.154 0.055 4 219.9 2.9 nd 126.7 5.3 158.5 2.65 nd 73.3 9.2 141.1 2.19 100 91.5 10.2 204.7 3.38 100 108.5 9R00465 Rat M N-EtFOSE 9R00466 Rat M N-EtFOSE 9R00471 Rat F N-EtFOSE 9R00472 Rat F N-EtFOSE 272 13 157.5 231 11.1 134.9 174 7.5 165.8 175 7.3 215.8 0.331 0.284 0.04 0.188 12 219.5 3.5 290 140.1 11.7 204.3 2.6 292 97.8 17.8 186.1 1.93 253 117.8 14.2 171.9 1.74 320 147.3 9R00467 Rat M PFOS 9R00468 Rat M PFOS 9R00473 Rat F PFOS 9R00474 Rat F PFOS 257 11.9 115.4 229 12.5 153 175 8.3 143.1 153 7.3 238.6 0.423 0.241 0.077 0.099 13.9 252 3.9 482 155.1 12 236.9 2.08 277 115.7 13.7 204.1 1.98 630 180.4 19.9 155.6 2 404 147.3 9G00045 9G00046 9G00051 9G00052 Gpig Gpig Gpig Gpig M Veh M Veh F Veh F Veh 296 11.3 175.2 298 11.9 252.7 274 11.9 180.3 289 14.2 222.1 0.123 0.089 0.188 0.17 9G00047 9G00048 9G00053 9G00054 9G00049 9G00050 9G00055 9G00056 Gpig Gpig Gpig Gpig M N-EtFOSE M N-EtFOSE F N-EtFOSE F N-EtFOSE 240 8.7 212.7226 8.5 269.1 230 9.6 176.4 236 8.9 218.3 Gpig M PFOS 215 7.2 Gpig M PFOS 240 10 Gpig F PFOS 221 8.4 Gpig F PFOS 237 7.4 (-) insufficient sample for analysis (nd) not detectable 153.2 222.1 127.8 171.6 - 0.013 0.126 0.195 . 0.074 0.031 0.016 1.1 314.7 1.19 nd 1.3 390.7 1.63 nd 1.2 550.6 1.65 nd 0.9 488 2.3 nd 349.3 - nd 0.7 379.3 1.71 nd 0.7 416.8 1.77 nd 1.5 478.1 1.28 nd 103 97 91 109 - 92 105 104 413- nd 0.9 460.8 1.27 nd 0.9 672.9 1.65 nd 1.4 531.6 1.52 nd 97 103 96 /& ? 142 T-7071.1 FR DT15 Appendix 10: Final Report for FOSA (T-7132.1). Title: Comparative Molecular Biology o f Perfluorooctanesulfonamide LFOSA, T- 7132) in Rats following four consecutive days o f dosing. Final Report February 18, 2004 Study Number: DT 15 Protocol Amendment Number*: 2. 3M M edical Department Study Number: T-7132.1 Study Director. Andrew Seacat Ph.D. Analytical laboratories: 1. Kendall B.Wallace, Ph.D. D.A.B.T. Professor, Dept of Biochemistry and Molecular Biology School of Medicine 10 University Drive Duluth, MN 55812-2496 2. Dr. Xin Lu Department o f Pharmacology and Physiology 601 Elmwood Ave, box 711 Rochester, New York 14642 Study Protocol Title: Comparative Molecular Biology o f Peroxisome Proliferation in Rats and Guinea Pigs Study Initiated: In-Life Start Date: November 16,1998 In-Life End Date: December 21, 1998 143 T-7071.1 FR DT15 Purpose: The purpose o f this study was to treat three male rats treated with either no compound (vehicle control) or40m g/kg/day perfluorooctanesulfonamide: C8F17S02NH2 (FOSA, PFOSA, FOSAmide, T-7132). The study was designed to evaluate the metabolism and certain toxicological effects, such as hepatic peroxisome proliferation, of this compound and to compare these effects to other compounds dosed at the same concentration.. Methods Test Materials: Perfluorooctanesulfonamide (FOSA, PFOSA, FOSAmide, C8F17S02NH2 NB # 120067-108, G. Moore 10/28/00, T-7132) was investigated. The vehicle control was propylene glycol. Dose Groups: Three male rats received propylene glycol as the vehicle control by oral gavage at a volume of 5 ml/kg body weight. Three male rats received a dose of 40 mg FOSA/Kg body weight, via oral gavage on days one through four o f the study, and were euthanized on day five. A suspension o f 8 mg/ml of FOSA in propylene glycol was prepared, and a volume o f 5 ml/kg was administered. This dosing regiment achieved a cumulative dose o f 160 mg/kg after four successive days of dosing. This dose was the same as the dose of PFOS administered under amendment 1 o f this protocol and is comparable to effective dose levels for hepatic peroxisome proliferation in rats o f PFOS found in the literature. The LD50 for FOSA is not known, but the cumulative dose o f FOSA administered under this protocol is below the LD50 for PFOS 251 mg/kg for PFOS in com oil, as a point o f reference. Method o fSpecimen Collection: The liver was removed as rapidly as possible after euthanasia. The livers were divided into ~ 1 g pieces and flash-frozen directly in liquid nitrogen in tared polypropylene (Nalgene) containers. The containers were moved to dry ice and weighed after all liquid nitrogen had evaporated. The tissue was stored at -70 C and shipped on dry ice. Sera: Up to 10 ml of blood was collected from each animal into glass serum tubes. Following clotting, the blood was centrifuged for 10 minutes at 1100 x g at 4 C. the sera was transferred to new tubes and be centrifuged again for 10 minutes at 1100 x g at 4 C. 144 T-7071.1 FR DT15 Two aliquots o f the serum sample (~ 0.75 ml) were saved for possible metabolite analysis. Specimen Handling: A 1-2 gram aliquot o f the liver samples that were flash frozen in liquid nitrogen were shipped in dry ice to the analytical laboratories listed according to the livers sample identification chart below to: Kendall B.Wallace, Ph.D. D.A.B.T. Professor, Dept of Biochemistry and Molecular Biology School of Medicine University of Minnesota 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 o f Ken Wallace Dept o f Biochemistry and Molecular Biology at the University o f MN. Another 1-2 gram aliquot o f the liver sample was sent to: Dr. Michael Wempe Laboratory o f M. W. Anders Department of Pharmacology and Physiology 601 Elmwood Ave, box 711 Rochester, New York 14642 Dr. Lin Xu performed the analysis o f these samples and provided a brief summary of the analysis of liver samples from rats given a range o f fluorocarbons. The parent and metabolites of the fluorocarbons were determined in liver samples by LC-MS/MS using previously published methods (Hansen et ah 2001). Liver Sample Identification Chartfo r T-7132.1 Sample # Anim al # Species Sex Dose Group 1 2 3 4 5 11111RRRRR0000000000777774444446352 RRRRRaaaaattttt MMMMM44cccoo0o0nnnmmtttrrrooogglll//kkgg//ddaayyFFOOSSAA 145 T-7071.1 FR DTI 5 6 1R00747 Rat M40mg/kg/dayFOSA 193 146 T-7071.1 FR DT15 Results and Discussion Average body weights o f the FOSA treatment group were significantly lower than the control group on days four and five (Table 1). Liver weights were not significantly different between the treated and control groups, but the liver weight as a percentage of body weight was significantly increased in the FOSA treatment group The results o f P450 content (Figure 1) and Acyl CoA oxidase activity (Figure 2) for the liver samples indicated that FOSA induced the expression o f these proteins and FOSA is therefore a hepatic peroxisome proliferator in rats. The concentrations o f the parent compound and metabolites in livers were measured (Table 2). The data show that PFOS was the major metabolite found in the livers o f rats given FOSA and FOSA V-glucuronide was identified as a minor metabolite. Approximately 0.3 percent o f the cumulative dose was present in the liver as either the parent compound, FOSA, or as the metabolite PFOS, one day after the last dose (Table 3). The low percentage of FOSA in the liver and the apparent low conversion o f FOSA to PFOS has also been noted in in-vitro microsomal and liver slice metabolism studies (Xu et al. 2003) which observed that FOSA was converted to PFOS in-vitro by liver slices at a low rate, but not by microsomes or cytosol. It is noteworthy that the control animals (1R00742,1R00743, and 1R00744) contained significant concentrations o f FOSA. Furthermore, no PFOS or FOSA JV-glucuronide were found in the control samples with a high background o f FOSA. A parallel analysis o f livers from Fischer 344 rats maintained in the University of Rochester Vivarium did not show detectable concentrations o f FOSA. These results suggest that the control liver samples for this study were contaminated at some point time ex-vivo by trace quantities o f FOSA, as no metabolism to PFOS or FOSA A-glucuronide had occurred in die control samples. 147 T-7071.1 FR DT15 Signatures: Report prepared by, Andrew M. Seacat, PhD, DABT Study Director Reviewed by, Daniel C. Hakes Sponsor representative Date Date fS 148 T-7071.1 FR DTI 5 References: Bradford, M. M. (1976). A rapid and sensitive method for the quantitation o f microgram quantities of protein utilizing the principle o f protein-dye binding. A nal Biochem 72,24854. Hansen, K. J., Clemen, L. A., Ellefson, M. E., and Johnson, H. O. (2001). Compoundspecific, quantitative characterization o f organic fluorochemicals in biological matrices. Environm ental Science a n d Technology 35,766-770. Lazarow, P. B. (1981). Assay o f peroxisomal beta-oxidation o f fatty acids. M ethods E nzym ol 72, 315-9. Poosch, M. S., and Yamazaki, R. K. (1986). Determination o f peroxisomal fatty acylCoA oxidase activity using a lauroyl-CoA-based fluorometric assay. Biochim Biophys A cta 884, 585-93. Wallace, K. B., Luebker, D. J., Butenhoff, J. L., and Seacat, A. M. (2001). Perfluorooctane sulfonate and 2-(N-ethylperfluorooctanesulfonamido)-ethyl alcohol are peroxisome proliferators in rats, but not guinea pigs. Toxicologist 6 0 ,348 Abstract ID: 1657. Xu, L., Seacat, A. M., Butenhoff, J. L., and Anders, M. W. (2003). Biotransformation o f N-Ethyl-N-(2-hydroxyethyl)perfluorooctanesulfonamide (N-EtFOSE) by rat liver microsomes, cytosol and slices. In Toxicological Sciences, Suppl., Vol. 72, p. 314. /96> 149 T-7071.1FR DTI 5 Tables fT#DCDSoaoaTTumbm11rl55pcpe,olaBeAn1roaAmsBdtenieyvciinomeuWdltMaoimevlgioeg#eilchndeaDcttalu#y(oBs2lPsaWearorfB)Ga,dimroLoolisoeuvigtepneyrgrW.sofei2dDBPaig/aWae1)hy_rt9eft/l1u0(L1oWro2dB(9)o/aW,1)cyT9t/h2a0ny1reos12dBiud9/aW2)lyf_W1o/3n0ea1igmh2dB(i9t/daW2)(ye.2T/4.(W0F1O)Sd2B(9/aAW2)y3,/5T0-1712L3/W223)(/g0in1)RWLaWt(s/%Bfo)loT(w9W)ing 231423111RRR000000777444cccooonnntttrrrooolll 250 259 260 264 272 10.73 3.9%10.62 269 278 285 292 295 10.82 3.7%10.87 256 260 267 268 276 10.70 3.9%10.65 Avg 258 266 271 275 281 10.75 3.8% 10.71 SD 10 11 13 15 12 0.06 0.14% 0.14 456657111RRR000000777444444FFF000OOOSSSmmmAAAggg///kkkggg///dddaaayyy 250 254 250 239 227 10.77 4.7%10.78 249 245 243 233 225 10.81 4.8%10.66 259 265 263 255 254 10.64 4.2%10.84 Avg 253 255 252 242 235 10.74 4.6% 10.76 SD 6 10 10 11 16 0.09 0.34% 0.09 P-value T-Test* 0.21 0.13 0.06 0.02 0.01 0.44 0.01 0.32 *T-Test (impaired, one tailed, equal variance). A P-value o f < 0.05 was considered significantly different from control. /?7 150 T-7071.1 FR DTI 5 Table 2 Liver Fluorocarbon Concentration Hepatic Concentrations o f Fluorocarbons and Fluorocarbon Metabolites in Livers o f Rats Given FOSA (40 Sample # Animal # 1 1R00742 2 1R00743 3 1R00744 4 1R00745 5 1R00746 6 1R00747 mg/kg/day) Weight o f liver sample Sex Treatment (g) M Control 0.4600 M Control 0.9236 M Control 1.1287 M FOSA 0.2402 M FOSA 0.7905 M FOSA 0.8305 of 0.0 0.0 0.0 163.2 214.3 202.8 FOSA (Ppm) FOSAA (ppm) FOSA Nglucuronide (ppm) 154.1 0.0 0.00 113.2 0.0 0.00 273.2 0.0 0.00 195.7 n.m. 0.44 174.1 n.m. 0.39 163.9 n.m. 0.34 Note: n.m. = not measured. /9S 151 T-7071.1 FR DT15 Table 3 Percent Fluorocarbon Dose in Liver Hepatic Percent o f Dosed Fluorocarbons and Fluorocarbon Metabolites in Livers o f Rats Given FOSA (40 mg/kg/day) for Four Days #SPaemrcpenletDAonsimeainl#LivDGerorosuepTD(mootgsa)el idPnoFsOeS** LP(piFvpOemrS) LP(miFvgOe)rSa(%in%sld)iPvoFesrOeSLF(PiOvPeSmrA) L(FmiOvgeS)rAi(%an%sld)iFvoOesrSeA 231111RRR000000777444243ccIcIIooonnntttrrrooo 0.00 0.00 0.00 Avg SD 0 0 0 154.1 113.2 273.2 180.17 83.12 456111RRR000000777444675FFFOOOSSSAAA666614044...238 666614044...238 221016243...832 221...137626 000...332575 111769435...197 211...179 000...223893 Avg 193.43 2.08 0.32 177.90 1.91 0.30 SD 26.81 0.29 0.05 16.24 0.18 0.02 152 T-7071.1 FR DTI 5 Figures Figure 1 Cytochrome P450 content in Liver T o tal C Y TP 450 C o n ten t in R at L iver F o llo w in g T re atm en t W ith 0.2000 FOSA 0.1500 E in Qu 0.1000 o 0 O c c 0.0500 Average of n=3; Error bars represent the standard error of the mean T Control ^ '--i V s-.' M 'ii u_ > K I8 I B 1 , FOSA 30 153 T-7071.1 FR DTI 5 Figure 2: Lauroyl CoA oxidase activity in liver LCoA-Oxidase Activity in Rat Liver Homogenate Following Treatment With FOSA 4! 154 Appendix 1 Cytochrome P450 content Absorbance Values Sam Treat p ie# meni 450 500 Contro 1 I -0.0366 -0.0312 Contro 0.0254 0.0220 2 f.*- 2 9 Contro 3 l -0.0144 -0.0172 4 FOSA -0.0404 -0.0456 0.1360 0.1008 5 FOSA 1 3 0.0059 6 FOSA 4 -0.013 protein cone. (uq/ul) 8.20 8.79 9.25 9.79 10.48 9.86 mg protein added n m o le s uM volu total CYTP CYTP me nmol 450/m 450 - 500 450 (mi) e 9 5 0.0054 0.059 3 0.178 0.0356 5 0.00333 0.037 3 0.110 0.0220 5 0.0028 0.031 3 0.092 0.0185 5 0.0052 0.057 3 0.171 0.0343 5 0.03518 0.387 3 1.160 0.2320 5 0.01894 0.208 3 0.624 0.1249 n=1 n=2 n=3 Averag e Stdev Sterror Varria nce ftest Ttest (unpaired, one tailed, unequal variance) P value Control 0.0356 0.0220 0.0185 0.0253 0.0091 0.0052 0.0001 0.50 Statisti cs FOSA 0.0343 0.2320 0.1249 0.1304 0.0989 0.0571 0.0098 0.02 0.10 P ro te in D e te rm in a tio n Standard Curve ul BSA 5 20 40 60 80 ul Lysis Buffer 95 80 60 40 20 Absorbance 0.4645 0.6167 0.7992 0.9413 1.0379 O 20 40 60 80 100 sa m p le Treatment 1A Control 1B Control 2A Control 2B Control 3A Control 3B Control 4A FOSA 4B FOSA ul 5 5 5 5 5 5 5 5 Absorbanc ug e protein mg/ml average slope intercep t 0.7799 42.064935 8.412987 8.2 0.0077 0.456 0.7635 39.935065 7.987013 0.0077 0.456 0.8004 44.727273 8.945455 8.790909 0.0077 0.456 0.7885 43.181818 8.636364 0.0077 0.456 0.8155 46.688312 9.337662 9.248052 0.0077 0.456 0.8086 45.792208 9.158442 0.0077 0.456 0.844 50.38961 10.07792 9.788312 0.0077 0.456 0.8217 47.493506 9.498701 0.0077 0.456 sample mg/m ul : neede 5A F O S A 5B F O S A 6A F O S A 6B F O S A d to g e t5 mg 5 0.8493 51.077922 10.21558 10.47792 0.0077 0.456 1 8.20 609.8 5 0.8695 53.701299 10.74026 0.0077 0.456 2 8.79 568.8 5 0.8305 48.636364 9.727273 9.855844 0.0077 0.456 3 9.25 540.7 5 0.8404 49.922078 9.984416 0.0077 0.456 4 9.79 510.8 5 10.48 477.2 6 9.86 507.3 Appendix 2: Lauroyl CoA Oxidase Activity Sample # average stdev %dev average stdev %dev 1 206,3 207.5 209.6 207.8 1.7 0.8% 1 123 120.3 120.2 121.2 1.6 1.3% 2 205.4 205 206.9 205.8 1.0 0.5% 2 122.9 121.7 121.5 122.0 0.8 0.6% 3 209.8 216.1 210.3 212.1 3.5 1.7% 3 126.3 125.9 124.4 125.5 1.0 0.8% 4 216.8 218.5 216.5 217.3 1.1 0.5% 4 124.3 121.9 121.7 122.6 1.4 1.2% 5 227.4 229.4 228.9 228.6 1.0 0.5% 5 124.3 123 123.3 123.5 0.7 0.6% 6 218.6 218.7 219.5 218.9 0.5 0.2% 6 131.1 129.3 125.4 128.6 2.9 2.3% nmoles H 2 0 2 produced/ 30 min: S am ple Control Difference 1 4.02 0.03 3.99 2 3.93 0.07 3.86 3 4.22 0.23 3.99 4 4.46 0.10 4.36 5 4.98 0.14 4.84 6 4.54 0.37 4.16 slope 21.692 21.692 21.692 21.692 21.692 21.692 nmoles H 2 0 2 produced/min/m g protein: nmoles H 2 0 2 tim e (min) ug protein Control FOSA 1 2 3 4 5 6 3.99 3.86 3.99 4.36 4.84 4.16 ' . 30 30 30 30 30 30 20 20 20 20 20 20 6.66 6.43 6.65 7.27 8.07 6.94 Control average (n=3) 6.6 FOSA 7.4 n=1 n=2 n=3 Average Stdev Sterror Control FOSA (nmoles H 2 0 2 produced/min/mg protein) 6.66 7.27 . 6.43 8.07 6.65 6.58 013 007 6.94 7.43 0.58 0.34 WC * H2O2 Standard Curve Data. HzCh Stock was 8.6 M nmnoles H 202 average SD % dev 0 109.7 112 113.8 111.8 2.1 1.8% 1.72 151.5 153.2 154.9 153.2 1.7 1.1% 3.44 202.4 206.3 199.3 202.7 3.5 1.7% 6.88 280.7 288.6 282.2 283.8 4.2 1.5% 13.76 412.9 410.7 408.3 410.6 2.3 0.6% H202 Standard Curve nm oles H202 Protein Standard Curve ul BSA ul Lysis Buffer 5 95 20 80 40 60 60 40 80 20 Absorbanc e 0.4464 0.6769 0.854 1.0135 1.037 sa m p le ul Absorbanc ug ug/ul average slope intercep e protein t 1 5 0.7814 34.435644 6.887129 7.054455 0.0101 0.4336 1 5 0.7983 36.108911 7.221782 0.0101 0.4336 sample ug/ul ul needed protein to get 20 ug 2 5 0.7956 35.841584 7.168317 7.617822 0.0101 0.4336 1 7.05 2.84 25 0.841 40.336634 8.067327 0.0101 0.4336 2 7.62 2.63 3 5 0.7963 35.910891 7.182178 7.369307 0.0101 0.4336 3 7.37 2.71 3 5 0.8152 37.782178 7.556436 0.0101 0.4336 4 7.88 2.54 4 5 0.8225 38.50495 7.70099 7.882178 0.0101 0.4336 5 8.38 2.39 4 5 0.8408 40.316832 8.063366 0.0101 0.4336 6 8.61 2.32 5 5 0.8592 42.138614 8.427723 8.379208 0.0101 0.4336 5 5 0.8543 41.653465 8.330693 0.0101 0.4336 6 5 0.8501 41.237624 8.247525 8.610891 0.0101 0.4336 6 5 0.8868 44.871287 8.974257 0.0101 0.4336 Appendix 11: Final Report for FOSAA (T-7071.1) Final Report Title: Comparative Molecular Biology o f Peroxisome Proliferation in Rats following four consecutive days o f Perfluorooctanesulfonamidoacetate: (FOSAA, T-7071) Study Number: D T15 Protocol Amendment Number*: 2. 3M M edical Department Study Number: T-7071.1 Study Director: Andrew M, Seacat Ph.D., DABT Analytical laboratories: 1. Kendall B.Wallace, Ph.D. D.A.B.T. Professor, Dept of Biochemistry and Molecular Biology School o f Medicine 10 University Drive Duluth, MN 55812-2496 2. Dr. Xin Lu Department o f Pharmacology and Physiology 601 Elmwood Ave, box 711 Rochester, New York 14642 Study Protocol Title: Comparative Molecular Biology o f Peroxisome Proliferation in Rats and Guinea Pigs Study Initiated: In-Life Start Date: November 16,1998 In-Life End Date: December 21, 1998 2/ Purpose The purpose of this study was to treat three male rats treated with either no compound (vehicle control) or 160 mg/'kg/day perfluorooctanesulfonamidoacetate: C8F17S02NHCH2COO- (FOSAA, M556, T-7071). The study was designed to evaluate the metabolism and certain toxicological effects, such as hepatic peroxisome proliferation, o f this compound and to compare these effects to other compounds dosed at the same concentration.. Test Materials Perfluorooctanesulfonamidoacetate: (FOSAA, M556, C8F17S02NHCH2C00- NB# 12837-84/30, from G. Moore 12/2000. 3M Medical department # T-7071) was investigated. The vehicle control was propylene glycol. Dose Groups Three male rats received propylene glycol as the vehicle control by oral gavage at a volume of 5 ml/kg body weight. Three male rats received a dose o f 160 mg FOSAA/Kg/day, via oral gavage on days one through four o f the study, and were euthanized on day five. A suspension o f 32 mg/ml M556 in propylene glycol was prepared, and a volume of 5 ml/kg was administered. This dosing regiment achieved a cumulative dose of 640 mg/kg M556 after four successive days o f dosing. This dose was comparable to the cumulative dose o f NEtFOSE which induced peroxisomal palmitoyl Co-A oxidase (PCOAO) activity in the rat in a 4 week feeding study with 300 ppm N-EtFOSE. Method of Specimen Collection The liver was removed as rapidly as possible after euthanasia. The livers were divided into ~ 1 g pieces and flash-frozen directly in liquid nitrogen in tared polypropylene (Nalgene) containers. The containers were moved to dry ice and weighed after all liquid nitrogen had evaporated. The tissue was stored at -70 C and shipped on dry ice. Sera: Up to 10 ml o f blood was collected from each animal into glass serum tubes. Following clotting, the blood was centrifuged for 10 minutes at 1100 x g at 4 C. the sera was transferred to new tubes and be centrifuged again for 10 minutes at 1100 x g at 4 C. Two aliquots of the serum sample (~ 0.75 ml) were saved for possible metabolite analysis. Specimen Handling A 1-2 gram aliquot o f the liver samples that were flash frozen in liquid nitrogen were shipped in dry ice to the analytical laboratories listed according to the livers sample identification chart below to: Kendall B.Wallace, Ph.D. D.A.B.T. Professor, Dept of Biochemistry and Molecular Biology School of Medicine University of Minnesota 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 of Biochemistry and Molecular Biology at the University of MN. Another 1-2 gram aliquot o f the liver sample was sent to: Dr. Michael Wempe Laboratory o f M. W. Anders Department of Pharmacology and Physiology 601 Elmwood Ave, box 711 Rochester, New York 14642 Dr. Lin Xu performed the analysis o f these samples and provided a brief summary o f the analysis o f liver samples from rats given a range o f fluorocarbons. The parent and metabolites of the fluorocarbons were determined in liver samples by LC-MS/MS using previously published methods (Hansen et al. 2001). Results and Discussion Average body weights o f the FOSAA treatment group were significantly lower than the control group on day five (Table 1). Liver weights were not significantly different between the treated and control groups, but the liver weight as a percentage of body weight was significantly increased in the FOSAA treatment group The results o f P450 content (Figure 1) and Acyl CoA oxidase activity (Figure 2) for the liver samples indicated that FOSAA induced the expression o f these genes and is therefore a good peroxisome proliferator in rats. The concentrations o f parent compound and metabolites in livers were measured (Table 1). The concentration o f the parent compound FOSAA in the liver was approximately 555 ppm, which is equivalent to about 0.24 % o f the cumulative dose o f FOSAA (Table 3). The data show that PFOS and FOSA (apparent) were the major metabolites found in the livers o f rats given FOSAA. The amount o f PFOS and FOSA in the liver represented approximately 0.07 and 0.14 percent o f the cumulative dose, respectively. If one subtracts the background levels o f FOSA in the control lives from the FOSA levels found in the FOSAA treatment group, the concentrations are decreased by approximately 50%. Therefore a quantitative interpretation o f the amount o f metabolism o f the parent compound to FOSA cannot be provided however the data qualitatively suggest that such metabolism occurred. It is noteworthy that the control animals (1R00742,1R00743, and 1R00744) contained significant concentrations o f FOSA. No PFOS was found in the control samples with a high background o f FOSA. These results suggest that the control liver samples were contaminated at some point time ex-vivo by trace quantities o f FOSA, as no metabolism to PFOS had occurred in the control samples. A parallel analysis o f livers from Fischer 344 rats maintained in the University o f Rochester Vivarium did not show detectable concentrations o f FOSA Z7& Signatures: Report prepared by, Andrew M. Seacat, PhD, DABT Study Director Reviewed by, Daniel C. Hakes Sponsor representative Date Date Tables Table 1 Biological Parameters D#SaTm15plBeoAdyniWmeailg#htD(BosWe),GLroivueprWeighDBd2ta/Wa(1yLt9eW/10)1Bd2a/W1y92/01Bd2a/W2y13/01dB2a/W2y24/01dB2a/W2y35/01L2/W23(/g0)1L(%W)/BW (g) (g) (g) (g) (g) 231243111RRR000000777444cccooonnntttrrrooolll Avg SD 250 259 260 264 272 10.73 3.9% 269 278 285 292 295 10.82 3.7% 256 260 267 268 276 10.70 3.9% 258 266 271 275 281 10.75 3.8% 10 11 13 15 12 0.06 0.14% 879908111RRR000000777454MMM111666555000555666mmmggg///kkkggg///dddaaayyy Avg ST-DTest* 254 263 264 258 250 10.74 4.3% 254 262 265 263 262 10.77 4.1% 267 270 267 247 227 10.79 4.8% 258 8 0.50 265 4 0.46 265 2 0.26 256 8 0.07 246 18 0.02 10.77 0.03 0.35 4.4% 0.33% 0.03 c*To-nTteroslt).(unpaired,onetailed,equalvariance,P<0.05consideredsignificantlydifferentfrom 'i . ^ v w i i ^ v / U i / U i t 111 -Iv i V C l Hepatic Percent o f Dosed Fluorocarbons and Fluorocarbon Metabolites in Livers o f Rats Given FOSAA (160 mg/kg/day) for Four Days #SampleA#nimal DGorosuepTe(Dmootgsa)lPid*(nmoFsgOe)S*LP(piFvpOemrS)PL(miFvgOe)rSa%dPil(ni%vsoFes)OreSLF(PiOvPeSmrA)(LFmiOvgeS)rAa%diFl(ni%vsoOes)SreA(FpOpSmA)A(LFmiOvgeS)rAAai(%Fn%sOld)iSvoAesrAe 231243111RRR000000777444cccooonnntttrrrooolll 0.00 0.00 0.00 0 0 0 154.1 113.2 273.2 0 0 0 SDAvg 180.2 83.1 978890111RRR000000777454FFFOOOSSSAAAAAA222444536395 22221201812921830180...947 2.56 0.88 1.09 0.12447.3 0.04235.7 0.05267.4 4.8 0.19 2.5 0.10 2.9 0.12 614.4 424.0 627.1 6.6 0.27 4.6 0.19 6.8 0.28 SDAvg 2452 2201 140.3 1.51 0.07 316.8 13 12 85.4 0.92 0.04 114.1 3.4 0.14 555.2 1.2 0.05 113.8 6.0 0.24 1.2 0.05 *(4*9M9o/5le5If3r)a=ct2io2n12PFmOgSP,FeO.gS.foinrdMo5s5e6.,Dose(mg)M556*(499MWofPFOS/MWM556)=2464.5* Figures Figure 1 Cytochrome P450 content in Liver Total C Y TP 450 C o n ten t in R at Liver F ollo w ing T re a tm e n t W ith FOSAA nmoles CYTP450/mg 0.2000 ! 0.1500 0.1000 0.0500 0.0000 Average of n=3; Error bars represent the standard error of the mean C on trol FOSAA Figure 2: Lauroyl CoA oxidase activity in liver LCoA-Oxidase Activity in Rat Liver Homogenate Following Treatment With FOSAA LCoA-Oxidase Activity (nmoles H202/min/mg) 14.00 -, 12.00 10.00 8.0 0 6.0 0 4.00 2.0 0 0.0 0 - Average of n=3; Error bars represent the Standard error of the mean. Control FOSAA T-7071.1 FR DTI 5 oca T-7071.1 FR DT15 A p p e n d ix 1 C y to c h ro m e P 4 5 0 co n ten t V a lu e s 450 1 -0.0366 2 0.02542 3 -0.0144 7 -0.0355 8 -0.0231 9 -0.0586 A bsorbance 500 -0.0312 0.02209 -0.0172 -0.0298 ^ ?': '? '^ :. i0 .0 2 2 ; ; .: v ':;/- ' -0.0262 protein cone, (ug/ul) 1 8.20 2 8.79 3 9.25 7 10.96 8 10.59 9 9.58 mg protein added 5 5 5 5 5 5 450 - 500 0.0054 0.00333 0.0028 0.0057 0.0011 0.0324 uM CYTP450 0.059 volum e (ml) 3 total n m o le 0.178 0.037 0.031 0.063 0.012 0.356 3 0.110 3 0.092 3 0.188 3 0.036 3 1.068 n m o le s C Y T P 450/m g 0.0356 0.0220 0.0185 0.0376 * 0.0073 0.2136 average (n=3) Contra 1 0.0253 FOSAA 0.0862 n=1 n=2 n=3 Average Control 0.0356 0.0220 0.0185 0.0253 Statistics FOSAA 0.0376 0.0073 0.2136 0.0862 173 / C T-7071.1 FR DTI 5 Stdev 0.0091 Sterror 0.0052 V ariance 0.0001 ftest 1 ite s t (unpaired, one tailed, uneqlual variance) p value 0.50 0.1114 0.0643 0.0124 0.01 0.22 tvd ? . ----- -- --- Standard Curve ul BSA 5 20 40 60 80 ul Lysis Buffer 95 80 60 40 20 Absorbance 0.4645 0.6167 0.7992 0.9413 1.0379 sample ul Absorbanc e ug protein mg/ml average slope intercep t 1A 5 0.7799 42.064935 8.412987 8.2 0.0077 0.456 1B 5 0.7635 39.935065 7.987013 0.0077 0.456 2A 5 0.8004 44.727273 8.945455 8.790909 0.0077 0.456 2B 5 0.7885 43.181818 8.636364 0.0077 0.456 3A 5 0.8155 46.688312 9.337662 9.248052 0.0077 0.456 3B 5 0.8086 45.792208 9.158442 0.0077 0.456 7A 5 0.8816 55.272727 11.05455 10.95584 0.0077 0.456 7B 5 0.874 54.285714 10.85714 0.0077 8A 5 8B 5 9A 5 0.8679 0.8597 0.8187 53.493506 10.6987 10.59221 0.0077 52.428571 10.48571 0.0077 47.103896 9.420779 9.581818 0.0077 0.456 0.456 0.456 0.456 sample mg/m 1 1 8.20 2 8.79 3 9.25 9B 5 0.8311 48.714286 9.742857 0.0077 0.456 87 10.96 10,59 9 9.58 ul needed to get 5 mg 609.8 568.8 540.7 456.4 472.0 521.8 average stdev %dev average stdev %dev 12 206.3 205.4 207.5 205 209.6 206.9 207.8 205.8 1.7 1.0 0.8% 0.5% 12 123 122.9 120.3 121.7 120.2 121.5 121.2 122.0 1.6 0.8 1.3% 0.6% 3 209.8 216.1 210.3 212.1 3.5 1.7% 3 126.3 125.9 124.4 125.5 1.0 0.8% 7 309.6 308.3 308.5 308.8 0.7 0.2% 7 136 131 128.2 131.7 4.0 3.0% 8 249.1 252.1 248.4 249.9 2.0 0.8% 8 130.5 133.2 128.1 130.6 2.6 2.0% 9 283.9 283.4 280.2 282.5 2.0 0.7% 9 128.7 131.9 125.6 128.7 3.2 2.4% nmoles H 2 0 2 p ro du ced/30 min: Sample Control Differenc e 1 4.02 0.03 3.99 2 3.93 0.07 3.86 3 4.22 0.23 3.99 7 8.68 0.52 8.16 8 5.96 0,47 5.50 9 7.47 0.38 7.09 slope 21.692 21.692 Intercep t 120.5 120.5 21.692 21.692 21.692 21.692 120.5 120.5 120.5 120.5 nmoles H 2 0 2 produced/min/mg protein: Control 1 2 n m o le s H202 3.99 tim e (min) ug protein 30 20 3 86 , .30 . 20 3 . 3.99 .30 . 20 FOSAA 7 8,16 30 20 8 5.50 30 2 0 .. 9 7.09 30 20 average (n=3) - 6.66 Control - 6.43 66 6.65 13.60 9.16 FOSAA 11.5 11.81 free n=1 n=2 n=3 Average Control FOSAA (nmoles H 2 0 2 produced/m n/mg protein) 6.66 13.60 643 9,16 6.65 11.81 6.58 11:53 22S h 1I H2O2 Standard Curve Data. H2 2 Stock was 8.6 M nmnoles H 202 average SD % dev 0 109.7 112 113.8 111.8 2.1 1.8% 1.72 151.5 153.2 154.9 153.2 1.7 1.1% 3.44 202.4 206.3 199.3 202.7 3.5 1.7% 6.88 280.7 288.6 282.2 283.8 4.2 1.5% 13.76 412.9 410.7 408.3 410.6 2.3 0.6% H202 Standard Curve Protein Standard Curve u lB S A ul Lysis Buffer 5 95 20 80 40 60 60 40 80 20 Absorbanc e 0.4464 0.6769 0.854 1.0135 1.037 sam p le ul Absorbanc ug ug/ul average slope intercep e protein t 1 5 0.7814 34.435644 6.887129 7.054455 0.0101 0.4336 1 5 0.7983 36.108911 7.221782 0.0101 0.4336 sample ug/ul ul needed protein to get 20 ug 2 5 0.7956 35.841584 7.168317 7.617822 0.0101 0.4336 1 7.05 2.84 25 0.841 40.336634 8.067327 0.0101 0.4336 2 7.62 2.63 3 5 0.7963 35.910891 7.182178 7.369307 0.0101 0.4336 3 7.37 2.71 3 5 0.8152 37.782178 7.556436 0.0101 0.4336 7 8.75 2.29 7 5 0.8708 43.287129 8.657426 8.748515 0.0101 0.4336 8 7.88 2.54 75 0.88 44.19802 8.839604 0.0101 0.4336 9 8 54 2.34 8 5 0.8226 38.514851 7.70297 7.878218 0.0101 0.4336 8 5 0.8403 40.267327 8.053465 0.0101 0.4336 9 5 0.8562 41.841584 8.368317 8.542574 0.0101 0.4336 9 5 0.8738 43.584158 8.716832 0.0101 0.4336 rtr C2 2 ? --~r~- v iiv ia u v ii iu u iv c i r r v / o A. Correlation of effects by different treatment groups to liver PFOS in rats. Correlation of effects to liver PFOS (ug/g) in Rats ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=M556-160mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=FOSA40mkg ------ Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE160mkd - .... Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) % BW dO Mean 140.3333 95.53118 Std Dev 85.44685 9.405515 Correlation 0.158821 Variable PFOS (ROC) (ug/g) % BW dO Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 00 0 106.2098 2.660886 Bivariate Dose Group (mg/Kg/da=FOSA40mkg Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 193.4333 26.80678 0.253937 % BW dO 93.07698 4.329203 Bivariate Dose Group (mg/Kg/da=N-EtFOSE160mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 1008.075 166.3236 0.662735 % BW dO 87.44432 4.602872 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 918.15 113.933 0.3006 % BW dO 85.28738 3.742806 Conretlation of BW to liver PFOS concentrations. Signif. Prob 0.8985 Number 3 Signif. Prob 1.0000 Number 6 Signif. Prob 0.8365 Number 3 Signif. Prob 0.3373 Number 4 Signif. Prob 0.6994 Number 4 - / v~<3' a/ ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=M556-160mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=FOSA40mkg ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE160mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Bivariate Dose Group (mg/Kg/da=M556-160mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 140.3333 85.44685 -0.12908 LW/BW ratio 0.043867 0.003308 Variable PFOS (ROC) (ug/g) LW/BW ratio Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 00 0 0.03818 0.002552 Bivariate Dose Group (mg/Kg/da=FOSA40mkg Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 193.4333 26.80678 -0.21723 LW/BW ratio 0.045793 0.003394 Bivariate Dose Group (mg/Kg/da=N-EtFOSE160mkd Variable Mepn Std Dev Correlation PFOS (ROC) (ug/g) 1008.075 166.3236 0.751977 LW/BW ratio 0.045166 0.003235 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 918.15 113.933 0.609934 LW/BW ratio 0.049007 0.003768 Signif. Prob 0.9176 Number 3 Signif. Prob 1.0000 Number 6 Signif. Prob 0.8606 Number 3 Signif. Prob 0.2480 Number 4 Signif. Prob 0.3901 Number 4 rigs, males and female combined. Correlation of effects to liver PFOS (ug/g) in Guinea Pigs % BW dO By PFOS (ROC) (ug/g) -z?e ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSA40mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE160mkd ------Bivariate Normal Ellipse P=0.5Q0 Dose Group (mg/Kg/da=N-EtFOSE40mkd ------Bivariate Normal Ellipse P=0,500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) % BW dO Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 0.014286 0.037796 0.592756 107.5171 5.69689 Bivariate Dose Group (mg/Kg/da=N-EtFOSA40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 100.4 22.36172 -0.20567 % BW dO 94.66277 0.909529 Bivariate Dose Group (mg/Kg/da=N-EtFOSE160mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 419.35 214.6788 -0.92559 % BW dO 91.83908 2.210568 Bivariate Dose Group (mg/Kg/da=N-EtFOSE40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 66.55 60.59266 0.63895 % BW dO 99.38514 0.513448 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 171.4857 116.5397 -0.30285 % BW dO 92.95562 5.748654 Signif. Prob 0.1607 Number 7 Signif, Prob 0.7943 Number 4 Signif. Prob 0.0744 Number 4 Signif, Prob 0.3611 Number 4 Signif. Prob 0.5091 Number 7 LW/BWratio By PFOS (ROC) (ug/g) ------ Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSA40mkd ------ Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE160mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE40mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) LW/BW ratio Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 0.014286 0.037796 -0.27546 0.041022 0.004556 Bivariate Dose Group (mg/Kg/da=N-EtFOSA40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 100.4 22.36172 0.032225 LW/BW ratio 0.034365 0.003385 Bivariate Dose Group (mg/Kg/da=N-EtFOSE160mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 419.35 214.6788 0.343072 LW/BW ratio 0.038328 0.00237 Bivariate Dose Group (mg/Kg/da=N-EtFOSE40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 66.55 60.59266 0.214886 LW/BW ratio 0.03445 0.002243 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 171.4857 116.5397 -0.21072 LW/BW ratio 0.036088 0.004271 Signif. Prob 0.5499 Number 7 Signif. Prob 0.9678 Number 4 Signif. Prob 0.6569 Number 4 Signif. Prob 0.7851 Number 4 Signif. Prob 0.6502 Number 7 K+ mmol/L By PFOS (ROC) (ug/g) /> < ? ------Bivariate Normal Ellipse P=0,500 Dose Group (mg/Kg/da=CONT ------Bivariate Normal Ellipse P=Q.500 Dose Group (mg/Kg/da=N-EtFOSA40mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE40mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) K+ mmol/L Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 0.025 0.05 0.918156 12.8 1.01653 Bivariate Dose Group (mg/Kg/da=N-EtFOSA40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 100.4 22.36172 -0.24593 K+ mmol/L 6.65 0.695222 Bivariate Dose Group (mg/Kg/da=N-EtFOSE40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 66.55 60.59266 -0.62394 K+ mmol/L 12.2 0.962635 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 148.2 106.5116 -0.25046 K+ mmol/L 4.825 1.746186 Signif. Prob 0.0818 Number 4 Signif. Prob 0.7541 Number 4 Signif. Prob 0.3761 Number 4 Signif. Prob 0.7495 Number 4 CHOL mg/dL By PFOS (ROC) (ug/g) ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=CONT ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSA40mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE40mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) CHOL mg/dL Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 0.016667 0.040825 -0.43116 40.08333 19.97853 Bivariate Dose Group (mg/Kg/da=N-EtFOSA40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 100.4 22.36172 -0.30166 CHOL mg/dL 71.75 30.43436 Bivariate Dose Group (mg/Kg/da=N-EtFQSE40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 66.55 60.59266 0.111675 CHOL mg/dL 34.375 23.75 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 148.2 106.5116 0.24662 CHOL mg/dL 50.875 21.58848 Signif. Prob 0.3933 Number 6 Signif. Prob 0.6983 Number 4 Signif. Prob 0.8883 Number 4 Signif. Prob 0.7534 Number 4 TRIG mg/dL By PFOS (ROC) (ug/g) ------Bivariate Normal Ellipse P=0.50Q Dose Group (mg/Kg/da=CONT ------Bivariate Normal Ellipse P=0.5Q0 Dose Group (mg/Kg/da=N-EtFOSA40mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=N-EtFOSE40mkd ------Bivariate Normal Ellipse P=0.500 Dose Group (mg/Kg/da=PFOS40mkd Variable PFOS (ROC) (ug/g) TRIG mg/dL Bivariate Dose Group (mg/Kg/da=CONT Mean Std Dev Correlation 0.016667 0.040825 -0.50738 128.1667 42.64466 Bivariate Dose Group (mg/Kg/da=N-EtFOSA40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 100.4 22.36172 0.198189 TRIG mg/dL 138.5 45.38355 Bivariate Dose Group (mg/Kg/da=N-EtFOSE40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 66.55 60.59266 -0.19932 TRIG mg/dL 123.25 24.04683 Bivariate Dose Group (mg/Kg/da=PFOS40mkd Variable Mean Std Dev Correlation PFOS (ROC) (ug/g) 148.2 106.5116 0.595121 TRIG mg/dL 106 19.74842 Signif. Prob 0.3042 Number 6 Signif. Prob 0.8018 Number 4 Signif. Prob 0.8007 Number 4 Signif. Prob 0.4049 Number 4