Document RjzgMgzz92KbO5Bkbxbp069Ra

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 perfhiorooctanesulfonamido ethanol (N-EtFOSE, T-6316), N-Ethyl perfluorooctanesulfonamide (N-EtFOSA, T-6868), Perfluorooctanesulfonamido acetate (FOSAA, T-7071), and/or Perfluorooctanesulfonamide (FOSA, T-7I32) 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 Location(s): 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 000014 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Table of Contents S u m m a r y ................................................................................................................................................................. 3 Methods................................................................................................................................................................... 7 DT15-B Protocol Procedures............................................................................................................................ 7 Amendment Number 1 Procedures...................................................................................................................7 Amendment Number 2 Procedures...................................................................................................................8 Analytical methods............................................................................................................................................. 9 R esults................................................................................................................................................................... 11 Biological Parameters....................................................................................................................................... 11 Liver Fluorochemical Concentrations............................................................................................................ 12 Clinical Chemistry.............................................................................................................................................13 Palmitoyl CoA oxidase (PCoAO) activity...................................................................................................... 13 C o n c lu s io n s ...........................................................................................................................................................16 Signature Page.............................................................................. 18 Summary Tables..................................................................................................................................................19 Table 1. Average Cumulative Dose.................................................................................................................19 Table 2. Summary of 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 of 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 Data.......................................................... 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 of 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 of 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 of effects by different treatment groups to liver PFOS in Guinea Pigs, males and female combined.......................................................................................................................................................... 185 O 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 of 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 of 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 of > 600 pg/g. 40 mkd FOSA and 160 mkd FOSAA significantly reduced body weight to 93% and 95 % of initial body weight, respectively, at average liver PFOS concentrations of 140 pg/g and 200 pg/g, respectively. Neither 40 mkd NEtFOSE of 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 of 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 of rats to 160 mkd N-Et-FOSE caused a doubling of the specific activity of lauryl CoA oxidase (LCoAO) and a 2-fold increase in the concentration of mRNA encoding for PCoAO in rat liver, but catalase activity and catalase mRNA were unchanged. Exposure of rats to 40 mkd PFOS caused a 2-fold increase in LCoAO activity and a 3 to 6-fold increase in PCoAO mRNA expression in rats. 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 of greater than 600 pg/Kg. PCoAO activity was not 3 CCOOIG 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 of acyl CoA oxidase activity occurred at relatively lower average liver PFOS concentrations of 193 p.g/g and 140 pg/g , respectively, with an equivalent or greater fraction of the total liver fluorochemical was contributed by the parent compound or the metabolite FOSA from the FOSAA treated rats. These data suggest that FOSA may be an equally potent peroxisome proliferator to PFOS, but not the N-acetyl metabolites of N-Et FOSE. In the guinea pig, potassium (K+) values were significantly reduced by 40 mkd PFOS and N-EtFOSA. Neither 40 mkd PFOS or N-EtFOSA, nor 40 mkd or 160 mkd N-EtFOSE caused peroxisome proliferation in either gender of guinea pigs. These results showed that PFOS, N-EtFOSE, FOSA and FOSAA all caused indications of peroxisome proliferation in rats, but not in guinea pigs. These results are in concordance with effects of classical peroxisome proliferators, in that the response is specific to certain species. c00017 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Introduction: The objective of this study was to investigate and compare the molecular mechanisms of peroxisome proliferation in rats and guinea pigs. Three compounds derived from perfluorooctane sulfonate and known to cause peroxisome proliferation in the rat were initially tested, 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 of evidence indicating that guinea pigs and primates are resistant to peroxisome proliferation. However, the molecular and biochemical mechanisms that differentiate the response of 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 of genes that are associated with peroxisome proliferation. 2. To measuring the hepatic activity of peroxisomal enzyme systems and fatty acid binding proteins. 3. To perform standard toxicity tests of serum clinical chemistry and to examine the liver for histological changes which may indicate an explanation for the species differences seen in response to these compounds. 4. To correlate any observed alterations of the above functions to liver and serum levels of perfluorosulfonamides and their metabolites. This study was part of a series of investigations designed to understand the molecular and biochemical mechanisms for the effects of these compounds observed invivo. This study was carried-out in collaboration with other investigators. Dr. Ken Wallace, University of Minnesota Duluth, who has been engaged in studies designed to understand the effects of these perfluorosulfonamides on mitochondrial bioenergetics, performed molecular and biochemical analyses of the induction of genes associated with peroxisomal proliferation and/or cell replication. Dr. Kris Hansen and Lisa A. Stevenson of 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 of 11/16/98 and an in-life end date of 11/20/98. Amendment number 1 had an in-life start date of 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 c00018 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 of 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 of each of 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: C8FI7S 0 3- K+, MW = 538.1 g/mole). 3. T-6316: N-ethylperfluorooctane sulfonamido ethanol, (narrow Range NEthyl Perfluorooctanesulfonamido ethyl alcohol), N-EtFOSE, EtFOSE, FC-10, Formula: C8F17S 0 2N(C2H5)CH2CH20H , 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 2NHC2H5, MW = 527.2). 5. T-7071: Perfluorooctanesulfonamido acetate, FOSAA, M556, (Formula C8F17S 0 2NHCH2COO`, MW - 556 g/mole) 6. T- 7132: Perfluorooctanesulfonamide , FOSA, PFOSA (abbreviation used by the 3M Environmental Lab), Formula C8F17SO2NH2, 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. C60 0 0 1 9 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 of 11/16/98 and an in-life end date of 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 of 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 of each test compound was prepared in 2% Tween 80 in a glass tissue grinder. The animals received four consecutive daily doses. The day of the first dose was designated day zero, thus the doses were administered on days 0,1,2 and 3 of 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 of amendment 1 was to add groups of 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 of 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 of 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 of the mouth to the guinea pigs on days 0 through day 3 of the study. This dose was comparable to the cumulative dose of 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 of the LD50. 7 C0Q020 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 of 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 of the study. The cumulative dose of PFOS delivered under protocol amendment #1 was --160 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 of 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 of these compounds in rats at equivalent doses. Amendment number 2 had an in-life start date of 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 of initiation were obtained from Fiarlan 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 of 5 ml/kg body weight on days zero through day 3 of the study. A larger volume of 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 of 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 of the study. A suspension of 8 mg/ml of FOSA in propylene glycol was prepared, and a volume of 5 ml/kg was administered by oral gavage to the rats. This dose achieved a cumulative dose of 160 mg/kg after four days of dosing. The dose of FOSA was the same as the dose of PFOS administered under amendment #1 of this protocol. The LD50 for FOSA is not known, but the cumulative dose of FOSA administered under this protocol was below the LD50 for PFOS of 251 mg/kg for PFOS in corn oil, as a point of 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 of the liver samples shipped in dry ice to the analytical laboratories listed in the protocol and amendments. The identification of each liver sample sent to each lab is listed in Appendix 2. cfcozi 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 of Ken Wallace Dept of Biochemistry and Molecular Biology at the University of MN by the following methods: Enzyme Sample Preparation - The enzyme fraction consisted of the 6,000 g supernatant of a 10% (wt/vol) homogenate of 0.5-1.0 g frozen liver tissue in 300 mM mannitol-10 mM HEPES-1 mM EGTA (pH 7.2). Protein concentration was estimated according to the method of Bradford using commercial bovine serum albumin as standard. L-CoA Oxidase Assay - The equivalent of ca., 5 pg/ml tissue homogenate was suspended in 60 mM KH2PO4-O.O2 % Triton X I00 (pH 7.4) containing 1 mMphydroxyphenylacetate (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 of 2 mM KCN in 100 mM sodium carbonate (pH 10.5). The concentration of H2O2 generated during the reaction was estimated from the fluorescence of PHAP as measured with an excitation wavelength of 317 nm and emission at 405 nm. The fluorescence was calibrated with commercial H2O2 and the results are expressed as nmol peroxide generated/min/mg mitochondrial protein. Protein was quantitated by the Bradford method. Catalase Assay - The activity of catalase was estimated by a modification of the original method published by Claiborne and Fridovich (J. Biol. Chem. 254, 424552, 1979), which is based on the direct measurement of 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 of the reaction was monitored at 240 nm for 5 min. Catalase activity was estimated from the initial linear rate ( E240=43.6 mM^cm'1) and expressed as units/mg protein (Table 1). One unit of activity is defined as that amount of enzyme which catalyzes the decomposition of 1 pmole of H2O2 per min. Northern Blot Analyses - Quantitation of mRNA for both acylCoA oxidase (ACoAO) and catalase were performed by Northern blot analysis of quick frozen liver samples from treated rats and guinea pigs. Approximately 1 g of 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 of the respective rat liver gene. mRNA 9 C00922 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 of the methods used and the results of 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 -107, 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 of FACT TOX 107). (Formula C8F,7S02N(C2H5)CH2C 0 0 ', N-EtFOSAA). narrow Range N-Ethyl Perfluorooctanesulfonamido ethyl alcohol (Formula C8FnS 0 2N(C2H5)CH2CH20H , N-EtFOSE) Perfluorooctanesulfonamidoacetate (Formula C8F,7S 0 2NHCH2COO' FOSAA) Perfuorooctane sulfonyl ethylamide (Formula C8Fi7S 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 of M. W. Anders Department of Pharmacology and Physiology, University of Rochester using previously published methods (Hansen et al. 2001). The results are reported in this report and integrated with the body weight and liver weight data. Certain other liver samples were sent to Covance in Madison WI and were analyzed for palmitoyl Co-A Oxidase activity as an indicator of peroxisome proliferation using a validated method based on published methods (Lazarow 1981). The principle of the assay is that in the presence of palmitoyl-Co-A, the third step of 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 C00023 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 of 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 of 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 of 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 C0Q024 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 of 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 of PFOS and FOSA fall within the experimental error inherent with the methods used. The percent of 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 of the concentrations of all fluorochemical species detected in the liver and designated TLPFOSX, and for the amount of PFOS itself in the liver. The amount (mg) of 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 of Rochester lab and the 3M Environmental lab (Appendix 6, B and D). In rats dosed with PFOS, the percent of PFOS dosed present in the liver as PFOS ranged from about 17 to 23 percent in both male and female rats, and was consistent between labs. In guinea pigs dosed with PFOS, the percentage of the total amount of 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 of Rochester so a comparison between labs for the fluorochemical content in the livers of 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 of the dose that was present in the liver as PFOSX was approximately 2-fold the percent of 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% of the TLPFOSX as PFOS in their livers. Conversely, the percentage of the dose that was present as PFOS in the liver of rats treated with 160 mkd FOSAA had high levels of FOSAA itself and FOSA, with low levels of PFOS. 12 C00025 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 (JC+) 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 of 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 of guinea pigs with 40 mkd PFOS. . 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 of four days of oral dosing of 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 of Toxicology meeting (Wallace et al. 2001). Acute exposure of rats to 160 mkd N-Et-FOSE caused a doubling of the specific activity of LCoAO and a 2-fold increase in the concentration of mRNA encoding for 13 c 00926 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 of 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 of 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 of rats with 160 mkd FOSAA caused significant increases in hepatic cytochrome P450 content and Acyl CoA oxidase activity (Appendix 11). The induced the expression of these of these proteins in liver of 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 of 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 of 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 of 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 of 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 of 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 of the control samples submitted for analyses at a given time, but not in other groups of control samples submitted at different times. The formation of PFOSA from PFOS treated animals was determined in all of the livers of all PFOS treated animals that were analyzed at the University of Rochester, and at the 3M environmental lab for the same samples, which 14 C00927 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1,1-7132.1 DT15-B had no background PFOS of PFOSA in the concurrent control from either lab. Clarification of the formation of the FOSA following treatment with PFOS should be obtained in further studies. Whatever its route of formation, FOSA was metabolized to FOSA A-glucuronide. JV-EtFOSE alcohol gives rise to a range of major and minor metabolites. FOSE alcohol could arise from the A-deethylation of JV-EtFOSE alcohol, and A-EtFOSAA could arise by the oxidation of the alcohol to the carboxylic acid. Glucuronidation of the parent JVEtFOSE alcohol would give the observed JV-EtFOSE alcohol glucuronides. FOSAA could be formed by the A-deethylation of A-EtFOSAA or by the oxidation of FOSE alcohol, or both. FOSA could be formed by the A-deethylation of A-EtFOSA or by the removal of the carboxymethyl group of 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 of the glycine moiety would give PFOS directly. The correlation of a few of 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 of > 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 % of initial body weight, respectively, at average liver PFOS concentrations of approximately 140 to 200 pg/g. A greater fraction of the total fluorochemical in the liver of 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 of 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 of the effect of 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 of > 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 Dunnetf s t-test Correlation of the clinical chemistry endpoints of potassium, cholesterol and triglycerides to liver PFOS concentrations in rats are not shown due to the limited number of 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 of 419 and 171 pg/g, respectively. A greater fraction of the total fluorochemical in the liver of the N-EtFOSE treated guinea pigs was present as the metabolites FOSAA and N-EtFOSAA than occurred in rats. Thus, these 15 G00028 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 of 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 of 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 of the N-EtFOSA treated guinea pigs. However, the 40 mkd N-EtFOSE treatment group guinea pig livers had a large fraction of 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 of 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 of the decreased body weight effect in guinea pigs for each of these dose groups. Given the lack of the liver responses in guinea pigs of either hepatomegaly or induction of peroxisome proliferating enzymes coupled with the fact that an Intraperitoneal injection of 100 mg/Kg PFOS caused death in guinea pigs bu not in rats (See DTI5 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 of the effect of 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 of 148 pg/g and lOOpg/g, respectively. In contrast, treatment of guinea pigs with 40 mkd N-EtFOSE had no effect on the serum potassium levels at average liver PFOS concentrations of approximately 45 pg/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 of 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 of acylCoA oxidase in rats in vivo, and 2) there is a marked difference in the '00929 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B response of 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. 17 C00030 SKP'l 1-6295,8, 1-6316.4, 4-6868.2, 1-7071.1,1-7132,1 DT15-B Signature Page Prepared by: .iJ viA lA J Andrew M. Seacat, Ph.D., DABT, Study Director ___ s /is - /o y Date 00931 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 = C u m u l a t i v e Dose (mg)______________________________________ DOSE GRO CONT N-EtFOSA40mkd N-EtFOSE160mkd N-EtFOSE40mkd PFOS4 Omkd M556-160mkd CONT FOSA40mkg N-EtF0SA4 Omkd N-EtFOSE16Omkd N-EtF0SE4 Omkd 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 c 00032 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B SPECIES Table 2. Summary of Body weights and BW changes SEX FM DOSE GRO BWDO BWD4(g) %BW DO IBWDO GP CONT (g) (g) N4 444 Mean 451 474 107 469 SD 234 223 6 246 N- N2 222 EtFOSA4 Omkd Mean 617 587 95 770 SD 18 25 1 1 N- N2 22 2 EtFOSEl60mkd Mean 253 233 92 255 SD 6 449 N- N2 222 EtF0SE4 Omkd Mean 635 629 99 736 SD 0 2 0 30 PF0S4 Omkd 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 SD Missing Missing N- N2 2 EtFOSA40mkd Mean 214 208 SD 8 4 N- N2 2 EtFOSEl60mkd Mean 201 175+ SD 5 1 N- N2 2 EtF0SE4 Omkd Mean 228 229 SD 6 3 PFOS40mkd N4 4 Mean 205 1 7 7 1" SD 14 19 1 Significantly different from control values by Dunnett's t-test Missing 2 97 2 2 87 3 2 101 4 4 8 61" 4 6 2 303 4 2 286 8 2 312 1 4 290 12 BWD4(g) %BW DO 44 503 110 240 6 22 726 94 41 22 233 92 10 1 22 734 31 4 429 235 3 246+ 18 7 297 18 3 2 3 5 1" 16 2 100 0 4 91 8 3 g 1" 9 7 107 2 3 931" 4 2 310 102 11 22 252t 29 2 316 1 4 251f 15 88 + 8 2 101 0 4 8 7t 3 20 (00033 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 %BW DO | CONT N8 Mean 108 SD 6 N-EtFOSA40mkd N 4 Mean 95f SD 1 N-EtFOSE160mkd N 4 Mean 92+ SD 2 N-EtFOSE4 Omkd N 4 Mean 99+ SD 1 PFOS4 Omkd N8 Mean gs1. SD 5 CONT N-EtFOSE40mkd N-EtFOSA4 Omkd M556-160mkd FOSA4 Omkg N-EtFOSE16Omkd N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean 11 106 3 4 101 2 4 100 3 3 961" 9 3 931" 4 4 PFOS4 Omkd SD N Mean SD 5 8 8 61" 3 Significantly different from control values by Dunnett's t-test 22 C0 Q9 3 4 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-EtFOSA4 Omkd N 4 4 Mean 0.034 0.008t SD 0.003 0.000 N-EtFOSEl60mkd N 4 0 Mean 0.038 Missing SD 0.002 Missing N-EtFOSE4 Omkd N 4 4 Mean 0.034 0 . 0081" SD 0.002 0.000 PFOS40mkd N8 4 Mean 0.036 0.008t 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-EtFOSA4 Omkd 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 o . o s o 1" 0.009 SD 0.004 0.000 ^Significantly different from control values by Dunnett's t-test 2C 0003 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 itg/g)__________________________________________________________________________ SPEC SEX DOSE PFOS FOSA FOSAA ETFOSAA FOSE EtFOSE NETFOSE IES _GRP __ ROC __ ROC __ R _ROC ALC _ROC glue ROC GP F CONT N 0 000 00 0 Mean NA NA NA NA NA NA NA SD NA NA NA NA NA NA NA N- N 2 200 00 0 EtFOSA 4Omkd Mean 104.6 0.6 NA NA NA NA NA SD 30.1 0.8 NA NA NA NA NA N- N 2 222 22 2 EtFOSE 160mkd Mean 350.2 16.2 217.9 388.2 28.7 1.9 6.1 SD 339.9 7.0 103.0 98.1 0.8 0.7 3.5 N- N 2 222 22 2 EtFOSE 4Omkd Mean 44.7 7.1 30.4 105.0 3.2 0.9 1.2 SD 45.3 3.9 6.2 27.5 1.4 0.1 0.2 PFOS N 3 300 00 0 4Omkd Mean 140.8 24.3 NA NA NA NA NA SD 110.5 37.5 NA NA NA NA NA M CONT N 1 000 00 0 Mean 0.1 NA NA NA NA NA NA SD NA NA NA NA NA NA NA N- N 2 200 00 0 EtFOSA 4Omkd Mean 96.3 0.5 NA NA NA NA NA SD 23.0 0.2 NA NA NA NA NA N- N 2 222 22 2 EtFOSE 160mkd Mean 488.6 8.2 129.4 261.6 16.0 2.3 2.0 SD 59.8 4.6 56.4 113.8 8.3 0.2 1.1 N- N 2 222 22 2 EtFOSE 4Omkd Mean 88.4 4.9 28.2 54.0 3.5 1.3 0.4 SD 84.0 0.3 8.6 1.6 1.6 0.5 0.1 PFOS N 4 400 00 0 4Omkd Mean 194.5 41.7 NA NA NA NA NA SD 131.8 33.1 NA NA NA NA NA R F CONT N 1 200 00 0 Mean 0.0 4 .4 NA NA NA NA NA SD NA 0.4 NA NA NA NA NA N- N 2 222 22 2 EtFOSE 160mkd Mean 891.9 18.1 67.5 201.9 47.2 6.4 0.2 SD 37.6 1.1 5.5 33.4 24.6 1.5 0.1 PFOS N 2 200 00 0 FOSA glue 0 NA NA 2 0.2 0.0 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 0.2 0.0 0 24 C00036 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 40mkd M556 Mean SD N 866.4 143.0 3 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 ^JA NA NA 22 12.1 2.5 0 3.8 1.2 0 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 C00037 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 4Omkd 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 4Omkd Mean 800.000 0.1 0.3 NA 0.1 NA SD 16.971 0.0 0.0 NA 0.0 NA 26 cnooas 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 GP R DOSE GRO CONT N-EtF0SA4 Omkd N-EtFOSEl60mkd N-EtF0SE4 Omkd PF0S4 Omkd 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 00 o PCOAO U 8 2 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 e1" 5.1 ^Significantly different from control values by Dunnett's t-test g 27 c 00039 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 of 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 of the study. Animals were not weighed in most cases on day one of the study, and in some cases on day three of the study. In those instances, the previous days' body weights were used for determination of dosing volume. Protocol amendment #2 stated the Dr. M. Wempe at the University of 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 of Rochester performed these metabolite analyses instead. Kidney weight was not obtained during necropsy under amendment number 2 on 3/5/99. Clinical chemistry was not performed on all serum samples. Histological evaluation of liver, kidney and testis were not performed. Serum perfluorosulfonamides and metabolite values were not obtained. Induction of 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). 90 C09040 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 N-EtFOSA N Mean SD N 4 19.6 7.7 2 4 0.043 0.005 2 2 4.3 0.1 2 2 0.006 0.001 2 44 2 19.7 0.039 4.6 10.1 0.003 0.9 22 2 40mkd N-EtFOSE 160mkd N-EtFOSE 40mkd Mean SD N Mean SD N 18.8 1.4 2 9.3 0.5 2 0.032 0.001 2 0.040 0.003 2 4.5 0.008 26.7 0.037 5.5 0.5 0.001 2.6 0.003 0.1 0 '0 22 0 Missing Missing 2 Missing Missing 2 8.6 0.1 2 0.037 0.001 2 Missing Missing 2 PFOS 40mkd M556I60mkd CONT Mean SD N 20.8 1.5 4 0.033 0.002 4 5.2 0.0 2 Mean SD N 13.8 6.9 Missing 0.035 0.003 Missing 4.1 0.1 Missing Mean SD N Mean SD Missing Missing 4 8.1 0.9 Missing Missing 4 0.038 0.003 Missing Missing 2 1.9 0.1 0.008 0.000 2 26.4 0.036 5.5 1.8 0.001 0.1 44 2 0.007 16.4 0.038 5.0 0.000 9.5 0.004 0.1 Missing 3 3 0 Missing 10.8 0.044 Missing Missing 0.0 0.003 Missing 2 77 2 0.009 12.1 0.041 2.9 0.000 1.4 0.003 0.0 FOSA N Missing Missing Missing Missing 3 3 0 40mkg N-EtFOSA 40mkd Mean SD N Mean Missing Missing 2 8.0 Missing Missing 2 0.038 Missing Missing 2 1.8 Missing 10.7 0.046 Missing Missing 0.1 0.003 Missing 2 22 2 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 40mkd 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 Mean 9.9 0.043 2.0 0.009 is .o1 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 0.052f 2.2 SD 0.7 0.001 0.1 0.000 1.2 0.004 0.0 Significantly different from control values by Dunnetf s t-test o o0o0 --4* 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 o .o o s 1 0.000 2 0.008+ 0.000 21 C00041 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. Study / Tube Animal se Record number # X dosed 11/16/98 11/19/98. 40 mkd PFOS, N-Et FOSE, FX-12. Dosed 19 3/1/99- 3/4/99 40 20 mkd PFOS; 160 mkd N-Et 21 22 23 FOSE; 24 vehicle control 25 tween 80 1 8R04032 M 2 8R04033 M 3 8R04041 F 4 8G0147 M 7 5 8G0147 M 8 6 8G0148 F 5 7 8G0148 F 6 8 8G0148 M 3 9 8G0148 M 4 10 8G 0149 F 1 11 8G 0147 M 9 12 8G 0148 M 0 13 8G 0148 F 7 14 8G 0148 F 9 15 8G0148 M 1 16 8G0148 M 2 17 8G0148 F 8 18 8G0149 F 0 9R00463 M 9R00464 M 9R00469 F 9R00470 F 9R00465 M 9R00466 M 9R00471 F Dose group Cont Cont Cont Cont Necropsy Specimen Amt Amt sent Date Type sent to to Covance Rocheste (9) r 1-19-01 11/20/98 liver 0.451 ~ 1 g Amt Amt sent to sent to 3M Univ Env MN lab (1- Duluth 19-01) (3/8/99) ~1 9 NA 11/20/98 liver 11/20/98 liver 11/20/98 liver 0.328 ~ 1 g 0.489 ~ 1 g 0.709 ~ 1 g . ~1 9 ~ ig ~ 19 NA NA NA Cont 11/20/98 liver 0.49 ~ 1 g ~ i g NA Cont 11/20/98 liver 0.544 ~ 1 g ~ 1 g NA Cont 11/20/98 liver 0.468 ~ 1 g ~ 1 g NA PFOS 11/20/98 liver 0.901 ~ i g ~1 g NA PFOS 11/20/98 liver 0.638 ~ 1 g ~ i g NA PFOS 11/20/98 liver 0.387 ~ 1 9 ~1 9 NA N-et FOSE N-et FOSE N-et FOSE N-et FOSE NE tF O S A NE tF O S A NE tF O S A NE tF O S A Cont 11/20/98 liver 11/20/98 liver 11/20/98 liver 11/20/98 liver 11/20/98 liver 11/20/98 liver 11/20/98 liver 11/20/98 liver 3/5/99 liver 0.374 ~ 1 g 0.817 ~ 1 g 0.484 ~ 1 g 0.786 ~ 1 g 0.57 ~ 1 g 0.538 ~ 1 g 0.483 ~ 1 g 0.85 ~ 1 g 1.025 ~ 1 g ~ 1 g NA ~1 g NA ~1 g NA ~1 g NA ~1 g NA ~1 g NA ~1 9 NA ~ 1 g NA ~ 1 g ~1 g Cont Cont Cont N-et FOSE N-et FOSE N-et FOSE 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 0.76 ~ 1 g 0.747 ~ 1 g 0.816 ~ 1 a 0.888 ~ 1 g 0.812 ~ 1 g 0.593 ~ l g ~ 1g ~ 1g - 19 ~ 1g ~1 g ~ 1g -1 g ~ 1g ~ 1g ~ 1g ~ 1g ~ 1g 29 C0004 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1,T-7132.1 DT15-B 2%; 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Dosed 2/19/01 160 mkd FOSAA, 40 mkd FOSA , Veh control 9R00472 F 9R00467 M 9R00468 M 9R00473 F 9R00474 F 9G0004 M 5 9G0004 M 6 9G0005 F 1 9G0005 F 2 9G0004 M 7 9G0004 M 8 9G0005 F 3 9G0005 F 4 9G0004 M 9 9G0005 M 0 9G0005 F 5 9G0005 F 6 1 1R00742 M N-et FOSE PFOS PFOS PFOS PFOS Cont Cont. Cont Cont N-et FOSE N-et FOSE N-et FOSE N-et FOSE PFOS PFOS PFOS PFOS Cont 2 1R00743 M 3 1R00744 M 7 1R00748 M 8 1R00749 M 9 1R00750 M 4 1R00745 M 5 1R00746 M 6 1R00747 M Cont Cont FOSAA FOSAA FOSAA FOSA FOSA FOSA 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 3/5/99 liver 2/23/01 liver 2/23/01 liver 2/23/01 liver 2/23/01 liver 2/23/01 liver 2/23/01 liver 2/23/01 liver 2/23/01 liver 2/23/01 liver 0.834 ~ 1 g ~ 1g ~ 1g 0.93 ~ 1 g 0.81 ~ 1 g 0.703 ~ 1 g 0.776 ~ 1 g 0.842 ~ 1 g ~ 1g ~ 1g ~ 1g ~ 1g ~ 1g ~ 1g ~ 1g ~1 g ~1 g ~ 1g 0.78 ~ 1 g 0.422 ~ 1 g 0.727 ~ 1 g ~1 g ~1 g ~1 g ~ i g ~1 g ~1 g 0.527 - 1 g -1 g -1 g 0.834 ~ 1 g ~ 1 g ~1 g 0.812 ~ 1 g ~ 1g ~ 1g 0.7 ~ i g ~ 1 g ~1 g 0.808 ~ i g 0.815 ~ 1 g ~ 1 g ~1 g ~1 g ~ 1 g 0.716 ~ 1 g ~ 1g ~ 1g 0.517 ~ 1 g ~1 g ~1 g NA ~ 1 g NA ~1 g NA ~ 1 g NA ~1 g NA ~ 1g NA ~1 g NA ~1 g NA ~ 1 g NA ~ i g NA ~ 1 g NA - 1 g NA ~ 1 g NA ~ 1 g NA ~1 g NA - 1 g NA ~ 1 g NA 2LJ3_____ N A ~1.g 30 C00943 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Appendix 3. Cumulative Dose Individual and Summary data. SPECIES SEX DOSE GRO GP F CONT ID 8G01485 8G01486 9G00051 9G00052 N Mean SD Cumulative 0 0 0 0 4 0.0 0.0 Dose (mg) N-EtFOSA40mkd 8G01488 8G01490 N Mean SD V0O0 96 101 2 3.9 N-EtFOSE160mkd 9G00053 9G00054 N Mean SD 166 160 2 163.4 4.3 N-EtF0SE4 Omkd 8G01487 8G01489 N Mean SD 103 102 2 102.2 0.6 PF0S4 Omkd 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-EtF0SE4 Omkd 8G01479 8G01480 115 120 00044 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B SPECIES SEX DOSE GRO ID N Mean SD PFOS4 Omkd 8G01483 8G01484 9G00049 9G00050 N Mean SD R F CONT 8R04040 8R04041 9R00469 9R00470 N Mean SD N-EtFOSA4 Omkd 8R04044 8R04045 N Mean SD N-EtFOSE16Omkd 9R00471 9R00472 N Mean SD N-EtFOSE4 Omkd 8R04042 8R04043 N Mean SD PFOS40mkd 8R04046 8R04047 9R00473 9R00474 N Mean SD M M556-160mkd SPECIES SEX DOSE GRO CONT 1R00748 1R00749 1R00750 N Mean SD ID 1R00742 1R00743 1R00744 8R04032 8R04033 9R00463 Cumulative 2 117.6 4.0 Dose (mg) 115 107 39 39 4 75.0 41.8 0 0 0 0 4 0.0 0.0 38 36 2 36.6 1.5 126 122 2 124.3 2.9 40 38 2 39.1 1.4 38 35 31 29 4 33.2 3.7 165 166 168 3 166.1 1.5 Cumulative 0 0 0 0 0 0 Dose (mg) SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B F0SA4Omkg N-EtF0SA4 Omkd N-EtFOSEl60mkd N-EtFOSE40mkd PFOS40mkd 9R00464 N Mean SD 0 7 0.0 0.0 1R00745 1R00746 1R00747 N Mean SD 40 39 41 3 40.0 1.3 8R04036 8R04037 N Mean SD 51 51 ,2 51.2 0.5 9R00465 9R00466 N Mean SD 184 175 2 179.5 6.3 8R04034 8R04035 N Mean SD 53 53 2 52.6 0.1 8R04038 8R04039 9R00467 9R00468 N Mean SD 49 50 45 43 4 46.8 3.2 33 C 00946 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B 34 C00047 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 GP SEX F DOSE GRO CONT ID 8G01485 8G01486 9G00051 9G00052 N Mean SD NEtFOSA40mkd 8G01488 8G01490 N Mean SD NEtFOSEl60mkd 9G00053 9G00054 N Mean SD NEtFOSE4 Omkd 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 8vOGU3 35 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B M SPECIES SEX SD CONT 8G01477 8G01478 9G00045 9G00046 N Mean SD NEtFOSA40mkd 8G01481 8G01482 N Mean SD NEtFOSEl60mkd 9G00047 9G00048 N Mean SD NEtFOSE40mkd DOSE GRO 8G01479 8G01480 ID N Mean SD PFOS4 Omkd 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 GvOO'^O 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 16.3 755 653 Missing Missing 2 704.0 72.1 753 748 2 750.5 3.5 Missing Missing 0 Missing Missing 725 754 BWD3 G 2 739.5 20.5 675 621 Missing Missing 2 648.0 38.2 194.8 754 662 296 298 4 502.5 240.2 729 723 2 726.0 4.2 240 226 2 233.0 9.9 712 756 BWD4 G 2 734.0 31.1 668 594 215 240 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 NEtFOSA4 Omkd 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 PFOS4 Omkd 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 OSGOOD 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 5.7 217 205 2 211.0 8.5 Missing Missing 0 Missing Missing 235 223 2 229.0 8.5 212 189 Missing Missing 2 200.5 16.3 258 263 247 218 215 210 201 4 211.0 7.4 210 205 2 207.5 3.5 174 175 2 174.5 0.7 227 231 2 229.0 2.8 200 178 175 153 4 176.5 19.2 250 262 227 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 G 250 269 256 297 295 279 294 7 277.1 19.4 3 265.G 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 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 3 -12.0 25.0 BW GAIN 22 26 20 24 12 19 19 7 20.3 5.0 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 FOSA40mkg 1R00745 1R00746 1R00747 N Mean SD 250 249 259 3 252.7 5.5 254 245 265 3 254.7 10.0 250 243 263 3 252.0 10.1 239 233 255 3 242.3 11.4 227 225 254 3 235.3 16.2 -23 -24 -5 3 -17.3 10.7 90.80 90.36 98.07 3 93.1 4.3 NEtF0SA4 Omkd 8R04036 8R04037 N Mean SD 305 300 2 302.5 3.5 Missing Missing 0 Missing Missing 371 367 2 369.0 2.8 306 304 2 305.0 1.4 310 309 2 309.5 0.7 5 9 2 7.0 2.8 101.64 103.00 2 102.3 1.0 NEtFOSE160mkd 9R00465 9R00466 N Mean SD 292 280 2 286.0 8.5 Missing Missing 0 Missing Missing 283 267 2 275.0 11.3 Missing Missing 0 Missing Missing 272 231 2 251.5 29.0 -20 -49 2 -34.5 20.5 93.15 82.50 2 87.8 7.5 N- 8R04034 312 Missing 375 314 316 4 EtF0SE4 Omkd 101.28 8R04035 311 Missing 378 318 315 4 N 2 022 2 2 101.29 2 O o o 4/1 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.I 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 NEtF0SA4 0 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 NEtFOSEl6 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 NEtFOSE4 0 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 SPECIES r> o o Jl 9G00056 N Mean SD CONT 8G01477 8G01478 9G00045 9G00046 N Mean SD NEtFOSA40 mkd 8G01481 8G01482 N Mean SD NEtF0SE16 Omkd 9G00047 9G00048 N Mean SD NEtF0SE4 0 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 5.20 3.90 Missing Missing 2 4.550 0.919 5.40 Missing 2 0.007 0.000 0.01 0.01 Missing Missing 2 0.006 0.001 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 41 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B R o 'J\ cn 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 F CONT 8R04040 8R04041 9R00469 9R00470 N Mean SD 9.00 8.80 7.60 7.10 4 8.125 0.922 0.04 0.04 0.04 0.04 4 0.038 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 NEtFOSEl6 Omkd 9R00471 9R00472 N Mean SD 7.50 7.30 2 7.400 0.141 0.04 0.04 2 0.042 0.001 NEtF0SE4 0 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 2.10 Missing 0 Missing Missing 0.01 1.80 2 1.950 0.212 1.70 0.01 2 0.009 0.001 0.01 42 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 FOSA4Omk g 1R00745 1R00746 1R00747 N Mean SD NEtFOSA4 0 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 iO O ^O 1.-60 Missing Missing 2 1.650 0.071 Missing Missing Missing 0 Missing Missing KW Missing Missing Missing 2.90 2.90 Missing Missing 2 2.900 0.000 Missing Missing Missing 0 Missing Missing 2.40 0.01 Missing Missing 2 0.009 0.000 Missing 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 0 Missing Missing 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 NEtF0SE16 Omkd 9R00465 9R00466 N Mean SD NEtFOSE40 mkd 8R04034 8R04035 N Mean SD PFOS4 Omk 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 o o v\ <1 2.450 0.071 Missing 0.008 0.000 Missing Missing 0 Missing Missing 2 . IQ 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 44 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 ii0 0 ^ 3 45 CD SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7I32.1 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 SOCO 3 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 o.i-i 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 16Omkd 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 4Omkd 8R04042 8R04043 N Mean SD NA NA 0 NA NA NA NA NA NA 00 NA NA NA NA PFOS 4Omkd 8R04046 NA 8R04047 9R00473 9R00474 N NA 967.50 765.30 2 NA NA 21.20 15.30 2 NA NA NA NA 0 GO0060 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 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B Mean SD 866.40 142.98 18.25 4 .17 NA NA M556 -160mkd CONT 1R00748 238.40 1R00749 1R00750 N Mean SD 1R00742 1R00743 1R00744 8R04032 8R04033 9R00463 9R00464 N Mean SD 81.90 100.70 3 140.33 85.45 NA NA NA NA NA 0.00 0.00 2 0.00 0.00 447.30 614.40 235.70 267.40 3 316.80 114.12 154.10 113.20 273.20 NA NA 2.60 2.80 5 109.18 113.59 424.00 627.10 3 555.17 113.77 NA NA NA NA NA NA NA 0 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 N-EtFOSA 40mkd 8R04036 8R04037 N Mean SD NA NA 0 NA NA NA NA NA NA 00 NA NA NA NA N-EtFOSE 160mkd 9R00465 9R00466 N 1241.70 1006.80 2 25.20 11.70 2 111.00 77.50 2 SOOO 3 NA NA NA NA NA 0 NA NA NA NA NA NA NA NA NA 0 NA NA NA NA NA 0 NA NA NA NA 0 NA NA 376.40 212.70 2 NA NA NA NA NA 0 NA NA NA NA NA NA NA NA NA 0 NA NA NA NA NA 0 NA NA NA NA 0 NA NA 13.90 10.30 2 NA NA NA NA NA 0 NA NA NA NA NA NA NA NA NA 0 NA NA NA NA NA 0 NA NA NA NA 0 NA NA 2.92 4.66 2 NA NA NA NA NA 0 NA NA NA NA NA NA NA NA NA 0 NA NA NA NA NA 0 NA NA NA NA 0 NA NA 0.24 0.29 2 NA NA NA NA NA 0 NA NA NA NA NA NA NA NA NA 0 NA NA 0.44 0.39 0.34 3 0.39 0.05 NA NA 0 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 4Omkd 8R04034 8R04035 N Mean SD NA NA 0 NA NA NA NA 0 NA NA PFOS 4Omkd 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 Z9003 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 N-EtFOSA 4Omkd ID 8G01485 8G01486 9G00051 9G00052 N Mean SD 8G01488 8G01490 N Mean SD TL PFOSX (ROC) (ug/g) Missing Missing Missing Missing 0 Missing Missing Missing 83.5 1 83.5 Missing TL PFOSX (ROC) (mg) Missing Missing Missing Missing 0 Missing Missing Missing 1.7 1 1.7 Missing % DOSE TL PFOSX (ROC ) (%) Missing Missing Missing Missing 0 Missing Missing Missing 1.6 1 1.6 Missing N-EtFOSE 160mkd N-EtFOSE 4Omkd PFOS 4Omkd 9G00053 9G00054 N Mean SD 8G01487 8G01489 N Mean SD 8G01491 8G01492 9G00055 1388.4 630.2 2 1009.3 536.1 198.8 186.5 2 192.6 8.7 Missing Missing 40.5 13.3 5.6 2 9.5 5.5 3.9 4.1 2 4.0 0.1 Missing Missing 0.3 8.0 3.5 2 5.8 3.2 3.8 4.0 2 3.9 0.1 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 GOLD C7> 50 CO SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B M CONT N-EtFOSA 4Omkd N-EtFOSE 160mkd N-EtFOSE 4Omkd PFOS 4Omkd 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 frscoua 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 S90G G 0 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 Missing 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-7071.1, T-7132.1 DT15-B C. Rat Liver PFOSX Analyses at the 3M Environmental Lab. (All units are |ig/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-EtFOSEl60mkd 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 PFOS4 Omkd 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 9R00464 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 t)0 'v 3 54 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DT15-B N-EtFOSEl60mkd 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 R SEX DOSE_GRO F CONT ID 8R04040 8R04041 9R00469 9R00470 N Mean SD TL PFOSX (3M)_ (ug/g) Missing Missing 0.26 0.48 2 0.4 0.2 TL PFOSX (3M)_ (mg) Missing Missing 0.0 0.0 2 0.0 0.0 % DOSE TL PFOSX (3M) (%) Missing Missing Missing Missing 0 Missing Missing % DOSE PFOS (3M) (%) Missing Missing Missing Missing 0 Missing Missing N-EtFOSE 9R00471 1408.00 10.6 8.4 3.6 160mkd 9R00472 1407.00 10.3 8.4 3.6 N2 2 2 2 Mean 1407.5 10.4 8.4 3.6 SD 0.7 0.2 0.0 0.0 O O O G3 55 SRPT T-6295.8, T-6316.4, T-6868.2, T-7071.1, T-7132.1 DTI5-B PFOS 4Omkd M55616Omkd CONT FOSA 4Omkg 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 3000 0 CO Missing 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 Missing 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 Missing 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 40mkd 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 CCO070 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