Document n9XQQ9zGBRBNnq1ZDge825MpG

PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 A#22kr1551 X. TRA^S^CRET Htv H At '* ( /. y'CTf. S' rr S )o^AI Xt u ^ Study Title /y/, /C1 vt-kyX PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats > LaboratoryProjectID: DuPont-13309 T f c s ' l -n Author: Eve Mylchreest, Ph.D. Study Completed on: December 19, 2003 Performing Laboratory: E.I. du Pont de Nemours and Company X * Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Sponsor: 3M Company 3M Center St. Paul, MN 55144-1000 and E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 Work Request Number: 14787 Service Code Number: 1569 Page 1 o f 139 0^ PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are consistent with the OECD Principles of Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM(98)17 and MAFF Japan Good Laboratory Practice Standards (59 NohSan No. 3850). Applicant / Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. and 3M Company 3M Center St. Paul, MN 55144-1000 Applicant / Sponsor:________________________________ '_______ _____ _ Applicant/Sponsor Representative Date 3 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats QUALITY ASSURANCE STATEMENT D uPont-13 309 Haskell Sample Number(s): 24921 Dates of Inspections: Protocol: July 11, 2003 Conduct: July 11,28,31, 2003; August 15, 2003; September 5,30 2003 Records, Reports: October 2,3,6-8,13-15,30-31 2003; November 3-7, 2003 Dates Findings Reported to: Study Director: July 11,29,31, 2003; August 18, 2003; September 10,30, 2003; October 10,16,17, 2003; November 6,7,10, 2003 Management: July 29,31, 2003; August 18, 2003; September 10,3^,^003; October 10,16,17, 2003; November 6,7,10,12, 2003 ' Reported by: Staff Quality Assurance Auditor Date PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Analytical Evaluations by: Senior Staff Chemist - '2.003 Date Biochemical Evaluations by: Shawn A. Gannon, B.S. Senior Staff Toxicologist /y- Vty - w } Date Approved by: Scott E. Loveless, Ph.D. Research Manager and Director t l - D e c -&*>;? Date Issued by Study Director: ,Ev^Mylchreest, Ph.D. Senior Research Toxicologist lR f Date S' PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ..2 QUALITY ASSURANCE STA TEM EN T............................................... .3 C E R T I F IC A T IO N ......................................................................................... ..4 LIST OF TA B LE S........................................................................................ ..6 LIST OF FIGURES....................................................................................... ..7 STUDY INFORM ATION......................................................... ................. ..8 STUDY PERSO N NEL................................................................................ ..9 SUM M ARY................................................................................................... 10 IN T R O D U C T IO N ......................................................................................... 13 O BJECTIV E.................................................................................................. 13 SPONSOR AND TEST FA CILITY ........................................................... 13 STUDY DESIGN.......................................................................................... A. Experimental Design........................................................................ B. Selection of Dose Levels................................................................. 13 13 14 MATERIALS AND M ETHODS................................................................ 14 ANALYTICAL............................................................................................................................... 14 A. Vehicle................................................................................................................................14 B. Test Substance....................................................................................................................14 IN-LIFE........................................................................................................................................... 17 A. Test Species........................................................................................................................17 B. Animal Husbandry............................................................................................................ 17 C. Quarantine and Pretest Period.............................................................................................19 D. Assignment to Groups - Randomization........................................................................... 19 E. In-life Observations.............................................................................................................19 F. Terminal Sample Collection and Sacrifice........................................................................ 19 BIOCHEMICAL TOXICOLOGY SAMPLE ANALYSES........................................................... 21 A. Tissue and Plasma Analysis............................................................................................... 21 STATISTICAL ANALYSES......................................................................................................... 23 RESULTS AND CONCLUSIONS................................................................................................ 24 ANALYTICAL DATA.................................................................................................................. 25 A. Test Substance Stability Analyses..................................................................................... 25 B. Test Substance Formulation Analyses............................................................................... 25 C. Analytical Conclusions...................................................................................................... 27 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 REPRODUCTIVE TOXICITY EVALUATIONS........................................................................ 28 IN-LIFE TOXICOLOGY................................................................................................................28 A. Maternal Observations....................................................................................................... 28 B. Reproductive D ata..............................................................................................................28 BIOCHEMICAL TOXICOLOGY................................................................................................. 30 A. Maternal, Fetal, and Pup Plasma........................................................................................ 30 B. Milk.....................................................................................................................................30 C. Amniotic Fluid....................................................................................................................30 D. Placenta...............................................................................................................................30 E. Embryo and Fetus...............................................................................................................30 CONCLUSIONS...... : ..................................................................................................................... 31 RECORDS AND SAMPLE STORAGE....................................................................................... 31 REFERENCES............................................................. 32 TABLES........................................................................................................................... 33 FIGURES....................................................................................................................................... 42 APPENDICES................................................................................................................................53 LIST OF TABLES Page TABLE 1 SUMMARY OF DOSING TEST FORMULATION ANALYSES................................................................... 35 TABLE 2 CONCENTRATION OF PFOA IN MATERNAL PLASMA DURING GESTATION AND P O STP A R TU M ....................................................................................................................................................................... 36 TABLE 3 CONCENTRATION OF PFOA IN FETAL AND PUP PLASM A ................................................................... 37 TABLE 4 CONCENTRATION OF PFOA IN M ILK............................................................................................................. 38 TABLE 5 CONCENTRATION OF PFOA IN AMNIOTIC FLUID.................................................................................... 39 TABLE 6 CONCENTRATION OF PFOA IN PLACENTA..................................................................................................40 TABLE 7 CONCENTRATION OF PFOA IN EMBRYO/FETUS...................................................................................... 41 7 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 LIST OF FIGURES Page FIGURE 1 MEAN BODY WEIGHTS OF FEMALE RATS DURING GESTATION....................................................44 FIGURE 2 MEAN BODY WEIGHTS OF FEMALE RATS DURING LACTATION................................................. 45 FIGURE 3 CONCENTRATION OF PFOA IN MATERNAL PLASMA DURING GESTATION AND POSTPARTUM............................................................................................................... 46 FIGURE 4 CONCENTRATION OF PFOA IN PUP PLASMA............................................................................................47 FIGURE 5 CONCENTRATION OF PFOA IN FETAL PLASMA ON GESTATION DAY 2 1 ...................................48 FIGURE 6 CONCENTRATION OF PFOA IN MILK............................................................................................................49 FIGURE 7 CONCENTRATION OF PFOA IN AMNIOTIC FL U ID ..................................................................................50 FIGURE 8 CONCENTRATION OF PFOA IN PLACENTA............................................................................................... 51 FIGURE 9 CONCENTRATION OF PFOA IN EMBRYO/FETUS.....................................................................................52 LIST OF APPENDICES Page APPENDIX A ANALYTICAL D A T A .......................................................................................................: .V ....................... 54 X APPENDIX B INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA * DURING GESTATION AND LACTATION............................................................................................................... 62 APPENDIX C INDIVIDUAL BODY WEIGHTS DURING GESTATION AND LACTATION................................76 APPENDIX D INDIVIDUAL BODY WEIGHT CHANGES DURING GESTATION AND LACTATION......... 102 APPENDIX E INDIVIDUAL REPRODUCTIVE D A T A ..................................................................................................112 APPENDIX F NUMBER OF PUPS DURING LACTATION............................................................................................118 APPENDIX G INDIVIDUAL LITTER CLINICAL OBSERVATIONS......................................................................... 121 APPENDIX H MEAN PUP WEIGHTS PER LITTER....................................................................................................... 123 APPENDIX I PLASMA AND TISSUE ANALYSIS........................................................................................................... 126 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13 309 STUDY INFORMATION 9th Collective Nomenclature: Octanoic acid, pentadecafluoro-, ammonium salt Svnonvms/Codes : Ammonium perfluorooctanoate FC-143 FLUORAD Brand Fluorochemical Surfactant (3M Company, Specialty Materials) C-8 Perfluorooctanoate, ammonium salt PFOA H-24921 Lot 332 (3M Specialty Materials) (Lot No.) Haskell Number: 24921 CAS Registry Number: 3825-26-1 Purity: 95.2% - 97.99% Straight chain: 77.6% Branched: 12.6% internal monomethyl (non-alpha) 9% isopropyl 0.2 % tert-butyl 0.1% gem-dimethyl 0.1% alpha monomethyl Known Impurities: C4(C3F7C 02' NH4+), 0.01% % C5(C4F9C 02` NH4+), 0.03% C6(C3FnC 0 2' N H /), 0.43% C7(C6F13C 0 2- NH4+), 0.57% C9(C8F17C 0 2- NH4+), 0.16% Monohydro APFO, 0.09% Monounsaturated APFO, 0.72% Undefined (possibly) substituted perfluorocyclo species, 0.2% cyclopentyl, and 0.1% cyclohexl Physical Characteristics: White solid Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. and 3M Company 3M Center St. Paul, MN 55144-1000 Study Initiated/Completed: July 8, 2003 / (see report cover page) In-Life Initiated/Completed: July 11, 2003 / August 22, 2003 ? PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats STUDY PERSONNEL Study Director: Eve Mylchreest, Ph.D. Management: Scott E. Loveless, Ph.D. Supervisor: Deborah L. Tyler Primary Technician: Joseph F. Aschiero Analytical Chemist: Janet C. Maslanka, B.S. Management: S. Mark Kennedy, Ph.D. Biochemical Toxicologist: Shawn A. Gannon, B.S. Management: Gary W. Jepson, Ph.D. Toxicology Report Preparation: Mary K. LaRoe Lisa G. Burchfield, A.A. Management: Nancy S. Selzer, M.S. Laboratory Veterinarian: Thomas W. Mayer, D.V.M., A.C.L.A.M. Management: Janice L. Connell, M.S., B.A. DuPont-13309 9 / PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 SUMMARY Twenty time-mated female Crl:CD(SD)IGS BR rats were dosed by oral gavage once daily at concentrations of 0, 3, 10, or 30 mg/kg/day of test substance on days 4-10, 4-15, or 4-21 of gestation (G), or from day 4G to day 21 postpartum (PP). Clinical observations and body weights were recorded on the day after rats arrived and daily until the end of the study. On days 10, 15, and 21G, 5 rats per group per time point were euthanized. For each female with visible implantation sites, the intrauterine location of each embryo/fetus and implantation type was recorded. Maternal viscera were examined grossly and blood samples were collected approximately 2 hours (+ 30 minutes) post-dose and were processed to obtain plasma samples. Embryos were collected on day 10G, and amniotic fluid, placentas, and embryos/fetuses were collected on days 15 and 21G. Fetuses were euthanized and fetal blood samples were collected on day 21G. The last 5 animals per group were designated for the lactational evaluation and were allowed to deliver and nurse their litters. On days 0, 3, 7, 14, and 21PP, offspring were individually handled and examined for abnormal behavior and appearance. Live pups in each litter were counted and weighed (sexes separate). On days 3, 7, 14, and 21PP, dams were anesthetized and milk and blood samples were collected. Maternal blood samples were collected approximately 2 hours (+ 30 minutes) post-dose. Randomly selected pups were weighed, euthanized and^lood samples were collected on days 3, 7, 14, and 21PP. s* Plasma, milk, amniotic fluid extract, and tissue homogenate (placenta, embryo, and fetus) supernatants were analyzed by high performance liquid chromatography - mass spectrometry (HPLC-MS). Clinical Observations and Mortality There were no test substance-related clinical observations in dams or litters. There was no mortality in maternal rats at any level tested. Body Weight and Body Weight Gain Mean body weight gain at 30 mg/kg/day was lower than control during gestation and was similar to control during lactation. Mean daily body weights at 30 mg/kg/day were lower than control throughout gestation and lactation. Mean body weights and body weight gain at 3 and 10 mg/kg/day during gestation and lactation were similar to the control group. PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Reproductive Data All animals were pregnant at scheduled sacrifice. All dams delivered a live litter. The number of implantation sites, resorptions, and live fetuses were comparable across groups that were sacrificed on either day 10, 15, or 21G. There were 2 small litters in the 30 mg/kg/day lactation group. Due to the intrinsic variability in litter size and the small number of litters evaluated (5 per group) the relationship of this finding to test substance administration is equivocal. Pup survival and pup weights during lactation were comparable across groups. Biochemical Toxicology The concentration of PFOA in maternal plasma appeared to be at steady state (means of 11.2, 26.8, and 66.6 jug/mL at 3, 10, and 30 mg/kg dose levels, respectively) over the range of time points sampled. The mean concentrations of PFOA in fetal plasma collected on day 21G were 5.9, 14.5, and 33.1 fig/mL at 3, 10, and 30 mg/kg dose levels, respectively. Pup plasma concentration decreased from day 3PP (means of 2.9, 5.9, and 12.0 jug/mL at 3, 10, and 30 mg/kg, respectively) to day 7PP (0.7, 2.8, and 4.9 jug/mL at 3.10, and 30 mg/kg/day, respectively, and were similar on days 7, 14, and 21PP at aletele levels. The concentration of PFOA in milk was at steady state (means of 1.1, 2.8, and 6.2 /rg/mL at 3, 10, and 30 mg/kg dose levels, respectively) from day 3PP to day 21PP at all dose levels. The concentration of PFOA in amniotic fluid was approximately four times higher on day 21G (means of 1.5, 3.8, and 8.1 jug /mL at 3, 10, and 30 mg/kg, respectively) than on day 15G (means of 0.6, 0.7, and 1.7 ig /mL at 3, 10, and 30 mg/kg, respectively). The concentration of PFOA in placenta was approximately two times higher on day 21G (means of 3.6, 9.4, and 24.4 xg /mL at 3, 10, and 30 mg/kg, respectively) than on day 15G (means of 2.2, 5.1, and 13.2 fig /mL at 3, 10, and 30 mg/kg, respectively). The day 10G embryo had the highest concentration of PFOA per gram of tissue (means of 1.4, 3.3, and 12.5 xg /mL at 3, 10, and 30 mg/kg, respectively) and the day 21G fetus had slightly lower levels (means of 1.3, 2.6, and 8.8 jug /mL at 3, 10, and 30 mg/kg, respectively). The day 15G embryo had a lower tissue concentration than tissue from day 10G or day 21G (means of 0.2, 0.5, and 1.2 /g /mL at 3, 10, and 30 mg/kg, respectively). /Z PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 In conclusion, under the conditions of this study, there was maternal toxicity (reduced body weight and weight gain) at 30 mg/kg/day. There were 2 small litters in the 30 mg/kg/day lactation group, a finding that was possibly test substance-related. Concentrations of PFOA in maternal plasma and milk were at steady state during the sampling interval. Steady state concentration in milk was approximately ten times less than the steady state concentration in maternal plasma. The concentration of PFOA in fetal plasma on day 21G was approximately half the steady state concentration in maternal plasma. The milk concentrations appeared to be generally comparable to the concentrations in pup plasma. Pup plasma concentrations decreased from day 3PP to day 7PP, and were similar on days 7, 14, and 21PP at all dose levels. PFOA was detected in placenta (days 15 and 21G), amniotic fluid (days 15 and 21G), embryo (days 10 and 15G), and fetus (day 21G). A /3 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INTRODUCTION The subject test substance, ammonium PFOA, is a surfactant used in industrial processes. In pharmacokinetic studies in adult rats, whole body elimination of the test substance was found to be much more rapid in females compared to males following oral dosing. Available data on the transport concentrations of the test substance when administered to pregnant females is limited and there is no existing data on the concentration of the test substance transported via the milk from lactating females.(1,2) OBJECTIVE The objective of this study was to determine the concentration of the test substance in maternal milk and blood plasma, placenta, amniotic fluid, embryo, fetus, pup, and fetal and pup plasma following repeated oral dosing of the dam using 3 graded dose levels during gestation and lactation. SPONSOR AND TEST FACILITY This study was co-sponsored by 3M Company, St. Paul, Minnesota, and E.I. du Pont de Nemours and Company, Wilmington, Delaware. The sponsor's approval was effective the date the sponsor authorized the work on the contract. The study was conducted at DuPont Haskell laboratory for Health and Environmental Sciences, E.I. du Pont de Nemours and Company, Newark^Delaware. STUDY DESIGN A. Experimental Design Test Formulation Dose Concentration Time-Mated Group (mg/kg/day)a (mg/mL)b Females I 0C 0 20 n3 0.6 20 m 10 2.0 20 IV 30 6.0 20 a Formulations of test substance in deionized water (NANOpure deionized water) were administered once daily by oral gavage at a dosing volume o f 5 mL/kg. b To achieve these concentrations o f active ingredient, the formulations were adjusted for sample purity (%). c The control group animals received NANOpure deionized water only at 5 mL/kg. 13 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Dose Group Subsets And Dosing Schedules Subseta A B C D Number of Animals 5 5 5 5 Dosing Period b (days) 4-10G 4-15G 4-21G 4G-21PP a Animals were randomly assigned to each subset, b G=gestation; PP=postpartum Terminal Sacrifice (days) 10G 15G 21G 21PP B. Selection of Dose Levels Dose levels selected for this study were based upon the results of a two-generation reproduction study in rats.2(3) Groups of 30 male and 30 female rats were dosed by oral gavage at 0, 1, 3, 10, or 30 mg/kg/day for approximately 70 days before cohabitation. Dosing of the P0 females continued throughout mating, gestation, and lactation or approximately 112 days total. While there was no test substance-related maternal toxicity observed at doses up to 30 mg/kg/day, a slight increase in offspring mortality, attributed to failure to thrive, was observed in Fi generation weanlings at 30 mg/kg/day. MATERIALS AND METHODS \ ANALYTICAL A. Vehicle The test formulation vehicle was NANOpure deionized water. B. Test Substance 1. Identification The test substance was obtained from 3M, St. Paul, Minnesota, and was assigned Haskell Laboratory Number PFOA upon receipt. Available information on the purity, composition, contaminants, synonyms, hazards, and hazardous material classification(s) was provided by the vendor and documented in the study records and report. 2. Purity The vendor reported purity was 95.2% - 97.99%. /s PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 3. Test Substance Stability The stability of the test substance over the course of the study was confirmed by purity analyses conducted near the beginning and end of the study. 4. Test Substance Administration, Preparation, and Sampling a. Administration The test substance was administered by oral gavage once daily at a dose volume of 5 mL/kg. Animals assigned to gestation subsets A, B, and C, were dosed for 7, 12, and 18 days respectively. Animals assigned to the lactation subset (D) were dosed for 40 days. Dose volumes were adjusted daily based on body weights. b. Preparation Test formulations of the test substance in the vehicle were prepared daily and stored at room temperature until used. The method of mixing the test substance with the vehicle is documented in the study records. c. Sampling Test formulations at concentrations of 0.6, 2.0, and 6.0 mg/mL of PFOA were prepared and collected for uniformity of mixing/concentration verification and stability analysis pshour room temperature) on July 11, 2003. On July 31, 2003 and August 21, 2003, test formulations at all levels were collected for concentration verification analysis. In addition, 0 mg/mL (control) samples were submitted for analysis with each set of samples. Samples submitted for analysis were analyzed the day they were received and/or when re analysis was indicated. 5. Analytical Methods a. Recovery Sample Analysis Concurrent with test formulations analyses, recovery of PFOA from spiked vehicle (NANOpure deionized water) was tested at the low level (0.6 mg/mL), at the mid level (2.0 mg/mL) and at the high level (6.0 mg/mL) to confirm the analytical method. A stock solution of PFOA was prepared in NANOpure deionized water. For all concentration levels, an appropriate aliquot of this solution to obtain 0.3 mg (low), 1 mg (mid), and 3 mg (high) of the test substance was added to 100 mL of NANOpure deionized water. All recovery samples were then mixed for dispersion of the test substance in the vehicle. The samples were then processed and analyzed in the same manner as the dosing samples at similar concentrations. b. Dosing Test Formulation Treatment Each dosing sample (0.5 mL) was diluted to 100 mL with NANOpure deionized water and mixed. The dosing samples were further diluted with NANOpure deionized water to an expected 15 /& PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13 309 concentration of approximately 0.000003 mg/mL (50 pL injections) or 0.00003 mg/mL (5 pL injections) prior to analysis. Before all final dilutions, the internal standard and the 0 mg/mL sample (initial dilution) was added to each test sample to give an equivalent final concentration of the internal standard and the matrix in all samples. Samples submitted for analysis were analyzed on the day of receipt and/or when re-analysis was indicated. c. Chromatographic Conditions LC parameters Instrument: Column: Flow Rate: Injection Volume: Column Temperature: Column Switch: Mobile Phase: Gradient: Time (min.) 0.0 0.9 1.0 6.0 6.1 7.0 Hewlett Packard Model 1100 HPLC Zorbax RX-C8, 2.1 mm x 150 mm, 5pm 0.4 mL/min 5 or 50 pL 30C 4.0 minutes 0.15% Acetic Acid/Acetonitrile % Acetonitrile 5 5 80 80 5 5 X M S parameters Instrument: Ionization mode: Capillary voltage: Cone Voltage: Source Temperature: Desolvation Temperature: Scan function: Micromass Quattro Micro Tandem MS Electrospray (ESI), negative ion -2.7 kV 15 V 120C 350C periluorooctanoic acid (PFOA): 413 m/z (parent) to 369 m/z (daughter) [l,2-di-13C] PFOA: 415 m/z (parent) to 369 m/z (daughter) Retention time of PFOA and [1,2-di-13C] PFOA: approximately 4.9 to 5.2 min (5 or 50 pL injections). d. Calibration and Quantitation A stock solution of the PFOA (separate sample used as analytical reference) was made in NANOpure deionized water. Appropriate aliquots of the stock were diluted with NANOpure deionized water to make calibration standards that bracketed the target concentration of the diluted sample solutions. Before these aliquots were brought to volume, an appropriate amount of internal standard ([l,2-di-13C] PFOA) was added. 16 /V PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Analysis of PFOA was by high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS). Negative-ion electrospray was used to generate negatively charged ions of PFOA. The ions were selected with the first MS quadrupole, collisionally dissociated using argon, and a fragment ion was monitored. [1,2-di- C] PFOA is used as an internal standard. Triplicate injections of the sample solutions and calibration standards solutions were made and peak areas were calculated electronically. The calibration curve was generated by regression analysis using the peak area ratio from the PFOA and the internal standard. Data for test formulations were compared to the calibration curves to evaluate the concentrations of PFOA. Test substance uniformity in the vehicle was evaluated by calculating the coefficient of variation (C.V. = standard deviation/mean x 100) of the measured concentrations of the duplicate samples (uniformity of mixing/concentration verification) for each dosing level. A coefficient of variation less than or equal to 10% is the standard criterion at Haskell Laboratory for acceptable distribution of the test substance throughout the solution. The mean result of the concentration verification samples (n = 2) for each dosing level was used to determine the concentration of the test substance for the respective dosing levels. Stability was evaluated by using the mean result of the uniformity of mixing/concentration verification samples as the baseline for comparing the corresponding stability resu^s. IN-LIFE A. Test Species The rat was selected for this study as it is a preferred species for reproductive toxicity studies and was the species used in a previous 2-generation reproduction study.(3) The Crl:CD(SD)IGS BR strain was selected based on consistently acceptable health status and on extensive experience with this strain at Haskell Laboratory. Eighty nulliparous, time-mated females were received on July 8, 2003 (60 female rats) and July 11, 2003 (20 female rats) from Charles River Laboratories, Inc., Raleigh, North Carolina, with an assigned birth date of May 5, 2003. The rats for this study were approximately 63 days old and were at day 1G upon arrival. Body weights on the day rats were mated (day 0G) were supplied by the vendor and are documented in the study records. B. Animal Husbandry 1. Identification Each rat was assigned a unique number and identified with an AVID Microchip implant by the supplier prior to shipping. Upon receipt, each animal was assigned a Haskell animal number. Both the Haskell animal number and unique vendor animal number were recorded on each cage 17 /? PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 card. A master list of unique vendor numbers, Haskell animal numbers, and corresponding unique AVID Microchip implant numbers are maintained with the study records. 2. Environmental Conditions Animal rooms were maintained at an acceptable temperature of 18-26C (targeted at 22-24C) and maintained at an acceptable relative humidity of 30%-70% (targeted at 40%-60%). Animal rooms were artificially illuminated (fluorescent light) on a 12-hour light/dark cycle (approximately 0600-1800 hours). 3. Housing All rats were housed individually in stainless steel, wire-mesh cages, suspended above cageboard until sacrifice or day 19G. Females selected for the lactation subset (D) were housed in polycarbonate pans containing bedding material on day 20G through day 21PP. 4. Feed and Water All rats were fed pelleted PMI Nutrition International, LLC Certified Rodent LabDiet 5002 ad libitum. All rats were provided with tap water from United Water Delaware ad libitum. 5. Health Monitoring Program X As specified in the Haskell Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to ensure that contaminant levels are below those that would be expected to impact the scientific integrity of the study: Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. Feed samples are analyzed for total bacterial, spore and fungal counts. Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Evaluation of these data did not indicate any conditions that affected the validity of the study. 18 /? PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 C. Quarantine and Pretest Period Rats were quarantined according to procedures outlined in Haskell Laboratory SOP LA003-P, and then released for the study upon approval of the Laboratory Animal Veterinarian or a designee. D. Assignment to Groups - Randomization Upon arrival, dams were ranked on the basis of day 0G body weights and assigned to control and experimental groups by random sampling from the ranked list. The distribution resulted in mean body weights for all groups that were not statistically different (p > 0.05). Animals in each group were then randomly assigned to each subset (A,B,C or D). E. In-life Observations 1. Clinical Observations Clinical observations were recorded on the day after arrival and daily until the end of the study. 2. Body Weights Body weights were recorded on the day after arrival and daily until the end of the study. Lactation Procedures - Subset D V The day when delivery was complete was designated day 0 postpartum. At each examination period (days 0, 3, 7, 14, and 21PP), offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded. Live and dead pups in each litter were counted by sex as soon as possible after delivery was complete and litter weights were recorded. Dead pups were discarded. F. Terminal Sample Collection and Sacrifice 1. Gestation Subsets A, B, and C On days 10, 15, and 21G, animals assigned to Subset A, B, and C, respectively, were euthanized by carbon dioxide inhalation. For each female with visible implantation sites, the intrauterine location of each embryo/fetus and implantation type (live or early resorption) were recorded. A gross examination of maternal viscera was done and blood samples from each dam were collected from .the vena cava approximately 2 hours ( 30 minutes) post-dose and processed to obtain plasma samples. On day 21G, fetuses were euthanized with an overdose injection (i.p.) of sodium pentobarbital and fetal blood samples were taken from a transverse cut made to the carotid artery. 19 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Gestation Subset Sample Collection and Sacrifice Schedule Terminal Subset Sacrifice Samples Collected A 10G Maternal Blood,3, Whole Embryosc B 15G Maternal Blood,3' Amniotic Fluid,c Placentas,0Whole Embryosb C 21G Maternal Blood,3 Amniotic Fluid,c Placentas,5Whole Fetuses,5Fetal Bloodd a Maternal blood samples (volume 0.5 mL per animal) in tubes containing heparin, were collected 2 hours ( 30 minutes) post-dose; other tissues were collected as soon as possible after maternal blood collection, b All embryos, fetuses, and placentas were pooled by litter. c Amniotic Fluid sample minimum volume approximately 100 ftL was pooled by litter. d Fetal blood sample minimum volume approximately 100 /aL collected in tubes containing heparin was pooled by litter. 2. Lactation Subset D a. Anesthesia Procedure Dams were removed from their litters approximately 1-2 hours before blood and milk samples were collected. Dams were placed in charged induction chambers (FLUOVAC anesthesia machine) and were anesthetized by isofluorane inhalation. When the dams appeared to be unconscious from the anesthesia, they were removed from the induction chambers and were checked for response to toe pinch. After samples were collected, dams were returned to holding cages or their home p?ht,where they were monitored while recovering from anesthesia. Dams were returned to their litters after recovery from anesthesia. b. Blood Sample Collection Sample Collection - Subset D Number of Pups for Pup Day Maternal Samples3 Terminal Sacrifice 5 Samples 3PP Milk,c Bloodd 1 per litter Bloode 7PP Milk,c Blood" 1 per litter Blood6 14PP Milk,c Blood" 1 per litter Blood6 21PPf Milk,c Blood" 2 per litter Blood6 a Samples were collected 2 hours ( 30 minutes) post-dose; other samples were collected as soon as possible after maternal blood collection, b Litter size permitting. c Maternal milk samples (minimum volume approximately 100 p.L). d Maternal blood sample volume 0.5 mL per animal collected in tubes containing heparin, e Pup blood sample minimum volume approximately 100 iL collected in tubes containing heparin, f Maternal Terminal Sacrifice 20 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Maternal Blood samples were collected from the orbital sinus (days 3, 7, and 14 PP) or vena cava (day 21PP) approximately 2 hours ( 30 minutes) post-dose. Fetal Randomly selected pups from these dams were weighed and euthanized on the same postpartum day with an overdose injection (i.p.) of sodium pentobarbital. Pup blood samples were taken from a transverse cut made to the carotid artery on days 3 and 7PP or from the vena cava on days 14 and 21PP. Maternal and pup blood samples were processed to obtain plasma samples. c. Milk Sample Collection Milk samples were collected after blood sample collection. Oxytocin was administered by intraperitoneal injection to promote milk ejection from the mammary gland. Approximately 2 to 5 minutes after injection, the dam's nipple area was washed with warm saline or water. Milk was collected from more than one mammary gland; mammary glands were changed frequently and were repeated as necessary. Milk was collected by applying pressure to the entire base of the gland, pushing milk toward the nipple and into a test tube. Pups were kept in polycarbonate pans with bedding during the milk collection prondure. On day 3PP litters were kept warm during the absence of the dam by the use of either a circulating hot water pad, heated gel packs, or heat lamp. d. Euthanasia After milk collection but before recovery from anesthesia, dams were euthanized by exsanguination. The remaining pups were euthanized with an overdose injection (i.p.) of sodium pentobarbital. Dam and pup carcasses were discarded. BIOCHEMICAL TOXICOLOGY SAMPLE ANALYSES A. Tissue and Plasma Analysis 1. Amniotic Fluid, Milk, and Plasma Plasma, milk, and amniotic fluid samples (pooled by litter), were processed by protein precipitation (PPT) using Isolute Array protein precipitation columns (Jones Chromatography, Lakewood, CO). A 0.5 [ig/mL solution of perfluorononanoic acid (Aldrich Chemicals, Milwaukee, WI) in acetonitrile (ACN) was used as an internal standard. Samples were thawed and a 20 /L aliquot of each sample was applied to the PPT array. Samples were precipitated by adding appropriate dilution rate volumes of ACN/intemal standard solution. Dilution rates ranging from 1:4 (60 fih of internal standard solution) to 1:80 (1580 juL of internal standard solution) were utilized to capture the sample concentrations within the range of the standard 21 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats D uPont-13 309 curve concentrations. The array was slowly eluted under vacuum into a 96-well receiver plate, centrifuged at -3000 rpm for 10 minutes, and the extracts were analyzed by high performance liquid chromatography - mass spectrometry (HPLC-MS). 2. Placenta and Embryo Placenta samples (pooled by litter) were coarsely chopped using scissors; embryo samples (pooled by litter) did not require this step. Aliquots (approximately 0.5 g) of the embryo samples or of the placenta coarse homogenate were transferred to another tube and homogenized further using a Branson ultrasonic probe sonifier in the presence of ACN in a ratio of approximately 4:1 (v:w) ACN:tissue. The homogenized sample was then centrifuged at -3000 rpm for 10 minutes. The supernatant was removed and taken to dryness by nitrogen convection, dissolved in internal standard solution (0.5 /rg/mL perfluorononanoic acid in ACN), and analyzed by HPLC-MS. 3. Fetus Fetus samples were pooled by litter and homogenized using a Waring blender in the presence of liquid nitrogen. Aliquots (approximately 0.5 g) of the homogenized samples were then transferred to another tube and homogenized further using a Branson ultrasonic probe sonifier in the presence of ACN in a ratio of approximately 4:1 (v:w) ACN:tissue. The homogenized sample was then centrifuged at -3000 rpm for 10 minutes. The supernatant was rqjjnoved and taken to dryness by nitrogen convection, dissolved in internal standard solution (0.5 )g/mL perfluorononanoic acid in ACN), and analyzed by HPLC-MS. 4. High performance liquid chromatography - mass spectrometry (HPLC-MS) a. Chromatographic Method Method: Column: Column temperature: Mobile phases: Gradient: Flow rate: Stop time: Injection volume: Plasma concentration analysis Waters Xterra MS C18, 2.1x30 mm, 2.5 /tm Ambient A: 50 mM Ammonium Acetate B: ACN Time (min) 0.0 0.5 10.0 10.9 11.0 %B 10 10 100 100 10 0.25 mL/min 11.0 min 5 iL 22 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 b. Mass Spectrometer Method System: Detector: Mode: Source: LM 1 resolution: HM 1 resolution Ion energy 1: Entrance: Collision: Exit: LM 2 resolution: HM 2 resolution: Ion energy 2: Multiplier (V): Capillary (kV): Cone (V): Extractor (V): RF lens (V): Source temperature: Desolvation temperature MS Method: Mode: Time: Chi: Ch2: Limit of Quantitation Waters 2790 Liquid Chromatograph, equipped with quaternary pump, column heater, and autosampler Quattro Micro Mass Spectrometer Multiple Reaction Monitoring (MRM) Negative Electrospray 10.0 10.0 1.0 5 2 5 14 14 2.0 650 2.0 -15 0 0 130C 350C MRM, 2 transitions 0-11 min 413.00 -- 369.00 Dwell: Collision energy: 469.00 -- 419.00 Dwell: Collision energy: 0.05 /ig/mL 0.25 sec 15 eV 0.25 sec 15 eV STATISTICAL ANALYSES For all quantitative data presented in the report, descriptive statistics were performed that included calculation of mean and standard deviation. 23 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 RESULTS AND CONCLUSIONS PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 ANALYTICAL DATA A. Test Substance Stability Analyses Samples of the test substance were analyzed near the beginning and end of the study. These analyses indicated that PFOA was stable over the course of the study. The average of the active ingredient was 91.9 1.0% and 96.6 2.1% for samples analyzed July 14, 2003 and September 4, 2003.(4) The PFOA was reported by the sponsor to be 95.297.99% pure. The difference between the sponsor reported purity and the experimental data are most likely due to analytical variability. B. Test Substance Formulation Analyses (Table 1, Appendix A) 1. Chromatography PFOA eluted from the HPLC column as a resolved peak with a retention time of approximately 4.9 to 5.2 minutes (5 ju.Lor 50 jaL injections) for the negative ion. Representative LC-MS/MS chromatograms are shown in Appendix A, Figures 2 (a - f). Test substance was not detected in the 0 mg/mL control formulation. [1,2-di- C] perfluorooctanoic acid used as an internal standard introduced a detectable peak in the control (0 mg/mL sample) in the stud^^This was determined by comparison of the NANOpure deionized water (diluent) used as a washln the LC/MS analysis and the NANOpure deionized water (diluent) with internal standard along with the 0 mg/mL sample (control) with internal standard. Refer to Appendix A, Figures 2 (a - c). 2. Recovery Samples Detailed analytical results of recovery samples are summarized in Appendix A, Table I. The variability of the analytical method was demonstrated by the coefficients of variation of the recovery results at each targeted dosing concentration (approximately 0.6, 2.0, 6.0 mg/mL) over the course of the study. The measured concentrations of PFOA for the 0.6 mg/mL level were 92.0% to 106.2% of nominal (mean percent recovery = 97.0 8.0%, C.V. = 8%). The measured concentrations of PFOA for the 2.0 mg/mL level were 83.0% to 100.2% of nominal (mean percent recovery = 92.6 8.8%, C.V. = 9%). The measured concentrations of PFOA for the 6.0 mg/mL level were 90.3% to 108.5% of nominal (mean percent recovery = 99.2 9.1%, C. V. = 9%). Based on this data, the analytical method performed satisfactorily over the entire concentration range for the study. 3. Uniformity of Mixing/Concentration and Stability Samples Analytical results from dosing solutions collected on July 11, 2003 and analyzed for uniformity of mixing/concentration verification and 5-hour room temperature stability are shown in Appendix A, Table II and Summary Table 1. 25 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 The following table summarizes the results for homogeneity/concentration verification and stability analyses for this sampling day of the PFOA preparation. Sample Day Sample Type Nominal mg/mL Measured a mg/mL Mean % Nominal C.V. Stability15 (%) % Nominal 11-July-2003 Uniformi ty/Concentration 0 0.60 2.0 6.0 NDC 0.613,0.558 2.21,2.07 6.68,6.30 97.7 107.0 108.2 7 102.0 5 102.0 4 104.0 a Mean results for the analysis of the duplicate samples, b Samples held 5 hours at room temperature. c Denotes none detected. Reported results are based on showing that the internal standard addition to the control caused a detectable peak for the test substance in the control. Shown in Figures 2 (a - c). The results for PFOA samples prepared and collected on July 11, 2003 indicated that the test substance was at the targeted concentrations ( 8.2% of nominal), adequately mixed (CV less than 10) and stable in the vehicle when held 5 hours at room temperature for all levels. Test substance was not detected in the 0 mg/mL samples. 4. Concentration Verification Samples Analytical results from dosing solutions collected on July 31, 2003 and August 21^003 and analyzed for concentration verification are shown in Appendix A, Table HI and Sumrhary Table 1. The following table summarizes the results for concentration verification analyses for both sampling days of the PFOA preparation. Preparation Day Nominal mg/mL Measured" mg/mL Average % Nominal CV % 31-July-2003b 21-Aug-2003C 0.60 2.0 6.0 0.6 2.0 6.0 0.497, 0.493 1.72, 1.69 4.88, 5.07 0.596, 0.613 1.90, 1.99 5.45, 5.04 82.5 85.5 83.0 100.7 97.5 87.5 1 1 3 2 3 5 a Duplicate samples per level were analyzed. C.V. calculated to verify uniformity o f mixture, b Reported results are the mean o f duplicate re-sampling of the original submitted samples because the original sample analysis was not acceptable due to standard curve preparation, c Reported results are from re-sampling o f the original submitted samples and dilution to a higher level for analysis (5pL injection) because the original sample analysis was not acceptable due to standard curve preparation. The results for samples prepared and collected on July 31,2003 indicated that the test substance was at the targeted levels ( 17.5% of nominal) and adequately mixed (CV less than 10) for all PFOA samples. Test substance was not detected in the 0 mg/mL samples. 26 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats D uPont-13 309 The results for samples prepared and collected on August 21, 2003 indicated that the test substance was at the targeted levels ( 12.5% of nominal) and adequately mixed (CV less than 10) for all PFOA samples. Test substance was not detected in the 0 mg/mL samples. C. Analytical Conclusions Results from the analysis of the PFOA dosing solutions during the study indicate that the test substance was mixed properly (CV less than 10), at the targeted levels ( 20% of nominal) and stable under the conditions of the study. Test substance was not found in the 0 mg/mL samples. A * 27 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 REPRODUCTIVE TOXICITY EVALUATIONS IN-LIFE TOXICOLOGY A. Maternal Observations (Figures 1 and 2, Appendices B, C, and D) There was no mortality at any level tested; all animals on study survived to scheduled sacrifice. There were no test substance-related clinical observations. Means and standard deviations fo r body weight and body weight gain data are in Appendices C and D. Mean body weight gain at 30 mg/kg/day was lower than control during days 4-10G (24.9g vs 34.5g for the control group) and days 10-15G (28.7g vs 33.8g in the control group). These reductions resulted in overall weight gain during gestation that was 10% lower than the control group (138.lg vs 152.7g for the control group) for days 4-21G. Mean body weight gain at 30 mg/kg/day was similar to control during lactation (11.2g vs 7.8g for the control group). However, mean daily body weights remained approximately 4% lower than control throughout gestation and lactation for this group. Body weights and body weight gain at 3 and 10 mg/kg/day during gestation and lactation were similar to the control group. \ Mean Body Weight Gain (grams) for Selected Intervals During Gestation Gestation Days 0 Dose (mg/kg/day) 3 10 30 4-10 34.5 8.28 10-15 33.8 7.16 4-21 152.7 15.76 Data presented: Means Standard Deviation 32.7 10.57 33.5 9.21 144.4 24.66 36.4 10.49 35.3 5.83 159.7 20.16 24.9 7.93 28.7 8.49 138.1 33.71 B. Reproductive Data 1. Gestation Subsets (Appendix E) All animals were pregnant at scheduled sacrifice. The number of implantation sites, resorptions, and live fetuses were comparable across groups that were sacrificed on either day 10G, 15G, or 21G. 28 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 2. Lactation Subset (Appendices F, G, and H) All animals delivered a live litter. There were no test substance-related clinical observations in litters during lactation. Pup survival and pup weights during lactation were comparable across groups; there was no test substance-related pup mortality during lactation. There were 2 small litters in the 30 mg/kg/day lactation group. Due to the intrinsic variability in litter size and the small number of litters evaluated (5 per group) the relationship of this finding to test substance administration is equivocal. Mean Standard Deviation Number of Pups Bom for Individual Litters Dose (mg/kg/day} 0 3 10 13 15 12 14 4 14 19 14 12 16 16 14 12 13 14 14.8 2.77 12.4 4.83 13.2 1.10 ^ X ' 30 13 3 6 11 13 9.2 4.49 3029 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 BIOCHEMICAL TOXICOLOGY A. Maternal, Fetal, and Pup Plasma (Tables 2-3, Figures 3-5, Appendix I) The concentration of PFOA in maternal plasma appeared to be at steady state over the range of time points sampled. The mean concentrations at steady state were 11.2, 26.8, and 66.6 pg/mL at 3, 10, and 30 mg/kg dose levels, respectively. The mean concentrations of PFOA in fetal plasma collected on day 21G were 5.9, 14.5, and 33.1 pg/mL at 3, 10, and 30 mg/kg dose levels, respectively. Pup plasma concentration decreased from day 3PP (means of 2.9, 5.9, and 12.0 pg/mL at 3, 10, and 30 mg/kg, respectively) to day 7PP (0.7, 2.8, and 4.9 pg/mL at 3, 10, and 30 mg/kg/day, respectively), and were similar on days 7, 14, and 21PP at all dose levels. B. Milk (Table 4, Figure 6, Appendix I) The concentration of PFOA in milk was at steady state from day 3PP to day 21PP at all dose levels. The mean concentrations at steady state were 1.1, 2.8, and 6.2 pg/mL at 3, 10, and 30 mg/kg dose levels, respectively. These concentrations are approximately ten times less than the steady state concentrations observed in maternal plasma. The milk concentrations appeared to be generally comparable to the concentrations in pup plasma. C. Amniotic Fluid (Table 5, Figure 7, Appendix I) The concentration of PFOA in amniotic fluid was approximately four times higher on day 21G (means of 1.5, 3.8, and 8.1 pg /mL at 3, 10, and 30 mg/kg, respectively) than on day 15G (means of 0.6, 0.7, and 1.7 pg /mL at 3, 10, and 30 mg/kg, respectively). D. Placenta (Table 6, Figure 8, Appendix I) The concentration of PFOA in placenta was approximately two times higher on day 21G (means of 3.6, 9.4, and 24.4 pg /mL at 3, 10, and 30 mg/kg, respectively) than on day 15G (means of 2.2, 5.1, and 13.2 pg /mL at 3, 10, and 30 mg/kg, respectively). E. Embryo and Fetus (Table 7, Figure 9, Appendix I) The day 10G embryo had the highest concentration of PFOA per gram of tissue (1.4, 3.3, and 12.5 pg/mL at 3, 10, and 30 mg/kg, respectively), and the day 21G fetus had slightly lower levels (1.3, 2.6, and 8.8 pg/mL at 3, 10, and 30 mg/kg, respective). The day 15G embryo had a lower tissue concentration than tissue from day 10G or day 21G (0.2, 0.5, and 1.2 pg/mL at 3, 10, and 30 mg/kg, respectively). 330 / PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 CONCLUSIONS Under the conditions of this study, there was maternal toxicity (reduced body weight and weight gain) at 30 mg/kg/day. The mean number of pups bom was lower than control at 30 mg/kg/day, a finding that was considered possibly test substance-related. Concentrations of PFOA in maternal plasma and milk were at steady state during the sampling interval. Steady state concentration in milk was approximately ten times less than the steady state concentration in maternal plasma. The concentration of PFOA in fetal plasma on day 21G was approximately half the steady state concentration in maternal plasma. The milk concentrations appeared to be generally comparable to the concentrations in pup plasma. Pup plasma concentrations decreased from days 3 to 7PP, and were similar on days 7, 14, and 21PP at all dose levels. PFOA was detected in placenta (days 15 and 21G), amniotic fluid (days 15 and 21G), embryo (days 10 and 15G) and fetus (day 21G). RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. A * 31 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 REFERENCES 1. Vanden Heuvel, J.P., Kuslikis, B.I., Van Rafelghem, M.J. and Peterson, R. E. (1991). Tissue Distribution, Metabolism, and Elimination of Perfluorooctanoic Acid in Male and Female Rats. J. Biochem. Toxicol. 6 920, 83-92. 2. DuPont Haskell Laboratory (2003). Perfluorooctanoic Acid: Toxicokinetics in the Rat. Unpublished report, DuPont-7473. 3. Butenhoff, J.L., Kennedy, G.L., Frame, S.R., O'Connor, J. and York, R.G. (2004). The reproductive toxicology of ammonium perfluorooctanoate (APFO) in the rat. From: Argus Laboratories (2002). Oral (gavage) 2-generation (one litter per generation) reproduction study of ammonium perfluorooctanoate (APFO) in rats. 4. DuPont Haskell Laboratory (2002). DuPont Haskell Laboratory Medical Research Project Number (MR) 5672. Unpublished Report. DuPont Haskell Laboratory Analytical Report Number DuPont-13707 and DuPont-13708. 5. Hazard Evaluation Division, Standard Evaluation Procedure, Toxicity Potential: Guidance for Analysis and Evaluation of Subchronic and Chronic Exposure Studies Paynter, O. E. et al., United States Environmental Protection Agency, Office of Pesticide Programs, Washington, D.C., 20406. EPA-540/9-85-020. (June 1985). V 6. Risk Assessment of Notified New Substances. Technical Guidance Document (XI/283/94EN), Chapter I, Sections 2.24 and 2.25. 1994. 32 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 TABLES * 3433 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats TABLES EXPLANATORY NOTES Abbreviations G LOQ = <LOQ = N.A. = PP S.D. = Gestation Limit of Quantitation, 0.05 pg/mL. Less than limit of quantitation Not applicable Postpartum Standard Deviation DuPont-13309 \ N <n 34 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 TABLE 1 SUMMARY OF DOSING TEST FORMULATION ANALYSES Sample Type Dosing Concentrations and Stability of PFOA (mg/mL) Uniformitv/Concentration July 11,2003 #1 Nominal: #2 Average Measured Conc.b Average Percent NominaIb Standard Deviation11 Coefficient of Variation11 0.6 0.613 (102.2)a 0.558 (93.0) 0.586 (97.7) 0.04 7% 2.0 2.21 (110.5) 2.07 (103.5) 2.14 (107.0) 0.10 5% 6.0 6.68 (111.3) 6.30 (105.0) 6.49 (108.2) 0.27 4% Stability 5-hour Room Temperature 0.612 (102.0) 2.04 (102.0) 6.24 (104.0) Concentration Verification July 31, 2003 #1 #2 Average Measured Conc.c Average Percent Nominal' Standard Deviation' Coefficient of Variation' 0.497 (82.8) 0.493 (82.2) 0.495 (82.5) 0.003 1% 1.72 (86.0) 1.69 (84.5) 1.71 (85.5) 0.02 1% \ 4-88 V (81.3) 5.07 (84.5) 4.98 (83.0) 0.13 3% August 21, 2003 #1 0.596 (99.3) 1.90 (95.0) 5.45 (90.8) #2 Average Measured Conc.d Average Percent Nominal*1 Standard Deviation*1 Coefficient of Variation*1 0.613 (102.2) 0.604 (100.7) 0.01 2% 1.99 (99.5) 1.95 (97.5) 0.06 3% 5.04 (84.0) 5.25 (87.5) 0.29 5% a Numbers in parentheses are the respective percent o f nominal values. b The average measured concentration, average percent o f nominal (in parentheses), standard deviation, and coefficient o f variation o f duplicate samples. c The average measured concentration, average percent o f nominal (in parentheses), standard deviation, and coefficient of variation o f the mean o f duplicate re-dilution o f the original samples, d The average measured concentration, average percent o f nominal (in parentheses), standard deviation, and coefficient of variation o f the mean o f re-dilution o f the original samples to higher level for analysis (5pL injection). 35 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 TABLE 2 CONCENTRATION (jig/mL) OF PFOA IN MATERNAL PLASMA DURING GESTATION AND POSTPARTUM 0 mg/kg Day Average S.D. 10G 15G 21G 3PP 7PP 14PP 21PP <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ NA NA NA NA NA NA NA Average <LOQ S.D. NA 3 mg/kg Average S.D. 8.53 15.92 14.04 11.01 10.09 9.69 9.04 1.06 12.96 2.27 2.11 2.90 0.92 1.01 11.19 2.76 10 mg/kg Average S.D. 23.32 29.40 34.20 22.47 25.83 23.79 28.84 2.15 14.19 6.68 2.74 2.07 2.81 5.15 26.84 4.21 30 mg/kg Average S.D. 70.49 79.55 76.36 54.39 66.91 54.65 64.13 8.94 3.11 14.76 17.86 11.82 11.63 1.45 66.64 9.80 V 36 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 TABLE 3 CONCENTRATION (jttg/mL) OF PFOA IN FETAL AND PUP PLASMA Sample Fetal Pup Pup Pup Pup Pay 21G 3PP 7PP 14PP 21PP 0 mg/kg ______3 mg/kg____ 10 mg/kg Average S.D. Average S.D. Average S.D. <LOQ <LOQ <LOQ <LOQ <LOQ NA NA NA NA NA 5.88 0.69 2.89 0.70 0.65 0.20 0.77 0.10 1.28 0.72 14.48 5.94 2.77 2.22 3.25 1.51 1.44 0.58 0.38 0.52 30 mg/kg Average S.D. 33.11 11.96 4.92 4.91 7.36 4.64 1.66 1.28 1.12 2.17 V V 37 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats TABLE 4 CONCENTRATION (jug/mL) OF PFOA IN MILK DuPont-13309 ___ P ay 0 mg/kg . _______ 3 m ^ g _______ 10 mg/kg Average S.D. Average S.D. Average S.D. 3PP 7PP 14PP 21PP <LOQ <LOQ 0.21 <LOQ NA NA NA NA 1.07 0.26 0.94 0.22 1.15 0.06 1.13 0.08 2.03 0.33 2.74 0.91 3.45 1.18 3.07 0.51 Average NAa 1.07 2.82 S.D. NA 0.09 0.60 a Average not calculated because 3 o f 4 values <LOQ. 30 mg/kg Average S.D. 4.97 1.20 5.76 1.26 6.45 1.38 7.48 1.63 6.16 1.06 \ N, is 3? PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 TABLE 5 CONCENTRATION Og/mL) OF PFOA IN AMNIOTIC FLUID 0 mg/kg Day Average S.D. 15G <LOQ NA 21G <LOQ NA 3 mg/kg__ Average S.D. 0.60 0.69 1.50 0.32 10 mg/kg Average S.D. 0.70 0.15 3.76 0.81 30 mg/kg Average S.D. 1.70 0.91 8.13 0.86 \ X* 39 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 TABLE 6 CONCENTRATION Qig/g) OF PFOA IN PLACENTA 0 mg/kg _________3 mg/kg_______ 10 mg/kg Day Average S.D. Average S.D. Average S.D. 15G <LOQ 21G <LOQ NA NA 2.22 1.79 3.55 0.57 5.10 1.70 9.37 1.76 30 mg/kg Average S.D. 13.22 24.37 1.03 4.13 c 40 4</ PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats D uP ont-13 309 TABLE 7 CONCENTRATION Qig/g) OF PFOA IN EMBRYO/FETUS Tissue Embryo Embryo Fetus 0 mg/kg . ____ 3 mg/kg____ 10 mg/kg Day Average S.D. Average S.D. Average S.D. 10G <LOQ NA 15G <LOQ NA 21G <LOQ NA 1.40 0.30 0.24 0.19 1.27 0.26 3.33 0.81 0.53 0.18 2.61 0.37 30 mg/kg Average S.D. 12.49 1.24 8.77 3.50 0.22 2.36 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 FIGURES V 42 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats FIGURES EXPLANATORY NOTES Abbreviations G LOQ = <LOQ = PP Gestation Limit of Quantitation, 0.05 pg/mL. Less than limit of quantitation Postpartum DuPont-13309 \ X 43 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats FIGURE 1 MEAN BODY WEIGHTS OF FEMALE RATS DURING GESTATION DuPont-13309 44 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats FIGURE 2 MEAN BODY WEIGHTS OF FEMALE RATS DURING LACTATION DuPont-13309 45 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 FIGURE 3 CONCENTRATION OF PFOA IN MATERNAL PLASMA DURING GESTATION AND POSTPARTUM Note: Concentration in the 0 mg/kg group = <LOQ (0.05 pg/mL). 4 746 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 FIGURE 4 CONCENTRATION OF PFOA IN PUP PLASMA (so o Co O 3PP 7PP 14PP 21PP Post-Partum Day Note: Concentration in the 0 mg/kg group = <LOQ (0.05 pg/mL). 47 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 FIGURE 5 CONCENTRATION OF PFOA IN FETAL PLASMA ON GESTATION DAY 21 49 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats FIGURE 6 CONCENTRATION OF PFOA IN MILK DuPont-13309 3 mg/kg - a--10 mg/kg -- 30 mg/kg 3PP 7PP 14PP 21PP Postpartum Day Note: Concentration in the 0 mg/kg group = <LOQ (0.05 pg/mL). * 49 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 FIGURE 7 CONCENTRATION OF PFOA IN AMNIOTIC FLUID Note: Concentration in the 0 mg/kg group = <LOQ (0.05 pg/mL). 5/ PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 FIGURE 8 CONCENTRATION (pg/g) OF PFOA IN PLACENTA 30.00 i 25.00 H Note: Concentration in the 0 mg/kg group = <LOQ (0.05 pg/mL). 51 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 FIGURE 9 CONCENTRATION (pg/g) OF PFOA IN EMBRYO/FETUS 18 1 16 - 14 - Day 10 Day 15 Gestation Day Day 21 Note: Concentration in the 0 mg/kg group = <LOQ (0.05 pg/mL). PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 APPENDICES N* 53 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 APPENDIX A ANALYTICAL DATA 54 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats D u P on t-13309 Table 1. Recovery of PFOA Added to Dosing Vehicle Sample mg/ml PFOA Percent Type Nominal Measured Nominal R ec o v er y (A) R ec o v er y(B) R e c o v e r y (C) 0.584 0.585 0.587 0.620 106.2 0.542 92.6 0.540 92.0 Mean: 97.0 8.0, C.V. 8% R ec o v er y (A) Recovery Recovery 1.95 1.953 100.2 1.95 1.618 83.0 1.96 1.854 94.6 Mean: 92.6 8.8, C.V.9% Rec o v er y (A) 5.84 6.336 108.5 Rec o v er y (B) 5.85 5.283 90.3 Recovery 5.87 5.799 98.8 Mean: 99.2 9.1, C.V.9% V (A) Processed with uniformity of mixing/concentration verification samples from dosing prepared on July 11, 2003. (B) Processed with concentration verification samples from dosing prepared on July 31, 2003. (C) Processed with concentration verification samples from dosing prepared on August 21,2003. 55 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Sample Type 11-July-2003 Uniformitv/Concentration Control mg/mL PFOA Nominal Measured 0.00 nd(A) Percent Nominal --- #1 0.60 0.613 102.2 #2 0.60 0.558 93.0 Mean{B): 0.5860.04 (97.7%) C.V. 7% #1 2.0 2.21 110.5 #2 2.0 2.07 103.5 Meanm : 2.14 0.10 (107.0%) C.V. 5% #1 6.0 6.68 111.3 #2 6.0 6.30 105.0 Meanm : 6.49 0.27 (108.2%) C.V. 4% 5 H o u r (C) 0.60 2.0 6.0 0.612 2.04 6.24 102.0 102.0 104.0 . (A) Denotes none detected. Reported results are based on showing that the internal standard addition to the control caused a detectable peak for the test substance in the control. Shown in Figures 2 (a - c). * (B) The average measured concentration, average percent o f nominal (in parentheses), standard deviation, and coefficient of variation o f duplicate samples. (C) Stability samples held 5 hours a room temperature. 56 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Table III. Concentration Verification of PFOA in Dosing solutions Preparation Day mg/mL PFOA Sample Type Nominal Measured 31-July-2003 Concentration Verification (A) Control 0.0 n d (A)B) Percent Nominal --- #1 0.60 0.497 82.8 #2 0.60 0.493 82.2 Mean(C): 0.495 0.003 (82.5) C.V. 1% #1 2.0 1.72 86.0 #2 2.0 1.69 84.5 Mean{C): 1.71 0.02 (85.5) C.V. 1% #1 6.0 4.88 81.3 #2 6.0 5.07 84.5 MeanC:>: 4.98 0.13 (83.0) C.V. 3% 21-Aug-2003 Concentration Verification(A) Control #1 #2 0.0 n d (B) 0.60 0.596 0.60 0.613 Mean(D): 0.604 0.01 C.V. 2% \ --- 99.3 102.2 (100.7) #1 2.0 1.90 95.0 #2 2.0 1.99 99.5 Mean(D): 1.95 0.06 (97.5) C.V. 3% #1 6.0 5.45 90.8 #2 6.0 5.04 84.0 Mean(D): 5.25 0.29 (87.5) C.V. 5% (A) The average measured concentration, average percent o f nominal (in parentheses), standard deviation, and coefficient of variation of duplicate samples. (B) Denotes none detected. Reported results are based on showing that the internal standard addition to the control caused a detectable peak for the test substance in the control. Shown in Figures 2 (a - c). (C) Mean result for analyses for duplicate re-sampling o f the original samples. Original analysis o f samples was not evaluated due to error in standard curve preparation. (D) Mean result for analyses for re-sampling o f the original samples and dilution to a higher level (5pL injection). Original analysis of samples was not evaluated due to error in standard curve preparation. 57 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Figure 1 Representative Analytical Calibration Curves DuPont-13309 Figure la: Calibration curve showing linear fit (line) to replicate peak area ratio (squares) for calibration solutions of PFOA diluted with NANOpure deionized water and matrix matched over a concentration range of 0.00051 to 0.00511 ppm. Figure lb: Calibration curve showing linear fit (line) to replicate peak area ratio (squares) for calibration solutions of PFOA diluted with NANOpure deionized water and matrix matched over a concentration range of 0.0050 to 0.0485 ppm. S7 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Figure 2 Representative LC/MS/MS Chromatography Chromatograms DuPont-13309 Figure 2a: Representative LC/MS chromatogram of NANOpure deionized water (samplecL(luent) used in the study. Negative ion/ perfluorooctanoic acid (PFOA) retention time is approximately 4.9 to 5.2 minutes. Figure 2b: Representative LC/MS chromatogram of NANOpure deionized water (sample diluent) used in the study with the internal standard. Negative ion/PFOA retention time is approximately 4.9 to 5.2 minutes. 59 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Figure 2 (continued) Representative LC/MS/MS Chromatography Chromatograms DuPont-13309 Figure 2c: Representative LC/MS chromatogram of 0.00 mg/mL control solution of PFO^with the internal standard. Negative ion/PFOA retention time is approximately 4.9 to 5.2*minutes. Figure 2d: Representative LC/MS chromatogram of PFOA analytical reference standard (0.00306 ppm) with the internal standard (0.0032 ppm). Negative ion/PFOA retention time is approximately 4.9 to 5.2 minutes. 60 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Figure 2 (continued) Representative LC/MS/MS Chromatography Chromatograms DuPont-13309 071103A31 Smooth(Mn,2x1) 100- PFOA 5.25 1320.24 15565 % ' MRM of 2 channels,ES413 >369 1.568e+004 ') ' P P l ... j....F f - f j I - f...I" 071 103A31 Sm ooth(Mn,2x1 100- 13CPFOA 5.24 1314.72 18613 % ? ,Vfflt P p lwW `^ V lW 1I" '| ' I1i1"I' l m in MRMof 2 channels,ES- 415 > 370 1.87 5e+004 6.00 "i t7i .0f0ni i r 11 I T8'P.0p0i T' Fr'ffW9.Y00*> i ( "i `i i r i10I'.|0m0 in Figure 2e: Representative LC/MS chromatogram of 0.6 mg/mL dosing solution diluted td'npminal concentration of 0.003 ppm of PFOA for negative ion /PFOA with retention time 5.2 minutes. The measured concentration of the representative solution is 0.613 mg/mL. Figure 2f: Representative LC/MS chromatogram of low recovery dosing solution (0.584 mg/mL) diluted to nominal concentration of 0.003 ppm of PFOA for negative ion /PFOA with retention time approximately 5.2 minutes. The measured concentration of the representative recovery solution is 0.620 mg/mL. PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 APPENDIX B X INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION 63 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION EXPLANATORY NOTES Note Day 21 = Gestation Day 21. Day 22 = Lactation Day 0. Day 43 = Lactation Day 21. A N. PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP I: 0 MG/KG/DAY ANIMAL NUMBER OBSERVATION DAYS ON WHICH SIGN WAS OBSERVED: 4,5,6,7,8,9,10,11, 12,13,14,15,16,17,18,19,20,21 7 SACRIFICED BY DESIGN 07/17/03 a : , , , , , ,10, Il II II t!! 9 SACRIFICED BY DESIGN 07/28/03 a: , , , , , , , , , , , , , , ,21 12 SACRIFICED BY DESIGN 07/28/03 a: , , , , , , , , ,21 15 SACRIFICED BY DESIGN 07/22/03 a : ! I l I I ! 1 / , , ,15................ 17 SACRIFICED BY DESIGN 07/22/03 a : I l I I t ! ! / , , ,15, , , , , , 20 SACRIFICED BY DESIGN 07/22/03 a: , , ,15, , , , , , 23 SACRIFICED BY DESIGN 07/22/03 a: , , ,15, ............ 31 SACRIFICED BY DESIGN 07/22/03 a: , , , , , , , , ,15............ ... 32 SACRIFICED BY DESIGN 07/17/03 a : , , , , , ,10, , , 34 SACRIFICED BY DESIGN 07/28/03 a : 1 1 1 1 1 1 1 / , , , , , , , , ,21 oy 36 SACRIFICED BY DESIGN 07/17/03 a : , , , , , ,10, , , 38 SACRIFICED BY DESIGN 07/28/03 a: , , , , , , ................ , , ,21 47 SACRIFICED BY DESIGN 07/17/03 a : , , , , , ,10, / 64 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP I: 0 MG/KG/DAY ANIMAL NUMBER: OBSERVATION DAYS ON WHICH SIGN WAS OBSERVED: 4 .5 .6 .7 .8 .9 .1 0 .1 1 .1 2 .1 3 .1 4 .1 5 .1 6 .1 7 .1 8 .1 9 .2 0 . 2 1 53 SACRIFICED BY DESIGN 07/28/03 59 SACRIFICED BY DESIGN 07/17/03 62 ALOPECIA BOTH FRONT PAW 66 ALOPECIA FACE ALOPECIA BOTH FRONT PAW 67 NO ABNORMALITIES DETECTED 75 NO ABNORMALITIES DETECTED 80 NO ABNORMALITIES DETECTED a: a: ,,,,,,,,,,,,,,,, ,21 ,10, , , , , ................................................................... a: 4 .5 .6 .7 .8 .9 .1 0 .1 1 .1 2 .1 3 .1 4 .1 5 .1 6 .1 7 .1 8 .1 9 .2 0 . 2 1 a: ,5 ,6 ,7 ,8 ,9 ,1 0 ,1 1 , , , , , , , , , , a: ,,,,,,,,,,,,,,, , 20,21 D uP ont-13 309 * / 65 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP I: 0 MG/KG/DAY ANIMAL NUMBER: OBSERVATION DAYS ON WHICH SIGN WAS OBSERVED: 22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43 62 ALOPECIA BOTH FRONT PAW ALOPECIA BOTH FRONT LEG SACRIFICED BY DESIGN 08/22/03 a 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.43 a , , , , , , , , , , , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43 a , ,,,,,,,,, , , ,, 4 3 66 ALOPECIA BOTH FRONT PAW ALOPECIA BOTH FRONT LEG SACRIFICED BY DESIGN 08/22/03 a 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42.43 a , , , , , , , , , , , 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43 a , ,,,,,,,, , , , , , , , , , ,, 4 3 67 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN08/22/03 a: ,43 75 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN08/22/03 a: ,43 80 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN08/22/03 a: \) ,43 / 66 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER OBSERVATION DAYS ON WHICH SIGN WAS OBSERVED: 4, 5,6,7,8,9,10,11,12 13,14,15,16,17,18 19,20,21 2 SACRIFICED BY DESIGN 07/28/03 4 SORE TOE BOTH FRONT PAW SACRIFICED BY DESIGN 07/28/03 22 SACRIFICED BY DESIGN 07/28/03 24 SACRIFICED BY DESIGN 07/28/03 25 SACRIFICED BY DESIGN 07/17/03 30 SACRIFICED BY DESIGN 07/22/03 40 SACRIFICED BY DESIGN 07/28/03 SACRIFICED BY DESIGN 07/22/03 44 SACRIFICED BY DESIGN 07/17/03 45 SACRIFICED BY DESIGN 07/22/03 50 SACRIFICED BY DESIGN 07/22/03 51 SACRIFICED BY DESIGN 07/22/03 54 SACRIFICED BY DESIGN 07/17/03 a: , , , , , , ,, a : 4, 5, , , , , , , a : - ............... a: - I l II ! ! ! a: - I l II ! 1 1 a : - , , , , ,10, a: / I l II ! ! / a: / , , , , , a: - I l I I ! ! ! a: - ........ 10, , a: - a: - < > ' ' - a: - I l I I ! /! a : ' , , , , ,10, Il II ! It Il ili II ! Itili lili! Itili , ,15, , , itili , ,15, , , , , ,15, , ,15, , ,15, , , ,,,,, , ,21 , ,21 , ,21 , ,21 , , ,21 , , , , /i fi / DuPont-13309 67 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats D uP ont-13 309 INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER: OBSERVATION DAYS ON WHICH SIGN WAS OBSERVED: 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 57 SACRIFICED BY DESIGN 07/17/03 58 SACRIFICED BY DESIGN 07/17/03 63 NO ABNORMALITIES DETECTED 69 NO ABNORMALITIES DETECTED 72 NO ABNORMALITIES DETECTED 74 ALOPECIA BOTH FRONT PAW 76 NO ABNORMALITIES DETECTED a: , , , , , ,10, a: , , , , , , 1 0 , , , , , , , , , , , a , ,18,19,20,21 IN 0 / 68 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER: OBSERVATION DAYS ON WHICH SIGN WAS OBSERVED: 22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43 63 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 69 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 72 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 74 ALOPECIA BOTH FRONT PAW SACRIFICED BY DESIGN 08/22/03 76 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 a : ............................................... , , , , ,43 a : ............................ ... , .......................... 43 a : . - , , , . , , , , , , , , , , , , , , ,43 a : 22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43 a: / / 43! f ! 1 ! t 1 t t t 1111 1t 1! a : , , , , , , , , , , , , , , , , , , , , ,43 0 / 69 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP III: 10 MG/KG/DAY ANIMAL NUMBER OBSERVATION 5 SACRIFICED BY DESIGN 07/22/03 8 SACRIFICED BY DESIGN 07/17/03 13 SORE TOE LEFT FRONT PAW SACRIFICED BY DESIGN 07/22/03 14 SACRIFICED BY DESIGN 07/28/03 16 SORE TOE BOTH REAR PAW SORE TOE BOTH FRONT PAW SACRIFICED BY DESIGN 07/17/03 18 ALOPECIA BOTH FRONT PAW SACRIFICED BY DESIGN 07/22/03 19 SACRIFICED BY DESIGN 07/17/03 21 ALOPECIA LEFT FRONT PAW SACRIFICED BY DESIGN 07/28/03 28 SACRIFICED BY DESIGN 07/28/03 33 SACRIFICED BY DESIGN 07/17/03 35 SACRIFICED BY DESIGN 07/28/03 DAYS ON WHICH SIGN WAS OBSERVED: 4, 5, 6,7,8,9,10,11,12,13,14,15,16,17,18,19 20,21 a : , , , , , , , , ,15, , , , a: a : 4, a: a: , , , ,10, , , , , , , , , , ,15, , , , /! I I ! ! Il II 11 ! - ' ,21 a : 4, 5, 6 ......... ! I l I I I I ! a : 4, 5, 6, , , , a : , , , ,10, a : 4, 5, 6,7,8,9,10, 11,12,13,14,15, a : ......... , , , ,15, , , , a : , , , ,10, ................ ' ' - a : , , , ,10, 11,12,13,14,15,16,17,18,19 20,21 a : ......... ,21 a: ill! /, ,21 a: , , , ,10, ! I l I I I I ! < a: ................... ! I l I I I I ! ,21 0 / DuPont-13309 70 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP III: 10 MG/KG/DAY ANIMAL NUMBER: OBSERVATION DAYS ON WHICH SIGN WAS OBSERVED: 4 ,5 ,6 ,7 ,8 ,9 ,1 0 ,1 1 ,1 2 ,1 3 ,1 4 ,1 5 ,1 6 ,1 7 ,1 8 ,1 9 ,2 0 , 2 1 39 SACRIFICED BY DESIGN 07/17/03 43 SACRIFICED BY DESIGN 07/22/03 46 SACRIFICED BY DESIGN 07/22/03 60 SACRIFICED BY DESIGN 07/28/03 64 NO ABNORMALITIES DETECTED 65 NO ABNORMALITIES DETECTED 68 NO ABNORMALITIES DETECTED 77 NO ABNORMALITIES DETECTED 79 NO ABNORMALITIES DETECTED a: 10, , , , , , , , , , , , , , , , , a: , , , , , , , , , , ,1 5 , , , , , , a: , , , , , , , , , , ,1 5 , , a: .......... , , , , , , ..................... 21 DuPont-13309 / 71 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP III: 10 MG/KG/DAY ANIMAL NUMBER: OBSERVATION DAYS ON WHICH SIGN WAS OBSERVED: 22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43 64 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 65 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 68 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 77 ALOPECIA NECK ALOPECIA BOTH FRONT PAW ALOPECIA BOTH FRONT LEG SACRIFICED BY DESIGN 08/22/03 79 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 a: a: a a: a: a: a: a: ,43 ,43 ,43 ,32,33, ,35,36,37,38,39,40,41,42,43 , , , , , , , , ,43 , , , , , , , , ,43 , , , ,43 / 72 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER OBSERVATION 1 SACRIFICED BY DESIGN 07/28/03 3 SACRIFICED BY DESIGN 07/17/03 6 SACRIFICED BY DESIGN 07/22/03 10 SACRIFICED BY DESIGN 07/17/03 11 SACRIFICED BY DESIGN 07/17/03 26 SACRIFICED BY DESIGN 07/22/03 27 SORE TOE BOTH FRONT PAW SACRIFICED BY DESIGN 07/28/03 29 SACRIFICED BY DESIGN 07/22/03 37 SACRIFICED BY DESIGN 07/28/03 42 SACRIFICED BY DESIGN 07/22/03 48 SACRIFICED BY DESIGN 07/22/03 49 SACRIFICED BY DESIGN 07/28/03 52 ALOPECIA BOTH FRONT LEG DAYS ON WHICH SIGN WAS OBSERVED: 4,5 6,7,8,9,10,11,12,13,14,15, 16, 17, 18,19,20 ,21 a: , #, ,, , , , r i > i i t , ,21 a : - , , , ,10, , , , , , < - - a : ! .......................15, - //< a: < , , , ,10, , , , , , - f1 - a: , , , ,10, , , , , , < // a : < ...................... 15, a : 4,5 6 ......................... a: ' ' ' ' ' 1 ' i i ' i a : > ............ , , ,15, , ' - /! ' , ' ' ,21 / a: / , , , , , , , , , , - - ,21 a : - ...................... 15, - - a : / , , , , , , , , ,15, / a: - , , , , , , , , , , / - ,21 a : t , , ,9,10,11,12,13,14,15, 16, 17, 18,19,20 ,21 fi / DuPont-13309 73 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER: OBSERVATION DAYS ON WHICH SIGN WAS OBSERVED: 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 52 ALOPECIA BOTH FRONT PAW SACRIFICED BY DESIGN 07/28/03 55 SACRIFICED BY DESIGN 07/17/03 56 SACRIFICED BY DESIGN 07/17/03 61 ALOPECIA BOTH FRONT PAW ALOPECIA BOTH FRONT LEG 70 NO ABNORMALITIES DETECTED 71 NO ABNORMALITIES DETECTED 73 NO ABNORMALITIES DETECTED 78 NO ABNORMALITIES DETECTED a: , , , , , , , , ,13,14,15,16,17,18,19,20,21 a: .................. , , , , , , , , , , , ,21 a: , . , , , ,10...................... ... , , a: 10, , , , , , , , , , , , , , , , , a: , , , , , , , , , , , ,16,17,18,19,20,21 a: , , , , , , , , , , , , , , ,19,20,21 DuPont-13309 0 / K 74 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL CLINICAL OBSERVATIONS AND MORTALITY DATA DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER: OBSERVATION 61 ALOPECIA BOTH FRONT PAW ALOPECIA BOTH FRONT LEG SACRIFICED BY DESIGN 08/22/03 70 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 71 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 73 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 78 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN 08/22/03 DAYS ON WHICH SIGN WAS OBSERVED: 22,23,24,25,26,27,28,29,30,31, 32,33,34,35,36,37,38,39,40,41,42,43 . . . . . . . . . .a: 22,23,24,25,26,27,28,29,30,31 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 9 4 0 4 1 4 2 . 4 3 . . . . . . . . . .a: 22,23,24,25,26,27,28,29,30,31 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 9 4 0 4 1 4 2 . 4 3 , , , , , , , , , ,a : , , , , , , , , , ,43 a : , ,43 a : ,43 a ,43 a ,43 / 75 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 APPENDIX C X% INDIVIDUAL BODY WEIGHTS DURING GESTATION AND LACTATION 76 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS DURING GESTATION AND LACTATION EXPLANATORY NOTES Note Day 21 = Gestation Day 21. Day 22 = Lactation Day 0. Day 43 = Lactation Day 21. Abbreviation = No data V 77 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP I: 0 MG/KG/DAY ANIMAL NUMBER DAY 4 DAY 5 DAYS ON TEST DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 7 249.1 263.8 271.7 272.4 281.3 285.0 297.1 9 274.7 284.8 285.3 290.9 291.5 300.0 301.0 12 255.5 266.1 264.6 259.8 271.2 276.0 281.0 15 247.8 263.8 265.1 268.8 278.4 287.3 283.5 17 257.9 266.0 265.1 273.3 273.9 281.9 287.9 20 266.5 274.6 281.7 288.2 291.3 299.6 304.4 23 233.0 250.8 259.9 263.5 262.8 268.8 271.0 31 256.9 263.9 268.7 275.8 278.3 284.4 289.4 32 266.1 272.6 275.8 281.7 284.2 295.3 304.0 34 262.8 269.3 273.8 276.1 282.0 288.8 293.5 36 246.2 250.4 256.2 260.9 271.3 275.7 284.3 38 250.6 257.2 260.0 267.1 271.7 281.2 283.7 47 231.7 248.2 238.0 242.1 263.2 278.5 285.9 53 260.4 267.5 272.1 279.7 283.8 290.1 294.5 59 257.5 261.8 264.8 266.3 271.4 274.9 288.6 62 266.5 271.0 276.2 274.7 282.2 285.5 288.5 66 245.4 243.8 268.7 276.0 279.7 285.6 291.6 67 270.5 277.2 286.2 283.2 295.4 299.0 301.8 75 249.1 258.4 267.1 267.0 277.4 282.5 285.9 80 246.3 251.4 254.4 256.2 265.8 266.8 267.6 GROUP MEAN S.D. S.E. N 254.7 11.55 2.58 20 263 .1 10 .60 2 .37 20 267.8 11.25 2.52 20 271.2 11.52 2.58 20 277.8 9.09 2.03 20 284.3 9.50 2.12 20 '89.3 9*.84 2.20 20 78 79 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP I: 0 MG/KG/DAY ANIMAL NUMBER DAY 11 DAY 12 DAYS ON TEST DAY 13 DAY 14 DAY 15 DAY 16 DAY 17 7 9 311.2 316.1 324.8 331.9 338.0 357.3 369.9 12 292.1 294.0 293.8 306.4 311.4 318.1 335.9 15 295.1 306.0 307.3 320.9 337.3 - - 17 294.7 297.4 303.5 310.7 320.0 - - 20 317.1 323.3 328.7 341.0 348.1 - 23 280.1 281.7 285.4 293.5 299.9 - - _ 31 302.0 303.6 307.0 313.6 319.8 - - 32 - - - - - - - 34 306.9 308.3 317.3 323.2 327.9 342.1 359.1 36 38 292.8 295.5 300.6 308.6 313.5 326.4 335.8 47 -_-- -- - 53 306.2 310.8 313 .1 322.1 320.2 336.8 346.8 59 - - - - - - - 62 296.7 299.5 306.6 315.1 321.9 327.0 342.8 66 300.4 297.5 308.0 313.7 319.1 328.6 348.6 67 312.1 312.8 322.5 329.7 338.8 352.1 373.9 75 293.3 293.3 302.5 313.9 314.4 324.2 343.0 80 277.6 278.8 287.4 296.2 301.4 308.9 320.8 GROUP MEAN S .D. S.E. N 298.6 11.16 2.88 15 301.2 12.18 3.14 15 307.2 12.73 3.29 15 316.0 12.73 3.29 15 322.1 13.79 3.56 15 332.1 15.00 4.74 10 1 4 7 .7 16*20 5.12 10 79 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP I: 0 MG/KG/DAY ANIMAL NUMBER DAY 18 DAY 19 DAYS ON TEST DAY 20 DAY 21 DAY 22 DAY 23 DAY 24 7 --- 9 390.1 406.1 426.6 450.8 12 345.7 355.7 381.8 407.1 - - - 15 - - - - _ - 17 - _ - - - - - 20 23 " _ --_ _ __ 31 - ~ -- 32 :: _ 34 374.8 382.9 404.1 433.2 - -- 36 - _ - - - - - 38 349.7 358.7 375.9 399.1 - -- 47 - - - - - - 53 351.5 359.0 373.5 396.9 - -- 59 - - - - - - - 62 356.3 370.6 391.2 400.8 284.6 297.0 293.1 66 359.7 382.6 397.3 410.3 426.5 305.5 322.2 67 396.2 416.5 434.1 414.6 293.0 317.9 328.7 75 367.7 381.7 399.8 415.6 295.1 300.0 316.9 80 335.0 355.0 373.7 380.5 277.2 278.5 291.5 GROUP MEAN S.D. S.E. N 362.7 19.56 6.19 10 376.9 21.43 6.78 10 395.8 21.35 6 .75 10 410.9 19.72 6.24 10 315.3 62.58 27.99 5 299.8 14.33 6.41 5 S 4.O.5 17*12 7.66 5 80 8/ PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP I : 0 MG/KG/DAY ANIMAL NUMBER DAY 25 DAY 26 DAYS ON TEST DAY 27 DAY 28 DAY 29 DAY 30 DAY 31 7 -- 9 -- -- - 12 15 - - - - -- 17 :20 - - - - - - 23 31 - - - 32 - - -- 34 36 - - - - - - - 38 47 _ - _ - - _ _ 53 -- - -- - 59 - - - _ - - - 62 307.4 307.4 311.5 308.6 322.4 320.6 335.0 66 331.1 320.0 333.2 328.6 338.3 338.9 351.2 67 331.9 343.2 356.6 349.9 347.8 351.6 358.6 75 311.1 315.7 316.6 323.7 321.1 326.9 341.8 80 297.1 298.7 310.8 303.9 320.5 318.4 329.4 GROUP MEAN S.D. S.E. N 315.7 15.29 6.84 5 317.0 16.76 7.50 5 325.7 19.48 8.71 5 322.9 18.22 8.15 5 330.0 12.38 5.54 5 331.3 13.88 6.21 5 '3*43.2 l!41.84 5.30 5 81 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP I: 0 MG/KG/DAY ANIMAL NUMBER DAY 32 DAY 33 DAYS ON TEST DAY 34 DAY 35 DAY 36 DAY 37 DAY 38 7 ------ 9- 12 15 _ - - - - - - 17 - - - - - - - 20 - -_ 23 31 ~ "" - 32 34 - -- 36 38 - ~ _ _ "- 47 53 - - - - - - - 59 - - _ - - - - 62 334.7 331.8 329.9 321.8 322.1 326.8 324.7 66 347.2 356.0 353.7 361.1 357.0 354.4 361.5 67 366.1 367.2 369.2 362.0 356.7 369.7 365.5 75 341.1 343.7 333.5 336.3 334.7 343.5 332.2 80 329.6 337.3 346.2 333.5 343.0 348.3 351.0 GROUP MEAN S.D. S .E. N 343.7 14.15 6.33 5 347.2 14.36 6.42 5 346.5 15.90 7.11 5 342.9 17.84 7.98 5 342.7 14.91 6.67 5 348.5 15.65 7.00 5 ^.0 17.92 8.02 5 82 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP I: 0 MG/KG/DAY ANIMAL NUMBER DAY 39 DAYS ON TEST DAY 40 DAY 41 DAY 42 DAY 43 7 - ---- 9 12 15 -- - - 17 _- - - - 20 - - - - 23 _- - - - 31 -- - -- 32 - - - - - 34 _- -- - 36 - - _ - - 38 -- - -- 47 - - - - 53 - - - - 59 -- - - - 62 321.0 322.4 320.3 316.2 306.9 66 355.1 355.6 352.8 345.6 341.7 67 349.7 350.0 343.3 338.5 336.2 75 315.5 317.0 318.2 320.5 312.0 80 338.7 332.3 336.3 334.1 318.4 GROUP MEAN S .D. S.E. N 336.0 17.36 7.76 5 335.5 16.87 7.54 5 334.2 14.85 6.64 5 331.0 12.33 5.52 5 323.0 15.21 6.80 5 83 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats D u P o n t-13 309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER DAY 4 DAY 5 DAYS ON TEST DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 2 266..8 271 .1 276,.8 283 .0 289..4 294 ,.7 296 .5 4 248..3 244 .2 257 .0 263 .,4 265..5 272 .9 271 .8 22 243 .1 247 .6 256 ,.0 263 .,9 247..3 269 ,.6 254 .4 24 233 .6 244..8 253 ,.6 261.,0 269..6 279..6 284 .6 25 266 .0 271 .8 276 .0 284..5 290..0 294 .8 315 .1 30 252 .1 254 .8 260,.0 264..8 266..9 274 .8 276 .8 40 245 .8 244 .2 250 .9 255 .2 261..7 268 .7 270 .9 41 286 .4 291 .5 296..6 306 .8 305 .8 321..6 325 .6 44 259 .7 261..7 272 .9 276 .5 276 .4 288..3 294 .9 45 253 ,.2 266 .5 271..8 275 .0 278..2 287 .4 291 .4 50 247 .8 261..7 267 .7 274 .0 276 .3 285..4 288 .1 51 269,.3 278 .3 281.,1 293 .7 297 .,4 313 .2 313 .4 54 263 ,.9 267..7 271.,5 274 .8 278. 0 285 .,8 288 .0 57 263 .0 269..6 278..6 280. 1 283 ..1 289 .6 297 .1 58 262 ,.5 274 .7 281.,4 286 .5 290 .3 301..0 308 .2 63 248..5 255..6 262 ..5 266. 5 278 .7 279..8 287 .7 69 268..5 275..7 277 .,7 279 .0 288 .2 287 ..3 293 .4 72 277 .2 279..5 288..5 290 .2 295 ..3 296 .,6 306 .0 74 255 .7 258..3 266..0 266. 0 271..7 271.,1 279 .6 76 234 .7 231,.7 244 .4 253 .5 260..8 265 .3 255 .7 GROUP MEAN S .D. S .E. N 257.3 13.55 3 .03 20 262.5 14.93 3.34 20 269.6 13.30 2.97 20 274.9 13.55 3 .03 20 278.5 14.32 3.20 20 286.4 14.71 3.29 20 'M.O 18 68 4.18 20 84 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER DAY 11 DAY 12 DAYS ON TEST DAY 13 DAY 14 DAY 15 DAY 16 DAY 17 2 308.2 307.7 313.6 324.1 327.2 337.6 356.3 4 280.5 285.6 289.8 297.3 297.6 311.8 327.4 22 284.7 290.2 288.9 301.5 305.9 304.9 327.6 24 297.3 300.9 303.8 316.3 314.9 332.0 342.6 25 - - - - - - - 30 290.1 298.4 299.6 313.4 326.0 - - 40 285.3 284.9 290.1 296.2 299.3 312.7 322.0 41 336.4 339.7 340.6 348.8 365.5 - - 44 - _ - - - - - 45 300.5 307.7 310.2 318.4 324.3 - - 50 299.9 299.5 309.8 315.7 321.7 - - 51 324.2 333.9 339.1 350.2 357.4 - - 54 ---- --- 57 ------ - 58 - - - - - - - 63 295.7 301.9 312.3 321.9 308.4 330.8 347.8 69 299.2 299.6 314.9 312.7 317.9 320.4 332.2 72 317.3 313.6 331.0 329.5 331.1 338.5 361.3 74 286.7 290.1 300.5 305.0 307.6 319.1 334.3 76 252.3 252.6 272.1 280.8 293.4 306.6 318.2 GROUP MEAN S .D. S.E. N 297.2 19.94 5.15 15 300.4 20.55 5.30 15 307.8 19.11 4.93 15 315.5 18.63 4.81 15 319.9 20.45 5.28 15 321.4 12.57 3.97 10 'HJ-o 14*. 53 4.59 10 85 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER DAY 18 DAY 19 DAYS ON TEST DAY 20 DAY 21 DAY 22 DAY 23 DAY 24 2 4 22 24 25 30 40 41 44 45 50 51 54 57 58 63 69 72 74 76 GROUP MEAN S.D. S.E. N 372.6 337.1 329.6 353.9 333.3 _ _ - _ - 373.0 336.9 369.5 349.1 329.0 348.4 17.90 5.66 10 387.4 353 .0 344.4 369.0 351.1 _ " : - 385.3 349.5 386.5 368.5 343.9 363.9 17.76 5.62 10 405.8 374.7 361.1 385.8 369.8 _ - - 415.1 355.2 409.2 391.4 357.7 382.6 22.23 7.03 10 423.0 389.5 385.8 405.7 385.3 - ~ - 429.9 357.2 418.8 398.7 372.3 396.6 23.15 7.32 10 - - - _ - - - 295.4 355.3 429.3 289.5 275.2 328.9 63.91 28.58 5 ---- : _ - - _ - 306.5 301.3 313.2 297.5 277.9 299.3 13.33 5.96 5 : _ - - 325.5 305.6 322.2 306.3 285.2 V '*>9.0 16.06 7.18 5 86 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER DAY 25 DAY 26 DAYS ON TEST DAY 27 DAY 28 DAY 29 DAY 30 DAY 31 2 4 -- - 22 - - - - 24 - - - 25 -- 30 -_ - - 40 -- - 41 - - - 44 -- - 45 - - 50 -- - 51 - - 54 - - - 57 _ - - 58 - - - 63 323.8 322.0 335.4 335.0 356.1 341.9 346.5 69 307.8 300.0 304.7 297.0 305.8 297.5 304.8 72 320.5 328.8 338.4 342.6 351.0 344.0 351.7 74 309.2 306.3 317.3 311.5 312.5 318.3 322.7 76 287.0 301.3 302.3 301.7 305.9 299.0 306.4 GROUP MEAN S.D. S.E. N 309.7 14.44 6.46 5 311.7 12.97 5.80 5 319.6 16.81 7.52 5 317.6 20.26 9.06 5 326.3 25.12 11.24 5 320.1 22.39 10.01 5 ''* .4 21.93 9.81 5 87 88 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats D u P on t-13 309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER DAY 32 DAY 33 DAYS ON TEST DAY 34 DAY 35 DAY 36 DAY 37 DAY 38 2- -- - 4- -- 22 24 25 - - - - - - 30 40 41 _ : --" 44 -- - -" - - 45 50 - - - - - - - 51 54 - _ _ _ _ - _ 57 - - - - - - 58 - - - - - - - 63 364.6 371.0 369.9 359.2 359.4 356.8 359.4 69 307.5 313.4 312.9 305.3 297.1 297.8 303.8 72 350.8 356.3 357.2 346.8 358.5 366.8 373.4 74 336.0 337.1 334.3 342.0 333.1 344.0 343.3 76 319.4 320.7 313.5 317.3 321.6 323.2 321.4 GROUP MEAN S .D. S .E. N 335.7 23.04 10.31 5 339.7 24.06 10.76 5 337.6 25.64 11.47 5 334.1 22.17 9.91 5 333.9 26.28 11.75 5 337.7 27.63 12.36 5 28.10 12.57 5 4 LO PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER DAY 39 DAYS ON TEST DAY 40 DAY 41 DAY 42 DAY 43 2 4 22 -- _- - 24 - - - - - 25 - - - - - 30 - - - - - 40 -- - - - 41 -- _ -- 44 -_ - -- 45 - - - - - 50 - - - - - 51 - - _ - 54 - - - - - 57 ---_ - 58 ---_ - 63 348.2 352.3 360.4 351.1 345.3 69 291.3 292.2 299.1 297.4 300.2 72 357.9 357.3 365.0 351.1 342.9 74 324.7 324.9 331.7 323,7 315.3 76 312.7 312.5 311.9 309.7 305.8 GROUP MEAN S.D. S.E. N 327.0 26.87 12.02 5 327.8 27.30 12.21 5 333 .6 29.02 12.98 5 326.6 24.22 10.83 5 321.9 20.99 9.39 5 `fO PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP III: 10 MG/KG/DAY ANIMAL NUMBER DAY 4 DAY 5 DAYS ON TEST DAY 6 DAY 7 DAY 8 DAY 9 DAY 10 5 260 .1 262 .6 265 .3 268 .1 269..4 277 .1 2 84 .1 8 249 .3 261 .7 265 .3 271 .8 280 ,.5 288 .0 295 .7 13 259 .5 266 .7 273 .7 277 .6 284 ,.7 291..9 297 .0 14 264 .8 265 .1 272 .9 278 .3 278 .5 282..8 290 .4 16 247 .3 258 .5 259..7 270 .9 280..4 283 .3 294 .9 18 269..0 283 .5 288..0 295 .8 298..3 309..4 317 .6 19 263 .9 273 .0 277 .3 280 .6 284..3 295 .1 300 .9 21 236 .1 243 .0 247 ,.7 251..2 253 .7 264,.9 263 ,.7 28 217 .6 246 .0 225 .3 238 .6 254 .7 270 .1 274..9 33 248..3 255 .3 257 ,.6 262 .9 264 .2 270 .9 270..9 35 261..4 266 .7 273 .8 278 ,.7 283 .1 292 ,.1 294 .7 39 240 .8 247 .8 249.,9 253 .7 255..0 262 .0 268..9 43 250 .4 263 .5 268 .4 276 .0 279..9 289..5 289 .8 46 261 .0 267 .4 273 .1 275 .0 283 .5 289,.7 293 ,.8 60 260 .1 279..8 285..0 290 .7 300.,8 309..3 313 .4 64 256 .0 258..9 268..7 269 .2 279 .,6 279 .,1 284..6 65 222 .4 247 .7 251..5 253 .0 258 .6 266 .,5 269 .4 68 250 .0 256 .3 267 .6 266 .7 274 .2 273 ,.9 274 .6 77 271 .3 277 .4 287 .0 266 .8 296 .8 301..8 301..2 79 260 ,.0 266 .3 274..0 275 .3 285..4 290 .3 297 .2 GROUP MEAN S.D. S.E. N 252.5 14.30 3.20 20 262 .4 11. 15 2 .49 20 266.6 15.00 3.35 20 270.0 13.52 3.02 20 277.3 14.15 3.17 20 284.4 13 .89 3.11 20 14*. 83 3.32 20 ?/ PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP III: IO MG/KG/DAY ANIMAL NUMBER DAY 11 DAY 12 DAYS ON TEST DAY 13 DAY 14 DAY 15 DAY 16 DAY 17 5 293.1 299.9 300.0 304.5 315.6 - - 8 13 302.0 308.2 313.9 325.4 334.2 _ - 14 296.3 297.7 303.5 308.6 319.2 328.9 334.3 16 - -- - - - - 18 328.2 328.8 338.4 341.2 360.3 - 19 21 274.9 277.6 287.0 292.3 292.7 308.0 319.3 28 282.2 287.2 290.6 297.9 302.6 316.9 329.1 33 -- - - - _ - 35 307.1 311.3 314.1 328.5 335.0 352.1 365.6 39 43 304.5 309.0 313.8 322.9 335.3 _ __ 46 306.1 306.5 313.6 326.6 328.4 - 60 315.1 330.3 331.3 340.9 348.0 366.0 376.9 64 291.3 297.8 309.1 309.0 318.7 321.9 342.3 65 276.5 278.5 290.2 293.2 300.3 307.7 328.8 68 281.6 284.7 291.1 299.0 305.0 310.0 323.3 77 311.4 315.9 321.9 326.7 339.3 350.5 366.8 79 302.9 318.3 325.5 332.8 341.5 351.4 369.5 GROUP MEAN S.D. S.E. N 298.2 15.12 3.90 15 303.4 16.58 4.28 15 309.6 15.80 4.08 15 316.6 16.87 4.36 15 325.1 19.40 5.01 15 331.3 21.77 6.88 10 .6 21*81 6.90 10 fi PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP III: 10 MG/KG/DAY ANIMAL NUMBER DAY 18 DAY 19 DAYS ON TEST DAY 20 DAY 21 DAY 22 DAY 23 DAY 24 5 8 13 14 16 18 19 21 28 33 35 39 43 46 60 64 65 68 77 79 GROUP MEAN S.D. S.E. N _ 349.7 332.2 339.9 382.3 - - 390.9 357.7 345.7 339.6 381.0 384.2 360.3 22.08 6.98 10 _ 359.7 : 339.4 352.8 406.2 - - 404.4 374.4 357.2 357.1 395.9 410.0 375.7 26.09 8.25 10 ~ _ 380.1 - 360.7 373.4 426.1 - _ - 424.6 389.4 379.6 372.3 408.8 427.4 394.2 25.22 7.98 10 _ _ 401.9 : 378.0 388.9 457.2 - - - 434.6 402.7 394.1 383.7 424.3 431.5 409.7 25.89 8.19 10 - : - - _ _ - - 419.2 302.5 276.4 316.0 326.8 328.2 54.26 24.26 5 -- __ - - - - 306.6 280.5 285.4 322.5 331.6 305.3 22.36 10.00 5 : - - _ - - - 312.2 285.3 293 .2 319.0 341.2 V X . 0 .2 22*. 09 9.88 5 ?3 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP III: 10 MG/KG/DAY ANIMAL NUMBER DAY 25 DAY 26 DAYS ON TEST DAY 27 DAY 28 DAY 29 DAY 30 DAY 31 5 --- --- - 8 13 _ _ _ _ _ _ _ 14 - _ - - -- 16 _ -- - - - - 18 - -- - - - - 19 - _ - _ - - - 21 _ - - - - - - 28 - - - - _ - - 33 - ~ - - - - 35 39 _ _ _ _ _ __ _ 43 - - - - - - - 46 - - - - - - 60 - - _ - - - - 64 322.3 321.1 331.7 340.7 331.6 344.5 346.9 65 286.1 288.0 297.3 293.0 299.3 300.2 298.3 68 294.2 297.5 301.7 304.4 304.6 302.9 315.5 77 317.8 332.4 334.3 329.5 331.8 342.5 346.6 79 343.3 349.3 359.7 355.1 350.2 365.6 365.7 GROUP MEAN S.D. S.E. N 312.7 22.94 10.26 5 317.7 25.08 11.22 5 324.9 25.72 11.50 5 324.5 25.59 11.45 5 323.5 21.16 9.46 5 331.1 28.50 12.75 5 ^4.6 27.13 12.13 5 14 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats D uP on t-13 309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP III: 10 MG/KG/DAY ANIMAL NUMBER DAY 32 DAY 33 DAYS ON TEST DAY 34 DAY 35 DAY 36 DAY 37 DAY 38 5 8 --- 13 - - - - - 14 -- - 16 - - _ -- 18 -_ _ 19 -- - 21 -- - 28 -- - 33 -- - 35 -- - 39 -- - 43 -- - 46 - - _ 60 - - - 64 354.7 365.8 359.4 362.9 352.4 364.3 366.5 65 258.0 298.2 300.9 311.2 305.6 317.0 318.7 68 321.9 325.8 323.0 310.7 315.5 323.6 322.8 77 356.5 360.7 363.0 349.0 359.7 368.0 364.4 79 375.6 378.5 378.3 369.0 374.8 381.9 376.8 GROUP MEAN S.D. S.E. N 333.3 46.33 20.72 5 345.8 33.00 14.76 5 344.9 31.89 14.26 5 340.6 27.99 12.52 5 341.6 29.68 13.27 5 351.0 28.84 12.90 5 S4 9 .8 27*01 12.08 5 94 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP III: 10 MG/KG/DAY ANIMAL NUMBER DAY 39 DAYS ON TEST DAY 40 DAY 41 DAY 42 DAY 43 5 8 13 - - - - - 14 - - - - _ 16 - - - - 18 - - - - - 19 - - - - - 21 - - -- - 28 - - - - 33 - - - - - 35 - - - - - 39 - - - - - 43 - - - - - 46 - - - - - 60 - - - _- 64 348.7 340.5 357.9 348.6 358.4 65 321.4 317.8 316.4 323.1 310.0 68 312.5 306.2 308.3 299.5 295.6 77 354.4 356.4 355.2 340.9 330.2 79 359.7 364.7 372.7 359.2 350.0 GROUP MEAN S.D. S.E. N 339.3 21.04 9.41 5 337.1 24.87 11.12 5 342.1 28.11 12.57 5 334.3 23.48 10.50 5 328.8 26.38 11.80 5 * n PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER DAY 4 DAY 5 1 263.4 265.6 3 271.9 279.6 6 256.9 262.6 10 259.2 260.0 11 269.7 273.6 26 234.1 243.8 27 248.3 231.9 29 248.3 251.4 37 253.8 255.3 42 253.8 243.7 48 268.2 274.3 49 272.8 277.0 52 257.1 260.9 55 254.4 260.8 56 246.5 253.0 61 241.4 254.4 70 256.5 256.9 71 250.6 250.1 73 261.3 262.2 78 253.9 251.4 GROUP MEAN S.D. S.E. N 256.1 10.04 2.24 20 258.4 11.99 2 .68 20 DAYS ON TEST DAY 6 DAY 7 DAY 8 267.4 281.1 268.5 265.5 274.2 243.6 217.3 259.0 263.0 245.2 275.2 276.6 257.6 266.9 255.7 268.5 262.2 259.9 266.4 264.3 272.7 291.6 268.9 273.3 274.9 250.5 245.7 264.5 269.5 247.0 281.3 283.6 266.6 272.4 263.1 257.9 263.6 259.6 267.8 266.9 278.5 285.4 271.5 273.7 278.9 248.0 246.2 265.7 269.4 253.3 287.7 286.4 269.8 275.4 257.5 289.5 274.2 265.7 274.7 272.9 261.9 14.08 3.15 20 267.1 11.60 2.59 20 271.2 12.43 2.78 20 DAY 9 DAY 10 282.8 299.0 279.3 278.2 283.7 255.8 262.0 277.5 279.7 262.6 295.3 289.7 275.4 286.5 269.0 287.0 276.0 266.7 276.5 267.6 277.5 11.24 2 .51 20 288.6 298.1 275.9 279.9 280.3 259.5 263.5 278.9 283.9 266.8 296.4 295.2 286.2 290.9 277.4 286.4 280.4 271.9 285.8 274.5 V 'agi.o 10*. 55 2.36 20 ?? PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grains) DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER DAY 11 DAY 12 DAYS ON TEST DAY 13 DAY 14 DAY 15 DAY 16 DAY 17 1 297.8 300.8 305.0 317.4 319.2 334.3 346.3 6 285.0 284.1 291.0 299.5 310.5 _ _ 10 -_ ----- 11 26 273.6 276.7 277.1 282.6 293.7 _ _ 27 277.0 281.3 286.8 297.8 299.6 315.5 326.5 29 293.9 297.2 301.3 309.0 316.2 - - 37 283.9 292.3 296.0 306.9 309.0 327.2 337.5 42 277.9 277.1 282.6 291.5 296.8 - - 48 307.5 314.3 316.4 330.1 336.7 - - 49 306.2 305.4 309.6 317.8 319.6 328.8 335.7 52 291.5 300.2 300.6 316.3 324.7 337.5 349.9 55 -- ----- 56 - - - - - - - 61 301.0 297.0 324.9 324.0 297.8 296.7 336.2 70 281.2 285.2 293.3 298.8 296.3 300.2 307.4 71 277.5 282.5 291.1 298.2 294.3 304.8 309.6 73 283.1 293.0 292.1 304.2 308.0 317.1 325.8 78 283.8 284.2 294.0 303.1 301.5 313.9 327.0 GROUP MEAN S.D. S.E. N 288.1 10.95 2.83 15 291.4 11.02 2.85 15 297.5 12.63 3.26 15 306.5 12.76 3.29 15 308.3 12.84 3.31 15 317.6 14.20 4.49 10 "90.2 13*. 96 4.42 10 V*97 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER DAY 18 DAY 19 DAYS ON TEST DAY 20 DAY 21 DAY 22 DAY 23 DAY 24 1 3 6 10 11 26 27 29 37 42 48 49 52 55 56 61 70 71 73 78 GROUP MEAN S .D. S.E. N 360.9 - 347.1 360.8 - 348.3 367.9 " 357.4 314.4 321.3 340.5 341.8 346.0 17.36 5.49 10 373.8 - 363.6 368.4 _ 364.5 383.3 - 383.3 326.4 335.9 359.5 363.2 362.2 18.42 5.82 10 395.0 - 379.3 391.8 - 375.8 410.9 - 399.7 329.1 347.1 370.0 376.3 377.5 24.61 7.78 10 414.2 - 413.6 421.5 - - 393.0 430.2 - 417.3 335.7 349.0 379.5 386.1 394.0 31.80 10.06 10 - - - - ~ 291.9 339.1 307.4 284.1 282.9 301.1 23.39 10.46 5 -- -- - _ " 305.1 296.8 290.8 288.8 290.6 294.4 6.69 .99 5 - - 314.1 297.1 299.7 291.0 288.7 V 'ass.i 9.98 4.46 5 *798 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER DAY 25 DAY 26 DAYS ON TEST DAY 27 DAY 28 DAY 29 DAY 30 DAY 31 1 3 6 10 11 26 27 29 37 42 48 49 52 55 56 61 70 71 73 78 GROUP MEAN S.D. S.E. N - - - - - - _ 311.1 300.6 300.5 301.3 286.8 300.1 8.66 3.87 5 - - _ - - - 319.2 293.4 297.6 305.3 303.6 303.8 9.82 4.39 5 - - _ ~ - - " _ 332.2 305.7 301.4 312.0 300.9 310.4 12.95 5.79 5 - - - - - - - - - _ 331.9 305.3 304.7 319.1 303.9 313.0 12.30 5.50 5 - - _ - - - " _ 330.7 309.1 306.9 323.3 299.9 314.0 12.64 5.65 5 - - _ - - - - _ 323.2 296.4 298.7 327.8 305.8 310.4 14.32 6.41 5 _ - __ - - - - _ 342.6 298.9 303.5 328.0 302.2 v >i.0 19.27 8.62 5 99 SCO PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER DAY 32 DAY 33 DAYS ON TEST DAY 34 DAY 35 DAY 36 DAY 37 DAY 38 1 3 6 10 11 26 27 29 37 42 48 49 52 55 56 61 70 71 73 78 GROUP MEAN S.D. S.E. N - - : - _ - - 356.1 306.0 305.2 334.3 311.7 322.7 22.11 9.89 5 - - _ - - - - 363.4 313.8 311.4 331.8 317.3 327.5 21.55 9.64 5 - - -- - _ - _- 353.9 311.5 307.8 331.1 311.0 323.1 19.54 8.74 5 - * - - 343.5 316.2 321.3 337.1 313.6 326.3 13.24 5.92 5 - - " - _ - 366.1 323.9 320.0 346.4 313.2 333.9 21.88 9.78 5 -- - - - _ - 358.8 312.6 309.2 341.9 321.3 328.8 21.06 9.42 5 - _ - : _ - 371.2 301.3 305.0 337.4 325.4 \ 'S38.1 28*30 12.66 5 100 /Of PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHTS (grams) DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER DAY 39 DAYS ON TEST DAY 40 DAY 41 DAY 42 1 3 6 10 - - - - 11 - - - - 26 -- - - 27 -- - - 29 -- - - 37 -- __ 42 - - - - 48 - - - - 49 __ _- 52 - - - - 55 - - - - 56 - - - - 61 358.4 355.3 355.2 342.5 70 296.5 299.4 302.6 298.9 71 307.2 307.8 304.8 307.2 73 332.8 333.6 330.7 321.3 78 314.1 315.1 309.4 304.6 DAY 43 - _ 345.0 301.1 298.8 312.4 303.9 GROUP MEAN S.D. S.E. N 321.8 24.36 10.89 5 322 .2 22.38 10.01 5 320.5 22.35 10.00 5 314.9 17.49 7.82 5 312.2 19.02 8.51 5 X 101 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 APPENDIX D V INDIVIDUAL BODY WEIGHT CHANGES DURING GESTATION AND LACTATION 102 /OS PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHT CHANGES DURING GESTATION AND LACTATION EXPLANATORY NOTES Note Day 21 = Gestation Day 21. Day 22 = Lactation Day 0. Day 43 = Lactation Day 21. Abbreviation = No data 103 /V PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHT CHANGES (grams) DURING GESTATION AND LACTATION GROUP I: 0 MG/KG/DAY ANIMAL NUMBER 4-10 10-15 DAYS ON TEST 15-21 22-25 25-29 29-36 36-43 7 48.0 9 26.3 37.0 112.8 - _ -- 12 25.5 30.4 95.7 - _-- 15 35.7 53.8 - - _ -_ 17 30.0 32.1 - - _ -- 20 37.9 43.7 - --- 23 38.0 28.9 - - -- - 31 32 .5 30.4 - _ - - - 32 37.9 - -- - -_ 34 30.7 34.4 105.3 - -_ - 36 38.1 - - - --- 38 33.1 29.8 85.6 - - - - 47 54.2 - - - - - - 53 34.1 25.7 I S .1 - - - 59 31.1 - - - - - - 62 22.0 33.4 IQ.9 22.8 15.0 -0.3 -15.2 66 46.2 27.5 91.2 -95.4 7.2 18.7 -15.3 67 31.3 37.0 75.8 38.9 15.9 8.9 -20.5 75 36.8 28.5 101.2 16.0 10.0 13.6 -22.7 80 21.3 33.8 79.1 19.9 23.4 22.5 -24.6 GROUP MEAN S.D. S.E. N 34.5 8.28 1.85 20 33 .8 7.16 1.85 15 90.2 13.13 4.15 10 0.4 54.28 24.27 5 14.3 6.22 2.78 5 12.7 8.89 3.98 5 N.^.7 4.28 1.91 5 104 /S PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHT CHANGES (grams) DURING GESTATION AND LACTATION GROUP I: 0 MG/KG/DAY ANIMAL NUMBER DAYS ON TEST 4-21 22-43 7 -- 9 176.1 - 12 151.6 - 15 - - 17 - - 20 - - 23 - - 31 - - 32 - - 34 170.4 - 36 - - 38 148.5 - 47 - - 53 136.5 - 59 - - 62 134.3 22.3 66 164.9 -84.8 67 144.1 43.2 75 166.5 16.9 80 134.2 41.2 GROUP MEAN S .D. S.E. N 152.7 15.76 4.98 10 7.8 53.00 23.70 5 X 105 />6 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHT CHANGES (grams) DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER 4-10 10-15 DAYS ON TEST 15-21 22-25 25-29 29-36 36-43 2 29.7 30.7 95.8 4 23.5 25.8 91.9 - - -- 22 11.3 51.5 79.9 - --- 24 51.0 30.3 90.8 - - - - 25 49.1 - - - --- 30 24.7 49.2 - - - - - 40 25.1 28.4 86.0 - - - - 41 39.2 39.9 - -- _- 44 35.2 - - - - - - 45 38.2 32.9 - - - - - 50 40.3 33 .6 - - - - - 51 44.1 44.0 - - - - - 54 24.1 - - - - - - 57 34.1 - - - - - 58 45.7 - - - - - - 63 39.2 20.7 121.5 28.4 32.3 3.3 -14.1 69 24.9 24.5 39.3 -47.5 -2.0 -8.7 3 .1 72 28.8 25.1 87.7 -108.8 30.5 7.5 -15.6 74 23.9 28.0 91.1 19.7 3.3 20.6 -17.8 76 21.0 37.7 78.9 11.8 18.9 15.7 -15.8 GROUP MEAN S .D. S.E. N 32.7 10.57 2.36 20 33.5 9.21 2.38 15 86.3 20.31 6.42 10 -19.3 58.24 26.05 5 16.6 15.56 6.96 5 7.7 11.39 5.09 5 \2-0 8*57 3.83 5 106 S67 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHT CHANGES (grams) DURING GESTATION AND LACTATION GROUP II: 3 MG/KG/DAY ANIMAL NUMBER DAYS ON TEST 4-21 22-43 2 156.2 4 141.2 - 22 142.7 - 24 172.1 - 25 - - 30 - - 40 139.5 - 41 - - 44 - - 45 - - 50 - - 51 - - 54 - - 57 - - 58 - - 63 181.4 49.9 69 88.7 -55.1 72 141.6 -86.4 74 143.0 25.8 76 137.6 30.6 GROUP MEAN S.D. S.E. N 144.4 24.66 7.80 10 -7 .0 59 .89 26 .78 5 107 /o3 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHT CHANGES (grams) DURING GESTATION AND LACTATION GROUP III: 10 MG/KG/DAY ANIMAL NUMBER 4-10 10-15 DAYS ON TEST 15-21 22-25 25-29 29-36 36-43 5 24.0 31.5 8 46.4 - - - - - - 13 37.5 37.2 - - - - - 14 25.6 28.8 82.7 - - - - 16 47.6 - -- - -- 18 48.6 42.7 -- -- 19 37 . 0 - -- - - - 21 27.6 29.0 85.3 - - - - 28 57.3 27.7 86.3 - - - - 33 22.6 - - - - - 35 33.3 40.3 122.2 - - - - 39 28.1 - - - - - - 43 39.4 45.5 - - - _- 46 32.8 34.6 - - - - - 60 53.3 34.6 86.6 - - - - 64 28.6 34.1 84.0 -96.9 9.3 20.8 6.0 65 47.0 30.9 93.8 -16.4 13.2 6.3 4.4 68 24.6 30.4 78.7 17.8 10.4 10.9 -19.9 77 29.9 38.1 85.0 1.8 14.0 27.9 -29.5 79 37.2 44.3 90.0 16.5 6.9 24.6 -24.8 GROUP MEAN S .D. S.E. N 36.4 10.49 2.35 20 35.3 5.83 1.51 15 89.5 12.19 3 . 86 10 -15.4 47.59 21.28 5 10.8 2.90 1.30 5 18.1 9.17 4.10 5 NJ-2.8 l*. 7 5 7.49 5 108 /* ? PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHT CHANGES (grams) DURING GESTATION AND LACTATION GROUP III: 10 MG/KG/DAY ANIMAL NUMBER DAYS ON TEST 4-21 22-43 5 -- 8 -- 13 - - 14 137.1 - 16 - - 18 - - 19 - - 21 141.9 - 28 171.3 - 33 - - 35 195.8 - 39 - _ 43 - - 46 - - 60 174.5 - 64 146.7 -60.8 65 171.7 7.5 68 133.7 19.2 77 153.0 14.2 79 171.5 23.2 GROUP MEAN S.D. S.E. N 159.7 20.16 6.37 10 0.7 34.85 15.59 5 X 109 // > PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHT CHANGES (grams) DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER 4-10 10-15 DAYS ON TEST 15-21 22-25 25-29 29-36 36-43 1 25.2 30.6 95.0 3 26.2 - -- -- - 6 19.0 34.6 - - --- 10 20.7 - - - --- 11 10.6 - - - - - - 26 25.4 34.2 - - -- - 27 15.2 36.1 114.0 - -- - 29 30.6 37.3 - _ - -- 37 30.1 25.1 112.5 - - - - 42 13.0 30.0 - - - - - 48 28.2 40.3 - _ --- 49 22.4 24.4 73.4 - - - - 52 29.1 38.5 105.5 - - - - 55 36.5 - - - - - - 56 30.9 - - - - - - 61 45.0 11.4 119.5 19.2 19.6 35.4 -21.1 70 23.9 15.9 39.4 -38.5 8.5 14.8 -22.8 71 21.3 22.4 54.7 -6.9 6.4 13.1 -21.2 73 24.5 22.2 71.5 17.2 22.0 23.1 -34.0 78 20.6 27.0 84.6 3.9 13.1 13.3 -9.3 GROUP MEAN S .D. S.E. N 24.9 7 .93 1.77 20 28.7 8.49 2.19 15 87.0 27.04 8.55 10 -1.0 23.48 10.50 5 13.9 6.79 3.03 5 19.9 9.57 4.28 5 ^24-7 8.76 3.92 5 // PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 INDIVIDUAL BODY WEIGHT CHANGES (grams) DURING GESTATION AND LACTATION GROUP IV: 30 MG/KG/DAY ANIMAL NUMBER DAYS ON TEST 4-21 22-43 1 150.8 - 3 -- 6 -- 10 - - 11 - _ 26 - - 27 165.3 29 - - 37 167.7 - 42 - - 48 - - 49 120.2 - 52 173.1 - 55 - - 56 - - 61 175.9 53.1 70 79.2 -38.0 71 98.4 -8.6 73 118.2 28.3 78 132.2 21.0 GROUP MEAN S.D. S.E. N 138.1 33.71 10.66 10 11 .2 35 .20 15 .74 5 t/Z PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 APPENDIX E INDIVIDUAL REPRODUCTIVE DATA ft3 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Individual Reproductive Data Explanatory Notes Notes Nidations = Implantations Five dams per group were sacrificed on day 10, 15, or 2IG. Abbreviations S.E. = Standard Error N = Number in Group . . . = No Data DuPont-13309 \N / hi113 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL REPRODUCTIVE DATA GROUP I: 0 MG/KG/DAY ANIMAL CORPORA ' NIDANUMBER LUTEA TIONS RESORPTIONS TOTAL EARLY LATE DEAD FETUSES 7 10 0 0 0 0 9 18 0 0 0 0 12 12 0 0 0 0 15 16 0 0 0 0 17 13 0 0 0 0 20 18 0 0 0 0 23 14 0 0 0 0 31 12 0 0 0 0 32 15 0 0 0 0 34 14 0 0 0 0 36 16 0 0 0 0 38 12 0 0 0 0 47 15 0 0 0 0 53 8 0 0 0 0 59 13 0 0 0 0 K/ 114 LIVE FETUSES TOTAL 10 18 12 16 13 18 14 12 15 14 16 12 15 8 13 DuPont-13309 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL REPRODUCTIVE DATA GROUP II: 3 MG/KG/DAY ANIMAL CORPORA -NIDANUMBER LUTEA TIONS RESORPTIONS TOTAL EARLY LATE DEAD FETUSES 2 15 2 2 0 0 4 13 0 0 0 0 22 13 0 0 0 0 24 13 0 0 0 0 25 13 0 0 0 0 30 13 0 0 0 0 40 13 0 0 0 0 41 17 0 0 0 0 44 14 0 0 0 0 45 14 0 0 0 0 50 14 0 0 0 0 51 16 1 1 0 0 54 14 0 0 0 0 57 17 0 0 0 0 58 14 0 0 0 0 / 115 LIVE FETUSES TOTAL 13 13 13 13 13 13 13 17 14 14 14 15 14 17 14 DuPont-13309 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL REPRODUCTIVE DATA GROUP III: 10 MG/KG/DAY ANIMAL CORPORA ' NIDA- NUMBER LUTEA TIONS RESORPTIONS TOTAL EARLY LATE DEAD FETUSES 5 17 0 0 0 0 8 15 0 0 0 0 13 14 0 0 0 0 14 12 0 0 0 0 16 14 0 0 0 0 18 16 0 0 0 0 19 16 0 0 0 0 21 13 2 2 0 0 28 12 0 0 0 0 33 15 0 0 0 0 35 16 0 0 0 0 39 12 0 0 0 0 43 15 0 0 0 0 46 14 0 0 0 0 60 14 0 0 0 0 / 116 LIVE FETUSES TOTAL 17 15 14 12 14 16 16 11 12 15 16 12 15 14 14 DuPont-13309 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL REPRODUCTIVE DATA GROUP IV: 30 MG/KG/DAY ANIMAL CORPORA - NIDANUMBER LUTEA TIONS RESORPTIONS TOTAL EARLY LATE DEAD FETUSES 1 13 0 0 0 0 3 15 0 0 0 0 6 12 0 0 0 0 10 14 0 0 0 0 11 17 0 0 0 0 26 13 0 0 0 0 27 13 0 0 0 0 29 14 0 0 0 0 37 13 0 0 0 0 42 13 0 0 0 0 48 12 0 0 0 0 49 13 0 0 0 0 52 15 0 0 0 0 55 9 0 0 0 0 56 16 0 0 0 0 { // / 117 LIVE FETUSES TOTAL 13 15 12 14 17 13 13 14 13 13 12 13 15 9 16 DuPont-13309 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 APPENDIX F NUMBER OF PUPS DURING LACTATION /* PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Number of Pups During Lactation Explanatory Notes DuPont-13309 Note One pup per litter was sacrificed on days 3, 7, and 14 for blood collection. Two pups per litter (litter size permitting) were sacrificed on day 21 for blood collection. Abbreviations S.D. = Standard Error S.E. = Standard Error N = Number of Litters in Group \ /ao119 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats ANIMAL # 62 66 67 75 80 Mean S.D. S.E. N ANIMAL # 63 69 72 74 76 Mean S.D. S.E. N ANIMAL # 64 65 68 77 79 Mean S.D. S.E. N ANIMAL # 61 70 71 73 78 Mean S.D. S.E. N NUMBER OF PUPS DURING LACTATION BORN GROUP: I DOSE : 0 MG/KG/DAY BORN ALIVE DAY 3 DAY 7 DAY 14 DAY 21 13 14 19 16 12 14.8 2.77 1.24 5 13 14 19 16 12 14.8 2.77 1.24 5 13 14 19 16 12 14.8 2.77 1.24 5 12 13 18 15 11 13.8 2.77 1.24 5 11 12 17 14 10 12.8 2.77 1.24 5 10 11 16 12 9 11.6 2.70 1.21 5 BORN GROUP: II DOSE: 3 MG/KG/DAY BORN ALIVE DAY 3 DAY 7 DAY 14 DAY 21 15 4 14 16 13 12.4 4.83 2.16 5 15 4 13 16 13 12.2 4.76 2.13 5 15 4 13 16 13 12.2 4.76 2.13 5 13 3 12 14 12 10.8 4.44 1.98 5 12 2 11 13 11 9.8 4.44 1.98 5 CO 11 1 10 11 10 4.28 1.91 5 BORN GROUP: III DOSE: 10 MG/KG/DAY BORN ALIVE DAY 3 DAY 7 DAY 14 DAY 21 12 14 12 14 14 13.2 1.10 0.49 5 12 13 12 14 14 13.0 1.00 0.45 5 12 13 12 14 14 13.0 1.00 0.45 5 11 12 11 13 13 12.0 1.00 0.45 5 10 11 10 12 12 11.0 1.00 0.45 5 9 10 9 11 11 10.0 1.00 0.45 5 BORN GROUP: IV DOSE: 30 MG/KG/DAY BORN ALIVE DAY 3 DAY 7 DAY 14 DAY 21 13 3 6 11 13 9.2 4.49 2.01 5 13 3 6 11 13 9.2 4.49 2.01 5 13 3 6 11 13 9.2 4.49 2.01 5 12 2 5 10 12 8.2 4.49 2.01 5 11 1 4 9 11 7.2 4.49 2.01 5 10 -a 3 8 10 7.8 3.30 1.48 4 DuPont-13309 a No pups. 120 /a/ PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 APPENDIX G INDIVIDUAL LITTER CLINICAL OBSERVATIONS * 121 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats INDIVIDUAL LITTER CLINICAL OBSERVATIONS ANIMAL LACTATION NUMBER DAY GROUP: I DOSE: 0 MG/KG/DAY SIGN INCIDENCE OBSERVATIONS 62 NO ABNORMALITIES DETECTED 66 NO ABNORMALITIES DETECTED 67 NO ABNORMALITIES DETECTED 75 NO ABNORMALITIES DETECTED 80 NO ABNORMALITIES DETECTED ANIMAL LACTATION NUMBER DAY GROUP: II DOSE: 3 MG/KG/DAY SIGN INCIDENCE OBSERVATIONS 63 NO ABNORMALITIES DETECTED 69 DAY 21 1 OF 1 SMALL WHOLE BODY LITTER SUMMARY: 1 SMALL WHOLE BODY 72 NO ABNORMALITIES DETECTED 74 NO ABNORMALITIES DETECTED 76 NO ABNORMALITIES DETECTED ANIMAL LACTATION NUMBER DAY GROUP: III DOSE: 10 MG/KG/DAY SIGN INCIDENCE OBSERVATIONS 64 NO ABNORMALITIES DETECTED 65 NO ABNORMALITIES DETECTED 68 NO ABNORMALITIES DETECTED 77 NO ABNORMALITIES DETECTED 79 NO ABNORMALITIES DETECTED ANIMAL LACTATION NUMBER DAY GROUP: IV DOSE: 30 MG/KG/DAY SIGN INCIDENCE OBSERVATIONS 61 NO ABNORMALITIES DETECTED 70 NO ABNORMALITIES DETECTED 71 NO ABNORMALITIES DETECTED 73 NO ABNORMALITIES DETECTED 78 NO ABNORMALITIES DETECTED 122 DuPont-13309 \ PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 APPENDIX H MEAN PUP WEIGHTS PER LITTER ^ PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Mean Pup Weights Per Litter Explanatory Notes DuPont-13309 Note One pup per litter was sacrificed on days 3, 7, and 14 for blood collection. Two pups per litter (litter size permitting) were sacrificed on day 21 for blood collection. Abbreviations S.D. = Standard Error S.E. = Standard Error N = Number of Litters in Group * as PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats ANIMAL # 62 66 67 75 80 Mean S .D. S.E. N ANIMAL # 63 69 72 74 76 Mean S.D. S.E. N ANIMAL # 64 65 68 77 79 Mean S.D. S.E. N ANIMAL # 61 70 71 73 78 Mean S.D. S.E. N MEAN PUP WEIGHTS (grams) PER LITTER DAY 0 GROUP : I DAY 3 DOSE: 0 MG/KG/DAY DAY 7 DAY 14 DAY 21 6.7 6.8 5.8 6.3 6.9 6.5 0.45 0.20 5 9.4 9.3 8.3 8 .7 10.4 9.2 0.80 0.36 5 14.3 13.7 13.6 13.9 15.7 14.2 0.87 0.39 5 26.4 27.1 22.8 24.1 26.5 25.4 1.84 0.82 5 43.6 37.9 38.7 41.3 46.1 41.5 3.40 1.52 5 DAY 0 GROUP: II DAY 3 6.9 7.3 6.7 6.1 6.7 6.7 0.43 0.19 5 9.7 11.7 9.6 8.6 9.7 9.9 1.13 0.50 5 DOSE: 3 MG/KG/DAY DAY 7 DAY 14 DAY 21 16.0 15.9 14.8 13.1 14.0 14.8 1.27 0.57 5 31.3 21.4 30.0 21.3 26.5 26.1 4.67 2.09 5 50.6 17.7 45.5 35.1 44.5 38.7 13.00 5.81 5 GROUP: III DAY 0 DAY 3 7.0 6.5 6.5 6.9 7.0 6.8 0.23 0.10 5 10.6 9.4 9.5 10.1 11.3 10.2 0.79 0.35 5 DOSE: 10 MG/KG/DAY DAY 7 DAY 14 DAY 21 17.0 14.2 15.5 13.8 17.3 15.6 1.59 0.71 5 33.6 27.6 29.7 25.4 32.9 29.8 3.48 1.56 5 54.9 41.8 44.5 41.2 53.0 47.1 6.42 2.87 5 GROUP : IV DAY 0 DAY 3 6.6 7.1 7.6 6.9 < 6.8 9.8 11.2 12.3 10.3 9.7 7.0 0.38 0.17 5 10.7 1.09 0.49 5 DOSE: 30 MG/KG/DAY DAY 7 DAY 14 DAY 21 15.3 15.6 19.9 16.9 14.1 16.3 2.23 1.00 5 24.2 15.1 38.8 30.4 27.4 27.2 8.69 3.89 5 44.4 -a 59.4 36.5 43.2 45.9 9.64 4.82 4 a No pups. 125 Mo DuPont-13309 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats D uP ont-13 309 APPENDIX I PLASMA AND TISSUE ANALYSIS 126 IV7 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats PLASMA AND TISSUE ANALYSIS EXPLANATORY NOTES Abbreviations G N.S. = LOQ = <LOQ = PP S.D. = *_ Gestation No Sample Limit of Quantitation, 0.05 pg/mL. Less than limit of quantitation Postpartum Standard Deviation Insufficient for sample reanalysis DuPont-13309 \ !Z3 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Concentration (/xg/mL) of PFOA in Maternal Plasma During Gestation and Postpartum Dose Day _(mg/kg)_ Concentration Average S.D. 10G 0 10 30 <LOQ <LOQ <LOQ <LOQ <LOQ 7.76 10.07 8.54 7.38 8.93 24.05 25.66 23.88 23.20 19.83 57.09 71.53 68.65 81.78 73.41 <LOQ NA 8.53 1.06 23.32 2.15 70.49 8.94 15G 10 30 <LOQ <LOQ <LOQ <LOQ <LOQ 10.61 8.71 39.04 11.32 9.94 47.27 30.22 9.15 24.08 36.26 81.42 81.62 79.10 81.33 74.29 <LOQ NA 15.92 12.96 29.40 14.19 79.55 3.11 /27 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Concentration (/xg/mL) PFOA in Maternal Plasma During Gestation and Postpartum (cont'd) Dose (mg/kg) 0 3 10 30 0 3 10 30 Concentration <LOQ <LOQ <LOQ <LOQ <LOQ 14.15 13.61 12.71 11.93 17.80 26.28 30.52 38.53 32.51 43.18 78.50 74.02 65.60 63.32 100.35 <LOQ <LOQ <LOQ <LOQ <LOQ 8.65 10.62 13.45 9.42 12.92 * 19.82 25.30 22.31 * 53.25 69.57 51.50 70.90 26.76 Average S.D. <LOQ NA 14.04 2.27 34.20 6.68 76.36 14.76 <LOQ NA 11.01 2.11 22.47 2.74 54.39 17.86 I3 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Concentration (/rg/mL) PFOA in Maternal Plasma During Gestation and Postpartum (cont'd) Dose (mg/kg) 0 3 10 30 0 3 10 30 Concentration <LOQ <LOQ <LOQ <LOQ <LOQ 9.34 7.79 10.37 8.02 14.93 23.38 24.45 25.89 26.72 28.72 64.97 * 84.10 60.58 57.98 <LOQ <LOQ <LOQ <LOQ <LOQ 9.68 10.22 8.38 9.35 10.82 19.52 26.27 23.61 23.16 26.38 50.73 42.05 56.48 50.69 73.30 Average S.D. <LOQ NA 10.09 2.90 25.83 2.07 66.91 11.82 <LOQ NA 9.69 0.92 23.79 2.81 54.65 11.63 /3/ PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Concentration (/xg/mL) PFOA in Maternal Plasma During Gestation and Postpartum (cont'd) Dose Day (mg/kg)_____ Concentration Average S.D. 21PP 0 3 10 30 <LOQ <LOQ <LOQ <LOQ <LOQ 10.35 7.99 9.68 9.05 8.12 24.34 23.45 28.76 35.68 31.97 63.27 62.86 <LOQ NA 9.04 1.01 28.84 5.15 64.13 1.45 64.30 66.10 * I3Z- PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Concentration (/g/mL) PFOA in Fetal Plasma on Gestation Day 21 Dose Day (mg/kg)_____ Concentration_____ Average S.D. 21G 0 3 10 30 <LOQ <LOQ <LOQ <LOQ <LOQ 5.52 7.06 5.53 5.38 5.91 12.19 14.90 15.96 15.51 13.84 37.32 37.09 32.27 25.90 32.96 <LOQ NA 5.88 0.69 14.48 1.51 33.11 4.64 DuPont-13309 /33 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Concentration (/xg/mL) PFOA in Pup Plasma Dose Day (mg/kg) 3PP 0 10 Concentration <LOQ <LOQ <LOQ <LOQ <LOQ 3.24 1.89 3.33 2.45 3.56 5.62 5.01 8.06 5.08 Average S.D. <LOQ NA 2.89 0.70 5.94 1.44 30 11.46 11.96 1.66 11.18 13.43 9.87 13.87 7PP <LOQ <LOQ NA 38.85a <LOQ <LOQ 0.78 0.41 0.65 0.20 0.84 0.57 10 3.38 2.77 0.58 2.28 3.02 2.03 3.13 30 4.22 4.92 1.28 <LOQ 5.15 6.61 3.70 DuPont-13309 * a Sample not used in calculation. Extraordinary high value is likely the result o f sample contamination. /3W PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Concentration (/rg/mL) PFOA in Pup Plasma (cont'd) Dose Day (mg/kg) Concentration Average S.D. 14PP 0 3 10 30 <LOQ <LOQ <LOQ <LOQ <LOQ 0.87 0.72 0.74 0.65 0.87 2.50 2.47 2.06 1.62 2.44 4.84 3.23 5.10 5.01 6.38 <LOQ NA 0.77 0.10 2.22 0.38 4.91 1.12 21PP 0 3 10 30 <LOQ <LOQ <LOQ <LOQ <LOQ 1.31 * 1.00 0.56 2.27 3.55 2.67 3.40 3.89 2.76 6.18 7.06 10.50 5.70 <LOQ NA 1.28 0.72 3.25 0.52 7.36 2.17 DuPont-13 309 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Concentration (jUg/mL) of PFOA in Milk Dose (mg/kg) 0 Average S.D. 3 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ NA Postpartum Day 7 14 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.21 <LOQ NA NAa NA 3 Average S.D. 0.81 0.92 1.41 0.93 1.28 1.07 0.26 0.75 1.10 0.90 1.20 0.90 1.12 0.84 1.09 1.31 1.22 0.94 1.15 0.22 0.06 10 Average S.D. 2.16 1.97 2.21 1.49 2.34 2.03 0.33 2.32 3.24 4.04 5.34 3.32 3.65 2.01 2.21 1.98 2.83 2.74 3.45 0.91 1.18 30 Average 'S.D. 5.32 6.33 4.97 5.20 3.04 4.97 1.20 6.55 4.91 7.47 5.52 4.34 5.76 1.26 a Average not calculated because 3 o f 4 values <LOQ. 5.95 4.55 7.73 6.14 7.87 6.45 1.38 DuPont-13309 21 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ NA 1.14 N.S. 1.01 1.17 1.18 1.13 . 0.08 V, 2.80 3.72 2.80 2.52 3.49 3.07 0.51 7.36 N.S. 9.83 6.36 6.36 7.48 1.63 135 134 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats DuPont-13309 Concentration (ig/mL) of PFOA in Amniotic Fluid Dose (mg/kg) Day 15 G Concentration Average S.D. 0 <LOQ <LOQ NA <LOQ <LOQ <LOQ <LOQ 3 0.25 0.60 0.69 0.22 1.63 0.32 <LOQ 10 0.73 0.70 0.15 0.60 0.49 0.88 0.79 30 0.99 1.70 0.91 1.24 3.25 1.29 1.73 Day 21 G Concentration Average S.D. <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ NA 1.28 1.50 0.32 2.00 1.29 1.29 1.66 2.97 3.76 0.81 4.00 4.89 2.98 3.98 7.73 0.86 8.67 7.54 7.32 9.37 /37 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Concentration (jUg/mL) of PFOA in Placenta Dose Tissue Day (mg/kg) Concentration Average S.D. 15G 0 <LOQ <LOQ NA <LOQ <LOQ <LOQ <LOQ 3 1.48 2.22 1.79 1.33 5.41 1.61 1.28 10 7.45 5.10 1.70 4.52 <LOQ 3.43 5.01 30 12.01 13.22 1.03 13.17 12.95 14.86 13.14 21G 0a <LOQ <LOQ NA <LOQ <LOQ <LOQ 3 3.58 3.55 0.57 2.88 3.65 3.21 4.45 10 6.90 9.37 1.76 8.49 11.14 10.91 9.40 30 29.85 24.37 4.13 22.28 27.16 19.43 23.12 a One control sample was used for method development. 131 DuPont-13309 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Concentration (/xg/mL) of PFOA in Embryo Dose Tissue Day (mg/kg) Concentration Average S.D. 10G 0 <LOQ <LOQ NA <LOQ <LOQ <LOQ <LOQ 3 1.12 1.40 0.30 1.58 1.49 1.05 1.76 10 2.95 3.33 0.81 4.49 3.59 3.29 2.31 30 11.51 12.49 3.50 12.33 10.15 18.50 9.95 15G 0 <LOQ <LOQ NA <LOQ <LOQ <LOQ <LOQ 3 0.19 0.24 0.19 0.13 0.57 0.17 0.14 10 0.55 0.53 0.18 0.61 0.27 0.48 0.76 30 1.39 1.24 0.22 0.97 1.48 1.31 1.06 138 131 DuPont-13309 PFOA: Lactational and Placental Transport Pharmacokinetic Study in Rats Concentration (/ag/mL) of PFOA in Fetus Dose Tissue Day_______ (mg/kg) Concentration Average 21G 0 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 3 0.95 1.27 1.65 1.15 1.26 1.35 10 a 2.61 2.69 2.79 2.06 2.89 30 7.23 8.77 12.28 S.D. NA 0.26 0.37 2.36 7.52 8.07 DuPont-13309 * a Sample lost due to misinjection. 139 NO K.