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D u P o n t-15302 Study Title Ammonium Perfluorooctanoate: Age Effect on the PFOA Plasma Concentration in Post-Weaning Rats Following Oral Gavage Test Guidelines: None applicable AUTHOR: Paul M. Hinderliter, Ph.D. Study Completed on: December 2,2004 Performing Laboratory: E.I. du Pont de Nemours and Company HaskellSMLaboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Laboratory Project ED: DuPont-15302 Work Request Number: 15339 Service Code Number: 1389 Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. and 3M Company 3M Center Building St. Paul, MN 55144-1000 Page 1of 52 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, except for the item documented below. The item listed did not impact the validity of the study. No conduct audit was conducted on this study, however, all activities were performed by experienced, trained personnel. All work was documented in the study records. Date -2 S 93 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage QUALITY ASSURANCE DOCUMENTATION DuPont-15302 Work Request Number: 15339 Study Code Number: 1389 The conduct of this study has been subjected to periodic Quality Assurance inspections. The dates of inspection are indicated below. Phase Audited Protocol: Audit Dates July 22,23,2004 Date Reported to Study Director July 23, 2004 Date Reported to Management July 23. 2004 Report/Records: October 28, 29, 2004 October 29. 2004 November 24, 2004 Date -3 & */ Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation o f data obtained from this study. Analytical Evaluation by: t Janet C. Maslanka, B.S. Analytical Chemist Date Approved by: Gary W. Jepson, Ph.D. Research Manager J - A/oS-ZOof Date Issued by Study Director: / ( Palhl'M. Hinderliter, Ph.D. (Research Toxicologist O t - E& c-j-ooH Date -4 - 31S Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT....................................2 QUALITY ASSURANCE DOCUMENTATION.......................................................................... 3 CERTIFICATION.............................................................................................................................4 STUDY INFORMATION................................................................................................................ 7 SUMMARY........................................................................................................................................ 8 A. Plasma......................................................................................................................................8 B. U rine........................................................................................................................................8 INTRODUCTION............................................................................................................................10 MATERIALS AND METHODS................................................................................................... 10 A. Test Substance.......................................................................................................................10 B. Test System............................................................................................................................10 C. Animal Husbandry................................................................................................................ 11 1. Housing............................................................................................................................11 2. Cage and Cage Rack Change..........................................................................................11 3. Environmental Conditions.............................................................................................. 11 4. Feed and W ater............................................................................................................... 11 5. Animal Health and Environmental Monitoring Program............................................. 11 D. Pretest Period.........................................................................................................................12 E. Assignment to Groups........................................................................................................... 12 F. Main Study.............................................................................................................................13 1. Animals............................................................................................................................13 2. Dose Preparation, Analysis, and R ates.......................................................................... 13 3. Sacrifice and Blood Sampling.........................................................................................13 4. Experimental Procedure.................................................................................................. 13 G. Supplemental Study.............................................................................................................. 14 H. Analytical Methods............................................................................................................... 14 1. Verification of Dose Solution.........................................................................................14 2. Extraction of PFOA from Plasma for LC/MS Analysis................................................ 14 3. Instrumentation............................................................................................................... 15 4. Chromatographic Methods..............................................................................................15 I. Statistical Analyses............................................................................................................... 16 RESULTS AND DISCUSSION..................................................................................................... 16 A. Dose Solution Analysis......................................................................................................... 16 B. Plasma Concentration o f PFOA............................................................................................16 1. Male R ats........................................................................................................................ 16 2. Female Rats..................................................................................................................... 16 3. Supplemental R ats.......................................................................................................... 17 C. Urine Concentration of PFO A ..............................................................................................17 CONCLUSIONS..............................................................................................................................17 RECORDS AND SAMPLE STORAGE.......................................................................................18 -5- 3JC, Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 REFERENCES................................................................................................................................. 19 TABLES............................................................................................................................................ 20 Table 1: Plasma and urine concentrations in male rats oral gavage with 10 or 30 mg/kg PFOA........................................................................................................22 Table 2: Plasma and urine concentrations in female rats oral gavage with 10 or 30 mg/kg PFOA........................................................................................................23 FIGURES.......................................................................................................................................... 24 Figure 1: Plasma concentration in rats following oral gavage with PFOA...........................25 Figure 2: Urine concentration in rats following oral gavage with PFOA.............................. 26 APPENDICES..................................................................................................................................27 Appendix A: Dosing Solution Analysis.................................................................................... 29 Appendix B: Plasma Concentration Data - M ales................................................................... 41 Appendix C: Plasma Concentration Data - Females................................................................ 44 Appendix D: Supplemental Plasma Concentrations................................................................ 47 Appendix E: Urine Concentration Data - Males...................................................................... 49 Appendix F: Urine Concentration Data - Females...................................................................51 -6 - 39 7 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 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 APFO 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 (C3F7CO2' NH4+), 0.01% C5 (C4F9CO2' NH4+), 0.03% C6 (C5F 11CO2' NH4+), 0.43% C7 (C6Fi3C02'NH4+), 0.57% C9 (CgFi7C 0 2' NH4+), 0.16% Monohydro APFO, 0.09% Monounsaturated APFO, 0.72% Undefined (possibly) substituted perfluorocyclo species, 0 .2 % cyclopentyl, and 0 .1% cyclohexyl Physical Characteristics: White solid Stability: The test substance appeared to be stable under the conditions of the study; no evidence o f instability was observed. Study Initiated/Completed: July 8, 2004 / (see report cover page) Experimental Start/Completion: August 9, 2004 / October 13 2004 -7- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 SUMMARY Perfluorooctanoate (PFOA) plasma concentrations were measured in immature (3, 4 or 5 weeks o f age) rats following single oral gavage doses at 10 and 30 mg/kg. Plasma concentrations varied significantly between experiments, especially when compared to a prior study with 4-week old animals. In the current study, male rat PFOA plasma concentrations are relatively constant in male rats across age groups, varying only with dose level. Female rat plasma concentrations decreased with increasing age, but was only statistically significantly at the 30 mg/kg dose level. PFOA concentrations in urine, while not normalized for total elimination, indicated more rapid urinary excretion in female rats at all ages. The plasma concentrations from the supplemental group, however, are significantly different from the main study levels for both male and female rats. The plasma concentration variability in 4-week old rats suggests an unknown physiological variability in animals younger than 5 weeks. As a result, the relationship between age and PFOA plasma concentration is undefined in 3-4 week-old rats following oral dosing with 10 or 30 mg/kg PFOA. A. Plasma PFOA plasma concentrations in male rats were not significantly different by sample time (at 2 and 24 hours) or by animal age (3, 4 or 5 weeks) at either 10 or 30 mg/kg, except at 2 hours and 5 weeks of age. The PFOA plasma concentrations in female rats were significantly lower at 24 hours post dosing (compared to 2 hours) at all ages and doses tested. Female plasma concentrations were also lower for 5 week-old rats than 3 and 4 week-old rats at both sample times and doses (p < 0.01). The PFOA plasma concentrations following a 30 mg/kg dose were approximately 1.5 to 4 times higher than those observed following a 10 mg/kg dose. In a prior study, male and female rat PFOA plasma concentrations were measured at 24 hours post-dosing following a single 10 mg/kg oral gavage dose. PFOA plasma concentrations are consistent with the current study for both sexes at 5 weeks of age but not at 4 weeks of age (3week old animals were not previously measured). The standard deviations are small in both studies, indicating that both are internally consistent. The only known variation between the two studies is whether the rats were fasted before dosing. The supplemental rats were added to examine the effect of fasting on the PFOA plasma concentrations in 4-week old rats. Fasting increased the PFOA plasma concentrations as expected but the nominal vales are different from either the current study or the prior study. Given the consistency in the 5-week old rat PFOA plasma concentrations, it appears that there is an unknown physiological variability in rats younger than 5 weeks which impacts the pharmacokinetics of PFOA. B. Urine There were significant differences in PFOA urine concentrations with age, dose, and sex. Female rats showed higher PFOA urine concentrations, and these increased with age. Male rats showed the opposite, with urine concentrations decreasing with age. Both sexes showed higher -8 - Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 (2.5-6.5 times) urine concentrations at 30 mg/kg compared to 10 mg/kg doses. No attempt was made to quantify urinary output (volume), and, because creatinine levels were not measured, adjustments for urine concentration could not be performed. -9 - ^60 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 INTRODUCTION Ammonium perfluorooctanoate (APFO) is a synthetic perfluorinated octanoic acid used as an industrial surfactant. Elimination of the disassociated anion, perfluorooctanoate (PFOA) in rats is sex-dependent and appears to be under hormonal regulation. PFOA elimination is much faster in female rats than in male rats, down-regulated by testosterone in both female and castrated male rats, and up-regulated by estradiol in the male rats.(1'4) A prior study conducted at DuPont Haskell Laboratory found sex-dependent PFOA elimination in 4-8 week old rats.(5) The sex difference was least at 4 weeks of age, at time period just prior to sexual maturation in the rat. The objective of the this study was to determine if 3-week old male and female rats eliminate PFOA from blood at a rate different than that observed in 4- or 5-week old rats. Male and female post-weaning rats at different ages were dosed with APFO, and the PFOA plasma concentrations were compared at 2 and 24 hours post-dosing. At 24 hours after dosing, the sex difference in PFOA elimination in sexually mature rats was easily distinguished via the plasma concentration ofPFOA.(6) Urine was collected for 24 hours and analyzed for PFOA concentration. This study was designed to evaluate limited kinetic endpoints exclusive of determination o f elimination rate constants. MATERIALS AND METHODS A. Test Substance APFO was obtained from 3M (St. Paul, MN) and assigned Haskell Laboratory Number H-24921 upon receipt. Available information on the purity, composition, contaminants, synonyms, hazards, and hazardous material classification(s) were provided by the vendor and documented in the study records and/or report. B. Test System Male and female Crl:CD(SD)IGSBR rats were obtained from Charles River Laboratories, Inc., Raleigh, North Carolina. Animals for groups I-IV arrived as litters with dams. The SpragueDawley rat was chosen for this study because of the extensive experience with this strain and its suitability with respect to longevity, sensitivity, and low incidence of spontaneous diseases. Furthermore, the Sprague-Dawley rat has been used previously for toxicokinetic testing of PFOA and other fluorinated test materials/2'5'8^ At the time o f dosing, rats were approximately 3, 4, and 5 weeks of age and the weight variation did not exceed 20% of the mean weight by dose group and sex. Information on the cage labels included the Haskell animal number and an individual identification number assigned to each rat. The individual identification number was placed on the tail of each rat by tattoo or tail mark. -10- fis / Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 C. Animal Husbandry 1. Housing Upon arrival at Haskell Laboratory, rats were removed from shipping cartons and housed in appropriate cages according to Standard Operating Procedures unless specified in the study records. Rats were maintained under quarantine for at least three days unless approved otherwise by the site veterinarian, had at least one recorded weight gain, and no abnormalities detected. After the quarantine period, rats were selected for study. During pretest, dams were housed with their litters in polycarbonate pens containing bedding. Throughout the dosing period with test substance, the rats were housed individually in appropriate cages according to the SOP. During the urine collection period, rats were housed in stainless steel wire mesh cages. 2. Cage and Cage Rack Change Due to the length of the study, no cage and cage rack changes were required. 3. Environmental Conditions Animal rooms were maintained at a temperature of 18-26C (targeted to 22-24C) and a relative humidity of 30-70% (targeted to 40-60%). Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. Unless judged by the study director or the laboratory veterinarian to have significantly affected the results o f the study, the relative humidity and temperature ranges in the housing rooms were recorded but were not included in the final report. 4. Feed and Water All rats were provided tap water ad libitum and fed PMI Nutrition International, LLC Certified Rodent LabDiet 5002 ad libitum. Rats were not fasted prior to dosing due to their age. 5. Animal Health and Environmental Monitoring Program As specified in the Haskell Laboratory animal health and environmental monitoring program, the following procedures were performed periodically to ensure that contaminant levels were below those that would be expected to impact the scientific integrity of the study: Water samples were analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. Samples from freshly washed cages and cage racks were analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed was used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations o f key contaminants, including -11 - Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer were not expected to impact the integrity of the study. The animal health and environmental monitoring program was administered by the attending laboratory animal veterinarian. Evaluation of these data did not indicate any conditions that affected the validity of the study. D. Pretest Period Upon arrival at Haskell Laboratory, all rats were housed in quarantine. The rats were: quarantined for at least 3 days. identified temporarily by cage identification. weighed at least 3 times during quarantine and once prior to initiation of exposures. observed with respect to weight gain and any gross signs of disease or injury. The rats were released from quarantine by the laboratory animal veterinarian or designee based on body weights and clinical signs. Rats that were accidentally killed or removed from study during the pretest period were discarded without necropsy. Rats that were found dead or were sacrificed in extremis during the pretest period were given a gross examination to check for the presence of disease. The results were not included in the final report unless considered significant to the evaluation of the study. E. Assignment to Groups Rats were selected for use on study based on adequate body weight gain and freedom from any clinical signs of disease or injury. They were distributed by computerized, stratified randomization into study groups as designated in the Study Design, so that there were no statistically significant differences among group body weight means. Rats for groups I-IV were also randomized by dam to ensure even distribution. The weight variation of selected rats did not exceed 20% of the mean weight. The first 5 rats in each group were designated for sacrifice at the 2 hours and the remaining rats in each group were designated for sacrifice at 24 hours. Each rat was assigned a unique 6-digit Haskell animal number and an individual cage identification number. The last 3 digits of the Haskell animal number was tattooed on the tail of each rat. The Haskell animal number and cage identification number were both included on the cage label. Rats that were not assigned to a test group, including the dams from groups I-IV, were released for other laboratory purposes or sacrificed by carbon dioxide asphyxiation and discarded without anatomic pathology evaluation, at the discretion of the study director. -12- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 F. Main Study 1. Animals The experiments were composed of the following animal groups: Group Male Female Dose Age* (mg/kg)c Weeks Days Number of Ratsb Male Female I II 10 3 21 10 10 III IV 30 3 21 10 10 V VI 10 4 28 10 10 VII VIII 30 4 28 10 10 IX X 10 5 35 10 10 XI XII 30 5 35 10 10 aAnimals were 1 day from the target age at the time of dosing. The actual age at dosing was documented in the study records and/or final report. b5/sex/group sacrificed at 2 and 24 hours post-dose for blood collection. 0Given as a single gavage dose 2. Dose Preparation, Analysis, and Rates The test substance was administered as a single oral gavage dose. This route was chosen because it is the route most commonly used for toxicity studies of APFO. APFO was mixed with NANOpure water to achieve the target dose concentrations. Dose levels o f 10 and 30 mg APFO/kg body weight were used with a dose volume of approximately 4 mL/kg body weight. Dose was prepared on the day of use or prior to the day of use and stored under appropriate conditions. HPLC-MS methods were used to confirm PFOA concentration in the dose solutions. 3. Sacrifice and Blood Sampling Rats were sacrificed by CO2 asphyxiation and blood collected from the vena cava or by cardiac puncture. 4. Experimental Procedure Rats were obtained from the vendor and administered PFOA at dose levels described above. Two hours after dosing, 5 rats/sex/group were sacrificed and whole blood collected. Whole blood samples were placed into EDTA tubes and maintained on wet ice. Plasma was separated by centrifugation and frozen at <-10C until analysis. The remaining 5 rats/sex/group were placed in metabolism cages for urine collection. Urine was collected for 24 hours after dosing. At 24 hours, all remaining rats were sacrificed and whole blood collected. Blood was handled and separated as above. - 13 - Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage No rats were found dead or were sacrificed in extremis. DuPont-15302 G. Supplemental Study Four additional male and four additional female rats were added to groups V and VI. Two of the male and two o f the female rats were fasted overnight for approximately 12 hours before dosing with test substance. Dose preparation and administration was the same as in the main study. All rats were sacrificed at 24 hours post dosing and whole blood collected. Blood was handled and separated as above. HPLC-MS method was used to determine plasma concentrations of PFOA. H. Analytical Methods 1. Verification of Dose Solution Methods for verification of dose solution are detailed in Appendix A. 2. Extraction of PFOA from Plasma for LC/MS Analysis Plasma samples were processed by protein precipitation (PPT) using Isolute Array protein precipitation columns (Jones Chromatography, Lakewood, CO). A 0.5 pg/mL solution of perfluorononanoic acid (Aldrich Chemicals, Milwaukee, WI) in acetonitrile (ACN) was used as an internal standard for quantitation o f PFOA. Plasma samples were thawed, and a 20 pL aliquot of each sample was applied to the PPT array. The plasma samples were precipitated by adding appropriate dilution rate volumes of ACN/intemal standard solution to the PPT array. Dilution rates, ranging from 1:10 (180 pL of internal standard solution) to 1:200 (3980 pL o f internal standard solution), were utilized in order to capture the sample concentrations within the standard curve parameters. The array was slowly eluted under vacuum into a 96-well receiver plate, centrifuged at -3000 rpm for 5 minutes, and extracts were analyzed by LC/MS. -14- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 3. Instrumentation 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: Waters 2795 Liquid Chromatograph, equipped with quaternary pump, column heater, and autosampler Quattro Micro Mass Spectrometer MRM Negative Electrospray 10.0 10.0 1.0 5 2 5 14 14 2.0 650 2.0 15 1.0 0 130C 350C MRM, 2 transitions 0-10.0 min 413.00->369.00 Dwell: Collision energy: 463.00->419.00 Dwell: Collision energy: 0.25 sec 15 eV 0.25 sec 15 eV 4. Chromatographic Methods Method: Column: Column temperature: Mobile phases: Gradient: Flow rate: Stop time: Injection volume: Plasma concentration analysis Waters Xterra MS C l8, 2.1x30mm, 2.5 pm Ambient A: 50 mM Ammonium Acetate B: Acetonitrile Time (min) 0.0 0.5 10.0 10.9 11.0 %B 5 10 100 100 10 0.25 mL/min 11.0 min 5 pL - 15- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 I. Statistical Analyses Group data is represented as Mean SD. Where appropriate, statistical significance was assessed by the Student's t-test. RESULTS AND DISCUSSION A. Dose Solution Analysis (Appendix A) The concentrations o f the test substance in the dose solutions were verified by LC/MS method as described in Materials and Methods, Section F. Standards for dose verification and the dose solutions were prepared by different personnel. The determined mean PFOA concentrations of the dose solutions were all within 20% of the targeted concentration (2.5 and 7.5 mg/mL, Appendix A). PFOA was stable in the vehicle for at least 5 hours. B. Plasma Concentration of PFOA (Tables 1-2, Figure 1, Appendices B-D) 1. Male Rats The PFOA plasma concentrations were not significantly different by sample time (at 2 and 24 hours) or by animal age (3, 4 or 5 weeks) at either 10 or 30 mg/kg, except at 2 hours and 5 weeks of age (p<0.01 based on the results of unpaired t-test at 99% confidence interval). In some groups the 24-hour post dosing concentrations were higher, but this was not unexpected due to the long plasma elimination of PFOA in male rats.(6) Individual PFOA plasma concentrations ranged from 21.00 to 48.85 pg/mL for the 10 mg/kg dose and from 49.51 to 153.89 pg/mL for the 30 mg/kg dose. PFOA plasma concentrations following a 30 mg/kg dose were approximately 2-3 times higher than those observed following a 10 mg/kg dose. 2. Female Rats PFOA plasma concentrations were significantly lower at 24 hours post dosing (compared to 2 hours) in all ages and doses tested. PFOA plasma concentrations were also lower at 5 weeks than at 3 or 4 weeks at both sample times and doses (p < 0.01). PFOA plasma concentrations following a 30 mg/kg dose were approximately 1.5 to 4 times higher than those observed following a 10 mg/kg dose. - 16- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 3. Supplemental Rats Individual PFOA plasma concentrations in the male rats were 64.95 and 30.00 pg/mL for the fasted and non-fasted animals, respectively. PFOA plasma concentrations in the female rats were 68.16 and 26.54 pg/mL for the fasted and non-fasted animals, respectively. In a prior study,(5) male and female rat PFOA plasma concentrations were measured at 24 hours post-dosing following a single 10 mg/kg oral gavage dose. PFOA plasma concentrations are consistent with the current study for both sexes at 5 weeks of age but not at 4 weeks of age (3week old animals were not previously measured). The standard deviations are small in both studies, indicating that both are internally consistent. The only known variation between the two studies is whether the rats were fasted before dosing. The supplemental rats were added to examine the effect of fasting on the PFOA plasma concentrations in 4-week old rats. Fasting increased the PFOA plasma concentrations as expected but the nominal vales are different from either the current study or the prior study. Given the consistency in the 5-week old rat PFOA plasma concentrations, it appears that there is an unknown physiological variability in rats younger than 5 weeks which impacts the pharmacokinetics of PFOA. C. Urine Concentration of PFOA (Tables 1-2, Figure 2, Appendices E-F) PFOA urine concentrations differed significantly with age, dose and sex (p < 0.01). Female rats had higher PFOA urine concentrations than males and the female PFOA urine concentrations increased with age. Male rats showed the opposite with urine concentrations decreasing with age. Both sexes had higher (2.5 to 6.5 times) PFOA urine concentrations at 30 mg/kg compared to 10 mg/kg doses. No attempt was made to quantify urinary output (volume) or standardize for creatinine levels. CONCLUSIONS Perfluorooctanoate (PFOA) plasma concentrations were measured in immature (3, 4 or 5 weeks of age) rats following single oral gavage doses at 10 and 30 mg/kg. Plasma concentrations varied significantly between experiments, especially when compared to a prior study with 4-week old animals. In the current study, male rat PFOA plasma concentrations are relatively constant in male rats across age groups, varying only with dose level. Female rat plasma concentrations decreased with increasing age, but was only statistically significantly at the 30 mg/kg dose level. PFOA concentrations in urine, while not normalized for total elimination, indicated more rapid urinary excretion in female rats at all ages. The plasma concentrations from the supplemental group, however, are significantly different from the main study levels for both male and female rats. The plasma concentration variability in 4-week old rats suggests an unknown physiological variability in animals younger than 5 weeks. As a result, the relationship between age and PFOA plasma concentration is undefined in 3-4 week-old rats following oral dosing with 10 or 30 mg/kg PFOA. Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 RECORDS AND SAMPLE STORAGE A sample of the test substance will be collected for archive purposes and retained at Haskell Laboratory, Newark, Delaware. 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. -18- "f t # Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 REFERENCES 1. Ohmori K., Kudo, H., Katayama, K., and Kawashima, Y. (2003). Comparison of the toxicokinetics between perfluorocarboxylic acids with different carbon chain length. Toxicology 184, 135-140. 2. Vanden Heuvel, J.P., Davis, J.W., Sommers, R., and Peterson, R.E. (1992) Renal excretion of perfluorooctanoic acid in male rats: inhibitory effect of testosterone. J. Biochem. Toxicol. 7,31-36. 3. Kudo, N., Suzuki, E., Katakura, ML, Ohmori, K., Noshiro, R., and Kawashima, Y. (2001) Comparison o f the elimination between perfluorinated fatty acids with different carbon chain length in rats. Chem. Biol. Interact. 134, 203-216. 4. Ylinen, M., Hanhijarvi, H., Jaakonaho, J., and Peura, P. (1989) Stimulation by oestradiol of the urinary excretion o f perfluorooctanoic acid in the male rat. Pharmacol. Toxicol. 65, 274277. 5. DuPont Haskell Laboratory (2003). Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage. Unpublished report, DuPont-13267. 6. DuPont Haskell Laboratory (2003). Perfluorooctanoic Acid: Toxicokinetics in the Rat. Unpublished report, DuPont-7473. 7. DuPont Haskell Laboratory.(1997). Hazard Characterization for Human Health C8 Exposure. Unpublished report. 8. 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, 83-92. - 19- -5V6 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 TABLES -20- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage TABLES EXPLANATORY NOTES ABBREVIATIONS: SD standard deviation DuPont-15302 -21 - Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Table 1: Plasma and urine PFOA concentrations in male rats dosed via oral gavage with 10 or 30 mg/kg PFOA Group Age Dose (weeks) (mg/kg) I 3 10 V 4 10 IX 5 10 III 3 30 VII 4 30 XI 5 30 Data expressed as lig/mL Plasma 2 Hours Post-Dose 24 H ours Post-Dose Mean SD Mean SD 41.87 39.92 26.32 4.01 4.45 6.89 34.22 42.94 40.60 7.89 5.33 3.69 120.65 117.40 65.66 12.78 18.10 15.53 74.16 100.81 113.86 18.23 13.18 23.36 Urine 24 Hours Post-Dose Mean SD 9.57 4.86 4.53 2.45 4.03 2.36 51.76 28.70 15.65 28.86 18.84 6.24 -22* t/3 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Table 2: Plasma and urine PFOA concentrations in female rats dosed via oral gavage with 10 or 30 mg/kg PFOA Group I V IX III VII XI Age Dose (weeks) (mg/kg) 3 10 4 10 5 10 3 30 4 30 5 30 Data expressed as ng/mL Plasma 2 Hours Post-Dose 24 H ours Post-Dose Mean SD Mean SD 37.87 29.88 33.23 5.77 12.15 7.41 13.55 18.98 1.36 3.83 7.01 0.87 84.86 80.67 56.90 10.51 14.10 29.66 51.43 28.01 3.42 13.61 9.90 1.95 Urine 24 Hours Post-Dose Mean SD 21.17 23.26 49.77 8.95 15.27 24.64 94.89 104.12 123.16 26.26 28.97 51.56 -23- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 FIGURES -24- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Figure 1: Plasma PFOA concentration in rats following oral gavage with PFOA l \ \ \ t 2 hours post dosing lXX>q 24 hours post dosing Age (weeks) a Male plasma at 10 mg/kg (groups I, V and IX). b Male plasma at 30 mg/kg (groups III, VII and XI). c Female plasma at 10 mg/kg (groups II, VI and X). d Female plasma at 30 mg/kg (groups IV, VIII and XII). -25 - Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Figure 2: Urine PFOA concentration in rats following oral gavage with PFOA 175-i 150- -g 125- O3). cof 10075- c8 oo c<u 50- 25- male EWsXJ female 53 Age (weeks) a 10 mg/kg (groups I, II, V, VI, IX and X). b 30 mg/kg (groups III, IV, VII, VIII, XI and XII). 2 6- - #7 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 APPENDICES -27- 4 f8 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage APPENDICES EXPLANATORY NOTES ABBREVIATIONS: LOQ SD SE % CV limit of quantitation standard deviation standard error percent coefficient of variation DuPont-15302 - 28- Ammonium Perfiuorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Appendix A Dosing Solution Analysis -29- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in PostWeaning Rats Following Oral Gavage ANALYSIS FOR H-24921 IN POSING SOLUTIONS Medical Research Project Number: Haskell Sample Number Analytical Report Number 15339 24921 DuPont-15302_an SUMMARY Dosing solutions at concentrations of 2.5, and 7.5 mg/mL of H-24921 were prepared and collected for uniformity of mixing/concentration verification and stability analysis (5hour loom temperature) on August 9,2004. In addition, 0 mg/mL (control) sample was submitted for analysis with the set of samples. Concentrations of H-24921 in separate dosing solutions were measured by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). The suspension vehicle for the study was Nanopure water. The results for H-24921 samples prepared and collected on August 9,2004 indicated that the test substance was at the targeted concentrations ( 16.9% of nominal), adequately mixed (CV less than 10) and stable in the vehicle when held 5 hours at room temperature for all levels. H-25425 was not detected in the 0 mg/mL (control) samples. -30- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 DuPont-153Q2_AN; Page 2 _______________________________________________________ SAMPLE SUBMITTAL Dosing solutions at concentrations of 2.5, and 7.5 mg/mL of H-24921 were prepared and collected for uniformity of mixing/concentration verification and stability analysis (5-hour room temperature) on August 9,2004. In addition, 0 mg/mL (control) sample was submitted for analysis with the set of samples. Samples submitted for analysis were analyzed the day they were received and/or when re analysis was indicated. The suspension vehicle for the study was Nanopure water. METHODS 1. Analytical Methods a. Recovery Sample Analysis Concurrent with dosing solutions analyses, recovery of H-24921 from spiked vehicle (Nanopure 9 water) was tested at the low level (2.5 mg/mL) and at the high level (7.5 mg/mL) to confirm the analytical method. A stock solution of H-24921 was prepared in Nanopure water. For all concentration levels, an appropriate aliquot of this solution to obtain approximately 1.25 mg (low), and 3.75 mg (high) of the test material was added to 100 mL of Nanopure* water. All recovery samples were then mixed for dispersion of the test material in the vehicle. The samples were then processed and analyzed in the same manner as the dosing samples at similar concentrations. b. Dosing Suspension Treatment Each dosing sample (0.5 mL) was diluted to 100 mL with Nanopure water and mixed to dissolve the H-24921 in the suspension. The dosing samples were further diluted with Nanopure water to an expected concentration of approximately 0.00003 mg/mL 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 the day the solutions were received and/or when re-analysis was indicated. -31 - Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 DuPont-15302_AN; Page 3 c. Chromatographie Conditions LC parameters Instrument: Column: Flow Rate: Injection Volume: Column Temperature: Column Switch: Mobile Phase: Gradient: Time (min.) 0.0 0.9 1.0 5.0 5.1 8.5 MS parameters Instrument: Ionization mode: Capillary voltage: Cone Voltage: Source Temperature: Desolvation Temperature: Scan function: Water Alliance 2795 HPLC Zorbax RX-C8,2.1 mm x 150 mm, 5pm 0.4 mL/min 20 fil 35C 3.0 minutes 0.15% Acetic Acid/Acetonitrile % Acetonitrile 5 5 80 80 5 5 Micromass Quattro Micro Tandem MS Electrospray (ESI), negative ion 2.7 kV 15 V 120" 350" PFOA: 413 m/z (parent) to 369 m/z (daughter) l,2-di-l3C PFOA: 415 m/z (parent) to 370 m/z (daughter) Retention Time of PFOA and l,2-di-13C PFOA: approximately 3.5 minutes (20 pL injections) d. Calibration and Quantitation A stock solution of Analytical Standard (APFO, H -22703-265,100% pure) was made in acetoni trilc. Appropriate aliquots of the stock were diluted with Naxtopure water to make calibration standards that bracketed the target concentration o f the diluted sample solutions. Before these aliquot were brought to volume, an appropriate amount of internal standard (1,2di-l3C periluorooctanoic acid) was added. Analysis o f H-24921 was by high-performance liquid chramatography/tandem mass spectrometry (LC-MS/MS). Negative-ion electrospray is used to generate negatively charged ions of PFOA, the ions are selected with the first MS quadrupole, collisionally dissociated using argon, and a fragment ion is monitored. 1,2-di- C perfluorooctanoic acid is used as an internal standard. Triplicate injection o f the sample solutions and calibration standards solutions were made and peak areas were calculated electronically. -32- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 PuPom-lS302_AN; Pige 4_______________________________________________________________ The calibration curve was generated by regression analysis using the peak area ratio from the analytical standard and the internal standard (refer to figure 1). Data for test solutions were compared to the calibration curves to evaluate the concentrations of the H-24921. 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 of 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 results. 33 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 DuPont-15302_AN;Page 5______________________________________________________________ ANALYTICAL RESULTS A. Chromatography H-24921 eluted from the HFLC column as a resolved peak with a retention time of approximately 3.5 minutes (20 pL injections) for the negative ion. Representative LC-MS/MS chromatograms are shown in Figures 2 (a - f). Test substance was not detected in the 0 mg/mL control. This was determined by a comparison of the diluent water with and without internal standard against the 0 mg/mL control containing internal standard. Refer to Figures 2 (a - c). B. Recovery Samples Detailed analytical results of recovery samples are summarized in Table I. The measured concentration of H-24921 for the 2.5 mg/mL level was 97.3% of nominal. The measured concentration of H-24921 for the 7.5 mg/mL level was 102.1% of nominal. Based on this data, the analytical method performed satisfactorily over the entire concentration range for the study. C. Uniformity of Mixing/Concentration and Stability Samples Analytical results from dosing solutions collected on August 9,2004 and analyzed for uniformity of mixing/concentration verification and 5-hour room temperature stability are shown in Appendix o , Table II and Summary Table 1. The following table summarizes the results for uniformity/concentration verification and stability analyses for this sampling day of the H-24921 preparation. Sample Day Sample Type Nominal mg/mL Measured ' mg/mL Mean % Nominal C.V. Stability0 m % Nominal 9-Aug-2004 Uniformity/tancentration 0 NDC _ __ 2.5 2.98,2.80 7 J 8.65,8.88 115-5 116.9 4 118.4 2 119.0 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 comparison of the diluent water with and without the intentai standard addition and the control with the internal standard addition. Shown in Figures 2 {a - C). The results far H-24921 samples prepared and collected on August 9,2004 indicated that the test substance was at the targeted concentrations ( 16.9% 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. E. Conclusion Results from the analysis of the H-24921 dosing solutions for 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. -34*2 S Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 ! PuPom-15302_AN;Page 6________________________________________________________________ Table L Recovery ofH-24921 Added to DosingVehicle Sample tasM H-24921 Percent Type Nominal Measured Nominal Recovery**5 2.52 2.45 97.3 Recovery**5 7.45 7.61 102.1 w Processed with uniformity ofmixing/concaitration verification samples fromdoting prepared on August9, 2004. m Reported result is from a duplicatepreparation offoe recovery sample. Theoriginalrecovery sample analyzed higherthanexpected due to aliquoterror and is notreported.. Sam ple Type 9-A ugust-2004 U niform itvfC oncentratkm CONTROL m a/rnL H -24921 N om inal M easured 0.00 N D (A ) P e rc e n t N om inal -- #1 2.5 2.98 119.1 n 2.5 _ m 111.8 M erur .- 2 . 8 9 . l 3 (ffj.5 % ) CV.4% # i I S 8.65 115.3 MS2 I S 118.4 M ean ).- 8 . 7 7 0 . 1 6 (116.9%) C.V. 2 % 5 HOUR 2.5 2 .9 6 118.4 I S 8.93 119.0 W Denotes none detected. Reported results are based on comparison o f the diluent water with and w ithout the internal standard addition and the control with the internal standard addition. Shown in Figures 1 (a - c), Theaveragemeasuredconcentration,averagepercentofnominal(in parentheses), standarddeviation,and coefficientof variationofduplicate samples. (Q Stabilitysampleshdd 5hours a roomtemperature. -35- 4 -2 < Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-lS302_AN;Page 7 Figure 1 Representative Analytical Calibration Curves DuPont-15302 Figure 1: Calibration curve showing linear fit (line) to replicate peak area ratio (squares) for calibration solutions of H-22703-265 (APFO Analytical Standard) diluted with Nanopure water and matrix matched over a concentration range of 0.005 to 0.051 ppm. -36- 427 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302_AN; Page 8 ____________ __________________________ Figure 2 Representative LC/MS/MS Chromatography Chromatograms DuPont-15302 study. Negative ion/PFOAretention time is approximately 3.5 minutes. study with the internal standard. Negative ion/PFOA retention time is approximately 3.5 minutes. -37- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-1S302_AN; Page 9 Figure 2 (continued) Representative LC/MS/MS Chromatography Chromatograms DuPont-15302 f Figure 2c: RepresentativeLC/MS chromatogram of0.00 mg/mLcontrol solution ofH-24921 withthe internal standard. Negative ion/PFOA retention time is approximately 3.5 minutes. Figure 2d: RepresentativeLC/MS chromatogramofH-22703-265 (APFO Analytical Standard) (0.030ppm) with the internal standard (0.0315 ppm). Negative ion/PFOA retention time is approximately 3.5 minutes. -38- -f29 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302_AN; Page 10 __________________________________ Figure 2 (continued) Representative LOMS/MS Chromatography Chromatograms DuPont-15302 concentration of0.03 ppm ofH-24921 for negative ion /PFOA with retention time 3.5 minutes. The measured concentration ofthe representative solutionis 8.65 mg/mL. 0 8 0 0 4 o 5 0 Sffl POIM M n.2*3) WR.'tSSJft; SC:t 3*2; S p t k 4 42 - 7.45 m g / m t APPO i .SI 4468.313 53598 MUM a l2 Channel*,ES413 > 3ft 5.383ft +004 ................ 060904C6C WR 1533ft, SC :13S ;S ftlk0 12 7.S m g/m l S-30 I1 -5 M133CC-.PFFOOA; 3487744J.870 6221712 U H M c><3 ch n n * l* ,E 3 415 > 370 8.304*004 Too T"** 400 Figure 2f: Representative LC/MS chromatogram ofhigh recovery dosing solution (7.45 mg/mL) diluted to nominal concentration of0.03 ppm ofH-24921 for negative ion /PFOA with retention time approximately 3.5 minutes. The measured concentration ofthe representative recovery solution is 7.61 mg/mL. -39- 4-30 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Table 1 Summary ofDosing Suspension Analyses Sample Type ___________________________ Dosing Concentrations and Stability o f H -2 4 9 2 1 (mg/mL) Unifoirohy/Conocntration August 9,2004 control Nominal: 2.5 0.0 7.5 NDa #1 n A verage M easu red C onc.c A v e ra g e P e r c e n t N o m in a l12 S tan d ard D eviation2 Coefficient o f V ariation2 Stability 5-hour Room Temperature 2.98 (119.1>b 2.80 (111.8) 18 HS45) *0.13 4% 2.96 (1184) 8.65 (115.2) 8 88 (118.4) 8.77 (116.9) 0.16 2% 8.93 (119.0) 1 Denotes none detected Reported results are based on comparison o f the diluent water with and without the internal standard addition and the control with the intim ai standard addition. Shown in figures 2 (a - c). b Numbers in parentheses are the respective percent o f nominal values. 2 The avenge measured concentration, average percent o f nominal (in parentheses), standard deviation, and coefficient o f variation o f duplicate samples. - 40*37 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Appendix B PFOA Plasma Concentration Data - Males -41 - 4r2< 9~ Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Group I I I I I III III III III III V V V V V VII VII VII VII VII IX IX IX IX IX XI XI XI XI XI Age (weeks) 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 Time Post Dose (hours) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Animal Number 1 2 3 4 6 8 10 12 15 16 51 53 54 58 61 52 55 56 57 60 99 100 103 105 107 96 97 98 101 102 Dose (mg/kg) 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 PFOA Plasma Concentration (pg/mL) 47.20 44.30 39.80 41.19 36.85 138.95 123.62 104.66 113.53 122.48 35.37 43.13 35.85 45.54 39.70 117.29 86.41 131.53 127.73 124.03 29.36 37.00 21.75 23.09 20.39 59.63 49.51 58.10 71.36 89.69 Mean 41.87 120.65 39.92 117.40 26.32 65.66 SD 4.01 12.78 4.45 18.10 6.89 15.53 -42- 433 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Group I I I I I III III III III III V V V V V VII VII VII VII VII IX IX IX IX IX XI XI XI XI XI Age (weeks) 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 Time Post Dose (hours) 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 Animal Number 9 13 14 18 24 17 19 21 22 25 62 63 65 66 70 64 67 68 71 72 108 109 110 115 116 106 111 112 113 114 Dose (mg/kg) 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 PFOA Plasma Concentration (Pg/mL)___ 35.23 21.00 36.13 42.23 36.49 65.30 76.80 80.05 98.90 49.73 40.04 42.68 47.32 48.85 35.80 112.17 106.94 108.70 96.60 79.64 37.05 39.60 40.52 39.05 46.80 98.30 98.70 103.53 153.89 114.88 Mean 34.22 74.16 42.94 100.81 40.60 113.86 SD 7.89 18.23 5.33 13.18 3.69 23.36 - 43- 3* < / Appendix C PFOA Plasma Concentration Data - Females -44- 43S Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Group II II II II II IV IV IV IV IV VI VI VI VI VI VIII VIII VIII VIII VIII X X X X X XII XII XII XII XII Age (weeks) 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 Time Post Dose (hours) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 Animal Number 26 27 31 33 38 28 30 32 35 36 73 74 75 77 78 76 80 82 83 84 122 124 126 127 128 117 118 120 121 125 Dose (mg/kg) 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 PFOA Plasma Concentration (gg/mL) 29.64 37.87 45.90 38.63 37.32 96.93 89.64 74.41 90.13 73.18 27.69 31.05 19.27 49.91 21.48 103.54 67.98 70.38 79.15 82.28 44.00 23.66 33.88 30.08 34.51 78.17 98.61 35.55 34.86 37.30 Mean 37.87 84.86 29.88 80.67 33.23 56.90 SD 5.77 10.51 12.15 14.10 7.41 29.66 -45*3C Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Group II II II II II IV IV IV IV IV VI VI VI VI VI VIII VIII VIII VIII VIII X X X X X XII XII XII XII XII Age (weeks) 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 Time Post Dose (hours) 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 Animal Number 40 44 46 49 50 37 39 42 43 45 79 81 89 91 94 86 87 88 90 92 130 131 132 133 137 129 134 135 136 138 Dose (mg/kg) . 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 PFOA Plasma Concentration (P&/mL)____ 8.82 13.70 13.76 19.38 12.08 56.25 51.05 42.29 71.43 36.13 16.63 23.20 24.04 7.67 23.39 37.70 39.31 21.62 17.32 24.09 2.49 0.92 0.36 1.00 2.02 0.61 5.53 4.33 4.33 2.30 Mean 13.55 51.43 18.98 28.01 1.36 3.42 SD 3.83 13.61 7.01 9.90 0.87 1.95 - 46- ^ 7 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Appendix D Supplemental PFOA Plasma Concentrations - 47- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 A nim al PFOA Plasm a C oncentration (pg/m L) M ean SD Fasted Male 1 Fasted Male 2 84.28 45.62 64.95 27.34 Fasted Female 1 Fasted Female 2 58.20 78.13 68.16 14.10 Non-Fasted Male 3 Non-Fasted Male 4 29.81 30.20 30.00 0.28 Non-Fasted Female 3 Non-Fasted Female 4 31.94 21.14 26.54 7.64 Note: All animals were 3 weeks of age and received a single 10 mg/kg dose. Plasma samples were collected 24 hours post-dosing. - 48- 437 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Appendix E Urine PFOA Concentration Data - Males - 49- 440 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Group I I I I I III III III III in V V V V V VII VII VII VII VII EX IX IX IX EX XI XI XI XI XI Age (weeks) 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 Animal Number 9 13 14 18 24 17 19 21 22 25 62 63 65 66 70 64 67 68 71 72 108 109 110 115 116 106 111 112 113 114 Dose (mg/kg) 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 PFOA Urine Concentration (pg/mL) 9.26 16.21 3.41 11.97 6.97 23.69 71.90 80.97 NS 30.48 0.72 3.62 5.45 5.85 7.01 16.32 22.55 10.99 58.17 35.47 2.78 4.04 2.35 2.85 8.10 11.24 13.75 25.61 17.46 10.16 NS = no urine sample was produced by the animal. Mean 9.57 51.76 4.53 28.70 4.03 15.65 SD 4.86 28.86 2.45 18.84 2.36 6.24 - 50- * -f/ Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Appendix F Urine PFOA Concentration Data - Females -51- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-15302 Group II II II II II IV IV IV IV IV VI VI VI VI VI VIII VIII VIII VIII VIII X X X X X XII XII XII XII XII Age (weeks) J 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 A nim al Number 40 44 46 49 50 37 39 42 43 45 79 81 89 91 94 86 87 88 90 92 130 131 132 133 137 129 134 135 136 138 Dose (mg/kg) 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 10 10 10 10 10 30 30 30 30 30 PFOA Urine Concentration (Pg/mL)___ 17.59 19.36 10.04 24.73 34.11 116.09 83.67 95.85 56.68 122.17 23.21 7.16 24.79 47.37 13.79 125.55 60.35 90.84 131.16 112.68 41.04 23.85 89.76 52.89 41.31 99.53 111.16 55.41 175.44 174.25 Mean 21.17 94.89 23.26 104.12 49.77 123.16 SD 8.95 26.36 15.27 28.97 24.64 51.56 -52- 443