Document mp5vyYnL4xBOGr5kLMDMoX8e4

DuPont-13267 -*2^ 6- /6S3> Study Title Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage A u t h o r : Xing Han, Ph.D. S t u d y C o m p l e t e d o n : December 15,2003 P e r f o r m i n g L a b o r a t o r y : E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 L a b o r a t o r y P r o j e c t I D : DuPont-13267 * 1 ,(0 Work Request Number: 14762 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 1 o f 34 c2 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards. Study Director: v Xing Han, Ph.D. Research Toxicologist RI. du Pont de Nemours and Company /(P-XVc Date -2 - 3 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage QUALITY ASSURANCE STATEMENT Haskell Sample Number(s): 24921 Dates of Inspections: Protocol: June 18, 2003 Conduct: July 8,9,21,28,30, 2003 Records, Reports: October 27-30, 2003 Dates Findings Reported to: Study Director: June 18, 2003; November 3,11, 2003 Management: November 3,11, 2003 DuPont-13267 Reported by: U ) ( m d a J . l i i j ________ W ondaK elly Quality Assurance Auditor Date Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Approved by Principal Research Toxicologist and Manager /.- iC ' S ' Date Approved by: Matthew S. BogdMffy. Ph; Research Manager Issued by Study Director: Xing Han, Ph.D. Research Toxicologist (2--j>e-C-Zt3 Date < s - ,)->c - ^ $ Date Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COM PLIANCE ST A T E M E N T .......................................2 QUALITY ASSURANCE ST A TE M EN T.............................................................................................. 3 C E R T IFIC A T IO N .........................................................................................................................................4 LIST OF T A B L E S .........................................................................................................................................6 LIST OF F IG U R E S ...................................................................................................................................... 6 LIST OF A PPEN D IC E S.............................................................................................................................. 6 STUDY INFO RM A TIO N............................................................................................................................ 7 STUDY PE R SO N N EL......................................................................... SUM M ARY.................................................................................................................................................... 10 A. Age Effect............................................................................................................................. 10 B. Sex Comparison.................................................................................................................... 10 INTRO DU CTIO N............................................................................................................ M ATERIALS AND M ET H O D S..............................................................................................................11 A. Test Substance....................................................................................................................... 11 B. Test System........................................................................................................................... 11 C. Animal Husbandry................................................................................................................ 12 1. Animal H ousing......................................................................................................................................................12 2. Environmental Conditions...................................................................................................................................... 12 3. Feed and Water......................................................................................................................................................... 12 4. Animal Health and Environmental Monitoring Program.................................................................................12 D. In-Life Methods..................................................................................................................... 13 1. Anim als...................................................................................................................................................................... 13 2. Dose Preparation, Analysis, and Rates................................................................................................................ 13 3. Sacrifice and B lood Sampling.............................................................................................................................. 13 4. Experimental Procedure..........................................................................................................................................13 E. Analytical Methods............................................................................................................... 14 1. Verification o f Dose Solution................................................................................................................................ 14 2. Extraction o f PFOA from Plasma for LC/MS Analysis................................................................................... 14 3. Instrumentation........................................................................................................................................................ 14 4. Chromatographic M ethods.....................................................................................................................................15 F. Statistical Analysis................................................................................................................ 15 RESULTS AND DISC U SSIO N ................................................................................................................15 A. Dose Verification.................................................................................................................. 15 B. Plasma Concentration of PFOA........................................................................................... 16 1. Male Rats................................................................................................................................................. 16 2. Female Rats............................................................................................................................................................... 16 3. Comparison between Male and Female Rats...................................................................................................... 16 CO NCLUSIONS........................................................................................................................................... 16 RECORDS AND SAM PLE S T O R A G E ...............................................................................................16 R E FER EN C ES............................................................................................................................................. 17 T A B L E S.......................................................................................................................................................... 18 FIG U R E S....................................................................................................................................................... 21 A P PE N D IC E S.............................................................................................................................................. 25 11 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage______________________________ DuPont-13267 LIST OF TABLES 1. Plasma concentration in rats 24 hours post-dose following oral gavage with lOmg/kg PFOA................................................................................................................. Page 20 LIST OF FIGURES Page 1. Plasma concentration in male rats 24 hours post-dose following oral gavage with lOmg/kg PFOA........................................................................................................................... 22 2. Plasma concentration in female rats 24 hours post-dose following oral gavage with lOmg/kg PFOA........................................................................................................................... 23 3. Plasma concentration in male and female rats 24 hours post-dose following oral gavage with 10 mg/kg PFOA......................................................................................................24 LIST OF APPENDICES Page A. Dose Verification Data................................................................................................................27 B. Plasma Concentration D ata........................................................................................................ 30 -6 - Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 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 FI-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 (C3F7COL N H /), 0.01 % C5(C4F9CO2' NH4+), 0.03% C6(QFuCOL NH4+), 0.43% C7(C6Fi3C 0 2' NH4+), 0.57% C9(C8Fi7C02' 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. -7 - & Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage 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 Experimental Start/Completion: June 30, 2003 / August 7, 2003 Study Initiated/Completed: June 27, 2003 / (see report cover page) DuPont-13267 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 STUDY PERSONNEL Study Director: Xing Han, Ph.D. Primary Technicians: LaRue A. Manning, B.A. Brian P. Shertz, A.A. Management: Matthew S. Bogdanffy, Ph.D., D.A.B.T. Gary W. Jepson, Ph.D. Toxicology Report Preparation: Lisa G. Burchfield, A.A. Management: Nancy S. Selzer, M.S. Laboratory Veterinarian: Thomas W. Mayer, D.V.M., Diplomate A.C.L.A.M. Management: Janice L. Connell, M.S., B.A., C.I.H. / Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 SUMMARY The relationship between age and plasma concentration of ammonium perfluorooctanoate (PFOA) in rats has been investigated in this study. Male and female rats between 4 and 8 weeks old were administered 10 mg/kg of PFOA by single oral gavage. The plasma concentration of PFOA at 24 hours post-dose was measured by LC/MS. The results showed that the plasma concentration of PFOA in rats was dependent on the age and sex of the rats. A. Age Effect In both male and female rats, the greatest age-related difference in PFOA plasma concentration was observed between 4 and 5 weeks of age. The 5-week old male rats had PFOA plasma concentrations that were 5.4-fold higher than the 4-week old male rats. In contrast, the PFOA plasma concentrations in the 5-week old female rats were 2.4-fold lower than in the 4-week old rats. PFOA levels in plasma were no longer significantly affected by age in rats of either sex that were older than 5 weeks (through study termination at 8 weeks). B. Sex Comparison At 4 weeks of age, male rats had PFOA plasma concentrations that were 2.7-fold higher than in females. The PFOA plasma concentrations in 5- to 8-week old male rats were 34.7- to 65.1-fold higher than that in the females of the same age. Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 INTRODUCTION Ammonium perfluorooctanoate (PFOA) is a perfluorinated octanoic acid salt used as an industrial surfactant. Elimination of PFOA in rats is sex-dependent and hormone regulated. PFOA elimination is much faster in female rats than in male rats,(l) down-regulated by testosterone in both female and castrated male rats/2,31 and up-regulated by estradiol in the male rats.(4) However, these studies were conducted on sexually mature rats. In the current study, male and female post-weaning rats at different ages were dosed with PFOA, and the plasma concentrations of PFOA at 24 hours post-dosing were compared. This post-dosing time point was chosen because 24 hours after dosing, the sex difference in PFOA elimination in mature rats can be easily distinguished via the plasma concentration of PFOA.(5) The objective of this study was to determine if immature male and female rats eliminate PFOA from blood at rates different than those observed in mature rats. This study was not designed to fully characterize the pharmacokinetics of PFOA in immature rats, and was not designed for use in determining definitive elimination rate constants. MATERIALS AND METHODS A. Test Substance PFOA (ammonium perfluorooctanoate, FC-143, Lot 332) 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) was provided by the vendor and documented in the study records and report. CAS Registry Number: 335-67-1 Molecular Weight: 414.1 Molecular Formula: C8F15O2H B. Test System Male and female Crl:CD(SD)IGSBR rats were obtained from Charles River Laboratories, Inc., Raleigh, North Carolina. The Sprague-Dawley 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.(2,5,6) At the time of dosing, rats were 4, 5, 6, 7, and 8 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 -11 - /=2- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 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. C. Animal Husbandry 1. Animal Housing Upon arrival at Haskell Laboratory, rats were removed from shipping cartons and housed in appropriate cages according to Standard Operating Procedures. Animals were maintained under quarantine for at least three days, had at least one recorded weight gain, and no abnormalities detected. After the quarantine period, rats were selected for study. Throughout the dosing period with test substance, the rats were housed individually in appropriate cages according to the SOP. 2. Environmental Conditions Animal room(s) 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 of the study, the relative humidity and temperature ranges in the housing rooms was recorded but not included in the final report. 3. Feed and Water All animals were provided tap water ad libitum and fed PMI Nutrition International, LLC Certified Rodent LabDiet 5002 ad libitum. Animals were fasted overnight for approximately 12 hours before dosing with test substance. Food was returned approximately two hours post dose. 4. Animal Health and Environmental Monitoring Program 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 - 12 - Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 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. D. In-Life Methods 1. Animals The experimental groups were as shown below: Group Age* Weeks Days Number of Rats Male Female I 4 28 II 5 35 III 6 42 IV 7 49 V 8 56 10 10 10 10 10 10 10 10 10 10 * Animals were 1 day from the target age at the time o f dosing. The actual age at dosing is documented in the study records. 2. Dose Preparation, Analysis, and Rates The test substance was administered to each rat as a single oral gavage. This route was chosen because it is the route most commonly used for toxicity studies of PFOA. PFOA was mixed with HPLC grade water to achieve the target dose concentrations. For all experiments, a single dose level was used equivalent to 10 mg PFOAfkg body weight, and the dose volume was approximately 4 mL/kg body weight. Dose solutions were prepared one day before the day of use and stored in a 4C refrigerator prior to use. LC/MS methods that were used for dose verification are described in Section E, Analytical Methods. 3. Sacrifice and Blood Sampling Rats were sacrificed by CO2 asphyxiation and exsanguinated by cardiac puncture. 4. Experimental Procedure Rats were administered PFOA as a single oral dose of 10 mg/kg. Twenty-four hours after PFOA administration, rats were sacrificed and whole blood samples were collected by cardiac puncture. Blood samples were all collected within 20 minutes of the desired sample time. Whole blood samples were placed into EDTA tubes and maintained on wet ice. Plasma was separated by centrifugation and frozen at <-10C until analysis. - 13- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 Kidneys and livers were collected at sacrifice and were flash frozen in liquid nitrogen and stored at <-60C for possible future biochemical analysis. E. Analytical Methods 1. Verification of Dose Solution PFOA standards (1.0, 0.5, 0.2, 0.1, and 0.05 pg/mL) were prepared in HPLC-grade water. The dose solution was diluted by 10,000-fold prior to the analysis by LC/MS method. 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 of 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 1:4 (60 pL of internal standard solution) to 1:50 (980 pL of 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. 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 (Y): Source temperature: Desolvation temperature: MS Method: Mode: Time: Chi: Waters 2790 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 0 0 130C 350C MRM, 2 transitions 0-10.0 min 4 1 3 .0 0 -->369.00 Dwell: 0.25 sec - 14 - Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Ch2: Collision energy: 463.00 -->419.00 Dwell: Collision energy: 15 eV 0.25 sec 15 eV 4. Chromatographic Methods Method: Column: Column temperature: Mobile phases: Plasma concentration analysis Waters Xterra MS C18, 2.1x30mm, 2.5 pm Ambient A: 50 mM Ammonium Acetate B: Acetonitrile Gradient: Time (min) 0.0 0.5 10.0 10.9 11.0 %B 10 10 100 100 10 Flow rate: Stop time: Injection volume: 0.25 mL/min 11.0 min 5 pL DuPont-13267 F. Statistical Analysis Group data is represented by mean, standard deviation, standard error, and percent coefficient of variation. Statistical significance was assessed by the Student's t-test. Rejection of data is based on Chauvenet's criterion.^ RESULTS AND DISCUSSION A. Dose Verification (Appendix A) The concentrations of the test substance in the dose solutions were verified by LC/MS method as described in Materials and Methods, Section E. 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 mg/mL, Appendix A). - 15- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage B. Plasma Concentration of PFOA (Table 1, Figures 1-3, Appendix B) DuPont-13267 1. Male Rats The plasma concentrations of PFOA in the male rats are shown in Table 1. Their relationship with the age of the male rats is plotted in Figure 1. The PFOA plasma concentration was 7.32 1.01 pg/mL (mean SD) in 4-weeks old male rats. In contrast, a higher PFOA concentration (39.24 3.89 pg/mL) was observed in the plasma of 5-week old male rats. The mean PFOA plasma concentrations in 6-,7-, and 8-week old male rats were in a range of 37.12 to 43.19 pg/mL, which, based on the results of unpaired t-test at 99% confidence interval, were not significantly different from the plasma concentration of PFOA in the 5-week old male rats. 2. Female Rats The plasma concentrations of PFOA in the female rats are shown in Table 1. Their relationship with the age of the female rats is plotted in Figure 2. The plasma concentration of PFOA in 4week old female rats was 2.68 0.64 pg/mL, which was significantly higher (p<0.01) than the PFOA concentrations (mean ranged from 0.57 to 1.18 pg/mL) in the elder females that were 5 to 8 weeks old. 3. Comparison between Male and Female Rats PFOA plasma concentrations in male and female rats are shown in Figure 3. PFOA plasma concentrations were higher in the males than in the females for all age groups. The most dramatic sex-dependent difference in PFOA concentration was observed for rats that were 5 weeks and older. The ratios of PFOA concentrations between male and female rats that were 5 to 8 weeks old were all greater than 34, whereas the ratio was only 2.7 for the 4-week old rats. CONCLUSIONS Age has a significant effect on the plasma concentrations of PFOA in both male and female rats following oral dosing with 10 mg/kg PFOA. The sex-related difference in PFOA plasma concentration is most pronounced for rats that are older than 5 weeks, with male rats having PFOA plasma levels more than 34-fold higher than female rats. 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. - 16- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage REFERENCES DuPont-13267 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, M., Ohmori, K., Noshiro, R., and Kawashima, Y. (2001) Comparison of 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 of perfluorooctanoic acid in the male rat. Pharmacol. Toxicol. 65, 274- 5. DuPont Haskell Laboratory (2003). Perfluorooctanoic Acid: Toxicokinetics in the Rat. Unpublished report, DuPont-7473. 6. 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. 7. Taylor, J.R. (1982). In An Introduction to Error Analysis. E.D. Commins, Editor, Oxford University Press. -17 - Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 TABLES - 18- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage TABLES EXPLANATORY NOTES ABBREVIATIONS: SD standard deviation DuPont-13267 - 19- o 2 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 Table 1: Plasma concentration in rats 24 hours post-dose following oral gavage with 10 mg/kg PFOA. Group Age (weeks) Males Mean SD Females Mean SD I 4 7.32 1.01 II 5 39.24 3.89 III 6 43.19 3.79 IV 7 37.12 4.07 V 8 38.55 5.44 2.68 1.13 1.18 0.57 0.81* 0.64 0.46 0.52 0.29 0.27* Data expressed as pg/mL. * According to Chauvenet's criterion,(7>the value o f PFOA plasma concentration obtained from rat 511 (2.66 pg/mL) was not included for the calculation o f the mean and the SD. -20- <3/ Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 FIGURES -21 - =2=2- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 Figure 1: Plasma concentration in male rats 24 hours post-dose following oral gavage with 10 mg/kg PFOA. - 22- <=23 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 Figure 2: Plasma concentration in female rats 24 hours post-dose following oral gavage with 10 mg/kg PFOA. Age (weeks) - 23- Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage D uP on t-13 267 Figure 3: Plasma concentration in male and female rats 24 hours post-dose following oral gavage with 10 mg/kg PFOA. Male --o -- Female Age (weeks) - 24- 25 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 APPENDICES -25 - -2 T 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-13267 - 26- o27 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 APPENDIX A Dose Verification Data -27- 8 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Number of Replicates Description Dose Solution I - Group I 1 250 ng/mL dilution - 3A 2 250 ng/mL dilution - 3A 3 250 ng/mL dilution - 3A 4 250 ng/mL dilution - 3B 5 250 ng/mL dilution - 3B 6 250 ng/mL dilution - 3B 7 250 ng/mL dilution - 3C 8 250 ng/mL dilution - 3C 9 250 ng/mL dilution - 3C Mean SD SE %CV Dose Solution II -- Group II 1 250 ng/mL dilution - 3A 2 250 ng/mL dilution - 3A 3 250 ng/mL dilution - 3A 4 250 ng/mL dilution - 3B 5 250 ng/mL dilution - 3B 6 250 ng/mL dilution - 3B 7 250 ng/mL dilution - 3C 8 250 ng/mL dilution - 3C 9 250 ng/mL dilution - 3C Mean SD SE % CV Dose Solution III - Group III 1 250 ng/mL dilution - 3A 2 250 ng/mL dilution - 3A 3 250 ng/mL dilution - 3A 4 250 ng/mL dilution - 3B 5 250 ng/mL dilution - 3B 6 250 ng/mL dilution - 3B 7 250 ng/mL dilution - 3C 8' 250 ng/mL dilution - 3C 9 250 ng/mL dilution - 3C Mean SD SE %CV mg/mL 2.64 3.01 2.70 2.83 2.60 2.84 2.80 2.75 2.78 2.77 0.12 0.04 4.45 2.29 2.06 1.93 2.20 2.22 1.87 1.77 1.86 1.85 2.01 0.19 0.06 9.47 2.69 2.65 2.62 2.59 2.82 2.55 2.60 2.73 2.76 2.67 0.09 0.03 3.32 - 28- *2? D uP ont-13 267 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Number of Replicates_________ Description____________ mg/mL Dose Solution IV -- Group IV 1 250 ng/mL dilution - 3A 2 250 ng/mL dilution - 3A 3 250 ng/mL dilution - 3A 4 250 ng/mL dilution - 3B 5 250 ng/mL dilution - 3B 6 250 ng/mL dilution - 3B 7 250 ng/mL dilution - 3C 8 250 ng/mL dilution - 3C 9 250 ng/mL dilution - 3C 2.49 2.47 2.32 2.51 2.51 2.43 2.63 2.62 2.56 Mean SD SE %CV 2.50 0.09 0.03 3.74 Dose Solution V - Group V 1 250 ng/mL dilution - 3A 2 250 ng/mL dilution - 3A 3 250 ng/mL dilution - 3A 4 250 ng/mL dilution - 3B 5 250 ng/mL dilution - 3B 6 250 ng/mL dilution - 3B 7 250 ng/mL dilution - 3C 8 250 ng/mL dilution - 3C 9 250 ng/mL dilution - 3C 2.28 2.19 2.11 2.18 1.78 2.07 1.85 2.20 2.21 Mean SD SE %CV 2.10 0.17 0.06 8.13 Dose Solutions I-V Mean SD SE %cv 2.41 0.34 0.11 14.19 DuPont-13267 - 29- 3 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 APPENDIX B Plasma Concentration Data - 30- 3/ Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Males Animal Number Group I 101 102 103 104 105 106 107 108 109 110 Mean SD SE %CV Group II 201 202 203 204 205 206 207 208 209 210 Mean SD SE %CV Group III 301 302 303 304 305 306 307 308 309 310 ' Mean SD SE %CV jig/mL 6.01 6.20 8.31 6.38 6.64 7.22 8.45 7.19 8.11 8.74 7.32 1.01 0.32 13.86 33.40 39.43 46.42 41.89 38.72 37.35 42.64 35.11 40.96 36.45 39.24 3.89 1.23 9.90 38.14 39.95 42.00 45.27 41.38 43.46 48.23 50.44 40.99 42.01 43.19 3.79 1.20 8.77 -31 - 3L2. DuPont-13267 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Males (continued) Animal Number Group IV 401 402 403 404 405 406 407 408 409 410 Mean SD SE %CV Group V 501 502 503 504 505 506 507 508 509 510 Mean SD SE %CV lig/mL 30.49 31.28 40.22 41.41 36.76 35.14 38.70 35.96 38.36 42.93 37.12 4.07 1.29 10.97 32.66 37.93 46.78 27.47 38.05 39.94 41.65 38.03 39.16 43.85 38.55 5.44 1.72 14.10 DuPont-13267 -32- 33 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Females Animal Number Group I 111 112 113 114 115 116 117 118 119 120 Mean SD SE %CV Group II 211 212 213 214 215 216 217 218 219 220 Mean SD SE % CV Group III 311 312 313 314 315 316 317 318 319 320 ' Mean SD SE %CV pg/mL 3.05 2.41 2.52 2.27 2.87 3.33 1.67 2.13 3.90 2.67 2.68 0.64 0.20 23.77 0.98 1.18 0.81 1.79 1.65 0.76 1.47 0.69 0.43 1.52 1.13 0.46 0.15 41.13 1.39 0.94 0.89 1.92 0.66 1.39 1.06 0.84 0.56 2.12 1.18 0.52 0.17 44.43 -33 - 31 DuPont-13267 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Females (continued) Animal Number Group IV 411 412 413 414 415 416 417 418 419 420 Mean SD SE % CV Group V 511* 512 513 514 515 516 517 518 519 520 Mean SD SE %CV pg/mL 0.66 0.70 0.21 0.51 0.75 0.43 1.09 LOQ LOQ 0.23 0.57 0.29 0.09 51.00 2.66 0.87 0.63 0.95 1.36 0.67 0.64 0.89 0.50 LOQ 0.81 0.27 0.09 33.17 DuPont-13267 * According to Chauvenet's criterion/71 the value o f PFOA plasma concentration obtained from rat 511 was not included for the calculation o f the mean and the SD. - 34- 3S