Document pmQ3VQrLByZKLQNoVByv24KLE

AR226-2813 Study Title Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Author: Xing Han, Ph.D. Study Completed on: December 15,2003 Performing Laboratory: 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 Laboratory Project ID: DuPont-13267 Work Request Number:! 1 Service Code Number: 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 34 Company Sanitized. Does not contain TSCA CBI 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 E.I. du Pont de Nemours and Company /<T-X W - 2.00^ Date Company Sanitized. Does not contain TSCA CBI 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)rr*id/i ) iS s J , Wonda IjJLKelly Quality Assurance Auditor Date -3 Company Sanitized. Does noi contain TSCA C B| 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 Approved by: /-r (Xtc.-ZOcQ' Date fZ .'-& c-z& d Date Issued by Study Director: Xing Han, PIlD. Research Toxicologist Date Company Sanitized. Does not contain TSCA CBS Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage TABLE OF CONTENTS G O O D LA BO R A TO R Y PR A C TIC E C O M PLIA NC E STA TEM EN T Q U A LITY A SSU R A N C E STA TEM EN T C E R T I F I C A T I O N ............................................... ........................................................ L I S T O F T A B L E S ........................... Z Z Z Z * ................................................................... L I S T O F F I G U R E S ........................ Z Z Z Z Z Z Z ................................................. L I S T O F A P P E N D I C E S ............. .............................................................................. S T U D Y I N F O R M A T I O N ................ Z Z Z Z Z .......................................................... S T U D Y P E R S O N N E L ....................... .................................................................... S U M M A R Y ...................................................Z Z Z Z Z ...................................................... A . A g e E f f e c t ..................................................... ........................................... B . S e x C o m p a r is o n ............... .......... I N T R O D U C T I O N .................. Z Z Z Z Z Z Z Z Z Z ........................................... M A T E R I A L S A N D M E T H O D S ........ Z Z Z Z Z Z Z Z Z Z '...................... A . T e st S u b sta n c e ........................................................... .................................. B . T e st S y s t e m ............................................................... ........................ C . A n im a l H u sb a n d r y ................................................ ................................... 1. Animal Housing.................................................... .................................. 2. Environmental Conditions.................................. ;....... 1 1 ...'................................... 3. Feed and Water..................................................... ............................................ 4. Animal Health and Environmental Monitoring Program....... ............. D . In -L ife M e th o d s ...................................................... ................................ 1. Animals............................................................. ..............: .................. 2. Dose Preparation, Analysis, and Rates..............................111.!...................... 3. Sacrifice and Blood Sampling............................... 4. Experimental Procedure........................................... .......................................... E . A n a ly tic a l M e t h o d s .............................................. 1 1 ................................................ 1. Verification ofD ose Solution..................................................................................... 2. Extraction o f PFOA from Plasma for LC/MS Analysis............................... 3. Instrumentation................................................... ......................... 4. Chromatographic Methods............................................. ........................................... F . S tatistical A n a l y s is ................................................ .................................. R E S U L T S A N D D I S C U S S I O N .......Z Z 'Z Z Z Z Z 'Z Z Z Z Z Z Z Z .......... A . D o s e V e r if ic a t io n .............................................. ................. B . P la sm a C on cen tration o f P F O A ....................................... . Z ................................ 1. Male Rats................................................ ............................. 2. Female R ats..................................................... .................................................. 3. Comparison between Male and Female Rats. ,, .................................... C O N C L U S I O N S ...................................................................;....................................................... R E C O R D S A N D S A M P L E S T O R A G E .......................................................................... R E F E R E N C E S ............................... ........................................................ T A B L E S ............................................ ................................................................................................ F I G U R E S .......................................Z Z ' Z Z ............................................................................... A P P E N D I C E S .......................... .................................................................................................... DuPont-13267 Page ..... 2 .... 3 .... 4 .... 6 .....6 .....6 ..... 9 .... 10 ....10 ...10 ...11 ... 11 ...11 ...11 ...12 .... 12 ........1122 ....12 ..13 ....13 ....13 ....13 ....13 ..14 ...14 ...14 ...14 ...15 .15 . 15 .15 .16 ...16 ..16 ..16 .16 16 17 18 21 25 Company Sanitized. Does not contain TSCA LIST OF TABLES 1. Plasma concentration in rats 24 hours 10 mg/kg PFOA................................. post-dose following oral gavage with Page ...20 LIST OF FIGURES ' fo m ^ P F O A 3!1TM in male raK 24 l,0"rS P0St' d0Se f0" Winoral gavage with Page 2' fema'e raB 24 h0UrS Pst-dose rd gavage with............ ^ ........................................................................ ................................ 23 3. Plasma concentration in male and female rats 24 hours post-dose following oral gavage with 10 mg/kg PFOA............................... S ^ LIST OF APPENDICES A. Dose Verification Data...... B. Plasma Concentration Data Page ....27 ...30 6- - --------------------------------------- - Company Sanitized. Does not contain TSCA CBf STUDY INFORMATION 9th Collective Nomenclature- Octanoic acid, pentadecafluoro-, ammonium salt Synonyms/Codes: Ammonium perfluoronntannatP Perfluorooctanoate, ammonium salt PFOA H-24921 t Haskell Number- 24921 CAS Registry Number-|^ J ^ | ^ Purity; Known Impuntici: Physical Characteristics: White solid Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. Company Sanitized. Does not contain TSCA CBI 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/Completinn- June 30, 2003 / August 7, 2003 Study Initiated/Completed June 27, 2003 / (see report cover page) Company Sanitized. Does not contain TSCA CBt 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. Company Sanitized. Does not contain TSCA CBf JumiviAK i ^ ie relatiQiiship between age and plasma concentration of ammonium perfluorooctanoate (P FA) m rate has been investigated in this study. Male and female rats between 4 and 8 weeks WOaT , i r stered mg/kg 0f PF0A by Si"8le 0ral H e plasma concen" ^ ^ Jpm t ^concentration of PFOA in rats wa"s dependenbtyoLnOthMe aSg-eTahnedrseesxuloKf the rats. plasma 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, toe PFOA rraattss. ^PPFFOAO lAev^ellst ImOpnlaSsm111a6 were no^longer signriaftiScaWntelrye a2f'f4e'cftoeldd bloywaegrethinanraitns tohfee4it-hweereskexoltdhat 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. Company Sanitized. Does not contain TSCA CB1 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 p p n a*1r S aCtant' Elimination f PFOA in rats is sex-dependent and hormone regulated. PFOA elimination is much faster in female rats than in male rats,(I) down-regulated by r (f r m botb femaleand castrated male rats,(2'3) and up-regulated by estradiol in the male rats. However, these studies were conducted on sexually mature rats. In the current study m e 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 S t y dlStlIlgulshed via tbe 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 han those observed in mature rats. This study was not designed to fully characterize the pharmacokinetics of PFOA m immature rats, and was not designed for use in determining definitive elimination rate constants. MATERIALS AND METHODS A, Test Substance PFOA (ammonium p e r f l u o r o o c t a n o a t M B B ^ i ^ v a s obtained from 3M (St. Paul, MN) and assigned Haskell Laboratory NumEr H-24921 up0Ifreceipt. Available information on the punty, 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: I | Molecular Weight: 414.1 Molecular Formula: B. Test System Male ^ d 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 th ' 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* eexlccecedt V2S0%o ^onf tthr ef 8m' e^an wWeeirgeAht b5y' dSo's7e' mgrdou8p Wan6deksSex.f aIgneformatthioenwoeni*thhet vcaagrieatliaobneldsid not -11 - - Company Sanitized. Does not contain TSCA CBt Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavaee DuPont-13267 mcluded the Haskell animal number and an individual identification number assigned to each rat. 1he 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 irector or the laboratory veterinarian to have significantly affected the results of the study the thermal report ^ 30(1 temperatUre ranges in the housing rooms was recorded but not included in j . Feed and Water AH animals were provided tap water ad libitum and fed PMI Nutrition International, LLC Cemfied Rodent LabO^t 5002 ad libitum. Animals were fasted overnight for approximately 12 hours before dosing with test substance. Food was returned approximately two hours post- 4. Animal Health and Environmental Monitoring Program As specified m 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- Company Sanitized. Does not contain TSCA CBf specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The wnnd these c n^niinants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. The animal health and environmental monitorial program is administered by the attendTM ^ r v S ; r Z m d y" " o f ^ d a u * * - oi i D. In-Life Methods 1. Animals The experimental groups were as shown below: GrouD Age* Weeks Days Number of Rats I 4 28 n 5 35 in 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 of 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 HPLC tra d e TM St coTMnonly 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 PFOA/kg body weight, and the dose volume was I " 6 a b dy weight. Dose solutions were prepared one day before the day of use and stored m a 4 C refrigerator prior to use. y LC/MS methods Methods. that were used for dose verification are described in Section E, Analytical 3. Sacrifice and Blood Sampling Rats were sacrificed by C 02asphyxiation 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 a d m M o n , rats were sacrificed and whole blood samples were collected by caxdiaf~ e Blood samples were all collected within 20 minutes of the desired sample time Whole blood " J " " ' pla" ? imo EDTA tubes and maintained on wet ice. Plasma was separated by centrifugation and frozen at <-10C until analysis. y Company Sanitized. Does not contain TSCA CBf Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage aat <-ol) C for possible fut"urUe"biKocdh1,aetmSiacaail faicnealay"sids.were flash frozen in H,3uid nitr<>en stored E. Analytical Methods 1. Verification of Dose Solution TM" S^ j'! ' 01, and 0-05 were prepared in HPLC-grade water. The e 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 Airay protein precipitation columns (Jones Chromatography, Lakewood, CO). A 0.5 /tg/mL solution of perfluorononanoic acid (Aldrich Chemicals, Milwaukee, WI) in acetonitrile (ACN) was used as S o u o t^ f l StT r qUantltf n f PF0A- Plasma samPles were thawed, and a 20 ^L aliquot of each sample was applied to the PPT array. The plasma samples were precipitated by addmg appropriate dilution rate volumes of ACN/intemal standard solution to the PPT array. , " ra es'.raiJging from 1:4 (60 of internal standard solution) to 1:50 (980 fiL of internal standard solution), were utilized in order to capture the sample concentrations withtii the ar CUI7 e 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 (V): Source temperature: Desolvation temperature: MS Method: Mode: Time: Chi: Waters 2790 Liquid Chromatograph, equipped with quaternary pump, column heater, and autosampler v Quattro Micro Mass Spectrometer MRM Negative Electrospray 10.0 10.0 1.0 5 2 5 14 14 2.0 26.500 15 0 0' 130C 350C MRM, 2 transitions 0-10.0 min 413.00 -> 369.00 Dwell: 0.25 sec Company Sanitized, Does not contain TSCA CB Ammonium Perfluorooctanoate: Age Effect on the lasma Concentration in Post-Weaning Rats Following Oral Gavage Ch2: Collision energy: 463.00 419.00 15 eV Dwell: 0.25 sec Collision energy: 15 eV Chromatographic Methods Method: Column: Column temperature: Mobile phases: Plasma concentration analysis Waters Xterra MS C18, 2.1x30mm, 2.5 /tm Ambient A: 50 mM Ammonium Acetate B: Acetonitrile Gradient: Flow rate: Stop time: Injection volume: Time (min) 0.0 0:5 10.0 10.9 11.0 0.25 mL/min 11.0 min 5 nL %B 10 10 100 100 10 Statistical Analysis Group data is represented by mean, standard deviation, standard ern variation. Statistical significance was assessed by the Student's t-tes on Chauvenet s criterion/7^ and percent coefficient of Rejection of data is based Dose Verification (Appendix A) RESULTS AND DISCUSSION Company Sanitized. Does not contain TS CA CBI Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Plasma Concentration of PFOA (Table 1, Figures 1-3, Appendix B) 1. Male Rats w i , h l r Z C f T n,ra!i0nS of PI;0A in the male are shown in Table 1. Their relationship with die age of the male rats ,s plotted in Figure 1. The PFOA plasma concentration was _ .01 Mg/mL (mean SD) in 4-weeks old male rats. In contrast, a higher PFOA mmeTaniepPFFOOAA pnlal sfm' 2a4c1o3nc8e9ntMratgi/omnLs )mWf6-,7-b,searnVde8d-wine^ek oPlldasmmaaleorfa5ts-wweeerke oinldamraanlegeraotsf.37Th1e2 to 43.19 ig/mL>which, based on the results of unpaired t-test at 99% confidence inteiwal 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 W v f / f C , e femaIe rats is plotted in Figure 2. The plasma concentration of PFOA in 4- PFOA^nn f ^ T 2 6 8 1 M Mg/mL' Which Was ^ ^ a n t l y higher (p<0.01) than the 8 w^eks oM mSan r3ngedfr m `57 t0 118 Mg/mL) in theeIder females *atw ere 5 to 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 amatic sex-dependent difference in PFOA concentration was observed for rats that were 5 ,o 8 w c S ,,f ' n " ,,n*t a 0f f 0A between male and female rats f t a 'e r e 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 concentranon 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 fats. RECORDS AND SAMPLE STORAGE NNeewwaarkri,TDneillaiw3aPrPeh, Co3rbalteIT'roranWMA?oautan'taainndRtheecoflrndaslMreaPnargt ewmile1nbt,eWstialimniendgtaotnH, Daseklealwl aLraeb. oratory, Company Sanitized. Doss not contain TSCA CBf 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' HeUVe1, J R ' Davis' J W ' Sommers, R,, and Peterson, R.E. (1992) Renal excretion o r r00Ctan01C 3Cld m male rats: inhibitory effect of testosterone. J. Biochem. Toxicol. 3. Kudo, N., Suzuki, E., Katakura, M., Ohmori, K., Noshiro, R,, and Kawashima, Y. (2001) Comparison ofthe elimination between perfluorinated fatty acids with different carbon chain length in rats. Chem. Biol. Interact. 134, 203-216. 4. Ylmen, M., Hanhijarvi, H., Jaakonaho, J,, and Peura, P. (1989) Stimulation by oestradiol of toe unnary excretion of perfluorooctanoic acid in the male rat. Pharmacol. Toxicol. 65, 274- 5' DuPont Haskell Laboratory (2003). Perfluorooctanni. A.iH- Toxicokinetics in the Rat Unpublished report, 1 a u 6. Vanden Heuvel, J.P., Kuslikis, B.I., Van Rafelghem, M.J., and Peterson, R.E. (1991). Tissue distnbuhon, metabolism, and elimination of perfluorooctanoic acid in male and female rats J. Biochem. Toxicol. 6, 83-92. ' 7. Taylor, J.R. (1982). University Press. In An Introduction to Error Analysis. E.D. Commins, Editor ' Oxford -17- : Company Sanitized. Does not contain TSCA CBt Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 TABLES -18- Company Sanitised. Does not contain TSCA CBt Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage m TABLES EXPLANATORY NOTES ABBREVIATIONS: SD standard deviation DuPont-13267 -19Company Sanitized. Does not contain TS C A CBI 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 n 5 39.24 3.89 m 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 /xg/mL. * According to Chauvenet's criterion,(7) the value of PFOA plasma concentration obtained from rat 511 (2.66 /xg/mL) was not included for the calculation of the mean and the SD. 9 _______________________________________________________________________________ -20- Company Sanitized. Does not contain TSCA CB Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 FIGURES -21 - Company Sanitized. Does not contain T S C A CBI 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. Age (weeks) -22Company Sanitized. Does not contain TS C A C B f 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. 45678 Age (weeks) -23- Company Sanitized. Does not contain TSCA CB! Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 Figure 3: Plasma concentration in male and female rats 24 hours post-dose following oral gavage with 10 mg/kg PFOA. -Male - D - Female Age (weeks) -24- Company Sanitized. Does not contain T S C A CBt Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 A PPENDICES -25- Company Sanitized. Doss not contain TSCA CBt Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage m APPENDICES ABBREVIATIONS: EXPLANATORY NOTES LOQ SD SE % CV limit of quantitation standard deviation standard error percent coefficient of variation DuPont-13267 -26Company Sanitized. Does not contain TSCA CBI Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 APPENDIX A Dose Verification Data -27Company Sanitized. Does not contain T5CA C81 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Number of Replicates____________Description___________ mg/mL 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 2.64 3.01 2.70 2.83 2.60 2.84 2.80 2.75 2.78 Mean SD SE %CV 2.77 0.12 0.04 4.45 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 2.29 2.06 1.93 2.20 2.22 1.87 1.77 1.86 1.85 Mean SD SE %CV 2.01 0.19 0.06 9.47 Dose Solution HI - Group HI 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 2.69 2.65 2.62 2.59 2.82 2.55 2.60 2.73 2.76 Mean SD SE %CV 2.67 0.09 0.03 3.32 DuPont-13267 -28Company Sanitized. Does not contain TS CA CBI 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- Com pany Sanitized. D oss not contain TS C A CB1 Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage DuPont-13267 APPENDIX B Plasma Concentration Data -30Company Sanitized. Does not contain TSCA CBI Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage M a le s Animal Number_________ ftg/mL Group I 101 102 103 104 105 106 107 108 109 110 6.01 6.20 8.31 6.38 6.64 7.22 8.45 7.19 8.11 8.74 Mean SD SE %CV 7.32 1.01 0.32 13.86 Group II 201 202 203 204 205 # 206 207 208 209 210 33.40 39.43 46.42 41.89 38.72 37.35 42.64 35.11 40.96 36.45 Mean SD SE % cv 39.24 3.89 1.23 9.90 Group HI 301 302 303 304 305 306 307 308 309 310 38.14 39.95 42.00 45.27 41.38 43.46 48.23 50.44 40.99 42.01 Mean SD SE %CV 43.19 3.79 1.20 8.77 DuPont-13267 -31 - Company Sanitized. Doss not contain TSCACB! 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 fig/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 - 32Company Sanitized. Does not contain TSCA CBI 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 El 311 312 313 314 315 316 317 318 319 320 Mean SD SE % cv ug/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 DuPont-13267 - 33Company Sanitized. D oes not contain TSCA GSI Ammonium Perfluorooctanoate: Age Effect on the Plasma Concentration in Post-Weaning Rats Following Oral Gavage Females (continued) Animal Number neJmL 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 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,*71the value of PFOA plasma concentration obtained from rat 511 was not included for the calculation of the mean and the SD. * - 34Company Sanitized. Does not contain TS CA CB!