Document EmXxGkQ6LM2Xwg3XjgjdoJO14

- i:'_i STUDY TITLE ! i Analysis of Pooled Human Sera and Plasma and Monkey Sera for Fluorocarbons Using Exygen Method ExM-023-071 1 DATA REQUIREMENTS OECD Principles of Good Laboratory Practice, ENV/MC/CHEM(98)17, November 26, 1997 .... STUDY DIRECTOR i Emily R. Decker i STUDY COMPLETED ON i October 30, 2002 PERFORMING LABORATORY / TESTING FACILITY xy Rone earoh 3058 Research Drive State College, PA 16801 _i] Phone: 814-272-1039 STUDY SPONSOR J 3M Environmental Laboratory Building 2-3E-09 St. Paul, MN 55133-3331 Phone: 651-778-6565 .J PROJECT Study Plan Number: ExP-023-082 _t Exygen Study Number: 023-082 Sponsor Study Number: E02-1071 Total Pages: 111 ! ! -! i i .... 'I i: iJ :: i '_" i L Exygen Study No.: 023-082 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number 023-082, entitled "Analysis of Pooled Human Sera and Plasma and Monkey Sera for Fluorocarbons Using Exygen Method ExM-023-071," conducted for 3M Environmental Laboratory, was performed in compliance with OECD Good Laboratory Practice Standards (as revised in 1997), ENV/MC/CHEM(98)17 by Exygen Research, with the following exceptions: 1. _ 8.3 (5): The computerized system of data generation did not provide for the retention of a full audit trail to show all changes or to associate all changes to data to a timed and dated electronic signature. 2. _ 6.2 (4): The stability of the test items under storage or the study test conditions was not known. Also the purity of C6 acid and THPFOS was not known. 3. _ 5.2 (3): The date of receipt of for the calf serum sample ID 0204718 was not documented. 4. _ 1.2.2 (g): The instrument used for the analysis has not been qualified. !(i En_ly_R. ]_cker Date Study Director Exygen Research William K. Reagan, Ph.D. Date Sponsor Representative 3M Environmental q I Exygen Research. Page 2 of 111 :"i ,i.,! ..../_ ""i I ii:'i ,>j .... ;-i i .i,:i'iil LZ_ :.: ! i 1 Exygen Study No.: 023-082 QUALITY ASSURANCE STATEMENT The Quality Assurance Unit of Exygen Research reviewed Exygen Study Number 023082 entitled, "Analysis of Pooled Human Sera and Plasma and Monkey Sera for Fluorocarbons Using Exygen Method ExM-023-071." All phases were reviewed for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Study Director and to management. Phase 1. Protocol Review 2. Extraction, Fortification 3. Raw Data, Draft Report Review 4. Final Report Review Date .Inspected Date Reported to Date Reported to Exygen Study Director Management 10/10/02 10114102 10/30/02 Date Reported to Sponsor 10/30/02 10115102 10125-28/02 10125102 10129/02 10125102 10130/02 10/30/02 10130/02 10130102 10/30/02 10/30/02 10/30/02 Naomi Lovallo Technical Lead-QA Date . Exygen Research. Page 3 of 111 :_,,,i :i I ,:,.:i i..i ,! _i:_:_ _i_ i ..... Exygen Study No.: 023-082 CERTIFICATION OF AUTHENTICITY This report, for Exygen Study Number 023-082, is a true and complete representation of the raw data for the study. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814) 272-1039 Study Director, Exygen E_I} R. ecker Date Scientist Exygen Research Exygen Research Facility Management: fd"ohn M. Flaherty Date Vice President Exygen Research Sponsor Study Monitor, 3M: William K. Reagan, Ph.D. Date 3M Environmental Exygen Research. Page 4 of 111 ...!. _I -:,_ ! , ..[ ., i I _,_iJ r_ _.] (i_!_1 i .... J ! Exygen Study No.: 023-082 STUDY IDENTIFICATION Analysis of Pooled Human Sera and Plasma and Monkey Sera for Fluorocarbons Using Exygen Method ExM-023-071 STUDY PLAN NUMBER: ExP-023-082 EXYGEN STUDY NUMBER: 023-082 SPONSOR STUDY NUMBER: E02-1071 TYPE OF STUDY: Residue TEST SYSTEM: HumanSerum, Human Plasma, and Monkey Serum TEST ITEMS: perfluorooctane sulfonate (PFOS), perfluorohexanoic acid (C6), perfluoroheptanoic acid (C7), pentadecafluorooctanoic acid (C8), heptadecafluorononanoic acid (C9), nonadecafluorodecanoic acid (C10), perfluoroundecanoic acid (C 11), perfluorododecanoic acid (C12), tetrahydroperfluorooctane sulfonate (THPFOS), and tetrahydroperfluorodecane sulfonate (THPFDS) SPONSOR: STUDY DIRECTOR: William K. Reagan- Sponsor Study Monitor 3M Environmental Building 2-3E-09 St. Paul, MN 55133-3331 Emily R. Decker Exygen Research Phone: (814) 272-1039 TESTING FACILITY: Exygen Research 3058 Research Drive State College, PA 16801 ANALYTICAL PHASE TIMETABLE: Study Initiation Date: Experimental Start Date: Experimental Termination Study Completion Date: Date: 10/09/02 10/15/02 10/23/02 10/30/02 Exygen Research. Page 5 of 111 7! i i:! I / ._ :,J I ,i - Exygen Study No.: 023-082 PROJECT PERSONNEL The Study Director for this project at Exygen Research was Emily R. Decker. The following personnel from Exygen Research were associated with various phases of the study: Name Paul Connolly Emily Decker Xiaoming Zhu Rickey Keller Title Technical Leader-LC/MS Scientist Technician Sample Custodian !'? i' (._| ....b I Exygen Research. Page 6 of 111 i :i I:_)t ::_ :):'} _, t','44 !_:'! J .... _,_ I_ :=J ! _ _ .! J.21 q ->?i Exygen Study No.: 023-082 TABLE OF CONTENTS Page TITLE PAGE ....................................................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT ............................. 2 QUAL1TY ASSURANCE STATEMENT .......................................................................... 3 CERTIFICATION OF AUTHENTICITY ........................................................................... 4 STUDY IDENTWICATION ............................................................................................... 5 PROJECT PERSONNEI ..................................................................................................... 6 TABLE OF CONTENTS .................................................................................................... 7 LIST OF TABLES ............................................................................................................... 8 LIST OF FIGURES ............................................................................................................. 9 LIST OF APPENDICES ................................................................................................... 10 1.0 SUMMARY ..........................................................................................I1......... 2.0 OBJECTIVE ............................................................................................................. 11 3.0 INTRODUCTION ............................................................................... ..................... 11 4.0 TEST SYSTEM ........................................................................................................ 11 5.0 TEST ITEMS ........................................................................................................... 12 6.0 DESCRIPTION OF ANALYTICAL METHOD ..................................................... 15 6.1 Extraction Procedure ....................................................................................... 15 6.2 Preparation of Standards and Fortification Solutions ...................................... 15 6.3 Chromatography .............................................................................................. 16 6.4 Instrument Sensitivity ...................................................................................... 16 6.5 Description of Instrument and Operating Conditions ..................................... 17 6.6 Quantitation and Example Calculation ........................................................... 17 7.0 EXPERIMENTAL DESIGN ................................................................................... 20 8.0 RESULTS .................................................................................................2.0......... 9.0 CONCLUSIONS ..................................................................................................... 21 10.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA .................... 21 1 1 0 RETENTION OF DATA AND SAM1 _l .F.S ............................................................ 21 Exygen Research. Page 7 of 111 i__ J ,_\_'_ iL _ !_Ii; i i i:_:] i:i Exygen Study No.: 023-082 Table I LIST OF TABLES Page Summary of Recoveries for Calibration Curve in Calf Serum Compared to Standards in Methanol .................................................................................... 23 Table II Table Ill Table IV Table V Table VI Summary of Recoveries for Calibration Curve in Human Plasma Compared to Standards in Methanol ............................................................................... 23 Summary of Recoveries for Laboratory Fortified Matrix Spikes ................... 24 Summary of Residues for Human Serum Samples ........................................ 26 Summary of Residues for Human Plasma Samples ....................................... 26 Summary of Residues for Monkey Serum Samples ....................................... 26 2J '! Exygen Research. Page 8 of 111 i"!: I _i , -- 'i Exygen Study No.: 023-082 Figure 1 Figure 2 Figure 3 Figure 4 Chromatogram LIST OF FIGURES Representing 0.1 ng/mL Calibration Page Standard ...................... 28 Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ID: 0204490 Spk J, Sponsor ]:13: TCR-674) .................................. 32 Chromatogram Representing a Human Plasma Sample (Exygen ID: 0204490, Sponsor 113:TCR-674) ................................................................................... 36 Chromatogram Representing a Sample Analyzed for Three Daughters for THPFDS 0Exygen ID: 0204292 Dup, Sponsor ID: X328-A) ......................... 40 i:::iJ i_,.=j Exygen Research. Page 9 of 111 Exygen Study No.: 023-082 LIST OF APPENDICES :_ Page Appendix A Study Plan ExP-023-082 (Exygen Study No. 023-082) and :.... Deviations .................................................................................................. 41 _,!_' .i Exygen Research. Page 10 of 111 i:i!I 1 ::i :_I -_ ._ ,:j i:.i !_-]: _ _kS! _, i Exygen Study No.: 023-082 1.0 SUMMARY Exygen Research conducted a quantitative screening on various human serum, human plasma, and monkey serum samples for the determination of perfluorooctane sulfonate (PFOS), perfluorohexanoate (C6), perfluoroheptanoate (C7), pentadecafluorooctanoate (C8), heptadecafluorononanoate acid (C9), nonadecafluorodecanoate (C10), perfluoroundecanoate (Cll), perfluorododecanoate (C12), tetrahydroperfluorooctane sulfonate (THPFOS), and tetrahydroperfluorodecane sulfonate (THPFDS) according to protocol ExP-023-082 (Appendix A). This screening was performed on an instrument that had not been used for routine fluorochemical analysis prior to this study. The method used for this study has not been validated at the levels reported for C8 and PFOS and not validated at any level for the other anions. These levels were completely dependent on instrument sensitivity. Recoveries for fortified samples are given in Tables I-III. Residues of each anion in human serum are summarized in Table IV. Residues of each anion in human plasma are summarized in Table V. Residues of each anion in monkey serum are summarized in Table VI. 2.0 OBJECTIVE The objective of this study was to screen human serum, human plasma, and monkey serum samples and quantitate to the lowest possible level according to instrument sensitivity. 3.0 INTRODUCTION This report details the results of the analysis for perfluorooctane sulfonate (PFOS), perfluorohexanoate (C6), perfluoroheptanoate (C7), pentadecafluorooctanoate (C8), heptadecafluorononanoate (C9), nonadecafluorodecanoate (C10), perfluoroundecanoate (Cll), perfluorododecanoate (C12), tetrahydroperfluorooctane sulfonate (THPFOS), and tetrahydroperfluorodecane sulfonate (TI-IPFDS) in human serum, human plasma, and monkey serum samples. The study was initiated on October 09, 2002, when the study director signed study plan number ExP-023-082. The experimental start date was October 15, 2002, and the experimental termination date was October 23, 2002. 4.0 TEST SYSTEM Pooled human serum samples were purchased by the sponsor from Sigma-Aldrich, Milwaukee, WI, Lampire Biological Laboratories, Pipersville, PA, Bioresource Exygen Research. Page 11 of 111 i !.!_,! ::, :_ :-ii I iili ._ .i,i:_, :::i!t _ =.._ 'i _i :_._ i_i i_I : _i i!i!t - Exygen Study No.: 023-082 Technology, Inc., Fort Lauderdale, FL, and Golden West Biologicals, Temecula, CA. Pooled monkey serum samples were purchased by the sponsor from Lampire Biological Laboratories, Pipersville, PA. Pooled human plasma samples were purchased by the sponsor from Lampire Biological Laboratories, Pipersville, PA, Bioresource Technology, Inc., Fort Lauderdale, FL, Golden West Biologicals, Temecula, CA, and Innovative Research, Inc. Southield, MI. In addition, blank matrix consisting of pooled human plasma collected in rural China was provided by the sponsor. Also, calf serum was purchased from Sigma-Aldrich by Exygen. Ex_,en ID 0203963 0203964 0203965 0204292 0204334 0204335 0204490 0204991 0204492 0204493 0204718 0204747 Sponsor ID Lot 020821 Lot 22K0965 Lot G0140604 X328-A TCR-684 TN-A-06332 TCR-674 TN-A-6337 TN-A-06333 TN-A-06336 NA TCR-683 Matrix Human Serum Human Serum . Human Serum Human Serum Human Plasma Monkey Serum Human Plasma Human Plasma Monkey Serum Monkey Serum Bovine (Calf Serum) Human Plasma Source BioResource Sigma-Aldrich Golden West Biologicals Lampire Golden West Biologicals Lampire 3M (plasma from rural China) Lampire Lampire Lampire Sigma-Aldrich Innovative Research Samples were received frozen on dry ice and then placed in frozen storage (<-10C) until samples were logged in by Exygen personnel. All records concerning sample receipt, processing and storage can be found in the raw data package associated with this study. 5.0 TEST ITEMS The analytical standards PFOS, C6, C7, C8, C9, C10, Cll, C12, THPFOS, and THPFDS were received at Exygen on September 30, 2002 from 3M Environmental Technology and Services. The available information for the reference material is listed below. The reference material was stored frozen. Compound PHAA (C6) TDHA (C7) PFOA (C8) PFNA (C9) C 10 C 11 C12 PFOS THPFOS THPFDS Exygen Research. Exygen Inventory No. SP0002086 SP0002091 SP0002087 SP0002085 SP0002090 SP0002093 SP0002089 SP0002084 SP0002088 SP0002092 Lot No. NB 117735-32 PU/07219EU 210002 H7568 R11K U 11N R24K 217 Q75-91 PMR-269-83 Purity (%) TBD 99.5 >97 >99 98 >99 96 86.9 Unknown 94.7 Expiration Date 01/01/10 01/01/05 07/19/07 07/19/07 12/01/10 07/19/07 12/01/10 08/31/06 06/07/05 08/22/12 Page 12 of 111 Exygen Study No.: 023-082 The molecular structures of the anions are given below. Name: PFOS Chemical Name: Perfluorooctanesulfonate Molecular Weight: 499, as shown F F F F /F/F/F/F . i F F_S03 F F F F ii:i,ii 'LI Name: C6 Chemical Name: Perfluorohexanoate ! Molecular Weight: 313, as shown !_-; F F 0 F F I F I FII 0. IF IF I F F F :__, i Name: CC7hemical Name: Perfluoroheptanoate J Molecular Weight: 363, as shown i_ F F F 0 .F F_O. __ F F F Name: C8 i Chemical Name: Pentadecafluorooctanoate ,, Molecular Weight: 413, as shown _ F F F 0 F O" F F F F Exygen Research. Page 13 of 111 "_'i ,_ "i'I ' E ,{ '[ :I -'] .._..:_ "] i li:,!,:_ "J _ii ' ._;) , Name: C9 Chemical Name: Heptadecafluorononanoate Molecular Weight: 463, as shown F F F F 0 Exygen Study No.: 023-082 F F_ 0 F F F F Name: C10 Chemical Name: Nonadecafluorodecanoate Molecular Weight: 513, as shown F F F F 0 F F_OJ' F J F J 'F l 'F [J " Ir IF Ir I I- I F F F F F Name: Cl1 Chemical Name: Perfluoroundecanoate Molecular Weight: 563, as shown F F F F F 0 F 0" F F F F F Name: C12 Chemical Name: Peffluorododecanoate Molecular Weight: 613, as shown F F F F F 0 F O" F_@_@_ F F F F F F Exygen Research. Page 14 of 111 ,.._:_ I '_ (:! !'_] L_iil t" t L.I i .l _'_t _:_ _1 __ . ,i _'_:l . Exygen Study No.: 023-082 Name: THPFOS Chemical Name: Tetrahydroperfluorooctane Molecular Weight: 427, as shown F F F H /F/F/F/H sulfonate F F_S 03" F F F H Name: Tt-IPFDS Chemical Name: Tetrahydroperfluorodecane Molecular Weight: 527, as shown sulfonate F _r F F F F F F H H F F__@_@_S03" F F F F H 6.0 DESCRIPTION OF ANALYTICAL METHOD Analytical method entitled "Method of Analysis for the Determination of Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFOS) and Pentadecafluorooctanoic Acid (PFOA) in Rat Liver, Serum and Urine" was used for this study. For this study, several modifications were made and have been documented in the protocol/protocol deviations. 6.1 Extraction Procedure a. Measure 2 mL of serum sample into a 15 mL disposable centrifuge tube and fortify, if appropriate. b, Add 5 mL of ACN and shake for -20 minutes on a wrist action shaker. c. Centrifuge tubes at -3000 rpm for ~ 5 minutes. Carefully decant supernatant into a 50 mL disposable centrifuge tube and add 35 mL of water. d. Load the sample onto a conditioned SPE column. Discard the eluate. Any analyte residues will be trapped on the SPE column at this point. e. Elute with 5 mL of methanol and then evaporate to less than 1 mL using a nitrogen evaporator. Bring final volume up to 1 mL with methanol. f. Analyze samples using electrospray LC/MS/MS. The volume of sample used and the volume of methanol used for elution were different than those cited in the method. This was done to allow for lower quantification limits for the anions in this study. Exygen Research. Page 15 of 111  i] i! i21 i .... i::_i:___;t _:_ _:i:J !!_:,!i_il .... _ i .... ! _'i!i Exygen Study No.: 023-082 6.2 Preparation of Standards and Fortification Solutions Individual stock solutions of all of the anions were prepared on October 02, 2002, as specified in method ExM-023-071. The stock standard solutions were prepared at a concentration of - 100 _tg/mL by dissolving ~10 mg of the standard (corrected for purity and salt content when appropriate) in methanol. From these solutions, a 1.0 _tg/mL mixed fortification standard solution was prepared by transferring the appropriate volume (~0.4 - 1 mL) of each of the stock solutions into a 100-mL volumetric flask and bringing the volume up to the mark with methanol. The 0.1 i.tg/mL mixed fortification standard was prepared by transferring 10 mL of the 1.0 pg/mL mixed fortification standard into a volumetric flask and bringing the volume up to 100 mL with methanol. A set of calibration standards were prepared by dilution in the following manner: Initial Conc. (ng/mL) 100 100 100 100 5.0 Volume (mL) 1 0.5 0.2 0.1 1 Diluted to (mL) 10 10 10 10 10 Final Conc. (ng/mL) 10.0 5.0 2.0 1.0 0.5 21.0 1 10 0.21 The stock standard solutions and all fortification and calibration standard solutions were stored in a refrigerator (6 + 2C) when not in use. 6.3 Chromatography Quantification was accomplished by electrospray LC/MS/MS analysis. An API 4000 Sciex system was used in this study because of its greater sensitivity and also because it had not been used for fluorochemical analysis prior to this study. Peaks were detected in the control matrices corresponding to some of the target anions, especially for C8. 6.4 Instrument Sensitivity The smallest standard amount injected during the chromatographic run had a concentration of 0.1 ng/mL, which corresponds to a concentration of 0.05 ng/mL (ppb) in the extracted samples. Residues were calculated below this level where the response of the anion was approximately three times the signal to noise ratio. The results were Exygen Research. Page 16 of 111 t _' _:! i .... i,' _-_, '[ ...._. '_ ii:t _--_ "l_!:] .._ i ,i ', !_i Exygen Study No.: 023-082 reported as Not Detected CN-D) if the response the signal to noise ratio and Not Quantifiable other-responses were reported. was approximately (NQ) was used for less than three times negative results. All 6.5 Description of Instrument and Operating Conditions Instrument: PE SCIEX AP14000 Biomolecular Mass Analyzer, (LC/MS/MS #8) SCW_,XTurbo Ion Spray Liquid Introduction Interface Turbo Ion spray temperature = 350 C Auxiliary gas flow = - 7.0 L/min Harvard Infusion Pump Computer: Dell OptiPlex GX 110 Software: PE Sciex Analyst 1.2 HPLC Equipment: HPLC Column: Column Temperature: Mobile Phase (A) : Mobile Phase (B) : Hewlett Packard fliP) Series 1100 HP Quat Pump HP Vacuum Degasser HP Autosampler I-IP Column Oven Genesis C-8, 5 cmx 2.1 mm i.d. x 4 g (Exygen ID: 71A) (JONESCHROMATOGRAPHY: Part No. FK5962E) 35C 2 mM Ammonium Acetate in Type I Water Methanol Time0.(0rnin) 2.0 5.0 9.0 9.5 14.0 14.5 20.0 %90A.0 90.0 I0.0 10.0 0.0 0.0 90.0 90.0 %I0.B0 10.0 90.0 90.0 100.0 100.0 10.0 10.0 Flow Rat0e.3(mL/min) 0.3 0.3 0.3 0.3 0.3 0.3 0.3 Injected Volume: Ions monitored : 15 gL Anion Parent ion Daughter ion CC67 331633 236199 C8 413 369 C9 463 419 C10 513 469 Cll 563 519 C12 613 569 PFOS 499 80 THPFOS 427 81 THPFDS 527 81 Dwell (secs) 00..11 0.1 0.1 0.1 0.1 0.i 0.1 0.1 0.1 Declustering Potential --2200 -20 -20 -20 -20 -20 -85 -65 -75 Collision Energ2L --1100 -10 -10 -10 -10 -10 -80 -60 -66 Exygen Research. Page 17 of 111 .... i F i :, :! _iI ':_'=_ _L,_u._""_ -__ i i ii_iit Exygen Study No.: 023-082 6.6 Quantitation and Example Calculation Fifteen microliters of sample or calibration standard were injected into the LC/MS/MS. The peak area was measured and the standard curve was generated (using 1/x weighted linear regression) by Analyst software using seven concentrations of standards prepared in methanol. The residue concentration for the samples was determined from the following equations: Use Equation 1 to calculate the amount of anion found (in ng/mL, based on peak area) using the standard curve (1/x weighted linear regression parameters) generated by the Analyst software program. Equation 1: Analyte found (ng/mL) = (peak area - intercept) slope Use Equation 2 to calculate the amount of analyte found (in ppb) Equation 2: Analyte found (ppb = ng/mL) = (analyte sfaomupnlde (vnogl/ummLe) (xmFLV) (mL) x DF FV = final volume DF = dilution factor For samples fortified with known amounts of analyte prior to extraction, use Equation 3 to calculate the percent recovery (ppb = ng/mL) Equation 3: Recovery (%) = [ total analyte found (ppb) - analyte found in control or sample (ppb)] x100 analyte added (ppb) Note: Any analyte found in the control was subtracted from analyte found. However, the response for the sample duplicate was not used. Exygen Research. Page 18 of 111 i! i_i! ' : .. _'_, _2i ?ii _-] 'Lt 1 _._ l,i_!_ _ I i ! _:' Exygen Study No.: 023-082 An example of a calculation using an actual sample follows: Human Serum Sample, Exygen ID 0204491 Spk J"(Data Set: 101702A), fortified with 0.5 ng/mL (calculation is using values for C6): Where: peak area = intercept = slope = dilution factor = ng/mL added (fort level) = avg. amt in controls = final volume = sample volume = 54636 632.277 59435.9 1 0.5 ng/mL 0 (Not detected) 1 mL 2 rnL From equation 1: Analyte found (ng/mL) = [54636- 632.2771 59435.9 = 0.9 ng/mL From equation 2: Analyte found (ppb) 0.9 ng/mL x 1 mL = 2 mL = 0.45 ppb (ng/mL) From equation 3: % Recovery = (0.45 ng/mL - 0 ng/mL) x 100 0.5ng/mL = 90% Note: This example calculation was done using rounded numbers, and therefore may be slightly different from the values shown in the raw data. Exygen Research. Page 19 of 111 t _:::i zLL_,I,i:_i :-q v- I ,_ d ii!]i ',:_ :] !_ _;s_l_i_ _i i ,_, i,;ii '::,_,_ Exygen Study No.: 023-082 7.0 EXPERIMENTAL DESIGN For the screening of each sample, duplicate extractions were performed. Also, each sample was fortified at 0.5 ng/mL and 5.0 ng/mL and then taken through the extraction procedure. Two calibration curves were also taken through the extraction procedure, one using calf serum and one using human plasma. These were treated as quality control fortifications in the data set and were not used for the calibration curve. Since there was residue detected in the samples for TI-IPFOS and THPFDS, an additional analysis in which a three-daughter ion confirmation was performed. 8.0 RESULTS There was no significant residue detected in the reagent blank analyzed with these samples. Also, there was no carry-over present for any of the anions in the instrument blanks (methanol washes) analyzed in the analytical sets, except for C8 and C9, and this is most likely contributed to those analytes being present in the instrumental system, particularly in the mobile phase. This is especially evident with the absence of the anions (except for C8 and C9) in the methanol wash analyzed after the injection of the 10 ng/mL calibration standard. All fortifications were at a level equal to or less than the 10 ng/mL standard. Since there was no carry-over observed after the injection of this standard, the carry-over present after proceeding injections would be minimal. A representative chromatogram of a standard prepared in methanol can be found in Figure 1. Rsuegcgoevsetreides rafnogre foorftif7ie0d% staomp1l3e0s%arwe ergeivernepoirntedT,abhloeswevI-eIrII.thisRemceotvheordies haosutnsoidte betehne validated at these low levels and some of the recoveries were outside of this range because the level of residue in the sample was significantly greater than the amount fortified, especially for C8 and PFOS. Example chromatograms of fortified samples are shown in Figure 2. Residues of each anion in human serum are summarized in Table IV. Residues of each anion in human plasma are summarized in Table V. Residues of each anion in monkey serum are summarized in Table VI. Example chromatograms of a human plasma sample are given in Figure 3. The detection of THPFDS in some of the samples warranted further investigation. The presence of THPFOS and THPFDS was confirmed with a reanalysis with additional daughter ion confirmation. A chromatogram detailing the three daughter ion confirmation of THPFDS is given in Figure 4. Exygen Research. Page 20 of 111 ii _ .... i ,I !! ?i !j _?_I ...i. Exygen Study No.: 023-082 9.0 CONCLUSIONS The quantitative screening of these serum and plasma samples produced levels of certain analytes at extremely low levels (< 100 ppt). These levels are based solely on the instrument sensitivity and not the method recovery. The results contained in this report should be evaluated as a quantitative screening. Contamination of these samples due to instrument conditions is very limited because the instrument used for the analyses had never been used for routine fluorochemical analysis prior to the initiation of this study. No carry-over was observed throughout the injections of the analytical sets, which was demonstrated with the absence of the target analytes in the methanol washes analyzed after the injection of the highest level of calibration standard (10 ng/mL). Two people took a set of 64 samples through the sample preparation procedure in approximately 10 hours and the analysis by LC/MS/MS took approximately 48 hours. 10.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE DATA The method used in this study has not been validated for C8 and PFOS at the levels given in this report and at any level for the rest of the anions. Residues were reported lower than the lowest calibration standard. 11.0 RETENTION OF DATA AND SAMPLES When the final report is complete, all original paper data generated by Exygen Research will be shipped to the sponsor. This does not include facility-specific raw data such as instrument logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the archives of Exygen Research for the lifetime of the product. Sponsor permission will be obtained before discarding. ,L:)! Exygen Research. Page 21 of 111 :I ,,i__i T r 4- d Exygen Study No." 023-082 TABLES Exygen Research. Page 22 of 111 _i _ i*'_',':t i ; ..... i,iI:/_ !:i1 _t L:J Exygen Study No.: 023-082 Table I Summary of Recoveries for Calibration Curve in Calf Serum Compared to Standards in Methanol %Recover), SamplelD XC101702-0 XCI01702-I XC101702-2 XC101702-3 XC101702-4 SponsorID NA NA NA NA NA Fort Level (ng/mL) C6 C7 C8 C9 C10 Cll C12 PFOS THPFOS THPFDS 0.0 ....... ** ii _ 0.2 i 85 120 163J 151 155 96 87 ** 148 140 I 0.5 198 111 127:132 135 110 111 ** 159 144 1.0 90 113 1091 124 112 111 116 ** 141 120 1.5 102 113 1211127 121 120 121 ** 157 138 XC101702-5 NA XC101702-6 NA 2.0 102 108 117] 122 115 109 110 ** 152 129 2.5 97 99 1111 107 103 98 ! 98 ** 141 122 XC101702-7 NA 5.0 95 ! 97 109 [ 108 104 1061 105 i ** 141 124 AVG: 96 109 122 124 121 107 107 ** 148 131 STANDARD DEVIATION: 6.2 8.2 19.1 15.0 18.6 8.2 11.5 ** 7.8 9.6 RELATIVE STANDARDDEVIATION: 6.5 7.5 15.6 12.1 15.4 7.6 10.7 ** 5.2 7.3 _:_ _,! i ., i _..i.:.i.'ii Table II Summary of Recoveries for Calibration Curve in Human Plasma Compared to Standards in Methanol % Recovery Sample ID XC101502-8 XC101502-9 XC101502-10 XC101502-11 XC101502-13 XC101502-15 Sponsor ID TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 TCR-674 Fort Level (ng/mL) 0.0 0.2 0.5 1.0 2.0 C6 C7 C8 _ _I 90 120 126 87 121! 146 96 1131 121 107 1141 129 C9 C10 C11 C12 PFOS i. 86 33 115 109 I ** 115 84 132 114 ** 104 90 110 108 ** 107 112 114 120 ** 5.0 95 97 I 106 97 98 101 104 ** AVG: 93 113 126 102 81 114 111 ** RELATIVE SSTTAANNDDAARRDD DDEEVVIIAATTIIOONN:: 1100..29 98..65 1114..54 1100..97 2395..28 191.9.3 56..62 **** THPFOS 142 160 151 150 127 146 12.4 8.5 THPFDS 133 142 123 137 113 130 11.6 9.0 ** Recovery not applicable because the residues detected in sample were significantly greater than the amount fortified. Exygen Research. Page 23 of 111 ::I ._ ....i :i_ ' r:i_"i ii:i " _i! _,,_, i{4! :_ ,iN _t _!_ =_J Exygen Study No.: 023-082 Table III Calf Serum Summary of Recoveries for Laboratory Fortified Matrix Spikes Sampled % Recovery Fort Level SponsorID (ng/mL) C6 C7 C8 C9 C10 Cll C12 PFOS THPFOS Tt-IPFDS 0204718SpkB NA 5.0 84 85 92 97 95 I 96 [100 ** I 118 AVG: 91 98 96 112 105 96 103 ** 131 STANDARD DEVIATION: 9.9 17.7 4.9 20.5 14.1 0.7 3.5 ** 18.4 RELATIVE STANDARD DEVIATION: 10.9 18.1 5.2 18.4 13.5 0.7 3.4 ** 14.0 108 i 117 12.7 10.9 Human Serum % Recovery Fort Level SampleID S_ponsorID (n _) C6 _ 8 C9 el0 Cll (212PFOSTI_FOS 0203963 SpkC Lot020821 3112411511161 ** I 228 0203964SpkD Lot22K0965 0_ 91t 96 [110 [ 107[ ** [ 133 0203965SpkE LotG0140604 0_311181139[1321 ** ] 265 0204292 Spk F X328-A 0_8 [ 129 [ 128 [ 131 [ ** [ 182 0203963SpkM Lot020821 5_6[131[137[134[ ** [ 321 0203964SpkN Lot22K0965 5_8 I 97 110711131 103 I 118 0203965Spko LotG0140604 0204292 SpkP X328-A 5_91110111711151 5.0 ** I 198 116 AVG: 83 111 197 129 114 124 125 ** 195 STANDARD DEVIATION: 24.3 17.6 113.1 32.6 14.0 17.7 17.8 ** 73.7 RELATIVE STANDARDDEVIATION: 29.3 15.9 57.3 25.3 12.4 14.3 14.2 ** 37.8 TI-IPFDS 198 117 149 149 168 109 121 113 141 31.4 22.3 ** Recovery not applicable because the residues detected in sample were significantly greater than the amount fortified. Exygen Research. Page 24 of 111 '_:':I "] _ _::J i ._I _] ::" : Exygen Study No.: 023-082 Table III (cont') Summary of Recoveries for Laboratory Fortified Matrix Spikes Human Plasma % Recovery SamplelD Fort Level SponsorID (ng/mL) C6 C7 C8 C9 C10 Cll C12PFO,' 0204334SpkH 0204490SpkJ 0204491SpkJ 0204747SpkG TCR-684 TCR-674 TN-A-6337 TCR-683 0.5 69 94 211 152 107 93 1103 I ** 0.5 78 78 73 84 90 91 I 96 I! ** 0.5 91 97 168 140 110 91 I 93 ** 0.5 89 124 406 182 143 1321 120 ** 0204334SpkR 0204490SpkU TCR-684 TCR-674 5.0 96 ::100 131 110 110 98 I100 ** 5.0 86:85 92 90 92 92 I 96 65 0204491SpkT TN-A-6337 5.0 97 I 96 109 101 951 86 I 88 ** 0204747SpkQ TCR-683 5.0 82 I 85 106 92 901 83 I 83 ** AVG: 86 95 162 119 105 96 97 ** STANDARD DEVIATION: 9.4 13.9 108.0 35.3 17.8 15.3 11.1 ** RELATIVE STANDARD DEVIATION: 11.0 14.7 66.7 29.7 17.0 16.0 11.4 ** THPFOS 122 107 113 159 114 109 113 111 119 17.0 14.3 THPFDS 113 102 116 146 112 102 106 94 111 15.7 14.1 Monkey Serum % Recovery S.m_.p Spo_orm&__mI._C_6__CZ7_C8 C9 Cl0ClXC12VmS T_FOS r_ms 0204335 SpkI TN-A-06332 0.5 0204492 Spk K TN-A-06333 0.5 0204493SpkL TN-A-06336 0.5 0204335 SpkS TN-A-06332 5.0 0204492 Spk U TN-A-06333 5.0 _ TN-A-06336 5.0 AVG: 109 97 106 101 106 102 105 ** STANDARD DEVIATION: 13.7 9.3 20.6 27.4 11.1 15.5 11.5 ** RELATIVE STANDARD DEVIATION: 12.5 9.6 20.4 27.2 10.5 15.2 10.9 ** 114 162 132 107 124 141 130 19.8 15.3 129 155 120 104 116 120 124 17.2 13.9 ** Recovery not applicable because the residues detected in sample were significantly greater than the amount fortified. Exygen Research. Page 25 of 111 i! -_ )_,_ ,'o_:,.j i !.:i_!,I :! ?z?q !_'t ii! ' i i !_i Exygen Study No.: 023-082 Table IV Summary of Residues for Human Serum Samples Amount Found (ng/mL) Sample ID 0203963 Sponsor ID Lot020821 0203963 Dup Lot020821 0203964 Lot 22K0965 0203964Dup 0203965 Lot22K0965 LotG0140604 0203965 Dup Lot G0140604 0204292 X328-A 0204292Dup X328-A C6 0.0261 C7 C8 C9 C10 ND 2.85 0.683 0.228 ND ND ND i 0,0940 ND ;0.0976 0.0687i 0.145 2.93 0.684 0.22 1,45 0,307 0,107 1.450.2760.108 5.93 0.508 0.214 0.08841 0,170 6.00 0.557 0.204 ND 10.115 3.58 0.634 0.189 ND 10,12 4.21 0.772 0.221 C11 0.371 0.373 0,0942 0.115 0.207 0.236 0.137 0.149 C12 0.0333 0.0282 0,0122 0.0172 0.0199 0,0184 0.0169 0.0490 PFOS THPFOS 32.0 I ND 33.3 I ND 8.64 I ND 8.951 ND 29.71 0.0440 29,3 I 0.0436 16.61 0.0221 20.61 0.0394 THPFDS 0.0743 0.0599 0.0402 0.0412 0.0846 0.0944 0.0791 0.0946 Table V Summary of Residues for Human Plasma Samples Amount Found (ng/mL) Sample ID 0204334 0204334Dup 0204490 0204490Dup 0204491 0204491Dup 0204747 _ S _10 _ TCR_06951 _4581 _67[0.03571 TN_50I _5810,03461 _ TCR-683 C11 C12 PFOS THPFOS THPFDS 14.8[ 0.0140[ 0.0539 [ ND[14.91 ND [0.0609[ ND[4.7810.01151 ND [ ND14.841 ND I lVD I I ND] 11.41 ND 10.02521 ND 112,61 ND [ __hiD [ ] 14.11 0.0193 I 0:0843 I Table VI Summary of Residues for Monkey Serum Samples Amount Found (ng/mL) Sample ID Sponsor ID 0204335 TN-A-06332 0204335DupTN-A-06332) 0204492 TN-A-06333 0204492DupTN-A-06333 0204493 TN-A-06336 0204493Du[ TN-A-06336 C6 C7 ND ND ND [ND ! ND ND ND ND ND ND ND ND C8 C9 C10 C11 C12 PFOS THPFOS THPFDS 1.25 3.24 0.286 0.540 0.03651 17.2 ND 0.0141 1.72 4.37 0.396 0.650 0.04641 22.4 ND NQ NQ 0.103 0.137 ND I 16.5 ND ND 0.0130 NQ NQ 0.0880 0.114 ND I 16.4 ND ND NQ NQ ND 0.09390.02271 14.8 ND ND NQ NQ ND 0.106 0.03211 18.1 ND ND ND = Not Detected NQ = Not Quantifiable (negative residue calculated) Exygen Research. Page 26 of 111 Exygen Study No.: 023-082 i ii 1 !=7 iii FIGURES ! -! l '? 1 'i i Exygen Research. Page 27 of 111 _+_++ t ;i 7+ ] + .... _'i_ _r_ _'i .!+t , Lug+ i! i t_'_ .+t !':-:J "'_ '_ 1 '.2.+1 .+t _!I i o Exygen Study No.: 023-082 Figure 1 Chromatogram Representing 0.1 ng/mL Calibration Standard 8,tool* Puk Niml: "CO" Id_u(ee): "313.0/'_60.0 _u I __ohnlmmM_*Inl:Niim'0*1:003"0120-11702.(100.!1"ng/mL)" AnIDn:ot"a8U1olnn:da_ld* s.l_t* l_*z, a llll_81111_.WIfI* Fi*: *l*mm eo_en_ra_t_, atulate4 co_, Aoq. n6tel &q* T_I,IS 0.100 O.I=T lOlt?/o= 01,11041 ml/nl, q/Jr, Pa 1_00_ / 1100, |vl_Lne ra_ozn Iloil* Tl_*=llo141 114.4L epe It=_ _b=e*hold i 57a.04 _= *L _n * r,x_ _ r._l ITI s.ll+. Iltdr.Ii J $*.1+ M*J_J+Cl ixp. I'*k Iiklol rxm. Adt, la_ioe Ilgl. vii, IrllglOI 3o.o lea a.$4 i_= - o.20 o,0l _ S.O0 4,0O = |.oo 1000 |00 7'OO Xoten+Lon TmI &+Ill Xltllh+: I ++4:g f_ml _.,t +t.., 1.53 1117 I.I$410003 I.]0 IlLS * Ooml + cp _n I 41.7o .t. SO0 400 0.6129 0oo ,a40s . . . 10.10 11.480_1 ' ' ' ' 'd" _llln_pkl Nlmll; _0170_.101 _4 mpl_ I_; *_tilrs_lll_ * I Peak/*lime: "07" MlUlI(OI): "803.0/310.0 m* _ikl; ' I_l_nl _ m onViO,will* II Comme_l: "G100302-0 (0.1 110_)* ArmollllJo_: *" 11_i= zn4n, I smile _-_1, lcle4*rd C_t:.ll_ld co_| &_q. _*i _q. _i*, o.110 101171o2 oI,110_] i_1:_. l_ 0S00+ I000. xo4L 4t1.<l, Xoi=l T_eshold_ x=o. _*l_old, i lgl+l:l<l 1'['1 Sell. mldl:_+l lep. _*Lehcl I_. I+, l,_io, I+* VI_, II_o1 _*l I041._ pl S31.4141 m_l 30.0 i*o I*71 l&m e.)o _" 0.01 _ 41.oo ." ]*00 let l_to_ T_ml z_. _1+. h_Lol 41.413 _o 4.00 ! __llJi_+ial hgt +i_llm I TLI_I 41. II 1|.I1|+001 i_I _pl 41.11 mill 7000 TOO0. 8100, I000, 55o0, moo. 4j00, 4000. 0000 _1OO0 2100 2000 _000 1000 0.71711 0 I 1 _ 4 i _Plolm*kpl*PdNlmlme:*:"G8"*101M70_1u.(1e0l)_: "4S1lm&0p.__00t.o0: "inS_tl"_aard" _onmmlnl: "cloo,lo:;i-e (0.1 n_ml.)" s*a_l* Zad_, 41 Al,,Mtll+ms: - i_1e Ty_., 8_ud._d co_*n_c._o_ 0.1oo ne/nlz, 11101 FH_: _+q. 'eJ+Jl+, e_,Ii_13 ' .,- TI mm ' ' ..... 9,8400 11.9741 11,e38 11.4812 41,821 ?.4111 0.Z04 O.SlP1 i 0 ? 0 0 Io 11 8.1 s| _olu _brH_old+ 14_.90 +pl r*_ _rel_old i 701.01 ,I;.i 3 8,_. WLd_K, e.241 s*p. x*tghe i 0.ol _ Ixp. P*,k _l_to+ _.0o Imp. +l_ll. t_c/ol 4.00 = I_. vii, 11_1o, 3.00 us+ llz_t/v* _T, Iro ---- ln_. _a_el Nillu&)+ * ue/_h_ i _LII*I l_ r_ _bl,,, 41.114,*00] 41.11 41.14 c_l in sin 41000 4000 3000 8OOO _00 ZOO0 1000 0.7314 _0.0076 .90_70 10100o0 111_07 1463_ 7 _..._ ) _0 4 3 48_00 7_"'027414712944198.071 0._500 1.0410 J TI_. mkn Exygen Research. Page 28 of 111 - -! F ' "_ i ..... ]" I : i,+, I i [ i - t++J , .._ _ ; ' i }. _:_.1 ..... o Exygen Study No.: 023-082 Figure I (cent) Chromatogram Representing 0.1 ng/mL Calibration Standard 8ample Name: "101702-101" 81mple ID: "_indmld" I Peak NINe: "G9* Mall(el): "49_,01419,0 Ianlu" OommianC "0100302-9 (0,1 r_/ml_" Armow_n: ' $ii_pII z_llXl 4 Ialll_o* Type, silage+4 Comenmrat calc,Xamed _q. Oltli lOml c._l 0.109 O.Oll 10117103 mllnl, iml/la+ O171 S000 Aoq. ?tSlll 0Z121143 111 OlO0 Il_IIhatlmLll_ed I r_ter, T Iklt ii TI_IIILoIII I IlO.lJ epl x,.l. hoot_l I l_ VlL_d_, 1o.o l_ Zqec_*d RT+ i*p. VLa_h, s i+ k/ihl:l I_p. _lak tal:/e, Exp. _1_. II_t;le, lltll. VII. lltiOl u.. htlCtv. I_1 |.li i_n _. o.20 O.OX 5.00 i.oo I ).00 Ue T. .< 2100. 2200+ 2OO0. II00+ 1900, PM*:N_ m iomefllng.w_r lI*n_t*.n_o_n_em TLie* aAce_l.l. Tlmr*.hold, llitghl_ i smart Tirol h**.lo + suei_a m 625503.1t?1li amcp I 2 .ol?e*oo] 4_pl e 9.0T _n 1400. 214O00C+O. +000, 9.1929 11.1111 " __ I00+ 0.717[ ; 4oa _ 8 299 1.97H 7.400 " 9o0 1 O S 4 0 9 7 | _m,. rnm Sample Name: '10|702-101* 5llmple IO: 'Slsndald" Peak miami: "010" Mlaml<eia):"113.0/489.0 atom" File: *llerum _menlnO.Wlll* Commen_ "0100302-41 (0.1 s*lmle Index. 4 Illlpl* _*| l_U4er4 Co_int rICLOnl celcul&_l Co_i 0.100 0.117 Acq. Dltel koq. TOll 10/17103 01134143 nglmL)* n41l_ Ill/Ill* IIII AmsolelJon: + 1990 10042 I 10 11 11.901 11,3912 N9uoedhiLZ_ied __eo_e_ i 1100 1T w_naov, Exl_eeted RTI _,p. WLd_h, ilep. ml/ah_* I_. kl:_. l_Cio, I_1_. v*l. le_o, ule l=lel=lve RT, Are_l ig_r_ T_IIII 3- .o sac l.ll i_i - 0.20 = o.ol _ 4.O0 :1 3.oo Iio - _1 oe e .le 3112 eo_nt i '11 J.31 I 1400 1300 1000 900 0,7171 10.4707 1 01970 4O0 8.094 200 1.089 Pllk Nllml: "_|1" Mill{Ill): 'IS6+.0/S19.9 Iamu* ]Olm_mpleanc .qli"a0m10l:0"3100211700120-,1101"nglm$1Lm)' pliaAnI0n1ola']lUl_odnl_: l"" lell_l i x_lll I lU_ll '171111 II;_l_dar4 CanIInt woOLen I o.1o0 =4111111, _40 ellI_lt_d CO_I 0.113 lUi/_U _cq. _it., 1olx71o3 300 &el. l/_l 0LIlllI3 111 lie0. XIiho_dtlodl _*a_or, [el _90. A=ea "_s_emhOldl _II.LS ._ 180, I_P tltl_dov, 1 .0 lee 940, smp. _d_h_ sm_..- J+_;_, _. ;-_:i. recto, Zz_. Val. hi:Let 0.20 o.o+. 4.00 ].00 _ 220. ; " leo. < 160- bc*n_ion ?trial _.11 nLu * 140 _*e, 3351 ee_ca I_a=c ?_e, 9.33 iln lrealild_ ?_iie_ 2 .llle*SO.7041 i_nol;1 u 112000 |O 90 40 I=_i; "_mm iO_mln_.wal" 0.7_04 200 1.821 2.34601__2.|09JS 2 3 l'lm*, mks 9.5079 1.246 l.S170 7.4941 7.03|0 78092 4.11679 I.I011% 4.444 4 O.9182 S I 7 9 9 Time, mUl 10.241_ It.1010 10 11 Exygen Research. Page 29 of 111 '", _ Li i 1 . .. ,.?_ ,_ gi_i_i iI i ;',_ d _!)l m' _ I - +,, i i , '_ ,.. L:; Exygen Study No.: 023-082 Figure 1 (cant) Chromatogram Representing 0.1 ng/mL Calibration Standard 18ampla Name: "101702-101' kmp_ ID: 'g_nda_* Paa# Name: "C12" Mex_ae): '015.Q/689.0 am' I C_en_ "01003024 (0.1 n_mk)* AnnotxUon: "* Jw]a JJ2de_j | scwontlcea_raty_pLeo,nl Ca2aul*ted o_l &oq, Dakel n.ha0l.=14O0 0.111 10117/02 .a/x& aglnL I _i40j A_I. _lill Olllllt) IV II_IL f J.ld I Yea = |_hL_ rMtar e IL voisl _hralhatd i 20.10 i 210 _.a Threlholda 1.00.98 Pk: *Nmm Nman_g.w_" 1_ WLndav, h'pN _.d 1TI |alp* Ilidgh, )0.0 e W.15 nin -- 0.20 a Sip. xeLga_, O.Ol ; zxlp. _ak P*a_Lo* !.00 _. _lJ. x.eto, i.ao : l_p. Val. _tLo, 3.OO UII hlatLv* xt* lit. l_p., ht_elon ?l_; Azeal Height * start t_, xaau*l 1.41 |l_*l 2.14_.e+ool 9.49 ash _lt_a 1 a118 nla Ind TIM, _.83 u_n 240 22O 2OO lie 140 120 100 0,7004 - 40 2O+ I.Z2_ 2.?140 I Peak Nine: "PFOS" Gommon_ 'G10080_4 S_tl t_ez, Ma_(aa): "499,_/80.0 8m" (0.1 nWmL_" AnnoMfon:" Imple _*, I_l_i=d co_*nm_a_ion, 0.10o ae/_ ill Calaul_g_ o_1 0.10_ ml/mr* Aoq. _gel I0/L7102 KI_ohdLL__edl r_l:o_ I _ea 100 _o_Ii _aaholdil llo.]? a A_** _.lho14_ I:0L.I) _p zxr,_ i_, lap. p ][xp. _.d ghi XILgKt, I_llk Itak_io| _q_. Ad_. llt_Ol l_ql. _II. IIaILOl 9.1= g._o o.oi 5.OO 1.4O ].OQ -- = 140 II 1 110 1 IO0 2218 4.727 .007_ 7.38 _,.. ,,m u., J.( 27 1.1 _S I_l J.ll_lg i led t_l 1 ._.lll*<)o] _p 9.30 _Ln 80 ,o 20 o _.,+r+ /_ +^ _PaII.__p_NaNtaal:me*:TH"P10F1O7]0"2-10M1"III(II)0: 1mp"_417IO,D:/B'$1ta.0ndIma_u"* Commen_ "0100_o2_ (0,1 _l/mb)" AlmotlUon: " l_m4lle Z_dexl | iiJp/.a Tylpl i It al_ll_l c_-e_ra_L_a* l.xoo ual.m Ca]L_lll;ed eoMn 0.101 _1:_. 120 &_l. Offal lOllTlOl _q. _L_m* Ollllll] IHI 110 _d[ILed i NoLle T_II_Xdl 7 .it _, Jk_ll _llh_Idl I 3S.$3 _a x_. IM_ChI l I I0 _tecl At i S*p. W/dthl l.ll _n -- 0.20 = 80 lip. _mtoht i o.ol I_qp. Ihl&k Itit_*ol 5.00 J_p. A_. IAtlol 4.00 r.xll. Vii. l_Lol 1.00 u.* l*_l_tv. ITI Io _ 70 _ ]0 Int. lrl_! i Nlau41 * [ la_*ntiol T_III I.ll 1411 _0 ..tv_, t.al_..ooa . hd _LUl l.ll ILa F_e: '_rum la_lnk_.w_l* ^^ Tlm*, mka ^ _j 0,9999 _0 0,0701 1.4701 l + ^^^, "A "_A, m"'_ :i AA 6,2883 A_ @ Tlml, mm __9"38S! .^._.i.^./_.A.+. i#VV (o 11s0_ _t_ (I Exygen Research. Page 30 of 111 +_+_ :+i ! :! [ I i_+!_'_ ,":'-+ Exygen Study No.: 023-082 Figure 1 (cont) Chromatogram Representing 0.1 ng/mL Calibration Standard i aml_l NRmt: '101702-10t" 8a_ I0: "SlandanP Peak Name: *THPFDS* Msu(Qm): "527+0/0t.0 ainu" C_mm_ "010030Z4 (OA ng/mL)" _tanBotollan: "" Fh: "mmm eonmc_ng.wllr o_l:_atLonl 0.1oo _/nl. 90 CllcuJla_e4 CO_J 0.000 &o4 o+11:11 10117102 151blllL II Aiq. ?l._J i 0_126141 I0 ItodLtiod, [.e 71 WOlll ThgllhOld i I.II Z_l 70 I_m. _,N_t,ll J IS, l? _l_ctov a 1o.0 l+e isXPp,l_, O _wiidthlRTI tOl..3:l2o iLn = 80 I_p. Peak IAl:Los Zxp. +_I_. hl:Lo, Ugsl_e. Iterlea+l;.l..ve lal:LIo_l1 5.0O 4.00 J3o.00 = So "-- 4S. dO, lltent[ou T_l S._O m,J.n _JS. Bl_ah_ I I._3e+O01 pe 3O lll_al_ ,rI.M _ s.lt nL_ End TLIWJ; J.5[ i_n 2_1o +..+ ^ A A A A ..,,.,+.++. A +J Exygen Research. Page 31 of 111 i:_i' '_'i , 1. ! r ,_ "_ / : _,_ _'_1 +,'+t ++ i .j _*+i _i{ ++ .j .:/j + ...=:+:'_.3 _.-3J : i+ Exygen Study No.: 023-082 Figure 2 Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ID: 0204490 Spk J, Sponsor ID: TCR-674) .ImRu_k vNl'nm_ w_."r m'M_u=l(e"l_): _m313_.G*_t4_S_e0m m_'...m.m_._r m_ _ s_ J.,.sPw _,_*=_ - .,Ilulpt e II_4n I 41 llLuqpl T3_el Corlolnt Ill: lone 1. OOQ_O nll/mL 74QD: cilal/+ltd e_l Acq* DILI:Ii D.O00 1011If01 iml/_ll. 7_oi : Ill,him l,_tor, uoLl. 'rl_*lhota, i A_oa _luelolaol41 I1_. _o<ll:Iml i lit VLIM_* g_.o =.<11IT , sep. WLdl:hl p, HIIlghl: I _. l_*k I_t+ios Ixp. l_l_. htLol Imp. vat. htio, UIe lel_tLve ILTI III.II 172.01 10.0 1.$4 0.3o 0.01 S.o0 4*00 1.oo I_ Int. Ty_, lml:enl;L_ T:_Ie i &re&l Hm_llhg I lltacl_ Tltml hd TL_I _.*1 1.52 11711 |.475*001 1.35 8.+Z =pl lqpl llm + id.u - : I=o_l;I II InN m.Ln ILia 0,4,Tr7 L 7.$4M _ LB207 2_0 t_oeas 1 _ _1 4 S II Sllml:,llK+i,_l: 'I01702-140" _I_ *S_" _ 'll_lo_w+l" s+,_ll* Index, suel* "_e., C omlDl_rl_Lonl 11CmllC(4 ml m4. m+ll &q. f_+n,el 41 oc [*000 II.000 10111102 03110121 _o/1111 no/l_L AM 1_14 m m. Nodif/_ls _o lAloI_ilIeI _"hrx_llrheolhoLldds s 150314+._.|ill cppl ]I1tT_. w_+slnodo_chI+ + 104.o i_ IS_ecp_.+<l _Idgh aTi, e0..?I0ll it :-p s.p.I.I +.ghl:, 0.oi zx_. _.k h_Lo+ 1.00 e _. _d_. Itll:l+o_ +t.oo ..... ,..,+..t.... = t.,q+, v*l. It+_l.ol 1.oo i11:1m1:o TLMI I.'71 8+La 1111 15133 oounml a .+J_m. 1.4211.oo3 +l + s_rl: _l.n,, + 1.4;3 ili.n IC4tt4Tml I.J] ilin "_0. 5_0, _0. _mo. 60C0- ,Ii_0. --" 3*OO _i_I: 7 8 II I0 It L3_S L] _ + I._ 11.941 _00 O. I.a3as _ , + isP.lm_c aN,i,.nm_ .",__S'"mutl7m<__+o'.4mt+m._o,_+oim:'us"_"r+,,,'.mm.om_,,,,." Iiiii L l Znd_l ICm_IIl:+l:L_o/nitl)l i II 1.00Q0 l_/id. Aoq. Dmtll _cq. T_M, 10111182 0]s_01r3 Jm woelteied, su,_hlna F_l_or i .................. uo+kl. ?t_elhold+ M_..llllooe hl i _ 14_.?0 l ::: Ira+ell ITI 1_,. Vdt_+ sre.l_l. . lhl.L,__lhl:tl ,_t., l.ll ii1_ o.Io _ 05.0.to0 ; Exit* VII* Itl:J.Ol ]*oo el lllltlv ItTl if 1.2_414 1.1014 1.ml4 8oc_.na int. _, AIIII might, lC.+m ?_** ra_t ?trait e. IIS75 1.t'l:ll+00l 1.77 9.t? +ornate ii _III m n4n _O(XI.00 ,JOg_._ 8om.no _0.oo 10_0,00 I 2 .71mi : &81sT1 4,s31m 4:_ , + S.2 s.Tsa7 7.a71 7.inlet Tim. re+,q +, , 3 4 S 6 ? e Tlm+, w_ , ,o iMP4 9._4tI.4 9 tO . 11,_7e_... II Exygen Research. Page 32 of 111 ,:.... ': 'I ' + 1_d i ++ _ +_ '-.-+J ++ _ Li + J o Exygen Study No.: 023-082 Figure 2 (cont') I,_ 'Or _lll(eo): ",IIUI._'41SI.Olln_" Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ID: 0204490 Spk J, Sponsor ID: TCR-674) co_.ntrltion, _=q. _te, _cq. 'rL_+ $.0 _II_ 1o/11/o2 0]ll0a2] |_u_l_ne ruc*r i l_IH c_ I1_. TILz..hold, _*ibold, luloo_bl I 1T MLu4_I l_*d IT+ c.*lcpu. l.=W.qlldth. con+, 51I* Illl_.ghg i Ixp. _l_. hmtol 1 110.15 5S0.J? 4 )0.0 5.11 o0.e.Io0o o.o[ 4.00 i_/.l.. 1.0_ l 1Jt_0,00 71mO_ 70G0_Oi _ ZnI. _'YI_ i HILil_ I b T_INI samx l.Sl)l+O0] J.25 . pl ll_. +o+om 11. II .+_._II_i i : 541m1_1_'101702-140" 54n_ll_'Spk" F]kl:'mlm+lsm_mif" P*_ i_nl: *10" M*_(w): "SItLOt4OLO*ram* lint* lmlUl _5 lmexe Type, Cmn+entr.Ctom, _c 48ff X.o o0 4/1,X, +llnLlm_ coral o.o0o I_ Inl+ icq. Olll:l i 10111101 m +eq. Tl_ll 01110ll3 All llod[ +JLl<lI siio 0J1229 mO1ll Tmllhold I 11.11 AIM _[ll_[dl 21_*05 all lira. hoo+_l | Zxpe=_.4 I_, Ip. v_l_, _ 5.11 n_ 0.20 =- sp*. M*lgb_, I_. I_llk It_CLOl Zxp. Kd'_. IL_gJ+Ol r._p. v.l. It_cto_ O.Ot l.oo 1.00 m.oo . _12_0. * 1 A_, znd _Pbl*, $1o50 oo_u_ci _ 2_0, 5.54 ILt= I_0 i i + e P i _o ++ e+s32s '1 i + ] i.,_j,:,:_ !J Camm_ "_Ou_ II11 I11 I_id T_Wml c_le_l*l_ml c_l J_*q. DI_II J_q. T+._I Spk J, O.Sppt+" ,_I_V_: _i _C o.ooo I0110101 0114012_ ng li/, 1 _0 |_h_n4 l'icl:o=, NOLll TI_III_Id I Xri _rnhotd_ w.ll. s,,o*_l_, lT wLnd_ t _Jq_ 1lld I+Jpl sep. Vtd_ l-p. u*tg_c i I 11.13 lZl.IS 6 30.0 !.15 0.15 o.oz _ll c_l l_ I_11 _ = l_ip. Ad:j. llgLol 1.00 11 r.xp. VaL. iim_o+ 3.00 ul* lltl=tvl 111 V+ xne..ry_l, ,_ _ , i*_n_t_ _L,w, 5.4J nt= -- ISlcm_irg_h_ _iINI t.d _t,,,,, I.?SL5*.OSO0I 5._0 _n _ p -aa 1 18C0 mm 1200 8OO 4OO O.64 o_ t i 8.41101 Z0A_7 so_a a.ss4+++.+:p4.,,o___Tss._,.3 _=ms_5. i _ i i -- a * a * _0 ft "viral, Exygen Research. Page 33 of 111 +_L_ _,: _ "1*! , + Exygen Study No.: 023-082 Figure 2 (cont') Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ID: 0204490 Spk J, Sponsor ID: TCR-674) IlSw, pler_'lolTo2-140- s*n_*lt_'St_" C_nm_'_2044imSpaJ.0Jpl_ _ s_.el, zada., 41L saa.el* _yp*l on++nc=atlOnl 1.00_0 _/_ CilGslaCed era. 0*000 a4/_ F_'m_m,_m_ql.v_r - _4C0, _:q, Tim, _difLi4, xo,* _ela_old ar_ _u:em_ld )_.l. saoo_ha, IT wlna_l 03,40,_) _i i 30.20 i 100.9| 10.0 _I _l elm IN *p. Vidtl_ i 0.20 _- s*p. II*Lvht I o.01 Imp. AdS. l_Lol 4.00 r,x_. v.l. largo, 3.00 IICO. IIBI. le_o. 11C0. 1._47T +.'+_l i_,+ +++++_+ !'+?15t _.:_ :.m,+ ....+_. i i !g _ i .. , $_lp 11 Zdell lalJ_ le _Ii Ccio1n_,1n1_tl_:r4il_ tCon_mi [C_* _tll J_lq. T_III * 411 (Ic 10..00o000 I0111102 63140111 _nlol//lal._t-, Inloudm_c,thie+dL+ne _o=, _o_l. _n:elhoZa, _'ll 14o.17 _1 r.. _*l_old, 12oi.11 _i let Vtnd_, 3o.0 i_ s*p. W_dl:_ I o._o = s*_. Ilei_thl:, o.01 Ix_. i_l_lc l&c@.ol 5.00 l_. Adl. 11_o* I.oo Imp. v.1. I_+o, ).oo U_* IlelatLve Itfl k .... ,_, l+lll mlv_m, l_l_m ?ml Ima +_i ),5141) mmgl u 1.13?l*O0i ql I.I13 I_I S.lO l_n 1114 1.7d 1,1_4 ImP4 1.41+4 1._ I._14 _,144 1.oe4 + 7_0+0 _0.O _.0 _.o 10m,0 *1 .......i.... z _ 'm_o4_lO _pk,L U i_" ..emWlor, c- sclo_lwen++_r*.l:/on, 1.0 Qcoo _II_L I_ J_q, Tl_ml O!114011l 11, lul_Mnl e*_o_l l** _o_e. _*lhatal ?.zg 1100 08 1000 ";r_,-;--"=:_, ,,., .. .xlll_ Id ITI l*ll i_II s*p. _tdtlt* o._0 _- Im_ lep. l_ght i 0.oi _ EX]II. IidJ, llaltIol 4.00 :I Ix'D. vii. l_/ol l,O0 UI* I*]**t[n Ill [ ........ _-,.. IInll_t.lhl: N*t sq:icl: ?LIi, _.i)IO(aln*0_lli apl _ : 1.17 i_11 IO0 Exygen Research. 9,1224 + .+- ._..... +._,,.,*.,_,_,+,_*_-_._,_ _ ,o,,m,_,,._._,, ,m LN4 J 2_.24 Tim+, mm Page 34 of 111 _:_ _ 1 t i r+'+_ [] o Exygen Study No.: 023-082 Figure 2 (cont') Chromatogram Representing a Fortified Human Plasma Sample at 0.5 ppb (Exygen ID: 0204490 Spk J, Sponsor ID: TCR-674) Peak H41mr:"TI_et:D$_ MIll(N); C_mtml: "_l_lO _lt J, 0.| _" s_X* radii ti s,,,eWX* '_l QC ' S27.0dl1,0 amy" _mot_orc - .__ cal_litml C,W_t O.O00 .e/it, Azq, _ml 10111101 1100 ,,1,1.,. p. tootle _llbJoll i t.lO _I fl_0 ]tml. hooCkl i it ;ind_ J Ix_ kl4 ITs |l_. Ilia_lll 9OO _0.0 t.32 o._o _ I00 stp. _liahc i IbW. leak I.dlt/OI b_, _1:). llkiol I_qp. VI[, l_k/Ol 0.01 I.O0 4.00 ]+oo = _0 i U_* Itq1*ci_ aTl tat. _+ lltlnk[_ A.... T_IIO I llo _I |.If ,m _ roll _+. __ mo s_.+++t.., s.s+ =t. " &31m 0._ 1._33._ 1.7.M0 i , :i s 4 5,3112dil s Tkn_I, rr,ln 77.0_47_i_ 7 i_i (_I -! Exygen Research. Page 35 of 111 I +" i_:"i i ' -] ++ - i .; : ! ,t_+?, i_ i+i].__;: j "_ ' i !4 4_ Exygen Study No.: 023-082 Figure 3 I _('1017ce.lacP I _ Nsm_ "C6 _u): _.._1_. "3t:+_.0 "Sa._" m_r Ullltm Za4oxl IiI_Ig _al co_el_cr +t _._n Caloulatld conl_ ACq. _l_ll A,_q. TLm++ li _ x/_ O.OOO LOlZilO2 01rOl+Z2 mllnb RI Chromatogram Representing Sample (Exygen ID: 0204490, Fa_'_i_mlmmml_waf _II _4_0+ a Human Plasma Sponsor ID: TCR-674) ?.M41 IIS_hl_imwf LI4, rN_arl SeLl. ?l_.ltso141 a,lP_ _*.hold, IvJI. sv,_ll _ tJ.l.ll S?2.0_ i ImNotl<l CP, lip. lScll_l_, IElpxp. . Il_lllilokhl: ll_i+.o. l__.p. PVdi__.. tIl__=+L,oo., Uu h).auL_ l_Pl I*Sl I.Zo IS..o0lO 4re,0.o0 lkl lnl:. _l, ]1o l,l_k elm ,_+ 11111 -- . = -- a_O, al_O, 2leo O_4_Z 24_0. _s33000 IKO. IlmO. II.CO_I 1.1)07 i Ill2 lO.lI_II O PaatNam_ '07" ka_N):"3na.C_tL0ml+ c_lcu t_t:ml cm_o_ &_cq. oll_e l_q. _J141 O.OOO 10/lII01 OlI01112 _I/i_ _I h_lo _b_eehOtd, m_Ji. llo<,_ha, _cll.I4 4 +Otld ITI II[+ l[d+_ 0 _p. alel_mm + Imp. _lk l_ttOl Ia_. Val. lt_eoa ue ItlacL_ I_i 1,71 i_1noe_o I;.oi 5.oo LO0 Io _. ht..l;L_ _, Xl L_rl_++ ?Lu, Ilia Xl_ i.tn nJ+n ]nt). _d:l. x_t, ./x+' _ _OoQ 2Sco i l a 4 S / 7 8 I0 II I !.97 I I_Xi _pII sCaomm,_Ix_+sg zldex_.on I _s]C.l__ 11NI& cA_4e.gqxq+._T_CJ__l1l,1_41co.., i0O11loll0lO].o2io_ o m_/.,_ t 3 i,oml.m_v.W" _r71 _co. Moil 'r_llholdl All "_hzl.h_Id,, I? wnd_, IXp. t_Ik h_le, 141.70 7of.st 30.0 _.ao _.oo use lleL_++v It?, _l ape I_ _ 4_002 _ as0 3000. osmao xeL+lh_1 a_cc _'LI. l 4 .I11+003 I.?I c p _ I.L. 20_0 - 80Q _OT 1 2 S 4 1 4 i I i 4 ? I I tO It 9.0174 I_e07t 9.4&II 73011 7N 7 8 i 10 l1 Exygen Research. Page 36 of 111 .+ i L ! '"I _" I , ,,+_ I I -3 12j _,.+-+< .! Exygen Study No.: 023-082 Figure 3 (cont') Chromatogram Representing a Human Plasma Sample (Exygen ID: 0204490, Sponsor ID: TCR-674) I_I :ha ql commwn_rlt clto_ll_+d &oq. _gl, xcq. TII+. 1onl cowl II Ilia 0.000 _0111102 06103+12 nI IIIU.. m ll'.LI_kl_I r _gog, _ lh+eth+ , 17 ULnav, lo.o m_ 2N0, a4a 1.411_! _. hak 14eLol 5.00 _ IN0 _1 Dill, _til. I_go_ ).00 1400 Xng. ']'ml_, mlmmlx l_1_hg i iml,<l'r.Li,ul, 5.410e.001 II. ) II =_I _ 400 _DO e,ammm_ '1_044_O" /_ncolallon: " co_*n_c_*aa, ilia+ 4_0, &ell+ _Igl Jteq, _:lJ_, 10/111_I 01,03,I= I I_JnebLng l,_l:or, 1 :*_ _h_eekold, 21a.09 ol_ gXlm_ _ed ITI Ill,. l'*ik _lgLol |.2_ ii_n -- S.O0 e l.l_S 1.8_10 [41 _'P4"IO 1.3424 $.ll_lXS 8 S 4 II I 7 l fl I l 9 10 It + l:.i_l_ ?Lsl_, s.2l lin I_0 ........ I00 0 ! 3 S 4 I 1411 tI_, II Cm_tmggamon, +Al1_=q*1_cg_ll c_+ Am. _Ill Nod1 tL.iml, l,l=ol_ flat=, NoLiI +_lI_mId _r** _tlho_d* ml_ IoI1o1.o1ooo3 01102112 bee * 2).11 _11.15 1+ v[nd_, _o_+d _+1 30.0 9.Is sep, _/gh=, .oh Ix_. _lk 14_1oi I.o0 tt_. Ad_i. ]Z_cL6, l.oo Oll llllgtvI ITI I_ _r/l_ _I I+ =_l i+o ln . * i _ lna_. T/pe, ll_ffl_g i II,t_*_ I.7111.ooi _p 471 411o GSlO6 _00 0+64?S. _ 7 l I0, 1.2807 2.1S,,_2li.4284 Ill7 432_i I . . . . . . . I. 7. 7.ell_ Tm_, mm 1.8417 I 1+1_' I0.311 l 1._12 II.ltlfl_ 11to!i04 tO II I++ I 1.97_. 11.3111 t 0 1 1 Exygen Research. Page 37 of 111 ..... + L+(..+: !.'t ! ? !"+_ -_ : _:*_it :_.' I ,_ b: f :I :: .,_1 t i :. _ ._ Exygen Study No.: 023-082 Figure 3 (cont') gl.,nWm+N_'10tTOl.la0" _m.,Cal_"_r,_e" PI.III Nln_ '_1 r kllll<el_ "IIIo+0t_ 0 Ira" Chromatogram Representing a Human Plasma Sample (Exygen ID: 0204490, Sponsor ID: TCR-674) R_'M+um_nmmm0.vt,+_ S_le 1_,'ip.i unX.,_m cax.lat*d cOMl AII. _tr.e i Aaq. +till 0.000 I01t8101 alt0l*l+J nll/llt. lm _+ied, l'+_hla_ll Y*lltor | ll_lll 'r_lslaol+d * &_ll _lllrllho 14 s mill. ImcChll i r';" wirdov f _,o 20.20 100.II )o.0 c_l _-,pl i_ Il_el_* =vI=ld,il<:hI ItTi l 90.1250 a.ta _ sep. bigh=l 0.01 It_. PuJt It_l:iol 5.00 100 _ Izx._lslD, . _V1A1tlo. IluLLIgtlL.oO, l UH lela_Lv* IT, 4L.O000 No X=_. "_yp*, u* _,*k I 110 140 _st,.,ti.n.,_,+rt,,t.,.,,,rt..,, m Tluml .Il,IA+.,. +.--"x",,+'0. : Ill& O+114711 .87M &+le2l O+4?+i IO+2_0 I171_ _ hklm_:"PFO_ kkm<Ng *4m._/l_.0 era" hmx* _y_** coM_t+rltion, *lcvla_ed C_l &eq* Dal_o I Aq. Ti_* Ul_mmm Ilia o.ooo 10110103 OllO]l_,_ rill IIii. I0111 1.714 I.E_4 lloil* 'rl_relkoldl 240,17 +'pl JLlrii "PI_1iklo _(I l I;I0_. 80 Iml t_m. _looCh,ll i 4 zz_e_*d ira-, p Width* |ep. Rolsrh i r,m. lhlllc IL*+I;iO, hp* Ad_* Itlltcol lXl_. vll. x+l_iOl _Jle fte_.l_:_ve ITl _,II 0.2o II,ILn 0*0L 5.0o * 0.oo = 3.0o NO _ leenton Till kial Kliohl= I _._5 1012_1 1.7)30+001 ILn _oII _ epe _ 1.k+4. 1._*I. 1+214. 1.0o4, _O+O. 7_00O+. ,,eoo.o. t._O7 _,.l'/'ee :l.37,rJ l+_2,11z ! I $ 4 0 i+0057 _'n'_ ?.07 1. II 7 8 11.3_07 9 to 11 O+I:_Q .ct,lr_Nam_.lOl10,Z-180" ,Silllqpkll[_.Sll,lllt#_. ,=_t_ reel,ram"'r-el_FO,S" kk,.ll<_): *4.Tt.0,tll.0 irmr Commlmr '0a_441)0" ,k,,-_++_" smmx* XMIXl 10 Fde;'m41_wltl" Co_lnl;raiOnl Ilia _E4 cs._z,m.?z..._._'lI'i_*, con=, I/nk0_.oo0 ml / i,,i,, mo J__qq.. _Tl_l;..ml ,, L010,10X30I1/002 1'11 l<luon<=llh_itle_dl , I'_I:o_ l _oi** "Pl_**laold i Alrel 't"l;_lshold i II..ul. _r_oo=hl + _4_e eel KTI sep. Wtdeh, Sip. Heigh i L'CO* Pe*k It*l:2o, I+,1_. X4_. It+,_iol I_xp. Vii. Ita_Lo, Uoe ieI*ive &T* _.1 7.11 30.03 I 30.0 1.$0 0.00 o.oi 0.00 0.00 :_.00 No CPll* In;,. ii II_II * = _ Ilglal:_, _ T.J,_ i X*/,ghl: * .,:._t+_.t,,,., ItlMl _P_,, 6.7].2.*0010 =p. 0.,_ .i. 0,10 _o dso 400 060 300 =co ll.S'/_l &31 6O 7.mNrl i 2 '+' - i - S s "_-- * s !0 *! T_*m, m Exygen Research. Page 38 of 111 !__ i J " ;ii ", _ _'"__*_ ?t i=J ''7 ] Exygen Study No.: 023-082 Figure 3 (cont') la,,mclm_'lOl?_e.lao I PMkNwe:_" l_ _944S_ _1(_=* Mau(_):l_7._Snl.oamu" Jvv_41_ " si_*tI*wZe TInyldN.*a eo_tracLoal c=2_1**d C_= X/A e.ooo ............... Acq. Tl'_a 06|O)_U xodilLmd* leoLs* "tbr **botd = _ru ,r_=_14 = I.|0 ILLJS nel_ III al_ =p,0 Chromatogram Representing a Human Plasma Sample (Exygen ID: 0204490, Sponsor ID: TCR-674) Rk*_n_r m.lll_ _ _0 aDO r,]q_ =idt I'? = llep. Wld_I I.]l i_lt - O.3o :s .slip HiLfh i 0.01 i ]_q). _ list.toe 5.00 _t3 _. J_tJ. ItAtLOI 1.oo Zzp. vax. it_=to= 3.00 = _0, ulJo I*IicLIO i_1 _ 1_' Ketahl: i llA I1:_ ?_til II& 1_0, liD, 1_3131 IOJl07 _0. _ .,,, ,._ Exygen Research. Page 39 of 111 i _+i "T"I _:,"':LI: 'i ] !j_ 1_,. '1":"_i' :_ .[ ) ..,i i Exygen Study No.: 023-082 Figure 4 Chromatogram Representing a Sample Analyzed Daughters for THPFDS (Exygen ID: 0204292 Dup, P*_ _ "T;tPF06* Mare(u): *S2?.Qtll.0 | ,:mnwm "_ .... _'' s.mpl, t_lu, co_ng=akLoa, _* An_I_ - X/_ Sponsor ID: X328-A). 1.2m3 ac,8c1qn. la='_r_l c_, loo04o72n1]9* M"e/_ IN _LfL_, I_h_g Ir_ear | 1H IN Iraia* "_l_..ho_d, O.J6 fpe ,_ 'rl_.._,J.d, a.is ,,_. 14o x VL_, 10.0 ._ lao dlp* Mll_;;_ I lp. He_lJhl: i item. h k h=_o, r, xp, _t. It_cLo, O*lO O.Ol S.oo _.oo _" 120 . 110 I uol telel.re RTI Jim Inc. TYPe, Ik,_l IAtoreCae*nCLO_ Tl_e 1$7.0177 __ o m xl:_.r_ _I_N, lnd ?lie. l.ll mln I.$1 i_i m for Three I_ lO.Farn _o ?.stoe IO,7S78 _0 . 10._ __ "I"HPFI_-I" M_m(_): "_17,0,T,07.0,,,_' u, 1__II Tryipde, _o_*n_lclon_ _*1c_1:_ c_, _q. D, eel i/a 0.225 lollZ/eS nglln_ 1oo, lga. Isv,v:hLng r_l_oc, xo_** ?hr_h_Id, _.a _hrtlho_, 1 O.n =pl 4.14 =pl _ WLnd_, lo.o lo it.xp_ c_d ITI s.p. ,*lm:l_, S,p. U.lghc, Imp. t_k It_Cio, Zlq,. _l_. {ulcLo, lx. vii. hcL_, _.t 1*1_L_ I_, L]L u_n 0.20 _" e.0_ S.oo 4.00 : 3.oo Io i II_I. II0: I:I_. m. 110. lO0. XeLg_Cl loire _, 1.914**oo_ I.II cP _t ibl l_d ?In., 1.51 iLn I_- 921_ .,.0.38o!I . Name: '71.1PF1_1.3" Mul,(iml: "S17._4117.0 ram*" ._.at cl/ulc_ o_, _l,q. D*It:l, &cq. Tilml _4_.I L._h i_ast_ r_eo_t IIoI* _..held_ Cl=o__t_:*T_Ch=lolnm,hold I Ii"k rinds, l_cc,d I_, sep. l,lde_, s_xilp).. hIN_lalkhcJ,l_l_tol r._. _d:l, It:t=. 0,244 101].1103 ].0047111 *. 1 o.ds v2l*lOt_ll lo.o t.]o 0,3_ 05..o001 4.00 .e i.a. Im _ idn - = 10 lit 1Ill7101 II II II Int* "/yt_l, Itct*Clou_ TIM, litco "r I.,M 1,14 ",PIM l Ila_il 1*30 II.n 9.11 nin *_ 9.54 x,Ln " I.I i" Exygen Research. a f T_le. nm @ b ,b I, LImQ4 5 Tim% m_ _ ?.I01_ 7.7S_ 0, .lO,_ ,//I]1 10 1"1 Page 40 of 111 ] :i .... ii,i_i _} ! ,, -,AA q i !_j Exygen Study No.: 023-082 APPENDIX A Exygen Study Plan ExP-023-082 (Exygen Study No. 023-082) and Deviations i i/ T Exygen Research. Page 41 of 111 -_ i :.-: _,_.i _ ) _ _:_." Exygen Study No.: 023-082 StudyPlanE: xP-023-082 ExygcnStudyNo.:023-082 STUDY PLAN _:. Study Title: ! L Analysis of Pooled Human Sera and Plasma and Monkey Sera _! for Fluorocarbons Using Exygen Method ExM-023-071 : Study Plan Number: ExP-023-082 Exygen Study Number: 023-082 performing Laboratory: Exygen Research 3058 Research Drive State College, PA 16801 Phone: (814) 272-1039 Study Sponsor: 3M Environmental Laboratory Building 2-3E-09 St. Paul, MN 55133-3331 Phone: (651) 778-6565 ,:.) :L'/L Exygen Research. Page I of 59 Page 42 of 111 Exyge13 Study No.: 023-082 ! " _,_vI_ : { : t i Study Plan: ExP-023-082 Exygen Study No.: ff23-082 I 1) EmilyIL Decker, Study Director, Exygen Research ti:_' * 2) William K. Reagen,SponsorStudy Monitor,3M -_ 3) ExygenResearch Quality Assurance Unit I i 1 il 'i I LJ i. , 1 ! I ' i i . i i : I '.i Exygen Research. Page 2 of 59 Page 43 of 111 Exygen Study No.: 023-082 StudyPlanE:xP-023-082 ExygcnStudyNo.:023-082 ). i >.:n I I .! I /::::.:I ',) ...L. I StudyTitleA:nalysiosf PooledHuman Scraand Plasmaand Monkey Serafor _, FluorocarboUnssingExygenMethodExM-023-071 _ StudyPlanNumber.ExP-023-082 ExygenStudyNumber.023-082 I _,,_ ! I I ! ' i>"! I [ 1 s : ThisstudpylanwasauditebdytheQualitAyssurancUenitofExygenResearch. Naomi Lovallo Dat_ TechnicIa.l_ad-QA APPROVALS _._!,_ _,, { _z_lly P_Decker, Study Director Exygen" Date [ ":"_ ,_ohnFlahextVyi,ce Pz6sidenFatc,ility Management Exygcn Date i'. William Rcagcn,Sponsor Study Monitor l 3M : ___..,t___ ,! " Exygen Research. Date I) i Page 3 of 59 Page 44 of 111 - Exygen Study No.: '023-082 -_::,! "! /i _ i ', ! 10/09/02 14:25 RSD IN = TECH 2-2E-01 * .88142311580 .. : I, .. ' ! ' ! i I,t0.215 P002 0 0 _ Study No.: e23,.0_ i " 1 1i " ' _ i" "!S_ly Tltl_ Analysis af Poolcd Human Sera and Plasma and Monkey Scra for Fluorocaxbo_UsingExygeuMethod]_tM-O_-071 i. . IlS_txuydgy_PlSmtuNd-y_NumbEs_.P042r3_-8o2s2 " oo .1"::I ! : :.. . ." " i i_ studyplanwas auditedby theQmdRyAsraL_ne%_ Tdt,ofEx_ma Rcs_ |I ,I I _,_.n,A__IT'... i1i ! o! _ROVALS .... i..i : {" _ .!::!!,.., 7 , I_hnFlahcrtTV, ic_Pre.sidc_lt_, n_ilityMmmgc=_at :I ;!"p !_m _ : :i_ _t_c=, Spons_ " Study Monitor D_t_ Darn = , :.t , ? o _" ! : I...,. ..... ." :" ' -. 'i" :' _I " I ' . _i.':'-," - :RECEIVED TIME Exygeri'Research. OCT. 9. 4:I3PM FRIHT TIrE OCT. 9. '.P_ 3 o.f_9 4:I4PM Page 45 of 111  Exygen Study No.: 023-082 i i i t ! ] -- -_ "1-" i;_iii '"'=_ _I i 5_.;,f :: i ' ....., j _._,_ !<_,I ! i .... i ..... Study Plan: ExP-023-082 Exygen Study No.: 013-082 TITLE PAGE ............................................................................................................................................... 1 DISTRIBUTION .......................................................................2................}........... i? STUDY PLAN APPROVAL ......................................;........................3................_...... TABLE OF CONTENTS .................................................................4................I., ......... INTRODUCTION ..................................................................................................................................... S }" o_a_cTrw....................................................s................... TESTING FACILITY ...................................................................8........................... STUDY DIRECTOR ..............................._................................................................8.......................... SPONSOR ............................................................................................................................................... 8 SPONSOR STUDY MONITOR ............................................................................................................ 9 t, PROPOSED EXPERIMENTAL START AND TERMINATION DATES .................................................... 9 COMMUNICATIONS ..................................................................9.................' .......... IDENTIFICATION AND IUSTIPICATION OF THE TF_Sr SYSTEM .............................9.......................... SAMPLE PROCUREMENt, RECEIFF AND RETENTION .....................................9.......................... SAMPLE IDENTIFICATION ........................................................................................................................ i0 ANALYTICAL PROCEDURE SUMMARY ..................................................1.0......................... MEI'HODS FOR CONTROL OF BIAS .....................................................1.2.......................... STATLSTICAL METHODS ...............................................................1.2.......................... _ STA_ .....................................................................1.2................, ......... REPORT ............................................................................................................................................... 12 SAFETYANDHEALTH............................................._.3...........i...... AblENDblENTS T0 STUDY PLAN ...................................................................................... .. ..........1.4...... DATA RECORD KEEPING ...................................................................................................................... 14 QUALITY ASSURA.NCH ...................................................................................................................... 15 RETENTION OF DATA AND ARCIHVINO .............................................................................................. 15 APPENDIX I ................................................................................................................................................... 16 "t i b ! ,.... Exygen Research. " Page 4 of 59 Page 46 of 111 Exygen Study No.: 023-082 '/' :!_ : A _ _,,_:,I !:"_ --.::_ ; : ' ii , " StudyPlan:ExP-023-082 Ex'ygenStudyNo.: 023-082 The purpose of this study is to perform analysis for perfluorooctane sulfonatc (PFOS), pcrfluorohexanoic acid (C6), peffluoroheptanoic acid (C7), pcnt_decafluorooctanoic acid (C8), hcptadccafluorononanoic acid (C9), nonadecafluorodecanoic acid (C10), perfluoroundecanoic acid (Cll), pcrfluorododccanoic acid (C12), tetrahydroperfluorooctane sulfonatc (THPFOS), and tetrahydroperfluorodcscualnfeonat(cTHPFDS) in pooled human serumand plasmaand monkey sereusingE,xygenmethodExM-023071 entitled '_Method of Analysis for the Determination of Pcrfluorohexanesulfon(aPtFeHS), Perfluorooctanesulfon(aPtFeOS) and PentadecafluorooctaAncoiide(PFOA) inRatLiver,Serum andUrine." The studywillbe auditedforcompliancewith OECD Principleosf Good LaboratoryPractice(asrevisedin 1997),F._NV/MC/CHEM(98)I7by the QualitAyssuranceUnitofExygenResearch. ...... . _i ' _i_:] : ,_ The test items are perfluorooctane sulfonatc (PFOS), perfluomhexanoie acid (C6), perfluoroheptanoic acid (C7), pentadeeafluorooctanoic acid (C8), heptadecafluorononanoie acid (C9), nonadecafluorodecanoic acid (C10), perfluoroundccanoic acid (Cll), perfluorododecanoie acid (C12), tetrahydroperfluorooctane sulfonate ffHPFOS), and tctrahydropcrfluorodcscualnfeonatCcIT-IPFDS)A.lltesittemswere received from the Sponsor. I, Name: PFOS Chemical Name: Perfluorooetanesulfonate i Molecular Weight: 499, as shown i F F F F ,. ] FF -J F. J F. [ F . F _S03 I F F F F i t i Exygen Research. Page 5 of 59 Page 47 of 111 i _,,i ". . , i :_:,} ! ! _:i!_ ' -:,:_I ,l :::l I i, _ t%_b!t;bt '!_'_!_ , ' ! ;ii 1 ! Exygen Study No.: 023-082 " Name: C6 Chemical Name: Peffluorohexanoieacid MoleculaWreight:313,asshown F F0 '--T-Fi"FI"-o" FFF Study Plan: ExP-023-082 Exygen Study No.: 023-082 Name: C7 Chemical Name: Pcrfluorohcptanoie acid Molecular Weight: 363, as shown FFF0 ", F i' 0o FFF i I i Name: C8 Chemical Name: Pentadecafluorooctanoicacid Molecular Weight: 413, as shown FFF0 F (_'t'_'t'_'t" "O" FFFF Name: C9 "- ChemicalName:Heptadccafluomnonaancoiidc Molecular Weight: 463, as shown F F F F0 F 0" FFFF Exygen Research. " Page 6 0f59 " i Page 48 of 111  Exygen Study No.: 023-082 i::] ; _' "7! ! i "; -_ !:_l'?i_ i I I i i'i_i !I. , i': i i_,_ _'-!I ! I I ,..... _ _ : i .._.,... _ .l "_ i t/.J '. " Study Plan:ExP-023-082 Exygen Study No.: 023-082 Name: ClO ChemicalName: Nonadecafluorodecanoicacrid MolecularWeight: 513, as shown FFFF0 "0" / I- / t"/I- /1" / FFFFF Name: Cll ChemicalName: Perfluoroundecanoicacid MolecularWeight: 563, as shown FFFFF0 0" F F_ FFFFF Name:C12 ChemicalName: Pcrfluorododccanoicacid Molecular Weight: 613, as shown FFFFF0 F O" FFFFFF Name"TttPFOS ChemicaNlame:Tctrahydropexfluorosoucltfaonneam MoleculaWreight:427,asshown FFFH /F/F/F/H F S03" FFFH Exygen Research. Page 7 of 59 Page 49 of 111 _:'_ I I _ ' : : Exygen Study No.: 023-082 - Study Plan: ExP-023-082 Exygen Study No.: 023-082 Name: THPFDS ChemicalName: Tetrahydroperfluorodecanseulfonate Molecular Weight: 527, as shown ,r,=,=,=,.F'FF F . ! F_503 I FFFFH ' .i The purpose of this study is to perform analysis on four different lots of ., pooled human serum, four different lots of pooled human plasma, and three lots of pooled monkey serum for the target fluoroeompounds using the . analytical method, "Method of Analysis for the Determination of i Perfluorohexanesulfonate (PFHS), Perfluomoctanesulfonate (PFOS) and I Pentadecafluorooctaaoic Acid (PFOA) inRat Liver, Serumand Urine." r. i !!__-'_il I __ : t :-_ _ E30x5y8geRneRseeasrecahrcDhrive State College, PA 16801 Phone: (814) 272-1039 Emily Decker Scientist ExygenResearch Phone: (814) 272-1039 emily.decker@exygen.eom 3M EnvironmentalLaboratory Building 2-3E-09 St. Paul, biN 55133-3331 Phone: (651) 778-6565 Exygen Research. 1. _o_8,o.:59 [ Page 50 of 111  Exygen Study No.: 023-082 /i 4;:'. ' i I i i I i : ,| .... i : !i:_ I I iJ3 1! _-l i i 1 I - StudyPhn:ExP-023 082 Exygcn Study No.: 023-082 WilliamReagen Building2-3E-09 StPaul, MN 55133-3331 Phone: (651) 778-6565 wkreagcn@mmm.com _ ,o _ It is proposed that the analytical portion of this study be conducted from October 14 to October 21, 2002. The actual experimental start and terminationdates will be included in the final report. i_?i_'._ :' _='" AllcommunicatiobnestweentheTestinFgacilitmye,thoddeveloperasn,dthe Sponsor wStiulldybeMdoirneictoter.d tChroomumghunthiceatSiotundsywDililrebcetofrul(loyrddoecsuigmneantete)dabnyd the StudyDirector. :_:, i 'i i ..... i.i.i.i.. _ Pooled human sera and plasma and monkey sera are used as the test systems i inthisstudyT.he matricewsillbeprovidebdythe sponsorT.hematricewsill bereprcstcant,ivoefthaftorwhichthiasnalytimceatlhodwasdesigned. ! Pooledhumanserumsampleswerepurchasebdy thesponsofrromSigmaAldrichM,ilwaukcsW,I,LampireBiologicLaalboratoriPeisp,ersvilPlAe,, BBiioorleosgoiucraTclTecsem,cchcnuollaCo,Ag.yI,nPco.o,FloerdmtoLnakuedyesredraulmeFs,La,mpalnedwseGroelpduerncWheasst ed bythesponsofrromLampireBiologicLaalboratoriPeisp,ersviPUAe.,Pooled human plasma samples were purchased by th_ sponsor from Lampire Page 9 of 59 Exygen Research. Page 51 of 111 - Exygen Study No.: 023-082 '! _, ' _ ! :! ; _:: '*_ '_ , ' :_'I i , StudyPlan:ExP-023-082 ExygcnStudyNo.: 023-082 Biological Laboratories, Pipcrsville, PA, Bioresouree Technology,/no., Fort Lauderdale, FL, Golden West Biologieals, Temecula, CA, and Innovative Research, Inc. Southield, NIL In addition, blank matrix consisting of pooled human plasma collected in rural China was provided by the sponsor. The samples will be used as received. The receipt and processing of the matrices will be documented in the final report and raw data associated with the study. The samples will be stored frozen at < -10*C. _.r__*.L?__ _ - _ ,: _a___L,Z_ I Prior to analysis, each sample will be assigned a laboratory sample reference number. The reference number will be unique and will distinguish each laboratory sample that is processed throughout the analytical procedure. Chromatographic data will be identified by the laboratory sample reference number, Samplestoragceonditionasnd locationwsillbe documentedthroughouthe validation. _ ReferenceE:xygen Method Ex.M-023-071"Method of Analysisfor the ij DeterminationfPerfluorohexanesulfo(nPaFtHeS),Perfluorooctanesulfonate (PFOS) and PentadecafluorooctaAncoiidc(PFOA) inRatLiver,Serum and Urine."A copyofthemethodcanbe foundinAppendixL The methodwill be modified as follows: Method Summary L Serum a. Measure 2 mL of serum sample into a 15 mL disposable centrifuge tube and fortify, if appropriate. b. Add 5 mid of ACN and shake for -20 minutes on a wrist action shaker. c. Centrifuge tubes at -3000 rpm for - 5 minutes. Carefully decant supernatant into a 50 mL disposable centrifuge tube and add 35 mL of i water. _ d. Load the sample onto a conditioned SPE column. Discard the eluate. :_.; Any analyte residues will be trapped on the SPE column at this point. e. Flute with 1 mL of methanol. Collect 1 mL of elute into a graduated !_t,ii f. A1n5amlyLzecesnatmripfulegse utsuibneg. electrospray LC/MS/MS. ,., If.CalibratioSntandards Exygen Research. Page 10 0f59 Page 52 of 111 i I ;':: ! :!i_i ....'. :' : --r ..,.. _ _!_ _ !-_I i Exygen Study No.: 023-082 _ Study Plan: ExP-023-082 Exygen Study No.: 023-082 In addition to the calibration standards described in Section 3.5.3, a set of calibration standards will be processed through the extraction procedure identical to the samples, using bovine serum and also a set using human plasma. The fortification of the standards before extraction is done according to the following table: Cone. Of Mixed Fortification Solution Fortification Volume of Volume (IXL) Control Sample (mL) Cone. of Extracted Calibration Standard (n_/mL) 1 400 10 100 10 200 10 300 10 400 100 50 100 100 (nfmL) 2.0 0.2 2.0 0.5 2.0 1.0 2.0 1.5 2.0 2.0 2.0 2.5 2.0 5.0 _!_ _.3 4 _ i; ' l i._..i.i.._ : ! B Trehsepontessetinagnd fathceilictyoncweniltlrateiostnabloifshantahleytereilnatioorndsehripto absestewsseetnhe tlhineeairnitsytruomf etnhte system. A standard curve should be constructed with at least five standards. matrixcontroslamples.Thisshouldbe accomplishebdy analysiosfa control The testinfgacilisthyouldalsoveriftyheendogenouslevelosfanalytienthe sampleforeachmatrixand examinatioonf theregionof analytreetention. The potentiaelxistsforinterferenfcreom fluorochemicailnstroducefdrom I dtiaertagryetamnaatelryiatlesa.nTdheothceor mepxooguenndosuswislolubrceesm. adSeamipnletsosaroelfuotrtiifoienadss with per tthhee method, and added to the matrices via a mieropipette. Fortified samples will be processed through the described procedures to ensure method accuracy and to cheek for bias. Recoveries should be between 70% and 130% of the fortified levels. The sponsor may accept occasional recoveries outside of this range. The relative standard deviation (RSD) for each fortification level as well as the overall RSD, should be less than or equal to 20%. Aranwy dmatoad.ifiMcaotdioifniscatitoonsthe daenemalyedticaslignmifeitchaondt bwyillthbeeSpdooncsuomr enotredStuidny thDeirsetcutdoyr will necessitate revision of the method. ExygenResearch. Page ll of 59 Page 53 of 111 --1 :' .. !;_:ii i ,,i i:'i?J ::*_ ';.I J ii_i_ ii _-J Exygen Study No.: 023-082 ". StudyPlan:ExP-023-082 ExygenStudyNo.: 023-082 Control of bias will be addressed by taking representative sub-samples from a homogeneous mixture of each matrix for untreated control samples, and by analyzing at least two levels of fortifications. _ " Statistics will be limited to those specified in the subject method and to the calculatioofnaveragerecoverieass,applicable. All aspects of this study shall be performed and reported in compliance with OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. The final report or data package (supplied to the Sponsor) shall contain a statement that the study was conducted in compliance with current and applicable GLP standards and will outline any deviations in thestudyfrom thosestandardsT.hisstatemenwtillbe signedby theStudy Director and Sponsor. "i ' I 1 '"" ] . __ 1 A final report will be prepared by the study director or their designee at the conclusion of the study. The report will include, but will not be limited to, the following: The name and address of the Study Director, Sponsor Monitor and of the testinfgacility. A statement of GLP compliance (any related documentation, such as chain-of-custody records, must be in the study records). The signed and dated statement by the Exygen Research Quality Assurance Unit regarding dates of study inspections and dates findings were reported to the Study Director and Management. Exygen Research. Page12 0f59 Page 54 of 111 Exygen Study No.: 023-082 i!__:_ :. (" _ "-,.. q ;, 4 . i J i i ! i "I I I ', ' : _::._ ,, "_ "- StudyPlan:ExP-023-082 ExygcnStudyNo.:023-082 A description of the exact analytical conditions employed in the study. If the subject method was followed exactly, it is necessary to include only a copy oftheanalyticmaelthod. Any modificatiotnosthismethod willbe incorporatiendtothereport.Ifthemethodisphoto-reducetdh,eproject numberandpagenumbermustbe includeodn eachpage. Any stepsconsiderecdriticail.,es.tepswherelittvlaeriatioinsallowable ordirectiomnusstbe followepdrecisely. The number of worker-hours or calendar days required to complete one set of samples. All results from all sets analyze& Identify all control and fortified saanmd pulnesiq, ueaniddeinntitfhiceatdioatna ntaubmlebeirncbluydseamsapmleplseet.number, fortification level, Representative chromatograms for each analytc in each matrix, including chromatograms of a standard and a control sample, and a chromatogram at a fortification level. The location of the analytc peaks will be clearly identified in all chromatograms. * Allcircumstancetshatmay haveaffectetdhequalitoyr integriotfy the datawillbe documentedinthereport. LAodcdaittioinosnwsohrecroerrreacwtiodnastaatondthteheffinianl arrleeppoortrtasmhatlol bbee ainrcthheivfeo&rm of an amendment by the Study Director. The amendment shall clearly identify tahlatt epraartt ioofnTsth.heereapmoernt dmtheant tiswibleilnbge aslitgenreeddaanndd tdhaeterdebaysotnhsefoSr ttuhdey Director and the Sponsor Study Monitor. i 4 _. !_'_! /ii "' Laboratory personnel will practice good sanitation and health habits. Any health condition of laboratory personnel that may be considered to adverselayffectthestudywillbe reportetdotheStudyDirector. Any injury to laboratory personnel occurring during the conduct of this study will be reported to the study director. Exygen Research. Page13 of 59 Page 55 of 111 J ii ,,*i_:i' ' 'l ! ' :k. i_it _i_:i ':']: =:._i1. .... Exygen Study No.: 023-082 - StudyPlan:ExP-023.082 ExygenStudyNo.:023-082 Every reasonable precaution shall be taken to prevent inadvertent exposure of personnel and the environment to the test or reference substance(s). All significant changes to the study plan outlined here will be expressed in writing, signed and dated by the Study Director and Sponsor Study Monitor. Amendments usually will be issued prior to initiation of study plan change. However,when a changeisrequirewdithoutsufficietnitmefortheissueof a writteanmendment,thatchangemay be effectevderballwyithsupporting documentatiosnignedand datedby theStudyDirectorand followedwitha writteanmendmentassoonaspossibleI.nthiscase,theeffectidvaeteofthe writteanmendmentwillbe thedateofthedocumentedchange.Copiesofthe signedamendmentswillbe appendedtoalldistributsetdudyplancopiesT.he originaalmendment willbe maintainewdiththeoriginasltudyplan. Any deviatiofnrsomthestudyplanorfromtheanalyticmaelthodasprovidedwill be documentedandreportepdromptlytotheSponsorStudyMonitor. _',i_-_ " '_:'_'_r_i'_ : 1 i : .....J J Recordstobe maintaineidncludtehe followin(gasappropriate): Sampletrackinsgheet(s) anpdrep atolof ntand(dstuofokr,*i oacdaolinb, .on) Samplereceiprtecordss,toragheistorya,ndchainsofcustody Description ofany modifications to the method Instrument run sheets, bench-sheets or logs Analytical data tables All chromatographic and instrumental conditions Sample extraction and analysis dates A complete listing of study personnel, signatures and initials Chronologicparlesentation ofallstudycorrespondence Any other documentation necessary for the reconstruction of the study Chromatograms- All chxomatograms will contain the following: Sample identification, date, Exygen study number, arrow or other indication of the area of interest, and injection number corresponding to the run. Additionally, fortifications will include the amount of analyte added and the sample number of the sample that was fortified. Page 140f59 Exygen Research. Page 56 of 111 ii .... : LJi ; !i ! i Exygen Study No.: 023-082 StudyPlan:ExP-023-082 ExygenStudyNo.: 023-082 Analyticalstandardchromatogramswill additionalliynclude the concentrati(oen.g.l,.tg/mL). As partofthedocumentatiotnhefollowinsgheetswillbeincludeidneach L analyticsaelt:a runsheetlistintghesamplestobe runin the set,and an !! instrument conditions sheet describing the instrument type and operating conditions. [ g, i The QA Unit of Exygen Research will inspect the study at intervals adequate to assure compliance with OLP's, and will report thefindings of audits to the StudyDirectoErx,ygen Management,andtheSponsorStudyMonitor. i I All hard copy raw data, including, but not limited to, the original chromatogramsw,orksheetsc,orrespondencaen,d resultsshallbe included withthedatapackagesubmittetdotheSponsor.Thesewillbe archivewdith theoriginasltudyplan,amendments,finarleporta,ndallpertinenitnformation fromtheSponsor. The testing facility shall keep all electronic raw data and any instrument, equipment, and storage logs for the lifetime of the product and shall obtain permission of the sponsor before discarding. .... ! ! ! Exygen Research. i. Page 15 of 59 Page 57 of 111 r. ! ! =-'7 ! i i ] :,!_! /.i :! _I ' i i ,I i : g Exygen Study No.: 023-082 Study PI_: ExP-023-O82 Exygen Study No.: 023-082 1-: I, [ i E_ APPENDIX I METHOD "Method of Analysis for the Determination of Perfluorohexanesulfonate (PFHS), Perfluorooctanesulfonate (PFOS) and Pentadecafluorooctanoic Acid (PFOA) in Rat Liver, Serum and Urine " f ;') , _1 Exygen Research. Page 16 of 59 Page 58 of 111 .t Exygen Study No.: 023-082 ! :. :--_r .; !!!;:i_'l i ., I.+] _,j '. '_ ) ' i 1 1 : i:i i I ,, i + '+'l 't o '} I } " Study Plan: ExP-023-082 Exygen Study No.: 023-082 Method of Analysis for the Determinatioonf Perfluorohexane.sulfonate (PF_S). Pcrfluorooctanesulfonam (PFO$) and Pcntadccafluorocctanoic Acid (PFOA) in Rat Uv_,s_un,_aU,in_ AUTHORS _AT_,Ss_r, July 30,2002 SPONSOR 3.1VMI edical Department Corporate Toxicology pER_ORM_G LA]3ORATQ._y Exygea Resemrch St3a0t5e8CRolelesgeae,rcPhAD1ri6v8e01 ! i ] I. :I ' i '. } , I,! +; i. TOTAL NUMBER OF PAGES 43 i i . i : =yg_x--enResearch. Page 17 of 59 Page 59 of 111 . Exygen Study No.: 023-082 ," :J r ._h L_ i :'_ :] i .- s I ! , : I -'=['" o " "StudyPlanE: xP-0_-082 Exy+SntudyNo.:1_23-0112 I ]hy_ Mc_od No:_1-l_3-071 . MANAGEMENTAPPROVAL : i t t <I ,:,i F.x_l_ li_._azch : ! A_hl 'i _i i t ,,_._t_-eL....- .t I Px-_id_t t : P.xyg_ II_arch t L,;I [ _,_, j I L2:,i : John L. Butenhoff S1mm_ Rcp_atafivn 3M blr.dical Depa.m_nt Dam Cotlxnatn Toxicology )7: !i c, " _-,J _xi%'_t _,smiir,.h Exygen Research. ; ! ! ,i .I | I l ; I Pag_2 of 43 ": i ," Page 18 0.#'59 Page 60 of 111 i i I .. _-. '::il ?i!:_ , i .... ; ,, : _i , _ : ' "_ ! i 1I { I I: i' .... !, i ; l I I :i I . Exygen Study No.: 023-082 $tudyPlan:ExP-023-082 Exygen StudyNo.:023-082 Kr/Ip=let_xlNo:_-01_071 TABLE OF CONTENTS : TITLE ............. ;.............. MANAGEMENT APPROVAL ........................... TABLE OF CONTEN_ LIST OF TABLP..S mz oer_o'ta._.......... I. _.TMlvlARY ....... 1 i 2 _[ ..... 3 _ ,-. 4 /i s !. 7 3. CI-II_VlZCAI_ AND SUFPr_-m_....................... 3.1. 1_ ..... 9 , 9 3.3. EqomM_rr ANDS_'L_. 3.4. SOLUnONS..... 3.5. Ig._,_.a_oN (_STANDARD SOL_/noNs ......... .--9 : 10 I 10 / 333.. PorlLfica_on Solutiom_.--... 333J1,3. SCtaloibcrkastioolnntSitoa.n.n.d..a..r..d.s _.__ 4. METHOD ........................... ........... 11 11 I 12 I 4.1. PLOW D_Ga_a ...................... 4.2. SA_an._Peoeessn_o...... 4.3. Ba_n'CH_-Jr ire ........... 12 t 12 13 4.4. $AMI_E _ON ...........................................1.3.. 4.4.1. Liver _ttt'acliom ....... 4.4.2. Sexum and Ufim_ Extraction .... _............ 13 13 4.41 SPE C_hmm Conditioning ._14 : 4.5.1.LDJMS/MS Sy_tmnandOpct-atingCondition.s.......... 14 i 4.5._=._ ........................................................... 4.5.2. Cah'bralion C_-ve _ 15 I 4.5__S.mpm._l_,.... 1, '! 4.6. _,_(::I_ _ .4.7, PI_n0RMANGS _ ..........................................1.7 ..... ._------, .......... 17 5. CALCULATIONS .......................................... 4.8. Tnvmgr_ma Kin. ANALY_m ............ 18 -- ...18 i I !. i L _, [_ L_ , ' ': I_ ! i: I' }; - ! ; iI " ! ExygenRe_,',ch Pa_ 3 of43 ,. Exygen Research. Page 19o/'59 Page 61 of 111 Exygen Study i'4o.: 023-082 " Study Plan: ExP-023-082 Exygen Study No.: 023-082 i! i -":- i _7 _ I, t : -=J : I I LIST OFTABLES Table 1. Recovery Summaryof PH_ in Rat/.2vet andSerum..................... 20 TableI RecoverSyumm_ ofPPOS i.RatLiverS,erumandUxine............2.1. i Tab_3.RecovezSyummaryofPFOA inRatLiv=,SerumandU_ne........ 22 I !' i :! I t ! r k i ' i | , ! ',1 I _: 1 ! ?'J i ' ] .F_aya__-_'_ Exygen Research. ! . i I ! : [ i Paio4of43 " , i: .i Page 20 of 59 Page 62 of 111 t .i :! _-+_ _I + :_Y:_:/i ::-_ ::!i L_+J !--] _J _ i i_ _.+:_ i | I .,. i i :_ :i i :. [ :: .._... i i I _: _' ] i'+ i Exygen Study No.: 023-082 " Study Plan:ExP-0"23-082 Exygen Study No.: 023-082 _SeaU_,d Nm_m_Tt T+TRrOF FIG_/RF__ F/gumI. C_R;a_.afCi m.v_eal-a_'B._.................. ,..__ I F/_z C_o.c_ for_os mr 3. Cah'b_onam, for_oA .................. Hgum4. Rce_tixmengenrt_astiCvle._..,.-.-.m..g..ramofa0.1ng/mLStanda_t .....2. 4 , ..__ l P 26 I F/grin5s. R_x_mtatt_sL_amatograomfa0.Ing/mLStandarCdontaininI_g05..26 t F/b,mm6. _tafiveChromatogramofa0.1 ng/mLStandardConta/n/ngFFOA..27 F/gum7. RepresentatCihvreomatograomfa0.5ng/mLStandarCdontaini_ng ..2.7 F/gum8. Repre_-ntatClhvrec_At_gmmofa0.5ng/mLStandarCdontainiPnFgOS...28 F/gu_ 9. R_pmsenmfiw Chromatogr_mof a 0.5 ng/mL StandardContaining PFOA.28 F/gum10.RepmaentaflCvhemmAtogramofa S.Ong/mLStandarCdonta/n/PnFgY_..29 i F/gumII.RepreaentatCihvmematogramofa 5.0 ng/mLStandarCdonm/ninPgFOS...29 I F/g_iz R_-_ _m_ of.59. .g_LS_d_ Co._.g _FoA3.0 I F/gum 13. RPe]bp'Bre_sentatCihvmematogramof a Reagent Blank Sample Analyzed fo.r.... 30 I] F/gum14. Represenra_C/hvmematogramofa Reagent Blank S_aple Analyzed for } FFOS .... 31 F/gum 15. RP_I:O.A_ntativCchmmatogntmof a Reagent Blank SampleAnalyzed for --.31 F/gum 16.Repwaentalive ChmmatogramofaControlLiveSrample Analyzed for _ .................................................... 32 F/gum 17. Repw._'ntative Chmmatogramof a Control LiverSample Amdyzed feeFFO&, ._..32. F/gum 18. Rf.(expPr]eFaOeAn...t..z...f.C.i.h.v..r.e.o...m.._..t..._..g...r..a.o.m.f..a...C..o...n..t...r..o.L.l..i..v...e..Sr..a...m..p..leAnalyzed 33 i: F/gum.19Rf.oerp_mmnt.a..l../vCehmm_t_gramoi; CantmlSen_ Sample Analyzed .33 !It F/gum20. Rgpre.umtativeChromato_tm of aControSlerum SampleAnalyzed I for PFOS. ---34 F/gum 21.Reixeumtative Clammatogramof a ControlSerum SampleAnalyzed for PFOK.......................................................... 34 F/gum 22. ReprmentativeC_ogrm_ of a ControUldne SampleAnalyzed Figure 23. fRoerpPrFesOeSnt..a..t.i.v..e..C..htomltogram of a ControlUrine SampleAnalyzed ...... 35 :i for PFOA .................. - ...................................... 35 I F/gum 24. ReIXe_. tafive Chmmamgramof a ControlLiver Sample Fortified t | at 10 ng/g with PFHS........................................... 36 I _tyg_aRmearda Page$ of43 ": t i, !i _i PI I !, [, I, Exygen Research. Page 21 of 59 Page 63 of 111 Exygen Study No.: 023-082 ; !_i'_ :_t . i: :" i i .t ":-" _::_';; i_i_ii_] : i !_:'_:_i I i,i,'i./ I , i ! ! i _! __!I_;' _! :,' i I! "ii''i ! " " Study Plan:ExP,-023-082 Exygcn S.tudyNo.: 023-082 E--'_cnMethodNo:P.z_.(I23-071 "s LISTOYFIGURF_(continued) f Pigum 25. RcprcaentativeChromatogrmxof a ControlLiver SampleFortified at 10ng/g with PI_S .... _36 Pigun:26. P.cpn:scntat/ve'Ch.mmatogrzmof a ControlLiar SampleFortified i at 10 ng/g with PFOA_, _37 I1 Figure2"7.RcprcscntaflvoChrom_ogramof a ControlLira SampleFortified ! g at50 nWg with Ptq_S ..................................... 37 Hgu_ 28. l_.semu.ivo C_omatogramof a ControlLiver SampleFortified ! at50 ng/g with l_OS ............. .38 " i Rgum 29. l_'presentattveChromatognm of a ControlLlver SampleForafied . at50 ng/g with FFOA.- ...... 38 Rgurc 30. Rcpresentaflve Chmmatogrunof a ControlScram SampleFortified ! at I0 ng/mLwith PtqtS....... ._.39 Hgum 31. Representative_grtm of a ControlSe_.unSamplePon_fied : t ,_ i at 10ng/mL with _ .......................... _ .................... 39 ;_ } l_&_n_3?. RepresentativeCh_nmogr_n of a ControlSmun SampleFortified ,_ -, x0.g/_. with_A ......................................... 4o _ i Iqgu_ 33. Ratc5p0rne.gs/o_nLt_twiviCtehh_romat.o..g..r..a.m..of a C_trol SczumSampleFortified _40 i Rgun: 34. Represmatal_vCe hromatogramof a ControlScram_ample l%rtifiod at50.ng/mL with FfOS .......................................... 41 at50ng/mL with PPOA ..... ,.......4.1. Rgum 36. RepresentativeChromatogramof a ControlUrine SampleFortified Figure37. aRattoIIp00rcnnsgEc//nmmtaLLtwwiviietCthhhPPrFFoOOmASa.t.og.r.a.m.o.f a ControlUrine SamplePot'lifted _._._42 .42 ': l_sum 38- Rate5p0rensg_/tmatLivweiCthhPrro-m.oa_tosramof a ControlU_m _amplc F_ified ,.43 l, Pigure 39. l_pmsentative Chmmatogramof a ControlUrineSamplePonified at50 nv_nL with PPOA...................................4.3......... : ,1 _ : I: , : _ '" ._/gm R_e_h Exygen Research. t _ . i Page6 of43 ": t Page 22 of 59 Page 64 of 111 o Exygen Study No.: 023-082 ii! il ,'3' it " Study Plan: EXP.023.082 Exygen Study No.: 023-082 Ji i _:',_ i:::!!! :l ![ _ _] i :. ' -_- : I. : : ..!..] :." i, [; [ , ' : i I i , I ' t, :LL'. : Exygea _d No:.ExM-02.3..071 1. SUMMARY report detailr a method of analy_ for t_dues of Pm_luomhexanesuifonate. (PFHS), Porfluorocctanesulfonate (PF'OS) and Pent___afluorooctanoio Acid (FFOA) in Rat Liver, Serum and Urine. Resi___,_ of I_HS, PFOS sad PFOA are extracted from each malxix with acetoni_8. The acetonit:file extract is _,.la_ to water and loaded onto a conditioned C18 solid phase extraction (SPE) cartridge. Analyte rv.sidues are eluted with 3 mL of methanol Quantification of PIfl_, PFOS and PFOA is accomplished by liquid chromatography/tandem mass spectron_tr7 (LC/MS/MS) malysis using multiple reaction monitoring _lt.M). The proposed limit of quantitatton (LOQ; the lowest fortification specified by ti_ : method which gives adequate recovery according to EPA. guidelines) for this i i" method is I0 ngig (paxts-per-billion) each for PI_IS, PFOS and PFOA. ] I I The theoretical limit of detection (I._D) will be based on the signal to noise ratio [ and will be at least greater than 3 times the level of noise, based on the instnmaontation system used. For all analytes, tim lowest analytical standard [, i' . corresponds to 0.1 ng/roL, i This method was developed using rat liver, sc_un and urine. Typical percent recoveries + standard deviations (at 10 and 50 ng/g) are shown below: | FL'eovttWelw(n.a_t/__)m PPI_ RcLiov,e_,ry inRat Lle_v'tellfi(cnaef.i/omnL) PFI_ RSn_c'ouvmezytnRat : 10 115% 9.9%(u,,,3) 10 1011%-I-4.7% (n=3) _oI,_w..l _ lnRaRt_IoA,vnw=y e.,Laeev_el, reols_tet=Se.r,u_my,, reoRsa_tUrine _ ' .I .t 50 88_t l._t (ha3) aO 120_ 2.1%(n,,3) 79'_ ta._ (u=3) ; Foa_ifl_ou PFiOnRk.aRt eLcIov.emy l_ttLioenvel PI_3RA_R,e_cnomve_r,/In PFOlA_tRt Ue.edonveeryln r (*e/_) (.e_L) I 10 98% 3.1%(n,_3)! 10 117_ 1.5%(n,,_t) 89% 2.5%(n-3) 30 94_ :1:2.3% (n=3) 50 111_, 4.0% (n-3) 87% 2.1% (n_3) Rcprmentafl',,,e cah'bration curves are shown in Figures 1-3. Representative [ chroma_ograms are shown in Figarm 4 to 39. ]_x_en Research Page 7of 43 ! Exygen Research. o, " Page 23 of 59 ' Page 65 of 111 Exygen Study No.: 023-082 ii ! i ! ,/_ ,fill:i! :;LiL...!i!:'. i : .,_"a f ,_ i "1. :'r _ I i i StudyPlan: ExP-023-082 Exygcn Study No.: 023-082 2. EXI_ERIMENThLCOM1)OLrblDS T/anstruclnt_ for PF/_, Eq=O$andPFOA arcgivcn below. ChcmicalName = Molecularwdght = Pcdluorohexmesulfonat= 399,m shovm 40_" _ is st_pl;edasthepotassiumsalt (C,P_SO.j'_, molecularwclght: 438 PFOS ChemicalNan_ Molecularwcight . = Pcrfluomoctanesuffonam = 499, as shown FF F FF F F O" PFO$ is supplicdas timpotassium salt (C_tvSO3"Km_ olecularweight = 538 ._o_ . "ChemicalName = l_ccaflucmocUmoic Acid _ wd_t -: ' 413, mshown FF I" O" P I I: ' i" t 1 ! t I '; , : " "i ! Exygen Research. i Page 24 of 59 Page 66 of 111 Exygen Study No.: 023-082 - StudyPlan: EXP.023.082 Exygen Study No.: 023-082 " '_i :" "-=-. ..,.-., j i:!!_! I LIJ !,4]i Ii _ _,:J I _t_,] I I ' II j '_,! ; ' i, I ! t : '_ - Kv/lleaMethedNe:Kv.M-t]_14171 : 3. O:mMICALS AND SUPPLIES i. 3.1. CnwmraT_ Chemical MethanctSde,Oa) /_mmoninm Acetate Water Acctonitrlln Grade B_.t2 l_agent TypeI HPLC Source mvtSdeaee JT Baker Exygen EM Science Catalo_ No. MX0475-1 0596-01 NA AX0145-1 Type I water= electrical resistivity,minimumof 16.67 M.O/cmat 23 C, from a Labconco WatezpmTM wodmtafloL 3.2._,_,_ l L Standard TCR Number Purlt7 (%) Source [ P(_uHrfSlu)orohexane.sulfonate Pedluomoctanesulfonate SE-036 SD-018 8949.3.969satrlal igsohmtcehrasi,n 3M 86.9 3M f i I i Pcntadccafluorooctanoic Lot No: 96 Akkich : , XddfPFOA) OS316DO Ch_-, !. 3.3. Equn'MmcrANDSmu,L_s Equipment Supplier Balance. analytical (display atleast 0.C001.g) 12.%mLLDFE nacrow mouth bottles Merrier Nalgene Disposable glass micropipets (50-100 & 100-200 ILL) Dr_mond (VWR) Tiasumi_r Tekmar Wristaction shaker Sorvall RC 5C plus Cenlxifuge Bun-eliScientific i :I Dupe,at ,- 50 mL disposablepolypmpyleue centrifugetubes VWR [ ' "I$ mL disposablepolypmpyleae _ge tubes VWR [ ' V'miprepvacuummanifold Supelco / r 036795 : S2e-mp PLadkeaVracEI6PcLcC(v1i8a)l tKCi1t8(ccaat#rtr5i1d8g1e-s3(p4a0r0t)# WAT HewWlctat-tPchackard ! ' ClassA pipets andvolumetricflasks Standardlab equipment(graduatedcyl_._, various suppliers ,_adaussuppliers I: t ; , dSltsapnods-aabloknteudbreospe-ticn.g)u. ardcartridgeholder(part Keystone Scientific #844017-40O) Hypercarbdrop-inguardcolumn(4 ram) (part# Keystone Scientific i 844017-400) ,t HPLCPump (LC-10AD) Shimadzu . I.C/MS/MS andHPLCs_tems As describedin section 4.5. .ExygeRnmem'ch Page9 of43 I Page 25 of 59 Exygen Research. Page 67 of 111 Exygen Study No.: 023-082 ';, "- StudyPlan: ExP-023-082 .Exygen Study No.: 023-082 ,.! iI , .... ,i-"_m-__ .... +::,:_ : '+i i , 'i _ ! I ' J !i_i(! _._+ _,_,_ '" _! i_ i.:i.l ! L,t) ,::_+ i_:'iS; _ tr, 1. In ordnrto avoid cont_!=.tlon, the use of disposshln lshware (centaine_, tubes, pipc_ etc.) is highly r_o_ i 2. Teflon orTeflon-Unedcontainersor equipmentshould not be used. 3. It may bnec___aryto check tho solvents (acetonitrile,methanol) for tho presenc_ of contaminants (especially I_OA) by LC/MS/IVISb_orn use. Certainlot numbershave be_nfound to be unsuitablofor use. t 4. Use disposable micropipcttes or pipettesto aliquot standard solutions i when pmpmlngstandardsand samples forextraction. 5. Equivalentmaterialsmaybo substitutedfor those specified in this method. : 3.4. SOL_XONS (1) 2 1_ ammonium _Ut_ tll WR_I" |$ pl'cpalP, d by weighing ' 1+ :: 0.L54 g of .mmonium acetat_aed dissolving in I.L of water, (2) Hypercarb filtered type I water is prepared by filtering type I water througha Hypercarbguardcolumn using a I-_LC pump at -2-3 mL/min. Baceefoturneiturislee,, twhennsh- th2e5 gmuLardofcatrytpreidIgewwaittehr,-t2h5en mbeYg.i.onf cHolPleLctCinggrathdee filteredtype I watereluatefor use in the extraction. Repeat _c wash after filtering-21. of water. t ' _ " Li , I. [ : Note: The _oremcntioned exmnp|c is pmvid_for _danc_. altca'nativevolumes may be preparedas long as the appropriateratios of the solvent to solute are maintained. 3.._. ]_ILEPA._I.TX0O NSqCA/TDASRODLUTIONS i 't ) !; . t,_na1_.]_Cticaa$li[tbarnadtiaorntS_,tanudaacrddsf-orTthhel'sce_pstnarnpdoasreds:sare preparedin methanoland are t: I used to calibratethe mspomo of thedetector used in the analysis. ._ + 2. LcoanbcoernatrtaotiroCynoscnotrrorel spSopnikdeinsg -to tTheheLseOQfoerntidfic5aXtio]LnsOQamandparmepuasreedd taot [ d_tnnninoanalyticalrecovery. Laboratorycontrol spikes arc preparedin i : oontrolmltTL_ .i" I 3. Malxix Spike_-These fortifications arc px_parcdby sp_cinginto the field : '" I samples at a known concentration. Matrix spikes are used to evaluate the effect of the sample matrix c_ anal_e.icraelcovery and are preparedat the i The analyst may vn.,_/theabsolute volumes of the standardsas long as the correct proportionsof solute to solvent are maintained. P .. .Exy_m_e,,_ o_ Page10of43 Exygen Research. Page 26 of 59 Page 68 of i 11 o Exygen Study No.: 023-082 i i : _,-_ ii,i!i _ 'i i :J ....i : _:i:,_, _ r_ i i ii 'i i . : l :i "',-, i ' i ' [ [II I ; , ' I _ : " Study Plan:EXP.23-082 , Exygen Study No.: 023-082 5d. Stoc.k_lutlon P_-pam individual stock solutiom at 100 lag/mL for PPHS, PFOS and PPOA by writhing out I0 nag of each analyticalstandard(correctedfor ; purity and if necessary, salt contenOand daqutoto 100 mL with methanol in separam 1O0-mLvolumetric flasks. Tan stock solutions On 125-mL I_v LDPE bottles) are to be stm_i in a refrigeratorat 2"C to 6"C and are stablefor a maximumperiodof one yearfrom the dateof preparation. [l JJ.2. Fortifumton $obaton_ a. Preparea mixed fortification standardat 1.0 p_'mL (11113n0g/mL) of : PPH3, PPOS madPPOAby adding 1.0 mL of each of the 100 pg/mL stock sohdom intoa IOOm/..volumetricflask:andbring upto volume ' with methanoL b. lh_-p_rn a mixed fortification standardat 0.I l_g/mL (1130ng/mL) of ; [ PPH3, PPOS and PFOA by diluting 10.0 mL of the 1.0 pg/mL mixrxl fot:tificationsolution to 100 mL with methanolin a volumetricflask, i Ex_ulc: one hundrai micro_tersof the 0.1 pg/mL sohtiou Ipikedinto 1 ! g of liver or I mL of sennn/mine is equivalentto a 10 ppb (10 ng/mL or ng/g_ fortification. , Storeall fortificationstandardsolutionsin a refrigerator(in 125-mLLDPE bottles) at 2_C to 6"C fora maximumpeflod of one year from the date of ". preparation. Note also that additionalconcentrationsmay be preparedif : n'e=_ary. , 3._J.C_r._on Standar_ preparedat 0.1, 0.7.,0-_, 1.0, 2.0 and 5.0 ng/mLin methanolvia dilutionof , , _the 0.1 I_mLcalmibixraedtiofonrsttaifnicdaatridonscsoolnuttaiionnin(sgePctPioHn33..PS.P2O.bS). and PFOA am" ' :i t @ The following is a typical example; additional concentrations may be I ! preparedu needed. Initial Cone. Volume Dilutetod FinaClone ng/mL) fmL) fmL) (n_rnL) 1130 5.0 I00 5.0 ', 100 lO 100 2.0 : I.o I.o I 5.0 I0.0 100 0.5 zo o.o " 1.0 10.0 100 0.1 ! The standardsmay be used for a period of one year On 125-mL LDPE bottles) whenstored rc_=atai (at 2"Cto6C). i I .F_tygeNn PageII of43 : [ ! Exygen Research. Page 27 o,/'59 Page 69 of 111 o Exygen Study No.: 023-082 t , " '. i. I I , Study Plan: ExP-023-082 Exygen Study No.: 0'23-082 + : i??! ii' _i i:i_/iii LI: --!:'_! i..,:! : ! i "J i t ,.. i : i t! i '! : i ' I 4. METHOD 4.1.Ft.owDrAGa,tM i The flow diagram of tho method Is given bdow, followed by a detailed | dcscciptionof each step. p MethodRow Dlaeram I Wdgh.1gofllwrorre.su1mmLofscnunorurine (fortify samples designatedas maUixspikes andlaborator7 controlspikes) ,I, Add 9mY.o.fwatertollve1r9omL ofwatertoserumandurineh.o,mogenize + R_.movoI mY.a.ndadd 5mL ofACN, shako +, _tl_ge Decant aupe.matanitnto 35 mL of wa_'r MadontocoJn,_tioncSdI_ J, _tStMS _aal_s 4.2. S_Pao_ "Per [iv= samples, plac_ fmzcmsamples in a food processor andhomogenize with dry lee. Thmaplm .samples in con_ and leave op_ in froz_ storage o's=might to sllow f_r C02 subIlm_t4ou. Seal and place tim samples in frozm storage be.low-lOOCuntil _ of extraction. No sample processing is needed for serum and urine sarapl_. Hewer=, frozcn serum and mine samplca must b_ allowed to eomplctdy thawto room tempe.returbneforeuse. 1 : [_ r I; i.! t: i t f[ ! { t i I 1 : "" I 1 | + Exygen Research. Page 28 of 59 Page 70 of 111  Exygen Study No.: 023-082 'i t _ ,. :.__. , : ":.3 ,. i i ] , i :! I ! _ i _ i ,_,_ ; _ i I i " i ! I i_ t I __-_ i : t " Study Plan: ExP-023-082 Exygen Study No.: 023-082 4.% "nA'rce(Scru_ P.wgeaMethodNo:P.xM-023-071 I,. A batch of samplesshould not containmorefilm 20 field samples. b. Each batch of samples analyzcd must include at lcaat one control (method blank using control match) and two matrix controls fordfled at t-.own concentrations(typically I0 and 50 ng/g for liver or ng/mL for serum and =ine)toredly_.edural _ve_y forthebat_ c. At least one field sample in each batch must tlso be separatelyfortified at a known concentration and carried tin'oughthe procedure to verify recovery. Additionalsamples in the batchmay also be fotified if desired. d. Allsamplerscquirdcuplicaitnejections. 4A. Sxt_t_Ex'r_crtoN 4.4.1. Lfwr _ra_n a. Weigh 1 8 of liver sample Into a 50 mL disposable centzhFugtcube and fortify, if appropriate. b. Add.waterto the samplefor a final volume of 9 mr- Captightly. c. Homogenize sampleusing a tissuemize_fo_-1 minute. d. Trander I m.L of thesample usingi disposable pipette into 15 mr.. dlzpcsabl= centrifuge tub,'. Add 3 mL of ACN and nh_k_ for -20 minutes on a wd_ action shaker. c. Ceutdfugc tubes at -3000 rpm for - 5 minutns. Ca_ful]y decant s_atant Water. into a 50 mL disposable centrifuge tube and add 35 mL of f, Load the sample onto a conditionedSPE column (for conditioningdetails. see section 4.4.3.). Discard the einate. Any analyt= residues will be g. tErlauptoI_wdiothn t2hme SLPoEfcmoleutmhannaotl thCisoplloeicntt.2. mL of elute into a graduated15 mL ccnt_fuge tube, h. Analyze samples usingelectmsprLaCy/MS/MS. : i, | [, i 1 , , i iI t | / i t 4.4.2. ,ferumand Urine Extmcaon I * a. Measure 1 mL of serum or urine sample into a _0 mL disposable centxL_getubeandfortify, if aI_propriate, t b. Add 19 mL ofwater tosample. Cap tightalnyd'vortfeoxr-I minute. , Thencontinuewith steps d-hin section4.4.1. . I ! ._._yge_n._trch Page13of43 "; i i Exygert Research. Page 29 of 59 "; Page 71 of 111 " Exygen Study No.: 023-082 , ..i , i i:;__! : _i:I:il i :::.i __ I ? ] :. i : :-% " , " Study Plan: ExP-023-082 Exygen Study No.: 023-082 ,. ]ExygeM= ethodN ExM-023-071 : b 4.4,3. SP[ Column Condftianln& I. I" Place the unconditionedSI_ colurrm_on the vacuum manifold. Conditionthe [ SPHcolumns by pasaing - 10 roT.of methanol throughthe column followed by - 5 mY..of water. The washes may be pulled through the SPE colmnn using vacuumat a flow rate of -1 drop/see ormay be allowedto pass through tlm columnunaided.Diacaxdall washes.Do not allowthecd,_ todry. _:._ __ _!_::_ _] !_:1 i':'j : ; i _ I [ .... { = : 4.5.QuAmrrrx_mN 4.3.1. LC/MS/MS _ and Opnmang C_ulh_.$ ', MassSpe_ Mi_ma_ quaumUlthn(Ma icmmass) [ hterfa,:= ElectmspmOyal,:_mms) [ Harvardinf_on pump (HxrvazdInslxuments)f,or tuning Compute. COMPAQProfessionalWockstationAP200 [ Software: W'mdows NT,Manlynx3.3 I I HPI..C: ttewlett Packard(FIP)Sed, ,es1100 J_eqmtPump HP VacutunDegasser , HP ColumnOven Note: A 4 x 10 mmhypexeazbdropin guard cartridgeIs attachedon-lineafter the purgevalve and before the sample injectorportto trapany maidue covt--;nanta thatmaybe in the mobile phaseand/orHPLCsystem. HPLC Column: GenesisC, (Jones Chromatography)2, .1 nun x 50 rm,n_4ix. Co]Lm_Tranpcxatm-3a5:=C Inj=ti_ Votume,:15_U _obile Phase (A): Mobile Phase (B): 2 mMAmmonium Acetatein Type] water Methanol T]rae _A 0.0 90 _B _'loRw.ate(rnY.Jmi_) 10 0.3 2.0 90 I0 0.3 5.0 I0 90 0.3 , 9.0 10 90 0.3 9._ 0 100 0.3 14.0 0 1130 0.3 t4_ 90 to 0.3 20.0 90 " 10 0.3 * i Iptemrfaoyrrbnae_n:eec..eCssoalurymtonsawdijtuhsdtitfhf=eteIaPtLdCimgerav.dsiieonntsi(ne.go.r2d.e1rxto3o0p)talmnd;.a, lsinosctroulummennts :[: '' E_ge._Re=eaxch Exygen Research. Pagn14of43 "; I, _ ! i F " Page 30 of 59 "' ! i ., Page 72 of 111 Exygen Study No.: 023-082 ! - Study Plan:ExP-(Y23-082 Exygen Study No.: 023-082 i - I -::. _' !ii ; i :I ! i : _ : ! _ _ _ .... ' _1 t I ,_ [ : i.:.I -_ : i i ( ', I ..... t _._ . r from differentmanufacturers(KeystoneBetasil Cm etc.) could be used,provided equivalentchromatographyis obtained. Ions monitored: Approximate t Analvte Mode TransitionMonitored Retention"rimo b PFOS Ne_gative 499--t 99 -8.8 rain PPOA Negative 413-_ 369 -8.6 rain I The retention _ may vary, on a day to day basis, dependingon the batch of mobilephaseeta Drifitnretentitoinmes(upto4-4 _) acc_3table withiann analy_carlun,U longu tlm_ coafinuetahrougthheentiraenallnhandthe _mdardaareincludeadtthebe_,,_,_g andend ofthemal_calnm Note: An alternative LC/MS/MS systemmy be used once dexmmstmtedto be equivalent. The mass spectromet_ is _ed foreachaual_ by infusing u - 1.0 pg/mL standardsolution (at 10 _ using m infusion pump) via a "T" intoa stream of mobile phase containing 50% methanol and 50% 2raM ammoniumacetatein water at 0.2 mI3min flow ram. Each analym is initially tuned for theparent ion and then tuned for_ production. Once the instt'umentis tuned,the optimized parametersare saved as a rune file. This tune file is then used during routine nnnlysis. 4.3.2. C_ibration CurveProcedure.v : (rangingfrom thelowestle,lel standardto thehighestkvd prepa.,_l)i,nto t_. ' : a. Inject the samealiquot(between10 to 50 ILL,)of eachcalibrationstandard , ! b. U_ wd_ted _ standarcdurvefsorquantimlcnL. ineerstandarcdurves I are generated for each analyte by linear regression using 1/x weighting of I peak area versus calibration standard concentration using Masslynx (or i ; eouqtuliiervablyensuostfi)ntwgaraensyasptepmro.pArinaytecoaultliiebrrtaetssti,taomnndaayrdbfeouenxcdtluodbeedasfrtaotmistitchael I ' : calculatiooftnhecalibratciuornveH. owcver, thetotal numberofcalibration I : standardsthat may be excluded must not exc,_l_ 20% of the total numberof i i s_dara, inj=te _ : [ I I c. T>h0e.99c2oz5r(eR.la"t>ionn.c9o8e5f)f.icIifenctaOlibRr)afotironcraelsibttrla_tifoanllcouurvtseisdgeethneersaetledimmiutsstt,hbeen t!. , [ appropriate steps must be taken'm adjust instrument opention, and tim ', standardosr the relevant set of samples shouldbe reanalyzcd. ; II .ExygeRne_earch t Page15of43 ! Exygen Research Page31 of 59 "; Page 73 of 111 . Exygen Study No.: 023-082 . Study Plan: ExP-023-082 Exygen Study No.: 023-082 i ! :- i " _L. _; '.i, J ' "2 ,..1 : ._ ' i i , ._ !!_i " ]' '1 ! '. ' ! .. ] ; tmMmlu_ No:/!xM_l Typicaclah'bmticottt_rv_forI_'H.%I_05 andPFOAcanbefoundial_iguttIs- 3. a Inject the same diqunt 0mtwtma 10 to 50 I_L) of each ttandard, sampl_, recovery, conlrol_ etc. into the LOMS/M3 sy.tmm. b. Staada_ corrmpoudizg to at ltatt five or more couctmtration Icvch (starting with the LOQ level or below) must be included in an analytical set. c. An tntim set of ca_hration standards should be injected at the begirming of a set followed by calibration standards interspersed appro_rmttcly every 5-10 samples (to t_count for a emceed set of extracted standards). As an altm-n_tlv_, an _tim set of cah'bration standm-ds may be included at the beginning and at the end of a sample set. In either case, calibration standards must be thefirst and _aatinjection In a sample seL cL The concentration of each sampledfortificafion/contzol is determined from the standard curve, based on the peak m_a of each analyt. Tim standard teapons_ should bracket responses of th_ residua found in each sample set. Results may bn quantitated up to 10% outside the curve by extrapolation. If ran_.. e. Fortification recoveries falling within 70 to 130% am considered acceptable. f. Samples must be stord xcfaigtm_d bctwcmt 20C to 60C until analysis. g. Samples In which eithe* no peaks mm detected or peskn lem than th,_ lowest caonnalc_ecntrazt_iotcnnfiofn ttiminmcalwibilrlatiboen rsetpaonrdteadrdsasa_r_ de(tnecoteddetaetc_tdh)e. c+oxSracmsppolnesdinign which peaks am detected at thecotr_ponding analym retention tinm that are less than tim LOQ and greater thin or equal to the lowest cmmmatratiem of the calibration standards will be repotted as NQ (not quantifiable). The analysis performed during the nmthod development included fortifications at 10 and _0 ng/g of _ in rat liver, 10 and 50 ng/mL of PFI_ in serum, l0 and 50 ng,/g of PFOS and FFOA in rat livex nnd 10 and 50 ng/mL of PFOS and PFOA in serum and urine.. Typtcal chromatosrams can be found in Figures 4-39. .E."yEPRal+ICm'ch Page 16of 43 ii t L f ! : [. I i _; _ [ _ [., I _: I i ! 'I t, t ! I "" i I _, i _ , ', I t Exygen Research Page 32 of 59 "; !' Page 74 of 111 Exygen Study No.: 023-082 -+3 + + - Study Plan:ExP-023-082 Exygen Study No.: 023-082 +-'. !_'+_I i:t : .,+ + _++J i L_ ; '_ _,_ :c _J L.... !_ ' ' ! +i _ . ; ; i !:! j _L_:.:+_ ] 1 "_'i_ ! t + 4.6. Acczrr_cz Omlm_ z TPhFeOAfo:llowing flteda must be met to enaure the Inmence of _, I'FOS and i 1. The chromatogrammust show a peak of a danghterion at 80 ainufrom a panmt of 399 ainu for PPHS, a daughte: ion at 99 mnufrom a pmcnt of 499 ainu for PFO$, and a daughterion at 369 ainu from a parentof 413 ainu x'_ PFO, A.. + _+ 2. Methodblanks umstnot contain analyte at levels g_mterthantheLOq. If a blankcontains the analyte at leveh greaterthan 10 ng/mL, then a new blank samplemust be obtainedand tl_ ent_ set m_t be re-e.xWacted. i 3. Recoveries of controlspikes and matrix spikes ('ifany) must be between [ 70-130% of their known values. If a control spike falls outside the acceptable limits, the eutim set.of samples should be re-extracted. Any 1 matrix spllm outside 70-130% should be evaluated by the analyst to ! determineff re-cxtractionis wan-anted. | 4. Any cah'l:_tinu stmdant found to be a statisticaloutlierby using the Huge Enur Test, may be excludedfrom the calculation of the calibrationcurve. However, the totalnumber of calibratsitaonndanh that couldbc excluded must not exceed 20% of the totalnumbero, f s_ndards injected. 3. The con_aticn coefficient (11)for calibrationcurv_ genera_d must be ; k0.99_ (R2 _.985). If calibrationms.Its fall outside these limits, then ; appropriate steps must be taken to adjust imtn_nent ol_ation, and the ,, t:' I smndL-dosr the relevant set of samples shouldbe =analTzed. -i i 6_ Rctcntion timca between standardsaud samples mint rtot drift nmm than I ! .4-4 % within an analytical n.m. If retention time drift e.xceed.qthislimit + within ananalytical me then the _t must be o-.anal_ed. , 4.7. I'_JrORMANCZ_ The following uvo criteriamust be peffonnsd once as a sDtem suitability teat, before the commencementof analysb, when using an instrumentationset-up that has notbeenusedforthimsethod. Ph'stCrit_'ion: Run a standard solution on _/M3 _onding to the estimated LOQ I (10 ng/mL) in matrix and obtaina signal to noise ratio fortheanalyte tran_tionof at least 9:1, comparedtoa reagentblank. If thiscriterion cannotbe met, optimize and change instram_ opcratingparamcters(or increase the injection volumc, if sppropdate). .Ew_enP.e_arch page17of43 , Exygen Research. " Page 33 of 59 Page 75 of 111 .4 - Exygen Study No.: 023-082 ,I _! :.:, !? _t =_._ ..._ _ +, 'i , " -i i :. i i_;i;i_ i_ J ' 1 _i_ t : _I I t ii_!i!' ; + [ ::i ! I .i i ! !, t " Study Plan: ExP-023-082 ,, Exygen Study No.: 023-082 P.xyl_nblahed No:HxM-023.071 Run a set of standards of five or mote coucentnaion levels, from at o_ below the LOQj up to the highest concentration lcvd to be included in the analysi_ C-__at_ a cahqrativn curvo fat the maly_ and obtain a linc_ regression with t _=fficient of detetmi_tion _a) of at least 0.985 for an_lytc. 4.8. TIb_ll_utlI_ IVORANt,LTSI$ One person can.take a _t of 20 samples through the sample prcpantion procedure in appmzlmn_|y 4 hours. The _ analysis of the set (containing 20 field semplcs, I matrix H mp_ 2 laboratory control spikes, 1 mat_ spil_ and 12 standard injections) _ tak= a_ately 14 hours. 5. C_.A.L_TIONS a. Use Equa_on 1 to calculate the amount of analytc found (in ng/mL, based on ppeaaxkamcat=xers.a) ) guensheargatedthbey sthtaenMdazrsdslFncuxrvseot_a(Ix/ex pwroeiggrhatmcd. linear xcgrcssion E __ation |; Anals'te found (agtmL) = (veak ere_,- ir+t_Xeept) xDI_ x aliquot factor alop_ DF = factor by which the final volume w_ diluted, if neccssary. Aliquot factor----9 for liver, 20 for sczum and urine b. FEoqruastaimonpl2estofocxatlicfiueldatewtihthe pkcn,coewnnt armecoouvnertsy. of analytn pzior to extraction, use _uation 2: :R_covcr(y_) = [ total analytc found (ng/mL) - analyte found in control (ng/mL)],xlO0 analyt*=4_-d(ngnff.) Note.: SubWact anal_ found in con_'ol (ng/mL) from anal_te found (ng/mL), if ng/mLin controils grcztcrthanLOQ. + t : + : !. ! _ I * I i [ i : ! ! * ! i , 1 : :t i': = !i ,1 ' -, !i 'i Exygen Research. ! Page 34 of 59 "' Page 76 of 111 ) :y, 3_ 4 t :. I i_'_,? i "!i:,,] -' . ==. i ] ,_ ; ', , L_j , ! '2 ..t Exygen Study No.: 023-082 _ $tudy Plan: ExP-023-082 Exygen Study No.: 023-082 hyl_a l_aod No: K,M_3-071 t : i c. UsnEqu_on3toeal_latntheammmotfmalytnfoundOnl_b) . _L_uatio3n: I AneJ_ _ouad(:r/,. nS_.) - ,mare rotma,'ne/mL'x, FV ,_t3 ! samploweight(,!)ot samplewfluma(mL) 1:_= ritualvolmm Por x_orttng purp,3__, sample_ In which either no peaks am detect=d or peaks Im than the loweat e,one=nlmtion of tlm cah'bradmt standards m detected at the con'enponding analyte retr.ation time will be t_portzd as bid (not d_teeted). Samples in which peaka _ detected at tim cmzcspond_ng analyte mt_ntion time ,. that nm leas than tim LOQ and grcatm- than or _xlual to the lowest con_ntration of ; / 6. SA_-_-.TY I TI_ analyst should read the material safe_ data sheets for all stan_ and | r_agcnts before lx_forming this method. Use tmiversal Iatcautions when ' handling standaxdz mad reagents, including wcflting in fume hood_ and wearing laboratory coats., safety glasses, stud gloves, t . .} l ; _ ! ! } _i:! i ! t !!i t :, i .IhTl_m_e___mdt Exygen Research. ! ' : , " I Pa_ 19of 43 , Page 35 of 59 "' : Page 7,7 of 111 o Exygen Study No.: 023-082 I ii J i i ;" __-.. '_!/ ; ...._. ; f .2 : j !! 1| " ; _i_'*_i_,,l : : ':..) . I _:;J l _*::_1 ! ! _' ' ;. i, : , i i " ! t _I ) | '2..] 1 ' _ Study Plan: ExP-023-082 ' F_,xygenStudy No.: 023-082 _,. Er/IPmId_=xlN_ _4P-_-071 ! ! i; Table 1. Recovery Sm'n'm_r 7 of PFHS in Ral Liver and Serum |: Recover/Summary of PFHS in Rat Liver I I. P 0201684 Spk5 020S1a6m84plSepIDk C i 10 Analy_ A1d0ded (ng/o) ii . Average: Standard OevlaUo.: 110 PercentR1e2c8overy _%) i 115 9._ SampleID 0o2201o68146Ssp8pk4kDE Analyte Added(ng/g) 5500 PercentRecovery _PyS) 101 -- oa Average: 98. Standard Deviation: 3.5 0201684 SpkF 50 94 Recovery Summary of PFHS In Rat Serum Sample ID 00220011668822 SSppkk;AB 0201682 SpkC Analyta Added (n_/mL_, 1100 10 Average: Standard Devlstlon: Percent Recovery([%) "111023 - ,,,,,.110 108 - 4.7 Sample ID '_o_se2spko 002200116688_2.SSppkkEF Analyte Added (ng/mL) so 5500 " Average: Standard Deviation: PercentRecovery {%) _o7 110242 111 9.6 I ! : i : (Y -. "ltt I ! = )! ' . ' _" ) , 1 i _p 2o,_.4,, i' t_yg'x---Pe.nesearch, " Page 36 of 59 "; Page 78 of 111 o Exygen Study No.: 023-082 . _.rl , Study Plan: ,_.P.-023-082 Exygcn Study No.: 023-082 .... ! .- . .._ i.:\! ::_4 ..... ) i l 'i I ;,:l ] ,_ M_Imd N= ]BxM-O'_-IO? ' Table 2,. Recover7 S,mmnr_' ofl_OS in Rat ][.,ITez'_Serlzm add Ux'izle [ Reco .v_.rySummary of PFOS In Rat Uver .J mn:pI_. .._.11,,_.-ded_,,_) e....nt.-=,,,.,/_) mo16884pkA 10 88 c_o1.4 SFdO( lO lO5 A'vemge: 98 5bmclardDeviation: 8.5 t SampleI0 Anal_e Added(n_/g} PercentRecoveryI_%} I '_Oleas4_ o 6o *o C_01884SpkE 150 aa ' o2oIe84 SpkV 5o U : Average: 18 i ' , ' i::3I, _-r_ ;'i'_ J j I ii ! I _ : , ' !27 Recovery Summary of PFOS In Rat Serum , san:pie It) (201682SpkA 0201682SpkB ,(_018825pkC A.alyte Added(nstmt]. 10 10 10 ii Average: percentR_:oveXt _) 80 85 99 i 88 (_0s1a6n8:2pSiepIDkD 0201822SpitE ,0201682SpkF Standa.-dDevlstlom AnalytoA5d0dedIns/mL) 5O 150 StandardDevlaUon: 9.8 Pe_o,nt R1e1c8overy_%_ 122 121 . 2.1 Averagt 120 Recovery Summary of PFOS In Rat Urine 0201682SpkA 10 98 O2O168S2pkB 1o 89 ,02o1682SpkC 1o 111 SampleID 1_.01682SpkO _01682 SpkE Am_dtymtelAddarddeDd(anv_lamlloLm} 50 50 Avarage: StandardDeviation: PercentR4e.7covery(%) 80 78 79 1.2 " I r i', T' | ; ' ' t i t " I L l ; ; , ; _XZ_ l_,_trch PaSeZt of 43 "; ! z ,, i I- Page 37 0.[59 .v Exygen Research. Page 79 of 111 1 ij /._:q ! !_,:#;\__/t ! '( :iii_ ..... .*: ! I - .i ?,_:'_ !,__,_ l "!J ; 1 '. I _:! Exygen Study No.: 023-082 .. StudyPlan:BxP-02.3-082 ExygcnStudyNo.: 023-082 1 Table 3. Reeovex7 $tmzmax7 of PFOA to Rat/Aver, Serum and Urine Recovery Summary of PFOA in Rat Liver sample IO 0201684 Spk A 0201684 _k C Anal_e Added (ng/gI 10 10 Avemg.e Standard DeviaUan: Percent Recmmry (%) 9Ji 99 ,91 3.1 020S1a8m84p.lSepIDkD 02ol e84 Spk z 0201884 Spk F An_l_ ABdOded (nglg) _ E0 Average: mancUo_,vl,Uo_ Percent R9e7cmmr_ _'/.) _ 94 94 _ : . ]' ; I" i ! t L :. Recovery Summary of PFOA In Rat Serum Sample IO Anal_4eAdded In_/mL} Percent Flecavar_(%) I O201662Spk A 10 118 _. n01662 Spk B 10 117 Z01682 Spk C 10 115 ; Average: 117 Standard Deviation: . 1.5 , Sample In 0201682 ,Spko Analytic Added (nz_/mt) 50 Percent Recover,./_'%1 107 t 0_01682 Spk E BO ! 12 o:zole82 S.k F SO 1 lS i: Standard DAevveiartaiogne:: 141.01 I I ! Recovery Summary of PFOA in RazeUrine Sample IO 0201882 Sp,k A Armlets Added In_lmL) 10 Percent Rs:over_,(%) 68 0201882 Spk B 10 91 0201882 Spk C 10 89 Average: 89 ; Standard D_tlon: 2.5 I |_ i Sample ID Anel_te Added (n_/mL) Percent Recmmry _%) I 0201682 Spk D 50 89 I 0201M2 8pk E E0 88 I 0201882 Spk F 50 85 A_mrage: 87 Standard DeMatlon: 2.1 t . .; "_ : !: .]],x,Jge1n_s_z_ . ?age 22 ot'43 i Exygen Research. Page38ofS9 "' Page 80 of 111 Exygen Study No.: 023-082 " Study Plan: ExP-023.082 Exygen Study No.: 023-082 -'7 i Figure 1. C_qlmrationCurve for PFHS r' _ LI i--* I :...._.1 : I I - i t _ I I J ; J_._n_ _ _t_ s_. _ _ Iq_l_4IJ1n_Iuu', Odl_ll_'_--I_1_ Wl_lhlh_ _ _ trllnl_ _ Ikma_l- X O,O......."..0.:._..".....-.1.'.n." "'t_i- "0" "9'_" "_'n" "_,_'=" "4:0 ",41:11"_. ' o I: i. I .l_gea Re._n_ Exygen Research. t ! I n , Page23 0f43 , Page39of59 Page 8"1of 111 Exygen Study No.: 023-082 L., i i ?_1)r I_L*I ':_ L1 I" ' ! i "" ! "I .,' I :;; Stud:/Plan: ExP-023-082 E_tygen Study No.: 023-082 , t; l_l_--ex Callbrati;nCum_for PFOS CCoomellll_cmlednt2ofnl_mtn_rrnPlFnaOllS_: 0.997909 Callbmt,lonctm_ 3211.30"xe 48.9285 Responsetype:ExlemalStd,AreI Cam tyl_: HEe_. Odg_'.:F_._axleW, eIQht_ l/x, _ls lrans:t_ne . [, "' : , .i ,_A ' t : _ _ ] -i ; 'i i I : . ! ............... Exygen Research. , ....... n_mt. 1 _' t l "i i l t ! "1 I. .i , / i Page 40 of 5_ "; r Page 82 of 111 , .:-] I I ,i ;" _! ' ]I i ! ) _, : Exygen Study No.: 023-082 "- Study Plan: ExP-ff23-082 EatygenStudy No.: 023-082 F-ardenlVIethoNdo:Er.bf-1_l-071 Fl_xre 3. Csllhraflon Curve for PFOA ! e. ! c,,_u,_ 1,am,_: o^ CCaaeliblfrldaetinooctnfuOnemt3e:1n2'd7n0a.9*txlo0+n.-93:9657954.S,_6 Respcr=teype:E_mal Sld,_ea CurvetypeL: inearO, riginF:.:_udeW, elghllnlg/x: ,Axislran_None ! F 1.66e5- I" a6'" _5''" "1'.o'""_"" _2o'"_"" "_o"" _:5"' ;,'.o"";s:i"""s:_g" !. !.:.i Exygen Research. ! i Page 41 o/59 "' Page 83 of 111 't ii:,3 ' i .._-7 -'--,'::. "_B i )_ ' " i' 'I i.'_ i Exygen Study No.: 023-082 . . StudyPlan:F__-(Y23-082 Exygen Study No.: 023-082 Figure4. RCeopnrteasinenintagPtilvqetCShromatogramof a 0.1 ngfmLStandard CNIW_,_LI mM_.m mll'l_lOI ImI1_ M m.,km,mm 1turin tcnm_ul n Itl_lll I ClumaIs, t LIII. L ............ J...._l 1_ .i .... I.._,JI,_2 _L,_i ......... t.ll_aj _,_ Figure5. Representative C]_omatogramof a 0.1ng/mL Standard Cmlml4, e.t nile. I,ImA ml t,l_S ummA.m Im (u_._al q m_l_ l_ lain _ sc_,_ u- " --- _ ' I,:! _. i t t t !, : : : 1 ......... N .... _ ...... - ,,... ,--.--'' } i 'i ; I. l z.. i 1 " Page 42 of 59 ": Exygen Research. Page 84 of 111 Exygen Study No.: 023-082 Study Plan: E_-023-082 + ExygcnStudNy o.:023-082 : i -i I : : ] . .... ii:*!LI Figure6. RepresentativeCbromatogramof a 0.1 nf/mL Standard _. Con_i-;.g PFOA _' oma_Ll_pma.m_m at_tmmam_ [: IIIqmtA4_l It IIt_ aldl _C,mMM I_' It_llal |Osmmlll I_1 '" J" i' k t % I: Figure 7. Representative Chromatogram Of a 0-_ ng/mL Standard Contain_g PFHS I t M_l_103 Im _Nh M MqMdl ghmmt _ I _&.i ' I, !,,i i ii'?!,ti:!,i ,_ -' % I I ' I " tm " .-'_,_,-'&"o. _ .,., .-..., " m .m ,u. ... | }. "l, .E."y_mP__'J__'._h .' , IS'aid2e7 of 43 ": t, 1! Page 43 of 59 : , i. Exygen Research Page 85 of 111 , i ee : *f ! 7,,'re, I ; i , o Exygen Study No.: 023-082 . Study Plan:ExP-0"23-082 Exygen Study No.: 023-082 Figure8. Representative Chromatogram of a 0.5ng/mLStandard ContainingPFOS _mb4, U NtJ. I_mA "--aPma _lm_WaW _n i. "" i" _" . . _-...z-_ . .:.. _ _j , _ ,., ...... _lgure 9. Representative Chromatogram Containing PFOA of a 0.5 ng/mL Standard. k i . i" i ! t t i . _ ...... ._,.-.--_--. ,_-_',;,._.._. _.m-:., __ ._. ,. _ - ._ .,, t I .i I I ExylpmP.,me.ah_ ." Exygen Research. P_l_28of43 ! . Page 44 of S9 "" Page 86 of 111  Exygen Study No.: 023-082 . i I : i; "_-" I; :! i t i[ _ ; {,._j- _,_. . Study Plan: E_tP-023-082 F.xygen $tudy No.: 0"23-082 Figure10. RepresentativeChromato_-a_ of a 5.0 ng/mL Standard ContainingPla'HS (:Rmm.I . U q_l.lm_ 11 mm_mh" (_.MI m&_ I&.bm.,tl_.rt.alml'attl:a:tlw/ Im)m mall aawm,u. "- m " .;-" .;,. _ " m " .',,. " 7kJ " tJ " .;- _" ff_" nC_.m l_fgure 11. Representative Chromatogram of a 5.0 ng/mL Standard Containing PFOS Cm_m-l. tll tql_L .BI_O_aml lq'08 _mktt_aq IiO Ile.ka._ I_tl_l tm_acam,_ _ _t' 4m_l__mt *m '_ _ p:" .' I i i i i i .. i i tI i1 : ; ; i , ti 1 l" 'r ! i I t "; _'_ _ R.nsnaz_ Exygen Research. L,,_) t ; I " " i o; Palie 29 of 43 , I Page 45 of 59 "; Page 87 of 111 I L_ ! i :_ _I '_"_ ;" _Lt I_ : Exygen Study No.: 023-082 Study Plan: ExP-023-082 Exygcn SU_dy No.: 023-082 * 12. RepresentativeChromatogramof a 5.0 ng/mLStandard ContainingPFOA C_IS'5, IIJIRIILmlP. IEOAmlPml _' "_" IL_I._ :l_llm I.CtMMB n "''U" : ! i. : '_. i: : L i 'i [_i_ ii_1,_ __' -"_t ] I I : I :Lj : i I " tm I,m m ,ui i_ lu_ Tm I,_ i Figure13. RepresentativeChromatogramof a ReagentBlankSample Analyzed for PFHS ' ,,,_._b,_ I_UMil/T _dt*--,,_ i rt: !.." 1" !. f|, ! 1 : : . t t h I --I " _._. _ ExlrlicuRc._ Exygen Research. .' i Palo30o_43 ' Page 46 of 59 "_ Page 88 of 111 f J :' I ,i i, ! : _i ! _,_ !t_i_* ! 'I Exygen Study No.: 023-082 " StudyPlanE:.xP-023-082 Exygen Study No.: 023-082 I ltxy_ ble.tl_ N_ Kv._-0P.._417! ! Figure14. R.Ae-p_r,_ezseendtfaotrilv_eOCShromatogramof a ReagentBl_k Sample II_IWW_,IU_ IoamaN, II Ilaq_t lll*J A IIIMMIICki'wl II* 11*4_.am =llal_ll 1 |* I ! ! i , .. ...... . ...... . . . .. ........ . r lq_re 15. RepresentativeChromatogramof aReagentBl_nlrSample Analyzedfor PFOA "' _ . ! [J I ' ._iPm ReseN Exygen Research. _ , i Pa|_31 of43 .Page 47 0f59 "' Page 89 of 111 Exygen Study No.: 023-082 i : "- StudyPlan: ExP-023-082 F._xygSctnudyNo.: 023-082 .... _ :, ..... , ' iq ! 'I i0 ...'. i '2 ] : , ?,'s-.,_ _'_'I _": ! ] _;?? [ l -: ) : : ! _i c.t 1 K."y&mMettmNdo:P.xM.023.0"/1 Figure 16. Representative Chromatogramof a ControlLiverSample Analyzed for PFHS _llql h ILt4M Mini allCh_ol I_. _,al_ill n gum _-Z-,. ._ --_-_, 1 mm I.u Un " am ' _ " -_ ' uo ' zm ' nun " lure ' 'ILu ' It_ ' 1,_ " 'l'Inu l_g_o 17o Repr_e_Nfive Cm'mnatogram Analyzed for PFOS OUlU4 Ug_ IIImllA o1'_, Control l.,h'ex Sample U.dl_m II1_la_ IMII_ "" i o 1 i _ [, P, I;I! a i i" ! _ F._ l_sca_h ! i _ -"! P_ge32 of43 . "" Exygen Research. " Page 48 of 59 "_ Page 90 of 111 . Exygen Study No.: 023-082 .i ii, .-. -71 , :- I : ; ' +'i I _-"J-" i !i _, _:,,!_ LJ iI +_ !i!i ' ii l 1 StudyPlan:_P-O23-OS2 Exygcn Study No.: 023-082 I_ ]_,_1 No:K,.M-C_-1_ lCigure18. Representative Chromatogram of a Control Liver Sample Analyzed for PFOA _gia _ nilnmA _ samuctl, 11_ I,Ile _ 4,_ _ ILM l'.l I_ el,mS 11101 SIAl _ ]Figure 19. Representative Chromatogram of a Control Serum Sample AnaI_.ed for PJ_E[S m , "+ '++"++ ' , +'++, ..... _ ,++-, -+, ..... ,, ,-+ ,+-+,--,-++- , -,-+ , : I L P : " _.. i _+. I I I. . .ExD_en1__-,__h "[ Exygen Research. Page33 of 43 " ! Page 49 0/'59 "; Page 91 of 111 i '_ ! s -, :'7 i .. ,! ! ..._.. !..q d , o Exygen Study No.: 023-082 _ .. Study Plan:E_tP-023-082 ]_xygenStudy No.: 023-082 _t_m/de_l_l 1_ BxM-023-071 : Figure20. RepresentativeChromatogramof a ControlSerumSample /_n_)yzedfor ]?FOS InOl,,.,,_, nlenltA _ol_ I _._i . i'**N , l q "_" _' -_-," _ "-_.." _' 7 _,,' _' ,-"-u''rl'.-. ..... : Figure 21. Representative Chromatogram of a Control Serum Sample i 1 Analyzed for ]PFOA '. i Ie _ . i;_ _i_ .._ "zm " -_- ' ,,._ " um am _r.m " ue " "_" ' w.,o'lt_ " E_ Re_uch ' , ! Pasn34of43 " ! | : Page 50 o/59 "" Exygert Research. Page 9_2of 111 I ....i _] , .. ::-i 'i ., '_" ,,i Exygen Study No.: 023-082 . Study Plan: ExP-0"23-082 Exygen Study No.: 0"23-082 I. lZxTIpMm _'daoNdo_IZ._ 1 i' l_lgure23.. RepresentativeC_aromatogramof a ControlUrineSample Analyzedfor PI_OS Imlm8 I1fla mm_A O_OMA.I:Naim_*_,abm} 7 II_d-Illl N_:Zl tP.nmmm_ IW_ d za_,_e U. *l - [ ! i _,_,_ _,_ _'_'_ I:_ i Figure 23. Representative Chromatogram /Lnalyr_xflor]L_OA of a Control Urine Sample ImtlW3 Lk'ie BlmakII li;oaam_aulmaIlMIl.al .I t,,d_ U,'_ i_ld_XlCSwn_B. _'_ .'., -i _ I l _-2 .... ! ;_..i_ ! ,r, I ........ _ . . ..,_._L _....'''"r. " JMI 3_0 4.00 _ _ ?._l m_ ItOm lone " I,',-, " _ it , : t E f .]_xg'yc_]_,,'_',,,',r.,5 ]E_,g3.o.o_t'43 " Exygen Research. Page 5..1of 59 ": Page 93 of 111 J ,. ,. 7! ! , ' 2;: !:!,i ,. : 72- I__::!ii_ , LL_ k_:i i '! : o Exygen Study No.: 023-082 '- StudPylanE: xP-023-082 Exygen StudyNo.: 023-082 EWZm McahodN_ _-C_O-071 : .Figure24. RepresentativeChromatogramof a ControlLiverSample Fortified at 10ngfgwith PFJHS . mmmL4 I,w_&w_, ! x_h,_mIP_.ma__m i i i ' _, m _ u. _,'-:-..' -'-_ it i Figure 25. RepresentativeChromatogramo.fa ControlLiver Sample I Fortified at 10ng/gwith PFOS IIlim4 LINt Ipk & 10ppb m.J_mma mucus1 I_tIMs #T i g I ; i .Ezy_R_m_.ch " Exygen Research. ol P_ _6of4_ , " Page 52 of S_ "; Page 94 of 111 Exygen Study No.: 023-082 l iL! i I '! ! i 7_/*i i;Li StudyPlanE: xP-0"23-082 ExygcnStudyNo.:023-082 ]Flgure26, RepresentativeChromatogramof a ControlLiverSample Fortifiedat 10ng/g withPFO_k _n " i i i_i:;i_)i : i t_ " w .--.s.'. _''w .-:--_ ;:i_. --tin " t*6 " io_ " 1tin '--r_ I, ]Figure27. RFoerptriel]seednatta5ti0venCg/hgrwomithatPoFgHraSmo-f a C"ontrolLiverSample i, mol_ u_e 81_ o. mllN _,/im-aoullt_ll t -I I I i ?, ! 1 2J i i .... J ,_ " she "-i,,m " -_-" si " -_,," s,,,t ' N " i_ " aoJo "i,'-- " ,,'n_ . Ii l 1 6 : ' :! , I _.,xyg_P__e_'__._,,r.,,.5 o' Pag,'3, 7of 4"4 i | " Page53of59 "; Exygen Research. Page 95 of 111 'i ""i + ,. u o I I _:i_i " _ t i , :i ' '. "_" 'L'i:_'L ' I J r_ .'''1 I ',:,j ! I r-/i+_ I l + ++ i_ : :_": : :.:_ Exygen Study No.: 023-082 , .. Study Plan: ExP-023-082 Exygen Study No.: 023-082 _'/glm Mctl_dNo:RxM4_.O-/i : Flgm"e 28. Representative Chromatogram of a Control Liver Fortifiedat 50 ng/gwithPFOS am_t_wu_n,se_ _ma_lt_m III us._4eeal_a _d ItrC.nhmlwA_mln;It- _ ahtilnm,4 _ Sample I _ " "+"-' _ " _ "tin " tin"" I_, ' -_ 'i a,.oK_,..1_ Flgure 29. Representative Chromatogram of a Control Liver Sample , Fortified at 50 ng/g with PFOA " mmm4 u_w t_ o. s_ m-_tttaum _4_t _ t! L +' _m*i : ;+ +: t !:;: I }. "'" i_ I 1 ' " LOI "" l_ ' s_s ' lJm '-+'ibs "" I_ " IJ ""l_i" "-'I 41 " I0_I " ll_01 ' ++11am ,, ! ' 1 : [ ._gea _se.arch Page38of43 ": [ 1 " Page 54 of 59 "; Exygen Research. Page 9;6of 111 ' i Exygen Study No.: 023-082 I "i :! .'" _i ; ?':J 52:,i _ Study Plan: ExP-0'23-082 Exygcn Study No.: 023.082 _TSm M_md l_o: E_t-_-0? 1 , "." Figure30. RepresentativeChromatogramof a ControlSerumSample Fortified at 10ng/mLwlth PFHS mmi,_ _mmmX_LM_ IIIIllbtla h lU__ _m_m I_W_ IQm_/IW. 1.1_1 Ik I !; r I i i :'i!_;_}!i_:'_:! e N ,*L 2'Ll .......... _._.:.r ,, .-':.=_. Figure3I. RepresentativeChromatogramof a ControlSerum Sample mDim mmvam_L i/S_mqoWrJ tifiedat 10 ng/mLwith PFOS _ n_-,i_mn' LC,_I_S_mN'4_m m _. ,_.,,. .--,_ .--_.._.--.._ .--_, .-.._ .'-_ ......... ,,.. ! i : _ -. i t i , i Bxlq_ Re.._u_ Exygen Research. " . i" Pall"39 of 43 "; i Page 55 of 59, .; Page 97 of 111 Exygen Study No.: 023-082 ii? ;_ ." | ; ' _"-;"" ii L 'L'I j ii..),,_i ..- '_'_',_{ ' t'i_v I i ; I '1 I Plan:ExP-023-082 Exygcn StudyNo.:023-082 PzYI_ MctbodNo:P.zM.023.071 Figure32. Representative Chromatogram of a ControlSerumSample Fortifiedat 10ng/mLwith PFOA nmm a'mma_&llm a_M'ial m" O/A,M I_,)d-a_ u:m:Sl Lonilu lit I_d|Clwm El Figure33. FortifiedRe5p0rensge/nmtaLtCiVheraPtmFaItLgSraatmCownitrholSerumSample _""''" _" "t,_= ,,_ .. ! I L s. oL ! F i " i t_ " .a. m - _ ._. ._, _. ._ - .;,,. _. a_-..., r'" ! l ._._gc_ _ _ago40 of 4.1 [ " Exygen Research. Page 56 of59 " Page 9:8 of 111 Exygen Study No.: 023-082 ('_i ". ; i I : " .-!" ' L] . Study Plan:ExP-O?.3-082 Exygen Study No.: 023-082 ; ?" Figure34. RepresentaflTeChromatogrnmof a ControlSermnSample "' ! Fortified at 50 ng/mL with PFOS L !_: mosmlmm alml_m p_ mlm#_t.lle Imi1_ _ID m._.amtr.sm_a:m_ n _l#|_lW. _i" i _ " i : , L : ] _..,.-.,._.-_ _ 1 i 2...! ,,, ' ,_- " 'a,m"a,m' ,chu_'_ _ " _,m" u,m"- _:-'" ' 1,',,.,.... Figure 3S. Representative Chromatogram of a Control Serum Sample or ed,,5t 0-JmLwith1'FOA , Rmm au_m _sl, mlt_ _tal I.ff_l_l$111, Ib-_lll ', II _N _._ t_ _ _- -_-'"I_ ': I .Exys_Re_u_h Exygen Research. ,. PaSo41 of43 i t " Page 57 of 59 "' Page 99 of 111 Exygen Study No.: 023-082 i ": _, " . ..... ,o _ .. 1 Study Plan: ExP-023-082 F..xygcnStudy No.: 023-082 ; ..._. -i i -"'_:'" . ! ,} i !)_ ,: 'i | Ii 'I '4 I Figure 36. Representative Chromat_gram of a Control Urine Sample orfified at 10 ng/mL _itla PFOS emmu_apk_,, lepl i ee..u_um e_k,m _, ' 't,_ "---- ' m " _o'S,',,-' Figure 37. lFleoprtrifeiseedntafait_e10 nCgh/mroTm.,awtoit_h-amPOAof a C"ontrol Urine Sample IIollli i_ la_,hll Imu _ Si:II, M_is,, - t '; t 1 :' --- 'i _ :.I ' i I t _ 1n, _._ _ .t_ _ _bo " _r. t_ too _e_ _ .]_x_ _ " 1 I_Ko4,?o.t'43 , I I ! Page 5_ ofb_ "; Exygen Research. Page 100 of 111 ..J ! 1 j L' , ;- i , , I [ i !_;_:i'i,;! i '_ Exygen Study No.: 023-082 " Study Plan:ExP-023-082 Exygcn Study No.: 023-082 lbtylpm_.thod N_ ExM_I F_nure38. RepresentativeChromatogramof a ControlUrine Sample Fortifiedat 50ng/mLwith PFOS s i K . ! i t i: .i !. , r ! Figure 39. FortlfiedR5e0prne_smeTn.t_at_ltVeY_aFwtOithhf a Control. Urine Sample ldmdllIClulu_ El* * I , 1I : ! I 1 ,i i =..,,I I : la, - _._ ,_-,_ ,'_ _ _, _ m, _ .-I... .-_ _"_'_ .]_-yg_Ro,r, ..s_m_ Exygen Research. I'a_ 43of43 Page.59 of S_ "; Page 101 of 111 Exygen Study No.: 023-082 i E,gen !i_] i ...:.. i "'" , _ ': i t I RES EA RC H _.PredRsee_ard_. ProvenResult. "-. - Page 1 of 1 ii, , -.__ i:_"t , _ :,! _\ '.. PRODTeOviCatOioLnNDuEmVbIeAr:T.1ION -- Date of Occurrence: 10115/02 ExygenStudyN; umber:.023-082 ProtocolNumber:.F.xP-023-082 '\ DESCRIPTION OF DEVIATION AnatytlcalPPocedurSeummary-.. CatlbrationStandards ..... Did not prepare:.e..x.t.ractedstandardsin humanptasmaat 1.S and2.5 ppbrevers. -_ : , :_i 1_.! '_<_t i :: _"I ...._.. ! ' ! I I I ! ,. i t ] t i : " i "1 : ACTIONS TAKEN '- for @xamole - deviation Issued. SOP revision, etc. Protocot devlaUonIssued.\ ',... ,,.... \o \ RecordedB Date: 0 IMPACT ON STUDY' No negaUveImpactbecauseontyhad tlmf.t_=sdampteavaltabteandthe tinearrangeof the curve wasstitt establishedwithout those two tevL_s.. \% i ",L NL_ PrincipalInvestigatorSigna I'Q Srtl_D, ire_r SIg'pa_re_..,_ " ManagementSignature (Sponsor) U:_FonnRsO_PTOCOL_DEV1ATION._doc :;,, Date ..-- Date , -- ' Date '". "..Data ',.. E.xygenQAU Review -... _dC. \ !_'1T/6_=,9/25/2002/5 Exygen Research. _" _3 058ResearcDhrive : 800281.3219 A _Tte F:C8o1l4le.2g7eP2,A.11061890%USA _n.com Page 102 of 111 + Exygen Study No.: 023-082 ?.-,_ E'gen :_ .. RESEARCH : i; . _PmciRse search. '....P..roveRnesults. i ' -, ' i .__. _:_ !j _"_:I ... ....I PROTOCOLDEVIATION '. Deviation Number. 2 Date of Occurrence: 10/17/02 Exygen _tudy Number. 023-082 Protocol Number:. ExP-07.3-082 \. DESCRIPTION OF DEVIATION Anatyttca[ Procedure....S..u. mmary-I. Method Summary.e. Etuted with 5 6_.Lof methanot and evaporated to tess than 1 mL udng a nitrogen evaporator and then brought flnk[, votume up to 1 mL with methanot. \, L .... '.. /_CTIONS TAKEN I" ",.. for example - deviation Issued= SOP revision, etc. ' Pmtocot deviation Issued_'.... \ !I"_'i,__ ' ' i:_+_!i , Recorded By:. /I ;MpACT ON STUDY Date: No negaUve impact because flnat C'_ncentt_aUonremained the same, but comptete etutlon of . the anatytes was estabttshed. _ Ii ",. ! Pdncl'pal "Inve,stlg_tor S/"yg'tn/'_'a'iture '" '. Date -- :! _' f! ! M,_gemant (Sponsor) Signature J Exygen Research. Date ", "...\Data Exygan OAU Review ".. _1)C /_/Ta/E'L X3058 ResearchDrive A _tate College,PA16801, USA _kT_8008-1248.12.732.119019 exygen.oom Page 103 of 111 Exygen Study No.: 023-082 .i _:_ R E S EA R C H ,PreciseResean:h. i , ' "... ProscmResults. i i :_ _ Ii _":_'i +_,!_ i : i_ I : Ii_'']I [ +' l . 1 PROTOCOLDEVIATION DeviationNumber:.3 Page 1 of 1 l " Date of Occurrence: throughoutstudy Exyge"n_tudy Number. 023-082 ProtocolNumber. ExP-023-082 I. '... DESCRIPTION OF DEVIATION 1.Anatyttcat:ProcedureSummary-MethodSection4.5.3.d---severat sampteswere quantified above10_ of'the curve. ".... 2.Ana(ytfca[ProCedureSummary-MethodSection4.5.3.e---the acceptancecrit_erfafor _ spike recoveriescoutd_qt atwa_ be met. p.CTIONS TAKEN t 1-2.Protocotdeviation_Iss_ldefo. r examols . deviation Issued. SOP revision, etP'-. ,. p- ; [ '_t ; J! ! i( _ , +' Ji _._i 1 J ! :i _ ; RecordedS_ "- _ Date: _:_L,'_. (J iMpACT ON STUDY 1.No ne_attveimpact becauseresiduewas..<than 15_ above the curvefor C6andC8for sampte 0204493SpkV andfor C8for samptes02039.65and0203965Dup. Atso,the samptesthat were quantifiedoutsidethe curvefor THPFOSand..,THPFDwSere ontyfortification recoveriesandthe maJorityof the recoverleswere > "lO0_. anionsat these levels. 2.Impact not determinedontydocumentedsince".thtsm_ ethodhasnot beenvatidated for these P . M_agement Signature/ (Sponsor) U :_Fo rm'_:l ROT OC O L"_EVIATION" doc _ Exygen Research. _ Dat_/'3_.,__ "- Date ", Date ExygenQAU Review ".. .Q _. I/+.,#'/#__ _ _t25_2002_5 _o58 R_arc_Or_ A _5tate CollegeP, A16801, USA ' ILT: 800.281-3219 P,814.272.1019 ex)gen.com Page 104 of 111 o Exygen Study No.: 023-082 '. o i?i:_ ',J.t _ i- i; i:_17! "i _ii:ii i"; .... i '1 ' : I. ' '.. i i 1' -- -, -;-. I i EgenRESEARCH _R_dseResearch. '- ProvenResu_ \ - PROTOCOL DEVIATION Page 1 of 1 \\. Exygen:Study Number: , - 023-082 DDateevioaftioOnccNuurrmenbceer:: 4thrculhout study Protocol Number:. ExP-023-08"2 - DESCRIPTION OF DEVIATION 1.Anatyticat.Pmcedure Summary-Method Section 4.5.3.g---severat samptes were quantified .- betow the cuiye and ND was used for when no peak was detected and NO.was used for negative re_tts. Artotherresuttws erereported. Z.AnatyticaPtrocedurSeummary-MethoSdecUo4n.5.3.c--a .seconsdetofcalibrationstandards { \\ wasnotanatyzedin"a...set.. .t -- A.CTIO NS TAKEbl l \. forexamoia- deviationissuedS. OPrevisione,tc. , ,,,, I 1-2.Prot_codteviationIs_Jed. z i , ._ \ % \ ; ..., Recorded B Date: ._ IMPACTONSTUDY il.i!_ 1.No negaUve Impact because no ctalm of. quanUtaUve accuracy has been established. i_ )..No negaUve Impact because the extracted, standards were anatyzed throughout the set which i irovlde a QC check on the lnll:iat catlbral:lon... I: t !i 11 ., ] ;i_"_! :1 PrtncipllInvestlgaloSr l(g_nature '.. Date i _agement(sponSsiogrn) ature I U:_Formi_PROTOCOL_DEVIAT1ON.d_oe Exygen Research. "'\_ Data ".....D..ate r Exygen QAU Review ".. M/_[. 16/_;#/0_--. i. _ 9/25/2002/5 _f i _3058 ResearchDrive ' A _Sm_ College, PA16801, USA _IL_T: 800.281.3219 F:814.272.1019 exygen.com Page 105 of 111 Exygen Study No.: 023-082 _i_L t, ! ; -7 [ -i I ,PrecisReeRsEeSarEcAhR. CH ProveRnesul_ \ I -". j '., Page 1 of 1 _' PROTOCOLDEVIATION :, DeviaUonNumber: 5 Date of Occurrence: throughoutztudy U -=r.. Exygen'_tudyNumber:.023-082 ProtocolNumber:. ExP-02.3-08Z _' ,?._ - DESCRIPTION OF DEVIATION 1.AnatyUcal."PmcedurSeummary-Met.hodSection4.5.1---the t.ranis|tionmonitored for PFQSwas ....! -. 499-> 80 i_ "... iE --. '.. _CTIONS TAKEN ! - l.o.erxamoled,eviatiolunued,_OPrevlsloeflt,c, : 1.Protocold. eviationt_u_d. - :, V:i il!:i't ; 'I ,J i ....! : _._ _ RecordedBy_---b_"'._ _ _ Date:/_-_//(_.,_' 1,Nonegative Impact. " " (] IMPACTIN STUOy "\"".\ Pdncipal_rw_s r Signa "_} _. " "_C'[/tPu"r_ / _nagement Signature,/" (Sponsor) "- .... ODatl/o_0/O,Z. \ _. Date , Date ExygenQAU Review "'. _'t C- Iol_d,'n.. ., ,. - t i i e Exygen Research. _3058 ResearcDhHve A _tate CollegeP,A16801,USA _LT: 800,281,3219 I" IKR 814.272.1019 exygen,com Page 106 of 111 Exygen Study No.: 023-082 _- -i m rJ-.l|_-d, ganc IIlIlllIPJIl|_ls _: _[i_.leh ..... ii'l_l._l_4'P - 0CT-3_'2_2 12:24 " Eenl_p ". P, _2/'_ _'_' "_ _ "i -_, I '_ " ! _":" " -- X \ " a" . I , ,__,. - - -- , II _"._.'."" -. " """'." .iP.R.O."T._.2.C..'.QL'DEVIATfOi'N I X : "...;..'.: :._i'O_mN, u_bi,__.' : .: pqo_._1 ,f._11"I1" "1 I , . , . .. , --,_ ,I/IF.x""y:""l'__t\IT',,.._.'u.:.d. yN'"umbe""0_'2""3-0a2"." ,:_I,,t_'"_.',_''k":_to"2; ' " ; P'roto"c'"o" rN"umTl:-lln-!"_-,.'-1:"173.1]82:", ! .] " " '_ "" ' DESCRII_ION'OFDEVIATION " ' "- _ ' I .. . : | ._ ._ . ".: ...: l__d n,:o.t.pre,n._+,__da._.m,;nd.a_,. . . ......, .. ..Imh,y'm,',a_plmmi _ 1.5lnd2._ pp. bte.rm.. / . . _ '......... I !. '_. "_..-..:." ..." 1" .. : "".'_. 7" , ' " " . " " .. i'" : '\!'" 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