Document Y9MwjaLNXJ5VoMkqRMOYo0VQO

3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 3M Medical Department Study: T-6295.22 Analytical Report: FACTTOX-160 LIMS E00-1668 Study Title Extended Recovery Study Followinga 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooctane SulfonateAcid Potassium Salt (PFOS; T-6295.22) in Cynomolgus Monkeys Analytical Laboratory Report Title Determination of the Presence and Concentration of PFOS in Serum and Liver Samples of Cynomolgus Monkeys Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date May 3, 2002 Performing Sera and Liver Analyses 3M EnvironmentaLlaboratory Building2-3E-09,935 BushAvenue St. Paul,MN 55106 Laboratories Sera and Uver Extractions PaceAnalyticaSl ervices,Inc.---Tier2Facility 1700Elm Street,Suite100 MinneapolisM, N 55414 Project Identification 3M Medical Department Study: 1"-6295.22 Covance In-Life Study:6329-268 Analytical Report: FACT TOX-160 3M LIMS NO. E00-1668 Total Number of Pages 113 3M Environmental Laboratory 3M Environmental Laboratory Page 1 Page 1 3M Medical Department Study: T-6295.22 3M Medical Department Study: 1"-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 Analytical Report: FACT TOX-160 LIMS E00-1668 This page has been reserved for specific country requirements. 3M Environmental Laboratory 3M Environmental Laboratory Parle 2 Page 2 3M Medical Department Study: T-6295.22 3M Medical DepartmentStudy:T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport: FACT TOX-160 LIMS E00-1668 GLP Compliance Statement Analytical LaboratoryReport Title: Determinationof the Presence and Concentration of PFOS in Serum and Liver Samples of CynomolgusMonkeys Study IdentificationNumbers: T-6295.22, FACT TOX-160, LIMS-E00-1668 This studywas conducted in compliance with United States EnvironmentalProtection Agency (EPA) Good Laboratory Practice (GLP) Standards 40 CFR Part 792, with the exceptionsin the bulleted list below. Exceptionsto GLP compliance: There were two study directors in this study. This studywas designed as four separate studies. The in-lifestudy phase was considered to end at the generation and shipment of specimens. The analytical study phase was considered to start at the receipt of these specimens for analysis. This resulted in havingtwo separate studydirectors, one for each phase of the same study. However, since the technical performance of each phase was entirely separate, no effect is expected from this exception. There were two in-life studies and two analytical studies that utilized the same test system. These studies includein-life studies Covance 6329-223 and Covance 6329-268 and analytical studies FACT-TOX-030 and FACT-TOX-160. The purityand stability of the reference standards are not included in this report, they are not known at this time. Andrew Seacat, Ph.D., Study Director John Butenhoff, Ph.D., Sponsor Representative Usa Clemen, Principal Analytical Investigator / ,2. zro _ Date Date William Reagen, Ph.D., Analytical Laboratory Manager .?M Environm_.ntal L_hor_torv 3M Environmental Laboratory Paoe 3 Page 3 3M Medical Department Study: T-6295.22 3M Medical Department Study: T-6295.2,?. Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport:FACT TOX-160 LIMS E00-1668 GLP Study--Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS in Serum and Uver Samples of CynomolgusMonkeys Study Identification Numbers: T-6295.22, FACT TOX-160, LIMS-E00-1668 This studyhas been inspected by the 3M Environmental LaboratoryQuality Assurance Unit (QAU) as indicated in the following table. The findings were reportedto the study directorand laboratory management. Inspection Dates 06/19/01 Phase I Protocol Date Reported to Management Study Director 06/19/01 06/19/01 09105/01 Extraction 09105/01 09105/01 09/11/01 02/01/02, 02/05/02-02/08/02, 02/18/02-02/22/02 02/21/02, 02/22/02 Analysis Data Draft report 09/12/01 02/26/02 02/26/02 09/12/01 02/26/02 02/26/02 .L QAU'Repre_entative Date 3M Environmental Laboratory 3M Environmental Laboratory Pacle 4 Page 4 3M Medical Department Study: T-6295.22 3M Medical DepartmentStudy:T-6295.22 Table of Contents Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport:FACT TOX-160 ElMS E00-1668 GLP Compliance Statement................................................................................................... 3 GLP Study- Quality Assurance Statement ............................................................................ 4 List of Tables .......................................................................................................................... 6 Study Personnel and Contributors......................................................................................... 7 Introductionand Purpose....................................................................................................... 8 Specimen Receipt and Maintenance...................................................................................... 9 Chemical Characterization of the Reference Substance ....................................................... 10 Sample Preparation and Analysis.......................................................................................... 11 Sera Analyses.................................................................................................................... 11 Liver Analyses ................................................................................................................... 11 Method Summaries ....................................................................................................... ....11 PreparatoryMethods .................................................................................................. 11 Analytical Methods ..................................................................................................... 12 Analytical Equipment.................................................................................................. 12 DeviationsAmendments ................................................................................................... 13 Data Quality Objectives and Data Integrity ............................................................................ 13 Data Summary, Analyses, and Results.................................................................................. 14 Summary of Quality ControlAnalyses Results.................................................................. 14 Statement of Data Quality ................................................................................................. 16 Summary of Sample Results............................................................................................. 16 Statistical Methods and Calculations ..................................................................................... 17 Statement of Conclusion........................................................................................................ 17 References ............................................................................................................................. 17 AppendixA: Control Matrices................................................................................................. 18 Appendix B: Protocol,Amendments, and Deviation(s).......................................................... 19 Appendix C: Extractionand Analytical Methods..................................................................... 40 ETS-8-4.2, "Extractionof Fluorochemical Compoundsfrom Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry," (15 pages) ............................................................ 41 ETS-8-6.0, "Extractionof Potassium Perfluorooctane-sulfonateor other Fluorochemical Compounds from Liver'for Analysis Using HPLC-Electrospray/Mass Spectrometry," (14 pages) .............................................................................................................................. 56 ETS-8-5.2, 'Analysis of Potassium Perfluorooctancesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry,"(11 pages) ........................ 70 ETS-8-7.0, "Analysisof Potassium Perfluorooctane-sulfonateor other Fluorochemicalsin Liver Extracts Using HPLC-Electrospray/Mass Spectrometry," (10 pages)........................... 81 Appendix D: Data Summary Tables ....................................................................................... 91 Appendix E: Data Spreadsheets ............................................................................................ 92 3M Environmental Laboratory 3M Environmental Laboratory Page 5 Page 5 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M MedicalDepartmentStudy:"1"-6295.22 AnalyticalReport:FACT TOX-160 LIMS E00-1668 Appendix F: Example Calculations............................................................... :......................... 107 Appendix G: Interim Certificate(s) of Analysis ....................................................................... 108 Appendix H: Report Signature Page ...................................................................................... 113 List of Tables Table 1. Study Timeline ......................................................................................................... 8 Table 2. CynomolgusMonkey Specimen Receipt for Study (#6329-268) ............................ 9 Table 3. Characterization of the Analytical Reference Substance in Study FACT-TOX-160 ......................................................................................................... 10 Table 4. Target Ions Monitored in 3M LaboratoryAnalyses.................................................. 13 Table 5. Determinationsof the LOQ For the ExtractedCurve in the Analyses of Serum and Liver Extracts................................................................................................. .... 14 Table 6. Uver Matrix Spike Recoveries ................................................................................. 15 Table 7. Sera Matrix Spike Recoveries ................................................................................. 16 Table 8. Characterization of the Control Matrices Used for Serum and Liver Analyses in Study FACT-TOX- 160 .............................................................................................. 18 Table g. Data Summary for PFOS in Serum FACT-TOX-160 - ptg/mL................................. 91 Table 10. Data Summary for PFOS in Liver FACT-TOX-160 - I.tg/g..................................... 91 3M Environmental Laboratory 3M Environmental Laboratory Page 6 Page 6 3M Medical Department Study: T-6295.22 3M Medical DepartmentStudy:T-6295.22 Study Personnel and Contributors Study Director Andrew Seacat, Ph.D. 3M Corporate Toxicology Building220-2E-02 St. Paul, MN 55144 Analytical Chemistry Laboratories Serum and Liver Analyses 3M EnvironmentaLl aboratory(3M Lab) LisaClemen, Principal Analytical Investigator Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport:FACT TOX-160 LIMS E00-1668 Sponsor 3M Corporate Toxicology 3M Medical Department Building220-2E-02 St. Paul, MN 55144 John Butenhoff, Ph.D., Sponsor Representative Serumand Liver Extractions Pace AnalyticaSl ervices,Inc.---Tier2Facility 3M Lab ContributingPersonnel Rhonda S. Dick* Kelly Dorweiler* Kristen J. Hansen Marlene M. Heying* Harold O. Johnson *Contractlabprofessionasl erviceemployees Location of Archives Ognjenka Krupljanin* Kelly J. Kuehlwein* Sally A. Linda* Bob W. Wynne* All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory and will be retained according to 40 CFR Part 792 requirements. The test substance r and analytical reference standard reserve samples, as well as the specimens pertainingto the analytical phase of this studyare archived at the 3M EnvironmentalLaboratory and will be retained according to 40 CFR Part 792 requirements, RM /::nvirnnmRntM LRhor_torv 3M Environmental Laboratory Paa_ 7 Page 7 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport:FACT TOX-160 LIMS E00-1668 Introduction and Purpose The purpose of the study is to determine the presence and concentration of PFOS in cynomolgus monkey sera and liver samples taken from Covance study# 6329-268, "Extended Recovery Study Followinga 26-Week Capsule Study with PerfluorooctaneSulfonic Acid Potassium Salt (PFOS; T6295.22) in Cynomolgus Monkeys." The animals from study# 6329-268 were previouslyassigned to a completedin-life Covance study# 6329-223, "26-Week Capsule ToxicityStudy with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295.7) in CynomolgusMonkeys" and a completed analyticalstudy# FACT-TOX-030, "Analytical LaboratoryReport from the 26-Week Capsule Toxicity Study with PerfluorooctanesulfonicAcid Potassium Salt (T-6295.7) in Cynomolgus Monkeys on the Determination of the Presence and Concentrationof Perfluorooctanesulfonate (PFOS) in Liver and Serum Samples". During study# 6329-223, the animals received 0.15 mg/kg/day of PFOS as a single daily capsule dose for at least 26 weeks followed by a 52-week recovery. At the end of the initial recovery the animals were transferred to a follow-up study (Covance 6329-268, T-6295-22) for evaluation of extended recovery. Animals were not treated in the follow-up study. The sara and liver samples for this study are the productof the in-life recovery study completed by Covance Analytical Research Laboratoriesunder study# 6329-268 (T-6295.22). Analyses of sera and liver samples were completed by the 3M Environmental Laboratoryunder study number FACTTOX-160 (E00-1668), and the resultsof these analyses are presented in this report. The analytical portionof this study was initiated on 27 August,2001. Table 1. Study Timeline 26 Weeks 52 Weeks Recovery In-LifeStudy:.Covance6329-223 AnalyticaSl tudy: FACT-TOX-030 3M Medical Study: 1"-6295.7 52 Weeks ExtendedRecovery In-LifeStudy:C. ovance6329-268 AnalyticaSl tudy:FACT-TOX-160 3M Medical Study: T-6295.22, 3M Environmental Laboratory 3M Environmental Laboratory PaQe8 Page 8 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:"1"-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport:FACT TOX-160 LIMS E00-1668 Specimen Receipt and Maintenance The 3M Environmental Laboratory received cynomolgus monkey specimens collected at the end of the in-life study #6329-268 from 09 May, 2000 to 15 March, 2001. All specimens were received frozen in good conditionon dry ice and were immediatelytransferred to storage at -50C 20C. Specimens that were extracted at Pace Tier2 were shippedfrozen on dry ice. Table 2. Cynomolgus Monkey Specimen Receipt for Study (#6329.223) Receipt Date 05/09/00 07/06/00 08/30/00 10/24/00 12/21/00 03/13/01 03/15/01 Tlmepolnt Week 9 Week 17 Week 25 Week 33 Week 41 Week 53 Week 53 Specimen II Serum Serum/Urine/Feces Serum Serum/Urine/Feces Serum Serurn/Udne/Feces Liver/Lung Kidney/Spleen Thyroid/Brain AbdominalFat Heart/Bile Serum Number Received 4 4/4/4 4 4/4/4 4 4/4/4 4/4 4/4 4/4 4 4/4 3 additionalvials from sample 105552 Control matrices used in sere and liver analyses performed during FACT-TOX-160 were obtained from commercial sources and are presented in Appendix A. Samples analyzed at the 3M Environmental Laboratorywill be stored and maintained at the laboratory accordingto 40 CFR Part 792 requirements. 3M Environmental Laboratory 3M Environmental Laboratory Paee 9 Page 9 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M MedicalDepartmentStudy: T-6295.22 AnalyticalReport: FACT TOX-160 LIMS E00-1668 Chemical Characterization of the Reference Substance Potassium perfluorooctanesuffonate (KPFOS) CAS Number: 2795-39-3 Chemical Formula: CeF17SO3K* Molecular Weight: 537.9 Chemical characterization informationon the reference substances KPFOS used in this study is presented in tabular form below. Table 3. Charecterization of the Analytical Reference Substances in Study FACT-TOX-160 Location 3M Lab Substance PFOS SD-018 THPFOS (SurrogateStandard) TCR-00017-055 Source 3M ExpirationDate Storage Condltlone ChemicalLot Number 08/31/06 Frozen -20C +/- 10C 217 Physical _a_on WhitePowder Purity 86.90/.0 ND- Notdetermined NA- NOteva]able *SeeCediftcaotefAnalysftrsomCe, ntreAr_ LabocatodIneAsppendGix. SynQuest ND Frozen -20C +/- 10C Q-75-91 WhitePowder NA 3M Environmental Laboratory 3M Environmental Laboratory PaQe10 Page 10 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:"1"-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport: FACTTOX-160 LIMS E00-1668 Sample Preparation and Analysis Sera As per the study protocol, all serum samples were analyzed in this phase study. Sera samples were extracted beginning on 31 August, 2001 usingan ion pairing reagent and methyl-tert-butyl ether (MtBE). Sample extracts were analyzed using high-performanceliquid chromatography-electrospray/tandemmass spectrometry(HPLC-ESMSMS) in the multiplereaction mode versus an extracted rabbitsara curve. PFOS levels were quantitated by external calibration. uver,am As per the study protocol, all liver samples were analyzed in this phase study. Liver samples were extracted beginningon 05 September, 2001 using an ion pairingreagent and methyl-tert-butyl ether (MtBE). Sample extracts were analyzed using high-performance liquid chrornatography-electrospray/tandemmass spectrometry (HPLC-ESMSMS) in the multiple reaction mode versus an extracted rabbit liver curve. PFOS levels were quantitated by external calibration. Method Summaries Following is a brief descriptionof the methods used duringthis analyticalstudy by the 3M Environmental Laboratory.Detailed descriptionsof the methods used in this study are located in Appendix C. . 3M Environmental Laboratory PREPAR.4 TORY METHODS ETS-8-4.2, "Extraction of Fluorochemical Compoundsfrom Serum for AnalysisUsing HPLCElectro,spray/MassSpectrometry" Analyticalsamples were extracted using an ion-pairingextraction procedure: An ion-pairing reagent was added to a laboratorysample and the analyte ion pair was partitionedinto methyltert-butyl-ether (MtBE). The MtBE extract was then removed and put into a nitrogenevaporator untildry. Each extracted laboratory sample was reconstitutedin 1.0 mL of methanol and passed through a 0.2 IJm nylonfilter using a 3 mL disposable plasticsyringe into glass autovials. RM EnvironmRntRl LRhnrRtotv 3M Environmental Laboratory Pan_. 1 1 Page 11 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 3M MedicalDepartmentStudy:"i"-6295.22 AnalyticalReport:FACT TOX-160 LIMS E00-1668 ETS-8-6.0, "Extractionof Potassium Perfluorooctane-sulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry" Liver samples were homogenized in water. An aliquot of each homogenate was spiked with THPFOS and extracted using an ion-pairingextraction procedure.An ion-pairing reagent was added to the sample and the analyte ion pair was partitionedinto MtBE. The extract was transferred to a centrifuge tube and put onto a nitrogenevaporatoruntil dry. Each extract was reconstitutedin 1.0 mL of methanol and passed througha 0.2 _m nylon filter, using a 3 mL disposable plastic syringe into glass autosampler vials. ANALY'nCALMETHODS ETS-8-5.2, "Analysisof Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0, "Analysis of Potassium Perfluorooctane-sulfonateor other Fluorochemicals in liver Extracts Using HPLC-Electrospray/Mass Spectrometry" The analyses were performed by monitoringone or more productions selected from a single primary ioncharacteristicof a particularfluorochemical using HPLC/ES/MS/MS. For example, molecularion 499, selected as the primary ion for PFOS (C8F_7SO3-)analysis, was fragmented to produce ion 99 (FSO3-). The characteristicion99 was monitored for quantitativeanalysis. ANALYTICALEQUIPMENT The followingis representative of the settings used duringthe analyticalphase of this study. Liquid Chromatograph: Hewlett-Packarde Series 1100 LiquidChromatograph system Analytical column: Keystone BetasilTM C182x50 mm (5 pro) Column temperature: 30C Mobile phase components: Component A: 2raM ammonium acetate Component B: methanol Flow rate: 300 IJL/rnin Injectionvolume: 10 pL Solvent Gradient: 9.0 minutes Time (minutes) 0.0 1.0 5.5 7.5 8.0 %B 10% 10% 95% 95% 10% 3M Environmental Laboratorv 3M Environmental Laboratory Paoe 12 Page 12 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport: FACTTOX-160 LIMS E00-1668 Mass Spectrometer: MicromassAPI/Mass Spectrometer Quattro I1" Triple Quadrupole system Software: Mass Lynx" 3.4 Cone Voltage: 30-70 V Collision Gas Energy: 20-50 eV Mode: ElectrosprayNegative Source BlockTemperature: 150C 10C Electrode: Z-spray AnalysisType: Multiple Reaction Monitoring (MRM) Table 4. Target Ions Monitored in 3M Laboratory Analyses Target Analyte PFOS Pdmary Ion(/u_J) I 499.0 ProductIon (/uuru) 99.0 THPFOS 427.0 80.0 IBevlationsLAmendments There were one amendment and seven deviations from the odginal protocol. Amendments and deviationsfrom the original protocol and methods are includedin the Appendix B. Data Quality Objectives and Data Integrity The following data qualityobjectives (DQOs) were indicated in the protocolfor this study: Linearity: The coefficient of determination (r_) equal to or greater than 0.985 for liver analyses and equal to or greater than 0.990 for sere analyses using 1/x weighting. Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standardin the calibrationcurve, defined as the loweststandard that is both 2 times the matrix blank and is calculated within 30% of the expected concentration. Acceptable Precision: Quality control samples are required to meet 25% precisionfor analyses, providedthey are quantitated within the selected calibrationrange. Acceptable Spike Recoveries: Matrix spikes and matrix spike duplicates requiredfor analysis of liver and sera samples should show spike recoverieswithin 70-130%. Confirmatory Methods: If a confirmatorymethod is used, an amendment to this protocolwill be written. Demonstration of Specificity: PFOS identification will be substantiated by chromatographic retention time (approximately 8.00 minutes), by the characteristic primary ion (499) and characteristic product ion (99). 3M Environmental Laboraton/ 3M Environmental Laboratory PaQe13 Page 13 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 3M MedicalDepartmentStudy:T-6295.22 AnalyticalReport:FACTTOX-160 LIMS E00-1668 Data Summary, Analyses, and Results Data qualityobjectives for the analytical phase of this studyoutlined in the 3M Environmental Laboratory protocol for FACT-TOX-160 (see Appendix B) were met withthe exceptions noted in this report. Summary of Quality Control Analyses Results Linearity: The coefficientof determination (r_) of the standard curve was _>0.985for liver analyses and _>0.990for sera analyses. Calibration Standards: Quantitation of the target analytes was based on linear regression analysis, 1/x weighted of one or two extracted matrix curves bracketing each group of samples, except as noted in the deviation summary. High and/or low pointson the curve may have been deactivatedto provide a better linear fit over the curve range most appropriateto the data. Low curve pointswithpeak areas less than two times that of the extraction blanks were deactivated to disqualifya data range that may have been significantlyaffected by backgroundlevels of the analyte. Occasionally,a single mid-range curve pointthat was an obviousoutlier may have been deactivated. Quantitation of each analyte was based on the response of one specific product ion(s) using the multipleresponse-monitoringmode of the instrument(see Appendix C, Analytical Methods). Limits of Quantitation (LOQ): The LOQ is equal to the lowestacceptable standard in the calibrationcurve that is within 30% of the theoretical value, and is at least two times the analyte peak area detected in the extraction blanks. Table 5. Determinations of the LOQ For the Extracted Curve in the Analyses of Serum and Liver Extracts Analyte II PFOS---Serum PFOS----Liver Method LOQ 4.92 ng/mL 12.6 ng/g Blanks: All blanks were below the lower limit of quantitationfor the compounds of interest. To simplify analyses that were complicated by endogenouslevels of fluorochemicals in unexposed monkey sera which were above the lower limitof quantitation, rabbitsera was selected as a suitable surrogate matrixand all rabbit sera blanks were withincriteria. Precision: precision was determined by analysis of CCVs in sera and was reproducibleto within 25%, precisionwas determined by analysis of CCVs in liver and was reproducibleto within 30%. Matrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of liver and sera samples and analyzed at the 3M EnvironmentalLaboratory(see tables 5 and 6). Two rabbit liver matrix spikes, extracted 09/05/01, were within criteriaand two monkey liver matrix spikes were not within :J:30%of the theoretical concentration when analyzed 09/17/01. The monkey liver matrix spikeswere not prepared at the appropriateconcentrationbased on endogenous levels present in the samples. Additional monkey liver matrix spikes and one monkey liver sample were re-extracted on 10/02/01 at levels appropriateto the endogenous 3M Environmental Laboratorv 3M Environmental Laboratory Paoe 14 Page 14 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M MedicalDepartmentStudy:%6295.22 AnalyticalReport:FACT TOX-160 LIMS E00-1668 levels. The re-extracted spikes did not meet criteriaand the extraction was suspect due to an inconsistentsample concentration, inconsistentmatrixspike recoveries, and high surrogate values - refer to deviations attached to this report for more information. Additional monkey liver matrix spikes and one monkey liver sample were re-extracted on 11/07/01. The extracted spikes and sample were in agreement with the initialextraction on 09/05/01. The rabbit liver matrix spike averages extracted with the samples on 09/05/01 and the monkey liver matrix spike averages extracted on 11/07/01 were within + 35% of the theoretical concentration. All sara matrix spike recoveries, evaluated versus extracted and unextracted curves, were within 25% of the theoretical concentration. Extraction efficiencyand absolute recovery were >100% in the sara matrix, based on average recoveries of 101%, 111%, 111%, and 116% versus an unextracted (solvent) curve. Table 6. Liver Matrix Spike Recoverles Matdx Extraction Date Analysis Date Type % Recovery Average Liver 09/05/01 09/10/01 Rabbit 09/17/01 Monkey 102/, 102% 298o/o 290% 102o 294% 10/02/01 Suspect Extractioninconsistent sample concentration saunrdrohgigahte 10/04/01 Initial Analysis 10/16/01 Reanalysis Monkey 2 ug/g Monkey 10 ug/g Monkey 2 ug/g 55% 15% 60% Too Dilute 30% -11% 35% NA 9% de1v0i/a2t4io/0nf1or Monkey 65% analysis 10 ug/g 43% 54% 10/24/01 Monkey 178% Redilution fromodginal sample 2 ug/g Monkey 10 ug/g 61% 2680/0 216% 119% 242% 11/07/01 11/15/01 Monkey 2 ug/g 100% 90% 95% Monkey 10 ug/g 73% 61% 67% 3M Environmental Laboratory 3114Environmental Laboratory Paoe 1fi Page 15 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:%. 6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticaRl eport:FACT TOX-160 LIMS E00-1668 Table 7. Sera Matrix Spike Recoveries Matdx Extraction Date Analysis Date Type % Average Recovery Sera 08/31/01 10/02/01 E0v9a/0lu6a/0te1d Monkey Versusan 25 ng/mL Unextracted Curve= Absolute Recovery Monkey 500 ng/mL 10104/01 VEevrasluuasatend Extracted Curve Monkey 2.0 ug/mL Monkey 10 ug/mL 10/04/01 Evaluated Monkey UVneerxsturasacnted 2.0 ug/mL Curve = Absolute Monkey Recovery 10ug/mL 118% 840/0 111% 112% 104% 99% 119/. 121% 113% 109% 115% 117% 101% 111% 102% 120% 111% 116% Surrogates: The surrogate (3"HPFOS)was added to all samples and standards. THPFOS was not used for quantitation,but was used to monitorfor gross instrumentfailure. The surrogate responseof each analyticalrun utilizingextracted matrixcalibrationcurves was verified to r determine that itdid not vary more than 50% from the mean withineach analytical run. All responses were within 50% except for analysis of liver dilutionson 10/24/01. These samples were >50%. These samples were considered suspect and re-extracted. Analysis of these re- extracts met surrogatecriteria requirements. of Data Qual_y It is not possibleto verify true recovery of endogenous analyte from tissueswithout radio-labeled reference material. The only measurementof accuracyavailable at this time, matrixspike studies, indicatesthat these data are quantitativeto 25% or greater in sara and 35% or greater in liver. Summary of Sample Results PFOS results (those obtained using lot# 217) have been corrected for purity of the analytical reference material. * Samples from Control Animals: No controlanimals were included in this study. ,, Samples from Dosed Animals: In general, PFOS levels found in sera of the test animals decreased over time. PFOS levels in male liverswere approximatelyhalf that determined in female livers. Detailed sample data tables are presented in Appendices D and E. 3M Environmental Laboratory 3M Environmental Laboratory Page 16 Page 16 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M MedicalDepartment Study:T-6295.22 AnalyticalReport:FACT TOX-160 LIMS E00-1668 Statistical Methods and Calculations Statistical methods were limited to the calculation of means and standard deviations. See Appendix F for example calculations used to generate the liver and serum sample data in FACT-TOX-160. Statement of Conclusion Under the conditionsof the present studies, the fluorochemical PFOS was observed in the serum and liver of all recovery study cynomolgus monkeysoriginallydosed with the test substanceduring the in-life phase of the study #6329-223 (TOX-030). References None 3M Environmental Laboratorv 3M Environmental Laboratory Paae 17 Page 17 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport:FACT TO)(-160 LIMS E00-1668 Appendix A: Control Matrices Table 8. Characterization of the Control Matdces Used for Serum and Liver Analyses in Study FACT-TOX-160 Control Matrix Source ExpiratioDnate StoragCe onditions ChemicaLlot# PhysicaDlescription Monkey Serum TCR-g9131-022 LampireBiological 01/01/'2010 Frozen-20"C+/- 10"C 111022515 MonkeSyerum Rabbit Serum TN-A-4511 Sigma-Aldrich 09/26/05 Frozen-200C+/-10C 99H8400 RabbiSt erum Rabbit Liver TCR-99131-046 I CovancLeaboratory NA Frozen-20C +/-10C F04053 RabbiLt iver .?M Fnvirnnmp.nt_l L_hnrRtn:v 3M Environmental Laboratory P_nA 1R Page 18 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 AnalyticalReport:FACT TOX-160 LIMS E00-1668 Appendix B: Protocol, Amendments, and Deviations 3M Environmental Laboratory 3M Environmental Laboratory Paae 19 Page 19 3M Medical Department Study: T-6295.22 3M Environmental Technology and Services PO Box 33331 St. Paul, MN 55133-3331 612 778 6442 Analytical Report: FACT-TOX- 160 LIMS E00-1668 Protocol #FACT-TOX-160 StudyTitle Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys ANALYTICALPHASEPROTOCOL Au_or LisaClemen Date: August 27, 2001 Performing Sera and Liver Analyses 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratories Sera and Liver Extractions Pace Tier2 Facility 1700 Elm Street, Suite 200 Minneapolis, MN 55414 Laboratory Project Identification FACT TOX-160 Covance In-life Study Number: 6329-268 3M Medical Department Study: T-6295.22 ET&SS LIMS: E00-1668 3M EnvironmentaLl aboratory 3M Environmental Laboratory Page 1of 9 Page 20 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol#FACT"-TO)1(-60 Study Identification Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooctane Sulfonie Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys Sponsor 3M Corporate Toxicology 3M Medical Department Building 220-2E-02 St. Paul, MN 55144 Sponsor Representative Study Director Principal Analytical Investigator (PAl) Phase Locations In vivo Testing Facility JohnButenhoff, Ph.D. 3M CorporateToxicology 3M Medical Department Building 220-2E-02 St. Paul, MN 55144 Telephone: 651-733-1962 Andrew Seacat, Ph.D. 3M Corporate Toxicology 3M Medical Department Building 220-2E-02 St. Paul, MN 55144 Telephone: 651-575-3161 Lisa Clemen Covance Laboratories, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Analytical Testing Laboratories (sera and liver analyses) 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 (sera and liver extractions) Pace Tier2 Facility 1700 Elm Street, Suite 200 Minneapolis, MN 55414 Proposed Study Timetable Experimental Start Date Experimental Completion Date 27 August 2001 29 April 2002 3MEnvironmentalLaboratory 3M Environmental Laboratory Page 2 of 9 Page 21 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol #FACT-TO)(-160 1. STUDY Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys. 2. PURPOSE This study is designed to continue the assessment ofperfluorooctanesulfonate (PFOS) levels in sera and liver during an extended recovery time of approximately one year following the daily administration of the test material by capsule to eynomolgus monkeys for at least 26 weeks and at least 52 weeks of recovery. The animals used in this study were previously assigned to a recovery phase of a completed study for at least 52 weeks. At the end of this initial recovery phase study Covance #6329223, a partial hepatectomy was conducted on the animals, they were allowed to recover from the surgical procedure, then transferred to this follow-up study for evaluation of recovery for an additional 52 weeks. The serum and liver ofcynomolgus monkeys will be analyzed for PFOS. Additional tissues or fluids may be analyzed at the discretion of the PAI or study director. The in-life portion of this extended recovery study was conducted at Covance Laboratories, study #6329-268. 3. REGULATORY COMPLIANCE This study will be conducted in accordance with the United States Environmental Agency Good Laboratory Practice Standards, 40 CFR 792. Protection 4. QUALITY ASSURANCE The 3M Environmental Laboratory Quality Assurance Unit will review the protocol and audit study conduct, data, and the final report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. 5. TEST MATERIAL Refer to Covance Laboratory protocol for study #6329-268. Animals were not treated during this study. The FACT TOX-160 study is an extended recovery study ofcynomolgus monkeys from Covance study 6329-223 (analytical work performed under 3M Environmental Study # FACT-TOX-030), where animals received 0.15 mg/kg/day of PFOS lot # 217 in a daily single capsule for at least 26 weeks, followed by a 52-week recovery period. 6. CONTROL MATRICES Types of control matrices and their source, physical description, storage requirements, and traceability numbers will be recorded in the raw data and included in the final report. L REFERENCEMATERIAL FOR FACT TOX-160 Potassium perfluorooctanesulfonate (KPFOS), CsFITSO3K*, CAS 2795-39-3 3M Environmental Laboratory 3M Environmental Laboratory Page 3 of 9 Page 22 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol #FACT-TOX-160 The target analyte is perfluorooctanesulfonate (PFOS), CsF_TSO3-. 8. TESTSYSTEM Cynomolgus monkeys were used as the test system, and were maintained and dosed as described in the original Covance protocol #6329-223 (TOX-030). Two male and two female monkeys which were dosed during study #6329-223 (TOX-030) were included in this extended recovery study and all were allocated to Group 1. These animals received no further treatment during the recovery study. 9. SPECIMENAND SAMPLE RECEIPT The 3M Environmental Laboratory received specimens of the following body tissues and fluids from the indicated points in the study from Covance Laboratories at the end of the inlife phase of the study. All specimens were frozen and packed on dry ice for shipping. Body tissue/fluid Serum - all animals Liver - all animals Collected 2, 4, 6, 8, 10, and 12 months following initiation of study At scheduled sacrifices At scheduled sacrifices Expected # of specimens -24-28 serum samples 4 Total number of test animals: 4 Modifications to the number of samples analyzed may be implemented at the discretion of the PAI and the study director. Specimens sent to 3M Environmental Laboratories were received and tracked according to the Sample Tracking System Standard Operating Procedure. Details of specimen inspection for damage, receipt, storage, identification, chain of custody, protocols and data will be presented in the phase report. 10. PREPARATORYMETHODS 10.1 ETS-8-6, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4, Extraction of Fluorochemical Compounds from Serum for Analysis Using HPLC-ElectrosprayRClass Spectrometry 10.3 If preparatory methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 3M Environmental Laboratory 3M Environmental Laboratory Page 4 of 9 Page 23 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol #FAC7"-TO)(-160 11. ANAL YTICALMETHODS 11.1 ETS-8-7, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry 11.2 ETS-8-5, Analysis of Potassium Perfiuorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Eleetrospray/Mass Spectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 12. DATA QUALITY OBJECTIVES The number of spikes/duplicates, use of surrogates,and information on other data quality indicators are included in the analytical methods. In addition, the following criteria will be met: 12.1 Linearity The coefficient of determination (r_) of the extracted liver standard curve must be equal to or greater that 0.985 using linear regression or quadratic fit. The coefficient of determination (r2) of the extracted serum standard curve must be equal to or greater that 0.990 using linear regression or quadratic fit. 12.2 Limits of Quantitation (LOQ) The LOQ will be equal to the lowest acceptable standard in the calibration curve, defined as the lowest standard that is both 2 times the matrix blank and is calculated within + 30% of the expected concentration. 12.3 Acceptable Precision Quality control samples are required to meet + 25% precision, provided they are quantitated within the selected calibration range. 12.4 Spike Acceptable Recoveries Matrix spikes and matrix spike duplicates required for analysis of liver samples should show spike recoveries within 70%-130%. ARer liver samples have been analyzed, sample concentrations will be evaluated. If a measured sample (s) concentration exceeded the calibration range of the method and was diluted with methanol into the validated calibration range, then additional matrix spikes will be prepared at the approximate measured concentrations of the samples then subsequently diluted with the same volume of methanol as the sample into the validated range of the calibration curve. These liver matrix spikes will be evaluated versus an extracted matrix calibration curve and an unextracted calibration curve. If the diluted liver matrix spikes do not show recoveries within 70-1305o versus the extracted matrix calibration curve, then the 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 9 Page 24 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol #FA C T- TOX- 160 sample concentrations, at the same dilution factor as the matrix spikes, will be corrected for the spike recovery. For serum matrix spikes, a comparison of extracted (matrix) versus unextracted (solvent) calibration standards will be performed at two concentrations spanning the validated calibration range of the method. This comparison will demonstrate the absolute recovery (recovery + serum matrix affect) ofanalyte from the matrix for determining the extraction efficiency of serum versus an unextracted calibration curve. After serum samples have been analyzed, sample concentrations will be evaluated. If a measured sample (s) concentration exceeded the calibration range of the method and was diluted with methanol into the validated calibration range, then additional matrix spikes will be prepared at the approximate measured concentrations of the samples then subsequently diluted with the same volume of methanol as sample into the validated range of the calibration curve. These serum matrix spikes will be evaluated versus an extracted matrix calibration curve and an unextracted calibration curve. If the diluted serum matrix spikes do not show recoveries within 70-130% versus the extracted matrix calibration curve, then the sample concentrations, at the same dilution factor as the matrix spikes, will be corrected for the spike recovery. 12.5 Use of Confirmatory Methods Ira conftrmatory method is used, an amendment to this protocol will be written. 12.6 Demonstration of Specificity PFOS identification will be substantiated by chromatographic retention time (approximately 8 minutes), by the characteristic primary ion (499) and the characteristic product ion (99). . Minor modifications to the Data Quality Objectives may be implemented at the discretion of the PAI. These will be documented in the raw data and the analytical report. 13. SuB-CONTRACTEDANALYSlS 13,1 All sera and liver extractions as detailed in this protocol will be performed at Pace Tier2, 1700 Elm Street, Suite 200, Minneapolis, MN 55414. 13,2 All sera and liver analyses as detailed in this protocol will be performed at 3M Environmental Laboratory, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 13.3 An amendment to this protocol will be written if extractions and analyses are performed at laboratories other than the 3M Environmental Laboratory or Pace Tier2. 14. STATISTICAALNALYSIS Statistical methods will be limited to the calculation of means and standard deviations. Examples of the calculations used in the analyses will be included in the analytical phase report. 3M Environmental Laboratory 3M Environmental Laboratory Page 6 of 9 Page 25 3M Medical Department Study: T-6295.22 r Analytical Report: FACT-TOX-160 LIMS E00-1668 Proto col #FA C 7"-TO)(- 160 15. REPORT A report of the results of the study will be prepared by 3M Environmental Laboratory. The report will include, but not be limited to, the following, when applicable: 15.1 Name and address of the facility performing the phase 15.2 Dates upon which the phase was initiated and completed 15.3 A statement of compliance by the PAl addressing any exceptions to Good Laboratory Practice Standards 15.4 Objectives and procedures as stated in the approved phase protocol, including any amendments to the original phase protocol 15.5 Identity, purity, stability and the solubility of the reference standard under conditions of use 15.6 A description of the methods used to conduct the test(s) 15.7 A description of the specimens 15.8 A description of any circumstances that may have affected the quality or the integrity of the data 15.9 The name of the PAl and the names of other scientists, professionals, and supervisory personnel involved in the phase 15. IOA description of the transformations, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement of the conclusions drawn from the analyses 15. llStatistieal methods used to evaluate the data, if applicable 15.12 15.13 The signed and dated reports of each of the individual scientists or other professionals involved in the phase, if applicable The location where raw data and the final report are to be stored 15.14 A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made, and the dates of any findings reported to the Study Director and Management If it is necessary to make corrections or additions to a final report alter it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The anaendment will clearly identify the part of the final report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 16. LOCATION OF RAW DATA, RECORDS, AND FINAL REPORT Original data, or copies thereof, will be available at 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the phase report. When the phase report is completed, all original paper data, including those items listed below, will be retained in the archives of 3M Environmental Laboratory. All corresponding 3M Environmental Laboratory 3M Environmental Laboratory Page 7 of 9 Page 26 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 Protocol #FACT"T- O)(- 160 training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained at the 3M Environmental Laboratory. 16.1 The following raw data and records will be retained in the study folder in the study/project archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 16.1.4 Raw data 16.1.5 Approved final report (original signed copy) 16.1.6 Electronic copies of data 16.2 The following supporting records will be retained separately from the study folder according to 3M Environmental Laboratory Standard Operating Procedures: 16.2.1 Training records 16.2.2 Calibration records 16.2.3 Instrument maintenance logs 16. 2.4 Standard Operating Procedures, Equipment Procedures, and Methods 17. SPECIMENRETENTION Specimens remaining aider the analytical by: Lisa Clemen 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Telephone: (651) 778-6176 phase is completed will be sent to and maintained 18. PROTOCOLAMENDMENTSAND DEVIATIONS Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. All changes to the protocol will be indicated in the final report. Any other changes will be in the form of written deviations, signed by the Study Director and Sponsor Representative and filed with the raw data. 3M Environmental Laboratory 3M Environmental Laboratory Page 8 of 9 Page 27 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol#FACT-TOX-f 60 19.A TTACHMENTS 19.1 Attachment A: Material Safety Data Sheets (MSDS) for Reference Standard 20. SIGNATURES Andrew_S"eacat, Ph.D., Study Director Date John Butenhoff, Ph.D., Sponsor Representative Date Lis_ Clemen, Principal Analytical Investigator Date 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 9 of 9 Page 28 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study Title Extended Recovery Study Following a 26-Week Capsule Toxicity Study (FACT-TOX-030) with Perfluorooetane Sulfonic Acid Potassium Salt (PFOS; T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 1 Amendment Date: April 4, 2002 Performing Laboratory 3M EnvironmentalTechnology& SafetyServices 3M Environmental Laboratory 935 Bush Avenue St. Paul, _ 55106 Laboratory Project Identification FACT TOX-160 Covance In-life Study Number: 6329-268 3M Medical Department Study: T-6295.22 ET&SS LIMS: E00-1668 RM Envirnnm_ntal Laboratory 3M Environmental Laboratory Page 29 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol FACT- TO)(- 160 Amendment No. 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOLREADS: Section 10 states that method ETS-8-6.0 will be used for extracting liver samples and section 12 of method ETS-8-6.0 references the use of equipment procedure AMDT-EP-22 for cleaning the tissue grinder. AMENDTOREAD: Section 12 of method ETS-8-6.0 changed to reference equipment procedure ETS-9-52.0 as of 11/07/01. REASON: Equipment procedure AMDT-EP-22 was replaced by equipment procedure ETS-9-52.0 on 11/07/01. Both cover the tissue grinder's cleaning procedure. 2. PROTOCOLREADS: Section 11 states that method ETS-8-7.0 will be used for analyzing liver extracts using HPLCElectrospray/Mass Spectrometry AMENDTOREAD: Method Modification Method: ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" Section modified: 10.3.2, 14.5.1, add sections 14.3.2-14.3.6 Effective date of modifications: July 22, 1999 Section 10.3.2 Method reads: Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. Modify method to read: Analyze a mid-rangcea, bration standard at least after every ten samples, with a minimum of one per batch. Section 14.5.1 Method reads: Continuing calibration verification percent recoveries must be within + 30% of the spiked concentration. Modify method to read: One continuing calibration verification per ten samples must show a percent recovery within +/-30% of the spiked concentration. 3M Environmental Laboratory 3M Environmental Laboratory Page 30 3M Medical Department Study: T-6295.22 Section 14.3.2 Method reads: NA Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol FACT- TOX- 160 Amendment No. 1 Modify method to read: The second (bracketing) calibration curve may be deactivated if instrumental drift affects the data. The first curve and acceptable calibration checks shall bracket usable data. Section 14.3.3 Method reads: NA Modify method to read: Calibration standards with peak areas less than 2 times the curve matrix blank should be deactivated to disqualify a data range that may be affected by background levels of the analyte. Section 14.3.4 Method reads: NA Modify method to read: Low or high curve points may be deactivated to optimize a linear range appropriate to the data. Section 14.3.5 Method reads: NA Modify method to read: A curve point may be deactivated if it deviates more than 30% from the theoretical value when the curve is evaluated over a linear range appropriate to the data. Section 14.3.6 Method reads: NA Modify method to read: A valid calibration curve must contain at least 5 active points. REASON: Method modifications are improvements/clarifications to the current method. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 31 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Protocol FACT- TOX- 160 Amendment No. 1 Amendment Approval Andrew Se_cat, Ph.D., Sponsor Representative/Study Director Lisa A. Clemen., Principal Analytical Investigator Date William K. Reagen, Ph.D., Performing Laboratory Management Date 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 32 3M Medical Department 3M Confidential Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Record of Deviation I. Identification Study/ Project No. FACT-TOX-160, E00-1668 Deviaiion type ......................s..oP ................. Method .......... Equipment PrOcedure " (Check one) X Protocol Other: Doc_ent number ......................................................................D...a..i.e..(.s.)....0..f.o..c..c.u.._ence ............. FACT-TOX- 160, E00-1668 09/05/01 II. Description Required procedure/process: ..P..rotocoi_FA_-Tox-_i6_O_EOO-i6dSstatesiMa_spikesdudma_x liver sami_lesshoulds'howS_ik7_e_ov_riewsithin70%-130%. spikeduplicates requiredt-or analysis Of "A_eriiver samples liave_beenanaiyzedl sample coneenirati0nSWiitbe evaluaied.If a measuredsample(s) .......... eoncentrati0nexceeded_ecaiii_raiJon range0fthe-meth0d _d wasdilutedwi_ methanol into the Validatcd ................. caiibrati0nrange,_en ad_ti0nalma_x _es-will hepreparedat _eappr0ximate measuredconcentrati0ns0f the samples then subsequently diluted with the same volume of methanol as the sample into the validated range of the 'eaiii_rationc_efTheseliver _Spikes-Wili beevaJuat_ed Versusanextracted matrix Calibration cu_;e and an _extractedc_ilibr-a_oncuweflf_e-ijil'uied liver ma_xsi_ikes do not siaow recoveries within 70-130% versus the extraciea marx caiii_raiionc_ve, _enfiie sampieconeenlrations, at the same dilution factor as the marx spikes, will be corrected for the si_i-kerec0ve_. .................. Actual procedure/process: "_e_696_ibi s-__ppiS-iO33-6SMma_spike/ma_xsp_edupiicate (lvis_sD) average recove_was .... 29_i%. ;i_heses_pies were not spiked at _eappr0priate leveih_reiati0n to endogenous levels present in _e sample. IlL Actions Taken (such as amendment issued, SOP revision, etc.) New matrix spikes were prepared at 2 ug/g & 10 ug/g then extracted on 10102/01. Recorded by ................................... ................. . . Date , 1,010, Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 of 1 Page 33 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS E00-1668 Record of Deviation L Identification Study / ProjectNo. FA...CT..:TOX-16..0.E,007.!_66.8.......................................... Deviation type SOP Method .... Equipment (Checkone) X Protocol Other: Document number FACT-TOX- 160,E00-1668 Date(s)of occurrence 10/04/01 It. Description Procedure Require d proced_e/process" ....................................................................................................... ProtocolFACT-TOX-160, E00-1668 states: Matrix spikes and matrixspike duplicates required for analysisof liver sables shouldshow spikereeover:ieswitllin70%2130%. " "AReriiversampieshavebeehanaiyzed, san_l-econceniiationswiiibeevaiuated. If a measured sample (s) " 'concen_a_onexcedde-dthe-c-a[ibraffoh-rah-geof_cmc_od-aad wasdiluted frith methanolinto the vaiidated..c..aiibrati0nrange,ihen-a_idii_onaf_t_xspikes wii-ii_eprep_ed-at&e approximate measured concentrati0ns'oethe Samplesthen subsequentlydiluted with the Samevolume of methanolas the sample into the validated range of the _-c_ibmti-_n-_e:....%ese-.i.ffer.`ff_-t_-_.fi_es-wil_.be-_eva_uaied...veresxutsra-_ciedmatrix Calibrationcurvearid an miex_acted Caiii_rati0n-cuffc.If the dilutediivcr_x spikes do n0(Sh0Wrecoveries within 70:130%versus the " e--xtractedim_eaiibratio//_-u/ve,iia-en _es_ple concenirati0ns,at _esamediiution factor as_e mairixspikes, wiiibe corrected for-thespikerecovet_............................... Actual procedure/process: The2-ug/gma_xsp_e/-r_t_ spike-dupiicate(-/(_S_SD) averagerecoverywas 35% and the'i0 ug/g MS/MSD was too dilute using a 1:500 dilutionfactor. IlL Actions Taken (such as amendment issued, SOP revision, etc.) The 10 ug]g MS/MSD was re-diluted using a 1:50 dilution and analyzed on 10/16/01. The 1:50 dilution of the 2 u_g MS/MSDs was also-reaJnalyzcdonio]] 6101 to co/lfir:m the original low recovery. " ................................................................................................................................................................................................. Rec0rdedby .................................................................................... Date ............ ............:..................l..,..o...!...o...t.......... Authorized by _ - 5p_cu,r i_r_,_',_'. _ot_ t_&,_,_ S_. _ir_..,_'_,'..13_kl't,_0 S_;tg._'_ Deviation No. 2 (,ssigucdby StudyDirectororProjecLteadat theend ofstudy orproject) Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 of 1 Page 34 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS E00-1668 Record of Deviation L "Identification Study / Project No. FACT-TOX-160, E00-1668 Deviation type SOP Method Equipment Procedure (Check one) X Protocol Other: D0cumentnumi0er ..............................................................D...a..t.e.(s)of 0ccu_ence ............... FACT-TOX-160, E00-1668 10/16/01 II. Description R.eq._ed pr0ced_e!process: Protocol FACT-TOX-160,E00-1668 staiesi-Ma_ sp_es-a-ildn;.atrixspikeduplicates required for analysis of liver samples should showsi_iicere-eoveries-wi-th7h0i %-130%. -Aher]ivers_pleshave been-_yzed,s_pie Co-ncenffafionWs iiii_eevaluated. If a measured sampie (s) ..... concentrationexceeded the calibration range of the method and was diluted with methanol into the validated 'c_ii_mfionrange, then addi(ibnaima_ Spikes_iibe prepared attheapproximate measured Concentrationsof _e samples then subsequentlydilutedwith the same volume of methanolas the sample into the validatedrange of the 'calii_tion c_e. _ese iiver_-sp_cs--_lbee:va luatecive_usahexffacted matrix calibrationcui_eand_ -unex_acted-calibrationcui_e_If_edfluiedlivermatrixspikes donot Showrecoverieswithin 70-130%versus the 'extra-ciedmatrix calibrati0n curve, then thes_l_ie-concentraii0ns, at the same dilution factor as the matrix spikes, will be eo=ected for the sp_erecovei_ ................. _Acma! procedu[e/pro tess" ............................................................................................................. The 2 ug/g and 10ug/g matrix spike/matrixspike duplicate(MS/MSD)average recoveriesWere9%and 52%. IlL Actions Taken (suchas amendmenitssued,SOPrevision,etc.) The MS/MSD andI05505M sample were all re-diluted from the original extracts at 1:50 then analyzed on 10/24/01. -Recorded by ..............................................:.................................................................. i Date AuthonzedT_ A. _C_,r_._ .......................y _ "7..-__ 4"/. _ _a_,,_. -S,_ 9. L,,_ _' gtof_,._orD _|rl?.,1__ ....,_,,., _ .1___ .................................... 13-11o I_l Da_./3.//o /" Deviation No. 3 (assignedby Study Directoror Project Lead atthe end of study or project) Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentationof Deviations Page 1of 1 Page 35 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS E00-1668 Record of Deviation Study / Project No. FACT-TOX-160, E00-1668 Deviation type SOP (Check one) X Protocol Document number FACT-TOX-160, E00-1668 L Identification Method Equipment Other: Date(s) of occurrence 10/24/01 II, Description Procedure Required procedure/process: 'Proioco]-FA_--TOX--l60,-E0(J-]668-s'_ates:Matrixsp_es _dmatr_ spike duplicatesrequired for analysis Of liversampies sfiould Showspike recoveries within 70%-130%. Xheriiver sampie-shave-b-een-_ai_ed,s_pie conceni_aiionswilt beevaluated. "If a measuredsa-mpie(s) .............. c0ncenixatZio-nexc-eededt]ac-ca]]bra-donS_o.Jf__geeme_od_dwas d]iuted with meihanolintothe Vaiidated...... ..e.a. fibrationrange, _eiaaa-difi0naf_t_ spikeswill _prep_ed at the appr0ximate measured concenffation_of the samplesthen subsequently diluted with thes_ne volume of methanol as the sample into the validated range of the calibration curve. These liver matrix spikes will be evaluatedversus an extracted matrix calibration curve and an "unexl_cted calibration c_e. if_e-diiui-ed liver matrix spiieesd0n0tSfiow recoveries within 70-130% Versusthe extractedmair_ eaiibrationC_e, thence s_p-le concentrations,at the same dilutionfactor as the matrix spikes, will becorrected forthe spike recovery. ............ Actual procedure/process: The 2 ug/g and 10 ug/g matrix spike/matrixspike duplicate (MS/MSD) average recoverieswere 1i9% and 242%1 ;i_ties_pie recove_ Wasnotco_isl:-entw-i_-theprevious analyses. "........................ III. Actions Taken (suchas amendmenitssued,SOPrevision,etc.) Thesamplesetwasre-extracteodn 11/07/01t,hendiluted1:50andanalyzed. ...R. ecorded by. ..............................................................................................................:..D...a.t.e........... Authorized b ..................................................................... Date ,_/,_t _p_n_r _,_re_r_c,-_ve.." "_o_n _n_ S-_b _)'il'_.)_'_r ". AI_I_I._ _-CI_ Deviation No. (assignedby Study Directoror ProjectLeadat the endof studyor project) Attachment A 3M Environmental Laboratory ETS-4-8.2 Dncumentatinn nf Deviatimn_ Page 1 of 1 Page 36 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX- 160 LIMS E00-1668 Record of Deviation L Identification '' Study / Project No. ..F..A : r0x:160,E00,_!.6..6..8........................................................................... Deviation type SOP Method Equipment Procedure ' (Checkone) X Protocol Other: Document numi_er .............................................................................D...a..t.e..(.s.i.....ofoccurrence FACT-TOX- 160, E00-1668 I 1/16/01 ............ II. Description Required procedure/process: "Prot0co] i_A_-TOX_l60,F.(_-i(i68-smteSiMa_ix-spikes an(tmatrix spike duplicates requiredfor analysis0f 'liver Samples should show spike rec0verieswitilin 70%- 130%. A_eriiver samples iaave bee-n-analyz'ed,-s_pie eoneen_aiions wiii be evaiuaied. If a measured sample(s) ....... "c-0nCehtratJonexceeded _eCaiibraiionratageoi_-[he-me_o-d-[L,[dwasdiihtedwi_ methano[ into the-vaiidateci ....... caii[;ration range, _en-aciditi-o_i_-s--p_es_I beprep-ared at [heapproximate nleasured C0ncentrati0nsof the samples then subsequently diluted with the same volume of methanol as the sample into the validated range of the 'cai-ib_d0ncu_e_ _i_heslei_;erh_trixspikes_ll-bee_;aiuatedversus an exh-acted matrix calibration cu_e and an .... unex_acted Calibrationc_e_-if _e-dii_Jted_ver ma_xsp_es d0n0t-show recoveries within 70-130% Versus tile -ex_acted matrix Calibration C)_ve,[hen-_esampie-concentratJ0ns, at [hesame diiution factor as the matrix spikes, wfli i_e corrected for the spike rec0very_ ................................ Actual procedure/process: '_e-i"O Ug/g matrixsp_lke/mat_ _il_eau_)lieate(i_s_sD)average recovery Was 67%. .................. IlL Actions Taken (such as amendment issued, SOP revision, etc.) The l 0 ug/g MS/MSD was not reanalyzed to confmn recovery since the 2 ug/g MS/MSD average recovery, _aalyzeddurir_g the Same run, was95%-and 'oi_jectives ...................................... Were witiain criteiSa-for_is_alyticai_ . ...................................................................................................... from +/- 30% to +/- 35% in the f'mal report. Wi_ _e stated criteria. Also, all CCVs The accuracyfor the iiver data _..................................................................................... and other data quality. in this study wili be changed Recorded by. ................................................................................................................ Date ..... ....... Authorized by<__ _I ....................... Date/_.._3./_ / Sp_,r P,_-_r_ti_., ". _ok_ _.n_ Deviation No. -_ Attachment A 3M Environmental Laboratory ETS-4-8.2 Documentation of Deviations Page 1 of 1 Page 37 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX-160 LIMS E00-1668 Record of Deviation I. Identification Study / Project No. Tox 160 Deviaii0n tYl_e.................................S..C...).P.... ...............-..X....M.. etiaod .............. Equipment (Checkone) Protocol Other: Document number Ets 8-7.0 Date(s) of occurrence 11-16-01 Procedure II. Description ......... Required procedure/process: According to section 11.1 The average of two standard curves Wi]l bepiotted by linear regression. .......... Actual procedure/process: 'i_0rthe data set m01fi i6a,-th-e_'ex-traciedcurve was-plottedusiiag-aquadraticfit. Alinear curve that metthe r_2 criteria and extended to 250 ppb (the level of the CCVs) could not be generated. This deviation was written. IlL Actions Taken (such as amendment issued, SOP revision, etc.) " -_ _"._m_Study co et syy / Project Poj ct Authorized by ................................................................. Date 3_c1<_ -_;,_t'_r i _ar_.tz .Se_,ceC,_,_-#,__l_/_,(Deviaaon No. b. 'Sp'_'f_'_'O_"_.,_fP,..3_.,_;_we.,'. "_'Okrx [_.1_'_l)_'_ (assignedby Study Direetoror Project Lead at the end of study or project) Attachment A ETS-4-8.1 Documentation of Deviations Page 1 of 1 3M Environmental Laboratory Page 38 3M Medical Department Study: T-6295.22 3M Confidential Analytical Report: FACT-TOX- 160 LIMS E00-1668 Record of Deviation I. Identification Study / Project No. FACT=TOXI=.6.0,. E00"!6.68 ....................................................................... Deviation type SOP Method Equipment Procedure (Che.ck.one.)................... X Protocol Documentnumber ................ FACT-TOX-160,E00-1668 Other: i I_ate(s) of occurrence ............................ ! 11116/01 II. Description .. ReqU'_edproecdm'e/process:............ ProtocolFACT-TOX-160,E00-1668 states: Matrix spikesandma_Sxspikeduplicatesrequired for analysisOf fiversamplesshouldsi_0w spikemc0ve_es within70%'i30%. ".................... After liver sampleshave been analyzed, sampleconeenwationswillbe evaluated. If a measm_i sample(s) concentrateixocneedetdhecaiibratriaonngeofthemethodandwasdilut_evditmhethanoilnitohevalidated calibratriaonnget,henadditiomnaatlrisxpikewsilbleprepareadttheapproximamteeasurecdoncentratoifotnhse sampletshensubsequendtillyutweidththesamevolumeofmethanoalsthesamplientothevalidatreadngeofthe CalibrationCurve. These livermatrix Spikeswill be evaluatedVersusan extractedmatrixcalibrationcan-reand an " unextractedealibration em-vel if _e dil_d liver malrixSpikesdo not show recoverieswithin70-130% versusthe extracted matrix calibradoncurve, then the sampleconcentrations,at the same dilution factor as the matrixspikes, will be correctedfor the si:fikerecovery. ....................... Actual proced_e/proccss: ................................................ 1) The 10 ug/g matrixspike/matrixspike duplicate(MS/MSD) averagerecovery was 67%. 2) NOl/S00 liver dilutionwas reported duringthe e0urse of _is study. T]ae1/500 liver diiuti0nwast0o dilute- because the wrong liver samplewas used for preparing that MS/MSD level. ..... IlL ActionsTaken. (such as amendment issued, SOP revision, etc.) 1) No liver values were corrected for this low recovery since one set of MS/MSDs were within criteria and the average recovery for this set of MS/MSDs were within the extended 65-135% criteria as stated in an cartier deviatio"n............................................... 2) Dilution was not reextractedlredilutedusing the correct samplesincethe Ii50 diluti0nwas withincriteriaand this I150dilution was used to showthat no effect wasobservedwhen liver dilutionswere needed. Recorded by Date Lisa A. Clemen d_ _" _ 04109102 0q 1o_1o2 Authorizedby ....... :Date sp or irc.or; Ana, DeviationNo. ? (assignedbyStud)D' irectororProjecLt eadat theendofstudyor project) 3M Environmental Laboratory Page 39 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:"1"-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport:FACTTOX-160 LIMS E00-1668 Appendix C: Extraction and Analytical Methods This appendix includesthe following methods: ET8-8-4.2, "Extractionof FluorochemicalCompoundsfrom Serum for AnalysisUsing HPLCElectrospray/MassSpectrometry," (16 pages) ETS-8-6.0, "Extractionof PotassiumPerfluorooctane-sulfonateor otherFluorochemical Compoundsfrom Liverfor Analysis usingHPLC-E/ectrospray/MassSpectrometry,"(14 pages) ETS-8-5.2, "Analysisof PotassiumPerfluorooctanesulfonateor Other Fluorochemicalsin Serum Extracts UsingHPLC-Electrospray/MassSpectrometry,"(11 pages) lETS-8-7.0,"Analysisof Potassium Peffluorooctane-sulfonateor otherFluorochernicalsin Uver ExtractsUsing HPLC-Electrospray/MassSpectrometry,"(10 pages) 3M Environmental Laboratory 3M Environmental Laboratory Page 20 Page 40 3M Medical Department Study: T-6295.22 3M ENVIRONMENTAL LABO_RATORy Analytical Report: FACT-TOX-160 r LIMS E00-1668 -=_,_C,.Wof 0_;2,_n;1 ,, METHOD EXTRACTION OF FLUOROCHEMICAL COMPOUNDSFROM SERUM FOR ANALYSIS USING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-8-4.2 Adoption Date: 03/01/99 Approved By: Effective Date: _.]_ I D { LabOratory Manager _'TZ,_. _ Group Leader Date o=/az-lo , Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the extraction offluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochernical surfactantsor other fluorinatedcompounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. 2.0 SUMMARYOFMETHOD 2.1 This performance-based method describes the procedure for extracting perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-tert-butyl ether (MtBE). An ion pairing reagent is added to the sample and the analyte-ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL of methanol, then filtered through a 0.2 _tmnylon filter using a 3-mL plastic Word 6/95 3M Environmental Laboratory _vtr.qr, ETS-8-4.2 t_t_n f_f I_l_rtaoh_mlt_nl_ Frnl'o _rllm Page 1 of 15 Page 41 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 syringe into glass autovials. (Application of this method to seven fluorochemicals was demonstrated: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and a surrogate standard {see 3. 0 Definitions }). 2.2 These sample extracts are analyzed following method ETS-8-5.2 or other appropriate method. 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F17SO3" 3.2 PFOSA: perfluorooctane sulfonylamide CsFt_SO2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CsFtvSO2N(CH2CH3)CH2CO2" 3.4 EtFOSE-OH: 2(N.-ethylperfluorooctane sulfonamido)-ethyl alcohol CsFITSO2N(CH2CH3)CH2CH2OH 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CsFzTSO2N(CH2CH3)H 3.6 M556: CsFITSOaN(H)(CH2COOH) 3.7 Surrogate standard TI-IPFOS: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 WARNINGS AND CAUTIONS 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 INTERFERENCES . 5.1 There are no interferences known at this time. _.0 EQUI]PMI_N'r 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 6.1.4 6.1.5 Nitrogen evaporator, Organomation Balance (+0.100 g) 7.0 Su AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposable pipettes, plastic or glass (or equivalent) 7.3 Polypropylene bottles, capable of holding 250 mL and 1 L (Nalgene or equivalent) 3M Environmental Laboratory w?..*--_.'__ ETS-8-4.2 _t_TTI ...... t.---.'-.1~ _____ o ..... Page 2 of 15 Page 42 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 7,4 Volumetric flasks, glass, type A 7.5 40 mL glass vials (I-CHEM or equivalent) : 7.6 7.7 Centrifuge tubes, polypropylene, 15 mL Labels 7.8 Bottle-top Dispenser - 3.0 to 10.0 mL or a 5-10 mL graduated pipette, glass 7.9 Syringes, eapablo of measuring 5 laL to 50 laL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 mL (B&D or equivalent) 7.12 Syringe filters, nylon, 0.2 _tm, 25 mm 7.13 Timer 7.14 Crimp cap glass autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 Reagent grade water, 1V[illi-QTM, Nanopure ITor equivalent 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na2CO3),J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaI-ICO3), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8,8 Serum or blood, frozen from supplier 8.9 Fluorochemieal standards 8.9.1 KPFOS (PFOS) (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.9.3 PFOSAA+H (PFOSAA) (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H CsFI3SO3H, [THPFOS]), molecular weight = 428 8.9.8 Other f/uorochemicals, as appropriate 3M Environmental Laboratory ETS-8-4.2 Page 3 of 15 Page 43 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 8.10 t 8.11 Reagent preparation NOTE: When preparing different volumes than listed in reagent, standard, or surrogate preparation,adjust accordingly. 8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL water, mix until all solids are dissolved. Store in a 1 L polypropylene (or equivalent) bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 rnL of 10 N NaOH solution into a l00 mL volumetric flask and bring to volume using water. Store in a 125 mL polypropylene (or equivalent) bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a I L volumetric containing 500 mL water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH. (Add the last 1.0 mL of NaOH slowly, as the pH changes abruptly.) Dilute to volume with water. Store in a 1 L polypropylene (or equivalent) bottle. 8.10.3.1 TBA requires a cheek prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na2CO3/NaHCO3): Weigh approximately 26.5 g of sodium carbonate (Na2CO3) and 21.0 g of sodium bicarbonate (NaHCO3) into a 1 L volumetric flask and bring to volume with water. Store in a 1 L polypropylene (or equivalent) bottle. Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorocherrticalstandards, as appropriate. Multicomponent fluorochernical standards are acceptable (for example, one working standard solution containing approximately 1.00 lug/mL of PFOS, PFOSA, PFOSAA, PFOSEA, M556 and EtFOSE-OH). 8.11.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. For standards with K + or other salts, multiply by a correction factor. For example, the molecular weight of KPFOS = 538, and the molecular weight of PFOS = 499. Calculate the correction factor by using the following equation: molecular wt. PFOS (499) molecular wt. KPFOS (538) = 0.9275 (Correction factor) 8.11.4 Bringto volume with methanol for a stock standard of approximately 1000 gg/mL. 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 lag/mL. ( l O001.Lg/ mL 5 mL ) = 50 I_gI mL 8.11.6 Dilute working standard I with methanol for a working standard 2 solution of approx. 5.0/ag/mL. 3M Environmental Laboratory ETS-8-4.2 Page '4 of i5 Page 44 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 50 p.glmL lOmL ) =5.oug/,,,L 8,11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 lag/mL. I50 l_g lIm0L0mL1.0 mL 1) = 0.500 /tg /mL 8.12 SurrogateTHPFOS stockstandardpreparation 8.12.1Weigh approximate5l0y-60mg ofsurrogatsetandarld-H,I-H2,-H,2-H, CsFI3SO_H(THPFOS) intoa 50 mL volumetrifclasakndrecordtheactuawleight. 8.12.2BringtovolumewithmethanoflorasurrogatsetockofapproximateIl0y00- 120rot rnL. 8.12.3Preparea surrogatweorkingstandardT.ransfearpproximateIlmyL ofsurrogate stocktoa I0mL volumetrifclasakndbringtovolumewithmethanolfora workingstandarodf 100/.tg/mLR.ecordtheactuavlolumetransferred. 9.0 SAMPLE HANDLING 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw at room temperature or in lukewarm water prior to extraction. 10.0 QUALITYCONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 10.1.2 10.1.3 Solvent Blanks: An aliquot of 1.0 mL methanol is used as a solvent blank. Method Blanks: Following this procedure, extract two 1.0 mL aliquots of water and use as method blanks. Matrix Blanks: Matrix blanks are prepared from one of three sources: 1) a study control nmtrix from a study control animal received with each sample set; 2) a commercially obtained sample of the same species as the study animals; or 3) a surrogate matrix, also obtained commercially, but of a different species than the study animal (e.g. if rabbit is used to generate standard curves and CCVs for a monkey or rat sera study). The matrix to use depends on what matrix is used for the curve. 10.1.3.1 Study control matrix curvewIf the study control matrix is used for the curve, prepare two (2) matrix blanks using the study control matrix. 10.1.3.2 Commercially obtained (same species) matrix curve--If the curve is prepared using commercially obtained matrix in the same species as the study animal, prepare two matrix blanks using this same commercially available matrix. 3M Environmental Laboratory ETS-8-4.2 Page 5 of 15 Page 45 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 t 10.2 10.3 10.1.3.3 Surrogate matrix curve--If a surrogate matrix is used for the curve and continuing calibration verification samples: a) prepare two (2) matrix blanks using the surrogate matrix. b) Also, prepare a matrix blank with each set of matrix spikes (a set is a matrix spike/matrix spike duplicate at one level) using a commercially available matrix of the same species as the study animals. Matrix spikes 10.2.1 Study Control Matrix Curve No matrix spikes are prepared. 10.2.2 Commercially obtained matrix curve (same species) No matrix spikes are prepared. 10.2.3 Surrogate matrix curve Matrix spikes are necessary if matrix is not available in the same species as the study animal and a surrogate matrix is used for the curve and CCV samples (e.g., rabbit sera may be used to generate standard curves for a monkey sera study, due to measurable levels of endogenous analyte in the monkey sera). Prepare and analyze matrix spike and matrix spike duplicate samples to verify the accuracy of the extraction for target analytes. 10.2.4 Prepare each MS and MSD at two (2) levels (usually a low and mid-range concentration) using a sample chosen by the analyst, typically sera from a control group animal received with each sample set. 10.2.4.1 If there is not sufficient sera available from the control group, prepare matrix spikes using commereiaUy available matrix from the same species as the study animal. 10r2.5 Prepare one matrix spike and matrix spike duplicate at each level listed in 10.2.4 _ per 20 samples, with a minimum of 2 matrix spikes per level per batch. If a batch includes more than 20 samples, additional spikes may be prepared at the same, low, or high range levels. 10.2.6 If more than 25% of the samples are <r.OQ, two matrix spikes should be prepared at approximately 2-5 times the expected LOQ. 10.2.7 If the majority of the samples are at or above the high range of the curve, additional matrix spikes should be prepared to approximate sample concentrations. Continuing calibration verifications (CCVs) 10.3.1 Prepare continuing calibration verification samples for instrument stability verification during analysis. Prepare and analyze CCVs for every assay run, regardless of the matrix type used to prepare the standard curve. 10.3.2 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve (i.e., either the study control matrix, commercial matrix of same species, or surrogate matrix). 3M Environmental Laboratory ETS-8-4.2 Page6 of 15 Page 46 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 10.3.3 The expected concentrations of the CCVs will fall within the range of the initial calibration curve; typically, the low to mid-range of the curve. 10.3.4 Prepare, at a minimum, two continuing calibration verifications, each at a different concentration, per group of 10 samples. For example, if a sample set = 34, eight (8) checks are prepared; four (4) at a low range and four (4) at a mid-range concentration. 10.3.5 Additional continuing calibration verifications may be included at the same, low, mid or high-range concentration. .11.0 CALmRA_O_ ANDSTANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL of appropriate serum to a 15 mL centrifuge tube. If commercially available sera is used, prepare two matrix blanks, using the volume of sera available for most study animals. 11.1.1.1 Depending upon study goals or if analyte-free commercial sera is not available, a surrogate matrix may be used for generation of the calibration standard. For example, rabbit sera may be used as a surrogate matrix for rat studies if quantitation of extremely low levels of the analyte is required. 11.1.2 If most sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract less than 0.50 mL of matrix. Record each sample volume on the extraction sheet. 11.1.3 While preparing a total of thirteen aliquots in 15-mL centrifuge tubes, mix or shake sera between aliquots. 11.1.4 Two 1-mL aliquots, or other appropriate volume, serve as curve matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1 at the end of this section to spike one standard curve, for a total of nine standards, two matrix blanks, and two method blanks. 11.1.5 11.1.6 Refer to validation report ETS-8-4.0 & ETS-8.5.0-V-I, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, continuing check, and sample add an appropriate amount of surrogate working standard to achieve a constant concentration that falls within the calibration curve range of 2.5 ng/mL-1000 ng/mL. Usually, samples are spiked for a constant concentration in all samples at approximately 500 ng/mL or other concentration as determined by the analyst. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.2 Page 7 of 15 Page 47 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Table 1 Approximate spiking amounts for standards and spikes t Using 1.0 mL of matrix Worl_ing standard Approxl final conc. of (approx. cone.) - lxL.., - analyte in matrix Blank 0.500 ppm ....... 0.500 ppm 5.00 ppm 5.00 ppm 5.00 ppm 50.0 ppm ,50.0 ppm 50.0 ppm 50.0 ppm *ppm=_t_mL 10 20 5 I0 20 5 ...... 10 15 20 " 0.005 ppm 0.010 ppm 0.025 ppm 0.050 ppm 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1.00 ppm 12.0 PROCEDURE 12.1 Obtain frozen samples and allow to thaw at room temperature or in lukewarm water. 12.2 Label a 15 mL polypropylene centrifuge tube with the study number, sample ID, date and analyst initials. See attached worksheet (Attachment A or simi/ar worksheet) for documenting the remaining steps. 12.3 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to the 15 mL polypropylene centrifuge tube. 12.4 Return unused samples to freezer after extraction amounts have been removed. 12.5 Record the initial volume on the extraction worksheet (Attachment A or similar worksheet). . 12.6 Spike all samples,.blanks and standards, that are ready for extraction with surrogate standard as desen'bed in 11.2. 12,7 Spike each calibration standard matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes if necessary and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH I0. If not, adjust accordingly. 12.10 To each sample, add 1 rnL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an bottle top dispenser or 5-10 mL graduated glass pipette, add 5 mL methyl-tertbutyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm for 20 minutes. 3M Environmental Laboratory ETS-8-4.2 Page 8 of i5 Page 48 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm or until layers are well separated. 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. .; 12.15 Remove 4.0 mL of the organic (top) layer to this clean 15 mL centrifuge tube. 1. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.17 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds, or longer ff needed. 12.19 12.20 Label a 1.5 mL glass autovial (or low-voinme autovial when necessary) with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. Attach a 25 mm, 0.2/am nylon mesh filter to a 3 mL syringe and transfer the sample from step 12.18 to this syringe. Filter into the labeled autovial. 12.21 12.22 Cap and store extracts at room temperature or at approximately 4 *C unti] analys_ Complete the extraction worksheet (Attachment A or similar worksheet) and include in the study binder. 13.0 DATAANALYSISANDCALCULATIONS 13.1 Calculations 13,1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of std concentrationof std ( p.g/ mL) = Finalconcentratio(np.g/ mI,) of PFOS in matrix mLof std + mL of surrogatestd + initialmatrixvolume( mL) 14.0 METHOD PERFORMANC_ ..... 14.1 The method detection limit (M.DL)is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachments B and C). At the discretion of the PAI, MDL may not be defined for some studies. 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 14.2.3 If surrogate matrix is used for curve and QC, matrix spike and matrix spike duplicate samples to verify extraction efficiency. Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.2 for method performance criteria. 3M Environmental Laboratory ETS-8-4.2 Page9 of 15 Page 49 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 15.0 POLLUTIONPREVENTIONAND WASTI_,MANAGEMENT 15.1 t Dispose of sample waste, flammable solvent waste and used glass pipette waste using proper methods and by placing each in their designated waste container. 16.0 RECORDS ,, ,, , ,, 16.1 Complete the extraction worksheet attached to this method (or a similar worksheet), and include in the study binder. 17.0 ATTACHMENTS i i 17.1 Attachment A, Extraction worksheet (a similar worksheet may be substituted for Attachment A) 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Ion Pair Standard Curves worksheet (a similar worksheet may be substituted for Attachment C) 18.0 REFERENCES 18.1 The validation report associated with this method is ETS-8-4.0 & 5,0-V-1. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 18.3 ETS-8-5.2, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-5.2, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" 20.0 REVISIONS, Revision Number Reason For Revision 1 Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section I2.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1.0 mL. 2 Section 1.1, 2.1 Eliminate 'potassium' from perfluorooctanesulfonate 8.9.1 Add K to PFOS 8.9.3 Add H to PFOSAA 10,2-10.2.4, 14.2.2 Add information about using surrogate matrix, clarify control matrix and spiking the curve, ehang_ spikes to every 20 samples 10.3 Clarify purpose of continuing calibration cheeks I 1. I Added wording about control animal sera, using and not using commercially available tissue In general, make less specific for equipment/supplies. Substitute water for Milli-Q, Nalgene or equivalent bottle may be used. Revision Date 04/02/99 3M Environmental Laboratory ETS-8-4.2 Page 10 o1"15 Page 50 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 Study # .... Matrix Box # Wk/Day ,, Date Spiked/ Analyst: _ .. ,. MS MSD Surrogate Std actual pg/mL # Extraction Worksheet ETS-8-4.2 FC-Mix approx. 0.5 lag/mL # FC-Mix approx. 5 _glmL # FC-Mix approx. 50 pg/mL #..... ,= ,=. ,. ,. , Comments LIMS E00-1668 Verified by: ,= , L.L ,. .= ,..,, . .. -- ,,, ,,, ,.. "Blank ,l'P",.#... Serum Extraction_Method ' ' Vorte_ 15 see ..... Pit.Jette Matrix. s_ike vAtti avort)briatc surro_te Pir)ette 1 mL of 0.5 M TBA, pH 1.0. vH = ,., : W FCMix ...... . ,. amount= ,, rnL ,,Date'& Initials l..i. [ Volume [ ml_ ....I , Std.# i , [ .... Pipette 2 mI_ of 0.25 Na2CO_0.25M NaH,CO_.buffer ,, DispenseR)i_tte 5 mL 9f methvi-t_uW! ether Dispenser'iI) _ ' ......... Shake 20 rain. ' Shaker ID #: std. # , .' .... "IN-A- Shaker spee_i': I . I .... .... CerltTif_g(__0-25 rain. Centrifu_.lD#: Centrifuge weed; Remove _ 4 mI_aliauot of oreavie laver. Put on Nitrogen ]_v_oorator to dryness , .... .. Add methanol, 'Volume mL TN-A-,','' Vor_e_0sec ' " . , , Filter usin_ a 3mL B-D svrin_with a 0.2urn filter into a 1.5 mL _utosam_[e vial Cont. Cal. Verifications used same matrix as for std curve. ,. I [ [ _ ,_ , l { ADt_endix A: Extraction Worksheet 3M Environmental Laboratory ETS-8-4,2 Pa_e 11 of 15 Page 51 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 MDL/LOQ talues for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (ng/mL) (ng/mL) Approximate concentrations to be used for preparing . the Standard Calibration Curve t PFOS " 1.74 5.55 5 n_/.rnLT1000 ng/mL PFOSA 1.51 4.79 5 ng/mL-1000 ng/mL PFOSAA 3.46 20.5 5 ng/mL--1000 n_mL EtFOSE-OH 11.4 36.2 5 ng/mL,-1000 n_mL M556 6.03 19.2 5 ng/mL--1000 ng/mL ,= PFOSEA 5.71 18.2 5 ng/mL-1000 ng/mL MDL/LOQ values in rat, bovine, monkey,and human serum,and monkeyplasma were not statistically determined. Two curves in each of these matriceswere extracted and analyzedwith the rabbit serum curves to determineequivalence. Responses in the rat, bovine,,monkey,and humanwere equivalent to the rabbit responses, therefore, their MDL and LOQ willbe the same values as determinedin rabbit serum. Pleasesee LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-1 for further information. AppendixB: MDL/LOQValuesand Summary 3M Environmental Laboratory ETS-8-4.2 Page 12of 15 Page 52 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 Compound: PFOS Prepared range Rabbit Serum of standards (ng/rnL) Full Range 0.995 - 978 Low Curve 4.94 - 248 High curve 1/X 97.8 - 978 0.995 - 978 LCR from curve (ngmL),. 24.8 - 978 4.94 - 248 % Recovery Range 83-108 85-104 97.8 - 978 4.94 - 978 85-106 94-111 RSD ' Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 Compound: PFOSA Prepared range Rabbit Serum of standards .... (n_n_) Full Range 0.993 - 976 LCR from curve (ngmL) 4.93- 976 Low Curve ..... High curve 4.93 - 97.6 L 24.8 - 976 4.93 - 97.6 , 24.8 - 978 1/X 0.993 - 976 4.93 - 976 % Recovery Range . . 88-103 87-105 93-102 94-103 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 Compound: PFOSAA Prepared range Rabbit Serum of standards ..... (ngmL) . Full Range 0.99I - 974 Low Curve 4.92 - 247 High curve 49.2 - 974 1/X 0.991 - 974 LCR from curve (ngmL) 24.7 -974 9.74 - 247 97.4 - 974 9.74 - 974 % R_overy Range 8I-I I 1 97-107 85-108 95-115 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Appendix B: MDL/LOQ Values and Summary 3M Environmental Laboratory ETS-8-4.2 Page 13 of 15 Page 53 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Compound: EtFOSE-OH Prepared range Rabbit Serum "" of standards (neCmL) Full Range 0.993 - 976 Low Curve 4.93 - 97.6 High curve 1/X 49.3 - 976 0.993 - 493 Compound. pFOSEA Prepared range Rabbit Serum of standards (ngr ) Full Range 0.993 - 976 Low Curve 4.93 - 248 High curve I/X 49.3 - 976 0.993 - 976 Compound: M556 Prepared range Rabbit Serum of standards (ngtmL) Full Range ,,, 0.993 - 976 Low Curve 4.93 97.6 High curve I/X 97.6 - 976 0.993 - 976 LCR from curve (ng(..mL) 49.3 - 976 % Recovery Range 77-110 RSD " Range I' 11.2-25.5 9.76 - 97.6 97-107 14.1-21.3 97.6 -976 9.76 - 976 90-109 86-111 11.5-19.6 11.1-21.2 LCR from curve (ng/mL) 24.8 - 976 9.76 - 248 49.3 -976 9.76 - 976 % Recovery Range . 96-I06 91-110 86-106 95-117 RSD Range 10.I-16.2 11.8-19.5 10.2-18.2 10.1-19.1 LCR from curve (ng/mL) 24.8 - 976 % Recovery Range .......... 88-106 9.76 - 97.6 100-105 RSD Range .4.82-17.9 ,, 5.95-18.2 97.6 - 976 9.76 - 976 81-111 97-110 5.11-9.74 4.77-19.5 Appendix B: MDL/LOQ Values and Summary 3M Environmental Laboratory ETS-8-4.2 Page 14 of 15 Page 54 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 EXAMPLE Ion Pair Standard Curves- Fluids Analyst(s) ;t Prep date(s): Analyte(s): Study Animal Matrix : Method/revision: Study number: Final solvent and TN: Blank fluid]identifier: [Record mah'ix used Io prepare curves, te.g., 'Rabbit,.' ID #XXX)} Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppnn FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: {Re.cord tile standards used to prepare the extracted cnrves. ) Actual concentrations of standards in the FC mix in methanol PFOS Std cone PFOSA Std cone PFOSAA Std cone EffOSE Std cone PFOSEA Std cone M556 Std cone pg/rnL 0.500 0.500 lag/mL 0.507 0.507 pg/mL 0.532 0,532 [ag/mL 0.501 0.50i p_/rn L 0.521 0.521 pg/mL 0.501 0.501 5.00 5.07 5.32 5.01 5.21 5.01 5.00 -1 _7 ' 5.32 5.00 |.,._07"_T _ - 50.0t Sb.1_k 5._11 5o.0.$ --_.L _._*'- 50.0 50.1 53.2 5.01 _M" _ll L_.. 50.1 . 5.21'"_.. _ ,,,9_., 52_1 L ,._, 52.1 5.01 _ r_k ._ 50.1 50.0 50.1 53.2 .... 50.1 52.1 5'0.1 All Ain't spiked.mL 0.010 0.020 0.005 0.0!i0 0.020 0.005 0.o10 0.015 0.020 All Final vol mE, 1.015 1.025 1.010 1.015 1.025 z.010 1.015 1.020 1.025 Calculated concentrations of standards in the sera matrix: Rabbit PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Surrogate All Final cone nghnL 4.93 9.76 Final cone ng/mL 5.00 9.89 Final cone ng/mL 5.24 10.4 Final cone ng/mL 4.94 9.78 Final cone ng/mL 5.01 9.93 Final c_nc ng/mL 5.13 10.2 Std cone ng/mL 100 Am_ spike.d mL 0.005 24.8 "q r-,_l 49.3 t 50.0_ 26.3 24.8 _ 57._&_ ,m..,d_.4,a 25.2 " _..,_.,f_d 25.8 Surrogate _ 'Final cone 97.6 _ _"_.9 -_ t I_g4,,,_ [[' 9_r8 i_'99_:_ AI-I,0_ ng/mL 248 _' 25'/_k _J _! _ Z'.4_. , 25_ _k258, 500 493 "t _'5_)'aL 735 746 t "_,--ff._,.,t. 782 4_4-" "_. _Y_ "_ _" _911_" 737 ._ J49 766 976 989 1038 978 993 1017 Validated range s - approximate concentrations ..... Serum Rabbit PFOS 5.00-1000 PFOSA '1 5.00-1000 , PFOSAA 5.00-1000 ,,. ng/mL [ ng/mL n_mL Bovine Estimates only,.. Use values for rabbit. Rat Estimates only. Use values for rabbit. Monkey & Plasma Human Estimates only. Use values for rabbit. Estimates only.. Use values for rabbit. EtFOSE-OH 5.00-1000 ng/mL . ..... PFOSEA 5.00-1000 ng/mL ..... M556 5.00-1000 ,,, ng/mL .. Attachment C: Ion Pair Standard Curves 3M Environmental Laboratory ETS-8-4.2 Page 15 of 15 Page 55 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 - ' -Ex_ 8_pyofOn'_lnJ 3M ENVIRONMENTAL t LABORATORY METHOD EXTRACTION OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTrrV.R FLUOROCr_MICAL COMPOUNDS FROM LIVER FOR ANALYSIS USING H.PLC- ELV.CTROSI'RAY/MAss SI'ECTROM_TRY Method Number: ETS-8-6.0 Author: Lisa Clemen, Robert Wymae Approved By: ' t Laboratory l_anager _' GroupLeader -d_,.c=h_ " Technical Reviewer Adoption Date: 0 3]'/-LIKe{ Revision Date:. ]O[_ Date Date o_/,,la,, Date .... 1,0 SCOPE AND APPLICATION 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemic,-d compounds from liver. 1.2 Applicable Compounds: Fluorochemica/surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. Word 6.0/95 3M Environmental Laboratory ETS-8-6.0 Page1 of 14 Page 56 3M Medical Department Study:T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 SUMMARY OF METHOD 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluoroehemical surfactants from liver, or other tissues, using an ion pairing reagent and methyl-tert-butyl ether ('MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 ee plastic syringe attached to a 0.2 lma nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 DEEtNTr]ONS 3.1 PYOS: perfluorooctanesulfonate (anion of potassium salt) C_FtTSO3 3.2 PFOSA: perfluorooctane sulfonylamide CsFiTSO2NI-_ 3.3 PFOSAA: perfluomoetane sulfonylamido (ethyl)acetate CsFtTSO2N(CH2CH3)CH2CO 2 3.4 EtFOSE-OH: 2(N-ethylperiluorooctane CtF_vSO2N(CH2CH,)CHxCH2OH sulfonamido)-ethyI alcohol 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CsFtTSO2N(CH2CH3)H 3.6 M556: CsFtTSO,N(H)(CH2COOH) 3.7 Surrogate standard: IH-1H-2H-2H perfluorooetane sulfonle acid 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use universal preeautlorm, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 INTERFERENCES 5.1 There are no interferences known at this time. 6.0 EQUIPMENT 6.1 The following equipment is used while performing this method. acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral I000 or IEC 6.1.4 Shaker, Eberbach or VWR. Equivalent equipme.nt is ETS-8-6.0 Page 2 of 14 3M Environmental Laboratory Page 57 3M Medical Department Study: T-6295,22, Analytical Report: FACT-TOX- 160 LIMS E00-1668 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) .: t 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorfor disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sampttle vials, Wheaten, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispenser-? 3.0 to 10.0 ml 7.11 Syringes, capable of measuring 5/_L to 50/.tL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 _tm, 25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- Qa-Mwater. Rinse syringes a minimum of 9 times with methanol; 3 rinses from 3 separate vials. 8.0" REAGENTS AND STA/_ARDS 8.1 Type I reagent grade water, Milli-Q TM or _lUiValent; all water used in this method should be Milli-Q TM water and be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate ('lqa2CO3),J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCO_), J.T. Baker or equivalent 8.6 Methyl-tert-butyl ether, Omnisolv, glass distilled or I-IPLC grade 8.7 Methanol, Omnisolv, glass distilled or I-IPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluoroehemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 3M Environmental Laboratory ETS-8-6.0 Page 3 of 14 Page 58 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 ,_. 8.11 8.12 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.I 0.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.10.5 PFOSEA (3M Specialty Chemical Division), molecular weight -- 527 8.11).6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.10.7 Surrogate standard: 4-H, perfluorooctane sulfonie acid (1-H,1-H, 2-H, 2-H CsF,_SO3I-I)molecular weight -- 428 8.10.8 Other fluorochemicals, as appropriate Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 ml'. Milli-Q TM water, m;x until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10 NNaOH 1:10. Measttre 10 mL of 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-Q TM water. Store in a 125 mI_,Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen"sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-Q TM water. Adjust to pH I0 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of NaOI-I, add slowly because the pH changes abruptly). Dilute to volume with Milli-Q TM water. Store in a I L Nalgene bottle. 8.11.3.1 TBA requires a cheek prior to each use to ensure pH -- 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na2COa/NaI-ICOa):Weigh approximately 26.5 g of sodium carbonate ('Na2C03)and 21.0 g of sodium bicarbonate (/,,laI2lCOs) into a 1 L volumetric flask and bring to volume with Milli- QTMwater. Store in a 1 L Nalgene bottle. Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multieomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFO S, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm 8.I2.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 3M Environmental Laboratory ETS-8-6.0 Page 4 of 14 Page 59 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 '_ 8.13 8.12,6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, C_F_3SO_H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 1.0 ml of surrogate stock to a 10 ml volumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 SAMPLE HANDING 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 QUALITY CONTROL 10.1 Matrix blanks and method blanks 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots ofMilli-Q TM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of liver homogenate following this procedure and use as matrix blanks. Refer to 11.1.6. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy oft.he extraction. 10.2.2 10.2.3 Prepare each spike using a'sample chosen by the analyst, usually a control liver received with each sample set. Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group of 10 samples. For example, ifa sample set = 34, four verifications are prepared and extracted. 3M Environmental Laboratory ETS-8-6.0 Page 5 of 14 Page 60 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-range of the initial t calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the ana/yst must quantitate using only the low end of the calibration curve (for example, 5 ppb- 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately 40 g of liver into a 250 mL Nalgene bottle containing 200 mLs Milh-Q TM water. Grind to a homogeneous solution. 11.1.2 If40 g is not available, use appropriate amounts of liver and water to ensure a 1:5 ratio. 11.1.3 Refer to 13.0 to calculate the actual density of liver homogenate and the concentration of solid liver tissue dispersed in 1.0 mL ofhomogenate solution. 11.1.5 Add 1 rnI_,ofhomogenate to a 15 mL centrifuge tube. Re-suspend solutionby shaking between aliquots while preparing a total of eighteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.6 TWO1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen samples, two matrix blanks, and two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 and ETS-8-7.0-V-1 or AttaebmentB, which fists the working ranges and the Linear Calibration Range ('LCR.)for calibration curves. 11.1.9 Use Attachment C as an aid in calculating the concentrations of the working standards. Refer to 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each working standard, blartk, or continuing verification, add appropriate amount of surrogate working standard for theconcentration to fall within the calibration curve range 5 ppb - 1000ppb. 3M Environmental Laboratory ETS-8-6.0 Page 6 of 14 Page 61 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 11.3 Extract spiked liver homogenates following 12.14-12.25 of this method. Use these ._ standards to establish each initial curve on the mass spectrometer. t Table 1 Approximate Spiking Amounts for Calibration Standards Vorking Standard (Approx. Cone.) - 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.'50 ppm 5.0 ppm .... 5.0 ppm 5.0 ppm 50 ppm _tl Approx. final cone. of PFOS in liver - Blank 2 0.005 ppm 4 0.010 ppm 10 0.025 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 1.00 ppm 12.0 PROCEDURE 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g of liver using a dissecting scalpel. This part oft.he procedure is best performed quickly, not allowing the liver to thaw. 12.3 Weigh the sample direetly into a taxed plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Return unused liver portions to freezer. . 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDl"-EP- 22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3M Environmental Laboratory ETS-8-6.0 Page7 of 14 Page 62 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-I668 12.11 Pipette 1.0 mL, or other appropriate volume, ofhomogenate imo a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sarnpule vial. Refer to attached worksheet for documenting the remaining steps. ,t 12.12 Pipette two 1 mL aliquots ofMilli-Q TM water to centrifuge tubes. These will serve as method blanks. 12.13 Spike all samples, including blanks and standards ready for extraction with surrogate standard as described in section 11.2. 12,14 Spike each matirx with the appropriate amount of standard as described in 11.1, or Table 1 of that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to ensure 0.5 M TBA reagent is at pH I0. If not, adjust aeeordingly. 12.17 To each sample, add 1 mI., 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-tert-butyi ether. 12.19 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. 12.21 Label a fresh 15 m.Lcentrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mI_,of the organic layer to the fresh 15 rnL centrifuge tube. 12.23 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.24 Add 1.0 mL to each centrifuge tube using a graduated pipette. 12.25 Vortex mix for 30 seconds. 12.26 Attach a 0.2 tara nylon mesh filter to a 3 co syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, - matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attached to this document, and tape in study notebook or include in study binder, as appropriate. 3M Environmental Laboratory ETS-8-6.0 Page8 of 14 Page 63 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 13.0 DATA ANALYSIS 13.1 Calculations: AND CALCULATIONS ,:_ 13.1.1 Calculate the average density of the liver homogenate by recording each mass of ten separate 1.0 mL aliquots of homogenate. Average density (mg/mL) = Average mass (mlz) of the aliquots 1.0 rn]L-aliquot 13.1.2 Calculate the amount of liver (mg) per 1.0 mL homogenate (or concentration of dispersed solid tissue per mL ofhomogenate equation; suspension) using the following of Liver x Average density* ofhomogenate (g of Liver + g of Water) * refer to 13.1.1 for details. (mg4'mL) 13.1.3 Calculate actual concentrations of PFOS and other fluorochemicals standards using the following equation: in calibration _tL of Standard x Concentration (_tg/mL) = Final Concentration (rtg/g or mg/kg) mg Liver/1 mL homogenate* of PFOS in Liver *refer to 13.1.2 for details. 14.0 METHOD PERFORMANCE 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit ofquantitation (LOQ) values (refer to Attachments B and t2). 14.2 The following quality control samples arc extracted the qualityofthe extractionandanalysis. 14.2.1 Method blanks and matrix blanks. with each batch of samples to evaluate 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and - precision of the extraction. 14.2.3 Continuing calibration verification samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-7.0 for method performance criteria. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 3M Environmental Laboratory ETS-8-6.0 Page 9 of 14 Page 64 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 16.0 RECORDS 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. t 17.0 TABLES, DIAGRAMS_ FLOWCHARTS_ ANDVALIDATION DATA 17.1 Attachment A, Extraction worksheet 17,2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard calculation and concentration worksheet 18.0 RZ_ZRZNCZS 18.1 The validatiornq_ortassociatewdiththismethod isETS-8-6.0& 7.0-V-1. 18.2 AMDT-EP-22, "RoutineMalntenanceofUltra-TurraTx-25" o 18.3 FACT-M-I.I, "Extractioonf'PFOS orOtherAnionicFluorochernicSaulrfaetantfsrom LiverforAnalysisUsingHPLC-Eleetrospray/MasSspectrometry" 19.0 A#FgCTED DOCUMENTS 19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluorochcmicals Mass Spectrometry" 20.0 REVISIONS Revision Number. Reason For Revision using HPLC-Eleetrospray Revision Date 3M Environmental Laboratory ETS-8-6.0 Page 10of 14 Page 65 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 Study # Matrix Box _ Wk/Day Date Spiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # FC Mix Std approx. 0.5 ppm actual ppm # FC Mix Std approx. 5 ppm actual ppm # --w m FC Mix Std approx. 50 ppm actual ppm # Comment's I ., .. --- , , .,. ,, m . , ,., ., i ,,. .,,, Bl_k Liver Homogenate: Std # Liver Extraction Method : ,- -,,, ............ Spike surrogate and Standard mix, Vortex 15 .Lee. Pi{_etter] mL of Liver Solution . Pipette I mL of'l'0.5 M TBA, pH 10. pH = '" Pipette 2 mL of 0.25 Na2CQ_/0.25M NaHCO_ Buffer Dispense 5ml of Meihyl-t-Butyl Ether Shake 20 rain .... - .. Centriful_e 20-25 rain. "' ., Remove a 4 mL aliquot of organic ]ayer ......... Put on Nitrogen Evapo_tor to dr_,n_ss Add !.0 mL of Meihanol - ......... Liv_ amount= . _ ...... Std. # . i m ill Std. # T_I-A- Shaker Spee'd'" _,' ., Centrifuge --Spie,d ,, Evaporator Temperature TN-A- .... Vortex'30 see. . ........ : Filter usin_ a 3ee B-D svrin_te with a 0.21_mSRI filter into autosamp!e vial Cont. Cal. Verifications used the same matrix as for the standard curve. ik Date & Initials ,- _.... ' : ......... . .. Attachment B: MDL/T_.OQ Values 3M Environmental Laboratory ETS-8-6.0 Page I 1 of 16 Page 66 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 MDL/LOQ values for rabbit liver Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (ppb) Approximate concentrations to be used for preparing the t Standard Calibration Curve PFOS ' 8.45 26.9 30 ppb- 1200 ppb PFOSA '3.50 11.1 1,2p.pb - 1200"ppb PFOSAA 24.6 7813 30 ppb - 1200 ppb EtFOSE-OH 108 345 60 ppb --900 ppb* ..... M556 82.3 262 69 ppb-..1200 ppb PFOSEA 33.9 .. 108 30 ppb- 1200 PPb MDL/LOQ values in rat, bovine, and monkey liver were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit liver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalent to the rabbit responses, therefore, their MDL and LOQ will be. assumed to be equivalent to those values as determined for the rabbit liver. Refer to LOQ Summary and MDL study in ETS-8-6.0 & 7.0-V-1 for further information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Compound: PFOS Prepared Range of ':LCIL:liOn?r,__R. ange of :.:LCR_iS_i_:i Range of mLiavlerirx sratnagnedoafrds avceurravgee .(ppb) (ng/mL) (ppb) (ng/mL) ::f.a."_./_":Sz.._._e. '- locwurvsted .:"_i!|_i_!_i_!i;!_!_high std "(ppl_:),,(n_.t_L)- (ppb) (ng/mL) t'_" l(_b_:_'[_'_ (ppb) (ng/mL) Rabbit 6,19 . 1237 12- 1200 -:.ii_1.2,1-02!"-. 6 - 300 '_ "12_->__-3"0:_0_>:60- I200 ._::LCR!_m_!i' '_,;-.-._:.g:_ch::",.8:".t,.:d'=!2,.:_.:- :_:(lipb),"' (ng/i_UI)=l "_ 7_. "_.z'_i-_"2_::_-,_-:,_;.+: _::_0::---1200_i._ Compound: PFOSA Liver Prepared range of matrix - standards (ppb) (n b/re.L) Rangeof average curve (ppb) (ng/mL) Y;2 .:...L;"C_,..R_"._::fr.'.om. Rangeof ..a..V..e..:.,.e'.u.../..ve- law std '" "5.":'_-"_---"",','- ..,(l_b)"(figh_)! (l?pb) (ng/mL) :.'.-.L:.:'__;_o'_Pm:;::__.,';_:.:;._.I;_ Rangeof -_'_:ft6_i_i _ ...lo...w...'s..t.d..;.._._.'.._-! 'high std _,i_i_.,;_._,_t_'_sfff_4':;ic.!_-;_!.:_ .i(pPb_:_(ng/mL)_l (ppb) (ng/mL) _-(_/pb):,(ngTmL),: Rabbit 6.19 - 1237 .1....2 - 1200 !.:=::.:-1.),.2. 'i . ". _ 12,-, 300 ,' ;_-!..:.....2....:..1_3.00::_6=0.--f1]200 . _v_id0:_._I:2(J0_'?. . Compound: PFOSAA Prepared Rangeof .LCg-frgm. Rangeof Liver rangeof matrix standards average avecurve curve lowstd curve (ppb) (ng/mL) (ppb) (nglrnL) (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.16 - 1232 12- 1200 30 -1200 : 30 - 900 -LCRfrom'. Rangeof 10Wstd: highs(d curve ": ._ curve (ppb) (ng/m.L):.[ (ppb) (ng/mL) 60 - 900 :._1 N/A LCR fron'L ...h.i.ghstd:..: ' : cuive:: ": "(ppb) (ng/mL) : ii::N/A _i AttachmentB: MDL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 Page 12of 16 Page 67 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Compound: EtFOSE-OH Prepared Range of ,_ Liver matrix range of standards average curve (ppb) (ng/rnL) (ppb) (ng/mL) Rabbit 6.17 - 1235 31 = 900 LCR from ave curve (ppb) (n,g/mL) 31 - 900 Range of low std curve (ppb) (ng/mL) N/A LCR from low std curve (ppb) (ng/mL) N/A Range of high std curve (ppb) (ng/mL) N/A " LCR from high std curve (ppb) ,(ng/mL) N/A Compound: PFOSEA Liver Prepared range of matrix standards Rabbit 6.17- 1235 Range of average curve 31 1200 . LCKfrom ave.em've.-i y. : -.' ' "...:'.:,:",:._",".... Range of low std curve " .'.-it ":_"_-i" -J . 31-,1_20 ,_._ " ::.... '._ ;' -,',':'3 N/A LCKfrom. Rangeof low std :" high std ' cur_ i_.._'. curve " " _" ; ,_-_.'.: 'I:N//_I;I:C:; "'_'" " ';'.. N/A LCR from - .- highstd 'i" , . . . _ '2:. _': ..:curve.,:-_._i,.: .,;:t:., - ":'!k,:._, :":-:NIA r: : " " "': " Compound: M556 Prepared Liver rangeof matrix standards (ppb) (nghnL) Rabbit 6.17- 1235 Range of average curve (ppb) (ntgrnL) 31 - 1200 ">"L_0rh_ . "..",Y'_I._';,..,.:,";':"_- -" -:/ave, :eurve':_ Range of low std , :,--:,:,.:_,_.._;_-,..- curvv ," .._ .:_,:.,.-y, ; ._ .4:.- r-(pl_.b);:(ng/i_I_): (ppb) (ng/mL) , : '_:i._::_."-'_"_, :_ 0_:_'.:-" NIA i.LCK:ifr0m.::?Range of "" ' ' _ "_" ' "" ' _ZliJ__ _-: high std . .' :."_i_:-_ _ "." _"" ":!._7".'"_',-& "_" -:.(ppb)..:(fi_ " , .,..o.4?.._,:._'_-;:_ :" "';"_': ":"":': , . ..-. .... curve (ppb) (nghnL) ....I_ fr6m::-, -_" "'"': " '_ _':: '; ::_highstdi:::_ _;,_('l_b)_,(fi_!!i :'_.,:_: _::,. :o:_; _., _,..:.:.N,,./A :, .,, ;.. . ..: . Attachment C: Standard Calculations 3M Environmental Laboratory ETS-8-6.0 Page 13of 14 Page 68 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Ion Pair Standard Curves - Tissue ';t Prep date(s): Analyte(s): Sample matrix: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Standard number: Equipment number: Final solvent and TN: Blank Hver/identifier: Actual concentrations PFOS PFOSA Std cone Std cone ug/mL ug/mL 0.500 0.500 0.500 0.500 0.500 0.500 '0.500 '().500 0.500 0.500 5.00 '5.00 "" 5.00 5.00 50.0 5.00 -5.00' 50.0 of standards in the FC mix PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone ug/mL ug/mL ug/mL 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500" 0.500 0.500 0.500 ' 0"500 0.500 0.500 0.5()0 5.00 5.00 5.60 5.00 5.00 50.0 5.00 5.00 50.0" 5.00 5.00 50.0 M556 Std cone ug/mL 0.500 0.500 0.500 0.500 0.500 ' '5.00 5.06 5.00 50.'0 Stdcone u.g/mL ._ ..... "" '"' All .... An_'tspiked mL 0.002 0.004 0.010 0.020 0.040 0.010 0.020 " 0.030 0.004 .-All Density g 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 Calculated ]PFOS Final cone ng/g 5.99 12.0 29.9 59.9 120 299 399 "hbS '1'198 concentrations of standards PFOSA Final PFOSAA Final cone EtFOSE Final cone ng/g rig/g 5.99 5.99 cone nK,'J_ 5.99 12.0 29.9 59.9, 120 299 599 12.0 29.9 59.9 120 299 599 __ 12.0 29.9 39.9 120 299 .... 599 .... 89 s 1198 895 1198 898 .... 1198 in the sample matrix PFOSEA Final M556 - Final cone cone ng/g n g/g 5.99 5.99 Std eoiac ng/g 12.0 12.0 .. 29.9 29.9 59.9 5'.9 120 120 299 299 399 599 1898 J I]'*_9S 895 fi98 Surrogate Std cone ng/mL 100 Surrogate Finaclone ng/mL 0.500 All Ain't spiked mL 0.O65 Validated ranges Liver Rabbit Bovine Rat 1' I " "Monkey - approximate concentrations PFOS PFOSA 5-1000 ppb 5-1000 ppb I Estimateson!y,userabbitvalues. Estimatesonly, use rabbitvalues. Estimates only, use rabbit va'lues. PFOSAA 5"1000ppb _ .... EtFOSE-OH 5-1000 ppb " '' POAA 5-1000 ppb PFOSEA 5-I000 ppb Attachment C: Standard Calculations 3M Environmental Laboratory ETS-8-6.0 Page 14 of 14 Page 69 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 3M E,NV1-RONMENT,ALLABORATORY .... t METHOD ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE OR OTHER FLUOROCHEMICALS IN SERUM EXTRACTS USING I'-IPL C-ELE CTROSPRAY]MASS SPECTROMETRY Method Number: ETS-8-5.2 Author: Lisa Clemen, Kris Hansen Approved By: Adoption Date: 03/01/99 Revision Date: _ ) ] [ 01 Laboratory Manager Group Leader Technical Reviewer Date / Date Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method describes using HPLC-electrospray/mass the analysis of serum extracts for fluorochemical spectrometry. surfactants 1.2 Applicable Compounds: Huorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-5.2 Page 1oFl I. Page 70 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 2.0 SUMMARYOF METHOD 2.1 ,_ Although supported by a validation for most commonly used matrices, this is a performance-based method. Careful attention should be paid to method QC as there is great variability in sera. This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II and Ultima triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization CESD, Atmospheric Pressure chemical Ionization (APED, Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II and Ultima triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass triple quadrupole systems (post 1998) utilize a '_-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II or Ultima triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use cautien with the voll_agecables for the probe. When engaged, the probe employs a voltage of approxfmately 5000 Volts. Word 6/95 3M Environmentat Laboratory ETS-8-5.2 Page 2 of 11 Page 71 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: ' 4.2.1 Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HPLC will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERfeReNCeS 5.1 To minimize interferences when analyzing samples, Teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.,0 EQUIPMENT 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Quattro II or Ultima triple quadrupole Mass Spectrometer equipped with an electrospray ionization source 6.1.2 HPI I00 or Agilent low pulse solvent pumping system, solvent degasser, column eompartrnent, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air or nitrogen system) %1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials and capped 15 rnL centrifuge tubes 8.0 REAGENTSANDS. TANDAR_.S 8.1 Reagents 8.1.1 Methanol_ HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be MiIli-QTM water or equivalent, and may be provided bYa Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.2. 3M Environmental Laboratory ETS-8-5.2 Page 3 of I 1 Page 72 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 9.0 SAMPLEHANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. t 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 QUALITYCONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed at least once during the course of the study to determine if contamination occurred during sample prep. 10.1.2 Analyze at least one solvent blank prior to each calibration curve. 10.1.3 Matrix blanks should be analyzed with each sample list that inehdes undiluted extracts. 10.2 Matrix Spikes 10.2.1 If curves and method QC are prepared in a surrogate matrix (e.g. curves in rabbit sera, samples are monkey sera), matrix spikes and matrix spike duplicates are prepared in blank sample matrix (e.g., monkey sera) and analyzed to verify extraction efficiency. 10.2.2 If curves and method QC are prepared in the same matrix as samples, no additional matrix spikes are required. 10.3 Continuing Calibration Verifications (CCVs) 10.3.1 Continuing cal_ration verifications are analyzed to verifT the continued accuracy of the calibration curve. 10.:3.2 Analyze two calibration standards (one at each of 2 levels) after every one to ten samples, with a minimum of two per batch and always finishing an injection sequence with at least two calibration standards. 11.0 CALIBRATIONANDSTANDARDIZATION 11.1 Analyze the extracted matrix calibration standards prior to each set of extracts. The curve will be plotted by linear regression, weighted l/x, not forced through zero, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements, perform routine maintenance, reextract samples, or reanalyze the standard curve. 11.3 For purposes of accuracy when quantitating levels of analyte at the limits of the curve range, it may be necessary to use either the low end or the high end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, it may be beneficial to generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of 3M Environmental Laboratory ETS-8-5.2 _1. _r_ _L-o ....... v"..,---_ Tr-.'_ T'?.Or_,fO Page4 of 11 Page 73 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 high concentration standards. It is also acceptable to break the linear range into a low curve and a high curve. If this is done, no more than one point should be used in common between the curves. For example, the low curve may include the following points: I, 5, 25, 100, 250 ,_ ppb and the high curve may include the points: 250, 500, 750, I000, 1250 ppb. t 12.0 PROCEDta_S 12.1 Acquisition Set up 12.1.1 On the MassLynx main page, set up a sample list name. Save the list as instrument designator letter, last 2 digits of test year-too-day, and a letter that will increase through the alphabet with each additional list for that day. Example Sample List: IYYMMDDa or D010712a I=instrument name (I3 for "Davey") YY=year of test (0I) MM=month of test (07) DD=day of test (12) a=first sample list (run) of the day (the next sample list will end with 'b,' the next 'c' and so on.) 12.1.2 Assign a f'flename using the instrument designator letter, the last 2 digits of year-tooday, and a 3-digit sequential file number that starts with 1 and increases by one for each filename. Example File Name: IYYMMDD,,,'_,, or D010712001 I=instrument name (13for "Davey") YY=year of test (13I) MM=month of test (07) DD=day of test (I2) ###=3-digit sequential file number starting with 1 through 999 (00I) 12.1.3 Also, as part of the sample list, assign a method (MS) for acquiring, an inlet file, a bottle number, an injection volume and sampl.e descriptions. To create a method, click on Method Editor button in the MS Status Pane and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. Also set the acquisition start and stop times. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.4 Typically the analytical batch run sequence begins with solvent blanks and a set of extracted matrix standards. 12.1.5 Sample extracts are analyzed with two CCVs injected every one to ten samples. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered sample extracts but may be included as such. 3M Environmental Laboratory ETS-8-5.2 Page 5 of 11 Page 74 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 12.2 Using the HPLC 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HPLC to the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 gJ-,injection 12'2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 10.0 minutes 12.2.2.4 Flow rate = 300 laL/min 12.2.2.5 Mobile Phase (program) Time ooorain. 1.00 rain. 5.50 win. 7.50 min. 8.00 rain. MeOH 10% 95% 95% 10% 2.0 mM Ammonium acetate_(inH_O) 90% 5% 5% 90% 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. The probe should be checked weekly. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 lal_Jminor as appropriate. Observe"dropletscoming out of the tip of the probe. 12.3.5 Turn on the nitrogen. A flue mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response_ 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 10 - 500 [xL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 3M Environmental Laboratory ETS-8-5.2 Page 6 of 11 Page 75 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 12.3.8 Print the tune page, MS file, HPLC parameters, sample list and the Microsoft Word summary page and store in the study binder with a copy taped into the instrument log. 12.3.9 Click on start button in the MassLynx main page (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Ensure start and end sample number includes all samples to be analyzed. 13.0 DATA A.NALYSIS AND CALCULATIONS 13.1 Calculations: 13,1.1 Calculate matrix spike percent recoveries using the following equation: ObserveRdesul-tMatriBxlankResult % Recovery. = 100 l X " Spiking Level 13.1.2 Calculate percent difference using the following equation: Expected Cone.- Calculated Cone. % Difference = x I00 Expected Cone. 13.1.4 Calculate actual concentration of PFOS, or other fluorochemical, in matrix ( tg/mL): .(Cone.of PFOS Calc.fromStd. Curve(nglmL) DilutionFactor) l J f(lnitial Volumeof Matrix (mL)+ mLof SurrogateStandard).l Final Volume( mL ) l lzg x lO00ng 14.0 METHOD PERFORMANC-_ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4,2, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values must be below the lowest active standard in the calibration curve. 14.3 Calibration Curves 14.3.1 The coefficient of determination value for the calibration curve must be 0.990 or better. 3M Environrnental Laboratory ETS-8-5.2 Page 7 of I I Page 76 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 14.3.2 All active calibration curve points must be within 25% of the theoretical value with the exception of the LOQ point, which may deviate up to 30%. 14.3.3 calibration standards with peak areas less than two times the curve matrix blank must be deactivated to disqualify a data range that may be affected by background levels of the analyte. 14.3.4 Low or high curve points may be deactivated to optimize a linear range appropriate to the data. 14.3.5 Not including low or high points dropped to optimize the linear range, curve points may be deactivated if they deviate more than 25% from the theoretical value when the curve is evaluated over a linear range appropriate to the data. 14.3.6 One point below the LOQ may remain active even if it deviates more than .-e30%or has a peak area less than two times the matrix blank; however, the LOQ will be defined at the lowest point with acceptable deviation. 14.3.7 A valid calibration curve must contain at least 5 active points above and including the LOQ. 14.4 Matrix Spikes 14.4.1 The average matrix spike percent recoveries should be within _+30% of the spiked concentration. Recoveries outside of this range should be discussed in the report. 14.5 Continuing Calibration Verifications (CCVs) 14;5.1 Continuing calibration samples within the linear range of the run must show a percent recovery within _-e.2%5 of the spiked concentration. If a CCV is outside of this recovery, subsequent data should not be accepted. Acceptable data must be bracketed by the curve and passing CCVs. 14.6 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 POLLUTIONPREVENTIONANDWASTEMANAGEMENT 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 The first page of each data packet generated for a study must have the following information included either in the header, in the footer or hand written on the page: study or project number, instrument, sample matrix and time point, date, and analyst. 16.2 A data packet includes the following: data review summary form, MassLynx quantify compound summary report for each target analyte, quantify calibration report for each target analyte (curve), method report, Word document listing set-up parameters, tune 3M Environmental Laboratory ETS-8-5.2 Page8 of 11 Page 77 3M Medical Department Study: T-6295.22 ' Analytical Report: FACT-TOX-160 LIMS E00-1668 method report, MassLynx scanning method report, HPLC method report, sample list, and quantity sample report (chromatogram). 16.3 For each analysis, after printing the tune method report, Word document listing, set-up :_ parameters, MassLynx scanning method report, and sample list, copy and tape into the instrument runlog. The original is maintained in the data packet. 16.4 On each page of the quantify compound report, quantify calibration report (curve), and quantify sample report (ehromatogram), the following information must be included either in the header, the footer, or hand written on the page: study or project number, instrument, method, calibration (the method and calibration are usually assigned the same name), analyst, and date. 16.5 The analyst must date and initial the first page in a packet as long as their initials and date are electronically included on each page. If initials and date are not electronically included, they must date and initial each page. 16.6 Summarize data using suitable software (e.g., Excel) for inclusion in the final report. See Attachment A for an example of a summary spreadsheet. 16,7 Back up electronic data to appropriate medium. Record the file name and location of backup electronic data in instrument log book. .17.0 TABLES,DIAGRAM.SF, LOWCHARTSA,NDVALIDATIONDATA 17.1 Attachment A: Data summaryspreadsheet. !8.0 REFEI_r_.CES..... 18.1 FACT-M-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluoroehemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.O & 5.0_V-1. !9.0 AFFECTEDDOCUMENTS ........... 19.1 ETS-8-4.2, ''Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory ETS-8-5.2 Page 9 of 11 Page 78 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 L/MS E00-1668 20.0 REVISIONS I I II III Revision Number. 1 Section Section plotting Section Section Reason For Revision 6.1.2 Clarification of HP1100 system components. 1I. I Average of two curves, not standard values, are used for linear regression and added the l/x weighting of the curve. 12.2.2.4 Clarification of solvent ramp. 17.1 Changed from attachment B to A. 2 I0.1 Clarified when blanks are run. 10.2 Added instructions for when.surrogate matrix is used. 10.3 Specify requirements for CCVs. 11.1 Requires only a calibration curve before the s.amples. 11,.2Clarify what to do if curve does not meet requirements.. 11.3 Allow to truncate the curve. I2.I.1 Clarifysample list ID. 12.1.3 Clarify typical run. I2.2 Changes to mobile phase gradient and specifyflow rate. 14.3 Change acceptable limits. Add specifies for acceptanceof calibration cur_e. 14.3.3 When to deactivate calibration standards based on blank response. 14.3.5 When to deactivate calibration standards within the linear range. 14.4 Modifies evaluation and use of matrix spikes. 14.5 Describes evaluation and use of CCVs. 14.5.1 Evaluation of CCVs. 16.0 Add requirements for records and documentation. Revision Date 04/02/99 Attachment A: Summary Spreadsheet 3M Environmental Laboratory ETS-8-5.2 Page 10 of 11 Page 79 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Humber: Instrument Software/Version: Filenara: R-Squared Value: Slope: Y Intercept: Dam of Extraction/Analyst: Date of Analysis/Analyst: Group Dose Sample# Concentration u_/mL ' Initial Vol. mL Dilution Factor Final Cone. u_i'mL ,, ,i . im Slooe: Taken from linear re_ression equation. Grou_/Dose: Taken from the study folder. Samvle#: Taken from the study folder. Concentration (ue./mL_: Taken from the MassLvnx integration Initial Volume (mL_: Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (u_mL_: Calculated by dividing the initial volume .m, summary. from the concentration Attachment A: Summary Spreadsheet 3M Environmental Laboratory ETS-8-5.2 Page 11 of 11 Page 80 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 3M ENVIRONMENTAL LABORATORY t METaOD ANALYSIS0F POTASSIUM PERFLUOROOCTANESULFONATE OR OTnR.R FLUOROCrrRMICALS IN LIVER EXTRACTS USING HPLC-ELE CTROSPRAY/MAss SPECTROMETRY Method Number: E'I_S-8-7.0 Author: Lisa Clcmcn, Glenn Langenburg Approved By: Adoption Date: _)7/7"L[t_ Revision Date: _ Laboratory__nagZ ''_'_'''*''- Group Leader A Technical Reviewer '-'_/D"a_t"a//_7" Date Date 1.0 SCOPE ANDAPPLICATION 1.1 Scope: This method is for the analysis of liver extracts for fluorochemical HPLC-eleetrospray/mass spectrometry. surfactants using 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: report. Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation Word 6/95 3M Environmental Laboratory ETS-8-7.0 Page 1 of 10 Page 81 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 4 2.0 SUMMARY OF METHOD 2.1 This method describes the analysis offluorochemical surfactants extracted from liver using I-1-PLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z ffi499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the selected parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These inehde but are not limited to: Eleetrospray Ionization ('ESI), Atmospheric Pressure chemical Ionization (APe D, Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). . 3.2 Electrospray Ionization 0gS, ES1): a method of ionization performed at atmospheric pressure, whereby ions in solution ar_ transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or an ion may be selected in the lirst quadrupole, fragmented in the collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 Conventional vs. Z-spray probe interface: The latest models ofMieromass Quattro II triple quadrupole (post 1998) utilize a "Z-spray" conformation. The spray emitted fi'om a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the eotmter electrode. Though the cortxeiguration is different, the methods ofoperatlon, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with other Z-spray systems, etc.) 3.5 Mass Lynx Software: System sofhvare designed for the specific operation ofthese Quattro II triple quadmpole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details refer to the manual specific to the instrument (Micromass Quattro II triple quadrupolc MassLynx or MassLynx NT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 3M Environmental Laboratory ETS-8-7.0 Page2 of 10 Page 82 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 4.1.2 When handling samples or so"vents wear appropriate protective gloves, eyewear, and elothing. t 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure of 400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP1100 will initiate auto, matic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferences when analyzing samples, Teflon shall not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 EQUIPMENT 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.i Mieromass Quattro II triple quadmpole Mass Spectrometer equipped with an electrospray ionization source. 6.1.2 HP1100 low pulse solvent pumping system, solvent degasser, eolunm compartment, and autosampler 7.0 SUPPLIES AND MATERIALS 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100 psi (house air system) 7.1.2 I-IPLC analytical column, specifics to be determined by the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8.0 REAGENTS AND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade orequivalent 8.1.2 MJlli-Q TM water (ASTM type I), all water used in this method should be ATSM type I, or equivalent, and be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour irtto a 2000 mL volumetric container containing 2000 mL Milli-Q TM water, mix until all solids are dissolved. Store at room temperature. 3M Environmental Laboratory ETS-8-7.0 Page3 of 10 Page 83 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 8.2 .; t Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 SAMPLE HANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples may be stored at room temperature, or refrigerated at approximately 4 C, until analysis can be performed. 10.0 QUALITY CONTROL 10,1 Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and.analyzed with each batch to determine con_mlnation or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration crave. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analytc. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty sample_. With a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lOW- range o the initial calibration curva. 10.3 Continuing Calibration Checks 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard every tenth sample, with a minimum of one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted matrix standards prior to and following each set of sample extracts. The average of two standard curves will be plotted by linear regression (y = mx + b), weighted I/x, not forced through the origin, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 3M Environmental Laboratory ETS-8-7.0 Page 4 of 10 Page 84 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a '_. calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This wiI1reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 PROC.EDURES 12.1 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 12.1.1.2 12.1.1.3 12.1.1.4 Assign a sample list filename using MO-DAY-last digit of year-increasing letter of the alphabet starting with a Assign a method (MS file)for acquiring Assign an HPLc program (Inlet file) Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then mass spectrometer headings and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. Refer to Micromass MassLyrtx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch run sequence begins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected standard aider every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. . 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 p.Linjecti6n 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 9 minutes 3M Environmental Laboratory ETS-8-7.0 Page 5 of 10 Page 85 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 12.2.2.4 Solvent ramp conditions Time MeOH 2.0 mM .. Ammonium acetate t 0.00 rain. '.... 40% 60% 1.0 rain. 40% 60% .... 4.5 min. 95% 5% 6.5 rain. 95% 5% 7.0 rain. 40% "" 60%- 9.0 mi. 40% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance of the Mieromass Quat_ro/I Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization S0uree," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Cheek the stainless steel eapillary at the end of the probe. Use an eyepiece to cheek the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operate to initiate source block and desolvat/on heaters. 12.3.6 Open the Inlet Editor. 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500 uL/rnin or as appropriate 12.3.6.30bsex_e droplets coming out of the tip of the probe. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-4001iter.,s/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 1.2.3.7.3 HPLC constant flow mode flow rate 10 - 500 btL/min 12.3.7.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150 12.3.7.6 Desolvation temperature 250 3M Environmental Laboratory ETS.8-7.0 Page6 of 10 Page 86 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. ._ 12.3.9 Click on start button in the Acquisition Control Panel (this may vary among t .MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 DATA ANALYSIS AND CALCULATIONS 13.1 Calculations: !3.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone. x 100 Expected Cone. 13.1.6 Calculate actual concentrations in matrix (_tg/g): (ng ofPFOS eale. from std. Curve x Dilution Factor) x 1 p.g (Initial Weight of Liver (_) 1000 ng Final Volume (mL) 14.0 METHOD PERFORMANCE 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Refer to ETS-8-6.0, Attachment B for a listing of current validated bIDL and LOQ values. 14.2 Solvent Blanks, Method Blanks and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks must be below the lowest standard in the calibration curve. 14.3 Calibration Curves 14.3.1 The ta value for the calibration must be 0.980 or better. - 14.4 Matrix Spikes 14.4,1 Matrix spike percent reeox;eries mustbe within + 30% of the spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within + 30% of the spiked concentration. 14.6 If criteria listed in the method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 3M Environmental Laboratory ETS-8-7.0 Page7 of 10 Page 87 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and:discussed in the text of the report. t 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date,dilution factor (if applicable), and analyst. 16.2 Print the tune page, _nple list, and acquisition method fi_omMassLynx to include in the appropriate study folder. Copy these pages and taPe into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted I/x, then print these graphs and store in the study folder 16.4 Print data integration summary, integration method, and ehromatograms from MassLynx and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.0+) and store in the study folder, refer to Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 TABLES_DIAGRAMSF, LOWCHARTSA,NDVALIDATIODNATA " 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18.0 REFERENCES 18.1 FACT-M-2.1, "Extraction of Potassium Perfluoro0ctanesulfonate or OflaerFluoroehernieal Compounds from Liver for Analysis Using I-IPLC-Eleetrospray/Mass Spectrometry" _ 18.2 ETS-9-24.0, "Operation and Maintenance of the Mieromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-6.0 & 7.0-V-1 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory ETS-8-7.0 Page 8 of I0 Page 88 3M Medical Department Study: T-6295.22 20.0 RgVlSlONS Revision Number t Reason For Revision Analytical Report: FACT-TOX-160 LIMS E00-1668 Revision Dat_.____e 3M Environmental Laboratory ETS-8-7.0 Page 9 of 10 Page 89 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 Laboratory Study # t Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept:" Date of Extraction/Analyst: Date of Analysis/Analyst: Group Dose sample# Concentration ng/g Initial Wt. g Dilution Factor J_'lnalCone. ug/g ,|., m ,J . - _lope: Talen from linearregressfon equalaon. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ng/g): Taken from the MassLynx integration summary. Initial Wt. (g): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/g): Calculated by dividing the initial volume from the concentration Attachment A: Summa_ Spreadsheet 3M Environmental Laboratory ETS-8-7.0 Page 10 of 10 Page 90 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport:FACT TOX-160 LIMS E00-1668 Appendix D: Data Summary Tables Table 9. Data Summary for PFOS In Serum FACT-TOX-160 pg/mL I Group Tlmepoint I Sex PFOSI_m.L Avemje, SD Group 1 Week9 Male 17.2 .824 n-_ Female 26.8 5.88 n=2 Week 17 Male .... Female 13.7n=21.13 20.3:t:2.37 n=2 Week25 Male Female 8.7 1.93 n_ 15.8 5.27 n--2 Week33 Male Female 8.10n=02.768 12.4+ 4.2 n=2 Week41 Male Female 5.65 = 0.303 n=2 10.4 4.37 n=2 Week53 Male Female 4.26 0.052 n=2 7.95 1.76 n=2 NOTEI:tisnotposstbtloeverifby'ue_ ofer_ anaJyf_reom__t__J-,_w_ithou_t refemrcemateria_l. o_y_ ofaccuracayvaJlaia_t thistheem, aVlSxpiws_udleins,dica_'_st fia dataarequar_atek,to30%orgmatan Table 10. Data Summary for PFOS in Liver FACT-TOX-16C .. ii PFOSIJg/g Group GroupI I Sex Male Avera_ SO 3.58 0.436 n=2 Female 923 2.04 n=2 ;_/g NOTE:I_Isnotposs_etoredlytreerecoveoryfe_ analytferomtissuews_hou_t refemrcemateria_l. n'__P_-___emoefanctcuracayvailabaletthislimem, atrix_ slud_si,ndicatefisatthedataarequantitattivoe35%orgreater. 3M Environmental Laboratory 3M Environmental Laboratory Page 21 Page 91 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:T-6295.22 Appendix E: Data Spreadsheets Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport:FACT TOX-160 LIMS E00-1668 3M Environmenta/ Laboratory 3M Environmental Laboratory Page 22 Page 92 3M Medical Depazh-_-_entStudy: T-6295.22 Study: FACT-TOX-160, E00-1668 Product Number(Test Substance): Matrix: Method/Revlsion: Analytical Equipment System Number:. InstrumentSoflwa_/Version: Filemme: R-Squared"Value: Slope: Y-Intercept: Dates 0 f Ex_"aefio_Ana_ysC Dates of Analysis/Analyst: Date of Date Reduction/Analysr Box: FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LI.h/[S E00-1668 Extended Recovery Study Following 26-Week Capsule Toxicity Stdy with Perfluorooetarte Sulfoni Acid Potassium Salt(PFOS; T-6295) in Cynomolgus Monkeys %6295.22 Monkey Serum ET$-8-4.2 & ET$-8-5.2 versus an extracted rabbit sets curve Amelia062498 Masslynx 3.4 SeeAttachments See AttachraeBB See Attachments See Attachments 09/07/01 RWW 09/13/01 MMH 09114101 MMH 01-042, 01-043 Sample Data MONKEY SERUM Group ]Dose Sample # PFOS Cone. Concentration of PFOS Mean ]PFOS RSD Std. l)ev. MethodBlk Matrix Blk WB09060I-H2OBIk-I WB090601.H20 Blk-2 RBS09060 I-Sofa Blk-I ng/mL, e_roL or % Roe 0.00 i <LOQ(O.00492ag/mL) 0.00 <LO_. (0.00492 ug/mL) 0.00 <LOQ (0.00492 u8/mL) u_/mL <LOQ MS/MSD RPD NA QC - 50 ppb 250 ppb RBS090601-SentBlk-2 0.00 MKS090601 -Sofa Blk-I 10.2 MKS090601-Sera Blk-2 7.71 MK._90601-50 ppb-MS 72.6 MKS09060 t-SOpp.b-MSD 64.8 MKS090601-250 ppb.-MS 279 <LOQ(O.00492ug/mL_ 0.0102 0.00771 129% 113,% 110% <LOQ , 0.00597 121% NA ! 0.00177 13/. Group I Week 9 MKS,090601-250ppb-MSD 25g 105505M 44.6 I05523M 45.g 105539F 76.8 ID5552F 44.8 101% 17.8 16.7 30.7 22.4 105% 17.2 26.6 , , S',/. 0.824 5.88 Group 1 Week 17 Group I Week 25 I05505M L 105523M I05539F I05552F I05505M I05523M 105539F 45.$ 14.52 37.5 12.93 48,4 22.0 77.3 18.6 36.7 I0.1 20.9 , ,, 7.33 50.8 19.5 13.7 1.13 20.3 2.37 8.70, 1.93 ..... Group I Week 33 105552F 105_05M 105523M I05539F _24.8 33.3 29. I 48.5 , 12.1 8.64 7.56 15.4 ,,15.8. & 10 5.27 0.768 Group I 105_52F 105505M 47.3 9.46 17.6 5.86 12.4 ....... 4.20 Week 41 105523M . 12.5 5.43 5.65 0.303 105539F 31.0 13.$ Group 1 Wt:_k 53 I05,552F .20.0 7,25 105505M I05523M 14.0 4.30 l 1.4 4.22 I05539F 14.7 9.19 10.4 4.37 ..... 4.26 0.052 PFOS = Perileorooctaneselfonate 105552F 19. ! .6.70 7.95 1.76 DDa,teteEvnt_eroedd/B/y_:: 0l9/_26O/01lrLA_C IAr.-t>q[ aS[v3. 3M Environmental Laboratory Page 93 3M Medical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX- 160 LIMS E00-1668 Study: FACT-TOX- 160, E00_1668 product Number(rest Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dotes of Analysis/Analyst: Dote ofDotJ ReductiordA:udyst: Box: Extended Recovery Study Following u 26-Week Capsule Toxicity Stdy with Pedluorooctane Sulfonic Acid potassium Sall(PFOS; %6295) in Cyt_omolgus Motakeys T-6295.22 Monkey Scram ETS-8-4.2 & ETS-8-5.2 vertus an extractedrabbitaera curve Amelia062498 M_sl'ynx 3.4 See Below See Attachments See Attachments SeeAttachments 09/07/01 RWW 09/13/01 MMH 09/14/01 MMH 01-O42, 01-043 Sample Data MONKEY SERUM Dose Group Method Blk Matrix Bik QC- 50ppb 250 ppb Group I Week 9 Group I Week 17 G_np 1'" Week25 Group I Week 33 Group I Week 41 Group I Sample _ WB09060i_H20 Blk-l' WBO9060I-H20 Blk-2 RBS09060 l.Sera Blk-I RBS090601-Sc{a BIk-2 MKS09060 l-Sort Blk-I MKS090601 -Sera Blk-2 MKS090601o50 ppb-MS MKS09060t-50 ppb-MSD MKS090601.250 ppb-MS MK$090601-250 _b-MSD 105505M 105523M 105539F I05552F 105505M 105523M I05539F 105552F 105505M , 105523M 105539F 105552F 105505M 105523M I05539F 105552F I05505M I05523M 105539F 105552F I05505M Week $3 PFOS ffiPeffluorooctanestdfonate I05539F 1_055555223FN4 VoL ExtrmacLtion "' 1 Verified Surrogate NA 1 NA I NA 1 NA 1 NA I NA 1 NA 1 NA 1 NA l NA 0.50 NA 0.55 NA 0.50 NA 0.40,_ NA 0.63 NA 0.58 NA 0.44 NA 0.83 NA 0.73 NA ..... O.57., NA 0.52 NA 0.41 NA .... 0.77 HA 0.77 NA 0.63 NA 1.00 NA 0.60 NA 0.46 NA 0.46 NA 0.55 NA 0.65 ! NA 0.32 NA 0.547 ' I NAA Dilution FPaFcOtoSr 1 1 1 1 1 1 i" I 1 1 200 200 200 200 200 200 200 20Q 200 200 200 2,00 200 2,00 200 200 200 200 200 200 200 200 220000 Cone.. nP_FJOmSL 0.00 o.oo 0.00 0.00 10.22 7.71 72.55 64.80 279.46 258,06 44.56 45.81 76.79 44.81 45.75 37.49 48.36 77.32 36.72 20.90 50.76 24.75 33.28 29.10 48.51 47.32 17.59 12.50 30.97 20.02 13.96 14.71 I119.4009 Filenome A010913049 ,.A010913050 A010913051 A010913052 A010913053 A010913054 A010913055 .A010913056 A010913057 A010913058 A010913088 A010913089 A010913090 , A010913091 A010913081 A010913082 A010913083 A010913084 A010913077 ,, A010913078 A010913079 A010913080 A010913070 A010913071 A010913075 A010913076 A010913066 A0109130fi7 A010913068 A010913069 A010913062 A010913064 AA001100991133006653 of PFOS uCg/omnLcenortro%tioRnue '_LOQ (0,00492 ug/mL) <LOQ {0.00492 ug/mL) <LOQ (0.00492 ng/mL) <LDQ (0.00492 ug/mL) 0.0102 0.00771 129% 113% 110% 101% 17.8 16.7 30.7 22.4 14.5 .!2.9 22.0 18.6 10. | .7.33 19.5 12.1 8.64 7..56 15.4 9.46 5.86 5.43 13.5 7.28 ! 4.30 9.19 ,,I 46.2720 PFOS uMg/emanL Std. Dee. _ ' MS/MRSSDDRPD <LOQ NA <LOO 0.00897 NA 0.00177 ,121% .... 13.0% 1.0_5% 17.2 8.24% 0.824 26.6 5.88 13.7 1.13 .... 20.3 2.37 8.70 1.93 15.8 5.27 ,8:10 12.4 0.768 4.20 5.65 0.303 10.4 4.37 47.2965 01.0.7562 Date Entercd_y: 09/26/01 LAC 3M Environmental Laboratory Page 94 3M Medical Department Study: T-6295.22 Study: FACT-TOX-160, E00-1668 Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduefiod/Analyst: Box: Sample Data CFovAaCnTce-#T6O32X%-126608 Analytical Report: FACT-TOX-160 LIMS E00-1668 Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluoroo_tane PotassiumSalI(PFOS;T-6295) in CynomolgusMonkeys T-6295.22 Monkey Serum ETS-g-4.2 & ETS-8-S.2 ve[sus an unextracted curve Amelia 062498 Masslynx 3.4 See Attachments See Attachments See Atlachmen_ See Attachments 08/31101 RWW 09/05/01, 09106101 MMH 09/06/01, 09/07/01 MMH 01-042 Sulfoni Acid MONKEY SERUM Group Dose Sample # PFOS Cone. Concentration of PFOS Method Blank Matrix Blank MKS083101 -H20 BIk-! MKS0g3101-H20 Blk-2 MKS083101-Semi Blk-I MKS083101-Sera BIk-2 n_/mL O.Ig 0.23 9.69 9.05 u_/mL or % Ree <LOQ (0.0012 up/m/,) <LOQ (0.0012 ng/mL) 0.00775 0.00724 QC MKS08310_-MS.-25 ppb 32.6 118'% MKS083101-MSD-25 _ 25.9 MLgOg3 IOI-MS-500 ppb 448 84% IIlea MKS083 I0 I-MSD-500 ppb 449 PI_6S= Perfluorooetaaesuffonate 112% LOQ = 1.0 ng/mL in standardcalculates to 1.2 np/mL in senon. LOQ - (i.0 ng/mL uncxt standard * 1.25 xt dil factor * I dilution factor)/lO00 = 0.0012 ug/mL in senma Mean PFOS u_/mL <LOQ 0,00750 101% I 11% Date Entered/By: Date Verified/By: 09112/01 LAC 16/6/01 mmh RSD Std. Dev. MS/MSD RPD NA 0.000362 34% O/o 3M Environmental Laboratory Page 95 3M Medical Department Study: T-6295.22 CFoAvaCnTee-#T6O3X29--126608 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-160, E00-1668 Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: File.name: R-SquaredValue: Slope: Y-intercept: Dates of Exu-acfion/Analyst: Dates of AnalysisAnalyst: Date of Data Reduction/Analyst; Box: Sample Data Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooclane Sulfonic Acid Polassium S_I_PFOS; %6295) in Cynomolgus Monkeys T-6295.22 MonkeySerum ETS-8-4.2 & ETS-8-5.2 versus an unextracted curve Amelia 062498 Masslynx 3.4 See Below See Attachments See AUachments See ARachments 08/3t/01 RWW 09/O_VO0l,9/06/01 MMH 09/06/01, 09/07/01MMH 01-042 MONKEY SERUM Group Dose Sample # Initial VUl. Extraction Dilution Surrogate Verified Method Blank MKS0g3101-1420 BIk-I mL Foe/or I 1.25 C_,ou.FameHd igh MKS083 IOI-H20 BIk-2 I 1.25 Confumed Hif_h Matrix Blank MKS08310 I-Sera BIk-1 l 1.25 Confirmed High MKS083101-Sere BIk-2 1 1.25 Confmned High QC MKS083101-MS-25 ppb I 1.25 Confirmed High MKS083101-MSD-25 ppb I MKS083101.MS-500 ppb I MKSO83101-MSD-500 ppb I PFOS = Pertluorooctanesulfonate 1.25 Confixmed High 1.25 2ridanalysis OK !.25 Continued High LOQ = 1,0 ng/mL in standard calculates to 1.2 ng/mL in senam. LOQ = (I.0 ng/mLuaext standard * 1.25 ext dil factor * 1 dilufon factor)/1000 = 0.0012 ug/mL in serum DateEntered/By: Date Verified/By: 09112/01 LAC 16/6/01 mmh PFOS Dilution Factor 1 I 1 I I I 1 1 PFOS Cone.. n_mL O.18 0.23 9.69 9.05 32.62 25.87 447.77 448.57 Filanamn A010906019 A010906020 A01090602 i A010906022 A010906023 A010906024 A010906025 A010906026 Concentration of FFOS u_/mL or % Re <LOQ (0.0012 ugJmL) <LOQ (0,0012 ng/mL_ 0.00775 0.00724 118% 84% I 11% 112% Mean PFOS u_/mL <LOQ 0.00750 101% 11I% RSD Std. Dev. MS/MSD RPD NA 0.000362 34% 0% 3M Environmental Laboratory Page 96 3M Medical Department Study: T-6295.22 Study: FACT-TOX-160, E00-1668 Product Number(Test Substance): Matrix: Method/Revision: Analytical Equipment System Number: hasmmaentSoRware/Version: Filename: R-Squared Value: Slope: Y-Intercept: Dates o f Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Analyst: Box: Sample Data FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E0O- 1668 Extended Recovery Study Following II 26-Week Capsule Toxicity Stdy with Perfluorooetane Sulfonic Acid Potassium Salt(PFOS; T-6295) in Cynomolgu$ Monkeys T-6295.22 Monkey Serum ETS-8-4.2 & ETS-8-5.2 versus an tmextracted ctu_e Amelia 062498 Masslynx 3.4 See Attachments See Attachments See Attachments See Attachments 10/02/01 RWW 10/04/01 MMIt 10/05/01 MMH 01-O46 MONKEY SERUM Group Dme Sample # PFOS Cone.. Concentration of PFOS Mean PFOS Method Blank WBI00201-I-L20 Blk=l n_/mL 0.00 _,/mL or % Ree <LOQ (0.00313 ug/mL) n_/mL Malrix Blank WBIOO201-H20 Blk-2 RBS10020 l-Sera BIk-I 0.(30 0.09 <LOQ (0.00313 u_/mL) <LOQ (0.00313 us/mL) <LOQ RBSI002OI-Sera Blk-2 MKS100201-Sera Blk-3 MKS 100201-Sera BIk-4 0.13 <LOQ (0.00313 n_nL} 7.23 0.00904 7.14 0,00893 <LOQ 0.00898 QC MKSI00201-MS-2 ug/mL 8.69 113% MKS 100201-MSD-2 u_/_:mL 8.34 MKSI002O I-MS-10 us/mL 45.6 109% 115'/o 111% MKS100201-MSD-10 u_/mL 46.5 PFOS = Perfluorooctanesulfoanmte 117% 116% LOQ = 2.50 ng/mL in standardcalculates to 3.13 ng/mL in serum. LOQ = (2.50 ng/mL unext standard* 1.25 ext dil factor * I dilution factor)/1000 - 0.00313 ug/mL in serum Date Entered/By: 10/11101 LAC o.te RSD St& Dev. MSLMSD RPD NA NA 0.0000795 4e/e 2% 3M Environmental Laboratory Page 97 3M Medical Department Study: T-6295.22 FACT-TOX-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-160, E00-1668 Product Numb_cM Substance); Matrix: Method/Revision: Analytical Equipment System Number:. Instrument Softw_re./Version: Filename: R-Squared Volta:: Slope: Y-Intercept: Dates of Extraction/Analyst: Dates of Analysis/Analyst: Date of Data Reduction/Aludyst: Box: Sample Data Extended Recovery Study Following a 26-Week Capsule Toxicity Stdy with Perfluorooctane Sulfonic Acid Potassium Sa]t(PFOS; T.6295) in Cynomolgus Monkeys T-4i295.22 Monkey Sermn ETS-8-.4.2& ETS-8-5.2 versus an unextracted curve Amelia 062498 Mnsslynx 3.4 See Below SeeAttachments See Attachments See Attachments 10/02/01 RWW 10/04/01 MMH 10/05/01 MMH 01-046 MONKEY SERUM Group Dose Samplo # Initial Vol. Extraction Dilution Surrogate Verified PFOS Dilution Method Bla_k WBIOO201.H20 Blk-I mL Factor I 1.25 WBIOO20I-H20 BIk-2 I 1.25 Matrix Blank RBS10020 I-Sara BIk-I 1 1.25 RBSl00201-Sera Blk-2 I 1.25 MKS 100201-Sara Blk-3 1 1.25 MKS100201-Sara Blk-4 I 1.25 Factor NA 1 NA 1 NA 1 NA i NA l NA 1 QC MKS 100201-MS-2 us/mL I 1.25 Out High 200 MKS10020 I-MSD-2 ns/RmL I 1.25 MKSI00201-MS-IO us/mL 1 1.25 MKS 10020 I-MSD- I0 ug/mL I 1.25 PFOS = Pedluorooctmmsulfonate NA 200 NA 200 NA 200 LOQ _ 2.50 ng/mL in standard c,.Iculatcsto 3.13 ng/mL in serum. LOQ = (2.50 ns/mL tmext standard* 1.25 eat (ill factor * I dilution factoO/lO00 = 0.00313 ughnL in serum Date E.ntemd/By: IO/1 !/01 LAC PFOS Cone,. n_/mL 0.00 0.00 0.09 0.13 7.23 7.14 8.69 8.34 45.57 46.54 Filename A011004082 A011004083 A011004084 A011004085 AO11004086 A011004087 AO11004088 A011004089 AO11004090 AO11004091 Concentration of PFOS u_/mL or % Re <LOQ (0.00313 ug/mL) <LOQ (0.00313 us/mL ) <LOQ (0.00313 us/mL) <LOQ (0.00313 ug/mL) 0.00904 0.00893 113/. 109% 115/o 117/. Mean PFOS u_/mL <LOQ <LOQ 0.00898 I 11% 116% RSD Std. Dee. MS/MSD RPD NA NA 0.0000795 ..... 4% 2% 3M Environmental Laboratory Page 98 3M Medical Department Study: T-6295.22 Stu_ FACT-TOX- 160. E00-1668 Producl NumberfTcst Substarwx): Matrix: Method/Revisioa: Ana_yUealEquipmem System Number: Inset Softwaze/Vemm: Fileamae: R-Squazed Value: $1ov: Y-Intercept: Datcs of Ex_Analyst: Dates of Analylitt/knalya: Date of Data Reduc_icm/Analyst: Box: Sample Data FACT-TOX-160 Covanc6e3#29-268 Analytical Report: FACT-TOX- 160 LIMS E00-1668 Ext_'ukd Rcro, very Stauly leollowin8 a 26-Weck Captulc Toxicity Potassium Sak(PFOS; T-6295) in Cymx_lgus Moldccylt T-6295,22 Mmlkey Scntm l:TS-I-4.2 & ETS-8-5.2 ver_s anexlxact_l rabbit sentcurve Amelia 062498 Maulyex 3.4 See Attadwamt_ Stdy with Perfl,_c See Anadmznu See Anaclmlents See Atuchncm* 10/02/01 RWW 10/04/01 MMH 10/05/01 MMH 01-046 Sulf_ Ae_ MONKEY SERUM Group Dote Sample # Method Blank Matrix Blank QC PFOS = pufluomectanemlf_mc WEll0020 I-H20 Blk-I WB 100201 -H20_BIk-2 RBSl00201-Sefa Dlk.l RBSI0020I -Sent sn,-2 MKS 100201-Sort Blk-3 MKS 100201 -Sofa BIk-4 MKS 100201-MS-2 us/mL MKS 100201-MSD*2 us/ptL MKS 100201 -MS-10 ul_mL MKS 100201 .MSD- I0u_'mL PFOS CelK. n[/mL 0.00 0.00 0.00 0.00 8.02 7.90 9.96 9.49 59.0 60.3 Concmtradea of PFOS _./mL er % Re <LOQ (0.00492 ug/mL) <LOQ {0.00492 u_mL) <LOQ (0.00492 u_/mL) <LOQ (0.00492 uy_L) 0.00802 0.00790 104% 99% 119% 121% Meam PFOS u[JmL <LOQ <LOQ 0.00796 102% 120% RSD Std. Dcv. MS/MSD RPD NA NA 0,0000849 5% 2% Da_ 10/11/01 LAC t 3M Environmental Laboratory Page 99 3M Medical Department Study: T-6295.22 FACr-TOX-I_0 Covance# 6329-268 Analytical Report: FACT-TOX- 160 LIMS E00-1668 study: FACT-TOX-160. E00-166| Produl Nurllbcl_T_lt Subt_am_): Matrix: M_aod/Revisian: Analytical F,,quipnt_t Systs_t Number: Inst_me_ Soltware/Verslon: Filename: R-Squared Value: Slope: Y-Im_cq Datesof_Analylt: Dat_ of Analysis/Analyst: Dale of Data Reduclkm/Aaalyst: Box: Sample Data Exlendcd Recovery Study Following 26-Week Capsule Toxicity Stdy with Pedluofe_'ta_ PotassiumSalt(PFOS;T-629_) in CyaomolgusMct_keys T-6295,22 MonkeySenun ETS-$-4.2 & ETS-8-5.2 wrsus an extracted_ scracurve Amelia 062498 Muslynx 3.4 See Below See Attachments See Attachaz_ See 10/02/01RWW 10/04/01 MMli 10/osJo I MMH 014146 Sulfmic Acid MONKEY SERUM Grot*p "' Dote SampLe # Meduxl Blank Matnx Blank QC PFOS _ Per_ulfonate WBI0020I-H20 BIk-1 WBI00201 -H20 81k.2 RBS 100201-Sela Blk-I RBSI00201-Scfa BIk-2 MKS 100201-Scsa BIk-3 MY_ 100201-Sera Blk-4 My_ ]0020 | .MS .2 UShlltL MKS 100201 -M$.D-2 u_/Ip_L MJ_ I0020I-MS-10 ug/mL MKS 100201-MSD-10 u_mL laitial Vol. nail I I I I I 1 | I I 1 Eatractlu Dilution Factor 1.00 | .00 1.00 1.00 1.0O 1.00 |.00 1.00 1.00 1.00 Da_: 10/I 1/01 LAC Surrogate Verif_l NA NA NA NA NA ,,,NA NA HA NA NA PFOS Dihttloa Factor I I I I I 1 200 200 200 200 PFOS tout. n_JmL 0.00 O.0_ 0.00 0.00 8.02 7.90 9.96 9.49 58.97 60.27 Hlenan_ A011004052 K0| |00401t3 A011004084 ;A011004085 A011004086 A011004097 A0| |004il$$ A011004089 A01J004090 A011004091 Cententratlon of PFOS U_l/mLor % Rec <LOQ (0.00492 us/mL) <LOQ (0.00492 ult,/mL} <LOQ (000492 uB/mL) <LOQ (0.00492 uffmL) 0.00802 0.00790 11}4% 99% 119% 121% Mean POS u|lmL <LOQ <LO_ 0.00796 102",4 120% RSD Std. Dev. MS/MSD RPD NA NA 00000849 5% 2% 3M Environmental Laboratory Page 100 3M Medical Department Study: T-6295.22 FACT-TOX-160 co,,.=,_6329-z_ Analytical Report: FACT-TOX-160 LIMS E00-1668 Study:PA_I'-TOX-160,EOO-l_dl Pi'odul/_umbcfl'I'_SubstaBcc): Matrix: MeUuxlfltcvisioQ: Ana/yliratFl _luipmcntSys_.mNmnbct: lnstmmclrl;_N_t_,c/Vcrsioa: Dateot'ExtlactJon/Analyil: Dale eltA" Nd_lil/Aanalyl4: Dale ofDILl R4xlu_io_AJulyl: _ lllOVeSllu_dy FolI(_IIaI2I6-WeekCapsu_To_eJlylSldy_th Pcrflt_'oectSu,l_le'orAtIi_I Potassium_dI(PFO_; T-6295)ils Cylx)lnolgtlMs Oldt)_ T-6295,22 Monkcy, Liw_r ETS-I-6.0 & ETS41-7.0vcma an c_raeted rabbitlivc'xcurve Amelia 0624911 Filcmm_: SI_ Alucluncnls Mar_ynx 3.4 09/05/01 RWW OgllO/I)l,09/13/01.09/17/01 Mb,IH 09/I 1/01,09/14/01,09/18/01 MM}t R-SquaredVaJll; Slope; Y*lnter_: BOxm: SAttachments SAltaChmcn_ SAIIJKhd_cn_ 01.O42 Sample Data MONKEY LIVER GrNp Dine Method BIk l_t_x l_k QC G_p I Saml_ # RBL090$0I-H20 BIk-I I_BL/)90_OI:_H2B0lk-2 RBL090501-LiverBIk-I t_L090_OI-Liv_ _k-2 RBL0905O2.300_ P.SL090501-300 ppb-MSD k,_KL0_501-300 p_I05505M-MS MIO.O_05t0-300_d_-I05505M-MSD 105_05M I05523M 105539F 1055521= PFOS Cs/c Comc. m_| 0.00 0.GO 0.00 o.oo 322 323 179 854 3251 31t6S 10675 77U Ccmce_rlltlial dPOS ul_l[or % Re_ <LOQ(0._29 n_J_ <L_ _0.G062u9g/ll) <LOQ(0.00629 uS/f,) <LOq (o.oo629 us/K) 102% 102% 29i% 291",% 3.25 3.97 10.7 7.79 Metal PFO$ uf/IL <3LOQ <t.oq 102% 294% 3.56 9.23 RSD Std. Ik.v. M_MSD RPD NA NA 0% 3% 0.436 2.04 5_Smrix_l}Lke_welsepni'_tcdatlheaplplropr_P_le.,Snadm. plcwillbere*_ikedm, th_opdatekvell, alalalcrdale. DaleEntea_l/Antly,=: 09112/010,9/26/01 LAC 3M Environmental Laboratory Page 101 3M Medical Department Study: T-6295.22 FACT-TOX-160 Covanc6e3#29-268 Analytical Report: FACT-TOX- 160 LIMS E00-1668 Sludy: FACT-TOX- 16fl. EIXI-1668 ProduclNumber(Te_ St/_taece): lVlatlix: I_hod/Revision: Analytical Equipment Syuem 1_ Soflwa_YVc_oR: Dalco[Exlrlglion/Ana_: _lC_Al_l_A_dy_l: Numbe_: Dale _" Da_ Rl_l,x:Uon/_dy,sl: I_/lgndcd Recovcly Sludy Fallowing I 26-Wl_.k C_I_II,, PO(_ tim _I(PFOS; T-6295) in Cynomollpts MonkeYs T-629_.22 Moalt_ Liver ETS4-6.0 & Ers_l.7._ _rsLul a_4e.xlrac4ed ril/M I llvel Tog_ily Amel_ 062498 MurJynx 3.4 09/05/('1 ]_WW 09110_,1, 09113101, 09117101 I,_1]1 OWl I_ 1, 09114/_1, 0_18/01 MMlI Stdy wilh P_fluorol_tal_ SLiIJ'oni Acid Filertamc: R-Squ_ucd Slope: Y-lu[c[c_; See Be.low V See AOJchmnU See Aitachl_nll 5_Att_hmcnts BOX#: 01-042 Sample Dais MONKEY LIVER C_p Dose Idctlu_ BIk _xBIk Sample # R_.O90501 ._ B_-I R_L09_501 -H20 BLk-2 RILL090501 -Liver BIk- I Surrolp_ V_riF_l NA NA NA IDilImJWL It 1,000 1.000 1.600 T_ 'd Mm o/[Jv_r | NA NA NA RBLOgOS01-Live_BIk.2 NA I._)0 NA (_ RBL_90$01-30_ pS_-_[S NA l,_00 HA P.BLO_501-300 p_-MSD NA 1._ NA _090501-300 pI_IOJ50._M-MS NA 1,0160 NA IvlKL090501-300 p_)-IO5505M-MSD NA 1.0160 NA Group i IO$505M NA 1.0160 NA IO$523M NA 1.06JO NA IO5539F NA 1.0155 NA 1055_2F NA 1.0664 NA PFOS = Pr.rlluo_rorml Mauixspikuwcren_s_dald_app_ptla_l*_. Sample will be m-sFi_d, altheapp_lpliatlm, els, lt ahlt_rdat_. PFOS _ nf,/g 0.00 0,_(} 0.00 0,00 321.51 322.711 13.93 83.43 66.07 |2.38 21.66 16.61 I'FO$ Dlk_loa Fa(1or 1 I 1 I 1 1 _0 JO 50 $0 .500 560 I'FOS Calc. Cone. n_| 0.00 0.00 0.00 000 322 323 1179 1154 32_| 3865 10675 7758 ]rll_mame A010910OI6 A010910017 A0109|(X}I| A010910019 A01091_(}22 AO10911XI23 AOI0 g 17016 A0109171117 A0109171118 A010917UI9 AOl0913020 AOI0913021 Comr._tratiem olrPFOS vF/J_ or % Roc <I.OQ (0.O11_29 ug/_) <I.O_ {0,00629 uN_) <LOQ (0.00_29 ul_/g) <1.OQ(0.OO629u#/g) 102% 102% 298% 290% 3,2_ 3.87 10,7 "/.79 Mmm PFOS u_/it <LOQ P.SD Std. Dev. MS/MSD RPD NA <L(_ ! NA IO2% I}'4 2_4% 3.56 9.23 3% 0.436 2.04 D_t_ Enteled/A_ 0WI2/01, 09_26/OI LAC 3M Environmental Laboratory Page 102 3M Medical Department Study: T-6295.22 FACT-TOX- 160 CovanceJ 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1668 $txly: FACT-TOX.I60. F_0-166! Pmd_l NuJab_est _): Matrix: MClhod/llcViliO_ Ai_tlytic_l_l d_e nl SyitcmNtmlk_r: In-qranznlSon_arsion: Datc ef Emracli_/Amdyst: Date e_'AnalFils/Analyll: Dale el_DitaR_Jcti_/AJnatlylt: Extended ;lecovety $1uCtFy'a_lm*infa 26-Wet Ca_ Toxicity$_dyw/OlPert/mreecane S.l_aic Acid Peu._ium Salt(l_t'_; T-6295) in C_lgus Menkeys T-6295,22 MonkeyLiver 12"1_.8-6.0A, _1"S-_7.0 _t'll_ all f,xlfaledrabbitliw[ UI*Ce At_n_li_a2498 Mauay_ 3.4 10_2_ I RWW 10/04_1 M/vfl-I 100_01 Iv_DI Filchme: R-SquareVdalue: Slope: Y-lntrcept: Box 1: See Allachnw_ts SeeAttachmenu See AOaChmenli Sec AIItaclm3eall 01-046 Samplc Data MONIGCY LIVER Gnmp Due BIk Sampbe# RBLIOO201-142E0lk-3 PFOS Cak. Co,_ nf/I 0.00 CHccatradu dPFOS ul/l or % 1_t <IJ3Q(0.0126uf/g) MaoixB[k QC RBLIOO2014120BIk-4 RBL 100201-LivB_IIt-I RBL100201.LiverBlk-2 I_.L 10020I-2 tlg/0-IOJ_OJM-MS 0.60 0.00 0.CO 4364 <LOQ(0.0126ug/R) <LOQ (0.012.6g/g) <LO_ (0.0126 ug/_) 55% MKLIO0201-_ut [/I-IO55OSM-MSD 3554 ld_Ll002Ol*10 US/_-IO$_JM-MS 9441 15% 60% MKI,IO020I.I 0 u_/a-105505M-MSD Group 1 105505M = Pm/l_mocum_u_oeXc 5761 3237 <LOQ(0.0126 u_/|) 3,24 I:_0 dilolimw_l_0odihl_ _exllr-_Lss_nHt-dtlul_lM I'JOluldlmaly_ 10/16/01. LAC 11/211/01 Mesa PFO$ ulll <LOQ _ 3J_ 60% ItSD Std+ I_-v. MSCMSDIIPD HA HA 112% NA DateEmeled/Amlyst: 10/I1_11 L&C Date Veril_d/Aaaly_: 12/1_OI Stud'/: FACT-TOX-I _, E00-1_,1 ProductNlallbe_Tel Sul_al_e): _dx: Al_lyli_l BqllJ]_llt_l Nmdb_. Ir_lalmx_merl_a: Datee(Ex?a'ac_/A_: Dateel'An_AMly10: Dale elfDala Rcdaftks_Aaaly_ me.,,.t,_ _ _ FelIo_i_ll 26-WellCpldl Toxkil_ Sl_ tillt Ptl(Ite_eal_ Sull'ookk_l Pvt -t't+'_ Salt(PFOST; -0295)in C_emoll_ Monkeyl T-629S,22 MeakeyLiver EWS-a-6.&0 ETS_-70 v_s_ anextracln_dl_biltiw.crw_ Alllteli0a624941 Rleltam_: See Allachml_tlt Mas_yu 3.4 R-Squmed Value: SeeAllachraentt 10/02/0R1WW Slope: .SeAeltachnaenU I0/16_0M1MH Y-ll.erc_: SeeAlt_hraents 10/16#01 _ Box#:. 01..046 Sampte Dam MONKEY LIVER G_p Dine Stmpk 0 PFOS Cdr. Ce_- Co_.mIrallm _r PFOS Me.am PFO$ I_B &td. Dev. MethodBIk ]t_L 100201-H20BIk-3 e_/I 0.00 _ or % I1_ Itl_ <LOQ (0.012u6g/g) MS/MSD UD RBLI00201-1120BIk,,4 0.00 <LOQ(0.0126e,V_ <LOQ NA Mmix _ RBL1002014Aw/Blk.I RBLI0020I-Li_ BIk.2 1,33 <_3Q (0.01/6 uS/g) 025 <LOQ (0.0126AV_ <L.OQ NA QC MIKLI00201-2_I_I-I05_05M-M_ 3_4 MY_1_O201-2 U_/A-IO$_OSM-MSD 2995 MK_I00101-10 ttg/l-1035OSM-MS 9719 29% -I I% 63% 9% 438% ,' _ 100201-10tq[/|-lO5505M.MSD 74456 4 I% 52% 42% Greap I PFOS- R:dl_renme _5505M 321_ 3.21 NOTE: DIItaWI_Uet witlun cntmi_ ldlldikl_dlmlal_lu,a_ml_filu_d I:S_ I_mm_lpn_limm_ _1 _a_l_ 1_4_1. LAC 11r_8_1 D_te E_Analtyt D_ Vcxifled/Am]ylt: IO(2_'1 LAC 17./1_-I mmh 3M Environmental Laboratory Page 103 3M Medical Department Study: T-6295.22 FACT-TOX- 160 Covance# 6329-268 Analytical Report: FACT-TOX- 160 LIMS E00-1 668 Study: FACT-TOX-160, E00-166| Preduct Numb_elt Suhllmu_): /vlaUi_ Method/l_-vialca: Alud_kad EquipnlemS! ystemNtmtb_. ImammemSoflwarc/Vcrlioo; Dateof Extracfio_/Aaudyil: DI_ of/tltalyals/Altaly_: Dateof Dlltl P_dlwlion/Analysl: Sample Dsta ExteNhx/I_ $1udyFollowing 2a-WeekCalmde To_'i ty Stdy w/lh Pefl]mmoctane SulfonicAcid I_tl_ium Salt0a_3_; T_295) in C'y_n_lguaMe_keyt T-6295.22 M_kfy Liwf ETS4-6.0 & _rS-lbT.0 _ aa cxtlataedraWoitliver curve Amelia1_2498 Mar_ 3.4 Fllenam_ R-_lUared Value: SecAtUtchmcnlts See Al_tachmcnu IO/0/d01 KWW 10/24401_ 10/26/01 _ 51oF_ Y-lllteln_: Box/: See AUaChmelats SCAU,_hmcnts 01.046 MONKEY LIVER Group Sliple a Method Blk Matr_ Blk QC RBLI002OI-H20 BIk-3 RBL100201-H20 Blk-4 RBL100201-Liv_ BLk-I RBL100201-Livcg 81k-2 MKLI00201-2 uM/I-IO550_M-MS MK{.1_201-2 u_/|-/05505M-MSD !OqC,L10020-1I0u_8-_SSOSM*MS l_gJLlO0201+10uI_/|-I05505M-MSD Glllp I PPOS= Pafltm_ctanesfuolnatc 10550JM pIros Ctk. Cm_ nf./! 0.60 O._ 0.00 0.1iO 14945 12530 38873 33522 ,1,1282 Cea_lllratim ef PlrOS ,,It8 of % Rec <LOQ(o.012fi uS/g) <LOQ(0 012fiu_8 ) <LOQ(0.0126 u_l ) <I.OQ{0.012t6rig) 178% 61% 268% 216% 11.28 Meam PFOS UI_IL <LOQ <LOQ 119% 242% RSD St/. _-v. MS/MSD RPD HA NA 9S% 21% D_t_EJ1/cfed/Pmalysl: Il_qtl LAC DateVt_dficd/Analysl: 12/I(_1m1mh Swdy: PACT-TOX-160,E00-166a Pmdm/Numbcs_'m Subm,_): MNrix: A_I Eq_pmem Sylem Number:. [nstmmatStaltwan_miea: Dine af _"_oa/AMlyst: Date ol'Amdyl_/AaalDl: Dalco(Data P_i_Amdysl: Sample Data ExtrudedReom_ StwdyF_lowiql at26-Wc_ Cap_k Toxicity_ Potawum SalI_FOS; T-6295)ia CynomollpmMookeyl 7-6295.22 ldmkeyLiver _'S-41-6,0& ETS-8-7,0"._c_sIm_m-actcdral_itilv_ ottn_ with Pcdlumeo_tanc SulfmucA_d _1098 Filcnam( S_ Attw.knamu Ma_y_ 3.4 11/07/01 RWW III15_1.11116/01 b_dl4 11/1_1, 11119/01MMH R-SquaraVlalue: Slope: Y-II_ Bo_#: SccA.aclunam _ Atlaclm_nts SeeAttachmcn_ 01-046 MONKEY LIVER Group Dine Samplea PFOS Cak. Co_ Cos_mtrat_a _ rlrO5 84eros PFOS RSD SId. Dcv. nl/I ul_ m+% ReC U_l MS/M_D RPD BIk RBLI I0701-H20 ]_k-I 0._0 <U3Q (0.A0639ul/10 P_I JO?Ol.l.1208,,..2 o.oo <L_Q <o.oo62_9VI0 R.BL110"m1-1120BIk-3 0.00 <LOQ (0.00629 us/8) 0 RBL110701-H20BIk4 0.00 _<LI3Q10.00629u_8 ) <LOQ NA BIk RBL110701_ BIk-I 0.00 <LOQ (0.00629 UlVI) R_I.I 1070l-Liver"BIk-2 0.00 <LOQ0}.006191_8 ) , RBLI 107014Av_BIk-3 0.00 <LOQ (0.00629Ul_/f,) 0 RBL110701_ BIk4 0.00 <LK_ (O.00629ul[/8) _ QC Grmp I pF_ta m _mllt MKL110'701-2vf/I-|05_0$M-MS MK_110"/01-2 uA/K-10JS05MAMSD I_d. I 10701-I0 ulVil.105_,OSM-Iv_ 11O7O-1IO U_|-IO550_M*MSD 105505M I 6328.44 6134.39 11136.7'8 1_7.49 4241.25 1oo% _ 73% 61% 4.24 95% 6?% 10% 17% Dsle Ellefed/Anal_a: I_le VelUl_/Amllylt I L/2_KI LAC 12/1_ | amh 3M Environmental Laboratory Page 104 3M Medical Department Study: T-6295.22 FACT-TOX-160 co,,c,_ 6329.268 Analytical Report: FACT-TOX-160 LIMS E00-1668 Study: FACT-TOX-I60, E00-1668 Pmdu_NnmbcrffcsSt ut:_Jmce): Marx: _: A_y_cal EquipmenSt ystemNumber: lnsmJmemSeflwamNemon: Dateo(Extr_tio_/Alalys_ DateofAnalys_AaaJyst: Dateo/Data I_ductJcm/Analy_ E_aead_l I_ StudyFollowius a 26-WeekCapsuleToxicity Stdy wilh PrJfluer_ PotassimnSait(PFOST;-6Z_5)in Cynemolt_Ms onkeys T-6295 22 Me,_'y Liver ETS-It-5.1&) _1"S4-7.0vcnmsanextractedrd_! livcr cttr_ Amclis062498 Ma.udynx3.4 10/02/01ItWW 10/04/01 _ 10/05/01 _ Filr_wc: R-SquaredValue: Slope: Y-Intercept: Box II: SeeBelow SeeAttaclmu:ts SceAttachmcn_ SeeAttachments 01-046 5ul/onicAcid Sample Data MONKEY LIVER Group Me_od Blk Matrix Blk QC Grmlp I PPOS = P_rll_lRmatc Sample ti Surref_ Verid'_d II_I.I_201.H2B01/t.3 NA !_8L10020I-H20 B"_-4 14A RBL10020i-LJvel Blk.I HA RSLI0020I_ Blk.2 NA MKLIO0301-2I_I-103505M-I_ NA MKLII_201-2uK/I-_1055051VI-IVISD NA _XLI_01-10 u_/g-IOS_OSM-MS NA I_,_L I00201*10uI_g-IO550JM-MSD NA 105505M NA lltltJalWL II 1.0000 1.0000 1.0_o0 I.(XI_O 1.0232 1.0232 1.0232 1.0232 1.0232 Total Mau d Liver | NA NA NA NA NA NA NA NA NA PFOS Coa_ nZ/_ 0.00 0.00 0.00 0,60 89.30 72.72 19.52 11.79 66.24 I'FOS Diluflom Teeter I 1 I I 50 50 _0 500 50 PFOS C*I_ Com_ n_| 0._ 0.00 0,00 0.00 4364 3554 9441 5761 3237 Filuam_ A0||004030 A011004031 A011004032 A011004033 _I I004030 A011004039 AO11004040 A011004041 A011004057 Coaccmtrstiou of PFOS m_=or % Rec <IJ_ _0.012_6tl/g) '_.OQ10.012u6_/R_ <LOQ(0.0126 u_g) 'd..O(Q0.012u6_'g) 55% 15% 60% <LOQ(00126 u_) 3.24 Meam PFOS ul/I <LOQ _OQ 3_% _0% RSD 51d. Dev. MS/MSD I_D NA NA 112% NA Date_.Aa,tF,/_ Dale Velifie_Anldy_. 10/I I_1 LAC 12/1/_01 Study: FACT.TOX.160. Eao-ImSS Produ_Nm_a_rfTmt_d_da__): Matrix: Melhed/Itt_isio_ Analyl_ EquipmealSyslcmNundx_. Inm..m,,eatSet_vare/Vertm: Da/_ 01f._t_arat;tiolt/AJ_]yll: DaU:el'Analys_AnMy_: Datee/Da_l P,_da_ma/Aadysl; S_mple Data Exten_d t_cove_y StudyFollowinl 26-Wedc_ Te_ia_y Stdywith _ Pmamum.%It_FOS; T-6295) in C_mmol0usMonkeys T.629_J.2_ _ ETS41-(,.0& ETS4-?,0 _ ancxuac_l rab_ liver Amcl_J962490 Vllenam_: Se_Belew Masdyax 3.t R-Squar_ Val_; S_ Aaachmems lO_D_dORI WW Siep_ See Altachments 10/1_1 _ I0/l(dOI _ Y-Imp: Box#: SeeAOaChments 01-O46 Selfeaic Arid MONKEY LIVER Greup Sample # Sun,ojate Vereled MethodBIk RI_ l(]O_Olq'120BIk-3 Mlgrix BIk RBLI00_01-H20 Blk-4 IU_LI0020l-l.._v_ Blk-I ItBLI00_0 l-Liv_ Blk-2 _..LI0_2A) 1-2tl_g-lO5 _ 5]t4-MS _I00201-2 II_/A-I0$5OSM*MSD M_I00201-I0 Itl_/g*10JS051Vl*IvlS IVl]I_I._L'201 -10 _,.-1005OJ_ Groep I I05505M PFO$ - Pernm_oa_ .NOTE: Dramwele itm within _ Idld_lutedmm_kawere _dilme_ NA NA NA NA NA NA NA NA NA 1:.5.)f_o_ lailhd Wt. Ir00_l 1.0000 1.0000 1./_0 1.0232 1.0232 1.0252 10252 1.0232 TeoJ Mm _ LJw_r | NA HA NA NA NA NA NA NA NA pi_S Cur. af_l 0.00 0.00 1.33 0.25 77.84 61.29 191.80 152.79 65.79 ex_ra_ ud anab,'_doa 10_/01. LAC 11/20_1 PI_OS DJlulJo_ Yac'_or I 1 1 I _0 5_ $0 $0 50 PtrOS Cak. Cme. ns/_ O.00 O.00 1.33 0.25 51104 2995 9719 7466 3215 Dm_ Emw.._VAm_m: 10/'29/01LAC Date Vmified/_ 12JI0_01tomb FIJeaame A.O1I01_030 AOII016031 A011016032 AO11016035 A01101611311 A011016039 A011016040 A011016041 A011016037 Ceecealratiea d pFOS tJ_z or % P-_ _,OQ (0.05|6ul/j) <LOQ{0.0126 u_/_ <LOQ(0.0126 u_/g) '_X)Q (0.0126 u_t 29% -I I% 63% 41% 3.21 Mean plros tJW8 <IJSQ <1.O9 9*/* 52% R_D Sad.De_. MS/MSD RPD NA NA 43V/* 42% 3M Environmental Laboratory Page 105 3M Medical Department Study: T-6295.22 FACT-TOg-160 Covance# 6329-268 Analytical Report: FACT-TOX-160 LIMS E00-1 668 Study: FAU-roTOX1-60.EO0-16611 ProductNumbedTat SubSume): MaldX_ Meth_VRcvisieu: Ain_)licaJEquipaleatSystemNumber: |_mtmcm SeR_tVm-_on: D_ of _aclion/_ya: Dateof _Aaalyit: Dateel Data Reda_on/Aaalylt: Extorted I_ StudyFollowinga 26-Wee_kCa_ Toxicity Sldywith Pclthmfmcta_ Potm_m S_t(PFOS;T-629S) in CynomolgtmMonkeys T-_295.22 MonkeyLtwr ETS4-t,.0 & ETS4-7.0 versusan exlxactcdrabbi liver cmvc Arabia 062498 Filcn,_0u: SocBelow MISSI_X3.4 |O_J/O| RWW 10/141/011_ 10t26/0 t MMH R.$quaredValue; Slope: Y4ale_: Bext: See An_hn_lu S_ AUacl*.m_u See Attw,hn_nU 01-046 SulfonlcAcid Sample Dram MONKEY LIVER Grmtp Dine Sampk # S411rrllllt_ Verified MfthodBIk RBLI00201.H20 BIk-3 Mat_x BIk QC RBLIOO2OI-H20BIk4 RBL 100201-IAvel"BLIkI- RBL100201-1AverBIk-2 MKLIU0201-2ulg/8-105505M-MS , _,.LI00201-2 u_/_- 05505M-MSD MIKLIUO201-1I0q_/II-105505M-MS MKLIGO2OII.0U_/|-IO550_tM-MSD GrmtpI T05505M PFOS- Perlluev_'t,mesulfoamc Out Hil,h Ot_ IqJ_h HA HA OutHiKh Out Hifah OI1 HJsh Ota HJ_h Out I-tilth NOTE: _l:om_i_lwilh_.vt_s_ Sampl_m_re-e_cled,_lm_l laJtlaJWit. g 1.0000 1.0000 1.0_0 1.0000 1.0232 1.0232 1.0232 1.0232 1.0232 Total Mau ot _ NzA NA NA NA NA NA NA NA NA PFO$ Cm. n0.f0/t0 000 0.00 0.00 305,113 256.41 795.50 6_6+Ofl 230._7 I;_0, amdltnalyledm111/16_1. LAC lit21/0! FFOS Dilution FacItor I I I 50 50 _0 50 50 ]PFOS CIl_ Come. n0.f0/z0 O.00 0.00 0,00 14945 12530 38873 33522 11282 Flkallm_ A011024030 A0i 102_d}31 AO11024032 AO11024033 AO11024038 AO11024039 AO110241140 A011024041 AO110244'137 ComtlmCratlom of PFOS <IJe3l/Q| (0e.0%| 26Rue_c/_) <LOQ(0.0126 ug/_) <LOQ(0.0126 u#g) <LC_ _0.0126ug/_ 178% 61% /.611% 216% 11.3 Meall PFOS wl/z <L_ <LO0 119% 242% I_D StdLBey, MS/MSD RPD NA NA 98% 21% Date E_Aaalylt: Date Vcvifetd/Amdyt I I_/0l LAC 17,/10/01mmh _a_: FACT-TOXo160, E0_|66a Pmdua Numb_r_t Sab_m_): Mm_: Method/Rcvi_em: Amb'UealEqeilm_al Sy_ Ntnab=z Inmmm_t Soawme/Ver_m: Dateof Extlalak_Allalyst: Dmc of Analysis/.Msaly_ D_eofData R_laaia_ARlyst Sampl* Data ExteadedP.gov_y SludyFello_n I 26-Wed_C___,I-.T_o0ddtyStd__lh Pcffluofeo_e _ A_I I_maiam _; T-6295) in C_momol_ Momk_ T-6295._2 Mm_ _TS-8-60 & L:'I'$4-7.0vcrmsu cartcted ntbbd5lvetcteve Madellae_ le_l Masslynx3.4 F'xlenmae: R.SqtmedVa_e: See Below See Auaehn'm._u I1_07/_1 RWW illlS/01. 11116OIMMH IIIIODI. 11119/01 _ Slo_: y.latefceplc BozD: See Altachal_l S_Atlaeh_ 01-046 MONKEY LIVER Grmtp Sample 8 Sit_mllmle Vcrlfuul l_tial Wt. Tottl Mala _L_c Meted BIk R_L | 1070i-lt20 Blk-I NS FLBL110701-H2B0 lk-2 NS RBL110701-1420BIk-3 _IA RBLI IUT01-H20BIk-4 qA MatrixBIk RBL| 10701-_ Blk-I NS RBL1107014Av_ BIk-2 N_ RBL110"70-1_ B_-3 31A GrNp I RBL II0"/01-IAve_BIk-4 NA I_LI I07UI-2 i[/It-105505[:d[_ lqA IdYLl 10701-2u_/g-IOS_OSM-MSD NA ),,G_UI I0_O1-10_4_8.105$OSM-M.S lqA 1,_r,l.i 1o-_1-io u_-|OS_M-_D NA 105505M NA 1.0_0 NEA 1,0000 NA 1.0000 NA I.C_O0 NA 0.9425 NA 0.9425 HA 0.9425 NA 0.9425 NA 1.0023 NA 1.0023 NA 1.0023 NIA 1.1_123 NA 1.002J NA N$ -/,t_ Spiral wilb 5en_ate NOTE: AverNlere_vel_ _'lhe 10t_| n_ttlixsplke,wal notwithint_ ,t./.30%re.very aslistedia _ _. A_ DaleI=meled/Amtly_: I |$26_11LAC DateV_il_d/AnaJy_: 12/10_l mmh PFOS Ceec. l0l.__/O! 0.430 0.00 0,130 0.00 0.00 0.00 0._0 126.86 122,97 237.28 213.64 85.02 roll_ _|_ PleOS DIImln FacI tor I ! I i I I I 50 50 50 50 50 PFOS Cak. Crate. i0a,(__ 0,00 0.00 0.00 0.00 0.00 0.00 0._ 6328 6134 11837 10_$7 4241 7114marne M011116031 MOII 116032 MOII 116033 M011116034 MOIl116038 MOll 116039 MOII 116040 M0t1116041 MOII 116046 MO]I116047 1,4011116o411 bdl_l|116049 M011116045 Colt_mnttiea dPFO_ <LlOalQ_(0O.0r0%_2i9_eul/I) <LOQ (0.0(029 tqt/g) <IX_ (0.00629al_ <LOQ(O.00_29uK/K) <LOQ (0.0062_9 <LO_ (0.00629 t_i) <LOQ(0.00629 ul_/f,) <LOQ(0.00629 u_ 100% 90% 73% 61% 4.24 Memt PI_OS if_I <LOQ _,J3Q 9J% 6"/% i_O Std. Dev, MS/MSD RPD NA lqA 10% |7'_ 3M Environmental Laboratory Page 106 3M Medical Department Study: T-6295.22 3MMedicalDepartmenSt tudy:T-6295.22 Analytical Report: FACT-TOX- 160 LIMS E00-1668 AnalyticaRl eportF: ACTTOX-160 LIMSE00-1668 Appendix F: Example Calculations Formula Used for SeraAnalysesin Study FACTTOX-160 AR (ng/mL) x DF x FV (mL) x 1.0 lag = Reported Concentration (pg/mL) EV (mL) lO00 ng Calculation Used for Group 1, Week 17,Animal ID 105505M 45.75 ng/mL x 200 x 1.0 mL x 1.0 ug = 14.5 lag/mL 0.63 mL 1000 ng AR-- Analytical result from MassLynx summary DF--- Dilution factor FV--Final extract volume (1.0 mL unless otherwise noted) EV--Volume of sera extracted FormulaUsedfor LiverAnalyses in StudyFACTTOX-160 AR (ng/g) x 9curve (]) x DF x 1.0 pg = Reported Concentration (pg/g) sample 1000 ng (]) _ curve is assumed to be: 1 g liver r 5 mL I-I20 Calculation Used for Group1, Week 53, Animal ID 105505M 66.07n_gx !_5mL x50 x _ =3.251=1dg 1.0160 g/5mL 1000 ng AR-- Analytical result from MassLynx summary curve--Density of the liver standard curve, assumed to be lg liver/5 ml water sample---Density of the liver sample (g sample/5 mL H20) DF--- Dilution factor 3MEnvironmentaLl aboratory 3M Environmental Laboratory Page23 Page 107 3M Medical Department Study: T-6295.22 3M Medical DepartmentStudy: T-6295.22 Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport: FACTTOX-160 LIMS E00-1668 Appendix G: Interim Certificate(s) of Analysis 3M Environmental Laboratory 3M Environmental Laboratory Paae 24 Page 108 3M Medical Department Study: T-6295.22 Analytical Report: FACT-TOX-160 LEviS E00- i 668 _O_k__Phone: CEntrE Analutical Laboratories. Inc. 3048 ResearchDrive StateCollege,PA16801 www.centrelab.com (814)231-8032 Fax:(814)231-1253or(814)231-1580 INTERIM CER TIFICA TE OF ANAL YSIS Revision 3 _JlgtCopyof Orfginal Centre Analytical Laboratories COA Reference #: 023-018A 3M Product: PFOS, Lot 217 Reference #: SD-018 -------[_C-_3u/0_ " Initial Date TestName Purity: 86.9% Specifications Result Purity _ i ii Appearance Identification NMR White Crystalline Powder 86.9% Conforms Positive Metals 0CP/'MS) I. Calcium 2. Magnesium 3. Sodium 4. Potassium 2 5. Nickel 6. Iron 1. 0.005wtJwt.% 2. 0.001wtJwt.% 3. 1.439wtJwt.% 4. 6.849wtJwt.% 5. <0.001wtJwt.% 6. 0.005wt./wt.% 7. Manganese 7. <0.001wtJwt.% Total % Impurity (NIVIR) Total %Impurity (LC/MS) i.91 wtJwt.% 8.41 wtdwt.% Total %Impurity (GC/MS) None Detected Related Compounds POAA 0.33 wt./wt.% Residual Solvents (TGA) None Detected -Ptmty by DSC Inorganic Anions OC) 1. C"hloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite ........... Not Applicable_ !. <0.015 wt./wt.% 2. 0.59 wt./wt.% 3. <0.040wtJwt.% 4. <0.009wtJwt.% 5. <0.006wtJwt.% 6. Phosphate =, 7. Sulfate* Organic Acids _(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA 6. <0.007wt./wt.% 7. 8.76 wtJwt.% I. <0.1 wtJwt.% 2. <0.1 wt./wt.% 3. 0.10 wt./wt.% 4. 0.28wtJwt.% Elemental Analysis*: 1. Carbon 1. Theoretical Value -- 17.8% 1. 12.48 wt./wt.% 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine 2. Theoretical Value = 0% 3. Theoretical Value -- 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% 2. 0.244 wtdwt.% 3. 1.74 wt./wt.% 4. 8.84 wt./wt.% 5. 54.1 wtdwt.% COA023-018A 3M Environmental Laboratory Page 1 of 3 Page 109 3M Medical Department Study: T-6295.22 ........... Analytical Report: FACT-TOX-160 T T'k Artl'l "lr_/'_tg't I ffl'l JI.aJLJ.VI_._ I-'_VV- 1 UUO '_rO"_'_k._ik Centr6 Analytical Laboratories, Inc. 3048ResearchDrive StateCollege,PA16801 www.centrelab.com Phone:(814)231-8032 Fax:(814)231-1253or (814)231-1580 INTERIM CER TIFICA TE OF ANAL YSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08/31/00 Expiration Date: 08/31/06 Storage Conditions: Frozen <- 10"(2 Re-assessment Date: 08/31/06 1Purity = 100% - (sum of metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.905%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.07% Purity = 100% - 13.07% = 86.9% ZPotassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials of low purity. No endotherm was observed for this sample. '=Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur " determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. _TFA I-IFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyrie acid Nonafluoropentanoic acid Pentafluoropropanoie acid _l'heoretieal value calculations based on the empirical formula, CsF17SO3K + (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018A 3M Environmental Laboratory Page2 of 3 Page 110 3M Medical Department Study: T-6295.22 -- Analytical Report: FACT-TOX-160 L_vIS E00-1668 CentrE Anakjtical LaboratopiEs. Inc. '_rO_k__Phone: 3048 Research Drive State College, PA 16801 www.centrelab.com (8142)31-603_ Fax(6: 142)31-1_o$r3(8142)31-1_60 INTERIM CER TIFICA TE OF ANAL YSIS Revision 3 Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total wt./wt. % 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average result from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average result from the C6 and C8 standard curves. PreparedBy: dQ,_/L_ Charles Simo_ Scientist, Centre Analytical Laboratories /ol_/i/o/ D_te .,_hn Flaherty / Date Laboratory Manager, Centre Analytical Laboratories COA023-018A 3M Environmental Laboratory Page 3 of 3 Page 111 3M Medical Department Study: T-6295.22 3M ENVIRONMENTAL LABORATORY Analytical Report: FACT-TOX-160 r r_Ac rmn '5_ Recorded By: LisaClemen C__ ]_ ...... 3M Environmental Laboratory ::Date 08,03,01 i o_}_,_1o, ,i I - Fo=_:rs-4-15.o ExactCopyol0riglnal _ Io_._D, Initial -Date Page 112 3M Medical Department Study: T-6295.22 3M MedicalDepartmentStudy:T-6295.22 Appendix H: Report Signature Page Analytical Report: FACT-TOX-160 LIMS E00-1668 AnalyticalReport: FACTTOX-160 LIMS E00-1668 Andrew Seacat, Ph.D., Study Director _---_atej John Butenhoff, Ph.D., Sponsor Representative Date LisaClemen,PrincipaIAnalytiIcnavlestigator Date William Reagen, Ph.D., Analytical Laboratory Manager Date 3M Environmental Laboratory 3M Environmental Laboratory Paae 25 Page 113