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3M Medical Department Study: T-6295.10 AR226-1030A003 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Study Title Oral (Stomach Tube) Developmental Toxicity Study of PFOS in Rabbits Analytical Laboratory Report Title Determination of the Concentration of Potassium Perfluorooctanesulfonate (PFOS) Fluorochemical in Rabbit Liver and Serum Specimens Data Requirement Not Applicable Author 3M Environmental Laboratory Analytical Phase Completion Date at signing Performing Laboratories Liver and Serum Extractions Liver and Serum Analyses Pace Analytical Services, Inc. Tier2 Division 1700 Elm Street, Suite 100 Minneapolis, MN 55414 3M Environmental Laboratory Building 2-3E-09, 935 Bush Avenue St. Paul, MN 55106 Project Identification 3M Medical D epartm ent Study: T-6295.10 Argus In-Life Study: #418-012 Analytical Report: FACT TOX-099 3M Environmental Laboratory Request No. U2400 Total Number o f Pages 127 3M Environmental Laboratory Page 1 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Concentration of Potassium Perfluorooctanesulfonate (PFOS) Fluorochemical in Rabbit Liver and Serum Specimens Study Identification Numbers: T-6295.10, FACT TOX-099, LRN-U2400 This study was conducted in compliance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations 21 CFR Part 58, with the exceptions in the bulleted list below. Exceptions to GLP compliance: There were two study directors in this study. This study was designed as two separate studies. The in-life phase was considered to end at the generation and shipment of specimens. The analytical study was considered to start at the receipt of these specimens for analysis. This resulted in having two separate study directors, 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 is a lack of an electronic data audit trail. There is no reference standard stability. The surrogate THPFOS was not characterized. On at least one occasion, data entries were not signed and dated on the date of entry ______________________ 9 ^ 4 - ________ Marvin T . Case, D .V .M ., Ph.D., Study Director Date 't/h*_T- John L. Butenhoff epresentatlve Analytical Investigator oi^lot/o / Date 3M Environmental Laboratory Page 3 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 GLP Study--Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Concentration of Potassium Perfluorooctanesulfonate (PFOS) Fluorochemical in Rabbit Serum and Liver Specimens Study Identification Numbers: T-6295.10, FACT TOX-099, LRN U2400, Argus 418-012 This study has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Dates Phase Date Reported to Management Study Director 10/16/00 Liver Extraction 10/20/00 10/20/00 11/08/00-11/10/00 Data 11/10/00 11/10/00 12/11/00-12/15/00 12/20/00-12/21/00 1/11/01-1/12/01,1/15/01, 1/18/01-1/19/01 Draft report Data/Draft Report 12/15/00 1/22/01 12/15/00 1/22/01 3M Environmental Laboratory Page 4 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Table of Contents GLP Compliance Statement................................................................................................ 3 GLP Study--Quality Assurance Statement........................................................................ 4 Study Personnel and Contributors.......................................................................................7 Introduction and Purpose.................................................................................................... 8 Test System.................................................................................................................... 8 Specimen Collection and Analysis..................................................................................9 Specimen Receipt and Maintenance.................................................................................. 9 Chemical Characterization of Reference Standard.............................................................10 Method Summaries..............................................................................................................10 3M Environmental Laboratory.........................................................................................10 Preparatory Methods................................................................................................. 10 Analytical Methods..................................................................................................... 11 Analytical Equipment................................................................................................. 12 Deviations........................................................................................................................ 13 Data Quality Objectives and Data Integrity......................................................................... 13 Data Summary, Analyses, and Results.............................................................................. 13 Summary of Quality Control Analyses Results............................................................... 13 Statement of Data Quality.............................................................................................. 14 Summary of Sample Results (3M Environmental Laboratory).......................................14 Statistical Methods and Calculations.................................................................................. 14 Statement of Conclusion (3M Environmental Laboratory)................................................. 15 References........................................................................................................................... 15 Appendix A: Chemical Characterization and Control Matrices........................................... 16 Appendix B: Protocol, Protocol Amendments, Deviations................................................. 17 Appendix C: Extraction and Analytical Methods.................................................................60 Appendix D: Data Summary Tables................................................................................... 112 Appendix E: Data Spreadsheets........................................................................................ 116 Appendix F: Example Calculations..................................................................................... 119 Appendix G: Interim Certificate of Analysis........................................................................120 Appendix H: Report Signature Page.................................................................................. 127 3M Environmental Laboratory Page 5 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 List of Tables Table 1. Rabbit Population Demographics for Study (Argus 418-012)............................. 8 Table 2. Characterization of the Analytical Reference Standard in Study FACT TOX-099 10 Table 3. Negative Ions Monitored in 3M Environmental Laboratory Analyses..................12 Table 4. Characterization of the Control Matrices Used for Liver and Sera Analyses in Study FACT TOX-099.............................................................................................16 Table 5. Characterization of Test Article.............................................................................16 Table 6. LOQ Values for PFOS Used in FACT TOX-099 Analyses by Method and Usage D a te s .......................................................................................................................112 Table 7. Average Concentration of PFOS (pg/g) in Rabbit Liver byDoseGroup.............. 112 Table 8. Average Concentration of PFOS (pg/mL) in Rabbit Serumby Dose Group..........113 Table 9. PFOS (pg/g) in Rabbit Liver by Individual Animal.................................................114 Table 10. PFOS (pg/mL) in Rabbit Serum by Individual Animal........................................ 115 3M Environmental Laboratory Page 6 BACK TO MAIN 3M Medical Department Study: T-6295.10 Study Personnel and Contributors Analytical Report: FACT TOX-099 LRN-U2400 Study Director Marvin T. Case, D.V.M. Ph.D. 3M Corporate Toxicology 3M. Center, Building 220-2E-02 St. Paul, MN 55144-1000 (651)733-5180 Sponsor 3M Corporate Toxicology 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 John L. Butenhoff, Ph.D., Sponsor Representative Analytical Chemistry Laboratories Serum and Liver Extractions Pace Analytical Services, Tier2 Division Serum and Liver Analyses 3M Environmental Laboratory Kris J. Hansen, Ph.D., Principal Analytical Investigator 3M Environmental Laboratory Contributing Personnel Lisa A. Clemen Rhonda Dick* Kelly J. Dorweiler* Sarah A. Heimdal* Marlene M. Heying* Harold O. Johnson Kelly J. Kuehlwein* Sally A. Linda* Bob W. Wynne* Richard Youngblom* 'Denotes contract laboratory professional personnel Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory. The analytical reference standard reserve samples, as well as the specimens pertaining to the analytical phase of this study are archived at the 3M Environmental Laboratory. 3M Environmental Laboratory Page 7 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Introduction and Purpose The purpose of the study is to determine the concentration of perfluorooctanesulfonate (PFOS) fluorochemical in rabbit liver and serum specimens taken from the Oral (Stomach Tube) Developmental Toxicity Study of PFOS in Rabbits. The analytical phase of this study was initiated on 10 June 1999 (date of protocol signing). Test System Five groups of female pregnant rabbits (a total of 110 rabbits) were used as the test system for the main part of the study. Additional female pregnant rabbits (a total of 19 rabbits) were assigned to one of the same five dosage groups for the satellite study. It is only the satellite study that provided toxicokinetic samples that were tested and reported here. The test article, PFOS, was administered orally via a stomach tube once daily to female rabbits on days 7 through 20 of presumed gestation (DG7 through DG20). The carrier for the test article was 0.5% Tween 80 in Reverse Osmosis Membrane Processed Deionized Water. Doses of 0 (carrier only), 0.1, 1.0, 2.5 and 3.75 mg/kg/day were administered at a dosage volume of 5 mL/kg, adjusted daily on the basis of weight. The test system species and strain selected was the New Zealand White Rabbits [Hra:NZW)SPF)J received from Covance Research Products, Inc., permanently identified using a Monel self-piercing ear tag. F0 female rabbits were approximately 5 months of age and weighed approximately 2.84-4.21 kg when received. There were 133 female rabbits initially acclimated. The quantity actually used for the study is given in Table 1. The rabbits in the main portion of the study were sacrificed and a gross necropsy of maternal rabbit tissues and fetuses was performed. See the Argus Final Report #418-012 for results. Toxicokinetic samples for laboratory analysis at 3M Environmental Laboratory were only obtained from the satellite study portion. Table 1. Rabbit Population Demographics for Study (Argus 418-012) Population Selected for Main Study Selected for Satellite Study Totals Group I (Control) 0 mg/kg/day 0 mg/mL Group II 0.1 mg/kg/day 0.02 mg/mL Group III 1.0 mg/kg/day 0.2 mg/mL Group IV 2.5 mg/kg/day 0.5 mg/mL Group V 3.75 mg/kg/day 0.75 mg/mL 110 22 22 22 22 22 19 3 5 3 3 5 3M Environmental Laboratory Page 8 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Specimen Collection and Analysis In the analytical study reported here, 19 maternal serum and 19 maternal liver specimens collected from rabbits on DG 21 were sent to the 3M Environmental Laboratory and analyzed for PFOS. In addition, 18 fetuses and 18 placentas were collected on DG21 from sacrificed rabbits. One doe was not pregnant at the time of sacrifice; therefore, no fetuses or placenta were collected from this rabbit. The number and type of specimens collected for analyses in the analytical phase of this study are presented below. Specimens Collected from Satellite Study Groups 1 through 5 (through 9/10/98): Maternal Serum Specimens-- 19 specimens Maternal Liver Specimens-- 19 specimens Fetuses-- 18 specimens Placentas-- 18 specimens Blood specimens were collected and centrifuged. Serum was then harvested and frozen at -60C to -78C until shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal were frozen and then stored at -60C to -78C until shipped to the 3M Environmental Laboratory. Fetuses and placentas were pooled per litter and stored frozen at -60C to -78C until shipped to the 3M Environmental Laboratory. Liver, sera, fetuses and placenta specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice. (Note: Although fetal and placenta tissues were collected at the same time as liver and serum, results from these analyses will not be included in this report. A separate report may be issued for fetal tissue data.) Sera and liver samples were extracted beginning on 16 October 2000 using an ion pairing reagent and methyl-terf-butyl ether (MtBE). Liver samples were homogenized prior to the extraction procedure. Sample extracts were analyzed using high-pressure liquid chromatographyelectrospray/tandem mass spectrometry (HPLC-ES/MS/MS) in the multiple response mode. PFOS levels were quantitated by external calibration. Analytical details are included in this report. Specimen Receipt and Maintenance The 3M Environmental Laboratory received serum, liver, fetuses and placenta specimens collected at DG21 of the in-life phase of this study FACT TOX-099 on 15 September 1998 from Argus Research Laboratories. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C 10C. Control matrices used in liver and sera analyses performed during TOX-099 were obtained from commercial sources and are presented in Appendix A (see Table 4). Samples analyzed at the 3M Environmental Laboratory will be maintained for a maximum period of 10 years and will be stored at the laboratory at -20C 10C. 3M Environmental Laboratory Page 9 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Chemical Characterization of Reference Standard Potassium Perfluorooctanesulfonate (KPFOS) CAS Number: 2795-39-3 Chemical Formula: C8F17S 03'K+ Molecular Weight: 537.9 Chemical characterization information on the reference standard, potassium perfluorooctanesulfonate, CAS #2795-39-3, is presented in Table 2 below. Table 2. Characterization of the Analytical Reference Standard in Study FACT TOX-099 Location 3M Environmental Laboratory Analytical Reference Standard Potassium Perfluorooctanesulfonate (KPFOS) SD009 Source 3M Specialty Chemicals Expiration Date 1/01/2010 Storage Conditions Ambient temperature* Chemical Lot Number 171 Physical Description Light-colored powder Purity 86.4% "Kept at ambient until 5/16/00, then moved to cold storage (-10C ) Method Summaries Following is a brief description o f the m ethods used during this analytical study by the 3M Environmental Laboratory. Detailed descriptions of the methods used in this study are located in Appendix C. As the present study progressed, more advanced methods evolved and earlier methods listed in the protocol were not used. Amendments to the protocol were written to cover method changes. These changes only improved the effectiveness of the method. Protocol, protocol amendments and deviations are included in Appendix B. 3M Environmental Laboratory Preparatory Methods ETS-8-4, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory Page 10 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACTTOX-099 LRN-U2400 Serum samples were extracted using an ion-pairing extraction procedure. An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into MtBE. The MtBE extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol, then filtered with a 0.2 pm nylon filter attached to a 3 mL plastic syringe into glass autovials. ETS-8-6, "Extraction of Potassium Perfluorooctanesulfonate 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-pairing extraction procedure. An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into MtBE. The extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol and passed through a 0.2 pm nylon filter, using a 3 mL disposable plastic syringe, into glass autosampler vials. Analytical Methods ETS-8-5, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorochemical using HPLC/ES/MS/MS. For example, molecular ion 499, selected as the primary ion for PFOS (C8F17S 0 3-) analysis, was fragmented to produce ion 99 (FS03-). The characteristic ion 99 was monitored for quantitative analysis. 3M Environmental Laboratory Page 11 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Analytical Equipment The analytical equipment settings used in the present analytical phase of this study varied slightly during data collection. The following is representative of the settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone BetasilTM C182x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2 mM ammonium acetate Component B: methanol Flow rate: 300 pl_/min Injection volume: 10 pL Solvent Gradient: 10.0 minutes Time (minutes) 0.0 1.0 5.5 7.5 8.0 %B 10% 10% 95% 95% 10% Mass Spectrometer:Micromass API/Mass Spectrometer Quattro IITMTriple Quadrupole system Software: Mass LynxTM 3.4 Cone Voltage: 60 V Collision Gas Energy: 45-50 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 3. Negative Ions Monitored in 3M Environmental Laboratory Analyses Target Analyte Primary Ion (amu) Product Ion (amu) PFOS 499.0 99.0 THPFOS 427.0 80.0 3M Environmental Laboratory Page 12 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACTTOX-099 LRN-U2400 Deviations It should be noted that as the analytical phase of this study progressed, method parameters were evaluated to improve analyses. Amendments to the protocol were written to address method changes. Deviations from the original protocol and methods are included in Appendix B. Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicated in the protocol for this study: Linearity: The coefficient of determination (r2) equal to or greater than 0.98 Lim its o f Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve and is at least two times the analyte peak area detected in the extraction blanks. Acceptable Precision: Precision is better than 30% for the method Acceptable Spike Recoveries: 70-130% Confirm atory Methods: None. Demonstration of Specificity: Specificity to be demonstrated by chromatographic retention time and mass spectral daughter ion characterization. Data Summary, Analyses, and Results Data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory protocol for FACT TOX-099 (see Appendix B) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results Linearity: The coefficient of determination (r2) of the standard curve was >0.98. Calibration Standards: Quantitation of PFOS was based on linear regression analysis (1/x weighted) of a single curve injected before or two extracted matrix curves bracketing, each group of samples. High or low points on the curve may have been deactivated to provide a better linear fit over the curve range most appropriate to the data. Low curve points with peak areas less than two times that of the extraction blanks were deactivated to disqualify a data range that may have been significantly affected by background levels of the analyte. Occasionally, a single mid-range curve point that was an obvious outlier may have been deactivated. Quantitation of PFOS* was based on the response of one specific product ion using the multiple response-monitoring mode of the instrument (see Appendix C, Analytical Methods). ` Isomers of P F O S were not chromatographically resolved. Quantitation was based on the sum of linear and all branched isomers. 3M Environmental Laboratory Page 13 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Limits o f Quantitation (LOQ): The LOQ was equal to 0.00426 pg/mL and 0.0214 pg/mL in sera and 0.00529 (jg/g and 0.0529 |jg/g in liver. See Table 6 for LOQs on specific analytical dates. Blanks: All blanks were below the lower limit of quantitation for the compounds of interest. Precision: Precision was determined by triplicate analysis of one extracted liver sample injected three times and two sera samples that were reproducible to within 5%. Matrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of samples and analyzed during analytical runs at the 3M Environmental Laboratory. Samples were spiked prior to extraction. Rabbit serum and liver samples for MS studies were obtained from commercial sources. After homogenation (liver) and prior to extraction, tissue samples were spiked with PFOS at approximately 250 ng/mL or 250 ng/g. All sera matrix spikes were within 10% of the theoretical concentration. Matrix spikes prepared in liver were compliant within 20% with one exception; one liver matrix spike was recovered at 156%. The average matrix spike recovery for the Group 1 samples was 138%. Surrogates: The surrogate (THPFOS) was added to all samples and standards prior to extraction. THPFOS was not used for quantitation, but was used to monitor for gross instrument failure. The surrogate response of each analytical run was monitored to determine that it did not vary more than 50% from the mean within each analytical run. No problems were observed with these data. Statem ent of Data Quality It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitative to approximately 30%; values for liver analysis may be biased high. Summary of Sample Results (3M Environmental Laboratory) Samples from Control Animals: PFOS w as detected in the sera and liver o f all anim als. Samples from Dosed Animals: In general, PFOS levels found in the sera and liver of the test animals increased with dose group. PFOS levels increased as dosage increased. Detailed sample data tables are presented in Appendices D and E. 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-099. 3M Environmental Laboratory Page 14 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Statement of Conclusion (3M Environmental Laboratory) Under the conditions of the present studies, the fluorochemical PFOS was observed in the liver and serum of all rabbits dosed with the test article during the in-life phase of the study. References Argus In-Life Final Report #418-012, "Oral (Stomach Tube) Developmental Toxicity Study of PFOS in Rabbits" 3M Environmental Laboratory Page 15 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACTTOX-099 LRN-U2400 Appendix A: Chemical Characterization and Control Matrices Table 4. Characterization of the Control Matrices Used for Liver and Sera Analyses in Study FACT TOX-099 Control Matrix Rabbit Serum TN-A4511 Rabbit Liver TCR99131-45 Source Expiration Date Storage Conditions Chemical Lot # Physical Description N/R-- not recorded Sigma Chemicals 9/2005 Frozen -20C (10C) 99H8400 Rabbit Serum Covance Labs N/R Frozen -20C (10C) F04051 Rabbit Liver Table 5. Characterization of Test Article Test Article Chemical Name KPFOS (Potassium Perfluorooctanesulfonate) Source 3M Toxicology Services, Medical Department Expiration Date 5/2000 Storage Conditions Room temperature Chemical Lot # 217 Physical Description Off-white powder Purity 86.9% a The test article is defined as the substance gavaged to the test system. 3M Environmental Laboratory Page 16 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACTTOX-099 LRN-U2400 Appendix B: Protocol, Protocol Amendments, Deviations 3M Environmental Laboratory Page 17 3M Medical Department Study: T-6295.10 3M Environmental Technology and Services PS61tO.2PB7ao7ux8l. 36M3434N32155133-3331 BACK TO MAIN Analytical Report: FACT TOX-099 LHN-U2400 Protocol UFACT-TOX-099 3M Study Title Oral (Stomach ToufbPeF)ODSevienloRpambebnittsal Toxicity Study PROTOCOL LiAsauCthleomren JunDe a8t,e1:999 3M Enviro3nMPmeEernnftovairlrmoTneinmchgennLotaalolbgLoyarb&aotrSoaartofyertyy Services 935 Bush Avenue St. Paul, MN 55106 LaboratoFryAPCrTo-jTeOcXt I-d0e99ntification U2400 3M Environmental Laboratory 3M Environmental Laboratory Page 1 o f 10 Page 18 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Protocol #FACT-TOX-099 Study Identification Oral (Stomach Tube) Developmental Toxicity Study of PFOS in Rabbits Test Material Perfluorooctane sulfonic acid potassium salt (T-6295) Sponsor 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 Sponsor Representative Marvin T. Case, D.V.M., Ph.D. 3M Toxicology Services Telephone: 651-733-5180 Facsimile: 651-733-1773 Study Director Kristen J. Hansen, Ph.D. 3M Environmental Technology and Safety Services Building 2-3E-09 651-778-6018 Study Location(s) In vivo Testing Facility Analytical Testing Laboratory Argus Research Laboratories, Inc. 905 Sheehy Drive, Building A Horsham, PA 19044 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 3M Environmental Laboratory 3M Environmental Laboratory Page 2 of 10 Page 19 BACK TO MAIN 3M Medical Department Study: T-6295.10___________________ ^ Analytical Report: FACT TOX-099 " mN-U2400 Protocol UFACT-TOX-099 Sub-Contract Laboratory Proposed Study Timetable Analytical Start Date Analytical Completion Date Advanced Bioanalytical Services, Inc, 15 Catherwood Road Ithaca, NY 14850 Battelle Memorial Institute 505 King Avenue Columbus, Ohio 43201-2693 June 08,1999 August 08,2000 1. S tudy Oral (stomach tube) developmental toxicity study o f PFOS in rabbits 2. Purpose This analytical study is designed to determine levels o f potassium perfluorooctanesulfonate (PFOS) in specimens of liver and serum of rabbits. The in-life portion o f this study was conducted at Argus Research Laboratories, study #418-012. All serum samples will be extracted and analyzed at Advanced Bioanalytical Services, Inc. and all liver samples extracted and analyzed at Battelle Memorial Institute. Additional analyses may be performed at the 3M Environmental Laboratory as methods are developed and validated.. If additional analyses are performed an amendment to this protocol will be written. 3. Regulatory Compliance AThdims isntiusdtryawtioilnl,bGe ocoodndLuacbtoerdaitnoraycPcorradcatinccees wStiatnhdtahredUs,nFitinedalSRtautlees2F1oCodFRan5d8D, wruigth the exception that analysis o f the test material mixture for concentration, solubility, homogeneity, and stability will not be conducted, and is the responsibility of the Sponsor. 4. Quality A ssurance The 3M Environmental Laboratory Quality Assurance Unit will review the protocol and audit study conduct, data, and results report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. The QA Unit at the sub-contract laboratory will audit their study conduct, data, and results report prior to submitting to the 3M Environmental Laboratory. 3M Environmental Laboratory 3M Environmental Laboratory Page 3 o f 10 Page 20 BACK TO MAIN 3M Medical Department Study: T-6295.10 ____________________________________________ Analytical Report: FACT TQX-099 LRN-U2400 Protocol #FACT-TOX-099 5. Test Material 5.1 Refer to Argus Research Laboratory protocol for study #418-012. 6. Control Matrices 6.1 Identification Rabbit liver and serum, traceability numbers will be recorded in the raw data and included in the final report 6.2 Source Argus Reseacrh Laboratories and/or Sigma Chemical 6.3 Physical Description Rabbit liver and serum 6.4 Purity and Stability Not applicable 6.5 Storage Conditions Frozen at -20 C 10 C or -5 0 C 10 C 6.6 Reserve Matrix A portion of the control matrix will be retained in the 3M archives for as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date o f the final test rule (if applicable). 6.7 Disposition Matrices will be retained at the 3M Environmental Laboratory per GLP regulation. Certain matrices (feces, urine, and blood) may be disposed after QAU verification. 6.8 Safety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 7. Reference Material 7.1 identifiestion Potassium perfluorooctanesulfonate (PFOS), lot #s 171,215, or 217 (equivalent lots) 7.2 Source 3M Specialty Chemicals 7.3 Physical Description White powder 7.4 Purity and Stability Purity = 99%, Stability to be determined by the Sponsor 7.5 Storage Conditions Room temperature 7.6 Reserve Material A reserve sample from each batch o f PFOS used in this study will be retained as long as the quality o f the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable). 7.7 Disposition Unused reference material will be retained for use by the 3M Environmental Laboratory and will be discarded when the quality o f preparation no longer affords evaluation. 7.8 Safety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 3M Environmental Laboratory 3M Environmental Laboratory Page 4 o f 10 Page 21 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Protocol #FA CT-TOX-099 R8.abbitsTweesrteSuysesdteams the test system and were maintained and dosed as described in ArgusLaboratories protocol #418-012. The rabbits will be given the test material or control once daily on days 7 through 20 of presumed gestation. See table 1 for dosage information. Table 1 Dosage Levels, Concentration, and Volumes Dosage Number of Dosage Group rabbits mg/kg/day 1 22+3* 0.0 2 22+5* 0.1 3 22+3* 1.0 4 22+3* 2.5 5 22+5* 3.75 ' Rabbits assignee to toxicokinetic evaluation. Concentration mg/mL 0.0 0.02 0.2 0.5 0.75 Dosage Volume mL/kg 5 5 5 5 5 9. Specimen and Sample Receipt The 3M Environmental Laboratory will receive specimens o f the following body tissues and fluids from the indicated points in the study. All specimens will be packed on dry ice for shipping. See table 2 for specimen information. Body tissue/fluid Table 2 Specimen Information Collected Serum - Dam animals Liver --Dam animals Toxicokinetic Collection - Day 21 Toxicokinetic Collection --Day 21 Fetus - Pooled litter animals Toxicokinetic Collection - Day 21 Placenta - Pooled litter animals Toxicokinetic Collection - Day 21 Expected # of specimens 1199 19 19 Total number of expected specimens: 76 Total number o f test animals: 104 Total number o f control animals: 25 Specimens sent to 3M Environmental Laboratories will be received and tracked according to applicable Standard Operating Procedures. 3M Environmental Laboratory 3M Environmental Laboratory Page 5 o f 10 Page 22 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TQX-099 LRN-U2400 Protocol UFACT-TOX-099 10. Preparatory Methods 10.1 FACT-M-1.1, Extraction o f Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLCElectrospray/Mass 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. 10.4 If analyses are sub-contracted to other laboratories, an amendment will be written to include their methods and copies of each method will be attached to this protocol. 11. Analytical Methods 11.1 FACT-M-2.1, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 11.2 ETS-8-5.1, Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/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. 11.4 If analyses are sub-contracted to other laboratories, an amendment will be written to include their methods and copies of each method will be attached to this protocol. 12. Data Quality Objectives The number o f spikes/duplicates, use o f 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 ^^0.98 12.2 Limits of detection / quantitation 12.2.1 Method Detection Limit (MDL) for PFOS a) Serum: 1.75 ppb b) Liver: 15 ppb 12.2.2 Limit o f Quantitation (LOQ) - Equal to the lowest acceptable standard in the calibration curve 12.3 Duplicate acceptable precision < 30% for the method 3M Environmental Laboratory 3M Environmental Laboratory Page 6 o f 10 Page 23 BACK TO MAIN 3M Medical Department Study: T-6295.10___________________ " Analytical Report: FACT TOX-099 -- ------------LRN-U2400 Protocol #FACT-TOX-099 12.4 Spike acceptable recoveries 70% -130% 12.5 Use of confirmatory methods Indeterminate samples will be re-analyzed using a confirmatory method. If a confirmatory method is used, an amendment to this protocol will be written. 12.6 Demonstration of specificity Chromatographic retention time, mass spectral daughter ion characterization. 13. Subcontracted Analysis 13.1 All analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3E-09,935 Bush Avenue, St. Paul, MN 55106, at Advanced Bioanalytical Services, Inc., 15 Catherwood Road, Ithaca, NY 14850, or at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. 13.2 An amendment to this protocol will be written if analyses are performed at laboratories other than 3M Environmental Laboratories, Advanced Bioanalytical Services, Inc., or Battelle Memorial Institute. 14. Statistical A nalysis Averages and standard deviations will be calculated. The statistical methods that will be used are described below: 14.1 Data transformations and analysis Data will be reported as the concentration (weight/weight or weight/vol) of PFOS or metabolite per tissue or fluid. 14.2 Statistical analysis Statistics used may include regression analysis of concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 15. Report A report containing all the results of the study will be prepared by the 3M Environmental Laboratory. If analyses are sub-contracted to other laboratories, each laboratory will prepare a report and submit it to the 3M Environmental Laboratory for inclusion in the 3M Environmental Laboratory report. Each report will include, but not be limited to, the following, when applicable: 15.1 Name and address of the facility performing the study 15.2 Dates upon which the study was initiated and completed 15.3 A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards 3M Environmental Laboratory 3M Environmental Laboratory Page 7 o f 10 Page 24 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TQX-099 LRN-U2400 Protocol UFACT-TOX-099 15.4 Objectives and procedures as stated in the approved protocol, including any changes in the original protocol 15.5 The test substance identification by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor 15.6 Stability and the solubility o f the test substances under the conditions of administration, if provided by the Sponsor 15.7 A description of the methods used to conduct the test(s) 15.8 A description of the test system 15.9 A description of any circumstances that may have affected the quality or the integrity o f the data 15.10 The name o f the Study Director and the names of other scientists, professionals, and supervisory personnel involved in the study 15.11 A description o f the transformations, calculations, or operations performed on the data, a summaiy and analysis o f the analytical chemistry data, and a statement o f the conclusions drawn from the analyses 15.12 Statistical methods used to evaluate the data, if applicable 15.13 The signed and dated reports o f each of the individual scientists or other professionals involved in the study, if applicable 15.14 The location where raw data and the final report are to be stored 15.15 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 IcahfmiatennigsdenmsewecneitsllswabrielylmtcolaedmaeraliykneitdhceoenrftroiefrcymtitohonefspaoanrrtaamodfdetinhtdieomnreespntotoriatsstruheaepdtoirbstybaeftthienergSitatuhmdaesynbDdeeiedrne,ctathocerc.erpeTathesedo,ntshfeor the amendment, and will be signed by the Study Director. 16. Location of Ra w Data, Records, and Final Report Original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance o f the final report. When the final report is completed, all original paper data, including those items listed below, will be retained in the archives o f 3M Environmental Laboratory for at least a period o f time as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 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: 3M Environmental Laboratory Page 8 o f 10 3M Environmental Laboratory Page 25 BACK TO MAIN 3M Medical Department Study: T-6295.10 -------A--n-a--l-y-t-ic--a-l--R--e--p-o--r-t:--F-LAFCTTTDO54X-0599 Protocol ttFACT-TOX-099 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 o f data 16.2 The following supporting records will be retained separately from the study folder in the archives 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. Specimen Retention Specimens will be maintained in the 3M Environmental Laboratory specimen archives for a period of time as specified by regulation or as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date o f the final test rule (if applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. 18. Protocol Amendments and deviations Planned changes to the protocol will be in the form o f written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the pinrothtoecfoilnaanl drewpoilrlt.bAe natytaocthheedr ctohatnhgeefsinwaillpl rboetoincotlh.eAflolrcmhaonfgwersittotetnhdeepvrioattoiocnosl,wsiigllnbeedibnyditchaeted Study Director and filed with the raw data. 19. A ttachments 19.1 Attachment A Preparatory and analytical methods 3M Environmental Laboratory 3M Environmental Laboratory Page 9 o f 10 Page 26 3M Medical Department Study: T-6295.10 20. S ig n a t u r e s N Marvin T. Case, D.V.M.,'IPshtL.D-., Sponsor Representative BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Protocol HFACT-TOX-099 1 9 ^ Date/ 99? Kristen J. Hansen,fJPh*.-D., 3M--Envi_ron_m_en_ta_l L_ab_o_rat_or_y _Stu_d_y Di_rector Date 3M Environmental Laboratory 3M Environmental Laboratory Page 10 o f 10 Page 27 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Study Title Oral (Stomach Tube) Developmental Toxicity Study o f PFOS in Rabbits PROTOCOL AMENDMENT NO. 1 Amendment Date: August 04,1999 3M EnvironPmeernftoarlmTienchgnLoalobgoyr&atSoarfyety Services 3M En9v3ir5oBnmusehntAavl eLnaubeoratory St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX099 LIRNU2400 3M Environmental Laboratory 3M Environmental Laboratory Page 28 BACK TO MAIN 3M Medical Department Study: T-6295.10 _____________________________________________ Analytical Report: FACT TQX-099 LRN-U2400 Protocol FACT-TOX099 Amendment No. 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL reads: The sub-contract laboratory assigned to extract and analyze the rabbit serum specimens is listed as: Advanced Bioanalytical Services, Inc. (ABS) 15 Catherwood Road ' Ithaca, NY 14850 AcoMnEtrNaDcteTdOtro:ead: The extraction and analysis of the rabbit serum specimens will be sub Pace Analytical Services, Inc. 1700 Elm Street, Suite 200 Minneapolis, MN 55414 Note: The serum specimens were sent to ABS on 6/15/99 and will be shipped directly from ABS to Pace Analytical Services in c/o Jon Berdahl. The specimens will be shipped on dry ice, overnight and will include the original chain of custody along with all paperwork listing storage conditions while at ABS. Also, Jon Berdahl at (612) 607-6451 will be notified prior to shipping these specimens. REASON: Since ABS hasn't performed a rabbit serum validation, the contract lab extracting and analyzing the serum specimens was changed to Pace Analytical Services who has completed a rabbit serum validation. 2. PROTOCOL reads: Section 10.0 and 11.0 list the following methods to use for extraction and analysis: FACT-M-1.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-4.1 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-2.1 "Analysis o f Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry" AMEND to read: The extraction and analytical methods are: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 3M Environmental Laboratory 3M Environmental Laboratory Page 29 BACK TO MAIN 3M Medical Department Study. T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Protocol FACT-TOX099 Amendment No. 1 ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" REASON: The FACT-M-1.1 and FACT-M-2.1, respectively were updated on 7/22/99 to ETS-8-6.0 and ETS-8-7.0. These methods were updated to replace the extraction solvent ethyl acetate with a different extraction solvent MTBE (methyl ten butyl ether), POAA and Monoester were removed from the standard mix, and M556 was added to the standard mix. The analytical method was updated to include linear regression with 1/x weighting and a few minor changes in the HPLC 1100 instrument parameters. 3. PROTOCOL reads: Sections 10.4 and 11.4 state that if analyses are sub-contracted to other laboratories, an amendment will be written to include their methods and copies o f each method will be attached to this protocol. AMEND TOread: The extraction and analytical methods for rabbit serum at Pace Analytical Services, Inc. are: CAG-SP-01.0 "Extraction o f Potassium Perfluorooctanesulfonate and Other Fluorochemical compounds from Serum for Analysis by LC/MS" CAG-CHR-01.0 "Analysis o f Potassium Perfluorooctanesulfonate and Other Fluorochemicals in Serum Extracts Using LC/MS" The extraction and analytical methods for rabbit liver at Battelle Memorial Institute are: "Method for Analysis o f Prefluorooctane Sulfonate (PFOS) in Rat Liver by LC/MS/MS, Version 1. 0" EREnvAiSroOnNm: eTnthael mLaebthoroadtsorliys.teTdhpirseavmioeunsdlymwenertewfaosrwexrtitrtaecntitooninanclduadneatlhyesims eptehrofodrsmtoedbeatftohlleo3wMed at the Pace Analytical Services, Inc laboratory and Battelle Memorial Institute. 4. PROTOCOL reads: Section 12.2.1 b) lists liver method detection limit as 15 ppb. AMEND TOREAD: The liver method detection limit is 8.50 ppb (ng/g). REASON: The validation supporting methods ETS-8-6.0 and ETS-8-7.0 included a lower method detection limit for PFOS. 3M Environmental Laboratory 3M Environmental Laboratory Page 30 BACK TO MAIN 3M Medical Department Study: T-6295.10_________________________________________________ Analytical Report: FACT TOX-099 " LRN-U2400 Protocol FACT-TOX099 Amendment No. 1 Amendment Approval Marvin Case, D.V.M., Ph.D., Sponsor Representative j^ l --- Kris J. Hansen, PhJD., Study Director ih. Ms Date 3M Environmental Laboratory 3M [Environmental Laboratory Page 31 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Study Title Oral (Stomach Tube) Developmental Toxicity Study of PFOS in Rabbits PROTOCOL AMENDMENT NO. 2 Amendment Date: 20 January 2000 Performing Laboratories Liver Analyses Serum Analyses CBSOoaStluteKmlliebnugMsA,eOmveHonrui4ae3l 2In0s1t-i2tu6t9e3 MP1a7i0cne0nAeEanlpianollySitsti,rceMaeltN,SSeru5vi5ti4ec1e2s40, 0Inc, Laboratory Project Identification ET&SS LRN-U2400 FACT TOX-099 Argus Study: 418-012 3M Medical Department Study: T-6295.10 3M Environmental Laboratory 3M Environmental Laboratory Page 32 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TQX-099 LRN-U2400 Protocol LRN-U2400 Amendment Number 2 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: The study director for the present study was identified in the protocol as Kristen J. Hansen, Ph.D.. Amend to read: The role of study director for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of 20 January 2000. The previous study director, Kristen Hansen, has been reassigned to the role of Principle Analytical Investigator. Reason: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58. 2. Protocol reads: The sponsor for the present study Was identified as Marvin T. Case, D.V.M., Ph.D. Amend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned, in this manner, personnel responsibilities and workload are more evenly balanced. 3. Protocol reads: 17. Specimen Retention: Specimens will be maintained in the 3M Environmental Laboratory specimen archives for a period of time as specified by regulation or as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. A mend to read: 17. Specimen Retention: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. Any specimens sent to sub-contract laboratories will be returned to the 3M Environ- mental Laboratory upon completion of analysis and submission of the sub contract laboratory(s) final report. Specimens analyzed at sub-contract laboratories will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility. Reason: To define in detail the appropriate disposition of specimens analyzed at subcontract laboratories. 3M Environmental Laboratory 3M Environmental Laboratory Page 33 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Protocol LRN-U2400 Amendment Number 2 4. Protocol reads: Section 16 states that the following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: Approved protocol and amendments; study correspondence; shipping records; raw data; approved final report (original signed copy); and electronic copies of data. Additionally, Section 16 states that supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: Training records; calibration records; instrument maintenance logs; Standard Operating Procedures, Equipment Procedures, and Methods; and appropriate specimens. Amend to read: Section 16 states: "The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis. Reason: To direct subcontract laboratories in the disposition of the items listed above. 5. Protocol reads: 2. Purpose This analytical study is designed to determine levels of potassium perfluorooctanesulfonate (PFOS) in specimens of liver and serum of rabbits. Amend to read: 2. Purpose This analytical study is designed to determine levels of potassium perfluorooctanesulfonate (PFOS) in specimens of liver, serum, and fetal tissues of rabbits. Reason: The study sponsor requested the addition of fetal tissue analyses. Note: At the time that this amendment was produced, a method for the determination of PFOS in fetal tissue is under development. When the method is approved and validated, another amendment will be written to address the use of the additional method. 3M Environmental Laboratory 3M Environmental Laboratory Page 34 3M Medical Department Study: T-6295.10 Amendment Approval BACK TO MAIN Analytical Report: FACT TQX-099 LRN-U2400 Protocol LRN-U2400 Amendment Number 2 John L. Butenhoff, PhD., Sponsor Representative Date fc Kristen L J H,ansenz,.P--h--D--.-,--O--u--t-g--o-i-n--g--S--t-u--d--y--D--i.r--e-c--t-o-r- Dat-e'ZOPO Marvin T. Case, D. V.M., PhD., Incoming Study Director Dater 3M Environmental Laboratory 3M [Environmental Laboratory Page 35 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Study Title Oral (Stomach Tube) Development Toxicity Study o f PFOS in Rabbits PROTOCOL AMENDMENT NO. 3 AmAeugnudsmt 1e6n,2t 0D0a0te: 3M EnvironPmeernftoarlmTeinchgnLoalobgoyr&atSoarfyety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-099 U2400 3M Environmental Laboratory 3M [Environmental Laboratory Page 36 BACK TO MAIN 3M Medical Department Study: T-6295.10_________________________________________________Analytical Report: FACT TQX-099 LRN-U2400 Protocol FACT-TOX-099 Amendment #3 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: 3M FACT TOX-099 PROTOCOL: Sub-Contract Laboratory heading under Study Location lists Battelle Memorial Institute. 2. PURPOSE--All liver samples will be extracted and analyzed at Battelle Memorial Institute. 13. SUB-CONTRACTEDANALYSIS--All analyses as detailed in this protocol will be performed at 3M Environmental Laboratory, Advanced Bioanalytical Services, or Battelle Memorial Institute. A R emmenovdetBoartteeallde :M emorial Institute as a subcontract laboratory under th e Study L o cation heading. 2. Change to state that all liver samples will be extracted and analyzed at 3M Environmental Laboratory. 13. Change to state that all analyses as detailed in this protocol will be performed at 3M Environmental Laboratory or Advanced Bioanalytical Services. R3MeaEsnovnir:onmental Laboratory is able to meet the analytical needs o f the study. 2. Protocol reads: 3M 10. FACT TOX-099 PROTOCOL: Preparatory Methods & 11. A nalytical --Methods The amended protocol states the liver methods are ETS-8-6.0 and ETS-8-7.0. AChmaenngde ttoosrpeecaidfy:: Use the most current version o f the methods listed in both sections. TRoeaspseocnif:y that the most current version o f the preparatory and analytical methods should be used during the course o f the study. 3M Environmental Laboratory 3M Environmental Laboratory Page 37 BACK TO MAIN 3M Medical Department Study: T-6295.10_________________________________________________Analytical Report: FACT TQX-099 LRN-U2400 Protocol FACT-TOX-099 Amendment # 3 3. Protocol reads: The amended 3M ANALYTICAL PROTOCOL FACT-TOX-099 (Amendment No. 2) states that Marvin T. Case replaces Kristen J. Hansen as the study director. Kristen J. Hansen was reassigned the role o f Principle Analytical Investigator. John L. Butenhoffwas reassigned the role o f Sponsor Representative. Amend to read: Kristen J. Hansen was reassigned to the role o f Principal (corrected spelling) Analytical Investigator. RCoerarsecotned: spelling o f Principal. Amendment Approval John L. Butenhoff, Ph.D., Sponsor Representative Martin T. Case, D. V.M., Ph.D., Study Director ihr+ Date 3M Environmental Laboratory 3M Environmental Laboratory Page 38 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TQX-099 LRN-U2400 Oral (Stomach Tube) DevelopSmtuedntyTToixtliecity Study of PFOS in Rabbits PROTOCOL AMENDMENT NO. 4 ASmepetenmdbmere2n5t, D20a0te0: Performing Laboratory 3M Environmental Technology & Safety Services 3M En9v3ir5oBnmusehntAalvLenaubeoratory St. Paul, MN 55106 LAabnaolryatitcoalryRePproortj:ecFtAICdTenTtOifiXc-a0t9io9 n 3M Laboratory Request No. U2400 3M Environmental Laboratory 3M Environmental Laboratory Page 39 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TQX-099 LRN-U2400 Protocol #FACT TOX-099 Amendment 4 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: Sub-contract Laboratories: The amended protocol (Amendment No. l) lists Pace Analytical Services, Inc. as the sub-contract laboratory for the extraction and analysis of rabbit sera specimens. The amended protocol (Amendment No. 3) states that all liver samples will be extracted and analyzed at 3M Environmental Laboratory. Section 2. Purpose--The amended protocol states that all serum samples will be extracted and analyzed at Pace Analytical Services, Inc. and all liver samples will be extracted and analyzed at 3M Environmental Laboratory. Section 13. Sub-Contracted Analysis: The amended protocol states that all analyses as detailed in this protocol will be performed at 3M Environmental Laboratories or at Pace Analytical, Inc. APamgeen3d, SttoudryeaLdoc:ations, Sub-Contract Laboratories: Add Pace Tier II as a sub-contract laboratory. 2. Purpose: Change to state that all liver and sera samples will be extracted at the Pace Tier II facility. All liver and sera samples will be analyzed at 3M Environmental Laboratory. 13.1 Sub-Contracted Analysis: Change to say that liver and sera extractions for this protocol will be performed at the Pace Tier II facility. Change to say that all liver and sera analyses as detailed in this protocol will be performed at 3M Environmental Laboratory. Reason: Pace Tier II and 3M Environmental Laboratory are able to meet the needs of the study. 2. Protocol reads: . lSaebocrtaitoonry1. 3. Sub-Contracted Analysis: N o SOPs are listed for the sub-contract Amend to read: Add to Section 13: Pace Analytical Services, Inc. (Pace--Tier II Facility, Minneapolis, MN) will be performing the extraction of samples for analyses of liver and sera specimens. All extracted samples will be shipped to the 3M Environmental Laboratory for analysis. Pace will follow all applicable 3M Environmental Laboratory Standard Operating Procedures and the following Pace Standard Operating Procedures: PSS-Admin-01 PSS-DC-05 PSS-OPS-Ol PSS-OPS-02 PSS-OPS-03 Quality System Lab Notebooks, Logbooks, and Phone Logs Use and Maintenance of Ultra-Turrax T25 Homogenizer Raw Data Hazardous Waste Disposal 3M Environmental Laboratory 3M Environmental Laboratory Page 40 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TQX-099 LRN-U2400 PSS-OPS-04 PSS-OPS-05 PSS-OPS-06 PSS-OPS-07 PSS-OPS-08 PSS-OPS-13 PSS-OPS-15 PSS-OPS-16 PSS-OPS-31 PSS-OPS-32 ETS-9-40 (3M Lab) PSS-OPS-37 PSS-MTR-01 PSS-MTR-02 PSS-MTR-06 PSS-MTR-08 PSS-MC-01 PSS-ARC-02 PSS-DM-03 Protocol #FACT TOX-099 Amendment 4 Use and Maintenance of N-Evap Analytical Evaporator Use of Compressed Gases in the Laboratory Use and Maintenance of NANOpure II Water Purification System Cleaning Non-disposable Volumetric Pipettes Laboratory Calculations Use and Maintenance of Lab Refrigerators, Freezers, and Ovens Glassware Cleaning Use and Maintenance of the Fisher Scientific Isotemp Freezer Use and Maintenance of Shakers (Use for Shaker Only) Operation, Maintenance and Calibration of Laboratory Centrifuges Operation and Maintenance of Shakers and Vortex Mixers (Use for Vortex Mixer Only) Operation, Maintenance, and Calibration of pH Meters and pH Electrodes Calibration of Certified Weight Set Calibration of Certified Thermometers/Thermocouples Verification of Calibration of the Eppendorf Pipettor Verification of Calibration and Use of Analytical Balances Purchasing of Laboratory Supplies Disposition of Archive Materials Statistical Evaluation of Data REASON: All extractions for liver and sera samples will be performed at Pace Tier II, using the above SOPs. 3. 3. Protocol Reads: Sectio n 13. S ub-C ontracted A nalysis: No methods are listed for the sub-contract laboratory. Under Preparatory Methods, ETS-8-4.1 is listed as the method for serum extraction. Under Analytical Methods, ETS-8-5.1 is listed as the method for serum analysis. The amended protocol (Amendment No. 1) lists ETS-8-6.0 and ETS-8-7.0 as the methods to use for extraction and analysis of liver samples. TAhmeefnodllotwoinRge3aMd:Environmental Laboratory methods will be used for liver and sera extractions and analyses: ETS-8-4 Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical 3M Environmental Laboratory 3M Environmental Laboratory Page 41 BACK TO MAIN 3M Medical Department Study: T-6295.10_______________________________ Analytical Report: FACT TQX-099 LRN-U2400 Protocol #FACT TOX-099 Amendment 4 ETS-8-5 ETS-8-6 ETS-8-7 Electrospray/Mass Spectrometry Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry TRoeaclsaorinfy: the methods to use for extraction and analyses of TOX-099 liver and sera samples. 3M Environmental Laboratory 3M Environmental Laboratory Page 42 BACK TO MAIN 3M Medical Department Study: T-6295.10_________________________________________________ Analytical Report: FACT TQX-099 LRN-U2400 Protocol #FACT TOX-099 Amendment 4 Amendment Approval John L. Butenhoff, Ph.D., Sponsor Representative Date 7 - m. Marvin T. Case, D.V.M., Ph.D., Study Director J A r b -.D9+at+eV- 3M Environmental Laboratory 3M Environmental Laboratory Page 43 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TQX-099 LRN-U2400 Record of Deviation I. Identification Study / Project No. Dev7iaVtioXn T- 0yp9e 9 (Check one) 4 /2 -0 /< ? - .................. SOP J^Method Equipment Procedure Protocol Other: Document Number Date(s) of occurrence rs -g -4 ./" ^ TS-Z-- /o //t> /D O II. Description: Required Procedure/process:________________ Type, Z yjJrCS t'S re ^ c /jV e /t^ or .6 L P g fu d j'cc. Actual Procedure/process: The 'h (P <L O ? tlfliin p u te . tAAXfctr DSld f r-flu S tx n A u a c t T i e r 2 t'c, Utt-Kkou/^.------------------- ----- ----------------- 1 III. Actions Taken: (such as amendment issued, SOP revision, etc.) Tht 77e r 2. fl.n o f i ^ c . U J a ic r S e n t-fo r -te s tin g vJf h e +jnj j d v p . r e ^ eftj''vi-j e- e d . S n n e ly * Recorded By IV. Impact on Study / Project I Date /o fe tfo o J T, jkt. ~sztM fy v u Z Z - Z Z Z J I hh turHoo L^. Authorized By (Study Director / Project Lead) Date Sponsor'. John Huk/thoPIr JVuci^ Dirt^W'. tlrtor*' Con, 3M EFnovrirmonEmTeSn-4ta-8l L.0aboratory Deviation No. (assigned by Study Directoror Project Lead at the end of study or project) 3M Environmental Laboratory Page 44 3M Medical Department Study: T-6295.10 October 30, 2000 BACK TO MAIN Analytical Report: FACT TQX-099 LRN-U2400 Pace Analytical Service, Inc. 1700ElmStreet, Suite200 Minneapolis, MN55414 Phone: 612.607.1700 Fax: 612.607.6444 H r. Wade Schell 3M Environmental Laboratory 935 Bush Ave. Bldg. 2-3E-09 S t . Pa u l. MN 55144 R E: Pace Pro jec t Number: 1037874 C lie n t Pro jec t ID : Water Q u a lity Checks-T2 , Dear Mr. Schell: Enclosed are the analytical results fo r sanple(s) received by the laboratory on October 18 . 2000. I f you have any questions concerning t h is re p o rt, please feel free to contact ee. Sincerely. v 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS This report shall not bereproduced, except infull, without thewrittenconsent of PaceAnalytical Services, Inc. Page 45 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TQX-099 LRN-U2400 P tn Analytical Stnlcas, lac. 1700ElmStreet, Suite200 Minneipolis, MN55414 Phone: 612.607.1700 Fax: 612.607.6444 DATE: 10/30/00 PAGE: 1 M Environmental Laboratory 35 Bush Ave. Idg. 2-3E-09 . t . Pa ul. HN 55144 Pace Project Number: 1037874 C lient Project ID : Water Q u a lity Checks-T2 \ttn : H r . Wade Schell ' hone: 612-778-5233 Solid results are reported on a wet weight basis ' ace Sample No: '.lie n t Sample ID : 102339926 T IE R I I WATER SAMPLE ' arameters Results Units Date Collected: Date Received: 10/18/00 10/18/00 M atrix: Water PRL Analyzed Analyst CAS# Footnotes tetal s ' Metals. Trace ICP Sodium Date Digested le t Chemistry S pe cific Conductance S p e cific Conductance pH, Water pH Anions, Ion Chromatography Chloride Total Organic Carbon Total Organic Carbon Method: EPA 6010 M> ug/1 1000 Method: EPA 12 0 .1 2 umhos/cm 1 Method: EPA 15 0.1 5.5 O .l Method: EPA 300.0 ND ng/1 1 k Hethod: SM 5310 ND mg/1 1 Prep Method: EPA 3010 10/28/00 8DA 7440-23-5 . 10/19/00 Prep Method: EPA 12 0 .1 10/18/00 JM1 10/18/00 JMlPrep Method: EPA 150.1 Prep Method: EPA 300.0 10/24/00 BKR Prep Method: SH 5310 10/20/00 BKR 7440-44-0 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS r This reportshall not bereproduced, except infull, without thewritten consent of PaceAnalytical Services, Inc. Page 46 3M Medical Department Study: T-6295.10 4RAMETER FOOTNOTES D Not Detected RLC Not Calculable Pace Reporting L 1 a 1 t BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 PasaAnalyticalSanieat, Ine. 1700ElmStreet, Suite200 Minneapolis, MN55414 Phone: 612.607.1700 Fax: 612.6075444 DATE: 10/30/00 PAGE: 2 Pace Project Nuaber: 1037874 C lie n t Project ID : Water Q u a lity Checks-T2 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS r Thisreportshall not bereproduced, except in full, withoutthewritten consent of PaceAnalytical Services. Inc. Page 47 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TQX-099 LRN-U2400 QUALITY CONTROL DATA Pact Analytical Services, Inc. 1700ElmStreet, Suite200 Minneapolis, MN55414 Phone: 612.607.1700 Fax: 612.607.6444 DATE: 10/30/00 PAGE: 3 it Envlromental Laboratory 135 Bush Ave. ,ldg. 2-3E-09 ,t . Paul. HN 55144 Para Project Nuafcer: 1037874 C lie n t Project ID : Water Q u a lity Checks-T2 ' \ttn: H r. Wade Schell ' hone: 612-778-5233 1C Batch ID : 51031 \nalys1s Method: EPA 12 0 .1 Xssoclated Pace Saeples: 102339926 QC Batch Method: EPA 12 0 .1 Analysis Description: S pe cific Conductance tETHOO BLANK: 102340106 tssoclated Pace S a b le s : ' araneter Specific Conductance 102339926 Units whos/ra Method Blank Result PRL 11 Footnotes SAMPLE DUPLICATE: 102340114 ' araneter Specific Conductance Units tahos/aa Dup. 102339926 Result 2.000 2.000 RPO Footnotes 0 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS f Thisreportshall not bereproduced, except Infull, without thewritten consent of PaceAnalytical Services. Inc. Page 48 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TQX-099 LRN-U2400 QUALITY CONTROL DATA Pace Analytical Sarvlcas, ine. 1700ElmStreet, Suita200 Minneapolis, MN55414 Phone: 612.607.1700 Fax: 612.607.6444 DATE: 10/30/00 PAGE: 4 :M Environmental Laboratory 35 Bush Ave. Idg. 2-3E-09 ,t . Paul, HN 55144 Pace Project Number: 1037874 C lie n t Project ID : Water Q u a lity Checks-T2 \ttn : H r. Wade Sdiell ' hone: 612-778-5233 1C Batch ID : 51032 \nalys1s Method: EPA 15 0 .1 \ssoc1ated Pace Samples: 102339926 QC Batch Method: EPA 15 0 .1 Analysis Description: pH. Water J1B0RAT0RY CONTROL SAMPLE: 102340130 ' araneter jH Units Spike LCS Cone. Result 5.000 5.000 Spike X Rec Footnotes 101 3AHPLE DUPLICATE: 102340122 ' arameter oH Units Dup. 102338563 Result 8.200 8.200 RPD Footnotes 0 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS r Thisreport shall not bereproduced, exceptinfull, without thewritten consent of PaceAnalytical Services, Inc. Page 49 3M Medical Department Study: T-6295.10 QUALITY CONTROL DATA BACK TO MAIN Analytical Report: FACT TQX-099 LRN-U2400 Pic i Analytical Snicas, Ine. 1700ElmStreet, Suite200 Minneapolis, MN55414 Phone: 612.607.1700 Fax: 612.607.6444 OATE: 10/30/00 PAGE: 5 -1 Environmental Laboratory 35 Bush Ave. Idg. 2-3E-09 t . P a u l, MN 55144 Pace Project Number: 1037874 C lie n t Project ID : Water Q u a lity Checks-T2 t t n : H r. Wade Schell hone: 612-778-5233 C Batch ID : 51059 nalysls Method: EPA 6010 ssodated Pace S n p le s : 102339926 QC Batch Method: EPA 3010 Analysis Description: Metals. Trace ICP E7HOO BLANK: 102340494 ssodated Pace Samples: 102339926 arameter odium Units ug/1 Method Blank Result PRL ND 1000 Footnotes ATRIX SPIKE & MATRIX SPIKE DUPLICATE: 102340510 102340528 Spike arameter Units 102339124 Cone. Matrix Spike Result odium ug/1 0 50000 61210 Matrix Spike Sp. Dup. X Rec Result 122 58410 Spike Dup X Ree RPD Footnotes 117 5 1 AB0RAT0RY CONTROL SAMPLE: 102340502 arameter Units odium ug/1 Spike LCS Spike Cone. Result ^ * Rec Footnotes 50000 59230 118 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS r Thisreport shall not bereproduced, exceptin full, without thewritten consent of PaceAnalytical Services, Inc. Page 50 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 QUALITY CONTROL DATA Pace Analytical Services, Inc. 1700ElmStreet, Suite200 Minneapolis, MN55414 Phone: S12.607.1700 Fax: 612.607.6444 DATE: 10/30/00 PAGE: 6 M Environmental Laboratory 35 Bush Ave . Idg. 2-3E-09 t . P a u l, MN 55144 Pace Project Number: 1037S74 C lie n t Project ID : Hater Q u a lity Checks-T2 ' .t t n : Mr, Wade Schell hone: 612-778-5233 1C Batch ID : 51065 n a lysls Method: EPA 300.0 \5Soc1ated Pace Samples: 102339926 QC Batch Method: EPA 300.0 Analysis Description: Anions. Ion Chromatography lETMOD BLANK: 102340734 .ssoclated Pace Samples: 102339926 ' arameter :hlor1de Units mg/1 Method Blank Result PRL 1.1 1 Footnotes IATRIX SPIKE & MATRIX SPIKE DUPLICATE: 102340742 102340759 Spike ' arameter Units 102339710 Cone. Matrix Spike Result Ihlorlde g/1 48.68 50 95.38 Matrix Spike Sp. Dup. X Rec Result 93.4 93.66 Spike Dup X Ree RPD Footnotes 90.0 4 2 .2 -AB0RAT0RY CONTROL SAMPLE 6 LCSD: 102340767 102340775 Spike LCS 'arameter Units Cone. Result v Spike LCSD X Rec Result ihlorlde mg/1 5.000 5.320 106 5.200 CM 3 Spike Dup X Rec RPD Footnotes 2.2 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS f Thisreportshall not bereproduced, except in full, withoutthewrittenconsent of PaceAnalytical Services, Inc. Page 51 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TQX-099 LRN-U2400 QUALITY CONTROL DATA Pace Analytical Services, Inc. 1700ElmStreet, Suite200 Minneapolis, MN55414 Phone: 612.607.1700 Fax: 612.607.6444 DATE: 10/30/00 PAGE: 7 H Environmental Laboratory 35 Bush Ave. Idg. 2-3E-09 t . Paul. HN 55144 Pace Project Nuaber: 1037874 C lie n t Project ID : Water Q u a lity Checks-T2 .ttn : H r. Wade Schell hone: 612-778-5233 1C Batch ID : 51099 analysis Method: SM 5310 \ssoc1ated Pace Sanples: 102339926 QC Batch Method: SH 5310 Analysis Description: Total Organic Carbon IETH0D BLANK: 102343464 xssoclated Pace Sanples: 102339926 Parameter 'otal Organic Carbon Units ng/1 Method Blank Result PRL ND 1 Footnotes IATRIX SPIKE & MATRIX SPIKE DUPLICATE: 102343472 1023434B0 Spike Parameter Units 603882358 Cone. Matrix Spike Result Total Organic Carbon ng/1 6.320 2.000 8.850 M atrix Spike Sp. Dup. X Rec Result 126 8.980 Spike Dup X Rec RPD Footnotes 133 5 3 ,3 .MORATORY C0N1R0L SAMPLE & LCSD; 102343506 102343514 Spike LCS ' araneter Units Cone. Result k Spike LCSD X Rec Result Total Organic Carbon ng/1 5.000 5.280 106 5.290 Spike Dup X Rec RPD Footnotes 106 0 SAMPLE DUPLICATE: 102343498 Parameter Total Organic Carbon Units ng/1 Dup. 102339926 Result ND ND RPD Footnotes NC 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS r This report shall not bereproduced, except infull, without thewritten consent of PaceAnalytical Services. Inc. Page 52 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACTTOX-099 LRN-U2400 Pace Analytical Stnricas, lac. 1700ElmStreet, Suite200 Minneapolis, MN55414 Phone: 612.607.1700 Fax: 612.607.6444 DATE: 10/30/00 PAGE: 8 P a i* Project Nuaber: 1037874 C lie n t Project ID : Water Q u a lity Checks-T2 UALITY CONTROL DATA PARAMETER FOOTNOTES ons1 stent w ith EPA guidelines unrounded concentrations are displayed and have been used to calculate t Rec and RPD values. 0 Not Detected C Not Calculable RL Pace Reporting H a l t ; PO Relative Percent Difference 4 .1] The NS was out o f our QC H a l t s but the MSD was acceptable. 2] Analyte Is found 1n the associated blank as well as In the sa*>1e (CLP B -Fla g ). .. ?. 3] The spike recovery was outside acceptance l l i l t s fo r the MS and /o r MSD due t o m atrix in te rfe re n c e. The LCS and/or LCSD were w ith in acceptance H i l t s showing th a t the laboratory 1s In control and the data 1s acceptable. 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS r Thisreport shall not bereproduced, except infull, without thewritten consent of PaceAnalytical Services, Inc. Page 53 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TQX-099 LRN-U2400 Pact Analytical Sendees, tot. 9608Loiret Blvd. Leime, KS66219 Phone: 913.599.5665 Fax: 913.599.1759 DATE: 10/24/00 PAGE: 1 INNESOTA LABORATORY 4CE ANALYTICAL SERVICES 700 ELM STREET SUITE 200 INNEAPOLIS, HN 55414 Pace Project Niaber: 6045897 C lient Project ID : SCIENCE SOUmONS/1037874 t t n : PROJECT MANAGER . hone: (612)544-3435 o lid results are reported on a wet weight basis ace Sanple No: lle n t Saeple ID : 603917352 T IE R I I HATER SAMPLE araweters Results Units Oate Collected: Date Received: 10/18/00 10/21/00 M atrix: Mater PRL Analyzed Analyst CAS# Footnotes .et Chesristry S ilic a . Total S ilic a R e s is tiv ity Resi s itiv i t y Method: EPA 375.3 ND ag/1 1 Method: 150000 otas/ca 100 Prep Method: EPA 375.3 10/23/00 HMG 7631-86-9 Prep Method: 10/22/00 JLC k 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS r This reportshall notbereproduced, except infull, withoutthewrittenconsent of PaceAnalytical Services, Inc. Page 54 3M Medical Department Study: T-6295.10 m ARAHETER FOOTNOTES O Not Detected Not Calculible ?<L Pace Reporting L i a i t BACK TO MAIN Analytical Report: FACT TQX-099 LRN-U2400 PaceAnalyticalSanlcat, Inc. 9608Loirtt Blvd. Lenexa, KS66219 Phone: 913.599.5665 Fax: 913.599.1759 DATE: 10/24/00 PAGE: 2 Pici* Project Ntaber: 6045897 ClUent Project 10: SCIENCE SOUmONS/1037874 v 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS r Thisreportshall notbereproduced, exceptinfull, withoutthewritten consent of PaceAnalytical Services, Inc. Page 55 3M Medical Department Study: T-6295.10 QUALITY CONTROL DATA BACK TO MAIN Analytical Report: FACT TQX-099 LRN-U2400 P tei Analytical Sanrieas, Ine. 9608 Loiret BM. Lenexa, KS66219 Phone: 913.5X.5665 Fax: 913.5X.1759 DATE: 10/24/00 PAGE: 3 MINNESOTA LABORATORY 'ACE ANALYTICAL SERVICES 1700 ELM STREET SUITE 200 MINNEAPOLIS, HN 55414 Pace Project Nuaber: 6045897 Client Project 10: SCIENCE SOLUTIONS/1037874 ' A ttn : PROJECT MANAGER ' hone: (612)544-3435 ' ' 1C Batch ID : 93019 Analysis Method: EPA 375.3 Associated Pace Saaples: . QC Batch Method: EPA 375.3 :; Analysis Description: S ilic a , Total 603917352 METHOD BLANK: 603919127 Associated Pace Saaples:. 603917352 ' araneter S ilic a Units ag/1 Method Blank Result PRL ND 1 Footnotes MATRIX SPIKE: 603919135 ' araneter silica Units g/l -ABORATORY CONTROL SAMPLE: 603919150 ' araaeter Units S ilic a ag/1 Spike 603886300 Cone. M atrix Spike Result 20.91 10 29.12 Spike t Ree Footnotes 82.1 Spike LCS i Cone. Result 10 5.830 Spike X Rec Footnotes 58.3 AMPLE DUPLICATE: 603919143 ' arameter Silica Units 9FI Dup. 603886300 Result 20.90 19.30 RPD Footnotes B 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS r Thisreportshall not bereproduced, except Inlull, withoutthewrittenconsent o1PaceAnalytical Services, Inc. Page 56 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TQX-099 LRN-U2400 raceAnalyticaT www.paulOt.tom Pact Analytical Servit, Ine. 9608LolntBM. Lenexa, KS66219 Phone: 913.599.5665 Fax: 913.599.1759 DATE: 10/24/00 PAGE: 4 Pao Project Nuaber: 6045897 Client Project ID : SCIENCE SOUfTIONS/1037874 ALITY CONTROL DATA PARAMETER FOOTNOTES . insistent w ith EPA guidelines unrounded concentrations are displayed and have been used t o calculate X Rec and RPD values. i Not Detected . Not Calculable - 0. Pace ReportingH i l t >0 R elative Percent Difference. v 3M [Environmental Laboratory REPORT OF LABORATORY ANALYSIS r Thisreportshall not bereproduced, except infull, withoutthewrittenconsent of PaceAnalytical Services, Inc. Page 57 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: F A C T T Q X -0 9 9 LR N -U 2400 Record of Deviation I. Identification Study/Project No. FACT-TOX-099 Argus 418-012 Deviation Type (Check one) SOP X Method Equipment Procedure Protocol Other: Document Number(s): EfS-8-7.0 Date(s) o f occurrence: 12/ 2 1 /0 0 II. Description: Required Procedure/process: Section 14.4.1 states the matrix spike percent recoveries must be within +/- 30% of the spiked concentration. Actual Procedure/process: The liver matrix spike (8S53F-MS-250ppb-3) percent recovery was 156% and the mean matrix spike/matrix spike duplicate percent recovery was 138%.________________________________ III. Actions Taken: (such as amendment issued, SOP revision, etc.) This deviation was written. Recorded By Date 01 / d /,oi IV. Impact on Study / Project 6} I MfifYTxcfs atklw kjt, htuotcl A u /tjL t he. a g g ira i fo fhe, Authorized By / p e rt(Study Director / Project Lead) > Q l/euoi Date t fa*** k St^ U i Sayonaor ` jJ Buio'i'of Oi/tdor- Mor/in Cor, 3M EFnovrirmonEmTeSn-4ta-8l L.0aboratory 3M Environmental Laboratory Deviation N o ._____ 2 _____ (assigned by Study Directoror Project Lead at the end ofstudy or project) Page 58 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical R eport: F A C T T O X -0 9 9 LkN-U2400 Record of Deviation I. Identification Study/Project No. FACT-TOX-099 Argus 418-012 Deviation Type (Check one) SOP X Method Equipment Procedure Protocol Other: Document Number(s): ETS-8-7.0 Date(s) o f occurrence: Entire study_________ II. Description: Required Procedure/process: Section 13.1.6 describes the calculations that should be used to convert extract concentration to matrix concentration. Actual Procedure/process: In order to accommodate purity information, the actual calculation used varied somewhat from that written in section 13.1.6. Two additional factors, salt correction and standard purity, were added. The first accommodates the mass difference between the analytical standard (C8F17S03K) and the target analyte (C8F17S03-), while the second addresses the purity o f the analytical reference material, determined after the study was completed. ..... ...... III. Actions Taken: (such as amendment issued, SOP revision, etc.) This deviation was written. Recorded By Date ^/xo/(fO IV. Impact on Study / Project The updated calculations accommodate new information and are an improvement. No adverse affect on the study._________________ jf,h 1^-fd^O/ifO___________________________ ___ ___ Authorized B y (StudyDirector / Project Lead) -0, Date 3M EFnovrirmRoneEmpTreStn-J4twa-8ltL.a0aWbora3tboLry 0u|t*UT Sludd-IWiV. Cast Deviation No. (assigned by Study DirectororProject Lead at the end of study or project) 3M Environmental Laboratory Page 59 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Appendix C: Extraction and Analytical Methods This appendix includes the following methods: ETS-8-4, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry," (14 pages) ETS-8-6, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry", (14 pages) ETS-8-5, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" (9 pages) ETS-8-7, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" (10 pages) 3M Environmental Laboratory Page 60 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: F A C T T O X -0 9 9 LR N -U 2400 3M Environm ental Laboratory M ethod E xtraction of P otassium P erfluorooctanesulfonate or Oth er Fluorochem ical compounds from Serum for Analysis U sing H PL C - E lectrospray/M ass Spectrom etry Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: `P i Approved By: O f ;$>--- Laboratory Manager U t-- ------- Group Leader Technical Reviewer Date DHatef u 3 3 of'/a Date U w .. 1.0 Scope and Application 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. Word6/95 3M Environmental Laboratory ETS-8-4.1 ExtractionofPFOS fromSerum Page l o f i 4 Page 61 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: F A C T T O X -0 9 9 LR N -U 2400 2.0 Summary of Method_______________________.__________________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-teri-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, Mtrheem55soa6vm,eadpnladenasdunprdrutothgoeantaetnosatalaynntdietariroodnge(psnaeeierv3ais.p0poDraarettfoiitrnioiutninoetdnilsidn)r.tyo.AMnEtaBiocEhn.pexTatihrraeincMgt irtsBeraEegceeonxnttsritsaitcautdtiedsdedinto1.0 mL of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-5.1 or other appropriate methods. 3.0 Definitions ______ __________________________________________ ;________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F,7S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide CgF,7S 02NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CgF,7S 0 2N(CH2CH3)CH2C 0 2' 3.4 ECt,FFO17SSE0-2ONH(C:H2(2CNH-e3t)hCyHlp2eCrfHlu20orHooctane sulfonamido)-ethyl alcoh. ol 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CgF17S 0 2N(CH2CH3)H 3.6 M556: CgFl7S 0 2N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooetcine sulfonic acid 4.0 Warnings and Ca u t i o n s _____________________________________________ 4.1 Health and safety warnings 4.1.1 Uhasnedulninigvearnsiaml parletcisasuuteio, nwsh, iecshpmecaiayllcyolnatbaoinraptaotrhyocgoeantss., goggles, and gloves when 5.0 Interferences_______________________________________________________________ 5.1 There are no interferences known at this time. 6.0 Equipment ______________________________________________________________ 6.1 Tachceepfotallbolwe.ing equipment is used while performing this method. Equivalent equipment is 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 3M Environmental Laboratory ETS-8-4.1 Extraction ofPFOS fromSerum Page 2 of 14 Page 62 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 Supplies and Materials_________________;__________ ___________________________ 7.1 Gloves 7.2 Eppendorfor disposable pipettes 7.3 Nalgene bottles, capable o f holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels . 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable o f measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Msepilalir-aQteTMviwaalst.er. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 8.0 Reagents and Standards___________________________ .______________________________ 8.1 TbeypMeiIllir-eQagTMenwt agtreardaenwdamtear,yMbeillpi-rQovTMideodr beqyuaivMalielnlit-;QaTllOwCatePrluussTMed sinystthemis method should 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na2C 03), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCOj), 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 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction ofPFOS fromSerum Page 3 of 14 3M Einvironmental Laboratory Page 63 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: F A C T T O X -0 9 9 LR N -U 2400 8.9.3 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 M 556(3M Specialty Chemical Division), molecular weight = 557 8.9.7 CSu8Frr,3oSga0t3eHs)tamndoalercdu:l4a-rHw,epiegrhftlu=or4o2o8ctane sulfonic acid (1-H,1-H, 2-H, 2-H 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8..10.1 d1IO0is0sN0olmsvoeLddi.buemSatkoheryredcrinoonxatiad1ienL(inNNgaaO5lg0He0)n:meWLboeMtitgliehll.ia-QppTMrowxiamteart,elmy i2x0u0ngtiNl aalOl sHo.liPdosuarreinto a 8.10.2 N1 NNasoOdHiusmolhuytidornoxinidtoe a(N1a0O0Hm)L: vDoilluumtee1tr0icNflNasakOaHnd1:d1i0lu. teMteoavsoulruem1e0 umsLingof 10 .Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 ' 0NoM1.0fa5iTOlulMiBsH-iQAnt,egTMatindraatdpwobpsauarltotoey1wrxl.aiLlmmySvabtmooteelrouclenyamiiu4unes4mteartitho1chyecL5do4prNnHomtagaLlceignhneoiannsfnuegg1lbf5e0aso0Ntt0eatblN(merT.auLBOpAMtHly)i:)l(.lWWi-DQheiiTMilgluehtwaeadatpodtpeivnrroo.gxlAutihmdmejaueltasewtsltyittomh1p6LH9ogf 8.10.3.1 nTeeBdAedreuqsuinirges1aNchNeacOkHprsioorluttoioena.ch use to ensure pH = 10. Adjust as 8.10.4 b0ap.i2cpa5rroMbxoinmsoaatdetieu(lNmy a2cH6a.r5Cb0go3no)afitnseto/osdoaidui1muLmcavbroibcluaomrnbaeottenria(cNtefalbajCuskfOfeaJrnad(Nnbdar2i2Cn1g0.0t3o/gNvoaofHlusComd0ei3u)w:miWtheMigihlliQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fs1lo.u1lou0rtoiocpnhpecmmoniEctaatFilnOsitnSagnEd1-Oa.0rHd0s.p)aprme aPcFceOpSta, b1l.e02(foprpmexaPmFOplSe,Ao,n0e.9w8o7rpkpinmg PstFanOdSaArdA, and 8.11.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 ExtractionofPFOS fromSerum Page4 of 14 3M Environmental Laboratory Page 64 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 8.11.7 aDppilruotxe. w0.o5r0kipnpgms.tandard 1 with methanol for a working standard 3 solution o f 8.12 Surrogate stock standard preparation 8.12.1 WC8FeingSh 0ap3Hprionxtoimaa5te0lym5L0-v6o0lummgetorficsuflrarsokgaatnedstraencodradrdth1e-Hac,1tu-Hal,w2-eHig,h2t.-H, 8.12.2 pBprmin.g to volume with methanol for a surrogate stock of approximately 1000-1200 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL o f surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard o f 100 ppm. Record the actual volume transferred. 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 to room temperature prior to extraction. 10.0 Quality Control_____________________ ;______________ ;__________;_____________ 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots o f Milli-QTM water following this procedure and use as method blanks. 10.1.3 Emxattrraixctbtlwanok1s..0 SmeLe 1a1li.q1u.4o.ts o f the serum following this procedure and use as 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Pmraetpraixrereecaecihvesdpiwkeituhseinacghassaammpplleesceht.osen by the analyst, usually the control 10.2.3 EAxdpdeitcitoendaclosnpcikenestrmataioynbsewinilcllfuadlledinatnhde mmaidy-rfaalnlgien othfethloewin-irtaianlgceaolifbtrhaetiionnitciaulrve. calibration curve. 10.2.4 Pmrienpiamruemonoefm2amtraixtrsixpiskpeikaensdpmerabtraitxcshp. ike duplicate per 40 samples, with a 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy o f the initial calibration curve. . 10.3.2 Prepare, at a minimum, one continuing check per group of 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. 3M Environmental Laboratory ETS-8-4.1 ExtractionofPFOS fromSerum Page 5 of 14 Page 65 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 10.3.4 The expected concentrations will fall within the mid-range o f the initial ctothhanaellinybinrtiahttieiaollnocwaculierbnvrdea.toioAfntdhcdeuitcrivaoelni.barlTashtpiioiskniescsunmrevcaeeys(sbfaoerryineixcfalutmhdepedlaen,tha5alyptspftabmll-uins1t0tqh0uepalpnobwti,t-arrtaaetnhugeserinogf 5 ppb - 1000 ppb). 11.0 Calibration and Standardization____________________________;________________ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11.1.2 If most sample volumes are less than 1.0 mL, extract standards with matrix vmoalturmixe. sReeqcuoarldtoeathche ssaammppllee vvoolluummeeso. nDthoeneoxtterxatcrtaiocnt lsehsseetthan 0.50 mL of 11.1.3 While preparing a total o f twenty aliquots in 15 mL centrifuge tubes, mix or shake between aliquots. 11.1.4 TTaeitywgtphhoitece1aeemlnnlydLstuoaasfnletidqhtahuirseodstssse,t,catotnwirdooaonmrt,hdtaeoctrorsainpxpcipkebnrelaot,rnpiankrtisiado,tunaep:n;vldioaclntauwdtmeos,epmt,wikseoeitnhrsgvotaedanambdslaoamrundnakttcsrsu.ilxrivsbetlesad,nfikonsr.Taatbolteal1o, f 11.1.5 RraenfgerestoanvdaltihdeatLioinneraerpCoratliEbrTaSti-o8n-4R.0an&geE(TLSC-R8)-5f.o0r-Vca-l1ib, rwahtiiocnhcluisrtvsetsh.e working 11.1.6 UsctaaslenibdAraatrttdaiosc.nhmsSteaeenndtSaDercdtasiso. nan1a3i.d0 itno ccaallccuullaattienagctthuealccoonncceenntrtraatitoionnssooffthPeFwOoSrikning 11.2 wT10oo0re0kaipcnhpgbss.ttaannddaarrdd, fbolranthke, ocronccoennttinrautiinogncthoefcakll, waditdhainppthroepcrailaitberaamtioonuncut rovfesurarnroggea5teppb11 3 Extract spiked matrix standards following 12.6-12.16 o f this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 6 of 14 Page 66 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix - - Blank 0.500 ppm 10 0.005 ppm 0.500ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 . 0.050 ppm 5.00 ppm . 20 0.100 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 1.00 ppm 12.0 Procedure_____________________________________________________ _____________ 12.1 wOabttearibnaftrho.zen samples and allow to thaw at room temperature or in a lukewarm 12.2 Vpoolrytperxompyixlefnoerc1e5ntsreicfuognedstu, bthee.n transfer 1.0 mL or other appropriate volume to a 15 mL 12.3 Return unused samples to freezer after extraction,amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Lwaobrkelshtheeettufobredwoictuhmtheentsitnugdythneurmembeari,nsianmg pstleepHs.), date and analyst initials. See attached 12.6 Sstpainkdeaardllassamdepslcersi,biendcliund1in1.g2b. lanks and standards, ready for extraction with surrogate 12.7 S1pinikteheaatcshecmtiaotnr,ixfowritthhetchaeliabprpartoiopnricautervaemsotaunndtaorfdsst.anAdlasrodparsepdaersecrmibaetdrixinsp1i1k.1es, oarndTable continuing calibration standards. 1 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 10. If not, adjust accordingly. 12.10 bTiocaerabcohnsaatembpuleff,eard. d 1 mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium 12.11 Using an Oxford Dispenser, add 5 mL methyl-terf-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are well separated. ETS-8-4.1 ExtractionofPFOS fromSerum Page 7 of 14 3M Environmental Laboratory Page 67 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL o f the organic layer to this clean 15 mL centrifuge tube. 12.16 Phouutresa.ch sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 12.17 Add 1.0 mL o f methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.1.9 Asytrtiancghea. 0F.i2lteprminntoylaon1.m5 emshLfgilltaesrstaouato3vciacl soyrrlinowge-vaonldumtraenasufetorvthiael swahmepnlenetocetshsiasry. 12.20 Lmaabtreilxt,hfeinaaultsoovlivaelnwt,itehxttrhaecsttioundydantuem, abnedr,aannaimlyasel sn)upmebrfeorramnidnggetnhdeeerx,tsraamctpiolne.timepoint, 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.22 nCootmebpoloetkeotrheinecxlturdaectiinonstwudorykbsihnedeetr,,aatstaacphperdotporitahties. document, and tape in the study 13.0 Data Analysis and Calculations_____________________________________________ 13.1 Calculations 13.1.1 cCaalilbcuralattioenacsttaunadl acrodnsceunstirnagtitohnesfoolfloPwFiOngS;, eoqruoatthioenr:applicable fluorochemical, in . mmLLofosftastnadnadradrd+ xmcLonocfesnutrrraotgioanteosftastnadnadradrd+ (iunieti/aml mLla_t_r_ix__v_o_l_u_m__e ;(_m__L_)___,,= Final Concentration (pg/mL) o f PFOS in matrix 14.0 Method Performance_______________________________________________________ 14.1 TfohresmpeectihfoicdMdeDteLctainond lliimmiitt o(Mf qDuLan) tiistaatnioanly(tLeOanQd)mvaalturiexs s(pseeeciAfictt.aRchemfeerntotsMBDaLndreCpo).rt 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2. Matrix spike and matrix spike duplicate samples to determine accuracy and precision o f the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve. 14.3 Refer to section 14 o f ETS-8-5.1 for method performance criteria. 15.0 Pollution Prevention and Waste Management________________________ 15.1 hSiagmhpBleTwUacsotentiasidniesrpso, saenddiunsbediohgalazsasrpdicpoenttteaiwnaersst,efilsamdimspaobsleedsionlvbernotkwenasgtleasiss dcoisnptoasineedrisn located in the laboratory. ETS-8-4.1 ExtractionofPFOS fromSemm Page 8 of 14 3M Environmental Laboratory Page 68 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 16.0 Records_____________ ;________________________________________________ ;______ 16.1 nCootmebpoleotkeothreinecxlturdaectiinonthweo3r-krsinhgeesttuadttyacbhineddetro, tahsisapmpertohpordia,taen. d tape in the study 17.0 Attachments___________________________________ ____________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 References________________________________;___________________________________ ' 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l. 18.2 FACT-M-3.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 Affected Documents________ ;______________ ,_________________________________ 19.1 EHTPSL-C8--E5.l1ec, tr"oAsnparalyysMis aosfsSSepruecmtroormOettrhye"r Fluid Extracts for Fluorochemicals using 20.0 Revisions ________________ :_______________________________________________ RNeuvmisbioern 1 Reason For Revision SSSeeeccctttiiiooonnn 111222...211137 FACihdnadaneldgveotdhluetomshienaicksleu1rd.s0epmseaeLmd;.pnloetstaodrjaugseteadtfrooroimnittieaml pveorluatmurees. less than 1.0 mL. ReDvaistieon 04/02/99 3M Environmental Laboratory . ETS-8-4.1 ExtractionofPFOS fromSerum Page 9 of 14 Page 69 BACK TO MAIN 3M Medical Department Study: T-6295.10 Extraction Worksheet ETS-8-4.1 Analytical Report: FACT TOX-099 LRN-U2400 Studv# Matrix Box# Wk/Day DateSpiked/Analyst Surrogate Std approx, ppm actual ppm # FC-Mix approx. 0.5 pm actual ppm # FC-Mix approx. 5 ppm actual ppm # CCV MS MSD FC-Mix approx. 50 ppm actual ppm # Comments --- -- -- - - - -. - -- - -- -- - - '- Blank Std# amount - Serum Extraction Method : Vortex 15 sec. Picette Matrix Volume mL Pipette 1mL of 0.5 M TBA, pH 10. pH= Std. # Pipette 2 mL of 0.25 Na2COV0.25M NaHCOt buffer Std. # Dispense 5 mL of methyl-t-butyl ether TO-A- Shake 20 min. Shaker speed: Centrifuae 20-25 min. Centrifuee speed: Remove a4mL aliauot of organic laver Put on Nitroeen Evanarator to drvness Temperature: Add methanol Volume mL TN-A- Vortex 30 sec. FCioltnert.uCsianla. Va 3ecricfiBc-aDtiosvnrsinueseewdisthamae0.m2uamtrifxiltaesrfinotrosatd1.c5umrvLe.autosamDle vial - - - - - - '- .- - - r -- - - mL ' Date & Initials Attachment A ETS-8-4.1 ExtractionofPFOS firmSerum Page 10 of 14 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 5ppb-1000ppb PFOSA 1.51 4.79 5 ppb - 1000 ppb PFOSAA 3.46 20.5 5 ppb - 1000 ppb EtFOSE-OH 11.4 36.2 5 ppb -1000 ppb M556 6.03 19.2 5 ppb -1000 ppb PFOSEA 5.71 18.2 5 ppb -1000 ppb MdeDteLrm/LiOneQd.vTalwueosciunrrvaets, binoveianceh, omfotnhkeseey,maantdrihcuesmwaenrseeerxutmra,catnedd manodnakneaylpylzaesdmwaitwhetrheenroatbsbtiattsisetriucmally curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbitresponses, therefore, theirMDL and LOQ will be the same values as determined in rabbit serum. Please see LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-l for further information. Attachment B: MDL/LOQ Summary ETS-8-4.1 ExtractionofPFOS fromSerum Page 11 of 14 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Compound: PFOS Prepared range Rabbit Serum (popfbs)ta(nndga/rmdLs ) Full Range Low Curve High curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 Compound: PFOSA Rabbit Serum Proefpsatraenddarardnsge (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 24.8 - 976 0.993 - 976 CRaobmbpitoSuenrdu:mPFOP(pSropeAfpbAsa)trae(nnddgar/armdnLsge) Full Range Low Curve High curve 1/X 0.991 - 974 4.92 - 247 49.2-974 0.991 - 974 LCR from curve (n(gp/pmbL) ) 24.8 - 978 4.94 - 248 97.8-978 4.94-978 % Recovery Range 83-108 85-104 85-106 94-111 LCcRurfvreom (n(gP/PmbL) ) 4.93 - 976 4.93 - 97.6 24.8-978 4.93 - 976 % Recovery Range 88-103 . 87-105 93-102 94-103 LCcRurfvreom (n(gP/PmbL) ) 24.7 - 974 9.74 - 247 97.4 - 974 9.74-974 %RReacnogveery 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RRaSnDge 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 AttachmentB: MDL/LOQ Summary ETS-8-4.1 ExtractionofPFOS from Serum 3M Environmental Laboratory Page 12 of 14 Page 72 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 ' LRN-U2400 Compound: EtFOSE-OH Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve 0.993 - 976 4.93 - 97.6 High curve 49.3 - 976 1/X 0.993-493 Compound: PFOSEA Rabbit Serum P(propefpbsa)trae(nnddgra/armdnLsge) Full Range 0.993-976 Low Curve 4.93 - 248 High curve 49.3 - 976 1/X 0.993-976 Compound: M556 Rabbit Serum Proefpsatraenddraardnsge (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993-976 LCcRurfvreom (n(gp/pmbL) ) 49.3 -976 9.76-97.6 97.6 - 976 9.76 - 976 LCcRurfvreom (n(gP/pmbL) ) 24.8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 LCcRurfvreom (n(gp/pmbL) ) 24.8 - 976 9.76-97.6 97.6 - 976 9.76 - 976 % RReacnogveery 77-110 97-107 90-109 86-111 % Recovery Range 96-106 91-110 86-106 95-117 % RReacnogveery 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RRaSnDge 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summary ETS-8-4.1 ExtractionofPFOS fromSerum 3M Environmental Laboratory Page 13 of 14 Page 73 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Ion Pair Standard Curves- Fluids Prep date(s): Standard number: ASanmalpyltee(ms)a:trix: Equipment number: FBilnaanlksfolluviedn/itdaenndtifTieNr:: 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: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Sutdg/cmoLne Sutdg/cmoLne Sutdg/cmoLne Sutdg/cmoLne Sutdg/cmoLne 0.500 0.507 0.532 0.501 0.521 0.500 0.507 0.532 0.501 0.521 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 M556 Sutdg/cmoLne 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 All Am't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All Final vol mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Calculated concentrations of standards in the sample matrix FinPngaF/lOmcoSLne 49..9736 24.8 4997..36 244983 . 735 976 FPinnFgaO/lmcSoLAne 59..0809 259508...190 255010 746 989 FPiFnngaO/lmScAoLnAe 51.02.44 251620..434 256234 782 1038 FEinntFga/lOmcSoLEne 49..9748 249497...848 244984 737 978 FPiFnn59gaO../l09mSc13EoLnAe 25.2 59250950..1213 749 993 FinnMga/l5mc5o6Lne SSuntrdgr/ocmogLnaete 51.01.32 100 2S.8 5110.23 258 756136 SFuinnrgar/lomcgoLantee 500 1017 Am'tmAsULpiked 0.005 VMaolnikdBHSReayeouRatrvmb&aueibtnamdiPnetlraasmnagesEEEE--ssss5ttttiaiii.mmmm0Pp0Faaaap-ttttO1eeeerss0ssSo0oooox0nnnnillllmyyyy....a|UUUUtssesseeee5cvvvvP.o0aaaaF0llllnuuOuu-ceee1eSssess0An0ffffoooo0trrrrrrrrraaaaatbbbb|ibbbboiiiittntt..5Ps.F0O0-S1A00A0 | Et5F.0O0S-1E0-0O0H | 5P.F0O0-S1E00A0 | 5.0M05-150600 Attachment C: Ion Pair Standard Curves ETS-8-4.1 ExtractionofPFOS fromSerum 3M Environmental Laboratory Page 14 of 14 Page 74 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 3M Environm ental Laboratory M ethod E xtraction of P otassium Perfluorooctanesulfonate or other F l u o r o c h e m ic a l C o m p o u n d s f r o m L iv e r f o r A n a l y s is u s in g HPLC- Electrospray/M ass Spectrom etry Method Number: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: LaboratoryyMan\ager i s , u __________ . Group Leader ----------- - (Tfl* A .d W tw Technical Reviewer Adoption Date: Revision Date: ~ Y 7 T- h <r Date Date Ol/mivt Date 1.0 Scope and Application 1.1 Sotchoepref:luTorhoischmeemthicoadl cisomfoprothunedexstfrraocmtiolniveorf.potassium perfluorooctanesulfonate (PFOS) or 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 Mvaalitdraictieosn: rRepaobrbt.it, rat, bovine, and monkey livers or other tissues as designated in the Word 6.0/95 ETS-8-6.0 ExtractionofPFOS fromLiver Page 1of 14 . 3M Environmental Laboratory Page 75 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 2.0 Summary of Method______________________________________________________ ' 2.1 Tr(PehaFigsOemnSt)eatohnrododtmhdeeertshcfyrluilb-o/eersroict-hhbeeumptyriloceactelhdseuurrrf(eaMfcottarBneEtxs)t.rfIranocmttihnligisvpmeoret,taohsrosidout,mhseerpveteirnsfslfuuluoesro,oruoosccithnaegnmeasincualifloosnncapatnaeibrieng pnesfitxailatttrrnretoarditgecaitdeorendndt.eh:edArvPoainFnupOgitooohrSnaMa,tpoP3tarBFicruEOcinn.SptgiATllar,dhesPrteaiygFcM.eOsnEtyStBraAiiEcsnhAgaeed,exxtEdartettartFdacaOtccttohiSsietEstdhr-reaOteoncsHasoafmn,e.0srPp.tr2FileteOdupatmtSenoEddnaAitynhc,leeoM1nn.a0t5nrf5miiafl6lutLye,gtraeemnintideouttnbohsueapgrnaalranoiorsldgsitasphatueeunttoovnitaolsa. 2.2 Tmheethseodsas.mple extracts are analyzed following method ETS-8-7.0 or other appropriate 3.0 Definitions_________________________________ ___________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8Fl7S 0 3 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F)7S 0 2N(CH2CH3)CH2C 0 2 3.4 ECt,FFO17SSE0-2ONH(C:H22(CNH-e3t)hCyHlp2eCrfHlu20orHooctane sulfonamido)-ethyl alcohol 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S 0 2N(CH2CH3)H 3.6 M556: C,F17S 0 2N(HXCH2COOH) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions__________________________________ ;________________' 4.1 Health and Safety Warnings: 4.1.1 Uhasnedulinnigvearnsaiml parletcisasuuteio, nwsh, iecshpmecaiayllcyonlatbaoinraptaotrhyocgoeantss., goggles, and gloves wh. en 5.0 Interferences______ ;________________________________________________________ 5.1 There are no interferences known at this time. ' 6.0 Equipment_______________________________________ '__________________ ;________ 6.1 Tachceepfotallbolwe.ing equipment is used while performing this method. Equivalent equipment is 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 1000 or IEC 6.1.4 Shaker, Eberbach or VWR ETS-8-6.0 ExtractionofPFOS fromLiver Page 2 of 14 3M Environmental Laboratory Page 76 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7.0 Supplies and Materials_______________________________________________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable o f holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A . 7.6 I-CHEM vials, 40 mL glass . 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor - 3.0 to 10.0 ml 7.11 Syringes, capable o f measuring 5 pL to 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer 7.16 Crimp cap autoviais and caps 7.17 Crimpers Note: PQriTMorwtoautesri.ngRginlasseswsyarrinegaensdabmotitnleims,urminsoef39ttimimeesswwiitthhmmeetthhaannooll,an3dri3ntsiems efrsowmit3h sMepiallria- te vials. 8.0 Reagents and Standards______________________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM 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 (Na^COj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-iert-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 . ETS-8-6.0 Extraction ofPFOS fromLiver Page 3 of 14 3M Environmental Laboratory Page 77 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.10.3 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 8.10.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.10.7 SCujFrrjojSgOatjeHs)tamndoalercdu:l4a-rHw,epiegrhftlu=or4o2o8ctane sulfonic acid (1-H.l-H, 2-H, 2-H 8.10.8 Other fluorochemicals, as appropriate 8.11 Reagent preparation pNrOepTaEra:tioWn,haednjpurstepaacrcionrgdlinargglyer. volumes than listed in reagent, standard, or surrogate 8.11.1 10 N sodium hydroxide QSfaOH): Weigh approximately 200 g NaOH. Pour into a . d1i0s0s0olmveLd.beSatkoerre cinonata1inLinNga5lg0e0nme bLoMttliel.li-QTM water, mix until all solids are 8.11.2 M110NilNlis-oNQdaTMiOumHwahstyoedlru.rtoiSxotinodriene(tiNonaaaO11H020)5:mmDLLilvuNotaelulg1me0enNteribNcoaftlOtalesH.k 1a:n1d0.diMluteeastuorveo1lu0mmeLuosifng 8.11.3 0o.f5TMBAteitnratobuaty1laLmvmolounmiuemtrihcycdornotgaeinninsuglf5a0t0e m(TLBAM)i:llWi-QeiTMghwaaptperro. xAimdjautsetlyto169 g pMoHfiNll1ia-0OQuHTMsi,nwagdaadtpesrpl.orowSxtlioymrbeaeticenalyau4s1e4LtthoNe5ap4lHgmecnLheaobnfogt1et0sleNa. bNruapOtlHy).(WDihluilteeatdodvinolgutmhee lwasitthmL 8.11.3.1 nTeeBdAedreuqsuinirges1aNcNheacOkHprsioolruttoioena.ch use to ensure pH = 10. Adjust as 8.11.4 b0aQ.pi2TMcpa5rrwoMbxaoitnmseaoratd.etieuS(lNmytoa2rcH6ea.ir5Cnb0goa3no)1afitLnseto/Nosdoaaidulig1mueLmnceavbrobibclouaotmrntlbaeeot.tenria(cNtefalb^aCuskfOfeajr)nad(Nnbdar2i2Cn1g0.0t3o/gNvoaoHflusComd0ei3u)w:miWtheMigihlli8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Pfs1lor.u1leou0prtaoirocepnhpoectmmohneiEctraatfFillnuOsitonaSrgnEod1c-aOh.0reHd0ms.p)iacpramel asPtcaFcneOdpSata,rbd1sl.e,0a2(fsoparpp.mepxraPompFrOpialSet,eA.o,nM0e.9uw8lto7ircpkopminmpgoPsntFaenOndtSaArdA, and 8.12.3 Weigh approximately 100 mg o f 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 (pg/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOS fromLiver Page4 of 14 Page 78 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dapilpurtoext.h0e.5s0topcpkmso. lution with methanol for a working standard 3 solution o f 8.13 Surrogate stock standard preparation 8.13.1 WCei,gFh,3aSp0p3rHoxinimtoatae5ly05m0-l6v0olmumg eotfrsicurfrlaosgkataensdtarnedcoarrdd t1h-eHa.lc-tHua,l2w-Hei,g2h-tH. , 8..13.2 Brpipnmg .to volume with methanol for a surrogate stock of approximately 1000-1200 8.13.3 Prsetpoacrketaosaur1r0omgaltevowlourmkeintrgicstfalansdkaradn.dTbrrainngsfteor vapolpurmoxeimwaitthelmy e1t.0hamnol loffosruarrogate working standard o f 10-20 ppm. Record the actual volume transferred. 9.0 Sample Handling_____________________________________________________________ 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 o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Easxtmraectht otwdobl1a.n0kms.L aliquots of Milli-QTM water following this procedure and use 10.1.3 Easxmtraactrtitxwbola1n.0ksm. LReafleiqrutoots11o.f1l.i6v.er homogenate following th-is procedure and use 10.2 Matrix spikes 10.2.1 Pthreepaacrceuraancdyaonfatlhyezeexmtraatrcitxiosnp.ike and matrix spike duplicate samples to determine 10.2.2 Prerceepiavreedewacithhsepaikche usasminpglea ssaemt ple chosen by the analyst, usually a control liver 10.Z.3 AEcaxdlpidbeirtciatotenidoancloscnpucirkevneest.rmataioynbsewinilcllfuadlledinatnhdemmaidy-rfaalnlgienothfethloewin-irtaianlgceaolifbtrhaetiionnitciaulrve. 10.2.4 mPrienpiamruemonoefm2amtraixtrsixpiskpeikaensdpmeratbraitxchsp. ike duplicate per 40 samples, with a 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy o f the initial calibration curve. 10.3.2 Porfe1p0arsea,matplaesm. inFiomruemxa,monpelec, oifnatinsauminpglecasleibt r=at3i4on, fvoeurrifvicearitfiiocnatsiaomnspalerepperregpraoruepd and extracted. ' 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOSfrom Liver Page 5 of 14 Page-79- 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 10.3.3 Pprreeppaarree tehaechincitoinatlicnuurivneg. calibration verification from the same matrix used to 10.3.4 The expected concentrations will fall within the mid-range of the initial ttcohhanaellinybinr5tiahtptieiapollbnocw-aculie1rbn0vrd0ea.0toiopAfnptdhbcde)ui.ctriavoleni.barTlashtpiioiskniescsunmrevcaeeys(sbfaoerryineixcfalutmhdepedlaent,ha5alyptspftabmll-uins1t0tqh0uepalpnobtwi,t-arrtaaetnhugesreinogf 11.0 Calibration and Standardization_____________________________________ 11.1 Prepare matrix calibration standards 11.1.1 mWLesigMh ialplip-QroTMximwaatteelry. 4G0rigndotfoliavehroimntoogaen2e5o0ums sLolNutailogne.ne bottle containing 200 11.1.2 Iraft4io0. g is not available, use appropriate amounts o f liver and water to ensure a 1:5 11.1.3 Rcoenfceernttora1t3io.0ntoofcsaollciudlalitveetrhteisascuteuadlisdpeenrssietyd oinf l1iv.0ermhLomofohgoemnaotgeeannadtethseolution. 11.1.5 Ahshodamdkoi1nggmenbLeeotowufsehesonomluaoltigiqoeunnoaitntsew1t5ohmaileL1p5crmeenpLtarrciifenunggteraitftuuobgtaeelst.oufbeei.gRhete-seunsp1emndL saolliquutiootns boyf 11.1.6 Two 1 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 o f this section, to spike, in duplicate, two standard curves, for a total o f eighteen samples, two matrix blanks, and two method blanks. 11.1.8 RwehfiecrhtloisvtsaltihdeatwioonrkrienpgorrtasnEgTesSa-n8d-6t.h0eaLnidneEaTr SC-a8l-ib7r.0a-tVio-n1RoarnAgett(aLcChmR)efnotrB, calibration curves. 11.1.9 UstasendAatrtdasc. hRmeefenrttCo 1as3.a0ntoaidcailncuclaaltceualacttiunagl tchoencceonntcreanttiorantsioonfsPoFfOthSeiwn ocrakliibnrgation standards. 11.2 Tpsuporbre-oalgOcahOtewOwopropkrbikn.igngstsatnadnadradr,dbfloarntkh,eocrocnocnetnintruaitnigonvetoriffiaclal twiointh,iandtdheapcparliobprraitaitoenacmuorvuentraonfge 5 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOS fromLiver Page 6 of 14 Page 80 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 11.3 sEtaxntrdaacrtdssptiokeedstalibvleirshhoeamcohgiennitaitaelscfuorlvloewonintghe12m.1a4s-s1s2p.2ec5troofmthetiesrm. ethod. Use these Table 1 Approximate Spiking Amounts for Calibration Standards Working 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 pi 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 Cpuetrafpoprmroexdimquaticeklyly1, ngootfallilvoewriunsgintghealdiviessretcotitnhgawsc.alpel. This part of the procedure is best 12.3 Weigh the sample directly into a tared 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 T2ak2e. the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP- 12.10 wCaeipgthhte, lsiavmerpIlDe ,andadtveoarntdexafnoarly1s5t isneictoianlds.s. Label the sampule vial with the study number, 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOS fromLiver Page 7 of 14 Page 81 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 12.11 Pipette 1.0 mL, or other appropriate volume, of homogenate into a 15 mL polypropylene cveianlt.rRifuefgeerttuobaet.tLacahbeedl twhoerckesnhtereiftufgoer tduobceuwmietnhttinhge tidheenrteimcaaliinnifnogrmstaeptiso.n as the sampule 12.12 mPeipthetotde btwlaonk1sm. L aliquots o f Milli-QTM water to ce;ntrifuge tubes. These will serve as 12.13 sStapnikdaeradllassadmepsclreisb, eindcilnudseincgtiobnla1n1k.s2.and standards ready for extraction with surrogate 12.14 ocSofpntihtkianetuesieanccgthicomanla,ibtfirorarxttiwohniethcstaatlhnibedraaarptdiposr.nopcuriravtee astmanoduanrtdos.f sAtalnsdoaprrdepasardeemscartirbiexdsipnik1e1s.1a,nodr Table 1 12.15 sVamorptelexsmfoirx 1th5esestcaonnddasr.d curve samples, matrix spike samples, and continuing calibration 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.17 To each sample, add 1 mL 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-butyl 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 mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mL of the organic layer to the fresh 15 mL 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 FAiltttearchinato0a.21p.5mmnLylgolnasms easuhtofvilitaelrotrolaow3 -cvcosluymrinegaeuatonvdiatrlawnhsfeenr tnheecessasmarpyl.e to this syringe. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst! 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 ExtractionofPFOS fromLiver Page 8 of 14 Page 82 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 13.0 Data Analysis and Calculations__________________________________________ 13.1 Calculations: 13.1.1 teCnalsceuplaartaeteth1e.0avmerLagaeliqduenotssitoyfohfotmheoglievneartheo. mogenate by recording each mass o f Average density (mg/mL) = Average mass ('mg') of the aliouots 1.0 mL aliquot 13.1.2 dCisaplecrusleadtestohliedatmisosuunetpoerf lmivLero(fmhgo)mpoegre1n.a0temsLushpoemnsoigoenn)autsein(ogrtchoenfcoelnlotrwaitniogn of equation: g of Liver x A(vgeorfaLgeivdeern+sitgy*o foWf haotmero)genate (mg/mL * refer to 13.1.1 for details. 13.1.3 sCtaanldcaurldasteuascintugatlhceonfoclelnotwraintigoneqsuoaftPioFnO: S and other fluorochemicals in calibration uL o f StmangdLarivdexr/C1omncLenhtroamtioognentuagte/*mL ) = oFfinPaFlOCSoninceLntirvaetrion (pg/g or mg/kg) referto 13.1.2 for details. 14.0 Method Performance_______________________________________________________ 14.1 TspheecmifiecthMoDd Ldeatencdtiloimn iltimo fitq(uManDtLita)tiisonan(LalOytQe )anvadlmueastr(irxefsepretcoifAict.taRcehfmeretnotsMBDaLnrdepCo)r.t for 14.2 Tthheeqfuoallloitwyionfgthqeuaelxittyraccotniotnroalnsdamanpalleyssaisr.e extracted with each batch of samples to evaluate 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and . precision o f the extraction. 14.23 oCfotnhteiniuniintigalccaalilbibrraatitoionnvceurirfviec.ation samples to determine the continued accuracy 14.3 Refer to section 14 o f ETS-8-7.0 for method performance criteria. 15.Q Pollution Prevention and Waste Management_______________________ - 15.1 hSiagmh pBlTe Uwacsotnetiasindeirssp,oasnedd uinsebdioghlaazsasrpdipceotntteawinaesrtse, ifsladmismpoasbelde sionlbvernokt ewnagstlaesiss cdoinsptaoisneedrsin located in the laboratory. 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOS fromLiver Page 9 of 14 Page 83 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 16.0 Records_____________________________________________________ ;________ . 16.1 oCroimncplluedteetihnethexet3ra-rcitniognswtuodrykbshinedeetra,ttaascahpepdrtooprthiaitsem. ethod, and tape in the study notebook 17.0 Tables. Diagrams. Flowcharts, and Validation 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 References_________________________________________________________________ 18.1 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l. 18.2 AMDT-EP-22, "Routine Maintenance o f Ultra-Turrax T-25" 18.3 FACT-M-1.1, "Extraction o f PFOS or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 19.0 Affected Documents___________________________________________________ 19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions RNuevmisbieorn. Reason For Revision ReDvaistieon 3M Environmental Laboratory ETS-8-6.0 ExtractionofPFOS fromLiver Page 10 of 14 Page 84^ 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Studv # Surrogate Std Matrix approx, ppm Box # actual ppm Wk/Dav # Date SpikedC/ACnValyst MS MSD FC Mix Std approx. 0.5 ppm actual ppm # FCMix Std approx. 5 ppm actual ppm # FC Mix Std approx. 50 ppm actual ppm # Comments . -------------- ------------- . - ------------ .- - Blank Liver Homogenate: Std# Liveramount" Liver Extraction Method : e- Date & Initials Soike surrogate and Standard mix. Vortex 15 sec. Pipette 1mLof Liver Solution .. Pipette lmLoft0.5M TBA.pHlO. pH - Std.# Pipette 2mL of0.25 NapCOy0.25M NaHCOi Buffer Std. # Dispense 5ml of Methyl-t-Butyl Ether TN-A-. Shake 20 min. Shaker Speed .... Centrifuge 20-25 min. Centrifuge Speed . RPuetmoonvNe aitr4oemeLn aliauot of organic layer Evaporator to drvness _________________________ ___________________________________ _________ Evaporator Temperature . Add l.OmL ofMethanol TN-A- VFCioltroetnerxtu.s3Ci0nagsle.acV.3ecrciBfi-cDatsiovnrisnguesewdiththae0s.a2mume mSRaItrfiixltearsifnotor athuetosstaamnpdlaervd-i-ac-lu--r-v-e--.-------------------------------------- --------------------.------ ------------- Attachment B: MDL/LOQ Values . ETS-8-6.0 ExtractionofPFOS fromLiver Page 11 of 16 3M Environmental Laboratory Page 85 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 MDL/LOQ values for rabbit liver Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 8.45 26.9 3 0 p p b - 1200 ppb PFOSA 3.50 11.1 12 ppb - 1200 ppb PFOSAA 24.6 78.3 30 ppb - 1200 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M556 82.3 262 60 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-l for further information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Compound: PFOS______________________________________ _________ ________ Liver matrix Prepared range of (psptba)nd(nagr/mdsL) Range of average (ppbc)u(rnvge/mL) LCR. fto'riu^; Range of vave`cifrv^:i; low std LCR from low tfdV . Range of high std (ppb)`(ng/mL)"' (ppbc)u(rnvge/mL) :(p. pbc)'(rbvg/eTri`L)v (ppbc)u(rnvge/mL) LCR from; higfr;std (pp:bc)u(rnVge/ml..) ; Rabbit 6.19-1237 12 -1200 :t2 V l2 0 p S 6 -3 0 0 :12'?1Q0V:, 60 - 1200 . \& f,- 1200 Compound: PFOSA Liver matrix Prepared range of (psptba)nd(nagr/dmsL) Range of average (ppbc)u(rnvge/mL) .avte".'*cu*r'-v*ed'::". (ppb) '(ni/mL) Range of low std (ppbc)u(rnvge/mL) HLGR/from:: Range of 1 Towstd ,' -- high std /.(.P*p`-b")cm(rytge/r*P|L)^ (ppbc)u(rnvge/mL) *:XhCigRhfrsotdm: ' (pp''cbu)(rnvga/it):;: Rabbit 6.19 -1237 12 - 1200 12,1200;1 12 - 300 ; 12-300 60 - 1200 '6 0 - 1200 . Compound: PFOSAA Liver matrix Prarenpgaeroedf ' Range of average (pSptba)nd(nagr/dmsL) (ppbc)u(rnvge/mL) Rabbit 6.16- 1232 12-1200 LGR.from. Range of LGRfrbm Range of ave curve (ppb) (ng/mL) , low std (ppbc)u(rnvge/mL) low std;.. : high std (ppbc)u(rngv/em.L) (ppbc)u(rnvge/mL) 30-1200 30-900 60-900 N/A LCR from, high std (ppbc)u(rnvge/mL) ' N/A Attachment B: MDL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 12 of 16 Page 8& 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Compound: EtFOSE-OH Prepared Range of Liver matrix range of average (psptba)nd(nagr/mdsL) curve (ppb) (ng/mL) Rabbit 6.17-1235 31 -900 LCR from ve curve (ppb) (ng/mL) 31-900 Range of low std curve (ppb) (ng/mL) N/A LCR from low std (ppbc)u(rnvge/mL) N/A Range of high std (ppbc)u(rnvge/mL) N/A LCR from high std (ppbc)u(rnvge/mL) N/A Compound: PFOSEA Prepared Range of Liver range of average matrix (psptba)nd(nagr/dmsL) (ppbc)u(rnvge/mL) Rabbit 6.17-1235 31 - 1200 LCR from ave. curve- (ppb).:(ng/mL) 31 - 1200- Range of low std (ppbc)ur(nvge/mL) N/A LCR from Range of (pplboc).uwr(nvsget/d.mL) high std (ppbc)u(rnvge/mL) N /A - ' N/A LCR from high std (ppbc)u(rnvge/m.-L) . N/A ' Compound: M556 Prepared Liver range of matrix (psptba)nd(nagr/mdsL) Rabbit 6.17-1235 Range of average (ppbc)u(rnvge/mL) 31 -1200 LCRifroni' Range of LCRfrom -: Range of LCR from avecuryi (ppb) (ng/mli)., low std (ppbc)u(rnvge/mL) Tow stdi. \,pb.c)u.(rhtgi/m'.L) high std (ppbc)u(rnvge/mL) high std (ppbc)u(rnvge/m. L).: 6 0 -20K.': N/A v - ' N//L7 ' ' N/A . ' N/A ` ' r Attachment C: Standard Calculations ETS-8-6.0 ExtractionofPFOS fromLiver 3M Environmental Laboratory Page 13 of 14 Page fr7- 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Ion Pair Standard Curves- Tissue PArneaplydtae(tse)(:s): Sample matrix: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FSuCrrmoigxasttedstadpparpopxr.o5x0. .010p0pmpp:m: SEtqaunidpamrdenntunmubmebre:r: FBinlaanl ksollivveenr/tidaenndtiTfiNer:: Actual concentrations of standards in theFC mix PFOS Std cone ug/mL PFOSA Std cone ug/mL PFOSAA Std cone ug/mL EtFOSE Std cone ug/mL PFOSEA Std cone 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 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 5.0 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 5.00 5.00 50.0 5.00 5.00 5.00 5.00 50.0 50.0 L/s O o Ti M556 Std cone ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 50.0 Std cone ug/mL All AU Am't spiked Density mL 0.002 0.1g67 0.004 0.167 0 .0 1 0 0.167 0.020 0.167 0.040 0.167 0 .0 1 0 . 0.167 0.020 0.167 0.030 0.167 0.004 0.167 Calculated concentrations of standards in the sample matrix PFOS Final cone n5g.9/9g 12.0 29.9 59.9 120 299 599 898 1198 PFOSA Final cone n5g.9/g9 12.0 29.9 59.9 120 299 599 898 1198 FPiFnOalScAonAe ng/g 5.99 12.0 29.9 59.9 120 299 599 898 1198 EtFOSE Final cone n5g.9/9g 12.0 29.9 59.9 120 299 599 898 1198 PFOSEA Final cone n5g.9/9g 12.0 29.9 59.9 120 299 599 898 1198 M556 Final cone ng/g 5.99 12.0 29.9 59.9 120 299 599 898 1198 Std cone ng/g Surrogate Std cone ng/mL too Surrogate Final cone ng/mL 0.500 All Am't spiked mL 0.005 ValidMBRLaooaRitvbnvaebiketndrieteyran' gesEEE--ss5sttt-aiii1mmmPp0Faaa0ptttO0eeersssSpoooopxnnnbilllmyyy,,,auuusssteeee5rrrcaa-aP1obbbF0bbbnO0iiicttt0SvevvpAaanapllluutubreeessas...tion5Ps-1F0O0S0ApApb E5t-F1O00S0Ep-OpbH 5-1P0O0A0Appb 5P-1F0O0S0EpApb . . Attachment C: StandardCalculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 14 of 14 Page 8fr 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 3M E nvironm ental Laboratory M ethod Analysis o f P otassium Perfluorooctanesulfonate or O ther Fluorochem icals in Serum Extracts U sing H PLC -Electrospray/M ass Spectrometry Method Number: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: Laboratory Manager -- Group Leader b /z .--------------- TeCchXntiiciAal RCellvAiTeUwMe_r_______ ^________________________________ Adoption Date: 03/01/99 Revision Date: Date Date onhufoi Date 1.0 Scope and Application_______________________________________________________ 1.1 uSscionpgeH: PTLhCis-emleectthroodspdreasyc/rmibaesss tshpeecatnraolmyseitsryo.f serum extracts for fluorochemical surfactants 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 tMheatvrailciedsa:tioRnabrebpito,rrt.at, bovine, monkey, and human serum, or other fluids as designated in Word 6/95 ETS-8-5.1 Analysis ofSerumExtractUsing ES/MS Page 1of9 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 2.0 Summary of Method_________________ ______ _______________________________ __ 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum or Tothheeranflauliydssi,s uissipnegrfHoPrmLCed-ebleycmtroosnpitroaryi/nmgaasssisnpgelcetrioonmecthrayr,aocrtesriimstiilcaor fsyaspteamrticauslaaprpropriate. fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. tAhdediidteionntiatlylyo,fsaamcopmlepsomunady bbey adnetaelcytziendgudsaiungghatetraniodnesmomf tahsespsapreecntrtoiomne.ter to further verify 3.0 Definitions___________________________ :____________________________________ 3.1 PtsiAneyrcttsemehtsrensofmuiasqrcpseuehasec.elshlrooeTiwcmchcPeiufcsorraerselisvaInsatocurnalriutioezmduaIesotoisbmonpuniheztte(aahArtroiiPecodcnpnsIoro)(et,AfslTsiiPoumhInrei)eirt:zem(aTdi.toehitso.oep,n:rMnEaboyilytce,urcuetonttrmcidol.iesazrpTisnrashagQveyvauiIacoaourntnituiorizmzouaast)Iti.Iisoootnnruiprp(ErcleoeSscqI,e)ups,asrAdoitnrbmuetpsho,oselapsenehderic 3.2 pTErhleeescssteurrocehs,pawrrghaeeydrIedobrnyoipzioalenttissoinanr(esEoplSruo,tdEiouSncIea)d:rebatymratehnteshfoeardprpeoldifctiaootnitoihzneaotgifaosanpsptherarosfnoegrvmeilaeedctitnartiycaacthlmfaioresglpdehd. edrricoplets. 3.3 dTdMspeehttaeeeecsccAisttfoeiPScdrIps.f.erQcAaIutgoramsnotitsnmernogaetlrIaeteIrtMisytore,niSlMpeiclmneatfiasqovsyruemaSlbydpeardteeuiicomsptncropr.olielmmosyiyenetsdaetterfemod(rMsbioaySrn)em,deTaeqsatusenictpdtopieocemnhdaoMwrrgiaatehssrseaqrStuiipoaesde(crm(utMr/pzoSo)ml/eaMenmtdSea)rssufs(obMrsseemSlqe/oMcurteeiSnv)te:ly 3.4 Conventional vs. Z-spray probe interface: The latest models o f Micromass Quattro II ntdemcfrrlooieioarmptcmeilntcceprottaaoelqmytdnupipeabaar.natlodetcbtrTihewuebhepaliioetsrochuelwooegntsrihhsotteyhhemtasohsoptegeaeneommorcenttoehusaan.renl(ferpotiH,gotoZhuaost-fertwhstrapee1e,trr9vibcaope9ouynar8nt,ss)ieosysZuinasn-dttplsigyeiplefmirftrwzethaesuriry,teraohneecu".ttosgc,ZimI.hmt)-nhspaipeltoahtrmnorae-eryescnt"tyuothsoscnotuovaednsenmsfnpdoostarcif(tomoihon.newpaav.tela,ieroyZcnano-tti.nsniioopfTnntorh,raahemyclelccecasooaoptumimnronaipnptnyeoogirentn,meieasnnnittadttssiemadarerede 3.5 MIv(IMertarissciisproolnLemsyq.anusAxasdlSQlrovuufepatrwotstlriaeoorsneIysI:sattreSrimeypsslseti.meqmCiulauasrdro.rrfetuFwnpotaolryrlemeMMdoaerassessigsdLLneyteynadnxixlfshooasrresteMhWteahissnepsdmLeocyawinnfsixuc9aNo5lpTsaepnrUeadcstieWiforinc'isntooGdfoutthwhideesesNi)en.TsQtr4uu.a0mtteront 4.0 Warnings and Cautions_____________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Uemseplcoayustiaonvowltiathgethoefvaoplptarogxeicmaabtleelsyf5o0r0t0heVporlotsb.e. When engaged, the probe 4.1.2 Wanhdecnlohtahnindgli.ng samples or solvents wear appropriate protective gloves, eyewear, ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 o f9 3M Page 90 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 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 HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences_____________________________________________________________ 5.1 Tstoormagineiomriazneyinptaerrtfeorfeinncsetsruwmheenntaatnioanlytzhiantgcsoammepsleins,ctoenfltoanctswhoituhldthneostabme pulseedorfoexrtsraamct.ple 6.0 Equipment__________________________________________________ ________ _ 6.1 mEqoudiipfimcaetniot nlisstiendtbheelroawwmdaatyabaes mmoetdhiofideddeinvioartdioenrst.o optimize the system. Document any 6.1.1 MeleicctrroomsparsasyQiuoantitzraotiIoIntrsiopulercqeuadrupole Mass Spectrometer equipped with an 6.1.2 HcoPm1p1a0r0tmloewntp, aunlsdeasuotlovseanmt ppluemr ping system, solvent degasser, column 7.0 Supplies and Materials___________ ;_____________________ ;_________________________ 7.1 Supplies 7.1.1 Hsyisgthemp)urity grade nitrogen gas regulated to approximately 100 psi (House air 7.1.2 HPLC 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 or equivalent 8.1.2 Meqiulliiv-aQleTMntw, aantedrm, aalyl wbaetperrouvsieddedinbtyhiasMmieltlhi-oQd TshOoCulPdlbues Msyisltlei-mQTMor owtahteerrvoerndor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 pTryeppiacraeldlydtuwroinmg tehtheoedxtbrlaacntikosn, tpwroocmedautrreix. bSlaeenkEsT, aSn-d8-e4i.g1h. teen matrix standards are 9.0 Sample Handling______________________________________________________________ 9.1 Farreesshtomreadtriinx csatapnpdeadrdaus taorveiaplrseporarceadpwpeidth1e5acmhLanceanlytrsiisfu. gEextturbaecsteudnsttial nadnaarldyssisa.nd samples 3M Enviionmental Laboratory ETS-8-5.1 Analysis ofSerumExtract Using ES/MS Page 3 of9 Page 91 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 9.2 Iafpapnraolxyimsisatweliyll 4beCd,eloaryaetdr, oeoxmtratcetmedpesrtaatnudraer,dusnatinldasnaamlypsilsescacannbbeepreerffroirgmereadt.ed at 10.0 Quality Control _____________________________________________________ _ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 eSaoclhvebnattbchlatnokds,emteremthionde bcolanntkams ainnadtmionatorirxcbalrarnykovsearr.e prepared and analyzed with 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Mrecaotrviexrsypeikffeicsiaernecpyr. epared and analyzed to determine the matrix effect on the 10.2.2 rMecaotrviexrsypfiokreedaucphliacnaatelystea.re prepared and analyzed to measure the precision and the 10.2.3 mAninailmyzuemaomfa2trsipxikspeiskpeearnbdamtcha.trix spike duplicate per forty samples, with a 10.2.4 Mrthaenatgirneiixotisfaptlhickeaeliinabinrtdaiatmliocanatlrciibuxrrasvptei.ioknAecdduduripvtlieio.cnaatel scpoinkceencotrnacteionntrsawtioilnl sfamllaiynftahlel imnitdh-eralnowge-of 10.3 Continuing Calibration Verifications 10.3.1 oCfotnhteincuailnibgrcaatiloibnracutirovne.verifications are analyzed to verify the c'ontinued accuracy 10.3.2 oAfnoanlyezpeear mbaitdc-hr.ange calibration standard after every tenth sample, with a minimum 11.0 Calibration and Standardization________________:____________________________ 11.1 Aa1v/nxea,rlanygozeteoftohfretcweedoxttsrhtaarcnotduedagrhmdzaceturrorixv, eusstsaiwnnigdlalMrbdeassppslLroiytotnrexdtoobraynoldtihnfeeoralrlsourweitgianrbgelseesaisoocnfhtw(syeat=reom.f eyx+trabc)t,sw. eTighheted 11.2 Istfatnhdeacrudrcvuerdvoee(sifnnoetcmeseseatrrye)qaunidrermeaennatsl,ypzee.rform routine maintenance or reextract the 11.3 urFErceaasoxgnlerairgbmtpeehruspaeosrtlipefilooo:tonnwhswweecesuchenuoiredgvrnfhevoaateccfitnco(tt5uehgnmrespoaipcpscfattbyhiilnniiwtbgggorhhtoa1eoctfn0ioqot0nhqun0ceuaecpansnupntttaribtrtvni)aate.datteiartToiraanndhtpghsispsetlfrroawronwtoxhidmliaallmenrr5vdaetesptdh.elpueslbycfoeut1foli0lannr1apaa0cpncl0byugtpeorepa,fobciafytnrtmaahattelahtyrysetitrebba,utnehgtdeeanadnnerectdtroehascelstuieanfrureavylaelr.to 3M Euvironn iei ital Laboratory ETS-8-5.1 Analysis of SerumExtract Using ES/MS Page4 of 9 Page 92 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 12.0 Procedures _______________________ ;____________________________________ __ 12.1 Acquisition Set up 12.1.1 aCfolfriiclaekcnqoaunmirseitnaurgst,inbaugntdMtotnyOpi-enDitAnhYesa-AmlacspqtluedisidgietiistoconrifpCytoeioannrtr-sso.almPapnleeln.uSmetbuerp, aasssaimgnpalemlisett.hoAds(sMignS) 12.1.2 MpTStSorIIoaoRR4csd9ssr(u.9LSecaiSytontnraeigovxoalneetGhmfaIerUoceranqtIghaDumRpoiEspdeeircntTcooitolOparincdtrikiDiomanntoAgeen)tiTmnhosfAorcaodasrMA.snmeCRIbsaf.QutMMitoUAt.noSInSfS/muMIeilaTntlSyIIstoOhcbinnaeeNsniAztcrafiocosutlqrimluoeuansceidutsnMediattdsliitloo.yaidonrScenecoeaaoelellsmneiMtancprptfoileopcoldrrrypmooeaampdalnro,taeiinaaoslgtsdnaedwnaaidntinitdodshenmtlaehlacetss MRM (Multiple Reaction Monitoring). . 12.1.3 Tstyanpdicaarldlys tahnedaennadlsytwiciathl baastecht oruf nexsterqaucteendcme batergiixnsstawnidtharadss.et o f extracted matrix 12.1.4 Samples are analyzed with a continuing calibration check injected after 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 iSannestat-rluyupsmttcehonentHsloiPdg1ebr1os0o0ak/p:apurtoopsraimatpelfeorraotpthtiemfaolllroewspinongsceo. nRdeictoiorndsaocrtuaatlccoonnddititioionnsstihnethe 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample =1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3M Environmental LdUuiatoiy ETS-8-5.1 Analysis of SerumExtractUsing ES/MS Page 5 of9 Page 93 3M M edical D epartm ent Study: T -6 2 9 5 .1 0 ; t, BACK TO MAIN Analytical Report: F A C T T O X -0 9 9 LR N -U 2400 12.3.3 tChheetcipk.thTehsetatiipnlsehssoustldeebl ecafplaitllwarityhant othjeagegneddoefdtgheesp. roIfbteh. eUtispeiasnfoeuynedpiteocebeto check unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 SaOpebtpsrHeorPxvLiemCdarptoeuplymlep1ts0tcomo"minOiunnt"ge.so.Suettothfethfelotwiptoof 1th0e-p5r0o0beu.LA/mllionwortoaseqauppilriborpartieatfeo.r 12.3.5 Taruorunnodnththeetinpitorof gtheen.prAobfein. eRmeaisdtjushstotuhlde tbipe eoxfpthelelepdrowbiethifnnoonmitrisotgiesnolbesaekrivnegd. 12.3.6 Tchhaenignestirnuomrednetrutoseosptthimesiezepatrhaemreesteprosnaste:the following settings. These settings may 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 plVmin 12.3.6.4 PHrPeLssCuries <o4p0er0abtianrg(cTohrirsepctalrya.)meter is not set, it is a guide to ensure the 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. 12.3.8 tParpinetdtihnetotutnhee pinasgtreu, mwietnhtiltosgp.arameters, and store it in the study binder with a copy 12.3.9 Uthseinagnathlyesicsroosfsb-fiololowgiccoaulnmteartreilceecst.rode in the ES/MS source is recommended for 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MbuattsosnL.ynExnsvuerresisotnarst, saenedaepnpdrosapmriaptleeMnuamssbLeyrninxcUluSdEeRs a'SllGsaUmIpDlEes).toPbreesasntahleyzsteadr.t 13.0 Data Analysis and Calculations______________ . _________________________ 13.1 Calculations: 13.1.4 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 C(pagl/cmulLa)t:e actual concentration o f PFOS, or other fluorochemical, in matrix tne of PFOS calc, from std. Curve x Dilution Factor) x 1 ue (Initial Volume of matrix (mLl + mL o f Surrogate Standard! 1000 ng Final Volume (mL) ETS-8-5.1 Analysis ofSerumExtract Using ES/MS Page6 of 9 3M EnvUui imbolai Laboratory Page 94 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACTTOX-099 LRN-U2400 14.0 Method Performance ___________________________________ ___________ _ 14.1 MMmaDettrLhioxadsnpDdeeLctiOeficQct.iovPnallLeuaiemsse.itse(Me EDTLS)-a8n-d4.L1,imAitttaocfhQmueannttitBa,tifoonr(aLlOisQtin) garoefmcuertrheondt,vaanliadlyattee,dand 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Sloowlveesnt tstbalnadnakrsd, minetthheodcablilbarnaktsi,onancdurmvaetrix blanks values are must be below the 14.3 Calibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 cMonatcreinxtrspatiikoenp. ercent recoveries are must be within 30% o f the spiked 14.5 Continuing Calibration Verifications 14.5.1 cCoonncteinnturiantgiocna.libration verification percent recoveries must be 30% o f the spiked 14.6 Ipafnercafrloyitrsemtr.ieaDdloioscntuetdmhieennsttyhasitlselmmacetatinhodondssaipnmerfpifloleersampraepnarncoepalrsyieazctetedioloonrgibosotnho'tekrm. aectt,iomnasinatsedneatnecremminaeydbbey the 14.7 Ifofodtantoataerdeotno btaebrleeps oarntdeddwischuensspederifnortmheatnecxet corfittehreiarhepaovret.not been met, the data must be 15.0 Pollution Prevention and Waste Management________________ _________________ 15.1 pSiapmetpteleweaxtsrtaecitswdaisspteosaenddifnlabmromkaenblgelsaoslsvceonnttiasindeisrps olosceadtiendhinigthhBe TlaUbocroantotrayin. ers, and glass 16.0 Records_____________________________________ '________________ ;______________ 16.1 haEinneataaecdlghyersprata.toigroenhgamennedethwraortdiet,dtesanfomorpnaletshtnueadpmyagem,eu:exsstttrhuaadcvtyieoontrhdpearftooejl,elocdtwilnuiuntimgonbineffrao,cratmocrqatu(iiiofsniatpiionpncllimucadebetlhdeo)e,dita,hnedr in the 16.2 Papripnrtotphreiattuensetupdaygefo, lsdaemr.plCe olipsty, tahnedseacpqaugiessitaionnd mtaeptehoindtofrtohme iMnsatrsusLmyennxt rtouninlocgl.ude in the 16.3 Pstloortethine tchaelisbtruadtyiofnoclduerrv.e by linear regression, weighted 1/x, then print these graphs and 16.4 Panridntstdoarteaiinnttehgersattuiodny sfuolmdemra. ry, integration method, and chromatograms, from MassLynx, 3M~[Environmental Laboratory ETS-8-5.1 Analysis ofSerumExtract Using ES/MS Page 7 of 9 Page 95 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Banadckloucpateiolenctorfobnaicckduaptaetloecatpropnroicprdiaattae.medium. Record in study notebook the file name 17.0 T a b l e s . D ia g r a m s . F l o w c h a r t s , a n d V a l id a t io n D a t a 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. ________________________ 18.0 R e f e r e n c e s _________________________________ ;_____________________________________________ 18.1 cFoAmCpTo-uMnd-4s.f1r,o"mExSterraucmtiofnoor fAPnoatlaysssiisuUmsiPnegrfHluPoLroCo-cEtaIencetsruolsfpornaayt/eMoarsOs tShpeercFtrluomoreotcrhyemical 18.2 EIoTnSiz-a9t-i2o4n./0M, "aOsspSepraetcitornomanedteMr QaiunattetnroanIEcetroipfltehqeuMadicrruopmolaesSs yAsttmemossp"heric Pressure 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. 19.0 A f f e c t e d D o c u m e n t s ____________________________________________________________________ 19.1 ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-EIectrospray/Mass Spectrometry" 20 .0 R e v is io n s _______________ ______________________ ;__________________________________________ _ NRuevmi1shieorn. pSSSSleeeeoccccttttttiiiiioooonnnnng 6111li.712n1...e11.22a.ACr2.Crvh4eelagaCrnrraglieagfesiredsciiafoofitrcfnioaotamwtnRinoooedanafctaHtosuadofrcPdnvsheloeFmdsl1ov,e0trennh0nRotesttBey1rsvs/attxtiamosenwimpAode.na.icrgodhmvtipanolguneoesnf,ttashr.eecuusrevde.for R04eD/v0ai2sti/eo9n9 3M Envtroi miei ilal Laboratory ETS-8-5.1 Analysis ofSerumExtract Using ES/MS Page 8of9 Page 96 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Laboratory Study# Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analyst: Date of Analysis/Analyst Group Sample# Concentration Dose ug/mL Initial Voi. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis ofSeramExtractUsing ES/MS . 3TJ1 Eiivtm niiiuiildl LdLiuialmy Page 9 of 9 Page 97 3M Medical Department Study: T-6295.10 Study #: FACT-TOX-099 BACK TO MAIN Analytical Report: FACT TQX-099 LRN-U2400 3M Environmental Lab --Method Modification Method: ETS-8-5.1 "Analysis ofPotassiumPerfluorooctanesulfonateor Other Fluorochemicalsin SeraExtracts UsingHPLC-Electrospray/Mass Spectrometry" Section modified: Effective date of modifications: 10.3.2,14.5.1, add sections 14.3.2-14.3.6 April 26,1999 Section 10.3.2______________________________ ________________________________ Method reads: 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one peT batch. Modify method to read: 10.3.2 Analyze a mid-range calibration standard at least after every ten samples, with a minimum of one per batch. Section 14.5.1_________________________________ ______________________________ Method reads: 14.5.1 Continuing calibration verification percent recoveries must be within 30% of the spiked concentration. ' Modify method to read: 14.5.1 At least one continuing calibration verification per ten samples must show a percent recovery within +/-30% of the spiked concentration. Section 14.3.2________________________________ ______________________________ Method reads: NA Modify method to read: 14.3.2 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. 3M Environmental Laboratory Page 98 BACK TO MAIN 3M Medical Department Study: T-6295.10 -A--n-a--l-y-t-ic--a-l--R--e--p-o--rt-:--FLARCNT-OTOSWX-B09"9 Study #: FACT-TOX-099 Section 14.3.3____________________________________________________ '______ Method reads: NA Modify method to read: 14.3.3 Calibration standards with peak areas less than 2 times the curve matrix blank should be deactivated to disqualify a datarange that may be affected by background levels of the analyte. Section 14.3.4_______________________________________________________________ Method reads: NA Modify method to read: 14.3.4 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: 14.3.5 A curve point may be deactivated if it deviates more than 30% from the theoretical value when the curve is evaluated over a linearrange appropriate to the data. Section 14.3.6____________________________________________________________ __ Method reads: NA Modify method to read: 14.3.6 A valid calibration curve must contain at least 5 active points. jC k Signature o f PAI and date ll/2-l/(r Signature o f Sponsor and date r i i> 0 SigLn%aturae o^f Study DTireCcto^r and date a 3 lh > -r,___________ 3M Environmental Laboratory Page 99 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 3M Environm ental Laboratory M ethod A nalysis o f P otassium Perfluorooctanesulfonate or O ther Fltjorochemicals in Liver E xtracts U sing H P L C -E lectrospray/M a ss Spectrom etry Method Number: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg Approved By: Y[Laboraftory MWager Adoption Date: f j 2X Revision Date: tf(\ 7 A ^ / r Date Group Leader Technical Reviewer 9-W /5? ) Date 0?//v/?9 Date 1.0 Scope and Application________ :_______________________________________________ 1.1 HScPoLpCe:-elTehctirsomspertahyo/dmiassfsosrptehcetraonmaleytrsiys. o f liver extracts for fluorochemical surfactants using 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation report. Word 6/95 ETS-8-7.0 Analysis ofLiver ExtractUsingES/MS Page 1of 10 3WtEi iviiuiiiiiental Laboratory Page 100 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 2.0 Summary of Method___________________________________________________ 2.1 THptadhenehPertaiLefsplocyCemrtzrim-enefedltgeluheduodcosrtbadriouynoodggsmcheptastaorceanntrrayeiinitb/soomduenrselaifsmntoshogsnemfasatpatahesensieca(nstPslgrsyeFoplslemOeeiscciSeottotre)nrfodaycmfn,plhuieoaaootrrrnereasr,oncimmtcttoehiir/oelfzianums=rtr.iitscch4yaeo9slr9ftsev.uamerpArfaiaadfrcsydttiaaitcthpnuioeptlsanrirodaepxlfellrtnyurit,aaoitstcreyato.emcodThfpfehalrmeeocsmiaocmmnalaalip,vylyosebusuriecnsuhdissiabnsyg 3.0 Definitions___________________________,________________________________________ 3.1 iAPtsneyrttcsemehtsrensofmuaisqrcpsueehasec.elshlrooTeiwmcchcPeiufcsorraerselisvaInsaotucrnalriuitoezmduaIesotoibsmnopuniehztte(aahArtroiiePcodncnpsIoro)(et,AfslTsiiPomuhInrei)eirt:zem(aTdit.oheitsoo.epn:nrMEobatyilyce,urcuenotttrdmicole.isarzpsTianrshagvQeyavuicIaoauorntunitiormizzouaa)stI.tiIisoootnnruiprp(EcrleeoSscqI,e)ups,asrAdoitrnbmuetpsho,osealpsenehderic 3.2 pETrhleeescssteurrocehs,pawrrghaeeydrIedobrnyoipzioalenttissoinanre(sEoplSruo,tdEiouSncIea)d:rebatymratehnteshfoeardprpeoldifcitaootnitoihzneaotgifaosanpsptherarofsnoegrvmeilaeedctitnartiycaacthlmafioresglpdehd. edrricoplets. 3.3 mMcThhaaaesrssAgssePSerpIlaeeQtccitoutirva(otemtmrd/ozee)t1te3racytntr,odiMrpsslueaabsqnssudeaqSaduprceeuoncplttllroiysoleimdomenetaetcecserstlels(.dpM.IeocSAnt)r,ssoTiamnrageenltesedereMliemscSteMimqvueaailsypyspbdSeeidpsecewmrciitmptrhloiontmywaeteodetdeqfrobur(ayMidomrSnua/psMsolSteo): danetdetchtieosne ofrraagnmieonntsmmayaybbe esealnecatlyedzeidnitnhethfeirssetcqounaddrquupaodlreu, pfroaleg.mented in the collision cell, 3.4 Conventional vs. Z-spray probe interface: The latest models o f Micromass Quattro II triple quadrupole (post 1998) utilize a "Z-spray" conformation. The spray emitted from a pdmnelrioearotceibntccreottoelmiydnsepaao.ntarttctThihebheoalogecruoeowngntihahetlhetathsopoeanetmrcehtoeeuan.rncfeoioH,gtnauhoeferwtraeae,trpvibeopeurnarttus,iosrZsinen-d.lsgiypfIftrwneharitrytehohnceutosg,cimhomthnpaievloatemnorneretstnituyothossnotuaaedsnmlspdcosaocft(nohiof.nweopv.reaemrZyna-aittsniitpooiortnnhna,ayeclitlccceioooasummnanippintmeoogrnne,eedannnttdss aarree compatible with other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation o f these Quattro IvinIesrttrsriiupomlneseq.nutAa(dlMlrvuiceprrooslimeosnayssssateQrmeusasi.tmtrCiolauIrrI.rterFnipotlrlyemMqouarasedsrLdueyptnaoxilleshMraesafWesrsLitnoydntohxweosmr9aM5nauasansldLsWypneixcniNdfiocTwtUosNtsheTre'4s.0 Guide). 4.n Warnings and Cautions______________________ - . ______________ 4.1 Health and Safety Warnings: 4.1.1 Uemsepcloayustiaonvowltiathgethoefvaoplptargoexicmaabtleelsyf5o0r0th0eVporlotbs.e. When engaged, the probe 3 M-Env ironmentalLa bo rato ry ETS-8-7.0 Analysis ofLiverExtractUsing ES/MS Page2 of 10 Page 101 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 4.1.2 Wanhdecnlohtahnindgli.ng samples or solvents wear appropriate protective gloves, eyewear, 4.2 Cautions: 4.2.1 pOrpeesrsautreetehxecseoeldvsen4t0p0ubmarp,sthbeelHowP1a1b00acwkipllrienssituiareteoafu4t0o0mbaatirc(s5h8u0t0dopwsin).. If the back 4.2.2 Do not run solvent pumps to dryness. 5.0 I n t e r f e r e n c e s ____________________________________________________________________________ 5.1 Tstoormagineiomriazneyinptaerrtfeorfeinncsetrsuwmheenntaatnioanlytzhinatgcsoammepsleins,cToenftlaocnt swhiathll tnhoet sbaemupsleedofrorexstarmacpt.le 6.0 Equipment__________________________________________________________ ;_________ 6.1 Emqoudiipfimcaetnitonlisstiendtbheelroawwmdaatyabaes mmoetdhifoideddienvioartdioenrst.o optimize the system. Document any 6.1.1 MeleicctrroomsparsasyQiounatitzraotiIoIntrsiopulercqeu. adrupole Mass Spectrometer equipped with an 6.1.2 HP1100 low pulse solvent pumping system, solvent degasser, column 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 HinPtLheCraanwaldyattiacal column, specifics to be determined by the analyst and documented 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8.0 Reagents and St a n d a r d s __________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent . 8.1.2 Milli-QTM 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 22a.m000mm0oMmniLuammMmialcloie-ntQaiutTMem. Pwaocauetetrarit,nemtsooixaluu2tni0ot0ni0l:amWllLesoivgloihdluasmpapreertrodixciismcsooalntvetealydin.0e.rS3tc0oo0rnegtaaitnrionogm temperature. ETS-8-7.0 Analysis ofLiver ExtractUsingES/MS Page 3 of 10 3M~Cn7Tronmental Laboratory Page 102 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 8.2 Standards 8.2.1 pTryeppiacraeldlydtuwroinmg tehtheoedxtbrlaacntkiosn, tpwroocmedautrriex. bRlaenfekrs,toanEdTeSig-8h-t6e.e0n. matrix standards are 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 Itefmanpaerlyastuisrwe,iollrbreefdrieglearyaetde,deaxttraapcptredoxsitmanadtealryd4sanCd, usanmtilpalensamlysaiys bcaenstboerepderaftorromoemd. 10.0 Quality Control___________________________________________ 10.1 Method Blanks and Matrix Blanks . '_________ 10.1.1 Seaoclvhebnattbchlatnokds,emteremthiondebcloanntkams, iannadtimonatorrixcabrlraynokvsear.re prepared and analyzed with 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Mrecaotrviexrsypeikffeicsiaernecpyr. epared and analyzed to determine the matrix effect on the 10.2.2 rMecaotrviexrsypfiokreedaucphliacnaatelystae.re prepared and analyzed to measure the precision and the 10.23 mAninailmyzuemaomfa2trsipxiskpeiskpeearnbdamtchat.rix spike duplicate per forty sample^. With a 10.2.4 rMthaeantgirneiixotisfaptlihckeaeliinabnirtdaiatmliocanatlrciibuxrrsavptei.ioknAe cdduduripvtlieioc.naatel scpoinkceencotrnacteionntrsawtioilnl sfamllaiynftahlel imnitdh-eralnowge- of 10.3 Continuing Calibration Checks 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy o f 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 o f sample extracts. Twheiegahvteedra1g/exo, nf otwt ofosrtcaenddtahrrdocuugrhvtehsewoirlilgbine,pulositntegdMbyaslsinLeyanrxroegrroetshseiornsu(iyta=blme xso+ftbw)a,re. 11.2 If the curve does not meet requirements perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 3M~Ei iviiumilei rtal Laboratory ETS-8-7.0 Analysis ofLiverExtractUsingES/MS Page4 of 10 Page 103 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 11.3 uFsoertphuerlpoowseesnodfoacfcthueraccayliwbrhaetnioqnucaunrtviteartianthgelrotwhalnevtehles foufllarnaanlygtee,oifttmheaystabnednaercdecsusarrvye.to rcrEeaaxglnairbgmeersapsotliifeoo:tnnhwwecuheciuregvnrhevtaecitno(t5egnmspoippsfttbhiinnitgggohto1ocf0oqt0nhu0ceaepnsnptttaibrtna)a.dtteiaTorandhpsispstfrarwonoxidmlialmrr5daesptd.epulbycet1oi0n1ap0cpc0bupropafbcayrnaaattlhtyretierb,tuhgtaeendnettrohaeltiefnoaeialr 12.0 P r o c e d u r e s ___________________________ ;________'______________ ____________________________ 12.1 Acquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Aletstseirgnofathsaemalpplhealbisettfsitlaenrtainmgewuistihnga MO-DAY-last digit o f year-increasing 12.1.1.2 Assign a method (MS file) for acquiring 12.1.1.3 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 RTaaspcopeqpaecruccrotiterspioaoirtmntiieaoeMtnateemommrnheeaittetshhoasoroediddsin.n.gcgAl)Isi.fcaMfkSoneoidStln/sIsMemoclanSeenictzihtniasoStsidtuIorRsnuiunmM(aStlehlionynedtgscAeloaecalrqlsIeeouaceniptsmepRidtrpieoolacopnloyorrcienadodgtine,nwtagarid)ontdlhodiprtmtaihMonaeneRsaSlsMlIttpRhores(4onM.d9mSuu9aclaottvsirpesioolenther fGraUgImDeEntTaOtioDnAinTfoArmAaCtQioUn ImSIaTy IbOeNcoflolercatdeddi.tiRoneafel rintfooMrmicartioomnaasnsdMMaRssMLy. nx 12.1.3 mTyaptriicxalsltyanthdearadnsa. lytical batch run sequence begins and ends with a set o f extracted 12.1.4 mSainfaotcmelnruipdteloveerdesrpaayorssetsseauinbnctlhhael.syaaznmeadplywlteei.tchSaaorrlcvyoeonnvtteirnbualaninndkgascrasehlinobourtaldctioobnnesviadenerarilfeyidczaesdtaimopneprilnieosjdebcicuteatdlmlysattayonbdeard 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 Saneta-luypsttchoenHsiPd1er1s00a/papurtoopsraimatpelfeorraotpthtiemfaolllroewspionngsceo. nRdeictoiorndsaocrtuaatlccoonnddititioionnsstihnethe instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 9 minutes . ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 5 of 10 Page 104 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACTTOX-099 LRN-U2400 12.2.2.4 Solvent ramp conditions Time MeOH 0.00 min. 1.0 min. 4.5 min. 6.5 min. 7.0 min. 9.0 mi. 40% 40% 95% 95% 40% 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 TIRorenifpiezlraettQoiouEnaTSdSoruu-9pr-co2ele4,".M0f,oa"rsOsmpSoeprreeacdttireootnamialesnt.derMFaititnetdenwainthceaonfAthtme MosipchreormicaPssreQssuuarttero II 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 tuChnheseatcitpkis.ftahTcehtosetratyiip,ndlseihssasosussltedemeblbeclafelpatihtllewaprityrhoabnteothajeangdegnrededpolefadtchgeeetshp.erIosfbtatehi.neUlteisspseissatnefoeeluycneadppitieolclaebretyo. check 12.3.4 Turn on the nitrogen. 12.3.5 hOepaetenrtsh. e tune page. Clicks on operate to initiate source block and desolvation 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/min or as appropriate 12.3.6.3 tOehxbepsteeilprlevodefdwtrhiotehpplnertoosbnceiotimrfonignoegnmoliuesattkoiisfnotghbaesretoripuvneoddftthhee tpiproobfe.thAe pfrinoebem. iRstesahdojuusldt be 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 Tchhaenignestirnumorednetrutoseosptthimesiezepathraemreesteprosnaste:the following settings. These settings may 12.3.7.1 Drying gas 250-400 liters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 12.3.7.3 HPLC constant flow mode flow rate 1 0 -5 0 0 pL/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 ETS-8-7.0 Analysis ofLiver ExtractUsing ES/MS Page 6 of 10 Laboratory Page 105 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 12.3.8 Ptarpinetdtihnetotutnhee pinasgter,uwmietnhtiltosgp.arameters, and store it in the study binder with a copy 12.3.9 MeCnladicskssaLomynnpsxlteavrneturbsmuiobtnteosrn,irniencfletuhrdeteoAsacapqllpursiaospmitripoialnetesCMtoonabtsresoLalynPnaaxlnyUezlesd(et.rh'iss Gmuaiydev)a.ryEanmsuornegstart and 13.0 Data Analysis and Calculations_______________________________________ 13.1 Calculations: 13.1.4 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 CEonxep.e-ctCeadlcCuolnatee.d Cone, x 100 13.1.6 Calculate actual concentrations in matrix (jxg/g): fag o f PFOS calc, from std. Curve x Dilution Factor') x 1 pg (Initial Weight of Liver fgl 1000 ng Final Volume (mL) 14.0 Method Performance_______________________________________________________ 14.1. MmanaedttrhLioxOdsQpDevecatieflucicte.iso.Rn eLfiemr itto(EMTDSL-8)-a6n.0d,LAimttaitcohfmQeunatnBtitfaotrioanli(sLtOinQg )oafrceumrreetnhtovda,liadnaatleydteM, aDnLd 14.2 Solvent Blanks, Method Blanks and Matrix Blanks 14.2.1 Sstoalnvdeanrtdbilnanthkes,cmaleibthroatdiobnlacnukrsv,ea. nd matrix blanks mu.st be below the lowest 14.3 Calibration Curves 14.3.1 The i2 value for the calibration must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within 30% o f the spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within 30% o f the . spiked concentration. 14.6 Ipafnercarfloyitrsemtr.ieaDdlioosctnuedtmhieennsttyhsaetlelmmaecttahinoodndsspainemrffpiolleremsarpaepnarcnoeapslryeizcaettdeio,lnoorgabroeothonekor.tamcteito,nms aaisndteentearnmceinmedaybybethe ETS-8-7.0 AnalysisofLiver ExtractUsing ES/MS Page 7 of 10 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 14.7 fIofodtantoateadreotno tbaeblreespoarntdeddiwschuesnsepderinfotrhmeatnecxet corfittehreiarehpaovret.not been met, the data must be 15.0 _P o l l u t i o n P r e v e n t io n a n d W a s t e M a n a g e m e n t ________ ___________________________ 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, ianntaelgyrsat.tion method, sample name, extraction date,, dilution factor (if applicable), and 16.2 Papripnrtotphreiattuensetupdaygef,olsdaemr.plCe olipsty, tahnedseacpqaugiessitaionndmtaeptehiondtofrtohme iMnsatrsusLmyennxt rtouninlocgl.ude in the 16.3 Pstloortetihnetchaelibstruadtiyonfoclduerrv.e by linear regression, weighted 1/x, then print these graphs and 16.4 Panridntstdoarteaiinnttehgersattuiodny sfuomldmera. ry, integration method, and chromatograms from MassLynx 16.5 ASutmtamchamrizeentdAatafoursianngesxuaimtapbllee sooffatwsaurmem(Eaxryceslp5re.0a+d)shaenedt.store in the study folder, refer to 16.6 Banadckloucpateiolenctorfobnaicckduaptaetloecatpropnroicprdiaattae.medium. Record in study notebook the file name 17.0 T a b l e s . D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a _____________________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18 0. R e f e r e n c e s _______________________________________________________________________________ 18.1 FACT-M-2.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 EIoTnSiz-a9t-i2o4n./0M, "aOsspSepraetcitornomanedteMr QaiunattetnroanIIcetroipfltehequMadicrruopmolaessSyAsttmemossp"heric Pressure 18.3 The validation report associated with this method is ETS-8-6.0 & 7.0-V -l 19.0 ,A f f e c t e d D o c u m e n t s ____________________________________________ ___ __________________ 19.1 ETS-8-6.0, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" ETS-8-7.0 Analysis ofLiverExtract Using ES/MS Page 8 of 10 3M Medical Department Study: T-6295.10 20.0 Revisions RNeuvmisbioern Reason For Revision BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 ReDvaistieon ETS-8-7.0 Analysis of Liver Extract Using ES/MS Page 9 of 10 Page 108 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Laboratory Study# Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: DDaattee ooff AExntarlaycstiiso/An/nAanlyaslyt:st: Croup Sample# Concentration Dose ng/g Initiael Wt. Dilution Factor Final Cone, ug/g Group/Dose: Taken from the study folder. SCaomncpelen#tr: aTtiaoknen(nfgr/ogm): thTeaksteundyfrfoomldtehre. 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 AttachmentA: Summary Spreadsheet ETS-8-7.0 Analysis ofLiver ExtractUsing ES/MS 3M Ertvtroiiinenlal Labuialuiy Page 10 of 10 Page 109 3M Medical Department Study: T-6295.10 Study #: FACT-TOX-099 BACK TO MAIN Analytical Report: FACT TOX-099 --------- r LRN-U" o ' 3M Environmental Lab --Method Modification Method: ETS-8-7.0"Analysis ofPotassiumPerfluorooctanesulfonate or Other FluorochemicalsinLiver ExtractsUsingHPLC-Electrospray/Mass Spectrometry" Section modified: Effective date of modifications: 10.3.2,14.5.1, add sections 14.3.2-14.3.6 July 22,1999 Section 10.3.2________________________________________________________________ Method reads: 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum o f one per batch. Modify method to read: 10.3.2 Analyze a mid-range calibration standard at least after every ten samples, with a minimum of one per batch. Section 14.5.1________________________________________________________________ Method reads: 14.5.1 Continuing calibration verification percent recoveries must be within 30% of the spiked concentration. Modify method to read: 14.5.1 At least one continuing calibration verification per ten samples must show a percent recovery within +/-30% of the spiked concentration. Section 14.3.2_________________________ ______________________________________ Method reads: NA Modify method to read: 14.3.2 TThhee fsiercsotncdur(vberaacnkdeaticncge)pctaalbibleracatiloibnrcautirovnecmhaecykbsesdheaallcbtirvaactkedetiufsianbstlreudmatean.tal drift affects the data. 3M Environmental Laboratory Page 110 BACK TO MAIN 3M Medical Department Study: T-6295.10 Study #: FACT-TOX-099 Analytical Report: FACT TQX-099 LRN-24 Section 1 4 . 3 . 3 __________________________________________________ ________ Method reads: NA Modify method to read: 14.3.3 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: 14.3.4 Low or high curve points may be deactivated to optimi: a linear range appropriate to the data. Section 14.3.5________________________________________________________________ Method reads: NA Modify method to read: 14.3.5 A curve point may be deactivated if it deviates more thin 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: 14.3.6 A valid calibration curve must contain at least 5 active points. Signature o f PAI and date Signature of Sponsor and date Signature ot Study Director and date 3M Environmental Laboratory Page 111 3M Medical Department Study: T-6295.10 Appendix D: Data Summary Tables BACK TO MAIN Analytical Report: FACTTOX-099 LRN-U2400 Table 6. LOQ Values for PFOS Used in FACT TOX-099 Analyses by Method and Usage Dates Method Effective Date LOQ Usage Dates Sera ETS-8-5.1 Liver ETS-8-7.0 4/26/99 7/22/99 pg/mL 0.0214 0.00426 pg/g 0.00529 0.0529 10/19/00 10/24/00, 11/7/00 11/1/00 12/21/00 NOTE: LOQ reported only for those data sets with values < LOQ. Table 7. Average Concentration of PFOS (pg/g) in Rabbit Liver by Dose Group Dose Group Mean PFOS in Liver Group 1 (Control) 0 mg/kg/day 0.239 Group 2 0.1 mg/kg/day 13.1 Group 3 1.0 mg/kg/day 133 Group 4 2.5 mg/kg/day 317 Group 5 3.75 mg/kg/day 416 NOTE: It is not possible to verily true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitative to 30% or greater. 3M Environmental Laboratory Page 112 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Table 8. Average Concentration of PFOS (pg/mL) in Rabbit Serum by Dose Group Dose Group Mean PFOS in Serum Group 1 (Control) 0 mg/kg/day 0.0690 Group 2 0.1 mg/kg/day 2.73 Group 3 1.0 mg/kg/day 23.8 Group 4 2.5 mg/kg/day 45.8 Group 5 3.75 mg/kg/day 88.9 NOTE: It is not possible to verify true recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitative to 30% or greater. 3M Environmental Laboratory Page 113 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Table 9. PFOS (pg/g) in Rabbit Liver by Individual Animal Group # Animal # Cone. PFOS pg/g Group 1 0 mg/kg/day Group 2 0.1 mg/kg/day Group 3 1.0 mg/kg/day 8553F 8554F 8555F 8556F 8557F 8558F 8559F 8560F 8561F 8562F 0.287 0.189 0.240 13.1 13.0 12.2 11.2 16.1 98.6 141 Group 4 2.5 mg/kg/day Group 5 3.75 mg/kg/day 8563F 8564F 8565F 8566F 8567F 8568F 8569F 8570F 8571F 158 297 360 295 432 401 436 346 466 NOTE: It is not possible to veritytrue recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrixspike studies, indicate that the data are quantitative to 30% or greater. 3M Environmental Laboratory Page 114 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Table 10. PFOS (pg/mL) in Rabbit Serum by Individual Animal Group # Animal # Cone PFOS pg/mL Group 1 8553F 0 .0 9 5 6 0 m g/kg/day 8554F 0 .0 4 1 1 Group 2 0.1 m g/kg/day 8555F 8556F 8557F 0 .0 7 0 3 2 .7 8 2 .9 2 8558F 2 .8 6 8559F 2 .6 4 Group 3 8560F 8561F 2 .4 4 22.1 1.0 m g/kg/day 8562F 2 5 .0 Group 4 8563F 8564F 2 4 .3 4 6 .5 2 .5 m g/kg/day 8565F 4 6 .3 Group 5 8566F 8567F 4 4 .5 100 3 .7 5 m g/kg/day 8568F 92.1 8569F 8 1 .6 8570F 88.1 8571F 8 2 .7 N O T E : It is not possible to verifytrue recovery of endogenous analyte from tissues without radio-labeled reference material. The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitative to 30% or greater. 3M Environmental Laboratory Page 115 3M M edical D epartm ent Study: T -6 2 9 5 .1 0 Appendix E: Data Spreadsheets BACK TO MAIN Analytical R eport: F A C T T O X -0 9 9 LR N -U 2400 3M Environmental Laboratory Page 116 BACK TO MAIN 3N1 Medical Department Study: T-6295.10 __ . -________ ___ __ . _ tA'ArCguTs-#T4U1X8--O01X2f Analytical R eport: F A C T T O X -0 9 9 I OKI I IO Anrs "' u" "u Study: Product Number(Tesl 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/Analysc DSaatme opflDeaDtaaRteaducdon/Analyst* RABBIT SERUM 418-012, Oral Developmental Toxicity Study of PFOS in Rabbits: 4 matrices T-6295 Rabbit Serum ETS-8-4.1 &ETS-8-5.1 Soup020199, Davey070799 Masslynx 3.4 See Below See Attachments See Attachments See Attachments 10/17/00 SAL 10/19/00, 10/24/00,11/07/00 KJH/MMH 10/23/00, 10/25/00.11/08/00 KJH/MMH Box#: 00-96 lot 171 Group D ose Method Blk Matrix Blk QC Group 1 Group 2 Group 3 Group 4 Group 5 Sam ple# 10170-H20 Blk-3 RRBB1SS011100711077-H00--2SS0eerrBaalBBk-ll4kk--34 RRBBSS1100117700-M-MSSD-2-25500ppppbb-3-3 88555543FF 8555F 88555576FF 8558F 8559F 8560F 8561F 88556623FF 8564F 8565F 8566F 8567F 8568F 8569F 8570F 857 IF E xtraction VoL m l. 11 1 1 l 1 1 11 l 1 1 i1 1 1 1 1 11 1 i 1 1 1 LOQ = Limit of Quantitation PFOS = Periluorooctanesulfonate Date Entered/By: 10/26/00.10/27/00,11/08/00 LAC Date Verified/ By: 11/08/00 KJH Purity Entered/Verified: 12/20/00 LAC. 12/27/00 mmh Surrogate PFOS Purity Verified Correction Factor NA NA 00..88664400 NA NA 00..88664400 NA NA NA NA NA NA NA 00..88664400 0.8640 NA NA NA 0.8640 0.8640 0.8640 NA NA 00..88664400 NA 0.8640 NA NA 0.8640 0.8640 NA NA NA 0.8640 0.8640 0.8640 NA 0.8640 NA 0.8640 NA 0.8640 NA 0.8640 NA 0.8640 a on 01/30/01. LAC 01/30/01 PFOS Dilution Factor 1 1 1 1 1 1 1 1 1 20 20 20 20 20 50 50 50 100 110000 200 200 200 200 200 PFOS Cone. ng/mL 0.00 0.00 0.00 02.5090 270 111 47.6 81.4 161 169 166 114513 557182 563 538 536 515 578 533 472 457180 F ilenam e (optional) DSO00O11100274002004 S001107021 DSO00O11100274002025 S001107023 SSS000000111000111999000111987 DD000011002244001187 D001024019 DD000011002244002210 DOOI024024 D001024025 D001024026 D001024027 D001024028 D00!024031 DO0I024O32 D001024033 D001024034 DD000011002244003358 C on cen tration of PFOS ug/m L or % Ree <LOQ (0.00426 ug/mL) <LOQ (0.00426 ug/mL) <LOQ (0.00426 ug/mL) <LOQ (0.00426 ug/raL) 104% 109% 000...000479105136 2.78 2.92 2.86 22..6444 2225..10 24.3 4466..35 44.5 100 92.1 81.6 8882..17 M ean PFOS ug /m L <LOQ (0.00426 ug/mL) <LOQ (0.00426 ug/mL) 107% 0.0690 2.73 23.8 45.8 88.9 RSD Std. Dev. M S/M SD RPD NA NA 4% 0.3092.753 7.10 0.194 6.25 1.49 2.40 1.10 8.45 7.51 ik U'o , ETS-'8J-5r.I Excel 97 3tfl Environm ental Laboratory TOX-099-Sera012-3B 01/30/2001 3:56 PM Page 117 BACK TO MAIN 3I\|I Medical Department Study: T-6295.10 ArAgUus-#i U418--0W12 Analytical Report: FACT TOX-099 Study: Product NumberTest Substance): Matrix: Method/Reviskm: Analytical Equipment System Number Instrument Softwarc/Vcrsion: Date of Extrac[ion/AnaJyst: Date of Analysis/Analyst Date of Data Reduction/Anxlyst: Sam ple Data 418-012, Oral Developmental Toxicity Study of PFOS in Rabbits T-6295 Rabbit Liver ETS-8-6.0 &. ETS-8-7.0 SMoausps0ly2n0x1939.4. Davey070799 10/16/00 SAL/KJK 10/25/00,11/01/00, 12/21/00 MMH 10/26XX), 11/02/00, 12/22/00 MMH/KJH Filename: R-Sqaared Value: Slope: Y-IntercepC Box #: 00-095 See Below See Attachments See Attachments See Attachments RABBIT LIVER lot 171 Group Dose Method Blk Matrix Blk QC Analyzed 12/21/00 AnalyzeQdC11X11/00 Group 1 Sample# 1016O-H2O Blk-3 10160-H20 BIk-4 RBL10160-Liver Blk-3 RBL10160-Liver Blk-4 8553F-MS-250 ppb-3 85S3F-MSD-250 ppb-3 8553F-MS-250 ppb-3 8553F-MSD-250 ppb-3 8553F 8554F 8555F Surrogate Verified NA NA NA NA NA NA NA NA NA NA NA Initial WL t 1.0000 1.0000 1.0000 1.0000 1.0074 1.0074 1.0074 1.0074 1.0074 1.0474 0.9981 Total Mass of Liver NgA..'........ NA NA NA NA NA NA NA NA NA NA PFOS Purity Correction Factor 0.8640 0.8640 0.8640 0.8640 NA NA NA NA 0.8640 0.8640 0.8640 PFOS Cone 0.00 0.00 0.00 0.00 804 698 608 584 335 229 278 PFOS Dilution Factor 1 1 1 I 1 1 1 1 1 1 1 PFOS Cale Cone 0n.*0/0i 0.00 0.00 0.00 465 360 271 247 287 189 240 Group 2 Group 3 Group 4 Croup 5 8556F 8557F 8558F 8559F 8560F 8561F 8562F 8563F 85 64F 8565F 8566F 8567F 8568F 8569F 8570F 8571F NA 1.0151 NA 0.8640 308 NA NA NA NA 0.9931 1.0386 1.0152 NA 0.8640 NA 0.8640 NA 0.8640 299 293 264 1.0263 NA 0.8640 382 NA NA NA 1.0586 1.0014 1.0398 NA 0.8640 NA 0.8640 NA 0.8640 242 328 381 NA NA NA 1.0057 1.0223 0.9907 NA 0.8640 NA NA 0.8640 0.8640 345 426 338 NA NA NA NA NA 1.0137 1.0494 1.0268 1.0328 0.9820 NA NA 0.8640 0.8640 NA 0.8640 NA 0.8640 NA 0.8640 506 487 519 413 529 50 50 13100 13003 50 12193 5500 11246 16069 500 98561 500 141282 500 158417 1000 296674 1000 360449 1000 294686 1000 431642 1000 400623 1000 436300 1000 345684 1000 465531 Original PFOS LOQ (30.6 n/g, 6.12 ng/g, 61.2 ng/g) updated to reflect purity information on 01/30/01. LAC 01/30/01 LOQ = Limit of Quantitation PFOS = Perfluorooctanesulfonate * QC analyzed ** QC analyzed 1112/0/211A/X0)0aarreeininssuuppppoorrttooffdGilruotiuopns1 for Group data. The 2 - 5 data and were within 8553-MS-250 ppb-3 spike 30% was criteria. above the 30% criteria, therefore, the group 1 data may be biased high. LAC 01/02AU Dale Entered/Analyst: 10/27/00, 11X13/00, 12/28/00 LAC Date Verified/Analyst; 11/06/00 KJH Purity Entered/Verified: 12/20/00 LAC, 12/27/00 mmh Filename D001221016 DOO1101005 D001221017 D001101006 D001221029 D 0 0 1221030 D01101042 DQO! 101043 D001221031 DOO1221032 D001221033 D001101018 D001101019 D001101020 D001101021 D 0 0 1101022 D001101025 D001101026 D001101027 D001101028 D001101029 D001101032 D001101033 D 0 0 1 101034 D 0 0 1 101035 D 0 0 1 101036 D 0 0 1 101039 Concentration Mean of PFOS PFOS ue/e or % Ree <LOQ (0.0529 ug/g) ** u&/e <LOQ (0.00529 ur/r) <LOQ (0.0529 ug/g) <LOQ (0.00529 156% ur/r) * ** ** <LOQ <LOQ 120% 138% 91% 83% * 87% 0.287 00..128490 **** ** 0.239 13.1 13.0 12.2 11.2 16.1 13.1 98.6 141 158 133 297 360 295 317 432 401 436 346 466 416 RSD Std. Dev. MS/MSD RPD NA NA 26% 9% 20.5 0.0490 13.8 1.81 23.2 30.8 11.8 37.4 10.9 45.5 ETS-8-7.0 Excel 97 3fil Environm ental Laboratory TOX-099-LiveiOl 2-3B 01/30/2001 3:55 PM Page 118 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT TOX-099 AR (ng/mL) x DF x FV (mL) x l.Q pg x PC= Reported Concentration (pg/m L) EV(m L) 1000 ng Calculation Used for Group 3, Animal ID 8561F 512 ng/mL x 50 x 1 mL x 1.0 ug x 0.864 = 22.1 pg/m L 1 mL 1000 ng AR-- Analytical result from MassLynx summary DF-- Dilution factor FV-- Final extract volume (1.0 mL unless otherwise noted) EV-- Volume o f sera extracted PC-- PFOS purity correction factor (86.4%) Formula Used for Liver Analyses in Study FACT TOX-099 A R (ng/g) x 3 cu rve(1) x DF x l.Q pg x PC = R eported C oncentration (pg/g) 9 sample 1000 ng (1) 3 curve is assumed to be: 1 g liver 5 mL H20 Calculation Used for Group 3, Animal ID 8561F 241.52 ng/g x l g / 5 m L x 5QQ x 1.0 gg x 0.864 = 98.6 pg/g 1.0586 g/ 5 mL 1000 ng AR-- Analytical result from MassLynx summary 3 curve-- Density o f the liver standard curve, assumed to be lg liver/ 5 ml water 9 sample-- Density o f the liver sample (g sample/ 5 mL FLO) DF-- Dilution factor PC-- PFOS purity correction factor (86.4%) 3M Environmental Laboratory Page 119 3M Medical Department Study: T-6295.10 Appendix G: Interim Certificate of Analysis BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 3M [Environmental Laboratory Page 120 BACK TO MAIN 3M M edical D ep artm ent Study: T -6 2 9 5 .1 0 Analytical Report: F A C T T O X -0 9 9 LR N -U 2400 Centre Analytical Laboratories, Inc. 3 0 4 8 R esearch D rive S tate C ollege, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS Centre Analytical LabRoervaitsoiornies1(C9O/7/A00R)eference #: 023-018B 3M Product: PFOS, Lot 171 Reference#: SD-009 Test N_a_m__e__________________ PuritSyp:ec8if6ic.4at%ions Purity1 Result 86.4% Appearance IMdeetnatlisfic(NIaCtMiPoRn/MS) White Crystalline Pcwder . -. ' Conforms Positive 4236751....... MMNPCISrooaioaacdtlnngakciusnigeusamelimsuniemusme2 Total % Impurity (NMR) T(LoCta/lM%S)Impurity 423651...... 600001......050003051005352747 wwwwwwtttttt......//////wwwwwwtttttt......%%%%%% 7. <0.001 wt./wt.% 1.00 wt./wt.% 10.60 wt./wt.% r ^ . Total % Impurity (GC/MS) None Detected PROelAatAed Compounds - . Residual Solvents (TGA) Purity by DSC ............... .. . . '. . . Inor1g.aniCchAlonriiodnes (IC) 2. Fluoride 34.. NBritormatiede 56.. NPhitorsitpehate 7. Sulfate4 Orga2431n.... icTNPHAFFFFcPAPBiAdAAs5 (IC) Elem1e. ntCalaArbnoanlysis": 4235.... NFHSuliyutldrfouorrrogingeenen 21.. TThheeoorreettiiccaall VVaalluuee == 01%7.8% 435... TTThhheeeooorrreeetttiiicccaaalll VVVaaallluuueee === 065%0.9%5% 0.30 wt./wt.% None Detected Not Applicable1 21.. <00..20715wwt./tw./wt.%t.% 43.. <<00..000490 wwtt..//wwtt..%% 56.. <<00..000076 wwtt..//wwtt..%% 7. 8.82 wt./wt.% 4231.... <<<000...111 wwwttt...///wwwttt...%%% <0.25 wt./wt.% 23451..... 501110..0267...11490w48wwtwwtt.../tt//www..//wwttt...%tt%%..%% COA023-018B Page 1of3 3M e nvironmental Laboratory- Page 121 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Centre Analytical Laboratories, Inc. k 3048 Research Drive State College, PA 16801 ^ Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B Date of Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum of metal impurities, 1.39% +LC/MS impurities, 10.60%+Inorganic Fluoride, 0.27%+NMR impurities, 1.00%+ POAA, 0.30%) Total impurity from all tests = 13.56% Purity = 100% -13.56% = 86.4% 2Potassium 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 s observed for this sample. 4Sulfur in inorganic the sample appears to be anion method conditions. coTnhveeartneidontoreSsOu4lt and hence agrees we detected u ll with the sing the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5TFA NHFFPBAA PFPA Trifluoroacetic acid NHoepntoafflluuoorroopbeunttyarnicoiacciadcid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CsFnSOsTC* (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). {/ "N COA023-018B 3M Environmental Laboratory Page2 of3 Page 122 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Centre Analytical! Laboratories, Inc. 3048 Research Drive State College, PA 16801 INTERIM CERTIFICATE OF ANALYSISPhone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 Centre Analytical Laboratories COA Reference #: 023-018B LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total wt./wt. % 1.03 1.56 6.38 1.63 10.60 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 response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. C ', Prepared By: Davui S. Bell Date S^jejrtist, Ce^tr^^jJ^lytical Laboratories Reviewed By: ?/(/U John Flaherty Date Laboratory Manager, Centre Analytical Laboratories COA023-018B 3M [Environmental Laboratory Page3 of3 Page 123 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Centre Analytical Laboratories. Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231 -1580 INTERIM CERTIFICATE OFANALYSIS Centre Analytical LabRoervaitsoiornies1(C9O/7/A00R)eference #: 023-018A 3M Product: PFOS,Lot217 Reference#: SD-018 Purity1Test N_a_m__e________|__________PuritSyp:ec8if6ic.a9t%ions '. "5;';;::V Result 86.9% AIdpepnetaifricaantcieon NMR Metals (ICP/MS) | - White Crystalline Powder .. ' . .. Conforms Positive 4627351....... MNMIPCSrooiaoaactdlnnagkcisuigneusamelimnsuiemusme2 . . . ' -: . TT(LooCttaa/llM%%S)IImmppuurriittyy (NMR) T(GoCta/lM%S)Impurity --------- ------------------------------ .. _. .; 1 .: . 4623751.......81..49<0<006113...0.0.00084ww..0000403tt1550909..AA11wwwwwvvwwttttttt.......A%At%//t/.www.AAvvtttttvv.....%%%%%tt..%% None Detected RRPOeeslAaidtAeudalCSoomlvpeonutnsd(sT-GA) Purity by DSC Inor2g1..aniFCclhuAloomrrioiddnees (IC) 34576..... BNPNShuriittoolrrfmsiaapttteiheed4aete Organic Acids5(IC) . " . . . -. . . .. " ^.. . :. . 0.33 wt.Avt.% NNootnAepDpelticeactbelde1 5247361....... <<<<<0800000.......7500000690001496750wwwwwwwtt..AAttttt.....AAAAAvvttvvvvv..%%ttttt.....%%%%% 2341.... NHTPFFFFPAPBAAA Elem42351.e.... ntFNHSCaluliayutAlrdrofbuornroogrianneglenyensis6: ' 231... TTThhheeeooorrreeetttiiicccaaalll VVVaaallluuueee ===1007%%.8% 45.. TThheeoorreettiiccaall VVaalluuee =*=650.9%5% 2341.... <<00..0012..0811 wwwwtttt....A/AAwvvvtttt....%%%% 43521..... 058114...2287..1444448wwwwwttt...tAtAA..AAvvvtttvv...%t%t%..%% COA023-018A Pace 1of3 3M Environmental Laboratory Page 124 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Centre Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 Purity = 100% - (sum of metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic F0.l3u3o%rid)e, 0.59%+NMR impurities, 1.93%+organic acid impurities, 0.38%+POAA, ToPtaulriimtyp=ur1it0y0%fro-m13a.l0l9te%sts==861.39.%09% 2Potassium is expected in this salt form and is therefore not considered an impurity. /*"*\ 3Purity by DSC is generally not applicable to materials oflow purity. No endotherm was 1 observed for this sample. 4Sulfur in inorganic athneiosnammpetlehoadppceoanrdsittoiobnes.coTnhveeratneidontoreSsOu4lt and hence detected u agrees well with the sing the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5THFFABA . NFPA PFPA THreipfltuaoflruooarcoebtuictyarciicdacid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CgFi7S03'K+(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR160). r^ - COA023-018A 3M Environmental Laboratory Page 2 of 3 Page 125 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Centre Analytical Laboratories. Inc. 3048 Research Drive : State College, PA 16801 Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTERIM CERTIFICATE OFANALYSIS 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 response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: _________ ?//*/<*> DScaivenidtisSt.,BCeelnl treAnalytical Laboratories Date/ / Reviewed By: ydohn FAlath/e^rt-yA ^ T * ______________ Dfa//t/e/0 $ Laboratory Manager, Centre Analytical Laboratories COA023-018A 3M Environmental Laboratory Page 3 o f3 Page 126 3M Medical Department Study: T-6295.10 Appendix H: Report Signature Page BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Marvin t . Case, D.V.M., Ph.D., Study Director P. John L. Butenhoff, Ph.D., Sponsor Representative Date o 2.^/0Tj/o/ Date Bill Reagen, Ph.D., Laboratory Manager 0 'A,r/o/ Date ` Kris J. Hansen, Ph.D., Principal Analytical Investigator e^/otr/oi Date 3M Environmental Laboratory Page 127