Document yr0gQB3Y6mL3EZDBre4REg93r

BACK TO MAIN 3M Medical Department Study: T-6295.10 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 Department Study: T-6295.10 Argus In-Life Study: #418-012 Analytical Report: FACT TOX-099 3M Environmental Laboratory Request No. U2400 Total Number of 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 Laboratoly 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) Regulations21 CFR Part 58, with the exceptions in the bulleted list below. Exceptions to GLP compliance: 0 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. 0 There is a lack of an electronic data audit trail. 0 There is no reference standard stability. 0 The surrogate THPFOS was not characterized. 0 On at least one occasion, data entries were not signed and dated on the date of entry Marvin T. Case, D.V.M.. Ph.D.. Study Director 9/55zku/ Date qAz. John L. Butenhoff, Ph.D., S ~ ~ n s o ~ ~ e ~ ~ e s e n f ~ ~ ~'Dvaete - Kds J. Hansen, Ph.D., Analyrical investigator ' ot/ot?lo I 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)Fluorochemicalin Rabbit Serum and Liver Specimens Study IdentificationNumbers: T-6295.10,FACT TOX-099, LRN U2400,Argus 418-012 lnspectlon Dates Phase 10/16/00 L i e r Extraction 1 1/08/w11/1o/oo Data 1 a1 1/00-12/15/00 Draft report 12/20/00-12/21/00 1/11/01-1/1~01,1/15/01, Data/Draft Report 1/I 8/01-1 /19/01 Date Reported to Management Study Director 10/20/00 10/20/00 11/10/00 . 1 1/1 o/oo 1 a1 5/00 la1 5/00 1/22/01 1/22/01 QAU Repres%tative Date 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........................................................................................................... I O 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. Characterizationof the Analytical Reference Standard in Study FACT TOX-099 10 Table 3. Negative Ions Monitored in 3M Environmental Laboratory Analyses ..................12 Table 4. Characterizationof the Control Matrices Used for Liver and Sera Analyses in Study FACT TOX-099 .......................................................................................... 16 Table 5. Characterizationof Test Article ........................................................................... 16 Table 6. LOQ Values for PFOS Used in FACT TOX-099 Analyses by Method and Usage Dates ................................................................................................................... 112 Table 7. Average Concentration of PFOS (pg/g) in Rabbit Liver by Dose Group ..............112 Table 8. Average Concentration of PFOS (pg/mL) in Rabbit Serum by Dose Group......... 113 Table 9. PFOS (vg/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 lnvestigafor 3M Environmental Laboratory Contributing Personnel Lisa A. Clemen Rhonda Dick' Kelly J. Dorweiler* Sarah A. Heimdal* Marlene M. Heying* Harold 0. Johnson Kelly J. Kuehlwein* Sally A. Linda* Bob W. Wynne* Richard Youngblom* "Denotes contract laboratoly professionalpersonnel 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) DevelopmentalToxicity 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 mUkg, adjusted daily on the basis of weight. The test system species and strain selected was the New Zealand White Rabbits [Hra:NZW)SPF)] received from Covance Research Products, Inc., permanently identified using a Monel@self-piercing ear tag. FO 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 MI8-012 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 mgikglday 0 rnglrnL Group II 0.1 rng/kg/day 0.02 mg/rnL Group 111 1.Omgkglday 0.2 malmL Group IV 2.5 mgkglday 0.5 rnglrnL Group V 3.75 mglkglday 0.75 rng/rnL 22 5 22 I 3 22 3 22 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 Ithrough 5 (through 9/10/98): Maternal Serum Specimens-I 9 specimens Maternal Liver Specimens-I 9 specimens Fetuses-I 8 specimens Placentas-I 8 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-teff-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 &0IC. 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 k1O"C. 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: CBFI7SOjK+ 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 *Kept at ambient until 5/16/00,then movedto cold storage (-10C) Method Summaries Following is a brief description of the methods 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 Envimnmental Laboratory PREPARATORMYETHOOS ETS-8-4, "Extraction of Potassium Perfluorooctanesulfonateor other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/MassSpectrometry" 3M Environmental Laboratory Page 10 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-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.OmL of methanol, then filtered with a 0.2 pm nylon filter attached to a 3 mL plastic syringe into glass autovials. 0 ETS-8-6, "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/MassSpectrometry" Liver samples were homogenized in water. An aliquot of each homogenatewas 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.O mL of methanol and passed through a 0.2 pm nylon filter, using a 3 mL disposable plastic syringe, into glass autosampler vials. ANALYTICAMLETHODS ETS-8-5, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicalsin Serum Extracts Using HPLC-ElectrospraylMass Spectrometry" 0 ETS-8-7, "Analysis of Potassium Perfluorooctanesulfonateor 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 (CsFI7SO3-) 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 ANAL YTlCAL 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@BetasilTMCI8 2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2 mM ammonium acetate Component B: methanol Flow rate: 300 pUmin Injection volume: 10 pL Solvent Gradient: 10.0 minutes Time (minutes) %6 0.0 10% 1 .o 10% 5.5 95% 7.5 95% 8.0 10% Mass Spectrometer: Micromass@APVMass Spectrometer Quattro 11'" Software: Mass LynxrM3.4 Cone Voltage: 60 V Collision Gas Energy: 45-50 eV Mode: Electrospray Negative Source Block Temperature: 150C *lOC Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Triple Quadrupole system Table 3. Negative Ions Monitored in 3M Environmental Laboratory Analyses Target Analyte I Primary Ion (mu) I ProductIon (mu) I PFOS I 499.0 I 1- 99.0 ~~ THPFOS 427.0 80.0 3M Environmental Laboratory Page 12 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-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 Limits of 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: 70-1 30% Confirmatory 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 20.98. Calibration Standards: Quantitation of PFOS was based on linear regression analysis ( l / 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 PFOS were not chromatographicallyresolved. Quantitationwas basedon 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 0 Limits of Quantitation (LOQ): The LOQ was equal to 0.00426 pg/mL and 0.0214 pg/mL in sera and 0.00529 pg/g and 0.0529 pg/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: Precisionwas 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 * I O % of the theoretical concentration. Matrix spikes prepared in liver were compliant within k20% with one exception; one liver matrix spike was recovered at 156%. The average matrix spike recovery for the Group 1 samples was 138%. 0 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. Statement 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) 0 Samples from Control Animals: PFOS was detected in the sera and liver of all animals. 0 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 M18-012,"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: FACT TOX-099 LRN-U2400 Appendix A: Chemical Characterization and Control Matrices Table 4. Characterization of the Control Matrices Used for Liver and Sera I I I I Analyses in Study FACT TOX-099 Control Matrix Rabbit SerumTNA- 4511 Rabbit LiverTCR- 99131-45 Source Sigma Chemicals Covance Labs Expiration Date 912005 NIR Storage Conditions Frozen -20C (*I OX) Frozen-20C (*IO"C) Chemical Lot # 99H8400 I Phvsical DescriDtion I Rabbit Serum I F04051 Rabbit Liver I N I R - n o t recorded Table 5. Characterization of Test Article I II II Chemical Name Test Articlea KPFOS (Potassium Pertluorooctanesulfonate) I Source I 3M Toxicolow Services. Medical Department I Expiration Date 512000 Storage Conditions Room temperature Chemical Lot# 217 I I I Physical Description I I Purity I Off-white powder 86.9% I 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: FACT TOX-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 PO Box 3333 I St. Paul. MN 55 133-3331 612 718 6442 BACK TO MAIN Analytical Report: FACT TOX-099 LHN-UZ400 Protocol #FACT-TOX-099 Study Title Oral (Stomach Tube) Developmental Toxicity Study of PFOS in Rabbits PROTOCOL Author Lisa Clemen Date: June 8,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-099 U2400 3M Environmental Laboratory 3M Environmental Laboratory Page 7 of 70 . 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. 3 M 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@) 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 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LHN-uZ~UT Protocol #FACT- 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. STUDY Oral (stomach tube) developmental toxicity study of PFOS in rabbits 2. PURPOSE This analytical study is designed to determine levels of potassium perfluorooctanesulfonate (PFOS)in specimens of liver and serum of rabbits. The in-life portion of 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. REGULATORCYOMPLIANCE This study will be conducted in accordance with the United StatesFood and Drug Administration,Good Laboratory Practices Standards,FinalRule 21 CFR 58, with the exception that analysis of the test material mixture for concentration, solubility, homogeneity, and stability will not be conducted, and is the responsibility of the Sponsor. 4. QUALITYASSURANCE 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 Standardsand 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 submittingto the 3M Environmental Laboratory. 3M Environmental Laboratory 3M Environmental Laboratory Page 3 of 10 Page 20 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400- Protocol #FACT-TOX-099 5. TESTMATERIAL 5.7 Refer to Argus Research Laboratory protocol for study M18-012. 6. CONTROLMATRlCES 6.7 IdentificationRabbit 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 Purify and Stability Not applicable 6.5 Storage Conditions Frozen at -20 "C f 10"C or -50 "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 of 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 samplesfor analysis. 7. REFERENCMEATERIAL 7.7 Identification 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 Stabirity Purity = 99%, Stability to be determined by the Sponsor 7.5 Storage Conditions Room temperature 7.6 Reserve Material A reserve sample fiom each batch of PFOS used in this study will be retained 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). 7.7 Disposition Unused reference material will be retained for use by the 3M Environmental Laboratory and will be discarded when the quality of 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 EnvironmentalLaboratory 3M Environmental Laboratory Page 4 of 10 Page 21 BACK TO MAIN 3 M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 PrOtOCOl #FA CT-TOX-099 8. TESTSYSTEM Rabbits were used as the test system and were maintained and dosed as described in ArgusLaboratoriesprotocol M18-012. 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 I Dosage Group Dosage Levels, Concentration, and Volumes I Number of rabbits I Dosage mg/kg/day I Concentration mg/mL 1 22+3' 0.0 0.0 2 22+S 0.1 0.02 3 22+3' 1.o 0.2 4 22-i-3' 2.5 0.5 5 22+5' 3.75 L- I I a Rabbits assignedto toxicokineticevaluation. I 0.75 I Dosagevolume 1 5 5 5 5 5 I I 9. SPECIMEANND SAMPLERECEIPT The 3M Environmental Laboratory will receive specimens of 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 tissudfluid Collected Serum -Dam animals Liver - Dam animals I F_ etus.-Po_ _ ole.d.~ l_ itte.r animals Placenta - Pooled litter animals Toxicokinetic Collection - Day 21 Toxicokinetic Collection - Day 21 Toxicokinetic Collection - Dav 21 I I Toxicokinetic Collection - Day 21 Expected ## of specimens 19 19 1 I 19 I19 Total number of expected specimens: 76 Total number of test animals: 104 Total number of control animals: 25 Specimens sent to 3M Environmental Laboratories will be received and tracked accordingto applicable Standard Operating Procedures. 3M Environmental Laboratory 3 M Environmental Laboratory Page 5 of 10 Page 22 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 PrOt0~0#l FACT-TOX-OBB 10.PREPARATORMYETHODS 10.1 FACT-M-1.1, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry 10.2 ETS-8-4.1,Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLCElectrosprayhlass Spectrometry 10.3 If preparatory methods other than those listed above are used, an amendment to this protocol will be written. Any deviationsfrom 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 Fluorochemicalsin Liver ExtractsUsing HPLCElectrosprayMass Spectrometry 11,2 ETS-8-5.I, Analysis of Potassium Pedluorooctanesulfonateor Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mas Spectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. Any deviations fkom 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 attachedto this protocol. 12.DATAQUALITY OBJECTIVES The number of spikedduplicates,use of surrogates,and informationon other data quality indicators are included in the analytical methods. In addition, the following criteria will be met: 12.1 Linearity 8 2 0.98 12.2 Limits of detection / quanfftation 12.2.1 Method DetectionLimit (MDL) for PFOS a) Serum: 1.75 ppb b) Liver: 15 ppb 12.2.2 Limit of Quantitation (LOQ) -Equal to the lowest acceptable standard in the calibration curve 12.3 Duplicate acceptable precision c 30% for the method 3M Environmental Laboratory 3M Environmental Laboratory Page 6 of 10 Page 23 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LHN-U240U PfOtOCOl #FACT-TOX-O99 72.4 Spike acceptable recoveries 70% - 130% 12.5 Use of confirmatory methods Indeterminate samples will be re-analyzed using a confirmatorymethod. If a confirmatory method is used, an amendment to this protocol will be written. 72.6 Demonstration of specificity Chromatographic retention time, mass spectral daughter ion characterization. 13.SUB-CONTRACTAENDALYSIS 13.7 All analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3E-09,935 Bush Avenue, St. Paul, MN 5 5 106, at Advanced BioanalyticalServices, Inc., 15 Catherwood Road, Ithaca, NY 14850, or at Battelle Memoria1Institute, 505 King Avenue, Columbus, Ohio 43201-2693. 73.2 An amendment to this protocol will be written if analyses are performed at laboratoriesother than 3M Environmental Laboratories, Advanced Bioanalytical Services,Inc., or Battelle Memorial Institute. 74.STATISTlCAL ANALYSIS Averages and standard deviations will be calculated. The statistical methods that will be used are described below: 74.7 Data transformations and analysis Data will be reported as the concentration (weightfweight or weightfvol) of PFOS or metabolite per tissue or fluid. 14.2 Statistical analysis Statistics used may include regression analysis of concentrationsover 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. 75. REPORT A report containing all the results of the study will be prepared by the 3M Environmental Laboratory.If analysesare 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: 75, f Name and address of the facility performing the study 75.2 Dates upon which the study was initiated and completed . 75.3 A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 7of 10 Page 24 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 P ~ O ~ O C#FOA/ CT-TOX-099 75.4 Objectivesand procedures as stated in the approved protocol, including any changes in the original protocol 75.5 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 75.6 Stability and the solubilityof the test substances under the conditionsof administration, if provided by the Sponsor 15.7 A description of the methods used to conduct the test@) 75.8 A description of the test system 75.9 A description of any circumstances that may have affected the quality or the integrity of the data 75.70 The name of the Study Director and the names of other scientists, professionals,and supervisorypersonnel involved in the study 75.7 7 A descriptionof the transformations, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement of the conclusions'drawnfrom the analyses 75.72 Statisticalmethods used to evaluate the data, if applicable 75.73 The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable 75.74 The location where raw data and the final report are to be stored 75.75 A statementprepared by the Quality Assurance Unit listingthe dates that study inspections and audits were made, and the dates of any findings reported to the Study Director and Management If it is necessary to make corrections or additions to a report after it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part of the report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 76.LOCATION OF RAW DATA, RECORDSA,ND FINAL REPORT Original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitateaudits of the study during its progress and before acceptanceof the final report. When the final report is completed, all original paper data, including those items listed below, will be retained in the archivesof 3M Environmental Laboratory for at least a period of time as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 76.7 The followingraw data and records will be retained in the study folder in the study/projectarchives according to 3M Environmental Laboratory StandardOperating Procedures: 3M Environmental Laboratory Page 8 of 10 3M Environmental Laboratory Page 25 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LHN-U2400 PrOtOCOl #FACT-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 (originalsigned copy) 16.1.6 Electronic copies of data 16.2 The following supportingrecords will be retained separately h m the study folder in the archives according to 3M Environmental Laboratory Standard Operating Procedures: 76.2.1 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 effectivedate of the final test rule (if applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. 18.PROTOCOL AMENDMENTASND DEWATIONS Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. All changesto the protocol will be indicated in the final report. Any other changes will be in the form ofwritten deviations,signed by the Study Director and filed withthe raw data 19. A~ACHMENTS 19.7 Attachment A Preparatory and analytical methods 3M Environmental Laboratory 3M Environmental Laboratory Page 9 of I O Page 26 3M Medical Department Study: T-6295.10 20. SIGNATURES /jJL?./.; /c& !i t& Mfu;in T. Case, D.V.M.P,h.D.,SponsorRepresentative BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 PrOtOCOl #FACT-TOX-O99 , 4/[u/f9 3M Environmental Laboratory Study Director Date - 3M Environmental Laboratory 3M Environmental Laboratory Page 70 of 70 Page 27 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Study Title Oral (StomachTube) DevelopmentalToxicity Study of PFOS in Rabbits PROTOCOL AMENDMENT NO. 1 Amendment Date: August 04,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX099 LIRN U2400 3M Environmental Laboratory 3M Environmental Laboratory Page 28 BACK TO MAIN 3M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 PrOtOCOl FACT-TOXO99 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 BioanalyticalServices,Inc. (ABS) 15 Catherwood Road Ithaca, NY 14850 AMENDTO READ: The extraction and analysis of the rabbit serum specimenswill be sub- contracted to: Pace Analytical Services,Inc. 1700Elm Street, Suite 200 Minneapolis, h4N 55414 Note: The serum specimens were sent to ABS on 6/15/99 and will be shipped directly from A B S to Pace Analytical Servicesin 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 specimenswas changed to Pace Analytical Services who has completed a rabbit serum validation. 2. PROTOCOL READS: Section 10.0 and 11.0 list the followingmethods to use for extraction and analysis: FACT-M-1.1 "Extraction of Potassium Perfluorooctanesulfonateor Other Anionic FluorochemicalSurfactantfrom Liver for AnalysisUsing HPLC-ElectrosprayIMas Spectrometry" ETS-8-4.1 "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLC-Electrosprayklass Spectrometry" FACT-M-2.1 "Analysis of Fluorochemicals in Liver Extracts Using HPLC-ElectrosprayIMass Spectrometry" ETS-8-51 "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-ElectrosprayIMassSpectrometry" AMENDTO READ: The extractionand analytical methods are: ETS-8-6.0 "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-ElectrosprayklassSpectrometry" 3M Environmental Laboratory 3M Environmental Laboratory Page 29 -3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLC-Electrospray/MassSpectrometry" 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 tert 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 l/x weighting and a few minor changes in the HPLC 1100 instrument parameters. 3. PROTOCOL READS: Sections 10.4 and 11.4state that 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. AMENDTO READ: The extraction and analytical methods for rabbit serum at Pace Analytical Services, Inc. are: CAG-SP-01.O "Extraction of Potassium Perfluorooctanesulfonateand Other Fluorochemicd compounds fromSerum for Analysisby LC/MS" CAG-CHR-01.O "Analysis of Potassium Perfluorooctanesulfonate and Other Fluorochemicalsin Serum Extracts Using LCMS" The extraction and analyticalmethods for rabbit liver at Elattelle Memorial Institute are: "Method for Analysis of PrefluorooctaneSulfonate (PFOS) in Rat Liver by LCiMSMS, Version 1 .o" REASON: The methods listed previously were for extraction and analysis performed at the 3M Environmental Laboratory. This amendment was written to include the methods to be followed 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 TO READ: The liver method detection limit is 8.50 ppb (ndg). REASON:The validation supportingmethods 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 -3M Medical Department Study: T-6295.10 Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 PrOtOCOl FACT-TOXO99 Amendment No. 1 a l w J G d - - Marvin Case, D.V.M.,Ph.D.,SponsorRepresentative mi5.44&43 m-42 flDate Kris J. Hansen, Ph.D., Study Director g f l ,.Iv% Date 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 31 3bJ 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 Battelle Memorial Institute Pace Analytical Services, Inc. 505 King Avenue 1700 Elm Street, Suite 200 Columbus, OH 43201-2693 Minneapolis, 55414 Laboratory Project Identification ET&SS LRN-U24100 FACT TOX-099 Argus Study: 418-012 3M Medical Department Study: T-6295.10 . 3M Environmental Laboratory 3M EinvironmentalLaboratory Page 32 BACK TO MAIN 3 l Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 PrOtOCOl LRN42400 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.. AMENDTO 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 identifiedl as Marvin T. Case, D.V.M., Ph.D. AMENDTO 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, personnelresponsibilities 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. AMENDTO 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 imalysis and submission of the subcontract laboratory(s) final report. Specimens analyzed at sub-contract laboratories will be returnedwith 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 EnvironmentalLaboratory 3M EinvironmentalLaboratory Page 33 BACK TO MAIN 3-M 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 andl 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 ofthe 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, arid 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 o.f liver, serum, and fetal tissues of rabbits. REASON: The study sponsor requestedthe 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 amendmentwill be written to address the use of the additional method. 3M EnvironmentalLaboratory 3M Einvironrnental Laboratory Page 34 3-M Medical Department Study: T-6295.10 Amendment Approval BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Protocol LRN-U2400 Amendment Number 2 John L. Butenhox Ph.D., Sponsor Representative Y Date A-0 Kristen J. Hansen,Ph.D., Outgoing Study Director //-E&& -woo Date Marvin T.Case, D. KM,Ph.D., Incoming Study Director MiL&. 9-4 Dad 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 of PFOS in Rabbits PROTOCOL AMENDMEINT NO. 3 Amendment Date: August 16,2000 Petforming Laboratory 3M Environmental Technology Bi Safety 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 3-M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-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. PURPOS-AIl liver samples will be extracted and analyzed at Battelle Memorial Institute. 13. SUB-CONTRACED ANALYSE-All analyses as d.etailed inthis protocol will be performed at 3M EnvironmentalLaboratory,Advanced BioanalyticalServices, or Battelle Memorial Institute. AMENDTO READ: Remove Battelle Memorial Institute as a subcontractlaboratoryunder the Study Location 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. REASON: 3M Environmental Laboratory is able to meet the analyticalneeds of the study. 2. PROTOCOL READS: 3M FACT TOX-099 PROTOCOL: IO. PREPARATMOETRHYODS& 71.ANALmCAL METHODS-The the liver methods are ETS-8-6.0and ETS-8-7.0. amended protocol states AMENDTO READ: Change to specify: Use the most current version of th.e methods listed in both sections. REASON: To specify that the most current version of the preparatory and analytical methods should be used during the course of the study. 3M Environmental Laboratory 3M IEnvironmental Laboratory Page 37 -3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 PrOtOCOl FACT-TOX-099 Amendment ##3 3. PROTOCOL READS: The amended 3M ANALYnCAL PROTOCOLFACT-TO)<-099(Amendment No. 2 ) states that Marvin T. Case replacesKristenJ. Hansen as the study director.KristenJ. Hansen was reassignedthe role of Principle Analytical Investigator.John L. Butenhoffwas reassignedthe role of SponsorRepresentative. AMENDTO READ: KristenJ. Hansen was reassignedto the role of Principal (correctedspelling) Analytical Investigator. REASON: Corrected spelling of Principal. Amendment Approval # 9& 2.*P Job; L. Butenhog Ph.D., SponsorRepresentative D%e dartin T. Case, D.V.M.,Ph.D., Study Director Date 3M EnvironmentalLaboratory 3M lfnvironmental Laboratory Page 38 3 M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Study Title Oral (Stomach Tube) Development Toxicity Study of PFOS in Rabbits PROTOCOL AMENDMENT NO. 4 Amendment Date: September 25,2000 Petforming Laboratory 3M Environmental TechnoIogy & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification Analytical Report: FACT ?'OX-099 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 TOX-099 LRN-U2400 Protocol #FACT TOX-099 Amendment 4 This amendment modifies the following portion(s)of the protocol: 1. PROTOCOL READS: SUB-CONTRACT blBORATORIES: The amended protocol (Amendment No. 1) lists Pace Analytical Services, Inc. as the subcontract laboratoiy for the extraction and analysis of rabbit sera specimens. The amended protocol (Amendment No. 3) states thalt 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 wiU be extracted and analyzed at 3M Environmental Laboratory. SEC77ON 13. SUB-CONTRACTEDANALYSIS:The anzended protocol states that all analyses as detailed in this protocol will be performed at 3M Environmental Laboratories or at Pace Analytical, Inc. AMENDTO READ: Page 3, Study Locations, subcontract Laboratories: Add Pace Tier II as a sub-contract laboratory. 2. Purpose: Change to state that all liver and sera saniples will be extracted at the Pace Tier I1 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 11and 3M Environmental Laboratory are alble to meet the needs of the study. 2. PROTOCOL READS: SEC77ON 13. SUB-CONTRACTEDANALYSlS: No SOPSare listed for the subcontract laboratory. AMENDTO READ: Add to Section 13: Pace Analytical Services, Inc. (Pace-Tier 11Facility, 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 Enviromnental Laboratory for analysis. Pace will follow all applicable 3M Environmental Laboratory Shndard Operating Procedures and the following Pace Standard Operating Procedures: PSS-Admin-01 PSS-DC-05 PSS-OPS-01 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 TOX-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-3 1 PSS-OPS-32 ETS-9-40 (3M Lab) PSS-OPS-37 PSS-MTR-0 1 PSS-MTR-02 PSS-MTR-06 PSS-MTR-08 PS S-MC-0 1 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 Maintenanceof NANOpure 11Water 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 Cdiiration 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 IJse of Analytical Balances Purchasing of Laboratory Supplies Disposition of Archive Materials StatisticalEvaluation of Data REASON: All extractions for liver and sera samples will be performed at Pace Tier II,using the above SOPS. 3. 3.PROTOCORLEADS: SECTION13. SUB-CO~ACEDANALYNSo~mSethods are listed for the sub-contract laboratory. Under Preparatory Methods, ETS-84.1 is listed as the method for serum extraction. Under Analytical Methods, ETS-8-5.1 is Wed 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. AMENDTO READ: The following 3M EnvironmentalLaboratory methods will be used for liver and sera extractions and analyses: ETS-8-4 Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical 3M Environmental Laboratory 3M Environmental Laboratory Page 41 BACK TO MAIN 3MMedical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Protocol #FACT TOX-099 Amendment 4 ElectrosprayMass Spectrometry ETS-8-5 Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-ElectrosprayMass Spectrometry ETS-8-6 Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Angysis Using HPLC-ElectrosprayMas Spectrometry ETS-8-7 Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts UssingHPLC-ElectrosprayMass Spectrometry REASON: To clarifythe methodsto use for extractionand analysi:~of TOX-099liver and sera samples. 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 42 -3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Protocol #FACT TOX-099 Amendment 4 Amendment Approval 9 A f. John L ButenhoB Ph.D., Sponsor Representative ocncub 5,. to& Date Marvin T. Case, D.V.M., Ph.D., Study Director Date 3M Envimnmental Laboratory 3M Environmental Laboratory Page 43 -3M Medical Department Study: T-6295.10 I Study / Project No. Deviation Type (Check one) Record of Deviation 1. Identification BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 0 SOP @Methoti 0 Equipment Procedure OProtocol 0 Other: Recorded By A 0 i Date , , hiEnvironmental Laboratoty Form EE-4-8.0 3M Environmental Laboratory .- Deviation No. 1 (assigned by Study Director or Pmject Lcad Y Ihc end of study or project) . Page 44 -3M Medical DepartmentStudy: T-6295.10 Analflical" mnv.pawlabs.com October 30, 2000 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Pace AnaIyW S~wlceIn~c,. 1?MElm Streel#Suite200 Minneapolis,MN 55414 Plhm: 61260Z l7cO Fax: 612.6076444 Hr. Hade Schell 3M Eminrmental Laboratory 935 Bush Ave. El$. 2-3E-09 st. Paul. HN 55144 RE: Pace Project )cder: 1037874 ~ . Client Project ID: Water Quality CheCLs-R Dear Hr. Schell: Enclosed are the analytical results for saaple(s) recelved by the llaboratory on October 18. 2000. I f you have any questions concerning this report. please feel free to contact me. Sincerely, Andrea Neaitz Project Hanager Enclosures 3M Environmental Laboratory ,- REPORT OF LABORATORY ANALYSIS This report shall not be reproduced, except in full. without the written consent of Pace Anallytical Services. Inc. Page 45 -3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 t%Mh / f l / U /s#N/c#$, /ne. 1100 Elm Sbzet, Suite 200 Minnsapalis, MN 55414 Phone: 612.607.17W Fm 612.607.6#44 DATE: 10/30/00 PAGE: 1 .H Envlromental Laboratory 0 5 Bush Ave. .ldg. 2-3E-09 ,t. Paul. HN 55144 Palce Project Nwber: 1037874 (Client Praject ID: Water Cuality Checks-TZ 4 t h Hr. Wade -11 'hone: 612-778-5233 , h l i d results am npwtcd a wct wtlght basis 'ace Sample No: : l i d Sarple ID: 102339926 TIER I1 MATER W E Date Collected: Date Received: 1O/l8/W 10/18/00 Matrix: Water 'arameters . . . . . . . . . . . . . . R_ eI s. u.l. ts..... u..n.it.s...-. P.R.L.-......A..n.a.ly.z.e.d. A..n.a.l.yst..U..S.#-....F..o.o.t.n.o.t.e.s. letal s Metals. Trace ICP sodim Date Digested let Chemlstry Specific Conductance Specific Conductance pH. Water PH Anions. Ion Chromatography . Chloride Total Organic Carbon Total Organic Carbon Method: EPA 6010 m ugn 1000 kthod: EPA 120.1 2 mhos/cl 1 Method: EPA 150.1 5.5 0.1 HeW: EPA3W.O m m 1 MeW: sn 5310 ' m m 1 Prep Method: EPA 3010 10/28/00 BDA 7440-0-5 , 10/19/w Prep Hethod: EPA 120.1 10/18/00 Jnl Prep Method: EPA 150.1 10/18/00 Jnl Prep Lthod: EPA 300.0 10/24/00 BKR Prep kthod: sn 5310 10/20/00 BKR 7440-44-0 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS This reportshall not be reproduced, exceptin full, without the wriien consentof PaceAnalyticalServices, Inc. Page 46 3M-Medical Department Study: T-6295.10 Analytical" ~ . p c ~ l 8 kco$rn. AJWErER -F 0 Not Detected C Not Calculable RL Pace Reporting Limit -:. . . .. . I . .,. . . : .- .-v . . . . BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Pace Analytiml Servl~esI,nc. 1760Elm Streel,Suite21x) Minneapolis,MN 55414 Phne: 612.W.17m Fax: 612.607.6444 DATE: 10/30100 PAGE: 2 Pace Project W r : 1037874 (:1ient Project ID: Water Qual1t y Checks-T2 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS This report shall not be reproduced, except in full, withoutthe written consent of Pace Analytical Services. Inc. Page 47 -3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 ptcr Analytical Sinices, /ne. 17w Elm Street Suite 2oLI Minneapolis,MN 55414 I QUAlIlY CONTROL DATA Phone: 612.607,lm Fax: 612.607.64f4 DATE: 10/30/00 PACE: 3 w Envlnr#rRal Laboratory 135 Bush Ave. #l&. 2-3E-09 t t . P w l , HN 55144 Paa! Project W r : 1037874 Gllent Project ID: Water Quality Checks-Ti! {ttn: Hr. Wade Schell 'how: 612.7786233 X Batch ID: 51031 inalysls Hethod: EPA 120.1 bsociated Paw Saqles: . QC Bat& Iwhod: EPA 120.1 . AMlytls Dexription: spcclfic c4m - 102339926 mw: 102340106 bsoclated Pace Saqles: 102339926 Heulbd 81ank '.a.r.a.m.e.t.e.r.......................u..n.it.s...........R.e.s.u..lt.....P.R.L..........F.o.o.tnotc?s ipecific Conductance Uhosla 1 1 A P E WPLICATE: 102340114 m. '.a.r.a.o.e.te.r.......................U.n.i.ts.........1.02.3.3.9.9.2.6...R..e.s.u.lt.......R.P.D......F.o.o.tnotes i ~ w i f i cConductance u h o s / a 2.000 2.OOO 0 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS This report shall not be reproduced, except in full, without the written consent of Pace Analytical Services, Inc. Page 48 3 M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Anal'icar wm.pac~?l8&s.com QwLmC l M l W L DATA h C 8 h8/fl/C8/SL?N/C#$,/ne. 1Mo Elm Stmt,Suite Minneapolis, MN 55414 Phone: 612.601.1700 Far 612.607.6444 ME: 10/30/00 PAGE: 4 :M Environental Laboratory 135 Bush Ave. .ldg. 2-3E.09 ,t. Paul, Wl 55144 Pace Project Nwber: 1037874 Client Project ID: Water Quality Checks-TZ dtn: Mr. Wade Schell ~IOIW: 612-778-5233 j Batch ID: 51032 inalysls Hetlmd: EPA 150.1 jssoclated Pace Saqles: 102339926 fi Batch MM: EPA 150.1 Analysis Desaiption: pH. Water ABORATORY CONTROL W E :10234ow Splke LCS Spl ke '.a..r.m..te..r.......................h.i..t .s.......C.o.n.c......R.e.s.u..lt......X..R..cc...F.o.o.t.nDtcs N 5.000 5.000 101 ;AHPLE OUPLICATE: 102340122 Dup. '.a.ra.m..e.t.er.......................u.n.i.ts.........1.0.2.3.3.85.6.3....R.e.s.u.l.t.......R.p.o.....F.o.o.tnotes M 8.200 8.200 0 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS This report shall not be reproduced, except in full, without the written consent of Pace Analytical Services, Inc. Page 49 -3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Analflicar w m r . p a ~ l ~ bcsor.n WITYcma DATA Pacn AnaWIcal Sewtces, /nc. 17M/m Sheet Suite200 Minneapolis, MN 55414 Phone: 612.607.1760 Far: 612.6076444 DATE: 10/30/00 PAGE: 5 'I Envlrolwntal laboratory 35 Bush Ave. Idg. 2-2-09 t. Paul, HN 55144 Palm Project NMber: 1037874 Cllent Project ID: Water Quality Checks-T;! ttn: nr. wade Scheil how: 612-778-5233 C Batch ID: 51059 nalysls Method: EPA 6010 ssodated Pace Saaples: QC Batch Ikthod: EPA 3010 Analysis Oescrlptlon: Hetals. Trace ICP 302339926 RHoDBlAtK: 102340494 ssoclated Pace Samples: 102339926 llcthod B1 ank a.r.a.m..e.te.r.......................u.n.i.t.s..........R..e.s.u.l.t .....P.R.L.........F.o.o.tnotes odi uo W/l H) 1000 ATRIX SPIKE a MlRIX SPIKE WPLICATE: 102340510 102340528 Hatrlx Matrix Splke Splke Sptke Spike Sp. Dup. Dup a.r.a.m.e.t.e.r.......................U.n.l.t.s........1.0.2.3.3.9.1.2.4..C.o.n.c......R..e.s.u.lt.....2..R.e.c...R.e.s.u.l.t .....X..R..e .c ...R.P.U...F..ootnotes dl M m 0 50000 61210 122 58410 117 5 1 PBDRAmRY COHlRoL SAHPLE: lo2740502 armter Units Splke Conc. LCS Result , Spl ke t Rec Footnotes ....................................................................... odium ug/l 50000 59230 118 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS This report shall not be reproducedl, except in full, without the written consent of Pace Analytical Services, Inc. Page 50 -3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 ptC8 &8/yt/U/ s8n?/ce$,/ne. Ifw Elm Street, Suite200 Minneqmh, MN 55114 Phone: 672607.l700 Fax: 612.607.6444 DATE: 10/30/00 PAGE: 6 M Environwntal Laboratory 35 Bush Ave. ldg. 2.3E-09 t. Paul, HN 55144 m: nr. wade setmil 'hon: 6l2-778-5235 a-. IC Batch ID: 51W nalysis Method: PA 300.6 ssodated Pace Samples: Pacr Project N-r: 1037874 Client Project ID: Hater QualityChecks-Tz 102339926 9c Bat& EPA 300.0 h l y s l s Description: Anions. Itrn mioDBLAM(: 102340734 m x i a t e d Pace Samples: 102339926 Hetw E1 ank '.a.r.a.m..et.e.r.......................U..n.i.ts...........R.e.s.u.l.t.....P.R.L..........F.o.o.tm1:es :hlor$de mn 1.1 1 lAlRIX SPIKE h HATRIX SPIKE DUPLICATE: 102340742 102310759 Hatrix Hatrix Spike Spike Spike !;pike Sp. Dup. Oup '.a.r.w..t.e.r..... ..................U..n.lt.s........1.0.2.3.39.7.1.0..C.o.n.c......R..e.s.u.lt.....1.: .R.e.c..R..e.su..lt......2..R.e.c...R.P.D...F..o.o.tn.o.tes :hlwide w 48.68 50 95.38 93.4 93.66 90.0 4 2.2 ABWATCRY CONlRoL WLE a LCSD: 102340767 102340775 Spl ke '.a.r.a.a.e.te.r......................U.n..it.5.......SC.op.ni.ck..e...Ru.;.eS.su.l.t..,....SZ.pR.l.ek.ce..RL.c.es.sou..lt......ZD.uR.p.e.c...R.w.....F.o.o.t.notes ihlorlde aon 5.000 5.320 106 5.200 lo4 2 2.2 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS This report shall not be reproduced, except in full, without the written consent of Pace AnalyticalSenrices, Inc. Page 51 BACK TO MAIN 3EMedical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 PIC8 h 8 l ~ f I ~S8o1nlcsr, Inc. 1lcQ Elm Sleet, Suile2~x1 Minneapolis, MN 55414 Phone: 612.607.1700 Fax: 612.607.6444 1 CUALIlY COKfRoL DATA DATE: 10/30/00 PAGE: 7 H Environmtal Laboratory 35 Bush Ave. la. 2-3E.09 t. Paul. HN 55144 Pace Project Nuber: 1037874 Client Project ID: Water Cuality Checks-T2 .ttn: Mr. Wade Schcll 'hone: 612-778-5233 1c Batch ID: 51099 nalysis Hethod: SM 5310 issociated Pace Sanples: 102339926 QC Batch Method: SH 5310 AMlySlS beXrlptfOn: Tatal Org(MniC C a I h IEMOBM: 102343464 bsoclated Pace Samples: 102339926 nethod E1 ank J.a.r.a.m..e.te.r.......................u.n.i.ts...........R..e.s.u.lt......P.R.L.........F.o.o.trotes b t a l Organic Carbon mn Kl 1 ATRIX SPIKE 6 WTRIX SPIKE DUPLICATE: 102343472 102343480 Matrix Matrix Spike Splke Splke Splke Sp. Dup. Dup '.ar.a.m.e.t.e.r.....................U..n.i.ts........6.0.3.8.8.2.35.8...C.on.c.......R.e.s.u.l.t .....t .R.e.c...R.e.s.u.l.t .....I..R.e.c....R.PD....F.o.o.t.n.otes rota1 'Jrganic Carbon wn 6.326 2.000 8.850 126 8.980 133 5 3.3 NORATCRY COHIRM.WLE 6 LCSD: 102343506 102343514 Spl ke Splke us Spike LCSD Dup '.a.ra.m..e.te..r.....................u.n.i.t.s.......C..o.x.....R..e.s.u.l.t., ....t..R.e.c...R.e.s.u.l.t......t.R..ec...R.P.D....F..o.o.tnotes iota1 Organic Carbon 5.000 5.280 106 5.290 106 0 ;AHPLE DUPLICATE: 102343498 w. '.a.ra.m..e.te.r......................u.n.i.t.s........1.0.2.3.3.99.%.....R.e.s.u.l.t......R.P.D......F.o.o.tn.otes Total Drganlc Carbon a011 No Kl Nc 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS This report shall not be reproduced. except in full, without the written consent of Pace Anallytical Services. Inc. Page 52 BACK TO MAIN 3M-Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Analflical" wm.prulabs.a m PIW Anrryflcrl Servtces, Inc. 1700Elm StW Suite2LxI M i m o l i a MN55414 Phone: 612.607.17' Far: 6126117.6444 ME: 10/30/00 PAGE: 8 Pace Project W r : 1037874 Cllent Project ID: Water QuallfyChecks-Ti! onslstent ulth EPA guldellnes unr#mded concentrations are dlsplapd and !we been used to calculate I Rec and RPD valys. 0 Not Detected C Not Cala 1ab1e RL . PD Pa= Rcportlw L f W t Relatlw P e e M f f m .:; .I] 2) .2] '3 31 ~ l r1,6uas out o f our QC limits hut ths IW was aaptable. AMlyte i s found ln the &rtcd blujr as bell as In the -1s [CLp 6-Flag). ihc splb recovery UIS outside Kccptmcc lldts for the I6 and /a* IW due to matrix interference. The LtS and/or LCSD yere wlthln rrrrcpturr limits slmulng that the laboratory I t -In control and the data i s acceptable. 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS This report shall not be reproduced, except in full, without the wriien consent of PaceAnalytical Services. Inc. Page 53 - 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Phona: 913.5995665 Fax: 913.5N.1759 ME: 10/24/00 PW: 1 Pace Project Ikdcr: 6045897 Client Project ID: SCIENCE W o ( s / 1 0 3 7 8 7 4 m: ttn: PRQIECT- (61215444435 '. -*' olld results am npsrtad on a vet wel& &Is ace Saaple No: 11ent Sarple ID: 603917352 TIER I1 MXR SlUlpLE (ktc collected: Date Received: 10/18/Oo 10/2l/W a..r.a.m...e.t.e.r.s..................... ........... Results units m .....I... I......... ........ ..... AMlyzed Analyst CAS# .....I.._. Matrix: Hater Footnotes ......I... ,et Chemistry Silica. Total Silica Resi s t i v l t y Resis itlvlty m Hethod: EFA375.3 mn 1 new: 15oooO ohsla 10 Prep Method: EPA 375.3 10/23/M) HI6 7631.86-9 Prep bthod: 10/22/00 JLC 3M Environmental Laboratory REPORT OF LABORATORY AWALYSIS This report shall not be reproduced, except In full. whou! the written consent of Pace Analytical Services, Inc. Page 54 3- M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 PlCr Analytld S r d m , Inc. !HM Loirsl Elvd. LmaKS66219 Phm: 913.599.5665 Fax: 913.599 1759 ME: 10/24/00 PAGE: 2 Paut Project lk&er: 6045897 Cllient Project ID: SCIENCE SOllJlIONS/1037874 ID Not ostscted K: Not Calculable '2L Pace Reporting L l r l t .'7 . . . . . . .,...... . . . : .. . .-. ,,<.:,.. ... . ....i 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS This report shal not be reproduced,Inceptin full. wkout the written consent of Pace Anabtical services, tnc. Page 55 3-M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Analytical" w.pe#i8b$.com PWITY COHlRoL DATA fit8 Anriyiicai SuMcus, he. 9w8 LoirefEM. LmeMa, h3 66219 Phone: 913.599.5665 Fax: 913.599.1759 ME: 10/24/00 Pm: 3 iINNESOTA uB(RANRY 'ACE ANALYTIUL SERVICES 1700 ELM STWl SUITE 200 iINNuWcIS. IWI 55114 Pace Project Nukr: 6045897 Client Project ID: SCIW samIoHs/l037874 4ttn: PRalEGTMwzR 'hone: (612)541-3435 .. ' 1~ Batch IO: 930U snalysir Method: EPA 375.3 4sssoclated Pace Saqler: QC Batch Method: EPA 375.3' ' ' *& ' ' ' Atialysls Dcscrlptlon: S l l l u . Total 603917352 jm#,BUM(: 60391927 Issodated Pace Sampler:. $039U352 HetW 81 a& '.a.r.a.a.e.t.e.r........................l.b.l.lt.s...........R.e.s..u.lt......P.m...........F.ootnotes Silica m m 1 (ATRIX SPIKE: 603919135 Hatrlx Spike Spike Spike '.a.ra.m..e.te.r.......................u..n.i.ts.........6.0.3.8.8.63.0.0...C.o.nc.......R..e.s.u.lt......t.R..e.c..f.o.o.t.mtes Slllca .on 20.91 10 29.12 82.1 ABORATORV CONlRoL !MPLE: 603919150 Splke Lcs splke y.a.r.a.m.e..t.e.r.......................u.n.i.t.s.......c.o.n.c......R..e.s.u.lt.......Z.R.e..c..F..o.otnotes jillca 19n 10 5.830 58.3 *LE WPLICATE: 603919143 Dup. '.a.r.am..e.te.r.......................h..i.t.s.........6.0.3.8.8.6.3.0.0...R.e.s.u.l.t.......R.w......F.o.otnotes . Silica .on 20.90 19.30 8 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS Thls reportshall not be reproduced,except in full, without the written consent oi Paca Analytical Selvices. Inc. Page 56 BACK TO MAIN 3- M Medical Department Study: T-6295.10 I Analytical Report: FACT TOX-099 LRN-U2400 m h e n Anrlytlc8l Srrvlcw, Inc. LOlrdBEvd. Lmexa, Ks 66219 Phone: 913.599..5665 Fax: 913.599.1759 DATE: 10/24/00 PAGE: 4 Pam Projcct W r : 6045697 C'llcnt Project ID: SCIENCE SOlUTIONS/1037874 Insistent with EPA guldellns UllPQnded conantrations am dlsplaved md haw! been used to calculate X Rec and RPD values. 1 wot oetectd wot Cllwlrbla . 3. Plcc~lngLMrlt :;! '0 <. I RelrtlyePercentMfferulce. . . . : .. .. . . . . - . . . <. . .. I .e.:_ . i' 2; , : . . . . . . 1 3M Environmental Laboratory REPORT OF LABORATORY ANALYSIS Thio report shall not bs reproduced, except In full. without the wilten consent of Pace Analytical Services. Inc. Page 57 BACK TO MAIN 3-M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Record of Deviation I. Identification I.- - - - ~ - IStudy / Project No. FACT-TOXqg9 -... ___ Argus 418-012 ___-.. ._ Deviation Type (Check one) 0 SOP -..___--__ -__ X Method I3Equipment Procedure OProtocol 0 Other: ---.__ -__ ~ -- Document Numbeds): ETS-8-7.0 i D a It e ( s ) of 1212iioo occurrence: 11. Description: IRequired Proc- edurdprocess: Section 14.4.1 states the matrix concentration. spike -____ percent recoveries must be ~- within +/- 30% of the spiked-- .-Actual Procedurdprocess: _.---___L--- - -T-he liver matrix spike (8553F-MS-250ppb-3) percent recovery was 156% and th.-e mean matr-ix____ ~ --s.--pikdmatrix spike duplicate percent recovery w_as 1_38%~ . - - ___ Ill. Actions Taken: (such as amendment issu.-e--d-,SOP revision,etc.) - . This deviation was written. . - 1.Recorded By rll- i Date . ... 4 1 Jqcf&t& m y L &add&. 3% hn.4 A+#?-&. A S X L f . 6 L V a , & PA& Ol/O2/Ol , Authorized By (StiCdy Director /Project Lead) Date k , h & __ -- 3M EnvironmentalLaboratory F o ET~S-4-8.0 3M Environmental Laboratory Deviation No. a (assigned by Study Director or Project Lead at the end of study or project) Page 58 3-M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LHN-U2400 Record of Deviation 1. Identiffcation No. Argus Study / Project FACT-TOX-099 18-1012 -...........I_.-_...-.....___._._-... .. 4 ..... _..__ .... ""..._l__ _I,_". .......... _._.___._ - ------.-. -. ._. Deviation Type 0 SOP X Method 0 Equipment Procedure (Check one) OProtocol a mer: _____________. ~ Document Number@): ETS-8-7.0 .. .. --I_ ......._ .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . i Date(s) of occurrence: j Entire study II. Description: Reauired Procedurdtmcess: - _ ~ - -_____ __._ - Section 13.1.6describesthe calculations that should be used to convert extract concentrationto matrix concentration. ___ -____. Actual Procedurdpro-cess: - ~ .-.___ -____ __----- .__ In order to accommodate purity information, the actual calculation used varied somewhat fiom that written in section 13.1.6. Two additional factors, salt correction and standard punty, were added. The first accommodates the mass difference between the analytical standard (C8F17SO3K)and the target analyte(C8F17S03-), while the second addresses the purity of the analytical-r_ef_erencematerial, determ_in_ed_a_fte_r the study was-c-o-mpleted. - - _- - .- 111. Actions Taken: _____-______-. . (.s.u.ch._.a.s_.a_.m._e.n_.d_.m_.e_.n_.t_.is_.s_ued,SOP revision, etc.) This deviation was written. -- .. I.- I IV. Impact on Study / Project ___I__---..-- ..... _ _....... __ The updated calculations accommodate new information and are an improvement. No adverse - .... . -____________ .. .___........ AuthorizedBy (StudyDirector /Project Lead) - - - .- .- - _ Date F o En~-4-8.0 3M Environmental Laboratory (assignedby Study Director OT Project Lead at tkad of study or project) 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, "Extractionof Potassium Perfluorooctanesulfonateor other FluorochemicalCompounds from Serum for Analysis using HPLC-Electrospray/MassSpectrometry,"(14 pages) 0 ETS-8-6, "Extraction of Potassium Perfluorooctanesulfonateor Other FluorochemicalCompounds from Liver for Analysis Using HPLC-Electrospray/MassSpectrometry",(14 pages) 0 ETS-8-5, "Analysis of Potassium Perfluorooctanesulfona1:eor Other Fluorochemicalsin Serum Extracts Using HPLC-ElectrospraylMass Spectrometry" (9 pages) 0 ETS-8-7, "Analysis of Potassium Perfluorooctanesu1fonat:eor other Fluorochemicalsin Liver Extracts Using HPLC-Electrospray/MassSpectrometry" (10 pages) 3M Environmental Laboratory Page 60 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 3M ENVIRONMENTALLABORATORY METHOD EXTRACTION OF POTASSIUMPEXFLJJOROOCTANESULFONATEFONATEOR OTHER FLUOROCHEMICALCOMPOUNDS FROM SERUM FOR ANALYSIS USING HPLCELECTROSPRAYMSAPSESCTROMETRY Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen,Glenn Langenburg Revision Date: q / a 7 m Approved By: Group Leader Technical Reviewer "f2c 1.9 Date (ly /;2& 4 Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate(PFOS) or other fluorochemical compounds h m serum. 1.2 Applicable compounds: Flumchemical surfactants or other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey,and human serum or other fluids as designated in the validation report. Word 6/95 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS frois Senun Page 1 of 14 Page 61 3 M 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 procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-tert-butyl ether (AWE). In this method, seven fluorochemicalswere extracted:PFOS,PFOSA,PFOSAA,EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 Definitioizs). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MBE. The MtBE extract is removed and put onto a nitrogen evaporatoruntil dry. Each extract is reconstituted in 1.O mL of methanol, then filteredthrough a 3 cc plastic syrkge attachedto 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) C,F,,SO;. 3.2 PFOSA perfluorooctane sulfonylamideC$,,SO,Nf-L, 3.3 PFOSAA: perfluomoctane sulfonylamido (ethyl)acetate C,F,,SO,N(CH,CH,)CH,CO~ 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonannido)-ethyl alcohol C*F,,SO,N(~,CH,)CCHZOH 3.5 PFOSEA: perfluomoctane sulfonyl ethylamide (:,F,,SO2N(CH2CH3)H 3.6 M556: C,F,,SO,N(H)(CH,COOH) 3.7 Surrogatestandard lH-lH-2H-2H perfluomoctane sulfonic acid 4.0 WARNINGS AND CAUTIONS 4.1 Health and safety warnings 4.1.1 Use universalprecautions, especiallylabomtory coats, goggles, and gloves when handling animal tissue, which may contairt pathogens. 5.0 INTERFERENCES 5.1 There are no interferencesknown at thistime. 6.0 EOUIPMENT 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrihge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 3 M IEnvironmental Laboratory ETS-84.1 Extraction ofPFOS from Serum 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 (2 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendarf or disposablepipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifbgetubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser- 3.0 to 10.0mC 7.9 Syringes, capable of measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe fdtm, 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 withmethanol and 3 times with Milli-Qm water. Rinse syringes a minimum of 5, times with methanol, 3 rinses from 3 separate vials. 8.0 REAGENTS AND STANDARDS 8.1 TbyepMeiIllri-eQa%e4nwt garteardaenwdamteary, hbGe lplir-oQvmideodr beqyuaivMal!Uen-tQ;aTllOwCatePrluussTedMsiynsttheims methodshould 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammoniumhydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (N%COJ, J.T.Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), 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 (3MSpecialty Chemical Division),molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction of PFOS from Serum Page 3 of 14 3M Einvironrnental Laboratory Page 63 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 8.93 PFOSAA (3MSpecialty Chemical Divisilon), molecular weight = 585 8.9.4 EtFOSE-OH(3M Specialty ChemicalDivision), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty ChemicalDivision),,molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (l-H,l-H, 2-H, 2-H C8F13S03Hm) olecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes thanlisted in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 10N sodium hydroxide (NaOH): Weigh approximately 200 gNaOH. Pour into a 1000mL beaker containing 500 mL Milli-Qm water, mix until all solids are dissolved. Store in a 1L Nalgene bottle. 8.10.2 1N sodiumhydroxide (NaOH): Dilute 10N NaOH 1:lO. Measure 10mL of 10 N NaOH solution into a 100mLvolumetric flask and dilute to volume using Milli-QTMwater. Storeina 125 mL Nalgene bottle. 8.10.3 ' 0.5 M tetrabutylammoniumhydrogen sulfate(TBA):Wei approximately 169g ofTBA into a 1L volumetriccontaining 500 mLMia-Ql ewater. Adjust to pH 10 using approximately44to 54 mL of 10N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-Qm water. Store in a 1L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as ' needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonatelsodiumbicarbonate buffer (Na&O,/NaHCO,): Weigh approximately26.5 g of sodium carbonate (Na$03)and 21.0 g of sodium bicarbonate (NaHCO,) into a 1L vo1umt:tric flask and bring to volume with MilliQmwater. 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 fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS,1.02 ppm PFOSA, 0.987 ppm PFOSAA,and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100mg of PFOS into a 100mL 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 solutionwith methanol for a working standard 1solution of approximately 50 ppm. 8.11.6 Dilute working standard 1with methanol for a working stkdard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Serum Page 4 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 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard 1-H,l-H, 2-H, 2-H, C8F13S03Hinto a 50 mL volumetric flask: and record the actual weight. 8.12.2 Bring to volume with methanol for a smogate stock of approximately 1000-1200 PPm. 8.12.3 Prepare a surrogateworking standard. Transfer approximately 1mL of surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard of 100 ppm. Record the actual volume transferred. 9.0 SAMPLHEANDLING 9.1 All samples are received h z e n and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperaturepncr to extraction. 10.0 OUALITYCONTROL 10.1 Solvent Blanks, Method blanks and matrix bllanks 10.1.1 An aliquot of 1.0 mL methanol is used a~13a solventblank. 10.1.2 Extract two 1.0 mL aliquots of h4illi-Qn' water followingthisprocedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquotsof the s m i followingthis procedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and miitrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usuallythe control matrix received with each sample set. 10.23 Expected concentrationsWill fall in the mid-range of theinitial calibrationcurve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicateper 40samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibrationcheck samples to ensure the accuracy of 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 continuingcalibrationcheck from the same matrix used to prepare the initial curve. 3M Ihvironmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum 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 w i t h the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100ppb, rather than 5 ppb - 1000 ppb). 11.o CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1mL of serum to a 15mC centrifige tube. '11.1.2 Ifmost sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract less than 0.50 mL of matrix. Record each sample volume on the extraction sheet, 11.1.3 While preparing a total of twenty aliquots in 15 mL centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1IIILaliquots,or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen standards, two matrix blanks, arid two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ICTS-8-5.0-V-1, which lists the working ranges and the Linear Calibration Range (LCR)for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each working standard, standard blank, for the or continuing check, concentration to fall add appropriate amount of surrogate Wjthin the calibration curve range 5 ppb - 1000 ppb. 113 Extract spiked matrix standards following 12.6-12.16 ofthis method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS frsm Serum Page 6 of 14 Page 66 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Working standard - (approx. conc.) 0.500 ppm 0.500 ppm 5.00 ppm 5.00 ppm 5.00 ppm 50.0 ppm 50.0 ppm 50.0 ppm 50.0 ppm PL Approx. final conc. of - analyte in matrix Blank 10 0.005 ppm 20 0.010 ppm 5 0.025 ppm 10 0.050 ppm 20 0.100 ppm 5 0.250 ppm 10 0.500 ppm 15 0.750ppm 20 1.OO ppm 12.0 PROCEDURE 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 Returnunused samples to fkeezer d e r extraction amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Label the tube with the study number, sample II), date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as describedin 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the Calibration curve standards. Also prepare matrix spikes and contiwing calibration standards. I 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 To each sample, add 1niL.0.5 M TBA and 2 mlL of 0.25Msodium carbonatdsodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-tert-butyl ether. 12.12 Cap each sampleand put on the shaker at a setting of 300 rpm,for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. ETS-8-4.1 Exbaction of PFOS froin Serum 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 15mL centrifuge tube with the same informationas in 12.5. 12.15 Remove 4.0 mL of the organic layer to this c l a i 15 mL centrifuge tube. 12.16 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.17 Add 1.0mL ofmethanol to each centrifbge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 02 pm nylon mesh filter to a 3 cc syringe and transferthe sample to this . syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst($ performing the extraction. 12.21 Cap and store extracts at room temperature or at approximately 4 "C until analysis. 12.22 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 DATAANALYSISAND CALCULATIONS 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL ofstandard x concentration of standard (UE / d l = . mL of standard +mL of surrogate standard + initialmatrix volume (d) Final Concentration (pg/mL) of PFOS ini matrix 14.0 METHODPERFORMANCE 14.1 The method detection limit (MDL)is analyte and matrix specific. Refer to MDL report for specificMDL and limit of quantitation (LOQ) values (see Attachments B and C). 14.2 The following quality controlsamples are extractedwith each batch of samples to evaluate the quality of 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 of the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method perfbrmance criteria. 15.0 POLLUTIOPNREVENTIOANND WASTEMANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction of PFOS froin Serum 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 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, a!;appropriate. 17.0 ATTACHMENTS 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDLJLOQ values and summary 17.3 Attachment CyCalibration standard concentrationworksheet 18.0 REFERENCES 18.1 The validation report associatedwith thismethod isETS-8-4.0 & 5.0-V-1. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 AFFECTEDDOCUMENTS 19.1 ETS-8-5.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number 1 Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1.0 mL. - Revision Date 04/02/99 3M Environmental Laboratory ETS-8-4.1 Extractionof PFOS from Serum Page 9 of 14 Page 69 3M Medical Department Study: T-6295.10 Extraction Worksheet E'rS-8-4.1 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 DatcSoiked/Analvst Vortex 30 sec. Filter using a 3cc B-Dsyringewith a 0.2pmfilter into a 1.5 mL autosamplevial Cont. Cal. Verificationsused same matrix as for std curve. Attachment A ETS-8-4.1 Extractionof PFOS frcm Serum I Page 10 of 14 Page 70 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 MDLLOQ values for rabbit serum Standard Calibration Curve MDLLOQ values in rat, bovine,monkey, and human serum, and monkeyplasma were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalentto the rabbitrespwes, therefore, theirMDL and LOQ will be lhe same values as determined in rabbit Senun. Please see LOQ Summary and MDL studyin ETS-84.0& 5.0-V-1for furtherinformation. AttachmentB:MDULOQ Summary 3RM-ntat-Caboratory ETS-8-4.1 Extraction of PFOS from Serum Page 11 of 14 Page 71 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Compound: PFOS I I Preparedrange I LCRfiom I %Recovery 1 RSD I Full Range - 0.995 978 24.8 - 978 4.67-11.0 Rabbit Serum Prepared range of standards (PPb) tng/mL) I FullRange 1. 0.993 -976 I 4.93 - 97.6 LCB Grom curve @Pb) (ndmL) 4.93 -976 4.93 - 97.6 % 1 88-103 0.993 - 976 - 4.93 976 I 5.10-14.7 I 9.85-14.7 5.08-13.9 5.10-14.5 Rabbit Serum of standards Full Range . LowCurve High curve 1K 0.991 - 974 - 4.92 247 - 49.2 974 0.991 - 974 curve 24.7 - 974 9.74 - 247 97.4 - 974 9.74 - 974 97-107 95-1 15 6.38-21.8 4.11-23.2 AttachmentB:MDYLOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 12 of 14 Page 72 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 - - Full Range I 1 I I P & e 0.993 976 4.93-97.6 49.3 976 9.76-97.6 77-110 97-107 High curve 49.3 - 976 97.6 - 976 I lix 1 0.993-493 I 9.76-976 I 86-111 I Compound: PFOSEA I I I Prepatedrange I LCRfiom %Recovery I I Rabbitserum I ofstandards I curve 1 Range 1 11.2-25.5 14.1-21q 11.5-19.6 11.1-21.2 I RSD I Range I I FullRange =me I= I 0.993-976 --493-248 I 49.3 -976 1 0.993 -976 I 24:8 - 976 1, I 9.76-248 I I 49.3 - 976 I I 9.76-976 I 96106 I 10.1-16.2 I 91-110 I 11.8-19.5 I 86-106 I 10.2-18.2 I 95-1:17 I 10.1-19.1 I Prepared range LCR from % Rabbit Serum of standards curve - Full Range Low Curve (PPb) (ng/mL) 0.993 - 976 4.93 - 97.6 (ppb) (ndmL) 24.8 - 976 - 9.76 97.6 -H 88-106 100-105 4.82-17.9 5.95-18.2 1 97.6-976 I 97.6-976 1 81-111 I 5.11-9.74 I 1 0.993-976 I 9.76-976 I 97-110 I 4.77-19.5 I AttachmentB:MDLnOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction ofPFOS from Serum 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 - FIuids Prep date(@: Standard number: Analyte(s): Equipment number: , Sample matrix: Final solvent and TN: Blank fluid/identifler: Methodlrevision: Target analyte(s): FC mix std approa 0.500ppm: FC mix std approx. 5.00 ppm: FC mix std approa 50.0ppm: Surrogate std approx. 100ppm: PFOS PFOSA PFOSAA EtFOSE PFOSEA NE56 All All Stdconc Stdconc Stdconc Stdconc Stdconc Strlconc Am't Finalvol Calculated concentrations of standards in the sample matrix - Validated ranges approximate concentrations PFOSAA 5.00-1 000 Bovine EtFOSE-OH PFOSEA Human Attachment C Ion Pair Standard Curves ETS-8-4.1 Extraction of PFOS from Serum 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 ENVIRONMENTLAALBORATORY EXTRACTIOONF POTASSIUM P E R @ L U O R O C ~ ~ ~ s ~ F O ONRAOlTTHER FLUOROCHEMICALCOMPOUNDSFROM LIVE:RFOR ANALYSIS USING HPLC- ELECTROSPRAYIRIASSPSIEC~ROMETRY Method Number: ETS-8-6.0 Adoption Date: 03 ) l L149 Author: Lisa Clemen, Robert Wynne Approved By: Revision Date: Laboratory Nianaier U Group Leader Technical Reviewer Date FWIK3 Date 071,y IF9 Date 1.0 SCOPEAND APPLICATION 1.1 Scope: Thismethod is for the extraction of potassiumperfluorooctanesulfonate(PFOS)or other fluorochemical compounds h m liver. 1.2 ApplicableCompounds: Fluorochemical surfactantsor other fluorinatedcompounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. Word 6.0195 ETS-8-6.0 Extraction of PFOS from Liver Page 1 of 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 SUMMARYOF METHOD 2.1 This method describes the procedure for extractingpotassium perfluorooctanesulfonate (PFOS)or other fluorochemicalsurfactants from liver, or other tissues, using an ion pairing reagent and methyl-tert-butyl ether (MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH,.PFOSEA,M556, and surrogate standard. An ion pairing reagent is added to the sarnple and the analyteion pair is partitioned into MtBE. The Mtl3E extract is transkred to a centrifugetube and put onto a nitrogen evaporatoruntil dry. Each extract is reconstituted in 1.O mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pmnylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 DEFINITIONS 3.1 PFOS: perfluorooctanesulfonate(anion of potassium salt) C$,,SO, 3.2 PFOSA perfluorooctane sulfonylamideCBF,,SOzNH, 3.3 PFOSAA: perfluorooctane sulfonylamido (ethy1)acetate C,F,,SOzN(CHzCH3)CHzC0, 3.4 , EtFOSE-OH: 2(N-ethylpduorooctane C$ ,SOzN(CHzCH3)CHzCHzOH sulfonamid0)-ethyl alcohol 3.5 PFOSEA pdwrooctane sulfonyl ethylamideC,F,,SOzN(CH,CH3)H 3.6 M556: C ,p ,SO2N(H)(CHZC00H) 3.7 Surrogate standard: lH-lH-2H-2H perfluorooctane sulfonic acid 4.0 WARNINGASNDCAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions, especiallylaboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 INTERFERENCES 5.1 There are no interferences known at this b e . 6.0 EOUIPMENT 6.1 The following equipment is used while performirig this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 Ultra-Turrax T25 Grinder for grinding liver samples Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR ETS-8-6.0 Extractionof PFOS from Liver 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 of holding 250 mL and 1L 7.5 Volumetric flasks,glass, type A 7.6 I-CHEMvials, 40 mL glass 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriatesize) 7.8 Centrifuge tubes, polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor - 3.0 to 10.0 ml 7.11 Syringes, capable of measuring 5 pL to 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposableplastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm,25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with MilliQ" water. Rinse syringes a minimumof 9 times with methanol, 3 rinses fforn 3 separate Vials. 8.0 REAGENTANSD STANDARDS 8.1 Type I reagent grade water, Milli-Qm or equivalent;all water used in this method should be M&Q* water and be provided by a Milli-Q TOC Plusm system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8;3 Tetrabutylammoniumhydrogen sulfate(TBA),Kodak or equivalent 8.4 Sodium carbonate (Na+20,), J.T.Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), J.T.Baker or equivalent 8.6 Methyl-tert-butyl ether, Omnisolv, glass distille:d or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen fromsupplier 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 ExtractionofPFOS from Liver Page 3 of 14 3M Ihvironmental Laboratory Page 77 3M Medical DepartmentStudy: 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 SpeciaItyChemical 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 S m g a t e standard:4-H, perfluorooctanesulfonic acid (1-H,l-H, 2-H, 2-H C$,,SO,H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 10N sodium hydroxide (NaOH): Weigh approximately200 g NaOH. Pour into a 1000mL beaker containing500 mLMilli-QTMwater, mix until all solids are dissolved. Store in a 1L Nalgene bottle. 8.11.2 1N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:lO. Measure 10mL of 10N NaOH solution into a 100mZ, volumetric flask and dilute to volume using Milli-QTMwater. Store in a 125mLNalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): W.eigh approximately 169 g of Tl3A into a 1 L volumetric containing 500 mL Milli-Qm water. Adjust to pH 10 using approximately44 to 54 mL of 10N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTMwater. Store in a 1L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1N NaOH soluticm. 8.11.4 0.25 M sodium carbonatdsodiumbicarbonatebuffer (Na.$O,/NaHCO,): Weigh approximately 26.5 g of sodium carbonate(Na&Od and 21.Og of sodium bicarbonate (NaHCOJ into a 1 L vo1ume:tric flask and bring to volume with Milli- Q" water. Store in a 1 L Nalgene bottle. 8.12 Standardspreparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemicalstandards, (asappropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.OO ppm PFOS,1.0'2 ppm PFOSA, 0.987 ppm PFOSAA,and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100mg of PFOS into a 100mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000ppm (CLdmL). 8.12.5 Dilute the stock solution with methanol for a working standard 1solution of approximately 50 ppm. ETS-8-6.0 ExtractionofPFOS from Liver Page 4 of 14 3M Environmental Laboratory 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 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60mg of surrogatestandard 1-H,l-H, 2-H,2-H, C,F,,SO,H into a 50 mlvolumetric flask and record the actual weight. 8.13.2 8.13.3 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 PPm. Prepare a surrogate working standard. Transfer approximately 1.0 ml of surrogate stock to a 10mlvolumetric flask and bring to volume with methanol for a working standard of 10-20ppm. Record the actual volume transferred. 9.0 SAMPLHEANDLING 9.1 All samples are received h z e n and must be kept kozenuntil the extraction is performed. 10.0 OUALITYCONTROL 10.1 Matrix blanks and method blanks 10.1.1 An aliquot of 1.0 mL methanol is used a;a solventblank. 10.1.2 Extract two 1.0mL aliquots of Milli-Q"` water followingthis procedure and use as method blanks. 10.1.3 Extract two 1.0mL aliquots of liver homogenate followingthis procedure and use as matrix blanks. Refer to 11.1.6. -. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually a control liver received with each sample set. 10.2.3 Expected concentrationswill fall in the inid-rangeof the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicateper 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum,one continuing calibrationverification sample per group of 10 samples. For example, if a sample set = 34, four verifications are prepared and extracted. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 5 of 14 Page?+ 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 10.3.3 Prepare each continuing calibration verification from the same matrix used to prepare the initial curve. 10.3.4 The expected concentrationswill fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of - the initial calibration curve. This is necessary if the analystmust quantitate using only the low end of the calibrationcurve (for example, 5 ppb - 100ppb, rather than 5 ppb 1000ppb). 11.0 CALIBRATION AND STANDARDIZATION 11.1 Prepare matrix calibration standards 11.1.1 Weigh approximately40 g of liver into a 250 mL Nalgene bottle containing 200 m L s Milli-QTMwater. Grind to a homogeneous solution. 11.1.2 If 40 g is not available,use appropriate amountsof liver and water to ensure a 1:5 ratio. 11.13 Refer to 13.0 to calculatethe actual density of liver homogenate and the concentration of solid liver tissue dispersed in 1.0 mL of homogenate solution. 11.1.5 Add 1mL of homogenateto a 15 mL centrifugetube. Re-suspend solutionby shakingbetween aliquotswhile preparing a total of eighteen 1mL aliquots of homogeneous solution in 15 XI&centrifugistubes. 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 Typicdly use the standard concentrationsand spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen samples, two matrix blanks, and `two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 andiETS-8-7.0-V-1 or AttachmentB, which liststhe working ranges and the Linear CalibrationRange (LCR)for calibration curves. 11.1.9 Use Attachment C as an aid in calculatingthe concentrationsof the working standards. .Referto 13.0 to calculate actual concentrations ofPFOS in calibration standards. 11.2 To each working standard, blank, or continuing verification, add appropriateamount of - surrogate working ppb 1OOOppb. standard for the concentration to fall within the calibration curve range 5 3M lfnvironmental Laboratory ETS-8-6.0 Extraction ofPFOS from Liver Page 6 of 14 - - 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 11.3 Extract spiked liver homogenates following 12.14-12.25of this method. Use these stand=& to estabkh each initial curve on the rnaS;jspectrometer. I Table 1 Working Standard (Approx-. Conc.) 0.50 m m as 0.50 ppm 0.50 pprn ' 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 pprn 50 PPm Pl Approx. finalconc. of PFOS in liver - Blank 2 0.005 p- p- m 4 0.010 ppm 10 0.025 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 30 4 12.0 PROCEDURE 12.1 Obtain ftozen liver samples. 12.2 Cut approximately 1 g of liver using a dissectkg :scalpel. This part of the procedure is best performed quickly, not allowing the liver to thaw. 123 Weigh the sample directly into a tared plastic sampulevial. 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 grinderprobe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sampleWith 2.5 mLswater using a pipette. 12.9 Take the grinder apart and clean it with methanoll after each sample. Refer to Ah4DT-EP22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3M Environmental Laboratory ETS-8-6.0 Extraction ofPFOS from Liver 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 appropriatevolume, of homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube With the identical information as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1mL aliquots ofMilli-QTMwater to cmtrifugetubes. These will serve as method blanks. 12.13 Spike all samples, including blanks and standardsready for extraction with surrogate standard as described in section 11.2. 12.14 Spike each m a t h with the appropriateamount of standard as described in 11-1,or Table 1 of that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.15 Vortex mix the standard c w e samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.17 To each sample, add 1mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonatdsodium 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 3500rpm,or until layers are well separated. 12.21 Label a fresh 15mL centrifugetube with the same information as in 12.10. 12.22 Remove 4.0mL of the organic layer to the fresh '15 mL centrifugetube. 12.23 Put each sample on the analyticalnitrogen evaporatoruntil dry,approximately 1to 2 hours. 12.24 Add 1.OmLto each centrifuge tube using a graduated pipette. 12.25 Vortex mix for 30 seconds. 12.26 Attach a 0.2pmnylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(:s) perfonring the extraction. 12.28 Cap and store extracts at room temperature or at approximately4 OC 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 . Extraction of PFOS from Liver 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 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.1 Calculate the average density of the liver h?mogenate by recording each mass of ten separate 1.O mL aliquots of homogenate. Average density (mg/mL) =Average mass (mg) of the aliauots 1.0mL aliquot 13.1.2 Calculate the amount of liver (mg) per 1.0 mL homogenate (or concentration of dispersed solid tissue per mL of homogena.te suspension) using the following equation: p of Liver x Average densiW of homopnate (me/xrL) (g of Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrationsof PFOS and other fluorochemicalsin calibration standards using the following equation: J.LLof Standard x Concentration (UE /d='Fi) nal Concentration(pglg or mgkg) mg Liver/ 1 mL homogenate* of PFOS in Liver *refer to 13.1.2 for details. 14.0 METHOD PERFORMANCE 14.1 The method detection limit (MDL) is d y t e and matrix specific. Refer to MDL report for specificMDL and limit of quantitation(LOQ) values (refer to Attachments B and C). 14.2 The followkg quality control samples are extract'edWith eachbatch of samples to evaluate the quality ofthe 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 of the extraction. 14.23 Continuing calibration verification samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-7.0 for method performance criteria. 15.0 POLLUTION PREVEh'TlON AND WASTE MANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 3M Environmental Laboratory ETS-8-6.0 Extraction ofPFOS from Liver 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 Completethe extractionworksheet attached to this method, and tape in the study notebook or include in the 3-ring studybinder, as appropriate:. 17.0 `TABLEDSI, AGRAMFSL,OWCHARATNSD,VALIDATION DATA 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDULOQ values and surrmiary 17.3 Attachment C, Calibrationstandardcalculation and concentrationworksheet 18.0 REFERENCES 18.1 The validation report associatedwiththis method js ETS-8-6.0 & 7.0-V-1. 18.2 AMDT-EP-22, "Routine Maintenance of Ultra-Twrax T-25" 18.3 FACT-M-1.1, "Extraction of PFOS or Other Anionic Fluorochemical Surfactants fiorn Liver for Analysis Using HPLC-ElectrosprayMas Spectrometry" ' 19.0 AFFECTEDDOCUMENTS 19.1 ETS-8-7.0, "Analysis of Liver Extracts for Fluomchemicalsusing HPLC-Electrospray Mass Spectrometry" 20.0 &VISIONS Revision Number. .. Reason For Revision Revision 3M EnvironmentalLaboratory ETS-8-6.0 Extraction of PFOS from Liver Page 10 of 14 Page 84- 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 IComments AttachmentB:MDLLOQ Values 3M Einvironmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver .._ _ Page 11 of 16 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Compound PFOS PFOSA PFOSAA EtFOSE-OH M556 PFOSEA MDL (ppb) 8.45 3.50 24.6 108 82.3 33.9 LOQ (ppb) 26.9 11.1 78.3 345 262 108 Linear Calibration Approximate concentrations to be used for preparing the - Standard Calibration Curve 30 ppb 1200 ppb 12ppb - 1200ppb 30ppb - 1200ppb 60 ppb - 900 ppb* 60 ppb - 1200ppb I 3Oppb- 1200ppb MDWLOQ values in rat, bovine, and monkey liverwen:not statisticallydetermined. Two curves in each of these matriceswere extracted and analyzedwith the rabbit liver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalentto 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 Bt 7.0-V-1for furtherinformation * EtFOSE-OHestimates only for MDL and LOQ. Did not meet criteria for validation. Attachment B: MDYLOQ Values ETS-8-6.0 Extractionof PFOS from Liver Page 12 of 16 3M EnvironmentalLaboratory - - 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 high std curve ' .highstd .: curve Attachment c Standard Calculations 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS fiom Liver Page 13 of 14 -- - 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Ion Pair Standard Curves - Tissue Prep date(s): Analyte(s): Sample matriP. Methodhevision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Standard number: Equipment number:' Final solventand T N Blank liverr'identifer: Actual concentrationsof standards in the FC mix Calculated concentrations of standards in the sample matrix 299 299 299 299 I 299 299 599 599 599 599 599 599 898 898 898 898 898 898 1198 1198 1198 1198 1198 1198 Rabbit Bovine Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS fiom Liver 3M Environmental Laboratory 0.500 Page 14 of 14 Page 88 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF POTASSIUM PJW?LUOROOC"ESULFONATE OR OTHER FLUOROCHEMICUIN SERUM]EXTRACTS USING HPLC-ELECTROSPRAYMASPSESCTROMETRY Method Number: ETS-8-51 Author: Lisa Clemen,Robert Wynne Approved By: Adoption Date: 03/01/99 Revision Date: q l * f l Laboratory Manager Group Leader L Technical Reviewer Date 4/w /99 Date Y h 0 b/f 4 Date 1.o SCOPE AND APPLICATION 1.1 Scope: This method describes the analysis of serum t:xtracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human sc:rum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 1 of 9 w- ratory Page 89 3 M 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 other fluids, using HPLC-electrospraylmass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ioln characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS)anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometerto further verify the identity of a compound by detecting daughter ions of the parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro I1 triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization @SI), Atmospheric Pressure chemical Ionization (APcI), Thennospray, etc. The ionizationprocess in these techniques occurs at atmosphericpressure(Le., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solutionare transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry,Mass Spectrometer(MS), Tandem Mass Spectrometer(MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupolemass selective detectors. Ions are selectivelydiscriminated by mass to charge ratio ( d z )and subsequently detected. A single MS may be employed for ion detection or a series ( M S I M S ) for more specific fragmentation information. 3.4 Conventional vs. %spray probe interface: The latest models of Micromass Quattro I1 triple quadrupole systems (post 1998) utilize a "2-spray" conformation. The spray emitted from a probe is orthogonalto the cone aperture. In the conventional conformationit is aimed directly at the cone aperture, after passing through a tcmtuous pathway in the counter electrode. Though the configuration is different,the methods of operation, cleaning, and maintenance are the same. However, 2-spray components and conventionalcomponents are not compatible with one another, but only with similar systems (Le., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operationof these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specificto the instrument (Micromass Quattro I1 triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 WARNINGSAND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solventswear appropriate protective gloves, eyewear, and clothing. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 of 9 3M EnvironmentattabetfttePp---- 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 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 EOUIPMENT 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source 6.1.2 HPllOO 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 nitrogen gas regulated to approximately 100psi (House air system) 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 Mini-Q"" water, all water used in this method should be Milli-Qm water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 SAMPLHEANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrihge tubes until analysis. ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 3 of 9 Page 91 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 9.2 f analysis will be L-.dyed, extracted standards an^ samples can be refrigerated at approximately4" C, or at room temperature, until analysis can be performed. 10.0 QUALITCYONTROL 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determinecontamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the . recovery efficiency. 10.2.2 Matrix spike duplicatesare prepared and analyzed to m e a w e the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicateconcentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 ContinuingCalibrationVerifications 10.3.1 Continuingcalibrationverifications are analyzed to veri@ the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. 11.0 CALIBRATIOANND STANDARDIZATION 11.1 Andyze the extractedmatrix standardsprior to and following each set ofextracts. The average of two standard curves will be plotted by linear regression (y = my + b), weighted l/x, not forced through zero, using MassLynx or other suitablesoftware. 11.2 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitatinglow levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attemptingto quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000ppb). This will reduce inaccuracy attributedto linear regression weighting of high concentration standards. ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 4 of 9 Page 92 3M Medical DepartmentStudy: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Time 0.00 min. 8.50 min. 11.O min. 12.0 min. MeOH 40% 90% 90% 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. ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 5 of 9 Page 93 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 12.3.3 12.3.4 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instmment uses these parameters at the followingsettings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 literskour 12.3.6.2 ESI nebulizing-gas 10-15 litemhour 12.3.6.3 HPLC constant flow mode, flow rate 10- 500 pL/min 12.3.6.4 Pressure 4 0 0bar (Thisparameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefilly 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 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Using the cross-flow counter electrode in the ESMS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriateMassLynx USER'S GUIDE). Press the start button. Ensure start and end samplenumber includes all samplesto be analyzed. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Backmund Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Exuected Conc. - Calculated Conc. x 100 Expected Conc. 13.1.6 Calculate actual concentrationof PFOS, or other fluorochemical,in matrix (Pg/m: {ne:of PFOS calc. from std. Curve x DilutionFactor) x 1 ug (Initial Volume of matrix (mL) f mL of Surrogate Standard) 1000ng Final Volume (mL) ETS-8-5.1 Analysis of Serum Extract Using ESMS Page 6 of 9 Page 94 3M Medical Department Study: T-6295.10 I BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 14.0 METHOPDERFORMANCE 14.1 Method Detection Limit (MDL)and Limit of Quantitation(LOQ)are method, analyte, and matrix specific. Please see ETS-8-4.1, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values are must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The ? value for the calibrationcurve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within k 30% of thdspiked concentration. 14.5 ContinuingCalibration Verifications 14.5.1 Continuingcalibration verificationpercent recoveriesmust be f 30% of the spiked concentration. 14.6 If criteria listed in this method performance section isn't met,maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteriahave not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 POLLUTION PREVENTION ANDWASTE MANAGEMENT 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibrationcurve by linear regression,weighted l/x,then print these graphs and store in the study folder. 16.4 Print data integration summary,integrationmethod, and chromatograms, fi-om MassLynx, and store in the study folder. ETS-8-5.I Analysis ofSerumExtract Using ESMS 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 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 TABLESD.IAGRAMSF.LOWCHARTANSD. VALIDATION DATA 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 REFERENCES 18.1 FACT-M-4.1, "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical compounds fiom Serum for Analysis Using HPLC-EIectrospray/Mass Spectrometry 18.2 ETS-9-24.0, ``Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/MassSpectrometerQuattro 11triple quadrupoleSystems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-1. 19.0 AFFECTED DOCUMENTS 19.1 ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum for Analysis Using HPLC-ElectrosprayMassSpectrometry" 20.0 REVISIONS Revision Number. 1 Reason For Revision Section 6.1.2 Clarification of H P l l O O system components. Section 11.l Average of two curves,not standard values, are used for plotting linearregression and added the llx weightingof the curve. Section 12.2.2.4 Clarification of solvent ramp. Section 17.1 Changed fiom attachment B to A. Revision Date 04/02/99 ETS-8-5.1 Analysis of Serum ExtractUsing E S N S Page 8 of 9 Page 96 3M Medical Department Study: T-6295.10 Laboratory Study # Study: Test Material Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument SoftwareNersion: Filename: R-Squared Value: Slope: Y Intercept Date of Extractiodhalyst: Date of AnalysidAnalyst BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 GroupDose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integrationsummary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initialvolume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using E W S Page 9 of 9 Page 97 BACK TO MAIN 3-M Medical Department Study: T-6295.10 Study #: FACT-TOX-099 Analytical Report: FACT TOX-099 LRN-U2400 3M Environmental Lab -- Method Modification Method: ETS-8-5.1 "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicalsin Sera Extracts Using HFLC-ElectrosprayMass 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 calibrationstandard after every tenth sample, with a minimum of one per batch. Modify method to read: 10.3.2 Analyze a mid-range calibrationstandardat least after every ten samples, witha minimum of one per batch. Section 14.5.1 Method reads: 14.5.1 Continuingcalibrationverificationpercent recoveries must be withinf 30% of the spiked concentration. Modify method to read: 14.5.1 At least one continuing calibrationverification 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)calibrationcurve may be deactivated if insfnmentaldrift affects the data. The first curve and acceptable calibration checks shall bracket usable &ta. 3kl Environmental Laboratory Page 98 BACK TO MAIN -3M Medical Department Study: T-6295.10 Study #: FACT-TOX-099 Analytical Report: FACT TOX-099 LRN-U2400 Section 14.3.3 Method reads: NA Modify method to read: 14.3.3 Calibration standards with peak areas less than 2 times the cmve matrix blank should be deactivated to &qual* 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 deactivatedto optimize a linearrange appropriate to the data. Section 14.3.5 Method reads: NA Modify method to read: 14.3.5 A curvepoint may be deactivated if it deviates more than 30% fiom the theoreticalvalue when the curve is evaluated over a linearm g e appropriateto the data. Section 14.3.6 Method reads: NA Modify method to read: 14.3.6 A valid caliiration curve must contain at least 5 active points. Lk 4 Signatureof PAI and date Ir/2t/VZ) I* Signatureof Sponsor and date I Signature of Study Director and date 3M Environmental Laboratory Page 99 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 3M ENVIRONMENTLAALBORATORY METHOD ANALYSIS OF POTASSIUM PERnUOROOCTANJ3SULFONATEOR OnaER EtUOROcHEMlCALS TN LIVEREXTIUCrSUSING HPLC-ELEXXROSPRAYMASSSPECTROMETRY Method Number: ETS-8-7.0 Author: LisaClemen, Glenn Langenburg Adoption Date: 0 712.2[qf Revision Date: Nk Approved By: Group Leader .4. Technical Reviewer 3 / (4/93 Date Date 1.0 SCOPE AND APPLICATION 1.1 Scope: This method is for the analysis of liver extracts for fluorochemicalsurfactantsusing HPLC-electrospray/mas spectrometry. . 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 of Liver ExtractUsing ES/MS Page 1 of 10 -nr - 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 This method describesthe analysis of fluorochemicalsurfactantsextracted from liver using HPLC-electrospray/massspectrometry,or similar system as appropriate. The analysisis performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate(PFOS) anion,m/z = 499. Additionally, samples may be analyzed using a tandem mass spectrometerto further verify the identity of a compound by detecting daughter ions of the selected parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: ElectrosprayIonization @SI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmosphericpressure (Le. not under a vacuum). 3.2 Electrospray Ionization @S, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the applicationof a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS),Tandem Mass Spectrometer ( M S M S ) : The API Quattro r[triple quadrupolemass spectrometer is equippedwithtwo quadrupole mass selective detectors and a collisioncell. Ions are selectivelydiscriminatedby mass to charge ratio ( d z )and subsequentlydetected. A single MS may be employed for ion detection or an ion may be selected in the first quadrupole,fiagmented in the collision cell, and these hgments may be analyzed in the second quadrupole. 3.4 Conventional vs. Zspray probe interface: The latest models of Micromass Quattro I1 triple quadrupole (post 1998)utilize a "Z-spray" conformation. The spray emitted fiom a probe is orthogonal to the cone aperture. In the conventional conformationit is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the confguration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatiblewith one another, but only with similar systems (Le. Z-spray components are compatiblewith other Z-spray systems,etc.) 3.5 Mass Lynx Software: System software designed for the specific operation ofthese Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details refer to the manual specific to the instrument (MicromassQuattro IItriple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 WARNINGS AND CAUTIONS 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 3hM- ttrtboratory ETS-8-7.0 Analysis ofLiverExtract Using ESNS Page 2 of 10 Page 101 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure of 400 bar (5800 psi). If the back pressure exceeds 400 bar, the H P l l O O will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFERENCES 5.1 To minimize interferences when analyzing samples, Teflon shall not be used for'sample storage or any part of instrumentationthat comes in contact with the sample or extract. 6.0 EOUIPMENT 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 Micromass Quattro IItriple quadrupoleMass Spectrometerequippedwith an electrospray ionization source. 6.1.2 H P l l O O low pulse solventpumping system,solvent degasser, column compartment, and autosampler 7.0 SUPPLIESAND MATERIALS 7.1 Supplies 7.1.1 High purity grade air regulated to approximately100psi (house air system) 7.1.2 HPLC analytical column, specificsto be determined by the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 ml centrifhge tubes 8.0 REAGENTSAND STANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-Q'" water (ASTMtype I),all water used in this method shouldbe ATSM type I, or equivalent, and be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately0.300 g ammonium acetate. Pourinto a 2000 mL volumetric container containing 2000 mL Mill;-Q" water, mix until all solids are dissolved. Store at room temperature. ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 3 of 10 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 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 SAMPLEHANDLING 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis, 9.2 If analysiswillbe delayed, extracted standards and samples may be stored at room temperature, or refkigeratedat approximately4' C,until analysiscan be performed. 10.0 QUALITY CONTROL, 10.1 Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each batch to determinecontaminationor canyover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and d y z e d to measure the precision and the recovery for each analyte. 10.23 Analyze a matrix spike and matrix spike duplicate per forty sample?. With a minimum of 2 spikesper batch. 10.2.4 Matrix spike and matrix spike duplicate concentrationswill fall in the mid-range of the initial calibrationcurve. Additional spike concentrationsmay fall in the lowrange of the initial calibration curve. 10.3 Continuing CalibrationChecks 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibrationstandard every tenth stample,with a minimum of one per batch. 11.0 CALIBRATION AND STANDARDIZATION 11.1 Analyze the extracted matrix standards prior to and following each set of sample extracts. The average of two standard curves will be plotted by linear regression (y =mx +b), weighted l/x, not forced through the origin, using MassLynx or other suitable software. 11.2 Ifthe curve does not meet requirementsperform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 3 M F- . - ETS-8-7.0 Analysis of Liver Extract Using ESMS Page 4 of 10 Page 103 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 11.3 For purposes ofaccuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from5 ppb to 100ppb rather than the full range of the curve (5 ppb to 1000ppb). This will reduce inaccuracy attributed to linear regressionweighting of high concentrationstandards. 12.0 PROCEDURES 12.1 AcquisitionSet up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last digit of year-increasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MSfile) for acquiring 12.1.13 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then mass spectrometerheadings and select SIR (Single Ion Recording)or MRM (Multiple Reaction Monitoring). Set IonizationMode as appropriateand mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRS. Save acquisition method. If MS/MS instrumentsare employed, additionalproduct ion hgmentation informationmay be collected. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.13 Typicallythe analyticalbatch run sequencebegins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuingcalibrationverificationinjected standard after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samplesbut may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HP1lOO/autosamplerat the following conditions or at conditionsthe analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook ' 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Jnject/sample = 1 12.2.2.3 Cycle time = 9 minutes ETS-8-7.0 AnaIysis of Liver Extract Using ESMS Page 5 of 10 Page 104 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 ' LRN-U2400 12.2.2.4 Solvent ramp conditions [ Time I MeOH I 2.0 mM I Ammonium acetate 60% 7.0 min. 40% 40% 60% 12.2.2.5 Press the "Start"button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenanceof the Micromass Quattro II Triple QuadrupoleMass SpectrometerFitted with an Atmospheric Pressure Ionization Source," for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 123.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. Ifthe tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 123.4 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operateto initiate source block and desolvation heaters. 12.3.6 Open the Inlet Editor. 123.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500 a m i n or as appropriate 12.3.6.3 Observe droplets coming out of the tip of the probe. A fine m i s t should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is abserved 12.3.6.4 Allow to equilibratefor approximately 10minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimizethe response: 12.3.7.1 Drying gas 250-400literdhow 12.3.7.2 ESI nebulizing gas 10-15 litershow 12.3.7.3 HPLC constant flow mode flow late 10-500 pL/min 12.3.7.4 Pressure C400 bar (This parametix 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 of Liver Extract Using ESMS Page 6 of 10 3Iw-1- tal Laboratory Page 105 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 123.9 Click on start button in the Acquisition Clmtrol Panel (this may vary among MassLynx versions, refer to appropriateEdassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 DATAANALYSIASND CALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = - Observed Result Backmowid Result x 100 Expected Result 13.1.5 Calculatepercent difference using the folllowingequation: % Difference = Exuected Conc. - Calculated Conc. x 100 Expected Conc. 13.1.6 Calculate actual concentrationsin matrix (pg/g): lnna of PFOS calc. fromstd. C u r e x Dilution Factor) flnitial Weinht oFLiver (E) Final Volume (d) x 1 UP 1000 ng 14.0 METHODPERFORMANCE 14.1 Method Detection Limit (MDL)and Limit of Quantitation(LOQ) are method, analyte, and matrix specific. Refer to ETS-8-6.0, Attachment B for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks and Matrix Bl.anks 14.2.1 Solvent blanks, method blanks, and matrix blanks must be below the lowest standard in the calibration curve. 143 Calibration Curves 14.3.1 The 3 value for the calibrationmust be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries must be within k 30% of the spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verificationpercait recoveries must be within f 30% of the spiked concentration. 14.6 If criteria listed in the method performance section are not met, maintenancemay be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. ETS-8-7.0 AnalysisofLiver ExtractlJsingESMS Page 7 of 10 Page 106 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 14.7 If data are to be reported when performance criteriahave not been met, the data mist be footnoted on tables and discussed in the text of the report. 15.0 POLLUTION PREVENTION AND WASTE MANAGEMENT 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or projlxt number, acquisition method, integrationmethod, sample name, extraction date,dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisitionmethod from MassLynx to include in the appropriatestudy folder. Copy these pages and tape into the instrument mlog. 16.3 Plot the calibrationcurve by linear regression, weighted l/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatogramsfrom MassLynx and store in the study folder. 16.5 Summarize data using suitablesoftware (Excel 5.W) and store in the study folder, refer to Attachment A for an example of a summary spreadsheet. 16.6 Back up electronicdata to appropriate medium. Record in study notebook the file name and location of backup electronicdata. 17.0 TABLES.DIAGRAMFSL. OWCHARTS, AND VALIDATION DATA 17.1 Attachment A: ETS-8-7.0Data summary spreadsheet 18.0. REFERENCES 18.1 FACT-M-2.1, "Extraction of PotassiumPerfluorcloctanesulfonateor Other Fluorochemical Compounds from Liver for Analysis Using HPLC-ElectrosprayMass Spectrometry" 18.2 ETS-9-24.0,"Operation and Maintenance of the ldicromassAtmospheric Pressure IonizationMass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validationreport associated withthis method is ETS-8-6.0 & 7.0-V-1 19.0 AFFEC~EDDOCUMENTS 19.1 ETS-8-6.0,"Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Liver or Fluid for Analysis Using HPLC-ElectrosprayMass Spectrometry" ETS-8-7.0 Analysis of Livcr.ExtractUsing ESMS Page 8 of 10 Page 107 3M Medical Department Study: T-6295.10 20.0 REVISIONS Revision Number Reason For Revi BACK TO MAIN Analytical Report: FACT TOX-099 LRN-LJ2400. Revision ETS-8-7.0 Analysis of Liver Extsact Using ESMS 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 # Dilution Factor Final Conc. udg Slope: 'I'akentrom linear regresson equation. Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis of LiverExtract U!hgESMS Page 10 of 10 Page 109 BACK TO MAIN -3M Medical Department Study: T-6295.10 Study #: FACT-TOX-099 Analytical Report: FACT TOX-099 LRN-U2400 3M Environmental Lab -- Method Modification Method: ETS-8-7.0 "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemicals in Liver Extracts Using HPLC-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 calibrationstandard after every tenth sample, with a minimumof one per batch. Modify method to read: 10.3.2 Analyze a mid-range calibrationstandard at least after (:very ten samples. with a minimum of one per batch. Section 14.5.1 Method reads: 14.5.1 Continuingcalibrationverificationpercent recoveries must be withinf 30% of the spiked concentration. Modify method to read: 14.5.1 At least one continuing calibrationverification per ten samplesmust 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)calibrationm e may be deaclivated if instrumentaldrift affects the data. The fist curve and acceptablecalibration checks shallbracketusable dats. 3M Environmental Laboratory Page 110 BACK TO MAIN - 3M Medical Department Study: T-6295.10 Study #: FACT-TOX-099 Analytical Report: FACT TOX-099 LRN-U2400 Section 14.3.3 Method reads: NA Modi& method to read: 14.3.3 Calibrationstandardswith peak areas less than 2 times the m e matrix blank shouldbe deactivatedto disqualifya data range that may be affectedby background levels of the analyte. Section 14.3.4 Method reads: NA Mod$ method to read 14.3.4 Low or high curve points may be deactivatedto optimise a linear range appropriateto the data. Section 14.3.5 Method reads: NA Modify method to read: 14.3.5 A curve point may be deactivated ifit deviates more than30%from the theoretical value whenthe 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 calibrationcurve must containat least 5 active points. Signature of PAT anddate Il/zs / u z ) Signature of Sponsor and date /3D& ;& Signature of Study Director and date 3M Environmental Laboratory Page 111 BACK TO MAIN 3M Medical Department Study: T-6295.10 Appendix D: Data Summary Tables Analytical Report: FACT TOX-099 LRN-U2400 Table 6. LOQ Values for PFOS Used in FACT TOX-099 Analyses by Method and Usage Dates I Method I EffectiveDate I LOQ I Usaae Dates 1 ETS-8-51 4/26/99 .. 0.0214 ....... ...... 0.00426 n/oo 1011...9.. /00 _.,.... 10/24/00, 11 ETS-8-7.0 7/22/99 ... ........ 0.00529 ........... 0.0529 11/1/00 . 12121/00 Table 7. Average Concentration of PFOS (pglg) in Rabbit Liver by Dose Group Dose Group I Mean PFOS in Liver Group 1 (Control) 0 mglkglday 0.239 Group 2 0.1 rngkglday 13.1 Group 3 1.O mglkglday 133 Group 4 2.5 mglkglday 2117 Group 5 3.75 rngkglday 416 NOTE: It is not possibleto verify true recoveryof endogenousanalytefrom tissues without radidabeledreferencematerial.The onb meastirementof accuracy availableat thistime, matrixspike studies, indicatethat tlie dataare quantitativeto *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 (pglmL) in Rabbit Serum by Dose Group I DoseGroup I Mean PFOSinSerum I Group 1 (Control) 0 mgkglday 0.0690 Group 2 0.1 mgkglday I Group 3 1.Omgkg/day Group 4 2.5 mgkg/day 2.73 23.8 45.8 Group 5 3.75 mgkglday 88.9 NOTE: It is not possibleto vemjtrue recovery of endogenous analytefrom tissues without radidabeledreferencematerial.The only measurement of accuracy availableat this time, matrixspike studies, iixlicatethat the data are quantitative to *30% or greater. 3M IEnvironmental Laboratory Page 113 3M Medical Department Study: T-6295.10 Table 9. PFOS (pg/g) in Rabbit Liver by Individual Animal BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 Group 2 0.1 mglkglday Group 3 1.O mglkglday Group 4 2.5 rnglkglday Group 5 3.75 mglkglday 8555F 8556F 8557F 8558F 8559F 8560F 8561F 8562F 8563F 8564F 8565F 8566F 8567F 8568F 8569F 8570F 8571F ~ 0.240 13.1 13.0 12.2 11.2 16.1 98.6 141 158 297 360 - NOTE:It is not possibleto verifytrue recovelyof endogenousanalytefrom tissues without radidabeledreferencematerial.The only measurement of accuracy available at this time, matrix spike studies, indicate that the data are quantitativeto *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 I O . PFOS (pg/mL) in Rabblit Serum by Individual Animal Group# I Animal# I Calnc PFOS unlmL Group 1 0 mglkglday 8554F 0.0956 0.0411 8555F 0.0703 Group 2 8556F 2.78 0.1 mglkglday 8557F 2.92 8558F 2.86 8559F 2.64 8560F 2.44 Group 3 8561 F 22.1 1 .O mglkglday 8562F 25.0 8563F 24.3 Group 4 8564F 46.5 2.5 mglkglday 8565F 46.3 8566F 44.5 Group 5 8567F 100 3.75 mglkglday 8568F 92.1 8569F 81.6 8570F 88.1 I 8571F 1 82.7 NOTE: It is not possible to veriry tlue recovery of endcgenous analyte from tissuesMthout radidabeledreferencematerial.The only measurementof accuracy available at this time, matrixspike studies, indicatethat the data are quantitativeto +30% or greater. 3M lhvironmental Laboratory Page 115 3M Medical Department Study: T-6295.10 Appendix E: Data Spreadsheets BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 3M Environmental Laboratory Page 116 BACK TO MAIN Medical Department Study: T-6295.10 FAcT-Tux-wy Argus# 418-012 Analytical Report: FACT TOX-099 L!?N-U240G Study: Product Number(Tcsf Subslance): Ma~rix: MethodRevision: AnalyticalEquipment SystemNumber: lnslnimcnt softwarcNerdon: Filename: R - S q u d Value: Slope: Y-lnterccp: Dates of Extraction/Analyn: Dater of AnalysirlAnalysc Dace of Data ReductionlAnalyrc Sample Data 418-012, Oral DevdopmentalToxicity Study of PFOS in Rabbits: 4 mauices T-6295 Rabbit Serum ETS-8-4.1 & ETS-8-5.1 SoupOZO199, Davey010799 Masslynx 3.4 See Below Sce Attachments Sce Auachments See Auadrmcnts 10/17100 SAL 10/19Xx), lOR4/00.11107/00 KJWMMH 10/23M). I O N 1 0 0 , l l B ~KJHlMMH Box* 00-% Group Dosc Method Blk Matrix BIk Qc I GI'OUD 2 sample # 10170-H20 BIk-3 10170-HZO BIk-4 RBS10170-S~nBlk-3 RBS1017C~SenB k - 4 RBS10170-MS-250 ppb-3 RBS10170-MSD2SO ppb-3 8553F I 8555F I 8556F Exhcthm VOL OIL 1 1 1 1 I I 1 1 I Sumogatc verified NA NA NA NA NA NA NA I NA I I NA I PFOSpuritY comctbn FvtW 0.8640 0.8640 0.8640 0.8640 NA NA 0.8640 0.8640 0.8640 PFOS DiLu6im Ftor I 1 I 1 I 1 1 I 11 I 20 I PFOS cone Il&hllL 0.00 0.M) 0.00 0.00 259 270 111 81.4 161 Fiknme (ope SO01107020 wOl024004 S001107021 DO01024005 S001107022 S00I 107023 S001019017 CMwatraIion of PFOS u w L or % Rec <LCQ (0.00426uglmL) 40Q (0.00426uglmL) <LOQ (0.00426ughnL) <LOQ (0.00426 ughnL) 104% 109% 0.0956 I s001019019 I I wO1024017 1 0.0703 2 7R M~M PFOS UBlmL cLOQ(0.00426 ughnL) cLOQ (0.00426udmL) 107% 0.0690 RSD std. Dev. M S M D RPD NA NA 4% 1 0.0273 Group 3 Gmup4 croup 5 8560F 8561F 8562F 8563F 8564F 8565F 8566F 8567F 85681 85693 8570F 8571F 1 NA 0.8640 20 141 DO01024021 2.44 1 NA 0.8640 50 512 DO01024024 22.1 1 NA 0.8640 50 578 DO01024025 25.0 I NA 0.8640 50 563 DO01024026 24.3 I NA 0.8640 100 538 DO01024027 46.5 I NA 0.8640 100 536 DO01024028 46.3 I NA 0.8640 100 515 DO01024031 44.5 1 NA 0.8640 200 578 DO01024032 100 1 NA 0.8640 200 533 wOI024033 92.1 1 NA 0.8640 200 472 DO01024034 81.6 1 NA 0,8640 200 510 W01024035 88.1 I NA 0.8640 200 478 DO01024038 82.7 2.73 0.194 6.25 23.8 I .49 2.40 45.8 1.10 8.45 88.9 7.51 A . 0 1/30/200 I 3 5 6 PM 3 Page 117 Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 I nat I tn.nn LniY-uc.+uu w 8553F-MS-250 ppb-3 NA Analyzed IU21Aw) 8553F-MSD-250 p~b.3 NA QC 8553F-MS-ZSO ppb-3 NA Analyzed IllDlAw) 8553F-MSDZH)e 3 NA Cmp 1 8553F NA 8554F NA 8555F NA c-2 8556F NA 8557F NA 8558F NA MS9F NA 856oF NA Group 3 8561F NA 856ZF NA 8563F NA croup 4 8564F NA 8565F NA 8566F NA Croup 5 8567F NA 8568F NA 8569F NA 857oF NA 857lF NA OriginllPFOSLOQ(30.6 n/g,6.12ng/g.61.2 ng/g)upducdmrcIlcclpurity 1.0074 NA I. m 4 NA I .an4 NA LOU74 NA 1.0074 NA 1.0474 NA 0.9981 NA 1.0151 NA 0.9931 NA 1.0386 NA 1.0152 NA 1.0263 NA 1.0586 NA 1.0014 NA 1.0398 NA 1.0057 NA 1.0223 NA 0.9507 NA 1.0137 NA 1.0494 NA 1.0268 NA 1.0328 NA 0.9820 NA informatiwonOl/3OXIl. LAC01130/01 NA NA NA NA 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 0.8640 804 698 608 584 335 229 278 M8 299 293 264 382 242 328 381 345 426 338 ' 506 487 519 413 529 I 465 DO01U1029 156% * I 360 DO0l221030 1ZM 138% 26% . 1 271 Do01101042 9152 I 247 WIlOlC43 83% 87% 9% .. I 287 wOIZZl031 0.287 .. I 189 DO01221032 0. I89 20.5 1 240 DO01221033 0.240 ** 0.239 0.0490 H) 13100 wO1101018 13.1 50 13003 Do01101019 13.0 50 12193 Do01101020 12.2 50 11246 DWIlOlO2l 11.2 50 I6069 Do01101022 16.1 13.8 13.1 1.81 500 98561 Do0llOlW 98.6 Mo 141282 wO1101026 141 500 158417 wO1101027 158 loo0 296674 wOllOlO28 297 23.2 133 30.8 loo0 w9 Do01101m 360 11.8 loo0 294686 wO1101032 295 loo0 431642 DO01101033 432 ?17 37 4 100 W23 DO01101034 401 loo0 436300 DO01101035 436 loo0 345684 Do01101036 346 10.9 IWO 465531 DO01101039 466 416 45.5 ETS-8-7.0 Excel 97 3 Environmental Laboratory 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 1.0 ug x PC= Reported Concentration (pg/mL) EV (mL) 1000 ng Calculation Used for Group 3, Animal ID 8561F 512 ng/mL x 50 x 1 mL x 1.0 pg x 0.864 = 22.1 pg/mL 1 mL 1OOOng AR- Analytical result from MassLynx summary DF- Dilution factor FV-Final extract volume (1.O mL unless otherwise noted) EV-Volume of sera extracted PC-PFOS purity correction factor (86.4%) Formula Used for Liver Analyses in Study FACT TOX-099 a AR (ng/g) x curve ('I x DF x 1.O pg a sample 1000 ng (l) a curve is assumed to be: 1 g liver 5 mL H20 x PC = R.eported Concentration (pg/g) Calculation Used for Group 3, Animal ID 8561F 241.52 ng/g x 1 g/ 5 mL 1.0586 g/ 5 mL x 500 x 1.0 pg X 0.864 = 98.6 pglg 1000 ng AR- Analytical result from MassLynx summary a curve-Density of the liver standard curve, assumed to be l g liver/ 5 ml water a sample-Density of the liver sample (g sample/ 5 mL,H20) DF- Dilution factor PC-PFOS purity correction factor (86.4%) 3M Ihvironmental 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 Medical Department Study: 1-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Cmtrc Analytical Laboratorks, Inc. 3048 Research Drive Phone: (814) 231-8032 State College, PA 16801 Fax: (814) 231-1253 or (814) 231-1580 INTERTM CERTIFICATE OF ANAIYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018B 3M Product: PFOS, Lot 171 Reference #: SD-OO!) Purity: 86.4% 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 2. 0.007 wt.lwt.% 3. 1.355 wt.lwt.% 4. 6.552 wt.lwt.% 5. 0.003 wt.lwt.% 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5 . Nitrite 1. co.015wt.lwt.% 2. 0.27 wt.lwt.% 3. c0.040 wt./wt.% 4. co.009 wt.lwt.% 1 2. PFPA 3. HFBA 4. NFPA Elemental Analysis': 1. Carbon2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine COA023-018B 2; 1 2. co.1wt.lwt.% 3. <0.1 wt.lwt?h 4. <0.25 wt./wt.% I Theoretical Value = 17.8% 1. 12.08 wt.lwt.% Theoretical Value = 0% 2. 0.794 wt.lwt.% Theoretical Value = 0% 3. 1.61 wt.lwt.% 4. TheoreticalValue= 5.95% 4. 10.1wt.lwt?h 5. TheoreticalVall e = GO% 5 . 50.4 wt.lwt.% Page 1 of 3 Page 121 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 P Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-1580 INTEMM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-01813 Date of Last Analysis: 08/31/00 ExpirationDate: 08131/01 Storage Conditions: Frozen 510C 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.OO%+ POAA, 0.30%) - Total impurity from all tests = 13.56% Purity = 100% 13.56% = 86.4% 'Potassium is expected in this salt form and is therefore not considered an impurity. 3purityby DSC is generally not applicable to materials of low purity. No endotherm was pl observed for this sample. 4Sulfurin the sample appears to be converted to so4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determinationin the elementalanalysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 'TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 'Theoreti I value calculations based on the mpirical finmula, CSFI~SO~X(M+W=538) This work was conducted under EPA Good Laboratory l?ractice Standards (40 CFR 160). COA023-018B 3M Environmental Laboratory Page 2 of3 . Page 122 BACK TO MAIN 3M Medical Department Study:T-6295.10 Analytical Report: FACT TOX-099 a\ LRN-U2400 CEntrE Analyticall Laboratories, Inc. 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 LCMS Purity Profile: c5 C6 c7 Total 1.56 6.38 ~ 1.63 10.60 Note: The C4 and C6 values were calculatedusing 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. - PreparedBy: Dayd S. Bell ?//O Date ical Laboratories db Date ntre Analytical Laboratories COAO23-018B 3M EEnvironrnental Laboratory Page 3 of 3 Page 123 3M Medical Department Study: T-6295.10 BACK TO MAIN Analytical Report: FACT TOX-099 LRN-U2400 le: (814) 231-8032 lzax: (814) 231-1253 or ( 8 i 4 j 231-1580 INTERIM CERTIFICATE 0 . FANALYSIS Revision 1(9/7/00) CentreAnalytical Laboratories COA Reference#: 023-018A 3M Product: PFOS,Lot 217 Reference #: SD-018 Purity: 86.99I0 Test Name I SnPrifirntir 2. Magnesium 3. Sodium 4. Potassiumz 5. Nickel 6 . Iron Positive 1 1. 0.005wt./wt.% I 2. 0.001 wt./wt.% 3. 1.439wt./wt?h 4. 6.849 wt./wt.% 5 . <0.001 wt.lwt.% COA023-018A 3M Einvironrnental Laboratory I. c0.015wt./wt.% 2. 0.59 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006wt./wt.% 6. <0.007wt./wt.% 7. 8.76wt./wt.% 1. CO.1 wt./wt.% I. Theoretical Valut: = 17.8% 2. TheoreticalValuc = 0% 2. 0.244 wt./wt.% Pam 1 of3 Page 124 BACK TO MAIN 3 M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 CmtrE Analytical Laboratories, Inc. 3048 Research Drive State College, PA 16801 P Phone: (814) 231-8032 Fax: (814) 231-1253or (814)231-1580 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COG Ref'erence#: 023-018A Date of Last Analysis: 08/31/00 Expiration Date: 08/3 1/01 Storage Conditions: Frozen 1-10"C Re-assessmentDate: 08131/01 'Purity = 100%- (sum of metal impurities, 1.45% +LC/FdS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMRimpurities, 1.93%+organicacid impurities, 0.38%+POAA, 0.33%) Total impurity from dl tests = 13.09% Purity = 100%- 13.09% = 86.9% zPotassiumis expected in this salt form and is therefore not consideredan impurity. 'Purity by DSC is generally not applicable to materials o.llowpurity. No endothermwas observed for tbis sample. 4Sulfurin the sample appears to be convertedto so4 and hence detectedusing the inorganic anion method conditions. The anion result agrees we11 with the sulfur odnettehremriensautilotsn, ithnethSeOe4leismneonttacloannsaidlyesriesd, laennidminpgucroitny.fidence. to this interpretation. Based 'TFA HFBA NFPA PITA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid PenWuoropropanoic acid %'heoretical value calculations based on the empirical formula, C8F17SO3-K' (MW-538) This work was conductedunder EPA GoodLaboratoryPractice Standards(40CFR 160). COAQ23-018A 3 M Environmental Laboratoty Page 2 of 3 Page 125 BACK TO MAIN 3 M Medical Department Study: T-6295.10 Analytical Report: FACT TOX-099 LRN-U2400 Centre Analytical Laboratories, lnc. 3048 Research Drive State College,FA 16801 f- Phone: (814) 231-8032 Fax: (814) 231-1253 or (814) 231-7580 INTERlM CERTIFICATE OF ANALYSIS Centre Analytical LaboratoriesCOA Reference#: 023-018A LCMS Purity Profile: Impurity c4 c5 C6 c7 Total wt./wt. % 1.22 I .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 vaIue was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: P//O/d Date &3 ' Date Laboratory Manager, Centre AnalyticaI Labmoratories COA023-018A 3 M Environmental Laboratory Page 3 of 3 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.VlM., Ph.D., Study Director Date . John L. Butenhoff, Ph.D., Sponsor Representative Bill Reagen, Ph.D., Laboratory Manager Date O J/u&/-I Date Kris J. Hansen, Ph.D., PrincipalAnalytical lnvestigator 0 =/u 5/0 1 Date 3M Einvironmental Laboratory Page 127