Document JJXagqzy6gvqnDyGrGrqXvYK2

3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Study Title Analytical Study 2(N-Ethylperfluorooctane sulfonamido)-ethanol in Two Generation Rat Reproduction Analytical Laboratory Report Title Determination of the Presence and Concentration of PFOS, M556, PFOSAA, and PFOSA in the Liver and PFOS, M556, PFOSAA, PFOSA, and EtFOSE-OH in the Sera of Crt:CD*BR VAF/Plus* Rats Exposed to EtFOSE-OH Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date At signing Performing Laboratories Sera Analyses Liver Analyses 3M Environmental Laboratory Building 2-3E-09, 935 Bush Avenue St. Paul, MN 55106 Battelle Memorial Institute 505 King Avenue Columbus, OH 43201-2693 Project Identification 3M Medical Department Study: T-6316.5 Argus In-Life Study: 418-009 Analytical Report: FACT TOX-013 3M Laboratory Request No. U2095 Total Number o f Pages 135 3M Environmental Laboratory Page 1 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 This page has been reserved for specific country requirements. 3M Environmental Laboratory Page 2 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 GLP Compliance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, M556, PFOSAA, and PFOSA in the Liver and PFOS, M556, PFOSAA, PFOSA, and EtFOSE-OH in the Sera of Crl:CCfBR VAF/Plus Rats Exposed to EtFOSE-OH Study Identification Number: T-6316.5, FACT TOX-013, LRN-U2095 This study was conducted in compliance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations 21 CFR Part 58, with the exceptions in the bulleted list below. All raw data, protocol, analytical report and samples for this study are retained in archives at the 3M Environmental Laboratory and will be retained for a period of at least ten years. The analytical phase completed at the 3M Environmental Laboratory was performed in accordance with 3M ET&SS Standard Operating Procedures. Exceptions to GLP compliance: There were two study directors in this study. This study was designed as two separate studies. The in-life phase was considered to end at the generation and shipment of specimens. The analytical study was considered to start at the receipt of these specimens for analysis. This resulted in having two separate study directors, one for each phase of the same study. However, since the technical performance of each phase was entirely separate, no effect is expected from this exception. . Some changes made in the standard preparation logs obscured the original entry, did not document the reason for the change and/or were not initialed and dated by the person making the change. The samples that were analyzed on 3/16/00 utilized standards that had an expiration date of 2/00. Liver values generated at contract laboratories were corrected by 3M Environmental Laboratory to reflect the official purity values from the COA. Revised final reports will be solicited from the contract laboratory and will be added as a report amendment at a later date. Expiration dates on some reagents and solutions were missing. Study Director Z Sponsor Representative Date ______ O^-fo Date 9, ZjO O O 3M Environmental Laboratory Page 3 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 GLP Study-- Quality Assurance Statement Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, M556, PFOSAA, and PFOSA in the Liver and PFOS, M556, PFOSAA, PFOSA, and EtFOSE-OH in the Sera of Crl:CD*BR VAF/Plus* Rats Exposed to EtFOSE-OH Study Identification Number T-6316.5, FACT TOX-013, LRN-U2095 This study has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management. Inspection Dates Phase Date Reported to Management Study Director 10/12/99 Extraction 10/26/99 10/26/99 6/5/00-6/14/00 Data 6/16/00 6/16/00 9/11/00-9/13/00 Draft report 9/14/00 9/14/00 QAU Represntetive 3M Environmental Laboratory Page 4 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table of Contents GLP Compliance Statement............................... 3 GLP Study--Quality Assurance Statement......... 4 Study Personnel and Contributors....................... ,7 Introduction and Purpose................................... Test System................................................. Specimen Collection and Analysis................. .8 .8 .9 Specimen Receipt and Maintenance.................. Chemical Characterization................................ Dose Confirmation Analyses......................... Method Summaries........................................... 3M Environmental Laboratory........................ Preparatory Method.................................. Analytical Method...................................... Analytical Equipment................................ Deviations.................................................... Data Quality Objectives and Data Integrity.......... .9 .10 .10 .11 .11 .11 .11 .11 ..12 ..12 Data Summary, Analyses, and Results.............. Summary of Quality Control Analyses Results Summary of Sample Results........................ Statistical Methods and Calculations................. Statement of Conclusion.................................. ..13 ..13 ..14 ..14 ..14 Appendix A: Chemical Characterization, Control Matrices and Dose Confirmation Analyses.............................................................................................................. Appendix B: Protocol................................................................................................................. Appendix C: Extraction and Analytical Methods.................................................................... 15 18 37 EJS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages)................................................................................................................................. .38 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorocherrucals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages)................. .52 Appendix D: Data Summary Tables...................................................................................... Appendix E: Data Spreadsheets............................................................................................. Appendix F: Example Calculations........................................................................................ .61 .64 .70 Appendix G: Contract Lab Report.......................................................................................... .71 Appendix H: Interim Certificate of Analysis........................................................................... .131 3M Environmental Laboratory Page 5 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix I: Report Signature Page................................................................................135 List of Tables Table 1. Test System Population Demographics and Dosage Levels for Study (418-009)...............................................................................................8 Table 2. Characterization of the Test Article in Study FACT TOX-013............................. 10 Table 3. Negative Ions Monitored in 3M Laboratory Analyses......................................... 12 Table 4. Deviation Summary for FACT TOX-013........................................................... 12 Table 5. Determinations of the LOQ in the Analyses of Serum Extracts........................... 13 Table 6. Characterization of the Control Matrices Used for Sera Analyses in Study FACT TOX-013......................................................................................15 Table 7. Characterization of the Control Matrices Used for Liver Analyses in Study FACT TOX-013......................................................................................15 Table 8. Characterization of the Analytical Reference Materials Used for Sera Analyses in Study FACT TOX-013.................................................................................. 16 Table 9. Characterization of the Analytical Reference Materials Used for Liver Analyses In Study FACT TOX-013.................................................................................. 16 Table 10. Tween Dosing Confirmation for Study In-life #418-009..................................... 17 Table 11. Tween Dosing Confirmation--Matrix Spikes for Study In-life #418-009.............. 17 Table 12. Reported Fiuorochemical Levels In Sera Analyses in Study FACT TOX-013.....61 Table 12. Reported Fiuorochemical Levels in Sera Analyses in Study FACT TOX-013 (continued)..................................................................................................... 62 Table 13. Reported Fiuorochemical Levels in Liver Analyses in Study FACT TOX-013.....62 Table 13. Reported Fiuorochemical Levels in Liver Analyses in Study FACT TOX-013 (continued)............................................................. 63 3M Environmental Laboratory Page 6 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Study Personnel and Contributors Study Director Marvin T. Case, D.V.M., Ph.D, Study Director 3M Corporate Toxicology - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN, 55144-1000 651-733-5180 Sponsor John L. Butenhoff, Ph.D., Sponsor Representative 3M Corporate Toxicology - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 Analytical Chemistry Laboratories Sera Analyses 3M Environmental Laboratory (3M Lab) Kristen J. Hansen Ph.D., Analytical Investigator Liver Analyses Battelle Memorial Institute Jon C. Andre, Ph.D., Analytical Investigator 3M Lab Contributing Personnel David R. Bamidge. Ph.D. Lisa A. Clemen Lisa Dick, Ph.D. Kelly J. Dorweiler Mark E. Ellefson Sara E. Estes Barb A. Gramenz Sarah A. Heimdal Cari S. Hewitt Marlene M. Heying Harold O. Johnson Kelly J. Kuehlwein Sally A. Linda Joseph C. Pilon Scott R. Post lan A. Smith Kathy M. Stock Anh-Dao Vo Bob W. Wynne Location of Archives All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory. The test substance and 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. Control sera and liver will be maintained at the contract lab along with the test substance. 3M Environmental Laboratory Page 7 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Introduction and Purpose The purpose of the study is to determine the presence and concentration of PFOS, PFOSA, PFOSAA, and M556 in liver samples and PFOS, PFOSA, PFOSAA, EtFOSEOH, and M556 in sera samples collected from rats exposed to EtFOSE-OH. This study was initiated on 1 October 1998. Test System Five groups of FO generation male and female rats and 3 groups of F1 generation male and female rats were used as the test system. Table 1 outlines the rat population demographics and dosage levels for study 418-009. On day 4 of lactation, litters were culled to four male and four female pups, where possible. On day 21 of lactation, 25 male and 25 female pups in Groups I, II, and III were selected for continued evaluation. F1 generation male and female rats were given appropriate dosages of the test article via gavage beginning on day 22 of lactation or postpartum through the day before sacrifice. The test system species and strain selected was the Crl:CCfBR VAF/Pluse (Sprague-Dawley) rat received from Charles River Laboratories, Inc., and assigned temporary numbers until assigned to the study. Rats were permanently identified using MoneP self-piercing ear tags when assigned to the study. FO generation rats were identified with ear tags. Pups were not identified during lactation, as parameters were evaluated in terms of the litter. At weaning, each F1 generation rat selected for continued observation was identified with a Monel*self-piercing ear tag. FO female rats were approximately 65 days of age and weighed approximately 179-229g when received. FO male rats were approximately 58-67 days of age and weighed approximately 223--331 g when received. Weight data are included in Argus Research Laboratories, Inc. final report (study number 418-009). Table 1. Test System Population Demographics and Dosage Levels for Study (418-009) Population Number of F0 Generation Rats per Sex Number of F1 Generation Rats per Sex Dosage (mg/kg/day) Dosage Group I (Control) Dosage GroupII Dosage GroupIII Dosage Group IV Dosage GroupV 35 35 35 35 35 25 0 (vehicle) 25 1 25 5 -- 10 -- 15 3M Environmental Laboratory Page 8 3M Medical Department Study: T631S.5 Analytical Report: FACT TOX-013 LRN-U2095 Specim en Collection and Analysis Sample specimens were collected by Argus (study 418-009) and sent to the 3M Environmental Laboratory for analysis. Liver and sera specimens were collected from F0 male rats at the completion of the cohabitation period and F0 female rats on day 21 postpartum. Liver specimens were collected from F0 generation litters, and stomach content specimens were collected from the F0 and F2 generation litters. The analysis of the stomach contents were not part of the scope of analysis determined by the study director. The number and type of specimens collected for analyses in the analytical phase of this study are presented below. Specimens Collected from Study Groups I through V (through 11/30/98): Serum Specimens--45 specimens Liver Specimens--65 specimens Blood specimens were centrifuged after collection. Serum was then harvested and immediately frozen on dry ice and maintained frozen at -70C until shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal were frozen and retained at -70C until shipped to the 3M Environmental Laboratory. Stomach content specimens were frozen at -20C until shipped to the 3M Environmental Laboratory. Liver, sera, and stomach content specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice. Sera and liver samples were extracted beginning on October 11, 1999 using an ion pairing reagent and methyl-tert-butyl ether (MtBE) for the sera and ethyl acetate for the liver samples. Liver samples were homogenized prior to the extraction procedure. Sample extracts were analyzed using high-performance liquid chromatography-electrospray/tandem mass spectrometry (HPLC-ESMSMS) in the multiple response monitoring mode. PFOS, PFOSA, PFOSAA, EtFOSE-OH, and M556 levels were quantitated by external calibration. PFOSEA was not analyzed due to inconsistent analysis and failed QC. Analytical details are included in this report. Specimen Receipt and Maintenance The 3M Environmental Laboratory received from Argus, serum, liver and stomach content specimens collected at predetermined time points during and at the end of thein-life phase of Argus study 418-009 on 8-4-98, 10-1-98 and 1-29-99. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C 10#C. Specimens that were analyzed at Battelle were shipped frozen on dry ice. Control matrices used in liver and sera analyses were obtained from commercial sources and are presented in Table 6 and 7. Samples analyzed at the 3M Environmental Laboratory will be maintained for a period of 10 years and will be stored at the laboratory at -20*C 10C. 3M Environmental Laboratory Page 9 3M Medical Department Study: TS316.5 Analytical Report: FACT TOX-013 LRN-U2095 Chemical Characterization EtFOSE-OH CAS Number: 1691-99-2 Chemical Formula: C8F17S 0 2N(CH2CH3)CH2CH20H Molecular Weight: 571.0 Chemical characterization information on the test article is presented in tabular form below. Chemical characterization information on the analytical reference materials used in this study is presented in tabular form in Appendix A (see Tables 8 and 9) and the interim Certificate of Analysis available in Appendix I. Table 2. Characterization of the Test Article in Study FACT TOX-013 Test Article Chemical Name Source Expiration Date EtFOSE-OH FM-3929 2(N-Ethylperfiuorooctane sul!bnamido)-ethanol 3M 05/2000 Storage Conditions Ambient temperature Chemical Lot # 30035, 30037, 30039 Physical Description Purity Waxy Solid To be determined* * The purity of the test article determ ined nominally by N M R a n a ly s t. Subsequent chem ical characterization is occurring and this analytical report w il be am ended to indicate the purity when a certificata of analysis is issued. Dose Confirmation Analyses The dose confirmation data w ere collected according to a method that was not fully validated. Dose confirmation analyses were performed on test article samples taken at the start of dosage, at 6 weeks, and at the end of dosage during the in-life phase of the study. Dose confirmation analyses were performed on 3 dose levels collected during the in-life phase of the study: the results are presented in Appendix A (see Tables 10 and 11). Dose confirmation was performed by diluting the Tween dose samples with Milli-Q water into the linear range of the instrument. For each sample, a matrix spike was prepared (at approximately 50-100% of the expected dose level). In all cases, samples were analyzed versus an unextracted curve using HPLC-ES/MS/MS. The instrumental parameters and analytical conditions described in ETS-8-5.1 were used for dose solution analyses. 3M Environmental Laboratory Page 10 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 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 are located in Appendix C. The methods and analytical equipment settings used by Battelle are presented in the Battelle final report (see Appendix G). 3M Environmental Laboratory Prepara tory Method ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry* Sera samples were extracted using an ion-pairing extraction procedure. An ion pairing reagent was added to the sample and the analyte ion-pair was partitioned into MtBE. The MtBE extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol, then filtered through a 3cc plastic syringe attached to a 0.2pm nylon filter into a glass autovial. Analytical Method ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum 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-ESMSMS. For example, molecular ion 499, selected as the primary-ion for PFOS (QF17S 0 3-) analysis, was fragmented further to produce ion 99 (FSQ,-). The characteristic product-ion 99 was monitored for quantitative analysis. Analytical Equipment The following equipment and parameters are representative of those used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard* Series 1100 Liquid Chromatograph system Analytical column: Keystone* BetasilTM Ce 2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM aqueous ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: 10 minutes Start at 40%B Hold at 40%B for 1 minute Increase to 95%B over 3.5 minutes 3M Environmental Laboratory Page 11 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Hold at 95%B for 2 minutes Return to 40%B over 0.5 minutes Hold at 40%B for 3 minutes Mass Spectrometer: Micromass* API/Mass Spectrometer Quattro H" Triple Quadrupole system Software: Mass Lynx'" 3.2 Cone Voltage: 20-60 V Collision Energy: 25-45 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Z-spray source Analysis Type: Multiple Reaction Monitoring (MRM) Table 3. Negative Ions Monitored In 3M Laboratory Analyses Target Analyte Primary ion (a m u ) Product Ion (a m u) PFOS 499.0 99.0 PFOSA PFOSAA 498.0 584.0 78.0 169.0 EtFOSE-OH 630.0 59.0 M556 THPFOS 556.0 427.0 78.0, 169.0 80.0 Deviations Deviations from the original protocol and methods are documented in the table below. Table 4. Deviation Summary for FACT TOX-013 Deviation Pipettewas used instead Oxforddispenser Date(s) of Occurrence 10/12/99 0.2--1.OmLof sample was used forextractioninsteadof 1.OmL. 10/12/99 Milk curd samples were not analyzed. Entire study Impact on Study Standards andsampleswereprepared identically. Noadverseimpactonstudy. Currantwork indicates that volumes 0.5 mLprovideresultsequivalentto1mL extractionvolumes. Resultsofsample volumes <0.5 mLhavenotbeenvalidated andwinbe markedinthedatatable. Nomilkcurddata isavailableforthefinal report Data Quality Objectives and Data Integrity The following data quality objectives (DQOs) were indicated in the method performance section of ETS-8-5.1, `Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry' : 3M Environmental Laboratory Page 12 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Linearity: The coefficient of determination (r2) equal to or greater than 0.980 Lim its o f Quantitation (LOQ): The LOQ for PFOS is 5.55 ppb, PFOSA is 4.79 ppb, PFOSAA is 20.5 ppb, EtFOSE-OH is 36.2 ppb, and M556 is 19.2 ppb. Acceptable Spike Recoveries: 70-130% Data Summary, Analyses, and Results With the exceptions noted in this report, data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory method ETS-8-5.1 (see Appendix C) and the Battelle final report (see Appendix G) were met. Although extraction and analysis were initiated in September 1998, the study was reprioritized and put on hold. Upon restarting the study, the decision was made to reextract and analyze the specimens. No data from the original analysis are included in this report. The data in this report reflect only that obtained from specimens extracted on, or after October 11,1999. Summary of Quality Control Analyses Results Linearity: The coefficient of determination (r2) of the standard curves werekO.980. Calibration Standards: Quantitation of the target analytes was based on linear regression analysis (1/x weighted) of 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 concentration range most appropriate to the data. All active curve points are accurate to within 70% of theoretical value. 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 outlier curve point may have been deactivated. Quantitation of each analyte was based on the response of one or more specific product ion(s) using the multiple response-monitoring mode of the instrument (see Appendix C). Limits of Quantitation (LOQ): The LOQ is equal to the lowest accepted standard in the calibration curve (defined as a standard with a concentration that is within 30% of the theoretical value, and which has at least two times the analyte peak area detected in the extraction blanks). Table 5. Determinations of the LOQ in the Analyses of Serum Extracts Analyte Method LOQ PFOS PFOSA PFOSAA EtFOSE-OH M556 5.55 ppb 4.79 ppb 20.5 ppb 36.2 ppb 24.9 ppb 3M Environmental Laboratory Page 13 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Blanks: All blanks were below the lower limit of quantitation for the compounds of interest. To simplify analyses that were complicated by endogenous levels of fluorochemicals in unexposed rat sera, rabbit sera was selected as a suitable surrogate matrix for standard curves. Precision: Precision was determined by analysis of MS/MSD and was reproducible to within 10% . Matrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of samples and analyzed during analytical runs. With the exception of M556, all sera matrix spikes were within 30% of the theoretical concentration. Both matrix spikes showed a recovery of 69% for the M556. These results were verified. Data quality objectives will be adjusted to reflect this recovery. Surrogates: The surrogate (THPFOS) was added to all samples and standards. THPFOS was not used for quantitation, but was used to monitor for gross instrument failure. The surrogate response of each analytical run was verified to determine that it did not vary more than 50% from the mean within each analytical run. Assuming spike recovery studies form a suitable indication of endogenous analyte recovery, sera data are quantitative to 30% for all analysis but M556; M556 data is quantitated to 31%. The validity of this assumption has not been verified by other techniques. Summary of Sample Results Samples from Control Animals: Low levels of PFOS, PFOSA, PFOSAA, EtFOSE-OH, and M556 were often detected in the sera and liver of the control animals. These levels were significantly lower than those found in the low dose test animals. Samples from Dosed Animals: In general, PFOS, PFOSA, PFOSAA, EtFOSE-OH, and M556 levels found in the sera and liver of the test animals increased with dose group. 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-013. Statement of Conclusion v Under the conditions of the present studies, PFOS, PFOSA, PFOSAA, EtFOSE-OH, and M556 were observed in the sera and liver of rats dosed with EtFOSE-OH during the in-life phase of the study. 3M Environmental Laboratory Page 14 3M Medical Department Study: T631S.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix A: Chemical Characterization, Control Matrices and Dose Confirmation Analyses Table 6. Characterization of the Control Matrices Used for Sera Analyses in Study FACT TOX-013 Location 3M Lab Control Matrix Rat Serum (TN-A-2001) Rabbit Serum (TN-A-2573) Source ExpirationDate Storage Conditions Chemical Lot # Physical Description N/R--not recorded Sigma 2010 Ambient 17H9306 Rat Serum Sigma 2010 Ambient 118H8418 Rabbit Serum Table 7. Characterization of the Control Matrices Used for Liver Analyses in Study FACT TOX-013 Location Battelle Memorial Institute Control Matrix Rat Liver Source ExpirationDate Storage Conditions Chemical Lot# Physical Description N/R--not recorded Harlan N/R N/R N/R Rat Liver 3M Environmental Laboratory Page 15 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 8. Characterization of the Analytical Reference Materials Used for Sera Analyses in Study FACT TOX- 013 Location 3M Lab Materials PFOS C/trSOv PFOSA C.FjjSOjNH, PFOSAA CjF^SO/mCH^HJ (CH|COOH)) EtFOSE-OH CjF^OjNtCH^lHj) CHiCHjOH M556 CtFjjSO^HH) (CH.CHJ) THPFOS* CJVFJ*Ofi Source Expiration Date Storage Condition Chemical Lot Number Physical Description Purity 3M Specialty Chemicals 08/31/01 Ambient temperafcjre N/R 01/01/2010 Ambient temperature 171 L-15700 Vit* aystallne powder 86.4% Light yeiow waxy soW TBD N/R 01/01/2010 Ambient temperature N8 112999-09 Tan waxy toW TBD 3M CP/PCP Division 01/01/2010 Ambient temperature 936 Amber waxy sofid TBD 3M 01/01/2010 Ambient temperatura N B 113047-80 White powder TBD ICN Blomedicals 01/2010 Ambient temperature 53406 Brown waxy solid NA N/R-- not recorded TBD-- to be determined NA-- not applicable Table 9. Characterization of the Analytical Reference Materials Location Materials S ource E x p ira tio n D ate S tora ge C o n d itio n s C h e m ic a l L o t Num ber P h y s lc a l D e s c rip tio n P u rity Battello Memorial InBtitute PFOS 3M M5S6 3M PFOSAA PFOSA THPFOS* 3M 3M ICN 08/31/01 01/01/2010 Ambient temperature Ambient temperature 171 NB 113047-80 W hits crystalline powder 86.4% ViMte powder TBD 2010 Amtent temperature 817 N/R TBD 01/01/2010 Ambient temperature L-15708 Light yellow waxy soid TBD N/R Ambient temperature 59000 N/R NA N /R -- not recorded TBD-- to be determined NA-- not applicable 3M Environmental Laboratory Page 16 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2 095 Table 10. Tween Dosing Confirmation for Study In-life #418-009 Group Do* Sample Number Expected Cone. MeasuredCone. EtFOSE (ng/mL) EtFOSE(ng/mL) Group 1--Control 0 mg/mL Group2--0.2 mg/mL Group3--1.0 mg/mL Group4--2.0 mg/mL Group5--3.0 mg/mL B-418-009-A, 06/08/98 B-418-009-A, 07/15/98 B-418-009-B, 06/08/98 B-418-009-B, 07/15/98 B-418-009-C. 06/08/98 B-418-009-C, 07/15/98 B-418-009-D, 06/08/98 B-418-009-0, 07/15/98 B-418-009-E, 06/08/98 B-418-009-E, 07/15/98 0.00 NA 200000 200000 1000000 100000 2000000 2000000 3000000 3000000 0.00 NA NA NA 1020000 942000 2190000 2750000 3060000 3640000 Homogeneity Samples-- 3.0 mg/mL B-418-009-A, 05/08/98 1of 6 T B-418-009-A, 06/08/98 3of6 M B-418-009-A, 06/08/98 5 of6 B 3000000 3000000 3000000 3250000 3690000 3790000 NA= Not applicable EtFOSE %Recovery Accuracy NA NA NA NA 102 94 110 138 102 121 108 123 126 Table 11. Tween Dosing Confirmation--Matrix Spikes for Study In-life #418-009 Sample Number Expected Cone. Measured Cone. EtFOSE % MS EtFOSE (ng/mL) EtFOSE (ng/mL) Recovery Accuracy B-418-009-B, 06/08/98-MS B-418-009-B, 07/15/98-MS B-418-009-C, 06/08/98-MS B-418-009-C. 07/15/98-MS B-418-009-D, 06/08/98-MS B-418-009-0, 07/15/98-MS B-418-009-E, 06/08/98-MS B-418-009-E, 07/15/98-MS 1200 1200 900 900 900 900 1100 1100 NA NA 818 826 910 733 973 1089 NA NA 91 92 101 81 88 99 B-418-009-A, 05/08/98 1 of 6 T-MS B-418-009-A, 06/08/98 3of6 M-MS 1100 1100 949 1053 86 96 B-418-009-A, 06/08/98 5of6 B-MS NA Notapplicable 1100 944 86 3M Environmental Laboratory Page 17 3M Medical Department Study: T6316.5 Appendix B: Protocol Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory Page 18 3M Medical Department Study: T6316.5 ' Analytical Repo^ ^ ? S f 9 l i - o ' * A 3M Environmental Laboratory____________ Protocol - Analytical Study 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction In-vivo study reference num ber: Argus 418-009 Study num ber: FACT 060998.1 Test substance: 2(N-EthyIperfIuorooctanesulfonamido)-ethanol (N-EtFOSE-OH) Name and address of Sponsor: Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144 - Name and address of testing facility: 3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-09 1 St. Paul, MN 55106 Experimental start date: Expected term ination date: December 31, 1998 Method numbers and revisions: . FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry Author: Lisa Clemen jllilz+x-flzz------------ ----------------------------- t+ r- Kris Hansen Date Study Director Marvin Case Sponsor Representative / b e t'/fa r Date ft 3M^Environmental Laboratory 1 Page 19 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 1.0 Purpose__________________________________________________________________ The analytical portion of this dosing study is designed evaluate the levels of perfluorooctane sulfonate (PFOS), or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol (NEtFOSE-OH) designated by the study director, in the liver of the parent and subsequent generations of the test system, or in the serum as necessary. The in life portion of this study was conducted at Argus Research Laboratories. 2.0 Reg ulatory Compliance_______ ___________________________________________ This study is conducted in compliance with the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report. 3.0 T est M a t e r ia l s ___________________ __ ____________________________________ 3.1 Test, control, and reference substances and matrices 3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217 3.1.2 Analytical reference substance m atrix: Rat liver and serum 3.13 Analytical control substance: None 3.1.4 Analytical control substance m atrix: Rat liver and serum 3.2 Source of materials - 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability information will be included in the final report 3.2.2 Analytical reference substance m atrix: Argus Research Laboratories; traceability information will be included in the final report 3.2.3 Analytical control m atrix: 3.2.3.1 Rat liver - Argus Research Laboratories; traceability information will be included in the final report; or Rabbit liver - Covance Laboratories; traceability information will be included in the final report 3.2.3.2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report 3 3 N um ber of test and control samples. Liver samples for testing were received from 40 test animals and 10 control animals. Serum samples will be tested at the discretion of the Study Director. 3.4 Identification of test and control sam ples: The samples are identified using the Argus Research Laboratories identifiers, which consist of a letter followed by the Argus project number, the animal number, the group designation, and the draw date. 3M Environmental Laboratory 2 Page 20 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3.5 Purity and strength of materials: Characterization of the purity and identity of the reference material is the responsibility of the Sponsor. 3.6 Stability of test m aterial: Characterization of the stability of the test material is the responsibility of the Sponsor. 3.7 Storage conditions for test materials: Test materials are stored at room temperature. Samples are stored at -20 1 0 C. 3.8 Disposition of test and/or control substances: Biological tissues and fluids are retained per GLP regulation. 3.9 Safety precautions: Refer to the material safety data sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 4.0 Experim ental - Overview_________________________ I___________________________ Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-009 are received for analysis of fluorine compounds. At the discretion of the Study Director, a series of analytical tests will be performed on select tissues. Initially, all liver samples will be analyzed for PFOS by electrospray/mass spectrometry (ES/MS). On the basis of findings from these analyses, additional sample matrices may be evaluated or other metabolites may be targeted. If additional analysis is performed, a protocol amendment will be written. 5.0 Experim ental - Analytical M ethods______________________ !______________________ 5.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 5.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.4 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry 6.0 Data Analysis _________________ _______________________________________ 6.1 Data transform ations and analysis: Data will be reported as the concentration (weight/weight) of fluoride per tissue or sample, or of PFOS per unit of tissue or fluid. 6.2 Statistical analysis: Statistics used may include regression analysis of the serum concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 3M Environmencal Laboratory 3 Page 21 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 7.0 M aintenance of Raw Data and Records________________________ _________ 7.1 The following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: 7.1.1 Approved protocol and amendments 7.1.2 Study correspondence 7.1.3 Shipping records 7.1.4 Raw data 7.1.5 Electronic copies of data 7.2 Supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: 7.2.1 Training records 7.2.2 Calibration records 7.2.3 Instrument maintenance logs 7.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 7.2.5 Appropriate specimens. 8.0 References___________________________________________________________ 8.1 3M Environmental Laboratory Quality System Chapters 1, 5 and 6 8.2 Other applicable 3M Environmental Laboratory Quality System Standard Operating Procedures 9.0 Attachments__________________________________________________________ 9.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- Electrospray/Mass Spectrometry 9.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.4 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLC- Electrospray/Mass Spectrometry 3M Environmental Laboratory 4 Page 22 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Study Title Combined Oral (Gavage) Fertility Development and-Perinatal/Postnatal Reproduction Toxicity Study o f N-EtFOSE in Rats PROTOCOL AMENDMENT NO. 1 Amendment Date: July 28,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-013 LERN U2095 3M Environmental Laboratory 3M Environmental Laboratory Page 23 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Protocol FACT-TOX-013 Amendment 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL reads: The proposed study completion date is listed as 12/31/98. AMEND to read: The proposed study completion data is 6/30/00. REASON: The proposed completion date was changed to allow time for analyzing all matrices o f interest. Amendment Approval Marvin Case Ph.D., Sponsor Representative J o <h k - Dat v Kris J. ansen Ph.D., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 24 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Protocol FA C T Tox-013 Amendment Number 3 3. Protocol reads: Disposition o f test and control substances: Biological tissues and fluids are retained per GLP regulation. Amend to read: Specimens will be maintained in the 3M Environmental Laboratory specimen archives. All specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion o f analysis and submission o f the sub-contract laboratory(s) final report. The specimens will be returned with the following documentation: the signed original chain of custody and records o f storage conditions while at the sub-contract facility. Reaso n: To define in detail the appropriate disposition o f specimens analyzed at subcontract laboratories. ' . Amendment Approval Marv Case, D.V.M., Ph.D., Sponsor Representative y Q de & U f fDate T/L & ------- ------------------------------------------------- Kristen J. Hansen, Ph.D., Previous Study Director Date Dale L. Bacon, PinT 3M Environmental Laboratory Management 3M Environmental Laboratory 3M Environmental Laboratory Page 30 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Study Title Analytical Study o f 2(N-Ethylperfluorooctanesulf>namido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 January 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS LRN-U2095 FACT TOX-013 Argus Study: 418-009 3M Medical Department Study: T-6316.5 3M Environmental Laboratory 3M Environmental Laboratory Page 31 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Protocol LRN-U2095 Amendment Number 4 This amendment modifies the following portion(s) of the protocol: 1. P rotocol reads: The study director for the present study was identified in the protocol as James K. Lundburg, Ph.D. Amend to read: The role of study director for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of 20 January 2000. The previous study director, James K. Lundburg, has been reassigned to the role of Principle Analytical Investigator. Reason: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58). 2. Protocol reads: The sponsor for the present study was identified as Marvin T. Case, D.V.M., Ph.D. A mend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. 3M Environmental Laboratory 3M Environmental Laboratory Page 32 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Protocol LRN-U2095 Amendment Number 4 Amendment Approval John L ButenhoffPhD., Sponsor Representative Date lA/V^n Marvin T. Case, D. V.M., Ph.D., Incoming Study Director lo Q -trtr Date & 3JVf Environmental Laboratory 3M Environmental Laboratory Page 33 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Study Title Analytical Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 5 Amendment Date: August 31, 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-013 ET&SS LRN U2095 Argus Study: 418-009 3M Medical Department Study: T6316.5 3M Environmental Laboratory 3M Environmental Laboratory Page 34 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Protocol F A C T TO X-013 Amendment No. 5 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL reads: The Principle Analytical Investigator for the present study was identified as James K. Lundberg, Ph.D. 2. Amend TO read: The role of Principle Analytical Investigator for the present study was reassigned to Kristen J. Hansen Ph.D. REASON: The role of Principle Analytical Investigator was reassigned due to availability of resources. . 3M Environmental Laboratory 3M Environmental Laboratory Page 35 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Protocol F A C T TOX-013 Amendment No. 5 Amendment Approval John L. Butenhoff, Ph.D., Sponsor Representative / Date rc M a h in T. Case, D.V.M., Ph.D,, Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 36 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix C: Extraction and Analytical Methods This appendix includes the following methods: ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages) ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages) 3M Environmental Laboratory Page 37 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory M ethod E x tra ctio n ,of P otassium P erfluoro o cta n esu lfo n a te o r O t h e r F l u o r o c h e m ic a l c o m po u n d s f r o m Seru m f o r Analysis U sin g HPLC- E lectro spra y /M ass Spectrom etry Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: ^ ^ Approved By: 01 -- F Laboratory Manager - Date Ir k ----------Group Leader Date LAtjfc. A Technical Reviewer ______________ _____________________ Date 1.0 S c o p e a n d A p p l i c a t i o n 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: 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 from Serum P ag el of 14 Page 38 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 2.0 S u m m a r y o f M e t h o d _____________ _______________________________________________ 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-ieri-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 |im nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-5.1 or other appropriate methods. 3.0 D e f i n i t i o n s ____________________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8FI7S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2CO2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F 17S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F I7S 0 2N(CH2CH3)H 3.6 M556: C8F17S 0 2N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W a r n i n g s a n d C a u t i o n s ________________ _____ _______________________________ 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 In t e r f e r e n c e s ___________ ____________________ ___ _____________________________________ 5.1 There are no interferences known at this time. 6.0 E q u i p m e n t _________________ __________________ ___ _________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 2 o f 14 Page 39 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 S u p p l i e s a n d M a t e r i a l s _________________ ____________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable o f measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials. 8.0 R e a g e n t s a n d S t a n d a r d s ___________ _____________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na2C 0 3), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCOj), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction of PFOS from Serum Page 3 of 14 3M Environmental Laboratory Page 40 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (l-H .l-H , 2-H, 2-H C8F,3S 0 3H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 mL o f 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L 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 carbonate/sodium bicarbonate buffer (NajCO/NaHCOj): Weigh approximately 26.5 g of sodium carbonate (NsqCO^ and 21.0 g o f sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent 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 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Serum Page 4 o f 14 3M Environmental Laboratory Page 41 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 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 l-H .l-H , 2-H, 2-H, C8F13S 0 3H into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. ' 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL o f surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard of 100 ppm. Record the actual volume transferred. 9.0 S a m p l e H a n d l i n g _________________ _____________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Qu a lity Co n t r o l _____________________________________________________ 10.1 Solvent Blanks, M ethod blanks and matrix blanks 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f the serum following this procedure and use as matrix blanks. See 11.1.4. 10.2 M atrix 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 the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing check per group o f 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 5 of 14 Page 42 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 10.3.4 The expected concentrations will 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 analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _________________________________________________ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11.1.2 If most sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract less than 0.50 mL o f 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 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two standard curves, for a total o f eighteen standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-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 o f the working standards. See Section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 6 o f 14 Page 43 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix -- - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 50.0 ppm 20 0.100 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 . 1.00 ppm 12.0 P r o c e d u r e ____________________________________ _______________________________________ 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 Return unused samples to freezer after 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 ID, 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 described in 11.2. 12.7 Spike each matrix with the appropriate amount o f standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL o f 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-ierr-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. ETS-8-4.1 Extraction o f PFOS from Serum Page 7 o f 14 3M Environmental Laboratory Page 44 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. 12.16 Put each sam ple on the analytical nitrogen evaporator until dry, approxim ately 1 to 2 hours. 12.17 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 pm nylon 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. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.21 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 D a t a A n a l y s i s a n d C a l c u l a t i o n s _______ _______________________________________ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration o f standard fug /mL-)__________________ = mL o f standard + mL o f surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) of PFOS in matrix 14.0 M e t h o d P e r f o r m a n c e _____________ ____________________ __________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachm ents B and C). 14.2 The following quality control samples are extracted with each batch o f 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 o f the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t __________________________ _ 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 from Serum Page 8 o f 14 3M Environmental Laboratory Page 45 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 16.0 Records________________________ ________________________________________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 A t t a c h m e n t s ______________________ _________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 173 Attachment C, Calibration standard concentration worksheet 18.0 R e f e r e n c e s __________ ______________________ ______________________________________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l. 18.2 FACT-M-3.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 a f f e c t e d D o c u m e n t s ----------------------------------------- ------------------------------------- 19.1 ETS-8-5.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R e v i s i o n s ____________________ _________________________________________ 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 Extraction of PFOS from Serum Page 9 of 14 Page 46 3M Medical Department Study: TS316.5 Analytical Report: FACT TOX-013 LRN-U2095 Extraction Worksheet ETS-8-4.1 Study # Matrix Box # Wk/Dav DateSpiked/Analyst CCV MS MSD Surrogate Std approx, ppm actual ppm # FC-Mix approx. 0.5 pm actual # ppm FC-Mix approx. 5 ppm actual ppm # FC-Mix approx. 50 ppm actual # ppm Comments Blank Std# --. ------ -------- amount = "" - mL ...... Serum Extraction Method _______________ 1........................................... ........................................... ft Initials----------- Vortex 15 sec. Pinette Matrix .... ...... ..................................................................................................................... Volume mL Pipette 1 mL of 0.5 M TBA, pH 10. pH = Std. # . .. Pipette 2 mL of 0.25 Na?COV0.25M NaHCOt buffer Std. # Dispense 5 mL of methyl-t-butyl ether TN-A- Shake 20 min. Shaker speed: ... . Centrifuge 20-25 min. Centrifuge speed: . .. ........... Remove a 4 mL aliquot of organic layer . Put on Nitrogen Evaporator to dryness Add methanol Volume Temperature: mL TN-A- Vortex 30 sec. _________________________ !________________________________________________ Filter using a 3cc B-D svringe with a 0.2um filter into a 1.5 mL autosample vial _. Cont Cal. Verifications used same matrix as for std curve. Attachment A 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 10 of 14 Page 47 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 MDL/LOQ values for rabbit serum Compound MDL (PPb) LOQ Linear Calibration Range (LCR) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 5 ppb - 1000 ppb PFOSA 1.51 4.79 5 ppb - 1000 ppb PFOSAA EtFOSE-OH 3.46 11.4 20.5 5 ppb - 1000 ppb 36.2 5 ppb - 1000 ppb M556 6.03 19.2 5 ppb - 1000 ppb PFOSEA 5.71 18.2 5 ppb - 1000 ppb MDL/LOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each o f these matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbit responses, therefore, their MDL and LOQ will be the same values as determined in rabbit serum. Please see LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-l for further information. Attachment B: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 11 o f 14 Page 48 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Compound: PFOS Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 Compound: PFOSA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 24.8 - 976 0.993 - 976 Compound: PFOSAA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.991 - 974 4.92 - 247 49.2 - 974 0.991 - 974 LCR from curve (Ppb) (ng/mL) 24.8 - 978 4.94 - 248 97.8-978 4.94 - 978 LCR from curve (ppb) (ng/mL) 4.93 - 976 4.93 - 97.6 24.8 - 978 4.93 - 976 LCR from curve (ppb) (ng/mL) 24.7 - 974 9.74 - 247 97.4 - 974 9.74 - 974 % Recovery Range 83-108 85-104 85-106 94-111 % Recovery Range 88-103 87-105 93-102 94-103 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Attachment B: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 12 of 14 Page 49 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Com pound: EtFOSE-OH Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93-97.6 49.3 - 976 0.993 - 493 LCR from curve (pph) (ng/mL) 49.3 - 976 9.76-97.6 97.6 - 976 9.76 - 976 % Recovery Range 77-110 97-107 90-109 86-111 Compound: PFOSEA Prepared range Rabbit Serum o f standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 248 49.3 - 976 0.993 - 976 LCR from curve (PPb) (ng/mL) 24.8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 % Recovery Range 96-106 91-110 86-106 95-117 Com pound: MS56 Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (PPb) (ng/mL) 24.8 - 976 9.76-97.6 97.6 - 976 9.76 - 976 % Recovery Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 13 of 14 Page 50 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.500 0.507 0.507 0.532 0.532 0.501 0.501 0.521 0.521 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 50.0 50.1 532 50.1 52.1 M556 Std cone ug/mL 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 All Am't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All Final vol mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Calculated concentrations of standards in the sample matrix PFOS Final cone ng/mL 4.93 9.76 24.8 49.3 97.6 248 493 735 976 PFOSA Final cone ng/mL 5.00 9.89 25.1 50.0 98.9 251 500 746 989 PFOSAA Final cone ng/mL 5.24 10.4 26.3 52.4 104 263 524 782 1038 EtFOSE Final cone ng/mL 4.94 9.78 24.8 49.4 97.8 248 494 737 978 PFOSEA M556 Final cone Final cone ng/tnL ng/mL 5.01 1 5.13 9.93 10.2 25.2 25.8 50.1 51.3 9 9 3 102 252 258 501 S13 749 766 993 1017 Surrogate Std cone ng/mL 100 Surrogate Final cone ng/mL 500 All Am't spiked mL 0.005 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA EtFOSE-OH PFOSEA M556 Rabbit 5.00-1000 | 5.00-1000 1 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 Bovine Estimates only. Use values for rabbit. Rat Estimates only. Use values for rabbit. Monkey & Plasma Estimates only. Use values for rabbit. Human Estimates only. Use values for rabbit. Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction of PFOS from Scrum 3M Environmental Laboratory Page 14 o f 14 Page 51 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory M ethod A n alysis q f P o ta ssiu m P erflu o ro o cta n esu lfo n a te o r O t h e r F l u o r o c h em ic a ls in Seru m E xtra cts U sin g H P L C -E lectro spr a y /M ass Spec tr o m etr y M ethod N um ber: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: vM Laboratory Manager -- Group Leader t j Z-*------------- - Technical Reviewer Adoption Date: 03/01/99 Revision Date: ^ `i / z c , Date 1 /U , Date oh I w Date 1.0 S c o p e a n d A p p l i c a t i o n __________ _______________________________________________________ 1.1 Scope: This method describes the analysis o f serum extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Com pounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 1 of 9 3M Environmental Laboratory Page 52 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 2.0 S u m m a r y o f M e t h o d _________________________________________________________________ 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions o f the parent ion. 3.0 D e f i n i t i o n s ___________ _________________________________________________________________ 3.1 A tm ospheric 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: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method o f ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models o f Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 M ass Lynx Software: System software designed for the specific operation o f these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W a r n i n g s a n d C a u t i o n s ____________________ __________________________________________ 4.1 Health and Safety W arnings: 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 solvents wear appropriate protective gloves, eyewear, and clothing. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 o f 9 Page 53 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back p ressu re exceeds 400 bar, the HP1100 w ill initiate au to m atic sh u td o w n . 4.2.2 Do not run solvent pumps to dryness. 5.0 In t e r f e r e n c e s _____________ ___________________________________________________________ 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 E q u i p m e n t __________________________ __________________________________________________ 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 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 S u p p l i e s a n d M a t e r i a l s __________________ _____________________________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (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 R e a g e n t s a n d S t a n d a r d s ____________________________ _________ ________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM 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 S a m p l e H a n d l i n g ___________________________________________________________ 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. 3M Environmental Laboratory ETS-8-5.1 Analysis o f Serum Extract Using ES/MS Page 3 of 9 Page 54 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Q u a l i t y C o n t r o l ____________________________________________________________________ 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 determine contamination 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 duplicates are prepared and analyzed to measure 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 duplicate concentrations will fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 103 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum of one per batch. 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _______________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set o f extracts. The average of two standard curves will be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting o f high concentration standards. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 4 of 9 Page 55 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 12.0 P r o c e d u r e s ________________________________________ _____________________ ______________ __ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit o f year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosam pler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1, 12.2.2 Set-up the HP 1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 jiL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrum ent Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 123.2 Check the solvent level in reservoirs and refill if necessary. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 5 of 9 Page 56 3M Medical Department; Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 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 o f the probe. Readjust the tip o f the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 1 0 -5 0 0 pL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 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 ES/MS 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 appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 D ata Analysis and Calculations__________ __________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration o f PFOS, or other fluorochemical, in matrix (pg/mL): fng o f PFOS calc, from std. Curve x Dilution Factor) x 1 u e (Initial Volume of matrix (mLl + mL o f Surrogate Standard) 1000 ng Final Volume (mL) 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 6 of 9 Page 57 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 14.0 M e t h o d P e r f o r m a n c e __________________ ____________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.1, Attachm ent 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 r2value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within 30% of the spiked concentration. 14.5 Continuing Calibration Verifications . 14.5.1 Continuing calibration verification percent recoveries must be 30% o f 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 die appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text o f the report. 15.0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _______________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s ___________________________________________ ___________________________________ 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 calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 7 of 9 Page 58 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see A ttachm ent 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 T a b l e s .D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a ____________________________ 17.1 Attachment A: 'ETS-8-5.1 Data summary spreadsheet. 18.0 R e f e r e n c e s ___________________________________ _______________________________________ 18.1 FACT-M-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. 19.0 A f f e c t e d D o c u m e n t s ______________ _____________ ___________________________________ 19.1 ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R e v i s i o n s ------------------ ------------------------------------------- Revision _ Number. Reason For Revision 1 Section 6.1.2 Clarification of HP1100 system components. Section 11.1 Average o f two curves, not standard values, are used for plotting linear regression and added the 1/x weighting of the curve. Section 12.2.2.4 Clarification o f solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 8 of 9 Page 59 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: - Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 9 of 9 Page 60 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix D: Data Sum m ary Tables Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013 Dosage Group Specimen 10 PFOS (pg/mL) 1 10097F 0.0394 1 10105F 0.0181 1 10106F 0.0258 1 10107F 1 10108F 0.0343 0.0253 1 9922M* 1 9930M* 0.0115 0.0134 1 9931M 0.00725 1 9932M 0.0162 1 9933M* 0.0156 II 10121F II 10126F 9.62 19.8 II 10136F 5.96 II 10140F 6.27 II 10142F 13.1 II 9961M* 34.8 II 9964M 30.4 II 9965M* 74.9 II 9967M 25.1 II 9970M* 38.9 III 10155F* 87.8 III 10156F III 10164F 76.1 49.6 III 10172F 68.4 III 10177F 42.2 III 9997M 108 III 9999M* 178 III 10001M 94.9 III 10002M III 10004M 113 130 IV 10187F IV 10194F 89.5 73.4 IV 10203F 126 IV 10211F 99.7 IV 10214F 98.3 IV 10019M 302 IV 10024M* 477 IV 10029M* IV 10033M IV 10034M 296 272 249 * - Tentative values, initial volume was <0.5 mL PFOSA (pg/mL) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) 0.0682 0.112 0.0663 0.0507 0.0665 0.0962 0.188 0.114 0.147 0.165 0.328 0.352 0.265 0.325 0.335 0.574 0.579 0.480 0.393 0.465 . 0.481 0.576 0.651 0.670 0.569 0.613 0.553 0.610 0.804 0.637 PFOSAA (pg/mL) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) 1.59 4.55 1.18 0.690 2.09 1.40 5.86 1.86 1.26 5.55 9.9 6.91 4.66 8.17 4.58 11.8 18.7 12.1 10.4 14.9 8.00 10.6 19.0 10.2 12.3 22.5 40.5 28.8 24.5 31.4 EtFOSE-OH (pg/mL) M666 (pg/mL) <LOQ (38.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) 1.86 4.19 0.952 1.15 2.45 4.69 5.18 4.54 3.44 6.11 43.3 22.3 18.0 17.0 17.9 29.1 73.6 25.1 38.1 37.8 39.0 25.6 39.6 28.8 33.8 71.3 102 94.9 90.9 56.7 3M Environmental Laboratory Page 61 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013 (continued) Doeage Group Specimen ID PFOS (pg/mL) V NR NR V NR NR V NR NR V NR NR V NR NR V 10042M 238 V 10044M 235 V 10045M 326 V 10051M 162 V 100M M 182 NR - Sample not received or reported * * Tentative velues, initial volume was <0.5 mL PFOSA (pg/mL) NR NR NR NR NR 0.791 0.972 0.897 0.574 0.669 PFOSAA (pg/mL) NR NR NR NR NR 25.2 20.7 26.8 14.0 15.8 EtFOSE-OH (pg/mL) NR NR NR NR NR <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) M556 (pg/mL) NR NR NR NR NR 62.4 55.6 93.8 30.7 55.5 Table 13. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-013 Doeage Group I I I I I I I I I I I I 1 1 1 II II II II II II II II II II II II II II II Specimen ID 10097F 10105F 10106F 10107F 10108F 9922M 9930M 9 9 3 1M 9932M 9933M 10097M 10105M 10106M 10107M 10108M 10121F 10126F 10136F 10140F 10142F 9961M 9964M 9965M 9967M 9970M 10121M 10126M 10136M 10140M 10142M PFOS (pg/g) 0.149 <LOQ 0.121 <LOQ <LOQ 0.585 0.816 0.836 1.04 1.01 0.281 0.242 0.226 0.221 0.251 25.1 22.9 39.8 23.7 22.1 116 102 89.9 80.7 87.3 M .7 67.8 53.7 28.0 71.5 PFOSA (pg/g) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 0.514 0.708 1.40 0.601 0.508 4.49 4.10 2.88 3.68 5.42 1.90 2.65 2.35 1.22 2.06 PFOSAA (pg/g) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 2.68 4.34 5.01 2.67 2.67 11.0 15.6 9.79 6.62 10.4 5.87 14.8 9.44 2.82 6.55 M558(pg/g) <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <L0Q <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 1.19 1.75 2.88 1.55 1.41 12.8 10.2 11.6 8.95 11.6 5.39 7.75 4.64 3.27 4.58 3M Environmental Laboratory Page 62 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U209S Table 13. Reported Fluorochemlcal Levels In Liver Analyses in Study FACT TOX-013 (continued) _____________________________ Dosage Group III III III III III III III III III III III III III III III IV IV IV IV IV IV IV IV IV IV IV IV IV IV IV V V V V V Spscltnsn ID 10155F 10156F 10164F 10172F 10177F 9997M 9999M 10001M 10002M 10004M 1015SM 10156M 10184M 10172M 10177M 10187F 10194F 10203F 10211F 10214F 10019M 10024M 10029M 10033M 10034M 10187M 10194M 10203M 1 0 2 1 1M 10214M 10042M 10044M 10045M 10051M 10054M PFOS (pg/g) PFOSA (pg/g) 102 130 179 119 105 415 234 498 257 386 89.0 219 203 188 153 164 240 344 255 264 831 791 556 781 556 226 277 448 457 344 1218 1356 1132 1063 1054 ~1 2.22 2.24 1.94 2.17 2.88 10.8 9.41 8.67 8.29 8.41 5.11 6.14 8.26 6.20 6.91 2.80 4.06 3.14 3.39 4.56 12.8 11.0 11.2 12.6 11.0 6.36 9.96 0.93 11.4 8.11 16.4 13.0 10.9 9.80 10.8 PFOSAA (pg/g) 15.6 20.2 22.7 11.9 20.0 73.5 28.6 85.2 34.2 64.9 12.0 27.3 294 33.7 17.1 31.1 51.5 49.5 46.6 51.4 122 148 86.2 129 135 27.4 45.5 76.0 56.2 60.0 188 206 150 157 165 **858 (pg/g) 7.17 7.64 8.41 5.87 8.16 63.5 39.3 66.4 38.0 54.6 11.5 33.3 43.1 29.9 31.4 19.8 26.8 26.6 27.5 29.3 84.5 97.5 78.2 117 82.2 39.9 39.5 67.8 65.4 64.5 128 152 133 118 161 3M Environmental Laboratory Page 63 3M Medical Department Study: T6316.5 Appendix E: Data Spreadsheets Analytical Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory Page 64 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 8 all S Z< Z< MX S io 27.4 OO351 2 s 5 5 1 = 5 S 5 ; 5 i SS 1 s Sm I i X 1So d c s i 5 s = Z fi 3 ---------------------- 2 1i fs! 5? h fi ** S3 = 30 3 S3 5 0S 0S 2 o S 5 o S 8 o 3X 5o e 5^^ -- s S 2 5s s e ? 3? S ES =SSp 5 *i 8S*Es 1 SS 32332' S U1 ii il s ihft 12 s SsSs' *8 |e 6 3 5 e I s tf 3a Ur Sal ns ili i III s jlillb llil }i e n Sd o8 8O SO 5 5 2 5 ? S s f i E n * S ^ k S s 5 3 S 3 S S 5 5 3 S 2 S S S i S S 5 ! 3 s 9 9, 1i -- - - -- ?o ed se os se ?d d 3o eS d5 gd So 8 -- S -- So d? d Sd 8d 5d $e 3d 8d 8d Pc Rd Sc Pd So 8o l i d i o s s 8a s s Sd 8 d S d 8 -- dS 33 .11 5 ke g! I SJ3ia * st SJi oc || ii I I S 5 ? 3 2 S DI 8 l 3 l o s 2S I S P S lI mO O Ouo o 3 1a * 1 * 3 L U. fc X 3 S. U. PP 1 L 1 ?3 !i 1 1 S S '- l s S !||i! s s S U IS iiil i lo V) V Sa *1S S il ii 5 8 h 7. l1 tifi f l |o o i?3 if! u s ifl Ifl- t * Ig II a i 9 $ X 5 3M Environmencal Laboratory g Page 65 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 ?*3 Kcpoductaa Stud; o( EifOSE-OH a iUu MM vdutM below05 bL. LAC01/31/00 Teals)vc 3M Environmental Laboratory Page 66 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 ,1 il Z< s t h ii s 3 P tn < 35 ffS = 3 ; Sb a1 b S ! t 5V* *w 9j ; S .V fti ^ a I * II I mu ttitt3 ! 51 S 5 S a s 5 g 3 S J S 2 2 r J ; - * - b ^ g b *1 *1 > w> 222 2- P5S3S 2 bb J 3 S S 2 2 J i l5 hiss 1 1 So 9o s 5 3 1 1 5 1 =s=sa 5 R S 53 j 5 a s 5 2 5 S E 3 = 3 * 3 2 o ~1 3; ; nbbb -- S 5 S It i2!* if u* aai - * g a -- -- -- oP c ?o 8e S ? o 9 9, 9>9 boes ^883 o ----o ? o s? a ob s e 9 b d l b l l b I b I I sb sb eb so so 1 3 3 3 3 o o ao a-- os ISacIfIJf Uj $UiU! l|staa A 8 113*5 13133i 3A i lISiIs2 mSS i2alolsosolloe ii* | Hl! |||SEili li S 5 * 5 3 i = Ssfilli 38 3 3; fi St 1 if mlI II: 111 1 1blliit II AM sn!i e il? }J f 1JJJ (XVIC/SODVl 3M Environmental Laboratory it l3 Page 67 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 A fp 411409. Two-GcacnAJGo RqrDdoctioB Study of EtTOSE-OH lo R*u EiFOSE-OH (T-Ali) NuobofTctt SobfUncv): 3M Environmental Laboratory i A 9s t ji Page 68 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 8 Taudvevahiet.iAhUlvnigratbtlovO-5mL. L A C O t ft lA D 3M Environmental Laboratory A Page 69 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT TOX-013 AR (ng/mL) * DF x SC x FV (mL) x 1.0 ng x PC = Reported Concentration (jig/mL) EV (m L)' lOOO ng Calculation Used for Group 4, Animal ID 10033M 340 ng/mL x 500 x 0.9275 x 1 mL x 1.0 pg x 0.864 = 272 pg/mL 0.5 mL 1000 ng AR-- Analytical result from MassLynx summary DF-- Dilution factor SC--PFOS salt correction constant (0.9275) FV-- Final extract volume (1.0 mL unless otherwise noted) EV--Volume of sera extracted PC-- PFOS purity correction factor (86.4%) 3M Environmental Laboratory Page 70 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix G: Contract Lab Report This appendix includes the following contract laboratory report: Battelle Memorial Institute, Study Number. N003604-D, 2 (N-Ethylfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction, (59 pages) 3M Environmental Laboratory Page 71 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 BIOLOGICAL SAMPLE ANALYSIS Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 . . . Putting Technology Ta Work FINAL REPORT 2 (N-Ethylfluorooctanesulfonamido)-ethanoI in Two Generation Rat Reproduction SPONSOR 3M Toxicology Services 3M Center Building 220-2E-02 S t Paul, MN 55144 Study Initiation: September IS, 1998 Testing Facility Battelle Memorial Institute SOS King Avenue Columbus, Ohio 43201-2693 Prepared By PatrickL. South, B.S. 3M Environmental Laboratory Page 72 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Batidle StudyNumber. N003604-D 3MEnvironmentalLaboratoryStudyNumber. FACT 060998.1 FINAL REPORT 2 (N-Ethylfluorooctanesulfonamido)-ethanol in Two Generation R at Reproduction Jod C. Andre, Ph-D. Battelle Principal Investigator RichardW. Slauter, PLD,, D .T. Battelle SeniorProgramDirector 3M Environmental, Laboratory u Page 73 3M Medical Department Study: TS316.5 Analytical Report: FACT TOX-013 LRN-U2Q95 Batidle StudyNumber. N003604-D 3MEnvironmentalLaboratory StudyNumber FACT 060998.1 2 (N-Ethylfluorooctanesulfonamido)-ethanol in Two Generation R at Reproduction EXECUTIVE SUMMARY Rat liver samples sent to Battelle by 3M Environmental Technology and Services were analyzedby the previously validated method "Method for Analysis of Potassium Pcrfluorooctanesulfonate (PFOS) in Rat Liver by LC/MS/MS". Samples were extracted and analyzed by High-Performance Liquid ChromatographyMass Spectroscopy (LC/MS/MS) for PFOS, M-556, PFOSAA, and PFOSA content only. Related flucrochemicals mentioned in the analytical method were not investigated. The results for the concentration determinations in the liver samples from this study are attached as appendices to this report Concentrations are reported as mass of analyte (pg) per gram ofliver tissue extracted. 3M Environmental Laboratory m Page 74 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Batidle StudyNumberN003604-D 3MEnvironmentalLaboratory StudyNumber FACT060998.1 QUALITY ASSURANCE STATEMENT This study was inspected by the Quality Assurance Unit and reports were submitted to the task leader, study director, and associated management as follows: Date Reported Date Reported to to Battelle Task Offiite Study Phase Inspected Inspection Date Leader/Battelle Director/ ______________ ______________ __________ Management . Management_____ Sample weights Sample homogenization Extraction Sample analysis 10/12/1999 10/12/1999 10/13/1999 10/13/1999 Quality Assurance Unit Battelle Memorial Institute Date 3M Environmental Laboratory IV Page 75 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle StudyNumber. N003604-D 3MEnvironmentalLaboratoryStudyNumber. FACT060998.1 GOOD LABORATORY PRACTICES COMPLIANCE STATEMENT Study Title: 2 (N-EthylfluorooctanesuIfonainido)-ethanol in Two Generation R at R eproduction This study was conducted in compliance with the Food and Drag Administration's Good Laboratory Practice Regulations (21 CFR 58), with the exception that the mass spectrometry data for the liver samples was collected and processed with the MassLynx software system (version 3.1), which was not fully validated. The study was listed on Battelle's Master List o f regulated studies. Jon C. Andre, P h D . . Battelle Principal Investigator Kris Hansen, P h D . Study Director 3M Environmental.Laboratory V Page 76 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 Table of Contents Executive Summary.....,,.................................... Quality Assurance Statement............................. Compliance Statement...................................... Table of Contents............................................. 1.0 Introduction............ ............................ 2.0 Reference Substances........................... 3.0 Receipt of Samples .......................... 4.0 Analysis of Samples ............................ 4.1 Summary of Method............... 4.2 Results.................................... 4.2.1 Quality Control.......... 4.2.2 Sample Results.......... 5.0 Conclusions......................................... 6.0 Acknowledgements.............................. 7.0 Specimen Storage andRecord Archives, L ist of Tables ' Table 1. Example of Instrument Parameters Used to Analyze Samples......................... Appendix A (Results) Summary Results for Rat liver Sample Analysis.................. '........................................ Appendix B (Daily Acceptance Criteria Summary) Daily Acceptance Criteria Summary............................................................... ............ Appendix C (Sample Inventory List) Sample Inventory List................ ............................................................................... Appendix D (Chromatograms) Representative Chromatograms.................................................................................... Appendix E (Protocol, Amendments, and Deviations) Protocol, Amendments, andDeviations.......................................................................... PFOS Purity Report Appendix F (PFOS Purity Report) Page iii iv .V vi ,,1 ..1 ..1 ..1 ..1 ..2 ..2 ..3 ...3 ...4 ...4 2 A -l B-l ,C-1 D -l .E-l .F-l 3M Environmental Laboratory vi Page 77 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Bsitelie StudyNumber N003604-D 3MEnvironmentalLaboratoryStudyNumber. FACT 060998.1 1.0 Introduction This report presents a description of the method used to analyze PFOS, M-556, PFOSAA, and PFOSA in rat liver samples from 3M StudyNumber FACT 060998.1 (TOX-013) and the results from this analysis. See Appendix E for a copy of the study protocol, amendments, andprotocol deviation reports (signed deviation reports were not available for inclusion here). ' 2.0 Reference Substances The analytical reference substances for this studywere supplied by 3M. The following lot number or tracking number designations apply: PFOS (lot 171), M-556 (TN-A-2203), PFOSAA (TN-A-1283) and PFOSA (L-15709). Note that based on information supplied to Battelle from 3M, PFOS has two equivalent names. The nam* appearing on the Material Safety Data Sheet andbottle label is potassium perfhioroalkyl yilfnnatff The name more commonly used by 3M in analytical methods and correspondence is potassium pCT-fl^t'rfyvtanrunlfnnuti The latter name will be used in this report. Sec Appendix F for purity data supplied by 3M to Battelle. The reference substances were stored atroom temperature. The surrogate standard was 1H,lH^H,2H-Perfluorooctane sulfonic acid, lot number 59909, supplied by ICN. The surrogate standardwas stored at roomtemperature. 3.0 Receipt of Samples Samples were received frozen and intact at Battelle, from 3M Environmental Technology and Services, in one batch on October 6, 1999. Samples were generated by Argus Research underprotocol number 418-009. See Appendix C for a copy of the inventory list The samples were stored at approximately -20C. 4.0 Analysis of Samples 4.1 Summary of Method Samples were analyzed by a previously validated method (Battelle study numberN003604-A). The current version of the method is attached to this report in Appendix E. Samples were analyzedby LG/MS/MS, and an example of the instrument parameters is listed in Table 1. Note that only PFOS, M-556, PFOSAA, and PFOSA (and the surrogate) were quantitated. The other related fluorochemicals, although present in the stock solutions, were not monitored. Quadratic regressions weighted 1/x were used to construct the calibration curves. 3M Environmental Laboratory 1 Page 78 3M Medical Cepartxent Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2 095 Battelle StudyNumber N003604-D 3MEnvironmental Laboratory StudyNumber. FACT 060998.1 Table 1. Example of Instrument Parameters Used to Analyze Samples LC /M S/M SSW cm AutoNumptcr HPLC |uni|>s Mas* spectrometer Analytical column Mobile phase components Gradient prolile Injection volume Flow Column temp HPLC pressure MS source Dcsnlvatioii .is Ncliuli/er as Source binds temp Dcsohation letup Cone voltaic Collision encr" v Collision "as Multiplier Resolution Ions monitored Total run time Approximate retention times: Make: Gilson Make: Gilson Make: Micromasa Model: 234 Models: 30S and 306 Model: Quattro LC with Z-spray source Keystone Betasil Cl 8, S|im, 2 x 5 0 mm. Part No. 055-701-2 Component A: Ammonium acctatc(2mM):methanol, 60:40, vrv Component B: Ammonium acetate(2mM)nnethanol, 5:95, vrv p n je, min %B Flow. mlAnin 0 0 0.3 1 0 0.3 ' 4.5 100 0.3 6 100 0.3 6.1 100 0.6 8.5 100 0.6 9 0 0.3 11 0 0.3 10 uL LC c o lu m n flow at start split to 20 nL/min into the MS Ambient Approximately 840 psi at gradient start Electrospray, Negative Ion Nitrogen at ~575 L/hr Nitrogen at -8 0 L/hr 140C 250*C 70 V for SS, PFOS 20 V for M-556, PFOSAA. PFOSA 40 eV Argon, gas cell, a t--2.5x10 mb 650 V 12.0 for M SI; 10.0 for MS2 427>81 MEM transition for SS 499>99 MEM transition for PFOS 556>78 MRM transition for M-556 584>169 MRM transition for PFOSAA 49S>78 MRM transition for PFOSA 11 minutes SS: 4 min PFOS: 4.2 min M-556: 4.4 PFOSAA: 4.5 PFOSA: 5 2 3M laboratory Page 79 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 4.2 Results Battelle StudyNumber. N003604-D 3MEnvironmentalLaboratoryStudyNumber FACT060998.1 4.2.1 Quality Control System suitability acceptance criteria were established during the method validation and are included in Appendix C, Section IX Acceptance Criteria. Relevant statistics from each sample set are provided in Appendix B. Representative chromatograms are given in Appendix D. 4.2.2 Sample Results The results of the sample analyses as well as a method detection limit determination are presented in Appendix A All liver samples were initially extracted as undilutedhomogenates. After the data were reviewed, dilutions of the homogenates were performed in order to bring analyte concentrations within the calibration range. The first analysis-thatprovided acceptable data for an analyte was used in reporting. Four extraction sets were required to provide data for each analyte. The limit of quantitation is defined as the concentration of the lowest standardwhich meets acceptance criteria for accuracy (25% RE). The notation BLOQ denotes "Below Limit of Quantitation" for samples that had concentrations lower than the theoretical concentration for the 0.13 pg/g calibration standard. The notationALOQ denotes "Above Limit of Quantitation" for samples thathad concentrations higher than the theoretical concentration for the 13 pg/g calibration standard. Samples that were initially ALOQ were diluted with blank liver homogenate andre extracted. Samples thatwere expected to be ALOQ were first dilutedwith blank liverhomogenate before extraction. The "Corrected PFOS Cone" presented in the results tables is the concentration found for the diluted sample multiplied by its dilution factor (final volume + sample homogenate volume). The method detection limit (MDL) ofPFOS was calculated to be 0.0173 pg/g from the analysis of 7 replicate preparations of 0.13 pg/g calibration standard. The MDL was calculated by multiplying the standard deviation of the found concentrations of the 7 reps by 3.143; the Signal-to Noise (S/N) ratio was calculated by dividing the mean found concentration ofthe 7 reps by their standard deviation. The method of MDL determinationwas providedby the Sponsor. 5.0 Conclusions All analyses met acceptance criteria unless otherwise noted. 3M Environmental Laboratory Page 80 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 6.0 Acknowledgements Acknowledgement of principal contributors participating in the performance of this study at Battelle is presented in the following list Participant T itle Jon C. Andre, PLD. Richard W. Slauter, Ph.D., DA.B.T. PatrickL. South, B.S. Gerke H. van der Zwaag, MS. Bobby C. Braswell, B.A. Cynthia J. Ryan, B.S. Battelle Principal Investigator Senior ProgramDirector Mass spectroscopist ' Sample preparation chemist Quality assurance auditor Lead quality assurance auditor 7.0 Specimen Storage and Record Archives All original paper H as well as electronic copies of data, will be forwarded to 3M for archival. Copies ofboth the final report and all original paper data generated in conjunction with this studywill be maintained by Battelle. All residual liver samples, extracts, andunused test article will be disposed of or returned to the Sponsor as directedby the Sponsor. 3M Environmental Laboratory 4 Page 81 3M Medical Department Study: T6316.5 __ ' Analytical Report: FACT TOX-013 . LRN-U2095 Battelle StudyNumber N003604-D 3MEnvironmentalLaboratory StudyNumber. FACT 060998.1 APPENDIX A -RESULTS 3M Environmental Laboratory . Page 82 3M Medical Department Study: TS316.5 Analytical Report: FACT TOX-013 LRN-U2 095 ' Battelle Study Number N003604-D 3M Environmental Laboratory Study Number: FACT 060993.1 rr o * . im m , m t M , m u BOX STUDY; AOSMnrr SOATWAAK w r n is : in r a t u v e a N0099040 MANUALLY CXCSLfT Seelbio Date Qrono Animal Number Sampln TYo P fOS Cono, 1 2 3 4 9 7 9 10 11 12 13 14 15 1 17 1 1 20 21 22 29 24 29 20 27 2 29 30 91 32 99 94 35 99 97 99 99 40 41 42 43 44 * 49 49 47 49 49 so S1 S3 S3 94 99 99 57 99 SB fio 92 83 94 as 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 9 a s8 9 1 1 1 1 1 2 2 2 *2 2 3 3 3 3 3 4 4 4 4 4 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 4 b lo o * seLCvy murr o f q uantttaticn 8822 0830 0831 8832 0093 88S1 0094 8888 0897 8870 0007 0000 io ao i 10002 10004 10019 10024 10029 10033 10034 10CVQ 10044 10049 10091 10094 10007 10109 10109 10107 10108 10139 10140 10142 10121 10128 10177 10155 10156 10104 10172 10157 10184 10203 10211 10214 10087 10106 10106 10107 10109 10139 10140 10142 10121 10128 10177 10195 10150 10164 10172 10167 10164 10203 10211 10214 Matama! M atan* Mamma! M eam al Mctnel Mamma! Mamma! Mamma! Matern Matern Metern Metern Mamma! Mamma! Metern Metern Matern Mamma! Mamma! Mamma! Mamma! Mamma! Mamma! Matama! Matamal Fat! Fata! Fata! Fata! Fata! Fatal FtCM Fatal Fata! Fata! Fatal Fatal Fatal Fata! Fatal Fata! Fata! Fata1 Fata! Fatal Matamal Mamma! Mamma! MammaJ Mamma! Matama! Mamma! Matama! Matama! Matama! Mamma! Mamma! Matama! Mamma! Mamma! Matama! Mamma! Matama! Mamma! Matama! -77E-Q1 0.446-01 9.88E-01 106HM 1.176*00 1.346*02 1.196*02 146*02 9.346*01 1.016*02 4306*02 2.716*02 9.798*02 4976*02 4476*02 .526*02 9.196*02 4436*02 9.046*02 4436*02 1416*01 1.876*01 1 16*03 1 3 6 *0 3 1 2 6 *0 3 1.7SE-01 BUM 1.406*01 BLOQ BLOQ 4.11 EH 2.746*01 4506*01 4906*01 2.66EHH U 1M 1.166*02 1.506*02 4076*02 1 0 6 *0 2 1.906*02 2.796*02 3.906*02 266*02 3.096*02 3.206*01 2.606-01 2.816-01 n s s E -o i 2.006-01 1 8 *0 1 346*01 4298*01 4336*01 7.966*01 1.776*02 1.036*02 236*02 2.366*02 2.106*02 2.526*02 316*02 L253 Analysts dete key: Normal tont October 13,1888 Undertine - Octobor 13 It M M d O c tet 18,1989 M S L U ttH M M " O d cb arX , 1888 Alt samples undiluted All samplaa undiluted All samplaa diluted All samples diluted M-3S0 C0IM p rt P ro sa* Cene, Ilfl/S PPQSA Cene, llfl/t BLOQ BLOQ BLOQ BLOQ BLOQ 18E*01 1.036*01 U C E itt fi.966HM 1.166*01 5 6 6 *0 1 3*936*01 4946*01 4906*01 4496*01 4496*01 4786*01 7326*01 1.176*02 4226*01 1 5 6 *0 2 1 2 6 *0 2 1 3 6 *0 2 1.196*02 1.918*02 BLOQ BLOQ 8LOQ BLOQ BLOQ 4666*00 1 .H -09 m esa 4166*00 U TS *sa 7.see*00 441 EHM .a 7 E *M 1 9 6 *0 1 4556*01 4556*01 4796*01 4336*01 BLOQ BLOQ BLOQ BLOQ BLOQ 4946*00 3.2TEU 0 |. W 0 5e*00 7.786*00 4146*01 1.136*01 4336*01 0 1 6 *0 1 4996*01 4996*01 4366*01 4716*01 4346*01 4456*01 BLOQ BLOQ BLOQ BLOQ BLOQ 1.106*01 1.506*01 in e - 4826*00 7.388*01 4526*01 i*a e o i 4496*01 ija e n a i* U 2M I 1js e n a 1 56 *0 2 ij o e - o* 4006*02 1.MCKJ3 1 7 6 *0 2 1.586*02 BLOQ BLOQ BLOa BLOQ BLOQ 4016*00 Z97Et<M Z 9 Z E -S 9 4906*00 4006*01 156*01 4026*01 4276*01 1.196*01 4116*01 4156*01 4366*91 4566*01 4146*01 BLOQ BLOQ BLOQ BLOQ BLOQ 4446*00 4826*00 8.3S E * 4576*00 1.406*01 1.716*01 1.H W 1 4736*01 4946*01 4376*01 4746*01 4366*01 7.006*01 4526*01 4006*01 BLOQ WLOQ BLOQ BLOQ BLOQ 4.496*00 4.106*00 Z flS ftS fl 3.506*00 6.426*00 1.096*01 ftJ lE iflQ AJZESfla 42S fl 4416*00 19C01 1.106*01 1.126*01 106*01 L106MM 1 .*< e *d 1 1306*01 U B E *fil 9.aoe*oo 1.M E-01 BLOQ BLOQ BLOQ BLOQ BLOQ 1.406*00 8.016-01 4056-01 n ia l Z&B&91 4886*00 46+O fl 2.24& 00 1.846M 4176*00 4906*00 4.066*00 H iE rflli 3.38&CQ 4506*00 BLOQ BLOQ BLOQ BLOQ BLOQ Z 3 S 6 -0 0 1 2 2 fc S B ZOSEN 1.S0EK zsa& oo 4916*00 5.116*00 4146*00 4296*00 5 206*00 S.3SEH 9.906*00 4996*00 w -E ta t 4116*00 3M Environmental Laboratory A-l Page 83 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 . . ' Battelle Study Num ber N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 METHOD DETECTION LIMIT (MDL) RESULTS STUDY: N003604-D ANALYSIS DATE AND INSTRUMENT ID: DATA ENTERED: SPREADSHEET SOFTWARE: 130ct99; 9053 Electronically and manually Excel 97 All cones in pg/g Calculated Concentration of Replicate 1 Calculated Concentration of Replicate 2 Calculated Concentration of Replicate 3 Calculated Concentration of Replicate 4 Calculated Concentration of Replicate 5 Calculated Concentration of Replicate 6 Calculated Concentration of Replicate 7 Mean Concentration Std. Dev. M -556 0.1269 0.1220 0.1433 0.1368 0.1670 0.1225 0.1190 0.1339 0.0170 Spike Level iiiiiiiiiiiiiin g g n m S/N n 0.1331 7.88 ^ il is LOQ (det from 10 x std.dev. "noise") LOQ (det from cal curve low std.) Curve Coeff of Determination Date analyzed . Method i 0.17005 0.1331 0.9978 130ct99 LC/MS/MS Kev 1 - Spike Level too high; Spike Level must be < lOx MDL 2 - Spike Level too low, Spike Level must be > MDL 3 - S/N too low, S/N must be > 5 4 - Coeff of Det of calibration curve unacceptable PFOSAA 0.1484 0.1281 0.1484 0.1605 0.1661 0.1513 0.1385 PFOSA 0.1167 0.1325 0.1521 0.1441 0.1490 0.1382 0.1413 0.1488 0.0128 0.1391 0.0119 0.1334 0.1315 IlllflliillP J I 11.66 11.74 msm^SSMmm: M I S S 0.12763 0.1334 0.11853 0.1315 0.9937 0.9891 130ct99 LC/MS/MS 130ct99 LC/MS/MS 3M Environmental Laboratory A-2 Page 84 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 _ LRN-U2095 Batidle StudyNumber. N003604-D 3MEnvironmental Laboratory StudyNumber FACT 060998.1 APPENDIX B-DAILY ACCEPTANCE CRITERIA SUMMARY 3M Environmental Laboratory Page 85 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 ' Battelle Study Number. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 PFOS e t ai IN RAT UVER STUDY NUMBER: N003804-0 DATA ENTERED: MANUALLY SOFTWARE: EXCEL 97 REGRESSX3N PARAMETBtt Anatvale Date OcU mt 13,1008 October 16,1808 October 1ft, 1808 October 30,1800 AnaM e PFOS M 466 PFOSM PPOSA PfO S UAt PFOSAA PFOSA p ros M-596 PFOSAA PFOSA PFOS PFOSA x *c o r -0.0164 4100640 0.00232 -156 4100368 000360 -250 a00383 a 0221 aoosTo -71. aooeis -153 x c o *r a ez7 0.0070 1.S0E+O4 1J2 0576 aoesi 1 .7 3 60 4 2.17 0.512 a06S3 1.236*04 2.07 1.666*04 Intercept 0.0772 410213 4100388 873 4100820 4100607 410Q321 -866 410805 4100268 41000603 722 410361 1.026*03 CoafTaf Determination 0864 0986 0984 0.988 0988 0986 0986 0980 0966 0964 0983 0.986 0987 0.988 Ceieiaantef Deviations One n p <* Cel pt 1 dueled One reo of Cal p|1 duded One rap of eel pt7 eluded One rep of eel pt 5 duded One rap of cal pt 7 oiduded One rep of pto 1 ,3 ,6 eluded One rep of pta 4 ,7 eluded 3M Environmental Laboratory B-l Page 86 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 . LRN-U2095 ' Battelle Study Number. N003604-D 3M Environmental Laboratory Study Num ber FACT 060998.1 PFOS t al IN RAT LIVER STUDY NUMBER: N003604-D DATA ENTERED: MANUALLY SOFTWARE: EXCEL 97 QC Result A nalysis Oats October 13,1969 AnM e PFOS M-556 PF08AA PFOSA October 18,1989 ... PFOS M-559 PFOSAA PFOSA October 18,1999 PFOS M-556 PFOSAA PFOSA October 20,1999 PFOS PFOSA QC Level. no/m L 10 3.3 0.7 0.19 10 3.3 0.7 0.18 10 3.3 - 0.7 0.18 10 3.3 0.7 0.18 10 3.3 0.7 0.18 10 3.3 0.7 0.18 10 3.3 0.7 0.16 10 3.3 0.7 0.18 10 3.3 0.7 0.18 10 M 0.7 0.18 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.18 10 3.3 0.7 0.18 %RSD %RE 6.6 3.8 7.9 3.1 4.7 11.7 3.0 11.5 8.3 5.1 8.7 14.5 16.9 9.2 5.8 4.6 -4 .0 4.8 6.4 -12.2 -2.4 2 .5 6 .6 -5.1 -1 .7 -10.4 -13.0 8.2 -4.3 *2.1 1.3 3.9 5.5 4.5 8.3 9.7 3.4 2.7 8.0 17.7 4.3 2.1 7.4 17.7 11.8 8.1 8.0 8.8 14.8 15.5 8.8 -2.7 -2.8 5.7 -3.7 4.5 -0.8 -2 .9 -7.0 15.8 -3.9 5 .4 4.4 13.7 8.7 11.7 7.0 15.1 14.7 17.0 21.2 28.8 14.7 11.4 15.1 21.3 16.7 11.3 12.6 I *.... 2.4 2.0 3.7 5.3 2.3 4.0 2.3 3.4 4.5 -1 .7 -20.3 -6.1 2.9 23.4 11.8 13.4 0.8 1 .8 20.6 -18.5 0.0 13.7 0.0 18.0 *4.0 -12.0 -21.7 16.0 -1.8 U 1.9 11.3 3M Environmental Laboratory B-2 Page 87 3 M .Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle StudyNumber N003604-D 3MEnvironmental LaboratoryStudyNumber FACT 060998.1 APPENDIX C-SAM PLE INVENTORY LIST ,3M Environmental Laboratory Page 88 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 .' Battelle Study Number. N003604-D 3M Environmental Laboratory Study Num ber FACT 060998.1 Study TOX-013, A rsu* 418-009. Sample Infemnagon fe r hum ant to H ltM la Sam p i* 1 2 3 4 s 8 7 8 9 10 11 12 13 14 IS 16 17 18 10 20 21 22 23 24 25 2a 27 28 29 30 31 32 33 34 35 38 37 38 39 40 41 42 43 44 45 48 47 48 49 so 51 52 53 54 55 58 57 58 50 80 81 62 63 64 65 v! uJI f oo 6u. u5. S am e l* c rip tion FO -9922-flrpl-M FO-9030-Ofpl-M F3-Se31-arp<-M FO -9932-grpl-M P3-9933-grpl-M F O -9961-grpll-M FO -9664-orpll-M FO-9965-oipH-M FC-9987-Qrpll-M FO-9970-orpH-M F0-S097-flrpm -M F0-B 999-0fpm -M FO -10001-tjrpllFM F0-100Q2-grpHI-M FO -10004-crpllU it F 0 -1 0 0 1 9 -crp tV -M FO -10024-prplV -M F0-1002om lV-M FO -10033-grplV -M FO-10034-prptV-W F0-10042-grpV-M FO-10044-grpV-M FO-10045-grpV-M F 0-10051-g rp V-M FO-10054-grpV-M FO-10097-grpl-F F0-10105-gtpl-F FO-10106-grpl-F F0-10107-grpl-F FO-10108-grpl-F F O -10138-orpll-F FO-10140-btpU-P FO -10142-grpll-F I F0-10121-orpll-F F0-10126-grpN-F FO-10177-prpm-P FO-10155-grpHt-F F O -l015& -grplU -F FO -10184-grpm -f1 FO -10172-grpW -F F C -1018 7-g rplV -F FO -10203-grplV -F FO-10 2 1 1 -g rp iV -F FO -10214-grplV -F FO-10007-OPpl-F FO-10105-grpl-F FO-10106-grpl-F FO-10107-grpl-F F 0 -1 0 1 0 6 ^ rp t-F FO -10136^jtpll-F FC-1014O-gtpU-F FG-10142-grpH-F FO-10121-grpH-F FO-10128-grpH-F FO -10177-fliplll-F F 0 -10 155-ffplll-F F0-10164-grplll-F FO -10172-grpM -F FO -10187-orplV -F F0-10194^rpfV -F i FO-10203-orpIV-F I F0-10211-qrplV-F i FO-10214-grplV-F 3 a m p l*T y p * ttetam al R a tU w Ktatem al R a tU w Aatamal R atU var lU tam al Rat LN *r 4atamai R a tU w itatam al Rat U v*r iatamai R a tU w d a te ra i R atU var datarm i R atU var datarne! R atU var da tarm i Rat U v*r idatam al Rat Uvar s ta rn a i R a tU w Maternal R a tU w Maternal R a tU w Matam al R a tU w Maternal R a tU w Matamal R a tU w Maternal R a tU w Matamal Ral U w Matamal Rat U w Maternal R a tU w Matamal R a tU w Maternai Rat U var Matamal R a tU w Fatal U w Fatal U w Fatal U w Fatal Uvar Fatal U w Fatal U w Fatal Uvar Fatal U w Fatal U w Fatal U w Fatal U w Fatal U w Fatal U w Fatal Uvar Fatal U w Fatal Uvar Fatal Uvar Fatal Uvar Fatal U w - Fatal Uvar Matamal R a tU w Matamal R a tU w Matamal R a tU w Matamal R a tU w Matom ai Rat U var Maternal R a tU w Maternal R a tU w Matamal R a tU w Maternal Rat U w Maternal Rat U w Matamal R a tU w Matamal R a tU w Matamal R a tU w Matamal Rat U w Matamal R a tU w Matamal R a tU w Matamal R a tU w Matamal R a tU w Maternal R a tU w M aternal Rat U w ' 3M Environmental Laboratory C-l Page 89 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2 095 Batidle StudyNumber N003604-D 3MEnvironmentalLaboratory StudyNumber FACT060998.1 APPENDIX D-KEPRESENTATIVE CHROMATOGRAMS 3M Environmental Laboratory Page 90 3M Medical Department Study: T6316.5 ' Analytical Report:- FACT TOX-013 LRN-U2095 ' Battelle StudyNumber N003604-D 3MEnvironmental Laboratory StudyNumber. FACT 060998.1 3M Environmental Laboratory D-l Page 91 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2 095 ' . Battello Study Number. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 3M Environmental .Laboratory D-2 Page 92 3M Medical Department Study: T6316.5 'Analytical Report: FACT TOX-013 LRN-U2095 ' Battelle Study Number. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 3M Environmental Laboratory D-3 Page 93 3M Medical Department Study: T6316.5 'Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number. N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3M Environmental Laboratory D-4 Page 94 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 . LRN-U2095 ' Battslie Study N um ber N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 .1 4 a i 3M Environmental Laboratory D-5 Page 95 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Batidle StudyNumber. N003604-D 3MEnvironmental Laboratory Study Number FACT 060998.1 APPENDIX E-PROTOCOL, AM ENDM ENTS, AND DEVIATIONS 3M Environmental -Laboratory Page 96 3M Medical Department Study: T6316.5 'Analytical Report: FACT TOX-013 . LRN-U2095 Battelle Study Number N003604-D 3M Environmental Laboratory StudyNumber. FACT 060998.1 rfrrt.T - 7"<0/t - D/~i 3M E n v ir o n m e n t a l L a b o r a t o r y _________________________ Protocol - Analytical Study 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction In-vivo study reference number: Argus 418-009 Study number: FACT 060998.1 Test substance: 2(N-Ethylperfluorooctanesulfonamido)-ethanol (N-EtFOSJB-OH) Name and address of Sponsor: ` Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 S t Paul, MN 55144 - Name and address of testing facility: 3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-09 . S t Paul, MN 55106 Experimental start date: Expected termination date: December 31,1998 Method numbers and revisions: __ FACT-M-1-0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- . Electrospray/Mass Spectrometry ' ' FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry Author. Lisa Clemen ' / . . 'j.rr'r fc . ' ' : Kris Hansen Study Director _______ i f i s l v i . Date Marvin Case Date Sponsor Representative i 1 3M Environmental Laboratory E-l Page 97 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 _ LRN-U2095 Battelle Study Humber. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 The analytical portion of this dosing study is designed evaluate the levels of perfluorooctane sulfrmte (PFOS), or another metabolite of 2(N-ethylperfluorooctanesulfonamido>ethanol (N- EtFOSE-OH) by the study director, in the liver of the parentand subsequent generations of the test system, or in the serumas necessary. The in life portion of this study was conducted at Argus ResearchLaboratories. 2.0 Regulatory Compliance_________________________________________________ This study is conducted in compliance with the Food andDrug Administration GoodLaboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report. 3:0 Tk?t Materials____ ____ ___________________________________ 3.1 Test, control, and reference substances and matrices ' 3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217 3.1.2 Analytical reference substance matrix: Ratliver andserum 3.1.3 Analytical control substance: Hone 3.1.4 Analytical control substance matrix: Ratliver andserum ' 3.2 Source of materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability information will be included in the final report 3.23 Analytical reference substance matrix: Argus ResearchLaboratories; traceability information will be included in the final report 3.2-3 Analytical control matrix: 3JL3.1 .Rat liver-Argus Research Laboratories; traceability information will be included in the final report; or Rabbit liver- Covance Laboratories; traceabilityinformationwill be included in the final report VTt ?. Rat serum - Sigma ("hemlca! Company; traceability informationwill be included in the final report 33 Number of test and control samples. Liver samples for testing were received from40 test animal and 10 control animals. Serum samples will be tested at the discretion of the Study Director. 3.4 Identification of test and control samples: The samples are identified using the Argus Research Laboratories identifiers, which consist of aletterfollowedby the Argus project number, the animal number, the group designation, and the drawdate.2 2 3M Environmental Laboratory E-2 Page '98 3M Medical Department Study: T6316.5 ^ . . . Analytical Report: FACT TOX-013 LRN-U2 095 Battelle Study Number. N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3.5 Purity and strength of materials: Characterization of the purity and identity of the reference material is the responsibility of the Sponsor. 3.6 Stability of test material: Characterization of the stability of the test material is die responsibility of the Sponsor. 3.7 Storage conditions for test materials: Test materials are stored atroomtemperature. Samples are stored at-20 10 C. 3jj Disposition of test and/or control substances: Biological tissues andfluids are retained per GLPregulation. 2 3 Safety precautions: Refer to the material safety data sheets of chemicals used. Wear appropriate laboratoryattire, andfollow adequate precautions forhandlingbiological materials andpreparing samples for analysis. - 4.0 Experimental - Overview ---------------------------------------------------------- ;-- Tissues fromanimals dosed as described in Argus Research Laboratories Protocol #418-009 are received for analysis of fluorine compounds. At the discretion of the StudyDirector, a series of analytical tests will be performed on select tissues. Initially, all liver samples will be analyzed forPFOS by electrospray/mass spectrometry (ES/MS). On the basis of findings fromthese analyses, additional sample matrices may be evaluated or other metabolites may be targeted. If additional analysis is performed, a protocol amendment will be written. 5.0 Experimental - Analytical Methods_______________________ _ 5.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or OtherAnionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry S 3 FACT-M-3.0, Extraction of PotassiumPerfluorooctanesulfonate or OtherAnionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 5j4 FACT-M-4.0, Analysis of Fluorochemicals In SerumExtractsUsing HPLCElectrospray/Mass Spectrometry 6.0 Data Analysis __________________ '__________________________ 6.1 Data transformations and analysis: Data will be reported as the concentration (weigfat/weight) of fluoride per tissue or sample, or of PFOS per unit of tissue orfluid. 6.2 Statistical analysis: Statistics used may include regression analysis of the serum concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be ' applied to evaluate statistical difference. 3 3M Environmental Laboratory E-3 Page 99 3M Medical Department Study. T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number. N003604-D 3M Environmental Laboratory Study N um ber FACT 060998.1 7.0 Maintenance orRaw Data ajtoRecords --------------------------------------------- 7.1 Thefollowing raw dataandrecords will be retained in the studyfolder in die archives ' according to AMDT-S-8: 7X1 Approved protocol and amendments ' 7X2 Study correspondence 7X3 Shipping records 7X4 Raw data - 7X5 Electronic copies of data 7.2 Supporting records tobe retainedseparately fromthe study folder in the archives according to AMDT-S-8 will include at least die following: 7X1 Training records 7X2 Calibrationrecords 7X3 Instrument maintenance logs . 7X4 Standard OperatingProcedures, EquipmentProcedures, andMethods 7X5 Appropriate specimens. 8.0 References_____ __________________________________________________ 8.1 3MEnvironmental Laboratory Quality System Chapters 1,5 and 6 8.2 Otherapplicable 3MEnvironmental Laboratory Quality System Standard Operating Procedures 9.0 Attachments_________:--------------------- -- --------------------------------------- 9.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants fromLiver forAnalysis Using HPLC-Electrospray/Mass Spectrometry 9.2 FACT-M-2D, Analysis ofFluorochemicals inliver Extracts Using HPLC- ' ' Electrospxay/Mass Spectrometry ' 93 FACT-M-3.0, Extraction of PotassiumPerfluorooctanesulfonate or OtherAnionic Surfactants from Serumfor Analysis Using HPLC-Electrospray/Mass Spectrometry 9.4 FACT-M-4.0, Analysis of Fluorochemicals in SerumExtracts Using HPLC- Electrospray/Mass Spectrometry . 3M Environmental Laboratory E-4 4 Page 100 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle StudyNumber N003604-D 3M Environmental Laboratory Study N um ber FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PXSFL'UOROOCTANXSTXLFONATX (FFOS)1NJAT1IV3ERBY;LC/MS/MS V a n io a L O . ' SfadvWei AMheffiaB Hervi&oaa to 6 e aethod "W ritten by: P a trick L South .D a ta : 7 9 * ^ ______ Approved by; _ C Andre, Pb-D. ,Bionatydcal O vgnbtry D ate G x ^ jJ r S I J H t f 3M Environmental Laboratory Page 1 of Id C-l E-5 Page 101 3M Medical Department Study: T6316.5 . . Analytical Report;: FACT TOX-013 . . LRN-U2095 BatteUc StudyNumber. N003604-D 3M Environmental Laboratory Study Number. F A C T 060998.1 I M E X H O P F O R A N A LY SIS O F PO TA SSIU M P IR F L U O R O O C T A N IS U L F O N A T I (PFO S) IN R A T U V X R B Y LC/M S/M S V crrfaaU . . StadiMaJ A natjat/D eies________________________ L Summary Bdacdo&vdBBlT^tofpotHtiiinpBdQonoctiBBnlfbfltfB&dnlttdfloBnchBUiii a t liver ii pedbcaaed. Callbratiaa Laudani reprepared by q jrin g blank iivac hrTrwgm att wilti sohret tealBtls 6mb w o h^$*odeaily-p*peed e x it. Tba cal&ntian Bated v e ta tte d witt. m rA iri befferai. and gm acad with after! man. Tbi orpnlcpbavi c e avspmtcdttidijim nd ieiurttanolfaBMljiiUbyi r /MS/MSi XL Pottos* ToCXtfB! . . Ibcnd in Spngna-Dawiey t Uvee. HL Samplxs See Chain afCuVDdjr record i f applicable. < IV . G XN E 2A I. IHSEB.UCTIOMS Calflaara all reqnfaedbalances accordinf to lbs SOP a t b ab n a mage. Makei - - ........................ Labd ill sandardand reagent solution ii^iecifled in inappropriate SOP. If jw t tattedts n i a asiatica for Udore talks. ba wan tha label tadndes tbs jatpanrim Ada and da^r nrunbertar which Ite solutlco was initially prepared. . '_ Sign on Ite S n a lp ^ c f this w etted to dgnUy that yonbevo M owed the method S fritta . ,n meinula u reagent ara am o * , and all eqoipacothas been property eaHbrato*.Ifyen ScvlasB fto m te method, d o ran e the change, n d obtain tha approval oft e w it avnagac, andy director, or axk leader a* anon aipavibla. . Initial and d ra all data entries on the page on whid they ware made. V only ana parana eatsa all data a a aingia dqr. Ite docomeaatlaa but ba aoada In a dngla loeadoo on dMt p j IfjenbifUe aodf naks --***-*--,. tba iddtkoal astica ro w be inirtaled and datedby the * i--Mh| Am BiOy* T > cam nr M ft tVmr--t "^1-- 1 rT H" >'1*" The method taw itattita m eal daonologkalorder.brtteaaqnane of tt^ a w ^ h a ilte e d Ir-K. .T^lvw to M Mafferm iate, galea tba a d e r te cenala ctMtke la tiarifleri, StodtairinbataedtarttedBialaaartteabahrvaleaaeattaiaedarnntattitttallbgria considered aspect. __ Ho conwsioawffl be nm delbrpttdty or O co n ee of any tatt ardetebat FFOSAA tT. [ f ia ta r repealer, or poridva-dh otartmans piatti ted& p m iH w^rftm wng wahxam. a f -- with Tefloa by the teat article hoald be laleiertarrt. ' V . M atzsuau S T * le l t e i l l reqnired ctenrieali, reagenti, and tren ti. U Tibia 1t e ihxunicna tta . O xck all labell tantady co ananre tbat all materiali i n oct atpbred and tfett thqr aia th pepar pnrbjr or giade. - Fagalof M 3M Environmental Laboratory C -2 E-6 Page 102 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 ' LRN-U2095 BattcUe Study Num ber N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 . M E T H O D FO B. A NA LY SIS O F PO TA SSIU M PE R FL U O K O O C TA N E SU LFO N A T I (PFO S) IN R A T LTVXR BY LC /M 3/M S . Vantaa LO . . StadvHea A-- h /P e te T aM eLM atarfab Materials . i:c Xii>[lu:r (al .ltli*ir Mm 1`j.c t .1 m 11 I'lltllv IVitif 1 Pnrewhiie Analytical Standard 3M Room Tamp 1 eHbeete IPPOS) S m ro(ita Standard 1CN Room Temp Sufahnnt Add A-S36 Analytical Standard 3M Room Temo . MJ70 Analytical Standard 3M Room Team PFCSAA Analytical Standard 3M Room Temo FFOSA Analytical 3M Room Standard Tamo PFCSEA A ialytical 3M Room i Standard Temp Bat Liver Matrix Hartan Spracoo- --20"C Ammonium Aretain. M otile Phare ***** RT NH3CO, Sodium Hydroxide, RcstcatPrep XT KaOH | n n tfitg Extract Prep : RT OBA). Srultiiwi CofaUMtte Extract Prep RT NaO, Extract Prep RT NaHCOi ESiyi Acetate Extract Prep RT l Mc&oool Mobile Phase, Stock*. W5 RT- M1U-Q Wiser Reagent Prep, MobDePhase M niipon ASTMTypel RT pH I Baffler pH octal RT calibration pS 10 Buffer pH meter RT RT menu Aetna Tempexamro. _ V L E q u ip m e n t See Table 2 Strali required natforpieces a t equipment. Ue the able to docuoent ectaet pie (e.f. nake, model) a t eqalpmoit. Chech ralibrarion ofefl eqiriprner t a^ulilm caflfaarioa (4 . baXizacei) to o s n it it wi|i,**rf ' P u e Jrfli 3 M 'Environmental Laboratory C-3 E-7 Page 103 3M Medical Department Study: T6316.5 Analytical Report:' FACT TOX-013 LRN-U2095 BatteUe StudyNumber N003604-D 3M Environmental Laboratory Study Num ber FACT 060998.1 M ETH O D F O R A NA LY SIS O F PO TASSIUM PE K FL U O R O O C T A N ISU L FO N A T X (PFO S) IN RA T L IV ER BY L G /M S/M S V enina L0 Study 5ou A aalw t/D atc Analytical Baiane Weighs Set Table X la d g a o t list* M:iT)tit:tctuicr Weigh Standaidi or Reagente rHKfrtii MmicI \m->N PipcQ&r ptpet Samples Plprtmr *nip--ooornpessr PtpetSample* . PipetEtOAc extacdcii Plpet Reagenti, WIS Eppeaderf Repealer Vortocer- 1 M x Samples ' Frecar (-20*C) Rdtigwatnr (1-9*0 CmtrtftHB Store QCl, Blank Liver Store Beffa-, Stocka Phase reparaban Test Tobes Csatriflige Tobes T a t Tabea Transport tabea Magnetic cirirr Orticai Shakir Uvcr ampia Evaporate Extracts Store QCj Stir matrix fvrw rt Sample* StodcweU Sdentine Blue Falcon Blue Palean Ettny Polypropylene, U taL Pdypropyiore, 15 mL Polypropylene, 12 x7Sasn ' 5 mL oolyproorlene SWS599 2096 . 2002 127-T160-36P E v ap o rar Evaporate Excaca Zytnatk TaibovapLV Syringe ra te a Filler Fm art . . . . ^nmfifwnin* tR meter Electrode VolmzwCck G aaA VobuneHlc Ptpea. C lin A Toasier Pipeta, Plaatic Grind ttw p^(i iiijfKi BtrflfarfH DetenxdzM Buffer pH Maka v^Hwi^ifl QOndoaa Make Volumetric . DOotfon T rm ifa Extracta to Cendfiipe FUten and LC Inserts HA HA Samoa HA HA HA HA 1 -1 3M Environmental Laboratory Page 4 of 16 C4 E-8 Page 104 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 . Battelie Study Number; N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 method for analysis of potassium p rB F U JO R O O C T A N IS U L F O N A T I (P F O S ) IN R A T L IV E R BY L O M S/M 3 V enial U Study Nau A m tnt/D etes V IL B l o c w u u i A . P re p a ra tio n o f 2 m M A m m onium A cetate \pdgh 0.1500 + 0.0020 g ofammonhnn acetate ndtBBMfhrID a 1000-aLvolumatilB flu k . Diuolvetho aolldhotteraidffim atovehm orttharater. Sdadccmejr banacd tor ana month aimed attoon tempeteme. . l m u rfra n m fa m cattle: Actual fla il votam * _ _ _ _ _ , Date of prependon:___________ \_______ Study K n : . B . p re p a ra tio n o f --29 % S odium H y d ro a id e S olution Weigh 200 a 2 f of toUarnbydnndde into abeaker. Add 300 aaL ofWnil-Qvnmrand sdxtodaotv. Cool lud oauiS? to i polypropylene bank Jar conge. Sohtdonnay be -- *rr I mtuirti at mem tm inriarare. . Actual meat o f S um hydradde: _ _ _ _ _ Actual volume MEU-Quater: Date of preparation: Study No: ______________ - C . P rep aratio n o f-2 -9 % S odintn H ydroxide S olution * iaa 10 mL of--29% Sodium Hydroxide Solution to a lOO-taLvctanetrie Saak aad dOua to volume with M IB -<2 an a*. Tramfcr to apoiyprepyianeb o te ft* auoga. Soludoe maybe cored to r i mnntha a room m npcri Unn. ^rvwi volume of--2994 K tO E eohtdon: _ _ _ _ _ _ Acnai fls il votasw Date of prepuriorc __ _ ------ fttiirfyNn; D . P re p a ratio n o f T e tra b u ty la n u n o n lu m H ydrogensaH ate (l'B A ) M,. S o lution , 0J5 (p H 1 0) ' pH Meter CaHhntintt pH bettor 7 pHbof&r. 10 pH reading:_______ pH reading:_______ Add 169 a lgtfTB A to-S O O aiLofVi& B .-Q 'w aiariaabeakat.A dhteteajH ia 10.00 a 0.02 uaiug-33-60 mL of29% Sodium Hydroxide Seindea, dDuta to 1000 aoLadth . and mix. Adjuat the pH tn 10.00 a 0 J 2 . ia g -2 J % 2 itO S m dm te " ta a iftr to a polypropylene bottle tor murage. Soindan nay be eaedtor ana monte Kond * nxun teaapenttnm. h s U b U lH je a tito S is d e lJ riS tllS O d U H b . * * * * pH 10.0 > 0.0 2 -with 2.9% SodiumHydroxide SohidonMntceamy. - PageS of 16 3M Environmental Laboratory C-S E-9 Page 105 3M Medical Department -Study: T6316.5 ' Analytical Report: FACT TOX-013 _ LRN-U2095 - Battelle Study Number: N003604-D 3M Environmental Laboratory Study Num ber FACT 060998.1 M ETH O D F O R A N A LY SIS O F PO TA SSIU M P X X F L rO RO O C TA N ESU LFO N A TE (P F O S )JN R A T L IV E R B Y L C /M S/M S V enina U Actual m as cfTBA: _ _ _ _ _ _ A ctndflsilW B ioe . Armai Anal pH: _ _ _ _ ^ _ Data ofprepaxaltac___________________ Study No: _ _ _ _ _ _ _ _ _ _ pH fhgm -hrrttnt and/oriqdhm in: . -' . . L P re p a ra tio n o f 0.25 M C a rb o n a ta B u ffer Weigh 26J a 0.1 gefjodiutn cartonate end11.0 a Oil gafoodhnaMiartnBelt and Baraka- a the lame 1000-mL vohnnctric flack. Diandre the gatnrtali falBM-Qitcr; ifiloto to votame with MUB-Q -water, mb; aad tarata: to a polypropylene botila be Kongo. Solution may bo uoedibrl month orbai Bond reftigecxted. ' Actualmaee of eodhum cartonate: _ _ _ _ _ _ w t i i / vflnw frtraHviwfa* Actual final volume: _________ Dale ofpreparatine: ___________________ Study N o:______________ * F . P re p a ra tio n o f M obile P h a je CampanealA: M ix together 600 mL of ImMennenniinn acetatetad <00 niL of n a *m n i Solution may be ueedf i * 1 month'whanacred et mom Imepantera. Aetna! volume of1 m M ammonium acetate:_______ e>L Actual votame of methanol: _ _ _ _ _ _ mL Dam ofpreparedopStudy No:______________ ComponentB: M ix together 30 mL of2 mM ammontata acetate md 930 mL of m e lim i Solution may be u aedfbrl monthwhoa acted et rocn temperatura. Actual volume of 1 mM atrnnnnhim acetate________n L f ^ --1 nrfmediaiml: nL Date cf prepamliw _ _ _ _ _ _ _ _ _ _ _ _ Study No: G . P re p a ra tio n o f S tock S u rro g ate S tan d ard a n d W orking S u rro g ata S ta n d a rd (W SS) ' 1. Stock Surrogate Standard (360,000 ng/mL): . Weigh 23 a 2 mg o f IH . IH . 2H. 2H.-p iflnnronctene phonic acid aad tram&r to a 100-mLvalnmetricflajk.DUaoiv In methanol Statata votone wi.v and m ix Store rctigcaicd. protecteditem UV tight Artnal W riaht; Actual DQudoa Yohnaoc Date afPrcpararinn:_________________ Study No: _ _ _ _ _ _ _ _ _ ' Page 5of 16 '3M Environmental Laboratory C-6 E-10 Page 106 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battello StudyNumber. N003604-D 3M Environmental Laboratory Study Number. FA C T 060998.1 M E T H O D F O R ANALYSIS O F PO TA SSIU M P IR F L U O R O O C T A N S U L F O N A T I (PFO S) IN R A T L IV ER B Y LC/M S/M S V anina L0 ' 5 ta 7 A a a h n t/D a tg 2. WSS (1000 ngfoL): Dflnm 100 >iLcfKock smagata Candan!B 23 mLwith raethaani rndndx- Aetna! Votan of StockInternal Standard: _ _ _____ Acciai DOmlco Vaiamo: ' Date afPiependen: ' H . P re p a ra tio n o f C aH bradon S olvent Stocka and W orW nf S tan d ard s X.imf.u 1 Stock LA Stock 1B Stock 2A Stock ZB Stock 3A Stock 3B Stock 4A Stock 4B Stock 3A Stock 3B Stock SA L Solvent Stocks . For each analyta neigh Iheapedfladamoimt ofS*ndinl(!ad^ndeadr weighed aa A aai B lepBotea) Hated la Tifala 3 andtxana&r into aepaate volumetrie "* Diaaolve In methanol, (Blote to votame with mmhenni, aed adxwalL Stata refrigerated, protected o n UV light. ' 2. Mixed Solvent Stock*: . P Y - fc innm ur f n d malyfeal andari HryUrate A aa Hmadla Tab lai and uaa& rbito a single volumetric Saak. Dlootva la mehmal, dData to volmne with methanol, and mix wdL Sana icOigcnted. protected ftetaUV light. Repeat die procea -withPepitene B so d a. V u m b d jahw sm adbara - ' 0 prepare tkm working andari* . . Data cf par.paiariog__________________ Study Ko: . 3. .. Working Standard! (WS): Dilute the mixed Kochi andworking candara with methanol aa^pedfied la Table 3 and mix wefl. . Pm ef ormaradon: S hikl* PFOS pros M-356 M370 M370 PROSAR PROSAR PROSA PROSA PROSEA T ah leX C xH b radon Sabrent Stock ad W erkt S trad arti V lii.it Am imi t.irj.ee Atti.il N.niiii.il 1 \nmiiMt \ 11.1f\ik4t sili. t-iil.lIVnl I ll.lt \nl. * "1* I Sii.it. or \ \ s .m i.) Imi.) `"'J -ni 1 30 a lm g ta*W BW s s m ____ 10 A M B 8 B 1 f f l M g - J U a 0.3 mg li fa w n t M ____ ____ - m m m ^ 1 30 x 1 ma I 13 a 0.3 ma m ____ ss____ ^ j W a i r **" m ____ ____ H i l f n 30 X 1 ma 10 i'll!lllll!!ll!lllll|[M 1000.000 1 23 X0-3 ma m m m -10 alllgyinaifflnq 2JO0.000 1 93.1m 46 X0.3 ma weaaraataSaSmaBSuma fmifxeibin 10 10 mrfial>wBl 3.00Q.000 H M U U lbOQ.000 30 X1 ma siM B s^ fig n es! 10 lllimipiffll |M 1000.000 23 XO J ma 6Siffi3agB5MgB 10 XJOQ.OOO 30 i 1 u t 10 M SHI 1000.000 Paga 7 of 16 3M Environmental Laboratory C-7 E-l 1 Page" 107 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 - LRN-U2 095 Battclle Study Num ber N003604-D 3M Environmental L aboratory S tudy N u m b e r F A C T 060998.1 M ETH O D T O R ANALYSIS O F PO TA SSIU M . PZ R FL D O R O O C T A N ISH L FO N A T C (PFO S) IN HAT LIVER. BY LC /M S/M S Venioa L0 . StndjNo-:_________' Analnt/Date: Stock SB Mixed Stock B WS l WS2 WS 3 WS 4 WS 3 WS 6 WS 7 FFOSEA. 23 a 0-5 m * |3a S t3t!ftSllaSi||B8 | Storta 1 thru 3 aiL !i 3 9 j S t eh M P W iiin Stock 1 don <KeuB 3 a L ByWtfingEafctnai each** Sh S M 8 S0 W W | Wftml Stock i m LM A Mfaced Stock 1 mL * B WS 1 WS 2 WS 3 i m L** 2mL l5fififtSS3MDdBa! U mL*" iMiffiMlWtIffTM WS 4 WS 3 2-3 mL*" tSHSSwiaicfcas 1 2 mL" io so 30 13 23 io 10 io io io w gnw nl pggggM l |y ||||ff l m ig lM a M M M M BifflB a M M S a iti weanroMBa m a rn a ' Weigh all analytical Jtandardato at lent the acarea 0.01m *. U volumetricorpodttvw ifigilar rmrnf rtprK)- Lsoaooo 300,000 250JW0 20 ,0 0 0 1 0 ,0 0 0 4000 2000 1000 300 200 i 1 P rep aratio n of C alib ratio n S tan d ard s and B lanlo 1. Liver homograna Prepare blank liver homogenaa la bulk by ureigbb* appnwimaoeiy 40 g of kutiV Hvt hun m300 mL N ilrae bod omninfat 300 mL cfMEB-Q water. rtrW I to a hooaogeneoiia mpenrinn. Ahqucr into approx 30 mL portico! Ibr fjwww (approx - 20*C ) e nrage. Actual M an a fliv o K raralvnlnmfrfwaten Darn of prep:__________;_______ . Study:_________________ -- p i*rmiTin densityofcalibrstioo/QC rnalibc M IX HOMOGENA3B THOROUGHLY ad determiaa the m m fa wlfflgfame o f 10 lepllcna wdghinp o f 1 mL portion of the THOROUGHLY M IXED homogenate. M IX HDMDGENATE IMMEDIATELY TOOK.TO BAS ALIQUOT REMOVAL. Rrpiicatr# M an 2, Liver CaHbodon Standards Prepare each Over calibration aandard by adding 0.43 m Laf undHutodBrer homogenate (STIRBOMOGEMA.TEWHILEA1IQUOTING)Intoa 13oL ,,oncQon tube and adding 30 >iL ofWS or MeCB. Prepare tripHcato cal Bgodards and 4 Mnin See Table 3 farveilmnea. Tin dUutadlivcr denaity la rnhaapproximately130mafaiL. MExuelL Fags Sof 16 3M Environmental Laboratory C-8 E-12 Page 108 3M Medical Department Study: T6316.5 Analytical Report': FACT TOX-013 LRN-U2095 Baltelle StudyNumber N003604-D 3M Environmental Laboratory Study Number. F A C T 060998.1 m t t h o d f o r a n a l y s is o f p o t a s s iu m .. ZRFLX JO ROO CTA NK STTLFON A TI (PFO S) D i R A T U V X R BY L C /M S/M S Venia 1.9 Study Mod ' Analrrt/D ale: . Cal SCitUl.iuU 1 2 3 4 5 < 7 Blank Nm ice WS 1 WS 2 WS 3 WS 4 WSJ WS 6 WS 7 MeOH Tables. C alib rato i Standard! Blanks Tarnet Voi Actual Vol Target l.-tL l ..L> [ fin a l Vnl n u l.) Actual liii.il Vnl n n l.l Nominai tu lli. (MlLHlIl ____ m m a r n ____ o j____ w a 2000 50 dto#5 titiW OJ E M B iiR U 1000 - 50 E h tito B d -2*5 fe jw w g M 400 50 P i oj loSeWtoM 200 30 fes M S t B ti o j kahaHBCSsf 100 30 . o j kaw s w a p 1 30 50 o j f l n U k 20 so to iS a S s ffii1 0.3 D ateafprepatadonofcalnds/blank:___________________________ e e V illin i.il l ii'ii. Ml" V 13 A 2A U 0M 033 0.13 J . P re p a ra tio n of Q uality C o n tro l L iv e r Sam ples (Q C i) 1. Quality Control Woridnf Standards . ' Dilute the Allowing jcunx volumes methanol la volumetricflasks a d m ix well. Prepare flesh wbca used. Actual vohunes are in parentheses. s.itif n i OC WS 1 OC WS 2 OC WS 3 OC WS 4 Mixed Stock A < Mixed Stock B OC WS1 OC WS 2 Tabla 4. OC WS pregaradoa Voi Source, m i. Fin.il Voi. mia t in a n iB s^ ^ tfoiiU w im cnaS a i230W HiinSIS|t < one. lili-Mil . 13.000 3000 1123 230 1 2. Preparation of Quality Control liv e r Simple* Prepare each QC In bulk by fiM nf die volumetric flask spproodmamljrhsirflill with Uvtx homogenate (STIR HOMOGENATE WHILE ALIQUCmNGl. adding lbs appropriata QC WS. m bdnt and dflntini to vchnaa with undiluted liver homosenate (STIR HOMOGENATE WHILE ALIQUCmNG). M IX THOROUGHLY tad dipmee X3-mL aUqncta bao polypropylene tubes and store at approximately -20*C. Date of QC prop:___________________Study. --- ---------------------- --K . P re p a ra tio n of M S C heck S ta n d a rd fo r System S u itab ility . Piper 130 jjL ofWS 2 a t--10.000 ngibnL and 2 J mL ofWSS at --1000 aa/mL la mm tbe ro* 50-mL volumeeric flask. Ufluta Covoluxas with MeOH and mfre. Paie 9 of 16 3M Environmental Laboratory C-9 E-13 P a g e '10 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battdle Study Number. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 M ETH O D F O R A N A LY SIS O F PO TA SSIU M . FXRFLUOROOCTANESTJLFONATX (FFO S) IN RA T L IV E R B Y L C /M S/M S . V in io a U> . ' Strut* Wo.; Anshrat/Dete; L . P re p a ra tio n o f hom ogenizer recovery Bver sam ples ' * To determine the recovo? from it homo|ealzricc prceu. enhornop u tted M ark llw r mil befhrtlfied la deplente at 3 cancentnclcn IcvtJa and honrogenteadaslbllow. TMa rinneaverf day that homnyrrriTi lltxt ofawdr males la nerthnaert W-r- n<g MnV guar tata aril a fA, 13 aT. polypropylene j-utiM iy tabea. Record iwigbts ofOvar. 3. Add 100 |iL o fWS 1,3, b x 14 (aee WS perdupficara toba) to prepon . giprrYriTOtriy , 0 ., ad 0.4 |Ig/g. 3. Vfnltiply fhe Tnnmr f llvg- in f by2 5 and add this BBT mL o famar. 4. Homoscaizeeodtlharmnpic.aBdrinehoaaoieolzerprobe'wiAaaotbbr 'im M r fM tg m a t tnnep3. addingtfaueTnhtanoreafandanmla. , 3. O ran borongudier with MOOH between sample. 4. Cap midvortex bomogeaateferuailn'extmctloi. M . P re p a ra tio n o f D ilatio n C heck Sam ple 1_ Place 133 mL ofrmdllrimd tver homogenate (STIRBCMOGEtlA'IB W HILE AUQ UO nNO ) bio a 13 mL exnacdcottbe a n iM d M jrL afSOxed Stodc A. 2. Dnnte30 pLefatep 1 eolation (VORTEX SOLUTION W HILE ALIQUOTING) -with 0.45 mL ofundiluted liver homogenate (STIR HOMOGENATE W HILE ALIQUOTING) in 3.13 mL extraction tribes. 3P T b ii sample ibrwiM be prepared 2br ertrurrioBonly oa days uhta iiudf smnplas win be dtaed and extracted. .' N . H om ogenization o f stu d y am pies . i. Place approximately 0.5 f efoahemogcaiaed atady sample UverbsoaU mL polyprepykne tube. Record weights of liver. ' 2. ' Multiply the mam ofliver in gby3L5 and add this many m Lrfwotec y Homogenize each liver ample, andrinae bomogeninerprobe with aeotber volume of water used In atap 2, addingTinao to hetaogt ntrrd sample. 4. ' dean hemegenfaoerwith MeOH between simples. 5, Cap and vortex homogenate hrr use In cxtricrtra. O. Analysis Standards,Blanks, QCs, andSamples 1. . 2. 3. . 4. 3! '' 7_ MDC UVES. HOMOGENATES THOROUGHLYBETORB ALIQUOTING and pipet 300 jrL c f each QC (4 irpllratre per level), aad otherample belag extracted hao 15-tnL polypropylene extraction tobes. Tbe cxl stria and blanka are already aliquoted. To the B lan ks-E 3 reps), add 100 >iL cfMeGH tad vertex. To the Blanks + IS (3 tepa) and to the remaining ample, add 100 uL WS3 sod vortex Add 05 m L rfO J M T B A (pH 10) loaQ tabee sod vertex bdefly. Add lm L a f 0^3 M carbonate butter and vortex briefiy. Add 15 mL of ethyl acetate. Piece the tnbea ddewqr* on fee oebital d o lo r Sta aetdng of M O tbr-2 0 mimar ____ Centriflige tubes at a *,rr*Tit o f3300 ipm fbr *20 TTirmtra to b patata layers. Transfer 2 mL at the top orpmic layer to a dean polypropylene aba. Pago 10 of 16 3M Environmental Laboratory C-10 E-14 Page 110 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 ' ' LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 method tor analysis of potassium P E R F L O R O O C T A N E S U L F O N A T I (FFOS) IN B A T LTVXB. B Y LC/MS/MS V e n in L0 . S trirM e i Aatijtt/Ottti-_________________________ 9. Evapore lo drynesi under otonfeu * i iBrtm of30*C 1brSOmlieene. 10. Recandone (h* raidae* In 300 jLafBcdeaol wi&vortmdaf. U . Syrinte-fiter xn>ct fato BnekmpletvhlifecM elyriK. Storiviale teftitented (up Id 1 month) IfLO M S/N O m illaotbapei&nadlhamma day. ignee 3 4 ; T~~" tn p m n n t i mhlHty WM dn n u nu tted M t j e l l i i l l i e , a Ve x tn e s of the c il ih , bienio, cad QCi mejrbe naeed t e op *>3 thy* ifte r t h r f r h r l H . 1 f r * " 1* * " " t r h e l d i t m n m i p n r i f t a c a p t h e M ie l t-- i n f l Timm i4 eel addalenfr p a n e te r a : . D a o QC o m e t p trp :______________________________ . P. L C / M S / M S Analyst* F0 Uselhe system condition qmdfied in TdblaJO?The candidanea rM d m deiiesued me be modified by the eoefcrstta produce accasatile peek A n e . LCMSMSwem T ib ie ! - DC/MS/MS CoedhSam .Vifiiviiniitci* m*i.t l'umpN M.hs S|iccfi'iintivr Aii.i1.vnc.il Aolimiii Mollili*Phase Oimimttaus CiMilicnt(iriitif* M ike- Model: ID : M a* M ik e Model: Model: ID: ID : KeystoneBeasti CIS, 5m - 2 X SOn o , P tn N a 005-701-2, S/N: CoiTip'TT1" ... . tleOC * A: Ammonium imtfattrmrrtamnl. 60:40, yy Camponeat B; Ammonium acetarmerbnal. 3:95. ttv T t a t min 0 1 ' .6 6-1 *-3 9 1 11 241 0 0 100 100 100 100 0 0 Flow. Ltarfn 03 03 03 03 06 06 03 03 * * litjcilton totum e l-loiv sp lit 10^ c LC column flow split te *30 u L/ain f idJvoni faro the MS M roa i liillltllll Ivlup IIP LC P u -m c A m b i e n t _________ 10 0 0 ta i * g a t o l a n ( P0 M> Siiiru* B e u im m * - K e te d v e Io n OCMlU.ltiiHl .ts N cltuli/cr "a* Nitrogen t 373 L/hr ( Nltrorai at L/br ( Sourcelllml*lenti) 140Cf *Q Uhrt Lthrt OcMiU.iti ii funi 230* C ( loue 70 V ( _ *20 V ( V)PP O S,-iS - ' V i PFOSA. FTOSAA, PFOSEA, M J56.M 570 C oIIMMII t'iH 'l"? eV) PFOS,TtP FO SA , PfOSAA. M-336, M370 eV> PFOSEA" 1`olltMmi .h M ultiplier Kcsoiliiioit Argon it *13 x 10~*mb p e cell 7 W i *650 V f Vi 12.0 t e MSI ( >. 10.0IhrMar (Dstout&u n ' ? V 11 r f 16 3M Environmental Laboratory c -u E-15 Page ill 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle StudyNumber N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 METHODFORANALYSIS OFPOTASSIUM PERFLUOROOCTANESULFONATX (PFOS) INRATLIVERBY LC/M&MS . VentoLO ' StudyMeu nalvm /Palc It lit wIll'i'lifiK U tl 4 2 7 X 1 MRM transition tx Surrogate Standard (SS) 493>99 MRM transitino HasPFOS 336>71 MRM transidost o M-556 J7C>1S9 MRM transitionftrM 370 34>169 MRM transitionIbrPFCSAA ' - 49t>73 MRM transition 4 PFOSA MRM Transition lhr PrOhldA 1Mi ll I lilt 'tills \|Hi MM.n.itv: 11 niluiires f SS: 4 noia ( arin) aria) - alt ik im ii tme* ": PFOS: 4J urin (_____ ma) 1 4 M : 43 d a ( aria) M370:4.6 min ( min) PFOSAA: 4.7 uria (_____ min) * PTOSA: 3.1 min f min) PFOSEA: 3.7 min f min) . . . * Parameters d ar may be cbanpd by the aimlytt. Actual veines in ( ). i The above cnadlttom iboold be dtabla tbr the Mkromass Quattro LC (SW 9033), Modifications may be necessary if m otherMlcromoie Quadra Saties spectrometer lam ed. Split the flow pcaKalam a via a Keystone BIO tee or siinilar device. 3. C.ItVrm. iC im --tjw n w w fw u d s | a tlA l* fw yrnw l CFYKify that the caHbndion Is mitablo by visual httpm tnn (os the tnnopact) dot a suitable mobile phase km !a sin accanttety determined. Reeoludon m qr Bead te be b itte r them dim used fcr snalyzbg samples. -' 4. standard. Tbo resulti should be compatible Id a recent Injection It avaSafata 3. Use an chromatography integration software system to m llert the 6 outputfrom the analysis. ' . . Loading Order: Sea the lnHhtg reportfrom (ha antnmatari chmtnstngiaphy Integtatlon suftwara tyncm. 7. Make ringle tqjccdorm of each cal standard, QC, study temple, orblank. M dta at least 4 injection of the Instrument chert standard. . a. Ran set rises should typically not exceed SOinjections (too to h tstnaari response roll-aff considerations. Longer tuna may be performed,but dmypoaaa ride ofyielding nraacceptablo curve resulta , VUL CALCULATIONS L Spreadsheet Software: ______________ _ _ Version _ _ 2. MS Analysis Software:,____________________ V errina_______ 3. "imi.re tbe average fa sn y of die Uvei homogenate (10 repe) In m gtaL. 4. Using the avenge density of the homogenate, calmimi la Over density (mg of liver per niL of diluted homogenate): UndUnted Bver density (zng/mL) (g ofliver x average density o fhom ot>& ay(g of liv er-fg ofwater) ' where g ofliver m d g o f w ear ate messes used to prepare bnHr homogenate; density cf water Is assnmed to be 1 g/tnL ' Diluted liver density (rog/rnL) - Undllnted density DUn Factor ' Page 12 of 16 3M Environmental Laboratory C-12 E-16 Page 112 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 -- LRN-U2095 . . Battelle Study Number N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 . . , - t 1 J . | ;, ' '' * . . ' M ETH O D F O R ANA LY SIS O F POTASSIUM P X R FL U O R .O O C T A N ISU L FO N A T I (PFO S) IN R A T LIV ER BY L C /M S/M S ' V erataaU 8tadv Wa.1 Aaahrat/Delg W h e re d ila fiK ln r-2 /l.i" L i l l i to aceocntibr 10%<2Iii aflSverbornofeaeta . In cel odaod QC matrices. . S. Calmhta the aftra! coocararadoa of?P08 ad etherflanredia ntrr ii k the apcnaioai of calltatkat aandarde end QC* by fa the area ataaalyt* a d dtoalaa >cton anlyCno Bver denaiiy corracticn). Uaa parity emtcdoaftrlPOSAAeady. 4. Calculate the acnraleoocsntraticnoii'FOS and otherflootocbenrical* to liver S r dr >nhr.Hiitaiidardi an d O C a a ith n o w c _ - C m fe|AnL.1 DOntadLiver dcartor (msfaL)x1000 mtft x ltr1 aartra 7 . a--~*m bg|ratlnnaaf the peat artaarf the test article endaurrom a E n d ed Me * etaioa. Fla* tnarmal ia c ja tio n i where petSumcd. Calculate Ac a c t coacsstiadecirfcnfc : liv e standard. ' ' j , r . w i . i . the teaicaaicn agnation idarin the peekreeparaemto (tea artd a /S S )tfaadi . m m h n lfM ri ' in leg grida couciueii tian 1 ttver h -ed ri fcrPPO&.M-g<L. M570, andPFCSAA. Calculate the retresriaa equation refedn* ihe peek an * of A yndyrrl m tear rrida mm^muition inBvertbr PEOSA pdPTOSEA. CTOS.M- 3 3 8 ,M 5 7 7 .H fc ri< * * -- hy adng ifa. n ra qata m d anl man harmal m ufarf- PFOSA end PFOSEA ire ntatrtiatrd withoot reference tn ihe rnrmrete fasdamel . enUbmion crave). Use a qaadradc regression e d |h c d 3/z yarigfai auJaded* h e a&aaabrtes. ' . 9. r*>TMt> ^rrm inrA aw ntrvrtnn f o r each injection of eallbadoa standard. QC. i d - ` ausple using the regression parameterx end the peak respoeaa ratios or erne. 10. rvL-nfam As relative error, avenge relative error, tieretard deviation, and relative Mandanl ' deviation Ibr aU QCa. rlm lai the relative errorft each IniectioQ ft calihrttirei Baiutard. iL r i W h v rtiviv m w recoveryibrthehom oecnizarrecoveryIhrtHlraHrma 12, rSivTviarive pairdard deviation ft tha CTOS to SS peakarea ratio atArcrepllratc ' bgectiona of the check aaodard. ' , IX . A co eftancx Cbtixrxa. A . M S C heck.S tandard (S y fte a S uitability) At least 3 injurious o f the MS Chet* Standard e a a provide a W SD eflO S orlaaaftrth a PTOS to S3 peak area ad o . ' B. ' * C a lib ra tio n S ta n d a rd i The percent relative en o n ftrr the ujum itiarion-level avenger rfthe raUbrnfan gaadardl thjviM rpw* dto fallowing ttrntrv 3M Environmental Laboratory Pasa 13 af 16 C-13 E-17 Page 113 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 _ LRN-U2095 Battelle StudyNumber. N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 method for analysis of potassium PXRFLIJOROOCTANKSULFONATE (FFOS) IN RATLIVERBY LC/MS/M8 V c n lo a U - . Stadie M ai TtbU 9. Arrnpnnre to it PFOS__________ I M -5 MJ70 PPQ6 AA PPOSA PPOSZA 2 0 O 3% ttL O O) roqjscixicn joajscuxn ingjsitLocn 20 03% it LO O 2 3 0 0 * t LOO) Us to 3 allbtadaa n nd ud iqjectioni saybe excluded float lb* ear, provided tfatfoM yecdemremsin! per fa d . Itenovri of la eolie f a t i Bay be dee If ^ g ro v il Is * 6ih *d . S b ottre level U-iam rod. the Mmpfa bracketedby the rentinlnt lib rarian reap afflb e ...-- m. --r TliecdJbiariaaenrvathaoVllam cecfOctatafdetenaiaUioBpf . -0 J 7 or better. 1 C. QCi naocoocentratien-fatiiveiifo perseti relativo error and percent rdPhreJtm fati dsvfaiais of tbe'QCi faM meerthe fbHowin Hinht: Tabi 10. QC Acceptance Umita ANALYTE pros 1 20 M-53d 1 20 MS70 20 "" PFOSAA 20 ` PFOSA PFOSEA __________ 2 _________ ! 23 Banov! of badMifati -?ahx fttsn the QC ealndadoni tmqrbe dose If accnopaniadlgr a li -iiiiMn ( t * , bansneti nnhfhncrinn orDteon'l Q fa t remit). H tbo rrerijo detBmJned oonceataion fbr any QC f a t i caestd tie m panetadL * ta b leader sixty director tioold bo notified. The nm may be repeated or portion efthe raoBaybecomidadececptabU. For example. If tbe tow QC M U the fated im if t w r t i, Bcopla may bo accepted Ufa fave enocentadoB bxaducedby * eHbrtloa andati and a mid-Uvei QC coocczeiarioa. D . H o m o gen izer R ecovery and M o tio n C h eck Sam ples ' T fa areale reenvwy crea the 3 fa ti of homoje n ia r recovery empie fa til A t ir f (be an.*!.*. *wtM &U -within the range of *76-130% hmhiaiva. P f a n t l of may bn dona If w rnm partti by iratrmahU . pip iliurifti t S en sitivity (L O Q s ) - Tbe validated lim iti of quantitation re nomimlly O.D |i( f f dt tot5FOS, M-334, MS70, ' endPFCSAA. . pjge 14 o f 16 3M Environmental Laboratory C-14 E-18 Page 114 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 B atteU e S tu d y N u m b e r N 0 0 3 6 0 4 -D 3M E n v iro n m en tal L aboratory S tudy N u m b er. F A C T 0 6 0 9 9 8 .1 METHODFORANALYSIS OFPOTASSIUM PEUrLTJOROOCTANXSTJLFONATX(TFOS) INRATLIVIBYLC/MSMS Vante U ' StmhrlTau . ` AaatjatfOales _ _ _ _ _ _ _ _ _ _ _ _ _ FePTOSA talPTOSEA taviU dtadIX X aoniailly 033 F/staelL j tati rfU i. * t a T k I w m . aniiailcam ifm j& A .aad ppn c p n i im. f^rrUtl itm ig h t a atnedoa. T h en lowar m n r m n a i f vilnM'wffl ba -- -withe * ran let, and may ba brindad in t a rrfrm W a if t a y m n t iccaptanca . i..Am y m w i niiwi m p t a a M d i m a n iu lturd a t a r a cm rrnlntlnaa W ow t a validated levai ^ n n ia aQ jO pgff) arts be appropriMatyfiagged. F. Spedfidty Tba 20% cfLO Q . T l bacnxpt r f t a calfhrarinnanwytm8 dftroncorreetiontaiaq eflfect on qn&dtuloBs.------, -- w .. 01 be ooniideted oe flldent evidence t a t b r a d a n t Body aampta n <|Bantxfledproperty. G . G eneral . IT above acceptance criteria Indiata ta t dril m atad la capable ofproducing ocaaiooal KKSioatxtte t a nonnid acceptance o taria oa validated iwtbod (13% netmalty). Wham faafisanl. plicate asatyaca Icrea t a Impact c it a occasional codicia. X . SJ3 LT3 hrd mpr of snrrvlthvgt ar ree flic onnetwcric drive. XL commotri 3M Environmental Laboratory Page 12 of 16 C-15 E-19 Page 115 3M Medical Department Study: T6316.5 .' Analytical Report: FAC^ R^ ^ 2 09.1 Battelk Study Number. N003604-D 3M E n v iro n m en tal L ab o rato ry S tudy N u m b e r F A C T 0609 9 8 .1 . M E T H O D F O R A N A LY SIS O F PO TA SSIU M prR yLX JO R O O C IA N X SU LFO N A TX TFOS) D i R A T U V I S BY L C /M S/M S V an taaU S ` _______________ X1L C onclu sion s TTTT s n a u z rm x s Anxfyzti Techidcal B eriew Q C X anew Data: D ate D ate D ate Date D ate D ate ' Dass 3M Environmental Laboratory Tata lrfl6 C -16 E-20 Page 116 3M Medical Department Study: T6316.5 ' Analytical .Report: FACT TOX-013 _ LRN-U2095 Battelle StudyNumber. N003604-D 3M Environmental Laboratory StudyNumber. FACT 060998.1 ' Study Tide ' Combined Oral (Gavage) Fertility Development and Perinatal/Poatnatal Reproduction Toxicity Study ofN-EtFOSE in Rats PROTOCOL AMENDMENT NO. 1. Amendment Date: July 28,1999 . Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 933 Bush Avenue . St Paul. MN 55106 Laboratory.Project Identification ET&SS FACT-TOX-013 URNU2095 3M Environment*! Laboratory 3M Environmental Laboratory E-21 Page 117 3M Medical Department Study: T6316.5 ' Analytical Report: FACT TOX-013 : LRN-U2095 -- Battelle StudyNumber N003604-D 1 3M Environmental Laboratory Study Num ber FACT 060998.1 Protocol FACT-TOX-C13 . Amendment 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL read s: The proposed study completion date is listed as 12/31/98. AMEND to read : The proposed study completion data is 6/30/00. REASON: The proposed completion date was changed to allow time for analyzing all matrices of interest. Amendment Approval Marvin Case PhDn Sponsor Representative Kris J. Hansen PhD., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory E -2 2 Page 118 3M Medical Department Study: T6316.5 .. Analytical Report:. FACT TOX-013 . .LRN-U2095 Battelle Study Number. N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 ' Study Tide - Combined Oral (Gavage) Fertility Development and Pcrinatnl/Poatnatal Reproduction Toxicity Study ofN-EtFOSE in Rata PROTOCOL AMENDMENT NO. 2 Amendment Date: September 10,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue S t Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-013 LIRNU2095 l 3M Environmental Laboratory 3M Environmental Laboratory E-23 Page 119 3M Medical Department Study. T6316.5 ' Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number. N003604-D 3M Environmental Laboratory Study Num ber FACT 060998.1 Protocol FACT-TQX-013 . Amendment 2 This amendment modifies the following portion(s) of the protocol: 1. P ro to col READS: The protocol states that liverwill be extracted and analyzed at the 3M Environmental Laboratory. AMEND to read : The liver specimens will be extracted and analyzed atBattelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. REASON: The liver specimens will be sent to Battelle Memorial Institute for extraction and . analysis to rime constraints in the 3M Environmental Laboratory. 2. PROTOCOL READS: The protocol states that serum specimens w ill be extracted and analyzed . following methods: FXCT-M-3.0, "Extraction ofPotassium Perfluorooctanesulfonate or OtherAnionic Surfactants from Serumfor Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-4.0, "Analysis ofFluorochemicals in SerumExtractsUsing HPLCElectrospray/Mass Spectrometry" AMEND to read : The serum specimens will be extracted and analyzed following methods: ETS-8-4.1, "Extraction ofPotassium Perfluorooctanesulfonate or OtherFluorochemical Compounds from Scrumfor Analysis Using HPLC-ElectrosprayMass Spectrometry" ETS-S-5J,."Analysis ofPotassiumPerfluorooctanesulfonate or OtherFluorochemical Compounds in SerumExtracts HPLC-ElectrosprayMass Spectrometry" REASON: The extraction and analytical methods FACT-M-3.0 andFACT-M-4.0, respectively, were updated on 04/27/99 to ETS-8-4.1 and ETS-8-5.1. 3M Environmental Laboratory 3M Environmental' Laboratory E-24 Page 120 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 ' LRN-U2095 Battelle StudyNumber. N003604-D 3MEnvironmental Laboratory StudyNumber FACT 060998.1 Protocol FACT-TOX-013 Amendment 2 PROTOCOL r e a d s: The protocol states that liver specimens will be extracted and analyzed following methods: FACT-M-1.0, "Extraction ofPotassiumPerfluorooctanesulfonate or OtherAnionic surfactants fromLiver for analysis Using HPLC-Electrospray/Maa Spectrometry" FACT-M-2.0, "Analysis ofFriuorochemicals in LiverExtractsUsing HPLC- . Electrospi&y/Mass Spectrometry" AMEND TOread: The liver specimens will be extracted andanalyzed following method: Method for Analysis ofPcrfluorooctane Sulfonate (PFOS) in Rat liverby LC/MS/MS, Version 1.0 Dma *rju` since die Ever extraction ar|d analysis was sub-contractedto Battelle Memorial Institute, this m vhnent was written to include their livermethods andtides. Amendment Approval A M PhD.-, Sponsor Representative fo b hk KristenJ. HansenPhD-, Study Director 3M Environmental Laboratory 3M Environmental Laboratory E-23 Date Page 121 3M Medical Department Study: T6316.5 ' Analytical Report: FACT TOX-013 LRN-U2095 Battelle StudyNumber N003604-D 3ME nvironm ental Laboratory StudyNumber FACT 060998.1 Study Tide Analytical Study 2(N-Ethylperfluorooctanesulfbnamido>eilumol in ' Two GenerationRatReproduction 3 ! PROTOCOL AMENDMENT NO. 3 Amendment Date: October 4,1999 . Performing Laboratory 3MEnvironmentalTechnology & Safety Service! 3M Environmental Laboratory 935 BushAvenue St Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-013 LIKNU2095 3M Environmental Laboratory 3M Environmental Laboratory E -2 6 Page 122 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number; N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 - 1 Protocol F A C T Tox-013 Am endm ent Num ber 3 This amendment modifies the following portlon(s) of the protocol: 1. Protocol Reads: Kristen J. Hansen, PLD. is the Study Director. Amend to Read: James K. Lundberg, Ph-D. is die Study Director. . Reason: Original study design bn changed due to availability of resources and James K. Lundberg will begin serving as the study director for'FACJT-TOX-013 as of 4 October 1999. 2. Protocol R eads: Section 7.1 states that the following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: Approved protocol and amendments; study correspondence; shipping records; raw data; and electronic copies of data. Additionally, 1 Section 73. states that supporting records to be retained separately from foe 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: t Section 7 states: "The original data, or copies thereof; will be available at foe 3M Environmental Laboratory to facilitate audits of foe study during its progress and before acceptance of foe final report. "When foe final report ia completed, all original paper data,' including: approved protocol and amendments, study couespondence, shipping records, raw data approved final report, and electronic copies of data will be retained in foe archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives of the facility performing each analysis. Reason: .. To direct subcontract laboratories in foe disposition of foe items listed above. 3M Environmental Laboratory 3M Environmental Laboratory E-27 Page 123 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 -- LRN-U2095 Battelle StudyNumber N003604-D 3M Environmental Laboratory StudyNumber FACT 060998.1 Protoco/ FACT Tax-013 Amendment Number3 3. P r o t o c o l r e a d s ; Disposition of test andcontrol substances: Biological tissues and fluids are retainedper GLP regulation. Am en d to read : Specimens 'will be maintained in tire 3MEnvironmentalLaboratory specimenarchives. All specimens sent to sub-contract laboratories will be returnedto the 3MEnvironmental Laboratory upon completion of analysis andsubmission ofthe sub-contract labontory(s) final report. The specimens will be returnedwiththe following documentation: tire original chain of custody andrecords of storage conditions while attire sub-contractfacility. a - Reason: To define in detail the appropriate disposition ofspecimen^analyzed at subcontract ' ' laboratories. Amendment Approval T C to L . Marv Case, D.V.M., PhJD., SponsorRepresentative y o d u . tv * * Date _ L J L = __ ;______;---------------------------js ts m Kristen J. Hansen, PhJD., Previous StudyDirector . Date ! Dale L. Bacon, 3M Environmental Laboratory Management 3M Environmental Laboratory 3M Environmental Laboratory E-23 Page 124 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 . . LRN-U2095 Battelle StudyNumber. N003604-D 3MEnvironmental Laboratory StudyNumber. FACT 060998.1 DEVIATION REPORT Battelle Study Number. N0036Q4-D 3M Environmental Technology and Services Study Number: FACT 060998.1 2 (N-Ethylpcrfluorooctaflcsulfonamidoy-ethanol in Two Generation Bat Reproduction TYPEOFDEVIATIONS: PROTOCOL . DATESOFDEVIATIONS: October18, 1999 NATUREOFDEVIATIONS: Someoftheanalytical methodacceptance criteriawerenotmetforthe LC/MS/MSanalyaiaconducted 180ct99atBattelle. Thesedeviationa(ate toprotocol-amendment2. Seebelowforaunmany. . An;il\n- FFOS M-556 M-556 M-556 M-556 PFOSAA PFOSAA OC3 exceeded20%error(-20.3%actual) OC2 exceeded 20%error (23.4%actual) OC3 exceeded20%RSD(21.2%actual) OC4 exceeded20%RSD (28.8%actual) Dilutionrecoveryexceeded 130%(131.5%actual with21.6%RSD) OC3 exceeded 20%error (-20.6% actual) OC4 exceeded 20%RSD (21.3% actual) CAUSEOFDEVIATIONS: Samplepreparationand/orLC/MS/MSvariabilitieaoverthecomaeofthe samplesetmayhave contributedtothedeviations. . IMPACTOFDEVIATIONSONTHESTUDY: The errantQCvalueswerebracketedbyacceptable QCconcentrationlevelswhichdemonstrate*that thecalibrationcurves generallyprovidedgoodaccuracy overthetestedrange. The dilutionrecovery forM-556was not consideredtoboexceedinglyhigh enough, at onlyapproximately 1.5%above thenormal acceptance level, to havesignificantlyimparted- thedata. '- CORRECTIVEACTION:Thisprotocol deviationsummarywas preparedforInclusionbnfoefinal report APPROVEDBY: JonC. Andre, Ph-D. BattellePrincipal Investigator Date KristenJ. Hansen, Ph.D. StudyDirector N003604-D Protocol Deviation. 1, Page 1 of 1 3M Environmental Laboratory E-29 Page 125 3M Medical Department- Study: T6316.5 Analytical Report: PACT TOX-013 LRN-U2095 Battelle StudyNumber N003604-D 3MEnvironmental Laboratory StudyNumber FACT 060998.1 DEVIATION REPORT Ttattelle. Study Number N003604-D ' 3M Environmental Technology and Services Study Number FACT 060998.1 2 (N-Ethylperfluorooctanesulfcmamido)-ethanol in Two Generation Eat Reproduction ' . T Y P E O F D E V IA T IO N S : PR O TO C O L D A T E O F D E V IA T IO N S : October 2 0 . 1999 N A T U R E O F D E V IA T IO N S : PFOS QC3 exceeded the 2 Q * R E method requirement (actual - 2 1 .7 * ) . The recovery nandard did not meet the 7 0 -1 3 0 * recovery requirement (actual* 5 . 0 * fo r PFOS and 4 .6 * fo r PFO SA ). Theae method deviation* relate to amendment 2 o f the atndy protocol. C A U SE OF D E V IA T IO N S : Sample preparation and/or L C /M S /M S variabilities over the coarse at the aample aet may have contributed to the QC deviation. Sample preparation error appear* to have been the cauio fo r the dilution recovery results. IM P A C T O F D E V IA T IO N S O N T H E S T U D Y : Tho orrant Q C value level waa bracketed by acceptable Q C level which demonatratea that (he calibration curvei generally provided good accuracy fo r study sample over the teated range. - . A comparison o f the reaulta obtained for the diluted study samples team 20O ct99 and previous reeulta that were slightly A LO Q (130ct99 and 180ct99) demonatrated good agreement between foe 2 xhia would indicate that the dilution o f the study samples was performed correctly 200ct99 so that do impact on foe quantitations occurred. C O R R E C TIV E A C T IO N :T hia protocol deviation aummaiy waa prepared for inclusion in the fin al report. APPROVED BY: Jon C . Andra, P h-D . - Battelle Principal Investigator Kristen I . Hansen, P h .D . Study Director N003604-D Protocol Deviation 2, Page 1 o f 1 3M Environmental Laboratory E-30 Page 126 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 ^ LRN-U2095 Battcllo StudyNumber N003604-D 3MEnvironmental Laboratory StudyNumber FACT 060998.1 DEVIATION REFQRX Battelle Study Number N003604-D 3M Environmental Technology and Services Study Number FACT 060998.1 2 (N-Ethylpcrfluorooctanesulfonamido)-ethaflol la Two Generation Sat Reproduction : TYPE OF DEVIATIONS: PROTOCOL DATES OF DEVIATIONS: October 12, 1999 through October 20,1999 NATURE OF DEVIATIONS: The lot number of PFOS used was not 217 as per i section 3.1.1 of the protocoL The source of reference substance matrix was not Argus j Research Laboratories as specified in section 3.2.2 of the protocoL Initial analyses of \ liver did not exclusively target PFOS as per section 4.0 of protocol. t j CAUSE OF DEVIATIONS: Only PFOS lot number 171 was available at Battelle. I Marian was the supplier of control rat livers used to prepare blanks, standards, and QCs for the analytical portion of the study. All 4 analytes of interest (PFOS, M-556, PFOSAA, and PFOSA) were monitored during each analysis. IMPACT OF DEVIATIONS ON THE STUDY: PFOS lot number 171 and Harlansupplied liver were both used for Battelle's validation of the analytical method (Battelle : study number N003604-A). These materials allowed achievement of the reported method xr^itanr criteria so that there is no impact on the study. The concurrent analysis of PFOS and metabolites was an efficiency improvement. CORRECTIVE ACTION: This protocol deviation report was prepared. APPROVED BY: Jon C. Andre, Ph.D. Battelle Principal Investigator Date Kristen J. Hansen, Ph.D. Study Director N003604-D Protocol Deviation 3, Page 1 of 1 3M Environmental Laboratory E-31 Page 127 3NLMedical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Battelle StudyNumber. N003604-D 3MEnvironmentalLaboratory StudyNumber FACT060998.1 APPENDIX F - PFOS PURITY REPORT 3M Environmental Laboratory Page 128 3M Medical Department Study: T6316.5 ETSS 2 3W Analytical Report: FACT TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number FACT 060998.1 S 651 778 4226 04/26/99 09:56 0 :02/03 N0:341 Tor From: Subject: Date: 3MSPECIALTY ADHESIVES * CHEMICALS ANALYTICAL LABORATORY Lisa Cernea (3-3368) - ET&5S- 2-03-09 Request # 5783 ' TomXestaer- (3-36331 - SA&C AnalyticalLab-236-2B-11 OuMiical CXxncUrizaan o f TOST-Baotd F luonxhtmlcah by 'H-t'&fR A Sputrotcayy March 24,1999: Preliminary report for FC-95 (PFOS), lot 171 SAMPLE DESCRIPTION^ , FC-95, lot 171 (PFOS), TN-A-0834;Nominal productC,FirS0j<-) K(+) (whitepowder) INTRODUCTION . This sample v u xubjecred to *H-NMR and l,F-NMR spectral analyses to determine the purity of the nominal product and tn characterize aa many impurity components aa possible. A portion of the sample was accurately weighed, spiked with a known amount of 1.4-bis(triflnorociethy0hen7are (p-HFX), and th^n totally dissolved in DMSO-d for subsequent analysis by NMR_ A 400 MHz 'H-NMR spectrum (# h57830.401) and a 376 MHz ''F-NMR spectrum (# Q7830.401) were acquired using a Varian UNITYplus 4C0 FT-NMR spectrometer. Use of the p-HFX internal standard was intended to permit the determination of the absolute weight percent concentrations of the assigned components without necessarily needing to Identify or quantify all the components in the sample mistum. K gsrars; The combined NMR spectral dTM were used to assign all of the major and most of the minor components in this sample as received. The qualitative and quantitative compositional results that were derived from the single trial NMR internal analyses are summarized in TABLE-1 on the following page. I have reported both relative and ihsolute weight p rr^n t concentrations. One possible reason that the absolute wt.% values add up to more 1004b may be due to the fact that 1assumed all of the components contained 8 carbons. If there were any shorter chain homologs present 7,6,5, etc. carbons), then the avenge compound molecular weights would have tw it somewhat less than those used in the calculations. In general, the " F-NMR technique U not ` particularly well suited for Identifying or quantifying small amounts of various fluorochemical homolog Impurity components the chains a n very short. A more complete characterization of any other fluorochemical homologs would require analysis by electrospray MS or a similar technique. Additional work would be required in an effort to positively verify the tentatively assigned components Hated la . TABLE-1 (denoted by possible). Small mounts of other unidentified impurities are also detected In the'NMR spectra, but additional work would be required in an effort to identify or quantify these other materials. Copies of the NMR spectra will be provided for you at a later date. If you have any questions about the results in this initial report for FC-93, lot 171. please let me know. I apologize for the delay In completing this initial work. Tom Xestner c W e k P a y te -JA * C A aly*aaU b- 234.11-11 KU&rac*:LCiJUtUXX/il 3M Environmental Laboratory r**iF-l Page 129 3M Medical Department Study: T6316.-5 Analytical Report: FACT TOX-013 .- LRN-U2095- Battelle StudyNumber N003604-D 3MEnvironmental Laboratory StudyNumber FACT060998.1 ETSS 2 3VI 651 77S 4226 . 04/26/59 09:56 0 :G3/03 N0;341 March 24,1999 . SAACAnalytical LxbRequest# 57830 - Inlfol Krem far FC-M. tot i n TABLE-1 Sample: FC-9S, lot 171 (FFOSXTN-A-0834 Overall Quantitative rnrnprnirma1 Results by vH/l,F-NMR Internal Standardizados Analyses S tru c tu ra l A erip im e n ta C Fj(C F j)-SOj(-) K<+) CNocmil chain; sanane z 7 tor ciUmlartoe purposes) NVOt A bsoluta W alfh t* (lin d e tria] measurement) 703% N M U K i IsIIt i W eight* . (sin tie trial measurement) 6 8 .6 * C F ^ C F ^ C F C C F jH C F O rS C -) K +) Cntam al aioootneibyl b ra n d ; assum a x+y 3, z a 0, A y * 0, for calculation purposes) 17 .7 * 173* . .(C FjJsC IH C FsJ.-SO sC -) K M (Isopropylbranch;assum e x * i fa rcalculadoparpse*) 103* 103* C J^ rC F IC F sF S C b i-) K M (Alpfat branch; x 6 for calcolitico purpotes) P o sato la F -S F s-C A rS O iM K M (assum e x * t for calculados purposes) C F K C F jJ^ C tC F jW C F Jj-S O , K M (Internal gem-dim ethyl branch: tssum e x+y 4 sad x * 0 for cakuliktiaa purposes) 33% 031% a i6 * 33* 036* 0 .1 6 * P o ssib le C F rS F rC ^ s rS O j K M (assum e z 7 for cslculsdon purposes) P robable C *H i.rS O j(-) K M (H ydrocarbon sulfonate salt; assume x S tor calculation purposes) (C F jJjC -C C F iJrS C -) K M (t-bw yi branch: assume z 4 tor calculation purpoass) . 0 .1 1 * 0 .0 3 1 * 0X327* 0.10% 0X330* 0X326* 3M Environmental Laboratory F-2 Page 130 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 Appendix H: Interim Certificate of Analysis 3M Environmental Laboratory Page 131 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018B 3M Product: PFOS, Lot 171 Reference #: SD-009 Test Name Purity1 Purity: 86.4%____________________ Specifications Result 86.4% Appearance White Crystalline Powder Conforms Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6. Iron 7. Manganese Total % Impurity (NMR) Total % Impurity (LC/MS) Total % Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic Acids 3(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis4: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Positive 1. 0.017 wt/wt.% 2. 0.007 wt/wt.% 3. 1.355 wtVwt.% 4. 6.552 wt7wt.% 5. 0.003 wt/wt.% 6. 0.004 wtJwt.% 7. <0.001 wt/wt.% 1.00 wt./wt.% 10.60 wtVwt.% None Detected 0.30 wt./wt.% None Detected Not Applicable3 1. <0.015 wt./wt.% 2. 0.27 wtiwt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6. <0.007 wt./wt.% 7. 8.82 wtVwt.% 1. <0.1 wt/wt.% 2. <0.1 wt/wt.% 3. <0.1 wt/wt.% 4. <0.25 wt/wt.% 1. 12.08 wt/wt.% 2. 0.794 wt/wt.% 3. 1.61 wt/wt.% 4. 10.1 wt/wt.% 5. 50.4 wt/wt.% COA023-018B 3M Environmental Laboratory Page 1 o f 3 Page 132 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum o f metal impurities, 1.39% +LC/MS impurities, 10.60%+Inorganic Fluoride, 0.27%+NMR impurities, 1.00%+ POAA, 0.30%) Total impurity from all tests = 13.56% Purity = 100% - 13.56% = 86.4% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the S 04 is not considered an impurity. 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, C8F 17S03"K+ (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018B 3M Environmental Laboratory Page 2 o f 3 Page 133 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B LC/MS Purity Profile: Im p u rity C4 C5 C6 C7 Total wt./wt. % 1.03 1.56 6.38 1.63 10.60 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: _________________________________________________ David S. Bell Date Scientist, Centre Analytical Laboratories Reviewed B y :_______ _______________________ ____ John Flaherty Date Laboratory Manager, Centre Analytical Laboratories COA023-018B 3M Environmental Laboratory Page 3 o f3 Page 134 3M Medical Department Study: T6316.5 Analytical Report: FACT TOX-013 LRN-U2095 A ppendix I: Report Signature Page ia.u 'C lA A .'^ rx MarvinT. Case D.V.M., Ph.D,,Study Director Date John L. Butenhoff, Ph.D., Sponsor Representative Date Dale L. Bacon, Laboratory Manager ^ fj,------ I W O fCrt'i PA*- o 3. Date O 3M Environmental Laboratory Page 135