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3M Medical Department Study: T-6316.5 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Report: FACT TOX-013 LRN-U2095 Study Title Analytical Study 2(N-Ethylperfluorooctanesu1fonamido)-ethanol in Two Generation Rat Reproduction Amended Analytical Laboratory Report 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/PIUS@Rats Exposed to EtFOSE-OH Data Requirement Not Applicable Author 3M Environmental Laboratory Study Completion Date MAayt s3ig1n,in2g001 Performing Laboratories Sera Analyses Liver Analyses 3M Environmental Laboratory Building2-3E-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 of Pages 143 3M Environmental Laboratory 3M Environmental Laboratory Page 1 Page 1 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 This page has been reserved for specific country requirements. R 3M Environmental Laboratory 3M Environmental Laboratory Page 2 Page 2 3M Medical Department Study: T-6316.5 3M Medical Department Study: T-63 16.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Study: 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:CD@BRVAF/Plus@ Rats Exposed to EtFOSE-OH Study Identification Number: T-631 6.5, 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/l 6100utilized 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. The analytical report from Battelle is not signed or dated by the Principal Analytical Investigator or laboratory management. The Quality Assurance Statement in the Battelle analytical report does not include the dates of the QA inspection activities or the dates reported to the Study Director and laboratory management. The Quality Assurance Statement is not signed. The Argus and Battelle analytical reports do not include the names of all the contributing personnel. Date Sponsor Representative Date 3M Environmental Labora tory 3M Environmental Laboratory Page 3 Page 3 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 3M Medical Department Study: T-6316.5 Analytical Study: 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@'BRVAF/PIUS@Rats Exposedto 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 10112/99 6/5/00 - 6114/00 9/11100 - 9113/00 Extraction Data Draft report 10/26/99 6116/00 911 4/00 10/26/99 6116/00 911 4/00 511 410 1 Amended report 511 410 1 511 410 1 QAU Repredntative Date 3M Environmental Laboratory 3M Environmental Laboratory Page 4 Page 4 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 3M Medical Department Study: T-6316.5 Table of Contents Analytical Study: FACT TOX-013 LRN-U2095 GLP Compliance Statement............................................................................................. 3 GLP Study-Quality Assurance Statement ...................................................................... 4 Study Personnel and Contributors.................................................................................... 7 Introduction and Purpose ................................................................................................. 8 Test System................................................................................................................. 8 Specimen Collection and Analysis ............................................................................... 9 Specimen Receipt and Maintenance ................................................................................ 9 Chemical Characterization................................................................................................ 10 Dose Confirmation Analyses ........................................................................................ 10 Method Summaries........................................................................................................... 1101 3M EnvironmentalLaboratory ...................................................................................... 11 PreparatoryMethod................................................................................................. 11 Analytical Method.................................................................................................... 11 Analytical Equipment............................................................................................... 11 Deviations .................................................................................................................... 12 Data Quality Objectives and Data Integrity....................................................................... 12 Data Summary. Analyses. and Results ............................................................................ 13 Summary of Quality Control Analyses Results............................................................. 13 Summary of Sample Results........................................................................................ 14 Statistical Methodsand Calculations................................................................................ 14 Statement of Conclusion.................................................................................................. 14 Appendix A: Chemical Characterization. Control Matrices and Dose Confirmation Analyses........................................................................................................................... 15 Appendix B:Protocol........................................................................................................ 1198 Appendix C: Extraction and Analytical Methods ............................................................... 2307 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry. (14 pages) ........................................................................................................................ 2308 ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-ElectrospraylMass Spectrometry. (9 pages)...................2502 Appendix D: Data Summary Tables ................................................................................. 2611 Appendix E: Data Spreadsheets ...................................................................................... 2644 Appendix F: Example Calculations ................................................................................... 70 Appendix G: Contract Lab Report .................................................................................... 71 Appendix H: Interim Certificate of Analysis ....................................................................... 137 Appendix I: Report Signature Page ......................................................................... 141 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 5 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 3M Medical Department Study: T-6316.5 Analytical Study: FACT TOX-013 LRN-U2095 Appendix J: Amendment 1 to FACT TOX-013 Final Report 14229 List of Tables Table 1. Test System Population Demographicsand 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 ln-life #418-009 .................................... 17 Table 11.Tween Dosing Confirmation-Matrix Spikes for Study In-life #418-009 ........... 17 Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013 ...621 Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013 (continued) ......................................................................................................... 6222 Table 13. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-013...6222 Table 13. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-013 (continued) ......................................................................................................... 6233 3M Environmental Laboratory 3M Environmental Laboratory Page 6 Page 6 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 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 c St. Paul, MN, 55144-1000 651-733-5180 P= Sponsor John L. Butenhoff, Ph.D., Sponsor Representative 3M Corporate Toxicology - Medical Department p" 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 Analytical Chemistry Laboratories Ser8 Analyses 3M Environmental Laboratory (3M Lab) I Kristen J. Hansen Ph.D., Analytical Investigator Liver Analyses Battelle Memorial Institute Jon C. Andre, Ph.D., Analytical investigator 3M Lab Contributing Personnel c 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 0. Johnson Kelly J. Kuehlwein Sally A. Linda Joseph C. Pilon Scott R. Post Ian A. Smith Kathy M. Stock Anh-Dao Vo Bob W. Wynne Location of Archives All original Laboratory. specimens Laboratory. substance. raw data, protocol, and analytical report have been archived at the 3M Environmental The test substance and analytical reference standard reserve samples, as well as the pertaining to the analytical phase of this study, are archived at the 3M Environmental Control sera and liver will be maintained at the contract lab along with the test A 3M Environmental Laboratory 3M Environmental Laboratory Page 7 Page 7 R3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 R Introduction and Purpose The purpose of the study is to determine the presence and concentration of PFOS, PFOSA, rl PFOSAA, and MS56 in liver samples and PFOS, PFOSA, PFOSAA, EtFOSEOH, and M556 in sera samples collected from rats exposed to EtFOSEOH. This study was initiated on 1 October 1998. P Five groups of FO generation mate and female rats and 3 groups of Fl 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. F" 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 Ill were selected for continued evaluation. Fl generation male and female rats were given appropriate dosages of the test article 111 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:Cl?`BR VAF/Pius@ (Sprague-Dawley) rat received from Charles River Laboratories, Inc., and assigned temporary numbers until assigned to the study. Rats were permanently identified using MonePself-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 Fl generation rat F" selected for continued observation was identified with a MonePself-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 (418-099) 3M Environmental Laboratory 3M Environmental Laboratory Page 8 Page 8 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 Sample specimens were collected by Argus (study 418-009) and sent to the 3M Environmental r" Laboratory for analysis. Liver and sera specimens were collected from FO male rats at the completion of the cohabitation period and FO female rats on day 21 postpartum. Liver specimens were collected from FO generation litters, and stomach content specimens were collected from the FO 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. I"" Specimens Collected from Study Groups I through V (through 11130/98): Serum Specimene-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 -70% 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. p" 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 M558 levels were quantitated by external calibration. PFCSEA 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 84-98, lo-l-98 and I-29-99. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C *IO%. 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 8 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% &IO%. 3M Environmental Laboratory 3M Environmental Laboratory Page 9 Page 9 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 Chemical Characterization EffOSE-OH CAS Number: 1691-99-2 Chemical Formula: CBF,rS02N(CH2CH,)CH,CH,0H Molecular Weight: 571 .O Chemical characterization information Chemical characterization information presented in tabular form in Appendix Analysis available in Appendix I. on the test article is presented in tabular form below. on the analytical reference materials used in this study is A (see Tables 8 and 9) and the interim Certificate of Table 2. Characte&ation of the Test Article in Study FACT TOX-013 IChemkal Name I I Source I FM-3929 2(N-Etiylperftuorooctane sulfonamido)ethanol 3M 1 Exdration Date 05J2000 1 StoraaeConditions 1 Ambient temperature Chemical Lot # 30035,30037,30039 Physical Description waxy solii l Tha purity of the test article clwa&rtzauan is oaming crr~ofan~~kissued. I To be determined' determined nominally by NMR analysisS. ubsequentchemical and thii analytical report wiPba amended to indicatethe putity whena - Do8econtinnatponAnaIyses The dose confirmation data were 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 resultsare presented in Appendix A (see Tables 10 and 1 I). pa Dose confirmation was performed by diluting the Tween dose samples with Mitli-Q water into the linear range of the instrument. For each sample, a matrix spike was prepared (at approximately XI-tOO% 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. P 3M Environmental Laboratory 3M Environmental Laboratory Page 10 Page 10 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 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). 3MtrvlmlLaboratory .- PREPARATORY METHOO l ETS-84.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemicat Compounds from Serum for Analysis using HPLC-ElectrospraylMass 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 .O mL of methanol, then filtered through a 3cc plastic syringe attached to a 0.2pm nylon filter into a glass autovial. ANALWCAL METNOD l ETS-8~8.1, "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 HPLCESMSMS. For example, molecular ion 499, selected as the primary-ion for PFOS (C,F,,S03-) analysis, was fragmented further to produce ion 99 (FSQ-). The characteristic product-ion 99 was monitored for quantitative analysis. ANAL~~CAL EQWMENT 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' BetasilTW C,B 2x50 mm (5 pm) p" Column temperature: Ambient Mobile phase components: Component A: 2mM aqueous ammonium acetate Component B: methanol Flow rate: 300 j.rL/min Injection volume: 10 uL Ic" Solvent Gradient: 10 minutes Start at 4O%B Hold at 40WB for 1 minute Increase to 95%B over 3.5 minutes 3M Environmental Laboratory 3M Environmental Laboratory Page 11 Page 11 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 R Hold at 95Or6B for 2 minutes Return to 40hB over 0.5 minutes Hold at 40r6B for 3 minutes Mass Spectrometer: Micromass@ API/Mass Spectrometer Quattro II"Triple Quadrupole system Software: Mass Lynx" 3.2 Cone Voltage: 20-80 V Collision Energy: 2545 eV Mode: Electrospray Negative Source Block Temperature: 150C &I 0C c Z-spray source Analysis Type: Multiple Reaction Monitoring (MRM) Table 3. Negative Ions Monitored in 3M Laboratory Analyses R EtFOSE-OH 630.0 M556 558.0 THPFOS 427.0 59.0 78.0, 169.0 80.0 I"r Deviations from the original protocol and methods are documented in the table below: Table 4. Deviation Summaw for FACT TOX-013 Deviation Pie was usad instead Oxforddiiser I0.2-l .OmL of for extraction sample instead was used of 1.OmL. Milk curd samples were not analyzed. Date(s) of Occurrence lOl12l99 Entire study Impact on Study I Standards and samplesware preparad identically. No adversa impact on study. Current work indicates that vdutnas 20.5 mL provide results equivalent to 1 mL extraction volumes. Resub of sample I volumes 46 mL have not been vaMat6U I andwilbemarkedinthedatatabk. No milk curd data is available for the final report Data Quality Objectives and Data Integrity prr The following data quality objectives (DQOs) were indicated in the method performance section of ETS-8-5.1 I `Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry*: 3M Environmental Laboratory 3M Environmental Laboratory Page 12 Page 12 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 l Linearity: The coefficient of determination (rZ) equal to or greater than 0.980 l Limits of Quantitation (LOO): The LOCI 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. l Acceptable Spike Recoveries: 70-I 30% Data Summary, Analyses, and Results With the exceptions noted in this report, data quality objectives for the analytical phase of this la 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 *rr included in this report. The data in this report reflect only that obtained from specimens extracted on, or after October 11, 1999. Summary of QuaMy Contml Anatyses Results l Linearity: The coefficient of determination (r*) of the standard curves werer0.980. l Calibration Standards: Quantitation of the target analytes was based on linear regression analysis (l/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). l Limits of Quantitation (LOQ): The LOCI 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 I-- ~PFOSAA 1 20.5 ppb I 1 EtFOSE-CH 1 36.2 ppb I I M556 I 24.9 ppb I 3M Environmental Laboratory 3M Environmental Laboratory Page 13 Page 13 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 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: 10%. Precision was determined by analysis of MS/MSD and was reproducible to within 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 prr spikes were within i30% 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 surrogateresponse 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 quantiiative to f30% 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 l Samples from Control Animals: Low levels of PFOS, PFOSA, PFOSAA, EtFOS&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. ila l Samples from Dosed Animals: In general, PFOS, PFOSA, PFOSAA, EtFOSEOH, 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 0 and E. Statistical Methods and Calculations Statistical Appendix TOX-013. methods were limited to the calculation of means and standard deviations. See F for example calculations used to generate the liver and serum sample data in FACT Statement of Conclusion 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 EtFOS60H during the in-life phase of the study. P-- 3M Environmental Laboratory 3M Environmental Laboratory Page 14 Page 14 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Appendix A: Chemical Characterization, Confirmation Analyses Control Matrices and Dose Table 6. Characterization of the Control Matrices Used for Sera Analyses in Study FACT TOX-013 I Control Matrix sowca Expiration Date Storage Conditions Chernloal Lot # Physical Description N/R-not racorded 3M Lab I Rat Serum I (TN-A-2001 1 siirna 2010 Ambient 17HD308 Rat !3enrm Rabbit Serum (TN-A-2573) I Sigma 2010 Ambient 118H8418 Rabbit Serum Table 7. Characterization of the Control Matrices - Used for Liver Analyses in Study FACT TOX-013 Location I Battelk Memorial Institute I I Rat Liver source Expiration Date Harlan N/R Storage Condiins N/R Chemical Lot # Physical Description N/R Rat Liver Nm-notracordad 3M Environmental Laboratory 3M Environmental Laboratory Page 15 Page 15 3M Medical Department Study: T-6316.5 3M Medical Department Study: T-63 16.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Study: 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 Source 3M Specially N/R NIR Chemicals Expiration Date 08/31/01 01/01/2010 01/01/2010 Storage Conditions Ambient temperature Ambient temperature Ambient temperature Chemical Lot 171 L-l 5709 NB 112999-99 Number Physical Description White crystalline powder Light yellow waxy solii Tan waxy solid Puritv 86.4% TBD TBD *Surrogate standard- 1H,l H,2H,2H-Tetrahydroperfluomctanesulfmic acid N/R-not recorded TBD-to be determined NA-not applicable 3M ICPiPCP Division 01/01/2010 Ambient temperature 936 Amber waxy solii TBD 3M 01/01/2010 NB 113047-80 White powder TBD Table 9. Characterization of the Analytical Reference Materials Used for Liver Analyses in Study FACT TOX-013 Battelle Memorial Institute ICN Biomedicals 01/2010 53406 Brown waxy solid NA Chemical Lot 171 NB 113047-80 617 Number L-l 5709 59909 PDheysscircipatlion Nhitapocvq@stadlliwnr Whk powder N/R Lwiaihxty yeslololiwd N/R Puritv I 86.4% I TBD 1 TBD 1 TBD 1 NA *Surrogate standard- 1H,l H,2H,2H-Tetrahydropertluorcoctanesulfcmic acid N/R-not recorded TBD-to be determined NA-not applicable 3M Environmental Laboratory 3M Environmental Laboratory Page 16 Page 16 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Group Dose Group I-Control 0 m@mL Sample Number Expected cont. EtFOSE hslmL1 Measumd Cont. EtFOSE EtFOSE (na!mL) I Ye Recovwy Accuracy I Group 2-0.2 m9imL Group 3-l .O m@mL Group 4-2.0 mglmL Group ".O m@mL Homogeneity Samples3.0 mghnL NA = Not applicable B418-009-E, 07/l!Y98 &416-009-A, 05iO6i96 1 of6T B418-009-A, 06KW98 3of6M I &418-009-A, 06fw98 5ofSB 364oooo 32!5oooo I 369oooo 3790000 121 108 123 126 I Table 11. Tween Dosing Confirmation-Matrix Spikes for Study In-life #418-809 I Sample Number Expected Cone. EtFOSE (nglml) Measured Cont. EtFOSE (n@mL) EtFOSE % MS Recovety Accuracy .c6lowS-MS I 1200 I NA I NA I B418-009-B, 07115t98-Ms 1 1200 ! NA I NA I 900 818 91 B-4184lO9-c. 07/15/9&Ms 900 826 92 B-416-009-D, 06/08&6-MS B-418-OosD, 07/15/964s &-418-009-E, -MS I I B-416-009-E. 07115&8-MS ! 1100 ! 1089 99 I B-418409-A, 0!5KW98 1100 GaA-Vn .3 I RR I 1 of6T-MS I - I B-418-009-A, OWOSl98 3of6M-MS 1100 1063 I B418-050o9f-6AB,-MS06KW98 1100 944 NA = Not appliiblt, 98 I 86 I 3M Environmental Laboratory 3M Environmental Laboratory Page 17 Page 17 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Appendix B: Protocol Analytical Analytical Study: FACT-TOX-013 LRN-U2095 Report: FACT TOX-013 LRN-U2095 3M Environmental Laboratory 3M Environmental Laboratory Page 18 Page 18 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 pr* Analytical Analytical Study: FACT-TOX-013 Re~~~~&~~~~ LRN-U2095 ,9/3 "Ws- 3M ENVIRONMENTALLABORATORY 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: Z(N-Ethylperfluorooctanesulfonamido)-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 Serv@s 935 Bush Avenue, Building 2-33-09 1 St. Paul, MN 55106 / Experimental start date: Expected termination date: December 31,1998 Method numbers and revisions: FACT-M-1.0, Extraction of Potassium Peffluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometrj FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospraybkss Spectrometry F" FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrosprayhkss Spectrometry cs FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLC- ElectrosprayIMass Spectrometry Author: Lisa Clemen FR -/la kl AZ Kris Hansen Study Director 4lrsl'rtl Date Marvin&e Sponsor Representative /4k/fw Date 4 3M/Environmental Laboratory 3M Environmental Laboratory Page 19 Page 19 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-L01R3 N-U2095 LRN-U2095 1.0 PURPOSE The analytical portion of this dosing study is designedevaluatethe levels of perfluorooctane sulfonate (PFOS), or anothermetabolite of 2(N-ethylpeffluorooctanesulfomunido)-ethanol (NFtFOSE-OH) designatedby 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 ResearchLaboratories. 2.0 REGuLAT~RYCOM~LI.~NCE This study is conducted in compliance with the Food and Drug Administration Good Laboratory . Practices regulation as statedin 21 CFR 58. Any exceptions will be noted in the final report. 3.0 TESTMATERIALS 3.1 Test, control, and reference substances and matrices p" 3.1.1 Anaiyticai reference substance: Potassium peffluorooctanesulfonate (PFOS), lot # 217 3.1.2 Analytical reference substance matrix: Rat liver and serum 3.13 Analytical control substance: None 3.1.4 Analytical control substance matrk Rat liver and serum 3.2 Source of materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceabihty information will be included in the final report 3.2.2 Analytical reference substance matrix: Argus ResearchLaboratories; traceability information will be included in the final report 3.2.3 Analytical contiol matrix: 3.23.1 Rat liver - Argus ResearchLaboratories; 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 Number of test and control samples. Liver samplesfor testing were received from 40 test animals and 10 control animals. Serum samples will be tested at the discretion of the Study Director. c" 3.4 Identification of test and control samples: The samples are identified using the Argus ResearchLaboratories 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 3M Environmental Laboratory 2 Page 20 Page 20 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-0L13RN-U2095 LRN-u2095 3.5 Purity and strength of materMs: Characterization of the purity and identity of the referencematerial is the responsibility of the Sponsor. 3.6 Stability of test material: 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 storedat -20 f 10 OC. 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 sheetsof chemicals used. Wear appropriatelaboratory attire, and follow adequateprecautions for handling biological materials and preparing sampIesfor analysis. 4.0 EXPERIMENTAL - Overview Tissues from animals dosedasdescribedin Argus ResearchLaboratories 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 selecttissues. Initially, all liver sampleswill be analyzedfor PFOS by electrospray/massspectrometry (ES/MS). On the basis of findings from theseanalyses,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 - Anaivtical Methods P 5.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Surfactants from Liver for Analysis Using HPLC-ElectrosprayIMas Spectrometry 5.2 FACT-M-2.0, Analysid of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 5.3 FACT-M-3.0, Extraction of Potassium Peffluorooctanesulfonate or Other Anionic Surfactantsfrom Serum for Analysis Using HPLC-ElectrosprayIMass Spectrometry 5.4 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrosprayIMass Spectrometry p" 6.0 DATA ANALYSIS 6.1 Data transformations and analysis: Data will be reported asthe concentration (weight/weight) of fluoride per tissue or sample, or of PFOS per unit of tissueor fluid. 6.2 Statistical analysis: Statistics usedmay include regressionanalysis of the serum concentrations over time, and standarddeviations calculated for the concentrations within each dosegroup. If necessary,simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 3M Environmental Laboratory 3M Environmental Laboratory 3 Page 21 Page 21 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-L01R3 N-U2095 LRN-u2095 7.0 ~~~~~N~CEOFRA~DATAANDRECORD~ 7.1 The following raw data and recordswill be retained in the study folder in the archives R according to AMDT-S-8: 7.1.1 Approved protocol and amendments p" 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 separatelyfrom 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 maintenancelogs p" 7.2.4 Standard Operating Procedures,Equipment Procedures,and Methods 7.2.5 Appropriate specimens. 8.1 3M Environmental Laboratory Quality System Chapters I,5 and 6 8.2 Other applicable 3M Environmental Laboratory Quality System StandardOperating Procedures Fan 9.0 ATTACHMENTS 9.1 FACT-M-LO, Extraction of Potassium Perfluorooctanesulfonateor 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 HPLCElectrosprayhkss Spectrometry 9.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/hkss Spectrometry 9.4 FACT-M-4.0, Analysis of Pluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry 3M Environmental Laboratory gs 3M Environmental Laboratory 4 Page 22 Page 22 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-L01R3 N-U2095 LRN-u2095 w Study Title Combined Oral (Gavage) Fertility Development an&PerinataL&ostnatal Reproduction Toxicity Study of N-EtFOSE in Rats PROTOCOL AMENDMENT NO. I Amendment Date: July 28,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identlffcation ET&SS FACT-TOX-013 LJRN U2095 3/U Environmental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory Page 23 Page 23 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 . Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-L01R3 N-U2095 LRN-u2095 Protocol FACT-TOX-073 Amendment I This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: The proposed study completion date is listed as 12/3l/98. AMEND TO READ: The proposed study completion data is 6/30/00. REASON: The proposed completion date was changedto allow time for analyzing all matrices of interest. Amendment Approval Marvin CasePh.D., SponsorRepresentative Ibis J. Hknsen Ph.D., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory Page 24 Page 24 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXL-0R13N-U2095 LRN-u2095 Combined Oral (Gavage)Fertility Development andPekaWPostnatal Reproduction Toxicity Study of N-EtFOSE in Rats PROTOCOL AMENDMENT NO. 2 Amendmenf Date: September lo,1999 Perfarming Laborafo# 3M Environmental `lk&nology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project IdenfMcafion w ET&SS FACT-TOX-013 LIRN u2095 - 3M Envimnn?ental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory Page 25 Page 25 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOi- LRN-U2095 LRN-u2095 Protocol FACT-7-0X-073 Amendment 2 This amendment modifies the following portlon(s) of the protocol: I. PROTOCOL READS: The protocol statesthat liver will be extracted and analyzed at the 3M Environmental Laboratory. AMEND 7'0 READ: The liver specimenswill be extracted and analyzed at Battelle Memo&l Institute, 505 King Avenue, Cohrmbus, Ohio 43201-2693. f?EASON: The liver specimenswill be sent to Battelle Memorial Institute for extraction and analysis due to time constraints in the 3M Environmental Laboratory. 2. PROTOCOL READS: The protocol statesthat serum specimenswill be extracted and analyked following methods: FACT-M-3.0, "Extraction of Potassium Petfluorooctanestionate or Other Anionic Suifactants fkom Serum for Analysis Using HPLC-Electrosprayhkss Spectrometry' FACT-M-4.0, `Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry" AMEND TO READ: The serum specimenswill be extracted and analyzed following methods: ET&8-4.1, "Extraction of Potassium Pefluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray Mass Spectrometq? ETS-84.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Serum Extracts HPLC-Electrospray Mass Spectrometry" i?EASON; `The extraction and analytical methods FACT-M-3.0 and FACT-M-4.0, respectively, were updatedon 04/27/99 to ETS-8-4.1 and ETS-8-5.1. 3M Environmenta/ Laboratory 3M Environmental Laboratory 3M Environmental Laboratory Page 26 Page 26 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Protocol FACT-TOX-013 Amendment 2 3. PROTOCOL READS: The protocol statesthat liver specimens will be extracted and analyzed following methods: FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic surfactants from Liver for analysis Using HPLC-Electrospray/Ma Spectrometry" FACT-M-2.0, "Analysis of Frluorochemicals in Liver Extracts Using HPLC- . Electrospray/Mass Spectrometry" AMEND TOREAD; The liver specimens will be extracted and analyzed following m&od: , M&hod for Analysis of Perfluorooctane Sulfonate (PFOS) in Rat liver by LCAWMS, Version 1.0 REASON: Since the liver extraction and analysis was sub-contractedto BatteUe Memorial Institute, this amendment was written to include their liver methods and titles. Amendment Approval ., Sponsor Representative Kristen J. Hansen Ph.D., Study Director P 3M Envirvtymmtel Laboratory 3M Environmental Laboratory 3M Environmental Laboratory sP9/g9 Date Page 27 Page 27 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOX-L01R3N-U2095 LRN-u2095 Study Title Analytical Study 2(N-Ethylperfluorooctauesulfomunido)-ethanol in Two GenerationRat Reproduction PROTOCOL AMENDMENT NO. 3 Amendment Date: octoher 4,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Labora tory ProJect ldentification ET&SS FACT-TOX-013 LIRN U2095 3M Environmental Laboratory cri 3M Environmental Laboratory Page 28 Page 28 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Protoco/ FACT Tax-013 Amendment Number 3 This amendment modifies the following portion(s) of the protocol: I. i'ROTOCoL READS: Kristen J. Hansen, Ph.D. is the Study Director. AME~VD TO READ: F" JamesK Lundberg, Ph.D. is the Study Director. REASON: Original study design has changed due to availability of resources and James K. Lundberg will begin serving asthe study director for FACT-TOX-013 as of 4 October 1999. 2. PROTOCOL READS: Section 7.1 statesthat the following ray data and records wilI 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, Section 7.2 statesthat supporting recordsto be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: Training records, calibration records; instrument maintenance logs; StandardOperating Procedures,Equipment Procedures,and Methods; and appropriatespecimens. AMEND TO READ: . Section 7 states:"The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress andbefore acceptanceof the final report. When the final report is completed, all original paper da@ including: approved protocol and amendments,study correspondence,shipping records, raw data, approved final report,`and electronic copies of datawill be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records,. instrument maintenance logs, standardoperating procedures,equipment procedures, and methods will be retainedin the archives of the facility performing eachanalysis. REASON: CI To direct subcontract laboratories in the disposition of the items listed above. 3M Environmenial Laboratory 3M Environmental Laboratory 3M Environmental Laboratory Page 29 Page 29 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LF!N-u2095 Pmfocol FACT Tax-013 Amendment Number 3 3. PROTOCOLREADS: Disposition of 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 of analysis and submission of the sub-contract laboratary(s) final report. The specimens will be returned with the following documentation: the signed m original chain of custody and records of storageconditions while at the sub-contract facility. REASON: To define in detail the appropriate disposition of specimens analyzed at subcontract laboratories. Amendment Approval Marv Case,D.V.M., Ph.D., Sponsor Representative Kristen J. Hansen, Ph.D., Previous Study Director Dale L. Bacon, Ph&y 3M Environmental Laboratory Managekent 3h4 Envhnmental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory Date Page 30 Page 30 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-0L13RN-U2095 LRN-II2095 Study Title Analytical Study of 2(N-Ethylperfluorooctanesulfwxunido)-ethauol in Two GenerationRat Reproduction PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 January2000 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 Labom tory 3M Environmental Laboratory 3M Environmental Laboratory Page 31 Page 31 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-0L13RN-U2095 LRx-u2095 Protocol lRN-U2095 AmendmentNumber 4 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: The study director for the present study was identified in the protocol as 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 11(. 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. AMEND TO READ: Tlie 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 atid workload are more evenly balanced. 3M Environmental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory Page 32 ..- Page 32 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Prutocol LRl%U2095 Amendment Number 4 Amendment Approval John L ButenAof Ph.D., Sponsor Representative Marvin T. Case,D. KM, Ph.D., Incoming Study Director Date fl 3M Environmental Laboratory 3M Environmental Laboratory 3M Environmental Laboratory ..-. _.- -- ._._. __ Page 33 Page 33 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Study Title Analytical Study of 2(N-Ethylpeffluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 5 Amendment Date: August 3I,2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project ldentificatlon FACT-TOX-0 13 ET&&S LRN U2095 Argus Study: 418409 3M Medical Department Study: T63 16.5 p" 3M Environmental Laboratory 3M Environmental Laboratory L" 3M Environmental Laboratory Page 34 Page 34 ,- 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-0L13RN-U2095 LRN-u2095 Protocol FACT TOX-073 Amendmeni No. 5 This amendment modifies the following portion(s) of the protocol: F- 7. PROTOCOL READS: The Principle Analytical Investigator for the present study was identified asJames K. Lundberg, Ph.D. 2. AMEND TO READ: The role of Principle Analytical Investigator for the present study was reassignedto K.&ten J. HansenPh.D. REASON: The role of Principle Analytical Investigator was reassigneddue to availability of resources. 3M Environmental Laboratory 3M Environmental #=-a Laboratory 3M Environmental Laboratory Page 35 Page 35 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Protocol FACT TOX-013 Amendment No. 5 Amendment Approval John L. Butenhos Ph.D., Sponsor Representative / Date TCG&L M., Ph.D., Study Director 3M Environmental Laboratory 3M Environmental Laboratory Ca 3M Environmental Laboratory Page 36 Page 36 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 Appendix C: Extraction and Analytical Methods This appendix includes the following methods: 4 ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-ElectrosprayNlass Spectrometry, - (14 paws) ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-ElectrosprayIMass Spectrometry, (9 pages) C 3M Environmental Laboratory 3M Environmental Laboratory Page 37 Page 37 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-0L13RN-U2095 LRN-u2095 3M ENVIRONMENTALLABORATORY METHOD F" EXTRACTIONOFPOTASSIUMPERFWOROOCTANESULFONATEOR OTEIZR FLUOROCHEMICAL COMPOUNDS FROM SERUM FOR ANALYSIS USING HPLC- ELECTROSPRAY/MASSSPECTROMETRY Method Number: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: q[a-/lqff Approved By: Laboratory Manager Group Leader R Date +W/YY Date &l/h/Y 4 Date 1.0 SCOPEANDAPPLICATION L** 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds f?om serum. 1.2 Applicable compounds: Fluorochemical surfactantsor other fluorinated compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum or other fluids asdesignated in the validation report. Word6/95 3M Environmental Laboratory 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 1 of 14 Page 38 Page 38 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXL-0R13N-U2095 LRN-u2095 2.0 SUMMARY OF METHOD 2.1 This method describesthe procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagentand methyl-tert-butyl ether @&BE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogatestandard(see3.0 Definiiiorts). An ion pairing reagentis added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporatoruntil dry. Each extract is reconstituted in 1.O mL of methanol, then filtered through a 3 cc plastic syringe attachedto a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-5.1 or other appropriate methods. 3.0 DEFINITIONS px" 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C,F,,SO, 3.2 PFOSA: perfluorooctane sulfonylamide C,F,,SO,NH, PI 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C,F,,SO,N(CH,CH,)CH,CO,' 3.4 EtFOSE-OH: Z(N-ethylperfluorooctane sulfonamide)-ethyl alcohol C,F,,SO,N(CH,CH,)CH,CH,OH 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C,F,,SO,N(CH,CH,)H 3.6 M556: C,F,,SOzN(H)(CH2COOH) - 3.7 Surrogate standard: lH-lH-ZH-2H perfluorooctane sulfonic acid p" 4.0 WARNINGS AND CAUTIONS 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when pa* handling animal tissue, which may contain pathogens. 5.0 INTERFERENCES 5.1 There are no interferencesknown at this time. 6.0 EOUIPMENT 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifkge, Mistral 1000or IEC 6.1.3 Shaker, Eberbachor VWR lQ+5- 3M Environmental Laboratory 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 2 of 14 Page 39 Page 39 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXL-0R13N-U2095 LRN-u2095 6.1.4 Nitrogen evaporator,Organomation 6.1.5 Balance (+ 0.100 g) 7.0 SUPPLIES AND MATERIALS 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capableof 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 p4 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0mL p* 7.9 Syringes, capableof measuring 5 PL.to 50 FL 7.10 Graduatedpipettes 7.11 Syringes, disposableplastic, 3 cc 7.12 Syringe filters, nylon, 0.2 l.trn,25 mm 7.13 Timer P-. 7.14 Crimp cap autovials and caps 7.15 crimpers Note: Prior to using glassware andbottles, rinse 3 times with methanol and 3 times with Mini-Q'" water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separatevials. 8.0 REAGENTS AND STANDARDS 8.1 Type I reaEnt grade water, Milli-QTM or equivalent; all water used in this method should Pi%+ be Milli-Q water andmay be provided by a Milh-Q TOC PlusTMsystem 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate(NqCOs), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCO,), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorocbemical 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 *cI 3M Environmental Laboratory Page 40 Page 40 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 Llw-u2095 FIS 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 89.4 EtFOSEOH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 89.6 M556 (3M Specialty Chemical Division), molecular weight = 557 89.7 Surrogate standard:4-H, perfluorooctane sulfonic acid (I-H,l-H, 2-H, 2-H C,F,,SO,H) molecular weight F 428 89.8 Other fluorochemicals, as appropriate 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 SO0mL Milli-QT" water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:lO. Measure 10 mL of 10 N NaOH solution into a 100mL volumetric flask and dilute to volume using Milli-Q'M water. Store in a 125mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammoniurn hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH lg using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of NaOH, add slowly becausethe pH changesabruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to eachuse to ensurepH = IO. Adjust as neededusing 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodiumbicarbonatebuffer (Na&O,MaHCO,): Weigh approximately 26.5 g of sodium carbonate(Ha&O,) and 21.0 g of sodium bicarbonate @JaHCO,)into a 1 L volumetric flask and bring to volume with Milli- Q" water. Store in a 1 L Nalgene bottle. ktandards preparation 8.11.1 PreparePFOS standardsfor the standardcurve. 8.11.2 Prepareother fluorochemical standards,as appropriate. Multicomponent fluorochemical standardsareacceptable(for example, one working standard solution containing 1 .OOppm PFOS, 1.02ppm PFOSA, 0.987 ppm PFOSAA, and 1.10ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standardof approximately 1000 ppm Q.&W. 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately SOppm. 8.11.6 Dilute working standard 1with methanol for a working standard2 solution of approx. 5.0 ppm. ETS-P-4.1 Extraction of PFOS from Serum Page 4 of 14 3M Env ronmental Laboratory Page 41 3M Environmental Laboratory Page 41 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 8.11.7 Dilute working standard 1 with methanol for a working standard3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogatestandard 1-H,l-H, 2-H, 2-H, C,F,,SO,H into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogatestock of approximately 1000-l 200 PPm. * 8.12.3 Preparea surrogateworking standard. Transfer approximately 1 mL of surrogate stock to a 10 mL volumetric flask andbring to volume with methanol for a working standardof 100 ppm. Record the actual volume transferred. 9.0 SAMPLEHANDLING 9.1 All samples arereceived frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperatureprior to extraction. 10.0 QUALITYCONTROL 10.1 Solvent Blanks, Method blanks and matrix blanks _- - T ;.I -.A.. _nl;qrc+ 10.1.2 Easxmtraectht otwdobl1a.n0kms.L aliquots of Milli-QTM water following this procedure and use F-- 10.1.3 Extract two 1.OmL aliquots of the serum following this procedure and use as matrix blanks. See11.1.4. p4 D.2 Matrix spikes 10.2.1 Prepareand analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepareeachspike u&g a sample chosenby the analyst, usually the control matrix received`with each sample set. 10.2.3 Expected concentrationswill 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 Prepareone matrix spike and matrix spike duplicate per 40 samples, with a p-4 minimum of 2 matrix spikes per batch. 0.3 Continuing calibration checks 10.3.1 Preparecontinuing calibration check samplesto ensurethe accuracy of the initial calibration curve. 10.3.2 Prepare,at a minimum, one continuing check per group of 10 samples. For example, if a sample set = 34, four checks arepreparedand extracted. 10.3.3 Prepareeachcontinuing calibration check from the samematrix used to prepare the initial curve. Fonmental Laboratory ETS-8-4.1 Exfraction of PFOS fromSerum 3M Environmental Laboratory Page 5 of 14 Page 42 PR 3M Envir L"* Page 42 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-0L13RN-U2095 LRN-u2095 10.3.4 The expectedconcentrations 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 necessaryif the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100ppb, rather than 5 ppb - 1000ppb). 11.0 CALIBRATIONANDSTANDARDIZATION 11.1 Prepare,matrix calibration standards 11.1.1 Transfer 1 mL of serum to a 15 mL centrifuge tube. 11.1.2 If most sample volumes are less than 1.OmL, extract standardswith matrix volumes equal to the sample volumes, Do not extract less than 0.50 nil, of matrix. Record eachsample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots in 15mL centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriatevolume, serve as matrix blanks. Typically use the standardconcentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standardcurves, for a total of eighteen standards,two matrix blanks, andtwo method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-1, which lists the working rangesand the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an iid in calculating the concentrationsof the working standards. SeeSection 13.0 to calculate actual concentrations of PFOS in calibration standards. (14 11.2 To eachstandard,blank, or continuing check, add appropriate amount of surrogate working standardfor the concentration to fall within the calibration curve range 5 ppb - 1000 ppb. 4 11.3 Extract spiked matrix standardsfollowing 12.6-12.16 of this method. Use these standards to establish eachinitial curve on the mass spectrometer. R 3M Environmental Laboratory 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 6 of 14 Page 43 Page 43 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 Approximate spiking amoun r standards and spikes 50.0 ppm 50.0 ppm 50.0 ppm 10 15 20 * 0.500 ppm 0.750 ppm 1.OOppm 12.0 PROCEDURE 12.1 Obtain frozen samples and allow to thaw at room temperatureor 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 beenremoved. 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 standardas describedin 11.2. 12.7 Spike eachmatrix with the appropriateamount of standardas described in 11.1, or Table 1 in that section, for the calibration curve standards. Also preparematrix spikes and continuing calibration standards. 12.8 Vortex mix the standardcurve samples,matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensurethe 0.5 M TBA reagentis at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 ml; 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-terl-butyl ether. 12.12 Cap eachsample 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 of PFOS from Serum Page 7 of 14 3M Environmental Laboratory Page 44 3M Environmental Laboratory Page 44 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-U2095 12.14 12.15 12.16 12.17 12.18 12.19 12.20 12.21 12.22 Label a fresh 15 mL centritige tube with the same information as in 12.9. Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. Put each sample on the analytical nitrogen evaporatoruntil dry, approximately 1 to 2 hours. Add 1.0 mL of methanol to eachcentrifuge tube using a graduatedpipette. Vortex mix far 30 seconds. Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1S mL glass autovial or low-volume autovial when necessary. Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date,and analyst(s)performing the extraction. Cap and store extracts at room temperatureor at approximately 4 "C until analysis. Complete the extraction worksheet, attachedto this document, and tape in the study notebook or include in study binder, asappropriate. 13.0 DATAANALYSISANDCALCULATIONS * 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standardsusing the following equation: mL of standardx concentration of standard(un /mLI = mL of standard+ mL of surrogatestandard+ initial matrix volume (mL) Final Concentration &&I&) of PFOS in matrix 14.0 METHODPERFORMANCE 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 (seeAttachments B and C). 14.2 The following quality control samples areextractedwith eachbatch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 POLL~TIONPREVENTIONANDWASTEMANAGEMENT 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glasspipette waste is disposedin broken glass containers located in the laboratory. ETS-8-4. I Extraction of PFOS from Serum Page 8 of 14 3M Environmental Laboratory Page 45 3M Environmental Laboratory Page 45 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-U2095 16.0 F&CORDS 16.1 Complete the extraction worksheet attachedto this method, and tape in the study notebook or include in the 3-ring study binder, asappropriate. 17.0 ATTACHMENTS 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standardconcentration worksheet 18.0 REFERENCES 18.1 The validation report associatedwith this method is ETS-8-4.0 & 5.0-V-l. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray,Mass Spectrometry" 19.0 AFFECTEDDOCUMENTS 19.1 ETS-8-5.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 REVISIONS Revision Number 1 ReasonFor Revision Section 12.21 Changedto include sample storageat room temperature. Section 12.13 Added the shakerspeed. Section 12.17Final volume is 1.OmL; not adjusted for initial volumes less than 1.OmL. Revision g&g 04/02/99 3M Environmental Laboratory 3M Environmental Laboratory ETS-S-4.1 Extraction of PFOS from Serum Page 9 of 14 Page 46 Page 46 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 Extraction Worksheet ETS-84.1 . Study # Matrix Box # may DateSpiked/Analyst ccv MS MSD Surrogate Std approx. mm actual pm # FC-Mix approx. 0.5 pm actual ppm # FC-Mix approx. 5 ppm actual wm # FC-Mix approx. 50 ppm actual wm # Comments Blank Serum Extraction Method I Std # I - : I - amount = I I mL Date & Initials Shake20 min. Centrifuge 20-25 min. Shaker meed: Centrifuge speed: Filter using a 3ccB-D svringewith a 0.2rtm filter into a 1.5mL autosamplevial Cont. Cal. Verifications used same matrix as for std curve. Attachment A 3M Environmental Laboratory 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS fkom Serum Page 10 of 14 Page 47 Page 47 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 . pa* MDL/LOQ values for rabbit serum Compound rPFOi3 L-- -- PFOSA MDL (ppb) 1.74 1.51 LOQ (ppb) 5.55 4.79 Linear Calibration Range (LCR) Approximate concentrations to be used for preparing the Standard Calibration Curve 5 ppb - 1000 ppb 5 rmb - 1000 th PFOSAA ii.46 20.5 5 ;pb - 1000 ;pb EtFOSE-OH 11.4 36.2 5 ppb - 1000 ppb M556 6.03 19.2 5 ppb - 1000 ppb PFOSEA 5.71 18.2 5 ppb - 1000 ppb MDL/LOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically determined. Two curves in each of these matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, monkey, and human were 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: MDLiLOQ Summary 3M Environmental Laboratory 3M Environmental Laboratory ETS-84.1 Extraction of PFOS from Serum Page 11 of 14 Page 48 Page 48 3M Medical Department Study: T-6316.5 3M Medical Department Study: T63 -6.5 Compound: * PFOS l/x 0.995 - 978 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 Compound: h PFOSAA l/X 0.991- 974 LCR from curve (Ppb) O&W 24.7 - 974 9.74 - 247 97.4 - 974 9.74 - 974 % Recovery Raw9 81-111 97-107 85-108 95-115 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Attachment B: MDULOQ Summary 3M Environmental Laboratory 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 12 of 14 Page 49 Page 49 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOXL-0R13N-U2095 LRN-u2095 Compound: ti EtFOSE-OH IFull Range I 0.99r3 - 976 I Low Curve I 4.93 - 97.6 IHigh curve 49.3 - 976 I l/X I 0.993 - 493 Compound: PFOSEA Rabbit Serum Prepared range of standards @pW (n&W Full Range 0.993 - 976 Low Curve I 4.93 - 248 High curve 49.3 - 976 l/X 0.993 - 976 Compound: M556 Rabbit Serum LCR from curve (ppb) 6x/~) 49.3 - 976 9.76 - 97.6 97.6 - 976 9.76 - 976 % Recovery Range 77-110 97-107 go-109 86-111 LCR from curve (PPb) (w/mL) 24.8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 % Recovery Range 96-106 I 91-110 86-106 9.5-l 17 RSD Range 11.2-25s 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 Attachment B: MDL/LOQ Summary 3M Environmental Laboratory 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 13 of 14 Page 50 Page 50 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOXL-0R13N-U2095 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: psi 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 Std cone PFOSA Std cone PFOSAA Std cone EtFOSE Std cone PFOSEA Std cone Wd 0.500 Ugly 0.507 Ug/mL 0.532 ug/mL 0.501 ug/mL 0.521 0.500 0.507 0.532 0.501 0.52i 5.00 -__I--5-.00 5.00 50.0 50.0 5.07 5.07 5.07 --- 50.1 50.1 5.32 5.32 5.32 53.2 1 53.2 5.01 _-- cil 5.01 5.21 5..-0' 1 ---I5- .2--1- 50.1 52.1 50.1 52.1 50.0 50.1 1 53.2 50.1 32.1 50.0 50.1 1 53.2 50.1 52.1 M556 Std cone VW0--.5.-0--1 0.501 5.01 -5.01 rs.ol---' 50.1 363 50.1 50.1 All AmY spiked mL 0.010 0.020 o.005 o.oio 0.020 0.005 0.010 0.015 0.020' AI1 Final vol mL 1.015 --- 1.025 1.010 1.015 - 1.025 1.-0-10 _1_.-015 1.020-1.025 Calculated concentrations of standards in the sample matrix Validated ranges Serum - Rabbit Bovine Rat Monkey 6%Plasma HWWl - approximate concentrations &OS PFOSA 5.00-1000 1 5.00-1000 ] Estimates only. Use values for rabbit. Estimates only. Use values for rabbit. Estimates only. Use values for rabbit. Estimates only. Use values for rabbit. PFOSAA 5.00-1000 EtFOSE-OH 1 5.00-1000 PFOSEA \ 5.00-1000 M556 1 5.00-1000 R IJ Attachment C: Ion Pair Standard Curves 3M Environmental Laboratory CI 3M Environmental Laboratory Extraction ETS-8-4.1 of PFOS from Serum Page 14 of 14 Page 51 Page 51 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 3M ENVIRONMENTALLABORATORY METHOD ANALYSISQF POTASSIXJMPERFL~OROOCTANESIJLFONAOTREOTFIER FLUOROCFIEMICALSIN SERUMEXTRACTSUSING HPLC-ELECTROSPRAY/MASS SPECTROMETRY Method Number: ETS-S-5.1 Author: Lisa Clemen, Robert Wyune Approved By: Laboratory Manager Adoption Date: 03/01/99 Revision Date: "Ila&fl 4/x, Date Group Leader +yu!-,c. TechnicaRl eviewer 1.0 SCOPEANDAPPLICATION 1.1 Scope: This method describes the analysis of serum extracts for fluorochemical surfactants using HPLC-electrospray/mas spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactantsor other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word (795 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 1 of 9 3M Environmental Laboratory Page 52 3M Environmental Laboratory Page 52 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOXL-0R13N-U2095 LRN-u2095 c 2.0 SUMMARYOF METHOD 2.1 This method describesthe analysis of fluorochemical surfactantsextracted Tom 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 asthe perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometerto further verify the identity of a compound by detecting daughter ions of the parent ion. 3.0 DEFINITIONS 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources,probes, and interfaces. These include but arenot limited to: Eleotrospray Ionization (ESI), Atmospheric Pressurechemical Ionization (APcI), Thermospray, etc. The ionization processin these techniques occurs at atmospheric pressure(i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure,whereby ions in solution aretransferredto the gasphasevia tiny chargeddroplets. These chargeddroplets areproduced 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 equippedwith quadrupole mass selective detectors. Ions are selectively discriminated by mass to chargeratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series(MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass 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 arethe 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 *a( compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx hasWindows 95 and WindowsNT 4.0 F versions. All versions are similar. For more details seethe manual specific to the instrument (Micromass Quattro II triple quadrupoleMassLynx or MassLynx NT User's Guide). 4.0 WARNINGSANDCAUTIONS 4.1 Health and Safety Warnings: Ipl 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 appropriateprotective gloves, eyewear, mm and clothing. 3M Environmental Laboratory Ya 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 2 of 9 Page 53 Page 53 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXL-0R1N3 -U2095 LRN-u2095 4.2 Cautions: 4.2.1 Do not operatesolvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressureexceeds400 bar, the HP 1100will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 INTERFEWNCE~ 5.1 To minimize interferenceswhen analyzing samples,teflon should not be used for sample storageor any part of instrumentation that comes in contact with the sample or extract. 6.0 EQUIPMENT 1" 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw dataas method deviations. 6.1.1 6.1.2 Micromass Quattro IT triple quadrupole Mass Spectrometerequipped with an electrospray ionization source HP1 100 low pulse solvent pumping system, solvent degasser,column compartment, and autosampler 7.0 SUPPLIESANDMATERIALS 7.1 Supplies 7.1.1 High purity grade nitrogen.gasregulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be.determinedby the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8.0 REAGENTSAND~TANDARDS 8.1 Reagents 8.1.1 Methanol, HPLC gradeor equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, andmay be provided by a Mini-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate,reagentgrade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteenmatrix standardsare preparedduring the extraction procedure. SeeETS-8-4.1. 9.0 SAMPLEHANDLING 9.1 Fresh matrix standardsarepreparedwith each analysis. Extracted standardsand samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 3M Environmental Laboratory 3M Environmental Laboratory ETS-S-5.1 Analysis of Serum Extract Using ES/MS Page 3 of 9 Page 54 Page 54 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOXL-01R3 N-U2095 LRN-u2095 9.2 If analysis will be delayed, extracted standardsand samples can be refrigerated at approximately 4" C, or at room temperature,unti1 analysis can be performed. 10.0 OUALITYCONTROL 10.1 Solvent Blanks, Method Blanks and Matrix BIanks 10.1.1 Solvent-blanks, method blanks and matrix blanks arepreparedand analyzed with eachbatch to determine contamiriation or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to eachcalibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes arepreparedand analyzedto determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are preparedand analyzed to measurethe precision and the recovery for eachanalyte. 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 andmatrix spike duplicate concentrationswill fall in the mid-range of the initial calibration curve. Additional spike concentrationsmay fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzedto verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standardafter every tenth sample, with a minimum of one per batch. 11.0 CALIBRATIONANDSTANDARDIZATION 11.1 Analyze the extracted matrix standardsprior to and following each set of extracts. The averageof two standardcurves will be plotted by linear regression(y = my + b), weighted l/x, not forced through zero, using MassLynx or other suitable software. 11.2 If the curve doesnot meet requirements, perform routine maintenance or reextract the standardcurve (if necessary)and reanalyze. 11.3 For purposesof accuracy when quantitating low levels of analyte, it may be necessaryto use the low end of the calibration curve rather than the full rangeof the standardcurve. Example: when attempting to quantitate approximately 10 ppb of analyte, generatea calibration curve consisting of the standardsfrom 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000ppb). This will reduceinaccuracy attributed to linear regressionweighting of high concentration standards. 3M Environmental Laboratory 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 4 of 9 Page 55 Page 55 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-L01R3 N-U2095 LRN-u2095 13 12.0 PROCEDURES 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 of year-samplenumber, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To createa method click on scanbutton 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 areemployed, additional product ion fragmentation information may be collected. SeeMicromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring), 12.1.3 Typically the analytical batch run sequencebegins with a set of extracted matrix standardsand endswith a set of extractedmatrix 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 arenot considered samplesbut may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list preparedin Section 12.1.1. 12.2.2 Set-up the HP 11OO/autosamplerat the following conditions or at conditions the analyst considers appropriatefor optimal response.Record actual conditions in the instrument logbook: Pa 12.2.2.1 Sample size = 10 PL injection 12.2.2.2 Inject/sample = 1 w 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = 12.2.2.5 Pressthe "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 3M Environmental Laboratory mm 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ESh4S Page 5 of 9 Page 56 Page 56 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-L01R3 N-U2095 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 replacethe stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or asappropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn 0x1the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instrument usestheseparametersat the following settings. These settings may changein order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour R 12.3.6.2 ES1nebulizing gas 1O-l 5 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 10 - 500 pL/.min 12.3.6.4 Pressure<400 bar (This parameter is not set, it is a guide to ensurethe 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 cablesto 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 electrodein the ES/MS sourceis recommended for the analysis of biological matrices. 12.3.1OClick on start button in the Acquisition Control Panel (this may vary among MassLynx versions, seeappropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 DATAANALYSISANDCALCULATIONS 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = ObservedResult - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cont. - Calculated Cont. x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix h&W ln(nnof PFOS talc. from std. Curve x Dilution Factor) x 1 ug (Initial Volume of matrix (mL) + mL of SurrogateStandard) 1000 ng Final Volume (mL) 3M Environmental Laboratory aar 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 6 of 9 Page 57 Page 57 3M Medical Department Study: T-6316.5 3M Medical c Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: FACT TOX-L01R3 N-U2095 LRN-u2095 14.0 METHOD PERFORMANCE 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. PleaseseeETS-8-4.1, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent*blanks, method blanks, and matrix blanks values are must be below the lowest standardin the calibration curve - 14.3 Calibration Curves 14.3.1 The ? value for the calibration curve must be 0.980 or better, R 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries aremust be within f 30% of the spiked concentration. 14.5 Continuing Calibration Verifications ( 14.5.1 Continuing calibration verification percent recoveries must be f 30% of the spiked p" concentration. 14.6 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samplesreanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data areto be reported when performance criteria have not beenmet, the data must be footnoted on tables and discussedin the text of the report. 15.0 POLLUTIONPREVENTIONANDWASTEMANAGEMENT 15.1 Sample extract waste and flammable solvent is disposedin high BTU containers, and glass pipette waste is disposedin broken glass containers located in the laboratory. 16.0 RECORDS 16.1 Each page generatedfor a study must have the following information included either in the headeror 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 R1 appropriate study folder. Copy these pagesandtape into the instrument runlog. 16.3 Plot the calibration curve by linear regression,weighted l/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 3M Environmental Laboratory - 3M Environmental Laboratory ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 7 of 9 Page 58 Page 58 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXL-0R1N3 -U2095 LRN-u2095 16.5 Summarize data using suitable software (Excel 5.0) and storein the study folder, see Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 TABLES,DIACRAMS,FLOWCHARTS.ANDVALIDATIONDATA F" 17.1 Attachment A: -ETS-8-5.1 Data surnrnaiy spreadsheet. LII 18.0 REFERENCES 18.1 FACT-M-4.1, "Extraction of Potassium Perfluorooctanesulfonateor Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrosprayh4ass Spectrometry p") 18.2 ETS-g-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" F- 18.3 The validation report associatedwith this method is ET!+8-4.0 & 5.0-V-l. 19.0 AFFECTEDDOCUMENTS 19.1 ETS-84.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mas Spectrometry" c" 20.0 &CVISIONS Revision Number. 1 ReasonFor Revision Section 6.1.2 Clarification of HP1 100 system components. Section 11.1 Average of two curves, not standardvalues, areused for plotting linear regressionand addedthe l/x weighting of the curve. Section 12.2.2.4 Clarification of solvent ramp. Section 17.1 Changed from attachment B to A. Revision pa& 04/02/99 3M Environmental Laboratory LI 3M Environmental Laboratory ETS-84.1 Analysis of Serum Extract Using ES/MS Page 8 of 9 Page 59 Page 59 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: FACT TOXL-0R13N-U2095 LRJsl-u2095 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: - Analytical Equipment System Number: Instrument SoftwarefVersion: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: slope: Taken from linear regression equauon. Group/Dose: Taken from the study folder. Sample#k Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration Initial Volume (mt): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cow. (ug/mL): Calculated by dividing the initial volume summruy. from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory 3M Environmental Laboratory Page 9 of 9 Page 60 Page 60 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 Appendix D: Data Summary Tables Table 12. Reported Fluorochemicai Levels in Sera Analyses in Study FACT TOX-013 l = Tentative values. hithl volume was 4.5 mL 3M Environmental Laboratory 3M Environmental Laboratory Page 61 Page 61 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT 10X-013 (continued) * = Tmtative vakma, initial vobma was Co.5 ml R 3M Environmental . Laboratory _ .-.--__ C 3M Environmental Laboratory Page 62 Page 62 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Table 13. Reported Fluorochemical kantinuedl Levels In Liver Analyses In Study FACT TOX-013 R 3M Environmental Laboratory 3M Environmental Laboratory Page 63 Page 63 3M Medical Department Study: T-6316.5 --- _ 3M Medical Department Study: T6316.5 Appendix E: Data Spreadsheets Analytical Analytical Study: FACT-TOX-013 LRN-U2095 Report: FACT TOX-013 LRN-u2095 3M Environmental Laboratory 3M Environmental Laboratory Page 64 Page 64 3M Medical Department Study: T-6316.5 FACT.TOX.013 Argud 418.009 Analytical Study: FACT-TOX-013 LRN-U2095 3M Environmental Laboratory lOlRF 0.00 515 OS.4 18.9 lOli7F ".iU 369 42.2 64.8 *s.* 999lU 0.x) 3-n 108 W99U" 0.30 159 178 IWXM 070 331 94.9 lmn2M 0.a 340 113 25.8 m I w NR lW2M O.&l 475 m44M 0.M 469 rw I 23s 235 NR I NR Page 65 3M Medical Department Study: T-6316.5 FACT-TOX-013 Ar@wU4lfHN Analytical Study: FACT-TOX-013 LRN-U2095 3M Environmental Laboratory Page 66 3M Medical Department Study: T-6316.5 FACT-TOX43 Ac~d41&00!3 Analytical Study: FACT-TOX-013 LRN-U2095 3M Environmental Laboratory Page 67 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 3M Environmental Laboratory 3M Environmental Laboratory Page 68 Page 68 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 3M Environmental Laboratory 3M Environmental Laboratory Page 69 Page 69 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Appendix F: Example Calculations Formula Used for Sera Analyses in Study FACT TOX-013 AR (ng/mL) x DF x SC x FV (mL) x 1.0 pg EVomao x PC = Reported Concentration (&mL) 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 &mL -iYFGX 1OOOng c AR-Analytical result from MassLynx summary DF-Dilution factor SC-PFOS salt correction constant (0.9275) R" FV-Final extract volume (1.OmL unless otherwise noted) EV-Volume of seraextracted PC-PFOS purity correction factor (86.4%) 3M Environmental Laboratory 3M Environmental Laboratory Page 70 Page 70 3M Medical Department Study: T-6316.5 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Study: 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-Ethylfluorooctanesu1fonamido)-ethanolin Two Generation Rat Reproduction, (65pages) 3M Environmental Laboratory 3M Environmental Laboratory Page 135 Page 71 3M Medical Department Study: T-6316.5 - BIOLOGICAL SAMPLE ANALYSIS Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Analytical Study: FACT-TOX-013 LRN-U2095 qpBaltelle d& . . . Putting Technology To Work FINAL REPORT 2 (N-Ethylfluorooctanesu1fonamido)-ethanolin Two Generation Rat Reproduction SPONSOR 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144 TestinP FaciliQ Battelle Memorial Institute 505 King Avenue Columbus, Ohio 43201-2693 Prepared By Patrick L. South, B.S. 3M Environmental Laboratory Page 72 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 FINAL REPORT 2 (N-Ethylfluorooctanesu1fonamido)-ethanolin Two Generation Rat Reproduction A Bathhe Principal Investigator Battelle Senior Program Director 11 3M Environmental Laboratory Page 73 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 2 (N-Ethy1fluorooctanesulfonamido)-ethanolin Two Generation Rat Reproduction EXECUTIVE SUMMARY Rat liver samples sent to Battelle by 3M Environmental Technology and Services were analyzed by the previously validated method "Method for Analysis of PotassiumPeriluorooctanesulfonate (PFOS) in Rat Liver by LC/MS/MS". Samples were extracted and analyzed by High-Performance Liquid Chromatography Mass Spectroscopy (LC/MS/MS) for PFOS, M-556, PFOSAA, and PFOSA content only. Related fluorochemicals 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 of liver tissue extracted. ... 111 3M Environmental Laboratory Page 74 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 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: Phase Inspected Inspection Date Date Reported to Battelle Task Leader/ Battelle Management Date Reported to Offsite Study Director/ Management Sample weights Sample homogenization Extraction Sample analysis Audit study file Audit final report Audit study file Audit final report 10/12/1999 10/12/1999 10/13/1999 10/13/1999 12/9/1999 12/9/1999 2/21/2001 2/21/2001 11/1/1999 11/1/1999 11/1/1999 11/1/1999 12/9/1999 12/9/1999 2/21/2001 2/21/2001 3/30/01 3/30/01 3/30/01 3/30/01 3/30/01 3/30/01 3/30/01 3/30101 Date kattelle Memorial Institute iv 3M Environmental Laboratory Page 75 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 GOOD LABORATORY PRACTICES COMPLIANCE STATEMENT Study Title: 2 (N-Ethylfluorooctanesu1fonamido)-ethanolin Two Generation Rat Reproduction This study was conducted in compliance with the Food and Drug 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 h l l y validated. The study was listed on Battelle's Master List of regulated studies. -.- n %c &eL, Jon C./q(ndre, Ph.D. Battellg Principal Investigator Marvin T. Case, DVM, Ph.D. Study Director i/ Date I,? Date h/ . V 3M Environmental Laboratory Page 76 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 . Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Table of Contents Executive Summary............................................................................................................................................. P..a. ge 111 QualityAssurance Statement............................................................................................................................... iv Compliance Statement.......................................................................................................................................... v Table of Contents................................................................................................................................................ vi 1.0 Introduction............................................................................................................................................. 1 2.0 Reference Substances ............................................................................................................................. 1 3.0 Receipt ofSamples................................................................................................................................. 1 4.0 Analysis of Samples ............................................................................................................................... 1 4.1 Summary ofMethod ................................................................................................................. 1 4.2 Results ....................................................................................................................................... 3 4.2.1 Quality Control ............................................................................................................ 3 4.2.2 Sample Results ............................................................................................................ 3 5.0 Conclusions............................................................................................................................................. 3 6.0 Acknowledgements................................................................................................................................. 4 7.0 Specimen Storage and Record Archives................................................................................................. 4 List of Tables Table 1. Example of InstrumentParameters Used to Analyze Samples............................................................. 2 Appendix A (Results) Summary Results for Rat liver SampleAnalysis......................................................................... A-1 Appendix B (Daily Acceptance Criteria Summary) Daily Acceptance Criteria Summary ...................................................................................... B-1 Appendix C (Sample Inventory List) Sample Inventory List........................................................................................................ C-1 Appendix D (Chromatograms) Representative Chromatograms ............................................................................................. D. 1 Appendix E (Protocol. Amendments. and Deviations) Protocol. Amendments. and Deviations...................................................................................... E-1 Appendix F (PFOS Purity Report) PFOS Purity Report ...................................................................................................................... F-1 vi 3M Environmental Laboratory Page 77 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: NOO3604-D 3M Environmental Laboratory Study Number: FACT 060998.1 1.O Introduction This report presents a description of the method used to analyze PFOS, M-556, PFOSAA, and PFOSA in rat liver samples from 3M Study Number FACT 060998.1 (TOX-0 13) and the results from this analysis. See Appendix E for a copy of the study protocol, amendments, and protocol deviation reports). 2.0 Reference Substances The analytical reference substances for this study were 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 0.-15709). Note that based on information supplied to Battelle from 3M, PFOS has two equivalent names. The name appearing on the Material Safety Data Sheet and bottle label is potassium perfluoroalkyl sulfonate. The name more commonly used by 3M in analytical methods and correspondence is potassium perfluorooctanesulfonate. The latter name will be used in this report. See Appendix F for purity data supplied by 3M to Battelle. The reference substances were stored at room temperature. The surrogate standard was lH,lH,2H,2H-Perfluorooctanesulfonic acid, lot number 59909, supplied by ICN. The surrogate standard was stored at room temperature. 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 under protocol 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 (Battellestudy number N003604-A). The current version of the method is attached to this report in Appendix E. Samples were analyzed by LC/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 l/x were used to construct the calibration curves. 1 3M Environmental Laboratory Page 78 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: NOO3604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Table 1. Example of InstrumentParametersUsed to Analyze Samples 2 3M Environmental Laboratory Page 79 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 4.2 Results Battelle Study Number: N003604-D 3M EnvironmentalLaboratory Study Number: FACT 060998.1 4.2.1 Quality Control System suitability acceptance criteria were established during the method validation and are included in Appendix E, 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 undiluted homogenates. After the data were reviewed, dilutions of the homogenates were performed in order to bring analyte concentrations within the calibration range. The first analysis that provided 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 standard which meets acceptance criteria for accuracy (25% RE; see Appendix E for definition). 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 notation ALOQ denotes "Above Limit of Quantitation" for samples that had concentrations higher than the theoretical concentration for the 13 pg/g calibration standard. Samples that were initially ALOQ were diluted with blank liver homogenate and re-extracted. Samples that were expected to be ALOQ were first diluted with blank liver homogenate before extraction. The "Corrected PFOS Conc" 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) of PFOS was calculated in Battelle study N003296Fto be 0.0173 pg/g fiom 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(SN)ratio was calculated by dividing the mean found concentration of the 7 reps by their standard deviation. The method of MDL determination was provided by the Sponsor. 5.0 Conclusions All analyses met acceptance criteria unless otherwise noted. 3 3M Environmental Laboratory Page 80 3M Medical Department Study: T-6316.5 Analytical Study: 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 Title Jon C. Andre, Ph.D. Richard W. Slauter, Ph.D., D.A.B.T. Patrick L. South, B.S. Gerke H. van der Zwaag, M.S. Battelle Principal Investigator Senior Program Director Mass spectroscopist Samplepreparation chemist 7.0 Specimen Storage and Record Archives See Appendix E, protocol amendment 3 for records archival information. All residual liver samples, extracts, and unused test article will be disposed of or returned to the Sponsor as directed by the Sponsor. 4 3M Environmental Laboratory Page 81 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental LaboratoryStudyNumber: FACT 060998.1 APPENDIX A -RESULTS 3M Environmental Laboratory Page 82 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Liboratory Study Number: FACT 060998.1 PFOS, M456, PFOSAA, PFOSA IN RAT LIVER EARELLE STUDY: N0036W-D SPREADSHEET SOFTWARE DATA ENTERED: MANUALLY Sample Dose GrOUL Animal Number 1 1 2 1 3 1 4 1 5 1 6 2 7 2 8 2 9 2 10 2 11 3 12 3 13 3 14 3 15 3 16 4 17 4 18 4 19 4 M 4 21 5 22 5 23 5 24 5 25 5 28 1 27 1 28 1 29 1 30 1 31 2 32 2 33 2 34 2 35 2 35 3 37 3 38 3 39 3 40 3 41 4 42 4 43 4 44 4 45 4 46 1 47 1 48 1 49 1 50 1 51 2 52 2 53 2 54 2 55 2 5.6 3 57 3 !% 3 59 3 60 3 61 4 62 4 63 4 84 4 65 4 BLOQ = BELOW LIMIT OF QUANllTATlON 9g22 9930 0931 9932 9933 9961 9gS4 9965 9967 9970 9997 9999 low1 10002 10004 10019 10024 10029 10033 10034 10042 10044 10045 10051 10054 10097 10105 10106 10107 10108 10136 10140 10142 10121 10126 10177 10155 10156 10164 10172 10167 10194 10203 10211 10214 10097 10105 10106 10107 10108 10138 10140 10142 10121 10126 10177 10155 10156 10184 10172 10187 10194 10203 10211 10214 Analysis data key: Normalfont = October 13,1999 Underline = October 16,1999 Bold = October 18,1999 Bold Underllne = October 20,1999 EXCEL 97 SampleType Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal PFOS Conc, p gIg M356 Conc, pg 1g 6.n~oi 9.44E-01 9.68E-01 1.2OE+00 ).17E+00 1.34E+02 1.I8E+02 l.C4E+O2 9.34EW1 1.01 E+OZ 4.80E+02 2.71 E+02 5.76E+02 2.97E+02 4.47E+02 9.62E+02 9.15E+02 6.43E+02 S.C4E+02 6.43+02 1.41 E+03 I.57E+03 I.31 E+03 I.23E+03 1.228+03 1.72E-01 BLOQ 1.40E-01 BLOQ BLOQ 4.61+01 2.74EMI 2.56EW1 2.90E+01 2.65E+01 12 1EM2 1.18E+02 1.5OEWZ 2.07E+02 1.38E+02 1.90E+02 2.78E+02 3.98+02 2.95E+02 3.068+02 3.25E-01 2.8OE-01 2.61E-01 2.56E-01 2.90E-01 6.21 E W I 3.24E+01 8.2BEWI 6.33E+01 7.85E+01 1.77E+O2 1.03E+02 2.531+02 2.35EW2 2.18E+02 2.62E+02 3.21 EM2 5.19EWZ 5.29E+02 3.98E+02 BLOQ BLOQ BLOQ moa BLOQ 1.28E+Ol l.OZE+Ol 1.16E+01 8.95E+00 l.lBE+Ol 6.35E+OI 3.93+01 6.64E+01 3.8OE+Ol 5.46+01 8.45E+01 9.75E+01 7.82+01 1.17E+02 8.228+01 1.28EW2 I.52E+02 1.33E+02 1.18E+02 1.61 E+OZ BLOQ BLoa BLOQ BLOQ BLOQ 2.66E+OO 1.55E+OO 1.41E+OO `1.1 9E+00 1.75POO 8.1 6E+OO 7.17E+00 7.64E+00 8.41E+00 5.87E+00 1.98E+01 2.68E+O1 2.66E+OI 2.75E+01 2.93E+01 BLOQ BLOQ BLOQ BLOQ BLOQ 4.64E+00 3.27E+00 4.56E+OO 5.39E+OO 7.75E+00 3.14E+01 1.15E+01 3.33E+01 4.31 E+01 2.99E+01 3.99E+01 3,95E+OI 6.78E+OI 6.54E+01 6.45E+OI All samples undiluted All samples undiluted All samples diluted All samples diluted PFOSAA Conc, pg1g moa BLOQ BLOQ BLOQ BLOQ 1,10E+01 1.56E+01 9.79E+00 6.62+00 1.04E+01 7.351+01 2.86E+01 8.52E+01 3.42E+OI 6.49E+01 I.Z2E+02 I.48E+02 8.62E+01 1.29E+02 1.35E+02 1.881+02 2.06E+02 1.50E+02 1.57E+02 1.65E+02 BLOQ BLOQ BLOQ BLOQ BLOQ 5.01E+00 2.67+00 2.67+00 2.68E+00 4.34E+00 Z.OOE+Ol 1.56E+01 2.02EW1 227E+01 1.19E+01 3.1 1E+OI 5.15E+01 4.958+01 4.66EWl 5.14E+OI BLOQ BLOQ BLOQ BLOQ BLOQ 9.44E+00 2.82E+OO 6.55+00 5.87E+00 I.46E+01 1.71 E+01 1.20+01 2.73+01 2.94E+01 3.37E+OI 2.74E+01 4.55E+01 7.60E+01 5.6ZE+01 6.00E+01 PFOSA Conc, pg1g moa BLOQ moa BLOQ BLOQ 4.49E+00 4.10E+00 2.88E+OO 3.86E+00 5.42E+00 1.06+01 9.41E+00 8.67E+00 8.29E+OO 8.41 E+00 1.26E+01 1.1 OE+01 1.12E+01 1.26E+O1 l.lOE+Ol 1.64E+01 1.30E+01 1.09+01 9.8OE+OO 1 .O8E+O1 BLOQ BLoa BLOQ BLOQ BLOQ 1.4OE+00 6.01E-01 5.06E-01 5.14E-01 7.08E-01 2.88E+00 2.22+00 2.24E+M) 1.94+00 2.17+00 2.80E+M) 4.06E+00 3.14E+00 3.398+00 4.56E+M) BLOQ BLOQ BLOQ BLOQ BLOQ 2.35E+OO 1.22E+00 2.DBE+00 1.90+00 2.65+00 6.91+00 5.11 E+OO 6.14E+00 6.26E+00 6.20E+OO 6.36E+00 9.96E+OO 9.93E+00 1.14E+01 8.11E+OQ A- 1 3M Environmental Laboratory Page 83 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: 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: All concs in pglg 130ct99; 9053 Electronically and manually Excel 97 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. Spike Level M-556 0.1269 0.1220 0.1433 0.1368 0.1670 0.1225 0.1190 0.1339 0.0170 0.133 1 PFOSAA 0.1484 0.1281 0.1484 0.1605 0.1661 0.1513 0.1385 0.1488 0.0128 0.1334 SM PFOSA 0.1 167 0.1325 0.1521 0.1441 0.1490 0.1382 0.1413 0.1391 0.0119 0.13 15 LOQ (det. from 10 x std.dev. "noise") LOQ (det. from cal curve low std.) Curve Coeff of Determination Date analyzed Method Kev -1 Spike Level too high; Spike Level must be < lox MDL 2 - Spike Level too low; Spike Level must be > MDL 3 - SM too low; SM must be > 5 -4 Coeff of Det of calibration curve unacceptable 0.17005 0.133 1 0.9978 130ct99 LC/MS/MS 0.12763 0.1334 0.9937 130ct99 LC/MS/MS 0.11853 0.13 15 0.9891 13Oct99 LC/MS/MS A-2 3M Environmental Laboratory Page 84 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: NOO3604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX B-DAILY ACCEPTANCE CRITERIA SUMMARY 3M Environmental Laboratory Page 85 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 PFOS et al IN RAT LIVER STUDY NUMBER: N003604-D DATA ENTERED: MANUALLY SOFTWARE: EXCEL 97 REGRESSION PARAMETERS October13,1999 One rep of Cal pt 1 excluded B-1 3M Environmental Laboratory Page 86 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 PFOS et al IN RAT LIVER STUDY NUMBER: N003604-D DATA ENTERED: MANUALLY SOFTWARE: EXCEL 97 Analysis Date October 13,1999 October 16,1999 October 18,1999 Analyte PFOS M-556 PFOSAA PFOSA PFOS M-556 PFOSAA PFOSA PFOS M-556 PFOSAA PFOSA PFOSA QC Level, nglmL 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 3.3 I 0.7 0.16 6.6 3.8 7.9 3.1 4.7 11.7 3.0 11.5 6.5 5.1 8.7 14.5 16.9 9.2 5.8 4.6 5.5 4.5 8.3 9.7 3.4 2.7 8.0 17.7 4.3 2.1 7.4 17.7 11.6 6.1 8.0 8.6 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 11.5 12.6 2.3 4.0 2.3 -4.0 -4.6 -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 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 -4.5 -1.7 -20.3 -6.1 -2.9 23.4 11.6 15.4 0.8 -1.8 -20.6 -1 8.5 0.0 13.7 0.0 16.0 -12.0 -21.7 -16.0 -1.6 6.5 1.9 11.5 B-2 3M Environmental Laboratory Page 87 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M EnvironmentalLaboratory StudyNumber: FACT 060998.1 APPENDIX C-SAMPLE INVENTORY LIST 3M Environmental Laboratory Page 88 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Study TOX-013, Argus 418009. Sample Informtion for shipment to Battelle c-1 3M Environmental Laboratory Page 89 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M EnvironmentalLaboratory Study Number: FACT 060998.1 APPENDIX D-REPRESENTATIVE CHROMATOGRAMS 3M Environmental Laboratory Page 90 3M Medical Department Study: T-6316.5 -- Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number:N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 & BQaLtCte9ll0e53 10 uL; N003684-D 16-0ct- 99! 10:55:3i ESI160ctO6 Srn (Mn, 1x2) Operator: PLI 2: MRM of 2 Channels ES- 427.00 > 81.O( 18' SURROGATE 4.43 0 ,,., ,.,, ,,,,I..,,,,.l,l,,,,l,,,,l,,,,I,,,, 160ct06 SmI(Mn, 1x2) 100- 4.20 PFOS %- ,,,, I,,,, ,, 2: MkM of 2 'ChannelsES- 499.00 > 99.0( I.79e: 0-, ~, , , , , , , I , , 1601306 SmI(Mn, l h ) % 5.28 5.87 , , , , , , , 1 , , , , 1 , , ~ , , 1 , , , , ~ , , , ,, ,, , I 3: MkM of 3lChannels ES556.00 > 78.0C 317 M-556 0: , , 16OctO6 , , Sm (Mn, Ih) '"4 PFOSAA 5.07 5.56 5.86 6.16 3:MkM of 3 Channels ES584.00 > 169.0C 0 ,,,, I60ct06 S 4.69 m 5.07 3: M M of 3 Channsels ES498.00 > 78.0C 43: 3M Environmental Laboratory rm , , , I , , , , , , 5.bO 5.77 6.bO 6.28 Time D- 1 Page 91 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 %%- , %- SURROGATE PFOS M-556 5.84er 1.08er 3.90e: 4.54 %- PFOSAA 100- PFOSA %- 1.06& D-2 3M Environmental Laboratory Page 92 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 D-3 3M Environmental Laboratory Page 93 3M Medical Department Study: T-6316.5 3attelle 2LC 9053 31- 30ct20 Sm (Mn, 1x2) 100- 3.94 %- Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Sample 3 10 uL; N003684-D SURROGATE 13-Oct-I 99! 16:01:4I Operator: PLI 2: MRM of 2 Channels ES- 427.00 > 81.O( 3.66s 0 ,I,, ,,. 30ct20 SmI(Mn, Ih) , , , . . ( . , 100- 4.22 %- PFOS 499.00 > 99.0( 6.02ed 0 I 1 II 1I I I , , ,3:,M M,of3,channels I I I I 1 II I 1I I I I 11 I I I 1 I II I 1 I I II 1 I I I I 19 I I 1 30ct20 SmI(Mn, I&) 100- 4.44 ES556.00 > 78.0( 34; %- 3.83 0 I S , . >,-, 30ct20 SmI(Mn, I&) 100- %- PFOSAA 4.63 3.65 PFOSA % 584.00 > 169.0( 87t 3M Environmental Laboratory D-4 Page 94 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 . Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 %- 3.39 %- %- %- ! %- SURROGATE PFOS M-556 PFOSAA PFOSA 5.94el 6.53el 1.86e! 1.18e! 2.85e! D-5 3M Environmental Laboratory Page 95 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: NOO3604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX E-PROTOCOL, AMENDMENTS, AND DEVIATIONS 3M Environmental Laboratory Page 96 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D - - &x 3M Environmental Laboratory Study Number: FACT 060998.1 fh-c f T'A bl =-??.- 3M ENVIRONMENTLAALBORATORY - PROTOCOLANALYTICASLTUDY 2(N-Ethylperfluorooctanesdfonamido)-ethanol in Two Generation Rat Reproduction In-vivostudy referencenumber: Argus 418-009 Study number: FACT 060998.1 Test substance: 2(N-Ethylpeffluorooctanesulfonamido)-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 St. Paul, MN 55106 Experimental start date: Expectedterminationdate: December 31,1998 Method numbers and revisions: FACT-M-1.0, Extraction of Potassium Peffluorooctanesulfonateor Other Anionic Surfactants from Liver for Analysis Using HPLC-ElectrosprayMas Spectrometry FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- i Electrospray/Mass Spectrometry ' FACT-M-3.0, Extraction of Potassium Peffluorooctanesulfonateor Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/h'lass Spectrometry FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLC- ElectrosprayMass Spectrometry Author: Lisa Clemen IhLA Kris Hansen Study Director q/l5/78 Date Marvin Case Sponsor Representative /O?/&Y Date E- 1 3M Environmental Laboratory 1 Page 97 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle StudyNumber: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 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)designatedby 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 REGULATORCYOMPLIANCE This study is conducted in compliance with the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptionswill be noted in the final report. 3.0 TESTMATERIALS 3.1 Test,control, and reference substancesand matrices 3.1.1 Analytical reference substance:Potassium perfluorooctanesulfonate (PFOS),lot # 217 3.1.2 Analytical reference substance matrix: Rat liver and serum 3.1.3 Analytical control substance: None 3.1.4 Analytical control substance matrix: Rat liver and serum 3.2 Source of materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability informationwill be included in the final report 3.2.2 Analytical referencesubstance matrix: Argus Research Laboratories; traceability information will be included in the final report 3.2.3 Analytical control matrix: 3.2.3.1 Rat liver -Argus Research Laboratories:traceabilityinfomation will be included in the frnal report; or Rabbit liver - Covance Laboratories; traceability information will be included in the final report 3.23.2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report 33 Number 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 Identificationof test and control samples: The samplesare 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. 2 3M Environmental Laboratory E-2 Page 98 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 3.5 Purity and strength of materials: Characterizationof 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 the responsibility of the Sponsor. 3.7 Storage conditions for test materials: Test materials are stored at room temperature. Samples are stored at -20 f 10 "C. 3.8 Dispositionof 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 - EXPERIMENTAOLverview Tissues from animals dosed as described in Argus Research LaboratoriesProtocol #418-009are 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/massspectrometry ( E S M S ) . On the basis of findings from these analyses, additionalsample matrices may be evaluated or other metabolites may be targeted. If additional analysis is performed, a protocol amendment will be written. 5.0 - EXPERIMENTALnalvticalMethods 5.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Surfactantsfrom Liver for Analysis Using HPLC-Electrosprayhlass Spectrometry 5.2 FACT-M-2.0,Analysis of Fluorochemicalsin Liver Extracts Using HPLCElectrosprayMass Spectrometry 5.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonateor Other Anionic Surfactantsfrom Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 5.4 FACT-M-4.0, Analysis of Fluorochemicalsin SerumExtracts Using HPLC- ElectrosprayMass Spectrometry 6.0 DATAANALYSIS 6.1 Data transformationsand analysis: Data will be reported as the concentration (weighvweight)of fluorideper 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 concentrationswithin each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 3 E-3 3M Environmental Laboratory Page 99 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 7.0 MAINTENANCEOFRAW DATAAND RECORDS 7.1 The followingraw 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 7.15 Electronic copies of data 7.2 Supporting records to be retained separately from the study folder in the archives accordingto AMDT-S-8will include at least the following: 7.2.1 Training records 7.2.2 Calibration records 72.3 Instrumentmaintenance logs 7.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 7.25 Appropriate specimens. 8.0 REFERENCES 8.1 3M Environmental Laboratory Quality System Chapters 1,5and 6 8.2 Other applicable 3M Environmental Laboratory Quality System Standard Operating Procedures 9.0 ATTACHMENTS 9.1 FACT-M-1.0, Extraction of PotassiumPerfluorooctanesulfonateor Other Anionic Surfactantsfrom Liver for Analysis Using HPLC-Electrospray/MasssSpectrometry 9.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC- ElectrosprayA4ass Spectrometry 9.3 FACT-M3.0, Extraction of Potassium Peffluorooctanesulfonateor Other Anionic Surfactantsfrom Serum for Analysis Using HPLC-Electrospray/Mas Spectrometry 9.4 FACT-M-4.0,Analysis of Fluorochemicals in SerumExtracts Using HPLC- Electrosprayh4ass Spectrometry E-4 3M Environmental Laboratory 4 Page 100 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M EnvironmentalLaboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASST[TM PERFLUOROOCTANESULFONATE(PFOS) IN RATLIVERBY LC/MS/MS Vaaion LO 3M Environmental Laboratory Page 1d16 E-5 Page 101 3M Medical Department Study: T-6316.5 .. Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 ? METHOD FOR ANALYSXS OF POTASSNM P E R F L U O R O O C T A N E S ~ O N A(P~FOS)IN RAT LIVERBYLc/Ms/116s Verrion LO Study No.: Annlyst/D.dc: L SUMMARY n. IIL N. ._ V. 3M Environmental Laboratory Page 2 of16 E-6 Page 102 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASsmM PTCRFLUOROOCTANESULFONATE(PFOS) IN RAT LIVER BY LC/MS/MS version 1.0 Study N u AndyJt/Jl.tc: , i i Peauomoctane Sulphonic Acid M-556 , I4570 PFOSAA PFOSA msEA RatLiw Methanol - I I vt EQUIPMENT SaTable 2 for all rquhdmajorpieces Ofcquipmcnt Usc the table to doaunenttpeachralpiax (e.& mak~m, odel) of equipment Check calibrarion ofall apipmcntnquiringcalibrarimtion(eg. babccs) to amre it is cumnt Page3 of16 E-7 3M Environmental Laboratory Page 103 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 \- METEOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESU.L.FONAveTrsEio(PnF1.O0 S) IN RAT LIVER BY LC/MS/MS . Stndr NC TCStTUbCS Transport* h&gIUtiCsrimr Orbital SbnLer Evaporate Extrac*l Store QCs Stir maaix Exmu Sampla Blue FaIan . l5mL Polypmwlcnc, 12 x75 mm SmL polypromilcnt 2002 127-Tl60-56F' HDmogmizer Griadliver PHmeter DaamineBII&rpH Electrode lktermbBII&rpH Vol~cFlaskS, MalreVOl~C QassA I Dilutions I J NA NA I I NA NA I NA NA I 1 i Page4d16 E-8 3M Environmental Laboratory Page 104 3M Medical Department Study: T-6316.5 . .. Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M EnvironmentalLaboratory Study Number: FACT 060998.1 ME'&OD FOR ANALYSIS OF POTASSIUM PJCR,FLTJOROOCTANESULFONAT(EPFOS) IN RAT LIVERBY Lc/Ms/Ms version LO Study No.: A4y#/Dote: va PROCEDURE A. Prepnration of 2 mMAmmonium Acetate Weigh0.1500_+0.0020gafammonium~andtraasfi?rtolaooOsrCvDhrmstric flask. Dissolve solid in water and dilute to volume with water. Solution may be used for onemonth stonxiatmom tcmpcrabac ~lnszsofrmmnnitrmacctate: AchlaldnalVOlLUE Dateofprepadon: Study No: B. Prep.ration of -29% Sodium Hydroxide Solution Weigh200 i 2 g of sodium hydmidc into abcalrer. Add 500 mL dMilliQwateraad mix to difsolvc Cool d trans6arn a polypropylene bottle ixnorngc Solutionmay bs stored for 6 months at room temperature. Actu?llmass ofsodium llyciroxide: AaualVOlumCMilli-Qwattr: Datc0fprrp;nntion: Study No: C. Preparation of -2.9% Sodium Hydroxide Solution Add 10 mL of-29% SodiumHydroxide Solutionto a lOO-mL wlumetricfksk and dilute to volumewith W-Qwater. Transfuto a polypropyk bottk for stomp. Solution may be s t o dfor6 monthsatmorntcmptlature. Actual volume of-29% NaOH solution: Actual final volume: Date ofprepamion: Study No: D. Preparation of TetrabutyhmmoniumHydrogensulfate(TBA) Solution, 0.5 M, @H 10) pHMttuCaliion pRbufher:7 pHbufher: 10 PHreading:@eradnig.. . Add 169 I1 gofTBA to-SO0 mL OfAGIli-QwaDxiuabeaker. AdjmttbcpHta 10.00 t 0.02 using4 5 - 6 0 mL d 2 9 % Sodim H y M & sol^^ dilute to lo00I& witb Milli-Qwata,andmixM~thapHm10.M)10.02using-z.9./o~oHaodmix Tmsfer to a polypmpylme boule for storage. Solution may k used for one month storedat room tempem- but the DEmust be checked urior to aeh use Adjustto pH 10.0 t 0.02 with 29% sodiumH-de Solution as masaxy. E-9 3M Environmental Laboratory Page 105 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METEOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONA~(PFOS) IN RAT LIVERBY LC/MS/MS vurion LO Study No.: ' AJlai~Itc ActoalmsssafTBA: ActnalfinaIvolUmC: . ActnalfhmlpH: Date ofprcpatioxt Study No: ~IHafter rrchcddng andlorreadjusting E. Preparation o f 0.25 M Carbonate Buffer Weigh 26.5 i 0.1gofsodium carbonateand 21.0 t 0.1 g ofsodium bicarbonateaod em&rtothcsame 1000-mLvolumehicflask.DkohrethematerinlsinMilli-qwtcr, dilateto vo1umewithMilli-Qwntcr. mix.aad himsf& to a polypropylenebottle for soorage. Soludon may be used for 1monthwhen s t o d r&igmtcd. Actualmassdsodiumcaltlonate: Actualmass ofsodiumbicarbonate: . ActuaIfinaIvol~: Date afprcpamioxt Study No: F. Preparation of MobiIe Phase &mponcnt A: Mix together 600 mL of2mMammo&m acetate aod 400 mL of mtthaaol Solution may be usedfor 1month whm StORd at room twpaahac Actualvolume afz mMsmmoniumaa$tc: mL . . Actualvolnmeofmethaool: mL Date ofprepamion: Study No: ComponentB: Mix to@cr 50 mL of2 mMammoniumacetateaud950 mL d metbaa01Solution may be used for 1monthwhea stored at mom tanpaatwe. Actual W l ~ n f ID2A4 ammopiumacetate: mL Actualvolumeofmcthanol: mL Date afprqmaiion: Study No: G. Prepamtion of Stock Surrogate Standard and Working Surrogate standard (WSS) 3M Environmental Laboratory Page 6 d l 6 E-10 Page 106 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUORO0CM"LFONATE (PFOS) IN RAT UNER BY LC/MS/MS Vvsion 1.0 Study No.: AndYSUDptc 2. wss (lo00ngImL.1: DilatelOOIJ.cbstocknmogatestandardto25mI.withmcthpnoland~. Actnal Volume afstock htunal standard: Ac~aulDilution ValDatedPnparation: E. Preparation of CalibrationSolvent Stock and Working Standards I. SolvultStDckr: . F o r d analytewdghthc specifitd a m & t o f s t a n d a r d ( i i weighedas A aad B I@-) ltted in Table 3 andtransftrintD scparare . volamctric flasks Dissolve in methanol, dilute to wlume with mcthanoi, a d mtcwdl. Sto~rcfiigaatcd,pmtcdedfiomWlight 2. Mixedsol~stocks: Pipet the specified amount of each analyficalStandardRqlicate A as listedin Table 5 a d tmnsfkr into a single volumetricflask Dissolve in rndmol, dilute to volume with methanol. andmix mil. Store re6ig.n;ue4 protectedfromW light Rcputtheprocsswith~licatcBstoclc; Themtecdsofventstockscac vscd toprrparetha workingstnndorplr Date ofpreparaton: Study No: 3. working StandardJ0: Dilutethe mised stocks andworkingstandnrdswith Wthaool assptcifitd in Table 3 and mix well. DaOe Cbpl'CpatiOS 3M Environmental Laboratory -7d16 E-1 1 Page 107 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERnUOROOCTANESULFONATE(PFOS) IN RAT LIVERBY Lc/Ms/Ms Venion 1.0 Study No.: Aml~atc: Weigh allanalyticalstandardsto at least the nearest 0.01 mg. ** Use volumuricorppsitive-displaamentpipct(s). I. Prepantion of Calibration Standards and Blanks 1. Livahomogenate Pnpanblank liwrhomogenate in bulk by weighingapproximntely40gof blankliverinto a 500 mL Nalgcncbottle containing200 mL 0fMilli-Q water. Grindto a homogeneous suspension. Aliquot into approx 30 mLportionsfor h z u l (approx -20C) storagt. AcamlMarsafliver: Achral wiume ofwotp: Date ofprcp: Study: Ddaminedalsi~ofealibrat~maDik MJXHOMOG~NATETHOROUGHLY and dtvrminethe in of 10 repiicatewcighings of 1mL partions afthcTHOROUGHLY MDED hom0genatL MIXHOMOGENATE IMMEDIATELY PRIOR TO EACH ALIQUOT REMOVAL. I I I I I I I I 1 I LivacalibEatianStandards pnparr each livacalibrationstambud by adding 0.45 mL of undilutedlivcr homogenate (STIR HOMOGENATE WHILE ALIQUOTING) into a 15 mI. d o n tube pnd adding 50 &of WS or MeOK Prrparetriplicate cal standardsand6blankrSceTable5forMiumes. Thedilutedliwdensiryis a s n r m t d t o b e ~ y 1 5 0 m g l m tMixwell Page80f16 E-I2 3M Environmental Laboratory Page 108 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLuoRoocTANESULFONATE (PFOS) IN RATLIVER BY LC/MS/MS v&M LO Study No.: \ An.lyStlDatc Table5. C d b d o a Standvdr andBl& Date ofpreparation of d ads/bk& J. Preparation of Quality Control Liver Samples (QCC) 1. Quality Contml Working Standards DiIutethefollowing sour^^ volumes methanol in volumceicflasksand mix d.Pnpankshwhenused Actualvolumessreinpareotheses. 2. -on of Quality ControlLiva Samples ReparreachQci n b u l k b y f i l l i n g t h e w l m ~ c f l a & a pW p ~kIl with undilutedliver homogcnac (STIR HOMOGENATEWHILE ALIQUOTING), adding the appropriateQC WS.mixing and dilutingtovolums withd u t d liver homgcoate (STIRHOMOGENATEWRILE ALJQUOTING). MIX THOROUGHLYand disptnse 2 . 5 4 . aliquotsinto polypropyltnetubes and store at approximately-2OOC Table 7. OC Prcnfirntinn Date ofQC pnp: study: K. Preparationof MS Check Standard for System Suitability Pipet 250 of WS 2 at-10.000 nglmL and2 5 mL of WSS at -IO00 n g h L in methim01 into thesame 5 0 - d volumCaicflask Dilute to volume withMdlH andmix Page9d16 E-13 3M Environmental Laboratory Page 109 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR aALY!SE OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) JN RAT LKVERBY u=IMsIMs L. Preparation of homogenizerrecovery liver samples Todetarmincthc~~~thehomogenization~~nnhomogenizcdblankliva w i l l k ~ m ~ t i c y c a t 3 ~ ~ c m I ~ a n d h o m o g c n i z t d a s f o Tlhlios w s n d s t o k done every day thathomogtnizationofsaudysampla is pcrfonnta 1. piaa appradmatdy0 3 gofunhomopepizedblank liverinto mchof6.15 mL polyplupybanh-ifagehlba. Rccordweightsafliva. 2. . Addl00~afwS1.3,aad4(onewSpaduplicatetubes)toprrpare fordfications a t w y 4, 0.8. and 0.4 pg/g 3. Multiply themass ofliverin g by 2 5 and addthis many mL ofwater. 4. Homogenizeeachliwsamplq andrinsehomogenizerpube with snotha VolUmeofwaterustd in stcp3. addingrinseto homogenized sample. 5. Clcanbomo~withMeoHbetwetnsamples 6. CapandVoltrxhomogermtefOrusein~aa M. Preparation of Dilution Check Sample 1. plaa 2.95 mL o f d u t e d liver homogenate (STIRHOMOGENATE WHILE ALIQUOTJNG) into a 15 mLcxmctiontubeandadd50pL ofMixui Stodc A 2. Dilute 50 pL ofstep 1 solution (VORTEX SOLunON WHILE ALIQUOTING) with 0.45 mL of undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING) in 3.U mL extradontubes 3. This sample should be prepandfor OmacfiOnonly on days when study samples willkdilutcdandcxtraad . . N. Homogenization of study samples 1. Placeappxr~ximatdy0.5 g ofnnhomogmizcdsi,udy sample liverinto a 15 mL polypropylentt u k Recordweights ofliva. 2 Multiply tile mass of liver ingby 2.5 andaddtbismany mt ofwale?. 3. HomogmizC tach h e r sample.and bhomogenizCrprobe with another volume ofwater wed in step 2. addingrime to homogenized sample. 4. Clamhomog~with~H~rampla. 5. Capandvortcxhomogmtcforuseinaeactioa. 0. Analysis Standards, Blanks, QCs,and Spmplu 1. MIXLIVERHOMOGE"ESTRoROUGHLY BEFORE ALIQUOTING andpipet 500 pL of each QC (4 replicatesper Iml). and other samples being pmaaed into 15-mLp01~pmpylcncomactiontubes The cal stds and blanks an alrcady aliquotd 2 TOthe B W - I S (3 npSh add 100& U L a f H a n d v m 3. To t h e B W + I S (3 andtothe Rmaiaingsamples, add 100& WSS and Vortff 4. Add 0.5 mL af0.5 MTBA ($3 10) to all tubesandvoncx briefly. 56. Add 1mLof0.2SMMbufkriindvowrtabrictly. Add 25 mL of ethyl accmtc. FYatx the tubes sidcways on the orbital shaker at a stting of300for-20 mimrta. 7. cmhifuge tubaat a settingof3500 rprnfor-u)mirmts to scpmatc layas 8. Transfer2 mL dthctop 0rganiClaytrto a cleanpolypmpylcnetllk Page 10 of 16 E-14 3M Environmental Laboratory Page 110 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M EnvironmentalLaboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE(PFOS) IN RATLNERBY Lc/Ms/Ms Version 1.0 Study No.: Adystmpte: ' E-1.5 3M Environmental Laboratory Page 111 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE(PFOS) IN RAT LIVER BY LC/MS/MS versiou LO Study Na: AO.lYSt/Dnte: 427>81 MRM masitionfor S m g a k Standard 499>99 MRM tnnsitionfor PFOS 55078 MRM &ansitionforM-556 -570>169 MRM hansitionfor M570 * 5&1>169MRM transitionforF'FOSAA 498>78 MRM transitionfor PFOSA 528169 MRM d t i o n forPFOSEA *11 minutes ( mia) ss: 4 min L m i n ) PFOS 4.3 min L m i n ) M-SS6: 4 5 d L d ) M570 4.6 min min) PFOSAA. 4.7 min L m i n ) PFOSAklmin(mia) PFOSEA: 5.7 min ( min) +PmmetasthatmnybechaugedbytheanalystActualvaluesin( ). 2 The above conditions should be suitablefor theMicmmass QunmoLC (SM 9053). Modifications may bc ncmsBlyifanothuh4icromassQuaam Series rpectrometeris uscd Splitthe flow postcolumn via a Keystone BIo-tceor similardevice. 3. Caliimtc themass spectmmeterusinga suitable nfumce compound,orveriij. that the calibrationis suitable by visual inspection(onthetunepage) that a suitablemobile phase ion is still acNTJtctydttermincd&solution may needto be higher than that used for analyzing samples. 4. To check the properperformance ofthe insenUnud,inject the instnmmt c h d standarbThe d t s should be comparableto a recent injection if availablt 5. U s e ~ s a u t o m a t t d c h m m a t o g r a p h y ~ ~ o n ~ s y s t r m t o c o ~ t h s outputtmm the analysis. 6. Logding orda:See the loading rrporthn the automated chromatD& in~onsoftwsrr~ 7. Makesingleinjcctionsdtachcalst&ard,QC. Jtuctysampl~orblankMake at least 4 irjectioruofthe cheek srandard 8. Run set- should typically not exceed80 injections dae to. e t response m l l d c o n s i ~ o n s .Laager nmsmay be performed, but thypose a risk ofyicldingnnacccp~lecurve rcsIll?s. Wa CALCULATIONS 1. sprradsh&tsoftwan: Version 2. MsAnalysissoftware: Version 3. Cakulatethcavuagedtnsitydthe liwhomogcnatc( l o w ) in mgimL 4. Using the avenge density of the homogenate. calculateia livadensity (mg ofliverpcrmLofdilutedhamogenate): Undiluted liwdensity (mdmL)= (gaflivaxaveragc densityofhamogenate)/(gof liw+gdwatet) when g ofliver and gofwater an masseswd to prrparebulkhomogcaatt; density of water isassumedtobe 1g/mL Dilutui liver density (mglmL)= Undiluteddensity + Diln Factor Page 12of16 E-16 3M Environmental Laboratory Page 112 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLuOROOCTANESULFONATE(PFOS) IN RAT LIVER BY LC/Ms/Ms V d o n LO Study No.: AariyruDuc whvcdiInEKxor-211.8= 1.1111toeccormtfor lo%& dliwhomoganme incalndandQCmarrim 5. c a l c u l a o t t h e i l c h l a l c o-~on(n&lL) afmand othernnorochemicalsin tbe auspauions ofcalibrationstand;lrdsand-by usiagthem a ~ o f a d y t easnd dilution factors ody (no liver density correction). Use purity cormxionfor W O W only. .. A. Ms Check Standard (System Suitablity) ss At least 3 injectionsofthe MS CheEk Stilndardmust pmvide a o/oRsD of lo./. or lessfor the PFOS to peak arca d o . B. CaIibration Standards The percentdative ~ m rfsor the concmtrdiion-level ay~agcsofthe caliimion standardc Jhouldmatthtfollowbg~ts: Page 13 of16 E-17 3M Environmental Laboratory Page 113 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCI`ANESULFONATE (PFOS) IN RAT LXVER BY LC/MS/MS Version LO Study No.: AnnlydDlltc: Table 10. OC Accmblnoc limits M-556 PFOSAA PFOSA PFOSEA Removal o f i n d i v i d d m h e h t h e QC Qlculationsfnaybe done ifacwmpaniedbya rrasonableexplanation(e.& instnanentmalfunctionor Dixon's Q test results). wmcavcragc dctaminedmnccnhationforanyQC lcvcl rrrrcQs thcacccpraacclimit, the taslcleadaastudydirectorshouldbe notified Them may k repeated or apanimofthe nm mgyk co&dmdocccpt?blc For -plq ifthe low QC Eailsthe stated reqr ` \ samplamaybePcceptedthathavem~tratonsbradretcdbythehighest~~ration smndard and a mid-lcvtl QC c0ncuWUion. D. Homogenizer Recovery and Dilution Chedt SampIes ' Thc ayu~gcrccovuyacrossthe 3 levels ofhomogenizerrsovuysamples as well that of . . the dilution check samples should EalIwithinthe rangeof70-130% inclusive. Rrmovpl of mdivuid outliersbmm thecalculations maybe done ifacmmpanicdby a reasonable aplanetiaa E. Sensitivity (LOQs) ' Thevalidatedlimits d#on andFFosAA are nominally0.13 vg/g each for PFOS, M-556,W O , Page 14 of16 E-18 3M Environmental Laboratory ! Page 114 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE(PFOS)IN RATLlVER BY Lc/Ms/Ms Yenion LO Stady No.: Ady#DabC: ForPFOSA andPFOSEA,thevalidatedLoQsare nominally 0.33 @ g d Dnc to the natnnattheheprrporationcbthe~onstandards,lowrrconcmatiaasdPFOSA8nd G. x M. 3M Environmental Laboratory Page 15 d16 E-I9 Page 115 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 METHOD FOR ANALYSIS OF POTASSIUM PERnUOR00CX'A"ULFNATE (PFOS) IN RAT LNER BY LC/M!YMS Version LO . Siudy Nar ' CONCLUSIONS QC RNim Date . Daa: Datr Datr Date: Date Daor Date: 3M Environmental Laboratory Page16d16 E-20 Page 116 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Study Title CombinedOral (Gavage) FertilityDevelopmentand PerinataVPostnatal ReproductionToxicity Study ofN-EtFOSE in Rats PROTOCOL AMENDMENT NO. I Amendment Date: July 28,1999 Performing Laboratory 3M EnvironmentalTechnology & SafetyServices 3M Environmental Laboratory 935 Bush Avenue St.Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-013 LIRN U2095 3M Environmental Laboratory E-21 3M Environmental Laboratory Page 117 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 PrOtOCOl FACT-TOX-Of 3 Amendment 1 This amendment modifies the following portion(s) of the protocol: 1. PROTOCORLEADS: The proposed study completion date is listed as 12131/98. AMENDToREAD: The proposed study completiondata is 6/30/00. REASONT: he proposed completiondate was changed to allowtime for analyzingall matrices of interest. Amendment Approval h m V & 776- A 0 Marvin Case Ph.D., SponsorRepresentative JO&d 1+%G Datd Kris J. Hhsen PhD.,'Study Director g IZ/55 Date 3M Environmental Laboratory 3M Environmental Laboratory E-22 Page 118 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 Study Title Combined Oral (Gavage)FertilityDevelopment and PerinataVPostnatal Reproduction Toxicity Study of N-EtFOSE in Rats PROTOCOL AMENDMENT NO. 2 Amendment Date: September 10,1999 Performing Laboratory 3M EnvironmentalTechnology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory praject /dentincation ET&% FACT-TOX-013 LIRN U2095 I 3M Environmental Laborafoty E-23 3M Environmental Laboratory Page 119 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 PrOtOCOl FACT-TOX-013 Amendment 2 This amendment modifies the following pottion(s) of the protocol: I. PROTOCORLEADS: The protocol statesthat liver willbe extracted and analyzedat the 3M Environmental Laboratory. AMENDTo READ: The liver specimenswill be extracted and analyzed at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693. REASON:The liver specimenswill be sent to Battelle Memorial Institute for extractionand analysis due to time constraints in the 3M Environmental Laboratory. 2. PROTOCORLEADS: The protocol statesthat serum specimens will be extracted and anal*ed following methods: FACT-M-3.0, "Extractionof PotassiumPerfluorooctanesulfonateor Other Anionic Surfactantsh m Serum for AnalysisUsing HPLC-ElectrosprayMass Spectrometry" FACT-M-4.0, "Analysis of Fluorochemicalsin Serum Extracts Using HPLCElectrosprayMass Spectrometry" AMENDTOREAD: The serum specimenswill be extracted and analyzed followingmethods: ETS-&;Q.l'%, xtraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds h m Serumfor AnalysisUsing HPLC-ElectrosprayMass Spectrometry" ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonateor Other Fluorochemical Compounds in Serum Extracts HPLC-ElectrosprayMass Spectrometry" REASON: The extraction and analyticalmethods FACT-M3.0 and FACT-M-4.0, respectively,were updated on 04/27/99 to ETS-84.1 and ETS-8-5.1. 3M Environmental Laboratory E-24 3M Environmental Laboratory Page 120 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 PrOtOCOl FACT-TOX-013 Amendment 2 3. PROTOCOL READS: The protocol states that liver specimens will be extracted and analyzed following methods: FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonateor Other Anionic surfactants fiom Liver for analysis Using HPLC-ElectrosprayMasSpectrometry" FACT-M-2.0, "Analysis of Frluorochemicalsin Liver ExtractsUsing HPLC- ElectrosprayMas Spectrometry" AMENDtoREAD: The liver specimenswill be extracted and analyzed followingmethod: Method for Analysis of PerfluomoctaneSulfonate(PFOS)in Rat liverby LC/MS/MS, Version 1.0 REASON: Sincethe liver extraction and analysiswas sub-contractedto Battelle Memorial Institute, this amendment was written to include their liver methods and titles. Amendment Approval Kristen J. Hansen Ph.D., Study Director 3M EnvirOnmental Laboratory E-25 3M Environmental Laboratory 3/2f7/99 Date Page 121 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 . 1 Study Title Analytical Study 2(N-Ethylperfluorooctanesulfoddo)-ethal in Two GenerationRat Repduction PROTOCOL AMENDMENT NO. 3 Amendment Date: October 4, 1999 Perfbnning Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project ldentiffcaffon ET&SS FACT-TOX-013 LIRN U2095 3M Environmental Laboratory E-26 3M Environmental Laboratory Page 122 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M EnvironmentalLaboratory Study Number: FACT 060998.1 . 2 Protocol FACT Tox-013 Amendment Number 3 This amendment modifiesthe following portion(s) of the protocol: 1. PROTOCORLEADS: Kristen J. Hansen, Ph.D. is the Study Director. AMENDTO READ: James K. Lundberg, Ph.D. is the Study Director. REASON: Original study design has changed due to availability of resources and James K. Lundberg will begin servingas the study director for'FACT-TOX-013as of 4 October 1999, 2. PROTOCORL EADS: Section 7.1 statesthat the followingray data and recordswill be retained in the study folder in the archives according to AMDT-S-8: Approved protocol and amendments; study correspondence;shippingrecords; raw data; and electroniccopies of data. Additionally, Section 7.2statesthat supportingrecords to be retained separatelyh m the study folder in the archives accordingto AMDT-S-8 will include at least the following: Trainingrecords; calibration records; instrument maintenance logs; Standard Operating Procedures, Equipment Procedures, and Methods; and appropriate specimens. AMENDTO READ: . Section 7 states: "The original data, or copies thereof, will be available at the 3M EnvironmentalLaboratory to facilitateaudits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M EnvironmentalLaboratory. All corresponding trainingrecords, calibrationrecords, btnunent maintenance logs,standard operating procedures, equipment procedures, and methods will be retained in the archives of the facilityperforming each analysis. REASON: To direct subcontract laboratories in the disposition of the items listed above. 3M Envirvnmental Laboratory 3M Environmental Laboratory E-27 Page 123 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M EnvironmentalLaboratory Study Number: FACT 060998.1 . 1 PIVtOCd FACT TOX-013 Amendment Number 3 3. PROTOCOL READS: Disposition of test and control substances: Biological tissues and fluids are retained per GLP regulation. AMENDTO READ: Specimenswill be maintainedin the 3M EnvironmentalLaboratoryspecimen archives. All specimens sent to sub-contractlaboratorieswillbe returnedto the 3M Environmental Laboratory upon completion of analysis and submissionof the sub-contractlaboratory(s) final report. The specimenswill be returned with the followingdocumentation: the signed original chain of custody and records of storage conditionswhile at the sub-contractfacility. REASON: To define in detail the appropriatedispositionof specimens analyzed at subcontract laboratories. Amendment Approval T L Marv Case, D.V.M., Ph.D., SponsorRepresentative v o u * [qqq Date ' &O /99? , Date Kristen J. Hansen, PhD., Previous Study Director Dale 'L. q f J & Bacon,M.3, M Environmental LaboratoryManagement 1015 / 9 9 Date Dke 3M EnvironmentalLaboratory E-28 3M Environmental Laboratory Page 124 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Study Tifle Analytical Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction PROTOCOL AMENDMENT NO. 4 Amendment Date: 20 January 2000 Performing Laboratory 3 M 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: 4 18-009 3M Medical Department Study: T-63 16.5 3M EnvironmentalLaboratory E-29 3M Environmental Laboratory Page 125 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 - APmrOetnOdCmOlenLtRNNum42b0e9r 54 This amendment modifies the following portion(s) of the protocol: I. PROTOCORLEADS: The study director for the present study was identified in the protocol as James K. Lundburg, Ph.D. AMENDTO READ: The role of study director for the present study was reassigned to Marvin T. Case, D.V.M., Ph.D., as of 20 January 2000. The previous study director, 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 Maivin T. Case, D.V.M., Ph.D. AMENDTO READ: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. REASON: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced. E-30 3M Environmental Laboratory 3M Environmental Laboratory Page 126 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 - APmreontodcmoel nLtRNNu-mUb2e0r945 Amendment Approval John L ButenhofPh.D., Sponsor Representative Date t A Wr Marvin T. Case, D. KM., Ph.D., Incoming Study Director l o F A , &-a Date 6 E-3 1 3M EnvironmentalLaboratory 3M Environmental Laboratory Page 127 3M Medical Department Study: T-6316.5 Analytical Study: 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 Laboratory935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-0 13 ET&SS LRN U2095 Argus Study: 418-009 3M Medical Department Study: T6316.5 3M Environ3mMeEntnavlirLoanbmoernattaolrLyaboratory E-32 Page 128 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 - Protocol FACT TOX-013 Amendment No. 5 This amendment modifies the following portion(s) of the protocol: 7. PROTOCOL READS: The Principle Analytical Investigator for the present study was identified as James K. Lundberg, Ph.D. 2. AMENDTO 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. E-33 3M Environmental Laboratoty 3M Environmental Laboratory Page 129 3M Medical Department Study: T-6316.5 Amendment Approval Analytical Study: FACT-TOX-013 LRN-U2095 Protocol FACT TOX-013 Amendment No. 5 (5 John L. ButenhoB Ph.D.,Sponsor Representative Date M h i n T.Case, D.V.M., Ph.D., Study Director W Date E-34 3M Environmental Laboratory 3M Environmental Laboratory Page 130 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 DEVIATION REPORT Battelle Study Number: N003604-D 3M Environmental Technology and Services Study Number: FACT 060998.1 2 (N-Ethy1perfluorowtanesulfonamido)-ethanol in Two Generation Rat Reproduction TYPE OF DEVIATIONS: PROTOCOL DATES OF DEVIATIONS: October 18, 1999 NATlJRE OF DEVIATIONS: Some of the analytical method acceptance criteria were not met for the LC/MS/MS analysis conducted 180ct99 at BatteIle. These deviations d a t e to protocol amendment 2. See below for summary. r PPOS M-556 M-556 M-556 M-556 PFOSAA PFOSAA I 1 QC3 exceeded 20%error (-20.3 % actual) QC2 exceeded 20%error (23.4%actual) QC3 exceeded 20%RSD (21.2%actual) QC4 exceeded 20%RSD (28.8% actual) Dilution recovery exceeded 130%(131.5%actual with 21.6%RSD) QC3 exceeded 20% error (-20.6%actual) QC4 exceeded 20%RSD (21.3 % actual) CAUSE OF DEVIATIONS: Sample preparation andor LC/MS/MS variabilities over the course of the sample set may have contributed to the deviations. IMPACT OF DEWATIONS ON THE STUDY: The errant QC values were bracketed by acceptable QC concentration levels which demonstrates that the calibration curves generally provided good accuracy over the twted range. The dilution recovery for M-556Was not considered to be exceedingly high enough,at only approximately 1.5% above the normal acceptance level, to have significantlyimpacted the data. CORRECTIVE ACTI0N:lXs protocol deviation summary was prepared for inclusion in the final report. APPROVED B Y -; r L Date I , Battile Principal investigator Study Director h 4 V # I k I: e 4 * bwhJpI,p Ivlcc avo/ l w w L . 2441 Date N003604-DProtocol Deviation 1, Page 1 of 1 E-35 3M Environmental Laboratory Page 131 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M EnvironmentarLaboratoryStudy Number: FACT 060998.1 DEVIATION REPORT Battelle Study Number: N003604-D 3M Environmental Technology and Services Study Number: FACT 060998.1 2 (N-E!thylperfluorooctanesulfomnido)-ethanol in Two Generation Rat Reproduction TYPE OF DEVIATIONS: PROTOCOL DATE OF DEVIATIONS: October20,1999 NATURE OF DEVIATIONS PFOS QC3 exceeded the 20% RE method requirement (actual -21.7%). The dilution recovery check standard did not meet the 70-130% recovery requirement (actuals 5.0% for PFOS and 4.6% for PFOSA). These method deviations relate to amendment 2 of the study protocol. CAUSE OF DEVIATIONS: Sample preparation andor LC/MS/MS variabilitiesover the course of the sample set may have contributed to the QC deviation. Sample preparation error appears to have been the cause for the dilution recovery results. IMPACT OF DEVIATIONS ON THE STUDY: The errant QC value level was bracketed by acceptable QC concentration levels which demonstrates that the calibration curves generally provided good accuracy for study samples over the tested range. A comparison of the results obtained for the diluted study samples from 200ct99 and previous results that were slightly-ALOQ (13Oct99 and 18Oct99) demonstrated good agreement between the 2 determinations. This would indicate that the dilution of the study samples was performed correctly 20Oct99 so that no impact on the quantitationsoccurred. CORREclTvE ACTI0N:This protocol deviation summary was prepared for inclusion in the final report. APPROVED BY: L c Gv& JofJC. Andre, Ph.D. Date Baitelle Principal Investigator A N003604-DProtocol Deviation 2,Page 1 of 1 E-36 3M Environmental Laboratory Page 132 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 - Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 DEVIATION REPORT Battelle Study Number: N003604-D 3M Environmental Technology and Services Study Number: FACT 060998.1 2 (N-Ethy1perfluorooctanesulfonamido)-ethanolin Two Generation Rat Reproduction TYPE OF DEMATIONS: PROTOCOL DATES OF DEVIATIONS: October 12, 1999through October 20,1999 N A m OF DEVIATIONS: The lot number of PFOS used was not 217 as per section 3.1.1 of the protocol. The source of reference substance matrix was not Argus 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. CAUSE OF DEVIATIONS: Only PFOS lot number 171 was available at Battelle. Harlan 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 hterest (PFOS,M-556, PFOSAA, and PFOSA) were monitored during each analysis. IMPACT OF DEVIATIONS ON THE STUDY: PFOS lot number 171 and Harlan- supplied liver were both used for Battelle's validation of the analytical method (Battelle study number NOO3604-A). These materials allowed achievement of the reported method acceptance 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: LCGik-dq Jon@. Andre,Ph.D. Batt&e Principal Investigator 2- 2-74 Date Study Director N003604-DProtocol Deviation 3, Page 1 of 1 E-37 3M Environmental Laboratory Page 133 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 APPENDIX F - PFOS PURITY REPORT 3M Environmental Laboratory Page 134 3M Medical Department Study: T-6316.5 ETSS 2 3U Analytical Study: FACT-TOX-013 LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 8 651 778 4226 ~ 04/26/99 09:56 @1 :02/03 N0:341 Ta; blnG s&iGc parr;. ~,~ 3M 3PECMLT.Y ADHESXVZS & CHEMICALSANALYTXCU, LABORATORY - Lisa Ckmn (8-5568) -ETUS- 24349 - - - Tom kstner (34633) SA&C Analytical Lab 236-2B-1I Rcquest # 57830 CIURIicd Chtaci2-n of POSF-Baed FtuorochrrmLaLv 8y 'R-NHRdi "F-NM. SpeCtTOSCan Match 24,1999 Retiminary report for FC-95 (PFOS), lot 171 . ~ ~ ~ ~ ~ hGom- indAprod-uct =OGFd8O,(-3) K(4+) (whitepowder) ODUCTIOY; 'Ibis sample was subjecrcd-to'H-NMR and '%NMR specaal analyses to determine the purity of the nominal product and to chanrcrerue aa many impuritycomponcnbM possible. A portion of the sample was accurately weighed, spiked with a known mount of 1,4-bis(trifluoromcthyhyl)bmzl?ne (pHFX),and then totally dissolved in D M S M for subsequent analysis by NMR A 400 MHz 'H-NMR Spectrum(b N7830.401)and a 376 h4Hz "F-NMR spectrum(# fS7830.401)were acquired using a Vdan UNITYpIus 400Fr-NMR sptctromcter. Use of the pHFX internal standard was intended LOpermit the derennination of the absolute weight percent concentrationsof the assignedcomponentswithout necessarily needing to identify or quanti6 all the components in the sample mixture. RzuYLm The combinedNMR spccrral dam were used to assign 311 of he major and most of the minor componentsin thi8 sample received. The qualitative and quantitalivc compositional r e s u l ~that w e n derived from the single Vial NMR internal sfandnrdization analyses are summarized in TABLE-1on the followingpage. I have reported both reiative and absalute weight percent concentrations. One possible rewon that the absoIuce wt.46 values add up to mom than 100% may be due to the fjcr that I assumed all of the components conrained 8 carbons. If thcre were any shorterchain homologs present (i-e.. 7.6.5, tcc. carbons). then the average coToudd molecular weights would have been samewhat less than those used in the calculations. In general, the ?-NMR technique is not POrticularly well suited for identifyingor quantifyingsmall amounts of various fluorochtmid homolog impurity components unless the chainr are very short. A more complete characterization of any other fluorochemical homologs woutd roquire analysis by elcctrospray MS or a similar technique. Additional work would be required in an effort LO positively verify the tentatively assigned components listed in TABLE- 1 (denoted by possible). Small amounts of other unidentified impurities ilfe also detected in the NMR SppetWa but additional work would be required in an effort to identify or qumriQ these other mrrids. Copies of the NMR spectra will be provided for you at a later date. If you have any questionsabout the ruultt in this initial report for FC-95,lot 171. piease let me know. I apologize for the delay in cnmpleting this initial work. 3M Environmental Laboratory PagC.1- F- 1 Page 135 3M Medical Department Study: T-6316.5 ETSS 2 3U Mdnh 24 1999 Analytical Study: FACT-TOX-013 - LRN-U2095 Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1 651 ?78 4226 04/26/99 09:56 # :03/03 N0:341 S A X AnalyticalLab R e p s t f 37830 tlal Rebort for FC-H. lot 174 , 3M Environmental Laboratory Page 136 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 R Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Appendix H: Interim Certificate of Analysis 3 3M Environmental Laboratory 3M Environmental Laboratory Page 131 Page 137 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 Purity' Test Name Qpearance :dentification VIetals. (IQ/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium' 5. Nickel 6. Iron 7. Manganese Iota1 % 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. Nitxate 5. Nitrite 6. Phosphate 7. Sulfate4 Organic Acids ' (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis? 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine COA023-018B 3M Environmental Laboratory 3M Environmental Laboratory mererence *: DU-UVY Purity: 86.4% Specifications White Crystalline Powder 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Result 86.4% Conforms Positive 1. 0.017 wt./wt.% 2. 0.007 wt./wt.% 3. 1.355 wt.hvt.% 4. 6.552 wt.lwt.`%o 5. 0.003 wt.lwt.% 6. 0.004 wt./wt.% 7. <O.OOl wt./wi.% 1.oo wt./wt.% 10.60 wt./wt.% I None Detected 0.30 wt.hvt.% None Detected Not Applicable3 1. co.015 wt./wt.% 2. 0.27 wt.lwt.% 3. co.040 wt./wt.% 4. <0.009 wt.lwt.% 5. -=0.006 wt.fwt.% 6. <0.007 wt./wt.?h 7. 8.82 wt.lwt.% 1. co.1 wt./wt.% 2. co.1 wt./wt.% 3. co.1 wt./wt.% 4. <0.25 wt./wt.% 1. 12.08 wt./wt.% 2. 0.794wt./wt.% 3. 1.61 wt./wt.% 4. 10.1 wtJwt.% 5. 50.4 wt./wt.% Page 1 of 3 Page 132 Page 138 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B Date of Last Analysis: 08/31/OO Expiration Date: OS/3I/O1 Storage Conditions: Frozen I-10C Re-assessmentDate: 08/31/01 `Purity = 100% - (sum of metal impurities, 1.39% +LC/MS impurities, 1O.CiO%+InorganicFluoride, 0.27%+NMR impurities, 1.OO%+PO&X, .0.30%) Total impurity from all tests = I3.56% Purity = 100% - 13.56% = 86.4% 2Potassiumis expectedin this salt form and is thereforenot considered an impurity. 3Purity by DSC is generalIy not applicable to materials of low purity. No endotherm was observed for this sample. `Sulfur in the sample appearsto be converted to SO, and hencedetectedusing the inorganic anion method conditions. The anion result agreeswell with the sulfur determination in the elemetital analysis, lending confidence to this interpretation. Based on the results, the SO4is not consideredan impurity. `TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid `Theoretical value calculations baaedon the empirical formula, C~FI~SOJX+ (MW=538) This work was conducted under EPA Good Laboratory Practice Standards(40 CFR 160). COA023-018B 3M Environmental Laboratory c 3M Environmental Laboratory Page 2 of3 Page 133 -. Page 139 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Analytical Study: FACT-TOX-013 Analytical Report: LRN-U2095 FACT TOX-013 LRN-u2095 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B LCYMS Purity Profile: Impurity c4 CS C6 Total wt./wt. O/o 1.03 1.56 6.38 10.60 1 m Note: The C4 and C6 values were calculated using the C4 and C6 standardcalibration curves, respectively. The C5 value was calculated using the averageresponsefactors from the C4 and C6 standardcurves. Likewise, the C7 value was calculated using the *r averageresponsefactors from the C6 and C8 standardcurves. PreparedBy: David S. Bell Date Scientist, Centre Analytical Laboratories Reviewed By: pl John Flaherty Date Laboratory Manager, Centre Analytical Laboratories m COA023-018B 3M Environmental Laboratory 3M Environmental Laboratory Page 3 of3 Page 134 Page 140 3M Medical Department Study: T-6316.5 3M Medical Department Study: T6316.5 Appendix I: Report Signature Page Analytical Analytical Study: FACT-TOX-013 Report: LRN-U2095 FACT TOX-013 LRN-u2095 A 7-L ., Ph.D., Study Director John L. Butenhoff, Ph.D., Sponsor Representative Dale L. Date Date , Date a 3M Environmental Laboratory F- 3M Environmental Laboratory Page 135 Page 141 3M Medical Department Study: T-6316.5 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Study: FACT TOX-013 LRN-U2095 Appendix J: Amendment 1 to FACT TOX-013 Final Report TOX-013 Final Study Report Amendment 1 Study number: TOX 013 Study title: Analytical Study 2(N-Ethylperfluorooctane sulfonamid0)-ethanolin Two Generation Rat Reproduction Study Director: Marvin T. Case, D.V.M., Ph.D. Amendment date: May 7,2001 Amendment number: 1 This amendment modifies the following portion of the final report: A final signed report from Battelle Memorial Institute, presenting the results for PFOS, PFOSAA, PFOSA, and M-556 levels in rat liver specimens, replaces the draft Battelle report in Appendix G. Liver results in this report are identical to those presented in the original TOX-013 report (Table 13, pages 22-23). As in the original liver data, the PFOS values reported in the Battelle report were corrected by 3M for purity of the reference standard material. The final Battelle report differs from the draft report in the following ways: All signature pages are signed and dated. The Quality Assurance Statement page has four additional audit dates added. 0 Table of Contents page numbers were corrected. 0 Two Battelle participants were eliminated from page 4, `Acknowledgements.' 0 The storage and archive instructions (page 4) are now found in the 3M TOX-013 protocol amendment 3. 0 Inclusion of 3M TOX-013 protocol amendments 4 and 5, thus changing the total number of pages. Minor wording changes. Other changes to the TOX-013 report include: The cover page was updated to reflect the total number of pages and the title was changed to say "Amended Final Report." The Table of Contents was updated to reflect the added amendment. The additional audit date of the Amended Analytical Laboratory Report TOXO13 was added to the Quality Assurance Statement. 3M Environmental Laboratory 3M Environmental Laboratory Page 135 Page 142 3M Medical Department Study: T-6316.5 3M Medical Department Study: T-6316.5 Analytical Study: FACT-TOX-013 LRN-U2095 Analytical Study: FACT TOX-013 LRN-U2095 Approved by: Kristen H. Hansen, Ph.D., Principal Analytical Investigator OS/!!O 10 1 Date 6 I Marvin T. Case, D.V.M., Ph.D., Study Director Date Bill Reagan, Ph.D., Environmental Laboratory Manager OJ+,/O, Date 3M Environmental Laboratory 3M Environmental Laboratory Page 135 Page 143