Document LpYbdDBxMajQ8ONrd5rZDdgYq

3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 Laboratory Request Number-U2279 A nalytical Laboratory Report FROM THE 26-Week Capsule Toxicity Study with Perfluorooctanesulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys ON THE Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Liver and Serum Samples Project Identification 3M Medical Department Study: T-6295.7 Covance In-Life Study: #6329-223 Analytical Study: FACT TOX-030 3M Laboratory Request No. U2279 Study Completion Date At signing Total Number o f Pages 233 3M Environmental Laboratory Page 1 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 GLP C o m p lia n c e S ta tem en t Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Study Title: Analytical Laboratory Report from the 26-Week Capsule Toxicity Study with Perfluorooctanesulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys on the Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Liver and Serum Samples Study Identification Number: FACT TOX-030, T-6295.7, Covance #6329-223 This study was conducted in compliance with United States Environmental Protection Agency Good Laboratory Practice (GLP) Standards 40 CFR Part 792, with the exceptions in the bulleted list below. All raw data and samples for this study are retained in archives at the 3M Lab and will be retained for a period of at least ten years. The analytical phase completed at the 3M Lab 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 study 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. On a few occasions, data were not recorded or corrected exactly as required by the GLPs. The 3M TOX 030 protocol states in the Regulatory Compliance section that "This study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practices Standards, 40 CFR 792, with the exception that analysis of the test material mixture for concentration, solubility, homogeneity, and stability will not be conducted, and is the responsibility of the Sponsor." Analyses were, however, completed on the concentration and homogeneity of the test material mixture, according to non-GLP validated methods, and are included in this report. As per the protocol, solubility and stability determinations were not conducted. Study Director }& i Date c'> Study Sponsor Date 3M Environmental Laboratory 3M Environmental Laboratory Page 2 Page 2 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 GLP Stu d y -- Q u a l ity A ssurance Sta tem en t Study Title: Analytical Laboratory Report from the 26-Week Capsule Toxicity Study with Perfluorooctanesulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys on the Determination of the Presence and Concentration of Perfluorooctanesulfonate (PFOS) in Liver and Serum Samples Study Identification Num ber FACT TOX-030, T-6295.7, Covance #6329-223 The analytical phase of this study has been inspected by the 3M Lab Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and management. In s p e c t io n D a t e s December 01/98 P hase Sample receipt Date Reported to M anagement S tu o y D ir e c t o r 1/17/00 1/17/00 March 19,22,23/99 Analysis 3/25/99 3/25/99 October 14/99 May 3,8-12,15-19,22-26,29-31/00, June 1,2,5,7,8/00 June 1,5,7,12-16/00 Extraction Data Draft report 10/20/99 6/14/00 6/16/00 10/20/99 6/14/00 6/16/00 September 14/00 Draft report 9/14/00 9/14/00 / / QAU Representative C( U Date , 3M Environmental Laboratory 3M Environmental Laboratory Page 3 Page 3 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 S tu d y P ersonnel and C ontributors Study Director Andrew M. Seacat, Ph.D. 3M Medical Department 3M Center, Building 220-2E-02 PO Box 33220 St. Paul, MN 55133-3220 (651)575-3161 Sponsor 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55133-3220 John L. Butenhoff, Ph.D., Sponsor Representative Analytical Chemistry Laboratory Liver and Serum Analyses 3M Environmental Technology and Safety Services (3M ET&SS) 3M Environmental Laboratory (3M Lab) Fluorine Analytical Chemistry Team (FACT) 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Kristen J. Hansen, Ph.D., Principal Analytical Investigator Contributing Personnel David R. Bamidge Lisa A. Clemen Kelly J. Dorweiler Mark E. Ellefson Sara E. Estes Barb A. Gramenz Sarah A. Heimdal Cari S. Hewitt Marlene M. Heying Harold O. Johnson Kelly J. Kuehlwein Sally A. Linda Michael D. Livingston Joseph C. Pilon Scott R. Post Ian A. Smith Anh-Dao Vo Bob W. Wynne In-life Testing Laboratory Covance Laboratories, Inc. 3301 Kinsman Boulevard Madison, W l 53704-2595 Peter J. Thomford, Ph.D., In-Life Phase Study Director 3M Environmental Laboratory 3M Environmental Laboratory Page 4 Page 4 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Table of Contents Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 GLP Compliance Statem ent........................................................................................................................... 2 GLP Study-- Quality Assurance Statement................................................................................................... 3 Study Personnel and Contributors.................................................................................................................. 4 Introduction and Purpose................................................................................................................................ 6 Test System ..............................................................................................................................................6 Specimen Collection and Analysis.......................................................................................................... 7 Specimen R eceipt............................................................................................................................................7 Dose Confirmation Analyses................................................................................................................... 8 Materials and M ethods.................................................................................................................................... 8 Chemical Characterization...................................................................................................................... 8 Method Summaries..................................................................................................................................8 Analytical Equipment................................................................................................................................9 Deviations............................................................................................................................................... 10 Data Quality Objectives and Data Integrity.................................................................................................. 10 Data Summary, Analyses, and Results........................................................................................................ 1 1 Summary of Quality Control Analyses Results....................................................................................11 Summary of Sample R esults................................................................................................................ 12 Statistical Methods and Calculations............................................................................................................ 12 Statement of Conclusion................................................................................................................................12 List of Attachm ents.........................................................................................................................................12 Attachment A: Control Matrix Characterization and Dose Confirmation Analyses...................................13 Attachment B: Protocol and Deviation Summary.........................................................................................15 Attachment C: Extraction and Analytical Methods...................................................................................... 43 Attachment D: Data Summary Tables........................................................................................................ 179 Attachment E: Data Spreadsheets............................................................................................................. 188 Attachment F: Example Calculations..........................................................................................................225 Attachment G: Interim Certificate of Analyses.......................................................................................... 226 Attachment H: Report Signature Page.......................................................................................................233 3M Environmental Laboratory 3M Environmental Laboratory Page 5 Page 5 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Introduction and Purpose CsFi7------- S------- O O Perfluorooctanesulfonate (PFOS) CAS Number 2759-39-3 Chemical Formula: CaF,7S03 Molecular Weight: 498.98 The purpose of the analytical phase of this study is to determine the presence and concentration of PFOS (CbF17S 0 3') in liver and serum specimens collected during the study of Cynomolgus monkeys orally dosed with perfluorooctane sulfonic acid potassium salt (T-6295). Test System The test system species and strain selected was the Cynomolgus monkey from Covance Research Products, Inc., identified using a collar tag. At the initiation of treatment, the Cynomolgus monkeys were young adult to adult, and weighed approximately 3-5 kg. Twenty-two male and 22 female Cynomolgus monkeys were used as the test system in the present study. Four groups of test animals were established according to dosage levels. Group 1 consisted of control Cynomolgus monkeys that did not receive the test substance, but received the equivalent amount o f lactose in gelatin capsules as that administered to the Group 4 animals. Groups 2, 3, and 4 were administered daily with 0.03 (low dose), 0.15 (mid dose), and 0.75 (high dose) mg respectively, of T-6295 per kg of body weight/day (mg/kg/day) triturated with lactose in gelatin capsules (see Table 1 for Dosage and Group Characteristics). Table 1. Dosage and Group Characteristics of Test System in Study T-6295.7 STUDY GROUP Number of A nimals Total Dosage Level Dosage Ratio (mg/kg/day) (w:w)a Total: Test System Group 1 (Control) Group 2 (Low Dose) Group 3 (Mid Dose) Group 4 (High Dose) 22 males 22 females 6 males 6 females 4 males 4 females 6 males 6 females 6 males 6 females 44* -- 12 0 8 0.03 12 0.15 12 0.75 -- -- 1:499 1:39 1:39 a Test substance triturated with lactose * 48 animals were included in the baseline sera collection, but 44 animals were assigned for treatment 3M Environmental Laboratory 3M Environmental Laboratory Page 6 Page 6 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 All treatment groups were dosed for a minimum period of 26 weeks. Sera specimens were collected from all test animals at various time points during the in-life phase of the 26-week study and sent to the 3M Lab for analysis (see Attachment D, Tables D-1a, D-1b). Four animals each from Groups 1,3, and 4 were designated as recovery group animals. Treatment was discontinued and the animals were monitored for elimination of compounds for one year post treatment. The recovery groups were observed after the cessation of treatment until February 25, 2000 (Week 79) for Group 1 and Group 4 recovery animals, and until March 7, 2000 (Week 80) for Group 3 recovery animals. Specimen Collection and Analysis In the analytical phase reported here, liver and sera specimens collected from all test animals were sent to the 3M Lab and analyzed for the presence of PFOS (some samples were analyzed to determine the presence of EtFOSE, PFOSA, POAA, PFOSEA, M556, PFOSAA, and the monoester; however, these data were collected for informational purposes only, and are not reported). Specimens other than serum and liver tissues were collected and received from Covance Laboratories (6329-223), but were not part of the current scope of analysis determined by the study director and sponsor. Additional analyses of feces are being completed and will be issued as an amendment to this final report. Blood specimens were centrifuged within one hour of collection. The serum was then harvested and stored in a freezer set to maintain specimens at -60 to -80C until shipped to the 3M Lab. Liver specimens collected from each animal were flash frozen in liquid nitrogen and then stored in a freezer set to maintain specimens at -60 to -80C until shipped to the 3M Lab. Liver and sera specimens were shipped to the 3M Lab frozen and on dry ice. Liver specimens from Group 3 (3/01/00) and Group 4 (9/22/99) recovery animals were collected via biopsy. Sera and liver samples were extracted using an ion-pairing reagent and methyl-tert-butyl ether (MtBE). Liver samples were homogenized prior to the extraction procedure. Sample extracts were analyzed using high-pressure liquid chromatography-electrospray/tandem mass spectrometry (HPLCES/MS/MS) in the multiple response mode. PFOS levels were quantitated by external standard calibration. Analytical details are included in this report. Specimens Collected from Study Groups 1 through 4 (through 2/25/99): Serum Specimens-- 550 specimens: 9-14 specimens/animal Liver Specimens-- 30 specimens Specimens Collected from the Recovery Group from 2/27/99 to 3/07/00: Serum Specimens-- 224 specimens: 18-20 specimens/animal Liver Specimens-- 12 specimens from Group 3 and Group 4 animals (8 via biopsy) S pecim en Receipt Specimens were received from Covance Laboratories periodically, during the in-life phase of this study, from August 1998 through March 2000. Specimens received were frozen and on dry ice. Specimens were logged in with the 3M Lab and transferred to freezers for storage at either -55C 1Q-20C or -20C 10C. Control matrices used in liver and sera analyses performed during TOX-030 were obtained from commercial sources and are presented in Attachment A (see Table A-1). Samples analyzed at the 3M Lab will be maintained for a period of 10 years and will be stored at the laboratory at -20C 10C. 3M Environmental Laboratory 3M Environmental Laboratory Page 7 Page 7 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-112279 Dose Confirmation Analyses Dose confirmation analyses were performed on lactose dose samples (1:39 and 1 499) collected on 8/11/98 during the in-life phase of the study: the results are presented in Attachment A (see Table A-2, A-3). The dose confirmation data were collected according to a method that was not fully validated. Dose confirmation was performed by diluting the lactose dose samples (1:39 - 1,000x and 1:499 1,000x) with Miili-Q water, then extracted using the ion-pair procedure, diluted 1:50 and 1:5 respectively into the linear range of the instrument. For each sample (top, middle, bottom), a matrix spike was prepared (approximately 5000 pg/g and 400 pg/g) by spiking the dose solution and then diluting and extracting as described above. 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. The average dose level measured was confirmed to be 99 27% of the target concentration. Matrix spikes were recovered at >60%. Materials and M ethods Chemical Characterization Table 2 presents information regarding characterization of the test substance used in the in-life phase of this study, and the analytical reference substance used in the analytical phase of this study. Table 2. Characterization of Test and Analytical Reference Substances in Study FACT TOX-030 C h e m ic a l N a m e Source Ex p ir a t io n D ate S t o r a g e CoNomoNs C h e m ic a l L o t N u m b e r P h y s ic a l D e s c r ip t io n P u r it y T est Substance K P F O S Potassium Perfluorooctanesuiforiate 3M Specialty Chemicals Div. 8/31/2001 Frozen <-10` C 217 FC-95, White crystalline powder 86.9% A nalytical Reference S ubstances K P F O S Potassium Perfluorooctanesulfonate 3M Specialty Chemicals Div. T H P FO S 1H.1H.2H.2Hperfluorooctanesulfonic add ICN Biomedics, Inc. 8/31/2001 1/01/2020 Frozen <-10"C Ambient temperature 171 59909 53406 White crystalline powder Brown powder Brown waxy solid 86.4% N/A N/A Reserve samples of the analytical reference substance will be stored at the 3M Lab for a period of 10 years, as will any reserve samples of test substance returned from the in-life phase of the study. Method Summaries Following is a brief description of the latest methods used during the analytical phase of this study by the 3M Lab. Detailed descriptions of the methods used in this analytical phase are located in Attachment C. As the present analytical phase of this study progressed, more advanced methods evolved and earlier methods were used with deviations until amendments to the protocol were written. Changes to the methods included the use of methyl-fert-butyl ether (MtBE) instead of ethyl acetate, curves plotted by linear regression weighted 1/x instead of unweighted curves, a reduction in the size of the analytical column from 100mm to 50mm, gradient changes, and faster HPLC cycle times. A summary of protocol and method deviations is presented in Attachment B (see Table B-1) of this report. 3M Environmental Laboratory 3M Environmental Laboratory Page 8 Page 8 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Preparatory Methods: ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or other Fluorochemical Compounds from Liver for Analysis using HPLC-Electrospray/Mass Spectrometry" Liver samples were homogenized in water. An aliquot of each homogenate was spiked with THPFOS and extracted using an ion-pairing extraction procedure. An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into MtBE. The extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol and passed through a 0.2 pm nylon filter, using a 3 cm3 disposable plastic syringe into glass autosampler vials. ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" Sera samples were spiked with THPFOS and extracted using an ion-pairing extraction procedure. An ion-pairing reagent was added to the sample and the analyte ion pair was partitioned into MtBE. The MtBE extract was removed and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol and passed through a 0.2 pm nylon filter, using a 3 cm3 disposable plastic syringe into glass autosampler vials. Analytical Methods: ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry" The analyses were performed by monitoring one or more product ions selected from a single primary ion characteristic of a particular fluorochemical using HPLC/ES/MS/MS. For example, molecular ion 499, selected as the primary ion for PFOS (CaF1?SOr ) analysis, was fragmented to produce ion 99 (FS 03-). Tne characteristic ion 99 was monitored for quantitative analysis. Analytical Equipment The actual analytical equipment settings used in the present analytical phase of this study varied slightly during actual data collection. The following is representative of the settings used during the analytical phase of this study. Liquid Chromatograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone Betasil" C,e2x50 mm (5 pm) Column temperature: Ambient Mobile phase components: Component A: 2mM ammonium acetate in water Component B: methanol Flow rate: 300 pUmin Injection volume: 10 pL Solvent Gradient: 10 minutes 3M Environmental Laboratory 3M Environmental Laboratory Page 9 Page 9 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Start at 10%B Hold at 10%B for 1.0 minute Increase to 95%B over 4.5 minutes Hold at 95%B for 2.0 minutes Return to 10%B over 0.5 minutes Hold at 10%B for 2.0 minutes Mass Spectrometer Micromass API/Mass Spectrometer Quattro II" Triple Quadrupole system Software: Mass Lynx" 3.2 Cone Voltage: 60V Collision Gas Energy: 40-60eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Electrode: Z-spray Analysis Type: Multiple Reaction Monitoring (MRM) Table 3. Negative Ions Monitored in FACT TOX-030 Target Analyte Primary Ion (amu) Product Ion (amu) PFOS THPFOS 499.0 j 99.0 427.0 ; 80.0 Deviations It should be noted that as the analytical phase of this study progressed, method parameters were evaluated to improve analyses. Earlier methods were used with deviations until amendments to the protocol were written. Deviations from the original protocol and methods are documented in the Attachment B (see Table B-1).* Da ta Q uality O bjectives and Data Integrity The following data quality objectives (DQOs) were indicated in the protocol for this study: Linearity: The coefficient of determination (r2) equal to or greater than 0.98 Limits of Quantitation (LOQ): The Method Detection Limit (MDL) for PFOS is 12 ppb for serum and 15 ppb for liver. The LOQ is equal to the lowest acceptable standard in the calibration curve. Duplicate/Acceptable Precision: Precision was reproducible to within 30% Spike/Acceptable Recoveries: 70-130% Confirmatory Methods: Indeterminate samples may be re-analyzed using a confirmatory method If a confirmatory method is used, an amendment to this protocol should be written. Demonstration of Specificity: Specificity to be demonstrated by chromatographic retention time and mass spectral daughter ion characterization. 3M Environmental Laboratory 3M Environmental Laboratory Page 10 Page 10 o 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-03 laboratory Request Number-U227 Report No. FACT TOX-030 Laboratory Request Number-U2279 Data Summary, A nalyses, and Results Data quality objectives for the analytical phase of this study outlined in the 3M Lab protocol for FACT TOX-030 (see Attachment B) were met with the exceptions noted in this report. Summary of Quality Control Analyses Results Linearity: The coefficient of determination (r2) of the standard curve was >0.985. Calibration Standards: Quantitation of the target analytes was based on linear regression analysis (unweighted - prior to 3/5/99, unweighted or 1/x weighted-3/5/99 to 3/19/99, and 1/x weighted-3/19/99 to end of the study) of two extracted matrix curves bracketing each group of samples, except as noted in the deviation summary. High or low points on the curve may have been deactivated to provide a better linear fit over the curve range most appropriate to the data. Low curve points with peak areas less than two times that of the extraction blanks were deactivated to disqualify a data range that may have been significantly affected by background levels of the analyte. Occasionally, a single mid-range curve point that was an obvious outlier was deactivated. Quantitation of each anaiyte was based on the response of one specific product ion using the multiple response-monitoring mode of the instrument (see Attachment C). Limits of Quantitation (LOQ): The LOQ is equal to the lowest acceptable standard in the calibration curve (defined as a standard within 30% of the theoretical value), and is at least two times the analyte peak area detected in the extraction blanks. This value does not exceed the validated LOQ of the method for data that is accepted (see Attachment D, Table D-6). Table 4. Determ ination o f PFOS LOQ in TOX-030 Analyses A n a t o e -M a tr ix LOQ PFOS-Sera PFOS-Liver 4 .3 9 -1 5 .2 ng/mL 2 6 .9 -6 0 .1 ng/g 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 monkey sera, rabbit sera was selected as a suitable surrogate matrix. Duplicate/Acceptable Precision: Precision was determined by analysis of MS/MSD and was reproducible to within 30%. Matrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of samples and analyzed during analytical runs at the 3M Lab. All sera matrix spikes were within 30% of the theoretical concentration. Matrix spikes prepared in liver were compliant within 30%, with the exception of one spike that was prepared with Day-393 samples and had a low recovery. The matrix spike was reextracted and the recovery was within 30% of the theoretical concentration. Spike/Acceptable Recoveries: Spike recoveries of 30% of expected values were achieved for all matrix spikes prepared in sera. With one exception (noted earlier), matrix spikes prepared in liver were within 30%. Use of 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. After 11/04/99, the surrogate response of each analytical run was verified to determine that it did not vary more than 50% from the mean within each analytical run. No problems were observed with these data. 3M Environmental Laboratory 3M Environmental Laboratory Page 11 Page 11 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2273 Report No. FACT TOX-030 Laboratory Request Number-U2279 Assuming spike recovery studies form a suitable indication of endogenous analyte recovery, data are quantitative to 30%. The validity of this assumption has not been verified by other techniques. Summary of Sample Results Samples from Control Animals: Low levels of PFOS were often detected in the sera and liver of the control animals. These levels were significantly lower than those found in the low dose test animals. Samples from Dosed Animals: In general, PFOS levels found in the sera and liver of the test animals increased with dose group. PFOS levels increased as dosage increased; significant differences between male and female PFOS levels were not observed in sera. However, Group 4 males had notably higher PFOS levels in liver samples than Group 4 females. Detailed sample data is presented in Attachments D and E. Sta tistic a l M ethods and Calculations Statistical methods were limited to the calculation of means and standard deviations. See Attachment F for example calculations used to generate the liver and serum sample data in TOX-030. Sta tem en t of C onclusion Under the conditions of the present analytical phase of this study, PFOS was detected in the sera and liver samples of Groups 2, 3, and 4 animals. The Control Group 1 animals showed minimal amounts of PFOS. PFOS levels increased as dosage increased; significant differences between male and female PFOS levels were not observed in sera. However, Group 4 (high dose) males had notably higher PFOS levels in liver samples than Group' 4 females. Data quality objectives for the analytical phase of this study outlined in the 3M Lab protocol for FACT TOX-030 (see Attachment B) were met with the exceptions noted in this report.* List of A ttachm ents Attachment A: Control Matrix Characterization and Dose Confirmation Analyses Attachment B: Protocol and Deviation Summary Attachment C: Extraction and Analytical Methods Attachment D: Data Summary Tables Attachment E: Data Spreadsheets Attachment F: Example Calculations A ttachm ent G: Interim Certificate of Analyses Attachment H: Report Signature Page 3M Environmental Laboratory 3M Environmental Laboratory Page 12 Page 12 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Attachment A C o ntro l M a trix C haracterization and D ose Confirmation A nalyses Tabla A-1. Characterization of the Control Matrices Used for Liver and Sera Analyses In Study FACT TOX-030 Control Matrix Source Expiration Date Storage Conditions Chemical Lot # Physical Description Control Matrix Source Expiration Date Storage Conditions Chemical Lot # Physical Description N/R-- not recorded Rabbit S erum Sigma Chemicals 01/01/2010 Frozen -20*0 118H8418 Rabbit Serum Rabbit Liver Coming Hazleton 12/01/1999 Frozen -20C F00007 Rabbit Liver Ra b bit S erum | M o nkey Serum Monkey Serum Sigma Chemicals ) Lampire Biological 01/01/2010 N/R Frozen -20C Frozen -50C 47H4641 111022515 Sierra Biomedical 01/01/2010 Frozen -20C #LY2N0 Rabbit Serum Monkey Serum Monkey Serum Rabbit Liver N/R 12/01/1999 Frozen -20"C N/R Rabbit Liver Rabbit Liver Coming Hazleton 01/01/2010 Frozen -20C F00005 Rabbit Liver Rabbit Liver Coming Hazleton 01/01/2010 Frozen -20C F00009 Rabbit Liver Monkey Serum N/R 01/01/2010 Frozen -20"C N/R Monkey Serum M onkey Liver Siena Biomedical 01/01/2010 Frozen -50*C N/R Monkey Liver 3M Environmental Laboratory 3M Environmental Laboratory Page A-1 Page 13 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Table A-2. Lactose Dose Verification (PFOS) for Study 6329-223-- 8/21/99 EXPECTED CONC. (ng/m L) MEASURED CONC. (ng/m L) %REC. FOR ng/m L Ex p e c t e d Calc. Co nc. (MS/S) M easured Calc. Conc. (pg/g) A verage S td D eviatio n %Rec. for pg/g 1:39 Dose (25000 p p m PFOS) Top Middle Bottom 580 500 500 479 650 422 j 83% I 130% I 84% 1:499 Dose (2000 p p m PFOS) Top Middle Bottom 410 404 400 305 287 272 74% i 71% I 68% 25000 20647 25000 32537 25000 I 21108 24764 6736 27% average / std. deviation= 83% 130% 84% 99% 27% 2000 2000 2000 1490 1425 1361 1425 64.5 5% average / std. deviation= 74% 71% 68% 71% 3% Actual MS Concentration-Actual background concentration, divided by expected, times 100 (Spiked too low which accounts for the wide differences in recovery) Table A-3. Lactose Dose Verification (PFOS-- Matrix Spikes) for Study 6329-223-- 8/21/99 Expected Conc. (ng/mL) Actual Conc. (ng/mL) %Rec. FOR ng/mL Calculated Conc. (pg/g) Expected Co n c . (pg/g) A ctual Calc. Co nc. (pg/g) Average STD D eviatxdn 1:39 D o s e (25000 p p m PFOS) MS Top Middle Bottom 604 524 524 507 84% 485 92% 438 83% 21858 24252 21889 10.8 12.5 12.5 1 :499 D o s e (2000 p p m PFOS) MS Top 606 475 78% 2320 j 9.77 Middle 600 447 75% 2217 i 9.92 Bottom 596 440 74% 2202 ! 10.0 * This value is an outlier and was not used in any calculations 12.1 -82.8 7.82 ! 22666 1374 6% 8.30 7.93 8.41 2246 64.5 3% %Rec. FOR pg/g A verage 112% -664* 63% I 87% 85% 80% 84% 83% 3M Environmental Laboratory 3M Environmental Laboratory Page A-2 Page 14 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Attachment B Protocol and D eviation S ummary Report No. FACT TOX-030 la b o r a to r y R e q u e s t N um be r-U 2 27 9 Report No. FACT TOX-030 Laboratory Request Number-U2279 Tabla B-1. Deviation Summary for FACT TOX-030 D e v ia t io n MtBE was used as an extraction solvent instead of ethyl acetate. Pipette was used instead of Oxford dispenser. Curves plotted by linear regression weighted 1/x rather than by linear regression as specified in the protocol. A second extracted matrix curve was not used to bracket samples. Recorded extraction method FACT-M -1.0 rather than FACT-M-1.1. Followed extraction method ETS-8-6.0 rather than FACT-M-1.1. Followed analytical method ETS-8-7.0 rather than FACT-M-2.1. Followed analytical method ETS-8-5.0 rather than FACT-M -4.1. Samples extracted using 0.5 mL rather than 1.0 mL due to insufficient sample. Followed extraction method ETS-8-4.0 rather than FACT-M-3.1. Matrix spikes were not spiked with standard (Used as blanks). Continuing calibration standards were not spiked with standard due to analyst error. Samples extracted using <0.5 mL due to insufficient initial sample volume. Dates of Occurrence 2/5/99, 2/9/99, 5/18/99.6/11/99 10/14/99 2/13/99,3/5(99, 3/1299, 3/1999, 3/2099, 3/2199,3/2399, 3 9 499, 3/25/99, 4 /7 9 9 ,4 /1 1 9 9 , 4/1299, 4/1799, 5/1999, 5/2299, 6 9 9 9 , 6/1499 3/5/99, 3 9 9 9 . 3/1599, 3/16/99, 5/19/99, 5/22/99. 10/2699, 1/21/00, 3/24/00, 4/27/00 6/1199 10/14/99. 10/25/99,1/19/00, 3/22/00 7/2999, 10/2099, 10/2299, 10/26/99, 10/2799, 1/28/00, 3/24/00, 3/2890 3 /0 5 9 9 ,3 9 8 9 9 10/2599 3/0299, 3/0399 11/3/99 11/3/99 2/599, 2 9 9 9 , 3 9 9 9 , 3 9 9 9 , 3/1099, 3/1299, 3/1599, 3/1699, 4/699,4/899, 8/2599, 11/399, 4/21/00 Impact on Study No negative impact on the study-- MtBE improved the absolute recoveries and shortened extraction time. No negative impact on the study. No negative impact on the study-- 1/x weighted curves improved the precision and accuracy of analysis. No negative impact on the study-- The accuracy of calibration checks analyzed every five to ten samples was monitored to ensure continued accuracy of the analysis. The QC provided by the calibration checks is sufficient and the data quality will not be adversely affected. No negative impact on the study--N ew method was followed, even though old method was recorded. No negative impact on the study-- New validated method provides improvements in precision and extraction time. No negative impact on the study-- New validated method provides improvements in precision, accuracy and analysis time. No negative impact on the study-- New validated method provides improvements in precision, accuracy and analysis time. No negative impact on the study-- Studies indicate that data quality is not jeopardized using 0.5 mL of sera. No negative impact on the study-- New validated method provides improvements in precision and extraction time. Adequate CX3 was prepared with the sample s e t unspiked samples pose no negative impact on the study. Mid-level curve standards were substituted as Q C for the non spiked calibration check standards; the unspiked calibration standards pose no negative impact to the study. Studies indicate that data accuracy and precision may be affected when sera samples less than 0.5 mL were extracted. Data reported from extraction of samples less than 0.5 mL is noted in the data tables. 3M Environmental Laboratory 3M Environmental Laboratory Page B-1 Page 15 3m M e d ic a l 3DMeEpnvairrotnmmeenntatl TSechtnuodlogyy: and Services T - 6 2PPOO9 5Bo.x7 233331 Report No. FACT TOX-030 1l;aboratory Request Number-U2279 St. Paul. M N 55133-333311 612 778 6442 Protocol #FACT-TOX-030 3M Study Title Perfluoroo2c6t-aWneeeSkCuCylfnoaonpmiscuoAllegcuTidsoxMPiocoitntayksseSiyutsumdySawltith(T-6295) in PROTOCOL Author Lisa Clemen Date: January 25, 1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT-TOX-030 3M Environmental Laboratory 3M Environmental Laboratory Page 1 of 9 Page 16 3m Medi ii ccaall Deeppaarrtt mmeenntt Stt uu ddyy:: TT-- 66229955 .. 77 R e p o rt N o. FACT TOX-030 la b o r a to r y R e q u e s t N um be r-U 2 27 9 Protocol #FACT-TOX-030 Study Identification Perfluoroo2c6t-aWneeeSkuClfoanpiscuAlecTidoxPioctitayssSiutumdySawltith(T-6295) in Cynomolgus Monkeys Test Material Perfluorooctane sulfonic acid potassium salt (T-6295) Sponsor 3M Toxicology Services - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000 Sponsor Representative Andrew Seacat, Ph.D. 3M Toxicology Services Telephone: 612-575-3161 Facsimile: 612-733-1773 Study Director Kristen Hansen, Ph.D. 3M Environmental Technology and Safety Services Building 2-3E-09 651-778-6018 Study Location(s) In vivo Testing Facility Analytical Testing Laboratory Covance Laboratories, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 3M Environmental Laboratory Building 2-3E-09 935 Bush Avenue St. Paul, MN 55106 Proposed Study Timetable Study Initiation Date Study Completion Date January 25,1999 January 25, 2000 3M Environmental Laboratory 3M Environmental Laboratory Page 2 of 9 Page 17 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol #FACT-TOX-030 1. Study Tcywneonmtyo-lsgiuxswmeoeknkceaypss.ule toxicity study with potassium perfluorooctane sulfonic acid (T-6295) in 2. Purpose This analytical study is designed to determine levels of potassium perfluorooctanesulfonate (PFOS) in the liver and serum of cynomolgus monkeys. Additional tissues or fluids may be analyzed. The in-life portion of this study was conducted at Covance Laboratories, study #6329223. 3. Regulatory Compliance This study will be conducted in accordance with the United States Environmental Protection Agency Good Laboratory Practices Standards, 40 CFR 792, with the exception that analysis of the test material mixture for concentration, solubility, homogeneity, and stability will not be conducted, and is the responsibility of the Sponsor. 4. Quality Assurance The 3M Environmental Laboratory Quality Assurance Unit will review the protocol and audit study conduct, data, and Final report to determine compliance with Good Laboratory Practice Standards and with 3M Environmental Laboratory Standard Operating Procedures. 5. Test Material 5.1 Refer to Covance Laboratory protocol for study #6329-223. 6. Control Matrices 6.1 Identification Monkey liver and serum and/or rabbit liver and serum, traceability numbers will be recorded in the raw data and included in the final report 6.2 Source Covance Research and/or Sigma Chemical 6.3 Physical Description Monkey liver and serum and/or rabbit liver and serum 6.4 Purity and Stability Not applicable 6.5 Storage Conditions Frozen at -20 C 10 C or -55 C 10 C 6.6 Reserve Matrix A portion of the control matrix will be retained in the archives for as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable). 6.7 Disposition Matrices will be retained per GLP regulation. Certain matrices (feces, urine, and blood) may be disposed after QAU verification. 3M Environmental Laboratory 3M Environmental Laboratory Page 3 of 9 Page 18 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol #FACT-TOX-030 6.8 Safety Precautions Refer to MSDS for chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis. 7. Reference Material 7.1 Identification Potassium perfluorooctanesulfonate (PFOS), lot #s 171,215, or 217 (equivalent lots) 7.2 Source 3M Specialty Chemicals 7.3 Physical Description White powder 7.4 Purity and Stability Responsibility of the Sponsor 7.5 Storage Conditions Room temperature 7.6 Reserve Material A reserve sample from each batch of PFOS used in this study will be retained as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable). 7 .7 Disposition Unused reference material will be retained for use by the 3M Environmental Laboratory and will be discarded when the quality of preparation no longer affords evaluation. 7.8 lSaaboferattyorPyraetctiareu, taiondnsfolRloewferadtoeqMuaStDe S for chemicals precautions for used. Wear appropriate handling biological materials and preparing samples for analysis. 8. Test S ystem Cynomolgus monkeys were used as the test system, and were maintained and dosed as described in Covance protocol #6329-223. Group 1 control animals did not receive the test substance. Groups 2, 3, and 4 received the test substance daily for 26 weeks, at concentrations of 0.02, 0.5, and 2.0 mg/kg/day, respectively. Refer to Covance protocol #6329-223 for tabular presentation of data. Two animals each from Groups 1, 3, and 4 were designated as recovery animals and were allowed at least a 13 week, which may be extended, recovery period after cessation of treatment. 9. Specimen and Sample Receipt The 3M Environmental Laboratory will receive homogeneity samples for dose analysis and specimens of the following body tissues and fluids from the indicated points in the study. All specimens will be packed on dry ice for shipping. 3M Environmental Laboratory 3M Environmental Laboratory Page 4 of 9 Page 19 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol nFACT-TOX-030 Body tissue/fluid Collected Expected # of specim ens Serum - all animals 7 days prior to treatment 616 from main 7 days post treatment study every two weeks during treatment 24 additional from and recovery recovery Urine and feces - recovery animals Day zero of recovery 24 urine 6, 30, and 90 (with a potential 180) days of recovery 24 feces Liver - all animals After termination of the study 44 Total number of test animals: 32 Total number of control animals: 12 Specimens sent to 3M Environmental Laboratories will be received and tracked according to applicable Standard Operating Procedures. 10. Preparatory Methods 10.1 FACT-M-l.O, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Fluorochemical Surfactant from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry 10.2 FACT-M-3.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Other Fluid for Analysis Using HPLCElectrospray/Mass Spectrometry 10.3 If preparatory methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 11. Analytical Methods 11.1 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry 11.2 FACT-M-4.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum or Other Fluid Extracts Using HPLC-Electrospray/Mass Spectrometry 11.3 If analytical methods other than those listed above are used, an amendment to this protocol will be written. Any deviations from these methods will be documented and included with the study data. 3M Environmental Laboratory 3M Environmental Laboratory Page 5 of 9 Page 20 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 Protocol IfFACT-TOX-030 12. D a t a Q u a l it y O b j e c t iv e s The number of spikes/duplicates, use of surrogates, and information on other data quality indicators are included in the analytical methods. In addition, the following criteria will be met: 12.1 Linearity r2 > 0.98 12.2 Limits of detection / quantitation 12.2.1 Method Detection Limit (MDL) for PFOS a) Serum: I2ppb b) Liver: 15 ppb 12.2.2 Practical Quantitation Limit (PQL) - Equal to the lowest standard in the calibration curve 12.3 Duplicate acceptable precision < 30% for the method 12.4 Spike acceptable recoveries 10% - 130% 12.5 Use of confirmatory methods Indeterminate samples will be re-analyzed using a confirmatory method. If a confirmatory method is used, an amendment to this protocol will be written. 12.6 Demonstration of specificity Chromatographic retention time, mass spectral daughter ion characterization. 13. Sub-Contracted Anal ysis 13.1 All analyses as detailed in this protocol will be performed at 3M Environmental Laboratories, Building 2-3E-09, 935 Bush Avenue, St. Paul, MN 55106. 13.2 An amendment to this protocol will be written if analyses are performed at laboratories other than the 3M Environmental Laboratory. 14. Statistical Analysis Averages and standard deviations will be calculated. The statistical methods that will be used are described below: 14.1 Data transformations and analysis Data will be reported as the concentration (weight/weight or weight/vol) of PFOS or metabolite per tissue or fluid. 14.2 cSotnacteinsttricataiol nasnoavleyrstiismeS,taantidstsictasnudsaerdd may include regression analysis of deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 3M Environmental Laboratory 3M Environmental Laboratory Page 6 of 9 Page 21 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol #FACT-TOX-030 15. Report A report of the results of the study will be prepared by 3M Environmental Laboratory. The report will include, but not be limited to, the following, when applicable: 15.1 Name and address of the facility performing the study 15.2 Dates upon which the study was initiated and completed 15.3 A statement of compliance by the Study Director addressing any exceptions to Good Laboratory Practice Standards 15.4 Objectives and procedures as stated in the approved protocol, including any changes in the original protocol 15.5 The test substance identification by name, chemical abstracts number or code number, strength, purity, and composition or other appropriate characteristics, if provided by the Sponsor 15.6 Stability and the solubility of the test substances under the conditions of administration, if provided by the Sponsor 15.7 A description of the methods used to conduct the test(s) 15.8 A description of the test system 15.9 oAf description the data of any circumstances that may have affected the quality or the integrity 15.10 The name of the Study Director and the names of other scientists, professionals, and supervisory personnel involved in the study 15.11 A description of the transformations, calculations, or operations performed on the data, a summary and analysis of the analytical chemistry data, and a statement of the conclusions drawn from the analyses 15.12 Statistical methods used to evaluate the data, if applicable 15.13 The signed and dated reports of each of the individual scientists or other professionals involved in the study, if applicable 15.14 The location where raw data and the final report are to be stored 15.15 A statement prepared by the Quality Assurance Unit listing the dates that study inspections and audits were made, and the dates of any findings reported to the Study Director and Management If it is necessary to make corrections or additions to a final report after it has been accepted, the changes will be made in the form of an amendment issued by the Study Director. The amendment will clearly identify the part of the final report that is being amended, the reasons for the amendment, and will be signed by the Study Director. 3M Environmental Laboratory 3M Environmental Laboratory Page 7 of 9 Page 22 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol #FACT-TOX-030 16. Location of Ra w Data, Records, and Final Report Original data, or copies thereof, will be available at 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including those items listed below, will be retained in the archives of 3M Environmental Laboratory for at least a period of time as specified by regulation, and as established by 3M Environmental Laboratory Standard Operating Procedures. 16.1 The following raw data and records will be retained in the study folder in the study/project archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.1.1 Approved protocol and amendments 16.1.2 Study correspondence 16.1.3 Shipping records 1 6 .1 .4 Raw data 16.1.5 Approved final report (original signed copy) 16.1.6 Electronic copies of data 16.2 The following supporting records will be retained separately from the study folder in the archives according to 3M Environmental Laboratory Standard Operating Procedures: 16.2.1 Training records 16.2.2 Calibration records 16.2.3 Instrument maintenance logs 16.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 17. Specimen Retention Specimens will be maintained in the laboratory specimen archives for a period of time as specified by regulation or as long as the quality of the preparation affords evaluation, but not longer than ten years following the effective date of the final test rule (if applicable), and as established by 3M Environmental Laboratory Standard Operating Procedures. 18. Protocol Amendments and deviations Planned changes to the protocol will be in the form of written amendments signed by the Study Director and the Sponsor's Representative. Amendments will be considered as part of the protocol and will be attached to the final protocol. All changes to the protocol will be indicated in the final report. Any other changes will be in the form of written deviations, signed by the Study Director and filed with the raw data. 3M Environmental Laboratory 3M Environms:ntal Laboratory Page 8 of 9 Page 23 3ra Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol #FACT-TOX-030 19. A ttachments 19.1 Attachment A Preparatory and analytical methods 20. Signatures T' ff- Andrew Seacat, Ph.D., Sponsor Representative Kristen Hansen, Ph.D., 3M Environmental Laboratory Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 9 of 9 Page 24 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 GLP Study Protocol Amendment Study Number: pAcT- Tox- ojo CoWrxL ( ,W - 2 2 1 Study Title: 2L Link Ccp.uL Tox r-L U J LI r \ .i ' S t u c b j b O i f h P FO S. IA C v y ' D I * s i g i l i I M n k ^ I Study Director: kru UQnJ6h Amendment Date: Oj'iji') Amendment Number: This amendment modifies the following portion of the protocol: SeoliDo 10 frtp w aW ^ Methods And -Sec-linn II 'A'laluylicxl NVeThods "TA-e.se. l i i l FACT- t n - l . l Ckn. FACT- fi-A I &5 ike scrum e .x lf& C -'h o n A n t i k c J m tiU cls. 7 h t hr<, W n u p a lt d ^ ETS- 8-H.O And. ETS - 8- 5. b. V\eJC u p d a lti -mel-hodf (ill L . ust or re-mAmivi^ Serum x^Tfic-Lons And Analyse, Approved by: tl [3 T > Date 3M Environmental Laboratory Page 25 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 26-Week Capsule Toxicity SStutduydwyitThitPleerfluorooctane Sulfonic Acid Potassium Salt (PFOS, T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 2 AmAepnrdilm29e,n1t9D99ate: Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-030 LIRN U2279 3M Environmental Laboratory 3M Environmental Laboratory Page 26 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 Protocol FAC T-TO X-030 Amendment 2 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL READS: In amendment 1, section 10 Preparatory Methods and Section 11 Analytical Methods were updated to ETS-8-4.0 and ETS-8-5.0 as the revised serum extraction and analytical methods. AMEND TO READ: These methods were revised on 4 /^ /9 9 to ETS-8-4.1 and ETS-8-5.1 which will be used for all future analyses. wREeiAgShOtiNng: The methods were for initial curves. revised for clarification and to include linear regression, 1/x Amendment Approval Andrew Seacat Ph.D., Sponsor Representative ________________________ ----------------------------------------------- Kris J. Hansen Ph.D., Study Director 3M Environmental Laboratory 3M Environmental Laboratory Date Page 27 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Study Title 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (PFOS, T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 3 Amendment Date: June 03, 1999 3M EnvironPmeernftoarlmTeinchgnLolaobgoyr&atSoarfyety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-030 LIRN U2279 3M Environmental Laboratory 3M Environmental Laboratory Page 28 3m Medical Department Study: T-6295.7 R e p o rt N o. FACT TOX-030 la b o r a to r y R e q u e s t N um be r-U 2 27 9 Protocol FA C T-TO X-030 Amendment 3 This amendment modifies the following portion(s) of the protocol: 1. PROTOCOL r e a d s : Section 10 Preparatory Methods and Section 11 Analytical Methods list FACT-M-1.0 and FACT-M-2.0 as the liver extraction and analytical methods. AMEND to read: These methods were revised on 06/03/99 to FACT-M-1.1 and FACT-M- 2.1 which will be used for all future analyses. REASON: The methods were revised for clarification and to expand on the list o f target analytes. Amendment Approval Andrew Seacat Ph.D., Sponsor Representative 7 Date Kris J. Hansen Ph.D., Study Director 3M Environmental Laboratory 3M Environmental Laboratory Date Page 29 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Study Title 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 4 Amendment Date: April 25, 2000 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification FACT Tox-030 ET&SS LRN-U2279 3M Environmental Laboratory 3M Environmental Laboratory Page 30 3m Medical Department Study: T-6295.7 R e p o rt N o. FACT TOX-030 la b o r a to r y R e q u e s t N um be r-U 2 27 9 Protocol LRN-U2279 Amendment Number 4 This amendment modifies the following portion(s) of the protocol: 1. Protocol reads: The sponsor for the present study was identified as Andrew Seacat, Ph.D. Amend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D. as of the date of signature approval of this protocol amendment. 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. 2. Protocol reads: On page 2 of the protocol, Kris Hansen is identified as the study director for the analytical phase of the study. Peter Thomford is also identified as a study director, but for the in-life phase of the study (see Covance Laboratories Protocol 6329-223). Amend to read: On page 2 of the protocol, Andrew Seacat will be identified as the study director, Kris Hansen will perform the duties of the principal analytical investigator, and Peter Thomford will be identified in the final report as the principal in-life investigator as of the date of signature approval of this protocol amendment. Reason: The original study design identified two study directors; one for the in-life phase of the study and one for the analytical phase of the study. The role of study director has been reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements. 3. Protocol reads: 10. Preparatory Methods: 10.1 FACT-M-1.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLCElectrospray/Mass Spectrometry" Amend to read: 10. Preparatory Methods: 10.1 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLCElectrospray/Mass Spectrometry" Reason: The method was revised to include extractions of other tissue types and the use of methyl-fe/t-butyl ether (MtBE) instead of ethyl acetate in the extraction process. 4. Protocol reads: 11. Analytical Methods: 3M Environmental Laboratory 3M Environmental Laboratory Page 31 3m Medical Department Study: T-6295.7 R e p o rt N o. FACT TOX-030 la b o r a to r y R e q u e s t N um be r-U 2 27 9 Protocol LRN-U2279 Amendment Number 4 11.1 FACT-M-2.1, "Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry" A m end to r ead: 11. Analytical Methods: 11.1 ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLCElectrospray/Mass Spectrometry" Reason: The method was revised to include curves plotted by linear regression weighted 1/x and a reduced cycle time from 13.5 minutes to 9.0 minutes. 5. P r o t o c o l r e a d s : 8. Test System Refer to the Covance protocol #6329-223 for tabular presentation of data. Two animals each from Groups 1, 3, and 4 were designated as recovery animals and were allowed at least a 13 week, which may be extended, recovery period after cessation of treatment. A m en d to r e a d : 8. Test System A tabular presentation of the test system data will be included in the final report for this project (FACT Tox-030). Four animals each (two/gender) from Groups 1, 3, and 4 were designated as recovery animals and were observed for a recovery period after cessation of treatment until study cut-off on March 7, 2000 (Week 80). The observation and analysis of the recovery group III animals beyond the cut-off date of this study will be reported in a new long-term recovery study. Reason: Additional animals were assigned to the recovery group following approval of the protocol for FACT Tox-030. A new study will report the long-term recovery of the remaining animals from recovery group III (mid-dose group). 6. P r o to c o l r e a d s : 9. Specimen and Sample Receipt: [Sample Receipt Table] Expected # of Specimens: Serum-all animals: 616 from main study, 24 additional from recovery Urine and feces-recovery animals: 24 urine, 24 feces Liver-all animals: 44 Collected: Urine and feces-recovery animals: Day 0, 6, 30, 90 (potential 180) Total number of test animals: 32 3M Environmental Laboratory 3M Environmental Laboratory Page 32 o CM 3m Medical Department Study: T-6295.7 Report No. FACT TOX- laboratory Request Number-U2 Protocol LRN-U2279 Amendment Number 4 A m end to read: 9. Specimen and Sample Receipt: [Sample Receipt Table] # of Specimens: Serum-all animals: 516 from main study, 280 additional specimens from recovery Liver-all animals: 42 (eight specimens via biopsy from recovery group) Total number o f test animals: 32 Total number o f animals: 48 (12 animals in control group) Note: 4 animals were not assigned to a study group. Day 0 serum specimens were collected. Specimens of body tissues and fluids other than serum and liver specimens will be collected and received from Covance Laboratories (6329-223 Study T-6295.7). Reason: Additional animals were assigned to the recovery group following approval of the protocol for FACT Tox-030. The recovery period has been extended for the recovery group. Specimen collection figures shown are through the end of the study 3/07/00. Amendment Approval John L. Butenhoff, Ph.D., Sponsor Representative Date Andrew Seacat, Ph.D., Incoming Study Director _K h ~ ^2------ Kristen J. Hansen, Ph.D., Outgoing Study Director Dale L. Bacon, Laboratory Manager Date 7JJW rA Date 3M Environmental Laboratory 3M Environmental Laboratory Page 33 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 26-Week Capsule Toxicity Study with PerSflutuordoyocTtaitnlee Sulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 5 AmAeugnudsmt 1e4n, t2D00a0te: 3M EnvironPmeernftoarlmTienchgnLoalobgoyr&atSoarfyety Services' " 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 LaborEaTto&rSySPFroAjCeTct-TIdOeXn-t0i3fi0cation Covance #6329-223 LIMS #U2279 3M Environmental Laboratory 3M Environmental Laboratory Page 34 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol TOX-030 Amendment 5 This am endm ent modifies the following portion(s) of the protocol: 1. PROTOCOL R e a d s : 2. P u r p o s e : This analytical study is designed to determine levels of potassium perfluorooctanesulfonate (PFOS) in the liver and serum of cynomolgus monkeys. Additional tissues or fluids may be analyzed. AMEND TO R e a d : Add that select feces samples will be analyzed for PFOS at Centre. REASON: Feces samples were added after the original protocol was written. 2. PEnrvoitrooncmoelntraleLaadbso:raStotruydy Locations(PAGE 2): Analytical Testing Laboratory:. 3M A m e n d TO r e a d : 3M Environmental Laboratory and Centre Analytical Laboratories Inc. (Centre), 3048 Research Drive, State College, PA 16801 REASON: Analyses o f feces are assigned to Centre, in addition to the analyses o f other tissues at the 3M Environmental Laboratory. 3. P r o t o c o l r e a d s : S e c t io n s 10. P r e p a r a t o r y M e t h o d s , 11. A n a l y t ic a l M e t h o d s a n d 12. D a t a Q u a l it y O b j e c t iv e s AF lmu oe rnodc hteom irc aela dR:esiAdudeds to in these sections Monkey/Rat the most Feces by current version of "Determination of LC/MS/MS," Centre method number 00M-023-003, with ar. LOQ in feces o f 10 ng/g. The method will incorporate an initial homogenization step immediately after adding the extraction solvent, using a micro- homogenizer, to allow for complete dispersion of the specimen in the solvent. REASON: analytical T o specify the validated m ethod to be used lim its. NOTE: LC/MS/MS is an ab b reviation for for feces analyses along w ith its "liquid chrom atography/m ass spectrom etry/m ass spectrom etry." 4 . Protocol reads: Section 10. Preparatory Methods and 11. 3M Analytical Methods A m e n d TO READ: Change to specify that the most current version of the methods listed should be used. mR EeAthSoOdNs :shToouldspbeeciufysedthdaut ritnhge most appropriate version the course o f the study. of the preparatory and analytical 3M Environmental Laboratory 3M Environmental Laboratory Page 35 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol TOX-030 Amendment 5 5. PisRtOheTOPCriOnLcipraelaAdnsa:lTythiceaalmInevnedsetdigaptroortofocorlth(Ae menetnirdemsteundty#.4) states that Kris J. Hansen, Ph.D. ACMenEtNreDfoTrOfreceeasda:nAaldydseEs.naksha Wickremesinhe, Ph.D. as the Principal In-Wcjn^ves^tigator at (4)/7.V/^^. , REASON: To specify the PAI at Centre for feces analysis. 6. Protocol reads: Section 16. Location of Raw Data, Records and Final Reports AEnmveirnodnmTOenrtaelaLda:bAordadtotrhieast, Centre w together ill w forward all ith copies o original study-specific raw data to f appropriate facility-specific raw 3M data applicable to this study. Centre will maintain a copy of the applicable study-specific raw data, protocol and analytical report in the Centre archives, as well as all original facility records. REASON: T o sp e c ify th e arch ival req u irem en t for th e p o rtio n o f th e data d e v e lo p e d by C en tre. 7. Protocol reads: Section 17. Specimen retention Adimreecntodr, TaOll fReEcAesDs:peAcidmdentshaotf after the analytical report this study will be returned on feces is signed by the study to 3M Environmental Laboratory. These specimens may then be discarded by written direction of the study director. Specimens of serum and urine may also be discarded by written direction of the study director after the analytical report for the 3M Environmental Laboratory analyses is signed by the study director. TRoeaspseocnif:y the handling of all biological fluids, and to define when the quality assurance verification is considered complete. 3M Environmental Laboratory 3M Environmental Laboratory Page 36 3m M<& 4 a DeP ^ ent 1 7410 884 9122 Report No. F A C ^ -0^gg gWM^SlS^/Ma M O 101S-':3331PM ECWENTLRAEBm2f-l3lEYT-0I9CAL* LA B14 231 laboratory i Nu^er-U2279 MO. 338 (7B2 Amendment Approval 5Protocol TQX-030 Amendment Joh_n_L. ButtnhoSJ PbU./S-p---o---M---,c--r' ve I ; .--at II*' 3MEnwonmantaf Laboratory 3M Environmental Laboratory !/ Page 37 *1- 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 26-Week Capsule Toxicity Study with PerSflutuodroyocTtaitnlee Sulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 6 ASmepetenmdbmeren1 1t ,D2 0a0te0 : 3M EnvironPmeernftoarlmTeinchgnLoalobgoyr&atSoarfyety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 LaborEaTto&rySSPFroAjCeTctTIdOeXn0ti3f0ication Covance #6329-223 LIMS #U2279 3M Environmental Laboratory 3M Environmental Laboratory Page 38 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol TOX 030 Amendment 6 This am endm ent modifies the following portion(s) of the protocol: l. Protocol read c8o.nceTnEtrSaTtiSonyss toef s m0 .::0G2,ro0u.5p,sa2n,d32,.a0nmd g4/kregc/deiavye,drethspeetcetsitvesulyb.stance daily for 26 weeks, at AGrmoeunpds 2t,o3r, eanadd :4 received the test substance daily for 26 weeks, at concentrations o f 0.03, 0.15, and 0.75 mg/kg/day. R T eesat ssuobns:tance doses were changed after protocol was written. The Covance protocol reflects the actual doses of test substance that were given. P rotocol reads: The amended protocol (Amendment Number 4, section 2.) states: On page 2 of the protocol, Andrew Seacat will be identified as the study director, Kris Hansen will perform the duties of the principal analytical investigator, and Peter Thomford will be identified in the final report as the principal in-life investigator as of the date of signature approval of this protocol amendment. APemteernTdhtoomrfoeraddw: ill remain as the Covance Study Director for the purpose o f issuing the Covance final report for the in-life phase of the study. SRteuadsyodnir:ectorship was not relinquished by Covance for the in-life phase of the study, since the animal experimental phase was completed before Amendment Number 4 was issued. Amendment Approval John L. Butenhoff, PH.D., Sponsor Representative c Date Andrew Seacat, Ph.D., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory Page 39 3m M e d e ^ ^ e B e p a r g g r i g j i t S g ^ Y A B 2 - ^ 0 # a 71, 410 804 RePrt No. FACT TOX-030 lanoratory Request Numb-^22 7SC)06 Study Title ProtoAcmoleTnOdmXe-0n3t 07 26-week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys Protocol Amendment No. 7 Amendment Date: September 14,2000 Performing Laboratory 3M Environmental Technology and Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-030 Covance #6329-223 LIMS #U2279 3M Environmental Laboratory 3m, Med&^0parl0ai33t -7 ! 410 8B4 gi22 Report No. FACT TOX-03 0 lacoratory Request Numbed,02$79 egg Amendment Approval ProtoAcnowl TnOdmXe-0n3t 07 John L. Butenhoff, Fh.D., Sponsor's Representative Andrew Seacat, Ph.D., Study Director Date (V /Daa>te 'I /* >jns' :;' i 3M Environmental Laboratory Sflf 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Attachment C Extraction and A nalytical M ethods Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 3M Environmental Laboratory 3M Environmental Laboratory Page C-1 Page 43 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03G laboratory Request Number-U227S 3M Environmental Laboratory M ethod E x tr a c tio n of P o tassium Perfluo ro o ctanesulfo nate or o th er F lu o r o c h em ic a l C o m po unds fro m L iver fo r A nalysis using HPLC- E lec tr o spra y/M ass Spec tr o m etry M ethod Num ber: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: Adoption Date: Revision Date: Lift ~Vvh ci Date Group Leader Technical Reviewer Date C/hVi Date 1.0 S c o p e a n d A p p l ic a t io n _____________________________________________________________________ 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 A pplicable C om pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 vMaalitdraitcieosn: rRepaobrbt.it, rat, bovine, and monkey livers or other tissues as designated in the Word 6.0/95 ExtractionEoTfSP-F8O-6S.0from Liver Page 1 of 14 3M Environmental Laboratory Page 44 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 S u m m a r y o f M e t h o d __________________________________________________________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver, or other tissues, using an ion painng reagent and methyl-/eri-butyl ether (MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard. An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-7.0 or other appropriate methods. 3.0 D e f in it io n s _____________________________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C3F17S 0 3 3.2 PFOSA: perfluorooctane sulfonylamide CgFpSOjNH-, 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8Fl7SO;N(CH;CH3)CH:CO, 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethy! alcohol C8F]7S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17SO:N(CH:CH3)H 3.6 M556: C8F17S 0 2N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W a r n in g s an d C a u t io n s______________________________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Uhasnedulinnigvearnsaiml palretcisasuuteio, nwsh, iecshpemcaiayllcyonlatbaoinraptoarthyocgoeantss., goggles, and gloves when 5.0 I n t e r f e r e n c e s _______________________________________________________________________________ 5.1 There are no interferences known at this time. 6.0 E q u ip m e n t ____________________________________________________________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral lOOOorlEC 6.1.4 Shaker, Eberbach or VWR ExtractionEoTfSP-F8O-6S.0from Liver Page 2 of 14 3M Environmental Laboratory Page 45 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance (sensitivity to 0.100 g) 7.0 S u p p l ie s a n d M a t e r ia l s ______________________________________________________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Volumetric flasks, glass, type A 7.6 I-CHEM vials, 40 mL glass 7.7 Plastic sampule vials, Wheaton, 6 mL (or appropriate size) 7.8 Centrifuge tubes^ polypropylene, 15 mL 7.9 Labels 7.10 Oxford Dispensor - 3.0 to 10.0 ml 7.11 Syringes, capable of measuring 5 pL to 50 pL 7.12 Graduated pipettes 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer 7.16 Crimp cap autovials and caps 7.17 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- QviTMals.water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate 8.0 R e a g e n t s a n d St a n d a r d s __________________________________________________ _ _ _ _______ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NaXOj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8 . 6 Methyl-fer/-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8 . 8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 ExtractionEoTfSP-F8O-6S.0from Liver Pane 3 of 14 3M Environmental Laboratory Page 46 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.10.5 PFOSEA (3\1 Specialty Chemical Division), molecular weight = 527 8.10.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.10.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (l-H .l-H , 2-H, 2-H CgF13S 0 3H) molecular weight = 428 8.10.8 Other fluorochemicals, as appropriate 8.11 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.11.1 d11i00s0sN0olsvmoeLddi.ubmeSatkoheryredcrinoonxatida1ienL(inNNgaaO5lg0He0n):meWLboeMtitgliehl.lia-QppTMroxwiamteart,elmy i2x0u0ngtilNaalOl sHo.liPdosuarreinto a 8.11.2 110NNsoNdaiOumH hsyodlurtoioxindein(tNoaaO1H0)0: mDLilvuotelu1m0eNtriNc afOlaHsk 1a:n1d0.diMluteeastuorveo1lu0mmeLuosifng Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g pooHff NT1Ba0OAuHsin,intaogddaaps1plorLowvxloiymlubametceealtyuris4ce4cttohonet5pa4HinmicnLhgaon5f0g0e1s0maNLbrNMuapOitlllHyi)-.Q(WD'Mihlwuilateeteatrdo.dAvinodgljuutmhsteetlowasitthmL Milli-QTM water. Store in a 1 L Nalgene bottle. 8.11.3.1 TBA requires a check prior to each use to ensure pH = 10. needed using 1 N NaOH solution. Adjust as 8.11.4 0ap.2p5roMximsoadtieulmy 2c6a.r5bgonoaftseo/sdoiduimumcabribcoanrbaotena(Nteab,Cuf0fe3r) a(nNda2X1.003/gNoafHsCod0i3u)m: Weigh bQiTMcarwboatneart.e S(NtoarHe CinOa,)1inLtoNaalg1 eLnevobloutmtlee.tric flask and bring to volume with Milli- 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard s1o.1lu0tipopnmcoEnttraOinSiEng-O1H.0.0) ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 B(prgin/mgLto).volume with methanol for a stock standard of approximately 1000 ppm 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. ExtractionEoTfSP-F8O-6S.0from Liver 3M Environmental Laboratory Page 4 of 14 Page 47 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 8.12.6 Dapiplurtoex.th5e.0stpopcmk .solution with methanol for a working standard 2 solution of 8.12.7 aDpiplurtoex.th0e.5s0topcpkmso. lution with methanol for a working standard 3 solution of 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg of surrogate standard l-H.l-H, 2-H, 2-H, C3F,3S0 3H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Brpipnmg .to volume with methanol for a surrogate stock of approximately 1000-1200 8.13.3 Prepare a surrogate working standard. Transfer approximately 1.0 ml of surrogate stock to workijig ast1a0ndmarl dvoolfum10e-t2r0icpfplams.k and bring to volume with Record the actual volume methanol for transferred. a 9.0 S a m p l e H a n d l in g ____________________________________________________________ _ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Q u a l it y C o n t r o l ______________________________________________________________ 10.1 M atrix blanks and method blanks 10.1.1 An aliquot of 1.0 mL methanol is used as a solvent blank. 10.1.2 aEsxtmraectht otwdobl1a.n0kms.L aliquots of Milli-QTM water following this procedure and use 10.1.3 Easxtmraacttritxwbola1n.k0sm. LReafleiqrutoots11o.f1.l6iv.er homogenate following this procedure and use 10.2 M atrix spikes 10.2.1 Pthreepaacrceuraancdyaonfatlhyezeexmtraatrcitxiosnp.ike and matrix spike duplicate samples to determine 10.2.2 Prerceepiavreedewacithhsepaikche usasminpglea sseatm. ple chosen by the analyst, usually a control liver 10.2.3 cAEaxdlipdbeirtcaitotenidoanlcocsnupcrikevnees.trmataioynbsewinilcllufadlledinatnhde mmaidy-rfaalnlgien othfethleowin-irtaianlgceaolifbtrhaetioinnitciaulrve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 C ontinuing calibration verifications 10.3.1 Pinrietpiaalrceacliobnrtaintiuoinngcucravleib. ration verification samples to ensure the accuracy of the 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group of 10 samples. and extracted. For example, if a sample set = 34, four verifications are prepared 3M Environmental Laboratory ExtractionEoTfSP-F8O-6S.0from Liver Page 5 of 14 Page 48 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U227S 10.3.3 Prepare each continuing calibration verification from the same matnx used to prepare the initial curve. 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of othnelyintihtieallocwaliebnrdatoiofnthceurcvaeli.brTahtiiosniscunrevcees(sfaorryeixfatmheplaen, a5lypsptbm-us1t0q0upapnbti,tartaethuesring than 5^ppb - 1000 ppb). 11.0 C a l ib r a t io n a n d S t a n d a r d iz a t io n ________________________________________________________ 11.1 Prepare m atrix calibration standards 11.1.1 WmLeisgMh ialplip-QroTMximwaatteelry. 4G0rigndoftolivaehroimntoogaen2e5o0ums sLolNutailogne.ne bottle containing 200 11.1.2 If 40 g is not available, use appropriate amounts of liver and water to ensure a 1:5 ratio. 11.1.3 Refer to 13.0 to calculate the actual density of liver homogenate and the concentration of solid liver tissue dispersed in 1.0 mL of homogenate solution. 11.1.5 Add 1 mL o f homogenate to a 15 mL centrifuge tube. Re-suspend solution by hshoamkoinggenbeeotuwseesnoluatliiqounoitns w15hmileLpcreenptarriifnuggeattuobtaels.of eighteen 1 mL aliquots of 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 tThyepeincadlloyfuthseistsheectsitoannd, atordspcoiknec,einntrdautpiolnicsataen,dtwspoikstinangdaamrdoucuntrsveliss,tefodrian tToatablleof1, at eighteen samples, two matrix blanks, and two method blanks. 11.1.8 wRhefiecrhtloisvtsaltihdeatwioonrkrienpgorrtasnEgeTsSa-n8d-6t.h0eaLnidneEarTCS-a8li-b7r.0a-tVio-n1RoarnAgett(aLcChRm)enfotrB, calibration curves. 11.1.9 sUtasnedAarttdasc. hRmeefenrt tCo a1s3.a0ntoaicdailncuclaaltceualacttiunagl cthoencceonntcreanttiorantsioonfsPoFfOthSeinwocrakliibnrgation standards. 11.2 sTuorreoagcahtewworokriknigngstsatnadnadradr,dbfloarnkth,eorcocnocnetnintruaitnigonvetoriffiaclal twiointh,iandtdheapcparliobprraitaitoenacmuoruventraonfge 5 ppb - lOOOppb. ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 6 of 14 Page 49 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 11.3 Extract spiked liver homogenates following 12.14-12.25 of this method. Use these standards to establish each initial curve on the mass spectrometer. Table 1 Approxim ate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm Hi Approx, final cone, of PFOS in liver - Blank 2 0.005 ppm 4 0 . 0 1 0 ppm 1 0 0.025 ppm 2 0 0.050 ppm 40 . 0 . 1 0 0 ppm 1 0 0.250 ppm 2 0 0.500 ppm 30 0.750 ppm 4 1 . 0 0 ppm 12.0 P r o c e d u r e ____________________________________________________________________________________ 12.1 Obtain frozen liver samples. 12.2 Cpuetrafporpmroexdimquatieclkyly1, gnootfallilvoewriunsgintghealdiviessretcotitnhgawsc.alpel. This part of the procedure is best 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Return unused liver portions to freezer. 12.6 Add 2.5 mLs o f water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Ta2k2e. the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP- 12.10 Cwaepighthte, lsiavmerpIlDe ,adndatevoarntdexafnoarly1s5t sineictoianlds.s. Label the sampule vial with the study number, ExtractionEoTfSP-F8O-6S.0from Liver 3M Environmental Laboratory Page 7 of 14 Page 50 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.11 Pipette 1.0 mL, or other appropriate volume, of homogenate into a 15 m l polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1 mL aliquots ofMilli-QTM water to centrifuge tubes. These will serve as method blanks. 12.13 Spike all sampjes, including blanks and standards ready for extraction with surrogate standard as described in section 1 1 .2 . 12.14 Spike each matirx with the appropriate amount of standard as described in 11.1, or Table 1 ocofnthtiantusiencgticoanl,ibfroartitohne cstaalnibdraartdios.n curve standards. Also prepare matrix spikes and 12.15 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.16 Check to ensure 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.17 To each sample, add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.18 Using an Oxford Dispenser, add 5 mL methyl-teri-butyl ether. 12.19 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information as in 12.10, 12.22 Remove 4.0 mL of the organic layer to the fresh 15 mL centrifuge tube. 12.23 hPouutrse.ach sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 12.24 Add 1.0 mL to each centrifuge tube using a graduated pipette. 12.25 Vortex mix for 30 seconds. 12.26 FAiltttearchinato0a.21p.5mmnLylgolnasms easuhtofvilitaelrotrolaow3-cvcolsuymrinegaeuatonvdiatrlawnhsfeenr tnheecessasmarpyl.e to this syringe. 12.27 mLaatbreixl ,thfienaalutsoovlviaelnwt, iethxtrthaectsiotunddyanteu,mabnedr,ananalimysat(lsn) upmerbfeorrmanindggtehnedeerx,trsaacmtiponle. timepoint, 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attached to this document, and tape in study notebook or include in study binder, as appropriate. ExtractionEoTfSP-F8O-6S.0from Liver 3M Environmental Laboratory Pace 8 of 14 Page 51 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s ___________________________________________________ __ 13.1 Calculations: 13.1.1 tCenalsceuplaartaeteth1e.0avmerLagaeliqduenotssityofohfotmheolgievnearthe.omogenate by recording each mass of Average density (mg/mL) = Average mass (mg) of the aliquots 1.0 mL aliquot 13.1.2 edCqiasuplaceutrilosanetd:e sthoelidamtiossuunet poefrlimveLr o(mf hg)ompeorge1n.0atmeLsuhsopmenosgieonna)tues(ionrgctohnecfeonltlroawtiionng of g of Liver x Average density* o f homogenate (mg/mL-) (g of Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 sCtaanlcdualradtes uacstinuagltchoenfcoelnlotrwaitniognesqoufaPtiFonO:S and other fluorochemicals in calibration uL of Standard x Concentration (ug /mL) = Final Concentration (ug/g or mg/kg) mg Liver/ 1 mL homogenate* of PFOS in Liver *refer to 13.1.2 for details. 14.0 M e t h o d P e r f o r m a n c e _______________________________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (refer to Attachments B and C). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Mpraetcriisxiosnpiokfethaendexmtraatcrtixionsp. ike duplicate samples to determine accuracy and 14.2.3 oCfotnhteiniuniintigalccaalilbibraratitoionnvceurrifviec.ation samples to determine the continued accuracy 14.3 Refer to section 14 of ETS-8-7.0 for method performance criteria. 15.0 P o l l u t io n P r e v e n t io n a n d W a st e M a n a g e m e n t _______________________________________ 15.1 ShloiacgmahtpeBldeTiwUnatcshoteentliaasbindoeirsrapst,oorsayend.d iunsebdiohglaazsasrdpicpoenttteaiwnearsst,e filsadmimspaobsleedsionlvbernotkwenasgtleasiss dcoisnptoasineedrsin ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory - Page 9 of 14 Page 52 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 16.0 R e c o r d s ________________________________________________________________________________________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 T a b l e s , D ia g r a m s , F l o w c h a r t s , a n d V a l id a t io n D a ta _______________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard calculation and concentration worksheet 18.0 R e f e r e n c e s ___________________________________________________________________________________ 18.1 The validation report associated with this method is ETS-8-6.0 & 7.0-V -l. 18.2 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 18.3 FLAivCerTf-oMr -A1n.1a,ly"sEisxtUrascintigonHoPfLPCF-OESlecotrroOstphrearyA/MniaosnsicSpFelcutorroomcehtermy"ical Surfactants from 19.0 A f f e c t e d D o c u m e n t s _______________________________________________________________________ 19.1 ETS-8-7.0, "Analysis o f Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R e v isio n s RNuevmisbieorn. Reason For Revision ReDvaistieon ExtractionEoTfPSF-8O-6S.0from Liver 3M Environmental Laboratory Page 10 of 14 Page 53 3m Medical Department Snudy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Study tr Matrix BWokx/DU ay Date Spiked/Analyst ccv MS MSD Surrogate Std approx, ppm actual ppm # - FC Mix Std FC Mix Std approx. 0.5 ppm approx. 5 ppm actual ppm actual ppm ## FC Mix Std approx. 50 ppm actual ppm U ft Comments ;1- --- - -- - - - i i Blank Liver Homogenate: Liver Extraction M ethod S td tf : - - Liver amount = SDike surrogate and S tandard m ix. V ortex 15 sec. Pipette 1 m L of Liver Solution Pipette 1 m L o f t0 .5 M T B A , pH 10. pH = Std. tt Pipette 2 m L of 0.25 N a->CO y0.25M N aH C O t Buffer Std. Dispense 5ml of M ethyl-t-Butyl Ether TN-A- Shake 20 min. Shaker Speed Centrifuge 20-25 mm. Centrifuge Speed Remove a 4 m L aliquot of organic laver Put on Nitrogen Evaporator to drvncss Evaporator Temperature A dd 1.0 m L o f M ethanol TN-A- Vortex FCiltoenr tu. s3Ci0nagsle.acV.3cecrifBi-cDatsiov nr isn guesewdiththae 0s.a2mu me mS RaItrNixitearsifnotor athu et o sstaamnpdlaervdi aclurve. - - Attachment B: MDL/'LOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction o fP F O S from L iv er - - - - - -------------------- ----------------------- j--------------------------------- - - - g D ate & Initials Page 11 of 16 Page 54 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 M DL/LOQ values for rabbit liver Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (ppb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 8.45 26.9 30 ppb - 1200 ppb PFOSA 3.50 11 .1 1 2 ppb - 1 2 0 0 ppb PFOSAA 24.6 78.3 30 ppb - 1200 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M556 82.3 262 60 ppb - 1 2 0 0 ppb PFOSEA 33.9 108 30 ppb- 1200 ppb MDL/LOQ values in rat, bovine, and monkey liver were not statistically determined. Two curves in each o f these-matrices were extracted and analyzed with the rabbit liver curves to determine equivalence. Responses in the rat, bovine, and monkey liver curves were equivalent to the rabbit responses, therefore, their MDL and LOQ will be assumed to be equivalent to those values as determined for the rabbit liver. R* eEfetrFtOoSLEO-OQHSeusmtimmaartyesaonndlyMfDorLMstDudLyainndELTOSQ-8.-6D.0id&no7t.0m-Vee-tl cfroirtefruiarthfoerr vinafloidramtiaotnio.n Compound: PFOS Liver matrix Prepared Range of LCR from Range of LCR from Range of LCR from range of average ave curve low std low std high std high std standards (ppb) (ng/mL) (ppbc)u(rnvge/mL) (ppb) (ng/mL) (ppbc)u(rnvge/mL) (ppbc)u(rnvge/mL) (ppbc)u(rnvge/mL) (ppbc)u(rnvge/mL) Rabbit j 6.19 - !237 12 -1200 12 - 1200 6 - 300 12 - 300 60 -1200 60 -1200 Compound: PFOSA Prepared Liver matrix srtaanndgearodfs (ppb) (ng/mL) Rabbit 6.19 - 1237 Range of average curve (ppb) (ng/mL) 12 - 1200 LCR from ave curve (ppb) (ng/mL) 12 - 1200 Range of low std curve (ppb) (ng/mL) 12 - 300 I.CR from low std curve (ppb) (ng/mL) 12-300 Range of high std (ppbc)uirnvge/mL) 60 - 1200 LCR from high std curve (ppb) (ng/mL) 60 - 1200 Compound: PFOSAA Prepared Range of Liver range of average matrix Rabbit standards i! (ppb) (ng/mL) 6.16-1232 (ppbc)u(rnvge/mL) 12 -1200 1 LCR from ave curve (ppb) (ng/mL) 30 - 1200 Range of low std curve (ppb) (ng/mL) 30 - 900 LCR from low std (ppbc)u(rnvge/mL) 60 - 900 Range of high std curve (ppb) (ng/mL) N7A LCR from high std curve (ppb) (ng/mL) N7A Attachment B: MDL'LOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 12 of 16 Page 55 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: EtFOSE-OH Prepared Range of Liver range of average matrix standards curve (ppb) (ng/mL) (ppb) (ng/mL) Rabbit 6.17 - 1235 3 1-900 LCR from ave curve (ppb) (ng/mL) 31-900 Range of low std curve (ppb) (ng.'mLl N/A LCR from low std curve (ppb) (ng/mL) N/A Range of high std curve (ppb) (ng/mL) N/A LCR from high std curve (ppb) (ng/mL) N/A Compound: PFOSEA Prepared Range of Liver range o f average matrix s t a n d a r d s curve (ppb) (ng/mL) {ppb) (ng/mL) Rabbit 6.17- 1235 - 31 - 1200 LCR from ave curve (ppb) (ng/mL) 31 - 1200 Range of low std curve (ppb) (ng/mL) N/A LCR from low std curve (ppb) (ng/mL) N/A Range of LCR from high std high std curve curve (ppb) (ng/m L ) --(ppb) (ng /m L ) N/A N/A Compound: M556 Prepared Liver range of matrix standards (ppb) (ng/mL) Rabbit 6.17- 1235 Range of average curve (ppb) (ng/mL) 31 - 1200 LCR from ave curve (ppb) (ng/mL) 60 - 1200 Range of low std curve (ppb) (ng/mL) N/A LCR from low std curve (ppb) (ng/ml.i N/A Range of high std curve (ppb) (ng/ml.) N/A LCR from high std curve (ppb) (ng/mL) N/A Artachmen: C: Standard Calculations ETS-8-6.0 Extraction ofPFOS from Liver 3M Environmental Laboratory Page 13 of 14 Page 56 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-C30 laboratory Request Number-U2279 Ion Pair Standard Curves - Tissue Prep date(s): ASanmalpyltee(ms)a: trix: SMTFFFuCCCaerrtrgmmmhoeoiiigtxxxdaa/ssstrnetttedddavsltyiaaasdtpppieopppa(nsprrr):ooop:xxxr...o05x5..0.05.000100pp0pppmmppmp::m: : ESFBtiqlanaunanidlkpasmlorivdlevneenrtn/uintdmuaembnnedtbrife:TireN:r:: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 5.00 50.0 50.0 50.0 50.0 50.0 M556 Std cone Std cone ug/mL ug/mL 0.500 0.500 0.500 0.500 0.500 5.00 ! 5.00 5.00 50.0 All Am't spiked mL 0.002 0.004 0.010 0.020 0.040 0.010 0.020 0.030 0.004 All Density 0.1g67 0.167 0.167 0.167 0.167 0.167 0.167 0.167 0.167 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Surrogate Final Final Final cone Final Final Final Std cone Std cone cone cone ng/g cone cone cone ng/g ng/mL n5.g9/9g n5.g9/9g 5.99 n5.g9/9g n5.g9/9g n5.g9'9g | . 100 12.0 12.0 12.0 12.0 12.0 12.0 29.9 29.9 29.9 29.9 29.9 29.9 Surrogate 59.9 59.9 59.9 59.9 59.9 59.9 Final cone 120 120 120 120 120 120 ng/mL 299 299 299 299 299 299 0.500 599 599 599 599 599 599 898 898 898 898 898 898 TT98 1198 1198 1198 1198 1198 All Am't spiked mL 0.005 Validated ranges - approximate concentrations L iv e r 1 PFOS i PFOSA PFOSAA Rabbit 5-1000 ppb j 5-1000 ppb j 5-1000 ppb Bovine Estimates only, use rabbit values. Rat Estimates only, use rabbit values. M onkey F.stimates only, use rabbit values. EtFOSE-OH 5-1000 ppb 11 POAA 5-1000 ppb i! PFOSEA 5-1000 ppb Attachment C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 14 of 14 Page 57 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 3M Environmental Laboratory M ethod A n a l y sis of P o tassium Perfluoro octaivesulfonate or O th er Flu o r o c h em ic a ls in L iv er E xtracts U sing H P L C -E lectrospray/M ass S pectrom etry M ethod Number: ETS-8-7.0 ojlziinAdoption Date: Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: Laboratory Manager A / A /A ---------- Group Leader _Te_c_h_n__ic_a_l__R_evriiel/wmetr*-___________________________ 7 A ^ /f ; Date ... Date O lh 'ih 'i Date 1.0 S c o p e a n d A p p l ic a t io n _______________________________________________________________________ 1.1 SHcPoLpCe:-elTehctirsomsperthayo/dmiassfsorsptehcetraonmaelytrsyis. of liver extracts for fluorochemical surfactants using 1.2 oAtpheprliicoanbizleabCleomcopmopuonudnsd: sF. luorochemical surfactants or other fluorinated compounds, or 1.3 rMepaotrrti.ces: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation Word 6/95 Analysis of LivEeTrSE-x8t-r7a.c0t Using ES/MS Page 1 ot'10 3M Environmental Laboratory Page 58 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 S u m m a r y o f M e t h o d _________________________________________________________________________ 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HpePrLfoCrm-eeledctbryosmproanyit/omrainsgs sapseicntgrolemieotnryc,hoarrascimteirliasrticsyostfeampaarstiacpuplarropflruiaotreo. chTehme iacnaal,lyssuicshisas the perfluorooctanesulfonate (PFOS) anion, m/z = 499. Additionally, samples may be danetaelcytziendg udsaiungghatetraniodnesmomf tahsesssepleeccttreodmpeatreernttoiofnu.rther verify the identity of a compound by 3.0 D e f in it io n s _____________________________________________________________________________________ 3.1 sAytsmteomssphaellroiwc PforresvsaurrioeuIsomnieztahtoiodns o(Af iPoIn)i:zaTthioenMbiycruotmiliazsinsgQvuaartitorousIIsotruirpcleesq, upardorbueps,olaend interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric tPercehsnsuiqrue ecshoecmciucrasl aItonatizmaotisopnhe(rAicPcpIr)e,sTsuhreerm(i.oes.pnraoyt ,uentdce.rTahveaciounuimza)t.ion process in these 3.2 pETrhleeescssteurrocehs,pawrrgaheyedrIedobrnyoipziolaenttissonianre(sEoplSru,otdiEouSncIe)ad:rebatymratehnteshfeoardprpeoldifctiaootnitoihzneatogifoasna pphearfsoervmiaedtinatyacthmaorsgpehdedrircoplets. strong electrical field. 3.3 TMhaessAPSIpeQcutraottmroetIIrytr, iMpleasqsuaSdpreucptroolemmeatesrs s(MpeSct)r,oTmaentedremis eMquaispspSedpewcittrhomtweoteqru(aMdrSu/pMolSe): mchaasrsgeserlaetcitoiv(emd/ze)teacntdorssuabnsdeqauceonltlliysidoenteccetlel.d.IoAnssianrgelseeMlecStimvealyy bdeisecmrimplionyaetdedfobryiomnass to detection or an ion may be selected in the first quadrupole, fragmented in the collision cell, and these fragments may be analyzed in the second quadrupole. 3.4 C onventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole (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 dnemloieratceitnccrottolemydnpeaa.nat tctTihebhelaeorcueowgntihhtehetahsopeanemrectoeuan.rnfeoiH,gthuaoferwtraee,trvibopeunrat,siossZinn-dlsgyipffrtwehariryteohncutosg,imhmthpaieloatnmoreresnttuythsosotueadsmnsdpsoacft(ohio.newp.veaeZryna-ttsiinipoornntah,ayelclccceoooaummnnipptneoogrnn,eeannnttdss aarree compatible with other Z-spray systems, etc.) 3.5 M ass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 ivnesrtsriuomnse.ntA(lMl vicerrosimonasssaQreusaitmtriolaIrI. trFiporlemqouraedrdueptaoilles MreafesrsLtoynthxeomr aMnuasaslLsypnexcifNicTtoUstheer's Guide). 4.0 W a r n in g s a n d C a u t io n s______________________________________________________________ 4.1 Health and Safety W arnings: 4.1.1 Uemseplcoayustiaonvowltiathgethoefvaoplptargoeximcaabtleelsy f5o0r0t0heVporlotsb.e. When engaged, the probe Analysis of LivEeTrSE-x8t-r7a.c0t Using ES/MS 3M Environmental Laboratory Page 2 of 10 Page 59 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. 4.2 Cautions: 4.2.1 Operate the solvent pumps below a back pressure of 400 bar (5800 psi). If the back pressure exceeds 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 I n t e r f e r e n c e s _____________________________________________________________________________ _ 5.1 To minimize interferences when analyzing samples, Teflon shall not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract. 6.0 Equipment__________________________________________________________________________ 6.1 mEqoudiipfimcaetnitonlisstiendtbheelroawwmdaatyabaes mmoetdhifoideddeinvioartdioenrst.o optimize the system. Document any 6.1.1 MeleicctrroomsparsasyQiuoantitzraotiIoIntrsiopulercqeu. adrupole Mass Spectrometer equipped with an 6.1.2 HP1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 S u p p l ie s a n d M a t e r ia l s ______________________________________________________________________ 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100 psi (house air system) 7.1.2 HinPtLheCraanwadlyattiacal column, specifics to be determined by the analyst and documented 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8.0 R e a g e n t s a n d S t a n d a r d s____________________________________________________________________ 8.1 R eagents 8 .1 .1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be ATSM tvyepnedoI,ror equivalent, and be provided by a Milli-Q TOC Plus system or other 8.1.3 Ammonium acetate, reagent grade or equivalent 8 .1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 2.0 mM ammonium acetate solution: Weigh approximately 0.300 g ammonium acetate. Pour into a 2000 mL volumetric container containing 2000 mL Milli-QTM water, mix until all solids are dissolved. Store at room temperature. Analysis of LivEeTrSE-x8t-r7a.c0t Using ES/MS Page 3 of 10 3M Environmental Laboratory Page 60 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. Refer to ETS-8-6.0. 9.0 S a m p l e H a n d l in g _______________________________________________________________ ______________ 9.1 aFrreesshtomreadtriinx csatapnpdeadrdasutaorveiaplrseporarceadppweidth1e5acmhl acnenaltyrsifius.geEtxutbraecstuedntsiltaanndaalrydssis.and samples 9.2 tIefmanpearlyastuisrew, iollrbreefdrieglearyaetded, eaxttraapcptreodxsitmanadtealryd4sanCd, usanmtilpalensamlysaiys bcaenstboerepderaftorromoemd. 10.0 Q u a l it y C o n t r o l ______________________________________________________________________ _ 10.1 M ethod B lanks'and M atrix Blanks 10.1.1 eSaoclhvebnattcbhlatnoksd,etmeremthionde bcolanntkams, iannadtimonatorrixcabrlraynokvsera.re prepared and analyzed with 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 rMecaotrviexrsypeikffeicsieanrecyp.repared and analyzed to determine the matrix effect on the 10.2.2 rMecaotrviexryspfiokreedaucphliacnaatelystea.re prepared and analyzed to measure the precision and the 10.2.3 mAninailmyzuemaomfa2trsipxikspeiskpeearnbdatmcha.trix spike duplicate per forty samples. With a 10.2.4 trMhaenatgirneixiotisfaplthickeaeliinabinrtdaiatlimocnaatlrciiubxrrvaspeti.ioknAe cdduduripvtlieio.cnaatel scpoinkceenctornacteionntrsawtioilnl sfamllaiyn ftahlel imnitdh-eralnogwe- of 10.3 Continuing Calibration Checks 10.3.1 oCfotnhteincuailnibgrcaatiloibnracutirovne.verifications are analyzed to verify the continued accuracy 10.3.2 oAnneaplyezrebaatmchid. -range calibration standard every tenth sample, with a minimum of 11.0 C a l ib r a t io n and S t a n d a r d iz a t io n __________________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of sample extracts. The average of two standard curves will be plotted by linear regression (v = mx + b), weighted 1/x, not forced through the origin, using MassLynx or other suitable software. 1 1 . 2 sItfatnhdeacrudrcvuerdvoee(sifnnoetcmeseseatrrye)qaunidrermeaenntaslypzeer.form routine maintenance or reextract the Analysis of LivEeTrSE-x8t-r7a.c0t Using ES/MS 3M Environmental Laboratory Page 4 of 10 Page 61 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. cEaxlaibmraptlieo:n wcuhrevneactotenmsipsttiinngg otof qthueansttaitnadtearadpspfrrooxmim5apteplby t1o0 1p0p0bpopfbanraatlhyetre,thgaennetrhaetefuall range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 P r o c e d u r e s ___________________________________________________________________________________ 12.1 A cquisition Set up 12.1.1 Set up the sample list. 12.1.1.1 Assign a sample list filename using MO-DAY-last digit of year-increasing letter of the alphabet starting with a 12.1.1.2 Assign a method (MS file) for acquiring 12.1.1.3 Assign an HPLC program (Inlet file) 12.1.1.4 Type in sample descriptions and vial position numbers 12.1.2 To create a method click on method in the Acquisition control panel then mass RasppeepacrctotrpioormniaeMtteeormnhaitesoasredisin.ngg)As. afSnuedltl sIsecolanenicztaisStiIuoRsnu(MaSllioyndgceloeallsIeocantpepRdreoacploorrinadgtienwgai)nthdormthMaeRsSsMItRos(4.M9Su9altovirpeloether GafrcUaqgIumDiseEintitToanOtiomDneAtihnTofoAdr.mAIaCftMQionUSI/mMSIaSTyIibOnesNtcrouflomlreecantdetdsdi.atiroReneeafmleripntlfoooyMremdica,rtaioodmndaiatsinsodnMaMlapRssrMLody.uncxt ion 12.1.3 Tmyaptriicxalsltyanthdearadnsa. lytical batch run sequence begins and ends with a set of extracted 12.1.4 aSfatmerpelveesrayreteannthalsyazmedplwe.ithSoalcvoennttinbulainnkgscashlioburladtiobne avnerailfyiczaedtiopneriniojdecictaedllysttaondard minoclnuidtoerdpaossssuibclhe. analyte carryover and are not considered samples but may be 12.2 U sing the Autosam pler 12.2.1 Set up sample tray according to the sample list prepared in Section 1 2 .1 .1 . 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 9 minutes Analysis of LivEeTrSE-x8t-r7a.c0t Using ES/MS 3M Environmental Laboratory Patte 5 of 10 Page 62 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.2.2.4 Solvent ramp conditions Time MeOH 0.00 min. 1.0 min. 4.5 min. 6.5 min. 7.0 min. 9.0 mi. 40% 40% 95% 95% 40% 40% 2.0 mM Ammonium acetate 60% 60% 5% 5% 60% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrum ent Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance of the Micromass Quattro II ITornipizlaetQiounaSdoruuprcoele,"MfoarssmSopreecdtreotamilest.er Fitted with an Atmospheric Pressure 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 tCuhnheseatcitpkis.ftahTcethosertyat'ii,pndlseihsssaosussltedemeblbeclaeflpatihtllewarpiytrhoabnteothajeangdegnrededpolefadctgheeetshp.eroIsfbtatehi.nelUetisspseisastnefeoeluycneadppitieolclaebreyto. check 12.3.4 Turn on the nitrogen. 12.3.5 Ohepaetenrsth. e tune page. Clicks on operate to initiate source block and desolvation 12.3.6 Open the Inlet Editor. 12.3.6.1 Set HPLC pump to "On" 12.3.6.2 Set the flow to 10 - 500 uL/min or as appropriate 12.3.6.3 Observe droplets coming out of the tip of the probe. A fine mist should be ethxepetilpleodfwthitehpnroobneitirfongoenmliesatkiisnogbasreoruvnedd the tip of the probe. Readjust 12.3.6.4 Allow to equilibrate for approximately 10 minutes. 12.3.7 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.7.1 Drying gas 250-400 liters/hour 12.3.7.2 ESI nebulizing gas 10-15 liters/hour 12.3.7.3 HPLC constant flow mode flow rate 10 - 500 pL/min 12.3.7.4 PHrPeLssCuries <o4p0er0abtianrg(cTohrirsepctalrya.)meter is not set, it is a guide to ensure the 12.3.7.5 Source block temperature 150 12.3.7.6 Desolvation temperature 250 Analysis of LivEeTrSE-x8t-r7a.c0t Using ES/MS Pase 6 of !0 3M Environmental Laboratory Page 63 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.3.8 Ptarpinedt tihnetotutnhee pinasgter,umwietnht iltosgp. arameters, and store it in the study binder with a copy 12.3.9 MeCnladicskssLaomynnpxslteavrneturbmsuiobtnteosrn,irniencfletuhrdeteoAsacapqllpursioaspimtripoialnteesCMtoonabtsresoLlaynPnaaxlnyeUzlesd(et.rh'iss Gmuaiydev)a.ryEanmsuornegstart and 13.0 D a t a A nalyses a n d C a l c u l a t io n s 13.1 Calculations: _________________________________________________ _ 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentrations in matrix (jag/g): (ng o f PFOS calc, from std. Curve x Dilution Factor) x 1 ug (Initial Weight of Liver (g) 1000 ng Final Volume (mL) 14.0 M e t h o d P e r f o r m a n c e ______________________________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Refer to ETS-8-6.0, Attachm ent B for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, M ethod Blanks and M atrix Blanks 14.2.1 Sstoalnvdeanrtdbilnanthkes,cmaleibthraotdiobnlacnukrsv,ea. nd matrix blanks must be below the lowest 14.3 Calibration Curves 14.3.1 The r value for the calibration must be 0.980 or better. 14.4 M atrix Spikes 14.4.1 Matrix spike percent recoveries must be within 30% of the spiked concentration. 14.5 Continuing Calibration Verification 14.5.1 Continuing calibration verification percent recoveries must be within 30% o f the spiked concentration. 14.6 If criteria listed in the method performance section are not met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. Analysis of LivEeTrSE-x8t-r7a.c0t Using ES-'MS 3M Environmental Laboratory Page 7 of 10 Page 64 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 14.7 fIofodtantoateadreotno tbaeblreespoarntdeddiwschuesnsepderifnorthmeatnecxet corfittehreiarehpaovret.not been met, the data must be 15.0 P o l l u t io n P r e v e n t io n a n d W a s t e M a n a g e m e n t ___________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s _______________________________________________________________________________________ 16.1 hEeaacdhepraogrehgaennderwartietdtenforona tshtuedpyagmeu: ststhuadvyeotrhperfoojleloctwninumg binefro,ramcqatuiiosnitiionnclmudeetdhoedit,her in the integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. ' 16.4 Panrdintstdoarteaininttehgersattuiodny sfuomldmera. ry, integration method, and chromatograms from MassLynx 16.5 Summarize data using suitable software (Excel 5.0+) and store in the study folder, refer to Attachment A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 T a b l e s . D ia g r a m s , F l o w c h a r t s , and V a l id a t io n D ata___________________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18.0 R e f e r e n c e s ___________________________________________________________________________________ 18.1 FACT-M-2.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 18.2 EIoTnSiz-a9t-i2o4n./0M, a"OsspSerpaetcitornomanedteMr QaiunattetnroanIcIetroipfltehqeuMadicrruopmolaessSyAsttmemossp"heric Pressure 18.3 The validation report associated with this method is ETS-8-6.0 & 7.0-V-l 19.0 A f f e c t e d D o c u m e n t s _______________________________________________________________________ 19.1 ETS-8-6.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Liver or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" Analysis of LivEeTrSE-x8t-r7a.c0t Using ES/MS 3M Environmental Laboratory Pane 8 of 10 Page 65 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 20.0 R ev isio n s Revision Number Reason For Revision Revision D ate ETS-8-7.0 Analysis of Liver Extract Using ES/'MS 3M Environmental Laboratory Pace 9 of 10 Page 66 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Laboratory Study # S tu d y : Test Material: Matrix/Final Solvent: _ Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analysh Date of Analysis/Analyst: Group Sample# Concentration Dose ng/g Initial Wt. g Dilution Factor Final Cone, "g/g Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ng/g): Taken from the MassLynx integration summary. Initial VVt. (g): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone, (ug/g): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet Analysis of LivEeTrSE-x8t-r7a.c0t Using ES/'MS 3M Environmental Laboratory Page 10 of !0 Page 67 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory M ethod E x tr a c tio n ,of P o tassium Perfluo ro o ctanesulfo nate o r O th er HPLC-F l u o r o c h e m i c a l c o m p o u n d s f r o m S e r u m f o r A n a l y s is U s i n g E lectro spray/M ass S pectro m etry M ethod Num ber: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: 7 /3 7/99 ___ 0 /JSApproved By: Laboratory Manager -- 7 /?> ' Date A i'^ IJ l -----------Group Leader Technical Reviewer /W7Date DCa't7e 9 s i li 1.0 S c o p e a n d A p p l ic a t io n ___________ ____________________________________________________________ 1.1 oSrcoopthee:r Tflhuiosrmocehtehmodicaisl fcoormthpeouenxtdrsacfrtioomn osefrpuomta. ssium perfluorooctanesulfonate (PFOS) 1.2 A pplicable com pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 tMheatvrailciedsa:tioRnabrebpito,rrt.at, bovine, monkey, and human serum or other fluids as designated in Word 6/95 ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 1 of 14 Page 68 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C3G laboratory Request Number-U2279 2.0 S u m m a r y o f M e t h o d ____________________________________________________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-ieri-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and the sample surrogate standard and the analyte ion (see pair 3is.0pDaretfiitnioitnieodn sin).toAMntBioEn. pairing reagent is added The MtBE extract is to removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 mTheethseodssa.mple extracts are analyzed following method ETS-8-5.1 or other appropriate 3.0 D e f in it io n s _____________________________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8Fl7S 0 3` 3.2 PFOSA: perfluorooctane sulfonylamide C8F]7SO-,NH-. 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 3N(CH3CH3)CH2C 0 3' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol CsF17S 0 2N(CH2CH3)CH3CH30H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamideCsFl?S 0 2N(CH3CH3)H 3.6 M556: C8Fl7S 0 3N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W a r n in g s a n d C a u t io n s ______________________________________________________________________ 4.1 H ealth and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 I n t e r f e r e n c e s _________________________________________________________________________________ 5.1 There are no interferences known at this time. 6.0 E q u ip m e n t __________________________________________________________________________ 6 .1 Tachceepfotallbolwe.ing equipment is used while performing this method. Equivalent equipment is 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6 .1 . 2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 2 of 14 Page 69 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) 7.0 S u p p l ie s a n d M a t e r ia l s _______________________________________________________________ 7.1 Gloves 7.2 Eppendorf ordisposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels * 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable o f measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 R e a g e n t s a n d S t a n d a r d s ____________________________________________________________________ 8 .1 TbeypMeilIlir-eQaTMgenwt agtreardaenwdamtear,yMbeillpi-rQovTMideodr beqyuaivMalielnlit-;QalTl OwCatePrluussTMed siynstthemis method should 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na^Oj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCOj), J.T. Baker or equivalent 8 . 6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8 . 8 Serum or blood, frozen from supplier 8.9 Fluorochem ical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight - 499 Extraction EoTf PSF-8O-4S.1from Scrum Page 3 of 14 3M Environmental Laboratory Page 70 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03C laboratory Request Number-U227S 8.9.3 PFOSAA (3\1 Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (l-H .l-H , 2-H, 2-H C8Fl3S 0 3H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a d1i0s0so0 lvmeLd.beSatkoerre cinonata1inLinNga5lg0e0nme LboMttliel.li-QTM water, mix until all solids are 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL oflO NMiNllai-OQHTMswolautteiro.n Sintotoreain10a01m25LmvoLluNmaelgtreinceflbaostktlae.nd dilute to volume using 8.10.3 NoM01.0fa5iTOlulMiBsH-iQAn,tgTMeaitdnradatpwobp'sauarltotoeywlrxl.iaLlmymSvatbmooteelrouclenyamiui4unes4mteartitoch1hye5Lcdo4prNnHomtagaLlceignhneoiannsfnuegg1lbef05aso0tNt0eatlbNe(mrT.auLBOpAtMHly):i)(l.lWWi-DQheiiiTMlgluehteawadtapodtpeivnrro.golxAutihmdmejaueltsaewtsltyittomh1pL6H9ogf 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as ... needed using 1 N NaOH solution. 8.10.4 0ap.2p5roMximsoadtieulmy 2c6a.5rbgonoafteso/sdoiduimumcabribcoanrbatoena(NteabjCuf0fe3r) a(nNda ,C 21 0.03/gNoafHsCod0i3u)m: W eigh bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with Milli- QTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8 .1 1 . 1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent sf1lo.u1luo0rtiopocpnhmecmoEnitctFaaOilnSsitnEagn-Od1aH.0r.d0)spaprme aPcFceOpSta, b1l.e02(foprpmexaPmFOplSe,Ao,n0e.9w8o7rpkpinmg PstFanOdSaArdA, and 8.11.3 tWheeiagchtuaaplpwreoixgihmt.ately 100 mg of PFOS into a 100 mL volumetric flask and record 8.11.4 B(prgi/nmgLto).volume with methanol for a stock standard of approximately 1000 ppm 8.11.5 Dapilpurtoextihmeastteolyck50sopluptmio.n with methanol for a working standard 1 solution of 8.11.6 aDpiplurtoex.w5o.r0kpinpgms.tandard 1 with methanol for a working standard 2 solution of Extraction EofTPSF-8O-4S.1from Serum Page 4 of 14 3M Environmental Laboratory Page 71 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.11.7 aDppilruotxe.w0o.5r0kipnpgmst.andard 1 with methanol for a working standard 3 solution of 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg o f surrogate standard l-H .l-H , 2-H, 2-H, C8Fl3S 0 3H into a 50 mL volumetric flask and record the actual weight. 8.12.2 pBprmin.g `to volume with methanol for a surrogate stock of approximately 1000-1200 8.12.3 Pstroecpkarteo aa s1u0rrmogLatveolwuomrektirnigc sfltaasnkdaarndd. bTrrinagnstfoervoaplupmroexiwmitahtemlyet1hmanLoloffosruarrogate working standard of 100 ppm. Record the actual volume transferred. 9.0 S a m p l e H a n d l in g ______________________________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Q u a l it y C o n t r o l ____________________________________________________________________________ 10.1 Solvent Blanks, M ethod blanks and matrix blanks 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 aEsxtmraectht otwdobl1a.n0kms.L aliquots ofMilli-QTM water following this procedure and use 10.1.3 Extract two 1.0 matrix blanks. mL See aliquots 11.1.4. o f the serum following this procedure and use as 10.2 M atrix spikes 10.2.1 Pthreepaacrceuraancdyaonfatlhyezeexmtraatrcitxiosnp.ike and matrix spike duplicate samples to determine 10.2.2 mPraetpraixrereeacecihvesdpiwkeithuseinagchassaammpplleesceht.osen by the analyst, usually the control 10.2.3 Expected concentrations will fall in the mid-range o f the initial calibration curve. Additional spikes maybe included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing check per group of 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Pthreepinariteiaelaccuhrvcoe.ntinuing calibration check from the same matrix used to prepare Extraction EofTPSF-O8-S4.f1rom Serum 3M Environmental Laboratory Page 5 of 14 Page 72 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 10.3.4 cTalhibereaxtpioenctceudrvcoe.ncAendtdriattioionnasl wspilikl efsallmwaiythbien itnhcelumdiedd-rtahnagt efaollf itnhethieniltoiawl-range of the initial calibration curve. This is necessary if the analyst must quantitate using to5hnpalnpybth-e low end of 1000 ppb). the calibration curve (for example, 5 ppb - 100 ppb, rather 11.0 C a l ib r a t io n a n d S t a n d a r d iz a t io n __________________________________________ ____ 11.1 Prepare m atrix 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 1.0 mL, extract standards with matrix mvoalturmixe. sReeqcuoarldtoeatchhe ssaammppllee vvoolluummeeso. nDthoeneoxttreaxctrtaiocnt lsehsesetth. an 0.50 mL o f 11.1.3 bWethwileeepnraepliaqruiontgs.a total of twenty aliquots in 15 mL centrifuge tubes, mix or shake 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two standard curves, for a total of eighteen standards, two matrix blanks, and two method blanks. 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V -1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 csUtaaslniebdAraartttdiaosc.nhmsSteaeenndtSaDercdtasiso. nan1a3i.d0 itno ccaallccuullaattienagctthuealccoonncceenntrtraatitoionnssooffthPeFwOoSrkining 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate w1 0o0r0kipnpgbs.tandard for the concentration to fall within the calibration curve range 5 ppb 11.3 tEoxtersatactblsipshikeedacmh aintriitxiasltcaunrdvaerdosnftohlelomwainssg s1p2e.c6t-r1o2m.1e6tero.f this method. Use these standards Extraction EofTPSF-8O-4S.1from Scrum 3M Environmental Laboratory Page 6 of 14 Page 73 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U2279 Table 1 Approxim ate spiking amounts for standards and spikes Using 1.0 mL of matrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix ` - - Blank 0.500 ppm 1 0 0.005 ppm 0.500 ppm 2 0 0 . 0 1 0 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 1 0 0.050 ppm 5.00 ppm 2 0 0 . 1 0 0 ppm 50.0 ppm 5 0.250 ppm 50.0 ppm 1 0 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 . 1 . 0 0 ppm 12.0 P r o c e d u r e ____________________________________________________________________________________ 12.1 wOabttearibnaftrho.zen samples and allow to thaw at room temperature or in a lukewarm 12.2 pVoolrytperxompyixlenfoerc1e5ntsreicfuognedst,ubthee. n transfer 1.0 mL or other appropriate volume to a 15 mL 12.3 Return unused samples to freezer after extraction amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 wLaobrkelshtheeettufobredwoictuhmtheentsitnugdythneurmembeari,nsinamg sptleepIsD. , date and analyst initials. See attached 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 1 1 .2 . 12.7 cS1opinniktitenhueaatincsgheccmtaioalitnbr,rixafotwirointthhesttachnaeldiabaprrpdarsti.oopnricautervaemsotaunndtaorfdss.tanAdlasrodparsepdaersecrmibaetdrixinsp1i1k.1es, oarndTable 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 bTiocaerabcohnsaatembpuleff,eard. d 1 mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium 12.11 Using an Oxford Dispenser, add 5 mL methyl-feri-butyl ether. 12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes. 12.13 sCeepnatrraitfeudg.e for 2 0 to 25 minutes at a setting of 3500 rpm, or until layers are well Extraction oEfTPSF-O8-S4.f1rom Serum Page 7 of 14 3M Environmental Laboratory Page 74 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5. 12.15 Remove 4.0 mL of the organic layer to this clean 15 mL centrifuge tube. 12.16 Phouutresa.ch sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 12.17 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Asytrtiancghe.a 0F.i2lteprminntoyloan1.m5 emshL fgilltaesrstaouato3vicacl soyrrlionwge-vaonldumtreanasufetrovtihael swahmepnlenetocetshsiasry. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.22 nCootmebpoleotke otrheinecxlturdaectiinonstwudoyrkbsihnedeetr,,aatstaacphperdotporitahties. document, and tape in the study 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s _______ ;________________________________________________ 13.1 Calculations 13.1.1 cCaalilbcuralatitoenacsttuanadl acrodnsceunstirnagtitohnesfoofllPowFOinSg, eoqruoatthioenr:applicable fluorochemical, in mLmLofosftastnadnadradrd+ xmcLonocfesnutrrraotigoanteosftastnadnadradrd+ (iungiti/aml Lm)a_t_r_ix__v_o_l_u_m__e__(m__L__)___= Final Concentration (pg/mL) of PFOS in matrix 14.0 M e t h o d P e r f o r m a n c e _____________________________________________________________________ 14.1 TfohrespmeectihfoicdMdeDteLctainond lliimmiitt o(Mf qDuLan) tiistaatnioanly(tLe OanQd) mvaalturiexs s(pseeeciAfict.tacRhemferentotsMBDaLndreCpo).rt 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction. 14.2.3 Cinoitniatilncuailnibgrcaatiloibnractuirovne.check samples to determine the continued accuracy o f the 14.3 Refer to section 14 o f ETS-8-5.1 for method performance criteria. 15.0 P o l l u t i o n P r e v e n t io n a n d W a s t e M a n a g e m e n t ____________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. Extraction EoTf PSF-8O-4S.1from Serum Page 8 of 14 3M Environmental Laboratory Page 75 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03Q laboratory Request Number-U2279 16.0 R e c o r d s ___________________________________________________________________________ 16.1 Cnootmebpoloetke otrheinecxlturdaectiinonthweo3r-krsinhgeesttuadttyacbhineddetro, tahsisapmpertohpordia,tae.nd tape in the study 17.0 A t t a c h m e n t s ____________________________________________________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 R e f e r e n c e s _______________________________________________________________________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l. 18.2 FACT-M-3.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 19.0 A f f e c t e d D o c u m e n t s ________________________________________________________________________ 19.1 HETPLS-C8--E5.l1e,ctr"oAsnparalyysMis aosfsSSepruecmtroormOettrhye"r Fluid Extracts for Fluorochemicals using 20.0 R e v is io n s _____________________________________________________________________________________ RNeuvmisbioenr 1 ' Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Sleescstitohnan121..017mFLi.nal volume is 1.0 mL; not adjusted for initial volumes ReDvaistieon 04/02/99 Extraction EofTPSF-8O-4S.1from Scnim 3M Environmental Laboratory Page 9 of 14 Page 76 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Extraction Worksheet ETS-8-4.1 Study # Surrogate Std Matrix Box rt approx, ppm actual ppm Wk/Dav U DateSpiked/Analyst CCV ! MS l MSD ! . i' -- j !1 i ! *i FC-Mix approx. 0.5 pm actual ppm # FC-Mix approx. 5 ppm actual ppm TUt FC-Mix approx. 50 ppm actual ppm # Comments -- _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ i1_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 1i i -. - - - - - - - - - - -- -- i -- -- Blank Std U amount = Serum Extraction Method : Vortex 15 sec. Pipette Matrix Volume mL Pipette 1mL of 0.5 M TBA, pH 10. pH = Std. U Pipette 2 mL of 0.25 Na;>COy0.25M NaHCOt buffer Std. #? Dispense 5 mL of methyl-t-butyl ether TN-A- Shake 20 min. Shaker speed: Centrifuge 20-25 min. Centrifuge speed: Rempve a 4 mL aliquot of organic laver Put on Nitrogen Evaporator to drvness Temperature: Add methanol Volume mL TN-A- Vortex 30 sec. FCioltnert.uCsianlg. Va e3rcicfiBc-aDtiosnvnsnugseedwisthamae0.m2uamtrifxiltaesr ifnotrosatd1.c5umrvLe.autosample via! - - - - - - -1 mL Date & Initials Attachment A Extraction EoTf PSF-8O-4S.1from Serum 3M Environmental Laboratory Page 10 of 14 Page 77 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 MDL/LOQ values for rabbit serum Com pound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 5 ppb - 1000 ppb PFOSA 1.51 4.79 5 ppb - 1000 ppb PFOSAA 3.46 20.5 5 ppb - 1000 ppb EtFOSE-OH 11.4 36.2 5 ppb - 1000 ppb M 556 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: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 11 of 14 Page 78 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: PFOS Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 Compound: PFOSA , Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 24.8 - 976 0.993 - 976 Compound: PFOSAA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.991 -974 4.92 - 247 49.2 - 974 0.991 - 974 LCR from curve (PPb) (ng/mL) 24.8 - 978 4.94 -248 97.8-978 4.94 - 978 % Recovery Range 83-108 85-104 85-106 94-111 LCR from curve (n(gP/PmbL) ) 4.93 - 976 4.93 - 97.6 24.8 - 978 4.93 - 976 % Recovery Range 88-103 87-105 93-102 94-103 LCR from curve (PPb) (ng/mL) 24.7 - 974 9.74 - 247 97.4 - 974 9.74 -974 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 Attachment B: MDL/LOQ Summary ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 12 of 14 Page 79 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: EtFOSE-OH Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range 0.993 - 976 Low Curve 4.93 - 97.6 High curve 49.3 - 976 1/X 0.993 - 493 Compound: PFOSEA Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range 0.993 - 976 Low Curve 4.93 - 248 High curve 1/X 49.3 - 976 0.993 - 976 Compound: M556 Prepared range Rabbit Serum of standards (ppb) (ng/mL) Full Range Low Curve High curve 1/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (n(gP/PmbL) ) 49.3 - 976 9.76-97.6 97.6 - 976 9.76 - 976 % Recovery Range 77-110 97-107 90-109 86-111 LCR from curve (n(gP/PmbL) ) 24.8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 % Recovery Range 96-106 91-110 86-106 95-117 LCR from curve (n(gp/pmbL) ) 24.8 - 976 9.76-97.6 97.6 - 976 9.76-976 % Recovery Range 88-106 100-105 81-111 97-110 RSD Range 11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2 RSD Range 10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1 RSD Range 4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5 Attachment B: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 13 of 14 Page 80 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U22 79 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: Method/revision: Target analyte(s): _ FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix SuPtdgF/cmOoSLne 0.500 0.500 5.00 55..0000 55550000....000 PFOSA Sut0dg.5/cm0o7Lne 0.507 5.07 5.07 5.07 5500..11 50.1 50.1 PEOSAA Sut0dg.5/cm3o2Lne 0.532 5.32 5.32 5.32 5533..22 53.2 53.2 EtFOSE Sut00dg..55/cm00o11Lne 55..0011 5.01 5500..11 50.1 50.1 PSFut0dgO.5/cmS2o1ELnAe 055...5222111 552.2.11 5522..11 52.1 M556 Sut0dg.5/cm0o1Lne 0.501 55..0011 5.01 55550000....1111 All All Am't Final voi spiked mL mL 0.0200 . 0 1 0 | ; 1.015 1.025 0.005 ! 1.010 0.0200 . 0 1 0 i 1.015 1.025 0.005 1.010 0.010 1.015 0.015 1.020 0.020 1.025 Calculated concentrations of standards in the sample matrix PFOS - PFOSA PFO SA A E tFO SE PFO SE A M 556 S urrogate Final cone Final cone Final cone Final cone Final cone Final cone Std cone ng/m L 4.93 9 .7 6 ng/m L 5 .0 0 9 .8 9 ng/m L 5.24 10.4 ng/m L 4.94 ; 9.78 ng/m L 5.01 9.93 ng/m L ; 5.13 ' 10.2 ng/m L 100 24.8 : 25.1 26.3 24.8 25.2 : 25.8 S urrogate 49.3 i 50.0 52.4 49.4 50.1 i 51.3 Final cone 97.6 98.9 104 97.8 99.3 102 ng/m L 248 . 251 263 248 252 ! 258 500 493 500 524 494 501 ! 513 735 ! 746 782 : 737 749 1 766 976 ; 989 1038 978 993 ! 1017 A ll A m 't spiked mL 0.005 Validated ranges - approximate concentrations S eru m PFOS PFOSA PFOSAA E tF O SE -O H PFOSEA M S56 R abbit 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 B ovine E stim ates only. U se values for rabbit. Rat E stim ates only. U se values for rabbit. M onkey & Plasm a E stim ates only. U se values for rabbit. H um an E stim ates only. Use values for rabbit. Attachment C: Ion Pair Standard Curves ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Page 14 of 14 Page 81 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory M ethod A n a ly sis q f Po tassium Perfluo ro o ctanesulfo nate or O th er F lu o r o c h em ic a ls in Ser u m E x tra cts U sing H P L C -E lectrospray/M ass Spectrom etry M ethod Num ber: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: Adoption Date: 03/01/99 Revision Date: Laboratory Manager Group Leader /-/? .--------------- TechnicaAl RCebvmietw<e-r Date Date Date 1.0 Sc o p e a n d A p p l ic a t io n _________________________________________________________________ 1.1 Sucsionpge:HPTLhCis-emleectthroodspdreasyc/rmibaesss tshpeecatnraolmyseitsryo.f serum extracts for fluorochemical surfactants 1.2 oAtpheprliicoanbizleabCleomcopmopuonudnsd:sF. luorochemical surfactants or other fluorinated compounds, or 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 1of 9 3M Environmental Laboratory Page 82 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 S u m m a r y o f M e t h o d ________________________________________________________ ________ __ 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. Tfluheoraoncahleymsiiscaisl,pseurcfohramsetdhebypemrfoluniotrooroincgtaanessiunlgfloeniaotne (cPhFarOaSct)erainsitoicn,omf a/zp=ar4t9ic9u.lar Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity o f a compound by detecting daughter ions of the parent ion. 3.0 D e f in it io n s ___________________________________________________________________________ _ 3.1 A tm ospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 E lectrospray Ionization (ES, ESI): a method o f ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 M ass Spectrom etry, M ass Spectrometer (MS), Tandem Mass Spectrometer (M S/M S): The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently dspeteeccifteicd.fraAgmsinengtlaetiMonS imnfaoyrmbaeteiomnp. loyed for ion detection or a series (MS/MS) for more 3.4 C onventional 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 cmnooamtincpotaemtnipabnalectiewblaietrhewstihothemsoeanmoeteha.enroHtZho-ewsrpe,rvbaeuyrt,soyZns-tlsyepmrwasiy,thectoscmi.m)piloanresnytsstaemnds c(oi.nev.,eZnt-isopnraaly ccoommppoonneennttss aarree 3.5 M ass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 (vMerisciroonms.asAslQl vuearttsrioonIsI atrriepsleimqiulaard.ruFpoorlemMoraessdLeytanixlsosreeMtahsesLmyannxuaNl TspUecsiefric'stGo uthideei)n.strument 4 .0 W a r n in g s a n d C a u t io n s _____________________________________________________________ 4.1 Health and Safety W arnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 4.1.2 aWndhecnlohthanindgl.ing samples or solvents wear appropriate protective gloves, eyewear, ETS-8-5.1 Analysis of Serum Extract Using ES.'MS Page 2 of 9 3M Environmental Laboratory Page 83 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 _I n t e r f e r e n c e s ._____________________________________________________________________ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract. 6 .0 E q u ip m e n t ______________________________________________________________________________ 6.1 mEqoudiipfimcaetnitonlisstiendthbeelroawwmdaatyabaes mmoetdhiofideddeinvioartdioenrst.o optimize the system. Document any 6.1.1 MeleicctrroomsparsasyQiuoantitzraotiIoIntrsiopulercqeuadrupole Mass Spectrometer equipped with an 6.1.2 cHoPm1p1a0r0tmloewnt,paunlsde asuotlovseanmt ppulemr ping system, solvent degasser, column 7 .0 S u p p l ie s a n d M a t e r ia l s ________________________________________________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes 8 .0 R e a g e n t s a n d St a n d a r d s ______________________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 pTryeppiacraeldlydtuwroingmtehtheoedxtbrlaacntikosn, tpwroocmedautrreix. bSleaenkEsT, aSn-d8-e4i.g1h. teen matrix standards are 9.0 Sa m p l e H a n d l in g _______________________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. Analysis of SerEumTSE-8x-t5r.a1ct Using ES/MS 3M Environmental Laboratory Page 3 of 9 Page 84 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 9.2 Iafpapnraolxyismisawteillyl 4be Cd,eloaryeadt ,roeoxmtratcetmedpesrtaantudraer,dusnatnildasnaamlypsliesscacannbbeepreerffroirgmereadt.ed at 10.0 Q u a l i t y C o n t r o l _____________________________________________________________________ 10.1 Solvent Blanks, M ethod Blanks and M atrix Blanks 10.1.1 Seaoclhvebnattcbhlatnokds,etmeremthionde bcolanntkams ainnadtimonatroirxcbalrarnykosvearr.e prepared and analyzed with 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Amninailmyzuema omfa2trsipxikspeiskpeearnbdamtcha.trix spike duplicate per forty samples, with a 10.2.4 tMheatirnixitisaplickaeliabnrdatmioantrciuxrvspe.ikAe ddduiptliiocnaatel scpoinkceencotrnacteionntrsawtioilnl sfamllaiyn ftahlel imnitdh-eralnowge- of range of the initial calibration curve. 10.3 Continuing Calibration Verifications 10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy of the calibration curve. 10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum o f one per batch. 11.0 C a l i b r a t i o n a n d St a n d a r d iz a t io n __________________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average o f two standard curves will be plotted by linear regression (y = my + b), weighted 1/x, not forced through zero, using MassLynx or other suitable software. 11.2 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels o f analyte, it may be necessary to cuEasxleaibmtrhpaetleiloo:nwwceuhnredvneoacftottehnmesipcstatiilnnibggrtoaotfiqothnueacnsuttraitvnaedtearraadtphspefrrrootxhmiamn5attphepelybfut1lo0l r1pa0pn0bgpeopfobafnrtahatelhyestetra,tnhgdaeannredtrhacetuefruavlel. range o f the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. Analysis of SerEumTSE-8x-t5r.a1ct Using ES/MS 3M Environmental Laboratory Page 4 of 9 Page 85 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.0 P r o c e d u r e s ____________________________________________________________________________ 12.1 Acquisition Set up 12.1.1 Cafolfriiclaekcnqoaunmirseitnaugrst,inbagnudtMtotnyOpi-enDiAtnhYesa-AlmacspqtluedisdigietiistoconrifpCytoeioannrtrs-so.almPpanleeln.uSmetbeurp, aasssaimgnplaemliestt.hoAds(sMigSn) 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the pSrIRodsu. ctSaiovne farcaqgumiseintitoantiomneitnhfoodr.mIaftiMonS/mMaSy binestcroullmecetnetds.arSeeeemMpilcoryoemd,aassdditional MMRasMsLy(MnxulGtiUplIeDREeaTcOtioDnAMToAniAtoCrQinUg)I.SITION for additional information and 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Set-up the HPllOO/autosampler at the following conditions or at conditions the iannsatrlyusmt ecnotnsloidgebrosoakp:propriate for optimal response. Record actual conditions in the 12.2.2.1 Sample size = 10 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 8.50 min. 11.0 min. 12.0 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 5 of 9 Page 86 3m Medical Department Study: T-6295.7 Report No. FACT T0X-C3C laboratory Request Number-U2279 12.3.3 Check the stainless steel capillary at the end o f the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary'. 12.3.4 SaOpebtpsrHeorPxvLiemCdartpoeuplymlept1s0tcomo"minOiunntg"es.o.Suettothf ethfelotwip toof 1th0e-p5r0o0beu.LA/mllionwortoaseqaupiplirborpartieatfeo.r 12.3.5 Taruomunoditththeetinpitorfogthene.prAobfein. eRmeaisdtjushstouthlde tbipe eoxfptehlelepdrowbiethifnnoonmitrisotgiesnolbesaekrivnegd. 12.3.6 Tchhaenignestirnuomrednetrutoseosptthimesiezepathraemreesteprosnaste:the following settings. These settings may 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 Iiters/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 ensure the HPLC is operating correctly.) 12.3.7 Cfuarrtheefur.llCy ognunideectththeepvroolbtaegientcoatbhleesotpoenthinegp.roIbnes.ert probe until it will not go any 12.3.8 tParpinedt tihnetotutnhee pinasgter,umwietnht iltosgp.arameters, and store it in the study binder with a copy 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis o f biological matrices. 12.3.10CbMulaitctsoksnLo.ynnEsxntasvruetrrbesuisottntaorsnt, asiennedthaeepnpdArcosaqpmruiiapstileteiMonnuamCssboLneyrtrnioxnlcUPluaSdnEeesRl a('tSlhl iGssaUmmIapDylEevs)a.troyPbraeemsasontnahgleyzsteadr.t 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s _________________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (pg/mL): fng of PFOS calc, from std. Curve x Dilution Factor) x 1 ug (Initial Volume of matrix (mL) + mL o f Surrogate Standard) lOOOng Final Volume (mL) Analysis of SerEuTmS-E8-x5tr.a1ct Using ES/MS 3M Environmental Laboratory Page 6 of 9 Page 87 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 14.0 M e t h o d P e r f o r m a n c e _______________________________________________________ ________ _ 14.1 mMMaeDttrLhioxadsnpDdeeLctieOfciQct.iovnPallLeuaiemsse.itse(Me EDTLS)-a8n-4d.1L,imAitttaocfhQmueannttitBa,tifoonr (aLlOisQtin) garoefmcuertrheondt, vaanliadlyattee,dand 14.2 Solvent Blanks, M ethod Blanks, and M atrix Blanks 14.2.1 Sloowlveesnt tstbalnadnakrsd, minetthheodcablilbarnaktsi,onancdurmvaetrix blanks values are must be below the 14.3 C alibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 M atrix Spikes 14.4.1 Mcoantcreinxtrspatikioenp. ercent recoveries are must be within 30% of the spiked 14.5 Continuing Calibration Verifications . 14.5.1 cCoonncteinnturiantgiocna.libration verification percent recoveries must be 30% of the spiked 14.6 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 Ifofodtantoataerdeotno btaebrleespoarntdeddwishcuenssepderifnortmheatnecxet corfittehreiarhepaovret.not been met, the data must be 1 5 .0 P o l l u t i o n P r e v e n t io n a n d W a s t e M a n a g e m e n t ___________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s _______________________________________________________________________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, ainntaelgyrsat.tion method, sample name, extraction date, dilution factor (if applicable), and 16.2 aPpripnrtotphreiattuensetupdaygef,olsdaemr.plCe olipsty, tahnedseacpqaugiessitaionnd mtaeptehoindtofrtohme iMnsatrsusLmyennxt rtounilnocgl.ude in the 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. Analysis of ScrEumTSE-8x-t5r.a1ct Using ES/MS 3M Environmental Laboratory Page 7 of 9 Page 88 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 16.5 Sum m arize data using suitable software (Excel 5.0) and store in the study folder, see A ttachm ent A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 T a b l e s . D ia g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a _____________________________ 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 R e f e r e n c e s ____________________________________________________________________________ 18.1 FACT-M-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 18.2 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems" 18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l, 19.0 A f f e c t e d D o c u m e n t s _________________________________________________________________ 19.1 ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R e v is io n s ______________________________________________________________________________ RNuevmi1sbieorn. Section 6.1.2 ClarificationRoeaf sHonP1F1o0r0Rseyvstiseimoncomponents. Section 11.1 Average of two curves, not standard values, are used for pSleoctttiionng 1li2n.e2a.r2.r4egCrleasrsiifoicnatainodn aodfdseodlvtehnet 1r/axmwp.eighting of the curve. Section 17.1 Changed from attachment B to A. R04eD/v0ai2sti/eo9n9 Analysis of SerEuTmSE-8x-t5r.a1ct Using ES/MS 3M Environmental Laboratory Page 8 of 9 Page 89 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Laboratory Study # TSteusdtyM: aterial: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument SoftwareAversion: Filename: R-Squared Value: Slope: DYaItneteorfcEepxtt:raction/Analyst: Date of Analysis/Analyst: Group Sample# Concentration Dose ug/mL Initial Voi. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 AnalysisofSerumExtractUsing ES/MS 3M Environmental Laboratory Page 9 of 9 Page 90 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory M ethod E x t r a c t i o n -o f P o t a s s i u m P e r f l u o r o o c t a n e s u l f o n a t e o r O t h e r Flu o r o c h em ic a l co m po unds fro m Serum fo r A nalysis U sing H P L C - E lectro spray/M ass Spec tr o m etry M ethod Number: ETS-8-4.0 Adoption Date: 3 / / / / 7 Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: Laboratory M anage/ 'kiTl-i-G---r--oyu/Ap vA -cLeader ' ------------------------------------------------------------------------------ 37 A Date m.3 / 1 Date c; y .4 y l i u s Technical Reviewer y /y Date 1.0 Sc o p e a n d A p p l ic a t io n 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 A pplicable com pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 tMheatvrailciedsa:tioRnabrebpito,rtr.at, bovine, monkey, and human serum or other fluids as designated in Word 97 SR-1 Extraction EofTPSF-8O-4S.0from Serum 3M Environmental Laboratory Page I of 13 Page 91 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2 .0 S u m m a r y o f M e t h o d __________________________________________________________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum using an ion pairing reagent and e5x.0tracmteLd:ofPFmOetSh,yPl-FfeOrfS-Abu, tPylFOethSeArA(M, EtBtFEO).SEIn-OthHis, PmFeOthSoEdA, s,eMve5n56fl,uaonrdocshuermroigcaatles were astnaanldyaterdio(nsepea3ir.0isDpeafrintiittiioonnesd).intAonMiotnBEp.airTihnge MreatBgeEntexistraadcdt eisdrteomthoevesdamanpdlepauntdonthteo a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 ml of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon Filter into glass autovials. 3.Q D e f i n i t io n s ______________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) CsFpSOj' 3.2 PFOSA: perfluorooctane sulfonylamide C8F 17SO2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S0 2N(CH2CH3)CH2C0 2 ' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C 8 F ,7 S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F |7S0 2 N(CH2CH3)H 3.6 M556: C8F 17S02N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W a r n in g s a n d C a u t io n s ________________________________________________________ 4.1 Health and safety warnings 4.1.1 hUasnedulinnigvearnsiaml aplreticsasuutei,onwsh,iecshpmecaiayllcyonlatabionraptaotrhyocgoeantss., goggles, and gloves when 5.0 I n t e r f e r e n c e s ___________________________________________________________________ 5.1 There are no interferences known at this time. 6.0 E q u i p m e n t _______ ____________________________________________________________________ 6.1 Tachceepfotallbolwe.ing equipment is used while performing this method. Equivalent equipment is 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) Extraction EofTPSF-8O-4S.0from Serum 3M Environmental Laboratory Patte 2 of 13 Page 92 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 7.0 Su p p l ie s a n d M a t e r ia l s __________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 ml 7.9 Syringes, capable of measuring 5 jjL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe Filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with sMepilalri-aQteTMviwalast.er. Rinse syringes a m---inimum of 9 times with methanol, 3 rinses from 3 8.0 R e a g e n t s a n d St a n d a r d s _____________________________________________________________ 8.1 TbeypMe iIllri-eQageMntwgartaedreanwdatmera,yMbiellpi-rQoTMvidoerd ebqyuaivMaleilnlit-;QalTl OwaCtePrluusseTMd isnysttheims method should 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (Na2C0 3 ), 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 Fluorochem ical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), m olecular weight = 570 Extraction EofTPSF-8O-4S.0from Serum Page 3 of 13 3M Environmental Laboratory Page 93 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557 8.9.7 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1 -H, 1-H, 2-H, 2-H CgF^SOaH) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation 8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 mL of 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using 8.10.3 0M.5illMi-QteTMtrawbautteyrl.amSmtoorneiiunmah1y2d5romgLenNsaullgfeantee (bToBttAle).: Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = needed using 1 N NaOH solution. 10. Adjust as 8.10.4 0a.p2p5roMximsoadtieulmy 2c6a.5rbgonoaftseo/sdoiduimumcabribcoanrabtoena(Nteab2CufOfejr) a(NndaiC21O.0j/Ng aoHf sCoCdibu)m: Weigh bicarbonate (NaHC0 3 ) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fsloulourtioocnhecmonictaailnsintagnd1a.0rd0spaprme aPcFceOpSta, b1l.e02(foprpmexaPmFOplSe,Ao, n0e.9w87orpkpinmg PstFanOdSaArdA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately the actual weight. 100 mg of PFOS into a 100 ml volumetric flask and record 8.11.4 B(prgin/mg lt)o. volume with methanol for a stock standard of approximately 1000 ppm 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. ETS-8-4.0 Extraction of PFOS from Scrum 3M Environmental Laboratory Page 4 of 13 Page 94 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 mg of surrogate standard 1-H, 1-H, 2-H, 2-H, C8F 13SO3H into a 50 ml volumetric flask and record the actual weight. 8.12.2 pBprmin.g .to volume with methanol for a surrogate stock of approximately 1000-1200 8.12.3 Prepare wstoorckkintog aastsa1un0rdrmaorgldavtooelfuwm1o0re0ktripincpgmfsl.atasRnkdeaacnroddr.dbrTtihrneagnatscofteuvraolalupvpmorleuomxwimeithtartamenlyestfhe1rarmneoldl.offosruarrogate 9 .0 __S a m p l e H a n d l in g ____________________________________________________________________ 9.1 All samples are-received frozen and must be kept frozen until the extraction is performed. 1 0 .0 Q u a l i t y C o n t r o l _____________________________________________________________________ 10.1 M ethod blanks and matrix blanks 10.1.1 aEsxtmraectht otwd obl1a.n0kms.l aliquots of Milli-QTM water following this procedure and use 10.1.2 Emxattrraixctbtlwanok1s..0 SmeLe aliquots 11.1.4, of the serum following this procedure and use as 10.2 M atrix spikes 10.2.1 Pthreepaacrceuraancdyaonfatlhyezeexmtraatrcitxiosnp.ike and matrix spike duplicate samples to determine 10.2.2 mPraetpraixrereeacecihvesdpikweithuseinacghassaammpplleesceht.osen by the analyst, usually the control 10.2.3 EcAaxdlpidbeirtcaitoteindoanlcocsnupcrikevnees.trmataioynbsewinilcl lfuadlle dinatnhde mid-range may fall in othf ethleo win-irtainalg ecaolifbtrhaetioinnitciaulrve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare and analyze continuing calibration check samples to ensure the accuracy of the initial calibration curve. If the percent difference between the initial curve alansdt athcececpotnatbilneucinhgecckh.eck differ by >30%, re-analyze samples analyzed after the 10.3.2 Prepare one continuing check per group of 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Pthreepinariteiaelaccuhrvcoe.ntinuing calibration check from the same matrix used to prepare 10.3.4 The expected concentrations fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial Extraction EofTPSF-8O-4S.0from Serum 3M Environmental Laboratory Pase 5 of 13 Page 95 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 calibration curve. This is necessary if the analyst must quantitate using only the 5lowppben-d 1o0f0t0heppcabl)i.bration curve (for example, 5 ppb - 100 ppb, rather than 11.0 C a l i b r a t i o n a n d S t a n d a r d iz a t io n _________________________________________________ __ 11.1 Prepare m atrix calibration standards 11.1.1 Transfer 1 mL of serum to a 15 mL centrifuge tube. 11.1.2 Ivmfoamlturomixste. ssRaemeqcupoalrledtvoeoatlchuhemsseaasmmapprelleelevvsooslluuthmmaeenso.1n.0DthomeLneo,xtetxerxatrtcartaicotcntstlsaehnsesdeatthr. dans w0.i5th0 mmaLtroixf 11.1.3 While preparing a total of twenty aliquots in 15 ml centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at ethigehetneednosfttahnidsasredcst,iotnw,otomsaptrikixe,bilnandkusp, laicnadtet,wtowmo esttahnoddarbdlacnukrsv. es, for a total of 11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 csUtaasleinbdAraatrttdiaoscn.hmsSteaeenndtSaDercdtasiso. nan1a3i.d0 itno ccaallccuullaattienagctthuealccoonncceenntrtraatitoionnssooffthPeFOwoSrkining 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 11.3 tEoxetrsatacbt lsipshikeeadchmaintritixialstcaunrdvaerdosnftohlelomwainssg s1p2e.c6t-r1o2m.1e6tero.f this method. Use these standards Extraction EofTPSF-8O-S4.0from Serum 3M Environmental Laboratory Page 6 of 13 Page 96 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 ml of matrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix ' - - Blank 0.500 ppm 10 0.005 ppm 0.500 ppm 20 0.010 ppm 5.00 ppm 5 0.025 ppm 5.00 ppm 10 0.050 ppm 5.00 ppm 20 0.100 ppm 5(1.0 ppm .5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 . 1.00 ppm 12.0 P r o c e d u r e ____________________________________________________________________________ 12.1 Obtain frozen samples and allow to thaw. 12.2 Vpoolrytperxompyilxenfoerc1e5ntsreifcuognedstu, bthe.en transfer 1.0 mL or other appropriate volume to a 15 mL 12.3 Return samples to freezer after extraction amount has been removed. 12.4 Record the volume on the extraction worksheet. 12.5 Lwaobreklshtheeettufobre dwoicthumtheentsitnugdythneurmembeari,nsinamg pstleepIsD. , date and analyst initials. See attached 12.6 sStpanikdearadll assamdepslcersi,biendcliund1in1.g2.blanks and standards, ready for extraction with surrogate 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table c1oinntitnhuatinsgecctaiolinb,rafotironthestacnaldiabrrdast.ion curve standards. Also prepare matrix spikes and 12.8 Vsaomrtpelxesmfoixr t1h5e ssetaconnddarsd. curve samples, matrix spike samples, and continuing calibration 12.9 Tbiocaerabcohnsaatembpuleff,eard. d 1 mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium 12.10 Using an Oxford Dispenser, add 5 mL methyl-terf-butyl ether. 12.11 Cap each sample and put on the shaker for 20 minutes. 12.12 Cseepnatrraitfeudg.e for 20 to 25 minutes at approximately 3500 rpm, until layers are well 12.13 Remove 4.0 mL of the organic layer to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. Extraction EofTPSF-8O-S4.0from Serum Page 7 of 13 3M Environmental Laboratory Page 97 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 12.14 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.15 Add 1.0 mL or other appropriate volume of methanol to each centrifuge tube using a gthreadeuxatrteadctipoinp.ette. Methanol volume to add equals the initial volume of sample used for Note: If the initial volume is less than 0.500 mL the final methanol volume will equal 1.0 mL. 12.16 Vortex mix for 30 seconds. 12.17 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.18 Lmaabtreilxt,hfeinaaultsoovlivaelnwt,itehxttrhaecsttioundydantuem, abnedr, aannaimlyastl(sn)upmebreforramnidnggetnhdeeerx,tsraamctpiolne.timepoint, 12.19 Cap and store extracts at approximately 4 C until analysis. 12.20 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s ________________________________________________ 13.1 Calculations 13.1.1 cCalaiblcrualtaiotenascttaunadlacrodnscuesnitnrgattihoensfoolfloPwFiOngS,eoqruaottihoenr: applicable fluorochemical, in mL of standard x concentration of standard (ug /mL)___________________ = mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) of PFOS in matrix 1 4 .0 M e t h o d P e r f o r m a n c e _______________________________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachm ents B and C). 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks. 14.2.2 pMraetcrisixiosnpiokfethaendexmtraatcrtixionsp. ike duplicate samples to determine accuracy and 14.2.3 Cinoitniatilncuailnibgrcaatiloibnractuirovne.check samples to determine the continued accuracy of the 15 .0 P o l l u t io n P r e v e n t io n a n d W a s t e M a n a g e m e n t _______________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. Extraction EofTPSF-8O-4S.0from Serum 3M Environmental Laboratory Page 8 of 13 Page 98 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 16.0 R e c o r d s ______________ _________________________________________________ _ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 A t t a c h m e n t s _______ ____________________________________________ ____________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 R e f e r e n c e s _______________________________________________________ __ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. 19.0 A ffec ted D o c u m en ts ____________________________________________________________ ____ 19.1 EHTPSL-C8--E5.l0ec, tr"oAspnraalyysMis aosfsSSepruecmtroormOettrhye"r Fluid Extracts for Fluorochemicals using 20.0 R e v is io n s ____________________ __________________________________________________________ RNeuvmisbioenr Reason For Revision ReDvaistieon Extraction EofTPSF-8O-4S.0from Scrum 3M Environmental Laboratory Page 9 of 13 Page 99 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Extraction Worksheet ETS-8-4.0 Study #/matrix Surrogate Std approx. ppm actual ppm # FC-Mix approx. 0.5 ppm actual ppm # FC-Mix approx. 5 ppm a#ctual ppm FC-Mix approx. 50 ppm actual ppm # Date and Initials for Std. or Comments .-- _ --- _ - - .. - - _ 1. - - -- - -- - - -- -- -- -- - - -- --- --- -- - -- --- --- --- --- --- ' Studv numher w acre the original worksheet is Inmteri Blank Std # amount = ml. Serum Extraction Method__________________________;________________________________________ Date & Initials V o rte x 15 sec.___________________________________________________________________________________________________________________________________ P ip e tte M a trix _________________________________________________V o lu m e ________________________m i______________________________________________ P ip e tte 1 m l o f 0 .5 M T B A , p H 10._______________________ S td . # _____________________________________________ P ipette 2 ml o f 0.25 N a.C O ,/Q .25M N aH CO x buffer Std. # D is p e n s e 5 m l o f m e th y l-t-b u ty l e th e r__________________________________ T N -A - _________________________________ S h a k e 2 0 m in .______________________________________________________________________________________________________ C e n trifu g e 2 0 -2 5 m in._________________________________C e n trifu g e sp e e d :____________________________________________________________________ R e m o v e a 4 m l. a liq u o t o f o rg a n ic la y e r_________________________________________________________________________________________________ ___ P u t o n N itro g e n E v a p o r a to r to d rv n e ss E v a p o ra to r #:___________________________ T e m p e ra tu re :________________________________________ A d d m e th a n o l___________________ V o lu m e m l_______________T N -A - _____________________________ V o rte x 3 0 se c . _____________________________________________________________________________________________ F ilte r u s in g a 3 c c R -D s y rin g e w ith a 0 .2 u m filte r in to a 1.5 m l a u to s a m p le vial_______________________________________________________ M S / M S D / ____ C o n t . C h e c k s : S p i k e d _________u L o f a _________p p m s td ( ______________________ ) f o r a f in a l c o n c e n t r a t i o n o l _____________p p m . M S / M S D u s e d s a m p l e _______________________ . C o n t. C h e c k s u s e d s a m e m a tr ix a s f o r s td c u r v e . S u r r o g a t e S t a n d a r d : S p i k e d _______ u L o f a _________p p m s td (______________________) to a ll s a m p le s , s t a n d a r d s , a n d b l a n k s Attachment A ETS-8-4.0 Extraction of PFOS from Scrum 3M Environmental Laboratory Page 10 of 13 Page 100 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03Q laboratory Request Number-U2279 M DL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 4.76 15.2 5 ppb - 1000 ppb PFOSA 2.89 9.19 5 ppb - 1000 ppb PFOS A A 2.94 9.33 5 ppb - 1000 ppb EtFOSE-OH 13.2 41.9 5 ppb - 1000 ppb M556 12.6 40.2 5 ppb - 1000 ppb PFOSEA 11.1 35.4 5 ppb - 1000 ppb MDL/LOQ values in rat, bovine, monkey, and human serum were not statistically determined. Two curves in each of these three matrices were extracted and analyzed with the rabbit serum curves to determine equivalence. Responses in the rat, bovine, and monkey were equivalent to the rabbit responses, therefore, their MDL and LOQ will be the same values as determined in rabbit serum. Pinlfeoarsme asteieonA. ttachment C (LOQ Summary) and MDL study in ETS-8-4.0 & 5.0-V-l for further Ion Pairing Extraction of Fluorochemicals from Serum and Analysis by API/M S(M S) Summary Table: Limits of Quantitation Compound PFOS PFOSA Matrix All All MDL 4.76 2.89 PFOSAA All 2.94 EtFOSE-OH* All 13.2* PFOSEA All 12.6 M556 All 11.1 * MDL and LOQ are estimates only for EtFOSE-OH. LOQ 15.2 9.19 9.33 41.9* 40.2 35.4 Low std 25.1 25.0 25.0 50.0 25.0 25.0 High std 1002 1000 998 1000 1000 1000 Attachment B: MDL/LOQ ETS-8-4.0 Extraction of PFOS from Serum 3M Environmental Laboratory Page I I of 13 Page 101 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: PFOS Prepared Serum range of matrix standards (ppb) (ne/mL) Rabbit 1 .0 0 - 1002 Range of average curve LCR from ave curve (ppb) (riR/mU (pob) (ng/mL) 1.00 -1002 25.1- 1002 Rloawngestdof curve (ppb) (ng/mL) 5.01-250 LCloRw fsrtodm curve (pob) (ng/mL) 5.01-250 Range of high std curve (ppb) ine/mL) 100-1002 LCR from high std curve (pob) (ng/mL) 100-1002 Compound: PFOSA Prepared Serum range of matrix (psptba)nd(nagr/mdsL) Rabbit 1.00- 1000 Range of average (ppbc)u(rnvge/mL) 1.00-1000 LCR from ave curve (ppb) (ng/mL) 10.0- 1000 Range of low std (ppbc)u(rnvge/mL) 5.00-100 LCR from low std (ppbc)u(rnvge/mL) 5.00-100 Range of high std (ppbc)u(rnvge/mL) 25.0 - 1000 LCR from high std (ppbc)u(rnvee/inL) 2 5 .0 - tOOG Compound: PFOSAA Prepared Serum matrix srtaanngdearodfs (ppb) (ng/mL) Rabbit 1.00 - 998 Range of average (ppbc)u(rnvge/mL) 1.00-998 LCR from ave curve (ppb) (ng/mL) 25.0-998 Range of low std curve (ppb) (ng/mL) 4.99 - 250 LCR from low std curve (ppb) (ng/mL) 9.98 - 250 Range of high std (ppbc)u(rnvge/mL) 49.9-998 LCR from high std (ppbc)u(rnvge/mL) 99.8 - 998 Compound: EtFOSE-OH Prepared Range of Serum range of average matrix standards (ppb) (ng/mL) 1j curve (ppb) (ng/mL) Rabbit j 1.00-1000 1.00 -1000 LCR from ave curve (pob) (ng/mL) 50.0- 1000 Range of low std curve (ppb) (ng/mL) 5.00 -100 LCR from low std curve (ppb) (ng/mL) 10.0 -100 Range of high std curve (ppb) (ne/mL) 50.0-1000 LCR from high std curve (ppb) (ng/mL) 100 -1000 Compound: PFOSEA Prepared Serum range of matrix standards (ppb) (ne/mL) Rabbit 1.00 - 1000 Range of acvuerravgee (ppb) (ng/mL) 1.00-1000 LCR from ave curve (ppb) (ng/mL) 25.0-1000 Range of low std curve (ppb) (ne/mL) 5.00 - 250 LCR from low std curve (ppb) (ne/mL) 5.00 - 250 Range of hicguhrvsetd (ppb) (ng/mL) 50 -1000 LCR from hciguhrvsetd (ppb) (ne/mL) 50 -1000 Compound: M556 Prepared Serum range of matrix (psptba)nd(nagr/dmsL) Rabbit 1.00 - 1000 Range of average curve (ppb) (ng/mL) 1.00-1000 LCR from ave curve (ppb) (ng/mL) 25.0-1000 Rloanwgestdof curve (ppb) (ng/mL) 5,00 -100 LCR from low std (ppbc)u(rnvge/mL) 5.00-100 Range of high std curve (ppb) (ng/mL) 100 -1000 LCR from high std curve (ppb) (ng/mL) 100 -1000 Attachment B: MDL/LOQ ETS-8-4.0 Extraction of PFOS from Serum 3M Environmental Laboratory Page 12 of 13 Page 102 3m Medical Department Study: T-6295.7 Report No. FACT T O X -0 3 0 laboratory Request Number-U2279 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: SAanmalpyltee(ms)a: trix: Equipment number: Final solvent and TN: Blank fluid/identifier: Method/revision: Target analyte(s): _ FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: APcFtuOaSl conPceFnOtSraAtionsPFoOf sStAaAndarEdtsFiOnStEhe FPCFOmSiEx A Std cone Std cone Std cone Std cone Std cone M556 Std cone ug/mL ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.507 0.532 ; 0.501 1 0.521 0.501 0.500 0.507 0.532 I 0.501 : 0.521 : 0.501 5.00 5.07 5.32 ; 5.01 i 5.21 ' 5.01 5.00 5.07 5.32 1 5.01 ! 5.21 ; 5.01 5.00 5.07 5.32 .....5 01 1 5.21 ; 5.01 50.0 50.1 53.2 50.1 i 52.1 ! 50.1 50.0 50.1 53.2 50.1 [ 52.1 1 50.1 5. ; 50.1 53.2 50.1 ! 52.1 I 50.1 50.0 : 50.1 53.2 50.1 52.1 1 50.1 All All Ami Final vol spiked mL mL 0.010 : i.oi5 0.020 1.025 0.005 1.010 0.010 1.015 0.020 1.025 0.005 1.010 0.010 1.015 0.015 1.020 0.020 1.025 Calculated concentrations of standards in the sample matrix FinnP49agF..l/97mOc36oLSne 4294..38 9479279374.35686 FPinnFagOl/mcSoLAne 5.00 9.89 i !1': 92592575805840..1.960190 1i 1 FPiFnn5ga1O.l/02mS.c44AoLnAe 5226..34 521260344 1708328 1 FEinnt4Fag.l/O9mc4SoLnEe j! 4929.47..488 ji! 942794.488 i 737 ! 978 : FPiFnn5gaO.l/0mS1cEoLnAe ;: 925.059..132 9925790954.13293 FinMa5l 5c6one ng/mL 5.13 10.2 25.8 ! 51.3 ! 102 258 i 513 ' 766 1 1017 SSuntrdgr1/o0cmo0gLnaete SFuinrarlocgoantee ng5/0m0L Am'tAmslLpliked 0.005 Validated ranges - approximate concentrations S eru m PFOS PFOSA PFOSAA R ab b it 25.1-1002 | 25.0-1000 | 25.0-998 B ovine E stim ates only. U se values for rabbit. Rat E stim ates only. U se values for rabbit. M onkev E stim ates only. Use values for rabbit. H um an E stim ates only U se values for rabbit. E tF O S E -O H PFOSEA | 50.0-1000 1 25.0-1000 M 5S6 | 25.0-1000 Attachment C: Ion Pair Standard Curves ETS-8-4.0 Extraction of PFOS from Scrum 3M Environmental Laboratory Page 13 of 13 Page 103 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03Q laboratory Request Number-U2279 3M Environmental Laboratory M ethod A n a ly sis of P o tassium Perflu o ro o ctan esu lfo n ate or O th er F luo r o ch em ic als in S erum E xtra cts U sing H P L C -E lectrospray/M ass Spectro m etry M ethod Number: ETS-8-5.0 Author: Lisa Clemen, Robert Wynne Approved By: Laboratory Manager i b i ---------- -- Group Leader ; fi Technical Reviewer Adoption Date: 7 / 1/-7 ;/ Revision Date: 11/ T 7 Date -?/' r - '\ Date lill'r , Date 1.0 S c o p e a n d A p p l ic a t io n _______________________________________________________________________ 1.1 uSscionpgeH: PTLhCis-emleectthroodspirsafyo/rmtahses analysis of extracts spectrometry. from serum for fluorochemical surfactants 1.2 oAtpheprliicoanbizleabCleomcopmopuonudnsd: sF. luorochemical surfactants or other fluorinated compounds, or 1.3 tMheatvraicliedsa:tioRnabrebpito,rtr.at, bovine, monkey, and human serum, or other fluids as designated in Word 97 SR-1 ETS-8-5.0 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 1 of 9 Page 104 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 S u m m a r y o f M e t h o d _________________________________________________________________________ 2.1 TuanshiainslgymsHisePtihLsoCpde-erdlfeeoscrctmrrioebsdepsrbaythyme/maoannsaistloysrspiinsegcotrfaofsmliuneogtrrloeyc,ihooenrmsciihmcaairllaascrutresfyraisscttteiamcnotsafseaxaptpraparrctoitcpeudrliaafrrteo.mTsheerum, fSlaumorpolcehsemmaicyaal,lssoucbhe aasnathlyezpedotuasssiniugmanpeArPflIu-oErSo/oMctSa/nMesSulsfyosntaetme (tPoFfOurSth)earnvioenri,fym/z= 499. compound identification. 3.0 D e f in it io n s _____________________________________________________________________________________ 3.1 AvatrmioousspmheerthicodPsreosfsiuorneizIaotnioinzabtiyounti(lAizPinIg): vTahrieouMsicsoroumrcaesss, pQruoabtetrs,o a1n3dsyisntteemrfsacaelslo. wThfoerse include but are not Jimited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 pErleescsturroes,pwrahyerIeobnyizioantiiozanti(oEnSo, cEcuSrIs):tharmouegthhotdheofprioondiuzcattiioonn opfetrifnoyrmcheadrgatedatdmroospplehtesriicn a strong electrical field. 3.3 TsMehlaeescsAtiPSvepIleQycutdraiotstcmrroiemItIrinysay, tsMetedambsssySamrpeaesecsqtrutooipmcpheeatdregrwe(itrMhatSqio)u,a(Tmdar/uzn)pdoaelnmedmMsausabssssesqeSluepecentcitvtlyreoddmeeteteetccettreodr(.Ms. ASI/oMsninsSg)al:ree MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 soCyrosthtneovmgeonsnt(aipolontsoatlt1hv9es9.c8Zo)n-useptiarlipazeyertpaurr"oeZ.b-seIpnirnatthyee"rcfcaoocnnevf:oernTmtihoaentialoalnte.csotTnmfhoeormdspearltsaiyoonfemMititiicsteradoimmfraeosdms dQairupeacrttotlrbyoeaIitIsthe ccoonnfeigauprearttiuorne,isafdteirffepraesnsti,ngthtehmroeutghhodastoorftoupouersaptiaotnh,wcaleyainninthge, acnodunmtearinetleecntarnocdee.arTe hthoeugshamthee. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 M ass L ynx Software: System software designed for the specific operation of these Quattro IvIesrysisotenms sa.reCsuimrrileanrt.lyFMorasmsLoryenxdehtaasilsWseinedtohwe sm9a5nuaanldsWpeicnifdiocwtosNthTe 4in.0stvruermsieonnts(.MAicllromass Quattro 13 MassLynx or MassLynx NT USER'S GUIDE). 4.0 W a r n in g s a n d C a u t io n s_____________________________________________________________________ 4.1 H ealth and Safety W arnings: 4.1.1 Use caution with the voltage cables for the probe. The probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. Analysis of SerEumTSE-8x-t5ra.0ct Using ES/MS 3M Environmental Laboratory Page 2 of 9 Page 105 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 4.2 Cautions: 4.2.1 Do not operate solvent pumps above capacity of 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5 .0 I n t e r f e r e n c e s ______________ ___________________________________________________________________ 5.1 Tstoormagine iomriazneyinptaerrtfeorfeinncsetrsuwmheenntaatnioanlytzhinatgcsoammepsleisn, ctoenfltoanctshwoituhldthneotsabme pulseedorfeoxrtsraamct.ple 6 .0 E q u ip m e n t _____________ ________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP1100 low pulse solvent pumping system and autosampler 7 .0 S u p p l ie s a n d M a t e r i a l s ________________________________________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8 .0 R e a g e n t s a n d S t a n d a r d s ______________________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.0. 9.0 S a m p l e H a n d l in g ________________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C until analysis can be performed. Analysis of SerEumTSE-8x-t5ra.0ct Using ES/MS 3M Environmental Laboratory Pago 3 of 9 Page 106 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03Q laboratory Request Number-U2279 10 .0 _Q u a l it y C o n t r o l _____________________________________________________________________ 10.1 Method Blanks and Matrix Blanks 10.1.1 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix Spikes 10.2.1 Analyze' a matrix spike and matrix spike duplicate per forty samples. With a minimum of 2 spikes per batch. 10.2.2 Eccauxlrpivbeerc.atteAidodnsdpciiutkiroevneca.olnscpeinkteractoinocnesnwtrialltifoanlls imn athyefamllidin-rtahnegeloowf-rthaengineitoiaf lthcealiinbirtaiatlion 10.2.3 See Sectipn 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 cAhnaanlgyeze(a 3m0i%d-)rainngpeeackaliabrreaatioocncusrtasn, draelradtiavfetetroetvheeryintiteinatlhstsaanmdparled. cIuf ravsei,gsntiofpicathnet rwuinll. bOenulysetdh.osTehseamrepmleasinainnaglyszaemdpbleesfomreustht ebelarsteaancacleypzteadb.le calibration standard 10.3.2 See Section 13 to calculate percent difference. 1 1 .0 C a l i b r a t i o n a n d S t a n d a r d iz a t io n ___________________________________________________ 11.1 Amneaanlyzoef ttwheoesxttarnadcaterdd vmaalutreisx, sattaneadcahrdsstapnrdioarrdtocoanndcefnotlrlaotwioinn,gweialclhbeseptlootfteedxtrbayctlisn. eaTrhe ' regression (r")for the calibration curve using MassLynx or other suitable software. 11.2 Tdihsecrre2tivoanluoef ftohretahneadlyastta asnhdoualpdpbroev0a.l9o8f0 tohre gPrreoajteecrt. LLeoawd.er values may be acceptable at the 11.3 Isftatnhdeacrudrcvuerdvoee(sifnnoetcmeseseatryre)qaunidrermeaennatsl,yzpee.rform routine maintenance or reextract the 11.4 uFEsoxeramtphuperleplo:owswesehnoednf oaafctcttuehmreapccatyilniwbgrhateotnioqqnuuacanuntrittviateatetrianatpghpelorrowthxialmenvatethleeslyofuf1l0lanrpaapnlbygteoe,foiaftntmhaleayytsetba,engdenanerecdreasctsuearravye.to craanligberaotfiotnhecucurvreveco(5nspipstbintgo o1f0t0h0epsptabn).daTrdhsisfrwoimll r5edppubcetoina1c0c0uprpacbyraattthreirbuthteadn ttohelinfuelalr regression weighting of high concentration standards. 12.0 Pr o c e d u r e s ____________________________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. Analysis of SerEumTSE-8x-t5ra.0ct Using ES/MS 3M Environmental Laboratory Page 4 of 9 Page 107 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.1.2 To create a method click on scan button in the Acquisition control panel and select StoIR49(9SionrgolethIeornaRppecrooprdriiantge)morasMseRs.MA. Sfueltl IsocnanizaistiounsuMalloydecoalsleacptepdroaplroinagtewaintdh mthaess SERs. Save acquisition method. If MS/MS instruments are employed, additional pMraosdsuLcytnioxnGfrUaIgDmEenTtOatiDonAiTnAforAmCaQtioUnISmITayIObNe cfoolrleacdteddit.ioSnaeel iMnfiocrrmomataiossn and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1, 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 juL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 13.5 minutes 12.2.2.4 Solvent ramp = . Time 0 . 0 0 min. 7.5 min. 1 1 . 0 min. 1 1.5 min. MeOH 40% 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrum ent Set-up 12.3.1 Refer to ETS-9-24.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 1 0 minutes. ETS-8-5.0 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 5 of 9 Page 108 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03G laboratory Request Number-U2279 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 123.6.1 Drying gas 250-400 liters/hour 12.3.6.Z ESI nebulizing gas 10-15 liters/hour 12.3.63 HPLC constant flow mode flow rate 10 - 500 juL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among bMuattsosnL.ynExnsvuerresisotnasr,t saenedaepnpdrosparmiaptleeMnuamssbLeyrnixncUluSdEeRs 'aSll GsaUmIDplEes).toPbreessanthaelyszteadr.t 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s ____________________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected CEonxep.e-ctCedalcCuolnatee.d Cone, x 100 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (|ig/ml): (ng of PFOS calc, from std. Curve x Dilution Factor) x 1 tig (Initial Volume of matrix (ml) + ml of Surrogate Standard) 1000 ng Final Volume (mL) 14.0 M e t h o d P e r f o r m a n c e _______________________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.0, Attachment B, for a listing of current validated MDL and LOQ values. ETS-8-5.0 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 6 of 9 Page 109 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 14.2 M ethod Blanks and Matrix Blanks 14.2.1 Mpoestshibolde bcloanntkasmainndatmioantroirxcbalrarnykosvewr.illVbaeluaensalayrzeedexwpeitchteedactho fsaalml bpeleloswettfhoerlowest standard in the calibration curve. 14.3 M atrix Spikes 14.3.1 Mexaptercixtedsptiokefsalalrwe iathnianlyze3d0%witohfetahcehspsaikmepdlecosnecteanntdratthioenp.ercent recoveries are 14.4 Continuing Calibration Checks 14.4.1 Continuing calibration checks are analyzed at a minimum of after every 10 samples with each sample set. The percent recoveries are expected to fall within 30% of the spiked concentration. 14.5 If any criteria listed in the method performance section isn't met, maintenance may be opanneratflhoyerstms.ueAmd lmolnaarctythieosnhssyesewtteiwmllitbahentddhoescasumammpelpenlsteerdreeasinunlattslhy-.ezeidnsotrruomtheenrt arcutniolongs, atshedemtearinmtienneadncbey ltohge, or 15.0 P o l l u t io n P r e v e n t io n a n d W a s t e M a n a g e m e n t _______________________________________ 15.1 pSiapmetptleewexatsrteacitswdaisspteosaenddinflabmromkeanblgelsaosslvceonnttiasindeirsspolosecdateind hinigthheBTlaUbocroatnotrayi.ners, and glass 16.0 R e c o r d s ______________________________________________________________________________________ 16.1 Store chromatograms in the study or project folder. Each chromatogram must have the fsotulldoywoinrgprionjfeocrtmnautmiobnerin, calcuqdueidsietiiothnermienththoed,hienatdegerraotriohnanmdetwhroidtt,ensaomnptlheencahmreo,meaxttorgarcatimon: date, dilution factor (if applicable), and analyst. 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Cstoorpey iinnsatprpurmoepnritarteunsltougdypafogledse,ri.ncluding instrument parameters and sample results, and 16.6 Summarize data using suitable software and store in the study folder. 16.7 Banadckloucpateiolenctorfonbiacckduaptaetloecatprponroicprdiaattae.medium. Record in study notebook the file name 17.0 T a b l e s , D ia g r a m s . F l o w c h a r t s , a n d V a l id a t io n D a t a ______________________________ 17.1 Attachment B: ETS-8-5.0 Data reporting spreadsheet. 17.2 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. Analysis of SerEumTSE-8x-t5ra.0ct Using ES/MS 3M Environmental Laboratory Page 7 of 9 Page 110 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 18.0 R e f e r e n c e s ______________________________________________________________________________ _ 18.1 IEoTnSiz-a9t-i2o4n./0M, a"sOspSepraetcitornomanedterMQauinattetnroanIIceSyosfttehme sM" icromass Atmospheric Pressure 19.0 A f f e c t e d D o c u m e n t s ____________________________________________________________________ 19.1 ETS-8-4.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R ev isio n s Revision Number. Reason For Revision Revision Date Analysis of SerEumTSE-8x-t5ra.0ct Using ES/MS 3M Environmental Laboratory Page 8 of 9 Page 111 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U2279 Attachment A Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: ` Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analvsis/Analvst: Group Sample# Concentration Dose ue/idL Initial Vol. ml. Dilution Factor Final Cone, iis/m l. Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration fug/mLl: Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. fue/mLI: Calculated bv dividing the initial volume from the concentration Analysis of SerEumTSE-8x-t5ra.0ct Using ES/MS 3M Environmental Laboratory Page 9 of 9 Page 112 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Einvironmental Laboratory M ethod E x tr a c tio n o f Po tassium perfluo ro o ctanesulfo nate or O th er A nio n ic F l u o r o c h e m ic a l Sur fa ctan ts fro m L iv er fo r A n a ly sis U sing H P L C -E lectro spray/M ass Spec tr o m etry M ethod Number: FACT-M-1.0 Author: Lisa Clemen Approved By: A doption Date: 5/? 6 / $ / R evision Date: yf/.-} Group Leader Date ly Technical AR-eviewer _______________________ .5'h l h i Y Date 1.0 S c o p e a n d A p p l ic a t io n _______________________________________________________________ 1.1 oStchoepr ef:luoTrhoicshmemetihcoadl siusrffoarcttahnetsexftrroamctiloivnero.f Potassium Perfluorooctanesulfonate (PFOS) or 1.2 A pplicable C om pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report. Microsoft 7.0.1/95 FACT-M-1.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 1 of 8 Page 113 3m Medical Department Study: T-6295.7 Report No. FACT TOX-G30 laboratory Request Number-U2279 2.0 Su m m a r y o f M e t h o d _____________________________________________________________ 2.1 Tfluhoisromcehtehmodicadlesscurrifbaecstahnotswfrtoomexltirvaecrt upsoitnagssiiounmppaeirrifnlugorreoaogcetnatnaensudlf5o.n0amteL(sPoFfOeSth) yolr other acetate. An ion pairing reagent is added to each sample and partitioned into ethyl acetate. Four mLs o f extract is removed to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL methanol then filtered through a 3 cc plastic syringe attached to a 0 .2 pm filter into glass autovials. 3.0 D e f in it io n s ____________________________________________________________________________ 3.1 None. 4.0 _W a r n in g s a n d C a u t io n s ___________________________________________________________ 4.1 H ealth and Safety W arnings: 4.1.1 Upastehougneivnesr. sal precautions when handling animal livers, they may contain 5.0 I n t e r f e r e n c e s ___________ ._____________________________________________________________ 5.1 There are no known interferences at this time. 6.0 E q u ip m e n t __________________________________________________________________________ 6.1 Tachceepfotallbolwe.ing equipment is..u...sed while carrying out this method. Equivalent equipment is 6.1.1 Ultra-Turrax T25 Grinder for grinding liver samples 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR 6.1.5 Nitrogen Evaporator, Organomation 6.1.6 Balance 7.0 Su p p lie s a n d M a t e r ia l s ____________________________________________________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 1 L 7.5 Glass, type A, volumetric flasks 7.6 40 mL glass I-CHEM vials 7.7 Plastic sampule vials, Wheaton, 6 mL 7.8 Polypropylene centrifuge tubes, 15 mL 7.9 Labels FACT-M-1.0 Extraction of PFOS from Liver Page 2 of 8 3M Environmental Laboratory Page 114 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 7.10 Syringes, capable o f measuring 10 pL to 50 pL 7.11 Glass, type A, volumetric pipettes 7.12 Graduated pipettes 7.13 Electronic pipettor, Eppendorf or equivalent 7.14 Timer 7.15 Disposable plastic 3 cc syringes 7.16 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.17 Crimp cap autovials Note: PQriTMor wtoatuesri.ngRginlasseswsyarrinegaensdabmotitnleims,urminsoef39ttiimmeesswwiitthhmmeetthhaannooll,an3dri3nsteims efrsowmit3h sMepiallria-te vials. 8.0 R e a g e n t s a n d St a n d a r d s ______________________________________________________________ 8.1 Reagents 8.1.1 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) 10N: weigh approximately 200 grams NaOH. Pour into a 1000 mL beaker containing 500 liters (L) Milli-QTM water, mix until all solids are dissolved. Store in a 1 L nalgene bottle. 8.1.2 Sodium Hydroxide (J.T Baker or equivalent), (NaOH) IN. Dilute 10N 1:10. dMileuatseutroe v1o0lummLeoufstihneg 1M0NilliN-QaOTMHwsaotelur.tioSntoirnetoinaa101025mmLLvonlaulmgeentericboftltalsek. and 8.1.3 Tetrabutylammonium hydrogen sulfate (Kodak or equivalent), (TBA) 0.5M: Weigh approximately 169 grams of TBA into a 1 L volumetric containing 500 L Milli-QTM water. Adjust to pH 10 using approximately 64 mL 10N NaOH and dilute to NvoalOumHebwecitahusMe itlhlie-QpHTMcwhaatnegre. sAadbrduNptalyO.HSstolorwe liny awh1iLlenaadldgeinnge tbhoettllaes.t 1 mL of 8 .1.3.1 TneBeAderdequusiinregs IaNchNeacOkHprsioorluttoioena.ch use to ensure pH = 10. Adjust as 8.1.4 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker or equivalent), a((NNndaa22dCCil00u33t/)eNatanodHvo2Cl1u0.0m3) ge0.ow2f5itMsho:dMiWuilmleii-gbQhiTMcaarpwbpoarntoeaxrti.em(SaNttoearlyHe 2Cin60.a53)1ginLotofnsaaolgd1eiLunmevobcloautrmtbleeo.tnraitceflask 8.1.5 PFOS (3M Specialty Chemical Division), molecular weight = 538. 8.1.6 Ethyl Acetate, Omnisolv, glass distilled or HPLC grade. 8.1.7 Methanol, Omnisolv, glass distilled or HPLC grade. 8.1.8 Liver and control liver, received frozen from testing laboratory. 8.1.9 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. 8.2 Standards 8.2.1 Prepare PFOS standards for the standard curve. FACT-M-1.0 Extraction of PFOS from Liver Paee 3 of 8 3M Environmental Laboratory Page 115 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.2.2 tWheeiagchtuaapl pwreoixgihmt.ately 100 mg of PFOS into a 100 mL volumetric flask and record 8.2.3 (Bnrgi/nmgLto).volume with methanol for a stock standard of approximately 1000 ppm 8.2.4 Dapilpurtoextimheastetolyck50sopluptmio.n with methanol for a working standard 1 solution of 8.2.5 aDpiplurtoex.th5e.0stpopcmk .solution with methanol for a working standard 2 solution of 8.2.6 aDpiplurtoex.th0e.5s0topcpkms.olution with methanol for a working standard 3 solution of 9.0 Sa m p l e H a n d l in g ______________________________________________________________________ 9.1 All livers are received frozen and must be kept frozen until the extraction is performed. 10.0 Q u a l it y C o n t r o l _______________________ ;____________________________________________ 10.1 M atrix Spikes 10.1.1 tPhreepaacrceuraancdyaonfatlhyezeexmtraatrcitxiosnp.ike and matrix spike duplicate samples to determine 10.1.2 Prepare each spike using liver chosen by the analyst, usually a control liver. 10.1.3 Expected concentrations will fall in the mid-range of the initial calibration curve. 10.2 C ontinuing Calibration Checks 10.2.1 Prepare and analyze continuing calibration check samples to determine the continued linearity of the initial calibration curve. 10.2.2 Ofonuer cchheecckksisarpereppraerpeadrepderangdroeuxptroacftteedn. samples. For example, if a sample set = 34, 10.2.3 pPrreeppatrheeeiancithiaclocnutrinveu.ing calibration check from the same liver homogenate used to 10.2.4 Tcuhrevee.xpected concentration will fall within the mid-range of the initial calibration 11.0 C a l ib r a t io n a n d St a n d a r d iz a t io n ________________________________________________ 11.1 Prepare Liver H om ogenate to Use for Standards 11.1.1 Weigh approximately 40 g of liver into a 250 mL Nalgene bottle containing 200 mLs Milli-QTM water. Grind to a homogeneous solution. 11.1.2 aIf14:05 graitsion. ot available, use appropriate amounts of liver and water in keeping with 1 1 .1 .3 See section 13.0 to calculate the actual density of liver. F A C T -M -1 .0 Extraction of PFOS from Liver Page 4 of 8 3M Environmental Laboratory Page 116 O G 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03 laboratory Request Number-U227 11.1.4 Add 1 mL o f homogeneous solution to a 15 mL centrifuge tube. Re-suspend homogeneous solution by shaking between aliquots while preparing a total of sixteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.5 Two 1 mL aliquots serve as matrix blanks. Use the standard concentrations and tsoptiaklinogf faomurotuenetns slaismtepdleisn. table 1 to spike, in duplicate, two standard curves for a ' Table 1 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm pL Approx, final cone, of PFOS in liver - Blank 4 0 . 0 1 0 ppm 2 0 0.050 ppm 40 0 . 1 0 0 ppm 10 . 0.250 ppm 2 0 0.500 ppm 30 0.750 ppm 4 1 . 0 0 0 ppm 11.1.1 See section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 Extract spiked liver homogenates following 12.14-12.24 of this method. Use these standards to establish each initial curve on the mass spectrometer. 12.0 P r o c e d u r e s ____________________________________________________________________________ 1 2 . 1 oOtbhtearintisfsruoezse.n liver samples. In spent tissue, note that the liver has not been packaged with 12.2 Cut approximately 1 g of liver using a dissecting scalpel. 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Gunrtiinldththeesasammpplelei.sPhuotmthoegegnreinoduesr. probe in the sample and grind for about 2 minutes, or 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Follow AMDT-EP-22. 12.10 Cap the sample and vortex for 15 seconds. FACT-M-1.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 5 of 8 Page 117 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.11 Pipette 1 mL homogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. (See Worksheet for documenting the remaining steps.) 12.12 sSepcitkioenliv1e1r.1hoormToagbelneat1e.s with the appropriate amount of PFOS standard as described in 12.13 Pinisptertutme tewntob1lamnkLs.aliquots of Milli-QTM water to centrifuge tubes. These will serve as 12.14 bAudfdfer1. mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate 12.15 Using a volumetric pipette, add 5 mLs ethyl acetate. 12.16 Cap each sample and put on the shaker for 20 minutes. 12.17 Centrifuge for 20 to 25 minutes, until layers are well separated. Set power on the centrifuge to approximately 3500 rpm. 12.18 Remove 4 mLs of organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with th same information as in 12.5. 12.19 hPouutresa.ch sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 12.20 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.21 Vortex mix for 30 seconds. 12.22 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial. ' 12.23 mLaabtreilxt,hfeinaaultsoovlivaelnwt,itehxttrhaecsttioundydantuem, abnedr,aannaimlysatl(sn)uwmhboerpaenrfdorgmenedderth, esaemxtprlaecttiiomne. point, 12.24 Cap and hold for electrospray mass spectrometry analysis. 12.25 Complete the worksheet and tape to page of study notebook. 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s ____________________________________________________ 13.1 Calculations: 13.1.1 eCqaulactuiolant:e the density of liver (mg) in 1.0 mL homogenate using the following g of Liver x Average weight of ten 1 mL aliquots (me) (g of Liver + g of Water) FACT-M-1.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 6 of 8 Page 118 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 13.1.2 fColalolcwuilnatge eaqcutuaatilonco:ncentrations of PFOS in calibration standards using the pL o f StmangdLarivdexr C/ o1nmceLnthroamtioonge(npagte/mL) = FoifnPaFl OCoSnicnenLtirvaetrion (pg/g or mg/kg) *Average weight of liver in solution as determined in 13.1.1, by weighing ten 1 mL homogenates of approximately 40 mg liver in 200 mL of Milli-Q water. 14.0 M e t h o d P e r f o r m a n c e _____________________________________________________________________ 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 15.0 P o l l u t io n P r e v e n t io n a n d W a s t e M a n a g e m e n t ______________________________________ 15.1 lhSoiacgmahtpeBldeTiwUnatcshoteenltiaasbindoeirsrapst,oorsayend.d iunsebdiohglaazsasrdpicpoenttteaiw'naerstse, filsamdimspaobseledsionlvbernotkwenasgtleasiss cdoisnptaoisneedrsin 16.0 R e c o r d s ______________________________________________________________________________________ 16.1 Complete the extraction worksheet and tape into the study notebook. 17.0 T a b l e s , D ia g r a m s , F l o w c h a r t s , a n d V a l id a t io n D a t a ______________________________ 17.1 The validation report associated with this method is FACT-M-1.0 & 2.0-V-l. 18.0 R e f e r e n c e s __________________________________________________________________________ 18.1 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 19.0 A f f e c t e d D o c u m e n t s ______________________________________________________________________ 19.1 FMAaCssTS-Mpe-c2tr, o"mAentrayl"ysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray 20.0 R e v is io n s Revision Number. Reason For Revision Revision Date FACT-M-1.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 7 of 8 Page 119 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Extraction Worksheet for FACT-M-1 Study # Sample Number set # PFOS PFOS approx. 0.5 ppm approx. 5 ppm actual ppm actual ppm #W #W PFOS approx. 50 ppm actual ppm #W Date and Initials for Std. - H ,0 Blank - -- - Liver Blank - - - - - - 1- - - - -- - -- - -- - - - - -- - -- - -- - --- - - -- - -- - -- - -- - - - - -- - -- 1Studv number where the original worksheet is located. Blank Liver Homogenate: Std# Ltvcr amount = g Liver Extraction Method Vortex 15 sec. : Date & Initials Pipette 1mL of Liver Solution Pipette 1 mL of t0.5 M TBA, pH 10. Std. # Pipette 2 mL of 0.25 Na2COy0.25M NaHCO-* Buffer Std. # Pipette 5 mL of Ethyl Acetate TN-A- Shake 20 min. Centrifuge 20-25 min. Centrifuge Speed Remove a 4 mL aliquot of organic laver Put on Nitrogen Evaporator to drvness Evaporator Temperature Add 1.0 mL of Methanol TN-A- Vortex 30 sec. Filter using a 3cc B-D svringe with a 0.2um SRI filter into a 1.5 mL autosample vial MS/MSD/___Cont. Checks: Spiked______uL of a ______ppm std (_______________ ) for a final concentration of _________PPm- MS/MSD used sample________________ , Cont. Checks used same homogenate as for std curve. FACT-M-1.0 Extraction of PFOS from Liver Page 8 of 8 3M Environmental Laboratory Page 120 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory M ethod A n a ly sis o f F luo r o c h em ic a ls in L iv e r E x t r a c t s U sing H P L C -E lectro spray/M a ss S pec tr o m etr y M ethod Num ber: FACT-M-2.0 Author. Lisa Clemen Approved By: /3 7 Laboratory Manager f/'sd'lv -- ---------- Group Leader T echVnj*i.caAl R (JlPvlk eviewer Adoption Date: Revision Date: /\j//) T 'A c / ? Date / VA Date h ll'it Date 1.0 S c o p e a n d A p p l ic a t io n ________________________________________________________________________ 1.1 Scope: This method is for the analysis of extracts of liver or other tissues for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 A pplicable Com pounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 1 of 8 Page 121 3m Medical Department Study: T-62 95.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 S u m m a r y o f M e t h o d ____________________________________________________________________ _____ 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HioPnLcCha-realecctterroisstpicraoyf/ma apsasrtsipcueclatrrofmlueotrroyc.hTehmeicaanla, lsyusicshiassptehrefopromtaedssibuymmonitoring a single vpeerrifflyuocroomocptoaunnesduildfoennatitfeic(aPtiFoOn.S) anion, M/Z= 499. Samples may also be screened to 3.0 D e f in it io n s __________________________________________________________________________________ ___ 3.1 None. 4.0 W a r n in g s a n d C a u t io n s ______________________________________________________________ _______ 4.1 H ealth and Safety W arnings: 4.1.1 Uinstoe cthaeutpioronbwe iDthOthNeOvToltTaOgeUcCaHbleTfHorEtPheRpOrBobEe,.tWherheenistrhieskvoolftaegleectcraibcalel sishopcluk.gged 4.2 Cautions: 4.2.1 oDvoerno4t00rubnasro, ltvheenHt Ppu1m10p0s wabilolvienictiaaptaecaituytoomf a4t0ic0 sbhaurt(d5o8w0n0. psi). If pressure goes 4.2.2 Do not run solvent pumps to dryness. 5.0 I n t e r f e r e n c e s __________________________________________________________________________________ 5.1 cToenfltoanctswhoituhldthneostabmepuleseodrfeoxrtrsaacmt.ple storage or any part of instrumentation that comes in 6.0 E q u ip m e n t ______________________________________________________________________________________ 6.1 Equipment listed below may be changed in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP1100 low pulse solvent pumping system and autosampler. 7.0 S u p p l ie s a n d M a t e r ia l s ______________________________________________________________________ 7.1 Supplies 7.1.1 Nitrogen gas, refrigerated liquid, regulated to approximately 100 psi. 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 R e a g e n t s a n d S t a n d a r d s______________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. Word 7.0.1/95 FACT-M-2.0 Analysis of Liver Extract Using ES/MS Page 2 of 8 3M Environmental Laboratory Page 122 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system. * 8.1.3 Ammonium acetate, HPLC grade or equivalent. 8.2 Standards 8.2.1 Typically one H20 blank, one liver blank, and seven liver standards are prepared during the extraction procedure. See FACT-M-1. 9.0 S a m p l e H a n d l in g _______________________________________________________________________ 9.1 Fresh liver standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 Iafnaanlyasliyssicsanwiblel bpeerdfoelramyeedd., extracted standards and samples may be refrigerated until 10.0 Q u a l it y C o n t r o l ________________________________________________________________ 10.1 M atrix Blanks and M ethod Blanks 10.1.1 Analyze a method blank and matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. 10.2.2 AcEuxdrpdveietc.itoendalcosnpcikenetcroanticoennstrwaitlilonfasllminaythfealml iind-trhaenlgoewo-rfathnegeinoitfiathlecainliibtiraalticoanlibcruartvieo.n 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a change ( mid-range calibration standard after 30%) in peak area occurs, relative to every tenth sample. the initial standard cIuf ravse,igsntoifpictahnet run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See section 13 to calculate percent difference. 10.4 System Suitability 10.4.1 aSsyssetsesmedsfuoirtaebaiclihtyru(ne..g. peak area, retention time and peak shape, etc.) will be 11.0 C a l ib r a t io n a n d S t a n d a r d iz a t io n ________________________________________________________ 11.1 Analyze the extracted liver standards prior to and following each set of extracts. The mean of two standard values, at each standard concentration, will be plotted by linear regression for the calibration curve using MassLynx or other suitable software. FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 3 of8 Page 123 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 11.2 The r2 value for the data should be 0.98 or greater. Lower values may be acceptable at the discretion of the analyst. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 12.0 P r o c e d u r e s ______________________________ ;___________________________________________ __ 12.1 A cquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 TmSIoaRsc.sreeSsa.etetAIaosmnciazenathtiiosodnucsMluiacokldlyeoncaosslcaleapcnptreboduptartiloaontneginawnthditehmAtahcseqsuStoiIsRi4ts9io.9nSocarovonetthrmeorel tpahpaopndre.ol parnidateselect 12.1.3 tThyepsieccaollnydthseetsoamf sptalendliasrtdbs.egins with the first set of liver standards and ends with 12.1.4 sSaammppllee.s Saroelvaennatlybzlaednkwsisthhoaulcdonbteinaunianlgyzceadlibpreartiioodniccahlelycktoinmjeocnteitdorafptoersseivbeleryanteanlythte carryover and are not considered samples but may be included as such. 12.2 Using the Autosam pler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1. 12.2.2 aSneta-luypsttchoenHsiPde1r1s00a/papurtoopsraimatpelfeorraotptthiemfaolllroewspinongsceo. nRdeictoiorndsaocrtuaatlccoonnddititioionnssthine the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time 0 . 0 0 min. 7.5 min. 1 1 . 0 min. 11.5 min. MeOH 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: soInftwthaisreinwstirthumae"nWt caoitninfigguforratiinolne,t tshtearrtu"nmmesussatgbeebseeftourep tohne t"hSetaerlet"ctbroustptornavis pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 4 o f 8 Page 124 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.3 Instrum ent Sep-up 12.3.1 Refer to AMDT-EP-31 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 tChheetcipk.thTehsetatiipnlsehssousltdeeblecaflpaitllwariythantothjeagegneddoefdtgheesp. rIofbteh.eUtispeiasnfoeuynedptioecbeeto check unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. aOpbpsreorxviemdartoeplylet1s0 cmominiuntgeso. ut of the tip o f the probe. Allow to equilibrate for 12.3.5 Taruorunnodnththeetinpitorof gthene.prAobfein. e mist should be expelled with no nitrogen leaking 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 LC constant flow mode flow rate 10 - 500 uL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the instrument is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log. 12.3.9 tUhseinagnatlhyesicsroosfsb-fiololowgiccoaulnmteartreilceecst.rode in the ES/MS source is recommended for 12.3.10 Click on start button in the Acquisition Control Panel. Press the start button at taonpaloyfzesdam. ple list. Ensure start and end sample number includes all samples to be 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s _________________________________________________________ 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 5 of 8 Page 125 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03G laboratory Request Number-U2279 13.1.3 Calculate actual concentration of PFOS anion in total liver (mg): )f ug PFOS anion calc, from std curve''] -V--------g---o--f--l-i-v1-e0--r0--0u--su-e-d-g--/f-o1--rm--a-gn--a--l-y-s--is--------- x T^otal mass of liver (g), 14.0 M e t h o d P e r f o r m a n c e _______________________________________________________________ _ 14.1 The method detection limit is equal to at least three times the baseline noise in the matrix blank. 14.2 The practical quantitation limit is equal to the lowest standard in the calibration curve. 15.0 P o l l u t io n P r e v e n t io n a n d W a s t e M a n a g e m e n t ______________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in hcoignhtaBinTerUs caorentlaoicnaetresd, ainndthgelalasbsopriapteotrtye.waste is disposed in broken glass containers. All 16.0 R e c o r d s _______________________________________________________________________________________ 16.1 Store chromatograms in the study folder. Each chromatogram should have the following information included either in the header or hand written on the chromatogram: study number, sample name, extraction date, and dilution factor (if applicable). 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and tape into appropriate study notebook. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Bloaccaktiounp oelfebctarcoknuipc edlaetcatrtoonaipcpdraotpar.iate media. Record in study notebook the file name and 17.0 T a b l e s , D ia g r a m s , F l o w c h a r t s , a n d V a l id a t io n D a t a _______________________________ 17.1 Attachment A: FACT-M-2 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-1.0 & 2.0-V -l. 18.0 R e f e r e n c e s ___________________________________________________________________________________ 18.1 AMDT-EP-31, "Operation of VG Platform Electrospray Mass Spectrometer" FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 6 of 8 Page 126 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 19.0 A f f e c t e d D o c u m e n t s _________________________________________________________________ __ 19.1 FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R e v is io n s ______________________________________________________________________________________ NRuevmisbieorn. - Reason For Revision ReDvaisteion FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 7 of 8 Page 127 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U2279 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: * Analytical Equipment System Number. Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Sample# Concentration Dose ug/mL Initial Vol. mL ' Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 8 of 8 Page 128 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory M ethod E x t r a c t i o n -o f P o t a s s iu m P e r f l u o r o o c t a n e s u l f o n a t e o r O t h e r F lu o r o c h em ic a l co m pounds fro m L iv er for A nalysis U sing H PL C -E lectrospray/M ass S pe ctro m etry M ethod Num ber: FACT-M-1.1 Author: Lisa Clemen, Glenn Langenburg Approved By: D/./1-- Laboratory Manager in L v fh --------- Group Leader C d s* A t h r u * Technical Reviewer Adoption Date: 05/26/98 Revision Date: ofela>hl /?../? ' Date C /tlO c) Date Date 1.0 S c o p e a n d A p p l ic a t io n ________________________________________________________________________ 1.1 Sotchoepref:luTorhoicshmemethicoadl cisomfoprotuhendesxtfrraocmtiolniveorf.potassium perfluorooctanesulfonate (PFOS) or 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 rMepaotrrti.ces: Rabbit, rat, bovine, and monkey liver or other liver as designated in the validation Microsoft 6.0/95 FACT-M-1.1 Extraction of PFOS from Liver 3M Environmental Laboratory Page 1 o f 15 Page 129 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Su m m a r y o f M e t h o d _________________________________________________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (5P.0FOmS)l oorfoetthheyrl fatcueotraotec.heImn itchaislsmfreotmhodli,veservheonmfolugoernoactheeumsiincaglsanwieorne pexatirraincgtedre:aPgFenOtSa,nd PFOSA, PFOSAA, EtFOSE-OH, POAA, PFOSEA, Dpaerftiintiiotinoends)in. toAnethioynl apcaeitrainteg. rFeaoguernmt ilsoafdedxetdratcot tahree asanmd pFlCe -a8n0d7tmheonanoaeslyteter i(osneep3a.i0r is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 ml of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3 .0 D e f in it io n s _____________________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8F,7S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide C8F,7S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8Fl7S 0 2N(CH2CH3)CH2C 02" 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F17S 0 2N(CH2CH3)CH2CH20H ' 3.5 POAA: perfluorooctanoate (anion of ammonium salt) C7FisCOO' 3.6 PFOSEA: perfluorooctane sulfonyl ethylamide C8F,7S 0 2N(CH2CH3)H 3.7 FC-807 monoester C8F i7S 0 2N(CH2CH3)CH2CH20 -P 0 3H) 3.8 Surrogate standard 1H,1H,2H,2H perfluorooctane sulfonic acid 4.0 W a rn in g s and C autions________________________________________________________________ 4.1 Health and safety warnings: 4.1.1 hUasnedulinnigvearnsaiml palretcisasuuteio, nits,measypeccoinatlalyinlapbaotrhaotgoernysc. oats, goggles, and gloves when 5.0 I n t e r f e r e n c e s _________________________________________________________________________ 5.1 There are no known interferences at this time. 6.0 E q u ip m e n t _____________________________________________________________________________ 6.1 Tachceepfotallbolwe.ing equipment is used while carrying out this method. Equivalent equipment is 6.1.1 Ultra-Turrax with T25 grinder attachment for grinding/dispersing/emulsifying 6.1.2 Vortex mixer, VWR, Vortex Genie 2 6.1.3 Centrifuge, Mistral 1000 or IEC 6.1.4 Shaker, Eberbach or VWR 6.1.5 Nitrogen evaporator, Organomation 6.1.6 Balance, ( 0.100 g) ExtractioFnAoCf TPF-MO-S1.f1rom Liver Page 2 of 15 3M Environmental Laboratory Page 130 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 7.0 Su p p l ie s a n d M a t e r ia l s __________________________ ___________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 ml and 1 L 7.4 Wheaton 6 ml Plastic Sampule Vials 7.5 Glass, type A, volumetric flasks 7.6 40 ml glass I-CHEM vials 7.7 Polypropylene centrifuge tubes, 15 ml 7.8 Labels 7.9 Syringes, capable of measuring 5 pL to 50 pL 7.10 Glass, type A, volumetric pipettes 7.11 Graduated pipettes 7.12 Electronic pipettor, Eppendorf or equivalent 7.13 Timer 7.14 Disposable plastic 3 cc syringes 7.15 Filters, nylon syringe filters, 0.2 pm, 25 mm 7.16 Crimp cap autovials Note: QPTMriowr atoteur.sinRgingsleasssywrainrgeeasnadmbointtimlesu,mrinosfe93titmimeesswwitihthmmeeththaannool,l 3anrdin3setsimfreosmw3ithseMpairlalit-e vials. 8.0 R e a g e n t s a n d St a n d a r d s _________________________________________________________ 8.1 ASTM Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC PlusTM system. 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate (TBA), Kodak or equivalent 8.4 Sodium carbonate (Na^CO^), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8 . 6 Ethyl acetate, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8 . 8 Liver tissue, frozen from supplier 8.9 Control matrix or blank matnx for standards, QC checks, blanks, etc. 8.10 Fluorochem ical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.10.2 PFOS A (3M Specialty Chemical Division), molecular weight = 499 8.10.3 PFOS.AA (3M Specialty Chemical Division), molecular weight = 585 ExtractioFnAoCf PTF-OMS-lf.lrom Liver Page 3 o f 15 3M Environmental Laboratory Page 131 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 571 8.10.5 POAA (3M Specialty Chemical Division), molecular weight = 431 8.10.6 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.7 FC-807 monoester (3M Specialty Chemical Division). FC-807 is a mixture of tmrioelsetceru,ladriewsteeirg,hatn=d 6m5o0noester fluorochemical components. The monoester 8.10.8 Surrogate Standard: 4-H, perfluorooctane sulfonic acid (l-H .l-H , 2-H,2-H C8FI3S 0 3H) molecular weight = 428 8.10.9 Other fluorochemicals, as appropriate 8.11 Reagent preparation 8.11.1 d11i00s0sN0olsmvoelddbi.uemaSktoehrryedcorinnoxtaaidi1neLin(NgNaa5Ol0gH0en)m:elWbMoetiitlgllehi-.QapTMprwoxaitmera, tmeliyx2u0n0tgil NalalOsoHli.dsPoaurer into a 8.11.2 1 N sodium hydroxide (NaOH): Dilute 10N NaOH 1:10. Measure 10 ml of 10N NaOH solution into a 100 ml volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 ml Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 pgrHam10s oufsiTngBAapipnrtooxaim1aLtevlyol4u4mteotr5i4c mcolnotafin10inNg N50a0OHmlaMndildlii-lQutTMe two avtoelru.mAedjwuistthto Milli-QTM water. While adding the last few m l's of NaOH, add slowly because the pH changes abruptly. Store in a 1 L Nalgene bottle. 8.11.3.1 nTeBedAedreuqsuiinrgesIaNcNheacOkHprsioolruttoioena.ch use to ensure pH = 10. Adjust as 8.11.4 0.25M Sodium approximately carbonate/sodium bicarbonate buffer 26.5 g of sodium carbonate (Na;C 0 3) (aNnda22C10.03/Ng a o HC03): W f sodium eigh bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with Milli- QTM water. Store in a 1 L nalgene bottle. 8.12 Standards 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent cflounotraoinchinegm1ic.a0l0 sptapnmdaPrFdOs aSr,e1a.0c2ceppptamblPeF(Oe.gS.Ao,n0e.9w8o7rkpipnmg sPtaFnOdSarAdAs,oaluntdio1n.10 ppm EtFOSE-OH.) 8.12.3 tWheeiagchtuaapl pwreoixgihmt.ately 100 mg of PFOS into a 100 ml volumetric flask and record 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/ml). 8.12.5 Dapilpurtoextihmeastteolyck50sopluptmio.n with methanol for a working standard 1 solution of ExtractioFnAoCf PTF-OMS-lf.lrom Liver Page 4 of 15 3M Environmental Laboratory Page 132. 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Preparer surrogate stock standard. Weigh approximately 50-60 mg o f surrogate astcatnudaal rwdei1g-hHt,.1-H, 2-H,2-H, C8F,3S 0 3H into a 50 ml volumetric flask and record the 8.13.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 0.5 ml o f surrogate ssttaoncdkatrodo5f010m-2l 0voplpumm.etRrieccfolradskthaendacbturianlgvtooluvmoleumtraenwsfietrhremde. thanol for a working 8.14 Liver homogenate preparation Note: The follo w in g procedure w ill be much easier to perform with frozen liver tissue. Prevent tissuefrom thawing; keep stored on ice until excising a portion o f it: 8.14.1 Weigh 40 g o f blank or control liver into a 250 ml Nalgene bottle containing 100 mis Milli-QTM water. Record the actual weight of liver and total volume o f water used. Grind the liver into a finely dispersed homogenate with an Ultra-Turrax T25 grinder (high speed for approximately 3 minutes or until sufficiently homogenized). Rinse grinder with an additional 100 ml of MilliQTM water, to bring the total volume of water added to 2 0 0 ml. 8.14.2 To determine the concentration of the blank liver homogenate, transfer ten 1.0 ml aliquots o f the homogenate to tared polypropylene tubes, and weigh each aliquot on a balance. The average density o f these aliquots is determined and then the concentration (g of liver/ml of homogenate) can be calculated as follows: 8.14.3 fgrams of liver] x fave, weight of 1 .0 ml of homogenate (density) (g/ml)] {[grams (g) of liver] + [grams (g) of water]} 8.14.3 Prepare sample livers as described in 8.3.1, but weigh out 1 g o f liver, homogenize with 2.5 ml of MilliQTM water, and rinse with another 2.5 ml of MilliQTM water. Use Wheaton 6 ml plastic sampule vials or appropriate receptacle. Rinse grinder unit after every sample with water and then with methanol. Label vials appropriately including study number, sample ID, liver weight, date, and analyst. Record all weights and volumes used. (Do not perform 8.3.2 fo r the sample liver homogenates). 3M Environmental Laboratory ExtractioFnAoCf TPF-MO-S1.f1rom Liver Page 5 ofl5 Page 133 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 9.0 _Sa m p l e H a n d l in g _____________________________________________________________ _______ 9.1 All livers are received frozen and must be kept frozen until the extraction is performed. 10.0 ___Q u a l i t y C o n t r o l _______________________________________________________________ 10.1 M atrix blanks and method blanks 10.1.1 Efoxltlroawctintgwothi1s.0prmolceadliuqrueoatsndofutsheealsivmerathroixmbolgaennkas.teS(peereSpeacrteidonin181..114.2.1. -2) 10.1.2 Emxettrhaocdt tbwlaonk1s.0. ml aliquots of Milli-QTM water following this procedure and use as 10.2 M atrix spikes 10.2.1 Pthreepaacrceurriancdyaonfatlhyezeexmtraatrcitxiosnp.ike and matrix spike duplicate samples to determine 10.2.2 rPerceepiavreedewacithhsepaikche suasminpglelivseetr. chosenby the analyst, usually the control liver 10.2.3 AcEaxdlpidbeirtciatotenidoanlcocsnpucrikevnees.trmataioynbsefainllcilnudtehde manidd-mraanygefaollfitnhethieniltoiawl-rcaanligberaotifotnhecuinrviteia. l 10.2.4 Prepare one matrix spike and one matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 aPtthhcreeceepcipnaotrinateitbaianllenucdcainhlaigebncrackalh.yteizocekncdcouinfrftveiner.ubiIynfg>thc3ae0l%pibe,rrarcteeianontnadcliyhfzfeeecrkseansmacemplbpeelsetwsanetoaenleynzthesuderiaenfitttheirealtahcceucrulavrseatcayndof 10.3.2 pPrreeppaarree aonnde ecxhtercakctpfeorugrrcohuepckosf.ten samples. For example, if a sample set = 34, 10.3.3 Pusreedpatroeperaecpharceonthtieniuniintigalcacluibrvrea.tion check from the same blank liver homogenate 10.3.4 The expected concentrations fall within the mid-range o f the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the plopwb).end of the calibration curve (e.g. 1 0 ppb - 1 0 0 ppb, rather than 1 0 ppb - 1 0 0 0 11.0 C a l i b r a t i o n a n d St a n d a r d iz a t io n __________________________________________________ 11.1 P re p a re liv e r h o m o g e n a te s ta n d a rd s 11.1.1 T ra n s fe r 1 m l a liq u o ts o f b la n k /c o n tro l liv e r h om og en ate prepared in 8 .1 4 .1 -2 to 15 m l ce ntrifug e tubes. 3M Environmental Laboratory ExtractioFnAoCf TPF-MO-S1.f1rom Li%'er Page 6 of 15 Page 134 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 11.1.2 If the volumes of sample liver homogenates are limited, extract standards with liver homogenate volumes equal to the sample volumes. Do not extract below 0.50 ml o f liver homogenate. Record the sample volume on the extraction sheet. 11.1.3 tWubheisle, mpriexpoarrisnhgaaketobtaeltwoefetnweanlitqyuaoltisq.uots of liver homogenate in 15 ml centrifuge 11.1.4 Two 1 ml, or appropriate aliquots, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table I (at the end of this asencdtitowno) tmoastpriixkeb,lainnkdsu.plicate, two standard curves, for a total of eighteen standards 11.1.5 Rtheefewrotroktinheg vraalnidgeastiaonndrLepinoretasrFCAalCibTra-tMion-l.Rl-aVn-g1e a(LndCRFA) fCorTc-Mali-b2r.a1t-iVon-1cuwrhviecsh. list 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 To each standard, blank, or QC check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 1 0 ppb - 1 0 0 0 ppb. 11.3 Extract spiked liver homogenate standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. Table 1 Approximate Spiking Amounts for Standards and Spikes Using 1.0 ml of Liver Working Standard (Approx. Conc.) pL ... Approx. final cone, of PFOS in liver - - Blank 0.500 ppm 4 0 . 0 1 2 ppm 0.500 ppm 10 0.030 ppm 0.500 ppm 2 0 0.060 ppm 0.500 ppm 40 0 . 1 2 0 ppm 5.00 ppm 1 0 0.300 ppm 5.00 ppm 2 0 0.600 ppm 5.00 ppm 30 0.900 ppm 50.0 ppm 4 1 . 2 0 ppm 50.0 ppm 6 1.80 ppm 12.0 P r o c e d u r e s ____________________________________________________________________________ 12.1 Obtain frozen liver samples and homogenize as described in 8.14.3. 12.2 Va o1r5temxlmpoixlyhpormopoygleennaetecefnotrri1f5ugseectounbde.s, then transfer 1 .0 ml or other appropriate volume to 12.3 Return liver homogenate samples to freezer after extraction amount has been removed. 3M Environmental Laboratory ExtractioFnAoCf PTF-MOS-If.lrom Liver Page 7 of 15 Page 135 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.4 Record the liver homogenate volume on the extraction worksheet. The final methanol volume will equal the initial homogenate volume. For example, if 1 ml of homogenate is transferred for extraction, then the final reconstitution methanol volume equals 1 ml. 12.5 wLaobrkelshtheeettufobredwoictuhmtheentsitnugdythneurmembeari,nliinvgerstIeDp,s.date, and analyst initials. See attached 12.6 dSepsickreibbeladnikn lSiveecrtihonom1o1g.1enoartTe aablilqeuIotisnwthitaht stehcetiaopnprfooprrtihaetecaalmiboruantitoonfcsutarnvdeasrtdanadsards. Also prepare matrix spikes and continuing calibration standards. 12.7 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in Section 11.2. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 To each sample, add 1 ml 0.5 M TBA and 2 ml of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 12.10 Using a volumetric pipette, add 5 ml ethyl acetate. 12.11 Cap each sample and put on the shaker for 20 minutes. 12.12 Centrifuge for 20 to 25 minutes at approximately 3500 rpm, until layers are well separated. 12.13 cTernatnrsiffeurge4 tmubl eo.fLoarbgealntichilsayfreers,hutsuinbge wa i5thmtlhgersaadmuaeteindfogrlamsastpioipneattse,into12a.5cl.ean 15 ml 12.14 hPouutresa.ch..s.ample on the analytical nitrogen evaporator until dry, approximately 2 to 3 12.15 peAxidptrdeattc1et..i0onMm. letohraanpoplrvooplruiamtee veoqluuamlsethoef mineittihaalnvoollutomeeacohf lcievnetrrhifoumgeogtuebneatuesuinsgeda fgorratdhueated 12.16 Vortex mix for 30 seconds. 12.17 FAitlttaecrhinato0.a2 1p.m5 mnlylgolnasms aesuhtofviilatelr(otor alo3wc-vcoslyurminegeauatnodvitarlawnshfeerntnheecseasmsapryle).to this syringe. 12.18 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) who performed the extraction. 12.19 Cap and store extracts at approximately 4 C until analysis. 12.20 Complete the extraction worksheet, attached to this document, and tape to page o f study notebook or include in study binder, as appropriate. 13.0 D a t a A n a l y s is a n d C a l c u l a t io n s ___________________________________________________ 13.1 Calculations: 13.1.1 Calculate actual concentrations o f PFOS, or other appropriate fiuorochemical, in calibration standards using the following equation: 3M Environmental Laboratory FACT-M-1.1 Extraction of PFOS from Liver Page 8 o f 15 Page 136 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 ml o f Standard x Concentration of Standard (ug/mQ______ = Concentration of Blank Liver Homogenate (g/ml) (see 8.14.2) Final Concentration (pg/g) of PFOS in Liver See Attachm ent D for a sample form to calculate the concentrations of standards. 14.0 M e t h o d P e r f o r m a n c e ________________________________________________________________ 14.1 TspheecimfiecthMoDd Ldeatnecdtiloimn iltimofitq(uManDtiLta)tiisonan(LalOytQe )anvdalumeastr(isxeespAetctiaficch. mReenftesr Bto aMndDLC)r.eport for 14.2 The following quality control samples are extracted with each batch of samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks 14.2.2 pMraetcriisxiosnpiokfethanedexmtraatcrtixiosnpike duplicate samples to determine accuracy and 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. ' 15.0 P o l l u t i o n P r e v e n t io n a n d W a s t e M a n a g e m e n t ____________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in hloicgahteBdTiUn tchoentlaabinoerrast,orayn.d used glass pipette waste is disposed in broken glass containers 16.0 R e c o r d s _______________________________________________________________________________ 16.1 Complete the extraction worksheet attached to this method, and tape into the study notebook or include into study binder, as appropriate. 17.0 A t t a c h m e n t s __________________________________________________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values 17.3 Attachment C, LOQ summary 17.4 Attachment D, Calibration standard concentration worksheet 18.0 R e f e r e n c e s ____________________________________________________________________________ 18.1 The validation reports associated with this method are FACT-M-1.1 and 2.1-V-l. 19.0 A f f e c t e d D o c u m e n t s _______________________________________________________________________ 19.1 FACT-M-2.1, "Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 3M Environmental Laboratory ExtractioFnAoCf TPF-MO-S1.f1rom Liver Page 9 of 15 Page 137 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03Q laboratory Request Number-U2279 2 0 .0 R e v is io n s _____________________________________________________________________ NRuevmisbieorn. 1 Reason For Revision 7Validation o f method to include flu o rochemicals, new A P I/M S (M S ) ReDvaistieon 08/01/98 systems, monkey liver cross validation, improvements to ion pairing extraction, M D L study, updates in record keeping and storing policies, etc. 3M Environmental Laboratory ExtractioFnAoCf PTF-OMS-l.flrom Liver Page 10 of 15 Page 138 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Study# Surrogate Std. Matrix approx, ppm Box# actual ppm #W Analyst/Date H.O BBllaannkk - FC Mix Std approx. 0.5 ppm actual ppm #W FC Mix Std aapctpuraolx. 5 ppppmm #W FC Mix Std Comments approx. 50 ppm a#cWtual ppm 1 Blank_______________ Std #____________________amount =____________________________________________________________ Extraction Mcthod/Revision:_____________________________________________________________________ Date & Initials_____ Add Surrogate, Vortex IS sec. ___________________________________________________________________________ Pipette Sample_______________________________________Volume________________ ml____________________________________ Pipette 1ml of 0.5 M TBA, pH 10. PH = Std. # Pipette 2 ml of 0.25 Na2COyQ.25M NaHCOi buffer__________ Std. tt _________________________________ Pipette S ml of ethyl acetate______________________________ TN-A- __________________________________ Shake 20 min._________________________________________ Shaker Speed________________________________________________ Centrifuge 20-25 min.___________________________________Centrifuge speed:_____________________________________________ Remove a 4 ml aliquot of organic layer________________________________________________________________________________ Put on Nitrogen Evaporator to dryness___________________ Temperature:__________________________________________________ Add methanol_____________ Volume ml__________ TN-A- _____________________________ Vortex 30 sec. ____ __________________________________________________________________________________ Filter usiMngSa/M3cScDB/-_D__syCroinngte. Cwihthecaks0:.2SppmikSeRdI_f_il_te_r__inutoLao1f.5am__l_a_u_t_ospapmmplestvdia(l_______________________________)_f_o_r_a__f_in_a_l__co__n_c_e_n_tr_a_t_i_o_n__o_f___ _________ppm. MS/MSD used sample________________ . Cont. Checks used same matrix as for std curve. Attachment A: Extraction worksheet ExtractioFnAoCfTP-FMO-S1.f1rom Liver Page 11 o f 15 3M Environmental Laboratory Page 139 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 M DL/LOQ values for Rabbit Liver: Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (PPb) Approximate Concentrations to be used for preparing the Standard Calibration Curve PFOS 1 1 .8 3 7 .4 3 8 p p b - 1 0 0 0 p p b PFOSA 6 .0 6 1 9 .3 2 0 p p b - 1 2 0 0 p p b PFOSAA 5 5 .7 177 18 0 p pb - 1 0 0 0 ppb EtFOSE-OH 5 8 .7 187 190 ppb - 1800 ppb POAA 2 3 .7 7 5 .5 7 6 p p b - 1 8 0 0 p p b PFOSEA Monoester 1 6 .2 5 1 .7 6 2 p p b - 1 2 0 0 p p b n/v n/v M o n o e s te r w as n o t d e te c ta b le /q u a n tifia b le at th e s p ik e d c o n cen tra tio n s. M DL/LOQ values for Rat Liver: Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (PPb) Approximate Concentrations to be used for preparing the Standard Calibration Curve PFOS 2 4 .7 7 8 .7 j 6 2 p p b - 1 2 0 0 ppb PFOSA PFOSAA EtFOSE-OH POAA PFOSEA Monoester 2 0 .7 n/v n/v n/v n/v n/v 6 5 .8 2 0 ppb - 1 2 0 0 ppb n/v 6 2 p p b - 1 2 0 0 p p b n/v 1 2 0 p p b - 1 2 0 0 p p b n/v 6 2 p p b - 1 2 0 0 p p b n/v 1 2 0 p p b - 1 2 0 0 p p b n/v M o n o e s te r w as n o t d e te c ta b le /q u a n tifia b le at th e s p ik e d c o n c e n tra tio n s . M DL/LOQ values for M onkey Liver: Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (PPb) Approximate Concentrations to be used for preparing the Standard Calibration Curve PFOS n/v n/v 5 9 p p b - 1 2 0 0 p p b PFOSA PFOSAA 2 7 .4 87 .1 2 8 p p b - 1 2 0 0 p p b n/v n/v 1 2 0 p p b - 1 2 0 0 p p b EtFOSE-OH n/v POAA n/v n/v 5 8 p p b - 1 2 0 0 p p b n/v 1 2 0 p p b - 1 2 0 0 p p b PFOSEA n/v n/v 1 2 0 p p b - 1 2 0 0 p p b Monoester n/v n/v M o n o e s te r w as n o t d e te c ta b le /q u a n tifia b le at the sp ike d c o n cen tra tio n s. n /v = N o t v a lid . U p o n a n a ly zin g the data, valu e d id n o t pass th e c rite ria set fo r th is c h a ra c te riza tio n . U n til fu rth e r a n a ly s is is c o m p le te d , use th e L C R to d e te rm in e th e ra n g e o f sta n d ard co n cen tratio n s fo r c a lib ra tio n cu rve p rep aratio n . A ttachm ent B: M D L /L O Q Values 3M Environmental Laboratory F A C T - M - 1.1 E xtraction o f PFO S from L iver Page 12 of 15 Page 140 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound PFOS Matrix Rabbit Bovine MDL LOQ 11.8 ppb 37.4 ppb ' n/d = not determined2 Approximate Linear Range1 StaLnodward !jHigh Standard 38 ppb 1000 ppb 60 ppb 1200 ppb Rat Monkey 24.7 ppb 78.7 ppb n/v = not valid3 62 ppb 59 ppb 1200 ppb 1200 ppb PFOSA Rabbit 6.06 ppb 19.3 ppb 20 ppb 1200 ppb Bovine n/d n/d 30 ppb 1200 ppb Rat 20.7 ppb 65.8 ppb 6 ppb 1200 ppb Monkey 27.4 ppb 87.1 ppb 6 ppb 1200 ppb PFOSAA Rabbit 55.7 ppb 177 ppb 180 ppb 1900 ppb Bovine n/d n/d 120 ppb 1200 ppb Rat n/v n/v 62 ppb 1200 ppb Monkey n/v n/v 120 ppb 1200 ppb EtFOSE-OH Rabbit 58.7 ppb 187 ppb 190 ppb 1800 ppb Bovine n/d n/d 120 ppb 1200 ppb Rat n/v n/v 120 ppb 1200 ppb Monkey n/v n/v 58 ppb 1200 ppb POAA Rabbit 23.7 ppb 75.5 ppb 76 ppb 1800 ppb Bovine n/d n/d 120 ppb 1200 ppb Rat n/v n/v 62 ppb 1200 ppb Monkey n/v n/v 120 ppb 1200 ppb PFOSEA Rabbit 16.2 ppb 51.7 ppb 62 ppb 1200 ppb Bovine n/d n/d 30 ppb 1200 ppb Rat n/v n/v 120 ppb 1200 ppb Monkey n/v n/v 120 ppb 1200 ppb Monoester Rabbit n/d n/d n/'a n/'a Bovine n/d n/d n/a n/a Rat n/d n/d n/'a n/'a Monkey n/d n/d n/a n/a e1x-cUespspiveerlyLiwmeiitgchhtotsheenswtahnedraerdthceuvravleuoerwaaffsewctitRhienpetahteabLiilniteyar&CRaleibprraotdiouncibRialintygev(aLluCeRs.) but did not 2 - Not determined refers to no sample was analyzed for this data. 3de-teNromtinvaatliiodnr.efers to data from the analysis failed to meet specific criteria for a valid MDL/'LOQ jC om pound L iv er M atrix R ab b it B o v in e Rat M onkey Prepared Range of Standards (p p b )(n g /g ) 5.95 - 1790 6.00 - 1200 6.22 - 1240 5.93 - 1190 PFOS Range of Average Curve (p p b )(n g /g ) 5.95 -1790 6.00 - 1200 L-__X__e_R__lf_r_o____ 1 R a n g e of LC R frani' Range of 1 1ffe! Low Std. Curve . Loti Std.:;7= H igh Std. Curve pH sim ilim (p p b )(n g /g ) 5.95 - 298 n/a 3 ;..(-pirp.69X;-d2j9?8/^ j n/a/*, (p p b )(n g /g ) 119-1790 n/a TSx 9? ___65_.._92_32__--_1_12_14_9_00___ii 622.r .1240 5913-9 : n/a n/a j . _. _n/a ! n/a n/a j n/a .. .n/a- : j : n/a Attachment C: LOQ summary 3M Environmental Laboratory FACT-M-1.1 Extraction o f P F O S from L iv er Page 13 o f 15 Page 141 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Com pound PFOSA ; 1 L iv er M atrix R abbit B o v in e Prepared Range of Standards (p p b )(n g /g ) 6.04 - 1810 5.95 - I 190 Range of Average Curve (p p b )(n g /g ) 6.0 4 - 1210 5 .9 5 -1 1 9 0 .L C R f r--o m-.z o m sm R ange of i L C R from y Range of I LGR/frptn L ow S td. L o\^ S td /V ( High Std. j.'H p iS td : Curve i --v. C u ry e iS i Curve (p p b )(n g/g) (pp^KPE/E^y: (P Pb)(ng/g) j[ppB}(ffge> 6 .0 4 - 302 ,r,A 2 i\^ Z 0 te i: 121 1 2 1 0 1m m m i n/a n/a Rat 6 .1 7 - 1240- 6.17 - 1240 n/a n/a M onkey Com pound L iv er M atrix 5.88 - 1180 Prepared Range of Standards (PPb)(ng/g) 5.38 - 1180 i PFO SA A (p p b )(n g /g ) j n/a ... .- -ttn / j S fi n/a i---------------------- 1 Range of ;bpC R Jj$m & Low Std. Curve m (p p b )(n g /g ) (P p b )(n g /g ) R abbit B o v in e 6.33 - 1900 5.99 - 1200 127 - 1900 120-1200 fflaaM B O M ^ '& m S S U n/a n/a n/a Rat M onkey 6.21 - 1240 5.92 - 1 180 62.1-1240 l l i p p i 59.2 - 1180 g P ]|jjg jg j i . n/a S H U fi ';i n/a n/a Com pound : E tFO SE -O H ; ii j| L iv er M atrix Prepared Range of Standards (P P b )(n g /g ) Range of ^ ange t5 L C K ^ ^ ^ ^ Range of Average Curve H igh Std. Curve |p | (ppb)(ng/g) ffiM flia llf (ppb)(ng/g) r r 'fp p B ^ ^ ^ i (PPb)(ng/g) R a b b it B o v in e Rat M onkey 5.96 - 1790 119-1790 aM i 5 .8 7 - 1 170 58.7 - 1 170 ! 6.09 -1220 122-1220 I S n 1 5.80-1160 [ 29 4 -1160 | ^ n /a n/a F l& f S S ffig g n/a n/a n/a V4 Compound]________ Liver Prepared M atrix Range of Standards (p p b )(n g /g ) ; POAA Range of Average Curve (p p b )(n g /g ) Range of pi n soei t o J Low Std. Curve (p p b )(n g /g ) S lgt .LRfroraaR Range of li.a ip w jstd ^ w High Std. Curve ! 'r (pp bM nJ'g)J (p p b )(n g /g ) R abbit B o v in e 6.06 - 1820 30.3 - 1820 sffiPSifaggg 30.3 - 606 -q a ie g r ^ t 303 - 1820 5.93 - 1190 59.3 - 1190 n/a i- i - i'ttv 'ify ^ r ' n/a Rat M onkey | 6.15-1230 5.86-1170 61i1-57--1i12730 m m s a n/a n/a j n/avt5ii^r; n/a n/a jk.v*-^s^APtvf Le :' C om pound 1 Liver Prepared M atrix Range of Standards (PPb)(ng/g) jH IPFOSEA (p p b )(n g /g ) ji ; R a n g e o f [.- L Q R frm l^ - R an ge o f [-.L C R irom - Low Std. Curve (p p b )(n g /g ) iE m ^ fe |' (ppb)C ng/gjS H|C?uhrSvted- (P P b )(n g /g ) np!(lfsiQpfp*iXi^p*&p&'Ei)r R ab b it B o v in e Rat 6.20 - 1860 5.92- 1180 6.14 - 1230 020 - 1240 r i g & g l 219236 --11213800 . ^ F 2 3 g r n/a n/a n/a n /a,:r * 2 g .-R t :F5 /a a r t y | ---n/a; -* - n/a | A'-^n/ahn/a l-.i.tjt/a y -. n/a j 1"-n /a -, -1 M onk ey 5.85 - 1170 58 5 -1170 -U ^ 1170t. n/a j n/a n/a ! n/a Monoester was not detcctablc/quamiliable in liver matrix for the concentration range of 4 94 - i450 ppb Attachment C: LOQ summary 3M Environmental Laboratory FACT-M-1.1 Extraction of PFOS from Liver Page 14 o f 15 Page 142 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Prep D atefs): j 11/2/98 A naiyst(s): IRWW-IAS Sam p le M atrix: [Monkey Liver M eth o d /R e v isio n FA CT-M -1.0 Target FC-Mix A nalyte(s): 1 Ion Pair Standard Curves--Tissues Study Number: [Cross Validation i Equipm ent Num ber: ; [F in a l S o lv e n t <& T N N u m b er: ;MeOH TN-A-2076 i Blank T issue/Identifier. Liver ! j FC M ix Std A pprox. 0.500 ppm: IW398-1004 FC M ix Std A p prox. 5.00 ppm:' [W398-1003 F C M ix S td A p p r o x . 50.0 ppm: !W 398-I002 S u r r o g a te S td A p p r o x . 16.5 ppm: ) j W398-989 Actual Concentrations o f Standards in the FC Mix PFOS PFOSA PFOSAA 1 EtFOSE Std Cone. ; Std Cone. Std Cone. Std Cone. ug/mL 1 ug/mL - ug/mL ug/mL 0.501 0.497 0.500 0.490 0.501 0.497 0.500 0.490 0.501 0.497 0.500 0.490 0.501 0.497 0.500 0.490 0.501 0.497 j 0.500 0.490 5.01 4.97 [ 5.00 4.90 5.01 4.97 5.00 4.90 5.01 4.97 : 5.00 4.90 50.1 49.7 ! 50.0 49.0 , i ! 1 POAA Std Cone. ug/mL 0.495 0.495 0.495 0.495 0.495 j 4.95 1 4.95 1 4.95 I 49.5 | I PFOSEA | Std Cone. . ug/mL 0.494 i 0.494 i 0.494 0.494 0.494 4.94 4.94 4.94 49.4 ; C alcu lated C on cen tration s o f S tan dards in the Sam ple M atrix. PFOS i PFOSA PFOSAA 1 EtFOSE Final Cone. "g/g 5.93 [ Final Cone. 1 ng/g 5.88 Final Cone. ng/g 1!1 Final Cone. ng/g 5.92 5.80 11.9 11.8 11.8 11.6 29.6 29.4 29.6 29.0 59.3 58.8 59.2 58.0 119 118 ! 18 116 296 294 296 290 593 588 592 580 889 882 888 870 1186 1176 1183 1160 POAA PFOSEA Final Cone. Final Cone. ng/g ng/g 5.86 5.85 i i.7 11.7 29.3 29.2 58.6 58.5 117 117 2 9 3 292 586 85 879 877 1 172 1169 ! t 1 1 ii ; ah Ain't 1 Spiked i mL ; 0.002 0.004 0.010 i 0.020 0.040 0.010 0.020 0.030 [ 0.004 i ; ! !i ir A ll Liver cone. g/ml [ 0.169 0.169 I 0.169 [ , 0.169 i 0.169 0.169 1 0.169 1 0.169 j 0.169 ! 1 i S u r r o g a te 1 Std Cone, i Am't ; Spiked ug/mL mL 16.50 ! 0.005 Surrogate1 Final Cone. ng/g 488.2 11 1 V alidated R anees -- ADDroxim ate C oncentrations L iv er PFOS PFOSA PFOSAA R a b b it 40 - 1000 ppb 20 - 1200 ppb 180- 1900 ppb B o v in e 60 - 1200 ppb 30 - 1200 ppb 120- 1200 ppb R at 60 - 1200 ppb 7 0 - 1200 ppb 60 - 1200 ppb M onkey 60 - 1200 ppb 9 - 1200 ppb 120- 1200 ppb E tF O S E -O H POAA 190 - 1800 ppb 80 - 1800 ppb 120- 1200 ppb 80 - 1200 ppb 120 - 1200 ppb 60 - 1200 ppb 60 - 1200 ppb 120 - 1200 ppb PFOSEA 60 - 1200 ppb 30 - 1200 ppb 120 120 -1200 -1200 ppb ppb i 1 Attachm ent D: Calibration standard concentration worksheet F A C T -M -l.l Extraction of PFOS from Liver 3M Environmental Laboratory P age 15 o f 15 Page 143 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory M ethod A n a ly sis o f F luo ro ch em icals in L iv er E xtracts U sing H P L C -E lectro spray/M ass S pec tr o m etry Method Number: FACT-M-2.1 Author: Lisa Clemen Approved By: Laboratory Manager /'v 7 ^ 7 Group Leader hT(echnical R___e_v_i_e__w__e__r______________________________________________ Adoption Date: 05/26/98 Revision Date: / ?/ T ? Date 6 / j },') Date Uni Date 1.0 Sc o p e a n d A p p l ic a t io n _________________________________________________________________ 1.1 Scope: This method is for the analysis of surfactants using HPLC-electrospray/mass extracts of liver spectrometry. or other tissues for fluorochemical 1.2 Applicable Compounds: Potassium perfluorooctanesulfonate, anionic fluorochemical surfactants, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, and monkey livers or other livers as designated in the validation report. Word 7.0.1/95 Analysis of LFivAeCrTE-xMtr-a2c.t1Using ES/MS 3M Environmental Laboratory Page 1 of 9 Page 144 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2 .0 _Su m m a r y o f M e t h o d ______________________________________________________________ 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HioPnLcCha-erlaecctterroisstpicraoyf/ma apsasrtsipcueclatrrofmlueotrroyc. hTehmeicaanla, lsyuscishiassptehrefopromtaedssibuymmonitoring a single vpeerrifflyuocroomocptoaunnedsuildfoennatitfeic(aPtiFoOn.S) anion, M/Z= 499. Samples may also be screened to 3 .0 __D e f in it io n s _________________________________________________________________________ 3.1 A tm ospheric Pressure Ionization (API): The Micromass platform systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These Iinocnliuzdateiobnut(AarPecnI)o,tJlhimerimtedostpor:aEyl,eecttcr.osTphraeyioIonniziazatitoionnp(rEoSceIs),s AintmthoesspehteercihcnPiqreusessuroecccuhresmaitcal atmospheric pressure (i.e. not under a vacuum). 3.2 E lectrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ionization occurs through the production of tiny charged droplets in a strong electrical field. 3.3 MThaessAPSIpepclatrtfoomrmetsryar, eMeqaussipSppedecwtritohmqeuteardr(uMpoSl)e, TmaanssdseemlecMtiavsesdSepteeccttorros.m Ieotenrs (aMreS/M S): selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MinfSormmaaytibone.employed for ion detection or a series (MS/MS) for more specific fragmentation 3.4 C onventional vs. Z-spray probe interface: The latest models of Micromass platform osyrtshteomgosn(aplotsot t1h9e9c8o)nuetialipzeertaur"eZ.-sIpnrtahye"ccoonnvfoenrmtioantiaolnc. oTnfhoermspartaioynemitiitsteadimfreodmdairpecrtolbyeatisthe cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one sapnroatyhesry,sbteumt osn, leytcw.)ith similar systems (i.e. Z-spray components are compatible with other Z- 3.5 MsaIIyresoatrssesimQmLusil.yaatnrtCr.xuoSFrIrooIefrMntwmtlayaosrrsMeeL:adysneSstxLyaisoyltrsnexmMseheasasotsshfLtWewymaninrxaednNoudwTaelssUisg9pSn5eEecadRinfdif'coSrWtGothiUntehdIesDopwiEencs)s.NtifriTucmo3e.p1netrva(eMtriosiincornoosfm. tahAseslslePvplealrtasftoiforonmrsm 4.0 W a r n in g s a n d C a u t io n s _________________________________________________________ 4.1 Health and Safety W arnings: 4.1.1 Uapspercoaxuimtioantewlyit5h00th0eVvoollttsa.ge cables for the probe. The probe employs a voltage of 4.1.2 aWndhecnlohthanindgl.ing samples or solvents wear appropriate protective gloves, eyewear, Word 7.0.1/95 Analysis of LFivAeCr TE-xMtr-a2c.t1Using ES/MS 3M Environmental Laboratory Page 2 of 9 Page 145 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 4.2 Cautions: 4.2.1 Dovoerno4t00rubnasro, ltvheenHt Ppu1m10p0s wabilolvienictiaaptaecaituytoomf a4t0ic0 sbhaurt(d5o8w0n0. psi). If pressure goes 4.2.2 Do not run solvent pumps to dryness. 5.0 I n t e r f e r e n c e ________________________________________________________________________ 5.1 sThooumldinnimotizbeeiunsteerdfefroernscaems pwlheesntoarnagaelyozrinagnsyapmaprtleosffionrstpreurmfleunotraotoiocntatnhoaat tceo(mPOesAiAn),cotenftlaocnt with the sample or extract. 6.0 E q u ip m e n t _____________________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP1100 low pulse solvent pumping system and autosampler. 7.0 Su p p l ie s a n d M a t e r ia l s _____________________________________________________ ;_________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi 7.1.2 HPLC column, specifics to be determined by the analyst. 7.1.3 Capped autovials or capped 15 mL centrifuge tubes. 8.0 R e a g e n t s a n d St a n d a r d s _____________________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. 8.1.2 MASilTliM-Q,TMTywpaetIerwaantedrm, Mayillbi-eQpTMrovwidaetedr,bayllawMatilelri-uQseTdOinCtPhlius smseytshtoemd .should be 8.1.3 Ammonium acetate, reagent grade or equivalent. 8.2 Standards 8.2.1 Typically one method blank, one matrix blank, and ten matrix standards are prepared during the extraction procedure. See FACT-M -1.1. 9 .0 Sa m p l e H a n d l in g _____________________________________________________________________ 9.1 Farreesshtomreadtriinx csatapnpdeadrdasutaorveiaplrsepoarrceadpwpeitdh1e5acmhLanceanlytsriisf.ugEextturbaecsteudnsttial nadnaarldyssisa.nd samples 9.2 If analysis will be delayed, extracted standards and samples may be stored at room temperature or refrigerated at 4 C until analysis can be performed. FACT-M-2.1 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 3 of 9 Page 146 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03G laboratory Request Number-U2279 10.0 Q u a l it y C o n t r o l ___________________________________________________________ ___________ 10.1 M atrix Blanks and M ethod Blanks 10.1.1 Analyze a method blank and matrix blank prior to each calibration curve. 10.2 M atrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate with each analysis. With a minimum o f 2 spikes per batch. 10.2.2 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the low-range of the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Achnaanlgyeze(a 3m0i%d-)rainngpeeackaliabrreaatioocncustrasn, drealradtiavfetetroetvheeriynitteinatlhstsaanmdparled. cIufravesi,gsntiofpicathnet run. Only those samples analyzed before the last acceptable calibration standard will be used. The remaining samples must be reanalyzed. 10.3.2 See section 13 to calculate percent difference. 11.0 C a l i b r a t i o n a n d S t a n d a r d iz a t io n ___________________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The average o f two standard curves will be plotted by linear regression (y = my + b), not forced through zero, using MassLynx or other suitable software. ' 11.2 sItfatnhdeacrudrcvuerdvoee(sifnnoetcmeseseatryre)qaunidrermeaenntasl,ypzee.rform routine maintenance or reextract the 11.3 For purposes o f accuracy when quantitating low levels of analyte, it may be necessary to use the low end o f the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a rrcaaegnlirgbeersaostiifoontnhewcuceurigvrhevtecino(5gnspoipsftbhinitggoho1cf0o0tnh0ceepsnpttabrn)a.dtiaTorndhsisstfarwnoidmllarr5despd.pubcetoina10cc0uprpabcyraatthtreirbuthteadn ttohelinfuelalr 12.0 Pr o c e d u r e s _____________________________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign afofrilaecnqaumireinugs,inagndMtyOp-eDiAnYsa-lmasptledidgeistcorifpyteioanrs-s.ample number, assign a method (MS) 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass F A C T -M -2.1 Analysis of Liver Extract Using ES/MS Page 4 of 9 3M Environmental Laboratory Page 147 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the sample list begins with the first set of liver standards and ends with the second set of standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the Autosam pler 12.2.1 Set up sample tray according to the sample list prepared in section 12.1.1. 12.2.2 Set-up the HP1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time =13.5 minutes 12.2.2.4 Solvent ramp = Time 0 . 0 0 min. 8 . 0 min. 1 1 . 0 min. 1 2 . 0 min. MeOH 40% . 90% 90% 40% 2.0 mM Ammonium acetate 60% 10% 10% 60% 12.2.2.5 Press the "Start" button. 12.3 Instrum ent Sep-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. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eye piece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out o f the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: FACT-M-2.1 Analysis of Liver Extract Using ES/MS Page 5 of 9 3M Environmental Laboratory Page 148 kOo 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03 laboratory Request Number-U227 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 LC constant flow mode flow rate 10 - 500 uL/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the instrument is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Print the tune page, with its parameters, and store it in the study binder with a copy taped into the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10toCploicfksaomn pstlaertlisbtu. ttEonnsiunrethsetaArtcaqnudiseitniodnsaCmonptlreonl uPmanbeelr. inPcrleusds etshealsltasratmbpultetsontoatbe analyzed. 13.0 D a t a A n a l y s is a n d C a l c u l a t i o n s __________________________________ 13.1 Calculations: 13.1.1 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.2 Calculate percent difference using the following equation: % Difference = Expected CoEnxep.e-ctCedalcCuolantee.d Cone, x 100 f13.1.3 Calculate actual concentration o f PFOS anion in total liver (mg): ug PFOS anion calc, from std curve^ V g of liver used for analysis 1000 ug / 1mg x Total mass of liver (g) 14.0 M e t h o d P e r f o r m a n c e ______________________________________________ . --------- 14.1 MmaettrhioxdspDeectiefcict.ionPlLeaimseitse(Me EDTLS) -a8n-d1.1L,imAitttaocfhQmueannttitBa,tifoonr (aLlOisQtin) garoefmcuerthreondt, vaanliadlyattee,dand MDL and LOQ values. 14.1 14.2 Solvent Blanks, Method Blanks, and M atrix Blanks 14.1.1 Solvent blanks, method blanks, and matrix blanks values are must be below the lowest standard in the calibration curve. 14.2 Calibration Curves 14.2.1 The r value for the calibration curve must be 0.980 or better. FACT-M-2.1 Analysis of Liver Extract Using ES/MS Page 6 of 9 3M Environmental Laboratory Page 149 CD O') 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03 laboratory Request Number-U227 14.3 Matrix Spikes 14.3.1 Mcoantcreinxtrspatikioenp. ercent recoveries are must be within 30% of the spiked 14.4 Continuing Calibration Verifications 14.4.1 Continuing calibration verification percent recoveries must be 30% of the spiked concentration. 14.5 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.6 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 P o l l u t i o n P r e v e n t io n a n d W a s t e M a n a g e m e n t ___________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and glass pipette waste'is disposed in broken glass containers. All containers are located in the laboratory. 16 .0 R e c o r d s _______________________________________________________________________________ 16.1 hEeaacdhepraogrehgaennderwartietdtenfoorna tshtuedpyagme:uststhuadvyeotrhperfoojlelcotwninumg binefro,ramcqautiiosnitiionnclmudeethdoedi,ther in the integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 sPtloortethine tchaelibstruadtiyonfoclduerrv.e by linear regression, weighted 1/x, then print these graphs and 16.4 Panridntstdoarteaiinnttehgersattuiodny sfuomldmera. ry, integration method, and chromatograms, from MassLynx, 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see A ttachm ent A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 17.0 T a b l e s . D ia g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a ____________________________ 17.1 Attachment A: FACT-M-2.1 Data reporting spreadsheet FACT-M-2.1 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 7 of 9 Page 150 3m Medical Department Study: T-6295.7 Report No. FACT T0X-C3G laboratory Request Number-U227S 18.0 R e f e r e n c e s _____________________________________________________________ 18.1 FACT-M -l.l, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical compounds from Serum for Analysis Using HPLC-E!ectrospray/Mass Spectrometry 18.2 IEoTnSiz-a9t-i2o4n./0M, a"OsspSepraetcitornomanedterMQaiunattetnroanIcIetroipfltehequMadicrruopmolaessSyAsttmemossp"heric Pressure 18.3 The validationTeport associated with this method is FA CT-M -l.l-V & 2.1-V-l. 19.0 A f f e c t e d D o c u m e n t s ________________________________________________________________ 19.1 UFAsinCgTH-MPL-lC.l-,E"lEecxttrroascptiroany/oMf aPsostaSspseiuctmroPmeertfrluy"orooctanesulfonate from Liver for Analysis 20.0 R ev isio n s_______________________________________________________________________________ RNuevmisbieorn. 1 Reason For Revision Section Section 611.1.1.2 Clarification o Average of two f HP 1100 system components. curves, not standard values, are used for plotting linear regression. Section 12.2.2.4 Clarification of solvent ramp. Section 17.1 Changed from attachment B to A. ReDvaistieon 05/04/99 FACT-M-2.1 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 8 of 9 Page 151 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U2279 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: " Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst Date of Analysis/Analyst: Group Sample# Concentration Dose ug/mL Initial Voi. mL ' Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Data Sheet FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 9 of 9 Page 152 3ra Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory M ethod E x t r a c t io n o f P o tassium Perfluo ro o ctanesulfo nate o r O th er F l u o r o c h em ic a l co m po unds fro m Serum o r O th e r Fluid fo r A nalysis U sing H P L C -E lectro spray/M ass S pectro m etry M ethod Num ber: FACT-M-3.1 Author: Lisa Clemen, Glenn Langenburg Adoption Date: 04/22/98 hiRevision Date: 10 ( ^ ? Group Leader (it Technical A ihRevieweri'ri.K *7h - ^ i f Date Date 1.0 Sc o p e a n d A p p l ic a t io n _______________________________________________________________ 1.1 oSrcoopthee:r This method is fluorochemical for the extraction compounds from of potassium perfluorooctanesulfonate serum or other fluid. (PFOS ) 1.2 A pplicable com pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey serum, rat whole blood, and rat milk curd. Word 6/95 Extraction of PFOSFAfrCoTm-MSe-3ru.1m and Other Fluids 3M Environmental Laboratory Page 1of 17 Page 153 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Su m m a r y o f M e t h o d _________________________________________________________________ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemicals from serum, blood, or milk curd using an ion pairing reagent and 5.0 ml o f ethyl acetate. In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, POAA, PFOSEA, and FC-807 monoester analyte ion (pseaeir3is.0pDaretfiitnioitnioednsin).toAenthiyolnapceatiartine.g reagent is Four ml o added to the sample and the f extract are removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 ml o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3.0 D e f in it io n s ____________________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C8F17S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8FI7S0,N(CH2CH3)CH2C 02' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F,7S 0 2N(CH2CH3)CH2CH20H 3.5 POAA: perfluorooctanoate (anion of ammonium salt) C7FlsCOO` 3.6 PFOSEA: perfluorooctane sulfonyl ethylamide C8F17S 0 2N(CH2CH3)H 3.7 FC-807 monoester C8F,7S0,N(CH2CH3)CH2CH20 -P 0 3H) 3.8 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4 .0 W a r n in g s a n d C a u t io n s ______________________________________________________________ 4.1 H ealth and safety warnings 4.1.1 Uhasnedulinnigvearnsaiml palretcisasuuteio, nwsh, iecshpemcaiayllcyonlatbaoinraptoarthyocgoeantss., goggles, and gloves when 5.0 I n t e r f e r e n c e s ________________________________________________________________________ 5.1 There are no known interferences at this time. 6.0 E q u ip m e n t _____________________________________________________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g) FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 2 of 17 Page 154 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 7.0 S u p p l ie s a n d M a t e r ia l s _______________________________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Electronic pipettor, Eppendorf orequivalent 7.4 Graduated pipettes 7.5 Nalgene bottles, capable o f holding 250 mL and 1 L 7.6 Volumetric flasks, glass, type A . 7.7 Volumetric pipets, glass, type A 7.8 I-CHEM vials^glass, 40 mL glass 7.9 Crimp cap autovials 7.10 Centrifuge tubes, polypropylene, 15 mL 7.11 Labels 7.12 Syringes, capable o f measuring 5 pL to 50 pL 7.13 Syringes, disposable plastic, 3 cc 7.14 Syringe filters, nylon, 0.2 pm, 25 mm 7.15 Timer Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Msepilalir-aQteTMviwalast.er. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 8.0 R e a g e n t s a n d St a n d a r d s _____________________________________________________________ 8.1 TbeypMeiIllir-eQagTMenwt agtreardeanwdamtera,yMbeillpi-rQovTMideodr beqyuaivMalielnlit-;QalTl OwCatePrluussTMed sinystthemis method should 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (N&,C03), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8 .6 Ethyl acetate, 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 Control matrix or blank matrix for purpose o f standards, QC checks, blanks, etc. 8.10 F luorocbem ical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid Page 3 of 17 3M Environmental Laboratory Page 155 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U227S 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.10.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 571 8.10.5 POAA (3M Specialty Chemical Division), molecular weight = 431 8.10.6 PFOSEA (3M Specialty Chemical Division), molecular weight = 527 8.10.7 FC-807 monoester (3M Specialty Chemical Division). FC-807 is a mixture of tmrioelsetceur,ladriewsteeirg,hatn=d 6m5o0noester fluorochemical components. The monoester 8.10.8 Surrogate standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H, 2-H C8F13S 0 3H) molecular weight = 428 8.10.9 Other fluorochemicals, as appropriate 8.11 Reagent preparation 8.11.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.11.2 I N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL of 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.11.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g o10f TuBsiAnginatpoprao1xiLmvaotelulym4e4trtioc 5co4nmtaLinoinfg1050N0 NmaLOMHilalni-dQdTMiluwteatteor.voAldujmusetwtoitphH Milli-QTM water. While adding the last mL of NaOH, add slowly because the pH changes abruptly. Store in a 1 L Nalgene bottle. 8.11.3.1 nTeBedAedreuqusiinregs1aNchNecakOpHrisoorlutotioenac.h use to ensure pH = 10. Adjust as 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NaXOj/NaHCOj): Weigh approximately 26.5 g of sodium carbonate (Na^COj) and 21.0 g of sodium bQiTMcarwbaotneart.e S(NtoareHiCn0a3)1inLtoNaalg1eLnevobloutmtlee.tric flask and bring to volume with Milli- 8.12 Standards preparation 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard s1o.1lu0tipopnmcoEnttFaOinSinEg-O1H.0.0) ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 8.12.3 tWheeiagchtuaaplpwreoixgihmt.ately 100 mg o f PFOS into a 100 ml volumetric flask and record 8.12.4 B(prgin/mg lt)o. volume with methanol for a stock standard of approximately 1000 ppm 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. Extraction of PFOFSACfrTo-mMS-3e.r1um or Other Fluid 3M Environmental Laboratory Page 4 of 17 Page 156 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.12.6 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.12.7 Dapilpurtoex.th0e.5s0topcpkmso. lution with methanol for a working standard 3 solution of 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, C ,FnS03H into a 50 ml volumetric flask and record the actual weight. 8.13.2 Bring to volume with methanol for a surrogate stock o f approximately 1 0 0 0 - 1 2 0 0 ppm. 8.13.3 Psurrerpoagraetea ssutorcrkogtaotea w50ormkilnvgolsutamnedtarridc. flTasrkanasnfedrbarpinpgrotxoimvoaltuemlye0w.5itmh lmoefthanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 Sa m p l e H a n d l in g ______________________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Q u a l i t y C o n t r o l _____________________________________________________________________ 10.1 M atrix blanks and method blanks 10.1.1 Esaxmtrpalcetstwdiolu1te.0d m1:L1 waliitqhuoMtsiloli-fQthTMe awpaptreorp)rfiaotlelomwaintrgixth(siserpurmoceodrubrleooadn,dwuisteh abslood matrix blanks. See 11.1.4. 10.1.2 Emxettrhaocdt tbwlaonk1s.0. ml aliquots of Milli-QTM water following this procedure and use as 10.2 M atrix spikes 10.2.1 Pthreepaacrceuraancdyaonfatlhyezeexmtraatrcitxiosnp.ike and matrix spike duplicate samples to determine 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range o f the initial calibration curve. Acadlidbirtiaotnioanl cspurikvees. may be included and may fall in the low-range of the initial 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare and analyze continuing calibration check samples to ensure the accuracy of the initial calibration curve. If the percent difference between the initial curve alansdt athcececpotnatbinleucinhgecckh.eck differ by >30%, re-analyze samples analyzed after the 10.3.2 Prepare one check per group o f ten samples. For example, if a sample set = 34, prepare and extract four checks. Extraction of PFOFSACfrTo-mMS-3e.r1um or Other Fluid 3M Environmental Laboratory Page 5 of 17 Page 157 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.3.3 Pthreepinairteiaelaccuhrvcoe.ntinuing calibration check from the same matrix used to prepare 10.3.4 Tcuhrevee.xpAedctdeidtiocnonacl esnptirkaetsiomnawyibllefainllclwuidtehdinththaet fmalildi-nratnhgeeloowf -trhaenigneitioafl tchaelibinriatitaiol n 5cloapwlipbberna-dtio1on0f0tc0huerpvpceab.l)i.bTrhaitsioins ncuecrevsesa(froyriefxtahme panlea,ly5spt pmbu-st1q0u0apnptibta,treauthseinrgthoannly the 11.0 C a l i b r a t i o n a n d St a n d a r d iz a t io n ________________________________________ ________ _ 11.1 Prepare m atrix calibration standards Note: Blood coagulates in air; therefore, minimize air contact until dilution. At this opofitnhte, dadildutTedBAmaatnrdixbsuafmfeprletotoeaecahchcetnutbrei.fuge tube as in step 12.9, then add 1.0 mL 11.1.1 Transfer 1 mL o f serum or 1 mL o f blood (blood is diluted 1:1 with Milli-QTM water) to a 15 mL centrifuge tube. The blood is similar in composition to milk curd and can be used in place o f milk curd for standard curves when extracting that matrix. . 11.1.2 vIRfoemlcuoomrsdtessthaeemquspaalmle tvpooletlhuvemosleausmmapereleolnevsotshluethmeaxentsr.1a.c0DtiomonnLo,stheeexextrtt.raaccttsbtaenldoawrd0s.5w0itmhLmoatfrmixatrix. 11.1.3 bWethwileeepnraepliaqruinotgs.a total of twenty aliquots in 15 ml centrifuge tubes, mix or shake 11.1.4 TTatywtphoiec1aelmnlydLuoasfletiqhtuhisoetssset,catonirdoanort,hdteocrosanppcikpeenr,otrpinartidaiotunepsvlioaclnaudtmes,ept,wiksoeinrsgvtaeanmadsaomrudnattcsruirxlivsbetelsad,nfkionsr.Taatbotleal1o, f eighteen standards and two matrix blanks. 11.1.5 Refer to validation reports FACT-M-3.1-V-1 and FACT-M-4.1-V-1, which list cthuervweos.rking ranges and the Linear Calibration Range (LCR) for calibration 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working cstaalnibdraartdios.n sSteaendSaercdtsio. n 13.0 to calculate actual concentrations of PFOS in 11.2 Tstoanedaacrhdsftoarndthaerdc,obnlcaennktr, aotrioQnCtochfaelclkw, iatdhdinatphpercoaplriibartaetaiomnocuunrtvoefrsaunrgreog5aptepbwo-1r0k0in0gppb. 11.3 tEoxetrsatactblsipshikeedacmh aintriitxiasltcaunrdvaerdosnftohlelomwainssg s1p2e.c6t-r1o2m.1e6tero.f this method. Use these standards Extraction of PFOFSACfrTo-mMS-3e.r1um or Other Fluid 3M Environmental Laboratory Page 6 of 17 Page 158 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Table 1 Approxim ate spiking amounts for standards and spikes using 1.0 ml of matrix Working standard (approx, cone.) *- pL Approx, final cone, of analyte in matrix - Blank 0.500 ppm 0.500 ppm 5.00 ppm 5.00 ppm 5.00 ppm 50.0 ppm 50.0 ppm 50.0 ppm 50.0 ppm 10 0.005 ppm 2 0 0 . 0 1 0 ppm 5 0.025 ppm 1 0 0.050 ppm 2 0 0 . 1 0 0 ppm 5 0.250 ppm 10 0.500 ppm 15 0.750 ppm 20 . 1 . 0 0 ppm Table 2 Approximate spiking amounts for standards and spikes using 0.5 ml o f m atrix__________________ Working standard (approx, cone.) pL Approx, final cone, of analyte in matrix - ... 0.500 ppm 5 Blank 0.005 ppm 0.500 ppm 1 0 0 . 0 1 0 ppm 5.00 ppm 2.5 0.025 ppm 5.00 ppm 5 0.050 ppm 5.00 ppm 50.0 ppm 50.0 ppm 10 2.5 0 . 1 0 0 ppm 0.250 ppm 5 0.500 ppm 50.0 ppm 7.5 0.750 ppm 50.0 ppm 1 0 1 . 0 0 ppm 12.0 P r o c e d u r e ______________________________________________ ___________________________ 12.1 Obtain frozen samples and allow to thaw. 12.2 pVoolrytperxompyixlenfoerc1e5ntsreicfuognedst,ubthee. nFtorranbslfoeord1.s0ammpLleosr, ortehmerovaepp0r.5opmriLateanvdolduimluetetotoa11.05 mmLL with Milli-QTM water. As soon after diluting as possible, pipet diluted blood into TBAbuffer mixture shown in step 12.9 and mix well. 12.3 Return samples to freezer after extraction amount has been removed. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 7 of 17 Page 159 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03G laboratory Request Number-U2279 12.4 Record the volume on the extraction worksheet. The final methanol volume equals the volume transferred from the sample. For example, if 0.5 mL is removed for a blood sample, the final methanol volume will equal 0.5 mL. 12.5 wLaobrkelshtheeettufobredwoictuhmtheentsitnugdythneurmembeari,nsinagmsptleepIsD. , date and analyst initials. See attached 12.6 Spike each matrix with the appropriate amount of standard as described in 11.1 or Table 1 or 2 in that section for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.7 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.8 sVaomrtpelxesmfoixr t1h5essetcaonnddarsd. curve samples, matrix spike samples, and continuing calibration 12.9 Tbiocaerabcohnsaatembpuleff,eard. d 1 mL 0.5 M TBA and 2 mL of 0.25 M sodium carbonate/sodium 12.10 Using a volumetric pipette, add 5 mL ethyl acetate. 12.11 Cap each sample and put on the shaker for 20 minutes. 12.12 sCeepnatrraitfeudg.e for 20 to 25 minutes at approximately 3500 rpm, until layers are well 12.13 Tcernatnrsiffeurge4 tmubLe.oLf oarbgeal nthicislafyreesrh, utusibnegwait5hmthLe gsraamdeuaintefdorgmlaastsiopnipaesttien, 1to2.5a.clean 15 mL 12.14 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.15 Add 1.0 mL or other appropriate volume o f methanol to each centrifuge tube using a tghreadeuxatrteadctipoinp.ette. Methanol volume to add equals the initial volume of sample used for 12.16 Vortex mix for 30 seconds. 12.17 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.18 mLaabtreilxt,hfeinaaultsoovlivaelnwt,itehxttrhaecsttioundydantuem, abnedr,aannailmysatl(sn)upmebrfeorramnidnggetnhdeeerx, tsraamctpiolne.timepoint, 12.19 Cap and store extracts at approximately 4 C until analysis. 12.20 Cnootmebpoleotke otrheinecxlturdaectiinonstwudorykbsihnedeetr,,aatstaacphperdotporitahties. document, and tape in the study Extraction of PFOFSACfrTo-mMS-3e.r1um or Other Fluid 3M Environmental Laboratory Page 8 of 17 Page 160 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 13.0 _D a t a A n a l y s is a n d C a l c u l a t io n s _______________________________________________ 13.1 Calculations 13.1.1 cCaalilbcuralattioenacsttaunadl acrodnsceunstinragtitohnesfoofllPowFOinSg, eoqruoatthioenr:applicable fluorochemical, in mmLLo fosftastnadnadradrd+ xmcLonocfesnutrrraotgioanteosftastnadnadradrd+ liungiti/aml Lm)a_t_r_ix__v_o_l_u_m__e__(m__L_)___= Final Concentration (pg/mL) of PFOS in matrix 14.0 M e t h o d P e r f o r m a n c e ________________________________________________________________ 14.1 TfohrespmeectihfiocdMdeDteLctainond lliimmiitt o(Mf qDuLan) tiistaatnioanly(tLeOanQd) mvaalturiexs s(pseeeciAfictt.acRhemfeerntotsMBDaLndreCpo).rt 14.2 The following quality control samples are extracted with each batch of samples to ensure the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve 15.0 P o l l u t io n Pr e v e n t io n a n d W a s t e M a n a g e m e n t ___________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s ___________________________________________________________________________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in study 3-ring binder, as appropriate. 17.0 A t t a c h m e n t s _________________________________________________________________________ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDL/LOQ values 17.3 Attachment C, LOQ Summary 17.4 Attachment D, Calibration standard concentration worksheet 18.0 R e f e r e n c e s ___________________________________________________________________ ________ 18.1 The validation reports associated with this method are FACT-M-3.1 & 4.1-V -l. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 9 of 17 Page 161 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 19.0 a f f e c t e d D o c u m e n t s _____________________________________________________________ 19.1 FHAPCLCT--EMle-4ct.1ro, sp"AranyaMlysaisssoSfpSeecrturommoertrOy"ther Fluid Extracts for Fluorochemicals using 20.0 R e v is io n s _________________________________________________________________ RNeuvmi1sbioern Validatio` n o f method to inRcleuadseon7 FflouroRroecvhiseimonicals, an additional matrix, nimewprAovPeIm/MenSt(sMtoS)iosnysptaeimrisn,gmexotnrkaecytiosner,uMmDcLrosstsudvayl,iduaptdioante,s in record keeping and storing policies, etc. ReDvaistieon 07/01/98 FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 10 of 17 Page 162 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Study ft - - 1- S am p le Number set ft H ,0 Blank Blank - FC-M ix approx. 0.5 ppm actual ppm #W - FC-M ix approx. 5 ppm actual ppm #W - - FC-M ix approx. 50 ppm actual ppm #W - Date and Initials for Std. or C om m ents -- * -- - '- --- - -- -- *- - - -- - --- -- - - -- - -- -- -- - - '* " -- - - - ' Study number where the original wortsheet is located. Blank std n Serum Extraction Method Vortex 15 sec Pipette Matnx Volume Pipette 1ml of 0.5 M TBA, pH 10. Std. # - amount = ml - mL Date & Initials Pipette 2 m l o f 0.25 N a?C O yO -25M N aH C O t buffer Std. # P ipette S ml o f ethyl acetate________________________________ T N -A - ___________________________________ Shake 20 min. C entrifu ge 2 0 -2 5 m in._______________________________ C entrifuge speed:_________________________________________________________________ R em o v e a 4 m l aliq uot o f organic layer_______________________________________________________________________________________________ Put on N itro g en E vaporator to d ryness E vaporator #:__________________________Tem perature:______________________________________ A d d m e th a n o l___________________ V o lu m e m l______________ T N -A - V ortex 3 0 sec.____________________________________________________________________________________________________________________________ FM__ilS_te/_rM_u_Ss_iDn_g/p_ap_m3_cC.c oMBn-StD./MCsyhSreiDncgkuess:weSditphsiaakme0 d.p2_ul_em____S__R_Iu_Lf_il_toe_fr_ai_n__to____a __1__.5. pmplma ustotds a(m__p l_e_v_i_al__________________)__f_o_r__a_f_i_n_a_l__c_o__n_c_e_n__tr_a_t_i_o_n__o__f Cont. Checks used same matrix as for std curve. Surrogate Standard: Spiked_____uL of a _____ ppm std (______________ ) to all samples, standards, and blanks Attachment A: Extraction worksheet FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 11 of 17 Page 163 3m Medical Department Study: T-62 95.7 Report No. FACT TOX-030 laboratory Request Number-U2279 M DL/LOQ values for Rabbit Serum: Com pound MDL LOQ Linear Calibration Range (LCR) (ppb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.38 4.39 5 ppb - 1000 ppb PFOSA 2.23 7.09 10 ppb - 1000 ppb PFOSAA 2.84 - 9.04 10 ppb - 1000 ppb EtFOSE-OH 3.90 12.4 15 ppb - 1000 ppb POAA 4.31 13.7 15 ppb - 750 ppb PFOSEA 1.09 3.48 25 ppb - 1000 ppb Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. M DL/LOQ values for Rat Serum: Compound M DL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.27 4.04 10 ppb - 1000 ppb PFOSA 2.14 6.81 25 ppb - 1000 ppb PFOSAA 2.32 7.38 10 ppb - 1000 ppb EtFOSE-OH 3.25 10.3 50 ppb - 1000 ppb POAA 1.20 3.81 5 ppb - 1000 ppb PFOSEA 1.84 5.86 ... 10 ppb - 1000 ppb Monoester | 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. M DL/LOQ values for Bovine Serum: Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 2.11 6.70 25 ppb - 1000 ppb PFOSA 5.04 16.0 25 ppb - 1000 ppb PFOSAA 2.34 7.45 260 ppb - 1000 ppb EtFOSE-OH 11.3 35.8 50 ppb - 1000 ppb POAA 4.64 14.8 15 ppb - 1000 ppb PFOSEA 3.71 11.8 15 ppb - 1000 ppb Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. No data is available for MDL or LOQ in Monkey Serum. Use validated Linear Calibration Range instead. Please see Attachment C (LOQ Summary) and MDL study in FACT-M-3.1 & 4.1-V-l for specifics. Attachment A: Extraction worksheEetxtraction of PFOFSACfrTo-mMS-3e.r1um or Other Fluid 3M Environmental Laboratory Page 12 of 17 Page 164 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 M DL/LOQ values for M onkey Serum: Com pound M DL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.38 4.39 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOS will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. PFOSA 2.23 ` 7.09 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOSA will be an estimate PFOSAA 2.84 9.04 only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOSAA will be an estimate EtFOSE-OH 3.90 12.4 only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for EtFOSE-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. POAA 4.31 13.7 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for POAA will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. PFOSEA 1.09 3.48 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for PFOSEA-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. M onoester 149 248 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for EtFOSE-OH will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. M DL/LOQ values for Rat W hole Blood: Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (PPb) Approximate Concentrations to be used for preparing the Standard Calibration Curve PFOS 1.25 3.96 5 ppb - 1000 ppb PFOSA 1.77 5.65 10 ppb - 1 0 0 0 ppb PFOSAA 17.3 55.0 55 ppb - 1 0 0 0 ppb EtFOSE-OH 7.89 25.1 MDL and LOQ are estimates only. No valid MDL was determinable from eMstDimLastteuodnyl.y.AnPyleqasueanrteifteartitoonFpAerCfTor-mMe-d3.1for&E4tF.1O-VSE-l-OfoHr will be an specifics. POAA 4.73 15.1 15 ppb - 1 0 0 0 ppb PFOSEA 24.2 77.1 80 ppb --1 0 0 0 ppb Monoester 58.0 185 MDL and LOQ are estimates only. No valid MDL was determinable from MDL study. Any quantitation performed for monoester will be an estimate only. Please refer to FACT-M-3.1 & 4.1-V-l for specifics. Please see Attachment C (LOQ Summary) and MDL study in FACT-M-3.1 & 4.1-V -l for specifics. Attachment A: Extraction worksheEetxtraction of PFOFAS CfrTo-mMS-3e.r1um or Other Fluid 3M Environmental Laboratory Page 13 of 17 Page 165 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Ion Pairing Extraction o f Fluorochemicals from Serum and Analysis by API/MS(MS) Summary Table: Limits o f Quantitation Compound Matrix MDL LOQ LAopwp rsotdx im a te 2linear range High std PFOS Rabbit 1.38 ppb 4.39 ppb 5 ppb 1000 ppb Bovine 2.11 ppb 6.70 ppb 25 ppb 1000 ppb - Rat 1.27 ppb 4.04 ppb 10 ppb 1000 ppb Monkey n/d n/d 25 ppb 1000 ppb PFOSA Rabbit 2.23 ppb 7.09 ppb 10 ppb 1000 ppb Bovine 5.04 ppb 16.0 ppb 25 ppb 1000 ppb Rat 2.14 ppb 6.81 ppb 25 ppb 1000 ppb Monkey n/d n/d 25 ppb 1000 ppb PFOSAA Rabbit 2.84 ppb 9.04 ppb 10 ppb 1000 ppb Bovine 2.34 ppb 7.45 ppb 263 ppb 1000 ppb Rat 2.32 ppb 7.38 ppb 10 ppb 1000 ppb Monkey n/d n/d 25 ppb 1000 ppb EtFOSE-OH Rabbit 3.90 ppb 12.4 ppb 15 ppb 1000 ppb Bovine 11.3 ppb 35.8 ppb 50 ppb 1000 ppb Rat 3.25 ppb ' 10.3 ppb 50 ppb 1000 ppb Monkey n/d n/d 10 ppb 1000 ppb POAA Rabbit 4.31 ppb 113.7 ppb 15 ppb 750 ppb Bovine 4.64 ppb 14.8 ppb 5 ppb 1000 ppb Rat 1.20 ppb 3.81 ppb 5 ppb 1000 ppb Monkey n/d n/d 5 ppb 1000 ppb PFOSEA Rabbit 1.03 ppb 3.48 ppb 25 ppb 1000 ppb Bovine 3.71 ppb 11.8 ppb 5 ppb 1000 ppb Rat 1.84 ppb 5.86 ppb 10 ppb 1000 ppb Monkey n/d n/d 5 ppb 1000 ppb Monoester1 Rabbit 149 ppb 474.0 ppb 250 ppb 1000 ppb Bovine 149 ppb 474.0 ppb 250 ppb 1000 ppb Rat 149 ppb 474.0 ppb 250 ppb 1000 ppb Monkey n/d n/d 100 ppb 1000 ppb 1. Values for monoester are estimates only. 2. Highest standard (approx. 1500 ppb) was excluded from final LCR and upper LOQ values due to poor R & R values and excessive weighting of the calibration curve. Compound: PFOS Smearturmix (psPrptarabenn)(pgdnaegar/remoddfLs) (pRpaabcv)nue(ngrrvage/gemoeLf) LCR from ave curve (ppbXng/mL) (pRplaobc)nwu(gnrvges/etmdoLf) (LppClboc)Ruw(nrgfvsr/etmodml_) (pRhpbacig)nu(hgnrvges/emtodLf) LCR from (phpbcig)u(hnrvgs/emtdL) Rabbit -4 .9 3 1 4 5 0 4 .9 3 - 1 4 5 0 4 9 .3 - 1 0 0 0 4 9 . 3 - 9 7 . 6 4.93 -9 7 .6 9 7.6 - 1450 9 7 .6 - 1000 Bovine 4 .93 - 1450 4 .9 3 - 1450 9 7 .6 - 1000 4.93 - 248 24.8 - 248 97.6 - 1450 9 7 .6 - 1000 Rat 4.9 3 - 1450 4.93 - 9 76 24.8 - 9 76 4.93 -248 9.76 - 248 97.6 - 1450 2 48 - 1000 Monkey 4 .9 3 - 1 4 5 0 4 .9 3 - 1 4 5 0 2 4 .8 - 1 0 0 0 2 4 .8 -4 9 3 24.8 - 493 97.6 - 1450 97.6 1000 Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 14 of 17 Page 166 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 Compound: PFOSA Serum Prepared matrix range of (psptabnXdnag/rmdLs) Rabbit 5.00- 1470 Range of average (ppbcXurnvg/emL) 5.00- 1470 Bovine 5.00- 1470- 5.00 - 1470 Rat 5.00- 1470 5.00- 1470 Monkey 5.00- 1470 5.00 - 1470 LCR from ave curve (ppbXng/mL) 9.89 - 1000 2 5 .1 - 1000 50.0 - 1000 98.9 - 1000 Range of low std (ppbcu)(rnvg/emL) 5.00-251 5.00-98.9 9.89-500 25.1 - 500 LCR from low std (ppbc)u(nrgv/emL) n/a n/a 25.1 -500 25.1 - 500 Range of high std (ppbc)u(nrvg/emL) 9 8 .9 - 1470 9 8 .9 - 1470 98.9 - 1470 9 8 .9 - 1470 LCR from high std (ppbcu)(rnvg/emL) 9 8 .9 - 1000 9 8 .9 - 1000 9 8 .9 - 1000 n/a Compound: PFOSAA Serum Prepared Range of matrix range of average (psptabn)(dnag/rmdLs) (ppbcXunrgv/emL) Rabbit 5 .2 0 - 1540 5.20 - 1540 Bovine 5 .2 0 - 1540 5 .2 0 - 1540 Rat 5 .2 0 - 1540 5.20 - 1540 Monkey 5 .2 0 - 1540 5 .2 0 - 1540 LCR from ave curve (ppbXng/mL) 104- 1000 263 - 1000 104- 1000 52.4- 1000 Range of low std (ppbcXurnvg/emL) 5.20-263 10.4-521 5.20-263 5.20-263 LCR from low std (ppbc)u(nrgv/emL) 10.4-263 n/a (1) 10.4-263 26.3 - 263 Range of high std (ppbcXurnvg/emL) 104- 1540 104- 1540 104 - 1540 104- 1540 LCR from high std (ppbc)u(nrgv/emL) 263 - 1000 263 - 1000 263 - 1000 263 - 1000 Compound: EtFOSE-OH Smearturmix sPrtaraennpgdaearreoddfs Raavcnuegrrvaegeoef (ppbXng/mL) (ppbXng/mL) Rabbit 4 .9 4 - 1450 4.9 4 - 1450 Bovine 4 .9 4 - 1450 4.9 4 - 1450 Rat 4 .9 4 - 1 4 5 0 4 .9 4 - 1 4 5 0 Monkey 4 .9 4 - 1 4 5 0 4 .9 4 - 1 4 5 0 LavCeRcfurrovme (ppbXng/mL) 49.4 - 1000 97.8 - 1000 4 9 4 - 1000 9 7 .8 - 1000 Rlaocnwugrvesetdof (ppbXng/mL) 4 .94 - 248 4 .9 4 -2 4 8 4 .94 - 248 4 .94 - 2 4 8 LClocRwurvfsretodm (ppbXng/mL) 9 .7 8 -2 4 8 4 .9 4 -2 4 8 n/a 9 .78 - 248 Rhaicgnuhgrvesetodf (ppbXng/mL) 97.8 - 1450 97.8 - 1450 97.8 - 1450 97.8 - 1450 LhCcigRuhrfvrseotdm (ppbXng/mL) n/a 248 - 1000 97.8 1000 n/a Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 15 of 17 Page 167 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: POAA Serum Prepared matrix range of (psptabnXdnagr/mdLs ) Rabbit 5.01 - 1480 Range of average curve (ppb)(ng/m L) 5.13 - 1510 Bovine 5.01 - 1480- 5 .13-1510 Rat 5.01 - 1480 5.13-1510 Monkey 5.01 - 1480 5.13-1510 LCR from ave curve (ppb)(ng/mL) 25.8 - 1000 102-1000 51.3-1000 102 - 1000 Range of low std (ppbc)u(nrgv/emL) 5.13-258 5.13-258 5.13-102 5.13-102 LCR from low std (ppbcXunrgv/emL) n/a 5.13-258 5.13 - 102 5.13 - 102 Range of high std (ppbc)u(nrgv/emL) 102 - 1510 102- 1510 102-1510 1 0 2- 1510 LCR from high std curve (P P b )(n g/'m L ) n/a 258 - 1000 1 0 2 - 1000 258 - 1000 Compound: PFOSEA Serum Prepared Range of matrix range of average (psptabn)(dnga/rmdLs) (ppbc)u(nrgv/emL) Rabbit 5.13- 1510 5.13 - 1510 Bovine 5.13 - 1510 5.13 - 1510 Rat 5.13 - 1510 5.13 - 1510 Monkey 5.13 - 1510 5.13 - 1510 LCR from ave curve (ppbXng/mL) 25.8- 1000 102 - 1000 51.3-1000 102 - 1000 Range of low std (ppbcXunrgv/emL) 5.'l3 -258 5.13 -258 5.13 - 102 5.13 - 102 LCR from low std (ppbcXunrgv/emL) n/a 5.13 -2 5 8 5.13 -1 0 2 5.13 - 102 Range of high std (ppbcXurnvg/emL) 102 - 1510 102 - 1510 102 - 1510 102 - 1510 LCR from high std (ppbcXurnvg/emL) n/a 258 - 1000 102 - 1000 258 - 1000 Compound: Monoester Serum Prepared Range of matrix range of average (psptabn)(dnag/rmdLs) (ppbc)u(nrgv/emL) Rabbit 4.94- 1450 9.78 - 978 LCR from ave curve (ppbXng/mL) n/a Bovine 4.94- 1450 97.8 - 1450 n/a Rat 4.94 - 1450 2 4 8 - 1450 2 4 8 - 1000 Monkey 4.94 - 1450 49.4 -1450 97.8 - 1000 In general, the chromatography for the monoester was very poor (broad peaks, high baseline). Curves for monoester in rabbit and bovine were unacceptable. Any quantitation performed with the monoester is only an estimate and should not be used for reliable, accurate data reporting. Attachment C: LOQ Summary Extraction of PFOFSACfrTo-mMS-3e.r1um or Other Fluid 3M Environmental Laboratory Page 16 of 17 Page 168 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Ion Pair Standard Curves - Fluids PASranemaplpydlteae(tmse)(a:st):rix: SEFBtiqlanaunanidlkpasmforlldeuvniendtnu/nitmduaebmnnedtbrief:TireN:r:: TMFFFSCCuCaerrtgrmmmhoeoiiigtxxxdaa/ssstrnttetedddavslitysdtaieaaopa(ppnspppp):rp:rrorooxoxx.x...055.5.0100.7000.7pp1ppppmmpmp:::m: W398-641 W398-640 W398-639 W398-605 Actual concentrations of standards in the PFOS PFOSA PFOSAA EtFOOHSESutdg/cmoLne Sutdg/cmoLne Sutdg/cmoLne Sutdg/cmoLne FCPOmAixA Sutdg/cmoLne PFOSEA Sutdg/cmoLne Monor este Sutdg/cmoLne 0.500 0.507 0.532 0.500 0.507 0.532 5.00 5.07 5.32 5.00 5.07 5.32 5.00 5.07 5.32 50.0 50.1 53.2 50.0 50.1 53.2 50.0 50.1 53.2 Ca50lc.0ulated 5c0o.1ncentrat5i3o.2ns of 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 standards 0.509 0.509 5.09 5.09 5.09 50.9 50.9 50.9 .50.9 in the ' 0.521 0.521 5.21 5.21 5.21 52.1 52.1 52.1 52.1 sample matrix 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 PFOS Final cone PFOSA Final cone ng/m L PFOSAA Final cone ng/m L E tFO SE Final cone ng/m L POAA Final cone ng/m L PFOSEA Final cone ng/m L M onoester Std cone ng/m L ng/m L 4.93 9 .7 6 24.8 49.3 97.6 248 493 735 976 5 .0 0 9 .8 9 25.1 5 0 .0 98.9 251 500 746 989 5.24 4.94 5.01 5.13 4~94 10.4 9.78 9.93 10.2 9.78 26.3 24.8 25.2 25.8 24.8 52.4 49.4 50.1 51.3 49.4 104 97.8 99.3 102 97.8 263 248 252 258 248 524 494 501 513 494 782 737 749 766 737 1038 978 993 1017 978 All spiAkemd'tmL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All VFomilnuLaml e 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 S urrogate Std cone ng/m L 2.64 S urrogate Final cone ng/m L 81.0 A ll A m 't spiked (m L) 0 .0 0 5 Validated ranges - approximate concentrations S era PFOS PFOSA PFOSAA R abbit B ovine R at M onkey 5-1000 ppb 25-1000 ppb 10-1000 ppb E stim ates only. 10-1000 ppb 25-1000 ppb 25-1000 ppb Use values for 10-1000 ppb 263-1000 ppb 10-1000 ppb R abbit E tF O SE -O H 10-1000 ppb 5-1000 ppb 50-500 ppb POAA 10-750 ppb 5-1000 ppb 5-1000 ppb PFOSEA 25-1000 ppb 5-1000 ppb 5-1000 ppb Attachment D: Ion Pair Standard CEuxrvtreasction of PFOFSACfrTo-mMS-3e.r1um or Other Fluid 3M Environmental Laboratory P ag e 17 o f 17 Page 169 3m Medical Department Study: T-6295.7 VReport N o . FACT TOX,-030 laboratory Request *F 3M Environmental Laboratory M ethod A na ly sis of P o tassium Perfluoro octanesulfonate o r O th er F lu o r o c h em ic a ls in Serum o r O th er Fluid E xtracts U sing H P L C -E lectrospray/M ass Spectrom etry M ethod Number: FACT-M-4.1 Author: Lisa Clemen, Glenn Langenburg Approved By: Adoption Date: 4/22/98 Revision Date: lo -i-ij- Laboratory Manager | y/v. Group Leader ---------- ' Technicaflt RevLilexw/rweri. Date Mw Date Date 1.0 S c o p e a n d A p p l ic a t io n ____________________________________________________________________ 1.1 Scope: This method is for the analysis of extracts from serum or blood for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, or monkey serum and rat whole blood or milk curd. Word 6.0.1/95 FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory Page 1 of 9 Page 170 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 S u m m a r y of M ethod_________________________________________________________________ __ 2.1 wTashhaiosplepmrbeotlpohrooidadt,edo.ersTmchriielbkeascnuatrhlydesuaissniianslgypsHeirsPfooLrfCmfl-euedolerbcoytcrhomseopmnriaictyoa/rlmisnaugsrsfaascsptienacngttlrseoemioxentrtarcycht,aeordracfsrtieomrmiislatsirecrsouymfsta,em particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be analyzed using an API/MS/MS system to further verify compound identification. 3.0 Definitions__________________________________________________________ 3.1 vAatrmioousspmh eerthi codPsr eosfsiuorneizIaotnioinz abt iyounti(lAizPinIg): The Micromass various sources, platform systems allow probes, and interfaces. for These iIanotmcnliuozdsapteihobenurti(cAaprPercenIs)os,utTrlhieme(riim.tee.odsntpoort:auEynl,edecettrcr.oasTvparhcaeuyiuoImonni)z.iazatitoionnp(rEoSceIs),s AintmthoesspehteercihcnPiqreusessuroecccuhresmaitcal 3.2 E lectrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ionization occurs through th production o f tiny charged droplets in a strong electrical field. 3.3 TMhaesAs PSIpepclatrtfoomrmetsryar,eMeqaussipSppedecwtriothmqeuteardr(uMpSol)e, Tmaanssd seemlecMtiavses dSepteeccttorros.m Ieotne rs (MS/MS): are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 soCcyorostnhtneeovmageponsent(raiptoluontrsoaet,lt1hav9fest9.ec8r2o)1pnu-aestpsiaslripiazneyegrtaputhrr"roeZo.b-useIgpnihrntahtayee"trocfcraootcnunevof:oeunrsTmtpihoaaenttihaloawlnteca.soytTnimfhnoeorthmdspeearlctsaioyoounfenmMtiteiriitscteeraldoeimcmftrareoosdmsddepai.lrapeTtcrfohtolrobymueagtihsththee cHoonwfiegvuerra,tiZo-nspisradyifcfoermenpto,ntehnetsmaenthdocdosnovfenotpieornaatliocno,mcpleoanneinntgs, aarnednmotaicnotmenpaantcibelearwe itthheosnaeme. sapnroatyhesry,sbteumt osn, leytcw.)ith similar systems (i.e. Z-spray components are compatible with other Z- 3.5 MsysatsesmLs.y nCxuSrroefntwtlyarMe:asSsLysytnexmhsaosftWwainredodwessig9n5eadndforWthinedsopwecsNifiTc operation of 3.1 versions. these platform All versions are similar. For more details see the manual specific to the instrument (Micromass Platform II or Quattro II MassLynx or MassLynx NT USER'S GUIDE). 4.0 W a r n in g s and C au tio n s_________________________________________________________________ 4.1 H ealth and Safety W arnings: 4.1.1 Uapspercoaxuimtioantewlyit5h00th0eVvoollttsa.ge cables for the probe. The probe employs a voltage of 4.1.2 Wanhdecnlohtahnindgli.ng samples or solvents wear appropriate protective gloves, eyewear, Word 6.0.1/95 Analysis of SerumFoArCFTlu-Mid-E4.x1tract Using ES/MS Page 2 of 9 3M Environmental Laboratory Page 171 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 4.2 Cautions: 4.2.1 DIfothneobt aocpkerparteesssuorlveeenxtcpeuedmsp4s0a0bboavre, ctahpeaHciPty11o0f04w00illbainri(t5ia8t0e0auptsoi)mbaatcicksphruetsdsouwren.. 4.2.2 Do not run solvent pumps to dryness. 5.0 I n t e r f e r e n c e s ______________________________________________________________________ 5.1 To minimize interferences when analyzing samples for perfluorooctanoate(POAA), teflon wshiothultdhenosat mbeplueseodr efoxrtrsaacmt. ple storage or any part o f instrumentation that comes in contact 6.0 E q u ip m e n t ' _____________ _____ ___________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler 7.0 S u p p l ie s a n d M a t e r ia l s _____________________________________________________________________ 7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi 7.1.2 HPLC analytical column, specifics to be determined by the analyst 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8.0 R e a g e n t s a n d St a n d a r d s_______________________________________ _ __________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water and may be provided by a Milli-Q TOC Plus system 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically one method blank, one matrix blank, and ten matrix standards are prepared during the extraction procedure. See FACT-M-3.1. 9.0 S a m p l e H a n d l in g ____________________________________________________________________________ 9.1 Farreesshtomreadtriinx csatapnpdeadrdasutaorveiaplrsepoarrceadpwpeitdh1e5acmhl acneanltyrsifisu.geEtxutbraecstuedntsiltaanndaalrydssis.and samples 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C until analysis can be performed. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory Page 3 of 9 Page 172 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.0 Q u a l it y C o n tro l______________________________________________________________________ 10.1 M ethod Blanks and M atrix Blanks 10.1.1 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 M atrix Spikes 10.2.1 Analyze a matrix spike and matrix spike duplicate per forty samples. With a minimum o f 2 spikes per batch. 10.2.2 Expected spike concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the low-range of the initial calibration curve. 10.2.3 See Section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 crAuhnnaa.nlOgyezne(lyat3mh0oi%ds-e)rsaiannmgpepealcekasliaabrnreaaatlyoiozcnecdusrtbasne, fdroearlraedttiahvfeetetlraosettvhaeecrciyneitpteitnaatlbhsletsaacnmadlapibrlerd.actIiufornavsesi,tgasnntoidfpaicratdhnet will be used. The remaining samples must be reanalyzed. 10.3.2 See Section 13 to calculate percent difference. 11.0 C a l ib r a t io n and S t a n d a r d iz a t io n _____________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of extracts. The mreegarnesosifotnw(or^stfaonrdtahredcvaalilbureast,ioant ecaucrhvestuasnidnagrdMcaosnscLeynntrxaotiroont,hwerillsubietapblloettseodftbwyarlien.ear 11.2 Tdihsecrre2tvioanluoefftohretahneadlyastat ashnodudldocbuem0e.n9t8e0doarpgprreoavtaelr.ofLtohwe ePrrovjaelcuteLs emada.y be acceptable at the 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.4 uFsoer tphuerploowsesenodf oacfctuhreaccayliwbrhaetnioqnucaunrtviteatriantghelrowthalnevtehles foufllanraanlygtee,oifttmheaystbanednaercdescsuarrvye.to rcEaaxnlaigbmerapotliefo:tnhwecuhcruevnrevaecto(t5enmspippsttbiinntggo to1of0q0thu0eapnspttaibtna).dteaTradhpsipsfrwrooximlilmr5aetpdepulybcet1oi0n1ap0cpc0bupropafbcaynraaattlhtyretierb,utghteeadnnettrohaetlienfuaelalr regression weighting of high concentration standards. 12.0 P r o c ed u res_____________________________________________________________________________ 12.1 A cquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. Analysis of SerumFoArCFTlu-Mid-E4.x1tract Using ES/MS Page 4 of 9 3M Environmental Laboratory Page 173 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording). Set Ionization Mode as appropriate and mass to 499 or oStahveer aacpqpuroispitriioantemmeathssoeds.. IAf MfuSll/MscaSninissturusumaellnytscoalrleecetmedplaolyoendg, waditdhittihoenaSlIpRrso.duct ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Tstyanpdicaarldlys athned aennadlsytwiciathl baastecthorfunexsterqacuteendcme batergixinsstawnidtharadss.et of extracted matrix 12.1.4 cSsaaamrmrpypolleve.serSaraoenlvdaennaartleybnzlaeodnt kcwsoinsthhsiodaueclrdoendbtesinaaumnianplglyezsceabdliubptremartiaiooydnbicceahlielnycckltuoidnmejedocntaeistdosruafcpthoe.rsseivbeleryanteanltyhte 12.2 Using the Autosampler 12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1. 12.2.2 Saneta-luypsttchoenHsiPd1er1s00a/papurtoopsraimatpelfeorraotptthiemfaolllroewspionngsce.onRdeictoiorndsaocrtuaatlccoonnddititioionnssthine the instrument logbook: 12.2.2.1 Sample size = 10 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 15 minutes 12.2.2.4 Solvent ramp = Time MeOH 0 . 0 0 min. 7.5 min. 1 1 . 0 min. 11.5 min. 45% 90% 90% 45% 2.0 mM Ammonium acetate 55% 10% 10% 55% Note: In this instrument configuration, the run must be set up on the electrospray software wHiPthWao"rWksataittiinogn.for inlet start" message before the "Start" button is pressed on the 12.2.2.5 Press the "Start" button. 12.3 Instrument Set-up 12.3.1 Refer to FACT-EP-3.0 for more details. 12.3.2 Check the solvent level in reservoirs and refill if necessary. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check tuhnesatitpis.faTchtoeryti,pdsihsaosusledmbbeleflatht ewpitrhobneo ajangdgreedpleadcgeetsh.e Isftathineletisps isstefeolucnadptiollabrey. Analysis of SerumFoArCFTlu-Mid-E4.x1tract Using ES/MS 3M Environmental Laboratory Page 5 of 9 Page 174 to o 3m Medical Department Study: T-6295.7 Report No. FACT TOXlaboratorv Request Number-U2 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 1 0 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may changein order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode flow rate 10 - 500 p.L/min 12.3.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7 Carefully guide the probe into the opening. Insert probe until it will not go any further. Connect the voltage cables to the probe. 12.3.8 Record tune parameters in the instrument log. 12.3.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis o f biological matrices. 12.3.10CMbulaitctsoksnLoyanntsxttoavpretorbsfuiostantomsn,psileneetlihasept.pArEconqpsruuiiasrtieetisMotnaarCtssaoLnnydtnreoxnl dUPaSsnaEmeRlp('ltSehiGnsuUmmIaDbyeErv)a.inryPclrauemdsseostnhagellstart samples to be analyzed. 13.0 D a t a A n a l y sis and C a lc u la tio n s 13.1 Calculations: __________________________________________ 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (jug/ml): (rig o f PFOS calc, from std. Curve x Dilution Factor) x 1 ug (Initial Volume of matrix (ml) + ml of Surrogate Standard) 1000 ng Final Volume (mL) 14.0 M e t h o d P er fo r m a n c e_______________________________________________________ __________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see FACT-M-3.1, Attachment A for a listing of current validated MDL and LOQ values. Analysis of SerumFoArCFTlu-iMd -E4x.1tract Using ES/MS Page 6 of 9 3M Environmental Laboratory Page 175 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 14.2 M ethod Blanks and M atrix Blanks 14.2.1 Mpstoaesntsdhibaorldedbcilnoanntthkaesmcaiannldaibtmiroaanttirooinrxccbaulrarrvnyeko.svewri.llVbaeluaensalayrzeedexwpeitchteedactoh fsaalml bpeleloswettfhoerlowest 14.3 M atrix Spikes. 14.3.1 Matrix spikes are analyzed with each sample set and the percent recoveries are expected to fall within 30% o f the spiked concentration. 14.4 Continuing Calibration Checks 14.4.1 wCoitnhtienaucihngsacmalpiblerasteito.nTchheecpkesrcaernetarneacloyvzeerdieast aarme einxipmecutmedotfoaffatellrwevitehriyn 103s0a%mpolfes the spiked concentration. 14.5 Ipferafnoyrmcreidteroina tlhisetesdysitnemtheamndetshaomdppleesrfroeramnaanlyczeedseocrtioonthiesrna'tctmioents, masadinetteenrmanicneedmbayy tbhee analyst. All actions will be documented in tKe instrument runlog, the maintenance log, or on the summary sheet with the sample results. 1 5 .0 P o l l u t io n P r e v e n t io n and W a s t e M a n a g e m e n t _______________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s________________________________________________________________________________________ 16.1 Sfotollroewcihnrgominafotormgraatmiosninintchluedsetuddeyitohrerprinojtehcet hfoeladdeerr. oErahcahncdhwrormittaetnogornamthemcuhsrtohmaavteogthraem: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Plot calibration curve by linear regression and store in the study folder. 16.3 Print sample list from MassLynx and tape into the instrument runlog. 16.4 Print data integration summary from MassLynx and tape into the instrument runlog. 16.5 Copy instrument runlog pages, including instrument parameters and sample results, and store in appropriate study folder. 16.6 Summarize data using suitable software and store in the study folder. 16.7 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data. 17.0 T a b l e s . D ia g r a m s . F l o w c h a r t s , a n d V a l id a t io n D a ta ________________________________ 17.1 Attachment A: FACT-M-4.1 Data reporting spreadsheet 17.2 The validation report associated with this method is FACT-M-3.1 & 4.1-V-l. Analysis of SerumFoArCFTlu-Mid-E4.x1tract Using ES/MS Page 7 of 9 3M Environmental Laboratory Page 176 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 18.0 R e f e r e n c e s _____________________________________________________________________________ 18.1 FACT-EP-3.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Platform Systems" 19.0 A ff e c t e d D o c u m en ts__________________________________________________________________ 19.1 FACT-M-3.1, `'Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum or Fluid for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R e v is io n s________________________________________________________________________________ Revision Number. * Reason For Revision Revision Date 1 Validation o f method to include 7flu o rochemicals addition o f whole 0 7 / 0 1 / 9 8 blood matrix, surrogate standard, new A P I/M S (M S ) systems, monkey sera cross validation, M D L study, updates in record keeping and storing policies, etc. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory Page 8 of 9 Page 177 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Attachment A Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: Group Sample# Concentration Dose ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. DFiinluatlioCnoFnea.c(tuogr:/mTLa)k:enCfarlocmulattheedsbtuyddyivfiodlidnegr.the initial volume from the concentration FACT-M-4.0 Analysis of Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory Page 9 of 9 Page 178 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Attachment D Data Summary Tables R e p o r t N o . FACT TOX-030 la b o r a to r y R e q u e st N um be r-U 2 27 9 Report No. FACT TOX-030 Laboratory Request Number-U2279 3M Environmental Laboratory 3M Environmental Laboratory Page D-1 Page 179 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Table D-1a. Sample Intervals and Types by individual Animal from Study 6329-223 Amm a l ID Group 1 (Contrai) 105508M 105517M 105519M 105520M 105526M 105527M 105529F 105530F 105531F 105535F 105544F 105549F G rou p II (Low Dose) Serum Samples Collected (Qty-- W eek) Liv e r S a m p le s C o llected (Da te ) 151 Samples 8 Samples 12-- 0, 1 .2 , 4. 6, 8 ,1 2 ,1 6 , 20. 24. 26, 27i 11-- 0, 1 ,2 ,4 , 6, 8 ,1 2 ,1 6 , 20, 24, 26 12-- 0 ,1 , 2 , 4 , 6 ,8 ,1 2 ,1 6 , 20, 24, 26, 27i 14-- 0 ,1 ,2 .4 , 6, 8,12,16, 20, 24, 26, 27, 27i, 27ii 14-- 0, 1 ,2 ,4 , 6, 8, 12, 16, 20, 24, 25, 27. 27i, 27ii 12-- 0, 1 ,2 .4 , 6, 8 .1 2, 16, 20, 24, 26, 27I 14--0, 1, 2 ,4 , 6, 8, 12,16, 20, 24, 26, 2 7 ,27i, 27ii 12-- 0 ,1 ,2 ,4 , 6 ,8 ,1 2 ,1 6 , 20, 24, 26, 27i 12-- 0, 1 ,2 ,4 , 6, 8, 12, 16. 20, 24, 26, 27II 12-- 0, 1 ,2 .4 , 6, 8 .1 2, 16, 20. 24, 26. 27ii 12--0, 1, 2, 4, 6, 8,12. 16, 20. 24. 26. 27i 14-- 0 ,1 ,2 ,4 , 6, 8 ,1 2 ,1 6 , 20, 24. 26. 27, 27I, 27il 99 Samples 2/25/99 2/25/99 2/25/99 Relumed to ooiony 3/07/00 Returned to colony 3/07/00 2/25/99 Returned to colony 3/07/00 2/25/99 2/26/99 2/26/99 2/25/99 Returned to ooiony 3/07/00 8 Samples Recovery Subgroup Serum Samples Co llected (Qty-- W eek) 72 Samples - : '-r '` l'f .v tl ' " 7" /':; ,r; 18-- 27iii, 28, 29. 30, 31, 35, 3 9 .4 3 , 47, 5 1 ,5 3 ,5 7 ,6 1 ,6 5 ,6 9 , 73,77, 79 18-- 27iil. 28, 29, 30, 31, 35, 3 9 ,4 3 , 47, 51,53,57,61,65, 69, 73,77.79 ' ' , , - 1', . - 18--27iii, 28, 29, 30, 31,35. 39, 43, 47, 51,53,57,61,65, 69,73, 77,79 , *'1 'y ' - "* ' ' ' 18--27iii, 28, 29, 30, 31, 35, 3 9 ,4 3 , 47, 5 1 ,5 3 ,5 7 ,6 1 ,6 5 , 69 ,73, 77, 79 -- ''W 8 S 105514M 105515M 105516M 105521M A O SSB ^k 1-- 0 12-- 0, 1,2, 4. 6, 8 ,1 2 ,1 5 , 20, 24, 26. 27i 12-- 0, 1, 2 ,4 , 6, 8. 12, 16, 20, 24, 26, 27li 12--0, 1 ,2 .4 , 6 ,8 , 12. 16, 20, 24, 26. 27li 12-- 0, 1 ,2 ,4 , 6 ,8 , 12, 16. 20. 24, 26, 27li 1-- 0 Baseline assigned 2/2699 2/26/99 2/26/99 2/2699 BaseOri|ibi)IV^Not assigned Returned to colony ' ' 1 t - .V ' : A y /iy A Returned to colony 105537F 105541F 11-- 0, 1,2 , 4, 6, 8,1 2, 16. 20, 24, 26 12-- 0, 1,2, 4,6, 8,12.16, 20, 24, 26, 27ii 1-- 0 2/25/99 2/2699 BasdiiflBflBSte assigned 'V r 1 I 'Returned to colony 105547F 105550F 1-- 0 12-- 0, 1. 2 ,4 ,6 , 8, 12, 16. 20, 24, 26, 27 12-- 0, 1 .2 , 4, 6 ,8 ,1 2 , 16, 20. 24. 26, 27 Bas&lfti(P$ft& assigned 2/2699 2/26/99 Returned to colony - , .y - " 27 Day 183 (2/23/99) 27i Day 184(2/25/99) ** Two samples 2/20/99 79 Sample on 2/23/00 27ii Day 185 (2/26/99) 79i Sample on 2/25/00 27iii Day 187 (2/28/99) 79il Sample on 2*25/00 N ote: Samples for Week 25 and Week 27 (Recovery Group) taken on same day (Day 183, 2/23/99) 3M Environmental Laboratory 3M Environmental Laboratory Page D-2 Page 180 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Table D-1b. Sample Intervals andTypes by Individual Animal from Study 6329-223 A n im a l ID Serum samples Collected (Qty-- W eek) LIVER SAMPLES COLLECTED (Da te) recovery Subgroup Serum Samples Collected (Qty-- W eek) Group III (Mid Dose) 105505M 105510M 105518M 105523M 105524M 10552BM 105532F 105538F 105539F 105545F 105548F 105552F Group IV (High Dosa) 105506M 105507M 105509M 105511M 10S512M 105522M 105533F 10S534F 105536F 105540F 105542F 105551F 152 Samples 14-- 0, 1,2 , 4, 6, 8 ,1 2 ,1 6 , 20, 24, 26, , 27, 27i, 27 12-- 0, 1, 2 ,4 , 6, 8, 1 2 ,1 6 , 20, 24, 26, 27 12-- 0, 1 ,2 ,4 , 6, 8, 12, 16, 2 0, 2 4, 26, 27 14-- 0, 1 ,2 , 4. 6, 8 ,1 2 ,1 6 , 2 0 ,2 4 , 2 6 ,2 7 ,2 7 1 , 27 12-- 0 ,1 ,2 .4 ,6 , 8 ,1 2 ,1 6 , 20, 24, 26, 27 12-- 0 ,1 ,2 ,4 , 6, 8 ,1 2 ,1 6 , 20. 24, 26,27 12--0 ,1 , 2 ,4 , 6, 8, 12,16, 20, 24, 26, 27i 12-- 0 ,1 , Z 4, 6 ,8 ,1 2 ,1 6 , 20, 24, 26, 27I 14-- 0, 1 .2 , 4, 6, 8, 12, 16. 20, 2 4, 2 6, 27,271, 27 12-- 0, 1,2 , 4, 6, 8 ,1 2 ,1 6 , 20, 24, 26, 27i 12-- 0, 1 ,2 ,4 , 6, 8, 12, 16, 20. 24, 26. 27i 14--0 ,1 ,2 , 4, 6, 8 ,1 2 ,1 6 , 20, 2 4, 2 6 , 27, 27i, 27ii 148 Samples 11--0 ,1 ,2 , 4 ,6 ,8 ,1 2 ,1 6 , 20. 24, " 12--0 , 1,2, 4 ,6 , 8 ,1 2 ,1 6 , 20. 24, 2 6 ,27i 9-- 0, 1 ,2 ,4 , 6 ,8 , 1 2 ,1 6 ,2 0 14-- 0 , 1, 2, 4, 6. 8, 1 2 ,1 6 , 20, 24, 26, 27, 27I, 27 12-- 0 , 1 , 2 , 4 , 6 , 8, 12, 16, 2 0 ,2 4 , 26, 27I 14--0 , 1 , 2 . 4 , 6, 8, 12, 16, 20, 24, 2 6 ,2 7 . 27i, 27 14--0 ,1 ,2 ,4 ,6 , 8 ,1 2,16, 20. 24, 26. 27, 2Ti, 27il 12--0 , 1 ,2 ,4 , 6, 8 ,1 2 , 16, 20, 24, 26.27 12--0 ,1 ,2 , 4, 6, 8, 12,16, 20.24, 2 6 ,27i 12-- 0, 1 ,2 ,4 , 6, 8, 12, 16, 20. 24. 26. 27 14--0 ,1 ,2 .4 , 6, 8 ,1 2 ,1 6 , 20. 24, 26. 27, 271,27 12-- 0, 1 ,2 ,4 , 6, 8, 12, 16, 20,24, 26,27 12 Samples 3/01/00 Biopsy 2/26/99 2/26/99 3/01/00 Biopsy 2/26/99 2/26/99 2125m 2125m 3/01/00 Biopsy 2/25/99 2/25/99 3/01/00 Biopsy 14 Samples none 2/25/99 none 9/22/99 Biopsy, 2/25/00 2 /2 5 /9 9 9/22/99 Biopsy. 2/25/00 9/22/99 Biopsy, 2/25/00 2/26/99 2125m 2125m 9/222/91925B/0io0psy. 2/26/99 72 Sam ples 18-- 271, 28, 29, 30, 31, 35, 39, 4 3, 47. 51,53, 5 7 ,6 1 .6 5 .6 9 ,7 3 .7 7 ,7 9 18-- 271, 28, 29, 30, 3 1 .3 5 , 39, 43, 4 7, 5 1 ,5 3 ,5 7 ,6 1 ,6 5 ,6 9 , 73. 7 7,79 . ; ` ' . 18-- 27iii, 28. 29. 30, 31, 35, 39, 43, 47, 5 1,5 3 ,5 7 , 6 1 ,6 5 ,6 9 , 73, 77, 79 18-- 27ill, 28, 29, 30, 31, 35, 39, 43, 47, 51,53,57, 6 1,6 5,69 , 73, 7 7,79 80 Sam ples ' ` ' 'T , 20-- 27III, 28. 29, 30. 31, 35. 39, 4 3. 47, 5 1 ,5 3 , 57. 61, 65, 69, 73, 77, 79, 79i, 79 20-- 27ill, 28, 29, 30, 3 1 .3 5 , 39. 43, 47, 51. 53. 57, 6 1 ,6 5 , 69. 73, 77, 79. 79i, 79 20--27iil, 2 8. 29, 30, 3 1 ,3 5 . 39. 43, 4 7, 51. 53. 57, 61. 65, 69, 73. 77, 79, 79i, 79 t -; ' -...... - 20-- 27iii, 28. 29. 30, 31.35, 39. 43, 47, 51,53. 57, 61, 65. 69. 73, 77. 79. 79i, 79 27 Day 183 (2/23/99) 27i Day 184 (2/25/99) '* Two samples 2/20/99 79 Sample on 2/23/00 27 Day 185 (2/26/99) 79i Sample on 2/25/00 27iii Day 187 (2/28/99) 79ii Sample on 2/25/00 Note: Samples for Week 26 and W eek 27 (Recovery Group) taken on same day (Day 183.2/23/99) 3M Environmental Laboratory 3M Environmental Laboratory Page D-3 Page 181 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-03Q laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Table D-2. Average PFOS Concentrations in Serum by Dosage Group and Week from Study FACT Tox-030 (Baseline- Day 185) Time Point LOQfcig/mU All PFOS values tig /m L G roup 1M GOrAomugpfeg1/dF Group O.O2VdMnofeQf Group OJUm2gFAtg/d Group Q.153mM0l(gfe! Group O.ISr3rvFfcefcJ Group 0.794fnMoAc^d Group O.754mF0%gftt Baseline LOQ 0.025 Average PFOS Std Deviation <LOQ NA Week 1 LOQ 0.025 Average PFOS Std Deviation <LOQ NA Week 2 LOQ 0.025 Week 4 LOQ 0.0152 Average PFOS Std Deviation Average PFOS Std Deviation <LOQ NA <LOQ - 1 Anomaly NA Week 6 LOQ 0.0152 Average PFOS Std Deviation <LOQ NA W eeks LOQ 0.0152 Average PFOS Std Deviation <LOQ -1 Anomaly NA W eek 12 LOQ 0.0152 Average PFOS Std Deviation 0.0383 -1 Anomaly 0.00811 W eek 16 LOQ 0.0152 Week 20 LOQ 0.0152 Week 24 LOQ 0.01S2 Average PFOS Std Deviation Average PFOS Std Deviation Average PFOS Std Deviation 0.0407 0.0110 0.0400 0.0120 0.0440 0.0101 W eek 26 LOQ 0.0152 Week 27 Average PFOS Std Deviation Average PFOS 0.0459 0.0143 0.0529 LOQ 0.024a Std Deviation 0.0145 Day 183 LOQ 0.0152 Average PFOS Std Deviation 0.117 0.0764 Day 184 LOQ 0.0152 Average PFOS Std Deviation 0.0233- 1 Anomaly NA Day 185 Average PFOS 0.0432 LOQ 0.0152 Std Deviation 0.00928 <LOQ: Less than Ihe lower Limit of (iiantitation <LOQ NA <LOQ NA <LOQ NA <LOQ NA <LOQ NA 0.0226 -2 Anomalies 0.00189 0.0263 -2 Anomalies 0.00362 0.0432 -2 Anomalies 0.00851 0.0504 0.0127 0.0426-1 Anomaly 0.00784 0.0506 0.0164 0.0416 0.0148 0.0533 0.0315 0.0352 0.00911 0.0546 0.0306 <LOQ NA 0.869 0.147 1.10 0.0835 3.20 0.577 3.61 0.430 4.73 0.432 6.69 0.578 11.2 2.44 12.3 1.40 14.5 3.06 15.8 1.41 15.9 5.54 -- -- -- -- -- -- <LOQ NA 0.947 0.110 1.10 0.0963 3.40 0.291 3.71 0.417 4.76 0.577 6.31 0.717 10.5 1.90 19.5 14.5 13.0 0.675 13.2 1.42 11.1 1.52 -- -- -- -- -- -- <LOQ NA 4.60 0.782 5.81 0.933 17.8 1.68 20.4 1.65 26.0 3.30 35.2 5.39 56.2 5.84 63.7 6.71 65.9 6.88 82.6 25.2 68.1 5.75 85.0 14.9 69.7 4.68 77.9 10.7 <LOQ NA 3.71 0.455 5.39 0.930 16.5 1.87 18.8 2.15 24.0 3.06 27.8 3.98 42.1 4.04 58.1 14.7 60.4 7.24 66.8 10.8 58.5 4.67 81.7 35.1 78.0 1Z0 84.3 23.3 <LOQ NA 21.0 1.57 26.9 3.54 95.3 70.4 94.5 8.07 109 18.3 122 23.9 189 15.9 144 10.9 215 24.9 173 36.5 194 8.93 249 46.8 259 110 294 22.0 <LOQ NA 20.4 2.71 22.0 3.25 92.7 39.6 90.1 7.11 107 11.8 117 11.7 162 19.3 156 21.8 174 20.9 171 22.2 160 23.9 230 40.3 245 29.2 321 170 3M Environmental Laboratory 3M Environmental Laboratory Page D-4 Page 182 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Tabla D-3. Average PFOS Concentrations in Serum by Dosage Group and Week from Study FACT TOX-030 (Recovery Group) Time Point LOQ (pg/mL) Day 187 LOQ 0.0152 Week 28 LOQ 0.0152 Week 29 LOQ0j0152 Week 30 LOQ 0.0152 Week 31 LOQ 0.0152 Week 35 LOQ 0.00655 Week 39 LOQ 0.00555 Week 43 LOQ 0.00555 Week 47 LOQ 0.00655 Week 51 LOQ 0.00555 Week 53 LOQ 0.00555 Week 57 LOQ 0.00655 Week 61 LOQ 0.00555 Week 65 LOQ 0.00555 Week 69 LOQ 0.00565 Week 73 LOQ 0.00556 Week 77 LOQ 0.00556 Week 79 LOQ 0.00555 AJI PFOS values ng/mL Average PFOS (mw -) Std Deviation Average PFOS (mow .) Std Dviation Average PFOS (paw.) Std Deviation Average PFOS (mow .) Std Deviation Average PFOS (mow .) Std Deviation Average PFOS (mow.) Std Deviation Average PFOS (mow.) Std Deviation Average PFOS (mow. i Std Deviation Average PFOS (mow.) Std Deviation Average PFOS (mow.) Std Deviation Average PFOS (mow.) Std Deviation Average PFOS (mow .) Std Deviation Average PFOS (mow.) Std Deviation Average PFOS (mow .) Std Deviation Average PFOS (mow .) Std Deviation Average PFOS (mow.) Std Deviation Average PFOS (mow .) Std Deviation Average PFOS (mow .) Std Deviation Group 1M Group 1F 0.0mg/kg/day 0.0300 -1 Anomaly NA 0.0371 0.0124 0.0457 0.0118 0.0430 0.00821 0.0665 0.00362 0.0742 0.000664 0.0313 0.000165 0.0303 0.000485 0.0358 0.000567 0.0368 0.0121 0.0459 0.00303 0.0723 0.00352 0.0391 0.0106 0.0509 0.000779 0.0319 0.00440 0.0368 0.0000923 0.0355 0.00221 0.0237 0.00333 0.0331 0.0086 0.0459 0.00323 0.0341 0.000403 0.0397 0.00311 0.0327 0.00526 0.0445 0.00385 0.0351 0.00449 0.0448 0.00210 0.0210 0.00365 0.0360 0.000522 0.0406 0.00313 0.0400 0.00301 0.0350 0.0115 0.0365 0.00284 0.0296 0.00535 0.0305 0.00167 0.0215 0.00296 0.0243 0.00355 Group 3M Group 3F 0.15m g/kg/day 79.1 4.67 39.5 41.4 84.6 1.51 86.1 3.59 75.8 2.18 69.9 11.4 65.2 4.60 62.0 10.6 56.6 4.77 79.6 43.8 84.5 12.0 74.4 9.53 60.1 3.16 51.7 7.29 45.7 1.12 58.1 0.249 48.3 3.69 42.6 6.70 37.9 2.62 35.1 13.2 46.2 3.30 36.7 6.24 30.2 2.36 32.3 1.34 31.6 5.98 38.2 0.283 32.9 0.0269 37.6 2.32 26.4 2.59 34.5 3.46 27.3 4.66 25.8 2.91 22.5 0.632 23.0 6.37 19.1 0.805 21.4 2.01 Group 4M Group 4F 0.75mg/kg/day 267 42.0 249 21.7 258 15.2 236 18.3 223 66.9 194 19.0 143 38.0 162 7.87 161 185 46.1 21.9 181 19.5 171 10.1 146 161 16.1 11.1 78.8 16.8 159 28.4 124 25.9 94.7 38.4 98.3 8.32 91.4 6.07 80.8 36.8 98.2 0.490 78.0 16.3 106 3.84 100 50.3 109 0.697 91.5 55.2 82.8 9.68 84.0 52.4 75.0 5.25 54.4 27.3 147 131 60.0 38.3 57.0 19.1 41.1 25.9 41.4 1.15 3M Environmental Laboratory 3M Environmental Laboratory Page D-5 Page 183 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 Laboratory Request Number-U2279 Table D-4a. PFOS Amounts Reported in Serum by Individual Animal (Baseline-Week 26) PFOS Re p o r te d WEEK0 (pO*ti) W eek 1 (petti) W eek 2 (uflimL) Week 4 Inoriti) Weeks Ui0frnL) W eek 8 (ligAnL) W eek 12 (moAiL) W eek 16 (p e ri) Group 1(Conta*> 105508M <LOQ 105517M <O Q 105619M <O Q 105520M <LOQ 105528M <LOQ 105S27M *<LOQ 105S29F <LOQ 105530F <LOQ 105531F *<LOQ <OQ <LOQ <LOQ <LOQ <00 <OQ <LOQ <-OQ <_OQ <LOQ <LOQ <O Q <O Q <LOQ < 0 0 <O Q <O Q <LOQ 105535F 105544F 105549F <LOQ <LOQ <-OQ <OQ <O Q <LOQ <OQ <LOQ K lC Group 1 (Low Do m ) 105513M 105514M 105515M 105516M 105521M <O Q <O Q <O Q <LOQ <LOQ 0.783 0.767 0.632 109 1.02 1.03 1.19 1.13 105525M 105537F 105541F 105543F 105546F 105547F 10S550F <LOQ <LOQ <LOQ <LOQ <LOQ <O Q <O Q 1.10 0929 0931 0.832 1.19 1.16 1 06 0.980 Group M fM klD oM ) 105505M 105510M 105518M 105623M 105524M 105528M 105532F 105536F 105539F 105545F 105546F 105552F <LOQ <L0Q <LOQ <LOQ <LOQ <O Q <LOQ <LOQ <OQ <O Q <LOQ <t_OQ 4.60 4.19 5.46 354 425 5.52 4.31 4.12 3.31 *3.60 3.72 3.12 6.07 4.82 7 01 456 624 6.15 461 6.67 4 65 5 28 6.42 4.73 Group (V (High Do m ) 105506M 105507M 105509M 105511M 105512M 105522M 105533F 105534F 105536F 105540F 105542F ^LOQ <LOQ <OQ <OQ <LOQ <LOQ <LOQ *<O Q *<O Q <O Q <LOQ 21 3 220 18.8 19.3 22.8 21.6 226 18.1 21.0 16.8 24 0 299 25.1 23.9 22.6 31.3 288 249 26.0 *23.5 19.5 199 105551F <OQ 20.1 18.2 indicates a sam ple w ith an extraction volume of <0 5 m l Shaded ceUs=moribund <OQ <LOQ <O Q <LOQ <LOQ <O Q < 0Q < 0Q <LOQ <LOQ <LOQ <O Q 2.87 2.75 3.13 4.03 3.73 3.07 3.55 3.27 17.0 17.2 20.2 176 15.4 19.2 16.6 18.7 14.3 18.7 15.7 14.9 236 72.6 54.8 48.4 77.4 822 141 145 64.1 67.2 78.4 60.2 <O Q < .0 0 <O Q <OQ <LOQ <O Q *<O Q *<O Q <tOQ <O Q <O Q <O Q 3.67 3.31 3.26 4.19 4.28 3.66 3.85 3.27 21.0 22.5 20.1 18.6 21.8 18.5 18.4 18.9 1B.4 22.7 18.4 16.1 91.0 828 923 93.7 105 102 90.0 83.3 89.8 90.8 102 820 <OQ <OQ <OQ <LOQ <LOQ 0.0385 0.0214 0.0244 0.0206 <OQ <OQ 0.0240 4.61 4.17 5.05 5.10 5.28 4.85 4.98 3.94 27.9 221 25.1 24.5 31.6 246 27.6 24.3 26.5 245 22.3 19.1 125 86.1 103 923 118 131 110 121 102 105 116 87.3 0.0381 0.0430 0.0438 <LOQ 0.0244 0.0423 <LOQ 0.0243 0.0254 <OQ 0.0238 0.0316 620 648 6.55 7 52 6.81 5.57 7.03 5.83 40.1 392 337 35.3 37.8 25.4 258 32.8 278 32.2 23.2 24.8 116 106 123 994 167 121 126 114 98.0 112 131 117 00344 0.0408 00423 00332 0.0320 0,0615 0.0438 0.0515 0.0314 <O Q <100 0.0462 10.4 9.47 10.1 14.8 11.1 .123 107 7.85 59.4 529 60.7 522 63.4 48 4 40.4 37 3 46.3 43.1 47 0 38.7 182 172 197 179 216 185 164 157 184 173 168 127 W eek 20 (petti) 0.0348 0.0419 0.0336 0 0300 0.0369 0.0631 0.0617 0 0579 0.0493 0.0263 0.0505 0.0570 12.5 10 7 11.9 14.1 11.0 11.5 14.3 41.1 ` 62 3 633 63.1 75.2 64.0 542 09.2 47.4 78.2 45.7 65.3 427 158 138 152 126 143 145 169 143 148 193 151 132 W eek 24 (petti) 0.0449 0 0486 0.0414 0.0283 0.0397 0 0613 0.0498 0 0497 0.0364 LOQ 0.0325 00446 14.7 10.9 14.1 184 13.4 13.7 127 122 77.4 65.2 58.8 597 69 6 648 63.1 486 69.9 63.5 56.1 61.7 198 186 -- 207 247 233 190 200 169 151 181 150 W eek 26 (pO/mL) 0 0496 0.0539 0.0417 0.0254 0.0376 0.0669 0.0613 0.0736 00431 00266 0.0433 00556 16.2 165 138 169 13.5 .148 130 11.4 92.9 129 69 5 72.1 69.5 624 634 590 72 2 85 7 645 556 -- 182 -- 142 148 221 203 187 155 177 165 142 3M Environmental Laboratory 3M Environmental Laboratory Page D-6 Page 184 3m Medical Department c - u d y : T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Table D-4b. PFOS Amounts Reported in Serum by Individual Animal (Days 183-Day 185 and Week 27) PFOS Repo r ted Da y 1S3 (ngiA) Day 184 (ppM.) Day 185 (kigftnL) WEEK 27 bi&mL) Group UCofrtroO 105506M -- @ -- 105517M -- -- -- 105519M 105520M 105528M 105527M 10S529F 105530F 105531F 105535F 105544F 105549F -- 0.171 -0.0626 -- 0.0756 -- -- -- -- 0.0310 & <LOQ 0.0233 @ 0.0416 @ -- -- @ 0.0287 -- 0.0366 0.0498 -- 0.0763 -- @ @ -- 0.0330 Group 1 (Low Do m ) 105514M -- -- 105515M -- -- @ 105516M -- _ @ 105521M -- -- @ 105537F -- -- -- 105541F -- -- @ 105547F -- -- @ 105550F -- -- @ Group III (Mid OoM) 105505M 74,4 66.4 70.3 105510M -- -- 105518M -- -- @ 105523M 95.5 73.0 854 105524M 105528M 105532F 105538F 105539F 105545F 10S548F 105552F -- -- -- -- 107 -- -- 56.9 -- -- @ @ 865 @ @ 69.5 @ @ -- -- 101 -- -- 678 Group IV (High Do m ) 10550eM -- -- -- 105507M -- -- 10S509M -- -- -- 105511M 216 181 309 105512M 105522M 105533F -- 282 258 @-- 336 278 265 441 105534F -- -- 105536F -- @ -- 105540F -- -- 105542F 201 224 201 105551F @ Sample colection date for Week 27 data * Sample colectad on 2/20/99 incicates a sample with an traction volume of <0.5 mL 0.0461 -- 0.0430 -- - 00695 -- 0.0635 00374 0.0318 0.0338 - 23.9 13.0 114 15.5 -- 126 953 11.2 -- 68.5 68.7 -- 60.6 74.7 54.3 565 58.0 65.1 - 196' 184 -- -- 202 -- 182 169 164 126 3M Environmental Laboratory Page D-7 3M Environmental Laboratory Page 185 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Table D-5a. PFOS Amounts Reported In Serum by Recovery Group Animal (Day 187- Week 47) PFOS Re p o r t e d D(a1y0/m18D7 Week 28 tup'll*.) Week 29 (pO/mL) W(EusEAKnL3)0 W(nefel4knL3|1 W eek 35 WEEK 39 MOW. (pptmL) WEEK 43 (uplrt.) Week 47 Group l(C o n tno*) 105520M <LOQ 105528M 0.0300 0.0284 -0 0459 0.0890 0.0639 105529F 105549F 0.0373 0.0541 0 0488 0.0372 0.0738 0.0747 Group I I (MM Po m ) 105505M 824 105523M 75.8 105539F 10.2 105552F 88.8 Group IV (HIglIi Do m ) 85.7 83.5 88.7 83.6 773 *742 77.9 61.9 105511M 105522M 105533F 105542F 237 297 269 248 233 264 249 223 175 270 207 180 Indica tes a sam ple w ith an extraction volum e o f <0.5 mL 0.0314 0.0312 0.0299 0.0306 68.4 61.9 69.9 54.5 116 170 156 167 00362 0.0354 0.0282 0.0453 60.0 53.2 111 48.6 129 194 201 170 0.0437 0.0480 0.0746 0.0698 93.0 76.1 81.2 67.7 167 195 164 178 0.0316 0.0466 0.0503 0.0514 62.4 57.9 56.8 46.5 135 158 169 154 0.0287 0.0350 0.0368 0.0367 465 449 58.3 58.0 669 90.7 179 139 0.0339 0.0370 0.0481 0.0436 50.9 45.7 47.4 37.9 105 142 104 92 Table D-5b. PFOS Amounts Reported in Serum by Recovery Group Animal (Week 5 1 - Week 79) PFOS Re p o r t e d WEEK 51 (jifl/mL) WEEK 53 (UP*"*-) WEEK 57 (ligAnD WEEK 61 luptmL) WEEK 65 {jiftnL) WEEK 69 tupmL) WEEK 73 (UPTnL) WEEK 77 (uptruL) WEEK 79 (upmL) G roup 1(ContrOl) 105520M 0.0213 105526M 105529F 0.0261 0.0344 105549F 0.0338 G roup III (Mid Do m ) 105505M 39.8 105523M 36.0 10553SF 44.5 105552F 25.8 G roup IV (Hlgl Do m ) 0 0269 0.0392 0.0375 0.0419 43.9 485 41.1 32.3 0.0289 0.0364 0.0472 0.0418 28.5 31.8 33.3 31.4 00319 0.0382 0.0433 0.046 27.4 35.9 38.0 38.4 0.0184 0.0236 0.0364 0.0357 32.9 329 39.3 36.0 0.0428 0.0383 0.0421 0.0379 0.0431 0.0269 0.0386 0.0345 24.6 28.3 36.9 32.0 240 30 6 27 9 23.7 0.0334 0.0259 0.0317 0.0294 22.1 23.0 27 5 18.5 0.0194 0.0236 00268 0.0218 197 185 228 19.9 105511M 105522M 105533F 105542F 67.5 122 87.1 95.7 548 107 986 97 9 66.5 B9.5 103 109 84.7 136 106 109 52.5 131 89.7 76.0 47.0 121 787 71 3 35.1 73.8 54.5 240 329 87.0 70.5 434 228 564 406 422 In d ica te s a sam ple w ith an extraction volum e of <0.5 m L Table D-6. LOQ Values Used In Analyses by Method and Usage Dates Methoo Effective Date LOQ Usage Dates Sera FACT-M-4.1 ETS-8-5.0 ETS-6-5.1 Liver FACT-M-2.0 FACT-M-Z1 ETS-8-7.0 10/10/98 3/01/99 4/26/99 5/26/S8 6/03/99 7/22/99 ng/m L 4.39 15.2 5.55 ng/g 30.0 37.4 26.9 1/25/9910 2/22/99 3/05/99(0 4/17/99 5/17/99 through the end of study 1/25/99 to 5/22/99 6/03/99 to 6/14/99 7/29/99 through the end of study 3M Environmental Laboratory Page D-8 3M Environmental Laboratory Page 186 3m Medical Department. Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-112279 Tabla D-7. PFOS Concentration* In Uver by Dosage Group DOSAGE Group COLLECTION Da t e PFOS C a l c u l a t e d Co n c e n t r a t io n (now Amount or PFOS a*) Group 1(Control) 105508M 2/25/99 2 Anomalies 105517M 2/25/99 143-1 Anomaly 105519M 2/25/99 91 10552OM -- 105S26M 105527M 105529F 10S530F 105531F 105535F 105544F 105549F N d fS tir - 2/25/99 222N11122o2566mmmmfer 2125m .y-:- . ' Nones.. : , ilniMawilirrj.g..y . 129 -- 128 112 87 97 _ Group II (Low Dose) 105514M 105515M 105516M 105521M 10S537F 105541F 105547F 105550F 222111222666mmm 22222111112222265666mmmmm 18237 22709 11417 16734 22818 24847 20102 20735 2 Anomalies 0.143 0.091 ----- 0.129 -- 0.128 0.112 0.087 0.097 --* . 18.2 22.7 11.4 16.7 22.8 24.8 20.1 20.7 Group III (Mid Dose) 105505M 105510M 105518M 105523M 105524M 105528M 105532F 105538F 105539F 105545F 105548F 105552F 3/01/00 Biopsy 2/26/99 2/26/99 3/012/01026Bmiopsy 22112265mm 2/25/99 3/0122/01102255Bmmiopsy 3/01/00 Biopsy 8252 42169 86173 10203 58673 48203 80421 49590 24728 66532 81376 17690 8.25 42.2 86 ' ' 10.2 58.7 48.2 80.4 49.6 24.7 66.5 81.4 17.7 Group IV (High Dose) 105506M 105507M M2M12f5llmilWft; "at t;;--.; -- 412474 105509M r 5N 5S p r--7 105511M 105512M 9/22/99 Biopsy 2125m 2125m 142465 23480 378723 105522M 9/222/91925Bmiopsy 137561 70781 105533F 9/222/91925Bmiopsy 175283 42668 105534F 105536F 22/12265/9m9 280575 256669 105540F 2/26/99 267328 105542F 105551F 9/222/91295Bmiopsy 2125m 421647 57895 287223 -- 412 -- 142 23.5 379 138 70.8 175 42.7 281 257 267 422 57.9 287 <LOQ: Less Sian the Lower Limit of Quantitation (26.9-37.4 ng/g) 3M Environmental Laboratory 3M Environmental laboratory Page D-9 Page 187 3m Medical Department Study: T - 6 2 9 5 . 7 3M Medical Department Study: T-6295.7 Attachment E Data S preadsheets Report No. FACT TOX-030 laboratory Request Number-U227S Report No. FACT TOX-030 Laboratory Request Number-U2279 3M Environmental Laboratory 3M Environmental Laboratory Page E-1 Page 188 3m Medical Department study: T-62 3& c ?TOX-30 Report No. FACT TOX-03 0 Covance#6329-223 laboratory Request Number-U2279 Study 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkeys Product Number(Tet Substance): T-6295 (PFOS) Matrix Monkey Sera Method/Revnion: FACT-M-3,1 & FACT-M-4.1 Analytical Equipment System Number Soup 020199 Instrument Software/Vernon: MassLynx 3 1 Filename: See Attachments R-Squarad Value See Attachments Slope See Attachments Y-Irrtercept See Attachments Date of Extraction/Analyst 02/09/99 RWW Date of Analysis/Analyst 02/13/99 MEE DSaatme opfleDaDtaaRtaeduction/Analyst 02/16/99 KJH BASELINE MONKEY SERA Group Doee Sauapls # PFOS Cone Coaeaa(ration of PFOS Meaa PFOS RSD Std. Dev. ag/osL ng/mL or % Ree ug/mL MS/MSDRPD Method Blk RBS02099-H20 Blk-1 RBS02099-H20 BIk-2 000 000 <LOQ <LOQ <LOQ NA Matrix Blk RBS02099-Sera Blk-1 0.00 <LOQ RBS02099-Sera Blk-2 0.00 <LOQ <LOQ NA QC *250 ppb MMKKSS0022009999-M-MSSD 299 285 121 % 115% 118% 5% Group 1 I05508M+ 1.28 <LOQ Control 105517M+ 1.32 <L0Q 0.0 mg/kg/day 105519M+ 3 16 <LOQ I05520M+ l 46 <LOQ I05526M+ KJ5527M+ I05529F+ 1.62 1 48 2.28 <LOQ <<LLOOQQ <!.OQ NA I05530F+ 113 <LOQ 10553IF - 1.73 <LOQ I05535F- 1 46 <LOQ I05544F l 60 <LOQ I05549F 5.42 <LOQ <LOQ NA Group 2 105513M+ 2.63 <LOQ Low-Dose 1055MM 1.59 <LOQ 0.03 mg/kg/day 110055551156MM+ 0.540 265 <<LLOOQQ 105521M+ 1.72 <L0Q 105525M* 1.90 <LOQ <LOQ <LOQ I05537F+ 1.33 <LOQ 105541F- 2.23 <LOQ I05543F+ 2.53 cLOQ I05546F-*- 2 50 <LOQ 05547F+ 1 69 <LOQ I05550F- 1.50 <LOQ <LOQ <LOQ Groap 3 I05505M 3 13 <LOQ Mid-Dose 105510M+ 1.04 <LOQ 0.15 mg/Vg/day 105518M+ 1.51 <LOQ I05523M+ 0.930 <LOQ I05524M+ II0055552382MF++ 1 82 2.69 3 13 <<LLOOQQ cLOQ <LOQ <L00 I05538F 2 25 <LOQ I05539F 2 15 <LOQ I05545F+ 3 27 cLOQ I05548F 360 <LOQ I05552F+ 2.02 cLOQ <LOQ <LOQ Groap 4 I05506M+ 2.19 <LOQ High-Dose I05507M 2.79 <LOQ 0 75 mg/kg/day I05509M 2.06 <LOQ I05511M 269 <LOQ I05512M+ 264 <LOQ 105522M+ 2.73 <LOQ <LOQ <LOQ I05533F+ 2.02 <LOQ I05534F+ 2.54 <LOQ I05536F+ 2.85 <L0Q I05540F+ I05S42F+ 3 45 1.94 <<LLOCQQ I05551F 3.52 <LOQ <LOQ <LOQ Limit of Quantitation (LOQ); 4.39 ng/mL Date Enterad/By 02/17/99 LAC Date Verified/ By: 03/23/99 OML, 06/07/00 PW Date punty corrected/verified 09/12/00 rrvnh, 09/12/00 LAC NA - Not applicable + Serum initial volume less than 0.50 mL, concentration* should be considered tentative 04/28/00 LAC PFOS " Pcrfluorooctaneaulfonate Extraction Volume Ratio * Initial Volume-Final Volume FACT-M-4 l Excel 97 3M Environmental Laboratory tox-030-sera223- l C 9/ 12/00 Page 189 3m Medical Department Study: T -6 2 % ^ c Y T O X - 0 3 0 Report No. FACT TOX-030 Covance#6329-223 laboratory Request Number-U2279 Study; 26 Week Capsule ToxicityStudy with PFC-S in Cynomolgus Monkeyi Product Number(7est Substance) T-6295 (PFOS) Matrix Monkey Sera MAneathlyotdic/R!eEvuqiuoinp:ment System Number FMAaCdTe-liMne-30411S0l9F8A&CTA-mMe-l4ia 1062498 Instrument Software/Venion: MaisLynx 3 1 Filename See Attachments R-Squared Value. See Attachments Slope: See Attachments Y-Intercept: See Attachments Date ofExtraction/Analyst: 02/05/99 SAH Date of AnaJysia/AnaJyst: Date of Data ReducUon/Analyst: 02/08/9, 02/10/99, 02/12/99, 02'1 */99 HOJ/DRB 02/10/99, 02/16/99. 02/2d'99 HOJ/DRB Sample Data WEEK 1 MONKEY SERA Groep Sample # PFOS Coflceutratioa Meas RSD Dose Cose of PFOS PFOS Std. Dev. af/naL ug/mL or % Ree MS/MSD RPD Method Blk RBS02059-H20 Blk-l RBS02059-H20 Blk-2 0.00 0.00 <LOQ <LOQ <LOQ NA Matnx Blk RBS02059-Sera Blk-l RBS02059-Sera Blk-2 0.00 000 <<L1OOQQ <LOQ NA QC - 250 ppb MMXKSS0022005599-M-MSSD 305 304 112233%% 123Y oy. Groep 1 I05508M 0.00 <LOQ Control 0.0 mg/kg/day 105517M+ 105519M I05520M 000 0.00 . 0.00 <<LLOOQQ <LOQ I05526M 0.00 <LOQ I05527M 000 ^LOQ <LOQ NA I05529F ooo <LOQ I05530F 0.00 <LOQ I05531F 0.00 <l o q I05535F o.oo <LOQ I05544FI05549F 00..0000 '[.OQ <LOO <LOQ NA Group 2 105514M 495 0 ~93 Low-Doae 105515M+ 383 0 ~6` 0 03 mg/kg/day 105516M 105521M 519 677 0 832 16.9 1 09 0 869 0 147 105537F 684 10 105541F+ 464 C929 I05547F 581 093! 1] 6 I05550F 519 0 832 0 947 0.110 Group 3 I05505M 287 4 60 Mid-Dose 0 15 mg/kg'day I05510M* 105518M 209 342 -5A4189 105523M 221 3.5-1 IC5524M 318 1 25 17 0 105528 M I05532F* 413 161 5 4 35l: 4 60 0 782 I05538F 257 4 !: I05539F 248 3 3! I05545F+ 184 3 69 II0055554582FF 213952 33 712: 3 71 0124.535 Group 4 I05506M 665 2l 3 High*Dose I05507M* 411 21.0 0 75 mg/kg/day 105509M* 351 18 8 105511M* 482 19 3 10551 2M 711 22 8 7 49 I05522M* 539 21 6 21 0 I 57 I05533F+ 564 22 6 I05534F* 451 18 1 I05536F* 393 21 0 I05540F- 419 6.8 I05542F 749 24 0 133 10555 IF* 377 20 ! 20 4 2 71 Limit of Quantitation (LOQ): 4 39 ng/mL Dete Entered/By 02/10/99, 03/02;99 LAC Date VenBed/ By 03/23/99 GML. Date punty correcled/vurified: 09/12/00 mmh. 09' 12/00 LAC NA " Not applicable * Serum ffutial volume lets than 0 50 ml., concentrations should be considered tentative 0 PFCS " PerCuorooctanerulfemale Extraction Volume Ratio = Initial Yolume/Finai Volume FACT-M-4 1 Excel 9 ' 3M Environmental Laboratory tox-030-seru223-l C 9/1 2/00 Page 190 3m Medical Deartment Study T-62^cf TOX-030 Report No. FACT TOX-030 Cov.nce#6329-223 laboratory Request Number-U2279 Study 26 Week Capsule Toxicity Study with PFOS ui CMtomolgus Monkey Product Number(Teit Subitanee): T-6295 (PFOS) Matrix Monkey Sera AMIFninlseeattrnhluyaomtmdic/eeaRnlteEvSuqoiufoitpnwm.aeren/tVSeyrantoenm' Number' MMSFeAaaedCsiAeTLlti-ytnManecx-h03m4311e10nA9ts8FAACATm-Meb-4a.1062498 R-Squared Value See Attachments Slope See Attachments DYa-ltneteorfcEepxtt.racbon/Analyst 0S2e/e0A9/t9ta9chJmCePnts Date of Analyau/Analyst: 02/15/99. 02/22/99 HOJ Dare of Data Reduction/Analyw. 02/19/99, 02/25/99 HOJ Sample Data WEEK 2 MONKEY SERA Graap Sample PFOS Cancantradan M taa RSD Dow Cone. af PFOS PFOS Std. Dev. ng/mL g/nsL ar % Ree ug/eaL MS/MSD RPD Method BQc Matnx BQc QC - 250 ppb RRRBSBBSS000222000999999---SHHe22r00a BBBfUllkcc---121 RBM30K2039092-0S9e9r-aMBSlk-2 M1CS02099-MSD 00..0000 000000 293 300 <LOQ <<LLOOQQ <1L18OVQ. 121% <LOQ NA <LOQ NA 120% 2V. Graap 1 105508M 4 09 <LOQ Control J05517M 3 07 <LOQ 0 0 mgkg/day I05519M* II00555S2206MM* 4 53 2 08 2.02 <LOQ <LOQ <LOQ 105527M* 1.89 <LOQ <LOO NA I05529F-* 0.520 <LOQ I100555533CIFF** I05535F 105344F* I05549F* 311..190202 0 380 0810 <<LLOOQQ <LOQ <LOQ <LOQ <LOQ NA LGowro-uDpos2e 105514M* 105515M* 557153 102 103 0.03 mg/kg/day I05516M- 492 1.19 7 63 05521M* 425 1 13 I 10 0 0835 105537F+ 105541F 105547F* I05350F+ 472426 527 550 1 19 1.16 01.90860 8 79 I 10 0.0963 Grasp 3 I05505M- 227 6.07 0 1M5 midg-D/kogs/eday I10055551180MM* 301 350 47 0812 I05523M 285 4 56 I05524M- 195 6 24 16 1 I05528M 384 6 15 5 81 0 933 [05532F* 173 4 61 105538F 416 6 67 I05539F* 232 4 65 I05545F 329 5.28 II0055554582FF* 322950 64.7432 5 39 0197.320 Graup 4 I05506M* 747 29 9 High-Dose 1O5507M* 626 25.1 0 25 mg/Vg/day 105509M 746 23.9 1055UM 706 22.6 105512M 976 31.3 13 1 05522M 899 28.8 26 9 3 54 105533F 778 24.9 I03534F* 650 26.0 105536F* 588 23.5 105540F* 105542F* 10555 IF 546 372 568 19 5 199 14 8 18.2 22 0 3.25 Limit of Quanbtabon (LOQ): 4.39 ng/mL Date Enlered/By 02/22/99. 03/02/99 LAC Date Verified/ By 03/23/99 GML Date purity correeted/venfied 09/12/00 mmh, 09//12/00 LAC NA Not applicable * Serum uutial volume leu than 0 50 mL. concentrations should be considered tentative 04/28/00 LAC PFCS * Perfluorooctanasulfonate Extraction Volume Ratio " Irubal Volwne/Final Volume FACT-M-4 1 Excel 97 3M Environmental Laboratory tox-030-KTa223-lC 9/ 12/00 Page 191 3m Medical Department Study T - 6 2 TOX-030 Report N o . FACT TOX-030 Covance# 6329-223 laboratory Request Number-U2279 SPtruoddyuct NumbcrfTest Subitanee) AMIFMRnin-lsaeSeatttnrrlhqiuyaoxutmmdiacie/eaeR:nldetEvVSqiosauiflaiutpwnem:areen/tVSerysrioonn Number SDWYDDSlaaa-aoItttEpmneeeetEeoo:oprfffKlceADEepxanD4ttt.arataayMcRtntaeisoOd/nAuN/cAntiaKnolaynEl/iyAtY:stn:aSlyEstR: A 26 Week Capsule Towaty Study with PFOS tn Cynomolgus Monkeys TMEMSSMeeT-ao6eae5dns2A-Aeik98Lltet5-itytn4ya(anePcc0SxhhF0eammO43nn1eeSd0lnn)9aEtt8ssnT,dSA-3m82-e5li0a 062498, A Soup 020199 0"0SS333ee//ee/000A82AV//tt999tt9aa99cc,,S0hh03Amm1//H2ee06nn/8R/tt/9ss9W99.W0KVJH25/D/9R9BHOJ/MEE/DRB CDrawtrep Staplea PFOS aCa/mneL aCg/amamLfaPwaFrnO%aSfloRaac Mtu PKFlOmSL M39/MtAR9SDDOevR.PD Method Blk Matrix B0t Matnx Blk QC - 250 ppb Groop1 0 0Cmogn/Vtr^o/lday 20.0L3Comwng-aD/pkogs/deay MCirda-aDpos3e 0 15 mg'kg'day 407H3Cigmnhg*a/Dpkgos/deay RBS03029-H20 Blk-1 MMRRR8BBKKSSSSS0000033333000002222299999-*---HSSSSee2eenn0nn BBQBBlIOllkkkkc-----12122 MMKKSS0033002299--SSeerna BBlIkk--43 MMMMJKCK)SCS1II1SI110II00S10100000000035500505535535530555553055555550251521220342302239497097259908694-MMMI-M9FFFFMMMMF-F-****M****MSSSDDS----2112 I055MM* 11I1I1I00000005555535555555552431140117567FMFFMMF++** I1IIIIIQ00I0000055555555555555552110322388053428MMMMMMFF+*-** 1I1100005555555544355892FFFF**** I1III1III000000Q0555355555555555251OI00333212793466MMMMMMFFF***+***** 1IIQ0055555354420IFFF*** 00..0000 ox o7721x17 762326 253 250 242 235 657463 791 667131 619683 611 98.1179 989746 108 111109871662 153 212613 63 7 646 101 no 9965.37 41 4 347185617873 55 9 118 222324970782023 335632 24 0 289384 150 <LOQ <<LLOOQQ <<<<LLLLOOOOOQQQ <119L00821O%%%0 <<<<<<<9LLLLLLL5OOOOOOO%QQQQQQQ 8 V <LOQ NA <LOQ NA <LOQ NA 101% !% 96% 3% NA <<<<LLLLOOOoQQQq <LOQ <LOQ NA 2 87 2343..07173335 3 20 0I.S571? 3.07 33.5257 3.40 0825931 1177..20 211057..624 9 47 19 2 1 7 168 16.6 187 111485377 14.9 165 111874 475228436486 774 738 861M244..1S12 95.3 70 4 6778.42 60.2 42 7 927 39 6 Land ofQuantuaoon (LOQ): PFOS 13 2 ng/mL Date punty DcDoaratreteecVtEeendrti/efvireeenddf//iBeBdyy*: 000393///]20128///099099,m0GVmM2hU6,/0999/1L2A/0C0 LAC NA - Not applicable * Scrum initial volume less than 0.50 mL, concentrations should be considered tentative 04>?8/00 LAC PFCS = Perfluorooctanesulfonate Extraction Volume Ratio - Initial Vohime/Final Volume EExTcSe-l89-5" 0 3M Environmer tal Laboratory tox-030-ser223-lC 9/ 12/00 Page 192 3m Medical Department Study T -62 TOX-030 Report No. FACT TOX-C30 Covance#6329-223 laboratory Request Number-U2279 Study. 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkeys Product NumberfTen Subtlance): T-6295 (PFOS) Matnx: MAInnseattrhluyomtdic/eRanletEvSmqoufoitnpwmareen/tVSeyrssitoemn Number: FRi-lSenqaumaree:d Value: SYl-oIpnetercept Date of Extraction/Analyst- DDSaaattmee oopffleADDnataaatyRtuaead/Aucntaiolyns/tA. nalyst: Monkey Sera AEMTmuSes-lL8ia-y40n6xO23a4n91d8aEAnT.dSS3-o8u2-p3.Q020199 See Attachments See Attachments See Attachments See Attachment 03/10/99 SAH 03/12/99, 03/21/99 MEE/DRB 03/ItW , 03/24/99 LAC/DRB WEEK 6 MONKEY SERA Grwup Daaa Samplea PFOS Mean RSD Cane. CfnL fPFOS ug/aLer%Ree PFOS Std.Dev. uf/nL MS/MSDRPD Method Blk Matnx BQc Matnx BQc QC - 250 ppb RRBBSS0033110099--HH2200 BBQOcc--12 RRBBSS0033110099--SSeerraa BBDDcc--12 MMKKSS0033110099--SSeerraa BBQQcc--12 MKS03109-Sera 31-3 MKS03109-Sera Blk-4 MMKJSS0031301099-M-MSSD-1-1 MMKKSS0033110099-M-MSSD-2-2 070704 000 4.19 2.56 21 9535 0.500 264 222544339 <LOQ <<LLOOQQ -LOQ NA <LOO <LOQ NA <LOQ <LOQ <LOQ 4<1L06O%O 101% <\,OQ NA 103% % 97% 100% 987'. 2% Graup 1 Control 00 mg/kgf'day I05508M* 105517M* 111000555555122906MMM* I05527M + I05529F+ I10035533301FF+ 105335F* I05544F* I05549F* 000 000 0016601000 949 1.56 34 3184 0 00 03.0504 <LOQ <LOQ <<<LLLOOOQQQ <<LLOOQO <LOQ NA <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ NA Craup 2 Low-Doae 0 03 mg/kg/day I[0055551145MM-** I05516M* I05521M* 105537F+ 1I0055554471FF* 105550F+ 3336 61 4312.58 42.7 4356-7r32 7 33.3617 3.26 4.19 :: 9 3 61 0 430 4.28 3 66 33.6257 3 71 0n4P: Craop 3 0 1M5 midg-D'kogs/eday Croup4 High-Doae 0.75 mg/kg/day I05505M 10551OM I03518M* I03523M+ III00103505555325233482BMMFF++* I05539F+ 1IID00555555445852FFF+ I05506M* I10055530079MM* 105511M 110035551222MM-V* I05533F I05534F+ I05336F* 1I000555555445201FFF* 87 4 6566 91 6712..79 6696911144 62455 6617 03 182 165 223340 211 203 213626 212779 220053 21.0 22.5 20.1 18.6 21.8 1188 54 :o a 8 1 5259 18 9 18 4 422.7 18 4 16 1 18 8 2!115 91.0 82 8 92 3 93.7 105 102 94 5 88057* 93 0 83 3 89 8 90 8 102 7 90 82.0 90 l 7 13 Limit of Quantitation (LOQ); PFOS* 15.2ng/mL Date Entered/By 03/19/99, 03/24A>9 LAC Data punty DcoartreecVteendf/iveetVnfiBedy 0049//1172//9090 mSmAHh,,0095//1129//0000LPAICO-PW PFCS " Perfluorooctaneaulfonate * Serum initial volume less than 0 30 mL, concentrations should be considered tentative 04/28/00 LAC NA Not applicable Extraction Volume Ratio " Initial Volume/Final Volume EETxcSe-l89-57 0 3M Environmental Laboratory tox-030-serm223-lC 9/ 12/00 Page 193 3m Medical Department Study: T -6 2 t o x -030 Report No. C ov*n ce#6329-223 laboratory Request Study 26 Week Capsule Toxicity Study with PFOS mCynomolgus Monkeys AMFMPInirnlseioeatttdnnrlhyuuaxotmmc:ditce/eaRN:nletuEvmSquobuifoeitpnwrfm:Tareeens/ttVSSeuyisbsitaoetnmmcNe)u:mber SRl-oSpqeu:ared Value: YDDDS-aaaaIttlmneeetoooeprffflcADeEepxnaDtattanlayeRtstaiiesod/nAu/cnAtainolyansi/tyA.sntalyst: 1SSS00MESSM*3VeeeeoT6oeee/etuSO0s2npAAsAA-39k58L/50tttteW-9tttty24yaaaa9(n0cPccc,0S1xhhhhF0Re9ammmmVO3rW9na.e0eeeSd)Wnnnn9)aEttt/t9ssssn/TJ9dCS,-3P08.32-/5100/99, 03/25/99 DRB 03/0&99.03/1V99, 03/24/99,0V26/99 DRB WEEK 8 MONKEY SERA GDroeunp Sample# PCFoOucS ng/mL Comutrsttou ug/moLf PoFrO%S Rac PMFeOaSu StdR.SDDer. ag/mL MS/MSD RPD Method Blk Matrix Blk Matrix Blk QC - 250 ppb MMRRRRMJBKBBKM3SKSSSSSKO0000S0S333333C0O0000033333333099999093-*--3--S9HSSHS9e-O-ee22MrMnan00SSBBBEBBD*fif2lllk1l-kktkc-1-----121221 00030211.0000971210004030 <<LLOOQQ <<<<6LLLL9OOOO%QQOQ 76V. <LOC> NA <LOQ NA <LOQ NA 72% 9% MMMMKKKKSSSS000033330003339399-9-MM--MMSSSSDD--2-3-23 221100881553 88772132%%%% 77V, 10% 76% 11% 0 0GCmrogon/utkrpgo/l1day III[I000000555555553555120122778906MMMMMM I11I000055S355552333930IFFFF J10055554494FF 9 43 2791n8620.o8083 16.0 911118458403605 <<LLOOQQ 0<0<<.LLL00O2OO318QQQ45 <LOQ 1Anomaly NA 0 0244 00<<..LL00OO2240QQ06 0.0226 - 2 Anomalies 0 08031689 Group 2 0 0L3omwg-/Dkogs/deay I05514M I1II1Ir000000055555555555555112345465I770IMMMFFFF 4343333311646744..13453821 44.6117 554435.998021845580 4^3 094I3d2 4 76 01.52717 0 1M5Gmirdog-uD/kpogs3/eday I1I1I100000003555335553555521202I38804532MMMMMMF 891242291 891M1601$76 279 222322254147.....116665 26.0 312307 305538F 106 243 I1100055555J344985FFF I05552F 685192922364 22214269.535i 24 0 12 7 306 0.7H5Cimgrhog-u/Dkpog4a/deay JI1I1IIW000000055335355555555330001213J96722134MMMMMMFF 8891111193821120275778a1 816251 911111220311113301 109 1168 37 1I100005553555544530!26FFFF 100 102 9m92 811170012563 107 111118 Limit of Quantitation CLOQ): PFOS 15 2 ng/tnL DDaateteVEenniereedd//BByy 0Q3B/M08//9999,0S3A/2HV, 9095,/01V1/2060/9P9IOL,AOCV2VOOA 05/24/00 PW Date punty corrected/'venfied 09/12/00 mmh, 09/12/00 LAC NPFAC=S N" oPt earpflpuloicraobolcetanesulfonaic Extraction Volume Raoo " Iretial Volume/Final Volume FACT TOX-030 Number-U22 79 EETxcSe-l89-57 0 3M Environmenta Laboratory tox-030-scTa223-lC 9; 12/oq Page 194 3m Medical Department S udy: T-62 9 F A C ? TOX-030 Report No. FACT TOX-030 C o v a n ce# 6329-223 laboratory Request Study: 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkeys MMPraeotidrhiuxoc;dt/NReuvnutbieoTnf:Tesi Substance): AIFRSYDnlin--aolsSIetatpnenrqletuyaueotmmraifccreeEeaenpldxtlEt:VnSqcoautlfuiitpowemn:a/erAenn/tVaSleyynssittoenm Number: DDSWaaattEmeeEoopffKlAeDanD1taa2layRUsMeisd/uAOcntNiaolKny/sAEt'nYalySstE: RA T-6295 (PFOS) 00S0MEMSSSS331oeeeeT//o/eeeue0uQS0n&sp5A-AAA3kL8///0te9ttt9-9ytttt42y999aaaan.0.,cccc0SRx1hhhh03e9ammWVmm33r9n/a0ee1eed2Wnn9nn5/ttE/tt/99ssssJT99CS.,P-00833-//512400//9999,, 0033//2265//9999 DDRRBB GDroocu*p Sample* PFOS nCgo/mucL. Ceu4tePuFtOtaSdeu ag/mL ar % Ree iPMigF/eOmanSL StdR.SDDev. MS/M3D RPD Method Blk Matrix Blk Matrix Blk QC 250 ppb 0 0GCmrogon/uktrpgo/ld1ay 0 0L3Comrwog-uD/kpogs2/deay MCirdo-uDpos3e 0 15 mg/kg/day MMRRRRMMBBBBKKMMSSKSSRSSJK0000SCS00333S30330S00000033003333333039990990393---3--39-9HSHSS99S--eee--2MM2errMMn00naaSSBSS5BBBDED--J1Ol1J2k-kk-t-2t-1----2112l2 MMKKSS0033003399-M-MS5D-3-3 11111I[I000000005555555555555555222021I3977S0690FMMMMMMF* I1I1100000555555555U34349154MFFFF 1III11000000555555555555241143176517MFMMFF** 1II000555555105050MMF I10053552138MM* 00300..0200030000 221111.809871113540 3322120620.39987 23110116..1443 19.0 294411384453381 47 4 4557 81 3364.89 488 143 135 133 88.0 <tOQ <<<<LLLLOOOOQOQQ <LOQ NA <1.00 NA <67877L96232O%%%%%Q 81% <LOQ NA 72% 9% 10% 76% 11% 0.0381 00.00443380 <0.L0O24Q4 0<0L4O2Q3 0 0383 - 1Anomaly 0 02108111 00..00324534 <LOQ 00..00233186 0.0263 -2 Anomalies 0 01033862 6.20 66765.8455518725 669 0856738 57.8033 631 0171147 433903.271 35.3 11I10000535553553223884F2MMF-* 87169(829 94 2 322755..848 328 35 2 31.5393 111100005555555534459582FFFF Group 4 1556M 0 7H5igmhg-D/kogs/deay Limit ofQuantitation (LOQ): PFOS- 1511I.I([I1II20I0000000000355535533n55S55555555g554230101345/302672m913214FFMMMMMFFFF-L+*- 771179001694 232247232828 8811136014.94369 911119206)63674 818090 681300006 128 114 Dale Entered/By 91118212.1460 111131217 0V08/99, 0^24/99, 0V26/99 LAC 27 8 122 I!4 314983 213969 :oo 117 Date Venfied/By: 08/18/99 SAH. 05/24/00 PW PFOS Perfluorooctanesuifonate Date punty c*orrSeecrtuedm/vienritiifaieldvolu0m9e/1l2e/a0s0thtraonnh0,5009m/1L2/,0c0onLcAeCntrations should be considered tentative 04/28/00 LAC NA Not applicable Extraction Volume Rato " [rubai Vohsne/Final Volume Number-U22 79 EETxcSe-l8-57 0 3M Environmental Laboratory tox-030-seii223-1C 9/12/00 Page 195 3m Medical Department Study: T- 6 29pj\cT TOX-030 Report No FACT TOX-030 Covince# 6329-223 laboratory Reques Number-U22 79 FQenimc:MMAISPntrnasueoattdtdrrhlyiyuuxo:tmc.dict/eRNn]eutEvmSiqaobuifeoitxpnwfm:Tueeens/ttVSSeuyrbsntaoelmaancNe)u:mber RSYDDDSl.a*aaaoStttlpmeeenqetuo:oopeafrfflrcAEeDeexdpanDtttrVaaaalacRtyltauiesoedtnuA/cAntainolaynls/yAts:tn:alyst. 26 Week Capsule Toxicity Study with PFOS in Cynomclgus Monkeys TMES-oT6ouS2np-9k850e-24y(0P0S1F9eaO9nnSd)ETS-8-5 0 MtstLyrutl 2 See Attachments oSSy0Seeyye1ceeii26yAAA/9/99ttt9ttt99aaa,c,ccSohhh0Aymmm3Hi/eee29/nnn4R/ttt/9sss9W99WdDrRBb/hoj WEEK 16 MONKEY SERA Group Sample* Doe* Method Blk Matrix Blk Matrix Blk QC- 250 ppb 0 0GCmrogon/utkrpgo/l1day LGorwa-uDpos2e 0.03 mg/kg/day 0 1M5Gmirdag-Du/kopgs3/eday 0 7H5Gigmrhag-uD/kpogs4/deay MMRRRRMMBBBBKKMMSSSKKSS$KK00000SS0333SS3300311I010133l222332221I9991192992---22-9--H9HSSS99S--eeM22--eMerMMrOOnanaSSBSSBBDBBBD--QIOtDI2I-k-kkc2cIc------12122l I10055551078MM*105519M+ III11CI0000005555555555555533222235060791FFMMMFF** II0055554449FF*I055I4M I11I0I00005555555555I24I53161M7MMFF*+ I10055555407FF 1I111IQI1100000000555355555555555553012221333503848289MMMMMMFFF* 111000555555454825FFF 1II111000000555535355553000112796212MMMMMM 1I11II00000055355355555533443563204FFFFFF* PCFKeOn/meS.L 0000212211171...0035899000659990000054 21.) 321089497 3255.57 2927II187.3...59776 88471367.374I 665639 289 233237209 343223762754522697 323 324213 227537 222259694686 222111349398115864 Cancaotratea ug/mo00L<<<<<<f9.777PLLLLLL00e0266FOO4OOOO3r****04OQOQQQO%84S Rac 0.0423 0000000<<.....0LL00000054O3643O31621313QQ5205842 91.0447 1104.91 675511I.92200U8...753749 654428304424 344736..133 4378.70 211111111117897865872627954743 116287 Memo PFOS og/naL <LOC LOO <LOQ 74% 83% 00407 0.0432 -2 Anomalies 11 2 105 56 2 42 1 189 162 StdR.SDDev. MS/MSD RPD NA NA NA 5% 16% 02071110 197 0 00851 221449 118901 510844 940549 815429 1! 9 193 Unit ofQuanmaoon (LOQ): PFCS - 15 2 ng/mL DDaateteVEenntefireedd//BByy:: 0038//0188//9999,0SAy H17,/9O9S,/0l&3'/O2O4,/9095/2L4A/0C0 PW PFOS - Perfluorooctanoulfonate Date punty corrStcetreudm/viennitfiiaeldv'.olu0m9e/1l1e/s0s0thnaunnh0,5009m/1L2/,0c0oLncAeCntrations should be considered tentative. 04/28/00 LAC NA " Not applicable Extraction Volume Ratio * Initial Votume/Final Volume EETxcSe-l89-5? 0 3M Environmental Laboratory tox-030-era223-1C 9/12/00 Page 196 3m Medical Departrr.ent Study T -629fcTOX-030 Report N o . FACT TOX-030 Covince# 6329-223 laboratory Request Number-U2279 Study: 26 Week Capsule Toxicity Study with PFOS in CynotnoJgus Monkeys MMASYPIFRDDnlir--naaolaseolSettapttntdeenrrhqletiuuyaeouxoo.mmtcidaiffetc//eeAeEeRaN:npdlxnettutEav:VrmSliaqysoacbuislftoeuiiitapnoiwef/m:nATv/eAenensa/nVttlaySSelsyuynt:sbsitots:etnam:ncNeu) mber Date ofData Reduction/Analyst TMESSM-eoT6ioeuaS2npsA-9k8L50et-ty42ya(n.P0c0Sxh)F9eamO39nneSdIn)EtsTS-8-5 0 See Attachments S0S13e'e'1ee/51AA/69/tt99ttaa9ccJ,h0Chm3mP//e2eSnn4Att/ss9H9 DRB 0V17/99,0V25/99 DRB WSaEmEpKle 2D0aUMONKEY SERA Grasp Sampte * Data pros CaocmrtraOaa nCgo/mmeL. ag/maLf PaFrO%S Rar Mean PFOS StdR.SDDev. ug/aaL MS/M3D RPD Method BUc Matrix Blk Matrix Blk QC - 250 ppb 0.0CCmrogon/utkrpgo/l1day LGorwa-uDpos2e 0.03 mg/kg/day 0 1M5Gmirdog-uD/kpoga3/eday 0 7H5Gimgrhag-s/Dkpog4s/deay MRRMRRBBRRBBKKRRSBSSBSSSBB000S0S003SS3330303111Q01331153355113559919933I99-*55--99-H-SHS99S-S-eMc2-M-2eeMrMjOr0aanSSaSSBDBBDBBB--Q*0l2-Il-lkkk2kIcc------1I222l I111(I0000005535533555321220I797068MMMMMM*11WI000555555553323I590FFFF* 1Q055554449FF* 111000355555111456MMM 111I00003555555S2344177IMFFF III[1000005555S5355510125S3030MMMMF 1III1111000000005553S55335553555522933445B4F2SS82M-MFFFFF*111111II1000000000555355355535355550523001317326962t3M4MFMMMMFF*+ III000555555445201FFF+ 0000030323312222....11.03090522100000......6369050000404203 253211989836634 838896.671 977931803650..129632 22245383? 3222222110251127051!3509044 332381851252 330029 323079343 441 333002 <<<<<<1989LLLLLL0180OOO3OOO%%%%QQOQQQ 00000000.......0000000653433633741610319899706 0000....0000554207963033 1102.57 114191 411U4M1..503 666323.331 677654464859572445...220372472 111111111355464442882359366 111593132 <LOQ NA <LOQ NA <LOQ NA 97% 13% 89% 2% 0 0400 02091.290 0 0504 02051227 12.3 12.3 19 5 7111l1144.447.560944 63 7 61075| 58 1 2154 47 144 710692 156 140 21 8 Limit of Quanatabcsi (LOQ) PFOS - 13 2 ng/mL Date punty DcDoaratreteecVEteemdri/efvireeednd/f/iBBeyyd::: 000938///110280//T99O99,0rSm1A/n1Hh7.,/90099V/11L72A//O0C00LPAWC NPFAO*S N" oPtearnflauloyrsoeodc, tnaontesauplpfloincaabtele a*CSaelrcuumlatienditiwalitvhooluutmseamlepalsethI0a3n503.05F0 mL, concentrations should be considered tentative 04/28/00 LAC Extraction Volume Ratio Initial Vohsne/Final Volume EETxcSe-l89-570 3M Environmental Laboratory tox*030-so*223-1C 9/12/00 Page 197 3m Medical Department Study T -629&C? TOX-030 Report N o . FACT TOX-030 Covance# 6329-223 laboratory Request Number-U2279 SPrruoddyu:ct NumbeifTest Substance) AMMnaeamthlyxot:dic/aRleEviqauiopnm: ent System Number Instrument Softwire/Vcraon: Filename: SRYl--oISpnqetue:racreepdt:Value: DDaattee ooffAExntarlaycstiios/nA/Ananlaylystst: WDSaatEmeEopfKlDea2Dt4aaRtaMeduOcNtioKn/EAYnalSysEt RA C m p Sample 0 Dew 26 Week Capsule Toxicity Study with PFOS in Cyr.omolgus Monkeys TEAM-Tm6oS2ne-9lk8i5ea-4y(0P60SF2eaO4nn9Sd8)EAT.SS-o8u-p5 0020)99 MSSS00S3V3/eeee1eea/ee162uA7AA/3AL9//ttt9t99ytttt9aaaa9nRcc,cc.x0hhhhWO3mmmm3V/W2eeee2l4nnnn0//Stttt/9ssss9A99H., 0043//022V/9999 DDRRBB/MEE PFOS PClo/wocl. g/m(Lprro%s Ree uPMgF/omObSL M3S/MtdR.SSDDDevR.PD Method Blk MatnxBlk QC - 250 ppb 0 0GCmrogon/utkrpgo/lIday 0 0L3Comwng-uD/pkogs2/eday 0.1M5Cmirdeg-u/Dkpogs/3deay 0 7H5Cigmhmg-D/*kOg4M/day RRBBSS00331I6699--HH22OO BBllkk--12 RRRJRRBRRRBBBSSBBB0S0SSSSS3030010031336331613I9II6696166-9-969S9S9--9--eMM-eM-MMrrMaaSSSSSSDDDBB---ll2-I3--kk23I--12 105508M III1110111(00I00050000055555555555555255555533714212432M7054690991MMFMFFMFFF+---1I111010II000005055355555555315I542513457416TMFM0IMMFF-F*** 1I1000555555011580MMM I11000555555222834MMMI1I1011000C0555555555S55533344229S58FFFFFF+*II11I111001I00000050505555333355555555325530001I432467269IF30FMMMMMM-FF-*** 1I0055553412FF* 000000 002220033116008 222122340 36 4 303 32222416445059..8447892 831.363264 815134 111433124 811430388 211159861164 2222132222111111221M03558032459380463141548532697935810780 <<<LLLOOOQQQ <89989L85360O%%%%%O 89% <LOQ <LOQ 92% B9% 90% NA NA 8% 8% 2% 00.00448469 0000000....0000000334464261919984347873 0 0440 001170?1 0<0..L1004O34.247Q56 0.0426 1Anomaly 0.01087484 1111140338...14497 12.7 122 14 5 231060 13.0 0561785 656736646665345799138689......186864726751 65 9 610884 60 4 712240 22221111M0043989670883079 2M 214196 1115851]0 174 2102 90 Lamt ofQuantitation (LOQ>. PFOS - 15.2ng/mL DDaateteVEenriifeireedd//BByy 0038//2148//9999 LSAACH, 05/24/00 PW Date punty corrected/venfied: 09/12/00mmh, 09/12/00 LAC PFOS PerUuorooctanesulfonate * Serum initial volume less than 0.50 mL. concentrations should be considered tentative 04/28/00 LAC MNA*-MNoontbaupnpdlicable Extraction Volume Rado " Snidai Volume/Final Volume ETS-S-5 0 Excit 97 3M Environmental Laboratory tox-030-iCTi223-lC 9/12/00 Page 198 3m Medical Department Study T -62^ * c f TOX-030 Report N o . FACT TOX-C30 Covnce# 6329-223 laboratory Request Number-U2279 Study: 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkeys Product Number(Test Substance) T-6295 (PFOS) Matrix MAInnesattrhluyomtdic/eRanletEvSuqooufoitpnwm.meinWt Seryssitoenm: Number: MEAMTmaoSsne-skb8Lae-yy40n6S0x2e3i4rn.a91d8.ESToSu-pS-052.00199 FUenante- See Attachments R*Squared Value: See Attachments SYl-oIpnetercept: See Attachments See Attachments DDaattee ooff EAxntaralycstiao/nA/Ananlyaslyt:st: 033/1/61/79^99,0R3W/2W1//9S9AHDRB/MEE DSaatme opfiDeaDtaaRtaeduction/Analyst 03/23/99. 03/22/99 DRB/MEE WEEK 26 MONKEY SERA Creep Dee* Sample * CPFeOaeS. tfmL Ceaceutratlen ef PFOS uf/oiL er H Ree Meiu nPgF/rOaSL RSD Std. Dev. MS/MSD RPD Method BDc Matra BDc QC - 250 ppb RBS03169-H2O Bik-1 RBS03169-H2O Blk-2 RS03169-Sera BIk-1 RBS03169-Scra Btk-2 RBS03169-MS-! RBS03169-MSD-1 000..000000 000 210 232 <LOQ <LOO <1.00 NA <LOQ <8i5o%o 94% <1.00 89% VA 10% RRBBS3003316196-9M-MSSD--22 229 193 9728%% 85% 17% RRBBSS03316196-9M-MSSD--33 211 213 8856%% 86% 1% Greup 1 0 0Cmogn/tkrgo/lday 105508M I05517MI05519M 05520M 30.9 2371..10 16.8 000.0419369 0.0417 0.0254 E05526M 105527M 4261.77 0.0376 0.0669 0 0459 00311.433 I05529F I05530F 4589.27 0.0613 0.0736 10553IF 30 1 00431 I05335F 166 0 0266 110055554449FF* 234167 00..00453336 0 0506 03021654 Greup 2 Low*Dose 0 03 mg/kg/day 105514M 1I005551165MM 110055533271FM-**105541F* I05547F 114513 131 n10:1 113 140 16.2 16.3 13.B 111634...389 15 8 8.91 1 41 13.0 108 I05550F+ 85 3 11.4 13 2 142 Greup 3 Mid-Dose 0 15 mglcg/day 105505M 05510M* I05518M* 105523M* 1I0I0055555252438M2MF*II0055553398FF 1I0055554485FF+ 105552F 215459 142 176 211105656900 221669 114452 92.9 6192.95 72.1 66652939.4504 82.6 30.4 25 2 72.2 85 7 6345.65 66 8 116038 HGigrhe-uDpos4e 105506M 105507M- 2M13 1M82 0 75 mg/kg/day 105509M 1ro05s5si12iMM 2M48 285 1M42 148 21 0 [05522M 298 221 173 36 5 110055553334FF 330480 210837 1I0055553460FF* I05542F 105551F* 221705856 163 115757 114623 171 13 0 22.2 Ltfnit of Quanmaon (LOQ) PPOS 13.2 n^mL DDaateteVEenrtiefrieedd//BByy:: 0038//2139//9999 LSAACH. 05/24/00 PW M * Moribund Date puitfy corrteted/venfied 09/12/00 mmh, 09/12/00 LAC PFOS 3 Perfluorocctaneauifonate * Serum initial volume leu than 0.50 mL. concentrations should be considered tentative 04.7&'00 LAC NA * Not applicable Extraction Volume Ratio " Initial Vohimc/Fmal Volume EExTcSe-l89-57 0 3M Environmental Laboratory tox-030-seTB223-IC 9/12/00 Page 199 3m Medical Departmen Study T -629fe \c ? t o x -030 Covance# 6329-223 Report No. FACT TOX-030 laboratory Request Number-U22 79 SPAMM(rtrnuuaoeatdttdrrlhyiuyaxoctn:idtce/aNnRltoeESmvqawbufcatifwpnfmTv*eu*n/1Vt SSeuyravbotictnamneNeu) mber T2MDME6-Taa6oSvWen2re-k9y8Le5e0-eyy47k(nP0SxICF7ea3a9Ornap93Sde)uElcTST-c8u-Scif1y Study with PFOS in Cynomoigua Monkeyi SYDFRli--aolSIetpnenqetau.eomvrfcweEe:dpxttV:raacluaoea/Anatyvt 0SSSS6eeee/eeea0AAAA1/tttt0ttttaaaa0cccchhhhSmmmmAeeeeLnnnn/tttKu!!JK DDSWaaattEmee EoopffKlADe aj2Dut7atayRtnMaet/dAOucnNtaiolKyni/EtAYnalSyiEt RA 0066//0067//0000,,0066//1145//0000,,00&6/1169//0000 [IAASS'M/MMMHH GDreoauep Method Blk Matnx Blk QC - 230 ppb Grasp 1 OQCmogn/ktrgo/lday Sample 9 RRBB0016S16S0000030316316300100191--1H9HM00W-2-2S-SM0-0eMcBrSnBaSlDlkBB-k5--ll3-6kk5--56 1I1111I00000005S335335533353O21343J789043IMMMFFFF pCraaocs. 0005033C3000033300000/lnL 334847609338...911561 CouccatraUoa g'oaLf prro%s Ree <LOQ <<LLOOQQ <11L22O123HC4 0.0461 000000......000000633463913337388034 Mean upfr/mosL <LOQ <LOQ 122H 0 0529 00416 MSS/MidR.S5DDD*vR.PD NA NA 194 02071443 0.3031458 0 0L3Goxwrno-guD/pkoge1/day Groap J 0 1M3imd-gD/kog/day 0.7H3Gigmrhog-uD/kpoge4/d*ay II1II01I1111111II00000O030000000055535535555553S333555O35533535552I2113124443U5C464858621838701MMMMMMMMFFMFFFFr* [III10000005353535335550I433J27O64IMMFFFF 21m1112386813737 i332233226064611^3i70409660: 433444687213494970 291 29111113123133...5062394 6688 37 13.9 11 ! 354384 n1.527 665373360844...133067 68 1 85.7445 58.5 476979 211111088669242946 194 846913 14 9 126 160 23 9 Limit ofQuanQtatKn (LOQ): PFOS NPFAOSN" oPtmapHpuloicraobolcetaneeulfonate 5.55 n|mL Dai punry cDDoaratrateecVEteendnt/fevireeednd1f/iBBeydy: Extraction Volume 0R00669a//t/Q0i1o&27=///0000Q0Im,m0Po6mJiW/lh1V,9/0o09l0u/1jn2Le/A-0T0CmELxaAltrVCacotliuomneVolume Ratio - ImbaJ Volum/FinaJ Volume cn-j-j o Excel 97 3M Environmental Laboratory tux-C36-im213-lC 9/12/00 Page 200 3m Medical Department Study T-62 9i^ c ?TOX-030 Report No. FACT TOX-030 Covmce# 6329-223 laboratory Request Number-U2279 Study: PMADDIDMFRSYSnilraa-naalo-saoeSettliatmtptdeeennrqnhleuuyatuxoooo:pmemct.adffifetlcr/eeAEeDeeaNR:ndplxnaetuDtEttavmVrSaliaqayosabRcutislfetaouietiixpwsnoedf/m.:nuATa/creeAnetnsia/ntotVlanSSyle/usyyAntbssinttsoeatnaml:ynscNte:)umber T2MAMSE6e-Tmo6aeWSsn2eAs-kl98Leiet5a-etyy4(ak0nPc60SxCFh2eamOa43nnp.9Se2ds8n)u.EtlsSeToSTu-o8px-05ta20r0y19S9tu, dAyMwiatdhePhnFeO0S4i1n09C8ynomolgus Monkeys 000SSS444eee//ee/e000A796AA///t9tt99ttt99a9aac,,cchhh00Rmmm44W//eee11Wnnn52ttt//sss9999,.0044//2170//9999., 0035//1147//9999.,0033//1178//9999 HHOOJJ//DDRRBB//MKJEHE/KJH DAY183 MONKEY SERA GDroewm? Method Blk Matrix Blk QC - 230 ppb S an|bl RBS04069-H20 BIk-1 RRBBSS0044006699--SHe2r0a BBl&k--12 RBMSX0S4006490*69S-eMrtSB-&1 -2 MMKKSS0044006699-M-MSSD--21 PFOS d0Cr.0m/b0eL. 01.4020 1225.47 224667 ag/tnrLfPeFrOHS Ree <LOQ <<LLOOOQ <1L02O*O4 19098%% uPgF/mOSL StdR.SDDrr. MS/MSD RPD NA NA NA NA 101% 3% 0.0CCmrogen/etkrpgo/l1day MICS04069-MSD-2 11I0I0005335555522246099MMFF+ 228 219287 626130 09127%1 000.000637213606 100% 0 IP 0 0533 000165096357.%163254 0.1M5Gmirdag-Dn/kotgs3/eday 11I0I0005355355502353392MMFF+- 343797781197 74,4 931630.793 83 0 81.7 411374.690 35 1 Groap 4 High-Dose 0.75 mg/kg/day 1I00555512I2MM I0055551432FF 97.1 194 214756 216 222058182 249 416888 230 417035 Lsmt ofQuantitation (LOQ): PFOS - 13 2 ng/ir.L Date punty DcDoarelreteecVtEcendrt/ievfticeerddi/f/BiBeydy:: 000489///111992///990990.r0Sn3mA/1Hh0,,/09b99e/,f1o02r5/e0/20006L/-90A98C/0L0ACTXR PFOS - Perfluorooctanesulfonate - Senim initial volume leu than 0.30 mL, concentrations should be considered tentabve 04/28/00 LAC NA * Not applicable Extraction Volume Ratio =Initial Volume/Final Volume EETxcSe-l89-57 0 3M Environmental Laboratory tox-030-era221-lC 9/12/00 Page 201 3m Medical Department Study: T - 6 2 t o x -030 Report N o . FACT TOX-030 Covinee# 6329-223 laboratory Request Number-U22 79 SPtruoddyu:ct Number(Test Subitanee) AMMInnsaeatttrnlhuyxotm.idce'alnlletEvSquouifoitpnwm:ireen/tVSeyrnstoenm Number FRSDDDYSil-aal-aoueSIttmpneneeqetaueooopmrafffclreAeEDee:pdxnaDtta:tVralaayaRctslatueiisedo/:unAc/Antiaonlnayl/syAtsntalyst: 26 Week Capsule Toxiaty Study with PFOS tn Cynoroolgus Monkeys TMA00MSSESS-44eeeeTm6oa//eeecS002nseAAA-6A79akl8i//5Letttat9-9tttt4yyaa9(a9e0.Pccnc,06Sh0hFhhxR2me4ammOm34Wnn/9e1Seeed28n2nWnn).Et/ttus9!!ST9oS,u-O8p-W053P00/19999, . A Madeline 0 4 1 09 05/14/99,05/17/99 8 HOJ/DRB/MEE/KJM 04/09/99, 04/15-99, 04/20/99, 05/17/99, 05/18/99 HOJ/DR3/KJH Group Sample* Dm Method Blk RRBBSS0044006699--HH22O0 BB0lkc--12 Matrix Blk QC - 250 ppb RRMMfBMMiSKKSJK00SCS4S400S0004406460049096066-9-69SS9-9-MeMe--MMnnSSSSBDBD--UO1-2-1c2M-2 O.QGCmroegna/tkrpgo/l1day 11110000555355522420699MMFF 0 1M5Gmirdog-uD/kpogs}/eday Group 4 0.7H5imghg-/Dkogs/deay Limn DfOiunnunonO-OQ) 1II000555555203339MMF I10055555121FM* PFOS- 15 JIII0n00S5j55'55m243223LMFF PFOS n0011222g.0244560/n.0624707aL 228 24213238.2063 866321991200 222164192420 ug/mfLPoFrO%S Ree <<<<1199LLLL0092OOOO28%%%%QOQO 000<...L00042O2138Q637 664 687196381550 322362654 aPgF/nOsSL NA NA 101% 100% 0.0233 ! Anomaly 0 0352 69 7 780 259 245 DDuueeVEenritfeireedd//BByy 00SVaIV9/9999,0S5A-1H0,-b9e9f,o0rVe2a0V/O99&OLOACTKR MSS/MtdR.SSDDDeRv.PD NA NA 3% 16% 002N059A911 671 411265.840 42112191.2095 PFOS-Pertluorooctinesulfonite Date punry c*onStcetmedi/tiveinnifbieiidv:ota0n9e/1l2e/00tmhimnh0,.3009/m12L/,0c0oLnAceCntricons should be coniideied muove 04/2&'00 LAC NA * Not applicable Extraction Volume Ratio Initial Volume/Finai Volume EETxcSe-l89-51 0 3M Environmental Laboratory tox-030-sen223-1C 9.'11'00 Page 202 C) 01 3m Medical Department Study: T -629jE*c?TOX-030 Report N o . FACT T0X-C3 Covance# 6329-223 laboratory Request Number-U227 MMAFSPIRnitrn-luaseoSeattdtdnnrqlhyuuyaxuo:tmcm:aditcre/eaeRN:nldetuEvVmSqtoasbuilfueoitpweinfm:Tareeens/ttVSSeuynbsitsoetnam.ncNe)u:mber SYDDDSlaa-aaoltttpmneeeetoeoo:prfffclDAeEepxanDttta:arlaayRctsateiisdo/unAc/nAdaonlnya/lsAytsntalyst: T26-6W29e5e(kPCFaOpSs)ule Toxicity Studywith PFOS inCynomolgus Monkeys EASSS000MMS444eeeeTmo/e//eeeu0S00nesAAAA7694klL//i/-tttet9a99ytttt4yaaa99a90n.ccc,c0.6Sx0hhhh0R2e4ammmm434Wnn//.91eeee12d85Wnnnn2,/tEttt/9ssss9ST99oS.,u-008p44-5//071.20070//1999999,, ,0055A//.11M74//a99d99e,.l00in55e//1107*4/91909998HHOOJJ/.'DDRRBB//KMJEHE/KJK DAY195 MONKEY SERA CDraatotp Staple* PFOS l/aL aCg/umarLfsaPatFrrOs%BSiwRei e uPMgF/emOanLS MSS/tMdR.SSDDDe*R.PD Limit of PFOS Method BOc Matnx Blk QC - 250 ppb 0 0GCmrogwn/tksrpgo/l1day 0.1M5Cmirdeg-eD/kpogs3/eday 0.7H5Gimgrhog-u/Dkpog4/sdeay Quantitation (LOQ): PFOS Perfiuocoocunesulfonau RHS04069-H2O Bflc-l RRBBSS0044006699--SHe2ra0 BBAlkM-2 RBMSJ0C4S006490-S69e-rMa SB-Q1c-2 MMMKKKSSS000444000666999--MM-MSSSOD--2-12 105520M 1II000555S5J224699MFF 105505M II0055552339MF I05552F* 15 211In1000g055555/55n52342t13L2M1FFM-+** Date pw.ty 104010 1022220654774604 228 <LOQ <<LLOOOQ <1L02O5O4 99% NA NA 10! % 108% 92% 100% 310 3637..13 23.0 0 0366 0.0498 00432 0.0763 0.0330 0 0546 575738 7805.34 77 9 867 322 6170.18 84 3 221112211603 324240071198 294 321 Dc+DoaratreSteeecVrEtueendmrit/feviireentdndit/f/iBiaBelydyv:::olu000m849e///211l529e///s909s909t,hm0Sa3Amn/1Wh00.,,5/09b099em/f1oLL2rA/e,0Cc00o6Ln/0Ac8eC/n0t0rioToXnRs should be NA NA 3% !6% 002019528 56 1 0 0306 137 10 7 27 6 233 7 49 2522.08 170 considered tentative 04/28/00 LAC EETxcSe-lg9-5T0 3M Environmental aboratory tox-030-sera223-1C 9/12/OC Page 203 3m Medical Department Study: T -62 3 = aC T T O X -030 Report No. FACT TOX-G3G Covance# 6329-223 laboratory Request Number-112279 Study: 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkeys Product N'umbertTcst Substance): T*6295 (PFOS) MMaettrhioxd: /Revision: Analytical Equipment System Numbci Instrument Software/Version: Filename: R-Squaxed Value: Monkey Sera EATmSe-l8ia-40.602a4n9d8.ESToSu-8p-052.00199, &. Madeline 041098 SMeaesAsLttyancxhm3.e2nts See Attachments Slope: Y-Inrcrcept: Date of Extraetion/Analyst: Date of Analysis/Analyst: DSaatme opfleDaDtaaRtaeduction/Analyst: See Attachments See Attachments 04/06/99 RWW 04-07/99. 04/11-99. 04/17/99, 05/14/99. 0517.99 HOJ DRB.MEE Kill 04/09/99. 04/15/99. 04/20/99. 05/17/99. 05 18:99 HOJ DRFVKJH DAYI87 MONKEY SERA Croup Dose Sample * PFOS Concentration Mean RSD Cone of PFOS ng/mL ug/mL or % Ree PFOS ug/mL Std. Dev. MS/MSD RPD Method Blk RBS04069-H20 Blk-1 RBS04069-H20 B2k-2 0.00 14.2 <LOQ <LOQ NA NA Matrix Blk RBS04069-Sera Blk-1 RBS04069-Sera Blk-2 0.00 12.7 <LOQ <LOQ NA NA QC - 250 ppb MKS04069-MS-1 MKS04069-MSD-1 254 246 102% 99% 101% 3% MMKKSS0044006699-M-MSSD-2-2 267 228 108% 92% 100% 16% Croup 1 Control 0 0 mg/kg/day Group 3 Mid-Dose 0.15 mg/kg/day I05520M 105526M I05529F I05549F+ 1I0055552035MM I05539F I05552F 13.6 22.1 39.1 31.7 699 662 63.7 498 0<.L03O0Q0 00..00534713 0.0300 - l Anomaly 0.0457 NA 25.9 00118 82.4 75.8 5 91 79.1 4 67 10.2 105 68.8 3-).5 41.4 Group 4 High-Dose 0.75 mg/kg/day 10551IM 105522M I05533F I05542F 225 185 289 253 237 297 269 248 15.7 267 42.0 5.90 258 15.2 Limit of Quantitation (LOQ): PFOS 15.2 ng'mL Date Entered/By: 04/19/99. 05/10/99. 05'`20 99 LAC Date Verified7By: 08/25/99 SAH. before 06/08-00 TKR Date purity corrected/verjfied: 09-12/00 nunh. 09/12 00 LAC PFOS - Perfluorooctanesulfonate - Semin initial volume less than 0.50 mL. concentrations should be considered tentative. 04 28 00 LAC EETxcSe-l8-957 0 3M Environmental Laboratory TOX-030-sera223-lC 0*M.V2000 Page 204 3m Medical Department Study: T -62C9 orvvincce#TO63X2-90-32023 Report N o . FACT TOX-C30 laboratory Request Number-U2279 PMSrtuoaddnyuxct NumberfTest Substance): TM26-6oW2n9ke5eeyk(PSCFeaOnpSs)ule Towcicy Study with PFOS in Cynomolgus Monkeys MAInnseattrlhuyomtidce/aRnlteEvSqmoutiotpwnmireen/Vt eSryssiotermv Number Filename EAMSTematSseAs-l8iLatUtya0nc06xh2am43n9ed28n.EtsSToSu-p8.50200199. & Madeline 041098 R-Squaxed Value: See Attachments DWSDYDSlaaaa-oltttEmpmeeeeEoooepifffKclADEeepxna2Dttta:8ralaayRctstMaetisod/nOAu/cnANtainolKyansl/EyeAsYnt alSysEt RA 0SS4ee/ee0AA8/tt9tt9aacchhRmmWeeWnnttss 0044//1161//9999..0044//1270//9999,,005V/1147//9999 DHROBJ//DMREBE//KKJJHH Crasf Dm * Sample PFOS ag/mL aCg/--aoiLfc--PeFrtOr%aSflReuec Meta PFOS StAR.3DDev. or/boL MS/M3D RFD Method Blk Matnx Blk QC - 250 ppb Cretip l 0.0Cmogn/tkrgo/lday Creep 3 0.1MSmid*gD/kogs/eday HCifihw-Dpoe4e 0.75 mg/kg/day RRRRBBBBSSSS00004444000018889999----SHHSee22rna00 BBBBQlIlkkkc----1122 MMMMKKKKSSSS00004444000088889999--MM--MMSSSSDD---2-I12 I1I100005555555524226990MPFM*-* 1I100035J355023339MMF*-* 105532F* I10100355555I23I2M3MF* I05542F 007722226.0066468810794000 222145066881...1.10844 635253389568 366 423 <<<<LLLLOOOOQCOQ 00001111..800000000534845162.7758%%%8742894 <LOQ NA <1.00 NA 106% 3% 103% 6% 0 0371 0 0430 000.311003918*72251841 8883.57 836 222634439 84 6 151 86 1 249 43.3196 827..177176 223 236 18.3 Limit ofQuanbtaoon (LOQ): PFOSPFOS * Perfluorooctanesulfonate 15 2 ng/mL Date punry cDDo*aratrSeteeecVrEtueendrnti/uefvirneeeidndb/f/aiBBelydyv:::olu000m498e///121l59e2//s/990s990t,hm0Sa5mnA/1Hh00,,5/09b099em/f1oLL2rA/e,0cC00o6Ln/0cA1eC/n0t0ratTioKnRs should be considered tenuave 04'2&/00 LAC EETxcSe-l89-57.0 3M Environmental Laboratory tox-03O.en223-IC 9/12/00 Page 205 3m Medical Departmen Study: T-62 9&cfTOX-O30 Report N o . FACT TOX-030 Covjnce# 6329-223 laboratory Request Number-U2279 MMAIPFSnitrnlsaueoeattdtdnrnhlyuyuaxo:mmtc:ditcee/aNR:nlteuEvSmqmobwfoetpwnr(m.vTereens/ttVSSeyunsbutsmetma:ncNeu)'mber: RSl-oSpqeu: arad Value: DDDYaaa-ItttneeeioooerfffcAEDepnxatatt:ralayRcnteaiod/Anu/cnAtiaonlnayl/iyAtsntalyst. A2TMEM6-Tm6oSWM2en-l9k8iLe5ae-ey4y(k0nP06SxCF2eaOa43nnp9tS2ds8)u.ElSeToSTu-o8px-50i2a0t0y19S9t.uAdyMwaitdhelPinFeO0S41in09C8ynomolgus Monkeys SSeeee AAttttaacchhmmeennttss 0SS4ee/ee08AA/tt9tt9aacchhRmmWeeWnnttss 0044//1116//9999., 0044/7107//9999,.0055//1147//9999 DHROBJ//DMREBE//KKJJHH Sample Data WEEK 29 MONKEY SERA CDreotuep Sample pros CO(/UmCL. Cm cmtrance vg/m*Lp rero%s Rcc uPM|F/moObLS StdR.SDDev. MS/MSD RPD Method BOc Matrix Blk QC 250 ppb RRRBBBSSS000444C0088S999---SHHe2r3Oa0 BBBDD&cc---2ll RMBMSKK0S4S0004840908-98S-9cM-rUaSSDB-01-c1*2 7.80 701 0.00 0226668070 <<LLOOQQ <LOQ <LOQ 110058%% <LOO NA <LOO NA 106% 3% MKS040S9-US-2 249 101% MKS04089-MSD-2 264 106% 103% 6% 0.0CCmorgnm/tkrgpo/llday iI10I0050555555252240969MFMF** 43648482...2771 0000....0000767643397980 0 0665 0 0742 5 45 00000080093664624 0.1M5Gmirdog-u/DkpagtJ/eday I10I10050555555250533529MMFF* 664511637 432 7747J2 761799 75 8 69 9 228188 116124 Gmg 4 0.7H5igmhg*/Dkgo/sdeay 1II00Q555555213213MMF 105542F 635548461490 221707075 180 223 194 300 696B93 19.0 Limit of Quantitation (LOQ): PFOS- 15.2 ng/mL Date Entered/0y 04/19/99,05/10/99 LAC Date Verified/ By 08/25/99 SAH, before 06/08/00 TXR Date punty coiTected/venfied: 09/l2/00mmh,09/12/00LAC PFOS Perfluofooctanesidfonate * Serum irutul volume less than 050 mL, concentrations should be considered tentative. 0478/00 LAC EExTcSe-l89-57 3 3M Environmental Laboratory tox-03O-scra223- l C 9*12/00 Page 206 3m Medical Department Study: T -62 9 p & c TOX-030 Report N o . FACT TOX-030 Covanct# 6329-223 laboratory Request Number-U2279 Study: 2dWeak Capsule Toxiaty Study with PFOS inCynomolgus Monkeys Product NumberfTest Substance). AMMInnseiatttnrhluyxomt:dic/eaRnletEvSqiosuifoitpwnm:aeren/tVSeyrnstoenm Number TAMEM-Tm6aoSs2ne-a9hk8L5ae-y4y(0nP06SxF2eaO34nn9S2d8).ESToSu-p8-05200199. k Madeline 041098 FDename: See Attachments RSYl-o-Slpnqetu.earreeedptValue: DDaattee ooffAExntraalcytsiotAn/nAanlyiisytit DSaatme opfleDaDtaaRtaeduction/AnaJyit: 0SSS044eeeeee//01AAA81//ttt99tttaa9a9ccc.hhh0Rmmm4W/eee1Wnnn7ttt/sss99.0V14/99 DRB/MEE/KJH 04/16/99,04/20/99,05/17/99 HOJ/DRB/KJH WEEK 30 MONKEY SERA GDraawi*p Method Blk Sample RRBBSS004400B899--HKI2O0 BBilkk--12 PFOS nC77jg.08w/10aacL ttC|/oaoafc<LepLeOrtrroQaSstSReaar <LOQ uPMP/emOanSL StdR.SDDev. MS/MSD RPD <LOQ NA Matnx Blk QC 250 ppb RBS04089-Sera BQc-1 RMBMKSK0SS40004480098B-99S--MeMnSSBD-1l-kI-2 MMKJSC0S4480989-M-MSSD-2-2 0022.6.66080700 226449 <LOQ <11110100058160%%%%0 <LOQ NA 106% 3% 103% 6% 0 0CCmrogen/Votrfgo/l1day 111I0000535555522426099MMFF 33221290.4551 0000..0000233391019264 00313 0 0303 00..00100.0061406855 0.1M5Gimrda*guD/kpogs3/eday Grwp 4 High-Do 0.75 mg/kg/day I05505W 11I10000055555555552321529312MFMMF* J10055553432FF 418 43524450* 346437161056 666198469 511114617576065 7.06 65 2 417.6.10 62 0 2160 65 143 162 347888607 Limit of Quantitation (LOQ): PFOS - 15.2 ng/mL DDaateteVEenrtiefireedd//BByy: Date punty conected'vmfied: 000894///211529///909909,m0S5mA/1Hh0.,/09b99e/f1oL2rA/e0CQ06L/OAVCOQ TKR PFOS " Perfluorooctanesulfonate * Serum initial volume less than 0 50 mL. concentrations should be considered tentative. 04>'2&'OO LAC EETxcSe-l8-951 0 3M Envi ronmental Laboratory tox-030-seim223-lC 9/12/00 Page 207 3m Medical Department Study T-62 9 & ,c ?TO X -030 Report No. FACT TOX-030 Covance# 6329-223 laboratory Request Number-U2279 ASMIPMntrnuseaoattdtdrrlhyiuyuox:tmc:ditc/eaRNnluteEmvSqiosbuifcoitrpnw^m:TaeerensI/tVSSeuynbsitsoetmarncNeu) mber 26 Week Capsule Toxicity Study with PFOS in Cynomoljtui Monkeys TAME-Tm6oS2ne-9lk8i5ae-4y(0.P06SF2eaOr4na9Sd8).ESToSu-8p-50200199. & Madeline 041098 MuiLynx 3.2 FRSDSYDDil-aaaa-loSeltttmpneeenqetauoooepmrafffclreeDEAee.pdrnaDtttar:VaalaycaRtsolauieosedt/:VAucAntainolaynlsy/tA:stn: alyst 000SSSS444eeee/ee/ee/01tA6AAA81///tttt999ttttaaa9a99cccc,,0h0hhhR4m4mmmW//1eee2eWn7nnn0tt/tt/ss9ss999.,0055//1174//9999 HDORJB/D/MREBEO/CKJJHH WEEK 31 MONKEY SERA GDraaeup Sample# nPCgFa/OmseSL Ceocaatrade* (/mefLPeFrOSS Rac PtgF/OasSl. M5S/MtdR.SSDDOrvR.PD Method BIk Matrix Blk QC - 250 ppb OOCCmrogan/aktrpgo/l1day 0.1M5Gmirdag-sD/kpogs3/eday 0 7H5Cimgrahgw-/Dkpog4s/deay RRRRMBBBBMSSKSSJO00S0C44440S000400S880t4999980----9HHSS-9M22ec-OOnnMSDBBSBBIDl--lkkk11c----1212 MMKKSS0044008899-M-MSSD-2-2 W1II0005555555542229609FMMF* 1I0055552035MM 1I0055555329FF*11I000055555555422312F13-MMF*- 770810 0022606680070 224694 2376..17 2254.35 325599981180 363599961888 <LOQ <<LLOOQO <11100580%%0 <LOQ <LOQ 106% NA NA 3% 110016%% 00.00335642 00.00425832 64510831...1062 103% 0 0358 0 0368 566 79.6 6% 000301021052598)67 84 4737 4535 80 211109721409 28 6 161 >85 42I1I6 981 Dale Entered/By 04/19/99 LAC PFOS - PerOuorooctanesulibmte Due punty Dc*oartrSeeecVrtueemdri/6vienednit/fiaiBeldyv::olu00m98e//12l25e//a09s09thmSamnAH0h,,50b09em/f1oL2r/,e0c00c6mL0Ac8eC0n0traTnKorRs should be considered tentative 04/28/00 LAC NA Not applicable Extraction Volume Ratio " Initial Volume/Final Volume EExTcSc-j89-750 3M Environmental Laboratory tox-030^oi223*lC 9/12/00 Page 208 3m Medical Department Study T - 6 2 3 & C ? TOX-O30 Report N o . FACT TOX-C30 Covanee# 6329-223 laboratory Request Number-U22 7S Study- 26 Week Capsule Toxicity Study with PFOS m Cynomolgus Monkeys Product NumbertTest Subitanee). T-6295 (PFOS) MMAnaeattrhliyxot:dic/aRleEvquuioipnment System Number: Instrument Softwere/Veroon. AMETmoSne-lk8iae-y406SI2e4aran9Bd ETS-8-3.1 MauLynx 3 3 Filename: R-Squared Value See Attachments See Attachments Slope: Y-Intercept DDaattee ooff EAxntariayc*ti*o/nA/Ananlaylsytst: Date of Data Reduction/Analyst 0SS8ee/ee2A5A/tt9ttaa9cchhRmmWeennWttss 09/28/99, 10/05/99, 06/14/00 MEE/MMH 09/29/99. 10/0^99. 06/15/00 MEE/IAS'MMH Sample Data WEEK 35 MONKEY SERA Creep Dose Sanpla 0 PFOS Caacentration Mean RSD Ceec. fPFOS PFOS SU. Dev. (/nL oc/aiLer H Ree mg/mL MS/MSD RPD Method Blk H20 Blk-1 H20 BOc-2 0.00 000 <LOQ <LOO <LOQ NA Matrix Blk QC - 250 ppb MRRaaKbb3bb0iitt8SS25ee9nn-MBBDISkc---12l MKS05289-MSD-1 00.0000 227 254 <LOQ <9L1O%O 102% <LOO NA 97% 11% MMKKSS00J8228599-M-MSSD-2-2 223596 19063%% 100% 7% Creep 1 Control 0.0 tr^A^/day 105520M I05526M 1I00555S4299FF 27 3 35 9 74.7 61.0 0.0437 660 0.0480 0 0459 0 00303 00..00764988 486 0 0723 0 00352 Creep J Mid-Dcee 0 15 mg/kg/diy I05505M* I05523M I05539F* I05552F- 118960 122 135 93 0 76 l 81 2 84 5 1U2 01 12 8 67 7 74 4 9 53 HCigrhe-eDpo4se 0.75 mg/kg/dey 105511M 1I0055552323MF 105542F 41? 486 444048 167 195 116748 181 171 511139394851 Lrrut of Quantitation (LOQ) PFOS - 5 33 n^mL Date Eniered/By: 1005/99, KV07/99, 06/19/00 LAC Date Verified/ By before 06/08/00 TKR PFOS Perflucrooctanesulfonate Date purity c*orrSeecrtuedm/viennitfiaieldvolum09e/1le2s/s00thmanm0h,3009m/1L2./0c0onLcAeCntrations should be considered tentadve 04/2800 LAC NA " Not analyzed, not applicable Extraction Volume Ratio Initial Volume/Final Volume EETxcSe-l89-57 1 3M Environmental Laboratory tox-030*erm223-1C 9/12/00 Page 209 3m Medical Department Study T -62SfccfT O X -030 Report No FACT TOX-C30 C ovance#6329-223 laboratory Reques Number-U2273 Study 26 Week Capsule Toweity Study with PFOS in Cynomolgus Monkey Product NumberiTest Substance) T-6295 (PFOS) Matrix: Monkey Sera MAInnseattrhluyomddc/eaRnletEvSuqoiuofitnpw.maeren/tVSeynsitoenm: Number AEMTmaSue-lL8ia-y40n6x123a4n93d8 ETS-8-5.1 Filename: See Attachments R.Squared Value See Attachments SYl-olpnetercept. SSeeee AAttttaacchhmmeennttss ' Date of Extraction/Analyst: Date of Analyst*/Analyst: Date of Data Reduction/Analyse 08/25/99 RWW 09/28/99, KV05/99. 06/14/00 MEE/MMH 09/29/99. lQ/06/99. 06/15/00 MEE/IAS/MMH Sample Data WEEK 39 MONKEY SERA Craap Date Sample* PFOS C a|/bieL. Concentration Of/aarLPeFrOHS Rc Mean RSD uPgF/OmSL MSS/tMd.SDDevR.PD Method Blk H 20B a-l H20 BOt-2 0.00 0.00 <LOQ <LOQ <LOQ NA Matrix Blk RRaabbbbiitt SSeerraa BBfllkc--12 0.00 0.00 <<LLOOQQ <1.0Q NA QC - 250 ppb MKS08259-MS-1 MKS05289-MSD-1 227 254 91% 102% 97% 11% MMKKSS0058228599-M-MSSD-2-2 223596 19063%% 100*4 7% Croup 1 Control 0 0 mgfcg/day 1I00555522C6MM I10055534299FF* 23 7 29 l 3215 47 00..00341666 0.0503 0.0514 27 00391 00106 1 53 0 0509 0 000779 MGirdo-uDpoeJe 0.15 mg/kg'day 1I0055552035MM++ 105539FI05552F- 321117 114742 62.4 5 26 579 568 60 1 314161 46.5 5) 7 7 29 0 7H5Gigmrhog-a/DVpoga4/eday 1I0055551221MM** II0055534323FF-- 227306 233380 113558 115649 11 0 1-16 616861 161 1! 1 Limit of Quantitation (LOQ) PROS - 5 55 ng'mL Dale Entered/By: 10/05/99. 10/07/99, 06/19/00 LAC Dale Verified/ By: before 06/08AW TKR Dale punty corrected^verified: 09/12/00 mmh. 09/12/00 LAC PFOS * Perfluorooctanesuifonate * Scrum mmal volume less than 0.30 mL, concentrations should be considered tentative. 04/28/00 LAC NA " Not analyzed, not applicable Extraction Volume Ratio * Initial Volumc'Final V olum e . ETS-8-5 1 Excel 97 3M Environmental Laboratory tox-030-sera223-lC 9/12/00 Page 210 3m Medical Department Study T -62 TOX-030 Report No. FACT TOX-C30 Covance# 6329-223 laboratory Request Number-U2279 StudyProduct Number{Te>t Substance) AMMnaeattnlhyxot:idc/aRleEvquuioinp.ment System umber Instrument Sofrware/Venion: 26 Week Capsule Toxicity Study with PFOS ui Cynomolgvu Monkeyi T-6295 (PFOS) MEAMTmoa5nue-lkL8iae-yy40n6xSi2e3a4ran93d8 ETS-8-5 l Filename RSDYl-a-oSItpneqelu.oearfcrEeedpxtt:VraaclOuoet:VAnalyst: Date of Anatywii/Analyst Date of Data Reduction/Analyst: See Attachments See Attachment See Attachments See Attachments 0089//2285//9999, R10W/0W5/99.06/14/00 MEE/MMH 09/29/99, 10/06/99, 0^15/00 MEE/1AS/MMH Sample Data WEEK 43 MONKEY SERA Groap Sample n Deea PFOS Cese. rtg/mL Concentration or PFOS ugtoL ar H Rec Mpreoans RSD Std. Dev. hr/qiL MS/MSD RPD Method Blk Matrix Blk QC - 250 ppb GCroonutrpol1 0.0 mg/kg/day MCirde-Depo#3 0.15 mgfcg/day HH2200 BBDfltt--12 Rabbit Sera Blk-1 Rabbit Sen BIk-2 MMKKSS0058228599-M-MSSD--1I MKS08259-MS-2 MKS05289-MSD-2 110055552260XM4 1I0055554299FF 10055552035MM I05539F I05552F* 0 00 000000 020270 254 256 239 21.5 2312..82 22.9 162 111426 116 <LOQ <<LLOOQQ <19L012O%VQ. <LOQ NA <LOQ NA 97% 11% 103% 96% 100% 7% 0.0287 00330 00..00336678 0 0319 . 0001203548140 0 0368 0 0000923 4446 95 5588.30 2.44 45 7 58 1 0012.4142299 HGigrho-eDpo4se 0 75 mg/kg/day 105511M 110055552323MF I05542F 167 226 447 347 9606.97 117399 214 78 8 1167.88 159 28 4 Limit of Quantitation (LOQ) PFOS * 5.55 ng/mL Date Entered/By 10/05/99, 10/07/99, 06/19/00 LAC Date Verified' By: before 06/08/00 TKR PFOS Perfluorooctanesulfonate Date purity c4orrSeecrtuemd/wuuntfiiaeldv:olum09e/1l2e/u00thtarnm0h.,5009m/1L2,/0c0onLcAenCtration* should be considered tentative 04/2&/00 LAC NA = Sot analyzed, not applicable Extracoon Volume Ratio Initial Volume/Fmal Volume ETS-B-S I Excel 97 3M Environmental Laboratory tox-030-era223-lC 9/lZ'00 Page 211 3m Medical Department Study: T -62 TOX-030 Report N o . FACT TOX-030 Covance# 6329-223 laboratory Request Number-U22 79 SPtruoddyu:ct Nuinber(Teit Substance) 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkeys T-6295 (PFOS) AMMlnnaaeatttrrlhiuyxomt.idce'alnllctEvSquoutfoitnpw.mareen/tVSeynsttoenm: Number Filename: METoSn-k8e-y4 Slearnad ETS-8-5 1 AMmasesliLay0n6x234.938 See Attachments R.Squired Value: SYDDl-aaolttpneaeteoorffcEAepxnttraalcytsitoAn/nAanlyaslyt:st: Sec Attachments SSeeee AAttttaacchhmmeenntts! 0089/7258//9999, R10W/0W5/99, 06/14/00 MEE/MMH DSWaatEmeEpofKleDDa4t7aa Reduction/Analyst; taMONKEY SERA 09/29/99, 10/06/99,06/15/00 MEE/IAS/MMH C risp Dm Sample a PFOS Csac. ag/mL Concentration of PFOS of/mLor % Rcc Mesa USD PFOS Std. Dev. ug/mL MS/MSD RPD Method Blk H20 B3c-1 H20 Bfc-2 OX OX <LOQ <1.00 <LOQ NA Matnx Blk Rabat Sera 30c-1 Rabbit Sen Bflc-2 ox ox <LOQ <LOO <LOO NA QC - 250 ppb MKS082S9-MS-1 MJCS05289-MSD-1 222574 19012%V. 97% 11% MXS0S259-MS-2 MKS05289-MSD-2 256 239 103% 96% 100*/. 7% Crisp 1 0.0Cmogn/tlcrogl/day [05520M I05526M 105529F I05549P 2275..74 36.0 32.6 0.0339 0 0370 0.0481 0.0436 6 23 0 0355 0.X7 02421 0.0459 0X323 C riap J 0 1M5 imdg-DAocgse/day I05505M 105523M 1I0055555329FF 111542 111148 445570.974 37 9 7 65 48 3 426 631.567907 Crap 4 Htgh-Dcac 0.75 mg/kg/day 105511M I05522M+ II0055553432FF 263 212 321727 105 21 0 110442 92 124 259 98 3 88 4372 Limit of Quanbtafion (LOQ) PFCS - 5 55 ntfmL Date Entered/By 10*05/99, 10/07/99. 06/19-00 LAC Date Verified/ By: before 06/08/00 TKR PFOS =*Perfluorooctaneiulfonate Date punty c*orrSeecrtuemd/winnitiaeldw: him09*/1le2u/Xthmanm0h,5009m/1L2,;XconLcAenCtrations hould be considered tentative. 04,7^00 LAC \ `A " Not analyzed, not applicable Extraction Volume Ratio * Initial Volume/Final Volume EExTcSe-l89-57 1 3M Environmental Laboratory tox-O30-tera223- 1C 9 /12/00 Page 212 3m Medical Department Study: T -623 & C ? TOX-030 Report N o . FACT TOX-C30 Covance# 6329-223 laboratory Request Number-U22 79 Study. Product Number{Test Subatance): AMMInnsaeatttrrlhuiyxomt.dice/aRnlteEvSuqoiufoitnpw.mareen/tVSeymstoenm: Number Filename R-Squared Value Slope: YD-ellneteorfceEpxtm: caon^Analyst: Date of Analysis/Analyst: Date of Data Reduction/Analyst 26 Week Capsule Toxicity Study with PFOS m Cynomolgui Monkeys TEM-T6oSn2-k98e5-y4(PS1FerOanSd)ETS-S-5 I AMmuesbLayn0x6234938 See Attachments See Attachments See Attachment! See Attachment! 0098//2285//9999, R10W/0W5/99, 06/14/00 MEE/MMH 09/29/99, 10/06/99, 06/15/00 MEE/1AS/MMH Sample Data WEEK 51 MONKEY SERA Grasp D*u Sample PFOS Cane. ng/mL Ceaceatretlen of PFOS ag/m LorH Rec Mean PFOS og/aL MSS/iMdR.3SDDDevR.PD Method BUc Matrix Blk QC - 230 ppb HH2200 BBllkk--21 Rabbit Sere Blk-1 MRaKbSb0d8S2e3r9e-MBlSk--12 MKS05239-MSCM 00.0000 0.00 02.2070 254 <LOQ <<LLOOQO <9L1O%O 102% <LOQ NA <LOQ NA 97% 11% MMKKS30058228599-M-MSSD-2-2 225369 19063%% 100% 7% Grasp I 0 0Cmogn/tkrgo/lday I05520M 105526M 1I0055554299FF 1193 53 324936 0.0213 00261 00..00334348 14 0 0 0237 0.00333 00341 0.010.014803 Croup 3 Mid*Doe* 0 15 mg/kg/day Gr*p 4 High*Doae 0 75 mg/Vg/day I03505M 103523M 1I0055535329FF* 10351 105522M II0055333432FF 119 108 15121 135 365 226817 39 8 36 0 4254.38 6.92 37 9 327.626 35 1 13.2 67 5 122 94 7 4308 64 87.1 95.7 9! 4 66 0657 Limit of Quantuaoon (LOQ): PFCS - 5.33 ngr'mL DDaateteVEenrtiefireedd//BByy:; b1e0f/o0r5e/9096,/0180//0070/9T9K, .0R6/19/00 LAC Date punty correcled/vtnfied: 09/12/00 mmh. 09/12/00 LAC PFOS " Perfluorooctanesulfonate * Serum initial toKune leu than 0 50 mL, concentration! should be considered tentative 04/28/00 LAC NA Not analyzed, not applicable Extraction Volume Ratio " Initial Vohune/Final Volume EETxcSe-l89-57 1 3M Environmental Laboratory tox-030-iera223-1C 9/12/00 Page 213 3m Medical Department Study: T -62Sfoc?t o x -030 Report N o . FACT TOX-030 Covance# 6329-223 laboratory Request Number-U2279 Study: 26 Week Capsule Toiaafy Study with PFOS ui C^nomolgui Monkeys Product Number(Test Substance) AMMInnaseatttrrhliuyxomt:dic/eaRnletEvSquouifeitpnwm.areen/tVSeyrnstoenm: Number TMESMo-Tao6uSan2pa-k908Le52-yy40n(1PSxl9Fe39aOnn3Sd)ETS-8-5 1 FRi-lSenqaumaree:d Value DDSYlaa-oIttpneeet:ooerffcAEepxntta:riaycmtio/An/nAanlyaslyt.st DSWaatmEeEopflKeDDaSt3aatRaMedOucNtioKn/EAYnalSysEt RA See Attachments SSSeeeeee AAAttttttaaaccchhhmmmeeennntttss] 111111///000348///999999., S1MA1''L1ll2//9999,, 1111//1282//9999 MIAMSH/1AS CDramup Sample * PFOS Caaceatratian nCge/nuciL. agriaaLf PaFrOHS Rec JO B a fc i StdRSDDev MS/MSD RPD Method Blk K20 Blk-5 H20 BQc-5 0180370 <LOQ <LOC <LOQ NA Matrix Blk QC - 250 ppb Rabbit Sera Blk-5 MRaKbbSi1t1S0e3r9a-MBlSk--56 MMMfKCK3S.S1l1111000333999--M-MMSSSDD-*5-5i 2.20 222339.978540054 <LOQ 0<.L02O3O7 0 0236 <LOO 0 0236 0 000N01A092453 111142%% 113% 2% 0.0GCmrogan/atkrpgo/l1day (I0055552206MM I100555S2499FF 33.6 446898 52.3 00.00329629 00.00431759 0 0331 02060.186 0 0397 007083211 MGirda*aDpos3e 0.15 mg/kg/day I05505M 3II000555555523239FMF+ 112019 18013 43 9 7.15 48 5 3421.13 46 2 36 7 6317.23400 Craup 4 High*Dose 0.75 mg/kg/day 110055552121MM* II0055553432FF 216377 224446 514078 997869 45 5 80 8 9R2 003644.99809 Limit of Quantitation (LOQ) PFOS * 5 55 ng/mL Date purity DcDoaratreteecVEteendrit/fevireeedrdi/f/BiBeyyd: 0b19e1/f1/o12r5/e0/90096m,/10m18/h/20,400/99T9/1K2LR/0A0CI.AC PNFAO"S N"oPtearpflpuloiccaobolcetanesulfonate * SAerluthmouingihtialalbveolleudmue lMeuS/tMhaSnD0-.65,0thmeLse, cwoenrceennotrtatsipoinksedshaonudldwbilel bceonussiedderuedbtleanntkatsiveLA0C4/2118//1050/9L9AC Extraction Volume Ratio * Initial Volume/Kwal Volume EETxcSe-l89-57 1 3M Environmental Laboratory tox-030-en223-lC 9/12/00 Page 214 IQ O 3ra Medical Department Study T -62SJVCf TOX-030 Report No. FACT T0X-G3 Covince# 6329-223 laboratory Request Nuraber-U2 27 Srudy" 26 Week Capsule Toxicity Study with PFOS in Cynomolpus Monkeys MPraotdriuxc:t Number(Teil Substance) AMInnseattrlhuyomtdice/aRnlteEvSquouifoitpnwmaeren/tVSeymstcetmv Number T-6295 (PFOS) MESMoTaouSsnps-k80Le-2yy40n1Sx19ea39rna5d ETS-8-5 1 Filename: See Attachments R-Squared Value: SYl-oIpnetercept See Attachments See Attachments See Attachments Date of Extraction/Analyst DDaattee ooff ADnataalyRsee/dAocn&aloyrsVt:Analyst. 111111///000348///999999,, S11A11//L1112//9999,, 1111//1282//9999 IMAMS H/1AS SWaEmEplKe D57ataMONKEY SERA GDreseuep Sample * PFOS nCge/mneL Concentration g/aeifLPnFrOHS Rec Mean oP(F/OmSU StdR.SDDev. MS/MSD RPD Method Bik Matrix Bik QC - 230 ppb H20 Bik-5 RabHbi2t 0SeBraikB-5lk-5 Rabbit Sera Bik-5 MMMM1KCKK-SS.SS11H11101CC30339399-9-M--MMMSSSSDD--56--65 021.8203070 2223399.875.40504 <<<LLLOOOQQQ 0<0L2O3Q7 011.101422%%36 <LOQ NA <l,OQ NA 0 0236 0 00001094253 113% 2% 0.0GCmroegnuAtrpgo/l1day I05520M [11000355555224699MFF 36.1 453258.194 0.0289 000...000344671428 16 1 0 0327 0 00526 0 0445 0 80063385 Cmup J Mid-Dose 0 15 mg/kg/day II0055550253MM I05539F I05552F 7719.14 83 1 78 4 2318.85 3313..43 30 : 72 8336 4 13 32.3 134 HGigrh*-uDpo4se 0 75 mg/kg/day I10I00555555213213MMF* I05542F* 166 222725138 6869.55 110039 78 0 106 2106 83 33 6834 Limit of Quantitation (LOQ): PFOS ' NPFAO"S N=oPtearptJpuloicraobolcetanesuJibnate 5 55 ng'tr.L Date punty Dc+DoaratrSeteeAecVrEltuteehnmdroti/e5uvirengetdinhdt/if/alBiBalebydyveolleudm0b1a9e1esf//1o1lMe25reu//S9009/0tM^h,m0aS1n8m1D/0/0h2-.6,054,/009t97h9m/Ke1Ls2LRe,'tAXcwCo)enLrceAennCtortatsipoinksedshaonudldwbilel bceonussiedderaesdbtelanntaktsiveLA0C4/7181//1050/9L9AC Extraction Volume Rato Initial Volumc'Final Volume EETxcSe-l89-57 1 3M Environmental Laboratory tox-030*era223-lC 9/12/00 Page 215 3m Medical Department Study T - 6 2 % ^ c ?TOX-030 Report N o . FACT TOX-030 Covince# 6329-223 laboratory Request Number-U22 79 SPtruodduyct Number<Te*l Substance) MMAnaeattrlhiyxot:idc/aRleEvqwuoipnment System Number: Instrument Software/Venion RFi-lSenqaumaree:d Value: SYl-oIpnet.ercept DDDSaaaatttmeee ooopfffleAEDxnDatatralaayctRxatWieodnAu/cAntainolyanls/ytA.stn:alyst: 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkeys ESTMMSoeT-oa6euSsn2pAi-k9L085et:y-ty0a4n(1cP.Sx1h9Fem93arOane3Sdn)tEsTS-S-5 1 SSSeeeeee AAAttttttaaaccchhhmmmeeennntttsss \11111///000843///999999,, SU1A1//1L112//9999,, 11W1/2128//9999 LMAMSH/IAS WEEK 61 MONKEY SERA GDroamsp Sample U aPCgFa/OanseSL. CoaceatraUe* mffmaLf PaFrOHS Rec uMPgFe/OaanSL MSS/UMRS3DDDevR.PD Method Blk Matrix Blk QC - 250 ppb Group 1 00 mg/kg/day H20 Blk-5 RabHbi2t 0SeBrIak-B5lk-3 Rabbit Sera Blk-5 MMKKSSl U1003399--MMSSD-6-6 MMKSSl111003399-M-MSSD-5-5 II0055552206MM- I10055552499FF 0 830 21.2070 22239.975.4504 380 4379.87 5347.07 <<LoOoQ NA A 8 <LOQ 00<..L002O233O76 <LOQ 0 0236 NA 0 000.10902453 111124%% 00..00338129 00..0044363 113% 2% 12.8 00351 0.40064849 0 0448 0.00210 MGirda-Dspos3e 0 15 mg/kg/day II0035550253MM II0055553592FF* 689845 9943 88 27 4 3358.09 38 4 31 6 051.7894982 38 2 0.283 0.7H5Gigmrhag-aD/pkoga4/eday 110053552U2MM 110055554323FF 322173761392 64 7 111030869 100 109 5500..23 00 669470 Limit of Quan&taQon (LOQ) PFOS - 5 55 ng/mL DDaateteVEenrtifeireedd//BByy:: b1e1f/o15re/9(9W, 0181//2040/9T9KLRAC Date punfy corrected/vcnfied 09/12/00 tnmh, 09/12/00 I.AC PNFAO-S S- oPtearpOpuliocraobolcetaneaulfonate - SAerluthmouingihtialalbveolleudmaeslMeaSs/tMhaSnD0-.63,0thmeLse. cwoenrceenntortatipotnksedshaonudldwbilel bceonusaiedderaesdbtleanntaktsiveLA0C4/1218//1050/9L9AC Extraction Volume Ratio " Initial Vohime/FinaJ Volume EEx7cSe-l89-57 1 3M Environmental Laboratory tox-030-sar223- 1C 9/ 12/00 Page 216 3m Medical Departme: Study T -6 29Pa c t t o x -030 Report N o . FACT TOX-030 Covance# 6329-223 laboratory Request Number-U22 79 Study 16Wsek CapsuleToxicityStudysnth PFOS in Cynomolgus Monkey* Product Numbr(Tcft Sobetance) AMMIFninlsaceatttnrrlhuiyaxomtmdicee/aR:nlttEvSiqoitufoitnpw.minen/Vt Stnywutmrm. Nu-nMt SRDYl-eo-SLlpeqnu:to*affrcaEdJptaVtraaeJuDao: /Analyst WDOSuaotEtmeEoopfKflDAean6Dta5alayRtiMatefd/AOacnNtaiolKyni/tEA. nYalySeEt RA SCSSSTRMMEmmmMe-Tuo6eubS/2n7eAAyAA-49kL8/5nttte-10yttt4yaaaa00n(.cccc0P1Sxhhhh4FSmmmm9a3rOAv9neeee3SLdnnnn)/ttttFKasssTJKS-8-5 1 0044//2275//0000,,0034//0217/A00.,0034//0218//0000,, 0055//0043//0000 IlAASS/MMH'ADV GDrouwmp Method BUt Matnx 01k QC - 250 ppb O.OGCmroagns/tkrpog/l1day Sample MRRBB0K04SS4S20020444446O222-44-H400H0--22SS-OS0eeerrBaaantlkkBBB--lll34kkk---433 MMMMMKKKKKSSSSS000004444422222444440000O>---SS-SSSeeaaearsnan-s--U-MMBMSJSSDSk---04-3434 IK1Q0333S3535522420699MMFF . 000tpC28g.3936or/7039m43os000eLs. U3332023166600 32111442....1577 Cla/aaocLfapourrtroHastioRuee 00<<<<.11109.LL0LL2212007O2900%709%%%QQ5Q050 0000....0000231336856447 aMpgr/esostaLs MSS/iMdR.SSDDDevR.TD <LOO MA cLOQ 0.00852 NA 001061.944 t07% 27% 123% 167%4 00210 0 0360 000.0010043356232 0.1M3Gimdra-gD/skepge3/edey 0.7H3Gigmrhag*s/Dkpoge4/dsay Lumi ofQuantitation (LOQ) PFCS PNFAO"S M" oPtearpflpulcicraobolcetaneeullbnate * 5.351rIeIIIOI0010g00C503-55f33m55355i253l420Ll235323M5M92FFMMFF**** Date punty 232164091438 33336292.0399 32 9 37 6 245363823474 85719263.1075 91 5 82 8 cDDe*aratretSeaceVtrEauendmrit/faviireansdnidt/fi/iaBBa]dyyv:.ota000m569e///001l322e///s000t000t,hm0Ta3nmK/0hR09,3/00090m/1L2I^/,0Ac0CoLn'CcASeCHntnooni Extraction Volume Rat " Initial Volume/Tinai Vohim 006269..51000..5113826..3782826209 ihouJd be considered tentative. 04/28/00 LAC EExTcSe-l89-57 1 3M Environmental Laboratory tox-030-Mft223-lC 9/12/00 Page 217 3m Medical Department Study T - 6 2 9 Tox-030 Report N o . FACT TOX-030 Covmncc# 329-223 laboratory Request Number-U2279 MMAFPISnirtnmlceuoeattddntrhlyyuuauo:tmmcdi-ct/eeaRNn:letuEvSmqiaouboficptnwrm:tTvetne/tsVSScyumwboormnunNceu)mber 12*64W29e5ek(PCFeOpScu)le Tonaty Study mth PFOS mCynomalguc Monkeys METoSn-kBe-4y SIennd ETS-B-5 1 RMSeucuby*A.t1yta0rc0wh6m9J93ent! DDDRSYSWl--uuaoulSpEeermsuqeoooE*:upfrffuKltDE*AeepdanD6trua.aV9lCcayafDtUtulrruMaaootdefntuuAe/OcpAnUNaMolycdKv*y/EAYjulySE. RA Sample * 0<0SSS4W4cee//ec22734AAA7/A'0t0ttttt00aaa..ccchhh0OSmm4mAS/^ee2eLtnnn7./'tKttO/*s0!JO0K., 0O4S/^3lA/0X0).. OOVSOWi/OOOO IlAASS'MMH'ADV opCfor/toeacsL. uCt/ouoLfcepourrtroHesttoRde< ipMmreoeLns MSS/tMdR.SSDDDevR.PD Method Bli MMru 31k QC . 250 ppb 0 0CCmroo^nuktrpfo/ld1ey 0.)M5Cmirdoj.yuDYpofSec*'day 0 7K3Cifmrho-guD/pko^e4deay MMMMMMRRKXBfKKKX00SiSSSS4SS4S002OO&04034044242044O40242422-2.OH4Q04H4400-00-0-.22SS.S.S--OSS0ScSeecUcrrrmnBvBnvan1.lMZ.MMkMBBBBk*S-llS3llS4SkkkkDD------434-433.43 1IIII101III10000000003003555555355535SS553S5523J220232345124035439992IM2MMMMMFFFFFF++ 000212333..133.913021709.36444050000 2U324443333549202544672.S9.1449113452 000000<<<<0111..942333770.LLL0101021200721140422432OOO.93?37..%.9140902797036%%3Q3QO03410904 <LOO NA <ioo 0.00852 0.01N06A1944 107% 27% 123% 00406 00400 264 34 5 St 0 75 0 00073670792551602.0.02420755%B334063014290103] Lima ofQuuttaMion aOQ): PFOS - 3.52 n*mL DDuueeVEenrilfeireedd//BByy: 0054//0032/-0000, 0T5X/0R9/00 LAOCS1I PFOS * PerflocroocUneruifontfe Dae puncyco*mSceurdum/vemnmfiaeldv:olu0m9/e12le/00tmhamh0, 5090/1m2L/0.0coLnAcCeniruon* should be considered tentative 04-2400 (.AC ETS-e-i l Eatel 97 3M Environmental Laboratory Ioi-430-em223-1C 9-1100 Page 218 3m Medical Department Study T - 6 2 9 :j>A(3r t o x -oo Report No. FACT TOX-030 Covante &329-22J laboratory Request Number-U2279 MYPUFRSDDDAlSSWilirut-nioaaa-luotlattoaSr*p**aEkdrdnuuluyoqyupooovxE:ztcdurnffinflttc/Kanc*EeDAKaN:npdlxnialtuD7vEt:a*VrSm3uqtaayRatoucbtointiaaciMlipnuadohrm:/:fonAmTOca/UAMnwN/kwnVySaKSVaynyuAE*aboneaYnamwi.yeSN*Eu):mRbAer CDrmoup Saapk > MahodBlk Manx Blk QC 230 ppt> 0.0CCmroo^nuktrpfo/dl1uy MMMMMMRRKXKBKBXKOSO3SSS3SS40040200040O42U44W3444M4!3JO212MO30CD344OC-44iJ-35H5O00OK002--25S--SS2---24S-32SSS0S99*M*Mrc*reFFmrr*BvrBn+na-Ml--MtkBBMMtBB--SlSl34llkkSSkDkD-----34-4-33443 02TMERMSS5S46t-Tu*a/*o*2bWS2an4AAyAASt-kL/l30fCBar-1iyty40aak0xa(rcrP0cruSthCIiShFaum3AzanOn*nnpua3caLSenafntTti)tiueuaElOaT*TSo-x-ic5it1y Study wih PFOS in Cynomolgua Monktyi 0044//22M7/J0.. 0O4iO/2l7.'O/0O0,. WOV/2OSl//O0O0,. 0OVVOOV*OO0 IlAASS'NiMH'ADY 000uCp22221331132.3.92427702413o13r93..../0054v17o0a0eLs. vC/N*o00LCf<<0000<<1I19...0LLLLp2000M21t2073OOa23OO279rr9H453%%HloQOQO5%Sn94I04sURteie upMtr/emoansL MSS/MtdR.SSDDDrR,PD <LOQ NA <LOO 0 0052 0 N116A944 107% 27% 113% 6% 0 0350 0.0345 00.3700021721*54 0. lM5CmxrIoj-ADupo/m3d*y 0.7H5Cimtrhoj-u/Dkpso/4wdy Lun* of Qumitaion (LOQ) PFOS - NPFAO*SNoPt*arpfpluliocraobolectai**uifonac 3 55 1I10000355S3555250332S9MFMF*+ (IrOI0o355s5S5s243i223iMFMF*np/mL Da* punty cDoDararSa*ccVtEretud4332nrml1!1t/072235tfvS47m103iieandnda/f/iiBBaaidyyv-otu000m4*9/0/*01322l/a//000a00a0,tm0hTaSmK23223752n07343W344Rh..0015.0791.W0590/1mL2LA/0.C0cCoLn5AcHCemne225li4?5c"'43rui ihould be 4211.1796.1163 5219073132l considered tentaive. EJdnctcn Votum* Rato (rubai S'olume/Final Votum* 04/21/00 LAC 3M Environmental Laboratory loa-430.*n223-IC 9/12/00 Page 219 3m Medical Department Study T - 6 2 9 Ac t TOX-030 Report N o . FACT TOX-030 Covane# 6329-223 laboratory Request Number-U22 79 SPtruoddyua Numbct<TMSubMance) MFMRA(ni-aMalSanxnqtayntxumot.aiodcrcalea.UldtEvVSiqaoauoifintfpwcim::wean/tVSeyrMnocnm: Number YDSl-otlupnUomf Erjpelracuon/AnMyM: DDSMmu*noopffiDAcunDalayRnteaad/AucntaiolynM/A:nniyM: T1EM000MRSSSS4446eeee-Tu6o///*aeee27b3'S2nnV574yAAAA-9kL//l/t3t00tte0-t!ttttyt400y00aaaka(n...ccc0cP1ShihhCh600SFmmmme9345aAO9n//ieeee2031tdSnnnno171tttt)K//i*E00eJ00TK..TSO0w-4Vlo-/O32eSla/l/Oy0O0S..tuO0d5Vy/00wM4/0*W0h PF05 in Cynomolgui IIAAS&MMH'ADV Monkeyi WEEK 77 MONKEY SERA GDrootup Sample pC|/roaoseLs. CocBefcLPreePtrOrHaSttReeee uMPiFteOahnSL MSS/iMdR.SSDDDevR.PD Method BU ManxBlk QC - 250 ppb 0 0CCroenetrpol1 0 1M5C*mrde-pDe'kopj*/)eday 0 2H5Cimtrbof-u/Dkpef/4adtay MMMMMMRRKXBKBKKXS00SSSSS045S404O0020020424444444244202320422O-44*4-044HH0-000-00S--2S--2--SS4SSOSeS0Teer*cOrBerrrBrae*--tltM--kMkBBBBMM--llS3lSU4kkSSkDD------433443--43 I11I11JI100I0I00000000055S5555SS555555SS3S553S542052M432132220539923I9FFMMMMFMFMFF--+**+*** 00012n3323.93307213(o309660645000 724411143111105661201102.131401093?92 000000<<<.1114.317.,920022LILLL0000112302710173t32O023OOO972.%.7139009594551%%5%O5QOQ4459?0 'LOO KA <LOQ OOOS52 SA 0 01061.944 10*% 2?% 123% 00296 0 0303 22 5 1)0 60 0 57 0 000065220I60S.064*7%313O360?763273 6331319)6831 Unul of OueiiUMxm(LOQ): PFOS = 555 np*mL PFOS * PcrfluoroocunevuifonMe Dmpunty cDoDMmmeScetVerEuedrmr/tiVfaiireentnddift//iiaBBcldyyv:olu000mV96/0e*1322l/e0**Oa00.tmhT05amK/t0RhW0. 005Q90/m1LAl,.0C0c'oCLnAScHCenttMion ihould be considered teniMive. 04/21/00 LAC \A - Not applicable Extraction Volume Rjkio" Inaiai VolumeTmal Volume ELTxSr-l#V-5T 1 3M Environmental Laboratory tox*03O-n223-1C 9*'12*00 Page 220 3m Medical Department Study T -6 2 9AcfrTox-3o Report No FACT TOX-030 cawi#6329-223 laboratory Reques Number-U2279 MSPMtruoaxdndhyxuoc:dt'KNruvmubuenr:tTrtt SubfUncc). RFAIni-nlaSeacnqlryauumtmaicremae:ld*EVSqoanlfurtpwenwrac/iVScymaeemn Number Slope DYDD-aaaIeaentoooafrffcEDAeapnantaactytRitoetadn/a/AAcatiaaoalnyl/yaA:an: aiya: 26 Week C^auit Toacity Study with PFOS u\ Cynomolju Monkey* RMTME-uTao6bSnn2yL9k-tSey-1y40n(0.PxS16Fe93KO9n.3iSd)ETS-J-5 i SSeeee AAttttaacchhmmeenntt*s SSeeee AAtttuadchamnceantti! 000444/-.717743///000000.. O034AS/'OL77I0//CO0OJ0K,. 0045//2091//0000.. OVOVOO IA5/MMH-ADV 0VO4AX) IAS SWaEmEpKle D79ataMONKEY CDrmaa#p Method Blk Manx Blk QC 250 ppb 0.0CCmroan^at^rpodl1ey 0. IMSCimtdc-^De^opde)eary 0.7H5Ci^mrhof-a/Dkpoj/e4deay Limit of Quanuuoca (LOQ) SPFEORSAMM-MMMMRRKX5KBKKK0SS03S3S4SSS40050O3010O0244144II404nS442430J1eOMH01IIC22333*00S4aC940i5503445e-4-4350m/3eS055355H33Kt00203503-04-5L3S3U)J--2p22S2-SL0-2224-25S3S3SSS-40-Ol3S0ee3992-91e-ee1eeeMMM1mMrFFIeFFrMrBMrrvrBDFFsaana*a-Ml--lM-BkaBMkBBM-eSI-l3Sll4kSpkSkDkD-Ou---O4-443nO4tycoDrDraaaeceiVeEd0e00pC1221133112322t23/111ri9c.21a.1v6337252916i303n54r6o7/3.f.mS093.e34102422o43b560s95i000reesidLd.f/i/BeBdyy: Cto/moofclepoarrtorHasttoRaee 00<<<<1I1900LLLL12200OOO9O72H97MHH3OQO3Q40 uMPKFe'ObanSL 'oq i.oo 000032 107% 123% 0000..2..111000091291222.909136754060 0 0215 0 0243 19 1 21 4 000369///001122///000000.m0T3m44K23/90230Rh9620.4/0090/1L2A/0C0-LCASHC 41 1 41 4 MSS/MUR.SSDDDevR.PD KA 001N061A944 27% 6% 0000046922211U00.43.59.3274230S00296099311565 PFOS Perflnorooctaiefulfonae - Serum inkial volume leea than Q.50mL. concimmiona ihould be coftfidmd tentajv* 04/79/00 LAC N A -N ct applicable Extraction Volume Rao " (nkial Volume.Fuul Volume CExTcSe-l1T-3 1 3M Environmental Laboratory iox-030-eera223- 1C 9-1: w Page 221 3m Medical Department Study: T - 62 9F5A .C7T -T O X -0 3 0 Report N o . FACT TOX-030 laboratory Request Number-U2279 Covance# 6329-223 SPtnuxdJyu.ct Numbcr(Tesl Substance): M atru: MAneathlyotdic/RalevEiqsjuoinp:ment System Number: Instrument Software/Venion: Date of Exiractinn/Anaiyst: Date of AnaJysis/Anaiyst: Dale SofaDmaptaleReDduacttaion/Analy *t MONKEY LIVER Week 27 Group Sample # Dose Method Blk RBL05189-H2O 81k-3 RBL0S189-H2O Blk-4 Method Blk RBL06119-H20 Blk-11 RBL06119-H20 Blk-12 Method Blk 05230-H20 Blk-5 0523O-H2O BUc-6 Matrix Blk RRBBLL0055I18899-L-LV\Tt Blk-3 BUt-4 Matrix Blk RBL061 19-Lvt Blk-11 RBL61 19-Lvr Blk-12 Matrix Blk RBLD5230-Lvr Blk-5 RBL05230-Lvr BUt-6 Matrix Blk MKL05230-Lvr Blk-5 MkL05230-Lvr Blk-6 QC - 250 ppb I05508M-MS 05/18/99 QC - 250 ppb I05508M-MSD 155I7M-MS 06/11/99 105517M-MSD QC - 250 ppb MKL0523O-MS-5 05/23/00 MKL05230-MSD- Group 1 10S5O8M 6/11/99 Control I05517M 6/11/99 0.0 mg/kg/day Q5508M 5/18/99 1055I7M 5/18/99 IC5519M 5/18/99 105527M 5/18/99 I05530F 10553 IF II0055553454FF Group 2 1055 UM Low.Dose 0.03 mg/kg/day IQ5515M 105516M I05521M I05537F I05541F 105547F ext 5/23AJO 105547F I05550F Group 3 I05510M Mid-Dose [Q5518M 0 15 rag/kg/day 105524M I05528M I05532F 10538F IQ5545F I05548F Group 4 High-Dose I05507M 105512M 0.75 mg/kg/day 105534F I05536F I0555545G1FF Limit of Quantitation (LOQ): PFOS * 30.0 ng/g NA Not analyzed. not applicable PFOS Perfluorooctanesulfonate 26 Week Capsule Toxicity Study with PFOS in Cyrnroolgos Monkey T-6295 <PFOS) Monkey Liver Filename See Attachment* FAAmCeTb-aM06-I24.O98k. FACT-M-2.0. 2.1. Madeline (Ml098. FTS-8- R-Squared DaseyOSlope Value: See Attachments See Attachments MassLynx 3.2 3.3 Y-tniervepi See Attachment* 05/l8<99. 06/11/99. 05/23/00 SAH/SRP/SKH/SAI. 05/19/99. 05/22/99, 06/09/99. 06/14/99, 07/2999. 06/U/99. 05/25/00 KJH/HOJ/MHH/SAH/lAS 05/20W. 05/25/99. 06/10/99. (W22/99, 08/04/99. 10/1299. 50-2MX) KJH/NOJ/Mh'Pl/MMH/lAS p r o s C m rn tn m Mean RSD Calc. Cone. 0.t0/0t m frt<orLrrOo%QaRee.______ PFOS "8/8 MSS/Mtd.SDDeKv.PD 0.454 <LOQ <LOQ NA 0.00 <LOQ NA NA <1.00 NA 0.00 <LOQ 1.64 <LOQ <1.00 NA 0.00 <LOQ 3.43 <LOO <1.00 NA 0.00 <LOQ NA 01.3.2951 <LNOAQ <LOO <LOQ . <t.oo NA NA 15.2 <LOQ 97 482 305 <1L74O%O 110% <LOO 142% NA 45% 300 101% 305 103% 102% 2% 239322 m98%% 104% 13% 564 0.564 ++ 143 0.143 477 0.477 119 0.119 R 91 0.091 22.2 129 0.129 0.121 0 0269 128 0.128 112 0.112 87 97 00..008977 0.106 001167H8 18237 182 22709 227 11417 16734 1161..74 27 0 17 3 4 66 22818 22.8 24847 24 8 282623 283 20102 20.1 9 73 20735 20.7 22.1 2.15 42169 42.2 86173 86 58673 58.7 33 1 48203 48.2 58.8 19 5 80421 80.4 49590 496 6861533726 66.5 81 4 21 4 69.5 149 412474 378723 412 379 396 62.3039 280575 281 256669 257 267328 267 5.00 287223 287 273 13 6 Date Exilerexi/By: 05/25W. O^IOW, 06/22/99. 10I2/V9 IAC Dale Verified/ By: 06/02/00 PJW. 06/05/00 TKR Date Punty correctedVerified/ By9/12/00 mmh. 09/12/00 I.AC Sample determined an outlier and waa not included in any calculation* Sample used for MS/MSD appear* to be spiked accounting for low recoveries. Sample 105507M will be used for the MS/MSD and 105518M cone. Vcnfied. Sample 105507 St IS will be rextractcd. R = Sample reextracted along with MS/MSD. This value *a* mi used in any calculation* * Sample determined an outlier and was not included in any calculations. Was extracted 05/25/00 FFAxcCeTl -9M7 -2.1 3M Environmental Laboratory tox-030-liver2231B.xls 9/12/00 5:59 PM Page 222 3m Medical Department Study: T-6295.7 3M Environmental Laboratory DDPMMAIDSntraaanusacotllatdtleeedrrhlyuuiytooox:mxctff:fitlc/eDAERaNnlxnaetutlEavSmraliaqoysRhcuifstoeleiiiwpsrnodf/m:nuAaT/rceeAnelnsia/nolVtlaynSSels/uyryAts:bssinttso:eatnaml:nycsNte:)u:mber: Sample Data FACT-TOX-030 Covance# 6329-223 ETS2MM1116o00-0To6au///SW111sn2p-s584k98le0///e5.99-9ey2y699k9n(0.P,,,Lx01CF11i19&v3aO0009pe.///2,Sr222sEAu)015&Tl///me99S93999Te.,,83loi-S11ax700Ei00c//E226it/252yR//499W9S999t,,Wu1d10y0//22w67i//t99h99P..F11O00//S2297i/n/999C9yIMnAoMSm/HMo/l1gMAuHsSMonFSRYkli--eolSIeypnnqsetaue:mracreee:pdt:Value: SSSSeeeeceec AAAAttttttttaaaacccchhhhmmmmeeeennnnttttssss M O NKGDErooYsuepL IV E R Day 393SaBmioplpes#y MMMMaa1111eett0000rrtt//ihi/h/21x1x2oo5445ddBB////9999llBB9999aanllnkkkk 25QppC1m-02/A2550/59p09p0mppb 0 7H5Gigmrhog-uD/pkog4s/deay RRRRRRMMRRBBBMBBBBBKMKLLLILLLKLL0ILIILIK10II500011L010I5052LII0001I011I512I444052202I00244045992509552555552999-5-F2-99555959F--HH-H25---9LL13H4---MLL29LL22M-3I22vv2MMM-vvOOFFOvvSrrMOSrrrrDSBB-BBBBBSBB1BD-lllll-llkIkllklkk2kk-kkk---2------1I1-1212112I212 Ca411111lP29c40473n038245NNNNFN18.7525g....029005416OAAA5AC9A42/18058802g64768So117453ne, uCro/ron<<<<<fco7588LLe4LLLNNNPNN111r97382n437OOOOOFAAAAA%%%%2%285tOQQQQQraSRtioeen. PMUFegOa/gnS MSS/MidR.SSDDDeRv.PD <LOQ NA <LOQ NA <LO0 NA <1.00 NA <LOQ NA 68% 30%- 85% 140 298 32551..8%744.4784 LimnofQuantitation(LOQ): PFOS=26.9ng/g DaleEnlered/By: 10/22/99. 10/25/99. 10/29/99LAC. 11/05/99GML DaleVerified/By:06rt)5/00TKR DalePurilycorrecledVcrified/By:09/12/00mmh.09/12/00LAC NA=Nol analyzed, nolapplicable MS/MSDsamplescxlracledan10/14/99werenai wiihinrecoverycriteria,andsampleswerereexiraeledan10/25/99 PFOS=Pcrfluoroocianesulfonair atahigherconcentration. TheearlierMS/MSDsampleswerenolspikedatahighenoughlevel inrelationtotheendogenousPFOSlevels ReexiraeledMS/MSDswerewithincriteria. LAC12/14/99 Tocalculatetheextracteddensity:(initial liverweighi/(initial liverweight+Milli QWateradded) TocalculatethePFOSCalc.Cone.: (PFOScone,ng/gXdensityofcurveliverhomogenateg/ml.)/extracteddensityg/mL)*PFOScorrectionfactordilutionfactor Report No. FACT TOX-030 laboratory Request Number-U2279 Page 223 FTS-8-7.0 Excel 97 lnx-030-liver223-IB.xls 9/12/00 4:00PM 3m Medical Department Study: T-6295.7 3M Environmental Laboratory FACT-TOX-030 Study: Product Num bcr( l est Substance): M atrix: M cthod/Revision: A nalytical Equipm ent System Num ber: Instrum ent Software/Version: Date o f Extraction/A nalyst: Date o f A nalysis/A nalysl: Date o f D ata R eduction/A nalyst: Sample Data 26 W eek C apsule Toxicity Study wilb PFOS in C ynotnolgus M onkeys T-6295 (PFOS) M onkey Liver ETS-8-6.0 & ETS-8-7.0 Davcy 070799 M assLynx 3 2 & 3.3 3/22/00 SAL 03/24/00, 03/27/00,03/28/00, 5/5/00 M M H , IAS 03/27/00, 03/28/00, 03/29/00, 5/8/00 M M H , IAS Monkey L ive r Wks 79 & 80 G roup Dose Sam ple # M cthod Blk M atrix Blk RBL03220-H 20-Blk-5 R BL03220-H 20-Blk-6 R B L 0 3 2 2 0 -liv erB lk -5 R B L 0 32 2 0-liv crB lk -6 M ICL03220-liverBlk-5 QC 250 ppb M KL03220-liverBlk-6 M KL03220-M S-250ppb-5-1 M KL03220-M SD-250ppb-5-2 M ICL03220-M S-250ppb-6-l M KL03220-M SD-250ppb-6-2 l05505-M S-50ppm -5-l l05505-M SD -50ppm -5-2 G roup 4 H igh-Dose 0.75 m g/kg/day 10551 1/M /W k79 105522/M /W k79 I05533/F/W k79 105542/F/W k79 G roup 3 M id-Dose 0.15 m g/kg/day I05505/M /W k80 I05523/M /W k80 I05539/F/W k80 I05552/F/W k80 Limit o f Q uantitation Limit (LO Q ): PFOS = 26.9 ng/g PFOS Calc. C one. ng/g 0.00 0.00 0.00 0.00 0.00 0.00 216 186 266 232 54408 47351 23480 70781 42668 57895 8252 10203 24728 17690 PFOS = Perfluorooctanesulfonate NA = N ot applicable Date Entered/A nalyst: Date V erified/A nalyst. Date Purity corrected V erified/ By: 04/05/00, 04/06/00, 5/10/00 M M H , CSH 06/05/00 TKR 09/12/00 mmh, 09/12/00 LAC C oncentration of PFOS u g /g o r */ R ee <LO Q (30.6 ng/g) <LO Q (30.6 ng/g) <LO Q (30.6 ng/g) <LO Q (30.6 ng/g) <LO Q (30.6 ng/g) <LO Q (30.6 ng/g) 72% 62% 89% 77% 95% 81% 23.5 70.8 42.7 57.9 8.25 10.2 24.7 17.7 M ean PFOS " g/g <LOQ <LOQ <LOQ 67% 83% 88% 47.1 50.3 9.23 21.2 RSD Std. Dev. M S/M SD RPD NA NA NA NA NA NA 15% 14% 17% 71.0 33.4 21.4 10.8 14.9 1.38 23.5 4.98 Report No. FACT TOX-C30 laboratory Request Number-U2279 Page 224 E T S -8 -7 .0 Excel 97 TO X -030-liver223-lB.xls 9/12/00 4 16 PM 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 Laboratory Request Number-U2279 Attachment F Example Calculations Formula Used for Sera Analyses in Study FACT TOX-030 AR (ng/mL) x DF x SC x FV (mL) x 1.0 pg = pg/mL x PC = Reported Cone (pg/mL) EV (mL) 1000 ng Calculation Used for Group 3, Week 1, Animal ID 105505M 287 ng/mL x 10 x 0.9275 x 1 mL x 1.0 pg = 5.32 pg/mL x 0.864 = 4.60 pg/mL 0.5 mL 1000 ng AR-- Analytical result from MassLynx summary DF-- Dilution factor SC-- PFOS salt correction constant (0.9275) FV-- Final extract volume (1.0 mL unless otherwise noted) EV-- Volume of sera extracted PC-- PFOS purity correction factor (86.4%) Formula Used for Liver Analyses In Study FACT TOX-030 AR (ng/g) x 8 curve ( 0 x SC x DF x 1.0 pg = pg/g x PC = [PFOS] sample (pg/g) d sample 1000 ng (l) d curve is assumed to be: 1 g liver 5 mL H2O Calculation Used for Group 3, Week 27, Animal tD 105510M 524 ng/g x 1 g/ 5 mL x 0.9275 x 100 x 1.0 pg = 48.8 pg/g x 0.864 = 42.2 pg/g 0.9963 g/ 5mL 1000 ng AR-- Analytical result from MassLynx summary d curve-- Density of the liver standard curve, assumed to be 1g liver/ 5 ml water d sample-- Density of the liver sample (1 g sample/ 5 mL H,0) SC-- PFOS salt correction constant (0.9275) DF-- Dilution factor PC-- PFOS purity correction factor (86.4%) 3 M Environm ental Laboratory 3M Environmental Laboratory P a g e F-1 Page 225 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Attachment G Intrim Certificate of Analyses Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-112279 3M Environmental Laboratory 3M Environmental Laboratory Page G-1 Page 226 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 INTERIM CERTIFICATE OF ANALYSIS Test N_Ca_me_ne_t_r_e_A__n_a_ly_t_i_ca_3l_M_L_aRP_brRe_oofePredvaaruiterscoiinttorS:cynipe:eeP#s1c8F:i(Cf96iOc/OS.a97SDt%/Ai,0o-0Ln0R)s1oe8tfe2r1e7nce #: 023-018A Purity1 Result 86.9% AIdpepnetaifricaNantcMieoRn Met2a1l..s (MCICaaPlgc/niMuemsSi)um Tota45367l.....%NIMPSIrmooioacdtnpnakiuusgesarmliinutyems(e2NMR) TRTP((GLOooeCltCtAaaa//tllMAMe%%dSSC))IImmomppuuprroiiuttyynds RPuersiidtyubalySDoSlvCents (TGA) Inor23g1...aniBCFclrhuAoloomnrriiiiodddneees (IC) Orga45671n..... icNNPTSAuhiFittcolrArfisadiaptttseehe34at(IeC) 243... NHPFFFPPBAAA Elem4231e.... ntNHCSaulaiytlrAdrfbuornoograngelenynsis6: 5. Fluorine White Crystalline Powder 24351..... TTTTThhhhheeeeeooooorrrrreeeeetttttiiiiicccccaaaaalllll VVVVVaaaaallllluuuuueeeee ===== 05061%%0.79%.58%% Conforms Positive 2435671.......81..4900<06<113....00.00084ww..0004030tt1559009..//11wwwwwwwwwttttttt......V%%////ttwwwwV.w/wtttwtt....%%%%%tt..%% None Detected NN0o.o3tn3AepwDpte.l/tiwceact.tb%elde1 4235671....... <<<<<0000008.......5000700916040059067wwwwwwwtt../tt/tttww...../////wwwwwtt..%ttt%tt.....%%%%% 4231.... <<00..0021..8011wwwwtttt../.7//wwwwtttt....%%%% 24351..... 0581I4...2287..1444484wwwtwwtt.../tt//w..ww//wwttt...%tt%%..%% COA023-018A 3M Environmental Laboratory Exact Copy of Original InLiAtiIa--i OatscaIizjc. Page 1 o f3 Page 227 3m Medical Department Study: T-6295.7 Report No. FACT TOX-G30 laboratory Request Number-112279 INTERIM CERTIFICATE OFANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A Date of Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 F0'P.l3uu3or%ritiyd)e=, 01.0509%%-+(NsuMmRoimf mpeutraitliiems,p1u.r9i3t%ies+, o1r.g45a%nic+aLcCid/MimSpuimriptiuers,it0ie.3s,88%.4+1P%O+AInAo,rganic ToPtaulriitmyp=ur1it0y0%fro-m1a3l.l09te%sts==861.39.%09% 2Potassium is expected in this salt form and is therefore not considered an impurity. o3PbuserirtvyebdyfoDrStChisissagmenpelrea. lly not applicable to materials of low purity. No endotherm was 4inSourlfgiairniicn athneiosnammpetlehoadppceoanrdsittioonbse.coTnhveeratneidontoreSsOul4taangdreheesnwceeldl ewteitchtetdheussiunlfgutrhe determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5THFFABA NFPA PFPA THreipfltuaoflruooarcoebtiuctyarciicdacid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CgFi7S03'K+(MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018A 3M Environmental Laboratory Page 2 o f3 Page 228 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 INTERIM CERTIFICATE OF ANALYSIS Centre Analytical Laboratories COA Reference #: 023-018A LC/MS Purity Profile: ImpCa4rity C5 TCCot76al wtV411w...732t232. % 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration cafruvoermrvaetgsh,eerreCessp4peoacnntidsveeCflay6c. sttoTarnhsdefarCrod5mcvutahrleuveeCsw.6 aaLsnikdcaeClwc8uislseat,atenthddeaurCsdi7ncgvuarthlvueeesa.wvearsacgaelcrueslaptoendseusfiancgtotrhse Prepared By: _D_a_v_i_d_S__. _B_e_l_l_________________ _D_a_t_e Scientist, Centre Analytical Laboratories Reviewed B y:_J_o_h_n_F_l_a_h_e_r_t_y_____._____________ _D_a_t_e Laboratory Manager, Centre Analytical Laboratories COA023-018A 3M Environmental Laboratory Page 3 of 3 Page 229 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 INTERIM CERTIFICATE OF ANALYSIS Revision 1(9/7/00) Centre Analytical Laboratories COA Reference #: 023-018B 3M Product: PFOS, Lot 171 Test Name Purity1 RefPeurreintSycpe:e#c8:if6icS.a4Dt%io-n0s09 Result 86.4% AIdpepnetaifricaNantMcieoRn TTTR(M(GLoooeeCtttClaataa423567/a/1llltMM.....e.l.%%%sdSS(NMPIMCSC)I)IIIrCmmmooiooaaacdtPnmlpppnagkci/uuuusngiepMusrrramleoimiiinsuStuttiyyyem)unsmd(e2NsM--R) PRPuOersAiitdyAubalySDoSlvCents (TGA) IOnrogra4232673511gn..........ainciTNNPHPFSBCcAuFlhFirihFuAttcoolPArlrBoifomnsadiaArptrAtitsieiioehedddn54aeees(tIe(CIC) ) 4. NFPA Elem2431e.... ntNHCaSluaiytAlrdrfbuornorogangelneynsis4: 5. Fluorine White Crystalline Powder 4231.... TTTThhhheeeeoooorrrreeeettttiiiiccccaaaallll VVVVaaaalllluuuueeee ==== 0051%%.79.58%% 5. Theoretical Value = 60% Conforms Positive 45672311.......10.0.60<0600100...0...005003w.w0050501t3402577t./.1/wwwwwwwwtwttttttt.V.VV..%.///t%www.www/wtttttt......%t%%%%%.% None Detected NN0o.o3tn0AewpDpte.l/tiwceact.tb%elde1 4235671....... <<<<<0800000.......2800000721040059076wwwwwwwtt..//tttttww...../////wwwwwtt..%%ttttt.....%%%%% 231... <<<000...111 wwwttt...///wwwttt...%%% 4. <0.25 wt/wt.% 231... 011..276.19084wwwt.tt/.w.//wwt.t%t..%% 4. 10.1 wt./wt.% 5. 50.4 wt./wt.% COA023-018B 3M Environmental Laboratory Exart Copy of Original Initial Data Page 1 o f3 Page 230 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03Q laboratory Request Number-U2279 INTERIM CERTIFICATE OFANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B Date of Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 '1P0u.6r0it%y +=In10o0r%gan-ic(sFumluoorfidme,et0a.l27im%p+uNriMtieRs, i1m.3p9u%rit+ieLsC, 1/M.00S%im+pPuOriAtiAes,, 0.30%) Total impurity from all tests = 13.56% Purity = 100% - 13.56% = 86.4% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials of low purity. No endotherm was observed for this sample. 4inSourlfguarniicn athneiosnammpetlheoadppceoanrdsittioobnse.coTnhveeartneidontoreSsOul4taangdreheesnwceeldl ewteitchtetdheussuinlfgutrhe determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5TNHFFFAPBAA PFPA HTNroeipnfltouafoflluruoooarrocoepbteuincttyaarcniicdoiacciadcid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CsFnSOsTC (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018B 3M Environmental Laboratory Page 2 of 3 Page 231 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 INTERIM CERTIFICATE OFANALYSIS Centre Analytical Laboratories COA Reference #: 023-018B LC/MS Purity Profile: ImpCCCCu5764rity Total w t6/11w...305386t % 1.63 10.60 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration cfruormvesth, ereCsp4eacntidveCly6.stTanhdeaCrd5 cvuarluveesw. aLsikcaelwcuislea,tethdeuCsi7ngvathlueeawvearsacgaelcrueslaptoendsuesfiancgtotrhse average response factors from the C6 and C8 standard curves. Prepared By: _DS_ca_ive_in_dt_iSs_t_.,_BC_ee_ln_lt_r_e_A__n._a_l_y_ti_c_a_l_L_a_b_o_r_a_tories _D_a_t_e Reviewed B y:_J_o_h_n_F_l_a_h_e_r_ty____________________ _D_a_t_e Laboratory Manager, Centre Analytical Laboratories COA023-018B 3M Environmental Laboratory Page 3 of 3 Page 23.2 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Attachment H Report Signature Page Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 ") TOO Andrew M. Seacat, P h tf., Study Director John L. Butenhoff, Ph.D., Sponsor Representative Date' / ' & /^ Date z*< 9 ? > 3M Environmental Laboratory 3M Environmental Laboratory Page H-1 Page 233