Document 9LONEGqjOZ8B8885QKZYBDZaV

O R IG IN A L 3M M edical Departm ent Study: T-6295.7 226- o?#! Report No. FACT TOX-030 Laboratory Request Num ber-U2279 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 Project dentification 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 L - O T3 m co ......U - O cD ro CO 3M Environmental Laboratory 10 1 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 GLP C o m p l ia n c e S t a t e m e n t Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-112279 Study Title: Analytical Laboratory Report from the 26-Week Capsule Toxicity Study with Perfluorocctanesuifonic 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 T h is 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 Y Y l.& u d y /n /e * Dte 7 Study Sponsor ftf f T Date ZCCO 3M Environmental Laboratory 3M Environmental Laboratory til Page 2 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-112279 GLP S tu d y -- Q u a l it y A ssu r an c e Statem 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 R eported to M anagement S tu d 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 I 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 3M Environmental Laboratory 3M Environmental Laboratory 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 Stu d y P er so nnel a n d 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 T e s tin g L a b o ra to ry 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 > ll3 Page 4 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 N u m b e r - U 2 2 79 Report No. FACT TOX-030 Laboratory Request Number-112279 ~ GLP Compliance Statement..................................................................................................................... 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................................................................................................... 11 Summary of Quality Control Analyses Results...............................................................................11 Summary o f 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 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Intro ductio n an d Purpose Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 ? Q 8 p 1 7 --------------- g ---------------- Q O PerfluorooctanesuHbnate (PFOS) CAS Number 2759-39-3 Chemical Formula: CgF^SOj Molecular W eight 498.98 The purpose of the analytical phase of this study is to determine the presence and concentration of PFOS (C a F ^O j-) in liver and serum specimens collected during the study of Cynomolgus monkeys orally dosed with perfluorooctane sulfonic acid potassium salt (T-6295). T est 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 of 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 G roup C haracteristics o f Test System in Study T-6295.7 STUDY GROUP N u m be r op A n im a ls To tal DOSAGE LEVEL Do sag e Ra t io (m g/kg/day) (w :w )a T otal: 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 Page6 P>*ge 6 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-Q30 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. Specim en C ollection 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 EIFOSE, 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-fert-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. Specim ens C ollected from Study G roups 1 through 4 (through 2/25/99); Serum Specim ens-- 550 specimens: 9-14 specimens/animal Liver Specim ens-- 30 specimens Specim ens C ollected from the Recovery G roup from 2/27/99 to 3/07/00: Serum Specim ens-- 224 specimens: 18-20 specimens/animal Liver Specim ens-- 12 specimens from Group 3 and Group 4 animals (8 via biopsy) S pecim en R eceipt 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 10-20C 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 P. 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-U2279 D ose Confirmation A nalyses 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 Milli-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%.. Ma ter ia ls and Meth o ds Chem ical 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. C haracterization o f Test and A nalytical Reference Substances in Study FACT TOX-030 C h e m ic a l N a m e Source E x p ir a t io n D a te Sto r a g e C o n d itio n s C h e m ic a l L o t N u m ber P h y s ic a l D esc r iptio n P u r ity T est S ubstance KPFOS Potassium Perfluorooctanesulfonate 3M Specialty Chemicals Div. 8131/2001 Frozen <-10*C 217 FC-95. W hite crystalline powder 86.9% A n a l y t ic a l FRe f e r e n c e S u b s t a n c e s KPFOS Potassium Perfluorooctanesulfonate 3M Specialty Chemicals Div. T H P F O S '1H ,1H .2H ,2H perfluorooctanesulfonic add ICN Blomedics, Inc. 8/31/2001 1/01/2020 Frozen <-10'C Ambient temperature 171 59909 53406 W hite crystalline powder Brown powder Brown w axy 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 Summ aries 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 P 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 M ethods: ETS-8-6.0, "Extraction of Potassium Perfiuorooctanesulfonate 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 urn nylon filter, using a 3 cm3 disposable plastic syringe into glass autosampler vials. ETS-8-4.1, "Extraction of Potassium Perfiuorooctanesulfonate 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 urn nylon filter, using a 3 cm3 disposable plastic syringe into glass autosampler vials. A nalytical M ethods: ETS-8-7.0, "Analysis of Potassium Perfiuorooctanesulfonate or other Fluorochemicals in Liver Extracts Using HPLC-Electrospray/Mass Spectrometry" ETS-8-5.1, "Analysis of Potassium Perfiuorooctanesulfonate 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 (CBF,7S 0 3-) analysis, was fragmented to produce ion 99 (FS03-). The 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. Liq u id C hrom atograph: Hewlett-Packard Series 1100 Liquid Chromatograph system Analytical column: Keystone Betasil" C 2x50 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 Vi Page 9 3m M e d ic a l D e p a r tm e n t S t u d y : T - 6 2 9 5 . 7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-1122 79 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 S pectrom eter 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 M onitored in FACT TOX-030 Target A nalyte Primary Ion (am u) Product ion (am u) PFOS THPFOS 499.0 427.0 99.0 80.0 D eviation s 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). D a t a Q u a lity O bjec tives and Da t a 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 L im its o f Q uantitation (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. D uplicate/Acceptable Precision: Precision was reproducible to within 30% Spike/Acceptable Recoveries: 70-130% C onfirm atory M ethods: Indeterminate samples may be re-analyzed using a confirmatory method. If a confirmatory method is used, an amendment to this protocol should be written. D em onstration o f S pecificity: Specificity to be demonstrated by chromatographic retention time and mass spectral daughter ion characterization. 3M Environmental Laboratory 3M Environmental Laboratory T II Page 10 Pa' 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 Da ta Sum m ary, 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 A nalyses R esults Linearity: The coefficient of determination (r2) of the standard curve was >0.985. C alibration 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 analyte was based on the response of one specific product ion using the multiple response-monitoring mode of the instrument (see Attachment C). Lim its o f Q uantitation (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 ly te -M a tr ix LOQ PFOS-Sera P FO S -Liver 4 .3 9 -1 5 .2 ng/m L 2 6 .9 -6 0 .1 ng/g B lanks: 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. D uplicate/Acceptable Precision: Precision was determined by analysis of MS/MSD and was reproducible to within 30%. M atrix 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 o f 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 'H S b Page 11 11 3m Medical Department Srudy: 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 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 Sam ple Results Sam ples from C ontrol Anim als: 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. Sam ples from Dosed Anim als: 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. St a tis tic a l M ethods and Calculatio ns 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 te m e n 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.* L ist of A ttachm ents A ttachm ent A: Control Matrix Characterization and Dose Confirmation Analyses A ttachm ent B: Protocol and Deviation Summary A ttachm ent C: Extraction and Analytical Methods A ttachm ent D: Data Summary Tables A ttachm ent E: Data Spreadsheets A ttachm ent F: Example Calculations A ttachm ent G: Interim Certificate of Analyses A ttachm ent H: Report Signature Page 3M Environmental Laboratory 3M Environmental Laboratory 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 A ttachment A C o n tr o l M a t r ix C haracterizatio n and Dose C onfirm ation A n alyses Table A-1. Characterization o f the Control Matrices Used fo r Liver and Sara Analyses In Study FACT TOX-030 Co ntro l Matrix Ra b b it S erum Ra bb it Serum Monkey Serum Monkey S erum Mo n k ey Serum Source Expiration Date Storage Conditions Chemical Lot # Physical D e scrip tio n Control Matr ix Sigma Chemicals 01/01/2010 Frozen -20` C 118H8418 Rabbit Serum Sigma Chemicals 01/01/2010 Frozen -20C 47H4641 Rabbit Serum Lampire Biological N/R Frozen -50"C 111022515 Monkey Serum Siena Biomedical 01/01/2010 Frozen -20 C #LY2N0 Monkey Serum Ra b b it L iver Rabbit L iver Ra b b it L iver Ra b b it Liver N/R 01/01/2010 Frozen -20' C N/R Monkey Serum Mo n k ey L iver Source Expiration Date Storage Conditions Chemical Lot # Physical D escriptio n N/R-- not recorded Coming Hazleton 12/01/1999 Frozen -20*C F00007 Rabbit Liver N/R 12/01/1999 Frozen-20C N/R Rabbit Liver Coming Hazleton 01/01/2010 Frozen -20` C F00005 Rabbit Liver Coming Hazleton 01/01/2010 Frozen -20' C F00009 Rabbit Liver Sierra Biomedical 01/01/2010 Frozen -50C N/R Monkey Liver 3M Environmental Laboratory 3M Environmental Laboratory 73 Z2. Page A-1 Pa' 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 Ex p e c te d CONC. (ng/mL) M easured CONC. (ng/mL) V .R E C . FOR ng/mL Expec ted Calc . Co nc. (PBfg) M easured Calc . Conc. (PQ/O) A verage S td D e v ia t io n 1:39 Dose (25000 p p m PFOS) Top M iddle Bottom 580 500 500 479 650 422 i 83% I 130% i 84% 25000 25000 25000 1:499 Dose (2000 p p m PFOS) Top M iddle Bottom 410 404 400 305 287 272 I 74% I 71% i 68% 2000 2000 2000 20647 32537 21108 24764 6736 27% average / std. deviation^ 1490 1425 1361 1425 I 64.5 5% average / std. deviation= Actual MS Concentration-Actual background concentration, divided by expected, times 100 (Spiked too low which accounts for the wide differences in recovery) V .R E C . f o r pgfg 83% 130% 84% 99% 27% 74% 71% 68% 71% 3% Table A-3. Lactose Dose Verification (PFOS-- Matrix Spikes) fo r Study 6329-223-- 8/21/99 Ex p e c te d Co n c . (ng/mL) A ctual C o nc. (ng/mL) %Rec . FOR ng/mL Calc u lated Conc. (ugfg) Expected Co nc. (ug/g) A ctual Calc . Co nc. (wg/g> A verage Std D e v ia t io n 1:39 D o s e (25000 p p m PFOS) MS Top M iddle Bottom 604 524 524 507 84% 485 92% 438 83% 21858 24252 21889 1:499 D o s e (2000 p p m PFOS) MS Top 606 475 78% 2320 M iddle 600 447 75% 2217 Bottom 596 440 74% 2202 * This value is an outlier and was not used in any calculations 10.8 12.5 12.5 9.77 9.92 10.0 12.1 -82.8 7.82 8.30 7.93 8.41 22666 1374 6% 2246 64.5 3% % Rec. FOR pg/g 112% -664* 63% 85% 80% 84% A verage 87% 83% 3M Environmental Laboratory 3M Environmental Laboratory Page A-2 14 3ra Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Attachment B P r o to c o l a n d D eviation S ummary Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Tabla B-1. Davtation Summary fo r FACT TOX-C3Q Deviatio n Dates of Occurrence Im pact on Study MtBE was used as an extraction solvent instead of ethyl acetate. Pipette w as used instead o f Oxford dispenser. Curves plotted by linear regression weighted 1/x rather than by linear regression as specified in the protocol. A second extracted m atrix curve was not used to bracket samples. Recorded extraction method FACT-M-1.0 rather than FACT-M-1.1. Followed extraction m ethod 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-fl-4.0 rather than FACT-M-3.1. M atrix 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. 2/5/99,2/9/99, 5/18/99.6/11/99 10/14/99 2/13/99,3/5/99,3/12/99, 3/19/99, 3/20/99, 3/21/99.3/23/99, 3/24/99, 3/25/99,4/7/99.4/11/99, 4/12/99. 4/17/99, 5/19/99,5/22/99,6/9/99,6/14/99 3/5/99.3/9/99. 3/15/99, 3/16/99. 5/19/99. 5/22/99,10/26/99,1/21/00, 3/24/00, 4/27/00 6/11/99 10/14/99,10/25/99,1/19/00,3/22/00 7/29/99,10/20/99, 10/22/99,10/26/99, 10/27/99, 1/28/00, 3/24/00, 3/28/00 3/05/99,3/08/99 10/25/99 3/02/99, 3/03/99 11/3/99 11/3/99 2/5/99,2/9/99, 3/2/99, 3/3/99, 3/10/99, 3/12/99,3/15/99.3/16/99, 4/6/99,4/8/99, 8/25/99, 11/3/99,4/21/00 No negative impact on the study-- MtBE im proved the absolute recoveries and shortened extraction time. No negative im pact 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 o f calibration checks analyzed every five to ten samples was m onitored to ensure continued accuracy of the analysis. The GC provided by the calibration checks is sufficient and the data quality w ill not be adversely affected. No negative impact on the study-- New m ethod w as followed, even though old method was recorded. No negative impact on the study-- New validated m ethod provides improvements in precision and extraction tim e. 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 o f sera. No negative im pact on the study-- New validated m ethod provides improvements in precision and extraction time. Adequate QC was prepared with the sam ple s e t unspiked samples pose no negative im pact on the study. Mid-level curve standards were substituted as QC for the nonspiked calibration check standards: the unsptked calibration standards pose no negative im pact to the study. Studies indicate that data accuracy and precision m ay be affected when sera samples less than 0.5 mL were extracted. Data reported from extraction o f samples less than 0.5 mL is noted in the data tables. 3M Environmental Laboratory 3M Environmental Laboratory 16 ZH Page B-1 Pa' 15 3m M e d ic a l D e p a rtm e n t S tu d y : 3M Environmental Technology and Services T-6295.7 Report No. FACT TOX-030 PPOOBBooxx] 33331 1l;aboratory Request Number-U2279 Si. Paul, 612 778 6M4N4255133-333311 Protocol UFACT-TOX-030 3M Study Title 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys 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 3mm MMeeddicicaall Deeppaarrttmmeenntt Sttuddyy:: T -- 6622955.. 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 u m b e r-U 2 2 7 9 Protocol #FACT-TOX-030 Study Identification 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (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 P 17 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol 0FACT-TOX-O3O 1. S tudy Twenty-six week capsule toxicity study with potassium perfluorooctane sulfonic acid (T-6295) in cynomolgus monkeys. 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 >027 Page 3 of 9 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol #FA C T-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 2 17 (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 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. 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 Pa> 19 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol #FACT-TOX-030 Body tissue/fluid Collected Expected # of specimens Serum - all animals Urine and feces - recovery animals Liver - all animals 7 days prior to treatment 7 days post treatment every two weeks during treatment and recovery Day zero of recovery 6, 30, and 90 (with a potential 180) days of recovery After termination of the study 616 from main study 24 additional from recovery 24 urine 24 feces 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-1.0, 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 -ee XI Page 5 of 9 Page^2 0 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol ttFACT-TOX-030 12. Data Quality Objectives 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 70% -130% 12.5 Use of confirmatory methods Indeterminate samples will be re-analyzed using a confirmatory method. If a confirmatory method is used, an amendment to this protocol will be written. 12.6 Demonstration of specificity Chromatographic retention time, mass spectral daughter ion characterization. 13. Sub-Contracted Anal ys/s 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. Sta tistical Anal ysis Averages and standard deviations will be calculated. The statistical methods that will be used are described below: 14.1 Data transformations and analysis Data will be reported as the concentration (weight/weight or weight/vol) of PFOS or metabolite per tissue or fluid. 14.2 Statistical analysis Statistics used may include regression analysis of concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference. 3M Environmental Laboratory 3M Environmental Laboratory Page 6 of 9 0 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 A description of any circumstances that may have affected the quality or the integrity of the data 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 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol UFACT-TOX-030 16. Location of Raw 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 16.1.4 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 Environmental Laboratory Page 8 of 9 3m 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 Andrew Seacat, Ph.D., Sponsor Representative ' Date (h a Kristen Hansen, Ph.D., 3M Environmental Laboratory Study Director /& ? /? * ' Date 3M Environmental Laboratory 3M Environmental Laboratory ^3 3 Page 9 of 9 Pa: 24 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 GLP Study Protocol Amendment Study Number: FACT- Tcc-JO CcVCniL (,121-223 Study Title: X U>k C c ^ L Toxci^ PF0 S a C^om.lgus Study Director. km klnJi* Amendment Date: Amendment Number: \ This amendment modifies the following portion of the protocol: 10 Prtpcr&Vtt^ MeW>od$ and i ha II ^ 'w l^ lic x i M ethods I V . Jtckons l u l F/CT-rn-l.l and FAtT-m-H.I Us i k t scrum t x W - k o n ftn 4 cuai^kcJ me.Ww<ls. Tht m tkkji hare, Ww ufJa-ltA V ETS- g-M.o 0r\^. ET-S- 5-5.0. TV- updfclti malkodj udl V wied V/- kke. remainmg 5rum x lra c lv o n ! C'lcJgSfiJ Approved by: iM s o Date 3M Environmental Laboratory 25 3V P 25 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 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. 2 Amendment Date: April 29,1999 Performing Laboratory 3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106 Laboratory Project Identification ET&SS FACT-TOX-030 LERN U2279 3M Environmental Laboratory 3M Environmental Laboratory '2 * 3 5 ' 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol FACT-TOX-030 Amendment 2 This amendment modifies the follow ing portion(s) o f the protocol: 1. PROTOCOL READS: In amendment l, 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 TOread: These methods were revised on 4/?9/'99 to ETS-8-4.1 and ETS-8-5.1 which will be used for all future analyses. Reason: The methods were revised for clarification and to include linear regression, 1/x weighting for initial curves. Amendment Approval Andrew Seacat Ph.D., Sponsor Representative _______________f> / ^ - l h ------Kris J. Hansen Ph.D., Study Director 3M Environmental Laboratory 3M Environmental Laboratory Date Pa 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 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 LERN U2279 3M Environmental Laboratory 3M Environmental Laboratory 2^ 37 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 u m b e r-U 2 2 7 9 Protocol FACT-TO X-030 Amendment 3 This amendment modifies the following portion(s) o f the protocol: 1. PROTOCOL reads: 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 TOREAD: These methods were revised on 06/03/99 to FACT-M-1.1 and FACT-M2.1 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 /3 o /ti Date Kris J. Hansen Ph.D., Study Director 3M Environmental Laboratory 3M Environmental Laboratory Date 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 Pa< 0 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 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-fert-butyl ether (MtBE) instead of ethyl acetate in the extraction process. 4. Protocol reads: 11. Analytical Methods: 3M Environmental Laboratory 3M Environmental Laboratory 3m Medical Department Study: T-6295.7 R e p o rt N o. FACT TOX -0 30 la b o r a to r y R e q u e s t N u m b e r-U 2 2 7 9 Protocol LRN-U2279 Amendment Number 4 11.1 FACT-M-2.1, "Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry" Amend to read: 11. Analytical Methods: 11.1 ETS-8-7.0, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Liver Extracts Using HPLCEiectrospray/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. Protocol reads: 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 mend to read: 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. Protocol reads: 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 num ber of test animals: 32 3M Environmental Laboratory 3M Environmental Laboratory $ *> /! Pa' 32 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol LRN-U2279 Amendment Number 4 Amend to read: 9. Specim en and Sample Receipt: [Sample Receipt Table] # o f Specim ens: Serum -all animals: 516 from main study, 280 additional specimens from recovery Liver-all animals: 42 (eight specimens via biopsy from recovery group) Total num ber o f te st anim als: 32 Total num ber o f anim als: 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. Butenhojf, Ph.D., Sponsor Representative /& * ? * > Date Andrew Seacat, Ph.D., Incoming Study Director / l / w 7 ^ - 2 ----------- Kristen J. Hansen, Ph.D., Outgoing Study Director //'Z T2*/tZ'tsnrz? Date --2- - 2'G77D ''D ate A -P A ------------------------ ---------------------------------- Dale L. Bacon, Laboratory Manager Date 3M Environmental Laboratory 3M Environmental Laboratory 3 *7 2 . Pa^r 3 3 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2 279 Study Title 26-Week. Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 5 Amendment Date: August 14, 2000 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 Covance #6329-223 LIMS #U2279 3M Environmental Laboratory 3M Environmental Laboratory 4 -* 3 34 3m Medical Department Study. T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol TO X-030 Amendment 5 This amendment modifies the follow ing portion(s) o f the protocol: PROTOCOL READS: 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. AMEND to Read: Add that select feces samples will be analyzed for PFOS at Centre. REASON: Feces samples were added after the original protocol was written. 2. PROTOCOL reads: S tudy L0CATI0NS(PAGE 2 ): Analytical Testing Laboratory:. 3M Environmental Laboratory AMEND TO read: 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 of other tissues at the 3M Environmental Laboratory. P r o to c o l r e a d s : S e c tio 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 ly tic a l M e th o d s a n d 12. D a ta Q u a lity O b jectives Amend to read: Add to these sections the most current version of "Determination.of Fluorochemical Residues in Monkey/Rat Feces by LC/MS/MS," Centre method number 00M-O23-OO3, with an LOQ in feces of 10 ng/g. The method will incorporate an initial homogenization step immediately after adding the extraction solvent, using a microhomogenizsr, to allow for complete dispersion o f the specimen in the solvent. Reason: To specify the validated method to be used for feces analyses along with its analytical limits. NOTE: LC/MS/MS is an abbreviation for "liquid chromatography/mass spectrometry/mass spectrometry." P r o to c o l r e a d s : S e c v o n 10. P r e p a r a to r y M eth o d s a n d 11. 3 M A n a ly tic a l Methods Amend to READ: Change to specify that the most current version of the methods listed should be used. REASON: To specify that the most appropriate version of the preparatory and analytical methods should be used during the course o f the study. Environmental Laboratory 3M Environmental Laboratory HH 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol TO X-030 Amendment 5 5. PROTOCOL READS: The amended protocol (Amendment #4) states that Kris J. Hansen, Ph.D. is the Principal Analytical Investigator for the entire study. AMEND TO r e a d : Add Enaksha Wickremesinhe, Ph.D. as the Principal I-hfeJnvestigator at ) T>'& Centre for feces analyses. ft REASON: To specify the PAI at Centre for feces analysis. 6. P r o to c o l r e a d s : Se c t io n 16. L o c a tio n o f Ra w D a ta , R e c o r d s a n d F in a l R eports AMEND t o r e a d : Add that Centre will forward all original study-specific raw data to 3M Environmental Laboratories, together with copies o f appropriate facility-specific raw 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: To specify the archival requirement for the portion of the data developed by Centre. 7. P r o t o c o l r e a d s : S e c t io n 17. S p e c im e n r e t e n t io n A m e n d TO READ: Add that after the analytical report on feces is signed by the study director, all feces specimens of this study will be returned 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. Reason: To specify the handling of all biological fluids, and to define when the quality assurance verification is considered complete. 3M Environmental Laboratory 3M Environmental Laboratory 3m SEP.15.200D0eP^119-:-t3^35Me1n t 89^13/00 0066::3311 ECWENTURfEfl PNALYTICflL 1L7A410 884 2-3E-G9 814 231 IS 9122^ lab o ra t o rRye p oRre <t fljN^o|. FFAAqqjj, 5 ^ ; - 0 ,5 N^giig'-;e r - U 2 2 7 9 MD. 338 P82 AmendmentApproval Protocol TQX-03Q Amondmont S 7 * jL d < fr /L -- _ John L . BtaohoJ FbJ5.,Spootor'iK8|>riiUiiI'V r fty . a u * t AndzvwSaciu, Ph.D., Study Director f/* 4 g Date 3 T m : m 'Wldomednhe, Fh.D., Principal In-IUe Inveitigitor Dat 1 3Mbjiuwcwvtmwt/ Laboratory 3M Environmental Laboratory S=uV Ii'*-1. in* \j \'r* 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U22 79 Study Tide 26-Week Capsule Toxicity Study with Perfluorooctane Sulfonic Acid Potassium Salt (T-6295) in Cynomolgus Monkeys PROTOCOL AMENDMENT NO. 6 Amendment Date: September 11,2000 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 Covanee #6329-223 LIMS #U2279 3M Environmental Laboratory 3M Environmental Laboratory 3%.H7 Pa 8 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Protocol TO X 030 Amendment 6 This amendment modifies the following portion(s) of the protocol: l . Protocol reads: 8. TEST System: 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. Amend to read: Groups 2, 3, and 4 received the test substance daily for 26 weeks, at concentrations o f 0.03, 0.15, and 0.75 mg/kg/day. Reason: Test substance doses were changed after protocol was written. The Covance protocol reflects the actual doses of test substance that were given. Protocol 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. Amend to read: Peter Thomford will remain as the Covance Study Director for the purpose of issuing the Covance final report for the in-life phase of the study. Reason: Study directorship 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 Date /) ? . Andrew Seacat, Ph.D., Study Director Date 3M Environmental Laboratory 3M Environmental Laboratory y .f Pa' 39 3m Medai'aBepargenBsait ' 5 - ^ ^ - 71 410 884 a ? Rep rt N O - FACT TOX-030 laboratory Request Numb-3?2 2 7SD0S Study Title Protocol TOX-030 Amendment 7 26-weok Capsule Toxicity Study with Pcrfluorooctane 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 LMS 152219 3M Environmental Laboratory 3m, M e d i ^ ^ S p a r B a a a t S t g M ^ M Tz-g--^-..7 1 410 884 9122 Report No. FACT TOX-030 laooratory Request Numbeyo.133$7 9 cxae Amendment Approval Protocol TOX-030 Amendment 7 ~ ~ ~ ~~__________________ John L. Butenhoff, Ph>., Sponsor's Representative Date f a ' i / U t L .j T h .J b & Andrew Seacat, Ph.D., Study Director f '_________ i / t i / 'S * . ' Date ) ;:i : *H>' ; $ 3M Environmental Laboratory ^50 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 A ttachment C Extractio n and A nalytical Methods 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 Pa' 3 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 io n o f P otassium P er flu o r o o cta n esu lfo n a te o r o t h e r F l u o r o c h e m ic a l C o m po u n d s f r o m L iv e r f o r A n a l y sis u sin g HPLC- E lectrospray/M ass Spec tr o m etr y M ethod N um ber: ETS-8-6.0 Author: Lisa Clemen, Robert Wynne Approved By: Laboratory Adoption Date: Revision Date: tOft ~V Date h7 Group Leader Technical Reviewer Date C / \ h(V Date 1.0 Scope and A pplication______________________________________________________ 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey livers or other tissues as designated in the validation report. Word 6.0/95 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver *52. Page 1 of 14 Pa. 44 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Summary of Method_________________________________________________ __ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver, or other tissues, using an ion pairing reagent and methyl-tert-butyl ether (MtBE). In this method, seven fluorochemicals can be extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard. An ion pairing reagent is added to the 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 Definitions___________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) CaF i7SOj 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CgF |7S 0 2N(CH:CH3)CH:CO, 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8Fi7S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8F |7S 0 2N(CH2CH2)H 3.6 M556: C8FnS 0 2N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions______________________________________________________ 4.1 Health and Safety W arnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences_______________________________________________________________ 5.1 There are no interferences known at this time. 6.0 Equipment___________________________________________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 Ultra-Turrax T25 Grinder for grinding liver samples Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral lOOOorlEC Shaker, Eberbach or VWR ETS-8-6.0 Extraction of PFOS From Liver Page 2 of 14 3M Environmental Laboratory ^453 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 Supplies and Materials______________________________________________ _ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 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- QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards_____________________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and be provided by a Milli-Q TOC PlusTM system 8.2 Sodium hydroxide (NaOH), J.T Baker or equivalent 8.3 Tetrabutylammonium hydrogen sulfate(TBA), Kodak or equivalent 8.4 Sodium carbonate (NaXOj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCOj), J.T. Baker or equivalent 8.6 Methyl-ierf-butyl ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Liver, frozen from supplier 8.9 Dry ice from supplier 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 ETS-8-6.0 Extraction o f PFOS from Liver 3M Environmental Laboratory Page 3 o f 14 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 (3M 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 (1-H,1-H, 2-H, 2-H C8F13S 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 10N 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 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 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. 8.11.3.1 TBA requires a check prior to each use'to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na^CO/NaHCOj): Weigh approximately 26.5 g of sodium carbonate (N a,C 03) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.12 S tandards 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 solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 4 o f 14 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 Weigh approximately 50-60 mg of surrogate standard l-H .l-H , 2-H, 2-H, CsF|3S 0 3H into a 50 ml volumetric flask and record the actual weight. 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 1.0 ml of surrogate stock to a 10 ml volumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 9.0 Sample Handling_____________________________________________________________ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 10.0 Q uality Control___________________________________________________________ 10.1 M atrix blanks and method blanks 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots of liver homogenate following this procedure and use as matrix blanks. Refer to 11.1.6. 10.2 M atrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually a control liver received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range o f the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration verifications 10.3.1 Prepare continuing calibration verification samples to ensure the accuracy o f the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing calibration verification sample per group of 10 samples. For example, if a sample set = 34, four verifications are prepared and extracted. 3M Environmental Laboratory ETS-8-6.0 Extraction o f PFOS from Liver Page 5 o f 14 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.3.3 Prepare each continuing calibration verification from the same matrix 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 the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5jipb - 1000 ppb). 11.0 Calibration and Standardization_____________________________________ _ 11.1 Prepare matrix calibration 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 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 o f liver homogenate and the concentration of solid liver tissue dispersed in 1.0 mL of homogenate solution. 11.1.5 Add 1 mL of homogenate to a 15 mL centrifuge tube. Re-suspend solution by shaking between aliquots while preparing a total of eighteen 1 mL aliquots of homogeneous solution in 15 mL centrifuge tubes. 11.1.6 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. 11.1.7 Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of eighteen samples, two matrix blanks, and two method blanks. 11.1.8 Refer to validation reports ETS-8-6.0 and ETS-8-7.0-V-1 or Attachm ent B, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.9 Use Attachm ent C as an aid in calculating the concentrations of the working standards. Refer to 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each working standard, blank, or continuing verification, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb - lOOOppb. ETS-8-6.0 Extraction o f PFOS from Liver 3M Environmental Laboratory Page 6 o f 14 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 Approximate Spiking Amounts for Calibration Standards Working Standard (Approx. Cone.) 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 0.50 ppm 5.0 ppm 5.0 ppm 5.0 ppm 50 ppm Hi 2 4 10 20 40 . 10 20 30 4 Approx, final cone, of PFOS in liver Blank 0.005 ppm 0.010 ppm 0.025 ppm 0.050 ppm 0.100 ppm 0.250 ppm 0.500 ppm 0.750 ppm 1.00 ppm 12.0 Procedure__________________________________________________________________ 12.1 Obtain frozen liver samples. 12.2 Cut approximately 1 g ofliver using a dissecting scalpel. This part of the procedure is best performed quickly, not allowing the liver to thaw. 12.3 Weigh the sample directly into a tared plastic sampule vial. 12.4 Record the liver weight in the study notebook. 12.5 Return unused liver portions to freezer. 12.6 Add 2.5 mLs of water to sampule vial. 12.7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, or until the sample is homogeneous. 12.8 Rinse the probe into the sample with 2.5 mLs water using a pipette. 12.9 Take the grinder apart and clean it with methanol after each sample. Refer to AMDT-EP22. 12.10 Cap the sample and vortex for 15 seconds. Label the sampule vial with the study number, weight, liver ID, date and analyst initials. 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver Page 7 of 14 P, 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, ofhomogenate into a 15 mL polypropylene centrifuge tube. Label the centrifuge tube with the identical information as the sampule vial. Refer to attached worksheet for documenting the remaining steps. 12.12 Pipette two 1 mL aliquots ofMilli-QTM water to centrifuge tubes. These will serve as method blanks. 12.13 Spike all samples, including blanks and standards ready for extraction with surrogate standard as described in section 11.2. 12.14 Spike each matirx with the appropriate amount of standard as described in 11.1, or Table 1 of that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.15 Vortex mix the standard 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-te/7-butyl ether. 12.19 Cap each sample and put on the shaker at a setting o f 300 rpm, for 20 minutes. 12.20 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. 12.21 Label a fresh 15 mL centrifuge tube with the same information as in 12.10. 12.22 Remove 4.0 mL of the organic layer to the fresh 15 mL centrifuge tube. 12.23 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.24 Add 1.0 mL to each centrifuge tube using a graduated pipette. 12.25 Vortex mix for 30 seconds. 12.26 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this synnge. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.27 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.28 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.29 Complete the extraction worksheet, attached to this document, and tape in study notebook or include in study binder, as appropriate. ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory 5? Page 8 o f H 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 13.0 Data Analysis and Calculations________________________________________ 13.1 Calculations: 13.1.1 Calculate the average density of the liver homogenate by recording each mass of ten separate 1.0 mL aliquots of homogenate. Average density (mg/mL) = Average mass (mg') of the aliquots 1.0 mL aliquot 13.1.2 Calculate the amount of liver (mg) per 1.0 mL homogenate (or concentration of dispersed solid tissue per mL of homogenate suspension) using the following equation: g o f Liver x Average density* of homogenate (mg/mL) (g of Liver + g of Water) * refer to 13.1.1 for details. 13.1.3 Calculate actual concentrations of PFOS and other fluorochemicals in calibration standards using the following equation: 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 Method Performance_______________________________________________________ 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 Matrix spike and matrix spike duplicate samples to determine accuracy and precision o f the extraction. 14.2.3 Continuing calibration verification samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-7.0 for method performance criteria. 15.0 Pollution Prevention and Waste Management_______________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory - Page 9 of 14 Pa< 5 2 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 16.0 Records___________________________________________________________________ _ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 T ables. Diagrams. Flowcharts, and Validation Data_____________________ _ 17.1 Attachment A, Extraction worksheet 17.2 Attachment B, MDLOQ values and summary 17.3 Attachment C, Calibration standard calculation and concentration worksheet 18.0 References_________________________________________________________________ 18.1 The validation report associated with this method is ETS-8-6.0 & 7.0-V -l. 18.2 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 18.3 FACT-M-1.1, "Extraction ofPFOS or Other Anionic Fluorochemical Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 19.0 Affected Documents_______________________________________________________ 19.1 ETS-8-7.0, "Analysis o f Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions Revision Number. Reason For Revision Revision Date ETS-8-6.0 Extraction ofPFO S from Liver 3M Environmental Laboratory 5 % < ./ Page 10 o f 14 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Study # Matrix Box # Wk/Day Date Spiked/Analyst ccv MS MSD Surrogate Std approx, ppm actual ppm # - FC Mix Std approx. 0.5 ppm actual ppm # FC Mix Std approx. 5 ppm actual ppm # FC Mix Std approx. 50 ppm actual ppm # Comments -- --- " --- - - . - - B lan k Liver Hom ogenate: S td tt Liver amount = L iver E x tra ctio n M eth o d S pike surrogate and S tan d ard m ix, V ortex IS sec. Pipette 1 m L o f Liver Solution Pipette J m L o f f 0 .5 M T B A , p H 10. pH = Std. # Pipette 2 m L o f 0.25 N a n C O V 0 .2 5 M N a H C O t B uffer Std. # Dispense 5m l o f M ethyl-t-B utyl Ether TN-A- Shake 20 min. Shaker Speed Centrifuge 20-25 m m . Centrifuge Speed Rem ove a 4 m L aliquot o f organic laver Put on N itrogen E vaporator to drvncss A dd 1.0 m L o f M ethanol Evaporator Tem perature T N -A - Vortex 30 sec. Filter using a 3cc B -D sv rin g e w ith a 0 .2 u m SR I filter into autosam ple vial Com. Cal. V erifications used the same matTix as for the standard curve. - A ttachm ent B: M DL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction of PFOS from Liver 5 5 -C Z - . - - - - - - g D ate & In itials Page 11 o f 16 54 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 MDL/LOQ values for rabbit liver Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (ppb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 8.45 26.9 30 ppb - 1200 ppb PFOSA 3.50 I I . 1 12 ppb - 1200 ppb PFOSAA 24.6 78.3 30 ppb - 1200 ppb EtFOSE-OH 108 345 60 ppb - 900 ppb* M556 82.3 262 60 ppb - 1200 ppb PFOSEA 33.9 108 30 ppb- 1200 ppb MDL/LOQ values in rat, bovine, and monkey liver were not statistically determined. Two curves in each 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. Refer to LOQ Summary and MDL study in ETS-8-6.0 & 7.0-V-l for further information * EtFOSE-OH estimates only for MDL and LOQ. Did not meet criteria for validation. Compound: PFOS____________________________________________________ Prepared Range o f LCR from Range of LCR from Range of Liver range of average ave curve low std low std high std matrix standards curve curve curve curve (ppb) (n g /m L ) (ppb) (n g/m L) (ppb) (ng/m L) (ppb) (ng/m L) (ppb) (n g/m L) (ppb) (ng/m L) Rabbit 6.19 - 1237 12 - 1200 12- 1200 6-300 12-300 60 - 1200 LCR from high std curve (ppb) (ng/m L) 60-1200 Compound: PFOSA Prepared Liver range of matrix standards (ppb) (ng/m L) Range of average curve (ppb) (n g /m L ) Rabbit 6.19 - 1237 12 - 1200 LCR from ave curve (ppb) (n g/m L) 12 - 1200 Range of low std curve (ppb) (ng/m L) 12-300 LCR from low std curve (ppb) (ng/m L) 12-300 Range of high std curve (ppb) (n g/m L) 60 - 1200 LCR from high std curve (ppb) (ng/m L) 60 - 1200 Compound: PFOSAA Prepared Range of Liver range of average matrix standards curve (ppb) (n g /m L ) (ppb) (n g /m L ) Rabbit 6 .1 6 - 1232 12 - 1200 LCR from ave curve (ppb) (n g /m L ) 3 0 - 1200 Range of low std curve (ppb) (n g/m L) 30 - 900 LCR from low std curve (ppb) (ng/m L) 60 - 900 Range of high std curve (p pb ) (ng.'m L) N/A LCR from high std curve (ppb) (ng/m L) N/A A ttachm ent B: M DL/LOQ Values 3M Environmental Laboratory ETS-8-6.0 Extraction o f PFOS from Liver 54- 3 Page ! 2 o f 16 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: EtFQSE-OH Prepared Range of Liver range of average matrix standards curve (ppb) (ng/m L ) (ppb) {ng/m L) Rabbit 6 .1 7 - 1235 31-900 LCR from ave curve (ppb) (ng/m L) 31-900 Range o f low sid curve (ppb) (ng.'m Ll N/A LCR from low std curve (ppb) (ng/m L) N/A Range of high std curve (ppb) (ng/m L) N/A LCR from high std curve (ppb) (ng/m L) N/A Compound: PFOSEA Prepared Range of Liver range of average matrix standards curve (ppb) (ng/m L ) (ppb) ing/m L) Rabbit 6 . 1 7 - 1235 - 31 - 1200 LCR from ave curve (ppb) (ng/m L) 31 - 1200 Range of low std curve (ppb) (ng/m t.) N/A LCR from low std curve (ppb) (ng/m L) N/A Range of high std curve (ppb) (ng/m L) N/A LCR from high std curve (ppb) (ng/m L) N/A Compound: M556 1 ; Liver Prepared range of j matrix standards (ppb) (n g/m L) 1 Rabbit 6 .1 7 - 1235 Range of average curve (ppb) (ng/m L) 31 - 1200 LCR from ave curve (ppb) (ng/m L) 60 - 1200 R^nge of low std curve (ppb) (ng/m L) N/A LCR from low std curve (ppb) (n g/m t.) N/A Range of high std curve (ppb) (n g /m l.) N/A LCR from high std curve (ppb) (ng/m L) N/A A ttachm ent C: Standard Calculations ETS-8-6.0 Extraction of PFOS from Liver 3M Environmental Laboratory 155 6V Paste 13 o f 14 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Ion Pair Standard Curves - Tissue Prep date(s): Analyte(s): Sample matrix: 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: Standard number: Equipment number: Final solvent and TN: Blank liver/identifier: 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 I 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 g 0.167 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 Final cone ng/g 5.99 PFOSA F in a l cone ng/g 5.99 PFOSAA EtFOSE Final cone Final ng/g cone ng/g 5.99 1 5.99 PFOSEA Final cone ng/g 5.99 M556 Final cone ng/g 5.99 Std cone ng/g i 12.0 12.0 12.0 12.0 12.Q 12.0 ; 29.9 29.9 29.9 29.9 29.9 29.9 59.9 59.9 59.9 59.9 59.9 59.9 120 120 120 120 120 120 299 299 299 299 299 299 599 599 599 599 599 599 898 898 898 898 898 898 1198 1198 1198 1198 1198 1198 S u rro g ate Std cone ng/mL 100 Surrogate Final cone ng/m L 0.500 All A m 't spiked mL 0.005 Validated ranges - approximate concentrations L iv er PFOS 1 PFOSA PFOSAA Rabbit 5-1000 ppb 5-1000ppb 5-1000 ppb B o v in e Estim ates only, use rabbit values. Rat Estim ates only, use rabbit values. M onkey Estim ates only, use rabbit values. EtFOSE-OH 5-1000 ppb POAA 5-1000 ppb PFOSEA 5-1000 ppb A ttachm ent C: Standard Calculations ETS-8-6.0 Extraction o f PFOS from Liver 3M Environmental Laboratory Page 14 o f 14 Pa 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 P otassium Per flu o r o o cta n esu lfo n a te o r O t h e r F lu o r o c h em ic a ls in L iv e r E x tra cts U sing H P L C -E lectrospray/M ass Spectro m etry Method Number: ETS-8-7.0 Author: Lisa Clemen, Glenn Langenburg Approved By: cy j Laboratory Manager ... lb * --------Group Leader AVu** A Technical Reviewer Adoption Date: c f / t i l n Revision Date: fjft 7 A ^ A /r Date Date o i/i-i/ri Date 1.0 Scope and A pplication_______________________________________________________ 1.1 Scope: This method is for the analysis of liver extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey liver, or other tissues as designated in the validation report. Word 6/95 ETS-8-7.0 Analysis o f Liver Extract Using ES/.MS Page 1 o f 10 3M Environmental Laboratory 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Summary of Method__________________________________________________ _ 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z = 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity of a compound by detecting daughter ions of the selected parent ion. 3.0 Definitions____________________________________________________________ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole mass spectrometer is equipped with two quadrupole mass selective detectors and a collision cell. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion 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 Conventional 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 directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details refer to the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 Warnings and Cautions________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory 3ft 67 Page 2 o f 10 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 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 HP 1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences____________________________________________________________ _ 5.1 To minimize interferences when analyzing samples, Teflon shall not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract. 6.0 Equipment___________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source. H P1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Materials_______________________________________________________ 7.1 Supplies 7.1.1 High purity grade air regulated to approximately 100 psi (house air system) 7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data 7.1.3 Capped autovials or capped 15 ml centrifuge tubes 8.0 Reagents and Standards______________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water (ASTM type I), all water used in this method should be ATSM type I, or equivalent, and be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.1.3.1 When preparing different amounts than those listed, adjust accordingly. 8.1.3.2 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. ETS-8-7.0 Analysis o f Liver Extract Using ES/MS Page 3 o f 10 3M Environmental Laboratory 3<L .S ' 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 Sample Handling______________________________________________________ _ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples may be stored at room temperature, or refrigerated at approximately 4 C, until analysis can be performed. 10.0 Quality Control____________________________________________________________ 10.1 Method Blanks'and Matrix Blanks 10.1.1 Solvent blanks, method blanks, and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples. With a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range of the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 10.3 Continuing Calibration Checks 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 every tenth sample, with a minimum of one per batch. 11.0 Calibration and Standardization___________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set of sample extracts. The average of two standard curves will be plotted by linear regression (y = mx +b), weighted 1/x, not forced through the origin, 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. ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 4 of 10 Pa<b 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. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting o f high concentration standards. 12.0 Procedures_________________________________________________________ _ 12.1 Acquisition 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 o f 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 spectrometer headings and select SIR (Single Ion Recording) or MRM (Multiple Reaction Monitoring). 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. Refer to Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM. 12.1.3 Typically the analytical batch run sequence begins and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration verification injected standard 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 pL injection 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle time = 9 minutes ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory :fat 7 o Page 5 o f 10 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 Instrument Set-up 12.3.1 Refer to ETS-9-24.0, "Operation and Maintenance of the Micromass Quattro II Triple Quadrupole Mass Spectrometer Fitted with an Atmospheric Pressure Ionization Source," 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 Turn on the nitrogen. 12.3.5 Open the tune page. Clicks on operate to initiate source block and desolvation heaters. 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 expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed 12.3.6.4 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 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 12.3.7.5 Source block temperature 150 12.3.7.6 Desolvation temperature 250 ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page o f 10 7^ 7/ 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 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 Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, refer to appropriate MassLynx User's Guide). Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations____________________________________ _ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentrations in matrix (pg/g): fng o f PFOS calc, from std. Curve x Dilution Factor) x 1 u.g (Initial Weight of Liver (g) 1000 ng Final Volume (mL) 14.0 M ethod Performance_______________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Refer to ETS-8-6.0, Attachment B for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks and M atrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks must be below the lowest standard in the calibration curve. 14.3 C alibration 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 C ontinuing 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. ETS-8-7.0 Analysis o f Liver Extract Using ES-'MS 3M Environmental Laboratory Page 7 o f 10 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management___________________________ _ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records_________________________________________________________________ _ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted I/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. 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 o f backup electronic data. 17.0 Tables. D iagrams, Flowcharts, and Validation Data________________________ 17.1 Attachment A: ETS-8-7.0 Data summary spreadsheet 18.0 References_________________________________________________________________ 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 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-8-6.0 & 7.0-V-l 19.0 Affected Documents_______________________________________________________ 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" ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory 04 73 Page 8 of 10 Pa 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 20.0 Revisions Revision Number Reason For Revision Revision Date ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory VS7V Page 9 of 10 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Re vision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date of Analysis/Analyst: G ro u p Dose S am p le # C o n c e n tra tio n ng/g In itia l W t. B D ilution Factor Final Cone, ug/g G roup/D ose: Taken from the study folder. Sam ple#: Taken from the study folder. C oncen tratio n (ng/g): Taken from the MassLynx integration summary. Initial W t. (g): Taken from the study folder. Dilution F acto r: Taken from the study folder. Final C one, (ug/g): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-7.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Wp 7 Page 10 of 10 Pa' 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 io n ,o f P otassium Per flu o r o o c ta n esu lfo n a te o r O t h e r F l u o r o c h e m ic a l com pounds fr o m Ser u m fo r A nalysis U sin g H P L C - E lectrospray/M ass Spectro m etry M ethod N um ber: ETS-8-4.1 Adoption Date: 03/01/99 Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: Q11 Laboratory Manager >44-- -- Group Leader ? /> '> ' Date H/Li/SO Date C ^ i-3^ A C -iL .ir-C ^ Technical Reviewer o ih u h 'i Date 1.0 Scope and A pplication 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. W ord 6/95 E T S-8-4.1 Extraction o f PFOS from Serum 3M Environmental Laboratory Page 1 of 14 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Summary of Method__________________________________________________ _ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum, or other fluids, using an ion pairing reagent and methyl-/eri-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 mL of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 2.2 These sample extracts are analyzed following method ETS-8-5.1 or other appropriate methods. 3.0 Definitions__________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) C8F,7S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide C8FnS 0 2XH; 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F|7S 0 7N(CH,CH3)CH2C 0 :' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol CgF 17S 0 2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamideCsF 17S 0 2N(CH2CH3)H 3.6 M556: C8F l7S 0 2N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 W arnings and Cautions ______________________________________________ 4.1 Health and safety warnings 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, which may contain pathogens. 5.0 Interferences_____________________________________________ _________ __________ 5.1 There are no interferences known at this time. 6.0 Equipment___________________________________________________________________ 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 ETS-8-4.1 Extraction of PFOS from Serum 3M Environmental Laboratory Vfc 77 Page 2 of 14 Pa' 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 upplies and Materials_______________________________________________ _ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable o f measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli-QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards______________________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and 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^COj), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHC03), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 ETS-8-4.1 Extraction of PFOS from Scrum Page 3 o f 14 3M Environmental Laboratory O f? * 3m Medical Department Study: T-6295.7 Report No. FACT TOX-Q30 laboratory Request Number-U2279 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 C8F uS0 3H) molecular weight = 428 8.9.8 Other fluorochemicals, as appropriate 8.10 Reagent preparation NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly. 8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle. 8.10.2 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL o f 10 N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g o f TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. .* 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na2C 0 3/NaHC03): Weigh approximately 26.5 g of sodium carbonate (N a X 0 3) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard o f approximately 1000 ppm (pg/mL). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. ETS-8-4.1 Extraction of PFOS from Serum Page 4 o f 14 3M Environmental Laboratory 7 6 79 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.12 Surrogate stock standard preparation 8.12.1 Weigh approximately 50-60 m g o f surrogate standard 2-H, 2-H, CgFnS 0 3H into a 50 mL volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. ` 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 mL o f surrogate stock to a 10 mL volumetric flask and bring to volume with methanol for a working standard oflOO ppm. Record the actual volume transferred. 9.0 Sample Handling______________________________________________________ ______ 9.1 All samples are received frozen and must be kept frozen until the extraction is performed. 9.2 Allow samples to thaw to room temperature prior to extraction. 10.0 Quality Control____________________________________________________________ 10.1 Solvent Blanks, Method blanks and matrix blanks 10.1.1 An aliquot o f 1.0 mL methanol is used as a solvent blank. 10.1.2 Extract two 1.0 mL aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.3 Extract two 1.0 mL aliquots o f the serum following this procedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare continuing calibration check samples to ensure the accuracy of the initial calibration curve. 10.3.2 Prepare, at a minimum, one continuing check per group of 10 samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. ETS-8-4.1 Extraction o f PFOS from Serum 3M Environmental Laboratory "H 8 0 Page 5 o f 14 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.3.4 The expected concentrations will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 C alibration and Standardization 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 mL o f serum to a 15 mL centrifuge tube. 11.1.2 If most sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract less than 0.50 mL of matrix. Record each sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots in. 15 mL centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table l, 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 Use Attachment D as an aid in calculating the concentrations o f the working standards. See Section 13.0 to calculate actual concentrations o f PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. E T S -8 -4 .1 Extraction o f PFOS from Serum 3M Environmental Laboratory "FZ- Sri Page 6 o f 14 3m Medical Department Study: T-6295.7 Report N o . 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 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 . 1.00 ppm 12.0 Procedure__________________________________________________________________ 12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath. 12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 Return unused samples to freezer after extraction amounts have been removed. 12.4 Record the initial volume on the extraction worksheet. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly. 12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL o f 0.25M sodium carbonate/sodium bicarbonate buffer. 12.11 Using an Oxford Dispenser, add 5 mL methyl-rerr-butyl ether. 12.12 Cap each sample and put on the shaker at a setting o f 300 rpm, for 20 minutes. 12.13 Centrifuge for 20 to 25 minutes at a setting of 3500 rpm, or until layers are well separated. ETS-8-4.1 Extraction o f PFOS from Scrum Page 7 of 14 3M Environmental Laboratory Pa> 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 Put each sample on the analytical nitrogen evaporator until dry, approximately 1 to 2 hours. 12.17 Add 1.0 mL of methanol to each centrifuge tube using a graduated pipette. 12.18 Vortex mix for 30 seconds. 12.19 Attach a 0.2 pm nylon mesh filter to a 3 cc syringe and transfer the sample to this syringe. Filter into a 1.5 mL glass autovial or low-volume autovial when necessary. 12.20 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.21 Cap and store extracts at room temperature or at approximately 4 C until analysis. 12.22 Complete the extraction worksheet, attached to this document, and tape in the study notebook or include in study binder, as appropriate. 13.0 Data Analysis and Calculations______;______________________________________ 13.1 Calculations 13.1.1 Calculate actual concentrations o f PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration of standard (ug /mL)__________________= mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) o f PFOS in matrix 14.0 Method Performance_______________________________________________________ 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 Attachments B and C). 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 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 14.3 Refer to section 14 of ETS-8-5.1 for method performance criteria. 15.0 Pollution Prevention and Waste Management________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. ETS-8-4.1 Extraction o f PFOS from Serum Page 8 o f 14 3M Environmental Laboratory 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 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. 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 ETS-8-5.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 R e v i s i o n s ____________________________________________________________ Revision Number 1 Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed/'" Section 12.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1.0 mL. Revision Date 04/02/99 ETS-8-4.1 Extraction of PFOS from Senim 3M Environmental Laboratory Page 9 of 14 3m Medical Department Study: T - 6 2 9 5 .7 Report No. FACT TOX-030 laboratory Request Number~U2279 Extraction Worksheet ETS-8-4.1 Study # Surrogate Std M atrix approx, Box # actual W k/D av # DateSpiked/Analyst j ppm ppm CCV ! MS i MSD | ! ! i1 1 i I 1 ( FC-Mix approx. 0.5 pm actual ppm U Tt FC-Mix approx. 5 ppm actual ppm U Tt FC-Mix approx. 50 ppm actual ppm # Comments Blank i 1 ! I ! i ! (i 1 | S td # -- --- - -- - 1: -- - - ---- am ount = S eru m E x tra ctio n M eth o d V o rtex 15 sec. P ipette M atrix P i p e t t e 1 m L o f 0 .5 M T B A , pH 10. p H = V o lu m e S td. # mL P ip ette 2 m L o f 0.25 N a ? C O y 0 .2 5 M N a H C O i b u ffer S td. tt D ispense 5 m L o f m eth y l-t-b u ty l e th er T N -A - Shake 20 mm.___________________________________________ Shaker speed: Centrifuge 20-25 min._________________________________ Centrifuge speed. Remove a 4 mL aliquot of organic laver__________________________________ Put on Nitrogen Evaporator to dryness_______________________ Temperature: Add methanol_____________ Volume mL_____________TN-A- Vortex 30 sec._______________________________________________________ Filter using a 3cc B-D syringe with a 0.2um filter into a 1.5 mL autosample vial Cont. Cai. V erifications used same matrix as for std curve. - - - - - - mL D ate & In itials Attachment A 3M Environmental Laboratory ETS-8-4.1 Extraction o f PFOS from Serum Page 10 o f l 4 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (PPb) (ppb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.74 5.55 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 M556 6.03 19.2 5 ppb - 1000 ppb PFOSEA 5.71 18.2 5 ppb - 1000 ppb M D L /L O Q values in rat, bovine, m onkey, and hum an serum , and m onkey plasm a w ere not statistically determ ined. T w o curves in each o f these m atrices w ere extracted and analyzed w ith the rabbit serum curves to d eterm in e equivalence. Responses in the rat, bovine, m onkey, and hum an w ere equivalent to the rabbit responses, therefore, their M DL and LO Q w ill be the sam e values as determ ined in rabbit serum . P lease see L O Q S um m ary and M D L study in E T S-8-4.0 & 5.0-V -l for further inform ation. Attachment B: MDL/LOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum Page 11 o f 14 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: PFOS Prepared range Rabbit Serum o f standards (ppb) (ng/m L) Full R ange Low Curve H igh curve 1/X 0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978 Compound: PFOSA . Prepared range Rabbit Serum o f standards (ppb) (ng/m L) Full R ange Low Curve H igh curve 1/X 0.993 - 976 4.93 - 97.6 24.8 -976 0.993 - 976 Compound: PFOSAA Prepared range Rabbit Serum o f standards (ppb) (ng/m L) Full R ange Low Curve H igh curve 1/X 0.991 -974 4.92 - 247 49.2 - 974 0.991 - 974 LCR from curve (PPb) (ng/m L ) 24.8 - 978 4.94 - 248 97.8 -9 7 8 4.94 - 978 LCR from curve (PPb) (ng/m L) 4.93 - 976 4.93 - 97.6 24.8 - 978 4.93 - 976 LCR from curve (PPb) (ng/m L) 24.7 - 974 9.74 - 247 97.4 - 974 9.74 - 974 % Recovery Range 83-108 85-104 85-106 94-111 % Recovery Range 88-103 87-105 93-102 94-103 % Recovery Range 81-111 97-107 85-108 95-115 RSD Range 4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5 RSD Range 5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5 RSD Range 4.18-10.6 6.38-21.8 4.33-12.5 4.11-23.2 A ttachm ent B: M DL/LOQ Summary 3M Environmental Laboratory ETS-8-4.1 Extraction of PFOS from Serum ^ S-7 Page 12 of 14 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: EtFQSE-OH Prepared range R abbit Serum o f standards (ppb) (ng/m L) Full Range Low Curve H igh curve l/X 0.993 - 976 4.93 - 97.6 49.3 - 976 0.993 - 493 Compound: PFOSEA Prepared range R abbit Serum o f standards (ppb) (ng/m L) Full Range Low Curve H igh curve l/X 0.993 - 976 4.93 -248 49.3 - 976 0.993 - 976 Compound: MS56 Prepared range R abbit Serum o f standards (ppb) (ng/m L) Full R ange Low Curve H igh curve l/X 0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976 LCR from curve (PPb) (ng/m L) 49.3 - 976 9 .7 6 -9 7 .6 97.6 976 9.76 - 976 LCR from curve (ppb) (ng/m L) 24.8 - 976 9.76 - 248 49.3 - 976 9.76 - 976 LCR from curve (ppb) (ng/m L ) 24.8 -976 9.76-97.6 97.6 - 976 9.76- 976 % Recovery Range 77-110 97-107 90-109 86-111 % Recovery Range 96-106 91-110 86-106 95-117 % 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 o f PFOS from Serum n > s-fr Page 13 o f 14 Pa' 0 3m Medical Department Study-. T-6295.7 Report N o . FACT TOX-C laboratory Request Number-U22 7_- Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifler: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PEOSAA EtFOSE PFOSEA Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.507 0.532 0.501 0.521 0.500 0.507 0.532 0.501 0.521 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 5.00 5.07 5.32 5.01 5.21 50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 r50.0 50.1 53.2 50.1 52.1 50.0 50.1 53.2 50.1 52.1 M556 Std cone ug/mL 0.501 0.501 5.01 5.01 5.01 50.1 50.1 50.1 50.1 All Ain't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All Final voi mL j 1.015 ! 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Calculated concentrations of standards in the sample matrix PFOS PFOSA Final cone Final cone n g/m L n g/m L 4.93 5.00 9 .7 6 9.89 24.8 25.1 49.3 50.0 1 97.6 9 8 .9 248 251 493 500 735 I 746 9 7 6 ' 989 NO>s i PFOSAA Final cone n g /m L 5.24 10.4 26.3 52.4 104 524 782 1038 EtFOSE PFOSEA M556 Final cone Final cone Final cone ng/mL n g /m L n g/m L 4.94 j 5.01 5.13 9 .7 8 r 9.93 ! 10.2 24.8 ; 25.2 25.8 49.4 1 50.1 ! 51.3 9 7 .8 ! 99.3 102 248 j 252 258 494 i 501 513 737 i 749 1 766 978 ! 993 1 1017 S u rro g a te Std cone n g /m L 100 S u rro g a te Final cone n g/m L 500 A ll A in 't spiked mL 0.005 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA EtFO SE-O H PFOSEA M556 Rabbit 5.00-1000 | 5.00-1000 j 5.00-1000 j 5.00-1000 | 5.00-1000 | 5.00-1000 Bovine Estimates only. Use values for rabbit. Rat Estimates only. Use values for rabbit. M onkey & Plasma Estimates only. Use values fo r rabbit. Human Estimates only. Use values for rabbit. A ttachm ent C: Ion Pair Standard Curves ETS-8-4.1 Extraction o f PFOS from Serum 3M Environmental Laboratory Page 14 o f 14 P1 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03Claboratory Request Number-U2279 3M Environmental Laboratory M ethod An a ly sis q f P otassium Perflu o ro o cta n esu lfo n a te o r O t h e r F l u o r o c h em ic a ls in Ser u m E x tra cts U sing H P L C -E lectrospray/M ass Spec tr o m etr y M ethod N um ber: ETS-8-5.1 Author: Lisa Clemen, Robert Wynne Approved By: 'll Laboratory Manager Group Leader U .. - __A Cb.rrK'- . Technical Reviewer Adoption Date: 03/01/99 Revision Date: /Lf z c . . Date Date Date 1.0 Scope and A pplication________________________________________________________ 1.1 Scope: This method describes the analysis o f serum extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 6/95 ETS-8-5.1 Analysis of Serum Extract Using ES/MS Page 1 of 9 3M Environmental Laboratory Pai 82 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Summary of Method_____________________________________________________ _ 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m lz- 499. 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 Definitions______________________________________________________________ ___ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI), Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e., not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method o f ionization performed at atmospheric pressure, whereby ions in solution are transferred to the gas phase via tiny charged droplets. These charged droplets are produced by the application of a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II triple quadrupole systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II triple quadrupole systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation o f these Quattro II triple quadrupole systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II triple quadrupole MassLynx or MassLynx NT User's Guide). 4.0 W a r n i n g s a n d C a u t i o n s ____________________________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory ^ 9/ Page 2 of 9 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 Interferences._______________________________________________________________ 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 Equipment___________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications inthe raw data as method deviations. 6.1.1 6.1.2 Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler 7.0 Supplies and Materials_______________________________________________________ 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 Reagents and Standards______________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent 8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor 8.1.3 Ammonium acetate, reagent grade or equivalent 8.2 Standards 8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1. 9.0 Sample Handling_____________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 3 of 9 Paqa^84 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed. 10.0 Quality Control ____________________________________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks 10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover. 10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve. 10.2 Matrix Spikes 10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency. 10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte. 10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum of 2 spikes per batch. 10.2.4 Matrix spike and matrix spike duplicate concentrations will fall in the mid-range o f the initial calibration curve. Additional spike concentrations may fall in the lowrange of the initial calibration curve. 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 of one per batch. 11.0 Calibration and Standardization____________________________________________ 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 use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory 93 Page 4 o f 9 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.0 Procedures____________________________________________________ _____________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the 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 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 = 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 o f Serum Extract Using ES/MS 3M Environmental Laboratory Page 5 of 9 Pagers 6 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 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 o f the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn off the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip o f the probe if no mist is observed. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 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 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 o f biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data analysis and Calculations_____________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (Hg/mL): (rig of PFOS calc, from std. Curve x Dilution Factor) x 1 pg (Initial Volume of matrix (mLl + mL of Surrogate Standard) lOOOng Final Volume (mL) ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory ? Page 6 of 9 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 14.0 Method Performance___________________________________________ _________ _ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-4.1, Attachment B, for a listing of current validated MDL and LOQ values. 14.2 Solvent Blanks, Method Blanks, and Matrix Blanks 14.2.1 Solvent blanks, method blanks, and matrix blanks values are must be below the lowest standard in the calibration curve 14.3 Calibration Curves 14.3.1 The r2value for the calibration curve must be 0.980 or better. 14.4 Matrix Spikes 14.4.1 Matrix spike percent recoveries are must be within 30% of the spiked concentration. 14.5 Continuing Calibration Verifications 14.5.1 Continuing calibration verification percent recoveries must be 30% of the spiked concentration. 14.6 If criteria listed in this method performance section isn't met, maintenance may be performed on the system and samples reanalyzed or other actions as determined by the analyst. Document all actions in the appropriate logbook. 14.7 If data are to be reported when performance criteria have not been met, the data must be footnoted on tables and discussed in the text of the report. 15.0 Pollution Prevention and Waste Management_______________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records____________________________________________________________________ 16.1 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. ETS-8-5.1 Analysis of Serum Extract Using ES/'MS 3M Environmental Laboratory Page 7 of 9 Pa- 8 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see A ttachm ent A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Data_______________________ __ 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet. 18.0 References____________________________________________________________ _ 18.1 FACT-M-4.1, "Extraction of Potassium 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 Affected Documents________________________________________________________ 19.1 ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions____________________________________________________________________ Revision Number. 1 Reason For Revision Section 6.1.2 Clarification of HP1100 system components. Section 11.1 Average of two curves, not standard values, are used for plotting linear regression and added the 1/x weighting of the curve. Section 12.2.2.4 Clarification o f solvent ramp. Section 17.1 Changed from attachment B to A. Revision Date 04/02/99 ETS-8-5.1 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory <&&97 Page 8 of 9 Pa' 9 3m Medical Department Study: T-6295.7 Report N o . FACT T O X - 0 3 0 laboratory Request Number-U2279 Laboratory Study # Study: Test Material: M atrix/Final Solvent: M ethod/R evision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analyst; Date o f Analysis/Analyst: G roup Dose S am ple# C o n c en tratio n u g/m L Initial Vol. mL D ilu tio n Factor Final Cone. u g/m L Slope: Taken from linear regression equation. G roup/D ose: Taken from the study folder. Sam ple#: Taken from the study folder. C o n cen tratio n (tig/m L): Taken from the MassLynx integration summary. Initial V olum e (m L): Taken from the study folder. D ilution F acto r: Taken from the study folder. F in al C one. (ug/m L ): Calculated by dividing the initial volume from the concentration Attachment A: Summary Spreadsheet ETS-8-5.1 Analysis of Scrum Extract Using ES/MS 3M Environmental Laboratory 9 Page 9 of 9 Pa> 9 0 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 io n s P otassium Perfluorooctanesulfonate or O th er Fluorochem ical com pounds from Serum for A nalysis U sing H PL C - E lectrospray/M ass Spectrom etry Method Number: ETS-8-4.0 Adoption Date: j/ r '' Author: Lisa Clemen, Glenn Langenburg Revision Date: Approved By: 0 ^ /5 - -- Laboratory Manager^ V, Date ---- f------------- ---------------------------------------------------------Group Leader .3/1 Date k4 Technical Reviewer 5 / / / `h Date 7 1.0 Scope and Application 1.1 Scope: This method is for the extraction of potassium perfluorooctanesuifonate (PFOS) or other fluorochemical compounds from serum. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report. Word 97 S R -1 3M Environmental Laboratory ETS-8-4.0 Extraction of PFOS from Serum Page 1 of 13 3m Medical Department Scudy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Summary of Method _________________________________________________ __ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from serum using an ion pairing reagent and 5.0 mL of methyl-ieri-butyl ether (MtBE). In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, PFOSEA, M556, and surrogate standard (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into MtBE. The MtBE extract is removed and put onto a nitrogen evaporator until dry. Each extract is reconstituted in 1.0 ml of methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 pm nylon filter into glass autovials. 3.Q Definitions _____________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) CsFnSO]' 3.2 PFOSA: perfluorooctane sulfonylamide CgFnSOzNH: 3.3 PFOSAA: perfluorooctane sulfonylamido (dthyl)acetate C gF nS C hN iC ^C fylC ^C C E ' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol CsF, 7SO2N(CH2CH3)CH2CH20H 3.5 PFOSEA: perfluorooctane sulfonyl ethylamide CgFnSC^NfCH^CH-OH 3.6 M556: CgF,7S02N(H)(CH2C 00H ) 3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions_____________________________________________________ 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 Interferences______________________________________________________________ 5.1 There are no interferences known at this time. 6.0 Equipment__________________________________________________________ 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) ETS-8-4.0 Extraction of PFOS from Serum 3M Environmental Laboratory 60 Paste 2 of 13 92 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 7.0 Supplies and Materials 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, 15mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 ml 7.9 Syringes, capable of measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe 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-Q ` water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards_____________________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-Q Mwater 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 (Na2CO), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHOEh), J.T. Baker or equivalent 8.6 Methyl-T-Butyl Ether, Omnisolv, glass distilled or HPLC grade 8.7 Methanol, Omnisolv, glass distilled or HPLC grade 8.8 Serum or blood, frozen from supplier 8.9 Fluorochemical standards 8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570 ETS-8-4.0 Extraction of PFOS from Serum 3M Environmental Laboratory Page 3 of 13 Pa> 1 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 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 C8F 13SO3H) 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 Milli-QTM water. Store in a 125 mL Nalgene bottle. 8.10.3 0.5 M tetrabutylammonium hydrogen sulfate (TBA): Weigh approximately 169 g of TBA into a 1 L volumetric containing 500 mL Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 mL of 10 N NaOH (While adding the last mL of NaOH, add slowly because the pH changes abruptly). Dilute to volume with Milli-QTM water. Store in a 1 L Nalgene bottle. 8.10.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.10.4 0.25 M sodium carbonate/sodium bicarbonate buffer (NaiC03/NaHC0 3 ): Weigh approximately 26.5 g of sodium carbonate (Na^COs) and 2 1.0 g of sodium bicarbonate (NaHCOs) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 8.11 Standards preparation 8.11.1 Prepare PFOS standards for the standard curve. 8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.11.3 Weigh approximately 100 mg of PFOS into a 100 ml volumetric flask and record the actual weight. 8.11.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/ml). 8.11.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm. 3M Environmental Laboratory ETS-8-4.0 Extraction of PFOS fromSerum jo a . Page 4 of 13 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 l-H.l-H, 2-H, 2-H, CgFnSChH into a 50 ml volumetric flask and record the actual weight. 8.12.2 Bring to volume with methanol for a surrogate stock of approximately 1000-1200 ppm. . 8.12.3 Prepare a surrogate working standard. Transfer approximately 1 ml of surrogate stock to a 10 ml volumetric flask and bring to volume with methanol for a working standard of 100 ppm. Record the actual volume transferred. 9.0 Sample Handling_____________________________________________________________ 9.1 All samples arrreceived frozen and must be kept frozen until the extraction is performed. 10.0 Quality Control____________________________________________________________ 10.1 Method blanks and matrix blanks 10.1.1 Extract two 1.0 ml aliquots of Milli-QTM water following this procedure and use as method blanks. 10.1.2 Extract two 1.0 mL aliquots of the serum following this procedure and use as matrix blanks. See 11.1.4. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and matrix spike duplicate per 40 samples, with a minimum of 2 matrix spikes per batch. 10.3 Continuing calibration checks 10.3.1 Prepare and analyze continuing calibration check samples to ensure the accuracy of the initial calibration curve. If the percent difference between the initial curve and the continuing check differ by >30%, re-analyze samples analyzed after the last acceptable check. 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 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. 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 3M Environmental Laboratory ETS-8-4.0 Extraction of PFOS from Serum Page 5 o f 13 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request N u m b e r - U 2 2 79 calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - IOOO ppb). 11.0 Calibration and Standardization_____________________________________ __ 11.1 Prepare matrix calibration standards 11.1.1 Transfer 1 m L 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 volumes equal to the sample volumes. Do not extract less than 0.50 mL of matrix. Record each sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots in 15 ml centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 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 of 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 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or continuing check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb 1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. 3M Environmental Laboratory ETS-8-4.0 Extraction of PFOS from Serum Page 6 of 13 Pa' 6 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U2279 Table 1 Approximate spiking amounts for standards and spikes Using 1.0 ml of matrix Working standard (approx, cone.) (iL Approx, final cone, of 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 5CT0 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 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. 12.3 Return samples to freezer after extraction amount has been removed. 12.4 Record the volume on the extraction worksheet. 12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2. 12.7 Spike each matrix with the appropriate amount of standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards. 12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds. 12.9 To each sample, add 1 mL 0.5 M TBA and 2 mL of 0.25M sodium carbonate/sodium bicarbonate buffer. 12.10 Using an Oxford Dispenser, add 5 mL methyl-feri-butyl ether. 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 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. 3M Environmental Laboratory ETS-8-4.0 Extraction of PFOS from Scrum ^ a / S Page 7 of 13 Pai 7 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 graduated pipette. Methanol volume to add equals the initial volume of sample used for the extraction. 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 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.19 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 Data Analysis and Calculations_____________________________________________ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration of standard (qg /mL)__________________= mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (pg/mL) of PFOS in matrix 14.0 M ethod Performance_______________________________________________________ 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 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 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction. 14.2.3 Continuing calibration check samples to determine the continued accuracy of the initial calibration curve. 15.0 Pollution Prevention and Waste Management________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 3M Environmental Laboratory ETS-8-4.0 Extraction of PFOS from Serum / ol Page 8 of 13 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request Number-U2279 16.0 Records_______________________________________________________________ 16.1 Complete the extraction worksheet attached to this method, and tape in the study notebook or include in the 3-ring study binder, as appropriate. 17.0 Attachments__________________________________________________________ 17.1 Attachment A. Extraction worksheet 17.2 Attachment B, MDL/LOQ values and summary 17.3 Attachment C, Calibration standard concentration worksheet 18.0 References___________________________________________________________ 18.1 The validation report associated with this method is ETS-8-4.0 & 5.0-V-l. 19.0 Affected Documents__________________________________________________ 19.1 ETS-8-5.0, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions Revision Number Reason For Revision Revision Date 3M Environmental Laboratory ETS-8-4.0 Extraction of PFOS from Scrum fo/ Page 9 of 13 Pa> 9 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Extraction Worksheet ETS-8-4.0 Study #/matrix - 1 Surrogate Std approx. ppm actual ppm # FC-Mix approx. 0.5 ppm actual ppm # - - FC-Mix approx. 5 ppm actual ppm # - - -------.- --- FC-Mix approx. 50 ppm actual ppm # - - Date and Initials for Std. or Comments - - - --- - - - -- 1Study nnm her wlacre the original work.sheet i.s-lncated______ Blank Std # amount = mL Serum Extraction Method Vortex 15 sec. : Date & Initials Picette Matrix .,, _ Volume _ m| , ......... ............. .. Pipette 1 ml of 0.5 M TBA, pH 10. Std. # Pipette 2 ml of 0.25 Na,CO,/0.25M NaHCO, buffer Std # Dispense 5 ml of methyl-t-butyl ether TN-A- Shake 20 min. Centrifuee 20-25 min. Centrifuge speed: Remove a 4 mL aiiauot of organic layer Put on Nitrogen Evaporator to drvness Evaporator#: Temperature: Add methanol Volume ml TN-A- Vortex 30 sec. F ilter u s in g a 3 c c B -D s y rin g e w ith a 0 .2 u m filter into a 1.5 ml a u to sa m p le vial M S/M SD /___ Coot. Checks'. S p ik ed ______ uL of a ______ ppm std (_______________ ) for a final concentration of _________ppm. M S/M SD used sa m p le ________________ . Cont. Checks used same matrix as for std curve. Surrogate Standard: S p ik e d _____ uL of a ______ ppm std (_______________ ) to all samples, standards, and blanks Attachment A 3M Environmental Laboratory ETS-8-4.0 Extraction of PFOS from Scrum Page IOof 13 Page^afOO 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 MDL/LOQ values for rabbit serum Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 4.76 15.2 5 ppb - 1000 ppb PFOSA 2.89 9.19 5 ppb - 1000 ppb PFOSAA 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. Please see Attachment C (LOQ Summary) and MDL study in ETS-8-4.0 & 5.0-V-l for further information. Ion Pairing Extraction of Fluorochemicals from Serum and Analysis by API/MS(MS) Summary Table: Limits of Quantitation Compound M atrix MDL PFOS All 4.76 PFOSA All 2.89 PFOSAA All 2.94 E tF O S E -O H * 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 A ttachm ent B: M D L/LO Q 3M Environmental Laboratory ETS-8-4.0 Extraction of PFOS from Serum t-/0? Page 11 o f 13 Pa' 101 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03Q laboratory Request Number-U2279 Compound: PFOS Prepared Serum range o f matrix standards (ppb) (ne/m L) Rabbit 1.00- 1002 Range of average curve fp p b ) (ng/m L) 1.00 - 1002 LCR from ave curve (ppb) (ne/m L) 25.1 - 1002 Range of low std curve (ppb) (n e/m L) 5.01 - 250 LCR from low std curve (ppb) (ne/m L) 5.01 -2 5 0 Range of high std curve (ppb) in e /m L ) 100- 1002 LCR from high std curve (pob) in e /m L ) 100 - 1002 Compound: PFOSA Prepared Serum range of matrix standards (ppb) (ng/m L) Rabbit 1.00 - 1000 Range of average curve (ppb) (ne/m L) 1.00 - 1000 LCR from ave curve (ppb) (n e/m L) 10.0-1000 Range of low std curve (ppb) (ne/m L) 5.00- 100 LCR from low std curve (ppb) (ng/m L) 5 .0 0 - 100 Range of high std curve (ppb) (ne/m L) 2 5 .0 - 1000 LCR from high std curve (ppb) (ne/m L) 25.0 - 1000 - Compound: PFOSAA Prepared Range of Scrum range of average matrix standards curve (ppb) (n g/m L) (ppb) (ng/m L) Rabbit i 1.00-998 1.00-998 LCR from ave curve (ppb) (ne/m L) 25.0 - 998 Range of low std curve (ppb) (ng/m L) 4.99 - 250 LCR from low std curve (ppb) (ng/m L) 9.98 - 250 Range of high std curve (ppb) (ng/m L) 49.9 - 998 LCR from high std curve (ppb) (ne/m L) 99.8 - 998 Compound: EtFOSE-OH Prepared Range of Serum range o f average matrix standards curve (ppb) (ng/m L) (ppb) (ng/m L) Rabbit 1.00- 1000 1.00- 1000 LCR from ave curve (ppb) (ne/m L) 50.0- 1000 Range of low std curve (ppb) (n e/m L) 5 .0 0 - 100 LCR from low std curve (ppb) (ne/m L) 10.0- 100 Range of high std curve (ppb) (ne/m L) 50.0 - 1000 LCR from high std curve (ppb) (ng/m L) 100 - 1000 Compound: PFOSEA Prepared Serum range of matrix standards (ppb) (ne/m L) Range of average curve (ppb) (ne/m L) Rabbit 1.00- 1000 1.00 - 1000 LCR from ave curve (ppb) (n e /m L ) 25.0- 1000 Range of low std curve (ppb) (ne/m L) 5.00 - 250 LCR from low std curve (ppb) (ng/m L) 5.00- 250 Range of high std curve (ppb) (ne/m L) 5 0 - 1000 LCR from high std curve (ppb) (ne/m L) 50 - 1000 Compound: M556 Prepared Serum range of matrix standards (ppb) (ne/m L) Rabbit 1.00 - 1000 Range of average curve (ppb) (ne/m L) 1.00 - 1000 LCR from ave curve (ppb) (ne/m L) 25.0- 1000 Range of low std curve (ppb) (ne/m L) 5.00 - 100 LCR from low std curve (ppb) (ne/m L) 5.00 - 100 Range of high std curve (ppb) (ne/m L) 100 - 1000 LCR from high std curve (ppb) (n e/m L) 100 - 1000 Attachment B: MDL/LOQ 3M Environmental Laboratory ETS-8-4.0 Extraction of PFOS from Serum nH- no Page 12 o f 13 Pa' 02 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03C laboratory Request Number-U2279 Ion Pair Standard Curves - Fluids Prep date(s): Standard number: Analyte(s): Equipment number: Sample matrix: Final solvent and TN: Blank fluid/identifier: Method/revision: Target analyte(s): FC mix std approx. 0.500 ppm: FC mix std approx. 5.00 ppm: FC mix std approx. 50.0 ppm: Surrogate std approx. 100 ppm: Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Std cone Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL ug/mL 0.500 0.507 0.532 ; 0.501 0.521 0.501 0.500 0.507 0.532 1 0.501 0.521 0.501 5.00 5.07 5.32 : 5.01 5.21 5.01 5.00 5.07 5.32 ! 5.01 5.21 j 5.01 5.00 5.07 5.32 5.01 5.21 : 5.01 50.0 50.1 53.2 50.1 52.1 " 1 50.1 50.0 50.1 53.2 50.1 52.1 50.1 50.0 50.1 53.2 50.1 52.1 j 50.1 50.0 50.1 53.2 50.1 52.1 1 50.1 All Ain't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 All Final voi mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Calculated concentrations of standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE PFOSEA M556 Surrogate Final cone Final cone Final cone Final cone Final cone Final cone Std cone ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL ng/mL 4.93 5.00 5.24 1 4.94 ! 5.01 1 5.13 100 9.76 ! 9.89 10.4 1 9.78 ; 9.93 10.2 24.8 ' 25.1 26.3 1 24.8 25.2 i 25.8 Surrogate 49.3 50.0 ! 52.4 j 49.4 50.1 I 5.3 Final cone 97.6 ! 98.9 104 j 97.8 99.3 102 ng/mL 248 251 i 263 j 248 252 258 500 493 ! 500 1 524 i 494 501 513 735 746 1 782 737 749 766 976 989 1038 ! 978 993 1017 All Amt spiked mL 0.005 Validated ranges - approximate concentrations Serum PFOS PFOSA PFOSAA EtFOSE-OH PFOSEA M556 Rabbit 25.1-1002 | 25.0-1000 | 25.0-998 | 50.0-1000 1 25.0-1000 | 25.0-1000 Bovine Estimates onlv. Use values for rabbit. Rat Estimates only. Use values for rabbit. Monkey Estimates only Use values for rabbit. Human Estimates only Use values for rabbit. Attachment C: Ion Pair Standard Curves ETS-8-4.0 Extraction of PFOS from Scrum 3M Environmental Laboratory irrt* m Page 13 o f 13 Pa' 3 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory M ethod A nalysis o f P otassium P erflu o r o o cta n esu lfo n a te o r O th er F l u o r o c h em ic a ls in S eru m E x tra cts Using H P L C -E lectrospray/M ass Spectrom etry Method Number: ETS-8-5.0 Author: Lisa Clemen, Robert Wynne Approved By: -----------.--ly - - -r " - --'-/ "-- 1 - ---Laboratory Manager -- -------- ~ Group Leader / ^ k fi Cli/rr-iv Technical Reviewer Adoption Date: b / j/ -7 :/ Revision Date: fry Date >/> f r - ) Date i h l rr i Date 1.0 Scope and Application_______________________________________________________ 1.1 Scope: This method is for the analysis of extracts from serum for fluorochemical surfactants using HPLC-electrospray/mass spectrometry. 1.2 Applicable Compounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds. 1.3 Matrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in the validation report. Word 97 SR-1 ETS-8-5.0 Analysis of Serum Extracr Using ES/MS 3M Environmental Laboratory T&brU2. Page I of 9 P 104 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Summary of Method_____________________________________________________ 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, m/z= 499. Samples may also be analyzed using an API-ES/MS/MS system to further verify compound identification. 3.0 Definitions_______________________________________________________________ __ 3.1 Atmospheric Pressure Ionization (API): The Micromass Quattro II systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These 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 Electrospray 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 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API Quattro II systems are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass Quattro II systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with some other Z-spray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these Quattro II systems. Currently MassLynx has Windows 95 and WindowsNT 4.0 versions. All versions are similar. For more details see the manual specific to the instrument (Micromass Quattro II MassLynx or MassLynx NT USER'S GUIDE). 4.0 Warnings and Cautions______________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cables for the probe. The probe employs a voltage of approximately 5000 Volts. 4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing. ETS-8-5.0 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory US Pase 2 of 9 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 HPl 100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences_______________________________________________________________ 5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment ______________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HPl 100 low pulse solvent pumping system and autosampler 7.0 Supplies and Materials______________________________________________________ 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 Reagents and Standards_____________________________________________________ 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 Sample Handling____________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 If analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C until analysis can be performed. ETS-8-5.0 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory ros- //y Pago 3 of 9 Pa> 0 6 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.0 Quality Control___________________________________________________ _ 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 Matrix 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 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 Sectipn 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the 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 Calibration and Standardization____________________________________________ 11.1 Analyze the extracted matrix 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 ( f )for the calibration curve using MassLynx or other suitable software. y 11.2 The r" value for the data should be 0.980 or greater. Lower values may be acceptable at the discretion of the analyst and approval of the Project Lead. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.4 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 Procedures_________________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. ETS-8-5.0 Analysis of Serum Extract Using ESA1S 3M Environmental Laboratory Page 4 of 9 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 SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set of extracted matrix standards and ends with a set of extracted matrix standards. 12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such. 12.2 Using the 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 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 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 40% 90% 90% 40% 2.0 raM 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. 12.3.3 Check the stainless steel capillary at the end of the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary. 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. ETS-8-5.0 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 5 of 9 3m Medical Department Study: T - 6295.7 Report No. FACT TOX-030 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 123.6.Z ESI nebulizing gas 10-15 liters/hour 123.6.3 HPLC constant flow mode flow rate 10 - 500 |i.L/min 123.6.4 Pressure <400 bar (This parameter is not set, it is a guide to ensure the HPLC is operating correctly.) 123.7 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. 123.9 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations_____________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (pg/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 rt>& //7 Page 6 of 9 P ag ^D 9 3m Medical Department: Study: T - 62 95.7 Report No. FACT TOX-030 laboratory Request Number-U2279 14.2 Method Blanks and Matrix Blanks 14.2.1 Method blanks and matrix blanks will be analyzed with each sample set for possible contamination or carryover. Values are expected to fall below the lowest standard in the calibration curve. 14.3 Matrix Spikes 14.3.1 Matrix spikes are analyzed with each sample set and the percent recoveries are expected to fall within 30% of the spiked concentration. 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 performed on the system and samples reanalyzed or other actions as determined by the analyst. All actions will be documented in the instrument runlog, the maintenance log, or on the summary sheet with the sample results-. 15.0 Pollution Prevention and Waste Management______________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records____________________________________________________________________ 16.1 Store chromatograms in the study or project folder. Each chromatogram must have the following information included either in the header or hand written on the chromatogram: 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 of backup electronic data. 17.0 Tables. Diagrams. Flowcharts, and Validation Data________________________ 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. ETS-8-5.0 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory rtrt M r Page 7 of 9 P, 10 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 18.0 References______________________________________________________________ 18.1 ETS-9-24.0, "Operation and Maintenance of the Micromass Atmospheric Pressure Ionizatvon/Mass Spectrometer Quattro II Systems" 19.0 Affected Documents_____________________________________________________ 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 e v is io n s Revision Number. Reason For Revision Revision Date ETS-8-5.0 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 8 of 9 3m Medical Department Study: T-6295.7 Report No. FACT T O X -030 laboratory Request Number-U2279 Attachment A Laboratory Study # Study: Test Material: M atrix/Final Solvent: M ethod/R evision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Anaiyst: Date of Analvsis/Analvst: G roup _ Dose S am ple# C o n c en tra tio n ue/m L Initial Vol. mL D ilution Factor Final Cone. ug/m L Slope: Taken from linear regression equation. G roup/D ose: Taken from the studv folder. Sam ple#: Taken from the studv folder. C o n cen tratio n (ug/m L): Taken from the MassLvnx integration summary. Initial V olum e (m L l: Taken from the studv folder. D ilution F a c to r: Taken from the studv folder. Final C one. (ug/m L): Calculated bv dividing the initial volume from the concentration ETS-8-5.0 Analysis of Serum Extract Using ES/MS 3M Environmental Laboratory Page 9 of 9 Pa 2 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory M ethod Extraction of P otassium perfluorooctanesulfonate or Other Anionic Fluo ro ch em ica l Surfactants from L iver for A nalysis U sing H PLC -E lectrospray/M ass Spectrom etry Method Number: FACT-M-1.0 Adoption Date: Author: Lisa Clemen Revision Date: L'/d Approved By: j_____ ' A / '"_/___ V,J__ Laboratory Manager ----------- 5 / z C / ? p Date % ------Group Leader d /ll / v f Date -- __ A- Q & fru .__ Technical Reviewer ..s' h i h i ? Date 1.0 Scope and Application______________________________________________________ 1.1 Scope: This method is for the extraction of Potassium Perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver. 1.2 Applicable Compounds: 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 am Page 1 of 8 3ra Medical Department Study: T - 6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Summary of Method________________________________________________ __ 2.1 This method describes how to extract potassium perfluorooctanesulfonate (PFOS) or other fluorochemical surfactants from liver using ion pairing reagent and 5.0 mLs of ethyl acetate. An ton pairing reagent is added to each sample and partitioned into ethyl acetate. Four mLs of 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 Definitions__________________________________________________________ _ 3.1 None. 4.0 Warnings and Cautions______________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use universal precautions when handling animal livers, they may contain pathogens. 5.0 Interferences_______________________________________________________________ 5.1 There are no known interferences at this time. 6.0 Equipment___________________________________________________________________ 6.1 The following equipment is used while carrying out this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 6.1.5 6.1.6 Ultra-Turrax T25 Grinder for grinding liver samples Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR Nitrogen Evaporator, Organomation Balance 7.0 Supplies and Materials______________________________________________________ 7.1 Gloves 7.2 Dissecting scalpels 7.3 Eppendorf or disposable pipettes 7.4 Nalgene bottles, capable of holding 250 mL and 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 3M Environmental Laboratory as, ixx Page 2 of 8 Page 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U2279 7.10 Syringes, capable of 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: 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 Reagents and Standards_________________________________________________ 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. Measure 10 mL of the 10N NaOH solution into a 100 mL volumetric flask and dilute to volume using Milli-QTM water. Store in a 125 mL nalgene bottle. 8.1.3 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 volume with Milli-QTM water. Add NaOH slowly while adding the last 1 mL of NaOH because the pH changes abruptly. Store in a l L nalgene bottle. 8.1.3.1 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using IN NaOH solution. 8.1.4 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker or equivalent), (Na2C 0 3/N aH C03) 0.25M: Weigh approximately 26.5 g of sodium carbonate (Na2C 0 3) and 21.0 g of sodium bicarbonate (NaHCO-,) into a 1 L volumetric flask and dilute to volume with Milli-QTM water. Store in a 1 L nalgene bottle. 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 3M Environmental Laboratory 1+4 123 Page 3 of 8 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 8.2.2 Weigh approximately 100 mg of PFOS into a 100 mL volumetric flask and record the actual weight. 8.2.3 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/mL). 8.2.4 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 8.2.5 Dilute the stock solution with methanol for a working standard 2 solution of approx. 5.0 ppm. 8.2.6 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 9.0 Sample Handling____________________________________________________________ 9.1 All livers are received frozen and must be kept frozen until the extraction is performed. 10.0 Q uality Control____________________ 10.1 Matrix Spikes 10.1.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction. 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 Continuing 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 One check is prepared per group of ten samples. For example, if a sample set = 34, four checks are prepared and extracted. 10.2.3 Prepare each continuing calibration check from the same liver homogenate used to prep the initial curve. 10.2.4 The expected concentration will fall within the mid-range of the initial calibration curve. 11.0 Calibration and Standardization___________________________________________ 11.1 Prepare Liver Homogenate 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 If 40 g is not available, use appropriate amounts of liver and water in keeping with a 1:5 ratio. 11.1.3 See section 13.0 to calculate the actual density of liver. FACT-M-1.0 Extraction o f PFOS from Liver Page 4 of 8 3M Environmental Laboratory 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 11.1.4 Add 1 mL of 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 spiking amounts listed in table 1 to spike, in duplicate, two standard curves for a total of fourteen samples. 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 0.010 ppm 20 0.050 ppm 40 0.100 ppm 10 0.250 ppm 20 0.500 ppm 30 0.750 ppm 4 1.000 ppm 11.1.1 See section 13.0 to calculate actual concentrations o f 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 Procedures_________________________________________________________________ 12.1 Obtain frozen liver samples. In spent tissue, note that the liver has not been packaged with other tissues. 12.2 Cut approximately l 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 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 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 m-izf Page 5 of 8 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 Spike liver homogenates with the appropriate amount of PFOS standard as described in section 11.1 or Table 1. 12.13 Pipette two 1 mL aliquots of Milli-QTM water to centrifuge tubes. These will serve as instrument blanks. 12.14 Add 1 mL 0.5 M TBA and 2 mL of the 0.25 M sodium carbonate/sodium bicarbonate buffer. 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 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 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 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. 12.24 Cap and hold for electrospray mass spectrometry analysis. 12.25 Complete the worksheet and tape to page of study notebook. 13.0 Data Analysis and Calculations______________________________________ ______ 13.1 Calculations: 13.1.1 Calculate the density of liver (mg) in 1.0 mL homogenate using the following equation: g of Liver x Average weight of ten 1 mL aliquots (me) (g of Liver + g of Water) FACT-M-1.0 Extraction o f PFOS from Liver 3M Environmental Laboratory t+4 726- Page 6 of 8 Pa' 18 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 13.1.2 Calculate actual concentrations of PFOS in calibration standards using the following equation: uL of Standard x Concentration (ug /mL) = Final Concentration (pg/g or mg/kg) mg Liver /1 mL homogenate of PFOS in Liver 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 Method Performance_________________________________________________ _ 14.1 The method detection limit is equal to half the lowest standard in the calibration curve. 15.0 Pollution Prevention and Waste Management_____________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records___________________________________________________________________ 16.1 Complete the extraction worksheet and tape into the study notebook. 17.0 Tables, Diagrams, Flowcharts, and Validation Data_______________________ 17*1 The validation report associated with this method is FACT-M-1.0 & 2.0-V -l. 18.0 References________________________________________________________________ 18.1 AMDT-EP-22, "Routine Maintenance of Ultra-Turrax T-25" 19.0 Affected Documents______________________________________________________ 19.1 FACT-M-2, " Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions Revision Number. Reason For Revision Revision Date FACT-M-1.0 Extraction of PFOS from Liver 3M Environmental Laboratory Page 7 of 8 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number~U2279 Extraction Worksheet for FACT-M-1 Study # 1 Sample Number set # H-0 Blank Liver Blank PFOS approx. 0.5 ppm actual ppm #W - - PFOS approx. 5 ppm actual ppm #W - - ---- -- -- --- --- -------- --- -- PFOS Date and approx. 50 ppm Initials actual ppm for Std. - - - ' Study num ber w here the original worksheet is located. Blank L iver H om ogen ate: Std # L iver E x tra ctio n M eth od L iver amount = ...S D ate & In itials V ortex 15 sec. P ipette 1 m L o f L iver Solu tion P ip ette 1 m L o f tO .S M T B A , pH 10. S td .# P ipette 2 m L o f 0 .2 5 N a 2 C O y 0 .2 5 M N aH C O ^ B uffer Std. # Pipette 5 m L o f E thyl A cetate T N -A - S h ak e 2 0 m in. C en trifu ge 2 0 -2 5 m in. C entrifuge Speed R em o v e a 4 m L a lio u o t o f organic laver Put an N itro g en E vaporator to d ryn ess E vaporator Tem perature A d d 1.0 m L o f M eth an ol T N -A - V ortex 3 0 sec. F ilter u sin g a 3 c c B -D sv rm g e w ith a 0 .2 u m SR I filter into a 1.5 m L a u to sa m p le vial M S/M SD /___ Cont. Checks: S p ik ed ______ uL o f a ______ ppm std (_______________ ) for a final concentration o f _________ ppm. MS/MSD used sa m p le________________ . 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 rw i z z 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory M ethod A naly sis o f F lu o ro c h em ic a ls in L iv er E x tra cts U sin g H P L C -E lectrospray/M ass Spec tr o m etr y Method Number: FACT-M-2.0 Author: Lisa Clemen Approved By: 0/ Laboratory Manager j/> h u Group Leader -- ---------- A Technical Reviewer . Adoption Date: 5 /0 1/9:? Revision Date: Date *>' / v K Date i'/n h x Date 1.0 Scope and Application________________________________________________________ 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 Applicable Compounds: 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 hac. Page 1 of 8 Pa< 21 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 S ummary of Method____________________________________________________ _ 2.1 This method describes the analysis of fluorochemical surfactants extracted from liver using HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0 Definitions__________________________________________________________________ 3.1 None. 4.0 Warnings and Cautions______________________________________________________ 4.1 Health and Safety Warnings: 4.1.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of 400 bar (5800 psi). If pressure goes over 400 bar, the HP1100 will initiate automatic shutdown. 4.2.2 Do not run solvent pumps to dryness. 5.0 Interferences_______________________________________________________________ 5.1 Teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 Equipment___________________________________________________________________ 6.1 Equipment listed below may be changed 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 Supplies and Materials______________________________________________________ 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 Reagents and Standards_____________________________________________________ 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 3M Environmental Laboratory Page 2 of 8 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request N u m b e r - U 2 2 79 8.1.2 MilJi-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 i n 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 If analysis will be delayed, extracted standards and samples may be refrigerated until analysis can be performed. 10.0 Q u a l i t y C o n t r o l _____________________________________________________________________________ 10.1 Matrix Blanks and Method 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 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 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the 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 System suitability (e.g. peak area, retention time and peak shape, etc.) will be assessed for each run. 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n _________________________________________________________ 11.1 Analyze the extracted 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 of 8 Pa 3 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 Acquisition 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 To create a method click on scan button in the Acquisition control panel and select SIR. Setlonization Mode as appropriate and mass to 499 or other appropriate. masses. A scan is usually collected along with the SIRs. Save method. 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 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 pL injection with a sample wash 12.2.2.2 Inject/sample = 1 12.2.2.3 Cycle tim e= 15 minutes 12.2.2.4 Solvent ramp = Time 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 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 electrosprav software with a "Waiting for inlet start" message before the "Start" button is 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 +^>/3<2- Page 4 of 8 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.3 Instrument 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 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 of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. 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 Using the cross-flow counter electrode in the ES/MS source is recommended for the analysis of biological matrices. 12.3.10 Click on start button in the Acquisition Control Panel. Press the start button at top of sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 D a t a A n a l y s i s a n d C a l c u l a t 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 E n v i r o n m e n t a l L a b o r a t o r y Page 5 of 8 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 13.1.3 Calculate actual concentration of PFOS anion in total liver (mg): f ug PFOS anion calc, fromstd curve'! V g of liver used foranalysis ) _ , ------------------------------------------------ x Total mass of liver (g) 1000 u g / 1 mg 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 i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t ______________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and 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 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 Back up electronic data to appropriate media. Record in study notebook the file name and location of backup electronic data. 17.0 T a b l e s , D i a g r a m s , F l o w c h a r t s , a n d V a l i d a t i o n D a t a _______________________________ 17.1 Attachment A: 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 +a^/3V Page 6 o f 8 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-112279 19.0 Affected D ocuments 19.1 FACT-M-1.0, "Extraction o f Potassium Perfluorooctanesulfonate from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 Revisions Revision Number. Reason For Revision Revision Date FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 7 of 8 Page- 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U22 79 Laboratory Study # Study: Test Material: Matrix/Final Solvent: M ethod/R evision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date of Extraction/Analyst: Date o f Analysis/Analyst: G roup Dose S am ple# C o n cen tratio n u g/m L Initial Vol. mL ' Dilution Factor Final Cone. u g/m L Slope: Taken from linear regression equation. G roup/D ose: Taken from the study folder. Sam ple#: Taken from the study folder. C o n cen tratio n (ug/m L): Taken from the MassLynx integration summary. Initial V olum e (m L): Taken from the study folder. Dilution F actor: Taken from the study folder. Final Cone. (ug/m L): 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 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 io 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 l u o r o c h e m ic a l com pounds fr o m L iv e r fo r Analysis U sin g H P L C -E lectrospray/M ass Spectrom etry M ethod N um ber: FACT-M-1.1 Author: Lisa Clemen, Glenn Langenburg Approved By: D / . / S --- Laboratory Manager {J n /c v ' U * ------------ Group Leader AA fJl/r Technical Reviewer Adoption Date: 05/26/98 Revision Date: O>) } 1^ /i/j ; Date 6/1 I'M Date 6 /ni/?? Date 1.0 Scope and A pplication________________________________________________________ 1.1 Scope: This method is for the extraction of potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from liver. 1.2 Applicable Com pounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey liver or other liver as designated in the validation report. M icrosoft 6.0/95 3M Environmental Laboratory FACT-M-1.1 Extraction o f PFOS from Liver trfe- /3? Page 1 of 15 3ra Medical Department Study-. T - 62 95.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Summary of Method______________________________________________ _ _ _______ 2.1 This method describes the procedure for extracting potassium perfluorooctanesulfonate (PFOS) or other fluorochemicals from liver homogenate using an ion pairing reagent and 5.0 ml of ethyl acetate. In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, POAA, PFOSEA, and FC-807 monoester (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into_ ethyl acetate. Four ml o f extract are 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 Definitions__________________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion of potassium salt) CgFl7SOj' 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F nS 0 2N(CH2CH2)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyI alcohol CSF, 7S 0 2N(CH2CH3)CH2CH20H 3.5 POAA: perfluorooctanoate (anion of ammonium salt) C7F |SCOO' 3.6 PFOSEA: perfluorooctane sulfonyl ethylamide CaF |7S 0 2N(CH2CH3)H 3.7 FC-807 monoester C8F l7S 0 2N(CH2CH2)CH2CH20 -P 0 3H) 3.8 Surrogate standard 1H,1H,2H,2H perfluorooctane sulfonic acid 4.0 Warnings and Cautions______________________________________________________ 4.1 Health and safety warnings: 4.1.1 Use universal precautions, especially laboratory coats, goggles, and gloves when handling animal tissue, it may contain pathogens. 5.0 Interferences_______________________________________________________________ 5.1 There are no known interferences at this time. 6.0 Equipment___________________________________________________________________ 6.1 The following equipment is used while carrying out this method. Equivalent equipment is acceptable. 6.1.1 6.1.2 6.1.3 6.1.4 6.1.5 6.1.6 Ultra-Turrax with T25 grinder attachment for grinding/dispersing/emulsifying Vortex mixer, VWR, Vortex Genie 2 Centrifuge, Mistral 1000 or IEC Shaker, Eberbach or VWR Nitrogen evaporator, Organomation Balance, ( 0.100 g) 3M Environmental Laboratory FACT-M-1.1 Extraction o f PFOS from Liver m. M Page 2 o f 15 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 7.0 Supplies and Materials_______________________________________________ _ 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 o f 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: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with Milli- QTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards____________________________________________________ 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 (NajCOj), 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 matrix for standards, QC checks, blanks, etc. 8.10 Fluorochemical standards 8.10.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.10.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499 8.10.3 PFOSAA (3M Specialty Chemical Division), molecular weight = 585 3M Environmental Laboratory FACT-M-l.l Extraction of PFOS from Liver Page 3 o f 15 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-1722 79 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 triester, diester, and monoester fluorochemical components. The monoester molecular weight = 650 8.10.8 Surrogate Standard: 4-H, perfluorooctane sulfonic acid (1-H,1-H, 2-H,2-H CgF nSOjH) molecular weight = 428 8.10.9 Other fluorochemicals, as appropriate 8.11 R eagent preparation 8.11.1 10N sodium hydroxide (NaOH): Weigh approximately 200g 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 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 grams of TBA into a 1 L volumetric containing 500 ml Milli-QTM water. Adjust to pH 10 using approximately 44 to 54 ml of 10N NaOH and dilute to volume with 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 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using IN NaOH solution. 8.11.4 0.25M Sodium carbonate/sodium bicarbonate buffer (NajCOj/NaHCOj): Weigh approximately 26.5 g of sodium carbonate (Na,COj) and 21.0 g of sodium bicarbonate (NaHCOj) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L nalgene bottle. 8.12 S tandards 8.12.1 Prepare PFOS standards for the standard curve. 8.12.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (e.g. one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOS A, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg of PFOS into a 100 ml volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/ml). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. 3M Environmental Laboratory FACT-M-l.l Extraction of PFOS from Liver tyr)*0 Page 4 o f 15 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 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 Prparera surrogate stock standard. Weigh approximately 50-60 mg o f surrogate standard 1-H,1-H, 2-H.2-H, CgF13S 0 3H into a 50 ml volumetric flask and record the actual weight. 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 of surrogate stock to 50 ml volumetric flask and bring to volume with methanol for a working standard of 10-20 ppm. Record the actual volume transferred. 8.14 Liver homogenate preparation Note: Thefollowing procedure will be much easier to perform withfrozen liver tissue. Prevent tissuefrom thawing; keep stored on ice until excising a portion o f it: 8.14.1 Weigh 40 g of 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 of 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 200 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 of these aliquots is determined and then the concentration (g of liver/ml of homogenate) can be calculated as follows: 8.14.3 fgrams la) ofliverl x lave, weight of 1.0 ml o f homogenate (density) ('g/ml)l {[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 of 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 8.3.2for the sample liver homogenates). 3M Environmental Laboratory F A C T -M -l.l Extraction ofPFOS from Liver Page 5 o f 15 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 9.0 Sample Handling___________________________________________________ 9.1 All livers are received frozen and must be kept frozen until the extraction is performed. 10.0 Quality Control______________________________________________________ _ 10.1 Matrix blanks and method blanks 10.1.1 Extract two 1.0 ml aliquots of the liver homogenate (prepared in 8.14.1-2) following this procedure and use as matrix blanks. See Section 11.1.2. 10.1.2 Extract two 1.0 ml aliquots of Milli-QTM water following this procedure and use as method blanks. 10.2 Matrix spikes 10.2.1 Prepare ahd analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.2.2 Prepare each spike using liver chosen by the analyst, usually the control liver received with each sample set. 10.2.3 Expected concentrations fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range of the initial calibration curve. 10.2.4 Prepare one matrix spike and one 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 and the continuing check differ by >30%, reanalyze samples analyzed after the last acceptable check. 10.3.2 Prepare one check per group of ten samples. For example, if a sample set = 34, prepare and extract four checks. 10.3.3 Prepare each continuing calibration check from the same blank liver homogenate used to prepare the initial curve. 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 calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (e.g. 10 ppb - 100 ppb, rather than 10 ppb - 1000 ppb). 11.0 Calibration and Standardization___________________________________________ 11.1 P repare liver homogenate standards 11.1.1 Transfer 1 ml aliquots of blank/control liver homogenate prepared in 8.14.1-2 to 15 ml centrifuge tubes. 3M Environmental Laboratory FACT-M-l.l Extraction of PFOS from Liver Page 6 o f 15 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 of liver homogenate. Record the sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots of liver homogenate in 15 ml centrifuge tubes, mix or shake between aliquots. 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 o f this section) to spike, in duplicate, two standard curves, for a total of eighteen standards and two matrix blanks. 11.1.5 Refer to the validation reports FACT-M -l.l-V-1 and FACT-M-2.1-V-1 which list the working ranges and Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or QC check, add appropriate amount of surrogate working standard for the concentration to fall within tfie calibration curve range 10 ppb - 1000 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. Cone.) pL Approx, final cone, of PFOS in liver - - Blank 0.500 ppm 4 0.012 ppm 0.500 ppm 10 0.030 ppm 0.500 ppm 20 0.060 ppm 0.500 ppm 40 0.120 ppm 5.00 ppm 10 0.300 ppm 5.00 ppm 20 0.600 ppm 5.00 ppm 30 0.900 ppm 50.0 ppm 4 1.20 ppm 50.0 ppm 6 1.80 ppm 12.0 Procedures_________________________________________________________________ 12.1 Obtain frozen liver samples and homogenize as described in 8.14.3, 12.2 Vortex mix homogenate for 15 seconds, then transfer 1.0 ml or other appropriate volume to a 15 ml polypropylene centrifuge tube. 12.3 Return liver homogenate samples to freezer after extraction amount has been removed. 3M Environmental Laboratory FACT-M-l.l Extraction of PFOS from Liver rsM \h3 Page 7 o f 15 Pa< 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 I ml of homogenate is transferred for extraction, then the final reconstitution methanol volume equals 1 ml. 12.5 Label the tube with the study number, liver ID, date, and analyst initials. See attached worksheet for documenting the remaining steps. 12.6 Spike blank liver homogenate aliquots with the appropriate amount of standard as described in Section 11.1 or Table I 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 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 Transfer 4 ml o f organic layer, using a 5 ml graduated glass pipette, to a clean 15 ml centrifuge tube. Label this fresh tube with the same information as in 12.5. 12.14 Put each sample on the analytical nitrogen evaporator until dry, approximately 2 to 3 hours. 12.15 Add 1.0 ml or appropriate volume of methanol to each centrifuge tube using a graduated pipette. Methanol volume equals the initial volume of liver homogenate used for the extraction. 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 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 Data Analysis and Calculations____________________________________________ 13.1 Calculations: 13.1.1 Calculate actual concentrations of PFOS, or other appropriate fluorochemical, in calibration standards using the following equation: 3M Environmental Laboratory FACT-M-l.l Extraction o f PFOS from Liver P ag e 8 o f 15 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 mi o f Standard x Concentration of Standard (ug/ml)_____ = Concentration of Blank Liver Homogenate (g/ml) (see 8.14.2) Final Concentration (pg/g) of PFOS in Liver See Attachment D for a sample form to calculate the concentrations of standards. 14.0 Method Performance_________________________________________________ _ 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 Attachments B and C). 14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality o f the extraction and analysis. 14.2.1 Method blanks and matrix blanks 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve. 15.0 Pollution Prevention and Waste Management_______________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 Records____________________________________________________________________ 16.1 Complete the extraction worksheet attached to this method, and tape into the study notebook or include into study binder, as appropriate. 1 7 .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 References_________________________________________________________________ 18.1 The validation reports associated with this method are FACT-M-1.1 and 2.1-V -l. 19.0 Affected Documents________________________________________________________ 19.1 FACT-M-2.1, "Analysis of Liver Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 3M Environmental Laboratory FACT-M-1.1 Extraction o f PFOS from Liver r^> ih^ Page 9 o f 15 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 ________________________________________________________________________ Revision Number. 1 Reason For Revision Validation o f method to include 7fluorochemicals, new API/MS(MS) systems, monkey liver cross validation, improvements to ion pairing extraction, MDL study, updates in record keeping and storing policies, etc. Revision Date 08/01/98 3M Environmental Laboratory FACT-M-1.1 Extraction o f PFOS from Liver r& iH t, Page 10 o f 15 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U227 9 Study# Matrix Box# Analyst/Date H ,0 Blank Blank Surrogate Std. approx, ppm actual ppm #W - FC Mix Std approx. 0.5 ppm actual ppm #W FC Mix Std approx. 5 ppm actual ppm FC Mix Std approx. 50 ppm actual ppm #W Comments B l a n k ______________________ S t d # ____________________________ a m o u n t = __________________________________________________________________________________ E x t r a c t i o n M e t h o d / R e v i s i o n : __________________________________________________________________________________________________ D a t e & I n i t i a l s A d d S u r r o g a t e . V o r t e x 15 s e e ._________________________________________________________________________________________________________________________ P i p e t t e S a m p l e ________________________________________________________V o l u m e _______________________ m l________________________________________________ P ip ette I m l o f 0.5 M T B A , pH 10. PH = S td. # ____________________________________________ P i p e t t e 2 m l o f 0 . 2 5 N a ? C O y Q . 2 5 M N a H C C b b u f f e r _______________S td . # ___________________________________________ P i p e t t e S m l o f e t h y l a c e t a t e ___________________________________________ T N - A - ____________________________________________ S h a k e 2 0 m m . ___________________________________________________________ S h a k e r S p e e d ________________________________________________________________ C e n t r i f u g e 2 0 - 2 5 m i n . __________________________________________________C e n t r i f u g e s p e e d :____________________________________________________________ R e m o v e a 4 m l a l i q u o t o f o r g a n i c l a y e r ______________________________________________________________________________________________________________ P u t o n N i t r o g e n E v a p o r a t o r t o d r y n e s s ____________________________T e m p e r a t u r e : ____________________________________________________________________ A d d m e t h a n o l ___________________ V o l u m e m l______________ T N - A - V o r t c x 3 0 s e c .___________________________________________________________________________________________________________________________________________ F i l t e r u s i n g a 3 c c B - D s y r i n g e w i t h a 0 2 u m S R I f i l t e r i n t o a 1.5 m l a u t o s a m p l e v ia l___________________________________________________________ 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 t d ( _____________________ ) f o r a F i n a l c o n c e n t r a t i o n o f _____________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 t r i x a s f o r s t d c u r v e . Attachment A: Extraction worksheet 3M Environmental Laboratory FACT-M-1.1 Extraction of PFOS from Liver /V7 Page 11 o i l 5 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 MDL/LOQ values for Rabbit Liver: Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (PPb) Approximate Concentrations to be used for preparing the Standard Calibration Curve PFOS 11.8 37.4 38 p p b - 1000 ppb PFOSA 6.06 19.3 20 ppb - 1200 ppb PFOSAA 55.7 177 180 ppb - 1000 ppb EtFOSE-OH 58.7 187 190 ppb - 1800 ppb POAA 23.7 75.5 76 ppb - 1800 ppb PFOSEA 16.2 51.7 62 ppb - 1200 ppb Monoester n/v n/v Monoester was not detectable/quantifiable at the spiked concentrations. MDL/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 24.7 78.7 62 ppb - 1200 ppb PFOSA 20.7 65.8 20 ppb - 1200 ppb PFOSAA n/v n/v 62 ppb - 1200 ppb EtFOSE-OH n/v n/v 120 ppb - 1200 ppb POAA n/v n/v 62 ppb - 1200 ppb PFOSEA n/v n/v 120 ppb - 1200 ppb Monoester n /v n /v Monoester was not detectable/quantifiable at the spiked concentrations. MDL/LOQ values for Monkey 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 59 ppb - 1200 ppb PFOSA 27.4 87.1 28 ppb - 1200 ppb PFOSAA n/v n/v 120 ppb - 1200 ppb EtFOSE-OH n/v n/v 58 ppb - 1200 ppb POAA n/v n/v 120 ppb - 1200 ppb PFOSEA n/v n/v 120 ppb - 1200 ppb Monoester n/v n/v Monoester was not detectable/quantifiable at the spiked concentrations. n/v = Not valid. Upon analyzing the data, value did not pass the criteria set for this characterization. Until further analysis is completed, use the LCR to determine the range of standard concentrations for calibration curve preparation. Attachment B: MDL/LOQ Values 3M Environmental Laboratory FACT-M-1.1 Extraction o f PFOS from Liver rss H i P age 12 o f 15 P a ' 40 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U22 79 BEST COPY AVAILABLE Compound PFOS M atrix Rabbit Bovine MDL LOQ 11.8 ppb 37.4 ppb n/d = not determined2 Approximate Linear Range1 Low S tan d a rd 38 ppb 60 ppb High Standard : I 1000 ppb 1200 ppb Rat Monkey 24.7 ppb 78.7 ppb n/v = not valid2 62 ppb 59 ppb 1200 ppb 1200 ppb PFOSA Rabbit Bovine Rat Monkey 6.06 ppb n/d 20.7 ppb 27.4 ppb 19.3 ppb n/d 65.8 ppb 87.1 ppb 20 ppb 30 ppb 6 ppb 6 ppb 1200 ppb 1200 ppb 1200 ppb 1200 ppb PFOSAA Rabbit Bovine Rat Monkey 55.7 ppb n/d n/v n/v 177 ppb n/d n/v n/v 180 ppb 120 ppb 62 ppb 120 ppb 1900 ppb 1200 ppb 1200 ppb 1200 ppb EtFOSE-OH Rabbit Bovine Rat Monkey 58.7 ppb n/d n/v n/v 187 ppb n/d n/v n/v 190 ppb 120 ppb 120 ppb 58 ppb 1800 ppb 1200 ppb 1200 ppb 1200 ppb POAA Rabbit Bovine Rat Monkey 23.7 ppb n/U n/v n/v 75.5 ppb n/d n/v n/v 76 ppb 120 ppb 62 ppb 120 ppb 1800 ppb 1200 ppb 1200 ppb 1200 ppb PFOSEA Rabbit Bovine Rat Monkey 16.2 ppb n/d n/v n/v 51.7 ppb n/d n/v n/v 62 ppb 30 ppb 120 ppb 120 ppb 1200 ppb 1200 ppb 1200 ppb 1200 ppb M onoester 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 1 - Upper Limit chosen where the value was within the Linear Calibration Range (LCR) but did not excessively weight the standard curve or affect Repeatability & Reproducibility values. 2 - Not determined refers to no sample was analyzed for this data. 3 - Not valid refers to data from the analysis failed to meet specific criteria for a valid MDL/LOQ determination. C om pound! L iver M atrix R ab b it B ovin e R at M onkey Prepared R ange of S tandards (p p b )(n g /g ) 5 95 - 1790 6.00 - 1200 6 .2 2 - 1240 5.93 - 1190 j PFOS R ange of A verage C urve (p p b )(n g/g) 5.95 - 1790 6.00 - 1200 6 .2 2 - 1240 5.93 - 1190 ^ C R /fro r a p i g e ra te R ange of L ow Std. C urve - L C R from (p p b )(n g/g) 62inT240 5.95 -29 8 n /a n /a ;n;s>r 298 n /a A ,, ZI n/a - n/a ! n/a I j lR ange of H igh S td . C urve (p p b X n g/g) 119-1790 /M a re n g o n/a y m if n /a n/a i : ".n/a . Attachment C: LOQ summary 3M Environmental Laboratory FACT-M-1.1 Extraction of PFOS from Liver T-H* U41 Page 13 o f 15 Pa' 41 3m Medical Department Szudy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 C om pound; L iver M atrix R ab b it B o v in e R at M onkey Prepared R ange of S ta n d a r d s (p p b )(n g /g ) 6.04- 1810 5.95 - 1190 6.17 - 12405.88- 1180 Compound! PFO SA R ange of A verage C urve (p p b )(n g/g) 6.04 - 1210 5.95 - 1190 6. 17-1240 5.38- 1180 PFOSAA i 1i 11 .tC R -fro m fe R ange of L ow S id . L C R T n im y 5L o j ^ t d . ;y Range of H igh Std. H H jheStd: C urve (p p b )(n g/g) 6.04-302 ..-.^ C u ry 'etfer 4; ( p p X ^ 2 30 r , J i l ^ ^ . C urve v fli$ e S L - (p p b )(n g/g) m w 121 - 1210 L S B p i R I g - 8 ME n/a n /a "/a n/a g f l|l n /a s - n /a 1 BEST COPY AVAILABLE C om pound L iver M atrix R abbit B ovin e R at M onkey Prepared Range of S ta n d a r d s (p p b )(n g /g ) 5.96 - 1790 5 .8 7 - 1 170 6.09 - 1220 5.80 - ! 160 E tF O S E -O H 5 3R a n g e o f jpgM R ttP & A verage n ^ a n | C urve HgBfcftTffmsBK* (p p b )(n g/g) 1 9 -1790 5 8 .7 - 1 170 1 2 2 - 1220 31S B S S H : 29 4 - 1160 Range of L ow Std. C urve (p p b )(n g/g) n /a n /a n /a n /a ii MBIR ange o f H igh S td . C urve K fp 'p ;B M to ( p p b ) ( n g /g ) 0< r*SS*f|SnM jj w n / a Hftioar*r v * n / a S a S s a S S 'c n /a n /a Compound! Liver Matrix Rabbit Bovine Rat Monkey ! !C o m p o u n d Liver Matrix Rabbit Bovine Rat Monkey POAA Prepared | Range of Range of Average Standards Curve (ppb)(ng/g) (ppb)(ng/g) 6.06 - 8 2 0 30.3 - 1820 5.93 - I 190 59.3 - 1 190 6.15 - 1230 61.5 - 1230 5.86 - 1 170 1 1 7 -1 1 7 0 ! ! PFOSEA Prepared Range of Standards (ppb)(ng/g) Range of Average Curve (ppb)(ng/g) 6 .2 0 - 1360 62.0 - 1240 5 .9 2 - 1180 29.6 - 1180 6.14 - 1230 123 - 1230 5.85 - 1170 58.5 - 1170 H/HnMltIT<7/0 w * & iiw raigwranffga Range of Low Std. Curve (ppb)(ng/g) 30.3 - 606 n/a b .LRifcori Range of . ito v i^ S td ^ S High Std. Curve (ppb)(ng/g) ^ 1 303 - 1820 ^.vF'!'i4lre16 n /a ^ id B S n /a n /a n /a n /a :.|^gAHi;; 1! 11 1 .Rft<>ndg Range of Low Std. p f f Curve (ppb)(ng/g) Range of ;.LGR5frdni; ?Lo\*Stf High Std. Curve l i s S r a f e (PPP)C?/^lS (ppb)(ng/g) iffiptyng/g) n /a y ? n/a; - - J n/a jCrinfa/':'.' n/a .y;:^n/a qtv-y. n/a 1yL ^n/tni-5 n /a - - n/a * - n /a ! ' n/a -, 1 1170 n /a n /a n/a n/a Monoester was not detectable/quanti liable in liver matrix for the concentration range of 4.94 - 1450 ppb A ttachm ent C: LOQ summary 3M Environmental Laboratory FACT-M-1.1 Extraction o f PFOS from Liver is Page 14 o f 15 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 (P re p D ate(s): j 11/2/98 ~ (A n aiyst(s): (R W W -IA S S a m p l e M a t r ix : 1Monkey Liver M e t h o d / R e v i s i o n FACT-M-! .0 T arget FC-Mix A n alyte(s): I | Ion Pair Standard Curves--Tissues i' Study N um ber: |C ro ss V alid atio n II 1 IE q u ip m e n t N u m b e r : > (F in a l S o l v e n t & TN N u m b e r : :MeOH TN-A-2076 (B lan k T issu e/ld en tifier: Liver i 11 1 ------------------------------ 1 FC M ix S t d A p p r o x . 0.500 ppm: iW398-1004 FC M ix S t d A p p r o x . 5.00 ppm:' (W398-1003 FC M ix S t d A p p r o x . 50.0 ppm: iW3 98-1002 S u r r o g a t e S t d A p p r o x . 16.5 ppm: W398-989 ; j j ! 1 ! i ~ !i '! ;! !i Actual Concentrations of Standards in the FC Mix PFO S PFO SA P F O SA A 1 E tF O SE Std Cone. j Std Cone. Std Cone, j Std Cone. ug/mL 0.501 0.501 0.501 0.501 0.501 5.01 5.01 5.01 50.1 : ug/mL " ug/mL 0.497 0.500 0.497 0.500 0.497 0.500 0.497 0.500 1 0.497 1 0.500 j 4.97 5.00 1 4.97 5.00 4.97 , 5.00 49.7 50.0 ug/mL 0.490 0.490 0.490 0.490 0.490 4.90 4.90 4.90 49.0 PO AA Std Cone. ug/mL 0.495 0.495 0.495 ! 0.495 i 0.495 1 4.95 ; 4.95 1 4.95 1 49.5 ! PFO SEA j Std Cone. ug/mL 0.494 i 0.494 1 0.494 0.494 0.494 4.94 4.94 4.94 49.4 i C a lc u la te d C o n c e n tr a tio n s o f S ta n d a r d s in th e S a m p le M a trix. PFO S i PFOSA P F O SA A 1 E tFO SE Final Cone. ! Final Cone. Fmal Cone. ! Final Cone. i ng/g ng/g 5.93 5.88 i ng/g i ng/g 5.92 5.80 11.9 11.8 118 11.6 29.6 29,4 29.6 29.0 59.3 58.8 59.2 58.0 119 118 118 116 296 294 296 290 593 588 592 580 889 882 888 870 1186 1176 1183 1160 PO A A Final Cone. ng/g 5.86 11.7 29.3 58.6 117 293 586 879 1172 PFO SE A Final Cone. ng/g 5.85 11.7 29.2 58.5 117 292 585 877 1169 1; ( A il Am't ! Spiked 1 mL : 0.002 ; 0.004 ; 0.010 ( 0.020 | 0.040 1 0.010 0.020 1 0.030 1 0.004 i A ll Liver cone. g/ml J ; 0.169 : ; 0.169 ; ( 0.169 ! ; 0.169 i | 0.169 0.169 0.169 1 0.169 ( 0.169 ! 1 1 S u rrogate A ll | Std Cone, i Am't ; Spiked ug/mL mL 16.50 ! 0.005 Su rrogate' Final Cone. ng'g 488.2 , j 1 V alid a ted R a n ees -- A p p ro x im a te C o n cen tra tio n s 1 L iver R ab b it B ovin e R at M onkey PFO S 40 - 1000 ppb 60 - 1200 ppb 60- 1200 ppb 60 - 1200 ppb PFO SA 20 - 1200 ppb 30 - 1200 ppb 70 - 1200 ppb 90 - 1200 ppb PFO SA A 180- 1900 ppb 120- 1200 ppb 60- 1200 ppb 120- 1200 ppb E tF O SE -O H 190- 1800 ppb 120 - 1200 ppb 120- 1200 ppb 60- 1200 ppb PO AA 80 - 1800 ppb 80- 1200 ppb 60 - 1200 ppb 120 - 1200 ppb PFO SE A 60 - 1200 ppb 30 - 1200 ppb 120 -1200 ppb 120- 1200 ppb i 1 Attachment D: Calibration standard concentration worksheet 3M Environmental Laboratory FACT-M-1.1 Extraction of PFOS from Liver rtr- 151 P a g e 15 o f 15 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 3M Environmental Laboratory Method A nalysis of F luorochemicals in L iv er E xtracts U sing H P L C -E lectrospray/M ass S pectrom etry Method Number: FACT-M-2.1 Author: Lisa Clemen Approved By: Adoption Date: 05/26/98 Revision Date: O t > ( o l Laboratory Manager Date Group Leader (h Technical Reviewer Date o ili Date 1.0 Scope and Application________________________________________________________ 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 Applicable Compounds: 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. W ord 7.0.1/95 FACT-M-2.1 Analysis o f Liver Extract Using ES/MS 3M Environmental Laboratory W> IS** Page 1 of 9 3ra 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 HPLC-electrospray/mass spectrometry. The analysis is performed by monitoring a single ion characteristic o f a particular fluorochemical, such as the potassium perfluorooctanesulfonate (PFOS) anion, M/Z= 499. Samples may also be screened to verify compound identification. 3.0 D e f i n i t i o n s ______________________________________________________________________________ _ _ _ _ _ 3.1 Atmospheric Pressure Ionization (API): The Micromass platform 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), Jhermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method o f ionization performed at atmospheric pressure, whereby ionization occurs through the production of tiny charged droplets in a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API platforms are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass platform systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with other Zspray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation of these platform systems. Currently MassLynx has Windows 95 and WindowsNT 3.1 versions. 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 Warnings and Cautions______________________________________________________ 4.1 Health and Safety Warnings: 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. Word 7.0.1/95 FACT-M-2.1 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory Page 2 of 9 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 4.2 Cautions: 4.2.1 Do not run solvent pumps above capacity of 400 bar (5800 psi). If pressure goes over 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 for perfluorooctanoate(POAA), teflon should not be used for sample storage or any part of instrumentation that comes in contact with the sample or extract. 6.0 E q u i p m e n t ______________________________________________________________________________________ 6.1 Equipment listed below may be modified in order to optimize the system. 6.1.1 Micromass Electrospray Mass Spectrometer 6.1.2 HP 1100 low pulse solvent pumping system and autosampler. 7.0 Supplies and Materials_____________________________________________ 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 Reagents and Standards____________________________________________________ 8.1 Reagents 8.1.1 Methanol, HPLC grade or equivalent. 8.1.2 ASTM, Type I water, 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-1.1. 9.0 Sample Handling____________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis. 9.2 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. FA C T -M -2.1 Analysis o f Liver Extract Using ES/MS 3M Environmental Laboratory Page 3 o f 9 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.0 Q u a l i t y C o n t r o l _______________________________________________________________ 10.1 Matrix Blanks and Method 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. 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 o f the initial calibration curve. 10.2.3 See section 13 to calculate percent recovery. 10.3 Continuing Calibration Checks 10.3.1 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the 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 i z a t i o n ________________________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set o f extracts. The average 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 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.3 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb of analyte, generate a calibration curve consisting of the standards from 5 ppb to 100 ppb rather than the full range of the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 P r o c e d u r e s _________________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using MO-DAY-last digit of year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. 12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. IfMS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass FACT-M-2.1 Analysis ofLiver Extract Using ES/MS Page 4 of 9 3M Environmental Laboratory 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 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 instrumeftt 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 0.00 min. 8.0 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 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 of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip of the probe. Readjust the tip of the probe if no mist is observed. 12.3.6 The 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 Pa' 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U227S 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. 12.3.10 Click on start button in the Acquisition Control Panel. Press the start button at top o f sample list. Ensure start and end sample number includes all samples to be analyzed. 1 3 .0 D a t a A n a l y s i s a n d C a l c u l a t i o n s ____________________________________________ 13.1 Calculations: 13.1.1 Calculate 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. 13.1.3 Calculate actual concentration of PFOS anion in total liver (mg): ug PFOS anion calc, fromstdcurve g ofliver used foranalysis 1000u g/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 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see ETS-8-1.1, Attachm ent B, for a listing of current validated 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 C alibration Curves 14.2.1 The r value for the calibration curve must be 0.980 or better. FACT-M-2.1 Analysis o f Liver Extract Using ES/MS Page 6 o f 9 3M Environmental Laboratory fct% 7 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 14.3 Matrix Spikes 14.3.1 Matrix spike percent recoveries are must be within 30% o f the spiked concentration. 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 i o n a n d W a s t e M a n a g e m e n t _______________________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and 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 Each page generated for a study must have the following information included either in the header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst. 16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog. 16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder. 16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder. 16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see A ttachm ent A for an example of a summary spreadsheet. 16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location of backup electronic data. 17.0 T a b l e s . D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a _______________________________ 17.1 Attachment A: 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 Pa' 1 5 0 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 FACT-M-1.1, "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 FA C T-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 FACT-M-1.1, "Extraction of Potassium Perfluorooctanesulfonate from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry" 20.0 R e v i s i o n s _______________________________________________________________________________________ Revision Number. 1 Reason For Revision Section 6.1.2 Clarification of HP1100 system components. Section 11.1 Average of two 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. Revision Date 05/04/99 FACT-M-2.1 Analysis ofLiverExtract Using ES/MS 3M Environmental Laboratory Page S of9 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-112279 Laboratory Study # Study: Test Material: Matrix/Final Solvent: Method/Revision: Analytical Equipment System Number: Instrument Software/Version: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analysf: Date o f Analysis/Analyst: Group Dose Sample# Concentration ug/mL Initial Vol. mL Dilution Factor Final Cone. ug/mL Slope: Taken from linear regression equation. Group/Dose: Taken from the study folder. Sample#: Taken from the study folder. Concentration (ug/mL): Taken from the MassLynx integration summary. Initial Volume (mL): Taken from the study folder. Dilution Factor: Taken from the study folder. Final Cone. (ug/mL): Calculated by dividing the initial volume from the concentration Attachment A: Data Sheet FACT-M-2.0 Analysis of Liver Extract Using ES/MS 3M Environmental Laboratory r5T Page 9 o f 9 3m Medical Department Study: T-6295.7 Report No. FACT TOX-30 laboratory Request Number-U2279 3M Environmental Laboratory M ethod E x t r a c t io n o f P otassium P erflu o ro o cta n esu lfo n a te o r O t h e r F l u o r o c h e m ic a l co m po u n d s fr o m Ser u m o r O t h e r F luid f o r An a ly sis U sin g H P L C -E lectrospray/M ass Spec tr o m etr y M ethod N um ber: FACT-M-3.1 Adoption Date: 04/22/98 Author: Lisa Clemen, Glenn Langenburg Revision Date: to Approved By: / ;///i-- Laboratory Manager /a/ i / n Date ------r----- i ------------ Group Leader "7 h - z i f / Date d A __________________ Technical Reviewer ` / a s f o Date 1.0 Scope and Application_______________________________________________________ 1.1 Scope: This method is for the extraction o f potassium perfluorooctanesulfonate (PFOS) or other fluorochemical compounds from serum or other fluid. 1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds. 1.3 M atrices: Rabbit, rat, bovine, and monkey serum, rat whole blood, and rat milk curd. W ord 6/95 FACT-M-3.1 Extraction o f PFOS from Serum and Other Fluids 3M Environmental Laboratory \^yb IUI Page 1 o f 17 P a' 153 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 fluorochemicals from serum, blood, or milk curd using an ion pairing reagent and 5.0 ml of ethyl acetate. In this method, seven fluorochemicals were extracted: PFOS, PFOSA, PFOSAA, EtFOSE-OH, POAA, PFOSEA, and FC-807 monoester (see 3.0 Definitions). An ion pairing reagent is added to the sample and the analyte ion pair is partitioned into ethyl acetate. Four ml of extract are 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 i n i t i o n s _____________________________________________________________ 3.1 PFOS: perfluorooctanesulfonate (anion o f potassium salt) C ,F17S 0 3' 3.2 PFOSA: perfluorooctane sulfonylamide C,F,7S 0 2NH2 3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate CgF l7S 0 2N(CH2CH3)CH2C 0 2' 3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F,7S 0 2N(CH2CH3)CH2CH20H 3.5 POAA: perfluorooctanoate (anion of ammonium salt) C7F15C 0 0 ` 3.6 PFOSEA: perfluorooctane sulfonyl ethylamide CaFl7S 0 2N(CH2CH3)H 3.7 FC-807 monoester C8F17S 0 2N(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 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 known interferences at this time. 6.0 E q u i p m e n t _____________________________________________________________________________________ 6.1 The following equipment is used while performing this method. Equivalent equipment is acceptable. 6.1.1 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR 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 1U3L Page 2 of 17 Pa' 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 7.0 Supplies and Materials__________________________________________ ___________ _ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Electronic pipettor, Eppendorf or equivalent 7.4 Graduated pipettes 7.5 Nalgene bottles, capable of 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 Milli-QTM water. Rinse syringes a minimum of 9 times with methanol, 3 rinses from 3 separate vials. 8.0 Reagents and Standards_____________________________________________________ 8.1 Type I reagent grade water, Milli-QTM or equivalent; all water used in this method should be Milli-QTM water and 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 (NajC03), J.T. Baker or equivalent 8.5 Sodium bicarbonate (NaHCOj), 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 Fluorocbemical 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 3M Environmental Laboratory 15*1 IL3 Page 3 of 17 P5 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 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 o f triester, diester, and monoester fluorochemical components. The monoester molecular weight = 650 8.10.8 Surrogate standard: 4-H, perfluorooctane sulfonic acid (l-H .l-H , 2-H, 2-H CgF13S 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 1 N sodium hydroxide (NaOH): Dilute 10 N NaOH 1:10. Measure 10 mL o f 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 g o f 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 and dilute to volume with 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 TBA requires a check prior to each use to ensure pH = 10. Adjust as needed using 1 N NaOH solution. 8.11.4 0.25 M sodium carbonate/sodium bicarbonate buffer (Na2C 0 3/N aH C03): Weigh approximately 26.5 g of sodium carbonate (Na^COj) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle. 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 solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.) 8.12.3 Weigh approximately 100 mg o f PFOS into a 100 ml volumetric flask and record the actual weight. 8.12.4 Bring to volume with methanol for a stock standard of approximately 1000 ppm (pg/ml). 8.12.5 Dilute the stock solution with methanol for a working standard 1 solution of approximately 50 ppm. FA C T -M -3.I Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory T9S- I U i Page 4 o f 17 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 Dilute the stock solution with methanol for a working standard 3 solution of approx. 0.50 ppm. 8.13 Surrogate stock standard preparation 8.13.1 Weigh approximately 50-60 mg of surrogate standard 1-H,1-H, 2-H, 2-H, CjFjjSOjH 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 1000-1200 ppm. 8.13.3 Prepare a surrogate working standard. Transfer approximately 0.5 ml of surrogate stock to a 50 ml volumetric flask and bring to volume with methanol for a working standard of 10-20 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. 10.0 Q u a l i t y C o n t r o l _____________________________________________________________________________ 10.1 Matrix blanks and method blanks 10.1.1 Extract two 1.0 mL aliquots of the appropriate matrix (serum or blood, with blood samples diluted 1:1 with Milli-QTM water) following this procedure and use as matrix blanks. See 11.1.4. 10.1.2 Extract two 1.0 ml aliquots of Milli-QTM water following this procedure and use as method blanks. 10.2 Matrix spikes 10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy o f the extraction. 10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set. 10.2.3 Expected concentrations will fall in the mid-range of the initial calibration curve. Additional spikes may be included and may fall in the low-range o f 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 and analyze continuing calibration check samples to ensure the accuracy of the initial calibration curve. If the percent difference between the initial curve and the continuing check differ by >30%, re-analyze samples analyzed after the last acceptable check. 10.3.2 Prepare one check per group o f ten samples. For example, if a sample set = 34, prepare and extract four checks. FACT-M-3.1 Extraction o f PFOS from Serum or Other Fluid 3M Environmental Laboratory 1SV !(ef' Page 5 o f 17 Pa' 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve. 10.3.4 The expected concentration will fall within the mid-range of the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end of the calibration curve (for example, 5 ppb - 100 ppb, rather than 5 ppb - 1000 ppb). 11.0 C a l i b r a t i o n a n d S t a n d a r d i z a t i o n ______________________________________________ 11.1 Prepare matrix calibration standards Note: Blood coagulates in air; therefore, minimize air contact until dilution. At this point, add TBA and buffer to each centrifuge tube as in step 12.9, then add 1.0 mL of the diluted matrix sample to each tube. 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 If most sample volumes are less than 1.0 mL, extract standards with matrix volumes equal to the sample volumes. Do not extract below 0.50 mL of matrix. Record the sample volume on the extraction sheet. 11.1.3 While preparing a total of twenty aliquots in 15 ml centrifuge tubes, mix or shake between aliquots. 11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end of this section, to spike, in duplicate, two standard curves, for a total of 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 the working ranges and the Linear Calibration Range (LCR) for calibration curves. 11.1.6 Use Attachment D as an aid in calculating the concentrations of the working standards. See Section 13.0 to calculate actual concentrations of PFOS in calibration standards. 11.2 To each standard, blank, or QC check, add appropriate amount of surrogate working standard for the concentration to fall within the calibration curve range 5 ppb -1000 ppb. 11.3 Extract spiked matrix standards following 12.6-12.16 of this method. Use these standards to establish each initial curve on the mass spectrometer. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory TS3 IU(- Page 6 o f 17 -15 8 3m Medical Department Study: T-6295.7 Report N o . 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 50.0 ppm 5 0.250 ppm 50.0 ppm 10 0.500 ppm 50.0 ppm 15 0.750 ppm 50.0 ppm 20 . 1.00 ppm Table 2 Approximate spiking amounts for standards and spikes using 0.5 ml of matrix Working standard pL Approx, final cone, of (approx, cone.) analyte in matrix - - Blank 0.500 ppm 5 0.005 ppm 0.500 ppm 10 0.010 ppm 5.00 ppm 2.5 0.025 ppm 5.00 ppm 5 0.050 ppm 5.00 ppm 10 0.100 ppm 50.0 ppm 2.5 0.250 ppm 50.0 ppm 5 0.500 ppm 50.0 ppm 7.5 0.750 ppm 50.0 ppm 10 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 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube. For blood samples, remove 0.5 mL and dilute to 1.0 mL with Milli-QTM water. As soon after diluting as possible, pipet diluted blood into TB Abuffer 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 Pa 9 3m Medical Department Study: T-6295.7 Report No. FACT TOX-03Q 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 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps. 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 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 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 Transfer 4 mL of organic layer, using a 5 mL graduated glass pipette, to a clean 15 mL centrifuge tube. Label this fresh tube with the same information as in 12.5. 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 graduated pipette. Methanol volume to add equals the initial volume of sample used for the extraction. 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 Label the autovial with the study number, animal number and gender, sample timepoint, matrix, final solvent, extraction date, and analyst(s) performing the extraction. 12.19 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. FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 8 o f 17 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 i s a n d C a l c u l a t io n s ______________________________________________ _ 13.1 Calculations 13.1.1 Calculate actual concentrations of PFOS, or other applicable fluorochemical, in calibration standards using the following equation: mL of standard x concentration of standard Cue /mL)_______________ _ = mL of standard + mL of surrogate standard + initial matrix volume (mL) Final Concentration (jig/mL) of PFOS in matrix 14.0 M e t h o d P e r f o r m a n c e _______________________________________________________________________ 14.1 The method detection limit (MDL) is analyte and matrix specific. Refer to MDL report for specific MDL and limit of quantitation (LOQ) values (see Attachments B and C). 14.2 The following quality control samples are extracted with each batch of samples to ensure the quality of the extraction and analysis. 14.2.1 Method blanks and matrix blanks 14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction 14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve 15.0 P o l l u t i o n P r e v e n t i o n a n d W a s t e M a n a g e m e n t _______________________________ 15.1 Sample waste is disposed in biohazard containers, flammable solvent waste is disposed in high BTU containers, and used glass pipette waste is disposed in broken glass containers located in the laboratory. 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 o f 17 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 19.0 Affected Documents 19.1 FACT-M-4.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry" 20.0 Revisions Revision Number 1 " Reason For Revision Validation of method to include 7 fluorochemicals, an additional matrix, new API/MS(MS) systems, monkey serum cross validation, improvements to ion pairing extraction, MDL study, updates in record keeping and storing policies, etc. Revision Date 07/01/98 FACT-M-3.I Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory 170 Page 10 of 17 J = g e --i-S ?" 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Study # Sample Number - - 1- set # H,0 Blank Blank .. FC-Mix approx. 0.5 ppm actual ppm #W - - FC-Mix approx. 5 ppm actual ppm #W - - FC-Mix approx. 50 ppm actual ppm #W - - Date and Initials for Std. or Comments -- - -- - -- - -- - -- - --- -- - -- - --- --- --- --- --- -- - --- --- --- - - 'Study number where the original wortahcsiis losatcd.____ Blank Std# - - amount = - - mL S e r u m E x t r a c t i o n M e t h o d _____________________________________ ;_________________________________________________________ D a t e & I n i t i a l s Vortex 15 sec.____________________________________________________________________________________________ Pipette Matrix__________________________________ Volume________________ m]_________________________________ Pipette 1 ml o f 0.5 M TBA, pH 10. Std. # Pipette 2 ml of0.2SNa2COy0.25MNaHCQ^ buffer Std.# Pipette 5 ml o f ethyl acetate________________________ TN-A- _______________________________ Shake 20 min.____________________________________________________________________________________________ Centrifuge 20-25 min._______________________Centrifuge speed:________________________________________________ Remove a 4 ml aliquot of organic layer_______________________________________________________________________ Put on Nitrogen Evaporator to dryness Evaporator #:___________________Temperature:_____________________________ Add methanol_____________ Volume ml__________ TN-A- ____________________ Vortex 30 sec.____________________________________________________________________________________________ Filter using a 3cc B-D syringe with a 0.2um SRI filter into a 1.5 ml autosample vial___________________________________ M S/M SD/___ Corn. Checks: S p ik ed ______ uL o f a ______ ppm std (_______________ ) for a final concentration o f _________ppm. M S/M SD used sa m p le________________ . Cont. Checks used same matrix as for std curve. Surrogate Standard: S p ik e d _____ uL o f 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 WrZ? m Page 11 o f 17 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 MDL/LOQ values for Rabbit Serum: Compound MDL LOQ Linear Calibration Range (LCR) (ppb) (PPb) Approximate concentrations to be used for preparing the Standard Calibration Curve PFOS 1.38 4.39 5 ppb - 1000 ppb PFOSA 2.23 7.09 10 ppb - 1 0 0 0 ppb PFOSAA 2.84 - 9.04 10 ppb - 1 0 0 0 ppb EtFOSE-OH 3.90 12.4 15 ppb - 1 0 0 0 ppb POAA 4.31 1 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 determ inable 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. MDL/LOQ values for Rat Serum: Compound MDL 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 - 1 0 0 0 ppb PFOSA 2.14 6.81 25 ppb - 1000 ppb PFOSAA 2.32 7.38 10 ppb - 1 0 0 0 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 - 1 0 0 0 ppb Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determ inable 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. MDL/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 - 1 0 0 0 ppb EtFOSE-OH 11.3 35.8 50 ppb - 1000 ppb POAA 4.64 14.8 15 ppb - 1 0 0 0 ppb PFOSEA 3.71 11.8 15 ppb - 1 0 0 0 ppb Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determ inable 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 worksheet FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory Page 12 of l 7 3m Medical Department Study: T-6295.7 Report No. FACT TOX-C30 laboratory Request N u m b er-1 1 2 2 7 9 MDL/LOQ values for Monkey Serum: Compound MDL 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 only. Please refer to FACT-M-3.1 & 4.1-V -l for specifics. EtFOSE-OH 3.90 12.4 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 -1 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. Monoester 149 248 MDL and LOQ are estimates only. No valid MDL was determ inable 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. MDL/LOQ values for Rat Whole 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 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 4.73 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. N o valid MDL was determ inable 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 worksheet FA C T -M -3.1 Extraction o f PFOS from Serum or Other Fluid 3M Environmental Laboratory M - 173 Page 13 o f 17 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 Approximate linear range3 Compound M atrix MDL LOQ Low std 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 E tF O S E -O H 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 M onoester1 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 m onoester are estim ates only. 2. H ighest standard (approx. 1500 ppb) was excluded from final LCR and upper LOQ values due to poor R & R values and excessive weighting o f the calibration curve. Compound: PFOS__________________________ Serum Prepared Range of LCR from Range of LCR from Range of LCR from matrix range of average ave curve low std low std high std high std standards curve curve curve curve curve (p pbX ng/m L) (p pb)(ng/m L) (p pbX ng/m L) (p pb X n g /m L ) (p pb X n g /m L ) (ppb X n g /m L ) (p p b X n g /m L ) Rabbit 4.93 - 1450 4.93 - 1450 49.3 - 1000 49.3 -9 7 .6 4.93 - 97.6 97.6 - 1450 9 7 .6 - 1000 Bovine 4.93 - 1450 4.93 - 1450 97.6 - 1000 4.93 - 248 24.8 - 248 97.6 - 1450 9 7 .6 - 1000 Rat Monkey 4.93 - 1450 4.93 - 976 24.8 - 9 7 6 4.93 - 1450 4.93 - 1450 24.8 - 1000 4.93 -248 24.8 -493 9.76 - 248 24.8 - 493 97.6 - 1450 97.6 - 1450 248 - 1000 97.6 1000 Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory \ f r l7 i Page 14 o f 17 66-- - 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: PFOSA Serum Prepared matrix range of standards (ppbXug/mL) Rabbit 5.00- 1470 Range of average curve (ppbXng/mL) 5.00 - 1470 LCR from ave curve (ppbXng/mL) 9 .8 9 - 1000 Range of low std curve (ppb)(ng/niL) 5.00-251 LCR from low std curve (ppb)(ng/mL) n/a Range of high std curve (ppbXng/mL) 9 8 .9 - 1470 LCR from high std curve (ppb)(ng/mL) 9 8 . 9 - 1000 Bovine 5 .0 0 - 1 470' 5.00 - 1470 25.1 -1 0 0 0 5 .0 0 -9 8 .9 n/a 9 8 .9 - 1470 98.9 - 1000 Rat 5 .0 0 - 1470 5.00 - 1470 5 0 .0 - 1000 9.89 - 500 25.1 - 5 0 0 9 8 .9 - 1470 9 8 . 9 - 1000 M onkey 5 .0 0 - 1470 5.00 - 1470 98.9 - 1000 25.1 - 500 25.1 - 500 9 8 .9 - 1470 n/a Compound: PFOSAA Serum matrix Prepared range of standards (p p b X n g /m L ) Range of average curve (p pb )(n g /m L ) Rabbit 5 .2 0 - 1540 5 .2 0 - 1540 LCR from ave curve (ppbX ng/m L) 104-1000 Range of low std curve (ppbX ng/m L) 5 .2 0 -2 6 3 LCR from low std curve (ppb X n g /m L ) 1 0 .4 -2 6 3 Range of high std curve (p pbX ng/m L) 1 0 4 - 1540 LCR from high std curve (ppb X n g /m L ) 263 - 1000 Bovine 5 .2 0 - 1540 5 .2 0 - 1540 263 - 1000 1 0 .4 -5 2 1 n /a (1 ) 104 - 1540 263 - 1000 Rat 5 .2 0 - 1540 5 .2 0 - 1540 10 4 -1 0 0 0 5.20 - 263 1 0 .4 -2 6 3 1 0 4 - 1540 263 - 1000 M onkey 5 .2 0 - 1540 5 .2 0 - 1540 52.4 - 1000 5 .2 0 - 2 6 3 26.3 - 263 1 0 4 - 1540 263 - 1000 Compound: EtFOSE-OH Serum Prepared Range of matrix range of average standards curve (ppb X n g /m L ) (p pb )(n g /m L ) Rabbit 4.94 - 1450 4.94 - 1450 LCR from ave curve (ppbX ng/'m L) 49.4 - 1000 Range of low std curve (ppb X n g /m L ) 4.94 - 248 LCR from low std curve (ppb X n g /m L ) 9.78 - 2 4 8 Range of high std curve C ppbXng/m L) 97.8 - 1450 LCR from high std curve (p pb X n g /m L ) n/a Bovine Rat Monkey 4.94 - 1450 4.94 - 1450 97.8 - 1000 4.94 - 248 4.94 - 1450 4.94 - 1450 494 -1 0 0 0 4 .9 4 -2 4 S 4.94 - 1450 4.94 -1450 9 7 .8 - 1000 4 .9 4 -2 4 8 4.94 - 248 n/a 9 .7 8 -2 4 8 97.8 - 1450 97.8 - 1450 97.8 - 1450 248 - 1000 97.8 1000 n/a A ttachm ent C: LOQ Summary FACT-M-3.1 Extraction o f PFOS from Serum or Other Fluid 3M Environmental Laboratory Jfahr ! 7 f Page 15 of 17 Pa> 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 Compound: POAA Serum matrix Prepared range of standards (ppbX ng/m L ) Rabbit 5.01 - 1480 Range of average curve (ppb)(ng/m L ) 5 .1 3 -1 5 1 0 LCR from ave curve (ppbX ng/m L) 25.8 - 1000 Range of low std curve (ppbX ng/m L) 5 .1 3 -2 5 8 LCR from low std curve (ppbX ng/m L ) n/a Range of high std curve (ppbX ng/m L) 102-1510 LCR from high std curve (ppb)(ng/m L ) n/a Bovine 5.01 - 1480" 5 .1 3 -1 5 1 0 1 0 2 -1 0 0 0 5 .1 3 -2 5 8 5 .1 3 -2 5 8 1 0 2 -1 5 1 0 2 5 8 - 1000 Rat 5.01 - 1480 5 .1 3 -1 5 1 0 5 1 .3 -1 0 0 0 5 .1 3 - 102 5 .1 3 - 1 0 2 1 0 2 - 1 5 1 0 1 0 2 - 1000 M onkey 5.01 - 1480 5 .1 3 -1 5 1 0 102 - 1000 5 .1 3 - 1 0 2 5.13 - 102 1 0 2 - 1510 258 - 1000 Compound: PFOSEA Seram matrix Prepared range of standards (ppbXng/mL) Range of average curve (ppb X n g /m L ) Rabbit 5 .1 3 - 1510 5.13 - 1510 LCR from ave curve (ppbXng/mL) 25.8- 1000 Range of low std curve (ppbXng/mL) 5.13 - 258 LCR from low std curve (P pb)(ng/m L) n/a Range of high std curve (p pb X n g /m L ) 102 - 1510 LCR from high std curve (p pbX ng/m L) n/a Bovine 5 .1 3 - 1510 5 .1 3 - 1510 102 - 1000 5.13 - 2 5 8 5.13 - 2 5 8 102 - 1510 258 - 1000 Rat 5 .1 3 - 1510 5.13 - 1510 5 1 .3 - 1000 5.13 - 102 5.13 - 102 102 - 1510 102 - 1000 M onkey 5.13 - 1510 5.13 - 1510 102 - 1000 5.13 - 102 5.13 - 1 0 2 102 - 1510 258 - 1000 Compound: Monoester Serum Prepared Range of matrix range of average standards curve (ppbX ng/m L ) (ppbX ng/m L ) Rabbit 4 .9 4 - 1450 9.78 - 978 LCR from ave curve (ppbX ng/m L) n/a Bovine 4.94 - 1450 97.8 - 1450 n/a Rat 4.94 - 1450 248 - 1450 248 - 1000 Monkey 4.94 - 1450 4 9 .4 - 1450 97 .8 - 1000 In general, the chromatography for the monoester was very poor (broad peaks, high baseline). Curves for m onoester in rabbit and bovine were unacceptable. Any quantitation perform ed with the m onoester is only an estimate and should not be used for reliable, accurate data reporting. Attachment C: LOQ Summary FACT-M-3.1 Extraction of PFOS from Seram or Other Fluid 3M Environmental Laboratory / 7<> Page 16 o f 17 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 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. (1500 ppm: W398-641 FC mix std approx. 5.00 ppm: W398-640 FC mix std approx. 50.0 ppm: W398-639 Surrogate std approx. 17.71 ppm: W398-605 Actual concentrations of standards in the FC mix PFOS PFOSA PFOSAA EtFOSE- POAA OH Std cone Std cone Std cone Std cone Std cone ug/mL ug/mL ug/mL ug/mL ug/mL PFOSEA Std cone ug/mL M onoeste r Std cone u g/m L 0.500 0.507 0.532 0.501 0.509 ' 0.521 0.501 0.500 0.507 0.532 0.501 0.509 0.521 0.501 5.00 5.07 5.32 5.01 5.09 5.21 5.01 5.00 5.07 5.32 5.01 5.09 5.21 5.01 5.00 5.07 5.32 5.01 5.09 5.21 5.01 50.0 50.1 53.2 50.1 50.9 52.1 50.1 50.0 50.1 53.2 50.1 50.9 52.1 50.1 50.0 50.1 53.2 50.1 50.9 52.1 50.1 50.0 50.1 53.2 50.1 50.9 52.1 50.1 Calculated concentrations ol standards in the sample matrix PFOS PFOSA PFOSAA EtFOSE POAA PFOSEA Monoester F inal Final co n e F inal co n e Final co n e F inal co n e F in al co n e S td cone cone n g/m L n g /m L ng/m L ng/m L n g /m L n g /m L n g /m L 4.93 1 5.00 9.76 9.89 24.8 25.1 49.3 50.0 97.6 1 98.9 248 j 25 493 i 500 735 746 976 989 5.24 4.94 5.01 5.13 4.94 i0.4 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 All Ain't spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020 i ' i j Final Volume mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025 Surrogate Std co n e ng/m L All A m 't sp ik e d (m L ) 2.64 1 0.005 Surrogate F inal cone nff/m L 8 1 .0 Validated ranges - approximate concentrations Sera PFOS PFOSA PFOSAA Rabbit 5-1000 ppb 10-1000 ppb 10-1000 ppb Bovine Rat 25-1000 ppb 10-1000 ppb 25-1000 ppb 263-1000 ppb 25-1000 ppb 10-1000 ppb Monkey Estimates only. U se values fo r Rabbit EtFOSE-OH 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 Curves FACT-M-3.1 Extraction of PFOS from Serum or Other Fluid 3M Environmental Laboratory fVb 7 7 Page 17 o f 17 P a ' "69 3m Medical Department Study: T-6295.7 Report N o . FACT TO} laboratory Requis 3M Environmental Laboratory M ethod A nalysis o f P otassium P er flu o r o o cta n esu lfo n a te o r O t h e r F l u o r o c h e m ic a l s in Seru m o r O t h e r F luid E x tra cts U sin g H P L C -E lectrospray/M ass Spectro m etry M ethod N um ber: FACT-M-4.1 Author: Lisa Clemen, Glenn Langenburg Approved By: Laboratory Manager j / f y 1>a . Group Leader --------------- h LlbnviiTechnical Reviewer Adoption Date: 4/22/98 Revision Date: ' J 'P ? ' Date Date ` k 'il'S Date 1.0 Scope and Application 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 M atrices: Rabbit, rat, bovine, or monkey serum and rat whole blood or milk curd. W ord 6.0.1/95 FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory - M - 1?S Page 1 of 9 Pag 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 2.0 Summary of Method______________________________________________ _ 2.1 This method describes the analysis of fluorochemical surfactants extracted from serum, whole blood, or milk curd using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the 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 D efinitions________________________________________________________________ __ 3.1 Atmospheric Pressure Ionization (API): The Micromass platform systems allow for various methods of ionization by utilizing various sources, probes, and interfaces. These include but are not limited to: Electrospray Ionization (ESI), Atmospheric Pressure chemical Ionization (APcI),Thermospray, etc. The ionization process in these techniques occurs at atmospheric pressure (i.e. not under a vacuum). 3.2 Electrospray Ionization (ES, ESI): a method of ionization performed at atmospheric pressure, whereby ionization occurs through th production of tiny charged droplets in a strong electrical field. 3.3 Mass Spectrometry, Mass Spectrometer (MS), Tandem Mass Spectrometer (MS/MS): The API platforms are equipped with quadrupole mass selective detectors. Ions are selectively discriminated by mass to charge ratio (m/z) and subsequently detected. A single MS may be employed for ion detection or a series (MS/MS) for more specific fragmentation information. 3.4 Conventional vs. Z-spray probe interface: The latest models of Micromass platform systems (post 1998) utilize a "Z-spray" conformation. The spray emitted from a probe is orthogonal to the cone aperture. In the conventional conformation it is aimed directly at the cone aperture, after passing through a tortuous pathway in the counter electrode. Though the configuration is different, the methods of operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e. Z-spray components are compatible with other Zspray systems, etc.) 3.5 Mass Lynx Software: System software designed for the specific operation o f these platform systems. Currently MassLynx has Windows 95 and WindowsNT 3.1 versions. 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 arnings and Cautions______________________________________________________ 4.1 Health and Safety Warnings: 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. Word 6.0.1/95 FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory Page 2 of 9 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 Interferences_______________________________________________________________ 5.1 To minimize interferences when analyzing samples for perfluorooctanoate(POAA), 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 Equipment _____________________________________ 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 Supplies and Materials______________________________________________________ 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 Reagents and Standards_____________________________________________________ 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 Sample Handling____________________________________________________________ 9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 ml centrifuge tubes until analysis. 9.2 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 o f Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory Page 3 of 9 3m Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 10.0 Quality Control_________________________________________________ 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 Matrix 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 o f 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 Analyze a mid-range calibration standard after every tenth sample. If a significant change ( 30%) in peak area occurs, relative to the initial standard curve, stop the 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 Calibration and Standardization____________________________________________ 11.1 Analyze the extracted matrix standards prior to and following each set o f extracts. The mean o f two standard values, at each standard concentration, will be plotted by linear regression (rJ)for the calibration curve using MassLynx or other suitable software. 11.2 The r2 value for the data should be 0.980 or greater. Lower values may be acceptable at the discretion o f the analyst and documented approval of the Project Lead. 11.3 If the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze. 11.4 For purposes of accuracy when quantitating low levels of analyte, it may be necessary to use the low end of the calibration curve rather than the full range of the standard curve. Example: when attempting to quantitate approximately 10 ppb o f analyte, generate a calibration curve consisting of die standards from 5 ppb to 100 ppb rather than the full range o f the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting of high concentration standards. 12.0 Procedures_________________________________________________________________ 12.1 Acquisition Set up 12.1.1 Click on start button in the Acquisition Control Panel. Set up a sample list. Assign a filename using letter-MO-DAY-last digit o f year-sample number, assign a method (MS) for acquiring, and type in sample descriptions. FACT-M-4.1 Analysis o f Serum or Fluid Extract Using ES/MS Page 4 of 9 3M Environmental Laboratory 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 SIR (Single Ion Recording). Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring). 12.1.3 Typically the analytical batch run sequence begins with a set o f 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 HP 1100/autosampler at the following conditions or at conditions the analyst considers appropriate for optimal response. Record actual conditions in the instrument logbook: 12.2.2.1 Sample size = 10 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 0.00 min. 7.5 min. 11.0 min. 11.5 min. MeOH 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 with a "Waiting for inlet start" message before the "Start" button is pressed on the HP Workstation. 12.2.2.5 Press the "Start" button. 12.3 Instrum ent 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 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. FACT-M-4.1 Analysis o f Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory 4 *3 /6 Z Page 5 of 9 P 174 3m Medical Department Srudy: T-6295.7 Report No. FACT TOX-030 laboratory Request Number-U2279 12.3.4 Set HPLC pump to "On". Set the flow to 10 - 500 uL/min or as appropriate. Observe droplets coming out of the tip of the probe. Allow to equilibrate for approximately 10 minutes. 12.3.5 Turn on the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. 12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response: 12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode flow rate 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 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. 12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button at top o f sample list. Ensure start and end sample number includes all samples to be analyzed. 13.0 Data Analysis and Calculations_____________________________________________ 13.1 Calculations: 13.1.4 Calculate matrix spike percent recoveries using the following equation: % Recovery = Observed Result - Background Result x 100 Expected Result 13.1.5 Calculate percent difference using the following equation: % Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone. 13.1.6 Calculate actual concentration of PFOS, or other fluorochemical, in matrix (jig/ml): fne o f PFOS calc, from std. Curve x Dilution Factor) x 1 ug (Initial Volume of matrix (ml) + ml o f Surrogate Standard! 1000 ng Final Volume (mL) 14.0 Method Performance_______________________________________________________ 14.1 Method Detection Limit (MDL) and Limit of Quantitation (LOQ) are method, analyte, and matrix specific. Please see FACT-M-3.1, Attachm ent A for a listing of current validated MDL and LOQ values. FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory f r t /*3 Page 6 of 9 P 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 Method blanks and matrix blanks will be analyzed with each sample set for possible contamination or carryover. Values are expected to fall below the lowest standard in the calibration curve. 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% of the spiked concentration. 14.4 C ontinuing 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 performed on the system and samples reanalyzed or other actions as determined by the analyst. All actions will be documented in the instrument runlog, the maintenance log, or on the summary sheet with the sample results. 15.0 P o l l u t i o n P r e v e n t io n a n d W a s t e M a n a g e m e n t _____________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass pipette waste is disposed in broken glass containers located in the laboratory. 16.0 R e c o r d s ____________________________________________________________________ 16.1 Store chromatograms in the study or project folder. Each chromatogram must have the following information included either in the header or hand written on the chromatogram: 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 of backup electronic data. 17.0 T a b l e s . D i a g r a m s . F l o w c h a r t s , a n d V a l i d a t i o n D a t a _______________________________ 17.1 Attachment A: 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. F A C T -M -4.1 Analysis of Serum or Fluid Extract Using ES/MS Page 7 of 9 3M Environmental Laboratory 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 f f e c t e d D o c u m e n t s _________________________________________________________________ __ 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 i s i o n s ______________________________________________________________________ Revision Number. 1 - Reason For Revision Validation o f method to include 7fluorochemicals addition o f whole blood matrix, surrogate standard, new API/MS(MS) systems, monkey sera cross validation, MDL study, updates in record keeping and storing policies, etc. Revision Date 07/01/98 FACT-M-4.1 Analysis of Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory ns Page 8 of 9 3m Medical Department Study: T-6295.7 Report N o . FACT TOX-030 laboratory Request Number-U2279 Attachment A Laboratory Study # Study: Test Material: M atrix/Final Solvent: M ethod/R evision: Analytical Equipment System Number: Instrument Sofrware/Version: Filename: R-Squared Value: Slope: Y Intercept: Date o f Extraction/Analyst: Date o f Analysis/Analyst: G roup Dose S am ple# C o n c en tra tio n ug/m L Initial Vol. mL D ilution Factor Final Cone. ug/m L Slope: Taken from linear regression equation. G roup/D ose: Taken from the study folder. Sam ple#: Taken from the study folder. C oncentration (ug/m L): Taken from the MassLynx integration summary. Initial V olum e (m L): Taken from the study folder. D ilution F acto r: Taken from the study folder. Final C one. (ug/m L): Calculated by dividing the initial volume from the concentration F A C T -M -4.0 Analysis o f Serum or Fluid Extract Using ES/MS 3M Environmental Laboratory Page 9 of 9 P 3m M e d ic a l D e p a r tm e n t S t u d y : T - 6 2 9 5 . 7 3M Medical Department Study: T-6295.7 A ttachment D Data Summary Tables R e p o rt N o. FACT TOX-030 la b o r a to r y R e q u e st N um ber-U 2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 3M Environmental Laboratory 3M E n v i r o n m e n t a l L a b o r a t o r y / k*? Page D-1 ^P ^g e -tT T 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-112279 BEST COPY AVAILABLE Table D-1a. Sample Intervals and Types by Individual Animal from Study 6329*223 A n n a l ID Group 1 (Control} 105508M 105517M 105519M 105520M 105526M 105527M 105529F 105530F 105531F 105535F 105544F 105549F Group U (Low Dose) Serum Samples Collected (Oty-- Week) Liver Samples Collected (Date) 151 Samples 8 Samples 12-- 0 .1 ,2 .4 ,6 , 8,12,16, 20. 24, 26, 27i 11-- 0, 1, 2, 4 ,6 ,8 , 12,16, 20, 24,26 12-- 0 .1 ,2 ,4 .6 ,8 ,1 2 ,1 6 , 20, 24, 2 6 .27i 1 4 -0 ,1 ,2 .4 ,6 ,8 ,1 2 .1 6 , 20, 24,26, 27, 27i. 27ii 14-- 0, 1 ,2 ,4 , 6,8 ,12 ,1 6 , 20.24, 2 6 ,2 7 ,27i, 2Tii 12-- 0, 1,2, 4.6, B, 12,16, 20, 2 4 ,2 6 .27i 14-- 0 ,1 ,2 , 4,6, 8,12.16, 20, 24, 26. 27, 27i. 27ii 1 2 -0 ,1 ,2 ,4 , 6,8,12,16, 20, 24, 26, 27i 12-- 0 ,1 ,2 ,4 , 6, 8,12.16. 20, 24, 26, 27ii 12-- 0, 1 ,2 ,4 , 6, 8,12, 16, 20, 24, 26, 27ii 12-- 0, 1.2. 4, 6, 8,12, 16, 20, 24, 26,275 14-- 0 ,1 ,2 ,4 , 6, 8,12,16, 20, 24. 26, 27, 27i, 27ii 99 Samples 2/25/99 2/25/99 2125m Relumed to oolony 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 Relumed to colony 3/07/00 8 Samples Recovery Subgroup Serum Samples Collected (Oty-- W eek) 72 Samples , ' . . : 1* 18-- 27, 28, 29, 30, 31, 35, 39, 43, 47, 51, 53, 57, 61, 65, 69, 73, 77, 79 18-- 27. 28,29, 30, 31, 35, 39, 43,47. 51.53,57,61,65, 69,73,77.79 :. X ' 18-- 27, 28, 29, 30, 31, 35, 39, 43,47, 51, 53, 57, 61, 65, 69, 73, 77. 79 -p . *1N.'v - <-C . *.,jri; !- /*- 18-- 27, 28, 29, 30. 31,35, 39, 43, 47, 51.53, 57. 61. 65. 69, 73. 77, 79 -- 1-- 0 Baeellne^CTiy-Not assigned Returned to colony 105514M 105515M 105516M 105521M 12-- 0, 1,2, 4 .6 ,8 ,1 2 .1 6 , 20, 24, 26, 27i 12-- 0, 1,2, 4 ,6 ,8 .1 2 ,1 6 , 20, 24, 26, 27ii 12-- 0. 1 ,2 ,4 , 6. 8, 12. 16, 20,24, 26, 27ii 12-- 0, 1 ,2 .4 , 6, 8, 12,16, 2 0 ,2 4 ,2 6 ,27ii 2/26/99 2/26/99 2/26/99 2/26/99 J- r 'Mi- ' - ' -1,'..!v' .-AJ.'1-'-'- - ' / 'V. 'rlT-1 1-- 0 B a s a 8 n ii|i]||jM )lp t assigned Relumed to colony 105537F 105541F 11-- 0, 1 .2 ,4 .6 ,8 ,1 2 ,1 8 . 20, 24, 26 12-- 0, 1,2, 4,6, 8,12.15, 20. 24. 26, 27ii 2/25/99 2/26/99 . 'rVilfk- 1-- 0 B a a e jS IW te S lo t assigned 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 ,1 6 , 20, 24, 26, 27 B aseM ^^^idtessJgned 2/2099 2/26/99 Relumed to colony --f . , . 27 Day 183 (2/23/99) 27I Day 184 (2/25/99) 27ii Day 185 (2/25/99) 27iii Day 187(2/28/99) " Two samples 2/20/99 79 Sample on 2/23/00 79 Sample on 2/25/00 79ii Sample on 2/25/00 Note: Samples forWeek 26 and Week 27 (Recovery Group) taken on same day (Day 183, 2/23/99) 3M Environmental Laboratory 3M Environmental Laboratory iPr / t r Page D-2 PacLe-crS'O 3m Medical Departmer. 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 Table D-1b. Sample Intervals and Types by Individual Animal from Study 6329-223 A nimal ID G roup III (M ld D o e e ) 105505M 105510M 105518M 105523M 105S24M 10552BM 105532F 105538F 105539F 105545F 105548F 105552F G roup IV (High Doee) 105506M 105507M 105509M 105511M 105512M 105522M 105533F 105534F 105536F 105540F 105542F 105551F Serum Samples Collected (Qty-- Week) 152 Samples 14-- 0 ,1 .2 ,4 ,6 .8 ,1 2 ,1 6 , 20, 2 4 ,2 6 ,, 27,271,2711 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 14-- 0 ,1 ,2 ,4 ,6 ,8 ,1 2 ,1 6 , 20,24, 26,27,271,27 12-t-Q, 1 .2 ,4 ,6 ,8 .1 2 ,1 6 , 20, 24, 26,27 1 2 -0 ,1 .2 ,4 ,6 , 8,12,16, 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, 2 6 ,27I 14-- 0 .1 ,2 ,4 ,6 ,8 ,1 2 ,1 6 . 20, 24, 26, 27,271,2711 12-- 0, 1,2, 4, 6. 8, 12,16, 20,24, 26, 27i 12-- 0 ,1 ,2 .4 ,6 ,8 ,1 2 ,1 6 , 20,24, 26.271 14-- 0, 1 ,2 ,4 , 6, 6,12,16, 2 0 ,2 4 ,2 6 .2 7 ,271,27 148 Samples 1 1 -0 ,1 ,2 , 4.6,8,12,16, 20.24, " 12-- 0 ,1 ,2 ,4 ,6 , 8,12,16, 20. 24, 26,271 9-- 0, 1 ,2 ,4 ,6 ,8 .1 2 ,1 6 ,2 0 14-- 0, 1, 2, 4, 6, 8, 12, 16, 20, 24,26,27, 27i, 27 12-- 0, 1 ,2 .4 , 6, 8, 12, 16, 20,24, 2 6 ,27i 14-- 0, 1 ,2 ,4 . 6, 8. 12, 16, 20.24,26.27.271,27 14-- 0 ,1 ,2 ,4 ,6 , 8,12,16, 20.24, 26.27,271,2711 12-- 0 ,1 ,2 , 4. 6, 8,12,16, 20,24, 2 6 ,27S 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, 12, 16, 20, 24, 26.27, 271.27u 12-- 0 ,1 .2 ,4 , 6, 8, 12, 16. 2 0 .2 4 .2 6 ,27it liver Samples Collected (DATE) 12 Samples 3/01/00 Biopsy 2/26/99 2/26/99 3/01/00 Biopsy 2/26/99 2/26/99 2/25/99 2/25/99 3/01/00 Biopsy "2125m 2125m 3/01/00 Biopsy 14 Samples none 2125m none 9/22/99 Biopsy. 2/25/00 2125m 5122m Biopsy. 2125100 9/22/99 Biopsy, 2125100 2/26/99 2/25/99 2125m 9/22/99 Biopsy. 2125100 2125m Recovery Subgroup Serum Sam ples I Collected (Qty-- w e e k ) | 72 Samples 18-- 27iii, 28, 29, 30, 31, 35, 39, 43, 47, 51,53, 57.61 .6 5 ,6 9 ,7 3 ,7 7 ,7 9 18-- 27iii, 28,29, 30, 31,35, 39, 43. 47, 5 1 ,5 3 ,5 7 ,6 1 ,6 5 ,6 9 ,7 3 . 77. 79 .V 'tw>. v rL v ,;-" . 18-- 27iii, 28. 29, 30, 31, 35, 39. 43. 47, 5 1 ,53 ,5 7 ,6 1 ,6 5 ,6 9 , 73, 77, 79 18-- 27iil, 28,29, 30, 31. 35. 3 9,43,47, 51,53.57, 61,65,69, 73, 77,79 80 Samples '' ' *, ' . 20-- 27iil, 28. 29, 30, 31, 35, 39, 43, 47, 51.53, 57.61,65.69, 73, 77.79, 79i, 79 20-- 27iii, 28,29, 30, 31,35, 39, 43.47, 51. 53, 57, 61.65, 69.73, 77,79, 79i, 79 20-- 27i, 28,29.30, 31, 35. 39. 43.47, 51, 53, 57, 61, 65. 69, 73. 77, 79, 791. 79 r 1'-"- v * * 20-- 271. 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 27ii Day 185 (2/26/99) 79i Sample on 2/25/00 27i Day 187(2/28/99) 79ii Sample on 2/25/00 Note: Samples for Week 26 and Week 27 (Recovery Group) taken on same day (Day 183. 2/23/99) 3M Environmental Laboratory 3M Environmental Laboratory Page D-3 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request N u m b e r - U 2 2 79 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 LOQfiiQtaL.) All PFOS values u g lm L G roup 1M OJDmg/ttQM Baseline Average PFOS <LOQ LOGO.025 Std Deviation NA Week 1 LOQ 0.025 Average PFOS Std Deviation <LOQ NA Week 2 tOO 0.025 Average PFOS Std Deviation <LOQ NA Week 4 LOQ 0.0152 Average PFOS Std Deviation <LOQ -1 Anomaly NA Week 6 Average PFOS <LOQ LOQ0.0152 Std Deviation NA Week8 LOQ 0.0152 Average PFOS Std Deviation <LOQ -1 Anomaly NA Week 12 LOQ.01S2 Average PFOS Std Deviation 0.0383 -1 Anomaly 0.00811 Week 16 LOQ 0.0152 Average PFOS Std Deviation 0.0407 0.0110 Week 20 LOQ 0.0152 Average PFOS Std Deviation 0.0400 0.0120 Week 24 LOQ 0.0152 Average PFOS Std Deviation 0.0440 0.0101 Week 26 Average PFOS 0.0459 LOQ 0.0152 Week 27 LOQ 0.0246 Std Deviation Average PFOS Std Deviation 0.0143 0.0529 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 the Lower Urn* of Quantitation G roup 1F OjOmgAcQM Group 2M O.O3mQft0f d Group 2F OiUmQfcoM Group 3M llS n ^ ltO U <LOQ <LOQ <LOQ <LOQ NA NA NA NA <LOQ NA 0.869 0.147 0.947 0.110 4.60 0.782 <LOQ 1.10 1.10 5.81 NA 0.0835 0.0963 0.933 <LOQ NA 3.20 0.577 3.40 0.291 17.8 1.68 <LOQ 3.61 3.71 20.4 NA 0.430 0.417 1.65 0.0226 -2 Anomalies 0.00189 4.73 0.432 4.76 0.577 26.0 3.30 0.0263 -2 Anomalies 0.00362 6.69 0.578 6.31 0.717 35.2 5.39 0.0432 -2 Anomalies 0.00851 0.0504 0.0127 0.0426-1 Anomaly 0.00784 0.0506 0.0164 11.2 2.44 12.3 1.40 14.5 3.06 15.8 1.41 10.5 1.90 19.5 14.5 13.0 0.675 13.2 1.42 56.2 5.84 63.7 6.71 65.9 6.88 82.6 25.2 0.0416 0.0148 15.9 ___1_1_.1___ 68.1 I 5.54 1.52 I 5.75 0.0533 0.0315 0.0352 0.00911 0.0546 0.0306 -- -- -- -- I- - 85.0 -- 14.9 -- 69.7 -- 4.68 -- 77.9 -- 10.7 Group 3F 0.19>nkaw <LOQ NA 3.71 0.455 539 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 Group 4M 0.75mg*^d Group 4F O.rSmofttgM <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 4fct M Page D-4 p 82 3m M e d i c a l D e p a r t m e n t S t u d y : T - 6 2 9 5 . 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-3. Average PFOS Concentrations in Serum by Dosage Group and Week from Study FACT TOX-030 (Recovery Group) Time Point LOQ {|ig/mL) Day 187 LOO 0.0152 Week 28 LOQ 0.0152 Week 29 LOQQ0152 Week 30 LOQ 0.0152 Week 31 LOQ 0.0152 Week 35 LOQ 0.00665 W eek 39 LOQ 0.00556 Week 43 LOQ 0.00555 Week 47 LOQ 0.00555 W eek 51 LOQ 0.00555 Week 53 LOQ 0.00555 W eek 57 LOQ 0.00555 Week 61 LOQ 0.00555 Week 65 LOQ 0.00565 Week 69 LOQ 0.00565 Week 73 LOQ 0.00555 W eek 77 LOQ 0.00656 W eek 79 LOQ 0.00655 A ll PFOS values Ufl/mL Average PFOS (pomu Std Deviation Average PFOS (wow) Std Deviation Average PFOS (i m ) Std Deviation Average PFOS (nemu Std Deviation Average PFOS (uomo Std Deviation Average PFOS (n>mu Std Deviation Average PFOS (uno Std Deviation Average PFOS (pewi Std Deviation Average PFOS (t^mu Sid Deviation Average PFOS (mw ) Std Deviation Average PFOS (now.) Std Deviation Average PFOS <uvn.) Std Deviation Average PFOS (new.) Std Deviation Average PFOS (pew.) Std Deviation Average PFOS (1*emu Std Deviation Average PFOS <m o w > Std Deviation Average PFOS (>*.) Std Deviation Average PFOS Std Deviation Group 1M Group 1F G roup 3M Group 3F O.Omg/kg/dlay 0.15mg/kg/day 0.0300 -1 Anomaly MA 0.0457 0.0118 79.1 4.67 39.5 41.4 0.0371 0.0124 0.0430 0.00821 84.6 1.51 86.1 3.59 0.0665 0.00362 0.0742 0.000664 75.8 2.18 69.9 11.4 0.0313 0.000165 0.0303 0.000485 65.2 4.60 62.0 10.6 0.0358 0.000567 0.0368 0.0121 56.6 4.77 79.6 43.8 0.0459 0.00303 0.0723 0.00352 84.5 12.0 74.4 9.53 0.0391 0.0106 0.0509 0.000779 60.1 3.16 51.7 7.29 0.0319 0.00440 0.0368 0.0000923 45.7 1.12 58.1 0.249 0.0355 0.00221 0.0237 0.00333 0.0459 0.00323 0.0341 0.000403 48.3 3.69 37.9 2.62 42.6 6.70 35.1 13.2 0.0331 0.0086 0.0397 0.00311 46.2 3.30 36.7 6.24 0.0327 0.00526 0.0445 0.00385 30.2 2.36 32.3 1.34 0.0351 0.00449 0.0448 0.00210 31.6 5.98 38.2 0.283 0.0210 0.00365 0.0406 0.00313 0.0360 0.000522 l 0.0400 0.00301 32.9 0.0269 26.4 2.59 37.6 2.32 34.5 3.46 0.0350 0.0115 0.0365 | 0.00284 27.3 4.66 25.8 2.91 0.0296 0.00535 0.0215 0.00296 0.0305 I 22.5 23.0 0.00167 ; 0.632 6.37 0.0243 I 19.1 I 21.4 0.00355 0.805 | 2.01 Group 4M Group 4F 0 .7 5m g lkg /d ay 267 4Z0 249 21.7 223 66.9 258 15.2 236 18.3 194 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 98.3 8.32 94.7 38.4 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 57.0 38.3 19.1 41.1 | 41.4 25.9 | 1.15 3M Environmental Laboratory 3M Environmental Laboratory J S fr/9 1 Page D-5 p 83 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-112279 Table D-4a. PFOS Amounts Reported in Serum by Individual Animal (Baseline-Week 26} PFO S Repo rted W eekO tW Fm U W eek 1 (moM J W eek 2 iMGfrnL) WEEK 4 W eeks W eeks W e e k 12 W EEK 16 toOfinLl G ro u p 1(C o n tra I t 105S08M *< O Q 10SS17M < O Q 105519M 0 .0 0 105520M < L O Q 105526M < 0 0 105SZ7M *<LC Q 10SS29F xLO Q 10S530F < O Q 105531F < L 0 Q 10S53SF XLO Q 105544F < tO Q 105548F < -O Q <0Q < 0 Q <LOQ <00 <LO Q <LO Q <LOQ <LOQ <0Q <LOQ < O Q <O Q <LO Q <LOQ < O Q < O Q <O Q < O Q < O Q < O Q < O Q <O Q < O Q < O Q G ro u p 1 (L o w Do m ) 10S513M 105S14M 105515M 105516M 105521M 105525M 105S37F 105541F 10S543F 105546F 105547F 105S50F *< 0 Q <LOQ < O Q <00 < L O Q <LO Q *<LO Q <LO O <LO Q xLO Q < O Q < O Q 0.793 0 7 6 7 0.832 1.09 1.10 0.929 0931 0.832 1.02 1.03 1.19 1.13 1.19 1.16 1 06 0.980 G ro u p *(M d D O M ) 105505M 105510M 105518M 10SS23M 105624M 10S52SM 105532F 105SM F 105539F 105545F 105648F 10SS52F <LOQ <LO Q LO O <LO Q *<LO Q LO Q <LO Q <LOQ <LOQ < 0 Q <0Q X I 4.60 4.19 5.48 3.54 4.2S 5.52 4.31 4.12 3.31 *3.69 372 3.12 6.07 4.82 7 01 456 6 24 6 15 4.61 867 4 65 528 *6.42 4.73 G ro u p IV (H ig h D o ) 105500M <LO Q 21.3 29.9 10S507M <LOQ 22-0 25.1 10S509M <O Q 18.8 23.9 10S511M <LO Q 19.3 22.8 10S512M *<l_O Q 228 31.3 105522M < 0 Q 21.6 288 105533F <LO Q 2 2 8 249 105&M F < L O Q 16.1 26.0 105536F *<LO Q 21.0 23.5 105S40F 105542F xLO Q < L O Q 16.8 24.0 19.5 199 105551F <LOQ 20.1 13.2 e indicates a sam ple w ith an extraction volume of <0 5 m l Shaded cells*m oriburd <LOQ < O Q < O Q < O Q <LOQ <LO Q < O Q < O Q <O Q <LO Q <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 17 6 15.4 19.2 16.6 18.7 14.3 18.7 .1 5 7 14.9 236 72.8 54.8 48.4 .77.4 8 2 2 141 *145 64.1 67.2 7 64 60.2 < O Q < O Q < 0 Q <O Q <LOQ < O Q < O Q *< O Q <O Q < O Q 4 .0 0 < O Q <00 <O Q <O Q <O Q <O Q 0.0385 0.0214 0.0244 00206 <O Q <O Q 0.0240 3.87 3.31 3.26 4.19 4.61 4.17 505 510 4.28 3.86 rt'- , 5.28 4 85 3.85 3.27 4.98 3.94 21.0 225 20.1 18.6 21.8 18.5 18.4 189 18.4 227 18.4 18.1 27.9 221 25.1 245 31.6 24.6 27.8 24.3 26.5 24.5 22.3 19.1 91.0 8 28 923 827 105 102 93.0 8 23 89.8 90.8 102 820 125 86.1 103 92.3 118 131 110 121 102 105 116 87.3 0.0381 0.0430 0.0438 <L0Q 00244 00423 <LO Q 0.0243 0.0254 <O Q 0.0238 0.0316 620 648 6.55 .7 5 2 6.81 5.57 7.03 583 40.1 392 337 35.3 37.8 25.4 25.8 32.8 278 32.2 232 24.3 116 106 123 994 167 .121 126 114 98.0 112 131 117 00344 0.0408 0.0423 0 0332 0.0320 00615 0.0438 0.0515 0.0314 < O Q <LO Q 0.0462 10.4 9.47 10.1 14.8 in 123 107 7.85 5 8 4 529 60.7 52.2 63.4 48.4 40.4 373 46.3 43.1 47.0 38.7 182 172 197 179 218 185 164 157 184 173 168 127 W eek 20 04prrL) 0.0348 0.0419 0.0336 0.0300 0.0369 0.0631 00617 0.0579 0.0493 0.0263 0.0505 0.0570 12.5 107 11.9 14.1 11.0 11.5 14.3 41.1 62.3 63.3 53.1 75.2 640 54.2 66.2 47.4 78,2 45.7 65.3 427 156 138 152 126 143 145 169 143 148 193 151 132 W eek 24 I W eek 26 (np/mL) J UiQ/mL) 0.0449 0.0486 0.0414 0.0283 0.0397 0 0613 0.0498 0 0 4 9 7 0.0364 *< O Q 0.0325 0.0446 0.0496 0.0539 0.0417 0.0254 0.0376 0.0669 0.0613 0.0736 00431 0.0266 0.0433 00556 14.7 1 0 .9 14.1 18.4 13.4 13.7 12.7 12.2 77.4 65.2 58.8 597 69.6 646 63.1 48.6 69 9 83.5 56.1 6 17 196 1B8 -- 207 247 233 190 200 169 151 181 15 16.2 16.5 138 .1 6 9 13.5 1 4 6 130 11.4 929 129 69 5 72.1 69.5 624 634 590 72.2 857 64 5 55.6 -- 182 -- 142 148 221 203 187 155 177 165 142 3M Environmental Laboratory 3M Environmental Laboratory Page D-6 p. 84 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-4b. PFOS Amounts Reported In Serum by Individual Animal (Days 183-Day 185 and Week 27) PFO S Repo r ted D a y 183 (MO*4J D A Y 184 (moM -i Day 185 C jg ftrl) W EEK 27 (p0/mL) G ro u p 1(C on e oO 105508M -- 105517M -- 105519M -- 105520M 0.171 10552BM 00626 O -- @ <LOQ 0.0233 -- -- -- 0.0366 0.0498 105527M 10S529F 105530F 105531F 105535F 1Q5544F 105549F -- 00756 -- -- -- -- 0.0310 @ 0.0418 -- -- 0.0287 -- 0.0763 -- -- 0.0330 G ro u p 1 (Low Oo m ) 105514M -- 6 -- 105515M -- -- @ 1Q5516M - -- @ 105521M -- -- @ 105537F -- -- -- 105541F -- -- @ 105547F -- -- @ 105550F -- -- @ G ro u p ID (MM D ose) 105505M 74.4 56.4 70.3 105510M -- -- @ 105518M -- -- 105523M 95.5 73.0 65.4 105524M 105528M 105532F 105S3BF 105539F 105545F 105548F 105552F -- -- -- -- 107 -- -- 56.9 -- -- @ a 86.5 69.5 @ @ -- 101 -- 67 8 G ro u p IV (H igh Do m ) 1055M M -- -- -- 105507M -- -- 10550PM -- -- -- 105511M 218 181 309 105512M -- -- 105522M 282 336 278 105533F 258 265 441 1Q5534F -- -- 105536F -- -- 10554F -- -- 1Q5542F 201 224 201 105551F - - @ @ Sample o ola cio n data* for W eek 27 data ' Sample oolectsd on 2OTB9 Indcales a sample with an extraction volume o f <0.5 r r l 0.0461 -- 0.0430 -- -- 0.0695 -- 0.0635 0 0374 0.0316 0.0336 -- 23.9 13.0 11.4 15.5 -- 126 953 11.2 -- 68.5 68.7 -- 60.0 74.7 54.3 565 -- 58.0 65.1 - 196* 164 -- -- 202 -- -- 182 169 164 -- 126 3M Environmental Laboratory 3M Environmental Laboratory A+ / fS Page D-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 Tabla D-5a. PFOS Amounts Reported In Serum by Recovery Group Animal (Day 187- Week 47) PFOS Repo r ted DAY 187 (uPAnl) W eek 28 lupins.) W eek 29 tue**-) WEEK 30 tiO IM .) W eek 31 lia n t) W eek 35 WEEK 39 (uqATLI (UPAnL) WEEK 43 (upAnt) WEEK 47 lupAnL) Group 1(Cootno*) 105520M <LOQ 105526M 0.0300 00284 0.0459 0.0890 0.0639 105529F 0.0373 0 0488 0.0738 105549F 0.0541 00372 0.0747 Group II (M id Doe*} 105505M 82.4 85.7 773 105523M 75.8 83-5 7 42 10S539F 10.2 88.7 77.9 1055SZF 68.8 33.6 61.9 Group IV (High Doae) 105511M 237 233 175 105S22M 297 264 270 105533F 269 249 207 105542F 24a 223 180 indicates a sample w ith an extraction volume of <0.5 nri. 0.0314 0.0312 0.029 00306 68.4 61.9 69.9 54.5 116 170 156 167 0.0362 0.0354 0.0282 0.0453 60.0 53.2 111 48.6 129 194 201 .1 7 0 0.0437 0 0480 0.0748 0.0698 93.0 76.1 81.2 67,7 167 195 164 178 0.0316 0.0466 0.0603 0.0514 62.4 57.9 56.8 46.5 135 158 169 154 0.0287 0.0350 0.0368 0.0367 46.5 44.9 583 58.0 66.9 90.7 179 139 0.0339 0.0370 0.0481 0.0436 50.9 45.7 47.4 37.9 105 1 4 2 104 92 Table D-5b. PFOS Amounts Reported In Serum by Recovery Group Animal (Week 5 1 -Week 79) PFOS Repo rted W eek 51 Imp*"*-) W eek 53 W eek 57 (upA *) Oi0frni-) Week 61 (upAnU Week 65 ) w eek 69 Week 73 (upAnt) (upfmL) (uPAnL) Week 77 (uOA"M WEEK 79 (upAnL) Group I (ConiroO 105520M 00213 105526M 0.0261 10552PF 0.0344 105549F 0.0338 Group III (M id Do m ) 0 0269 00392 0.0375 0.0419 0.0289 0.0364 0.0472 0.0418 105505M 105523M 105539F 105552F 39.8 36.0 445 25.8 43.9 43.5 41.1 32.3 28.5 31.8 333 31.4 Group IV (High Dope) 105S11M 67.5 548 66.5 105522M 122 107 89.5 105533F 671 986 103 105542F 95.7 979 109 Indicates a sample w ith an e xtra cto r volume o f <0 5 m l 00319 0.0362 0.0433 0.046 27.4 35.9 38.0 36.4 64.7 136 106 106 0.0184 0.0236 00364 0.0357 32.9 329 393 36.0 .5 2 .5 131 89.7 76.0 0.0428 0.0383 0.0421 0.0379 0.0431 0.0269 0.0388 0.0345 24.5 283 369 32.0 240 3 0 6 279 23.7 470 121 787 71.3 35.1 73.5 545 240 0.0334 -0.0259 0.0317 0.0294 22.1 23.0 .2 7 5 18.5 32.9 870 70.5 43.4 0.0194 0.0236 0.0268 0.0218 197 185 226 199 228 5 64 406 42.2 Table D-8. LOQ Values Used In Analyses by Method and Usage Dates Method Sera FACT-M-4.1 ETS-8-5.0 ETS-6-5.1 Uver FACT-M-2.0 FACT-M-21 ETS-8-7.0 Effective Date 10/10/98 3/01/99 4/26/99 5/26/98 6/03/99 7/22/99 LOQ ng/mL 4.39 15.2 5.55 nwV 30.0 37.4 26.9 Usage Dates 1/25/99 to 2/22/99 3/05/99 to 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 w * /? ? 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Report No. FACT TOX-030 laboratory Request N u m b e r - U 2 2 79 Report No. FACT TOX-030 Laboratory Request Number-U2279 JJJ oa 3 I < ooCL f-- C US C3 Tab) D-7. PFOS Concentrations In Uver by Dosage Group Dosage Group COLLECTION Date PFOS Calculated Concentration imwi amount of PFOS (Mo/fl) Group 1(ControO 105508M 105517M 105519M 105S20M 105S26M 1055Z7M 105529F 105530F 105531F 105535F 1Q5544F 105549F 2/25/99 2/25/99 2/25/99 : 2/25/99 Non**-- 2/25/99 2/26/99 2/26/99 2/25/99 .Nones.-./ Group II (Low Dose) 2 Anomalies 143-1 Anomaly 91 . . tfiUBSi^.r - S ffife : t --- -- 129 -- 128 112 87 97 a a w fc u - - 105514M 10551SM 105516M 105521M 105537F 105541F 105547F 105550F 2/26/99 2/26/99 2/26/99 2/26/99 2/25/99 2/26/99 2/26/99 2/26/99 18237 22709 11417 16734 22818 24847 20102 20735 G roup Hi (M id Does) 105505M 105510M 105518M 105523M 105524M 10552SM 105532F 105538F 105539F 105545F 105548F 105552F 3/01/00 Biopsy 2/26/99 2/26/99 3/01/00 Biopsy 2/26/99 2/26/99 2/25/99 2/25/99 3/01/00 Biopsy 2/25/99 2/25/99 3/01/00 Biopsy 8252 42169 86173 10203 58673 48203 80421 49590 24728 66532 81376 17690 Group IV (H igh Do m ) 105506M 105507M 105509M 105511M 105512M 105522M 105533F 105534F 105536F 105540F 105542F 105551F 2/25/99 9/22/99 Biopsy 2/25(00 2/25/99 9/22/99 Biopsy 2/2590 9/22/99 B iopsy 2/25/00 2/26(99 2/25/99 2/2S>99 9/22/99 Biopsy 2/25/00 2/26/99 W S* fefcs- 412474 -- 142465 23480 378723 137561 70781 175283 42668 280575 256669 267328 421647 57895 287223 <LOQ: Less than the Lower Limit of Quantitation (26.9-37.4 ng/g) 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 8.25 42.2 66 10.2 58.7 48.2 80.4 49.6 24.7 66.5 81.4 17.7 412 -- 142 23.5 379 138 70.8 175 42.7 281 257 267 422 57.9 287 3M Environmental Laboratory 3M Environmental laboratory Page D-9 p. 3m Medical Department Study: T-6295.7 3M Medical Department Study: T-6295.7 Attachment E Data Spreadsheets Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-112279 3M Environmental Laboratory 3M Environmental Laboratory J5&r Page E-1 3m Medical Department study: T-62 3&C?TOX-030 Report N o . FACT TOX-030 Study Product NumbeKTett Subctance): Matruc Mcthod/Revision: Analytical Equipment System Number Instrument Softwara/Vemon; Filename: R-Squared Value Slope Y-Intercept Date o f Extraction/Analyet. Date o f Analyau/Analyat Date of Data Reduction/Analyil Covince#6329-223 laboratory 26 Week Capsule Toxicity Study with PFOS m Cynomolgm Monkeys T-6295 (PFOS) Monkey Sen FACT-M-3.1 & FACT-M-4 1 Soup 020199 ManLynx 3 I See Attachment! See Attachment! See Attachment! See Attachments 02/09/99 RWW 02/13/99 MEE 02/16/99 KJH Request Number-U2279 Sample Data BASELINE MONKEY SERA Group Doe Method BUc Matrix Blk QC - 250 ppb Group 1 Control 0.0 m^kg/day Sample 0 RBS02099-H20 Blk-1 RBS02099-H20 BQc-2 RBS02099-Sera Btk-1 RBS02099>Ser* Blk-2 MKS02099-MS MKS02099-MSD I05508M+ 105517M+ 105519M+ pros Coac. f/mL o.oQ 0 00 0.00 0.00 299 285 \ .28 1.32 316 C o aeaatraciaa of PFOS ag/mL or % Rte <LOQ <LOO <LOQ <LOQ 121% 115% <LOQ <LOQ <LOQ Maa PFOS u|/m L <LOO <LOO 118% USD Std. Dev. MS/MSD RPD NA NA 5% Group 2 Low-Doae 0 03 mg/kg/day Groap 3 Mid-Doe 0 .15 mg/Vg/day I03520M+ 105526M+ W5527M+ I05529F+ I05330F+ 105531 F t I05535F-*I05544F I05549F 105513M+ 105514M 105515M+ I05516M 105521M+ W552SM-*I05537F+ 105541F+ I05543F+ I05546F+ I05547F+ I05550F-*- I05505M I05510M+ 105518M+ 105523M+ 105524M+I05528M+ I05532F+- 1 46 1 62 1 48 2.28 1.13 1.73 1 46 1.60 5.42 2.63 1.59 0.540 2.65 1.72 1.90 1 33 2.23 2.53 2.50 1 69 1.50 3.13 1.04 1.51 0.930 1 82 269 3 13 <LOQ <LOQ <LOO <LOQ <LOQ <LOQ <LOQ <LOQ <1-00 <LOQ cLOQ <LOQ <LOQ <LOQ <LOO <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOO <LOQ <1.00 <LOQ <LOQ <LOQ <LOO NA NA <LOQ <LOQ <LOO Group 4 High-Doe 0.75 mg/ty'day I05538F I05539F I05545F+ I05548F HM5J2F* I05506M+ 105507M [05509M 103511M 105512M+ I05522M+ I05533F+ 105534F+ 2 25 215 3 27 360 102 2.19 2.79 2.06 269 2.64 2.73 2.02 2.54 <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ < io o <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <I,OQ <LOQ <LOQ <LOQ I05536F+ I05540F+ I0S542F+ 105551F L m t of Quantitation (.OQ): 4.39 ng/mL 2.85 3.45 1.94 352 Data Entred/By <LOQ <LOQ <LOQ <LOQ 02/17/99 LAC <LOQ <LOQ Dale Verified/ B y 0303/99 QML, 06/07/00 PW Data punty corractad/verified: 09/12/00 mmh, 09/12/00 LAC NA " Not applicable PFOS " Periluorooctaneaulfonate + Senati initiai volume lets than 0 50 mL, concentrations hould be considered tentative 04/28/00 LAC Extraction Volume Rabo - bubal Volume/Fmal Volume FACT*M-4.1 Exeel 97 3M Environmental Laboratory tox-03O-sera223-l C it7 9/12/'00 3m Medical Department Study T-62^c?TOX-030 Report No. FACT TOX-03 0 Covance#6329-223 laboratory Request Number-U2279 Study: Produc t Number(Te*t Substance) Matrix Method/Revuion: Analytical Equipment System Number Instrument Softwire/Version: Filename R-Squared Value; Slope: Y-lntercept: Data o f Extraction/Analyst: Date of Analyia/Analyst. Data of Data Reduction/Analyst: Sample Data 26 Week Capsule Toxicity Study with PFCS in Cynomolguj Monkeys T-6293 (PFOS) Monkey Sera FACT-M-3 l A, FACT-M-4 I Madeline 041098 & Amelia 062-199 MaaaLynx3.1 See Attachments See Attachments See Attachment* See Attachment* 02/05/99 SAH 02/08/9. 02/10/99,02/12/99, 02'1 ~/99 HOJ'DRB 02/10/99. 02/16/99. 02/26/99 HOJ/DRB WEEK ! MONKEY SERA Groap Dose Method Blk Matnx BUc QC - 250 ppb Groap 1 Control 0 0 mg/kg/dty Group! Low-Dose 0 03 mg/kg'day Group! Mid-Dose 0.15 mg'kg'day Group 4 High-Dose 0.75 mg/kg/day Sample # RBS02059-H20 Blk-I RBS02059-H20 Blk-2 RBS02059-Sera Blk-1 RBS02059-Sera Blk-2 MKS020S9-M5 MXS02059-MSD I05508M 105517M+ 105519M I05520M I05526M I05527M I05529F I05530F I0553IF I05535F 05544F105549F 105514M IOS515M+ [5516M 105521M 105537F 105541F+ I05547F I05550F 105505M I05510M* 105518M 105523M I05524M I05528M T05532F* I05538F I5539F 105545F+ I05548F I05552F I05506M I05507M* 1O5S09M-* 10551IM 105512M I05S22MIQS533F* I05534F* I05536F* I05540F* I05542F 10555 IF - PFOS Cone. ag/mL ooo 0 00 000 000 305 304 000 000 0.00 0.00 0.00 0.00 000 0.00 0.00 0.00 0.00 0.00 495 383 519 677 684 464 581 519 28? 209 342 221 318 413 161 251 248 184 232 195 665 411 351 482 711 539 564 451 393 419 749 37? Ceaceotratioo of PFOS ug/mL or % Ree <LOQ <LOQ <LOQ <LOQ 123% 123% <LOQ <LOQ <LCQ <LOQ <LOQ <LOQ <LOQ <LOQ '-I.W <LOQ :LOQ <LOQ 0 "93 0 ?6" 083: t 09 1 10 C 929 0 931 0 832 4 60 -1 19 5 48 3.5-1 1 25 5 52 4 3! 4 I2 3 31 3 69 3 ?2 3 12 21.3 22.0 18 8 193 22 8 21 6 22 6 18 1 21 0 16 8 24 0 20 ! Meas PFOS ft/BL <LOQ < tO Q 123% RSD Std. Dev. MS/MSD RPD NA NA 0% <LOQ NA <LOQ 0.869 0.947 4.60 3 ?1 21 0 20 4 NA 16.9 0147 11 6 0110 17.0 0 782 12.3 0 455 7 49 15: 133 2 71 Limit of Quantitation (LOQ): 4 39 ng/mL NA " Not applicable PFOS * Perfluorooctancaulfonate Date Entered/By 02/10/99.03/02*99 LAC Date Verified/By 03/23/99 QML. Date punty correcled/vunfied: 09/12/00 mmh. 09'1 2/00 LAC - Serum suual volume less than 0 50 mL, concentrations should be considered tentative 0 Extraction Volume Ratio Initial Volume/Fmal Volume FACT-M-4 Excel 97 3M Environmental Laboratory tox-030-sera223-lC & f 9/12,00 fJ2~a^e--'T9V 3m Medical Department Study T-62 3&C? TOX-030 Report No. FACT TOX-030 Covnce#6329-223 laboratory Request Number-U2279 Study Product Numbei<Te*t Substance) Matrix Method/Revision. Analytical Equipment Syetcm Number Irueminent Software/Vennon Filename R-Squared Value. Slope Y-Intercept: Date of Extraction/Analyst. Date of Analyin/Analyst Date of Data Reduction/Analyst Sample Data 26 Week Capiule Toxtcjly Study with PFOS in Oynornolgus Monkey* T-629J (PFOS) Monkey Sera FACT-M-3 1 A. FACT-M-4.1 Madeline 041098 A Amelia 062498 MassLynx. 3 1 See Attachments See Attachments See Attachments See Attachments 02/09/99 JCP 02/13/99, 02/22/99 HOJ 02/19/99, 02/23/99 HOJ WEEK 2 MONKEY SERA G rasp Dew Method BUc Matrix BQc QC - 250 ppb Group 1 Control 0.0 mg/Vg/day Croup 2 LowDose 0.03 mg/kg/day Group 3 Mid-Dose 0 15 mg'leg'day Staple RSB2099-H2O Blk-1 R8S02099-H20 Blk-2 RB302099-Sen Btk-I RB902099-Sera BOc-2 MKS02099-MS MKS02099-MSD I03308M 103517M I03319M* I05520M* I03526M I05327M* 105529F- 10533CF* I0333IF* I05335F 105544FI03349F* I053MM+ 103515M+ 105516M+ I05521M* 103537F+ 10354 IF J05547F+ I03350F+ I03303MI05510M 0551IM* I03323M I03324M* (05328M 03332F* PFOS Cane. " 'L 0.00 000 000 000 293 300 4 09 3 07 4 53 2 08 2.02 L.89 0.520 1.02 1 90 3.12 0 380 0810 575 513 492 425 446 722 527 530 227 301 330 285 195 384 173 Cencentrada #f PFOS K/u L k H Ree <LOQ <LOQ <LOQ <LOQ 118% 121% <LOQ <,OQ <LOQ <LOQ <LOQ <LOO <LOQ <LOQ <LOQ CLOQ <LOQ <LOQ 1 02 1 03 119 1 13 1 19 1.16 l 06 0.980 6.07 4 82 7 01 4 56 6 24 6 15 4 61 Mean PFOS urAuL <LOO <LOQ 120% RSD Std. Dev. MS/MSD RPD NA NA 2% <LOQ NA <LOQ 1 to 1 10 NA 7.61 0 0835 879 0.0963 16 1 ? 81 0933 Group 4 High-Dose 0 73 mg/Wg/day J05S3SF 103539F* I05545F I05548F* I05552F I05506M* 105507M* 103309M 10531IM 105512M J05522M 105333F I03534F-* I05536F* W5S4F* IG5542F* 10555 IF 416 232 329 320 295 747 626 746 706 976 899 778 650 588 546 372 568 6 67 4 65 5 28 642 17.2 4 73 5 39 0 930 29 9 25.1 23.9 22.6 31.3 13 1 28 8 26 9 3 54 24.9 26.0 25.5 195 199 14 8 18.2 22 0 3 25 Limit of Quantitation (LOQ)- 4 39ngm L NA * Not applicable PFCS * Perfluorooctanesulfonate Date Entered/By 02/22/99, 03/02/99 LAC Date Venfied/ By 03/23/99 GML Date purity corrected/venfted 09/12/00 mmh, 09//12/00 LAC * Serum initial volume less than 0 30 mL, concentrations should be considered tentative 04/28/00 LAC Extraction Volume Ratio " Initial Volume/Final Volume FACT-NM 1 Excel 97 3M Environmental Laboratory tox~030-sera223-1C -W /9 9 9/12/OQ 3m Medical Department Study: T - 6 2 xox-Q30 Report No. Covanct#<329-223 laboratory Request Study Produci NumberfTast Subatance): Matrix: Method/Rcviaian: nalyticaJ Eqiopment System Number Irutmmcnt Softwvc/Venion Filename: R-Squared Value Slope Y-Intercept. Otte of Extracbaft/nalyst: Date of Analyeii/Analyit: Date of Data Reducon/Aniiy*t: Sunple Data 76 Week Capsule Toooty Study with PFOS tn Cynomolgus Monkeys T-6295 (PFOS) Monkey Sera ETS-8-4 0 ind E n-8-50 Madeline 04)098, Amelia 06249, A Soup 020199 MassLynx 3 1and 3 2 See Attachments See Attachments See Attachments See Attachments 0V02W SAH/RWW 03/05/99.0V0g/99,03/25/99 HOi/MEE/DRB rt3/08.99, 03/26/99 KJH/DRB WEEK 4 MONKEY SERA Cnay Dw Sample pros CWK. C iacaw tiilii af pros Maas PFOS R3D Std. Dev. ag/mJL ar % Rae fl/mL MS/MSD RPD Method 81k RBS03029-H20 Blk-] 0.00 <LOQ RBS03029-H20 BOc-2 0.00 <LOQ <LOQ NA Matm BOc RBS0329-S Blk-J 000 <LOQ RBS03029-Sen BUc-2 0.00 <LOQ <LOQ NA Manx Blk MKS03029-Sen Blk-1 7.21 <LOQ MKS03029-Sen B0c-2 7.17 <LOQ MKSQ3029-Sen Blk-3 726 <LOQ MJCS03C29-Ser BJk-4 6.32 <LOO <LOO NA QC - 250 ppb MJCS03029-MS-1 233 102% MKS03C29-MSD-1 250 101% 101% 1% MKS03029-MS-2 242 98% MKS03029-MSD-2 235 95% 96% 3% Giaap 1 I0550SM 5 43 <LOQ Control 105517M* 6.76 <LOQ OOmp/kg/day IQ5519M* 7.91 <LOQ I05520M* 6 11 <LOQ I05526M 673 <LOQ 105527M-*- 198 <LOO 8 V na 105529F* 6 63 <LOQ W5530F* 611 <LOQ 10333IF 9 19 <LOQ 105533F* 8 17 <LOQ 105544F 996 <LOQ I05549F* 174 <LOQ <LOQ NA Creep 2 I05314M- 108 2B7 Low-Dose 105S1SM 172 2 75 0.03 mg/kg/day I05S16M 10532IM* 196 101 3 13 18 1 403 320 0577 103537F* 186 3.73 J05J4IF+ I05547F 153 221 3.07 3.55 8 53 I03S50F* 163 3.27 3.40 0 291 Crawp 3 (05505M* 63 7 17.0 Mid-Dote 0 15 mg/kg/day 105S10M* I05518M-* 646 101 17.2 20.2 I05323M no 17.6 105324M I05328M- 963 93 7 15.4 9 47 19.2 r g 168 (05532F- 41 4 166 I03538F 117 187 I05339F- 338 143 105545F+ 46.7 18.7 I05548F* 783 157 11 4 I05332F+ 559 149 163 ! 87 Cnap 4 105306M* 118 236 High*Dose I03307M* 272 72.6 0.75 mg/kg/day I03509M* 27 3 548 105311M+ 24 2 48.4 105512M* 290 77.4 738 [0S522M* 308 *1.2 95.3 704 IO333F+ 35 2 141 103334F+ 363 145 105536F* 240 64.1 I05340F* 838 67.2 I03S42F* 29 4 78 4 42 7 I0555F* 130 60.2 92 7 396 Limit of Quanauaon (IOQ): PFOS M U ng/mL Date Entcred/By: 03/08/99,03/76/99 LAC Date Vcnfied/ By: 0*73/99 GML, Dite purify conecte<J/ven6ed- 09/12/Q0mirth, 09/12/00 LAC NA - Not applicable * Serum initial volume leas than 0.30 mL, concentrations should be considered tentative 04/28/00 LAC PFOS * Perfluorooetanetulfonate Extraction Volume Ratio (rubai Vohune/Futal Volume FACT TOX-Q30 Number-U2279 ETS-8-5 0 Excel 9" 3M Environmental Laboratory tox-030>tcra223*lC |VfZ o o 9/12/00 -- fcige 19-g-- 3m Medical Department study: T - 6 2 ^ c t o x -030 Report No. FACT TOX-03 0 Covnce#6329-223 laboratory Request Number-U2279 Study: Product NumbeKTest Substance): Matrix: Method/Revifcon Analytical Equipment System Number: instrument Software/Venion: Filename'. R-Squared Value: Slope Y-lntereept Date of Extraction/Analyst Data of AnalyticAnalyst: Data of Data Reduction/Analyst: 26 Week Capsule Toxiaty Study with PFOS in Cynamo'-gui Monkeys T-6295 (PFOS) Monkey S en ETS-8-4 Oand ETS-8-5 0 Amelia 061498 A. Soup 010199 MassLynx 3 1 and 3 2 See Attachments See Attachment* See Attachments See Attachments 01/10/99 SAH 03/12/99.03/21/99 MEE/DRB O l/liW . 03/24/99 LAC/DRB Sample Data WEEK 6 MONKEY SERA Gruup Dea# Method BUc Matrix Blk Matrix Blk QC - 230 ppb Sample a RBS03109-H2O B i-1 RBS03109-H20 Bk-2 RBS03109-Sera Bft-1 RBS03109-Sera BDt-2 MKS03109-Sere BOc-1 MXS03)09*Sen BOc-2 MKS03I09-SeraBQc-3 MKS03109-Sera Blk-4 MK.S03109-MS-1 MICS03109-MSD-1 MK303109-MS-2 MKS03109-MSD-2 PFOS Cune. itgfmL OX 7 74 OX 4.19 2.56 2 55 1.93 0.5X 264 233 243 249 CanceMnUon #f PFOS u f/a L ar % Ree <LOQ <LOQ <LOQ <lOQ <LOQ <LOQ <LOQ < ioo 106% 101% 97% 100% 8 V Mean PFOS ug/aL -LOO RSD Std. Dev. MS/M3D RPD NA NA <l,OQ 103% 9854 NA 4% 2% Gruup 1 Control 0.0 mg/k^day Graup 2 Low-Doe# 0 03 mgfkj^day Group 3 Mid-Dose 0.13 mg/kg/day Group 4 ILgh-Dote 0.73 mg/kg/day I05508M* I03317MIQ5319M* 103320M 103326M I03327M* IOS529F+ 103530F* 103331F I05335F* I05544F* I03549F+ I05314M* I0313MI05316M* I03521M* 10337F+ I03341F IQ5547F+ I03550F* 105305M I05S10M* I03518M* 105323M+ I03524M+ 03S28MI05332F* rassiSF I0S539F-*10543F 105S48F+ I05332F I03306M+ 103307M* 105309M 1053UM 105512M* I05522M* 105533F I03334F* I05J36F* (05540F I5342F* I03331F OX OX ox 1 60 0 610 9 49 1.56 3 38 4.24 0.00 OX 3.54 36 6 33 1 32.5 4) 8 42 7 45 7 36 4 32 7 87 4 56 I 669 61 9 72.7 61 6 61.2 94 3 61 4 94 5 61 3 57 0 182 163 230 234 211 203 232 166 179 227 203 205 <LQQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ 367 3 31 3.26 4.19 4.28 3 66 365 3.27 21 0 22.5 20.1 186 218 18 5 18 4 VS 9 18 4 22.7 184 16 I 910 82 8 92 3 93.7 105 102 93.0 83 3 89 8 90 8 102 82.0 <LOO NA <LOQ 3 6! 3 71 20 4 188 9.1 5 90 1 NA 119 0 430 U2 0 417 8 09 l 6.5 II 4 2 15 8 54 8.07 790 7 11 U ntf of Quanatation(LOQ): PFO S- 13.2 t^m L PFOS * Perfhiorooctanesulfonate NA * Not applicable Date Entcred/By 03/19/99,03/24/99 LAC Date Verified/ By 08/17/99 SAH, 05/19/00 PICVPW Date punty correeted/ven&ed 09/12/00 mmh, 09/12/00 LAC Serum initial volume less than 0 30 mL, concentrations should be considered tentative 04/28/00 LAC Extraction Volume Ratio " Initiai Volume/Final Volume ETS-8-3 0 Excel 97 3M Environmental Laboratory tox-030-scra223-lC iP^Al 9/12/00 ^Pjige--dTS-? 3m Medical Department Study T -62 TOX-030 Report No. Study Product NumberfTest Subita): Matrix. Method/Revuion: Analytical Equipment System Number Instrument Sortware/Vemon Filename; R-Squared Value: Slope: Y-lntcrcept Oste of Extracborv/Analyst Oate of Analysa/Analyst Date of Data Reduction/Analyst: Simple Data Cov.nce# 6329-223 laboratory 26 Week Capsule Toxicity Study with PFOS tn Cynomoigus Monkeys T-6295 (PFOS) Monkey Sen ETS-8-4 0 and ETS-8-5 0 Soup 020199 M JLynx 3 1and 3.2 See Attachments See Attaehmenta See Attachments See Attachments 03/03/99 RWW/JCP 03/05/99,0V09/99, 0V10/99. 0V25/99 DRB 03/08/99.0yIV99,03/24/99, 03/26/99 DRB Request WEEK I MONKEY SERA Gtwp Sample# PFOS Mean RSD Dm * C ata ofp ro s PFOS Std. Drr. ng/mL utfadLerS Ree of/aL MS/MSD RPD Method Blk RBS03Q39-K1O BDt-L 000 <LOQ RBS03039-H20 Blk-2 000 <LOO <LOO NA Matrix Blk Matrix Blk RB503039*Sen BDt-1 RBSO3039-Sera BDt-2 MKSQ3039-SenBQt-l 000 000 3 23 <LOQ <LOO <LOQ <LOQ NA QC - 230 ppb MKS03039-Sen BSt-2 MXS03039-MS-I 2.10 174 <LO<5 49S rA 8 NA MKS03039-MSD-1 191 76% 72% 9% MXS03039-MS-2 MKS0339-MSD-2 205 183 n% 73% 77% 10% MJC503039-MS-3 181 72% MKS03039-MSD-3 203 81% 76% 11% Gravy 1 I0350IM 943 <LOQ Control 105517M 768 <LOQ 0 0 mg/kg/day [05519M 120 <LOQ (05520M 9 03 <LOQ I05526M 11.0 <LOQ 105527M 28.8 0.0383 <LOQ 1Anomaly NA [03329F 16.0 0.0214 10533F 183 00244 (0333IF 180 0.0206 103333F 143 <LOQ 103544F 946 <LOQ 836 I03549F 130 9.Q240 0 0226 - 2 Anomalies 000189 Grovp 2 1033 4M 343 461 Low-Do* I03313M 31 2 4.17 0.03 mg/kg/day I03316M 10352IM 44 I 31 8 5.05 5 10 9 14 4 73 0 432 I03537F 46.1 528 10554IF I05547F I0550F 36.3 373 34 4 483 498 3.94 12.1 4 76 0 577 Group 3 I3503M 122 27.9 Mid-Dose 0.13 mg/kg/day IQ551QM I055I8M 1Q5323M 82.9 94 1 91 6 22.1 25.1 24.5 105S24M 118 31.6 12 7 105528M 107 24.6 26.0 3.30 105532F 86 l 27 6 105538F 106 243 105339F 82 6 265 I0545F 122 245 0554SF 69 4 221 12 7 IOJ552F 59.3 19 I 24.0 3 06 Crvup 4 I03506M 122 125 High-Do*e 105507M 83 8 86.1 0.73 mg/kg/day I05509M IQ5511M 117 89 B 103 92.3 103512M [03322M 115 127 111 131 16 7 109 (83 I03533F 107 110 W55J4F 98 1 121 t05536F 100 102 I05540F 102 105 1QS42F 10535IF 113 99.2 1)6 873 11 1 107 II 8 Umit of Quanoution (LOQ): PFOS 13 2 ng/mL Date Entered/By 03/08/99.03/24/99,03/26/99 LAC Dau Venfici/ By 08/18/99 SAH. OS/21/00 PJO. OS/23'00 . 05/24/00 PW Date punry conecied/venfied 09/12/00 menh, 09/12/00 LAC PFOS " PerUuoTOoctanesulfonate NA * Not applicable Extraction Volume Rado - Initial Volume/Final Volume FACT TOX-030 Number-U2279 ETS-8-5 0 Excel 1 3M Environmental Laboratory tox-030-*crt223-lC 1920A. 9/12/00 3m Medical Department Study T -6 2 9 f i \ c f TOX-030 Report N o . FACT TOX-030 Covance#6329-223 laboratory Request Number-U22 79 Study: Product NurnberfTesl Substance): Matrix: Methodttevuion: Analytical Equipment System Number . Instrument Software/Venion 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkey T-6295 (PFOS) Monkey Sen ETS-8-4.0andrS-8.5.0 Soup 020199 MauLynx 3 2 Filename' R-Squared Value: Slope Y-Intercept: Date of Extraction/Analyst Date of Analysin'AnalystDate of Data Reduction/Analyst: See Attachments See Attachment! See Attachments See Attachments 0*03/99 RWW/JCP 03/05/99, 03/09/99,0*10/99,03/25/99 ORB 03/08*99, 0*15/99,03/24/99,03/26*99 ORB Sample Data WEEK 12 MONKEY SERA Graup Oaw Method Blk Matrix Blk MatnxB&c QC - 250 ppb Creep 1 Control 0 0 mg/kg/day Craup 2 Low-Dose 0.03 mg/kg/dey Croup J Mid-Dose 0.13 mgOcg/day Croup 4 High-Dose 0.75 mg/tyday Sample# RBS03039-H20 Blk-1 RBS03039-H20 Blk-2 RBS03Q39-Sm Blk-1 RfiS03039-Sera Blk-2 MKS03039-Sen Blk-I MKS03039-Scra Blk-2 MKS0X39-MS-1 MKS03039-MSD-1 MKS03039-MS-2 MKS3039-MSD-2 MKS03039-MS-3 MKS03039-MSD-3 I03508M 105517M [05519M I05520M I05526M 105527M 105529F-* IO5530F 103531F I05535F 103564F 103349F 103514M I0515M I05516M 105521M+ 105537F 10354IF I05547F* 105550P I05505M I05510M I03518M I0S523M* 105524M 10S328M I05532F 103538F-* J05539F 305545F 103548F 103332F (03506M I03507M 105509M 10551IM 105512M I05322MI05533F 10S534F I03536F+ 103540F-* 103542F I05551F PFOS ng/aaL 000 000 0.00 0.00 323 2.10 174 191 203 185 181 M3 309 269 32.8 12.7 21 3 30 1 11 4 164 190 943 \%\ 233 41 8 445 47 4 45 I 578 34.8 36.9 488 143 135 133 R8.0 118 79 2 869 94 2 101 109 796 77 4 103 864 116 83.9 149 880 109 100 63 6 800 128 H4 (pros aa/hL w H R w <LOQ <LOQ <LOQ <LOO <LOQ <LOQ 69% 76% 82% T3% 72% 11% 0.0381 0.0430 0 0438 <LOQ 0.0244 00423 <LQQ 0.0243 Q.0254 <LOQ 0.0238 0.0316 6.20 648 6 55 7.32 681 537 7.03 583 40.1 39 2 33.7 35.3 37.8 25 4 25 8 328 271 32 2 23.2 24 8 116 106 123 994 167 121 126 IU 98.0 112 131 117 O O Mean PFOS ug/mL <LOQ <LOQ 72% 77** 76% RSD 5 t4 Dev. MS/MSD RPD NA NA NA 9% 10% 11% 0 0383 1Anomaly 21 1 0 00811 0.0263-2 Anorrakes 69 611 13.8 0 00362 8.63 0 578 11 4 07)7 153 35 2 539 143 27 8 3.98 196 121 23 9 10.0 117 11.7 Limit of Quantitation (LOQ): PFOS - 13.2 n*/mL Date Entercd/By- 0*01/99,0*24/99.0*76/99 LAC Daw Vended/ By: 0*11/99 SAH, 05/24/00 PW Dste purity corrected/verified: 09/12/00 nsnh. 09/12/00 LAC PFOS Perfluoeooctanesulfonale * Serum initial volume less than 0.50 mL, concentrations should be considered tentative 04/28/00 LAC NA " Not applicable Extraction Volume Ratio - Initial Vohsnc/Final Volume ETS-S-5 0 Excel 97 3M Environmental Laboratory tox-030.sm22>]C M 2< > 3 9/12/00 JPage- 19S- 3m Medical Department Study T-62 9jvcT1TOX-030 Report N o . FACT TOX-030 Covance# 6329-223 laboratory Request Number-U2279 Study: Product NumberfTert Substance); Matrix: Mcthod/Rxvinosv. Analytical Equipment System Number IFnrsetnraurmnee:nt Softwue/Vmum. R-Squared Value Slope: Y-lntercept Date o f Extraction/Analyst. Date o f Analysis/Analyst: Date of Data Reduction/Analyst: 26 Week Capsule Toxicity Study wtth PFOS m Cynomolgus Monkeys T-6293 (PFOS) Monkey Sen ETS-8-4 0 and ETS-8-5 0 Soup 020199 MaatLynx 1.2 See Attachments See Attachments See Attachments See Attachments V12/99 SAH/RWW 03/1*99,0*19/99 DRB/HOJ 03/16/99,0*2499 DRfi Sample Data WEEK 16 MONKEY SERA G rf Dm* Method Blk Matrix BUc Matrix Blk QC- 230 ppb Great 1 Control 0 0 mg/kg/dey G reat 2 Low*Dose 0.03 mgrkg/day G reat* Mid-Dose 0 15 mg/kg/day Sangle* RfiS03129*H2O Blk*1 RBS03129-H2O BDc-2 R3S03129-5* Blk-1 RfiS03129*Sera Blk-2 MKS03I29-Scra Blk-I MKS03I29*Sera Blk-2 MKS03I29-MS-I MKS03I29-MSD-1 MKS03129-MS-2 MKS03129-MSD-2 I03308M 1035l7Mr 105519M* 10S320M 105326M 103327M I05329F IQ5530F* 103531F 105535F+ 103344F* 105549F 10334M I05515M* 103316M I0J521M* 105537F 105541 F* I05J47F 103350F 105505M* IQ5510M I05518M I05523M 105524M 105528M 103332F 105338F 103339F 105545F 103548F 105552F PFOS Ceac. (AnL 0.00 000 000 0.00 150 76 189 199 199 235 21.5 20.4 21.1 207 199 384 335 257 213 9.37 Il 6 289 77.7 47.3 81.7 S3 1 76 4 69 2 668 53 9 222 330 379 326 475 362 252 279 347 323 323 241 ef pros ughaLerKRec <LOQ <LOO <LOQ <UX5 <LOQ <LOO 72% 76% 76% 90% 00344 0.0408 0.0423 0.0332 0.0320 0.0615 00438 00515 0 0314 <LOQ <LOQ 0.0462 10.4 9.47 IQ.l 14.8 11.1 12.3 10.7 7.83 394 52.9 607 52 2 63 4 484 40.4 37.3 463 43.1 47.0 31.7 Maaa PFOS og/naL <LOC <i.OQ <LOQ 74% 83% 0 0407 0 0432 -2 Anomalies 11 2 I0S 56 2 42.1 JUD StdL Dnr. MS/M3D RPD NA NA NA 5% 16% 27 i 0 0110 19.7 0 00851 21 9 244 18 1 1.90 10.4 5 84 9 59 404 Great 4 I05506M 273 182 Ksgh-Dose 105307M 257 172 0 73 mg/kg/day I05509M 296 197 105511M 268 179 I05512M I05522M 296 254 216 185 8 42 (89 13 9 105533F 245 164 105534F 196 137 103336F* (84 184 (03540F 238 173 105542F 231 168 119 10555 IF 191 127 162 193 Uimt of Quanttticn (LOQ): PFOS - 13 2 ng/mL Date Entered/By: 03/0*99,0*17/99,03/2*99 LAC Date Verified/ By: 0*1*99 SAX, 0*16/00,05/2*00 PW Date punry corrected/ymfied: 09/12/00 mmh, 09/12/00 LAC PFOS Perfiuoeooctancsulfonate * Serum initial volume lesi than 0.30 mL. concentrations should be considered tentative. 04/28/00 LAC NA - Not applicable Extraction Volume Ratio * Initial Vohame/Final Volume ETS-8-5 0 Excel 97 3M Environmental Laboratory tox-030-aer*22*lC R7 ay 9/12/00 ^ E a g e 1 9 fe 3m Medical Department Study T -62 9 r v c t o x -030 Report No. FACT TOX-030 Covince# 6329-223 laboratory Request Number-U2279 Study: Product Numberdest Substance) M itra: Method/Reviixm: Analytical equipment System Number Instrument Softwaxe/Venion: Filename: R-Squared Value Slope: Y-Intercept Date of Extraction/Analyst: Dite of AnalyticAnalyst Date of Data Reduction/Analyst 26 Week Capsule Toxicity Study wjtfi PFOS in CynomoJgu* Monkeys T-6295 (PFQS) Monkey Sera ETS-8-4.0 and ETS-8-S 0 Soup 020199 MauLynx 3 1 See Attachment* See Attachments See Attachment* See Attachments yiVW JCP/SAH 0VI6/99,03/24/99 DRB 0VI7/99,0173/99 DRB Sampl* Data WEEK 10 MONKEY SERA Graap Dea Method BUc Matrix Btk Matrix BD QC - 230 ppb Group 1 Control 0 0 mgfcg/day Sample R3S03159-K20 BUc-1 RBS31S9-H20 BIk-2 RBS031J9-Sen Bik-I RBS03159-Sen BIk-2 MK503J59-Scn BUc-J MKS63159-Sen B&*2 RBSQ3159-MS-I RBS031W-MSD-1 RBS03139-MS-2 RBS03I39-MSD-2 . 10S38M I033J7M I03319MI05320M 03326M I0527M* 105529F 105530F 10333IF* J05535F 103544F* 105549F PFOS ag/iuL 0.00 000 0.00 0.00 0.00 0.00 256 226 219 223 30.4 31.4 19.3 23.2 300 31.5 316 38 3 283 194 29 6 39.1 fpros l/n L a rH Ret <LOQ <UXJ <LOQ <LOO <LOQ <LOQ 103% 91% 88% 90% 0.0341 0.0419 0.0336 0.0300 0.0369 0.0631 0.0617 0.0579 0.0493 0.0263 0.0303 0.0570 pros uft/nL <100 <LOQ <LOQ 97% 89% RSD StA Drv. MS/M3D RPD NA NA NA 13% 2% 0 0400 29.9 00120 0.0304 25 2 00127 Group 2 Low-Doe 0.03 mgAg/day Greupi 2 Mid-Doae 0 15 mg/kg/day 103514M 10531SM J055I6M 10332IM I05337F 105541F I05547F I03330F I03503M 10351OM I03318M I05323M I03324M I05528M I0S332F IQ353F MS539F* 105S45F 10534SF 103352F 886 86.7 101 98.3 70.2 73.2 966 359 237 253 ' ` 248 319 220 223 255 201 210 ni 204 154 123 10.7 11.9 14.1 11.0 it 3 14.3 41.1 62.3 633 63.1 752 64.0 542 69 2 47.4 78.2 45.7 65 3 42.7 II 4 12.3 140 146 12.3 1.79 74 4 193 145 105 63 7 6.7J 25 4 58.1 147 Croup I03306M 381 138 High-Dote 103307M+ 282 138 0 73 mg/kg/day I03309M 315 132 I055UM 352 126 03312M 309 143 7 62 103S22M 302 145 144 10.9 I05333F 393 169 I03334F 303 143 103536F+- 274 146 I03340F 441 193 105J42F+ 302 151 140 103331F 330 132 156 21 8 Limit of Quanoutaon (LOQ) PFOS - 13.2 ng/mL Daw Entered/By: 03/0699,03/17/99 La c Date Venfied/ By: 08/18/99 SAH, OVI7/00 PW Date punty coirected/venfied: 09/12/00 trenh, 09/12/00 LAC PFOS PerfiuorooctanesuUbnaLe ' Calculated without sample 105350F NA - Not analyzed, not appbcabic * Serum ouhal volume las* than 0 30 mL, concntrateos should be considered tentative D47S/00 LAC Extraction Volume Rabo Initial Vohone/Finai Volume ETS-8-5 0 Excel 97 3M Environmental Laboratory tox-0J0*scsi22>1C 9/12/00 3m Medical Department Study T - 6 2 9vc? TO X-030 Report N o . FACT T O X -030 Covane# <329-223 laboratory Request Number-U2279 Study: Product NunibcrCTat Subitine*) Mitro: Method/Revunon: Analytical Equqsnent System Number Ixvstnsnent Sofrmit/Verucrtv Filmarne: R-Squared Value: Slope: Y-Intercept Date of Extraction/Analyst Date of Analysu/Analyvt Date of Data Reduction/Analyst Sampio Data 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkeys T-6295(PFOS) Monkey Sen ETS-8-4.0 and ETS-8-3 0 Amelia 06249 A. Soup 020199 MmLynx VI See Attachments See Attachments See Attachments See Attachments 3/16/99 RWW/SAH 03/17/99,03/20/99,0V2V99 DRB/MEE 0*23/99.0V2V99,04/02/99 DRS WEEK 24 MONKEY SERA Graap L*m Method BDc MamxBUc QC- 230ppb Group 1 Control 0 0 mg/kg/day G n#l Low-Dose 0 03 mg/kg/day G nupi Mid-Doae 0.i5mg/kg/day Group 4 High-Do* 0 75 mg/kg/day Sample* RBS03169-H2O Blk-1 RBS03169-H2 Bik-2 RBS03169-Sera BIk-1 RfiS03169-Scra BDc-2 RBS03169-MS-I RBS3I69-MSD-I RBS03169-MS-2 RBS031-MSD-2 RBS03169-MS-3 RBS03169-MSD-3 I05508M 105317M 105519M 103320M 105526M I05527M* 015MF 105530F I0353IF 105535F-- 105544F* 103349F I053I4MI05S15MMSS16M 105521M* I05537F 10354IF I05547FI05350F I05505M 105510M 105518M 105523M 203324M 105328M*13532F+ I0538F 105539F I05545F 101548F J05532F+ 105506M 105507M I03309M 105311M 105512M 105322M+ 103533F I05534F-* 103536F* 103540F I05342F [05531F* PFOS ng/mL 000 000 0.00 000 218 236 231 213 224 220 364 303 238 194 248 36.7 40.4 242 259 8.36 162 33 4 115 85 4 132 141 134 133 848 108 251 186 191 164 201 153 M3 153 142 )68 196 139 287 234 M 258 309 233 280 225 207 218 240 131 af PFOS g/mLr% Sac <LOQ <LOO <LOQ <LOO 88% 95% 93% 86% 90% 89% 0.0449 0.0486 0.0414 0.0283 0.0397 0.0613 0.0498 0.0497 0.0364 <LOQ 00325 0.0446 14.7 10 9 14.1 184 134 13.7 127 122 77 4 632 388 39.7 69.6 648 631 486 696 635 561 61 7 198 188 M 207 247 233 19Q 200 169 131 181 150 Mean PFOS ug/mL <LOQ cLOO 92% 89% 90% 0 0440 0.0426 1Anomaly 14 5 13.0 65 9 60.4 215 174 RSD StdL D*v. M3/MSD RPD NA NA 8% 8% 2% 177 00101 18 4 0.00784 110 306 5 18 0 675 104 688 120 7.24 116 24 9 12 0 20.9 Lana of Quantnatkm(lOQk PFOS- 15.2 ng/mL Date Entaed/By: 03/2V99 LAC Date Vented/ By: 08/1899 SAH, 0*24/00 PW Date punty conrected/venfied: 09/12/00 nunh. 09/12/00 LAC PFOS PerthiorooctjnesuUbnate * Smuri intu volume less than 0.30 mL. concentradoras should be considered tentative 04/2S/00 LAC M - Moribund NA - Not applicable Extraction Volume Ratio Irubai Vohune/Fuu) Volume ETS-S-50 Excel 97 3M Environmental Laboratory tox-030>sen223-!C fr r 9/12/00 ^^B ag o --1-9-8---- ` 3m Medical Department Study T - 6 2 ^ c f TOX-030 Report No. FACT TOX-030 Covine*# 6329-223 laboratory Request Number-U2279 Study: Product NumbertTTeit Subitane). Matrix 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkeys T-6295 (PFOS) Monkey Sera Hcthod/Revuuon: Analytical Equipment System Number: Instrument Soflrwere/Veraion: Filename: R*Squared Value: Slope ETS-8-4.G and ETS-S-5.0 Amelia 062498. Soup 020199 M uiLyru 3.1 See Attachments See Attachments See Attachments Y*lnteTcept: See Attachments Date of Extraction/Analyw. Date of Analyaa/AnalyiC 3>16/99 RWW/SAH 03/17/99,03/21/99 DRB/MEE Date of Data Reduction/Analyn 03/23/99, 03/22/99 DRB/MEE Sample Data WEEK 2 6 MONKEY SERA G rasp EWm Sample a 1 r pros Ceac. CeuceutraUen ef PFOS tff/mL er % Ree M taa PFOS ag/mL RSD Std. Dev. MS/MSD RFD Method BDc Matnx BDc QC - 250 ppb RB303169-H2O Blk-1 RBS03169-H2O Blk-2 RB303169-Sera Bik-1 RBS03169-Sen BIk-2 RBS03169-MS-1 RBS03169-MSD-l RB303169-MS-2 RBS03169-MSD-2 RBS03169-MS-3 RBS03169-MSD-3 0.00 0.00 000 000 210 232 229 193 111 213 <LOQ <LOO <LOQ <LOO 85% 94% 92* 78* 85* 86* 'l-OQ <LOQ 89% 85% 86% NA NA 10% 17% 1* Graup 1 Control 0 0mgftg/day I05308M I05517M03319M 05520M I05526M I05527M I05329F I03530F 105531F 30.9 31.0 27.1 16.8 26.7 41.7 48.2 59 7 30 1 0.0496 0.0539 00417 0.0254 0.0376 0.0669 0.0613 0.0736 00431 0 0459 31.3 0 0143 I05533F 166 00266 I05344F-* 105349F 21.6 34.7 0.0433 0.0536 0 0506 .12 5 0 0164 Group 2 Low-Dose 0 03 mg/kg/day 1055 HM I05515M I05516M 103321M* 10S537F* 10554 IF* I05547F 10555QF* 151 143 131 112 101 113 140 B3 3 16.2 16.5 13 B 8.91 169 15 8 1 41 13.3 14.8 13.0 IQ 8 11.4 13 2 1 42 Group 3 Mul-Dcee I05505M 05510M* 255 149 92.9 129 0 15 mg/kg/day I03518M* I03523M* I05524M* 10552BM 05532F 05538F I05539F I03545F I03548F* 105332F 42 176 160 156 209 150 216 269 145 142 69 5 72.1 69 5 30.4 62.4 82 6 25.2 63 4 59 0 72.2 85 7 64 5 16.3 55.6 668 10.8 Group 4 High-Dose 0 73 mg/kg/day I03506M (055Q7M* W5509M 105511M 1D5312M T05522M 105533F 103S34F I05J36F* 105540F I03542F 10555 IF* M 213 M 248 285 298 340 308 155 278 206 163 M 182 M 142 UB 21 0 221 173 36 203 187 133 177 163 13.0 142 171 22.2 Lnut of Quantnanon (LOQ): ?FOS - 15.2 ng'mL Date &ntered/By: 03/23/99 LAC Date Verified/ By: 08/19/99 SAH, 03/24/00 PW M = Moribund Data punty correctcd/vari&ed 09/12/00 mmh, 09/12/00 LAC PFOS =*Perftuorooctanesulfonate 4 Scrum initial volume lees than 0.50 mL, concentrations should be considered tentative 04/28/00 LAC NA 3 Not applicable Extraction Volume Rano * Initial Vohune/Final Volume ETS-8-5 0 Excel 97 3M Environmental Laboratory tox-030-aer*223~IC j^ r Z 0 7 9/ 12/00 3m Medical Department Study T -62 9ikc? t o x -030 Report N o . FACT TOX-030 Covuice# 6329-223 laboratory Request Number-U2279 Study: Product Numbar(T*ft Svbataact) Matrix: Methed/Renmon: Analytical Equipment Syatcm Numbcr Inetjumeiit SoftwirWVvmon Fdanaaet: ASquared Value. Sop: Y*latercapt: Data of Extraeboo/Anaiyvt' Data of Aailyma/AJtalyit Data of Data Raducton/AAilyat 26 Week Capela Toxicity Stody with PTCS m Cyneenol|u> Monkey* 1*6293 (PPOS) Monkey Sara E75-8-4 I and ETS-B-3 I Davey07Q799 MamLynx 3 3 See Attackmante Sat Attachment Sea Attachment See Attachment 06,-0]/00 SAL/WK 06/06/00, 06/14/00,06/160 IAS/MMH 06/07/00,06/15/00,06/19/00 1AS/MMH Sample Data WEEK 27 MONKEY SERA Graap Daac Method BUt Matrix Bik QC - 230 ppb Creep 1 Control 0 0 mpikp/day Semple Q60HVH2O Blk-J 060I0-K2O BIk-6 R5S06010-Sen Blk-5 RBS060IQ-Scra Btk-6 I03519U-MS-3 1055I9M-MSD-5 103508M 103519M I03S27M 1Q5S30F I05531F 103533F 1033*47 pros Caec. tf-L 0.00 000 000 000 330 333 33 1 49.3 *0.1 73.1 43 1 366 38.9 Caecaatratlaa of p r o s ap/aaLer% Jte <LOQ <LOO <LOQ <LOO 13136 13296 00461 0.0430 0.0693 0.0635 0.0374 00318 0 0338 Meaa pros u^mL <LOQ <LQQ 122H 00329 0 0416 RSD SUL Dev. MS/MSD RPD NA NA 194 27 4 00143 353 0.0141 Creep 1 I03514M 111 339 Lov-Doet I035I3M IS? 130 0 03 rn^kp/day I03316M 163 111 34 8 103521M 223 15.3 13 9 5 54 105341F 181 12.6 103347F 137 933 13? I03550F 161 2 11.2 1! I 1.32 Creep J Mid-Doe* 0.13 mp/kp/day I03SI0M 1033ISM I03324M 103S2SM J03532F 316 316 279 3*4 230 61.5 68? 606 844 74 7 68 1 3.75 34 3 10533SF 103343F 10354IF 260 26? 300 363 380 7 99 63.1 5*5 4 67 Greepd High-Do* 0.73 mpAg/day 103S06M* 105507M 105312M I03534F W5536F I05340F 10333 IF 450 424 464 419 389 3?7 291 196 114 4 6) 202 194 8 93 112 169 164 149 126 160 23 9 Limit of Quanoaaoa (LOQ): PfOS "5.31 nf/mL Dal Eatered/By: 06/07/00,06/19/00 LAC Data Verified/ By. 06/08/00 PIW Dele punty corrected/venfied: 09/12/00 mmh, 09/12/00 LAC PFOS *Parfluoroocnncaulfonate NA * Not applicable Extractan Volume Ratio *(tubal VolumWFinal Volume Extraction Volume Ratio = Initial Volume Tinal Volume ETS-8-S 0 Excel?? 3M Environmental Laboratory tox-030*im22J*lC m or 9/J2/00 _*ere"2TT 3m Medical Department Study T-62 9&c? TOX-030 Report No. FACT TOX-030 Covance# 6329-223 laboratory Request Number-U2279 Study: Product NumberfTest Substance) Matnx: Method/Revision: Analytical Eqtapntent System Number Instrument Softwwe/Vemon Filename: R-Squared Value. Slope. Y -Intercept Date of Extraction'Analyst Date of Analysa/Analyst Date of Data Reduction/Analyst: Sample Data 26 Week Capsule Toxicity Study with PFOS m Cynomoigu* Monkeys T-6293 (PFOS) Monkey Sen ETS-8-4.0 and ETS-8-S 0 Amelia 062491, Soup 020199. A Madehne 041098 MaasLynx 12 See Attachments See Attachments See Attachments See Attachments 04/0699 RWW 04/07/99,04/12/99,04/17/99, 0614*9.0S/I7/99 HOJ/DRB/MEE/KJH 04/09/99,04/13/99,04/20/99,03/17/99,03/11/99 HOJ/DR&/1CJH DAY 183 MONKEY SERA Ci aap Dow Method Blk Matrix EUk QC 230 ppb Craap 1 Control 0.0 mg/tyday Sample 0 RBS04069-H20 Btt-1 RBS04069-H20 Blk-2 RBS04069*Scra Blk-1 RBS04069*Sera BDt-2 MXS04069-MS-I MKS04069-MSD-1 MKS04069-MS-2 MKS04069-MSD-2 103320M I0J526M+ I03329F 103J49F PFOS ac/aaL 000 14.2 0.00 t27 234 246 267 228 128 297 660 21 3 C--raH aflsa a# PFOS gW LarHRac <LQQ <LOO <LOQ 'LOO 1023 99% 108S 91% 0.171 60626 0.0756 00310 Mura PFOS ui/n>L NA NA 101% 100% 0117 00333 RSD Sid. Dev. MS/M3D RPD NA NA 3% 16% 653 0.0764 39.2 00313 Grasp 3 Mid-Dow 0.13 mg/fcg/day 103303M+ [03323M I03539F* 787 479 103332F 391 Grap4 High-Dose 10331IM* 103522M 97.1 194 0.73 mg/kg/day I03533F 245 IOSS42F 176 limit of Quanbtitton(LOQ): PFOS- 132 ng/mL Dale Entered/By: Dale Verified/ By: Dale punty corrected/verified 74.4 93 3 107 56.9 216 282 258 201 04/19/99,03/10/99,03/20/99 LAC 08/19/99 SAH, before 06/0600 TKR 09/12/00 mmh, 09/12/00 LAC 850 81.7 249 230 176 149 430 35 1 188 46.8 175 403 PFOS - Perfluorooctanesulfonate NA " Not applicable - Serum initial votuma leu then 0.30 mU concentrations should be considerad lentatrve 04/28/00 LAC Extraction Volume Rabo 3 Initial Volume/Fmal Volume ETS-8-5 0 Exeel 97 3M Environmental Laboratory lox-OSO-urallVlC ^ j& e Z O < i 9/12/00 3m Medical Department Study T-62 9&C TOX-030 Report No. FACT TOX-030 Covane# 6329-223 laboratory Request Number-U2279 Study: Product NumbeifTest Subitanee). 26 Week Capsule Tosoaty Study with PFOS m Cynomolgus Monkeys T-6295 (PFOS) Matrix. Monkey Sen Method/Revuion: Analytical Equipment System Number Instrument Software/Version Filename: ETS-t-4.0 and ETS-8-5 0 Ameha 062498. Soup 020199. A Madeline 041098 MaaaLynx3.2 See Attachments R-Squared Value: Slope See Attachments See Attachments Y-Intercept: Date of Extraction/Analyst See Attachments 04/06/99 RWW Date of Analysis/Analyst Date of Dan Reduction/Analyst 04/07/99.04/12/99.04-17/99. 0VI4/99.05/17/99 H0J/DR3/MEE/KJH 04/09/99, 04/13/99.04/2*99,0VI7/99,05/18/99 HOJ/DRB/KJH Sample Data DAY 184 MONKEY SERA Greup Deee Sample PFOS ng/mL C asm ttiH oi ef PFOS ug/mL er % Ree Mean PFOS RSD StdL Dev. MS/MSD RPD Method Bik RBS04069-H20 Blk-1 0.00 <LOQ Matnx Blk QC - 250 ppb RBS04069-H2O BDc*2 RBS04069*Sen Blk~l RBSO4069*Sen BQc-2 MKS04069-MS-I MKS04069*MSD* l MKS04069-MS-2 142 000 12.7 254 246 267 <LOO <LOQ <LOO 102% 99% 108% NA NA 101% NA NA 1% MKS04O69-MSD-2 228 92% 100% 16% Creep 1 Control 0.0 mg/kf/day 105320M I0S526M 105529F I03549F 12.3 23.2 436 280 <LOQ 0.0233 0.04)6 0.0287 0.0233 ! Anomaly 0 0352 NA 25 9 000911 Giwp3 Mid*Dose 0 15mg/V^'day WH0SM I05523M I05539F 62\ 692 810 66.4 730 865 677 69 7 4.68 154 I05532F* 390 695 780 12.0 Creup 4 High-Dose 105511M I05522M 192 264 111 336 42.5 259 110 0.75 mg/kg/day I05533F 242 265 11.9 I05S42F 210 224 245 29 2 Limn of Quantitation (LOQ) PFOS " 152 ng/mL Date Entered/By: 04/19/99,QV1Q/99,05/2<V99 LAC Date Verified/ By: 08/74/99 SAH, befte 0*08/00 TKR Date punty conected/vetified: 09/12/00 mmh. 09/12/00 LAC PFOS * Perfluorooctanesuifomte NA - Not applicable * Serum initial volume less than 0.30 mL, concentrations should be considered tentative 04/28/00 LAC Extraction Volume Ratio " Initial Volume/Finai Volume ETS-8-5.0 Excel 97 3M Environmental Laboratory tox-030.era223-lC y * zio 9:1roo 3m Medical Department Study T -6 2 9j5vc? TOX-030 Report N o . FACT TOX-030 Cov.nct# 6329-223 laboratory Request Number-U2279 Study Product NumbeifTest Substance): Matrix: Method/Revmon Ajialyncal Equipment System Number Instrument Software/Vemon: Filename: R-Squared Value: Slope: Y-lntexeept Date of Extraction'Analyst Date ofAnalysis/Analyst Date of Data Reduction/Analyst: Samp) Data 26 Week Capsule Toxicity Study w*ith PFOS in Cjnomoljfus Monkeys T-629S(PFOS) Monkey Sen ETS-A-4 0 and FTS-8-5.0 Amelia 062498. Soup 020199, A Madebne 041098 M&ssLynx 3.2 See Attachments See Attachments See Attachments See Attachments 04/06/99 RWW 04/07/99,04/11/99.04/17/99,0V14/99,05/17/99 HOJ.DRB/MEE/KJH 04/09/99,04/1V99, 04/20/99.0Vl7/99.oyiR'99 HOJ-'DRB/KIH DAY185 MONKEY SERA Crmap S ta p le d PFOS CaacaatradM Meas RSO Dm * Cene t/saL af PFOS tta L ar% R*e PFOS ue/mL Std. Dm . MS/MSD RPD Method Blk RJ9504Q69-H2O Bflc-1 000 <LOQ RBS04069-K20 BIk-2 1418 <LOO NA NA Matrix BOc QC - 250 ppb RBS04069-Sera BBt-1 RfiS04069-Sera Blk-2 MKS04069-MS-1 MKSCM069-MSD-1 MKS04069-MS-2 000 1274 254 246 267 <LOQ <LOO 10254 9954 10894 NA 101% NA 3% MKS04069-MSD-2 228 92H 100% 16% Creep 1 Control 0.0 mfAqpday 105520M 105S26M 05329F 32.0 37.3 63.1 0 0366 0.0498 0.0763 0.0432 21.5 0 00928 561 05S49F 23.0 0 0330 0 0546 00306 Cp3 Mid-Dose 1035O5M I05323M 553 778 70.3 137 85 4 7 ?9 107 0.15 m*/k*/day 105339F 867 101 276 I055S2F+ 322 67.8 84 3 233 G rf 4 105511M 216 309 749 Huh-Dose 10S521M- U5 271 294 22.0 0 75 mg/kg/day 105533F+ 220 441 528 I05543F+ 121 201 321 170 Limit of Quanotaoon aOQ): P F O S -15 2 n*AnL Date Entered/By: 04/19/99,05/10/99 LAC Dale Verified/ By: 08/25/99 SAN. before 06/01-00 TXR Date purty correctcd/vtnficd: 09/12/00 mmh, 09/12/00 LAC PFOS PerfiuoroecUncsulfonate * Serum initial volume lest than 0.30 mL, concentnbonj should be considered tentative 04/28/00 LAC ETS-8-5 0 Excel 97 3M Environmental Laboratory tox-030>sera223-1C 9 / I2 '0 0 3m Medical Department Study: T - 6 2 99kC TTO X-030 Report No. FACT TOX-030 Cov#nce#6329-223 laboratory Request Number-U2279 Study: Product N'umberfTest Substance): Matrix: Mcthod/Revision; Analytical Equipment System Number. Instrument Software/Version: Filename: R-Squaxed Value: Slope. 26 Week Capsule Toxicity Study with PFOS in Cynomolgus Monkeys T-6295 (PFOS1 Monkey Sera ETS-8-4.0 and ETS-S-5.0 Amelia 06249S. Soup 020199. Sl Madeline 04)098 MassLynx 3.2 See Attachments See Attachments See Attachments Y-fntercept: Date of Extnction/Analysc Date o f Analysis/Analyst: See Attachments 044)6/99 RWW 04 07/99. 04/lZ;99. 04/17/99. 05.'14*99. 05-17.99 HOJ DRR- MEE KJM Date of Data Reduction/Analyst: 04.09.99.04/15/99. 04/20/99,05/17/99.05`18:99 HOJ DRn K ill Sample Data DAYI87 MONKEY SERA Group Dose Method Blk Matrix Blk QC - 250 ppb Sample RBS04069-H20 Blk-I RBS04069-H2O BIk-2 RBS04069*Sera BIk-l RBS040i9-Sa Blk-' MJCS04069.MS-1 MKS04069-MSD-1 MKS04069-MS-2 MKS04069-MSD-2 PFOS Coite. ng/mL 000 14.2 000 12.7 25* 246 267 228 Concentration of PFOS ug/mL or % Ree <LOQ <LOQ <LOQ <LOQ 102% 99% 108% 92% Mean PFOS ug/mL MA NA 101% 100% RSO Std. Dev. MS/MSD RPD NA na 3% 16% Group l Control 0 0 mg/kg'day Group 3 Mid-Dose 0.15 mg/kg/day I05520M 13.6 <LOQ 105526M 22.1 0.0300 0.0300 > 1 Anomaly NA I05529F 39.1 0.0373 2S.9 I05549F+ 31.7 0.0541 0.0457 0.0118 105505M 699 82.4 5 91 105523M 662 75.8 79.1 4 67 I05539F 63.7 10.2 105 I05552F 498 68.8 39.5 414 Group 4 High-Dose 0.7S mg/kg/day 1055! IM 105522M 105533F I05542F 225 185 289 253 237 297 269 248 15.7 267 42.0 5.90 258 15.2 Limit o f Quantitation (LOQ). PFOS - 15.2 ng'mL Dale Entered/By: 04/19/99. 05/10/99. 05/20-99 LAC Date Verified'By: 08/25/99 SAH. before 06 0 00 TKR Date purity correeted/verified: 09/12/00 mmh, 09-'l2'00 LAC PFOS Perfluorooctanesulfonate * Strum initial volume less than 0.50 ntL. concentrations should be considered lentatrve. 04.28-00 LAC ETS-8-5 0 Excel 97 3M Environmental Laboratory T O X '030-sera223-1C 2)2- 09 I.V2000 age -5-e-3- 3m Medical Department Study: T -62 9&\ctTOX-030 Report N o . FACT TOX-030 Covance# 6329-223 laboratory Request Number-U2279 Study Produet NumbeTtTeit Substance): MatnX Method/Renaon: Analytical Equipment Syitfm Number Instrument Software/Venum: Filename R-Squared Value: Slope Y-Intercept: Date of Extraction/Analyst 26 Week Capsule Towary Study with PFOS m Cynomolfus Monkeys T-6295 (PFOS) Monkey Sen ETS-8-4 0 and ETS-8*5 0 Amelia 062498, Soup 023199, & Madeline 041098 MassLynx 3 2 See Attachments Sec Attachments See Attachments See Attachments 04/0199 RWW Date of Analysis/Analyst Date ofData Reduction/Analyst 04/11/99.04/17/99.05/1499 DRB/MEE/KJH 04/16/99,04/20/99,05/17/99 HOJ/DRB/KJH Sample Data WEEK 2 MONKEY SERA Cramp Dm # Sw fl PFOS gfaL C--c-- Brailaa TPFOS C /aaL rH R c Mom PFOS ng/mL USD Sid. Dev. MS/MSD RPD Method Blk Matrix Blk QC-230ppb Cravp 1 Control 0.0 mg/kg/day CrMpJ Mid-Doac 0. IS mg/kg/day Crauf 4 High-Doee 0.73 mg/kg/day RBS04089-H20 Btic-t RBS04CC9-H2O BIk-2 RBS04089-Sn BZk-1 RBS040a9-Sen&ac-2 MKS04089-MS-1 MKS04089-MSD-1 MKS04089-M5-2 MKS040S9-MSD-2 10S320M I03526M* I05S29F* 105S49F* 103303M+ I03J23M+ 105539F- 10S532F1033UM* I05322M W33J3F I05542F 7.80 701 000 0 680 260 267 249 264 11.4 26.4 26.8 20.0 481 311 288 333 336 659 366 423 <LOQ <LOO <LOQ <LOO 103% 108% 101% 106% 0.0284 00459 0.0488 00372 83.7 833 88.7 836 233 264 249 223 <LOO <1.00 106% 103% 0037! 0.0430 846 86 1 249 236 NA NA 3% 6% 33 5 0 0124 19 1 0 00821 1 78 131 4.16 339 8.71 21.7 7.76 18.3 Lima of Quanbtabon (LQQ): PFOS IS 2 ng/mL Date Entercd/By: 04/19/99, 03/10/99 LAC Date Verified/ By: 017V99 SAH, before 06/0100 TKR Date punry conected/venficd: 09/12/00 mmh, 09/12/00 LAC PFOS * Perfluorooctanesuifonate Serum initial volume leis than 0.50 mL, concentrations should be considered tentauve 04/2100 LAC ETS-8-3.0 Excel 97 3M Environmental Laboratory tox-03O-sera223-IC 2 !3 9/11*00 3m Medical Department Study T - 6 2 9 f o c f TOX-030 Report No. FACT TOX-030 Covance# 6329-223 laboratory Request Number-U2279 Study: Product NumberfTest Subitanee): Matrix: Mcthod/Renuoit Analytical Equipment System Number Instrument Softwwe/Versum: Filename: R-Squared Value: Slope: Y-buereept: Date of Extraction/Analyst Data of Analyna/Anaiyst Date of Data Reduction/Analyst Sample D*U 26 Week Capsule Toxlaty Study with PFOS in Cynemolgus Monkeys T-6295 (PFOS) Monkey Sera ETS-8-4 0 and ETS-8-5 0 Amelia 062498. Soup 020199, & Madeline 041098 MassLynx 3 2 See Attachments See Attachments See Attachments See Attachments 04/08/99 RWW 04/11/99, 04/17/99,05/14/99 DRB/MEE/XJH 04/16/99,04/20/99,05/17/99 HOJ/DRB/KJH WEEK 29 MONKEY SERA Croup Duae Sample PFOS Co k oriL fPFOS C/aaL a r % Ree Mean PFOS ug/mL RSD SU. Dev. M3/MSD RPD Method Blk RBS040W-H3OB&-1 RBS04089-H30 BIk-2 7.80 7.01 <LOQ <LOQ LOQ VA Matrix BUc RBS04089-Sen Blk-l 0.00 <LOQ QC - 230 ppb RBS04089-Sera Bft-2 MKS040I9-MS-1 MKS04089-MSD-I 0 680 260 267 <LOO 105% I08H 'LOO 106% NA 3% MKS04089-MS-2 249 101% MKS04089-MSD-2 264 106% 103% 6% CTMpl Control 0.0 mp/kg/day I05520M105S26M 10J529F* 105J49F 42.2 447 38.7 68.1 0.0690 0.0639 0.073B 0.0747 00665 00742 545 000362 0894 0000664 Croup J Mid-Dose 105305M 105J23M* 636 417 77J 288 74 2 75 8 2 18 O.I5mp/kp/day I05339F 613 779 16 2 I03332F 432 619 699 11.4 C rw ^4 High-Doee 0.73 mg/kg/day (03311M 103322M 105533F 105542F 360 384 641 549 175 300 270 223 669 207 9.83 180 194 19.0 Limit of QuanCtaotm (LOQ): PFOS -15.2 nf/mL Dale Entcred/By 04/19/99,OV10/99 LAC Date Verified/ By 08/23/99 SAH. before 06/08/00 TKR Date punty ccxrected/venfied: 09/12/00 mmh, 09/12/00 LAC PFOS Perfiuorooctanesulfonate * Serum initial volume less than 030 mL, concentrations should be considered tentaove. 04/28/00 LAC ETS4-50 Excel 97 3M Environmental Laboratory iox-030>scra22>lC Zi 3m Medical Department Study T -62 9 \C TOX-030 Report N o . FACT TOX-030 Covince# 6329-223 laboratory Request Number-U2279 Study. Product NumbesfTest Substwee) Mttttx: Method/Revisum: Analytic) Eqispmoil System Number Instrument Softwaie/Vemon. Filename R-Squared Value: Slope Y-lntercept: Dele o f Extracoon/Analyst: Date o f Analysis/Analyst: Date of Data Reduction/Analyst Suipl Data 26 Week Capcule Toxicity Study with PFOS m CynamolRus Monkeys T-6295 (PFOS) Monkey Sen ETS-8-4 0 and ETS-8-5 0 Amelia 062498. Soup 020199, & Madeline 041098 MaaaLynx 3.2 See Attachments See Attachments See Attachments See Attachments 04/08/99 RWW 04/11/99,04/17/99.05/14/99 DRB/MELXJH 04/16/99,04/20/99,05/17/99 HOJ/DRB/XJH WEEK 31 MONKEY SERA Craup Data Method BDc Matrx Blk QC - 250 ppb Cmf 1 Control 00mg/kg/day Creap 3 Mid-Dow 0.15 mg/kg/day Creep 4 High-Oose Sample RBS04089-H2O Blk-i RBS04089-H20 Bc-2 RES04089-Sen Blk-I &BS040t9-Sera Blk-2 MKS04089-MS-I MKS04089-MSD-1 MKS04089-MS-2 MKS04089MSD-2 105520M 105S26M 105529F 05549FI05505M I03523M 10S539FI055J2F* 103511M I05522M PFOS Case aglmL 780 701 000 0680 260 267 249 264 27.1 36.7 24.3 25 5 591 398 580 291 398 668 CaacaatrUaa ef PFOS iM L a r H Rac <LOQ <LOO <LOC <LOO 105% 108% 101% 106% 0.0362 0.0354 0.0282 0.0433 60.0 53.2 111 48.6 129 194 M ae PFOS U0EbL <LOO <LO0 106% 103% 0 0358 0 0368 566 79.6 161 K3D S lR D rr. MS/MSD RPD na NA 3% 6% 1.38 0 00036? 32 9 00121 8 43 4 rr 550 43 8 28.6 46.1 075 mg/kg/day I05533F 105542F+ 591 398 201 118 170 185 21 9 PFOS Perthiorooctanesulfonate NA - Not applicable Date Entered/By 04/19/99 LAC Date Veh6ed/ By: 08/25/99 SAH. before 06 08,00 TKR Date punfy corrected/venfied: 09/12/00 mmh. 09/12/00 LAC * Serum w ot) volume leas than 0 30 mL, concentration* should be considered tentative 04/28/00 LAC Extraction Volume Ratio Inibal Volume/Ftnal Volume ETS-B-5.0 Excel 97 3M Environmental Laboratory (ox-030-sen223-lC Z f* t 2 ! $ Pa< 3m Medical Department Study T - 6 2 TOX-030 Report N o . FACT TOX-030 Covane# 6329-223 laboratory Request Number-U2279 Study Product NumberfTeat Substance): Matrix: Method/Revuicn Aivaiyncal Equipment System Number: Instrument Software/Vcroon. Filename. R-Squared Value Slope: Y-Intereept: 26 Week Capsule Toxicity Study with PFOS m Cynomolgua Monkeys T-6293 (PFOS) Monkey Sen ETS-8-4 I and ETS-8-5.1 Amelia 062*9 MauLynx 3.3 See Attachments See Attachments See Attachments See Attachments Date of Extncoon/Analyst: Date of AnalyiMb'Analyst Date of Data Reduction/Analyst 08/23/99 RWW 09/28/99. 10/03/99, 06/14/00 MEE/MMH 09/29/99. 10/06/99, 06/15/00 MEE/IAS'MMH Sample Data WEEK 35 MONKEY SERA Creep D*ee Sample # PFOS Ceac. agftaL Ceaceatratlon *f pros uf/mL e r H Ree Mean PFOS (M L RSD SU. Dr*. MS/M3D RPD Method BOc Matrix Blk QC - 250 ppb Cretap t C ontri 0.0 mg/fcg/day H20 filk-1 H20 BBc-2 Rabbit S en BIk-1 Rabbit S en B0c-2 MK308259-MS-1 MK.505289-MSD' 1 MKS08259-MS-2 MKS05289-MSD-2 103320M 105526M I05329F I03349F 000 000 0.00 OX 227 254 236 239 27.3 35 9 74.7 61.0 <LOQ <LOO <LOQ <LOO 91% 102% 103% 96% 0.0437 0 0480 0.0748 0.0698 <LOQ <LOQ 97% 100% 00459 0.0723 NA NA 11% 7% 660 0.00303 486 0.00352 Creep J Mid-Dose 0.13 mg/kg/day Creep 4 High-Dose 0.75 mg/kg/day I03303M + 103523M J03539F* I05552F105511M 10S522M J0J533F 05542F 186 190 122 135 417 486 408 444 93.0 14.1 76 t 84 5 120 812 12 8 67.7 74 4 9.53 167 108 195 18! 19 5 164 5.94 178 171 10 1 Umjt of Quantitation (LOQ): PFOS 5.33 ng/mL Da Eniered/By: 10/03/99, 10/07/99, 06/19/00 LAC Date Verified/ By before 06/08/00 TKR Date punty conected/venfied 09/12/00 mmh. 09/12/00 LAC PFOS PerfHiarooctaneauUbnate NA " Not analyzed, not appbcabta * Serum initial volume leu than 0 30 mL, concentrations should be considered tentative 04/28/00 LAC Extraction Volume Ratio * Irubad Volume/Fimi Volume ETS-8-5 1 Excel 97 3M Environmental Laboratory tox-030-ier*223-tC I f i p f U < 0 9/12/00 3m Medical Department Study T -62 3.cTTOX-030 Report N o . FACT TOX-C30 Cov*nce# 6329-223 laboratory Request Number-U22 79 Study: Product Number{Tett Substance) Matrix: Metho/Revia Analytical Equipment System Number Instrument Software/Version: Filename: R-Squared Value Slope Y-lnterccpc: Date of ExtracticsVAnalyst Date of Anafym/Analyst: Date of Data Reduction/Analyst Sample Data 26 Week Capsule Toxicity Study uith PFOS m Cynomoigus Monkeys T-6295 (PFOS) Monkey Sera ETS-8-4 1 and ETS-8-5.1 Amelia 062498 MassLynx 3.1 See Attachments See Attachments See Attachments See Attachments 08/23/99 RWW 09/2S/99, 10/05/99. 06/14/00 MEE/MMH 09/79/99. 10/06/99. 06/15/00 MEE/IAS/MMM WEEK 39 MONKEY SERA Cmp Sample 4 PFOS Ceucesitratlen Mean RSD Deea Method Blk H20 Blk-1 Ceuc. a tlu L 0.00 ef PFOS u f/a a L e r H Rec <LOQ PFOS '-i- SM. Oct. MS/MSD RPD H 20 Blk-2 0.00 <LOQ *UX3 NA Matrix Blk Rabbit Sera Blk-1 0.00 <lOQ Rabbit Sera BUc-2 0.00 <IDQ <l.OQ VA QC - 250 ppb MKS08259-MS-1 227 91% MKS0S289-MSD-1 254 102% 97% 11% MKS08259-MS-2 256 103% MKS05289-MSD-2 239 96% 100% 7% Croup 1 I05320M 23.7 0.0316 27 1 Control 105326M 29.1 0.0466 0 0391 0.0106 0.Q m^Tcg/day W5529F 31 4 0.0503 1.53 105349F+ 23 7 00314 00509 0.000779 Group 3 105505M* 311 62.4 5 26 Mid-Doce I05523M + 217 57 9 60 1 3 16 0.13 mg/kg/day I05339F* 142 56.8 14.1 105552F-* 174 465 51 7 7.29 Group 4 I03511M + 270 L35 11 0 High-Doee I05522M+ 236 158 U6 16 1 0 75 m/ykg/day 105533F* 338 169 686 105342F- 210 154 61 11 l Limit of Quantitation (LOQ): PFOS - 5 53 ng/mL Date Entered/By. 10/05/99. 10/07/99, 06/19-00 I.AC Dale Verified/ By: before 06/08/00 TK.R Date puny eorrected/venfied: 09/12/00 mmh, 09/12*00 LAC PFOS Perftuorooctanesuifonate * Serum initial volume less than 0.50 mL, concentrations should be considered tentative. 04/28/00 LAC NA " Not analyzed, not applicable Extraction Volume Ratio " Initial Volume-Final Volume ETS-8-5 1 Excel 97 3M Environmental Laboratory lox-03O-eim223-lC 2% 217 9/12/00 3m Medical Department Study: T - 6 2 % ^ c ' TOX-030 Report No. FACT TOX-030 Covarne# 6329-223 laboratory Request Number-U2279 StudyProduct NuRiberCTeit Subatance) Matrix: Method/Revuion. Analytical Equipment System Number 26 Week Capsule Tenacity Study with PFOS in Cynomolgus Monkeys T-6293 (PFOS) Monkey Sen E7S-8-4 ; and ETS-8-5 l Amelia 062498 Instrument Softwve/Vemon: Filename R-Squared Value. Slope, YIntercept: Date of Extraction/Analyst MasiLynx 3 3 See Attachment! See Attachment! See Attachments See Attachment! 08/25/99 RWW Date of Aitatysia/Analyst Date of Data Reduction/Analyst 09/28/99, 10/05/99, 06/14/00 MEE/MMH 09/29/99, 10/06/99,06/15/00 MEE/IAS/MMH Sample Data WEEK 43 MONKEY SERA Graup Dae Method Bc Matrix BUc QC - 250 ppb Sample a H 20 Blk-l H20 BIk-2 Rabbit S en Bflr-1 Rabbit Ser BBc-2 MJCS08239-MS-I MK3052S9-MSD-1 MK308259-MS-2 MKS05289-M5D-2 PFOS Ceac. nf/mL 000 000 ooo 000 227 234 236 239 Cencaacratten rp ro s tfgftni, ar H Ree <LOQ <LOO <LOQ <LOO 91% 102% 103% 96% Mean PFOS *fL <LOO <LOQ 97% 100% RSD Std. Dev. MS/MSD RPD NA NA US 7% Graap 1 103320M 21.3 0.0287 13.B Control 0.0 mg(kg/day 105526M 10SS29F 105549F 21.8 32.2 22.9 0.0330 0.0368 0.0367 0 0319 0 0368 0.00440 0231 0 0000923 Graap Mid-Dota 0.15 mg/Vg/day I0305M 05523M I05539F 10552F* 162 112 146 116 46 $ 2 44 44 9 45 7 1.12 58.3 0 429 58.0 38 1 0249 Graap 4 High-Do 0 73 mg/lcg/day 105511M 105522M 105333F I05542F 167 226 447 347 66.9 21.4 90.7 788 16 8 179 178 139 139 28 4 Limit of Quantitation (LOQ): PFCS * 5 55 ngfaL Date Entered/3y. 10/0S/99, 10/07/99, 06/19<`00 t.AC Date Verified' By before 06/08/00 TK.R Date punty correctedAenfied: 09/12/00 mmh, 09/12/00 LAC PFCS * Perfluorooctanesuironatc NA 3 Not analyzed, not applicable + Serum initial volume le u than 0.30 mL. concentration should be considered tentative 04/28/00 LAC Extracoon Volume Rano " Initial Vohime-'Final Volume ETS-8-J \ Excel 97 3M Environmental Laboratory tox-030-iera223*1C U F c z if r 9M2/00 3m Medical Department Study T - 6 2 % ^ c f TOX-030 Report N o . FACT TOX-030 Covance# 6329-223 laboratory Request Number-U2279 Study: Product Number(Teat Subatanca) Mitnx: Method/Reviaion. Analytical Equipment System Number Instrument Softwarc/Version: Filename: R-Squared Value: Slope Y-lntercept Date of ExtrnctioiVAnalyst: Date of AnaJyva/Analyst: Date of Data Reduction/Analyst: 2d Week Capsule Toxicity Study with PFOS in Cynomolpis Monkey* T-6295 (PFOS) Monkey S en ETS-8-4 1and ETS-S-5 1 Amelia 062498 M*iaLyrtt33 See Attachments See Attachments See Attachments See Attachment! 08/25/99 RWW 09/28/99, 10/05/99,06/14/00 MEE/MMH 09/29/99, 10/06/99, 06/15/00 MEE/IAS'MMH Sample Data WEEK 47 MONKEY SERA Groap Dee# Method BUc Matrix BUc QC - 250 ppb Sample 0 H 20 BOc-1 H 20 Bflc-2 Rabbit S e n 3 Uc-1 Rabbit S en BOc-2 MK.508259-MS-1 MKS052R9-MSD-1 MXS08259-MS-2 MKS05289-MSD-2 pros Cerne. s|te L 000 000 000 OX 227 234 236 239 Concentration of pros S |/ n L e r Mi Rcc <LOQ <LOO <LOQ <LOO 91% 102% 103% 96% Mean pros "i l <LOQ <LOO 97% 100% RSD Std. Dev. MS/MSD RPD NA NA 11% 7% Croup 1 Control O.Omgfcg/day Groap 3 Mid-Dow 0.15 mgfcg/day 105520M (05526M 105529F 103549F I05505M I05523M I03339F I05352F 234 27.7 360 32.6 132 114 ns 114 0.0339 00370 0.0481 0.0436 50.9 457 47 4 37 9 0.0355 0439 48 3 42 6 6 23 0.00221 7 04 0X323 7 65 3.69 157 6 70 G rssp 4 High-Doee 0.75 mg/kg/day 1055UM I05322M+ I0S533F 105542F 263 212 312 277 105 21.0 142 124 259 104 8 47 92 98 3 8 32 Limit of Quantitation (LOQ) PFOS 3 35 ng'mL Date Emred/By 1005/99. 10/07/99. 06/19/00 LAC Date Verified/ By before 06/0B/X TKR Date punty corrected/verified: 09/12/00 mmh, 09/12'X LAC PFOS * Pertiuorooctanesulfonate NA Not analyzed, not applicable * Serum uubal volume lesa than 0 SOmL. concentrations should be considered tentative. D4/28/00 LAC Extraction Volume Ratio " Initial Volume/Fuul Vohime ETS-8-5 I Excel 97 3M Environmental Laboratory tox-030-*era223-lC 2& 2 .U 9/12/00 3m Medical Department Study T - 6 2 $ & C ? TOX-030 Report No. FACT TOX-030 Covanee# 6329-223 laboratory Request Number-U22 7 9 Study. Produci Number<Tesf Subitanee): Matnx. 2b Weak Capsule Twocity Study with PFOS in Cynomolgua Monkeys T-6295 (PFOS) Monkey Sen Method/Revuioft. Analytical Equtpmcnt System Nurnbcr Inatrument Softw w W emoni Filettarne R-Squared Vahie Slopc: Y-Intereept: Date of Exoaction/Analyi*: Date of A naty/Anaiyit ETS-8-4 1 and ETS-8-5 l Ameba 062498 MassLynx 3 3 See Attachments See Attachments See Attachments See Attachments 08/75/99 RWW 09/28/99,10/05/99, 06/14/00 MEE/MMH Da of Dati Raductaon/Analyit: 09/29/99, 10/06/99, 06/15/00 MEE/IAS/MMH S*m p ltD al* WEEKS1 MONKEY SERA Graap D*ee Method Blk Matnx 31k QC -230ppb Sample * H20 Blk-1 H20 Blk-2 Rabbit S an BDc-1 Rabbit S en Blk-2 MKS08259-MS- 1 MKS03289-MSD-1 MK30S259-MS-2 MKS05289-MSD-2 PFOS Cene. hr/ b L 0.00 000 0.00 0.00 227 254 256 239 C ea ceatratlea a f PFOS ag/mL ar H Ree <LOQ <LOQ <LOQ <LOO 91% 102% 103% 96% Meea PFOS BRrtuL <LOQ iLOQ 97% 100% R5D S td Dev. MS/M3D RJPD NA NA 11% 7% Graap 1 Control 0.0 n^/kg/day I05520M 105326M [QS529F 105549F 13.3 195 34.3 29.6 0.0213 0 0261 0 0344 0.0338 00237 0 034) 14 0 0 00333 MB 0000403 Group 3 Mid-Do 0.15 nqpfcg/day 105505M 10J523M I05339F I05532F- 119 108 111 52 398 6.92 36.0 37,9 262 44.5 376 25 8 35.1 13.2 Gnap 4 High*DoM 0.75 m^Vg/day 10551IM* 105522M 105533F I05542F 135 365 261 287 67 5 40 6 122 94 7 384 87.1 665 95 7 91 4 607 Limit of QuintttMon (LCQ): PFOS - 5.35 tigftnl Dale Entered/By: 10/05/99, 10/07/, 06/19.00 LAC Date Verified/ 8 y. before 06/0S/O0 TKR Date punty corrected/venfied: 09/12/00 mmh, 09/12*00 LAC PFOS * Perfluofooctaneaulfonate * Serum initial volume lesa than 0 50 mL, ccncentraoons should be considered tentative. o a a a w LAC NA * Not analyzed, not appheable Extraction Volume Ratio * Initial Vohime/Final Volume ETS-8-5 I Excel 97 3M Environmental Laboratory lox-030-sera223- 1C 3m Medical Department Study T - 6 2 9 f i \ c t TOX-030 Report N o . FA C T T O X - 0 3 0 CovanceM 6329-223 laboratory Request Number-U2279 Study: Product Number(Test Substance) Matrix: Method/Reusion. Analytical Equipment System Number Instrument Software/Veroon: Filename: R-Squared Value: Slope: Y-lntercept. Date of Extraction/Analyst: Date of Analytu/Analyse Date of Data Reduction/Analyst: 26 Week Capsule Toxjary Study with PFOS in Cynomalgu* Monkeys T-6295 (PFOS) Monkey Sera ETS-8-4 lan d ETS-8-5 1 Soup020199 MauLynx 3 3 See Attachment! See Attachments See Attachments See Attachments lt/03/99 SAL U/04/99. U /ll/99. 11/18/99 LAS U'08/99, 11/12/99. 11/32/99 MMH/US Sample Data WEEK S3 MONKEY SERA Craup Date Sample * PFOS Cane. nt/uL Ceaceatnden rrros f/u L ar H Rec Mean PFOS u*L RSD Std Dev. MS/MSD RPD Method Blk Matrix Blk QC - 250 ppb Civifi 1 Control 0.0 mg/kg/day H 20 Blk-5 H 20 Bfle-5 Rabbit S e n Blk-5 Rabbit Sera Blk-5 MK311039-MS-6 MK3U039-MSD-6 MK311039-MS-5 MK311039-MSD-3 I05520M I05526M 103329F 105S49F 0 830 1 07 2.20 2.50 29 5 29 4 374 380 33.6 48.9 46.8 52.3 <LOQ <LOO <LOQ <LOQ 0.0237 0 0236 112% 114% 0.0269 00392 0.0375 00419 <LOQ <LOQ 0 0236 113% 0.0331 0 0397 N'A NA 0.192 0.0000453 2% 26.1 0.0086 7.82 0 00311 G ruf 5 Mid-Dose 0.15 m^kg/day 105505 M I05523M 105339F I05552F+ 109 121 103 81 43 9 7.15 48 5 46 2 3.30 41.1 17 0 323 36 7 624 Craup 4 High-Dose 0 ?5 mg/kg/day 105511M 105522M* I05533F 105542F 137 261 246 244 54.8 45 5 107 80 8 36.8 986 0 499 979 9ft 1 0 490 Limit of Quantitation (LOQ) PFOS " 5 55 nj'mL Date Entered/By: 11/15/99, 11/34/99 LAC Date Verified/ By: before 06/08/00 TX.R Dale purity correcied/verified. 09/12/00 mmh, 09/12/00 IAC PFOS " Periluarooctaneiulfonatc ** Although labeled as M&MSD-6. these were not spiked and will be used as blanks LAC 11/15/99 NA Not applicable - Serum iracial volume leu than 0 50 mL, concentrations should be considered tentative 04/28/00 LAC Extraction Volume Ratio " Initial Volumc'KinaJ Volume ETS-8-5 l Excel 91 3M Environmental Laboratory lox-030-ere223- 1C 221 3m Medical Department Study T -62 SACf TOX-030 Report N o . FACT TOX-C30 Covinee# 6329-223 laboratory Request Number-U22 7 9 Study Product NumberfTeit Substance) Matnx: Method/Rewuon Analytical Equipment System Number Instrument Software/Vertiorv Filename: R-Squared Value: Slope Y-lntercept: Dele of Extraction/Analyst Oats of Analysis/Analyst: Date of Data Reduction/Analyst. Sample Data 26 Week Capsule Toxicity Study with PFOS in Cynomolfrus Monkeys T-6295 (PFOS) Monkey Sera ETS-8-4 1 and ETS-8-5 1 5oup020l99 MataLyiui 3 3 Sec Attachments See Attachments See Attachments See Attachments ! 1/03/99 SAL 11/04/99, 11/11/99. 11/1*99 IAS 11/0*99, 11/12/99, 11/22/99 MMH/1AS WEEK 57 MONKEY SERA Croup Date Method Blk Matnx Blk QC - 230 ppb Sample H20 Blk-5 H 20 Btt-5 Rabbit S en Blk-5 Rabbit S en Blk-5 -- MKS11039-MS-6 MKS11039-MSD-6 MKS11039-MS-5 MK311039-MSD-5 PFOS Cane. ng/aL 0830 107 2.20 2.50 29 5 29 4 374 380 Concentration of PFOS ag /aL or Mi Ree <LOQ <LOO <LOQ <LC30 0.0237 0.0236 11254 114% Mean PFOS g/aL <LOO <1.00 0 0236 113% RSD Std. Dev. MS/MSD RPD NA NA 0 192 0 0000453 2% G iu ri Control 0.0 mg/tyday Group.) Mid-Dose 0.15 mg/kg/day Croup 4 High-Dose 0 75 mg/kg/day I03S20M 103526M I05529F 1Q5549F I05503M I05523M 105539F 105352F 105511M I05S22M* I05533F I05542F* 36.1 45 4 58.9 52.1 711 79 4 83 1 78.4 166 221 258 271 0.0289 0.0364 0.0472 0.04)8 28.5 31.8 33.3 31.4 66.5 895 103 109 0 0327 0.0445 30 2 32.3 78.0 106 16! 0 00526 8 65 0 00385 7 83 2 36 4n 1 34. 20 8 163 3 63 3 84 Limn of Quantitation (LOQ): PFOS * 5 35 n*mL Date Entered/By 11/15/99, 11/24/99 LAC PFOS * Perthioroocianesulfonate NA Not applicable Date Verified/By before 0 *0*00 TTCR Dele purity conrcted/venfied 09/12/00 mmh, 09/12/00 LAC ** Although labeled as MS/MSD-6, these were not spiked end will be used as blanks. LAC 11/15/99 * Serum initial volume leu than 0 30 mL, concentrations should be considered tentative 04/7*00 LAC Extraction Volume Ratio * Initial Vohime'Final Volume ETS-8-5 1 Excel 97 3M Environmental Laboratory tox-030-scn223-IC Z L2Z . 9/12/00 Pa< 3m Medical Department Study T " 6 2 %^C?TOX-O30 Report No. FACT TOX-030 C o v u ic e # 329-223 laboratory Request Number-U2279 Study: Product NumbetfTest Substance) Matnx: Mcthod/Revuion Analytical Equipment System Number Instrument Softwaie/Veraon Filename R-Squared Value: Slope Y-Intercept: Date of Extraction/Analyst: Date of Analyte/Analyst. Date of Data Reduction/Analyst Sample Data 26 Week Captuk Tonoty Study with PFOS in Cynomolgua Monkeys T-629J (PFOS) Monkey Sere ETWM.l and cTS-8-5 1 Soup020199 MaitLyru 3 3 See Attachments See Attachments See Attachments See Attachments 11/03/99 SAL 1W04/99, IUU/99. IW18/99 IAS 11>08/99, 11/12/99, 11/22/99 MMH/IAS WEEK 61 MONKEY SERA C reep Deee Method Blk Matrix Blk QC - 250 ppb Cnupi Control 0.0 mg/kg/day Creeps Mid-Doae 0 15 mg/tyday Creep 4 High-Dcee 0.75 mg/kg/day Sample H2Q 31-5 H20 BSc-5 Rabbit Sera Bik-5 Rabbit Sera Blk-5 MKSI1039-MS-6 MK.51I039-MSD-6 MKSI1039-MS-5 M1CS11039-MSD-5 10552044* 10552M I05529F 105549F I05505M I05523M 1Q553SF I03352F 105511M 105322M I05533F I03542F PFOS Cene. eg/eiL 0 830 1 07 2.20 2.50 29.5 29.4 374 380 39.8 47.7 54 0 57 7 684 895 94 8 93 8 162 339 271 273 Ceeceetratlee efPFOS eg /eiL erH Rcc <LOQ <LOO <LOQ <LOO 0.0237 00236 112% 114% 0.0319 0.0382 0.0433 0.046 27.4 35.9 3B.0 38 4 64.7 136 108 109 Mean PFOS eg/eiL <LOO <LOC 0 0236 113% 00351 0 0448 31 6 38 2 100 109 R3D SU. Dev. MS/MSD RPD NA NA 0.192 0 0000453 2% 12.8 0.00449 468 0.00210 189 5.98 0.742 0.283 50.2 50.3 0640 0 697 Umit o f Quantitation (LOQ) PFOS * 5 55 ng/rnL Date Entered/By: 11/15/99, 11/24/99 LAC Date Verified/ By before 06/08/00 TKR Date punty corrected/wified' 09/12/00 mmh. 09/12/00 LAC PFOS " Perthiocooctenesulforuite ** Although labeled MS/MSD-6, these were not ipiked and will be uaed as Wanks LAC 11/15/99 NA - Mot applicable - Serum initial wohnne leas than 0.30 mL, concentration should be considered tentative 04/28/00 LAC Extraction Volume Redo Initial VolumeTmal Volume ETS-8-5 1 Excel 97 3M Environmental Laboratory tax-030-ee223-lC -23 9/12/00 gafe"2T^ 3m Medical Department Study t " 6 2 9 ac3 t o x -<o Report N o . FACT TOX-030 Covance# 6329-223 l a boratory Request Number-U2279 Study: Product NajnbarfTeet Sebeare*) 16 Week Cpenle Toxuty Study with PFOS in Cynomotgua Monkey* T-629S (PFOS) Mitm. Method/Rrvuton. Monkey Sera ETS-l-4.1 uid ETS-l-i l Analytic! Equipment Syetem Numbei Ruby 100699 lrurtnun*nt SoftwuWVcmen. Filename: Mamiyiu 3.3 So* Attachment* R-Squamd Value: Slop*: Y'lntarccpt 5m Attachment* Sm Attachment* Sa* Attachment* Data of Emncbae/AJulyet: Dita ofAtiaiyM/Analy 04/34/00 sal/w k 04/33/00, 04/37/00.04/28/00, 03/03/00 lAS/MMH-'ADV Dot* of Data Redaction/Aiudyet 04,'17/do, 03/01/00, 03/01/00, 03/04/00 IAS Sample Data WEEK 65 MONKEY SERA Granp Sample# PFOS CeaceftlrmtlM Mean RSD Dos* Coac. of p ro s pros Std. Dev. |ta L u|/m L*r % Rec ag/aiL MS/MSD RPD Method Blk 04240-H10 Blk-3 0424O-H2O Blk-4 0.300 0390 <LOQ <LOO *LOQ NA Matrac Blk RBS0424O-Sen Blk-1 RBS04240.S*ra Blk*4 MKSCW2*0-S*ra Blk-3 0.930 2 73 864 <LOQ <LOO 0.007SO <LOO NA 16.9 QC - 230 ppb MXS04240'San BOe-4 MKS04240'S*ra-MS-3 no no 0.0093 122% 0.00832 0.00144 MKS04240*San*MSI>3 MKS04240'S*ra-MS-4 UKS04240>$ra-MSD-4 336 306 326 92H JI9H 12774 |0?H 123% 27S 6% Creep l Control 103330X4 12.7 0.0IS4 17 4 I03336M I4.| 0.0236 0 02)0 000363 0.0 mf'Vg/day I05339F 31.3 0.0364 1 43 10S349F 24.7 0.0357 0 0360 0000322 Group 3 103303X4 201 32.9 0.0820 Mid'Doe* 0.13 mtyday 505S23M I03339F 193 344 329 32.9 0.0269 393 6 18 1QSS52F 261 360 37.6 2.32 Greap 4 High-Don 0.73 m^kg/day (03311X4* 103522*4* I05S33F* I05342F* 234 587 465 324 32.5 60.3 i n 91 3 332 19.7 11.7 76.0 82 8 968 Limit ofQuantitation (LOQ) PFOS * 5.35 |'m i Date Ent*md/By: 03/03/00, 03/09/00 LAC'CSH Dote Verifiad/By: 06/02/00 TKR Data punty conrctKi/vmfiod: 09/12/00 mmh. 09/12/00 LAC PFOS PerfiuorooctanMiilfonat* * Senon mibal volnm* leer than 0.30 mL, concentration ihould be eonadcrcd tentative. 04-18/00 LAC NA Not applicable Extraction Volnm* Ratio m Initial Voluma/Finai Volume ET5*8*5.1 Eacejrr 3M Environmental Laboratory tox030-a*fi223-IC 2> 2 J i 9/12/00 3m Medical Department Study T - 6 2 9 t o x -030 Report No. FACT TOX-030 C o n n e r* 029-223 laboratory Request Number-U2279 Study: Mar:Product Numbcr(Te* Subaunt t) M cthodriltvincn: Anetyiral Equipment Synem Number Instrument S o ftw a A 'rm o n FUeamne: R-Squared Value U Week Capsule Tojaaty Study T-4293 (PFOS) Monkey Sara ETS-S-4 l aid ETS--5 I Ruby I0M99 MmaLynx 3 3 Sat Attachments Sea Attachments PFOS m Cyrvomoljus Monkeys Slope: Y-Intercept: D a* of EanetKM/Aiudya D ao o f Anatyua/Attaly* Dma o f D a n Reduction/Analy: SaaipU Data Sm Mtachmeno Sea Attachments M W SAUKJK (0H4i/T2Tv'oOoO,,QwVrrO/Ql.OQO..OmV/OzUsO-oOo..004*/0034//0000IiAaSs/m muadv WEEK 69 MONKEY SERA G rasp Dose Sam ple# pros Cooc. Ceoceoirotfo# of pros C /m L o r* Rec Mean pros ot/m L RSD Sid. Dev. MS/MSD RPD Method m k M anttBIk 04240-H20 Bik-3 04240.H2O Blk4 RBS0426.Sam Blk-3 0 300 0 390 0.930 <LOQ <LOO <LOQ <LOO SA QC UO ppb JIB3 0 0 4 6 -S e n BOt-4 MKSO4340-Sen Btk-3 MKSM240-Sara Blk-4 MX304246-S*r~MS-3 MKS04240-SctvMSD -V MKStM240-Sm-MS-4 MXSO4240-3er-MSD-4 2.73 1 no 310 234 304 324 <LOO 0 00730 0.00934 I22H 92% 1I9H 127% <tO O 000152 107% 123% NA 14 9 0.00144 27% 4% Group 1 Control 0 0 m0i/iy IP3J20M* S324M 10JJJ9F 103S49F 22.2 24.5 323 244 0.042 0.03S3 0 0421 0 0379 0 0404 90100 7.71 0.003I3 7.32 0 00301 G roupJ Mid.Dom 0.1 3 mft/kf/day 10SS05M I05S13M (0J539F 1033S2F 341 J7I 439 40 34.4 9S0 S 3 2*4 239 349 10 0 320 3* 5 3.44 Croup 4 Hifh-Ootc 0 7} m |/kf/day 10331IM ta22M * I05533F I05M 7 341 344 499 323 470 424 111 4 0 32.4 7S.7 700 71.3 730 523 tu n * o f Q um nunon flOQ}: PFOS * 5.33 s* m L Dae Emarod/By: OVOJ/OO, OVOT/OC LACCS1I Da* Venilad/ By. 0M2/0Q TKR D a# p u n y cometed/venfiad 09/123)0 mmh, 09/(2/00 LAC PFOS - Parfloeroocunafulfonai * Sanan sutxal volume teas than OJO mL. concentrations should be eonaidettd lentnsv 04*2SCn i. AC E T S -S V l Excel r 3M Environmental Laboratory lot-OTOmraTTVlC zV f a s s ' 912.00 3m Medical Department Study T - 6 2 9 ^ a c ^ t o x -030 Report N o . FACT TOX-Q30 Covmnce# 6329-223 laboratory Request N u mber-U2279 Study: Product NMmber(Te* Subaanca): Manx Mthod/R*vuioa: Analytical Equipment Syaem Number Inarommi Softm eW iraon: FtltMRM: R-Squared Value: 2d Week Cepeule Tosctfy Study vmh PFOS in Cynomolgua Monkey* T-4295 (PFOS) Monkey Sere ETS-M.I end ETS-0-5 1 Ruby 100499 MamLyiu 31 See Mtachmcnu See Attachment* Slope: Y.lrtcrcep: Dae of EjsmtaoB/Anafy* Dae of Analyse/Aaatya See Attachment* See Aaarhmeau 44//2214**00,oSAcrL//oXoJX.od^a/oo,os/oyooiasmmk*adv Dae of Dae Reduction/Anelya: 04m*O.CS*t.*0.05*1/00. 0SW0Q IAS S n p lt DaU WEEK 73 MONKEY SERA Creep Date Saapte PFOS Ceee. e/aL Ceeceatrattoe r pro* emL or H Rcc Meet PFOS it l RSD Sttf. Dev. MS/MSD RPD Mahod Blk W24O-H20 Blk*3 04240-H30 Blk-4 QJCO 0 390 <LOQ <LOQ <LOQ NA Manx Blk RBS04340*San BUt*3 0.930 <LOQ QC-2ppb RBS042*0*Sen Blk-4 MKS04340*Sere Blk*3 MKS04240-Sere Blk-4 MKS04240-Sere*MS*3 MKS04340*Sere*MSD'3 MXS04240-Sere*MS-4 MKSM240-Scr*-MSD-4 2 7J 164 110 310 234 30 326 LOO 000750 0 00954 123% 93H 11956 12756 <LOO OOOI52 10?% 12356 NA 149 0 00144 2756 456 Creep 1 Cotaml 0.Qmgfcf(dlay I03520M+ I03524M W5S25F 10354F 22.1 17.6 12.4 2*7 9.0431 0.0249 0.0314 0.0345 0 0350 0.0345 321 0.01(5 7 76 0.002S4 Creeps Mid*Doea 0. lSmg/k*/d*y 10550SM 105323M+ 105539F WS5S2F* 143 1 110 131 24.0 17.1 306 r j 444 27.9 11.3 23.7 259 2.91 Creep 4 Hiph-Doee 10351|M I03522M- 405 374 33 1 50.2 731 54 4 37 3 0.75 mp/kf/day I05533F I05542F* 32d 237 345 340 19 1 131 Lund orQ uaaioa (LOO) PFOS - 525 ntfmL Dae Eacred/By: OStiVOO. 03/09/00 LACC5JI Dae Vended/ By 04/02/00 TKR PFOS " Ptrfluoroociaaealfona* Da puny eorrected/venfied' 09/13/00 omh, 09/12/00 LAC - Serum initial volume leae than 0.30 mL. concentraren* ehould be considered tintaive. M/2S/00 I aC na Not ^iphcabie E/dracaoo Volume Rabo bubal Veluma/Fuial Volume BTS-t-yl Excel 97 3M Environmental Laboratory s-03O-Mn223*IC aw 3m Medical Department Study T -6 2 9 t o x -ojo Report N o . FACT TOX-030 Cornice# 6329-223 laboratory Request Number-U22 7 9 StofProduct NiunbcifT* 5ub*0 Mans: Matod/JUvuaon: Analytical Equipment Syuem Number (nearoRMni Sdftwva/Vcrvon: Filman*. R-Squanrd Valsa: Slop Y-lntarcpt Dm of Extraction/Andy Dtfa or Analym/AnaiyK: Dm ofDaaRtducnwAnely*: Ssaspic Date 1* 'Vttk Capmk Toscfty SroOy ni PFOS tn Cynomolpra Monkryi T-629J(PFOS> Monkey Sec* ET3-0-U ETS-l-5 l Ruby 100699 ManLynx 3 3 S** Aflachrnrau 3m Attachmeaie Sa* AttacJoMau Sm Attachment* 04/00 XAI-TCIC 040*00. 04/77-00. 04/2*00. OVOVOO (ASMMH.ADV 04/77/00. OVOI/OO. 0*01/00. 04*04/00 IA3 WEEK 77 MONKEY SERA C rn p Dm* Method BQl Matrix B(k QC-TMppb Croapl Control OOmf/lt^/day Cnap} Mtd-DoM 0 lSm^k|/dsy Croups Hifb-Doua 0.74 m t / t y r t a 04340-H30 Blk-3 M24&-H20 Blfc-4 RBSCM240-Scn Blk*l RS30474O3cra BUM MKS04340*Sara BlkO MKS&42*0-Sera BOM MK30240-3ra-M30 MK3042*0-3ru-MSO-1 MKSOO*0-W*-M3-4 MX300*0-SrvM3D-4 105S2CM*rcssaiM* M5S29F 4549F+ 450544+ S523M+ I04539F+ 0S352F+ I054I1M H59522M 105S13F I0SS42F- PFOS Case 0300 0 390 0930 2.7S 1.64 110 310 236 306 326 12.7 11.9 21.9 132 120 101 101 70.J 424 311 463 60 C tK iatniM of pros t/uLor H Rue <LOQ <LOO LOQ <LOO 0.00730 00093 122% 92% 119% 127% 0.0334 0.0239 0.031? 0.02*4 211 230 273 10 3 319 7.0 70.3 43 4 Mean PFOS rL LOO <LOO 0 00052 UP% 23% 0 0296 0 0303 22 3 230 600 57 0 BSD Sid. D*v. MS.'MSD RPO KA NA 16.9 0 00144 27% 6% 100 0 00333 S44 0 00167 200 0 632 :?? 6.37 630 30.3 33.6 19 1 Umil of Quauaxn (LOQ): PFOS * S 53 a*TnL Daa Entmrt/By OVOVOO, 0V09/00 LACCSII DM* VmAtJ B r 06MQO TKR Dm pumy eonrcud/vtnflrd: 09/12/U0 mmli. OT/iiitt laC PFOS Pcrfluorooctmcvulfan* * Scran utttiatvofofMl*aathm 0.50 mL. conc*ntrjoni ihould be cotunOcred tentative. 04/20/00 LAC Na * Nal appbcabk Extraction Volumt Rmjo - laaiaJ Vohune'Fuial Volume ETS.I-4.1 Excel F* 3M Environmental Laboratory tox-030-a*ra223-lC 2 .2 7 9-1200 3m Medical Department Study T - 6 2 9 5Ack TOX-030 Report N o . FACT TOX-030 Covann# 4329 223 laboratory Request Number-U2279 Study Product HumbertTot Subsana). Manx: MWiod/ltivMisn: AiutytacaLEqmpmsel Sysem Number U Watk. Capeuli Toaeity Study with PFOS u\ Cynomolus Monkeyi T-S(PF03) Monkey S ei ET3-t-4.l and ETS-9-J 1 Ruby 100499 (luasrumani SoAmee/VenBorv Filaums: R'SqwvH Value Slope: Y-JMareept D*a ofEJCncttotVAnaiys: Dae or Anelym^Anatyer ManLyni 3-3 See Aoachmnu Set Attachment* Sea Attachment* See Attachment* 0(M4//224V/00O0.wSArrLto/Kot.t04/21/00.ovoyooiasamh/adv Dau ofDaia Redaction/Anafyil: 04/27/00. 01*1/00. 0VOJ/OO. 01*4/00 IA5 SxatpWDsta WEEK 7 MONKEY SERA Group Doae Method Blk Madia 81k QC-M0pb leapt* 04240'H20 Blk-J MJ40-H2O Blk-4 Rfi304240-Sera Hlk*3 RBS04240Sera BOt-4 MKS643A&>SaraBlkO MKS04240.Sen Blk-4 MK3CM240-3erfr-MS-3 MK3<*2*0-Sari.M5D-J MKS0424P-Sera-MS-4 MXSOOaO-Seru-MSP-4 pro* Coac. ac/al> 0 300 0 390 0930 2.73 *.W 11 0 310 23 304 32 Cenceatrattoa or p ro s l l / u l e r H Rec <LOQ <LOO <LOO <1.00 9.00730 0 00934 122% 92% 119% 127% Mm PFOS <1.00 <1.00 OOOI32 107% 12334 BSD Sid- Dev. MS/MSD RPD NA NA 1 9 000144 27% 6% Croap 1 Control 0.0 mpftp/day Graap) Mid-Doaa 0 IS mg/k^dey MS530M WS2SM W I9F W5S49F OttSOSM IOS$23M 103S39F IC3332F 1 3 17.9 22.3 l 132 III 142 13* 0.0194 0 023 0 03! 0.021* 19.1 1*3 22* 199 00213 0.0243 19 1 :i 4 111 0 0029 14 0.00333 4.22 0*03 9.40 2.01 Gruap4 Kiph-Doae O.TSm^k^dey 1053U-3M I05S22-IM104U3-IF 105542-lF 293 3 242 233 221 63 1 394 41 1 23 9 40 2-19 422 414 M3 Luna of QuamilaOon(LOQ) PFOS * 5 35 apiwL Dale Emered/By 05/03/00, 03/09/00 LACCSH D*c Verified By OdWOO TKR Dar punty carrecud/verifled 09/12/00 mmh. 09/13/00 LAC PFO S - Parfiuoreocimaauirons* Na Net applicable * Serum initial volume leat than 0.3OmL rciKemmiona ehouid be (onnderad irm sw . c*/00 LAC Eancum Volume A* bubal Volume?uial Volume CTS-l-J I Excel >7 3M Environmental Laboratory 1O1-030 amTTMC V f\ 2 .2 .* ' 3m Medical Department Study: T - 6 2 9 5 .7 FACT-TOX-030 Covance# 6329-223 Report N o . laboratory Request FACT T O X -030 Number-U2279 Study; Product Nuraber(Tesi Substance): Matrix: Method/Revision: Analytical Equipment System Number: Instrument Softwaxe/Verswn: Date of Extracuon/Anaiyii: Dale of Analysis/Analyst: Date of Data Reducum/Analysi: Sam ple D ala 26 Week Capsule Toxicity Study with PFOS m Cyrmmoigu* Monkey > T-6295 (PFOS) Monkey Liver Filename See Attachments F'ACT-M-I.O & FACT-M-2.0. 2.1. ETS-8-/R.Squared Value: See Attachment* Amelia 062498. Madeline 011098. DaveyO Slope See Attachments M anLyni 3.2 A 3.3 Y-Intercept Sec Attachments 05/! 8/99. 06/11/99. 05/23/00 SAH/SR P/SHF>SA!. 05/19/99, 05/22/99. 06/09/99. 06/14/99. 07/296*9. 06/14/V9. 05/25/00 KJH/HOJ/MEFVSAH/IAS 05/20W . 05/25/99, 06/10/99. 06/22/99. 08/04/99. 10/126, 50-2MX) KJH/HOJ/MEF>MMH/lAS MONKEY LIVER Week 27 Croup Dom Sample# Method Btk RBL05189-H2O8lk-3 RBL05189-H2O Blk-4 Method Blk RBL0119-H2O BUt-11 RBL06U9-H20 Blk-12 Method Blk 05230-H20 Bli-5 0523O-H2O BUt-6 Matrix Blk RBL05I89-L\t Blk-3 RBL05189-Lvr Blk-4 Matrix Blk R B t0 6 1 1 9 .L v T B U c.ll RBL061 19-Lvt Blk-12 Matrix Blk RBL0523O-LVT Blk-J RBL05230-Lvr Blk-6 Matrix Blk MKL0523O-Lvr Blk-5 MkL0523O-Lvr BUc-6 Q C - 250 ppb I05508M-MS 05/18/99 105508M-MSD QC - 250 ppb I055I7M-MS 06/11/99 105517M-MSD QC 250 ppb MKL05230-MS-5 05/23/00 MKL05230-MSD-5 Group 1 I055O8M 6/11/99 Control IQ5517M 6/11/99 0.0 mg/kg/day Q5508M 5/18/99 IQ5517M 5/18/99 105519M 5/18/99 105527M 5/18/99 I05530F 10553 IF I05535F 105544F Group 2 1Q5514M Low-Dose 105515M 0.03 mg/kg/day 105516M 105S21M 105537F 10554IF I05547F ext. 5/23/00 IQ5547F 105550F Group 3 [055I0M Mid-Dine IQ5518M 0.15 rag/kg/day 105524M 105528M I05532F 105538F 1Q5545F 105548F Group 4 105507M High-Dote 105512M 0.75 rag/kg/day IQ5534F I05536F IQ5540F 10555 IF Limit of Quantitation (LOQ): PFOS 30.0 ng/g NA Not analyzed, nut applicable PPOS Perflunnxxtaneaulfoaatc PPOS Calc. Cone. C oucM tnU lao rrvos Mean PFOS RSD SUL Dev. 1/1 -- f t e r % Rac. 0.00 <LOQ UR/* MS/MSD RPD 0.454 <LOQ <l o q NA 0.00 <LOQ NA NA <1.00 NA 0.00 <LOQ 1.64 <LOQ <LOQ NA 0.00 <LOQ 3.43 <LOQ 0.00 <LOQ <1.00 NA NA NA 1.25 <LOQ <LOO NA 0.391 15.2 <LOQ <LOQ <1.00 NA 9.7 <L0Q <LOO NA 482 1745k * 305 U 0* 142*. 45% 300 101 305 103% 102% 2% 332 111% 292 98% 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 o . t u 87 0.087 168 97 0.097 0.106 0 .0 P 8 18237 18 2 22709 22.7 11417 16734 11.4 16.7 27 0 17.3 4.66 22818 22.8 24847 248 282623 283 20102 20735 20.1 20.7 9 73 22.1 2.15 42169 42.2 R6I73 86 58673 58.7 33 i 48203 48.2 58.8 19 5 80421 80.4 49590 49.6 66532 81376 66.5 81 4 21 4 69.5 149 412474 412 6.03 378723 379 396 23 9 28Q575 281 256669 257 267328 267 5.00 287223 287 273 13 6 Date EctcreVBy. 05/23/99.06/KV99. 06/22/99. 1 0 1 2 6 l^ C Date Verified/ By:06/02/00 PJW. 06/05/00 TKR Date Punty correctedVcnfkd/ By09/11/00 mmh, 09/12/001.AC --* Sample determined an outlier and waa not included in any caJculaixm*. " Sample used for MS/MSDappear! to be spiked accounting for low reoneriev Sample 105507 it 18 will be rextmcied. Simple 105507M will be uaed for the MS/MSD and 105518M c>mic. VeriFted. R * Sample reexiracted along with MS/MSD. This value *a> nut uted in any calculation * Sample determined an mitlicT and waa not included in anv calculation*. Was rccitmctcd 0503/00 FACT-M-2.1 Excel 97 3M Environmental Laboratory tox*03<Miver223-l B.xls 9/12/00 3m Medical Department Study: T-6295.7 CJ 2 M d < O d (3B d ffdt ffu cSodrOdit)r dj K) Vo FACT-TOX-030 Covance# 6329-223 S tu d y: Pruduct N um ber(Tesl Substance): M atrix: M elhod/Re vision: Analytical E quipm ent System Number: Instrum ent Softw are/V ersion: Date o f Extraction/A nalyst: Date o f Analysis/A nalyst: Dale o f D ata R cduction/A nalyst: Sample Data 26 W eek Capsule T o x ic ity S tudy w ith PFOS in C ynom olgus M o nkeys T-6295 (PFOS) M onkey Liver Filenam e: ETS-8-6.0 A ETS-8-7.0 R -Squared Value: Soup 020199. Am elia 062490 Slope: M assLynx 3.2 A 3.3 Y -Intercept: 10/14/99,10/25/99 SEE/RW W 10 /1 5 /9 9 .1 0 /2 0 /9 9 , 10/22/99, 10 /2 6 /9 9 , 10/2 7 /9 9 M M H /IA S 10/18/99.10/21/99, 10/25/99.10/27/99. 10/29/99 IAS/M M H Sec Attachm ents See A ttachm ents See A ttachm ents See A ttachm ents M ONKEY LIV E R Day 393 Biopsy G roup Dos* Sam ple # M ethod Blk 10/14/99 R BLI0I49-H 2O B lk-ll R B L 10 1 4 9 - H 2 0 B lk -12 M ethod Blk 10/25/99 R B L I0259-H 2O Blk-1* RBL102S9-H 2O Blk-2 M atrix Blank 10/14/99 RBLI0M 9-Lvr B lk-ll R B L I0 I4 9 -L v r Blk-12 M atrix Blank R B L 1 0 2 5 9 -L vt Blk-1 10/25/99 R BL10259-Lvr Blk-2 M K L 10259-L vr Blk-1 M K L 10259-L vr Blk-2 Q C-250 ppm 10/25/99 25 ppm A 500 pph I05542F-M S-1 105542F-M SD -1 M KLI0259-M S-2 M KL10259-M SD-2 G roup 4 10551IM H igh-D ose 0.75 m g/kg/day I05522M I05533F J05542F L im it o f Q uantitation (L O Q ): PFOS = 26.9 ng/g N A = N ot a na lyred . not applicable PFOS = Pcrfluorouctanesuliunaie PFOS Calc. C one. ngfe NA 3.58 8.48 NA NA 0.00 0.00 NA 2.05 NA C oncentration of PFOS ur/r o r % Rec. NA <L00 <LOQ NA NA <LOQ <LOQ NA <LOQ NA M ean PFOS <LOQ <LOQ <l o q <LOQ <LOQ RSD Std. Dev. M S/M SD R PD NA NA NA NA NA 198121 145974 521 498 79% 58% 87% 83% 68% 8 5 '4 30%. 5% 142465 137561 75283 421647 142 138 175 422 2.48 140 3.47 58.4 298 174 Dale Entered/By: 1 0/22/99.10/25/99. 10/29/99 L A C . 11/05/99 G M L Date V e rifie d/ B y: 06/05/00 T K R D a le P u rity corTectedVerified/ B y :0 9 /1 2/00 m m h . 0 9 /1 2/IX) L A C M S /M S D sam ples extracted on 10/14/99 were not w ith in recovery criteria, and samples were teextracled on 10/25/99 at a higher co ncentration. The earlier M S /M S D sam ples w ere n o t spiked at a high enough level in relation to the endogenous PFOS levels. R eextracted M S /M S D s w ere w ith in c rite ria . L A C 12 /1 4 /9 9 Report No. FACT TOX-030 laboratory Request Number-U2279 To calculate the extracted density: (in itia l liv e r w e ig h t/(in ilia l liver w eig ht + M illi Q W ater added) T o calculate the PFOS C alc. Cone.: (PFO S cone, ng/g X density o f curve live r homogenate g /m L)/extracted density g /m t.) PFOS correction factor * d ilu tio n factor bWJ\\ ETS-X-7.0 Excel 97 tnx-03U -liver223-IB .xls 9/12/00 a no p m 3m Medical Department Study: T-6295.7 U) 2 m <H3MOb n3> b rt U> fPJ tr o Msu rt 0 b C Vo FACT-TOX-030 S tu d y : Product N um ber(T est Substance): M atrix: M ethod/R evision: A nalytical E quipm ent System N um ber: Instrum ent Softw are/V ersion: D ate o f Extraction/A nalyst: D ate o f A nalysis/A nalyst: D ate o f D ata R eduction/A nalyst: Sample Data 26 W eek C ap su le T oxicity S tu d y w ith PFO S in C yn o m o lg u s M onkeys T-6295 (PFO S) M onkey L iver E T S -8-6.0 & E T S -8-7.0 D avcy 070799 M assL y n x 3.2 & 3.3 3/22/00 SAL 0 3 /2 4 /0 0 ,0 3 /2 7 /0 0 ,0 3 /2 8 /0 0 ,5 /5 /0 0 M M H , IA S 03/27/00, 0 3 /2 8 /0 0 ,0 3 /2 9 /0 0 ,5 /8 /0 0 M M H , IA S M onkey Liver W ks 79 & 80 G roup D ose S am ple H M elhod Blk M atrix Blk R B L 03220-H 20-B lk-5 R B L 03220-H 20-B lk-6 R B L 03220-liverB lk-5 R B L 03220-livcrB lk-6 M K L 03220-livcrB lk-5 QC 250 ppb M K L 03220-liverB lk-6 M K L03220-M S-250ppb-5-l M K L03220-M SD -250ppb-5-2 M K L 03220-M S-250ppb-6-1 M K L03220-M SD -250ppb-6-2 l05505-M S-50ppm -5-l l05505-M SD -50ppm -5-2 G roup 4 H igh-D ose 0.75 m g/kg/day 10551 |/M /W k 7 9 I05522/M /W k79 105533/F/W k79 105542/F/W k79 G roup 3 M id-D ose 0.15 m g/kg/day 10S505/M /W k80 I05523/M /W k80 I05539/F/W k80 105552/F/W k80 L im it o f Q u antitation L im it (L O Q ): PFO S = 26.9 ng/g PFOS C alc. C onc. n g /g 0.00 0.00 0.00 0.00 0.00 0 .0 0 216 186 266 232 54408 47351 23480 70781 42668 57895 8252 10203 24728 17690 C o n cen tratio n of PFOS u g /g o r */ R e e <L O Q (30.6 ng/g) < L O Q (30.6 ng/g) <L O Q (30.6 ng/g) <L O Q (30.6 ng/g) < L O Q (30.6 ng/g) < L O Q (30.6 ng/g) 72% 62% 89% 77% 95% 81% 23.5 70.8 4 2 .7 57.9 8.25 10.2 24.7 17.7 M ean PFOS u g /g <LOQ <LOQ <LOQ 67% 83% 88% 47.1 50.3 9.23 21.2 RSD S td. D ev. M S/M SD R PD 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 PFOS = Perfluorooctanesulfonate NA = N ot applicable D ate E ntered/A nalyst: D ate V erified/A nalysl: D ate P urity corrected V erified/ By: 0 4 /0 5 /0 0 , 0 4 /0 6 /0 0 , 5 / 10 /0 0 M M H , C S H 0 6 /0 5 /0 0 TK.R 09/12/00 m m h, 09/12/00 LA C Report No. FACT TOX-C30 laboratory Request Number-U2279 to ' ETS-8-7.0 Excel 97 T O X -030-liver223-lB .xls 9/12/00 4:16 PM 3m Medical Department Study : T-6295.7 3M Medical Department Study: T-6295.7 A ttachment F Ex a m p l e Ca lc u la t io n s Report No. FACT TOX-030 laboratory Request Number-U2279 Report No. FACT TOX-030 Laboratory Request Number-U2279 Formula Used for Sera Analyses In Study FACT TOX-030 AR (ng/mL) * DF * SC * JFV (mL) * 1.0 pg = pg/mL x PC = Reported Cone (pg/mL) EV (mL) 1000 ng Calculation Used fo r Group 3, Week 1, Animal ID 105505M 287 ng/mL x 10 * 0.9275 * 1mL * 1.0 jig = 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 <5curve ( 1) x SC x DF x 1.0 pg = pg/g x PC = [PFOS] sample (jig/g) d sample 1000ng m d curve is assumed to be: 1g liver 5 mL H2O Calculation Used for Group 3, Week 27, Animal ID 105510M 524 ng/g x l g / 5 m L * 0.9275 * 100 * 1.0 jig = 48.8 jig/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 lg 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%) 3M Environmental Laboratory 3M Environmental Laboratory Page F-1 P 225 3m M e d ic a l D e p a r tm e n t S tu d y : T - 6 2 9 5 . 7 3M Medical Department Study: T-6295.7 Attachment G Intrim C ertificate of A nalyses R e p o rt N o. FACT TOX-030 la b o r a to r y R e q u e st N um ber-U 2279 Report No. FACT TOX-030 Laboratory Request Number-112279 3M Environmental Laboratory 3M Environmental Laboratory Zi3> Page G-1 Pa' 6 3ra 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 #: 023-018A 3M Product: PFOS, Lot 217 Reference#: SD-018 ____________________ Purity: 86.9%____________ Test Name Specifications Purity1 Result 86.9% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium1 5. Nickel 6 . Iron 7. Manganese Total %Impurity (NMR) Total %Impurity (LC/MS) Total %Impurity (GC/MS) Related Compounds POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6 . Phosphate 7. Sulfate4 Organic Acids5 (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis4: 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine White Crystalline Powder 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Conforms Positive 1. 0.005 wtVwt.% 2 . 0.001 wt./wt.% 3. 1.439 wt./wt.% 4. 6.849 wt./wt.% 5. <0.001 wt./wt.% 6 . 0.005 wt./wt.% 7. <0.001 wt/wt.% 1.93 wt./wt.% 8.41 wt./wt.% None Detected ' 0.33 wt./wt.% None Detected Not Applicable4 1. <0.015 wt/wt.% 2. 0.59 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6 . <0.007 wt./wt.% 7. 8.76 wtVwt.% 1. <0.1 wt7wt.% 2 . <0.1 wt./wt.% 3. 0.10 wt/wt.% 4. 0.28 wt7wt.% 1. 12.48 wt./wt.% 2. 0.244 wt./wt.% 3. 1.74 wt./wt.% 4. 8.84 wt./wt.% 5. 54.1 wt./wt.% COA023-018A 3M Environmental Laboratory Exact Copy of Original IA1-- cTiliri.x. Initial Data zas Y Page 1 of 3 27 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 Date of Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum o f metal impurities, 1.45% +LC/MS impurities, 8.41%+Inorganic Fluoride, 0.59%+NMR impurities, 1.93%+organic acid impurities, 0.38%+POAA, 0.33%) Total impurity from all tests = 13.09% Purity = 100% - 13.09% = 86.9% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO* and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity. 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CgFi7S0 3 "K+(MW=5 3 8 ) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018A 3M Environmental Laboratory Page 2 o f 3 Pai 2 2 8 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: Impnrity C4 C5 C6 C7 Total w t/w t % 1.22 1.33 4.72 1.14 8.41 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: ______________________________ ____ David S. Bell Date Scientist, Centre Analytical Laboratories Reviewed By: ______________________________ ____ John Flaherty Date Laboratory Manager, Centre Analytical Laboratories COA023-018A 3M Environmental Laboratory Page 3 o f 3 Pa' 29 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 Reference#: SD-009 _____________________Purity: 86.4%____________________ Test Name Specifications Purity1 Result 86.4% Appearance Identification NMR Metals (ICP/MS) 1. Calcium 2. Magnesium 3. Sodium 4. Potassium2 5. Nickel 6 . Iron 7. Manganese Total %Impurity (NMR) Total %Impurity (LC/MS) Total %Impurity (GC/MS) Related Compounds - POAA Residual Solvents (TGA) Purity by DSC Inorganic Anions (IC) 1. Chloride 2. Fluoride 3. Bromide 4. Nitrate 5. Nitrite 6 . Phosphate 7. Sulfate4 Organic Acids 1 (IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA Elemental Analysis": 1. Carbon 2. Hydrogen 3. Nitrogen 4. Sulfur 5. Fluorine | White Crystalline Powder 1. Theoretical Value = 17.8% 2. Theoretical Value = 0% 3. Theoretical Value = 0% 4. Theoretical Value = 5.95% 5. Theoretical Value = 60% Conforms Positive 1. 0.017wt7wt.% 2. 0.007 wtVwt.% 3. 1.355 wtVwt.% 4. 6.552 wt./wt.% 5. 0.003 wt./wt.% 6 . 0.004 wt./wt.% 7. <0.001 wt./wt.% 1.00 wt./wt.% 10.60 wt./wt.% None Detected 0.30 wtVwt.% None Detected Not ApplicableJ 1. <0.015 wt./wt.% 2. 0.27 wt./wt.% 3. <0.040 wt./wt.% 4. <0.009 wt./wt.% 5. <0.006 wt./wt.% 6 . <0.007 wt./wt.% 7. 8.82 wt./wt.% 1. <0.1 wt./wt.% 2 . <0.1 wt./wt.% 3. <0.1 wt./wt.% 4. <0.25 wt./wt.% 1. 12.08 wt./wt.% 2. 0.794 wt./wt% 3. 1.61 wt./wt.% 4. 10.1 wt./wt.% 5. 50.4 wt./wt.% COA023-018B 3M Environmental Laboratory Exact Copy of Original - lL- eft l u L -, Initial " "Data Page 1 o f3 Pa' 30 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 #: 023-018B Date o f Last Analysis: 08/31/00 Expiration Date: 08/31/01 Storage Conditions: Frozen <-10C Re-assessment Date: 08/31/01 'Purity = 100% - (sum o f metal impurities, 1.39% +LC/MS impurities, 10.60%+Inorganic Fluoride, 0.27%+NMR impurities, 1.00%+ POAA, 0.30%) Total impurity from all tests = 13.56% Purity = 100% -13.56% = 86.4% 2Potassium is expected in this salt form and is therefore not considered an impurity. 3Purity by DSC is generally not applicable to materials o f low purity. No endotherm was observed for this sample. 4Sulfur in the sample appears to be converted to SO* and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO* is not considered an impurity. 5TFA HFBA NFPA PFPA Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid 6Theoretical value calculations based on the empirical formula, CgFnSOslC4 (MW=538) This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160). COA023-018B 3M Environmental Laboratory Z& ZZSr Page 2 of 3 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 #: 023-018B LC/MS Purity Profile: Impurity C4 C5 C6 C7 Total wL/wt % 1.03 1.56 6.38 1.63 10.60 Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves. Prepared By: ______________________________ ____ David S. Bell Date Scientist, Centre Analytical Laboratories Reviewed B y :______________________________ ____ John Flaherty Date Laboratory Manager, Centre Analytical Laboratories COA023-018B 3M Environmental Laboratory 30 3; Page 3 o f 3 232