Document 8N1kV6grrBDOYya8mgpY5jYm
3MGeneral Offices
3M Center
St. Paul, MN 55144-1000 612 733 1110
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September 5, 2001
Document Processing Center (7407) Office of Toxic Substances U.S. Environmental Protection Agency 401 M Street, SW Washington, DC 20460 Attn: TSCA Section 8(e) Coordinator
3?3
Dear Section 8(e) Docket Coordinator:
Re: TSCA 8(e) Supplemental Notice on Sulfonate-based Fluorochemicals
With this letter, 3M is providing final reports and other supplemental information related to previous TSCA Section 8(e) notifications. Many of the enclosed items are analytical reports providing blood serum and liver levels o f test materials for which the in-life report referring to administered doses has already been submitted to the 8(e) docket. In other cases where the 8(e) notification consisted of preliminary data, we are submitting a final study report.
All o f the enclosed items are already in EPA's possession and available in TSCA Docket AR-226. We believe, however, that placing these items in the 8(e) docket may allow for more convenient access to information directly related to previous 8(e) notifications by 3M.
The table below lists the enclosed items and references the study or data which already has been the subject o f an 8(e) notification by 3M:
Attached Submission
Related Study/Data Already Filed Under 8(e)
1. Amended Analytical Study, 2(N-Ethylperfluorooctane
Combined Oral (Gavage) Fertility,
sulfonamido)-ethanol in Two Generation Rat Reproduction, Developmental and Perinatal/Postnatal
Determination of the Presence and Concentration of PFOS, Reproduction Toxicity Study of N-
M556, PFOSAA, and PFOSA in the Liver and PFOS,
EtFOSE in Rats, 3M Reference No. T-
M556, PFOSAA, PFOSA and EtFOSE-OH in the Sera of 6316.5, June 30, 1999, full report
Crl:CDBR VAF/Plus Rats Exposed to EtFOSE-OH, 3M submitted February 15, 2000 to ~
Reference No. T-6316.5, Analytical Report TOX-013,
supplement^arlier filing
rg
LRN-U2095, June 11, 2001.
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TSGA Section 8(e) Docket Coordinator Page 2
Attached Submission
Related Study/Data Already Filed Under 8(e)
2. Analytical Laboratory Report, Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate (CAS Number: 2759-39-3) in the Serum and Liver of Sprague-Dawley Rats Exposed to PFOS via Gavage, Laboratory Report No. U2006, Requestor Project No. 3M TOX 6295.9, October 27, 1999.
3. Report Amendment 1, Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rats, Argus Research Laboratories, Inc., Protocol 418-008, Sponsor's Study No. 6295.9, April 13, 2000.
Combined Oral (Gavage) Fertility, Developmental and Perinatal/Postnatal Reproduction Toxicity Study of PFOS in Rats, Argus Research Laboratories,
Inc., Sponsor's Study No. 6295.9, June 10, 1999, full report submitted February 15, 2000 supplementing earlier filing
4. Analytical Report, Determination of the Presence and Concentration of Perfluorooctanesulfonate, Perfluorooctanesulfonylamide, M556, and M570 in the Liver and Sera Samples, 3M Environmental Laboratory Ref. No. U2636, TOX-028, February 23, 2001
13-Week Dietary Study ofN-Methyl Perfluorooctanesulfonamido Ethanol (N-MeFOSE) in Rats, 3M Ref. No. T6314.1, Covance Study No. 6329-225, dated June 30, 2000, Section 8(e) filing July 24, 2000
5. Analytical Laboratory Report, Determination of the Concentration of PFOS, PFOSA, PFOSAA, and EtFOSEOH in the Sera and Liver of CrhCDBR VAF/Plus Rats Exposed to N-EtFOSE, 3M Environmental Laboratory Report No. TOX-098, Laboratory Request No. U2402, 3M Ref. No. T-6316.7, February 6, 2001.
Final Report, Oral (Gavage) Developmental Toxicity Study of 2(NEthylperfluorooctanesulfonamido)ethanol in Rats, 3M Reference No. T6316.7, December 17, 1998, submitted to Section 8(e) docket per letter of August 21, 2000
6. Analytical Laboratory Report on the Determination of the Presence and Concentration of Potassium Perfluorooctanesulfonate (PFOS) or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol (NEtFOSE) in Liver and Serum Specimens, 3M Environmental Laboratory Report No. TOX-097, Laboratory Request No. U2452, 3M Ref. No. T-6316.8, February 8, 2001
Final Report, Oral (Stomach Tube) Developmental Toxicity Study of NEtFOSE in Rabbits, 3M Reference No. T-6316.8, January 11, 1999, submitted to Section 8(e) docket per letter of August 21, 2000
7. Final Report, Alexander, B., Mortality Studies of Workers Employed at the 3M Decatur Facility, University of Minnesota, April 26, 2001.
Preliminary data submitted to Section 8(e) docket in letter of December 15, 2000
, TSA Section 8(e) Docket Coordinator Page 3
Attached Submission
Related Study/Data Already Filed Under 8(e)
8. Final Report, Acute Oral Toxicity Screen with T-3290CoC in Albino Rats, Safety Evaluation Laboratory, Riker Laboratories, Inc., Project No. 0882AR0362, 3M Reference No. T-3290 (40 % K+PFOSAA in 3 % EtOH, 17 % IPA and 40 % H20, L-6778, F-6873, Lot 501), November 5, 1982 [Bibliography entry in Docket AR-226, final report was to be moved to TSCA 8(e) docket]
Acute Oral Toxicity Screen with T3290CoC in Albino Rats, Safety Evaluation Laboratory, Riker Laboratories, Inc., Project No. 0882AR0362, 3M Reference No. T3290 (40 % K+PFOSAA in 3 % EtOH, 17 % IPA and 40 % H20, L-6778, F6873, Lot 501), November 5, 1982, submitted to Section 8(e) docket in August 21,2000 self-audit letter (which erroneously refers to rabbits rather than rats)
9. Giesy, J.P., and K. Kannan, Accumulation of Perfluorooctanesulfonate and Related Fluorochemicals in Fish Tissue, Michigan State University, June 20, 2001.
Preliminary data submitted to Section 8(e) docket May 26, 1999
10. Giesy, J.P., and K. Kannan, Accumulation of Perfluorooctanesulfonate and Related Fluorochemicals in Mink and River Otters, Michigan State University, June 20, 2001.
11. Giesy, J.P., and K. Kannan, Perfluorooctanesulfonate and Related Fluorochemicals in Oyster, Crassostrea Virginica, From the Gulf of Mexico and Chesapeake Bay, Michigan State University, June 20, 2001.
12. Giesy, J.P. and K. Kannan, Perfluorooctanesulfonate and Related Fluorochemicals in Fish-Eating Water Birds, Michigan State University, June 20, 2001.
13. Giesy, J.P. and K. Kannan, Accumulation of Perfluorooctanesulfonate and Related Fluorochemicals in Marine Mammals, Michigan State University, June 20, 2001.
If you have any questions about this submission, please contact me at (651)737-4795.
Enclosures
Manager, 3M Corporate Product Responsibility
3M Medical Department Study: T-6316.5
3M M edical D epartm ent Study: T -6 3 1 6 .5
Analytical Study: FACT-TOX-013 LRN-U2095
A nalytical Report: F A C T T O X -0 1 3 LR N -U 2095
Study Title
Analytical Study 2(N-Ethylperfluorooctane sulfonamido)-ethanol In Two Generation Rat Reproduction
Amended Analytical Laboratory Report
Determination of the Presence and Concentration of PFOS, M556, PFOSAA, and PFOSA in the Liver and PFOS, M556, PFOSAA, PFOSA, and EtFOSE-OH in the Sera of Crl:CDBR VAF/Plus Rats Exposed to EtFOSE-OH
Data Requirement
Not Applicable
Author
3M Environmental Laboratory
Ml?
Study Completion Date May 31,2001
Performing Laboratories
Sera Analyses
Liver Analyses
3M Environmental Laboratory Building 2-3E -09, 935 Bush Avenue
St. Paul, M N 55106
Battelle M em orial Institute 505 King Avenue
Columbus, O H 43201-2693
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Project Identification
3M Medical Department Study: T-6316.5 Argus In-Life Study: 418-009
Analytical Report: FACT TOX-013 3M Laboratory Request No. U2095
Total Number o f Pages
143
& e P A - CIT S
000811825P
QQGB11B25P
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Analytical Report: FACT TOX-013 LRN-U2095
BfflW This page has been reserved for specific country requirements.
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3M Medical Department Study: T-6316.5
GLP Compliance Statement
Analytical Study: FACT TOX-013 LRN-U2095
Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, M556, PFOSAA, and PFOSA in the Liver and PFOS, M556, PFOSAA, PFOSA, and EtFOSE-OH in the Sera of Crl:CDBR VAF/Plus Rats Exposed to EtFOSE-OH
Study Identification Number: T-6316.5, FACT TOX-013, LRN-U2095
This study was conducted in compliance with United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations 21 CFR Part 58, with the exceptions in the bulleted list below. All raw data, protocol, analytical report and samples for this study are retained in archives at the 3M Environmental Laboratory and will be retained for a period of at least ten years. The analytical phase completed at the 3M Environmental Laboratory was performed in accordance with 3M ET&SS Standard Operating Procedures.
Exceptions to GLP compliance:
There were two study directors in this study. This study was designed as two separate studies. The in-life phase was considered to end at the generation and shipment of specimens. The analytical study was considered to start at the receipt of these specimens for analysis. This resulted in having two separate study directors, one for each phase of the same study. However, since the technical performance of each phase was entirely separate, no effect is expected from this exception.
Some changes made in the standard preparation logs obscured the original entry, did not document the reason for the change and/or were not initialed and dated by the person making the change.
The samples that were analyzed on 3/16/00 utilized standards that had an expiration date of 2/00.
Liver values generated at contract laboratories were corrected by 3M Environmental Laboratory to reflect the official purity values from the COA. Revised final reports will be solicited from the contract laboratory and will be added as a report amendment at a later date.
Expiration dates on some reagents and solutions were missing.
The analytical report from Battelle is not signed or dated by the Principal Analytical Investigator or laboratory management.
The Quality Assurance Statement in the Battelle analytical report does not include the dates of the QA inspection activities or the dates reported to the Study Director and laboratory management. The Quality Assurance Statement is not signed.
The Argus and Battelle analytical reports do not include the names of all the contributing
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3M Medical Department Study: T-6316.5
Analytical Study: FACT TOX-013 LRN-U2095
GLP Study-- Quality Assurance Statement
Analytical Laboratory Report Title: Determination of the Presence and Concentration of PFOS, M556, PFOSAA, and PFOSA in the Liver and PFOS, M556, PFOSAA, PFOSA, and EtFOSE-OH in the Sera of Crl:CDBR VAF/Plus Rats Exposed to EtFOSE-OH
Study Identification Number: T-6316.5, FACT TOX-013, LRN-U2095
This study has been inspected by the 3M Environmental Laboratory Quality Assurance Unit (QAU) as indicated in the following table. The findings were reported to the study director and laboratory management.
Inspection Dates
Phase
Date Reported to Management Study Director
10/12/99
Extraction
10/26/99
10/26/99
6/5/00-6/14/00
Data
6/16/00
6/16/00
9/11/00-9/13/00
Draft report
9/14/00
9/14/00
5/14/01
Amended report
5/14/01
5/14/01
QAU Representative
3 O'-n- t l , 3 o Q i Date
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Table of Contents
Analytical Study: FACT TOX-013 LRN-U2095
GLP Compliance Statement............................................................................................... 3
GLP Study--Quality Assurance Statement....................................................................... 4
Study Personnel and Contributors......................................................................................7
Introduction and Purpose................................................................................................... 8 Test System.................................................................................................................... 8 Specimen Collection and Analysis.................................................................................9
Specimen Receipt and Maintenance..................................................................................9
Chemical Characterization.................................................................................................... 10 Dose Confirmation Analyses............................................................................................10
Method Summaries............................................................................................................... 11 3M Environmental Laboratory..........................................................................................11 Preparatory Method..................................................................................................... 11 Analytical Method........................................................................................................ 11 Analytical Equipment................................................................................................... 11 Deviations......................................................................................................................... 12
Data Quality Objectives and Data Integrity.......................................................................... 12
Data Summary, Analyses, and Results............................................................................... 13 Summary of Quality Control Analyses Results................................................................13 Summary of Sample Results............................................................................................14
Statistical Methods and Calculations....................................................................................14
Statement of Conclusion...................................................................................................... 14
Appendix A: Chemical Characterization, Control Matrices and Dose Confirmation Analyses................................................................................................................................ 15
Appendix B: Protocol............................................................................................................ 18
Appendix C: Extraction and Analytical Methods..................................................................37
ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages).............................................................................................................................38
ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemlcals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages).................... 52
Appendix D: Data Summary Tables.................................................................................... 61
Appendix E: Data Spreadsheets......................................................................................... 64
Appendix F: Example Calculations......................................................................................70
Appendix G: Contract Lab Report..................................................................................... 71 .
Appendix H: Interim Certificate of Analysis........................................................................ 13 7
Appendix I: Report Signature Page....................................................................................141
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Analytical Study: FACT TO X-013
LRN-U2095
Appendix J: Amendment 1 to FACT TOX-013 Final Report
142
List of Tables
Table 1. Test System Population Demographics and Dosage Levels for Study (418-009)....................................................................................................... 8
Table 2. Characterization of the Test Article in Study FACT TOX-013 ...............................10 Table 3. Negative Ions Monitored in 3M Laboratory Analyses........................................... 12 Table 4. Deviation Summary for FACT TOX-013...............................................................12
Table 5. Determinations of the LOQ in the Analyses of Serum Extracts............................13 Table 6. Characterization of the Control Matrices Used for Sera Analyses in
Study FACT TOX-013 .............................................................................................15
Table 7. Characterization of the Control Matrices Used for Liver Analyses in Study FACT TOX-013 ............................................................................................. 15
Table 8. Characterization of the Analytical Reference Materials Used for Sera Analyses in Study FACT TOX-013.........................................................................................16
Table 9. Characterization of the Analytical Reference Materials Used for Liver Analyses in Study FACT TOX-013.........................................................................................16
Table 10. Tween Dosing Confirmation for Study In-life #418-009...................................... 17
Table 11. Tween Dosing Confirmation-- Matrix Spikes for Study In-life #418-009............17
Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013... 61 Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013
(continued).............................................................................................................62 Table 13. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-013...62 Table 13. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-013
(continued)............................................................................................................ 63
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Analytical Report: PACT TOX-013 LRN-U2095
Study Personnel and Contributors
Study Director
Marvin T. Case, D.V.M., Ph.D, Study Director - 3M Corporate Toxicology - Medical Department
3M Center, Building 220-2E-02 St. Paul, MN, 55144-1000 651-733-5180
r - Sponsor
John L. Butenhoff, Ph.D., Sponsor Representative 3M Corporate Toxicology - Medical Department 3M Center, Building 220-2E-02 St. Paul, MN 55144-1000
_ Analytical Chemistry Laboratories
Sera Analyses 3M Environmental Laboratory (3M Lab) _ Kristen J. Hansen Ph.D., Analytical Investigator
Liver Analyses Battelle Memorial Institute Jon C. Andre, Ph.D., Analytical Investigator
3M Lab Contributing Personnel
David R. Bamidge. Ph.D. Lisa A. Clemen Lisa Dick, Ph.D. Kelly J. Dorweiler Mark E. Ellefson Sara E. Estes Barb A. Gramenz Sarah A. Heimdal Cari S. Hewitt Marlene M. Heying
Harold O. Johnson Kelly J. Kuehlwein Sally A. Linda Joseph C. Pilon Scott R. Post Ian A. Smith Kathy M. Stock Anh-Dao Vo Bob W . Wynne
Location of Archives
All original raw data, protocol, and analytical report have been archived at the 3M Environmental Laboratory. The test substance and analytical reference standard reserve samples, as well as the specimens pertaining to the analytical phase of this study, are archived at the 3M Environmental Laboratory. Control sera and liver will be maintained at the contract lab along with the test substance.
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Analytical Report: FACT TOX-013 LRN-U2095
Introduction and Purpose
The purpose of the study is to determine the presence and concentration of PFOS, PFOSA, PFOSAA, and M556 in liver samples and PFOS, PFOSA, PFOSAA, EtFOS&OH, and M556 in sera samples collected from rats exposed to EtFOSE-OH. This study was initiated on 1 October 1998.
T o st System
Five groups of FO generation male and female rats and 3 groups of F1 generation male and female rats were used as the test system. Table 1 outlines the rat population demographics and dosage levels for study 418-009.
On day 4 of lactation, litters were culled to four male and four female pups, where possible. On day 21 of lactation, 25 male and 25 female pups in Groups I, II, and III were selected for continued evaluation. F1 generation male and female rats were given appropriate dosages of the test article via gavage beginning on day 22 of lactation or postpartum through the day before sacrifice.
The test system species and strain selected was the CitCCPBR VAF/Plus* (Sprague-Dawley) rat received from Charles River Laboratories, Inc., and assigned temporary numbers until assigned to the study. Rats were permanently identified using MoneF self-piercing ear tags when assigned to the study. FO generation rats were identified with ear tags. Pups were not identified during lactation, as parameters were evaluated in terms of the litter. At weaning, each F1 generation rat selected for continued observation was identified with a Monel*seif-piercing ear tag. FO female rats were approximately 65 days of age and weighed approximately 1 7 9 -2 2 9 g when received. FO male rats were approximately 5 8 -6 7 days of age and weighed approximately 223-331 g when received. Weight data are included in Argus Research Laboratories, Inc. final report (study number 418-009).
Table 1. Test System Population Demographics and Dosage Levels for Study (418-009)
P op u latio n
Num ber of F0 G en eratio n R ats
per Sex
N um ber o f F1 G en eratio n R ats
per Sex
Dosage (mg/kg/day)
Dosage Group I (Control) Dosage Group II Dosage Group III
Dosage Group IV
Dosage Group V
35
35 35 35 35
25 0 (vehicle)
25 1 25 5 -- 10
_ 15
pm *
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Analytical Report: PACT TOX-013 LRN-U2095
Specim en C ollection and A nalysis
Sample specimens were collected by Argus (study 418-009) and sent to the 3M Environmental Laboratory for analysis. Liver and sera specimens were collected from F0 male rats at the completion of the cohabitation period and F0 female rats on day 21 postpartum. Liver specimens were collected from F0 generation litters, and stomach content specimens were collected from the F0 and F2 generation litters. The analysis of the stomach contents were not part of the scope of analysis determined by the study director. The number and type of specimens collected for analyses in the analytical phase of this study are presented below.
Specimens Collected from Study Groups I through V (through 11/30/98): Serum Specim ens-- 45 specimens Liver Specimens-- 65 specimens
Blood specimens were centrifuged after collection. Serum was then harvested and immediately frozen on dry ice and maintained frozen at -70C until shipped to the 3M Environmental Laboratory. Liver specimens collected from each animal were frozen and retained at -70C until shipped to the 3M Environmental Laboratory. Stomach content specimens were frozen at -20C until shipped to the 3M Environmental Laboratory. Liver, sera, and stomach content specimens were shipped to the 3M Environmental Laboratory frozen and on dry ice.
Sera and liver samples were extracted beginning on October 1 1 ,1 9 9 9 using an ion pairing reagent and methyl-ferf-butyl ether (MtBE) for the sera and ethyl acetate for the liver samples. Liver samples were homogenized prior to the extraction procedure. Sample extracts were analyzed using high-performance liquid chromatography-etectrospray/tandem mass spectrometry (HPLC-ESM SM S) in the multiple response monitoring mode. PFOS, PFOSA, PFOSAA, EtFOSE-OH, and M556 levels were quantitated by external calibration. PFOSEA was not analyzed due to inconsistent analysis and failed QC. Analytical details are included in this report.
Specimen Receipt and Maintenance
The 3M Environmental Laboratory received from Argus, serum, liver and stomach content specimens collected at predetermined time points during and at the end of thein-life phase of Argus study 418-009 on 8 -4 -9 8,1 0-1-98 and 1-29-99. All specimens were received frozen on dry ice and were immediately transferred to storage at -20C 10C. Specimens that were analyzed at Battelle were shipped frozen on dry ice.
Control matrices used in liver and sera analyses were obtained from commercial sources and are presented in Table 6 and 7. Samples analyzed at the 3M Environmental Laboratory will be maintained for a period of 10 years and will be stored at the laboratory at -20C 10C.
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Analytical Report: FACT TOX-013 LRN-U2095
Chemical Characterization
EtFOSE-OH CAS Number: 1691-99-2
Chemical Formula: CgF17S 0 2N(CH2CH3)CH2CH20 H Molecular Weight: 571.0
Chemical characterization information on the test article is presented in tabular form below. Chemical characterization information on the analytical reference materials used in this study is presented in tabular form in Appendix A (see Tables 8 and 9) and the interim Certificate of Analysis available in Appendix I.
Table 2. Characterization of the Test Article in Study FACT TOX-013
Test Article
Chemical Name
Source Expiration Date
EtFOSE-OH
FM -3929 2(N-Ethyfperfluorooctane sulfbnam ido)-ethanol
3M
05/2 0 0 0
Storage Conditions
Am bient tem perature
Chemical Lot#
Physical Description
30035, 30037, 30039 W axy Solid
Purify
To be determ ined*
* The purity of the test article determined nominally by NMR analysis. Subsequent chemical characterization is occurring and this analytical reportwill be amended to indicate the purity when a certificate ofanalysis is issued.
Doss C onfirm ation A nalyses
T h e d o s e c o n firm a tio n d a ta w e re c o lle c te d a c c o rd in g to a m e th o d th a t w a s n o t fully v a lid a te d . Dose confirmation analyses were performed on test article samples taken at the start of dosage, at 6 weeks, and at the end of dosage during the in-life phase of the study.
Dose confirmation analyses were performed on 3 dose levels collected during the in-life phase of the study: the results are presented in Appendix A (see Tables 10 and 11).
Dose confirmation was performed by diluting the Tween dose samples with Miili-Q water into the linear range of the instrument. For each sample, a matrix spike was prepared (at approximately 5 0-100% of the expected dose level). In all cases, samples were analyzed versus an unextracted curve using HPLC-ES/MS/MS. The instrumental parameters and analytical conditions described in ETS-8-5.1 were used for dose solution analyses.
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Analytical Report: FACT TOX-013 LRN-U2 095
Method Summaries
Following is a brief description of the methods used during this analytical study by the 3M Environmental Laboratory. Detailed descriptions of the methods used are located in Appendix C. The methods and analytical equipment settings used by Battelle are presented in the Battelle final report (see Appendix G).
3IM Environm ental Laboratory
Preparatory Method
ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemicai Compounds from Serum for Analysis using HPLC-Electrospray/Mass Spectrometry"
Sera samples were extracted using an ion-pairing extraction procedure. An ion pairing reagent was added to the sample and the analyte ion-pair was partitioned into MtBE. The MtBE extract was transferred to a centrifuge tube and put onto a nitrogen evaporator until dry. Each extract was reconstituted in 1.0 mL of methanol, then filtered through a 3cc plastic syringe attached to a 0.2pm nylon filter into a glass autovial.
Analytical Method
ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry"
The analyses were performed by monitoring one or more product ions selected from a Single primary ion characteristic of a particular fluorochemicai using HPLC-ESMSMS. For example, molecular ion 499, selected as the primary-ion for PFOS (Q F 17S 0 3-) analysis, was fragmented further to produce ion 99 (FSQ,-). The characteristic product-ion 99 was monitored for quantitative analysis.
Analytical Equipment
The following equipment and parameters are representative of those used during the analytical phase of this study.
Liquid Chrom atograph: Hewlett-Packard* Series 1100 Liquid Chromatograph system Analytical column: Keystone* BetasilTM C182x50 mm (5 pm) Column temperature: Ambient Mobile phase components:
Component A: 2mM aqueous ammonium acetate Component B: methanol Flow rate: 300 pL/min Injection volume: 10 pL Solvent Gradient: 10 minutes
Start at 40%B Hold at 40% B for 1 minute Increase to 95%B over 3.5 minutes
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Hold at 95% B for 2 minutes Return to 4 0 % 8 over 0.5 minutes Hold at 40% B for 3 minutes
Mass Spectrom eter: Micromass* API/Mass Spectrometer Quattro 11TMTriple Quadrupole system Software: Mass Lynx" 3.2 Cone Voltage: 2 0-60 V Collision Energy: 2 5 -4 5 eV Mode: Electrospray Negative Source Block Temperature: 150C 10C Z-spray source Analysis Type: Multiple Reaction Monitoring (MRM)
Table 3. Negative Ions Monitored in 3M Laboratory Analyses
Target Analyte
Primary Ion (AMU)
Product Ion (a m u )
PFOS
499.0
99.0
PFOSA PFOSAA EtFOSE-OH
498.0 584.0 630.0
78.0 169.0 59.0
M556 THPFOS
556.0 427.0
78.0, 169,0 80.0
D eviations Deviations from the original protocol and methods are documented in the table below:
Table 4. Deviation Summary for FACT TOX-013
Deviation
0ate(8) of Occurrence
Impact on Study
Pipette was used instead Oxford dispenser
0.2-1 .OmL of sample was used for extraction instead of 1.OmL.
Milk curd samples were not analyzed.
10/12/99 10/12/99 Entire study
Standards and samples were prepared identically. No adverse impact on study.
Current work indicates that volumes 0.5 mL provide results equivalent to 1 mL extraction volumes. Results of sample volumes <0.5 mL have not been validated and wiK be marked in the data table.
No milk curd data is available for the final report
Data Quality Objectives and Data Integrity
The following data quality objectives (DQOs) were indicated in the method performance section of ETS-8-5.1, "Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry":
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Analytical Report: FACT TOX-013 LRN-U2095
Linearity: The coefficient of determination (r2) equal to or greater than 0.980
Lim its o f Quantitation (LO Q ): The LOQ for PFOS is 5.55 ppb, PFOSA is 4.79 ppb, PFOSAA is 20.5 ppb, EtFOSE-OH is 36.2 ppb, and M556 is 19.2 ppb.
Acceptable Spike Recoveries: 70-130%
Data Summary, Analyses, and Results
With the exceptions noted in this report, data quality objectives for the analytical phase of this study outlined in the 3M Environmental Laboratory method ETS-8-5.1 (see Appendix C) and the Battelle final report (see Appendix G) were met. Although extraction and analysis were initiated in September 1998, the study was reprioritized and put on hold. Upon restarting the study, the decision was made to reextract and analyze the specimens. No data from the original analysis are included in this report. The data in this report reflect only that obtained from specimens extracted on, or after October 11,1999.
Summary of Quality Control Analyses Results
Linearity: The coefficient of determination (r2) of the standard curves were0.980.
Calibration Standards: Quantitation of the target analytes was based on linear regression analysis (1/x weighted) of two extracted matrix curves bracketing each group of samples. High or low points on the curve may have been deactivated to provide a better linear fit over the concentration range most appropriate to the data. Ail active curve points are accurate to within 70% of theoretical value. Low curve points with peak areas less than two times that of the extraction blanks were deactivated to disqualify a data range that may have been significantly affected by background levels of the analyte. Occasionally, a single outlier curve point may have been deactivated. Quantitation of each analyte was based on the response of one or more specific product ion(s) using the multiple response-monitoring mode of the instrument (see Appendix C).
Lim its o f Quantitation (LO Q ): The LOQ is equal to the lowest accepted standard in the calibration curve (defined as a standard with a concentration that is within 30% of the theoretical value, and which has at least two times the analyte peak area detected in the extraction blanks).
Table 5. Determinations of the LOQ in the Analyses o f Serum Extracts
A nalyte
M ethod LO Q
FFOS PFOSA PFOSAA EtFO SE-O H M 556
5.55 ppb 4 .7 9 ppb 2 0 .5 ppb 36.2 ppb 2 4 .9 ppb
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Analytical Report: PACT TOX-013 LRN-U2 095
Blanks: All blanks were below the lower limit of quantitation for the compounds of interest. To simplify analyses that were complicated by endogenous levels of fluorochemicals in unexposed rat sera, rabbit sera was selected as a suitable surrogate matrix for standard curves.
Precision: Precision was determined by analysis of M S/M SD and was reproducible to within 10%.
M atrix Spikes: Matrix spikes and matrix spike duplicates were extracted with each set of samples and analyzed during analytical runs. With the exception of M556, all sera matrix spikes were within 30% of the theoretical concentration. Both matrix spikes showed a recovery of 69% for the M556. These results were verified. Data quality objectives will be adjusted to reflect this recovery.
Surrogates: The surrogate (THPFOS) was added to all samples and standards. THPFO S was not used for quantitation, but was used to monitor for gross instrument failure. The surrogate response of each analytical run was verified to determine that it did not vary more than 50% from the mean within each analytical run.
Assuming spike recovery studies form a suitable indication of endogenous analyte recovery, sera data are quantitative to 30% for all analysis but M556; M 556 data is quantitated to 31% . The validity of this assumption has not been verified by other techniques.
Sum m ary o f Sam ple R esults Sam ples from Control Anim als: Low levels of PFOS, PFOSA, PFOSAA, EtFOSE-OH, and
M 556 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, PFOSA, PFOSAA, EtFOSE-OH, and M556 levels found in the sera and liver of the test animals increased with dose group. Detailed sample data tables are presented in Appendices D and E.
Statistical Methods and Calculations
Statistical methods were limited to the calculation of means and standard deviations. See Appendix F for example calculations used to generate the liver and serum sample data in FACT TOX-013.
Statement of Conclusion
v
Under the conditions of the present studies, PFOS, PFOSA, PFOSAA, EtFOSE-OH, and M556 were observed in the sera and liver of rats dosed with EtFOSE-OH during the in-life phase of the study.
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Appendix A: Chemical Characterization, Control Matrices and Dose Confirmation Analyses
Table 6. Characterization o f the Control Matrices Used for Sera Analyses in Study FACT TOX-013
Location
3M Lab
Control Matrix
Rat Serum (TN-A-2001)
Rabbit Serum (TN-A-2573)
Source Expiration Date Storage Conditions Chemical Lot # Physical Description N/R--not recorded
Sigma
2010
Ambient 17H9306 Rat Serum
Sigma
2010
Ambient 118H8418 Rabbit Serum
Table 7. Characterization o f the Control Matrices Used fo r Liver Analyses in Study FACT TOX-013
Location
Battello Memorial Institute
Control Matrix
Rat Liver
Source Expiration Date Storage Conditions Chemical Lot # Physical Description N/R--not recorded
Harlan N/R N/R N/R
R a tU v e r
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Table 8. Characterization of the Analytical Reference Materials Used for Sera Analyses in Study FACT TOX-013
L o catio n
45M L ab
M a te ria ls
PFOS
C ,, F 17S 0 3 .
PFOSA c bf 17s o 2n h 2
PFOSAA CbF17S 02N((CH2CH3)
(C H 2C O O H ))
Source Expiration Date
3 M Specialty C hem icals
08/3 1/01
N /R 0 1/0 1/20 1 0
N /R 0 1/0 1/20 1 0
Storage Conditions
Chemical Lot Number
Physical Description
A m bient tem perature
A m bient tem perature
171 L -15709
W h ite crystalline powder
Light yellow w axy solid
A m bient tem perature NB 112999-99 Tan w axy solid
Purity
8 6.4%
TBD
TBD
` Surrogate standard-- 1H,1 H,2H,2H-Tetrahydroperfluorooctanesulfonic acid N/R-- not recorded TBD--to be determined NA-- not applicable
E tF O S E -O H CbF17S 0 2N(CH2CH3)
c h 2c h 2o h
3M IC P /P C P Division 0 1/0 1/20 1 0
A m bient tem perature
936
A m b er w axy solid TBD
M 556 c bf 17s o 2n ((H)(CHjCOOH))
3M
0 1 /0 1 /2 0 1 0
A m bient tem perature
THPFOS* c bh 4f 13s o 3h
IC N B iom edicals 0 1/2 01 0
A m bient tem perature
N B 113047-80
53406
W h ite pow der TBD
Brown w axy solid NA
Table 9. Characterization of the Analytical Reference Materials Used fo r Liver Analyses in Study FACT TO X-013
L o c a tio n
B a tte lle M em o rial In s titu te
M a te ria ls
PFOS
M 556
PFOSAA PFOSA THPFOS*
Source
3M 3M 3M 3M
Expiration Date
Storage Conditions
Chemical Lot Number
Physical Description
0 8 /3 1 /0 1
0 1/0 1/20 1 0
A m bient tem perature
A m bient tem perature
171 NB 113047-80
/Vhite crystalline powder
W h ite pow der
2010 A m bient tem perature
617
N /R
0 1 /0 1 /2 0 1 0
A m bient tem perature
L -1 5 7 0 9
Light yellow w axy solid
Purity
8 6.4%
TBD
TBD
TBD
` Surrogate standard-- 1H,1H,2H,2H-Tetrahydroperfluorooctanesulfbnic add N/R--not recorded TBD--to be determined NA-- not applicable
IC N N /R A m bient tem perature 59909
N /R NA
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Analytical Report: FACT TOX-013 LRN-U2095
Table 10. Tween Dosing Confirmation for Study In-life #418-009
Group Dose
Sample Number
Expected Cone. Measured Cone. EtFOSE (ng/mL) EtFOSE (ng/mL)
Group 1-- Control 0 mg/mL Group 2--0.2 mg/mL Group 3-- 1.0 mg/mL Group 4-- 2.0 mg/mL Group 5--3.0 mg/mL
Homogeneity Samples-- 3.0 mg/mL
B-418-009-A, 06/08/98 B-418-009-A, 07/15/98 B-418-009-B, 06/08/98 B-418-009-B, 07/15/98 B-418-009-C, 06/08/98 B-418-009-C, 07/15/98 B-418-009-D, 06/08/98 B-418 -0 0 9 -0 ,0 7 /1 5 /9 8 B-418-009-E, 06/08/98 B-418-009-E, 07/15/98
B-418-009-A, 05/08/98 1 0f6T
B-418-009-A, 06/08/98 3of6M
B-418-009-A, 06/08/98 5of6B
0.00 NA 200000 200000 1000000 100000 2000000 2000000 3000000 3000000
3000000
3000000
3000000
0.00 NA NA NA 1020000 942000 2190000 2750000 3060000 3640000
3250000
3690000
3790000
NA = Not applicable
EtFOSE % Recovery Accuracy
NA NA NA NA 102 94 110 138 102 121
108
123
126
Table 11. Tween Dosing Confirmation-- Matrix Spikes for Study In-life #418-009
Sample Number
Expected Cone. EtFOSE (ng/mL)
Measured Cone. EtFOSE (ng/mL)
EtFOSE % MS Recovery Accuracy
B-418-009-B, 06/08/98-MS B-418-009-B, 07/15/98-MS B-418-009-C, 06/08/98-MS B-418-009-C, 07/15/98-MS B-418-009-D, 06/08/98-MS B-418-009-0,07/15/98-M S B-418-009-E. 06/08/98-MS B-418-009-E, 07/15/98-MS
1200 1200 900 900 900 900 1100 1100
NA NA 818 826 910 733 973 1089
NA NA 91 92 101 81 88 99
B-418-009-A, 05/08/98 1 of6T-M S
B-418-009-A, 06/08/98 3of6M-MS
B-418-009-A, 06/08/98 5 Of 6 B-MS
1100 1100 1100
949 1053 944
86 96 86
NA * Not applicable
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BACKTOMAIN
Analytical Report: FACT TOX-013 LRN-U2095
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Analyt i c m
BACKTOICAIN
)/^
3M Environmental Laboratory____________
P ro to c o l - A n a lytical Stu d y
2(N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction
In-vivo study reference num ber: Argus 418-009 Study num ber: FACT 060998.1 Test substance: 2(N-Ethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE-OH)
Name and address of Sponsor:
Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144
-
Name and address of testing facility: 3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-09 * St. Paul, MN 55106
Experimental start date: Expected term ination date: December 31,1998 M ethod num bers and revisions:
FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry
FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry
FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry
FACT-M-4,0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry
Author: Lisa Clemen
--------- ----------------------5_l%
Kris Hansen
Date
Study Director
Marvin Case Sponsor Representative
Date
3M-^Environmental Laboratory
3MEnvironmental Laboratory
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AnalytrcdX KBpui l :
xwa- uxj
LRN-U2095
1.0 Purpose
_________________________________________________________
The analytical portion of this dosing study is designed evaluate the levels of perfluorooctane sulfonate (PFOS), or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol (NEtFOSE-OH) designated by the study director, in the liver of the parent and subsequent generations of the test system, or in die serum as necessary.
The in life portion of this study was conducted at Argus Research Laboratories.
2.0 Regulatory Compliance_________________________________________________ ____
This study is conducted in compliance with the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report.
3.0 Test Materials_________________________________________________ _______________
3.1 Test, control, and reference substances and matrices
3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217
3.1.2 Analytical reference substance matrix: Rat liver and serum
3.1.3 Analytical control substance: None
3.1.4 Analytical control substance matrix: Rat liver and serum
3.2 Source o f materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability information will be included in the final report
3.2.2 Analytical reference substance matrix: Argus Research Laboratories; traceability information will be included in the final report
3.2.3 Analytical control matrix:
3.23.1 Rat liver - Argus Research Laboratories; traceability information will be included in the final report; or
Rabbit liver - Covance Laboratories; traceability information will be included in the final report
3.2.3.2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report
3.3 Number o f test and control samples. Liver samples for testing were received from 40 test animals and 10 control animals. Serum samples will be tested at the discretion of the Study Director.
3.4 Identification o f test and control samples: The samples are identified using the Argus Research Laboratories identifiers, which consist of a letter followed by the Argus project number, the animal number, the group designation, and the draw date.
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c
Study:
u
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' Analyticar Repuxu:
i u a -v j
T.TDT>,T,,TTOnQC:
3.5 Purity an d strength of materials: Characterization of the purity and identity of the reference material is the responsibility of the Sponsor.
3.6 Stability of test material: Characterization of the stability of the test material is the responsibility of the Sponsor.
3.7 Storage conditions for test materials: Test materials are stored at room temperature. Samples are stored at --20 10 C.
3.8 Disposition of test and/or control substances: Biological tissues and fluids are retained per GLP regulation.
3.9 Safety precautions: Refer to the material safety data sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis.
4.0 Experimental - Overview__________________________I______________________ _
Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-009 are received for analysis of fluorine compounds. At the discretion of the Study Director, a series of analytical tests will be performed on select tissues.
Initially, all liver samples will be analyzed for PFOS by electrospray/mass spectrometry (ES/MS). On the basis of findings from these analyses, additional sample matrices may be evaluated or other metabolites may be targeted. If additional analysis is performed, a protocol amendment will be written.
5.0 Experimental - Analytical M ethods
5.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry
5.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry
5.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Seram for Analysis Using HPLC-Electrospray/Mass Spectrometry
5.4 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry
6.0 Data Analysis pm* 6.1 D ata transform ations and analysis: Data will be reported as the concentration
(weight/weight) of fluoride per tissue or sample, or of PFOS per unit of tissue or fluid.
6.2 Statistical analysis: Statistics used may include regression analysis of the serum concentrations over time, and standard deviations calculated for the concentrations within each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference.
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Analytrcax Kepun.:
i u a -u u
LRN-U2095
fSWSHl .
--
r* &*< Simse (mm.
7/.0V Maintenance of Raw Data and Records
7.1 The following raw data and records will be retained in the study folder in the archives according to AMDT-S-8:
7.1.1 Approved protocol and amendments
7.1.2 Study correspondence
7.1.3 Shipping records
7.1.4 Raw data
7.1.5 Electronic copies of data
7.2 Supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following:
7.2.1 Training records
'
7.2.2 Calibration records
7.2.3 Instrument maintenance logs
7.2.4 Standard Operating Procedures, Equipment Procedures, and Methods
7.2.5 Appropriate specimens.
8.0 References
8.1 3M Environmental Laboratory Quality System Chapters 1,5 and 6 8.2 Other applicable 3M Environmental Laboratory Quality System Standard Operating
Procedures
9.0 Attachments
9.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry
9.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry
9.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry
9.4 FACT-M-4.0, Analysis of Fluorochemicals in Seram Extracts Using HPLCElectrospray/Mass Spectrometry
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' Analytical tepore: AUX IUA-U1J LRN-U2 095
Study Title Combined Oral (Gavage) Fertility Development and'Perinatal/Postnatal
Reproduction Toxicity Study o f N-EtFOSE in Rats
PROTOCOL AMENDMENT NO. 1
Amendment Date: July 28,1999
Perform ing Laboratory 3M Environmental Technology & Safety Services
3M Environmental Laboratory 935 Bush Avenue
S t Paul, MN 55106
Laboratory Project Identification ET&SS FACT-TOX-013 LIRNU2095
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Analytxcax Keporc : ali iua-u u LRN-U2095
Protocol FACT-TOX-013 Amendment 1
T h is am endm ent m o d ifies the fo llo w in g p o rtio n (s) o f th e p rotocol:
1. PROTOCOL r e a d s : The proposed study completion date is listed as 12/31/98.
AMEND TO r e a d : The proposed study completion data is 6/30/00.
REASON: The proposed completion date was changed to allow time for analyzing all matrices o f interest.
Amendment Approval
Marvin Case Ph.D., Sponsor Representative
Kris J.
a Study Director
J o ^ tU ! 4 <?f
Dat/
Date
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A n a l y t i c a l t v c y u i u , l ' n u i x\uxx w j -- -
LRN-U2 095
Study Title Combined Oral (Gavage) Fertility Development and Perinatal/Postnatal
Reproduction Toxicity Study o f N-EtFOSE in Rats
PROTOCOL AMENDMENT NO. 2
Amendment Date: September 10,1999
Performing Laboratory 3M Environmental Technology & Safety Services
3M Environmental Laboratory 935 Bush Avenue
St. Paul, MN 55106
Laboratory Project Identification ET&SS FACT-TOX-013 LIRNU2095
* - 3M Environm ental Laboratory
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LRN-U2095
Protocol FA CT-TO X-013 Amendment 2
T h is am endm ent m odifies th e fo llo w in g p o rtio n (s) o f th e pro to co l:
1. PROTOCOL r e a d s : The protocol states that liver will be extracted and analyzed at the 3M Environmental Laboratory.
AMEND to r ea d : The liver specimens will be extracted and analyzed at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693.
REASON: The liver specimens will be sent to Battelle Memorial Institute for extraction and analysis due to time constraints in the 3M Environmental Laboratory.
2. PROTOCOL r e a d s : The protocol states that serum specimens w ill be extracted and analyzed following methods:
FACT-M-3.0, "Extraction o f Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-4.0, "Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry"
AMEND to r ea d : The serum specimens will be extracted and analyzed following methods:
ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray Mass Spectrometry" ETS-8-5.1, "Analysis o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Serum Extracts HPLC-Electrospray Mass Spectrometry"
REASON: The extraction and analytical methods FACT-M-3.0 and FACT-M-4.0, respectively, were updated on 04/27/99 to ETS-8-4.1 and ETS-8-5.1.
3M Environmental Laboratory
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Analytical Repul i; irtuX x u x j LRN-U2095
Protocol FACT-TOX-013 Amendment 2
3 . P r o t o c o l r e a d s : The protocol states that liver specimens will be extracted and analyzed following methods:
FACT-M-1.0, "Extraction o f Potassium Perfluorooctanesulfonate or Other Anionic surfactants from Liver for analysis Using HPLC-Electrospray/Mas Spectrometry" FACT-M-2.0, "Analysis o f Frluorochemicals in Liver Extracts Using HPLCElectrospray/Mass Spectrometry"
AM END t o r e a d : The liver specimens will be extracted and analyzed following method:
Method for Analysis o f Perfluorooctane Sulfonate (PFOS) in Rat liver by LC/MS/MS, Version 1.0
REASON; Since the liver extraction and analysis was sub-contracted to Battelle Memorial Institute, this amendment was written to include their liver methods and titles.
Amendment Approval
A fo \.
Marvin Case Ph.D., Sponsor Representative
b it--
._____________
Kristen J. Hansen Ph.D., Study Director
_ 3 M Environm ental Laboratory
3M Environmental Laboratory
3M Environmental Laboratory
Date
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Analytical Report: r a u i v a -u u LRN-U2095
Study Title Analytical Study 2(N-Ethylperfluorooctanesulfonamido)-ethanol in
Two Generation Rat Reproduction
PROTOCOL AMENDMENT NO. 3
Amendment Date: October 4,1999
Performing Laboratory 3M Environmental Technology & Safety Services
3M Environmental Laboratory 935 Bush Avenue
St. Paul, MN 55106
Laboratory Project Identification ET&SS FACT-TOX-013 LIRNU2095
_ 3M Environmental Laboratory
3M Environmental Laboratory
3MEnvironmental Laboratory
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Analytical Keport : f a u t m - u u LRN-U2095
Protocol FACT Tox-013 Amendment Number 3
T h is am endm ent m o d ifies th e fo llo w in g p o rtio n (s) o f th e pro to co l:
1. Pro to co l R ea d s:
Kristen J. Hansen, Ph.D. is the Study Director.
A mend to Read:
James K. Lundberg, Ph.D. is the Study Director.
Reason:
Original study design has changed due to availability o f resources and James K. Lundberg . will begin serving as the study director for FACT-TOX-013 as o f 4 October 1999.
2. Pro to c o l R e a d s :
-
Section 7.1 states that the following raw data and records will be retained in the study folder
in the archives according to AMDT-S-8: Approved protocol and amendments; study
correspondence; shipping records; raw data; and electronic copies o f data. Additionally,
Section 7.2 states that supporting records to be retained separately from the study folder in
the archives according to AMDT-S-8 will include at least the following: Training records;
calibration records; instrument maintenance logs; Standard Operating Procedures, Equipment
Procedures, and Methods; and appropriate specimens.
A m end to Read:
Section 7 states: "The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits o f the study during its progress and before acceptance o f the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, dripping records, raw data, approved final report, and electronic copies o f data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument maintenance logs, standard operating procedures, equipment procedures, and methods will be retained in the archives o f the facility performing each analysis.
Reason:
To direct subcontract laboratories in the disposition o f the items listed above.
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Analytical Report: jj'a u t t u a -uj.3 LRN-U2095
Protocol FACT Tox-013 Amendment Number 3
3. Protocol reads:
Disposition o f test and control substances: Biological tissues and fluids are retained per GLP
regulation.
A mend to read:
Specimens will be maintained in the 3M Environmental Laboratory specimen archives. All specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion o f analysis and submission o f the sub-contract laboratory(s) final report. The specimens will be returned with the following documentation: the signed original chain of custody and records o f storage conditions while at the sub-contract facility.
Reason:
To define in detail the appropriate disposition o f specimens analyzed at subcontract
laboratories.
-
Amendment Approval
Marv Case, D.V.M., Ph.D., Sponsor Representative
Date
-ti* -- ___________________________________________________ t o / s m
Kristen J. Hansen, Ph.D., Previous Study Director
Date
Dale L. Bacon, PhrBT 3M Environmental Laboratory Management
3M Environmental Laboratory 3M Environmental Laboratory
3MEnvironmental Laboratory
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' Analyticctx ncpuit: r LKN-U2095
Study Title Analytical Study o f 2(N-Ethylperfluorooctanesulfpnamido)-ethanol in
Two Generation Rat Reproduction
PROTOCOL AMENDMENT NO. 4
Amendment Date: 20 January 2000
Performing Laboratory 3M Environmental Technology & Safety Services
3M Environmental Laboratory 935 Bush Avenue
St. Paul, MN 55106
Laboratory Project Identification ET&SS LRN-U2095 FACT TOX-013 Argus Study: 418-009
3M Medical Department Study: T-6316.5
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' Analytical Keport: raw tua-uxj LRN-U2095
Protocol LRN-U2095 Amendment Number 4
This am endm ent m odifies the following portion(s) of the protocol:
1. P r o t o c o l r e a d s : The study director for the present study was identified in the protocol as James K. Lundburg, Ph.D. A mend to read: The role of study director for the present study was reassigned to Marvin T. Case, D .V.M ., Ph.D., as of 20 January 2000. The previous study director, James K. Lundburg, has been reassigned to the role of Principle Analytical Investigator. Reason: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 CFR Part 58).
2. Protocol reads: The sponsor for the present study was identified as Marvin T. Case, D .V.M ., Ph.D. A mend to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced.
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Analytical Report: FACT TOX-013 LRN-U2095
Protocol LRN-U2095 Amendment Number 4
mm, '
Amendment Approval
John L ButenhoffPhD., Sponsor Representative
Date
I Marvin T. Case, D. V.M., PhD ., Incoming Study Director
J O .--
eh$rL Date &
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Analytical Report: f a c t t u i -u u LRN-U2095
Study Title Analytical Study of 2(N-Ethylperfluorooctanesulfonamido)-ethanol in
Two Generation Rat Reproduction
PROTOCOL AMENDMENT NO. 5
Amendment Date: August 31, 2000
Performing Laboratory 3M Environmental Technology & Safety Services
3M Environmental Laboratory 935 Bush Avenue
St. Paul, MN 55106
Laboratory Project Identification FA CT -T O X -013
ET&SS LRN U2095 Argus Study: 418-009 3M Medical Department Study: T6316.5
r" 3M Environmental Laboratory
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Analytiuai
BA C K TO M A IN
LRN-U2 095
" Protocol FACT TOX-013 Amendment No. 5
mt This am endm ent m odifies the following portion(s) of the protocol:
- 1. PROTOCOL READS: The Principle Analytical Investigator for the present study was identified as James K. Lundberg, Ph.D.
2 . AMEND TO r e a d : The role of Principle Analytical Investigator for the present study was reassigned to Kristen J. Hansen Ph.D.
*. REASON: The role of Principle Analytical Investigator was reassigned due to availability of resources.
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Analytical Report: FACT TOX-013 LRN-U2095
Protocol FACT TOX-013 Amendment No. 5
Amendment Approval
a' John L. Butenhoff, Ph.D., Sponsor Representative
Date
rc ^
Marvin T. Case, D. V.M., Ph.D., Study Director
Date
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Analytical Report: FACT TOX-013 LRN-U2095
Appendix C: Extraction and Analytical Methods
This appendix includes the following methods: ETS-8-4.1, Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry, (14 pages) ETS-8-5.1, Analysis of Potassium Perfluorooctanesulfonate or Other Fluorochemicals in Serum Extracts Using HPLC-Electrospray/Mass Spectrometry, (9 pages)
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Analyticax Kepuu-:
i u a -u u
LRN-U2095
3M E Lnvironmental^I | I'j 131 I J A l r l l ITI Ivi ^ JL aboV/ ratJL.oryA.
jw*a
M ethod
pm 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 em ic a l co m po u n d s fr o m Seru m fo r An a ly sis U sin g HPLC-
E lectro spra y /M ass Spec tr o m etr y
fmm
M ethod N um ber: ETS-8-4.1
Adoption Date: 03/01/99
Author: Lisa Clemen, Glenn Langenburg
Revision Date:
Approved By:
-0 Laboratory Manager
,****,
1 i_
Date
U t-- -------
WS Group Leader
Date
A
Technical Reviewer
..... dv/ W w 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.
1.2 Applicable compounds: Fluorochemical surfactants or other fluorinated compounds.
1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum or other fluids as designated in the validation report.
Word 6/95
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Analytical K e p u n : rii i u a -uio LRN-U2095
2.0 Summary of M ethod___________________________________________________________ 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-iert-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 m i. o f methanol, then filtered through a 3 cc plastic syringe attached to a 0.2 jum 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 o f potassium salt) C8F17S 0 3` 3.2 PFOSA: perfluorooctane sulfonylamide C8F17S 0 2NH2
3.3 PFOSAA: perfluorooctane sulfonylamido (ethyl)acetate C8F17S 0 2N(CH2CH3)CH2CO2'
3.4 EtFOSE-OH: 2(N-ethylperfluorooctane sulfonamido)-ethyl alcohol C8F 17S 0 2N(CH2CH3)CH2CH20 H
3.5 PFOSEA: perfluorooctane sulfonyl ethylamide C8FI7S 0 2N(CH2CH3)H
3.6 M556: C8FnS 0 2N(H)(CH2C 0 0 H )
3.7 Surrogate standard: 1H-1H-2H-2H perfluorooctane sulfonic acid
4.0 Warnings and Cautions_____________________________________________________ __ 4.1 H ealth 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 Vortex mixer, VWR, Vortex Genie 2 6.1.2 Centrifuge, Mistral 1000 or IEC 6.1.3 Shaker, Eberbach or VWR
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Analytical Keport: f a c t t u a -u u LRN-U2095
6.1.4 Nitrogen evaporator, Organomation 6.1.5 Balance ( 0.100 g)
7.0 Supplies and Materials________________________________________________________ 7.1 Gloves 7.2 Eppendorf or disposable pipettes 7.3 Nalgene bottles, capable of holding 250 mL and 1 L 7.4 Volumetric flasks, glass, type A 7.5 I-CHEM vials, glass, 40 mL glass 7.6 Centrifuge tubes, polypropylene, 15 mL 7.7 Labels 7.8 Oxford Dispenser - 3.0 to 10.0 mL 7.9 Syringes, capable o f measuring 5 pL to 50 pL 7.10 Graduated pipettes 7.11 Syringes, disposable plastic, 3 cc 7.12 Syringe filters, nylon, 0.2 pm, 25 mm 7.13 Timer 7.14 Crimp cap autovials and caps 7.15 Crimpers Note: Prior to using glassware and bottles, rinse 3 times with methanol and 3 times with
Milli-QTM water. Rinse syringes a minimum o f 9 times with methanol, 3 rinses from 3 separate vials.
8.0 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 (NajCOj), 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 Fluorochem ical standards
8.9.1 PFOS (3M Specialty Chemical Division), molecular weight = 538 8.9.2 PFOSA (3M Specialty Chemical Division), molecular weight = 499
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8.9.3 PFOSAA (3M Specialty Chemical Division), molecular weight - 585
8.9.4 EtFOSE-OH (3M Specialty Chemical Division), molecular weight = 570
8.9.5 PFOSEA (3M Specialty Chemical Division), molecular weight = 527
8.9.6 M556 (3M Specialty Chemical Division), molecular weight = 557
8.9.7 Surrogate standard: 4-H, periluorooctane sulfonic acid (l-H .l-H , 2-H, 2-H C8F 13S 0 3H) molecular weight = 428
8.9.8 Other fluorochemicals, as appropriate
8.10 Reagent preparation
NOTE: When preparing larger volumes than listed in reagent, standard, or surrogate preparation, adjust accordingly.
8.10.1 10 N sodium hydroxide (NaOH): Weigh approximately 200 g NaOH. Pour into a 1000 mL beaker containing 500 mL Milli-QTM water, mix until all solids are dissolved. Store in a 1 L Nalgene bottle.
8.10.2 1 N sodium hydroxide (NaOH): Dilute 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 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 o f 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 (Na2C03/N aH C 03): Weigh approximately 26.5 g of sodium carbonate (N a^O j) and 21.0 g o f sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with MilliQTM water. Store in a 1 L Nalgene bottle.
8.11 Standards preparation
8.11.1 Prepare PFOS standards for the standard curve.
8.11.2 Prepare other fluorochemical standards, as appropriate. Multicomponent fluorochemical standards are acceptable (for example, one working standard solution containing 1.00 ppm PFOS, 1.02 ppm PFOSA, 0.987 ppm PFOSAA, and 1.10 ppm EtFOSE-OH.)
8.11.3 Weigh approximately 100 mg o f 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 (ug/mL).
8.11.5 Dilute the stock solution with methanol for a working standard 1 solution o f approximately 50 ppm.
8.11.6 Dilute working standard 1 with methanol for a working standard 2 solution of approx. 5.0 ppm.
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8.11.7 Dilute working standard 1 with methanol for a working standard 3 solution of approx. 0.50 ppm.
8.12 Surrogate stock standard preparation
8.12.1 Weigh approximately 50-60 mg o f surrogate standard 1-H,1-H, 2-H, 2-H, C8F,3S 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 o f 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 o f 100 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, M ethod blanks and m atrix blanks
.a-
--poJLt
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.
9.2 M atrix spikes
10.2.1 Prepare and analyze matrix spike and matrix spike duplicate samples to determine the accuracy of the extraction.
10.2.2 Prepare each spike using a sample chosen by the analyst, usually the control matrix received with each sample set.
10.2.3 Expected concentrations will fall in the mid-range 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 o f 2 matrix spikes per batch.
0.3 Continuing calibration checks
10.3.1 Prepare continuing calibration check samples to ensure the accuracy o f the initial calibration curve.
10.3.2 Prepare, at a minimum, one continuing check per group o f 10 samples. For example, if a sample set - 34, four checks are prepared and extracted.
10.3.3 Prepare each continuing calibration check from the same matrix used to prepare the initial curve.
It
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10.3.4 The expected concentrations will fall within the mid-range o f the initial calibration curve. Additional spikes may be included that fall in the low-range of the initial calibration curve. This is necessary if the analyst must quantitate using only the low end o f the calibration curve (for example, 5 ppb --100 ppb, rather than 5 ppb - 1000 ppb).
11.0 Calibration and Standardization ___________________________________________
11.1 P repare m atrix 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 o f twenty aliquots in 15 mL centrifuge tubes, mix or shake between aliquots.
11.1.4 Two 1 mL aliquots, or other appropriate volume, serve as matrix blanks. Typically use the standard concentrations and spiking amounts listed in Table 1, at the end o f this section, to spike, in duplicate, two standard curves, for a total o f eighteen standards, two matrix blanks, and two method blanks.
11.1.5 Refer to validation report ETS-8-4.0 & ETS-8-5.0-V-1, which lists the working ranges and the Linear Calibration Range (LCR) for calibration curves.
11.1.6 Use Attachment D as an aid in calculating the concentrations o f the working standards. See Section 13.0 to calculate actual concentrations o f PFOS in calibration standards.
11.2 To each standard, blank, or continuing check, add appropriate amount o f 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.
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Analytical Report: FACT TOX-013 LRN-U2095
Table 1
Approximate spiking amounts for standards and spikes
Using 1.0 mL of m atrix
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 P r o c e d u r e __________________________________________________________________________________
12.1 Obtain frozen samples and allow to thaw at room temperature or in a lukewarm waterbath.
12.2 Vortex mix for 15 seconds, then transfer 1.0 mL or other appropriate volume to a 15 mL polypropylene centrifuge tube.
12.3 Return unused samples to freezer after extraction amounts have been removed.
12.4 Record the initial volume on the extraction worksheet.
12.5 Label the tube with the study number, sample ID, date and analyst initials. See attached worksheet for documenting the remaining steps.
12.6 Spike all samples, including blanks and standards, ready for extraction with surrogate standard as described in 11.2.
12.7 Spike each matrix with the appropriate amount o f standard as described in 11.1, or Table 1 in that section, for the calibration curve standards. Also prepare matrix spikes and continuing calibration standards.
12.8 Vortex mix the standard curve samples, matrix spike samples, and continuing calibration samples for 15 seconds.
12.9 Check to ensure the 0.5 M TBA reagent is at pH 10. If not, adjust accordingly.
12.10 To each sample, add 1 mL 0.5 M TBA and 2 mL o f 0.25M sodium carbonate/sodium bicarbonate buffer.
12.11 Using an Oxford Dispenser, add 5 mL methyl-terf-butyl ether.
12.12 Cap each sample and put on the shaker at a setting of 300 rpm, for 20 minutes.
12.13 Centrifuge for 20 to 25 minutes at a setting o f 3500 rpm, or until layers are well separated.
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12.14 Label a fresh 15 mL centrifuge tube with the same information as in 12.5.
12.15 Remove 4.0 mL o f 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 o f 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 o f standard x concentration o f standard fug /m D __________________ = mL o f standard + mL o f surrogate standard + initial matrix volume (mL)
Final Concentration (pg/mL) o f 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 M DL report for specific MDL and limit o f quantitation (LOQ) values (see Attachm ents B and C).
14.2 The following quality control samples are extracted with each batch o f samples to evaluate the quality of the extraction and analysis.
14.2.1 Method blanks and matrix blanks.
14.2.2 Matrix spike and matrix spike duplicate samples to determine accuracy and precision of the extraction.
14.2.3 Continuing calibration check samples to determine the continued accuracy o f the initial calibration curve.
14.3 Refer to section 14 o f ETS-8-5.1 for method performance criteria.
15.0 P o l l u t io n P r e v e n t io n a n d W a 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.
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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 summaty 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.
18.2 FACT-M-3.1, "Analysis o f Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry"
19.0 A f f e c t e d D o c u m e n t s __________ ;___________________________________________________________
19.1 ETS-8-5.1, "Analysis of Serum or Other Fluid Extracts for Fluorochemicals using HPLC-Electrospray Mass Spectrometry"
20.0 Revisions___________________ :________________________________________________
Revision Number
1
Reason For Revision Section 12.21 Changed to include sample storage at room temperature. Section 12.13 Added the shaker speed. Section 12.17 Final volume is 1.0 mL; not adjusted for initial volumes less than 1,0 mL.
Revision Date
04/02/99
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Extraction Worksheet ETS-8-4.1
Study# Matrix Box# Wk/Day
DateSpiked/Analyst CCV MS MSD
Surrogate Std approx, ppm actual ppm #
FC-Mix approx. 0.5 pm actual ppm #
FC-Mix approx. 5 ppm actual ppm #
FC-Mix approx. 50 ppm actual ppm #
Comments
--. ------
-
--
--
----
--
--
Blank
Std #
amount =
Serum Extraction Method
s
Vortex 15 sec.
Pinette Matrix
Volume
mL
Pipette 1 mL of 0.5 M TBA, pH 10. pH =
Std. #
Pipette 2 mL of 0.25 Na?COV0.25M NaHCCh buffer
Std. #
Dispense 5 mL of methyl-t-butyl ether
TN-A-
Shake 20 min.
Shaker soeed:
Centrifuge 20-25 min.
Centrifuge speed:
Remove a 4 mL aliauot of organic layer
Put on Nitrogen Evaporator to drvness
Temperature:
Add methanol
Volume
mL TN-A-
Vortex 30 sec.
Filter using a 3cc B-D svrinee with a 0.2um filter into a 1.5 mL autosamole vial Cont. Cal. Verifications used same matrix as for std curve.
-
-
-
-
-
-
-
-
-
-
-
-
-
mL
Date & Initials
Attachment A 3M Environmental Laboratory
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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 p p b - 1000ppb
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
MDL/LOQ values in rat, bovine, monkey, and human serum, and monkey plasma were not statistically
determined. Two curves in each of these matrices were extracted and analyzed with the rabbit serum
curves to determine equivalence. Responses in the rat, bovine, monkey, and human were equivalent to the rabbit responses, therefore, their MDL and LOQ will be the same values as determined in rabbit
serum.
Please see LOQ Summary and MDL study in ETS-8-4.0 & 5.0-V-l for further information.
Attachment B: MDL/LOQ Summary 3M Environmental Laboratory
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Analytical Report: FACT TOX-013 LRN-U2095
Compound: PFOS Prepared range
Rabbit Serum of standards (ppb) (ng/mL)
Full Range Low Curve High curve
1 /X
0.995 - 978 4.94 - 248 97.8 - 978 0.995 - 978
LCR from curve (PPb)
(ng/mL)
24.8 - 978
4.94 - 248
9 7 .8 -9 7 8
4.94 - 978
% Recovery Range
83-108 85-104 85-106 94-111
RSD Range
4.67-11.0 5.34-12.0 4.84-9.80 4.60-10.5
Compound: PFOSA
Prepared range Rabbit Serum of standards
(ppb) (ng/mL)
Full Range Low Curve High curve
1 /X
0.993 - 976 4.93 - 97.6
24.8 - 976 0.993 - 976
LCR from curve (PPb)
(ng/mL)
4.93 - 976
4.93 - 97.6
24.8 - 978
4.93 - 976
% Recovery Range
88-103 87-105 93-102 94-103
RSD Range
5.10-14.7 9.85-14.7 5.08-13.9 5.10-14.5
Compound: PFOSAA
Prepared range Rabbit Serum of standards
(ppb) (ng/mL)
Full Range
0 .9 9 1 -9 7 4
LCR from curve (PPb)
(ng/mL)
24.7 - 974
% Recovery Range
81-111
RSD Range
4.18-10.6
Low Curve
4.92 - 247
9.74 - 247
97-107
6.38-21.8
High curve
49.2 - 974
97.4 - 974
85-108
4.33-12.5
pm*
1 /X
0.991 - 974
9.74 - 974
95-115
4.11-23.2
Attachment B: MDL/LOQ Summary
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ETS-8-4.1 Extraction o f PFOS from Serum
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Analytical Keport: F A U T T U A - U H LRN-U2095
Compound: EtFOSE-OH Prepared range
Rabbit Serum of standards (ppb) (ng/mL)
Full Range Low Curve High curve 1/X
0.993 - 976 4.93 - 97.6 49.3 - 976 0.993 - 493
LCR from curve (ppb)
(ng/mL) 49.3 - 976
9.76 - 97.6
97.6 - 976
9.76 - 976
%Recovery Range
77-110 97-107 90-109 86-111
Compound: PFOSEA Prepared range
Rabbit Serum of standards (ppb) (ng/mL)
Full Range Low Curve High curve 1/X
0.993 - 976 4.93 - 248 49.3 - 976 0.993 - 976
LCR from curve (PPb)
(ng/mL) 24.8 - 976
9.76 - 248
49.3-976
9.76 - 976
%Recovery Range
96-106 91-110 86-106 95-117
Compound: M556 Prepared range
Rabbit Serum of standards (ppb) (ng/mL)
Full Range Low Curve High curve 1/X
0.993 - 976 4.93 - 97.6 97.6 - 976 0.993 - 976
LCR from curve (Ppb)
(ng/mL) 24.8 - 976
9.76-97.6
97.6 - 976
9.76 - 976
%Recovery Range
88-106 100-105 81-111 97-110
RSD Range
11.2-25.5 14.1-21.3 11.5-19.6 11.1-21.2
RSD Range
10.1-16.2 11.8-19.5 10.2-18.2 10.1-19.1
RSD Range
4.82-17.9 5.95-18.2 5.11-9.74 4.77-19.5
--> Attachment B: MDL/LOQ Summary
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Analyuxuax Kepuru: Iftli 1UA-UX-3 LRN-U2095
Ion Pair Standard Curves - Fluids
Prep date(s):
Standard number:
Analyte(s):
Equipment number:
Sample matrix:
Final solvent and TN:
Blank fluid/identifier:
Method/revision:
Target analyte(s): _
FC mix std approx. 0.500 ppm:
FC mix std approx. 5.00 ppm:
FC mix std approx. 50.0 ppm:
Surrogate std approx. 100 ppm:
Actual concentrations of standards in the FC mix
PFOS PFOSA PFOSAA EtFOSE PFOSEA
Std cone Std cone Std cone Std cone Std cone
ug/mL ug/mL
ug/mL
ug/mL ug/mL
0.500
0.507
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
50.0 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 Amt spiked mL 0.010 0.020 0.005 0.010 0.020 0.005 0.010 0.015 0.020
All Final vol
mL 1.015 1.025 1.010 1.015 1.025 1.010 1.015 1.020 1.025
Calculated concentrations of standards in the sample matrix
PFOS 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 4.94 5.01 5.13 100
9.76 9.89 10.4 9.78 9.93 10.2
24.8 25.1 26.3 24.8 25.2 25.8 Surrogate
49.3 50.0 52.4 49.4 50.1 51.3 Final cone
97.6 98.9
104
97.8 99.3
102 ng/mL
248 251 263 248 252 258 500
493 500 524 494 501 513
735 746 782 737 749 766
976 989 1038 978 993 1017
All Ain't spiked
mL
0.005
Validated ranges - approximate concentrations
Serum
PFOS
PFOSA
PFOSAA EtFOSE-OH PFOSEA
M556
Rabbit
5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000 | 5.00-1000
Bovine
Estimates only. Use values for rabbit.
Rat Estimates only. Use values for rabbit.
Monkey & Plasma Estimates only. Use values for rabbit.
Human
Estimates only. Use values for rabbit.
"
Attachment C: Ion Pair Standard Curves
ETS-8-4.1
Extraction o f PFOS from Serum
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Analytical Report: FACT TOX-013 LRN-U2095
3M Environmental Laboratory
M ethod
A n a ly sis o f P o ta ssiu m P er flu o r o o c ta 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 Seru m E x tra cts U sin g H P L C -E lectrospray/M ass Spectro m etry
- M ethod N um ber: ETS-8-5.1
Author: Lisa Clemen, Robert Wynne
Approved By:
t J ! l i -- Laboratory Manager
Group Leader
AL.
....
S* A ' Technical Reviewer
Adoption Date: 03/01/99 Revision Date:
^ y 2. c f Date
Date oh/ u / m
Date
1 .0 S c o p e a n d A p p l ic a t io n ____________________________________________________________________
1.1 Scope: This method describes the analysis o f serum extracts for fluorochemical surfactants using HPLC-electrospray/mass spectrometry.
1.2 Applicable C om pounds: Fluorochemical surfactants or other fluorinated compounds, or other ionizable compounds.
1.3 M atrices: Rabbit, rat, bovine, monkey, and human serum, or other fluids as designated in
the validation report.
^ Word 6/95
ETS-8-5.1 Analysis o f Serum Extract Using ES/MS
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Analytical Report: r a u t t u -uis LRN-U2 095
2.0 S u m m a r y o f M e t h o d ______________________________________________________ __________________
2.1 This method describes the analysis o f fluorochemical surfactants extracted from serum or other fluids, using HPLC-electrospray/mass spectrometry, or similar system as appropriate. The analysis is performed by monitoring a single ion characteristic of a particular fluorochemical, such as the perfluorooctanesulfonate (PFOS) anion, m/z= 499. Additionally, samples may be analyzed using a tandem mass spectrometer to further verify the identity o f a compound by detecting daughter ions o f the parent ion.
3.0 D e f in it io n s
_____________________________________________________________________________
3.1 A tm ospheric Pressure Ionization (API): The Micromass Quattro II triple quadrupole systems allow for various methods o f 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 o f a strong electrical field.
3.3 Mass Spectrometry, Mass Spectrom eter (MS), Tandem Mass Spectrom eter (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 o f operation, cleaning, and maintenance are the same. However, Z-spray components and conventional components are not compatible with one another, but only with similar systems (i.e., Z-spray components are compatible with some other Z-spray systems, etc.)
3.5 M ass Lynx Software: System software designed for the specific operation of 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 in g s a n d C a u t io n s _________________________________________________________________ 4.1 H ealth and Safety W arnings:
4.1.1 Use caution with the voltage cables for the probe. When engaged, the probe employs a voltage o f approximately 5000 Volts.
4.1.2 When handling samples or solvents wear appropriate protective gloves, eyewear, and clothing.
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Analytical Report: f a c t t o x -u i 3 LRN-U2095
4.2 Cautions:
4.2.1 Do not operate solvent pumps above capacity o f 400 bar (5800 psi) back pressure. If the back pressure exceeds 400 bar, the HP 1100 will initiate automatic shutdown.
4.2.2 Do not run solvent pumps to dryness.
5.0 I n t e r f e r e n c e s _______________________________________________________________________________
5.1 To minimize interferences when analyzing samples, teflon should not be used for sample storage or any part o f instrumentation that comes in contact with the sample or extract.
6.0 E q u ip m e n t ____________________________________________________________________________________
6.1 Equipment listed below may be modified in order to optimize the system. Document any modifications in the raw data as method deviations.
6.1.1 6.1.2
Micromass Quattro II triple quadrupole Mass Spectrometer equipped with an electrospray ionization source
HP 1100 low pulse solvent pumping system, solvent degasser, column compartment, and autosampler
7.0 S u p p l ie s a n d M a t e r ia l s ________________________________________ :____________________________
7.1 Supplies 7.1.1 High purity grade nitrogen gas regulated to approximately 100 psi (House air system)
7.1.2 HPLC analytical column, specifics to be determined by the analyst and documented in the raw data.
7.1.3 Capped autovials or capped 15 mL centrifuge tubes
8.0 R e a g e n t s a n d S t a n d a r d s _________________________________________ ;_________________________ 8.1 Reagents
8.1.1 Methanol, HPLC grade or equivalent
8.1.2 Milli-QTM water, all water used in this method should be Milli-QTM water or equivalent, and may be provided by a Milli-Q TOC Plus system or other vendor
8.1.3 Ammonium acetate, reagent grade or equivalent
8.2 Standards
8.2.1 Typically two method blanks, two matrix blanks, and eighteen matrix standards are prepared during the extraction procedure. See ETS-8-4.1.
9.0 S a m p l e H a n d l in g ________________________'_______________________,____________________________
9.1 Fresh matrix standards are prepared with each analysis. Extracted standards and samples are stored in capped autovials or capped 15 mL centrifuge tubes until analysis.
^
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Analytical Keport: f a u t t u a -u u LRN-U2095
9.2 I f analysis will be delayed, extracted standards and samples can be refrigerated at approximately 4 C, or at room temperature, until analysis can be performed.
10.0 Q u a l it y C o n t r o l __________________________________________________________________________ 10.1 Solvent Blanks, Method Blanks and Matrix Blanks
10.1.1 Solvent blanks, method blanks and matrix blanks are prepared and analyzed with each batch to determine contamination or carryover.
10.1.2 Analyze a method blank and a matrix blank prior to each calibration curve.
10.2 Matrix Spikes
10.2.1 Matrix spikes are prepared and analyzed to determine the matrix effect on the recovery efficiency.
10.2.2 Matrix spike duplicates are prepared and analyzed to measure the precision and the recovery for each analyte.
10.2.3 Analyze a matrix spike and matrix spike duplicate per forty samples, with a minimum o f 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 o f the initial calibration curve.
10.3 Continuing Calibration Verifications
10.3.1 Continuing calibration verifications are analyzed to verify the continued accuracy o f the calibration curve.
10.3.2 Analyze a mid-range calibration standard after every tenth sample, with a minimum o f one per batch.
11.0 C a l ib r a t io n a n d S t a n d a r d iz a t io n _____________________________________________________ 11.1 Analyze the extracted 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), weighted 1/x, not forced through zero, using MassLynx or other suitable software.
11.2 I f the curve does not meet requirements, perform routine maintenance or reextract the standard curve (if necessary) and reanalyze.
11.3 For purposes o f accuracy when quantitating low levels o f analyte, it may be necessary to use the low end o f the calibration curve rather than the full range o f 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 o f the curve (5 ppb to 1000 ppb). This will reduce inaccuracy attributed to linear regression weighting o f high concentration standards.
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Analyticax Keporc: c x x u a - u u LRN-U2 095
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 o f year-sample number, assign a method (MS) for acquiring, and type in sample descriptions.
12.1.2 To create a method click on scan button in the Acquisition control panel and select SIR (Single Ion Recording) or MRM. Set Ionization Mode as appropriate and mass to 499 or other appropriate masses. A full scan is usually collected along with the SIRs. Save acquisition method. If MS/MS instruments are employed, additional product ion fragmentation information may be collected. See Micromass MassLynx GUIDE TO DATA ACQUISITION for additional information and MRM (Multiple Reaction Monitoring).
12.1.3 Typically the analytical batch run sequence begins with a set o f extracted matrix standards and ends with a set o f extracted matrix standards.
12.1.4 Samples are analyzed with a continuing calibration check injected after every tenth sample. Solvent blanks should be analyzed periodically to monitor possible analyte carryover and are not considered samples but may be included as such.
12.2 Using the Autosam pler
12.2.1 Set up sample tray according to the sample list prepared in Section 12.1.1.
12.2.2 Set-up the 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 = 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.
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Analytrcax Keporc:
i u a -u u LRN-U2095
12.3.3 Check the stainless steel capillary at the end o f the probe. Use an eyepiece to check the tip. The tip should be flat with no jagged edges. If the tip is found to be unsatisfactory, disassemble the probe and replace the stainless steel capillary.
12.3.4 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 orr the nitrogen. A fine mist should be expelled with no nitrogen leaking around the tip o f the probe. Readjust the tip o f the probe if no mist is observed.
12.3.6 The instrument uses these parameters at the following settings. These settings may change in order to optimize the response:
12.3.6.1 Drying gas 250-400 liters/hour 12.3.6.2 ESI nebulizing gas 10-15 liters/hour 12.3.6.3 HPLC constant flow mode, flow rate 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 of biological matrices.
12.3.10Click on start button in the Acquisition Control Panel (this may vary among MassLynx versions, see appropriate MassLynx USER'S GUIDE). Press the start button. Ensure start and end sample number includes all samples to be analyzed.
13.0 D a t a A n a l y s is a n d C a l c u l a t io n s ______________ ;___________________________________ 13.1 Calculations:
13.1.4 Calculate matrix spike percent recoveries using the following equation:
% Recovery = Observed Result - Background Result x 100 Expected Result
13.1.5 Calculate percent difference using the following equation:
% Difference = Expected Cone. - Calculated Cone, x 100 Expected Cone.
13.1.6 Calculate actual concentration o f PFOS, or other fluorochemical, in matrix (pg/mL):
fag o f PFOS calc, from std. Curve x Dilution Factor) x 1 pg (Initial Volume o f matrix (mL>) + mL o f Surrogate Standards 1000 ng
Final Volume (mL)
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Analycicax Report: ihui iua-u u LRN-U2 095
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 o f Quantitation (LOQ) are method, analyte, and
matrix specific. Please see ETS-8-4.1, A ttachm ent B, for a listing o f 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% o f the spiked concentration.
14.5 Continuing Calibration Verifications ,
14.5.1 Continuing calibration verification percent recoveries must be 30% o f the spiked concentration.
14.6 I f 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 o f the report.
15.0 P o l l u t i o n P r e v e n t io n a n d W a s t e M a n a g e m e n t ______________________________________ 15.1 Sample extract waste and flammable solvent is disposed in high BTU containers, and glass
pipette waste is disposed in broken glass containers located in the laboratory.
16.0 R e c o r d s _____________________________________________________________________________________ 16.1 Each page generated for a study must have the following information included either in the
header or hand written on the page: study or project number, acquisition method, integration method, sample name, extraction date, dilution factor (if applicable), and analyst.
16.2 Print the tune page, sample list, and acquisition method from MassLynx to include in the appropriate study folder. Copy these pages and tape into the instrument runlog.
16.3 Plot the calibration curve by linear regression, weighted 1/x, then print these graphs and store in the study folder.
16.4 Print data integration summary, integration method, and chromatograms, from MassLynx, and store in the study folder.
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16.5 Summarize data using suitable software (Excel 5.0) and store in the study folder, see Attachment A for an example o f a summary spreadsheet.
16.6 Back up electronic data to appropriate medium. Record in study notebook the file name and location o f backup electronic data.
17.0 T a b l e s . D i a g r a m s . F l o w c h a r t s , a n d V a l id a t io n D a t a _______________________________ 17.1 Attachment A: ETS-8-5.1 Data summary spreadsheet.
18.0 R e f e r e n c e s _________________________________________________________________________________ 18.1 FACT-M-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical
compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry
18.2 ETS-9-24.0, "Operation and Maintenance o f the Micromass Atmospheric Pressure Ionization/Mass Spectrometer Quattro II triple quadrupole Systems"
18.3 The validation report associated with this method is ETS-8-4.0 & 5.0-V -l.
19.0 A ffec ted D o c u m en ts
19.1 ETS-8-4.1, "Extraction o f Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry"
20.0 R e v is io n s
Revision Number.
1
Reason For Revision Section 6.1.2 Clarification of HP1100 system components. Section 11.1 Average o f two curves, not standard values, are used for plotting linear regression and added the 1/x weighting o f 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
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BACKTOMAIN
Analytical Keport: c a u i t u a -u u LRN-U2 095
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/Analyst:
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: Summary Spreadsheet
ETS-8-5.1
Analysis o f Serum Extract Using ES/MS
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Analytical Report: FACT TOX-013 LRN-U2095
A p p en d ix D: D ata Su m m ary T a b les
Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013
Dosage Group
Specimen ID
PFOS (pg/mL)
PFOSA (pg/mL)
PFOSAA (pg/mL)
EtFOSE-OH (pg/mL)
M566 (pg/mL)
1 10097F
0.0394
1 10105F
0.0181
1 10106F
0.0258
1 10107F 1 10108F
0.0343 0.0253
1 9922M* 1 9930M*
0.0115 0.0134
1 9931M
0.00725
1 9932M
0.0162
i 9933M*
0.0156
II 10121F II 10126F
9.62 19.8
II 10136F II 10140F
5.96 6.27
II 10142F II 9961M * II 9964M
13.1 34.8 30.4
II 9965M *
74.9
II 9967M
25.1
II 9970M *
38.9
III 10155F* III 10156F
87.8 76.1
III 10164F
49.6
III 10172F
68.4
III 10177F
42.2
III 9997M
108
III 9999M * III 10001M III 10002M
178 94.9 113
III 10004M
130
IV 10187F IV 10194F
89.5 73.4
IV 10203F
126
IV 10211F
99.7
IV 10214F
98.3
IV 10019M IV 10024M*
302 477
IV 10029M*
296
IV 10033M IV 10034M
272 249
* = Tentative values, initial volume was <0.5 mL
<LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb) <LOQ (4.79 ppb)
0.0682 0.112 0.0663 0.0507 0.0665 0.0962 0.188 0.114 0.147 0.165 0.328 0.352 0.265 0.325 0.335 0.574 0.579 0.480 0.393 0.465 0.461 0.576 0.651 0.670 0.569 0.613 0.553 0.610 0.804 0.637
<LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb) <LOQ (20.5 ppb)
1.59 4.55 1.18 0.690 2.09 1.40 5.86 1.86 1.26 5.55
9.9 6.91 4.66 8.17 4.58 11.8 18.7 12.1 10.4 14.9 8.00 10.6 19.0 10.2 12.3 22.5 40.5 28.8 24.5 31.4
<LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb)
<LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb)
<LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb) <LOQ (24.9 ppb)
1.86 4.19 0.952 1.15 2.45 4.69 5.18 4.54 3.44 6.11 43.3 22.3 18.0 17.0 17.9 29.1 73.6 25.1 38.1 37.8
39.0 25.6 39.6 28.8 33.8 71.3 102 94.9 90.9 56.7
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Analytical Report: FACT TOX-013 LRN-U2095
Table 12. Reported Fluorochemical Levels in Sera Analyses in Study FACT TOX-013 (continued)
Dosage G roup
Specimen ID
PFOS (pg/mL)
V NR
NR
V NR
NR
V NR
NR
V NR
NR
V NR
NR
V 10042M
238
V 10044M
235
V 1004SM
326
V 10051M
162
V 10054M
182
NR * Sample not received or reported
* = Tentative values, initial volume was <0.5 mL
PFOSA (pg/mL)
NR NR NR NR NR 0.791 0.972 0.897 0.574 0.669
PFOSAA (pg/mL.)
NR NR NR NR NR 25.2 20.7 26.8 14.0 15.8
EtFOSE-OH (pgftnL)
NR NR NR NR NR <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb) <LOQ (36.2 ppb)
M556 (pg/mL)
NR NR NR NR NR 62.4 55.6 93.8 30.7 55.5
Table 13. Reported Fluorochemical Levels in Liver Analyses in Study FACT TOX-013
Dosage Group
I I 1 1 1 1 l 1 I l 1 l i 1 l II il il il il ll il II ll ll ll ll ll II
II
Specimen ID
10097F 10105F 10106F 10107F 10108F 9922M 9930M 9931M 9932M 9933M 10097M 10105M 10106M 10107M 10108M 10121F 10126F 10136F 10140F 10142F
9961M 9964M 9965M 9967M 9970M 10121M 10126M 10136M 10140M 10142M
PFOS(pg/g)
0.149 <LOQ 0.121 <LOQ <LOQ 0.585 0.816 0.836
1.04 1.01 0.281 0.242 0.226 0.221 0.251 25.1 22.9 39.6 23.7 22.1 116 102 89.9 80.7 87.3 54.7 67.8 53.7 28.0 71.5
PFOSA (pg/g)
<LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <1.00 <LOQ <10Q <LOQ <LOQ <LOQ <LOQ <LOQ 0.514 0.708
1.40 0.601 0.508 4.49 4.10 2.88 3.88 5.42
1.90 2.65 2.35 1.22 2.06
PFOSAA <pg/g)
<LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <UOQ <LOQ <LOQ <LOQ <LOQ
2.68 4.34 5.01 2.67 2.67 11.0 15.6 9.79 6.62 10.4 5.87 14.6 9.44 2.82 6.55
M556(pg/g)
<LOQ <LOQ <UOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ <LOQ
1.19 1.75 2.86 1.55 1.41 12.8 10.2 11.6 8.95 11.6 5.39 7.75 4.64 3.27 4.58
.aa a s
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Analytical Report: FACT TOX-013 LRN-U2095
Table 13. Reported Fluorochemical Levels In Liver Analyses in Study FACT TOX-013 (continued)
Doug* Group
III III III III III III III III III III III III III III III IV IV IV IV IV IV IV IV IV IV IV IV
IV IV IV V V V V V
Spadnw n ID
10155F 10156F 10164F 10172F 10177F 9997M 9999M 10001M 10002M 10004M 10155M 10156M 10184M 10172M 10177M 10187F 10194F 10203F 10211F 10214F 10019M 10024M 10029M 10033M 10034M 10187M 10194M 10203M 10211M
10214M 10042M 10044M 1004SM 10051M 10054M
p f o s (pg/g)
102 130 179 119 105 415 234 498 257 386 89.0 219 203 188
164 240 344 255 264 831 791 556 781 556 226 277 448 457 344 1218 1356 1132 1063 1054
PFOSA (pg/g)
PFOSAA (pg/g)
2.22
2.24
1.94
2.17
2.88
10.8 9.41
8.67
8.29
8.41 5.11
6.14
6.26 6.20
6.91
2.80
4.08
1
3.14
3.39
4.56
12.8
11.0
11.2
12.6
11.0
6.36
9.96
9.93
11.4
8.11
16.4
13.0
10.9
9.80
I 10.8
15.6 20.2 22.7 11.9 20.0 73.5 28.6 85.2 34.2 64.9 12.0 27.3 29.4 33.7 17.1 31.1 51.5 49.5 46.6
51.4 122 148 86.2 129 135 27.4 45.5 76.0 56.2 60.0 188 206 150 157 165
M 5S 6(P 04)
7.17 7.64 8.41 5.87 8.16 63.5 39.3 66.4 38.0 54.6 11.5 33.3 43.1 29.9 31.4 19.8 26.8 26.6 27.5 29.3
84.5 97.5 78.2 117 82.2 39.9 39.5 67.8 65.4 64.5
128 152 133 118 161
past
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Appendix E: Data Spreadsheets
BACKTOMAIN
Analytical Report: FACT TOX-013 LRN-U2095
: 3M Environmental Laboratory
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Analytical Report: FACT TOX-013 LRN-U2095
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BACK TO MAIN
FACT-TOX-013 Argus# 418-009
Sfltdy: Prod*U N i mbef(Tesl Substance): Matrix: McKxSiRcviofl: Analytical Equipment System Number Instrument SoitwareA'enioo: Filename:
R-StfJMvdViltG-
Slope Y-Intocept Dates nTExtraction/Analyst Dales of Amtyiii/Analyin:
Atgtu 418-009. Two-Generation Reproduction Study of ElFOSE-QH in Rats EtFOSB-OH (T-63165) Rat Seram ETS-8-4.I 4ETS-8-5.1 Davey 070799. Soup 020199 MassLytu 3 J Sec lifting to the right S ee A ttachm ents See Attachment* S e e A tttc ta n e c ii 1/12/W RWW 10/14/99,10/20/99.10/22/99,03/16/TO MMHAAS
Date of Data Radaclkm/Analyst:
10/1*99,10/21/99, KV25/99,03/17/00.03/23A MMH/lAS
Sample Dal
RATSERA F6____________
Gcwp Do k Method Elk Matrix Bk QC-lOOfpb C rew pl Cannot O.Omg/fcgiiay 0 rog/m L
GrMip2 1 mg/kg/ti*y 0.Ir tty r L
GranpS 5 mgAgAUy l.Qmg/mL
Group 4 (0.0 mgAg/day
2.0mg/mL
G toupj 15.0 mg/fcjidxy
3.0mg/mL
Snraple
10129-H2O Blk-1 10129-H2O BQl-2 RBS10129-Sen BA-1 RBS10129-Sen Bk-2 RBS 10129-MS-1 RBS10129-MSD-1
10097F m o st* 10106F 10107F 10I08F 9922M** 993<W-* 9931M 9932M 9933M10121F 10126F 10136F 1014QF 10142F 9961M** 9964M 996SU *' 9967M 9970M10155F** 10I56F 10164F 10172F 10I77F 9997M 9999MIG001M 10002M 10004M 10I87F 0194F 10203F 102UF I02MP 10019M 10024M** 10029M* 10033M 10034M NR NR NR NR NR 1042M I0044M 1004M 10051M 10054M
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I 1
1
1
l 1 0.70 0.7 0.50 0.60 .60 0.40 0.40 oso 050 0.40 0.70 0 so 1.00 1.00 0.60 0.40 0.50 0.20 0.50 0.40
0.40 0.60 0.70 0.60 0.70 0.70 0J0 0.70 0.60 0.50
0.60 0.80 0.70 0.80 0.70 0.60 OJO 0.40 0.50 0.50 NR NR NR NR NR 0.80 080
OJO TOO 0.90
prosa Caw.
HW* 0.00 1.57 0.00 1J7
220 215 1.46 1.28
1.15 1.44 1.14 2.41
1.89 144 1.41 133 47.7 56.0 66.3 50.7 39.9 38.5 94.2 217 73.6 66.2 131 211 185 195 235 402 174 336 236 233 276 461 456 536 398 368 166 244 402 318
NR NR NR NR NR 633 778 448 574 602
Cam tati itjun *f PFOSA
a g t e L or % Rc
cLOQ <LOO cLOQ <UX)
89* 87*
cLOQ <LQQ -cLOQ cLOQ
cLOQ <LQQ cLOQ <LOQ cLOQ <LOQ
0.0682 0.112 0.0663 0.0507 0.0665 03)962 0.188 0.114 0.147 ai6S 0328 0.352 0.265 0J25 0.335 0574 0579 0.480 0J93 0.465 0.461 0.576 0.651 0.670 0569 0.613 0553 0.610 0.804 0.637 NR NR NR NR NR
0.791 0.972 0.897 0.374 0.669
Mra FFOSA *W L <LOO <LOO
88%
<LOQ
<LOQ
0.0727
0.142
0J21
0.498
0585
0.643
NR
0.780
USD SM -D cr. MS/MSD RD
NA NA 3*
NA NA
NA NA
31.7 0.0231
26.4 0.0376
10.3 0.0331
15.8 0.0786
14.1 0.0826
14.8 0.0951
NR NR
208 0.163
Luot of Qucitation (LQQ): PFOS * $55 nj/mL, PFOSA = 4.79 ppb, PPOSAA 205 ppb, EtFOSE=36.2 ppb, PINK Sample not received nor repaced.
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"* Tentative varies, InitljJ volume below 0.5 oL. LAC 08/31/00
nalytical Study: FACT-TOX-013 LRN-U295
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3M Medical Department Study: T6316.5
Analytical Report: FACT TOX-013 LRN-U2095
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3M Environmental Laboratory
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3M Medical Department Study: T-6316.5
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BACK TO ICAIN
Analytical Report: FACT TOX-013 LRN-U2095
FACT-TOX-013 Argus#418-009
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3M Environmental Laboratory
3MEnvironmental Laboratory
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3M Medical Department Study: T-6316.5
3M Medical Department Study: T6316.5
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Analytical Report: FACT TOX-013 LRN-U2095
Tentative vahiet, initial vfltamebe1Ow0.5mL. LAC08/31/00
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BACKTOMAIN
Analytical Report: FACT TOX-013 LRN-U2095
Appendix F: Example Calculations
Formula Used for Sera Analyses in Study FACT TOX-013
AR (ng/mL) x DF x SC x FV (mL) x 1.0 pg x p c = R eported C oncentration (pg/mL) EV(mL) 100"ng
Calculation Used for Group 4, Anim al ID 10033M 340 ng/mL x 500 x 0.9275 x 1 mL x 1.0 pg x 0.864 = 272 pg/mL 0.5'mU 10(J0"Hg~
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%)
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Analytical Study: FACT TOX-013 LRN-U2095
Appendix G: Contract Lab Report
This appendix includes the following contract laboratory report:
Battelle M em orial Institute, Study Number: N003604-D, 2 (N-Ethylfluorooctanesulfonamido)-ethano! in Two Generation Rat Reproduction, (65 pages)
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BIOLOGICAL SAM PLE ANALYSIS Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FA C T 060998.1
C lB 3 ll6 ll6 Puttins Technology To Work
FINAL REPORT
2 (N-Ethylfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction
SPONSOR
3M Toxicology Services 3M Center
Building 220-2E-02 St. Paul, MN 55144
Testing Facility
Battelle Memorial Institute 505 King Avenue
Columbus, Ohio 43201-2693
Prepared Bv
Patrick L. South, B.S.
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Battelle Study Number: N003604-D 3M Environmental Laboratoiy Study Number: FACT 060998.1
FINAL REPORT
2 (N-EthylfluorooctanesuIfonamido)-ethanoI in Two Generation R at Reproduction
Battelle Senior Program Director
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ii
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BACK TOM AIN
Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
2 (N-Ethylfluorooctanesulfonamido)-ethanol in Two Generation R at Reproduction
EXECUTIVE SUMMARY
Rat liver samples sent to Battelle by 3M Environmental Technology and Services were analyzed by the previously validated method "Method for Analysis of Potassium Perfluorooctanesulfonate (PFOS) in Rat Liver by LC/MS/MS". Samples were extracted and analyzed by High-Performance Liquid Chromatography Mass Spectroscopy (LC/MS/MS) for PFOS, M-556, PFOSAA, and PFOSA content only. Related fluorochemicals mentioned in the analytical method were not investigated.
The results for the concentration determinations in the liver samples from this study are attached as appendices to this report. Concentrations are reported as mass of analyte (fig) per gram of liver tissue extracted.
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Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998. t
QUALITY ASSURANCE STATEMENT
This study was inspected by the Quality Assurance Unit and reports were submitted to the task leader, study director, and associated management as follows:
Phase Inspected
Inspection Date
Date Reported to Battelle Task Leader/ Battelle Management
Date Reported to Offsite Study Director/ Management
Sample weights Sample homogenization Extraction Sample analysis Audit study file Audit final report Audit study file Audit final report
10/12/1999 10/12/1999 10/13/1999 10/13/1999 12/9/1999 12/9/1999 2/21/2001 2/21/2001
11/1/1999 11/1/1999 11/1/1999 11/1/1999 12/9/1999 12/9/1999 2/21/2001 2/21/2001
3/30/01 3/30/01 3/30/01 3/30/01 3/30/01 3/30/01 3/30/01 3/30/01
Date
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Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
GOOD LABORATORY PRACTICES COMPLIANCE STATEMENT
Study Title. 2 (N -Ethylfluorooctanesulfonam ido)-ethanol in Two G eneration R at Reproduction
This study was conducted in compliance with the Food and Drug Administration's Good Laboratory Practice Regulations (21 CFR 58), with the exception that the mass spectrometry data for the liver samples was collected and processed with the MassLynx software system (version 3.1), which was not fully validated. The study was listed on Battelle's Master List o f regulated studies.
Battelle Principal Investigator
M arvin T. Case, DVM, Ph.D. Study Director
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V
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Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
Table of Contents
Page Executive Summary........................................................................................................................................iii Quality Assurance Statement......................................................................................................................... iv Compliance Statement....................................................................................................................................v Table of Contents...........................................................................................................................................vi 1.0 Introduction........................................................................................................................................1 2.0 Reference Substances........................................................................................................................ 1 3.0 Receipt of Samples............................................................................................................................ 1 4.0 Analysis of Samples.......................................................................................................................... 1
4.1 Summary of Method..............................................................................................................1 4.2 Results..................................................................................................................................3
4.2.1 Quality Control........................................................................................................3 4.2.2 Sample Results........................................................................................................3 5.0 Conclusions........................................................................................................................................3 6.0 Acknowledgements............................................................................................................................ 4 7.0 Specimen Storage and Record Archives............................................................................................. 4
List of Tables Table 1. Example of Instrument Parameters Used to Analyze Samples..........................................................2
Appendix A (Results) Summary Results for Rat liver Sample Analysis............................................................................................ A-l
Appendix B (Daily Acceptance Criteria Summary) Daily Acceptance Criteria Summary............................................................................................................. B-l
Appendix C (Sample Inventory List) Sample Inventory List.................................................................................................................................... C-l
Appendix D (Chromatograms) Representative Chromatograms..................................................................................................................... D-l
Appendix E (Protocol, Amendments, and Deviations) Protocol, Amendments, and Deviations.......................................................................................................... E-l
Appendix F (PFOS Purity Report) PFOS Purity Report.......................................................................................................................................F-l
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1.0 Introduction This report presents a description of the method used to analyze PFOS, M-556, PFOSAA, and
PFOSA in rat liver samples from 3M Study Number FACT 060998.1 (TOX-013) and the results from this analysis. See Appendix E for a copy of the study protocol, amendments, and protocol deviation reports).
2.0 Reference Substances The analytical reference substances for this study were supplied by 3M. The following lot number or
tracking number designations apply: PFOS (lot 171), M-556 (TN-A-2203), PFOSAA (TN-A-1283) and PFOSA (L-15709). Note that based on information supplied to Battelle from 3M, PFOS has two equivalent names. The name appearing on the Material Safety Data Sheet and bottle label is potassium perfluoroalkyl sulfonate. The name more commonly used by 3M in analytical methods and correspondence is potassium perfluorooctanesulfonate. The latter name will be used in this report. See Appendix F for purity data supplied by 3M to Battelle. The reference substances were stored at room temperature.
The surrogate standard was 1H,1H,2H,2H-Perfluorooctane sulfonic acid, lot number 59909, supplied by ICN. The surrogate standard was stored at room temperature.
3.0 Receipt of Samples Samples were received frozen and intact at Battelle, from 3M Environmental Technology and
Services, in one batch on October 6,1999. Samples were generated by Argus Research under protocol number 418-009. See Appendix C for a copy of the inventory list. The samples were stored at approximately -20C.
4.0 Analysis of Samples
4.1 Summary of Method
Samples were analyzed by a previously validated method (Battelle study number N003604-A). The current version of the method is attached to this report in Appendix E. Samples were analyzed by LC/MS/MS, and an example of the instrument parameters is listed in Table 1. Note that only PFOS, M-556, PFOSAA, and PFOSA (and the surrogate) were quantitated. The other related fluorochemicals, although present in the stock solutions, were not monitored. Quadratic regressions weighted 1/x were used to construct the calibration curves.
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Table 1. Example of Instrument Parameters Used to Analyze Samples
LC/MS/MS Svstem
Autosampler HPLC pumps VIass spectrometer Analytical column Mobile phase components Gradient profile
Injection volume Flow Column temp HPLC pressure MS source Desob ation as Nebulizer gas Source block temp Desolvation temp Cone voltage
Collision energy Collision gas Multiplier Resolution Ions monitored
Total run time Approximate retention times:
Make: Gilson
Model: 234
Make: Gilson
Models: 305 and 306
Make: Micromass
Model: Quattro LC with Z-spray source
Keystone Betasil C 18,5 pun, 2 x 50 mm, Part No. 055-701 -2
Component A: Ammonium acetate(2mM):methanol, 60:40, v:v
Component B: Ammonium acetate(2mM):methanol, 5:95, v:v
Time, min
%B Flow. mL/min
0 0 0.3 1 0 0.3
4.5 100 0.3 6 100 0.3 6.1 100 0.6
8.5 100 0.6 9 0 0.3 11 0 0.3
10 UL
LC column flow at start split to 20 pL/min into the MS
Ambient
Approximately 840 psi at gradient start
Electrospray, Negative Ion
Nitrogen at ~575 L/hr
Nitrogen at ~80 L/hr
140C
250C
70 V for SS, PFOS
20 V for M-556, PFOSAA, PFOSA
40 eV Argon, gas cell, at ~ 2 .5 x l0 mb
650 V
12.0 for MSI; 10.0 for MS2
427> 81 MRM transition for SS
499>99 MRM transition for PFOS
556>78 MRM transition for M-556
584> 169 MRM transition for PFOSAA
498>78 MRM transition for PFOSA
11 minutes
SS: 4 min
PFOS: 4.2 min
M-556: 4.4
PFOSAA: 4.5
PFOSA: 5
2
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4.2 Results
Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
4.2.1 Quality Control
System suitability acceptance criteria were established during the method validation and are included in Appendix E, Section IX Acceptance Criteria. Relevant statistics from each sample set are provided in Appendix B. Representative chromatograms are given in Appendix D.
4.2.2 Sample Results
The results of the sample analyses as well as a method detection limit determination are presented in Appendix A. All liver samples were initially extracted as undiluted homogenates. After the data were reviewed, dilutions of the homogenates were performed in order to bring analyte concentrations within the calibration range. The first analysis that provided acceptable data for an analyte was used in reporting. Four extraction sets were required to provide data for each analyte.
The limit of quantitation is defined as the concentration of the lowest standard which meets acceptance criteria for accuracy (25% RE; see Appendix E for definition). The notation BLOQ denotes "Below Limit of Quantitation" for samples that had concentrations lower than the theoretical concentration for the 0.13 pg/g calibration standard. The notation ALOQ denotes "Above Limit of Quantitation" for samples that had concentrations higher than the theoretical concentration for the 13 pg/g calibration standard. Samples that were initially ALOQ were diluted with blank liver homogenate and re-extracted. Samples that were expected to be ALOQ were first diluted with blank liver homogenate before extraction. The "Corrected PFOS Cone" presented in the results tables is the concentration found for the diluted sample multiplied by its dilution factor (final volume + sample homogenate volume).
The method detection limit (MDL) of PFOS was calculated in Battelle study N003296F to be0.0173 pg/g from the analysis of 7 replicate preparations of 0.13 pg/g calibration standard. The MDL was calculated by multiplying the standard deviation of the found concentrations of the 7 reps by 3.143; the Signal-to-Noise (S/N) ratio was calculated by dividing the mean found concentration of the 7 reps by their standard deviation. The method of MDL determination was provided by the Sponsor.
5.0 Conclusions
All analyses met acceptance criteria unless otherwise noted.
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6.0 Acknowledgements
Acknowledgement of principal contributors participating in the performance of this study at Battelle is presented in the following list.
Participant
Title
Jon C. Andre, Ph.D. Richard W. Slauter, Ph.D., D.A.B.T. Patrick L. South, B.S. Gerke H. van der Zwaag, M.S.
Battelle Principal Investigator Senior Program Director Mass spectroscopist Sample preparation chemist
7.0 Specimen Storage and Record Archives
See Appendix E, protocol amendment 3 for records archival information. All residual liver samples, extracts, and unused test article will be disposed of or returned to the Sponsor as directed by the Sponsor.
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APPENDIX A -RESULTS
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Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
PFOS, M-S56, PFOSAA, PFOSA IN RAT LIVER
BATTELLE STUDY:
N003604-D
SPREADSHEET SOFTWARE:
DATA ENTERED:
MANUALLY
EXCEL 97
Sample
Dose Group
Animal Number
1 1 9922 2 1 9930 3 1 9931 4 1 9932 5 1 9933 a 2 9961 7 2 9964 8 2 9965 9 2 9967 10 2 9970 11 3 9997 12 3 9999 13 3 10001 14 3 10002 15 3 10004 16 4 10019 17 4 10024 18 4 10029 19 4 10033 20 4 10034 21 5 10042 22 5 10044 23 5 10045 24 5 10051 25 5 10054 28 1 10097 27 1 10105 28 1 10106 29 1 10107 30 1 10108 31 2 10136 32 2 10140 33 2 10142 34 2 10121 35 2 10126 36 3 10177 37 3 10155 38 3 10156 39 3 10164 40 3 10172 41 4 10187 42 4 10194 43 4 10203 44 4 10211 45 4 10214 46 1 10097 47 1 10105 48 1 10106 49 1 10107 50 1 10108 51 2 10136 52 2 10140
53 2 10142 54 2 10121 55 2 10126 56 3 10177 57 3 10155 58 3 10156 59 3 10164 60 3 10172 61 4 10187 62 4 10194 63 4 10203 64 4 10211 65 4 10214
BLOQ = BELOW LIMIT O F QUANTITATION
Analysis date key:
Sample Type
Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal
Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Fetal Maternal Maternal Maternal Maternai Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal
PFOS Cone,
pg/fl
6.77E-01 9.44E-01 9.68E-01 1.20E+00 1.17E+00 1.34E+02 1.18E+02 1.04E+02 9.34 E+01
1.01 E+02 4.80E+02 2.71 E+02 5.76E+02 2.97E+02 4.47 E+02 9.62E+02 9.15E+02 6.43 E+02 9.04E+02 6.43 E+02 1.41E+03 1.57E+03 1.31 E+03 1.23E+03 1.22E+03 1.72E-01
BLOQ 1.40E-01
BLOQ BLOQ 4.61 E+01 2.74E+01 2.56E+01 2.90E+01 2.65E+01 1.21 E+02 1.18E+02 1.50E+02 2.07E+02 1.38 E+02 1.90E+02 2.78E+02 3.98E+02 2.95E+02 3.06E+02 3.25E-01 2.80E-01 2.61 E-01 2.56E-01
2.90E-01 6.21 E+01 3.24E+01 8.28E+01 6.33E+01 7.85E+01 1.77E+02 1.03E+02 2.53E+02 2.35 E+02 2.18E+02 2.62E+02 3.21 E+02 5.19E+02 5.29E+02 3.98E+02
M-556 Cone,
pg/g
BLOQ BLOQ BLOQ BLOQ BLOQ 1.28E+01 1.02E+01 1.16E+Q1 8.95E+00 1.16E+01 6.35E+01 3.93E+01 6.64E+01 3.80E+01 5.46 E+01 8.45 E+01 9.75E+01 7.82E+01 1.17E+02 8.22E+01 1.28 E+02 1.52E+02 1.33E+02 1.186+02 1.616+02 BLOQ BLOQ BLOQ BLOQ BLOQ 2.86E+00 1.55E+00 1.41E+00 f 1.19E+00 1.75E+00 8.16E+00 7.17E+00 7.64E+00 8.41E+Q0 5.87E+00 1.98E+01 2.68E+01 2.66E+01 2.75E+01 2.93E+01 BLOQ BLOQ BLOQ BLOQ
BLOQ 4.64E+00 3.27E+00 4.56E+00 5.39E+00 7.75E+00 3.14E+01 1.15E+01 3.33E+01 4.31 E+01 2.99E+01 3.99E+01 3.95E+01 6.78E+01 6.54E+01 6.45E+01
PFOSAA Cone,
pg/g
BLOQ BLOQ BLOQ BLOQ BLOQ 1.10E+01 1.56E+01 9.79E+00 6.62E+00 1.04E+01 7.35E+01 2.86E+01 8.52E+01 3.42E+01 6.49E+01 1.22E+02 1.48E+02 8.62E+01 1.29E+02 1 .35E+02 1.88E+02 2.0SE+02 1.50E+02 1.57E+02 1.65E+02 BLOQ BLOQ BLOQ BLOQ BLOQ 5.01 E+00 2.67E+00 2.67E+00 2.68E+00 4.34E+00 2.00E+01 1.56E+01 2.02E+01 2.27E+01 1.19E+01 3.11 E+01 5.15E+01 4.95E+01 4.66E+01 5.14E+01
BLOQ BLOQ BLOQ BLOQ
BLOQ 9.44E+00 2.82E+00 6.55E+00 5.87E+00 1.46E+01 1.71 E+01 1.20E+01 2.73E+01 2.94 E+01 3.37E+01 2.74E+01 4.55E+01 7.60E+01 5.62E+01 6.00E+01
PFOSA Cone,
pg/g
BLOQ BLOQ BLOQ BLOQ BLOQ 4.49E+00 4.10E+00 2.88E+00 3.86E+00 5.42E+00 1.08E+01 9.41 E+00 8.67E+00 8.29E+00 8.41 E+00 1.28E+01 1.10E+01 1.12E+01 1.26E+01 1.10E+01 1.64E+01 1.30E+01 1.09E+01 9.80E+00 1.08E+01 BLOQ BLOQ BLOQ BLOQ BLOQ 1.40 E+00 6.01 E-01 5.08E-01 5.14E-01 7.08E-01 2.88E+00 2.22E+00 2.24E+00 1.94E+00 2.17E+00 2.80E+00 4.06E+00 3.14E+00 3.39E+00 4.56E+00 BLOQ BLOQ BLOQ BLOQ
BLOQ 2.35E+00 1.22E+00 2.06E+00 1.90E+00 2.65 E+00 6.91 E+00 5.11 E+00 6.14E+00 6.26E+00 6.20E+00 6.36E+00 9.96E+00 9.93E+00 1.14E+01 8.11 E+00
Normal font = October 13,1999 Underline = October 16,1999 Bold - October 18,1999
Bold Underline - October 20,1999
All sam ples undiluted All sam ples undiluted All sam ples diluted All sam ples diluted
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Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD DETECTION LIMIT (MDL) RESULTS STUDY: N003604-D ANALYSIS DATE AND INSTRUMENT ID: DATA ENTERED: SPREADSHEET SOFTWARE:
130ct99; 9053 Electronically and manually Excel 97
All cones in (ig/g
Calculated Concentration o f Replicate 1 Calculated Concentration o f Replicate 2 Calculated Concentration o f Replicate 3 Calculated Concentration o f Replicate 4 Calculated Concentration o f Replicate 5 Calculated Concentration o f Replicate 6 Calculated Concentration o f Replicate 7
Mean Concentration Std. Dev.
M -556 0.1269 0.1220 0.1433 0.1368 0.1670 0.1225 0.1190
0.1339 0.0170
Spike Level
0.1331
MDL determined S/N
0.05345 7.88
Valid
Yes
LOQ (det. from 10 x std.dev. "noise") LOQ (det. from cal curve low std.)
0.17005 0.1331
Curve Coeff of Determination
0.9978
Date analyzed Method
130ct99 LC/MS/MS
Key 1 - Spike Level too high; Spike Level must be < lOx MDL 2 - Spike Level too low; Spike Level must be > MDL 3 - S/N too low; S/N must be > 5 4 - Coeff o f Det o f calibration curve unacceptable
PFO SA A 0.1484 0.1281 0.1484 0.1605 0.1661 0.1513 0.1385
0.1488 0.0128
0.1334
0.04011 11.66
Yes
0.12763 0.1334
0.9937
130ct99 LC/MS/MS
PFO SA 0.1167 0.1325 0.1521 0.1441 0.1490 0.1382 0.1413
0.1391 0.0119
0.1315
0.03725 11.74
Yes
0.11853 0.1315
0.9891
130ct99 LC/MS/MS
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APPENDIX B-DAELY ACCEPTANCE CRITERIA SUM MARY
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PFO S e t al IN R A T LIV E R
STU D Y NUMBER: N003604-D
DATA ENTERED: MANUALLY
SO FTW ARE:
EXCEL 97
REGRESSION PARAMETERS
Analysis Date October 13, 1999
October 16, 1999
October 18, 1999
October 20, 1999
Analyte
PFOS M-556 PFOSAA PFOSA
PFOS M-556 PFOSAA PFOSA
PFOS M-556 PFOSAA PFOSA
PFOS PFOSA
X ' Coeff
-0.0164 -0.00646 0.00232
-155
-0.00389 8.88E-06 0.00360
-250
0.00393 0.0221 0.00370 -71.9
0.00615 -153
X Coeff
Intercept
1.88 0.627 0.0970 1.50E+04
-0.0772 -0.0213 -0.00398
-872
1.82 0.578 0.0931 1.73E+04
-0.00920 -0.00807 -0.00321
-986
2.17 0.312 0.0853 1.23E+04
-0.0905 -0.00269 -0.000603
-722
2.07
-0.0381
1.68E+04 -1.02E+03
Coeff of Determination
0.984 0.998 0.994 0.989
0.999 0.998 0.998 0.990
0.985 0.984 0.993 0.996
0.997 0.999
Comments/ Deviations
One rep of Cal pt 1 excluded One rep of Cal pt 1 excluded One rep of cal p t7 excluded One rep of cal pt 5 excluded
One rep of cal pt 7 excluded One rep of pts 1, 3, 5 excluded
One rep of pts 4, 7 excluded
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PFOS et al IN RAT LIVER
STUDY NUMBER: N003604-D
DATA ENTERED: MANUALLY
SOFTWARE:
EXCEL 97
QC Results Analysis Date
October 13, 1999
Analyte PFOS
M-556
PFOSAA
PFOSA
October 16,1999
PFOS M-556 PFOSAA PFOSA
October 18, 1999
PFOS M-556 PFOSAA PFOSA
October 2 0 ,199 9
PFOS PFOSA
QC Level, ng/m L
10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16
10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16
10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16 10 3.3 0.7 0.16
10 3.3 0.7 0.16 10 3.3 0.7 0.16
%RSD
6.6 3.8 7.9 3.1 4.7 11.7 3.0 11.5 6.5 5.1 8.7 14.5 16.9 9.2 5.8 4.6
5.5 4.5 8.3 9.7 3.4 2.7 8.0 17.7 4.3 2.1 7.4 17.7 11.6 6.1 8.0 8.6
8.7 11.7 7.0 15.1 14.7 17.0 21.2 28.8 14.7 11.4 15.1 21.3 16.7 11.5 12.6 6.4
2.4 2.0 3.7 5.3 2.3 4.0 2.3 3.4
%RE
-4.0 -4.6 -6.4 -12.2 -2.4 2.5 -6.6 -5.1 -1.7 -10.4 -13.0 8.2 -4.3 -2.1 1.3 3.9
14.8 15.5 8.8 -2.7 -2.8 5.7 -3.7 4.5 -0.8 -2.9 -7.0 15.8 -3.9 5.4 -4.4 13.7
-4.5 -1.7 -20.3 -6.1 -2.9 23.4 11.6 15.4 0.8 -1.8 -20.6 -18.5 0.0 13.7 0.0 16.0
-8.0 -12.0 -21.7 -16.0 -1.6
6.5 1.9 11.5
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APPENDIX C-SAMPLE INVENTORY LIST
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Study TOX-013, Argus 418-009. Sample Information for shipment to Battelle
Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
43
44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
Sample Description F0-9922-orpl-M
FO-9930-grpl-M F0-9931-grpl-M F0-9932-grpl-M FO-9933-gipl-M F0-9961-grpll-M F0-9964-grpll-M F0-9965-grpll-M F0-9967-grpll-M F0-9970-grpll-M F0-9997-grplll-M F0-9999-grplll-M FO-10001-grplll-M F0-10002-grplll-M F0-100O4-qrplll-M F0-10019-grplV-M F0-10024-grplV-M FO-10029-grplV-M F0-10033-grplV-M F0-10034-grplV-M F0-10042-grpV-M F0-10044-grpV-M FO-10045-grpV-M F0-10051-grpV-M F0-10054-grpV-M FO-10097-flrpl-F F0-10105-grpl-F F0-10106-grpl-F F0-10107-grpl-F F0-10108-grpl-F F0-10136-grpll-F F0-10140-grpll-F FO-10142-grpll-F FO-10121-grpH-F FO-10126-prpH-F F0-10177-grpUI-F F0-10155-grplll-F F0-10156-grplll-F F0-10164-grplll-F F0-10172-grplil-F F0-10187-grplV-F F0-10194-prplV-F
F C M 0 20 3-grp lV -F
FO-10211-grplV-F F0-10214-grplV-F FO-10097-grpl-F F0-10105-grpl-F FO-10106-grpl-F FO-10107-grpl-F FO-10108-grpi-F FO-10136-grpll-F FO-10140-prpll-F FO-10142-grpH-F F0-10121-grpll-F FO-10126-grpH-F F0-10177-grplll-F F0-10155-qrplll-F F0-10156-grplll-F FO-10164-grplH-F FO-10172-grplll-F F0-10187-grplV-F F0-10194-grplV-F F0-10203-grplV-F FO-10211-grplV-F F0-10214-grplV-F
Sample Type Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Uver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Liver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Liver Fetal Uver Fetal Liver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Uver Fetal Liver Fetal Liver Fetal Uver Fetal Uver
Fetal U ver
Fetal Uver Fetal Liver Maternal Rat Uver Maternal Rat Liver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Liver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Uver Maternal Rat Liver Maternal Rat Uver
!
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APPENDIX D-REPRESENTATIVE CHROMATOGRAMS
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APPENDIX E-PR O TO C O L, AM ENDM ENTS, AND DEVIATIONS
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fir r c . - 7-/t - l)/-$
3M Environmental Laboratory
Protocol - Analytical Study 2(N -Ethylperfluorooctanesulfonam ido)-ethanol in
Two G eneration R at Reproduction
In-vivo study reference number: Argus 418-009
Study number: FACT 060998.1 Test substance: 2(N-Ethylperfluorooctanesulfonamido)-ethanol (N-EtFOSE-OH)
Name and address of Sponsor:
Marvin Case 3M Toxicology Services 3M Center Building 220-2E-02 St. Paul, MN 55144
Name and address of testing facility: 3M Environmental Technology and Services 935 Bush Avenue, Building 2-3E-09 St. Paul, MN 55106
Experimental start date: Expected termination date: December 31,1998 Method numbers and revisions:
FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry
FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC-
Electrospray/M ass Spectrom etry
' FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry
FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry
Author: Lisa Clemen
Kris Hansen Study Director
Date Marvin Case
Date
Sponsor Representative
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_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
1.0 P urpo se _________________________________________________________________________ The analytical portion of this dosing study is designed evaluate the levels of perfluorooctane sulfonate (PFOS), or another metabolite of 2(N-ethylperfluorooctanesulfonamido)-ethanol (NEtFOSE-OH) designated by the study director, in the liver of the parent and subsequent generations of the test system, or in the serum as necessary.
The in life portion of this study was conducted at Argus Research Laboratories.
2.0 Reg ulato ry C o m pliance___________________________________________________________ This study is conducted in compliance with the Food and Drug Administration Good Laboratory Practices regulation as stated in 21 CFR 58. Any exceptions will be noted in the final report.
3.0 Te st M aterials_____________________________________________________________________ 3.1 Test, control, and reference substances and matrices
3.1.1 Analytical reference substance: Potassium perfluorooctanesulfonate (PFOS), lot #217
3.1.2 Analytical reference substance matrix: Rat liver and serum 3.1.3 Analytical control substance: None 3.1.4 Analytical control substance matrix: Rat liver and serum 3.2 Source of materials 3.2.1 Analytical reference substance: 3M Specialty Chemical Division; traceability
information will be included in the final report 3.2.2 Analytical reference substance matrix: Argus Research Laboratories;
traceability information will be included in the final report 3.2.3 Analytical control matrix:
3.2.3.1 Rat liver - Argus Research Laboratories; traceability information will be included in the final report; or Rabbit liver - Covance Laboratories; traceability information will be included in the final report
3.23.2 Rat serum - Sigma Chemical Company; traceability information will be included in the final report
3 3 Number of test and control samples. Liver samples for testing were received from 40 test animals and 10 control animals. Serum samples will be tested at the discretion of the Study Director.
3.4 Identification of test and control samples: The samples are identified using the Argus Research Laboratories identifiers, which consist of a letter followed by the Argus project number, the animal number, the group designation, and the draw date.
2
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Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
3.5 Purity and strength of materials: Characterization of the purity and identity of the reference material is the responsibility of the Sponsor.
3.6 Stability of test material: Characterization of the stability of the test material is the responsibility of the Sponsor.
3.7 Storage conditions for test materials: Test materials are stored at room temperature. Samples are stored at -20 10 C.
3.8 Disposition of test and/or control substances: Biological tissues and fluids are retained per GLP regulation.
3.9 Safety precautions: Refer to the material safety data sheets of chemicals used. Wear appropriate laboratory attire, and follow adequate precautions for handling biological materials and preparing samples for analysis.
4.0 E xperim ental - Overview_________________________________________________
Tissues from animals dosed as described in Argus Research Laboratories Protocol #418-009 are received for analysis of fluorine compounds. At the discretion of the Study Director, a series of analytical tests will be performed on select tissues.
Initially, all liver samples will be analyzed for PFOS by electrospray/mass spectrometry (ES/MS). On the basis of findings from these analyses, additional sample matrices may be evaluated or other metabolites may be targeted. If additional analysis is performed, a protocol amendment will be written.
5.0 E xperim ental - Analytical Methods________________________________________
5.1 FACT-M-1.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Liver for Analysis Using HPLC-Electrospray/Mass Spectrometry
5.2 FACT-M-2.0, Analysis of Fluorochemicals in liver Extracts Using HPLCElectrospray/Mass Spectrometry
5.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry
5.4 FACT-M-4.0, Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry
6.0 D ata Analysis______________________________________________________________________
6.1 Data transformations and analysis: Data will be reported as the concentration (weight/weight) of fluoride per tissue or sample, or of PFOS per unit of tissue or fluid.
6.2 Statistical analysis: Statistics used may include regression analysis of the serum . concentrations over time, and standard deviations calculated for the concentrations within
each dose group. If necessary, simple statistical tests, such as Student's t test, may be applied to evaluate statistical difference.
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_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
7.0 M a in ten a n c e o f R aw D a ta a n d Rec o r d s_______________________________________ 7.1 The following raw data and records will be retained in the study folder in the archives
according to AMDT-S-8: 7.1.1 Approved protocol and amendments 7.1.2 Study correspondence 7.1.3 Shipping records 7.1.4 Raw data 7.1.5 Electronic copies of data 7.2 Supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: 7.2.1 Training records 7.2.2 Calibration records 7.2.3 Instrument maintenance logs 7.2.4 Standard Operating Procedures, Equipment Procedures, and Methods 7.2.5 Appropriate specimens.
8.0 References ____________________________________________________________________ 8.1 3M Environmental Laboratory Quality System Chapters 1,5 and 6 8.2 Other applicable 3M Environmental Laboratory Quality System Standard Operating
Procedures
9.0 A ttachm ents___________________________________________________________________
9.1 F A C T -M -l.O , Extraction o f Potassium Perfluorooctanesulfonate or Other A nionic
Surfactants from Liver for Analysis Using HPLC-Electrospray/M ass Spectrom etry 9.2 FACT-M-2.0, Analysis of Fluorochemicals in Liver Extracts Using HPLC-
Electrospray/Mass Spectrometry 9.3 FACT-M-3.0, Extraction of Potassium Perfluorooctanesulfonate or Other Anionic
Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry 9.4 FACT-M-4.0, Analysis of Ruorochemicals in Serum Extracts Using HPLC-
Electrospray/Mass Spectrometry
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_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
V ersion 1.0 ' Study No.s
Analyst/Sate: _
Revisions to the method
D ru e o l'R e v is io n
R e v ised bv
A pprov cd bv
IBI^B^^BIB^BHBB
BIB^Bk^^^Bh^^^Bb b b b ^ b b b b h b b b b B B B ^ B B B i BBBBBH^HB b b b b b b b BflHBHHB^BHBHBB iB B B B B B b b b b b b b b b
W ritten by:
Patrick L. South
_________ D ate:
'
Approved by:
C<
J d t l c . A n d re ,. P h D .
_____________ D ate: Gm j j J t
f
O>
;er, B io n a ly d c a l C h e m istry
3MEnvironmental Laboratory
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1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
Version L0
Study No.: Analyst/Date: _
L Su m m a ry
The extraction and analysis o f potassium perfluorooctanesulfonate and related fluorochemicals in rat liver is performed. Calibration standards are prepared by spiking blank liver homogenate w ith solvent standards tram two independently-prepared stocks. The calibration standards are fortified with surrogate standard, buffered, and extracted with ethyl acetate. The organic phases are evaporated to dryness and reconstituted in methanol for analysis by LC/MS/MS.
EL P u r p o se
To extract and analyze potassium perfluorooctanesulfonate and related fiuorochemical compounds found in Sprague-Dawley ra t liver.
in . Samples
See Chain o f Custody records if applicable.
IV. General Instructions
Calibrate all required balances according to the SOP on balance usage.
M ake equivalent dilutions w hen the volume needed varies from the volum e stated in the
method.
Label ail standard and reagent solutions as specified in the appropriate SOP. I f you intend to
reuse a solution for future tasks, be sure tire label includes the preparation date and study
number for which the solution was initially prepared.
Sign on the final page afth is method to signify that you have followed the method as written,
all materials and reagents are current, and all equipment has been properly calibrated. If you
deviate from the method, document the change, and obtain the approval o f the unit manager,
study director, or task leader as soon as possible.
Initial and date all data entries on the page on which they were made. If only one person enters all data on a single day, the documentation may be marfe in a single location on that page.. I f multiple staff make entries, the additional entries must be initialed and dated by the
person malting tire entry.
Line-outs or NA denotes "Not Applicable".
The method is written in general chronological order, but the sequence ctf steps may be altered
if the analyst deems it appropriate, unless the order for certain activities is specified.
Stocks will be used fo r the duration o f the study unless consumed or unless stability is
considered suspect
No correction will be made for purity o r salt content o f any test article but PFOSAA.
a Use glass volumetric, Eppendorf repeater, or positive-displacement pipets for dispensing
methanolic solutions.
a C ontact with Teflon by the test article should b e minimized.
.
V. Materials
See Table 1 for all required chemicals, reagents, and solvents. Use Table 1 fo r documentation. Check all labels carefolly to ensure that all materials are not expired and that they are the proper purity o r grade.
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Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
. METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
. V ersion 1.0
Study N<w___________________________ A n a ly it/D a te :_____________________________
M aterials
Use
Potassium Perfluorooctanesulfbnate (PFOS) lH ,nU K t2H Perfluorooctane Sulphonic A dd
M-536
. MS70
PFOSAA
PFOSA
PFOSEA
R at Liver
Analytical Standard
Surrogate Standard
Analytical Standard Analytical Standard Analytical Standard Analytical Standard Analytical Standard
M atrix
Ammonium Acetate, N ILC O ,
Sodium Hydroxide, NaOH
Tetrabutylammonium Hydrogensulfate (TBA),
rCH,(CH,),LN<HSCM Sodium Carbonate, NajCOj Sodium Bicarbonate, NaHCOi Ethyl Acetate
Mobile Phase Reagent Prep E xtra Prep
E xtra Prep E xtra Prep E xtra Prep
T ab let. M ateriali
Supplier
Grade or
P uri tv
3M
ICN
3M 3M 3M 3M 3M Harlan
SpragueDawley
Storage Temp Room Temp
Room Temp
Room Temp Room Terno Room Temp Room Temp Room Temp --20C
RT
RT
RT
Lot o r ID
RT RT RT
Methanol M illi-Q Water
pH 7 Buffer pH 10 Buffer
Mobile Phase, Stocks, WS
Reagent Prep, Mobile Phase
pH meter calibration pH meter calibration
Millipore
ASTM Typel
RTRT
RT
RT
I
RT means Room Temperature.
V L E q u ipm en t
See Table 2 for all required major pieces o f equipment Use the table to document the actual piece (e.g. make, model) o f equipm ent Check calibration of all equipment requiring calibration (e.g. balances) to ensure it is current
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METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
- - Version 1.0
S tudy N o .:___________________________
Analyst/Daie:_______________________
Equipm ent Analytical Balance
Weight Set
Use W eigh Standards or
Reagents Calibrate Balance
Table 2. Equipm ent M anufacturer
Model.
XLm-SN
1
Pipettor
Pipet Samples
Pipettor
Pipet Samples
Pipettor Pipettor
Pipet EtOAc extraction phase Pipet Reagents, WIS
Eppendorf - Repeater
Vortexer-
M ix Samples
Freezer (-20C)
Refrigerator a-9Q
Centrifuge
Store QCs, Blank Liver
Store Buffer, Stocks
Phase separation
Test Tubes Centrifuge Tubes
Test Tubes Transport tubes Magnetic stirrer Orbital Shaker
E vaporator
Liver sample homogenization Extract Samples
Evaporate Extracts
Store QCs
Stir matrix Extract Samples
E vap orate E xtracts
Stockwell Scientific Blue Falcon
Blue Falcon
Elkay
Polypropylene, 15 mL
Polypropylene, 15 mL
Polypropylene, 12 x 7 5 mm 5 mL
polypropylene
Z yxn ark
Turbovap L V
SW8599 2096 . 2002
127-T160-56P
Syringe Filters
Filter Extract
Homogenizer pH meter Electrode
Volumetric Flasks, Class A
Volumetric Pipets, Gass A
Transfer Pipets. Plastic
Grind liver Determine Buffer pH Determine Buffer pH
M ake Volumetric Dilutions
M ake Volumetric Dilutions
Transfer Extracts to Centrifuge Filters and LC Inserts
NA NA Samco
NA NA
NA NA
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_ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LTVER BY LC/MS/MS
V e n a n 1.0 Study No.:
A n aly jt/D ate:
VEL PROCEDURE
A. Preparation of 2 mM Ammonium Acetate
Weigh 0.1300 + 0.0020 g o f ammonium acetate and transfer to a 1000-mL volumetric flask. Dissolve the solid in w ater and dilute to volume with water. Solution may be used fo r one month stored a t room temperature.
Actual mass o f ammonium a c e ta te :___________ Actual final v o lu m e :__________ D ate o f prep aratio n :______________________ Study No: ____________
B. Preparation of~29% Sodium Hydroxide Solution
Weigh 200 2 g of sodium hydroxide into a beaker. Add 500 mL o f MOli-Q w ater and m ix to dissolve. Cool and transfer to a polypropylene bottle for storage. Solution may be stored for 6 months a t room temperature.
Actual m a o f sodium hydroxide: _ _ _ _ _ _ Actual volume M illi-Q w a t e r __________ D ate o f prep aratio n :______________________ Study N o :________________
C. Preparation of --2.9% Sodium Hydroxide Solution
'
A dd 10 mL o f--29% Sodium Hydroxide Solution to a 100-mL volumetric flask and dilute to volume w ith M illi-Q water. Transfer to a polypropylene bottle for storage. Solution m ay be stored fo r 6 m onths a t room temperature.
Actual volume o f ~29% NaOH so lu tio n :___________ Actual final volume: __________ D ate o f prep aratio n : _________________________ _ Study N o :________________
'
D. Preparation of Tetrabutylammonium Hydrogensulfate (TBA) Solution, 0.5 M, (pH 10)
pH Meter Calibration
pH buffer 7 pH buffer 10
pH reading:________ pH reading:________
Add 1 6 9 i Ig a fT B A to -S O O m L a fM illi-Q w a te rin a b e a k e r. Adjust the pH to 10.00
t 0.02 using -55-60 mL o f 29% Sodium Hydroxide Solution, dilute to 1000 m L wife
. Milli-Q water, and mix. Adjust the pH to 10.00 * 0.02 using -2.9% NaOH and mix. Transfer to a polypropylene bottle for storage. Solution may be used for one month stored at room temperature, b ut the pH m ust be checked n rio r to each use. Adjust to
pH 10.0 i 0.02 with 2.9% Sodium Hydroxide Solution as necessary.
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METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
' Version 1.0 Study No.: `
A nalyst/D ate:_____________________________
Actual m ass o f T B A :___________ Actual final v o lu m e :_________ . Actual final p H : ___________ Date o f p rep aration:_____________________ Study N o :________________ pH after techedring and/or readjusting:_________
E . P re p a ra tio n o f 0.25 M C a rb o n a te B uffer
Weigh 26.5 1 0.1 g o f sodium caihonate and 21.0 * 0.1 g o f sodium bicarbonate and
transfer to the same 1000-mL volumetric flask. Dissolve the materials in M illi-Q water, dilute to volume w ith Milli-Q water, mix, and transfer to a polypropylene bottle for storage. Solution may be used for 1 month when stored refiigerated.
Actual m ass o f sodium carbonate: __________ Actual m ass o f sodium bicarbonate:__________ Actual final volume: __________ Date o f prep aratio n :_____________________ Study N o :________________
F . P re p a ra tio n o f M obile P h ase
Component A: M ix together 600 mL of 2 mM ammonium acetate and 400 mL of methanoL Solution may be used fo r 1 month when stored at room temperature.
Actual volum e o f 2 m M ammonium ace ta te:________ mL Actual volume o f methanol: ________ mL D ate o f prep aratio n :_____________________
Study N o :________________
Component B: M ix together 50 mL of 2 mM ammonium acetate and 950 mL of methanoL Solution may be used for 1 month when stored at room temperature.
Actual volume o f 2 niM ammonium acetate:
Actual volume of methanol:
ml.
Date o f preparation:_____________________
Study N o :________________
mL
G . P re p a ra tio n o f S tock S u rro g ate S tan d ard a n d W orking S urrogate S ta n d ard (W SS)
1. Stock Surrogate Standard (250,000 ng/mL):
W eigh 25 a 2 mg o f 1H, 1H, 2H, 2H,-pcrfluorooctane sulphonic a d d and transfer to a 100-mL volumetric flask. Dissolve in methanoL dilute to volume with m ethanol, and mix. Store refiigerated, protected fiom U V light
Actual W eig h t:_________________ Actual Dilution V olum e:____________________
D ate o f P reparation:___________________ Study No: __________________
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Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
V ersion 1.0
S tudy N o .:____________ ;______________ A naiyst/D ate:_____________________________
2. WSS (1000 ng/mL):
D ilute 100 /JL ctf stock, surrogate standard to 25 mL with m ethanol and mix..
Actual Volum e o f Stock Internal S tandard:____________ Actual D ilution V olum e:____________________ D ate o f P re p a ra tio n :___________________
H . Preparation of Calibration Solvent Stocks and Working Standards
Standard
Stodc 1A Stodc IB Stock 2A Stodc 2B Stock 3A Stock 3B Stock 4A Stock 4B Stodc SA Stodc SB Stodc 6A
1. Solvent Stocks:
F o r each analyte weigh the specified amount of standard (independently weighed as A and B replicates) listed in Table 3 and transfer into separate volum etric flasks. Dissolve in methanol, dilute to volume w ith methanol, and m ix well. Store refrigerated, protected from UV light.
2. M ixed Solvent Stocks:
.
P ipetthe specified amount of each analytical standard Replicate A as listed in
Table 3 and transfer into a single volumetric flask. Dissolve in methanol, dilute
to volum e w ith methanol, and m ix well. Store refrigerated, protected from UV
lig h t Repeat the process with Replicate B stocks. Themixedsolventstocksare - - usedtopreparethe working standards.
D ate o f p re p ara tio n :_____________________ Study N o :________________
3. W orking Standards (WS):
Dilute the mixed stocks and working standards with m ethanol as specified in Table 3 and m ix well.
D ate o f preparation:__________________________________
Source
PFOS PFOS M-SS6 M-556 M570 M570 FFOSAA PFOSAA PFOSA PFOSA PFOSEA
Table 3. Calibration Solvent Stocks and W orking Standards
Target
Actual Amount
Target
Actual
A m ount Analytical Std. Final Vul Final Vol.
Studi, or WS
imL)
(mL)
50 x 1 mg
10
25 0.S mg it.::
10
'
1
SO x 1 mg g g a S is s S s S m K S
10
25 t 0.5 mg i r>i
50 x 1 mg *
Esc:i f H'lp;j| .m g *
10 10
. cs?r. 't-'SS j
25 * 0.5 mg 93 x 1 mg
46 x 0.5 mg 50 x 1 mg
fell0: B:i3wSf
mg
10
10
10 :Vii:i:
10 ^5Hs:
:?
25 x 0.5 mg
mg*
10
50 x 1 m g ___________ S S _
10
Nominal Conc
(n/mL> 5,000,000 2,500,000
5,000,000 2J00.000 5,000,000 2,500,000 5.000,000 2.500,000
5.000.000 2,500,000 5.000,000
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Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM
PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
V ersion 1.0
-
S tudy N o .:__________
A nalyst/D ate:_____________________________
Stock 6B Mixed Stock A Mixed Stock B WS I
W S2
WS3 WS 4 WS 5 WS6 WS 7
FFOSEA
Stocks 1 thru 6 Rep A
Stocks I thru 6RepB
Mixed Stock A
Mixed Stock B
WS 1 W S2 WS 3 W S4 W S5
25 0.5 mg
5 mL each**
5 mL each**
1 mL**
-------.n iL e a c h
ty [ip.:
7> mL _
1 mL**
mL
2 mL** 2 mL** 2.5 mL** 2.5 mL** 2 mL**
~
mL
mL
l
10
*Ks:r.:
tin 2.500.000
50
SS:luf!:::: ' :H?ltj:Vtur.t
500,000
50 ~>*wftJ?!i:f.H:: 250,000 >M>;2 52*
25 :xtrssHx?:::;i:-: i5'y.>i 20,000
fincans:::s:::s::;:
25 SUHilwSttttRl 10,000 Bt::trPrj; :H:;H:*::iT:
10 4000
10 g S S I s S
2000
10
:t"HJt!:
1000
10 500
10 ~j*&**!;Ifi *
200
* Weigh all analytical standards to a t least the nearest 0.01 mg. ** Use volumetric or positive-displacement pipers).
L Preparation of Calibration Standards and Blanks
l. Liver homogenate
Prepare blank liver homogenate in bulk by weighing approximately 40 g o f
blank liver into a 500 mL Nalgene bottle containing 200 mL of Milli-Q water. Grind to a homogeneous suspension. Aliquot into approx 30 mL portions for frozen (approx -2 0 Q storage.
Actual M ass o f L iven_____________ Actual volume o f w ater____________ D ate o f p r e p : ____________________
S t u d y : ______________________
Determine density o f calibradon/QC matrix:
M IX HOMOGENATE THOROUGHLY and determine the mass in milligrams o f 10 replicate weighings o f 1 mL portions of the THOROUGHLY MIXED homogenate. M K HOMOGENATE IMMEDIATELY PRIOR TO EACH ALIQUOT REMOVAL.
Replicate# Mass (inai
a
2. Liver Calibration Standards
Prepare each Ever calibration standard by adding 0.45 mL o f undiluted liver homogenate (STIR HOMOGENATE WHILE ALIQUOTING) into a 15 mL extraction tube and adding 50 pL o f WS or MeOH. Prepare triplicate cal standards and 6 blanks. See Table 5 for volumes. The diluted liver density is assumed to be approximately 150 mglmL. M ix well.
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_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LCM S/M S
Vernon 1.0 Study No.:
Anxlyst/Date: _
Cal Std/Blank
1 2 3 4 5 6 7 Blank
Source
W Sl WS 2 WS 3 W S4 WS 5 WS 6 WS 7 MeOH
T ab les. Calibration Standards and Blanks
T ara et Voi Actual Vol
Target
Actual Nominal
0-L)
(aL)
Final Vol Final Vol Cone
(mL)
(mL)
(ns/m U
50 0.5 2000
50 n s g g p i
50 xri: 50 tgri |t 50 1k^SSH:*3 rt:
0.5 0.5 0.5 0.5
1000 ;:Sb: :hi 400
200
100
50 [X " i-j *st1' "
0.5
50
50 0.5 20
50
1 0.5
<: <
0
Nominal Cone (ua/u) 13 6.6 2.6 1J 0.66 0.33 0.13 0
Date o f preparation o f cal stds/blank:______________________________
J. Preparation of Quality Control Liver Samples (QCs)
1. Quality Control Working Standards
Dilute the following source volumes methanol in volumetric flasks and mix well. Prepare fresh when used. Actual volumes are in parentheses.
Soin ID OC WS 1 QCW S2 QC WS 3 QCW S4
Z
Source Mixed Stock A Mixed Stock B
QC WS 1 QCW S2
w.
-W
T abled. QC WS Preparation
Vol Source. mL
Final Vol. mL
mm
IC
--? .
50 (
)
Preparation o f Quality Control Liver Samples
Cone, mi/mL 15,000 5000 1125 250
Prepare each QC in bulk by filling the volumetric flask approximately h a lf full with undiluted liver homogenate (STIR HOMOGENATE WHILE
ALIQUOTING), adding the appropriate QC WS, mixing, and diluting to volume w ith undiluted Uver homogenate (STOR. HOMOGENATE WHILE
ALIQUOTING). M IX THOROUGHLY and dispense Z5-m L aliquots into polypropylene tubes and store at approximately -2 0 C
T able 7. Q C Preparation
QC
Source
Vol Source. mL
Final Vol. m L Cone. n*a/mL Cone. U!T2
1 OCWS 1
llS H t S iiiM iS i
1500 1 10
2 QC WS 2
500 3.3
3
QCWS 3
Z5f `
112.5
0.7
4
OCWS 4
Z5f
)
25 0.16
Date o f QC prep:____________________ Study.______________________
K. Preparation of MS Check Standard for System Suitability
Pipet 250 itL o f WS 2 a t--10,000 ng(mL and Z 5 mL of WSS at -1000 ng/mL in
methanol into the same 50-mL volumetric flask. Dilute to volume with MeOH and mix.
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BACK TOMAIN
Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
Version L0 . Study N o .:______________________________
A nalyst/D ate:________________________________
L. Preparation of homogenizer recovery liver samples
.
M.
To determine the recovery from the homogenization process, nnhom ogeaized blank liver will be fortified in duplicate a t 3 concentration levels and homogenized as follows. This needs to be done every day that homogenization o f study samples is performed.
1. Place approximately 0.3 g o f unbom ogenized blank liver into each o f 6 ,1 5 mL
polypropylene centrifuge tubes. Record weights o f liver.
X A dd 100 pL o f WS 1 ,3 , and 4 (one WS per duplicate tubes) to prepare
- fortifications at approximately 4, 0.8, and 0.4 pg/g.
3. Multiply the mass ofliver in g by 2.5 and add this manym L ofwater.
4. Homogenize each liver sample, and rinse homogenizer probe with another
volume of w ater used in step 3, adding rinse to homogenized sample.
.
5. Clean homogenizer w ith MeOH between samples.
6. Cap and vortex homogenate for use in extraction.
Preparation of Dilation Check Sample
1. Place 2.95 mL o f undiluted liver homogenate (STIR HOMOGENATE WHILE
ALIQUOTING) into a 15 mL extraction tube and add 50 pL o f M ixed Stock A.
X Dilute 50 pL of step 1 solution (VORTEX SOLUTION WHILE
ALIQUOTING) with 0.45 mL of undiluted liver homogenate (STIR.
HOMOGENATE WHOLE ALIQUOTING) in 3 ,1 5 mL extraction tubes.
3. This sample should be prepared for extraction only on days when study samples
will be diluted and extracted.
.-
N. Homogenization of study samples
1. Place approximately 0.5 g o f unhom ogenized study sample liver into a 15 mL polypropylene tube. Record weights ofliver.
X Multiply the mass of liver in g by X5 and add this m anym L of water. 3. Homogenize each liver sample, and rinse homogenizer probe with another
volum e o f w ater used in step X adding rinse to homogenized sample. 4. Clean homogenizer with MeOH between samples. 5. Cap and vortex homogenate for use in extraction.
O . Analysis Standards, Blanks, QCs, and Samples
1. M IX LIVER HOMOGENATES THOROUGHLY BEFORE ALIQUOTING
andpipet 500 /jL o f each QC (4 replicates per level), and other samples being
extracted into 15-mL polypropylene extraction tubes. T he cal stds a n d blanks
are already aliquoted.
X To the B la n k s -IS (3 reps), add 100 jL o f MeOH and vortex. 3. T o the Blanks + IS (3 reps) and to the remaining samples, add 100 jL W SS and
vortex. 4. Add 0.5 mL o f 0.5 M T B A (pH 10) to all tubes and vortex briefly. 5. Add 1 mL o f 0X5 M carbonate buffer and vortex briefly. 6. Add 2 J mL of ethyl acetate. Place the tubes sideways on the orbital shaker at a
' setting o f 300 for -2 0 minutes. 7. Centrifuge tubes at a setting o f3500 rpm for -2 0 minutes to separate layers. 8. Transfer 2 mL o f the top organic layer to a dean polypropylene tube.
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BACK TOTIAIN
Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
Version 1.0 Study N o.:___________________________
Anaiyjt/Date: ______________________________
9. Evaporate to dryness tm der nitrogen at a setting o f 30*C f o r - 6 0 minutes. 10. Reconstitute the residues in 500 mL o f methanol with vortexing. 11. Syringe-filter extracts into antosnmplcr vials for analysis. Store vials refrigerated
(up to 1 month) if LC/MS/MS will not be performed the same day. Since 3-day room temperature extract stability was demonstrated during validation, the - extracts o f the cal stds, blanks, and QCs may be reused for up to 3 days after ' their initial preparation if held at room temperature (recap the vials if reusing).
Date of cal std/blank extract prep:_________________________ Date of QC extract prep :_________________________________
P. LC/MS/MS Analysis
.
tfP y fa fo
8 .
1. Use the system conditions specified in Table ys. The conditions which are
designated may be m odified by the analyst to produce acceptable peak shape.
LC/MS/MS System
Table 8 - LC/M S/M S Conditions
A utosam pfcr HPLC Pumps Mass Spectrom eter Analytical column Mobile Phase C om ponents G radient profile
Injection volume Plirn split
M a k e :_____
Model:
ID:
Make: _____
Model:
ID:
Make:
Model:
ID:
Keystone Betasil C18, 5m. 2 x 50 mm. Part No. 055-701-2,
S/N: :L o c
Component A Ammonium acetate methanol, 60:40, vrt Comoonent B: Ammonium acetate:methanol. 5:95. vy
'
Tim e, min
%B Flow. mL/min
0 0 0.3
1 0 0.3
4.5 100 0.3
6 100 0.3
6.1 100 0.6 8.5 100 0.6
9 0 0.3 '
11 0 0.3
10 uL (
______________________ :_________________
LC column flow split to *30 ML/min ( uL/m inl into the M S a t run start
Column Temp
Ambient
lU'LC Pressure
1000 psi at gradient s ta rti
osi)
'
MS Source
Electrosnrav. Negative Ion
Desolvation as Nebulizer as Source Block Temp DcmiIs ation Temp
Nitrogen at 575 L/hr (
Nitrogen at 80 L/hr (
*140C(
o
*250Cf
a
L/hr) L/hr)
Cone voltage
*70 V ( ____
*20 v r
V) P F O S , - V) PFOSA, PFOSAA PFOSEA M-556, M570
Collision energy
*40 eV (___ eV) PFOS, I t PFOSA PFOSAA, M-556, M570
Collision as
*30 eV (
eV) PFOSEA
1 Argon at *2.5 x HT* mb gas cell (
mb)
M ultiplier Restitution
I 650 V ( ___ Y)___ ____ .____________________________________ ____________ 1
1 *12.0 for M SI (
): *10.0 for M S2
)
jv/ouajkcto " s r" ; * fe e ffir lV f
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BACK TOM AIO
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM
PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
Version 1.0
Study N o .:___________________________
Analyst/Date:_______________________
Ions m onitored
Total run time A pproxim ate retention times:
427>81 MRM transition for Surrogate Standard (SS)
1 499>99 MRM transition for PFOS
1 556>78 MRM transition for M-556
5 7 0 1 6 9 MRM transition for M570
1 584>169 MRM transition for PFOSAA
1 498>78 MRM transition for PFOSA
526>169 MRM transition for PFOSEA
*11 minutes (
SS: 4 min ( PFOS: 4.3 min (
min) min) ___ m in)
M-556: 4 a m in ( ____min)
M 570:4.6 min ( __ min)
PFOSAA: 4.7 m in e
min)
PFOSA: 5.1 min (______ min)
PFOSEA: 5.7 m in 1
min)
* Parameters that may be changed by the analyst Actual values in ( ).
2. The above conditions should be suitable for the Micromass Quattro L C (S/N
9053). Modifications may be necessary if another Micromass Quattro Series
spectrometer is used. Split the flow post-column via a Keystone BIO-tee or
sim ilar device.
3. Calibrate the mass spectrometer using a suitable reference compound, o r verity
that the calibration is suitable by visual inspection (on the tune page) that a
suitable mobile phase ion is still accurately determined. Resolution may need to
be higher than that used for analyzing samples.
-'
4. To check the proper performance o f the instrument, inject the instrument check
standard. The results should be comparable to a recent injection if available.
5. Use an automated chromatography integration software system to collect the
output from the analysis.
6. Loading O rder See the loading report from the automated chromatography
integration software system.
7. Make single injections of each cal standard, QC, study sample, or blank. Make
at least 4 injections of the instrument check standard. 8. Run set sizes should typically not exceed 80 injections due to instrument
response roll-off considerations. Longer runs may be performed, but they pose a
risk o f yielding unacceptable curve results.
VUL CALCULATIONS
1. Spreadsheet Software: ____________________ V e rsio n ________ 2. MS Analysis Software:_______________________V ersio n ________ 3. Calculate the average density of the liver hom ogenate (10 reps) in mg/mL. 4. Using the average density o f the homogenate, calculate its liver density
(mg of liver per mL of diluted homogenate):
Undiluted liver density (mg/mL) - (g o f liver x average density o f hom ogenate)/(g o f liver + g of water)
where g of liver and g o f water are masses used to prepare bulk homogenate; density of water is assumed to b e l g/mL.
Diluted liver density (mg/mL) " Undiluted density + Diln Factor
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Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
' V ersion 1.0 Study N o .:_____________________________
A n aly st/S ate:_______________________________
W here diln fa cto r--2/1.8 * 1.1111 to account for 10% diln o f liver hom ogenate in cal std and QC matrices.
5. Calculate the actual concentration (ng/mL) ofPFOS and other flnorochemicals in the suspensions of calibration standards and QCs by using the mass of analytes and dilution factors only (no liver density correction). Use purity correction for FFOSAA only.
6. falmtnti the actual concentration of PFOS and other flnnmriiMniraR hi liw r fn r rim calibration standards and QCs as follows:
Conc(|ig/g) " Cone (ng/mL) * Diluted Liver density (mg/mL) x 1000 m g/g x 1 0 pg/ng
7. Assure that the integrations o f the peak areas of the test article and surrogate standard are correct Flag manual integrations where performed. Calculate the exact concentration o f each
.- liver standard.
8. Calculate the regression equation relating the peak response ratio (test ardde/SS ) o f each calibration standard (y-axis) to test article concentration in liver (x-axis) for PFOS, M-556, M570, and FFOSAA. Calculate the regression equation relating the peak area of r h calibration standard to test article concentration in liver for PFOSA and PFOSEA. PFOS, M 356, M 570, and FFOSAA are quantitated by using the surrogate standard as an internal standard: PFOSA and PFOSEA are quantitated without reference to the surrogate fexternal
standard calibration curve). Use a quadratic regression weighted V x, origin excluded, for
all analytes.
9. Calculate a determined concentration for each injection of calibration standard, QC, and ' sample using the regression parameters and the peak response ratios or areas.
10. Oatentare the relative error, average relative error, standard deviation, and relative standard deviation for all QCs. Calculate die relative error for each injection of calibration standard.
11. Calculate the average recovery fo r die hnnuigenm r recovery fnTtifirarirma
12. Calculate the relative standard deviation for the PFOS to SS peak area ratio o f the replicate * in jectio n s o f the check, standard.
IX.
a c c e p t a n c e C r it e r ia
A. MS Check Standard (System SuitabDity)
At least 3 injections of the M S Check Standard must provide a %RSD of 10% or less for the PFOS to SS peak area ratio.
B. Calibration Standards
The percent relative errors for the concentration-level averages of the calibration standards should meet die following limits:
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BACK TOTIAIN
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
Version L0 Study No.:
Analyst/Date: _
T able 9. Calibration Standard Acceptance limits
ANALYTE
% Error
PFOS
20 0 5% at LOO)
M-556
20 0 5 % at LOO)
M570
20 0 5 % at LOO)
PFOSAA
20 05% at LOO)
PFOSA
20 0 5% at LOO)
PFOSEA
25 i30% atL 001
Up to 5 calibration standard injections may be excluded from the curve, provided that one injection remains p er level. Removal o f a n entire level may be done if approval is obtained. I f an entire level is-.removed, the samples bracketed by the remaining calibration range will be - considered acceptable. The calibration curve should have a coefficient of determination of 0.97 or better.
C. QCs
The concentration-level average percent relative errors and percent relative standard deviations of the QCs should meet the following limits:
Table 10. QC Acceptance limits
ANALYTE
%
PFOS
20
M-556
20
M570
20
PFOSAA
20
PFOSA
20
PFOSEA
25
Removal of individual values from the QC calculations may be done if accompanied by a reasonable explanation (e.g_, instrument malfunction or Dixon's Q test results).
If die average determined concentration for any QC level exceeds the acceptance limit, the task leader or study director should be n otified . T he ran may be repeated o r a portion o f the run may be considered acceptable. For example, if the low QC fails the stated requirements, samples may be accepted that have concentrations bracketed by die highest calibration standard and a mid-level QC concentration.
D. Homogenizer Recovery and Dilution Check Samples
The avenge recovery across the 3 levels o f homogenizer recovery samples as well as that of the dilution check samples should fill within the range o f 70-130% inclusive. Removal of individual outliers from the calculations may be done if accompanied by a reasonable explanation.
E. Sensitivity (LOQs)
The validated limits o f quantitation are nominally 0.13 pg/g each for PFOS, M-556, M570, and PFOSAA.
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BACK TOM AIN
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LC/MS/MS
V enioB 1.0 S tudy N o .:_____________________________
. ' Anaiyrt/Date: _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
For PFOSA and PFOSEA, the validated LOQs are nominally 0.33 pg/g each. Due to the nature of die preparation of die calibration standards, lower concentrations o f PFOSA and PFOSEA will be carried through the extraction. These lower concentration values will be evaluated with each run set, and may be included in the rgressions if they meet acceptance criteria. I f they are included, study samples which are quantitated to have concentrations below the validated level (nominally 0.33 pg/g) will be appropriately flagged.
F. Specificity
The method suffers from endogeneous matrix interferences a t levels sometimes exceeding 20% o f LOQ. The intercept o f die calibration curve appears to offer some correction for any - effect on quantitations. Acceptai performance (error) o f the lowest used standard, therefore, will be considered sufficient evidence that bracketed study samples are quantified properly.
G. General
The above acceptance criteria indicate that this method is capable o f producing occasional errors outside the normal acceptance criteria of a validated method (15% normally). Where indicated, replicate analyses lessen die impact of these occasional outliers.
X. RESULTS
See attached hard copy o f spreadsheet or see file on network drive.
XL C o m m e n t s
3MEnvironmental Laboratory
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T? t n
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BACK TATIAIN
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
METHOD FOR ANALYSIS OF POTASSIUM PERFLUOROOCTANESULFONATE (PFOS) IN RAT LIVER BY LGMS/MS
Version U> Study No.: -
A nalyit/D ate___
X3L C onclusions
XHL S i g n a t o r e s
A nalysts
________________________________________________ . - - _______________________________________
________________________________________________ ________________________________________________
Technical Review
________________________________________________ '_________________________________________
QC Review
Date: ' D ate
D ate D ate
D ate D ate
D ate
Date
3MEnvironmental Laboratory
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BACKTOM AIN
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
Study Title Combined Oral (Gavage) Fertility Development and Perinatal/Postnatal
Reproduction Toxicity Study ofN-EtFOSE in Rats
PROTOCOL AMENDMENT NO. 1
Amendment Date: July 28,1999
Performing Laboratory 3M Environmental Technology & Safety Services
3M Environmental Laboratory 935 Bush Avenue
S t Paul, MN 55106
Laboratory Project Identification ET&SS FACT-TOX-013 LIRNU2095
3 M Environm ental Laboratory
3MEnvironmental Laboratory
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BACK TOTIAIN
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
Protocol FACT-TOX-013 Amendment 1
This amendment modifies the following portion(s) of the protocol:
1. PROTOCOL READS: The proposed study completion date is listed as 12/31/98.
A m e n d TO READ: The proposed study completion data is 6/30/00.
R e a s o n : The proposed completion date was changed to allow time for analyzing all matrices of interest.
Amendment Approval
Marvin Case Ph.D., Sponsor Representative
Kris J.
Ph.D., Study Director
g/2-/^5 Date
3M Environm ental Laboratory
3MEnvironmental Laboratory
r?
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BACK TOM AIN
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
Study Title
Combined Oral (Gavage) Fertility Development and Perinatal/Postnatal Reproduction Toxicity Study ofN-EtFOSE in Rats
PROTOCOL AMENDMENT NO. 2
Amendment Date:
September 10,1999
Performing Laboratory
3M Environmental Technology & Safety Services 3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106
Laboratory Project Identification
ET&SS FACT-TOX-013 LIRNU2095
i
3 M Environm ental Laboratory
3MEnvironmental Laboratory
r? 0*2
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BACK TOM AIN
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
Protocol F A C T-TO X -013 Amendment 2
This amendment modifies the following portion(s) o f the protocol:
1. P r o t o c o l READS: The protocol states that liver will be extracted and analyzed at the 3M Environmental Laboratory.
AM END TO read: The liver specimens will be extracted and analyzed at Battelle Memorial Institute, 505 King Avenue, Columbus, Ohio 43201-2693.
REASON: The liver specimens will be sent to Battelle Memorial Institute for extraction and analysis due to time constraints in the 3M Environmental Laboratory.
2 . PROTOCOL r e a d s : The protocol states that serum specimens will be extracted and analyzed following methods:
FACT-M-3.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic Surfactants from Serum for Analysis Using HPLC-Electrospray/Mass Spectrometry" FACT-M-4.0, "Analysis of Fluorochemicals in Serum Extracts Using HPLCElectrospray/Mass Spectrometry"
A M END t o r e a d : The serum specimens will be extracted and analyzed following methods:
ETS-8-4.1, "Extraction of Potassium Perfluorooctanesulfonate or Other Fluorochemical Compounds from Serum for Analysis Using HPLC-Electrospray Mass Spectrometry" ETS-8-5.1, "Analysis ofPotassium Perfluorooctanesulfonate or Other Fluorochemical Compounds in Serum Extracts HPLC-Electrospray Mass Spectrometry"
R EASO N: The extraction and analytical methods FACT-M-3.0 and FACT-M-4.0,
respectively, w ere updated on 04/27/99 to ETS-8-4.1 and E T S-8-5.1.
3M Environm ental Laboratory
3MEnvironmental Laboratory
t: D/i
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BACK TOTIAIN
Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
Protocol F A C T -T O X -013 Amendment 2
3 . PROTOCOL r e a d s : The protocol states that liver specimens will be extracted and analyzed following methods:
FACT-M-1.0, "Extraction of Potassium Perfluorooctanesulfonate or Other Anionic
surfactants from Liver for analysis Using HPLC-Electrospray/Mas Spectrometry"
FACT-M-2.0, "Analysis ofFrluorochemicals in Liver Extracts Using HPLC-
.
Electrospray/Mass Spectrometry"
A M EN D TO r e a d : The liver specimens will be extracted and analyzed following method:
Method for Analysis of Perfluorooctane Sulfonate (PFOS) in Rat liver by LC/MS/MS, Version 1.0
REASON: Since the liver extraction and analysis was sub-contracted to Battelle Memorial Institute, this amendment was written to include their liver methods and titles.
Amendment Approval
*
__ _________
Marvin case Ph.D.-, Sponsor Representative
h p f.l isp.
f o b fc k Kristen J. Hansen Ph.D., Study Director
3M Environmental Laboratory
3MEnvironmental Laboratory
T? O C
Date
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BACK TOMAIN
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
Study Title Analytical Study 2(N-Ethylperfluorooctanesulfonamido)-ethanol in
Two Generation Rat Reproduction
PROTOCOL AMENDMENT NO. 3
Amendment Date: October 4,1999
Performing Laboratory 3M Environmental Technology & Safety Services
3M Environmental Laboratory 935 Bush Avenue St. Paul, MN 55106
Laboratory Project identlfcation ET&SS FACT-TOX-013 LIRNU2095
3MEnvironmental Laboratory 3MEnvironmental Laboratory
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_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
* i*
Protocol FACT Tox-013 Amendment Number 3
This amendment modifies the following portion(s) of the protocol:
1. Protocol Reads: Kristen J. Hansen, Ph.D. is the Study Director.
Amend to Read: James K. Lundberg, Ph.D. is the Study Director.
Reason: Original study design has changed due to availability of resources and James K. Lundberg will begin serving as the study director for'FACT-TOX-013 as of 4 October 1999.
2. Protocol Reads: Section 7.1 states that the following raw data and records will be retained in the study folder in the archives according to AMDT-S-8: Approved protocol and amendments; study correspondence; shipping records; raw data; and electronic copies of data. Additionally, Section 7.2 states that supporting records to be retained separately from the study folder in the archives according to AMDT-S-8 will include at least the following: Training records; calibration records; instrument maintenance logs; Standard Operating Procedures, Equipment Procedures, and Methods; and appropriate specimens.
Amend to Read: Section 7 states: "The original data, or copies thereof, will be available at the 3M Environmental Laboratory to facilitate audits of the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including: approved protocol and amendments, study correspondence, shipping records, raw data, approved final report, and electronic copies of data will be retained in the archives of the 3M Environmental Laboratory. All corresponding training records, calibration records, instrument m aintenance logs, standard operating procedures, equipm ent procedures, and methods will be retained in the archives ofthe facility performing each analysis.
Reason: To direct subcontract laboratories in the disposition of the items listed above.
3 M Environm ental Laboratory
3MEnvironmental Laboratory
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_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
Protocol FACT Tox-013 Amendment Number 3
3. Protocol reads: Disposition of test and control substances: Biological tissues and fluids are retained per GLP regulation.
A m end to read:
Specimens will be maintained in the 3M Environmental Laboratory specimen archives. All specimens sent to sub-contract laboratories will be returned to the 3M Environmental Laboratory upon completion of analysis and submission of the sub-contract laboratory(s) final report The specimens will be returned with the following documentation: the signed original chain of custody and records of storage conditions while at the sub-contract facility.
Reason: To define in detail the appropriate disposition of specimens analyzed at subcontract laboratories.
Amendment Approval
T G to L Marv Case, D.V.M., Ph.D., Sponsor Representative
Date
& L J k ----
- t O / Z f U------------------- ^ ___________ ;______________________________
Kristen J. Hansen, Ph.D., Previous Study Director
Date
1 Dale L. Bacon, PfcB., 3M Environmental Laboratory Management
!f f s h 1
Date
3 M Environm ental Laboratory
3MEnvironmental Laboratory
T ? TO
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BACK TOM AIN
Study Title Analytical Study o f 2(N-Ethylperfluorooctanesulfonamido)-ethanol in
Two Generation Rat Reproduction
PROTOCOL AMENDMENT NO. 4
Amendment Date:
20 January 2000
Performing Laboratory 3M Environmental Technology & Safety Services
3M Environmental Laboratory 935 Bush Avenue
St. Paul, M N 55106
Laboratory Project Identification
ET&SS LR N-U 2095 FACT TOX-013
Argus Study: 418-009 3M M edical Department Study: T -6 3 16.5
3M Environmental Laboratory
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Protocol LRN-U2095 Amendment Number 4
This amendment modifies the following portion(s) of the protocol:
1. P r o t o c o l r e a d s : The study director for the present study was identified in the protocol as James K. Lundburg, Ph.D. A m end to read: The role of study director for the present study was reassigned to Marvin T. Case, D.V.M ., Ph.D., as of 20 January 2000. The previous study director, James K. Lundburg, has been reassigned to the role of Principle Analytical Investigator. Reason: The role of study director was reassigned in an effort to ensure compliance with Good Laboratory Practice Standards that outline study personnel requirements (refer to 21 C FR Part 58).
2. P rotocol r ea d s: The sponsor for the present study was identified as Marvin T. Case, D.V.M ., Ph.D. A m end to read: The role of sponsor for the present study was reassigned to John L. Butenhoff, Ph.D., as of 20 January 2000. Reason: To ensure that the study director does not also carry the duties of study sponsor, the sponsor role was reassigned. In this manner, personnel responsibilities and workload are more evenly balanced.
o i l r --__ ________ ___ _____I $ _ L _________ i ____ _ ,,
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Amendment Approval
BACK TOTIAIN
Protocol LRN-U2095 Amendment Number 4
John L ButenhoffPhD ., Sponsor Representative
Fste*t r y to ,
Date
fh k A /M A M arvin T. Case, D. V.M., P hD ., Incoming Study Director
J.P_R-Uaju (h^r
Date V
3 M Environmental I ahnratnrv
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BACK TOM AIN
Study Title Analytical Study o f 2(N-Ethylperfluorooctanesulfonamido)-ethanol in
Two Generation Rat Reproduction
PROTOCOL AMENDMENT NO. 5
Am endm ent Date: August 31,2000
Perform ing Laboratory 3M Environmental Technology & Safety Services
3M Environmental Laboratory 935 Bush Avenue
St. Paul, MN 55106
Laboratory P roject identification FACT-TOX-013
ET&SS LRN U2095 Argus Study: 418-009 3M Medical Department Study: T6316.5
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- Protocol FACT TOX-013 Amendment No. 5
This amendment modifies the following portion(s) of the protocol:
1. PROTOCOL r e a d s : The Principle Analytical Investigator for the present study was identified as James K. Lundberg, Ph.D.
2 . AMEND TO r e a d : The role of Principle Analytical Investigator for the present study was reassigned to Kristen J. Hansen Ph.D.
REASO N: The role of Principle Analytical Investigator was reassigned due to availability of resources.
3 M Fm/ironmantal Laboratorif
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Amendment Approval
BACK TOM AIN
Protocol FA C T TOX-013 Amendment No. 5
John L. Butenhoff, Ph.D., Sponsor Representative
; T C *u
Marvin T. Case, D.V.M., Ph.D., Study Director
Date Date
3 M Fnwirnnmontal I ahnratnrv
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BACK TOM AIO
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
DEVIATION REPORT
Battelle Study Number: N003604-D 3M Environmental Technology and Services Study Number: FACT 060998.1
2 (N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction
TY PE O F D EVIATIO NS: PRO TO CO L
D A TE S O F DEVIATIO NS:
O ctober 18, 1999
N A T U R E O F D E V IA T IO N S: Som e o f the analytical m ethod acceptance criteria w ere not m et for the L C /M S /M S analysis conducted 1 8 0 c t9 9 at B attelle. T hese deviations relate to protocol am endm ent 2 . S ee b elow for sum m ary.
A nalvte
A cceptance criterio n n o t m et
PFOS M -556 M -556 M -556 M -556
PFO SA A PFOSAA
Q C 3 ex ceed ed 2096 error (-2 0 .3 % actual) Q C 2 exceeded 2096 error (23.4% actual) ( Q C 3 exceeded 20% R S D (21.2% actual) Q C 4 exceeded 20% R S D (28.8% actual) D ilution recovery exceeded 130% (131.5% actual w ith 21.6% R SD ) Q C 3 exceeded 20% error (-20.6% actual) Q C 4 exceeded 20% R S D (21.3% actual)
C A U SE O F D E V IA T IO N S: Sam ple preparation and/or L C /M S /M S variabilities over the course o f the sam ple set m ay have contributed to the deviations.
IM P A C T O F D E V IA T IO N S O N T H E ST U D Y : T he errant Q C values w ere bracketed by acceptable QC concen tration le v e ls w h ich dem onstrates that th e calibration curves generally provided good accuracy over tho tested range. T he dilution recovery for M -556 w as not considered to be ex ceed in gly high en ou gh , at o n ly approxim ately 1.5% above the norm al acceptance lev el, to have sign ifican tly im pacted the data.
C O R R E C T IV E A C T IO N :T his protocol deviation sum m ary w as prepared for inclusion in the final report.
APPROVED BY:
Cv
Jon ( t j A ndre, P h .D . B attelle Principal Investigator
Z .- Z .l- 'O i
D ate
f a d . V/Wk
Ja m ea-K . L w ndbar g , P h rD . Study D irector
f r w r U K T . f r s t> V * \ f t p J
I / fa tS .I k Q *- f D ate
N 003604-D Protocol Deviation 1, Page 1 o f 1
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BACK TATIAIN jk
_ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
DEVIATION REPORT
Battelle Study Number: N003604-D 3M Environmental Technology and Services Study Number: FACT 060998.1
2 (N-Ethylpeifluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction
TY PE O F DEVIA TION S: PR O TO C O L
D A TE O F DEVIA TION S:
O ctober 20, 1999
N A TU R E O F D EV IA TIO N S: P F O S QC3 exceeded the 20% R E m ethod requirem ent (actual -2 1 .7 % ). T he dilution recovery check standard did not m eet the 70-130% recovery requirem ent (actuals 5.0% for PFO S and 4.6% for PFO SA ). T hese m ethod deviations relate to am endm ent 2 o f the study protocol.
C A U SE O F D EV IA TIO N S: Sam ple preparation and/or L C /M S/M S variabilities over the course o f the sam ple set m ay have contributed to the QC deviation. Sam ple preparation erro r appears to have been the cause for the dilution recovery results.
IM P A C T O F D E V IA TIO N S O N T H E STUD Y: T he errant Q C value level w as bracketed by acceptable Q C concentration levels w hich dem onstrates that the calibration curves generally provided good accuracy fo r study sam ples over the tested range.
A com parison o f the results obtained fo r the diluted study sam ples from 20O ct99 and previous results th at w ere Slightly L O Q (1 3 0 c t9 9 and 180ct99) dem onstrated good agreem ent betw een the 2 determ inations. T his w ould indicate that the dilution o f the study sam ples w as perform ed correctly 20O ct99 so that no im pact on the quantitations occurred.
C O R R E C T IV E A C TIO N : T his protocol deviation sum m ary w as prepared for inclusion in the final report.
APPROVED BY:
B attelle Principal Investigator
t-T /h o l
D ate
Study D irector
j Zholiii
D ate
N003604-D Protocol Deviation 2, Page 1 of 1 E-36
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BACK TOMAIN
- Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
DEVIATION REPORT
Battelle Study Number: N003604-D 3M Environmental Technology and Services Study Number: FACT 060998.1
2 (N-Ethylperfluorooctanesulfonamido)-ethanol in Two Generation Rat Reproduction
TYPE OF DEVIATIONS: PROTOCOL
DATES OF DEVIATIONS: October 12, 1999 through October 20,1999
NATURE OF DEVIATIONS: The lot number of PFOS used was not 217 as per section 3.1.1 of the protocol. The source of reference substance matrix was not Argus Research Laboratories as specified in section 3.2.2 of the protocol. Initial analyses of liver did not exclusively target PFOS as per section 4.0 of protocol.
CAUSE OF DEVIATIONS: Only PFOS lot number 171 was available at Battelle. Harlan was the supplier of control rat livers used to prepare blanks, standards, and QCs for the analytical portion of the study. All 4 analytes of interest (PFOS, M-556, PFOSAA, and PFOSA) were monitored during each analysis.
IMPACT OF DEVIATIONS ON THE STUDY: PFOS lot number 171 and Harlansupplied liver were both used for Battelle's validation of the analytical method (Battelle study number N003604-A). These materials allowed achievement of the reported method acceptance criteria so that there is no impact on the study. The concurrent analysis of PFOS and metabolites was an efficiency improvement.
CORRECTIVE ACTION: This protocol deviation report was prepared.
APPROVED BY:
C C LhI
Jon(G. Andre, Ph.D. Battelle Principal Investigator
z- zz^ Date
James K. Lundberg, Ph.B. Study Director
N003604-D Protocol Deviation 3, Page 1 of 1
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Battelle Study Number: N003604-D 3MEnvironmental Laboratory StudyNumber: FACT 060998.1
APPENDIX F - PFOS PURITY REPORT
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_ Battelle Study Number: N003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
1^651 778 4226
04/26/99 09:56 0 : 02/03 NO:341
I 2I From: Subject:
Date:
3M SPECIA LTY ADHESIVES & CHEM ICALS ANALYTICAL LABORATORY
Lisa Clemen - (8-5568) - ET&SS- 2-03-09
Request # 57830
Tom K estner - (3-5633) - SA&C Analytical Lab 236-2B-11
Chtmical Characterization ofPOSF-Based Fluorochemicals by `H-NMR & lfF-NMR Spectroscopy
M atch 2 4 ,1 9 9 9 : Prelim inary report for FC-95 (PFOS), lot 171
SAM PLE DESCRIPTION FC-95, lot 171 (PFO S), T N -A -0834; N om inal product = C F irS 0 3(-) K(+) (white pow der)
IN T R O D U C T IO N :
T his sam ple was su b jected to 'H -N M R and 1SF-N M R spectral analyses to determ ine the purity o f the nom inal product and to characterize as m any impurity components as possible.
'AL:
A portion o f the sam ple was accurately weighed, spiked with a known amount o f 1,4-bis(tiifluorom ethyl)benzene (p-HFX), and then totally dissolved in DMSO-d for subsequent analysis by NMR. A 400 M H z 'H -N M R spectrum (# h57830.401) and a 376 M H z t9F-NM R spectrum (# (57830.401) were acquired u sin g a V arian UNITYplus 400 FT-NM R spectrometer. Use o f the p-HFX internal standard was intended to perm it the determination o f the absolute weight percent concentrations of the assigned components without necessarily needing to identify or quantify all the components in the sample mixture.
RESULTS:
The combined N M R spectral data were used to assign all of the major and m ost o f the minor com ponents in this sample as received. The qualitative and quantitative compositional results that were derived from the single trial NM R internal standardization analyses are summarized in TABLE-1 on the following page. 1 have reported both relative and absolute w eight percent concentrations. One possible reason that the absolute wt.% values add up to more than 100% may be due to the fact that I assumed all of the components contained 8 carbons. If there were any sh o rter ch ain h o m o l o g s p r e s e n t ( i . e . , 7, 6, S, e re . c a r b o n s ) , th e n th e a v e r a g e c o m p o u n d m o l e c u l a r w e i g h t s w ould h ave been som ew hat less than those used in the calculations. In general, the f*F-NMR technique is not particularly well suited for identifying or quantifying small amounts of various fluorochemical hom olog impurity components unless the chains are very short. A more complete characterization of any other fluorochem ical homologs would require analysis by electrospray MS or a similar technique.
A dditional w ork would be required in an effort to positively verify the tentatively assigned com ponents listed in T A B L E -1 (denoted by possible). Sm all amounts o f other unidentified impurities are also detected in the N M R spectra, but additional w ork would be required in an effort to identify or quantify these other m aterials.
Copies o f the N M R spectra will be provided for you at a later date. If you have any questions about the results in this initial report for FC-95, lot 171. please let me know. I apologize for the delay in completing this initial work.
Tom Kestner
e: Rick Payfer - SAAC Analytical L a b -2 3 6 -2 B -l!
Fila iUfcrcncs: LC37S30CC061
3MEnvironmental Laboratory
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ETSS 2 3W
March 24,1999
_ Battelle Study Number: N 003604-D 3M Environmental Laboratory Study Number: FACT 060998.1
^ 651 778 4226
04/26/99 09:56 0 :0 3 /0 3 NO:341
SA&C Analytical Lab Request # 37830 Initial R e a o rt fo r FC -95. lo t 171
"
TABLE-1
S am p le: FC-95, lot 171 (PFOS), TN-A-0834 O verall Q uantitative C om positional R esults by 'H /`*F-NMR Internal Standardization A nalyses
S tru ctu ral Assignments
C F3( C F ^ - S 0 3(-) K(+) (Normal chain; assume x =7 for calculation purposes)
NMR Absolute Weight% Concentradona
(initie trial measurement)
70.3%
NMR Relative Weight* Concentrations
(staile trial measurement)
68.6%
C F ^ C F J i -CFCCFjK C F jJj-SO jM K(+)
(Internal mooomethyl branch; assume x+y 5, a * 0, A y * 0, for calculation purposes)
17.7%
173%
(C Fj)jCF-(C Fj)*-SOj(-) X(+) (Isopropyl branch; assume x =3 for calculation purposes)
10.3%
10.2%
Q Fji-CF(CFi)-SOi(-) K M (Alpha branch; assume x * 6 for calculation purposes)
Possible F-SFi-C JV S O jM K(+) (assume x * 3 for calculation purposes)
C F jK C F^-C C C FjJ H C F ^ - S O jH K(+) (Internal gem-dimethyl branch: assume x+y - 4 and x * 0 for
calculation purposes)
3.3% 0.37% 0.16%
3.2% 036% 0.16%
P ossible C F j-S F rC Jv -S O ji-) K(+) (assume x * 7 for calculation purposes)
Probable C iH j rS O j(") K(+) (Hydrocarbon sulfonate salt; assume x a 3 for calculation purposes)
(CF3)3C-<CFj)x-S03(-) K(+) (t-butyl branch; assume x * 4 for calculation purposes)
. 0.11% 0.031% 0.027%
0.10% 0.030% 0.026%
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BACKTOMA IN
Analytical Report: FACT TOX-013 LRN-U2095
Appendix H: Interim Certificate of Analysis
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BACKTO MAIN
Analytical Report: FACT TOX-013 LRN-U2095
INTERIM CERTIFICATE OF ANALYSIS
Revision 1(9/7/00)
C entre A nalytical L aboratories C O A R eference #: 023-018B
3M Product: P F O S ,L o tl7 1
R eference#: SD -009
________________________ P u r ity : 8 6 .4 %
Test Name
Specifications
PurityJ
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 Acids5(IC) 1. TFA 2. PFPA 3. HFBA 4. NFPA
Elemental Analysis6: 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. 0.017 wt./wt.% 2. 0.007 wt/wt.% 3. 1.355 wt/wt.% 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 wt./wt.% None Detected Not Applicable4
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
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B A C K T O MAIN
Analytical Report: FACT TOX-013 LRN-U2 095
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
P urity = 100% - (sum o f metal impurities, 1.39% +LC/MS impurities, 10.60%+Inorganic Fluoride, 0.27%+NMR impurities, 1.00%+ POAA, 0.30%)
Total impurity from all tests = 13.56% Purity = 100% -13.56% = 86.4%
2Potassium is expected in this salt form and is therefore not considered an impurity.
3Purity by DSC is generally not applicable to materials o f low purity. No endotherm was observed for this sample.
4Sulfur in the sample appears to be converted to SO4 and hence detected using the inorganic anion method conditions. The anion result agrees well with the sulfur determination in the elemental analysis, lending confidence to this interpretation. Based on the results, the SO4 is not considered an impurity.
5TFA HFBA NFPA PFPA
Trifluoroacetic acid Heptafluorobutyric acid Nonofluoropentanoic acid Pentafluoropropanoic acid
^Theoretical value calculations based on the empirical formula, CsFi7S0 3 *K+ (MW=538)
This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160).
COA023-018B
3M Environmental Laboratory
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1 -- ' --
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BACKTOM AIN
Analytical Report: FACT TOX-013 LRN-U2095
INTERIM CERTIFICATE OF ANALYSIS
Centre Analytical Laboratories COA Reference #: 023-018R
LC/MS Purity Profile:
Im purity C4 C5 C6 C7
Total
wt./wt. % 1.03 1.56 6.38 1.63 10.60
Note: The C4 and C6 values were calculated using the C4 and C6 standard calibration curves, respectively. The C5 value was calculated using the average response factors from the C4 and C6 standard curves. Likewise, the C7 value was calculated using the average response factors from the C6 and C8 standard curves.
Prepared By: ______________________________
____
David S. Bell
Date
Scientist, Centre Analytical Laboratories
Reviewed B y :_________________________ ;_____
____
John Flaherty
Date
Laboratory Manager, Centre Analytical Laboratories
COA023-018B 3M Environmental Laboratory
3MEnvironmental Laboratory
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,3M M edical D epartm ent Study: T-6316.5 3M Medical Department Study: T6316.5
BACKTOMAIN
Analytical Report: FACT TOX-013 LRN-U2095
Appendix I: Report Signature Page
% /yuy^
Marvin T. Case D.V.M., Ph.D., Study Director
Date
John L. Butenhoff, Ph.D., Sponsor Representative
Date
^
far is
IM /"
Ffil
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3M Medical Department Study: T-6316.5
Analytical Study: FACT TOX-013 LRN-U2095
Appendix J: Amendment 1 to FACT TOX-013 Final Report
TO X-013 Final Study Report Am endm ent 1
Study number: TOX 013
Study title: Analytical Study 2(N-Ethylperfluorooctane sulfonamido)-ethanol in Two Generation Rat Reproduction
Study Director: Marvin T. Case, D.V.M., Ph.D. Amendment date: May 7, 2001
Amendment number: 1
This amendment modifies the following portion of the final report:
A final signed report from Battelle Memorial Institute, presenting the results for PFOS, PFOSAA, PFOSA, and M-556 levels in rat liver specimens, replaces the draft Battelle report in Appendix G.
Liver results in this report are identical to those presented in the original TOX-013 report (Table 13, pages 22-23). As in the original liver data, the PFOS values reported in the Battelle report were corrected by 3M for purity of the reference standard material.
The final Battelle report differs from the draft report in the following ways: All signature pages are signed and dated. The Quality Assurance Statement page has four additional audit dates added. Table of Contents page numbers were corrected. Two Battelle participants were eliminated from page 4, `Acknowledgements.'
T h e storage and archive instructions (page 4) are now found in the 3 M T O X -0 1 3
protocol amendment 3. Inclusion of 3M TOX-013 protocol amendments 4 and 5, thus changing the total
number of pages. Minor wording changes.
Other changes to the TOX-013 report include: The cover page was updated to reflect the total number of pages and the title was changed to say "Amended Final Report." The Table of Contents was updated to reflect the added amendment.
The additional audit date of the Amended Analytical Laboratory Report TOX013 was added to the Quality Assurance Statement.
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Approved by:
BACK TAM AIN
Analytical Study: FACT TOX-013 LRN-U2095
Kristen H. H ansen, P h.D ., Principal Analytical Investigator
O S-/3 0 /0 1
D a te
M arvin T. C ase, D .V .M ., P h.D ., Study Director
D a te
Bill R e a g a n , P h .D ., E n v iro n m e n ta l L a b o ra to ry M a n a g e r
D a te
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