Document k9yZXnD32LDNr46JX5dEGaYeB

f i k k U . in s ' 497 Supplement 1 DuPont-11418 TRADE SECRET Study Title H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 Laboratory Project ID: DuPont-11418 Te st Gu idelin es: U.S. EPA Health Effects Test Guidelines OPPTS 870.3100 (AUG-1998) Auth o r : Susan A. MacKenzie, V.M.D., Ph.D., D.A.B.T. Study C om pleted o n: August 1,2003 S upplem ent 1 C om pleted o n : March 19 ,2 0 0 4 P erfo rm ing L a bo rato ry: E.I. du Pont de Nemours and Company Haskell Laboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050 Work Request Number Service C ode Num ber Page 1 of 97 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This supplement was conducted in compliance with U.S. EPA TSCA (40 CFR part 792) Good Laboratory Practice Standards, which are consistent with the OECD Principles of Good Laboratory Practice (as revised in 1997) published in ENV/MC/CHEM(98)17 and MAFF Japan Good Laboratory Practice Standards (59 NohSan Number 3850). Applicant / Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Director: XAsiJL*'__________ Susan A MacKenzie, V.M.D., Ph.D., D.A.B.T. Senior Research Toxicologist Date Applicant / Sponsor:____________________________________________ __________ DuPont Representative Date -2Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations QUALITY ASSURANCE STATEMENT Haskell Sample Number(s): 25425 Dates of Inspections: Supplement No. 1: March 1-2,2004 Dates Findings Reported to: Study Director: March 5, 2004 Management: March 5,2004 Reported by: Staff Quality Assurance Auditor Supplement 1 DuPont-11418 Date -3 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 CERTIFICATION We, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Approved by: Scott E. Loveless, Ph.D. Management [S . M A fc 'Zc& ' f Date Issued by Study Director: ShaA A ssA (a Oc, r^A/'A^JL^___________ Susan A. MacKenzie, V.M.D., PhD ., D.A.B.T. Senior Research Toxicologist Date . 4Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 TABLE OF CONTENTS Page GOOD LABORATORY PRACTICE COM PLIANCE ST A T E M E N T....................................... 2 QUALITY ASSURANCE ST A T E M E N T .................................................... 3 CERTIFIC ATIO N ........................................................................................................................................ 4 STUDY IN FO R M A T IO N ...........................................................................................................................6 REASON FO R SUPPLEM ENT 1 ............................................................................................................ 7 SU M M A R Y ....................................................................................................... 8 LIST OF TABLES TA BLE 1 PLA SM A P FO A C O N C E N T R A T IO N S .....................................................................................................................10 TA BLE 2 SU M M A R Y O F BLOOD TO TA L FLU O RIN E A N A L Y S IS .............................................................................. 11 LIST OF APPENDICES A PPEN D IX A A N A LY TIC A L R E P O R T ...................................................................................................................................... 12 A PPE N D IX B IN D IV ID U A L ANIM AL BLO O D FLU O R IN E................................................................................................95 -5 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations STUDY INFORMATION 9th Collective Nomenclature' Svnonvms/Codes: H-25425 Haskell Number: 25425 GAS Registry Numbe: Supplement 1 DuPont-11418 Stability: The test substance appeared to be stable under the conditions o f the study; no evidence o f instability was observed. Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Study Initiated/Completed: October 3,2002 / (see report cover page) In-Life Initiated/Completed: October 15,2002 / April 15,2003 -6 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 REASON FOR SUPPLEMENT 1 As described in protocol amendments 1,6, 8, and 9, the sponsor requested analysis o f plasma samples collected at the time of necropsy for concentration of perfluorooctanoic acid (PFOA), and analysis of selected whole blood samples, collected prior to and during the dosing phase of the study, for total fluorine. This supplement contains the results of the analyses which were conducted by Emily R. Decker of Exygen Research, State College, PA (PFOA concentration) under the supervision o f John M. Flaherty, and Ward R. Gibson of Jackson Laboratory, Deepwater, NJ (total fluorine concentration), under the supervision of Robert M. Good. -7 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 SUMMARY Four groups of young adult male and female Crl:CD(SD)IGS BR rats were administered H-25425 in 0.05% methylcellulose in deionized water by gavage for approximately 90 days (92 days in males and 93 days in females), at dosages of 0 ,1 0 ,1 0 0 , or 1000 mg/kg/day. Selected animals from each dosage group were designated for evaluations o f subchronic toxicity, recovery from subchronic toxicity, or reproductive toxicity. Under the conditions of this study, the NOEL for subchronic toxicity endpoints was 100 mg/kg/day in male and female rats, based on reversible, minimal to mild thyroid follicular hypertrophy, observed in the 1000 mg/kg/day groups. Under the conditions of this study, the NOEL for reproductive endpoints was 1000 mg/kg/day, based on a lack of effects on any reproductive endpoint in Pi or Fj generation animals exposed to the highest dosage tested. Blood was collected at necropsy, at the end of dosing and after a one-month recovery period (control and high-dose rats only), for analysis of perfluorooctanoic acid (PFOA). PFOA was extracted from plasma by protein precipitation in acetonitrile. The compound was quantified by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using selected reaction monitoring (SRM). Quantification was performed using extracted calibration standards containing an internal standard. The lower limit o f quantitation (LLOQ) for this method is 10 ppb. Blood was collected at selected timepoints throughout the dosing phase of the study for analysis of total fluorine. Samples were analyzed from males and females in all dose groups on test day 22, from high-dose males and females on test day 89, and from high-dose males on test days -4 (pretest), 3, and 9. Whole blood was analyzed for total fluorine by the Wickbold torch combustion method and an ion selective electrode. The limit of detection for the method is 0.5 ppm. Results o f both o f these analyses are presented in this supplemental report. The results o f the PFOA and total fluorine analyses are summarized in Tables 1 and 2, respectively. The details of the analyses o f PFOA, which were conducted by Exygen Research, are reported in Appendix A. Individual animal total fluorine data are reported in Appendix B. PFOA concentrations, measured at the end of the dosing period, were below the LLOQ in all control males and in all females dosed with up to 100 mg/kg/day. At the end o f the dosing period, the mean concentration in male rats was 51.7 ppb in the 10 mg/kg/day group, 147 ppb in the 100 mg/kg/day group, and 3621 ppb in the 1000 mg/kg/day group. After a one-month recovery period, the mean concentration in the 1000 mg/kg/day male group was 400 ppb. Females dosed with 1000 mg/kg/day had a mean concentration of 51.7 ppb at the end of dosing. All females in this dose group had concentrations below the LLOQ at the end o f the recovery period. Concentrations in control males and females were below the LLOQ at the end of the recovery period. -8Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Low, but measurable, levels of total fluorine were present in blood from all but one control males and all control females on test day 22. Mean values were 1.18 ppm in males and 1.38 ppm in females. A dose-related increase in concentration was observed in both sexes on test day 22. Male mean values were 1.31 ppm in 10 mg/kg/day males, 1.86 ppm in 100 mg/kg/day males, and 4.74 ppm in 1000 mg/kg/day males. Mean female values were 1.54 ppm in 10 mg/kg/day females, 1.91 in 100 mg/kg/day females, and 3.32 ppm in 1000 mg/kg/day females. In male rats dosed with 1000 mg/kg/day, concentrations were below the limit o f detection for all rats prior to dosing, but demonstrated increasing concentrations over time, with no plateau evident near the end o f the dosing period (test day 89). In female rats dosed with 1000 mg/kg/day, total fluorine levels did not change from test day 22 to 89, and blood concentrations were lower than in males on both test days. .9. Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 TABLE 1 PLASMA PFOA CONCENTRATIONS DESIGN DOSAGE (mg/kg/day) MEASURED CONCENTRATION (ppb) MALE FEMALE MEAN S.D. n MEAN S.D. n End of Exposure3 0 10 100 1000 <10.0* 51.7 147 3621 0 10 <10.0* 19.4 10 <10.0* 172 10 <10.0* 846 10 51.7 0 10 0 10 0 10 25.1 10 End of 1-Month Recovery15 0 <10.0* 0 10 <10.0* 1000 400 305 10 <10.0* 0 10 0 10 * All values below the lower limit of quantitation (10.0 ppb). n = number of samples S.D. = standard deviation a test day 92 (males) or 93 (females) b test day 126 (males and females) - 10Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 TABLE 2 SUMMARY OF BLOOD TOTAL FLUORINE ANALYSIS DAYS ON TEST Group I / n 0 mg/kg/day BLOOD FLUORINE (ppm)a Group i n / IV Group V / V I 10 mg/kg/day 100 mg/kg/day Group v n / v m 1000 mg/kg/day MALES -4 b b b <0.50* 0(5) 3 b b b 1.65 0.16(5) 9 b b b 3.59 0.26(5) 22 89 FEMALES 22 89 1.18 0.44(5) b 1.38 0.40(5) b 1.31 0.36(5) b 1.54 0.22(5) b 1.86 0.31(5) b 1.91 0.39(5) b 4.74 0.45(5) 9.86 0.66(5) 3.32 0.34(5) 3.30 0.62(5) Data arranged as: Mean Standard deviation (Number o f values included in calculation) a W here individual value was < 0.5,0.5 was used to calculate the mean and standard deviation, b Samples not evaluated for this time point. * All values below the lim it o f detection (0.50 ppm)._______________________________________ - 11 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 APPENDIX A ANALYTICAL REPORT - 12Company Sanitized. Does not contain TSCA CB1 H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 STUDY TITLE H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations DATA REQUIREMENTS Analytical Method Requirements STUDY DIRECTOR Susan A. MacKenzie ANALYTICAL PHASE COMPLETED ON November 19,2003 PERFORMING LABORATORY / TESTING FACILITY Exygen Research 3058 Research Drive State College, PA 16801 Phone: 814-272-1039 E. I du Pont de Nemours and Company (DuPont) P.O.Box 50 Newark, DE 19714-0050 USA PROTECT Exygen Study Number 008-342 Sponsor Study No.: DuPont-11418 Total Pages: 81 - 13Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 O O P LABORATORY PRACTICE COMPLIANCE STATEMENT Exygen Study Number 008-342, entitled ' `H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations," conducted for DuPont, was performed in compliance with US EPA TSCA (40 CFR part 792) by Exygen Research. Exygen Research Susan A. Mackenzie Study Director DuPont S. Mark Kennedy Sponsor Representative DuPont / Exygen Research Date Date Page 2 of 81 -14Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 QUALITY ASSURANCE STATEMENT Exygen Research's Quality Assurance Unit reviewed Exygen Study Number 008-342, entitled, "H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with OneGeneration Reproduction Evaluations". AH phases were reviewed for conduct according to Exygen Research's Standard Operating Procedures, the Study Protocol, and all applicable Good Laboratory Practice Standards. All findings were reported to the Study Director and to management. Phase 1. Protocol Review Date Insoected Date Reported Date Reported to to Exygen Principal Management Investigator Date Reported to Study Director and Snonsor Management 10/08/03 10/08/03 10/23/03 11/13/03 2. Standard Preparation, Fortification, and Extraction 10/08/03 3. Raw Data & Draft Analytical Report Review 10/27,28,29, 30/03 4. Raw Data and Final Analytical Report Review 11/18/03 10/10/03 11/06/03 11/18/03 10/23/03 11/12/03 11/19/03 11/13/03 11/13/03 11/19/03 Lydia I/Shafter QualityAssurance Audit) Ma Date Exygen Research Page 3 of 81 - 15Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 CERTIFICATION OF AUTHENTICITY This report, for Exgyen Study Number 008-342, is a true and complete representation of the raw data for the study. Submitted by: Exygen Research 3058 Research Drive State College, PA 16801 (814)272-1039 Principal Investigator, Exygen: Decker Scientist/Brincipal Investigator Exygen Research n[nf(C> Date Exygen Research Facility Management: John M. Flaherty Vice President Exygen Research //d ? ? Date Study Director, DuPont: Susan A Mackenzie DuPont Date Sponsor Representative, DuPont: S. Mark Kennedy DuPont Exygen Research 2i~/Oov'~Zoa2 Date Page 4 of 81 -16Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 STUDY IDENTIFICATION H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations SPONSOR STUDY NUMBER: DuPont-11418 EXYGEN STUDY NUMBER: 008-342 TYPE OF STUDY: Residue SAMPLE MATRIX: Rat Plasma TEST SUBSTANCE: Pentadecailuorooctanoic acid (PFOA) SPONSOR: E. I. du Pont de Nemours and Company P.O.Box 50 Newark, DE 19714-0050 Phone: (302) 366-5255 STUDY DIRECTOR: Susan A. MacKenzie DuPont TESTING FACILITY: ANALYTICAL PHASE TIMETABLE: Exygen Research 3058 Research Drive State College, PA 16801 Study Initiation Date: 10/03/02 Analytical Start Date: 10/03/03 Analytical Termination Date: 10/09/03 Analytical Report Completion Date: 11/19/03 Exygen Research Page 5 of 81 - 17Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 PROJECT PERSONNEL The Study Director for this project at DuPont was Susan A. MacKenzie. The following personnel from Exygen Research were associated with various phases of the study: Name Paul Connolly Emily Decker Karen Risha Amy Sheehan Shawn Robb Title Technical Lead-LC/MS Scientist Scientist Associate Scientist Sample Custodian Exygen Research Page 6 of 81 -18Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity Supplement 1 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations___________________ DuPont-11418 Exygen Study No.: 008-342 PROJECT PERSONNEL The Study Director for this project at DuPont was Susan A. MacKenzie. The following personnel from Exygen Research were associated with various phases of the study: Name Paul Connolly Emily Decker Karen Risha Amy Sheehan Shawn Robb Title Technical Lead-LC/MS Scientist Scientist Associate Scientist Sample Custodian Exygen Research Page 6 of 81 - 19Company Sanitized. Does not contain TSCA CBI /"*n H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 TABLE OF CONTENTS TITLE PAGE......................................................................................... 1 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT............................... 2 QUALITY ASSURANCE STATEMENT............................................................................... 3 CERTIFICATION O F AUTHENTICITY................................................................................ 4 STUDY IDENTTFICATTON................................................................. 5 PROJECT PERSONNEL...................................................................... 6 TABLE OF CONTENTS.......................................................................................................... 7 LIST OF TABLES.......................................................................................................................7 LIST OF FIGURES...................................................................................... 8 LIST OF APPENDICES............................................................................................................8 1.0 SUMMARY.........................................................................................................................9 2.0 OBJECTIVE.........................................................................................................................9 3.0 INTRODUCTION...............................................................................................................9 4.0 TEST SYSTEM................................................................................................................... 9 5.0 REFERENCE MATERIAL.............................................................................................. 10 6.0 DESCRIPTION OF ANALYTICAL M ETHOD......................................... 11 6.1 Extraction Procedure.......................................................................................................11 6.2 Preparation of Standards and Fortification Solutions..................................................11 6.3 Chromatography............................................ 12 6.4 Instrument Sensitivity.....................................................................................................12 6.5 Description o f Instrument and Operating Conditions..................................................12 6.6 Quantitation and Example Calculation........................................................................ 13 7.0 EXPERIMENTAL DESIGN............................................................................................ 15 8.0 RESULTS........................................................................................................................... 15 9.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE QUALITY OR INTEGRITY OF THE DATA......................................................................................... 15 10.0 CONCLUSIONS.............................................................................. 15 11.0 RETENTION OF DATA AND SAMPLES..................................................................15 LIST OF TABLES Table L Summary of PFOA (ppb) in Rat Plasma Control Samples............................... 17 Table IL Summary of Recoveries o f PFOA (%) in Rat Plasma Samples........................18 Table DDL Summary of Residue Found for PFOA (ppb) in R at Plasma Samples.............19 Exygen Research Page 7 of 81 - 20Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 LIST OF FIGURES Figure 1. Typical Calibration Curve for PFOA.................................................................24 Figure 2. Chromatogram Representing a Calibration Standard for PFOA at 10 ppb with ~ 5 ng/mL of^C -PFO A ............................................................................. 25 Figure 3. Chromatogram Representing Control Rat Plasma for PFOA with - 5 ng/mL of ,3C-PFOA (Exygen ID: 0302239 Control A, Set: 100303A).. 26 Figure 4. Chromatogram Representing Control Rat Plasma Fortified at 10 ppb with PFOA a n d - 5 ng/mL of ,3C-PFOA (Exygen ID: 0302239 Spk A, Set: 100303A )..............................................................................................................27 Figure 5. Chromatogram Representing Rat Plasma Sample for PFOA with ~ 5 ng/mL of I3C-PFOA (Exygen ID: 0302026, Sponsor ID: 665053, Set: 100303A)..28 LIST OF APPENDICES Page Appendix A Study Protocol DuPont-11418 (Exygen Study No. 008-342) and Amendments and Deviation..........................................................................29 Appendix B Analytical Method ExM-008-276 Revision 1, "Method of Analysis for the Determination of Pentadecafluorooctanoic Acid (PFOA) in Serum by LC/MS/MS" .................................................................. ................................ 61 Exygen Research Page 8 of 81 -21 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-34-2 1.0 SUMMARY Exygen Research (Exygen) analyzed samples of rat plasma for residues of PFOA in according to protocol DuPont-11418 (Appendix A) using the analytical method ExM008-276 Revision 1 (Appendix B). The limit of quantitation for this method is 10 ppb, which was established at Exygen in the method validation study 008-277. Residues of PFOA in the rat plasma samples ranged from < 10.0 ppb to 4800 ppb. D ie average recovery standard deviation for PFOA in fortified rat plasma samples was 100% 11.2% . 2.0 OBJECTIVE The objective of this analytical phase was to analyze rat plasma samples received at Exygen using the method ExM-008-276 Revision 1 entitled "Method o f Analysis for the Determination o f Pentadecafluorooctanoic A d d (PFOA) in Serum by LC/MS/MS". 3*0 INTRODUCTION This report details the results of the analysis of rat plasma samples for PFOA using the analytical method ExM-008-276 Revision 1. The study DuPont-11418 was initiated on October 03, 2002, when the study director signed protocol number DuPont-11418. The analytical start date was October 3, 2003, and the analytical teimination date was October 9,2003. 4.0 TEST SYSTEM The control rat plasma used for control samples and laboratory control fortified samples was received from DuPont on 10/01/03. One hundred nineteen rat plasma samples were received at Exygen on September 23, 2003. All samples were logged in by Exygen personnel upon receipt and placed in frozen storage ( -20C). Sample login and chain of custody information can be found in the raw data package associated with this study. Storage records will be kept at Exygen Research and a true copy o f the storage records will be furnished upon request. Exygen Research Page 9 of 81 -22Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 5.0 REFERENCE MATERIAL The analytical standard pentadecafluorooctanoic acid (PFOA) was received at Exygen on March 05, 2003 from Critical Path Services. The control article (internal standard) nperfluorooctanoic acid (1,2 di-,3C) was received at Exygen on Februaiy 20, 2003 from DuPont. The available information for the reference material is listed below. The analytical standard was stored in a refrigerator and the control article was stored at room temperature. Compound PFOA 13C-PFOA Exygen Inventory No. SP2516 SP2492 Lot No. GVS-Nr.6V 06687 NA Purity (%) Expiration Date 97 96.35 03/05/04 01/31/05 The structure of perfluorooctanoic acid (PFOA) and the internal standard are given below. PFOA Chemical Name Molecular weight = = perfluorooctanoic acid 414 FF F F C -PFO A Chemical Name Molecular weight perfluorooctanoic acid, [l,2-di-I3C] 416 Exygen Research FF F F Page 10 of 81 -23Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity Supplement 1 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations____________________ DuPont-11418 Exygen Study No.: 008-342 6.0 DESCRIPTION OF ANALYTICAL METHOD 6.1 Extraction Procedure 1. Measure 0.1 mL of sample into 1 mL eppendorf centrifuge tubes (fortify with analyte as needed, close lid, and vortex ~ 10 seconds). 2. Add enough acetonitrile containing internal standard at 0.005 pg/mL (accounting for fortification volume) to make extraction volume 500 pL and vortex for ~ 10 seconds. 3. Centrifuge for ~ 10 minutes at - 14,000 rpm. 4. Analyze samples using electrospray LC/M SM S. 6.2 Preparation of Standards and Fortification Solutions The stock standard solution of PFOA was prepared on September 17, 2003, at a concentration of 100 pg/mL by dissolving approximately 10 mg of the standard (corrected for purity) in methanol. The stock standard solution of l3C-PFOA was prepared on September 17, 2003, at a concentration o f 100 pgrtriL by dissolving approximately 10 mg of the standard (corrected for purity) in methanol. A fortification solution of PFOA at 10 pg/mL was prepared by taking 10 mL of the stock standard solution and bringing the volume up to 100 in acetonitrile. A 1.0 pg/mL fortification solution of 13C-PFOA was prepared by taking 1.0 mL of the stock and bringing the _ volume up to 100 mL with acetonitrile. r" A set of standards containing PFOA with internal standards was prepared by dilution of the 10.0 pg/mL PFOA solution and the 1.0 pg/mL l3C-PFOA solution in the following manner. These solutions were used to fortify the samples and the extracted calibration standards. Initial Cone. (Up/mLY 10.0 10.0 10.0 Volume (mL) Diluted to (mL) 10.0 1.0 1002 0.1 1002 Final Cone. (jig/mL)1 1.0 0.1 0.01 20.5 mL of the 1.0 pg/mL I3C-PFOA solution was added prior to making final volume to give a concentration o f0.005 pg/m L o f u C-PFOA in each solution. The calibration standards are processed through the extraction procedure, identical to the samples. The extracted standards are assigned a two-week expiration date from the date of extraction based on refrigerator stability data obtained during the method validation study (Exygen study: 008-277). The fortification of the standards before extraction is done according to the following table: Exygen Research Page 11 of 81 - 24Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Cone. Of PFOA Fortification Solution (ug/mL) 0.01 0.01 0.1 0.1 0.1 Fortification Volume (pL) 100 200 50 100 200 Volume of Control Sample (mL) 0.1 0.1 0.1 0.1 0.1 Cone, of Extracted Calibration Standard (ppb) 10 20 50 100 200 The extraction solution was prepared by taking 0.5 mL of the 1.0 pg/mL 13C-PFOA solution and bringing the volume up to 100 mL with acetonitrile. The dilution solution was prepared by taking 0.5 mL of the 1.0 pg/mL ,3C-PFOA solution and bringing the volume up to 100 mL with 50:50 methanobwater. The stock standard solution and all fortification/calibration standard solutions were stored in a refrigerator (4 2C) when not in use. Documentation of standard preparation can be found in the raw data associated with this report. 6.3 Chromatography Quantification of PFOA was accomplished by analysis using electrospray LC-MS/MS. The retention time of PFOA was - 2.0 min., with no significant interfering peaks (< 20% of the LOQ standard) in the control samples corresponding to the analyte retention times. 6.4 Instrument Sensitivity The smallest amount of PFOA injected during the chromatographic run was equivalent to lOppb. 6.5 Description of Instrument and Operating Conditions A PE Sciex LC-MS/MS coupled to a Hewlett Packard HPLC system was used. Data acquisition and processing were performed using Analyst 12 software. Detailed operating conditions are listed below: Mass Spec: Interface: Computen Software: HPLC: PE SCIEX API 4000 Biomolecular Mass Analyzer SCIEX Turbo Ion Spray Liquid Introduction Interface Turbo Ion Spray temperature = 350 C with N2 at ~7 L/min Dell Optiplex GX110 PE Sciex Analyst: Version 1.2 Hewlett Packard (HP) Series 1100 Exygen Research Page 12 of 81 -25Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 HP Quat Pump HP Vacuum Degasser HP Autoinjector HP Column Compartment Filter Cartridge(s): 2 Keystone Hypercarbs in tandem, exiting pump HPLC Column: Genesi s Cg (Jones Chromatograhpy), 2.1 mm x 50 nun, 4ji Column Temp. : 30 C Mobile Phase (A): 2 mM Ammonium Acetate in water Mobile Phase (B): Methanol Time 0 3.0 3.5 3.7 7.0 7.5 9.0 9.5 12.0 Injection Vol.: 5 pL Ions monitored: %A 40 40 0 0 0 40 40 40 40 Flow CmL/minl 60 0.3 60 0.3 100 0.3 100 0.5 100 0.5 60 0.5 60 0.5 60 0.3 60 0.3 Analvte PFOA 13C-PFOA Mode Transition Monitored Negative 413 - 3 6 9 Negative 415 -370 6.6 Quantitation and Example Calculation Five microliters of sample or calibration standard were injected into the LC-MS/MS. The peak area was measured and the standard curve was generated by linear regression using 1/x weighting o f the ratio analyte peak area/intemal standard peak area versus the ratio of the concentration of analyte/concentration of internal standard using Analyst 1.2 (or equivalent) software system. The residue concentration for PFOA was determined from tire following equations: Equation 1 was used to calculate the amount of analyte found (in ppb or ng/mL, based on ratio of analyte peak area to internal standard peak area) using the standard curve (linear regression parameters) generated by the Analyst software program. Equation!;, Analyte found (ppb) = Analyte Peak area/IS peak area) - interceptllx IS cone. (ng/mL) x DF slope Exygen Research Page 13 of 81 -26Company Sanitized. Does not contain TSCA CB1 H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 For samples fortified with known amounts of analyte prior to extraction, Equation 2 was used to calculate the percent recovery. Equation 2: Recovery (%) = Analyte found (ppb) - ave. Analvte found in control (publ X 100 Amount Analyte added (ppb) For field samples fortified with known amounts o f analyte prior to extraction. Equation 3 was used to calculate the percent recovery, Equation 3: Recovery (%) = Analvte found fppbl - avp. Analvte found in sample (nobl x 100 Amount Analyte added (ppb) An example of a calculation using an actual sample follows: Rat plasma sample Exygen ID 0302239 Spk A (Set: 100303A), fortified at 10 ppb with PFOA. Where: analyte peak area = 60581 IS peak area = 143204 IS cone. (ng/mL) = 4.167 ng/mL intercept = -0.0104953 slope = 0.176029 dilution factor (DF) = 1 ppb added (fort level) = 10 amt found in control = ND From equation 1: Analyte found (ppb) = r(60581/143204) - -0.01049531 x 4.167 x 1 0.176029 = 10.3 ppb From equation 2: % Recovery = (10.3 ppb - 01 x 100 10 ppb = 103% Note: This example calculation was done using rounded numbers, and therefore may Be slightly different from the values shown in the RAW DATA. Exygen Research Page 14 of 81 -27Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 7.0 EXPERIMENTAL DESIGN Each analytical set was extracted according to method ExM-008-276 Revision 1 and consisted of a reagent blank, one matrix control, two laboratory matrix controls fortified at known concentrations, one field sample fortified at a known concentration, 19-20 field samples, and one field sample extracted in duplicate. 8.0 RESULTS The PFOA found in the control rat plasma samples are listed in T able I. Individual recoveries for PFOA in the laboratory fortified rat plasma samples are detailed in T able n . The average percent recoveries standard deviation for PFOA in the rat plasma samples was 100% 11.2%. Individual results for the PFOA in the rat plasma samples are given in Table in . Residues o f PFOA in the rat plasma samples ranged from < 10.0 ppb to 4800 ppb. A typical calibration curve for PFOA and representative chromatograms are given in Figures 1-5. 9.0 CIRCUMSTANCES THAT MAY HAVE AFFECTED THE QUALITY OR INTEGRITY OF THE DATA There are no known circumstances that have affected the quality or integrity of the data. 10.0 CONCLUSIONS The rat plasma samples were successfully analyzed according to method ExM-008-276 Revision 1. 11.0 RETENTION OF DATAAND SAMPLES When the final report is complete, all original paper data generated by Exygen Research will be shipped to the sponsor. This does not include facility-specific raw data such as instrument logs. Exact copies of all raw data, as well as a signed copy of the final analytical report and all original facility-specific raw data, will be retained in the Exygen Research's archives for the period of time specified fn 40 CFR Part 792. Retained samples of reference substances are archived by the sponsor. Exygen Research Page 15 of 81 -28 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 00S-342 TABLES Exygen Research Page 16 of 81 - 29Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Table I. Summary o f PFOA (ppb) in Rat Plasm a Control Samples Sponsor ID 669160 Control A 10/03/03 669160 Control A 2 10/03/03 669160 Control A 10/07/03 669160 Control A 2 10/07/03 669180 Control A 10/08/03 669180 Control A 2 10/08/03 PFOA <ngftn.L)..... _ ND ND ND ND ND ND Exygen Research Page 17 of 81 -30Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Table H . Summary o f Recoveries of PFOA (% ) in Rat Plasma Sam ples Sponsor ID 669160 (CCS A) 10/03/03 668160 (LCS B)10/03/03 665047 {Matrix Spk) 10103/03 669160 (LCS A2) 10/03/03 669160 (LCS B2)10/93/03 665074 {Matrix Spk) 10/03/03 669160 (LCS A) 10/07/03 669160 (LCS 6)10/07/03 665096 (Matrix Spk) 10/07/03 669160 (LCS A2) 10/07/03 669160 (LCS B2)10/07/03 685096 (Matrix Spk) 10/07/03 669180 (LCS A2) 10/08/03 669180 {LCS 62)10/08/03 665215 (Matrix Spk) 10/08/03 669180 {LCS A2) 10/08/03 669180 (LCS 62)10108/03 665242 (Matrix Spk) 10/08/03 PFOA (ppW 10.3 951 1030 10.2 987 1090 10.1 1040 5570 10.3 633 907 9.72 1060 1060 9.72 1020 1070 Ameunt Fortlfled (PP*>) 10.0 1000 1000 10.0 1000 1000 10.0 1000 1000 10.0 1000 1000 10.0 1000 1000 10.0 1000 1000 AVERAGE: STDDEV: % RSD: Recovery <%> 103 95 103 102 99 107 101 104 121 103 63 91 97 106 106 97 102 103 100 11.2 11.1 Exygen Research Page 18 o f 81 -31 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Table m . Summary o f Residue Found for PFOA (ppb) in Rat Plasma Sam ples Sponsor ID 665047 665047 Dup 665049 665070 665130 665076 665015 665068 665148 665016 665086 665098 665149 665081 665159 665043 665064 665121 665106 665008 665053 665074 665074 Dup 665128 665080 664993 665131 665060 664995 665136 665033 665122 PFOA (ppb) < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 10.0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 10.0 51.8 20.2 20.0 52.5 87.7 39.6 58.1 54.9 74.3 43.0 34.5 731 Exygen Research Page 19 of 81 -32Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Table i n (coni'). Summary o f Residue Found for PFOA (ppb) in Rat Plasma Samples Sponsor PFOA ___________________!2 __________________________________ fe e * )_______________ 665155 592 665073 549 665089 547 665119 636 665137 1010 665103 645 665143 500 665054 640 665035 665160 665096 665096 Dup 665062 665107 665152 665120 664999 665026 665162 665027 665158 665002 665067 665095 665031 665146 665062 665138 665112 665007 665211 868 4170 4380 4340 3640 2790 4800 3620 4090 4000 2270 2470 966 109 82.2 771 85.0 257 236 579 525 387 N P _____ Exygen Research Page 20 o f 81 -33Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Table III (cont'). Summary o f Residue Found for PFOA (ppb) in Rat Plasma Samples Sponsor PFOA IP____________________________ (PPb) 665271 ND 665271 Dup ND 665178 ND 665317 ND 665230 ND 665225 ND 665174 ND 665319 ND 665221 ND 665278 ND 665201 ND 665197 ND 665256 ND 665323 ND 665276 ND 665228 ND 665260 ND 665189 ND 665204 ND 665327 ND 665308 < 1 0 .0 665215 < 1 0 .0 665215 Dup < 10.0 665277 < 10.0 665241 ND 665236 < 1 0 .0 865324 < 1 0 .0 665332 < 1 0 .0 665239 < 1 0 .0 665182 < 10.0 665268 < 10.0 665295 < 10.0 Exygen Research Page 21 o f 81 -34Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Table III (cont'). Summary of Residue Found for PFOA (ppb) in Rat Plasma Samples Sponsor ID 665251 665300 665340 665250 665341 665199 665238 665250 665170 665234 665242 666242 Dup 665183 665261 665291 665337 665333 665200 665310 665326 665288 665282 665275 665229 665237 665209 665335 665303 665328 665299 PFOA (PPb) < 10.0 < 1 0 .0 < 1 0 .0 < 10.0 < 10.0 < 1 0 .0 < 1 0 .0 < 1 0 .0 < 1 0 .0 63.4 36.9 35.8 37.1 66.0 35.3 43.8 20.8 33.4 101 80.2 < 10.0 < 1 0 .0 ND < 1 0 .0 ND < 10.0 < 1 0 .0 < 10.0 ND < 10.0 Exygen Research Page 22 o f 81 -35Company Sanitized. Does not c o n ta in TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 FIGURES Exygen Research Page 23 of 81 -36Company Sanitized. Does not contain TSCA C8I H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Figure 1. Typical Calibration Curve for PFOA 100303A.rclb (PFOA): "Linear" Regression ("1 /x"w aiQ hling):y=O .176O 29X+-0.0l04953(f = 0.8998331) Exygen Research Page 24 o f 81 -37Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Figure 2. Chromatogram Representing a Calibration Standard for PFOA at 10 ppb with ~ 5 ng/mL o f 13C-PFOA Exygen Research Page 25 of 81 -38Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Figure 3. Chromatogram Representing Control Rat Plasma for PFOA with ~ 5 ng/mL of 13C-PFOA (Exygen ID: 0302239 Control A, Set: 100303A) Mt.e...ae..... .0T..M.l..w.#.>iewe* U/IM.M/H* Kw/ak to w tlwariwto. m Mmiwi Mt tutoi MM. *Mk b U t l to > t timo l l m toU lln B i 104 i.e ee.iesi 1.0 4i..eMi I im j vto*t an IeMn(NHMIK*2'.S"&0ir*1itooto:,tawto-<ia- nwltoPiMMMr" UVtoUMIOew. rt M. ZM0I X0/C4M M*. TlMl M tU lH Ni MfrtUlaMy iMUn 1* U l toi Ilamw. iBMnMtttotoUi i 1 w IT Vtftfew tM an iw m m I art 1.1 kl wm.. wtotorttuoi 4l..11 I . t o t t o i 1.8* 1 9 . Q . MUtol 4.00 MMm.tokinl.iwIm (t* i. 3.to IW. Ht i MmmMw MttolAM TtMi l. H tot ton i UIIN M t totlM i V .lto .m tot M ilto i ' aln w* t x* Exygen Research Page 26 of 81 -39Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Figure 4. Chromatogram Representing Control Rat Plasm a Fortified at 10 ppb with PFOA and - 5 ng/mL o f 13C-PFOA (Exygen ID: 0302239 Spk A, Set: 100303A) Exygen Research Page 27 of 81 -40Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Figure 5. Chromatogram Representing Rat Plasma Sample for PFOA with - 5 ng/mL of i3C-PFOA (Exygen ID! 0302026, Sponsor ID: 665053, Set: 100303A) Exygen Research Page 28 o f 81 -41 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 APPENDIX A Study Protocol DuPont-11418 (Exygen Study No. 008-342) and Amendments and Deviation Exygen Research Page 29 of 81 -42Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 DuPoat-11418 H-25425: Subchronic Toxicity 90-Day GavageStudyin Ratswith One-GeneratiotiReproduction Evaluations Protocol { t HaskcDAnimal Welfare CommitteeNi Exyges Research 1 Page 30 o f 81 -43Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: SubdxrcoricToxicity 90-DcyCaveggStudyin Rats withOofi-GcasritioaReproductiveEvaluation! TABLEOF CONTENTS INTRODUCTION OBJECTIVE ., SPONSORANDTEST FACILITY. REGULATORYCOMPLIANCE, STUDY DESIGNMATERIALS ANDMETHODS.. A. TestSubstance., B. Test Species. C. AnimalHusbandry-- D. PretestPeriod.. E. F. TestSubstance Preparationand Administration G. BodyWeights.. H. Food Consumption andFoodEfficiency L Detailed CliniealObservationsandMortality. I. Ophthalmology.. K. Nenrobelmvioral Evaluation ,, L. Clinical PathologyEvaluation. M. TotalFluorine LevelEvaluation N. Reproductive Assessment O. Anatomic Pathology. P. Biochemical Analysis STATISTICAL ANALYSES- SAFETYAND HOUSEKEEPING RECORDS AND SAMPLE RETENTION REFERENCES. PROTOCOLAPPENDIX-... SIGNATURES.. DttPopt-H418 Page a*##>** * m ,,3 ,,5 ,,J ...7 ...8 ...9 --9 -9 .10 .10 .11 ,.12 .13 .14 .16 .21 ..22 -24 ,,24. ...25 .26 -.27 ! i Exygen Research 2 Page 31 o f 81 -44Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: SubchronicToxidty 90-Day Gsvigc StudyInRati with QnoOatcitliOtt Reproductive Evtduatiom . DuPont-11418 ' INTRODUCTION The teatsubstance, H-25425 is as a fabric protector.. The dose levels forthis study were selectedbased ondietoxicity andan analysis ofHood fluorine concentrationsfrom a range-finding studyin which rata wo dosedby oral gavagewith 1 ,10.1DO,-or1000mgfeg/day forapproximately45 days, No compound-relatedeffects on bodyrigid; clinicalsigns oftoxicity, organ weights, or gross pathology lesions were observed in rets in any dose group. Blood fluorine levels appeared to have readied a plateauwithin 1-2weeks ofdosingwith 1000mg/lcgfday,and levels remained very lowthroughoutthe entire exposureperiod. Therefore,the limit dosage o f1000mg/kg/daymay be a no-observed-effectlevel(NOEL) ormay produce mildtoxicity. The 10 and 100mgfleg/day dosages are expected to be NOBLs. OBJECTIVE The objectiveofthis studyinto evaluatethepotential subehronic andreproductive toxicity of H-25425 whenadministeredby gavageto male andfemalerets. A recoverygroup willalso be includedto investigatethereversibility ofanyobserved toxicological effects. Theoral route of administration waiiselected as themostefficientwayto deliver anaccurate dosage. SPONSORAND TESTFACOTY This study is sponsoredby the DuPontChemical Solutions Enterprise (DCSE), E.1 duPont deNemours and Company,Wihnington,Delaware. The sponsor's approvalwas effectivethedate the sponsorauthorized dmWorkAuthorizationForm. The studywillbe conductedat Haskell LaboratoryforHealth and Environmental Sciences,EX du Pont de Nemoursand Company, Newark; Delaware. The testsubstance characterization was performed at Regional Analytical Services (RAS)at DuPontJackson Laboratories, Deepwater, NX REGULATORY COMPLIANCE Thestudywill be conducted in compliancewithU.S.EPATSCA (40 CFRpart792) Good Laboratory Practice Standards, whichareconsistentwith the OECDPrincjpks ofGood Laboratory Practice (as revised in 1997)publishedin ENV/MCfCHBM(98)l7 andMATT Japan GoodLaboratotyPractice Standards (59NdhSanNo. 3850).<w) Areas ofnoncompliancewill be documented in thefinal report. TbeSO-dayitudy designcomplieswithfoeU.S. EnvimnmeolslProtection Agency (EPA) Office ofPrevention, Pesticides,and Toxic Substances (OPPTS) 8703100 test guidelines.TM 3 ! }! rI ! 1 Exygen Research Page 32 o f 81 -45Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 K-2425: SubchronicToxicity 90-Dsy Osvtgc Studyin Ritwith OofrGmcniionReproductiveEvtluadons DuPout-lMlS STUDYDESIGN The study design is as follows: Number/Group I I I n SO SO 0(Control) 0 in IV 40 40 10 2 V VI 40 40 100 20 v n VM 50 SO 1000 200 s. Weightoften aituteaceAgd in nulttaxgjj^MwKcijtot g& b. Suspendons willbo edhutcd fo r ep j Five ofthe 40 or50 mts/sex/dose willbeslatted on study approximately 70 daysafter study start forthe rest ofthe rats, and winbe designatedfor 10-dayhepaticbiochemical analysis. I ! Study Parameters Frequency BodyWeight Day 0 andweekly thereafter Food Consumption Day 0 and weekly thereafter Detailed CEmcnl Observations Day 0 andweekly thereafter MortaKty/MoribundityChecks Twice daily Clinical Pathology* Week7 and Week 14 Ophthalmoscopic Evaluations* Pretest and Week 13 NeurobehavioralEvaluations* Pretest and Wed: 13 Biochemical Analysis Subchronic Week 14 Recovery Week 17 Satellite (day 10) After 10 days ofdosing Reproductive Assessments Pi adults WeekU-18 Fi weanlings Week 13-18 Vaginal Smears(Pi Females) Week7-5 Developmental landmarks (Fi adults) Week 19-23 Necropsy Subchronic Week 14 Pi adults Week 12-18 Weanlings. Week 17-18 Recovery Week 18 Fi adults Week 23-25 Blood fluorine animals Week 26 * Clinicalpathology, ophthalmoscopic, and neurobehavioralevaluations may also be conductedonrecovery animalanearthe end ofthe recoveryphase, if necessary, S i1 A Exygen Research Page 33 of 81 - 46Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: Subetaric Toxicity StHUay Gavsgt StttdyinRatswith One-GenerationReproductive Evaluations DuPont-11418 MATERIALS AND METHODS A. TestSubstance The test jubitaacewas supplied bythe aonsormdwas assigned HasloeOLaboratory Number 2542S. The sponsorreportedpurity w a i||H B 9 h e sponsoris responsible forcharacterization ofthetestsubstance. Ik e test substancecharacterizationwasperfematd at DuPontRegional Aualytictl Services,anon-GUP laboratory. The characterization was conducted in compliance with IS09002 regulations and consideredvalidandsufficient forthepurposes ofthiB study. Availableinformation on die parity, composition, contaminants,synonyms, CASregistry number; basic physicalproperties, hazards, andhazardous materialciasrification(s) will be providedbythe sponsor on the HaskellLaboratory SampleEvaluation form. Stability ofthe teat substance willbe confirmed by analyses nearthebeginning andend ofthe study. B. Test Species Male andfemale Cd:CD*(SD)IGSBR.tab willbeobtained fiem CharlesRiver Laboratories, Inc. Thelocationofthe supplier(city/state)willbe documentedin the studyrecords and final report The Crl:CD(SD)lGSBR rathas beenselectedon thebases oftensive experience with this strain atHaskellLaboratory, and its suitability withrespectto longevity,hardiness, and low incidence ofspontaneous diseases. Approximately352 rah (176males and 176females)willbe receivedapproximately 2 weeks prior to study start An addithmsl40rats (20 milesand20 females) writhe received as a separate shipmentapproximately S9 day alterstudy start, and willbe addedto the test groups (5 rats/sex/group) and designated for the 10-day hepaticbiochemical analysis, C. Animal Husbandry 1. Housing a. Subchronic Study, Piemating Dosing Period,Postweanlng Period AHratswiB behoused individually in stainless steel, wire-mesh cages suspended above cage boards. t I Exygen Research J Pago 34 of 81 -47Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygeti Study No.: 008-342 H-25425: Subcbionio Tonoity 90-DyGavnw Study in Rite with Ons-Gtnsrtlion Rcprcxiuctlvc Evaluations DuPont-11418 b. Reproductive Study All nute ratswillboboused individually during non-mating periods in stainless steel, wite-mesh cages. x CohabitationPeriod Alltats wiHbe housedas breedingpairs in stainless steel, wire-meshcages. At Hieend of the cohabitationperiod, females withoutevidenceofcopulation willbe housedindividually in polycarbonate pans. 11 Gestation Period Days 0-20: Females will bohousedindividually in stainless steel, wire-mesh cages. Day 20-Delivery: Females will be housed individually in polycarbonate pans. iii Lactation Period Adultfemales willbe housedwith their titters in polycarbonate pans. 2. CageRackPositioning Cage racks with wire mesh cages will be relocated within the animal room and cages on the racks will be repositioned atleast every otherweek: > for animals designated for subchianic evaluation, up to approximately 70 days foranimals designated forreproductive assessment, and up to sacrifice (approximatelypostnatal day 60) forFI generation rats. 3 . Environm ental C onditions Animalrooms will bemaintained ata temperature of22 3C and a relative humidity of 50%4 20%. Animal momswill be artificially illuminated (fluorescent light) on an approximate 12-hour lighl/darie cycle. 4. WaterandFood All rats, exceptwhen fasted, willheprovided tap waterand pelletedPMXNutrition International, EIC Certified RodentLabDiet* 5002 ad libitum. Exygen Research Page 35 o f 81 . -48Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: SubchrotUcToxicity 90-Dsy Gavage Studyin Rats with Onc-GgacntmReproductiveBvaduationj DuPont-11418 5. Animal HealthMonitoring As specified in the HaskellLaboratory animal healthand environmentalmonitoring program, the followingprocedures areperformedperiodically to assure thatcontaminant levels are below those thatwouldho expectedto impactthe scientific integrity ofthe study: - Water samples are analyzedfbrtotalbaotraal counts, and Urnpresence of coliforms, lead, and other contaminants. * Feed samples are analyzed fortotal bacterial, spore and fungal counts. * Samples fiom freshly washed cageaand cage racks are analyzed to ensure adequate sanitation bythe cagewashera. Certified animal feed is used, guaranteedby the manufacturerto meet specifiednutritional requirements andnot to exceed stated maximum concentrations oflay contaminants, including specifiedheavymetals, aflatoxin, chlorinatedhydrocarbons, and organophosphates. The presenceof these contaminants below the maximum concentrationstatedby the manufacturer would not be expected to impactthe integrity ofthe study. Husanimal health aod environmentalmonitoringprogram is administered by the attending laboratory animBl veterinarian. Data aremaintained separately fiom study records and may be included in the final report at the discretion ofthe study director. D. Pretest Period Upon arrival atHaskell Laboratory,all rats will be housed one percage, sexes separate, in quarantine. The rats will be: quarantined for atleast 5 days. * assigneda unique 6-digit Haskell number,the last3 digitsofwhich will be tattooed on the animal tail. * weighed atleaat 3times during quarantine. * observedwithrespectto weightgainand any gross signs ofdisease or injury. * given an ophthalmology examination. Therats will bereleasedfiom quarantineby the laboratoryanimalveterinarian ordesignee on the basis of acceptable bodyweights and olinicalsigns. Rats dunwe accidentally trilled or removed fiom study duringthe pretestperiodwillbe discarded withoutnecropsy. Ratsthat ire founddead orsacrificedis extremis during thepretest periodwill be sent to Pathology and given a gross examinationto cheek forthe presence of disease. Dependentuponthese findings, further diagnostic procedures maybe employed atthe 7 f 1 | | g f j j t | F | [ ! ' \ Exygen Research Page 36 of 81 -49Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: SubchiocicToxicity 90-Ds.y Osvaxc Study in Rats wife Onc-Ocacrstton Reproductive Evaluations , DuPont-11418 discretion ofdie study director, a pathologist, or thelaboratory animal veterinarian. -Theresults will not berepotted in the final report unless considered significantto the evaluation ofthe study. E. Assignmentto Groups Rats ofeach sex will be selected foruse onstudy 1 the basis ofadequate body weightgain and freedom from any ophthalmologic! abnormalities or clinical signs ofdiseaseor injury. They will be distributedby computerized, stratified randomizationinto study groupsas designated in the StudyDesign, sothatthereare no statistically significant differences among group body weight meanswithin a sex. The weightvariation ofselected rats willnot exceed 20%ofthe mean weight for each sex. Each grotqt ofstudy animals, assignedpriorto study start, will consist ofthree or four subgroups: the first 10rats/sex/dose will be designated forevaluation ofsnbehroniptoxicity; the next 5 rats/sex/dose willbe designated forcollection ofMoodforpossible analysis offluorine; the next 20 rats/sex/dosewill be designated forreproductive assessment; the remaining 10rats/sex/dose (controland high-dose groups only)willbe designatedfor recovery assessment. An additional subgroup (5 rats/sex/dose) will be receivedas a separate shipment andwill be addedto each dose group approximately 70 days afterstudystart, forevaluation ofhepatic biochemical analysis following a 10-day exposure. Therats designated far possible fluorine snnlyiis mayreceive otherevaluations, at the discretion ofthe study director, if considered valuablein interpretation ofreversibility ofeffects. Each animal will be assigned aunique 6-digitHaskell animalnumber and m individual cage identificationnumber. Thelast 3 digits ofthe Haskell animalnumberwill be tattooed onthe tail ofeach rat The Haskellanimalnumberand cage identification numberwill bothbe included on the cage label. ?ups from thereproductive assessmentwill be tattooed whenthey are 21 days old. A listofcorrespondingnumbers fin the pupswill be identified in study records. At study start (test day 0) the rats will be between 6 and 8 weeks ofage. On test day 0, when possible,rats with bodyweights thatare notwithin 20% ofthemean within asex will be removed from study andreplacedwith tats having body weights withinthat range (subject to the same selection criteria ss the original rats). Therats that are addedto file study for 10-day biochemical evaluation willbe the same approximate ageat the startoftheirexposure phase as the otherrats were at study start Rats thathave notbeen assignedto a testgroup or whichhave been removedfromstudy ontest day 0 forout-of-range body weights,will bereleased forother laboratorypurposes or be sacrificed by carbon dioxide asphyxiation and discarded withoutpathologicalevaluation, at the discretion ofthe study director. Therats designated fortin 10-daybiochemioalanalysis will arrive separately, approximately 59 days after study start Theywillbe handled the same as tile otherrats during the quarantine and pre-testperiods, exceptthey will not receive an ophthshnologicalor neurobehavioral examination. They will be distributed by computerized,stratifiedrandomization into study ! i Exygen Research ' Page 37 of 81 -50Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 /*****''. i i . ....... i .. i : | | , : i 1 H-2542J; Sebchroak Toxicity 9043ayGCTagc Study to Rats with Ona-Oaientim Reproductive Bvslurtioas , PaPont-11418 groups (5/sex/dose), so thatthere are no statistically significantdifferences amonggroup.body . weightmeans withina sex when this subsetofrats is compared amongthemselves. . F. Test Substance Preparation andAdministration H-25425 willbe suspendedin 0.5% mefhylcellulose in deionizedwater. Dosing suspensions of die test substance will be prepared daily and stored atroom temperature until used Themethod ofmixing the testsubstance withthe vehicle will be documented in the study records. Animals designated for evaluation of subdnonic toxicity, recovery,and possible analysis ofblood for fluorine will be dosed daily by gavage forat least 90 days. Animals designated for 10-dsy biochemical evaluation willbe dosed daily by gavage for 10 days, starting approximately 70 days after study start Animals designated for reproductive evaluationswill be dosed dailyby gavage forapproximately 70 days, and then dailyduring the 14-daymatingperiod. TheFt male rats will continue to be dosed followingthe cohabitationperioduntilsacrifice. Pregnant femaleswillbe dosed duringthe three-weekgestation period. Pregnant femalesin the process ofdelivery or showing signs ofdelivery will notbe dosed. From gestation day 18 until delivery, dosevolumes will be basedon the gestation day 18 bodyweights. Lactatmgfemales and femaleswith no evidence ofcopulation will be dosed untilpups are weaned onpostpartum day 21. Thevolume ofH-25425 given to eachrat(5 mL/kg)willbe based on the mostrecently recorded bodyweight. Male and female control animalB will be similarly treated with 0.5% methylodlulose at the same dose volume as used in the other groups. Samples ofeach test dosing suspensionwill be collected three times during the study: nearthe beginning, nearthe middle(approximately test day 45)and nearthe endofthe exposurephase of the study. Ifnecessary, additional samples maybe collected at the discretionofthe studydirector or designee. Analysis ofthe firstsampling willverifyhomogeneity, concentration (average of homogeneity samples), and roomtemperature stability. The subsequent samplingswill address concentration. Samples willbe submitted, shortly afterpreparation, to the Analytical Group of Environmental Sciences (ES) at Haskell Laboratory. On days simples are collected, the suspensions remaining after dosing willbe refrigeratedfor additional analysis if required. Refrigerated suspensionswillbe discarded ifno additionalanalysis is necessary. G. Body Weights Duringthe study, all rats win be weighedonce eachweek unless experimental findings or special scheduling situations warranta change intheweighing schedule. In addition, rats designated for neurobehavioralevaluations, undergoing functional observationalbatteryand motoractivity assessments, will beweighed on the days ofthoseobservations. Female rats designated for reproduction assessmentwillbe weighedon gestationdays 0,7,14,18, and21 and lactation days 0,7,14, and 21. Weaned Ft ratswill beweighedweeklyuntil approximately postnatal day 60. EL Food Consumption andFood Efficiency During the study, the amountof food consumedby eachrat overthe weighing interval willbe . determined by weighing each feeder atthe beginning and end ofthe Intervaland subtracting the 9 Exygen Research Page 38 of 8L -51 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: SubcHranicToxicity 90-Dsy GavagcStudy m RatswithQnfrgencrstion ReproductiveEvalmtiajn . DuPont-11418 final weight andthe amountofpillage fromthe feederduring the intervalfrom (heinitial weight From these measurements, mean dailyfood consumption overthe interval will be determined. Fromthe food consumption andbodyweightdata, the mean dailyfoodefficiency will be calculated. Food consumption for the ratsbeing sampled for blood fluorine will only be j collected duringthe exposure period. f i Duringthe reproductive assessment, foodconsumptionwill be determined as follows for designated rats: Premating Dosing period- individualfoodconsumptionwillbe determined weekly,ending approximately testday 70. ! Cohabitationperiod,beginning on approximately test day 70 and formates following cohabitationperiod- food consumptionwill notbe determined. Gestationperiod- individual food consumption willbe determined ongestation days 0,7, 14, and21. lactation period-food consumption will not be determined. Post-weaningFI rats - individual food consumption will be determined weekly, ending on approximately postnatal day 60. 1 r I 1 Detailed ClinicalObservations andMortality Duringthetest period, cage-rite examinations to detectmoribund or deadrats and abnormal behaviorand/orappearance among rats willbe conductedat leasttwice dailythroughoutthe ii !I study. Moribundrate willbe sacrificed. Atevery weighing, each rat willbe individually handled and examined forabnormal behavior andappearance. DetaBed clinicalobservations in a standardizedanna will also be evaluatedonrats designated for lubchronic toxicity and recovery. (I The detailed clinicalobservations will include (butare not limited to) evaluation offor, skin, ! eyes, mucous membranes, occurrence ofsecretions and excretions, autonomicnervous system . i activity(Iacrimafion, piloerectkm, and unusualrespiratorypattern), changes in gait,posture, responseto handling,presence ofclonic, tonic, stereotypical, or bizarrebehavior. Any abnormal clinicalsigns noted will be recorded. Priorto sacrifice ofmoribundrats, ifpossible, and at the discretionofthe clinical pathologist and/or study director,blood samples forhematology and clinical chemistrymeasurements will be collected for clinicalpathology evaluations. J. Ophthalmology Two ophthalmological examinations will be conductedby a veterinary ophthalmologist The pretest examinationwillbe performedon all tats received forthe study(exceptthosedesignated for 10-dayhepatic biochemicalevaluation),priorto assignmentto groups. Anyrats with preexisting ophthalmologic!abnormalitiesmaybe nliminntrri.from consideration for use in the study. Surviving rsts designated for subchronic toxicity (10rsts/sex/dose) andrecovery (10 rats/sex in controland high dose groups) evaluationswill be examinedpriorto the final to Exygen Research Page 39 o f 81 -52Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: Subchronic Toxicity 90-DayOavsgsStudyInRitwith Oae-OnaCTtioaReproductiveEvahntloiu DuPoaMl41S sacrifice. Both eyes ofeach rat will be examinedby focalillumination Budindirectophthalmoscopy, th e eyes wifibe examinedin subdued light after mydriasis has been produced. A third ophtbalmologicalexaminationmaybe performed onrats designated forrecovery prior to sacrifice if compound-related eye lesions bicobservedin animals sacrificed at the end of thetest period. A report offindings from eachophthalmology examinationwill be submittedby the examining veterinarian. K. Neurobehavioral Evaluation Forall the followingassessments, the experimenterwill be unaware ofthe group designation of the animal. Priorto initiation oftestsubstanceadministration andthen during week 13 oftest substance administration, sensorymotortaction and motoractivity will be evaluated on animals designatedforrecovery (control andhighdose). Sensory motorJunction andmotor activity will also be evaluatedon animals designatedforthe subchronic toxicity evaluation subset (10 animala/sex/dosc)in the low- endmid-dose groups. Sensorymotor fonction.and motor activity assessmentsmay also beperformed on recoveryanimals at theend ofthe one-month recoveryperiod. Spare rata will alsobe evaluatedforfoe functional observationalbattery(FOB) and motoractivity priorto study atari. 1. SensoryMotorFunction Simple assessments ofsensorymotorfunction (grip strength, response to approach/touch, tail pinch, andsharp auditory stimulus) willbemade. Fore- andhind-limb grip strength will be measuredby a strain gauge device (ChatiUcnDigitalForce gauge). Due to technical difficulty ofevaluating pupil sizein albino rata underambientlightingconditions, pupil size will be evaluatedby assessmentofpupillaryresponse. Pupillaryresponse to a beam oflight will be measured immediatelypriorto removingthe rate from the motoractivity chambers. 2. MotorActivity Motoractivity (MA) will be assessedfollowingtire sensory motor fonctionevaluation onthe sameday. fiats wifibe Individuallytestedin oneof30 nominally identical, automated activity monitors (Coulboura). Groupswill be counterbalanced aoroaathe monitors and time ofday to the ftllest extentpossible. The infraredmonitoring device enables measurementof2 dependent variables, duration ofmovementandnumberofmovements. A continuous movement is counted as onemovementregardless ofduration. Eachtest eessitmwiUbe 60 minutes in duration, and the results willbe expressed fordie total session as well as for 6 successive 10-minute blocks. Presence ofdefecstion andurination onthe cageboardsbelowthe motor activity cages will also be evaluated following eachmotoractivity session. Exygen Research I Page 40 of 81 -53Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: SubchionicToxicity 90-DeyQaviigB Studyto IUBwithPiOmtSoaReproductiveEvalustioi . DuPont-11418 L. ClinicalPathologyEvaluation A clinical pathology evaluationwillbe conducted on rata designated for subcbronic toxicity during week7 andweek 14 (prior to necropsy).A clinical pathology evaluationwillbe conducted onrats deaignated forrecovery near theend ofthe.recoveryphase. Coagulation . parameters andplasma and urine fluoride will not be determinedduring week7. The day before collectionofblood samplesforthe clinicalpathology evaluation,the rata willbe placed in metabolism cages. Theserati will be fisted overnight{at least 15 hours) and urinewill be collected fiom eachrat. Blood samples for hematology and clinical chemistry measurementswill thenbe collected fiom the orbital sinusofeachratwhilethe rat is under lightcarbon dioxide anesthesia. Blood samples forcoagulation parameters and plasmafluoride will he collected fiom each rat at sacrifice fiom the abdominal vena cum, while flusrat is under carbon dioxide anesthesia. Bloodsamples willbe evaluated for quality by visual examination priorto analysis. Additionalblood, collectedfiomIho vena cava attho final sacrifice, will bo placed in a serum tube, processedto serum, and fiozen at -70 to -80"C. Serum maybe used foradditionaltesting as documented byprotocolamendment, or willbe discarded whentee finalreport issues. Bane marrow smears willbepreparedat thefinal saerifico fiom all rats sacrificed at that timepoint, and willbeevaluated ifwarrantedby experimental findings. Atthe discretion oftoe study director orclinicalpathologist, blood samples for clinicalpathology tests maybe collected fiom animals showingclinical evidence oftoxicity or sacrificed in a trtm a . 1. Hematology B it f Rodblood cell count Red cell distribution width Hemoglobin Absolutereticulocyte count Hematocrit White blood cell count Mean corpuscularvolume Differentialwhiteblood cell count Mean corpuscularhemoglobin Platelet count Mean corpuscularhemoglobin concentration Microscopic blood smear examination Hew methylene-blue-stained blood smears will be preparedfiom all hematology samples and willbe evaluated,ifrequired, to substantiate orclarify the results ofhematology findings. 2. CoagulationParameters The following coagulationparameterswill be determinedat sacrifice only: Prothrombintime : Activated partialthromboplastintime I i 12 Exygen Research Page 41 of 81 -54Company Sanitized. Does not c o n ta in TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Bxygen Study No.: 008-342 H-25425: SubchronicToxicity 9043yO*yas Study ia Hsu'rilhOna-CteiieiatinnRapBxtuctivc Evaluations . 3. Serum Chemistry The following serum chemistry parameters willbe determined: Aspartate aminotransferase Alanine aminotransferase Sorbitol dehydrogenase Alkaline phosphatase Total bilirubin Urea nitrogen Creatinine Cholesterol Triglycerides Glucose Total protein Albumin Globulin Calcium Inorganic phosphorus Sodium Potassium Chloride Plasma fluoride (sacrifice only) DuPont-1 H IS 4. Urinalysis The following urinalysis parameterswill be determined: Quality Color Clarity Volume Osmolality pH Urobilinogen Blood Glucose Protein Bilirubin Ketone Microscopicurine sediment examination Urine fluoride (sacrifice only**) BDTAwillbeplacedinurine vials priorto collectionofthisurine specimen M Total Fluorine Level Evaluation On testday -4 (pre-bleed), and test days 3,9,22,34,55,76, and 89, blood(approximately 0.5 mL) willbe collected ftomthe orbital sinus ofdesignatedanimals (5 rsta/sex/dose), while the animals are underlight carbondioxide anesthesia, forpossible total blood fluorine analysis. On the day ofblood collection (excepttest day -4), blood willbe collectedform the animals 2hours i (30 minutes)afterdosing. The blood willbe collected in plastie tubes containingBDTA while onice andstored fiozen. During the lastweek oftho approximately 90-day exposure period the animals winbeplaced in metabolism cages forcollection offeces andurine. Urine andfoce? willbe collecteddaily at 24-hourintervals feomeach animaL The exacttimeperiod ofcollection ofurine and feceswill be documented alongwiththototalvolume ofurine obtained. Urine and feceswill be storedfrozenuntil analyzed. The decisionto analyze fi>rblood fluorine andtho method for determiningbloodfluorine willbe documentedin the study records. Tbetypeof I kineticanalysis to be used on blood fluorine datawillbe determined after study startandwillbe addressedin protocol amendment 13 I Exygen Research Page 42 o f 81 -55 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-2542S: SubchronicToxicity 90-Dsy Garag Studyin Ran withOaBGaigaaon Reproductive Evaluattoiia . DuPoal-11418 Blood will alsobecollected for possible total blood fluorine analysis at3,7,11,18, 25,3 6 ,50, 71, and 92 days postdasing (afterthe last dose)forbothmale and female animals ofeach dose group. Animalswill be sacrificed at the endofthe approximately 90-day recoveryperiod. The blood, urine, and fecal samples collectedpostdoringmaybe analyzed as needed to answer - questions regarding distributionand metabolism ofthe test substanco. N. ReproductiveAssessment 1. Bleeding a. Startofcohabitation Afterapproximately 10 weeks ofexposureto thetest substance. b. Duration ofcohabitation period Until evidence ofcopulation is observed (designated as day 0 of gestation), oruntil 2 weeks have elapsed. c. Evidence ofcopulation Once daily, each female willbe examinedforthe presence ofan inttavagmalcopulationplug or sperm in the vaginal lavage sample. The day evidence ofcopulation is observed is designatedas day O ofgestation. d Cohabitation Eachfemalewillbe continuallyhousedona-1:1 baas with a randomly selected male ofthe same doeagelevel, inthe male's cage. Onfile daycopulationis confirmed,the female willbe transferred backto individualcage housing. Based on the outcome ofthe first msriog and at the discretion ofthereproductive toxicologist, the females maybe cohoused againin themanneroutlinedabove, except with differentmales, to produce a secondset oflitters. 2. Estrous Cycle Evaluation Vaginal smean will be collected daily fromall Pi female rats in order to determine the stages of the eatroui cycle. Vaginalsmears willbe collectedbeginning threeweeks prior to mating, and continuing until copulationis confirmed, orthe cohabitationperiod has ended. Vaginalsmears will also bo collected ftomallPi parental female rats at the time ofsacrificeto determine fiu stage ofestrouscycle. ! j J j I j j r 14 Exygen Research Page 43 of 81 -56Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: Subchronic Toxicity 90-Day Gavagg Study in Rat wIthOno-Ganqadan Reproductive Evaluations . Dnfont-11418 3. GestationProcedures Afterbeing tnmsfencdintopolycarbonatepans (on day 20 ofgestation formated females, orat the end ofthe cohabitation period for fannies withoutevidence ofcopulation), femalerats will be observed at leasttwice daily>rsigns ofdelivery and pups. 4. LactationProcedures The day when delivery is complete is designatedday Opos^iaitum. At each examination period, pups win be individually handledandexamined for abnormalbehaviorand appearance; any dead, missing; or abnormalpups-willberecorded. a. Day 0 postpartum Live and dead pupsin each litterwillbe counted as soonas possible afterdelivery is completed. Live pups in each litterwill be individually weighed. b. Day 4 postpartum Litters willbe culledrandomly to 8 (4/sex when possible). Extra pups will be euthanatized (by decapitation) and discarded withoutpathologicalexamination. Litters ofeightpupa or fewerwill notbe reduced. Littercounts will be determinedpriorto and after culling and individualpup weights will bedeterminedpriorto culling. c. Days 7 and 14 postpartum Pups in each litterwillbe countedby sexand individually weighed d Day21 postpartum (Weaning) Pupa in each litterwillbe countedby sexand individually weighed (3 of4 rats/sex/litterwill then be sacrificed and grossly examined on postnatal day 21). Randomly selectedweanlings (ouefrex/tttter)winbe placed in individual cages, monitored for attainment ofdevelopmental landmarks. 5. PostWeaning and DevelopmentalLandmarks Developmentallandmarks IntheFi generation maleandfemale rats (one/sex/litter) will be monitored on a dailybasisuntil criterionis achieved. Body weight and food consumptionwillbe determined weekly untiltermination (approximately postnatal day 60). Animals will also be weighed ontee daythe developmentallandmarkis achieved. a. Body Weightand FoodConsumption Bodyweightand foodconsumption willbe determinedweeklyuntil termination (approximately postnatalday 60). b. VaginalPatency Female rats willbe examinedbeginning on test day 0 (postnatal day 21) until achievement,or postnatal day43, whichevercomes first. Bodyweight willbe recorded onthe day of achievement is Exygen Research Page 44 of 81 -57Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: SubctamlcToxleity 90-DayGayage Scaly m RjCi with Qne-Gctienujcn ReproductiveEvaluation) . DsFeuHM U c. Preputial Separation -- Male ratswillbe examinedboginning on test day 14 (postnatal day 35) until achievement, or postnatal day 55, whichever comes fust Bodyweightwillberecorded on the day of achievement^ o. ' 1. Pretest See Section D. Pretest Period. ; 2. Rats Designated forSubchionic ToxicityEvaluation All rats founddead, accidentallykilled, saaificed in extremis, or sacrificedby designwill undergo a gross andmicroscopicevaluation. All rata removed from atudy (oxceptfor out-ofrange body weightontest day 0) willbe sentto Pathologyfor grossevaluation end collection of tissues. Ratswill be euthanatizedby catbondioxide anesthesia and exsanguinsfion. Rati sacrificedby designwillbe fasted after3 pan. onthe afternoonbeforetheir scheduled sacrifice (atleast 15 hours). A final sacrifice willbeperformed oo surviving rats followingthe final clinicalpathology evaluation. The order ofsacrifice for scheduled deaths willbe random among alltreatmentgroupswithin a sex. Blood samples for coagulationparametersand plasma Buoride evaluationwill be collected formfire abdominalvena cava ofeach ratat sacrifice, while the rat is under carbon dioxideanesthesia. - j L I1 f \ , \ j | t | i f Exygen Research 16 ! Page 45 of 81 -58Comparty Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: SubchrotixToxicity 90-Pay Gavags StudyinfUUwithOee-GeugatiroKepmductivt Eyiluatioe_______________ DuPont-11418 The followingtissues willbe collected from ruts which an found dead or accidentally killed (tissue integritypermitting), sacrificed lit extremis, removed from study (except out-of-rarige body w eight o n te st day 0), o r sacrificed by design. Piewiive System fiver esophagus CmfipyimliffSystem heart aorta MMgttofo)wri System Skeletal muscle fcm uriknee jo in t item um duodenum jejunum Ileum Htmatepcfclfc System spleen thymus ReprodugfreSasn M ale cecum colon rectum mandibularlymph node mesenteric lymphnode bone marrow" testes epididymides prostate salivary glands seminal vesicles pancreas Endocrine System Female Urinary System kidneys urinary bladder pituitary gland thyroid gland parathyroid glands adrenal gUnda ovaries uterus mammary glands W seefigM ous Resniratorv System lungs trachea nose Nervous-System brain [including cerobnun, cerebellum, medulla/pons) spinal cord (cervical, skin eyes (including retina and optic nerve) gross observation* larynx trad-thoracic, lum ber) nhaivnx sdetie nerve a Bone msmiw wig lx reflected with the famir and temcm. b QrouobiicTittoram ideaiuecropiy forwMchlilJtojathologylJttOtippropriale (e-g, fluid, ruffled flu, udm W aguihm depats) will generallynotbe collected. All tissues'trill beplaced in the appropriate fixative. Sets sacrificed by design willhavethe following organsweighed: liver, kidney, adrenalglands, thymus, brain, spleen, heart, thyroid(weighed alter fixation) and ovaries and uterus (female) or testes and epididymides(male). Relativeorgan weights (percentoffinal bodyweight; ratioto brainweight)will be calculated. Thudbodyweights determinedjustpriorto necropsy willbe used inthe assessment oforgan weightchanges. Organsfrom rets founddead, sacrificed in extremis, or accidentallykilled maybeweigh! at die discretionofthe pathologistor study director. Bonemarrow smears will be prepared atthe final sacrifice fromall surviving rati andwillbe. evaluated ifwarrantedby experimental findings. All tissues collected fromrata designated for subchronic toxicityevaluation in thehigh- couoentration andcontrol groups, and finmrats thatare found dead or accidentally killed (tissue integritypermitting), or are sacrificed,in extremis, will be timberprocessed to slides, stained with hematoxylin endeosin, and examined microscopically. Gross lesions and any target organs from 17 Exygen Research I Page 46 of 81 > -59Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 JU-25425: Subdmmio Toxicity 9M)yOavagcStudyfaRmYrithOM-aencation ReproductiveEvaluation , DuPont-11'll 8 rata in the low- andinteimedixte-eoncmtration groupwill be evaluated microscopically, , Selected gross observations forwhich amicroscopic diagnosiswould not be additive (o.g., { osteoarthritis, pododermatitis, tail chronic dennatiti, calculus, and deformities ofthe teeth, toe, tail, or earpinna) willbe saved, butwill generally notbeprocessedformicroscopic evaluation, j - - Tissues fromrats removed hum atudy will notbe processed for microscopic evaluation unless j considered nooeaaaiy bythe study directororpathologist. Additionalprocedures to identify and/orclarify histologicfeatures oflesions maybe performed atthe discretion ofthepathologistand willbe documented in the final report 1 3. Rata Designated forPossibleAnalysis ofTotalBloodfluorine Levels Any rats designated for possible analysis ofbloodfluorine thatare found dead,accidentally 1 killed, or aresacrificed in extremis willbo discarded withoutpathologicalevaluation. At the end ofthe approximately 90-daypost-dosingperiod, theserats (j rats/sex/dose) willbe sacrificed by carbon dioxide anesthesia and exsanguination andthe followingtissue will be collected and weighed: liver, kidney, testes, and fit (approximately 1 gram). These tissues willbe placed in plastic freezerbags andstored in die fieezeruntil analysis oftotal fluorine levels, if needed. 4. Rats Designated forRecovery Any rats designated forrecovery evaluationsthatare founddead, accidentally Jailed, or are sacrificed lit extremis will be givena pom pathological evaluation. The sametissues collected forthe subchronic animals atthe approximatelytest day 90 sacrifice will be collected for all recovery animalaand saved in the appropriate fixative. Onemouthafterthe final dose, rats designatedforrecovery(10 rats/sex in control andhigh dose groups)will be sacrificed and undergo a gross examination. Trasuea willbe collected and groaalesions and target organs, identified in animalssacrificed at the endofdie approximately 90-day exposureperiod,will be examined microscopically. S. Rats Designated for ReproductiveAssessment _______________Sacrifice Schedule ___________ Animal Adult Males Schedule Aftersiring litters (atdiscretion ofreproductivetoxicologist) PregnantFemales On dayo fweaning litters NonpregnantFemales With pregnant females Weanlings 3 of4 lata/aex/litterwill bo sacrificedonpostnatal day 21. Fi Adults Atpostnatal day 60_________ Exygen Research IS I Page 47 of 81 -60Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-2S421: Subehronic Toxicity 9a-DyGryga StudyInRan widi dnc^lmnztioaRaproducllveBvtlUBaona . DuPont-lMlS a. Pj Adults Ail Pi parental ratswiUbe sacrificedby carixm dioxide anesthesia and exsanguination, andwill receive gross pathological examination, including the females that die orare sacrificed i t extremispriorto the endoflactation and those forwhichmating did not result in production of offspring. The uteri ofall cohabited femaleswill be examinedfor the presence and numberof implantation sites. Sperm parametersforthe first 10survivingPi malesnimals sacrificed bydesignin each treatmentgroup willbe evaluated. Therightepididymis willbe removed, andthe right cauda epididymiswill be weighed. Spam willhe collected fromthe right cauda epididymis and percentmotility andmorphology willbe determined. The loft epididymis andtestiswill be frozen in liquidnitrogen and storedbetween -65C and -85C Kirspenn and spermatid counts, respectively, The followingtissues andall gross lesions willbe collected from all Pi adults (including those that die anytimeprior to the scheduledsacrifice) andpreserved in appropriate fixative for possible ftiturc bistopathologicalexamination. [ f j | f ! ; ' : Tissues Collected from Pi Adults Male `Female EM hSro Testis" Ovaries Thyroid , Epididymis" Uterus(withoviducts) Grosslesions" i Prostate Vagina Pituitary ! SeminalVesicles i CoagulatingGlsnd a Testesand epktidymlda collectedirom milen ts will beplaced m BouirTs loiudtm. Ai] other tissues (reproductive endnon-reproductive)collected frommile ted fcmile rats will b<placedin formalin, b (hostlesions observed atnecropsy forwhichMitopsthology is not appropriate orwould aot be sdditivo will generallysotbe collected. I Exygen Research 19 Page 48 of 81 -61Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity Supplement 1 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations____________________DuPont-11418 Exygen Study No.: 008-342 H-2S425: Subchronfe Toxicity 9M )ay Oavsgo Study to Ran -withOn&Ocncmioa RwroductlyoEvsluntioiu , PuPrnit-11418 Reproductive organsofanimals with impairedreproductive performance (e.g., failure to . conceive or deliverhealthy offspring, oreffects on estrous cyclicity or spermparameters) Willbe evaluatedmicroscopically. Gross (tions will also be evaluatedmicroscopically atthe discretion ofthe study directororpathologist. b. Offspring Offspringthat are found deadduring the lactation periodwill undergo agross pathological evaluation. c. Ft Weanlings Three Fi weanlings/sex/litter (randomlyselected),litter size permitting, will be sacrificed by carbon dioxide asphyxiation midundergo a gross pathological evaluation on postnatal day 21. i All grosslesions will be preservedforpossible future histopathologtcal examination. d. Fi Adults All Ft generationratswill be sacrificedby carbondioxide asphyxiation and exsanguination, and will receive a gross pathologicalexamination,including Ft rats that die or are sacrificed in extremis prior to postnatal dayfiO. Selectedtissues tutdall gross lesions will be preservedin appropriate fixative for possible fiiturehistopathologtcal examination. The followingtable liststissues to be collected and/or weighed (tissue integrity permitting): Mite Testes Epididymides Seminal Vesicles (with coagulatingglands) Prostate Otgan Wrights fromFiAdults Fetoaltt Both Sexes Uterus (withoviductssad cervix) Thyroid (afterfixation) Liver Brain Tissues Collected fromFi Adults Male Esoofilft Bo.fthS.ClWi Testis* Ovaries Thyroid ; Epididymal* Uterus (with oviducts) Grossissions* Prostate Vagina Pituitary Seminal Vendes CosRulatinaGland a Ttw and apUhdyaddw UlactedSommato rati w ait* placid In Boulrisadutioa.AH otherlissea (reproductivesadnoa-iepteduetlv) coUoctadSonia u k and female rats will ha placed in fimoaVs. b Gioaslcaioniobacryad at noaopsy forwhich Hitopathologyla not appropriato or wouldnotbe addMw will jcnenlty notbe catteeted. 1 I Exygen Research Page 49 of 81 -62Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 025425: SubchicnicToxicity 90-Day Oiragc Srodyin Raiswith`Oi.Gcraniion Reproductive Evaluations . DuPont-11418 Histopathologywill be evaluated in reproductive organa, at the discretionofthestudy directoror reproductivetoxicologist P. BiochemicalAnalysis 1. Ratsdesignated fix Biochemical Analysis Biochemicalanalysis (hepatic peroxisome proliferation)will be evaluated in rats sacrificed attar 10 days ofdotting (10-dosebiochemical subgroup), at the approximately 90-day sacrifice (lubciamnctqxicitysabgwup), and attire endofthe recoveryphaseoftits Andy Recovery subgroup). Sauries fin biochemical evaluation,will be taken firm the first 5rats/sex/doae designated for subchronic toxicity evaluation and fiomthe first 5ratl/sex/dose designated for recovery. An additional40 Cri:CD(SD)IGSBR rats(5 rata/setfdose) will be obtained fiomCharles River Laboratories,Inc. approximately 59 d^yaafterstudy start forthe restofthe rats, and designated for lOriay biochemical analysis. Theserats willbe dosed dailywiththe testsubstance by oral gavagefor 10 days,starting soon afterthereproduction subgroup has beenremoved formating Rats willbe fisted after3 p.ra. on the afternoonbefore their scheduled sacrifice (atleast IS hours). Onfhel l* day, survivingrats willbe euthanatized by carbon dioxideanesthesia and exsanguination. Procedures for animalhusbandly, environmental conditions, waterand food, pretestperiod (no ophthalmologyexamination), assignmentto groups, body weights,food consumption, and food efficiency in tiierats designated for 10-daybiochemical analysis willbethe same as forrats designated for subchronic toxicity evaluation. No opbthalmologjcal orneurobehaviaral evaluations or detailed clinical observations willbe conducted These rats will be considered part ofthe samedose groups as the rats startedat study start; however, data from these animals will be repotted separatelyfiom datafiom the restofthe rats in eachgroup. liven willbe collected and weighed, andrelative organweights (percentoffinalbody weight) trill be calculated. Knalbodyweightsdeterminedjustpriorto necropsy will tie usedin the assessment o ftiverweight changes, 2. BiochemicalAnalyais Atnecropsy, a portion ofthe liver will beplaced in liverhomogenizationbuffer andmaintained on ice farpreparationofhepatic peroxisomes. Hepatic peroxisomes will bepreparedin accordancewith DuPont Haskdl Standard Operating Procedure (SOP)BT015-P. Peroxisomal [1-oxidationactivitywfflbe determinedusing [14C]pafaritoyl CoA asthesubstmte according to S O ^ flB M ijrh e proteancontentofthe peroxisomeswillbemeasured before and afteranalysesby the Stand method. Final calculations trill be based on the past-assayprotein determination. Exygen Research ( I Page SOo f 81 -63Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425; Subckroofo Toxicity 90-Pay Oavago Study InRan wlthOne-Ooagation Reproductive Evaiuattau , PttPom -W t | STATISTICALANALYSES . Significant will bejcdged alp <0-05. Males aiuifhMle* will be analyzed separately. The j methtKlforkiiaeticanalysjiwjUteitotBnnii^aftetJtudy startimddcKSjmcntedmatudy records. ; In-lifodatafiomihe 10-daybioclieaiical analyrij subgroupwill onlybe compared amongthese ! subgraqra. Statistical Methodsfor Snbchronk Toxicity Aiiotmenti Method afStet dealAnatolia Parameter PreUmlnaiyTeit IfpreW naay tnatb oot IfpndtafaK yteatla tiflfdfleanfc rim ifieut Body Weight Body Weight Gain Food Consumption Food Effieiwtcy 6-Oxldatwu Organ Weight Teatftr lack oftrend^ Sequential application1'*o f the Joaekhoea'Terpftn trend teal?* Preliminary ieeta for pairwise conpaiiaon _____________________ :_______ Q E______________ Lflvet**test for homogeneity***nd Sbapirc^WOkteitP^foT nnrmaht^ One-way eojflyaiiof Kradad-W altiatnl"1) vadarW 1*fottowedwith M owed with Dumb a m a e tfa W ,! toriTM Clinical Pxt&ology* Leva*teat for bocnogenetty** and ShH dro-W fiktM ^^lbr Bormilitv* Ooo-way anityaU of Knnkil-WaUia teatTM varianceTM foBowadwith fbSosmd withDunn's Dunntt!afeidm w M otor Activity* Lcwm'i teat far homogeneity** end Sbajdro-W afcteit^foT normalit Repeatedmeasure! ituUyaiao fvarUoce followed tyconftaata^ Sequentialapplication0*o f the jbaQkfceeK-Terpttn tread teat^ Chip Stasggth Footipky Survival B aittetfeteat^fa feomofeaBityofvarieras One-way analylif of v a risW " followedwith DuaMtfl tosi1 Khakd-Waliia W u* M owed with Dunn't teatP^ IncidenceofO talcil Obeemtioni ISOidODMOf CphthataolegiM! CfceervitfotM N on Cochmn-Annitnge testf a trend01*' Incidenceo fFOB D eaorirttvoP ttw utan Ind k ^o fM tew co p te N on Le*twa_____________ None a FthwlMcompiiinRiindssioeialedpieGminuyteitatnenlyeondnctBdU'tlistutlbrlaekaflnndls IgaU cut 1> Ifdw ShapIro-WUl:teeth notdenlfleintbutLovt1ted I*<lgiilflcioi,t rob vendon ofDuomtfi (eat ' willbeused. e Te*dayandblockU-iiitBUainterval)witibouidiitepeat*diaeaitetictoes. d Wheninutdivi&ialobservationixrecord!asbeinglessthana certainvalue,calculationsereperformedon halttherecordedvalue. 7otextnpIekifbUiiubInbnpartedea<ai>d.aSiiiBed(braiiyea]oiililiona performedwit*thatbilirubindata. a Iftheincidence b dc<significant;bat asignificanttack offit occurs,then Fiahec'aexacttest^'1witha Bcothnari eometloa la used. 22 Exygen Research Page 51 o f 81 H-25425: Subchrorc Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-23423; SuM&oaieToxicity PO-DnyOnvatoStudyto Kaa with Qac-GencntioK Reproductive Evaluations . DuPont-11418 The followingtable Liststhe indices ofreproductive function thatwill be calculatd'foxthe P] and F| adultt. SUUaticxl Methods for Reproductive Auessaient Parameter PretiminarvTejt . MettodofStatbidealAnatada Ifpnnninatytetiaiiat Ifpnliadmrytestis . ibnriffcanT dntficant BodyWeight BodyWeight(tan FoodCcoaumpdoo GsstatioaLength Testforlickoftraod^ SeqnenlUapplication" o f theJnackfceeroTetwtratrendt&P* Prelhninarytests fir pdkwtecomparison OR? OrganWeight JxaflnOtiSM SitsNumb* IayUntUiomEfftckacy MeteNumberofPupsPerUtter BstooutBertAh*o foeoa! btorvsl BitrousCycloLength Leveae'stestfor honiogenrity^and Shipiro-WBkterf^ftr KxmnUty* One-wayaatiyaiiof Knalcal-Waliteat011 vidant015foUowadwith fbUowadwithDram'i DumetfataatM> teat"4 Pwpetisl Separation IncidenceofClinical Obaervatiraa NumberofFemaleCycling Normally Matingta la FecundityIndex Fertilityfade* GestationIndex ViaMBtyIndex LactationIndia MamPupWeights (CovMktes: Unr ilagidt ztfo) AoogenitdDisttnoe (Comtes: popwslghQ SexRatio Nano Nom CocfaflKfAmiitigotestforbond01* Iiaoar contest afloat squintinsM04 DuniTi terf"* a PiiratacantfitriK m i and aiaocU tedpttlliniiiiiyteittare oUyconducted if the teatfor lack oftrend il significant b If tbo Shtpito-Wilk totlacotalgiKfictn! butlcvcaofr Ite iaslgalficai^ arobuat verston ofDoanttf* tart will o Iftb incidence is notiignifieaz)tTbtitftcI{nifioaxtlackoffiCoocm,tbeDFj8hear,exacttec^>11wifita Bonftm nl axreettai Uuied. I i 23 Bxygen Research Page 52 of 81 - 65- ^msmWmiOmA mm not contain TSCACBS H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: Sutxdtroide Tradefty 90-DayQsvsee Sttuty in H ia wtth Onc-qeaaratkm Rcpreducllv Bvstuulona . D uPoaU H lt Foresch parameter analyzed with a trend test, the testwillbe applied to the data sequentially. If a significant dose-response is detected,dsia fiom the top doie group will be excluded andthe test repeateduntilno significanttrend ia detected.01 For litterparameters, the proportion of affected fetusespnr litter orthe littermeanwillbomodu the experimental unit forstatistical evaluation.01>- Where the data wetied andthe standardlargeample version of Jonekheere's test is not applicable, exactp values will be calculatedturag permutation methodology.051 Additionalstatistical testewill be used, and otherparameters analyzed,if deemed necessary. The tatistiealmethods need foranalysis will bedocumented in die final report. SAFETY AND HOUSEKEEPING Goodhousekeeping procedurewillbepracticedto avoid contamination ofthe neatdosing material and potential health hazards. To avoid skin, contact, gloves will be worn when handling die teat substance. Animal carcasses, foces,andunused dietwill bo incinerated. RECORDSANDSAMPLE RETENTION All originalrecords ortale copies oforiginalrecords (sponsor-supplied characterization oftest substance) will be retained at Haskell Laboratory,E l. da Pontde Nemours and Company, Newark, Delaware or atIronMountainRecordsManagement,200 ToddsLane, Wilmington, Delaware. Laboratory-specific or site-specificrecords such anpersonnel files and equipment records will beretained atthe ftcility wheretheworkwas done. Preserved wet dsaues, embedded tissues, histological slides, blood smears andbtniemsxrow smears willberetained at Haskell Laboratory. Asampleofthe test substancewill be collected for archivepurposes and retained at Haskell Laboratory. Exygen Research 24 Page 53 o f 81 H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: Subchronic Toxicity 90-Day G avua Study ri Ran with Oiw-QancjKia*]tpro<todvaBrluaaoni PuPooi-1141 REFERENCES 1. EPA/FIFRA GoodLaboratory Practice Standards (40 CFR160). (1989). 2. OECD PrintiplesofGood M oratory Fracricc (as reviaod in 1997, published in ENV/MC/CHEM(98)17 (OCDE/GD(92)32). 3. MAFF Japan GoodLaboratoryPractice Standards (59NohSanNo. 3850). 4. United StaleEnvironment! ProtectionAgency (HPA), Office ofPrevention, Peatiridea, and Toxic Substance* (OPPTS)Hsilth Effect*TestGuidelines, OPPTS 870.3100 90-DayOral Toxicity in Rodents (AUG-1998). 5. Bradford, M. M. (1976), A rapid ind sensitive tocdiod forthe quantification ofmiraogram quantities ofproteinutilizingtheprincipleofprotein-dye binding. Anal. Biochem. 72, 248-254. 6. Draper,NR. and Smith, H. (1981). Applied RegreaaionAnalysis, Sedition, pp 266-273. Wiley, New York. 7. Selwyn, M R (1995). Theuse oftrendtosts to determine *no-observable-effectlevel in animalsafety studies. Journal o fthe American College o fToxicology 14(2), 158-168, 8. Joackheere, AJt. (1954). A diatribution-fteo K-samplotest againstordered alternatives. Biometrika 4X, 133-145. 9. Levene, H. (I960). Robusttest for equality ofvariances. CorUrilnrfiiwii to Probability and Statistics (1. Olkin, ed.), pp 278-292. Stanford University Press, Palo Alta 10. Shapiro, S.S. and Wilt., M.B, (1965). An analysis ofvariance test for normality (complete samples), Biometrika 52, 591-611. 11. Snedecor, G.W. and Cochran, W.G.(1967). StatisticalMethods, 6* edition, pp 246-248 and 349-352. The Iowa State University Press, Iowa. 12. Dunnett, C.W. (1955). A multiple comparisonprocedure forcomparing severaltreatments with a control. J. Ames, Statist Assoc. 50,1096-1121. 13. Kruskal, W.H. and Wallis, W.A. (1952). Use ofranks in one-criterionanalysis ofvariance. J.A tner. Statist Assoc. 47,583-621. 14. Dunn,O J. (1964). Multiple contrasts using rank sums. Technometrics 6,241-252. 15. Hocking, R. A. (1985). The Analysts o fUnear Models. Broolca/Cole, Monterey. 16. Bartlett, M. S. (1937). Some examples ofstatistical methodsofresearch in agriculture and appliedbiology. J. R oyal Stasis. Soc. SappL 4,137-170.. 17. Fiaher, RA. (1985). StatisticalMethodsfo r Research Workers, 13* edition. Haflhor, New York. 18. Dempattt, A. P., Selwyn,M.R., Patel, C.M., andRoth, A. J. (1984). Statistical and Computational Aspects ofMixedModel Analysis. The Journal o fthe Royal Statistical Society, Series C (Applied Statistics). 33(2), 203-214. 19. Mehta, D. andPatel N. (1995). StatXact 3 forWindows, CytelSoftwardCorporation, Cambridge, MA. 25 f 1 I | | f | i j t I i | t f j j j 'F ! Exygen Research Page 54 o f 81 -67- net sontatfi jjaW i CfH H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425? Subdrnniie Toxicity 9Q-]^y<Hvm8<SOK>yfa)Uawilh(>>^ennaionBjproditttlvEvhatkin3 ,, DuPnM1418 Study Function Study Director PROTOCOLAPPENDIX Study Pffiwnnsl Susan A. MacKentie Senior Research Toxicologist Analytical Assessment JanetC. Mulanka Analytical Chemist i Neurobehaviorsi Evaluation: Linda A. Malloy Neuiobehaviotsd Toxicologist Biochemical Analyses: JofcnC. O'Cotmor Biochemical Toxicologijt Clinical PathologyEvaluation: Nancy E. Everds Clinical Pathologist AnatomiePathology Evaluation: John F. Hansen Anatomical Pathologist 1! Reproductive Assessments: RobertM. Parker Reproduction Toxicologist I i Initiation ofTost SubstanceAdministration October 15,2002 Scheduled Sacrifice (final) April. 2003 Exygen Research 36 I Page 55 o f 81 ; - 68- ^ S p iS ff Dm s. H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H*2542i: SubdiionicToxicity GavagcStodyin R*a with Opo-OanerctioaRrpnxfoctm Evaluationa Apfrmeilty:_ SIGNATURES Prfwnyfai HirelwthSwjiijoUMKtTVUiTqkrnVuttt ' ^ 4 4 d t---- lieferthLtotar A^ndhwdMt'wMa^ht DuPont-U418 Ptt* Dm cuts XMe I ^J r * * f i8ta.ettX.l0MH 67- OoDwr-tl'Zaa'X- I Exygen Research - 27 Page 56 of 81 -69Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-2S425: SubeltronlcToxicity 9D-Pay Gavage Study in B aa rrlth O ne-G aittailcm StpraifoctiveByahatiom_________ _________ DuPoot-11418 ' Theprotocolit amendedasiollowsp 1. Rats designatedforevaluationof 10-dayhepaticbiochemicalanalysiswillbereceived approximately79days alterstudystartanddosingwiltbegin approximately90days afterstudystart, changesreferences to timingofreceiptanddosingof theseratson: Page5, Materials andMethods,B,TestSpecies,paragraph 2. Pge8,Mtenali andMetfiixlJ,E AssignmenttoGroups,paragraphs2 and6, Page 9,MaterialsandMethods,F.TatSubstancePreparationandAdmimstratioa,paragraph 1. Page21,MaterialsandMethods,P.BiochemicalAnalysis, 1. RatsDesignated forBiochemical Analysis,paragraph2. Rationale: Thetimingofthisanalysiswaschangedtoaccommodate wortschedules dotingthe holidays. 2. Page 13,Materula endMethods,M.TotalFluorineLevelEvaluation, ChangeheadertoreadTotalFluorineandPerfluorooctanoicAcidLevelEvaluations. Addthefollowingparagraphs atthe andofthe section: "Bloodwinbe collectedftomthetraincavasi necropsy,fromaErata designatedfor subchromctoxicityevaluationandalljnS designatedforrecoveryevaluation. Plasmawillbo preparedandstoredfcrrenat-80 to-20'Canfil analyzedforconcentrationof petlluoroootanolc sold(PFOA). Plasmawillbe evaluatedfromcontrolandhigh-doserats. The decisionto evaluatelow- andintennadintedosegroupswillbebaaedonresultsinthehigh-dose group. Plasmasampleswinbe extractedbyorganicsolventproteinprecipitationusingt protein precipitationcolumn, andthenanalyasdforPFOAby LC-MS-MS." Rationale:Abnainrasdecisionwasmadeto evaluate PFOAphuma levels atthe endofthe dosing period and alterrecovery. _ f r j j l f jr f j i ! ! u Approwdfcy*, O l-C dP MmmCCITffOOooaawof n^irtmlrvriretoobtht ,.SxHa J i.T n t lk g iz -I - h a y aaga. ?.Alm/-a2- Exygen Research Page 57 o f 81 -70Company Sanitized. Does not contain TSCA C8I H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: SuWuomeToxicity 9&-DeyGarage Study inRats withOnc-GaacotiattRamcdactivcBvalaatjmg________ _______ DuPoal-lHIB ProtocolxmendmentNo. 1, item2, is amendedas follows (changes bolded): Page 13, M aterials smdM ethods,M .TotalFluorine Level Evaluation, * Change header to read TotalFluorine and Perfluorooctatoic A dd Level Evaluations. Addthe followingparagraphs atthe end ofthe section: "Blood-willbe collected from the vote cave at necropsy, from all rats designated for aubdrtumc toxicity evaluation andall rats designated for recovery evaluation. Plasma willbe preparedandstoredfrozen at-80 to -20'C until analyzed forconcentration of perfhioroactanoic acid (PFOA). Plasmawill be evaluated from control end high-doserats. Thededsion to evaluatelow- andintermediate-dose groupswill be baaed on results in the high-dose group. Plasma samples w ill h e extracted by organic solventprotein precipitation uring a protein precipitation column, and then analyzed fo r PFOA by LC-MS-MS. R esults a f analysis wfll be reported in a supplem entary re p o rt" Rationale: Due to timing on analyticalmetfiocia development the resoles of these analyses w ill notbe available when the mainreportissues, but will issue later, as a supplem ent -71 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 H-25425: SubchronicToxicity SO-DsyGarage Studyin Ratwith Ono-Gmcraiion ReproductiveErnlmniom i PuTowUHli ProtocolAmendmentNo. 8 Protocol amendmentNo. 6, is amendeda follow(changes bolded): Page 13. Materials and Methods, M.Total FluorineLevelEvaluation, Changeheader to read Total FluorineandPerfluarooctanaic Acid LevelEvaluations, Addthe following paragraphs at foecod ofth section: "Blood vrill be collected from foe vaie cava et necropsy, fromall rats designated for subchronic toxicity evaluation and all rats designated forrecoveryevaluation. Plasmawill be prepared and stored frozen at-80 to -20C tmtilanalyzed forconcentration ofpecOiurooctaaoic acid(PFOA). Plasma samples willbe extractedby organic solventprotein precipitation and then analyzed for PFOA by an appropriate method atExygen Research (3058 Research Drive, State College,PA 16801). Remits ofanalysiswillbe reported in a supplementary report" Reason: To changethe evaluation to all sabchronic toxicityandrecovery group rats, change the methodofextraction and analysis, and add the name andaddrcsBoffoelaboratory performing the PFOAanalysis. ---------- z S-SXh>3 Exygen Research Page 59 of 81 -72Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 RESEARCH kPtedse Research. Proven Results. Page _1_ of _1_ PROTOCOL DEVIATION \ DevMlon Number: 1 Date of Occurrence: 10/07-08/03.10/08/03 . ExygehStudy Number. 008-342 . Protocol Number DuPont-11418 1 . Amendtrumt 8, Analytical W eU u ^ ^ H o n 4 .5 .< eS ro ^ ^ S ^ reco w ry o f 63X for Exygen sam ple ID0302238 Spk 82 a t 1000 ppb, 2. Amendment 8. Analytical Method Section 4.S.4.e~acceptcd recovery o f 119* far Exygen sample ID0302047 Spk C a t 1000 ppb. \ \ AQTIQB?-TAKN ey ampiadaUWIanImimJ. SOPwvMan ria 1-2. Protocol Deviation Issued. \ \ Recorded Datei:: l l j r / c a IMPACT 1_________ 1. No negative Impact because the LUX} fortification and th e laboratory fortified field sample w ere w ithin th e acceptable range for thkanalytlcat set. 2. Nor acceptable range for this analytical set. control fortified samples were within the \ wOMMAf:__ iStfAi/cfrC-af;--------------- Pdfib^al tebeiUgator Signature StuStsfwy DiAirvectorQSigfnbatnurte/^et^Vu i \ \ la te \ Date y * * --- *, ,, , V, fManagement Slgnitur " /`l i t Date fSoonaofi -- \ ExygenQAUReview'. LTIS l l \ l t l o U.AFomaWROTOCOl.OEVWTIONeoo \W li1 Exygen Research 16801, USA V exfSoCom Page 60 of 81 -73Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 APPENDIX B Analytical Method ExM-008-276, Revision 1 ``Method of Analysis for the Determination of Pentadecafluorooctanoic Acid (PFOA) in Serum by LC/MS/MS" Exygen Research Page 61 of 81 -74Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 RESEARCH Predse Research. \ Prcven Rssute. mm Method ofAnalyste for the Determination ofPerfluoraoctanoic Add (PFQA) in Serum \ by LC/MS/MS - Revision 1 AUTHOR i \' Emily Decker II BATE ESTOP v. September 24,2003 \\ \ SPONSOR \ DuPont Haskell kaboracoryfor Health & Environmental Sciences \ P.O.Box 50 Newark,DE 19714-0050 USA DuPonfStudyNumber DuPont-13090 PERFORMING LABORATORY Exygdt Research 3058 ResearchDrive State CoUegyPA 16801 \ METHOD NUMBER ExM-008-276 R evlon 1 TOTAL NUMBER OF PA&ES 20 t.* s Exygen Research BiygeruDm Page 62 of 81 -75Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 MANAGEMENT APPROVAL I 11 Seeker "Sdentisi Exygen Research Exygen Research _________T -5 t -Z o c3 S. Made Kennedy T Date Sponsor Representative DuPont 5( i \ BxygeoResessch Exygen Research i i Page 2 of 20 * X II, f Page 63 of 81 -76Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 D u P o n t-11418 Exygen Study No.: 008-342 TABLE OF CONTENTS OTLE..........................................~................................................................... .-- MANAGEMENT APPROVAL TABLE'OF CONTENTS LIST OF TABLES LIST OF FIGURES. 1. SUMMARY 2. EXPERIMENTALCOMPOUNDS 3. CHEMICALS AND SUPPLIES 3.1. C hem icals 3.2. Standards 3 3 . E q uipm ent a n d Supplies 3 .4 . Solutions 3.5. Prepa ra tio n o f Standards a n d F o rtifica tio n So lu tio n s............................... 9 3 .5 .1 . Sto ck so lu tio n 3 3 2 . F o rtifica tio n Solutions ................................................................................- 9 II3 .5 .3 . C a l ib ra tio n sta n d a rd s u tu M m ifilliM O n H M n u H K iu m iim liM li 10 METHOD. 4 .1 . F low D ia g ra m 4 2 . S AMPLEPROCBSSINa.. 10 10 10 43. Sa m ple P repa ra tio n 11 4 .4 . Extra ctio n 11 4.5. Q u antitation 43.1. LC/MS/MS System and Operating Conditions 11 11 4.52. T une F il e P aram eters 12 4 3 .3 . C a libra tio n P rocedures 12 4 3 .4 . Sam ple A n a ly sis..- ........... 13 4.(5. Acceptance C r u e r ia - .1 4 4 .7 . Perform ance C riteria .. 14 4 .8 . T im e Beq u ir bo f o r A n a l y sis. 15 51 CALCULATIONS. 15 6. SAFETY.. HIIUH 15 Exygen Research "Exygen Research Page 3 of20 Page 64 o f 81 -77Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 LIST OF TABLES i<i Table I. Recovery ofEFOA from Fortifications In Monkey Serum..........................16 LIST OF FIGURES Figure l. Calibration curve for PFOA in ControlMonkey Serum............... ................ 17 Figure 2. Representative Chromatogram of a Control Monkey Serum Sample for PFOA with ~ 5 ng/mL of "C-PFOA-............. - ..................................-..... 18 Figure 3. Representative Chromatogram of a 10 ppb Standard for PFOA in Control Monkey Serum with - 5 ng/mL of ,3C-EFOA........... ............................... 19 Figure A. Representative Chromatogram of ControlMonkey Seram Fortified at 10 ppb with PFOA and with ~ 5 ng/mL of 13C-PFOA...... ..................................... 2D Exygen Research Exygen Research - P tge4cf20 { i Page 65 of 81 -78Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 1. SUMMARY This report details a method of analysis for perfluoiooctanoic acid (PFOA) in serum. IPFOA is extracted torn serum by protein precipitation in acetonitrile. Quantification of PFOA is accomplished by liquid chiomatography/tandem miss spectrometry (LC/MS/MS) analysis using selected reaction monitoring (SRM). The chemicalformula ofPFOA Is given in section 2 of this method. The lower limit of quantitation (LLOQ) for this method is 10 ppb for PFOA in serum. Quantification is performed using extracted calibration standards containing an internal standard. This method was developed using monkey serum. The overall percent recovery standard deviation for PFOA in monkey serum at 10,100, and 1000 ppb was 94% 6.9% (Table I). A representative calibration curve forPFOA in monkey serum is shown in Figure 1, Representative chromatograms for PFOA in monkey serum are shown in Figures 2to4. I ExygenRtseuxh Exygen Research Page Serf20 I 1 Page 66 o f 81 -79Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No,: 008-342 2. EXPERIMENTAL COMPOUNDS The structure of petfluorooctanoic acid (PFOA) and the internal standard are given below, PFOA ChemicalName = Molecularweight * perfluorooctanoic acid 414 r " C-PFO A Chemical Name Molecular weight pcrfluorooctanoic acid, [1,2-di- 3C] =416 5 s , Hxygcn Rcsurcb Exygen R esearch r t: " Page 6 o f20 Page 67 of 81 - 80Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 3. CHEM ICALS AND SUPPLIES 3 .1 . C h e m ic a l s Chemical Methanol (MeOH) Acetonitrile (ACN) Ammonium Acetate OmniSdlv Water Grade HPLC HPLC Reagent HPLC Source EM Science EM Science Sigma-Aldrich EM Science Catalog No. JT9093-2 AXB145-1 : A-7330 WX0004-1 Standards Standard perUuorooctanoie add (EPOA) porfluoiooctanoic acid, LotNumber Purify Source aHil- (ft) - -- 21002 1 Oakwood I Products NA 1 & DuPont i i ,^****,N, Exygen Research Exygett Research * Psge7of20 i | Page 68 of 81 -81 Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity Supplement 1 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations____________________ DuPont-11418 Exygen Study No.: 008-342 1 3 3 . E q u ipm e n t and S uppls E autam ent Balance, analytical (display at least0.0001 g) Centrifuge 2 mLeppendorfcentrifoge tubes Disposable micropipets (50-100uL, 100-200uL) Class A pipets and volumetric flasks Hypercarb drop-in guard column (4 mm) (pat #844017-400) Stand-alone drop-in guard cartridgeholder 125-mLLDPEnarrow-mouth bottles 2 mL clea HPLC vial ldt (cat# 5181-3400) Standard lab equipment (graduated cylinders, disposable tubes etc.) LC/MS/MS aridHPLC systems Supplier Mettler Eppendorf VWR ' Drummond (VWR) various suppliers Keystone Keystone Nalgene HP various suppliers As described in section 4.5. Note: Equivalent materials may be substituted for those specified In this method if they can be shown to produce satisfactoryresults. Notea: 1. hi order to avoid contamination, the use of disposable labware is highly recommended (tubes, pipets, etc.). r 2. FIFE or PTEE lined containers or equipment, including PTEB-lined HPLC vials for the HPLC antosamplermust naibe used. 3. It is necessary to check the solvents (methanol) for tho presence of ccmtaminants by LC/MS/MS before usa Certain lot numbers have been found to be unsuitable for use. 4. Use disposable micropipets or pipets to aliquot standard solutions to make calibration standards and sample fortifications. 5. The hypercarb cartridges should be changed when the system contaminants (elevatedbaseline)move to within 1minute from the elution ofPFOA. 3 .4 . S o lu tio n s (I) 2 mM ammonium acetate solution is prepared by weighing 0.15 g of ammonium acetate and dissolving in 1L of CtamiSolv Water. Note; The aforementioned example is provided for guidance, alternative volumes 5 maybe prepared as long as the ratiosofthe solvent to solute are maintained. > !i Exygen Reoeirch PijcBoFM J i j Exygen Research i Page 69 o f 81 - 82Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 3.5. Preparation o r Standards and Fortification Solutions Analytical standards an used foe time purposes: 1. Calibration Standards - These standards an prepared in control monkey serum and are used to calibrate the response of the detector usedIn the analysis. 2. Laboratory Control Spikes - These fortifications are usually prepared at concentrations corresponding to the LLOQ and lOx 1XOQ and are used to determine analytical recovery. Laboratory control spikes are prepared in control monkey serum. i 3. Matrix Spikes - These fortifications are prepared by spiking into die field samples at known concentrations. Matrix spikes are used to evaluate the effect of the sample matrix on analytical recovery. The absolute volumes of the standards maybe varied by the analyst as long as the correct proportions of solute to solvent are maintained. Alternate concentrations may be prepared as long as the preparation and concentration are accurately recorded in the raw data. Note also that additional concentrations may be prepared if necessary. : 3.5.1. Stock solution Prepare individual stock solutions of - 100 pg/mL of PFOA and I3C- y.-- PFOA by weighing out - 10 mg of analytical standard (corrected for purity) and dilute to 100 m L with methanol in a 100-m L volumetric flask. ! Bach stock solution fin 125-mL LDPE bottles) is to be stored in a refrigerator at 2C to 6'C and is stable for a maximum period of 1 year from the date of preparation. 35.2. FortificationSolutions a. Prepare a fortification standard of 10.0 pgftnL for PFOA by diluting 10,0 mL of the PFOA solution described in 3.5.1 to 100 mL with ' acetonitrile in a volumetric flask. b. Prepare a fortification standard of 1.0 pg/mL for n C-FFOA by diluting 1.0 mL of the l3C-PFOA solution described in 35.1 tit 100 mL with acetonitrile in a volumetric flask. c. Prepare a fortification standard of 1.0 pg/mL for PFOA containing 0.005 pgWL of " C-SFOA bydiluting 10.0 mL of the PFOA solution described in 355,a and 05 mL ofthe 13C-FFOAsolution described in 35.25 to 100mL with acetonitrile in a volumetric flask d. Prepare a fortification standard of 0.1 pg/mL for PFOA containing 0.005 pg/mL erf n C-PFOA by diluting 1.0 mL of the PFOA solution described in 3.5.2.a and 05 mL of the^C-PFOA solution described in 355.b to 100mLwith acetonitrile in a volumetric flask. e. Prepare a fortification standard of 0.01 pgftnL for PFOA containing 0.005 pg/mL of l5C-PFOAby diluting 0.1 mL of the PFOA solation Bxygoo Research ' Page 9 of20 Exygen Research Page 70 of 81 -83Company Sanitized. Does not contain TSCA C B I H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 described in 3.5.2.a and 0.5 mL of the uC-PFOAsolution described in 3.5.2.b to 100mL with acetonitrile in a volumetric flask. Store all fortification standard solutions in a refrigerator (in 125-noLLDPE bottles) at 2C to 6C for s maximum period of 1 year from the date of preparation, after which rime it is necessary to make new standards'tising the stock solution. 3 .5 3 . Calibration Standards The calibration standards are processed through die extraction procedure, identical to the samples. The fortification of die standards before I extraction Is done according to the following table: Cone. Of Mixed Fortification Solution (lig/mL) 0.01 0.01 0.1 0.1 0.1 Fortification Volume of Volume (pL) Control Sample (mL) 100 0.1 200 0.1 so 0.1 100 0.1 200 0.1 Cone, of Extracted Calibration Standard (ppb) 10 20 50 100 200 ; ; 4. METHOD 4.1. FlowDiagram The flow diagram of the method is given below, followed by a detailed description of each step. Method Flow Diagram Measure 0.1 mL ofsample (using disposable micropipette) (fortify samples when designated] 4* 4 Protein precipitation in acetonitrile LC/MS/MS analysis 4 2 . SampleProcessing No sample processing is needed for serum samples. However, frozen samples must be. allowed to completely thaw, un-aided, at mom temperature. Samples ! 1. ExygenRcscueh Tage 10of20 Exygen Research Page 71 of 81 -84Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 stored refrigerated should also be allowed to equilibrate to room temperature. AJ] samples mustbe thoroughly mixedbefore being sampled for extraction.. 4.3. Sa m pl e P repa r a tio n a. Each batch of samples extracted (typically 30 or less) must include at least one reagent control (acetonitrile blank), one matrix control (method blank), and two matrix controls fortified at known concentrations to verify procedural recovery for the batch. b. At least one sample perbatch should be extractedin duplicate c. At least one sample extracted should be separately fortified at a known concentration and carried through the procedure to verify recovery. Additional matrix spikes may be performed at the sponsor's request. 4 .4 . E x t r a c t io n 1. Mesante 0.1 mL of sample into 2 mL sppendorf centrifuge tubes (fortify with analyte as needed, closelid, end vortex -1 0 seconds). 2. Add enough acetonitrile contsining internal standard at 0.005 pg/mL (accounting for fortification volume) to make extraction volume 500 |oL and vortex for-1 0 seconds. 3. Centrifuge for -1 0 minutes at - 14,000 rpm. 4. Analyze samples using electrospray LOMS/MS. 4 5 . QUANTITATION 4S I. LCMS/MSSystemandOperatingConditions Instrument: PE SCIEX API 4000 BiomolecularMass Analyzer SCIEXTurbo Ion Sprayliquid Introduction Interface Computen Dell OptiFlexGX 110 Software: PE Scicx Analyst 1.2 HPLC Equipment HewlettPackard (HP) Series 1100 QuatpumpG1311A VacuumDegasser G1322A Autoinjector G1313A Column CompartmentG1316A Note: Two 4 x 10 mm hypetcarb drop-in guard cartridges (Keystone, part # 844017-400) are attached in-line after the purge valve and before the sample iqjector port to trap any residue contaminants that may be in the mobile phase and/orHPLC system. Exygen Reje*rch -Page 11 of20 | | | I j j L 1 , 1 ; j ! i!, 1. i r i t j ; I ^ Exygen Research Page 72 o f 81 -85Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 HPLC Column: Genesis C, (Jones Chromatography), 2.1 mm x 50 mm, 4p Column Temperature: 30 C i Injection Volume: 3 (iL Mobile Phase (A): 2 mM Ammonium Acetate in OmniSolv Water Mobile Phase (B): Methanol j Time &A % B . Flow (mL/min) 0.0 40 60 03 i 3 40 60 03 J 33 0 100 0.3 3.7 0 100 03 I 70 100 03 7.5 40 60 0.5 : 9 40 60 03 93 40 60 03 12 40 60 0.3 It may be necessary to adjust the HPLC gradient in order to optimize instrument performance. Ions monitored: Analyte PFOA ,3C-PFOA Mode Transition Monitored Negative 413 - 4 369 Negative 413 - 370 Approximate Retention Time -2.8 min. -2.8 min. The retention times may vary, on a day-to-day basis, depending on the batch of mobile phase etc. Drift in retention times is acceptable within an analytical run, as long ss the drift continues through the entire analysis and the standards are interspersed throughout the analytical tun. Note: An alternative LC/MS/MS system may be used once demonstrated to be equivalent. 43.2. Time file Parameters 1: | | | The mass spectrometer is tuned for the analyte by infusing a - 1 JtgtaL standard J solution of PFOA (at 10 pL/min, using an infusion pump) via a "1" into a stream of mobile phase containing 60% methanol and 40% 2mM ammonium acetate at j 03 mL/min flow rate. The analyte la initially tuned for the parent ion and then | tuned for the product ion. Once the instromenl Is toned, the optimized parameters | are saved as a tune file. This tune file is then used during routine analysis. i 4.5.3. Calibration Procedures ' .; a. Iqject the same aliquot (3 |tL) of each calibration standard into the LC/MS/MS. [, Exygen Research ` Page 12of 20 Exygen Research Page 73 of 81 -86Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 b. Use linear standard curves for quantitation. linear standard curves are generatedfor each analyte by linearregression using 1/x weighting of the ratio analyte peak area/lmemal standard peak area versus the ratio of the concentration of aoalytafconcentration of internal standard using Analyst 1.2 (or equivalent) software system. Any calibration standard found to be a j. j; 1 - statistical outlier by using the appropriate-outlier test (e.g. Huge OutlierTest), n may be excluded from the calibration curve. However, the total number of | calibration standards that could be excluded must not exceed 20% of the total r number of standards injected and at leaat one calibration standard at the I LLOQ must be retained. ; l c. The correlation coefficient (r) for calibration curves generated must be 0.992S (t2 0.98$). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or therelevant setof samples should be reanalyzed. ; j 4.5.4. Sample Analysis a. Inject the same aliquot (5 (it) of each standard, sample, recovery, control, etc. into theLC/MSWdS system. j j I b. Standards corresponding to at least five or more concentration levels must be 5 included in an analytical set. ; c. An entire set of calibration standards should be injected at the beginning of a set followed by calibration standards interspersed every 5-10 samples (to : i account for a second set of standards). As an alternative, an entire set of calibration standards may be included at the beginning and at the end of a sample se t In either case, calibration standards must be the first and last injection in a sampleset l- (L The concentration of each sample/fortificalion/control is determined from the | standard curve, based on the peak ares of each analyte. The standard responses shouldbracketresponses ofthe residue found in each sample Bet If ' necessary, dilute the camples in 5(hS0 metbanobwater to give a response j. within the standard curve range. y e. Fortification recoveries falling within 85 la 115% (80 to 120% for levels at the IXOQ) are considered acceptable. f. Extracted samples must be stored refrigerated between 2C to 6C until analysis. g. Samples in which no peaks are detected die. signal: noise ratio < 3:1) at the corresponding analyte retention times will be reported as ND (not detected). Samples In which peaks are detected at the corresponding analyte retention Exygen Research 'Page 13 of 20 Exygen Research Page 74 o f 81 -87Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 times but are less titan the lowest concentration of the calibration standards (10 ppb where LLOQ 10ppb) will be reported as <10 ppb. _ 4 .6 . Ac c ept a n c e Cr it e r ia The following criteria must be met to ensure the presence of PFOA and MC-PPOA: 1. The chromatograms mustshow the following peaks: PFOA: a daughterion at 369 amu from a parent of 413 amu 13C-PFOA: a daughterIon at 370 amu from a parent of413 amu 2. Any analyte present in method blanks must be at least -fold lower than the LLOQ. Any analyte present in the reagent blank must be at least 5fold lowerthan the LLOQ. 3. Recoveries of lab control spikes and matrix spikes must be between 85115% (80-120% for levels at the LLOQ) of their known values. Any method fortification (lab control spike) foiling outside the acceptable limits warrants re-extraction of the entire analytical set Any matrix spike outside die acceptable range should be evaluated by foe analyst to determine if re-extraction Is warranted. 4. Any calibration standard found to be a statistical cottier by using an appropriate outlier test may be excluded from the curve. However, foe total number of calibration standards that could be excluded must not exceed 20% of foe total number of standards injected and at least one calibration standard atthe LLOQ mostbe retained. 5. The correlation coefficient (r) for calibration curves generated must be 50.992S (r* 50.985). If calibration results fall outside these limits, then appropriate steps must be taken to adjust instrument operation, and the standards or the relevant set ofsamples should be reanalyzed. 4 .7 . P e r fo r m a n c e C w t e m a The following two criteria must be performed as a system suitability test, before the commencement of analysis when using an instrumentation set-up that has not been used for this method. First Criterion: Run a standard solution on LG/MS/MS corresponcEng to the estimated LLOQ and obtain a signal-to-nolse ratio of at least 9:1, compared to a reagent blanle If this criterion cannot be met, optimize and change instrument operating param eters. E xygaaR cunth Exygen Research ' Pge 14 o f20 Page 75 of 81 -88Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Second Criterion: Run a set of standards of five or mote concentration levels, spanning a range slatting at or below the LLQQ up to the highest concentration level to be'included in the analysis. Generate a calibration curve for each analyte and obtain a linear regression with a coefficient of determination (r2) of at least 0.985. Once this criterion is met, samples may be analyzed with standards interspersed. 4 .8 . T im e R e q u ir e d fo b A n a ly sis i A set of 35 samples (1 reagent control, 1 matrix control, 1 laboratory spike, 2 laboratory control spikes, and 30 samples) can be taken through die extraction I procedure in approximately 8 hours by one person. The LC/MS/MS analysis (standards and 35 samples) will take approximately 9 hours. 5. CALCULATIONS a. Use Equation 1 to calculate the amount of analyte found (in ng/mL, based on peak area) using die standard curve (linear regression parameters) generated by the Mass Lynx software program. Equation 1; A nalyte found (ppb) = (AnalyteftakyrM /IS peak a n a l- Intercepts xIS cone. (ngtaL) dope b. For samples fortified with known amounts of analyte prior to extraction, use Equation 2 to calculate the percent recovery. I : E a w te l; Recovery (%)= Analvte found (pub)- avg. Analyte found in control ftmb) x 100% AmountAnalyte added (ppb) 6. SAFETY The analyst should read the material safety data sheets for all standards and reagents before performing this method. Use universal precautions when handling standards and reagents, including working in fume hoods and wearing laboratory coats, safety glasses, and gloves. Use blood-home pathogen handling precautions when handlingserum. 1 l Exygen Research 'P age 15 of 20 1 Exygen Research Page 76 of 81 -89Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 T able I. Recovery of PFOA from Fortifications In M onkey Serum SummitryofPFOARecoveriesfor 10ppbFortiEe*tion in Monkey Serum Exypo ID 0300808SpkA 030&808SpkB 030030! SokC Spanar Extraction Analysis Boit Uvei ID Dais D ue (rob) Lot4QK7400 0S0/3 5/3/3' Lat40K74Q0 os/mo mom 10 10 Lot40E7400 5/3/03 05/30/03 10 AVERAGE: STANDARDDEVIATION: RELATIVESTANDARDDEVIATION: Recovery fS 94 98 96 LI 12 i Summary ofFFOA Rrcoverics for 100 ppb FortffiraUon in Monkey Serum E iyta Span* Extraction Analysis ForLLwet % Recovery ID ID Dato Data Cm*) t 4t 0300808SpkD 0300808SpkE 0300801SdJcF Lot40K740Q 05/3093 Lflt40K740Q 0500/03 Lot40K7400 0S/30/D3 QSBOm 100 Gsatm 100 05/30/03 100 AVERAGE: 92 90 93 92 STANDARDDEVIATION: 1.5 RELATIVESTANDARDDEVIATION: L7 Summary ofPFOA Recoveriesfor 1000ppbFortification in MonkeySerum fV Spout* Extraction Analyst* F ort Level % Recovery ID ID Date Due (PI*) 0300808 SpkO Lot4QK340a 05/30/03 0930/03 1000 107 0300808 Spk H UH40K7400 05/30/03 0930/03 1000 95 0300108 Spk1 LOC4QK74C0 05/30/03 05/30/03 1000 81 A vouas 94 STANDARDDEVIATION: UO J RELATIVE STANDARD DEVIATION: ZXS OVERALLAVERAGE! 94 ! OVERALLSTANDARDDEVIATION! 9 OVERALLRELATIVESTANDARDDEVIATION! 7J j ti Exygea Research Exygen Research 'Pago 16 of 20 Page 77 of 81 -90Company Sanitized. Does not contain TSCA CBi H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Figure 1. C alibration curve fo r PFOA in C ontrol M onkey Serum C630Qaiiu*fPai^njflMrmgnBMp/x,wriaMno):T*>04)4asxi4x+a.oiaoge3{r*iasMWq s J1 b I Exygen Resarch Exygen Research 'T ag e 17 of 20 f \ Page 78 of 81 -91 Company Sanitized. Does not contain TSCA CB1 H-25425: Subchronic Toxicity Supplement 1 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations____________________ DuPont-11418 Exygen Study No.: 008-342 Figure 2. R epresentative Chrom atogram o f a C ontrol Monkey Serum Sam ple fo r PFOA w ith - 5 ngfaiL of "C-PFO A L- Exygen Research Exygen Research 'F ags 18 of 20 Page 79 of 81 -92Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.t008-342 Figure 3. Representative Chrom atogram of a 10 ppb Standard for PFO in Control M onkey Serum w ith ~ 5 n g /m t'o f " C- Exygen Research Exygen Research Page 19 o f 20 Page 80 of 81 -93Company Sanitized. Does not contain TSCA CBI H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 Exygen Study No.: 008-342 Figure 4. Representative Chromatogram of Control Monkey Serum Fortified at 10 ppb with PFOA and With - 5 ngfrnL of ^CPFOA I ^a^iaaijij^aw^iKjigiCTp flSSS. ta - 5TS" s i. & I" t&Was.s.r*M.-ta "ss.- ** a i r Tl Is - "n j[--.'- 'j - ,,`"",,`"V. `TT' Exygen Reseucb Exygen Research ' Pogs 20 o f20 Page 81 of 81 -94Company Sanitized. Does not contain TSCA C8I H-25425: Subchronic Toxicity 90-Day Gavage Study in Rats with One-Generation Reproduction Evaluations Supplement 1 DuPont-11418 APPENDIX B INDIVIDUAL ANIMAL BLOOD FLUORINE -95Company Sanitized. Does not contain TSCA C8I .) ) H-25425: Subchronic Toxicity 90-Day G avage Study in R ats with O ne-G eneration R eproduction Evaluations___________ Blood Fluorine Fg Day -4 Blood Fluorine ppm Day -4 Male, Group I, 0 mg/kg/day 665023 a 665072 - 665151 - 665164 - 665154 - - M ale, Group III, 10 mg/kg/day 665132 - 665156 - 665061 - 665145 - 665019 - - Male, Group V, 100 mg/kg/day 665048 - 665030 - 665028 - 665052 - 665150 - - Male, Group VII, 1000 mg/kg/day 665084 <0.05 <0.5 665142 0.05 <0.5 665118 0.16 <0.5 665108 0.19 <0.5 665024 <0.05 <0.5 Blood Fluorine m Day 3 - - - 0.89 0.84 0.82 0.75 0.95 Individual Animal Blood Fluorine Blood Fluorine ppm Day 3 Blood Fluorine Hg Day 9 Blood Fluorine ppm Day 9 Blood Fluorine Fg Day 22 - - - <0.05 - - - 0.77 - - - 0.59 - - - 0.71 - - - 0.31 - - - 0.72 - - - 0.65 - - - 0.44 - - - 0.91 - - - 0.58 - - - 0.94 - - - 0.54 - - - 1.31 - - - 0.81 - - - 0.87 1.87 1.59 3.87 1.68 1.56 1.47 3.56 2.05 1.48 1.53 3.20 1.78 1.60 2.03 3.56 2.26 1.75 1.07 3.76 2.18 Blood Fluorine ppm Day 22 <0.50 1.51 1.47 1.43 0.97 1.43 1.38 0.77 1.77 1.22 1.85 1.62 2.38 1.67 1.77 5.26 4.36 5.15 4.26 4.68 Blood Fluorine Fg Day 89 _ - - 5.00 3.43 4.37 4.62 3.27 Supplement 1 DuPont-11418 Blood Fluorine ppm Day 89 _ - _ _ - _ _ _ - - 10.96 9.96 9.59 9.50 9.28 Company Sanitized. Does not contain TSCA CBI A dash indicates that samples were not evaluated for the respective time point - 96- H-25425: Subchronic Toxicity 90-D ay G avage Study in R ats w ith O ne-G eneration Reproduction Evaluations___________________ Blood Fluorine Fg Day -4 B lood Fluorine ppm Day -4 Female, Group II, 0 mg/kg/day 665176 665283 665297 665301 665269 Female, Group IV, 10 mg/kg/day 665218 665309 665177 665202 665235 Female, Group VI, 100 mg/kg/day 665313 665233 665216 665266 665270 Female, Group VIII, 1000 mg/kg/day 665302 665179 665287 665172 665194 Blood Fluorine Fg Day 3 - - - - - - - - Individual Animal Blood Fluorine Blood Fluorine ppm Day 3 Blood Fluorine Fg Day 9 Blood Fluorine ppm Day 9 Blood Fluorine Fg Day 22 - - - 0.99 - - - 0.68 - - - 0.42 - - - 0.42 - - 0.57 - - - 0.92 - - - 0.72 - - - 0.66 - - - 0.66 - - - 0.63 - - - 0.68 --- 1.07 - - - 0.85 --- 1.07 - - 0.69 ----------- 1.58 1.07 1.73 1.60 1.25 Blood Fluorine ppm Day 22 2.07 1.27 1.16 1.07 1.34 1.86 1.65 1.45 1.30 1.44 1.50 2.43 2.00 2.07 1.54 3.38 3.34 3.82 3.19 2.88 Blood Fluorine Fg Day 89 _ _ - _ - - 1.23 1.34 1.10 1.83 1.23 > Supplement 1 D uP ont-11418 Blood Fluorine ppm Day 89 _ _ . - _ - - 2.93 3.03 2.64 4.13 3.75 Company Sanitized. Does not contain TSCA CBi A dash indicates that samples were not evaluated for the respective time point. - 97-