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AR226-2817 DuPont-15372 TRADE SECRET Study Title H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substance in the Analysis of Rat Serum Lipids Author: Denise Hoban, B.A, MLT (ASCP) Study Completed on: October 13,2004 Testing Facility: E.I. du Pont de Nemours and Company HaskellSMLaboratory for Health and Environmental Sciences Elkton Road, P.O. Box 50 - Newark, Delaware 19714-0050 U.S.A. Laboratory Project ID : DuPont-15372 Sponsor: E.I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A. Page 1 o f 29 Company Sanitized. Does not contain TSCAC31 H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 CERTIFICATION I, the undersigned, declare that this report provides an accurate evaluation of data obtained from this study. Issued by Study D irector: Denise Hoban, B.A, MLT (ASCP) Staff M edical Technologist and Supervisor i3 P e r 200^ Date 2e -- -- lB " H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 TABLE OF CONTENTS Page C E R T IF IC A T IO N .......................................................................................................................................... 2 ST U D Y IN FO R M A TIO N ..............................................................................................................................4 SU M M A R Y ........................................................................................................................................................ 6 O B JE C T IV E ......................................................................................................................................................7 EX PER IM EN TA L D E SIG N ........................................................................................................................ 7 M A TER IA LS AND M E T H O D S ................................................................................................................ 8 A. Test Substances........................................................................................................................ 8 B. Phosphate Buffered Saline (PBS)............................................................................................ 8 C. Experimental Procedures..........................................................................................................9 R E SU L TS AND D IS C U S S IO N ................................................................................................................ 12 A. Clinical Chemistry Analyses..................................................................................................12 C O N C L U S IO N S ........................................................................................................................................... 12 R E C O R D S AND SAM PLE S T O R A G E ................................................................................................ 12 T A B L E S ............................................................................................................................................................13 TABLE 1 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-24921 IN PBS.............................................................................................................................................................. *5 TABLE 2 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-24921 IN SERUM................................................................................................................................... TABLE 3 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-26313 IN PBS........................................................................................................................................... TABLE 4 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-26313 IN SERUM................................................................................................................................... TABLE 5 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-26225 IN PBS........................................................................................................................................... TABLE 6 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-26225 IN SERUM.................................................................................................................. 20 A P P E N D IC E S ................................................................................................................................................21 APPENDIX A CLINICAL CHEMISTRY DATA........................................................................................................ 22 -3 CO' jaiATS! H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 STUDY INFORMATION 9th Collective Nomenclature: Octanoic acid, pentadecafluoro-, ammonium salt Synonyms/Codes: Ammonium perfluorooctanoate Perfluorooctanoate, ammonium salt PFOA H-24921 Cl Haskell Number: 24921 CAS Registry Number: Composition Purity: Physical Characteristics: White solid Stability: The test substance appeared to be stable under the conditions of the study; no evidence of instability was observed. ;not contain ,TsCA C3 -4 - Company Sanitteea. b * H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 STUDY INFORMATION (Continued) Substance Tested: APFO Svnonvms/Codes: Ammonium perfluorooctanoate Haskell Number: 26313 CAS Registry Number: Known Impurities: Physical Characteristics: Water white to slightly opaque liquid Stability: The test substance appeared to be stable under the conditions of the study; no evidence o f instability was observed. Substance Tested: APFO Haskell Number: 26225 Known Impurities: Physical Characteristics: White solid Stability: The test substance appeared to be stable under the conditions of the study; no evidence o f instability was observed. Study Initiated/Completed: July 29, 2004 / (see report cover page) Experimental Start/Termination: July 29, 2004/July 30, 2004 *TSCACB\ - 5 - San'ui--.--sUd. Do' CpmpanV H-24921, H-26313, andH-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 SUMMARY The objective o f this study was to evaluate whether or not the in vitro addition of H-24921, H-26313, and H-26225 to pooled normal fasted rat serum interfered with the determination of any o f the following clinical pathology parameters: triglycerides, total cholesterol, high-density lipoprotein cholesterol, or non-high-density lipoprotein cholesterol. The test substance was added to fresh phosphate buffered saline (PBS) or pooled normal rat serum. Subsequently, serial dilutions were performed. Dilutions in PBS and serum, and negative controls o f PBS and serum, were analyzed for triglyceride, total cholesterol, HDL cholesterol, and non-HDL cholesterol. Neither H-24921, H-26313, nor H-26225, at concentrations up to 720 fig/mL, interfered with the determination of concentration of triglycerides, total cholesterol, HDL cholesterol, or non-HDL cholesterol in phosphate-buffered saline or in pooled normal rat serum. -6- H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 OBJECTIVE The objective of this study was to evaluate whether or not the in vitro addition of H-24921, H-26313, or H-26225 to pooled normal fasted rat serum interfered with the determination o f any o f the following clinical pathology parameters: triglycerides, total cholesterol, high-density lipoprotein cholesterol, or non-high-density lipoprotein cholesterol. EXPERIMENTAL DESIGN The following experimental design was conducted for each of the 3 test substances. Negative control samples were analyzed for each part of the study. Concentration of Test Substance Dilution Gig/mL) PBS Pooled Normal Rat Serum 1 0.0 V (PBS Negative Control) V (Serum Negative Control) 2 720.0 V V 4 360.0 V V 5 180.0 V V 6 90.0 V V 7 45.0 V V 8 22.5 V V 9 11.3 V V 10 5.6 V V 11 2.8 V V 12 1.4 V V 13 0.7 V V - 7 - contain TSCACfi, Gerr.psny San:.iizeG- vs not H-24921, H-26313, and H-26225: .. Evaluation of tbe Interference of the Test Substances in the Analysis of Rat Serum Lipids MATERIALS AND METHODS DuPont-15372 A. Test Substances Test Substances: The test substances were assigned the following Haskell Laboratory numbers: Part A: H-24921 for APFO PartB: H-26313 for APF Part C: H-26225 for APFOo| Q H g H-262:25 is made by combining H-26203 and H-26204. A: H-24921 [APFO^ DMSO. , a solid, was mixed with B: H-26313 [APFO a liquid, was mixed with DMSO. C: H-26203 and H-26204^olids, were combined to form H-26225 [ A P F O O H V Stock solutions for the highest concentration were made with H-26203 and H-26204 using .DMSO. The stnr.k solutions were mixed together at a ratio of _______ resulting in the stock "solution for H-26225. Dilutions were not analyzed to verify concentration and stability Purity/Assay: Confirmation of identity and purity of the test substance were conducted on a recent study, and were not conducted on this study/1,2) Stock Concentration: 14,400 /zg/mL (or 14.4 mg/mL) in volumes of approximately 1 mL Vehicle: dimethylsulfoxide (DMSO) Storage Conditions: The test substances were stored at normal room temperature and humidity prior to preparation o f the solutions. B. Phosphate Buffered Saline (PBS) Manufacturer: Fluka BioChemika Expiration Date: April 2009 -8Qprii?3^- Sa* ,,aioTSCftCBl H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids Lot Number: Physical State: clear, colorless liquid Storage Conditions: normal room temperature and humidity Known Contaminants: none DuPont-15372 C. Experimental Procedures Animal Care and Housing: Stock male Crl:CD(SD)IGS BR rats previously obtained from Charles River Laboratories, Inc were used in this study. The Crl:CD(SD)IGS BR male rat was selected because it was used in a previous study with these test substances. All rats were housed in stainless steel, wire-mesh cages suspended above cage boards. Animal rooms were maintained at a temperature of 22 3C and a relative humidity o f 50 20%. Animal rooms were artificially illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. All rats were provided tap water ad libitum. All rats were fed PMI Nutrition International, LLC Certified Rodent LabDiet 5002 except when fasted. Rats were fasted overnight prior to sample collection. As specified in the Haskell Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to ensure that contaminant levels are below those that would be expected to impact the scientific integrity of the study: Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants. * Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers. Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations o f key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence o f these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study. -9- H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Evaluation o f these data did not indicate any conditions that affected the validity of the study. Blood Source and Collection Site: Descending aorta or the abdominal vena cava from healthy fasted stock Crl:CD(SD)IGS BR rats approximately 8 weeks of age or older. Blood was collected while rats were under carbon dioxide anesthesia. After blood collection, rats were euthanized and discarded. Blood Handling: Approximately 6-8 mL of whole blood was collected from each rat and placed into commercially available serum separator collection tubes. Blood was processed to serum after centrifugation at 2500 rpm for 15 minutes and pooled. PBS Negative Control: PBS without test substance was tested as the PBS negative control. Serum Negative Control: Rat serum (pooled) without test substance was used as the serum negative control. Addition of Test Substances to PBS or Serum: For each part o f the study, the test substance was added to fresh PBS or serum as follows: test substance at 14,400 /xg/mL was diluted 1:2 in both PBS and serum (concentrations of 7200 /xg/mL). These working solutions were diluted using 1:10 dilutions to achieve concentrations o f 720 /xg/mL in PBS or serum. Subsequently, serial dilutions were performed according to the Experimental Design. The test samples in PBS and in serum were prepared at approximately the same time. The test samples in PBS and in serum were gently mixed prior to incubation at room temperature. Incubation, Processing, and Analysis: Test samples in PBS and serum, and negative controls were incubated at ambient temperature for approximately 10 minutes. Immediately after incubation, the samples were frozen at approximately -80C in plastic tubes until the analysis was run (at least overnight, but not more than 3 days). Prior to analysis, the samples were thawed, placed on the Olympus AU 640 clinical chemistry analyzer and analyzed in triplicate (one tube analyzed 3 times) by standard procedures for the following parameters: -10- H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 Triglyceride Method: Enzymatic (lipases, kinase, oxidase, and peroxidase), with colorimetric detection (Olympus Diagnostics, CAT # OSR6116) Total cholesterol Method: enzymatic (esterase and oxidase), with colorimetric detection (Olympus Diagnostics, CAT # OSR6133) High-density lipoprotein cholesterol (HDL) Method: Accelerator Selective Detergent methodology, with colorimetric detection (Equal Diagnostics, CAT #6121) Non-high-density lipoprotein cholesterol (non-HDL) Method: Calculated Calculations: Mean values were calculated for the clinical chemistry parameters for all control and test samples. Calculation of interference at each concentration o f test substance was not conducted, because there was no interference detected. -11 - H-24921, H-26313, andH-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 RESULTS AND DISCUSSION A. Clinical Chemistry Analyses (Tables 1-6, Appendix A) The values for the measured lipid concentrations did not change despite a thousand-fold change in test substance concentration. Therefore, the test substances did not have any effect on the measurement of triglycerides, total cholesterol, HDL cholesterol, and non-HDL cholesterol in pooled normal rat serum or in phosphate buffered saline. CONCLUSIONS Neither H-24921, H-26313, nor H-26225, at concentrations up to 720 /xg/mL, interfered with the determination of concentration of triglycerides, total cholesterol, HDL cholesterol, or non-HDL cholesterol in phosphate-buffered saline or in pooled control rat serum. RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, and the final report will be retained at Haskell Laboratory, Newark, Delaware, or at Iron Mountain Records Management, Wilmington, Delaware. REFERENCES 1. DuPont Haskell Laboratory unpublished data 2. DuPont Haskell Laboratory unpublished data - 12- H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 TABLES inlSC A Cm 13- nf*s5 tto COTOP3^ San*1*6 H-24921, H-26313, and H-26225: ....... Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids TABLES ABBREVIATIONS: EXPLANATORY NOTES PBS CHOL TRIG HDL non-HDL phosphate buffered saline cholesterol triglycerides high-density lipoprotein cholesterol non-high-density lipoprotein cholesterol DuPont-15372 - 14- O*r ! \ t scPkC0' not go H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 TABLE 1 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-24921 IN PBS SERIAL DILUTIONS NEGATIVE (Mg/mL) CONTROL 720 360 180 90 45 22.5 11.3 5.6 2.8 1.4 0.7 CHOL (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 TRIG (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 HDL (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 non-HDL (mg/dL) 0 0 0. 0 0 0 0 0 0 0 0 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Data arranged as: Mean Standard deviation - 15 - Sanitized. Coes not fcOUU'mT6CA HBI Company  H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids___________________ # DuPont-15372 TABLE 2 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-24921 IN SERUM SERIAL DILUTIONS NEGATIVE Gig/mL) CONTROL 720 360 180 90 45 22.5 11.3 5.6 2.8 1.4 0.7 CHOL (mg/dL) 54 51 53 53 54 54 55 57 55 54 54 54 0.00 0.58 0.58 0.00 0.00 0.58 2.00 3.06 1.00 0.00 0.00 1.00 TRIG (mg/dL) 48 46 48 49 49 49 50 49 49 49 49 49 0.58 0.58 0.58 0.58 0.00 0.00 1.53 0.58 1.00 0.00 0.00 0.58 HDL (mg/dL) 23 21 22 23 23 23 24 23 23 23 23 23 0.58 0.00 0.00 0.58 0.00 0.00 1.53 0.58 0.58 0.58 0.58 0.58 non-HDL (mg/dL) 31 30 31 30 31 31 31 33 32 31 31 31 0.58 0.58 0.58 0.58 0.00 0.58 0.58 3.21 0.58 0.58 0.58 0.58 Data arranged as: Mean Standard deviation s nAc TS - 16- % H-24921, H-26313, and H-26225 : Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids # DuPont-15372 TABLE 3 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-26313 IN PBS SERIAL DILUTIONS NEGATIVE (jig/mL) CONTROL 720 360 180 90 45 22.5 11.3 5.6 2.8 1.4 0.7 CHOL (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 TRIG (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 HDL (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 non-HDL (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Data arranged as: Mean Standard deviation - 17- ) H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids D uPont-153 72 TABLE 4 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-26313 IN SERUM SERIAL DILUTIONS NEGATIVE (jig/mL) CONTROL 720 360 180 90 45 22.5 11.3 5.6 2.8 1.4 0.7 CHOL (mg/dL) 54 52 53 54 54 55 55 55 56 55 54 55 0.00 0.58 0.00 0.58 0.58 1.53 0.58 0.58 1.15 0.00 0.00 2.08 TRIG (mg/dL) 49 47 48 48 49 49 49 49 49 49 49 50 0.58 0.58 0.00 0.00 0.58 1.00 1.73 0.00 0.58 0.58 0.58 1.15 HDL (mg/dL) 23 21 22 23 23 23 24 23 23 23 23 24 0.58 0.00 0.00 0.00 0.00 0.58 0.58 0.58 0.58 0.58 0.00 0.58 non-HDL (mg/dL) 31 31 31 31 31 31 32 31 32 32 31 32 0.58 0.58 0.00 0.58 0.58 1.15 0.58 0.58 0.58 0.58 0.00 1.53 Data arranged as: Mean Standard deviation - 18- S` Cot? DO,'f5C` o\cotl CBfc * m # H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids_______________________________________________ DuPont-15372 TABLE 5 SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-26225 IN PBS SERIAL DILUTIONS NEGATIVE______________________________ (jttg/mL) CONTROL 720 360 180 90 45 22.5 11.3 5.6 2.8 1.4 0.7 CHOL (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 TRIG (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 HDL (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 non-HDL (mg/dL) 0 0 0000000000 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 Data arranged as: Mean Standard deviation - 19- CotnPan^ H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis o f Rat Serum Lipids_______________________________________________ DuPont-15372 TABLE SUMMARY OF CLINICAL CHEMISTRY PARAMETERS FOR SERIAL DIUTIONS OF H-26225 IN SERUM SERIAL DILUTIONS NEGATIVE (ixg/mL) CONTROL 720 360 180 90 45 22.5 11.3 5.6 2.8 1.4 0.7 CHOL (mg/dL) 54 51 53 54 54 54 54 55 54 55 54 54 0.00 0.58 0.00 0.58 0.00 0.58 0.58 1.15 0.58 1.15 0.58 0.58 TRIG (mg/dL) 49 47 48 49 49 49 49 49 49 50 49 49 0.00 0.00 0.58 0.58 0.58 0.58 0.00 1.00 0.00 0.58 0.00 0.00 HDL (mg/dL) 23 21 22 23 23 23 23 23 23 23 23 23 0.00 0.58 0.00 0.00 0.00 0.00 0.00 0.58 0.00 0.00 0.58 0.00 non-HDL (mg/dL) 31 31 31 31 31 31 31 32 31 32 32 31 0.00 0.58 0.00 0.58 0.00 0.58 0.58 1.00 0.58 1.15 0.58 0.58 Data arranged as: Mean Standard deviation - 20- Coirtpany S -t s A r.Bi % H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 APPENDICES __________________________________________________________________________________________ Company Sanitized. Does not contain TSCA CBS H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 APPENDIX A Clinical Chemistry Data -22- Sanitized. Does not contain T30ACBI Compsny H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids CLINICAL CHEMISTRY DATA EXPLANATORY NOTES ABBREVIATIONS : CHOL TRIG HDL non-HDL PBS cholesterol triglycerides high-density lipoprotein cholesterol non-high-density lipoprotein cholesterol phosphate buffered saline . DuPont-15372 -23- rSCACB' Company Sanitized, t c s s not comam 4 H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 Clinical Chemistry Data for H-24921 in PBS CHOL (mg/dL) Run 1 2 3 Negative Control 0 0 0 720 Mg/mL 0 0 0 360 Mg/mL 0 0 0 180 Mg/mL 0 0 0 90 Mg/mL 0 0 0 45 Mg/mL 0 0 0 22.5 Mg/mL 0 0 0 11.3 Mg/mL 0 0 0 5.6 Mg/mL 0 0 0 2.8 Mg/mL 0 0 0 1.4 Mg/mL 0 0 0 0.7 Mg/mL 0 0 0 TRIG (mg/dL) Run 1 2 3 Negative Control 0 0 0 720 Mg/mL 0 0 0 360 Mg/mL 0 0 0 180 Mg/mL 0 0 0 90 Mg/mL 0 0 0 45 n g / m L 0 0 0 22.5 Mg/mL 0 0 0 11.3 Mg/mL 0 0 0 5.6 Mg/mL 0 0 0 2.8 Mg/mL 0 0 0 1.4 Mg/mL 0 0 0 0.7 Mg/mL 0 0 0 HDL (mg/dL) Run 1 2 3 Negative Control 0 0 0 720 Mg/mL 0 0 0 360 Mg/mL 0 0 0 180 Mg/mL 0 0 0 90 Mg/mL 0 0 0 45 Mg/mL 0 0 0 22.5 Mg/mL 0 0 0 11.3 Mg/mL 0 0 0 5.6 Mg/mL 0 0 0 2.8 Mg/mL 0 0 0 1.4 Mg/mL 0 0 0 0.7 Mg/mL 0 0 0 non-HDL (mg/dL) Run 1 2 3 Negative Control 0 0 0 720 Mg/mL 0 0 0 360 Mg/mL 0 0 0 180 Mg/mL 0 0 0 90 Mg/mL 0 0 0 45 M g/mL 0 0 0 22.5 Mg/mL 0 0 0 11.3 Mg/mL 0 0 0 5.6 Mg/mL 0 0 0 2.8 Mg/mL 0 0 0 1.4 Mg/mL 0 0 0 0.7 Mg/mL 0 0 0 -24- H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 Clinical Chemistry Data for H-24921 in Serum CHOL (mg/dL) Negative Run Control 1 54 2 54 3 54 720 Mg/mL 52 51 51 360 MS/mL 52 53 53 180 Mg/mL 53 53 53 90 Mg/mL 54 54 54 45 Mg/mL 54 55 54 22.5 Mg/mL 53 57 55 11.3 Mg/mL 60 56 54 5.6 Mg/mL 55 56 54 2.8 Mg/mL 54 54 54 1.4 Mg/mL 54 54 54 0.7 Mg/mL 54 55 53 TRIG! (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 1 49 47 47 49 49 49 48 49 48 49 49 49 2 48 46 48 48 49 49 51 50 50 49 49 50 3 48 46 48 49 49 49 50 49 49 49 49 49 HDL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 1 24 21 22 23 23 23 22 2 23 21 22 22 23 23 25 23 23 23 23 23 24 24 24 23 24 3 23 21 22 23 23 23 24 23 23 23 24 23 non-HDL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 1 30 31 31 30 31 31 31 37 32 31 31 31 2 31 30 31 31 31 32 32 32 32 30 31 31 3 31 30 30 30 31 31 31 31 31 31 30 30 Company Sanitized. Does not contain TSCA cbi H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids______________________________________________ DuPont-15372 Clinical Chemistry Data for H-26313 in PBS CHOL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 10 0 0 0 00 0 0 0000 20 0 0 0 00 0 0 0000 30 0 0 0 00 0 0 0000 TRIG (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 10 0 0 0 00 0 0 0000 20 0 0 0 00 0 0 0000 30 0 0 0 00 0 0 0000 HDL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 10 0 0 0 00 0 0 0000 20 0 0 0 00 0 0 0000 30 0 0 0 00 0 0 0000 non-HDL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 2 2 . 5 Mg/m L 1 1 . 3 Mg/mL 5 . 6 Mg/mL 2 . 8 Mg/m L 1 . 4 M g/m L 0 . 7 M g/mL 10 0 0 0 00 0 0 00 00 20 0 0 0 00 0 0 0000 30 0 0 0 00 0 0 0000 contain -26- San.-'TM *1 GoinpaW H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids________________________________________________DuPont-15372 Clinical Chemistry Data for H-26313 in Serum CHOL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 1 54 51 53 54 54 55 55 55 55 55 54 53 2 54 52 53 53 55 56 56 54 57 55 54 57 3 54 52 53 54 54 53 55 55 55 55 54 56 TRIG (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 1 48 46 48 48 49 48 47 49 49 49 49 49 2 49 47 48 48 49 50 50 49 50 49 49 51 3 49 47 48 48 48 49 50 49 49 50 50 51 HDL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/inL 1 23 21 22 23 23 23 23 23 23 23 23 23 2 24 21 22 23 23 24 24 23 24 23 23 24 3 23 21 22 23 23 23 24 24 23 24 23 24 non- HDL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 1 31 30 31 31 31 32 32 32 32 32 31 30 2 30 31 31 30 32 32 32 31 33 32 31 33 3 31 31 31 31 31 30 31 31 32 31 31 32 contain TSCA C g --------------Sonetead. Boarnff -27- Company - %# # H-24921, H-26313, and H-26225: Evaluation o f the Interference of the Test Substances in the Analysis of Rat Serum Lipids_______________________________________________ DuPont-15372 Clinical Chemistry Data for H-26225 in PBS CHOL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 pg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 10 0 0 0 00 0 0 0000 20 0 0 0 00 0 0 0000 30 0 0 0 00 0 0 0000 TRIG (mg/dL) Negative Run Control 10 20 30 720 Mg/mL 0 0 0 360 Mg/mL 0 0 0 180 Mg/mL 0 0 0 90 Mg/mL 0 0 0 45 Mg/mL 0 0 0 22.5 Mg/mL 0 0 0 11.3 Mg/mL 0 0 0 5.6 Mg/mL 0 0 0 2.8 Mg/mL 0 0 0 1.4 Mg/mL 0 0 0 0.7 Mg/mL 0 0 0 HDL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 10 0 0 0 00 0 0 0000 20 0 0 0 00 0 0 0000 30 0 0 0 00 0 0 0000 non-HDL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 10 0 0 0 00 0 0 0000 20 0 0 0 00 0 0 0000 30 0 0 0 00 0 0 0000 nelCOn1' A GO9^ -28- 0 0 ^^ ' --------------- H-24921, H-26313, and H-26225: Evaluation of the Interference of the Test Substances in the Analysis of Rat Serum Lipids DuPont-15372 Clinical Chemistry Data for H-26225 in Serum CHOL (mg/dL) Negative Run Control 1 54 2 54 3 54 720 Mg/mL 51 52 51 360 Mg/mL 53 53 53 180 Mg/mL 53 54 54 90 Mg/mL 54 54 54 45 Mg/mL 54 55 54 22.5 Mg/mL 54 54 55 11.3 Mg/mL 56 56 54 5.6 Mg/mL 54 54 55 2.8 Mg/mL 54 56 54 1.4 Mg/mL 55 54 54 0.7 Mg/mL 54 54 55 TRIG (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 1 49 47 47 48 49 48 49 49 49 49 49 49 2 49 47 48 49 49 49 49 50 49 50 49 49 3 49 47 48 49 48 49 49 48 49 50 49 49 HDL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 Mg/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 1 23 21 22 23 23 23 23 23 23 23 23 23 2 23 21 22 23 23 23 23 24 23 23 22 23 3 23 20 22 23 23 23 23 23 23 23 23 23 non- HDL (mg/dL) Negative Run Control 720 Mg/mL 360 Mg/mL 180 Mg/mL 90 Mg/mL 45 Mg/mL 22.5 g/mL 11.3 Mg/mL 5.6 Mg/mL 2.8 Mg/mL 1.4 Mg/mL 0.7 Mg/mL 1 31 30 31 30 31 31 31 33 31 31 32 31 2 31 31 31 31 31 32 31 32 31 33 31 31 3 31 31 31 31 31 31 32 31 32 31 32 32 -29- Company Sanitized. Does not contain TSCA CBI