Document ZJRBb79xa28z2opQK1LNKrEKd

ftRl%-0 2 3 5 3M Environmental Laboratory Final Report- Analytical Study Single-Dose Intravenous Pharmacokinetic Study of T-6052 in Rabbits In-Vivo Study Reference Number: HWI#6329-134 Study Number: AMDT- 111 694.1 Test Substance: FC-120 (T-6052) Name and Address o f Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology & Services 935 Bush Avenue St. Paul, MN 55106 Method Numbers and Revisions: AM DT-M -1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AM DT-M -2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer AM DT-M 4-0, Extraction o f Fluorochemicals from Rabbit Liver AM DT-M 5-0, Analysis o f Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry AM DT-M -8-0, Analysis of Fluoride Using the Skalar Segmented Flow Analyzer With Ion Selective Electrode AM DT-M -14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum Initiation Date: See attached protocol Author: James D. Johnson Approved By: James D. Jri son 'Study Din Completion Date 000802 l 1.0 SUM M AR Y The liver samples at 48 hours after single intravenous administration o f FC-120 (T-6052) were analyzed by combustion for total organic fluorine. T-6052 is a 0.02% solution o f FC-120. Only the 200 mg/kg (10 ug/kg) and 1000 mg/kg (50 ug/kg) samples had detectable organic fluorine: 48 and 106 ug/whole liver, respectively. These trivial amounts o f organic fluorine are a reflection o f the low doses. There is a marker for dermal absorption studies, if the doses are higher than used in this study. 2.0 INTRODUCTION________________________________ ___________________ The liver and serum samples from HWI#6329-134 were available for analysis. This compound is perfluorodecanesulfonate (ammonium salt). There is not expected to be any biotransformation o f this compound and the pharmacokinetics and disposition are expected to be similar to that found for perfluorooctanesulfonate. The tissues at 48 hours were analyzed by combustion for total organic fluorine and by electrospray mass spectrometry for perfluorodecanesulfonate anion. The data were to be analyzed to provide data for the assessment o f a subsequent dermal absorption study. The high dose is just 50 ug/kg. T-6052 is a 0.02% solution o f FC-120, which is 25% solids. 3.0 TEST MATERIALS_______________________________________________ _ 3.1 Test, Control, and Reference Substances and Matrices 3.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 3.1.2 Analytical Reference Matrix: Bovine liver and bovine serum 3 .1 3 Analytical Control Substance: None 3.1.4 Analytical Control Matrix: Bovine liver and bovine serum 3.2 Source o f Materials: 3MICP/PCP Division for FC-95, bovine liver from grocery store, bovine serum from Sigma Chemical Company 3.3. Purity and Strength of Reference Substance: Responsibility o f Sponsor. 3.4 Stability o f Reference Substance: To be determined by Sponsor. 3.5 Storage Conditions for Test Materials: Room temperature for FC-95. For biological samples the storage is -2010 C. 000803 2 3.6 Disposition o f Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required for studies longer than 28 days. This study is in parallel with a 28 day absorption study, so all tissues will be retained. 4.0 EX PER IM EN TA L -O verview ______________________________ _ Serum and tissues from animals dosed as described (HWI#6329-134), were available for analysis for fluorine compounds. Since perfluorodecanesulfonate anion is not biotransformed, the analysis was accomplished with combustion and subsequent analysis for fluorine. The fluorine data are related directly to perfluorodecane sulfonate anion concentration. Additional analysis o f liver samples with electrospray mass spectrometry provides evidence that the perfluorodecanesulfonate anion is present. Data from these analysis will be used to assess the extent of dermal absorption in a subsequent study (HWI#6329-135). 5.0 EXPERIM ENTAL - METHODS______________________________ ___ 5.1 AM DT-M -1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 5.2 AM DT-M -2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer 5.3 AM DT-M -4-0, Extraction o f Fluorochemicals from Rabbit Liver 5.4 AM DT-M -5-0, Analysis o f Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 5.5 AM DT-M -8-0, Analysis of Fluoride Using the Skalar Segmented Flow Analyzer With Ion Selective Electrode 5.6 AM DT-M -14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum 6.0 DATA ANALYSIS____________________________________________________ The data (Skalar) is attached for combustion analysis. The total organic fluorine in liver at 48 hours after an intravenous dose o f FC-120 was nondetected for the control, 0.1 ug/kg, and 1.0 ug/kg groups. The total organic fluorine measured for 000804 3 the 10 ug/kg and 50 ug/kg rabbits were 48 and 106 ug/whole liver, respectively. Electrospray mass spectrometry (see attached) confirmed the presence o f perfluorodecanesulfonate (m/z=599). Other data was collected using the Dorhman organic halide analyzer, Orion ion analyzer (liver and serum), Skalar segmented flow analyzer with ion selective electrode (serum), and electrospray mass spectrometry (liver) - see appendices. This data, although supportive, in the opinion o f the Study Director is not required to reach the conclusion stated here and therefore is not discussed in detail. 6.1 Circumstances that May Have Affected the Quality o f the Data: The problem with this analysis is that there is not nearly enough fluorine in the liver after these intravenous doses because the doses are too low. 7.0 CONCLUSION_______________________________________________ _ This pharmacokinetic study is not useful in terms o f providing data for the assessment o f a dermal absorption study. The perfluorodecanesulfonic acid anion expected is not observed in liver except for a trace at the highest dose (50 ug/kg). If the dermal absorption study doses are high enough, there is a marker. 8.0 M AINTENANCE OF RAW DATA AND RECORDS_________________ 8.1 Raw Data and Data: Raw data, approved protocol, approved final report, appropriate specimens, and electronic data will be maintained in the AMDT archives. 9.0 APPENDICES________________________________________________________ 9.1 Protocol and Amendments 9.1.1 Protocol and Final Report: HWI#6329-134, "Single-Dose Intravenous Pharmacokinetic Study o f T-6052 in Rabbits" (Protocol type TP8084.PK for dosing o f animals, tissue collection, etc.) 9.1.2 Analytical protocol AMDT-111694.1 9.2 Signed Reports from Individual Scientists: None 9.3 Quality Assurance Unit Statement: See attached 000805 4 9.2 Signed Reports from Individual Scientists: None 9.3 Quality Assurance Unit Statement: See attached 9.4 K ey Personnel Involved in the Study: See attached 9.5 M aterials and Equipment: See methods 9.6 Solutions, Reagents, and Standards: See methods 9.7 Sample Preparation: See methods 9.8 Quality Control Practices: See methods 9.9 Test Methods: See Protocol AMDT-111694.1 9.10 Instrument Settings: See methods 9.11 Data: See attached. 9.11.1 Summary and raw data; ug F in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 9.11.2 Summary and raw data; analysis o f liver extracts using electrospray mass spectrometry. 9.11.3 Summary and raw data; ug F in whole liver as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. 9.11.4 Summary and raw data; ppm F* in serum as determined by thermal extraction followed by analysis using Orion ion analyzer. 9.11.5 Summary and raw data; ppm F' in serum as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. 000806 5 9.1.1 Final Report: HWI#6329-134, "Single-Dose Intravenous Pharmacokinetic Study of T-6052 in Rabbits" (Protocol type TP8084.PK for dosing of animals, tissue collection, etc.) G00S07 HAZLETOfM WISCONSIN P O S T O F F I C E B O X 75 4 5 MA D I S O N , Wl 5 3 70 7 - 7 5 4 5 Sponsor: 3M St. Paul, Minnesota ,1 C O R N IN G Cornpany FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6052 in Rabbits Author: Steven M. Glaza Study Completion Date: February 1, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-134 Page 1 of 24 P h o n e 6 0 3 - 2 4 I _- 4 4 7 1 EX P R E S S -MAIL D E L I V E R Y : 3301 KINSMAN BLVD. F a x 603 M A D I S O N . Wl 000808 53 7 04 Page 2 of 24 QUALITY ASSURANCE STATEMENT HWI 6329-134 This report has been reviewed by the Quality Assurance Unit of Hazleton Wisconsin, Inc., in accordance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations, 21 CFR 58.35 (b) (6) (7). The following inspections were conducted and findings reported to the Study Director and management. Written status reports of inspections and findings are issued to Hazleton management monthly according to standard operating procedures. Inspection Dates From To Phase Date Reported to Date to Study Director Management 11/05/94 11/11/94 01/10/95 01/30/95 11/05/94 11/11/94 01/10/95 01/30/95 Protocol Review Animal Observation Data/Report Review Report Rereview 11/08/94 11/11/94 01/10/95 01/30/95 12/10/94 12/10/94 02/10/95 02/10/95 Representative, Quality Assurance Unit Date 00080 Page 3 of 24 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study of T-6052 in Rabbits HWI 6329-134 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Experimental Start Date Experimental Termination Date T-6052 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 John L. Butenhoff, PhD 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 November 11, 1994 November 13, 1994 ooosio Page 4 of 24 KEY PERSONNEL HWI 6329-134 Acute Toxicology Steven M. Glaza Study Director Manager Francis (Bud) W. McDonald Study Coordinator Patricia Padgham In-life Supervisor Rose M. Bridge Report Supervisor Quality Assurance Sherry R. W. Petsel Manager Laboratory Animal Medicine Cindy J. Cary, DVM Dipl ornate, ACLAM Supervisor Anatomical Pathology Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data 000811 Page 5 of 24 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and Control Materials Test System Procedures Results Discussion Signature Reference Table 1 Individual Body Weights(g) 2 Individual Clinical Signs Appendix A Protocol TP8084.PK HWI 6329-134 Page 2 3 4 g 7 7 7 8 g U II 11 II 12 13 u 15 000812 Page 6 of 24 SUMMARY HWI 6329-134 This study was done to assess the level of systemic exposure of T-6052 when administered by intravenous injection to rabbits. Female Hra:(NZW)SPF rabbits were assigned at random to five groups (one/group). On Day 0, the animals received a single intravenous injection of the vehicle (sterile water for injection) or 2, 20, 200, or 1,000 mg of T-6052/kg of body weight (Groups 1 through 5, respectively). The dose volume was 0.5 mL/kg for Groups 1 through 4 and 1.02 mL/kg for Group 5. Clinical observations were conducted at approximately 0.5, 2, 4, 24, and 48 hours after intravenous injection. Body weights were determined just before test material administration (Day 0). A blood sample (approximately 4 mL) was collected from an auricular artery or marginal ear vein of the animals at 2-, 4-, 6-, 8-, 12-, and 24-hours post-injection. In addition, at the time of experimental termination (48-hours post-injection), approximately 20 mL of blood was obtained from each animal. All samples were centrifuged, separated into serum and,cellular fractions, and sent to the Sponsor. Approximately 48 hours post-injection, the animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not done, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected and sent frozen to the Sponsor after termination of the in-life phase. All five animals appeared normal throughout the study. 000813 Page 7 of 24 OBJECTIVE HWI 6329-134 The objective of this study was to assess the level of systemic exposure to the test material, T-6052, when administered as a single intravenous injection to rabbits. REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and Drug Administration's Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58, with the exception that analysis of the test mixtures for concentration, homogeneity/solubility, and stability was not conducted. All procedures used in this study were in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study did not unnecessarily duplicate any previous work. TEST AND CONTROL MATERIALS Identification The test material was identified as T-6052 and described as clear, colorless liquid. The control material was Sterile Water for Injection, USP (Abbott Laboratories, Lot No. 86-748-DM-02; Exp. March 1, 1996), and was described as a clear, colorless liquid. Purity and Stability The Sponsor assumes responsibility for test material purity and stability determinations (including under test conditions). A sample of the test material/vehicle mixtures for concentration, solubility, homogeneity, and stability analyses was not taken before administration as this was not requested by the Sponsor. The purity and stability of the USP grade control material were considered to be adequate for the purposes of this study. Storage and Retention The test material was stored at room temperature. The control material was stored refrigerated. Any unused test material will be returned to the Sponsor after completion of all testing according to Hazleton Wisconsin (HWI) Standard Operating Procedure (SOP). Any remaining vehicle may be used for other testing and will not be discarded after issuance of the final report. 000814 Page 8 of 24 Safety Precautions HWI 6329-134 The test and control material handling procedures were according to HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPF strain were received from HRP, Inc., Kalamazoo, Michigan on October 5, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing After receipt, the animals were acclimated for a period of at least 7 days. During acclimation and throughout the study, the animals were individually housed in screen-bottom stainless steel cages in temperature- and humiditycontrolled quarters. Environmental controls for the animal room were set to maintain a temperature of 19 to 23#C, a relative humidity of 50% 20%, and a 12-hour 1ight/12-hour dark lighting cycle. In cases where variations from the required temperature and humidity conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal husbandry and housing at HWI complied with standards outlined in the "Guide for the Care and Use of Laboratory Animals" . 1 Animal Diet The animals were provided access to water ad libitum and a measured amount of Laboratory Rabbit Diet HF #5326, PMI Feeds, Inc. The feed is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. Samples of the water are periodically analyzed by HWI. There were no known contaminants in the feed or water at levels that would have interfered with or affected the results of the study. Selection of Test Animals The animals were identified by animal number and corresponding ear tag and were selected at random based on health and body weight requirements. 000815 Page 9 of 24 Study Design HWI 6329-134 Female animals weighing from 2,813 to 3,031 g at initiation of treatment were placed into the following study groups: GrouD 1 (Control) 2 3 4 5 Treatment * T-6052 T-6052 T-6052 T-6052 Dose Level ima T-6052/kal 0 2 20 200 1,000 Dose Volume imL/ka) 0.5 0.5 0.5 0.5 1.02 Number of Anima' 1 1 1 1 1 * Sterile Water for Injection, USP. Justification for Species Selection Historically, the New Zealand White albino rabbit has been the animal of choice because of the large amount of background information on this species. PROCEDURES Dose Preparation and Administration The test material was diluted with Sterile Water for Injection to achieve a specific concentration for each dose level in Groups 2 through 4. The test material was administered undiluted at the 1,000 mg/kg dose level, using the bulk density of 0.98 g/mL to determine the dose volume. An individual dose of each respective test solution or control was calculated for each animal based on its body weight on the day of treatment. The respective test solution was administered by intravenous injection into a marginal ear vein. The dose was given as a slow push (approximately 30 to 60 seconds in duration). The prepared test solutions were stored at room temperature until administered. After administration, any remaining test solutions were discarded. Reason for Route of Administration Intravenous injection is an acceptable route to assess systemic exposure. Observations of Animals Clinical observations were conducted at approximately 0.5, 2, 4, 24, and 48 hours after intravenous injection. Body weights were determined just before test material administration (Day 0). 000816 Page 10 of 24 Sample Collection HWI 6329-134 A blood sample (approximately 4 mL) was collected from either ear via the catheterization of the auricular artery or from the marginal ear vein of all animals at 2, 4, 6, 8, 12, and 24 hours post-injection. At the time of necropsy (approximately 48-hours post-injection), approximately 20 mL of blood was obtained from the posterior vena cava of each animal. All samples were stored at room temperature until centrifuged and separated into serum and cellular fractions. The blood samples were then stored in a freezer set to maintain a temperature of -20*C 10*C until shipped to the Sponsor. Pathology At termination of the experimental phase (approximately 48-hours post-injection), animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, and exsanguinated. An abbreviated gross necropsy examination was not conducted, however, tissues were collected. The whole liver, bile, and both kidneys from each animal were collected and immediately placed on dry ice, then frozen by placing in a freezer set to maintain a temperature of -20*C 10*C. After tissue/bile collection, the animals were discarded. Shipment of Tissues After completion of the in-life phase the blood samples, livers, bile, and kidneys were sent frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.E. & P.C., Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN, 55106). The Sponsor is responsible for the retention and disposition of the samples. HWI does not accept any responsibility for the analysis of the samples collected in this study nor are these results presented in this report. Statistical Analyses No statistical analyses were required by the protocol. Location of Raw Data. Records, and Final Report The raw data, records, and an original signed copy of the final report will be retained in the archives of HWI in accordance with HWI SOP. 000817 Page 11 of 24 RESULTS Body Weights Individual body weights at initiation are in Table 1. HWI 6329-134 Clinical Observations Individual clinical signs are in Table 2. All five animals appeared normal throughout the study. Pathology All animals survived to termination of the experimental phase and were not examined grossly when sacrificed. DISCUSSION The level of systemic exposure of T-6052 was evaluated in female albino rabbits when administered as a single intravenous injection at levels of 0, 2, 20, 200, and 1,000 mg/kg. All animals appeared normal throughout the study following administration of this material. SIGNATURE Steve___ _____ Study Director Acute Toxicology Date REFERENCE 1. NIH Publication No. 86-23 (revised 1985). 2.-lA s 000818 Page 12 of 24 GrouD 1 2 3 4 5 Table 1 Individual Body Weights (g) Dose Level (mq/kq) Sex Animal Number 0 Female F52548 2 Female F52549 20 Female F52559 200 Female F52566 1,000 Female F52567 Dav 0 3,031 2,921 2,813 2,912 2,853 HWI 6329-134 000819 GrouD Dose Level imq/kq) 10 Sex Female Page 13 of 24 Table 2 Individual Clinical Signs HWI 6329-134 Animal Number F52548 Observation Appeared normal _________ Hour 0.5 2 4 24 /// 48 2 2 Female F52549 Appeared normal // 3 20 Female F52559 Appeared normal / 4 200 Female F52566 Appeared normal / 5 1,000 Female F52567 Appeared normal / / Indicates condition exists. 000820 Page 14 of 24 APPENDIX A Protocol TP8084.PK HWI 6329-134 000821 HAZLETON WISCONSIN P O S T O F F I O F. R O X 7 5 't 5 MA D I S O N , Wl h ;) ; 0 / ' i>A'> Page 15 of 24 .1 C O R N IN G <.(u1i()iliiy Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.PK Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6052 in Rabbits Date: November 9, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-134 P h o n e >i -j r \ F* H t: S \ ! -X 1' rI 000822 Page 16 of 24 STUDY IDENTIFICATION TP8084.PK Page 2 Single-Dose Intravenous Pharmacokinetic Study of T-6052 in Rabbits HWI No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6329-134 T-6052 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 John L. Butenhoff, PhD 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.0. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Week of November 7, 1994 Week of November 7, 1994 Week of December 12, 1994 G0023 Page 17 of 24 TP8084.PK Page 3 1. Study Single-Dose Intravenous Pharmacokinetic Study in Rabbits 2. Purpose To assess the level of systemic exposure when the test material is administered as a single intravenous injection to rabbits 3. Regulatory Compliance This study will be conducted in accordance with the following Good Laboratory Practice Regulations/Standards/Guidelines with the exception that analysis of the test material mixtures for concentration, solubility, homogeneity, and stability will not be conducted: [ ] Conduct as a Nonregulated Study [X] 21 CFR 58 (FDA) [ ] 40 CFR 160 (EPA-FIFRA) ( ] 40 CFR 792 (EPA-TSCA) [ ] C(81)30 (Final) (OECD) [ ] 59 Nohsan No. 3850 (Japanese MAFF) [ ] Notification No. 313 (Japanese MOHW) All procedures in this protocol are in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. 4. Quality Assurance The protocol, study conduct, and the final report w1ll.be audited by the Quality Assurance Unit in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedures (SOPs) and policies. 5. Test Material A. Identification T-6052 B. Physical Description (To be documented in the raw data) C. Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under test conditions). Samples of test material/vehicle mixture(s) for concentration, solubility, homogeneity, and stability analyses will be taken before administration if requested by the Sponsor. These samples (if taken) will be sent to the Sponsor after experimental termination for possible analysis. 000824 Page 18 of 24 TP8084.PK Page 4 D. Storage Room temperature E. Reserve Samples Reserve samples will not be required for this study. F. Retention Any unused test material will be discarded after issuance of the final report, unless directed otherwise by the Sponsor. G. Safety Precautions As required by HWI SOPs and policies 6. Control Material A. Identification Sterile water for injection B. Physical Description Clear, colorless liquid C. Purity and Stability The purity and stability of this USP grade material is considered to be adequate for the purposes of this study. D. Storage Refrigerated E. Reserve Samples See Section, 5. E. Reserve Samples F. Retention Any remaining control material may be used for other testing and will not be discarded after issuance of the final report. 6. Safety Precautions As required by HWI SOPs and policies 7. Experimental Design A. Animals (1) Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP, Inc. (3) Aae at Initiation Adult 000825 Page 19 of 24 TP8084.PK Page 5 (4) Weight at Initiation 2.5 to 3.5 kg (5) Number and Sex 5 females (6) Identification Individual numbered ear tag (7) Husbandry (a) Housing Individually, in screen-bottom stainless steel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds, Inc.). The food is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. (c) Water Ad libitum from an automatic system. Samples of the water are analyzed by HWI for total dissolved solids, hardness, and specified microbiological content and for selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the food or water that would interfere with this study. (e) Environment Environmental controls for the animal room will be set to maintain a temperature of 19*C to 23*C, a relative humidity of 50% 20%, and a 12-hour light/12-hour dark cycle. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to HWI SOPs. An adequate number of extra animals will be purchased so that no animal in obviously poor health is placed on test. (9) Justification for Species Selection Historically, the New Zealand White albino rabbit has been the animal of choice because of the large amount of background information on this species. Page 20 of 24 Dose Administration TP8084.PK Page 6 ill Test Grouos Group 1 2 3 4 5 Dose Level (mq/kg)* 0 (Control) 2 20 200 1000 Number of Females 1 1 1 1 1 a The dose volume will be 0.5 ml/kg for Groups 1-4 and approximately 1.0 mL/kg of body weight (depending on the bulk density of the test material) for Group 5. C. Dosing Procedures (1) Dosing Route Intravenous injection into a marginal ear vein over approximately 30 to 60 seconds. (2) Reason for Dosing Route Intravenous injection is an acceptable route to assess systemic exposure. (3) Dosing Duration Single dose (4) Dose Preparation The test material will be diluted with sterile water for injection to achieve a specific concentration for each dose level in Groups 1-4. The test material will be administered undiluted at the 1,000 mg/kg dose level, using the bulk density to determine the dose volume. Individual doses will be calculated based on the animal's body weight taken just before test material administration. The prepared test mixtures will be stored at room temperature until administration. D. Observation of Animals (1) Clinical Observations The animals will be observed for clinical signs of toxicity at approximately 0.5, 2.0, 4.0, 24, and 48 hours after treatment. 000827 Page 21 of 24 TP8084.PK Page 7 (2) Body Weights Just before test material administration. (3) Sample Collections (a) Frequency 2, 4, 6, 8, 12, 24, and 48 hours post-injection (b) Number of Animals All (c) Method of Collection Blood samples (approximately 4 ml) will be collected from either ear via the catheterization of the auricular artery or from the marginal ear vein at 2, 4, 6, 8, 12, and 24 hours post-injection. Approximately 20 mL of blood (actual volume to be documented in the raw data) will be obtained from the posterior vena cava of each animal at the time of necropsy (48 hours post-injection). Approximately 20 ml of blood will be collected from moribund animals during the study, also, if possible. The samples will be stored at room temperature and then centrifuged, and the separate serum and cellular fractions stored in a freezer set to maintain a temperature of -20*C 10*C. The separated serum and cellular fractions will be sent frozen to the Sponsor after experimental termination. Samples will be shipped to: James D. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 \ James D. Johnson will be notified by telephone at (612) 778-5294 prior to the shipment of the samples. Termination (1) Unscheduled Sacrifices and Deaths Any animal dying during the study or sacrificed in a moribund condition, will be subjected to an abbreviated gross necropsy examination and all abnormalities will be recorded. Animals in a moribund condition will be anesthetized with sodium pentobarbital, bled via the vena cava, and exsanguinated. 000828 Page 22 of 24 TP8084.PK Page 8 (2) Scheduled Sacr1fice At approximately 48 hours post-injection, animals surviving to termination will be anesthetized with sodium pentobarbital, bled via the vena cava, and exsanguinated. An abbreviated gross necropsy examination will not be done, however, tissues will be collected. (a) Sample Collection The whole liver and bile from each animal dying during the study, sacrificed in a moribund condition, or surviving to termination will be collected. Both kidneys from each animal will also be collected. The tissues will be placed on dry ice immediately after collection and then placed in a freezer set to maintain a temperature of -20*C 10*C. The tissues (liver, bile, kidneys) will be sent frozen on dry ice to the Sponsor after experimental termination; The samples will be shipped to the person listed in Section 7.D.(3).(c). The Sponsor is responsible for the retention and disposition of the samples. F. Statistical Analyses No statistical analyses are required. 8. Report A final report including those items listed below will be submitted. Description of the test and control materials Description of the test system Procedures Dates of experimental initiation and termination Description of any toxic effects Gross pathology findings/gross pathology report (if applicable) 000829 Page 23 of 24 TP8084.PK Page 9 9. Location of Raw Data. Records, and Final Report Original data, or copies thereof, will be available at HWI to facilitate auditing the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including those item listed below will be retained in the archives of HWI according to HWI SOP. Protocol and protocol amendments Dose preparation records In-life records Body weights Dose administration Observations Sample collection records Pathology Records Study correspondence Final report (original signed copy) The following supporting records will be retained at HWI but will not be archived with the study data. Animal receipt/acclimation records Water analysis records Animal room temperature and humidity records Refrigerator and freezer temperature records Instrument calibration and maintenance records 000830 Page 24 of 24 TP8084.PK Page 10 PROTOCOL APPROVAL John L. Butenhoff, PhD Sponsor's Representative 3M Steven H. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. - / lca L i .^ 3 -- .-- ( Representative Quality Assurance Unit Hazleton Wisconsin, Inc. {6329-134.protdskl) , //- /T-<7y Date ________________ - Date " A /?/ Date 000331 9.1.2 Analytical protocol AMDT-111694.1 000832 3M Environmental Laboratory________________________ Protocol - Analytical Study Single-Dose Intravenous Pharmacokinetic Study of T-6052 in Rabbits In-Vivo Study Reference Number: HWI#6329-134 Study Number: AM DT-111694.1 Test Substance: FC-120 (T-6052) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55106 Proposed Initiation Date: July 25, 1995 Proposed Completion Date: August 25, 1995 Method Numbers and Revisions: AM DT-M -1-0, Thermal Extraction o f Fluoride by means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AM DT-M -2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer AMDT -M-4-0, Extraction o f Fluorochemicals from Rabbit Liver AM DT M-5-0, Analysis o f Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry AM DT-M -8-0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode AMDT-M-14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum Author: James D. Johnson Approved By: es D. Jottfison >tudy Director Date John Butenho.ff, PhD Date Sponsor Representative 000833 1.0 PURPOSE This study is performed in order to provide pharmacokinetic data for the assessment o f a subsequent dermal absorption study (HWI#6329-135). 2.0 TEST MATERfALS________________________________________ __ 2.1 Test, Control, and Reference Substances and Matrices 2.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 2.1.2 Analytical Reference Matrix: Bovine liver and bovine serum 2.1.3 Analytical Control Substance: None 2.1.4 Analytical Control Matrix: Bovine liver and bovine serum 2.2 Source of Materials: 3M ICP/PCP Division (2.1.1), grocery store (2.1.2, 2.1.4liver), Sigma Chemical Company (2.1.2, 2.1.4-serum) 2.3 Num ber of Test and Control Samples: Liver and serum from 4 test animals and 1 control animal. Other biological tissues (kidney, bile, cellular fraction) will be available for analysis if deemed appropriate by the Study Director. 2.4 Identification o f Test and Control Samples: The samples are identified using the HWI animal identification number which consists of a letter and five digit number, plus the tissue identity, and day identity (serum). 2.5 Purity and Strength o f Reference Substance: To be determined by Sponsor. 2.6 Stability o f Reference Substance: To be determined by Sponsor. 2.7 Storage Conditions for Test Materials: Room temperature (2.1.1), -20 10C (2.1.2, 2.1.4). Test and Control samples will be received according to AMDT-S-10-0. 2.8 Disposition o f Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required for studies longer than 28 days. This study is in parallel with a 28 day dermal absorption study so all tissues will be retained. 2.9 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting the samples. 000834 2 3.0 EXPERIM ENTAL - Overview The tissues from animals dosed as described (HWI#6329-134), are available for analysis for fluorine compounds. At the discretion o f the Study Director, a series o f analytical tests can be performed. The screening for fluoride in liver via combustion (See Methods--next Section) is the appropriate analysis to present definitive data for fluorine in the liver. Electrospray mass spectrometry will be performed in order to confirm the presence o f specific molecules. The material being studied is a perfluorodecanesulfonic acid salt (Ammonium). This material is not expected to be biotransformed. If the material is similar to perfluorooctanesulfonic acid anion, it will be persistent in the liver. Analysis o f liver for total organic fluorine will provide information as to the extent o f persistence. 4.0 EXPERIMENTAL - Methods _________________________________ __ 4.1 Liver and Serum screening methods: (attached) 4.1.1 AMDT-M-1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 4.1.2 AMDT-M-2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer 4.1.3 AMDT-M-4-0, Extraction o f Fluorochemicals from Rabbit Liver 4.1.4 AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 4.1.5 AMDT-M-8-0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 4.1.6 AMDT-M-14-0,Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum 000835 3 5.0 DATA ANALYSIS 5.1 Data Reporting: Data will be reported as a concentration (weight/weight) o f fluoride per tissue or fluid, or as FC-120 (electrospray mass spectrometry) per unit o f tissue or fluid. Statistics used, at the discretion o f the Study Director, may include averages and standard deviations from different dose groups. If necessary, simple statistical tests such as the Student's t test may be applied to determine statistical difference. 6.0 MAINTENANCE OF RAW DATA AND RECORDS___________________ 6.1 Raw Data and Records: Raw data, approved protocol, appropriate specimens, approved final report, and electronic data will be maintained in the AMDT archives. 7.0 REFERENCES______________________ 7.1 AMDT-S-10-0, Sample Tracking System 8.0 ATTACHMENTS__________________________________________________ 8.1 AMDT-M-1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 8.2 AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 8.3 AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver 8.4 AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 8.5 AMDT-M-8-0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 8.6 AMDT-M-14-0,Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum 0 0 0 8 3 6 ,4 3M Environmental Laboratory Method Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer - Liver M ethod Identication Number: AMDT-M-1 Revision Number: 0 Adoption Date: / - i ^ C" Revision Date: None Author Rich Youngblom Approved by: Software: MS Word 5.1a Affected Documents: AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 1G0GS37 i.o SCOPE . APPLICABLE COMPOUNDS. AND MATRiCFS 1.1 Scope: This method is for the operation of a Dohrmann DX2000 when it is used to extract fluoride from various matrices. The fluoride is typically collected in TISAB solution for analysis with an ion selective electrode. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Biological tissues, particularly liver. 2.0 KEYW ORDS_________________________________________ 2.1 Fluoride, fluorine, extraction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS________________________________________ 3.1 Glassware and exhaust gases can be extremely hot. 3.2 Glassware is fragile, broken glass may cause injuries. 3.3 Pressurized gases, proper compressed gas handling practices required. 3.4 Solvent based samples may flash, may need to allow them to dry down before starting run. 3.5 Potential biohazards due to the biological matrices. Use appropriate personal protective equipment. 4.0 SUPPLIES AND MATERIALS______________________________________ _ 4.1 Compressed Oxygen, Hydrocarbon free, regulated to 30 PSI. 4.2 Compressed Helium, High Purity Grade, regulated to 45 PSI. 4.3 Quartz glass sample boat with TeflonTM tubing, Dohrmann 890-097 or equivalent. 4.4 Quartz glass combustion tube, Reliance Glass G-9405-012 or equivalent. 4.5 Orion 940999 Total Ionic Strength Adjustment Buffer (TISAB I I ) or equivalent. 4.6 Sample collection vials, HDPE. 4.7 Milli-QTM water 4.8 Polystyrene pipettes. 4.9 Activated Charcoal, E. Merck 2005 or equivalent. 4.10 Hamilton Syringe or equivalent. 4.11 Miscellaneous laboratory glassware 5.0 EQ UIPM ENT ________________________________________________ ___ 5.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer, modified for fluoride extraction. 5.2 IBM compatible 386 or 486 computer. 5.3 DX2000 software, version 1.00, modified for fluoride extraction. 5.4 Excel Spreadsheet, version 5.0 or greater 6.0 INTERFERENCES___________________________________________________ 6.1 Sample size is limited to approximately 150 mg, depending on sample moisture content. This may vary from matrix to matrix. 2 000838 7.0 SAM PLE HANDLING 7.1 Samples are not to be handled with bare hands. Fluoride may leach from the skin to the sample. Use forceps or probe to transfer tissues. 7.2 Samples of liver are cut from frozen liver and placed in a tared and labeled weigh boat. Use a clean scalpel and cutting board. The cutting board and scalpel should be cleaned with water, methanol, or methanol-water solution after each liver is cut. 8.0 CALIBRATION AND STANDARDIZATION_______________ 8.1 Preparation of Calibration Standards 8.1.1 The standards required for each project will need to be appropriate for that individual project. Refer to protocol for that project. 8.1.2 Typically 50-500 ppm FC-95 in methanol standards are used. 8.1.3 For rabbit liver studies, use beef liver as the matrix. Cut a piece of frozen beef liver (100 150 mg) and weigh it in a labeled and tared weigh boat. 8.2 Calibration - Overview The normal calibration is the fluoride curve (AMDT-M-2). However, if an optional spiked liver curve is required the procedure listed below is used. 8.2.1 A calibration curve for the DX2000 is generated by spiking samples with known standards and combusting them using the same methods and matrix type as the samples to be tested. 8.2.2 Typically, three replicates of each standard and five concentrations of standards will be spiked. 8.2.3 Standard curve will be plotted as Mass Spiked F (ug) on the x-axis and Standard Mass Recovered F (ug) on the y-axis. Generate a regression curve and calculate the equation for the line and the r^ value. 8.2.4 Mass Spiked F (ug) = (Amount spiked in mL) x ( Cone, of standard in ppm) x (0.6004)* *FC-95 is 60.04% F therefore 0.6004 is the factor used to convert FC-95 to F 8.2.5 Standard Mass Recovered F (ug) = (TISAB volume in mL) x (Orion reading in ppm) 8.3 Calibration - Procedure 8.3.1 S tart Up 8.3.1.1 Run 2 or more Clean Cycles when starting instrument each day. More clean cycles may be used if the previous samples contained high concentrations of fluoride. 8.3.2 Blanks 8.3.2.1 Prepare sample using the same methods and type of matrix as the test sample. 8.3.2.2 For rabbit studies, use beef liver as the matrix. Prepare at least 3 samples of beef liver (100 - 150 mg) for blanks. 8.3.2.3 Put sample in Dohrmann boat. Combust each sample as described in section 9.0 and analyze sample according to method AMDT-M-2 for the ion selective electrode analysis. 3 000839 8.3.2.4 For rabbit studies, the meter reading for a blank sample should be 0.03 ppm or lower before proceeding with the calibration. Bum samples until this limit is reached, or until in the judgement of the operator the reading is stable with respect to historical readings (previous 48 hours). 8.3.2.5 For non-rabbit studies, the blank readings should reach a predetermined ion concentration before proceeding with the calibration. 8.3.2.6 It may be necessary to mix approximately 50 mg of charcoal with the sample to aid combustion. 8.3.3 Standard Curve 8.3.3.1 Weigh out at least 15 matrix samples (5 standards with 3 replicates each) in tared and labeled weigh boats. For rabbit studies, weigh 100-150 mg beef liver samples. Record weights in study data. Store the matrix samples on dry ice or ice packs to keep them frozen until used. 8.3.3.2 Place weighed beef liver sample in Dohrmann sample boat. 8.3.3.3 Start with the lowest standard concentration. Using a Hamilton syringe, eject a fixed quantity of the standard on or in the matrix. For rabbit studies, use 4 uL of standard and eject it on or in the beef liver. 8.3.3.4 At least 3 replicates should be used for the lowest standard concentration; more replicates may be used at the discretion of the analyst. 5.3.3.5 Combust the sample as described in section 9.3 and analyze according to AMDT-M-2. 8.3.3.6 Run all 15 standards. If one replicate is significantly different from the other two replicates, run another sample for that standard. Indicate in data that the new replicate replaces the old replicate and that the new replicate will be used to calculate the regression curve. 8.3.3.7 When all standards have been run, calculate the r2. r2 must be at least 0.95. Ifitis n o ta t least 0.95, consult with supervisor. 8.3.3.8 A new standard curve should be run when the combustion tube or sample matrix is changed. New standard curve may also be run at the discretion of the analyst. 8.4 Storage Conditions for Standards 8.4.1 Storage requirements for standards are dependent on the individual standards used. Typically, standards are stored at room temperature in plastic screw top bottles. 8.4.2 New FC-95 standards should be prepared at least once a month. 9.0 PROCEDURES_________________ ' ________ 9.1 Typical Operating Conditions: 9.1.1 Combustion tube temperature = 950C. 9.1.2 Oxygen and Helium flow = 50 cc/minute. 9.1.3 Vaporization/Drying time = 240 seconds. 9.1.4 Bake time = 300 seconds. 9.2 S tart Up Procedure: 9.2.1 If the program is not started, start the EOX program on the PC. 9.2.2 Open the SYSTEM SETUP window. 9.2.3 Put the furnace module and the cell in the READY mode. 9.2.4 Close the SYSTEM SETUP window. 4 000840 9.2.5 When the oven has reached the READY temperature, run the CLEAN BOAT program found in the CELL CHECK menu. 9.2.6 See AMDT-EP-3 for details of the Dohrmann software. 9.3 Sample Extraction Procedure: 9.3.1 Open the SAMPLE HATCH and place the sample in the BOAT. It may be necessary to mix approximately 50 mg of charcoal with the sample to aid combustion. If this is done, charcoal should also be mixed in while establishing the baseline and when generating the standard curve 9.3.2 Close SAMPLE HATCH. 9.3.3 Add appropriate volume of TISAB solution or 1:1 TISAB:Milli-QTM water mixture to a labeled sample collection vial. Typically 0.6 mL to 15 mL are used. For rabbit studies, use 1.0 or 2.0 mL of 1:1 TISAB:Milli-QTM water mixture. 9.3.4 Place the vial so that the tip of the COMBUSTION TUBE is in the TISAB at least 0.25 inches. Gases released during pyrolysis must bubble through the TISAB. 9.3.5 Run the EOX-SOLIDS program found in the RUN menu. 9.3.6 When the EOX program is finished, remove the collection vial from the combustion tube. 9.3.7 If undiluted TISAB was used to collect the sample, add an equal volume of Milli-QTM water to the TISAB to make 1:1 TISAB:Milli-QTM. 9.3.8 Rinse the end of the combustion tube with Milli-QTM water and wipe with a KIMWIPE to remove any TISAB remaining on the tube. 9.3.9 Open the sample hatch and remove any remaining ash from the boat. Ash can be removed with a cotton tipped applicator or vacuumed out. It may be necessary to scrap particles off the bottom with a spatula or other similar device. A drop of Milli-QTM water may be added to the boat to aid in the Clean Cycle. 9.3.10 Close the hatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample is ready for analysis by ion selective electrode (AMDT-M-2). 9.4 Sample Calculations 9.4.1 Use the standard curve to calculate the sample value. 9.4.2 Sample Mass Recovered F (ug) = (TISAB vol in mL) x fOrion reading in ppm - intercept^ (Slope) 10.0 VALIDATION 10.1 Quality Control 10.1.1 Daily Start Up Check Samples: Once the standard curve is established, each day of analysis is started by analyzing QC samples. The QC samples are to be the same as the lowest concentration spiked samples used to generate the standard curve. Each concentration must be done in triplicate unless the first two replicates are within 20% of the standard curve, then a third replicate is not necessary. 10.2 Precision and Accuracy: See method development analysis and sample analysis in Fluoride Notebooks 2,3, and 5. Precision and accuracy varies when analyzing samples o f different matrices and different reference compounds. 10.3 O ther Validation Parameters: NA 5 000841 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1 For the standard curve, use regression analysis in Excel, version 5.0 or greater. 11.1.2 To calculate the fluoride contraction in the sample, see method AMDT-M-2. 11.2 Analyzing the Data 11.2.1 must be at least 0.95 or greater. "Outliers" may be excluded if two of the three replicates are within 20% of each other and the outlier is greater than 200% of the average of those two or less than 50% of the average of those two. Any such outliers should be pointed out in the data and noted in the Final Report along with the reason it was considered an outlier. 12.0 ATTACHM ENTS________________________________________ _ None 13.0 REFERENCES __________________________ 13.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer Operator's Manual (Manual 915349, revision B, December 1993) 13.2 AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 13.3 AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 REVISIONS____________________________________________________ ___ Rvision Number Reason for Change Rvision Date 6 000842 3M Environmental Laboratory Method Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer Method Identification Number: AMDT-M-2 Revision Number: 0 Adoption Date: Revision Date: None Author: Rich Youngblom Approved By: Grodt/ Leader 1/ /J Date Quality Assurance Date Software: MS Word 5.1a Affected Documents: AMDT-M-1 Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer 1 000843 1.0 SCOPE . APPLICABLE COM POUNDS. AND MATRICES___________ 1.1 SCOPE: This method is for the calibration and operation of an Orion EA940 Expandable Ion Analyzer. 1.2 APPLICABLE COMPOUNDS: Fluoride. 1.3 APPLICABLE MATRICES: Liquid samples in an appropriate buffer solution. Preferred pH of 6.0. 2.0 KEYW ORDS__________________________________________ 2.1 Fluoride, fluorine, ion selective electrode 3.0 PRECAUTIONS______________________ ______________________ ___ 3.1 No hazards identified with this method. 4.0 SUPPLIES AND MATERIALS____________________________ 4.1 Orion 940999 Total Ionic Strength Adjustment Buffer II (TISABII) or equivalent. 4.2 Orion Model 900001 electrode filling solution (AgCl) or equivalent. 4.3 Orion 940907 100 ppm fluoride standard or equivalent. 4.4 Milli-QTM water or equivalent. 4.5 Magnetic stir bars. 4.6 Lab tissues. 4.7 Sample collection vials. 4.8 Plastic 100 mL volumetric flasks. 4.9 Polystyrene pipettes. 4.10 Miscellaneous laboratory glassware. 5.0 EQUIPM ENT_______________________________________________ ________ 5.1 Orion Model EA940 Expandable Ion Analyzer or equivalent. 5.2 Orion Model 960900 Solid State Combination Fluoride electrode or equivalent. 5.3 Magnetic Stir Plate. 5.4 IBM compatible 386 or 486 computer (only needed if using Orion 3E software). 5.5 Orion RS232 interface cable (only needed if using Orion 3E software). 5.6 Microsoft Excel 5.0 (only needed if using Orion 3E software). 6.0 INTERFERENCES__________________________________________________ 6.1 It is recommended that the pH be at or near 6.0. A 1: l mixture of TISAB and sample/MilliQTM water will generally bring sample to pH of 6.0. 6.2 Sample temperature may effect fluoride measurement. It is recommended that the sample be at room temperature as the standards were when the meter was calibrated. 6.3 The rate the samples are stirred at should be consistent with the rate the standards were stirred. -2 000844 6.4 Air bubbles trapped under electrode can give erroneous readings. Make sure no air is trapped under electrode. 7.0 SAM PLE HANDLING__________________________________ _ _ _ _ _ 7.1 No special handling necessary. 8.0 CALIBRATION AJVILSTANDARPIZATION___________________ 8.1 Preparation of Calibration Standards 8.1.1 Measure 50 mL of TISAB II into 5 100 mL plastic volumetric flasks. 8.1.2 Label the flasks as 0.05,0.1,0.5, 1.0, and 1.5 ppm F-, along with the date and your initials. 8.1.3 Pipette 0.05, 0.1, 0.5,1.0, and 1.5 mL of 100 ppm fluoride standard into the appropriately labeled flasks. 8.1.4 Add approximately 30 mL of Milli-QTM water to each flask. 8.1.5 Shake the flasks to mix the solutions. 8.1.6 Eliminate air bubbles from the flasks by tipping the flasks on their sides and rolling the air in the flasks over the air bubbles. 8.1.7 Bring the volume in the flasks up to the 100 mL mark with Milli-QTM water. 8.1.8 Invert and shake the flasks for the final mixing. 8.1.9 Record standards in Standards Log Book. 8.2 Calibration 8.2.1 If necessary, remove tape from electrode filling hole. 8.2.2 Invert probe to wet top seal. 8.2.3 Eject a few drops of filling solution from bottom of electrode to wet lower seal. 8.2.4 Fill the electrode with filling solution. 8.2.5 The meter and the F- electrode are typically calibrated by direct measurement with no blank correction, using standards with concentrations of 0.05, 0.1, 0.5, 1.0, and 1.5 ppm F-, following the manufacturer's instructions. 8.2.6 Record the slope in the appropriate log book. 8.2.7 Clean the electrode by rinsing with Milli-QTM water and wiping the sides down with lab tissues. 8.3 Storage Conditions for Standards 8.3.1 Calibration standards are stored at room temperature. 9.0 PROCEDURES__________________________________________________ _ 9.1 Calibration and Measurement, Standard method: 9.1.1 The sample to be measured needs to be mixed with TISAB using the proportions recommended by the TISAB manufacturer. 9.1.2 Place a stir bar in the sample and place the sample on the stir plate. 9.1.3 Allow the sample to mix for a few seconds before inserting the electrode. When the electrode is inserted, make sure there are no air bubbles trapped under the electrode. 9.1.4 The sample should be the same temperature as the calibration standards and stirred at the same rate as the calibration standards. 9.1.5 When the readings have stabilized, record the reading in the appropriate log book. 3 00084 9.2 Calibration And Measurement, Using Orion 3E Software: 9.2.1 Calibration: 9.2.1.1 Follow steps 8.2.1 to 8.2.4. 9.2.1.2 Press Function Key #8 (F8). 9.2.1.3 The computer screen will ask you to confirm the number of standards to be used, concentration of the standards, and whether or not a blank is to be included in the calibration. Make any necessary changes to the information presented and click on CONTINUE. 9.2.1.4 Place the electrode in the first standard on the stir plate and click on CONTINUE. 9.2.1.5 Observe the readings on the graphic display on the computer. When the readings have stabilized, press ACCEPT READING. 9.2.1.6 Repeat step 9.2.1.4 and 9.2.1.5 for the remaining standards. 9.2.1.7 After the final standard, the computer will display the slope of the curve, as well as the intercept and correlation. Record the slope, intercept, and correlation in the appropriate log book and click on CONTINUE. The calibration data is automatically copied to C:\Orion\Data\Calib.txt. 9.2.2 D ata Spreadsheet: 9.2.2.1 Select either NEW or OPEN from the FILE menu to open a new or existing spreadsheet to store data in. 9.2.2.2 Record the name of the spreadsheet used in the appropriate log book. 9.2.3 Fluoride Measurement: 9.2.3.1 Follow steps 9.2.1 through 9.2.4 9.2.3.2 Enter the name of the sample in the appropriate place on the screen. 9.2.3.3 Click on the NEW SAMPLE button 9.2.3.4 When the readings have stabilized, click on the RECORD button and write the result in the appropriate log book. 10.0 VALIDATION_________________________________ _________________ _ 10.1 Quality Control: 10.2 Precision and Accuracy 10.3 O ther Validation Parameters According to Reference 13.2, the range o f detection is 0.02 ppm fluoride up to a saturated solution of fluoride. 11.0 DATA ANALYSIS___________________________________________________ 11.1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 12.0 ATTACHM ENTS____________________________________________________ None 13.0 REFERENCES______________________________________________________ 4 000846 13.1 Orion Model EA940 Expandable Ion Analyzer Instruction Manual, Orion Research Incorporated, 1991. 13.2 Orion Model 960900 Solid State Combination Fluoride Electrode Instruction Manual, Orion Research Incorporated, 1991. 14.0 REVISIONS_______________________________________________ __ Revision Number Reason for Change Revision Date 5000847 3M Environmental Laboratory M ethod Extraction of Fluorochem icals from Rabbit Livers SOP Identification Number: AMDT-M-4 Revision Number: 0 Adoption Date: Revision Date: None Author Dave Christenson/Cynthia Weber Approved By: Software: MS Word, 6.0 Affected Documents: M-5, Analysis of Rabbit Extract for Fluorochemicals Using Electrospray Mass Spectroscopy. 000848 1 1,0 SCOPE._________________________________________ _ 1.1 Scope: This method is for the extraction of fluorochemicals from rabbit livers. Ethyl acetate is used to extract fluorochemicals from the livers for analysis by electrospray mass spectroscopy. 1 .2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Rabbit Livers. M . J K E X -W D R D S ______________________ _____________________________ _ 2 .1 Fluorochemicals, rabbit livers, electrospray mass spectrometer, fluorinated compounds, extraction. 3d PRECAUTIONS_________________ ________________ 3 .1 Use gloves when handling the rabbit livers, they may contain pathogens. 4 .0 SU PPLIES AND M ATERIALS_______________________________ 4.1 Supplies 4 .1 .1 Syringe, capable of measuring 100 jiL 4 .1 .2 Eppendorf type or disposable pipets 4 .1 .3 Gloves 4 .1 .4 Plastic grinding tubes 4 .1 .5 Plastic centrifuge tubes, 15 mL 4 .1 .6 Labels 4 .1 .7 Nitrogen 4 .1 .8 Timer 4 .1 .9 Filters, Titan nylon syringe filters, 0.2 (im. 4 .1 .1 0 Analytical pipets: glass volumetric pipets. 4 .1 .1 1 Disposable plastic 3 cc syringes. 4 .1 .1 2 Crimp cap autovials. 4.2 Reagents 4 .2 .1 Aqueous Ammonium Acetate (Aldrich), approx. 250 ppm: Prepare a 2500 ppm aqueous solution of ammonium acetate by adding 250 mg ammonium acetate to a 100 mL volumetric flask and dilute to volume with Milli-Q water. Dilute this solution 1.TO for a 250 ppm solution. 4 .2 .2 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker), (NajCOa/NaHCOs) 0.25 M: Weigh 26.5 g of sodium carbonate (Na^O a) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with Milli-Q water. 4 .2 .3 Dilute acetonitrile solution, dilute acetonitrile 1:1 with Milli-Q water. 4 .2 .4 Ethyl Acetate 4 .2 .5 Methanol 4 .2 .6 Milli-Q water 4 .2 .7 1H,1H,2H,2H - perfluorooctanesulfonic acid (Aldrich) 4 .2 .8 FC-95 (3M Specialty Chemical Division) 00084^ 2 g.Q EQUIPMENT--------------------------------------- 5 .1 Ultra-Tunrax T25 Grinder for grinding liver samples. 5 .2 Vortex mixer 5 .3 Centrifuge 5 .4 Shaker 5 .5 Analytical Evaporator <.0 IN T E R FE R E N C E S______________________ 6 .1 There are no known interferences at this time. 7.0 SA M PLE H A N D LIN G ______ _______________________________________ 7 .1 The rabbit livers are received frozen, and must be kept frozen until the extraction is performed. 8.0 C A L IB R A T IO N AND ST A N D A R D IZ A T IO N _________________ _ 8 .1 Preparation of Internal Standards 8 .1 .1 Prepare an internal standard of approximately 12 ppm 1H,1H,2H,2Hperfluorooctanesulphonic acid to be added to each liver sample. 8 .1 .2 Weigh at least 0.1 g of lH,lH,2H,2H-perfluorooctanesulphonic acid into a 100 mL volumetric flask. Record the actual weight. 8 .1 .3 Bring it up to volume with methanol, this is the stock standard. 8 .1 .4 To a 250 mL volumetric flask, add 3 mLs of the stock standard and bring to volume with Milli-Q water. Calculate the actual concentration of the standard. actual mg perfluoroctane- subhonic acid X 3 mL = 0.1 L 250 mL actual concentration, ppm 8 .2 Prepare FC-95 Anion Standards 8 .2 .1 Prepare FC-95 standards for the standard curve. 8 .2 .2 Weigh approximately 100 mg of FC-95 into a 100 mL volumetric flask. Record the actual weight. 8 .2 .3 Bring up to volume with dilute acetonitrile. 8 .2 .4 Dilute the solution with dilute acetonitrile 1:10 for a solution of approximately 100 ppm. Dilute this solution 1:10 with dilute acetonitrile for a solution of approx. 10 ppm. 8 .2 .5 Use the 10 ppm solution to make working standards with values close to 5.0 ppm, 1.0 ppm and 500 ppb. 8 .3 Prepare Beef Liver Homogenate to Use for Standards 8 .3 .1 Weigh 40 g of Bovine liver into a 250 mL Nalgene bottle containing 200 mLs Milli-Q water. Grind to a homogenous solution. 8 .3 .2 Add 1 mL of the solution to a 15 mL centrifuge tube. Prepare a total of eight 1 mL aliquots of the solution in 15 mL centrifuge tubes. Be sure to re suspend solution by shaking it between aliquots. 000630 3 8 .3 .3 Spike seven of the 1 mL aliquots with the following amounts of working standards in step 9.12 of the procedure. One 1 mL aliquot serves as the blank. Working Standard (Approximate Cone.) 500 ppb 500 ppb 500 ppb 500 ppb 1 PPm 5 ppm 5 ppm uL - H5<5 2 3 4 5S 2 330 Approximate final concentration of FC-95 in liver Blank 0.292 ppm 0.584 ppm 0.877 ppm 1.16$ ppm 2.924 ppm 5.848 ppm 8.772 ppm 8 .4 Calculate the actual value of the standards: uL of standard x concentration fin ppm) = final concentration (ppm) 171 mg liver /1 ml homogenate of FC -95 in liver Average weight of bovine liver in solution as determined by weighing 1 mL homogenates of 40 mg liver in 200 mL of Milli-Q water. The amount of FC-95 is reported as equivalents ofFC-95 potassium salt. 8 .5 Calibration 8 .5 .1 Extract the spiked beef liver homogenate following 9.13 to 9.23 of this method. Use these standards to establish your curve on the mass spectrometer. 8 .5 .2 Alternatively, a standard curve may be generated using ratios of responses of the perfluorooctansulfonate anion and the internal standard anion versus concentration of the perfluonooctanesulfonate anion. 8 .6 Storage Conditions for Standards 8 .6 .1 New standards are prepared with each analysis. Standards are stored in covered plastic centrifuge tubes until the analysis on the mass spectrometer is performed. 8 .7 Storage Conditions for Standards 8 .7 .1 Beef liver homogenates may be frozen after preparation. 9.0 PR O C ED U R E S___________________________________________________ 9 .1 Obtain frozen liver samples. In spent tissue, note that the liver has not been packaged with other tissues. 9 .2 Use a dissecting scalpel and cut off approximately 1 g of liver. 9 .3 Weigh the sample directly into a tared plastic grinding tube. 9 .4 Record the liver weight in the study note book. 9 .5 Put a label on the vial with the study number, weight, rabbit ID, date and analyst inidals. 000651> 4 9 .6 Add 2.5 mLs water. 9 .7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, until the sample is a homogeneous solution with no large chunks. 9 .8 Rinse the probe off into the sample with 2.5 mLs water using a pipet. 9 .9 Take the grinder apart and clean it with methanol after each sample. Follow AMDT-EP-22. 9 .1 0 Cap the sample and vortex for 15 seconds. 9 .1 1 Pipet 1 mL into a 15 mL centrifuge tube. Label the centrifuge tube with the identical information as the grinding tube. (See AMDT-M-4 Worksheet for documenting the remaining steps.) 9 .1 2 Spike the beef liver homogenates with the appropriate amount of FC-95 standard as described in 8.3. 9 .1 3 Spike the samples and beef liver homogenates with 100 uL of internal standard. 9 .1 4 Add 1 mL of the sodium carbonate/sodium bicarbonate buffer and 1 mL ammonium acetate. 9 .1 5 Using an analytical pipet, add 5 mL ethyl acetate. 9 .1 6 Cap the sample and vortex 20 to 30 seconds. 9 .1 7 Put them in the shaker for 20 min. 9 .1 8 Centrifuge for 20 to 25 minutes, until the layers are well separated. Set the power on the centrifuge to 25. 9 .1 9 Remove 4 mLs of the top organic layer to a fresh 15 mL centrifuge tube with a 5 mL graduated glass pipet. Transfer the label to the fresh tube. 9 .2 0 Blow the sample down on the analytical evaporator to near dryness with nitrogen, approximately 30 to 40 minutes. 9 .2 1 Bring the remaining sample up in 1 mL dilute acetonitrile with an analytical pipet. 9 .2 2 Vortex 15 seconds. 9 . 2 3 ' Transfer the sample to a 3 mL syringe. Attach a 0.2 Jim nylon mesh filter, and filter the sample into a fresh centrifuge tube or a autovial. Label the tube or vial with the study number and animal number. 9 .2 4 Cap and hold for analysis by electrospray mass spectroscopy. 9 .2 5 Complete AMDT-M-4 worksheet and attach to page of study notebook. 1 0 .0 V A L ID A T IO N _____________________________________________ 1 0 .1 Quality Control - not applicable 1 0 .2 Precision and Accuracy- not applicable 10 .3 Other Validation Parameters- not applicable 11.0 D A TA A N A L Y SIS_________________________________________ 11.1 None 1 2 .0 A TT A C H M EN TS__________________________________________ 1 2.1 Worksheet AMDT-M-4 1 3 .0 R E FE R E N C E S_____________________________________________ 1 3.1 AMDT-EP-22 Routine Maintenance of Ultra-Turrax T-25 1 4 .0 R E V ISIO N S________________________________________________ Revision Number Reason for Change Revision Daffi. 000652 5 BEST COPY AVAII ABLE Worksheet AMDT-M-4 Study # _ .. _ _ _ _ 1 Sample Number set # Blank Fiver FC-95 approx 0.5 ppm actual ppm #W 100 uF 200 uF 300 u l 400 uF . _ _ _ _ _ ... _ _ FC-95 approx 1 ppm actual ppm #W _ . - . 500 uF _ _ _ _ _ _ . _ FC-95 approx. 5 ppm actual ppm #W - _ 200 uF TOO uF _ Date and Initials for Std. _ _ 1study number rhere the original worksheet is located and nlace a conv.___________________ ...........................1 - .. ,, - L - ........................ 1 - _________ 1 ______ __ Liver Extraction Process;------------------------------------------------------------------------ Date & Initials Pinet 1 mT, n f T.iver Solution----------------------------------------------------------------;------- Pinet 100 uF o f 12 nnm Internal Standard____________ StiL J--------.----------------- Vortex 15 see-------------------------------------------------------------------------------------------- Pinet 1 mf, n f 250 nnm Ammonium Acetate___________ SttLS-------------------------- Pinet 1 mL of 0.25 N*,CQi/0.2SM.NaHCQ,.Buffer__________________- - ,, ...Z Einet 5 mL of Ethyl Acetate------------------------------------------------------------Vortex 20-10 sec-------------------------------------------------------------------------- Shake 20 min_____________________________________________________________ Centrifuge 20-25 min______________________________________________________ Remove a.4-mL alinunt nf organic layer________________________________ Blow down tn near drvness r<0 2S m U with N-________________________________ Arid 1 m: n f 1:1 Acetnnitrile/FFO_____________________ I N__________________ Vortex 15 we_____________________________________________________________ Filter using a Tcc B-P syringe with a 0.2um SRI filter into a 1.5 mL autnsamnle vial G0085 3M Environmental Laboratory_____ __________________ __ M ethod Analysis of Rabbit Liver Extract for Fluorochem icals using Electrospray Mass Spectroscopy SOP Identification Number: AMDT-M-5 Revision Number: 0 Adoption Date: .--'Y r" Revision Date: None Author Dave Christenson/Cynthia Weber Software: MS Word, 6.0 Affected Documents: M-4, Extraction of Fluorochemicals from Rabbit Livers 000854 l 1.0 SCO PE_____________________________________________________________ _ 1.1 Scope: This method is for the analysis of extracts of rabbit liver or other tissues or fluids for fluorochemicals using the electrospray mass spectrometer. The analysis is performed by single ion monitoring of FC-95 anion, M/Z= 499, the internal standard M/Z = 427, and other appropriate masses. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1 .3 Matrices: Rabbit Livers (samples), Beef Liver (standards), other tissues and fluids. 2 .0 K E Y W O R D S _______________________________ _____________________________ 2 .1 Fluorochemicals, fluorinated compounds, electrospray mass spectroscopy, mass spectrometer, rabbit livers. 3 .0 P R E C A U T IO N S _______________ _______________________ ___________________ 3 .1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 3 .2 Do not run the pump above it's capacity of 4000 psi. If pressure goes over 4000 psi stop and release pressure. The peak tubing may be plugged. Troubleshoot back to find the plug and replace the plugged tubing. See AMDT-EP-15 3 .3 Do not run the pump to dryness. 4T . 0 S UV - P P L I E S nA uN uD iMn An Tx jEu Ri \ iIr A& LL/Su 4.1 Supplies 4 .1 .1 Nitrogen gas regulated to 140 psi. 4 .1 .2 Fluofix column or equivalent. 4 .1 .3 100 uL or 250 uL flat dp syringe for sample injection. 4.2 Reagents 4 .2 .1 Dilute acetonitrile mobile phase, dilute acetonitrile 1:1 with Milli-Q water. 4 .2 .2 Milli-Q water, all water used in this method should be Milli-Q water. 5.0 E Q U IPM E N T 5 .1 VG Trio 2000 Electrospray Mass Spectrometer or equivalent. 5 .2 ISCO Syringe Pump 5 .3 Spectraphysics AS300 Autosampler 5 .4 100 uL Assembly 5 .5 Autovials or capped centrifuge tubes. 6.0 IN T E R FE R E N C E S 6 .1 There are no known interferences at this time. 7.0 SA M PLE H ANDLING 7 .1 Keep the extracted samples in capped 15 mL centrifuge tubes or in capped autovials until ready for analysis. oooass 2 8 .0 C A L IB R A T IO N A N D S T A N D A R D I Z A T I O N ___________________ ____ 8.1 Preparation of Calibration Standards 8 .1 .1 Seven beef liver standards and one blank beef liver are prepared during the extraction procedure. (See AMDT-M-4, section 8.0) 8.2 Calibration 8 .2 .1 Run the seven beef liver standards twice, starting with the lowest standard to obtain the standard curve. 8 .2 .2 Typically one standard is run after each 5 to 7 samples. Choose a standard in the same range of concentration as the samples. 8.3 Storage Conditions for Standards 8 .3 .1 Fresh standards are prepared with each analysis. Standards are stored in covered plastic centrifuge tubes until the analysis on the mass spectometer is performed. Samples and standards are NOT refrigerated. 8.4 Storage Conditions for Beef Liver Homogenates 8 .4 .1 Beef liver homogenates may be frozen after preparation. 9 .0 P R O C E D U R E ___________________________________________________________ 9 .1 Initial Set-up 9 .1 .1 Set software to "Operate on", Ion Mode ES'. 9 .1 .2 Record backing pressure in the instrument log. 9 .1 .3 Fill the solvent cylinder with mobile phase. 9 .1 .4 Set the pump to "Run". Set the flow to 1000 uL/min. Observes droplets coming out of the tip of the probe. The pressure should be at 1700 to 1800 psi. 9 .1 .5 Check the fused silica at the end of the probe. Use an eye piece to check for chips. The tip should be flat with no jagged edges. If any chips are found cut off the tip of the silica with a column cutter and pull the silica through to the appropriate length. 9 .1 .6 Check your nitrogen supply. Turn on the nitrogen. There should be no nitrogen leaking around the tip of the probe. A fine mist should be coming out of the tip. 9 .1 .7 Carefully guide the probe into the opening. Insert it until it won't go any further. Connect the voltage cable to the probe. 9 .1 .8 Go to the "Editor" page, and set Ionization Mode to ES', and the appropriate masses to 427 and 499. 9 .1 .9 If it is not in single ion mode go to "Option" and set SIR. 9 .1 . lOStart Acquisition. Assign a file name, MO-DAY-YR + letter. Record it in the log book. 9 .1 .1 1 Run the beef liver samples first, running each standard twice at the beginning of the run.. Run a QC check by running one standard after every 5 to 7 samples. 9.2 Manual Injection 9 .2 .1 Draw 150 uL of sample into a syringe. Inject the sample into the rheodyne injection port. Inject slowly. Record the sample ID in the log book. 9 .2 .2 Turn the valve to "On". 9 .2 .3 Wait two minutes, and inject the next sample. 9 .2 .4 Record the scan number for each sample in the logbook. 000656 . 6 9.3 Using the Autosampler 9 .3 .1 Set up sample tray A, B, or C. 9 .3 .2 Record the samples and their positions in the instrument log book. Up to 17 vials may be in each run. 9 .3 .3 Set-up the sampler: 9 .3 .3 .1 Push the sample button 9 .3 .3 .2 Set sample loop size = 100 uL 9 .3 .3 .3 Set inject/sample = 2 9 .3 .3 .4 Set Cycle time = 0 9 .3 .3 .5 Name the file: Livers 9 .3 .3 .6 Identify the tray used 9 .3 .3 .7 Add the samples to Queue by pressing "Enter" 9 .3 .3 .8 Press "Run" to start 10.0 V A L ID A T IO N ____________________________________________________ 10.1 Quality Control 1 0 .1 . IRun a standard every 5 to 7 samples. If a significant change( 50%) in peak height occurs stop the run. Only the samples before the last acceptable standard will be used. The remaining samples will be reanalyzed. 10.2 Precision and Accuracy 1 0 .2 . lSee Method Validation Report number AMDT-M-5.0.VI 10.3 O ther Validation Param eters 1 0 .4 Refer to Method Validation Report Number AMDT-M-5.0.VI 11.0 DATA ANALYSIS__________ ;______________________________________ 11.1 11.2 Calculations Plot the standard curve, using the mean of the two values obtained for each standard. 1 1 .2 . IRead peak heights or areas for the samples from the printout. Use linear regression to determine the sample concentrations. 1 1 .2 .2 Calculate the mg of FC-95 anion, or other fluorochemical in the total rabbit liven mg FC-95 anion in the total rabbit liver = mg FC-95 anion from std. curve gms of liver used for analysis x Total mass of liver, gms 1 1 .3 Make a results table and enter it in the study book. 1 1 .4 Print a chromatogram for each sample, with the peaks labeled with the sample or standard ID. Write the study number on the printout, initial, date, and put it in the study folder. Staple all chromatograms together and number pages. 000857 4 12.0 A T T A C H M E N T S None 13.0 R E F E R E N C E S 13.1 AMDT-EP-17 1 4 .0 R E V IS IO N S _________________________________________________ Revision Number Reason for change Revision Date G00S585 3M Environmental Laboratory M ethod Analysis of Fluoride Using the Skalar Segmented Flow Analyzer With Ion Selective Electrode Method Identification Number: AMDT-M-8 Adoption Date: Revision Number: 0 Revision Date: None Author: Deb Wright / Cynthia Weber Approved By: ^ u p U ad e r^ /A/fs Date Quality Assurance Date Software: IBM MS Word, 6.0 Affected Documents: AMDT-EP-26, Operation and Maintenance of the Skalar Segmented Flow Analyzer 000859 j S68PY AVAILABLE 1.0 SCO PE______________________________________________________________ 1.1 This method is for the analysis for fluoride, thermally extracted from samples using the Dohrmann DX2000 (AMDT-M-1), and collected in TISAB for analysis with an Ion Selective Electrode (ISE). The analysis is performed using the Skalar Segmented Flow Analyzer with ISE. 1.2 Samples can be tissues, serum, biological material, or other materials extracted on the Dohrmann. 2.0 K EYW O RD S_______________ ____________________________________ ____ 2 .1 Skalar, segmented flow, fluoride. 3.0 PRECAUTIONS _________________________________ _____________ 3 .1 Follow standard laboratory safety practices. 4 . 0 S U P P L IE S A N D M A T E R IA L S ______________________ __________________ 4.1 Supplies 4 .1 .1 Sample cups, 4 mL plastic cups with caps 4 .1 .2 Autopipets, oxford or equivalent with plastic tips 4 .1 .3 Polypropylene volumetric flasks, 100 mL 4 .1 .4 Cartridge components, refer to the Skalar Methods for components and part numbers. 4 .1 .5 Sample prefilters, Evergreen 4.2 Reagents 4 .2 .1 Brij 35, 30% S.F.A.S. Detergent 4 .2 .2 TISAB II buffer solution: Purchase TISAB II from Orion. To 1 liter of TISAB II add 2.5 mL or 100 ppm fluoride solution and 1 mL Brij. 4 .2 .3 Sampler rinsing solution: Dilute TISAB II 1:1 with Milli-Q water. 4 .2 .4 Nitric acid solution for decontamination, 1 N (lab grade): Slowly add 64 mLs concentrated nitric acid (HN03) to 250 mLs of Milli-Q water. Bring the volume up to 1 L with Milli-Q water. 4.3 Standards 4 .3 .1 Stock solution, 100 ppm F: purchased from Orion. 4 .3 .2 Intermediate standard, 10 ppm: Dilute 10 mLs of stock solution to 100 mLs with Milli-Q water. Use polypropylene volumetric flasks. 4 .3 .3 Working standard: Make up the following working standards by adding the volumes of intermediate or stock standard indicated on the table, using oxford or pumpmate pipets, to 50 mLs of TISAB and diluting to 100 mLs __________________ with Milli-Q water._______________________________________________ Working Standard mLs of Stock Standard mLs of Intermediate Standard 0.015 ppm - 0.15 0.03 ppm - 0.3 0.06 ppm - 0.6 0.09 ppm - 0.9 0.12 ppm - 1.2 0.15 ppm - 1.5 0.3 ppm 0.3 - 0.6 ppm 0.6 - 0008602 5,0 EQUIPMENT_____________________________________________ _____ 5 .1 Skalar Segmented Flow Auto Analyzer Sans"*1' System equipped with ISE M IN T E R FE R E N C E S_________________________________________ 6 .1 High concentrations of alkalinity, chloride, phosphate, sulfate or iron can cause interferences. 7.0 SA M PL E HANDLING___________________________________ __________ 7 .1 Samples should be stored in polyethylene bottles. Samples should be analyzed within 30 days. 8.0 C A L IB R A T IO N AND ST A N D A R D IZ A T IO N _____________________ 8.1 Preparation of Calibration Standards 8 .1 .1 Prepare calibration standards as in section 4.3. 8.2 Calibration 8 .2 .1 The standards are analyzed at the beginning of the run. 8.3 Storage Conditions for Standards 8 .3 .1 Standards are stored in capped polypropylene volumetric flasks. New standards are prepared at a minimum of every six months, or as necessary. 9.0 PR O C E D U R E _______________________________________________________ 9.1 Start Up Procedure 9 .1 .1 Clamp down the pumpdecks, air bars and sampler-pump tubing. 9 .1 .2 Put the fluoride electrodes in the electrode chamber. 9 .1 .3 Turn on the power of the sampler, pumps, offset potentiometer and heating bath. 9 .1 .4 Put the reagent-lines in the appropriate bottles. 9 .1 .5 Turn on the interface, computer, display and printer. Make sure you turn on the interface before the computer. 9 .1 .6 Let the system stabilize for approximately 30 minutes. 9.2 Starting a Run 9 .2 .1 Create a sample table by selecting FILES, TABLE, and CREATE, type in the name of the file, and press ENTER. 9 .2 .2 Print the sample table, inserted in the system table by pushing ESC, PRINT, GROUP 1. This will print the entire run. 9 .2 .3 Dial the sampler settings to the appropriate number of samples, number of seconds for sample wash, and number of seconds for the sample. 9 .2 .4 Fill the sample tray with the standards, samples, washes and drifts. IW and FW/RUNOUT cups on the sampler do not need to be filled. 9 .2 .5 Set the baseline. 0008613 IEST COPY AVAI1ABLE 9 .2 .5 .1 Select GRAPHICS, REALTIME. If you cannot get real-time, you may be in the Data Handling Panel. Switch to the Analysis Panel by selecting CONTROL PANEL and pushing F7. 9 .2 .5 .2Use the small screwdriver for the offset potentiometer to set the base line. Adjust the baseline until it is approximately 3/4 inch from the bottom of the screen. 9 .2 .5 .3Check the highest standard and adjust the gain, if necessary, with the interface screw #3. 9 .2 .6 Go to CONTROL PANEL, and to analysis panel. Deselect the analysis that will not be run. (Select or deselect analysis by pressing ENTER.) Press Tab to return to the Analysis Panel. 9 .2 .7 Press the spacebar to bring up the local menu. 9 .2 .8 Select START to start the analysis. 9 .2 .9 Type your ID (initials), the sample table which you created under 9.2.1 (or press ENTER for choices), choose running with or without the system table and select START ANALYSIS. 9 .2 .1 0 After starting the software, start the sampler. Make sure that the sampler is set to the right number of samples and that the sample/wash/air times are OK. 9 .2 .1 1 Select GRAPHICS, REAL TIME to view the progress of the analysis. 9 .3 Loading and Printing the Data-File 9 .3 .1 Go to CONTROL PANEL, press the spacebar to bring up the local menu and select LOAD. Select AUTOCALCULATION and enter the filename (or highlight the file to be printed and press ENTER). 9 .3 .2 To view the calibration curve, go to GRAPHICS, CALIBRATION CURVE. 9 .3 .3 To print the high level curve, push PRINT SCREEN. 9 .3 .4 To print the low level screen, push ESC to get out of graphics. Select SETTINGS. Change the max y value to approximately 900. Go to CAL CURVE and press ESC, and Enter. Press PRINT SCREEN. 9 .3 .5 Return to SETTINGS and change the max value back to 4095, go to EDIT, press ENTER and PRINT SCREEN to print sample peaks. 9 .3 .6 To print the results go to CONTROL PANEL, SPACEBAR, OUTPUT, OUTPUT. Select PRINTER for the Epson or PRN for the Laser. 9.4 Shutdown 9 .4 .1 Put all the reagent-lines in Milli-Q water. 9 .4 .2 Let the system rinse for approximately 30 minutes. 9 .4 .3 After the system has rinsed completely, turn off the sampler, pump and offset potentiometer. Turn off the heating bath on weekends. Leave liquid in the lines. 9 .4 .4 Take the electrode out and soak in 100 ppm F overnight. 9 .4 .5 Release the pump-decks, air bars and sampler pump-tubing. 9 .4 .6 Select FILES, press ALT F and select QUIT to exit the program. 9 .4 .7 On Friday, turn off the computer, display and interface for the weekend. 10.0 V A L ID A T IO N _____________________________________________________ 1 0 .1 Quality Control 10.1. IRun a standard (mid to high concentration) every 10 samples. If a significant change in peak height occurs, only the samples before the last acceptable standard will be used. The remaining samples will be reanalyzed. 000862 4 10.2 Precision and Accuracy 1 0 .2. lSee Method Validation Report number AMDT-M-8.0.V1 10.3 Other Validation Parameters 1 0 .4 Refer to Method Validation Report Number AMDT-M-8.0.V1 11.0 DATA A N ALYSIS _________________________________ 11.1 Calculations 11.1. IThe standard curve is plotted by the Skalar software. 1 1 .1 .2 All calculations are done by the Skalar software, r2 should be 0.995 or better. 1 1 .2 Prepare spreadsheets to summarize data. Include sample volume, weights used etc. 1 1 .3 Write the study number on the printouts, initial, date the printout, and bind together with all package documents and place in the study folder. Make a copy of the summary sheet and tape into the study notebook. Back up all data and spreadsheets onto study disk and backup disks. 1 1 .4 Electronic Data 1 1 .4 .1GLP studies: Electronic data is copied onto the Study floppy disk for each study, and also data is copied onto a floppy disk that is stored in the lab. 1 1 .4 .2 Other studies: All data is copied onto a floppy disk that is stored in the lab. 12.0 A T T A C H M E N T S______________________________________________ _ None 13.0 R E FE R E N C E S____________________!_________________________________ 1 3 .1 AMDT-M-1, Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 1 3 .2 Skalar Methods, #335, Skalar Methods Manual 1 3 .3 AMDT-EP-26, Operation and Maintenance of the Skalar Segmented Flow Analyzer 14.0 REVISIONS Revision Number Reason for change Revision Date 000863 5 3M Environmental Laboratory M ethod Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer - Serum Method Identification Number: AMDT-M-14 Revision Number: 0 Adoption Date: / o - 3 -V s'" Revision Date: None Author: Rich Youngblom Approved by: ^roup Leader ' / / 4 ' % / ?s Date Quality Assurance 9-z?-7v- Date Software: MS Word 5.1a Affected Documents: AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 000864 1.0 SCOPE . APPLICABLE COMPOUNDS. AND MATRICES 1.1 Scope: This method is for the operation of a Dohrmann DX2000 when it is used to extract fluoride from various matrices. The fluoride is typically collected in TISAB solution for analysis with an ion selective electrode. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Biological fluids, particularly serum. 2.0 K E Y W O R D S _____________________________________________________________ 2.1 Fluoride, fluorine, extraction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, DX2000, fluorochemicals. 3.0 P R E C A U T IO N S ________________________________________ ___ 3.1 Glassware and exhaust gases can be extremely hot. 3.2 Glassware is fragile, broken glass may cause injuries. 3.3 Pressurized gases, proper compressed gas handling practices required. 3.4 Solvent based samples may flash, may need to allow them to dry down before starting run. 3.5 Potential biohazards due to the biological matrices. Use appropriate personal protective equipment. 4.0 S U P P L IE S A N D M A T E R IA L S_______________ ___________________________ 4.1 Compressed Oxygen, Hydrocarbon free, regulated to 30 PSI. 4.2 Compressed Helium, High Purity Grade, regulated to 45 PSI. 4.3 Quartz glass sample boat with TeflonTM tubing, Dohrmann 890-097 or equivalent. 4.4 Quartz glass combustion tube, Reliance Glass G-9405-012 or equivalent. 4.5 Orion 940999 Total Ionic Strength Adjustment Buffer (TISAB I I ) or equivalent. 4.6 Sample collection vials, HDPE. 4.7 Milli-QTM water 4.8 Polystyrene pipettes. 4.9 Activated Charcoal, E. Merck 2005 or equivalent. 4.10 Hamilton Syringe or equivalent. 4.11 Miscellaneous laboratory glassware 5.0 E Q U IP M E N T _____________________________________________________________ 5.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer, modified for fluoride extraction. 5.2 IBM compatible 386 or 486 computer. 5.3 DX2000 software, version 1.00, modified for fluoride extraction. 5.4 Excel Spreadsheet, version 5.0 or greater 6.0 IN T E R F E R E N C E S ________________________________________________ ___ 6.1 Sample size is limited to approximately 100 jil. This may vary from matrix to matrix. 000865 2 7.0 SAM PLE HANDLING 7.1 Samples are to be handled with plastic pipettes. A new pipette is to be used for each sample. 8.0 CALIBRATION AND STANDARDIZATION____________________ __ 8.1 Preparation of Calibration Standards 8.1.1 The standards required for each project will need to be appropriate for that individual project. Refer to protocol for that project. 8.1.2 Typically 50-500 ppm FC-95 in methanol standards are used. 8.1.3 For rabbit serum studies, use beef serum as the matrix. 8.2 Calibration - Overview The normal calibration is the fluoride curve (AMDT-M-2). However, if an optional spiked serum curve is required the procedure listed below is used. 8.2.1 A calibration curve for the DX2000 is generated by spiking samples with known standards and combusting them using the same methods and matrix type as the samples to be tested. 8.2.2 Typically, three replicates of each standard and five concentrations of standards will be spiked. 8.2.3 Standard curve will be plotted as Mass Spiked F (ug) on the x-axis and Standard Mass Recovered F (ug) on the y-axis. Generate a regression curve and calculate the equation for the line and the r^ value. 8.2.4 Mass Spiked F (ug) = (Amount spiked in mL) x ( Cone, of standard in ppm) x (0.6004)* *FC-95 is 60.04% F therefore 0.6004 is the factor used to convert FC-95 to F 8.2.5 Standard Mass Recovered F (ug) = (TISAB volume in mL) x (Orion reading in ppm) 8.3 Calibration - Procedure 8.3.1 Start Up 8.3.1.1 Run 2 or more Clean Cycles when starting instrument each day. More clean cycles may be used if the previous samples contained high concentrations of fluoride. 8.3.2 Blanks 8.3.2.1 Prepare sample using the same methods and type of matrix as the test sample. 8.3.2.2 For rabbit studies, use beef serum as the matrix. 8.3.2.3 Put serum blank in Dohrmann boat. Combust sample as described in section 9.0 and analyze sample according to method AMDT-M-2 for the ion selective electrode analysis. 8.3.2.4 For rabbit studies, the meter reading for a blank sample should be 0.03 ppm or lower before proceeding with the calibration. Bum samples until thus limit is reached, or until in the judgement of the operator the reading is stable with respect to historical readings (previous 48 hours). 8.3.2.5 For non-rabbit studies, the blank readings should reach a predetermined ion concentration before proceeding with the calibration. 8.3.2.6 It may be necessary to mix approximately 50 mg of charcoal with the sample to aid combustion. 000866 3 8.3.3 Standard Curve 8.3.3.1 If beef serum is frozen, thaw at least enough to complete the standard curve analysis for the day (=30 mL). 8.3.3.2 Pipette 100pL of beef serum into Dohrmann sample boat. 8.3.3.3 Start with the lowest standard concentration. Using a Hamilton syringe, eject a fixed quantity of the standard on or in the matrix. For rabbit studies, use 4 uL of standard and eject it on or in the beef serum. 8.3.3.4 At least 3 replicates should be used for the lowest standard concentration; more replicates may be used at the discretion of the analyst. 8.3.3.5 Combust the sample as described in section 9.3 and analyze according to AMDT-M-2. 8.3.3.6 Run all 15 standards. If one replicate is significantly different from the other two replicates, run another sample for that standard. Indicate in data that the new replicate replaces the old replicate and that the new replicate will be used to calculate the regression curve. 8.3.3.7 When all standards have been run, calculate the r^. r^ must be at least 0.95. If it is not at least 0.95, consult with supervisor. 8.3.3.8 A new standard curve should be run when the combustion tube or sample matrix is changed. New standard curve may also be run at the discretion of the analyst. 8.4 Storage Conditions for Standards 8.4.1 Storage requirements for standards are dependent on the individual standards used. Typically, standards are stored at room temperature in plastic screw top bottles. 8.4.2 New FC-95 standards should be prepared at least once a month. 9.0 PROCEDURES 9.1 Typical Operating Conditions: 9.1.1 Combustion tube temperature = 950C. 9.1.2 Oxygen and Helium flow = 50 cc/minute. 9.1.3 Vaporization/Drying time = 240 seconds. 9.1.4 Bake time = 300 seconds. 9.2 Start Up Procedure: 9.2.1 If the program is not started, start the EOX program on the PC. 9.2.2 Open the SYSTEM SETUP window. 9.2.3 Put the furnace module and the cell in the READY mode. 9.2.4 Close the SYSTEM SETUP window. 9.2.5 When the oven has reached the READY temperature, run the CLEAN BOAT program found in the CELL CHECK menu. 9.2.6 See AMDT-EP-3 for details of the Dohrmann software. 9.3 Sample Extraction Procedure: 9.3.1 Open the SAMPLE HATCH and pipette 100|iL o f sample into the BOAT. It may be necessary to mix approximately 50 mg of charcoal with the sample to aid combustion. If this is done, charcoal should also be mixed in while establishing the baseline and when generating the standard curve. 9.3.2 Close SAMPLE HATCH. 000867 4 9.3.3 Add appropriate volume ofTISAB solution or 1:1 TISAB:Milli-QTM water mixture to a labeled sample collection vial. Typically 0.6 mL to 15 mL are used. For rabbit studies, use 1.0 or 2.0 mL of 1:1 TISAB:Milli-QTM water mixture. 9.3.4 Place the vial so that the tip of the COMBUSTION TUBE is in the TISAB at least 0.25 inches. Gases released during pyrolysis must bubble through the TISAB. 9.3.5 Run the EOX-WATER program found in the RUN menu. 9.3.6 When the EOX program is finished, remove the collection vial from the combustion tube. 9.3.7 If undiluted TISAB was used to collect the sample, add an equal volume of Milli-QTM water to the TISAB to make 1:1 TISAB:Milli-QTM. 9.3.8 Rinse the end of the combustion tube with Milli-QTM water and wipe with a KIMWEPE to remove any TISAB remaining on the tube. 9.3.9 Open the sample hatch and remove any remaining ash from the boat. Ash can be removed with a cotton tipped applicator and/or vacuumed out. It may be necessary to scrap particles off the bottom with a spatula or other similar device. A drop of Milli-QTM water may be added to the boat to aid in the Clean Cycle. 9.3.10 Close the hatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample is ready for analysis by ion selective electrode (AMDT-M-2). 9.4 Sample Calculations 9.4.1 Use the standard curve to calculate the sample value. 9.4.2 Sample Mass Recovered F (ug) = (TISAB vol in mL) x /Orion reading in ppm - intercept^ (Slope) 10.0 VALIDATION______________________________________________________ 10.1 Quality Control 10.1.1 Daily Start Up Check Samples: Once the standard curve is established, each day o f analysis is started by analyzing QC samples. The QC samples are to be the same as the lowest concentration spiked samples used to generate the standard curve. Each concentration must be done in triplicate unless the first two replicates are within 20% of the standard curve, then a third replicate is not necessary. 10.2 Precision and Accuracy: See method development analysis and sample analysis in Fluoride Notebooks 2,3, and 5. Precision and accuracy varies when analyzing samples of different matrices and different reference compounds. 10.3 O ther Validation Parameters: NA 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1 For the standard curve, use regression analysis in Excel, version 5.0 or greater. 11.1.2 To calculate the fluoride contraction in the sample, see method AMDT-M-2. oc 000868 11.2 Analyzing the Data 11.2.1 r2 must be at least 0.95 or greater. "Outliers" may be excluded if two of the three replicates are within 20% of each other and the outlier is greater than 200% of the average of those two or less than 50% of the average of those two. Any such outliers should be pointed out in the data and noted in the Final Report along with the reason it was considered an outlier. 12.0 ATTACHM ENTS____________________________________________________ None 13.0 REFERENCES_______________________________________ _______________ 13.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer Operator's Manual (Manual 915349, revision B, December 1993) 13.2 AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 13.3 AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 R EVISIO NS ____________________________________________________ Revision Number Reason for Change Revision Date 000869 6 9.3 Quality Assurance Unit Statement 000870 Attachment D GLP Study Quality Assurance Statement Original t: Study Director Study Title: Single-dose Intravenous Pharm acokinetic Study of T-6052 in Rabbits Study Number: AMDT-111694.1 Name of Auditor: KariRambo This study has been inspected by the Quality Assurance Unit as indicated in the following table. The findings were reported to the study director and management. Inspection Dates From Iq 10/13/95 10/19/95 Phase_____________ Final Report Date Inspection Reported to Management Study Director 10/19/95 10/19/95 XAU Auditor / 9S ' BEST COP! AVAILABLE 0008711 9.4 Key Personnel Involved in the Study 000872 3M Environmental Laboratory Key Personnel Thermal extraction followed by analysis using Orion ion analyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plummer Analysis of liver extracts using electrospray mass spectrometry: Jim Johnson Dave Christenson Thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode: Jim Johnson Deb Wright 'R ich Youngblom Deann Plummer Documentation and Reporting: Jim Johnson Rich Youngblom Quality Assurance Unit: Gale Van Buskirk Cynthia Weber Rari Rambo 000873 9.11 Data 000S74 9.11.1 Summary and raw data; ug F` in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. This data, although supportive, in the opinion of the Study Director is not required to reach the conclusion stated in Final Report Section 6.0, and therefore is not discussed in detail. 000875 Summary of Combustion Data - Liver AMDT-111694.1, H W I6329-134 As Referenced in Final Report section 6.0 D A TA A N A L Y S IS Total ug Fluoride in Whole Liver Mean per Dose Group Control Group 2.0 mg/kg dose (T6052) 20.0 mg/kg dose (T6052) 200 mg/kg dose (T6052) 1000 mg/kg dose (T6052) ug 26.3 17.9 17.0 35.1 77.7 000876 RPT134L.XLS FC120 PK ID LiverBlank-1 Liver Blank-2 Liver Spike-1 Liver Spike-2 Liver Spike-3 F52548-1 F52548-2 F52548-3 F52549-1 F52549-2 F52549-3 F52559-1 F52559-2 F52559-3 F52566-1 F52566-2 F52566-3 F52567-1 F52567-2 F52567-3 Liver Blk-1 Liver Blk-2 Liver Spk-1 Liver Spk-2 Liver Spk-3 Liver Spk-4 Liver Spk-5 Liver Spk-6 % rcvry 103% 92% 84% 80% 90% 92% 94% 108% 94% Actual ppm Fin liver (W/W) 0.355 0.181 1.16 1.34 1.24 0.328 0.337 0.207 0.179 0.159 0.264 0.277 0.222 0.211 0.298 0.372 0.333 0.853 0.98 0.772 0.133 0.108 1.10 0.980 1.06 2.72 3.24 2.43 Average ppm Fin liver (W/W) 0.291 0.200 0.237 0.334 0.867 liver burned (grams) 0.109 0.140 0.135 0.105 0.102 0.106 0.127 0.116 0.101 0.131 0.124 0.107 0.138 0.125 0.143 0.139 0.133 0.137 0.101 0.108 0.112 0.124 0.110 0.139 0.132 0.105 0.101 0.117 Whole liver weight (grams) 90.3 90.3 90.3 89.3 89.3 89.3 71.9 71.9 71.9 105.1 105.1 105.1 89.7 89.7 89.7 Total F- in whole liver (ug) 26.3 17.9 17.0 35.1 77.7 Dosage (mg/kg) 0.0 2.0 20.0 200 1000 Page 1 000877 9.11.2 Summary and raw data; analysis of liver extracts using electrospray mass spectrometry. 000878 HWI# 6329-134 Study: Protocol Number: Test Material: Matrix: R Squared Value: Response Factor Amount: Analyst: Date: Method: Instrument: LA BBA SE File: Single-Dose Intravenous Pharmacokinetic TP8084.PK T-6052 in Rabbits (FC 120) Uver Screening N/A DLC 4M/95 Fisons VG 2000 Electrospray M S 040495C ?cv\Aaif\S. A- I (O- 3 1- ^ 5 7X M Group Dose Group 1: Omg/kg * Sterile Water Group 2: 2 mg /kg ** Group 3: 20 mg/kg Group 4: 200 mg/kg Group 5: 1000 mg/kg Sample # F52548 Ion Count Extracted wt Dilution Area * g factor N.D. 1.0036 1 Concentration p g /g " Total m ass of liver g N.D. 90.344 Total amount of FC-95 per liver mg N.D. % of FC-95 F52549 F52559 F52566 F52567 N.D. N.D. $ $ 1.0055 1.0026 1 1.0027 1 1 1 1 N.D. N.D. 89.284 71.921 105.089 89.664 N.D. N.D. $ a Positive response for Ion monitored. * SIR of M 598 4 599 ** The concentration was calculated by using the standard curve and multiplying the result by 4/5. The 4/5 factor Is the result of a miscalculation in applying formula 8.4 in Method AMDT-M-4-0. 137 mg of liver was used in this calculation rather than 171 mg. The concentrations in the standard curve are therefore 5/4 larger than they should be. By multiplying the calculated concentration in the standard curve by 4/5, the correct result Is obtained. 000879 IA&6A9E froy*x ,, I , [JEST COPY AVAILABLE !>a mp I e d I c 1 i u lj p e r a t a r rl I c o ^o j? S (L Run d a te 05-08-1995 09*34*56 U e rs io n * 1 P r i n t e d on 05 0 8 - 1 9 9 5 AT 0 9 * 3 6 * 1 4 3 t r a i c| h t L i n e F i t f o r e e d t h r o u c| h 0 r i q i n , *# 134 A -2 32*- Ccapouent 1:1 710803 ft R JO1100 ./ / / iQi 137171 ,4 1.6 DEL AMOUNT Component 1 = EXTERNAL STANDARD CAL IB RAT IO N AREA 1 0.4000 > 0.0000 3 1.6000 4 4.0000 C` 0 .0000 lj 12.0000 74739 91007 137121 137036 501406 710039 Y - SLOPE X INTERCEPT ri i"5 o = 5. 90 3 IE 0 4 * A m o u n t Am o i.i n t 1 .6 7 14 E -05 * A re a K a q u a r e d := 0.9 7 2 2 + 0 .OOOOE+Ou * 0 .0 0 0 0E + 0 0 12 000880 000881 copy' ? File:040495C LAB-BASE - The MS Data System 04/04/1995 13:53 Sample:HUI 1 6329-134; FC-120 (PK) BABBIT LIMER EXTRACTS 000882 I File:040495C LAB-BASE - The MS Data System 04/04/1995 13:53 Sample:HUI # 6329-134; FC-120 CPK) RABBIT LIVER EXTRACTS A -5 000883 File:040495C LAB-BASE - The MS Data System 04/04/1995 13:53 Sample:HUI ft 6329-134; FC-120 (PK) BABBIT LIUER EXTRACTS A -U 000884 9.11.3 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. 000(385 Kj*iu-> is m RE: 6329-134 LIUER SAMPLES HMDT 111684.1 Date of Analysis: 3-30-95 Analyst: DDlil The samples are burned in the Dohrman at 950 C using between 0.1 and 0.2 grams of the liuer. The gas is collected in 1.0 mL of 1:1 TISAB/Milli-Q water then an additional 2 mL of 1:1 TISAB/Milli-Q is added to allow for sufficient uolume for Skalar analysis. The samples are then analyzed on a Skalar Segmented Flow Analyzer using the Ion Specific Electrode (ISE) Method. TISAB buffer is added to each sample as it proceeds through the system. The sample then goes through a heated mining coil before the potential between the ion selectiue electrode and the reference electrode is measured. The signal is amplified and related to the fluoride concentration. The instrument was calibrated in the ranges of 0.015 - 0.15 ppm and 0.15 - 1.50 ppm fluoride. The standard curue for the high range was plotted using the inuerse logarithm option. The standard curue for the low range is linear. All standards and samples were then calculated by the Skalar software using these curues. All results below 0.0001 ppm appear on the raw data as #.####. A quality control standard was analyzed euery 10 samples to check for accuracy and drift. Raw data is taken from the appropriate calibrated range of the Skalar printout and summarized on an Excel spreadsheet. The final results are adjusted for the collection uolume and any subsequent dilutions. 000886 SUMMARY OF 6329-134 LIVER SAM PLES AMDT 111694.1 GROUP 1 Dose L evel: 0 GROUP 2 Dose L evel: 2 mg/kg GROUP 3 Dose L evel: 20 mg/kg GROUP4 Dose L evel: 200 mg/kg GROUPS Dose Level: 1000 mg/kg F52548-1 F52548-3 F52549-1 F52549-2 F52549-3 F52559-1 F52559-2 F52559-3 F52566-1 F52566-2 F52567-1 F52567-2 F52567-3 ND ND 0.017 ND 0.016 ND 0.015 ND 0.019 0.02 0.05 0.04 0.04 3.0 0.1057 3.0 0.1160 3.0 0.1008 3.0 0.1309 3.0 0.1239 3.0 0.1074 3.0 0.1377 3.0 0.1246 3.0 0.1426 3.0 0.1393 3.0 0.1374 3.0 0.1007 3.0 0.1084 000887 P 134-LSUM ND ND 90.3439 ND ND 90.3439 ND 0.51 89.2836 45 ND ND 89.2836 ND 0.39 89.2836 35 ND 71.9209 ND 0.33 ND 71.9209 24 ND 71.9209 ND 0.39 0.46 105.0891 41 0.52 105.0891 55 1.14 89.6636 102 1.26 1.19 89.6636 113 1.17 89.6636 105 ND ND ND 48 106 BEST COPY AVAILABLE Page 1 134-LXLS O O O Gb QO QC U 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Tracer 1.50 1.24 83% Drift 1.50 1.27 85% Wash 0.016 S tdl 0.015 0.020 131% Std2 0.03 0.03 85% Std3 0.06 0.06 99% Std4 0.09 0.09 98% Std 5 0.12 0.13 105% Std 6 0.15 0.15 98% Std 7 0.30 0.29 96% Std 8 0.60 0.61 101% Std 9 1.20 1.24 103% Std 10 1.50 1.47 98% Drift 1.50 1.31 88% Wash 0.016 Blk-IA 0.02 3.0 0.1087 0.61 Blk-IB ND 3.0 0.1087 ND Blk-2A ND 3.0 0.1399 ND Blk-2B ND 3.0 0.1399 ND Spk-IA 0.05 3.0 0.1347 1.18 Spk-IB 0.05 3.0 0.1347 1.15 Spk-2 0.06 3.0 0.1047 1.66 Spk-3 0.06 3.0 0.1019 1.65 F52548-1 ND 3.0 0.1057 ND F52548-3 ND 3.0 0.1160 ND Drift 1.50 1.28 85% Wash 0.016 F52549-1 F52549-2 F52549-3 F52559-1 F52559-2 F52559-3 F52566-1 F52566-2 F52567-1 0.017 ND 0.016 ND 0.015 ND 0.019 0.02 0.05 3.0 0.1008 0.51 3.0 0.1309 ND 3.0 0.1239 0.38 3.0 0.1074 ND 3.0 0.1377 0.33 3.0 0.1246 ND 3.0 0.1426 0.39 3.0 0.1393 0.52 3.0 0.1374 1.14 Page 1 BEST COPYAVAILABLE 90.3439 90.3439 ND ND 0.004 0.004 0.004 0.004 63.00 63.00 63.00 63.00 0.15 0.15 0.15 0.15 0.16 105% 0.16 103% 0.17 115% 0.17 111% 89.2836 89.2836 89.2836 71.9209 71.9209 71.9209 105.0891 105.0891 89.6636 45 ND 34 ND 24 ND 41 55 102 37 F52567-2 0.04 3.0 0.1007 1.26 38 Drift 1.50 1.26 84% 39 Wash 0.016 40 F52567-3 0.04 3.0 0.1084 1.17 41 Drift 1.50 1.28 85% 42 Wash 0.016 89.6636 113 89.6636 105 BEST COPY AVAILABLE 1995-03-30 13:29 Output of : 950330B1 Software : version 6.1 cl990,93 Operator : ddw Date of the Analysis : 1995-03-30 11:18 Analysis File Name : C:\SKALAR\DATA\950330B1 Fluoride 1.5 Calibration order = Inverse Logarithm S lo p e : s = #.##### Result = 6 x - cl 4 dddadS 0 10cL s i x = corrected value of the sample cl = corrected value of the concentration 1 s Slope of the electrode a2 = al = aO = -0.00000 0.00085 -1.13248 Fluoride L Calibration order = 2 C o r r e la tio n : r = 0 .9 9 6 6 5 Result = a2 * x + al * x + aO a2 a al a aO a 0.00000 0.00031 0.01615 Sampler Type Number Sample Time Wash Time Air Time Take up sPecial needle Height SA1000 1 50 sec. 120 sec. 1 sec. Single None 70 mm. Diluter needle Height dilution Factor dilution Volume Resample Dilution runs : 80 : 10 : 2.5 :1 :1 mm ml. User file : Reproces : No . TXT 000890 1995-03-30 13:29 Output of : 950330B1 F l uoride 1. 5 Path number Signal type Decolor system Number diLute Resample dil Threshold diG output Window event 3 Debubbled Yes 0 No No 4095 0 Off sl sTandard Ignore s2 sTandard Ignore s3 sTandard Ignore s4 sTandard Ignore s5 sTandard Ignore s6 sTandard 0.150 s7 sTandard 0.300 s8 sTandard 0.600 s9 sTandard 1.200 slO sTandard 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed stArt ignore 60 Sec eNd ignore 120 Sec Measure window 75 % Filter No Regeneration No formula : output : ##.### Fluoride L Path number Signal type Decolor system Number diLute Resample dil Threshold diG output Window event 0 Debubbled No 0 No No 4095 0 Off 000cS9l 1995-03-30 13:29 OutPut of : 95033OBI si sTandard 0.015 s2 sTandard 0.030 s3 sTandard 0.060 s4 sTandard 0.090 s5 sTandard 0.120 s6 sTandard 0.150 s7 sTandard Ignore s8 sTandard Ignore s9 sTandard Ignore slO sTandard Ignore Order : 2 Dimension : PPM start Value : 500 DU trigger Limit 1800 Sec Peak shape Pointed stArt ignore 60 Sec eNd ignore 120 Sec Measure window 75 % Filter No Regeneration No formula : c 4 :=c3 output : #.#### 000392 1995-03-30 13:29 OutPut of : 950330B1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dii Weight Ch Result F Cor. Valu Time wt iw Initial Wash 1 1.000 3 0.074 4 0.0161 0 128 00 65 0 1t Tracer 1 1 . 0 0 0 3 1.242 2167 2313 212 4 0.8874 2167 0 2d Drift 1 1 . 0 0 0 3 1.274 2207 2370 388 4 0.9072 2207 0 0 3w Wash 1 1.000 3 0.074 4 0.0161 0 181 574 000 4 si Standard 1 1 1.000 3 0.075 4 0.0196 11 192 747 11 0 0 5 s2 Standard 2 1 1.000 3 0.078 4 0.0255 30 212 911 30 0 0 6 S3 Standard 3 1 1.000 3 0.096 4 0.0594 137 320 1086 13 7 0 0 7 S4 Standard 4 1 1.000 3 0.113 4 0.0883 226 410 1264 226 0 0 8 s5 Standard 5 1 1.000 3 0.138 4 0.1257 338 524 143 7 338 0 0 9 s6 10 s7 11 s8 12 s9 Standard 6 1 Standard 7 1 Standard 8 . 1 Standard 9 1 1.000 3 0.153 4 0.1465 1.000 3 0.289 4 0.2901 1.000 3 0.605 4 0.5171 1.000 3 1.239 4 0.8854 399 586 1612 399 0 0 797 992 1787 797 0 0 1362 1568 1963 1362 0 0 2163 2386 2137 2163 00 13 slO Standard 10 1 1.000 3 1.466 4 1.0359 2460 2694 2312 2460 00 14 d Drift 1 1 . 0 0 0 3 1.313 2256 2440 2487 4 0.9317 2256 00 15 w Wash 1 1.000 3 0.074 4 0.0161 0 184 2668 0 00 Page 1 of 000893 1995-03-30 13:29 OutPut of : 950330B1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dil Weight Ch Result F Cor. Valu Time 16 u BLK 1-A 1 1.000 3 0.077 4 0.0220 19 200 2836 19 0 0 17 u 18 u BLK 1-B BLK 2-A 1 1.000 3 Absen A -39 138 3012 4 0.0041 -39 0 0 1 1.000 3 too 1 > -27 148 3242 4 0.0078 -27 0 0 19 u BLK 2-B 1 1.000 3 0.068 4 0.0029 -43 128 3360 -43 0 0 20 u SPK 1-A 1 1.000 3 0.092 4 0.0530 117 288 3536 117 0 0 21 u 22 u SPK 1-B SPK 2 1 1.000 3 0.092 4 0.0517 1 1.000 3 0.095 4 0.0581 113 280 3714 113 0 0 133 298 3888 133 0 0 23 u SPK 3 1 1.000 3 0.094 4 0.0559 126 288 4059 126 0 0 24 u F52548-1 1 1.000 3 0.072 4 0.0121 -13 145 4236 -13 0 0 25 u F52548-3 1 1.000 3 A b sen A -23 132 4412 4 0.0090 -23 0 0 26 d Drift 1 1.000 3 1.282 2216 2368 4588 4 0.9117 2216 0 0 27 w Wash 1 1.000 3 0.074 4 0.0161 0 149 4771 000 28 u F52549-1 1 1.000 3 0.074 4 0.0171 3 152 4904 300 29 u F52549-2 1 1.000 3 Absen A -22 128 5113 4 0.0094 -22 0 0 30 u F52549-3 1 1.000 3 0.073 4 0.0155 -2 148 5285 -2 0 0 31 u F52559-1 1 1.000 3 0.073 4 0.0140 -7 144 5461 -7 0 0 Page 2 of 000894 1995-03-30 13:29 OutPut of : 950330B1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dil Weight Ch Result F Cor. Valu Time 32 u F52559-2 1 1.000 3 0.073 4 0.0152 -3 148 5606 -3 0 0 33 u F52559-3 1 1.000 3 Absen A -31 120 5813 4 0.0066 -31 0 0 34 u F52566-1 1 1.000 3 0.075 4 0.0186 8 160 5982 800 35 u F52566-2 1 1.000 3 0.078 4 0.0242 26 178 6163 26 0 0 36 u F52567-1 1 1.000 3 0.092 4 0.0520 114 266 6338 114 0 0 37 u F52567-2 1 1.000 3 0.087 4 0.0422 83 236 6513 83 0 0 38 d Drift 1 1.000 3 1.262 2192 2346 6689 4 0.8998 2192 00 39 w Wash 1 1.000 3 0.074 4 0.0161 0 154 6871 000 40 u F52567-3 1 1.000 3 0.087 4 0.0422 83 236 7038 83 0 0 41 d Drift 1 1.000 3 1.282 2217 2368 7213 4 0.9122 2217 0 0 42 w Wash 1 1.000 3 0.074 4 0.0161 0 150 7450 000 wt rw RunOut Wash 1 1.000 3 0.074 4 0.0161 0 152 7688 0 00 Page 3 of 000895 Pathno'- Raw data of 950330B1 : Fluoride 1.5 E s o E x i t 1 F i= H e lp 1 C r tl- P - E d it p e a k s I *t>OuJ T6 000896 4000 R au d a ta o f 9 5 0 3 3 0 8 1 : F lu o r id 1 .5 IT tu 3 ? nP'i} -t"2> I ^OJVAJVA^l] V ---- 1------ ,------ 1------ 1------ 1-- 2423 T in e E s c " E x i t I F l " H lp ! C r t i - P - E d it p e a k s i i -3JU3"t . 4 -3? iir 6798 00089? Raw data of 950330B1 : Fluorids 1.5 E sc = E x it ! F I " H e lp I C r tl- P E d lt p e a k s ! (DuJ'T ' I ' 000898 000899 C o n c e n t r a t io n 000900 9.11.4 Summary and raw data; ppm F` in serum as determined by thermal extraction followed by analysis using Orion ion analyzer. This data, although supportive, in the opinion of the Study Director is not required to reach the conclusion stated in Final Report Section 6.0, and therefore is not discussed in detail. 000901 HW I6329-134 AMDT 111694.1 Dohrmann Serum Analysis Analysis Dates: 07/31/95 - 08/2/95 All serum samples were thermally extracted by a modified Dohrmann DX2000 Organic Halide Analyzer and collected in a 1:1 milli Q water and TISAB solution. The samples were measured on an Orion EA940 expandable ion analyzer. The Dohrmann was calibrated using 34ppm, 40ppm, 62ppm, lOOppm, 124ppm, 250ppm, and 500ppm FC-95 standards. The Orion was calibrated by direct measurement with no blank correction using 0.05ppm, O.lppm, 0.5ppm, l.Oppm and 1.5ppm F standards. The slope, intercept, and correlation were recorded in die appropriate logbook. A summary table is included, showing the ppm F" in each sample (see page 2). The summary table also shows the actual Orion readings. An initial calibration curve with standard deviation, %RSD, R2 value and equation of the line is on pages 3 and 4. Pages 5 and 6 show the excel spreadsheet that was generated when the samples were being analyzed. The Dohrmann FC95 calibration curve was not used to generate the data. Page 1 of 6 G 0G S02 S T U D Y # 6329-134 S E R U M FC120 PK HWI 6329-134 Fluoride concentration in rabbit serum (ppm F-) Dosage: Sample 0 mg/kg F52548 2 mg/kg F52549 20 mg/kg F52559 200 mg/kg F52566 1000 mg/kg F52567 2 hour 0.453 0.372 0.357 0.400 0.402 4 hour 6 hour 0.359 0.334 0.329 0.322 0.283 ` 0.532 0.282 0.539 0.367 0.427 8 hour 0.411 0.384 0.291 0.436 0.519 12 hour 24 hour 48 hour 0.383 1.70 0.678 0.353 0.68 0.642 0.290 0.584 0.594 0.296 0.564 0.718 0.327 0.684 0.560 Actual Orion Reading (ppm F-) Dosage: Sample 2 hour 0 mg/kg F52548 0.0226 2 mg/kg F52549 0.0186 20 mg/kg F52559 0.0179 200 mg/kg F52566 0.200 1000 mg/kg F52567 0.0201 4 hour 0.0180 0.0164 0.0142 0.0141 0.0183 6 hour 0.0167 0.0161 0.0266 0.0270 0.0214 8 hour 0.0206 0.0192 0.0145 0.0218 0.0260 12 hour 24 hour 48 hour 0.0191 0.0848 0.0339 0.0177 0.0340 0.0321 0.0145 0.0292 0.0297 0.0148 0.0282 0.0359 0.0164 0.0342 0.0280 000903 2 NORMAN SERUM CURVE 1 Sample ID 34-ppm-1 34-ppm-2 34-ppm-3 40-ppm-1 40-ppm-2 40-ppm-3 40-ppm-4 62-ppm-1 62-ppm-2 62-ppm-3 100-ppm-1 100-ppm-2 100-ppm-3 124-ppm-1 124-ppm-2 124-ppm-3 250-ppm-1 250-ppm-2 250-ppm-3 500-ppm-1 500-ppm-2 500-ppm-3 Actual Sample reading Qty (ppm F-) (mLor g) 0.07175 0.1 0.05614 0.1 0.06462 0.1 0.08668 0.1 0.06728 0.1 0.05939 0.1 0.06385 0.1 0.07291 0.1 0.0753 0.1 0.07839 0.1 0.0902 0.1 0.1026 0.1 0.1126 0.1 0.1371 0.1 0.1451 0.1 0.1617 0.1 0.3217 0.1 0.2447 0.1 0.3078 0.1 0.4438 0.1 0.4584 0.1 0.4888 0.1 TI5AB snatvoi (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mLFC95 Cene FC95 % Attuai spfcsd sokfttft recovery ppm F- (PP*n) (ug/ltg) msample 0.004 34 176% 1.4350 0.004 34 138% 1.1228 0.004 34 158% 1.2924 0.004 40 180% 1.7336 0.004 40 140% 1.3456 0.004 40 124% 1.1878 0.004 40 133% 1.2770 0.004 62 98% 1.4582 0.004 62 101% 1.5060 0.004 62 105% 1.5678 0.004 100 75% 1.8040 0.004 100 85% 2.0520 0.004 100 94% 2.2520 0.004 124 92% 2.7420 0.004 124 97% 2.9020 0.004 124 109% 3.2340 0.004 250 107% 6.4340 .0.004 250 82% 4.8940 0.004 250 103% 6.1560 0.004 500 74% 8.8760 0.004 500 76% 9.1680 0.004 500 81% 9.7760 Masa spiked (ugF-) 0.0817 0.0817 0.0817 0.0961 0.0961 0.0961 0.0961 0.1489 0.1489 0.1489 0.2402 0.2402 0.2402 0.2978 0.2978 0.2978 0.6004 0.6004 0.6004 1.2008 1.2008 1.2008 Masa recoveredl l l l l i p i l (usF-) 0.1435 STDEV: 0.11228 %RSD: 0.12924 0.17336 STDEV: 0.13456 %RSD: 0.11878 0.1277 0.14582 STDEV: 0.1506 %RSD: 0.15678 0.1804 STDEV: 0.2052 %RSD: 0.2252 0.2742 STDEV: 0.2902 %RSD: 0.3234 0.6434 STDEV: 0.4894 %RSD: 0.6156 0.8876 STDEV: 0.9168 %RSD: 0.9776 0.015629 12 0.024057 17 0.005495 3.6 0.022443 11 0.025096 8.5 0.082072 14 0.045915 5.0 SE R U M C U R V E 1 N O R M A N (07/25/95) 000304 SERUM CURVE 1 NORMAN (07/25/95) O O O oen 1 -- 0.0000 -- 0.2000 -- 0.4000 " 1 i 0.6000 -- --i -- 0.8000 MASS SPIKED (ug) ---- -----1.0000 ------------- 1-- ..-- .... 1.2000 1.4000 STUDY# 6329-134 SERUM 96 Sample ID BLANK-1 BLANK-2 SPIKE 62-1 SPIKE 62-2 SPIKE250-1 SPIKE250-2 SPIKE250-3 F52548-2HR F52549-2HR F52559-2HR F52566-2HR F52567-2HR 62PPM-3 62PPM-4 250PPM-3 250PPM-4 BLANK-1 BLANK-2 BLANK-3 BLANK-4 BLANK-5 SPIKE 62-1 SPIKE 62-2 SPIKE 250-1 SPIKE 250-2 SPIKE 250-3 SPIKE 250-4 BLANK BLANK F52548-4HR F52549-4HR F52559-4HR F52566-4HR F52567-4HR F52548-6HR F52549-6HR F52559-6HR 62-PPM-1 62-PPM-2 250-PPM-1 250-PPM-2 250-PPM-3 BLANK F52566-6HR F52567-6HR F52548-8HR F52549-8HR F52559-8HR Actual reading (ppm F-) 0.0830 0.0314 0.0635 0.0644 0.157 0.239 0.240 0.0226 0.0186 0.0179 0.0200 0.0201 0.0560 0.0834 0.215 0.240 0.0722 0.0386 0.0317 0.0340 0.0247 0.0658 0.0802 0.200 0.204 0.253 0.185 0.0796 0.0237 0.0180 0.0164 0.0142 0.0141 0.0183 0.0167 0.0161 0.0266 0.0542 0.0722 0.101 0.159 0.251 0.786 0.0270 0.0214 0.0206 0.0192 0.0145 Sample Qty (mL or g) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 TISAB final vol m 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 spiked 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 one. FC9 solution (ppm) 62 62 250 250 250 62 62 250 250 62 62 250 250 250 250 62 62 250 250 250 % recovery (ug/ug) 85% 86% 52% 80% 80% 75% 112% 72% 80% 88% 108% 67% 68% 84% 61% 73% 97% 33% 53% 84% Actual ppm F~ in sample 1.66 0.627 1.27 1.29 3.15 4.78 4.80 0.453 0.372 0.357 0.400 0.402 1.12 1.67 4.29 4.80 1.44 0.772 0.633 0.680 0.493 1.32 1.60 4.00 4.07 5.07 3.69 1.59 0.475 0.359 0.329 0.283 0.282 0.367 0.334 0.322 0.532 1.08 1.44 2.01 3.17 5.03 15.7 0.539 0.427 0.411 0.384 0.291 Mass spiked 0.15 0.15 0.60 0.60 0.60 0.15 0.15 0.60 0.60 0.15 0.15 0.60 0.60 0.60 0.60 0.15 0.15 0.60 0.60 0.60 Mass recovered ("3 F*> 0.166 0.0627 0.127 0.129 0.315 0.478 0.480 0.0453 0.0372 0.0357 0.0400 0.0402 0.112 0.167 0.429 0.480 0.144 0.0772 0.0633 0.0680 0.0493 0.132 0.160 0.400 0.407 0.507 0.369 0.159 0.0475 0.0359 0.0329 0.0283 0.0282 0.0367 0.0334 0.0322 0.0532 0.108 0.144 0.201 0.317 0.503 1.57 0.0539 0.0427 0.0411 0.0384 0.0291 000306 5 STUDY# 6329-134 SERUM 98 i Sample ID F52566-8HR F52567-8HR F52548-12HR F52549-12HR F52559-12HR F52566-12HR F52567-12HR 62-PPM-1 62-PPM-2 250-PPM-1 250-PPM-2 BLANK BLANK BLANK BLANK BLANK 62-PPM-1 62-PPM-2 250-PPM-1 250-PPM-2 250-PPM-3 250-PPM-4 250-PPM-5 BLANK BLANK F52548-24HR F52549-24HR F52559-24HR F52566-24HR F52567-24HR F52548-48HR F52549-48HR F52559-48HR F52566-48HR F52567-48HR BLANK BLANK BLANK BLANK BLANK 62-PPM-1 62-PPM-2 250-PPM-1 250-PPM-2 Actual reading (ppm F-) 0.0218 0.0260 0.0191 0.0177 0.0145 0.0148 0.0164 0.0744 0.0915 0.284 0.339 0.0485 0.0471 0.0295 0.0304 0.0301 0.0583 0.0803 0.186 0.175 0.201 0.247 0.228 0.105 0.0205 0.0848 0.0340 0.0292 0.0282 0.0342 0.0339 0.0321 0.0297 0.0359 0.0280 0.0613 0.0396 0.0383 0.0412 0.0319 0.0786 0.0934 0.304 0.269 Sample Qty (mL or g) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 TISAB final vol (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 spiked 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 one. FC9 solution (ppm) 62 62 250 250 62 62 250 250 250 250 250 62 62 250 250 % recovery (ug/ug) 100% 123% 95% 113% 78% 108% 62% 58% 67% 82% 76% 106% 125% 101% 90% Actual ppm Fin sample 0.436 0.519 0.383 0.353 0.290 0.296 0.327 1.49 1.83 5.68 6.79 0.970 0.942 0.590 0.607 0.602 1.17 1.61 3.72 3.50 4.02 4.94 4.56 2.10 0.410 1.70 0.680 0.584 0.564 0.684 0.678 0.642 0.594 0.718 0.560 1.23 0.792 0.766 0.824 0.638 1.57 1.87 6.08 5.38 Mass spiked (ugF-) 0.15 0.15 0.60 0.60 0.15 0.15 0.60 0.60 0.60 0.60 0.60 0.15 0.15 0.60 0.60 Mass recovered (ug F-) 0.0436 0.0519 0.0383 0.0353 0.0290 0.0296 0.0327 0.149 0.183 0.568 0.679 0.0970 0.0942 0.0590 0.0607 0.0602 0.117 0.161 0.372 0.350 0.402 0.494 0.456 0.210 0.0410 0.170 0.0680 0.0584 0.0564 0.0684 0.0678 0.0642 0.0594 0.0718 0.0560 0.123 0.0792 0.0766 0.0824 0.0638 0.157 0.187 0.608 0.538 000907 Lo 9.11.5 Summary and raw data; ppm F' in serum as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. This data, although supportive, in the opinion o f the Study Director is not required to reach the conclusion stated in Final Report Section 6.0, and therefore is not discussed in detail. 000308 SV-rt-O- RE: 6329-134 SERUM SAMPLES AADnMaatDelyTostf1:A1nD1a6Dl9yW4s.i1s: 8/8/95 aTTnhhaeelygszaaemsdpisolencsoalaleSrecktaebludarrninSe2de.g0inmmtehLnetoeDdf o1Fh:1lromTwaISAnAnaBatl/y9Mz5ie0llri-CQusuiwnsaigntegthr.0e.1TIo0hnemSsLpaeomcfpitfhliecesEsaleerrecutmrtho.edne (ISE) Method. TISAB buffer is added to each sample as it proceeds through the system. The asioamnmpsplieflieleedcthtiaevnnedgeroleeelcasttrteohddroetouagtnhhdeatfhlhueeoarreitdefeedrecmonincxeicnegenltecrocaittlrioobnde.efoirsemtheeaspuorteedn.tiaTlhbeetswigeneanl tihse The instrument was calibrated in the ranges of 0.015 - 0.15 ppm and 0.15 -1.50 ppm fluoride. The standard curve for the high range was plotted using the sintavenrdsaerdlosgaanridthsmamopptleiosnw. Tehreetshteanndcaarldcuclautrevde bfoyrtthhee Slokwalraarnsgoeftwisalrineeuasr.ingAltlhese curves. All results below 0.0001 ppm appear on the raw data as #.####. A quality control standard was analyzed every 10 samples to check for accuracy and drift. Raw data is taken from the appropriate calibrated range of the Skalar printout and summarized on an Excel spreadsheet. The final results are adjusted for the collection volume and any subsequent dilutions. G 0 0 l)09 GROUP 1 Dose Level : 0 SUMMARY OF 6329-134 SERUM SAMPLES AMDT 111694.1 Sample ID Fluoride in Fluoride in Fluoride in Fluoride in Fluoride in Fluoride in Fluoride in Sample Sample Sample Sample Sample Sample Sample (ppm) (ppm) (ppm) (ppm) (ppm) (PPm) (ppm) 2 hr 4 hr 6 hr 8 hr 12 h r 24 h r 48 h r F52548 0.79 0.46 0.43 0.77 0.44 2.26 0.88 GROUP 2 Dose Level : 2 mg/kg F52549 0.52 0.55 0.40 0.57 0.39 0.91 0.83 GROUP3 F52559 0.52 0.39 0.75 0.35 ND 0.87 0.76 Dose Level : 20 mg/kg GROUP 4 F52566 0.72 0.34 0.86 0.56 0.31 0.77 0.94 Dose Level : 200 mg/kg GROUP5 F52567 0.62 0.51 0.65 0.66 0.42 0.89 0.72 Dose Level : 1000 mg/kg 134S-RPT.XLS 000910 Page 1 3 SUMMARY OF 6329-134 SERUM SAMPLES AMDT 111694.1 H*;X c l Z V l W Uum. BEST COPYAVAILABLE F52548-2 0.04 2.0 0.10 0.79 F52549-2 0.03 2.0 0.10 0.52 F52559-2 0.03 2.0 0.10 0.52 F52566-2 0.04 2.0 0.10 0.72 F52567-2 0.03 2.0 0.10 0.62 F52548-4 0.02 2.0 0.10 0.46 F52549-4 0.03 2.0 0.10 0.55 F52559-4 0.02 2.0 0.10 0.39 F52566-4 0.02 2.0 0.10 0.34 F52557-4 0.03 2.0 0.10 0.51 F52548-6 F52549-6 F52559-6 F52566-6 F52567-6 0.02 0.02 0.04 0.04 0.03 2.0 0.10 2.0 0.10 2.0 0.10 2.0 . 0.10 2.0 0.10 0.43 0.40 0.75 0.86 0.65 F52548-8 0.04 2.0 0.10 0.77 F52549-8 0.03 2.0 0.10 0.57 F52559-8 0.02 2.0 0.10 0.35 F52566-8 0.03 2.0 0.10 0.56 F52567-8 0.03 2.0 0.10 0.66 F52548-12 0.02 2.0 0.10 0.44 F52549-12 0.02 2.0 0.10 0.39 F52559-12 ND 2.0 0.10 ND F52566-12 0.02 2.0 0.10 0.31 F52567-12 0.02 2.0 0.10 0.42 F52548-24 0.11 2.0 0.10 2.26 F52549-24 0.05 2.0 0.10 0.91 F52559-24 0.04 2.0 0.10 0.87 F52566-24 0.04 2.0 0.10 0.77 F52567-24 0.04 2.0 0.10 0.89 F52548-48 0.04 2.0 0.10 0.88 F52549-48 0.04 2.0 0.10 0.83 F52559-48 0.04 2.0 0.10 0.76 F52566-48 0.05 2.0 0.10 0.94 F52567-48 0.04 2.0 0.10 - 0.72 134S-SUM.XLS 000311 Page 1 3 SERUM CURVE 1 7-31-95 NORMAN Sample ID Spk 34-1 Spk 34-2 Spk 34-3 Spk 40-1 Spk 40-2 Spk 40-3 Spk 62-1 Spk 62-2 Spk 62-3 Spk 100-1 Spk 100-2 Spk 100-3 Spk 124-1 Spk 124-2 Spk 124-3 Spk 250-1 Spk 250-2 Spk 250-3 Spk 500-1 Spk 500-2 Spk 500-3 Skalar Result (ppm) DI.TISAB mL FC 95 Cone final voi Solution FC 95 Soin (mL) Spiked (ppm) Mass Spiked (ug F-) Average % Mass Recovery Recovered (ug F-) 0.09 2.0 0.004 34.00 0.07 2.0 0.004 34.00 0.08 0.15 188% 0.08 2.0 0.004 34.00 0.08 2.0 0.004 40.00 0.07 2.0 0.004 40.00 0.10 0.15 155% 0.07 2.0 0.004 40.00 0.09 2.0 0.004 62.00 0.09 2.0 0.004 62.00 0.15 0.18 121% 0.09 2.0 0.004 62.00 0.11 2.0 0.004 100.0 0.12 ' 2.0 0.004 100.0 0.24 0.24 99% 0.13 2.0 0.004 100.0 0.16 2.0 0.004 124.0 0.17 2.0 0.004 124.0 0.30 0.34 115% 0.18 2.0 0.004 124.0 0.33 2.0 0.004 250.0 0.26 2.0 0.004 250.0 0.60 0.61 102% 0.32 2.0 0.004 250.0 0.47 2.0 0.004 500.0 0.49 2.0 0.004 500.0 1.20 0.99 82% 0.52 2.0 0.004 500.0 SERUM CURVE 1 (NORMAN) 7-31-95 y = 0.7743X + 0.086 R1 = 0.98*1 MASS SPIKED (ug) SERCRV1N.AVE 000912 Page 1 SERUM CURVE 1 7-31-95 NORMAN Sample ID Spk 34-1 Spk 34-2 Spk 34-3 Spk 40-1 Spk 40-2 Spk 40-3 Spk 62-1 Spk 62-2 Spk 62-3 Spk 100-1 Spk 100-2 Spk 100-3 Spk 124-1 Spk 124-2 Spk 124-3 Spk 250-1 Spk 250-2 Spk 250-3 Spk 500-1 Spk 500-2 Spk 500-3 Skalar DI:TISAB mLFC95 Cone Mass Mass % Result final voi Solution FC 95 Soin Spiked Recovered Recovery (ppm) (mL) Spiked (ppm) (ug F-) (ug F-) 0.09 2.0 0.004 34.00 0.08 0.17 211% STANDARDDEVIATION : 0.2450 0.07 2.0 0.004 34.00 0.08 0.13 163% % RSD : 12.9998 0.08 2.0 0.004 34.00 0.08 0.16 191% 0.08 2.0 0.004 40.00 0.10 0.16 164% STANDARDDEVIATION : 0.0826 0.07 2.0 0.004 40.00 0.10 0.14 147% % RSD : 5.3307 0.07 2.0 0.004 40.00 0.10 0.15 154% 0.09 2.0 0.004 62.00 0.15 0.18 120% STANDARD DEVIATION : 0.0263 0.09 2.0 0.004 62.00 0.15 0.18 119% % RSD : 2.1670 0.09 2.0 0.004 62.00 0.15 0.18 124% 0.11 0.12 2.0 0.004 100.0 0.24 0.21 88% STANDARDDEVIATION : 0.1138 2.0 0.004 100.0 0.24 0.24 100% % RSD : 11.4530 0.13 2.0 0.004 100.0 0.24 0.27 110% 0.16 2.0 0.004 124.0 0.30 0.32 108% STANDARDDEVIATION : 0.0778 0.17 2.0 0.004 124.0 0.30 0.34 114% % RSD : 6.7516 0.18 2.0 0.004 124.0 0.30 0.37 124% 0.33 0.26 2.0 0.004 250.0 0.60 0.67 111% STANDARD DEVIATION : 0.1318 2.0 0.004 250.0 0.60 0.52 87% % RSD : 12.9196 0.32 2.0 0.004 250.0 0.60 0.65 108% 0.47 0.49 2.0 0.004 500.0 1.20 0.94 78% STANDARDDEVIATION : 0.0442 2.0 0.004 500.0 1.20 0.99 82% % RSD : 5.3672 0.52 2.0 0.004 500.0 1.20 1.04 87% SERUM CURVE 1 (NORMAN) 7/31/95 y * 0.7743X + 0.086 RJ= 0.9762 SERCRV1N.SUM 000313 Page 1 134S-A.XLS 1995-08-08 10:58 OutPut of : 950808A1 Operator : DDW Date o f the Analysis : 1995-08-08 07:01 Analysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950808A1 1 Tracer 1.50 2 Drift 1.50 3 Wash 4 Standard 1 0.015 5 Standard 2 0.03 6 Standard 3 0.06 7 Standard 4 0.09 8 Standard 5 0.12 9 Standard 6 0.15 10 Standard 7 0.30 11 Standard8 0.60 12 Standard 9 1.20 13 Standard 10 1.50 14 Drift 1.50 15 Wash 16 SERUM BLK1 17 SERUM BLK 2 18 SPK62-1 19 SPK62-2 20 SPK 250-1 21 SPK 250-2 22 SPK 250-3 23 F52548-2 24 F52549-2 25 F52559-2 26 Drift 1.50 27 Wash 28 F52566-2 29 F52567-2 30 SPK 62-3 31 SPK 62-4 32 SPK 250-1 33 SPK 250-2 34 BLK 1 1.44 1.47 ND 0.015 0.03 0.06 0.09 0.12 0.15 0.28 0.62 1.24 1.46 1.53 ND 0.12 0.05 0.09 0.10 0.28 0.20 0.25 0.04 0.03 0.03 1.53 ND 0.04 0.03 0.08 0.11 0.24 0.28 0.10 96% 98% 97% 99% 103% 100% 99% 100% 93% 103% 103% 97% 102% 102% 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 Page 1 000314 ST COPYAVAILABLE 2.46 1.04 1.86 0.004 62.00 0.15 1.90 0.004 62.00 0.15 5.58 0.004 250.0 0.60 3.98 0.004 250.0 0.60 5.08 0.004 250.0 0.60 0.79 0.52 0.52 0.19 125% 0.19 128% 0.56 93% 0.40 66% 0.51 85% 0.72 0.62 1.61 0.004 62.00 0.15 2.28 0.004 62.00 0.15 4.86 0.004 250.0 0.60 5.56 0.004 250.0 0.60 2.03 0.16 108% 0.23 153% 0.49 81% 0.56 93% 134S-A.XLS 35 BLK2 0.06 2.0 0.10 36 BLK 3 0.04 2.0 0.10 37 BLK4 0.05 2.0 0.10 38 Drift 1.50 1.55 103% 39 Wash ND 40 BLK 5 0.04 2.0 0.10 41 SPK 62-1 0.09 2.0 0.10 42 SPK62-2 0.11 2.0 0.10 43 SPK 250-1 0.25 2.0 0.10 44 SPK 250-2 0.25 2.0 0.10 45 SPK 250-3 0.30 2.0 0.10 46 SPK 250-4 0.23 2.0 0.10 47 BLK 0.12 2.0 0.10 48 BLK 0.04 2.0 0.10 49 F52548-4 0.02 2.0 0.10 50 Drift 1.50 1.56 104% 51 Wash ND 52 F52549-4 0.03 2.0 0.10 53 F52559-4 0.02 2.0 0.10 54 F52566-4 0.02 2.0 0.10 55 F52557-4 0.03 2.0 0.10 56 F52548-6 0.02 2.0 0.10 57 F52549-6 0.02 2.0 0.10 58 F52559-6 0.04 2.0 0.10 59 SPK 62-1 0.08 2.0 0.10 60 SPK 62-2 0.10 2.0 0.10 61 SPK 250-1 0.11 2.0 0.10 62 Drift 1.50 1.57 105% 63 Wash ND 64 SPK 250-2 0.20 2.0 0.10 65 SPK 250-3 0.31 2.0 0.10 66 BLK 0.12 2.0 0.10 67 F52566-6 0.04 2.0 0.10 68 F52567-6 0.03 2.0 0.10 69 Drift 1.50 1.57 105% 70 Wash ND STGOOO Page 2 1.11 0.88 0.95 0.84 1.86 0.004 62.00 0.15 2.29 0.004 62.00 0.15 4.94 0.004 250.0 0.60 4.98 0.004 250.0 0.60 5.94 0.004 250.0 0.60 4.56 0.004 250.0 0.60 2.34 0.80 0.46 0.19 125% 0.23 154% 0.49 82% 0.50 83% 0.59 99% 0.46 76% s BEST COPY AVAILABLE 0.51 0.43 0.40 0.75 1.57 0.004 62.00 0.15 2.06 0.004 62.00 0.15 2.21 0.004 250.0 0.60 0.16 105% 0.21 139% 0.22 37% 3.96 0.004 250.0 0.60 6.16 0.004 250.0 0.60 2.38 0.86 0.65 0.40 66% 0.62 103% 134S-B.XLS 1995-08-08 14:27 OutPut of : 950808B1 Operator : DDW Date of the Analysis : 1995-08-08 10:58 Analysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950808B1 1 Tracer 1.50 1.47 98% 2 Drift 1.50 1.48 99% 3 Wash ND 4 Standard 1 0.015 0.015 97% 5 Standard 2 0.03 0.03 99% 6 Standard 3 0.06 0.06 104% 7 Standard 4 0.09 0.09 100% 8 Standard 5 0.12 0.12 98% 9 Standard 6 0.15 0.15 101% 10 Standard 7 0.30 0.28 94% 11 Standard 8 0.60 0.62 103% 12 Standard 9 1.20 1.23 103% 13 Standard 10 1.50 1.47 98% 14 Drift 1.50 1.49 99% 15 Wash ND 16 F52549-8 0.03 2.0 17 F52559-8 0.02 2.0 18 F52566-8 0.03 2.0 19 F52S67-8 0.03 2.0 20 F52548-12 0.02 2.0 21 F52549-12 0.02 2.0 22 F52559-12 ND 2.0 23 F52566-12 0.02 2.0 24 F52567-12 0.02 2.0 25 SPK62-1 0.10 2.0 26 Drift 1.50 ' 1.48 99% 27 Wash ND 28 SPK 62-2 0.12 2.0 29 SPK 250-1 0.33 2.0 30 SPK 250-2 0.39 2.0 31 F52548-8 0.04 2.0 32 BLK 0.07 2.0 33 BLK 0.07 2.0 34 BLK 0.04 2.0 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 Page 1 9TC 000 jtST COPY AVAILABLE 0.57 0.35 0.56 0.66 0.44 0.39 ND 0.31 0.42 1.95 0.004 62.00 0.15 0.20 131% 2.49 0.004 62.00 0.15 6.68 0.004 250.0 0.60 7.84 0.004 250.0 0.60 0.77 1.43 1.36 0.84 0.25 167% 0.67 111% 0.78 131% 3>H ^.1" 35 BLK 0.04 36 BLK 0.04 37 SPK62-1 0.08 38 Drift 1.50 1.49 99% 39 Wash ND 40 SPK 62-2 0.11 41 SPK 250-1 0.21 42 SPK 250-2 0.21 43 SPK 250-3 0.23 44 SPK 250-4 0.28 45 SPK 250-5 0.25 46 BLK 0.15 47 BLK ND 48 F52548-24 0.11 49 F52549-24 0.05 50 Drift 1.50 1.49 99% 51 Wash ND 52 F52559-24 0.04 53 F52566-24 0.04 54 F52567-24 0.04 55 F52548-48 0.04 56 F52549-48 0.04 57 F52559-48 0.04 58 F52566-48 0.05 59 F52567-48 0.04 60 BLK 0.08 61 BLK 0.06 62 Drift 1.50 1.49 99% 63 Wash ND 64 BLK 0.06 65 BLK 0.06 66 BLK 67 SPK 62-1 0.05 0.10 68 Drift 1.50 1.48 99% 69 Wash ND 000317 134S-B.XLS 2.0 0.10 0.80 2.0 0.10 0.82 2.0 0.10 1.54 0.004 62.00 0.15 0.15 103% 2.0 0.10 2.16 0.004 62.00 0.15 0.22 145% 2.0 0.10 4.28 0.004 250.0 0.60 0.43 71% 2.0 0.10 4.12 0.004 250.0 0.60 0.41 69% 2.0 0.10 4.68 0.004 250.0 0.60 0.47 78% 2.0 0.10 5.66 0.004 250.0 0.60 0.57 94% 2.0 0.10 5.06 0.004 250.0 0.60 0.51 84% 2.0 0.10 2.90 2.0 0.10 ND 2.0 0.10 2.26 2.0 0.10 0.91 2.0 0.10 0.87 2.0 0.10 0.77 2.0 0.10 0.89 2.0 0.10 0.88 2.0 0.10 0.83 2.0 0.10 0.76 2.0 0.10 0.94 2.0 0.10 0.72 2.0 0.10 1.58 2.0 0.10 1.13 2.0 0.10 1.21 2.0 0.10 1.23 2.0 0.10 1.06 2.0 0.10 1.93 0.004 62.00 0.15 0.19 129% BEST COPY AVAILABLE Page 2 1995-08-08 10:58 OutPut of : 950808A1 Software : version 6.1 cl990,93 Operator : DDW Date of the Analysis : 1995-08-08 07:01 Analysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950808A1 n i 4v Fluoride 1.5 Calibration order = Inverse Logarithm Slope : s = #.##### [x -cl i ------sJ x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 = al = aO = -0.00000 0.00074 -1.18614 Fluoride L Calibration order = 2 Correlation : r = 0. 99946 Result = a2 * x a + al * x + aO a2 = al = aO = -0.00000 0.00030 0.00019 Sampler Type Number Sample Time Wash Time Air Time Take up sPecial needle Height SA1000 1 50 sec. 120 sec. 1 sec. Single None 70 mm. Diluter needle Height dilution Factor dilution Volume Resample Dilution runs : 80 : 10 : 2.5 :1 :1 mm ml. User file : Reproces : No . TXT 000318 10 1995-08-08 10:58 OutPut of : 950808A1 Fluoride 1. Path number Signal type Decolor system Number diLute Resample dil Threshold diG output Window event :3 : Debubbled : Yes :0 : No : No : 4095 :0 : Off si sTandard : Ignore s2 sTandard : Ignore s3 sTandard : Ignore s4 sTandard : Ignore s5 sTandard : Ignore s6 sTandard : 0.150 s7 sTandard : 0.300 s8 sTandard : 0.600 s9 sTandard : 1.200 slO sTandard : 1.500 Order : Inverse Logarithm Dimension : PPM start Value 500 DU trigger Limit 1800 Sec Peak shape Pointed stArt ignore 60 Sec eNd ignore 120 Sec Measure window 75 % Filter No Regeneration No formula : output : ##.### Fluoride L Path number Signal type Decolor system Number diLute Resample dil Threshold diG output Window event 0 Debubbled No :0 : No : No : 4095 :0 : Off 0003X9 1995-08-08 10:58 OutPut of : 950808A1 si sTandard 0.015 s2 sTandard 0.030 S3 sTandard 0.060 s4 sTandard 0.090 s5 sTandard 0.120 s5 sTandard 0.150 s7 sTandard Ignore s8 sTandard Ignore s9 sTandard Ignore SlO sTandard Ignore Order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed stArt ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula : c4: =c3 output : #.#### 00320 1995-08-08 10:58 OutPut of : 950808A1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time wt iw Initial Wash 3 0.065 65 1t Tracer 3 1.436 209 2d Drift 3 1.468 383 3w Wash 3 0.065 620 4 si Standard 1 3 0.071 734 5 s2 Standard 2 3 0.077 909 6 S3 Standard 3 3 0.093 1081 7 s4 Standard 4 3 0.109 1259 8 s5 Standard 5 3 0.129 1433 9 s6 Standard 6 3 0.156 1609 10 s7 Standard 7 3 0.279 1783 11 s8 Standard 8 3 0.617 1959 12 s9 Standard 9 3 1.239 2133 13 slO Standard 10 3 1.460 2307 14 d Drift 3 1.532 2483 15 w Wash 3 0.065 2725 16 u SERUM BLK 1 3 0.133 2834 17 u SERUM BLK 2 3 0.088 3008 18 u SPK 62-1 3 0.112 3182 19 u SPK 62-2 3 0.113 3360 20 u SPK 250-1 3 0.279 3536 21 u SPK 250-2 3 0.199 3710 22 u SPK 250-3 3 0.254 o 3886 23 u F52548-2 3 ^4060 24 u F52549-2 3 0.076 4234 25 u F52559-2 3 0.076 4408 26 d Drift 3 1.533 4584 27 w Wash 3 0.065 4825 28 u F52566-2 3 0.080 4926 29 u F52567-2 3 0.078 5109 30 u SPK 62-3 3 0.104 5284 31 u SPK 62-4 3 0.126 5463 32 u SPK 250-1 3 0.243 5636 33 u SPK 250-2 3 0.278 5811 34 u BLK 1 3 0.117 5985 35 u BLK 2 3 0.090 6161 36 u BLK 3 3 0.084 6337 37 u BLK 4 3 0.085 6511 38 d Drift 3 1.548 6687 39 w Wash 3 0.065 6917 40 u BLK 5 3 0.083 7035 41 u SPK 62-1 3 0.112 7212 42 u SPK 62-2 3 0.126 7388 43 u SPK 250-1 3 0.247 7562 44 u SPK 250-2 3 0.249 7738 45 u SPK 250-3 3 0.297 7912 46 u SPK 250-4 3 0.228 8087 47 u BLK 3 0.128 8263 48 u BLK 3 0.082 8437 49 u F52548-4 3 0.074 8613 50 d Drift 3 1.557 8787 51 w Wash 3 0.065 9019 52 u F52549-4 3 0.076 9136 53 u F52559-4 3 0.073 9305 Ch Result F Time 4 0.0002 4 0.4779 4 0.4793 4 0.0002 4 0.0146 4 0.0297 4 0.0618 4 0.0896 4 0.1185 4 0.1507 4 0.2466 4 0.3715 4 0.4644 4 0.4790 4 0.4816 4 0.0002 4 0.1231 4 0.0521 4 0.0931 4 0.0950 4 0.2470 4 0.1908 4 0.2317 4 0.0397 4 0.0259 4 0.0259 4 0.4816 4 0.0002 4 0.0360 4 0.0311 4 0.0805 4 0.1141 4 0.2239 4 0.2460 4 0.1013 4 0.0554 4 0.0442 4 0.0473 4 0.4819 4 0.0002 4 0.0419 4 0.0931 4 0.1146 4 0.2272 4 0.2282 4 0.2569 4 0.2136 4 0.1170 4 0.0402 4 0.0228 4 0.4821 4 0.0002 4 0.0277 4 0.0193 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page 1 of 2 000321 1995-08-08 10:58 Output of : 950808A1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time Ch Result F Time 54 u 55 u 56 u 57 u 58 u 59 u 60 u 61 u 62 d 63 w 64 u 65 u 66 u 67 u 68 u 69 d 70 w wt rw F52566-4 3 0.072 F52557-4 3 0.075 F52548-6 3 0.074 F52549-6 3 0.073 F52559-6 3 0.081 SPK 62-1 3 0.102 SPK 62-2 3 0.118 SPK 250-1 3 0.124 Drift 3 1.571 Wash 3 0.065 SPK 250-2 3 0.198 SPK 250-3 3 0.308 BLK 3 0.130 F52566-6 3 0.083 F52567-6 3 0.078 Drift 3 1.572 Wash 3 0.065 RunOut Wash 3 0.065 9488 9664 9834 10013 10190 10364 10540 10715 10889 111 00 11239 11415 11591 11764 11940 12115 12351 12590 4 0.0169 4 0.0254 4 0.0216 4 0.0201 4 0.0377 4 0.0783 4 0.1032 4 0.1107 4 0.4823 4 0.0002 4 0.1899 4 0.2627 4 0.1190 4 0.0431 4 0.0323 4 0.4823 4 0.0002 4 0.0002 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page 2 of 2 000322 C a lib ra tio n curve o f 950808ft! : F lu o rid e L Order : 2 r : 0.99946 Calibration curve of 950808ft! - Fluoride 1.5 C o n c e n tra t io n Order : Inverse Logarithn s : 5894 00032# 000925 000926 000927 1995-08-08 14:27 Output of : 950808B1 Software : version 6.1 cl990,93 Operator : DDW Date of the Analysis : 1995-08-08 10:58 Analysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950808B1 T \\' U^Mi ) t e Vi St ^. \ Fluoride 1.5 Calibration order = Inverse Logarithm S lo p e : s = .***** Result = 10L s J x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 = al = aO = -0.00000 0.00069 -1.21965 Fluoride L Calibration order = 2 C o r r e la tio n : r = 0.99948 Result = a2 * x a + al * x + aO a2 = al = aO = 0.00000 0.00023 0.00544 Sampler Type Number Sample Time Wash Time A i r Tijne Take up special needle Height SA1000 1 50 sec. 120 sec. 1 sec. Single None 70 mm. Diluter needle Height dilution Factor dilution Volume Resample Dilution runs 80 mm 10 2.5 ml 1 1 User file : Reproces : No . TXT 000328 1995-08-08 14:27 Output of : 950808B1 Fluoride 1. Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 3 Debubbled Yes 0 No No 4095 0 Off si sTandard Ignore s2 sTandard Ignore S3 sTandard Ignore s4 sTandard Ignore s5 sTandard Ignore s6 sTandard 0.150 s7 sTandard 0.300 s8 sTandard 0.600 s9 sTandard 1.200 slO sTandard 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed stArt ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula : output : ## .### Fluoride L Path number Signal type Decolor system Number dilute Resample dil Threshold diG output Window event 0 Debubbled No 0 No No 4095 0 Off 000929 1995-08-08 14:27 Output of : 950808B1 si sTandard 0.015 s2 sTandard 0.030 S3 sTandard 0.060 s4 sTandard 0.090 s5 sTandard 0.120 s6 sTandard 0.150 s7 sTandard Ignore s8 sTandard Ignore s9 sTandard Ignore slO sTandard Ignore Order : 2 Dimension : PPM start Value 500 DU trigger Limit 1800 Sec Peak shape Pointed stArt ignore 60 Sec eNd ignore 120 Sec Measure window 75 9 Filter No Regeneration No formula : c4:=c3 output : #.#### 000930 1995-08-08 14:27 Output of : 950808B1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time wt iw Initial Wash 3 0.060 1t Tracer 3 1.468 2d Drift 3 1.480 3w Wash 3 0.060 4 si Standard i 3 0.064 5 s2 Standard 2 3 0.071 6 S3 Standard 3 3 0.089 7 s4 Standard 4 3 0.107 8 S5 Standard 5 3 0.126 9 s6 Standard 6 3 0.155 10 S7 Standard 7 3 0.281 11 s8 Standard 8 3 0.616 12 s9 Standard 9 3 1.230 13 SlO Standard 10 3 1.468 14 d Drift 3 1.487 15 w Wash 3 0.060 16 u F52549-8 3 0.071 17 u F52559-8 3 0.066 18 u F52566-8 3 0.071 19 u F52567-8 3 0.073 20 u F52548-12 3 0.068 21 u F52549-12 3 0.067 22 u F52559-12 3 0.064 23 u F52566-12 3 0.065 24 u F52567-12 3 0.067 25 u SPK 62-1 3 0.112 26 d Drift 3 1.480 27 w Wash 3 0.060 28 u SPK 62-2 3 0.132 29 u SPK 250-1 3 0.334 30 u SPK 250-2 3 0.392 31 u F52548-8 3 0.076 32 u BLK 3 0.095 33 u BLK 3 0.092 34 u BLK 3 0.078 35 u BLK 3 0.076 36 u BLK 3 0.077 37 u SPK 62-1 3 0.098 38 d Drift 3 1.489 39 w Wash 3 0.060 40 u SPK 62-2 3 0.119 41 u SPK 250-1 3 0.214 42 u SPK 250-2 3 0.206 43 u SPK 250-3 3 0.234 44 u SPK 250-4 3 0.283 45 u SPK 250-5 3 0.253 46 u BLK 3 0.150 47 u BLK 3 0.061 48 u F52548-24 3 0.123 49 u F52549-24 3 0.080 50 d Drift 3 1.488 51 w Wash 3 0.060 52 u F52559-24 3 0.079 53 u F52566-24 3 0.076 65 210 386 628 737 911 1085 1261 1437 1613 1787 1961 2137 2311 2487 2729 2840 3010 3188 3362 3534 3712 3878 4063 4235 4413 4589 4824 4939 5113 5289 5459 5642 5814 5990 6164 6340 6514 6690 6929 7040 7216 7390 7565 7741 7915 8089 8241 8439 8610 8788 9015 9138 9310 Ch Result F Time 4 0.0054 4 0.7420 4 0.7466 4 0.0054 4 0.0145 4 0.0297 4 0.0621 4 0.0904 4 0.1172 4 0.1512 4 0.2577 4 0.4307 4 0.6533 4 0.7417 4 0.7496 4 0.0054 4 0.0283 4 0.0174 4 0.0281 4 0.0328 4 0.0220 4 0.0195 4 0.0134 4 0.0156 4 0.0208 4 0.0977 4 0.7468 4 0.0054 4 0.1247 4 0.2919 4 0.3253 4 0.0387 4 0.0716 4 0.0682 4 0.0419 4 0.0399 4 0.0412 4 0.0770 4 0.7503 4 0.0054 4 0.1081 4 0.2072 4 0.1997 4 0.2231 4 0.2589 4 0.2377 4 0.1451 4 0.0066 4 0.1129 4 0.0457 4 0.7498 4 0.0054 4 0.0437 4 0.0387 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 '0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page 1 of 2 000331 1995-08-08 14:27 OutPut of : 950808B1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time Ch Result F Time 54 u 55 u 56 u 57 u 58 u 59 u 60 u 61 u 62 d 63 w 64 u 65 u 66 u 67 u 68 d 69 w wt rw F52567-24 3 0.079 F52548-48 3 0.079 F52549-48 3 0.077 F52559-48 3 0.075 F52566-48 3 0.080 F52567-48 3 0.074 BLK 3 0.099 BLK 3 0.085 Drift 3 1.487 Wash 3 0.060 BLK 3 0.088 BLK 3 0.088 BLK 3 0.084 SPK 62-1 3 0.111 Drift 3 1.482 Wash 3 0.060 RunOut Wash 3 0.060 9487 9662 9837 10013 10187 10361 10539 10706 10886 11127 11236 11412 11586 11760 11935 12166 12410 4 0.0446 4 0.0439 4 0.0417 4 0.0378 4 0.0471 4 0.0358 4 0.0788 4 0.0564 4 0.7496 4 0.0054 4 0.0607 4 0.0614 4 0.0532 4 0.0963 4 0.7475 4 0.0054 4 0.0054 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page 2 of 2 000932 C a lib ra tio n curve o f 950808B1 - F lu o rid e 1.5 Order : Inverse Logarithn s : 5911 0 0 0 7 C a lib ra tio n curve o f 950808B1 - F lu o rid e L Order - 2 r * 0.99948 000333 000334 000935 Raw d a ta o f 9508Q8B1 : F lu o rid e 1.5 4095 r3t 5I 8935 T iiie Esc=Exit 1 Fl=Help ! Crtl-P=Edit peaks ! 13935 0 O 0 J3 6