Document n96BRoevrz5vGMQRNzGQnx53w

3M Environmental Laboratory m % - o ix Final Report- Analytical Study Single-Dose Intravenous Pharmacokinetic Study of T-6054 in Rabbits In-Vivo Study Reference Number: HWI#6329-138 Study Number: AM DT-122094.2 Test Substance: FC-129 (T-6054) 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 o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode Initiation Date: See attached protocol Author: James D. Johnson G 0041G l 1.0 SUMMARY Liver samples at 48 hours post intravenous dose o f FC-129 (T-6054) in rabbits were analyzed for total organic fluorine and perfluorooctanesulfonate. After an intravenous dose o f FC-129 in rabbits, there is a detectable increase o f total organic fluorine in liver at 48 hours post dose at doses ranging from 0.128 to 12.8 mg/kg. A substantial amount o f this total organic fluorine is in the form o f perfluorooctanesulfonate. This pharmacokinetic study shows that there is a convenient marker (perfluorooctanesulfonate) for assessing the extent o f dermal absorption. 2.0 INTRODUCTION------------------ ------------------------------------------------------------ This study was performed in order to provide data for the assessment a subsequent dermal absorption study (HWI#6329-133). Knowing the disposition o f an intravenous dose o f FC-129, facilitates interpretation o f a dermal absorption study. Analysis o f liver and serum samples from rabbits dosed intravenously with FC-129 for total organic fluoride (combustion analysis) and specific compounds (electrospray mass spectrometry) provide data as to whether fluorinated compounds are in the liver at 48 hours post dose and whether perfluorooctanesulfonate is present. Perfluorooctanesulfonate is a very good marker for a dermal study since previous work has shown it to be persistent in liver and serum in rabbits (biological half life >1 month). 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 o f Reference Substance: Responsibility o f Sponsor. 3.4 Stability o f Reference Substance: To be determined by Sponsor. 000417 2 3.5 Storage Conditions for Test Materials: Room temperature for FC-95. For biological samples the storage is -20+10 C. 3.6 Disposition of 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 EXPERIM ENTAL - Overview________________________________________ _ Serum and tissues from animals dosed as described (HWI#6329-138), were available for analysis for total organic fluorine and fluorinated compounds. The samples were analyzed by combustion and/or electrospray mass spectrometry to the extent necessary to provide sufficient data for the interpretation o f a second study on the extent o f dermal absorption o f FC-129 (HWI#6329-133). 5.0 EX PER IM EN TA L-M ETH O D S____________________________ __________ 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 o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 6.0 DATA ANALYSIS___________________________________________________ The data are attached. For combustion analysis for total organic fluorine, the rabbits dosed with 0, 0.128, 0.64, 1.28, and 12.8 mg/kg had 18 (below the level o f practical quantitation), 61, 118, 164, and 2239 ug/whole liver, respectively. The body weight 000418 3 o f the 12.8 mg/kg rabbit (F52752) was 2.7 kg. Thus, the total dose given this rabbit was 34.6 mg. The amount o f total organic fluorine in this dose is 19 mg. The total organic fluorine in liver after the intravenous dose in rabbit F52752 represents 11.8% o f the dose. The data for an electrospray mass spectrometry analysis is attached. There is detectable perfluorooctanesulfonate in all treated rabbits. The high dose rabbit (F52752) had about 335 ug perfluorooctanesulfonate present in liver at 48 hours. If the 34.6 mg dose is expressed as FC-95, (that is, assuming 100% biotransformation) the dose is 31.8 mg. The 335 ug present in whole liver in this animal represents 1.05% o f the dose. This represents a substantial amount o f biotransformation o f FC-129 to perfluorooctanesulfonate at 48 hours. Other data was collected using Skalar segmented flow analyzer with ion selective electrode (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 Affect the Quality o f the Data: The problem with this analysis is that in a pharmacokinetic study reported separately (HWI#6329-159), there was a delay in perfluorooctanesulfonate concentration observed in serum that indicated an unexplained distribution pattern. The level dropped dramatically at 12 and 24 hours but returned to a maximum at 48 hours with a subsequent decay in serum levels that indicated a biological half-life o f > 1 month. This delay in perfluorooctanesulfonate levels may be shifted to a later time when perfluorooctanesulfonate is resulting from biotransformation. There may actually be a higher level o f perfluorooctanesulfonate in liver at a later time period and this study only went to 48 hours. This would indicate that perfluorooctanesulfonate is an even better marker for a dermal absorption study than would be indicated by this data. 7.0 CONCLUSION_______________________________________________________ After an intravenous dose o f FC-129 in rabbits, there is a detectable increase o f total organic fluorine in liver at 48 hours at doses ranging from 0.64 to 12.8 mg/kg. A substantial amount o f this total organic fluorine is in the form o f perfluorooctanesulfonate. This pharmacokinetic study shows that there is a convenient marker for assessing the extent o f dermal absorption. 000419 4 8.0 MAINTENANCE 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. M . APPENDICES___________________________________________________ _ 9.1 Protocol and Amendments 9.1.1 Protocol and Final Report: HWI#6329-138 "Single-Dose Intravenous Pharmacokinetic Study o f T-6054 in Rabbits" (Protocol type TP8084.PK for dosing o f animals, tissue collection, etc.) 9.1.2 Analytical protocol AMDT-122094.2 9.2 Signed Reports from Individual Scientists: None 9.3 Quality Assurance Unit Statement: See attached 9.4 Key Personnel Involved in the Study: See attached 9.5 Materials 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-122094.2 9.10 Instrument Settings: See methods 000420 5 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. 000421 6 9.1.1 Protocol and Final Report: HWI#6329-138 "SingleDose Intravenous Pharmacokinetic Study of T-6054 in Rabbits" (Protocol type TP8084.PK for dosing of animals, tissue collection, etc.) 000422 HAZLETON WISCONSIN POST OFFICE BOX 754 5 M A D I S O N , Wl 53 70 7 754 5 Sponsor: 3M St. Paul, Minnesota I CORNING Company \ FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6054 in Rabbits Author: Steven M. Glaza Study Completion Date: February 24, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Pro.iect Identification: HWI 6329-138 Page 1 of 24 P h o n e 6 0 8 - 2 4 I - 4 4 71 EXPRESS-MAIL DELIVERY: 3301 KINSMAN ....... BLVD. F ax 6 0 8-2 4 1- 7 2 2 7 M A D I S O N , Wl - 53 70 4 000423 Page 2 of 24 HWI 6329-138 QUALITY ASSURANCE STATEMENT 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 12/09/94 12/09/94 Protocol Review 12/19/94 12/19/94 Animal Observation 02/02/95 02/02/95 Data/Report Review 12/09/94 12/19/94 02/02/95 01/10/95 01/10/95 03/10/95 Date 000424 Page 3 of 24 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study of T-6054 in Rabbits HWI 6329-138 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Experimental Start Date Experimental Termination Date T-6054 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 (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 December 17, 1994 December 19, 1994 000425 Page 4 of 24 HWI 6329-138 KEY PERSONNEL Acute Toxicology Laboratory Animal Medicine Steven M. Glaza Study Director Manager Cindy J. Cary, DVM Dipl ornate, ACLAM Supervisor Francis (Bud) W. McDonald Study Coordinator Anatomical Pathology Patricia Padgham In-life Supervisor Rose M. Bridge Report Supervisor Quality Assurance Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data Sherry R. W. Petsel Manager 000426 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 6329-138 Page 2 3 4 6 7 7 7 8 9 11 11 11 11 12 13 14 15 000427 Page 6 of 24 HWI 6329-138 SUMMARY This study was done to assess the level of systemic exposure of T-6054 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 0.128, 0.64, 1.28, or 12.8 mg of T-6054/kg of body weight (Groups 1 through 5, respectively). The dose volume was 0.5 mL/kg for all groups. 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. 000428 Page 7 of 24 OBJECTIVE HWI 6329-138 The objective of this study was to assess the level of systemic exposure to the test material, T-6054, 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-6054 and described as an amber 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 was 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. 000429 Page 8 of 24 HWI 6329-138 Safety Precautions 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 November 16, 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. 000430 Page 9 of 24 Study Design HWI 6329-138 Female animals weighing from 2,702 to 2,891 g at initiation of treatment were placed into the following study groups: Group (Control) 2 3 4 5 Treatment * T-6054 T-6054 T-6054 T-6054 Dose Level (mg T-6054/kq) 0 0.128 0.64 1.28 12.8 Dose Volume (mL/kg) 0.5 0.5 0.5 0.5 0.5 Number of Animals 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. 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). 000431 Page 10 of 24 HWI 6329-138 Sample Collections 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. 000432 Page 11 of 24 RESULTS Body Weights Individual body weights at initiation are in Table 1. HWI 6329-138 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-6054 was evaluated in female albino rabbits when administered as a single intravenous injection at levels of 0, 0.128, 0.64, 1.28, and 12.8 mg/kg. All animals appeared normal throughout the study following administration of this material. SIGNATURE Acute Toxicology Date REFERENCE 1. NIH Publication No. 86-23 (revised 1985). 000433 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 F52792 0.128 Female F52793 0.64 Female F52750 1.28 Female. F52751 12.8 Female F52752 Dav 0 2,784 2,805 2,891 2,702 2,783 HWI 6329-138 000434 GrouD Dose Level ima/kal 10 Sex Female Page 13 of 24 Table 2 Individual Clinical Signs Animal Number F52792 Observation Appeared normal 0.5 HWI 6329-138 Hour 2 4 24 /// 48 2 0.128 Female F52793 Appeared normal / / 3 0.64 Female F52750 Appeared normal ///// 4 1.28 Female F52751 Appeared normal //// 5 12.8 Female F52752 Appeared normal //// Indicates condition exists. 000435 Page 14 of 24 APPENDIX A Protocol TP8084.PK HWI 6329-138 000436 z~ HAZLETON WISCONSIN O S T O F f I C f 13 O X / 5 4 5 AD I SOM. Wl Fj .V/O / / S 4 5 Page 15 of 24 I CORNING Company Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.PK Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6054 in Rabbits Date: December 13, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Pro.iect Identification: HWI 6329-138 T1 h O n o ti 0 y I -1 4 4 / 1 (. X f* K l s t', M A i ( 1 I VE. RY 000437 330 1 M M S M A N LV0 T d X ti'J vi ! ?.7 M A ! i ! ' I N V;i !t) Page 16 of 24 STUDY IDENTIFICATION TP8084.PK Page 2 Single-Dose Intravenous Pharmacokinetic Study of T-6054 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 6329-138 T-6054 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (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 December 17, 1994 December 19, 1994 January 23, 1995 000438 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 will 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-6054 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. 000439 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 returned to the Sponsor after completion of the in-life phase of the study. 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. G. 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) Aoe at Initiation Adult 000440 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) Ar.cl imat ion 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. 000441 Page 20 of 24 i B. Dose Administration TP8084.PK Page 6 (1) Test Groups Grouo Dose Level (mq/kq)a Number of Females 1 0 (Control) 1 2 0.128 1 3 0.64 1 4 1.28 1 5 12.8 1 a The dose volume will be 0.5 mL/kg of body weight. 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. 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. (2) Body Weights Just before test material administration. (3) Sample Collections (a) Freouencv 2, 4, 6, 8, 12, 24, and 48 hours post-injection 000442 Page 21 of 24 TP8084.PK Page 7 (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 on dry ice 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 or his alternate will be notified by telephone at (612) 778-5294 prior to the shipment of the samples. E. 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 (via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. Tissues, as described in section E. Termination, (3) Sample Collection, will be collected. 000443 Page 22 of 24 TP8084.PK Page 8 (2) Srheduled Sacrifice At approximately 48 hours post-injection, animals surviving to termination will be anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. An abbreviated gross necropsy examination will not be done, however, tissues will be collected. (3) 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 (if applicable) Gross pathology report (if applicable and requested by the Study Director) 000444 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 Shipping 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 ) 000445 Page 24 of 24 TP8084.PK ) Page 10 PROTOCOL APPROVAL John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department Date Steven M. G l a z a 5 Study Director Acute Toxicology Hazleton Wisconsin, Inc. ) _________ K Z r t e A V Date Representative Quality Assurance Unit Hazleton Wisconsin, Inc. ____________ (6329-138.protdsk2) __________ n a * > f Date 000446 9.1.2 Analytical protocol AMDT-122094.2 000447 3M Environmental Laboratory Protocol - Analytical Study Single-Dose Intravenous Pharmacokinetic Study of T-6054 in Rabbits In-Vivo Study Reference Number: HWI#6329-138 Study Number: AM DT-122094.2 Test Substance: FC-129 (T-6054) 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 AM DT-M -4-0, Extraction o f Fluorochemicals from Rabbit Liver AM DT-M -5-0, Analysis of 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 Author: James D. Johnson Approved By: //la m e s D^^pftnson [ / Study E lector Z. Date John Butenhoff, PhD Date Sponsor Representative 000448 1.0 PURPOSE This study is performed in order to provide information necessary to assess the extent o f dermal absorption o f FC-129 (T-6054) in a subsequent dermal absorption study, HW I#6329-133. The study is designed to provide information as to whether FC-129 and its metabolites are detectable in liver and other tissues, either as total organic fluorine or as specific compounds when the FC-129 is administered as an intravenous dose; and to ascertain whether perfluorooctanate will provide a marker for dermal absorption. 2.0 TEST MATERIALS_________________________________ _________________ 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 Number o f 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 of Test and Control Samples: The samples are identified using the HWI animal identification number which consists o f a letter and five digit number, plus the tissue identity and day identity (serum). 2.5 Purity and Strength of Reference Substance: To be determined by Sponsor. 2.6 Stability of 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. 000449 ) 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. 3.0 EXPERIMENTAL - Overview______________________ ____________ The tissues and serum from animals dosed as described (HWI#6329-138), are available for analysis for fluorine compounds. At the discretion of 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. For confirmation o f the presence o f specific compounds in tissues and serum, electrospray mass spectrometry can be used. 4.0 EXPERIM ENTAL - Methods______________________________ 4.1 Liver and Serum screening methods: (attached) 4.1.1 AM DT-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 o f 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 000450 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-95 (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 standard 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 o f an Orion EA940 Expandable Ion Analyzer 8.3 AMDT-M-4-0, Extraction o f Fluorochemicals from Rabbit Liver 8.4 AMDT-M-5-0, Analysis o f 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 000451 4 3M Environmental Laboratory Method Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer - Liver Method Identification Number: AMDT-M-1 Revision Number: 0 Adoption Date: / ~ Revision Date: None Author: Rich Youngblom Approved by: Quality Assurance 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 000452 1 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 tissues, particularly liver. 2.0 K EYW O RDS ____________________________________________________ _ 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. 000453 2 7.0 SAMPLE 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 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 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. 000454 3 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. 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. 000455 4 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 (Orion reading in ppm - intercept (Slope) 10.0 V A L I D A T I O N ___________________________________________________ 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 000456 5 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 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 ATTACHMENTS _____________________________________________ None 13.0 REFERENCES ____________________________________________________ 13.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer Operator's Manual (Manual 915- 349, 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___ 000457 6 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 [/ /J _________ / A / / s Date Quality Assurance / o - t - i r" Date Software: MS Word 5.1a Affected Documents: AMDT-M-1 Thermal Extraction of Fluoride by Means of a Modified Dohnnann DX2000 Organic Halide Analyzer 000458 1 1.0 SCOPE . APPLICABLE COMPOUNDS. 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 o f 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.Q SUPPLIES AND M ATERIALS______________________________________ 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 1:1 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. 000459 2 6.4 Air bubbles trapped under electrode can give erroneous readings. Make sure no air is trapped under electrode. 7.0 SAMPLE HANDLING_______________________________________________ 7.1 No special handling necessary. 8.0 CALIBRATION AND STANDARDIZATION __________________ 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. 000400 3_ 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 Data 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 Other Validation Parameters According to Reference 13.2, the range of detection is 0.02 ppm fluoride up to a saturated solution of fluoride. 11.0 DATA ANALYSTS___________________________________ _______________ 11.1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 12.0 ATTACHM ENTS_______________________________ _________________ _ None 13.0 REFERENCES_________________________________ ___ __ _________ 000461 4. 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 000462 5 3M Environmental Laboratory M ethod Extraction of Fluorochemicals 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. 000463 1.0 SCOP E -------------------------------------------------------------------- 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. 2.0 K EY W O R D S________________________ ____________________________ 2 .1 Fluorochemicals, rabbit livers, electrospray mass spectrometer, fluorinated compounds, extraction. M PR E C A U T IO N S_________________________________________ ___ 3 .1 Use gloves when handling the rabbit livers, they may contain pathogens. 4 .0 SU PPL IE S 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 p.m. 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 1:10 for a 250 ppm solution. 4 .2 .2 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker), (NaiCOj/NaHCCy 0.25 M: Weigh 26.5 g of sodium carbonate (NajCC^) 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) 000464 2 5.0 EQUIPMENT--------------------------------------------- 5 .1 Ultra-Turrax T25 Grinder for grinding liver samples. 5 .2 Vortex mixer 5 .3 Centrifuge 5 .4 Shaker 5 .5 Analytical Evaporator 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 The rabbit livers are received frozen, and must be kept frozen until the extraction is performed. & 0 C A L IB R A T IO N AND STA 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- sulphonic acid X 3mL = 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. 000465 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 506 ppb 500 ppb 5 ppb 1 PPm 5 ppm 5 PPm______ uL Kx5 20 30 4 560 2 300 Approximate final concentration of FC-95 in liver Blank 0.292 ppm 0.584 ppm 0.877 ppm 1.168 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 ppml = 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 of FC-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 perfluorooctanesulfonate 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 ini dais. 000466 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. Id Cap die 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 (im 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. 10.0 VALIDATION 1 0 .1 Quality Control - not applicable 1 0 .2 Precision and Accuracy- not applicable 1 0 .3 Other Validation Parameters- not applicable 11.0 DATA ANALYSIS________________________________________ 11.1 None 12.0 A TT A C H M EN TS___________________ 12.1 Worksheet AMDT-M-4 ______________ 13.0 R EFER E N C E S______________________________________ ___ 1 3 .1 AMDT-EP-22 Routine Maintenance of Ultra-Turrax T-25 14.0 R E V ISIO N S________________________________________________ Revision Number Reason for Change Revision Date 000467 Worksheet AMD.TrM-4 Study # _ .. _ _ _ 1 Sam ple Number set # Blank I.iver FC-95 approx 0.5 ppm actual ppm #W 100 uL 200 uL .100 u L 400 uL - _ . . _ _ _ _ _ _ _ FC-95 approx 1 ppm actual ppm #W _ _ . . 500 ul- _ _ _ . ,, . _ _ _ F C -9 5 approx. 5 ppm actual ppm #W . 200 ul. .100 u l. D ate and Initials for Std. _ _ _ _ _ 1' stu d v n u m b e r w h ere th e orig in al w o rk sh eet is located an d n lace a conv. __ _____________ I ________________ 1__________ _________ ___________________1___________________ T.iver Fxtracrinn Process- Date & Initials P inet 1 m l, o f T.iver Solution P inet 100 uT. o f 12 nnm Internal Standard Std. # V ortex 15 sec Pinet 1 m l, o f 0.25 Na-CO./O 25M N aH CO , Buffer P inet 5 m l. o f F.thvl A cetate Vortex 20-10 sec Shake 20 min. C entrifune 20-25 min R em ove a 4 mT. aliouot o f organic laver B lo w d o w n to n e a r d rv n ess f< 0 25 mLT w ith N , A dd 1 m- o f 1-1 A ceto n itrile/H -O TN# _V ortex 15 sec F ilte r u sin g a l e e R -D sy rin g e w ith a (V2um SRI filte r in to a i .5 m l. au to sam n le vial____________________________ 000468 6 3M Environmental Laboratory________________________ _ Method Analysis of Rabbit Liver Extract for Fluorochem icals using Electrospray Mass Spectroscopy SOP Identification Number: AMDT-M-5 Revision Num ber: 0 Adoption Date: - C -7 r ' Revision Date: None Author: Dave Christenson/Cynthia Weber Approved By: Software: MS Word, 6.0 Affected Documents: M-4, Extraction of Fluorochemicals from Rabbit Livers 000469 ] 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 KEYWORDS __________________________________________________ _ 2 .1 Fluorochemicals, fluorinated compounds, electrospray mass spectroscopy, mass spectrometer, rabbit livers. 3.0 PR EC 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. 4 .0 SUPPLIES AND M ATERIALS___________________________________ 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 tip 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 TE R FE R EN C E S__________________________________________________ 6 .1 There are no known interferences at this time. 7.0 SAM PLE HANDLING______________________________________________ 7 .1 Keep the extracted samples in capped 15 mL centrifuge tubes or in capped autovials until ready for analysis. 000470 2 8.0 C A L IB R A TIO N AND STA N D A R D IZA TIO 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 PR O C ED 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. 000471 34 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 10.2. lSee Method Validation Report number AMDT-M-5.0.VI 10.3 Other Validation Parameters 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. 11.2. 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 liver: 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 ED. Write the study number on the printout, initial, date, and put it in the study folder. Staple all chromatograms together and number pages. 000472 4, 12.0 ATTACHM ENTS None 13.0 REFERENCES . ------ --- . 13.1 AMDT-EP-17 14.0 R E V ISIO N S_____________________________________________ Rvision Number Reason for change Rvision Date 000473 1 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: Software: IBM MS Word, 6.0 Affected Documents: AMDT-EP-26, Operation and Maintenance of the Skalar Segmented Row Analyzer 000474 l 1.0 SCOPE____________________________________________________________ _ 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 RDS____________________________________________________ _ _ 2 .1 Skalar, segmented flow, fluoride. 3.0 PR EC A U T IO N S_____________________________________________________ 3 .1 Follow standard laboratory safety practices. 4 .0 SUPPLIES AND M ATERIALS____________ .__________ _____________ 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 - 0.5 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 000475 ? 5.0 E Q U IPM E N T ______________________________________________ __ 5 .1 Skalar Segmented Flow Auto Analyzer Sans"1" System equipped with ISE 6.0 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 PLE 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 STA 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. 000476 3 9 .2 .5 .1 Select GRAPHICS, REAL TIME. 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 .2 Use 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 . 11 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 _____________________________________________________ 10.1 Quality Control 1 0 .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. 000477 4 10.2 Precision and Accuracy 10.2. ISee Method Validation Report number AMDT-M-8.0.V1 10.3 Other Validation Parameters 10.4 Refer to Method Validation Report Number AMDT-M-8.0.V1 11.0 d a t a a n a l y s i s ________________________ _______________ _ 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. 11.2 Prepare spreadsheets to summarize data. Include sample volume, weights used etc. 11.3 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. 11.4 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 TTAC H M EN TS______________________________________ ____________ None 13.0 R EFER 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 000478 5v 9.3 Quality Assurance Unit Statement 000479 Attachment D GLP Study Quality Assurance Statement Study Title: Single-dose Intravenous Pharm acokinetic Study of T-6054 in Rabbits Study Number AMDT-122094.2 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 Jq 10/14/95 10/19/95 Phase_____________ Final Report Date Inspection Reported to M anagew n Study Director 10/19/95 10/19/95 BEST COPY AVAILABLE QAU Auditor Am v Date G004S0 l 9.4 Key Personnel Involved in the Study 000481 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 'Rich Youngblom Deann Plummer Documentation and Reporting: Jim Johnson Rich Youngblom Quality Assurance Unit: Gale Van Buskirk Cynthia Weber Kari Rambo 000482 * 9.11 Data 000483 9.11.1 Summary and raw data; ug F` in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 000484 I Summary of Combustion Data - Liver AMDT-122094.2, H W I6329-138 As Referenced in Final Report section 6.0 DATA AN ALYSIS Total ug Fluoride in Whole Liver Mean per Dose Group g Control Group 18.0 0.128 mg/kg dose (T6054) 60.5 0.64 mg/kg dose (T6054) 118 1.28 mg/kg dose (T6054) 164 12.8 mg/kg dose (T6054) 2239 000485 RPT138.XLS FC129 PK ID Liver Blank-1 Liver Blank-2 Liver Blank-3 Liver Spike-1 Liver Spike-2 F52750-1 F52750-2 F52750-3 F52793-1 F52793-2 F52793-3 F52792-1 F52792-2 F52792-3 F52751-1 F52751-2 F52751-3 F52752-1 F52752-2 F52752-3 Liver Blank-1 Liver Blank-2 Liver spike 63-1 Liver spike 63-2 Liver spike 126-1 Liver spike 126-2 Liver spike 126-3 % rcvry 97% 95% 97% 96% 95% 82% 83% Actual ppm Fin liver (W/W) 1.11 0.371 0.253 1.04 0.860 1.71 1.53 1.54 0.839 0.840 0.959 0.253 0.234 0.187 2.64 2.34 2.37 23.3 24.5 23.0 0.364 0.272 0.985 1.06 2.38 1.76 2.31 Average ppm Fin liver (W/W) 1.59 0.879 0.225 2.45 23.6 Liver burned (grams) 0.145 0.137 0.128 0.141 0.168 0.118 0.121 0.130 0.141 0.125 0.123 0.141 0.129 0.148 0.108 0.133 0.133 0.143 0.142 0.137 0.134 0.109 0.148 0.137 0.121 0.142 0.109 Whole liver weight (grams) 73.9 68.8 80.2 66.8 94.9 Total Fin whole liver (F9) 118 60.5 18.0 164 2239 Dosage (mg/kg) 0.64 0.128 0.0 1.28 12.8 Page 1 000486 < 9.11.2 Summary and raw data; analysis of liver extracts using electrospray mass spectrometry. 000487 t -o HWI# 6329-138 Study: Protocol Number: Test Material: Matrix: R Squared Value: Response Factor Amount: Analyst: Date: Method: Instrument: LA B B A SEFlIe: Single-Dose Intravenous Pharmacokinetic T P 8 0 8 4 .P K T-6054 in Rabbits (FC -129) Liver 0 .9 8 2 6 1.92EOO DLC 4 /6 /9 5 AMDT-M-4 Fisons VG 2000 Electrospray MS 040695A A-l A-l l(> 2 - ^ 5 clc Group Dose Group 1: 0 mg/kg Group 2: 0.128 mg /kg Group 3: 0.64 mg/kg Group 4: 1.28 mg/kg Group 5: 12.8 mg/kg Sam p le# F52792 Ion Count Extracted wt Dilution Ratio * 9 factor 0.0245 1.2562 1 Concentration pg/g ** Total m ass of liver 9 0.0299 80.1586 Total amount of FC-95 per liver mg 0.002 F52793 0.2346 1.3227 1 0.2722 68.8400 0.019 F52750 0.4983 1.2397 1 0.6169 73.9156 0.046 F52751 0.5016 1.2835 1 0.5998 66.7527 0.040 F52752 2.7225 1.1852 1 3.5254 94.8862 0.335 * Ratio of M499 Ion Count/M-427 Ion Count **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. 000488 I HWI# 6329-138 Cong 0.4 0.8 1.2 1.6 4 8 12 499 Ion Count 427 Ion Count m S S I.C./IW427 I.C 9091 74303 0.1224 63720 112333 0.5672 117051 183223 342495 654597 774077 146156 145956 135711 157098 128461 0.8009 1.2553 2.5237 4.1668 6.0258 Sample # 499 Ion Count 427 Ion Count M"499 I.C./M-427 I.C. F52792 F52793 F52750 F52751 F52752 2399 12826 41969 41908 256267 97837 54664 84226 83555 94129 0.0245 0.2346 0.4983 0.5016 2.7225 000489 t 6.0258 A R E A 4.1668 M e t h o d C :D L C L I V E 1 Sample Operator Run date 07-26-1995 16:12:19 version: 35 Printed on 07-26-1995 AT 16:12:32 Straight Line Fit forced through Origin. Component tt'.l 2.537 1.2553 .8009 .5672 .1224 LEVEL AMOUNT .8 1.6 AMOUNT Component 1 -- EXTERNAL STANDARD CALIBRATION AREA 8 1 0.4000 2 0.8000 3 1.2000 4 1.6000 5 4.0000 6 8.0000 7 12.0000 0 1 1 1 ---- 3 4 6 MT Y SLOPE X + INTERCEPT Area = 5.2126E-01 * Amount Amount = 1.9184E+00 * Area R squared = 0.9826 + 0. O O O O E + O O + 0.OOOOE+OO Are*-/ /t_c m A- r&s_ l0/<*/*S Dct 000490 i -Ir > V A q t3 2 R - ,a | CF^ - '^ 5 ) |3L ( f c -'S')} 000491 A-5 000492 Mr * <*** ( t c - i v i ) Fi le!040695A LAB-BASE - The MS Data System 06/04/1995 08:14 j(j) > J? JZ <* 000493 ! *" h2 3 F ile:040695A LAB-BASE - The MS Data System Sample:H1IT tt CUBO-tGGrCmpd 610I W WD io\%lx> s 06/04/1995 08:14 000494 000495 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. 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. 000496 RE: 6329-138 LIUER SAMPLES RMDT 122094.2 Date of Analysis: 4-20-95 Analyst: DDLU The samples are burned in the Dohrman at 950 C using betmeen 0.1 and 0.2 grams of the liuer. The gas is collected in 1.0 mL of 1:1 TISRB/Milli-Q Luaterthen an additional 1 mL of 1:1 TISRB/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 w as plotted using the inuerse logarithm option. The standard curue for the low range is linear. All standards and samples w ere then calculated by the Skalar softw are using these curues. Rll 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 Eucel spreadsheet. The final results are adjusted for the collection uolume and any subsequent dilutions. 000497 SUMMARY O F 6329-138 LIVER SAM PLES AMDT 122094.2 000498 GROUP 1 Dose Level :0 F52792-1 F52792-2 F52792-3 GROUP2 F52793-1 Dose Level :0.128 mg/kg F52793-2 F52793-3 GROUP3 Dose Level :0.64mg/kg F52750-1 F52750-2 F52750-3 GROUP4 Dose Level :1.28 mg/kg F52751-1 F52751-2 F52751-3 GROUP5 Dose Level :12.8 mg/kg F52752-1 F52752-2 F52752-3 0.02 ND ND 0.07 0.06 0.06 0.11 0.11 0.11 0.16 0.17 0.17 0.42 0.76 0.73 2.0 0.1409 0.27 2.0 0.1287 N D 2.0 0.1481 N D 80.1586 22 ND 80.1586 N D 80.1586 N D 2.0 0.1410 1.00 2.0 0.1253 0.99 2.0 0.1229 1.01 68.8400 1.00 68.8400 68.8400 69 68 70 2.0 0.1176 1.95 2.0 0.1206 1.85 2.0 0.1303 1.73 73.9156 1.84 73.9156 73.9156 144 137 128 2.0 0.1078 3.02 2.0 0.1332 2.58 2.0 0.1328 2.62 66.7527 202 2.74 66.7527 172 66.7527 175 2.0 0.1429 5.91 94.8862 560 2.0 0.1419 10.67 9.05 94.8862 1012 2.0 0.1370 10.58 94.8862 1004 ND 69 136 183 859 K S T COPYAVAILABLE 138LIVER.SUM Page 1 S b ( U f> C T K \ 1995-07-06 15:57 OutPut of:950420A1 Operator :D D W Date oftheAnalysis :1995-04-20 07:12 AnalysisFileName :C:\SKALAR\DATA\HWIDATA\LIVERS\950420A1 138LIVER.XLS 000499 1 Tracer 1.50 1.46 97% 2 Drift 1.50 1.47 98% 3 Wash ND 4 Standard 1 0.015 0.015 98% 5 Standard2 0.03 0.03 101% 6 Standard 3 0.06 0.06 101% 7 Standard4 0.09 0.09 100% 8 Standard 5 0.12 0.12 99% 9 Standard6 0.15 0.15 100% BiEST COPY AVAILABLE 10 Standard 7 0.30 0.28 94% 11 Standard8 0.60 0.61 102% 12 Standard 9 1.20 1.23 103% 13 Standard 10 1.50 1.47 98% 14 Drift 1.50 1.48 99% 15 Wash ND 16 BLK-1 0.10 2.0 0.1445 1.36 17 BLK-2 0.03 2.0 0.1365 0.48 18 SPK-1 < 19 SPK-2 20 50-1 0.09 0.09 0.11 2.0 0.1410 1.26 0.004 63.00 2.0 0.1677 1.02 0.004 63.00 2.0 0.1176 1.95 73.9156 144.06 21 50-2 0.11 2.0 0.1206 1.85 73.9156 136.68 22 50-3 23 93-1 0.11 2.0 0.1303 1.73 73.9156 128.20 0.07 2.0 0.1410 1.00 68.8400 68.55 24 93-2 0.06 2.0 0.1253 0.99 68.8400 68.46 25 93-3 0.06 2.0 0.1229 1.01 68.8400 69.57 26 Drift 1.50 1.47 98% 0.15 0.18 0.15 0.17 118% 113% 27 Wash ND 28 92-1 29 92-2 30 92-3 31 51-1 0.02 2.0 0.1409 0.27 80.1586 21.73 ND 2.0 0.1287 ND 80.1586 N D ND 2.0 0.1481 ND 80.1586 ND 0.16 2.0 0.1078 3.02 66.7527 201.87 lo. Page 1 32 51-2 0.17 2.0 33 51-3 0.17 2.0 34 52-1 0.42 2.0 35 52-2 0.76 2.0 36 52-3 0.73 2.0 37 BLK-1 0.04 2.0 38 Drill 1.50 1.51 100% 39 Wash ND 40 BLK-2 0.02 2.0 41 SPK-63-1 0.09 2.0 42 SPK-63-2 0.09 2.0 43 SPK 126-1 0.15 2.0 44 SPK 126-2 0.14 2.0 45 SPK 126-3 0.14 2.0 46 Drift 1.50 1.50 100% 47 Wash ND 000500 138LIVER.XLS 0.1332 0.1328 0.1429 0.1419 0.1370 0.1334 2.58 2.62 5.91 10.67 10.58 0.55 66.7527 66.7527 94.8862 94.8862 94.8862 172.39 174.92 560.42 1012.39 1004.27 0.1092 0.1484 0.1370 0.1212 0.1439 0.1085 0.44 1.21 1.32 2.40 1.91 2.53 0.004 0.004 0.004 0.004 0.004 63.00 63.00 126.00 126.00 126.00 0.15 0.18 0.15 0.18 0.30 0.29 0.30 0.28 0.30 0.27 119% 119% 96% 91% 91% BEST COPY AVAILABLE Page 2 1995-04-20 09:36 OutPut of : 950420A1 Software : version 6.1 cl990,93 Operator : DDW Date of the Analysis : 1995-04-20 07:12 Analysis File Name : C:\SKALAR\DATA\HWIDATA\LIVERS\950420A1 A ^ b T 12.2.0 **.1, Hcox t3 z q -j-ss Fluoride 1.5 Calibration order = Inverse Logarithm Slope : s = #.##### Result x - cl s] x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 = al = aO = -0.00000 0.00065 -1.24984 Fluoride L Calibration order = 2 Correlation : r = 0.99991 Result = a2 * x2 + al * x + aO a2 = al = aO = 0.00000 0.00020 0.00010 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 000501 I 1995-04-20 09:36 OutPut of : 950420A1 Fluoride 1. Path number : 3 Signal type : Debubbled Decolor : Yes system Number : 0 diLute : No Resample : No dil Threshold : 4095 diG output :0 Window event : 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 000502 1995-04-20 09:36 Output of : 950420A1 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 : c4 :=c3 output : #. #### 000503 1995-04-20 09:35 OutPut of : 950420A1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time Ch Result F Time wt iw Initial Wash 3 0.056 1t Tracer 3 1.457 2d Drift 3 1.470 3w Wash 3 0.056 4 si Standard 1 3 0.063 5 s2 Standard 2 3 0.070 6 S3 Standard 3 3 0.087 7 s4 Standard 4 3 0.106 8 s5 Standard 5 3 0.128 9 s6 Standard 6 3 0.155 10 s7 Standard 7 3 0.282 11 s8 Standard 8 3 0.614 12 s9 Standard 9 3 1.232 13 slO Standard 10 3 1.467 14 d Drift 3 1.483 15 w Wash 3 0.056 16 u BLK-1 3 0.112 17 u BLK-2 3 0.071 18 u SPK-1 3 0.105 19 u SPK-2 3 0.103 20 u 50-1 3 0.124 21 u 50-2 3 0.122 22 u 50-3 3 0.123 23 u 93-1 3 0.093 24 u 93-2 3 0.088 25 u 93-3 3 0.088 26 d Drift 3 1.467 27 w Wash 3 0.056 28 u 92-1 3 0.065 29 u 92-2 3 0.063 30 u 92-3 3 0.061 31 u 51-1 3 0.163 32 u 51-2 3 0.172 33 u 51-3 3 0.174 34 u 52-1 3 0.422 35 u 52-2 3 0.757 36 u 52-3 3 0.725 37 u BLK-1 3 0.074 38 d Drift 3 1.507 39 w Wash 3 0.056 40 u BLK-2 3 0.067 41 u SPK-63-1 3 0.106 42 u SPK-63-2 3 0.106 43 u SPK 126-1 3 0.151 44 u SPK 126-2 3 0.143 45 u SPK 126-3 3 0.143 46 d Drift 3 1.495 47 w Wash 3 0.056 wt rw RunOut Wash 3 0.056 65 210 384 626 733 907 1085 1261 1436 1611 1785 1959 2135 2311 2485 2727 2836 3011 3187 3363 3538 3714 3886 4060 4238 4414 4588 4740 4936 5111 5285 5463 5637 5813 5989 6163 6339 6515 6689 6930 7034 7214 7390 7566 7740 7914 8090 8324 8565 4 0.0001 4 0.7942 4 0.8006 4 0.0001 4 0.0147 4 0.0302 4 0.0603 4 0.0904 4 0.1190 4 0.1505 4 0.2602 4 0.4429 4 0.6965 4 0.7988 4 0.8066 4 0.0001 4 0.0986 4 0.0326 4 0.0889 4 0.0857 4 0.1146 4 0.1115 4 0.1130 4 0.0702 4 0.0623 4 0.0621 4 0.7988 4 0.0001 4 0.0191 4 0.0143 4 0.0117 4 0.1586 4 0.1686 4 0.1705 4 0.3468 4 0.5053 4 0.4918 4 0.0368 4 0.8183 4 0.0001 4 0.0238 4 0.0898 4 0.0902. 4 0.1456 4 0.1375 4 0.1371 4 0.8123 4 0.0001 4 0.0001 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 1 000504 < BEST COPY AVAILABLE Calibration curue of 950420A1 : Fluoride L ------------------ 1 Channel .a Measured: 0.1842330 6 0.0012M8i A! / / C 'H0 /Y V. H M c / // / a4 (J c / 0 0 / / // / / /f / T, / / / 0.0001048 - 0 Order : 2 / Measured iii r : 0.99931 900 000505 1.7262884 "FST COPY AVAILABLE C a lib ra tio n cu rve o f 95042001 Measured- K : F lu o rid e 1 .5 O .O Sg^SgPli^tileT 0.0562547 jir Order Measured Inverse Logarithm 6205 4095 000506 BEST COPY AVAILABLE 000507 BEST COPY AVAILABLE 409b Raw d a t a o f 9 5 0 4 2 0 A 1 : F l u o r i d e _________ 1Pathno:-- 3--- -,T-iae- { I- - ------ 1 .5 ' -- i i i umi i i H iiiiiiiH W i-- M B -- M B fililH ifM M H fW ! I O& A l vyvj t^ n /' "1 7 \I I I !\ 0 1373 T itte Esc=Exit 1 Fl=Help i Crtl-P=Edit peaks 1 3998 000508 4U9b I Raw d a t a o f 9 5 0 4 2 0 A 1 : F l u o r i d e 1 . 5 3998 flffsa 252 BEST COPY AVAHADLE iI M3 3 A aj .A A A /\ / \ 'J Vj \\ A & -V* A i\ n /I Is f i, U 1U VJ VJ 3998 T irte Esc=Exit i Fl=Help ! Crtl-P=Edit peaks V/\ V\ 6G23 000S09 ( iI i 4035 ! 1 ~~ Vs i Raw d a t a o f 9 5 0 4 2 0 A 1 : F l u o r i d e 1 . 5 BEST COPTIAVAILABLE I! 11 3 I 5- T ine Ese=Exit i Fl=Help ! Crtl-P=Edit peaks ! j 9248 000510