Document QM4NegreReRMbgOLkarj3XmJ6

A l W - o 157 3M Environmental Laboratory__________________________ Final Report- Analytical Study Single-Dose Intravenous Pharmacokinetic Study of T-6053 in Rabbits In-Vivo Study Reference Number: HWI#6329-136 Study Number: AMDT-010495.1 Test Substance: FC-99 (T-6053) Name and Address of 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: AMDT-M-1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AMDT-M-2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer AMDT-M-4-0, Extraction o f Fluorochemicals from Rabbit Liver AMDT-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 Approved By: o 07 1.0 SUMMARY Rabbit liver were analyzed by combustion for fluorine content at 48 hours after individual rabbits were dosed intravenously with a range o f single doses o f FC-99 (one rabbit/dose group). The fluorine was detected after a single dose o f 20 ug/kg, but not after a 0.8, 4, 10 ug/kg dose at 48 hours postdose. Doses are calculated with respect to the C8 solids. The perfluorooctanesulfonate salt form o f FC-99 does not change the availability o f total organic fluorine in whole liver after intravenous injection. The analysis o f liver for total organic fluorine will provide a convenient marker to assess the dermal absorption o f FC-99. 2.0 INTRODUCTION____________________________________________________ This study was designed to provide information as to whether the perfluoro octanesulfonate anion goes to the liver and other tissues after a dose o f T-6053 (a 0.04% solution o f FC-99 solids) is administered in an intravenous dose; and to ascertain the change in concentration with time after dose in serum and liver. FC-99 is a solution o f perfluoroalkylsulfonates: perflourooctanesulfonate ( about 20%), C4 - C7 perfluoroalkylsulfonates (about 5%), and water (75%). The sulfonates are as the diethanolamine salt. It is known from studies done previously with rats, that the half-life o f perfluoro octanesulfonate anion is quite long (>1 month). It was shown in a previous study that the half-life o f perfluorooctanesulfonate is >1 month in rabbits after an intravenous dose (HWI#6329-159). It is not expected that the half-life in rabbits would be different with just the salt form o f the perfluorooctanesulfonate changed, as it is comparing FC-95 (potassium salt) with FC-99. It was noted in the previous intravenous study with the perfluorooctanesulfonate that the serum levels lagged and even decreased after the injection and then returned to a maximum at 48 hours. It was clear from the 28 day study (HWI#6329-159) that the perfluorooctane sulfonate would provide a marker for dermal studies for which there was reasonable expectation that perfluorooctanesulfonate would be formed as a biotransformation product. It will therefore also be a useful marker for the assessment o f the dermal absorption o f FC-99. 003765 000002 2 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. 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. 4.0 EX PER IM EN TA L -O verview _____________________________ Serum and tissues from animals dosed as described (HWI#6329-136) were available for analysis for fluorine compounds. Since perfluorooctanesulfonate anion is not biotransformed, the analysis was accomplished with combustion and subsequent analysis for fluorine. The fluorine data are related directly to perfluorooctanesulfonate concentration. The fluorine analysis o f serum collected at different time intervals after dosing provides data which can be interpreted pharmacokinetically. The data are to provide evidence that perfluorooctanesulfonate and/or total organic fluorine in liver can be used to assess dermal absorption o f FC-99. 5.0 EXPERIMENTAL - METHODS_______________________________________ 5.1 AM DT-M -1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 5.2 AMDT-M-2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer 0037G B 000003 3 5.3 AMDT-M-4rO, Extraction o f Fluorochemicals from Rabbit Liver 5.4 AMDT-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 from combustion analysis o f liver for total organic fluorine are attached. The liver at 48 hours post dose indicates that only the 250 mg/kg dose o f T-6053 resulted in significant total organic fluorine in the liver (35 ug in total liver). This is a 0.04% solution o f an aqueous solution in which the perfluorooctanesulfonate salt is about 20%. This corresponds to a dose in terms o f perfluorooctanesulfonate salt as about 0.02 mg/kg. (The control value is estimated to be about 15 ug total in the whole liver, but the value is below the quantitation limit and is extrapolated from below the calibration o f the instrument.) This result is as expected from a previous study in rabbits when perfluorooctanesulfonate as the potassium salt was injected and only doses above 0.6 mg/kg resulted in significant amounts o f total organic fluorine in the liver. There is enough o f the perfluorooctanesulfonate present to indicate that if appreciable amounts o f FC-99 are absorbed in a dermal absorption study, it will be observed. Other data was collected using electrospray mass spectrometry and 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 there is too short a time period used for the study. In other studies, it has been observed that there is a lag in serum concentrations until 48 hours. There didn't appear to be any advantage in doing serums for this study, since the time period was too short and in view o f the results from study HWI#6329-159. 003767 OOOOOl 4 7,0 CONCLUSION The perfluorooctanesulfonate salt form (diethanolamine) o f FC-99 does not change the availability o f total organic fluorine in whole liver after intravenous injection. The analysis o f liver for total organic fluorine will provide a convenient marker to assess the dermal absorption o f FC-99. However a larger dose will be required. 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. 9J) APPENDICES_________________________________________________________________ 9.1 Protocol and Amendments 9.1.1 Protocol and Final Report: HWI#6329-136 "Single-Dose Intravenous Pharmacokinetic Study o f T-6053 in Rabbits" (Protocol type TP8084.PK for dosing o f animals, tissue collection, etc.) 9.1.2 Analytical protocol AMDT-010495.1 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-010495.1 9.10 Instrument Settings: See methods 003768 00000,7 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. 003769 000006 6 9.1.1 Protocol and Final Report: HWI#6329-136 "Single-Dose Intravenous Pharmacokinetic Study of T-6053 in Rabbits" (Protocol type TP8084.PK for dosing of animals, tissue collection, etc.) 003770 000007 HAZLETON WISCONSIN POST OFFICE BOX 7545 MADI SON. Wl 5 3 707- 7 545 Sponsor: 3M St. Paul, Minnesota .) C O R N IN G Company FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6053 in Rabbits Author: Steven M. Glaza Study Completion Date: March 31, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Pro.iect Identification: HWI 6329-136 003771 Page I of 25 Phone 608-241-4471 EXPRESS-MAIL DELIVERY: 3301 KINSMAN BLVD Fax . MADISON. OOOOOS 6 0 8 - 2 4 1 -7 2 2 7 Wl 53 7 0 4 Page 2 of 25 QUALITY ASSURANCE STATEMENT HWI 6329-136 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 Date Reported to Date to _________ Phase_________ Study Director Management 12/09/94 12/27/94 02/15/95 03/30/95 12/09/94 Protocol Review 12/27/94 Sample Collection 02/15/95 Data/Report Review 03/30/95 Report Rereview 12/09/94 12/27/94 02/15/95 03/30/95 01/10/95 01/10/95 03/10/95 04/10/95 Cecilia M. Danner Representative, Quality Assurance Unit Date 003772 OOOOOii Page 3 of 25 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study of T-6053 in Rabbits HWI 6329-136 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Experimental/In-1ife Start Date Experimental/In-life Termination Date T-6053 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 27, 1994 December 29, 1994 003773 OOOOK) Page 4 of 25 KEY PERSONNEL HWI 6329-136 Acute Toxicology Steven M. Glaza Study Director Manager Laboratory Animal Medicine 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 003774 000011 Page 5 of 25 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compli ance Test and Control Materials Test System Procedures Results Discussion Signature Table 1 Individual Body Weights (g) 2 Individual Clinical Signs Appendix A Protocol Deviations Protocol TP8084.PK HWI 6329-136 Page 2 3 4 6 j 7 7 8 9 11 11 11 12 13 14 15 16 003775 OOOOlii Page 6 of 25 HWI 6329-136 SUMMARY This study was done to assess the level of systemic exposure of T-6053 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 10, 50, 125, or 250 mg of T-6053/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 in-life 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. 003776 000013 Page 7 of 25 HWI 6329-136 OBJECTIVE The objective of this study was to assess the level of systemic exposure to the test material, T-6053, 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-6053 and described as a clear, colorless liquid. The control material was Sterile Water for Injection, USP (Abbott Laboratories, Lot No. 86-748-DM-02; Exp. March 1, 1996), and was described as a clear, colorless liquid. Purity and Stability The Sponsor assumes responsibility for test material purity and stability determinations (including under test conditions). A sample of the test material/vehicle mixtures for concentration, solubility, homogeneity, and stability analyses was not taken before administration as this was not requested by the Sponsor. The purity and stability of the USP grade control material were considered to be adequate for the purposes of this study. Storage and Retention The test material was stored at room temperature. The control material was stored refrigerated. Any unused test material was returned to the Sponsor after completion of all in-life 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. 003777 000014 Page 8 of 25 Safety Precautions HWI 5329-136 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 30, 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 23C, a relative humidity of 50% 20%, and a 12-hour 1ight/12-hour dark lighting cycle. In cases where variations from these conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad l ib it u m 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. 003778 000017 Page 9 of 25 Study Design HWI 6329-136 Female animals weighing from 2,572 to 2,836 g at initiation of treatment were placed into the following study groups: GrouD 1 (Control) 2 3 4 5 Treatment * T-6053 T-6053 T-6053 T-6053 Dose Level imo T-6053/kal 0 10 50 125 250 Dose Volume (mL/kq) 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 seconds to 2.5 minutes 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. 003W9 Body weights were determined just before test material administration (Day 0). ooooio Page 10 of 25 Sample Collection HWI 6329-136 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 -20C 10C until shipped to the Sponsor. Pathology At termination of the in-life 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 placed in a freezer set to maintain a temperature of -20C 10C.' 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. 003750 000017 Page 11 of 25 RESULTS Body Weights Individual body weights at initiation are in Table 1. HWI 6329-136 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 in-life phase and were not examined grossly when sacrificed. DISCUSSION The level of systemic exposure of T-6053 was evaluated in female albino rabbits when administered as a single intravenous injection at levels of 0, 10, 50, 125, and 250 mg/kg. All animals appeared normal throughout the study following administration of this material. SIGNATURE Steven M. Glaza Study Director Acute Toxicology Date 003781 000018 GrouD 1 2 3 4 5 Page 12 of 25 Table 1 Individual Body Weights (g) Dose Level (mq/kqV Sex Animal Number 0 Female F52856 10 Female F52857 50 Female F52861 125 Female F52862 250 Female F52863 Initial 2,833 2,802 2,572 2,746 2,836 HWI 6329-136 003782 000013 GrouD Dose Level (mq/kq) 10 Sex Female Page 13 of 25 Table 2 Individual Clinical Signs HWI 6329-136 Animal Number F52856 Observation Appeared normal Hour 0.5 2 4 24 48 / 2 10 Female F52857 Appeared normal / 3 50 Female F52861 Appeared normal / / / / 4 125 Female F52862 Appeared normal / / / 5 250 Female F52863 Appeared normal / / / / / / Condition existed. 037S3 OOOO^O Page 14 of 25 APPENDIX A Protocol Deviations Protocol TP8084.PK HWI 6329-136 0Q37S4 OOOCtl Page 15 of 25 Protocol Deviations HWI 6329-135 ___________ Protocol___________ Page 8, 7. Experimental Design, C. Dosing Procedures, (1) Dosing Route. Intravenous injection into a marginal ear vein over approximately 30 to 60 seconds. ________ Actual Procedure Animal Nos. F52856 (Group 1), F52861 (Group 3), and F52862 (Group 4), had a dosing duration of 157, 91, and 99 seconds, respectively. These deviations are not considered to have had an adverse effect on the outcome of the study. 0037S5 000033 HAZL.ETOIM WISCONSIN P OS T OFFI CE BOX 7 5 4 5 M A D I S O N . Wl 5 .1 / 0 / / 5 'I 5 Page 16 of 25 a C O R N IN G Company Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.PK Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6053 in Rabbits Date: December 13, 1994 Performing Laboratory. Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-136 G037S6 P h o n e 6 0 8 2 4 I 4 4 7 t EXPRESS MAIL DELIVERY 3101 KINSMAN BLVD Fax 608 2 4 1 / 2 2 / MADISON Wl 53704 0000*23 STUDY IDENTIFICATION TP8084.PK Page 2 Single-Dose Intravenous Pharmacokinetic Study of T-6053 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 6329-136 T-6053 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 27, 1994 December 29, 1994 February 2, 1995 003787 OOOO24 Page 18 of 25 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-6053 B. Physical Description (To be documented in the raw data) C . Purity and Stability The Sponsor assume? 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. C03*7S8 oooo^rj Page 19 of 25 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. 0037S9 (3) Aae at Initiation Adult ) 0000% Page 20 of 25 (4) Weight at Initiation 2.5 to 3.5 kg TP8084.PK Page 5 (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 (PHI 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 1ight/12-hour dark cycle. (f) Acclimation At least 7 days / (8) Selection of Test Animals Based on health and body weight according to HWI SOPs. An adequate number of extra animals will be purchased so that no animal in obviously poor health is placed on test. (9) Justification for Soecies Selection Historically, the New Zealand White albino rabbit has been 0 0 3 V 9 0 the animal of choice because of the large amount of background information on this species. ooooa? Page 21 of 25 TP8084.PK Page 6 Dose Administration ill Test Groups Group Dose Level (mq/kgl* Number of Females 1 0 (Control) 1 2 10 1 3 50 1 4 125 1 5 250 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 003791 (a) Frequency 2, 4, 6, 8, 12, 24, and 48 hours post-injection GOGOLS Page 22 of 25 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. 003792 ) 000029 Page 23 of 25 TP8084.PK Page 8 (2) Scheduled 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) 003793 000030 Page 24 of 25 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 003794 000031 Page 25 of 25 TP8084.PK 1 Page 10 PROTOCOL APPROVAL John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department Date Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. ) Representative Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-136.protdsk2) Date Date C03795 ; oooo;jy 9.1.2 Analytical protocol A M D T -010495.1 003796 000033 3M Environmental Laboratory_____________________ ___ Protocol - Analytical Study Single-Dose Intravenous Pharmacokinetic Study of T-6053 in Rabbits In-Vivo Study Reference Number: HWI#6329-136 Study Number: AMDT-010495.1 Test Substance: FC-99 (T-6053) 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 o f Rabbit Liver Extract for Fluorochmicals Using Electrospray Mass Spectrometry AM DT-M -8-0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode Author: James D. Johnson Approved By: /j m e s D. Jdfmson [ / Study Director /fer Date John Butenhoff, PhD Sponsor Representative Date 003797 000034 i LO PURPOSE To assess the level o f systemic exposure when T-6053 is administered as a single intravenous injection to rabbits. This study is designed to provide information as to whether the perfluorooctanesulfonate anion does go to the rabbit liver and other tissues when the material is administered in an intravenous dose and to ascertain the change in concentration with time after dose in serum and liver. 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 o f Materials: 3M ICP/PCP Division (2.1.1), grocery store (2.1.2, 2.1.4 liver), Sigma Chemical Company (2.1.2, 2.1.4 serum) 2.3 Num ber 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 o f 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 o f Reference Substance: To be determined by Sponsor. 2.7 Storage Conditions for Test Materials: Room temperature (2.1.1), -20 10C (2.1.2, 2.1.4). Test and Control samples will be received according to AMDT-S-10-0. 2.8 Disposition 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 dermal absorption study so all tissues will be retained. 0 3 /SB 000035 2 2.9 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting the samples. 3.0 EXPERIM ENTAL - Overview________________________________________ The tissues from animals dosed as described (HWI#6329-136), are available for analysis for fluorine compounds. At the discretion o f the Study Director, a series o f analytical tests can be performed. The screening for fluoride in liver via combustion (See M ethods-next Section) is the appropriate analysis to present definitive data for fluorine in the liver. Since perfluorooctanesulfonate anion is not biotransformed, fluorine content w ill be an accurate estimate o f the concentration o f the compound. 4.Q EX PER IM EN TA L -M ethods_________________________________________ 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 AM DT-M -2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer 4.1.3 AM DT-M -4-0, Extraction o f Fluorochemicals from Rabbit Liver 4.1.4 AM DT-M -5-0, Analysis o f Rabbit Liver Extract for Fluorochmicals Using Electrospray Mass Spectrometry 4.1.5 AM DT-M -8-0, Analysis o f Fluoride Using the Skalar Segmented Analyzer with Ion Selective Electrode 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 of tissue or fluid. Statistics used, at the discretion o f the Study Director, may include regression analysis o f serum concentrations with time and averages and standard deviations o f concentrations for different dose groups. If necessary, simple statistical tests such as the Student's t test may be applied to determine statistical difference. 003799 000030 3 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 AM DT-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 AM DT-M -4-0, Extraction o f Fluorochemicals from Rabbit Liver 8.4 AMDT-M-5-0, Analysis o f Rabbit Liver Extract for Fluorochmicals Using Electrospray Mass Spectrometry 8.5 AM DT-M -8-0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode C03800 0000374 3M Environmental Laboratory Method Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer - Liver M ethod Identification Number: AMDT-M-1 Revision Number: 0 Adoption Date: Revision Date: None Author: Rich Youngblom Approved by: Software: MS Word 5.la 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 003801 1 000038 1.-0 SCOPE , APPLICABLE COMPOUNDS. AND M ATRfCES 1.1 Scope: This method is for the operation of a Dohrmann DX2000 when it is used to extract fluoride from various matrices. The fluoride is typically collected in TISAB solution for analysis with an ion selective electrode. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Biological tissues, particularly liver. 2.0 KEYW ORDS____________________________________________________ 2.1 Fluoride, fluorine, extraction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS____________________________________________________ 3.1 Glassware and exhaust gases can be extremely hot. 3.2 Glassware is fragile, broken glass may cause injuries. 3.3 Pressurized gases, proper compressed gas handling practices required. 3.4 Solvent based samples may flash, may need to allow them to dry down before starting run. 3.5 Potential biohazards due to the biological matrices. Use appropriate personal protective equipment. 4.0 SUPPLIES AND MATERIALS_________________________________________ 4.1 Compressed Oxygen, Hydrocarbon free, regulated to 30 PSI. 4.2 Compressed Helium, High Purity Grade, regulated to 45 PSI. 4.3 Quartz glass sample boat with TeflonTM tubing, Dohrmann 890-097 or equivalent. 4.4 Quartz glass combustion tube, Reliance Glass G-9405-012 or equivalent. 4.5 Orion 940999 Total Ionic Strength Adjustment Buffer (TISAB I I ) or equivalent. 4.6 Sample collection vials, HDPE. 4.7 Milli-QTM water 4.8 Polystyrene pipettes. 4.9 Activated Charcoal, E. Merck 2005 or equivalent. 4.10 Hamilton Syringe or equivalent. 4.11 Miscellaneous laboratory glassware 5.0 EQUIPM 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. 003S02 2 000030 7.0 SAM PLE HANDLING 7.1 Samples are not to be handled with bare hands. Fluoride may leach from the skin to the sample. Use forceps or probe to transfer tissues. 7.2 Samples of liver are cut from frozen liver and placed in a tared and labeled weigh boat. Use a clean scalpel and cutting board. The cutting board and scalpel should be cleaned with water, methanol, or methanol-water solution after each liver is cut. 8.0 CALIBRATION AND STANDARDIZATION_______________________ __ 8.1 Preparation of Calibration Standards 8.1.1 The standards required for each project will need to be appropriate for that individual project. Refer to protocol for that project. 8.1.2 Typically 50-500 ppm FC-95 in methanol standards are used. 8.1.3 For rabbit liver studies, use beef liver as the matrix. Cut a piece of frozen beef liver (100 150 mg) and weigh it in a labeled and tared weigh boat. 8.2 Calibration - Overview The normal calibration is the fluoride curve (AMDT-M-2). However, if an optional spiked liver curve is required the procedure listed below is used. 8.2.1 A calibration curve for the DX2000 is generated by spiking samples with known standards and combusting them using the same methods and matrix type as the samples to be tested. 8.2.2 Typically, three replicates of each standard and five concentrations of standards will be spiked. 8.2.3 Standard curve will be plotted as Mass Spiked F (ug) on the x-axis and Standard Mass Recovered F (ug) on the y-axis. Generate a regression curve and calculate the equation for the line and the r^ value. 8.2.4 Mass Spiked F (ug) = (Amount spiked in mL) x ( Cone, of standard in ppm) x (0.6004)* *FC-95 is 60.04% F therefore 0.6004 is the factor used to convert FC-95 to F 8.2.5 Standard Mass Recovered F (ug) = (TISAB volume in mL) x (Orion reading in ppm) 8.3 Calibration - Procedure 8.3.1 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. 003S03 30 0 0 0 4 0 83.2.4 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 P-. 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. 003804 4 000041 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 VALIDATION_______________________________________________________ 10.1 Quality Control 10.1.1 Daily Start Up Check Samples: Once the standard curve is established, each day of analysis is started by analyzing QC samples. The QC samples are to be the same as the lowest concentration spiked samples used to generate the standard curve. Each concentration must be done in triplicate unless the first two replicates are within 20% of the standard curve, then a third replicate is not necessary. 10.2 Precision and Accuracy: See method development analysis and sample analysis in Fluoride Notebooks 2,3, and 5. Precision and accuracy varies when analyzing samples of different matrices and different reference compounds. 10.3 O ther Validation Parameters: NA 003S05 5 00004:2 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 ATTACHM ENTS___________________________________________________ None 13.0 REFERENCES____________________________________________________ 13.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer Operator's Manual (Manual 915349, revision B, December 1993) 13.2 AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 13.3 AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 REVISIONS _________________________________________________ _ Revision Number Reason for Change Revision Date___ 003S06 6 000043 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 L/ /J -- Date /it Quality Assurance Date Software: MS Word 5.1a Affected Documents: AMDT-M-1 Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer 003807 1 000044 1.0 SCOPE . APPLICABLE COM POUNDS. AND M A TR IC ES 1.1 SCOPE: This method is for the calibration and operation of an Orion EA940 Expandable Ion Analyzer. 1.2 APPLICABLE COMPOUNDS: Fluoride. 1.3 APPLICABLE MATRICES: Liquid samples in an appropriate buffer solution. Preferred pH of 6.0. 2.0 KEYW ORDS____________________________________ _____________________ 2.1 Fluoride, fluorine, ion selective electrode 3.0 PRECAUTIONS_______________________________________________________ 3.1 No hazards identified with this method. 4.0 SUPPLIES AND MATERIALS_________________________________________ 4.1 Orion 940999 Total Ionic Strength Adjustment Buffer II (TISABII) or equivalent. 4.2 Orion Model 900001 electrode filling solution (AgCl) or equivalent. 4.3 Orion 940907 100 ppm fluoride standard or equivalent. 4.4 Milli-QTM water or equivalent. 4.5 Magnetic stir bars. 4.6 Lab tissues. 4.7 Sample collection vials. 4.8 Plastic 100 mL volumetric flasks. 4.9 Polystyrene pipettes. 4.10 Miscellaneous laboratory glassware. 5.0 EQUIPM ENT_________________________________________________________ 5.1 Orion Model EA940 Expandable Ion Analyzer or equivalent. 5.2 Orion Model 960900 Solid State Combination Fluoride electrode or equivalent. 5.3 Magnetic Stir Plate. 5.4 IBM compatible 386 or 486 computer (only needed if using Orion 3E software). 5.5 Orion RS232 interface cable (only needed if using Orion 3E software). 5.6 Microsoft Excel 5.0 (only needed if using Orion 3E software). 6.0 INTERFERENCES____________________________________________________ 6.1 It is recommended that the pH be at or near 6.0. A 1:1 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. 003S0S 2 000045 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 J Pipette 0.05,0.1, 0.5, 1.0, and 1.5 m L 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. 003S09 3 000040 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 necessaty 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 o f 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 Param eters According to Reference 13.2, the range of detection is 0.02 ppm fluoride up to a saturated solution of fluoride. 11.0 DATA ANALYSIS___________________________________________________ 11.1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 12.0 ATTACHM ENTS____________________________________________________ None 13.0 REFERENCES_______________________________________________________ 003810 4 000047 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 C03S H 5 00004*3 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: 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 003812 00004 0 l I tO-SCOPE_____________________________________________ 1.1 Scope: This method is for the analysis of extracts of rabbit liver or other tissues or fluids for fluorochemicals using the electrospray mass spectrometer. The analysis is performed by single ion monitoring of FC-95 anion, M/Z= 499, the internal standard M/Z = 427, and other appropriate masses. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1 .3 Matrices: Rabbit Livers (samples), Beef Liver (standards), other tissues and fluids. 2.0 K EYW O RDS____________________________________________________ ___ 2 .1 Fluorochemicals, fluorinated compounds, electrospray mass spectroscopy, mass spectrometer, rabbit livers. 3.0 PRECAUTIONS______________________________________________________________ 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 T E R FE R E N 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 readyforanalysis. 003813 000030 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 PRO C ED U RE______________________________________________________ 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. a 9 .2 .2 Turn the valve to "On". G O v io X 'i 9 .2 .3 Wait two minutes, and inject the next sample. 9 .2 .4 Record the scan number for each sample in the logbook. O O O O *""1 3 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 injeci/sample = 2 9 .3 .3 .4 Set Cycle rime = 0 9 .3 .3 .S 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 10.1. IRun a standard every 5 to 7 samples. If a significant change( 50%) in peak height occurs stop the run. Only the samples before the last acceptable standard will be used. The remaining samples will be reanalyzed. 10.2 Precision and Accuracy 1 0 .2 .1 See Method Validation Report number AMDT-M-5.0.V1 10.3 Other Validation Parameters 1 0.4 Refer to Method Validation Report Number AMDT-M-5.0.V1 11.0 DATA ANALYSTS_________________________________________________ 11.1 11.2 Calculations Plot the standard curve, using the mean of the two values obtained for each standard. 1 1 .2 . IRead peak heights or areas for the samples from the printout. Use linear regression to determine the sample concentrations. 1 1 .2 .2 Calculate the mg of FC-95 anion, or other fluorochemical in the total rabbit 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 11.3 Make a results table and enter it in the study book. 11.4 Print a chromatogram for each sample, with the peaks labeled with the sample or standard ID. Write the study number on the printout, initial, date, and put it in the study folder. Staple all chromatograms together and number pages. 003S15 00005 4 12.0 ATTA CH M EN TS None 13.0 REFERENCES 13.1 AMDT-EP-17 14.0 REVISIONS Revision Number Reason for change Revision Date 003S16 000053 5 3M Environmental Laboratory Method Analysis of Fluoride Using the Skalar Segmented Flow Analyzer With Ion Selective Electrode Method Identification Number: AMDT-M-8 Adoption D ate: Q~<r-f j - 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 Flow Analyzer 003817 000054 i Id . 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 KEYW 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 I I 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 03 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 ^03818 2 00005 __________ L Im u __________ 1.5 ppm 1.2 1.5 - 2A EQUIPMENT__________________________________________ 5 .1 Skalar Segmented Flow Auto Analyzer Sanspiul System equipped with ISE M IN TERFERENCES_______________________________________________________ 6 .1 High concentrations of alkalinity, chloride, phosphate, sulfate or iron can cause interferences. 7.0 SAMPLE HANDLING_____________________________________________________ 7 .1 Samples should be stored in polyethylene bottles. Samples should be analyzed within 30 days. &.Q CAURRA.TJQN AND..NTANPARP^ATIQN_____________________ S. 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 ED 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. 003819 3 00005G 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 .10 After starting the software, start the sampler. Make sure that the sampler is set to the right number of samples and that the sample/wash/air times are OK. 9 .2 .1 1 Select GRAPHICS, REAL TIME to view the progress of the analysis. 9.3 Loading and Printing the Data-File 9 .3 .1 Go to CONTROL PANEL, press the spacebar to bring up the local menu and select LOAD. Select AUTOCALCULATION and enter the filename (or highlight the file to be printed and press ENTER). 9 .3 .2 To view the calibration curve, go to GRAPHICS, CALIBRATION CURVE. 9 .3 .3 To print the high level curve, push PRINT SCREEN. 9 .3 .4 To print the low level screen, push ESC to get out of graphics. Select SETTINGS. Change the max y value to approximately 900. Go to CAL CURVE and press ESC, and Enter. Press PRINT SCREEN. 9 .3 .5 Return to SETTINGS and change the max value back to 4095, go to EDIT, press ENTER and PRINT SCREEN to print sample peaks. 9 .3 .6 To print the results go to CONTROL PANEL, SPACEBAR, OUTPUT, OUTPUT. Select PRINTER for the Epson or PRN for the Laser. 9.4 Shutdown 9 .4 .1 Put all the reagent-lines in Milli-Q water. 9 .4 .2 Let the system rinse for approximately 30 minutes. 9 .4 .3 After the system has rinsed completely, turn off the sampler, pump and offset potentiometer. Turn off the heating bath on weekends. Leave liquid in the lines. 9 .4 .4 Take the electrode out and soak in 100 ppm F overnight. 9 .4 .5 Release the pump-decks, air bars and sampler pump-tubing. 9 .4 .6 Select FILES, press ALT F and select QUIT to exit the program. 9 .4 .7 On Friday, turn off the computer, display and interface for the weekend. 10.0 V A L ID A T IO N _____________________________________________________ 10.1 Quality Control 10.1. IRun a standard (mid to high concentration) every 10 samples. If a significant change in peak height occurs, only the samples before the last acceptable standard will be used. The remaining samples will be reanalyzed. 003820 4 000057 10.2 Precision and Accuracy 10.2. ISee Method Validation Report number AMDT-M-8.0.V1 10.3 Other Validation Parameters 1 0 .4 Refer to Method Validation Report Number AMDT-M-8.0.V1 11.0 d a t a a n a l y s i s _______________________________________________ ___ 11.1 11.2 11.3 11.4 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. Prepare spreadsheets to summarize data. Include sample volume, weights used etc. 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. 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 TTA C H M EN TS___________________________________________________ None 13.0 R EFER E N C E S _________________________________________________ 13.1 AMDT-M-1, Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 13.2 Skalar Methods, #335, Skalar Methods Manual 1 3.3 AMDT-EP-26, Operation and Maintenance of the Skalar Segmented How Analyzer 14.0 REVISIONS Revision Number Reason for change Revision Date 003821 000058 9.3 Quality Assurance Unit Statement 003822 .00005J Attachment D GLP Study Quality Assurance Statement ppifcSto; QAU - I Study Title: Single-dose Intravenous Pharm acokinetic Study of T-6053 in Rabbits Study Number: AMDT-010495.1 Name of Auditor: KariRambo This study has been inspected by the Quality Assurance Unit as indicated in the following table. The findings were reported to the study director and management. Inspection Dates From Iq 10/13/95 10/19/95 Phase_____________ Final Report Date Inspection Reported to Management Study Director 10/19/95 10/19/95 003823 l 000000 9.4 Key Personnel Involved in the Study 003824 000001 3M Environmental Laboratory Key Personnel Thermal extraction followed by analysis using Orion ion analyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plummer Analysis o f 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 003825 o u o o c;;: 9.11 Data 003826 000063 9.11.1 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 003827 000004 Summary of Combustion Data - Liver AMDT-010495.1, H W I6329-136 As Referenced in Final Report section 6.0 D ATA A N A L Y S IS Total ug Fluoride in Whole Liver Mean per Dose Group Control Group 10 mg/kg dose (T6053) 50 mg/kg dose (T6053) 125 mg/kg dose (T6053) 250 mg/kg dose (T6053) ug 15.1 12.7 25.5 17.7 34.7 003828 00000.1 R P T 136L.XLS FC99 PK ID liver spk 63-1 liver spk 63-2 liver spk 126-1 liver spk 126-2 liver spk 253-1 liver spk 253-2 liver blank-1 F52856-1 F52856-2 F52856-3 F52857-1 F52857-2 F52857-3 F52861-1 F52861-2 F52861-3 F52862-1 F52862-2 F52862-3 liver blk 1 liver blk 2 liver spk 1 liver sp k 2 F52863-1 F52863-2 F52863-3 S P K 63-1 S P K 63-2 S P K 126-1 S P K 126-2 % rcvry 88% 96% 91% 89% 84% 83% 97% 94% 95% 96% 81% 82% Actual ppm Fin liver (W /W ) 1.08 0.970 2.06 2.16 4.16 3.44 0.314 0.250 0.172 0.160 0.150 0.150 0.192 0.186 0.311 0.390 0.237 0.211 0.219 0.412 0.359 1.24 0.857 0.503 0.426 0.395 0.940 0.912 1.76 2.18 Average ppm F- liver in liver burned (W /W ) (gram s) 0.1224 0.1499 0.1327 0.1242 0.122 0.1462 0.1499 0.194 0.136 0.1263 0.1383 0.164 0.141 0.1428 0.1038 0.296 0.1466 0.1198 0.222 0.1366 0.1456 0.1053 0.136 0.105 0.1157 0.1179 0.1667 0.441 0.1199 0.1386 0.1232 0.1525 0.1593 0.1393 0.1136 W hole liver w eight (gram s) 77.8 77.8 77.8 77.4 77.4 77.4 86.1 86.1 86.1 79.5 79.5 79.5 78.5 78.5 78.5 T otal F- in w hole liver (ug) 15.1 12.7 25.5 17.7 34.7 D o sa ge (m g/kg) 0 10 50 125 250 Page 1 003829 oooooo 9.11.2 Summary and raw data; analysis of liver extracts using electrospray mass spectrometry. 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. 003830 000067 HW I# 6329-136 Study: Protocol Number: Test Material: Matrix: R Squared Value: Response Factor Amount: Analyst: Date: Method: Instrument: LABBA SE File: Single-Dose Intravenous Pharmacokinetic TP8084.PK T-6063 in Rabbits (FC-99) Liver 0.9816 1.00E-05 DLC 4/6/95 AMDT-M-4 Fisons VG 2000 Electrospray MS 040695A -\ <3oaWw> A- I 4&ro<^ti A ) .02vUskjerv~-- U/2/35 Group Dose Group 1: Omg/kg Group 2: 10 mg /kg Group 3: 50 mg/kg Group 4: 125 mg/kg Group 5: 250 mg/kg Sam ple# F52856 Ion Count Extracted wt Dilution g factor 20514 1.0361 1 Concentration ug/g * Total m ass of liver 9 0.1587 77.8299 Total amount of FC-95 per liver mg 0.012 F52857 8628 1.0019 1 0.0690 77.4061 0.005 F52861 7353 1.1609 1 0.0508 86.1239 0.004 F52862 27068 1.0462 1 0.2074 79.5049 0.016 F52863 29919 1.1937 1 0.2009 78.5453 0.016 * 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 than171 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. 003831 OOOOCS A 'Z > AfrA q s G32R- ,a | Cp ^ - '3 5 ) L ` '3 * (jFc- ^ ) CS? n oo o O O 90000 A-a 000070 tw o:* >3^-1 ( ^ F c : ^ 00 n o o 000071 fr- Y n m n Q File:040695A LAB-BASE - The MS Data System 06/04/1995 08:14 A-5 00007, in cn ao eon o File1040695A LAB-BASE - The MS Data System 06/04/1995 08:14 A-, n r? X on o o o o <OtfS4 M e t h o d C :D L C L I V Sample ' Operator Run date 06-23-1995 09:19:00 version: 16 Printed on 06-23-1995 AT 09:19:13 Straight Line Fit forced through Origin. A 388722 R E rl 187049 127117 67071 LEVEL AMOUNT 1.2 AMOUNT Component 1 = EXTERNAL STANDARD CALIBRATION AREA 1 0.8000 67071 2 1.2000 127117 3 1.6000 187049 4 4.0000 388722 Y * SLOPE * X + INTERCEPT Area - 9.9824E+04 * Amount Amount - 1.0018E-05 * Area R squared = 0.9816 + 0.0000E+00 + 0.0000E+00 o4cfe?5A 003837 000074 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. 003838 0 0 0 0 9 .7 RE: 6329-136 LIUER SAMPLES RMDT 02495.1 Date of Rnalysis: 4-6-95 Analyst: DDUJ 7)nl< ?rQ au} The samples are burned in the Dohrman at 950 C using between 0.1 and 0.2 grams of the liuer. The gas is collected in 1.0 mL of 1:1 TISRB/Milli-Q water then an additional 2 mL of 1:1 TISAB/Milli-Q is added to allow for sufficient uolume for Skalar analysis. The samples are then analyzed on a Skalar Segmented Flow Rnalyzer using the Ion Specific Electrode (ISE) Method. TISAB buffer is added to each sample as it proceeds through the system. The sample then goes through a heated mining coil before the potential between the ion selectiue electrode and the reference electrode is measured. The signal is amplified and related to the fluoride concentration. The instrument was calibrated in the ranges of 0.015 - 0.15 ppm and 0.15-1.50 ppm fluoride. The standard curue for the high range was plotted using the inuerse logarithm option. The standard curue for the low range is linear. All standards and samples were then calculated by the Skalar software using these curues. All results below 0.0001 ppm appear on the raw data as #.####. A quality control standard was analyzed euery 10 samples to check for accuracy and drift. Raw data is taken from the appropriate calibrated range of the Skalar printout and summarized on an EKcel spreadsheet. The final results are adjusted for the collection uolume and any subsequent dilutions. 003839 0 0 0 ()7 (> P* ' ^ ^ GROUP 1 Dose L evel: 0 GROUP2 Dose L e v e l: 10 m g/kg GROUP 3 Dose L evel: 50 mg/kg GROUP4 Dose L evel: 125 mg/kg GROUPS Dose L evel: 250 mg/kg F52856-1 F52856-2 F52857-1 F52857-2 F52857-3 F52861-1 F52861-2 F52861-3 F52862-1 F52862-2 F52862-3 F52863-1 F52863-2 F52863-3 ND 0.03 0.08 0.11 ND ND ND 0.02 ND ND ND 0.02 0.03 0.02 00007 o o Co GO P SUMMARY OF 6329-136 LIVERS A M D T 02495.1 i M j &QfBS&SS& ttS T m m iu m 3.0 0.1360 ND 3.0 0.1263 0.68 3.0 0.1410 1.79 3.0 0.1428 2.37 3.0 0.1038 ND 3.0 0.1466 ND 3.0 0.1198 ND 3.0 0.1366 0.49 3.0 0.1456 ND 3.0 0.1053 ND 3.0 0.1360 ND 3.0 0.1199 0.62 3.0 0.1386 0.55 3.0 0.1232 0.52 ND 77.8299 77.8299 77.4061 1.39 77.4061 77.4061 86.1239 ND 86.1239 86.1239 79.5049 ND 79.5049 79.5049 78.5453 0.56 78.5453 78.5453 ND 53 139 184 ND ND ND 42 ND ND ND 49 43 41 ND 108 ND ND 44 Page 1 v/ 1995-07-10 10:23 OutPut o f :950406C1 Operator : DDW Date of the Analysis : 1995-04-06 13:33 Analysis File Name : C:\SKALAR\DATA\HWIDATA\LIVERS\950406C1 136-LIV.XLS BEST COPY AVAILABLE 1 Tracer 2 Drift 3 Wash 4 Standard 1 5 Standard 2 6 Standard 3 7 Standard 4 8 Standard 5 9 Standard 6 10 Standard 7 11 Standard 8 12 Standard 9 13 Standard 10 14 Drift 15 Wash 16 F52856-1 17 F52856-2 18 BLANK 19 F52857-1 20 F52857-2 21 F52857-3 22 F52861-1 23 F52861-2 24 F52861-3 25 F52862-1 26 Drift 27 Wash 28 F52862-2 29 F52862-3 30 LIVER BLK1 31 LIVER BLK 2 32 LIVER SPK 1 1.50 1.49 99% 1.50 1.51 101% 0.00 0.015 0.015 101% 0.03 0.03 99% 0.06 0.06 100% 0.09 0.09 101% 0.12 0.12 99% 0.15 0.15 100% 0.30 0.29 96% 0.60 0.61 102% 1.20 1.24 103% 1.50 1.46 97% 1.50 1.58 105% ND ND 3.0 0.1360 ND 77.8299 ND 0.03 3.0 0.1263 0.68 77.8299 53 0.06 1.0 0.1499 0.39 0.08 3.0 0.1410 1.79 77.4061 139 0.11 3.0 0.1428 2.37 77.4061 184 ND 3.0 0.1038 ND 77.4061 ND ND 3.0 0.1466 ND 86.1239 ND ND 3.0 0.1198 ND 86.1239 ND 0.02 3.0 0.1366 0.49 86.1239 42 ND 3.0 0.1456 ND 79.5049 ND 1.50 1.56 104% ND ND 3.0 0.1053 ND 79.5049 ND ND 3.0 0.1360 ND 79.5049 ND ND 3.0 0.1050 ND ND 3.0 0.1157 ND 0.05 3.0 0.1179 1.37 0.004 63.00 Page 1 0.15 0.16 107% 003841 00007S 136-LIV.XLS 23 LIVER SPK 2 34 F52863-1 35 F52863-2 36 F52863-3 37 SPK 63-1 38 Drift 39 Wash 40 SPK 63-2 41 SPK 126-1 42 SPK 126-2 43 Drift 44 Wash 0.06 3.0 0.1667 1.07 0.02 3.0 0.1199 0.62 78.5453 49 0.03 3.0 0.1386 0.55 78.5453 43 0.02 3.0 0.1232 0.52 78.5453 41 0.06 3.0 0.1525 1.20 1.50 1.56 104% ND Multi 3.0 0.1593 * Multi 3.0 0.1393 * Multi 3.0 0.1136 * 1.50 1.54 103% ND 0.004 63.00 0.004 63.00 ** ** ** * Samples # 40-42 had multiple peaks due to insufficient sample in cup. Disregard results for these three samples. DDW 0.15 0.18 117% 0.15 0.18 121% < > ' .0 0 0 0 O Q CO 00 io X- BEST COPY AVAILABLE Page 2 995-04-06 16:07 OutPut of : 950406C1 Software : version 6.1 cl990,93 Operator : DDW Date of the Analysis : 1995-04-06 13:33 Analysis File Name : C:\SKALAR\DATA\950406C1 OOu) "Mn t a i A * c>t S.' ho x 6 -3 2 . ^ - r Fluoride 1.5 Calibration order = Inverse Logarithm S lo p e : s = #.##### r x - cl -I ------ Result = 10*- s -> x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 = al = aO = -0.00000 0.00093 -1.24069 Fluoride L Calibration order = 2 C o r r e la tio n : r = 0.99928 Result = a2 * x 8 + al * x + aO a2 = al = aO = 0.00000 0.00025 0.00292 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 003843 OOOO'SO 1995-04-06 16:07 OutPut of : 950406C1 Fluoride 1.5 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 : 0 Signal type : Debubbled Decolor : No system Number : 0 diLute : No Resample : No dil Threshold : 4095 diG output :0 Window event : Off 003844 000081 1995-04-06 16:07 OutPut of : 950406C1 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 : #.#### Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dii Weight Ch Result Dii F Cor. Valu Time wt iw Initial Wash 1t Tracer 2d Drift 1 1.000 3 0.057 4 0.0029 1 1.000 3 1.489 4 1.0431 1 1.000 3 1.508 4 1.0550 1 0 280 65 0 000 1 2284 2571 211 0 2284 0 0 1 2302 2595 387 0 2302 0 0 3w Wash 1 1.000 3 0.057 4 0.0029 1 0 0 300 629 000 4 si Standard 1 1 1.000 3 0.064 4 0.0152 1 0 48 348 735 48 0 0 5 s2 Standard 2 1 1.000 3 0.071 1 102 402 912 4 0.0296 0 102 0 0 6 s3 Standard 3 1 1.000 3 0.089 1 211 512 1080 4 0.0601 0 211 0 0 7 s4 Standard 4 1 1.000 3 0.109 1 313 616 1261 4 0.0906 0 313 0 0 8 s5 Standard 5 1 1.000 3 0.129 1 404 708 1436 4 0.1193 0 404 0 0 0 0 3 8 4 5 9 s6 Standard 6 1 1.000 3 0.154 1 497 804 1612 4 0.1502 0 497 0 0 10 s7 Standard 7 1 1.000 3 0.287 4 0.2868 1 0 863 1178 1786 863 0 0 00008 11 s8 Standard 8 1 1.000 3 0.609 1 1387 1716 1962 12 s9 Standard 9 13 slO Standard 10 14 d 15 w 16 u 17 u 18 u 19 u 20 u 21 u 22 u 23 u 24 u 25 u Drift Wash F52856-1 F52856-2 BLANK F52857-1 F52857-2 F52857-3 F52861-1 F52861-2 F52861-3 F52862-1 26 d Drift 27 w Wash 28 u F52862-2 29 u F52862-3 30 u LIVER BLK 1 31 u LIVER BLK 2 32 u LIVER SPK 1 33 u LIVER SPK 2 4 U. 1 1.000 3 1.240 4 0.8957 1 1.000 3 1.459 4 1.0247 1 1.000 3 1.582 4 1.1018 1 1.000 3 0.057 4 0.0029 3 1.000 3 0.189 4 0.0142 3 1.000 3 0.212 4 0.0285 1 1.000 3 0.088 4 0.0586 3 1.000 3 0.313 4 0.0841 3 1.000 3 0.373 4 0.1129 3 1.000 3 0.178 4 0.0070 3 1.000 3 0.178 4 0.0067 3 1.000 3 0.189 4 0.0142 3 1.000 3 0.201 4 0.0221 3 1.000 3 0.186 4 0.0119 1 1.000 3 1.555 4 1.0843 1 1.000 3 0.057 4 0.0029 3 1.000 3 0.183 4 0.0103 3 1.000 3 0.183 4 0.0098 3 1.000 3 0.185 4 0.0113 3 1.000 3 0.181 4 0.0090 3 1.000 3 0.256 4 0.0540 3 1.000 3 0.265 U i JB/ u u 1 2053 2402 2136 0 2053 0 0 1 2256 2616 2312 0 2256 0 0 1 2372 2668 2485 0 2372 0 0 1 0 296 2717 0 000 3 44 342 2834 0 44 0 0 3 98 400 3009 0 98 0 0 1 206 512 3181 0 206 0 0 3 292 600 3360 0 292 0 0 3 384 696 3536 0 384 0 0 3 16 320 3676 0 16 0 0 3 15 320 3869 0 15 0 0 3 44 352 4053 0 44 0 0 3 74 384 4235 0 74 0 0 3 35 345 4411 0 35 0 0 1 2346 2657 4587 0 2346 0 0 1 0 312 4751 0 000 3 29 340 4934 0 29 0 0 3 27 336 5104 0 27 0 0 3 0 33 340 5286 33 0 0 003846 3 24 329 5461 0 24 0 0 3 190 496 5637 000083 0 190 0 0 3 2O0A8 512A 5807 % 34 u F52863-1 35 u F52863-2 36 u F52863-3 37 u SPK 63-1 38 d Drift 39 w Wash 40 u SPK 63-2 41 u SPK 126-1 42 u SPK 126-2 43 d Drift 44 w Wash wt rw RunOut Wash 3 1.000 3 0.206 4 0.0247 3 1.000 3 0.207 4 0.0253 3 1.000 3 0.201 4 0.0215 3 1.000 3 0.269 4 0.0612 1 1.000 3 1.563 4 1.0896 1 1.000 3 0.057 4 0.0029 3 1.000 3 Multi 4 0.3527 3 1.000 3 Multi 4 0.3518 3 1.000 3 Multi 4 0.3406 1 1.000 3 1.544 4 1.0776 1 1.000 3 0.057 4 0.0029 1 1.000 3 0.057 4 0.0029 u zuo u u 3 84 384 5985 0 84 0 0 3 86 384 6153 0 86 0 0 3 72 368 6336 0 72 0 0 3 215 512 6510 0 215 0 0 1 2354 2644 6688 0 2354 0 0 1 0 288 6861 0 000 3 M 1020 1360 7103 ) 0 1020 0 3M 1018 1388 7160 > ( 0 1018 0 0 11 3 M 992 1392 7344 0 992 0 ) H |^ S 1 2336 2752 7558 0 2336 0 0 1 0 448 7743 0 000 1 0 1405 8033 0 000 003847 000084 Calibration curve of 95040601 : Fluoride 1.5 Order Inverse Logarithm s : 3129 Calibration curve of 9504G6C1 : Fluoride L -CharmeI C Measure .5278708 i C oncen tration : 0 . 0 0 4 4 3 9 4 1 8 )!! (J03845 ,b<|)()0Sil ii !! \ ci 0* I *J *I c 9 -I (W<J 0 0 0.0029232 y y u-+" Order iir Measured 4095 r : 0.99928 4095 Pathno:-- 3 Raw data of 950406C1 Fluoride 1.5 fine: Q Ualue: ~280 I f l S n M ' M \ 9) : ij 3 II! t IIII ! II IMI !i i I ( I I I C0384S O jO O O S (> II II II 1 III! II I I I I i I Ml 1 i 1 l 11 1 ll II 1I i 11 H1 i1 11 1 9<* n, A ll i 11 1 1 H i-J !l WII i u 4 ^ < -a J G> /l <l5`\JnA\rJi'( i f \J! t!l\J!1I ]1\\J11 i i Si 1 1 li(' iilI i1 V .<, ^ IP l\ f 1 ft Ip 1! <p. I ' ) l i L \ i T ' _ , 75 I I Vzl 2&I T 0 T iwe Esc=Exit i Fi=Help i Crtl-P=Edit peaks ) Raw data of 9504Q6C1 : Fluoride 1.5 4095 iUI I ine: 386Q Ualue- f f i Z E s m n 5000 4, 8 D4 II 4 ^ v' ! & !\ h Vv p ^ A . *' .-r 11'! / ,-vJ i I! J I I! I r| l4J,v..vV` I/ I! If iI .1 V 1I ASJ \J 0 -I I 3860 T T ine Esc=fcxit ; Fi=Heip i urti-l^fcdit peaks ; (}0385< ()(M )0 i5 f 8860 I tV