Document qknMVv1KEVp0zDjm5bervQLBn

f y i - o r o o - /3 7 < f Attachments to Letter to C. Auer dated May 25,2000 / & T o d o o o o c n ) Toxicology Studies and Other Informmatiaonn oo nh PrFvOAA* ^ < v Pharmacokinetic Studies PfcWi-OyfiS&rtf J) 1) Final Report, Analytical Study, Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits, 3M Ref. No. LI 3492 (45% perfluorooctanoic acid tetrabutylammonium salt in water and isopropyl alcohol), Study No. AMDT120694.1, November 28, 1995; report includes Final Report, Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits, Hazelton Wisconsin, Inc., Study No. H W I6329-151, February 1, 1995 2) Final Report, Analytical Study, Single-Dose Absorption/Toxicity Study of T-6067 T6068, and T-6069 in Rabbits, 3M Environmental Technology & Services, Study No. AMDT-011095.1, November 28, 1995; report includes Final Report, Single-Dose Absorption/Toxicity Study of T-6067, T-6068, and T-6069 in Rabbits, Hazelton Wisconsin, Inc., Study No. HWI 6329-152, May 26,1995; T-6067 is LI 3492, 45% perfluorooctanoate tetrabutyl ammonium salt in water and isopropyl alcohol; T-6068 is fabric treated with high level of L13492, 45% perfluorooctanoate tetrabutyl ammonium salt in water and isopropyl alcohol; T-6069 is fabric treated with low level of LI 3492, 45% perfluorooctanoate tetrabutyl ammonium salt in water and isopropyl alcohol 3) ADME studies with FC-143-UC a) Final Report, Absorption of FC-143-14C in Rats After a Single Oral Dose, Riker Laboratories, Inc., January 14, 1980 b) Final Report, Synthesis and Characterization of FC-143-14C, Commercial Chemicals Division and Riker Laboratories, Inc., January 23,1980 c) Final Report, Extent and Route of Excretion and Tissue Distribution of Total Carbon-14 in Male and Female Rats After a Single IV Dose of FC-143-I4C, Riker Laboratories, Inc., February 7, 1980 ^ d) Final Report, Extent and Route of Excretion of Total Carbon-14 in Pregnant Rats After a Single Oral Dose of Ammonium 14C-Perfluorooctanoate, Riker Laboratories, Inc., July 1,1983 e) Final Report, Enhanced Elimination of FC-95-14C and FC-143-14C in Rats with Cholestyramine Treatment, Riker Laboratories, Inc., March 14,1980 4) Final Report, Single-Dose Intravenous Pharmacokinetic Study of T-6564 in Rabbits, Coming Hazelton Inc., Study No. CHW 6329-186, April 18, 1997 5) Final Report, 5-Daily Dose Dermal Absorption/Toxicity Study of T-6564 in Rabbits, Coming Hazelton, Inc., Study No. CHW 6329-185, April 28, 1997 6 4* - 3M Environmental Laboratory Final Report- Analytical Study Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits In-Vivo Study Reference Number: HWI#6329-151 Study Number: AMDT-120694.1 Test Substance: L -13492 (T-6067) 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 M ethod 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 A M D T-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 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 AM DT-M -14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum Initiation Date: See attached protocol Author: James D. Johnson Approved By: 'Z*v/?S Completion Date ojjft S ? 2- 1.0 SUMMARY Tissue and serum samples were analyzed for total organic fluorine after single intravenous doses to individual rabbits in order to collect information in support o f a dermal absorption study. Serum samples were analyzed at 2, 4, 6, 8,12, 24, and 48 hours post dose. There is a rapid decrease in serum level o f total organic fluorine with time. The biological half-life is on the order o f 4 hours. There is very little total organic fluorine present in liver or serum at 48 hours post intravenous dose o f L -13492 (T-6067). The pharmacokinetic study suggests that interpretation o f dermal absorption o f L -13492 (to be done and reported in a separate study) will be more difficult. It is unlikely that the serum levels at 48 hours and the liver samples at 28 days will contain any detectable organic fluorine whatever the extent o f dermal absorption. 2.0 IN TR O D U C TIO N ______________________________________________________ This study was designed to provide information as to whether L -13492 when administered as a single intravenous dose results in total organic fluoride increase in whole liver at 48 hours post dose or in serum at 2, 4, 8, 12, 24, or 48 hours. This information is to be acquired in order to interpret a subsequent dermal absorption study with the same compound (HWI#6329-152, AM DT-011095.1). The dermal absorption study will be reported separately. 3.0 TEST M ATERIALS____________________________________________________ 3.1 Test, Control, and Reference Substances and M atrices 3.1.1 A nalytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 3.1.2 A nalytical Reference M atrix: Bovine liver and bovine serum 3.1.3 Analytical Control Substance: None 3.1.4 A nalytical Control M atrix: Bovine liver and bovine serum 3.2 Source o f M aterials: 3M ICP/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. OQ253J 2 3.5 Storage Conditions for Test M aterials: Room temperature for FC-95. For biological samples the storage is -2010 C. 3.6 D isposition o f Test and Control Substances: 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 EXPER IM EN TA L-O verview Serum and tissues from animals dosed as described (HW I#6329-151) were available for analysis by combustion with subsequent analysis by selective ion electrode or by electrospray mass spectrometry. The tissue and serum samples were analyzed for total organic fluorine to collect information in order to support a dermal absorption study. The extent o f analysis will be at the discretion o f the Study Director. The samples arise from a previous study (HW I#6329-151) in which single rabbits were dosed intravenously with L -13492 at doses ranging 4 to 24 mg/kg. (See attached protocol for dosing, tissue collection, etc.) In the final report from HWI on this study the dosing is from 4 to 24 mg/kg. In the original protocol, the dosing is to 160 mg/kg. A rabbit dosed at 40 mg/kg died 5 minutes after injection. N o tissues were saved. 5.0 EXPERIM ENTAL - M ETHODS 5.1 A M D T-M -1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 5.2 A M D T-M -2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer 5.3 A M 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 5.6 A M D T-M -14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum G253f 6.0 DATA ANALYSIS The data are attached for total organic fluorine analysis o f liver at 48 hours post dose. The total organic fluorine for whole liver for control animals, 4 mg/kg, 16 mg/kg, and 24 mg/kg intravenous doses o f a 45% solution are 20 ug, 43 ug, 66 ug, and 54 ug. Each data point represents one animal. An animal dosed with 40 mg/kg died 5 minutes after injection. The data are attached for serum concentrations o f total organic fluorine at 2, 4, 6, 8, 12, 24, and 48 hours post dose. What is immediately apparent is that the concentrations decrease rapidly with time and are non-detectable at 48 hours. The biological half-life appears to be about 4 hours (see pages 84-86). It appears likely that this salt o f perfluorooctanoate is rapidly eliminated from female rabbits. It is not known if there is a sex difference in the elimination o f the perfluorooctanoate in rabbits as is observed with rats. 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 Circum stances that M ay Affect the Q uality o f the Data: The problem with this analysis is that there is not a good biological marker and thus the pharmacokinetic study does not provide a good avenue for the interpretation o f a dermal absorption study 7.0 CO NCLUSIO N______________________________________________________ There is very little total organic fluorine present in liver at 48 hours post intravenous dose o f L-13492. The pharmacokinetic study suggests that interpretation o f dermal absorption o f L-13492 (to be done and reported in a separate study) will be very difficult. 8.0 M AINTENANCE OF RAW DATA AND RECORDS__________________ 8.1 Raw Data and Data: Raw data, approved protocol, approved final report, appropriate specimens, and electronic data will be maintained in the AMDT archives. 00253^ 4 9.0 APPENDICES 9.1 Protocol and Am endm ents 9.1.1 Protocol and Final Report: HWI#6329-151 "Single-Dose Intravenous Pharmacokinetic Study o f T-6067 in Rabbits" (Protocol type TP8084.PK for dosing o f animals, tissue collection, etc.) 9.1.2 Analytical protocol AM DT-120694.1 9.2 Signed Reports from Individual Scientists: None 9.3 Q uality Assurance Unit Statement: See attached 9.4 K ey Personnel Involved in the Study: See attached 9.5 M aterials and Equipment: See methods 9.6 Solutions, Reagents, and Standards: See methods 9.7 Sample Preparation: See methods 9.8 Quality Control Practices: See methods 9.9 Test Methods: See Protocol AMDT-120694.1 9.10 Instrum ent Settings: See methods 9.11 Data: See attached. 9.11.1 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 9.11.2 Summary and raw data; ppm F* in serum as determined by thermal extraction followed by analysis using Orion ion analyzer. 9.11.3 Summary and raw data; ppm F' in serum as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. 9.11.4 Summary and raw data; analysis o f liver extracts using electrospray mass spectrometry. 0025 9.1.1 Protocol and Final Report: HWI#6329-151 "Single-Dose Intravenous Pharmacokinetic Study o f T-6067 in Rabbits" (Protocol type TP8084.PK for dosing o f animals, tissue collection, etc.) 00253^ . HAZLETON WISCONSIN P I J M O f F I <' B O X 7 B 4 6 T / i A n r . o r j 'A'i -, , ; / o ' / 6 4 l> Sponsor: 3M St. Paul, Minnesota CO RN IN G ( ru h v FINAL REPORT Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits Author: Steven M. Glaza Study Completion Date: February 1, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-151 Page 1 of 28 Phone 608241-447] EXPRESS -MAIL DELIVER) F a x 6 0,8 - 2 4 1 - 7 2 2 7 0 0 I K I H L M A I I B L V D M A D 1S O N W I 0253$ Page 2 of 28 QUALITY ASSURANCE STATEMENT HWI 6329-151 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 11/27/94 12/09/94 12/28/94 01/17/95 01/30/95 11/27/94 12/09/94 12/28/94 01/19/95 01/30/95 Protocol Review Sample Collection Protocol Amendment Data/Report Review Report Rereview 11/27/94 12/09/94 12/28/94 01/19/95 01/30/95 12/10/94 01/10/95 01/10/95 02/10/95 02/10/95 Lilia M. Danner Representative, Quality Assurance Unit _______ h s Date * Page 3 of 28 STUDY IDENTIFICATION Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits HWI 6329-151 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Experimental Start Date Experimental Termination Date T-6067 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 John L. Butenhoff, PhD 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.0. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Haileton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 December 9, 1994 December 11, 1994 002540 Page 4 of 28 HWI 6329-151 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 Thomas E. Palmer, PhD Anatomical Pathologist Rose M. Bridge Report Supervisor Quality Assurance Sherry R. W. Petsel Manager Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data 00254/ Page 5 of 28 CONTENTS Quality Assurance Statement Study Identification Key Personnel Summary Objective Regulatory Compliance Test and Control Materials Test System Procedures Results Discussion Signature Reference Pathology Report Table 1 Individual Body Weights (g) 2 Individual Clinical Signs Appendix A Protocol Deviation Protocol TP8084.PK Protocol Amendment No. 1 HWI 6329-151 Page 2 3 4 6 7 7 7 8 9 11 11 11 11 12 13 14 15 16 17 27 0025-yL. Page 6 of 28 HWI 6329-151 SUMMARY This study was done to assess the level of systemic exposure of T-6067 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 4, 16, 24, or 40 mg of T-6067/kg of body weight at a dose volume of 0.5 mL/kg. 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 surviving animals at 2-, 4-, 6-, 8 -, 12-, 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 surviving animal. All samples were centrifuged, separated into serum and cellular fractions, and sent to the Sponsor. The one animal (40 mg/kg) found dead during the study was necropsied but tissues were not saved. Animals surviving to the end of the 48-hour post-injection period 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 surviving to termination were collected and sent frozen to the Sponsor after termination of the in-life phase. The animals treated at 0, 4, 16, and 24 mg/kg appeared normal throughout the study. The animal treated at the 40 mg/kg dose level died within 5 minutes of dosing. At necropsy, the lungs in this animal were diffusely dark red. This change was attributed to acute death and postmortem change. O02S4* if;* Page 7 of 28 HWI 6329-151 OBJECTIVE The objective of this study was to assess the level of systemic exposure to the test material, T-6067, 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/solubi1 ity, 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-6067 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 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. 00254^ Page 8 of 28 HWI 6329-151 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 230C , 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. 00254 Page 9 of 28 HWI 6329-151 Study Design Female animals weighing from 2,511 to 2,781 g at initiation of treatment were placed into the following study groups: Group 1 (Control) 2 3 4 5 Treatment * T-6067 T-6067 T-6067 T-6067 Dose Level ima T-6067/kq) 0 4 16 40 24 Dose Volume (mL/kal 0.5 0.5 0.5 0.5 0.5 Number of Anima 1 1 1 1 1 * Sterile Water for Injection, USP. Justification for Species Selection Historically, the New Zealand White albino rabbit has been the animal of choice because of the large amount of background information on this species. PROCEDURES Dose Preparation and Administration The test material was diluted with Sterile Water for Injection to achieve a specific concentration for each dose level. 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). C02S4 Page 10 of 28 HWI 6329-151 Sample Collection Blood samples (approximately 4 mL) were collected from either ear via the catheterization of the auricular artery or from the marginal ear vein of all surviving 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 surviving 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 The animal found dead immediately after dosing (40 mg/kg) was subjected to an abbreviated gross necropsy examination and any abnormalities were recorded. No tissues were collected from this animal. At termination of the experimental phase, surviving animals were anesthetized with sodium pentobarbital and exsanguinated without necropsy however tissues were collected. The whole liver, bile, and both kidneys from each animal surviving to termination were collected and immediately placed on dry ice, then frozen by placing in a freezer set to maintain a temperature of -20*C 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. 00254)1 Page 11 of 28 RESULTS Body Weights Individual body weights are in Table 1. HWI 6329-151 Clinical Observations Individual clinical signs are in Table 2. The animals treated at 0, 4, 16, and 24 mg/kg appeared normal throughout the study. The animal treated at 40 mg/kg died within 5 minutes of dosing. Pathology The animals treated at 0, 4, 16, and 24 mg/kg survived to termination of the experimental phase and were not examined grossly when sacrificed. The animal treated at 40 mg/kg died on the day of treatment and was necropsied. The lungs in this animal were diffusely dark red. This change was attributed to acute death and postmortem change. DISCUSSION The level of systemic exposure of T-6067 was evaluated in female albino rabbits when administered as a single intravenous injection at levels of 0, 4, 16, 24, and 40 mg/kg. The animals treated at 0 , 4, 16, and 24 mg/kg appeared normal throughout the study. Administration of this material at 40 mg/kg resulted in the death of the animal. SIGNATURE Steven M. Glaza Study Director Acute Toxicology REFERENCE 1. NIH Publication No. 86-23 (revised 1985). 00254$ Page 12 of 28 HWI 6329-151 PATHOLOGY REPORT There was one Group-4 female rabbit treated at 40 mg/kg that died on Day 0 and was necropsied. At necropsy, the lungs in this animal were diffusely dark red. This change was attributed to acute death and postmortem change. The liver, bile, and both kidneys from this animal were inadvertently not collected as required by protocol. After necropsy, the animal was discarded. Pathologist (6329-151.slh) 122294 Page 13 of 28 GrouD 1 2 3 4 5 Table 1 Individual Body Weights (g) Dose Level (mq/kql Sex Animal Number 0 Female F52762 4 Female F52763 16 Female F52768 40 Female F52769 24 Female F52770 Dav 0 2,781 2,770 2,735 2,511 2,778 HWI 6329-151 CO2550 Dose Level Group (mq/kq) 10 24 3 16 4 40 5 24 Sex Female Female Female Female Female Page 14 of 28 Table 2 Individual Clinical Signs HWI 6329-151 Animal Number F52762 F52763 F52768 F52769 F52770 Observation _________ Hour 0.5 2 4 24 Appeared normal // Appeared normal Appeared normal // Dead (=5 minutes post-injection) Appeared normal / 48 Indicates condition exists. - Not applicable. 00255 Page 15 of 28 HWI 6329-151 APPENDIX A Protocol Deviation Protocol TP8084.PK Protocol Amendment No. 1 C025SJL Page 16 of 28 Protocol Deviation HWI 6329-151 Protocol Page 8 , 7. Experimental Design, E. Termination, (2) Scheduled Sacrifice, (a) Sample Collection. The whole liver and bile from each animal dying during the study, sacrifice in a moribund condition, or surviving to termination will be collected. Both kidneys from each animal will also be collected. ________ Actual Procedure The liver, bile, and kidneys from the animal that died on test (Group 4, No. F52769) were inadvertently not collected at necropsy. This deviation is not considered to have had an adverse effect on the outcome of the study. 00255* HAZLETON WISCONSIN P O S T O M l C f - B O X / fi 4 S M A D I S O N . W l >33 70 ? 75 4 5 Page 17 of 28 C O R N IN G {,on>|*iny Sponsor: 3M St. Paul, Minnesota PROTOCOL TP8084.PK Study Title: Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits Date: December 6 , 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-151 i Pn : V -, w I I .1 i O ^ - f- ri f -I , N'M -,M .%;j n V I) M a I)1- N 00255^ Page 18 of 28 STUDY IDENTIFICATION TP8084.PK Page 2 Single-Dose Intravenous Pharmacokinetic Study of T-6067 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 6329-151 T-6067 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 John L. Butenhoff, PhD 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 December 9, 1994 December 11, 1994 January 13, 1995 Page 19 of 28 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 -6067 B. Physical Description (To be documented in the raw data) C. Purity and Stabilitv 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. ) C02Ss6 Page 20 of 28 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. 6 - 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 Animals (1) Soecies Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP, Inc. (3) Age at Initiation Adul t CQ2SSf Page 21 of 28 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 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 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. C0255J Page 22 of 28 TP8084.PK Page 6 B. Dose Administration (1) Test Groups Group 1 2 3 4 5 Dose Level -ijogZkqil 0 (Control) 4 16 40 160 Humber of Females 1 1 1 1 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. Page 23 of 28 TP8084.PK Page 7 (2) Body Weights Just before test material administration. (3) Sample Collections (a) Frequency 2, 4, 6, 8 , 12, 24, and 48 hours post-injection (b) Number of Animals All (c) Method of Collection Blood samples (approximately 4 mL) will be collected from either ear via the catheterization of the auricular artery or from the marginal ear vein at 2 , 4, 6, 8 , 12, and 24 hours post-injection. Approximately 20 mL of blood (actual volume to be documented in the raw data) will be obtained from the posterior vena cava of each animal at the time of necropsy (48 hours post-injection). Approximately 20 mL of blood will be collected from moribund animals during the study, also, if possible. The samples will be stored at room temperature and then centrifuged, and the separate serum and cellular fractions stored in 3 freezer set to maintain a temperature of -20*C 10*C. The separated serum and cellular fractions will be sent frozen to the Sponsor after experimental termination. Samples will be shipped to: James D. Johnson 3M E.E. 4 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. Page 24 of 28 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. (a) Sample Collection The whole liver and bile from each animal dying during the study, sacrificed in a moribund condition, or surviving to termination will be collected. Both kidneys from each animal will also be collected. The tissues will be placed on dry ice immediately after collection and then placed in a freezer set to maintain a temperature of -20*C 10*C. The tissues (liver, bile, kidneys) will be sent frozen on dry ice to the Sponsor after experimental termination. The samples will be shipped to the person listed in Section 7.D.(3).(c). The Sponsor is responsible for the retention and disposition of the samples. ) F. Statistical Analyses No statistical analyses are required. 8 . Report A final report including those items listed below will be submitted. Description of the test and control materials Description of the test system Procedures Dates of experimental initiation and termination Description of any toxic effects Gross pathology findings (if applicable) Gross pathology report (if applicable and requested by the Study Director) ) Page 25 of 28 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 Page 26 of 28 TP8084.PK Page 10 PROTOCOL APPROVAL John L. Butenhoff, PhD Sponsor's Representative 3M Acute Toxicology ) Hazleton Wisconsin, Inc. - ), presentat:iive Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-151.protdskl) / 2. - tZ.-CjLj Date Date 'L A v Date J CQ25SJ Page 27 of 28 HAZLETON WI S CONS I N POST OFFICE MADI SON W I 5B3 7O0X7 - 77554455 a CO R N IN G Company PROTOCOL TP8084.PK Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits HWI 6329-151 Sponsor 3M Toxicology Services 220-2E-02 3M Center St. Paul, MN 55144 Sponsor's Representative John L. Butenhoff, PhD Contractor Hazleton Wisconsin, Inc 3301 Kinsman Boulevard Madison, WI 53704 Study Director Steven M. Glaza Amendment No. 1 This amendment modifies the following portion of the protocol: Effective December 9, 1994 1. Page 6. 7. Experimental Design: B. Dose Administration; (11 Test Groups. Due to the death of the Group 4 animal (40 mg/kg), modify the dose level for the Group 5 animal as indicated by the following underlined change: Group Dose Level (mg/kg)* 1 0 (Control) 24 3 16 4 40 5 24 a The dose volume will Number of Females 1 1 1 1 1 0.5 ml/kg of body weight Phone o0-S 24 : f XPREr mail n - : \ MAN BLVD bb 4 % , M .-VI Amendment No. 1 ') Page 28 of 28 PROTOCOL APPROVAL HWI 6329-151 Page 2 John L. Butenhoff, PhD Sponsor's Representative 3M Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. 0~ ) ipresentative Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-151.Ami.dsk2) -H '9" Date ___________ Date -- ;____V v J 4S' Date J CQZ5&f 9.1.2 Analytical protocol AM DT-120694.1 3M Environmental Laboratory______________________ _ Protocol - Analytical Study Single-Dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits In-Vivo Study Reference Number: HWI#6329-151 Study Number: A M DT-120694.1 Test Substance: L-13492 (T-6067) 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 Com pletion Date: August 25, 1995 M ethod 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 A M D T -M -2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer A M 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 A M D T-M -8- 0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode AM DT-M 14-0, Therman Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum Author: James D. Johnson Approved By: John Butenhoff, PhD Sponsor Representative Date LO ,,PU R P O SE To assess the level o f systemic exposure when the test material L -13492 (T-6067) is administered as a single intravenous injection to rabbits. 2.0 T EST M ATERIALS____________________________________________________ 2.1 Test, Control, and Reference Substances and M atrices 2.1.1 A nalytical R eference Substance: FC-95, lot 161 or 171. They are equivalent. 2.1.2 Analytical R eference M atrix: Bovine liver and bovine serum 2.13 Analytical Control Substance: None 2.1.4 Analytical Control Matrix: Bovine liver and bovine serum 2.2 Source o f M aterials: 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 N um ber o f Test and Control Samples: Liver and serum from 3 test animals and 1 control animal. One animal died after injection o f a 40 mg/kg dose, and was not replaced. 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 M aterials: Room temperature (2.1.1), -20 10C (2.1.2, 2.1.4). Test and Control samples w ill be received according to A M D T -S-10-0. 2.8 Disposition o f Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required for studies longer than 28 days. This study is in parallel with a 28 day dermal absorption study so all tissues will be retained. 2.9 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting the samples. 2 3.0 EXPERIM ENTAL - Overview The tissues from animals dosed as described (HWT#6329-151), 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. 4 .0 -EXPE R IM E N T A L ^ M e th a d i___________________________________________ 4.1 Liver and Serum screening methods: (attached) 4.1.1 A M D T-M -1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann D X 2000 Organic Halide Analyzer-Liver 4.1.2 A M D T-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 A M 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 Flow Analyzer with Ion Selective Electrode 4.1.6 AM DT-M -14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum 5.0 DATA ANALYSTS______________________________________________________ 5.1 Data Reporting: Data will be reported as a concentration (weight/weight) o f fluoride per tissue or fluid, or as FC-143 (electrospray mass spectrometry) per unit o f tissue or fluid. 6.0 M AINTENANCE OF RAW DATA AND RECORDS 6.1 R aw Data and Records: Raw data, approved protocol, appropriate specimens, approved final report, and electronic data will be maintained in the AM DT archives. 7.0 REFERENCES 7.1 AM DT-S-10-0, Sample Tracking System 81Q 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 A M D T-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 AM DT-M -5-0, Analysis o f Rabbit Liver Extract for Fluorochmicals Using Electrospray Mass Spectrometry 8.5 AM D T-M -8-0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 8.6 AM DT-M -14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum 002574? 3IVf Environmental Laboratory M ethod 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: -- roup Lead' L Quality Assurance /g- / - 9 T 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 1 1.0 SCOPE . APPLICA BLE C O M PO UND S. AND M ATRICES 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 E Y W O R D S_________________________________________________________ 2.1 Fluoride, fluorine, extraction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, DX2000, fluorochemicals. 3.0 PR E C A U TIO N S_______________________________________________________ 3.1 Glassware and exhaust gases can be extremely hot. 3.2 Glassware is fragile, broken glass may cause injuries. 3.3 Pressurized gases, proper compressed gas handling practices required. 3.4 Solvent based samples may flash, may need to allow them to dry down before starting run. 3.5 Potential biohazards due to the biological matrices. Use appropriate personal protective equipment. 4.0 SU PPLIE S AND M ATERIALS________________________________________ 4.1 Compressed Oxygen, Hydrocarbon free, regulated to 30 PSI. 4.2 Compressed Helium, High Purity Grade, regulated to 45 PSI. 4.3 Quartz glass sample boat with TeflonTM tubing, Dohrmann 890-097 or equivalent. 4.4 Quartz glass combustion tube, Reliance Glass G-9405-012 or equivalent. 4.5 Orion 940999 Total Ionic Strength Adjustment Buffer (TISAB I I ) or equivalent. 4.6 Sample collection vials, HDPE. 4.7 Milli-QTM water 4.8 Polystyrene pipettes. 4.9 Activated Charcoal, E. Merck 2005 or equivalent. 4.10 Hamilton Syringe or equivalent. 4.11 Miscellaneous laboratory glassware 5.0 E Q U IPM E N T __________________________________________________________ 5.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer, modified for fluoride extraction. 5.2 IBM compatible 386 or 486 computer. 5.3 DX2000 software, version 1.00, modified for fluoride extraction. 5.4 Excel Spreadsheet, version 5.0 or greater 6.0 IN T E R FE R E N C E S____________________________________________________ 6.1 Sample size is limited to approximately 150 mg, depending on sample moisture content. This may vary from matrix to matrix. 2 QQ257JL 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 C A L IB R A T IO N AND STA N D A R D IZA TIO N __________________________ 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. 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. 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 ofTISAB solution or 1:1 TISAB:Milli-QTM water mixture to a labeled sample collection vial. Typically 0.6 mL to 15 mL are used. For rabbit studies, use 1.0 or 2.0 mL of 1:1 TISAB:Milli-QTM water mixture. 9.3.4 Place the vial so that the tip of the COMBUSTION TUBE is in the TISAB at least 0.25 inches. Gases released during pyrolysis must bubble through the TISAB. 9.3.5 Run the EOX-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 KIMWEPE to remove any TISAB remaining on the tube. 9.3.9 Open the sample hatch and remove any remaining ash from the boat. Ash can be removed with a cotton tipped applicator 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 VALIDATIO N 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 Other Validation Parameters: NA 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 P- 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 A T TA C H M EN TS_____________________________________________________ None 13.0 R E F E R E N C E S________________________________________________________ 13.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer Operator's Manual (Manual 915349, revision B, December 1993) 13.2 AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 13.3 AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 R E V ISIO N S__________________________________________________________ Revision Number Reason for Change Revision Date 6 ' V0 Q 2 5 7 3M Environmental Laboratory M ethod Fluoride M easurement by Means o f 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/ /? A//S Date Quality Assurance Date Software: MS Word 5.1a Affected Documents: AMDT-M-1 Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer 1 00257^ 1.0 SCOPE , APPLICABLE C O M PO UND S. 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 K E Y W O R D S_________________________________________________________ 2.1 Fluoride, fluorine, ion selective electrode 3.0 P R E C A U T IO N S______________________________________________________ 3.1 No hazards identified with this method. 4.0 SU PPLIE S A N D 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 laboratoiy glassware. 5.0 E Q U IPM E N T _________________________________________________________ 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 IN T E R FE R E N C E S____________________________________________________ 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. 2 00257 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. SJ-CALIBRAXIQN A]VP-SIANDARDIZATrON_______________________ 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 PR O C E D U R E S_________________________________________________________ 9.1 Calibration and Measurement, Standard method: 9.1.1 The sample to be measured needs to be mixed with TISAB using the proportions recommended by the TISAB manufacturer. 9.1.2 Place a stir bar in the sample and place the sample on the stir plate. 9.1.3 Allow the sample to mix for a few seconds before inserting the electrode. When the electrode is inserted, make sure there are no air bubbles trapped under the electrode. 9.1.4 The sample should be the same temperature as the calibration standards and stirred at the same rate as the calibration standards. 9.1.5 When the readings have stabilized, record the reading in the appropriate log book. 3 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 V A L ID A T IO N ____________________ __ _________________________________ 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 D A TA ANALYSIS____________________________________________________ 11.1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 12,0 A TTA C H M EN TS___________ __________________________________________ None 13.0 REFERENCES 4 0 0 2 5 3 ff 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 R EV ISIO N S__________________________________________________________ Revision Number Reason for Change Revision Date C Q Z S si 3M Environmental Laboratory Method Extraction of Fluorochem icals from Rabbit Livers SOP Identification Number: AMDT-M-4 Revision Number: 0 Adoption Date: Revision Date: None Author: Dave Christenson/Cynthia Weber Approved By: roup Leader /o -y i-ts Date Quality Assurance /fr-3 /-T r Date Software: MS Word, 6.0 Affected Documents: M-5, Analysis of Rabbit Extract for Fluorochemicals Using Electrospray Mass Spectroscopy. 1.0 SC O PE l.l Scope: This method is for the extraction of fluorochemicals from rabbit livers. Ethyl acetate is used to extract fluorochemicals from the livers for analysis by electrospray mass spectroscopy. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Rabbit Livers. M KEYWQRDg____________________________________________ 2.1 Fluorochemicals, rabbit livers, electrospray mass spectrometer, fluorinated compounds, extraction. M P R E C A U T IO N S ______________________________________________ 3.1 Use gloves when handling the rabbit livers, they may contain pathogens. 4 .0 S U P P L IE S A N D M A T E R IA L S______________________________________ 4.1 Supplies 4 .1 .1 Syringe, capable of measuring 100 pL 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 pm. 4 .1.10 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), (Na^Oj/NaHCC^) 0.25 M: Weigh 26.5 g of sodium carbonate (Na2C 03) 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) C025S 5Ji -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 F E R E N C E S ______________________ 6.1 There are no known interferences at this time. 7.0 SA M P L E H A N D L IN G ________________________________________________ 7.1 The rabbit livers are received frozen, and must be kept frozen until the extraction is performed. 8.0 C A L IB R A T IO N A N D S T A N D A R D IZ A T IO N _______________________ 8.1 Preparation of Internal Standards 8 .1 .1 Prepare an internal standard of approximately 12 ppm 1H,1H,2H,2Hperfluorooctanesulphonic acid to be added to each liver sample. 8 .1 .2 Weigh at least 0.1 g of lH,lH,2H,2H-perfluorooctanesulphonic acid into a 100 mL volumetric flask. Record the actual weight 8 .1 .3 Bring it up to volume with methanol, this is the stock standard. 8 .1 .4 To a 250 mL volumetric flask, add 3 mLs of the stock standard and bring to volume with Milli-Q water. Calculate the actual concentration of the standard. actual mg perfluoroctane- sulphonic acid X 3 mL = 0.1 L 250 mL actual concentration, ppm 8.2 Prepare FC-95 Anion Standards 8 .2 .1 Prepare FC-95 standards for the standard curve. 8 .2 .2 Weigh approximately 100 mg of FC-95 into a 100 mL volumetric flask. Record the actual weight. 8 .2 .3 Bring up to volume with dilute acetonitrile. 8 .2 .4 Dilute the solution with dilute acetonitrile 1:10 for a solution of approximately 100 ppm. Dilute this solution 1:10 with dilute acetonitrile for a solution of approx. 10 ppm. 8 .2 .5 Use the 10 ppm solution to make working standards with values close to 5.0 ppm, 1.0 ppm and 500 ppb. 8 .3 Prepare Beef Liver Homogenate to Use for Standards 8 .3 .1 Weigh 40 g of Bovine liver into a 250 mL Nalgene bottle containing 200 mLs Milli-Q water. Grind to a homogenous solution. 8 .3 .2 Add 1 mL of the solution to a 15 mL centrifuge tube. Prepare a total of eight 1 mL aliquots of the solution in 15 mL centrifuge tubes. Be sure to re suspend solution by shaking it between aliquots. C 02SS^ 8 .3 .3 Spike seven of the 1 mL aliquots with the following amounts of working standards in step 9.12 of the procedure. One 1 mL aliquot serves as the blank. Working Standard (Approximate Cone.) 500 ppb 500 ppb 500 ppb 500 ppb 1 ppm 5 ppm 5 PPm______ uL 10 2 300 400 500 2 3 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 ppm) = final concentration (ppm) 171 mg liver*/ 1 ml homogenate of FC -95 in liver *Average weight of bovine liver in solution as determined by weighing 1 mL homogenates of 40 mg liver in 200 mL of Milli-Q water. The amount of FC-95 is reported as equivalents 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 P R O C E D 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 1g 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 initials. 4 G025SJT 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. 9.10 Cap the sample and vortex for 15 seconds. 9.11 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.) 9.12 Spike the beef liver homogenates with the appropriate amount of FC-95 standard as described in 8.3. 9.13 Spike the samples and beef liver homogenates with 100 uL of internal standard. 9.14 Add 1 mL of the sodium carbonate/sodium bicarbonate buffer and 1 mL ammonium acetate. 9.15 Using an analytical pipet, add 5 mL ethyl acetate. 9.16 Cap the sample and vortex 20 to 30 seconds. 9.17 Put them in the shaker for 20 min. 9.18 Centrifuge for 20 to 25 minutes, until the layers are well separated. Set the power on the centrifuge to 25. 9.19 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. 9.20 Blow the sample down on the analytical evaporator to near dryness with nitrogen, approximately 30 to 40 minutes. 9.21 Bring the remaining sample up in 1 mL dilute acetonitrile with an analytical pipet. 9.22 Vortex 15 seconds. 9.23 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. 9.24 Cap and hold for analysis by electrospray mass spectroscopy. 9.25 Complete AMDT-M-4 worksheet and attach to page of study notebook. 1 0 .0 V A L ID A T IO N __________________________________ 10.1 Quality Control - not applicable 10.2 Precision and Accuracy- not applicable 10.3 Other Validation Parameters- not applicable 11.0 D A T A A N A L Y S IS ______________________________ 11.1 None 12.0 A T T A C H M E N T S ________________________________ 12.1 Worksheet AMDT-M-4 13.0 R E F E R E N C E S ______________ ____________________ 13.1 AMDT-EP-22 Routine Maintenance of Ultra-Turrax T-25 14.0 R E V IS IO N S _____________ RevisioNn umber Reason for Change Revision Dale 002580 Worksheet AMDT-M-4 Study # _ _ _ _ _ _ _ 1 Sam ple N um ber set # B lank L iver F C -9 5 approx 0.5 ppm actual ppm #W _ 100 III. 200 uI. 300 uL 400 uI. _ _ _ _ _ _ _ _ _ _ F C -95 approx 1 ppm actual ppm #W _ . 500 uL * _ . _ . _ . FC -95 approx. 5 ppm actual ppm #W _ D ate and In itials for Std. _ 200 uL 300 uL - _ . _ 1studv n u m b er w here th e original w orksheet is located and o lace a conv. _______ I_______ _________ _________ _________1 j 1 T.iver E x tractio n Process- D ate ft. Initials P in et 1 m l. o f T.iver S o lu tio n Pinet 100 u l. o f 12 nnm Internal Standard Std. # V ortex 15 sec Pinet 1 m l. o f 250 nnm A m m onium A cetate Std # P inet 1 m l. o f 0.25 N a,C O ,/0 .2 5 M N aH C O , B uffer P in et 5 m l. o f F.thvl A cetate V ortex 20-30 sec. Shake 20 m in. _ C entrifiipe 20-25 min. R em ove a 4 m l. alim iot o f organic laver B lo w d o w n to n e a r d rv n e ss C<0.25 m l.I w ith N , A d d 1 m- o f 1-1 A c e t n n i t r i l e / H ,n TN# V ortex 15 sec i i lt e r u s in g a 3 c c B -D s v r in n e w ith a Q .2iim S R I f ilte r in to a 1 5 m L a u tn s a m n le v ial______________________________ i6 3M Environmental Laboratory Method Analysis of Rabbit Liver Extract for Fluorochem icals using Electrospray M ass Spectroscopy SOP Identification Number: AMDT-M-5 Revision Number: 0 Adoption Date: 1- i s-- 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 C025Sy 1.0 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 E Y W O R D S__________________________________________________________ 2.1 Fluorochemicals, fluorinated compounds, electrospray mass spectroscopy, mass spectrometer, rabbit livers. 3.0 P R E C A U T IO N S _______________________________________________________ 3.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 3.2 Do not run the pump above it's capacity of 4000 psi. If pressure goes over 4000 psi stop and release pressure. The peak tubing may be plugged. Troubleshoot back to find the plug and replace the plugged tubing. See AMDT-EP-15 3.3 Do not run the pump to dryness. 4 .0 SU P P L IE S AND M A T E R IA L S_____________________________________ 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 IP M 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 F E R E N C E S ___________________________________________________ 6.1 There are no known interferences at this time. 7.0 SA M P L E H A N D L IN G ________________________________________________ 7.1 Keep the extracted samples in capped 15 mL centrifuge tubes or in capped autovials until ready for analysis. C025J 8.0 C A L IB R A T IO N AND S T A N D A R D IZ A T IO N _______________________ 8.1 Preparation of Calibration Standards 8.1.1 Seven beef liver standards and one blank beef liver are prepared during the extraction procedure. (See AMDT-M-4, section 8.0) 8.2 Calibration 8.2.1 Run the seven beef liver standards twice, starting with the lowest standard to obtain the standard curve. 8 .2 .2 Typically one standard is run after each 5 to 7 samples. Choose a standard in the same range of concentration as the samples. 8.3 Storage Conditions for Standards 8 .3 .1 Fresh standards are prepared with each analysis. Standards are stored in covered plastic centrifuge tubes until the analysis on the mass spectometer is performed. Samples and standards are NOT refrigerated. 8.4 Storage Conditions for Beef Liver Homogenates 8.4.1 Beef liver homogenates may be frozen after preparation. 9.0 P R O C E D U R E ________________________________________________________ 9.1 Initial Set-up 9 .1 .1 Set software to "Operate on", Ion Mode ES\ 9 .1 .2 Record backing pressure in the instrument log. 9 .1 .3 Fill the solvent cylinder with mobile phase. 9 .1 .4 Set the pump to "Run". Set the flow to 1000 uL/min. Observes droplets coming out of the tip of the probe. The pressure should be at 1700 to 1800 psi. 9 .1 .5 Check the fused silica at the end of the probe. Use an eye piece to check for chips. The tip should be flat with no jagged edges. If any chips are found cut off the tip of the silica with a column cutter and pull the silica through to the appropriate length. 9 .1 .6 Check your nitrogen supply. Tum 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. 9.1.11 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. G0 2 5 9 1 ? 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 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 10.2.1 See Method Validation Report number AMDT-M-5.0.V1 10.3 Other Validation Parameters 10.4 Refer to Method Validation Report Number AMDT-M-5.0.V 1 11.0 D A T A A N A L Y S IS___________________________________________________ 11.1 Calculations 11.2 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 liven mg FC-95 anion in the total rabbit liver = mg FC-95 anion from std. curve gms of liver used for analysis x Total mass of liver, gms 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. ** 12.0 ATTACH M ENTS None 13.0 REFERENCES 13.1 AMDT-EP-17 14.0 REVISIO NS Revision Number Reason for change Revision Date 3 M Environmental Laboratory Method A nalysis of Fluoride Using the Skalar Segm ented Flow A nalyzer 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: 0rpup Leader / 7 /A/?s Date Quality Assurance Date Software: IBM MS Word, 6.0 Affected Documents: AMDT-EP-26, Operation and Maintenance of the Skalar Segmented Flow Analyzer LQ. SCOPE_____________________________________ __ 1.1 This method is for the analysis for fluoride, thermally extracted from samples using the Dohnmann 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. 1.0.KEYWHRPg_______________________________________________ __ 2.1 Skalar, segmented flow, fluoride. 3.0 P R E C A U T IO N S _______________________________________________________ 3.1 Follow standard laboratory safety practices. 4 .0 SU P P L IE S A N D M A T E R IA L S____________________________________ __ 4.1 Supplies 4 .1 .1 Sample cups, 4 mL plastic cups with caps 4 .1 .2 Autopipets, oxford or equivalent with plastic tips 4 .1 .3 Polypropylene volumetric flasks, 100 mL 4 .1 .4 Cartridge components, refer to the Skalar Methods for components and part numbers. 4 .1 .5 Sample prefilters, Evergreen 4.2 Reagents 4 .2 .1 Brij 35, 30% S.F.A.S. Detergent 4 .2 .2 TISAB II buffer solution: Purchase TISAB II from Orion. To 1 liter of TISAB II add 2.5 mL or 100'ppm fluoride solution and 1 mL Brij. 4 .2 .3 Sampler rinsing solution: Dilute TISAB II 1:1 with Milli-Q water. 4 .2 .4 Nitric acid solution for decontamination, 1N (lab grade): Slowly add 64 mLs concentrated nitric acid (HN03) to 250 mLs of Milli-Q water. Bring the volume up to 1L 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 pipers, 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 - 6.15 0.03 ppm - 0.3 6.06 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 - 1.2 ppm ____________ U . P P T ____________ 1.2 1.5 - 5.Q .EQUIPMENT___________________________________ 5.1 Skalar Segmented Flow Auto Analyzer Sansptu' System equipped with ISE M IMTEREERENCES__________________________________ 6.1 High concentraons 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. 8.0 C A L IB R A T IO N AND S T A N D A R D IZ A T IO N ______________________ 8.1 Preparation of Calibration Standards 8 .1 .1 Prepare calibration standards as in section 4.3. 8.2 Calibration 8.2.1 The standards are analyzed at the beginning of the run. 8.3 Storage Conditions for Standards 8 .3 .1 Standards are stored in capped polypropylene volumetric flasks. New standards are prepared at a minimum of every six months, or as necessary. 9 .0 P R 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. \ 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 wh^rh 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 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. 4 602591^ 10.2 Precision and Accuracy 10.2. ISee Method Validation Report number AMDT-M-8.0.VI 10.3 Other Validation Parameters 10.4 Refer to Method Validation Report Number AMDT-M-8.0.VI 11.0 D A T A A N A L Y S IS_____________________________________________ _____ 11.1 Calculations 11.1. IThe standard curve is plotted by the Skalar software. 11.1.2 All calculations are done by the Skalar software, r2 should be 0.995 or 11.2 11.3 11.4 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 T T A C H M E N T S _____________________________________________________ None 13.0 R E F E R E N C E S _______________________________________________________ 13.1 AMDT-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 13.3 AMDT-EP-26, Operation and Maintenance of the Skalar Segmented Flow Analyzer 14.0 REVISIO NS Revision Murker Reasonfare-hangs Revision Date 3M Environmental Laboratory M ethod Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer - Serum Method Identification Number: AMDT-M-14 Revision Number: 0 Adoption Date: / o - 3 -*7 C" Revision Date: None Author: Rich Youngblom Approved by: roup Leader J% / Date 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 -t.e .&P2 "/afe* 00259g 1.0 SCOPE . APPLICABLE COM POUNDS. AND M ATRICES 1.1 Scope: This method is for the operation of a Dohrmann DX2000 when it is used to extract fluoride from various matrices. The fluoride is typically collected in TISAB solution for analysis with an ion selective electrode. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Biological fluids, particularly serum. 2.0 K E Y W O R D S__________________________________________________________ 2.1 Fluoride, fluorine, extraction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, DX2000, fluorochemicals. 3.0 P R E C A U T IO N S______________________________________________________ 3.1 Glassware and exhaust gases can be extremely hot. 3.2 Glassware is fragile, broken glass may cause injuries. 3.3 Pressurized gases, proper compressed gas handling practices required. 3.4 Solvent based samples may flash, may need to allow them to dry down before starting run. 3.5 Potential biohazards due to the biological matrices. Use appropriate personal protective equipment. 4.0 SU PPL IE S A N D M ATERIALS______________________________________ 4.1 Compressed Oxygen, Hydrocarbon free, regulated to 30 PSI. 4.2 Compressed Helium, High Purity Grade, regulated to. 45 PSI. 4.3 Quartz glass sample boat with TeflonTM tubing, Dohrmann 890-097 or equivalent. 4.4 Quartz glass combustion tube, Reliance Glass G-9405-012 or equivalent. 4.5 Orion 940999 Total Ionic Strength Adjustment Buffer (TISAB I I ) or equivalent. 4.6 Sample collection vials, HDPE. 4.7 Milli-QTM water 4.8 Polystyrene pipettes. 4.9 Activated Charcoal, E. Merck 2005 or equivalent. 4.10 Hamilton Syringe or equivalent. 4.11 Miscellaneous laboratory glassware 5.0 E Q U IP M E N T _______________________________________________________ 5.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer, modified for fluoride extraction. 5.2 IBM compatible 386 or 486 computer. 5.3 DX2000 software, version 1.00, modified for fluoride extraction. 5.4 Excel Spreadsheet, version 5.0 or greater 6.0 IN T E R F E R E N C E S__________________________________________________ 6.1 Sample size is limited to approximately 100 |il This may vary from matrix to matrix. 2 C Q 2 fffl 7.0 SAMPLE HANDLING 7.1 Samples are to be handled with plastic pipettes. A new pipette is to be used for each sample. 8.0 C A L IB R A T IO N AND STA N D A R D IZA TIO N ___________________________ 8.1 Preparation of Calibration Standards 8.1.1 The standards required for each project will need to be appropriate for that individual project. Refer to protocol for that project. 8.1.2 Typically 50-500 ppm FC-95 in methanol standards are used. 8.1.3 For rabbit serum studies, use beef serum as the matrix. 8.2 Calibration - Overview The normal calibration is the fluoride curve (AMDT-M-2). However, if an optional spiked serum curve is required the procedure listed below is used. 8.2.1 A calibration curve for the DX2000 is generated by spiking samples with known standards and combusting them using the same methods and matrix type as the samples to be tested. 8.2.2 Typically, three replicates of each standard and five concentrations of standards will be spiked. 8.2.3 Standard curve will be plotted as Mass Spiked F (ug) on the x-axis and Standard Mass Recovered F (ug) on the y-axis. Generate a regression curve and calculate the equation for the line and the r^ value. 8.2.4 Mass Spiked F (ug) = (Amount spiked in mL) x ( Cone, of standard in ppm) x (0.6004)* *FC-95 is 60.04% F therefore 0.6004 is the factor used to convert FC-95 to F 8.2.5 Standard Mass Recovered F (ug) = (TISAB volume in mL) x (Orion reading in ppm) 8.3 Calibration - Procedure 8.3.1 Start Up 8.3.1.1 Run 2 or more Clean Cycles when starting instrument each day. More clean cycles may be used if the previous samples contained high concentrations of fluoride. 8.3.2 Blanks 8.3.2.1 Prepare sample using the same methods and type of matrix as the test sample. 8.3.2.2 For rabbit studies, use beef serum as the matrix. 8.3.2.3 Put serum blank in Dohrmann boat. Combust sample as described in section 9.0 and analyze sample according to method AMDT-M-2 for the ion selective electrode analysis. 8.3.2.4 For rabbit studies, the meter reading for a blank sample should be 0.03 ppm or lower before proceeding with the calibration. Bum samples until 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 If beef serum is frozen, thaw at least enough to complete the standard curve analysis for the day (=30 mL). 8.3.3.2 Pipette 100|iL of beef serum into Dohrmann sample boat. 8.3.3.3 Start with the lowest standard concentration. Using a Hamilton syringe, eject a fixed quantity of the standard on or in the matrix. For rabbit studies, use 4 uL of standard and eject it on or in the beef serum. 8.3.3.4 At least 3 replicates should be used for the lowest standard concentration; more replicates may be used at the discretion of the analyst. 8.3.3.5 Combust the sample as described in section 9.3 and analyze according to AMDT-M-2. 8.3.3.6 Run all 15 standards. If one replicate is significantly different from the other two replicates, run another sample for that standard. Indicate in data that the new replicate replaces the old replicate and that the new replicate will be used to calculate the regression curve. 8.3.3.7 When all standards have been run, calculate the r^. r^ must be at least 0.95. If it is not at least 0.95, consult with supervisor. 8.3.3.8 A new standard curve should be run when the combustion tube or sample matrix is changed. New standard curve may also be run at the discretion of the analyst. 8.4 Storage Conditions for Standards 8.4.1 Storage requirements for standards are dependent on the individual standards used. Typically, standards are stored at room temperature in plastic screw top bottles. 8.4.2 New FC-95 standards should be prepared at least once a month. 9.0 PR O C E D U R E S____________________ ________________________________ 9.1 Typical Operating Conditions: 9.1.1 Combustion tube temperature = 950C. 9.1.2 Oxygen and Helium flow = 50 cc/minute. 9.1.3 Vaporization/Drying time = 240 seconds. 9.1.4 Bake time = 300 seconds. 9.2 Start Up Procedure: 9.2.1 If the program is not started, start the EOX program on the PC. 9.2.2 Open the SYSTEM SETUP window. 9.2.3 Put the furnace module and the cell in the READY mode. 9.2.4 Close the SYSTEM SETUP window. 9.2.5 When the oven has reached the READY temperature, run the CLEAN BOAT program found in the CELL CHECK menu. 9.2.6 See AMDT-EP-3 for details of the Dohrmann software. 9.3 Sample Extraction Procedure: 9.3.1 Open the SAMPLE HATCH and pipette lOOpL of sample into the BOAT. It may be necessary to mix approximately 50 mg of charcoal with the sample to aid combustion. If this is done, charcoal should also be mixed in while establishing the baseline and when generating the standard curve. 9.3.2 Close SAMPLE HATCH. 4 00260^ 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 mJL are used. For rabbit studies, use 1.0 or 2.0 mL of 1:1 TISAB:Milli-QTM water mixture. 9.3.4 Place the vial so that the tip of the COMBUSTION TUBE is in the TISAB at least 0.25 inches. Gases released during pyrolysis must bubble through the TISAB. 9.3.5 Run the EOX-WATER program found in the RUN menu. 9.3.6 When the EOX program is finished, remove the collection vial from the combustion tube. 9.3.7 If undiluted TISAB was used to collect the sample, add an equal volume of Milli-QTM water to the TISAB to make 1:1 TISAB:Milli-QTM. 9.3.8 Rinse the end of the combustion tube with Milli-QTM water and wipe with a 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 and/or vacuumed out. It may be necessary to scrap particles off the bottom with a spatula or other similar device. A drop of Milli-QTM water may be added to the boat to aid in the Clean Cycle. 9.3.10 Close the hatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample is ready for analysis by ion selective electrode (AMDT-M-2). 9.4 Sample Calculations 9.4.1 Use the standard curve to calculate the sample value. 9.4.2 Sample Mass Recovered F (ug) = (TISAB vol in mL) x /Orion reading in ppm - intercept) (Slope) 10.0 V A L ID A T IO N ________________________ 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 Other Validation Parameters: NA 11.0 DATA A N A LY SIS__________________________________________ 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. G0260JU 11.2 Analyzing the Data 11.2.1 must be at least 0.95 or greater. "Outliers" may be excluded if two of the three replicates are within 20% of each other and the outlier is greater than 200% of the average of those two or less than 50% of the average of those two. Any such outliers should be pointed out in the data and noted in the Final Report along with the reason it was considered an outlier. 12.0 A TTA C H M E N T S_____________________________________________________ None 13.0 R EFE R E N C E S________________________________________________________ 13.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer Operator's Manual (Manual 915349, revision B, December 1993) 13.2 AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 13.3 AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 R E V ISIO N S___________________________________________________________ Revision Number Reason for Change Revision Date 6 C0260J 9.3 Quality Assurance Unit Statement 0026040 Attachment D GLP Study Quality Assurance Statement Study Title: Single-dose Intravenous Pharmacokinetic Study of T-6067 in Rabbits Study Number: AMDT-120694.1 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 11-09-95 11-13-95 Phase_____________ Final Report Date Inspection Reported to Management Study Director 11-13-95 11-13-95 / / ' / 3 >V Date C02S0J* 9.4 Key Personnel Involved in the Study 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 V C Q ZG Q p r 9.11 Data C02G0 9.11.1 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. Summary of Combustion Data - Liver AMDT-120694.1, HWI 6329-151 As Referenced in Final Report section 6.0 D A T A A N A L Y S IS Total ug Fluoride in Whole Liver Mean per Dose Group Control Group 4.0 mg/kg dose (T6067) 16 mg/kg dose (T6067) 24 mg/kg dose (T6067) ug 19.7 42.9 66.4 54.4 002610 R P T 1 5 1 l.X L S L13492 PK ID liver blk 1 liver blk 2 liver blk 3 liver spike 1 liver spike 2 liver spike 3 F52762 -1 F52762 - 2 F52762 - 3 F52763 -1 F52763- 2 F52763- 3 F52768-1 F52768-2 F52768-3 F52770-1 F52770-2 F52770-3 liver spike 4 liver spike 5 Liver blank % rcvry 91% 94% 84% 96% 87% Actual ppm Fin liver (W/W) 0.501 0.286 0.262 1.08 1.00 0.985 0.384 0.245 0.242 0.732 0.483 0.524 0.727 0.794 0.803 0.806 0.775 0.776 1.07 1.19 0.228 Average ppm Fin liver (W/W) 0.290 0.580 0.775 0.786 liver burned (grams) 0.145 0.150 0.136 0.127 0.143 0.129 0.120 0.136 0.142 0.111 0.146 0.133 0.123 0.133 0.115 0.130 0.134 0.142 0.136 0.111 0.123 Whole liver weight (grams) 67.7 67.7 67.7 73.9 73.9 73.9 85.7 85.7 85.7 69.2 69.2 69.2 Total Fin whole liver (P9) 19.7 42.9 66.4 54.4 Dosage (mg/kg) 0.0 4.0 16 24 C 0 2 S li 9.11.2 Summary and raw data; ppm F` in serum as determined by thermal extraction followed by analysis using Orion ion analyzer. G0261jb H W I6329-151 AMDT 120694.1 Dohrmann Serum Analysis Analysis Dates: 07/27/95 - 07/31/95 All serum samples were thermally extracted by a modified Dohrmann D X 2000 Organic Halide Analyzer and collected in a 1:1 milli Q water and TISAB solution. The samples were measured on an Orion EA940 expandable ion analyzer. The Dohrmann was calibrated using 34ppm, 40ppm, 62ppm, lOOppm, 124ppm, 250ppm, and 500ppm FC-95 standards. The Orion was calibrated by direct measurement with no blank correction using 0.05ppm, O.lppm, 0.5ppm, l.Oppm and 1.5ppm F standards. The slope, intercept, and correlation were recorded in die appropriate logbook. A summary table is included, showing the ppm F ` in each sample (see page 2). The value o f "N D " has been entered for any sample with an Orion reading o f below 0.05. An initial calibration curve with standard deviation, %RSD, R2 value and equation o f the line is on pages 3 and 4. Pages 5 and 6 show the excel spreadsheet that was generated when the samples were being analyzed. Pages 7 and 8 show the same spreadsheet with "N D " inserted where the Orion reading is below 0.05. tu a w t* . aM w i ^ er^ Page 1 o f %1) T .6 . ^ L13492 PK Dosage: Omg/kg Dosage: 4 mg/kg Dosage: 16 mg/kg Dosage: 469-mg/kg BP- uH < e S T U D Y # 6329-151 SERUM HWI 6329-151 Fluoride concentration in rabbit serum (ppm F-) Sample F52762 F52763 F 52768 F52770 2.0 hour 4.0 hour 6.0 hour 8.0 hour 12.0 hour 24.0 hour 48.0 hour 1.25 5.84 ND 3.56 2.98 1.22 ND 4.09 3.25 2.27 1.89 2.01 1.41 ND 14.9 8.05 4.39 2.87 2.72 ND ND 41.0 19.4 12.7 8.08 4.40 ND ND 00261^ 10.00 151CHART.XLS Chart 12 4 mg/kg ?Vr I/ - i v s r Cone. 1.00 10 15 20 25 time (hours post dose) fT9 2 0 0 100.0 OO 10.0 1.0 il 151CHART.XLS Chart 11 16 mg/kg Ai ir- n -v 468 time (hours post dose) 10 12 Page 1 100.0 10.0 1.0 I Cone. 151CHART.XLS Chart 10 2 4 mg/kg IV h <\-'s 468 time (hours post dose) 10 12 NORMAN SERUM CURVE 1 Sample IO 34-ppm-1 34-ppm-2 34-ppm-3 40-ppm-1 40-ppm-2 40-ppm-3 40-ppm-4 62-ppm-1 62-ppm-2 62-ppm-3 100-ppm-1 100-ppm-2 100-ppm-3 124-ppm-1 124-ppm-2 124-ppm-3 250-ppm-1 250-ppm-2 250-ppm-3 500-ppm-1 500-ppm-2 500-ppm-3 Actual Sample reading Qty (PPm F*> (mL or o) 0.07175 0.1 0.05614 0.1 0.06462 0.1 0.08668 0.1 0.06728 0.1 0.05939 0.1 0.06385 0.1 0.07291 0.0753 0.1 0.1 0.07839 0.0902 0.1026 0.1 0.1 0.1 0.1126 0.1 0.1371 0.1 0.1451 0.1 0.1617 0.3217 0.1 0.1 0.2447 0.3078 0.1 0.1 0.4438 0.4584 0.4888 0.1 0.1 0.1 TISA8 final voi (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 $p$ed 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 Cone FC95 % solution recovery (ppm) ....<& 34 176% 34 138% 34 158% 40 180% 40 140% 40 124% 40 133% 62 98% 62 101% 62 105% 100 75% 100 85% 100 94% 124 92% 124 97% 124 109% 250 107% 250 82% 250 103% 500 74% 500 76% 500 81% Actual ppm F in sample 1.4350 1.1228 1.2924 1.7336 1.3456 1.1878 1.2770 1.4582 1.5060 1.5678 1.8040 2.0520 2.2520 2.7420 2.9020 3.2340 6.4340 4.8940 6.1560 8.8760 9.1680 9.7760 Mass spiked (03 F-> 0.0817 0.0817 0.0817 0.0961 0.0961 0.0961 0.0961 0.1489 0.1489 0.1489 0.2402 0.2402 0.2402 0.2978 0.2978 0.2978 0.6004 0.6004 0.6004 1.2008 1.2008 1.2008 Mass recovered (U0 F") 0.1435 STDEV: 0.11228 %RSD: 0.12924 0.17336 STDEV: 0.13456 %RSD: 0.11878 0.1277 0.14582 STDEV: 0.1506 %RSD: 0.15678 0.1804 STDEV: 0.2052 %RSD: 0.2252 0.2742 STDEV: 0.2902 %RSD: 0.3234 0.6434 STDEV: 0.4894 %RSD: 0.6156 0.8876 STDEV: 0.9168 %RSD: 0.9776 0.015629 12 0.024057 17 0.005495 3.6 0.022443 11 0.025096 8.5 0.082072 14 0.045915 5.0 SERUM CURVE 1 NORMAN (07/25/95) 'tr C 0261 b SERUM CURVE 1 NORMAN (07/25/95) 1.4000 S T U D Y # 6329-151 SERUM Sample ID BLANK-1 BLANK-2 BLANK-3 BLANK-4 BLANK-5 BLANK-6 62-PPM-1 62-PPM-2 62-PPM-3 62-PPM-4 62-PPM-5 62-PPM-6 250-PPM-1 250-PPM-2 250-PPM-3 F52762-2.0HR F52763-2.0HR F52768-2.0HR F52770-2.0HR F52762-4.0HR F52763-4.0HR F52768-4.0HR F52770-4.0HR 62-PPM-1 62-PPM-2 250-PPM-1 250-PPM-2 62-PPM-3 SERUM BLANK SERUM BLANK SERUM BLANK SERUM BLANK SERUM BLANK SPIKE 62-1 SPIKE 62-2 SPIKE 62-3 SPIKE 250-1 SPIKE 250-2 SPIKE 250-3 SERUM BLANK SERUM BLANK F52762-6HR F52763-6HR F52768-6HR F52770-6HR F52762-8HR F52763-8HR F52768-8HR Actual reading (ppm F-) 0.0507 0.0355 0.0344 0.0308 0.0353 0.0290 0.0856 0.110 0.0835 0.0778 0.0746 0.0718 0.190 0.213 0.223 0.0626 0.205 0.746 2.05 0.292 0.163 0.402 0.971 0.189 0.135 0.221 0.246 0.130 0.0458 0.0352 0.0340 0.0358 0.0350 0.0628 0.0689 0.0715 0.155 0.261 0.217 0.0936 0.0289 0.0222 0.114 0.220 0.636 0.178 0.094 0.144 Sample Qty (m Lorg) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 TISAB final vol (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 Cone. FC95 spiked solution (ppm) 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 62 62 62 62 62 62 250 250 250 0.004 0.004 0.004 0.004 0.004 62 62 250 250 62 0.004 0.004 0.004 0.004 0.004 0.004 62 62 62 250 250 250 % recovery (ug/ug) 115% 148% 112% 104% 100% 96% 63% 71% 74% 254% 181% 74% 82% 174% 84% 92% 96% 52% 87% 72% Actual ppm Fin sample 1.01 0.710 0.689 0.617 0.707 0.581 1.71 2.20 1.67 1.56 1.49 1.44 3.80 4.27 4.47 1.25 4.09 14.9 41.0 5.84 3.25 8.05 19.4 3.78 2.70 4.42 4.92 2.60 0.916 0.703 0.680 0.715 0.700 1.26 1.38 1.43 3.10 5.22 4.34 1.87 0.577 0.445 2.27 4.39 12.7 3.56 1.89 2.87 Mass spiked (ug F-) 0.15 0.15 0.15 0.15 0.15 0.15 0.60 0.60 0.60 0.15 0.15 0.60 0.60 0.15 0.15 0.15 0.15 0.60 0.60 0.60 Mass recovered (ug F-) 0.101 0.0710 0.0689 0.0617 0.0707 0.0581 0.171 0.220 0.167 0.156 0.149 0.144 0.380 0.427 0.447 0.125 0.409 1.49 4.10 0.584 0.325 0.805 1.94 0.378 0.270 0.442 0.492 0.260 0.0916 0.0703 0.0680 0.0715 0.0700 0.126 0.138 0.143 0.310 0.522 0.434 0.187 0.0577 0.0445 0.227 0.439 1.27 0.356 0.189 0.287 S T U D Y # 6329-151 SERUM Sample ID F52770-8HR F52762-12HR F52763-12HR SPIKE 62-1 SPIKE 250-1 SPIKE 250-2 F52768-12HR F52770-12HR F52762-24HR F52763-24HR F52768-24HR F52770-24HR F52762-48HR F52763-48HR F52768-48HR F52770-48HR BLANK-1 BLANK-2 SPIKE 62-1 SPIKE 62-2 SPIKE250-1 SPIKE250-2 SPIKE250-3 Actual reading (ppm F-) 0.404 0.149 0.101 0.0920 0.187 0.225 0.136 0.220 0.0609 0.0704 0.0442 0.0387 0.0282 0.0264 0.0260 0.0357 0.0830 0.0314 0.0635 0.0644 0.157 0.239 0.240 Sample Qty (mL or g) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 TISAB final vol (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 Cone. FC95 spiked solution ..<PPm> 0.004 0.004 0.004 62 250 250 0.004 0,004 0.004 0.004 0.004 62 62 250 250 250 % recovery (ug/ug) 124% 62% 75% 85% 86% 52% 80% 80% Actual ppm Fin sample 8.08 2.98 2.01 1.84 3.74 4.51 2.72 4.40 1.22 1.41 0.883 0.775 0.564 0.527 0.520 0.715 1.66 0.627 1.27 1.29 3.15 4.78 4.80 Mass spiked (ug F-) 0.15 0.60 0.60 0.15 0.15 0.60 0.60 0.60 Mass recovered (ug F-) 0.808 0.298 0.201 0.184 0.374 0.451 0.272 0.440 0.122 0.141 0.0883 0.0775 0.0564 0.0527 0.0520 0.0715 0.166 0.0627 0.127 0.129 0.315 0.478 0.480 S T U D Y # 6329-151 SERUM Sample ID BLANK-1 BLANK-2 BLANK-3 BLANK-4 BLANK-5 BLANK-6 62-PPM-1 62-PPM-2 62-PPM-3 62-PPM-4 62-PPM-5 62-PPM-6 250-PPM-1 250-PPM-2 250-PPM-3 F52762-2.0HR F52763-2.0HR F52768-2.0HR F52770-2.0HR F52762-4.0HR F52763-4.0HR F52768-4.0HR F52770-4.0HR 62-PPM-1 62-PPM-2 250-PPM-1 250-PPM-2 62-PPM-3 SERUM BLANK SERUM BLANK SERUM BLANK SERUM BLANK SERUM BLANK SPIKE 62-1 SPIKE 62-2 SPIKE 62-3 SPIKE 250-1 SPIKE 250-2 SPIKE 250-3 SERUM BLANK SERUM BLANK F52762-6HR F52763-6HR F52768-6HR F52770-6HR F52762-8HR F52763-8HR F52768-8HR Actual reading (ppm F-) 0.0507 0.0355 0.0344 0.0308 0.0353 0.0290 0.0856 0.110 0.0835 0.0778 0.0746 0.0718 0.190 0.213 0.223 0.0626 0.205 0.746 2.05 0.292 0.163 0.402 0.971 0.189 0.135 0.221 0.246 0.130 0.0458 0.0352 0.0340 0.0358 0.0350 0.0628 0.0689 0.0715 0.155 0.261 0.217 0.0936 0.0289 0.0222 0.114 0.220 0.636 0.178 0.094 0.144 Sample Qty (mL org) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 TISAB final vol (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 Cone. FC95 spiked solution (ppm) 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 62 62 62 62 62 62 250 250 250 0.004 0.004 0.004 0.004 0.004 62 62 250 250 62 0.004 0.004 0.004 0.004 0.004 0.004 62 62 62 250 250 250 % recovery (ug/ug) 115% 148% 112% 104% 100% 96% 63% 71% 74% 254% 181% 74% 82% 174% 84% 92% 96% 52% 87% 72% Actual ppm Fin sample 1.01 ND ND ND ND ND 1.71 2.20 1.67 1.56 1.49 1.44 3.80 4.27 4.47 1.25 4.09 14.9 41.0 5.84 3.25 8.05 19.4 3.78 2.70 4.42 4.92 2.60 ND ND ND ND ND 1.26 1.38 1.43 3.10 5.22 4.34 1.87 ND ND 2.27 4.39 12.7 3.56 1.89 2.87 Mass spiked (ug F-) 0.15 0.15 0.15 0.15 0.15 0.15 0.60 0.60 0.60 0.15 0.15 0.60 0.60 0.15 0.15 0.15 0.15 0.60 0.60 0.60 Mass recovered (ug F-) 0.101 ND ND ND ND ND 0.171 0.220 0.167 0.156 0.149 0.144 0.380 0.427 0.447 0.125 0.409 1.49 4.10 0.584 0.325 0.805 1.94 0.378 0.270 0.442 0.492 0.260 ND ND ND ND ND 0.126 0.138 0.143 0.310 0.522 0.434 0.187 ND ND 0.227 0.439 1.27 0.356 0.189 0.287 S T U D Y # 6329-151 SERUM Sample ID F52770-8HR F52762-12HR F52763-12HR SPIKE 62-1 SPIKE 250-1 SPIKE 250-2 F52768-12HR F52770-12HR F52762-24HR F52763-24HR F52768-24HR F52770-24HR F52762-48HR F52763-48HR F52768-48HR F52770-48HR BLANK-1 BLANK-2 SPIKE 62-1 SPIKE 62-2 SPIKE250-1 SPIKE250-2 SPIKE250-3 Actual reading (ppm F-) 0.404 0.149 0.101 0.0920 0.187 0.225 0.136 0.220 0.0609 0.0704 0.0442 0.0387 0.0282 0.0264 0.0260 0.0357 0.0830 0.0314 0.0635 0.0644 0.157 0.239 0.240 Sample Qty (mL or g) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 TISAB final vol (mL) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 mL FC95 Cone. FC95 spiked solution (ppm) 0.004 0.004 0.004 62 250 250 0.004 0.004 0.004 0.004 0.004 62 62 250 250 250 % recovery (ug/ug) 124% 62% 75% 85% 86% 52% 80% 80% Actual ppm Fin sample 8.08 2.98 2.01 1.84 3.74 4.51 2.72 4.40 1.22 1.41 ND ND ND ND ND ND 1.66 ND 1.27 1.29 3.15 4.78 4.80 Mass spiked (ug F-) 0.15 0.60 0.60 0.15 0.15 0.60 0.60 0.60 Mass recovered (ug F-) 0.808 0.298 0.201 0.184 0.374 0.451 0.272 0.440 0.122 0.141 ND ND ND ND ND ND 0.166 ND 0.127 0.129 0.315 0.478 0.480 G0263jf 9.11.3 Summary and raw data; ppm F` in serum as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. UCu3'" /*/qS RE: 6329-151 SERUM SAMPLES RMDT 120694.1 Date of Rnalysis: 8/2/95 Analyst: DDLU The samples are burned in the Dohrman at 950 C using 0.10 mL of the serum. The gas is collected in 2.0 mL of 1:1 TISRB/Milli-Q water. The samples are then analyzed on a Skalar Segmented Flow Analyzer using the Ion Specific Electrode (ISE) Method. TISAB buffer is added to each sample as it proceeds through the system. The sample then goes through a heated mining coil before the potential between the ion selectiue electrode and the reference electrode is measured. The signal is amplified and related to the fluoride concentration. The instrument was calibrated in the ranges of 0.015 - 0.15 ppm and 0.15 - 1.50 ppm fluoride. The standard curue for the high range was plotted using the inuerse logarithm option. The standard curue for the low range is linear. All standards and samples were then calculated by the Skalar software using these curues. All results below 0.0001 ppm appear on the raw data as # . # # # # . A quality control standard was analyzed euery 10 samples to check for accuracy and drift. Raw data is taken from the appropriate calibrated range of the Skalar printout and summarized on an Excel spreadsheet. The final results are adjusted for the collection uolume and any subsequent dilutions. SUMMARY OF 6329-151 SERUM SAMPLES AMDT 120694.1 OCko W iV 5 Dose Level: 0 F52762 1.56 6.18 ND 3.96 3.44 1.43 0.55 Dose Level: 4 mg/kg F52763 4.10 3.44 2.49 2.24 2.51 1.60 0.46 Dose Level: 16 mg/kg F52768 15.6 8.08 4.72 3.36 2.94 0.96 0.57 2*f Dose Level: JHne/kg _ F52770 32.9 20.2 14.8 9.26 4.62 0.80 0.82 * v-s, Ol'C^OC-^ 1 oryri 6329151 S.SUM Page 1 C-O^G^ SERUM CURVE 1 7-31-95 NORMAN licu *|l-WS S am ple ID Spk 34-1 Spk 34-2 Spk 34-3 Spk 40-1 Spk 40-2 Spk 40-3 Spk 62-1 Spk 62-2 Spk 62-3 Spk 100-1 Spk 100-2 Spk 100-3 Spk 124-1 Spk 124-2 Spk 124-3 Spk 250-1 Spk 250-2 Spk 250-3 Spk 500-1 Spk 500-2 Spk 500-3 Skalar Result (ppm ) DLTISAB mL FC 95 Cone final voi Solution FC 95 Soin (m L ) Spiked (ppm ) M ass Spiked (ug F-) M ass % Recovered Recovery (ug F-) 0.09 0.07 0.08 2.0 0.004 34.00 0.08 0.17 211% STANDARD DEVIATION : 0.2450 2.0 0.004 34.00 0.08 0.13 163% % RSD : 12.9998 2.0 0.004 34.00 0.08 0.16 191% 0.08 0.07 0.07 2.0 0.004 40.00 0.10 0.16 164% STANDARD DEVIATION : 0.0826 2.0 0.004 40.00 0.10 0.14 147% % RSD : 5.3307 2.0 0.004 40.00 0.10 0.15 154% 0.09 0.09 0.09 2.0 0.004 62.00 0.15 0.18 120% STANDARD DEVIATION : 0.0263 2.0 0.004 62.00 0.15 0.18 119% % RSD : 2.1670 2.0 0.004 62.00 0.15 0.18 124% 0.11 0.12 0.13 2.0 0.004 100.0 0.24 0.21 88% STANDARD DEVIATION : 0.1138 2.0 0.004 100.0 0.24 0.24 100% % RSD : 11.4530 2.0 0.004 100.0 0.24 0.27 110% 0.16 0.17 0.18 2.0 0.004 124.0 0.30 0.32 108% STANDARD DEVIATION : 0.0778 2.0 0.004 124.0 0.30 0.34 114% % RSD : 6.7516 2.0 0.004 124.0 0.30 0.37 124% 0.33 0.26 0.32 2.0 0.004 250.0 0.60 0.67 111% STANDARD DEVIATION : 0.1318 2.0 0.004 250.0 0.60 0.52 87% % RSD : 12.9196 2.0 0.004 250.0 0.60 0.65 108% 0.47 0.49 0.52 2.0 0.004 500.0 1.20 0.94 78% STANDARD DEVIATION : 0.0442 2.0 0.004 500.0 1.20 0.99 82% % RSD : 5.3672 2.0 0.004 500.0 1.20 ' 1.04 87% SERUM CURVE 1 (NORMAN) 7/31/95 y = 0.7743X + 0.086 Rs= 0.9762 SERCRV1N.SUM TPage 1 00262 3 6329151S.XLS 1995-08-02 13:20 OutPut of : 950802B1 Operator : DDW Date of the Analysis : 1995-08-02 08:38 Analysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950802B1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 r > 18 19 r o 20 m 21 22 0 0 23 1. 24 25 1 26 I * 27 1 28 m 29 ?v 30 Tracer Drift Wash Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 Standard 9 Standard 10 Drift Wash SERUM BLK 1 SERUM BLK 2 SERUM BLK 3 SERUM BLK 4 SERUM BLK 5 SERUM BLK 6 SPK62-1 SPK 62-2 SPK 62-3 SPK 62-4 Drift Wash SPK 62-5 SPK 62-6 SPK 250-1 1.50 1.50 0.015 0.03 0.06 0.09 0.12 0.15 0.30 0.60 1.20 1.50 1.50 1.50 1.48 1.48 ND 0.017 0.03 0.06 0.09 0.12 0.15 0.28 0.62 1.23 1.47 1.49 ND 0.07 0.05 0.05 0.04 0.05 0.04 0.11 0.12 0.10 0.09 1.47 ND 0.09 0.09 0.20 98% 99% 111% 92% 101% 101% 99% 100% 93% 103% 102% 98% 99% 98% 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 2.0 0.10 Page 1 1.39 0.97 0.96 0.80 0.96 0.78 2.17 0.004 62.00 0.15 2.45 0.004 62.00 0.15 1.94 0.004 62.00 0.15 1.89 0.004 62.00 0.15 0.22 146% 0.25 165% 0.19 131% 0.19 127% 1.84 0.004 62.00 0.15 1.74 0.004 62.00 0.15 3.96 0.004 250.00 0.60 0.18 124% 0.17 117% 0.40 66% 31 SPK 250-2 0.22 32 SPK 250-3 0.23 33 F52762-2 0.08 34 F52763-2 0.21 35 F52768-2 0.78 36 F52770-2 1.65 37 F52762-4 0.31 38 Drift 1.50 1.47 98% 39 Wash ND 40 F52763-4 0.17 41 F52768-4 0.40 42 F52770-4 1.01 43 SPK 62-1 0.26 44 SPK 62-2 0.15 45 SPK 250-1 0.23 46 SPK 250-2 0.25 47 SPK 62-3 0.15 48 SERUM BLK 1 0.06 49 SERUM BLK 2 0.04 50 Drift 1.50 1.42 95% 51 Wash ND 52 SERUM BLK 3 0.05 53 SERUM BLK 4 0.04 54 SERUM BLK 5 0.04 55 SPK 62-1 0.07 56 SPK 62-2 0.08 57 SPK 62-3 0.08 58 SPK 250-1 0.17 59 SPK 250-2 0.28 60 SPK 250-3 0.23 61 BLK 1 0.11 62 Drift 1.50 1.38 92% 63 Wash ND 64 BLK 2 0.04 65 BLK 3 0.02 66 F52762-6 ND 6329151S.XLS 2.0 0.10 4.40 0.004 250.00 0.60 0.44 73% 2.0 0.10 4.54 0.004 250.00 0.60 0.45 76% 2.0 0.10 1.56 2.0 0.10 4.10 2.0 0.10 15.58 2.0 0.10 32.94 2.0 0.10 6.18 2.0 0.10 3.44 2.0 0.10 8.08 2.0 0.10 20.18 2.0 0.10 5.18 0.004 62.00 0.15 0.52 348% 2.0 0.10 2.97 0.004 62.00 0.15 0.30 199% 2.0 0.10 4.52 0.004 250.00 0.60 0.45 75% 2.0 0.10 5.08 0.004 250.00 0.60 0.51 85% 2.0 0.10 2.93 0.004 62.00 0.15 0.29 197% 2.0 0.10 1.20 2.0 0.10 0.90 2.0 0.10 0.92 2.0 0.10 0.88 2.0 0.10 0.80 2.0 0.10 1.47 0.004 62.00 0.15 0.15 99% 2.0 0.10 1.62 0.004 62.00 0.15 0.16 109% 2.0 0.10 1.65 0.004 62.00 0.15 0.17 111% 2.0 0.10 3.38 0.004 250.00 0.60 0.34 56% 2.0 0.10 5.64 0.004 250.00 0.60 0.56 94% 2.0 0.10 4.62 0.004 250.00 0.60 0.46 77% 2.0 0.10 2.15 2.0 0.10 2.0 0.10 2.0 0.10 Page 2 0.74 0.47 ND Sample 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 Skater Sample ' liii F52763-6 F52768-6 F52770-6 F52762-8 F52763-8 F52768-8 F52770-8 Drift Wash F52762-12 F52763-12 SPK 62-1 SPK 250-1 SPK 250-2 F52768-12 F52770-12 F52762-24 F52763-24 F52768-24 Drift Wash F52770-24 F52762-48 F52763-48 F52768-48 F52770-48 Drift Wash 1.50 1.50 1.50 cicalar iii 6329151S.XLS PPMM M Bp iM 0.12 2.0 0.24 2.0 0.74 2.0 0.20 2.0 Oil 2.0 0.17 2.0 0.46 2.0 1.43 95% ND 0.17 2.0 0.13 2.0 0.12 2.0 0.21 2.0 0.24 2.0 0.15 2.0 0.23 2.0 0.07 2.0 0.08 2.0 0.05 2.0 1.39 93% ND 0.04 2.0 0.03 2.0 0.02 2.0 0.03 2.0 0.04 2.0 1.43 95% ND 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 ffS S S G 'D Page 3 2.49 4.72 14.80 3.96 2.24 3.36 9.26 3.44 2.51 2.48 0.004 62.00 0.15 4.18 0.004 250.00 0.60 4.80 0.004 250.00 0.60 2.94 4.62 1.43 1.60 0.96 0.25 166% 0.42 70% 0.48 80% 0.80 0.55 0.46 0.57 0.82 1955-08-02 13:20 OutPut of : 95000211 Software : version 8.1 cl990,93 Operator : SDK Date of tie Analysis : 1995-08-02 08:38 Analysis File Xaie : C:\S1ALAB\DATA\BBIDATA\SBEDII\950802B1 Fluoride 1.5 Calibration order = Inverse Logaritfaa Slop : t - t.tfftf r X - Cl Sesult = 10L s x = corrected value of the saiple cl ^ corrected value of the concentration 1 s = Slope of the electrode a2 * -0.00000 al = 0.00070 a0 = -1.23789 Fluoride L Calibration order : 2 Coitthtioa : r = O.iSSiS Sesult = a2 * x* t al ` x + aO a2 = -0.00000 al = 0.00025 aO = -0.00103 Saipler lype :SA1000 Buiber :1 Saiple lite :50 sec. Vash Tiie :120 sec. Air Tiie :1 sec. Tabe up :Single special :Bone needle Beigbt :70 u . Diluter needle Beigbt : 80 u dilution Factor : 10 dilution Voluie : 2.5 il. Besaiple :1 Dilution runs : 1 User file : Beproces : Bo . TIT c o o *MqS Ak d t WjCXA' .1 H ^ x b izq -eS) Ss/w 'v. co2q 7 If95-08-12 13:20 Output of : 950802B1 fluoride 1. Patii ouiber Signal type Decolor systei Suiter dilute Sesaiple dii Threshold diS output Wiodov event 3 Dehuhhled Tes 0 So So 4095 0 Off si sTaodard Ignore s2 standard Ignore $3 sTaodard Ignore s4 sTaodard Ignore s5 sTaodard Ignore s6 sTaodard 0.150 s7 sTaodard 0.300 s8 sTaodard 0.500 s9 sTaodard 1.200 slO sTaodard 1.500 Order : Inverse Logarithi Dimension : PPM start Falue : 500 DO trigger Liiit : 1800 Sec Peak shape : Pointed stArt ignore : 80 Sec eSd ignore : 120 Sec Measure viodov : 75 1 Filter : So Segeneratioo : So fonila : output : FI.IFF Fluoride L Path nuiher Signal type Decolor systei Suiter dilute Sesaiple dii Threshold diG output Kindow event 0 Dehuhhled So 0 So So 4095 0 Off 00263 1995-08-02 13:20 OatPot of : 15000211 si sTandard 0.015 s2 standard 0.030 S3 standard 0.050 si standard 0 .0 1 0 s5 standard 0 .1 2 0 si standard 0.150 s7 standard Ignore s8 standard Ignore s9 standard Ignore slO standard Ignore Order : 2 Diiension : PPM start Value : 500 DO trigger Liiit : 1800 Sec Peak shape : Pointed stArt ignore : (0 Sec eld ignore : 120 Sec Measure vindov : 75 t filter : lo legeaeration : lo foriOla : c4:=c3 output : t t l l l 02G 3 1995-08-02 13:20 OntPnt of : 9500(211 Pigolili 1.5 m Ploorii L m Poi Tpp Ident Ck le s a lt P T in Ck l sa it p Tin vt iv Initial liasli 3 0.058 1 t Tracer 3 1.475 2 d Drift 3 1.481 3V Wash 3 0.058 4 1 Standard 1 3 0.065 5 s2 Standard 2 3 0.069 5 S3 Standard 3 3 0.086 7 S4 Standard 4 3 0.105 8 s5 Standard 5 3 0.126 5 s6 Standard 6 3 0.156 10 s7 Standard 7 3 0.280 11 s8 Standard 8 3 0.618 12 s9 Standard 9 3 1.228 13 slO Standard 10 3 1.469 14 d Drift 3 1.489 15 1 Wash 3 0.058 15 u SIIOK 8LI 1 3 0.091 17 u S i m BLI 2 3 0.080 18 u S I N K 11 3 3 0.079 19 u S110K BLI 4 3 0.075 20 u SEBUM BLI 5 3 0.079 21 u SilOH BLI 6 3 0.075 22 u SPI 62-1 3 0.118 23 g SPI 62-2 3 0.129 24 u SPI 62-3 3 0.109 25 u SPI 62-4 3 0.107 26 d Drift 3 1.474 27 V Vast 3 0.058 28 a SPI 62-5 3 0.106 29 g SPI 62-6 3 0.102 30 ti SPI 250-1 3 0.198 31 u SPI 250-2 3 0.220 32 u SPI 250-3 3 0.227 33 u P52762-2 3 0.096 34 a P52763-2 3 0.205 35 u F52768-2 3 0.779 36 a P52770-2 3 1.647 37 g P52762-4 3 0.309 38 d Drift 3 1.474 39 V Vasti 3 0.058 <0 u P52763-4 3 0.172 41 u F52768-4 3 0.404 42 u P52770-4 3 1.009 43 u SPI 62-1 3 0.259 44 a SPI 62-2 3 0.154 45 g SPI 250-1 3 0.226 46 u SPI 250-2 3 0.254 47 u SPI 62-3 3 0.152 48 u S U O BLI 1 3 0.086 49 u SI10M BLI 2 3 0.078 50 d Drift 3 1.418 51 V Vasti 3 0.058 52 u SE!OH BLI 3 3 0.078 53 u SEBUM BLI 4 3 0.077 65 4 f.fill 209 4 0.3996 385 4 0.3995 624 4 U H I 735 4 0.0167 911 4 0.0275 1085 4 0.0606 1262 4 0.0912 1436 4 0.1187 1612 4 0.1503 1786 4 0.2336 1962 4 0.3348 2136 4 0.3960 2310 4 0.3997 2486 4 0.3994 2700 4 1.1*11 2836 4 0.0696 3011 4 0.0486 3187 4 0.0481 3363 4 0.0400 3533 4 0.0479 3711 4 0.0388 3887 4 0.1087 4063 4 0.1225 4237 4 0.0972 4414 4 0.0945 4588 4 0.3996 4829 4 * .# * 4939 4 0.0921 5113 4 0.0870 5287 4 0.1849 5465 4 0.2000 5639 4 0.2048 5810 4 0.0782 5989 4 0.1902 6165 4 0.3598 (339 4 0.3919 6515 4 0.2471 6(89 4 0.3996 6918 4 U H I 7039 4 0.1647 7214 4 0.2828 7389 4 0.3833 7567 4 0.2232 7740 4 0.1483 7914 4 0.2041 8090 4 0.2203 3266 4 0.1467 8439 4 0.0599 8616 4 0.0448 8790 4 0.3999 9016 4 I H M 9139 4 0.0462 9315 4 0.0441 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Pag 1 of 2 0 002S3(f 1995-08-02 13:20 OutPnt of : 05080211 Fluorido 1.5 m Fluoride l m Pos Tip Ideat Ch lault F Tiu C8 Beault F Tiae 54 u SKBOH BOI 5 3 0.075 9491 4 0.0400 55 u SPI 62-1 3 0.093 9665 4 0.0734 56 u SPI 62-2 3 0.098 9841 4 0.0809 57 u SPI 62-3 3 0.099 10016 4 0.0825 58 u SPI 250-1 3 0.169 10191 4 0.1621 5 u SPI 250-2 3 0.282 10366 4 0.2347 60 u SPI 250-3 3 0.231 10539 4 0.2071 61 u BLI 1 3 0.117 10715 4 0.1074 62 d Drift 3 1.382 10890 4 0.3997 63 V Wash 3 0.058 11011 4 i.HH 64 u BLI 2 3 0.074 11242 4 0.0369 65 u BLI 3 3 0.068 11414 4 0.0236 66 u P52762-6 3 0.063 11588 4 0.0122 67 u F52763-6 3 0.131 11766 4 0.1244 68 u F52768-6 3 0.236 11940 4 0.2099 69 u F52770-6 3 0.740 12116 4 0.3546 70 1 F52762-8 3 0.198 12292 4 0.1849 71 tt F52763-8 3 0.121 12(66 4 0.1121 72 u F52768-8 3 0.168 126(0 4 0.1614 73 u F52770-8 3 0.463 12817 4 0.3001 74 d Drift 3 1.428 12990 4 0.3999 75 V Naso 3 0.058 13230 4 f.flif 76 u F52762-12 3 0.172 13341 4 0.1647 77 u F52763-12 3 J.132 13517 4 0.1256 78 11 SPI 62-1 3 0.130 13691 4 0.1238 79 u SPI 250-1 3 0.209 13867 4 0.1924 80 11 SPI 250-2 3 0.240 14043 4 0.2121 81 u F52768-12 3 0.152 14217 4 0.1469 82 u F52770-12 3 0.231 14391 4 0.2072 83 u F52762-24 3 0.092 14561 4 0.0714 84 u F52763-24 3 0.098 14743 4 0.0802 85 11 F52768-24 3 0.079 1(918 4 0.0479 86 d Drift 3 1.388 15091 4 0.3998 87 V Bash 3 0.058 15223 4 u t n 88 u F52770-24 3 0.075 15444 4 0.0400 89 u F52762-48 3 0.070 15618 4 0.0277 90 u F52763-48 3 0.067 15790 4 0.0231 91 u F52768-48 3 0.070 15964 4 0.0285 92 u F52770-48 3 0.076 16142 4 0.0412 93 d Drift 3 1.432 16318 4 0.3999 94 V Basil 3 0.058 16545 < t . t m at rw BuuOut Wash 3 0.058 16793 4 I.SHI 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 2 of 2 C0263f C a lib ra tio n curve o f 950802B1 : Flu o rid e L uoi^e^^uaouoQ 0 ti Order : 2 Measured 900 r 0.99908 n 02630 1.7683598 -f C a lib ra tio n curve o f 950802B1 ' Fluoride 1.5 Concentrat ion I 0 .0 5 7 8 2 5 3 r' 0 Measured Order : Inverse Logarithm 6068 4095 C Q 263jT Raw d a ta o f 950802B1 ' F lu o r id e 1 .5 Esc=Exit i F i= H e ip Crtl-P=Edit p eaks i G 0263g C02GJ9 lihUli!:!: 3 4095 - Raw data of 950802B1 : Fluoride 1.5 Tine: 19016 1 Ualue: 1 176 1D"elta: [64 iA ID 3- \> i'. 'I t\ ft it JlMlljUl A .& \i\ m i 8935 ii i T ine tsc=Exit M=Heip Crtl-P=Edit peaks ! i *QH I fl 111 13935 CO2540 n 00264/ 9.11.4 Summary and raw data; analysis o f liver extracts using electrospray mass spectrometry. This data, although supportive, in the opinion o f the Study Director is not required to reach the conclusion stated in Final Report Section 6.0, and therefore is not discussed in detail. 'J&rvu^v A"t> OCC O ' o-*7\ VJO - &J AA- r Af e LG: # . 'Ttvr-S ^nX>Y . ^ yiece. -slv ^ ^ <,cre-t^--i 'Car' 'O.aV qLOO^^^0^ * ^ . tA' HV2> ic#V o v ^ a^c5 < 0* o o s^eeeoi (^6r File:080195F LAB-BASE - The MS Data System Sample:HUItt 6329-151: L13492 CPK) 01/08/1995 14:58 4oJT--a= k 3 2 9 - lS l Cwvp^ M.OOE : SCnI $" CpK0 File:080195E Sample : LAB-BASE - The MS Data System 01/08/1995 14:22 A-3 O File:080195E Sample : LAB-BASE - The MS Data System 01/08/1995 14:22 t.0 Doi Io File:080195E Sample : LAB-BASE - The MS Data System 01/08/1995 14:22 te s <*D CM O O File:080195E Sawple ' 080195EJ324 (8.640) 1001 3 6 9 .0 0 LAB--BASE -- The MS Data System 01/08/1995 14:22 COMBINE:(321 to 326)-(((284 to 302)+1369 to 394))*1.000T~ 2272 JCF S - 4 1 3 .0 0 3 7 0 .0 0 4 1 4 .0 0 / -419.00 5 8 1 .0 0 5 9 2 .0 0 X 628.. 00  3 2 ^ -15( U W 2- Q P^ ) File:080195G LAB-BASE - The MS Data System 01/08/1995 15:37