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3M Environmental Laboratory Final Report- Analytical Study Single-Dose Dermal Absorption/Toxicity Study of T-6052 in Rabbits In-Vivo Study Reference Number: HWI#6329-135 Study Number: AMDT-020795.1 Test Substance: FC-120 (T-6052) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology & Services 935 Bush Avenue St. Paul, MN 55106 Method Numbers and Revisions: AM DT-M -1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AM DT-M -2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer AMDT- M -4-0, Extraction o f Fluorochemicals from Rabbit Liver AMDT- M -5-0, Analysis o f Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry AM DT-M -8-0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 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: 000630 1.0 SUMMARY Samples o f liver at 28 days post dermal doses o f FC-120 (T-6052) were analyzed by combustion for total organic fluorine. Even the highest dose o f 1000 mg/kg (50 ug/kg) resulted in no organic fluorine at practical quantitation levels. There is a trace o f some fluorine detectable if one uses just relative meter readings. Electrospray mass spectrometry was able to detect m/z=599 ion which is the perfluorodecanesulfonate anion. The doses were too low to assess dermal absorption with this test method. 2.0 INTRODUCTION____________________________________________ _ The pharmacokinetic study for FC-120 was not successful. The highest dose was 50 ug/kg. Thus, in this study, if there was a substantial amount o f organic fluorine present at 28 days it would indicate that a significant absorption o f FC-120 had occurred. However, in the event o f very little organic fluorine at 28 days, it would not be possible to make an assessment o f dermal absorption. Liver samples and serum samples were available for analysis o f total organic fluorine and perfluorodecanesulfonate anion. The samples were analyzed. Because the doses are quite low (high dose 50 ug/kg), it was not expected that fluorine would be detected. 3.0 TEST MATERIALS __________________________________ ___________ 3.1 Test, Control, and Reference Substances and Matrices 3.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 3.1.2 Analytical Reference Matrix: Bovine liver, bovine serum, rabbit serum 3 .1 3 Analytical Control Substance: None 3.1.4 Analytical Control Matrix: Bovine liver, bovine serum and rabbit serum 3.2 Source o f Materials: 3MICP/PCP Division for FC-95, bovine liver from grocery store, bovine serum from Sigma Chemical Company, rabbit serum from AM DT-110394.1 (HWI#6329-123) control group animals. 3.3. Purity and Strength of Reference Substance: Responsibility o f Sponsor. 3.4 Stability o f Reference Substance: To be determined by Sponsor. 000631 --= 2-- 3.5 Storage Conditions for Test Materials: Room temperature for FC-95. For biological samples the storage is -2010 C. 3.6 Disposition Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required for studies longer than 28 days. 4.0 EXPERIMENTAL - Overview_______________________________________ The tissues from animals dosed as described (HWI#6329-135), were available for analysis for fluorine compounds. At the discretion o f the study director, a series o f analytical tests could be performed. The screening for fluoride in liver via combustion was the most likely analysis to present definitive data for absorption if the pharmacokinetic test (IV administration) was positive for fluorine in the liver. Other available tests were electrospray mass spectroscopy. However, if the definitive results could be obtained with combustion analysis alone, only the liver samples would be analyzed and any other tests would be for confirmation. 5.0 EX PER IM EN TA L-M ETH O D S_______________________________________ 5.1 AM DT-M -1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 5.2 AM DT-M -2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer 5.3 AM DT-M -4-0, Extraction o f Fluorochemicals from Rabbit Liver 5.4 AM DT-M -5-0, Analysis o f Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 5.5 AMDT-M-8-0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 5.6 AM DT-M -14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum 000332 6.0 DATA ANALYSIS The data from combustion analysis are attached. There does not appear to be any total organic fluorine above the practical quantitation limit for any o f the liver samples at 48 hours post intravenous dose for any o f the dosing regimens. Even the 50 ug/kg dose is not above the practical quantitation limit. However, if one uses just the meter readings and compares those reading to the values for the controls, there is possibly a trace o f fluorine being detected. Electrospray mass spectrometry analysis is attached. The electrospray was able to detect perfluorodecanesulfonate anion in the 50 ug/kg dose livers. The amount present was not quantitated. In view o f the failure o f the pharmacokinetic study (HWI#6329-134) to show a good marker for FC-120 at a dose o f 50 ug/kg, it is not reasonable to make an assessment o f the extent o f dermal absorption from this study at the same dose level. Other data was collected using Skalar segmented flow analyzer with ion selective electrode (see appendices). This data, although supportive, in the opinion o f the Study Director is not required to reach the conclusion stated here and therefore is not discussed in detail. 6.1 Circumstances that May Have Affected the Quality o f the Data: The problem with this analysis is that the pharmacokinetic study did not provide a good marker for dermal absorption because the dose level was too low. The dermal study is not at a higher dose. 7.0 CONCLUSION________________________________ ____________________ This study does not provide a useful assessment o f dermal absorption o f FC-120. There is not a useful marker. 8.0 MAINTENANCE OF RAW DATA AND RECORDS_________________ 8.1 Raw Data and Data: Raw data, approved protocol, approved final report, appropriate specimens, and electronic data will be maintained in the AMDT archives. 000333 9.0 APPENDICES 9.1 Protocol and Amendments 9.1.1 Protocol and Final Report: HWI#6329-135: "Single-Dose Dermal Absorption/Toxicity Study o f T-6052 in Rabbits" (Protocol type TP3016.AB for dosing o f animals, tissue collection, etc.) 9.1.2 Analytical protocol AMDT-020795.1 9.1.3 Amendment to Analytical Protocol AMDT-020795.1 9.2 Signed Reports from Individual Scientists: None 9.3 Quality Assurance Unit Statement: See attached 9.4 Key Personnel Involved in the Study: See attached 9.5 Materials and Equipment: See methods 9.6 Solutions, Reagents, and Standards: See methods 9.7 Sample Preparation: See methods 9.8 Quality Control Practices: See methods 9.9 Test Methods: See Protocol AMDT-020795.1 9.10 Instrument Settings: See methods 9.11 Data: See attached. 9.11.1 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 9.11.2 Summary and raw data; analysis o f liver extracts using electrospray mass spectrometry. G0G34 9.11.3 Summary and raw data; ppm F' in serum as determined by thermal extraction followed by analysis using Orion ion analyzer. 9.11.4 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. GG 0 6 - 3 5 6-- 9.1.1 Protocol and Final Report: HWI#6329-135: "Single-Dose Dermal Absorption/Toxicity Study o f T-6052 in Rabbits" (Protocol type TP3016.AB for dosing o f animals, tissue collection, etc.) 000636 HAZLETON WISCONSIN POST OFFI CE BOX 7545 MA D I S O N . WI 53 7 0 7 - 754 5 Sponsor 3M Toxicology Service Medical Department St. Paul, Minnesota a C O R N IN G Company FINAL REPORT Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6052 in Rabbits Author: Steven M. Glaza Study Completion Date: June 27, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-135 Phone 608 241-4471 E X P R f S S M A II n F IIV R Y Page 1 of 41 330 1 P IIJ!,M A N B IV f> 000637 Fax CO M A P I S ON. V\ Page 2 of 41 HWI 6329-135 QUALITY ASSURANCE STATEMENT This report has been reviewed by the Quality Assurance Unit of Hazleton Wisconsin, Inc., in accordance with the Food and Drug Administration (FDA) Good Laboratory Practice Regulations, 21 CFR 58.35 (b) (6) (7). The following inspections were conducted and findings reported to the Study Director and management. Written status reports of inspections and findings are issued to Hazleton management monthly according to standard operating procedures. Inspection Dates From To Phase Date Reported to Date to Study Director Manaaement 12/21/94 01/30/95 02/02/95 03/29/95 03/29/95 06/27/95 12/21/94 01/30/95 02/02/95 03/29/95 03/29/95 06/27/95 Protocol Review Protocol Amendment Body Weight Data/Report Review Data Review Report Rereview 12/22/94 01/30/95 02/02/95 03/29/95 03/29/95 06/27/95 01/10/95 02/10/95 03/10/95 04/10/95 04/10/95 07/10/95 Quality Assurance Unit 2?-?sr Date 0G0G38 Page 3 of 41 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6052 in Rabbits HWI 6329- Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Study Initiation Date Experimental (In-life) Start In-life End Date Experimental Termination Date Study Completion Date T-6052 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 December 30, 1994 January 5, 1995 February 2, 1995 June 27, 1995 June 27, 1995 00GG33 Page 4 of 41 HWI 6329-135 KEY PERSONNEL Acute Toxicology Laboratory Animal Medicine Steven M. Glaza Study Director Manager Cindy J. Cary, DVM Dipl ornate, ACLAM Supervisor Francis (Bud) W. McDonald Study Coordinator Anatomical Pathology Patricia Padgham In-life Supervisor Rose M. Bridge Report Supervisor Quality Assurance Sherry R. W. Petsel Manager Thomas E. Palmer, PhD Anatomical Pathologist Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data G O G o 0 Page 5 of 41 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 and Mean Body Weights (g) 2 Individual Clinical Signs 3 Individual Dermal Irritation Scores 4 Individual Pathology Comments 5 Individual Animal Tissue Weightsand Bile Volumes Appendix A Protocol TP3016.AB Protocol Amendment No. 1 HWI 6329-135 Page 2 3 4 6 8 8 8 9 10 13 13 14 14 15 16 17 19 23 25 27 28 40 00GG41 Page 6 of 41 SUMMARY HWI 6329-135 This study was done to assess the systemic absorption/toxicity and relative skin irritancy of T-6052 when applied to the skin of rabbits. The study was conducted using three male and three female acclimated rabbits of the Hra:(NZW)SPF strain for each treatment group. GrouD Dose Level Number of Animals Test Material (mq/kq) Males Females 1 (Control) 2 3 4 Distilled water T-6052 T-6052 T-6052 0a 2 200 1,000 3 3 3 3 3 3 3 3 a Administered at a dose volume of 2.0 mL/kg. The back of each rabbit was clipped free of hair and a single dose of the respective material at the indicated dose level was administered to the skin of the rabbits. The treatment sites remained intact. The area of application was covered with a gauze bandage secured with paper tape around all edges and overwrapped with Saran Wrap and Elastoplast tape to provide an occlusive dressing for a 24-hour exposure period. Clinical observations were conducted predose and at approximately 1, 2.5, and 4 hours after test or control material administration. Additional clinical observations and twice a day mortality checks were conducted daily thereafter for 28 days. Body weights were determined on Day -8 for randomization purposes, before test or control material administration (Day 1), and at in-life termination (Day 29). The initial dermal irritation reading was made before test or control material administration (recorded as the Day 1 reading). Subsequent readings of dermal irritation were made approximately 30 minutes after bandage removal (Day 2) and on Days 4 and 8. Blood samples were collected from a marginal ear vein of the animals before in-life initiation (Day 1), approximately 24-hours postdose (Day 2), on Days 4, 8, 15, and 22. In addition, at the time of necropsy on Day 29, approximately 20 mL of blood was obtained from each animal. All samples were centrifuged and separated into serum and cellular fractions. All animals were euthanized at termination of the in-life phase and necropsied. The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from one male and one female in each group were collected at necropsy and weighed (volume only determined for bile). The blood samples (serum and cellular fractions), livers, bile, dermal application sites, and kidneys were sent frozen to the Sponsor after termination of the in-life phase. 000<l>4:2 Page 7 of 41 HWI 6329-135 Application of T-6052 did not result in any test material-related changes in body weight gain or macroscopic findings at necropsy. All animals appeared clinically normal throughout the study. No dermal irritation was observed at the dermal scoring intervals as a result of the application of distilled water or T-6052 at any of the dose levels. 0GGG43 Page 8 of 41 OBJECTIVE HWI 6329-135 The objective of this study was to assess the systemic toxicity/absorption and relative skin irritancy of a test material when applied to the skin of 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 material mixture prepared for the Group 2 animals for concentration, homogeneity/solubility, and stability was not conducted. All procedures used in this study are in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study did not unnecessarily duplicate any previous work. TEST AND CONTROL MATERIALS Identification The test material was identified as T-6052 and described as a clear, colorless liquid. The control material was distilled water 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). Analysis of the test material mixture prepared for the Group 2 animals for concentration, homogeneity/solubility, and stability was not conducted or requested by the Sponsor. The purity and stability of the control material were considered to be adequate for the purposes of this study. Storage and Retention The test and control materials were stored at room temperature. A reserve sample of each test and control material was taken and will be retained in a freezer set to maintain a temperature of -20C 10'-for 10 years in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedure (SOP). Any unused test material was returned to the Sponsor after completion of the in-life phase according to HWI SOP. Any remaining control material is retained for other testing and will not be discarded after issuance of the final report. GC44 Page 9 of 41 Safety Precautions HWI 6329-135 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 procured from HRP, Inc., Denver, Pennsylvania on December 28, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing After receipt, the animals were acclimated for a period of at least 7 days. During acclimation and throughout the study, the animals were individually housed in screen-bottom stainless steel cages in temperature- and humiditycontrolled quarters. Environmental controls for the animal room were set to maintain a temperature of 19 to 23C, a relative humidity of 50% 20%, and a 12-hour light/12-hour dark lighting cycle. In cases where variations from these conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad 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 placed into study groups using a stratified body weight randomization program. The randomization body weights were determined on Day -8. The weight variation of the animals for each group of eah sex selected for the study did not exceed 2 standard deviations of the mean weight, and the mean body weights for each group of each sex were not statistically different at the 5% probability level. One female animal (No. F53409) was replaced in the study prior to treatment due to poor health. This animal was replaced with another female (No. F52982) which was treated in the same manner. 000b45 Page 10 of 41 Study Design HWI 6329-135 Animals weighing from 2,052 to 2,471 g at initiation of treatment were placed into the following study groups: Group Dose Level Number of Animals Test Material (mq/kq) Males Females 1 (Control) 2 3 4 Distilled water T-6052 T-6052 T-6052 0a 2 200 1,000 3 3 3 3 3 3 3 3 a Administered at a dose volume of 2.0 mL/kg. 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 Preparation of Exposure Area On the day before test material application, the back and, if necessary (to obtain unblemished skin), the flanks of each rabbit was clipped free of hair. The clipped area made up approximately 20% of the total body surface area. The intact skin of the test sites was inspected for interfering lesions, irritation, or defects that would preclude the use of any of the animals. The animals were clipped on Days 8 and 29 to aid in visualizing the application sites. Dose Administration All animals received a single administration of the respective test or control material. The day of treatment was designated as Day 1. Group 1 . An individual dose (2.0 mL/kg) was calculated and measured based on each animal's body weight on the day of treatment. The control material (distilled water) was applied evenly to the test~-site at a rate of approximately 0.04 mL/cm . Oi>4:i> Page 11 of 41 HWI 6329-135 Groups 2. 3. and 4 . For the Group 2 animals (2 mg/kg), the test material (T-6052) was mixed with distilled water to a concentration of 200 mg/mL and applied at a dose volume of 0.01 mL/kg. The mixture was stored at room temperature until administered. The test material was administered undiluted to the test sites of the Groups 3 and 4 animals (200 or 1,000 mg/kg, respectively) using the average bulk density of 0.98 g/mL to determine the dose volume for each dose level (0.20 and 1.02 mL/kg, respectively). An individual dose of the respective test material or test material mixture was calculated for each animal based on its body weight on the day of treatment. The area of exposure for the 2, 200, and 1,000 mg/kg dose levels was 4, 25, and 100 cm2, respectively. The approximate rate of application ranged from 0.006 to 0.024 mL/cm. Each area of application was covered with a 10-cm x 10-cm gauze bandage secured with paper tape around all edges and overwrapped with Saran Wrap and Elastoplast tape to provide an occlusive dressing. Collars were used to restrain the animals during the 24-hour exposure period. Approximately 24 hours after test or control material application, the restraining collars and bandages were removed and any residual test material was removed with tap water and disposable paper towels. Reason for Route of Administration The dermal route is a potential route of exposure in humans. Observations of Animals Clinical observations were conducted predose and at approximately 1, 2.5, and 4 hours after test or control material administration. Additional clinical observations and twice a day mortality checks (morning and afternoon) were conducted daily thereafter for 28 days. Body weights were determined for randomization purposes on Day -8, before test material administration (Day 1), and at in-life termination (Day 29). The initial dermal irritation reading was made before test or control material administration according to the Draize1 technique (recorded as the Day 1 reading). Subsequent readings of dermal irritation were made approximately 30 minutes after bandage removal (Day 2) and on Days 4_and 8. 000647 Page 12 of 41 HWI 6329-135 Sample Collections Blood samples (approximately 4 mL) were collected from a marginal ear vein of all animals before experimental initiation (Day 1). Subsequent collection of blood was conducted approximately 24-hours postdose (Day 2), and on Days 4, 8, 15, and 22. In addition, at the time of necropsy on Day 29, approximately 20 mL of blood was obtained from the posterior vena cava of each animal. All samples were centrifuged and separated into serum and cellular fractions. These samples were then stored in a freezer set to maintain a temperature of -20C 10C until shipped to the Sponsor. Pathology At termination of the experimental phase (Day 29), animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, exsanguinated, and necropsied in random order. The sites of test and control material application were washed with lukewarm tap water before the necropsy procedure. All animals were subjected to an abbreviated gross necropsy examination and any abnormalities were recorded. The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from the first male and female in each group were collected. The tissue samples were weighed (volume only determined for bile) and immediately placed on dry ice, then placed in a freezer set to maintain a temperature of -20C 10C. After necropsy, the animals were discarded. Shipment of Blood. Bile, and Tissues After experimental termination, the blood samples (serum and cellular fractions), livers, bile, dermal application sites, 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), along with their corresponding weights or volumes. The Sponsor is responsible for the retention and disposition of the samples. HWI does not accept any responsibility for the analysis of the tissue 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. G00o48 Page 13 of 41 HWI 6329-135 RESULTS Body Weights Individual and mean body weights are in Table 1. All animals exhibited body weight gains from Day 1 to Day 29. Clinical Observations Individual clinical signs are in Table 2. All animals appeared normal throughout the study. Dermal Irritation Individual dermal irritation scores are in Table 3. The control material produced no dermal irritation. No dermal irritation was observed in the animals treated with T-6052 at any of the dose levels. Pathology Individual animal pathology comments are presented in Table 4. Individual animal tissue weights and bile volumes are in Table 5. There were no lesions observed in any of the animals. Page 15 contains a pathology report by the study pathologist. DISCUSSION The acute systemic absorption/toxicity and relative skin irritancy of T-6052 were evaluated in male and female albino rabbits when administered as a single dermal application. Application of this material did not result in any dermal irritation or test material-related in-life clinical effects. There were no effects on body weight gain or macroscopic findings at necropsy. 049 Page 14 of 41 SIGNATURE HWI 6329-135 Acute Toxicology Date REFERENCE 1. Draize, J. H., "Acute Dermal Toxicity (Single Exposure)," In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics - Dermal Toxicity, Association of Food and Drug Officials of the U.S., pp. 54-56 (1959). COGtoO Page 15 of 41 HWI 6329-135 PATHOLOGY REPORT There were six rabbits (three males and three females) each from four dose levels euthanized and necropsied at the termination of the study. The test material, dose level, day of death, and gross observations recorded for each animal are in the Individual Pathology Comments that follow this report. At necropsy, there were no visible lesions in any of the animals. The liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from one male and one female in each group were collected. The tissue samples were weighed (volume only determined for bile), frozen, and sent to the Sponsor. After necropsy, the animals were discarded. (6329-135.slh) 031695 ____ 6 - z 7 - ^ ' S -- Date 000651 Animal Number F52979 F52972 F52973 Mean Page 16 of 41 HWI 6329-135 Table 1 Individual and Mean Body Weights (g) Male Randomization Dav -8 _______________ Day 1 29 ______________ Female Random- Animal ization _____Dan Number Dav,-8 1 29 Group 1 (Control) - Distilled Water (0 mq/ko) 1,903 2,090 1,921 2,072 2,362 2,211 2,559 3,041 2,818 F52976 F52983 F52975 2,151 2,116 2,043 2,259 2,296 2,265 2,681 2,863 2,671 1,971 2,215 2,806 2,103 2,273 2,738 F52990 F52997 F52986 Mean 2,095 2,031 2,034 2,053 GrouD 2 - T-6052 (2 mq/kq) 2,351 2,205 2,332 2,863 2,928 2,816 F529828 F52994 F53410 1,885 2,022 2,220 2,296 2,869 2,042 2,145 2,261 2,471 2,292 2,681 2,914 2,875 2,823 F52996 F52992 F52984 Mean 2,190 1,889 1,950 2,010 GrouD 3 - T-6052 f200 mq/kq) 2,302 2,052 2,257 2,897 2,729 2,993 F52989 F52993 F52977 2,097 1,993 2,049 2,204 2,873 2,046 2,316 2,234 2,323 2,291 2,874 2,651 2,687 2,737 F52980 F52978 F52991 Mean 1,936 2,181 2,108 2,075 GrouD 4 - T-6052 (1.000 mq/kq) 2,184 2,384 2,351 2,637 3,063 3,142 F52995 F52987 F52988 2,131 2,140 2,188 2,306 2,947 2,153 2,249 2,274 2,423 2,315 2,644 2,817 3,015 2,825 a Animal No. F53409 was originally selected by the randomization program for use in the study but was replaced prior to treatment with No. F52982 due to poor health. G0G52 Sex Male Female Page 17 of 41 Table 2 Individual Clinical Signs HWI 6329-135 Animal Number Observation 1-4 Hours Dav (Day 1) 2 throuah 29' Grouo 1 (Control) - Distilled Water (0 ma/kal F52979 F52972 F52973 Appeared normal Appeared normal Appeared normal // / // F52976 Appeared normal / F52983 Appeared normal / / F52975 Appeared normal / / Male Female GrouD 2 - T-6052 (2 mq/kq) F52990 Appeared normal / F52997 Appeared normal / F52986 Appeared normal / F52982 Appeared normal / F52994 Appeared normal / F53410 Appeared normal / / / / / / / Condition existed. 000553 Sex Male Female Page 18 of 41 HWI 6329-135 Table 2 (Continued) Individual Clinical Signs Animal Number Observation 1-4 Hours Dav (Day 1) 2 throuoh 29 GrouD 3 - T-6052 (200 mq/kq) F52996 Appeared normal / F52992 Appeared normal / F52984 Appeared normal / F52989 Appeared normal // F52993 Appeared normal // F52977 Appeared normal // Male Female GrouD 4 - T-6052 (1,000 mq/kq) F52980 Appeared normal / F52978 Appeared normal / F52991 Appeared normal / F52995 Appeared normal / F52987 Appeared normal F52988 Appeared normal / / / / / / / Condition existed. 000i>54 Page 19 of 41 HWI 6329-135 Table 3 Individual Dermal Irritation Scores Group 1 (Control) - Distilled Water (0 mg/kg) Dermal Reaction Erythema Edema Atonia Desquamation Coriaceousness Fissuring Males________ Study Day _ L _ 2 _ _4_ 8 Animal No. F52979 0000 0000 0000 0000 0000 0000 _______ Females Studv Dav 1 _2_ 4 _8_ Animal No. F52976 0000 0000 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52972 0000 0000 0000 0000 0000 0000 Animal No. F52983 0000 0000 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52973 0000 0000 0000 0000 00 0 0 0000 Animal No. F52975 0000 0000 0000 0000 0000 0000 G00o55 Page 20 of 41 HWI 6329-135 Table 3 (Continued) Individual Dermal Irritation Scores Group 2 - T-6052 (2 mg/kg) Dermal Reaction ________ Males________ Study Dav______ 1 _ 2_ 4 8 Animal No. F52990 _______ Females ______ Study Day 1 _2_ 4 8 Animal No. F52982 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0 00 0 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52997 0000 0000 0000 0000 0000 0000 Animal No. F52994 0000 0 0 .0 0 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52986 0000 0000 0000 0000 0000 0000 Animal No. F53410 0000 0000 0000 0000 0000 0000 000656 Page 21 of 41 HWI 6329-135 Table 3 (Continued) Individual Dermal Irritation Scores Group 3 - T-6052 (200 mg/kg) Dermal Reaction Males Study Dav _ L _2_ 4 J8_ Animal No. F52996 Females Study Dav 1 _2_ 4 _8_ Animal No. F52989 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 00 0 0 00 00 0000 00 00 00 00 0000 0000 0000 0000 0000 0 00 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52992 0000 0000 0000 0 0 0 0' 0000 0000 Animal No. F52993 0000 0000 0000 0000 0 0 0 0 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52984 00 0 0 00 00 00 0 0 00 0 0 00 0 0 0000 Animal No. F52977 0000 0000 0000 0000 0000 0000 G 00657 Page 22 of 41 HWI 6329-135 Table 3 (Continued) Individual Dermal Irritation Scores Group 4 - T-6052 (1,000 mg/kg) Dermal Reaction ________ Males________ Study Dav______ JL _2_ 4 _8_ Animal No. F52980 _______ Females ______ Study Day JL _2_ 4 8 Animal No. F52995 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 00 00 00 00 0000 0000 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52978 0000 0000 0000 0000 0000 0000 Animal No. F52987 0000 0000 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52991 0000 0000 0000 0000 0000 0000 Animal No. F52988 0000 0000 0000 0000 0000 0000 000*G58 Animal Number F52979 F52972 F52973 F52976 F52983 F52975 F52990 F52997 F52986 F52982 F52994 F53410 F52996 F52992 F52984 F52989 F52993 F52977 Page 23 of 41 Table 4 Individual Pathology Comments Test Dav Sex Died Sacrificed NecroDSV Observation GrouD 1 (Control 1 - Distilled Water (0 ma/kal MM- 29 No visible lesions. 29 No visible lesions. MFF- 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. F- 29 No visible lesions. GrouD 2 - T-6052.12. mq/kq) M- 29 No visible lesions. MMF- 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. F- 29 No visible lesions. F- 29 No visible lesions. GrouD 3 - T-6052 (200 mq/kq) MMMFFF_ 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visiblfr lesions. 29 No visible lesions. 29 No visible lesions. HWI 6329-135 Not applicable. 000659 Page 24 of 41 Table 4 (Continued) Individual Pathology Comments HWI 6329-135 Animal Test Dav Number Sex Died Sacrificed Necrosv Observation GrouD 4 - T-6052 H.OOO ma/ko) F52980 F52978 F52991 F52995 F52987 F52988 M M M F F F 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. - Not appiicable. GOOoGO Sex Male Female Male Female Male Female Page 25 of 41 Table 5 Individual Animal Tissue Weights and Bile Volumes HWI 6329-135 Animal Number ____________Weight (g)_____________ Dermal Appli- Liver Kidneys cation Site Bile Volume (mLi Group 1 (Control) - Distilled Water fO mo/kol F52979 91.387 - 0.593 0.9 F52972 83.176 16.196 0.358 1.15 F52973 76.442 - 0.800 1.2 F52976 61.443 - 0.445 0.8 F52983 98.109 - 0.393 0.9 F52975 87.984 14.617 0.608 0.4 Group 2 - T-6052 (2 mg/kg) F52990 81.033 - 0.281 0.4 F52997 92.537 - 0.421 0.5 F52986 83.745 16.924 0.632 1.1 F52982 84.395 - 0.568 1.1 F52994 83.650 15.618 0.432 1.4 F53410 91.541 - 0.386 1.1 Group 3 - T-6052 (200 mg/ko) F52996 78.540 - 0.673 0.46 F52992 80.845 - 0.511 1.31 F52984 89.277 15.044 0_,421 0.75 F52989 98.425 16.883 0.837 0.7 F52993 88.925 - 0.848 0.55 F52977 72.840 - 0.526 0.9 - Not appiicable. OOCoGl Sex Male Female Page 26 of 41 Table 5 (Continued) Individual Animal Tissue Weights and Bile Volumes HWI 6329-135 Animal Number ____________ Weight fa)_____________ Dermal Appli Liver Kidnevs cation Site GrouD 4 - T-6052 n.000 ma/kal Bile Volume * F52980 F52978 F52991 83.049 84.235 88.738 15.630 - 0.896 0.547 0.439 1.0 1.6 0.9 F52995 F52987 F52988 63.956 82.019 83.911 14.923 - 0.508 0.287 0.875 0.25 1.35 1.2 - Not applicable. GG0662 Page 27 of 41 HWI 6329-135 APPENDIX A Protocol TP3016.AB Protocol Amendment No. 1 000<S63 HAZLETON WISCONSIN POST OFFICE BOX 7545 M A D I S O N , Wl 5 3 7 0 7 7 54 5 Page 28 of 41 a CORNING Company Sponsor: 3M Toxicology Service Medical Department St. Paul, Minnesota PROTOCOL TP3016.AB Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6052 in Rabbits Date: December 30, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-135 P h u n < i, o s - ' 4 1 4 <: / i f X P R I 5 S M A M I M l I VI li \ G0664 A 3 II ! K I N S M A N H I b F i \ O H 7 4' m n 11I <"lM tA/ I STUDY IDENTIFICATION TP3016.AB Page 2 Single-Dose Dermal Absorption/Toxicity Study of T-6052 in Rabbits HWI No. Test Material Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6329-135 T-6052 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 January 5, 1995 February 2, 1995 March 16, 1995 60065 Page 30 of 41 1. Study Single-Dose Dermal Absorption/Toxicity Study in Rabbits TP3016.AB Page 3 2. Purpose To assess the systemic absorption and toxicity and relative skin irritancy of a test material when applied to the skin of rabbits 3. Regulatory Compliance This study will be conducted in accordance with the following Good Laboratory Practice Regulations/Standards/Guidelines: [ ] 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-6052 B. Physical Description (To be documented in the raw data) C. Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under test conditions). D. Storage Room temperature ) 00C6S6 Page 31 of 41 TP3016.AB Page 4 E. Reserve Samples Reserve sample(s) of each batch/lot of test and control materials will be taken for this study. The test and control material reserve samples will be stored at HWI in a freezer set to maintain a temperature of -20*C 10*C for 10 years per HWI SOP. The Sponsor will be contacted after 10 years for disposition in accordance with the appropriate regulatory Good Laboratory Practices. F. Retention Any unused test material will be returned to the Sponsor after completion of the in-life phase of the study. G. Safety Precautions As required by HWI SOPs and policies 6. Control Material A. Identification Distilled water B. Physical Description ) Clear, colorless liquid C. Purity and Stability The purity and stability of this manufactured material is considered to be adequate for the purposes of this study. D. Storage Conditions Room temperature 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. ExDerimental Design A. Animals (1) Species Rabbit ; f?\ Strain/Source Hra:(NZW)SPF/HRP, Inc. >G0oS7 Page 32 of 41 TP3016.AB Page 5 (3) Age at Initiation Adult (4) Weight at Initiation 2.0 to 3.0 kg (5) Number and Sex 12 males and 12 females (6) Identification Individual numbered ear tag (7) Husbandry (a) Housing Individually, in screen-bottom stainless steel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds, Inc.). The food is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. (c) Water Ad libitum from an automatic system. Samples of the water are analyzed by HWI for total dissolved solids, hardness, and specified microbiological content and for selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the food or water that would interfere with this study. (e) Environment Environmental controls for the animal room will be set to maintain a temperature of 19*C to 23*C, a relative humidity of 50% 20%, and a 12-hour light/12-hour dark cycle. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to HWI SOPs. An adequate number of extra animals will be purchased so that no animal in obviously poor health is placed on test. The animals will be placed into study groups using a stratified body weight randomization program within nine days of study initiation. G00GG8 Page 33 of 41 TP3016.AB Page 6 (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. B. Dose Administration (11 Test Groups GrnuD Test Material Dose Level Number of Animals (mq/kq) Males Females 1 (Control) 2 3 4 Distilled water T-6052 T-6052 T-6052 0* 2** 200 1000 3 3 3 3 3 3 3 3 * To be administered at a dose volume of 2.0 mL/kg ** To be administered at a dose volume of .01 mL/kg (2) Preparation of Exposure Area On the day before test material application, the back and, if necessary (to obtain unblemished skin), the flanks of each rabbit will be clipped free of hair. The shaved area will constitute approximately 20% of the total body surface area. The treatment sites (intact skin) will be inspected for interfering lesions, irritation, or defects that would preclude the use of any of the animals. The animals will be clipped as needed throughout the study. (3) Dose Administration All animals will receive a single administration of the respective test or control material. The day of treatment will be designated as Day 1. The dose for each animal in Group 2 will be diluted with distilled water and applied at a dose volume of .01 mL/kg. The respective dose for each animal in Groups 3 and 4 will be applied undiluted. All doses in Groups 1-4 will be based on the animal's body weight just before administration and will be spread onto the area of exposure in a thin and uniform a layer. The area of application (Groups 1-4) will be covered with a 10-cm x 10-cm gauze bandage secured with japer tape around all edges and overwrapped with Saran Wrap and Elastoplast* tape to provide an occlusive dressing*. The rabbits will be collared during the 24-hour application period. (4) Reason for Route of Administration The dermal route is a potential route of exposure in humans. 0069 Page 34 of 41 TP3016.AB Page 7 (5) Removal of Test Material Approximately 24 hours after test or control material application the bandages and collars will be removed and the residual test material will be removed using water or an appropriate solvent, if necessary. C. Observation of Animals (1) Clinical Observations For clinical signs before test or control material administration and for clinical signs and mortality at approximately 1, 2.5, and 4 hours after test material administration (Day 1) and daily thereafter for clinical signs, and twice daily (a.m. and p.m.) for mortality for at least 28 days. Observations may be extended when directed by the study director. (2) Reading of Dermal Irritation Before test or control material administration the initial dermal irritation reading will be made and recorded as the Day 1 reading (Attachment I). Additional dermal irritation readings will be made approximately 30 minutes after bandage removal (Day 2) and on Study Days 4 and 8. Individual dermal irritation records will be maintained for each animal. (3) Body Weights For randomization, before test or control material application (Day 1), on Day 29, and at unscheduled death (when survival exceeds 1 day) (4) Sample Collections (a) Frequency Before initiation (Day 1), approximately 24 hours post-dose (Day 2), Days 4, 8, 15, 22, and at experimental termination (Day 29) (b) Number of Animals All 000670 Page 35 of 41 TP3016.AB Page 8 (c) Method of Collection Blood samples (approximately 4 mL) will be collected from the marginal ear vein of either ear on Days 1, 2, 4, 8, 15, and 22. 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 sacrificed in a moribund condition or sacrificed at the time of necropsy (Day 29). The samples will be stored at room temperature and then centrifuged, and the separate serum and cellular fractions stored in a freezer set to maintain -20*C 10*C. The separated serum and cellular fractions will be sent frozen on dry ice to the Sponsor after experimental termination. Samples will be shipped to: James D. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James D. Johnson or alternate will be notified by telephone at (612) 778-5294 prior to the shipment of the samples. D. Pathology (1) Unscheduled Sacrifices and Deaths Any animal dying during the study or sacrificed in a moribund condition will be subjected to an abbreviated gross necropsy examination and all abnormalities will be recorded. Animals in a moribund condition will be anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. Tissues, as described in section D. Pathology, (3) Sample Collection, will be collected. After necropsy, the animals will be discarded. (2) Scheduled Sacrifice At termination of the experimental phase (Day 29), surviving animals will be anesthetized with sodium pentobarbital (via injection in the juarginal ear vein), bled via the vena cava, exsanguinatedr and subjected to an abbreviated gross necropsy examination. The animals will be necropsied in random order and all abnormalities will be recorded. G0071 Page 36 of 41 TP3016.AB Page 9 (3) Sample Collection The sites of test and control material application will be washed with lukewarm tap water prior to the necropsy procedure. The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from the first male and female necopsied in each group will be collected and immediately placed in a freezer set to maintain a temperature of -20*C 10*C. After necropsy, the animals will be discarded. The tissues (liver, bile, dermal application site, 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.C.(4).(c). The Sponsor is responsible for the retention and disposition of the samples. E. 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 Tabulation of mortality data by sex and dose level Description of any toxic effects/dermal irritation Tabulation of mean body weights by sex and dose level Gross pathology findings/gross pathology report 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 Anatomical pathology records Sample collection records Shipping records Study correspondence Final report (original signed copy) G00G72 Page 37 of 41 TP3016.AB Page 10 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 ) ) GGG673 Page 38 of 41 PROTOCOL APPROVAL TP3016.AB Page 11 John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department /-J"- ^ ~ Date Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. presentative Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-135.protdsk2) A ./ Date 000674 Page 39 of 41 TP3016.AB ) Page 12 Attachment 1 Scoring Scale for Acute Dermal Reactions Erythema 0 - None 1 - Slight 2 - Moderate 3 - Severe Edema 0 - None 1 - Slight (barely perceptible to well defined by definite raising) 2 - Moderate (raised approximately 1 mm) 3 - Severe (raised more than 1 mm) Atonia 0 - None 1 - Slight (slight impairment of elasticity) 2 - Moderate (slow return to normal) 3 - Marked (no elasticity) Desouamation 0 - None 1 - Slight (slight scaling) 2 - Moderate (scales and flakes) 3 - Marked (pronounced flaking with denuded areas) Coriaceousness 0 - None 1 - Slight (decrease in pliability) 2 - Moderate (leathery texture) 3 - Marked (tough and brittle) Fissurino 0 - None 1 - Slight (definite cracks in epidermis) 2 - Moderate (cracks in dermis) 3 - Marked (cracks with bleeding) GG0o75 Page 40 of 41 HAZLETON WISCONSIN POST OFFICE BOX 7545 M A D IS O N . Wl 53707 7545 a CORNING Company PROTOCOL TP3016.AB Single-Dose Dermal Absorption/Toxicity Study of T-6052 in Rabbits HWI 6329-135 Sponsor Contractor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 Sponsor's Representative Study Director John L. Butenhoff, PhD Steven M. Glaza Amendment No. 1 This amendment modifies the following portions of the protocol: Effective January 24, 1995 At the request of the Sponsor, the weights of tissues collected and the volume of bile collected will be documented in the raw data. These weights and volumes will be included with the sample shipment. Modify the following sections of the protocol to include these additions. 1. Page 9. 7. Experimental Design: D. Pathology; 131 Sample Collection. Modify the second sentence in the first and second paragraphs of this section with the following underlined additions: The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from the first male and female necropsied in each group will be collected, weighed (volume only determined for bile), and immediately placed in a freezer set to maintain a temperature of -20*C 10*C. The samples and their corresponding weights or volumes will be shipped to the person listed in Section 7.C-J4).(c). 2. Page 9. 8. Report. Add the following to this section: Individual animal tissue weights and bile volumes P h o n e C. o B ? 4 i 4 4 / i E X P R E S S MAM fil I IV FRY 'IIO I K I N S MA N III VII Fax 6o h :><ii /? ? ; MADISON Wl !> :i 1 O 4 Page 41 of 41 Amendment No. PROTOCOL AMENDMENT APPROVAL HWI 6329-135 Page 2 John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department Date Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. & ) Representative/ Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-135.Ami.dsk2) Date _________ ^ - 7^*7T Date 9.1.2 Analytical protocol AMDT-020795.1 000878 3M Environmental Laboratory_____________________ _ Protocol - Analytical Study Single-Dose Dermal Absorption/Toxicity Study o f T-6052 in Rabbits In-Vivo Study Reference Number: HWI#6329-135 Study Number: AMDT-020795.1 Test Substance: FC-120 (T-6052) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55106 Proposed Initiation Date: July 25, 1995 Proposed Completion Date: August 25,1995 Method Numbers and Revisions: AM DT-M -1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AM DT-M -2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer 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 D T-M -14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum Author: James D. Johnson Approved By: John Butenhoff, PhD Sponsor Representative 000679 Date 1.0 PURPOSE This study is designed to provide information as to whether FC-120 (T-6052) is dermally absorbed. The analytical aspect o f this study is to determine fluorinecontaining compounds in the liver and serum o f rabbits. By comparison o f the data obtained after dermal absorption with that obtained after intravenous injection, assessment o f the extent o f dermal absorption can be performed. 2.0 TEST MATERIALS__________________________________________________ _ 2.1 Test, Control, and Reference Substances and Matrices 2.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 2.1.2 Analytical Reference Matrix: Bovine liver, bovine serum and rabbit serum 2.1.3 Analytical Control Substance: None 2.1.4 Analytical Control Matrix: Bovine liver, bovine serum and rabbit serum 2.2 Source o f Materials: 3MICP/PCP Division (2.1.1), grocery store (2.1.2, 2.1.4 liver), Sigma Chemical Company (2.1.2, 2.1.4 bovine serum), AMDT 110394.1 (Hwi#6329-123) control group animals (2.1.2, 2.1.4 rabbit serum) 2.3 Num ber o f Test and Control Samples: Tissues and fluids from 18 test animals and 6 control animals. Tissues and fluids include liver, kidney, serum, cellular fraction, dermal application site and bile. Analysis o f these tissues will be at the discretion o f the Study Director. 2.4 Identification o f Test and Control Samples: The samples are identified using the HWI animal identification number which consists o f a letter and five digit number, plus the tissue identity and day identity (serum). 2.5 Purity and Strength of Reference Substance: To be determined by Sponsor. 2.6 Stability o f Reference Substance: To be determined by Sponsor. 2.7 Storage Conditions for Test Materials: Room temperature (2.1.1), -20 10C (2.1.2, 2.1.4). Test and Control samples will be received according to AMDT-S-10-0. 2.8 Disposition of Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required for studies longer than 28 days. OOOoi 2.9 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting the samples. 3.0 EXPERIM ENTAL - Overview__________________________________ The tissues from animals dosed as described (HWI#6329-135), are available for analysis for fluorine compounds. At the discretion o f the Study Director, a series o f analytical tests can be performed. The screening for fluoride in liver via combustion (See Methods--next Section) is the appropriate analysis to present definitive data for fluorine in the liver. To confirm the identity o f fluorine-containing compounds present in liver (if any at 28 days) and serum at various intervals, electrospray mass spectrometry may be selected as one o f the analytical techniques employed. Not all o f the tissues and fluid samples will be analyzed. When sufficient data has been collected to meet the objectives o f the study in the opinion o f the Study Director, analysis will cease. 4.0 EXPERIM ENTAL - Methods______________ _________________________ _ 4.1 Liver and Serum screening methods: (attached) 4.1.1 AMDT-M-1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 4.1.2 AMDT-M-2-0, Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer 4.1.3 AMDT-M-4-0, Extraction o f Fluorochemicals from Rabbit Liver 4.1.4 AMDT-M-5-0, Analysis o f Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 4.1.5 AMDT-M-8-0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 4.1.6 AMDT-M-14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum OOOGS1 5.0 DATA ANALYSIS 5.1 D ata Reporting: Data will be reported as a concentration (weight/weight) o f fluoride per tissue or fluid, or as FC-95 (electrospray mass spectrometry) per unit o f tissue or fluid. Statistics used, at the discretion o f the Study Director, may include regression analysis o f serum concentrations with time and averages and standard deviations o f concentrations for different dose groups. If necessary, simple statistical tests such as Student's t test may be applied to determine statistical difference. 6.0 MAINTENANCE 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 AMDT archives. 7.0 REFERENCES______________________ 7.1 AM DT-S-10-0, Sample Tracking System 8.0 ATTACHMENTS_________________________ ___________________________ 8.1 AM DT-M -1-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 8.2 AM DT-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 Fluorochemicals Using Electrospray Mass Spectrometry 8.5 AM DT-M -8-0, Analysis o f Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 8.6 AM DT-M -14-0, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Serum ~ 000682 3M Environmental Laboratory Method Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer - Liver Method Identification Number: AMDT-M-1 Revision Number: 0 Adoption Date: Revision Date: None Author: Rich Youngblom Approved by: 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 000o83 1 1.0 SCOPE . APPLICABLE COMPOUNDS. AND MATRICES 1.1 Scope: This method is for the operation of a Dohrmann DX2000 when it is used to extract fluoride from various matrices. The fluoride is typically collected in TISAB solution for analysis with an ion selective electrode. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Biological tissues, particularly liver. 2.0 KEYW O RDS_________ _______________________________________ ___ 2.1 Fluoride, fluorine, extraction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS_______________________________________________ _ 3.1 Glassware and exhaust gases can be extremely hot. 3.2 Glassware is fragile, broken glass may cause injuries. 33 Pressurized gases, proper compressed gas handling practices required. 3.4 Solvent based samples may flash, may need to allow them to dry down before starting run. 3.5 Potential biohazards due to the biological matrices. Use appropriate personal protective equipment. 4.0 SUPPLIES AND MATERIALS_______________________________________ 4.1 Compressed Oxygen, Hydrocarbon free, regulated to 30 PSI. 4.2 Compressed Helium, High Purity Grade, regulated to 45 PSI. 4.3 Quartz glass sample boat with TeflonTM tubing, Dohrmann 890-097 or equivalent. 4.4 Quartz glass combustion tube, Reliance Glass G-9405-012 or equivalent. 4.5 Orion 940999 Total Ionic Strength Adjustment Buffer (TISAB I I ) or equivalent. 4.6 Sample collection vials, HDPE. 4.7 Milli-QTM water 4.8 Polystyrene pipettes. 4.9 Activated Charcoal, E. Merck 2005 or equivalent. 4.10 Hamilton Syringe or equivalent. 4.11 Miscellaneous laboratory glassware 5.0 EQ UIPM ENT________________________________________________ _ 5.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer, modified for fluoride extraction. 5.2 IBM compatible 386 or 486 computer. 5.3 DX2000 software, version 1.00, modified for fluoride extraction. 5.4 Excel Spreadsheet, version 5.0 or greater 6.0 INTERFERENCES_________________________ ^ ________________________ 6.1 Sample size is limited to approximately 150 mg, depending on sample moisture content. This may vary from matrix to matrix. 000884 2 7.0 SAMPLE HANDLING 7.1 Samples are not to be handled with bare hands. Fluoride may leach from the skin to the sample. Use forceps or probe to transfer tissues. 7.2 Samples of liver are cut from frozen liver and placed in a tared and labeled weigh boat. Use a clean scalpel and cutting board. The cutting board and scalpel should be cleaned with water, methanol, or methanol-water solution after each liver is cut. 8.0 CALIBRATION AND STANDARDIZATION____________________ 8.1 Preparation of Calibration Standards 8.1.1 The standards required for each project will need to be appropriate for that individual project. Refer to protocol for that project. 8.1.2 Typically 50-500 ppm FC-95 in methanol standards are used. 8.1.3 For rabbit liver studies, use beef liver as the matrix. Cut a piece of frozen beef liver (100 150 mg) and weigh it in a labeled and tared weigh boat. 8.2 Calibration - Overview The normal calibration is the fluoride curve (AMDT-M-2). However, if an optional spiked liver curve is required the procedure listed below is used. 8.2.1 A calibration curve for the DX2000 is generated by spiking samples with known standards and combusting them using the same methods and matrix type as the samples to be tested. 8.2.2 Typically, three replicates of each standard and five concentrations of standards will be spiked. 8.2.3 Standard curve will be plotted as Mass Spiked F (ug) on the x-axis and Standard Mass Recovered F (ug) on the y-axis. Generate a regression curve and calculate the equation for the line and the r^ value. 8.2.4 Mass Spiked F (ug) = (Amount spiked in mL) x ( Cone, of standard in ppm) x (0.6004)* *FC-95 is 60.04% F therefore 0.6004 is the factor used to convert FC-95 to F 8.2.5 Standard Mass Recovered F (ug) = (TISAB volume in mL) x (Orion reading in ppm) 8.3 Calibration - Procedure 8.3.1 Start Up 8.3.1.1 Run 2 or more Clean Cycles when starting instrument each day. More clean cycles may be used if the previous samples contained high concentrations of fluoride. 8.3.2 Blanks 8.3.2.1 Prepare sample using the same methods and type of matrix as the test sample. 8.3.2.2 For rabbit studies, use beef liver as the matrix. Prepare aUeast 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. GOO685 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. 8J.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 S tart Up Procedure: 9.2.1 If the program is not started, start the EOX program on the PC. 9.2.2 Open the SYSTEM SETUP window. 9.2.3 Put the furnace module and the cell in the READY mode. 9.2.4 Close the SYSTEM SETUP window. 000686 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 Rim the EOX-SOLIDS program found in the RUN menu. 9.3.6 When the EOX program is finished, remove the collection vial from the combustion tube. 9.3.7 If undiluted TISAB was used to collect the sample, add an equal volume of Milli-QTM water to the TISAB to make 1:1 TISAB:Milli-QTM. 9.3.8 Rinse the end of the combustion tube with Milli-QTM water and wipe with a KIMWIPE to remove any TISAB remaining on the tube. 9.3.9 Open the sample hatch and remove any remaining ash from the boat. Ash can be removed with a cotton tipped applicator or vacuumed out. It may be necessary to scrap particles off the bottom with a spatula or other similar device. A drop of Milli-QTM water may be added to the boat to aid in the Clean Cycle. 9.3.10 Close the hatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample is ready for analysis by ion selective electrode (AMDT-M-2). 9.4 Sample Calculations 9.4.1 Use the standard curve to calculate the sample value. 9.4.2 Sample Mass Recovered F (ug) = (TISAB vol in mL) x (Orion reading in ppm - intercept) (Slope) 10.0 VALIDATION_______________________________ ______________________ 10.1 Quality Control 10.1.1 Daily Start Up Check Samples: Once the standard curve is established, each day of analysis is started by analyzing QC samples. The QC samples are to be the same as the lowest concentration spiked samples used to generate the standard curve. Each concentration must be done in triplicate unless the first two replicates are within 20% of the standard curve, then a third replicate is not necessary. 10.2 Precision and Accuracy: See method development analysis and sample analysis in Fluoride Notebooks 2,3, and 5. Precision and accuracy varies when analyzing samples o f different matrices and different reference compounds. 10.3 O ther Validation Parameters: NA 00068V 5 11.0 DATA ANALYSIS 11.1 Calculations 11.1.1 For the standard curve, use regression analysis in Excel, version 5.0 or greater. 11.1.2 To calculate the fluoride contraction in the sample, see method AMDT-M-2. 11.2 Analyzing the Data 11.2.1 r2 must be at least 0.95 or greater. "Outliers" may be excluded if two of the three replicates are within 20% of each other and die outlier is greater than 200% of the average of those two or less than 50% of the average of those two. Any such outliers should be pointed out in the data and noted in the Final Report along with the reason it was considered an outlier. 12.0 ATTACHM ENTS___________________________________________ ___ None 13.0 REFERENCES_________________________________________________ _ 13.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer Operator's Manual (Manual 915349, revision B, December 1993) 13.2 AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 13.3 AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 REVISIONS________________________________________________________ Revision Number Reason for Change Revision Date OO0 8 8 6 3M Environmental Laboratory Method Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer M ethod Identification Number: AMDT-M-2 Revision Number: 0 Adoption Date: Revision Date: None Author: Rich Youngblom Approved By: C Grorft/ Leader iy L /J --- y/fs 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 0G0GS9 1 1.0 SCOPE . APPLICABLE COMPOUNDS. AND M ATRICES 1.1 SCOPE: This method is for the calibration and operation of an Orion EA940 Expandable Ion Analyzer. 1.2 APPLICABLE COMPOUNDS: Fluoride. 1.3 APPLICABLE MATRICES: Liquid samples in an appropriate buffer solution. Preferred pH of 6.0. 2.0 KEYW ORDS______________________________________ ___________ ___ 2.1 Fluoride, fluorine, ion selective electrode 3.0 PRECAUTIONS ____________________________________________ 3.1 No hazards identified with this method. 4.0 SUPPLIES AND MATERIALS______________________ ________________ 4.1 Orion 940999 Total Ionic Strength Adjustment Buffer II (TISABII) or equivalent. 4.2 Orion Model 900001 electrode filling solution (AgCl) or equivalent. 4.3 Orion 940907 100 ppm fluoride standard or equivalent. 4.4 Milli-QTM water or equivalent. 4.5 Magnetic stir bars. 4.6 Lab tissues. 4.7 Sample collection vials. 4.8 Plastic 100 mL volumetric flasks. 4.9 Polystyrene pipettes. 4.10 Miscellaneous laboratory glassware. 5.0 EQUIPM ENT_______________________________________________________ 5.1 Orion Model EA940 Expandable Ion Analyzer or equivalent. 5.2 Orion Model 960900 Solid State Combination Fluoride electrode or equivalent. 5.3 Magnetic Stir Plate. 5.4 IBM compatible 386 or 486 computer (only needed if using Orion 3E software). 5.5 Orion RS232 interface cable (only needed if using Orion 3E software). 5.6 Microsoft Excel 5.0 (only needed if using Orion 3E software). 6.0 INTERFERENCES___________________________________________________ 6.1 It is recommended that the pH be at or near 6.0. A 1:1 mixture of TISAB and sample/MilliQTM water will generally bring sample to pH of 6.0. 6.2 Sample temperature may effect fluoride measurement. It isjecommended 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. 00G G 90 2 6.4 Air bubbles trapped under electrode can give erroneous readings. Make sure no air is trapped under electrode. 7.0 SAM PLE HANDLING_________________________________ 7.1 No special handling necessary. 8.0 CALIBRATION AND STANDARDIZATION_______________ _ 8.1 Preparation of Calibration Standards 8.1.1 Measure 50 mL of TISAB II into 5 100 mL plastic volumetric flasks. 8.1.2 Label the flasks as 0.05, 0.1,0.5, 1.0, and 1.5 ppm F-, along with the date and your initials. 8.1.3 Pipette 0.05, 0.1, 0.5, 1.0, and 1.5 mL of 100 ppm fluoride standard into the appropriately labeled flasks. 8.1.4 Add approximately 30 mL of Milli-QTM water to each flask. 8.1.5 Shake die 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 die final mixing. 8.1.9 Record standards in Standards Log Book. 8.2 Calibration 8.2.1 If necessary, remove tape from electrode filling hole. 8.2.2 Invert probe to wet top seal. 8.2.3 Eject a few drops of filling solution from bottom of electrode to wet lower seal. 8.2.4 Fill the electrode with filling solution. 8.2.5 The meter and the F- electrode are typically calibrated by direct measurement with no blank correction, using standards with concentrations of 0.05,0.1,0.5,1.0, and 1.5 ppm F-, following the manufacturer's instructions. 8.2.6 Record the slope in the appropriate log book. 8.2.7 Clean the electrode by rinsing with Milli-QTM water and wiping the sides down with lab tissues. 8.3 Storage Conditions for Standards 8.3.1 Calibration standards are stored at room temperature. 9.0 PROCEDURES_______________________________________________________ 9.1 Calibration and Measurement, Standard method: 9.1.1 The sample to be measured needs to be mixed with TISAB using the proportions recommended by the TISAB manufacturer. 9.1.2 Place a stir bar in the sample and place the sample on the stir plate. 9.1.3 Allow the sample to mix for a few seconds before inserting the electrode. When the electrode is inserted, make sure there are no air bubbles trapped under the electrode. 9.1.4 The sample should be the same temperature as the calibration-standards and stirred at the same rate as the calibration standards. 9.1.5 When the readings have stabilized, record the reading in the appropriate log book. 000o91 3 9.2 Calibration And Measurement, Using Orion 3E Software: 9.2.1 Calibration: 9.2.1.1 Follow steps 8.2.1 to 8.2.4. 9.2.1.2 Press Function Key #8 (F8). 9.2.1.3 The computer screen will ask you to confirm the number of standards to be used, concentration of the standards, and whether or not a blank is to be included in the calibration. Make any necessary changes to the information presented and click on CONTINUE. 9.2.1.4 Place the electrode in the first standard on the stir plate and click on CONTINUE. 9.2.1.5 Observe the readings on the graphic display on the computer. When the readings have stabilized, press ACCEPT READING. 9.2.1.6 Repeat step 9.2.1.4 and 9.2.1.5 for the remaining standards. 9.2.1.7 After the final standard, the computer will display the slope of the curve, as well as the intercept and correlation. Record the slope, intercept, and correlation in the appropriate log book and click on CONTINUE. The calibration data is automatically copied to C:\Orion\Data\Calib.txt. 9.2.2 Data Spreadsheet: 9.2.2.1 Select either NEW or OPEN from the FILE menu to open a new or existing spreadsheet to store data in. 9.2.2.2 Record the name of the spreadsheet used in the appropriate log book. 9.2.3 Fluoride Measurement: 9.2.3.1 Follow steps 9.2.1 through 9.2.4 9.2.3.2 Enter the name of the sample in the appropriate place on the screen. 9.2.3.3 Click on the NEW SAMPLE button 9.2.3.4 When the readings have stabilized, click on the RECORD button and write the result in the appropriate log book. 10.0 VALIDATION______________________________________________________ 10.1 Quality Control: 10.2 Precision and Accuracy 10.3 O ther Validation Parameters According to Reference 13.2, the range of detection is 0.02 ppm fluoride up to a saturated solution of fluoride. 11.0 DATA ANALYSIS___________________________________________________ 11.1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 12.0 ATTACHM ENTS_______________ _____________ ______________________ None 13.0 REFERENCES______________________________________________________ 4 13.1 Orion Model EA940 Expandable Ion Analyzer Instruction Manual, Orion Research Incorporated, 1991. 13.2 Orion Model 960900 Solid State Combination Fluoride Electrode Instruction Manual, Orion Research Incorporated, 1991. 14.0 REVISIONS__________________________________________________ _ Revision Number Reason for Change Revision Date 000G93 5 3M Environmental Laboratory Method Extraction of Fluorochem icals from Rabbit Livers SOP Identification Number: AMDT-M-4 Revision Num ber: 0 Adoption Date: /<a - ? /-* Revision Date: None Author Dave Christenson/Cynthia Weber Approved By: 'yroupLeader / J /0 -3 -2 S Date Quality Assurance /a - h ir Date Software: MS Word, 6.0 Affected Documents: M-5, Analysis of Rabbit Extract for Fluorochemicals Using Electrospray Mass Spectroscopy. 000894 1,0 SCOPE__________________________________________ _ 1.1 Scope: This method is for the extraction of fluorochemicals from rabbit livers. Ethyl acetate is used to extract fluorochemicals from the livers for analysis by electrospray mass spectroscopy. 1 .2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1 .3 M atrices: Rabbit Livers. zjLEmwmm______________________ _ 2 .1 Fluorochemicals, rabbit livers, electrospray mass spectrometer, fluorinated compounds, extraction. 3.QPRECAUTIONS ________________________________ 3 .1 Use gloves when handling the rabbit livers, they may contain pathogens. 4 .0 SU PPL IE S AND M ATERIALS_____________________________________ 4.1 Supplies 4 .1 .1 Syringe, capable of measuring 100 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 Jim. 4 .1 .1 0 Analytical pipets: glass volumetric pipets. 4 .1 .1 1 Disposable plastic 3 cc syringes. 4 .1 .1 2 Crimp cap autovials. 4.2 Reagents 4 .2 .1 Aqueous Ammonium Acetate (Aldrich), approx. 250 ppm: Prepare a 2500 ppm aqueous solution of ammonium acetate by adding 250 mg ammonium acetate to a 100 mL volumetric flask and dilute to volume with Milli-Q water. Dilute this solution 1:10 for a 250 ppm solution. 4 .2 .2 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker), (Na2C 03/NaHC03) 0.25 M: Weigh 26.5 g of sodium carbonate (N a^O j) and 21.0 g of sodium bicarbonate (NaHCOa) 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) G0S5 2 5.0 EQUIPMENT____________________________ 5 .1 Ultra-Turrax T25 Grinder for grinding liver samples. 5 .2 Vortex mixer 5 .3 Centrifuge 5 .4 Shaker 5 .5 Analytical Evaporator 6.0 IN T E R FE R E N C E S_______ _____________ 6 .1 There are ho known interferences at this time. 7.0 SA M PLE H A N D LIN G ______________________________________________ 7 .1 The rabbit livers are received frozen, and must be kept frozen until the extraction is performed. 8.0 CALIBRATION AND STANDARDIZATION ________________ 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. 0GGG96 3' 8 .3 .3 Spike seven of the 1 mL aliquots with the following amounts of working standards in step 9.12 of the procedure. One 1 mL aliquot serves as the blank. Working Standard (Approximate Cone.) 50 ppb 500 PPb 500 ppb 500 ppb lppm 5 ppm 5 ppm uL 100 20 300 400 500 2 300 Approximate final concentration of FC-95 in liver Blank 0.292 ppm 0.584 ppm 0.877 ppm 1.168 ppm .924 ppm 5.848 ppm 8.772 ppm 8 .4 Calculate the actual value of the standards: uL of standard x concentration (in 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 PR 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 T-g of liver. 9 .3 Weigh the sample directly into a tared plastic grinding tube. 9 .4 Record the liver weight in the study note book. 9 .5 Put a label on the vial with the study number, weight, rabbit ID, date and analyst initials. GOG >97 -4~ 9 .6 Add 2.5 mLs water. 9 .7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, until the sample is a homogeneous solution with no large chunks. 9 .8 Rinse the probe off into the sample with 2.5 mLs water using a pipet. 9 .9 Take the grinder apart and clean it with methanol after each sample. Follow AMDT-EP-22. 9 .1 0 Cap the sample and vortex for 15 seconds. 9 .1 1 Pipet 1 mL into a 15 mL centrifuge tube. Label the centrifuge tube with the identical information as the grinding tube. (See AMDT-M-4 Worksheet for documenting the remaining steps.) 9 .1 2 Spike the beef liver homogenates with the appropriate amount of FC-95 standard as described in 8.3. 9 .1 3 Spike the samples and beef liver homogenates with 100 uL of internal standard. 9 .1 4 Add 1 mL of the sodium carbonate/sodium bicarbonate buffer and 1 mL ammonium acetate. 9 .1 5 Using an analytical pipet, add 5 mL ethyl acetate. 9 .1 6 Cap the sample and vortex 20 to 30 seconds. 9 .1 7 Put them in the shaker for 20 min. 9 .1 8 Centrifuge for 20 to 25 minutes, until the layers are well separated. Set the power on the centrifuge to 25. 9 .1 9 Remove 4 mLs of the top organic layer to a fresh 15 mL centrifuge tube with a 5 mL graduated glass pipet. Transfer die label to the fresh tube. 9 .2 0 Blow the sample down on the analytical evaporator to near dryness with nitrogen, approximately 30 to 40 minutes. 9 .2 1 Bring the remaining sample up in 1 mL dilute acetonitrile with an analytical pipet. 9 .2 2 Vortex 15 seconds. 9 .2 3 Transfer the sample to a 3 mL syringe. Attach a 0.2 Jim nylon mesh filter, and filter the sample into a fresh centrifuge tube or a autovial. Label the tube or vial with the study number and animal number. 9 .2 4 Cap and hold for analysis by electrospray mass spectroscopy. 9 .2 5 Complete AMDT-M-4 worksheet and attach to page of study notebook. 10.0 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 Param eters- not applicable 11.0 D A TA A N ALYSIS_____________________________ 11.1 None 12.0 A TT A C H M EN TS_______________________________ 1 2 .1 Worksheet AMDT-M-4 13.0 R EFER E N C E S__________________________________ 13.1 AMDT-EP-22 Routine Maintenance of Ultra-Turrax T-25 14,0 REVISIONS Revision N u m ber Reason for Chanee Revision Pats 00069S f<ijjftimR IMfctV "IKS'* $%8r' Worksheet AMPT-M-4 S tudy # . _ _ . . . _ 1 Sam ple N um ber set # B lan k T.iver FC -95 approx 0.5 ppm actual #W ppm . ________ 1 0 0 u l,________ 2 0 0 uT. 300 uL 4 0 0 uT, - . _ . _ FC -95 approx 1 ppm actual #W ppm _ - . _ . 500 uT. . . _ - ._ _ -,, FC -95 approx. 5 ppm actual ppm #W . _ Date and Initials for Std. _ 200 uL 300 uT. . _ * _ _ _ _ _ 1stu d v n u m b er w here th e original w o rk sh eet is located y id n lace a conv. T.iver E x tra c tio n P ro c ess- D ate R / in itials P in e t 1 mT. o f T .iver S o lu tio n P inet 100 uT. o f 12 nnm internal S tandard S td. # V ortex 15 sec P inet 1 mT, o f 250 nnm A m m onium A cetate Std # P in et 1 m l, o f 0.25 N a,,C O -/0.25M N aH C O , B u ffer P inet 5 mT, o f F thvl A cetate V ortex 20-30 sec S hake 20 m in C entrifiipe 20 -2 5 m in R em ove a 4 m i, alinnot o f n m anic laver B low do w n to n ear drvness f< 0.25 m L l w ith N , A dd 1 m: o f 11 A ceto n itrile/H ,0 V ortex 15 sec TN# F i l t e r u s i n g a 3 c c B - D s y r i n g e w ith a 0 .2 u m S R I f i l t e r in to a 1 5 m l , a n t o s a m n l e v ia l________________________________ 000699 <T 3M Environmental Laboratory_____________________ _ Method Analysis of Rabbit Liver Extract for Fluorochemicals using Electrospray Mass Spectroscopy SOP Identification Number: AMDT-M-5 Revision Number: 0 Adoption Date: -C -'ir' 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 / 000700 -l 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 EYW O RDS_____________________________________________________ 2 .1 Fluorochemicals, fluorinated compounds, electrospray mass spectroscopy, mass spectrometer, rabbit livers. 3.0 PR EC A U T IO N S_________________________________________ 3.1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 3 .2 Do not run the pump above it's capacity of 4000 psi. If pressure goes over 4000 psi stop and release pressure. The peak tubing may be plugged. Troubleshoot back to find the plug and replace the plugged tubing. See AMDT-EP-15 3 .3 Do not run the pump to dryness. 4 .0 SUPPLIES AND M A T ER IA LS___________________________________ 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 EQUIPM ENT 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 INTERFERENCES 6 .1 There are no known interferences at this time. 7.0 SA M PLE HANDLING______________________________________________ 7.1 Keep the extracted samples in capped 15 mL centrifuge tubes or in capped autovials until ready for analysis. OOO 701 2 8.0 C A L IB R A T IO N AND ST A N D A R D IZ A T IO N __________________ 8.1 Preparation of Calibration Standards 8 .1 .1 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 PROCEDURE _______________________________________________ 9.1 Initial Set-up 9 .1 .1 Set software to "Operate on", Ion Mode ES\ 9 .1 .2 Record backing pressure in the instrument log. 9 .1 .3 Fill the solvent cylinder with mobile phase. 9 .1 .4 Set the pump to "Run". Set the flow to 1000 uL/min. Observes droplets coming out of the tip of the probe. The pressure should be at 1700 to 1800 psi. 9 .1 .5 Check the fused silica at the end of the probe. Use an eye piece to check for chips. The tip should be flat with no jagged edges. If any chips are found cut off the tip of the silica with a column cutter and pull the silica through to the appropriate length. 9 .1 .6 Check your nitrogen supply. Turn on the nitrogen. There should be no nitrogen leaking around the tip of the probe. A fine mist should be coming out of the tip. 9 .1 .7 Carefully guide the probe into the opening. Insert it until it won't go any further. Connect the voltage cable to the probe. 9 .1 .8 Go to the "Editor" page, and set Ionization Mode to ES', and the appropriate masses to 427 and 499. 9 .1 .9 If it is not in single ion mode go to "Option" and set SIR. 9 .1 . lOStart Acquisition. Assign a file name, MO-DAY-YR + letter. Record it in the log book. 9 .1 .1 1 Run the beef liver samples first, running each standard twice at the beginning of the run.. Run a QC check by running one standard after every 5 to 7 samples. 9.2 Manual Injection 9 .2 .1 Draw 150 uL of sample into a syringe. Injecfthe sample into the rheodyne injection port. Inject slowly. Record the sample ID in the log book. 9 .2 .2 Tujrii 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. -3 000702 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 "Enter1 9 .3 .3 .8 Press "Run" to start 10.0 V A L ID A T IO N __________________________________________ 10.1 Quality Control 1 0 .1 . IRun a standard every 5 to 7 samples. If a significant change( 50%) in peak height occurs stop the run. Only the samples before the last acceptable standard will be used. The remaining samples will be reanalyzed. 10.2 Precision and Accuracy 10 .2 . lSee Method Validation Report number AMDT-M-5.0.VI 10.3 O ther Validation Param eters 1 0 .4 Refer to Method Validation Report Number AMDT-M-5.0.V1 11.0 DATA ANALYSIS____________________________________________ ___ 11.1 11.2 Calculations Plot the standard curve, using the mean of the two values obtained for each standard. 1 1 .2 . IRead peak heights or areas for the samples from the printout. Use linear regression to determine the sample concentrations. 1 1 .2 .2 Calculate the mg of FC-95 anion, or other fluorochemical in the total rabbit liven mg FC-95 anion in the total rabbit liver = mg FC-95 anion from std. curve gms of liver used for analysis x Total mass of liver, gms 1 1 .3 Make a results table and enter it in the study book. 1 1 .4 Print a chromatogram for each sample, with the peaks labeled with the sample or standard ID. Write the study number on the printotruinitial, date, and put it in the study folder. Staple all chromatograms together and number pages. 000703 4~ 12.0 ATTACHM ENTS None 13.0 INFERENCES______________________________ 13.1 AMDT-EP-17 14.0 R EV ISIO N S_________ ___________________________________ . Revision Number Reason for change Revision Date 000704 3M Environmental Laboratory Method Analysis of Fluoride Using the Skaiar Segmented Flow Analyzer W ith Ion Selective Electrode Method Identification Number: AMDT-M-8 Adoption Date: Revision Number: 0 Revision Date: None Author: Deb Wright / Cynthia Weber Approved By: /^ Quality Assurance /A /?s Date Date Software: IBM MS Word, 6.0 Affected Documents: AMDT-EP-26, Operation and Maintenance of the Skalar Segmented Flow Analyzer OOO 70S l 1.0 SCO PE________________________________________________________ 1.1 This method is for the analysis for fluoride, thermally extracted from samples using the Dohrmann DX2000 (AMDT-M-1), and collected in TISAB for analysis with an Ion Selective Electrode (ISE). The analysis is performed using the Skalar Segmented Flow Analyzer with ISE. 1.2 Samples can be tissues, serum, biological material, or other materials extracted on the Dohrmann. 2.0 K EY W O R D S_______________________________________________ ___ 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 PPL IE S AND M ATERIALS_______________________________ _____ 4.1 Supplies 4 .1 .1 Sample cups, 4 mL plastic cups with caps 4 .1 .2 Autopipets, oxford or equivalent with plastic tips 4 .1 .3 Polypropylene volumetric flasks, 100 mL 4 .1 .4 Cartridge components, refer to the Skalar Methods for components and part numbers. 4 .1 .5 Sample prefilters, Evergreen 4.2 Reagents 4 .2 .1 Brij 35, 30% S.F.A.S. Detergent 4 .2 .2 TISAB II buffer solution: Purchase TISAB II from Orion. To 1 liter of TISAB II add 2.5 mL or 100 ppm fluoride solution and 1 mL Brij. 4 .2 .3 Sampler rinsing solution: Dilute TISAB I I 1:1 with Milli-Q water. 4 .2 .4 Nitric acid solution for decontamination, 1 N (lab grade): Slowly add 64 mLs concentrated nitric acid (HN03) to 250 mLs of Milli-Q water. Bring the volume up to 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 pipets, to 50 mLs of TISAB and diluting to 100 mLs __________________with Milli-Q water._______________________________________________ Working Standard mLs of Stock Standard mLs of Intermediate Standard 0.015 ppm 0.03 ppm - - 0.15 0.3 0.06 ppm - 0.6 0.09 ppm -- 0.9 0.12 ppm - 1.2 0.15 ppm - 1.5 0.3 ppm 0.6 ppm 0.3 0.6 - i 0 0 0 7 0 6 2= 1.2 ppm __________ L5 PPm__________ 1.2 1.5 - - 5.0 EQUIPM-M 1________________________________________________ 5 .1 Skalar Segmented Flow Auto Analyzer Sans"1" System equipped with ISE $.0 INTERFERENCES_________________________________ _ 6 .1 High concentrations of alkalinity, chloride, phosphate, sulfate or iron can cause interferences. 7.0 SA M PLE HANDLING _____________________________________________ 7 .1 Samples should be stored in polyethylene bottles. Samples should be analyzed within 30 days. 8.0 C A L IB R A T IO N AND STA N D A R D IZ A T IO N _____________: ._ 8.1 Preparation of Calibration Standards 8 .1 .1 Prepare calibration standards as in section 4.3. 8.2 Calibration 8 .2 .1 The standards are analyzed at the beginning of the run. 8.3 Storage Conditions for Standards 8 .3 .1 Standards are stored in capped polypropylene volumetric flasks. New standards are prepared at a minimum of every six months, or as necessary. 9.0 PR O C E D U R E _____________________________________________________ 9.1 Start Up Procedure 9 .1 .1 Clamp down the pumpdecks, air bars and sampler-pump tubing. 9 .1 .2 Put the fluoride electrodes in the electrode chamber. 9 .1 .3 Turn on the power of the sampler, pumps, offset potentiometer and heating bath. 9 .1 .4 Put the reagent-lines in the appropriate bottles. 9 .1 .5 Turn on the interface, computer, display and printer. M ake 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 appropriatejiumber 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, REALTIME. If you cannot get real-time, you may be in the Data Handling Panel. Switch to the Analysis Panel by selecting CONTROL PANEL and pushing F7. 9 .2 .5 .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 .3 Check the highest standard and adjust the gain, if necessary, with the interface screw #3. 9 .2 .6 Go to CONTROL PANEL, and to analysis panel. Deselect the analysis that will not be run. (Select or deselect analysis by pressing ENTER.) Press Tab to return to the Analysis Panel. 9 .2 .7 Press the spacebar to bring up the local menu. 9 .2 .8 Select START to start the analysis. 9 .2 .9 Type your ID (initials), the sample table which you created under 9.2.1 (or press ENTER for choices), choose running with or without the system table and select START ANALYSIS. 9 .2 .1 0 After starting the software, start the sampler. Make sure that the sampler is set to the right number of samples and that the sample/wash/air times are OK. 9 .2 .1 1 Select GRAPHICS, REAL TIME to view the progress of the analysis. 9.3 Loading and Printing the Data-File 9 .3 .1 Go to CONTROL PANEL, press the spacebar to bring up the local menu and select LOAD. Select AUTOCALCULATION and enter the filename (or highlight the file to be printed and press ENTER). 9 .3 .2 To view the calibration curve, go to GRAPHICS, CALIBRATION CURVE. 9 .3 .3 To print the high level curve, push PRINT SCREEN. 9 .3 .4 To print the low level screen, push ESC to get out of graphics. Select SETTINGS. Change the max y value to approximately 900. Go to CAL CURVE and press ESC, and Enter. Press PRINT SCREEN. 9 .3 .5 Return to SETTINGS and change the max value back to 4095, go to EDIT, press ENTER and PRINT SCREEN to print sample peaks. 9 .3 .6 To print the results go to CONTROL PANEL, SPACEBAR, OUTPUT, OUTPUT. Select PRINTER for the Epson or PRN for the Laser. 9.4 Shutdown 9 .4 .1 Put all the reagent-lines in Milli-Q water. 9 .4 .2 Let the system rinse for approximately 30 minutes. 9 .4 .3 After the system has rinsed completely, turn off the sampler, pump and offset potentiometer. Turn off the heating bath on weekends. Leave liquid in the lines. 9 .4 .4 Take the electrode out and soak in 100 ppm F overnight. 9 .4 .5 Release the pump-decks, air bars and sampler pump-tubing. 9 .4 .6 Select FILES, press ALT F and select QUIT to exit the program. 9 .4 .7 On Friday, turn off the computer, display and interface for the weekend. 10.0 V A L ID A T IO N _____________________________________________________ 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. 0 0 0 708 _j4^, 10.2 Precision and Accuracy 10.2. lSee Method Validation Report number AMDT-M-8.0.VI 10.3 O ther Validation Parameters 10.4 Refer to Method Validation Report Number AMDT-M-8.0.VI 11.0 DATA A N A LY SIS___________ _____________________________ ___ 11.1 11.2 11.3 11.4 C a lc u la tio n s 1 1 .1 .1 The standard curve is plotted by the Skalar software. 1 1 .1 .2 All calculations are done by the Skalar software, r2 should be 0.995 or better. Prepare spreadsheets to summarize data. Include sample volume, weights used etc. Write the study number on the printouts, initial, date the printout, and bind together with all package documents and place in the study folder. Make a copy of the summary sheet and tape into the study notebook. Back up all data and spreadsheets onto study disk and backup disks. Electronic Data 1 1 .4 .1GLP studies: Electronic data is copied onto the Study floppy disk for each study, and also data is copied onto a floppy disk that is stored in the lab. 1 1 .4 .2 Other studies: All data is copied onto a floppy disk that is stored in the lab. 12.0 A TT A C H M EN TS___________________________________________________ None 13.0 R E FE R E N C E S______________________________________________________ 13.1 13.2 13.3 AMDT-M-1, Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver Skalar Methods, #335, Skalar Methods Manual AMDT-EP-26, Operation and Maintenance of the Skalar Segmented Flow Analyzer 14.0 R EV ISIO N S________________________ ________________ _______________ Revision Number Reason for change Revision Date 000709 3M Environmental Laboratory Method 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: f o - 3 -*/ r " Revision Date: None Author: Rich Youngblom Approved by: ^Group Leader * /o /s / YS Date Quality Assurance Date Software: MS Word 5.1a Affected Documents: AMDT-M-2 Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 000710 1.0 SCOPE . APPLICABLE COMPOUNDS, AND MATRICES 1.1 Scope: This method is for the operation of a Dohrmann DX2000 when it is used to extract fluoride from various matrices. The fluoride is typically collected in TISAB solution for analysis with an ion selective electrode. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Biological fluids, particularly serum. 2.0 KEYW ORDS_____________________________________________________ __ 2.1 Fluoride, fluorine, extraction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS_______________ _____________________________ ________ 3.1 Glassware and exhaust gases can be extremely hot. 3.2 Glassware is fragile, broken glass may cause injuries. 3.3 Pressurized gases, proper compressed gas handling practices required. 3.4 Solvent based samples may flash, may need to allow them to dry down before starting run. 3.5 Potential biohazards due to the biological matrices. Use appropriate personal protective equipment. 4.0 SUPPLIES AND 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 EQ UIPM ENT________________________________________________________ 5.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer, modified for fluoride extraction. 5.2 IBM compatible 386 or 486 computer. 5.3 DX2000 software, version 1.00, modified for fluoride extraction. 5.4 Excel Spreadsheet, version 5.0 or greater 6.0 INTERFERENCES___________________________________________________ 6.1 Sample size is limited to approximately 100 jil. This may vary from matrix to matrix. 000711 2 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 CALIBRATION AND STANDARDIZATION________________________ _ 8.1 Preparation of Calibration Standards 8.1.1 Th 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, o f standard in ppm) x (0.6004)* *FC-95 is 60.04% F therefore 0.6004 is the factor used to convert FC-95 to F 8.2.5 Standard Mass Recovered F (ug) = (TISAB volume in mL) x (Orion reading in ppm) 8.3 Calibration - Procedure 8.3.1 S tart Up 83.1.1 Run 2 or more Clean Cycles when starting instrument each day. More clean cycles may be used if the previous samples contained high concentrations of fluoride. 8.3.2 Blanks 8.3.2.1 Prepare sample using the same methods and type of matrix as the test sample. 8.3.2.2 For rabbit studies, use beef serum as the matrix. 8.3.2.3 Put serum blank in Dohrmann boat. Combust sample as described in section 9.0 and analyze sample according to method AMDT-M-2 for the ion selective electrode analysis. 8.3.2.4 For rabbit studies, the meter reading for a blank sample should be 0.03 ppm or lower before proceeding with the calibration. Bum samples until thus limit is reached, or until in the judgement of the operator the reading is stable with respect to historical readings (previous 48 hours). 8.3.2.5 For non-rabbit studies, the blank readings should reach a predetermined ion concentration before proceeding with the calibration. 8.3.2.6 It may be necessary to mix approximately 50 mg of charcoal with the sample to aid combustion. 000712 3 8.3.3 Standard Curve 8.3.3.1 If beef serum is frozen, thaw at least enough to complete the standard curve analysis for the day (=30 mL). 8.3.3.2 Pipette 100JJ.L of beef serum into Dohrmann sample boat. 8.3.3.3 Start with the lowest standard concentration. Using a Hamilton syringe, eject a fixed quantity of the standard on or in the matrix. For rabbit studies, use 4 uL of standard and eject it on or in the beef serum. ' 8.3.3.4 At least 3 replicates should be used for the lowest standard concentration; more replicates may be used at the discretion of the analyst. 8.3.3.5 Combust the sample as described in section 9.3 and analyze according to AMDT-M-2. 8.3.3.6 Run all 15 standards. If one replicate is significantly different from the other two replicates, run another sample for that standard. Indicate in data that the new replicate replaces the old replicate and that the new replicate will be used to calculate the regression curve. 8.3.3.7 When all standards have been run, calculate the r^. r^ must be at least 0.95. If it is not at least 0.95, consult with supervisor. 8.3.3.8 A new standard curve should be run when the combustion tube or sample matrix is changed. New standard curve may also be run at the discretion of the analyst. 8.4 Storage Conditions for Standards 8.4.1 Storage requirements for standards are dependent on the individual standards used. Typically, standards are stored at room temperature in plastic screw top bottles. 8.4.2 New FC-95 standards should be prepared at least once a month. 9.0 PROCEDURES_________________________ __________________________ 9.1 Typical Operating Conditions: 9.1.1 Combustion tube temperature = 950C. 9.1.2 Oxygen and Helium flow = 50 cc/minute. 9.1.3 Vaporization/Drying time = 240 seconds. 9.1.4 Bake time = 300 seconds. 9.2 S tart Up Procedure: 9.2.1 If the program is not started, start the EOX program on the PC. 9.2.2 Open the SYSTEM SETUP window. 9.2.3 Put the furnace module and the cell in the READY mode. 9.2.4 Close the SYSTEM SETUP window. 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 100pL 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. 000713 4 9.3.3 Add appropriate volume ofTISAB solution or 1:1 TISAB:Milli-QTM water mixture to a labeled sample collection vial. Typically 0.6 mL to 15 mL are used. For rabbit studies, use 1.0 or 2.0 mL of 1:1 TISAB:Milli-QTM water mixture. 9.3.4 Place the vial so that the tip of the COMBUSTION TUBE is in the TISAB at least 0.25 inches. Gases released during pyrolysis must bubble through the TISAB. 9.3.5 Run the EOX-WATER program found in the RUN menu. 9.3.6 When the EOX program is finished, remove the collection vial from the combustion tube. 9.3.7 If undiluted TISAB was used to collect the sample, add an equal volume of Milli-QTM water to the TISAB to make 1:1 TISAB:Milli-QTM. 9.3.8 Rinse the end o f 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 VALIDATION___________________________ __________________________ 10.1 Quality Control 10.1.1 Daily Start Up Check Samples: Once the standard curve is established, each day of analysis is started by analyzing QC samples. The QC samples are to be the same as the lowest concentration spiked samples used to generate the standard curve. Each concentration must be done in triplicate unless the first two replicates are within 20% of the standard curve, then a third replicate is not necessary. 10.2 Precision and Accuracy: See method development analysis and sample analysis in Fluoride Notebooks 2,3, and 5. Precision and accuracy varies when analyzing samples of different matrices and different reference compounds. 10.3 O ther Validation Param eters: NA 11.0 DATA ANALYSIS_________________________________________ 11.1 Calculations 11.1.1 For the standard curve, use regression analysis in Excel, version 5.0 or greater. 11.1.2 To calculate the fluoride contraction in the sample, see method AMDT-M-2. 000714 5 11.2 Analyzing the Data 11.2.1 must be at least 0.95 or greater. "Outliers" may be excluded if two of the three replicates are within 20% of each other and the outlier is greater than 200% of the average of those two or less than 50% of the average of those two. Any such outliers should be pointed out in the data and noted in the Final Report along with the reason it was considered an outlier. 12.0 ATTACHM ENTS _______________ ______________________________ _ None 13.0 REFERENCES____________________________________________________ __ 13.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer Operator's Manual (Manual 915349, revision B, December 1993) 13.2 AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 13.3 AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 REVISIONS _______________________________________________________ Revision Number Reason for Change Revision Date 000715 6 .1.3 Amendment to Analytical Protocol AMDT-020795.1 000716 Attachment I GLP Study Protocol Amendment Study Number: AMDT-020795.1 Study Tide: Single-Dose Dermal Absorption /Toxicity Study of T-6052 in Rabbits Study Director James D. Johnson Amendment Date: November 8, 1995 Amendment Number: 1 This amendment modifies the following portion of the protocol: AMDT-M-14-0 specifies using bovine serum for the blanks in the thermal extraction. However, rabbit serum from the control animals (AMDT-110394.1) was used because the bovine serum blanks were higher than the samples. Approved by: 00071V 9.3 Quality Assurance Unit Statement 000718 Attachment D GLP Study Quality Assurance Statement Study Title: Single-dose Derm al Absorption/Toxicity Study of T-6052 in Rabbits Study Number AMDT-020795.1 Name of Auditor: Kari Rambo 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 I q ____ Phase__________ 10/13/95 10/19/95 Final Report Date Inspection Reported to Management Study Director 10/19/95 10/19/95 BEST COPY AVAILABLE /Qt QU Auditor /c-r)-9S Date 000719" 9.4 Key Personnel Involved in the Study 000720 3M Environmental Laboratory Key Personnel Thermal extraction followed by analysis using Orion ion analyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plummer Analysis o f liver extracts using electrospray mass spectrometry; Jim Johnson Dave Christenson Thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode: Jim Johnson Deb Wright ' Rich Youngblom Deann Plummer Documentation and Reporting: Jim Johnson Rich Youngblom Quality Assurance Unit: Gale Van Buskirk Cynthia Weber Kari Rambo 000721 9.11 Data 000722 9.11.1 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 000723 Summary of Combustion Data - Liver AMDT-020795.1, H W I6329-135 As Referenced in Final Report section 6.0 DATA ANALYSIS Total ug Fluoride in Whole Liver Mean per Dose Group** ug Std. Dev. Control Group 16.7 + 5.9 2.0 mg/kg dose (T6052) 12.6 + 3.2 200 mg/kg dose (T6052) 18.3* + 4.2 1000 mg/kg dose (T6052) 24.1 + 4.2 * Calculated as the mean of triplicate samples from each of three male and three female rabbits. One outlier omitted. Value of re-analyzed sample included in this report. 000724 RPT135L.XLS FC120 AB ID Liver Blk-1 Liver Blk-2 Liver Spk-1 Liver Spk-2 Liver Spk-3 Liver Spk-4 Liver Spk-5 Liver Spk-6 Liver blank-3 Liver blank-4 F52972-1 F52972-2 F52972-3 F52973-1 F52973-2 F52973-3 F52979-1 F52979-2 F52979-3 Liver Biank-1 Liver Blank-2 Liver Spike-1 Liver Spike-2 Liver Spike-3 Liver Spike-4 Liver Blank-A Liver Spike-5 Liver Spike-6 Liver Spike-7 F52975-1 F52975-2 F52975-3 F52976-1 F52976-2 F52976-3 F52983-1 F52983-2 F52983-3 F52986-1 F52986-2 F52986-3 F52990-1 F52990-2 F52990-3 F52997-1 F52997-2 F52997-3 % rcvry 80% 90% 92% 94% 108% 94% 80% 89% 79% 77% 88% 90% 92% Actual ppm Fin liver (W/W) 0.133 0.108 1.10 0.980 1.06 2.72 3.24 2.43 0.393 0.320 0.360 0.317 0.315 0.240 0.243 0.269 0.214 0.171 0.166 0.276 0.131 0.864 1.35 0.863 0.965 0.000 1.02 0.993 1.04 0.165 0.135 0.116 0.135 0.125 0.294 0.153 0.151 0.132 0.149 0.147 0.145 0.162 0.196 0.195 0.220 0.243 0.123 Average ppm Fin liver (W/W) 0.331 0.251 0.183 0.138 0.185 0.145 0.147 0.184 0.195 liver burned (grams) 0.112 0.124 0.110 0.139 0.132 0.105 0.101 0.117 0.119 0.121 0.107 0.103 0.132 0.148 0.150 0.131 0.119 0.148 0.133 0.114 0.135 0.141 0.100 0.138 0.121 0.132 0.130 0.138 0.134 0.136 0.135 0.139 0.150 0.141 0.127 0.134 0.116 0.128 0.125 0.108 0.135 0.145 0.120 0.107 0.114 0.116 0.150 Whole liver weight (grams) 82.5 82.5 82.5 75.8 75.8 75.8 90.6 90.6 90.6 86.4 86.4 86.4 60.8 60.8 60.8 96.5 96.5 96.5 82.8 82.8 82.8 80.4 80.4 80.4 91.7 91.7 91.7 Total F- in whole liver (ug) 27.3 19.0 16.6 12.0 11.2 14.0 12.2 14.8 17.9 Dosage (mg/kg) 0.0 0.0 0.0 0.0 0.0 0.0 2.0 2.0 2.0 Page 1 000725-* RPT135LXLS FC120 AB ID F52982-1 F52982-2 F52982-3 F52994-1 F52994-2 F52994-3 F53410-1 F53410-2 F53410-3 Liver Blank-1 Liver Blank-2 Liver Spike-1 Liver Spike-2 Liver Spike-3 Liver Spike-4 Liver Spike-5 Liver Spike-6 Liver Spike-7 F52984-1 F52984-2 F52984-3 F52992-1 F52992-2 F52992-3 F52996-1 F52996-2 F52996-3 F52977-1 F52977-2 F52977-3 F52989-1 F52989-2 F52989-3 F52993-1 F52993-2 F52993-3 liver blank-1 liver spike-1 liver spike-2 liver spike-3 liver spike-4 liver spike-5 liver spike-6 liver spike-7 liver spike-8 liver spike-9 liver spike-10 % rcvry 65% 65% 71% 85% 96% 90% 80% 72% 75% 67% 77% 77% 88% 152% 81% 88% 88% Actual ppm Fin liver (W/W) 0.136 0.153 0.113 0.121 .0.105 0.113 0.114 0.130 0.102 0.207 0.130 0.911 0.747 0.751 1.02 1.13 1.04 0.946 0.216 0.224 0.160 0.173 0.205 0.247 0.843 0.272 2.07 0.207 0.180 0.173 0.217 0.249 0.192 0.174 0.199 0.153 0.312 0.741 0.928 0.670 0.826 0.934 1.27 1.52 0.829 1.32 1.12 Average ppm Fin liver (W/W) 0.134 0.113 0.115 0.200 0.209 1.06 0.187 0.219 0.175 liver burned (grams) 0.147 0.105 0.116 0.130 0.151 0.148 0.129 0.119 0.146 0.106 0.102 0.108 0.131 0.144 0.125 0.129 0.131 0.128 0.112 0.104 0.131 0.121 0.115 0.136 0.134 0.115 0.142 0.140 0.126 0.128 0.106 0.134 0.135 0.141 0.129 0.147 0.148 0.146 0.123 0.151 0.141 0.124 0.105 0.151 0.148 0.101 0.118 Whole liver weight (grams) 82.4 82.4 82.4 82.9 82.9 82.9 91.0 91.0 91.0 88.5 88.5 88.5 80.6 80.6 80.6 77.8 77.8 77.8 72.0 72.0 72.0 97.9 97.9 97.9 88.3 88.3 88.3 Total F- in whole liver (ug) 11.0 9.37 10.5 17.7 16.8 82.5* 13.5 21.5 15.5 -* Dosage (mg/kg) 2.0 2.0 2.0 200 200 200 200 200 200 Sample reanalyzed below Page 2 000726 RPT135L.XLS FC120 AB ID F52996-1 F52996-2 F52996-3 F52978-1 F52978-2 F52978-3 Blank liver 1 Blank liver 2 Blank liver 3 Blank liver 4 Blank liver 5 Blank liver 6 Liver Spike-1 Liver Spike-2 Liver Spike-3 Liver Spike-4 Liver Spike-5 F52980-1 F52980-2 F52980-3 F52991-1 F52991-2 F52991-3 F52987-1 F52987-2 F52987-3 F52988-1 F52988-2 F52988-3 F52995-1 F52995-2 F52995-3 liver blank-1 liver spike-1 liver spike-2 liver spike-3 liver spike-4 liver spike-5 liver spike-6 liver spike-7 liver blank-2 % rcvry 130% 84% 87% 82% 77% 72% 81% 82% 77% 83% 84% 81% Actual ppm Fin liver (W/W) 0.291 0.244 0.422 0.259 0.224 0.225 0.411 4.45 2.01 0.340 0.277 0.404 1.37 1.09 0.831 0.955 0.784 0.289 0.235 0.231 0.330 0.270 0.238 0.204 0.604 0.230 0.249 0.490 0.340 0.453 0.315 0.266 0.168 0.862 0.820 0.847 0.905 2.03 1.89 1.75 0.266 Average ppm Fin liver (W/W) 0.319 0.236 0.252 0.279 0.346 0.360 0.345 liver burned (grams) 0.136 0.141 0.103 0.148 0.141 0.143 0.129 0.146 0.149 0.137 0.145 0.114 0.143 0.117 0.158 0.130 0.150 0.133 0.148 0.158 0.134 0.147 0.120 0.122 0.150 0.150 0.118 0.138 0.109 0.147 0.136 0.113 0.146 0.126 0.150 0.146 0.129 0.123 0.135 0.141 0.131 Whole liver weight (grams) 77.8 77.8 77.8 83.2 83.2 83.2 82.0 82.0 82.0 86.9 86.9 86.9 81.2 81.2 81.2 83.2 83.2 83.2 63.1 63.1 63.1 Total F- in whole liver (ug) 24.8** 19.6 20.7 24.3 28.0 29.9 21.8 Dosage (mg/kg) 200 1000 1000 1000 1000 1000 1000 "Repeat of above analysis Page 3 0 0 0 7 2 7 __ 9 .1 1 .2 Summary and raw data; analysis of liver extracts using electrospray mass spectrometry. 000728 HWI # 6329-135 - *>., ^ .y** A " I ^Arv*. ^ . u/*' J'<T D-cC- Study: Protocol Number: Test Material: Matrix: R Squared Value: Response Factor Amount: Analyst: Date: Method: Instrum ent: LABBASE File: Single-Dose Dermal Absorption TP3016.AB T-6052 in Rabbits (FC-120) Liver Screening N/A DLC 4/6/95 Fisons VG 2000 Electrospray MS 040695B jtoW V Jk ^XAee^eX^ M Group Dose Sample # Ion Count Extracted wt Dilution A rea* 9 factor Group 1: Omg/kg " F52975 F52976 F52983 F52972 F52973 F52979 N.D. re-extract re-extract N.D. N.D. N.D. Group 2: 2 m g/kg *** F52986 F52990 F52997 F52982 F52994 F53410 N.D. N.D. re-extract N.D. N.D. N.D. Group 3: 200 mg/kg *** F52984 F52992 F52996 F52977 F52989 F52993 $ $ re-extract $ N.D. N.D. Group 4: 1000 mg/kg *** F52978 F52980 F52991 F52987 F52988 F52995 $ $ $ $ $ re-extract i t - 'i -*TC ftp ^er-ir-v*. C on cen tratio n pg/g **** Total mass of liver a Total amount of FC-95 per liver mg % of FC-95 , * SIR Monitoring of M 599 and 598. $ = Positive response for ion monitored. ** Administered at a dose volume of 2.0 mL/kg. *** Administered at a dose volume of 0.01 mL/kg. -- ****The concentration was calculated by using the standard curve and multiplying the result by 4/5. The 4/5 factor is the result of a miscalculation in applying formula 8.4 In Method AMDT-M-4-0. 137 mg of liver was used in this calculation rather than 171 mg. The concentrations in the standard curve are therefore 5/4 larger than they should be. By multiplying the calculated concentration in the standard curve by 4/5, the correct result is obtained. 000729 ri C Q ^crvv^J^^^v^ L/-<5>!%,*,_ fyi<_ I 4 6 t 5 5 P , `s* u i> v l * - t - a 2 . q ~ i ^ s sn- : S 'T'I 1k Q ^ BEST COPY AVAILABLE File:040695B LAB-BASE - The MS Data System Sample:HUI # 6329-135; COMPOUND FC-120 (AB) CM- 5991 06/04/1995 10:56 I I }' I ji I 0 Min ooo ______ 15.0_________ 20.0 F10 NOT DEFINED 25.0 30.0 LA&ftteE Fil O u 03^5 ^ v-V'UX* ( ,3 2 9 - 1 3 5 " sfrsh ^ &LT- s m m u mMethod DLCLIV Sample DLCLIV Operator DLC R u n d a t e 0 5 - 0 3 - 1 9 9 5 11: 3*3: 36 V e r s i o n : t Printed on 05-03-1995 AT 11:39:53 Straight Line Fit forced through Origin, im r.'i-ltVnSrJA> n'11a A-*f An siO,*irj--iinnnn Q\J'QJQj 4IAU Jllbb ,Ji 14.10 ft 1 0n jE V E L AMOUNT Component 1 EXTERNAL STANDARD CALIBRATION AREA 1 0.4000 31156 2 0.8000 77583 3 1.2000 83946 4 1.6000 160773 5 4.0000 166388 6 8.0000 449783 7 12.0000 565003 -- Y= SLOPE * X + INTERCEPT Area = 5 .0 1 5 9 E + 0 4 * A m o u n t Amount 1.993 7E-05 * Area R squared = 0.9467 + 0.0000E+00 + O.OOOE+ i40i 000732 9.11.3 Summary and raw data; ppm F in serum as determined by thermal extraction followed by analysis using Orion ion analyzer. This data, although supportive, in the opinion 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. 000733' HW I6329-135 AMDT 011095.1 Dohrmann Serum Analysis Analysis Dates: 08/02/95 - 08/18/95 All serum samples were thermally extracted by a modified Dohrmann DX2000 Organic Halide Analyzer and collected in a 1:1 milli Q water and TISAB solution. The samples were measured on an Orion EA940 expandable ion analyzer. The Dohrmann was calibrated using 34ppm, 40ppm, 62ppm, lOOppm, 124ppm, 250ppm, and 500ppm FC-95 standards for serum curve 1. The same FC-95 standards were used to calibrate the Dohrmann with the exception of 250ppm and 500ppm for serum curve 2. 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). An initial calibration curve with standard deviation, %RSD, R2 value and equation of the line is on pages 8 - 1 1 . Pages 3 - 7 show the excel spreadsheet that was generated when the samples were being analyzed. The Dohrmann FC-95 initial calibration curve was not used to generate the data in the spreadsheet. Any Orion reading below 0.05 is below the Orion calibration and should be considered an estimate. The FC-95 initial calibration curve 1 was spiked into bovine serum. Bovine serum was used as blanks and QC check samples on 08/02/95 and 08/03/95. However, due to problems with blanks having higher readings than the samples, serum curve 2 was analyzed using rabbit serum from study # 6329-123 rabbit # F52346. On days 08/15/95 - 08/18/95 rabbit serum from study # 6329-123 rabbits F52346, F52335, and F52332, were used for blanks and QC check samples We were unable to located serum samples for Day 2 through Day 4. This study was discontinued after day 8 because nothing was found in the first day of sampling. Page 1 o f 11 000734 STUDY# 6329-135 SERUM FC 120 AB HWI 6329-135 Fluoride concentration in rabbit serum (ppm F-) Group 1 Dosage: 0 mg/kg Sample F52972 F52973 F52979 F52975 F52976 F52983 Day 1 0.533 0.498 0.551 0.517 0.479 0.506 Day 8 0.311 0.178 0.490 0.436 0.323 0.286 Day 15 0.553 0.859 0.612 0.674 0.501 0.503 Day 22 0.582 0.712 0.561 0.524 0.472 0.677 Group 2 Dosage: 2 mg/kg Sample F52986 F52990 F52997 F52982 F52994 F53410 Day 1 1.45 0.650 0.444 0.466 0.446 0.388 Day 8 0.338 0.277 0.305 0.283 1.27 0.697 Day 15 0.393 0.463 0.36 0.298 0.346 0.354 Day 22 0.610 0.466 0.529 0.438 0.620 0.677 Group 3 Dosage: 200 mg/kg Sample F52984 F52992 F52996 F52977 F52989 F52993 Day 1 0.594 0.550 0.568 0.602 0.642 0.642 Day 8 0.694 0.526 0.845 1.10 1.57 0.612 Day 15 0.271 0.281 0.257 0.274 0.641 0.777 Day 22 0.620 0.636 0.712 0.685 0.814 0.580 Group 4 Dosage: 1000 mg/kg Sample F52978 F52980 F52991 F52987 F52988 F52995 Day 1 0.762 1.01 1.07 0.886 0.549 0.491 Day 8 0.449 0.566 0.709 0.630 0.478 0.513 Day 15 0.777 0.807 0.752 0.700 0.668 0.57 Day 22 0.449 0.516 0.581 0.335 0.249 0.275 000735 2 STUDY# 6329-135 SERUM Sample ID BLANK BLANK BLANK BLANK BLANK 62-PPM-1 62-PPM-2 250-PPM-1 250-PPM-2 BLANK BLANK BLANK F52986-DAY1 F52990-DAY1 F52997-DAY1 F52982-DAY1 F52994-DAY1 F53410-DAY1 62-PPM-1 250-PPM-1 SERUM BLANK-1 SERUM BLANK-2 SERUM BLANK-3 SERUM BLANK-4 SERUM BLANK-5 SERUM BLANK-6 SERUM BLANK-7 SERUM BLANK-8 SERUM BLANK-9 SERUM BLANK-10 SERUM BLANK-11 SERUM BLANK-12 SERUM BLANK-13 SERUM BLANK-14 SERUM BLANK-15 SERUM BLANK-16 SERUM BLANK-17 SERUM BLANK-18 SERUM BLANK-19 SERUM BLANK-20 62PPM-1 62PPM-2 62PPM-3 250PPM-1 250PPM-2 F52984DAY1 Actual reading (ppm F-) 0.0613 0.0396 0.0383 0.0412 0.0319 0.0786 0.0934 0.304 0.269 0.0903 0.0527 0.0326 0.0723 0.0325 0.0222 0.0233 0.0223 0.0194 0.0760 0.246 0.207 0.176 0.107 0.0842 0.0745 0.0856 0.0755 0.0691 0.0615 0.0657 0.0592 0.0672 0.0703 0.0674 0.0655 0.0507 0.0434 0.0403 0.0398 0.0375 0.0969 0.0902 0.0869 0.262 0.270 0.0297 Sample TISAB mL FC95 Cone. FC95 % Actual Qty final vol spiked solution recovery ppm F- (mL or g) (mL) (ppm) (ug/ug) in sample 0.1 2.0 1.23 0.1 2.0 0.792 0.1 2.0 0.766 0.1 2.0 0.824 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 0.004 0.004 0.004 62 62 250 250 106% 125% 101% 90% 0.638 1.57 1.87 6.08 5.38 1.81 1.05 0.652 1.45 0.650 0.444 0.1 2.0 0.1 2.0 0.466 0.446 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 0.004 62 250 0.004 0.004 0.004 0.004 0.004 62 62 62 250 250 102% 82% 130% 1-21% 117% 87% 90% 0.388 1.52 4.92 4.13 3.52 2.15 1.68 1.49 1.71 1.51 1.38 1.23 1.31 1.18 1.34 1.41 1.35 1.31 1.01 0.868 0.806 0.796 0.751 1.94 1.80 1.74 5.24 5.40 0.594 Mass spiked (ug F-) 0.149 0.149 0.600 0.600 0.149 0.600 0.149 0.149 0.149 0.600 0.600 Mass recovered (ug F-) 0.123 0.0792 0.0766 0.0824 0.0638 0.157 0.187 0.608 0.538 0.181 0.105 0.0652 0.145 0.0650 0.0444 0.0466 0.0446 0.0388 0.152 0.492 0.413 0.352 0.215 0.168 0.149 0.171 0.151 0.138 0.123 0.131 0.118 0.134 0.141 0.135 0.131 0.101 0.0868 0.0806 0.0796 0.0751 0.194 0.180 0.174 0.524 0.540 0.0594 000736~^= STUDY# 6329-135 SERUM Sample ID F52992DAY1 F52996DAY1 F52977DAY1 F52989DAY1 F52993DAY1 F52978DAY1 F52980DAY1 F52991DAY1 F52987DAY1 62PPM-1 250PPM-1 8/15/95 Blank Serum-1 Blank Serum-2 Blank Serum-3 F52988-day1 F52995-day1 F52972-day1 F52973-day1 F52979-day1 F52975-day1 F52976-day1 F52983-day1 40ppm-1 100ppm-1 serum blank serum blank serum blank spike 34-1 spike 34-2 spike 100-1 spike 100-2 spike 100-3 F52972 DAY 8 F52973 DAY 8 F52979 DAY 8 F52975 DAY 8 F52976 DAY 8 F52983 DAY 8 F52986 DAY 8 F52990 DAY 8 F52997 DAY 8 F52982 DAY 8 SPIKE 62-1 SPIKE 62-2 SPIKE124-1 Actual reading {ppm F-) 0.0275 0.0284 0.0301 0.0321 0.0321 0.0381 0.0507 0.0533 0.0443 0.0890 0.264 Sample TISAB mL FC95 Cone. FC95 % Actual Qty final vol spiked solution recovery ppm F- (mL or g) (mL) (ppm) (ug/ug) in sample 0.1 2.0 0.550 0.1 2.0 0.568 0.1 2.0 0.1 2.0 0.602 0.642 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 0.004 62 250 120% 88% 0.642 0.762 1.01 1.07 0.886 1.78 5.28 Mass spiked (ug F-) 0.149 0.600 Mass recovered (ug F-) 0.0550 0.0568 0.0602 0.0642 0.0642 0.0762 0.101 0.107 0.0886 0.178 0.528 0.0401 0.0365 0.0279 0.0275 0.0246 0.0266 0.0249 0.0276 0.0259 0.0240 0.0253 0.0388 0.0982 0.0201 0.0192 0.0147 0.0431 0.0435 0.0928 0.0905 0.0984 0.0155 0.00864 0.0245 0.0218 0.0161 0.0143 0.0169 0.0139 0.0153 0.0142 0.0343 0.0853 0.106 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 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 0.004 0.004 40 100 0.004 0.004 0.004 0.004 0.004 40 40 100 100 100 0.004 0.004 0.004 62 62 124 81% 82% 90% 91% 77% 75% 82% 46% 115% 71% 0.803 0.731 0.558 0.549 0.491 0.533 0.498 0.551 0.517 0.479 0.506 0.776 1.96 0.403 0.383 0.294 0.863 0.870 1.86 1.81 1.97 0.311 0.173 0.490 0.436 0.323 0.286 0.338 0.277 0.305 0.283 0.685 1.71 2.11 0.096 0.240 0.096 0.096 0.240 0.240 0.240 0.149 0.149 0.298 0.0803 0.0731 0.0558 0.0549 0.0491 0.0533 0.0498 0.0551 0.0517 0.0479 0.0506 0.0776 0.196 0.0403 0.0383 0.0294 0.0863 0.087 0.186 0.181 0.197 0.0311 0.0173 0.0490 0.0436 0.0323 0.0286 0.0338 0.0277 0.0305 0.0283 0.0685 0.171 0.211 000737 STUDY# 6329-135 SERUM Sample ID SPIKE124-2 Blank F52994-day 8 F53410 day 8 F52984 day8 F52992 day 8 F52996 day 8 F52977 day 8 F52989 day 8 F52993 day 8 F52978 day 8 F52980 day 8 40ppm-1 124ppm-1 Blank F52991 day 8 F52987 day 8 F52988 day 8 F52995 day 8 F52972 day 15 F52973 day 15 F52979 day 15 F52975 day 15 F52976 day 15 F52983 day 15 40 ppm-1 124 ppm-1 serum blank serum blank spike 40-1 spike 40-2 spike 40-3 spike 124-1 spike 124-2 spike 124-3 serum blank F52986 DAY15 F52990 DAY15 F52997 DAY15 F52982 DAY15 F52994 DAY15 F53410 DAY15 F52984 DAY15 F52992 DAY15 F52996 DAY15 F52977 DAY15 Actual reading (ppm F-) 0.134 0.0722 0.0634 0.0348 0.0347 0.0263 0.0422 0.0550 0.0407 0.0306 0.0272 0.0283 0.0530 0.124 0.0608 0.0354 0.0315 0.0239 0.0257 0.0277 0.0430 0.0306 0.0337 0.0250 0.0251 0.0452 0.123 0.0259 0.0199 0.0373 0.0390 0.0448 0.122 0.134 0.116 0.0302 0.0196 0.0231 0.0180 0.0149 0.0173 0.0177 0.0135 0.0141 0.0129 0.0137 Sample TISAB mL FC95 Cone. FC95 % Actual Qty final voi spiked (mL or g) (mL) solution recovery ppm F(ppm) (ug/ug) in sample 0.1 2.0 0.004 124 90% 2.67 0.1 2.0 1.44 0.1 2.0 0.1 2.0 0.1 2.0 1.27 0.697 0.694 0.1 2.0 0.1 2.0 0.526 0.845 0.1 2.0 1.10 0.1 2.0 0.814 0.1 2.0 0.612 0.1 2.0 0.544 0.1 2.0 0.566 0.1 2.0 0.004 40 110% 1.06 0.1 2.0 0.004 124 84% 2.49 0.1 2.0 1.22 0.1 2.0 0.709 0.1 2.0 0.630 0.1 2.0 0.478 0.1 2.0 0.513 0.1 2.0 0.553 0.1 2.0 0.859 0.1 2.0 0.612 0.1 2.0 0.674 0.1 2.0 0.501 0.1 2.0 0.503 0.1 2.0 0.004 40 94% 0.903 0.1 2.0 0.004 124 83% 2.47 0.1 2.0 0.518 0.1 2.0 0.398 0.1 2.0 0.004 40 78% 0.745 0.1 2.0 0.004 40 81% 0.781 0.1 2.0 0.004 40 93% 0.896 0.1 2.0 0.004 124 82% 2.45 0.1 2.0 0.004 124 90% 2.68 0.1 2.0 0.004 124 78% 2.33 0.1 2.0 0.605 0.1 2.0 . 0.393 0.1 2.0 0.463 0.1 2.0 0.360 0.1 2.0 0.298 0.1 2.0 -- 0.346 0.1 2.0 0.354 0.1 2.0 0.271 0.1 2.0 0.281 0.1 2.0 0.257 0.1 2.0 0.274 Mass spiked (ug F-) 0.298 0.096 0.298 0.096 0.298 0.096 0.096 0.096 0.298 0.298 0.298 Mass recovered (ug F-) 0.267 0.144 0.127 0.0697 0.0694 0.0526 0.0845 0.110 0.0814 0.0612 0.0544 0.0566 0.106 0.249 0.122 0.0709 0.0630 0.0478 0.0513 0.0553 0.0859 0.0612 0.0674 0.0501 0.0503 0.0903 0.247 0.0518 0.0398 0.0745 0.0781 0.0896 0.245 0.268 0.233 0.0605 0.0393 0.0463 0.0360 0.0298 0.0346 0.0354 0.0271 0.0281 0.0257 0.0274 000738 STUDY# 6329-135 SERUM Sample ID SPIKE 40-1 SPIKE 124-1 serum blank F52989 day15 F52993 day 15 F52978 day 15 F52980 day 15 F52991 day 15 F52987 day 15 F52988 day 15 F52995 day 15 F52972 day 22 F52973 day 22 40ppm spike-1 40ppm spike-2 124ppm spike-1 F52979 day 22 F52975 day 22 F52976 day 22 F52983 day 22 F52986 day 22 F52990 day 22 F52997 day 22 F52982 day 22 F52994 day 22 F53410 day 22 40ppm spike-1 124 ppm spike -1 62ppm spike-1 serum blank F52984 day 22 F52992 day 22 F52996 day 22 F52977 day 22 F52989 day 22 F52993 day 22 40ppm spike-1 124 ppm spike -1 serum blank serum blank spike 40-1 spike 40-2 spike 40-3 spike 40-4 spike 124-1 spike 124-2 Actual reading (ppm F-) 0.0373 0.152 0.0465 0.0785 0.0389 0.0389 0.0404 0.0376 0.0350 0.0334 0.0285 0.0291 0.0356 0.0722 0.0460 0.110 0.0280 0.0262 0.0236 0.0339 0.0305 0.0233 0.0264 0.0219 0.0310 0.0339 0.0645 0.130 0.0898 0.0405 0.0310 0.0318 0.0356 0.0343 0.0321 0.0290 0.0616 0.116 0.0221 0.0172 0.0235 0.0333 0.0343 0.0371 0.0562 0.0910 Sample TISAB mL FC95 Cone. FC95 % Actual Qty final vol spiked (mL or g) (mL) solution recovery ppm F(ppm) (ug/ug) in sample 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 0.004 40 124 78% 102% 0.746 3.04 0.931 1.57 0.777 0.777 0.807 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.004 0.004 0.004 40 40 124 0.004 0.004 0.004 40 124 62 0.004 0.004 40 124 0.004 0.004 0.004 0.004 0.004 0.004 40 40 40 40 124 124 150% 96% 74% 134% 88% 121% 128% 78% -49% 9% 71% 77% 38% 61% 0.752 0.700 0.668 0.570 0.582 0.712 1.44 0.919 2.20 0.561 0.524 0.472 0.677 0.610 0.466 0.529 0.438 0.620 0.677 1.29 2.61 1.80 0.810 0.620 0.636 0.712 0.685 0.641 0.580 1.23 2.32 0.442 0.344 0.471 0.666 0.686 0.743 1.12 1.82 Mass spiked (ug F-) 0.096 0.298 0.096 0.096 0.298 0.096 0.298 0.149 0.096 0.298 0.096 0.096 0.096 0.096 0.298 0.298 Mass recovered (ug F-) 0.0746 0.304 0.0931 0.157 0.0777 0.0777 0.0807 0.0752 0.0700 0.0668 0.0570 0.0582 0.0712 0.144 0.0919 0.220 0.0561 0.0524 0.0472 0.0677 0.0610 0.0466 0.0529 0.0438 0.0620 0.0677 0.129 0.261 0.180 0.0810 0.0620 0.0636 0.0712 0.0685 0.0641 0.0580 0.123 0.232 0.0442 0.0344 0.0471 0.0666 0.0686 0.0743 0.112 0.1 &2_ ----- ~ _ 000739 STUDY# 6329-135 SERUM Sample ID spike 124-3 spike 124-4 SPIKE100-1 SPIKE100-2 SPIKE100-3 BLANK BLANK F52978 DAY22 F52980 DAY22 F52991 DAY22 F52987 DAY22 F52988 DAY22 F52995 DAY22 SPIKE 40-1 SPIKE 100-1 Actual reading (ppm F-) 0.205 0.0799 0.0659 0.0855 0.0851 0.0454 0.0222 0.0225 0.0258 0.0290 0.0168 0.0125 0.0138 0.0337 0.0850 Sample TISAB mL FC95 Cone. FC95 % Actual Qty final vol spiked (mL or g) (mL) solution recovery ppm F(ppm) (ug/ug) in sample 0.1 2.0 0.004 124 138% 4.10 0.1 2.0 0.004 124 54% 1.60 0.1 2.0 0.1 2.0 0.1 2.0 0.004 0.004 0.004 100 100 100 55% 71% 71% 1.32 1.71 1.70 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.1 2.0 0.908 0.444 0.449 0.516 0.581 0.335 0.1 2.0 0.249 0.1 2.0 0.1 2.0 0.004 40 70% 0.275 0.673 0.1 2.0 0.004 100 71% 1.70 Mass spiked (ug F-) 0.298 0.298 0.240 0.240 0.240 0.096 0.240 Mass recovered (ug F-) 0.410 0.160 0.132 0.171 0.170 0.0908 0.0444 0.0449 0.0516 0.0581 0.0335 0.0249 0.0275 0.0673 0.170 000 740 1F- NORMAN SERUM CURVE 1 Sample ID 1-ppm-1 1-ppm-2 1-ppm-3 3-ppm-1 D-ppm-2 0-ppm-3 0-ppm-4 2-ppm-1 2-ppm-2 2-ppm-3 00-ppm-1 00-ppm-2 00-ppm-3 24-ppm-1 24-ppm-2 24-ppm-3 50-ppm-1 50-ppm-2 :50-ppm-3 00-ppm-1 00-ppm-2 00-ppm-3 Actual Sample TISAB mL FC95 Cone. FC95 % Actual Mass Mass reading Qty final voi spiked solution recovery ppm F- spiked recovered (ppm F-) [mL or g) (mL) (ppm) (ug/ug) in sample (ug F-) (ug F-) 0.07175 0.05614 0.06462 0.1 0.1 0.1 0.08668 0.06728 0.05939 0.06385 0.1 0.1 0.1 0.1 0.07291 0.0753 0.07839 0.1 0.1 0.1 0.0902 0.1026 0.1126 0.1 0.1 0.1 0.1371 0.1 ,0.1451 0.1 0.1617 l0.1 0.3217 0.2447 0.3078 0.1 0.1 0.1 0.4438 0.4584 0.4888 0.1 0.1 0.1 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 2.0 0.004 34 176% 1.4350 0.0817 0.1435 STDEV (34ppm): 0.0156 34 138% 1.1228 0.0817 0.11228 %RSD: 12 34 158% 1.2924 0.0817 0.12924 AVERAGE: 0.128 40 180% 1.7336 0.0961 0.17336 STDEV (40ppm): 0.0241 40 140% 1.3456 0.0961 0.13456 %RSD: 17 40 124% 1.1878 0.0961 0.11878 AVERAGE: 0.139 40 133% 1.2770 0.0961 0.1277 62 98% 1.4582 0.1489 0.14582 STDEV(62ppm): 0.00549 62 101% 1.5060 0.1489 0.1506 %RSD: 3.6 62 105% 1.5678 0.1489 0.15678 AVERAGE: 0.151 100 75% 1.8040 0.2402 0.1804 STDEV (100ppm): 0.0224 100 85% 2.0520 0.2402 0.2052 %RSD: 11 100 94% 2.2520 0.2402 0.2252 AVERAGE: 0.204 124 92% 2.7420 0.2978 0.2742 STDEV (124ppm): 0.0251 124 97% 2.9020 0.2978 0.2902 %RSD: 8.5 124 109% 3.2340 0.2978 0.3234 AVERAGE: 0.296 250 107% 6.4340 0.6004 0.6434 STDEV (250ppm): 0.0821 250 82% 4.8940 0.6004 0.4894 %RSD: 14 250 103% 6.1560 0.6004 0.6156 AVERAGE: 0.583 500 74% 8.8760 1.2008 0.8876 STDEV (500ppm): 0.0459 500 76% 9.1680 1.2008 0.9168 %RSD: 5.0 500 81% 9.7760 1.2008 0.9776 AVERAGE: 0.927 O O w; ! < SERUM CURVE 1 NORMAN (07/25/95) STUDY# 6329-135 SERUM AVERAGE MASS RECOVERED (ug) 1.4000 Sample ID Actual Sample TISAB mL FC95 Cone. FC95 % reading Qty final vol spiked solution recovery Ippm F-) (mL or g) (mL) (ppm) (ug/ug) SPIKE 34-1 0.0330 0.1 2.0 0.004 34 81% SPIKE 34-2 0.0479 0.1 2.0 0.004 34 117% SPIKE 34-3 0.0430 0.1 2.0 0.004 34 105% SPIKE 40-1 0.0682 0.1 2.0 0.004 40 142% SPIKE 40-2 0.0459 0.1 2.0 0.004 40 95% SPIKE 40-3 0.0508 0.1 2.0 0.004 40 106% SPIKE 62PPM-1 0.0422 0.1 2.0 0.004 62 57% SPIKE 62PPM-2 0.0441 0.1 2.0 0.004 62 59% SPIKE 62PPM-3 0.0690 0.1 2.0 0.004 62 93% SPIKE 62PPM-4 0.0922 0.1 2.0 0.004 62 124% SPIKE 100PPM-1 0.0962 0.1 2.0 0.004 100 80% SPIKE 100PPM-2 0.159 0.1 2.0 0.004 100 132% SPIKE 100PPM-3 0.0774 0.1 2.0 0.004 100 64% SPIKE 100PPM-4 0.113 0.1 2.0 0.004 100 94% SPIKE 100PPM-5 0.0930 0.1 2.0 0.004 100 77% SPIKE 100PPM-6 0.100 0.1 1 2.0 0.004 100 83% SPIKE 124PPM-1 0.149 0.1 2.0 0.004 124 100% SPIKE 124PPM-2 0.150 0.1 2.0 0.004 124 101% SPIKE 124PPM-3 0.140 0.1 2.0 0.004 124 94% 000 743 5 Actual Mass Mass ppm F- spiked recovered in sample (ug F-) (ug F-) 0.660 0.957 0.861 0.0817 0.0817 0.0817 0.0660 0.0957 0.0861 STDEV (34ppm): %RSD: AVERAGE: 0.0152 18 0.0826 1.36 0.917 1.02 0.0961 0.0961 0.0961 0.136 0.0917 0.102 STDEV (40ppm): %RSD: AVERAGE: 0.845 0.882 1.38 1.84 0.149 0.149 0.149 0.149 0.0845 0.0882 0.138 0.184 STDEV (62ppm): %RSD: AVERAGE: 0.0234 21 0.110 0.0472 38 0.124 1.92 3.17 1.55 2.25 1.86 2.00 0.240 0.240 0.240 0.240 0.240 0.240 0.192 0.317 0.155 0.225 0.186 0.200 STDEV (100ppm): %RSD: AVERAGE: 0.0560 26 0.213 2.97 3.00 2.80 0.298 0.298 0.298 0.297 0.300 0.280 STDEV (124ppm): %RSD: AVERAGE: 0.0108 3.7 0.293 SERUM CURVE 2 NORMAN (08/15/95) STUDY# 6329-135 SERUM ooC 9.11.4 Summary and raw data; ppm F in serum as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. This data, although supportive, in the opinion o f the Study Director is not required to reach the conclusion stated in Final Report Section 6.0, and therefore is not discussed in detail. OOO 745" &QU> RE: 6329-135 SERUM SAMPLES AMDT 20795.1 Date of Analysis: 8/16, 8/21, and 8/22/95 Analyst: DDW 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 TISAB/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 mixing coil before the potential between the ion selective 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 .0 1 5 -0 .1 5 ppm and 0.15 - 1.50 ppm fluoride. The standard curve for the high range was plotted using the inverse logarithm option. The standard curve for the low range is linear. All standards and samples were then calculated by the Skalar software using these curves. All results below 0.0001 ppm appear on the raw data as #.####. A quality control standard was analyzed every 10 samples to check for accuracy and drift. Raw data is taken from the appropriate calibrated range of the Skalar printout and summarized on an Excel spreadsheet. The final results are adjusted for the collection volume and any subsequent dilutions. 000746^ COPY AVAILABLE SUMMARY OF 6329-135 SERUM SAMPLES AMDT 020795.1 A/u t a m s . i "S S ko-la^-'i^ATc GROUP 1 Dose Level : 0 GROUP 2 Dose Level : 2 m g/kg GROUP3 Dose Level : 200 m g/kg GROUP4 D ose Level : 1000 m g/kg F52972 F52973 F52979 F52975 F52976 F52983 1.04 0.98 1.02 0.88 0.84 0.96 0.51 ND 0.78 0.69 0.52 0.46 0.88 1.36 1.00 1.09 0.82 0.91 0.05 0.05 0.04 0.03 0.03 0.05 F52986 F52990 F52997 F52982 F52994 F53410 1.78 0.75 0.47 0.49 0.56 0.46 0.54 0.46 0.61 0.50 1.85 1.06 0.25 0.33 0.23 0.12 0.33 0.27 0.04 0.03 0.04 0.03 0.04 0.05 F52984 F52992 F52996 F52977 F52989 F52993 0.81 0.71 0.62 0.73 0.71 0.70 1.05 0.96 1.29 1.65 1.26 0.98 0.14 0.17 0.18 0.22 2.07 1.12 0.05 0.05 0.05 0.05 0.04 0.04 F52978 F52980 F52991 F52987 F52988 F52995 0.81 1.25 1.31 1.03 1.02 0.92 0.82 0.90 1.22 1.09 0.84 0.86 1.17 1.12 1.06 0.94 0.95 0.77 0.03 0.04 0.03 0.02 0.02 0.03 135S-SUM.XLS 000747 P~aga.C-,- 135S-A.XLS 1995-08-16 14:08 OutPut of : 950816A1 Operator : DDW Date of the Analysis : 1995-08-16 08:58 Analysis File Name : C:\SKALAR\DATA\HWIDATA\SERUM\950816A1 1 Tracer 1.50 1.47 98% 2 Drift 1.50 1.49 99% 3 Wash ND 4 Standard 1 0.015 0.016 109% 5 Standard 2 0.03 0.03 94% 6 Standard 3 0.06 0.06 100% 7 Standard 4 0.09 0.09 99% 8 Standard 5 0.12 0.12 102% 9 Standard 6 0.15 0.15 99% 10 Standard 7 0.30 0.29 95% 11 Standard 8 0.60 0.61 102% 12 Standard 9 1.20 1.22 101% 13 Standard 10 1.50 1.48 99% 14 Drift 1.50 1.49 99% 15 Wash ND 16 |>PK62-1 0.13 2.0 0.10 17 SPK 250-1 0.35 2.0 0.10 18 SPK 250-2 0.32 2.0 0.10 19 BLANK 0.12 2.0 0.10 20 BLANK 0.07 2.0 0.10 21 BLANK 0.04 2.0 0.10 22 F52986-1 0.09 2.0 0.10 23 F52990-1 0.04 2.0 0.10 24 F52997-1 0.02 2.0 0.10 25 F52982-1 0.02 2.0 0.10 26 Drift 1.50 1.48 98% 27 Wash ND 28 F52994-1 0.03 2.0 0.10 29 F53410-1 0.02 2.0 0.10 Page 1 000748 3tST COPY AVAILABLE 2.59 6.92 6.44 2.41 1.37 0.82 1.78 0.75 0.47 0.49 0.004 0.004 0.004 62.00 250.0 250.0 0.15 0.60 0.60 0.56 0.46 0.26 0.69 0.64 174% 115% 107% \P _L<3VV s n - feebLOs'?Z (5 135S-A.XLS 30 SPK 62-1 31 SPK 250-1 32 BLANK SERUM 33 BLANK SERUM 34 BLANK SERUM 35 BLANK SERUM 36 SPK 62-1 37 SPK 62-2 38 Drift 1.50 39 Wash 40 SPK 62-3 41 SPK 250-1 42 SPK 250-2 43 F52984-1 44 F52992-1 45 F52996-1 46 F52997-1 47 F52984-1 48 F52993-1 49 F52978-1 50 Drift 1.50 51 Wash 52 , {=52980-1 53 F52991-1 54 F52987-1 55 SPK 62-1 56 SPK 250-1 57 Drift 1.50 58 Wash 0.10 0.29 0.06 0.05 0.06 0.05 0.14 0.11 1.49 ND 0.12 0.25 0.27 0.04 0.04 0.03 0.04 0.04 0.03 0.04 1.49 ND 0.06 0.07 0.05 0.11 0.26 1.50 ND 99% 99% 100% 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 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 0.10 000749 5 Page 2 1.96 5.88 1.25 1.04 1.13 0.97 2.80 2.29 0.004 0.004 62.00 250.0 0.004 0.004 62.00 62.00 0.15 0.60 0.15 0.15 2.39 5.08 5.42 0.81 0.71 0.62 0.73 0.71 0.70 0.81 0.004 0.004 0.004 62.00 250.0 250.0 0.15 0.60 0.60 0.20 0.59 131% 98% 0.28 0.23 188% 154% 0.24 0.51 0.54 160% 85% 90% 1.25 1.31 1.03 2.10 0.004 62.00 0.15 0.21 141% 5.14 0.004 250.0 0.60 0.51 86% BESTCOPYAVAILABLE 135S-B.XLS 1995-08-21 12:30 O utPut of : 950821A1 O perator : DDW D ate of the Analysis : 1995-08-2107:55 A nalysis File Nam e : C:\SK A LA R\D A TA \H W ID A TA \SERU M \950821A 1 1 Tracer 1.50 1.47 98% 2 D rift 1.50 1.48 99% 3 Wash ND 4 Standard 1 0.015 0.014 96% 5 Standard 2 0.03 0.03 99% 6 Standard 3 0.06 0.06 104% 7 Standard 4 0.09 0.09 99% 8 Standard 5 0.12 0.12 99% 9 Standard 6 0.15 0.15 101% 10 Standard 7 0.30 0.28 93% 11 Standard8 0.60 0.62 103% 12 S tandard 9 1.20 1.22 102% 13 S tandard 10 1.50 1.47 98% 14 D rift 1.50 1.54 103% 15 W ash 16 S p i U M B L K l ND 0.08 2.0 0.10 17 SERU M B L K 2 18 SERU M B L K 3 19 F52988-1 20 F52995-1 21 F52972-1 22 F52973-1 23 F52979-1 24 F52975-1 0.07 0.06 0.05 0.05 0.05 0.05 0.05 0.04 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 25 F52976-1 0.04 2.0 0.10 26 Drift 1.50 1.53 102% 27 Wash ND 28 F52983-1 0.05 2.0 0.10 29 - SPK40-1 0.07 2.0 0.10 . Page 1 00075 M M COPYAVAILABLE 1.53 1.34 1.12 1.02 0.92 1.04 0.98 1.02 0.88 0.84 0.96 1.34 0.004 40.00 O.IO 0.13 140% 135S-B.XLS 30 SPK 100-1 0.14 2.0 0.10 31 BLK-1 0.03 2.0 0.10 32 BLK-2 0.03 2.0 0.10 33 BLK-3 0.02 2.0 0.10 34 SPK 40-1 0.06 2.0 0.10 35 SPK 40-2 0.06 2.0 0.10 36 SPK 100-1 0.13 2.0 0.10 37 SPK 100-2 0.12 2.0 0.10 38 D rift 1.50 1.54 103% 39 W ash ND 40 SPK 100-3 0.14 2.0 0.10 41 BLK 0.03 2.0 0.10 42 F52972-8 0.03 2.0 0.10 43 F52973-8 ND 2.0 0.10 44 F52979-8 0.04 2.0 0.10 45 F52975-8 0.03 2.0 0.10 46 F52976-8 0.03 2.0 0.10 47 F52983-8 0.02 2.0 0.10 48 F52986-8 0.03 2.0 0.10 49 F52990-8 0.02 2.0 0.10 50 D rift 1.50 1.53 102% 51 W ash ND 52 t #52997-8 0.03 2.0 0.10 53 F52982-8 0.02 2.0 0.10 54 SPK 62-1 0.05 2.0 0.10 55 SPK 62-2 0.11 2.0 0.10 56 SPK 124-1 0.14 2.0 0.10 57 SPK 124-2 0.17 2.0 0.10 58 BLK 0.10 2.0 0.10 59 F52994-8 0.09 2.0 0.10 60 F53410-8 0.05 2.0 0.10 61 F52984-8 0.05 2.0 0.10 62 D rift 1.50 1.55 103% 63 W ash ND 64 F52992-8 0.05 2.0 0.10 , 65 F52996-8 0.06 2.0 0.10 Page 2 000751" 2.83 0.69 0.64 0.49 1.29 1.23 2.50 2.44 0.004 100.0 0.004 0.004 0.004 0.004 40.00 40.00 100.0 100.0 0.24 0.10 0.10 0.24 0.24 2.70 0.69 0.51 ND 0.78 0.69 0.52 0.46 0.54 0.46 0.004 100.0 0.24 0.28 118% 0.13 0.12 0.25 0.24 134% 128% 104% 102% 0.27 113% 0.61 0.50 1.10 2.27 2.75 3.42 2.07 1.85 1.06 1.05 0.004 0.004 0.004 0.004 62.00 62.00 124.0 124.0 0.15 0.15 0.30 0.30 0.11 0.23 0.27 0.34 74% 152% 92% 115% BESTCOPYAVAILABLE 66 F52997-8 0.08 2.0 0.10 67 F52989-8 0.06 2.0 0.10 68 F52993-8 0.05 2.0 0.10 69 F52978-8 0.04 2.0 0.10 70 F52980-8 0.05 2.0 0.10 71 SPK 40-1 0.08 2.0 0.10 72 SPK 124-1 0.17 2.0 0.10 73 BLK 0.09 2.0 0.10 74 D rift 1.50 1.54 102% 75 W ash ND 76 F52991-8 0.06 2.0 0.10 77 F52987-8 0.05 2.0 0.10 78 F52988-8 0.04 2.0 0.10 79 F52995-8 0.04 2.0 0.10 80 F52972-15 0.04 2.0 0.10 81 F52973-15 0.07 2.0 0.10 82 F52979-15 0.05 2.0 0.10 83 F52975-15 0.05 2.0 0.10 84 F52976-15 0.04 2.0 0.10 85 F52983-15 0.05 2.0 0.10 86 D rift 1.50 1.57 105% 87 W ash ND 88 , SPK 40-1 0.08 2.0 0.10 89 SPK 124-1 0.17 2.0 0.10 90 D rift 1.50 1.53 102% 91 W ash ND 000752 II Page 3 1.22 1.09 0.84 0.86 0.88 1.36 1.00 1.09 0.82 0.91 1.52 3.30 0.004 0.004 40.00 124.0 0.10 0.30 0.15 0.33 158% 111% BESTCOPYAVAILABLE 135S-C.XLS 1995-08-21 16:58 O utPuf of : 950821B1 O perator : DDW D ate of the A nalysis : 1995-08-21 12:29 Analysis File Nam e : C:\SKALA R\D A TA \H W ID A TA \SERU M \950821B1 1 Tracer 1.50 1.49 99% 2 D rift 1.50 1.48 99% 3 Wash ND 4 Standard 1 0.015 0.016 105% 5 Standard 2 0.03 0.03 95% 6 Standard 3 0.06 0.06 102% 7 Standard 4 0.09 0.09 101% 8 Standard 5 0.12 0.12 98% 9 Standard 6 0.15 0.15 100% 10 Standard 7 0.30 0.28 93% 11 Standard 8 0.60 0.62 103% 12 Standard 9 1.20 1.22 102% 13 Standard 10 1.50 1.47 98% 14 Drift 1.50 1.44 96% 15 W ash ND 16 SFlUM B L K 1 0.03 2.0 17 SER U M B L K 2 0.02 2.0 18 SPK 40-1 0.04 2.0 19 SPK 40-2 0.04 2.0 20 SPK 40-3 0.05 2.0 21 SPK 124-1 0.14 2.0 22 SPK 124-2 0.14 2.0 23 SPK 124-3 0.14 2.0 24 BLK 0.03 2.0 25 F52986-15 0.01 2.0 26 Drift 1.50 1.41 94% 27 W ash ND 28 F52990-15 0.02 2.0 29 F52997-15 0.01 2.0 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 Page 1 -CSdOOO BESTGOPY AVAILABLE 0.63 0.37 0.81 0.86 1.03 2.76 2.88 2.77 0.25 0.004 0.004 0.004 0.004 0.004 0.004 40.00 40.00 40.00 124.0 124.0 124.0 0.10 0.10 0.10 0.30 0.30 0.30 0.08 0.09 0.10 0.28 0.29 0.28 85% 90% 107% 93% 97% 93% 0.33 0.23 S t U.0-2. -t c v i'iz .ii. c^rar 135S-C.XLS 30 F52982-15 0.01 2.0 0.10 31 F52994-15 0.02 2.0 0.10 32 F53410-15 0.01 2.0 0.10 33 F52984-15 0.01 2.0 0.10 34 F52992-15 0.01 2.0 0.10 35 F52996-15 0.01 2.0 0.10 36 F52977-15 0.01 2.0 0.10 37 SPK 40-1 0.04 2.0 0.10 38 D rill 1.50 1.43 96% 39 Wash ND 40 SPK 124-1 0.19 2.0 0.10 41 BLK 0.07 2.0 0.10 42 F52989-15 0.10 2.0 0.10 43 F52993-15 0.06 2.0 0.10 44 F52978-15 0.06 2.0 0.10 45 F52980-15 0.06 2.0 0.10 46 F52991-15 0.05 2.0 0.10 47 F52987-15 0.05 2.0 0.10 48 F52988-15 0.05 2.0 0.10 49 F52995-15 0.04 2.0 0.10 50 D rift 1.50 1.45 97% 51 W ash ND 52 ,F$2972-22 0.05 2.0 0.10 53 F52973-22 0.05 2.0 0.10 54 SPK 40-1 0.10 2.0 0.10 55 SPK 40-2 0.07 2.0 0.10 56 SPK 124-1 0.14 2.0 0.10 57 BLK 0.06 2.0 0.10 58 F52979-22 0.04 2.0 0.10 59 F52975-22 0.03 2.0 0.10 60 F52976-22 0.03 2.0 0.10 61 F52983-22 0.05 2.0 0.10 62 Drift 1.50 1.42 95% 63 Wash ND 64 F52986-22 0.04 2.0 0.10 65 F52990-22 0.03 2.0 0.10 Page 2 -^S dO O O 0.12 0.33 0.27 0.14 0.17 0.18 0.22 0.86 0.004 40.00 0.10 0.09 90% 3.82 1.43 2.07 1.12 1.17 1.12 1.06 0.94 BEST COPYAVAILABLE 0.95 0.77 0.91 1.02 2.03 1.31 2.78 1.19 0.82 0.68 0.65 0.91 0.004 0.004 0.004 40.00 40.00 124.0 0.10 0.10 0.30 0.20 0.13 0.28 212% 136% 93% 0.88 0.69 135S-C.XLS 66 F52997-22 0.04 2.0 0.10 67 F52982-22 0.03 2.0 0.10 68 F52994-22 0.04 2.0 0.10 69 F 53410-22 0.05 2.0 0.10 70 SPK 40-1 0.09 2.0 0.10 71 SPK 124-1 0.17 2.0 0.10 72 SPK 62-1 0.12 2.0 0.10 73 BLK 0.06 2.0 0.10 74 D rift 1.50 1.47 98% 75 W ash ND 76 F52984-22 0.05 2.0 0.10 77 F52992-22 0.05 2.0 0.10 78 F52996-22 0.05 2.0 0.10 79 F52977-22 0.05 2.0 0.10 80 F52989-22 0.04 2.0 0.10 81 F52993-22 0.04 2.0 0.10 82 SPK 40-1 0.09 2.0 0.10 83 SPK 124-1 0.15 2.0 0.10 84 D rift 1.50 1.46 97% 85 W ash ND i SStdO O O Page 3 0.73 0.57 0.89 0.92 1.79 3.36 2.42 1.24 0.004 0.004 0.004 40.00 124.0 62.00 0.10 0.30 0.15 0.18 0.34 0.24 186% 113% 163% 0.94 0.91 0.96 0.93 0.88 0.79 1.71 2.92 0.004 0.004 40.00 124.0 0.10 0.30 0.17 0.29 178% 98% EST COPY AVAILABLE 135S-D.XLS 1995-08-22 09:05 O utPut of : 950822A1 O perator : DDW D ate of th e Analysis : 1995-08-22 06:49 Analysis File Nam e : C:\SKALA R\D A TA \H W ID A TA \SERIIM \950822A 1 Sample * Sample ID Sk.il.u Standard ippm) Refill ippm) Dl TISAB Qtv Spinpl Keimen final vol i mLj '.'.V .'.'.V .V .'.V .'.'.V /.'.'.V .V .V .V .'.V .'.V .V .V .V .V .'.V .V .V .V .1 1 Tracer 1.50 2 D rift 1.50 3 Wash 4 Standard 1 0.015 5 Standard 2 0.03 6 Standard 3 0.06 7 Standard 4 0.09 8 Standard 5 0.12 9 Standard 6 0.15 10 Standard 7 0.30 11 S ta n d a rd s 0.60 12 Standard 9 1.20 13 Standard 10 1.50 14 D rift 1.50 15 W ash 16 SERU M B L K 1 17 SIUM B L K 2 18 S P K 40-1 19 SPK 40-2 20 SPK 40-3 21 SPK 40-4 22 SPK 124-1 23 SPK 124-2 24 SPK 124-3 25 SPK 124-4 26 D rift 1.50 27 W ash 28 SPK 100-1 29 SPK 100-2 30 SPK 100-3 1.46 1.48 ND 0.016 0.03 0.06 0.09 0.12 0.15 0.28 0.62 1.23 1.47 1.54 ND 0.05 0.03 0.04 0.06 0.06 0.06 0.08 0.13 0.28 0.12 1.53 ND 0.11 0.13 0.13 97% 98% 104% 94% 104% 101% 97% 101% 92% 104% 102% 98% 102% 102% 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 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 Page 1 -9 S 0 0 0 Actual ppm Fm Sam ple mLFC95 V' ***C***o**n**e ** s Solution FC 93 Soin (ppm) Mass Spiked cpr^K Mass % Recovered Recovery ,,&&?) v, - . BEST COPY AVAILABLE 0.94 0.67 0.86 1.14 1.15 1.22 1.66 2.57 5.56 2.34 0.004 0.004 0.004 0.004 0.004 0.004 0.004 0.004 40.00 40.00 40.00 40.00 124.0 124.0 124.0 124.0 0.10 0.10 0.10 0.10 0.30 0.30 0.30 0.30 0.09 0.11 0.12 0.12 0.17 0.26 0.56 0.23 90% 119% 120% 127% 56% 86% 187% 79% 2.12 2.63 2.56 0.004 0.004 0.004 100.0 100.0 100.0 0.24 0.24 0.24 0.21 0.26 0.26 88% 110% 106% Sam ple Sample n># '.V.ViVi'.V/.'.V.W.VA' 31 B LK 32 131.K 33 F52978-22 34 F52980-22 35 F52991-22 36 F52987-22 37 F52988-22 38 Drift 39 W ash 40 F52995-22 41 BLANK TISAB 42 SPK 100-1 43 D rift 44 Wash Stellar 1.50 1.50 'ftcstili 0.08 0.04 0.03 0.04 0.03 0.02 0.02 1.53 ND 0.03 ND 0.11 1.55 ND 135S-D.XLS % DTTISAB <2tj Sampf Recovery CmL), riVA-.V/.V,V.7AV.V '.w.m w w -;*?-//; 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 0.10 102% 2.0 0.10 2.0 0.10 103% 000757 5 li Page 2 BEST COPY AVAILABLE SERUM CURVE 1 7-31-95 NORMAN UUtO HtX 3 '\U R Z o - i S-J -llS Oo-sv Sample ID Spk34rl Spk 34-2 Spk 34-3 Spk 40-1 Spk 40-2 Spk 40-3 Spk 62-1 Spk 62-2 Spk 62-3 Spk 100-1 Spk 100-2 Spk 100-3 Spk 124-1 Spk 124-2 Spk 124-3 Spk 250-1 Spk 250-2 Spk 250-3 Spk 500-1 Spk 500-2 Spk 500-3 Skalar Result (ppm) DI:T1SAB mL FC 95 Cone final voi Solution FC 95 Soln (mL) Spiked (ppm) M ass Spiked (ug F-) Average M ass Recovered (ug F-) % Recovery 0.09 . 2.0 0.004 34.00 0.07 2.0 0.004 34.00 0.08 0.15 188% 0.08 2.0 0.004 34.00 0.08 2.0 0.004 40.00 0.07 2.0 0.004 40.00 0.10 0.15 155% 0.07 2.0 0.004 40.00 0.09 2.0 0.004 62.00 1210.09 2.0 0.004 62.00 0.15 0.18 % 0.09 2.0 0.004 62.00 0.11 2.0 0.004 100.0 0.12 2.0 0.004 100.0 0.24 0.24 99% 0.13 2.0 0.004 100.0 0.16 0.17 0.18 2.0 0.004 124.0 2.0 0.004 124.0 0.30 0.34 115% 2.0 0.004 124.0 0.33 2.0 0.004 250.0 0.26 2.0 0.004 250.0 0.60 0.61 102% 0.32 2.0 0.004 250.0 0.47 0.49 0.52 2.0 . 2.0 2.0 0,004. 0.004 0.004 500.0 500.0 500.0 1.20 0.99 82% SERUM CURVE 1 (NORMAN) 7-31-95 y <0.7743x + 0.086 R2 = 0.9881 MASS SPIKED (ug) SERCRV1N.AVE 000 753-- SERUM CURVE 1 7-31-95 NORMAN Sample ID Spie 34-1 Spie 34-2 Spie 34-3 Spie 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 (mL) Spiked (ppm) M ass Spiked (ug F-) Mass % Recovered Recovery (ug F-) 0.09 2.0 0.004 34.00 0.08 0.17 211% STANDARD DEVIATION : 0.2450 0.07 2.0 0.004 34.00 0.08 0.13 163% 0.08 2.0 0.004 34.00 0.08 0.16 191% % RSD : j 12.9998 0.08 0.07 0.07 0.09 0.09 0.09 0.11 0.12 0.13 0.16 0.17 0.18 0.33 0.26 0.32 0.47 0.49 0.52 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% 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% 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% 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% 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% 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 RJ= 0.9762 SERCRV1 N.SUM 000759 SERUM CURVE 2 8-16-95 NORMAN 5ESTCOPY AVAILABLE S PK 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 62-4 S PK 100-1 SPK 100-2 SPK 100-3 SPK 100-4 SPK 100-5 SPK 100-6 S P K 124-1 SPK 124-2 S PK 124-3 0.05 0,07 0.06 0.10 0.07 0.08 0.05 0.07 0.09 0.12 0.14 0.20 0.11 0.16 0.14 0.14 0.19 0.23 0.19 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 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 34.00 3 4 .0 0 34.00 40.00 40.00 40.00 62.00 62.00 62.00 62.00 100.0 100.0 100.0 100.0 100.0 100.0 124.0 124.0 124.0 0.08 0.10 0.15 0.24 0.30 0.12 150% 0.16 167% 0.17 112% 0.30 123% 0.40 134% SERUM CURVE 2 8-16-95 yI.21I2xM>.OI9t i SERCRV2N.AVE SERUM CURVE 2 8-16-95 NORMAN COPY AVAILABLE SPK 34-1 S P K 34-2 S P K 34-3 S P K 40-1 S P K 40-2 S P K 40-3 S P K 62-1 S P K 62-2 S P K 62-3 S P K 62-4 SPK 100-1 SPK 100-2 SPK 100-3 SPK 100-4 SPK 100-5 SPK 100-6 SPK 124-1 SPK 124-2 SPK 124-3 0.05 0.07 0.06 0.10 0.07 0.08 0.05 0.07 0.09 0.12 0.14 0.20 0.11 0.16 0.14 0.14 0.19 0.23 0.19 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 0.004 0.004 0.004 . 34.00 34.00 34.00 0.004 0.004 0.004 40.00 40.00 40.00 0.004 0.004 0.004 0.004 62.00 62.00 62.00 62.00 0.004 0.004 0.004 0.004 0.004 0.004 100.0 100.0 100.0 100.0 100.0 100.0 0.004 0.004 0.004 124.0 124.0 124.0 0.08 0.08 0.08 0.10 0.10 0.10 0.15 0.15 0.15 0.15 0.24 0.24 0.24 0.24 0.24 0.24 0.30 0.30 0.30 0.11 0.14 0.12 0.20 0.13 0.15 0.10 0.14 0.18 0.25 0.27 0.40 0.23 0.32 0.27 0.27 0.38 0.45 0.37 132% 169% 150% STANDARD DEVIATION : % RSD : 0.1837 12.2150 204% 139% 159% STANDARD DEVIATION : 0.3354 % RSD : 20.0349 66% 92% 122% 167% STANDARD DEVIATION : 0.4333 %RSD : 38.7472 114% 168% 95% 135% 114% 114% STANDARD DEVIATION : 0.2535 % RSD : 20.5563 126% 151% 126% STANDARD DEVIATION : % RSD : 0.1454 10.8282 SERUM CURVE 2 (NORMAN) 8-16-95 y =I. I992X + 0.0179 R* = 0.7*65 A U U. * * A 1( < O X )0 0.15 0. 0 0. 5 oe 10 0.25 o.:10 MASS SPI[K ED (u g ) SERCRV2N.SUM 000761 1995-08-16 14:10 OutPut of : 950816A1 S oftw are : v e rsio n 6.1 cl9 9 0 ,9 3 O perator : DDW D ate of th e A n aly sis : 1995-08-16 08:58 A n a l y s i s F i l e Name : C:\SKALAR\DATA\HWIDATA\SERUM\950816A1 T tf'l-'iS. I H u;^ 6 TZ.9 -I35 I-vU F lu o rid e 1.5 C a lib ra tio n o rd er = In v erse L ogarithm Slope : s = #.####* R esult = 1 0 L s J x = c o rre c te d v a lu e o f th e sam ple c l = c o rre c ted value of the co n cen tratio n 1 s = Slope o f th e e le c tro d e a2 = al = a0 = -0 .0 0 0 0 0 0 .0 0 0 6 5 -1 .1 5 7 0 6 F lu o rid e L C alib ratio n order = 2 C orrelation : r = 0.99717 R e s u l t = a 2 * x a + a l * x + aO a2 = al = aO = 0 .0 0 0 0 0 0 .0 0 0 1 8 0 .0 0 9 1 2 Sam pler Type Number Sam ple Time Wash Time A ir T im e Take up sp ecial n eed le H eight SA 1000 1 50 s e c . 120 s e c . 1 sec. S ingle N one 70 mm. D ilu ter n eedle H eight d ilu tio n F actor d i l u t i o n Volume Resam ple D ilu tio n runs : 80 : 10 : 2.5 :1 :1 mm m l. User f i l e : R e p r o c e s : No . TXT 000762 1995-08-16 14:10 Output of : 950816A1 F lu o rid e 1. 5 Path number S ignal type D ecolor sy ste m Number diL ute Resam ple d il T hreshold diG o u tp u t Window e v e n t :3 : D ebubbled : Yes :0 : No : No : 4095 :0 : O ff F luoride L s i sTandard ; Ignore s2 sT andard : Ig n o re s3 sT andard : Ig n o re s4 sT andard : Ig n o re s5 sTandard : Ignore s6 sT andard : 0 .1 5 0 s7 sT andard : 0 .3 0 0 s8 sT andard : 0 .6 0 0 s9 sT andard : 1 .2 0 0 slO sT an d ard : 1 .5 0 0 O rder : In v erse Logarithm D i m e n s i o n : PPM s t a r t V alue : 5 0 0 DU tr ig g e r L im it 1800 Sec Peak shape : Pointed stA rt ignore : 60 Sec eNd i g n o r e : 120 Sec M easu re w indow : 75 % F ilter : Mo R e g e n e r a t i o n : No form ula : o u tp u t : ##.### P ath number S ignal type D ecolor sy ste m Number d ilu te Resam ple d il T hreshold diG o u tp u t Window e v e n t 0 D ebubbled No 0 No No 4095 0 O ff 000763 1995-08-16 14:10 OutPut of : 950816A1 s i sTandard 0 .0 1 5 s2 sT andard 0 .0 3 0 s3 sT andard 0 .0 6 0 S4 s T a n d a r d 0 .0 9 0 s5 sT andard 0 .1 2 0 s6 sT andard 0 .1 5 0 s7 sT andard Ig n o re s8 sT andard Ig n o re S9 s T a n d a r d I g n o r e slO sT an d ard Ignore O rder : 2 D i m e n s i o n : PPM s t a r t V alue : 5 0 0 DU tr ig g e r L im it : 1800 Sec Peak shape : P ointed stA rt ignore : 60 Sec eNd ig n o r e : 120 Sec M e a su re w indow : 75 % F ilter : No R egeneration : No form ula : c4 :=c3 o u tp u t : #. #### 000764 1995-08-16 14:10 OutPut of : 950816A1 F lu o rid e 1.5 F luoride L PPM PPM Pos Typ Id e n t Ch R e s u lt F Time w t iw I n i t i a l Wash 3 0 .0 7 0 1t T racer 3 1.472 2d D r if t 3 1.486 3w Wash 3 0 .0 7 0 4 si S tan d ard 1 3 0.074 5 s2 S tan d ard 2 3 0.081 6 s3 S tan d ard 3 3 0.100 7 s4 S tan d ard 4 3 0.117 8 s5 S tan d ard 5 3 0.137 9 s6 S tan d ard 6 3 0.154 10 s7 S tan d ard 7 3 0.285 11 s8 S tan d ard 8 3 0.614 12 s9 S tan d ard 9 3 1.217 13 slO S ta n d a r d 10 3 1 .4 7 9 14 d D r if t 3 1.490 15 w Wash 3 0 .0 7 0 16 u SPK 6 2 - 1 3 0.142 17 u SPK 2 5 0 - 1 3 0 .3 4 6 18 u SPK 2 5 0 - 2 3 0 .3 2 2 19 u BLANK 3 0 . 1 3 6 20 u BLANK 3 0 . 1 0 5 21 u BLANK 3 0 . 0 8 8 22 u F52986-1 3 0.117 23 u F52990-1 3 0.086 24 u F52997-1 3 0.078 25 u F52982-1 3 0.079 26 d D r if t 3 1.477 27 w Wash 3 0 .0 7 0 28 u F52994-1 3 0.081 29 u F53410-1 3 0.078 30 u SPK 6 2 - 1 3 0 .1 2 2 31 u SPK 2 5 0 - 1 3 0 . 2 9 4 32 u BLANK SERUM 3 0 . 1 0 1 33 u BLANK SERUM 3 0 . 0 9 5 34 u BLANK SERUM 3 0 . 0 9 8 35 u BLANK SERUM 3 0 . 0 9 3 36 u SPK 6 2 - 1 3 0 .1 4 8 37 u SPK 6 2 - 2 3 0 .1 3 2 38 d D r if t 3 1.485 39 w Wash 3 0 .0 7 0 40 u SPK 6 2 - 3 3 0 .1 3 6 41 u SPK 2 5 0 - 1 3 0 . 2 5 4 42 u SPK 2 5 0 - 2 3 0 .2 7 1 43 u F52984-1 3 0.088 44 u F52992-1 3 0.085 45 u F52996-1 3 0.082 46 u F52997-1 3 0.086 47 u F52984-1 3 0.085 48 u F52993-1 3 0.085 49 u F52978-1 3 0.088 50 d D r if t 3 1.488 51 w Wash 3 0 .0 7 0 52 u F52980-1 3 0.101 53 u F52991-1 3 0.103 65 207 383 619 729 907 1081 1258 1434 1608 1784 1958 2133 2308 2484 2726 2830 3011 3187 3361 3536 3710 3886 4062 4236 4410 4586 4825 4934 5110 5288 5462 5635 5810 5986 6161 6335 6510 6684 6911 7034 7210 7382 7557 7734 7910 8086 8258 8434 8608 8785 9004 9131 9311 Ch R e s u l t F Time 4 0.0091 4 1.8335 4 1.8558 4 0.0091 4 0.0164 4 0.0281 4 0.0601 4 0.0894 4 0.1221 4 0.1488 4 0.3400 4 0.7483 4 1.4687 4 1.8442 4 1.8617 4 0.0091 4 0.1296 4 0.4211 4 0.3892 4 0.1206 4 0.0686 4 0.0410 4 0.0889 4 0.0377 4 0.0234 4 0.0247 4 1.8413 4 0.0091 4 0.0281 4 0.0228 4 0.0978 4 0.3525 4 0.0625 4 0.0519 4 0.0566 4 0.0485 4 0.1400 4 0.1145 4 1.8539 4 0.0091 4 0.1194 4 0.2971 4 0.3208 4 0.0407 4 0.0356 4 0.0310 4 0.0364 4 0.03564 0.0348 4 0.0407 4 1.8587 4 0.0091 4 0.0625 4 0.0654 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page 1 of 2 000765 1995-08-16 14:10 OutPut of : 950816A1 F lu o rid e 1.5 PPM F luoride L PPM Pos Typ Id e n t Ch R e s u lt F Tim e Ch R e s u l t F Time 54 u 55 u 56 u 57 d 58 w w t rw F52987-1 3 0.095 SPK 6 2 - 1 3 0 . 1 2 7 SPK 2 5 0 - 1 3 0 .2 5 7 D r if t 3 1.503 Wash 3 0 .0 7 0 RunOut Wash 3 0 .0 7 0 9486 9662 9838 10012 10247 10487 4 0.0517 4 0.1051 4 0.3017 4 1.8831 4 0.0091 4 0.0091 0 0 0 0 0 0 Page 2 of 2 0 0 0 7 6 6 __ 0.2692961 Calibration curve of 950816A1 ' Fluoride L Channel 0 Measured: Order : 2 lT Measured r : 0.99717 900 000767 BEST COPYAVAII ABLE Calibration curve of 950816A1 : Fluoride 1.5 -Charme I -3- Measured: b ~ 'ESRIiHH a B B 'oT727gj D elta: 1.8541875 C o n c e n tr t io n 0.0636451 J Measured Order : Inversa Logarithm s : 5671 4035 000768 000769 BEST E8PY AVAILABLE 000 770 000771 1995-03-21 12:30 OutPut of : 950821A1 S oftw are : v e rsio n 6.1 cl990,93 O perator : DDW D ate o f th e A n aly sis : 1995-08-21 07:55 A n a l y s i s F i l e Name : C:\SKALAR\DATA\HWIDATA\SERUM\950821A1 Q c o *|ts! b T 10 hu)!d t*JZ 2C o t r <r F lu o rid e 1.5 C a lib ra tio n o rd er = In v erse L ogarithm Slo p e : s = #.##### R esult = r x- ------------- 10 L s -* cl I x= c o r r e c t e d v a lu e o f th e sam ple c l = c o rre c ted value of the c o n cen tratio n 1 s = Slope of th e e le c tro d e a2 = al = aO = -0 .0 0 0 0 0 0 .0 0 0 7 1 -1 .2 0 0 6 1 F luoride L C alib ratio n order = 2 C o r r e la tio n : r = 0.99953 R e s u l t = a 2 * x a + a l * x + aO a2 = al = aO = -0 .0 0 0 0 0 0 .0 0 0 2 5 0 .0 0 7 7 1 Sam pler Type Number Sam ple Time W ash Time A ir Time Take up sp ecial needle H eight SA1000 1 50 s e c . 120 s e c . 1 sec. S ingle None 70 mm. D ilu ter needle H eight d ilu tio n F actor d i l u t i o n Volume Resam ple D ilu tio n runs : 80 : 10 :2.5 :1 :1 mm m l. User f i l e : R e p r o c e s : No . TXT 000772 1995-08-21 12:30 Output of : 950821A1 F lu o rid e 1. P ath number S ig n al type D ecolor sy ste m Number diL ute Resam ple d il T hreshold diG o u tp u t Window e v e n t 3 D ebubbled Yes 0 No No 4095 0 O ff F luoride L s i sTandard Ignore s2 sT andard Ig n o re S3 s T a n d a r d I g n o r e s4 sT andard Ig n o re s5 sT andard Ig n o re s6 sT andard 0 .1 5 0 s7 sT andard 0 .3 0 0 s8 sTandard 0 .6 0 0 s9 sT andard 1 .2 0 0 slO sT andard 1 .5 0 0 O rder : In v erse L ogarithm D i m e n s i o n : PPM s t a r t V alue : 5 0 0 DU tr ig g e r L im it : 1800 Sec Peak shape : P ointed stA rt ignore : 60 Sec eNd ig n o r e : 120 Sec M easure window : 75 % F ilter : No R egeneration : No form ula : o u tp u t : ##.### P ath number S ignal type D ecolor sy ste m Number diL ute Resam ple d il T hreshold diG o u tp u t Window e v e n t 0 D ebubbled No 0 No No 4095 0 O ff 000773 1995-08-21 12:30 Output of si sT andard 0 .0 1 5 s2 sT andard 0 .0 3 0 s3 sT andard 0 .0 6 0 s4 sT andard s5 sT andard 0 .0 9 0 0.120 s6 sT an dard 0 .1 5 0 s7 sT andard Ig n o re s8 sT andard Ignore s9 sT andard Ig n o re slO sT andard Order : 2 Ignore D i m e n s i o n : PPM s t a r t V alue : 5 0 0 DU tr ig g e r L im it : 1800 Sec Peak shape : P ointed stA rt ignore : 60 Sec eNd ig n o r e : 120 Sec M easu re w indow : 75 F ilte r : No R egeneration : No form ula : c4:=c3 o u tp u t : #.#### 950821A1 000774 1995-08-21 12:30 OutPut of : 950821A1 F lu o rid e 1.5 F luoride L PPM PPM Pos Typ Id e n t Ch R e s u lt w t iw I n i t i a l Wash 3 0 .0 6 3 1t 2d T racer D rift 3 1.468 3 1.482 3w Wash 3 0 .0 6 3 4 si S tan d ard 1 3 0.066 5 s2 S tan d ard 2 3 0.073 6 s3 S tan d ard 3 3 0.090 7 s4 S tan d ard 4 3 0.106 8 s5 S tan d ard 5 3 0.128 9 s6 S tan d ard 6 3 0.156 10 s7 S tan d ard 7 3 0.279 11 s8 S tan d ard 8 3 0.619 12 s9 S tan d ard 9 3 1.224 13 SlO S ta n d a r d 10 3 1 .4 7 1 14 d D r if t 3 1.539 15 w Wash 3 0 .0 6 3 16 u SERUM BLK 1 3 0 . 0 9 8 17 u SERUM BLK 2 3 0 . 0 9 2 18 u SERUM BLK 3 3 0 . 0 8 6 19 u F52988-1 3 0.083 20 u F52995-1 3 0.081 21 u F52972-1 3 0.084 22 u F52973-1 3 0.082 23 u F52979-1 3 0.083 24 u F52975-1 3 0.080 25 u F52976-1 3 0.079 26 d D r if t 3 1.529 27 w Wash 3 0 .0 6 3 28 u F52983-1 3 0.082 29 u SPK 4 0 - 1 3 0 . 0 9 2 30 u SPK 1 0 0 - 1 3 0 .1 4 7 31 u BLK-1 3 0 .0 7 5 32 u BLK-2 3 0 .0 7 4 33 u BLK-3 3 0 .0 7 0 34 u SPK 4 0 - 1 3 0 . 0 9 1 35 u SPK 4 0 - 2 3 0 . 0 8 9 36 u SPK 1 0 0 - 1 3 0 . 1 3 3 37 u SPK 1 0 0 - 2 3 0 . 1 3 1 38 d D r if t 3 1.540 39 w Wash 3 0 .0 6 3 40 u SPK 1 0 0 - 3 3 0 .1 4 2 41 u BLK 3 0 . 0 7 5 42 u F52972-8 3 0.071 43 u F52973-8 3 0.064 44 u F52979-8 3 0.077 45 u F52975-8 3 0.075 46 u F52976-8 3 0.071 47 u F52983-8 3 0.070 48 u F52986-8 3 0.071 49 u F52990-8 3 0.070 50 d D r if t 3 1.525 51 w Wash 3 0 .0 6 3 52 u F52997-8 3 0.073 53 u F52982-8 3 0.070 T im e Ch R e s u l t F Time 65 208 382 570 728 903 1083 1259 1435 1609 1783 1958 2133 2309 2483 2723 2832 3012 3184 3360 3534 3710 3885 4060 4234 4410 4583 4810 4933 5111 5285 5460 5632 5810 5988 6162 6336 6512 6684 6902 7037 7207 7384 7558 7736 7909 8085 8257 8437 8609 8785 9012 9136 9310 4 0.0077 4 0.5962 4 0.5992 4 0.0077 4 0.0144 4 0.0298 4 0.0624 4 0.0891 4 0.1184 4 0.1509 4 0.2480 4 0.3935 4 0.5438 4 0.5968 4 0.6121 4 0.0077 4 0.0765 4 0.0672 4 0.0559 4 0.0510 4 0.0462 4 0.0518 4 0.0488 4 0.0508 4 0.0440 4 0.0420 4 0.6099 4 0.0077 4 0.0481 4 0.0672 4 0.1414 4 0.0347 4 0.0322 4 0.0246 4 0.0643 4 0.0617 4 0.1251 4 0.1221 4 0.6123 4 0.0077 4 0.1352 4 0.0347 4 0.0256 4 0.0090 4 0.0388 4 0.0347 4 0.0261 4 0.02314 0.0271 4 0.0231 4 0.6090 4 0.0077 4 0.0303 4 0.0248 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page 1 of 2 000775 = 1995-08-21 12:30 OutPut of : 950821A1 F lu o rid e 1. 5 F luoride L PPM PPM Pos Typ Id e n t Ch R e s u lt F Time Ch R e s u l t F T im e 54 u 55 u 56 u 57 u 58 u 59 u 60 u 61 u 62 d 63 w 64 u 65 u 66 u 67 u 68 u 69 u 70 u 71 u 72 u 73 u 74 d 75 w 76 u 77 u 78 u 79 u 80 u 81 u 82 u 83 u 84 u 85 u 86 d 87 w 88 u 89 u 90 d 91 w wt rw SPK 6 2 - 1 3 0 .0 8 5 SPK 6 2 - 2 SPK 1 2 4 - 1 3 0.124 3 0.144 SPK 1 2 4 - 2 3 0 .1 7 1 BLK 3 0 . 1 1 6 F52994-8 3 0.109 F53410-8 3 0.085 F52984-8 3 0.084 D r if t 3 1.552 Wash 3 0.0 6 3 F52992-8 3 0.082 F52996-8 3 0.091 F52997-8 3 0.102 F52989-8 3 0.090 F52993-8 3 0.082 F52978-8 3 0.078 F52980-8 3 0.080 SPK 4 0 - 1 3 0 . 0 9 8 SPK 1 2 4 - 1 3 0 .1 6 6 BLK 3 0 . 1 0 6 D r if t 3 1.536 Wash 3 0 .0 6 3 F52991-8 3 0.089 F52987-8 3 0.085 F52988-8 3 0.079 F52995-8 3 0.079 F52972-15 3 0.080 F52973-15 3 0.093 F52979-15 3 0.083 F52975-15 3 0.085 F52976-15 3 0.078 F52983-15 3 0.080 D r if t 3 1.574 Wash 3 0 .0 6 3 SPK 4 0 - 1 3 0 . 0 9 8 SPK 1 2 4 - 1 3 0 . 1 6 5 D r if t 3 1.534 Wash 3 0 .0 6 3 RunOut Wash 3 0 .0 6 3 9488 9664 9838 10014 10189 10365 10539 10715 10888 11124 11239 11415 11589 11766 11938 12108 12290 12466 12642 12816 12990 13232 13341 13517 13683 13867 14042 14218 14392 14566 14740 14916 15092 15334 15441 15619 15792 16032 16267 4 0.0549 4 0.1135 4 0.1373 4 0.1659 4 0.1037 4 0.0927 4 0.0532 4 0.0527 4 0.6153 4 0.0077 4 0.0479 4 0.0646 4 0.0823 4 0.0629 4 0.0488 4 0.0410 4 0.0452 4 0.0756 4 0.1606 4 0.0894 4 0.6115 4 0.0077 4 0.0612 4 0.0544 4 0.0420 4 0.0432 4 0.0442 4 0.0679 4 0.0501 4 0.0547 4 0.0410 4 0.0454 4 0.6204 4 0.0077 4 0.0761 4 0.1601 4 0.6110 4 0.0077 4 0.0077 ooooooooooooooooooooooooooooooooooooooo Page 2 000776 Calibration curve of 950821A1 : Fluoride L Order - 2 r 0.99953 000777- Calibration curve of 950821A1 : Fluoride 1.5 Order : Inverse Logarithn s 5886 000778 4095 - Raw data of 950821A1 : Fluoride 1.5 Tine: I Q__ ! Ualue: 1 736 1Delta: TP A & 1 2 klU t t i f i t i'CvXAjVIViU. V1 4 a"-J y J \j V \J VJ \J V 0 T ine Esc=Exit ! Fi=Help ! Crtl-P=Edit peaks ! 5000 000779 Esc=Exit i Fl=Help ! Crtl-P=Edit peaks 1 G0078* 4095 - Raw data of 950821R1 : Fluoride 1.5 ; Tine: 1 8935 ! Ualue- 1 888 1Delta"-TM kVr A IV 3 -I A' 1 * i/1 tip x> A* A J U A VjUVJVMAAJVM v \J \J \J \] \,V i"[ A^ J Vi V /V jV 'u 8935 T ine Esc=Exit 1 Fl=Help ! Crtl-P=Edit peaks 1 13935 000781 000782 1995-08-21 16:58 O utP ut o f : 950821B1 S oftw are : v e rsio n 6.1 cl990,93 O perator : DDW D ate of th e A n aly sis : 1995-08-21 12:29 A n a l y s i s F i l e Name : C:\SKALAR\DATA\HWIDATA\SERUM\950821B1 <QCko a )isV\S> HuJX. Sm u C -)35 X. - ) F lu o rid e 1.5 C a lib ra tio n o rd e r = In v erse Logarithm Slo p e : s = #.##### [ x - c l -| -------------sJ x = c o rre c te d v a lu e o f th e sam ple cl = c o rrected value of the c o n cen tratio n 1 s = Slope of th e e le c tro d e a2 = al = aO = -0 .0 0 0 0 0 0 .0 0 0 6 8 -1 .1 9 9 9 7 F luoride L C alib ratio n order = 2 C o r r e la tio n : r = 0.99960 R esult a2 = al = aO = = a2 * xa + a l -0 .0 0 0 0 0 0 .0 0 0 2 5 0 .0 0 3 0 7 * x + aO Sam pler Type Number Sam ple Time Wash Time A ir T im e Take up sP ecial n eedle H eight SA1000 1 50 s e c . 120 se c . 1 sec. S ingle None 7 0 mm. D ilu ter n eedle H eight d ilu tio n F actor d i l u t i o n Volume Resam ple D ilu tio n runs : 80 : 10 : 2.5 :1 :1 mm m l. User f i l e : R e p r o c e s : No . TXT 000783 1995-08-21 16:58 Output of : 950821B1 F lu o rid e 1. 5 P a th number S ig n al type D ecolor sy ste m Number diL ute Resam ple d il T hreshold diG o u tp u t Window e v e n t 3 D ebubbled Yes 0 No No 4095 0 O ff F luoride L s i sTandard Ignore s2 sT andard Ig n o re s3 sT andard Ign o re s4 sTandard Ignore s5 sT andard Ign o re s6 sT andard 0 .1 5 0 s7 sTandard 0 .3 0 0 s8 sTandard s9 sTandard 0 .6 0 0 1.200 slO sT andard 1 .5 0 0 O rder : In v erse Logarithm D i m e n s i o n : PPM s t a r t V alue 5 0 0 DU tr ig g e r L im it 1800 Sec Peak shape P ointed s t A r t ig n o r e : 60 Sec eNd ig n o r e :120 Sec M easure window : 75 % F ilter : No R egeneration : No form ula : o u tp u t : ##.### P ath number S ig n al type D ecolor sy ste m Number diL ute Resam ple d il T hreshold diG o u tp u t Window e v e n t 0 D ebubbled No 0 No No 4095 0 O ff OOO 784 1995-08-21 16:58 OutPut of : 950821B1 s i sTandard 0 .0 1 5 s2 sT andard 0 .0 3 0 s3 sT andard 0 .0 6 0 s4 sT andard 0 .0 9 0 s5 sT andard 0 .1 2 0 s6 sT andard 0 .1 5 0 s7 sT andard Ig n o re s8 sT andard Ig n o re s9 sT andard Ig n o re slO sT andard Ignore O rder : 2 D i m e n s i o n : PPM s t a r t V alue : 5 0 0 DU tr ig g e r L im it : 1800 Sec Peak shape : Pointed s t A rt ignore : 60 Sec eNd ig n o re : 120 Sec M easure window : 75 % F ilter : No R egeneration : No form ula : c4: =c3 o u tp u t : #.#### 000785 1995-08-21 16:58 OutPut of : 950821B1 F lu o rid e 1.5 F luoride L PPM PPM Pos Typ Id e n t Ch R e s u lt F Time w t iw I n i t i a l Wash 3 0 .0 6 3 1t T ra c e r 3 1.488 2d D r if t 3 1.478 3w Wash 3 0.0 6 3 4 si S tan d ard 1 3 0.068 5 s2 S tan d ard 2 3 0.074 6 s3 S tan d ard 3 3 0.091 7 s4 8 s5 S tan d ard 4 3 0.109 S tan d ard 5 3 0.129 9 s6 S tan d a rd 6 3 0.156 10 s7 S tan d ard 7 3 0.279 11 s8 S tan d ard 8 3 0.620 12 s9 S tan d ard 9 3 1.224 13 slO S ta n d a r d 10 3 1 .4 7 2 14 d D r if t 3 1.435 15 w Wash 3 0 .0 6 3 16 u SERUM BLK 1 3 0 . 0 7 5 17 u SERUM BLK 2 3 0 . 0 7 0 18 u SPK 4 0 - 1 3 0 .0 8 0 19 u SPK 4 0 - 2 3 0 .0 8 1 20 u SPK 4 0 - 3 3 0 .0 8 6 21 u SPK 1 2 4 - 1 3 0 . 1 4 5 22 u SPK 1 2 4 - 2 3 0 .1 5 0 23 u SPK 1 2 4 - 3 3 0 .1 4 5 24 u BLK 3 0 . 0 7 6 25 u F52986-15 3 0.067 26 d D r if t 3 1.408 27 w Wash 3 0.0 6 3 28 u F52990-15 3 0.069 29 u F52997-15 3 0.067 30 u F52982-15 3 0.064 31 u F52994-15 3 0.069 32 u F53410-15 3 0.068 33 u F52984-15 3 0.065 34 u F52992-15 3 0.065 35 u F52996-15 3 0.066 36 u F52977-15 3 0.066 37 u SPK 4 0 - 1 3 0 .0 8 1 38 d D r if t 3 1.434 39 w Wash 3 0.0 6 3 40 u SPK 1 2 4 - 1 3 0 .1 9 1 41 u BLK 3 0 . 0 9 7 42 u F52989-15 3 0.118 43 u F52993-15 3 0.088 44 u F52978-15 3 0.089 45 u F52980-15 3 0.088 46 u F52991-15 3 0.086 47 u F52987-15 3 0.083 48 u F52988-15 3 0.083 49 u F52995-15 3 0.079 50 d D r if t 3 1.454 51 w Wash 3 0 .0 6 3 52 u F52972-22 3 0.083 53 u F52973-22 3 0.085 65 208 384 625 733 911 1085 1261 1435 1611 1787 1960 2135 2311 2485 2662 2837 3013 3186 3362 3536 3714 3888 4062 4238 4411 4586 4732 4938 5109 5285 5459 5635 5811 5987 6163 6334 6513 6686 6924 7036 7212 7386 7559 7736 7911 8083 8263 8437 8613 8787 9024 9137 9313 Ch R e s u lt F Time 4 0.0031 4 0.6348 4 0.6322 4 0.0031 4 0.0157 4 0.0284 4 0.0612 4 0.0909 4 0.1181 4 0.1507 4 0.2517 4 0.4063 4 0.5703 4 0.6308 4 0.6213 4 0.0031 4 0.0314 4 0.0184 4 0.0406 4 0.0431 4 0.0515 4 0.1381 4 0.1441 4 0.1386 4 0.0326 4 0.0127 4 0.6146 4 0.0031 4 0.0164 4 0.0115 4 0.0058 4 0.0167 4 0.0137 4 0.0070 4 0.0083 4 0.0090 4 0.0110 4 0.0431 4 0.6211 4 0.0031 4 0.1851 4 0.0715 4 0.1036 4 0.0561 4 0.0585 4 0.0559 4 0.0530 4 0.04694 0.0474 4 0.0385 4 0.6263 4 0.0031 4 0.0457 4 0.0511 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page 1 of 2 000 1995-08-21 16:58 OutPut of : 950821B1 F lu o rid e 1.5 F luoride L PPM PPM Pos Typ Id e n t Ch R e s u lt F Time 54 u 55 u 56 u 57 u 58 u 59 u 60 u 61 u 62 d 63 w 64 u 65 u 66 u 67 u 68 u 69 u 70 u 71 u 72 u 73 u 74 d 75 w 76 u 77 u 78 u 79 u 80 u 81 u 82 u 83 u 84 d 85 w wt rw SPK 4 0 - 1 3 0 .1 1 7 SPK 4 0 - 2 3 0 .0 9 3 SPK 1 2 4 - 1 3 0 .1 4 6 BLK 3 0 . 0 9 0 F52979-22 3 0.080 F52975-22 3 0.077 F52976-22 3 0,076 F52983-22 3 0.082 D r if t 3 1.419 Wash 3 0 .0 6 3 F52986-22 3 0.082 F52990-22 3 0.077 F52997-22 3 0.078 F52982-22 3 0.074 F52994-22 3 0.082 F53410-22 3 0.083 SPK 4 0 - 1 3 0 .1 0 8 SPK 1 2 4 - 1 3 0 . 1 6 8 SPK 6 2 - 1 3 0 .1 3 1 BLK 3 0 . 0 9 1 D r if t 3 1.469 Wash 3 0 .0 6 3 F52984-22 3 0.083 F52992-22 3 0.082 F52996-22 3 0.084 F52977-22 3 0.083 F52989-22 3 0.082 F52993-22 3 0.079 SPK 4 0 - 1 3 0 .1 0 6 SPK 1 2 4 - 1 3 0 .1 5 2 D r if t 3 1.461 Wash 3 0 .0 6 3 RunOut Wash 3 0 .0 6 3 9489 9662 9838 10010 10186 10365 10537 10713 10886 11120 11237 11409 11583 11762 11936 12114 12288 12462 12638 12812 12985 13227 13337 13511 13685 13858 14035 14211 14379 14559 14735 14976 15210 Ch R e s u lt F Time 4 0.1017 4 0.0655 4 0.1391 4 0.0593 4 0.0411 4 0.0341 4 0.0324 4 0.0453 4 0.6175 4 0.0031 4 0.0438 4 0.0343 4 0.0363 4 0.0284 4 0.0445 4 0.0460 4 0.0893 4 0.1627 4 0.1211 4 0.0621 4 0.6301 4 0.0031 4 0.0469 4 0.0455 4 0.0479 4 0.0467 4 0.0440 4 0.0397 4 0.0857 4 0.1459 4 0.6280 4 0.0031 4 0.0031 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 0 0 0 7 8 7 __ Calibration curve of 950821B1 : Fluoride L 000788 Calibration curve of 950821B1 : Fluoride 1.5 Channel 3 Measured 1.7656142 0.0631072 jir' I Measured Order : Inverse Logarithm r 6199 4095 000789 000790 000791 0 0 0 732- Rau data of 950821B1 : Fluoride 1.5 Esc=Exit ! Fl=Help ! Crtl-P=Edit peaks P r i n t o u t o f Sam ple T ab le : 6329135D I n s e r t e d in System T ab le o f : g ro u p 1 0 iw 1t 2d 3w 4 si 5 s2 6 s3 7 s4 8 s5 9 s6 10 s7 11 s8 tn A I n i t i a l Wash T racer D rift W ash S tandard 1 S tandard 2 Standard 3 Standard 4 S tandard 5 S tandard 6 Standard 7 Standard 8 1 1.0000 1 1.000 1 1.000 1 1.000 1 1.000 1 1.000 1 1.000 1 1.000 1 1.000 1 1.000 1 1.000 1 1.000 000793 1995-08-22 09:05 Output of : 950822A1 S oftw are : v e rsio n 6.1 cl990,93 O perator : DDW D ate of th e A n aly sis : 1995-08-22 06:49 A n a l y s i s F i l e Name : C:\SKALAR\DATA\HWIDATA\SERUM\950822A1 'Hzs.VtS V ^ T 2x>-79S.J KuJJ 43^<^l3S F lu o rid e 1.5 C a lib ra tio n o rd e r = In v e rse Logarithm Slo p e : s = #.##### [ x - c l -| -------------s -* x = c o rre c te d v alu e o f th e sam ple cl = c o rre c ted value of the c o n cen tratio n 1 s = Slope o f th e e le c tro d e a2 = al = aO = -0 .0 0 0 0 0 0 .0 0 0 7 5 -1 .1 8 7 3 4 F luoride L C alib ratio n order = 2 C o rre la tio n : r = 0 .9 9 9 1 7 R e s u l t = a 2 * x* + a l * x + aO a2 = al = aO = -0 .0 0 0 0 0 0 .0 0 0 2 7 0 .0 0 5 7 7 Sam pler Type Number Sam ple Time W ash Time A ir T im e Take up sp ecial n eed le H eight SAXUUU 1 50 s e c . 120 se c . 1 sec. S ingle None 7 0 mm. D ilu ter n eedle H eight d ilu tio n F actor d i l u t i o n Volume Resam ple D ilu tio n runs : 80 : 10 :2.5 :1 :1 mm m l. User f i l e : R e p r o c e s : No . TXT 000 794 ` 1995-08-22 09:05 OutPut of : 950822A1 F lu o rid e 1. 5 P a th number S ig n al type D ecolor sy ste m Number diL ute Resam ple d il T hreshold diG o u tp u t Window e v e n t 3 D ebubbled Yes 0 No No 4095 0 O ff F luoride L s i sTandard Ignore s2 sT andard Ig n o re s3 sT andard Ig n o re s4 sT andard Ig n o re s5 sT andard Ignore s6 sT andard 0 .1 5 0 s7 sT andard 0 .3 0 0 s8 sTandard 0 .6 0 0 s9 sT andard 1 .2 0 0 SlO sT a n d a rd 1 .5 0 0 O rder : In v erse Logarithm D i m e n s i o n : PPM s t a r t V alue ; tr ig g e r L im it : Peak shape ; 5 0 0 DU 1800 Sec P o in te d stA rt ignore ; 60 Sec eNd ig n o re ; 120 Sec M easu re w indow ; 75 F ilte r ; No % R egeneration ; No form ula : o u tp u t : ##.### P a th number S ig n al type D ecolor sy ste m Number diL ute Resam ple d il T hreshold diG o u tp u t Window e v e n t 0 D ebubbled No 0 No No 4095 0 O ff 0 0 0 795 1995-08-22 09:05 Output of : 950822A1 s i sTandard 0 .0 1 5 s2 sT andard 0 .0 3 0 s3 sTandard 0 .0 6 0 s4 sT andard 0 .0 9 0 s5 sT andard 0 .1 2 0 s6 sTandard 0 .1 5 0 s7 sT andard Ig n o re s8 sTandard Ignore s9 sT andard Ig n o re slO sT andard Ignore O rder : 2 D i m e n s i o n : PPM s t a r t V alue 5 0 0 DU tr ig g e r Lim it 1800 Sec Peak shape P ointed stA rt ignore 60 Sec eNd ig n o re 120 Sec M easu re w indow 75 % F ilter No R egeneration : No form ula : c4:=c3 o u tp u t : #.#### 000 796 1995-08-22 09:05 OutPut of : 950822A1 F lu o rid e 1.5 F luoride L PPM PPM Pos Typ Id e n t Ch R e s u l t F Tim e w t iw I n i t i a l Wash 3 0 .0 6 5 65 1t T racer 3 1.461 208 2d D r if t 3 1.475 383 3w Wash 3 0 .0 6 5 616 4 si S tan d ard 1 3 0.069 729 5 s2 S tan d ard 2 3 0.075 907 6 s3 S tan d ard 3 3 0.093 1083 7 s4 S tan d ard 4 3 0.111 1259 8 s5 S tan d ard 5 3 0.129 1433 9 s6 S tan d ard 6 3 0.157 1609 10 s7 S tan d ard 7 3 0.277 1785 11 s8 S tan d ard 8 3 0.621 1959 12 s9 S tan d ard 9 3 1.227 2134 13 slO S t a n d a r d 10 3 1 .4 6 9 2309 14 d D r if t 3 1.536 2484 15 w Wash 3 0 .0 6 5 2722 16 u SERUM BLK 1 3 0 . 0 8 5 2837 17 u SERUM BLK 2 3 0 . 0 7 8 3011 18 u SPK 4 0 - 1 3 0 . 0 8 3 3187 19 u SPK 4 0 - 2 3 0 .0 9 0 3361 20 u SPK 4 0 - 3 3 0 .0 9 0 3533 21 u SPK 4 0 - 4 3 0 . 0 9 2 3710 22 u SPK 124-1 3 0 .1 0 5 3886 23 u SPK 1 2 4 - 2 3 0 . 1 3 8 4059 24 u SPK 1 2 4 - 3 3 0 . 2 7 8 4236 25 u SPK 1 2 4 - 4 3 0 . 1 2 9 4412 26 d D r if t 3 1.533 4584 27 w Wash 3 0 .0 6 5 4825 28 u SPK 1 0 0 - 1 3 0 . 1 2 1 4936 29 u SPK 1 0 0 - 2 3 0 .1 4 1 5112 30 u SPK 1 0 0 - 3 3 0 .1 3 7 5286 31 u BLK 3 0 . 1 0 1 5460 32 u BLK 3 0 . 0 8 0 5635 33 u F52978-22 3 0.078 5811 34 u F52980-22 3 0.082 5987 35 u F52991-22 3 0.076 6151 36 u F52987-22 3 0.073 6335 37 u F52988-22 3 0.070 6511 38 d D rift 3 1.532 6685 39 w Wash 3 0 .0 6 5 6927 40 u 41 u F52995-22 3 0.074 7037 n n K i & 7 91 n 42 u SPK 1 0 0 - 1 3 0 .1 2 1 7386 43 d D r if t 3 1.550 7561 44 w Wash 3 0 .0 6 5 7803 wt rw RunOut Wash 3 0 .0 6 5 8036 Ch R e s u l t F Time 4 0.0058 0 4 0.7660 0 4 0.7723 0 4 0.0058 0 4 0.0156 0 4 0.0281 0 4 0.0623 0 4 0.0911 0 4 0.1167 0 4 0.1512 0 4 0.2587 0 4 0.4443 0 4 0.6732 0 4 0.7697 0 4 0.8009 0 4 0.0058 0 4 0.0472 0 4 0.0334 0 4 0.0432 4 0.0570 0 0 4 0.0575 4 0.0609 4 0.0829 4 0.1283 0 0 0 0 4 0.2595 0 4 0.1172 4 0.7994 0 0 4 0.0058 4 0.1059 0 0 4 0.1317 4 0.1278 0 0 4 0.0755 0 4 0.0390 0 4 0.0334 4 0.0424 4 0.0291 0 0 0 4 0.0249 0 4 0.0169 0 4 0.7989 0 4 0.0058 0 4 0.0268 0 4* ---------------- 0- 4 0.1059 4 0.8082 0 0 4 0.0058 0 4 0.0058 0 Page 1 of 1 -T>V.rl.Y_T\S>A& 0 0 0 197-- Calibration curve of 950822(11 Fluoride L Order : 2 r : 0.99917 GOO 798 1.6923981 Calibration curve of 950822A1 : Fluoride 1.5 - M e a s u r e d ^ L 6____ C o n c e n t r a t i o n : 1 0 . 0 6 5 6 4 9 6 1 D e l t a : C o n c e n tra i io n 0.0649727 *" Ir Measured Order : Inverse Logarithm s : 5996 4095 000 ?99 000800 Rau data of 950822A1 : Fluoride 1.5 Esc=Exit ! Fi=Help Crtl-P=Edit peaks * G0083T