Document XzxgyX81q28oNEg66a21qeXyw

3M Environmental Laboratory Final Report- Analytical Study Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits In-Vivo Study Reference Number: HWI#6329-137 Study Number: AMDT-022195.1 Test Substance: FC-99 (T-6053) Name and Address of Sponsor: 3M SCD Division 367 Grove Street S t Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology & Services 935 Bush Avenue St. Paul, MN 55106 Method Numbers and Revisions: AMDT-M-1-0, Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry AMDT-M-8-0, Analysis of Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode Initiation Date: See attached protocol Author: James D. Johnson Approved By: mes D. J ison Study Dir^ tor /l/zz/ Completion Date 003851 01)0001 . If 133 1 1.0 SUMMARY After dermal application of FC-99 (T-6053), tissue and serum samples were collected (HWI#6329-137). The liver at 28 days was combusted and analyzed for total organic fluorine. The results of this analysis showed no differences between the controls and treated rabbits including the high dose which were rabbits treated with 250 mg/kg (T-6053). This formulation is a diethanolamine salt of perfluorooctanesulfonate. Twenty percent of these solids are the C8. T-6053 is a solution that is 0.04% of FC-99 solids. The dose for the high group was 20 ug/kg with respect to C8 perfluorooctanesulfonate. The dose was below detection limits of the method even if all of the samples were absorbed. 2,0 INTRODUCTION This study was performed in order to provide data for the assessment of dermal absorption of FC-99. Data from other forms of perfluorooctanesulfonate (FC-95, the potassium salt) were available (HWI#6329-130, HWI#6329-159). A pharmacokinetic study (HWI#6329-136) had shown that perfluorooctanesulfonate would be a convenient marker for FC-99 if the doses administered were high enough. 3.0 TEST MATERIALS_______________________________________________ 3.1 Test, Control, and Reference Substances and Matrices 3.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 3.1.2 Analytical Reference Matrix: Bovine liver and bovine serum 3.1.3 Analytical Control Substance: None 3.1.4 Analytical Control Matrix: Bovine liver and bovine serum 3.2 Source of M aterials: 3MICP/PCP Division for FC-95, bovine liver from grocery store, bovine serum from Sigma Chemical Company. 3.3. Purity and Strength of Reference Substance: Responsibility of Sponsor 3.4 Stability of Reference Substance: To be determined by Sponsor 3.5 Storage Conditions for Test Materials: Room temperature for FC-95. For biological samples the storage is -20+10 C. 003852 000002 2 3.6 Disposition of Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required for studies longer then 28 days. 4.0 EXPERIMENTAL - Overview__________ ________________________ __ Tissue and serum samples were available for analysis from HWI#6329-137. Liver samples collected at 28 days post dose were analyzed for total organic fluorine by combustion and selective ion electrode. The data were used to assess the extent of dermal absorption. 5.0 EXPERIM ENTAL-M ETHODS 5.1 AMDT-M-1-0, Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 5.2 AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 5.3 AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver 5.4 AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 5.5 AMDT-M-8-0, Analysis of Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 6.0 DATA ANALYSIS_______________________________________________ The data from combustion and subsequent combustion analysis for total organic fluorine of liver samples from rabbits dosed dermally with FC-99 is attached. The data from livers at 28 days post dose show no differences between dosing groups. Rabbits were dosed at 0, 10, 125, 250 mg/kg (0, and about 0.8, 10, and 20 ug/kg with respect to the C8 perfluorooctanesulfonate). The formulation of FC-99 is a diethanolamine salt of perfluorooctanesulfonate with about 25% solids. Twenty percent of these solids are the C8. T-6053 is a 0.04% solution of FC-99 solids. The dose for the high group was 20 ug/kg with respect to the perfluorooctanesulfonate. In previous studies, FC-95 has been shown to be persistent in rabbits after an intravenous dose (HWI#6329-159), not dermally absorbed at doses of 0.30 mgdcg (HWI#6329-130), and suitable as a marker to assess dermal absorption 0032 000003 3 (HWI#6329-159). In a pharmacokinetic study of FC-99 (HWI#6329-136), it was concluded that perfluorooctanesulfonate would be a useful marker for assessment of dermal absorption of FC-99. Since FC-99 contains perfluorooctanesulfonate the same as FC-95 but just a different salt form (FC-95 is the potassium salt), it is consistent that no dermal absorption appears to occur at these dose levels of T-6053. However, if all of the 20 ug/kg dose were absorbed, the method would not have been able to detect it. Since T-6053 is the same preparation of FC-99 used in the pharmacokinetic study, a direct comparison is appropriate. Other data was collected using electrospray mass spectrometry and Skalar segmented flow analyzer with ion selective electrode (see appendices). This data, although supportive, in the opinion of the Study Director is not required to reach the conclusion stated here and therefore is not discussed in detail. 6.1 Circumstances that May Affect the Quality of the Data: The circumstances that may affect this data analysis is that a longer term pharmacokinetics study on this salt form of perfluorooctanesulfonate was not performed. The dose was very low (below detection limits). However, it seems unlikely that there would be a substantial difference in the pharmacokinetics of two salt forms at 28 days especially with respect to deposition in liver. 7.0 CONCLUSION__________________________________________________ There does not appear to be dermal absorption of FC-99 (T-6053) at dermal dose levels at 20 ug/kg. The dose was below the detection limit of the method. 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. 9.0 APPENDICES__________ 9.1 Protocol and Amendments 9.1.1 Protocol and Final Report: HWI#6329-137 "Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits" (Protocol type TP3016.AB for dosing of animals, tissue collection, etc.) 9.1.2 Analytical protocol AMDT-022195.1 003854 000004 4 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 M aterials and Equipment: See methods 9.6 Solutions, Reagents, and Standards: See methods 9.7 Sample Preparation: See methods 9.8 Quality Control Practices: See methods 9.9 Test Methods: See Protocol AMDT-022195.1 9.10 Instrum ent Settings: See methods 9.11 Data: See attached. 9.11.1 Summary and raw data; ug F" in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 9.11.2 Raw data; analysis of liver extracts using electrospray mass spectrometry. 9.11.3 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. 003855 000005 5 9.1.1 Protocol and Final Report: HWI#6329-137 "Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits" (Protocol type TP-3016.AB for dosing of animals, tissue collection, etc.) 003856 000606 HAZLETOFM WISCONSIN POST OFFI CE BOX 754 5 MA D I S O N . Wl 5 3 / 0 7 - 7 5 4 5 i C O R N IN G Company Sponsor: 3M Toxicology Service Medical Department St. Paul, Minnesota 5 FINAL REPORT Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits Author: Steven M. Glaza Study Completion Date: July 11, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Pro.iect Identification: HWI 6329-137 Phone 608-241-4471 E X P R E S S - MAI L DELI VERY Page 1 of 45 330 1 KINSMAN BLVD 000007 F a x 6 0_8 - 2 4 I - 7 2 2 7 MADISON, W I 53 704 Page 2 of 45 HWI 6329-137 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 Manaoement 12/21/94 01/25/95 01/30/95 02/10/95 04/10/95 04/20/95 04/20/95 07/10/95 12/21/94 01/26/95 01/30/95 02/10/95 04/10/95 04/20/95 04/20/95 07/10/95 Protocol Review Protocol Amendment Protocol Amendment Necropsy Protocol Amendment Data/Report Review Data Review Report Rereview 12/22/94 01/26/95 01/30/95 02/10/95 04/10/95 04/20/95 04/20/95 07/10/95 01/10/95 02/10/95 02/10/95 03/10/95 05/10/95 05/10/95 05/10/95 08/10/95 Representative, Quality Assurance Unit 7-/ / i s ' Date 003858 ooooos Page 3 of 45 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits HWI 6329-137 Test Material Sponsor Sponsor's Representative Study Director Study Locations T-6053 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.0. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Initial Test: Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Replacement Animal: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 Study Timetable Study Initiation Date Experimental (In-life) Start Date In-life End Date Experimental Termination Date Study Completion Date December 30, 1994 January 13, 1995 February 17, 1995 July 11, 1995 July 11, 1995 nnoor q tuuooJ OOOGOfJ Page 4 of 45 KEY PERSONNEL HWI 6329-137 Acute Toxicoloov Quality Assurance Steven M. Glaza Study Director Manager Sherry R. W. Petsel Manager Francis (Bud) W. McDonald Study Coordinator Patricia Padgham In-life Supervisor Laboratory Animal Medicine Cindy J. Cary, DVM Dipl ornate, ACLAM Supervisor Rose M. Bridge Report Supervisor Anatomical Patholoov Toxicoloov SuDDort Thomas E. Palmer, PhD Anatomical Pathologist Kathy Myers Manager Calvin L. Horton Supervisor Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data 003860 O O O O IO Page 5 of 45 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 Deviations Protocol TP3016.AB Protocol AmendmentNo. Protocol AmendmentNo. 1 2 HWI 6329-137 Page 2 3 4 6 8 8 8 9 10 13 14 14 14 15 16 17 19 23 25 27 28 29 41 44 003861 O00011 Page 6 of 45 SUMMARY HWI 6329-137 This study was done to assess the systemic absorption/toxicity and relative skin irritancy of T-6053 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 (Control) 2 3 4 Distilled water T-6053 T-6053 T-6053 Oa 10 125 250 3 3 3 3 3 3 3 3* a Administered at a dose volume of 2.0 mL/kg. * One animal sacrificed on Day 2 due to possible broken back and replaced with another female animal. 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 -9 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 experimental 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, a 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 tissues from the female dosed at 00386 250 mg/kg and sacrificed on Day 2 were collected but were not weighed. 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. 000012 Page 7 of 45 HWI 6329-137 Application of T-6053 did not result in any test material-related changes in body weight gain. All animals appeared clinically normal throughout the study with the exception of one Group 4 female animal that was sacrificed on Day 2 due to an injury (possible broken back). This animal was replaced in the study and the replacement animal appeared 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-6053 at any of the dose levels. There were no test material-related lesions observed at necropsy. 003860 000013 Page 8 of 45 OBJECTIVE HWI 6329-137 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-6053 and described as a clear, colorless liquid. The control material was distilled water and was described as a clear, colorless liquid. Purity and Stabilitv 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 all in-life testing according to HWI SOP. Any remaining control material is retained for other testing and will not be discarded after issuance of the final report. 000014 Page 9 of 45 Safety Precautions HWI 6329-137 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. The replacement animal was received from HRP, Inc., Kalamazoo, Michigan on January 11, 1995 and was maintained at the Hazleton Wisconsin facility at 3301 Kinsman Boulevard, 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 -9. The weight variation of the animals for each group of each sex selected for the study did not exceed 2 standard deviations of the mean weight, and the mean AA'?j3iTl:; body weights for each group of each sex were not statistically different at the 5% probability level. One female animal (No. F53454) was replaced after test material exposure due to a possible broken back. This animal was replaced with another female animal (No. F53504). OOOOI-j Page 10 of 45 Study Design HWI 6329-137 Animals weighing from 2,311 to 2,609 g at initiation of treatment were placed into the following study groups: Group Dose Level Number of Animals Test Material (mg/ko) Males Females 1 (Control) 2 3 4 Distilled water T-6053 T-6053 T-6053 0a 10 125 250 3 3 3 3 3 3 3 3* a Administered at a dose volume of 2.0 mL/kg. * One animal sacrificed on Day 2 due to a possible broken back and replaced with another female animal. This animal weighed 2,352 g at initiation. 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 0.05 mL/cm. 003866 000016 Page 11 of 45 HWI 6329-137 Groups 2, 3. and 4 . For the Group-2 animals (10 mg/kg), the test material (T-6053) was mixed with distilled water to a concentration of 100 mg/mL and applied at a dose volume of 0.1 mL/kg. This 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 (125 or 250 mg/kg, respectively) using the average bulk density of 0.975 g/mL to determine the dose volume for each dose level (0.13 and 0.26 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 10, 125, and 250 mg/kg dose levels was 4, 16, and 25 cm2, respectively. The approximate rate of application ranged from 0.02 to 0.06 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 -9, 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. Sample Collections 00386V 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 000017 Page 12 of 45 HWI 6329-137 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, a 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 for all animals, with the exception of the animal sacrificed and necropsied on Day 2, were weighed (volume only determined for bile). The samples were immediately placed on dry ice (initial test animals only), 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 in-life 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. oooois Page 13 of 45 RESULTS HWI 6329-137 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 with the following exception: One Group 4 female (No. F53454) treated with T-6053 at 250 mg/kg appeared to have injured its back at the time of the 24-hour blood collection. This animal was sacrificed, necropsied, and replaced with No. F53504. The replacement animal 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-6053 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. The necropsy of the Group 4 female (No. F53454), treated with T-6053 at 250 mg/kg and sacrificed on Day 2, revealed a fracture of the first lumbar vertebra. There were no lesions observed in any of the remaining animals. Page 15 contains a pathology report by the study pathologist. 003869 01)0019 Page 14 of 45 DISCUSSION HWI 6329-137 The acute systemic absorption/toxicity and relative skin irritancy of T-6053 were evaluated in male and female albino rabbits when administered as a single dermal application. There were no test material-related changes in body weight gain or in-life clinical findings at any of the dose levels. No test material-related dermal irritation was observed during the study. SIGNATURE 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). 000020 Page 15 of 45 PATHOLOGY REPORT HWI 6329-137 There were six rabbits (three males and three females) each from four dose levels of 0, 10, 125, and 250 mg/kg of body weight euthanized and necropsied at the termination of the study. One female (Animal No. F53454) dosed at 250 mg/kg of body weight was sacrificed on Day 2 because of an apparent broken back. This animal was replaced with another female (Animal No. F53504). 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 sacrificed at study termination. The liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from each of these 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. The tissues from the female (dosed at 250 mg/kg) sacrificed on Day 2 were collected but were not weighed. After necropsy, the animals were discarded. The animal sacrificed on Day 2 had a fracture of the first lumbar vertebra. This finding correlated with the clinical observation of an apparent broken back. Pathologist (6329-137.slh) 040695 Date 003871 OOOO1 Animal Number F53464 F53441 F53451 Mean F53447 F53463 F53452 Mean F53453 F53458 F53446 Mean F53459 F53445 F53439 Mean Page 16 of 45 Table 1 Individual and Mean Body Weights (g) HWI 6329-137 Male_________________ Random ization Dav Dav -9 1 29 _______________ Female Random Animal ization Number Dav -9 Dav 1 29 Group 1 (Control) - Distilled Water (0 ma/kal 2,210 2,253 2,285 2,378 2,413 2,536 2,923 3,030 3,058 F53461 F53455 F53442 2,428 2,347 2,437 2,597 2,484 2,456 2,891 2,929 3,097 2,249 2,442 3,004 2,404 2,512 2,972 Group 2 - T-6053 10 ma/kal 2,241 2,207 2,085 2,539 2,464 2,362 2,977 2,937 2,777 F53444 F53448 F53460 2,358 2,476 2,411 2,338 2,486 2,516 2,798 2,944 2,972 2,178 2,455 2,897 2,415 2,447 2,905 Group 3t - T-6053 125 ma/kal 2,275 2,265 2,156 2,497 2,425 2,323 2,944 2,863 2,743 F53443 F53467 F53450 2,434 2,476 2,427 2,544 2,581 2,537 2,844 2,965 3,003 2,232 2,415 2,850 2,446 2,554 2,937 Group 4 - T-6053 250 ma/kal 2,221 2,090 2,206 2,338 2,311 2,380 2,742 2,798 2,942 F53449 F53456 F53454* F53504c 2,297 2,220 2,424 NA 2,390 2,336 2,609 2,352 2,660 2,782 2,627b 2,939 2,172 2,343 2,827 2,314 2,359 2,794 NA Not avai Table. a Animal No. F53454 was originally selected by the randomization program for use in the study and was treated. This animal was sacrificed after completion of the exposure period due to a possible broken back and was replaced with No. F53504. The body weights for No. F53454 are not included in the Day 1 or Day 29 group means. 0G3S72 b Day 2 body weight. c Replacement animal (not included in group assignment randomization). 0 0 0 0 * - * * Sex Male Female Page 17 of 45 Table 2 Individual Clinical Signs HWI 6329-137 Animal Number Observation 1-4 Hours Dav (Day 1) 2 3 through 29 Group 1 (Controll - Distilled Water (0 mq/kq) F53464 Appeared normal // / F53441 Appeared normal // F53451 Appeared normal / / F53461 Appeared normal // / F53455 Appeared normal // / F53442 Appeared normal // Male Female F53447 F53463 F53452 F53444 F53448 F53460 GrouD 2 - T-6053 Ojg/kq) Appeared normal / Appeared normal / Appeared normal / Appeared normal / Appeared normal / Appeared normal / / / / / / / / / / / Condition existed. 003873 00003 Sex Male Female Page 18 of 45 Table 2 (Continued) Individual Clinical Signs Animal Number Observation 1-4 Hours (Dav 1) 2 Group 3 - T-6053 (125 mq/kq) F53453 Appeared normal // F53458 Appeared normal // F53446 Appeared normal // F53443 Appeared normal // F53467 Appeared normal // F53450 Appeared normal // HWI 6329-137 Dav 3 throuoh 29 / / / / / / Male Female GrouD 4 - T-6053 (250 mq/kol F53459 Appeared normal / F53445 Appeared normal / F53439 Appeared normal / F53449 Appeared normal F53456 Appeared normal / F53454 Appeared normal Possible broken back (at time of 24-hour bleeding interval) Moribund sacrifice - F535043 Appeared normal / / / / - / / / / / / / / Condition existed. - Condition not evident. a Animal No. F53504 replaced Animal No. F53454. 003874 000024 Page 19 of 45 HWI 6329-137 Table 3 Individual Dermal Irritation Scores Group 1 (Control) - Distilled Water (0 mg/kg) Dermal Reaction Males________ Study Dav _2_ _4_ _8_ _______ Females Studv Dav JL _2_ 4 8 Animal No. F53464 Animal No. F53461 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F53441 0000 0000 0000 0000 0000 0000 Animal No. F53455 0000 0000 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F53451 0000 0000 0000 0000 0000 0000 Animal No. F53442 0000 0000 0000 0000 0000 0000 003875 ooooaa Page 20 of 45 HWI 6329-137 Table 3 (Continued) Individual Dermal Irritation Scores Group 2 - T-6053 (10 mg/kg) Dermal Reaction Males Study Dav 1 _2_ 4 _8_ Females Study Dav 1 _ 2_ 4 8 Animal No. F53447 Animal No. F53444 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 Animal No. F53463 0000 0000 0000 000 0 0000 0000 Animal No. F53448 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 Animal No. F53452 000 0 0000 0000 0000 000 0 0000 Animal No. F53460 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 003876 0000& Page 21 of 45 HWI 6329-137 Table 3 (Continued) Individual Dermal Irritation Scores Group 3 - T-6053 (125 mg/kg) . Males________ Studv Dav Dermal Reaction _ L _2_ 4 8 _______ Females Study Dav _1_ _2_ 4 8 Animal No. F53453 Animal No. F53443 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 Animal No. F53458 0000 0000 0000 0000 0000 0000 Animal No. F53467 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 Animal No. F53446 0000 0000 0000 0000 00 0 0 0000 Animal No. F53450 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 003877 OOOO7 Page 22 of 45 HWI 6329-137 Table 3 (Continued) Individual Dermal Irritation Scores Group 4 - T-6053 (250 mg/kg) Dermal Reaction Males Studv Dav J L _2_ 4 JL Animai No. F53459 Females Studv Dav 12 4 8 Animai No. F53449 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Erythema Edema Atonia Desquamation Coriaceousness Fissuring Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 Animai No. F53445 0000 0000 0000 0000 0000 0000 Animai No. F53439 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 Animai No. F53456 0000 00 0 0 0000 0000 0000 0000 Animai No. F534543 00 __ 00 00 00 00 00 - Animai No. F53504b Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 00 0 0 0000 0000 0000 0000 a Animal replaced with No. F53504. b Replacement animal for No. F53454. - Not applicable. 003878 OOOO'S Animal Number F53464 F53441 F53451 F53461 F53455 F53442 Page 23 of 45 Table 4 Individual Pathology Comments ______Test Day Sex Died Sacrificed Necropsy Observation Group 1 (Control 1 - Distilled Water (0 ma/kal M 29 No visible lesions. M 29 No visible lesions. M 29 No visible lesions. F 29 No visible lesions. F 29 No visible lesions. F 29 No visible lesions. HWI 6329-137 o 3 a7T F53447 F53463 F53452 F53444 F53448 F53460 M M M F F F Group 2 - T-6053 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 appl icable. 003879 000039 Page 24 of 45 HWI 6329-137 Table 4 (Continued) Individual Pathology Comments Animal Test Dav Number Sex Died Sacrificed Necropsy Observation GrouD 3 - T-6053 (125 mq/kq) F53453 M - 29 No visible lesions. F53458 M - 29 No visible lesions. F53446 M - 29 No visible lesions. F53443 F - 29 No visible lesions. F53467 F53450 F F - 29 No visible lesions. 29 No visible lesions. F53459 F53445 F53439 F53449 F53456 F534543 F53504b M M M F F F F GrouD 4 - T-6053 (250 mq/kq.l - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. - 29 No visible lesions. 2 The first lumbar vertebra is fractured. - 29 No visible lesions. - Not applicable. a Animal replaced with No. F53504. b Replacement animal for No. F53454. 003880 000030 Sex Male Female Page 25 of 45 Table 5 Individual Animal Tissue Weights and Bile Volumes HWI 6329-137 Animal Number Liver Weight (g) Dermal Appli Kidnevs cation Site Bili Volume GrouD 1 (Control) - Distilled Water (0 ma/kai F53464 F53441 F53451 F53461 F53455 F53442 87.407 114.827 89.123 73.175 94.647 91.590 - 16.722 - 16.740 - 0.468 0.331 0.394 0.371 0.512 0.391 0.3 2.5 0.3 0.7 1.0 1.5 Male Female F53447 F53463 F53452 F53444 F53448 F53460 GrouD 2 - T-6053 (10 mq/kg) 81.754 - 0.213 83.148 15.119 0.357 82.687 - 0.535 70.587 - 0.719 74.702 - 0.361 92.881 15.925 0.368 2a 0.6 0.3 0.7 0.7 0.7 - Not applicable. a Volume not measured to tenths of a ml. 0038S1 000031 Sex Male Female Table 5 (Continued) Individual Animal Tissue Weights and Bile Volumes Animal Number ___ Weight (q)_____________ Dermal Appii- Liver Kidnevs cation Site Bile Volume (mLl GrouD 3 - T-6053 1125 ma/kal F53453 96.249 - 0.612 1.5 F53458 77.755 - 0.390 0.5 F53446 83.574 17.429 0.321 0.7 F53443 79.767 - 0.602 0.6 F53467 78.684 15.937 0.467 0.7 F53450 79.782 - 0.580 2a Male Female F53459 F53445 F53439 F53449 F53456 F53454b F53504c Grouo 4 - T-6053 (250 ma/kal 79.255 15.567 0.372 97.381 - 0.270 83.548 - 0.349 73.983 - 0.391 77.804 14.412 0.572 NA - NA 83.081 - 1.253 0.4 0.5 0.5 0.4 1.0 NA 1.1 Not applicable. a Volume not measured to tenths of a mL. b Moribund sacrifice on Day 2 (01/14/95) due to possible broken back. Tissues were collected, however, their corresponding weights or volumes were not taken since this requirement became effective on 01/24/95. Animal replaced with No. F53504. 0038S2 c Replacement animal for No. F53454. 00003 Page 27 of 45 HWI 6329-137 APPENDIX A Protocol Deviations Protocol TP3016.AB Protocol Amendment No. 1 Protocol Amendment No. 2 0038S3 000033 Page 28 of 45 Protocol Deviations HWI 6329-137 Protocol Page 8, 7. Experimental Design, C. Observation of Animals, (4) Sample Collections, (c) Method of Collection, Second Sentence. 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). ________Actual Procedure______ Blood was inadvertently not collected at the time of necropsy from the female animal (No. F53454) treated at 250 mg/kg that was sacrificed on Day 2 due to an apparent broken back. Page 8, 7. Experimental Design, D. Pathology, (1) Unscheduled Sacrifices and Deaths, Second Sentence. 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. The female animal (No. F53454) treated at 250 mg/kg was sacrificed on Day 2 due to an apparent broken back using an overexposure to carbon dioxide. Page 9, 7. Experimental Design, D. Pathology, (3) Sample Collection, Second Sentence. 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 and immediately placed in a freezer set to maintain a temperature of -20C 10C. The size of the dermal application site collected at necropsy was inadvertently not documented for the female animal (No. F53454) treated at 250 mg/kg that was sacrificed on Day 2 due to an apparent broken back. Also, the tissues collected from the animals in the initial test were placed on dry ice (during transportation) prior to being stored in a freezer. 003884 These deviations are not considered to have had an adverse effect on the outcome of the study. 000004 HAZLETON WISCONSIN MP OA SD TI S OONF.F WI Cl E 5 B3 O7 0X7 - 77 55 4455 Page 29 of 45 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-6053 in Rabbits Date: December 30, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-137 0038S5 000035 Ph One 6 08-241 4471 EXPRESS-MAIL DELIVERY 330 1 kinsman blvn Fax 608 241 7227 MA D I S ON Wl 53 70 4 Page 30 of 45 STUDY IDENTIFICATION TP3016.AB Page 2 Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits HWI No. 6329-137 Test Material T-6053 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative ) 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 Study Director Steven M. Glaza Hazleton Wisconsin, Inc. P.0. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Study Location Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date January 13, 1995 February 10, 1995 March 24, 1995 0038S6 ) ooooa< Page 31 of 45 " TP3016.AB ) Page 3 1. Study Single-Dose Dermal Absorption/Toxicity Study in Rabbits 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) (OECO) [ ] 59 Nohsan No. 3850 (Japanese MAFF) [ ] Notification No. 313 (Japanese MOHW) All procedures in this protocol are in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. ^ 4. Quality Assurance The protocol, study conduct, and the final report will be audited by the Quality Assurance Unit in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedures (SOPs) and policies. 5. Test Material A. Identification T-6053 B. Physical Description (To be documented in the raw data) C. Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under test conditions). D. Storage Room temperature 003837 ) 000037 Page 32 of 45 - TP3016.AB Page 4 E. Reserve Samples Reserve sample(s) of each batch/!ot 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. Experimental Design A. Animals (1) Species Rabbit C038S8 (2) Strain/Source Hra:(NZW)SPF/HRP, Inc. 000038 Page 33 of 45 " ') (3) Aae at Initiation Adult TP3016.AB Page 5 (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 HW1 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 003889 stratified body weight randomization program within nine ) days of study initiation. 000039 Page 34 of 45 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 (1) Test Groups GrouD Test Material Dose Level Number of Animals (mq/kq) Males Females 1 (Control) 2 3 4 Distilled water T-6053 T-6053 T-6053 0* 10** 125 250 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 3 3 3 3 (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 gaper 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. 003890 ; 0000*30 Page 35 of 45 TP3016.A8 ) 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 1). 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 003891 000041 Page 36 of 45 " 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 marginal ear vein), bled via the vena cava, exsanguinated, and subjected to an abbreviated gross necropsy examination. The animals will be necropsied in random order and all abnormalities will be recorded. 003892 000042 Page 37 of 45' 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) * ' 000043 Page 38 of 45 ' 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 ) 003894 ) 000044 PROTOCOL APPROVAL TP3016.AB Page 11 John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department /" < ' Date Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. M u . L 'ft. gresentative Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-137.protdsk2) Date Date 003895 000045 Page 40 of 45J 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) Desquamation 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) Fissuring 0 - None 1 - Slight (definite cracks in epidermis) 2 - Moderate (cracks in dermis) 3 - Marked (cracks with bleeding) ) 003896 000046 Page 41 of 45 HAZLETON WISCONSIN PMOA SDTI S OONF.F IWCl E 5 B3 7O0X7 77554455 a CORNING Company PROTOCOL TP3016.AB Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits HWI 6329-137 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative John L. Butenhoff, PhD Contractor Hazleton Wisconsin, Inc 3301 Kinsman Boulevard Madison, WI 53704 Study Director Steven M. Glaza Amendment No. 1 This amendment modifies the following portions of the protocol: Effective January 13, 1995 1. Page 6. 7. Experimental Design; B. Dose Administration; (11 Test Groups. The test material mixture could not be prepared at the concentration needed to utilize a dose volume of .01 mL/kg. Modify the double asterisked footnote (**) in this section with the following underlined change: ** To be administered at a dose volume of JL0 mL/kg 2. Page 6. 7, Experimental Design; B. Dose Administration; (3) Dose Administration. The test material mixture could not be prepared at the concentration needed to utilize a dose volume of .01 mL/kg. Modify the third sentence in this section with the following underlined change: The dose for each animal in Group 2 will be diluted with distilled water and applied at a dose volume of ^JO mL/kg. 003897 P h o n e 608 241-4 471 EXPRESS MAIL DELIVERY 3301 KINSMAN BLVD 000047 Fax 603 24 I 72 2 ! MA D I S O N . Wl 5 3 70 4 Page 42 of 45 Amendment No. 1 ') HWI 6329-137 Page 2 Effective January 17, 1995 3- Pane 6. 7. Experimental Design; B. Dose Administration: (31 Dose Administration. Animal No. F53454 (Group 4 female) was sacrificed on Day 2 due to an injury (apparent broken back). Add the following as the second paragraph to this section: Due to the sacrifice on Day 2 of one Group 4 female (Animal No. F53454) because of an injury (apparent broken back), a replacement animal will be treated at the same dose level in the same manner as for the other animals in the study. The observations (clinical observations, reading of dermal irritation, body weights and sample collections) and the pathology of the animals (unscheduled sacrifices and deaths, scheduled sacrifice, and sample collection) will be conducted in the same manner as for the other animals in the study. 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. 4. Page 9. 7. Experimental Design; D. Pathology; (31 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 -20C 10C. The samples and their corresponding weights or volumes will be shipped to the person listed in Section 7.C.(4).(c). 5. Page 9. 8. Report. Add the following to this section: Individual animal tissue weights and bile volumes 003898 ) 000048 Amendment No. 1 ) Page 43 of 45~ PROTOCOL AMENDMENT APPROVAL HWI 6329-137 Page 3 2. John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department z/s<~Af Date Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. ^ ) f a c A (L Representative/ Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-137.Aml.dsk2) Date Date 003899 j 0000-19 2^ Page 44 of 45 HAZLETON WISCONSIN OA SD lI S O0NI.f - WI Cl f !>D: O/ 0* / !so s .1 CO RNIN G Compmly PROTOCOL TP3016.AB Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits HWI 6329-137 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative John L. Butenhoff, PhD Contractor Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 Study Director Steven M. Glaza Amendment No. 2 This amendment modifies the following portion of the protocol: Effective January 20, 1995 1. Page 2. Study Location. The replacement animal was treated and maintained at the main facility of Hazleton Wisconsin, Inc., 3301 Kinsman Boulevard, instead of at Hazleton Wisconsin's Acute Studies Laboratory, 3802 Packers Avenue, since the replacement animal was received and acclimated in this facility. Replace this section with the following: Study Location (Initial Test) Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Study Location (Replacement Animal) Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 003300 000050 P h o n e 6 0 3 2 <t : : a / l E X P R E S S M A I I <rI!7 f Rv <i0 ! K I M S M A M B [ V 0 F a x r;0 o 24 1 /2 2/ MADlS O N ! p !/0A Amendment No. 2 ) Page 45 of 45 HWI 6329-137 Page 2 PROTOCOL AMENDMENT APPROVAL 2. John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department Date f- U Steven M. Glaza Q Study Director ) Acute Toxicology Hazleton Wisconsin, Inc. Date Representative Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-137.Am2.dsk3) Date 003901 ; 000051 9.1.2 Analytical protocol AMDT-022195.1 003902 000032 3M Environmental Laboratory_______________________ Protocol - Analytical Study Single-Dose Dermal Absorption/Toxicity Study of T-6053 in Rabbits In-Vivo Study Reference Number: HWI#6329-137 Study Number: AMDT-022195.1 Test Substance: FC-99 (T-6053) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55106 Proposed Initiation Date: July 25, 1995 Proposed Completion Date: August 25, 1995 Method Numbers and Revisions: AMDT-M-1-0, Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry AMDT-M-8-0, Analysis of Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode Author: James D. Johnson Approved By: C03S03 //J a m e s 'D/Johmson s ' Study Dfiirreeccttor John Butenhoff, PhD Sponsor Representative Date 000053 1 l-.Q. P U R P O S E This study is designed to provide information as to whether FC-99 (T-6053) is dermally absorbed. The analytical aspect of this study is to determine fluorinecontaining compounds (biotransformation products) in the tissue and serum of rabbits at various times post dose dermal application of FC-99. The data are to be used to make an assessment of the extent of dermal absorption of FC-99. 2.0 TEST MATERIALS 2.1 Test, Control, and Reference Substances and Matrices 2.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 2.1.2 Analytical Reference Matrix: Bovine liver and bovine serum 2.1.3 Analytical Control Substance: None 2.1.4 Analytical Control Matrix: Bovine liver and bovine serum 2.2 Source of M aterials: 3M ICP/PCP Division (2.1.1), grocery store (2.1.2, 2.1.4 liver), Sigma Chemical Company (2.1.2, 2.1.4 serum) 2.3 Num ber of Test and Control Samples: Tissues and fluids from 18 test animals and 6 control animals. One animal was replaced on day 2 (animal F53454 in the 250 mg/kg dose group was replaced with animal F53504). Tissues and fluids include liver, kidney, serum, cellular fraction, dermal application site and bile. Analysis of these tissues will be at the discretion of the Study Director. 2.4 Identification of Test and Control Samples: The samples are identified using the HWI animal identification number which consists of a letter and five digit number, plus the tissue identity and day identity (serum). 2.5 Purity and Strength of Reference Substance: To be determined by Sponsor. 2.6 Stability of Reference Substance: To be determined by Sponsor. 2.7 Storage Conditions for Test Materials: Room temperature (2.1.1), -20 10C (2.1.2, 2.1.4). Test and Control samples will be received according to AMDT-S-10-0. 003804 000054 2 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. 2.9 Safety Precautions: Refer to appropriate MSDS. Wear appropriate laboratory attire. Use caution when handling knives for cutting the samples. 3,0 EXPERIMENTAL -Overview________________________ ______ The tissues from animals dosed as described (HWI#6329-137), are available for analysis for fluorine compounds. At the discretion of the Study Director, a series of 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. When sufficient data has been collected to meet the objectives of the study in the opinion of the Study Director, analysis will cease. 4.0 EXPERIM ENTAL-M ethods_______________________________________ 4.1 Liver and Serum screening methods: (attached) 4.1.1 AMDT-M-1-0, Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 4.1.2 AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 4.1.3 AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver 4.1.4 AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 4.1.5 AMDT-M-8-0, Analysis of Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 5.0 DATA ANALYSTS________________________________________________ 5.1 Data Reporting: Data will be reported as a concentration (weight/weight) of fluoride per tissue or fluid, or as FC-95 (electrospray mass spectrometry) per unit of tissue or fluid. Statistics used, at the discretion of the Study Director, may include 003905 000055 3 regression analysis of serum concentrations with time and averages and standard deviations of 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 Raw Data and Records: Raw data, approved protocol, appropriate specimens, approved final report, and electronic data will be maintained in the AMDT archives. 7.0 REFERENCES____________________ 7.1 AMDT-S-10-0, Sample Tracking System 8.0 ATTACHMENTS_________________________________________________ 8.1 AMDT-M-1-0, Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver 8.2 AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer 8.3 AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver 8.4 AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochemicals Using Electrospray Mass Spectrometry 8.5 AMDT-M-8-0, Analysis of Fluoride Using the Skalar Segmented Flow Analyzer with Ion Selective Electrode 003906 000056 4 3M Environmental Laboratory Method Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer - Liver M ethod Identification Number: AMDT-M-1 Revision Number: 0 Adoption Date: Revision Date: None Author: Rich Youngblom Approved by: Quality Assurance /3Date 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 o f a Modified Dohrmann DX2000 Organic Halide Analyzer 003907 1 000057 1.0 SCOPE . APPLICABLE COMPOUNDS. AND MATRICES 1.1 Scope: This method is for the operation o f 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 KEYWORDS__________________________________________ 2.1 Fluoride, fluorine, extraction, pyrolysis, ionization, ion selective electrode, Dohrmann, halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS___________ ____________________________ 3.1 Glassware and exhaust gases can be extremely hot. 3.2 Glassware is fragile, broken glass may cause injuries. 3.3 Pressurized gases, proper compressed gas handling practices required. 3.4 Solvent based samples may flash, may need to allow them to dry down before starting run. 3.5 Potential biohazards due to the biological matrices. Use appropriate personal protective equipment. 4.0 SUPPLIES AND MATERIALS__________________________________ 4.1 Compressed Oxygen, Hydrocarbon free, regulated to 30 PSI. 4.2 Compressed Helium, High Purity Grade, regulated to 45 PSI. 4.3 Quartz glass sample boat with TeflonTM tubing, Dohrmann 890-097 or equivalent. 4.4 Quartz glass combustion tube, Reliance Glass G-9405-012 or equivalent. 4.5 Orion 940999 Total Ionic Strength Adjustment Buffer (TISAB I I ) or equivalent. 4.6 Sample collection vials, HDPE. 4.7 Milli-QTM water 4.8 Polystyrene pipettes. 4.9 Activated Charcoal, E. Merck 2005 or equivalent. 4.10 Hamilton Syringe or equivalent. 4.11 Miscellaneous laboratory glassware 5.0 EQUIPMENT_________________________________________________ 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. 003908 000058 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 o f 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 STANDARDrZATION___________________ __ 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 o f 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 o f each standard and five concentrations o f 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 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 o f fluoride. 8.3.2 Blanks 8.3.2.1 Prepare sample using the same methods and type o f matrix as the test sample. 8.3.2.2 For rabbit studies, use beef liver as the matrix. Prepare at least 3 samples o f 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. 003909 000059 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 o f 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 o f 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 o f the standard on or in the matrix. For rabbit studies, use 4 uL o f standard and eject it on or in the beef liver. 8.3.3.4 At least 3 replicates should be used for the lowest standard concentration; more replicates may be used at the discretion o f the analyst. 8.3.3.5 Combust the sample as described in section 9.3 and analyze according to AMDT-M-2. 8.3.3.6 Run all 15 standards. If one replicate is significantly different from the other two replicates, run another sample for that standard. Indicate in data that the new replicate replaces the old replicate and that the new replicate will be used to calculate the regression curve. 8.3.3.7 When all standards have been run, calculate the P-. P- 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 o f 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 N ew FC-95 standards should be prepared at least once a month. 9.0 PROCEDURES___________________________________________________ 9.1 Typical Operating Conditions: 9.1.1 Combustion tube temperature = 950C. 9.1.2 Oxygen and Helium flow = 50 cc/minute. 9.1.3 Vaporization/Drying time = 240 seconds. 9.1.4 Bake time = 300 seconds. 9.2 Start Up Procedure: 9.2.1 If the program is not started, start the EOX program on the PC. 9.2.2 Open the SYSTEM SETUP window. 9.2.3 Put the furnace module and the cell in the READY mode. 9.2.4 Close the SYSTEM SETUP window. 003910 OOOOkO 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 o f 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 o f 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 o f TISAB solution or 1:1 TISAB:Milli-QTM water mixture to a labeled sample collection vial. Typically 0.6 mL to 15 mL are used. For rabbit studies, use 1.0 or 2.0 mL o f 1:1 TIS AB:Milli-QTM water mixture. 9.3.4 Place the vial so that the tip o f the COMBUSTION TUBE is in the TISAB at least 0.25 inches. Gases released during pyrolysis must bubble through the TISAB. 9.3.5 Run the EOX-SOLIDS program found in the RUN menu. 9.3.6 When the EOX program is finished, remove the collection vial from the combustion tube. 9.3.7 If undiluted TISAB was used to collect the sample, add an equal volume o f 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 or vacuumed out. It may be necessary to scrap particles o ff the bottom with a spatula or other similar device. A drop o f 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 U se the standard curve to calculate the sample value. 9.4.2 Sample Mass Recovered F (ug) = (TISAB vol in mL) x (Orion reading in ppm - intercept^ (Slope) 10.0 VALIDATION 10.1 Quality Control 10.1.1 Daily Start Up Check Samples: Once the standard curve is established, each day o f analysis is started by analyzing QC samples. The QC samples are to be the same as the lowest concentration spiked samples used to generate the standard curve. Each concentration must be done in triplicate unless the first two replicates are within 20% o f 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 Other Validation Parameters: NA 003911 (jO UO bl 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 r^ must be at least 0.95 or greater. "Outliers" may be excluded if two o f the three replicates are within 20% o f each other and the outlier is greater than 200% o f the average o f those two or less than 50% o f the average o f those two. Any such outliers should be pointed out in the data and noted in the Final Report along with the reason it was considered an outlier. 12.0 ATTACHMENTS________________________________________________ None 13.0 REFERENCES__________________________________________________ 13.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer Operator's Manual (Manual 915349, revision B, December 1993) 13.2 AMDT-M-2 Fluoride Measurement by Means o f an Orion EA940 Expandable Ion Analyzer 13.3 AMDT-EP-3 Routine Maintenance o f a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 REVISIONS____________________________________________________ Revision Number Reason for Change Revision Date 003912 0000G2 6 3M Environmental Laboratory Method Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer Method Identification Number: AMDT-M-2 Revision Number: 0 Adoption Date: Revision Date: None Author: Rich Youngblom Approved By: Grorfr/ Leader is /J ------- /ty a/Z -S Date Quality Assurance Date Software: MS Word 5.1a Affected Documents: AMDT-M-1 Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer 003913 000003 1 1.0 SCOPE . APPLICABLE COMPOUNDS. AND MATRICES 1.1 SCOPE: This method is for the calibration and operation o f an Orion EA940 Expandable Ion Analyzer. 1.2 APPLICABLE COM POUNDS: Fluoride. 1.3 APPLICABLE M ATRICES: Liquid samples in an appropriate buffer solution. Preferred pH o f 6.0. 2.0 KEYWORDS________________________________________________ 2.1 Fluoride, fluorine, ion selective electrode 3.0 PRECAUTIONS_______________________________________ _ 3.1 N o hazards identified with this method. 4.0 SUPPLIES AND MATERIALS___________________________________ 4.1 Orion 940999 Total Ionic Strength Adjustment Buffer II (TISABII) or equivalent. 4.2 Orion Model 900001 electrode filling solution (AgCl) or equivalent. 4.3 Orion 940907 100 ppm fluoride standard or equivalent. 4.4 Milli-QTM water or equivalent. 4.5 Magnetic stir bars. 4.6 Lab tissues. 4.7 Sample collection vials. 4.8 Plastic 100 mL volumetric flasks. 4.9 Polystyrene pipettes. 4.10 Miscellaneous laboratory glassware. 5.0 EQUIPM ENT___________________________________________ 5.1 Orion Model EA940 Expandable Ion Analyzer or equivalent. 5.2 Orion Model 960900 Solid State Combination Fluoride electrode or equivalent. 5.3 Magnetic Stir Plate. 5.4 IBM compatible 386 or 486 computer (only needed if using Orion 3E software). 5.5 Orion RS232 interface cable (only needed if using Orion 3E software). 5.6 Microsoft Excel 5.0 (only needed if using Orion 3E software). 6.0 INTERFERENCES_______________________________________ 6.1 It is recommended that the pH be at or near 6.0. A 1: l mixture o f TISAB and sample/MilliQTM water will generally bring sample to pH o f 6.0. 6.2 Sample temperature may effect fluoride measurement. It is recommended that the sample be at room temperature as the standards were when the meter was calibrated. 6.3 The rate the samples are stirred at should be consistent with the rate the standards were stirred. 003914 2 0000G4 6.4 Air bubbles trapped under electrode can give erroneous readings. Make sure no air is trapped under electrode. 7.0 SAMPLE HANDLING_____________________________ 7.1 No special handling necessary. 8.0 CALIBRATION AND STANDARDIZATION_____________________ __ 8.1 Preparation of Calibration Standards 8.1.1 Measure 50 mL o f 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 o f 100 ppm fluoride standard into the appropriately labeled flasks. 8.1.4 Add approximately 30 mL o f Milli-QTM water to each flask. 8.1.5 Shake the flasks to mix the solutions. 8.1.6 Eliminate air bubbles from the flasks by tipping the flasks on their sides and rolling the air in the flasks over the air bubbles. 8.1.7 Bring the volume in the flasks up to the 100 mL mark with Milli-QTM water. 8.1.8 Invert and shake the flasks for the final mixing. 8.1.9 Record standards in Standards Log Book. 8.2 Calibration 8.2.1 If necessary, remove tape from electrode filling hole. 8.2.2 Invert probe to wet top seal. 8.2.3 Eject a few drops o f filling solution from bottom o f 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 o f 0 .0 5 ,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 M easurem ent, 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. 003915 3 000005 9.2 Calibration And M easurement, 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 o f standards to be used, concentration o f 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 o f the curve, as well as the intercept and correlation. Record the slope, intercept, and correlation in the appropriate log book and click on CONTINUE. The calibration data is automatically copied to C:\Orion\Data\Calib.txt. 9.2.2 Data Spreadsheet: 9.2.2.1 Select either NEW or OPEN from the FILE menu to open a new or existing spreadsheet to store data in. 9.2.2.2 Record the name of the spreadsheet used in the appropriate log book. 9.2.3 Fluoride Measurement: 9.2.3.1 Follow steps 9.2.1 through 9.2.4 9.2.3.2 Enter the name o f the sample in the appropriate place on the screen. 9.2.3.3 Click on the NEW SAMPLE button 9.2.3.4 When the readings have stabilized, click on the RECORD button and write the result in the appropriate log book. 10.0 VALIDATION__________________________________________________ 10.1 Quality Control: 10.2 Precision and Accuracy 10.3 Other Validation Parameters According to Reference 13.2, the range o f detection is 0.02 ppm fluoride up to a saturated solution o f fluoride. 11.0 DATA ANALYSIS______________________________________________ 11.1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 12.0 ATTACHMENTS_______________________________________________ None 13.0 REFERENCES 00393.6 4 00006 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 003917 0000G7 5 3M Environmental Laboratory Method Extraction of Fluorochemicals from Rabbit Livers SOP Identification Number: AMDT-M-4 Revision Number: 0 Adoption Date: Revision Date: N one Author: Dave Christenson/Cynthia Weber Approved By: Software: MS Word, 6.0 Affected Documents: M-5, Analysis o f Rabbit Extract for Fluorochemicals Using Electrospray Mass Spectroscopy. 003918 OOOGGS i 1.0 SCOPE__________________________________________________ ____ 1 .1 Scope: This method is for the extraction o f 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. 2.0 KEYW ORDS______________________________________________ _ 2 .1 Fluorochemicals, rabbit livers, electrospray mass spectrometer, fluorinated compounds, extraction. 3.tQPRECAUTIONS_________________________________________ 3 .1 Use gloves when handling the rabbit livers, they may contain pathogens. 4.0 SUPPLIES AND MATERIALS__________________________________ 4.1 Supplies 4 . 1 . 1 Syringe, capable o f measuring 100 uL 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 um. 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 100 mLs Milli-Q water. Dilute this solution 1.TO for a 250 ppm solution. 4 . 2 . 2 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker), (Na2C03/NaHC03) 0.25 M: Weigh 26.5 g o f sodium carbonate (Na2C 0 3) and 21.0 g o f sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with Milli-Q water. 4 . 2 . 3 Dilute acetonitrile solution, dilute acetonitrile 1:1 with Milli-Q water. 4 . 2 . 4 Ethyl Acetate 4 . 2 . 5 Methanol 4 . 2 . 6 Milli-Q water 5.0 EQ U IPM EN T_____________________________ 5 .1 Ultra-Turrax T25 Grinder for grinding liver samples. 5 .2 Vortex mixer 5 .3 Centrifuge 5 .4 Shaker 003919 0000U9 2 5 . 5 Analytical Evaporator 6.0 INTERFERENCES____________________ 6 .1 There are no known interferences at this time. 7.0 SAM PLE HANDLING_______________________________________ ___ 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 o f approximately 12 ppm 1H,1H,2H,2Hperfluoroctanesulphonic acid to be added to each liver sample. 8 . 1 . 2 Weigh at least 0.1 g o f lH,lH,2H,2H-perfluoroctanesulphonic acid. Record the actual weight. 8 . 1 . 3 Bring it up to volume with 100 mLs o f methanol, this is the stock standard. 8 . 1 . 4 Dilute 3 mLs o f the stock standard to 250 mLs final volume with Milli-Q water. Calculate the actual value of the standard. actual mg perfluoroctanesulphonic acid 0.1 L X 3 mL = 250 mL actual value, 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 o f FC-95 and record the actual weight. 8 . 2 . 3 Bring up to volume with dilute acetonitrile in a 100 mL volumetric flask. 8 . 2 . 4 Dilute the solution with dilute acetonitrile 1:10 for a solution of approx. 100 ppm. Dilute this solution 1:10 with dilute acetonitrile for a solution of approx. 10 ppm. 8 . 2 . 5 U se 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 o f 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 o f eight 1 mL aliquots of the solution in 15 mL centrifuge tubes. Be sure to resuspend solution by shaking it between aliquots. 8 . 3 . 3 Spike seven o f 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. CC39RO 000070 3 Approximate Cone, ug FC-95/ g liver Blank 400 ppb 800 ppb 1.2 ppm 1.6 ppm j PPm 8 ppm 12 ppm Working Standard (Approximate Cone.) 500 ppb 500 ppb 500 ppb 500 ppb 1 PPm 5 ppm 5 PPm uL 100 200 300 400 500 200 300 8 .4 Calculate the actual value o f the standards: uL o f standard x mg/L x 1L/1000000 uL = mg FC-95 ______ mg FC-95 x .(organic layer recovery factor) = mg/mL FC-95 137 mg liver*/l mL solution 5 equivalents in liver mg/mL x 1000 = mg/L = ppm FC-95 in liver Average weight of bovine liver in solution. The amount o f FC-95 is reported as equivalents o f FC-95 potassium salt. 8.5 Calibration 8 . 5 . 1 Extract the spiked beef liver homogenate following 9.13 to 9.23 o f this method. Use these standards to establish your curve on the mass spectrometer. 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 spectometer is performed. 8 .7 Storage Conditions for Standards 8 . 7 . 1 B eef liver homogenates may be frozen after preparation. 9.0 PROCEDURES__________________________________________________ 9 .1 Obtain frozen liver samples.In spent tissue, note that the liver has not been packaged with other tissues. 9 .2 Use a dissecting scalpel and cut off approximately 1 g o f liver. 9 .3 Weigh the sample directly into a tared plastic grinding tube. .. 9 .4 Record the liver weight in the study note book. v U ii9 2 1 000071 4 9 .5 Put a label on the vial with the study number, weight, rabbit ID, date and analyst initials. 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 o f FC-95 standard as described in 8.3. 9 . 1 3 Spike the samples and beef liver homogenates with 100 uL o f internal standard. 9 . 1 4 Add 1 mL o f the sodium carbonate/sodium bicarbonate buffer and 1 mL ammonium acetate. 9.15 Using an analytical pipet, add 5 mL ethyl acetate. 9 . 1 5 Cap the sample and vortex 20 to 30 seconds. 9 . 1 6 Put them in the shaker for 20 min. 9 . 1 7 Centrifuge for 20 to 25 minutes, until the layers are well separated. Set the power on the centrifuge to 25. 9 . 1 8 Remove 4 mLs o f the top organic layer to a fresh 15 mL centrifuge tube with a 5 mL graduated glass pipet. Transfer the label to the fresh tube. 9 . 1 9 Blow the sample down on the analytical evaporator to near dryness with nitrogen, approximately 30 to 40 minutes. 9 . 2 0 Bring the remaining sample up in 1 mL dilute acetonitrile with an analytical pipet. 9 . 2 1 Vortex 15 seconds. 9 . 2 2 Transfer the sample to a 3 mL syringe. Attach a 0.2 nm 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 3 Cap and hold for analysis by electrospray mass spectroscopy. 9 . 2 4 Complete AMDT-M-4 worksheet and attach to page o f study notebook. 10.0 VALIDATION______________ 10.1 Quality Control 10.2 Precision and Accuracy 10.3 Other Validation Parameters 11.0 DATA ANALYSIS__________ 11.1 None 12.0 ATTACHM ENTS____________ 1 2 . 1 Worksheet AMDT-M-4 13.0 REFEREN CES______________ 13.1 AMDT-EP-22 14.0 REVISIONS_________________ Revision Number Reason for Change Revision Date 003922 000072 5 Worksheet AMDT-M-4 Study # _ - _ . 1 Sam ple N um ber set # Blank Liver approx 0.5 ppm actual ppm #W . in n iff. 200 nL 200 uL 400 nL . _ . . . _ _ _ _ - _ _ F C -95 approx 1 ppm actual ppm #W . . . 500 uL _ . _ . _ _ _ _ approx. 5 ppm actual ppm #W . _ _ _ _ 200 uL 2 0 0 iiT. _ _ _ _ . _ . _ - _ D ate and Initials fo r Std. 1 s tu d v n iim tw r w'h e re th e o rig in a l w o rk s h e e t is lo c a te d a n d n la c e a c o n v .____ I1 1I L iv e r E x tr a c tio n P ro c e ss:_________ _________________ D a te & Tnitials____ P inet 1 m L n f L iver Solution P in e t 100 u L n f 12 n n m in te rn a l S ta n d a rd _____________ S td # V ortex 15 sec. P in e t 1 mT, n f 2 5 0 n n m A m m o n iu m A c e ta te ____________ S t d . # ____________ P inet 1 m L o f 0.25 Na-,CO,,/0 25M N aH C O , B uffer P in et 5 mT. n f F.thvl A cetate .Vortex 20-20 sec. S hake 20 m in. C entrifiipe 20-25 m in R e m o v e a 4 mT. a lin u n t n f o rg a n ic 1av<r B low dow n to near drvness C O .25 m L i w ith N, A dd 1 m: n f L I A ceroni tri le/H -,0 v V o rte x 15 see ___ I N # ____________ ;ilt r u s in g a 3 c c B -D s y rin g e w ith a ().2um S R I f ilte r in to a 1 5 m L a u to s a m n le v ia l______________________________ 003923 000073 6 3M Environmental Laboratory_____________________ Method Analysis of Rabbit Liver Extract for Fluorochemicals using Electrospray Mass Spectroscopy SOP Identification Number: AMDT-M-5 Revision Number: 0 A doption Date: - C - `j Revision Date: N one Author: Dave Christenson/Cynthia Weber Approved By: Software: MS Word, 6.0 Affected Documents: M-4, Extraction o f Fluorochemicals from Rabbit Livers 003924 000074 l M . .SCOPE______________________________________________________ 1 .1 Scope: This method is for the analysis o f extracts o f 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 A pplicable Compounds: Fluorochemicals or other fluorinated compounds. 1 .3 M atrices: Rabbit Livers (samples), Beef Liver (standards), other tissues and fluids. 2.0 KEYWORDS____________________________________________________ 2 .1 Fluorochemicals, fluorinated compounds, electrospray mass spectroscopy, mass spectrometer, rabbit livers. 3.0 PRECAUTIONS 3 .1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk o f electrical shock. 3 .2 Do not run the pump above it's capacity o f 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 MATERIALS 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 EQUIPMENT 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. 0,0 INTERFERENCES 6 .1 There are no known interferences at this time. 7.0 SAMPLE HANDLING 7 .1 Keep the extracted samples in capped 15 mL centrifuge tubes or in capped autovials until ready for analysis. 0039 000075 2 8.0 CALIBRATION AND STANDARDIZATION__________________ 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 o f 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 o f the tip o f the probe. The pressure should be at 1700 to 1800 psi. 9 . 1 . 5 Check the fused silica at the end o f 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 o f 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 o f the probe. A fine mist should be coming out o f the tip. 9 . 1 . 7 Carefully guide the probe into the opening. Insert it until it w on'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-DA Y-YR + letter. Record it in the log book. 9 . 1 . 1 IRun 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 o f sample into a syringe. Inject the sample into the rheodyne injection port. Inject slowly. Record the sample ID in the log book. 9 . 2 . 2 Turn the valve to "On". 9 . 2 . 3 Wait two minutes, and inject the next sample. C0 3 9 2 6 9 . 2 . 4 Record the scan number for each sample in the logbook. OOOOr f> 3 9 .3 Using the Autosampler 9 . 3 . 1 Set up sample tray A, B, or C. 9 . 3 . 2 Record the samples and their positions in the instrument log book. Up to 17 vials may be in each run. 9 . 3 . 3 Set-up the sampler: 9.3.3.1 Push the sample button 9.3.3.2 Set sample loop size = 100 uL 9.3.3.3 Set inject/sample = 2 9.3.3.4 Set Cycle time = 0 9.3.3.5 Name the file: Livers 9.3.3.6 Identify the tray used 9.3.3.7 Add the samples to Queue by pressing "Enter" 9.3.3.8 Press "Run" to start 10.0 VALIDATION______________________________________________ ___ 10.1 Quality Control 1 0 . 1 . IRun a standard every 5 to 7 samples. If a significant change( 50%) in peak height occurs stop the run. Only the samples before the last acceptable standard will be used. The remaining samples will be reanalyzed. 10.2 Precision and Accuracy 1 0 . 2 . lS e e Method Validation Report number AMDT-M-5.0.V1 10.3 Other Validation Parameters 1 0 . 4 Refer to Method Validation Report Number AMDT-M-5.0. VI 11.0 DATA ANALYSIS_____________________________________________ 11.1 11.2 Calculations Plot the standard curve, using the mean o f 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. 11.2'. 2Calculate the mg of FC-95 anion, or other fluorochemical in the total rabbit liver: mg FC-95 anion in the total rabbit liver = m e FC-95 anion from std. curve gms o f liver used for analysis x Total mass o f liver, gms 11.3 11.4 Make a results table and enter it in the study book. Print a chromatogram for each sample, with the peaks labeled with the sample or standard ID. Write the study number on the printout, initial, date, and put it in the study folder. Staple all chromatograms together and number pages. 003927 000077 4 12.0 ATTACHMENTS None 13,.0-BEE-ERENCES______________________________ 1 3 . 1 AMDT-EP-17 14.0 REVISIONS__________________________________________ Rvision Number Reason for change Rvision Date 003928 000078 5 3M Environmental Laboratory Method Analysis of Fluoride Using the Skalar Segmented Flow Analyzer With Ion Selective Electrode Method Identification Number: AMDT-M-8 Adoption Date: / j - s '- t r ' Revision Number: 0 Revision Date: N one Author: Deb Wright / Cynthia Weber Approved By: ( 7 * U,V / ------- Quality Assurance /A/Ts Date Date Software: IBM MS Word, 6.0 Affected Documents: AM DT-EP-26, Operation and Maintenance o f the Skalar Segmented Flow Analyzer 003929 000079 l LftJSCQPS__________________________________ _________ 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 KEYWORDS_____________________________________________________ 2 .1 Skalar, segmented flow, fluoride. 3.0 PRECAUTIONS_________________________________________________ , 3 .1 Follow standard laboratory safety practices. 4.0 SUPPLIES AND MATERIALS________________________________ _ 4.1 Supplies 4 . 1 . 1 Sample cups, 4 mL plastic cups with caps 4 . 1 . 2 Autopipets, oxford or equivalent with plastic tips 4 . 1 . 3 Polypropylene volumetric flasks, 100 mL 4 . 1 . 4 Cartridge components, refer to the Skalar Methods for components and part numbers. 4 . 1 . 5 Sample prefilters, Evergreen 4.2 Reagents 4 . 2 . 1 Brij 35, 30% S.F.A.S. Detergent 4 . 2 . 2 TISAB II buffer solution: Purchase TISAB II from Orion. To 1 liter of TISAB II add 2.5 mL or 100 ppm fluoride solution and 1 mL Brij. 4 . 2 . 3 Sampler rinsing solution: Dilute TISAB II 1:1 with Milli-Q water. 4 . 2 . 4 Nitric acid solution for decontamination, 1 N (lab grade): Slowly add 64 mLs concentrated nitric acid (H N 03) to 250 mLs of Milli-Q water. Bring the volume up to 1 L with Milli-Q water. 4.3 Standards 4 . 3 . 1 Stock solution, 100 ppm F: purchased from Orion. 4 . 3 . 2 Intermediate standard, 10 ppm: Dilute 10 mLs of stock solution to 100 mLs with Milli-Q water. Use polypropylene volumetric flasks. 4 . 3 . 3 Working standard: Make up the following working standards by adding the volumes o f intermediate or stock standard indicated on the table, using oxford or pumpmate pipets, to 50 mLs o f TISAB and diluting to 100 mLs ___________________ with Milli-Q water. ______________________________________________ Working Standard mLs o f Stock Standard mLs o f Intermediate Standard 0.015 ppm - 0.15 0.03 ppm - 0.3 0.06 ppm - 0.6 0.09 ppm - 0.9 0.12 ppm - 1.2 0.15 ppm - 1.5 0.3 ppm 0.3 - 0.6 ppm 0.6 - 003930 OOOOfcO 2 ___________ L2 ppm__________ 1.5 ppm 1.2 1.5 - - JLEQUIPMENT______________________________________ 5 . 1 Skalar Segmented Flow Auto Analyzer Sanspius System equipped with ISE ^ . I NTERFERENCES__________________________________ 6 . 1 High concentrations o f alkalinity, chloride, phosphate, sulfate or iron can cause interferences. 7.0 SAMPLE HANDLING__________________________________________ 7 .1 Samples should be stored in polyethylene bottles. Samples should be analyzed within 30 days. 8JLCALIB,R.A-TIQN.ANIL.STANDA.RDIZA1IQN_______________ 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 o f every six months, or as necessary. 9.0 PROCEDURE___________________________________________________ 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 appropriate number o f 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. vO d^. 000081 3 9 . 2 . 5 . 1 Select GRAPHICS, REAL TIME. If you cannot get real-time, you may be in the Data Handling Panel. Switch to the Analysis Panel by selecting CONTROL PANEL and pushing F7. 9 . 2 . 5 . 2 Use the small screwdriver for the offset potentiometer to set the base line. Adjust the baseline until it is approximately 3/4 inch from the bottom o f the screen. 9 . 2 . 5 . 3Check the highest standard and adjust the gain, if necessary, with the interface screw #3. 9 . 2 . 6 Go to CONTROL PANEL, and to analysis panel. Deselect the analysis that will not be run. (Select or deselect analysis by pressing ENTER.) Press Tab to return to the Analysis Panel. 9 . 2 . 7 Press the spacebar to bring up the local menu. 9 . 2 . 8 Select START to start the analysis. 9 . 2 . 9 Type your ID (initials), the sample table which you created under 9.2.1 (or press ENTER for choices), choose running with or without the system table and select START ANALYSIS. 9 .2 .1 0 After starting the software, start the sampler. Make sure that the sampler is set to the right number of samples and that the sample/wash/air times are OK. 9 . 2 . 1 1 Select GRAPHICS, REAL TIME to view the progress o f 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 o f 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 VALIDATION_________________________________________________ 10.1 Quality Control 1 0 .1. IRun a standard (mid to high concentration) every 10 samples. If a significant change in peak height occurs, only the samples before the last acceptable standard will be used. The remaining samples will be reanalyzed. 003932 ` 000082 4 10.2 Precision and Accuracy 1 0 . 2 . lS e e Method Validation Report number AMDT-M-8.0.VI 10.3 Other Validation Parameters 1 0 . 4 Refer to Method Validation Report Number AMDT-M-8.0. V 1 11.0 DATA ANALYSIS______________________________________ __ 11.1 11.2 11.3 11.4 Calculations 1 1 . 1 . IThe standard curve is plotted by the Skalar software. 1 1 . 1 . 2 All calculations are done by the Skalar software, r2 should be 0.995 or better. Prepare spreadsheets to summarize data. Include sample volume, weights used etc. Write the study number on the printouts, initial, date the printout, and bind together with all package documents and place in the study folder. Make a copy of the summary sheet and tape into the study notebook. Back up all data and spreadsheets onto study disk and backup disks. Electronic Data 1 1 . 4 . 1GLP studies: Electronic data is copied onto the Study floppy disk for each study, and also data is copied onto a floppy disk that is stored in the lab. 1 1 . 4 . 2 Other studies: All data is copied onto a floppy disk that is stored in the lab. 12.0 ATTACHMENTS_______________________________________________ None 13.0 REFERENCES__________________________________________________ 13.1 13.2 13.3 AMDT-M-1, Thermal Extraction o f Fluoride by Means o f a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver Skalar Methods, #335, Skalar Methods Manual AMDT-EP-26, Operation and Maintenance of the Skalar Segmented R ow Analyzer 14.0 REVISIONS____________________________________________________ Revision Number Reason for change Revision Date 003933 000083 5 9.3 Quality Assurance Unit Statement 003934 000084 Attachment D GLP Study Quality Assurance Statement Study Title: Single-dose Derm al A bsorption/Toxicity Study o f T-6053 in Rabbits Study Number: AMDT-022195.1 Name o f Auditor: KariRambo This study has been inspected by the Quality Assurance Unit as indicated in the following table. The findings were reported to the study director and management. Inspection Dates From la 10/14/95 11/03/95 Phase_______________ Final Report Date Inspection Reported to Management Study Director 11/03/95 11/03/95 QAU Auditor //' 3' ?5~ Date 003935 000085 l 9.4 Key Personnel Involved in the Study 003936 000086 3M Environmental Laboratory Key Personnel Thermal extraction followed by analysis using Orion ion analyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plummer Analysis of liver extracts using electrospray mass spectrometry: Jim Johnson Dave Christenson Thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode: Jim Johnson Deb Wright ` Rich Youngblom Deann Plummer Documentation and Reporting: Jim Johnson Rich Youngblom Quality Assurance Unit: Gale Van Buskirk Cynthia Weber Kari Rambo 003937 000087 9.11 Data 003938 00008S 9.11.1 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 003939 01)0089 Summary of Combustion Data - Liver AMDT-022195.1, H W I6329-137 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 20.2 + 7.9 10 mg/kg dose (T6053)** 18.5 + 8.7 125 mg/kg dose (T6053) 16.5 + 4.0 250 mg/kg dose (T6053) 17.2 + 6.6 Calculated as the mean of triplicate samples from each of three male and three female rabbits. **One outlier not included in mean. 003940 000090 RPT137L.XLS FC99 % ID rcvry SPK 63-1 95% SPK 63-2 96% SPK 126-1 81% SPK 126-2 82% LIVER BLK 1 LIVER BLK 2 F53442-1 F53442-2 F53442-3 F53464-1 F53464-2 F53464-3 F53455-1 F53455-2 F53455-3 liver blank-1 liver blank-2 liver spike-1 93% liver spike-2 90% liver spike-3 90% F53441-1 F53441-2 F53441-3 F53451-1 F53451-2 F53451-3 F53461-1 F53461-2 F53461-3 F53460-1 F53460-2 F53460-3 liver blank-1 liver blank-2 liver spike-1 90% liver spike-2 63% liver spike-3 97% liver spike-4 95% liver blank-3 F53447-1 F53447-2 F53447-3 F53452-1 F53452-2 F53452-3 LIVER BLK 1 LIVER BLK 2 LIVER SPK 1 92% LIVER SPK 2 100% Actual ppm Fin liver (w/w) 0.940 0.912 1.755 2.180 0.373 0.265 0.365 0.231 0.189 0.194 0.170 0.162 0.210 0.145 0.177 0.436 0.200 1.231 0.960 0.950 0.399 0.271 0.232 0.211 0.235 0.181 0.193 0.165 0.168 0.182 0.202 0.181 0.604 0.277 1.013 0.875 1.450 1.397 0.265 0.233 0.152 0.170 0.143 0.152 0.146 0.202 0.131 1.128 1.074 Average ppm F- Liver in liver burned (w/w) (grams) 0.1525 0.1593 0.1393 0.1136 0.1216 0.1145 0.1460 0.262 0.1132 0.1596 0.1240 0.175 0.1447 0.1223 0.1375 0.178 0.1558 0.1126 0.1099 0.1376 0.1140 0.1420 0.1440 0.1388 0.301 0.1435 0.1343 0.1398 0.209 0.1152 0.1413 0.1552 0.175 0.1277 0.1316 0.1230 0.188 0.1370 0.1265 0.1361 0.1326 0.1342 0.1087 0.1011 0.1028 0.1518 0.1007 0.185 0.1376 0.1161 0.1295 0.147 0.1159 0.1309 0.1580 0.1622 0.1240 0.1402 Whole Total Fliver in whole weight liver (grams) (WJ) 90.68 23.75 86.57 15.17 93.31 16.57 114.12 34.35 88.41 18.47 72.71 12.73 90.52 17.03 79.96 14.80 80.42 11.85 Page 1 Dosage (mg/kg) 0 0 0 0 0 0 10 10 10 SPIKE DATA FOR 63 AND 126 PPM FC95 COPIED FROM 6329136L ENDING QC DDW 4-5-95 003941 000091 FC99 ID F53463-1 F53463-2* F53463-3 F53444-1 F53444-2 F53444-3 F53448-1 F53448-2 F53448-3 F53446-1 F53446-2 F53446-3 F53453-1 F53453-2 F53453-3 F53458-1 F53458-2 F53458-3 F53443-1 F53443-2 F53443-3 F53450-1 F53450-2 F53450-3 F53467-1 F53467-2 F53467-3 F53439-1 F53439-2 F53439-3 F54445-1 F54445-2 F54445-3 F54459-1 F54459-2 F54459-3 Liver Blank-2 Liver Blank-3 Liver Spike-1 Liver Spike-2 Liver Spike-3 Liver Spike-4 Liver blank-4 F53449-1 F53449-2 F53449-3 % rcvry 83% 81% 90% 96% Actual ppm Fin liver (w/w) 0.696 1.869 0.170 0.629 0.068 0.163 0.188 0.146 0.184 0.187 0.140 0.183 0.246 0.145 0.285 0.503 0.160 0.190 0.175 0.175 0.194 0.164 0.171 0.153 0.222 0.203 0.178 0.119 0.139 0.390 0.200 0.151 0.242 0.140 0.150 0.142 0.432 0.148 1.096 1.149 0.932 1.306 0.277 0.248 0.173 0.163 Average Whole Total F- ppm F- Liver liver in whole in liver burned weight liver (w/w) (grams) (grams) (p-9) 0.1285 0.433 0.0152 81.40 35.28 0.1283 0.1190 0.287 0.1399 69.27 19.85 0.1348 0.1252 0.172 0.1199 72.33 12.47 0.1155 0.1169 0.170 0.1332 82.78 14.07 0.1164 0.1196 0.225 0.1479 94.51 21.30 0.1513 0.1133 0.284 0.1332 76.79 21.84 0.1001 0.1461 0.181 0.1238 76.45 13.87 0.1082 0.1303 0.163 0.1270 76.89 12.53 0.1072 0.1365 0.201 0.1087 77.56 15.61 0.1004 0.1366 0.216 0.1419 81.71 17.63 0.1328 0.1320 0.198 0.1261 95.81 18.97 0.1324 0.1479 0.144 0.1273 78.24 11.26 0.1177 0.1255 0.1328 0.1143 0.1063 0.1466 0.1110 0.1079 0.1284 0.194 0.1257 70.80 13.76 0.1495 Dosage (mg/kg) 10 10 10 125 125 125 125 125 125 250 250 250 250 * Outlier not included in average. Page 2 RPT137L.XLS 003942 OOOO^ RPT137L.XLS FC99 ID F53504-1 F53504-2 F53504-3 F53456-1 F53456-2 F53456-3 F53454-1 F53454-2 F53454-3 Liver Blk-1 Liver Blk-2 Liver Spike-1 Liver Spike-2 Liver Spike-3 Liver Spike-4 % rcvry 94% 108% 104% 94% Actual ppm Fin liver (w/w) 0.138 0.141 0.166 0.188 0.730 0.220 0.126 0.111 0.215 0.146 0.140 Average ppm Fin liver (w/w) 0.148 0.379 0.151 Liver burned (grams) 0.1274 0.1368 0.1116 0.1017 0.1398 0.1258 0.1304 0.1414 0.1245 0.1149 0.1029 0.1074 0.1378 0.1052 0.1200 Whole liver weight (grams) 82.31 77.10 93.04 Total Fin whole liver (pg) 12.22 29.25 14.02 Dosage (mg/kg) 250 Replacement for F54454 250 250 Replaced on Day 2 by F53504 Page 3 003943 000093 9.11.2 Raw data; analysis o f liver extracts using electrospray mass spectrometry. This data, although supportive, in the opinion o f the Study Director is not required to reach the conclusion stated in Final Report Section 6.0, and therefore is not discussed in detail. 003944 000094 " f *5 00394 003947 000097 9.11.3 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. This data, although supportive, in the opinion of the Study Director is not required to reach the conclusion stated in Final Report Section 6.0, and therefore is not discussed in detail. 003948 000098 RE:6329-137 LIUER SAMPLES HMDT 22195.2 Date of Analysis: 5 -2 -9 5 Analyst: DDLD The samples are burned in the Dohrman at 950 C using between 0.1 and 0.2 grams of the liuer. The gas is collected in 1.0 mL of 1:1 TISRB/Milli-Q w a te r then an additional 1 mL of 1:1 TISAB/Milli-Q is added to allow for sufficient uolume for Skalar analysis. The samples are then analyzed on a Skalar Segmented Flow Analyzer using the Ion Specific Electrode (ISE) Method. TISRB buffer is added to each sample as it proceeds through the system. The sample then goes through a heated mining coil before the potential between the ion selectiue electrode and the reference electrode is measured. The signal is amplified and related to the fluoride concentration. The instrument was calibrated in the ranges of 0.015 - 0.15 ppm and 0.15 - 1.50 ppm fluoride. The standard curue for the high range was plotted using the inuerse logarithm option. The standard curue for the low range is linear. All standards and samples were then calculated by the Skalar softw are using these curues. All results below 0.0001 ppm appear on the raw data as # .# # # # . A quality control standard was analyzed euery 10 samples to check for accuracy and drift. Raw data is taken from the appropriate calibrated range of the Skalar printout and summarized on an Excel spreadsheet. The final results are adjusted for the collection uolume and any subsequent dilutions. 003949 % '^ BEST COPY AVAILABLE SUMMARY of 6329-137 LIVER SAMPLES AMDT 22195.2 \sKiLU l i O / T O GROUP 1 Dose Level : 0 F53442-1 F53442-2 F53442-3 F53464-1 F53464-2 F53464-3 F53455-1 F53455-2 F53455-3 F53441-1 F53441-2 F53441-3 F53451-1 F53451-2 F53451-3 F53461-1 F53461-2 F53461-3 0.02 ND ND ND ND ND ND ND ND 0.03 0.02 ND ND ND ND ND ND ND GROUP 2 Dose Level : 10 mg/kg F53460-1 F53460-2 F53460-3 F53447-1 F53447-2 F53447-3 F53452-1 F53452-2 F53452-3 F53463-1 F53463-2 F53463-3 F53444-1 F53444-2 F53444-3 F53448-1 F53448-2 F53448-3 ND ND ND ND ND ND ND ND ND 0.06 ND ND ND ND ND ND ND ND F53446-1 F53446-2 F53446-3 F53453-1 F53453-2 F53453-3 F53458-1 ND ND 0.02 0.02 0.02 0.03 0.04 2.0 0.1460 0.33 2.0 0.1132 N D 2.0 0.1596 N D 2.0 0.1240 N D 2.0 0.1447 N D 2.0 0.1223 N D 2.0 0.1375 N D 2.0 0.1558 N D 2.0 0.1126 N D 2.0 0.1388 0.39 2.0 0.1435 0.23 2.0 0.1343 N D 2.0 0.1398 N D 2.0 0.1152 N D 2.0 0.1413 N D 2.0 0.1552 N D 2.0 0.1277 N D 2.0 0.1316 N D 90.6763 29.6 ND 90.6763 ND 90.6763 ND 86.5739 ND ND 86.5739 ND 86.5739 ND 93.3090 ND ND 93.3090 ND 93.3090 ND 114.1164 45.1 0.21 114.1164 26.1 114.1164 N D 88.4088 N D ND 88.4088 ND 88.4088 N D 72.7104 ND ND 72.7104 ND 72.7104 ND ND ND ND 23.7 ND ND 2.0 0.1230 N D 2.0 0.1370 N D 2.0 0.1265 N D 2.0 0.1007 N D 2.0 0.1376 N D 2.0 0.1161 N D 2.0 0.1295 N D 2.0 0.1159 N D 2.0 0.1309 N D 2.0 0.1285 0.95 2.0 0.1516 N D 2.0 0.1283 N D 2.0 0.1190 N D 2.0 0.1399 N D 2.0 0.1348 N D 2.0 0.1252 N D 2.0 0.1199 N D 2.0 0.1155 N D 90.5154 ND N D 90.5154 N D 90.5154 N D 79.9589 ND ND 79.9589 ND 79.9589 ND 80.4220 N D N D 80.4220 N D 80.4220 ND 81.3971 77.7 0.32 81.3971 ND 81.3971 ND 69.2675 ND ND 69.2675 ND 69.2675 ND 72.3334 ND ND 72.3334 ND 72.3334 ND ND ND ND 25.9 ND ND 2.0 0.1169 N D 2.0 0.1332 N D 2.0 0.1164 0.30 2.0 0.1196 0.29 2.0 0.1479 0.21 2.0 0.1513 0.37 2.0 0.1133 0.75 HWI-137L.SUM 82.7817 N D N D 82.7817 N D 82.7817 24.5 94.5134 27.2 0.29 94.5134 20.2 94.5134 34.9 76.7873 57.9 ND 27.4 003950 0001(ftp1 SUMMARY of 6329-137 LIVER SAMPLES AMDT 22195.2 GROUP 3 Dose Level: 125 mg/kg F53458-2 F53458-3 F53443-1 F53443-2 F53443-3 F53450-1 F53450-2 F53450-3 F53467-1 F53467-2 F53467-3 0.02 ND ND ND ND ND ND ND 0.02 ND ND GROUP4 Dose L evel: 250 mg/kg F53439-1 F53439-2 F53439-3 F53445-1 F53445-2 F53445-3 F53459-1 F53459-2 F53459-3 F53449-1 F53449-2 F53449-3 F53504-1 F53504-2 F53504-3 F53456-1 F53456-2 F53456-3 F53454-1 F53454-2 F53454-3 ND ND ND ND ND ND ND ND 0.02 ND ND ND ND ND ND ND 0.06 ND ND ND ND 2.0 0.1332 0.26 2.0 0.1001 N D 2.0 0.1461 N D 2.0 0.1238 N D 2.0 0.1082 N D 2.0 0.1303 N D 2.0 0.1270 N D 2.0 0.1072 N D 2.0 0.1365 0.23 2.0 0.1087 N D 2.0 0.1004 N D 0.34 76.7873 19.8 76.7873 N D 76.4455 N D N D 76.4455 N D 76.4455 N D 76.8935 N D N D 76.8935 N D 76.8935 N D 77.5604 17.6 N D 77.5604 N D 77.5604 N D 2.0 0.1366 N D 2.0 0.1419 N D 2.0 0.1328 N D 2.0 0.1320 N D 2.0 0.1261 N D 2.0 0.1479 N D 2.0 0.1479 N D 2.0 0.1273 N D 2.0 0.1177 0.39 2.0 0.1284 N D 2.0 0.1257 N D 2.0 0.1495 N D 2.0 0.1274 N D 2.0 0.1368 N D 2.0 0.1116 N D 2.0 0.1017 N D 2.0 0.1398 0.82 2.0 0.1258 N D 2.0 0.1304 N D 2.0 0.1414 N D 2.0 0.1245 N D 81.7065 ND ND 81.7065 ND 81.7065 ND 95.8121 ND ND 95.8121 ND 95.8121 ND 78.2415 ND N D 78.2415 N D 78.2415 30.3 70.7962 ND ND 70.7962 ND 70.7962 N D 82.3142 N D N D 82.3142 N D 82.3142 N D 77.1014 N D 0.27 77.1014 62.9 77.1014 N D 93.0439 N D N D 93.0439 N D 93.0439 N D 25.9 ND ND ND ND ND ND ND ND 21 ND BEST COPY AVAILABLE HWI-137LSUM 003951 000101 Page 2 1995-06-13 08:39 OutPut of : 950502A1 Operator : DDW Date of the Analysis : 1995-05-02 08:48 Analysis File Name : C:\SKALAR\DATA\HWIDATA\LIVERS\950502A1 HWI-137LXLS 1 Tracer 1.50 1.46 97% 2 Drift 1.50 1.48 99% 3 Wash 4 Standard 1 0.015 0.01 97% 5 Standard 2 0.03 0.03 108% 6 Standard 3 0.06 0.06 95% 7 Standard 4 0.09 0.09 101% 8 Standards 0.12 0.12 101% 9 Standard 6 0.15 0.15 103% BEST COPY AVAILABLE 10 Standard 7 0.30 0.29 95% 11 Standard 8 0.60 0.61 102% 12 Standard 9 1.20 1.23 103% 13 Standard 10 1.50 1.47 98% 14 Drift 1.50 1.51 100% 15 Wash 16 LIVER BLK 1 0.00 0.00 2.0 0.1216 i 17 LIVER BLK 2 0.00 2.0 0.1145 e 1189 2 CJ21 (cn2223 F53442-1 F53442-2 F53442-3 F53464-1 F53464-2 F53464-3 0.02 0.00 0.01 0.00 0.01 0.00 2.0 0.1460 0.33 90.6763 29.56 2.0 0.1132 0.06 90.6763 5.13 2.0 0.1596 0.12 90.6763 11.02 2.0 0.1240 0.06 86.5739 5.17 2.0 0.1447 0.13 86.5739 11.61 2.0 0.1223 0.04 86.5739 3.82 ^24 F53455-1 0.01 2.0 0.1375 0.10 93.3090 8.96 25 F53455-2 0.01 2.0 0.1558 0.07 93.3090 6.71 26 Drift 1.50 1.48 99% 27 28 Wash F53455-3 0.00 0.01 2.0 0.1126 0.19 93.3090 18.07 29 LIVER BLK 1 0.02 2.0 0.1099 0.41 K*31 30 LIVER BLK 2 LIVER SPK 1 0.00 0.08 2.0 0.1376 0.05 2.0 0.1140 1.40 0.004 63.00 0.15 0.16 105% rJ Page 1 jt/i .1 003952 000102 4_tc>r?l. 32 LIVER SPK 2 33 F53441-1 34 F53441-2 35 F53441-3 36 F53451-1 37 F53451-2 38 Drift 1.50 39 Wash 40 F53451-3 41 F53461-1 42 F53461-2 43 F53461-3 44 F53460-1 45 F53460-2 46 F53460-3 47 F53447-1 48 F53447-2 49 F53447-3 50 Drift 1.50 51 Wash 52 F53452-1 53 F53452-2 54 F53452-3 55 B L K 1 56 B L K 2 57 SPK 1 58 SPK 2 59 F53463-1 60 F53463-2 61 F53463-3 62 Drift 1.50 63 Wash 64 F53444-1 65 F53444-2 66 F53444-3 67 F53448-1 0.08 0.03 0.02 0.01 0.01 0.01 1.50 0.00 0.01 0.01 0.01 0.01 0.00 0.01 0.00 0.00 0.00 0.01 1.52 0.00 0.01 0.00 0.00 0.01 0.00 0.08 0.09 0.06 0.01 0.01 1.52 0.00 0.04 0.00 0.01 0.00 100% 101% 101% 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 003953 000103 Va HWI-137L.XLS 0.1420 0.1388 0.1435 0.1343 0.1398 0.1152 1.09 0.39 114.1164 45.05 0.23 114.1164 26.08 0.19 114.1164 22.09 0.10 88.4088 8.85 0.14 88.4088 11.97 0.004 63.00 0.15 0.16 103% 0.1413 0.1552 0.1277 0.1316 0.1230 0.1370 0.1265 0.1007 0.1376 0.1161 0.15 88.4088 13.26 0.15 72.7104 10.68 0.10 72.7104 6.95 0.08 72.7104 5.64 0.07 90.5154 6.48 0.16 90.5154 14.67 0.05 90.5154 4.87 0.07 79.9589 5.88 0.07 79.9589 5.35 0.10 79.9589 7.99 ST COPY AVAILABLE 0.1295 0.1159 0.1309 0.1580 0.1622 0.1240 0.1402 0.1285 0.1516 0.1283 0.12 80.4220 9.69 0.05 80.4220 4.16 0.06 80.4220 4.79 0.10 0.03 1.35 1.27 0.95 81.3971 77.66 0.15 81.3971 12.46 0.09 81.3971 7.36 0.004 0.004 63.00 63.00 0.15 0.15 i 0.17 110% 0.18 117% 0.1190 0.1399 0.1348 0.1252 0.61 0.00 0.11 0.05 Page 2 69.2675 69.2675 69.2675 72.3334 42.26 0.00 7.71 3.70 HWI-137LXLS 68 F53448-2 0.00 2.0 0.1199 0.00 72.3334 0.00 69 F53448-3 0.00 2.0 0.1155 0.07 72.3334 4.88 70 Drift 1.50 1.53 102% 71 Wash 0.00 72 inOut Wash 0.00 i BEST copy AVAILABLE Page 3 003954 HWI137L2.XLS 1995-06-13 09:41 OutPut o f : 950502B1 Operator : DDW Date of the Analysis : 1995-05-0212:41 Analysis File Name : C:\SKALAR\DATAVHWIDATAVLIVERS\950502B1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Oo f3 1 CD2 $3 (A4 25 26 ~ 27 X 28 X 29 wl 30 Tracer Drift Wash Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 Standard 9 Standard 10 Drift Wash F53446-1 F53446-2 F53446-3 F53453-1 F53453-2 F53453-3 F53458-1 F53458-2 F53458-3 F53443-1 Drift Wash F53443-2 F53443-3 F53450-1 1.50 1.50 0.015 0.03 0.06 0.09 0.12 0.15 0.30 0.60 1.20 1.50 1.50 1.50 1.49 1.49 0.00 0.01 0.03 0.06 0.09 0.12 0.15 0.29 0.61 1.24 1.47 1.51 0.00 0.01 0.01 0.02 0.02 0.02 0.03 0.04 0.02 0.01 0.01 1.48 0.00 0.02 0.01 0.02 99% 99% 95% 108% 96% 100% 102% 103% 95% 102% 103% 98% 101% 98% 2.0 0.1169 0.22 2.0 0.1332 0.15 2.0 0.1164 0.30 2.0 0.1196 0.29 2.0 0.1479 0.21 2.0 0.1513 0.37 2.0 0.1133 0.75 2.0 0.1332 0.26 2.0 0.1001 0.11 2.0 0.1461 0.16 2.0 0.1238 0.37 2.0 0.1082 0.15 2.0 0.1303 0.33 Page 1 00010 BEST COPY AVAILABLE 82.7817 82.7817 82.7817 94.5134 94.5134 94.5134 76.7873 76.7873 76.7873 76.4455 18.41 12.43 24.46 27.18 20.19 34.86 57.88 19.83 8.59 12.24 76.4455 76.4455 76.8935 28.16 11.16 25.14 c rcff> tO t J> O HWI137L2.XLS 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 C56 C57 ^61 62 63 64 65 66 F53450-2 F53450-3 F53467-1 F53467-2 F53467-3 F53439-1 F53439-2 Drift Wash F53439-3 F53445-1 F53445-2 F53445-3 F53459-1 F53459-2 F53459-3 BLK2 BLK 3 SPK 1 Drift Wash SPK 2 SPK 3 SPK 4 LIVER BLK F53449-1 F53449-2 F53449-3 F53504-1 F53504-2 F53504-3 Drift Wash F53456-1 F53456-2 F53456-3 1.50 1.50 1.50 0.01 0.00 0.02 0.00 0.00 0.00 0.01 1.52 0.00 0.03 0.01 0.00 0.01 0.01 0.00 0.02 0.02 0.00 0.06 1.48 0.00 0.06 0.07 0.08 0.00 0.01 0.00 0.00 0.00 0.00 0.00 1.46 0.00 0.00 0.06 0.01 101% 99% 97% 2.0 0.1270 0.16 76.8935 12.11 2.0 0.1072 0.09 76.8935 6.74 2.0 0.1365 0.23 77.5604 17.61 2.0 0.1087 0.08 77.5604 6.14 2.0 0.1004 0.09 77.5604 6.95 2.0 0.1366 0.06 81.7065 5.14 2.0 0.1419 0.13 81.7065 10.59 2.0 0.1328 0.49 81.7065 40.11 2.0 0.1320 0.21 95.8121 19.74 COPY AVAILABLE 2.0 0.1261 0.07 95.8121 6.23 2.0 0.1479 0.13 95.8121 12.44 2.0 0.1479 0.15 78.2415 11.53 2.0 0.1273 0.06 78.2415 4.55 2.0 0.1177 0.39 78.2415 30.31 2.0 0.1255 0.34 2.0 0.1328 0.00 2.0 0.1143 1.12 0.004 63.00 0.15 0.13 85% 2.0 0.1063 1.13 2.0 0.1466 0.94 2.0 0.1110 1.42 2.0 0.1079 0.03 0.004 0.004 0.004 63.00 63.00 63.00 0.15 0.15 0.15 2.0 0.1284 0.12 70.7962 8.27 2.0 0.1257 0.00 70.7962 0.00 2.0 0.1495 0.06 70.7962 4.45 2.0 0.1274 0.00 82.3142 0.00 2.0 0.1368 0.00 82.3142 0.00 2.0 0.1116 0.00 82.3142 0.00 0.12 80% 0.14 91% 0.16 104% 2.0 0.1017 0.01 77.1014 1.06 2.0 0.1398 0.82 77.1014 62.87 2.0 0.1258 0.12 77.1014 9.44 Page 2 *>01000 HWI137L2.XLS 67 F53454-1 68 F53454-2 69 F53454-3 70 BLK 1 71 BLK2 72 SPK 1 73 SPK2 74 Drift 1.50 75 Wash 76 SPK 3 77 SPK 4 78 Drift 1.50 79 Wash wt RunOut Wash 0.00 2.0 0.1304 0.00 93.0439 0.00 0.00 2.0 0.1414 0.00 93.0439 0.00 0.01 2.0 0.1245 0.08 93.0439 7.77 0.00 2.0 0.1149 0.02 0.00 2.0 0.1029 0.00 0.08 2.0 0.1074 1.56 0.10 2.0 0.1378 1.38 1.49 100% 0.004 0.004 63.00 63.00 0.15 0.15 0.00 0.19 2.0 0.1052 3.52 0.16 2.0 0.1200 2.73 1.52 101% 0.004 0.004 126.00 126.00 0.30 0.30 0.00 0.00 0.17 111% 0.19 126% 0.37 122% 0.33 108% 000107 Oo CO CO tn ^3 is BEST COPY AVAILABLE 1995-05-03 08:38 Output of : 950502A1 Software : version 6.1 cl990,93 Operator : DDW Date of the Analysis : 1995-05-02 08:48 Analysis File Name : C:\SKALAR\DATA\HWIDATA\LIVERS\950502A1 M b l o7.t.\ Fluoride 1.5 Calibration order = Inverse Logarithm Slope : s = #.##### [ * - C1 ] Result = 10 L s J X = corrected value of the sample cl = corrected value of the c o n c e ntration 1 s = Slope of the electrode a2 = al = aO = -0.00000 0.00066 -1.20681 Fluoride L Calibration order = 2 Correlation : r = 0.99926 Result = a2 * x 2 + al * x + aO a2 = al = aO = 0.00000 0.00024 -0.00641 Sampler Type Number Sample Time Wash Time Air Time Take up sPecial needle Height SA1000 1 50 sec. 120 sec. 1 sec. Single None 70 mm. Diluter needle Height dilution Factor dilution Volume Resample Dilution runs :80 :10 :2 .5 :1 :1 mm ml. User file : Reproces : No . TXT 003958 oooios 1995-05- 03 08:38 Output of : 950502A1 Fluoride 1.5 Path number Signal type Decolor system Number diLute Resample dil Threshold diG output Window event 3 Debubbled Yes 0 No No 4095 0 Off si sTandard Ignore s2 sTandard Ignore s3 sTandard Ignore s4 sTandard Ignore s5 sTandard Ignore s6 sTandard 0.150 s7 sTandard 0.300 s8 sTandard 0.600 s9 sTandard 1.200 slO sTandard 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed stArt ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula : output : ##. ### Fluoride Path number Signal type Decolor system Number diLute Resample dil Threshold diG output Window event :0 : Debubbled : No :0 : No : No : 4095 :0 Off 003959 000109 1995-05-03 08:38 OutPut of : 950502A1 si sTandard 0.015 s2 sTandard 0.030 s3 sTandard 0.060 s4 sTandard 0.090 s5 sTandard 0.120 s6 sTandard 0.150 s7 sTandard Ignore s8 sTandard Ignore s9 sTandard Ignore slO sTandard Ignore Order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed stArt ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula : c4:=c3 output : #.#### 0Q3960 OOOilO 1995-05-03 08:38 OutPut of : 950502A1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Ch Result F Time Ch Result F Time wt iw Initial Wash 3 0.062 65 1t Tracer 3 1.458 211 2d Drift 3 1.481 386 3w Wash 3 0.062 626 4 si Standard 1 3 0.071 738 5 s2 Standard 2 3 0.079 910 6 s3 Standard 3 3 0.091 1084 7 s4 Standard 4 3 0.112 1261 8 s5 Standard 5 3 0.133 1437 9 s6 Standard 6 3 0.154 1611 10 s7 Standard 7 3 0.285 1787 11 s8 Standard 8 3 0.611 1961 12 s9 Standard 9 3 1.234 2135 13 slO Standard 10 3 1.465 2311 14 d Drift 3 1.505 2485 15 w 16 u Wash 3 0.062 2721 LIVER BLK 1 3 0.061 A 2835 17 u LIVER BLK 2 3 0.060 3018 18 u F53442-1 3 0.075 3187 19 u F53442-2 3 0.066 3360 20 u F53442-3 3 0.069 3536 21 u 22 u F53464-1 3 0.066 F53464-2 3 0.069 3708 3879 23 u F53464-3 3 0.066 4063 24 u F53455-1 3 0.067 4237 25 u F53455-2 3 0.067 4411 26 d Drift 3 1.479 4587 27 w Wash 3 0.062 4812 28 u F53455-3 3 0.069 4925 29 u LIVER BLK 1 3 0.074 5109 30 u LIVER BLK 2 3 0.066 5291 31 u 32 u LIVER SPK 1 3 0.105 LIVER SPK 2 3 0.103 5463 5641 33 u 34 u F53441-1 3 0.077 F53441-2 3 0.072 5815 5987 35 u F53441-3 3 0.070 6167 36 u 37 u 38 d F53451-1 F53451-2 Drift 3 0.068 3 0.068 3 1.502 6335 6515 6689 39 w 40 u 41 u 42 u Wash F53451-3 F53461-1 F53461-2 3 0.062 3 0.069 3 0.069 3 0.067 6927 7029 7214 7386 43 u F53461-3 3 0.067 7563 44 u F53460-1 3 0.066 7740 45 u 46 u F53460-2 3 0.069 F53460-3 3 0.066 7908 8088 47 u F53447-1 3 0.066 8262 48 u F53447-2 3 0.067 8440 49 u 50 d F53447-3 3 0.067 Drift 3 1.517 8616 8790 51 w 52 u 53 u Wash F53452-1 F53452-2 3 0.062 3 0.068 3 0.066 9029 9135 9314 4 #.#### 4 0.7832 4 0.7926 4 #.#### 4 0.0145 4 0.0325 4 0.0567 4 0.0908 4 0.1213 4 0.1492 4 0.2708 4 0.4552 4 0.6976 4 0.7863 4 0.8025 4 #.#### 4 #.#### 4 #.#### 4 0.0238 4 0.0032 4 0.0097 4 0.0037 4 0.0097 4 0.0027 4 0.0066 4 0.0056 4 0.7919 4 #.#### 4 0.0109 4 0.0224 4 0.0034 4 0.0798 4 0.0776 4 0.0274 4 0.0164 4 0.0130 4 0.0070 4 0.0078 4 0.8013 4 #.#### 4 0.0106 4 0.0114 4 0.0061 4 0.0051 4 0.0044 4 0.0111 4 0.0034 4 0.0037 4 0.0046 4 0.0058 4 0.8078 4 #.#### 4 0.0078 4 0.0030 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 003961 000111 1995-05-03 08:38 OutPut of : 950502A1 Fluoride 1.5 Fluoride L PPM Pos Typ Ident Ch Result F Time 54 u 55 u 56 u 57 u 58 u 59 u 60 u 61 u 62 d 63 w 64 u 65 u 66 u 67 u 68 u 69 u 70 d 71 w wt rw F53452-3 3 0.066 BLK 1 3 0.068 BLK 2 3 0.066 SPK 1 3 0.107 SPK 2 3 0.110 F53463-1 3 0.094 F53463-2 3 0.070 F53463-3 3 0.067 Drift 3 1.519 Wash 3 0.062 F53444-1 3 0.081 F53444-2 3 0.062 F53444-3 3 0.068 F53448-1 3 0.066 F53448-2 3 0.064 F53448-3 3 0.066 Drift 3 1.531 Wash 3 0.062 RunOut Wash 3 0.062 9485 9661 9839 10016 10191 10367 10537 10715 10889 11126 11239 11425 11593 11765 11941 12118 12291 12529 12766 PPM Ch Result F Time 4 0.0039 4 0.0078 4 0.0025 4 0.0834 4 0.0887 4 0.0613 4 0.0116 4 0.0058 4 0.8085 4 #.#### 4 0.0363 4 #.#### 4 0.0075 4 0.0032 4 #.#### 4 0.0039 4 0.8135 4 #.#### 4 #.#### 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Page 2 of 2 003962 00011^ Calibration curve of 350SG2A : Fluoride 1 0039S3 OOOll'* ------ Calibration curve of 950502A1 : Fluoride 1.5 ----------! 1.8131529 i i! 0.0621156 i i Measured Order : Inverse Logarithm i 5894 4095 003964 000114 Raw data of 95502A1 : Fluoride 1.5 139 m Esc=Exit ; Fl=Help Crtl-P=Edit peaks ! 003965 000115 n Pattino4095 1 Rau data of 950502A1 Fluoride 1.5 -Tine: B O S B - ^fclue: 799 Delta: ri t i H3 I 5 i A' I\ \ I \- ! \J 1373 -------,-- T ine Ese=xi t i Fl=Help i Crtl-P=Edit peaks ,c, TO t i O-V Z.'2- I J 3998 003966 OOOIK ! 4095 1 I Raw data of 95G5G2A1 : Fluoride t.S ~4Q? i*rjMjSSI"64 2<r i! m M3 "V V2- \ i\ iS \ Z Z*\ z? A . A 3 l\ /\ 3998 T ine Esc=Exit I Fi=Help Crt1-P=Edii peaks ! S S -5 ^ -*-? 8823 003967 00 o r? Raw data of 35502R1 - Fluoride .S Esc=Exit ! Fl=Help Crtl-P-Edit peaks ' 00()ll> ZO Rau data of 9b0502Al : Fluoride i.b Ese=xit Fl=Help Crtl-P=EcJit peaks \ 00011-> Raw data of 950502A1 : Fluoride 1.5 003970 oooiao 1995-05-03 09:05 OutPut of : 950502B1 Software : ve rsion 6.1 c l 9 9 0 ,93 Operator DDW Date of the Analysis 1995-05-02 12:41 Analysis File Name C:\SKALAR\DATA\HWIDATA\LIVERS\950502B1 012 \ QTZ''in ' Fluoride 1.5 Calibration order = Inverse Logarithm Slope : s = *.*#### r x - cl I ------- Result = 10 L s -* x = corrected value of the sample C1 = corrected value of the co nc entration 1 s = Slope of the electrode a2 = al = aO = -0.00000 0.00068 -1.24604 Fluoride L Calibration order = 2 Correlation : r = 0.99932 Result = a2 * x a + al * x + aO a2 = al = aO = 0.00000 0.00021 -0.00305 Sampler Type Number Sample Time Wash Time Air Time Take up special needle Height SA1000 1 50 sec. 120 sec. 1 sec. Single None 70 mm. Diluter needle Height dilution Factor dilution Volume Resample Dilution runs :80 :10 :2.5 :1 :1 mm ml. User file : Reproces : No . TXT 003971 oooi~i 1995-05-03 09:05 Output of : 950502B1 Fluoride 1. 5 Path number Signal type Decolor system Number diLute Resample dil Threshold diG output Window event 3 Debubbled Yes 0 No No 4095 0 Off si sTandard Ignore s2 sTandard Ignore s3 sTandard Ignore s4 sTandard Ignore s5 sTandard Ignore s6 sTandard 0.150 s7 sTandard 0.300 s8 sTandard 0.600 s9 sTandard 1.200 slO sTandard 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed stArt ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula : output : ##. ### Fluoride L Path number Signal type Decolor system Number diLute Resample dil Threshold diG output Window event :0 : Debubbled : No :0 : No No : 4095 :0 : Off 003972 000122 o ? Vl > 1995-05-03 09:05 Output of : 950502B1 si sTandard 0.015 s2 sTandard 0.030 s3 sTandard 0.060 s4 sTandard 0.090 s5 sTandard 0.120 s6 sTandard 0.150 s7 sTandard Ignore S3 sTandard Ignore s9 sTandard Ignore slO sTandard Ignore Order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed stArt ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula : c4 :=c3 output : #. #### C03973 OOOl^.'i "2 H 1995-05-03 09:05 OutPut of : 950502B1 Fluoride 1.5 Fluoride L PPM Pos Typ Ident Ch Result F Time wt iw Initial Wash 3 0.057 1t Tracer 3 1.486 2d Drift 3 1.491 3w Wash 3 0.057 4 si Standard 1 3 0.064 5 s2 Standard 2 3 0.073 6 S3 Standard 3 3 0.087 7 s4 Standard 4 3 0.107 8 s5 Standard 5 3 0.131 9 s6 Standard 6 3 0.154 10 s7 Standard 7 3 0.286 11 s8 Standard 8 3 0.609 12 s9 Standard 9 3 1.235 13 slO Standard 10 3 1.467 14 d Drift 3 1.514 15 w Wash 3 0.057 16 u F53446-1 3 0.064 17 u F53446-2 3 0.062 18 u F53446-3 3 0.066 19 u F53453-1 3 0.066 20 u F53453-2 3 0.065 21 u F53453-3 3 0.071 22 u F53458-1 3 0.079 23 u F53458-2 3 0.066 24 u F53458-3 3 0.060 25 u F53443-1 3 0.063 26 d Drift 3 1.476 27 w Wash 3 0.057 28 u F53443-2 3 0.068 29 u F53443-3 3 0.062 30 u F53450-1 3 0.068 31 u F53450-2 3 0.062 32 u F53450-3 3 0.060 33 u F53467-1 3 0.065 34 u F53467-2 3 0.060 35 u F53467-3 3 0.060 36 u F53439-1 3 0.060 37 u F53439-2 3 0.062 38 d Drift 3 1.515 39 w Wash 3 0.057 40 u F53439-3 3 0.073 41 u F53445-1 3 0.064 42 u F53445-2 3 0.060 43 u F53445-3 3 0.062 44 u F53459-1 3 0.063 45 u F53459-2 3 0.060 46 u F53459-3 3 0.068 47 u BLK 2 3 0.068 48 u BLK 3 3 0.056 49 u SPK 1 3 0.091 50 d Drift 3 1.483 51 w Wash 3 0.057 52 u SPK 2 3 0.089 53 u SPK 3 3 0.094 65 211 387 628 737 913 1089 1264 1440 1613 1788 1964 2138 2312 2488 2723 2829 3004 3191 3360 3539 3713 3891 4065 4237 4405 4587 4826 4941 5113 5289 5463 5636 5817 5987 6162 6337 6516 6688 6927 7038 7214 7384 7564 7738 7918 8090 8266 8436 8616 8790 8930 9141 9317 PPM Ch Result F Time 4 #.#### 4 0.8506 4 0.8536 4 #.#### 4 0.0143 4 0.0324 4 0.0576 4 0.0896 4 0.1221 4 0.1490 4 0.2673 4 0.4541 4 0.7300 4 0.8407 4 0.8662 4 #.#### 4 0.0130 4 0.0100 4 0.0172 4 0.0172 4 0.0158 4 0.0279 4 0.0427 4 0.0172 4 0.0056 4 0.0117 4 0.8457 4 #.#### 4 0.0228 4 0.0079 4 0.0213 4 0.0100 4 0.0047 4 0.0155 4 0.0043 4 0.0045 4 0.0043 4 0.0092 4 0.8668 4 #.#### 4 0.0326 4 0.0136 4 0.0041 4 0.0096 4 0.0109 4 0.0037 4 0.0228 4 0.0215 4 #.#### 4 0.0640 4 0.8492 4 #.#### 4 0.0603 4 0.0688 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 003974 00014 1995-05-03 09:05 OUtPut of : 950502B1 Fluoride 1.5 Fluoride L PPM Pos Typ Ident Ch Result F Time 54 u 55 u 56 u 57 u 58 u 59 u 60 u 61 u 62 d 63 w 64 u 65 u 66 u 67 u 68 u 69 u 70 u 71 u 72 u 73 u 74 d 75 w 76 u 77 u 78 d 79 w wt rw SPK 4 3 0.100 LIVER BLK 3 0.059 F53449-1 3 0.061 F53449-2 3 0.056 F53449-3 3 0.060 F53504-1 3 0.056 F53504-2 3 0.054 F53504-3 3 0.057 Drift 3 1.459 Wash 3 0.057 F53456-1 3 0.058 F53456-2 3 0.087 F53456-3 3 0.061 F53454-1 3 0.057 F53454-2 3 0.057 F53454-3 3 0.060 BLK 1 3 0.058 BLK 2 3 0.057 SPK 1 3 0.103 SPK 2 3 0.111 Drift 3 1.494 Wash 3 0.057 SPK 3 3 0.185 SPK 4 3 0.164 Drift 3 1.518 Wash 3 0.057 Runout Wash 3 0.057 9493 9665 9841 10013 10189 10361 10539 10711 10889 11044 11237 11413 11593 11763 11933 12108 12288 12463 12641 12817 12991 13222 13345 13517 13691 13890 14166 PPM Ch Result F Time 4 0.0786 4 0.0018 4 0.0075 4 #.#### 4 0.0047 4 #.#### 4 #.#### 4 #.#### 4 0.8366 4 #.#### 4 0.0007 4 0.0570 4 0.0077 4 #.#### 4 #.#### 4 0.0052 4 0.0009 4 #.#### 4 0.0839 4 0.0954 4 0.8550 4 #.#### 4 0.1815 4 0.1599 4 0.8686 4 #.#### 4 #.#### 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 003975 OOOJLSW ! 0.196063 Calibration curve of 95G502B1 : Fluoride L Channel 0 Measured- Concent ration: -0.003050 Qrder : 2 0.S9932 003976 00013 1.7637298 Calibration curve of 95G5G28 : Fluoride 1.5 " .... .--I I i /+ V C II tl c 0 Q /f Y i0.0567536 ; 0 ii Measured Order ' Inverse Legarithr. 8070 4035 003977 000137 7S 4095 Raw data of 35502B1 : Fluoride 1.5 Esc=xit ! Fi=Heip 5 Crti-P=tdit peaks i 003978 0001-S ! -1095 taw data o f 95050281 : Fluoride 1.5 T 4W K m m -y7r-m ^m eT il'? '1 il U a lu e A iA\ / !! 1 J) 1j t J V, ! \! Hs % \i \j \% -ve. 1373 T ine sc=Exit I F=HeIp ! Crtl-P=Edit peaks i 773998 003979 0001^9 30 ! 4095 Raw da la of 95050281 Fluoride 1.5 Si P3i jn . v\ \ri %X T* V v._./ "'--.,1 *'* -3`2" ^ -JH -35 3998 T iwe sc=Exit ! Fi=Help I Crtl-P=Edit peaks 77 i 6623 003980 OOOl.'iO "1095 1 Raw data of 950502B1 Fluoride 1 5 RRRP, ikiSiWI 3620 IfeliFH 64 6 \ U a 1. u e HO y\ l l <u- q(- < 1 !\ j ^ 45 A A 1* / ' V. 0 6823 Tiiie sc=Exit ! Fl=Hel,o 1 Crtl-P=Edit peaks ! 5^ i\ i{ i)\ ^ f\ 9248 0039S1 0 0 0 1:3 1 ! 4095 Raw data of 950S02B1 : Fluoride 1.5 & i! r-Ib o II !I \ ,! \ t. aK 0 9248 % * 5? ufi K* Time Lse=Lxii i rl=HeIp 1 Crtl-P=Edit peaks ts w: (/I i1p,72 0039S2 000132 4095 1 Raw data of 3S050281 : Fiuoride 1.5 ii 3 8 j 4 (g* ( A < o v> /' 11 i/\ li. , '-J t un yfj \ i ! 11873 T iiie Esc=E;<it i Fl=Help ! Crtl-P=Edit peaks i -,,i 14498 0039S3 000133