Document GEQp32jj5LXb5RemELd26wyq

3M Environmental Laboratory WX&OX7S Final Report- Analytical Study Single-Dose Dermal Absorption/Toxicity Study of T-6051 and T-6054 in Rabbits In-Vivo Study Reference Number: HWI#6329-133 Study Number: AMDT-013195.1 Test Substance: FC-129 (T-6051 and T-6054) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology & 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: ames D. Study Dir " A * / ? * ____ Completion Date 000269 1.0 SUMMARY Samples of liver from rabbits administered FC-129 (T-6054) or FC-129 treated fabric (T-6051) were analyzed at 28 days post dermal administration for total organic fluorine and perfluorooctanesulfonate. The results show that for the highest liquid formulation dose group (12.8 mg/kg) there is on the average about 0.2% of the dose in whole liver at 28 days. Thus, dermal administration of FC-129 at higher levels results in some dermal absorption. 2.0 INTRODUCTION______________________ __________________________ Two studies were performed on FC-129. A pharmacokinetic study (HWI#6329138) and this dermal absorption study (HWI #6329-133). The pharmacokinetic study showed that perfluorooctanesulfonate is a useful marker to assess the dermal absorption of FC-129. Liver, serum, and other tissues were available for analysis by combustion for total organic fluorine and electrospray mass spectrometry for analysis of specific molecules such as perfluorooctanesulfonate. By obtaining and then analyzing data from rabbits at 28 days post dermal dose, information for the assessment of the extent of dermal absorption of FC-129 is provided in this study. 3.0 TEST MATERIALS______________________________________________ 3.1 Test, Control, and Reference Substances and M atrices 3.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 3.1.2 Analytical Reference M atrix: Bovine liver and bovine serum 3.1.3 Analytical Control Substance: None 3.1.4 Analytical Control M atrix: Bovine liver and bovine serum 3.2 Source of Materials: 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. 000270 2 3.5 Storage Conditions for Test Materials: Room temperature for FC-95. For biological samples the storage is -2010 C. 3.6 Disposition of Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required for studies longer than 28 days. 4.0 EXPERIMENTAL - Overview___________________________________ The tissues from animals dosed as described (HWI#6329-133), were available for analysis for fluorine compounds. At the discretion of the Study Director, a series of analytical tests could be performed. The screening for fluoride in liver via combustion was the most likely analysis to present definitive data for absorption. Other available tests were electrospray mass spectroscopy and gas chromatography/mass spectrometry for metabolites. Liver samples were analyzed by both combustion and electrospray. Data were then analyzed to assess the extent of dermal absorption. 5.0 EXPERIMENTAL - METHODS____________________________________ 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 are attached. The level of total organic fluorine in whole liver for the control, 0.128, 1.28, and fabric doses are below the practical quantitation limit for this method. Just using meter readings and extrapolating from the standard curve, the values are on the order of 16 ug/whole liver. The 12.8 mg/kg dermal dose however, results in detectable amounts of organic fluoride in whole liver at 28 days 000271 3 post dose. The values range from below the practical quantitation limit for one of the 6 rabbits (F52895) to 75 ug/whole liver for rabbit F52889. The average is 45 ug/whole liver with a standard deviation of 21 ug. Electrospray mass spectrometry data are in agreement with the combustion data; there is very little perfluorooctanesulfonate in the groups other than the 12.8 mg/kg group. Small detectable amounts are observed in the 1.28 mg/kg and fabric groups; however, these are estimated to be on the order of 17 ug/whole liver or less. For the 12.8 mg/kg dose group, perfluorooctanesulfonate is detected in all rabbit liver samples at 28 days post dermal dose. The amounts are estimated to range from 25 to 85 ug/whole liver (mean of 48 ug/whole liver). The rabbit that showed 75 ug/whole liver of total organic fluorine for combustion analysis (F52889), had 56 ug/whole liver perfluorooctanesulfonate. From the pharmacokinetic study on FC-129 (HWI#6329-138), it is known that a good portion of the intravenous dose will be biotransformed to perfluorooctanesulfonate. It is known that the half-life of perfluorooctanesulfonate in rabbits is >1 month. Thus, if FC-129 is dermally absorbed a portion of it will appear in liver at 28 days as perfluorooctanesulfonate. Fifty ug perfluorooctanesulfonate/whole liver is 0.2% of the dose for the 12.8 mg/kg rabbits assuming a body weight of 2 kg and expressing the dose in potassium perfluorooctanesulfonate equivalents (FC-95). After an intravenous dose of 12.8 mg/kg a rabbit had 1.05% of the dose in liver at 48 hours. For comparison, 1.05% with a biological half-life of 30 days would be approximately 0.5% of the dose at day 28. Thus, estimated levels after an intravenous dose of 12.8 mg/kg would be 0.5% of the dose in whole liver and estimated levels after dermal administration of 12.8 mg/kg would be 0.2% of the dose in whole liver if the levels are compared at 28 days. Other data was collected using 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 Have Affected the Quality of the Data: The problem with this analysis is that the extent of biotransformation of the rest of the fluorinated compounds in the liver at 48 hours in the pharmacokinetic study (HWI#6329-138) to perfluorooctanesulfonate is not known. There could be considerable biotransformation of the several percent of dose that fluorinated molecules other than perfluorooctanesulfonate represent. However, the 1.05% of the dose observed at 48 hours still indicates that perfluorooctanesulfonate is a sensitive marker to assess biotransformation of this compound. At 28 days, the value could 000272 4 be somewhat higher than the above estimate of 0.5% of the dose due to delayed metabolism. If this were true, the value for dermal absorption has by definition (since it is measured at 28 days) a built in compensation for this delay and the value of 0.2% of the dose after dermal administration is being compared with a percentage of dose from the intravenous dose that is too low. 7i0 CONCLUSION____________________________________ There is evidence of dermal absorption in rabbits of FC-129 after dermal administration of a 12.8 mg/kg dose. 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#6329-133: "Single-Dose Dermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits" (Protocol type TP3016.AB for dosing of animals, tissue collection, etc.) 9.1.2 Analytical protocol AMDT-013195.1 9.2 Signed Reports from Individual Scientists: None 9.3 Quality Assurance Unit Statement: See attached 9.4 Key Personnel Involved in the Study: See attached 9.5 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 000273 5 9.9 Test Methods: See Protocol AMDT-013195.1 9.10 Instrum ent Settings: See methods 9.11 Data: See attached. 9.11.1 Summary and raw data; ug F* in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 9.11.2 Summary and raw data; analysis o f liver extracts using electrospray mass spectrometry. 9.11.3 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Skalar segmented flow analyzer with ion selective electrode. 000274 6 9.1.1 Protocol and Final Report: HWI#6329-133: "Single-Dose Dermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits" (Protocol type TP3016.AB for dosing of animals, tissue collection, etc.) 000275 HAZLETOfM WISCONSIN P O S T O F F I C E BOX 7545 MADISON, Wl 53707-75 4 5 Sponsor: 3M St. Paul, Minnesota 4 C O R N IN G Compnny FINAL REPORT Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits Author: Steven M. Glaza Study Completion Date: June 16, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-133 Page 1 of 46 P h o n e 608-241-4471 EXPRESS- MAI L DELIVERY 3o 3^ 0J 1I NXiIl M* S M A M 000276 B LVD Fax 608-241-72 2 7 MADISON. Wl 53704 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 Studv Director Manaaement 12/08/94 12/28/94 01/09/95 01/30/95 03/17/95 03/17/95 06/14/95 06/16/95 12/09/94 12/28/94 01/09/95 01/30/95 03/21/95 03/21/95 06/15/95 06/16/95 Protocol Review Dose Administration Protocol Amendment Protocol Amendment Data/Report Review Data Review Report Rereview Report Rereview 12/09/94 12/28/94 01/09/95 01/30/95 03/21/95 03/21/95 06/15/95 06/16/95 01/10/95 01/10/95 02/10/95 02/10/95 04/10/95 04/10/95 07/10/95 07/10/95 Ia Cecilia M. Darner Representative, Quality Assurance Unit Date 000277 Page 3 of 46 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits HWI 6329-133 Test Materials 1. T-6054 2. T-6051 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. 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.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Study Director Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Study Location Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Study Timetable Study Initiation Date Experimental (In-life) Start Date In-life End Date Experimental Termination Date Study Completion Date December 13, 1994 December 28, 1994 January 25, 1995 June 16, 1995 June 16, 1995 000278 Page 4 of 46 KEY PERSONNEL HWI 6329-133 Acute Toxicology Laboratory Animal Medicine Steven M. Glaza Study Director Manager Cindy J. Cary, DVM Diplomate, ACLAM Supervisor Francis (Bud) W. McDonald Study Coordinator Anatomical Pathology Patricia Padgham In-life Supervisor Thomas E. Palmer, PhD Anatomical Pathologist Rose M. Bridge Report Supervisor Quality Assurance Sherry R. W. Petsel Manager Jack Serfort/ Deborah L. Pirkel Supervisors Necropsy Anne Mosher Supervisor Pathology Data 000279 Page 5 of 46 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 Deviation Protocol TP3016.AB Protocol Amendment No. 1 Protocol Amendment No. 2 HWI 6329-133 Page 2 3 4 6 8 8 8 9 10 13 13 14 14 15 16 18 20 25 27 29 30 31 42 45 000280 Page 6 of 46 SUMMARY HWI 6329-133 This study was done to assess the systemic absorption/toxicity and relative skin irritancy of T-6054 and T-6051 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 Femal 1 (Control) 2 3 4 5 Sterile water T-6054 T-6054 T-6054 T-6051 Oa 0.128 1.28 12.8 b 3 3 3 3 3 3, 3 3 3 3 a Administered at a dose volume of 2.0 mL/kg. b Administered as a 10-cm x 10-cm section of test material (fabric). 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 in-life initiation (Day 1), approximately 24-hours postdose (Day 2), on Days 4, 8, 15, and 22. In addition, at the time of necropsy on Day 29, approximately 20 mL of blood was obtained from each animal. All samples were centrifuged and separated into serum and cellular fractions. All animals were euthanized at termination of the in-life phase and necropsied. The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from one male and one female in each group were collected at necropsy and weighed (volume only determined for bile). The blood samples (serum and cellular fractions), livers, bile, dermal application sites, and kidneys were sent frozen to the Sponsor after termination of the in-1ife phase. 000281 Page 7 of 46 HWI 6329-133 Application of T-6054 and T-6051 did not result in any test material-related changes in body weight gain or macroscopic findings at necropsy. All animals appeared clinically normal throughout the study with the exception of one female animal treated with T-6054 at 1.28 mg/kg that exhibited weakened hind limbs the last 22 days of study. This animal was also noted as having small feces on Day 8. These findings are considered to be due to an injury incurred during the sample collection procedures and are not considered to be test material-related. The control material and test material T-6051 did not produce any dermal irritation. No dermal irritation was observed as a result of T-6054 at a dose level of 0.128 or 1.28 mg/kg. T-6054 produced very slight dermal irritation in five animals at the 12.8 mg/kg dose level. 000282 Page 8 of 46 OBJECTIVE HWI 6329-133 The objective of this study was to assess the systemic toxicity/absorption and relative skin irritancy of test materials 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 mixtures prepared for the Groups 2, 3, and 4 animals for concentration, homogeneity/solubility, and stability was not conducted and the original test material usage log can not be located although a copy is retained in the study file. 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 materials were identified and described as follows: Identification Physical Description T-6054 T-6051 Amber liquid White plastic sheets The control material was Sterile Water for Injection, USP (Abbott Laboratories, Lot No. 86-748-DM-02; Exp. March 1, 1996), and was described as a clear, colorless liquid. Purity and Stability The Sponsor assumes responsibility for test material purity and stability determinations (including under test conditions). Analysis of the test material mixtures prepared for the Groups 2, 3, and 4 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 materials were stored at room temperature. The control material was stored refrigerated. A reserve sample of each test and control material was 000283 Page 9 of 46 HWI 6329-133 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 phase according to HWI SOP. Any remaining control material is retained for other testing and will not be discarded after issuance of the final report. Safety Precautions The test and control material handling procedures were according to HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPF strain were procured from HRP, Inc., Kalamazoo, MI, on December 14, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing After receipt, the animals were acclimated for a period of at least 7 days. During acclimation and throughout the study, the animals were individually housed in screen-bottom stainless steel cages in temperature- and humiditycontrolled quarters. Environmental controls for the animal room were set to maintain a temperature of 19 to 23C, a relative humidity of 50% 20%, and a 12-hour 1ight/12-hour dark lighting cycle. In cases where variations from these conditions existed, they were documented and considered to have had no adverse effect on the study outcome. Animal Diet The animals were provided access to water ad 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 000284 Page 10 of 46 HWI 6329-133 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 body weights for each group of each sex were not statistically different at the 5% probability level. One female animal (No. F52890) was replaced in the study prior to treatment due to poor health. This animal was replaced with another female (No. F52877). Study Design Animals weighing from 2,157 to 2,508 g at initiation of treatment were placed into the following study groups: GrouD Dose Level Number of Animals Test Material (mq/kq) Males Females 1 (Control) 2 3 4 5 Sterile water T-6054 T-6054 T-6054 T-6051 Oa 0.128 1.28 12.8 b 3 3 3 3 3 3 3 3 3 3 a Administered at a dose volume of 2.0 mL/kg. b Administered as a 10-cm1 x 10-cm section of test material (fabric). 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 test sites (intact skin) were 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. 000285 Page 11 of 46 HWI 6329-133 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 (sterile water for injection) was applied evenly to the test site at a rate of approximately 0.05 mL/cm2. Groups 2, 3, and 4 . For the Groups 2, 3, and 4 animals (0.128, 1.28, 12.8 mg/kg, respectively), the test material (T-6054) was mixed with sterile water for injection to a concentration of 99, 990, and 9,920 mg/mL, respectively, and applied at a dose volume of 0.01 mL/kg. The mixtures were stored at room temperature until administered. An individual dose of the respective test material mixture was calculated for each animal based on its body weight on the day of treatment. For all three groups, the area of exposure was 4 cm2 and the approximate rate of application was 0.006 mL/cm2. Group 5 . The test material (T-6051) was applied to each animal's skin as a 10-cm x 10-cm section of material that was moistened with distilled water. 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. The only exception to this was the Day 8 erythema score for one female animal (No. F52889) in Group 4 was inadvertently not recorded. 000286 Page 12 of 46 Sample Collections HWI 6329-133 Blood samples (approximately 4 mL) were collected from a marginal ear vein of all animals before experimental initiation (Day 1). Subsequent collection of blood was conducted approximately 24-hours postdose (Day 2), and on Days 4, 8, 15, and 22. In addition, at the time of necropsy on Day 29, approximately 20 mL of blood was obtained from the posterior vena cava of each animal. All samples were centrifuged and separated into serum and cellular fractions. These samples were then stored in a freezer set to maintain a temperature of -20C 10C until shipped to the Sponsor. Pathology At termination of the experimental phase (Day 29), animals were anesthetized with sodium pentobarbital, bled via the posterior vena cava, exsanguinated, and necropsied in random order. The sites of test and control material application were washed with lukewarm tap water before the necropsy procedure. All animals were subjected to an abbreviated gross necropsy examination and any abnormalities were recorded. The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from the first male and female in each group were collected. The tissue samples were weighed (volume only determined for bile) and immediately placed on dry ice, then placed in a freezer set to maintain a temperature of -20C 10C. After necropsy, the animals were discarded. Shipment of Blood. Bile, and Tissues After experimental termination, the blood samples (serum and cellular fractions), livers, bile, dermal application sites, and kidneys were sent frozen (on dry ice) to the Sponsor (James D. Johnson, 3M E.E. & P.C., Bldg. 2-3E-09, 935 Bush Avenue, St. Paul, MN, 55106), along with their corresponding weights or volumes. The Sponsor is responsible for the retention and disposition of the samples. HWI does not accept any responsibility for the analysis of the tissue samples collected in this study nor are these results presented in this report. Statistical Analyses No statistical analyses were required by the protocol. Location of Raw Data. Records, and Final Report The raw data, records, and an original signed copy of the final report will be retained in the archives of HWI in accordance with HWI SOP. 000287 Page 13 of 46 Body Weights RESULTS HWI 6329-133 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 exception of one female animal (No. F52900) treated with T-6054 at 1.28 mg/kg that exhibited weakened hind limbs during the last 22 days of study. This animal also had small feces on Day 8. These findings are considered to be due to an injury incurred during the sample collection procedures and are not considered to be test material-related. Dermal Irritation Individual dermal irritation scores are in Table 3. The control material and test material T-6051 produced no dermal irritation. No dermal irritation was observed in the animals treated with T-6054 at a dose level of 0.128 or 1.28 mg/kg. T-6054 produced slight to moderate erythema reactions at Days 2 and 4 only in five animals at the 12.8 mg/kg dose level. Pathology Individual animal pathology comments are presented in Table 4. There were no lesions observed in any of the animals. Page 15 contains a pathology report by the study pathologist. DISCUSSION The acute systemic absorption/toxicity and relative skin irritancy of T-6054 and T-6051 were evaluated in male and female albino rabbits when administered as a single dermal application. Application of the these materials did not result in any test material-related effects on in-life clinical findings, body weight gain or macroscopic findings at necropsy. The control material and test material T-6051 did not produce any dermal irritation. No dermal irritation was observed with T-6054 applied at a dose level of 0.128 or 1.28 mg/kg. T-6054 produced very slight dermal irritation in five animals at the 12.8 mg/kg dose level. 000288 Page 14 of 46 SIGNATURE HWI 6329-133 Steven M. Glaza Study Director Acute Toxicology Q 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). 000289 Page 15 of 46 PATHOLOGY REPORT HWI 6329-133 There were six rabbits (three males and three females) each from five dose levels euthanized and necropsied at the termination of the study. The test material, dose level, day of death, and gross observations recorded for each animal are in the Individual Pathology Comments that follow this report. At necropsy, there were no visible lesions in any of the animals. The liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from the first male and female in each group were collected. The tissue samples were weighed (volume only determined for bile), frozen, and sent to the Sponsor. After necropsy, the animals were discarded. (6329-133.slh) 031095 (r/-vr Date 000290 Page 16 of 46 Table 1 Individual and Mean Body Weights (g) HWI 6329-133 Animal Number Male_________________ Random ization Dav Dav -9 1 29 _______________ Female Random Animal ization Dav Number Dav -9 1 29 Group 1 (Control) - Sterile Water for Injection (0 ma/ka) F52885 F52873 F52898 2,292 2,204 2,169 2,483 2,321 2,366 2,853 2,745 2,745 F52967 F52878 F52883 2,204 2,331 2,274 2,368 2,475 2,499 2,863 2,978 2,985 Mean 2,222 2,390 2,781 2,270 2,447 2,942 F52887 F52893 F52897 Mean 2,272 2,204 2,338 2,271 Group 2 - T-6054 (0.128 ma/kal 2,235 2,260 2,423 2,621 2,603 2,913 F52901 F52876 F52882 2,281 2,270 2,127 2,306 2,712 2,226 2,340 2,358 2,323 2,340 2,839 2,842 2,814 2,832 F52965 F52891 F52892 Mean 2,248 2,077 2,261 2,195 Group 3 - T-6054 (1.28 ma/ka1 2,369 2,383 2,333 2,966 2,757 2,614 F52884 F52900 F52968 2,351 2,210 2,166 2,362 2,779 2,242 2,508 2,460 2,289 2,928 2,588 2,822 2,419 . 2,779 F52886 F52880 F52899 Mean 2,286 2,161 2,353 2,267 Group 4 - T-6054 (12.8 mq/kq) 2,417 2,219 2,393 2,793 2,525 2,741 F52889 F52894 F52895 2,292 2,160 2,219 2,343 2,686 2,224 2,357 2,489 2,462 2,436 2,853 2,837 2,791 2,827 000291 Animal Number F52879 F52963 F52874 Mean Page 17 of 46 HWI 6329-133 Table 1 (Continued) Individual and Mean Body Weights (g) Male_________________ Random ization Dav Dav -9 1 29 _______________ Female Random Animal ization Number Dav -9 Day 1 29 GrouD 5 - T- 6051 n o -cm X 10-cm Sectionl 2,278 2,173 2,048 2,398 2,258 2,265 2,923 2,622 2,495 F52877a F52888 F52966 2,021 2,191 2,264 2,157 2,273 2,322 2,782 2,752 2,792 2,166 2,307 2,680 2,159 2,251 2,775 a Animal No. F52890 was originally selected by the randomization program for use in the study but was replaced with No. F52877 due to poor health. 000292 Page 18 of 46 HWI 6329-133 Table 2 Individual Clinical Signs Animal Sex Number Observation 1-4 Hours Day . (Dav 1) 2 - 7 8 9 - 2 9 Group 1 (Control) - Sterile Water for In.iection (0 ma/kal Male F52885 F52873 F52898 Appeared normal Appeared normal Appeared normal /// / // / /// Female F52967 F52878 F52883 Appeared normal Appeared normal Appeared normal // / /// / /// Group 2 - T-6054 (0.128 ma/kal Male F52887 F52893 F52897 Appeared normal Appeared normal Appeared normal / // / // / /// Female F52901 F52876 F52882 Appeared normal Appeared normal Appeared normal / /// / // / /// Group 3 - T-6054 (1.28 mg/kg) Male F52965 F52891 F52892 Appeared normal Appeared normal Appeared normal / /// / // / /// Female F52884 Appeared normal / /// F52900 Appeared normal Weakened hind limbs Small feces / - - /_ - - // /- - F52968 Appeared normal / /// / Condition existed. - Condition not evident. 000293 Page 19 of 46 Sex Male Female Animal Number F52886 F52880 F52899 F52889 F52894 F52895 Table 2 (Continued) Individual Clinical Signs Observation 1-4 Hours (Day 1) Group 4 - T-6054 (12.8 mo/kal Appeared normal Appeared normal Appeared normal / / Appeared normal Appeared normal Appeared normal HWI 6329-133 Day 2 - 7 8 9 - 29 /// / / / // / Group 5 - T-6051 (10-cm x 10-cm Sectionl Male F52879 F52963 F52874 Appeared normal Appeared normal Appeared normal /// / /// / / Female F52877 F52888 F52966 Appeared normal Appeared normal Appeared normal / // /// / /// / Condition existed. 000294 Page 20 of 46 Table 3 Individual Dermal Irritation Scores HWI 6329-133 Group 1 (Control) - Sterile Water for Injection (0 mg/kg) Dermal Reaction Males Study Dav 1 2 4 _8_ Females Study Dav 1 _2_ 4 _8_ Animal No. F52885 Animal No. F52967 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 000 0 0000 000 0 0 00 0 0 00 0 00 0 0 0000 0000 0000 0000 00 0 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52873 000 0 0000 000 0 000 0 000 0 0 00 0 Animal No. F52878 0000 0000 000 0 00 0 0 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52898 0 00 0 000 0 000 0 0 00 0 0 00 0 000 0 Animal No. F52883 0000 00 0 0 0000 00 0 0 00 00 0000 00029S Page 21 of 46 Table 3 (Continued) Individual Dermal Irritation Scores HWI 6329-133 Dermal Reaction Erythema Edema Atonia Desquamation Coriaceousness Fissuring Group 2 - T-6054 (0.128 mg/kg) Males Study Dav 1 _2_ 4 8 Females Study Dav 12 48 Animal No . F52887 Animal No. F52901 0000 000 0 0000 000 0 0000 000 0 00 00 00 00 00 0 0 0000 00 00 00 00 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No . F52893 0000 0000 0000 000 0 000 0 000 0 Animal No. F52876 00 00 00 00 00 00 00 00 00 0 0 00 00 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No . F52897 0000 0000 0000 0000 000 0 000 0 Animal No. F52882 00 00 00 00 0000 00 00 00 00 00 0 0 000296 Page 22 of 46 Table 3 (Continued) Individual Dermal Irritation Scores Group 3 - T-6054 (1.28 mg/kg) HWI 6329-133 Dermal Reaction Erythema Edema Atonia Desquamation Coriaceousness Fissuring Males________ Studv Dav _L _2_ 4 8 Animal No. F52965 0000 0000 0000 0000 0000 0000 _______ Females Study Dav _1 2 4 _8_ Animal No. F52884 000 0 0000 000 0 000 0 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52891 0000 0000 0000 0000 0000 0000 Animal No. F52900 000 0 0000 000 0 0000 000 0 000 0 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52892 0000 0000 0000 0000 0000 0000 Animal No. F52968 0000 0000 0000 0000 000 0 0000 000297 Page 23 of 46 Table 3 (Continued) Individual Dermal Irritation Scores HWI 6329-133 Group 4 - T-6054 (12.8 mg/kg) Dermal Reaction Males________ Studv Day _2_ 4 8 _______ Females Studv Dav _2_ 4 _8_ Animal No. F52886 Animal No. F52889 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 011 0000 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52880 0110 0000 0000 0000 0000 0000 Animal No. F52894 0100 0000 0000 0000 0000 00 00 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52899 0 110 0000 0000 0000 0000 0000 Animal No. F52895 02 10 0000 0000 0000 0000 0000 - Value not recorded. 000298 Page 24 of 46 Table 3 (Continued) Individual Dermal Irritation Scores HWI 6329-133 Group 5 - T-6051 (10-cm x 10-cm Section) Dermal Reaction Males Studv Dav 1 _2_ 4 _8_ Animal No. F52879 Females Studv Dav _ L _2_ 4 8 Animal No. F52877 Erythema Edema Atom'a Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 0000 0000 00 0 0 0000 0000 0000 Erythema Edema A t o m 'a Desquamation Coriaceousness Fissuring Animal No. F52963 0000 0000 0000 0000 0000 0000 Animal No. F52888 0000 00 00 0000 00 0 0 0000 00 00 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F52874 0000 0000 0000 0000 0000 0000 Animal No. F52966 0000 0000 0000 0000 0000 00 0 0 000299 Animal Number F52885 F52873 F52898 F52967 F52878 F52883 F52887 F52893 F52897 F52901 F52876 F52882 F52965 F52891 F52892 F52884 F52900 F52968 Page 25 of 46 Table 4 Individual Pathology Comments HWI 6329-133 ______ Test Dav Sex Died Sacrificed NecroDsv Observation GrouD 1 (Controll - Sterile Water for Injection (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. GrouD 2 - T-6054 (0.128 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. GrouD 3 - T-6054 (1.28 ma/kal MMMFFF_ 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 applicable 000300 Page 26 of 46 HWI 6329-133 Table 4 (Continued) Individual Pathology Comments Animal Test Dav Number Sex Died Sacrificed NecroDsv Observation GrouD 4 - T -6054 112.8 ma/kal F52886 M 29 No visible lesions. F52880 M 29 No visible lesions. F52899 M 29 No visible lesions. F52889 F 29 No visible lesions. F52894 F 29 No visible lesions. F52895 F 29 No visible lesions. GrouD 5 - T-6051 (10-cm x 10-cm Section) F52879 M 29 No visible lesions. F52963 M 29 No visible lesions. F52874 M 29 No visible lesions. F52877 F 29 No visible lesions. F52888 F 29 No visible lesions. F52966 F 29 No visible lesions. - Not applicable. 000301 Page 27 of 46 Table 5 Individual Animal Tissue Weights and Bile Volumes HWI 6329-133 Weight (a) Animal Dermal Appli Bili Sex Number Liver Kidnevs cation Site Volume GrouD 1 fControl) - Sterile Water for In.iection (0 ma/kal Male F52885 70.634 - 0.960 1.0 F52873 75.513 16.417 0.866 1.6 F52898 74.433 - 0.645 0.9 Female F52967 62.517 - 0.777 2.1 F52878 63.222 16.808 0.890 2.0 F52883 75.418 - 0.618 2.5 GrouD 2 - T-6054 0.128 ma/kal Male F52887 71.149 - 0.784 1.1 F52893 64.537 13.509 0.836 0.6 F52897 73.622 - 0.604 1.5 Female F52901 71.908 14.610 0.640 1.7 F52876 66.359 - 1.179 1.2 F52882 77.959 - 1.337 1.6 GrouD 3 - T-6054 (1.28 ma/kal Male F52965 69.880 18.191 0.452 1.8 F52891 65.695 - 0.525 1.1 F52892 69.216 - 0.949 1.1 Female F52884 65.907 - 0.590 1.2 F52900 61.937 - 0.710 1.0 F52968 67.347 12.513 0.866 1.3 - Not applicable. 000302 Sex Male Female Male Female Page 28 of 46 HWI 6329-133 Table 5 (Continued) Individual Animal Tissue Weights and Bile Volumes Animal Number Liver Weioht (q) Dermal Appli Kidnevs cation Site Bili Volume GrouD 4 - T-6054 f12.8 ma/kal F52886 77.219 - 0.857 0.6 F52880 59.985 12.254 0.634 0.8 F52899 89.883 - 1.391 0.4 F52889 69.288 - 0.953 0.5 F52894 75.086 - 1.000 1.4 F52895 59.174 15.603 0.635 0.9 GrouD 5 - T-6051 (10-cm x 10-cm Section) F52879 89.874 - 0.884 1.0 F52963 71.814 - 1.535 0.6 F52874 71.657 15.784 0.990 0.6 F52877 71.284 - 1.154 0.6 F52888 65.633 - 1.231 0.5 F52966 68.719 15.855 0.898 1.5 - Not applicable. 000303 Page 29 of 46 HWI 6329-133 APPENDIX A Protocol Deviation Protocol TP3016.AB Protocol Amendment No. 1 Protocol Amendment No. 2 000304 Page 30 of 46 Protocol Deviation HWI 6329-133 Protocol Page 7, 7. Experimental Design, C. Observation of Animals, (2) Reading of Dermal Irritation, Second Sentence. Additional dermal irritation readings will be made approximately 30 minutes after bandage removal (Day 2) and on Study Days 4 and 8. ________ Actual Procedure______ The Day 8 erythema score was inadvertently not recorded for one Group 4 female (No. F52889). This deviation is not considered to have had an adverse effect on the outcome of the study. 000305 HAZLETON WISCONSIN P O S T OFFICE BOX 7 54 5 MA D I S O N , Wl 5 3 7 0 7 / 5 'I 5 Page 31 of 46 il C O R N IN G Company Sponsor: 3M St. Paul, Minnesota PROTOCOL TP3016.AB Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits Date: December 13, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-133 Phone 5 0 741 4 4 I E :< V ft P. S S y Al l . D E L I V E R Y 000306 I O: Page 32 of 46 STUDY IDENTIFICATION TP3016.AB Page 2 Single-Dose Dermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits HWI No. Test Materials Sponsor Sponsor's Representative Study Director Study Location Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date 6329-133 1. T-6054 2. T-6051 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.O. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.O. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 December 28, 1994 January 25, 1995 March 8, 1995 000307 page 33 of 46 1. Study Single-Dose Dermal Absorption/Toxicity Study in Rabbits TP3016.AB Page 3 2. Purpose To assess the systemic absorption and toxicity and relative skin irritancy of test materials when applied to the skin of rabbits 3. Regulatory Compliance This study will be conducted in accordance with the following Good Laboratory Practice Regulations/Standards/Guidelines: [ ] Conduct as a Nonregulated Study [X] 21 CFR 58 (FDA) [ ] 40 CFR 160 (EPA-FIFRA) [ ] 40 CFR 792 (EPA-TSCA) [ ] C(81)30 (Final) (OECD) [ ] 59 Nohsan No. 3850 (Japanese MAFF) [ ] Notification No. 313 (Japanese MOHW) All procedures in this protocol are in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. 4. Quality Assurance The protocol, study conduct, and the final report will be audited by the Quality Assurance Unit in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedures (SOPs) and policies. 5. Test Materials A. Identification 1. T-6054 2. T-6051 B. Physical Description 1. (To be documented in the raw data) 2. (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 000308 Page 34 of 46 TP3016.AB Page 4 E. Reserve Samples Reserve sample(s) of each batch/lot of test and control materials will be taken for this study. The test and control material reserve samples will be stored at HWI in a freezer set to maintain a temperature of -20*C 10*C for 10 years per HWI SOP. The Sponsor will be contacted after 10 years for disposition in accordance with the appropriate regulatory Good Laboratory Practices. F. Retention Any unused test materials 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 (2) Strain/Source Hra:(NZW)SPF/HRP, Inc. 000309 Page 35 of 46 TP3016.AB Page 5 (3) Age at Initiation Adult (4) Weight at Initiation 2.0 to 3.0 kg (5) Number and Sex 15 males and 15 females (6) Identification Individual numbered ear tag (7) Husbandry (a) Housing Individually, in screen-bottom stainless steel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PMI Feeds, Inc.). The food is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. (c) Water Ad libitum from an automatic system. Samples of the water are analyzed by HWI for total dissolved solids, hardness, and specified microbiological content and for selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the food or water that would interfere with this study. (e) Environment Environmental controls for the animal room will be set to maintain a temperature of 19*C to 23*C, a relative humidity of 50% +20%, and a 12-hour 1ight/12-hour dark cycle. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to HWI SOPs. An adequate number of extra animals will be purchased so that no animal in obviously poor health is placed on test. The animals will be placed into study groups using a stratified body weight randomization program within nine days of study initiation. 000310 Page 36 of 46 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 5 Distilled water T-6054 T-6054 T-6054 T-6051 0* 0.128 1.28 12.8 ** 3 3 3 3 3 3 3 3 3 3 * To be administered at a dose volume of 2.0 mL/kg ** To be administered as a 10.0-cm x 10.0-cm piece of test material (fabric) (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 with an electric clipper. 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 respective doses for the animals in Groups 1, 2, 3, and 4 will be based on the animal's body weight just before administration and spread onto the area of exposure in a thin and uniform layer. The Group 1, 2, 3, and 4 materials will be applied undiluted. The Group 5 material will be applied as a 10.0-cm x 10.0-cm piece of the test material moistened with distilled water. The area of application (Groups 1-5) will be 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. The rabbits will be collared during the 24-hour application period. ; 000311 Page 37 of 46 TP3016.AB Page 7 (4) Reason for Route of Administration The dermal route is a potential route of exposure in humans. (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 (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 000312 Page 38 of 46 TP3016.AB Page 8 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. (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 1Q*C. After necropsy, the animals will be discarded. 000313 Page 39 of 46 TP3016.AB Page 9 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) 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 000314 Page 40 of 46 PROTOCOL APPROVAL TP3016.AB Page 10 $ <K John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department Date Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. ___ f i L J l ________________ Representative Quality Assurance Unit Hazleton Wisconsin, Inc. (6329-133.protdsk2) _________ \7r Date Date ii-is-ftf 000315 Page 41 of 46 TP3016.AB J Page 11 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) Fissurinq 0 - None 1 - Slight (definite cracks in epidermis) 2 - Moderate (cracks in dermis) 3 - Marked (cracks with bleeding) 000316 Page 42 of 46 HAZLETON WISCONSIN POST OFFICE BOX /545 M A D I S O N . W I 53707 754 5 i C O R N IN G Company PROTOCOL TP3016.AB Single-Dose Dermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits HWI 6329-133 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 December 23, 1994 In order to obtain a measurable amount of test material (T-6054) for application in Groups 2, 3, and 4, the test material will be diluted with sterile water for injection and applied at a common dose volume of .01 ml/kg. Modify the following two sections of the protocol (protocol amendment items #1 and #2) to indicate these changes. Paoe 6. 7. Experimental Desiqn: B. Dose Administration: U V Test Groups Add the fol1owi ng shaded additions to this sectiori: Group Test Material Dose Level Number of Animals fmq/kq) Males Females 1 (Control) 2 3 4 5 Distilled water T-6054 T-6054 T-6054 T-6051 0* 0.128?** 1.28*** 1*2*.8*** 3 3 3 3 3 3 3 3 3 3 * To be administered at a dose volume of 2.0 mL/kg ** To be administered as a 10.0-cm x 10.0-cm piece of test material (fabric) *** T.Q be!;'adminiStered'.a't a'dose volume'of .01 mL/kg P h o n e 608 2 4 1 4 4 7 1 E X P R E S S MAIL DELIVERY 000317 .(3 0 i K I N S M A N ft l v I) he- <> U ft ' `I I > 2 2 7 M A I) I -i O rj W I ?3 7 0 4 Page 43 of 46 Amendment No. 1 HWI 6329-133 Page 2 2. Paoe 6. 7. Experimental Design; B. Dose Administration: (3) Dose Administration. Delete the fourth sentence in this section and then add the following as the third and fourth sentences to this section. The control material (Group 1) will be applied undiluted. The dose for each animal in Groups 2, 3, and 4 will be diluted with sterile water for injection and applied at a dose volume of .01 mL/kg. Effective December 28, 1994 Sterile water for injection will replace distilled water as the control material based on the fact that sterile water for injection will also be the vehicle in the test mixtures for Groups 2, 3, and 4 (see protocol amendment items #1 and #2). Modify the following three sections of the protocol to indicate this change. 3. Page 4. 6. Control Material: A. Identification. Replace distilled water with the following: Sterile Water for Injection 4. Page 4. 6. Control Material: D. Storage Conditions. Replace Room temperature with the following: Refrigerated 5. Page 6. 7. Experimental Design: B. Dose Administration: H I Test Groups. Modify the table in this section with the following shaded change: Group Test Material Dose Level Number of Animals (mo/kq) Males Females 1 (Control) 2 3 4 5 Sterile water .T-6054 T-6054 T-6054 T-6051 0* 0.128*** 1.28*** 12.8*** ** 3 3 3 3 3 3 3 3 3 3 * To be administered at a dose volume of 2.0 mL/kg ** To be administered as a 10.0-cm x 10.0-cm piece of test material (fabric) *** To be administered at a dose volume of .01 mL/kg ) 000318 Page 44 of 46 Amendment No. 1 PROTOCOL APPROVAL HWI 6329-133 Page 3 2. John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department Date Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. Date _____ ________________________________ Representative Quality Assorance Unit Hazleton Wisconsin, Inc. (6329-133.Aml.dsk2) _______ / ! % - ? r ~ Date ; Page 45 of 46 HAZLETON WISCONSIN P O S T OF F t CT BOX 7545 ) M A D I S O N , W l M / 0 7 75 4 5 i C O R N IN G Company PROTOCOL TP3016.AB Single-Dose Oermal Absorption/Toxicity Study of T-6054 and T-6051 in Rabbits HWI 6329-133 Sponsor Contractor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, WI 53704 Sponsor's Representative Study Director John L. Butenhoff, PhD Steven M. Glaza Amendment No. 2 This amendment modifies the following portions of the protocol: Effective January 24, 1995 At the request of the Sponsor, the weights of tissues collected and the volume of bile collected will be documented in the raw data. These weights and volumes will be included with the sample shipment. Modify the following sections of the protocol to include these additions. 1. Page 8. 7. Experimental Design; D. Pathology; (31 Sample Collection. Modify the second sentence in the first paragraph of this section with the following underlined addition: The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from the first male and female necropsied in each group will be collected, weighed (volume only determined for bile), and immediately placed in a freezer set to maintain a temperature of -20*C 10*C. 2. Page 9. 7. Experimental Design; D. Pathology; (31 Sample Collection. Modify the second sentence in the second paragraph of this section with the following underlined addition: The samples and their corresponding weights or volumes will be shipped to the person listed in Section 7.C.(4).(c). Phone 60 E X P R E S S ,v -i i r . . ;Rv 000320 I D l> I fc . IS 'V A >t H ! y I ) Fax M ADISON .* a . M l 7 2 2 7 W I 5X704 Page 46 of 46 Amendment No. 2 3. Page 9, 8, Report. Add the following to this section: Individual animal tissue weights and bile volumes HWI 6329-133 Page 2 PROTOCOL AMENDMENT APPROVAL John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department Date Steven M. Glaza 0 Study Director Acute Toxicology .) Hazleton Wisconsin, Inc. /p u < c~\ Representat>i/v(e Quality Assutirriance Unit Hazleton Wisconsin, Inc. C6329-133.Am2.dsk2) Date Date ) 000321 9.1.2 Analytical protocol AMDT-013195.1 000322 3M Environmental Laboratory_________ Protocol - Analytical Study Single-Dose Dermal Absorption/Toxicity Study of T-6051 and T-6054 in Rabbits In-Vivo Study Reference Number: HWI#6329-133 Study Number: AMDT-013195.1 Test Substance: FC-129 (T-6051 and T-6054) 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 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: Q- /j/m es D. JMnson ^ tu d y Director ft/3*/IS Date 000323 John Butenhoff, PhD Sponsor Representative Date l 1.0 PURPOSE This study is designed to provide information as to whether FC-129 (T-6051 and T-6054) is dermally absorbed. The analytical aspect of this study is to determine fluorine-containing compounds (biotransformation products) in the tissue and serum of rabbits at various times post dose dermal application of FC-129. 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 M atrix: Bovine liver and bovine serum 2.2 Source of M aterials: 3M ICP/PCP Division (2.1.1), grocery store (2.1.2, 2.1.4liver), Sigma Chemical Company (2.1.2, 2.1.4-serum) 2.3 Num ber of Test and Control Samples: Tissues and fluid from 24 test animals and 6 control animals. Tissues and fluids include liver, 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. 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. 000324 2 3.0 EXPERIMENTAL - Overview The tissues from animals dosed as described (HWI#6329-133), 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. To confirm the identity of fluorine-containing compounds present in liver (if any at 28 days) and serum at various intervals, electrospray mass spectrometiy may be selected as one of the analytical techniques employed. Not all of the tissues and fluid samples will be analyzed. 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 EXPERIMENTAL - Methods 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 ANALYSIS 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 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. 000325 3 6.0 MAINTENANCE OF RAW DATA AND RECORDS 6.1 Raw Data and Records: Raw data, approved protocol, appropriate specimens, approved final report, and electronic data will be maintained in the AMDT archives. 7.0 REFERENCES____________________ 7.1 AMDT-S-10-0, Sample Tracking System 8.0 ATTACHMENTS_________________________________________________ 8.1 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 000326 4 3M Environmental Laboratory Method Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer - Liver Method Identification Number: AMDT-M-1 Revision Number: 0 Adoption Date: Revision Date: None Author: Rich Youngblom Approved by: Software: MS Word 5 .la 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 000327 1 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 M atrices: 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 EQUIPM ENT____________________________________________________ 5.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer, modified for fluoride extraction. 5.2 IBM compatible 386 or 486 computer. 5.3 DX2000 software, version 1.00, modified for fluoride extraction. 5.4 Excel Spreadsheet, version 5.0 or greater 6.0 INTERFERENCES_______________________________________________ 6.1 Sample size is limited to approximately 150 mg, depending on sample moisture content. This may vary from matrix to matrix. 000328 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 STANDARDIZATION________________________ 8.1 Preparation of Calibration Standards 8.1.1 The standards required for each project will need to be appropriate for that individual project. Refer to protocol for that project. 8.1.2 Typically 50-500 ppm FC-95 in methanol standards are used. 8.1.3 For rabbit liver studies, use beef liver as the matrix. Cut a piece 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. 000329 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 r^ must be at least 0.95. If it is not at least 0.95, consult with supervisor. 8.3.3.8 A new standard curve should be run when the combustion tube or sample matrix is changed. New standard curve may also be run at the discretion 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. 000330 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 ofTISA B 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 TISAB: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 COrion reading in ppm - intercept^ (Slope) 10.0 VALIDATION_____________________________________________ _ 10.1 Q uality Control 10.1.1 D aily 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 O ther Validation Parameters: NA 000331 s 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 tw o 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_____________________________________________________ Rvision Number Reason for Change Rvision Date 000332 6 3M Environmental Laboratory Method Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer Method Identification Number: AMDT-M-2 Revision Number: 0 Adoption Date: Revision Date: None Author Rich Youngblom Approved By: Grodt/ Leader W /j ------- Date Quality Assurance / - / - f i" Date Software: MS Word 5.1a Affected Documents: AMDT-M-1 Thermal Extraction o f Fluoride by Means o f a Modified Dohimann DX2000 Organic Halide Analyzer 000333 1 1.0 S C O P E . A P P L IC A B L E C O M P O U N D S . A N D M A T R IC E S 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 1:1 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. 000334 2 BEST COPYAVAILABLE 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 ofT ISA B 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 ppmF-, 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 0.05, 0.1, 0.5, 1.0, and 1.5 ppm F-, following the manufacturer's instructions. 8.2.6 Record the slope in the appropriate log book. 8.2.7 Clean the electrode by rinsing with Milli-QTM water and wiping the sides down with lab tissues. 8.3 Storage Conditions for Standards 8.3.1 Calibration standards are stored at room temperature. 9.0 PROCEDURES___________________________________________________ 9.1 Calibration and 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. 000335 3 9.2 Calibration And Measurement, Using Orion 3E Software: 1 H f IIII.HIILE 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 o f 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 ANALYSTS_______________________________________________ 11.1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 12.0 A T T A C H M E N T S ____________________________________ _________________________ None 13.0 R E F E R E N C E S ________________________________________________________________ 000336 4 13.1 Orion Model EA940 Expandable Ion Analyzer Instruction Manual, Orion Research Incorporated, 1991. 13.2 Orion Model 960900 Solid State Combination Fluoride Electrode Instruction Manual, Orion Research Incorporated, 1991. 14.0 REVISIONS__________________________________ __________________ Revision Number Reason for Change Revision Date 000337 5 3M Environmental Laboratory Method Extraction of Fluorochemicals from Rabbit Livers SOP Identification Number: AM DT-M -4 Revision Number: 0 Adoption Date: / o 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. 000338 1 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 A pplicable Com pounds: Fluorochemicals or other fluorinated compounds. 1 .3 M atrices: Rabbit Livers. ?,.Q K E Y W O R D S__________________________________________________________ 2 .1 Fluorochemicals, rabbit livers, electrospray mass spectrometer, fluorinated compounds, extraction. 2JLJRECAUTIQNS__________________________________ 3 .1 U se 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 pL 4 . 1 . 2 Eppendorf type or disposable pipets 4 . 1 . 3 Gloves 4 . 1 . 4 Plastic grinding tubes 4 . 1 . 5 Plastic centrifuge tubes, 15 mL 4 . 1 . 6 Labels 4 . 1 . 7 Nitrogen 4 . 1 . 8 Timer 4 . 1 . 9 Filters, Titan nylon syringe filters, 0.2 pm. 4 . 1 . 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 o f ammonium acetate by adding 250 mg ammonium acetate to a 100 mL volumetric flask and dilute to volume with Milli-Q water. Dilute this solution 1:10 for a 250 ppm solution. 4 . 2 . 2 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker), (Na^Oj/NaHCOj) 0.25 M: Weigh 26.5 g o f sodium carbonate (N a^ O j) 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 4 . 2 . 7 1H,1H,2H,2H - perfluorooctanesulfonic acid (Aldrich) 4 . 2 . 8 FC-95 (3M Specialty Chemical Division) 000339 2 -EQUIPMENT_________________________ 5 .1 Ultra-Turrax T25 Grinder for grinding liver samples. 5 .2 Vortex mixer 5 .3 Centrifuge 5 .4 Shaker 5 .5 Analytical Evaporator 6.0 IN TE R FER EN C ES____________________ 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 C A LIB R A TIO N AND STANDARDIZATION ___________________ 8 .1 Preparation of Internal Standards 8 . 1 . 1 Prepare an internal standard o f approximately 12 ppm 1H,1H,2H,2Hperfluorooctanesulphonic acid to be added to each liver sample. 8.1.-2 Weigh at least 0.1 g o f lH,lH,2H,2H-perfluorooctanesulphonic acid into a 100 mL volumetric flask. Record the actual weight. 8 . 1 . 3 Bring it up to volume with methanol, this is the stock standard. 8 . 1 . 4 To a 250 mL volumetric flask, add 3 mLs o f the stock standard and bring to volume with Milli-Q water. Calculate the actual concentration o f the standard. actual mg perfluoroctane- sulphonic acid X 3 mL = 0.1 L 250 mL actual concentration, ppm 8 .2 Prepare FC-95 Anion Standards 8 . 2 . 1 Prepare FC-95 standards for the standard curve. 8 . 2 . 2 Weigh approximately 100 mg o f FC-95 into a 100 mL volumetric flask. Record the actual weight. 8 . 2 . 3 Bring up to volume with dilute acetonitrile. 8 . 2 . 4 Dilute the solution with dilute acetonitrile 1.TO for a solution o f approximately 100 ppm. Dilute this solution 1:10 with dilute acetonitrile for a solution o f 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 of Bovine liver into a 250 mL Nalgene bottle containing 200 mLs Milli-Q water. Grind to a homogenous solution. 8 . 3 . 2 Add 1 mL of the solution to a 15 mL centrifuge tube. Prepare a total o f eight 1 mL aliquots o f the solution in 15 mL centrifuge tubes. Be sure to re suspend solution by shaking it between aliquots. 000340 3 8 . 3 . 3 Spike seven o f the 1 mL aliquots with the following amounts o f working standards in step 9.12 o f the procedure. One 1 mL aliquot serves as the blank. Working Standard (Approximate Cone.) 500 ppb 500 ppb 500 ppb 500 ppb 1 PPm 5 ppm 5 ppm uL 100 2 300 400 500 200 3 Approximate final concentration o f FC-95 in liver Blank 0.292 ppm 0.5 84 ppm 0.877 ppm 1.168 ppm 2.924 ppm 5.848 ppm 8.772 ppm 8 .4 Calculate the actual value o f the standards: uL o f standard x concentration fin ppm) = final concentration (ppm) 171 mg liver* / 1 ml homogenate of FC -95 in liver Average weight o f bovine liver in solution as determined by weighing 1 mL homogenates o f 40 mg liver in 200 mL o f Milli-Q water. 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 . 5 . 2 Alternatively, a standard curve may be generated using ratios o f responses o f the perfluorooctansulfonate anion and the internal standard anion versus concentration o f the perfluorooctanesulfonate anion. 8 .6 Storage Conditions for Standards 8 . 6 . 1 New standards are prepared with each analysis. Standards are stored in covered plastic centrifuge tubes until the analysis on the mass spectrometer is performed. 8 .7 Storage Conditions for Standards 8 . 7 . 1 B eef liver homogenates may be frozen after preparation. M.P-BQCEPURES____________ ___________________________ 9 .1 Obtain frozen liver samples. In spent tissue, note that the liver has not been packaged with other tissues. 9 .2 U se 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. 9 .5 Put a label on the vial with the study number, weight, rabbit ID, date and analyst initials. 000341 4 9 .6 Add 2.5 mLs water. 9 .7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, until the sample is a homogeneous solution with no large chunks. 9 .8 Rinse the probe off into the sample with 2.5 mLs water using a pipet. 9 .9 Take the grinder apart and clean it with methanol after each sample. Follow AMDT-EP-22. 9 . 1 0 Cap the sample and vortex for 15 seconds. 9 . 1 1 Pipet 1 mL into a 15 mL centrifuge tube. Label the centrifuge tube with the identical information as the grinding tube. (See AMDT-M-4 Worksheet for documenting the remaining steps.) 9 . 1 2 Spike the beef liver homogenates with the appropriate amount 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 . 1 5 Using an analytical pipet, add 5 mL ethyl acetate. 9 . 1 6 Cap the sample and vortex 20 to 30 seconds. 9 . 1 7 Put them in the shaker for 20 min. 9 . 1 8 Centrifuge for 20 to 25 minutes, until the layers are well separated. Set the power on the centrifuge to 25. 9 . 1 9 Remove 4 mLs 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 . 2 0 Blow the sample down on the analytical evaporator to near dryness with nitrogen, approximately 30 to 40 minutes. 9 . 2 1 Bring the remaining sample up in 1 mL dilute acetonitrile with an analytical pipet. 9 . 2 2 Vortex 15 seconds. 9 . 2 3 Transfer the sample to a 3 mL syringe. Attach a 0.2 Jim nylon mesh filter, and filter the sample into a fresh centrifuge tube or a autovial. Label the tube or vial with the study number and animal number. 9 . 2 4 Cap and hold for analysis by electrospray mass spectroscopy. 9 . 2 5 Complete AMDT-M-4 worksheet and attach to page o f study notebook. 10.0 V A LID A TIO N _______________________________ 1 0 . 1 Quality Control - not applicable 1 0 . 2 Precision and Accuracy- not applicable 1 0 . 3 Other Validation Parameters- not applicable 1 1 .0 DATA ANALYSIS___________________________ 11.1 None 12.0 ATTACHM ENTS_____________________________ 1 2 .1 Worksheet AMDT-M-4 1 3 .0 R E FE R E N C E S_______________________________ 1 3 . 1 AMDT-EP-22 Routine Maintenance o f Ultra-Turrax T-25 1 4 .0 REV ISIO N S____________ Revision Number Reason for Change Revision Date 0003 5 Worksheet A M D T-M -4 Study # . 1 Sample Number set # Blank l iver FC-95 approx 0.5 p p m actual ppm #W 100 ul. 200 ul. 500 uL 400 uL _ _ _ _ _ _ _ _ _ _ FC-95 approx 1 p p m actual p p m #W . - . 500 uT, _ . _ _ . _ _ * _ FC-95 approx. 5 p p m actual p p m #W . . . _ 200 u L 500 uT, _ _ Date and Initials for Std. _ _ _ _ . * _ 1studv n u m b e r where the original worksheet is located and nlace a conv. ________________ T ____ " I__________ _________ 1__________________ ___________________ T,iver Extraction Process- Date h Initials Pinet 1 mT, of T.iver Solution Pinet 100 u L of 12 n n m Internal Standard Std. # Vortex 15 sec Pinet 1 ml. of 250 n n m A m m o n i u m Acetate Std # Pinet 1 ml, of 0.25 N a , C O . / 0 . 2 5 M N a H C O , Buffer Pinet 5 mT. of F.thvl Acetate Vortex 20-50 sec Shake 2 0 min. Centrifuge 20-25 mi n R e m o v e a 4 ml. aliouot of organic laver B l o w d o w n to near drvness f<0 25 ml.l with N, A d d 1 m- of l-I Acernnirrile/H,0 TN# Vortex 15 sec. Filter usinp a 5cc B - D svrinoe with a 0 . 2 u m SRI filter into a 1.5 ml. autosamnle vial___________________________ 000343 6 3M Environmental Laboratory_____________________ _ Method A n alysis o f R ab bit L iver E xtract for F lu oroch em icals using E lectrosp ray M ass S p ectroscop y SOP Identification Number: AMDT-M -5 Revision Number: 0 Adoption Date: 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 000344 l L O - s a o p E __________________________________________________________ 1 .1 Scope: This method is for the analysis of 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 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1 .3 M atrices: Rabbit Livers (samples), Beef Liver (standards), other tissues and fluids. 2 .0 K E Y W O R D S ___________________________________________________________________ 2 .1 Fluorochemicals, fluorinated compounds, electrospray mass spectroscopy, mass spectrometer, rabbit livers. 3 .0 P R E C A U T I O N S _______________________________________________________________ 3 .1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk o f electrical shock. 3 .2 D o 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 D o not run the pump to dryness. 4 . 0 S U P P L I E S A N D M A T E R I A L S __________________________________________ 4.1 Supplies 4 . 1 . 1 Nitrogen gas regulated to 140 psi. 4 . 1 . 2 Fluofix column or equivalent. 4 . 1 . 3 100 uL or 250 uL flat tip syringe for sample injection. 4.2 Reagents 4 . 2 . 1 Dilute acetonitrile mobile phase, dilute acetonitrile 1:1 with Milli-Q water. 4 . 2 . 2 Milli-Q water, all water used in this method should be Milli-Q water. 5 .0 E Q U I P M E N T ________________________________________________________________ 5 .1 VG Trio 2000 Electrospray Mass Spectrometer or equivalent. 5 .2 ISCO Syringe Pump 5 .3 Spectraphysics AS300 Autosampler 5 .4 100 uL Assembly 5 .5 Autovials or capped centrifuge tubes. 6 .0 I N T E R F E R E N C E S ____________________________________________________________ 6 .1 There are no known interferences at this time. 7 .0 S A M P L E H A N D L I N G __________________________________________________ _____ 7 .1 Keep the extracted samples in capped 15 mL centrifuge tubes or in capped autovials until ready for analysis. 000345 2 BESTCOPYAVAILABLE 8 .0 C A L I B R A T I O N A N D S T A N D A R D I Z A T I O N ____________________ 8.1 Preparation of Calibration Standards 8 . 1 . 1 Seven beef liver standards and one blank beef liver are prepared during the extraction procedure. (See AMDT-M-4, section 8.0) 8.2 Calibration 8 . 2 . 1 Run the seven beef liver standards twice, starting with the lowest standard to obtain the standard curve. 8 . 2 . 2 Typically one standard is run after each 5 to 7 samples. Choose a standard in the same range 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 Hom ogenates 8 . 4 . 1 B eef liver homogenates may be frozen after preparation. 9.0 PRO CED URE___________________________________________________ 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 of the probe. Use an eye piece to check for chips. The tip should be flat with no jagged edges. If any chips are found cut off the tip 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 of the probe. A fine mist should be coming out of the tip. 9 . 1 . 7 Carefully guide the probe into the opening. Insen 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 E S \ and the appropriate masses to 427 and 499. 9 . 1 . 9 If it is not in single ion mode go to "Option" and set SIR. 9 . 1 . lOStart Acquisition. Assign a file name, MO-DAY-YR + letter. Record it in the log book. 9 . 1 . 1 1 Run the beef liver samples first, running each standard twice at the beginning of the run.. Run a QC check by running one standard after every 5 to 7 samples. 9 .2 Manual Injection 9 . 2 . 1 Draw 150 uL 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. 9 .2 .4 Record the scan number for each sample in the logbook. 000346 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 "Enter1 9.3.3.8 Press "Run" to start 1 0 .0 V A L I D A T I O N ____________________________________________________ 10.1 Quality Control 1 0 . 1 . IRun a standard every 5 to 7 samples. If a significant change( 50%) in peak height occurs stop the run. Only the samples before the last acceptable standard will be used. The remaining samples will be reanalyzed. 10.2 Precision and Accuracy 1 0 . 2 . 1 See Method Validation Report number AMDT-M-5.0.VI 1 0 .3 Other Validation Parameters 1 0 . 4 Refer to Method Validation Report Number AMDT-M-5.0.V1 1 1 .0 D A T A A N A L Y S I S _________________________________________________________ 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. 1 1 . 2 . 2 Calculate the mg o f FC-95 anion, or other fluorochemical in the total rabbit liven mg FC-95 anion in the total rabbit liver = mg FC-95 anion from std. curve gms of liver used for analysis x Total mass 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 ED. Write the study number on the printout, initial, date, and put it in the study folder. Staple all chromatograms together and number pages. 000347 4 1 2 .0 A T T A C H M E N T S ________________________________________________ None 13..LEEEEBENCES_________________________________ 1 3 . 1 AMDT-EP-17 14.0 REVISION S__________________________________________ Revision Number Reason for change Revision Date 000348 5 3M Environmental Laboratory Method A n alysis o f F lu orid e U sin g the S k alar S egm en ted F low A n alyzer W ith Ion S elective E lectrod e M ethod Identification Number: AM DT-M -8 Adoption Date: / p - T - i r " Revision Number: 0 Revision Date: N one Author: Deb Wright / Cynthia Weber Approved By: Gripup Leader / ) /" ----- -- Quality Assurance /A /?s Date Date Software: IBM MS Word, 6.0 Affected Documents: AM DT-EP-26, Operation and Maintenance o f the Skalar Segmented Flow Analyzer 000349 l BEST espy JtVMLHHE L.O-S.CQPE_______________________________________ ___ 1 .1 This method is for the analysis for fluoride, thermally extracted from samples using the Dohrmann DX2000 (AMDT-M-1), and collected in TISAB for analysis with an Ion Selective Electrode (ISE). The analysis is performed using the Skalar Segmented Flow Analyzer with ISE. 1 .2 Samples can be tissues, serum, biological material, or other materials extracted on the Dohrmann. 2 .0 K E Y W O R D S ________________________________________________________ ___ 2 .1 Skalar, segmented flow, fluoride. 3 .0 P R E C A U T I O N S _______________________________________________ _ 3 .1 Follow standard laboratory safety practices. 4 . 0 S U P P L I E S A N D M A T E R I A L S ____________________________________ _ 4.1 Supplies 4 . 1 . 1 Sample cups, 4 mL plastic cups with caps 4 . 1 . 2 Autopipets, oxford or equivalent with plastic tips 4 . 1 . 3 Polypropylene volumetric flasks, 100 mL 4 . 1 . 4 Cartridge components, refer to the Skalar Methods for components and part numbers. 4 . 1 . 5 Sample prefilters, Evergreen 4.2 Reagents 4 . 2 . 1 Brij 35, 30% S.F.A.S. Detergent 4 . 2 . 2 TISAB II buffer solution: Purchase TISAB II from Orion. To 1 liter o f 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 o f 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 - 000350 1.2 ppm 1.5 ppm BEST COPY AVAILABLE 1.2 1.5 5.0 E Q U IP M E N T 5 . 1 Skalar Segmented Flow Auto Analyzer Sans System equipped with ISE 6.0 IN T E R F E R E N C E S 6 . 1 High concentrations o f alkalinity, chloride, phosphate, sulfate or iron can cause interferences. 7.0 S A M P L E H A N D L IN G 7 .1 Samples should be stored in polyethylene bottles. Samples should be analyzed within 30 days. 8 .0 C A L I B R A T I O N A N D S T A N D A R D I Z A T I O N __________________________ 8 .1 Preparation of Calibration Standards 8 . 1 . 1 Prepare calibration standards as in section 4.3. 8.2 Calibration 8 . 2 . 1 The standards are analyzed at the beginning o f the run. 8 .3 Storage Conditions for Standards 8 . 3 . 1 Standards are stored in capped polypropylene volumetric flasks. New standards are prepared at a minimum of every six months, or as necessary. 9 .0 P R O C E D U R E _________________________________________________________________ 9 .1 Start Up Procedure 9 . 1 . 1 Clamp down the pumpdecks, air bars and sampler-pump tubing. 9 . 1 . 2 Put the fluoride electrodes in the electrode chamber. 9 . 1 . 3 Turn on the power o f 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 o f 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 o f 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. 000351 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 . 3 Check the highest standard and adjust the gain, if necessary, with the interface screw #3. 9 . 2 . 6 Go to CONTROL PANEL, and to analysis panel. Deselect the analysis that will not be run. (Select or deselect analysis by pressing ENTER.) Press Tab to return to the Analysis Panel. 9 . 2 . 7 Press the spacebar to bring up the local menu. 9 . 2 . 8 Select START to stan 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, stan the sampler. Make sure that the sampler is set to the right number o f 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. 1 0 .0 V A L I D A T I O N _______________________________________________________________ 10.1 Quality Control 1 0 . 1 . lRun 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. 000352 4 10.2 Precision and Accuracy 1 0 . 2 . l S 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.VI 1 1 .0 D A T A A N A L Y S I S ______________________________________________ 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 o f 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. 1 2 .0 A T T A C H M E N T S _____________________________________________________________ None 1 3 .0 R E F E R E N C E S ___________________________________________________________ 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 o f the Skalar Segmented Flow Analyzer 1 4 .0 R E V I S I O N S ___________________________________________________________________ Revision Number Reason for change Revision Date 000353 5 9.3 Quality Assurance Unit Statement 000354 Attachment D GLP Study Quality Assurance Statement Study Title: Single-dose Dermal Absorption/Toxicity Study of T-6051 and T-6054 in Rabbits Study Number AMDT-013195.1 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 F lam 12 10/12/95 10/19/95 Phase_______________ Final Report Date Inspection Reported to Management study Director 10/19/95 10/19/95 BESTCOPY AVMIAKE Date 000355 9.4 Key Personnel Involved in the Study 000356 3M Environmental Laboratory Key Personnel T h erm al extraction follow ed by analysis using O rion ion analyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plummer A nalysis o f liver extracts using electrospray m ass spectrom etry: Jim Johnson Dave Christenson T h erm al extraction follow ed by analysis using Sk alar segm en ted flow an alyzer w ith ion selective electrode: Jim Johnson Deb Wright Rich Youngblom Deann Plummer D ocu m en tation and R eporting: Jim Johnson Rich Youngblom Q u a lity A ssu ran ce U nit: Gale Van Buskirk Cynthia Weber Kari Rambo 000357 9.11 Data 000358 9.11.1 Summary and raw data; ug F' in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 000353 Summary of Combustion Data - Liver AMDT-013195.1, H W I6329-133 As Referenced in Final Report section 6.0 DATA AN ALYSIS Total ug Fluoride in Whole Liver Mean per Dose Group* ug Std. Dev. Control Group 16.2 + 5.5 0.128 mg/kg dose (T6054) 11.1 + 2.0 1.28 mg/kg dose (T6054) 17.1 + 2.6 12.8 mg/kg dose (T6054) 45.2 + 20.5 Fabric Exposure (T6051) 14.7 + 2.7 ^Calculated as the mean of triplicate samples from each of three male and three female rabbits. 000360 RPT133L.XLS FC129 AB ID liver blank-3 liver spike-4 liver spike-5 liver spike-6 liver spike-7 liver spike-8 liver spike-9 liver blank-4 F52873-1 F52873-2 F52873-3 F52885-1 F52885-2 F52885-3 F52898-1 F52898-2 F52898-3 liver blank-1 liver blank-2 liver spike-1 liver spike-2 F52878-1 F52878-2 F52878-3 F52883-1 F52883-2 F52883-3 F52967-1 F52967-2 F52967-3 F52887-1 F52887-2 liver blank-1 liver spike-1 liver spike-2 liver spike-3 liver spike-4 liver spike-5 liver spike-6 F52887-3 F52893-1 F52893-2 F52893-3 % rcvry 83% 83% 102% 94% 84% 83% 91% 95% 69% 85% 83% 109% 101% 101% Actual ppm Fin liver (W/W) 0.230 0.950 1.06 1.28 1.00 2.34 2.49 0.328 0.245 0.334 0.329 0.449 0.222 0.306 0.139 0.131 0.103 0.228 0.298 1.36 0.995 0.286 0.230 0.176 0.211 0.242 0.179 0.158 0.194 0.200 0.252 0.189 0.281 0.968 0.973 0.875 1.29 1.11 1.26 0.173 0.168 0.147 0.175 Average ppm Fin liver (W/W) 0.303 0.326 0.124 0.231 0.211 0.184 0.205 0.163 liver burned (grams) 0.140 0.132 0.118 0.121 0.142 0.108 0.101 0.116 0.121 0.107 0.104 0.150 0.114 0.108 0.150 0.148 0.146 0.152 0.134 0.101 0.145 0.105 0.150 0.151 0.115 0.107 0.135 0.144 0.116 0.108 0.108 0.128 0.129 0.108 0.132 0.143 0.128 0.138 0.121 0.148 0.133 0.135 0.135 Whole liver weight (grams) 74.2 74.2 74.2 69.2 69.2 69.2 73.3 73.3 73.3 70.2 70.2 70.2 73.8 73.8 73.8 61.5 61.5 61.5 70.6 70.6 70.6 63.5 63.5 63.5 Total F- in whole liver (ug) 22.4 22.5 9.11 16.2 15.6 11.3 14.5 10.4 Dosage (mg/kg) 0.0 0.0 0.0 0.0 0.0 0.0 0.128 0.128 Pagel 000361 RPT133L.XLS FC129 AB ID F52897-1 F52897-2 F52897-3 F52876-1 F52876-2 F52876-3 F52882-1 F52882-2 F52882-3 F52901-1 F52901-2 F52901-3 Liver blk 1 Liver blk 2 Liver blk 1 Liver blk 2 Liver blk 3 Liver spk-1 Liver spk-2 Liver spk-3 F52965-1 F52965-2 F52965-3 F52891-1 F52891-2 F52891-3 F52892-1 F52892-2 F52892-3 F52884-1 F52884-2 F52884-3 F52968-1 F52968-2 F52968-3 F52900-1 F52900-2 F52900-3 F52880-1 F52880-2 F52880-3 F52886-1 F52886-2 F52886-3 F52899-1 F52899-2 F52899-3 % rcvry 92% 89% 83% Actual ppm Fin liver (W/W) 0.156 0.174 0.179 0.117 0.155 0.146 0.112 0.154 0.169 0.126 0.127 0.142 0.331 0.218 1.22 0.322 0.280 1.10 0.869 0.864 0.235 0.205 0.216 0.239 0.197 0.239 0.333 0.270 0.273 0.206 0.255 0.230 0.299 0.261 0.242 0.335 0.390 0.259 0.487 0.548 0.579 0.611 0.728 0.490 0.428 0.366 0.340 Average ppm Fin liver (W/W) 0.169 0.139 0.145 0.132 0.219 0.225 0.292 0.231 0.267 0.328 0.538 0.610 0.378 liver burned (grams) 0.134 0.113 0.123 0.149 0.119 0.138 0.146 0.130 0.112 0.140 0.143 0.112 0.112 0.137 0.146 0.159 0.139 0.127 0.156 0.145 0.125 0.163 0.151 0.114 0.144 0.128 0.132 0.134 0.102 0.146 0.101 0.135 0.145 0.116 0.121 0.147 0.115 0.115 0.117 0.142 0.138 0.137 0.114 0.113 0.103 0.115 0.126 Whole liver weight (grams) 72.4 72.4 72.4 65.2 65.2 65.2 76.1 76.1 76.1 70.5 70.5 70.5 68.7 68.7 68.7 64.5 64.5 64.5 68.7 68.7 68.7 65.0 65.0 65.0 66.6 66.6 66.6 61.4 61.4 61.4 59.6 59.6 59.6 75.3 75.3 75.3 89.1 89.1 89.1 Total F- in whole liver (ug) 12.3 9.09 11.0 9.29 15.0 14.5 20.1 15.0 17.8 20.1 32.1 46.0 33.7 Dosage (mg/kg) 0.128 0.128 0.128 0.128 1.28 1.28 1.28 1.28 1.28 1.28 12.8 12.8 12.8 Page 2 000362 R P T 133L.XLS FC129 AB ID liver blank 1 liver blank 2 liver blank 3 liver spike-1 liver spike-2 liver spike-3 liver spike-4 liver spike-5 liver spike-6 F52889-1 F52889-2 F52889-3 F52894-1 F52894-2 F52894-3 F52895-1 F52895-2 F52895-3 F52874-1 F52874-2 F52874-3 F52879-1 F52879-2 F52879-3 F52963-1 F52963-2 F52963-3 F52877-1 F52877-2 F52877-3 F52888-1 F52888-2 F52888-3 F52966-1 F52966-2 F52966-3 Liver spike-7 Liver spike-8 Liver spike-9 Liver spike-10 Liver spike-11 Liver spike-12 Liver spike-13 Liver spike-14 Liver spike-15 % rcvry 87% 0.7786 0.7852 0.9656 0.8486 82% 60% 71% 75% 98% 103% 101% 78% 107% 102% Actual ppm Fin liver (W/W) 0.265 0.327 0.220 0.921 1.05 0.823 1.06 0.859 0.978 0.812 0.896 1.59 0.835 0.794 0.917 0.307 0.523 0.276 0.331 0.249 0.173 0.168 0.194 0.185 0.216 0.175 0.154 0.222 0.190 0.160 0.158 0.211 0.142 0.321 0.277 0.167 0.881 1.05 0.775 0.990 1.15 1.16 2.16 2.46 2.39 Average ppm Fin liver (W/W) 1.10 0.849 0.368 0.251 0.182 0.182 0.191 0.170 0.255 liver burned (grams) 0.125 0.111 0.123 0.143 0.112 0.144 0.138 0.149 0.127 0.114 0.113 0.141 0.127 0.119 0.118 0.151 0.109 0.154 0.144 0.122 0.145 0.134 0.105 0.147 0.130 0.123 0.135 0.119 0.118 0.141 0.144 0.104 0.139 0.123 0.131 0.132 0.102 0.103 0.146 0.150 0.136 0.133 0.110 0.131 0.130 Whole liver weight (grams) 68.7 68.7 68.7 74.0 74.0 74.0 58.1 58.1 58.1 69.7 69.7 69.7 89.1 89.1 89.1 70.2 70.2 70.2 69.9 69.9 69.9 64.2 64.2 64.2 67.6 67.6 67.6 Total F- in whole liver (ug) 75.4 62.8 21.4 17.5 16.3 12.8 13.3 10.9 17.3 Dosage (mg/kg) 12.8 12.8 12.8 Fabric Fabric Fabric Fabric Fabric Fabric Page 3 000363 9.11.2 Summary and raw data; analysis of liver extracts using electrospray mass spectrometry. 000364 HWI #6329-133 Study: Protocol Number: Test Material: Matrix: R Squared Value: Response Factor Amount: Analyst: Date: Method: Instrument: LABBASE FILE Single Dose Dermal Absorption TP3016.AB T-6054 &T-6051 in Rabbits (FC-129) Liver 0.9889 1.15E-05 DLC 4/3/95 AMDT-M-4 Fisons VG 2000 Electrospray MS 040395D A- I A' \ -^rrx^U A10-31-^ Group Dose Group 1: Sterile Water 2.0 mL/kg Group 2: 0.128 mg /kg Group 3: 1.28 mg/kg Group 4: 12.8 mg/kg Group 5: Fabric Sample # F52883 F52898 F52885 F52967 F52873 F52878 Ion Count Extracted wt Dilution Area a factor N/D N/D N/D N/D N/D N/D Concentration pg/g* Total m ass of liver a Total amount of FC-95 per liver mg N/D .N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D F52893 F52887 F52901 F52876 N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D N/D F52892 F52891 F52965 F52884 11413 14922 8191 12572 1.0139 1.0615 1.1217 1.0881 1 1 1 1 0.1033 0.1290 0.0670 0.1060 68.707 64.487 68.667 64.989 0.007 0.008 0.005 0.007 F52895 F52899 F52894 F52880 F52886 F52889 51584 58481 127876 58961 84958 93984 1.117 1.1894 1.0236 1.0931 1.0915 1.0615 1 1 1 1 1 1 0.4237 0.4511 1.1462 0.4949 0.7142 0.8124 58.094 89.078 73.983 59.624 75.341 68.670 0.025 0.040 0.085 0.030 0.054 0.056 F52879 F52877 F52874 F52963 F52888 16734 26249 N/D 9735 N/D 1.2566 1.0106 1.0772 1 1 1 0.1222 0.2383 N/D 0.0829 N/D 89.125 69.913 70.161 0.011 0.017 N/D 0.006 N/D * The concentration was calculated by using the standard curve and multiplying the result by 4/5. The 4/5 factor is the result of a miscalculation in applying formula 8.4 in Method AMDT-M-4-0. 137 mg of liver was used In this calculation rather than171 mg. The concentrations In the standard curve are therefore 5/4 larger than they should be. By multiplying the calculated concentration In the standard curve by 4/5, the correct result Is obtained. 000365 HWI #6329-133 Study: Protocol Number: Test Material: Matrix: R Squared Value: Response Factor Amount: Analyst: Date: Method: Instrument: LABBASE RLE Single Dose Dermal Absorption TP3016.AB T-6054 & T-6051 in Rabbits (FC-129) Liver 0.9946 9.10E-06 DLC 4/4/95 AMDT-M-4 Fisons VG 2000 Electrospray MS 040495B Group Dose Group 2: 0.128 m g/kg Group 3: 1.28 mg/kg Group 5: Fabric * Sample # F52897 F52882 Ion Count Extracted wt Area 9 18736 N/D 1.1033 1.1025 Dilution factor 1 1 Concentration p g /g ** Total mass of liver g 0.1236 N/D 72.433 76.060 Total amount of FC-95 per liver mg 0.009 N/D F52900 16432 1.3161 1 0.0909 61.356 F52968 27181 1.2011 1 0.1648 66.642 0.006 0.011 F52966 8521 1.1457 1 0.0542 67.632 0.004 * Administered as a 10.0 cm x 10.0 cm piece of test fabric ** The concentration was calculated by using the standard curve and multiplying the result by 4/5. The 4/5 factor is the result of a miscalculation in applying formula 8.4 in Method AMDT-M-4-0. 137 mg of liver was used in this calculation rather than171 mg. The concentrations in the standard curve are therefore 5/4 larger than they should be. By multiplying the calculated concentration in the standard curve by 4/5, the correct result is obtained. 000366 Flue C'4-oi4-d`5 ^plc-r Method DLCLIV BEST COPY AVAILABLESample DLCLIV Operator DLC R u n d a t e 0 5-0'3-1995 0 6 : 4 9 : 4 4 V e r s i o n : 12 Printed on 05-09-1995 AT 06:49:56 Straight Line Fit forced through Origin. A-3 4 3 # -/ V 3 &-/3>*f= 6324-''?^ "KC.-"ITV *TT1:-T1 s 132859 - 47743 mm LEVEL AMOUNT Component 1 EXTERNAL STANDARD CALIBRATION AREA 1 0.4000 47743 0.3000 85693 3 1 .2000 132059 Y= SLOPE INTERCEPT Area Amount = R squared = 1 . 0988E-<-05 * 9.1012E-6 * 0.9946 Amount Area 0.0000E+00 0.OOOOE+OO 000367 FiLE : 632<t-i33 Fc-iZ<t f~cUpUV Me!-, ned D L L l .I.v Sample LCLIU perator DLC BEST COPV AVAILABLE Run u t e 0 5 - 0 9 - 1 9 9 5 0 / s24*45 Uer 5 ior lG P r i n t e d on 0 5 - 0 9 - 1 9 9 5 RT 0 7 = 2 4 : 5 6 S t r a i g h t Line Fit f o r c e d thro uq h 0 r ig in . o F Cwpownt 1 A K182142 l A II - 74869 +/ / .4 .8 AflNf 1.2 EUEL AMOUNT Component 1 = EXTERNAL STANDARD CALIBRATION AREA 1 0.4000 33695 2 0.8000 74069 3 1.2000 102142 Y = SLOPE INTERCEPT Area Am ount = R squared = 8.7189E+04 * Amount 1 .1469E-05 * Area 0.9889 + 0.0000E+00 + 0.0000E+00 000368 A-fc A*JT G32^ - \33 R>l 2^ ExactCoPMof Original * * 69C 000 -Ol 7d rt Iwtr- ^ .r ^ . C4JUS.-VJc. File:040395D LAB-BASE - The MS Data System Sample'.6329-133 BABBIT LIVER EXTRACTS; FC-129 CAB) * 03/04/1995 14:00 000370 File:040395D LAB-BASE - The MS Data System Sample:6329-133 RABBIT LIUER EXTRACTS; FC-129 CAB) 03/04/1995 14:00 000371 v* File:040395D LAB-BASE - The MS Data System Sample:6329-133 RABBIT LIVER EXTRACTS; FC-129 (AB) 03/04/1995 14:00 000372 o * #* r. hooo rtoJI 4 3 2 ^ - 0 3 s fe32l - l43 4>32*t ~ '33 Awrwva.ls /rvuJi^$-e*? F52^*7 F Si-'fCio F52i4>S F 52 SZ7. opyofOrlglMi Dlc_ Initial C /22?S 000374 4 * File:040495B LAB-BASE - The MS Data System 04/04/1995 10:07 Sample:6329-133 CFC-1293 6329-143 CFC-1367F] RABBIT LIUER EXTRACTS 0 0 0 3 VS 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 section 6.0 and therefore is not discussed in detail. 000376 RE: 6329-133 LIUER SAMPLES RMDT 13195.1 Date of Analysis: 3/28, 3/29 and 3/30/95 Rnalyst: DDLU U O .c O '( ifc l^ S The samples are burned in the Dohrman at 950 C using between 0.1 and 0.2 grams of the liuer. The gas is collected in 1.0 mL of 1:1 TISRB/Milli-Q water then an additional 2 mL of 1:1 TISAB/Milli-Q is added to allow for sufficient uolume for Skalar analysis. The samples are then analyzed on a Skalar Segmented Flow 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 software using these curues. All results below 0.0001 ppm appear on the raw data as # .# # # # . A quality control standard was analyzed euery 10 samples to check for accuracy and drift. Raw data is taken from the appropriate calibrated range of the Skalar printout and summarized on an Excel spreadsheet. The final results are adjusted for the collection uolume and any subsequent dilutions. 000377 SUMMARY of 6329-133 LIVER SAMPLES AMDT 013195.1 BEST COPY AVAILABLE 000378 ** This is only a partial study. AH samples were not saved from the Dohrmann analysis. DDW F52884-1 F52884-2 F52884-3 ND ND ND 3.0 0.1463 ND 3.0 0.1011 ND 3.0 0.1345 ND GROUP 3 Dose Level : 1.28 mg/kg F52891-1 F52891-2 F52891-3 F52892-1 F52892-2 ND ND ND 0.02 ND 3.0 0.1140 ND 3.0 0.1443 ND 3.0 0.1284 ND 3.0 0.1322 0.43 3.0 0.1342 ND F52900-1 F52900-2 F52900-3 ND ND ND 3.0 0.1474 ND 3.0 0.1153 ND 3.0 0.1150 ND F52965-1 F52965-3 0.02 0.02 3.0 0.1246 0.40 3.0 0.1510 0.31 F52968-1 F52968-2 F52968-3 ND ND ND 3.0 0.1448 ND 3.0 0.1159 ND 3.0 0.1211 ND 64.9889 ND ND 64.9889 ND 64.9889 ND 64.4869 ND ND 64.4869 ND 64.4869 ND 0.20 68.7067 29 68.7067 ND 61.3555 ND ND 61.3555 ND 61.3555 ND 0.36 68.6665 68.6665 28 21 66.6415 ND ND 66.6415 ND 66.6415 ND ND ND 15 ND 25 ND 1 133-L.SUM Page 1 SUMMARY of 6329-133 LIVER SAMPLES AMDT 013195.1 BEST COPY AVAILABLE GROUP 4 Dose Level: 12.8 mg/kg F52889-1 F52889-2 F52889-3 F52894-1 F52894-2 F52894-3 F52895-1 F52895-2 F52895-3 0.04 0.05 0.10 0.05 0.06 0.05 0.03 0.03 0.02 GROUP 5 Dose Level: 10.0 cm x 10.0 cm fabric F52874-1 F52874-2 F52874-3 F52879-1 F52879-2 F52888-1 F52963-2 F52963-3 0.02 0.02 0.02 0.02 ND ND ND ND 3.0 0.1139 1.17 3.0 0.1125 1.34 3.0 0.1410 2.18 3.0 0.1272 1.20 3.0 0.1183 1.43 3.0 0.1185 1.17 3.0 0.1513 0.62 3.0 0.1091 0.87 3.0 0.1537 0.44 3.0 0.1438 0.46 3.0 0.1221 0.50 3.0 0.1454 0.35 3.0 0.1338 0.34 3.0 0.1050 ND 3.0 0.1438 ND 3.0 0.1228 ND 3.0 0.1345 ND 68.6699 1.56 68.6699 68.6699 73.9834 1.27 73.9834 73.9834 58.0944 0.65 58.0944 58.0944 80 92 149 89 105 87 36 51 26 69.6893 0.44 69.6893 69.6893 32 35 25 0.17 89.1246 30 89.1246 ND ND 64.2262 ND ND 70.1610 ND 70.1610 ND 107 94 38 31 16 ND ND ^cooo 133-L.SUM Page 2 L133-1.XLS 000380 1 Tracer 1.50 1.20 80% 2 Drift 1.50 1.23 82% 3 Wash ND 4 Std 1 0.015 0.016 104% 5 Std 2 0.03 0.03 101% 6 Std 3 0.06 0.06 97% 7 Std 4 0.09 0.09 99% 8 Std 5 0.12 0.13 104% 9 Std 6 0.15 0.15 98% 10 Std 7 0.30 0.29 98% 11 Std 8 0.60 0.60 101% 12 Std 9 1.20 1.23 102% 13 Std 10 1.50 1.47 98% 14 Drift 1.50 1.25 83% 15 Wash ND 16 Blk-1 ND 3 17 Blk-2 ND 3 18 Spk-1 ND 3 19 Spk-2 ND 3 20 Spk-3 0.06 3 21 F52900-1 ND 3 0.1474 22 F52900-2 ND 3 0.1153 23 F52900-3 ND 3 0.1150 24 F52968-1 ND 3 0.1448 25 F52968-2 ND 3 0.1159 26 Drift 1.50 1.26 84% 27 Wash ND 28 F52968-3 ND 3 0.1211 29 F52884-1 ND 3 0.1463 30 F52884-2 ND 3 0.1011 31 F52884-3 ND 3 0.1345 32 F52892-2 ND 3 0.1342 33 F52548-2 ND 3 34 F52888-1 ND 3 0.1438 35 F52963-2 ND 3 0.1228 36 F52963-3 ND 3 0.1345 I Page 1 BESTCOPYAVAILABLE 61.3555 61.3555 61.3555 66.6415 66.6415 66.6415 64.9889 64.9889 64.9889 68.7067 64.2262 70.1610 70.1610 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.004 0.004 0.004 63 63 63 0.15 ND 0.15 ND 0.15 0.17 0% 0% 112% I- loZZ n L133-1.XLS 37 Blk ND 3 38 Drift 1.50 1.26 84% 39 Wash ND BESTCOPY AVAILABLE 000381 Page 2 A 000382 1 Tracer 1.50 1.21 81% 2 Drift 1.50 1.23 82% 3 Wash ND 4 Std 1 0.015 0.016 108% 5 Std 2 0.03 0.03 98% 6 Std 3 0.06 0..0578 7 Std 4 0.09 0.09 100% 8 Std 5 0.12 0.12 104% 9 Std 6 0.15 0.15 98% 10 Std 7 0.30 0.29 98% 11 Std 8 0.60 0.60 101% 12 Std 9 1.20 1.23 102% 13 Std 10 1.50 1.47 98% 14 Drift 1.50 1.25 83% 15 Wash ND 16 Blk-1 0.07 3.0 17 Blk-2 0.02 3.0 18 Blk-3 0.02 3.0 19 Spk-1 0.05 3.0 20 Spk-2 0.05 3.0 21 Spk-3 0.05 3.0 22 F52891-1 ND 3.0 0.1140 ND 23 F52891-2 ND 3.0 0.1443 ND 24 F52891-3 ND 3.0 0.1284 ND 25 F52892-1 0.02 3.0 0.1322 0.43 26 Drift 1.50 1.25 83% 27 Wash ND 28 F52965-1 0.02 3.0 0.1246 0.40 29 F52965-3 0.02 3.0 0.1510 0.31 30 Drift 1.26 31 Wash ND Page 1 BEST COPY AVAILABLE 64.4869 64.4869 64.4869 68.7067 ND ND ND 29.31 0.004 0.004 0.004 63.00 63.00 63.00 68.6665 68.6665 27.78 21.15 0.15 0.15 0.15 0.16 0.15 0.15 Samplo Skater Skater L133-3.XLS % DTUSAB QtySanipia g e : 000383 1 Tracer 1.50 1.25 83% 2 Drift 1.50 1.28 85% 3 Wash 0.02 4 Std 1 0.015 0.018 123% 5 Std 2 0.03 0.03 88% 6 Std 3 0.06 0.06 100% 7 Std 4 0.09 0.09 98% 8 Std 5 0.12 0.12 104% 9 Std 6 0.15 0.15 98% 10 Std 7 0.30 0.29 96% 11 Std 8 0.60 0.61 101% 12 Std 9 1.20 1.24 104% o 13 6 14 15 CO 16 Std 10 Drift Wash Blk-1 1.50 1.46 97% 1.50 1.31 87% 0.02 0.02 3.0 GO 17 CO 18 Blk-2 Blk-3 0.02 3.0 ND 3.0 19 Spk-1 0.06 3.0 20 Spk-2 0.05 3.0 21 Spk-3 0.05 3.0 22 Spk-4 0.06 3.0 23 Spk-5 0.06 3.0 24 Spk-6 0.06 3.0 25 F52874-1 0.02 3.0 0.1438 0.46 26 Drift 1.50 1.27 84% 27 Wash 0.02 28 F52874-2 0.02 3.0 0.1221 0.50 29 F52874-3 0.02 3.0 0.1454 0.35 30 F52879-1 0.02 3.0 0.1338 0.34 31 F52879-2 ND 3.0 0.1050 ND 32 F52889-1 0.04 3.0 0.1139 1.17 33 F52889-2 0.05 3.0 0.1125 1.34 34 F52889-3 0.10 3.0 0.1410 2.18 35 F52894-1 0.05 3.0 0.1272 1.20 36 F52894-2 0.06 3.0 0.1183 1.43 1 Page 1 BEST COPY AVAILABLE 69.6893 32.13 0.004 0.004 0.004 0.004 0.004 0.004 63.00 63.00 63.00 63.00 63.00 63.00 0.15 0.15 0.15 0.15 0.15 0.15 0.17 113% 0.15 100% 0.16 107% 0.18 120% 0.19 124% 0.18 117% 69.6893 69.6893 89.1246 89.1246 68.6699 68.6699 68.6699 73.9834 73.9834 34.93 24.59 30.17 ND 80.31 91.74 149.47 88.64 105.44 V </0 U 37 F52894-3 0.05 3.0 0.1185 1.17 38 Drift 1.50 1.28 85% 39 Wash 0.02 40 F52895-1 0.03 3.0 0.1513 0.62 41 F52895-2 0.03 3.0 0.1091 0.87 42 F52895-3 0.02 3.0 0.1537 0.44 43 Drift 1.50 1.31 87% 44 Wash 000384 I <%> Page 2 73.9834 86.91 58.0944 58.0944 58.0944 36.17 50.64 25.74 BEST COPY AVAILABLE 1995-03-28 17:30 OutPut of : 950328F1 Software : version 6.1 cl990,93 Operator : DDW Date of the Analysis : 1995-03-28 15:28 Analysis File Name : C:\SKALAR\DATA\950328F1 I3 \\ S .I Ho~H G-3D-\ -\13 Fluoride 1.5 Calibration order = Inverse Logarithm S lo p e : s = #.##### x - cl C O Result = 10a s X = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 al = aO -0.00000 0.00092 -1.24810 Fluoride L Calibration order = 2 C o r r e la t io n ; r = 0.99716 Result = a2 * X + al * x + aO a2 = al = aO = 0.00000 0.00022 0.00604 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 m m 10 2.5 m l . 1 1 User file : Reproces : No . TXT 000385 1995-03-28 17:30 OutPut of : 950328F1 F l uoride 1. 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 000386 1995-03-28 17:30 Output of : 950328F1 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 S lO 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 M e a s u r e w i n d o w 75 % Filter No Regeneration No formula : c4 :==c3 output : #.#### 000387 1995-03-28 17:30 OutPut of : 950328F1 Page 1 of Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dii Weight Ch Result F Cor. Valu Time wt iw Initial Wash 1 1.000 3 0.056 4 0.0060 0 219 00 65 0 1t 2d Tracer Drift 1 1.000 3 1.199 2065 2282 203 T V - .74 0.9756 2065 0 0 1 1.000 3 1.230 2096 2312 380 "TU x. \.T4 0.9981 2096 0 0 3w Wash 1 1.000 3 0.056 4 0.0060 0 214 557 000 4 si Standard 1 1 1.000 3 0.062 4 0.0156 43 258 732 43 0 0 5 s2 Standard 2 1 1.000 3 0.070 4 0.0303 106 322 907 106 0 0 6 S3 Standard 3 1 1.000 3 0.088 4 0.0579 214 432 1081 214 0 0 7 S4 Standard 4 1 1.000 3 0.109 4 0.0893 325 544 1257 325 0 0 8 s5 Standard 5 1 1.000 3 0.135 4 0.1250 440 661 1432 440 0 0 9 s6 Standard 6 1 1.000 3 0.152 4 0.1469 506 728 1606 506 0 0 10 S7 Standard 7 1 1.000 3 0.293 4 0.2979 895 1121 1781 895 0 0 11 s8 Standard 8 1 1.000 3 0.604 1410 1641 1956 4 0.5548 1410 00 12 s9 Standard 9 1 1.000 3 1.229 2095 2332 2132 4 0.9974 2095 00 13 S10 S t a n d a r d 10 1 1.000 3 1.473 2335 2576 2305 4 1.1797 2335 0 0 14 d Drift 1 1.000 3 1.248 2113 2338 2482 -Tn/ I-*-f 4 1.0106 2113 00 15 w Wash 1 1.000 3 0.056 4 0.0060 0 226 2661 000 000388 1995-03-28 17:30 OutPut of : 950328F1 Fluoride 1.5 Fluoride L Page 2 of 3 PPM PPM Pos Typ Ident 16 u Dil Weight Ch Result F Cor. Valu Time BLK 1 1 1 . 0 0 0 3 too e < 14 240 2779 4 0.0091 14 0 0 ''belvCM".. JiO-'S* (\rtsbcr\) ,'-or Wb 17 u BLK 2 1 1 . 0 0 0 3 Absen A -32 194 3007 4 #.#### -32 0 0 Pb 18 u SPK 1 1 1 . 0 0 0 3 Absen A -23 202 3182 4 0.0011 -23 0 0 Pb 19 u 20 u SPK 2 SPK 3 1 1 . 0 0 0 3 Absen A -33 192 3357 4 #.#### -33 0 0 1 1 . 0 0 0 3 0.087 4 0.0566 209 436 3533 209 0 0 Pb 21 u F52900-1 1 1 . 0 0 0 3 too 1 > 17 242 3765 4 0.0098 17 0 0 Mb 22 u F52900-2 1 1 . 0 0 0 3 too e < -1 224 3820 4 0.0058 -1 0 0 ub 23 u F52900-3 1 1 . 0 0 0 3 A b sen A -33 192 4057 4 #.#### -33 0 0 Jjb 24 u F52968-1 1 1 . 0 0 0 3 0.058 4 0.0087 12 236 4184 12 0 0 Pb 25 u 26 d F52968-2 Drift 1 1 . 0 0 0 3 Absen A -32 192 4407 4 #.#### -32 0 0 jjb 1 1 . 0 0 0 3 1.259 2124 2348 4581 -r<S-~l."2_ 4 1.0187 2124 0 0 27 w 28 u 29 u Wash F52968-3 F52884-1 1 1 . 0 0 0 3 0.056 4 0.0060 0 224 4751 000 1 1.000 3 too e < 32 256 4871 4 0.0131 32 0 0 1 1 . 0 0 0 3 0.061 4 0.0133 33 256 5089 33 0 0 Vb .03^3 }jb alcf /* 30 u F52884-2 1 1.000 3 0.061 4 0.0135 34 256 5282 34 0 0 UT 31 u F52884-3 1 1 . 0 0 0 3 0.061 4 0.0135 34 256 5435 34 0 0 03, / 000389 1995-03-28 17:30 OutPut of : 950328F1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dil Weight Ch Result F Cor. V a lu T im e 32 u F52892-2 1 1.000 3 0.061 4 0.0144 38 2 6 0 5 6 3 5 38 0 0 33 u F52548-2 1 1.000 3 0.061 4 0.0147 39 2 6 0 5 8 0 5 39 0 0 34 u F52888-1 1 1.000 3 0.061 4 0.0140 36 2 5 6 5 9 8 3 36 0 0 35 u F52963-2 1 1.000 3 0.061 4 0.0140 36 2 5 6 6 1 5 9 36 0 0 36 u F52963-3 1 1.000 3 0.061 4 0.0140 36 2 5 6 6 3 3 3 36 0 0 37 u 'F52^63-A 1 >SAi!> 1.000 3 Absen A -23 1 9 6 6 5 0 6 4 0.0011 -23 0 0 38 d Drift 1 1.000 3 1.257 2122 2 3 4 0 6 6 8 4 4 1.0172 2122 0 0 39 w Wash 1 1.000 3 0.056 4 0.0060 0 218 6858 00 0 wt rw RunOut Wash 1 1.000 3 0.056 4 0.0060 0 0 7159 00 0 Page 3 of 3 -V .W*5Z- JJb OHIO MO /J P ,m . A S t> ,oH l A J0 1 O-iS /J T .o il' /J l OZ IS ' h> . l 000390 Raw data o f 950328F1 : Fluorlda 1.5 Esc"Exit i Fl=Help ! Crtl-P-Edit peaks I 000391 i Rau data of 950328F1 : Fluoride 1.5 Esc-Exit ! FI-Help ! Crtl-P=Edit peaks 000392 Concentration 000393 i Concantrat ion 000394 1995-03-29 12:46 OutPut of : 950329A1 Software : version 6.1 cl990,93 Operator : DDW Date of the Analysis : 1995-03-29 10:24 Analysis File Name : C:\SKALAR\DATA\950329A1 3> S i K od ji (,32.^ Fluoride 1.5 Calibration order = Inverse Logarithm S lo p e : s = #.##### Result = 6 x - cl C tttata 10 s i x = corrected value of the sample cl = corrected value of the concentration 1 s = Slope of the electrode a2 al = aO = -0.00000 0.00087 -1.21638 Fluoride L Calibration order = 2 C o r r e la t io n : r = 0.99645 Result = a2 * x + al * x + aO a2 = al = aO = 0.00000 0.00020 0.00843 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 000395 1995-03-29 12:46 OutPut of : 950329A1 F luoride 1. 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 000396 1995-03-29 12:46 OutPut of : 950329A1 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 S10 sTandard Ignore Order : 2 Dimension : PPM start Value : 500 DU trigger Limit : 1 8 0 0 Sec Peak shape : Pointed stArt ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula : c 4 :=c3 output : #.#### 000397 1995-03-29 12:46 OutPut of : 950329A1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dii Weight Ch Result F Cor. Valu Time wt iw Initial Wash 1 1.000 3 0.061 4 0.0084 0 216 00 65 0 1t Tracer 1 1.000 3 1.209 2068 2285 208 4 1.1307 2068 0 0 2d Drift 1 1.000 3 1.234 2091 2308 382 4 1.1510 2091 0 0 3w Wash 1 1.000 3 0.061 4 0.0084 0 218 555 000 4 si Standard 1 1 1.000 3 0.065 4 0.0162 37 258 732 37 0 0 5 s2 Standard 2 1 1.000 3 0.073 4 0.0294 96 320 901 96 0 0 6 s3 Standard 3 1 1.000 3 0.091 4 0.0578 208 436 1086 208 0 0 7 s4 Standard 4 1 1.000 3 0.112 4 0.0902 320 552 1260 320 0 0 8 s5 Standard 5 1 1.000 3 0.136 4 0.1244 425 660 1436 425 0 0 9 s6 Standard 6 1 1.000 3 0.152 4 0.1470 489 728 1608 489 0 0 10 s7 Standard 7 1 1.000 3 0.293 4 0.3207 893 1136 1784 893 0 0 11 s8 Standard 8 1 1.000 3 0.603 4 0.6246 1415 1664 1957 1415 0 0 12 s9 Standard 9 1 1.000 3 1.228 2085 2342 2131 4 1.1457 2085 0 0 13 slO S t a n d a r d 10 1 1.000 3 1.474 4 1.3519 2308 2570 2309 2308 0 0 14 d Drift 1 1.000 3 1.247 2103 2356 2483 4 1.1617 2103 00 15 w Wash 1 1.000 3 0.061 4 0.0084 0 256 2663 000 Page 1 of 3 T O - 1-i- T V - \ T, 000398 1.995-03-29 12:46 TM o, 3503$ST copy avaiuubil of 3 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dii W e i g h t Ch R e s u lt F Cor. V alu Time _ "''buk'-'-'O*'-'- 16 u 17 u BLK 1 BLK 2 1 1.000 3 0.100 4 0.0709 1 1.000 3 0.070 4 0.0230 255 512 2829 255 0 0 *2ou> 68 321 3010 68 0 0 -risi, -ST 18 u 19 u BLK 3 SPK 1 1 1.000 3 0.067 4 0.0186 1 1.000 3 0.086 4 0.0503 48 300 3185 48 0 0 32*>.8 180 432 3357 180 0 o +s1* ifcS i IP l w- 20 u SPK 2 1 1.000 3 0.087 4 0.0519 186 436 3533 +|jl 186 0 0 **ssn - m 21 u SPK 3 1 1.000 3 0.085 4 0.0485 173 422 3711 ,, 173 0 0 ** 22 u F52891-1 1 1.000 3 Abs e n A 13 260 3883 4 0.0111 13 0 0 .0 33*3 23 u F52891-2 1 1.000 3 A b s e n A 13 258 4058 4 0.0111 13 0 0 .013*3* 24 u F52891-3 1 1.000 3 0.064 4 0.0142 28 272 4231 28 0 0 .0^2 (a 25 u F52892-1 1 1.000 3 0.067 4 0.0188 49 292 4410 49 0 0 4 26 d Drift 1 1.000 3 1.252 4 1.1653 2107 2348 4584 TV-.. 2107 0 0 27 w Wash 1 1.000 3 0.061 4 0.0084 0 240 4747 00 0 28 u 29 u 30 d F52965-1 F52965-3 Drift 1 1.000 3 0.066 4 0.0168 1 1.000 3 0.065 4 0.0155 1 1.000 3 1.263 4 1.1752 40 280 4938 40 0 0 34 274 5110 34 0 0 2118 2358 5284 2118 0 0 < iPH feS' iiT i 31 w Wash 1 1.000 3 0.061 4 0.0084 0 240 5455 000 000399 1995-03-29 12:46 OutPut of : 950329A1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dll Weight Ch Result F Cor. Valu Time wt rw RunOut Wash 1 1.000 3 0.061 4 0.0084 0 256 5759 000 Page 3 of 000400 z Esc=Exit ! Fl-Help ! Crtl-P-Edit peaks I 000401 BEST COPY AVAILABLE Raw data of 350329! : Fluoride 1.5 ----------------------- ------ 1-----------,-----------1-----------1---------- 1-----------1---------- 1---------- I-----------1----------- 2423 Tine 6798 Esc=Exit ! Fl=Help ! Crtl-P=Edit peaks i 000402 Concentrt ion 000403 Concentrt ion 000404 1995-03-30 10:59 OutPut of : 950330A1 Software : version 6.1 cl990,93 Operator : DDW Date of the Analysis : 1995-03-30 08:43 Analysis Pile Name : C:\SKALAR\DATA\950330A1 OOlO ~1))a \ q 5 C 32.^1 h 3 j Fluoride 1.5 Calibration order = Inverse Logarithm Slop e : s = #.##### 6 x - cl C 0 aSadaa Result = 10a s i x corrected value of the sample cl = corrected value of the concentration 1 s Slope of the electrode a2 a al a aO a -0.00000 0.00088 -1.12348 Fluoride L Calibration order = 2 C o r r e la t io n : r = 0.99781 Result = a2 * x + al * x + aO a2 a al a aO = 0.00000 0.00033 0.01842 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 000405 L 1995-03-30 10:59 OutPut of : 950330A1 Fluoride 1.5 Path number : 3 Signal type : Debubbled Decolor : Yes system Number : 0 diLute : No Resample : No dil Threshold : 4095 diG output :0 Window event : Off si sTandard Ignore s2 sTandard Ignore S3 sTandard Ignore s4 sTandard Ignore s5 sTandard Ignore s6 sTandard 0.150 s7 sTandard 0.300 s8 sTandard 0.600 s9 sTandard 1.200 slO sTandard 1.500 Order : Inverse Logarithm Dimension : PPM start Value : 500 DU trigger Limit : 1800 Sec Peak shape : Pointed stArt ignore : 60 Sec eNd ignore : 120 Sec Measure window : 75 % Filter : No Regeneration : No formula : output : ##.### Fluoride L Path number Signal type Decolor system Number diLute Resample dil Threshold diG output Window event :0 : Debubbled : No :0 : No : No : 4095 :0 : Off 000406 1995-03-30 10:59 OutPut of si s T andard 0 015 s2 sTandard 0 030 s3 sT a n d a r d 0 060 s4 sTa n d a r d 0 090 s5 sTandard 0 120 s6 sTandard 0 150 s7 sTandard Ignore s8 sTandard Ignore s9 sTandard Ignore S10 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 : #.#### 950330A1 000407 1995-03-30 10:59 OutPut of : 950330A1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dii Weight Ch Result F Cor. Valu Time wt iw Initial Wash 1 1.000 3 0.075 4 0.0184 0 155 00 65 0 1t Tracer 1 1.000 3 1.248 2101 2274 211 4 .8362 2101 0 0 2d Drift 1 1.000 3 1.279 2139 2329 386 4 0.8533 2139 0 0 3w Wash 1 1.000 3 0.075 4 0.0184 0 208 569 00 0 4 si Standard 1 1 1.000 3 0.075 4 0.0184 0 209 755 00 0 5 s2 Standard 2 1 1.000 3 0.079 4 0.0264 24 234 906 24 0 0 6 s3 Standard 3 1 1.000 3 0.096 4 0.0599 124 336 1084 124 0 0 7 s4 Standard 4 1 1.000 3 0.113 4 0.0882 207 420 1262 207 0 0 8 s5 Standard 5 1 1.000 3 0.137 4 0.1248 313 528 1435 313 0 0 9 s6 Standard 6 1 ' 1.000 3 0.154 4 0.1472 377 594 1612 377 0 0 10 s7 Standard 7 1 1.000 3 0.287 4 0.2835 753 976 1785 753 0 0 11 s8 Standard 8 1 1.000 3 0.606 1308 1540 1962 4 0.4990 1308 0 0 12 s9 Standard 9 1 1.000 3 1.242 2093 2338 2137 4 0.8327 2093 0 0 13 slO S t a n d a r d 10 1 1.C00 3 1.461 2383 2636 2312 4 0.9645 2383 0 0 14 d Drift 1 1.000 3 1.306 2173 2392 2487 4 0.8686 2173 0 0 15 w Wash 1 1.000 3 0.075 4 0.0184 0 220 2729 00 0 000408 1995-03-30 10:59 Output of : 950330A1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dil Weight Ch Result F Cor. Valu Time 16 u blk 1 1 1.000 3 0.076 4 0.0204 6 224 2822 600 17 u blk 2 1 1.000 3 0.074 4 0.0161 -7 208 3013 -7 0 0 18 u blk 3 1 1.000 3 0.072 4 0.0115 -21 192 3183 -21 0 0 19 u spk 1 1 1.000 3 0.095 4 0.0572 116 328 3363 116 0 0 20 u spk 2 1 1.000 3 0.091 4 0.0505 96 304 3536 96 0 0 21 u spk 3 1 1.000 3 0.093 4 0.0539 106 312 3706 106 0 0 22 u spk 4 1 1.000 3 0.097 4 0.0606 126 330 3888 126 0 0 23 u spk 5 1 1.000 3 0.098 4 0.0623 131 332 4063 131 0 0 24 u spk 6 1 1.000 3 0.096 4 0.0589 121 320 4235 121 0 0 25 u F52874-1 1 1.000 3 0.077 4 0.0221 11 208 4410 11 0 0 26 d Drift 1 1.000 3 1.265 2122 2316 4587 4 0.8456 2122 0 0 27 w Wash 1 1.000 3 0.075 4 0.0184 0 192 4766 000 28 u F52874-2 1 1.000 3 0.076 4 0.0204 6 196 4935 600 29 u F52874-3 1 1.000 3 0.075 4 0.0171 -4 184 5116 -4 0 0 30 u F52879-1 1 1.000 3 0.074 4 0.0151 -10 176 5274 -10 0 0 31 u F52879-2 1 1.000 3 0.073 4 0.0131 -16 169 5464 -16 0 0 Page 2 of 3 000409 1995-03-30 10:59 OutPut of : 950330A1 Fluoride 1.5 Fluoride L PPM PPM Pos Typ Ident Dil Weight Ch Result F Cor. Valu Time 32 u F52889-1 1 1.000 3 0.088 4 0.0444 78 261 5640 78 0 0 33 u F52889-2 1 1.000 3 0.091 4 0.0501 95 276 5812 95 0 0 34 u F52889-3 1 1.000 3 0.122 4 0.1023 248 426 5990 248 0 0 35 u F52894-1 1 1.000 3 0.091 4 0.0508 97 274 6164 97 0 0 36 u F52894-2 1 1.000 3 0.094 4 0.0562 113 288 6340 113 0 0 37 u F52894-3 1 1.000 3 0.089 4 0.0464 84 258 6514 84 0 0 38 d Drift 1 1.000 3 1.276 2136 2308 6688 4 0.8519 2136 0 0 39 w Wash 1 1.000 3 0.075 4 0.0184 0 170 6872 00 0 40 u F52895-1 1 1.000 3 0.081 4 0.0314 39 208 7034 39 0 0 41 u F52895-2 1 1.000 3 0.082 4 0.0317 40 208 7212 40 0 0 42 u F52895-3 1 1.000 3 0.077 4 0.0227 13 180 7388 13 0 0 43 d Drift 1 1.000 3 1.310 4 0.8708 2178 2344 7565 2178 0 0 44 w Wash 1 1.000 3 0.075 4 0.0184 0 165 7796 000 wt rw RunOut Wash 1 1.000 3 0.075 4 0.0184 0 138 8040 000 Page 3 of 3 000410 BEST COPY AVAILABLE Rau data of 950330A1 : Fluorid 1.5 Esc=Ex it ! F 1"He1b i Crtl-P=Edit peaks 1 Q Q M ?~ r& n (( 1 *5 000411 3 R m u cfjtie o f 950330A1 Fluoride 1.5 BEST COPYAVAILABLE 4000 2423 Tine Esc=Exit I FIHelp ! Crt1-P-Edit peaks I 0798 000412 Pcithno ' 000413 000414 Concentrt ion 000415