Document QXqEZqrr9N5QL9DLEdn7b73b6

3M Environmental Laboratory A R Z26-O O f| Final Report- Analytical Study Single-dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits In-Vivo Study Reference Number: HWI#6329-130 Study Number: AMDT-020895.1 Test Substance: FC-95 (T-6049) 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 Initiation Date: See attached protocol Author: James D. Johnson Approved By: D.Johnson 'Study Dil / l / f / j> Completion Date 000352 O O O O O l of fi~ i 1.0 SUMMARY Samples of rabbit liver at 28 days post dermal administration of 0, 0.003, 0.06, and 0.30 mg/kg FC-95 (T-6049) were analyzed by combustion for total organic fluorine. There was no detectable level of total organic in any of the treated groups above that observed in control rabbit liver. Since in a pharmacokinetic study (HWI#6329-159), it was established that perfluorooctanesulfonate is persistent in rabbit liver with a biological half-life in excess of one month and that perfluorooctanesulfonate will serve as a good marker for absorption of FC-95, it is concluded that detectable dermal absorption does not occur at these dose levels. These dose levels are very low and possibly too low to make an assessment of dermal absorption. 2.0 INTRODUCTION________________________________________________ FC-95 is the potassium salt of perfluorooctanesulfonic acid. In a previous pharmacokinetic study (HWI#6329-159), it was shown that the biological half-life is >1 month. It is persistent in serum and liver after an intravenous dose. From previous studies in rats, the molecule is not known to biotransform or conjugate. Thus, analysis by combustion with subsequent measurement of fluoride will estimate the amount of perfluorooctanesulfonate present. At later times, the concentration in liver is higher than the amount in serum on the average. For use as a marker to assess dermal absorption, the level of total organic fluorine in liver is therefore a more sensitive test at 28 days post dose. 3.0 TEST MATERIALS 3.1 Test, Control, and Reference Substances and Matrices 3.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 3.1.2 Analytical Reference Matrix: Bovine liver and bovine serum 3.1.3 Analytical Control Substance: None 3.1.4 Analytical Control Matrix: Bovine liver and bovine serum 3.2 Source of Materials: 3M ICP/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 000353 000002 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 Samples of tissue and serum were available from HWI#6329-130 for analysis for total organic fluorine. The samples were combusted and analyzed for total organic fluorine. The data were assessed for extent of dermal absorption of FC-95. 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 6.0 DATA ANALYSTS The data from combustion analysis at 28 days post dermal administration of FC-95 (T-6049) are attached. The dosing material T-6049 is a 0.06% solution of FC-95. Doses in this analysis are expressed as the FC-95 equivalents. The liver was combusted and subsequently analyzed for total organic fluoride. There is no evidence of dermal absorption at any dose level for the groups of rabbits in the control, 0.003 mg/kg, 0.06 mg/kg, and 0.30 mg/kg groups. In all cases the amount of organic fluorine was below the practical quantitation levels and apparently below the detection limits. Electrospray mass spectrometry did detect perfluorooctanesulfonate anion, but again the results are nebulous and below the practical quantitation limits. There does not appear to be dermal absorption of FC-95 at these dose levels. 000354 000003 3 6.1 Circumstances That May Have Affected the Quality of the Data: No problems with this analysis are recognized that could affect the conclusion. However, the doses administrated are so low that even if all the dose had been absorbed it would be difficult to quantitate. In the higher dose group, 2 kg rabbits would receive only 600 ug total. For dermal dosing with analysis at 28 days and a half life on the order of a month, there would be less than 300 ug present in liver even if all the absorbed perfluorooctanesulfonate had gone to the liver. 7.0 CONCLUSION________________________________________________ FC-95 is not dermally absorbed at 0.30 mg/kg dose. This is a very low dose and possibly too low to make an assessment of dermal absorption. 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#6329-130 "Single Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits (Protocol type TP-3016.AB for dosing of animals, tissue collection, etc.) 9.1.2 Analytical protocol AMDT-020895.1 including methods. 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 000355 000004 9.8 Quality Control Practices: See methods 9.9 Test Methods: See Protocol AMDT-020895.1 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 of liver extracts using electrospray mass spectrometry. 000356 000005 5 9.1.1 Protocol and Final Report: HWI#6329-130 "Single Dose Dermal Absorption/Toxicity Study o f T-6049 in Rabbits (Protocol type TP3016.AB for dosing o f animals, tissue collection, etc.) 000357 OOOOOG HAZLETON WISCONSIN POST O F F I C E BOX 7545 M A D I S O N . Wl 5 3 7 0 7 - 7 5 4 5 Sponsor: 3M Toxicology Service Medical Department St. Paul, Minnesota i C O RNING Company FINAL REPORT \ Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits Author: Steven M. Glaza Study Completion Date: June 27, 1995 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-130 P h o n e 608-241-4471 EXPRESS-MAIL DELIVERY Page 1 of 42 330 KINSMAN BLVD 000358 000007 Fax 603-241-7227 M A D I S O N . Wl 53704 Page 2 of 42 QUALITY ASSURANCE STATEMENT HWI 6329-130 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 Management 12/21/94 01/10/95 01/24/95 04/03/95 04/03/95 06/27/95 12/21/94 01/10/95 01/24/95 04/13/95 04/13/95 06/27/95 Protocol Review Animal Observation Protocol Amendment Data/Report Review Data Review Report Rereview 12/22/94 01/10/95 01/24/95 04/14/95 04/14/95 06/27/95 01/10/95 02/10/95 02/10/95 05/10/95 05/10/95 07/10/95 Assurance Unit (*'21-f-T Date 000359 ooooo-s Page 3 of 42 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits HWI 6329-130 Test Material Sponsor Sponsor's Representative Study Director Study Location Study Timetable Study Initiation Date Experimental (In-life) Start Date In-life End Date Experimental Termination Date Study Completion Date T-6049 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 (612) 733-1962 Steven M. Glaza Hazleton Wisconsin, Inc. P.0. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 December 30, 1994 January 7, 1995 February 10, 1995 June 27, 1995 June 27, 1995 000360 OOOOUJ Page 4 of 42 HWI 6329-130 KEY PERSONNEL Acute Toxicology Laboratory Animal Medicine Steven M. Glaza Study Director Manager Cindy J. Cary, DVM Dipl ornate, ACLAM Supervisor Francis (Bud) W. McDonald Study Coordinator Anatomical Pathology Patricia Padgham In-life Supervisor Thomas E. Palmer, PhD Anatomical Pathologist Rose M. Bridge Report Supervisor Qualitv Assurance Sherry R. W. Petsel Manager Jack Serfort/ Deborah L. Pirkel Supervi sors Necropsy Anne Mosher Supervisor Pathology Data 000361 000010 Page 5 of 42 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 Weights and Bile Volumes Appendix A Protocol Deviations Protocol TP3016.AB Protocol Amendment No. 1 HWI 6329-130 Page 2 3 4 6 8 8 8 9 10 13 14 14 14 15 16 17 19 23 25 27 28 29 41 000362 000011 Page 6 of 42 SUMMARY HWI 6329-130 This study was done to assess the systemic absorption/toxicity and relative skin irritancy of T-6049 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 Distilled water T-6049 T-6049 T-6049 0a 5 100 500 3 3 3 3 3 3* 3* 3 a Administered at a dose volume of 2.0 mL/kg. * One animal sacrificed on Day 2 due to possible broken back and replaced with another female animal. The back of each rabbit was clipped free of hair and a single dose of the respective material at the indicated dose level was administered to the skin of the rabbits. The treatment sites remained intact. The area of application was covered with a gauze bandage secured with paper tape around all edges and overwrapped with Saran Wrap and Elastoplast tape to provide an occlusive dressing for a 24-hour exposure period. Clinical observations were conducted predose and at approximately 1, 2.5, and 4 hours after test or control material administration. Additional clinical observations and twice a day mortality checks were conducted daily thereafter for 28 days. The only exceptions to this were on Days 4 and 6 when the afternoon mortality check was inadvertently not conducted for the two replacement animals. Body weights were determined on Day -4 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, 9, 16, and 23. Replacement animals were bled on Days 1, 2, 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 000363 000012 Page 7 of 42 HWI 6329-130 and weighed (volume only determined for bile). The blood samples (serum and cellular fractions), livers, bile, dermal application sites, and kidneys were sent frozen to the Sponsor after termination of the in-life phase. Application of T-6049 did not result in any test material-related changes in body weight gain. All animals appeared clinically normal throughout the study with the exception of one Group 2 and one Group 3 female animals that were sacrificed on Day 2 due to injury (possible broken backs). These animals were replaced in the study and the replacement animals appeared normal throughout the study. No dermal irritation was observed at the dermal scoring intervals as a result of the application of distilled water or T-6049 at any of the dose levels. There were no test material-related lesions observed at necropsy. 000364 000013 Page 8 of 42 OBJECTIVE HWI 6329-130 The objective of this study was to assess the systemic toxicity/absorption and relative skin irritancy of a test material when applied to the skin of rabbits. REGULATORY COMPLIANCE This study was conducted in accordance with the U.S. Food and Drug Administration's Good Laboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58, with the exception that analysis of the test material mixture prepared for the Group 2 animals for concentration, homogeneity/solubility, and stability was not conducted. All procedures used in this study are in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study did not unnecessarily duplicate any previous work. TEST AND CONTROL MATERIALS Identi fication The test material was identified as T-6049 and described as a clear, colorless liquid. The control material was distilled water and was described as a clear, colorless liquid. Purity and Stability The Sponsor assumes responsibility for test material purity and stability determinations (including under test conditions). Analysis of the test material mixture prepared for the Group 2 animals for concentration, homogeneity/solubility, and stability was not conducted or requested by the Sponsor. The purity and stability of the control material were considered to be adequate for the purposes of this study. Storage and Retention The test and control materials were stored at room temperature. A reserve sample of each test and control material was taken and will be retained in a freezer set to maintain a temperature of -20C 10 for 10 years in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedure (SOP). Any unused test material was returned to the Sponsor after completion of the in-life phase according to HWI SOP. Any remaining control material is retained for other testing and will not be discarded after issuance of the final report. 000365 000014 Page 9 of 42 Safety Precautions HWI 6329-130 The test and control material handling procedures were according to HWI SOPs and policies. TEST SYSTEM Test Animal Adult albino rabbits of the Hra:(NZW)SPF strain were procured from HRP, Inc., Denver, Pennsylvania on December 28, 1994 and maintained at the Hazleton Wisconsin facility at 3802 Packers Avenue, Madison, Wisconsin. Housing After receipt, the animals were acclimated for a period of at least 7 days. During acclimation and throughout the study, the animals were individually housed in screen-bottom stainless steel cages in temperature- and humiditycontrolled quarters. Environmental controls for the animal room were set to maintain a temperature of 19 to 23C, a relative humidity of 50% 20%, and a 12-hour 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 Tibi turn 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 -4. The weight variation of the animals for each group of each sex selected for the study did not exceed 2 standard deviations of the mean weight, and the mean body weights for each group of each sex were not statistically different at the 5% probability level. Two female animals (Nos. F53398 and F53355) were replaced after they were treated due to possible broken backs. These animals were replaced with two other female animals (Nos. F52999 and F53351) which were treated in the same manner. 000366 000015 Page 10 of 42 Study Design HWI 6329-130 Animals weighing from 2,018 to 2,625 g at initiation of treatment were placed into the following study groups: Group Dose Level Number of Animals Test Material (mq/kq) Mal es Femal 1 (Control) 2 3 4 Distilled water T-6049 T-6049 T-6049 Oa 5 100 500 3 3 3 3 3 3* 3* 3 a Administered at a dose volume of 2.0 mL/kg. * One animal sacrificed on Day 2 due to possible broken back and replaced with another female animal. This animal weighed 2,625 g at initiation. Justification for Species Selection Historically, the New Zealand White albino rabbit has been the animal of choice because of the large amount of background information on this species. PROCEDURES Preparation of Exposure Area On the day before test material application, the back and, if necessary (to obtain unblemished skin), the flanks of each rabbit was clipped free of hair. The clipped area made up approximately 20% of the total body surface area. The intact skin of the test sites was inspected for interfering lesions, irritation, or defects that would preclude the use of any of the animals. The animals were clipped as needed throughout the study to aid in visualizing the application sites. Dose Administration All animals received a single administration of the respective test or control material. The day of treatment was designated as Day 1. Group 1. An individual dose (2.0 mL/kg) was calculated and measured based on each animal's body weight on the day of treatment. The control material (distilled water) was applied evenly to the test site at a rate of approximately 0.05 mL/cm. 000367 O O O O IC Page 11 of 42 HWI 6329-130 Groups 2, 3, and 4 . For the Group-2 animals (5 mg/kg), the test material (T-6049) was mixed with distilled water to a concentration of 500 mg/mL and applied at a dose volume of 0.01 mL/kg. The mixture was stored at room temperature until administered. The test material was administered undiluted to the test sites of the Groups 3 and 4 animals (100 or 500 mg/kg, respectively) using the average bulk density of 0.99 g/mL to determine the dose volume for each dose level (0.10 and 0.51 mL/kg, respectively). The area of exposure for the 5, 100, and 500 mg/kg dose levels was 4, 25 (16 for replacement animal), and 100 cm2, respectively. The approximate rate of application ranged from 0.006 to 0.014 mL/cm2. An individual dose of the respective test material or test material mixture was calculated for each animal based on its body weight on the day of treatment. 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. The only exceptions to this were on Days 4 and 6 when the afternoon mortality check was inadvertently not conducted for the two replacement animals. Body weights were determined for randomization purposes on Day -4, 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. 000368 000017 Page 12 of 42 Sample Collections HWI 6329-130 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, 9, 16, and 23. Replacement animals were bled on Days 1, 2, 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 for all animals, with the exception of the animal sacrificed and necropsied on Day 2, 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 the collected 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. 000369 oooois Page 13 of 42 Body Weights RESULTS HWI 6329-130 Individual and mean body weights are in Table 1. All animals exhibited body weight gains from Day 1 to Day 29. Clinical Observations Individual clinical signs are in Table 2. All animals appeared normal throughout the study with the following exceptions: One Group 2 female (No. F53398) treated with T-6049 at 5 mg/kg appeared to have injured its back at the time of the 24-hour blood collection. This animal was sacrificed, necropsied, and replaced with No. F52999. The replacement animal appeared normal throughout the study. One Group 3 female (No. F53355) treated with T-6049 at 100 mg/kg was found prior to completion of the exposure period with its leg caught in the cage floor, appeared to have injured its back, and was sacrificed. Since this animal did not receive its full test material exposure, the animal was discarded without a gross necropsy examination. The animal was replaced with No. F53351. The replacement animal appeared normal throughout the study. Dermal Irritation Individual dermal irritation scores are in Table 3. The control material produced no dermal irritation. No dermal irritation was observed in the animals treated with T-6049 at any of the dose levels. Pathology Individual animal pathology comments are presented in Table 4. Individual animal tissue weights and bile volumes are in Table 5. The treated skin in two animals dosed at 0 mg/kg, two animals dosed at 5 mg/kg, and one animal dosed at 500 mg/kg was diffusely red or had multiple red areas of variable size. These findings are not considered to be test material-related. There were no lesions observed in any of the remaining animals. Page 15 contains a pathology report by the study pathologist. 000370 000010 Page 14 of 42 HWI 6329-130 DISCUSSION The acute systemic absorption/toxicity and relative skin irritancy of T-6049 were evaluated in male and female albino rabbits when administered as a single dermal application. There were no test material-related changes in body weight gain or in-life clinical findings at any of the dose levels. No test material-related dermal irritation was observed during the study. There were no test material-related lesions observed at necropsy. SIGNATURE Steveni M. G l a z a Study Director Acute Toxicology Date REFERENCE 1. Draize, J. H., "Acute Dermal Toxicity (Single Exposure)," In: Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics - Dermal Toxicity, Association of Food and Drug Officials of the U.S., pp. 54-56 (1959). 000371 0 0 0 0 .0 Page 15 of 42 PATHOLOGY REPORT HWI 6329-130 There were six rabbits (three males and three females) each from four dose levels of 0, 5, 100, and 500 mg/kg of body weight euthanized and necropsied at the termination of the study. One female (Animal No. F53398) dosed at 5 mg/kg of body weight was sacrificed on Day 2 because of an apparent broken back. This animal was replaced with another female (Animal No. F52999). 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, the treated skin in some animals was diffusely red or had multiple red areas of variable size. This included two animals dosed at 0 mg/kg, two animals dosed at 5 mg/kg, and one animal dosed at 500 mg/kg. There were no visible lesions in any of the remaining animals. The liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from each of these animals, and both kidneys from one male and one female in each group were collected. The tissue samples were weighed (volume only determined for bile), frozen, and sent to the Sponsor. The tissues from the female dosed at 5 mg/kg and sacrificed on Day 2 were collected but were not weighed. After necropsy, the animals were discarded. The animal sacrificed on Day 2 had multiple dark red areas of variable size within the skeletal muscle of the lumbar-sacral region of the spinal column. These findings correlated with the clinical observation of an apparent broken back. Pathologist (6329-130.slh) 03/22/95 Date 000372 000021 Animal Number F53358 F53359 F53353 Mean F53402 F53364 F53352 Mean F53360 F53400 F53346 Mean F53365 F53354 F53347 Mean Page 16 of 42 Table 1 Individual and Mean Body Weights (g) HWI 6329-130 Male_________________ Random ization Dav Dav -4 1 29 _______________ Female Random Animal ization Number Dav -4 Dav 1 29 Group 1 (Controll - Distilled Water (0 ma/kal 2,066 2,030 2,136 2,077 2,231 2,093 2,312 2,816 2,670 3,012 F53404 F53405 F53356 2,200 2,426 2,110 2,212 2,833 2,245 Group 2 - T--6049 (5 ma/kal 2,286 2,474 2,224 2,328 2,997 2,911 2,685 2,864 2,288 2,244 2,102 2,211 2,382 2,364 2,018 2,938 2,923 2,622 F53399 F53349 F533983 F52999 2,095 2,376 2,331 2,485 2,255 2,828 2,319 Group 31 - T-6049 n O O ma/kal 2,122 2,492 2,419 2,625 2,413 2,580 2,919 - 3,272 2,924 2,157 2,076 2,115 2,194 2,122 2,211 2,943 2,696 2,694 F53350 F53355b F53397 F53351 2,320 2,280 2,145 2,059 2,409 2,297 2,245 2,253 2,996 - 2,706 2,606 2,116 2,176 2,778 2,175 Group 4. - T-6049 (500 ma/kal 2,302 2,769 2,126 2,325 2,103 2,276 2,478 2,152 2,951 3,008 2,646 F53403 F53000 F53345 2,170 2,241 2,306 2,270 2,268 2,341 2,860 2,914 2,951 2,185 2,302 2,868 2,239 2,293 2,908 a Animal No. F53398 was originally selected by the randomization program for use in the study and was treated. This animal was sacrificed after completion of the exposure period due to a possible broken back and was replaced with No. F52999. The body weights for No. F53398 are not included in the group means. b Animal No. F53355 was originally selected by the randomization program for use in the study and was treated. This animal was sacrificed before completion of the exposure period due to a possible broken back and was replaced with No. F53351. The body weights for No. F53355 are not included in the group means. Not applicable. 000373 ooooaxi Sex Male Female Page 17 of 42 Table 2 Individual Clinical Signs HWI 6329-130 Animal Number Observation 1-4 Hours Dav (Day 1) 2 3 throuah 29 GrouD 1 (Control 1 - Distilled Water (0 ma/kal F53358 Appeared normal / F53359 Appeared normal // F53353 Appeared normal // F53404 Appeared normal / F53405 Appeared normal F53356 Appeared normal // / Male Female GrouD 2 - T-6049 (5 ma/kal F53402 Appeared normal / F53364 Appeared normal / F53352 Appeared normal / F53399 Appeared normal / F53349 Appeared normal F53398 Appeared normal Possible broken back (at time of 24-hour bleeding interval) Moribund sacrifice / - F529993 Appeared normal / / / / / / - / / / / / / / / / / Condition existed. - Condition not evident. a Animal No. F52999 replaced Animal No. F53398. 000374 00003 Page 18 of 42 Table 2 (Continued) Individual Clinical Signs HWI 6329-130 Animal Sex Number Observation 1-4 Hours Dav (Day 1) 2 3 throuah 29 GrouD 3 - T-6049 (100 mq/kq) Male F53360 Appeared normal / / F53400 Appeared normal // / Female F53346 F53350 F53355 Appeared normal Appeared normal Appeared normal Leg caught in cage floor (possible broken back) Moribund sacrifice // / - _/ -/ / / F53397 Appeared normal / F533 51a Appeared normal // / Male Female F53365 F53354 F53347 F53403 F53000 F53345 GrouD 4 - T-6049 (500 mq/kq) Appeared normal / Appeared normal Appeared normal Appeared normal / Appeared normal Appeared normal / / / / / / / / / / / / / Condition existed. - Condition not evident. a Animal No. F53351 replaced Animal No. F53355. 000375 0000^4 Page 19 of 42 Table 3 Individual Dermal Irritation Scores HWI 6329-130 Group 1 (Control) - Distilled Water (0 mg/kg) Dermal Reaction Males________ Study Dav 1 _2_ 4 _8_ Animal No. F53358 _______ Females Studv Dav _2_ 4 _8_ Animal No. F53404 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F53359 0000 0000 0000 0000 0000 0000 Animal No. F53405 0000 0000 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F53353 0000 0000 0000 0000 0000 0000 Animal No. F53356 0000 0000 0000 0000 0000 000 0 000376 OOOOM Page 20 of 42 Table 3 (Continued) Individual Dermal Irritation Scores HWI 6329-130 Group 2 - T-6049 (5 mg/kg) Males Study Dav Dermal Reaction _1_ 2 4 8 Females Studv Dav 1?4 8 Animal No. F53402 Animal No. F53399 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 000 0 0000 Animal No. F53364 Animal No. F53349 Erythema Edema Atonia Desquamation Coriaceousness Fi ssuring 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 Animal No. F53352 Animal No. F53398a Erythema Edema Atonia Desquamation Coriaceousness Fi ssuring 0000 0000 0000 0000 0000 0000 00 00 _ 00 00 00 00. Animal No. F52999b Erythema Edema Atonia Desquamation Cori aceousness Fissuri ng 0000 0000 0000 000 0 000 0 000 0 a Animal replaced with No. F52999. b Replacement animal for No. F53398. - Not applicable. 000377 0000^6 Page 21 of 42 Table 3 (Continued) Individual Dermal Irritation Scores HWI 6329-130 Group 3 - T-6049 (100 mg/kg) Dermal Reaction Males Studv Dav 1248 Animal No. F53360 Females Studv Dav 1 _2_ 4 8 Animai No. F53350 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring Animal No. F53400 0000 00 00 0000 0000 0000 0000 Animai No. F53355a 0. 00000- - - - Animal No. F53346 Animai No. F53397 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 Animai No. F53351b Erythema Edema Atonia Desquamation Coriaceousness Fissuring 0000 0000 0000 0000 0000 0000 Animai replaced with No. F53351. Replacement animal for No . F53355. - Not applicable. 000378 0000*27 Page 22 of 42 HWI 6329-130 Table 3 (Continued) Individual Dermal Irritation Scores Group 4 - T-6049 (500 mg/kg) Dermal Reaction Males________ Study Day 1 _ 2 _ 4 _8_ _______ Females Study Dav 124 8_ Animal No. F53365 Animal No. F53403 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 00 0 0 00 0 0 00 0 0 0000 00 0 0 00 00 0000 0000 0000 0000 0000 0000 Animal No. F53354 Animal No. F53000 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 00 0 0 00 0 0 00 0 0 00 0 0 0000 00 0 0 0000 0000 0000 0000 0000 0000 Animal No. F53347 Animal No. F53345 Erythema Edema Atonia Desquamation Coriaceousness Fissuring 00 0 0 0000 0000 00 0 0 0000 00 0 0 0000 0000 0000 0000 0000 0000 000379 OOOO^S Animal Number Sex F53358 F53359 F53353 M M M F53404 F53405 F53356 F F F F53402 F53364 F53352 F53399 M M M F F53349 F F53398a F F52999b F Page 23 of 42 HWI 6329-130 Table 4 Individual Pathology Comments Test Dav Died Sacrificed NecroDsv Observation GrouD 1 (Control) - Distilled Water (0 ma/kq) 29 No visible lesions. 29 No visible lesions. 29 The skin has multiple red areas within the dermal application site, up to 3 cm x 2 cm. Collected with routine section. 29 No visible lesions. 29 No visible lesions. 29 The treated skin area appears diffusely dark red. Collected with routine section. Group 2 -- T-6049 (5 mo/ka) 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 The skin has diffusely dark red areas along the entire dorsal lumbar and thoracic region. Collected with routine section. 29 The skin has multiple red areas on the application site up to 1 cm in diameter. Collected with routine section. 2 There are multiple dark red areas within the skeletal muscle of the lumbar sacral region surrounding the spinal column, up to 1.5 x 2.0 cm. 29 No visible lesions. - Not applicable, a Animal replaced with No. F52999. b Replacement animal for No. F53398. 000380 oooorj Page 24 of 42 Table 4 (Continued) Individual Pathology Comments Animal Test Dav Number Sex Died Sacrificed NecroDsv Observation Group 3 - T-6049 (100 ma/ka) F53360 M - 29 No visible lesions. F53400 M - 29 No visible lesions. F53346 M - 29 No visible lesions. F53350 F - 29 No visible lesions. F53397 F - 29 No visible lesions. F53351 F - 29 No visible lesions. HWI 6329-130 F53365 F53354 F53347 F53403 F53000 M M M F F - _ F53345 F _ GrouD 4 - T-6049 f500 ma/ka1 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 No visible lesions. 29 The skin has multiple red areas on the application site, up to 1 cm x 3 cm. Collected with routine section. 29 No visible lesions. - Not appiicable. 0003S1 000030 Sex Male Female Male Female Page 25 of 42 Table 5 Individual Animal Tissue Weights and Bile Volumes HWI 6329-130 Animal Number ____________ Weight (q)_____________ Dermal Appli Liver Kidnevs cation Site Bili Volume GrouD 1 (Control 1 - Distilled Water (0 ma/kal F53358 F53359 81.381 73.857 - 0.783 0.761 1.4 0.8 F53353 F53404 F53405 F53356 77.411 15.761 0.644 70.277 12.585 0.620 78.270 - 0.775 74.687 - 0.915 Group 2 - T-6049 (5 mq/kq) 0.6 1.8 1.1 0.5 F53402 78.731 14.611 0.468 2.3 F53364 F53352 F53399 F53349 84.099 67.613 65.818 77.596 - 14.211 0.641 0.706 0.546 0.489 1.8 1.8 0.8 0.9 F53398a - - - - F52999b 87.510 0.660 0.5 - Not applicable. a Moribund sacrifice on Day 2 (01/08/95) due to possible broken back. Tissues were collected, however, the corresponding weight or volume was not taken since this requirement became effective on 01/24/95. Animal replaced with No. F52999. b Replacement animal for No. F53398. 000382 000031 Sex Male Female Page 26 of 42 Table 5 (Continued) Individual Animal Tissue Weights and Bile Volumes HWI 6329-130 Animal Number _____ ______ Weight (al_____________ Dermal Appli Liver Kidnevs cation Site GrouD 3 - T-6049 (100 ma/kal Bili Voi urne F53360 F53400 F53346 F53350 F53397 F53351 76.347 74.958 75.414 74.050 74.882 80.686 15.873 - 15.120 - 0.802 0.395 0.570 0.784 0.801 0.279 0.9 1.6 1.3 1.4 0.9 0.5 Male Female F53365 F53354 F53347 F53403 F53000 F53345 GrouD 4 - T-6049 (500 ma/kal 82.598 - 0.441 70.968 15.234 0.540 83.696 - 0.716 70.624 15.427 0.910 79.617 - 0.714 81.341 - 0.633 1.8 1.3 2.0 1.4 0.8 1.8 Not appiicable. 000383 oooooy Page 27 of 42 HWI 6329-130 APPENDIX A Protocol Deviations Protocol TP3016.AB Protocol Amendment No. 1 000384 000033 Page 28 of 42 Protocol Deviations HWI 6329-130 Protocol Page 7, 7. Experimental Design, C. Observation of Animals, (1) Clinical Observations, First Sentence. 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. ________Actual Procedure_____ On Day 4 and 6, the afternoon mortality check was inadvertently not conducted for the two replacement animals (Group 2, No. F52999, and Group 3, No. F53351). Page 7, 7. Experimental Design, C. Observation of Animals, (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). The body weight of the Group 2 animal that was sacrificed on Day 2 was inadvertently not documented. These deviations are not considered to have had an adverse effect on the outcome of the study. 000385 000034 HAZLETON WISCONSIN P O S T O F F I C E 8 O X 7S-I5 M A D I S O N . Wl 5 3 7 0 7 7 5-15 Page 29 of 42 -i CORNING Company Sponsor: 3M Toxicology Service Medical Department St. Paul, Minnesota PROTOCOL TP3016.AB Study Title: Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits Date: December 30, 1994 Performing Laboratory: Hazleton Wisconsin, Inc. 3301 Kinsman Boulevard Madison, Wisconsin 53704 Laboratory Project Identification: HWI 6329-130 Phone 608 24 I 44 /i E X P R E S S M A U DELIVERY }30 I 000386 KI NS MAN B ! VD Fax MADISON 000035 6 0 8 2-1 I - 7 2 2 7 Wl 53704 Page 30 of 42 TP3016.AB ) Page 2 STUDY IDENTIFICATION Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits HWI No. Test Material 6329-130 T-6049 Sponsor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 Sponsor's Representative John L. Butenhoff, PhD 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN. 55133-3220 ) (612) 733-1962 Study Director Steven M. Glaza Hazleton Wisconsin, Inc. P.0. Box 7545 Madison, WI 53707-7545 (608) 241-7292 Study Location Hazleton Wisconsin, Inc. Building No. 3 3802 Packers Avenue Madison, WI 53704 Proposed Study Timetable Experimental Start Date Experimental Termination Date Draft Report Date January 7, 1995 February 4, 1995 March 18, 1995 ; 000387 00003 Page 31 of 42 1. Study Single-Dose Dermal Absorption/Toxicity Study in Rabbits TP3016.AB Page 3 2. Purpose To assess the systemic absorption and toxicity and relative skin irritancy of a test material when applied to the skin of rabbits 3. Regulatory Compliance This study will be conducted in accordance with the following Good Laboratory Practice Regulations/Standards/Guidelines: [ ] Conduct as a Nonregulated Study [X] 21 CFR 58 (FDA) [ ] 40 CFR 160 (EPA-FIFRA) [ ] 40 CFR 792 (EPA-TSCA) [ ] C(81)30 (Final) (OECD) [ ] 59 Nohsan No. 3850 (Japanese MAFF) [ ] Notification No. 313 (Japanese MOHW) All procedures in this protocol are in compliance with the Animal Welfare Act Regulations. In the opinion of the Sponsor and study director, the study does not unnecessarily duplicate any previous work. Quality Assurance The protocol, study conduct, and the final report will be audited by the Quality Assurance Unit in accordance with Hazleton Wisconsin (HWI) Standard Operating Procedures (SOPs) and policies. 5. Test Material A. Identi fication T-6049 B. Physical Description (To be documented in the raw data) C . Purity and Stability The Sponsor assumes responsibility for purity and stability determinations (including under test conditions). D. Storage Room temperature ) 000388 000037 Page 32 of 42 TP3016.AB Page 4 E. Reserve Samples Reserve sample(s) of each batch/lot of test and control materials will be taken for this study. The test and control material reserve samples will be stored at HWI in a freezer set to maintain a temperature of -20'C 10*C for 10 years per HWI SOP. The Sponsor will be contacted after 10 years for disposition in accordance with the appropriate regulatory Good Laboratory Practices. F. Retention Any unused test material will be returned to the Sponsor after completion of the in-life phase of the study. G. Safety Precautions As required by HWI SOPs and policies 6. Control Material A. Identification Distilled water B. Physical Description Clear, colorless liquid C. Purity and Stability The purity and stability of this manufactured material is considered to be adequate for the purposes of this study. D. Storage Conditions Room temperature E. Reserve Samples See Section 5. E. Reserve Samples F. Retention Any remaining control material may be used for other testing and will not be discarded after issuance of the final report. G. Safety Precautions As required by HWI SOPs and policies 7. Experimental Design A. Animals (1) Species Rabbit (2) Strain/Source Hra:(NZW)SPF/HRP, Inc. 000389 ooooos Page 33 of 42 ( 3 ) Aae at Initiation Adult TP3016.AB Page 5 (4 ) Weiaht at Initiation 2.0 to 3.0 kg ( 5 ) Number and Sex 12 males and 12 females (6 ) Identification Individual numbered ear tag ( 7 ) Husbandry (a) Housing Individually, in screen-bottom stainless steel cages (heavy gauge) (b) Food A measured amount of Laboratory Rabbit Diet HF #5326 (PHI Feeds, Inc.). The food is routinely analyzed by the manufacturer for nutritional components and environmental contaminants. (c) Water Ad libitum from an automatic system. Samples of the water are analyzed by HWI for total dissolved solids, hardness, and specified microbiological content and for selected elements, heavy metals, organophosphates, and chlorinated hydrocarbons. (d) Contaminants There are no known contaminants in the food or water that would interfere with this study. (e) Environment Environmental controls for the animal room will be set to maintain a temperature of 19*C to 23*C, a relative humidity of 50% +20%, and a 12-hour 1ight/12-hour dark cycle. (f) Acclimation At least 7 days (8) Selection of Test Animals Based on health and body weight according to HWI SOPs. An adequate number of extra animals will be purchased so that no animal in obviously poor health is placed on test. The animals will be placed into study groups using a stratified body weight randomization program within nine days of study initiation. 000390 000039 Page 34 of 42 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) Distilled water 2 T-6049 3 T-6049 4 T-6049 0* 5** 100 500 3 3 3 3 3 3 3 3 * To be administered at a dose volume of 2.0 mL/kg ** To be administered at a dose volume of .01 mL/kg (2) Preparation of Exposure Area On the day before test material application, the back and, if necessary (to obtain unblemished skin), the flanks of each rabbit will be clipped free of hair. The shaved area will constitute approximately 20% of the total body surface area. The treatment sites (intact skin) will be inspected for interfering lesions, irritation, or defects that would preclude the use of any of the animals. The animals will be clipped as needed throughout the study. (3) Dose Administration All animals will receive a single administration of the respective test or control material. The day of treatment will be designated as Day 1. The dose for each animal in Group 2 will be diluted with distilled water and applied at a dose volume of .01 mL/kg. The respective dose for each animal in Groups 3 and 4 will be applied undiluted. All doses in Groups 1-4 will be based on the animal's body weight just before administration and will be spread onto the area of exposure in a thin and uniform a layer. The area of application (Groups 1-4) will be covered with a 10-cm x 10-cm gauze bandage secured with gaper tape around all edges and overwrapped with Saran Wrap and Elastoplast* tape to provide an occlusive dressing. The rabbits will be collared during the 24-hour application period. (4) Reason for Route of Administration The dermal route is a potential route of exposure in humans. ) 000391 000040 Page 35 of 42 TP3016.AB ) Page 7 (5) Removal of Test Material Approximately 24 hours after test or control material application the bandages and collars will be removed and the residual test material will be removed using water or an appropriate solvent, if necessary. C. Observation of Animals (1) Clinical Observations For clinical signs before test or control material administration and for clinical signs and mortality at approximately 1, 2.5, and 4 hours after test material administration (Day 1) and daily thereafter for clinical signs, and twice daily (a.m. and p.m.) for mortality for at least 28 days. Observations may be extended when directed by the study director. (2) Reading of Dermal Irritation Before test or control material administration the initial dermal irritation reading will be made and recorded as the Day 1 reading (Attachment 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, 9, 16, 23, and at experimental termination (Day 29) (b) Number of Animals All ) 000392 000041 Page 36 of 42 TP3016.AB Page 8 (c) Method of Collection Blood samples (approximately 4 mL) will be collected from the marginal ear vein of either ear on Days 1, 2, 4, 9, 16, and 23. Approximately 20 ml of blood (actual volume to be documented in the raw data) will be obtained from the posterior vena cava of each animal sacrificed in a moribund condition or sacrificed at the time of necropsy (Day 29). The samples will be stored at room temperature and then centrifuged, and the separate serum and cellular fractions stored in a freezer set to maintain -20*C 10*C. The separated serum and cellular fractions will be sent frozen on dry ice to the Sponsor after experimental termination. Samples will be shipped to: James D. Johnson 3M E.E. & P.C. Bldg. 2-3E-09 935 Bush Avenue St. Paul, MN 55106 James D. Johnson or alternate will be notified by telephone at (612) 778-5294 prior to the shipment of the samples. D. Pathology (1) Unscheduled Sacrifices and Deaths Any animal dying during the study or sacrificed in a moribund condition will be subjected to an abbreviated gross necropsy examination and all abnormalities will be recorded. Animals in a moribund condition will be anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, and exsanguinated. Tissues, as described in section D. Pathology, (3) Sample Collection, will be collected. After necropsy, the animals will be discarded. (2) Scheduled Sacrifice At termination of the experimental phase (Day 29), surviving animals will be anesthetized with sodium pentobarbital (via injection in the marginal ear vein), bled via the vena cava, exsanguinated, and subjected to an abbreviated gross necropsy examination. The animals will be necropsied in random order and all abnormalities will be recorded. ) 000393 00004a Page 37 of 42 TP3016.AB Page 9 (3) Sample Collection The sites of test and control material application will be washed with lukewarm tap water prior to the necropsy procedure. The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from the first male and female necopsied in each group will be collected and immediately placed in a freezer set to maintain a temperature of -20*C 10*C. After necropsy, the animals will be discarded. The tissues (liver, bile, dermal application site, kidneys) will be sent frozen on dry ice to the Sponsor after experimental termination. The samples will be shipped to the person listed in Section 7.C.(4).(c). The Sponsor is responsible for the retention and disposition of the samples. E. Statistical Analyses No statistical analyses are required. 8. Report A final report including those items listed below will be submitted. Description of the test and control materials Description of the test system Procedures Dates of experimental initiation and termination Tabulation of mortality data by sex and dose level Description of any toxic effects/dermal irritation Tabulation of mean body weights by sex and dose level Gross pathology findings/gross pathology report 9. Location of Raw Data, Records, and Final Report Original data, or copies thereof, will be available at HWI to facilitate auditing the study during its progress and before acceptance of the final report. When the final report is completed, all original paper data, including those item listed below will be retained in the archives of HWI according to HWI SOP. Protocol and protocol amendments Dose preparation records In-1ife records Body weights Dose administration Observations Anatomical pathology records Sample collection records Shipping records Study correspondence Final report (original signed copy) 000394 000043 Page 38 of 42 TP3016.AB Page 10 The following supporting records will be retained at HWI but will not be archived with the study data. Animal receipt/acclimation records Water analysis records Animal room temperature and humidity records Refrigerator and freezer temperature records Instrument calibration and maintenance records T ) ) 000395 000044 Page 39 of 42 PROTOCOL APPROVAL TP3016.AB Page 11 2 - jT 4V&A.sCurff John L. Butenhoff, PhD Sponsor's Representative 3H Toxicology Service Medical Department IDate Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. Ci ) X4esentative '1 4, Quality Assurance UhT Hazleton Wisconsin, Inc (6329-130.protdsk2) Date M s o fa Date ) 00039G 000043 Page 40 of 42 J TP3016.AB ) Page 12 Attachment 1 Scoring Scale for Acute Dermal Reactions Erythema 0 - None 1 - Slight 2 - Moderate 3 - Severe Edema 0 - None 1 - Slight (barely perceptible to well defined by definite raising) 2 - Moderate (raised approximately 1 mm) 3 - Severe (raised more than 1 mm) Atonia 0 - None 1 - Slight (slight impairment of elasticity) 2 - Moderate (slow return to normal) 3 - Marked (no elasticity) Desouamation 0 - None 1 - Slight (slight scaling) 2 - Moderate (scales and flakes) 3 - Marked (pronounced flaking with denuded areas) Coriaceousness 0 - None 1 - Slight (decrease in pliability) 2 - Moderate (leathery texture) 3 - Marked (tough and brittle) Fissurina 0 - None 1 - Slight (definite cracks in epidermis) 2 - Moderate (cracks in dermis) 3 - Marked (cracks with bleeding) 000397 00004; Page 41 of 42 HAZLETON WISCONSIN POST OFFI CE BOX 7545 MADI SON. Wl 53707 75 4 5 a CORNING Company PROTOCOL TP3016.AB Single-Dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits HWI 6329-130 Sponsor Contractor 3M Toxicology Service Medical Department 3M Center, Bldg. 220-2E-02 P.0. Box 33220 St. Paul, MN 55133-3220 Hazleton Wisconsin, Inc 3301 Kinsman Boulevard Madison, WI 53704 Sponsor's Representative Study Director John L. Butenhoff, PhD Steven M. Glaza Amendment No. 1 This amendment modifies the following portions of the protocol: Effective January 10, 1995 1. Page 6. 7. Experimental Design; B. Dose Administration: (31 Dose Administration. Animal Nos. F53355 (Group 3 female) and F53398 (Group 2 female) were sacrificed on Day 2 due to injury (apparent broken backs). These animals will be replaced in this study. Replacement animals will be dosed, bled, and necropsied (includes tissue collection) at the same time as the animals in another 3M sponsored study (6329-137). Add the following as the second paragraph to this section: Due to the sacrifice on Day 2 of one Group 2 female (Animal No. F53398) and one Group 3 female (Animal No. F53355), replacement animals will be treated at the respective dose levels in the same manner as for the other animals in the study. The observations (clinical observations, reading of dermal irritation, and body weights) and the pathology of the animals (unscheduled sacrifices and deaths, scheduled sacrifice, and sample collection) will be conducted in the same manner as for the other animals in the study. The method of blood sample collection will be in the same manner as for the other animals in the study. The frequency of blood collection will be as follows: Before initiation (Day 1), approximately 24 hours post-dose (Day 2), Days 4, 8, 15, 22, and at experimental termination (Day 29). P h_q n e 6 0 8 2 4 1 4 4 71 EXPRESS- MAI L DELIVERY 3 J0 1 KINSMAN BlVO 000398 F a x 6 q.8_-_2jLL;J. 2 0 ( ) O < j 4 >? MADISON Wl 53704 Page 42 of 42 Amendment No. HWI 6329-130 Page 2 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. 2. Page 9. 7. Experimental Design: D. Pathology; (31 Sample Collection. Modify the second sentence in the first and second paragraphs of this section with the following underlined additions: The whole liver, bile, an approximate 1-cm x 1-cm section of the dermal application site from all animals, and both kidneys from the first male and female necropsied in each group will be collected, weighed (volume only determined for bilel. and immediately placed in a freezer set to maintain a temperature of -20*C 10*C. The samples and their corresponding weights or volumes will be shipped to the person listed in Section 7.C.(4).(c). 3. Page 9. 8. Report. Add the following to this section: Individual animal tissue weights and bile volumes PROTOCOL AMENDMENT APPROVAL John L. Butenhoff, PhD Sponsor's Representative 3M Toxicology Service Medical Department Date Steven M. Glaza Study Director Acute Toxicology Hazleton Wisconsin, Inc. O Date ________ d uo-s Representative Quality Assurance Unit Hazleton Wisconsin, Inc. ) Date 2.-0 2 *?r~ 000399 (6329-130.Ami.dsk2) 00004S 9.1.2 A n a l y t i c a l p r o t o c o l A M D T - 0 2 0 8 9 5 . 1 000400 00004!! 3M Environmental Laboratory Protocol - Analytical Study Single-dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits In-Vivo Study Reference Number: HWI#6329-130 Study Number: AMDT-020895.1 Test Substance: FC-95 (T-6049) Name and Address of Sponsor: 3M SCD Division 367 Grove Street St. Paul, MN 55106 Name and Address of Testing Facility: 3M Environmental Technology and Services 935 Bush Avenue St. Paul, MN 55106 Proposed Initiation Date: July 25, 1995 Proposed Completion Date: August 25, 1995 Method Numbers and Revisions: AMDT-M-1-0, Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer-Liver AMDT-M-2-0, Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer AMDT-M-4-0, Extraction of Fluorochemicals from Rabbit Liver AMDT-M-5-0, Analysis of Rabbit Liver Extract for Fluorochenmicals Using Electrospray Mass Spectrometry Author: James D. Johnson Approved By: 000401 000050 l 1.0 PURPOSE The purpose of this study is to assess the dermal absorption of FC-95 (T-6049) in rabbits after a single dermal dose of FC-95. 2.0 TEST MATERIALS 2.1 Test, Control, and Reference Substances and Matrices 2.1.1 Analytical Reference Substance: FC-95, lot 161 or 171. They are equivalent. 2.1.2 Analytical Reference Matrix: Bovine liver and bovine serum 2.1.3 Analytical Control Substance: None 2.1.4 Analytical Control Matrix: Bovine liver and bovine serum 2.2 Source of Materials: 3M ICP/PCP Division (2.1.1), grocery store (2.1.2, 2.1.4 liver), Sigma Chemical Company (2.1.2, 2.1.4 serum) 2.3 Number of Test and Control Samples: Tissues and fluids from 18 test animals and 6 control animals. One animal was replaced on day 2 (animal F53398 in the 5 mg/kg dose group replaced by animal F52999). Tissues and fluids include liver, kidney, serum, cellular fraction, dermal application site and bile. Analysis of these tissues will be at the discretion of the Study Director. 2.4 Identification of Test and Control Samples: The samples are identified using the HWI animal identification number which consists of a letter and five digit number, plus the tissue identity and day identity (serum). 2.5 Purity and Strength of Reference Substance: To be determined by Sponsor. 2.6 Stability of Reference Substance: To be determined by Sponsor. 2.7 Storage Conditions for Test Materials: Room temperature (2.1.1), -20 10C (2.1.2, 2.1.4). Test and Control samples will be received according to AMDT-S-10-0. 2.8 Disposition of Specimens: Biological tissues and fluids will be retained per GLP Regulation for the time period required 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. 000402 000051 2 3.0 EXPERIMENTAL - Overview Rabbits were dosed with T-6049 in a control group, 5 mg/kg, 100 mg/kg, and 500 mg/kg dermally. (See HWI#6329-130 for dosing, animal weights, tissue collection, etc.) Tissues and serum are available from these animals for analysis of total organic fluorine content. At the discretion of the Study Director samples will be analyzed to the extent necessary to provide information to assess the extent of dermal absorption. 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 Fluorochenmicals Using Electrospray Mass Spectrometry 5.0 DATA ANALYSIS_________________________________________________ 5.1 D ata Reporting: Data will be reported as a concentration (weight/weight) of fluoride per tissue or fluid, or as FC-95 per tissue or fluid. Statistics used, at the discretion of the Study Director, may include serum averages and standard deviations of concentrations for different dose groups. If necessary, simple statistical tests such as Student's t test may be applied to determine statistical difference. 6.0 MAINTENANCE OF RAW DATA AND RECORDS__________________ 6.1 Raw Data and Records: Raw data, approved protocol, appropriate specimens, approved final report, and electronic data will be maintained in the AMDT Archives. 000403 0 0 0 0 .% 3 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 Fluorochenmicals Using Electrospray Mass Spectrometry 000404 0000.% 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 5.1a Affected Documents: AMDT-M-2 Fluoride Measurement by Means of an Orion EA940 Expandable Ion Analyzer AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 000405 1 000054 1.0 SCOPE . APPLICABLE COMPOUNDS. AND MATRfCES 1.1 Scope: This method is for the operation of a Dohrmann DX2000 when it is used to extract fluoride from various matrices. The fluoride is typically collected in TISAB solution for analysis with an ion selective electrode. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1.3 Matrices: Biological tissues, particularly liver. 2.0 KEYWORDS____________________________________________________ 2.1 Fluoride, fluorine, extraction, pyrolysis, ionization, ion selective electrode, Dohrmann* halide, DX2000, fluorochemicals. 3.0 PRECAUTIONS_________________________________________________ 3.1 Glassware and exhaust gases can be extremely hot. 3.2 Glassware is fragile, broken glass may cause injuries. 3.3 Pressurized gases, proper compressed gas handling practices required. 3.4 Solvent based samples may flash, may need to allow them to dry down before starting run. 3.5 Potential biohazards due to the biological matrices. Use appropriate personal protective equipment. 4.0 SUPPLIES AND MATERIALS__________________________________ 4.1 Compressed Oxygen, Hydrocarbon free, regulated to 30 PSI. 4.2 Compressed Helium, High Purity Grade, regulated to 45 PSI. 4.3 Quartz glass sample boat with TeflonTM tubing, Dohrmann 890-097 or equivalent. 4.4 Quartz glass combustion tube, Reliance Glass G-9405-012 or equivalent. 4.5 Orion 940999 Total Ionic Strength Adjustment Buffer (TISAB I I ) or equivalent. 4.6 Sample collection vials, HDPE. 4.7 Milli-QTM water 4.8 Polystyrene pipettes. 4.9 Activated Charcoal, E. Merck 2005 or equivalent. 4.10 Hamilton Syringe or equivalent. 4.11 Miscellaneous laboratory glassware 5.0 EQUIPMENT__________________________________________________ 5.1 Rosemount Dohrmann DX2000 Organic Halide Analyzer, modified for fluoride extraction. 5.2 IBM compatible 386 or 486 computer. 5.3 DX2000 software, version 1.00, modified for fluoride extraction. 5.4 Excel Spreadsheet, version 5.0 or greater 6.0 INTERFERENCES_____________________________________________ 6.1 Sample size is limited to approximately 150 mg, depending on sample moisture content. This may vary from matrix to matrix. 000406 2 00005.1 7.0 SAMPLE HANDLING 7.1 Samples are not to be handled with bare hands. Fluoride may leach from the skin to the sample. Use forceps or probe to transfer tissues. 7.2 Samples of liver are cut from frozen liver and placed in a tared and labeled weigh boat. Use a clean scalpel and cutting board. The cutting board and scalpel should be cleaned with water, methanol, or methanol-water solution after each liver is cut. 8.0 CALIBRATION AND STANDARDIZATION 8.1 Preparation of Calibration Standards 8.1.1 The standards required for each project will need to be appropriate for that individual project. Refer to protocol for that project. 8.1.2 Typically 50-500 ppm FC-95 in methanol standards are used. 8.1.3 For rabbit liver studies, use beef liver as the matrix. Cut a piece of frozen beef liver (100 150 mg) and weigh it in a labeled and tared weigh boat. 8.2 Calibration - Overview The normal calibration is the fluoride curve (AMDT-M-2). However, if an optional spiked liver curve is required the procedure listed below is used. 8.2.1 A calibration curve for the DX2000 is generated by spiking samples with known standards and combusting them using the same methods and matrix type as the samples to be tested. 8.2.2 Typically, three replicates of each standard and five concentrations of standards will be spiked. 8.2.3 Standard curve will be plotted as Mass Spiked F (ug) on the x-axis and Standard Mass Recovered F (ug) on the y-axis. Generate a regression curve and calculate the equation for the line and the r^ value. 8.2.4 Mass Spiked F (ug) = (Amount spiked in mL) x ( Cone, of standard in ppm) x (0.6004)* *FC-95 is 60.04% F therefore 0.6004 is the factor used to convert FC-95 to F 8.2.5 Standard Mass Recovered F (ug) = (TISAB volume in mL) x (Orion reading in ppm) 8.3 Calibration - Procedure 8.3.1 Start Up 8.3.1.1 Run 2 or more Clean Cycles when starting instrument each day. More clean cycles may be used if the previous samples contained high concentrations of fluoride. 8.3.2 Blanks 8.3.2.1 Prepare sample using the same methods and type of matrix as the test sample. 8.3.2.2 For rabbit studies, use beef liver as the matrix. Prepare at least 3 samples of beef liver (100 - 150 mg) for blanks. 8.3.2.3 Put sample in Dohrmann boat. Combust each sample as described in section 9.0 and analyze sample according to method AMDT-M-2 for the ion selective electrode analysis. 000407 3 OOOOGG 8J.2.4 For rabbit studies, the meter reading for a blank sample should be 0.03 ppm or lower before proceeding with the calibration. Bum samples until this limit is reached, or until in the judgement of the operator the reading is stable with respect to historical readings (previous 48 hours). 8.3.2.5 For non-rabbit studies, the blank readings should reach a predetermined ion concentration before proceeding with the calibration. 8.3.2.6 It may be necessary to mix approximately 50 mg of charcoal with the sample to aid combustion. 8.3.3 Standard Curve 8.3.3.1 Weigh out at least 15 matrix samples (5 standards with 3 replicates each) in tared and labeled weigh boats. For rabbit studies, weigh 100-150 mg beef liver samples. Record weights in study data. Store the matrix samples on dry ice or ice packs to keep them frozen until used. 8.3.3.2 Place weighed beef liver sample in Dohrmann sample boat. 8.3.3.3 Start with the lowest standard concentration. Using a Hamilton syringe, eject a fixed quantity of the standard on or in the matrix. For rabbit studies, use 4 uL of standard and eject it on or in the beef liver. 8.3.3.4 At least 3 replicates should be used for the lowest standard concentration; more replicates may be used at the discretion of the analyst. 8.3.3.5 Combust the sample as described in section 9.3 and analyze according to AMDT-M-2. 8.3.3.6 Run all 15 standards. If one replicate is significantly different from the other two replicates, run another sample for that standard. Indicate in data that the new replicate replaces the old replicate and that the new replicate will be used to calculate the regression curve. 8.3.3.7 When all standards have been run, calculate the r^. r^ must be at least 0.95. If it is not at least 0.95, consult with supervisor. 8.3.3.8 A new standard curve should be run when the combustion tube or sample matrix is changed. New standard curve may also be run at the discretion of the analyst. 8.4 Storage Conditions for Standards 8.4.1 Storage requirements for standards are dependent on the individual standards used. Typically, standards are stored at room temperature in plastic screw top bottles. 8.4.2 New FC-95 standards should be prepared at least once a month. 9.0 PROCEDURES______________________________ 9.1 Typical Operating Conditions: 9.1.1 Combustion tube temperature = 950C. 9.1.2 Oxygen and Helium flow = 50 cc/minute. 9.1.3 Vaporization/Drying time = 240 seconds. 9.1.4 Bake time = 300 seconds. 9.2 Start Up Procedure: 9.2.1 If the program is not started, start the EOX program on the PC. 9.2.2 Open the SYSTEM SETUP window. 9.2.3 Put the furnace module and the cell in the READY mode. 9.2.4 Close the SYSTEM SETUP window. 000408 OOOO.77 9.2.5 When the oven has reached the READY temperature, run the CLEAN BOAT program found in the CELL CHECK menu. 9.2.6 See AMDT-EP-3 for details of the Dohrmann software. 9.3 Sample Extraction Procedure: 9.3.1 Open the SAMPLE HATCH and place the sample in the BOAT. It may be necessary to mix approximately 50 mg of charcoal with the sample to aid combustion. If this is done, charcoal should also be mixed in while establishing the baseline and when generating the standard curve. 9.3.2 Close SAMPLE HATCH. 9.3.3 Add appropriate volume of TISAB solution or 1:1 TISAB:Milli-QTM water mixture to a labeled sample collection vial. Typically 0.6 mL to 15 mL are used. For rabbit studies, use 1.0 or 2.0 mL of 1:1 TISAB:Milli-QTM water mixture. 9.3.4 Place the vial so that the tip of the COMBUSTION TUBE is in the TISAB at least 0.25 inches. Gases released during pyrolysis must bubble through the TISAB. 9.3.5 Run the EOX-SOLIDS program found in the RUN menu. 9.3.6 When the EOX program is finished, remove the collection vial from the combustion tube. 9.3.7 If undiluted TISAB was used to collect the sample, add an equal volume of Milli-QTM water to the TISAB to make 1:1 TISAB:Milli-QTM. 9.3.8 Rinse the end of the combustion tube with Milli-QTM water and wipe with a KIMWIPE to remove any TISAB remaining on the tube. 9.3.9 Open the sample hatch and remove any remaining ash from the boat. Ash can be removed with a cotton tipped applicator or vacuumed out. It may be necessary to scrap particles off the bottom with a spatula or other similar device. A drop of Milli-QTM water may be added to the boat to aid in the Clean Cycle. 9.3.10 Close the hatch. 9.3.11 Run the CLEAN BOAT program. 9.3.12 Sample is ready for analysis by ion selective electrode (AMDT-M-2). 9.4 Sample Calculations 9.4.1 Use the standard curve to calculate the sample value. 9.4.2 Sample Mass Recovered F (ug) = (TISAB vol in mL) x (Orion reading in ppm - intercept") (Slope) 10.0 VALIDATION 10.1 Quality Control 10.1.1 Daily Start Up Check Samples: Once the standard curve is established, each day 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% of the standard curve, then a third replicate is not necessary. 10.2 Precision and Accuracy: See method development analysis and sample analysis in Fluoride Notebooks 2,3, and 5. Precision and accuracy varies when analyzing samples of different matrices and different reference compounds. 10.3 O ther Validation Parameters: NA 000409 5 OOOO.j 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 of the three replicates are within 20% of each other and the outlier is greater than 200% of the average of those two or less than 50% of the average of those two. Any such outliers should be pointed out in the data and noted in the Final Report along with the reason it was considered an outlier. 12.0 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 of an Orion EA940 Expandable Ion Analyzer 13.3 AMDT-EP-3 Routine Maintenance of a Modified Dohrmann DX2000 Organic Halide Analyzer 14.0 REVISIONS_____________________________________________________ Revision Number Reason for Change Revision Date 000410 6 OOOO.TJ 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: CL^ux Grodt/ Leader /j ----- " M/Z-s Date Quality Assurance Date Software: MS Word 5.1a Affected Documents: AMDT-M-1 Thermal Extraction of Fluoride by Means of a Modified Dohrmann DX2000 Organic Halide Analyzer ^00411 1 OOOOCiO 1.0 SCOPE . APPLICABLE COMPOUNDS. AND MATRICES 1.1 SCOPE: This method is for the calibration and operation of an Orion EA940 Expandable Ion Analyzer. 1.2 APPLICABLE COMPOUNDS: Fluoride. 1.3 APPLICABLE MATRICES: Liquid samples in an appropriate buffer solution. Preferred pH of 6.0. 2.0 KEYWORDS___________________________________________________ 2.1 Fluoride, fluorine, ion selective electrode 3.0 PRECAUTIONS________________________________________________ 3.1 No hazards identified with this method. 4.0 SUPPLIES ANT) MATERIALS____________________________ 4.1 Orion 940999 Total Ionic Strength Adjustment Buffer II (TISABII) or equivalent. 4.2 Orion Model 900001 electrode filling solution (AgCl) or equivalent. 4.3 Orion 940907 100 ppm fluoride standard or equivalent. 4.4 Milli-QTM water or equivalent. 4.5 Magnetic stir bars. 4.6 Lab tissues. 4.7 Sample collection vials. 4.8 Plastic 100 mL volumetric flasks. 4.9 Polystyrene pipettes. 4.10 Miscellaneous laboratory glassware. 5.0 EQUIPM ENT___________________________________________ 5.1 Orion Model EA940 Expandable Ion Analyzer or equivalent. 5.2 Orion Model 960900 Solid State Combination Fluoride electrode or equivalent. 5.3 Magnetic Stir Plate. 5.4 IBM compatible 386 or 486 computer (only needed if using Orion 3E software). 5.5 Orion RS232 interface cable (only needed if using Orion 3E software). 5.6 Microsoft Excel 5.0 (only needed if using Orion 3E software). 6.0 INTERFERENCES_______________________________________ 6.1 It is recommended that the pH be at or near 6.0. A 1:1 mixture of TISAB and sample/MilliQTM water will generally bring sample to pH of 6.0. 6.2 Sample temperature may effect fluoride measurement. It is recommended that the sample be at room temperature as the standards were when the meter was calibrated. 6.3 The rate the samples are stirred at should be consistent with the rate the standards were stirred. 000412 2 OOOOC1 6.4 Air bubbles trapped under electrode can give erroneous readings. Make sure no air is trapped under electrode. 7.0 SAMPLE HANDLING_________________________________ 7.1 No special handling necessary. 8.0 CALIBRATION AND STANDARDIZATION________________________ 8.1 Preparation of Calibration Standards 8.1.1 Measure 50 mL o f TISAB II into 5 100 mL plastic volumetric flasks. 8.1.2 Label the flasks as 0.05, 0.1, 0.5, 1.0, and 1.5 ppm F-, along with the date and your initials. 8.1.3 Pipette 0.05, 0.1, 0.5, 1.0, and 1.5 m L of 100 ppm fluoride standard into the appropriately labeled flasks. 8.1.4 Add approximately 30 mL of Milli-QTM water to each flask. 8.1.5 Shake the flasks to mix the solutions. 8.1.6 Eliminate air bubbles from the flasks by tipping the flasks on their sides and rolling the air in the flasks over the air bubbles. 8.1.7 Bring the volume in the flasks up to the 100 mL mark with Milli-QTM water. 8.1.8 Invert and shake the flasks for the final mixing. 8.1.9 Record standards in Standards Log Book. 8.2 Calibration 8.2.1 If necessary, remove tape from electrode filling hole. 8.2.2 Invert probe to wet top seal. 8.2.3 Eject a few drops of filling solution from bottom of electrode to wet lower seal. 8.2.4 Fill the electrode with filling solution. 8.2.5 The meter and the F- electrode are typically calibrated by direct measurement with no blank correction, using standards with concentrations of 0.05, 0.1,0.5, 1.0, and 1.5 ppm F-, following the manufacturer's instructions. 8.2.6 Record the slope in the appropriate log book. 8.2.7 Clean the electrode by rinsing with Milli-QTM water and wiping the sides down with lab tissues. 8.3 Storage Conditions for Standards 8.3.1 Calibration standards are stored at room temperature. 9.0 PROCEDURES___________________________________________________ 9.1 Calibration and Measurement, Standard method: 9.1.1 The sample to be measured needs to be mixed with TISAB using the proportions recommended by the TISAB manufacturer. 9.1.2 Place a stir bar in the sample and place the sample on the stir plate. 9.1.3 Allow the sample to mix for a few seconds before inserting the electrode. When the electrode is inserted, make sure there are no air bubbles trapped under the electrode. 9.1.4 The sample should be the same temperature as the calibration standards and stirred at the same rate as the calibration standards. 9.1.5 When the readings have stabilized, record the reading in the appropriate log book. 000413 OOOOta 9.2 Calibration And Measurement, Using Orion 3E Software: 9.2.1 Calibration: 9.2.1.1 Follow steps 8.2.1 to 8.2.4. 9.2.1.2 Press Function Key #8 (F8). 9.2.1.3 The computer screen will ask you to confirm the number of standards to be used, concentration of the standards, and whether or not a blank is to be included in the calibration. Make any necessary changes to the information presented and click on CONTINUE. 9.2.1.4 Place the electrode in the first standard on the stir plate and click on CONTINUE. 9.2.1.5 Observe the readings on the graphic display on the computer. When the readings have stabilized, press ACCEPT READING. 9.2.1.6 Repeat step 9.2.1.4 and 9.2.1.5 for the remaining standards. 9.2.1.7 After the final standard, the computer will display the slope of the curve, as well as the intercept and correlation. Record the slope, intercept, and correlation in the appropriate log book and click on CONTINUE. The calibration data is automatically copied to C:\Orion\Data\Calib.txt. 9.2.2 D ata Spreadsheet: 9.2.2.1 Select either NEW or OPEN from the FILE menu to open a new or existing spreadsheet to store data in. 9.2.2.2 Record the name of the spreadsheet used in the appropriate log book. 9.2.3 Fluoride Measurement: 9.2.3.1 Follow steps 9.2.1 through 9.2.4 9.2.3.2 Enter the name of the sample in the appropriate place on the screen. 9.2.3.3 Click on the NEW SAMPLE button 9.2.3.4 When the readings have stabilized, click on the RECORD button and write the result in the appropriate log book. 10.0 VALIDATION__________________________________________________ 10.1 Quality Control: 10.2 Precision and Accuracy 10.3 O ther Validation Param eters According to Reference 13.2, the range of detection is 0.02 ppm fluoride up to a saturated solution of fluoride. 11.0 DATA ANALYSTS______________________________________________ 11.1 Calculations None necessary. 11.2 Analyzing the Data None necessary. 12.0 ATTACHMENTS_______________________________________________ None 13.0 REFERENCES 000414 4 oooog;j 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 000415 5 0000G4 3M Environmental Laboratory Method Extraction of Fluorochemicals from Rabbit Livers SOP Identification Number: AMDT-M-4 Revision Number: 0 Adoption Date: Revision Date: None Author: Dave Christenson/Cynthia Weber Approved By: roup Leader /0-3)-tS Date Quality Assurance Date Software: MS Word, 6.0 Affected Documents: M-5, Analysis of Rabbit Extract for Fluorochemicals Using Electrospray Mass Spectroscopy. 000416 l 000005 1.0 SCOPE_______________________________________________________ 1.1 Scope: This method is for the extraction of fluorochemicals from rabbit livers. Ethyl acetate is used to extract fluorochemicals from the livers for analysis by electrospray mass spectroscopy. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1 .3 M atrices: Rabbit Livers. 2.0 KEYWORDS__________________________________________________ 2 .1 Fluorochemicals, rabbit livers, electrospray mass spectrometer, fluorinated compounds, extraction. 3.0 PRECAUTIONS____________________________________________ _ 3 .1 Use gloves when handling the rabbit livers, they may contain pathogens. 4.0 SUPPLIES AND MATERIALS___________________________________ 4.1 Supplies 4 . 1 . 1 Syringe, capable of measuring 100 jiL 4 . 1 . 2 Eppendorf type or disposable pipets 4 . 1 . 3 Gloves 4 . 1 . 4 Plastic grinding tubes 4 . 1 . 5 Plastic centrifuge tubes, 15 mL 4 . 1 . 6 Labels 4 . 1 . 7 Nitrogen 4 . 1 . 8 Timer 4 . 1 . 9 Filters, Titan nylon syringe filters, 0.2 Jim. 4 . 1 . 1 0 Analytical pipets: glass volumetric pipets. 4 . 1 . 1 1 Disposable plastic 3 cc syringes. 4 . 1 . 1 2 Crimp cap autovials. 4.2 Reagents 4 . 2 . 1 Aqueous Ammonium Acetate (Aldrich), approx. 250 ppm: Prepare a 2500 ppm aqueous solution of ammonium acetate by adding 250 mg ammonium acetate to a 100 mL volumetric flask and dilute to volume with Milli-Q water. Dilute this solution 1:10 for a 250 ppm solution. 4 . 2 . 2 Sodium carbonate/Sodium Bicarbonate Buffer (J.T. Baker), (NajCOj/NaHCC^) 0.25 M: Weigh 26.5 g of sodium carbonate (Na^Oj) and 21.0 g of sodium bicarbonate (NaHC03) into a 1 L volumetric flask and bring to volume with Milli-Q water. 4 . 2 . 3 Dilute acetonitrile solution, dilute acetonitrile 1:1 with Milli-Q water. 4 . 2 . 4 Ethyl Acetate 4 . 2 . 5 Methanol 4 . 2 . 6 Milli-Q water 4 . 2 . 7 1H,1H,2H,2H - perfluorooctanesulfonic acid (Aldrich) 4 . 2 . 8 FC-95 (3M Specialty Chemical Division) 000417 2 oooogc; 5.0 EQUIPMENT______________________________ 5 .1 Ultra-Turrax T25 Grinder for grinding liver samples. 5 .2 Vortex mixer 5 .3 Centrifuge 5 .4 Shaker 5 .5 Analytical Evaporator 6.0 INTERFERENCES____________________ 6 .1 There are no known interferences at this time. 7.0 SAMPLE HANDLING___________________________________________ 7 .1 The rabbit livers are received frozen, and must be kept frozen until the extraction is performed. 8.0 CALIBRATION AND STANDARDIZATION_____________________ 8 .1 Preparation of Internal Standards 8 . 1 . 1 Prepare an internal standard of approximately 12 ppm 1H,1H,2H,2Hperfluorooctanesulphonic acid to be added to each liver sample. 8 . 1 . 2 Weigh at least 0.1 g of lH,lH,2H,2H-perfluorooctanesulphonic acid into a 100 mL volumetric flask. Record the actual weight. 8 . 1 . 3 Bring it up to volume with methanol, this is the stock standard. 8 . 1 . 4 To a 250 mL volumetric flask, add 3 mLs of the stock standard and bring to volume with Milli-Q water. Calculate the actual concentration of the standard. actual mg perfluoroctane- sulphonic acid X 3 mL = 0.1 L 250 mL actual concentration, ppm 8 .2 Prepare FC-95 Anion Standards 8 . 2 . 1 Prepare FC-95 standards for the standard curve. 8 . 2 . 2 Weigh approximately 100 mg of FC-95 into a 100 mL volumetric flask. Record the actual weight. 8 . 2 . 3 Bring up to volume with dilute acetonitrile. 8 . 2 . 4 Dilute the solution with dilute acetonitrile 1:10 for a solution of approximately 100 ppm. Dilute this solution 1:10 with dilute acetonitrile for a solution of approx. 10 ppm. 8 . 2 . 5 Use the 10 ppm solution to make working standards with values close to 5.0 ppm, 1.0 ppm and 500 ppb. 8 .3 Prepare Beef Liver Homogenate to Use for Standards 8 . 3 . 1 Weigh 40 g of Bovine liver into a 250 mL Nalgene bottle containing 200 mLs Milli-Q water. Grind to a homogenous solution. 8 . 3 . 2 Add 1 mL of the solution to a 15 mL centrifuge tube. Prepare a total of eight 1 mL aliquots of the solution in 15 mL centrifuge tubes. Be sure to re suspend solution by shaking it between aliquots. 000418 00006^ 8 . 3 . 3 Spike seven of the 1 mL aliquots with the following amounts of working standards in step 9.12 of the procedure. One 1 mL aliquot serves as the blank. Working Standard (Approximate Cone.) - 500 ppb 500 ppb 500 ppb 500 ppb 1 ppm 5 ppm 5 PPm______ uL - 100 200 300 400 500 200 300 Approximate final concentration of FC-95 in liver Blank 0.292 ppm 0.584 ppm 0.877 ppm 1.168 ppm 2.924 ppm 5.848 ppm 8.772 ppm 8 .4 Calculate the actual value of the standards: uL of standard x concentration fin ppm") = final concentration (ppm) 171 mg liver*/1 ml homogenate of FC -95 in liver Average weight of bovine liver in solution as determined by weighing 1 mL homogenates of 40 mg liver in 200 mL of Milli-Q water. The amount of FC-95 is reported as equivalents of FC-95 potassium salt. 8 .5 Calibration 8 . 5 . 1 Extract the spiked beef liver homogenate following 9.13 to 9.23 of this method. Use these standards to establish your curve on the mass spectrometer. 8 . 5 . 2 Alternatively, a standard curve may be generated using ratios of responses of the perfluorooctansulfonate anion and the internal standard anion versus concentration of the perfluorooctanesulfonate anion. 8 .6 Storage Conditions for Standards 8 . 6 . 1 New standards are prepared with each analysis. Standards are stored in covered plastic centrifuge tubes until the analysis on the mass spectrometer is performed. 8 .7 Storage Conditions for Standards 8 .7 .1 Beef liver homogenates may be frozen after preparation. 9.0 PROCEDURES________________________________________________ 9 .1 Obtain frozen liver samples. In spent tissue, note that the liver has not been packaged with other tissues. 9 .2 Use a dissecting scalpel and cut off approximately 1 g of liver. 9 .3 Weigh the sample directly into a tared plastic grinding tube. 9 .4 Record the liver weight in the study note book. 9 .5 Put a label on the vial with the study number, weight, rabbit ID, date and analyst initials. 000419 OOOOOS 4 9 .6 Add 2.5 mLs water. 9 .7 Grind the sample. Put the grinder probe in the sample and grind for about 2 minutes, until the sample is a homogeneous solution with no large chunks. 9 .8 Rinse the probe off into the sample with 2.5 mLs water using a pipet. 9 .9 Take the grinder apart and clean it with methanol after each sample. Follow AMDT-EP-22. 9 . 1 0 Cap the sample and vortex for 15 seconds. 9.1 1 Pipet 1 mL into a 15 mL centrifuge tube. Label the centrifuge tube with the identical information as the grinding tube. (See AMDT-M-4 Worksheet for documenting the remaining steps.) 9 .1 2 Spike the beef liver homogenates with the appropriate amount of FC-95 standard as described in 8.3. 9 .1 3 Spike the samples and beef liver homogenates with 100 uL of internal standard. 9 .1 4 Add 1 mL of the sodium carbonate/sodium bicarbonate buffer and 1 mL ammonium acetate. 9 .1 5 Using an analytical pipet, add 5 mL ethyl acetate. 9 .1 6 Cap the sample and vortex 20 to 30 seconds. 9 . 1 7 Put them in the shaker for 20 min. 9 . 1 8 Centrifuge for 20 to 25 minutes, until the layers are well separated. Set the power on the centrifuge to 25. 9 . 1 9 Remove 4 mLs of the top organic layer to a fresh 15 mL centrifuge tube with a 5 mL graduated glass pipet. Transfer 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 |im nylon mesh filter, and filter the sample into a fresh centrifuge tube or a autovial. Label the tube or vial with the study number and animal number. 9 . 2 4 Cap and hold for analysis by electrospray mass spectroscopy. 9 .2 5 Complete AMDT-M-4 worksheet and attach to page of study notebook. 10.0 VALIDATION_______________________________ 1 0 .1 Quality Control - not applicable 1 0.2 Precision and Accuracy- not applicable 1 0 .3 Other Validation Parameters- not applicable 11.0 DATA ANALYSIS___________________________ 11.1 None 12.0 ATTACHMENTS_____________________________ 12.1 Worksheet AMDT-M-4 13.0 REFERENCES_______________________________ 13.1 AMDT-EP-22 Routine Maintenance of Ultra-Turrax T-25 14.0 REVISIONS____________ Revision Number Reason for Change Revision Date 000420 oooocy 5 Worksheet AMDT-M-4 S tu d y # _ _ _ _ _ _ _ 1 S a m p le Num ber set # B la n k L iv e r FC -9 5 a p p ro x 0 .5 p p m a c tu a l ppm #W _ 100 uT, 200 uT. 200 u L 400 u L _ _ _ _ _ . _ FC -9 5 approx 1 ppm actual p p m #W _ _ 500 u L _ _ _ _ _ _ _ _ _ . _ FC -9 5 approx. 5 ppm a c tu a l ppm #W D a te and In itials fo r S td . . _ 200 u L 200 uT. _ _ _ _ _ _ _ 1 s tu d v n u m b e r w h e re th e o rig in a l w o rk s h e e t is located a n d n lace a c o n v . ___________________1___________________1______________________ ] _____________________ 1_____________________ I.iv e r E x tra c tio n Process- D a te A in itials P in et 1 m l. o f L iv e r S o lu tio n P in e t 10 0 u l- o f 12 nnm In ternal Standard S td . # V o rte x 1 5 sec Pin e t 1 m l, o f 250 nnm A m m o n iu m Acetate Std # P in e t 1 m l o f 0 .2 5 N a ,C O ,/ 0 .2 5 M N a H C O , B u ffe r Pin e t 5 m l, o f E th v l Acetate V o rte x 2 0 -S 0 sec. S h ake 20 m in . C e n trifu g e 2 0 -2 5 m in R e m o v e a 4 m l, a lio u o t o f o rg a n ic lave r B lo w d o w n to near d rvness K 0 .2 5 m l/l w ith N ,, A d d 1 m - o f 1 1 A c e to n itrile /FLO V o rte x 1 5 sec TN# F i l t e r u s i n g a 2 c c B - D s y r i n g e w i t h a 0 .2 t i m S R I f i l t e r i n t o a 1 .5 m l . a u t o s a m n le v i a l ______________________________ 000421 000070 6 3M Environmental Laboratory_____________________ Method Analysis of Rabbit Liver Extract for Fluorochemicals using Electrospray Mass Spectroscopy SOP Identification Number: AMDT-M-5 Revision Number: 0 Adoption Date: 1- d s-- Revision Date: None Author: Dave Christenson/Cynthia Weber Approved By: Software: MS Word, 6.0 Affected Documents: M-4, Extraction of Fluorochemicals from Rabbit Livers 000422 000071 1 1.0 SCOPE_________________________ .______________ _________________ 1.1 Scope: This method is for the analysis of extracts of rabbit liver or other tissues or fluids for fluorochemicals using the electrospray mass spectrometer. The analysis is performed by single ion monitoring of FC-95 anion, M/Z= 499, the internal standard M/Z = 427, and other appropriate masses. 1.2 Applicable Compounds: Fluorochemicals or other fluorinated compounds. 1 .3 Matrices: Rabbit Livers (samples), Beef Liver (standards), other tissues and fluids. 2 .0 K E Y W O R D S _____________________________________________________________ 2 .1 Fluorochemicals, fluorinated compounds, electrospray mass spectroscopy, mass spectrometer, rabbit livers. 3.0 PRECAUTIONS_________________________________________________ 3 .1 Use caution with the voltage cable for the probe. When the voltage cable is plugged into the probe DO NOT TOUCH THE PROBE, there is risk of electrical shock. 3 .2 Do not run the pump above it's capacity of 4000 psi. If pressure goes over 4000 psi stop and release pressure. The peak tubing may be plugged. Troubleshoot back to find the plug and replace the plugged tubing. See AMDT-EP-15 3 .3 Do not run the pump to dryness. 4 .0 S U P P L IE S A N D M A T E R IA L S _______________________________________ 4.1 Supplies 4 .1 .1 Nitrogen gas regulated to 140 psi. 4 .1 .2 Fluofix column or equivalent. 4 .1 .3 100 uL or 250 uL flat rip syringe for sample injection. 4.2 Reagents 4 .2 .1 Dilute acetonitrile mobile phase, dilute acetonitrile 1:1 with Milli-Q water. 4 .2 .2 Milli-Q water, all water used in this method should be Milli-Q water. 5 .0 E Q U IP M E N T __________________________________________________________ 5 .1 VG Trio 2000 Electrospray Mass Spectrometer or equivalent. 5 .2 ISCO Syringe Pump 5 .3 Spectraphysics AS 300 Autosampler 5 .4 100 uL Assembly 5 .5 Autovials or capped centrifuge tubes. 6 .0 IN T E R F E R E N C E S _______________________ 6 .1 There are no known interferences at this time. 7 .0 S A M P L E H A N D L IN G ___________________________________________________ 7 .1 Keep the extracted samples in capped 15 mL centrifuge tubes or in capped autovials until ready for analysis. 000423 000072 2 8.0 CALIBRATION AND STANDARDIZATION_____________________ 8 .1 Preparation of Calibration Standards 8 .1 .1 Seven beef liver standards and one blank beef liver are prepared during the extraction procedure. (See AMDT-M-4, section 8.0) 8 .2 Calibration 8 .2 .1 Run the seven beef liver standards twice, starting with the lowest standard to obtain the standard curve. 8 .2 .2 Typically one standard is run after each 5 to 7 samples. Choose a standard in the same range of concentration as the samples. 8 .3 Storage Conditions for Standards 8 .3 .1 Fresh standards are prepared with each analysis. Standards are stored in covered plastic centrifuge tubes until the analysis on the mass spectometer is performed. Samples and standards are NOT refrigerated. 8 .4 Storage Conditions for Beef Liver Homogenates 8 .4 .1 Beef liver homogenates may be frozen after preparation. 9.0 PRO CED U R E___________________________________________________ 9.1 Initial Set-up 9 .1 .1 Set software to "Operate on", Ion Mode ES`. 9 .1 .2 Record backing pressure in the instrument log. 9 .1 .3 Fill the solvent cylinder with mobile phase. 9 .1 .4 Set the pump to "Run". Set the flow to 1000 uL/min. Observes droplets coming out of the tip of the probe. The pressure should be at 1700 to 1800 psi. 9 .1 .5 Check the fused silica at the end of the probe. Use an eye piece to check for chips. The tip should be flat with no jagged edges. If any chips are found cut off the tip of the silica with a column cutter and pull the silica through to the appropriate length. 9 .1 .6 Check your nitrogen supply. Turn on the nitrogen. There should be no nitrogen leaking around the tip of the probe. A fine mist should be coming out of the tip. 9 .1 .7 Carefully guide the probe into the opening. Insert it until it won't go any further. Connect the voltage cable to the probe. 9 .1 .8 Go to the "Editor" page, and set Ionization Mode to ES', and the appropriate masses to 427 and 499. 9 .1 .9 If it is not in single ion mode go to "Option" and set SIR. 9 .1 . lOStart Acquisition. Assign a file name, MO-DAY-YR + letter. Record it in the log book. 9 .1 .1 1 Run the beef liver samples first, running each standard twice at the beginning of the run.. Run a QC check by running one standard after every 5 to 7 samples. 9.2 Manual Injection 9 .2 .1 Draw 150 uL of sample into a syringe. Inject the sample into the rheodyne injection port. Inject slowly. Record the sample ID in the log book. 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. 000424 00()07;j 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 10.0 V A L ID A T IO N _________________________________________________ 10.1 Quality Control 1 0 .1 . IRun a standard every 5 to 7 samples. If a significant change( 50%) in peak height occurs stop the run. Only the samples before the last acceptable standard will be used. The remaining samples will be reanalyzed. 10.2 Precision and Accuracy 1 0 .2 .1 See Method Validation Report number AMDT-M-5.0.V1 10.3 O ther Validation Param eters 1 0 .4 Refer to Method Validation Report Number AMDT-M-5.0.V1 11.0 DATA ANALYSIS_____________________________________________ 11.1 11.2 Calculations Plot the standard curve, using the mean of the two values obtained for each standard. 1 1 .2 . IRead peak heights or areas for the samples from the printout. Use linear regression to determine the sample concentrations. 1 1 .2 .2 Calculate the mg of FC-95 anion, or other fluorochemical in the total rabbit liver: mg FC-95 anion in the total rabbit liver = mg FC-95 anion from std. curve gms of liver used for analysis x Total mass of liver, gms 11.3 Make a results table and enter it in the study book. 11.4 Print a chromatogram for each sample, with the peaks labeled with the sample or standard ED. Write the study number on the printout, initial, date, and put it in the study folder. Staple all chromatograms together and number pages. 000425 000074 4 12.0 ATTACHMENTS None 13.0 REFERENCES 13.1 AMDT-EP-17 14.0 REVISIONS Revision Number Reason for change Revision Date 000426 000075 5 9.3 Quality Assurance Unit Statement 000427 000071 Attachment D GLP Study Quality Assurance Statement Study Title: Single-dose Dermal Absorption/Toxicity Study of T-6049 in Rabbits Study Number: AMDT-020895.1 Name of Auditor: KariRambo This study has been inspected by the Quality Assurance Unit as indicated in the following table. The findings were reported to the study director and management. Inspection Dates From IQ 10/13/95 10/19/95 Phase_____________ Final Report Date Inspection Reported to Management Study Director 10/19/95 10/19/95 QU Auditor /-/9-9<r Date 000428 000077 l 9.4 Key Personnel Involved in the Study 000429 3M Environmental Laboratory Key Personnel Thermal extraction followed by analysis using Orion ion analyzer: Jim Johnson Deb Wright Rich Youngblom Deann Plummer Analysis of liver extracts using electrospray mass spectrometry: Jim Johnson Dave Christenson Documentation and Reporting: Jim Johnson Rich Youngblom Quality Assurance Unit: Gale Van Buskirk Cynthia Weber Kari Rambo 000430 OOOOTj 9.11 Data 000431 0000S0 9.11.1 Summary and raw data; ug F` in whole liver as determined by thermal extraction followed by analysis using Orion ion analyzer. 000432 OOOOftl Summary of Combustion Data - Liver AMDT-020895.1, HW I6329-130 As Referenced in Final Report section 6.0 DATA ANALYSIS Total |ig Fluoride in Whole Liver Mean per Dose Group* pg Std. Dev. Control Group 22.4 + 5.7 5.0 mg/kg dose (T6049) 17.6 + 4.9 (0.003 mg/kg)** 100 mg/kg dose (T6049) 15.8 + 2.6 (0.06 mg/kg)** 500 mg/kg dose (T6049) 22.3 + 2.2 (0.30 mg/kg)** Calculated as the mean of triplicate samples from each of three male and three female rabbits. *Test material is a 0.06% solution of T6049, actual dose in parenthesis. 000433 OOOOftte % rcvry 106% 99% 91% 102% Actual ppm Fin liver (W/W) 0.835 0.287 1.140 1.200 0.474 0.281 0.305 0.212 0.249 0.198 0.684 0.244 0.254 0.232 0.205 0.181 0.272 0.129 1.111 1.096 0.402 0.320 0.283 0.336 0.209 0.197 0.149 0.136 0.157 0.275 0.285 0.335 0.257 0.241 0.267 0.180 0.242 0.280 0.238 0.209 0.155 0.167 0.172 0.328 0.284 0.266 0.174 0.238 0.291 0.230 Average ppm Fin liver (W/W) 0.353 0.220 0.394 0.206 0.335 0.247 0.147 0.298 0.255 0.234 0.200 0.222 0.241 0.253 liver burned (grams) 0.1138 0.1347 0.1403 0.1248 0.1141 0.1270 0.1322 0.1431 0.1458 0.1367 0.1279 0.1186 0.1230 0.1441 0.1096 0.1355 0.1165 0.1550 0.1237 0.1408 0.1346 0.1164 0.1370 0.1143 0.1174 0.1142 0.1518 0.1645 0.1434 0.1097 0.1140 0.1085 0.1685 0.1418 0.1401 0.1390 0.1010 0.1437 0.1014 0.1021 0.1290 0.1340 0.1396 0.1283 0.1296 0.1477 0.1375 0.1273 0.1367 0.1128 Whole liver weight (grams) 78.27 74.69 70.28 77.41 81.38 78.86 78.73 84.10 67.61 77.60 65.82 75.41 74.05 74.88 Total F- in whole liver (ug) 27.65 16.40 27.68 15.93 27.27 19.52 11.61 25.09 17.26 18.15 13.20 16.75 17.87 18.93 RPDosage (mg/kg) 0 0 0 0 0 0 5 5 5 5 5 100 100 100 Page 1 000434 FC95 AB ID F53400-1 F53400-2 F53400-3 F53360-1 F53360-2 F53360-3 Liver blk 1 Liver blk 2 Liver spk 1 Liver spk 2 F53347-1 F53347-2 F53347-3 F53365-1 F53365-2 F53365-3 F53345-1 F53345-2 F53345-3 F53354-1 F53354-2 F53354-3 F53403-1 F53403-2 F53403-3 F53000-1 F53000-2 F53000-3 F52999-1 F52999-2 F52999-3 F53351-1 F53351-2 F53351-3 liver spk-3 liver spk-4 liver spk-5 liver spk-6 liver spk-7 % rcvry 95% 95% 94% 89% 71% 119% 100% Actual ppm Fin liver (W/W) 0.157 0.191 0.165 0.144 0.209 0.151 0.432 0.366 1.389 1.266 0.344 0.268 0.195 0.206 0.289 0.154 0.261 0.251 0.335 0.420 0.364 0.216 0.323 0.344 0.340 0.307 0.272 0.301 0.243 0.236 0.211 0.193 0.188 0.203 1.079 0.997 3.467 5.930 4.330 Average ppm Fin liver (W/W) 0.171 0.168 0.269 0.216 0.282 0.333 0.336 0.293 0.230 0.195 liver burned (grams) 0.1418 0.1233 0.1356 0.1420 0.1057 0.1309 0.1137 0.1019 0.1036 0.1132 0.1221 0.1073 0.1415 0.1301 0.1212 0.1486 0.1438 0.1499 0.1352 0.1120 0.1091 0.1272 0.1126 0.1375 0.1378 0.1407 0.1384 0.1257 0.1369 0.1040 0.1218 0.1419 0.1449 0.1210 0.1312 0.1355 0.1253 0.1215 0.1401 Whole liver weight (grams) 74.96 76.35 83.70 82.60 81.34 70.97 70.62 79.62 87.51 80.69 Total F- in whole liver (ug) 12.80 12.86 22.50 17.88 22.96 23.66 23.71 23.36 20.13 15.71 RPT130L.XLS Dosage (mg/kg) 100 100 500 500 500 500 500 500 5 100 000435 Page 2 0000S4 9.11.2 Summary and raw data; analysis o f liver extracts using electrospray mass spectrometry. 000436 0000 S HWI# 6329-130 Study: Protocol Number: Test Material: Matrix: R Squared Value: Response Factor Amount: Analyst: Date: Method: Instrument: LABBASE File: Single-Dose Derma) Absorption TP3016.AB T-6049 in Rabbits (FC 95) Liver 0.9955 1.27E-05 DLC 4/3/95 AMDT-M-4 Fisons VG 2000 Electrospray MS 040395A/B to- -^5 Group Dose Group 1: 0 mg/kg Distilled Water Group 2: 5 mg /kg * Group 3: 100 mg/kg * Group 4: 500 mg/kg Sample # F53404 F53356 F53359 F53358 F53405 F53353 Ion Count Area N/D N/D N/D 6571 N/D N/D Extracted wt 9 1.0422 1.1273 1.2009 1.0779 1.2166 1.0734 Dilution factor 1 1 1 1 1 1 Concentration pg/g " Total mass of liver g N/D N/D N/D 0.0618 N/D N/D 69.156 73.403 72.193 69.308 77.164 75.915 F53402 F53352 F53399 F53349 F53364 6645 58373 N/D N/D 22296 1.1472 1.1648 1.1776 1.0143 1.0259 1 1 1 1 1 0.0587 0.5080 N/D N/D 0.2203 76.184 81.324 63.494 76.006 80.666 F53400 F53360 F53350 F53346 F53397 12849 11111 18253 N/D N/D 1.3817 1.054 1.3119 1.3537 1.2903 1 1 1 1 1 0.0943 0.1069 0.1410 N/D N/D 73.828 74.574 72.534 73.666 73.063 F53403 F53347 F53000 F53345 F53365 F53354 59686 N/D 34395 4623 N/D N/D 1.0511 1.0447 1.1242 1.2143 1.0281 1.194 1 0.5756 69.012 1 N/D 83.100 1 0.3101 77.255 11 0.0386 77.250 N/D 66.252 1 N/D 80.146 Total amount of FC-95 per liver mg N/D N/D N/D 0.004 N/D N/D 0.004 0.041 N/D N/D 0.018 0.007 0.008 0.010 N/D N/D 0.040 N/D 0.024 0.003 N/D N/D * Rabbits F52999 (G2) and F53351 (G3) were not analyzed ** 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. 000437 O O O O bti r-l .cqoSTS.V A-ui * 32^-130 tC-^ p'o V- M e t h o>3 D L C L I V Sample DLCLIV Operator DLO R u n d a t e 0 5 - 0 9 - 1 9 9 5 0 6 : 4 6 : 5 2 V e r s i o n : 11 Printed on 05-09-1095 AT 06:47:07 Straight Line Fit forced through Origin. A -/. Q.J bZ428 il<i Mndini ".EVEL AMOUNT Component 1 EXTERNAL STANDARD CALIBRATION AREA 1 0.4000 2 9 977 z 0 . 0 0 0 6 2 4 2 0 1.2000 9S 59 4 4 1.6000 124122 Y- SLOPE * X + INTERCEPT Area Amount = P. s q u a r e d = 7.8924E-!-G4 * 1.2670E-05 * 0.9955 Amount Area 0.0 000E +00 0.E+0 000438 000087 HvC ^ E\eeW s^>n^ >WL^ F C ~l ^ r0 t Gve s*y*^le >Wi>iiA*jr^rtc,<L ( scavl. ^ 2t^(o^ 3 3 5 3 ^ <^ciucJ au^lw^L ru ^A o ^ ^ aW^vouj/ \ io/i%h^> d l x . GoaAou(VvS peuqt'S A"3> 4Woo<^U .-10 J JC c 000439 Fi le :040395a LAB-BASE - The MS Data System Sample :CABBQPOL- RABBIT LIVER -EXTRACTS- \o/r*/ss oui 03/04/1995 09:06 A-H- 000440 Bt^ -aWi File:040395A LAB-BASE - The MS Data System SampleICABBQPOL BABBIT LIVJEB EXTRACTS- io/i%l<\s Ouc X c 03/04/1995 09:06 C A -5 000441 Fi le 1040395A LAB-BASE - The MS Data System 03/04/1995 09:06 H V c cw 000442 F ile:040395A LAB-BASE - The MS Data System SampleIGABBOPOL BABBIT -H U E R EXTBAGT8 ioft*hr Cx_^ 03/04/1995 09:06 A% c A~T- 000443 File:040395A LAB-BASE - The MS Data System 03/04/1995 09:06 4-2 000444 c F ile :040395A LAB-BASE - The MS Data System 03/04/1995 09:06 r'4 0I" < 000445 File:040395A LAB-BASE - The MS Data System Sample:CARDOPOL BABBIT LIUEIt EXTRACTS '%<* l<vr bt-e 03/04/1995 09:06 -IO 000446 c File: 040395B LAB-BASE - The MS Data System 03/04/1995 10 :53 no xw . w xfc . w j F10 NOT DEFINED