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Diana Lead Data Entry Coding Form: BatesBegin DUP040005095 BatesEnd: I jDoc Type Protocol (amendment) Year Date'| TotalPages J j1993 Month Date: DecControl# 281 12 Attmnf?# 2 j10 Day Date: | 271 Title Protocol Amendment No 1 - Absolute Bioavailability of Soil Lead in Microswine Soils AEC 3-3. AEC 9-3, Reference. AEC 10-1 jPrimary Author Last Name Ghantous ISecondary Authors. j jFirst Name, Middle Initial, Hanan N j 1Publication. Primary MEDICAL RESEARCH PROJECT NO 9727-001 Recipient (s): J I CC's: P E Ross T S Bingman, K. McCleary Quality Assurance Names Stated: Matthew S Bogdanffy, Ph D Doc Problems^ 1Case Produced In Steven Thomas Produced By DPT Dup/Cross Ref #'s 1 j Source. Responsive to Plaintiffs 1st Re j Doc Control Comments: Disk. Box 1 January 2002, SH001001 - SH001002 Obj. Coder Nam | D Green | Obj. Data Entry Nam | D Green j ObjDataDate | 8/22/2002 N 26928 Diana Lead Data Entry Coding Form: BatesBegin- DUP040005096 BatesEnd' DUP040005097 TotalPages 1 jDoc Type: | Protocol (Amendment) | Year Date' |1994 Month Date- DecControl#' I 281.01 I 2 Attmnt?# 0 1 j10 Day Date: j18 Title Protocol Amendment No. 2 - Absolute Bioavailability of Soil Lead in Microswine Soils AEC 3-3, AEC 9-3, Reference. AEC 10-1 Primary Author Last Name. Ghantous First Name. Middle Initial.! Manan N. Secondary Authors. Publication. Primary MEDICAL RESEARCH PROJECT NO 9727-001 Recipient (s): CC's. T S Bingman. K McCleary Quality Assurance P E Ross Names Stated. Matthew S Bogdanffy Doc Problems jCase Produced In | Steven Thomas Produced By | DPT Source: Responsive to Plaintiffs 1 st Re j Dup/Cross Ref #'s Doc Control Comments Disk, Box 1 January 2002, SH001001 - SH001002 Obj Coder Nam | D Gieen Obj Data Entry Nam j D Green ObjDataDate | 8/22/2002 ^ 2692&01 Protocol Amendment No. 2 Absolute Bioavailability of Soil Lead in Microswine: Soils AEC 3-3, AEC 9-3, and AEC10-1 page 1 of 2 TO: MEDICAL RESEARCH PROJECT NO. 9727-001 October 18,1994 The original protocol is amended as follows: 1. Add the following to Materials and Methods, section F, Diet Preparation and Analysis: `The contaminants, if any, in the sucrose to be used for this study were considered. Because the sucrose chosen for use was originally intended for human consumption, it was felt that any contaminants would be within acceptable ranges. However, the diet (containing sucrose) will be analyzed for lead content and the results from die analysis will be presented in the final report.' This addition, required by Good Laboratory Practices, was erroneously omitted from the original protocol, 2. The original protocol states that blood samples will be refrigerated until the time of preparation for analysis. This is changed to state that blood samples will be frozen (approximately -20C) until preparation for analysis. This change was made because blood samples will be held until all time points have been collected, then sent to Jackson Labs for analysis. 3. Add the following to Materials and Methods, section J, Sacrifice ofAnimals: `Animals that are sacrificed in extremis or accidentally killed will be neeropsied and examined for cause of death.' This addition is made to explain the procedure for animals that are sacrificed in extremis or accidentally killed. 4. The original protocol stated that each microswine would receive an oral dose of test soil which contained approximately 17 g lead. This is changed to state that each inicrpswine would receive an oral dose of test soil which contained approximately 17 mg lead. This change is made to correct a typing error. 5. The amount of lead contained in each soil is changed from 520,570, and 585 ppm to 1070,1120, and 1210 ppm for AEC 3-3, AEC 9-3, and AEC 10-1, respectively. This change was made based on data received after the sponsor sent the soils for reanalysis of lead concentrations. 6. Inprotocol Amendment#!, reasons for two changes were not presented. Regarding the change describing animal grouping, random animal selection replaced selection by age in order to enhance the study design. Regarding the addition of animal examination and recording of clinical signs concurrent with the collection of body weights, this was always the intent and was erroneously not included in the original protocol. 7. The results of this study will be included in a final report written for Medical Research Projects (MRs) 9727,9740, and 9905. These three projects were absolute bioavailability studies of soil lead in microswine; each project used different test soils. This change is made in order to present the results more efficiently and in a timelier manner. Protocol Amendment No. 2 Absolute Bioavailability of Soil Lead in Microswine: Soils AEC 3-3, ABC 9-3, end ABC 10-1 page 2 of2 8. The intravenous dose of Pb Acetate administered in this study was intended to be used in comparison with the test soils labeled AEC 3-3, AEC 9-3, and AEC 10-1. The dose of the Pb Acetate was calculated to be equivalent to the dose of lead received by animals orally gavaged with the leadcontaining test soils. The intravenous dose of Pb Acetate used in MR 9740 was designed to accomplish a similar task (at the same dose of lead per animal); however, due to technical error, the Pb Acetate dose was incorrectly prepared. Therefore, the Pb Acetate dose group from MR 9727 will be used in comparison with the test soils in MR 9740. This change is made in order to correct a mistake in a cost-effective manner. Approved by: lanan N. Ghantous, Ph.D. Study Director n/fl! (date) ce: T.S. Bingman K. MeCleary Quality Assurance PE. Ross Biochemical Toxicology and Risk Analysis (date) DUP040005097 Diana Load Data Entry Coding Form: BatesBegin. DUP040005098 Doc Type. Protocol BatesEnd: DUP040005106 Year Date- 1993 TotalPages jMonth Date DecCcntrol#: | 281 02| 9 Attmnt?# 1 0 101 jDay Date: 11 j Title Protocol - Absolute Bioavailability of Soil Lead in Microswine: Soils AEC 3-3, AEC 9-3, AEC 10-1 Reference: Primary Author Last Name I Ghantous Secondary Authors Primary Medical Research Project 9727-001 Recipient (s): First Name, Middle Initial.] Hanan N Publication: CC's. T S Bingman. K McCleary, R C. Rhea, P E Ross Names Stated Stine-Haskell Animal Welfare Committee, Corporate Remediation Group of DuPont Chemicals, Bellevue Corporate Center, Wilmington. Delaware. Haskell Laboratory, Newark. Delaware, Aldrich, Milwaukee. Wisconsin Yucatan MirrnswinP Charlps Riypr I ahnratnrios Inc Windham Mainn Guido for Carp and Doc Problems' Case Produced In Steven Thomas jDup/Cross Ref #'s id.Produced By: | DPT Source: | Responsive to Plaintiffs 1st Re Doc Control Comments Disk, Box 1, January 2002. SH001001 - SH001002 Obj. Coder Nam I D Green J bj *~>ata ^am | D- Green | j 1ObjDataDate 8/22/2002 N 26928.02 ABSOLUTE BIOAVAILABILITY OF SOIL LEAD IN MICROSWINE: SOILS AEC 3-3, AEC 9-3, AND AEC 10-1 MEDICAL RESEARCH PROJECT NO. 9727-001 PROTOCOL Stine-Haskell Animal Welfare Committee No. CBT51-P DUP040005098 Protocol Absolute Bioavailability of Soil Lead in Microswines Soils AEC 3-3, AEC 9-3, and AEC 10-1 TABLE OF CONTENTS page 2 OBJECTIVE......... ....................................................................... ..........................,......... ,3 SPONSOR AND TEST FACILITY...___ __________________________ ____________ 3 STUDY DESIGN.........---------------------------............................... ........................... . 3 MATERIALS AND METHODS.....__________...----------------- ------------------------------- 4 A. Test Materials.......................................................--..................................... 4 B. Test Species.......................................................................................................74 C*. Pretest Procedures 4 D. Assignment to Groups5 E. Animal Husbandry,,.......... .--........................... 5 F. Diet Preparation and Analysis G. Body Weights................... 5 H. Dosing........................... ..6 L Blood Sampling............................................................................... ................. J. Sacrifice of Animals ............. ........................................................ . 6 K. Blood Analysis.......... .......................................................................................... ...6 L. Data Analysis ........ ............................................................. .--......... 7 M. Statistical Methods........................................................ ....................... . 7 SAFETY AND HOUSEKEEPING _________________________ 7 RECORDS AND SAMPLE RETENTION...............__..........................___...... 7 APPENDIX I................................................... ..................... ______8 A, Study Personnel................... 8 B. Study Dates,.................................................................. . 8 DUP040005099 Protocol Absolute Bioavailability of Soil Lead in Microswine: Soils AEC 3-3, AEC 9-3, and AEC 1(1-1 page3 OBJECTIVE The objective of this study is to determine the absolute bioavailability of lead from three different soils in microswine to aid in developing a site specific-clean-up goal. SPONSOR AND TEST FACILITY This study is sponsored by the Corporate Remediation Group of DuPont Chemicals, E. L du Pont de Nemours and Company, Bellevue Corporate Center, Wilmington, Delaware. The study will be conducted at Haskell Laboratory for Toxicology and Industrial Medicine, E. I. du Pont de Nemours and Company, Newark, Delaware, in accordance with all applicable Good Laboratory Practice Standards. The sponsor's approval was effective the date the sponsor signed the Medical Research (MR) project. STUDY DESIGN The study design is as follows: twenty male microswine will be used a predosing blood sample will be drawn from each animal for levels of background lead fifteen microswine will be dosed orally by gavage (5 microswine/soil) with test soils containing 520,570, or 585 ppm lead (approximately 17 grams/microswine) five microswine will be given a single intravenous injection of lead acetate solution containing lead-dose equivalent to that contained in the gavage dose approximately 0.5 ml of blood will be drawn from each microswine at 0.5,1,2,4, 8,12, 24,48,72,96,120,144,192, and 240 hours after dosing. blood will be collected in lead-free tubes containing heparin and stored at 4C until analysis at sacrifice, liver and bone (both cortical and trabecular as well as whole femur) will be collected, weighed, and frozen at -20C DUP040005100 Protocol Absolute Bioavailability of Soil Lead in Microswine: Sods AEC 3-3, AEC 9-3, and AEC10-1 page 4 MATERIALS AND METHODS A, Test Materials The test materials will be supplied by Aldrich, Milwaukee, Wisconsin. The lead acetate trihydrate will be assigned Haskell Laboratory sample no. 20202. The lead/soils will be assigned Haskell Laboratory sample number as follows: AEC 3-3 will be assigned no. 20366; AEC 9-3 will be assigned no, 20367; and AEC 10-1 will be assigned no. 20368. Characterization and mineralogy data of the test soils will be maintained by the sponsor. B. Test Species Male Yucatan microswine, approximately 30 days of age, with body weights in the range of approximately 3 to 6 kilograms will be acquired from Charles River Laboratories, Inc., Windham, Maine, The weanling Charles River microswine has been selected on the bases of its low body weight, its suitability as a model of soil-lead ingestion by children, and its suitability with respect to longevity, hardiness, sensitivity, and low incidence of spontaneous disease. C. Pretest Procedures Microswine will be housed according to the Guide for Care and Use of Laboratory Animals (NIH Publications No. 86-23, Revised 1985). The quarantine period will be approximately two weeks, during which microswine will be fed an optimized diet (once per day) of microswine diet (Charles River) and transferred gradually to Teklad basal mix (95%) for microswine. Microswine will be provided deionized water ad libitum, weighed three limes, and observed with respect to eating habits, weight gain, and any signs of disease or injury. Animals that die or are sacrificed in extremis during the quarantine period will be necropsied to check for the presence of disease. Animals will be released by the laboratory veterinarian at the end of the quarantine period. Haskell Laboratory has an animal health monitoring program which consists of periodic food and water analyses for contaminants, sampling freshly washed cages and cage racks for bacteria, and sampling non-study animals housed in the study room for pathogens. DUP040005101 Protocol Absolute Bioavailability of Soil Lead in Microswines Soils AEC 3-3, AEC 9-3, and AEC 10-1 pageS The program is monitored and administered by the laboratory veterinarian; data are D. Assignment, to Groups Healthy animals will he assigned to groups according to age. Microswine will be housed individually. Each animal will be assigned its own Haskell animal number and permanently identified by a number marked on the microswine's ear. E. Animal Husbandry After assignment to groups, animals will be identified with a cage card and housed in stainless steel cages. All microswine will be provided optimized test diets of approximately 340 gram/microswine/day. Deionized water will be provided ad libitum. Animal rooms will be targeted at a tempeature of 25 2C and a relative humidity of 50 10%. F. Diet Preparation and Analysis During the test period, microswine in each group will be fed Swine Basal mix (95%) (TD 93034), These diets will be custom formulated by their manufacturers so that each diet contains complete normal nutrition for each species. The diets will contain <0.1 ug Pb/g. Sucrose will be added to the diets and thoroughly mixed for a period of time adequate to assure homogeneous distribution in the diet Once prepared, diets will be refrigerated until used or discarded. G. Body Weights Microswine will be weighed twice per week unless experimental findings warrant a more infrequent weighing schedule. DUP040005102 Protocol Absolute Bioavailability of Soil Lead in Microswine: Soils AEC 3-3, ABC 9-3, andAEC 10-1 page 6 H. Dosing Microswine will be dosed intravenously via die ear vein. Sterile water will be used as a vehicle. Oral doses will be administered by gavage. Deionized water will be used as a vehicle. The route of administration of the lead-contaminated soil will be used by oral gavage since ingestion of soil lead activity presents one of the principle direct pathways for exposure to children. Intravenous administration of lead acetate will be performed in order to calculate area under the curve (AUC) and absolute bioavailability of lead. I. Blood Sampling Approximately 0.5-1.0 ml of microswine venous blood will be drawn from each designated animal at 0.5,1,2,4,8,12,24,48,72,96,120,144,192, and 240 hours after dosing. Blood will be collected into a lead-free tube containing heparin and refrigerated until the time of preparation for analysis. J. Sacrifice ofAnimals At the end of the experiment, the animals will be humanely euthanatized. Animals will be necropsied, liver and bone will be collected, weighed, frozen at approximately -20C, and stored for potential future analyses. K. Blood Analysis The blood lead concentration will be analyzed by graphite furnace atomic absorption spectroscopy (GFAA), at DuPont Chambers Works, E. I. du Pont de Nemours and Company, Deepwater, New Jersey. Dosing solutions, diets, and drinking water will also be analyzed. DUP040005103 Protocol Absolute Bioavailability of Soil Lead in Microswlne: Soils ABC 3-3, AEC 9-3, and AEC 10-1 page 7 L. Data Analysis The blood lead concentrations determined by atomic absorption will be used to calculate AUC (area under blood concentration versus time curve) and absolute bioavailability of lead. Individual animal data as well as the mean for five animals in each group will be reported. M. Statistical Methods Body weights and body weight gain will be analyzed by one-way analysis of variance. Appropriate statistical analysis will be performed on mean AUC values between oral and intravenous administrations. SAFETY AND HOUSEKEEPING Good housekeeping practices will be used to avoid contamination of work areas and potential health hazards. Gloves will be worn when handling formulation, blood, animals, animal tissues, or analytical standards. Animal carcasses, feces, and diet will be incinerated. RECORDS AND SAMPLE RETENTION All original records will be retained at Haskell Laboratory or at the Records Management Center, E. I. du Pont de Nemours and Company, Wilmington, Delaware. DUP040005104 Protocol Absolute Bioavailability ofSoil Lead in Microswine: Soils AEC 3-3, AEC 9-3, and ABC 10-1 APPENDIX! pageS A. Study Personnel Study Sponsor: Corporate Remediation Group of DuPont Chemicals E. I. du Pont de Nemours and Company Bellevue Corporation Center Wilmington, Delaware" 19809 Contact: Timothy S. Bingman, D.A.B.T. Consultant (302) 571-7761 Study Director: Hanan N. Ghantous, Ph.D. Research Toxicologist B. Study Dates Proposed Experiment Start: October, 1993 Proposed Sponsor Approval: Date the Sponsor signed the MR Project Proposed Experiment Completion: November, 1993 Proposed Report Issue Date: April, 1994 DUP040005105 Protocol Absolute Bioavailabilityof Soil Lead in Microswine: Soils AEC 3-3, AEC 9-3, and AEC lifll-1 MEDICAL RESEARCH PROJECT NO. 9727-001 page 9 Approved: Study Director Matthew S. Bog' Manager cc: P.E. Ross T.S. Bingman (REO-PIT) K. McCleary R.C. Rhea it In I ft (date) DUP040005106 Protocol Amendment No. 1 Absolute Bioavailability of Soil Lead in Microswine: Soils AEC 3-3, AEC 9-3, and AEC 10-1 page 1 of 1 TO: MEDICAL RESEARCH PROJECT NO. 9727-001 The following changes are made to the original protocol: page 4 MATERIALS AND METHODS C. Pretest Procedures paragraph 4, sentence 1 Delete '...and sampling non-study animals housed in the study room for path ogens.' This study will not use non-study animals to test for pathogens. page 5 MATERIALSAND METHODS D. Assignment to Groups paragraph 1, sentence 1 Delete sentence 1. Replace with 'Healthy animals will he selected by computerized stratifiedrandomization such that there are no statistically significant differences among group body weight means.' page 5 MATERIALS AND METHODS F. Diet Preparation and Analysis paragraph 2, sentence 1 Change '...normal nutrition for each species.' to '...normal nutrition for this species.' This was a clerical error. page 5 MATERIALS AND METHODS G. Body Weights paragraph 1 Add the following sentence: 'Concurrent with the collection of body weights, microswine will Study Director cc: P.E. Ross K.McCleary T.S. Bingman Quality Assurance DUP040005095