Document G54vZJ6zbDjJmM0v4EbXOLBav

DuPont HLR 81-94 Protocol #3: Soils FM (Fort Madison) and PLW1 Protocol Absolute BloiyallabUtty otSotl Lead in Microswine: Solb Identited as Fort Malison and PVW-l page 1 of 9 ABSOLUTE BIOAYA1LABXLTTY OF SOIL LEAD IN MICROSWINE: SOILS IDENTIFIED AS FORTMADISON AND PLW-I MEDICAL RESEARCH PROJECT NO. 9905-001 PROTOCOL Stine-Haskcll Animal Welfare Committee No. CBTS1-P -57 DUP040004289 DuPont HLR 581-94 Protocol #3: Soils FM (Fort Madison) and PLW1 Protocol Ab^uCe QioayaJIabUlly ofSoil L<ad (aMkros>via: Soils IdenliOed as ForiMadison and PLW-l TABLE OF CONTENTS page 2 OBJECTIVE....____ ,,--3 SPONSOR AND TEST FACILITY_______________ .__________________________ 3 STUDY DESIGN MATERIALS AND METHODS---------- -------- ------ ----- -------...______ --TM.,,4 A. Test Materials----TM..--.................... ------ -- .----4 C. Pretest Procedures ...... -.............----..........----..........--....... 4 D. Assignment to Groups -----...TM.--TMTM--5. E. Animal Husbandry ..TMTM-.-.-.-...TM-..-.TM.-.TM.--.--.----,,5 F. Diet Preparation and Analysis.-TM....--..,-.---- G. .Body Weights Fa DOSID^Wh imm............. .mh .w --.i--..mh ....h h h ..h --.mm.-^..im.mh mm..iw .--w --.w ^ W..H.-WH.W.WMWww.<..i..i^w.i L Blood Sampling ---.........- .----TM-------------- TM.6 .J. Sacrifice of Aniffliil5.-..MM.MWM-...Mw--<...m....h w ...i-..w .mw m--mm--.6 L. Data Analysis------.TM-TM----....--...... --.--........ ........TMTM.TM--TM.....7 M- Statistical MethodsTM.TM.--..... ..................--.. ................................. -.---7 SAFETY AND HOUSEKEEPING----------------- -7 RECORDS AND SAMPLE RETENTION------------------------------- 7 APPENDIX!--_________ ._____________________________ __ __________ 8 A-- Study Personnel --..8 B. Study Dates.-..-TM-..,.,....-....TM...TMTM.....-...TM..TMTM..TM..--TM.-,.TMTM...TM.......8 -58- D U P040004290 DuPont HLR 581-94 Protocol #3: Soils FM (Fort Madison) and PLW1 Protocol Absolute BioavanabQUyofSoil Lead Ja Micronrine: Soils Identified mFcrt Madison and PLW-1 page 3 OBJECTIVE The objective of this study is to determine the absolute bioavailability of lead from two 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 ofDuPont Chemicals, E. L du Pont de Nemours and Company, Bellevue Corporate Center, Wilmington, Delaware. The study Will be conducted at Haskell Laboratory forToxicology and Industrial Medicine, E. L 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 designIs as follows: fifteen male microswine will be used; a predosing blood sample will be drawn from each animal for levels of background lead**, ten microswine will be dosed orally by gavage (five microswine/soil) with test soils containing 500 ppm (Port Madison) or 4,000 ppm (PLW-1) lead (approximately 17 mg of lead/microswine); five microswine will be given a single intravenous injection of lead acetate 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 approximately -20C until analysis; and at sacrifice, liver and bone (both cortical and trabecular as well as Whole femur) will be collected, weighed, and frozen at approximately -20G. -59- DUP040004291 DuPont HLR 581-94 Protocol #3: Soils EM (Fort Madison) and PLW1 Protocol Absolute lHoavariability ofSoil Lead ja Micronviae: Soils IdeaUGedas Fort Madison tadPLW-l page 4 MATERIALS AND METHODS A. Test Materials s, Lead acetate will be supplied by Aldrich, Milwaukee, Wisconsin. The test soils will be supplied by the sponsor. Upon arrival to Haskell Laboratory, each test substance will be identified with a Haskell Laboratory sample number. Characterization and mineralogy data of the test soils will be maintained by the sponsor. B. Test Species Male Yucatan microswine, approximately 30 days ofage, 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 mtcroswjne-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 ofLaboratory Animals (NIH Publications No. 86-23. Revised 198SY The quarantine period will be approximately two Weeks, during which microswine will be fed an optimized diet (once per day) ofmicroswine diet (Charles River) and transferred gradually to Teklad basal mix (95%) for microswine. Microswine will be provided deionized watered libitum, weighed three times, and observed with respect to eating habits, weight gain, and any signs ofdisease 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 water analyses for contaminants and sampling freshly washed cages and cajge racks for bacteria and for pathogens. The potential effects of dietary contaminants have been considered and, on the basis ofthe manufacturer's data, are believed to be within acceptable ranges. ,60, DUP040004292 DuPont HLR 581-94 Protocol #3; Soils FM (Fort Madison) and PtWl Protocol Absolute BioavaiUbllUy ofSoil Lead in Microswine: Soils MenUOed as Fort Madison and PLW-t page 5 The program is monitored and administered by the laboratory veterinarian; data are maintained separately from study records. D. Assignment to Groups Healthy animals will be selected by computerized stratified-iandomizationsuch that there are no statistically significant differences among group body weight means. Microswine will be housed individually. Each animal will beassigned 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 microswinewill 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 temperature of 252,C and a relative humidity of 5010%. F. Diet Preparation and Analysis During the test period, microswine in each group will be fed Swine Basal mix (95%) (TD 93034). This diet will be custom formulated by the manufacturerso that it contains complete normal-nutrition for this species. The diet will contain < 0.1 ug Pb/g. Sucrose will be added to the diet and thoroughly mixed for a period oftime adequate to assure homogeneous distribution in the diet Once prepared, the diet 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. Concurrent with the collection of body weights, microswine will be individually examined and clinical observations will be recorded. -61- DUP040004293 DuPont HLK 581-94 Protocol #3: Soils FM (Fort Madison) and PLW1 Protoqsl AbsoluLeBio^vaUabUltyof$ollLadlnM[cro^wlne: Soils IdcntlOtd as PortMadison aod PLW-1 page 6 BL Dosing ^ Microswine mil be dosed intravenously via the 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 ofadministration of the lead-contaminated soil will be by oral gavage since soil ingestion presents one of the principle direct pathways for exposure to children. Intravenous administration oflead acetate will be performed in order-to calculate area under the curve (AUC) and absolute bioavailability of lead. I. Blood Sampling Approximately 0.2-04 ml ofVenous blood will be drawn from each designated animal at 04,1,2,4,8,12,24,48,72,96,120,144,192, and 240 hours after dosing. Blood will be collected into a lead-free tube containing heparin and frozen (approximately -20C) until the time ofpreparation 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 spectroscopy (GFAA), at DuPont Chambers Works, E. L du Pont de Nemours and Company, Deepwater, New Jersey. Dosing solutions, diets, and drinking water will also be analyzed. -62 DUP040004294 DuPont HLR 581-94 Protocol #3; Soils FM (Fort Madison) and PLW1 Protocol Absolut* BIolvaHjMUtj ofSoil Lead In Mlcrorwine: Solti MenUtted is Fort Uaitsou rad M.W-J L. Data Analysis poC<7 lead. Individual animal data as well as the mean for six animals in each group will be reported. M. Statistical Methods Body weights and body weight gain will be analyzed by one-way analysis ofvariance. 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 Haskett Laboratory, Jackson Laboratory (DuPont Chambers Works) or at the Records'Management Center, E. L du Pont de Nemours and Company, Wilmington, Delaware. 63- DUP040004295 DuPont HLR 581-94 Protocol #3: Solis FM (Fort Madison) and PLW1 Protocol Absolute BloavaUabttHy ofSod toad In Mlcronrinc: Soils MenUljed uPertMalltpn end PLW-l APPENDIX I pjgcg A. Study Personnel Study Sponsor: Corporate Remediation Group of DuPont Chemicals E. L du Pont de Nemours and Company Bellevue Corporation Center Wilmington, Delaware 19809 Contact: Timothy S. Bingman, D.A.B.T, Consultant . (412) 262-9110 Study Director Hanan N. Ghantous, Ph.D. Research Toxicologist B. Study Dates Proposed Experiment Start June, 1994 Proposed Experiment Completion: July, 1994 Proposed Report Issue Date: January, 1995 -64- DUP040004296 DuPont HLR 581-94 Protocol #3: Soils FM (Fort Madison) and PLW1 Protocol Absolute BloavalUtilltly ofSol! Lead InMIcroswioe: Soils IdenUOed as Fort Madison and PLW-I MEDICAL RESEARCH PROJECT NO. 9905-001 page 9 Approved: Hanaj/N. Ghantous, Ph,D. Study Director (date) Matthew S. Bogdaifty, J1A.B.T. Manage Biochemical Toxicology and Risk Analysis s-/f fw (date) cc: P.E. Ross T.S. Bingman (REO-P1T) K.McCleary Quality Assurance Study Technician Pathology Steward -65- DUP040004297 Diana Lead Data Entry Coding Form: BatesBegin: DUP040004298 BatesEnd: TotalPages Doc Type j Protocol (Amendment) | Year Date- j1994 j Month Date-. | DecControl#- 238.01 1 Attrnnt?# 0 10 j Day Date' j 191 Title Protocol #3 (Amendment] - Absolute Bioavailability of Soil Lead in Microswine Soils Identified as Fort Reference Madison and PLW-1 Primary Author Last Name.l Ghantous First Name Middle Initial. Hanan N. Secondary Authors: Dupont Publication: Primary Medical Research Project No 9905-001 Recipient (s) CC's TS Bingman K McCleary, Quality Assurance. P.E Ross Names Stated. Haskell Doc Problems Case Produced In j Steven Thomas | Produced By j DPT Dup/Cross Ref #'s I Source11 Responsive to Plaintiff's 1st Re Ob], Coder Nam j D Green Obj Data Entry Nam I D. Green ObjDataDate | 8/21/2002 N 26887.01 DuPont HLR 581-94 Protocol #3 (Amendment); Soils FM (Fort Madison) and PLW1 Protocol Amendment No. 1 Absolute Bfoavallabitily ofSoit Lead tn Mlcrosyrlnc; Soils Idcntitied u Fort Madison and PLYf-l page loti TO: MEDICAL RESEARCH PROJECT NO. 9905-001 October 18.1994 The original protocol is amended as follows: 1. Add the following to Materials and Methods, section E, Diet Preparation andAnalysir. *011; contaminants, ifany, 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 he within acceptable ranges. However, the diet (containing sucrose) will be analyzed for lead content and the results from the analysis will be presented in the final report' This addition, required by GoodLaboratory Practices, was erroneously omitted from the original protocol, 2. The Haskell Sample Nos. assigned to the test substances were 20659,20658, and20202for Fort Madison (FM), PLW-1, and Pb Acetate, respectively. The sample numbers forthe two test soils were not availablewhen the original protocol was written. 3. In Materials and Methods, section L, Data Analysis, it is erroneously stated that individual animal data as well as the mean for six animals in each group will be repotted, this is changed to state that individual animal data as well as the mean for five animals in each group will be repoitcd. the numberof animals in each group was incorrectly stated in the original protocoL 4. The results ofthis study will be included In afinal report written forMedicalResearch Projects (MRs) 9727,9740, and 9905. these three projects were absolute bioavailahility studies ofsoil lead in miccoswine; each project used different lest soils, this change is made in orderto present the results more efficiently and in a timelier manner. Additional Information: the intravenous dose of Pb Acetate administered in tins study was intended to be used in comparison with the test soils labeled FortMadison (FM) and PLW-1. the dosage ofdie Pb Acetate was calculated to be equivalent to the dosage oflead receivedby animals orally dosed with the leadcontaining test sods. the intravenous dose ofPb Acetateused in MRs 9727 and 9740 were designed to accomplish a similar task; however, dueto technical etror, the Pb Acetate dose for MR.9740 was incorrectly prepared, the Pb Acetate dose group from MR 9727 will be used in comparison with die test soils in MR 9740. this infotmation is being provided to explain why MRs 9727 and MR 9740 were sharing a Pb Acetate dose group, while MR9905 was not KMoOeary Quality Assurance PH. Ross Study Director Biochemical Toxicology and Risk Analysis -66- DUP040004298