Document eveYJeyn66VgrvkjZ6ZDow3be

Protocol #2: Soils PLW and EC1 DuPont HLR 581-94 Protocol Absolute Bloareliability ofSoil Lead In Mlcrotwinet Soils PLW and ECI page 4 MATERIALS AND METHODS A. Test Materials Lead acetate will be supplied by Aldrich, Milwaukee, Wisconsin. The test soils will be supplied by the sponsor. Upon arrival to Haskell Laboratory, each testsubstance will be identified with a Haskell Laboratorysample number. 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 ofapproximately 3 to 6 kilograms veil 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 ofsoil-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 he 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 times, and observed with respect to eating habits, weight gain, and any signs ofdisease orinjury. Animals that die or are sacrificed in extremis during the quarantine period will be necropsied to check for the presence ofdisease. 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 and sampling freshly washed cages and cage racks for bacteria for pathogens. -50- DUP040004282 Protocol #2: Soils PLW and EC1 DuPont HLR 581-94 Protocol Absolute BtawaUablUty ofSoil Lead !n Microswine: Soils Pf.W ond.ECl pajrS 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-randomization such that there are no statistically significant differences among group body weight means. Microswine will be housed individually. Each animal will be assigned its own Haskell animal number and permanently identified by a number marked on the mioroswine's ear. E. Animal Husbandry After assignment to groups, animals will be identified with a cage card and housed in stainless.steel cages. All raicroswine will be provided optimized testdiets of approximately 340 gram/microswinc/day. Deionized water will he provided ad libitum. Animal rooms will be targeted at a temperature of252C 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 @5%) (TD 93034). These diets will be custom formulated by their manufacturers so that each diet contains complete normal nutrition for this 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. Concurrent with the collection of body weights, microswine will be individually examined and clinical observations will be recorded. -51- DUP040004283 Protocol #2: Soils PLW and EC1 DuPont HLR 581-94 Protocol Absolute BloavmUabUJty ofSoli Lead la Mteroewtae: Soils PLW and EC1 page 6 H. Dosing Microswine will be dosed intravenously via the ear vein. Sterile waterwill 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 by oral gavage since ingestion ofsoil lead activity 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 oflead. L BloodSampling Approximately 0.2-0.5 ml ofvenous blood will be drawn from each designated animal at. 05,1,2,4,8,12,24,48,72,96,120,144,192, and 240 hours after dosing. Blood will be collected into a lead-free tube containing heparin and frozen (approximately -20Q until the time of preparation for analysis. J. Sacrifice ofAnimals At tire end ofthe experiment, the animals will be humanelyeuthanatized. Animals will be necropsied, liver and bpne 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, NewJersey. Dosing solutions, diets, and drinking waterwill also be analyzed. -52- DUP040004284 Protocol #2; Soils PLW and EC1 DuPont HLR 581-94 Protocol Absolute Bloavallabllity of Soil Lead In MIcroswJnci Soils PLW and EC2 P*c 7 L. Data Analysis The blood lead concentrations determined by atomic absorption will be used to calculate AfJC (area under blood concentration versns time curve) and absolute bioavailability of 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 analysed 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 ofWork areas and potential health hazards. Gloves will be wom 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, Jackson Laboratory (DuPont Chambers Works) or at the Records Management Center, E. L du Pont de Nemours and Company, Wilmington, Delaware. -5.3- DUP040004285 Protocol #2: Soils PLW and EC1 DuPont SLR 581-94 Protocol Absolute BJoavattabUHyofSoil Lcxd fa MIcrocwiae: Solb PLW end ECl APPENDIX t pate 8 A. Study Personnel Study Sponsor. Corporate Remediation Group ofDuPont Chemicals E, t du Pont de Nemours and Company Bellevue Corporation Center Wilmington, Delaware 19809 Contact: Timothy S. Bingman, D.A-B.T. Consultant (412)257-7761 Study Director: Hanan N. Ghantous, Ph.D. Research Toxicologist B, Study Dates Proposed Experiment Start: November, 1993 Proposed Sponsor Approval: Date the Sponsor signed Che MR Project Proposed Experiment Completion: December, 1993 Proposed Report Issue Date; June, 1994 .54- DUP040004286 Protocol #2: Soils PLW and EC1 DuPontHLR 581-94 Protocol Absolute BloavaUabtlity ofSoil Lead In Microswine Soils PLW and ECl MEDICAL RESEARCH PROJECT NO. 9740-001 page 9 Approved: 1/Hanan N. Ghantous, PhJD. Study Director (date) Matthew S. Bogdanffy, e Ca .B.T. Manager BiochemicalToxicology and Risk Analysis ///if/h (date) cc: P.E. Ross T.S. Bingman (REO-PII) K-McCteary Quality Assurance Study Technician Pathology Steward -55- DUP040004287 Protocol #2 (Amendment); Soils PLW and EC1 DuPont HLR 581-94 Protocol Amendment No. 1 Absolute BloavullabllUy ofSoli Lead In Mkrorwine: Salts PLW and EC1 page 1 of I TO: MEDICAL RESEARCH PROJECT NO. 9740001 October 18,1994 The original protocol is amended as follows: 1. Add the following to Materials andMethods, section E, Diet Preparation andAnalysis: The 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 be within acceptable ranges. However, the diet (containing sucrose) will be analyzed for lead content and die results from the analysis will be presented in the final report.' This addition, required by Good Laboratory Practices, was erroneously omitted from the original protocol. 2. The results ofthis studywill be included in a final report written forMedical Research Projects (MRs) 9727,9740, and9905. These three projects were absolute bioavailaKlity studies of soil lead in microswine; each project used different test soils. This change is made in order to presentthe results more efficiently and in atimelier manner. 3. The intravenous dose of Pb Acetate administered in this study was intended to be used in comparison with thetest soils labeled PLW and ECI. Thedosage ofthe Pb Acetate was calculated to be equivalentto thedosage oflead received by animals orally dosed with the lead-containing test soils. Due to technical error, the Pb Acetatedose was incenectly prepared. However, the Pb Acetate dose group from MR9727 was designed to accomplish the same task and at the same dose of lead per animal. Therefore, the Pb Acetatedose group from MR.9727 Will be used in comparison with the groups orally dosed with test soils in MR 9740. This change is madein order to correct a mistake in a cost-effective manner. Study Director cc: T.S. Bingman K-McCteary Quality Assurance P.E.Ross Manager i/1^ Biochemical Toxicology and Risk Analysis -56- DUP040004288