Document npebzdzg4OQpK6jeL5RbXZYk2

DU PONT CENTRAL RESEARCH AND DEVELOPMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE -/f. 7 TO: TIMOTHY BINGMAN 0 / DU- rONlh-AGRICULTURAbLb-mR&OEOUCTS E4D2-32-2-G- -Pn: f V DATE; MAY 17* 1993 PROJECT MR 9660 PART I TITLE: ABSOLUTE BIOAVAILABILITY OF SOIL LEAD IN RATS AND MICROSWINE Authority is requested to undertake the study identified above and described as foliovs: To conduct a two part study in tats and microsvine to determine the absolute, bioavailability of soil lead. Details are in the study protocol, provided under separate cover. This study will be billed in 9 equal installments beginning June, 1993. Forecast month and year Amount requested $ 104,700 Experiment to start: May, 1993 Previously authorized $ Experiment to end: August, 1993 Expended through / / $ Final report to issue: February, 1993 General Ledger & Subaccount to Request for study made by: T. Bingman Charged /OS IBSr' yoo TESTING _X_ RESEARCH ___ CONSULTING __ (Check appropriate classification) If this request meets with your approval, please enter the GENERAL LEDGER AND SUBACCOUNT TO BE CHARGED, the CLASSIFICATION, and have it PROPERLY AUTHORIZED. Proposed by: Cost Code 19 23 24 25 30 31 32 Total (%') 41.1 30.2 1.0 27.8 Approved by: Authorized: PLEASE RETURN AUTHORIZED COPY DIRECTLY TO: LAURA WALLS, FINANCIAL SERVICES, HASKELL LABORATORY HG/amb s'ln-lu Hanan N. Ghantous This is a ISCA 8(d) chemical, (check if appropriate) DuPont HLR 671-93 SOIL LEAD NE 571-93 mpany Lustrial Medicine 60-001 Date TO: FROM: HANAN N. GHANTOUS CR&D Haskell Laboratory (302) 451-0834 | For Attention For Information --p3ZT7i OTH 'J Forwarded per your Request 6-Mf\M _) u. po^i: /3?0 k) I f(\ 150(7 77// DUP040000520 N 25663.01 DuPont HLR 671-93 Study Title ABSOLUTE BIOAVAILABILITY OF SOIL LEAD IN RATS AND MICROSWINE Laboratory Project ID Haskell Laboratory Report No. 671-93 Author Hanan N. Ghantous, Ph.D. Study Completed on December 9,1993 Performing Laboratory E. I. du Pont de Nemours and Company Haskell Laboratory for Toxicology and Industrial Medicine Elkton Road, P. O. Box 50 Newark, Delaware 19714 Medical Research Project No. 9660-001 Page 1 of 142 DUP040000521 Absolute Bioavailability of Soil Lead in Rats and Microswine DuPont HLR 671-93 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT This study was conducted in compliance with U. S. EPA TSCA Good Laboratory Practice Standards (40 CFR 792) except for the deviations documented and explained in Appendix B. The deviations did not affect the integrity of the study. Submitter: E, I. du Pont de Nemours and Company Sponsor: Corporate Remediation Group of DuPont Chemicals E. I. du Pont de Nemours and Company Study Director; Date Ian N, Ghantous, Ph.D. Research Toxicologist Biochemical Toxicology and Risk Analysis iihi& Company Representative: -2- DUP040000522 Absolute Bioavailability of Soil Lead in Rats and Microswine_________________________ ______ _________________ DuPont HLR 671-93 GENERAL INFORMATION Substance Tested (Intravenous Administration): Synonyms /Codes : Haskell Sample No. : Purity: C.A.S. Registry No.: Acetic acid, lead(2+) salt, trihydrate H-20202 Aldrich Cat. No. 31,651-2 Lot #KY08221CY Lead acetate trihydrate Lead(II) acetate trihydrate Lead acetate Acetic acid, lead(2+) salt 20202 99.999% 6080-56-4 Substance Tested (Oral Administration): Synonyms /Codes: Haskell Sample No.: Purity: CA.S. Registry No.: Soil, from former DuPont Works, Washington State, contaminated with lead H-20204 Soil contaminated with approximately 1,000 ppm lead from former DuPont Works in Washington State Soil contaminated with approximately 1,000 ppm lead from Washington State Soil with lead 20204 N/A None Available Sponsor: Corporate Remediation Group of DuPont Chemicals E. I. du Pont de Nemours and Company Bellevue Corporate Center Wilmington, Delaware 19809 Sponsor Contact: Timothy S. Bingman, D.A.B.T. (412) 257-7761 3 DUP040000523 Absolute Bioavailability of Soil Lead in Rats and Microswine DuPont HLR 671-93 GENERAL INFORMATION (Continued) Study Initiated/Completed: 05-17-93 / 12-09-93 Experiments Initiated/Completed: Rats 5-18-93/5-28-93 (Acute I.V.) 6-08-93/6-18-93 (Acute Oral) 5-18-93/6-28-93 (Subchronic Feed & I.V.) 6-08-93/7-19-93 (Subchronic Feed & Oral) Microswine 8-10-93/8-20-93 (Acute I.V.) 8-06-93/8-16-93 (Acute Oral) 6-12-93/7-23-93 (Subchronic Feed & I.V.) 6-15-93/7-26-93 (Subchronic Feed & Oral) -4- DUP040000524 Absolute Bioavailabiliiy of Soil Lead in Rats and Microswine ____ DuPont HLR 671-93 TABLE OF CONTENTS GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT........ ........ ..2 GENERAL INFORMATION............................ ............................. .......... .............3 SUMMARY ........ .... ..... ................ ................................. ..... ............... .......... .8 SIGNATURE PAGE............................................... ............. ... ............................10 QUALITY ASSURANCE DOCUMENTATION.......... .... ...... .... .... ...... ....... ...11 STUDY PERSONNEL .......... .......................... .................... ......... ................12 INTRODUCTION............................................ ..................... ..................... ....... 14 OBJECTIVE ................................. ........ .... ........................ ............. ..... ........ ..14 MATERIALS AND METHODS.............. ...... ................... .... .......... ..................14 A. Test Substance...................... ........ ...... ..... ..... ........ ............................14 B. Test Species ......... ........................ .................................................. 15 C. Animal Husbandry......................... ......... .................... .....................15 D. Pretest Period........................................... ....................................... ..16 E. Assignment to Groups and Study Start......--------- ---------------------- 16 F. Diet Preparation, Administration, and Sampling ................... ....... 17 G. Dose Preparation, Administration, and Sampling................... ........ 18 H. Blood Sampling .............................. .................. ........... ...... ...... .........19 I. Body Weights, Clinical Observations, and Mortality....................... 19 J. Food Consumption and Intake of Test Substance.............. ........ ,.... 19 K. Sacrifice of Animals .....................................,................... ....... .......... 20 L. Data Analysis ....................................... ...... ... .................... ... ...20 RECORDS AND SAMPLE RETENTION................ ........................... ........... ....20 RESULTS AND DISCUSSION.............. ........................ .................................... 21 A. Diet Samples..................................... ......... ......... ... ..................... 21 B. Dosing Solutions.......... ................................................ .......... ...........21 C. Blood Lead Concentrations, Area Under the Curve, and Absolute Bioavailability............... ......................... .......... ........ ......... 22 1. Single Dose Administration (Acute).................................. ....... 22 2. Single Dose Administration (Subchronic)............................... .23 D. Body Weights, Body Weight Gains, Food Consumption, Intake of Test Substance, and Clinical Observations...................................24 CONCLUSION........... ..................................... ............................. >........ ........... 24 REFERENCES................. ....... ................................ .... ........................ ..... ......... 25 -5- DUP040000525 Absolute Bioavailabitiiy of Soil Lead in Rats and Mieroswine ____________________________________ DuPont HLR 671-93 TABLE OF CONTENTS (Continued) TABLES. .26 1. Concentration of Lead in Intravenous and Oral Gavage Dosing Solutions ...................... .......... ,................ ....... ......... ........27 2, Homogeneity of Lead in Diet Prepared for Rats.............. .... ....28 3. Homogeneity of Lead in Diet Prepared for Mieroswine.... ........29 4. ........ 30 5. Mean Blood Lead Concentration in Male Rats (Acute)............ ........ 31 6. Mean Blood Lead Concentration in Male Rats (Subchronic)............32 7. Mean Blood Lead Concentration in Male Mieroswine (Acute)........33 8. Mean Blood Lead Concentration in Male Mieroswine (Subchronic)..................................................... ..............34 9. Absolute Bioavailability in Rats and Mieroswine ............. 35 10. Area Under the Blood Concentration-Time Curve for Mieroswine (Acute)................ 36 11. Area Under the Blood Concentration-Time Curve for Mieroswine (Subchronic).......... ......37 12. Mean Body Weights of Male Rats (Acute)............... 38 13. Mean Body Weights of Male Rats (Subchronic).... ....... ..39 14. Mean Body Weights of Male Mieroswine (Acute)............. 40 15. Mean Body Weights of Male Mieroswine (Subchronic)................... .41 16. Mean Body Weight Gains of Male Rats (Acute)........ ...... 42 17. Mean Body Weight Gains of Male Rats (Subchronic)..................... ,.43 18. Mean Body Weight Gains of Male Mieroswine (Acute)....................44 19. Mean Body Weight Gains of Male Mieroswine (Subchronic)........ ..45 20. Mean Daily Food Consumption by Male Rats (Acute).............. 46 21. Mean Daily Food Consumption by Male Rats (Subchronic).............47 22. Mean Daily Food Consumption by Male Mieroswine (Acute)...... ,.48 23. Mean Daily Food Consumption by Male Mieroswine (Subchronic) 49 24. Mean Daily Intake of Lead by Male Rats (Subchronic)............ 50 25. Mean Daily Intake of Lead by Male Mieroswine (Subchronic)-------51 26. Summary of Clinical Observations in Male Rats (Acute)................. 52 27. Summary of Clinical Observations in Male Rats (Subchronic) ,...53 -6- DUP040000526 Absolute Bioavailability of Soil Lead in Rate and Microswine______ __________________ __________________________ DuPont HLR 671-93 TABLE OF CONTENTS (Continued) TABLES (Continued) 28. Summary of Clinical Observations in Male Microswine (Acute)....................................... 54 29. Summary of Clinical Observations in Male Microswine (Subchronic)................. ......... ... 55...,.. FIGURES 56 1. Mean Blood Lead Concentration in Male Rats (Acute) ..57 2. Mean Blood Lead Concentration in Male Rats (Subchronic)....., ..58 3. Mean Blood Lead Concentration in Male Microswine (Acute)....,.................................................,-- ___ 59 4. Mean Blood Lead Concentration in Male Microswine (Subchronic).,................................................ ................. 60 APPENDICES................................................. ........ .................................... ...61 A. Protocol and Protocol Amendments................. ...... ...... ........... ...62 B. GLP Deviations............................................... ................... ........ ...80 C. Blood Lead Concentration Before Intravenous or Oral Dosing ...81 D. Blood Lead Concentration After Intravenous or Oral Dosing.. ...84 E. Blood Lead Concentration During the 30-Day Feeding Phase.. ...94 F. Individual Body Weights................ ....................................... .... ...99 G. Individual Clinical Observations and Mortality------------ ------- .110 H. Analysis of Lead in Blood, Soil/ and Associated Materials ........ .128 -7- DUP040000527 Absolute Bioavaiiability of Soil Lead in Hats and Microswine DuPont HLR 671-93 SUMMARY Objectives of this study were: to determine the absolute bioavailability of lead in test soil, to determine if subchronic exposure to lead-contaminated soil influences lead bioavailability, and to compare the bioavailability in two model species (rat and microswine). The bioavailabilities of both a single dose (acute) and a single dose afterfeedingfor 30 consecutive days (subchronic)'wove determined. For single-dose (acute) administration, 5 male juvenile microswine and 25 male juvenile rats were each gavaged with test soil (17 g soil/microswine, 1 g soil/rat). Six additional microswine and 25 additional rats were each given a single intravenous injection of lead acetate solution containing a lead dose equivalent to that of the gavage dose. For the subchronic part of the study, 12 male microswine and 50 male rats were provided with optimized diets of approximately 340 g/microswine/day or 21.5 g/rat/day of a special purified diet mixed with test soil (5% w/w) for 30 days. Microswine and rats were then each divided into two groups of equal size and given either a single intravenous or oral (gavage) dose. Blood was drawn from both groups of microswine and rats at 0.5,1,2,4,8,12,24, 48,72,96,120,144,192, and 240 hours after dosing. The blood samples were analyzed for lead content, and the absolute bioavaiiability was determined as the ratio of the area under the blood-lead versus time curve (AUC) for the oral /intravenous groups. Findings from this study were: Differences between AUCs of the gavage group and the intravenous group were statistically significant in the acute rat and microswine groups. Differences between AUCs of the gavage and intravenous groups were significant in the subchronic rat groups but not the subchronic microswine groups. However, assessment of bioavaiiability after subchronic exposure appeared to be inaccurate due to the apparent confounding effect of bioaccumulation of lead in blood. Single-dose rats absorbed only 4.14% of the lead dose while the single dose microswine absorbed 20.90%. -8- DUP040000528 Absolute Bioavailability of Soil Lead in Rats and Microswine Absolute bioavailability results were: acute rats subchronic rats acute microswine Subchronic microswine DuPont HLR 671-93 4.14% 27.83% 20,90% 47.13% In conclusion, based upon (1) the fact that microswine appear to provide a more conservative estimation of bioavailability than rats, and (2) the difficulty in interpreting subchronic data due to bioaccumulative effect, acute administration to microswine appears to be the most appropriate model for lead bioavailability in children. -9- DUP040000529 Absolute Bioavailability of Soil Lead in Rats and Microswine______________ ________ ______________________ SIGNATURE PAGE DuPont HLR 671-93 Authored by: Tlanan N. Ghantous, Ph.D. Research Toxicologist Biochemical Toxicology and Risk Analysis Approved by: Matthew S. Bogdanffy, Ph.D.fD.A.B.T. Manager Biochemical Toxicology and Risk Analysis Reviewed and Approved for Issue by Study Director: Date Report Issued: Hainan N. Ghantous, Ph.D. Research Toxicologist Biochemical Toxicology and Risk Analysis 9, /9P j -10 - DUP040000530 Absolute Bioavailability of Soil Lead in Rats and Microswine DuPont HLR 671-93 QUALITY ASSURANCE DOCUMENTATION Patel's) of Inspection: Conduct - 6/14,17/93,7/6,7,13,16,19,23/93,8/9/93 Records, Report(s) - 5/25/93,7/7-9/93,8/9,10/93, 11/19,22,23,27-30/93,12/1-4,6-8/93 Patel's) Findings Reported to: Study Director - 7/9/93,9/1/93,12/4,8,9/93 Management - 7/19/93,9/1/93,12/9/93 Reported by: ___ -iWu, Robert C. Rhea, B.S. Auditor Quality Assurance ixkht (date) - li - DUP040000531 Absolute Bioavailability of Soil Lead in Rats and Microswine______________________________________ DuPont HLR 671-93 STUDY PERSONNEL The following individuals participated in the conduct of the study: Study Director: Hanan N. Ghantous, Ph.D. Manager: Matthew S. Bogdanffy, Ph.D. Primary Technician: Christine M. Loeffler Assisting Technicians: Michele Aschiero, A.A.S. Walter C. Moll Benjamin Robinson, B.A. Report Preparation: John D. Oldham, Jr., B.S. The following individuals were responsible for the gross pathology examinations and tissue collection from the microswine: Manager: Nancy C. Chromey, Ph.D. Pathologist: Paul E. Ross, D.V.M. Coordinator Pathology Operations: William J. Lynam, A.S. Pathology Steward: Merralyn K. Vaillancourt, B.S. The following individuals were responsible for lead analysis 0ackson Lab): Coordinator: Keith A. McCleary, Ph.D. Technicians: Billy R. Cooper Ralph Postorivo Michael Sisitka -12 - DUP040000532 Absolute Bioavailability of Soil Lead in Rats and Microswine DuPont HLR 671-93 STUDY PERSONNEL (Continued) The following individuals were responsible for quality assurance: Coordinator: Joseph C. Haxnill Auditor: Robert C. Rhea, B.S. The health status of the animals on study was assessed by the attending Laboratory Veterinarian, Charles E. Cover, V.M.D., with the assistance of James G. Aftosmis, V.M.D. and Carolyn S. VanPelt, D.V.M., Ph,D. Assistance with statistical analysis was provided by John W. Green, Ph.D., Ph.D. -13 DUP040000533 Absolute Bioavailability of Soil Lead in Rats and Microswine DuPont HLR 671-93 INTRODUCTION Recently, DuPont conducted a study aimed at developing a suitable animal model for determining site-specific bioavailability of lead-contaminated soil. The initial phase of the study, performed previously, was designed to establish the proper dose levels to be used in subsequent phases. Results from this previous phase indicated that subsequent testing should be performed using diet formulations containing 5% of lead-contaminated soils with lead concentrations ranging between 375 and 1500 mg/kg.W A second phase of the study, which is the subject of this report, was designed to establish the absolute bioavailability of lead from the site's soil in both rats and microswine using either single dose or subchronic dosing followed by single- Rats and microswine were used in order to choose the correct animal model in relevance to humans. Weis and LaVeiled summarized the considerations to be addressed when choosing an animal model to determine the bioavailability of lead. OBJECTIVE The objective of the study was to determine the absolute oral bioavailability of lead from soil in rats and microswine following a single oral (gavage) dose. Absolute oral bioavailability is also to be determined in these species following a subchronic exposure to lead-contaminated soils that are mixed with feed. These results will assist development of a site-specific clean-up goal by providing soilspecific data on gastrointestinal absorption. MATERIALS AND METHODS The original protocol and protocol amendments are in Appendix A. A. Test Substance Lead acetate trihydrate was supplied by Aldrich, Milwaukee, Wisconsin, and assigned Haskell sample number 20202. The lead-contaminated soil (lead/soil) was supplied by the sponsor and assigned Haskell sample number 20204. - 14 - DUP040000534 Absolute Bioavailability of Soil Lead in Rats and Microswine DuPont HLR 671-93 Characterization and mineralogy data of the lead/soil are maintained by the sponsor. B. Test Species Ninety juvenile male Crl:CDBR rats, born approximately 4/19/93, were received from Charles River Laboratories, Inc., Kingston, New York, on 5/11/93. On 5/12/93, these male rats weighed between 33.3 and 65.4 g. Sixty additional juvenile male Crl:CDBR rats, born approximately 5/10/93, were received from Charles River Laboratories, Inc., Kingston, New York, on 6/01/93. On 6/02/93, these additional male rats weighed between 34.6 and 58.2 g. The Crl:CDBR rat was selected on the bases of extensive experience with this strain and its suitability with respect to hardiness, longevity, sensitivity, and low incidence of spontaneous disease. Twelve juvenile male Yucatan microswine, born approximately 4/24/93, were received from Charles River Laboratories, Inc., Windham, Maine, on 6/03/93. On 6/04/93, these male microswine weighed between 3795 and 7008 g. Twelve additional juvenile male Yucatan microswine, born approximately 6/22/93, were received from Charles River Laboratories, Windham, Maine, on 7/29/93. On 7/30/93, these additional male microswine weighed between 3843 and 7688 g. The male Yucatan microswine was selected on the bases of its low body weight, its reported 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. Animal Husbandry During the pretest period, rats and microswine were housed according to the Guidefor Care and Use ofLaboratory Animals.W After assignment to groups, rats and microswine were housed in stainless steel cages. Rat cages were suspended above cage boards; microswine cages were bedded with recyclable, biodegradable shredded paper (Enviro Dri). To improve housing conditions, microswine were provided with recreational equipment (balls) in their cages and occasionally rewarded with Oreo cookies. Animal rooms were targeted at a temperature of 232C for rats and 252C for microswine and a relative humidity of 5010%. On two occasions, the relative humidity of rooms housing rats was beyond 60%; however, these deviations were brief and did not affect the study. Animal rooms that housed rats were artificially illuminated (fluorescent light) on a 12-hour light/dark cycle. Animal rooms that housed microswine were artificially illuminated (fluorescent light) on -15 DUP040000535 Absolute Bioavailability of Soil Lead in Rais and Microswine DuPont HLR 671-93 a 12-hour light/dark cyde with additional natural light entering through a window at one end of the room. Haskell Laboratory has an animal health monitoring program which consists of periodic food analysis for contaminants and sampling freshly washed cages and cage racks for bacteria. The program is monitored and administered by the laboratory veterinarian; data are maintained separately from study records. All parameters were found to be within acceptable ranges. Dosing solutions, prepared diets, components of diets, deionized water, Oreo cookies, and microswine bedding were analyzed for lead content by DuPont Chemicals at DuPont Chambers Works, Deepwater, New Jersey, D. Pretest Period Rats and microswine were quarantined for at least six days. During the pretest period, rats were fed an optimized diet (once per day) of Purina Certified Rodent Chow #5002 meal (PCRC) and transferred gradually to Teklad basal mix (95%) for rats (TD 92242) combined with sucrose (5%; sucrose obtained from Acme Markets, Inc.). Microswine were fed an optimized diet (once per day) of microswine diet (Charles River) and transferred gradually to Teklad basal mix (95%) for microswine (TD 93034) combined with sucrose (5%). For rats and microswine, transfer to Teklad basal mix and sucrose was completed by the fifth day of quarantine. Rats and microswine were provided deionized water ad libitum. Rats and microswine were weighed three times during the quarantine period and observed daily with respect to eating habits, weight gain, and any signs of disease or injury. One microswine (Pretest ID #6) was sacrificed in extremis because of loss of appetite and, consequently, severe weight loss; no Signs of infectious disease were found at necropsy. E. Assignment to Groups and Study Start After the quarantine period, rats and microswine were grouped as follows: 50 of the 90 rats received on 5/11/93 were selected for study use on the bases of adequate body weight gain and freedom from any clinical signs of disease or injury. The selected rats were divided by computerized, stratified randomization into two groups of 25 rats each. These groups were designated as Groups I and m The remaining 40 rats were removed from the room and used on other studies. -16 DUP040000536 Absolute Bioavailability of Soil Lead in Rats and Microswine DuPont HLR 671-93 50 of the 60 rats received on 6/01 /93 were divided by computerized, stratified randomization into two groups of 25 rats each. These groups were designated as Groups n and IV. In addition, due to a technical error, 5 of the remaining 10 rats were randomly selected to be bled for the 12hour timepoint for Group H. The remaining 5 rats were removed from the room and used on other studies. all of the microswine received on 6/03/93 were divided by computerized, stratified randomization into two groups of six microswine each. These groups were designated as Groups V and VII* the remaining 11 (of 12) microswine received on 7/29/93 were divided by computerized, stratified randomization into one group of six microswine (designated as Group VI) and one group of five microswine (designated as Group VUI). In addition, one microswine from each group was fed one hour before dosing; data from these animals are presented separately (at the end of Section C.2). Groups of rats and microswine were exposed to lead in the following manner: Group Number Species Approximate Days Fed Composition Experiment Age at Start of Lead in Diet Dosing of Dosing Start Date Experiment (days') Before Dose Method Material I Rat 5/18/93 II Rat 6/08/93 m Rat 5/18/93 IV Rat 6/08/93 V Microswine 6/12/93 VI Microswine 8/10/93 VH Microswine 6/15/93 vm Microswine 8/06/93 29 0 (Acute) I.V. Lead Acetate 29 0 (Acute) Oral Lead in Soil 29 30 (Subchronic) I.V. Lead Acetate 29... 30 (Subchronic) Oral Lead in Soil 49 30 (Subchronic) I.V. Lead Acetate 49 0 (Acute) I.V. Lead Acetate 52 30 (Subchronic) Oral Lead in Soil 45 0 (Acute) Oral Lead in Soil F. Diet Preparation, Administration, and Sampling Subchronic rats and microswine, designated to be fed at least 30 days prior to dose administration, received diets containing test soil contaminated with approximately 1,000 ppm lead. Diets were prepared by combining Teklad basal mix for the particular species with the lead-contaminated soil. The basal mix was designed for use at the 95% level, in conjunction with 5% of the test substance (or sucrose). These ingredients were mixed in a high-speed mixer for three minutes. On the day of dose administration and until sacrifice, rats and microswine received diets containing sucrose. Diets were prepared by combining Teklad -17 - DUP040000537 Absolute Bioavailability of Soil Lead in Rats and Microswine ___________________________________________ DuPont HLR 671-93 basal mix (95%) for the particular species with sucrose (5%). These ingredients were mixed in a high-speed mixer for three minutes. All diets were refrigerated until used or discarded. During the study, rats were provided approximately 21.5 g/day of test diet and microswine were fed approximately 340 g/day of diet. Rats and microswine were provided deionized water ad libitum. Prior to the start of any of the experiments (5/17/93), samples were collected from the top, middle, and bottom of the mixing bowl containing diet prepared with lead/soil. These samples were analyzed to measure concentration and homogeneity. Due to a technical error, samples from feed jars were not collected and sent for analysis of lead concentration. Because the samples collected from the mixing bowl were found to be at the proper concentration and homogeneously mixed (see Results and Discussion) and because the lead in the samples used in this study was not expected to degrade, this did not affect the integrity of the study. G. Dose Preparation, Administration, and Sampling Oral Dose. Rats designated for oral (gavage) administration (acute or subchronic) received a 2.0 ml suspension of test soil (containing approximately 1,000 ppm lead) and deionized water. Each rat received approximately 1 g test soil within the 2.0 ml dose. Microswine designated for oral (gavage) administration (acute or subchronic) received a 30.0 ml suspension of test soil (containing approximately 1,000 ppm lead) and deionized water. Each microswine received approximately 17 g test soil within the 30.0 ml dose. For rats and microswine, the suspension was constantly stirred until all animals were dosed. Oral dosing suspensions were analyzed for lead concentration and the measured values were used for calculating lead bioavailability (Table 1). Due to an error, oral dosing suspensions from the rat subchronic groups were not sent for analysis. Intravenous Dose. Rats and microswine designated for intravenous administration (acute or subchronic) received a 0.1 ml solution of lead acetate and sterile water. The solution was prepared to contain a lead dose equivalent to that contained in the oral (gavage) dose for each species. Rats were dosed intravenously via the tail vein; microswine were dosed intravenously Via the ear vein. - 18 - DUP040000538 Absolute Bioavailability of Soil Lead in Rais and Microswine DuPont HLR 671-93 Intravenous dosing solutions were analyzed for lead concentration and the measured values were used for calculating lead bioavailability (Table 1). Due to an error, intravenous dosing suspensions from the rat subchronic groups were not sent for analysis. H. Blood Sampling Approximately 0.1-0.2 ml of rat venous blood and approximately 0.2-0.5 ml of microswine venous blood were drawn from designated animals at 0.5,1,2,4,8, 12,24,48,72,96,120,144,192, and 240 hours after dosing. At each timepoint, all microswine were bled; however, only four or five rats were bled at each timepoint. The groups of 25 rats were randomly divided into five subgroups of five rats each. Each subgroup was bled at two or three non-consecutive timepoints. Because of a death and a tail injury during the first day of bleeding, two subgroups within Group HI had only four rats (see Body Weights, Clinical Observations, and Mortality section). Due to an error, no blood was collected from rats at 12 hours after the oral (gavage) dose. In order to analyze blood at this timepoint, five rats were randomly selected from those not originally designated for use on the study. These rats were bled, dosed orally, and bled again 12 hours later. All blood samples were stored at 4C and sent to DuPont Chambers Works for analysis. I. Body Weights, Clinical Observations, and Mortality All rats and microswine were weighed and carefully examined for clinical signs twice each week. During the bleeding procedure for Group IH (subchronic intravenous group), one rat was accidentally killed and a second rat, which sustained a tail injury, was removed from the study. J. Food Consumption and Intake of Test Substance The amount of food consumed by each rat or microswine fed for 30 consecutive days was determined weekly. From these values and body weigh data, intake of the test substance was determined. -19 - DUP040000539 Absolute Bioavailability of Soil Lead in Bats and Microswine DuPont HLR 671-93 K. Sacrifice of Animals At the end of the experiment, rats bled at 240 hours after dose administration were humanely euthanatized by carbon dioxide asphyxiation, then dissected. The liver and femurs from rats that were dosed at study start were collected, weighed, and frozen. The liver, kidneys, femurs, spleen, and brain from the subchronic rats Were also collected, weighed, and frozen. Tissues, other than blood, collected at the time of sacrifice Were stored for potential future analyses. Analyses of these tissues is not part of this project. At the end of the experiment, all surviving microswine were weighed, humanely euthanatized by intravenous injection of Euthanasia-V solution, then given a gross pathological examination. The livers and femurs from microswine that were dosed at study start were collected, weighed, and frozen. The liver, kidneys, femurs, spleen, and brain from the subchronic microswine were collected, weighed, and frozen. Tissues, other than blood, collected at the time of sacrifice were stored for potential future analyses. Analyses of these tissues is not part of this project. L. Data Analysis Blood lead concentration was analyzed by graphite furnace atomic absorption spectroscopy (GFAA) at DuPont Chambers Works, Deepwater, New Jersey. These concentrations were used to calculate AUC (area under blood concentration versus time curve) and the absolute oral bioavailability of lead. Mean body weights, body weight-gains, and food consumption were analyzed statistically using the One-Way Analysis of Variance and Dunnett's tests. Blood lead concentrations from rats were analyzed statistically using nonlinear regression analysis. Blood lead concentrations versus time curves (AUCs) from microswine were analyzed statistically using the Student t-test. Statistical significance was judged at alpha = 0.05. RECORDS AND SAMPLE RETENTION All original records are retained at Haskell Laboratory, Jackson Laboratory (DuPont Chambers Works), or at the Records Management Center, E. I. du Pont de Nemours and Company, Wilmington, Delaware. - 20 DUP040000540 Absolute Bioavailability of Soil Lead in Rats and Microswine DuPont HLR 671-93 RESULTS AND DISCUSSION A, Diet Samples Samples of diet containing lead-contaminated soil were collected at the time the diet was prepared to verify concentration and homogeneity. Homogeneity in the diet was evaluated by calculating the coefficient of variation (C.V. == standard deviation/mean x IDO) of the average concentrations measured in samples collected from the top, middle, and bottom of the mixing vessel. For the rat diet, the mean standard deviation of the homogeneity values expressed as the percent of nominal were: 84.66 6.43,905.29, and 74+2, for the top, middle, and bottom samples, respectively. The C.V. was 9.8% (Table 2). For the microswine diet, the mean standard deviation of the homogeneity values expressed as the percent of nominal were: 89.46+3.26,87.4+3.55, and 94.2+6.88, for top, middle, and bottom samples, respectively. The C.V. was 3.85% (Table 3). The measured concentration of lead in the bottom samples of the rat diet were lower than expected. However, since the C.V. was less than 10%, the diet mixture was considered homogeneous. (Historical data at Haskell Laboratory indicates that homogeneous diets yield a C.V. of 10% or less.) There were no apparent differences between the top, middle, or bottom samples in the measured concentrations of lead in the microswine diet nor between the top and middle samples of the rat diet, demonstrating that the mixing conditions resulted in a homogeneous distribution of lead in the diets. Stability testing of the soils were not performed. U.S. EPA Publication SW-846, Methodsfor the Analysis ofSolid Waste, specifies a holding time for solid material samples of six months with proper refrigeration for samples being analyzed for lead. Since the lead in the samples used in this study was not expected to degrade during the study period, stability testing was not necessary. Samples of Teklad basal mix (rat and microswine), sucrose mixed diets, sucrose, Oreo cookies, swine bedding, deionized water, and lead-contaminated soil were also analyzed. Results are provided in Table 4. Results of these analyses demonstrated that the nominal 1,000 ppm soil was actually 840.9 ppm and no lead was present in the other samples. 6. Dosing Solutions Dosing solutions were analyzed and the measured lead concentrations were used to calculate the bioavailability of lead. Results are provided in Table 1. 21 - DUP040000541 Absolute Bioavailability of Soil Lead in Rats and Microswine DuPont HLR 671-93 C. Blood Lead Concentrations, Area Under the Curve, and Absolute Bioavailability Mean blood lead concentrations are listed in Tables 5-8. Individual animal blood lead concentrations before dosing, after dosing, and during the 30-day feeding phase are listed in Appendices C-E. Area under the blood lead concentration versus time curve (AUC) for lead was determined by trapezoidal rule using the terminal elimination rate constant (13) to extrapolate from the last blood concentration value (Figures 1-4). Absolute bioavailability was determined as the ratio of area under the blood-lead concentration versus time curve for the oral/intravenous groups in comparison to the dosage administered (Table 9). AUCoralx Dosejnfxavenous % Absolute Bioavailability = - ---------- ---------- AUCjntravenous x Uoseorai x 100 1. Single Dose Administration (Acute) Rats. Area under the mean blood concentration-time curve was determined for rats. Individual AUCs were not determined since only four samples (or less) were drawn from each rat due to the size of the animals used for this study (juveniles). Instead, AUC was evaluated using the group mean data for each timepoint. The mean AUC for the intravenous dose (Group I) was 534.32 pg(hr)/ml and the mean AUC for the oral (gavage) dose (Group II) was 10.81 jig(hr)/ml. The percent absolute oral bioavailability was 4,14%. The difference between the AUCs of the intravenous and the oral (gavage) doses was significant (Figure 1). Microswine. Individual animal AUCs were determined for the microswine and are reported in Table 10. The mean AUC for the intravenous dose (Group VI) was 54.09 pg(hr)/ml and the mean AUC for the oral (gavage) dose (Group VUI) was 14.98 pg(hr)/ml. The percent absolute oral bioavailability was 20.90%. The difference between AUCs of the oral (gavage) and intravenous doses was statistically significant (Figure 2). In both species, the observed Cmax of the intravenous dose was at the first timepoint (0.5 hr). The Cmax of the oral (gavage) dose was at 8.0 hrs for rats and at 12.0 hrs for microswine. - 22 DUP040000542 Absolute Bioavailability of Soil Lead in Rats and Microswine DuPont HLR 671-93 2. Single Dose Administration (Subchronic) After 30 consecutive days of feeding, predosing blood lead levels were higher than predosing levels in the single administration groups of rats and microswine (Appendix C). Rats. Similar to the single dose administration group, due to the small number of samples drawn from each animal, the individual areas under the blood concentration-time curve were not determined. Instead, AUCs were determined using the group mean for each timepoint. The mean AUC for the intravenous dose (Group HI) was 457.94 pg(hr)/ml and the mean AUC for the oral (gavage) dose (Group IV) was 62.22 pg(hr)/ml. The percent absolute oral bioavailability was 27.83%. The difference between the AUC of the oral (gavage) and the intravenous dose was significant (Figure 3). Microswine. Individual animal AUCs were determined for the microswine and are reported in Table 11. The mean AUC for the intravenous dose (Group V) was 96.95 pg(hr)/ml and the mean AUC for the oral (gavage) dose (Group VH) was 70.02 |ig(hr)/ml. The percent absolute oral bioavailability was 47.13%. However, there were no significant differences between AUCs of the oral (gavage) and intravenous doses (Figure 4). In both species, the observed Cmax of the intravenous dose was at the first timepoint (0.5 hr). The Cmax of the oral (gavage) dose was at 8.0 hrs for rats and at 4.0 hrs for microswine. The apparent absolute oral bioavailability was greater following subchronic exposure compared to acute exposure. However, this finding appears to be an artifact resulting from the remobilization of lead which had bioaccumulated in tissues during subchronic exposure. Therefore, the oral bioavailability determined after subchronic exposure appears to be inaccurate. Rats and microswine were provided with diets of approximately 21.5 g/rat/day or 340 g/microswine/day at approximately 11:00 a.m. The microswine consumed their diet within one hour while the rats did not consume all their diet until the next day. Therefore, on the day of study start, when the oral (gavage) orintravenous doses were administered (approximately 6:00 a.m.), the rats were considered fed animals while the microswine were fasted for at least 15 hours. Data presented in Sections C.l and C.2 suggest that decreased bioavailability in rats could be related to the presence of food in the G-I. tract. To determine if increased bioavailability of soil lead in microswine relative to rats is related to the presence of food in the G.I. tract at the time of gavage - 23 - DUP040000543 Absolute Bioavailability of Soil Lead in Rats and Microswine_________________________________________ DuPont HLR 671-93 dosing, two microswine were fed their optimized diet one hour before receiving the intravenous or oral (gavage) dose. The AUC for the microswine administered the intravenous dose was 184.86 and for the microswine administered the oral (gavage) dose was 13.05. Calculated absolute bioavailability was 5.33%, which was similar to the acute rat bioavailability (4.14%). The AUC of the intravenous dose of the fed microswine was much higher than the AUC of the intravenous group of the fasted microswine. The AUC of the oral (gavage) dose did not decrease as expected. These results could be due to the deprivation of food which alters the lead distribution rather than affecting absorption. Aungst and Fung, 1981,(4) suggested that alterations in tissue and blood concentration of peptides or amino acids with high affinity for lead could be a causative factor. D. Body Weights, Body Weight Gains, Food Consumption, Intake of Test Substance, and Clinical Observations Exposure to lead produced no statistically significant effects on mean body weights (Tables 12-15, Appendix F), body weight gains (Tables 16-19), or food consumption (Tables 20-23) in either species. Mean daily intake values are listed in Tables 24 and 25. No deaths or compound-related clinical observations (Tables 24-27, Appendix G) of toxicity were observed in any group. A few rats had swollen tails or were missing the ends of the tails, due to drawing blood from tail veins. A few other rats had ruffled fur or skin sores. Microswine were salivating before feeding time. CONCLUSION___ In conclusion, on the bases of species sensitivity and interpretability of the single dose assay results, these results suggest that the determination of the bioavailability of lead in soil following single-dose gavage administration to microswine is a reasonable model, of the methods examined, for determination of lead bioavailability in children. Based on this assay, the test soil exhibited an absolute oral bioavailability of 20.90%. - 24 - DUP040000544 Absolute Bioavailability ol Soil Lead in Rats and Mkroswine DuPont HLR 671-93 REFERENCES 1. Subchronic Oral Toxicity: 30-Day Feeding Study in Male Rats and MiniPigs with Lead. Duane M. Smith, Jr., Ph.D. Louisiana State University School of Medicine. 1992. 2. Characteristics to Consider When Choosing an Animal Model for die Study of Lead Bioavailability. Christopher P. Weis and James M. LaVelle. Chemical Spedation and Bioavailability, 3 (314) 1991, pages 113-119. 3. U.S. Department of Health and Human Services, Public Health Service, National Institute of Health. Revised 1985. Guidefor the Use and Care of Laboratory Animals. 4. Intestinal Lead Absorption in Rats: Effects of Circadian Rhythm, Food, Undernourishment, and Drugs Which Alter Gastric Emptying and G.I, Motility. Bruce J. Aungst and Ho-Leung Fung. Research Communications in Chemical Pathology and Pharmacology. Vol. 34, No.3 1981. - 25 - DUP040000545 DuPont HLR 671-93 CO CM c>Mwo-! DUP040000546 1 rH N & hJ JB G O P* r\ w w a hi % o Cm 0 G 2 < <z> G O 3 5 B 2 D w j<! ft C/5 z 2O og p5 S ft B8 3g yg 2 eg oo UQ m >p a4 45 45 d dS Q i 44 i 0 4^4s 4d (j) N HI "H rl d 44 O 0) "H 45 tt tJ rH rH aa HO * Od |I 11 1i n3 45 d ........ d >3 0 i <w 30 tj Ot aa f' C4 d VO Ho !1 !i 11 45 d <2 _,, 45 od (3 o i d o d o > ft Vi rH 45 d vh HO rH rH 0> 6> dd rl -rl CQ CQ O rl a s 53 O 53 O d -H co d >- o 54 53 O 0 53 ft a CO r*> d 0 d o > ft 44 rH 45 d 44 HO rH rH 0 tn -rdH -drl co CO t M HH H ft H> HH H d ft ,, fl od Vh o CD Vi ft d do on cd n o CO C3 3 h 45 45 d dS 0 a 44 <0 <d r- M rl *rl -H d H0 -ri 45 43 rH rl aa HO OO ro 0 -* N rH "H -H fl HO 0 -H 4> 43 HH aa HO * oo ro 43 45 d d >a 2 a 44 30 o> o> aa t- C4 HH . CSI VO rH rH 2* 21 aa m vo 00 rH H 00 H rl 4a> ijHj 45 td) OH d O M 405 ^ 43 ft g3 V0>l h 45 iS t D H fl o 45 d u i 45 0d ft o s-H d o d > fot Vl rH 45 du HO o rrl d o 9 o --. 53 0 dM co d wO 44 53 O O 53 ft d CO -- d o d o >o Vl H 45 d Vl HO g r*. C4 o m 9 H i rH rl t bi dd H rl CO CQ H rH t? tt dd H "rl CO CQ ji! 45 O AU i * HH >H O >H H 53* M H ft> 45 H S CQ O PS o H ft> d,, O ft vi d CD O .r4t1i d_ 0 ft 44 d CD O Vl CD M0 r0H> 44 -hV(3i 4n5s n03 od COO 44 5U3 45 o -rl 43 *H 45 45od nu 44 53 d aa dmo O-H 53 >0 45 45 M> 4V5l Vol 4O4 d 44 DUP040000547 COIO'. Isno ei X *> e o H CO <n O flj ot O . 3 Q rl so IjO C4 -1 +vo1 vo +o1 o +o1 o'? 01 00 <J> a 1 1 rH C0 +1 1 I1I CJ* CO CHO 3 3 3 S 11I1 |1 1 CO C4 i\no C4 +o1 in o o H -oH o rco TABLE 2. HOMOGENEITY OF LEAD IN DIET PREPARED FOR RATS ft's 38 3 *o Ss 3* 44 o 3 o ri P 3 P 3 03 a5H5Si3 H SO O <# o in r> vo a> co co co o m 3 HI 3 H3 a o aa I3S& forl tJ H0 p p o CO a n <o Ok ii d? 3 0ri p 3H >3 44 0 P 33H "oH 44 44 3 O O 00 CM t DUP040000548 OcOv tN vc &>J J*ocB* csw Q io \o 00 <0 Cj gj MAVrl a CO +v1o "I in co +1 o <.** 00 u> -H OJ O' 1 0CO\ r> 00 oil a CO 00 VO r CO H H CO +1 +1 +i +! co o a t- i" H to a> CO r* s O' ** TABLE 3. HOMOGENEITY OF LEAD IN DIET PREPARED FOR MICROSWINE 9 <d iSiS' |1 [ VD \0 O P o 2 , CO VO j T* m s 1 <H O ao *rl 4J flj (4 4J a , 8* O in CO t- tH c j in co o in oo m in ri < in ** o m r-4 ft Pi SK 3& CQ ft O EH --I <d a rl s ao 4J 40 O <a>o co a o rl 4J IHd U mo *> fl H -OH <4-1 <4-4 o o o> svi DUP040000549 TABLE 4. MISCELLANEOUS LEAD CONCENTRATIONS cCOt H-ID>'! so a X c o p* 3 o d o s rl 44 <D O oo >* oooooo 03 d o o rl rl 0 (4 xn q 44 fl Unj Sq TJ ,, 0) S NH AA d Oo -rl O a) o oH q d rl 1 qo rl Pi -- A O 44 a rl 8) s t!J am 44 a q HHtg!> -qH -Mrl (n0 fral! -<rdl ao o PQ n q qa n n ou o Hj Hq qR o q 40 4! -rl x3n oM q E4 q Enr > W &A A in v a 0 44 -H s rl r-f d o rl 44 O q 44 oq 8. DUP040000550 p CO CTs i MEAN BLOOD LEAD CONCENTRATION (pg/ml) IN M ALE RATS (Acute) Group I: Single Intravenous Dose Group II: Single Oral Dose o3 nID CP 3 Q A p I P IS 4 ri O O O) h A OCSf VO 00 CD CD * I ...............' | oiooHionnmooHoooo H , OOOOHOQO OO O w I O O O O 04 < A 04 04 04 "Vf in 04 V0 O CO 04 CO 00 00 040404"*H04'^'0'*<*>V00404HO w OOOOHCOHOOOOOOOO s ooooooooooooooo a P0 P p A PA g a %I 01 04 rH 001 AJ p 3 04 p mri op g ao 04 0VOV VO O VO in VO CO H O' r> r*l . I HVI^IOiOlflOIMflbrlOP*Ots Q I O VO 00 O' VO 04 m O' O' m 00 t** 00 CO o * I OV0V0inV004C0H04r4OOOOO H I t 4 -H 04 8 p$ rHOOOOOinOQOOOOOO ...............o Ii HomVOr0i4m00v0o0 vortmp04HVOn0'4OifCf4l Oid O 1 II VKOH C> 04 CO CV CO 04 04 H r-4 H Wp -g O OH p 44 2 H p a I m gao a HP &a oil H j l>1 m on) mooooooooooooo II aI) O H 04 <# 00 04 00 04 VO O -'tft 04 OH o s* I1 p ft H 04 O' 00 04 # 00 HHH04 r 3 P -H o3 3-rl <0 A TABLE 5. DUP040000551 TABLE 6. MEAN BLOOD LEAD CONCENTRATION (pg/ml) IN M ALE RATS (Subchronic) Group III: Single Intravenous Dose A fter Feeding fo r 30 Consecutive Days Group IV : Single Oral Dose A fter Feeding for 30 Consecutive Days eCins e cr\cocsici<novin<oinaotnF*t-<ct o .ooooooooooooooo > H * I.............................................................. . ooooooooaooo o o o CU 8 ei O i r* w> r> t -i rf v> a\ <n pj csi nf <n l OOOOHHOOOOOOOOO jooooooooooooooo . i mHsooMnaocjooomHcjo p. ii hofnicHiifotoriinriao> nooinHweoomnmijnhpvi>in^ W, H i oonionciiHonoooooo H H 01 Do riomooooinooiOoooo oPi p to m in <n m to r* o> *#10 06 r* O OOP-OVOCOlOHfOrHOOOO O HHH 2 O m HH E* O A HI O O 0) on ,100000000000000 J,Or-IC,'<#P*><*a>0|tOOTfC*0 2 rlHHO <0 DUP040000552 TABLE 7. MEAN BLOOD LEAD CONCENTRATION (pg/ml) IN M ALE MICROSWINE (Acute) Group V I: Single Intravenous Dose Group V III: Single Oral Dose co O '. g so & * lOOOMMHCIrtNOriHHNO H H , OOOOOOOOOOOOOOO H j ooooooooooooooo H > ft D O OOHrtiOf^Old'iWMWN'Jrl CD OOOOOOHOOOOOOO o ooooooooooooooo . t to oo r* co r> co co vo in to vn ** e* p | wa>rtiflo<n>ijcoinNinifiwu . I OlOhWriOOrfOHHOOOO I OOOOOOOOOOOOOOO03 I......................................................................................... M > b Do m o r^-nflowOin*OuOiTCf4inCo'4HV>OVOftiNnt0OftOfl\OO\irDH o VO co in CO CO CO CO C* CO H rt O o OHrHOOOOOOOOOOOO BH &c m o H , in ooo o o o o o o o o o oJ XJ 2 O f I Cl <* 00 rt Mtof hCoVOicOlf*l*oCS-I do n, HHHC1 m to DUP040000553 CO S' TABLE 8. M EAN BLOOD LEAD CONCENTRATION (pg/ml) IN M ALE MICROSW INE (Subchronic) Group V: Single Intravenous Dose A fter Feeding fo r 30 Consecutive Days Group V II: Single Oral Dose A fter Feeding for 30 Consecutive Days I VO r-f VO C* r-4 IO H O VO O <) VO d IO Q . in**v0E0ico<0V0V0inrodC4c4c , ooooooooooooooo H H CQ ] ooooooooooooooo > Pi 8 cjHH*#ov-5r<nrtovco<n0\Hrt oomnnvo'jMHNffl ioin n m h HHMflrtNMMCJHriHHHH OOOOOOOOOOOOOOO a iI iTnifnHdirf>indr'*f*o*o'su*oiopvi'*j*ino\fmip'xnosif*l I OOHOVOTfOtNIMHi-irlHOO WI I OfOHHOOOOOOOOOOO Pi oD o moooor'coc^cNiininino^f'O (0 N rt in Cl d> H H (O ^MlO IO M(0 ** fO H iH o o o o o o o o o 8 Hw A oua a <D a ,1000000 0 0000000 loHN^aniKDriioo^no l , i-i 01 ** r* o\ d *4 o\ 3, HrlHM w DUP040000554 TABLE 9. ABSOLUTE BIO AVAILABILITY IN RATS A N D MICROSWINE A A to CCrHIOO rH 44 1 >3 O * A A 0 44 O VO o 3i es >-) sc H3 A0 -H 3 3 0 ffl-toxS 3 A ' rl 0 cs A to A to 44 & >3 44 O O o Oh 3 ft to a o 3 0 to to >i O 0 rH o *3 o t3o 3_ H to -iH 44 A a3 5* XJ >3 3 to 3 >i to ft 303 Oft H tt) H 44 -H H3 Ora HI H <0 S Hf fO rH 00 r> CJ o to <3\ H -* * o r> o ** CO CO in 3 o to XJ tn 44 44 3 0O 3 O to <3 xi 3 "3 3 3 3 3 -H <r> 0 >3 0)44 "3 333 rt 0 44 -H a oH H 4J 0 -- to *3 to to 44 -- 0 rf 3 0 to XJ 44 O 3 X) O3 w< >o--i o H 3 0 to x 44 o 3 XJ g3 I CQ ' >* 33 H *rl n XJ 3H 0 to o rl to A 3 "H A S. 4J rH to O <3 A 3 -u rl to to O 44 JJ W& too *3 O <3 44 A XJ 3 3 & OO >3 3 >-- rH A fa a to -H 44 A 0 3 3 <3 3 -rl to & rH icno I OP to to a *3 fa to >3 t fa X! -rl 3 X> 44 44 vi Pi OO rl "H aa 0 fa 3 o 5 -rl 3> 3 44 0 no 0o PP oo PQ o p >i I >3 4J -H rl A to rH 0 to fa >3 O 44 to to -rhl HH 3rl -3rl ca c q rH rl bi t 33 -rf *rl 01 01 rH 3& -rl CQ rl 44 X) 3 *rl rH to 3 a rl fa > '3 3 O rl -Orl $ H -rl 44 tn XI ra 3 3 "rl rl rH rl 3 "rl &.H a fa O' tOo <3 O -ri XJ A M il to AA 33 H 31 0O xi O 0 S3 0>3 rl to X) a3 3 H 44 *H & 44 & -rl 3 O 3 rt O a to-H O to oo o a-H 3 XJ -aH XJ DUP040000555 rt th e . ft <H fO ft 0 rt ft u ft u SH -rt rt D j* H 0> ft h co v rt rt -rt ft Q tl 00 O H HM aa ft C0 ft rt ft rt 8 c-q t- m N* 6 H ft 0 rt ft rt AREA UNDER THE BLOOD CONCENTRATION-TIME CURVE (AUC) FOR MICROSWINE (Acute) Group V I: Single Intravenous Dose Group V III: Single Oral Dose 4 ft C* H O VO <M rH rH h.fc > ft >- rt W H 0) H rt H BlH naN .Oft >i rt rH a rt H o rt o> rt D rt O *4 o irt m D O m 0 ' ft m& =L CO -rt H ft -rt OJ ft <0 rt JJ t ft N rt oo rt rt oooo 00 00 00 00 3 n -V'* ft rt ft a rt rt jl 1# rH ft & O ft rt n -rt rt m ft o m 0 rt 4J rt -rt >-rt & o rt *-rl S) a-ri ctoo rt rt co a a 4J 0 rt o rt rt 0 ft o rt*! a 0* rt <H -- * a co in in m co 0 rt . r rt n a in ft *9 Cl CH 00 VO rt o o ft ft Uu < ft vo vo <n a\ oo vo vo vo t -I *# !> ft rt MH ft Q ft a o <. o ft < H> 0> =t ft rt rt rt HQ n i! ft Oft rt at ft ft ft D O PS 0 rt rt rrl rl tn 01 rrtl -rrtl ft O O rt ft rt rt rt rH 0> o rt at >- aa CO CO a ft o o rt a ft ft csi co* in vo o oo o o VO VOVO VO VO rt rt 0o !M VH ft rt rl 4) O rl rt i ft rt > ft Q rt oa ft O D D O ft rt rt! rt 0 ft > A III rt rH rt rt >0 rt 01 rt rt rH ft ft ft rt rt rH 4J ft -H SS l! 01 H CQ TABLE 10. DUP040000556 om \ 0 Vi 0 VO 3 43 >i Pi 0) f 44 H rl .<-> H ft |m| to ^ 43 0 3 X 44 o o +* 0 rl -rl 3 s 3 & 44 t o P-, 3 a**. 3|5 a >0-H 0 0 44 a D ri 0 ov in co o* r U fe 43 ~ 44 0 0 0 44 a 30 moooonn h AREA UNDER THE BLOOD CONCENTRATION-TIME CURVE (AUC) FOR MICROSWINE (Subchronic) Group V: Single Intravenous Dose A fter Feeding fo r 30 Consecutive Days Group V II: Single Oral Dose A fter Feeding fo r 30 Consecutive Days I VI 43 H H m L > o^noh m vp in vo i> t" e> =ts N. in r* 0> Vi A VO <n bi 0Q3 >3Q 03 00 >i 3 > * 1-10 0 '--ri & 3 0 poP a =L 0M -o0H So 44 -H 3 ... HO Vi si*m in 0 43 .*** o -rl CJ > 44 H ^ m vo ooo r- r- c* r> C' r* rl u in I 4O3 A3 -OH C--Q oa ao OA Q w3 0 0 4TO4 0 3 -ri 3 CQ w3 3 rl 10 4$3 43 44 Ou 44 % ou U >*rl OH Cn 0 aO 0 0 atoo 0 30 55 S o 3<0 .43 > 0) 3. oo co vo o in in vo oo vo in vo H vo oo n co in o m co vo ri rl rl 30 0 03 0 O >0 fi MH 44 0 33 NO 44 *03 . 0O *3 m 5 00 44 404 O 0 A rl 33 TV II TO -rl 403 0 0 O W & OP 00 i--1 HI C# Cn 43 3 0 0 Pi 0 A 30 rl 0 3 0 44 33 HI -H "-44 43 <3 3 03 03 TO 44 -rl 0 h N in Tf in >o oooooo in in in in in in 33 O0 <3 44 0 0 43 30 r0l 44 1 44 O d-rj > 44 0 3 OO OO D P O 44 -H 3 << P<0 3 0 <3 -H .3 O 030 3 0 0 SS 43 a A ri 3 a> 3 r10l 44 03 -rl 0 03 'x TABLE 11. DUP040000557 TABLE 12. M EAN BODY WEIGHTS (g) OF M ALE RATS (Acute) Group I: Single Intravenous Dose tCoO IMSS PH hJ 53 *- ts o Pas P is co co .* H HO rl in CO HI H H *W ^ 'i in . t PI O H oo co in HH HO 10 CO t> <n in co woo *it VO 00 xf oo co in HH 0c -~- gM o P. 0O0 --Pi 0S3 H 1 ec SM3 a o m co 9H O O IS Q H a am) 4) A IS a> u (0 P. ao> Xi on Pi a H a 0 4hJ 10 rl a) d g nj 'H <0 .p CQ 10 as DUP040000558 TABLE 13. MEAN BODY WEIGHTS (g) OF MALE RATS (Subchronic) Group III: Single Intravenous Dose After Feeding for 30 Consecutive Days Group IV: Single Oral Dose After Feeding fo r 30 Consecutive Days o tVxO a a VP a o (Ll 3 Q OOCJOHft^l >O H OO H o d in in oo r- o rl rl rl H H pi CJ S** S.P VP VP .V* VP HOOdOMMO m d t H p H m o to 00 to CO d VO o d rl h c j n n HH O Ho rl CU *> da 20BJ SP1i 8 H g s In 25 w o 8 o e COW w Ei **d WE< H Xo d Pi % 05 CP op 00 d VO Cl o\ oo co oo d d vo io rH H d d Cl d VP VP V. -.V* VP O d d t> d VO vo oo ** t> ov rco <n -tf r> o d rH p H d d d rt O C* ^ p H 00 in p H H d d cn xf CO 0<0) J I M d JaJ) Po S' u *mH *oifHt -u <0 -H <D d t n5 m JJ w <0 a\ DUP040000559 o9soooowdna 0 tror M OI* aPn>. OM "4 SR 1 ! H a. CO CO * o 3 < H- CJ Pl fHtO O& Oa V- t> y tHt- H $ Orr co -J o\ to -o oo - to to H- C3 0 0 ** to O 41 to W l W <D MHM *. U1 W OMUI 4 to * rr Q) ff (a o< H to *4 *0 Oi C4O C0O0 w01 o in 00 . t * co to cn UtoMoo oMo to in * < OH H H s 03 IT * Oot Oo>1 *ct c pj s g z << 03 o cd a CD 5 *+*'sSZ. <. re o V a M ES I aO offi 0-30 0re3 a01 Cfoot0 `oPI > 5 s s z PI P' r> afre** a e OP3 a X r* $a a 4s > CtoD TABLE 15. M EAN BODY WEIGHTS (g) OF MALE MICROSWINE (Subchronic) Group V: Single Intravenous Dose A fter Feeding fo r 30 Consecutive Days Group V II: Single Oral Dose A fter Feeding fo r 30 Consecutive Days CO On i prf J X C 3 Q re o M h ri <n n <r* a oo to o\ ** co HO oo oo t- a> oo h co H HO > HQ fO 00 o o o o o WMIMONMO Mftwo^oa f> VO OOH<(M| (OOOOIOHMM rt H ri H o >O o t-i Pc dO a ft 0rt --ft g H i wz a a EC 01 01 w 1 SIS 8 H g Pc Pi < <0 > W rl Ifl rl |H w o n o m tn MOOOOOO\Orl HH '% ** -> * ' bnrtMOoo *> CO P- CO <*1 r-i * inrionoo A O T> tj oo o\ o VO 00 CO OV O H CO HHH O P> ^ H ffl IO H H N N 1*1 CO 0m) JH 4l d 0) u dft d rl d fMot u a rl d 0 -rl 41 flj -rl > d d h (0 40(110 I DUP040000561 TABLE 16. M EAN BODY WEIGHT GAINS (g) OF M ALE RATS (Acute) Group I: Single Intravenous Dose t0rHo-I1 In SO <h2J5 as P a Csn Q to.* to in to H HO O . to to fSI HO 00 . r- c n i P> rl. H "<# in csi Pi O Pi 05 Pi O 55 O H Ej g S3 W O U Et co <n fH w ^* !3 O t" o rl 11 o t" aa> a <D .P 4J 0P) u id Pi P rl 'CaJ) 4J M o aat h n rl P0 rl 4J rl 5> tJ <d 1 U oa id 3 DUP040000562 TABLE 17. M EAN BODY WEIGHT GAINS (g) OF M ALE RATS (Subchronic) Group III: Single Intravenous Dose A fter Feeding for 30 Consecutive Days Group IV : Single Oral Dose A fter Feeding fo r 30 Consecutive Days eo C*5 X C O eu 3 O H r o u co {> O HI O o rl inoomonw rl rl w 'w >-- ' w v r ** <o ci in cn aj o in in H HO HO rl 0, rj da 2& ga H I1 W o a o o 01 <n H e? a H * 0 PS 01 p. *! O 10 10 H 0 I< f* r- o 'P* c* c> in ID rl Cl C4 P- CO in in in ro d d r ** ri oo in rl I rIl NI NI PII ^I o t> "<# h i m rl N W PI U(0 U a u Pi -aH a u oft M a -ri a 0 "rl 4J 10 -rl S d * * X(0i 01 DUP040000563 DuPont HLR 671-93 1016. 153 485. 272 * .g5p,S> Q M) - OCQ >hH :H> Z cm o* 2oo loo 00 i-3 CQ a H H HO > ww l A H >O 10 01 HI in ot 04 ri CO CO m n trt CO <n ir 0. ^ Q ft gS H I w su o & co CQ Qg t- o rl o r- ca fClO) J4J Grt ft V) n o S' rt a -rt Ort 1O0 >4 0 *rot c! 44 CQ 9 DUP040000564 TABLE 19. M EAN BODY W EIGHT GAINS (g) OF M ALE MICROSWINE (Subchronic) Group V: Single Intravenous Dose A fter Feeding for 30 Consecutive Days Group V II: Single Oral Dose A fter Feeding fo r 30 Consecutive Days CO <3i> 3 Q O rl (O ffl IO cn to e4 oo rlrlNrl (*) HO HO >O in co inc m c m cm * 4 44 4 4 WM ^ H t- co w m oo m o <ri ot> H HH O >o h Dg 2ft fftt 2< > a 0 w 1 uft 0 w 1 H g fffttt < Bt nia Ih m < Eh ft a O n) Hfloionn CM Cl rM r* Cl rH UVO 00 CM CM O o\ *9 in o r* <n C" ** CO 0 H H rl :H pi pi t* * eo w r* h <m n IIIIII o r- r-4 oo in rM CM CM CO trat a) A jj a au) aftt a0> 4VJi Oufot a H a 0pi 4J -H >0 0 s 0 1 O to 111 DUP040000565 TABLE 20. M EAN D A ILY FOOD CONSUMPTION (g) BY MALE RATS (Acute) Group I: Single Intravenous Dose 46 COOt CrV-A-l PS) rJ ip cos H % O o 1 **'* <0 < M H e H >-- W' rl HO ao u> at HI HI ip ft o <w 0 HO 0 * O CSI M Op -O' m H rl II Pi -~ DS oa ft o-- S H Ej WM |M w 5S ** r- o HI ii o r >1 0 d >>s a fl) to 01 A 4J a u N. & 13 rMJ in 0) Li rl 0 CM 0. >1 HH H rl 0 IP u a> -H ip VI o A X 0n 0j. ft 0 m -rl IP ao -H & -rl ip 0 0 SJ > H 0 V on t ft <8 n 0 Vi W Is (8 IP W a ip O0 a as Pi DUP040000566 TABLE 21. M EAN D AILY FOOD CONSUMPTION (g) BY MALE RATS (Subchronic) Group III: Single Intravenous Dose A fter Feeding fo r 30 Consecutive Days Group IV: Single Oral Dose A fter Feeding for 30 Consecutive Days CO d rH O O VO H a& E *0 c I -rl >0 D vo w in h ci m3 o HO rH H'O O W rH 1 o O 90 CPP> PI O * r> oh o o 41 & H H M Cl CM 44 O & tJ 41 (0 n HO HO HO H Pi -> O2P5 ~A& J A O t> VOHlOri H H rl H H H CO O hO^ CO P t -I C4HOO rH CM CM CM CM CM to eo*i Si C M H ffl in H rH CM CM PI sf II I I I I oc MHOin rH CM CM P> io H ci rH a> 41 g g M a 43 4H1 ad> MH3 s u A h N (0 at Xt a 41 at p$ DUP040000567 TABLE 22. M EAN DAILY FOOD CONSUMPTION (g) BY MALE MICROSWINE (Acute) Group V I: Single Intravenous Dose Group V III: Single Oral Dose GROUP (ppm) COIO'* rH vp i<-slj X -<-> c o ft* 3 H H HO > HO" w o VO CO CO CO CO CO H t> < --t , vo ** .. VO ov >- >o--* P CO 00 vo H Cl CO CO z pi oH CO CO H P> o H Ej < pi 1 1 g fi Z o r*. Pt O ZM & *ds 0 d -H a O h -oH *8>. di a a o a 0 CO A P a0) M H 0 P 10 a H d Vt 0 a 1 d pon a n X! PH *S sa -H 0 *d -H > -rl O P * -Hm P 0 a> H irf d Id 0 N at rt * a H at P Pra 8 at 9 DUP040000568 TABLE 23. MEAN D AILY FOOD CONSUMPTION (g) BY MALE MICROSWINE (Subchronic) Group V: Single Intravenous Dose After Feeding for 30 Consecutive Days Group V II: Single Oral Dose A fter Feeding for 30 Consecutive Days CO <T> fcs SO j a HO HO >O CHI CO CO * ri t' (<) b o o o ,r o h so r- h O 01 00 00 00 00 co co co co co co CO CO CO CO CO CO >O o ri Oc O5 IPi t6 ft O --' B H I zw I a EC W CO i] 99 az H 9 rt 00<. Id r"*d iT*. dTS d----d dd^ a co Pi h ri cj CO o o o o Ci 00 SO SO in Hi CO IO 00 00 CO 00 Ci CO CO CO CO CO (O CO CO CO 00 CO p>HHC0inH I r-I i CIH CI CIO I* a t> h > ri oo in 1-1 <H CH CO o Sf S3 "-s c -H CoD 0n H 01 a a o H CO I 40-> ifdt rl p H X0n 14H0J JdCp3 H 9u * a S3 ri S3 ri -ri > o 4J * Id ft dp -rl 5) Pu .3 S3 S3 S3 n 1 M0 1 aId rl a rl a TO oMo dg 2rl w} DUP040000569 TABLE 24. ftiors tr>"l SO hJ te loa % Q l> t> CM CM h* tH _ to co co co CM oooooo c m r> r to o> in tf so o ro c m so in ro to MEAN DAILY INTAKE (mg LEAD/kg BODY WEIGHT/day) OF LEAD BY M ALE RATS (Subchronic) Group III: Single Intravenous Dose After Feeding for 30 Consecutive Days Group IV : Single Oral Dose A fter Feeding for 30 Consecutive Days WH s^o rhtcojsccimtsroi oooooo N> CM CO CM t" CO CO c m ^ so r- ro h so in to to to Eh ww MM KSj Eh O 55 O h m h in h I It H Ic m Ic m ro I I o r> h j h co in HI CM CM ro a ra a n (0 A ril tJ 0) a u o 0 M0 id ! P XfX oin DUP040000570 TABLE 25. CO i MEAN DAILY INTAKE (mg LEAD/kg BODY WEIGHT/day) OF LEAD BY MALE MICROSW INE (Subchronic) Group V: Single Intravenous Dose A fter Feeding fo r 30 Consecutive Days Group V II: Single Oral Dose A fter Feeding for 30 Consecutive Days ts Q r> vo *f 05 05 H H HHH O O O '* O O S** 05 O <o m vo oo t" m co oi t"*5 cH 10 >*. OOMO^fTjM 05 H r-1 H H r-l O *OW O NOm* O O loooeono eo in <n 05 05 H H cl H E* TO 01 J*0* EM< Q 5o3 O^HOJIAH I HI CI5 0I5 nI *#I o c- t j i in T-l 05 05 CO a 00) J1 la u0 10 a a rt a 0 *3 .ou 0au a a 0H U <H0 > 0 *0 a 13 *1 4<J0 01 10 s DUP040000571 0m\ S hj K cOw Jfa 3 Q o3 3 CO H <3 35 w < CO 2 0 P 1 w co pa q O 03 s os3 Q*vi. 2 1k--J4 <*-r& Jb TM 3O U o34 >C* *-H fTt5o3i tiof 00 w H JH-H 235*5 oO0Sh o0O0h CO O O VMO >J pa is oH CQ H0 h w HM in H 0000 l q o co n H fO in rl m & -rl u e > olg g gS 0 H CO CQ H a Q g H O H B i< H H fa El Ej Ei HI O > fa M H (ti OJ J M fa W iQ CQ Q fa PS O CQ D O O M fa CQ CQ N 10 DUP040000572 x * a o 00 M P TABLE 27. SUMMARY OF CLINICAL OBSERVATIONS IN M ALE RATS (Subchronic) : Group III: Single Intravenous Dose A fter Feeding fo r 30 Consecutive Days Group IV : Single Oral Dpse A fte r Feeding for 30 Consecutive Days > in hn H H W> H (N i I OOHO lO O ^ C> ri tH rH u> n o h H H 01 01 H S CQ P H Uh ofe W hH | 301 m p O CM o M P$ CQ CQ OO H P OW P H <d tino DUP040000573 s o CL. TABLE 28. SUMMARY OF CLINICAL OBSERVATIONS IN M ALE MICROSWINE (Acute) i Group V I: Single Intravenous Dose i i Group V III: Single Oral Dose ! H HH > in H > 10 o nJ o t-f in DUP040000574 TABLE 29. SUMMARY OF CLINICAL OBSERVATIONS IN MALE MICROSWINE (Subchronic : Group V: Single Intravenous Dose A fter Feeding for 30 Consecutive Days Group V II: Single Oral Dose A fter Feeding fo r 30 Consecutive Days j fa > H O W tv H 5i Hi si W Oi 05 O H si fa O 05 fa p 5 i i i i i i i i i i i i ! H H > VO > VO rCoSI a a) c<No o. o I imn i DUP040000575 DuPont HLR 671-93 \co CO W o PM DUP040000576 p> < pM UHHM w *O K) P*>. M AaO> 'KJ> M** hK>* 00 *> K> H o hJJ 0 ooooooooooooo Wig* Dffi- n 0 HH* 4 a. t *s o *3 H Hi H g oicnoiinam(n<n(noio\aio\OMn ooooooooooooooo 8 PHHHHKHHh h h h k h m P 0 PPM PH* O O O o O O P wp M M u t<a Hu ppiowiodwouomcDM mu o iniwfcvii>iooiHOooooo UlCOtOOOOOOOOOOOOOl < H O Hi ra 0 <co0 oo oo oo oo co co co a> co co co oo oo a> oo OOOOOOOOOOOOOOO W W b> U> W U U> U> U> U> W W W W U) OOOOOOOOOOOOOOO o o o o o o to O P* oooo o OMPWPfctOOPOCDWMOO WWOOOUIMOHWWUIWWOO p oa *d <; H H H BLOOD LEAD CONCENTRATION (^ig/ml) IN MALE MICROSWINE AFTER INTRAVENOUS OR ORAL DOSING Values from microswine fed one hour before dosing a ss o s ffi r1 SJ >o0v V0O3 DUP040000577 APPENDIX E. BLOOD LEAD CONCENTRATION DURING THE 30-DAY FEEDING PHASE O z & >ww*> W W W OJ WU)UIUUO)UWU)WWUWO)WUUU)U)U MMBMMMHHHHHHMHHHdOOOOOOOO Ul U U HO VO 00 ~J Ol Ol iH WMHOlOOOHOi Ol Is. W M H Sam ple n o t s u b m itte d f o r a n la y s is . mta CO o o o o oo ooo o o oooooooOoooo toI JI oooooooo o. OOOOOOOO Oo 100 000 0 0,5 H o w o o H P O O O l- O ,p* OI oowuioun h -J oui< *k. H cn OV Ol VO VO oo Ki o\ m w w so HI s o o o o oo o 0 O P* O P* O M 0fc1 O!>lWMlUO1 MPHUIWO oooooooo o O *- O O O O j- O J, o o UOOlslioOllOUl1* VO 4 uoMonuifra O ->3 o PPM w o 00 to J I 0 w H u i <Ool oOoOo O O O O OO O PWWMHOOOl M 00 Ol 00 VO U) O O O o O OOOOOOOO O O O O O H H H* H H O H UIOUI^O.WVOIOVO'JHWVOOOU **-Ul*k.~JOlOlt0tOO1UltO00ttt0tO M VO W * M tJ H Ol to CD 0. 0. ooooo Oooooo *4 o to w U1 Ol Ol 00 00 Ol 1-* VO Ol tk ooo oooo ooo o o o o o H* O o o O O O O O o Ol to Ol tk Ol M-OO Sl Ol-vJ oi oo oi -vd vo Ol 00 H1 Ik Ol OOlkO tk. oo Ol l-v VO Vo Ol o 3H o U1 SM t *H Ik cn a c 0u &* X r* 0'MO1 VOOo DUP040000579 BLOOD LEAD CONCENTRATION <jig/ml) IN M ALE RATS DURING THE 30-DAY FEEDING PHASE Group IV : Single Oral Dose A fter Feeding for 30 Consecutive Days fe. ^ ^ k *k fe. MBHNMMHh'HHHHHHHPObOObO O in^wiopou)a)^<nuii^w(OHo<oQ)'Joiui^wiOH oooooooo ooooooooo o o oo o ooooooooooooooooooooooooo ooooooooooooooooooooooooo oooo'Jooouiuio>'4ooooooo\cooooovo 4 fMcd 2M a2 HM o ooooooooooooooooooooooooo O O O O O M O O O O I-* O + H H H H O M W 0l H OlH H M VO si (J1 a >J Ol W O |k H >1(0 :*>> O >] k > H a cv utn a w u> a s] >] io w w tii oi h (n si m w 3 K Ho $ OOOOOOOOOOOOOOOOOOOOOOOOO O O I-* O O O O O O O O O H* l- O H H> O O O HOlOUHWUUItDtkWWWWUlOHOlHMUlOlfflW'J oMH^bisis]otik>Ms]s]|jiOHsis]u^(n^ai)oia n4 w w o H M S3 o ooooooooooooooooooooooooo . * b * OH^OOOHOOMHOOOOOOMOHHHOOOH o\wo<iMfio^t.omouiu c j i vo oacneno toto moo ui to s]UlMJO\UIUIUiaWi>blb)OOlOsI^WlOsl<eUIO *4 sH uW SCJ H w.J a3 a e 13 o 3 33 IT* 53 ON sHq vwo DUP040000580 D U R IN G TH E 30-DAY FEEDING PHASE Group V: Single Intravenous Dose A fter Feeding for 30 Consecutive Days oi in ui yi ui in oooooo o\ ui U) to h % oooooo ooooo M 0 0> ts> O Ia> *4 00 * O 00 poo poo HHHWH A UI U U M OUIOU^IW <o o o o o o |_1 |_1 o M M H in oi p oooo OOP o o o P Ui o OI W >U A A J CB s> * Mg H U) a ooo WH Hn IUMM*0AhWM in u> u i u i to p *8 a o *3** a r1 5s o PW DUP040000581 DuPont H > wa Hu n Wg *H - ** 3M |a ^H Mu o? u> w w Q H Z o W M M VWO to o ot ** uo> o j 702 0.023 0.130 0.206 0.169 703 0.005 0.080 0.217 0.137 704 0.057 0.170 0.261 0.209 705 0.010 0.180 0.258 0.227 706 0.007 0.290 0.201 0.173 SO2 c*o > 3w Z a a Tl z 3 a 03 O a < o ffi 3 DuPont HLR 671-93 DUP040000583 IN D IVID U AL BODY WEIGHTS (g ) OF MALE BATS Group !: Single Intravenous Dose 100 UI CP UI ip UI cp UI CP UcoI UI CP UI IP UI IP UI CP UI CP UI IP UI CP UI CP UI CP UI CP UI IP UI CP UI to UI CP UI CO UI CP UI (p UI CP UI CP UI CP t H ttj OM S5 d > 3 hi OI to OtoI OI to OI to OtoI OI to Ol to OI to OI to tOoI OtoI OtoI OI to OI to OI to OtoI OI to tOoI OI to OI to OI to OI to OI to Oi to OI to 5 b* M to CO 00 cn 00 00 4 00 4 S3 SI OI <4 UI 4 p 4 CP 4 to 4 M 4 OI cp OI 00 Oi 4 OI Ol OI UI Oi OI CP OI to OI M 4fc *3 00 to \D u> so UI CoO 4 00 CtoO 00 CP H 4 4 CO 4 4 to GO as 4 Ol CD 4 00 to CD 4 Ol O 4 CO oo CP 4 OI O to 4 CO CO to 06 W 4-j ip cr> vo co co o c p oo co c p o o \ h co w c p o. oo ui in o H* H H H M M H H* HH M l- H h* O h1 H hMS H W M W M (O O U O O U) U K) V (O M U H O W W H Ol "J M M W H U1 H M U Ol M Ol OH SO H W U1 Jfe M /** O^OtUMOtO^UIUlUICDOl 10 * lO bO VO VO H 06 UI >3 00CP HHKHHHh'HPHPHHH.MHHHHHHMHHH1 WWikOHOOlWCnWUlWWlOWMKJH'JWMHUlWWU 'JHWO'JHfrUlWsJUimoOffl^VOOMCOWW'JMH M IO Ol O) n ] CO Ul O U Ul W U) U) Ul W W W O W H U U) CO A u to to co to co to to to Co to co to ta to to to to to to OOOt) 0 008 0 ODOOO d d d d d d ^ UPI HS3 PP^ ^ #S HCP 'S ,<*s ^ Sd d dS dS i.p .j >e>o dd>>d.p^dp do* d> pd dk> d> >d w d> d> d>- >d i< * K H W oi * Ki *4 k ! * CP 4 K h * *{ * K h { * *>. k J h * * K >4 H* o oo *4 4 4 oo oo 00 4 4 4 4 4 4 4 4 4 4 00 >*3 *4 aS o3r* frfi4 53 <x\ VCOO DUP040000584 g ia UI CO ui CO UI CO UI CO UI CO UI CO UI co Ul Co UI CO UI CO Ul CO Ul CO Ul CO Ul CO Ul CO Ul CO UI CO Ul CO Ul CO Ul CO Ul co Ul CO Ul CO Ul CO Ul CO -O -O *0 4 4 >o >0 <4 >4 *4 4 4 4 "4 -4 >4 *4 >3 -si *4 >4 >4 >4 >4 >4 H 6 > 3 Hi W to 09 "0 oCO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO CO co cO co o -o *o >o si Ol Ol Ol Ol Ol Ol Ol Ol Ol Ol Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul * > CO to p VO Ol UI #* CO to p o VO co >4 Ol Ul CO to p o pi m C0 *3 PP >4 Co SI o d s4 vo 01 co <-4 o 01 o *4 a> oa 09 *4 a> *4 co 01 vp >4 06 >4 (kOUlPOOUOlOOMWOWH'JM'OHOO'JOlMOl P >4 VO sioi>J)Ui'Jo\a)om<hiW(woooooDWioutkOiH UWWWWPH0PHUUP)ffiPU(l)POPOOP OOP )OP 'JHP VOPW O'JOOlJWUIOUOiivJOtiMWWaiOlWCDttO'J VO Cp CO tO tO 00 Ul *4 00 Ul VO Ul P VO P CO U-JtSHUUIUUVOH CO 0 PPPPPPPPPPPPPPPPPRPPPPPPP 1 NlOXBlIiflOOlfcWMWOOOWOOsnoWPMUlOHaOOl Ol HJ OUO Ol -4 VOM^UOO^^^'JM^OHOUIO Ul co to to co to co to Co co to to co to Co to a oootiooe a w a o o ea Pov Rw <* r. uP>-~s^'OPvPoV'~'PWoPv#P^--- aP ^ P a e*p v. > uJt>< >a uJ> oJl* [et* eJt e>> to a> o c. o t.o aj> .o i o i o. v u>> ^a ci o *> tJpj p hj(B'OHjKKjK|KKKiU1)<KWOlKOUlWKl4Ml<^t> >4 *4 *4 SI >4 SI >4 >0-0 SI >4 SI -4 P DuPont HLR 671-93 DUP040000585 IN D IV ID U A L BODY WEIGHTS (g) OF MALE RATS Group III: Single Intravenous Dose A fter Feeding for 30 Consecutive Days 213. 536311 NW RS (DAY g171 U1 C71 cn cn C7I cn cn cn tn tn cn tn tn tn in tn cn tn tn tn tn tn tn tn tn 8 CO CO CO CO CO CO to CO CO to to to CO CO to to CO to to to CO to to to to to PC PC PC PC PC PC PC PC PC PC Pc PC Pc Pc PC PC PC PC p i PC PC PC PC PC PC PC CO CO CO CO CO to CO to CO to to CO to to to to to to to to to to to to to to M M O o o o o o o o o o to to CO CO CO CO CO CO CO CO 00 00 00 00 M 0 CO 00 >4 PC cn CO to M o CO 00 4 p i cn A CO to M o CO 00 >3 PC & tH o33 KtoS M MM O 4 CD >0 CO 4 CO >4 00 4 CO OD CO >0 00 00 00 4 00 00 co 00 o 00 O O CO M 00 cn 4 to M 4 to o PC o M PC PC o Cn Pc *4 M 4 M PC O WHM'OOIOPJ'IpH0lt)I(>S>H'4W#OWHteOlft P> ta to M M M M M M MUM M M M M M M M M M MO^VOMOUOM10OCOUMSOPUCWO MOMOMUtoIoOaOPWCMO PUOUMIkMU1O0 --ia. P10 looawaouiiui^fkOcooi^uxsMoooawMOoKtk o co q mmmmmmmo mmmmj mmh mmmM'mmm-mo m p c to to tn to cn A to a tn A m to A A to co a tn tn cn ^ cr\ CO M PC 0 CO A PC UMtouiotoAuinowoMpcn U1 CO CD 4 4 M 00 00 m m A o p i oc Pi to cn tn oo pc to to tn CO 4 MM MMM M M oo to tn pc oo cn oo m oo 4 in tn in in tn 4 0 Pc to to to M M M M MMM M M MM M M M M p i tn 4 g o p c to tn oc p c in tn 4 4 4 ob MOOUlOMOCnUIMOinOMO top to m 4 pc pc to 4 to A to A to tn M 00 PC A M o lOMM MlOM to to in oo to m oo o 0 to co A A tn to CO PC 00 M A 4 4 M M M to M M M to to M M to to to to OOlOMUUOPPcKIPPP# 44COOCtOCO(*MOUIlODi400 (DAOItnUOMPOUIOncOWOl in to M to to to to to A PC O M to M to O 00 A PC CO 0 00 00 PI A to O Q0 M to to to to toM M to to to M to to to to O P to A PO CO to M M co to (O to A CO cn PC Pc 4to t A M M M os cn CP A mauoMOiopctocoiouiioppio to A 0 4 M A M 4 tO M tO to tO 10 tO pc co to a tn to oc co tn co tn to co A oooioiointn to to toto to to to to to to to to to to to to to in*4 A P U) A cn A M Pi PC in 44 *4 p c into m o cn tn p c o vo a a a in 4COs4M4AOllDAtOCO0tnGDM to co to 4 to M 284. to to to to to to to oo o A in oc tn 4 M oc to inUAdcoitnt04 4Pcoo CnOOMtOOCP^COUOtOPMP pi4McoAin4tnppocnpi0to to to A cj % of-r 53 5 (Tv vaC*8PD DUP040000586 536311 VI o00i 00 in 00 Ol 00 o VO in 00 oi 00 o Co Ul 00 Ol 00 o >0 Ul 00 Cl 00 o\ Ul 00 Ol 00 o Ul Ul oo oi 00 *>> Ul Ul Ol oo o 00 Ul 00 Ol oo o to Uool Ol 00 M in oo Ol 03 o Ul 00 Ol to VO VO Ul 00 Ol to VO co Ul oo Ol to VO SI Ul oo Ol to VO 0\ Ul 03 Ol to VO Ul Uool Ol to VO o Ul 00 Ol to VO u> Ul 00 Ol to VO to Ul 10 Ol to VO l-l Ul 00 Ol to VO Ul 00 Ol to 00 VO ui Oi Ol to 00 00 ui 00 Ol to 00 si ui 00 Ol to 00 Ol 0o0 to to to <n to 00 to CO to si 00 o to o oo 00 -3 Ol to in Ul tv CO M V to Q\ to to si to 10 oo 00 Ol to Ul si to 00 to Ol 00 to 0S0i to oo vo to a> M to Ol 00 to VO si to VO o to 0tv0 CO o si to t- H Ol t-1 VO ui M> VO to si Ol si 00 Ol u> o ui to o> to to to to to co ho> tcoo oo v*>o. vOo aVO> two HO*vO#VOM totoooootototocococoiooooitooi >3OlfflOHOlOlsIOOO'JHOVOW VO H O O femu>HUltOU si O Ol H a H VO CO |j VO H vo ui w to u> 00 00 in w to to W 00 U) w 3 00W to VO O to H to tO CO CO CO tO tO tO CO U U> Oi 03 03 03 03 OSOHWCmniBHtOOOtOMHW It) w in oo vo o o o oi to to o in Co oi to to ui H si to to *J si CO si oi to in its |- 00 ca CO a to>COO tCOO (0t0lO(j)<os 2> * O 00 UI t-v Ol U si to Ul u Kj CO ca a Ul Ul OO Ol to 00 00 U CO 03 00 00 00 u *"N o u> o si O MWPMtvUtO^ a uft i . *S3 ftt o .Cft O ftto fOt l fOt l fMt .f0t 0 fOt ftto V O Jojjf si to 00 Oft l <n Ul >U*l* to Ul to 00 03 00 03 Ul 03 UUll S^ Ol to U VP o CO H H Ol to UUll -- U> M Ol 00 03 UI 03 M to 00 Ul H 00 oi 00 ui 00 .* 00 to 03 ui Ul u> Ul is UUll s^ Ol VO M o to OMMiOiO MUUtOOltOlniM-vcoHOO 00 fCut Oi H 9S |2 N*4 * < ka cn s 3 r* s% ow 3 I Ova3en ao O ffi H cn 0to0 > o 52 w CD cL w nm H00 e3ra ST CHO o o Ui no3 VfntI Ew# 3 CO 00 a co tk DuPont HLR 671-93 .8 Y ,5 3 25 ,1 6 67 31 44 85 68 01 20 28 0Y DUP040000587 IN D IV ID U A L BODY WEIGHTS (g) OF MALE RATS Group IV : Single Oral Dose A fte r Feeding for 30 Consecutive Days .5 .9 .1 .7 .8 .0 .2 .8 .1 .8 .5 .3 .2 104 aa Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul Ul in Ul Ul Ul to to tO to tO tO U> to to to to to to to to to to to to to to to to to to H > Kl 4 4 4 4 4 >4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 lO to to to tO W to to to to to to to to to to to to to to to to to to to s M HI 00 to VO vo VO VO vo VO VO VO VO VO CD 00 00 00 00 09 00 00 00 00 4 4 4 4 4 3 VO 00 4 01 Ul .fe- to to 1--1 VO 00 4 Ol Ul fe to to H O VO 00 4 Ol Ul * H 00 VO VO 0k 4 4 O 09 VO 4 VO Q\ 00 4 00 4 00 4 00 4 00 4 VO 4 00 00 . * VO VO 4 Ul I-* to to Ul Ol Ok Ul to Ol VO 4 m 4 Q Ol to 4 t>OHlOUIUU)U to o\ 00 |_A ^ppp pp pp p OOM'OUMOMOHIOHOJOlOWlOOWOlOOIOHaiO *4 o\ to in to to # to o> to vo 4 vo 01 4 co 4 to u p m o o vo -a VOVOHWOOMOlHCOO\01[OCn'JU1M(DOOtOPU'JWOIffl to UUlHUMUIU^M<kHUUWMUHH*>tJUIHW UIUHOROtUaxlbOtMVMOUlUIslHQUIOWOiO (UUIOCI^(>IHO>HK)UWa>U>UIPU>OiK}a)OtJU)btv) 4 Aa4utks]|)i>j^muinuiuifroiui(Mk4iio)(ii)^ O....W....a...W....'.J...^..W....O....l.M....'l.M....H....U...NJ...t.-.i.H....O...t..O....0..1..O...U...U...O...H...C...O. mWMWHOWWUMOWMWMOCOOtkOWOOWl'JOlU HHHHHHUHHPKHHHh'HHHMHHHBHH sO..].O.a..O..w..lU..w.K...UM..i .f4l.U..o.l.O..w.l.W..w..V..f.i.O.c.dl.^.aU...Mu...i.^o..O..>.O.i..av..J.a.'f.l.O.a..l.U.w..l.O.s.i.O.4.U..-nJV.oOu.firO.oo OOlOUIOSlOIOWUO'lOtO'JUOO^VICOMOOVD^O MHMMHHMMMHUHMUWHMMHHWHHHH OOOMOl'J'OHHH'OOfflMOOOOOOWWOWMWVO ui ui w *u> w ^ at o <oino\4huh<ui touu>a\ in4toa 0 0^0 0 4 3 4WO\(k^:UVi>JIO(& ( AOtM WI CI) > OiJUI tOK>tOHVtOHtOtON>tOh>tOKllOtOtOK>tOtOlOtOtOtOtOtO VO HtP>H..m.W...A.(..k.U.(..(i.Ho...i.s.i.fm..r.o.t..o..O.>..UJ...MlM...>yiMOo\Wa\lOMWHWouOfrOh(`iO(k(aBiMowU mI*s.) *MtO.OUK>OWtoWMtotJttfoWWtOWstno0stO1WWtot.tIOO.WUtOmIKH>OftocIW|tO.lOOtOUMtOlW>to)U"KJ> lUtMO It|OO.tt.Oo-JOKt>OOIOWO10 .t.O...M....H...O...<...k..t.O....H...I.k..t..h..t.O....M...U....>..m....O...t.O....H...W....^..^..W...1...0..0...g..o..t. H o H fe M4 to m t*o a % o S3 VwIO DUP040000588 .3 .0 .0 .0 .6 .0 .4 .2 .9 .5 .6 .0 .3 .2 ,0 .7 .5 .2 .0 .0 .8 ,3 .9 .5 .7 tUttkooojl VO tUtuook>ll CD tttU*ooooI tooo0l #k Utol tttkoooj ttUkoodl VO to U-to0l ttoo t- UtoI ttooJ o Utol tVo] CVOO UI u> kj to CD CO tUtoojl CD *J tUtkoojl CD Ol Utol to CO UI cUtnoaJl co utU*o0)l tdoo Uktodl ca CD to ttUoodl CO ttUkoodl CO O tUoI kj to >1 VO tUol Utol -0toJ00-t*-0odj tUol t<o1 kj Ol tUol tkod s] Ul to a% o taot oi t<oi to to o Ol to 05 to to Ul < tt oo>0 to *3 CD to CD Ul to VOOl to t0o5 to Utol to CD <3 to CD O to VOOl to oH1i to oVOi to 01 ,4k. t*o0 o\ to UI h* to Ol CD to t oj to tOol ttoo to to Ul Ol UMWMIOMIOIOWIOWMWWMIOMIOWMMMWWIO COD 0'J0 ^Hw*kc0o0 cJn0o0 oVOoOtio00in4^>t4JHs HO uCOc0s0 ^0Ds0n0 oVOuOilsViOwVOo1H0 *^0'>J0 'JPH'lOW^W'JUOW'JslOtiffl^loaUOUMMOl o<n WMWHWMMAWOMWWOWHIaOoWaWCOMWOHW^MWMWIOHIOOWOIOHWHUWWOMlVU U M M b) K> >1 M W p O) 00 W U W Ol 1)0 U> fk ^ i)> (O W O) Ql Ul MOOUOW'JOlWUlHbOlflUlMIOWWUlWWOOUUI co m w to a d du co d lO lO lO tO tO UIOMWWMWWWWWWW tO tO tOtOijHO^-.~..-N HVOWOMOOHOOHOMW^-JO I ui h a> to h{.Kpd.Kdit.H* Jd pj0W1HBHUmI'J(OtlnWWtkUO1i'O40lsMIOOH)NO>ltKJjUOViOOi Utol kk^ to Ul to to Ok Ol * >3 to CtoO M ca d a *! H{ Otli >* d > Hi tUol V utoi w utoi s** Oi Oi to d d vfcOo*^pt a>*s toK> K> cda d > t< cdo Oi ^ H!k to d > VO Hi ttoo Ok Ul Htoi VO ^ ttoo o to Oi d d bHj to O J ca d --k d > Hi dca <o-s d > X tUol *-** to o H ~J cda dSi* Hi to to CO CD ' w* 'k"' to to to 03 03 iV* s<* to CO VO to to to CO CD CO kk* w to CD o Z d!P w3 Kca Ia 0to0 lMO to Ul C0O0 <k M >a lg ^ <J aD S` P ?s9aS> LoW Oo S sg >5 *S1' V was, nO pMa *J 030 g* gO> jp*; "S S rton re> <` a a! w a % . ** a 5 0N53 50 CO DUP040000589 IN D IV ID U A L BODY WEIGHTS (g) OF M ALE MICROSWINE Group V I: Single Intravenous Dose 106 % H oas W9 apKca* W *4 to to M to o H * * u> 00 H -d * v 6\ <n o cn o -J VO cn o ot to 00 H* cn M w VO 00 O o W vo c j i ~j si >J a\ <JoUlK)OP>] h in >4 VO 10 fcJ oo to oo -J w to O W CO 'J OT Sj HUIOWHO M P Ol Ol P W OiWWOlOO OoT Hoi o|kt aW Aa axti 0 W 00 tO O VO 00 o o o o o j H O ea. 0 & ffi c CSl-1vJ\ wV> DUP040000590 IN D IV ID U A L BODY WEIGHTS (g) OF MALE MICROSWINE 107 K> to to to to >3 o> Ul t*> Ul s3 si to Ul 10 S3 to Ul Ol <1 M w 10 IO si Ol u> CO 01 0 Ol si Ol o o CD CO Ul to wto Ul to H Ol *-* si Ul Ol 00 Ol 00 Ol V0 to S3 w si to Ul l- to to Ul 0 to to >3 CO s3 00 Ol Ul 10 to to si Ul o M 00 Ul to Ul Ul Ol si HO a % s 33 DUP040000591 IN D IV ID U A L BODY WEIGHTS (g) OF M ALE MICROSWINE Group V: Single Intravenous Dose After Feeding fo r 30 Consecutive Days 108 oag H gg O VO 00 "4 Ol U1 J* C*O P4 HHHP MWOOHO O 10 Ul VO O VO P4 Ol 00 00 Vo * WOUIti^O 0to0 I-4 P4 P4 P4 P4 10 VO P4 P4 to P4 VO VO to 00 O 00 00 00 U Ol Ok Ul P to VO Co Ol w to P4 P4 P4 P4 P4 P4 U> O P4 to to to POPPtlO l*> >4 O 00 O O W W 4 Ol U P P4 P4 P4 P4 P4 P4 P P M W l*> 00 PPUIWWP O lfl tO P O 4 * to Ol vo Ol OJ cn oo vo P4 P4 H P4 P4 P4 PPIOWUW UI p4 Ul > UI 00 00 P4 00 OO to Ol 00 00 VO VO OI to P4 P4 O VO 00 <1 01 UI 4 in oi Ol d in own w u h u ownowowoi o mfeHSi tn 00 01 Ol Ol Ol Ol 00 <4 00 to to to #* Ol vo VO U Ul to 4 p> to -4 00 -4 00 "4 Ol to O -4 O Ok O P4 OI VO vo P4 O -4 Ol 01 Ol Ol VO Ol "4 *4 Si Si Ol 00 Ol 00 VO P Ol if* VO *4 00 to OPM014P VO 4 00 00 CP 00 VO 00 00 ^ Ol P4 ui oo Ui to P4 p4 o ^4 ui in vo Co 0 -4 00 VO VO 00 0* 00 4 to 00 si p4 00 VO O tO 01 Cn 00 P4 * P4 I-4 O 00 VO VO VO VO 00 to W tk 4 tl4 00 O H Ol P W U tn O H W 4 P4 P4 p4 P4 P4 HfflOOOO 4 o o own 0 vo to to ** p 4 01 to to w p 4 Co 4 P4 P4 P4 p P4 00 to P4 to ui c0ovs S3-r tn 01 M3 CO DUP040000592 IN D IV ID U A L BODY WEIGHTS (g) OF M ALE MICROSWINE Group V II: Single Oral Dose After Feeding for 30 Consecutive Days 109 HPPHMK ta OvUltMOUH =tfc HHPHHH NOKOH6 tkOOHtOVOtO w . oi w to <n Ol U O O M to 00 HWHKKHHHtHOOt* w oi to o > in cn oi v o b o *J o Oi to HH H H H H fctOMHWH oi *> oi in to OdlU^OH OtMb)OH Oi in HHHHMM *>. to u to > to OI V l>i to OI to to o to l H H MO W 0i V M HHH M 1 # to t to i to V to OI -0 l 01 OI tO > H H OI O O OI H to M t M M 1 :t 01 Ol i^Oi tO M dt *3 00 01 01 01 >1 01 HOOOVOOOOOCDOtoI O t> OI o to >0 o 1 J sj 'j 00 *4 t Oi 00 * V Ol v oo in oo Ct oi oOiodowo l 00 00 J to 00 00 >-> DJ OO to Ol Ol 01 1 V to t o Oo to to <1 1 00 00 SI to 00 OlOOOtkli Ol 00 V l 00 >1 Oi M OJ Ol 00 -J M O 00 00 00 VO 00 o ui oo t to oi t- M * Ol >4 SI OiHOPMd M 1 1-1 001 tMo wu odoi ow vo >1 00 VO 01 V Ol >4 W W W Ol p H VO VO VO O VO HslOOOOOW 0 O(0V CWD OWV 0M5 O< KH I-* HH t- O O SO H O 00 OI OO *J oi o 01 oi o to oi co * o in to o > 0l-0 to to in cd 0 rK1 01 ?vis Oi DUP040000593 eD :s APPENDIX G. IN D IV ID U A L CLINICAL OBSERVATIONS A N D MORTALITY IN D IVID U AL CLINICAL OBSERVATIONS AN D MORTALITY IN MALE RATS Group I: Single Intravenous Dose U00! U1 W tUol Ul t*l Of OJ Ul u> Ul u tUol Ul to ON to OtoN ON to ON to ON to ON to ON to ON to ON to ON NO ON 00 O-JN On ON ON a ON ON w ON to On h a tO >a O *4 B wb 4 H H O B *4 bd a 50 14 b...... a a H o z B a 4 b > 4 -J to z . o o to > ha 4 z Ho o 14 g bg to H 14 >3 H aa B CO CO ha O 14 Z 13 B 3 O M i3 to M 13 b b > SI to Z >O O^ > ha 4 z Ho oa aa a5 t* WH 4 3 H b 14 t4 to xa Oh Ha Z 13 14 3 O B i3 CO M 3 b b > 14 -J co Z >o ato ^> Ha 4 z HO oM ga bP b ah i4 t3 H b.B 14 a a nb a to Z 13 14 3O B 3 aw >3 b a 4 >0 az oo. to g ha 4 z Ho oa ag a gH BH *4 iH3 bW aa a ha O 14 Z 1143 *3 O M 13 aH 3 b b 4 M o az >o oJO .g ha 4 Z HO oa Bg b gp ah 4 *H3 b to t4 a a Hb OB Z 13 B 3 O 14 13 an i3 b b > *0 aa >Q o to to B H 4 B H> oa Hw b, a> 4 H b a a aH o Z 3 H a 13 b & i4 03 aZ o> O JO g ha 4 z Ho oB ag b gb ah * i3 H ba aa a Hb ow z a3 i3 o M 13 aa *3 b a > 14 xj az o5 JO > ha 4Z HO oM ga bg b ah K 13 N aa aa a Hb ow Z J3 a 13 o B 13 am 13 b B 4 xj o a a a to 4 > i3 H O Z O >4 WH CBO tJOa 00 to *a &a P 4 a c*Os-d aa r** 00 w * ffi JO t* 3:8 a *4 ON < VOOJ DUP040000595 <UwnI t-oo CO <wtU-oonI *4 tud>Uo3*ii UI VoU\I too ui UI iaji\ t-oo UI 03 to*o0\ 03 UI 03 ot>o0\ to Ui 03 tCMo0l UU)I otoj o a SHJ fi s>to> oa HnH gOf oHw4 ogaa og UH XOHHHI HCHO CO OH co o>H oat> hehhn gtSsSoa og HH >4 *H3 CHOHO HtCHoO HtoHHpo4 >aottOgo H0 2 og tXo 1HH3 CHOOHO HOCHO co H H>HOtOH*o4 *tHd*O444 >4 g to * X tHHoo H>towH*o4 a^>OOtao OH 2K Og to H x HHOCO >HCH3O HO 03 H >o H4 o OH H OH *3 > O tXo tHo SN tHo CO 03 03 h aH to a >o o^ H HH4 taOo oH 2K Og W X >H3 COHO HCHO H to ah co a *oH o^ H to N4 Oa OH 2 to H t>o H X *3 HCOO HHCO H to GH to a O> O, H h4 H 2& oa Ho 2S og Xto HH OH HCHO CWO O OH a 4H 30 H 0 33 HH O a >CH33O tOHH>o-33 a H>3 HC O33 tOHo3 a 3 CHO 3 a H>3 a H3 0H3I OiHO3 3 CH3O a H>3 a HHI a Hh i C*H3O O>H3 3O H 03 HHI HC3o OHHI HI to 3 to 3 O t>Xo XO> >Xto >XP XO> O &X O > X O X> to X> 00 00 <3 <3 HO1 to- -0 >3 HCouO tO < >3 H O a oO Oh* Oto *4 H 0H3 to tCHoO ^O O >X ae COHO >Ct>*O3 O K 2 H a P >X f Uv .3 VoO DUP040000596 IN D IV ID U A L CLINICAL OBSERVATIONS AND MORTALITY IN MALE RATS Group I: Single Intravenous Dose 113 OOJl o* t0o0 Ul Ol u> ttoo 00 tv Ol to t<on 00 W Ul WOl to 00 to UwI t(on 00 UI U) oto 00 Ul U> o t>oJ tp .2HWI d * to > n2 toao H >4 09 aH Sto* 22 d x09 >3 dh tdwo .. - aHa tHwoI 3 d > < 00 to a o ot >9 HW 2*No4 aowg d Kt* 09 X HHt3 taao tao H ad 2 HwJ HI O 2to ma3 3 d a > Hi <s to a o> ot 2g o2ahaw Saogg(tHo3 09 H Mi HI H d2to t2O H0 o2 2 HI 3 20 2to 2HI 3 U d > wok> aot 2 h 4 >tao HO oa ag d 5tr?* 09 H Mi mH3 td2o 2to Hd <0 09 2 0m39 HI O t0o9 m23 3d d > X to to to to >SOfl O O 2 *4 2 d 09 2 H*4 PM H d2 OH 2 H> d 2d >m3 d g*4 H2 09 d Mi d t2o 0H2 3 2 to HI d > i *-* t>Oo to o2 2H 2 *4 W o2dH W t2>o >3 Mi d Hd t2o H 0 a M3 2 0 3 d > Mi M 00 >O Q2 22 H 2 09 H it# O2d U 02 >3 Mi d Hd 02 H 0 2 3 02 3 d >Mi 03 o0w 2 H5<> 53 O 2 o *sa WH to 2 aoo u> u> o 00 22 to 81 u ow d> S0M3tl {3 00 HHH OOO t-i o 00 to to g* fl-fHi tag ax $0 (VTvI 1p* vo OO DUP040000597 SACRIFICED BY DESIGN TEST DAY 10 s UI U1 UI UI UI UI UI UI UI o j u> o j OJ OJ OJ u> OJ OJ w J -4 -j -4 J -o *j 1} o j 00 OJ OJ OJ OJ OJ OJ OJ oU1 cn UI UI UI UI UI UI in t* 00 OI UI fe OJ to H 411: 2 03 2 CQ 2 co 2 03 &i to 2 to 2 to 2 to 2 aw M CO co H t) ow 24 M 4O W4 to H 4a O > X M O C3 W HI 03 to H t) mw 24 W 4O H4 to M 4a O > X H t w M 03 to HO to HI 2 ti HI 4O M4 03 n 4a o > X -4 ew M to to ha to H 24 H 4o M4 to H 4a a >1 X h J OH HI to to H tl to M 24 HI 4O M4 to H 4 tJ p > X M O O HI Hi to to H CJ to h i 2a HI 4Q HI 4 to HI 4O O > X o OH W to to H tJ to HI 2S HI 4O M4 to HI 4 tl P > h! >4 OM HI to to HO to HI 23 HI 4O H4 to HI 4D P > Hi 0W HCCII 1 5 HI i! O HI McMa mtHa 24 a fig CO CO w4 JO 3? a* Qz JS HH3-4 <M- .tr* 0 Cfi 3 CW nn o cIT* *t z a og O re3 "O I0w033 So3 zco > z a . tr* Z s >e-< IB sa I a :0HC3 a as 5 HSW1*> DUP040000598 IN D IV ID U A L CLINICAL OBSERVATIONS A N D MORTALITY IN MALE RATS Group II: Single Oral Dose 537367 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN TEST DAY 7 Wiinn4 oo\ iaiinnn4\ Ul o^iu>inn>4 iinn Ti>nOn4 TiiSi1nnnO4I >iiinnn4 <Mn oiiiCnnn4Tl imiiinnnn4 TO a gO O to g hw to 3 HO ow wa ag to H K *9 H to to to TO TO H to to to 3O to *3 TO to >3 O to g k! 4 TO a gO o to g MU to g HO O to to 3 og to H *4 >3 H to to to TO TO H to m 3 *3 to *3 O to 3 TO to 3 to to g * SI TO 3 gO O to g H to to a Ho O to to a to g to H * *3 H to to to TO TO H to m a >3 n 3 O to >3 TO to 3 to to gHI M O TO gO O^ to g H to to a Ho o to to a og to H H >3 H to to to TO TO HO O to aM M >3 O to >3 TO to >3 to to g Hi >3 TO gO Q^ to g H to to a HO o m tSo og to h Kj >3 H to to to TO TO H to m a 3 m t3 o to M TO to 3 to to g Hi 4 to a go ow to g H to to a Ho O to ws to M to h Hi 13 H to to to TO TO H to to 3M to 3 O to >3 TO to 3 to to g h! M O to a go n^ .w g H to to a Ho O to to s to g to H Hi H to to to TO TO H to ow a 3 * to 3 O to >3 TO to 3 to to g * o to a go o to g H to to a Ho O to to s og to M 4 *3 H to to to TO TO HO to a J3 to >3 O to >3 TO to *3 O to g * -4 O to TO to to <! g *3 H O a O to Wca Hto Hi TO to H3 t3 a e g 4 a % oTwOtT<gO 8" to xj vio 03 DUP040000599 IN D IV ID U A L CLINICAL OBSERVATIONS AND MORTALITY IN MALE RATS Group II: Single Oral Dose 116 Oicn Ul Ul Ul Ul Ul Ul Ww Oi Oi b> u 4 >1 *4 <4 4 4 <1 01 oi oi w Oi Oi w *4>4 >4 4 >1 <3\ CT> tou> H O o> 00 a!op> aO 5H>H4 a8O ow 3 O gjj >c q oa 5Q0 ohwM4 Soaaa a ox a o> o 50 HoWah aoaaK d oc q ao h a owH aoa wa ag >ao50 Howhw oa OSaa ag a>o oa 50 hw aa owaH 2aa aa o o^ hw50 a a owH Sws aH >W4 HHH dw W CQ CHOQ OW ah w hi o m a CQ W 1-3 U ah 4 hH aw W CQ a ha ow a >3 w ho W >3 Oi w 3 t> a4 HHhI aW01 w01 ha ow a 3w Hi O W HI aw 3a a *4 hH H aw W CQ a ha w a 3 W3 wOi3 aw 3 a a4 hHHI aw wa a ha Qw a >3 3 wO W +3 aw 3 a a *4 HhHi aw wa a ha 9W a H| 3 Ow W >3 aw *3 a a 4 +hH3 aw wa a ha oaa >w3 3 Ow W HI aw 3 a a 5p* 4 a > 4 a >* a > 4 a > 4 a *4 a 4 4 i- O 4 *4 *4 O a a w a < g HI Hi a2 o *4 a cq Ha nW >C3Q aP 4 OW t>* CQ CQ 915 39 a *< a c O X r1 .53 SJ wSD DUP040000600 IN D IV ID U A L CLINICAL OBSERVATIONS AN D MORTALITY IN MALE RATS Group III: Single Intravenous Dose A fter Feeding for 30 Consecutive Days ui U) Ol to ID Ul u> o\ Ul w Ol Ul Uo\l Ul w Ol Ul Ul Ol to to to to to so so 00 00 00 UI ui H Ol SJ to Si CO p to H so 00 J Ol m C>oO zo pd h >Ed W 25 nH Og Wo 3g WH Kj H a Hw W CO CO HC O Cd Z *3 OM4 W >3 CO W HO a> K fMe CO CO CO CO >00.0 O 5d Sd Jd Sd W M w H w w td Z nwHO*3 ow> Wow a waw 4tvzOaa*J co W H Ml O Z to .s^ i3 W CO H3 a> 4 > H CO Z >O Ow Sd h >Ed 4 Z HO 02 Wa g WH s4 3 awH W CO CO h a <zD Ws H *3 O W *3 C O3 Wa a > * ** H to >o oZ w Sd > Hw zW HO OMa g23 t d4 h*3 awH W CO CO ah ow Z *3 *wo3 W >to3 Hwa a > *4 H to Z >O Ow Sd hw Ho o>Ezd 2 aW 3g td H KH a wH W to CO oh aw Z Hw 3O WH CO h Wa a > 4 u> Ul Sd Z WO qJ KEd WZ ao *4 Sd 3g 3h *3 01 H HW qa 4 to aw >w3 *w3 CO O 33 a> Wa o to to CO >QQ QWW Sd W W H oH4 >nJfl ZWa Wa WW td K aw CO H 2Z *3 W to 3 >a 4 u H o w to M W <! HI H 2z tO tO tO CD 00 00 lo w .>. 00 Ul H 5.iS3S0sswaEssWi,'Si:i oW HW tO H mcot tSod mo *a>4 8 k HH tn co B* OH dS*3q no3 Xr1 {3 Cl a w DUP040000601 IN D IV ID U A L CLINICAL OBSERVATIONS A N D MORTALITY IN MALE RATS Group III; Single Intravenous Dose After Feeding for 30 Consecutive Days 01 oi Ol Ol 01 w w w o> <A> H ot 01 <n o Ol to to to to to V-JO VO oi VO OI VO VO w *s CQ GQ CQ M tn CQ >0 0Sd 0Sd 20 0Sd0Sd Sd M M MM Ho m a h i m a ss HH > OM O > H 3 H O M 6 f >a OH Fg W X KM H ** O tn tn M CQ CQ H H2 OQ 2 t3 M CQ >9 O > Hi H tn tn M >o2 o Sd 0 Sd M Ho M *3 M H> O H >3 H F> OH F W XK H o tn M CQ CQ H H2 O CD 2 *3 M tn 3 O > 4 Ok H tn tn tn M >mo2 G F Sd 0 Sd > M H < o M H Ma >3 M. O H 3 M FF> OM H F M CQ _ XK o--H ... M CQ CQ H H2 oa 2 3 M CQ 3 a > xj tk H CQ tn CQ H >OQ2 O W Sd 0 Sd M M HO HFSt HM> Ot:Ht3 Ma HI F > h CQ F MW XQ _ K a OF h CQ MO CQ CQ M H H Sd 2 fi) ' * * Q 2 -tsa^ >3 M CQ *3 a > * k H CQ M CQ M >200 O 0 Sd M Sd M M HOF M M 3 H H _>O O 3 H M>FM OH g F Sd W,, XK H O ta M Ta tn H H2 O 2 3 M to HI O > x! *k H O w tn M Sd <5 > HH! 2 2 lO to to to H t- H> J- H *k HH >. O MPH -O O O 10 t- H CD O O tMO *H O COao M hpa M CO Sd *S HMHHH *k #k HH H Ok ^ OHH MHM M**H Oto* co co Mpal t3 Sg O xi a %o S3 frf*i 5*3 a\ MI VO 03 DUP040000602 536305 NO ABNORMALITIES DETECTED SACRIFICED BY DESIGN TEST DAY 41 Ol Ol on ot Ol OJ OJ OJ OJ OJ o <n <n OJ <n OJ 0J O) OJ 0J ooo 0J to o Ol Ol OJ 0J OJ OJ to to vo VO VO 00 5! 5HJ fr*1 4k 01 CO 01 01 >000 o 53 53 S3 53 tfl W M H 4 03 Z *4 H > tfl > oo oo M 54 54 tfl d 03 xt a w 01 H o z 9 M 01 9 O > * OJ ut 53 Z WO Qg <3 & HZ ao si s3 g o fc4 KH 9 01 H +3 W a at o k! o 14 9 >9 tfl tfl at o 3 >9 tfl oO is o at z >o o^ wg HW 4 Z HO ow tfl 2 ag 03 H * >9 H tJ tfl tfl ta 01 HU O tfl Z 19 tfl *9 O tfl 9 at tfl 9 a o g * OJ Ol ai Z go ow 53 g HW 4 Z Hs nw tfl s O gH 03 H Kfl 9 H OJ tfl tfl at at HU O tfl Z >9 tfl 19 O tfl 19 01 tfl 9 t> tJ g * at Z go o Jd g H 03 4 Z Ho ow tfl o> 03 H *< 9 H a tfl n at at Ho ow Z *9 tfl >9 O tfl 19 01 tfl 3O a g <V H ai at oi >QH Q 53 g tfl g H^ K 4 g tfl h mo o wa oS tm* Ed G2 o b z at H! a tfl at H a z >9 M 01 >9 o g Hj *> M 9 2 >a n fl 53 fl Hf >4 tfl ha o tfl *4 og 50 03 Hj a tfl at H a z 9 tfl at >9 p g t< M o 03 at at <! g HI w C| Z tO M M M *>> fe OJ OJ U Ol (Jl Ol 0M0 > O OJ to O 4 03 H at 53 tfl at 53 9 wg ag *< Oawt Pa>t tVfl t-3 wa Qz 2 -I ao "d on > 5'd ro t-4 E'g 3Q sd oC O03 7(9 f& <4 53 rt rl Z wt .> o5o 'Zo ol-t gg ooj Og no >H 9 tr* CD 13 f2 o? ^fit r* s w a > Hon 0 Ogfit a t"* PS oSni vo OJ DUP040000603 120 S?z OI OI CQ 01 CQ Ol CQ CQ M MO OI OI ot Ot SS CQ CQ CQ CQ H POI OI OI OI CQ CQ CQ CQ O V> OO 00.... -..j -- -O0j E< 4fc IwNb-4s<Ha3-t "cn d:> S' r 92. n a fterH* co S3 * O a Ha aW 0C>soQ 0CsBQo 2Wq H0 B t3 B a 0>a *.0 Hhi aB cao a 0 0aaH ,gBa co a It* 0 0a aH >aa aa H0 0aq gs H> O H |3 a t* > w0 HO B0q 5g2 H0 0aq 2 2g H 0a 0sa 0g S WH{ H*3 a SS WH *H aHj h3 ah kJ hi 3Z r> O W oD Mfd Vrti 553 s '. >z3 q a B Cq q Hn M CQ qB H cc oq q Ha W CQ aq Ha cq qa Ha cq W cq 3 l CQ h q Oa >M3 H 1M3 0 >3 qCQ H 3 ChO Oa a3 CQ *3 Ha O ChQ q 0a ac 3o3 a >a3 0aq3 CQ aah a3 w 3 qaaa 0 1aq3 ha 0a a3 cq aa ah 0Ha >q Hj >q* qqq > > > a < * HCQ <J1 H #* MM Oa CaaQ <! a *3 H 2 S *Tl 4 2 <*> c3n o!>-*1 a s oC_Q oB no r* f3ts d n V**a Sifw> * O *Jj taetl 5 CO K00> 0M0 SI CQ HI $ Cl S a #M>> H 8S at c q o a as tig 3 ak vb CQ DUP040000604 SACRIFICED BY DESIGN TEST DAY 41 UI OI tn UI in UI UI UI UI 09 U> 09 09 u> w 09 09 09 -4 *4 <1 *4 -J o -4 -4 *4 09 w w 09 w w u> 09 09 00 W 09 to 09 H1 00 o 4 CO *4 >1 09 --", -4 oi 4 UI OW 0H3 | H iOtMtndo tHwn*o4 a d> < si; 03 03 S* u d* O4 z Js od>< d c o* a3s re.I*** d r> 0t* Oi-i og rse r* Ow r>e-t rreen CWO 3 > sMP3u* d 09 St ' W CO > o on 3 d s S3<r3rceen O ** re r> 0 4CgO Z S >t-1 :W 3 d 3 o 3 5r<3 o\ v0o5 DUP040000605 :> Z(71 (71 (71 (71 (71 (71 (71 (71 (71 03 U> 03 03 03 03 03 03 03 W *4 <1 J sl Sl si Si sl si SJ 03 07 03 03 03 03 03 03 03 VO to VO H VO O 00 VO Co 00 00 *4 00 -... <n -- 00 (71 00 - -------gt t* CO Z >O Ot H4 ZU oBH tf ggOw td h X 73 tJ HB M CO 0) HB Z0 B *2 B >a n B HI CO B HI Z O >X 03 Oi CO Z >o o BB H 4 Z& oHBO O gga xtd h 73 BB MH CO CO HB Z0 B t3 H 73 o a >a (0 B 3 fc> B gX 03 (71 CO Z >o n^ a BH gZEd HO aaa gag txd ha aH caHo CO ha oa z 73 a a o B HI CO B *3 a a gX ik M CO Z >O O^ ag >h4 tZd HO aM ag ag WH X 73 Ba mH CO aCO h Z0 B HI B HI O H 73 CO B a a a gX 03 CO Co Z >o ot B h>4 gZEd ow ato O gag UH X 73 a aH B CO CO ma 0B zh B 73 O BH CO B 73 a a X> 03 00 CO Z >O a 74H oBaH ogZEd agg td H X 73 a aH B CO aCO h ow Z 73 H 73 O W 73 CO H Hi a CO CO >Q ao aS H 4 a HH Om a0 a 73 Cn>O oZ^ a oaHh o>Ezd a ag Ux CaO d _a-q- wa wa HM 0z a m 73 ^ w a 73 ah X *3 awH a co CO ha 0a z 73 a 13 o a 73 aa 73 a aaa >X >X >X 03 03 CO - a CO Z >O o^ ha aHao ogEzd agg td H X 73 a Na aa xa ha Zow HI a 73 n a 73 aa 73 a a >X M Oa a a a < > 73 H oz Oa ctado aHa 0to0 a h i ss CO CO 0to0 s* tag ax f>f*, o 4 S 3 d 0CM2/5*. to* O g r1 n n z A) 5? sg (AoD > re r1 OOd (dfitl *rrTeeJ > a> .* 00 Si- ' 03 o <*) > z goownr3e niPs* a w re d 4s HI I % co dc a o3 i v0o3 DUP040000606 IN D IV ID U A L CLINICAL OBSERVATIONS AN D MORTALITY IN MALE RATS Group IV : Single Oral Dose A fter Feeding for 30 Consecutive Days 123 Ul Ul Ul Ul Ul Ul Ul w O) 03 03 03 03 03 *4 -J 4 >4 4 4 o> W 03 03 03 03 03 VO 03 \D CO VO *4 VoO\ SO io VO in...... ..... o...... "W 03 S3 gO O_ ta g Hw 4 S3 NO o2 Wg pg WH Hi 13 H Pw W in 01 ha aw S3 gM >3 O M 33 01 W 33 P P g 03 Ul CQ S3 gn O_ H &W 4 S3 HO ow 2g pg WH Hi <3 H PH W CO CO HP ow S3 33 H *3 O W 33 to 14 33 P P Kg f 01 S3 gO O^ $4 g HW 4 S3 HO oM 2g Pg WH Hi 33 H Pw W 01 01 HP W S3 33 14 33 O W 33 01 14 3 P P g H 01 g Og O^ to g H OS 4 S3 HO o2 14 g Pg bd H{ h 33 _H PM 14 01 01 HP W S3 33 M 3M3 O 33 01 14 33 P P g Hi lO3-* 01 ss go a^ to g h as 4 S3 HO o14 2g pg W S3 Og O_ to g h as awH4 Ss3 2g pg CQ S3 ng Ow 54 g h as 4 S3 HO oM 2g pc W H W H tt H Hi 13 K >3 Hi 33 HH H -p w --PM --P 14 W CQ 14 CQ 14 01 01 CQ CQ Hp HP HP 14 O 14 w S3 33 S3 33 S3 *3 14 H H 3 O i3 O 3 O W 33 W 33 W 33 in m 01 14 CQ H *3 P 33 P 33 P PPP gg g H{ 3< Hi o> OI oi 00 00 Ui 0W UHl 1 H fi 0 4 0O1f Hta M in to >3 -- p p Hgi 8 * in oi 8* P Ki ds o a a IT* S3 0> \ OI DUP040000607 N D IVID U AL CLINICAL OBSERVATIONS AND MORTALITY IN MALE MICROSWINE Group V I: Single Intravenous Dose 124 to to to M to 0 \bt>* ' ... 0H0 -J* CO o> h oZ wE C>O n OHf5e MW >co OZ OH ,EW >SO Z G O_ a H Eb) CO >oaH oZ ^Etd >co OaH z OwEW 4Z Ho Qa 0M 5g 0Hnw4 aW> 4 H aM aZOgg 4 Z gH 1a4 Og 4Z Howa Oa g 4Z HoMa Oagg WH 4 >3 U HM tt CO W H tJ OW Z >3 M 3 O wh co w >3 O W* aw CO H oz *3 M CO WH >4 *3 H t> W 14 CO CO wB oa Z >3 14 *134 Oi3 Co Cd Hi a WH 4 HI awH W CO CO h a CD M Z >3 M >3 O W HI c3o Ma W* *H3 *W >H3 awH~ H aw 14 CO W CO sCo h CO h a OH O M Z >3 M Z H14I >3 O 3 O CM3O HWaI MH C3O Wa a > P E *4 B > * a > K a > h! a *E< HMH M t- ooOooo - Ow CteOl a *" HI sH Z O 4 cWo SHd W CO a HI 3-a +>a4 Stigi ow >t* co CO o s. O 9* US 6 O * o> MWD DUP040000608 2 DuPont HLR 671-93 125 -too oto* tgoi to (tUo *Tos3 SS S g re Co CO ttJ S3 d 8 2C5O tts>z>ax*l s0 *4 0C1d H SiW C*3Q sSNH n * rOC*O ^U a >+$ %w 8$: at at g* $$ t> * DUP040000609 IN D IVID U AL CLINICAL OBSERVATIONS AN D MORTALITY IN MALE MICROSWINE Group V: Single Intravenous Dose A fter Feeding for 30 Consecutive Days 126 M H O Vo 00 -4 OV "Oi--- MU CO CO >oJHOHBa4O H*>tH<fOa3e*S 01 St >oto o > h Hoa4 Oaatsot aH oa> zo_ a ah oHaa aogKawst aa o> t* a Ha OaHa aoh<>HHI oat>o aHaE* HHaaaO >H<HoasI aa otOahaHao f<>HaHHo*; B4 BMCO aHO CM3O 3 >B4 HH Ka h hi *a< aHoaa a_aaHoi HcaH2o5 fOl haai aaa aHoi aah >ao33i aa oha aha aCa0 oHa 0aH1i 3 ahi aaa SH53 aaHH a* oaa H2a .. Oaaaa aHaH HI 2Ha B> a *} >a>4 *>a4 a> Hi >a4 MH #k H H... 00B1 aHto to Ul JO t-v H00 U(0l BtO C4O -a9 *4 M 8S p* t* HM P* K CO CO aJO * fO1i3 a S3 3? S B *4 <SJI\ v0b DUP040000610 IN D IV ID U A L CLINICAL OBSERVATIONS AN D MORTALITY IN MALE MICROSWINE 127 o|_\l |in4 pft- w|4 |tao t>osHHOHb4aa g2O^g02OWS cn 2 OHnH>tha4 OW2O^>a2 bE t>oHH*HOHaa4 htgHHO2<!Ha*I 0> 2o oosHHHb4a OW2aSg_> 0>OH4 O^2 aW HQ Ho aS bg H0>Osa 0b>> h< HObH4 H>O2HI *W HHHI t*d HHHI ta4 OHth02HtHnn HaHOHMtHHnI Hb cO2bHHn 0HHHII H0 OHHHObHH tbHa 02H HcHnI H t*a4 hHH ta4 hHHI tha! H0H0b 0H HD 00HbH 0H Hb bH0H0 2 0HHHI bHHOHHI 2 >HH03 OaWHHb 2 0HH +>b4 x>O{ >bk ! b>* b> * 4 pi- fHt f> H h* tH*. ftH Ot0Hsaa 5! HHI 22 O0til HHSd to to 0H 0HI in ft! tl O > 4 Ota t>* p 0H t4n S I c 1J So3 r* JSS o\ 3voi> DUP040000611 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AND ASSOCIATED MATERIALS basal m ix (95%) fo r rats (TD 92242) was submitted for analysis. Zrftt* a fafrai a 2a ssa CO >% a o 4 a ah"4 a SDtflaonffl> CT. aoco OD. 2Bg. g sr 3 sr 4af3t* car CO ft ato** 09 Su ctaon** ctaor*>* cn a ctaoo** ctao*o.* iaton** ctaoo** t<aoz*>* 09 09,09 09 09 09 09 09 aa,** SCua^ Ea *a aa* 3 a 4aa00arfa4r41* <4 M CO ePa> a ao os* ao s* o aa oa as*CO 4 43 o w4* Ia ao ISa CO aa a* a.sau EL < 34. CO I--O >4* a1 44a. 3 a aaa Wa 14" na* o 36 aH* n A a ft ft ft ft ft OO o co ft o co ft o co ft o a> ft o CO ft fG3t OCfOt OCfOt 43*- EOd. m 5L *4 a4* SLf jtMot E^L O O2 O % S o^ >3 % caw a a 09 ft a 09 sfi a a*5 a co CP) co jae S3 s. *& to** 0aft 1to** a *5 Cai ^a/5h/3 to** f3ot Itaao** 4 a 1 5L 5.? > * >C*O*t CO aa* ft 3g* G )* *> > 2 es. >C*OK S aa ST e * 8 a5* a14. a, a a* 3 e SWa Stro>3** aCl . ar< a14. >4 CJUO a>4 aa. a* o8. * 09 g 2* 1*3 sft rto<t wa o s** ACL Aa* aa '4- *to4*"* a a S* 09 sr B a a 43 1to4** s2 o c l Cl o E?09 2ET. cao 33 Wft ri a 43 oto** . 3Ct> CO CfratOt* u p &a a 3. 3 2. aa a tSoi ap. sa? 0K9> AK> a 43 o M KJ a* >4 CO O CL 2CL " t8 sr a a b. a. 3 CaO a t> 43 a a->cBo Ci a a 2a. " CO Oaco4 "SOs\a' ^o o*4 a^ CO Ona >4 >ar4 a&rftt>* a* a* CO >fOf3tottn*** ^53 a sl 0*2. o3 BL a CL EaO. tao** a CO 09 r* C"<ht C> U DUP040000612 0\ VO c3 & FIGURE 1. MEAN BLOOD LEAD CONCENTRATION IN MALE RATS (Acute) Time (hours) XT [u m rr..r |m in r o oo (pn/g-ri) noiiujjuaouoo pusqpoojg DUP040000613 DuPont H -58- a a> DUP040000614 DuPont H DUP040000615 DuPont H LR 671-93 o e .'9P (pn/Sii) uopuijn9ouo3 puaq poojg DUP040000616 eou, s Q (<M0 DUP040000618 APPENDIX A. PROTOCOL AN D PROTOCOL AMENDMENTS cCnTi \ VO - i C! s co 3 D ABSOLUTE BIOAVAILABILITY OF SOIL LEAD IH HATS AMP MICROSWIKE MEDICAL RESEARCH PROJECT HO. 9660 PROTOCOL CO CO APPENDIX A. PROTOCOL AND PROTOCOL AMENDMENTS Stine-Haskell Animal Welfare Coamiittee Mo. CBT51-P DUP040000619 APPENDIX A. PROTOCOL AN D PROTOCOL AMENDMENTS eOnS 2- so .as&1-1 ABSOLUTE BIOAVAILABILITY OF SOIL LEAD IN RATS AND MICROSWINE foet3Q MEDICAL RESEARCH PROJECT HP. 9660 OBJECTIVE The objective of this study Is to determine the absolute bioavailability of lead from Soil in rats and 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, Bellevue Corporate Center, Wilmington, DE 19809. 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 Practices. The sponsor's approval was effective the date the sponsor signed the Medical Research (MR) project. , * STUDY DESIGN A. Single Dose Administration o Fifty male rats and ten male microswine will be used. o A predosing blood sample will be drawn from each animal for levels of background lead. o Twenty-five rats and five microswine will be dosed orally (gavage) with test soil containing 1,000 ppm lead (approximately 2 g soil/rat and 17 g soil/microswine). o Twenty-five rats and five microswine will be given a single intravenous injection of lead acetate solution containing lead-dose equivalent to that contained in the gavage dose. DUP040000620 APPENDIX A. PROTOCOL AND PROTOCOL AMENDMENTS to IvNe - 3 a*4 53 Single,Dose Administration (continued) c o o Approximately 0.1-0.3 ml of blood will be drawn from the rats (each s rat will be used to collect three blood samples only) and Q approximately 0.5 ml of blood will be drawn from each microswine at 0.S, 1, 2, 4, a, 12, 24. 48. 72. 96, 120, 144, 192, and 240 hours after dosing- .......... ..... ---....~j~-....-- o Blood samples will be collected in lead-free tubes Containing EDTA and stored at 4C until analysis. o At sacrifice, liver and bone (both cortical and trabecular as well as whole femur) will be collected, weighed, and frozen at -20"C. B. Single Dose Administration After Feeding For 30 Consecutive Days o Fifty male rats and ten male microswine will be used. o A predosing blood sample will be drawn from each animal for levels of background lead, followed by one sample/week for the duration of 30 days. o All animals will be provided with optimized diet of approximately 21.5 g/rat/day or 340 g/microswine/day of a special purified diet mixed with test soil containing 1,000 ppm lead, 5Z w/w, for 30 days. 1 Jg o After 30 days, blood samples will be drawn from each animal for levels of background lead and the animals will be divided into two groups. o Twenty-five rats and five microswine will be dosed orally (gavage) with test soil containing 1,000 ppm lead (approximately 1 g soil/rat and 17 g soil/microswine). o Twenty-five rats and five microswine will be given a single intravenous injection of lead acetate solution containing lead-dose equivalent to that contained in the gavage dose. o Approximately 0.1-0.3 ml of blood will be drawn from the rats (each rat will be used to collect three blood samples only) and approximately 0.5 ml of blood will be drawn from microswine at 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 192. and 240 hours after dosing. o Blood samples will be.collected in lead-free tubes containing EDTA and stored at 4"C until analysis. . o At sacrifice, liver, kidney, brain, spleen, and bone (both cortical and trabecular as well as whole femur) will be collected, weighed, and frozen at -20C. DUP040000621 DuPont HLR 671-93 -4- MATERIALS AND METHODS A. Test Material The test materials will be supplied by Aldrich, Milwaukee, WI 53733, U.S.A. The lead acetate trihydrate will be assigned Haskell Laboratory No. 20202. The lead/soil will be assigned Haskell Laboratory No. 20204. Characterization and mineralogy data of the test soils will be maintained by the sponsor. B. Test Species H Male Crl;CDBR rats, approximately 21 days of age, with body weights in the 2w range of approximately 40 tp 65 grams will be acquired from Charles River Q2 Laboratories, Inc., Kingston, New York. The CrlsCDGBR rat has been selected on the bases of extensive experience with this strain and its suitability with 2w respect to hardiness, longevity, sensitivity, and low incidence of spontaneous ' t 2 < disease. 1 Ou 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 BO Laboratories, Inc., Wilmington, Massachusetts. The weanling Charles River pP$i microswine has been selected on the bases of its low body weight, its Q suitability as a model of soil-lead "ingestion by children, and its suitability 2 with respect to longevity, hardiness, sensitivity, and low incidence of < spontaneous disease. o u B C. Pretest Procedures g Rats and 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 one week, during which rats X will be fed optimized diet (once per day) of irradiated Purina Certified a 2 [XI &Pi < DUP040000622 tOnI' r-t - 5 - '.O 2 x Rodent Chow (PCRC) #5002 meal and transferred gradually to Teklad basal mix "oS (951) for rats. Microswine will be fed optimised diet (twice per day) of % microswine diet (Charles River) and transferred gradually to Teklad basal mix Q (951) for microswine. Rats and microswine will be provided deionized water ad libitum, weighed three times, and observed with respect to eating habits, weight gain, and any signs of disease or injury. During the test period, all rats and microswine will be fed the diet of their respective treatment groups. 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. The program is monitored and administered by the laboratory veterinarian! data are maintained separately from study records, 1 S* 1 D. Assignment to Groups Healthy animals will be assigned to groups by computerized, stratified randomization. Rats will be housed individually while microswine will be housed two per cage. Each animal will be assigned its own Haskell animal number, and permanently identified by a number marked on the rat's tail or the microswine's ear. APPENDIX A. PROTOCOL AND PROTOCOL AMENDMENTS DUP040000623 APPENDIX A. PROTOCOL AND PROTOCOL AMENDMENTS i tV-O - 6 c.ja X E. Animal Husbandry &Dco3 After assignment tip groups, animals will be identified with a cage card and housed in stainless steel cages- All rats and microswine will be provided optimized test diets ofapproximately 21,5-g/rat/day and 340 g/microswine/day. Deionized water will be provided ad libitum. Animal rooms will be targeted at a temperature of 23 2*C and a relative humidity of 50 +101. F. Diet Preparation and Analysis During the test period, rats and microswine in each group will be fed the following purified diets: O Eat Basal mix (95Z) (TD 92242) o Swine Basal mix (952) (TD 93034) CO to , These diets will be custom formulated by their manufacturers so that each diet contains complete normal nutrition for each species when the diet is diluted (weight/weight) with test soil containing 1,000 ppm lead or with sucrose. These diets are sucrose-free so that the soil added will replace the sucrose rather than diluting the complete mimed diet. The diets will contain < 0.1 ug Pb/g. - ------- <------- ------------- Lead/soil or sucrose will be added to the diets and thoroughly mixed for a period of time that is adequate to assure homogeneous distribution in the diet. Once prepared, diets will be refrigerated until used. Sucrose mixed diets will be given to all animals during quarantine and the single dose administration group (Group A in Study Design). The lead/soil mixed diet will be given to the other group for 30 days (Group B in Study Design), After 30 days. Group 8 animals will be given sucrose-mixed diets for the rest of the study period. DUP040000624 APPENDIX A. PROTOCOL AND PROTOCOL AMENDMENTS 69 7 Before the beginning of the study, samples will be collected from the diet prepared with lead/soil. These samples will be analyzed to verify 3 concentration and homogeneity in the test diets. Q Homogeneity samples will be collected from the top, middle, and bottom of the diet mixer. Samples will be frozen until analyzed. Once during the study, samples will be collected from randomly selected feed jars to verify concentration of lead in the feeders. Samples will be collected from each species. Backup samples will be taken on the same day from other feeders in the same species and analyzed if needed. Feeder samples and backup samples will be frozen on the day of collection. G, Body Weights All rats and microswine will be weighed twice per week unless experimental findings warrant a more infrequent weighing schedule. H. Food Consumption and Intake of Test Material The amount of food consumed by each microswine will be determined twice per week throughout the study. The amount of food consumed by each rat will be determined weekly throughout the study. From these determinations and body weight data, average test animal daily food consumption, and intake of test material will be calculated.I. I. Dosing Rats will be dosed intravenously via the tail vein, while microswine will be dosed via the ear vein. Sterile water will be used as a vehicle for intravenous administration. Oral doses to both species will be administered by gavage. Deionized water will be used as a vehicle. DUP040000625 APPENDIX A. PROTOCOL AN D PROTOCOL AMENDMENTS C\ r[V>-O4. - 8 Pi PC the route of administration of the lead-contaminated soil will be by oral G gavage and/or feeding since ingestion of soil lead activity presents one of the S principle direct pathways for exposure to children. Intravenous administration Q Of lead acetate will be performed in order to calculate area under the curve (AUC) and-absolute-bioavailability of lead. J. Blood Sampling Approximately 0.1-0.3 ml of rat venous blood and 0.3-1 ml of microswine venous blood will be drawn from each designated animal at 0, 0.5, i, z, 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 EDTA and refrigerated until the time of preparation for analysis. K. Sacrifice of Animals At the end of the experiment, the animals will be humanely euthanized. Animals will be necropsied, liver and bone will be collected from the single dose administered animals (Group A in Study Design) and liver, bone, kidney, spleen, and brain will be collected from the animals fed lead/soil for 30 days (Group B in Study Design). All tissues will be weighed, frozen at approximately -20"C, and stored for potential future analyses. L. Blood Analysis The blood lead concentration will be analyzed by graphite furnace atomic absorption spectroscopy (GFAA), at DuPont Chambers Works, Dosing Solutions, diets, and drinking water will also be analyzed, DUP040000626 DuPont HLR 671-93 -9- M. 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. N. Statistical Methods Body weights, body weight gain, and food consumption will be analysed by one-way analysis of variance. Appropriate statistical analysis will be Cfl performed on mean AUC values between oral and intravenous administrations. H Zw SAFETY AND HOUSEKEEPING m0Z Good housekeeping practices will be used to avoid contamination of work I N. <% areas and potential health hazards. Gloves will be worn when handling 1 formulation, blood, animals, animal tissues, or analytical standards. Animal Ou carcasses, feces, and diet will be incinerated. 8 8 RECORDS AND SAMPLE RETENTION P* Q Z All original records will be retained at Haskell Laboratory or at the < Records Management Center, E. I. du Pont de Nemours and Company, Wilmington, oh 4 Delaware. u og s < X D Z < DUP040000627 DuPont HLR 671-93 - 10 PROTOCOL APPENDIX I MEDICAL RESEARCH PROJECT BO. 9660 Study Personnel and Study Oates STUDY PERSONNEL C/3 H Study Sponsors Corporate RemediationGroup of DuPont Chemicals zm E. I. du Pont de Nemours and Company Bellevue Corporate Center I Wilmington, Delaware 1980$ S Contact: Timothy S. Bingman,D.A.B.T. Consultant I (302)571-7761 -3 0 Study Directors Hanan N, Ghantous, Ph.D, u Research Toxicologist 1 i cu ^ < g STUDY DATES Qz c Proposed Experiment Start: May, 1993 0hJ Proposed Sponsor Approval: Date the Sponsor signed the MR Project u Proposed Experiment Completion: August, 1993 1 Proposed Report Issue Date: February, 1994 < X pz g Pi < DUP040000628 COON 11 SO pj X AESOHJTE BIOAVAILABILITY OF SOIL LEAD IB RATS AMD HICROSWIHE eo D MEDICAL RESEARCH PROJECT HO. 9660 PROTOCOL Approved: jjaiian N. Ghantpus Study Director ~>s> Matthew S. B(jgdanffy Manager Biochemical Toxicol Date shills s sJaM cc: P. E. Ross G. :S. Elliott T. S. Bihgman (REO-PIT) K. Me Cleary M. S. Bogdanffy R. C. Rhea APPENDIX A, PROTOCOL AND PROTOCOL AMENDMENTS DUP040000629 APPENDIX A. PROTOCOL AND PROTOCOL AMENDMENTS O' SO & h4 X DUPONT CENTRAL RESEARCH AND DEVELOPMENT Uos HASKELL LABORATORY FOR TOXICOLOGY % AND INDUSTRIAL MEDICINE D June 28, 1993 TO; MEDICAL RESEARCH PROJECT NO. MR 9660-001 ABSOLUTE BIOAVAILABILITY OF SOIL LEAD IN RATS AND MICRCSWINE PROTOCOL AMENDMENT tl The protocol is amended ss follows; 1. page 2, Study Design, Section A. Single Dose Administration, replace 'Fifty male rats and ten male microswine will be used.' with 'Fifty male rats and twelve male microswine will be used.* Reason: To obtain better statistical analysis of results. 2. Page 2, Study Design, Section A. Single Dose Administration, replace 'Twenty-five rats and five microswine will be dosed orally...* with "Twenty-five rats and six microswine will be dosed orally...'. Reason: TO obtain better statistical analysis of results. 3. Page 2, Study Design, Section A. Single Dose Administration, replace Twenty-five rats and five microswine will be given...* with "Twenty-five rats and six microswine will be given.,.". t Wf f** i Reason: To obtain better statistical analysis of results. k. Page 3, Section B. Single Dose Administration After Feeding For 30 Consecutive Days, replace "Fifty male rats and ten male microswine..." with Fifty male rats and twelve male microswine...' Reason: To obtain better statistical analysis of results. 5. Page 3, Section B. Single Dose Administration After Feeding For 30 Consecutive Days, replace "Twenty-five rats and five microswine will be dosed orally.,." with "Twenty-five rats and six microswine will be dosed orally.. Reason: To obtain better statistical analysis of results. 6. Page 3, Section B. Single Do'se Administration After Feeding For 30 Consecutive Days. replace "Twenty-five rats and five microswine will be given..." with "twenty-five rats and six microswine will be given...'. Reason: To obtain better statistical analysis of results. DUP040000630 DuPont HLR 671-93 7. Page A, Pretest Procedures, second paragraph, delete "irradiated" from first sentence. Reasoni The animals are not being feed irradiated chow. The study is being conducted in Building 1 and irradiated chow is not received there. 8. _PageJS, second sentence, replace '(twice per day)" With "(once a day)". Reason: More convenient for individual food consumption calculations. 9. Page 5, Section D. Assignment to Groups, second sentence, replace 'housed two per cage" with "housed individually". Reason: Animals will be housed Individually to calculate individual food consumption. 10. Page 6, Section E. Animal Husbandry, second paragraph should read "Animal rooms will be targeted at a temperature of 23 + 2'C for tats and 25 + 2"C f2 for microswine and a relative humidity of 50 + 102," Reason: Micrpswine should be kept at a higher temperature than rats. 2 11. Page 6, Section E. Animal Husbandry, after second paragraph, insert the o following paragraph: Animal rooms that will house rats will be artificially illuminated (fluorescent light) on a 12-hour light/.dark cycle. Zw Animal rooms that will house microswine will be artificially illuminated ' }g 2 (fluorescent light) on a 12-hour light/dark cycle with additional natural < light being available through a window at one end of the room, 1 hJ O Reason: Microswine do not need to be on a 12 hour light cycle. The animals u should be housed in conditions similar to a barn. 12. Page 2, Section A, Single Dose Administration. "Blood from the single dose go administered group of rats Was collected in lead-free tubes containing EDTA. Heparin containing tubes were used for collecting blood from the Ch other groups. Q Reason: EDTA caused complications in analyzing blood lead by atomic 2 absorption. _i 13. Page 6, Section E. Animal Husbandry, add the following paragraph O Microswine were provided with recreational equipment (balls) in their u cages. g Reason: To improve housing conditions. Balls were added after consulting with o Linda Panepinta, Swine Research Consultant. pi 14. Page 6, Section F. Diet Preparation and Analysis, the following should be added "Microswine were given Oreo cookies after blood sampling. Reason: Linda Panepinta, Swine Research Consultant, suggested the < administration of Oreo cookies after blood sampling as a reward. x 5zw 6 Pi DUP040000631 mOis 15. Page 7, first paragraph, the following sentence should be added "Diet Co samples will be refrigerated, not frozen until analysis. tus Reason: Analysis was done within a short period of time after sampling. Q 16. 'Due to technical error, the 12 hr. interval blood collection was not taken ___for the single dose gaVage group of rats. Five male rats from the animals hot used on the study were randomly selected, prebled, dosed by gavage, and bled after 12 hrs. Reason: Technical error. 17. Page 8, Section K. Sacrifice of Animals. Replace the final sentence with Tissues from rats bled at the 240 hr. time point will be saved at sacrifice. Reason: Tissues from 5 rats only are needed for statistical analysis. Date Approved: cc: P. E. Ross G. S. Elliott T, S, Bingman (REO-PIT) K. Me Cleary M. S. Bogdanffy R. C. Rhea l--h. nan N. Ghantous Study Director Matthew S. B&gdan Manager Biochemical Toxicology seliMt /* <> /i 93 to APPENDIX A. PROTOCOL AND PROTOCOL AMENDMENTS DUP040000632 APPENDIX A. PROTOCOL AND PROTOCOL AMENDMENTS 9m\ so Pd X Protocol Amendment No, 2 f9tSoa Q Absolute BioavaiUbility of Soil Lead in Rats and Microswine TO: MEDICAL RESEARCH PROJECT NO. 9660-001 page 1 of 3 The following changes are made to the original protocol: page 2 STUDY DESIGN A. Single DoseAdministration first and third bullets Change '...and ten male microswine...' to '...and nine male microswine...' in the first bulleted item. Change '...and five microswine will be dosed orally (gavage)...' to '...and four microswine will be dosed orally (gavage)...' in the third bulleted item. One microswine became ill and was sacrificed in extremis. page 4 MATERIALS AND METHODS A. Test Material paragraph 1, sentence 1 Replace the first sentence with: 'Lead acetate will be supplied by Aldrich, Milwaukee, WI 53733, US.A. and the test soils will be supplied by the sponsor.' This sentence was written incorrectly. It was always the intent to receive the lead acetate from Aldrich and the test soils from the sponsor. page 7 MATERIALS AND METHODS H. Food Consumption and Intake of Test Material paragraph 1, sentence 1 Change '..food consumed by each microswine will be determined twice per week...' to '...food consumed by each microswine will be determined weekly.,.' For easier data comparison, microswine and rats were maintained on the same schedule for food consumption determination. DUP040000633 tfi Os StNS Pi 4 as u h Protocol Amendment No. 2 6O Absolute Bioavailability of Soil Lead in Rats and Microswine page 2 of 3 O3h D page 7 MATERIALS AND METHODS I. Dosing paragraph 1 Add the following sentences: 'One microswine designated for intravenous administration and one microswine designated for oral (gavage) administration will be fed one hour before dosing. Data from these animals will be reported separately.' The microswine ate all of their food immediately, while the rats tended to have food remaining in their food jars up to the time of dosing. Therefore, microswine were considered 'fasted' while the rats were considered 'fed.' To evaluate ifbeing 'fed' made a difference, one microswine from each type of single dose administration was fed one hour before dosing. page 8 MATERIALS AND METHODS J. Blood Sampling paragraph 1 Insert the following sentence after the original first sentence: 'For microswine in the single dose administration group, syringes will be washed with heparin before blood collection.' The blood collected from microswine that received a single dose after feeding for 30 consecutive days was clotting. The heparin wash was added to prevent this from occurring in the single dose administration group. f5 , APPENDIX A. PROTOCOL AND PROTOCOL AMENDMENTS DUP040000634 <n Protocol Amendment No. 2 Absolute Biosvariability of Soil Lead in Rata and Microswine page 3 of 3 page9 RECORDS AND SAMPLE RETENTION paragraph 1, sentence 1 Change'-will be retained at Haskell Laboratory or at the Records Management Center,-' to '-will be retained at Haskell Laboratory, Jackson Laboratory (DuPont Chambers Works), or at the Records Management Center,...' Records and samples from die analyses for lead will be maintained at Jackson Laboratory. Approved by: \ j Hanan N. Ghantous, Ph.D. Study Director (date) cc: T.S. Bingman K.A. McCleary RCRhea Manager APPENDIX A. PROTOCOL AND PROTOCOL AMENDMENTS DUP040000635 CO SVO! Ch J4 .X4s4 o p-i 3 Q co Z OHH H Hi > (3 Q Ck >-J O m x>-H P Z w Pk Jh a p r"< t 3N 60 c o 0) .434 VVVi ,3 CO p 60 3 JuS Vi ai ,3 p4-4 OooI 'S3 3 VO OVOn 52 5 3 T4V-34 P 3pH w g 4o*4 4p4 P *0 P T3 44 4-4 O 3 P 3 3u I a 3 4o4 <y v G ft ,3 M "fl a Ck 44 C3A 3 cBu w** 3p 4>44 CO **3H <*4 P *u3 eg *235 ti cpo P3 S SP rts *3 Pc flj 2 32 tl 4, Wp 423 5*4 *p* Ck p3Jj T3 .S Pco S COA >0 s Ck o 606' 0 *3H j<Uf S*4 4>1 CpJ >? 3O PS 2p T>3v M3 CA 4*CH3A c s s ai <y *G 7c3i S3 --u3< G Iuu OO CA G *<QD ' 8 ft G CA ft Cl *ft 4h5-# G6*H0 73 ft 'fl <y OG ooo O ofht ' 4 P ft 73 <a> %1 H34 P*NH 8K ^ T3 Ck p og Sg CO 'tl 2P 04 t pu 2 P 55 * 73 tl -C-3A CL. .2 3 3 5pC 3 ts P ^ S^ 4 4-- pp? .323 *fht 5 ra 4-4 o 3 "ccSj ,2w;j 3 ,3 m & o* ^ n>p> xcw>Hj> 4g ft 73 J4_s ^14 3P J3v Ck hJ 0 1 fi sI? 4? 60 r3t '-p' -s X 2 Ck Ck < 's Ck p*p 3 P c4ao4 p > ^*CJ P *3 434 CO 3 co cPo .c*2o" s-3pS 1 Nm I s DUP040000636 APPENDIX C. BLOOD LEAD CONCENTRATION BEFORE INTRAVENOUS OR ORAL DOS z2 0 11 w PQ PQ < PS T3 C 3MA TSI 1 C3 CD DUP040000637 BLOOD LEAD CONCENTRATION (^ig/ml) IN MALE RATS BEFORE INTRAVENOUS OR ORAL DOSING .0 ,0 ,0 0 0 0 0 12-hour bleed in a d v e rte n tly m issed. F ive e x tra anim als (re p o rte d here) were s e le c te d to be dosed and b le d a f t e r 12 h o u rs . ,H0I1 IN vo Pi HI H AO fO co m ro vo in rt m ov m vo H ClSlNrlOmrl^ <71 d a OlfitDWOOO o nm vo n oo in vo vo VO p. in wf vo in ** d oo in >-w H H O O H O H OHO o o c? o o o o O OH o esou ft O O o o o o D o o ooo oo oo o o o oo js O rl N Cl in VO o 00 <71 O rl d ro -<* in VO IN oo ci O H d ro in Q ft o o o o o o o O O H rl H rH rH rl H rH H H d d d d d d o sf 'l *0 Tf 1# 'I *# f *f 1# V# m o <f*f ^ of * --* oo >1 >1 H H H O o H Q VO ilo rl in <n o 00 VO o o CO o <71 r* o vo oo vo ci QO in vo O [N in o O tN o pi d 0o0 o<# vo o Cl vo vo vo oo <6 Cp- to>o o d ro l> H ci in ro m oo o <71 VO o H 00 o ft O0fDt O 0ft1 o* o o o oooooooo o o ft * oo 00 00rH CN| CO in id r> <71 O H d ro ^ in vo N Cl o H d ro in o o o o o o o H H H H H rl HHHHdddd d d co ro oo CO ro ro ro ro ro ro rn ro ro ro ro co co co co co to <0 <o CO ro a V oooom A HOOOH LJ o o o o o n *1(11 OO p OOS O rl N COl ^O C5 U p> in r~ i> p- <*in<ovotNcndvocovocovodcO*f owioriio^aooo H Cl O PI fl O Cl ^f H N H H H H M H H O O O O H OHOOOOOOOO OOO OODfftt ooooooooooooooooooooooooo H d <*i *f in u> r* co <t i o h d c*> * in vo tN oo oi r-i d ro # in OOOOOOOOOHHHHHHHHHHnPmpmd ddddddddddddddddddddddddd OOOOI0O10 014QOClClOU)O10PIP|inWNinOO rlOOOOOOO^HinrlHHrl rIOrlHOHHrlOO H OOOOOOOOOOOOOOOOOOOOOOOOO ft OOOOOOOOOOOOOOOOOOOOOOOOO I* HMci^inionamoHnniMnioMoioHM<nfin fn OOOOOOOOOridrlrlHWHHHHPinP(PI HrtrlHHHHrlHHHdrlHHrlHHHHHHrlHpl (fl DUP040000638 B L O O D L E A D C O N C E N T R A T IO N (|ig /m l) IN M A L E M IC R O S W IN E B E FO R E IN T R A V E N O U S O R O R A L D O S IN G 0. 0. 0, 0. 0 CO1ts-OINt NO C4 X -4- H H H in o o o oooo ooooo > ooooo COi csu CH Bo tH d co in P5 o o o o Q 0 CO CO 00 00 to H H > o\ C\ in 00 O' in vo H t# vo OS! in H H H rl d d Pi o 8 tH CO If) vo 8 oooooo r- f' T- VO t O VO l> H > O d O ts* H O O O O tH O O Pi O O O O OO DO05 nJ oooooo vo vo vo vo vo vo m rl in o CO <n o 00 tf > d d tH HI o H Pi o o o O o O oD K o rl d CO in vo oooooo in If) in in m in tOHS P(u0 Pra rl d rl <d a '0d n0M a VI 1 1 P0i CO CO DUP040000639 APPENDIX D. BLOOD LEAD CONCENTRATION AFTER INTRAVENOUS OR ORAL DOSIN tc3sMn zo s < >w 0) pa I pa C/3 < P4 CO DUP040000640 co OIN SIpNOH W H rlfflrt j r>f mxf t"* in <# 01 x* Xto? X#CO O ICiOOrHOOOO & >4 X OGw cj'OiinHinrHrjtinHcj'xffoinH M CM rH P CH O <M Cl <N C* IN rH d M H iHl H H rl rl rl rl rj H rl H ri H (4 g G SO M> O p| ) n 91 > V? M xiciflnMinxiplrio(ioo in mi H wrtHoownrtffioiociHmo p| rl rl H pi H ri H H H H H ri --rH <0 toinri<oioeoMDHHnipmxf 'JOCUOWOKMOIOOMHOO 4CWU C 10 rH O M p xf 00 H CO xf o O OS Xl W rlriOriHHHriHHrl.OHH HHHriripIPlriHrlrlrlpiri 01 n w ri Mn lii n o oo ^ h o io COXMfthxJoOnfiriHHHOO 4PH1c VO M H w> oi x# to r* r* 011> to io oi x* c> to w O O O Pi H Q O 1-i 1-1 O O O pH ri rHrHrHrHrHHrHrHrHrHrHrHrHrH 00 O O H Pi <0 n oo X# O 00 M Ifl 01 tO 0pH0 rini pHH to MO 10 o to csi co o o o CH 00 ri in to xf 00 tO VO IN 00 pH to in OpOOOOOOOOOQOO HrlPiripipirlririrlpIpiHH 3O -<># inooooooooooooo OHCiOOOPlrJOOPiVOOOriO HCHNVoOiCJxfOlX# rl pi Pi Pi <0 raH <0 4H rH O fA tCoO -a0H0 0M p>H. 0 CO a erol pH a u a >1 pH *} pH a <d g o a<0 H o 00 SH o 44 no os in fc 4J 4H a<0 a G *o u >> ri QuM a *0" *0 raH s r1GH0 rH <d CQ > > 4 u DUP040000641 BLOOD LEAD CONCENTRATION (pg/ml) IN MALE RATS AFTER INTRAVENOUS OR ORAL DOSING Group II: Single Oral Dose here) were mooHinorotoffiin'jMDCNia) to N rl (*l in {OH^f(*)rfr>rlrlHH Cl pH vo OOOHCIHOOOOOOOO in oooooooooooooo o *rl 41 a <0 10 pH a a m o m h in <4 n n m in h cun ^ OHCJOMOCJriHCJMnHM d d pH e sPodh Q M M M -!t N M C* H H ft [" rl O 00 O Sun H |v W O WCIflMnOftftftldftriHO 10 O >1 4) p| I O OOOCICIOOOOOOOO W oooooooooooooo cu <0 *d ri a> 43 a (0 oX H *H (O M 'J HOlO M> H to to HOHOHOHftHHMrlHW ciddcici-'ftpc~ icMcicicicMcici # o > t>o i( ft h o lo o o m in OIMIfl m !* (*l H ft m o CO O o o <rl in H O O o ro o o o o wa& oooooooooooooo 10 * d t o i-i a> d o to p a> tji o c< d TO H d O H H O pH d O pH pH H O pH d dd d d *** d d d d d d d d r* H to to 00 O to in TO Pft Cl Cl VO O d ci d in us d h in in to a> to h to H ^ OOOOtOrHOOOOOOOO Pi (0 X0) > (0 s-> O -rl aA pH H (d -H 0 a8 10 n <x0 H a 'd X0 <0 <0 d 0) H >0 H a Ot H CO to w OOOOOOOOOOOOOO Ao, (0. o 'i in c h io o t i mo w c TO H O fl rl rl O O O O ri rl O O O c>M M M n CJ M M Cl Cl N ftl M 41 O rt a a> Qa -h ft. X o in (i h o w in <o o cl (i ri ft HdincfttocitflrtinciioHo O O O O d pH rH O O O O O O O w oooooooooooooo Pi (0 HWffl*llOOC|H(OlOC|pHO TO CSI iH O O O O O O O O O o o o d cl d d d r* d d d d d d d d 55 ^ Oa Ht M A M 3! 8S inoooOooooooooo OrtMfflNMOPIW0lO pH CM r TO Cl TO -Of HrlHM DUP040000642 BLOOD LEAD CONCENTRATION (pg/ml) IN MALE RATS AFTER INTRAVENOUS OR ORAL DOSING Group III: Single Intravenous Dose After Feeding for 30 Consecutive Days a 1 a 4 5 sam ples. O '. VO in pa H in H P H o r- a> MO <n o ti WH 00 CO o f4 PM X COS3U Q 1in n in H J IO w o to dH i to n 1 04 1 CO 1 MH i to to 1 C4 i to 1 trl 1 CO H G 0 03 033 I in in vo o tsi t> t> to o\ i> m io \p Cl t'ti^W^OrlrlOOOOO aH pa rt rl rl H s I pm y 01 noMHiflocinHioocjrin a 4G3 H tototototototOtototototototO 0 to ro rt ** oo oo <* r* H m er** Ma ^ aAiH OH 4 4 O If) rl QOHO9 pm 3 01 H OOOrirlrlrlprfHrtOri to to to to to to to to to ro to to to to 1 *H 43 CQ J! 43 3 43 n So Oi o H co <n H d .< io to in vo HMONninMHHOHOOO w Hrlrlrl ato 0^HMOOOViM*>bTtOI 01 dC4C40tHHH00r-lrtr400 to to tototOtotototototototOtO n r* in vo ri in ^ n oo r> to t> c- ^ p>a4 H d4 o MO V3 4 o n n o o o o a 3 ri to oo Tf m in u> oo h <n in to h o o 0} dHOOOOOOOOOOOO nfflnnMrtrtnwrtrtnnn J! 43 u8 44 0 3 i M H I a 43 fc a-- O <a H O 3--M3 W 8 inooooooooooooo <rt d T* r- C\ CU Ol Of rl rl rl W I to 8 *4 8E n 44 DUP040000643 BLOOD LEAD CONCENTRATION (pg/ml) IN MALE RATS AFTER INTRAVENOUS OR ORAL DOSING Group IV: Single Oral Dose After Feeding for 30 Consecutive Days 0 0 r'C'inmcoddovaSnioeoeoinin tCtPNo-i-.v( VD >-J h i m ^ w n mu \o o n n M fli m OOOOrl0O*fOO0OO0 HAPi oooooooooooooo do 4rJl t rdl X ri d <ft in w h d *1 in in n in d ft o c o co ft H# Hi fti Tf "ft -ft! "ft "ft "ft "ft "ft "ft ri lOit Cl , ,03 D oi fluo h ci o in o o VO r* d O C* VO rl O d O O "ft <0 CO a>i O O O rl rl O rl rl O O O O o W" w oooooooooooooo A nt 01 OlflfflrlffiOIOnriOIOHlon CO 'J Tf "ft ^ "ft tf "ft "ft "ft "ft rHl a>s aOs -H A oinio^oaxnn'fooo^oiin "ft ri to H so in d oo o r- vo d vo to OrlOrlrlOrlOOOOOOO H3 oooooooooooooo a 0< rl o r> co Tj o m r> oo "ft p* o d CO H"ft H"ft H"ft O"ft r"lftri"Hft "pft H"ft O"ft H"ft "rflt "Oft H"ft r"flt At inooraaoodd<0"ftri0"ftinp> 4 4ifl in oi n q a h in o o ^ u Cl OOOrlrlOrlOOOOOOrl H OOOOOOOOOOOOOO 2 a to vo o\ "# o to vo qv "ft o m "ft vo o\ 03 rl O O O rl rl O O O rl rl O O O <0 u rdl SH Jj H uOnu 0co0 00 p <T\ 00 "ft oo d d rl O O in vo c\ vo rl d 00 p o o vo 01 rl 00 fO HI o o rl .rl rH o o o 'ft oft o -ft oft ft O ft O o o o Ooo o o o m d 00 rl to in d CO rl m in rl d 00 o "ft o "ft ft ft o ft o ft fot o "ft O ft o ft ft ft o ft ft d as A O -u H d rl a do o inooooooooooooo o h d "ft rdid""*ooicd''0ovd""ffttodi"oft rl H rl Cl DUP040000644 BLOOD LEAD CONCENTRATION (^ig/ml) IN MALE MICROSWINE AFTER INTRAVENOUS OR ORAL DOSING Group V I: Single Intravenous Dose be an e rro r; n o t in clu d e d in mean b io a v a ila b ility c a lc u la tio n . id CD cri> \ptDNH OOMO*OisOiOnOooOOOiO)nrHoo0\ in 6 p) M O rt H O ri P< O M d pH o a tAo K tos is <o U IO U U VMS to u to U) IS ID m oooooooooooo o o to ft3* <8 P O O O O O O O d Cl so HONO'ftOrlOtON in CO CO p H CO <o d d d H H rH O O Aw to oooooooooooooo m ionionioniontonoinioniontonoinoinointonoin tototototototototo oooooooooooindin r <n d in d co -v d fO O rt CO Hi *! n CO <1 H H H H 9 CNCIrHO ooooo ooooo 01 t| Hf Hl Hl Hf H| H| HI H| -cl H| m oooooooooooooo tptototototovpbtdtoto to to <d ooooo ooooo co <n cb in <n o c- r*> in ci ot> ao ni co a> o d d d H# h * co co CO H# p-l h H o H w iHHOOOOOOOOOOOO 04 0} cocococOcococococococococOcO OOOOOOOOOOOOOO H ooooido o o Ao o o Ac> a> IO HI IO O C r( Ifl CO 00 f> Ot H CO fli h in to n (O h i ot n h m H o rHrHOOOOOOHOOOOO dodododododododododododododo to to HSo5 Hueh A--Ha 5 fl 8S 0 0 00000000000 t #'*.# I :l oHoiHico'cHidHHitjCb'cOiH'OPOlHlHIOirtHibji pH pH pi d gf M vh tauj) <ad> tj mfoVHtI a> 33 pH p H $ id A DUP040000645 BLOOD LEAD CONCENTRATION (pg/ml) IN MALE MICROSWINE AFTER INTRAVENOUS OR ORAL DOSING Group V III: Single Oral Dose Value d e riv e d from re a n a ly s is o f sample. CO ON NO C4 X to+s* $ n> Q io io oo < in in 01 ri n eo A in n oHCNiint>HOr'H5jicsi<jNfooio< OOOOOrHHOOOOOOO oooooooooooooo oin oin oin oin oin oin oin oin oin ot o ot o ot o ot o ot o <DcbOodoeoaocDaocDcococD<Dco MiftMOlOHjTOfflOOCiflrt O TO TO TO ON p C ci H CO C4 CO t H CO OOOOOOOOOOOOOO M OOOOOOOOOOOOOO >to4 OOOOOOO'OTII'ONIOO"* m o ooooooooooooo cD co oo oo co ao aO co oo aO co ao ao co OooHoHO^Nr*00-tC**)-HTOcNOnO'i<fNcOnTTOOriodtWM> iC4D CNI ooooooooooo o o o M oooooooooooooo >4 Oh TO NoMoo o oM oN ots oM oM oPJ o<1oMoMod 00 00 00 CD CD 00 00 00 00 00 00 00 CD 00 id C>cnr*U>ONC)**HUOTH'S*T-IC'I OO rH CJ oOn Ot* Ci-Dt t> CoD Wo o CD rol 00 CJ H oooooooooooooo >041 H ri rl rl rl rl rl rl rl rl rl rl rl rl TO OOOOOOOOOOOOOO 00 00 oo 00 00 00 00 00 oo oo 00 00 00 00 8 HOEh Ana gs OH O El TO O O O O O O O O O O O O rl N ^ 00 d^jooriioo^ppio rld'fPftd'ION'J 0 DUP040000646 BLOOD LEAD CONCENTRATION (^ig/rnl) IN M ALE MICROSWINE AFTER INTRAVENOUS OR ORAL DOSING Group V: Single Intravenous Dose A fter Feeding fo r 30 Consecutive Days CO In v OanSoOtOnOniOAOnOoO'o^iCnOmOt'AHCrIl M cooHovHmooiriTjiinTjicocscj VO eS g hHOHHHOOOOOOOO SC -w OG 2 \o to (O IO W IO w o io to IO V to IS CQ o oooooooooooo o in in in in in in m in in in in in in in 3Ph oooooooowinnnioo Q ' c m <n c m pH ** v i*. c m ot u> ov c m m3 VOfO rOCM CO CO CM pHCQCM_p*H_CQpHrH CMCMrHHOOOOOOOOOO in in in in m in m m m m in in in in CQ oooooooooooooo in in m in in in in in in in in in in in o o o o o o o o <n o c m in r> o\ OtrlCOOldOtHMOH HI ri CO to H OtWCICirlrlrHOOHrlPlO O cu oooooooooooooo ^ ^^ ^ "!# # w o o o oooo o oo o oo o in in m to in in in in in in inin in in m ooooooooHincqtOTjui in oo in p oovo **. in o o r> o c m m W r> m CM <0 so CM VO CM CO CO CM CM H S CM O H O OO O O O O o O O o aICQ coooiocoofoocoocoocooiorocoocooofocoocoocoo in m in in in in in in m in in in in in o CM ooooOooOot>l^p'inO o r> c m o v c d c m c m in r* c m in so H 1 W MrlOOHrlNCJCMCMHOOH H CU HOOOOOOOOOOOOO 5 CM CM CM CM CM CM CM CM CM CM CM CM CM CM CO OOOOOOOOOOOOOO in in in m in in m in in in m in in in oino^MoOopoqoNMOivOocnMnHAncinMnOttnai H CO O H P* f*> CM CM CO H CQ CM CM pH pH P< r^COrHOOOOOOOOOOO rl rl rl rl H pi rt H H rt rl rl H p| CQ OOOOOOOOOOOOOO in in m m in in in m in in in m in in mooooooooooooo O pH CM Ml CD CM * CM VO O Hf CM o pH CM M C" CT> CM M OV M pH pH pH CM DUP040000647 OvPCHIOV vo f4 oCQ CM p M CQ CQ rl H CO rf CM p vo voin O Ml O CQ 1 Cl p in CO CQ in W * > * *CM CM CO CQ r4 rH rl CM CM rl H rl rl rl . o o o oo o O O o o o o o o ft *4 VoO oto oIO VO o vo o ovo IoO ovo ovo o10 voo ovo voo voo CQ p p p p p p p p p p p p p p o fiSnJ ooajinoinn^Honnooo Q co <n in p voinoioooo\oovocQr4 rlHCttSHflNrlrlrtrlpIHrl jwp . oooo.o poooooo o o mo ion ion om ion ion oin oin oin ion ion oin oin om p p p p p p p p p pp p p p CQ OOOIOfflMIp. Tj VO CM p H o h p vo vo to nnon njp c m 'h i N M rl PI (il H (1 H M rl 1 HI r4 r4 ...... . . . . | . . . oooooooooo ooo to - fi 'a-**'*'#'#"* ^ ** ** n# M oooooooooooooo p p P p ppp p p p p p p p ft BLOOD LEAD CONCENTRATION (ng/ml) IN MALE MICROSWINE AFTER INTRAVENOUS OR ORAL DOSING Group V II: Single Oral Dose After Feeding for 30 Consecutive Days CQ o c m c m p c m^ vb o ^ r-t to p* m o h n h o ri o vo n o\ m rl M H (O (*)Cl d fll H H rlrl O O O O O O OO O O o o o O O o cooocoocoocoocoocoocoocoocootoocoocootocoo CM P P P P P P P P P P P P P P W 3 ft p rq p c m ffliooci^ffnopHH to r.tH.M..H.n.n.nntn.H.H.O.H. o. oooooooooooooo CM CM CM CM PI PI CM CM CM CSI CM CM CM c m o o o o o o o ooo o o o o PPPPPPPPPPPPPP 3 inMopnoicj^voo ininTjHibinpM"#rio ft Ct fl PI H (O M Cl M H rl H H rl r| OOOOOOOOOOOOOO to i roPl oPrl oPH oPrl oPrl oPrl oPrl oPH oPrloPrloPrloPHoPrloPrl >0 JoJ uo T--oCoJI A 4O4 4CJ COM 4J S3 VH *p mooooooooooooo orinnact-iIiCcMn'fnPOioCMo'^OcViMol >a0 OH rl rl rl N H CJ DUP040000648 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AND ASSOCIATED MATERIALS <ir. StNO as Analysis ofPb in Blood, Soil, and Associated Materials in Support ofHaskell Lab Medical Research Project No. MR 9660 Author Keith A. McCleary Study Completed: 8/30/93 Performed By: DuPont Specialty Chemicals Trace Metals Analysis Group Quality Control Laboratory Chambers Works Deepwater, NJ 08023 Signature:1 frcEj XI Keith A- McCleary, Ph.D? I DUP040000649 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AN D ASSOCIATED MATERIALS cn O'. IVNO t* X Summary The purpose ofthis study was to determine the amount oflead that was contained in samples associated with MR9660. There were essentially three type of samples: blood (fat and microswine), aqueous (drinking water, dosing sohidons), and solid (soil; bedding, etc.). Blood samples were prepared by dilution with a modifier solution (to digest sample and break viscosity) and analyzed using Graphite Furnace Atomic Absorption Spectrometry (GFAAS). Aqueous and solid samples were prepared using microwave assisted digestion and analyzed using Inductively Coupled Plasms Atomic Emission Spectometry (ICP-AES). Generally, all samples were run in duplicate and average answers reported. In the case ofthe blood, each sample and duplicate was spiked and checked for recovery. Ifthe spike recovery was greater than 10% In error, a one point standard additions calculation was perforated. Greater than 1000 samples were analyzed throughout the duration ofthis study. Methodology The descriptions listed below are summaries of the actual methods used to analyze samples. The actual methods are documented in the Procedures Notebook, archived at QCL and copies are available upon request. ` Sample Preparation: Blood Blood samples were received in small polyethylene vails. Approximate sample volume was 100-300 uL. 100 uL ofblood is diluted to 1.0 mL with modifier Sohrtioa The modifier solution is a solution of nitric acid (digests sample), Triton X-100 (surfactant, breaks viscosity), methanol (antifoaming agent), and ammonium phosphate (GFAAS modifier) in water. Sample Analysis: Blood Blood samples are analyzed using Graphite Furnace Atomic Absorption Spectrometry (GFAAS). In GFAAS, a 20 microliter volume ofsample + modifier is injected into a small graphite tube. This tube is electromagnetically heated to dry, char, and atomize the sample. A hollow cathode lamp (HCL) shines light corresponding to the atomic emission ofPb (283.3 nm) through the tube. As free Pb atoms are generated by the sample atomization, they absorb the radiation, attenuating the amount of signal from the HCL. The degree ofattenuation ofthe HCL radiation is proportional to the concentration of Pb in the sample. Calibration standards of 10,20 ,and .30 ngftnL are prepared by serial dilution ofa 1000 ug/mL stock. Each blood sample is analyzed twice. Each sample and duplicate is then spiked with lOng Pb and reanalyzed to check for recovery. If recovery is less than 90% or greater than 110%, a one point standard addition calculation is DUP040000650 APPENDIX H, ANALYSIS OF LEAD IN BLOOD, SOIL, AND ASSOCIATED MATERIALS 131 m(TI. R\o es X performed to determine the amount ofPb in the sample. Each result is the average ofthe two replicates Sample Preparation:. Aqueous Samples Samples that are primarily aqueous are prepared by acidifying 10.00 mL ofsample with 2.00 mL concentrated HN03. The sample is then diluted to a final volume of20.00 ml. and analyzed by ICP-AES. Sample Preparation: Solid Samples Solid Samples are prepared by microwave assisted digestion. 0.1 - 0.2 g ofsample are digested with 5.00 mL concentrated HN03 in a Teflon lined pressure vessel. The vessels are placed in a microwave digestion apparatus and cooked for 10 minutes at 300@W power. The samples are allowed to cool and are then filtered into a 50 mL volumetric flask and diluted to volume with deionized Water. Samples are analyzed for Pb content by ICP-AES. Sample Analysis : Aqueous and Solid Samples These samples are analyzed by Inductively Coupled Plasma - Atomic Emission Spectrometry (ICP-AES). The prepared samples are pumped into an sample introduction system where a fine spray ofaerosol is generated. This sample aerosol flows along a stream of argon gas into the plasma. The plasma is a highly energetic discharge sustained in argon by applied radio-frequency power in excess of 1000 W. This discharge is energetic enough to desolvate, atomize, and excite the sample species to radiative emission. The intensity ofthe characteristic emission wavelength ofPb (220.35 nm) is proportional to the amount ofPb in the Sample. Results The results for all samples submitted under MR9660 are listed immediately following this page. DUP040000651 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AND ASSOCIATED MATERIALS li/8 /9 3 Pane 1 of 11 DUP040000652 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AND ASSOCIATED MATERIALS 12/8/93 Page 2 o f 11 DUP040000653 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AN D ASSOCIATED MATERIALS 12/8/93 Pago 3 of 11 CO OIN VO CA nJ X Ii iI i I. i. I .co.no r> rs;vim tv-iUp^.psvK rvjr T>.jr^ Slf|i'; <oliSico ico tpj< r-'l5f'ini<0'f*s`c0 `*--fc^jto *HjcD'jrN. OcotfOcotOicolOtto'.ic.oi-eo <o.f (J'O' O i O vO'O U ` nlnlnico QiU'Ului Iit CDT..JOfH tf)iCffNl I "*.*! mcoin UIO U lir- I in to <N:f* o'u CD oiCunD r cgOo'c.uCoDn.*cOoj I':cCuDo!lunii1.a)cooiin<1 ^iunti'miopw*oji lGO)l CiNN!0'3rt>.Ct3 ; CCOD'iN r>..>0)\c<S>Ii0;.)N|.- P.r*vIliri :<r *:o'| ; { r~-r- r- I ! ;I i! .0iO|Ot.O`O:O o. ;i!iij;r iK o**O*to..<iNngj.iiC^eMg*Io!NoiilonWiwiglniiuiiCnoN{i ninin co n co;.fo..-iirt`!o;rt>' ti' < ilii'(to| *C-O|Cl C0Ojo rO*tr|c'o'ClOO 1 r0cvo ccoojiic<fnoitiior; -ic^i.coj^ij`r,.j to j n i co i co coin in| u`u u;o uioroO'CJ Ol<J<QiO QtQ Q< ccosii i jirK-^oi1 ;co | to j<o ] co 1'li i,r-l|r0ci>rN-ic.<M^ j to i.COtCO utoloio :i U31 I; co !*.; COO lOCOjtOCOj ulUiOl IlCoO ioco; JU:CJ. j, f<sn, |i rwv<t rw> rr*.| cr*y :iCs^.|..;c m 1 ' * r | } I {;.*- ,cs;l^| If*-* jicuoilueoiuin, cuo ojlnu coin UlU ()! j 'r-f* *- | *"!*- :iO j aCCONIOCuO* iji i[o . t i.o 0*00. <10*c0os:.co too:.cOo.cro- QU u u-g ui.U u .nco. in roieo' to lUOfUiO`U 0*0 OiU DUP040000654 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AND ASSOCIATED MATERIALS 12/8/93 Pago 4 of 11 DUP040000655 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AN D ASSOCIATED MATERIALS 12/6/93 Pago 6 of 11 i\ DUP040000S56 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AND ASSOCIATED MATERIALS 12/8/93 Pago 6 of 11 CO tsso BiJS s !I 1;i il ^1}^ 9{CO oj im Nils 5]? a aEi 010 QiOlQ Q <uft 1M#I. u>| 0*0 bio Air^l ft * A{101CCNV'CNIprCOjlOM j[< Ail a <r fti a id in |i o CO o --* 0iI 3S"CL' tr j*r OIQO X ps.,r..r> rs. .r. r* Et CD - CO- Q< O^:'|O0, 0-0 <D\r* aO'O*o 1tIO^.0f1IiQ0v). iCr^Djlofl {I olo j.<S> ni^.!0iN r.V * !'*. *r ^ i.o; 0i0:Q|Q:0 a ?!<?. Otft cv}jc*s*t{ ;.!ft Jftl^f I 10 !I CNHft 2 <N <01 i<Bito'<-i|fflliitij( o-|<N olo rO!?t!i5wO: i^AlO'|l.O:!-rvvl:Oiit !|*i0frtI{O0t `tr*.-itrc^.MlI^|ftU*-!-*.|i.iV^ N'CO OfQ OiQiQiQiOlOlOlQlQlQlQlQIO fOt|iOMirp^**lifAtil!.s*"c'-:iiTrtrl!rt {i0ft1ia ** fx <& I o OIO rv.iNjfNiKir-lN r*% I I'! I .': i 2 2 2,0':0|.010 'Ol*" -- *- *-j-- OtOiU'U CJ O U 0:3>3^3iU3 3 UtcIc'UcI c l -a. DUP040000657 12/8/93 Page 7 of 11 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, A N D ASSOCIATED MATERIALS roHv IN VO Pl-lt S3 1\\ , ' ' ! 1!!i 1! !!); . * : : :` i ; : ; ! : !; :i : f1j:!I! 1 1n ! I:: 1!i1||i. ; ! 1 1 ! -j : ! ' j j |....I'......... -- - Jill ;1 : ! t .i i! i ii ii !i !i nil i 'i i i | IIIi j j ! i j t 1 1 1 -- ! ! . j ! i i; m i Sliji !1: ;i M! t\ CO .105 iv? ;c *j 'Oltr!V iNi iCJHOlO Amo , *-i c n AICNi to K,to to A; CO j. CD ^1 1 : ! 1 i i 1. ; M ` ! 3 1 ?! j o' a. U5 i in VV v f !1 : 1 i ! : f (' I !; | i 1: 0^.0 o-o o <N!.CN |Sample tnirj sialsi Oi*SiSIS 0(0 * <c n ;<n a n tO|D:f>- jiilisds ISiStSISlS IO. CN n o CN (to O CN 05 c jS i j ; i nn CL (Sample i CN c n CN i CN j.CN lb co 4idjr-i O -I CN(<N I o Q OlOiOi CO O to ; A Vj Vt i *r i"<r CN CNICN CN c n CN o S2 tfol- CN CN Vl*r *r. o OlO Q O ato* ar to ar*. fcNo i ca ja. 1i i >| to lojlO'lO-lfl( o o o o o djdtdioi CL\ wSmi. in 'oV in c n rr o 0(0 Q O cn o *r to o 6 5 to CN CN oo oQQ CN CN AjA CN A E A *-i c n to 6 to CNj O*fj*r OlO NT' ft oa 3! 05 9 CO to <*> o <Ao\I o V- o*r o^r ? o V- CN CN CN CN CN eb A CN *4 o O CN CN a W o * O 3 ^r o tfo-- ft. to * r* CN l CO 03 to to to- Nr T *r; CD o N 0> O "T CN s? cb CN V i| aa Qo CO cinn ttoo to CN to c CN rv CN v CN CIVN CfvN 4 in O e a Oa ? a :n t o 5 O o CN CIOO ft r" AQ nto CN CN CN CN CN 1 3en CN 'ft O CN CN O NT a. o s * * ft N *+ i* 4 oo ib sr QQooO CN CD to to to IO VVV s to A to A s to A s'aft fs ^r a CD 3 A ft a ib CNNT o CN A 1 CN tCoO fi.to ca to CO CO S Nf to ? A w tb CN * ooooa DUP040000658 12/8/93 Page 8 o f 11 coau C /4) s< 02 E 5 2 Q Pi <pm o tcon < D Z C JT pm Oto Ooo' hCJQ ZPH Q <w4 u* O tiC--oOi * *< E Xtm D w2 Dm Dm c ,5 r) O in .60 CM pH 09 to 0) * ' CM 0) CrLf- i CM CM O'O) CM Cf> CM CM O) CM o> CL- ' *"lfN in to Et tOoiltOn lQiOO O: to o 01.0(0 O O- -i CM CM o r r O to ce O to COlMf m o> o in CO O) (O oCM CM **' CM CO d <0 10 cs. m , !. o--CCOO-lMOr CD CM PH CO to d n O' CM o tn ooo T M1 CMiCM [CMl CMlCM 1 cMlnutlioloi tOn lOmlrOinroinitn!-- 5 PM oLuIiiOmttsn *7 O i o o in o b tn 9 o OiOtO OiOiOi C oo a o o o o CO CO PH CM CM tn oCO Olif) CMitn CM tn PH CO o PH CM CO 05 CO * PM O : Z:- <!; UJ, .ft.1-, So!; enr ta.r .COIC ac JaO. , CM CM o a. r* CM CO 1- *7 tn Ph ( CO1 Oto Oin 4O0 o o Ol to1- O O o 0`0(0` C0D) 0CD> $ C0O5 0C5O CD 0) wm CM CO 4o0 oto oin Otn oin otn o (3 O O O Ci .1 f CO 6"0 0033 CO!*!N` . ^ t pH CM CD 05 CM m* CO o O O 0:0 *"! o o O O d OCM CoM oio o o *T CM ton otn IOOIIIOO o tn o O O do O o PM CM PM CM t- CM CM a O O OIO o o ^. o o CO d Mm*Ttf :I!mmTPp too ltuo> 0(0 p9h I!crd 0(0 i-^ jcLofico mtuonito (9.01(9 a. ! : i CD :; coL PM CM CMVP*M ICM c m! C0M- I-CMM* 1 COM' CM CM CM a. oIf) CM O otCnO u0n!0in (0otn- PC ICO CD ton t0n1t.t0n1.m0 (iion O, tn PM o mo ton OIf) O o CO o o O (3,0 O O OiOIOiO O C3 0(0010 o tn CO i p* o: <0 0) CM oo 05 CM jO CM PH' ; ^:pH O CO 05 <o CO <o CO o ICO/O ;i O.i c m .1 r P CM tn -ICOJPM CPMD CO -0)1 a .*-i CO ; .O CL | in IO U5 if) tn: o! O o 0(010 ai CM CO * tn <bi oto o o o O! to O O o o 0(01 cl 0C5O CIfM) mCM tn'coun; C"O P**MO^V; CSi ' -| CAL r- Ph pH PH *F" - p- PH pH PH pHlPHj CM CO mice; Ea s Oto oto oIf) tonlitoni (ft <5 o o o O'01 . Oco CD MP 1.00 pHi 09 i = JCOL - --, . >2 A A 1 o 3 acc. 1 t0c.. ? ?! ba s n Id in St 0 a O o ,o ; , - - CM PM CM CM CM oin oin tOnulOn-Otn oto otn OIO tnun o tn oIf) 8 in O 010(0 O O W oio:o o O CO O oCM i0n0 05 ain 0) - -- : 33 33 3 i . Ui leQu. Z s& IOO O toao 1 O0 O o t0o oIO a toton O cr> Ph tn CCOMiOtn in 05 aCO oo o05 otn om r> o O <o CD 1 CD y* CO.IC**D* CO to CO CD CD o! tO PM CO o tn otnitOn Oin mO om O OIO O OIO PCMO ro CM CCOM to e* CCOM CM CD CD cbio P CD o O to coU otn O tn otn <5inP OtO O'O O O CHPD o o pH o 0. V o o If) CM o CD Otn O 01+T3 }j.,iTn0 i,ICTO to0n-1uo:n0il1ton0 CD OIO t(OOo OIO T3 *0 "S' 3 DUP040000659 12/8/93i i Page 9 of APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AND ASSOCIATED MATERIALS j i!! i: ; 1!! [\ ! i! ! : i i ; .i : ; 1 \ i i . I r. -j- - ... :i 1j : i; 1 i! i 1! ' \ i: i! fs as CCNO ICrNs ICCOD.C-Nrtf o fs rs CN CO fs M as c ;i jQl !| . a. E0 v> * *IT *Wt'*Wr:i ors X CN O T^T coiv pOsIOr-% xlx T:Ti Ofsi'p-Os ii x;x. CON CN as | CN as CN OS CN ors CN ofs s rs 4 fs s CD Ofs X XX X XX _J n CD cn <y O CO s CN fs CO <*o Ps CN CNCNNT ** o CN * CN cca Ja3. 1; 00 o* !;i CD' j` orsj ,!rcsn fCsV.|CfsNIjCfsN CrsN | Sample CN CO:4iii s Ors -Ofs IiOfs.Jj pOs:'Ofs; X X, XrX!X:Xi sfs fs fN. <oi4j Ois.fi0rs.1ff.s0 s- xix XIX !.X X -J CO ID CN rco <0I|rIAsj CO 1 CN I CO 00*,!0 <0 CN CN CN CO QiO CN1jr- CO co 1 !CD.) j i.aja. 1 1 1 ; 0* -Cf V; .10-j V'j-C ii , i CQ| olo a i 8CEO fs csl rs Cor-0Nio..r4%lo1fs|lC.oirDv j X X xlxlx-x! ~j.CN CO 14 ,Ofs .fs- iOs IOrs fs fs XIX xix X X 8OS a CO *CPN *i. CNjCN PN ..rrss oo CN oCO CO oCN |Pb, ng/mL j 1 . IcDc CL Ul CccL UccJ CL cUacJ. UJ SaC. UJ CaL: sa. CN CO 4 ID CO E0 ps Ors fos ofs fos CO X X X X X X 10_1 "a> E fs CO <n ; fs o r*% .ace. UlUl c cca. a. UJ CC CL Ul CaC. uriui cc Uc CL: O. aE ros CN ors CO Ops 4 ors | 0fs :0fs. CO X' X X X X-X 1 o|d o o o o ~fCN ofs ros CO fos rs ors CD Ors xlx X X X X aos oCO oo fos o oas CN CCON SO CCON CCNO CO CO CN CO 52 ors CN fs CO ors ors rs . ors X X. X X X X 558 as a o 4 0V CN .oNT CN oCoNr 0NCNT OCNNf o CN 1 s rs CO ois 1 OIS CO oPs XX XXX CN fs A s fs Oass as isliolce ! ofs CNjcOl4 ofslfoslors o|S ors X XI xlx X X rs CN CO jps col CNj~ CN CO CN CN NT 2!o CO a X .V0 oCN CON oCN 0 CN o CN CN 1 iCN <0 aps QIS g fs ors rs 'XXXX XX 3 8 oCN 55 CN CN CN CN 1 t CN CN CN CN CN CN I *> CN CO Ofs Ofs ros ors Ofs XXX XX X S|2 CN CN CN i CN o CN CN CNNf CN NCN 'P CN C7Nt CN s Ofs CN ofs CO r4os Ois COfsD XX X X X X CCNN CN rs OCN CN fos a CN fs CPO: | CN *0*4 5 sfs ros ofslofs fos xixX X XX rs CN oCN CN CN CN oCN CN CN CN CN CN CN s 10 sOrs IN fOs CO of4s ofs fs 'X X X X X X fs CO as CoN fs CN CN CfsO 8OCO oCO o o oCO On so sOfs X CN oPs X m ofs X s ofs X. X rs rs fs N ps fs P? S 8CN CO ofs ors ors rs ofs fs XX XX XX i i i ii j DUP040000660 {S a m p le 12/8/93 Page 10 of VI as .oe txoJ < D2 B 2 D B l-S Uo 0055 < Q 2 ! O 05 Doo" xJ 2 D Cw xJ b O 05 CD >xJ* < 2 < as * Q cb2wu < jI i 1i i* > J .. _ : i i i -'-I- 1 1 I I :i !j ! 1 i i i i i _i or cs 9) 05 05 E0 05 <o 4 ?3 05 CN CO CO C: J3 Q i*r4 01 icos^iv a-T TIT Sr.P { x..v.| *:coi --N r>i4i a'OIOto Ol.O'O V' ` count' ccru-;u.:u_ Uilbl.lb! ,J 1 i i iI .KDlfMiX. in >c o si.olioiv csto .| "i 1 ! --'tn eo CO 05 CN <N CO CN i ! i .1 j | CN CO 05 CO <N CS -- IfN i 1 i1 i 5*'NT v;*r t -- |CN co i 4 i OjO ooio 00 0 coloia u.! u_ Ib.lbilb mJ <0*C0 eoir* cs to a 05 o! c. X3 CL CN. CN ` tN CN CN C5;05!C5 5> C) 4ifNtC0 4 in OjOlO o o a I CO ibi.ibiib lb. u. Pn . 05 -- r*. 0* r> ITJ o Ooo O' CN CN sr CN 5 ?j CN CO 4 S! oooo CO lb 0 lb lb ib 2! CD 05 0 m n <0 CN cs N CS 0 o cf- CO CN .0* CN CN <S CN CN CN 15 *- t 4 cs co 4 in OOoOo 0 0 a CO CD 0 lb u. u. u. ib r*s ur> 05 C j S un in 05 Qu V V V LU u> U1 UJ lb 0 E E ec Q. <c a. e 'CL Ea. CN co it O) O ooO o a eo 00 CO 00 (0 u. Ux lb lb Ux CNvfS it CN o* CN :C* S "let oio CO o 4 o in CO-CD CD CO CD Ibltt. lb u. lb ;|N r CO 05 05 VV 05105 05 05 05 o?d d o "j CN CO 05 oOa fOa lb lb o CD lb o a u. o CO .lb 000 0 0 * *yf o s CO. It o 05 O 000a lb lb lb. lb ib 05 r* CO CN CO CN CO CN CO 4 05 ooo O CO 0 0 u. u. lb a. lb CN CN IN CS CN rv f*s fN IN CN CO 4 05 Ooooa 0 0 0 a ib lb lb ib u. K o CO 0 0 CN CO 0 ec CN cs CN CN CN s*" CN CO 4 0 Oo o0 O 00 lb UL lb U. Ib ] i O 0i O cs CN CO?-- CN 1 l 0 0 00 0 05 05 Ol o 0 CS CO i 0 . o o 0 lb 2 0 lb CO ib a ib A 0 |N CN o PC 0 0 0 It T e Sf r o s eo o 4 o 0 0 u. CO lb 2 o ib o ib 0 o o0 w 0 CO eo 05 o CN s B CN O CN cs CO 4 0 Ooa o 0000 lb lb u. ib lb 05 0 IN IN 05 0 0 0 O* IN ' CD 0 CD * O CN o CO O sr s 0 lb 0 lb 0 U. 0U. 0 lb DUP040000661 APPENDIX H. ANALYSIS OF LEAD IN BLOOD, SOIL, AN D ASSOCIATED MATERIALS 12/8/93 Page 11 o f 11 E C| 1 j 1 :i ' I ii :i 1 l_ in f* W .-1 I CV V 0 CD o> a s' ICO n 0 to tv n Jpi d d V d 0 dd Oi 3; J3.! 0.. Cfl 0 59 tv UJ c. 5 o UJ 0 CD CO c UJ UJ U1 CD 50 0 0 00 v UJ cv th in CD ft. O o a o o o UJ CD CD to CO CD CO cc UJ UJ m UJ UJ UJ -J tv n P* <0 CD n to CD cv n j: <N cv CD e CO tv cv CO S' 00 O n o to o 05 ID 0 at 05 flO .o cv 0 c' jS a. 0' 0 0' 0-0 0. 0 010 0*0-0; '0 CL tvlcn 4!ib:tbi E o OiO OlO'Oj n to CD CD CDICS'CD <A UJ uiiui 010.0 J at CD E ao :=> CD OJ o cv n d JO <V o 0)105 i 10; didi 11 d a. i Mj CO 00 CD 00 lOO f 00 1 0 0 0 0i0'0j Q. i tv n m E. 0) CD 0 J* ? O; d S' a. CJ 1 1 0 0 0 0 0V a 5 n o s vn cb 0.0 0 CD CD CO CO CO co w UJ UJ UJ UJ UJ: UJ <0 f*H to 0 co:K tv V .a* 1 c d a. icv.tvicvevicv CM* i!<jiq)i.0>[<n qji i^jrvinj^iio cbi O 0!0l.0`0 OI IcOlCOItOiCOiO CD iUJiUJlUJ UJ.*UJ UJ 0*.i CV;*~ tv o0jUOVd0|iWdjix n d !h ! i1 ! tv w fVTtVICV tv r* fs r^THr* p* 5 cv o tbf4iib O rO' O CD CD CD C04C0ICD CD UJ UJ umuiui UJ CD r** f 05 0 0 CN 0>t*5 cv *> d did d o* i :j 0ooooo * 01 tv tv cv tv cv tv o cv o to o s to o 0 o to UJ to UJ to UJ 0 Ul 0 UJ 0 Li fs fs COJtO cv 0 tv 0 -rr'CV o o d did o d T 1 CD CD CD 0 CD 0 99999 9 tv tn ib 0 ooooo o CO UJ D UJ to UJ CD UJ to UJ 0 UJ 05 o fs 'fs fl* 0 tv 0 A o od odo <0 05 0 in at D .-- to o d o 0 ojo d i I 0 o 0 o!o o tv cv cv TV lev tv 1 cv s 1 4in ss 0 o CD w UJ 010 0 0 m m TV cv 0 .0* to 0 o oo6dd !' 9 os CD'j.GO 00 CD tv s 4 in CD oo oO CD UJ CD UJ CD' CD UJiUJ CD UJ CD UJ CV cv cv cv cv tv itv n 4 Osss to UJ UJ UJ UJ UJ 0 vr <0 0 0 0 0 cv cv tv tv cv tv tv tb s s 0 o0o o 0 0 .0 0 CD CD LU UJ UJ 0 0 0 to tniflb fs. CN (CV o tv o to tv ID n CT 0 CO cv d cv <v A0 in tv 0 cv A rs 0 CV a tv d CV cv od 0d 1a d. CL UJ UJ UJ UJ u j .u j ; 0 tc a. AC CL cc ft. cc CL ce.;<e: a.-a.! a tv n 0 ?bi cot E 0 ifi o CD UJ o CD UJ o CO UJ o CO UJ O o: CO CD ` UJ LU . A to fin ib ID in J2 o ojo d o d 1 evitb tb CD v> o CD UJ 0-0 CD :<0 UJ UJ CO UJ O CD UJ o CO UJ tv n 4 0 cb oooooo to CD CD o CO CD UJ UJ UJ UJ UJ Ui cv tv tv cv cv cv tv tb 4 ib cb ooo oo o CD 0 0 o to 0 0 UJ UJ 0 UJ UJ DUP040000662 To: MRHLE 9660 From: Hanan N. Ghantous HC^ Study Director Notebooks used in this study: E-78163, E-78163-AA, -BA, -CA, -DA E-78164, E-78164-AA, -BA, -CA, -DA Haskell Laboratory Report No.: 671-93 Name of Report: Absolute Bioavailability of Soil Lead in Rats and Microswine Distribution of Report: Timothy Bingman (6 bound, 1 unbound, all single-sided) Hanan Ghantous (1 bound, double-sidedD)~" t\C- i'Xlupfc!'b Medical Research Project No.: 9660-001 Haskell Sample No.: 20202 and 20204 Haskell Test Code: 467 Haskell Report No.: 671-93 COMPLETE THIS PORTION AFTER REPORT IS SIGNED, BUT BEFORE REPORT IS SENT TO INFORMATION SERVICES Does the summary from this report qualify for the ICARE database? If yes, who made the electronic submittal? If yes when was the electronic submittal sent? yes ./_/ TO: INFORMATION SECTION PLEASE DESTROY ALL OLD FORMS HASKELL LABORATORY TEST SUBSTANCE EVALUATION FORM i. Fill out a tost substance evaluation formas completely as possible for each test substance submitted ii. If a test substance is being submitted for mutagenicity testing, pay special attention to Sections 6, 8 and 9. Hi. Unless otherwise requested, test substances will be returned to submitter within six months after the final report is written. Haskell No.: rUOtKQlk (to be assigned by Haskell Personnel) 1, Chemical Abstracts nomenclature: d ei-vo, A C. >' Ad lot-p) noiLt" ______________________ _____________ ______ _________________ _________ . .. _______ Ct-yseAI _________________________ `bfr'jrz Chemical Abstracts Registry No.: U OH O 2. Other name(s): ............ .......... 3. Cost Center identification code and/or lot number: K V 0 2 231 C Y 4. Structural formula: 2>U3s0 (a:) Molecular weight373>33 5. Composition of mixtures by % (include all additives and solvents):__a / { ft 6. Purity% (a) Impurities present: N ffr 7. Physical characteristics (a) Colon uM't <- (b) ForauLik t'cjeJolt'd ' m.p.iJldJL. W b.p. at 760 mm Hg.: 43%o \Pq (L . (el Density: (f)pH: 0.0 fit StX (g) Vapor pressure at 25 degrees C: \Pt 37 degrees C: (h) Vapor density UNKNOWN (attach curve if available) (i) Maximum process temp. ii Iff in presence/absence of air. (delete one) DUP040000664 TO: INFORMATION SECTION PLEASE DESTROY ALL OLD FORMS i. Fill out a lest substance evaluation form as completely as possible for each test substance submitted ii. If a test substance is being submitted for mutagenicity testing, pay special attention to Sections 6,8 and 9. iii. Unless otherwise requested, test substances will be returned to submitter within six months after the final report is written. Haskell No.: <gt(?ao4 (to be assigned by Haskell Personnel) 1. Chemical Abstracts nomenclature: hJA ______ . . . Chemical Abstracts Registry No.: Kjf\ 2. Other namefs): /-& AD fKoti AA Ta /AT" O WITH' I0OO A <;xT^ XA2 w ) h%Y\WC~~ro/Q STAtg . 3. Cost Center identification code and/or lot number: AAA . ............ . 4. Structural formula: (a) Molecular weight A/A 5. Composition of mixtures by % (include all additives and solvents):__ /W A 6. Purity AJ(\ % (a) Impurities present: A/ A . Physical characteristics: fj (\ (a) Colon , (d) b.p. at 760 mm Hg.: & f A (g) Vapor pressure at 25 degrees C:_ fKNOWN (attach curve if available) (i) Maximum process temp.______ (b) Form: S&Lid (c) m.p.: )A (e) Density: AyA 37 degrees C: , (:i) pH: A-> !A(h) Vapor density N(\ in presence/absence of air. (delete one) DUP040000665 LOUISIANA STATE UNIVERSITY MEDICAL CENTER 1542 Tulane Avenge New Orleans, LA 70112-2822 Telephone: (504) 568-4750 FAX: (504) 568-4633 Department of Surgery May 3, 1993 Dr. Hanan Ghantous Stein Laboratory (DuPont) Building l Elkton Road Newark, DE 19714 Dear Dr. Ghantous: Please find enclosed 2 small keys for the lock on the container of 19.5 Kg DuPont test soil "P" that I am shipping to you via 2day Federal Express per Tim Bingman's (DuPont) request. This test soil is from our original stock of test soil obtained from Hazen Research, Inc., Project # 7910-08. D 1542 Tulane Avenue New Orleans, LA 70112 (504) 568-4763 School of Allied Health Professions School of Graduate Studies School of Dentistry School of Medicine in New Orleans School of Medicine in Shreveport DUP040000666 School of Nursing TEST REFERENCE NO. .-RF- 011-139-000-2 (### ### ### #) *NAME ..NM- Acetic Acid; lead(2+) salt, trihydrate *REGISTRY NO. ,.RN- 6080-56-4 (######-##-#, zero-filled) *SYNONYMS .-SY- Lead acetate trihydrate / Lead(II) acetate trihydrate / Lead acetate / Acetic acid, lead(2+) salt SPONSORING DEPT, -DE- CHEM (see DE word list) MEDICAL RESEARCH SAMPLE NO. ..MR-MR-9660-001 --SN- 20202 SAMPLE R'CD DATE .-SA- 930511 TEST TYPE ..TT-999 MISCL (see TT wordlist) COMMENTS ..CO- Distribution: K. D. Dastur (1) W. J. Brock (1) E. L. Holloway (1) R. V. Daum (1) Other Codes: (TYMMDD) Aldrich Cat. No. 31,651-2 Lot# KY08221CY LMR MSB/'HNG Sample Room Report Nos. (HL-#-YY D+.) HASKELL REPORT HR-HL-- - (Outside rpts) ..HR-HLO-- . (Shell rpts) ..HR-SR- CLINICAL REPORT -CRA- . PATHOLOGY REPORT - PEA-- DOCUMENT NUMBER . .DN- . Date (YYMMDD. ) . . Notebook & Pages (E-# P. #-#. ) . . . + Input a "D" one space after the Haskell Report No. if and only if the report includes a chemical which is a TSCA 5(D) chemical.* * Computer entered Completed (__ ) 1/16/87 DUP040000667 TEST REFERENCE NO. . .RF- (### ### ### #) *NAME ..NM- Soils, Washington State, contaminated with lead *REGISTRY NO. ..RN- NA (ft#####-##-#, zero-filled) *SYNONYMS ..ST- Soil contaminated with 1000 ppm lead from a site in Washington State / Soil contaminated with 1000 ppm lead from Washington Statew / Soil with lead SPONSORING DEPT, ..DE- CHEM (see DE word list) MEDICAL RESEARCH SAMPLE NO. ..MR-MR-9660-001 .,SN- 20204 SAMPLE R'CD DATE ..SA- 930512 TEST TYPE ..TT-999 MISCL (see TT wordlist) COMMENTS . .CO- Distribution: K. D. Dastur (1) W. J. Brock (1) E. L. Holloway (1) R. V. Daum (1) Other Codes; (YTMMDD) NA (Note: NA was not defined by the sponsor) LMR RWR/HNG Sample Room Report Nos. (HL-#-YY D+.) HASKELL REPORT ..HR-HL- (Outside rpts) .-HR--HLO-- - (Shell rpts) * HR-SR- CLINICAL REPORT CRA" * PATHOLOGY REPORT - PRA-- - DOCUMENT NUMBER .-DN- Date (YTMMDD.) . ** Notebook & Pages (E-# P. #-#.) * . + Input a "D" one space after the Haskell Report No. if and only if the report includes a chemical which is a TSCA 8(D) chemical- * Computer entered Completed (__ ) 1/16/87 DU P040000668 Two completed and signed copies of this Declaration must be handed to the operator TRANSPORT DETAILS This shipment is within the limitations prescribed for (delete non-applicable) PASSENGER AND CARGO AIRCRAFT Rt X Airport of Departure /Je u J Airport of Destination PP/Ldbet/^/d WARNING Failure.to comply in all respects with the applica ble Dangerous Goods Regulations may be in breach of the applicable law, subject to legal penalties. This Declaration must not, in any cir cumstances, be Completed and/or signed by a consolidator, a forwarder or an IATA cargo agent. Shipment type (delete non-applicable) NON-RADIOACTIVE X NATURE AND QUANTITY OF DANGEROUS GOODS Dangerous Goods Identification 1 Proper Shipping Name Lead Compounds, Soluble, N .0.S. (Lead Contaminated vO 1. 1 l POISON Class | UN or 1 or Divi- j ID sion | No. . .j 6.1 j UN |2291 1 j 1 i 1 ! i SUbsi-i diary! Risk j 1 i |2 | i type of packing plastic drums lOKg each x iii 1 j Inst, i j 1 i iY619 | i III iii . H Limited Quantity Additional Handling Information 1 ] 1 I 1 1 11 iiii iii . Iiii !ii. . 24Hr Emergency phone # {504)361-5284 I hereby declare that the contents of this consignment are fully and accurately described above by proper shipping name and are classified, packed, marked and labelled, and are in all respects in the proper condition for transport by air according to the applicable International and National Government Regulations. Name/Title of Signatory AS S t . Duane M Smith Jr.Ph.D./Prof Place and Date LSUMGy 1542 Tulane Ave^N Signature (see'warning ab * xt5/i: 49^r Ic 4 R^sed>r?8;DUP040000669 NumCaps THIS IS THE ENTRY SCREEN FOR NEW COMPOUNDS THAT HAVE BEEN RECEIVED. ALL ENTRIES MUST FIT WITHIN THEIR AREA. HASKELL NUMBER=20202 AMOUNT RECEIVED 125.200 FOR UNITS USE- SAMPLE WAS RECEIVED 05/11/93 SEF DATE 05/07/93 SUBMITTER CHRISTINE LOEFFLER LOCATION HASKELL DEPT. CHEM TEST ARTICLE LEAD ACETATE LOT# KY08221CY CAS# 6080-56-4 CODE# CAT#31,651~2(ALDRICH N.B. HAZARD.CODE HAZARDS COMMENTS MATERIAL-WHITE, CRYSTALLINE SOLID ZDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD? 3 USE "PAGE-DOWN" TO FLIP TO NEXT PAGE 3 3 USE "ESC" TO ABORT 3 mDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDY NumCaps THERE IS SPACE FOR TWO SYNONYMS-EACH MUST FIT ON A LINE LEAD(II) ACETATE TRIHYDRATE, 99.999% 9th Cl NAME ZDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDODDDDDDDDODD? 3 USE "CTRL-END" TO ACCEPT ALL FIELDS AND EXIT 3 3 USE "ESC" TO ABORT 3 @DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDODDDDDDDDDDDDDDDDDDDDDY DUP040000671 NumCaps THIS IS THE ENTRY SCREEN FOR NEW COMPOUNDS THAT HAVE BEEN RECEIVED. ALL ENTRIES MUST FIT WITHIN THEIR AREA. HASKELL NUMBER:20204 AMOUNT RECEIVED COMMENT FOR UNITS USE- SAMPLE WAS RECEIVED 05/12/93 SEF DATE 05/12/93 SUBMITTER TIM BINGMAN LOCATION REO-PIT DEPT. CHEM TEST ARTICLE CAS# SOIL W/LEAD LOT# CODE# N.B. HAZARD CODE 2291 HAZARDS POISON COMMENTS 1 OF 2 *11,199 GRM GROSS, 2 OF 2 =10,774 GRM GROSS ZDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD? 3 USE "PAGE-DOWN" TO FLIP TO NEXT PAGE 3 3 USE "ESC" TO ABORT 3 @DDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDY NumCaps THERE IS SPACE FOR TWO SYNONYMS-EACH MUST FIT ON A LINE SOIL CONTAMINATED W/ 1000 PPM LEAD FROM WASHINGTON STATE 9th Cl NAME ZDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDDD? 3 USE "CTRL-END" TO ACCEPT ALL FIELDS AND EXIT 3 3 USE "ESC" TO ABORT 3 @DDDDDDDDDDDDDDDDDDDDDDDDDDDDDODDDODDDDDDDDDDODDDDDDDDDDDDY DUPONT CENTRAL RESEARCH AND DEVELOPMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE Date: M'/f? TO: STUDY DIRECTOR, // /? / (3US QUARANTINE RELEASE FORM The shipment of animals described below has been examined, meets the health status criteria set forth by the Manager, Animal Resources, and is suitable for study. DATE RECEIVED: l/a-l/n__________________________ s p e c ie s: /^cr* (J/jrATfoJ /%/&>P/6S Bd/e*) & HASKELL ANIMAL NUMBERS: * /7 -<?8 SUPPLIER: fyMies_____PWeiL LMe/sm&ss , we. TO BE USED ON STUDY: . RELEASED FROM QUARANTINE: _______ r/r/?3 CharULeB rT. '-cover, VMD, ACLAM Manager, Animal Resources DUP040000673 CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE ccs Quality Assurance Dates (n -10-^3 TO: . STUDY DIRECTOR, G>W QUARANTINE RELEASE FORM The shipment of animals described below has been examined, meets the health status criteria set forth fay the Consultant, Animal Resources, and is suitable for study. DATE RECEIVED: _________ \3 _______________ . 5pECIES ; 0\ cT1 M\c.HoSu)iAHL D.Onl HASKELL ANIMAL NUMBERS: S~IU ___________ . ' V .... '...... . SUPPLIER: Qjon/? 1^ li.'ug/iL TO BE USED ON STUDY: frtL hln ___ , RELEASED FROM QUARANTINE: <x" O-aI /'v^|-/s c^'t. rot- 3 Ses No'ie book 7Tf/Vj ri^- DUP040000674 CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORA1ORY FOR, TOXICOLOGY AND INDUSTRIAL MEDICINE cc: Quality Assurance Date: U~T-S 3 TO: . STUDY DIRECTOR, BouOoua Co In OL.^ou5 QUARANTINE RELEASE FORM The shipment of animals described below has been examined, meets the health status criteria set forth by the Consultant, Animal Resources, and is suitable for study. DATE RECEIVED: SPECIES: CilL'CT) Lo - I lofi 3 A93 HASKELL ANIMAL NUMBERS : ,<T3 ~7MQS SUPPLIER: C Mcx*L\ TVv-g_ L-o Jq q R. o^foC.i' e3 TO BE USED ON STUDY: RELEASED FROM QUARANTINE: <*hk'3 ji /Mm cfuifeU'Mc- DUP040000675 CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE Date: TO: STUDY DIRECTOR, QUARANTINE RELEASE FORM The shipment of animals described below has been examined, meets the health status criteria set forth by the Consultant, Animal Resources, and is suitable for study* DATE RECEIVED: s pec ies : 90rr> f`d'cWA sUi/s , `///`tffk HASKELL ANIMAL NUMBERS: SUPPLIER: __ flfMteS / fove U&o /Zj &s &l sf. /A'C TO BE USED ON STUDY: RELEASED FROM QUARANTINE: ^ Only o of 9o yiais uuof 7Qi /r*. ai^J suz//?aj#f $. ifO j,#/k uw rtf'.UfitejQ&eJl of yfat "Mm eiLd -fan . Charles E. Cover, V.M.D. Consultant, Animal Resources DUP040000676 CENTRAL RESEARCH AND DEVELOPMENT DEPARTMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE cc: Quality Assurance Date: TO: . STUDY DIRECTOR, Ha-Kian) fiUcuJfrOt/^ QUARANTINE RELEASE FORM The shipment of animals described below has been examined, meets the health status criteria set forth by the Consultant, Animal Resources, and is suitable for study. DATE RECEIVED: SPECIES: ________ V\ /IH LCxSbllfaQ ^ ____________________ HASKELL ANIMAL NUMBERS : 53Ca2>t0l -- 63lo?>SC> SUPPLIER: QlraJlAefi TO BE USED ON STUDY: _________ RELEASED FROM QUARANTINE: __ _______________ _______ Se E l 3 To iil oe-i'c^.fvtoA t'oTZ- ^eWcu-j-e c rO^:'^' v^Oa.Oo.t\oAo\e O^i d>f *2l-eA<?oa^ c Tn O s [o )^3 Q DUP040000677 MELLIAB..BJ5RQRT.S; WRITING AIDS, (EAsaaft r_jLu.m mis sheet sdJtH iHa Unal report.) Please use the names, codes, synonyms and composition supplied below when writing your reports. When pathology or clinical pathology work is scheduled, pass a copy of this form on to the appropriate, persons in those groups. MR-9660-001 SAMPLE # 20204 CHEMICAL SAME USED FOR HASKELL LAB'S CENTRAL FILES; (Use in the 'MATERIAL TESTED" section) Soils, Washington State, contaminated with lead NAME TO BE USED IH THE REPORT; H-20204 OTHER CODES : NA (Note: NA was not def ined by the sponsor) SUBMITTER 1S NOTEBOOK NO. : None submitted SYNONYMS: Soil contaminated with 1000 ppm lead from a site in Washington State Soil contaminated with 1000 ppm lead from Washington State Soil with lead CAS REGISTRY NUMBER: None available PURITY; NA (Note: NA was not defined by the sponsor) COMPOSITION: NA (Note: NA was not defined by the sponsor) CONTAMINANTS: NA (Note: NA was not defined by the sponsor) PREPARED BY: Paul W. Grube DATE: 2 June 1993 DUP040000688 Protocol Amendment No. 2 Absolute Bioavailability of Soil Lead in Rats and Microswine page 1 of 3 TO: MEDICAL RESEARCH PROJECT NO. 9660-001 The following changes are made to the original protocol: page 2 STUDY DESIGN _ A. Single Dose Administration first and third bullets Change '...and ten male microswine...' to '...and nine male microswine...' in the first bulleted item. Change '...and five microswine will be dosed orally (gavage)...' to '...and four microswine will be dosed orally (gavage)...' in the third bulleted item. One microswine became ill and was sacrificed in extremis. page 4 MATERIALS AND METHODS A. Test Material paragraph 1, sentence 1 Replace the first sentence with: 'Lead acetate will be supplied by Aldrich,, Milwaukee, WI 53733, U.S.A. and the test soils will be supplied by the sponsor.' This sentence was written incorrectly. It was always the intent to receive the lead acetate from Aldrich and the test soils from the sponsor. page 7 MATERIALS AND METHODS H. Food Consumption and Intake of Test Material paragraph 1, sentence 1 Change '...food consumed by each microswine will be determined twice per week...' to '...food consumed by each microswine will be determined weekly...' For easier data comparison, microswine and rats were maintained on the same schedule for food consumption determination. DUP040000689 Protocol Amendment No. 2 Absolute Bioavailability of Soil Lead in Rats and Microswine page 2 of 3 page 7 MATERIALS AND METHODS I. Dosing paragraph 1 Add the following sentences; 'One microswine designated ______ for intravenous administration and one microswine designated for oral (gavage) administration will be fed one hour before dosing. Data from these animals will be reported separately.' The microswine ate all of their food immediately, while the rats tended to have food remaining in their food jars up to the time of dosing. Therefore, microswine were considered 'fasted' while the rats were considered 'fed.' To evaluate if being 'fed' made a difference, one microswine from each type of single dose administration was fed one hour before dosing. page 8 MATERIALS AND METHODS J. Blood Sampling paragraph 1 Insert the following sentence after die original first sentence: 'For microswine in the single dose administration group, syringes will be washed with heparin before blood collection.1 The blood collected from microswine that received a single dose after feeding for 30 consecutive days was clotting. The heparin wash was added to prevent this from occurring in the single dose administration group. DUP040000690 Protocol Amendment No. 2 Absolute Bioavailability of Soil Lead in Rats and Microswine page 3 of 3 page 9 RECORDS AND SAMPLE RETENTION paragraph 1, sentence 1 Change '...will be retained at Haskell Laboratory or at the Records Management Center,...' to '...will be retained at Haskell Laboratory, Jackson Laboratory (DuPont Chambers Works), or at the Records Management Center,...' Records and samples from the analyses for lead will be maintained at Jackson Laboratory. Approved by: Study Director Manager Biochemical Toxicology and Risk Analysis cc: T.S. Bingman K.A. McCleary R.C. Rhea DUP040000691 Memorandum To: From: Date: Subject: Cc: Hanan Ghantous, H-l Keith A, McCleary, Jackson Lab December 8,1993 Final Report, Lead in Blood Analysis C. J. Hensler, Jackson Lab M. S. Bogdanfiy, Haskell Toxicology R. C. Rhea, Haskell Quality Assurance R. L. Sohocinski, QCL Enclosed please find three copies ofthe final report for the analysis ofPb in blood as conducted by the Du Pont Specialty Chemicals' Trace Metals Analysis Group as part of Medical Research Project No, MR 9660: "Absolute Bioavailability of Soil Lead in Rats and Microswine". A copy of this report is to be kept with those materials relating to the analyses performed (SOP's, methods, and copies of sample logs) under GLP guidelines here at Jackson Lab. Ifyou have any questions, please feel free to call me (Ducom 540-2467). DUP040000692 Analysis ofPb in Blood, Soil, and Associated Materials Support ofHaskell Lab Medical Research Project No. MR 9660 Author Keith A. McCleary Study Completed: 8/30/93 Performed By: DuPont Specialty Chemicals Trace Metals Analysis Group Quality Control Laboratory Chambers Works Deepwater, NJ 08023 Signature: Keith A. McCleary, Ph.D. l`T3 DUP040000693 Summary The purpose of this study was to determine the amount of lead that was contained in samples associated with MR9660. There were essentially three type of samples: blood (rat and microswine), aqueous (drinking water, dosing solutions), and solid (soil, bedding, etc.). Blood samples were prepared by dilution with a modifier solution (to digest sample and break viscosity) and analyzed using Graphite Furnace Atomic Absorption Spectrometry (GFAAS). Aqueous and solid samples were prepared using microwave assisted digestion and analyzed using Inductively Coupled Plasma Atomic Emission Spectometry (ICP-AES). Generally, all samples were run in duplicate and average answers reported. In the case ofthe blood, each sample and duplicate was spiked and checked for recovery. Ifthe spike recovery was greater than 10% in error, a one point standard additions calculation was performed. Greater than 1000 samples were analyzed throughout the duration ofthis study. Methodology The descriptions listed below are summaries of the actual methods used to analyze samples. The actual methods are documented in the Procedures Notebook, archived at QCL and copies are available upon request. Sample Preparation: Blood Blood samples were received in small polyethylene vails. Approximate sample volume was 100-300 uL. 100 uL of blood is diluted to 1.0 mL with modifier solution. The modifier solution is a solution ofnitric acid (digests sample), Triton X-100 (surfactant, breaks viscosity), methanol (antifoaming agent), and ammonium phosphate (GFAAS modifier) in water. Sample Analysis: Blood Blood samples are analyzed using Graphite Furnace Atomic Absorption Spectrometry (GFAAS). In GFAAS, a 20 microliter volume of sample + modifier is injected into a small graphite tube. This tube is electromagnetically heated to dry, char, and atomize the sample. A hollow cathode lamp (HCL) shines light corresponding to the atomic emission ofPb (283.3 nm) through the tube. As free Pb atoms are generated by the sample atomization, they absorb the radiation, attenuating the amount of signal from the HCL. The degree of attenuation ofthe HCL radiation is proportional to the concentration ofPb in the sample. Calibration standards of 10, 20 ,and 30 ng/mL are prepared by serial dilution of a 1000 ug/mL stock. Each blood sample is analyzed twice. Each sample and duplicate is then spiked with lOng Pb and reanalyzed to check for recovery. Ifrecovery is less than 90% or greater than 110%, a one point standard addition calculation is DUP040000694 performed to determine the amount of Pb in the sample. Each result is the average ofthe two replicates. Sample Preparation: Aqueous Samples Samples that are primarily aqueous are prepared by acidifying 10.00 mL of sample with 2.00 mL concentrated HN03. The sample is then diluted to a final volume of20.00 mL and analyzed by ICP-AES. Sample Preparation: Solid Samples Solid Samples are prepared by microwave assisted digestion. 0.1 - 0.2 g of sample are digested with 5.00 mL concentrated HN03 in a Teflon lined pressure vessel. The vessels are placed in a microwave digestion apparatus and cooked for 10 minutes at 300@W power. The samples are allowed to cool and are then filtered into a 50 mL volumetric flask and diluted to volume with deionized water. Samples are analyzed for Pb content by ICP-AES. Sample Analysis : Aqueous and Solid Samples These samples are analyzed by Inductively Coupled Plasma - Atomic Emission Spectrometry (ICP-AES). The prepared samples are pumped into an sample introduction system where a fine spray of aerosol is generated. This sample aerosol flows along a stream of argon gas into the plasma. The plasma is a highly energetic discharge sustained in argon by applied radio-frequency power in excess of 1000 W. This discharge is energetic enough to desolvate, atomize, and excite the sample species to radiative emission. The intensity of the characteristic emission wavelength ofPb (220.35 nm) is proportional to the amount ofPb in the sample. Results The results for all samples submitted under MR9660 are listed immediately following this page. DUP040000695 DUP040000696 Sample la-101 la-102 la-103 a-104 |a-105 |a-106 la-107 a-108 a-109 la-111 |a-112 Lead <5 <5 <5 <5 44 10 50 CO CO 03 o CO r-- T~" r* r* r-- r T" 1I <0 CD VO VO fa-116 | a-116 la-118 la-119 15 12 10 <0 CN 10 O r- CN CM i cb VO cbvs la-122 |a-125 CN CM CO <5 <5 o c o CD CN oCO CO t" CN 00 i ii f>- o oCM >" t"* CO r-* CO c>i CN TCNt < CO 10 * CN C/5 O >< C0O3 OCL c 03 "iCQ Q& oO L'L 8 '0 -O U Sample 1 0 2 -0 .5 1 0 6 -0 .5 1 1 6 -0 .5 1 2 2 -0 .5 1 0 8 -1 1 0 9 -1 1 1 2 -1 1 2 3 -1 1 2 4 -1 1 0 1 -2 1 0 7 -2 1 1 1 -2 1 1 5 -2 1 0 5 -4 1 1 3 -4 1 1 4 -4 1 2 1 -4 1 0 3 -8 1 1 8 -8 1 1 9 -8 1 2 5 -8 Lead Ug/mL 3 9 .8 ! 6 8 .9 5 4 .6 4 9 .2 1 8 .6 2 4 .6 6 0 ,5 2 2 .8 3 4 .1 1 5 .1 1 9 .1 1 6 .6 2 .6 2 0 .3 4 .5 6 .5 T* CO T"* CO r- co CO CO CO; d Sample 104-12 107-12 111-12 115-12 Lead ug/mL 6.2 8.5 0.8 1.97 A 108-24 A109-24 A ll 2-24 A 123-24 6.4 6.6 5.2 A 1 0 3 -4 8 A 1 1 7 -4 8 A ll 8 -4 8 A 1 1 9 -4 8 A 1 2 5 -4 6 i 1 A 1 0 5 -7 2 [A ll 3 -7 2 A ll 4 -7 2 A 1 2 0 -7 2 A 1 2 1 -7 2 A 1 0 2 -9 6 A 1 0 6 -9 6 A 1 1 0 -9 6 A ll 6 -9 6 A 1 2 2 -9 6 0.3 10.3 13.2 4.1 0.4 3.8 1.6 0.5 2.4 Oo CM CN T" r* 1 CO ** o CN r* << A 1 0 9 -1 2 0 A ll 2 -1 2 0 A 1 2 3 -1 2 0 12/8/93 Page 1 of 11 0.9 0.7 CN: r-- CO r--. CM r- CO d .t-- Tmm CO r-- CO <: < CN 03 f COi oo << Sample A101-144 Le 1ug A107-144 A ll 1-144 A ll 3-144 A ll 7-192 A 118-192 A 119-192 A 1 25-192 A105-240 A ll 3-240 A 114-240 |A120-240 A121-240 STABILITY A105-240S A113-240s Co-<fN0r A120-240s A 1 21-240s REPEATS A 107-12 A ll 7-48 A l l 8-48 1 i 4 r-- r-v CM CN so CN A 1 21-240(6/7) Sample Pb, ng/mL |B201-PRE 1B202-PRE B203-PRE IB204-PRE 1B205-PRE B206-PRE B207-PRE ! IB208-PRE ! IB209-PRE B210-PRE B211-PRE | IB212-PRE IB213-PRE CT"D CO CM 34 105 26 32 42 23 QUof1cr.l T-- CCMQ CQMQ B215-PRE IB216-PRE IB217-PRE 20 26 1X3 y-- CN LU CC r 00 CO [B219-PRE IB220-PRE jB221-PRE IB222-PRE [B223-PRE IB224-PRE fB225-PRE <5 <5 <5 *1* CM <r~o <o CO CO CM CO CM T-- CN CQQN CN CQ CCMQ CN CQ 89 oe ZP 69 Comprehensive Blood Report PB_BL_CP.XLS Sample Pb, ng/mL Sample Pb, ng/mL B2 21-0.5 B220-0.5 B214-0.5 B213-0.5 B205-0.5 LO CM 24 o? CO CN B202-24 B206-24 B210-24 B216-24 B222-24 1 125 55 30 B216-1 B202-1 B222-1 B206-1 22 B209-48 23 B208-48 59 B223-48 B224-48 39 n p r-- 03 U3 CO o 03 CO CM CM r~ 00 CM t P,Si CN CN 03 6 *o.*1 1i0 Nrr-.1-t"1 ' B208-2 B224-2 B209-2 B212-2 B223-2 CO 10 in CO p* 59 t-- B207-4 B201-4 ! B203-8 8217-8 B218-8 B219-8 B225-8 I 197 1 155 290 320 530 250 230 r* CO * -- CM o CN r-1 rCN 03 CmN r* o B204-72 B207-72 B211-72 B215-72 B203-96 B217-96 B218-96 B219-96 B225-96 B205-120 B213-120 B220-120 B221-120 CO o CO 1X3 <o 36 15 82 336! 74 22 32 17 CQ CQ CQ rx 1 0CM) rt o cn CQ CONQ CN CM 1 V*1 OCN 0 j | | Sample P -- B202-144 <oT1* o B210-144 B216-144 B222-144 CN CN CNi CN CN 05 o B204-192 B207-192 B211-192 B215-192 B208-240 B223-240 *o* o < 0Tf CN1 CM CM Q. oC OO' .1 ir~ 11-1-7400pre 11-1-7401 pre 11-1-7402pre 11-1-7404pre < < < 111-7400-12 ! 11-1-7401-12 ri>"i 11-1-7403-12 -- -- DUP040000697 12/8/93 Page 2 of 11 26 001 99 S9 85 08 [801 66 96 .C0O0 CM i0 jSample Pb, ng/mL Sample Comprehensive Blood Report PB_BL_CP.XLS Pb, ng/mL Sample Pb, ng/mL Sample j Pb, ng/mL Sample 1005 CCOO <100 00 05 05 10 CO 10 CoCOO a |C301 |C303 |C304 C305 IC306 |C307 |C309 |C310 ! IC313 [C314 |C315 |C316 ! 10 r- C303 5/27 C304 5/27 C305 5/27 307 5/27 C308 5/27 C310 5/27 C311 5/27 C312 5/27 C313 5/27 | 314 5/27 34 315 5/27 C316 5/27 C317 5/27 0 CO 10 CO a CO 0CO5 C301 6/3 237 C303 6/3 132 C304 6/3 C305 6/3 C306 6/3 77 307 6/3 90 C308 6/3 C309 6/3 C310 6/3 124 C311 6/3 312 6/3 C313 6/3 70 C314 6/3 62 316 6/3 317 6/3 1 | | co o C301 6/10 122 C303 6/10 83!|C304 6/10 94 305 6/10 C306 6/10 112 307 6/10 175! C308 6/10 195 C309 6710 122 C310 6/10 92 26 C312 6/10 46 C313 6/10 77 314 6/10 54 315 6/10 (316 6/10 C317 6/10 r- CO o 10 CO ian5 CO 10 CO 54 C301 6/17 C303 6/17 304 6/17 C305 6/17 |C306 6/17 IC307 6/17 |C308 6/17 64 C309 6/17 70 [310 6/17 311 6/17 128 C312 6/17 56 313 6/17 44 |C314 6/17 C315 6/17 38 316 6/17 56 317 6/17 1 10 10 10 CTM**; r*' CM CO 'TC"O CCMO <CNO CCMO O aoa |C319 |C320 |C321 |C325 CO CO 10 CO 319 5/27 320 5/27 321 5/27 C322 5/27 1 323 5/27 < 2 IC324 5/27 24 C325 5/27 10 <0 103 319 6/3 320 6/3 121 C321 6/3 . 59. C322 6/3 132 323 6/3 324 6/3 64 325 6/3 1 63 C319 6/10 89 C320 6/10 321 6/10 48 C322 6/10 36 323 6/10 38 C324 6/10 325 6/10 CO <0 CM 'CrO1 0150 0r~5 CCOJ 64 56 320 6/17 C321 6/17 322 6/17 323 6/17 324 6/17 78 325 6/17 <5 rP*N --1 oci --- -ad. 100 106 95 79 66 98 57 77 82 69 66 87 53 37 69 lO Ct --O ..... DUP040000698 12/8/93 Page 3 of 11 cDo mm* & mJk 03o O > 0) Xl 03O cno o CO in 00 Sample [C321 0.5 JC32Q 0.5 C314 0.5 |C313 0.5 |C305 0.5 IC316 1.0 |C322 1.0 |C310 1.0 10308 2.0 IC324 2.0 IC309 2.0 IC312 2.0 |C323 2.0 |C304 4.0 |C307 4.0 IC311 4.0 |C315 4.0 1C303-8 IC317-8 IC319-8 IC325-8 C305-12 C313-12 IC320-12 IC321-12 14.2 19.4 32.5 15.1 20.7 18.3 17.5 4.3 12,2 9.7 6.6 12.2 4.4 4.6 3.2 3.9 4.5| 5.3 5.8 4.2 6.8 "P0" CcQ r3~ CO GMOo OnO Mk 80 I'O \V 0 Sample Comprehensive Blood Report PB BL CP.XLS Pb,ug/mL Sample Pb,ug/mL 4* co ISO CO 1o (SO C306-24 C310-24 C316-24 C322-24 i C308-48 C309-48 C323-48 C324-48 C301-72 C304-72 C307-72 C311-72 C315-72 ;h 2.1 0.7 2.2 1.2 7.9 6.9 2,8 0.7 SJ CO b CO a GO O CO U1 C301-192 1 C304-192 C307-192 C311-192 C308-240 C312-240 C323-240 C324-240 N) fO* CD N3 o .1 0.74 0,53 0.43 0.45 0.28 0.64 0.37 0.58 1.72 oO C303-96 C317-96 C319-96 C325-96 0.9 CO a> C305-120 C313-120 C314-120 C321-120 mJkO 0.6 0.5 0.7 C306-144 C310-144 C316-144 C322-144 0.7 0,6 1.2 0.5 12/8/93 Page 4 o f 11 mmX o 0) 1O Sample Pb, ug/mL (fiat Gavage DOSING SOLUTION (GROUP B 311.9 (GROUP B 310.9 |DEIONIZED WATER <5 (1000 PPM Pb SOIL 840.9 SUCROSE <5 to>o fi) (Swine IV Single Dose Samp) 116.6mg/g 126.8mg/g 1swine gavage 17/16/93 30 Day .425 mg/g 11a 0, ,348 mg/g P> CO CD fl> Comprehensive Blood Report PB BL CP.XLS Sample Pb, ug/mL SUCROSE DIET SAMPLE <5 ! Sample Pb, mg/kg #1 top of mixer 5/17/93 & 4* 4* o CD 03 to O cr CD AA or OREO COOKIES <5 TEKLAD BASAL SWINE < 5 PURINA RAT CHOW <5 1 SWINE BEDDING Di H20 Pb ACETATE (RAT IV SOLN) 1.27% (400mg/20mL) Swine IV 30 Day Solution 118.5 mg/g Swine single gavage 8/93 0.339mg/g 0.347mg/g fi) O) a aat 4* fi> CO 03 fti)o fi) CnD 03a >4 03 G) at 4* O U" CD #1 middle of mixer 5/17/93 42 ..... 47 #1 bottom mixer 5/17/93 37 38 top mixer 6/17/93 43.6 44 middle mixer 6/17/93 42.3 45.7 43.1 bottom mixer 6/17/93 45.6 -P- p b> go OJ 4* OJ Cl 03 Q) fiC)D 44.51 12/8/93 Page 5 of 11 0077 00 <o ccnn L9 81 89 180 U 66 08 601 69 98 Comprehensive Blood Report PR BL CP.XLS Sample Pb, ng/mL Sample Pb, ng/mL Sample Pb, ng/mL Sample Pb, ng/mL Sample Pb, ng/mL NJVoo m3 A <71 CD 00 CO C401-PRE C403-PRE C404-PRE jC405-PRE |C406-PRE (C407-PRE IC408-PRE C409-PRE (C410-PRE C411-PRE [C412-PRE (C413-PRE <5 <5 <5 <5 <5 <5 <5 <5 T3mO0 "3m00 cn V 4Ml*h o O -t00um00 IC416-PRE IC417-PRE (C419-PRE C420-PRE IC421-PRE [C422PRE IC423-PRE IC424-PRE IC.425-PRE <5 <5 <5 <5 <5 <5 A <71 A Ol <71 cn CO >4 --Pi*o 4!-->*o 4o* ro 4* 0) 0) --* >g D401 6/17 0402 6/17 0403 6/17 D404 6/17 I 0405 6/17 0406 6/17 0407 6/17 0408 6/17 0409 6/17 0410 6/17 0411 6/17 0412 6/17 0413 6/17 0415 6/17 0418 6/17 0419 6/17 0420 6/17 0421 6/17 0422 6/17 0423 6/17 0424 6/17 0425 6/17 CJ1 sj o> <<7711 --Jt 00 07 t sio 44*. <71 CD --k 03 -*4 103 0401 6/24 j 102 D402 6/24 147 0403 6/24 95 0404 6/24 0405 6/24 | 0406 6/24 1 145 0407 6/24 83 0408 6/24 135 D409 6/24 0410 6/24 0411 6/24 0412 6/24 67 0413 6/24 67 0414 6/24 0415 6/24 0416 6/24 123 0417 6/24 0418 6/24 15 0419 6/24 0420 6/24 0421 6/24 0422 6/24 0423 6/24 22 D424 6/24 D425 6/24 0N33 03 00 V 00 <>JslI <N731 *034 w D O CD Sj CCOO Nj CO 0401 7/1 86 0402 7/1 0403 7/1 54 10404 7/1 56 0405 7/1 124 0407 7/1 D4Q8 7/1 117 D409 7/1 101 0410 7/1 0411 7/1 32 0412 7/1 0413 7/1 37 0414 7/1 0415 7/1 D416 7/1 0417 7/1 56 0418 7/1 27 0419 7/1 37 0420 7/1 [0421 7/1 24 D422 7/1 101 0423 7/1 0424 7/1 D425 7/1 | ! [' I 0sj) <>741 <c7n1 m ImDAV N7 *4 00 120 0401 7/8 D402 7/8 89 0403 7/8 0404 7/8 120 0405 7/8 126 0406 7/8 109 0407 7/8 64 0408 7/8 157 D409 7/8 D410 7/8 96 0411 7/8 i 50 36 0413 7/8 | 53 0414 7/8 34 0415 7/8 129 D416 7/8 D4177/8 j 45 D418 7/8 "4 5 D419 7/8 | 105 D420 7/8 96 D421 7/8 1 D422 7/8 82 D423 7/8 I 135 0424 7/8 0425 7/8 4* 03 03 o 88 76 155 62 75 66 63 52 60 59 124 52 * 4 CO CCOO v 12/8/93 Page 6 of 11 Pb.no/mL Sample jpr CO3 :rf* Comprehensive Blood Report PB BL CP.XLS Sample Tp3CO3 rVol ,r0o> cCnO ro r3po p A ro CD mm*- 4N>-3 0033 K43* CND3 pp CO 09 4* CCDD O3 10405-0.5 |D413-0.5 |D414-0.5 10420-0.5 D421-0.5 4sj^ po c4n* op 0405-12 D414-12 69 D420-12 D421-12 ro 101 D405-120 < 5 D413-120 <5 D420-120 <5 0421-120 78 38 09 O il 09 68 10402-1.0 ID406-1.0 |D410-1.0 |D416-1.0 ID422-1.0 CNJO 00 0402-24 D406-24 115| D410-24 79 0416-24 D422-24 j CO .ISO a .i--p*Op D401-144 102 123 0407-144 i 0411-144 D415-144 64 cn N3 CP ID408-2 10409-2 ID412-2 1D423-2 D424-2 <n KO)3 o60> 84 0408-48 53 D409-48 D412-48 0423-48 D424-48 1 | CD 0OJ0 CoO> NSp0o 1 Ml D406-192 D410-192 103 D416-192 469 D422-192 | 39 rcno OP O 98 861 D404-4 ID407-4 ID411-4 10415-4 | ibbA .mm*' P pD 'Ml ro 120 D401-72 0404-72 D407-72 109 65 D415-72 1 CO D408-240 14 D409-240 74 D412-240 25 D423-240 0424-240 38 167 65 45 cn f0O0 pp N> COo cAn CJ P cp 1D403-8 D417-8 D418-8 D425-8 124 0403-96 D417-96 160 0418-96 D 4 19-96 133 D425-96 <5 <5 12/8/93 Page 7 of 11 Comprehensive Blood Report PB BL CP.XLS C0fl3 *03 CD Pb, ng/mL Sample Pb, ng/mL Sample Pb, ug/mL Sample Pb, ug/mL Sample Pb, ng/mL S ZVO z:ro 880 810 canO r37n a at 3Tm77 |G502-PRE(a) G503-PRE|a) G505-PRE(a) |G506-PRE(a| SJ 00 ISOP 00 30 G501 7/7 G502 7/7 G503 7/7 34 G504 7/7 G505 7/7 G506 7/7 139' G501-0.5 152 G502-0.5 225 G503-0.5 135 G504-0.5 173 G505-0.5 195 G506-0.5 1 ! j Ni 0o0o 0N>3 7.35 G501-8 1.4 G502-8 2.731 G503-8 0.99 [G504-8 G505-8 G506-8 O' p CO -p CO 0.31 G501-72 G502-72 G503-72 G504-72 G505-72 1.12 G506-72 p. COo NJO3 69 122 G 240 G G G 376 G ccnnOo cPOnO- cano g> 0> 03 03 o O) crio CJol 03 03 O Ual en cno CJlO cn foo> IG502 6/16 IG503 6/16 |G506 6/16 16501 6/23 IG502 6/23 IG5Q3 6/23 IG504 6/23 |G506 6/23 |G501 6/30 IG502 6/30 IG503 6/30 |G504 6/30 IG506 6/30 _a> o> o Q aot 3Ta33t rn S 129 G501 PRE(b) 257 323 duplicate 150 G5Q3 PRE(b) 195 G504 PRE(b) G506 PRE(b) 100 190 150 50 140 100 258 347j 266 155 147 193 r0oo01 "303m $ 06 i mmk cn 203 G501-1 201 G502-1 185 G503-1 194 G504-1 G505-1 G506-1 143 G501-2 G502-2 G5Q3-2 G504-2 G505-2 G506-2 G501-4 G502-4 G503-4 G504-4 G506-4 | |i_ I 1 3.04 G501-12 G502-12 0.98 G503-12 0.31 G504-12 2.33 G505-12 103' G506-12 1.17 G501-24 0.32 G502-24 G503-24 0.28 G504-24 1.34 G505-24 3.19 G506-24 1i 0.76 G501-48 0.39 G502-48 0.37 G503-48 0.29 G504-48 1.22 G505-48 1.93 I 4 fCJlo uoi 10of0k0> cn l 1s0> p CaO> 0.27 G501-96 G502-96 G503-96 0.19 G504-96 0.31| G505-96 1.95 G506-96 0.24 G501-120 | 0.22 G502-120 0.24 G503-120 0.11 G504-120 1 I 0.24 G505-120 0.82| G506-120 0.361 G501-144 | 0.25 L 0.65 G503-144 0.07 G504-144 0.16 G5Q5-144 0.52 G506-144 ,03 fO (NNJ133 03 >Ns3l C0JlO3 G 227 G G 110 225 G 554 G 239 R 147 G 112 193 G 438 G G iG _ 57 G 206 G 115 369 C 03 03 |(a) denotes predose prior to weekly testing (b) denotes predose prior to hourly testing G G G G G 12/8/93 Page 8 of 11 fO CsjO 4* O) CO sj 09l III 36 681 691 Comprehensive Blood Report PB BL CP.XLS Sample Pb, ng/mL Sample Pb, ng/mL Sample Pb, ng/mL Sample Pb, ng/mL Sample Pb, ng/mL 1 I cn Sj X j1N5o0 mX -os>iX 3X0I m IH701-PRE H702-PRE H703-PRE IH704-PRE IH705-PRE SJ o H701-PRE 23 H703-PRE 57 H704-PRE H705-PRE H706-PRE j sJX X H701-4 119 145 H703-4 168 224 H705-4 255 H706-4 O0 .1f* p1o -p- CO nJ sJX 1CJ)0 00 Vj ,>cdo 153 H701-48 142 H702-48 H703-48 366 H704-48 275 H705-48 304 263 H701-144 | 302 H702-144 200 H703-144 i H704-144 164 H705-144 H706-144 128 163 132 SJX p.o CN3O 00 Q |H701 6/23 |H702 6/23 H703 6/23 IH705 6/23 |H706 6/23 1 190 H701-0.5 130 H702-0.5 H703-0.5 170 H704-0.5 180 H705-0.5 290 H706-0.5 csJXno .100 255 H701-8 177 H702-8 180 H703-8 200 H704-8 130 203 H706-8 I 340 H701-72 268 H702-72 322 H703-72 268 H704-72 160 H705-72 133 H706-72 159 H701-192 224 H702-192 114 H703-192 204 H704-192 181 H705-192 ! 254 mmk o 127 130 157 --i CCOO o >OX4O rCOo H701 6/30 |H702 6/30 |H703 6/30 IH704 6/30 H705 6/30 H706 6/30 cSjOnX 206 H701-1 206 H702-1 217 H703-1 261 H704-1 258 201 H706-1 ICCSOOO siX 1N>0 aii H701-12 112 282 H703-12 210 H704-12 190 H705-12 242 H706-12 ls> 267 H701-96 316 H702-96 234 H703-96 187 H704-96 255 H705-96 143 H706-96 ! 172 H701-240 H702-240 H703-240 176 H704-240 H7Q5-240 168 H706-240 118 mmXN3 sJX o H701 7/7 |H702 7/7 IH703 7/7 |H704 7/7 IH705 7/7 IH706 7/7 193 169 H702-2 137 H703-2 209 H704-2 227 H705-2 173 H706-2 222 H701-24 207 H702-24 142 H703-24 170 H704-24 258 H705-24 347 H706-24 318 H701-120 122 220 H702-120 146 o> 216 H703-120 224 H704-120 See Note 203 H705-120 183 CO H706-12Q | 181 nl C0 OO 12/8/93 Page 9 of 11 Tlo 00 03 Sample F801 PRE |F802 PRE |F803 PRE IF804 PRE 30 m Pb, ng/mL (Sample F801-12 F802-12 <5 F803-12 <5 F804-12 <5 F805-12 00 00 Tl Tl CO 000 O CO 03 CO CO vl 03 *4 -p>. TJ j F801 0.5 F802 0.5 |F803 0.5 [F804 0.5 |F805 0.5 ! <5 <5 F801-24 !F802-24 F803-24 F804-24 F805-24 Tl-0n CO CD 03 mJOk 1 IF802-1 IF803-1 IF804-1 ' N3 N5 19 F802-48 F803-48 F805-48 1 |F8Q1-2 IF802-2 IF803-2 1F804-2 IF805-2 CO F801-72 10 F802-72 F803-72 59 F804-72 28 F8Q5-72 03 03 Tl "TJ0 00 00 O CeJ1 1 IF8Q2-4 IF803-4 IF804-4 *n -n 00 o 0o0 F801-96 49 85 F803-96 57 F804-96 52 fO CO <b IF801-8 IF802-8 F803-8 IF8Q4-8 1F805-8 79 F801-120 78 F802-120 65 F803-120 F804-120 74 F805-120 CJ1 "a-0 3 CO If"* 86 98 zz 96 LZ Comprehensive Blood Report PB BL CP.XLS -j cn Sample 133 F801-144 73 F802-144 118 F803-144 96 F804-144 115 F805-144 74 F801-192 F802-192 F803-192 74 F804-192 105 F805-192 Pb, ng/mL Repeat 206 F803-48 162| F801-144 147 F802-144 727 F803-144 F804-144 F805-144 Pb, ng/mL ( 65.1 14.4 21.2 9.9 21.9 38.5 N3 00 COw 03 23 33 25 a CO 00 >j KS <0 CJ1 CO 03 w4 F801-240 F802-240 229 F803-240 F804-240 F805-240 22 13 rTOo N> O) 4* O f* 4> 23 36 N3 O O CJ1 CO 63 00 55 30 12/8/93 Page 10 of 11 Comprehensive Blood Report PB BL CP.XLS TO3" C5 r3_ N3 CO ho O0" C CD .MM*. 3" b r" p GO ho o o CO Sample Pb, ng/mL Sample mm* A 03 4n 03 03 E601-PRE E601-4 31m 33m 0m5 rTJoo 27 E602-4 | E603-PRE 603-4 | E604-PRE E605-PRE | 10003m3 TO E606-4 rn -i0c*10 03mo 4* Sample j 9.8 601 -48 E602-48 | 6.3 E603-48 4.1 !E604-48 clotted E605-48 i ' 5.3 E606-48 1 CN5O 0m3 fO. 00 b> 0m3 0m5 1003 0 CJl [Sample (E601-0.5 (E602-0.5 IE603-0.5 [E604-Q.5 IE601-1 E602-1 IE603-1 IE604-1 IE605-1 IE606-1 9 CJl p 01 IN) mJk k> GO 0O3l Pb, ug/mL E601-8 [ 22.3 E602-8 1.85 E603-8 2.07 E605-8 0.41 E606-8 ! 2.02 E601-12 12.6 E602-12 1,14 E603-12 E604-12 E605-12 0.3 E606-12 1.28 m o>0 Mk I 1S5 E601-2 IE6Q2-2 |E603 2 1E604-2 | E605-2 E606-2 11.2 E602-24 0.53 E603-24 0.29 E604-24 1.16 E605-24 E606-24 0.47 ! I E601-72 0.27 602-72 0.47 E603-72 0.39 E604-72 0.24 E605-72 0.34 606-72 | 1 0.91 E601-96 0.31 E602-96 0.37 E603-96 0.47 0.26 E605-96 606-96 0m5 o C03O E601-12Q 0.45 E602-120 0.35 [E603-120 0.42 E604-120 E605-120 0.2 E606-120 U 'O 990 seo 810 j O Nw5 Sample Pb, ng/mL REPEATS Pb, ug/mL 0.39 E601-144 i E601-0.5 i 22.35 0.23 IE602-144 267 E601-1 21.51 *13 fr c: <o 2~3r r~ ^4 0.32! 603-144 136 E601-2 0.25 E604-144 nV I i1qW 1 165 REPEATS 00 CO 00 CO K) m rn O) 03 OO CJl Mi CO 4* N5 4^ 600 E606-144 101 601-4 2.6 E602-4 1 0.3 0.44 148 E603-4 0-4 1.98 E602-192 E604-4 0.46 0.48 [E603-192 E605-4 0.21 Q.32 E604-192 102 E606-4 0.431 p E605-192 32 E606-192 106 961 0.27 E601-240 | 0.37 E602-240 fE6Q3-240 0.23 E604-240 0.02 E605-240 0.18 E606-240 O) CO 05 86 109 125| 0.34 0.19 0.14 0.14 0.05 12/8/93 Page 11 o f 11 1 ABSOLUTE BIOAVAILABILITY OF SOIL LEAP IN RATS AND MICROSWINE MEDICAL RESEARCH PROJECT NO, 9660 PROTOCOL j Stine-Haskell Animal Welfare Committee Mo. CBT51-P DUP040000707 -2- ABSOLUTE BIOAVAILABILITY OF SOIL LEAD IN RATS AND MICROSWINE MEDICAL RESEARCH PROJECT NO, 9660 OBJECTIVE The objective of this study is to determine the absolute bioavailability of lead from soil in rats and 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, Bellevue Corporate Center, Wilmington, DE 19809. The study will be conducted at Haskell Laboratory for Toxicology and Industrial Medicine, E. 1. du Pont de Nemours and Company, Newark, Delaware, in accordance with all applicable Good Laboratory Practices. The Sponsor's approval was effective the date the sponsor signed the Medical Research (MR) project. STUDY DESIGN A. Single Dose Administration o Fifty male rats and ten male microswine will be used. o A predosing blood sample will be drawn from each animal for levels of background lead. o Twenty-five rats and five microswine will be dosed orally (gavage) with test soil containing 1,000 ppm lead (approximately 1 g soil/rat and 17 g soil/microswine). o Twenty-five rats and five microswine will be given a single intravenous injection of lead acetate solution containing lead-dose equivalent to that contained in the gavage dose. DUP040000708 -3 - Single Dose Administration (continued) o Approximately 0.1-0,3 ml of blood will be drawn from the rats (each rat will be used to collect three blood samples only) and 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. 0 Blood samples will be collected in lead-free tubes containing EDTA and stored at 4C until analysis, 0 At sacrifice, liver and bone (both cortical and trabecular as well as whole femur) will be collected* weighed, and frozen at -20C. B. Single Dose Administration After Feeding For 30 Consecutive Days o Fifty male rats and ten male microswine will be used. o A predosing blood sample will be drawn from each animal for levels of background lead, followed by one sample/week for the duration of 30 days. o All animals will be provided with optimized diet of approximately 21.5 g/rat/day or 340 g/microswine/day of a special purified diet mixed with test soil containing 1,000 ppm lead, 5% w/w, for 30 days. o After 30 days, blood samples will be drawn from each animal for levels of background lead and the animals will be divided into two groups. o Twenty-five rats and five microswine will be dosed orally (gavage) with test soil containing 1,000 ppm lead (approximately 1 g soil/rat and 17 g soil/microswine). o Twenty-five rats and five microswine will be given a single intravenous injection of lead acetate solution containing lead-dose equivalent to that contained in the gavage dose, o Approximately 0.1-0.3 ml of blood will be drawn from the rats (each rat will be used to collect three blood samples only) and approximately 0.5 ml of blood will be drawn from microswine at 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 192, and 240 hours after dosing, o Blood samples will be collected in lead-free tubes containing EDTA and stored at 4C until analysis. o At sacrifice, liver, kidney, brain, spleen, and bone (both cortical and trabecular as well as whole femur) will be collected, weighed, and frozen at -20C. DUP040000709 -4- MATERIALS AMP METHODS A. Test Material The test materials will be supplied by Aldrich, Milwaukee, WI 53733, U.S.A. The lead acetate trihydrate will be assigned Haskell Laboratory No. 20202. The lead/soil will be assigned Haskell Laboratory No* 20204. Characterization and mineralogy data of the test soils will be maintained by the sponsor. B. Test Species Male Crl:CDBR rats, approximately 21 days of age, with body weights in the range of approximately 40 to 65 grams will be acquired from Charles River Laboratories, Inc., Kingston, New York, The Crl:CDBR rat has been selected on the bases of extensive experience with this strain and its suitability with respect to hardiness, longevity, sensitivity, and low incidence of spontaneous disease. 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., Wilmington, Massachusetts, 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 Rats and microswine will be housed according to the Guide for Care and Ilse of Laboratory Animals. (NIH Publications No. 86-23, Revised 1985) The quarantine period will be approximately one week, during which rats will be fed optimized diet (once per day) of irradiated Purina Certified DUP040000710 5 Rodent Chow (PCRC) #5002 meal and transferred gradually to Teklad basal mf* (952) for rats. Microswine will be fed optimized diet (twice per day) of microswine diet (Charles River) and transferred gradually to Teklad basal mix (952) for microswine. Rats and microswine will be provided deionized water ad libitum, weighed three times, and observed with respect to eating habits, weight gain, and any signs of disease or injury. During the test period, all rats and microswine will be fed the diet of their respective treatment groups. Animals that die or are sacrificed in extremis during the quarantine period will be necrapsled 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. The program is monitored and administered by the laboratory veterinarian; data ate maintained separately from study records. D. Assignment to Groups Healthy animals will be assigned to groups by computerized, stratified randomization. Rats will be housed individually while microswine will be housed two per cage. Each animal will be assigned its own Haskell animal number, and permanently identified by a number marked on the rat's tail or the microswine's ear. DUP040000711 -6 E. Animal Husbandry After assignment to groups, animals -will be identified with a cage card and housed in stainless steel cages. All rats and microswine will be provided optimized test diets of approximately 21.5 g/rat/day and 340 g/microswine/day. Deionized water will be provided ad libitum. Animal rooms will be targeted at a temperature of 23 + 2C and a relative humidity of 50 + 102. F. Diet Preparation and Analysis During the test period, rats and microswine in each group will be fed the following purified diets; o Rat Basal mix (95Z) (TD 92242) o Swine Basal mix (952) (TD 93034) These diets will be custom formulated by their manufacturers so that each diet contains complete normal nutrition for each species when the diet is diluted (weight/weight) with test soil containing 1,000 ppm lead or with sucrose. These diets are sucrose-free so that the soil added will replace the sucrose rather than diluting the complete mixed diet. The diets will contain < 0.1 ug Pb/g. Lead/soil or sucrose will be added to the diets and thoroughly mixed for a period of time that is adequate to assure homogeneous distribution in the diet. Once prepared, diets will be refrigerated until used. Sucrose mixed diets will be given to all animals during quarantine and the single dose administration group (Group A in Study Design). The lead/soil mixed diet will be given to the other group for 30 days (Group B in Study Design), After 30 days, Group B animals will be given sucrose-mixed diets for the rest of the study period. DUP040000712 -7 - Before the beginning of the study, samples will be collected from the diet prepared with lead/soil.. These samples will be analyzed to verify concentration and homogeneity in the test diets. Homogeneity samples will be collected from the top, middle,, and bottom of the diet mixer. Samples will be frozen until analyzed. Once during the study, samples will be collected from randomly selected feed jars to verify concentration of lead in the feeders. Samples will be collected from each species. Backup samples will be taken on the same day from other feeders in the same species and analyzed if needed. Feeder samples and backup samples will be frozen on the day of collection. G. Body Weights All rats and micro swine will be weighed twice per week unless experimental findings warrant a more infrequent weighing schedule. H. Food Consumption and Intake of Test Material The amount of food consumed by each microswine will be determined twice per week throughout the study. The amount of food consumed by each rat will be determined weekly throughout the study. From these determinations and body weight data, average test animal daily food consumption, and intake of test material will be calculated.I. I. Dosing Rats will be dosed intravenously via the tail vein, while microswine will be dosed via the ear vein. Sterile water will be used as a vehicle for intravenous administration. Oral doses to both species will be administered by gavage. Deionized water will be used as a vehicle. DUP040000713 The route of administration of the lead-contaminated soil will be by oral gavage and/or feeding 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. J. Blood Sampling Approximately 0.1-0.3 ml of rat venous blood and 0.5-1 ml of micro swine venous blood will be drawn from each designated animal at 0, 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 EDTA and refrigerated until the time of preparation for analysis. K. Sacrifice of Animals At the end of the experiment, the animals will be humanely euthanized. Animals will be necropsied, liver and bone will be collected from the single dose administered animals (Group A in Study Design) and liver, bone, kidney, spleen, and brain will be collected from the animals fed lead/soil for 30 days (Group B in Study Design). All tissues will be weighed, frozen at approximately -20C, and stored for potential future analyses. L. Blood Analysis The blood lead concentration will be analyzed by graphite furnace atomic absorption spectroscopy (GFAA), at DuPont Chambers Works. Dosing solutions, diets, and drinking water will also be analyzed. DUP040000714 9 M. 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. N. Statistical Methods Body weights, body weight gain, and food consumption 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. DUP040000715 - 10 PROTOCOL APPENDIX I MEDICAL RESEARCH PROJECT NO, 9660 Study Personnel and Study Dates STUDY PERSONNEL Study Sponsor; Corporate Remediation Group of DuPont Chemicals E. I. du Pont de Nemours and Company Bellevue Corporate 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 STUDY DATES Proposed Experiment Start; Proposed Sponsor Approval; Proposed Experiment Completion; Proposed Report Issue Date; May, 1993 Date the Sponsor signed the MR Project August, 1993 February, 1994 DUP040000716 ABSOLUTE BIOAVAILABILITY OF SOIL LEAD IN RATS AND MICROSWINE MEDICAL RESEARCH PROJECT NO. 9660 PROTOCOL Approved: jljanan N. Ghantous Study Director Date 6 Ini15 Matthew S. B(. Manager Biochemical T.oxicol cc: P. E. Ross G. S. Elliott T. S. Bingman (REO-PIT) K. Me Cleary M, S. Bogdanffy R. C. Rhea DUP040000717 DUPONT CENTRAL RESEARCH AND DEVELOPMENT HASKELL LABORATORY FOR TOXICOLOGY AND INDUSTRIAL MEDICINE June 28, 1993 TO: MEDICAL RESEARCH PROJECT NO. MR 9660-001 ABSOLUTE BIOAVAILABILITY OF SOIL LEAD IN RATS AND MICROSWINE PROTOCOL AMENDMENT #1 The protocol is amended as follows: 1. Page 2, Study Design, Section A. Single Dose Administration, replace "Fifty male rats and ten male microswine will be used." with "Fifty male rats and twelve male microswine will be used." Reason: To obtain better statistical analysis of results, 2. Page 2, Study Design, Section A. Single Dose Administration, replace "Twenty-five rats and five microswine will be dosed orally..," with "Twenty-five rats and six microswine will be dosed orally...". Reason: To obtain better statistical analysis of results. 3. Page 2, Study Design, Section A, Single Dose Administration, replace "Twenty-five rats and five microswine will be given..." with "Twenty-five rats and six microswine will be given...". Reason: To obtain better statistical analysis of results, 4. Page 3, Section B, Single Dose Administration After Feeding For 30 Consecutive Days, replace "Fifty male rats and ten male microswine..." with "Fifty male rats and twelve male microswine... " Reason: To obtain better statistical analysis of results. 5. Page 3, Section B. Single Dose Administration After Feeding For 30 Consecutive Days, replace "Twenty-five rats and five microswine will be dosed orally..." with "Twenty-five rats and six microswine will be dosed orally.,.". Reason: To obtain better statistical analysis of results. 6. Page 3, Section B. Single Dose Administration After Feeding For 30 Consecutive Days, replace "Twenty-five rats and five microswine will be given..." with "Twenty-five rats and six microswine will be given...". Reason: To obtain better statistical analysis of results. DUP040000718 7. Page 4, Pretest Procedures, second paragraph, delete " irradiated" from first sentence. Reason: The animals are not being feed irradiated chow. The study is being conducted in Building 1 and irradiated chow is not received there. 8. Page 5, second sentence, replace "(twice per day)" with "(once a day)". Reason: More convenient for individual food consumption calculations. 9. Page 5, Section D. Assignment to Groups, second sentence, replace "housed two per cage" with "housed individually". Reason: Animals will be housed individually to calculate individual food consumption. 10, Page 6, Section E. Animal Husbandry, second paragraph should read "Animal rooms will be targeted at a temperature of 23 + 2C for rats and 25 + 2C for microswine and a relative humidity of 50 + 10Z." Reason: Microswine should be kept at a higher temperature than rats. 11, Page 6, Section E, Animal Husbandry, after second paragraph, insert the following paragraph: Animal rooms that will house rats will be artificially illuminated (fluorescent light) on a 12-hour light/dark cycle. Animal rooms that will house microswine will be artificially illuminated (fluorescent light) on a 12-hour light/dark cycle with additional natural light being available through a window at one end of the room. Reason: Microswine do not need to be on a 12 hour light cycle. The animals should be housed in conditions similar to a barn, 12, Page 2, Section A. Single Dose Administration, "Blood from the single dose administered group of rats was collected in lead-free tubes containing EDTA. Heparin containing tubes were used for collecting blood from the other groups. Reason: EDTA caused complications in analyzing blood lead by atomic absorption, 13, Page 6, Section E. Animal Husbandry, add the following paragraph "Microswine were provided with recreational equipment (balls) in their cages. Reason: To Improve housing conditions. Balls were added after consulting with Linda Panepinta, Swine Research Consultant. 14, Page 6, Section F. Diet Preparation and Analysis, the following should be added "Microswine were given Oreo cookies after blood sampling. Reason: Linda Panepinta, Swine Research Consultant, suggested the administration of Oreo cookies after blood sampling as a reward. DUP040000719 15. Page 7, first paragraph, the following sentence should be added "Diet samples will be refrigerated, not frozen until analysis. Reason! Analysis was done within a short period of time after sampling. 16. "Due to technical error, the 12 hr. interval blood collection was not taken for the single dose gavage group of rats. Five male rats from the animals not used on the study were randomly selected, prebled, dosed by gavage, and bled after 12 hrs. Reason! Technical error. 17. Page 8, Section K. Sacrifice of Animals. Replace the final sentence with "Tissues from rats bled at the 240 hr. time point will be saved at sacrifice. Reason: Tissues from 5 rats only are needed for statistical analysis. Date Approved: p. E. Ross G. S. Elliott T. S. Bingman (REO-PIT) K. Me Cleary M, S. Bogdanffy R. C. Rhea Biochemical Toxicology DUP040000720 DuPont Central Research and Development CC: H. Ghantous - CR&D, Haskell June 9, 1995 TO: Tim Bingman-SPEC FROM: Matthew S. Bogdanffy - CR&D, Haskell 1-1712-A SUBJECT: Response to Comments from die Washington Department of Health ' Dr. Ghantous has reviewed the comments on the lead bioavailability study prepared by the Washington State Department of Health. Seven points were raised in their letter to Mike Blum, Department of Ecology, which we wish to address. A summary of our responses are presented below: 1. Inaccuracies in calculating the doses given to the study animals. In the documentation supporting the DOH memo, it is stated that "the author then mistakenly assumed that the 30 ml suspension weighed 47 g (i.e. 30 g of water plus 17 g soil) in the calculated dose. However, mixing soil with water results in a suspension with a volume greater than 30 ml. Thus, 30 ml of this 47 g suspension weighed less than 47 g, and the actual dose of lead received by each animal was less than the dose used in the calculations by the author." The reviewer then goes on to demonstrate, hypothetically, that such an error would result in a decrease in the final calculated bioavailability. In the absence of an understanding of the nature of the test materials, the reviewer would have been correct on this point The volume added to the overall dose suspension contributed by the volume of the admixed soil was not taken into account in the dose calculations and would have, theoretically, resulted in an overestimation of the true bioavailability. To examine the validity of this theoretical point. Dr. Ghantous returned to the lab and conducted an experiment in which 17 grams of soil (the same soil used in the subject bioavailability study [Haskell Laboratory Report 671-93]) was mixed with 30 mis of water. The methods for weighing and mixing the soil were similar to those used in the bioavailahility study except that in the latter, and as correctly noted by DOH, the investigators mixed a bulk amount of soil in water and then withdrew individual doses from this suspension. The volume of the resulting suspension in Dr. Ghantous's experiment was measured. The volume was approximately 30 mis (limit of accuracy was + 1 ml). Dr. Ghantous's observations suggested that this negligible volume increase was a result of the ultrafine powder-like consistency of the soil. These soils were sifted to a mesh of approximately 250 microns. DUP040000721 6/9/95 Page 2 The entire soil/water mixture was then drawn into a tarred syringe, in a manner similar to that used in the study, and weighed. The weight of the mixture was 41.6 grams. This is less than 47 grams and, since mass is conserved, this suggested some loss of materi al on the walls of the mixing vessel. This was confirmed by observation. Therefore, preparation of the dosage in this manner would have lead to negligible loss of dosage volume, but an 11.5% loss of soil weight (1.96 grams per dosage). However, as noted above, the investigators prepared the dose mixture in bulk. Therefore the soil loss noted in the paragraph above would only apply to the last dosage removed from the mixing vessel. Thus, given that a) the volume change induced by soil addition to water is negligible, and b) enough dose solution was prepared for 10 animals, but only five dosages were removed, the point made by the DOH regarding inaccuracies in calculating the doses given to the study animals becomes moot. Nevertheless, we thank the DOH reviewer for assisting us in evaluating the data. Regarding the density of the i.v. dose solution, Dr, Ghantous also looked into this question experimentally in response to the comments of the reviewer. The density of the dose prepared by weighing 3.527 g of lead acetate trihydrate and dissolving it in 10 tnls of water (approximately 35% solution) was 1.2 g/ml. Therefore, given that the density of water is 1.0, the difference between 1.2 and 1.0 (i.e. 0.2 g/ml) is due to the added lead acetate. A calculation of proportions can then be made to estimate the density of the i.v. dose solution. Appendix H contains measured values of 116.6 mg/g of water (116.6 mg/ml of water, or 11.7%) and 126.8 mg/g of water (126.8 mg/ml of water, or 12.7%). Using the 11.7% value as an example, 35%/0.2 = 11.7%/X; X = 0.06. Therefore, the density of the i.v. lead acetate solution equals 1.0 + 0.06, or 1.06. This value is very near the assumed density of 1.0 and, therefore, has no material effect on the final calculations. 2, 3. The small number ofmicroswine used in each experiment. The high inter-animal and intra-animal variability in blood lead concentration over time. The DOH reviewer notes that "It is difficult to have confidence in studies that looked at so few animals," The reviewer goes on to support this with discussion around tine variability in the blood lead measures. As a scientist, the term confidence implies statistical interpretations. Large mfraanimal variability is common in pharmacokinetic data. The revieweris directed to any of a number of texts and literature references on pharmacokinetics and bioavailability for discussions on these points1. Clearly, increasing the numbers of microswine per group could reduce the mteranimal variability. The question is how many microswine per group would have to be added and, importantly, whether the statistical "pay-off' achieved by increasing the group size justifies the ethical issues of increased animal usage. We considered these arguments in the study design. Our study was also subjected to review by our in-house animal welfare committee. Consultation 1 For a recent article, see Prelusky, D. B., Hartin, K. E., Trenholm, H. L,, and Miller, J. D. (1988). Pharmacokinetic fate of "C-Iabeled deoxynivalenol in swine. Fundam. Appl. Toxicol., 10, 276-286. DUP040000722 6/9/95 Page 3 with our statistician indicated that increasing the numbers of microswine per group by 2 would yield little, if any, increase in statistical power. This suggests that a large increase in group size would be necessary to off-set the interanimal variation. It is our opinion that such a large increase could not be justified against the ethical issues. Furthermore, recent swine bioavailability studies conducted by the 0. S. EPA used three swine per group2. 4,5. The lack ofscientifically soundjustificationfor choosing one particular experiment out of all the Phase land Phase II studies on which to base the "absolute bioavailability" figure. The lack ofjustificationfor applying abioavailabilityfigure derivedfrom acute dosing experiment to a chronic exposure scenario. The choice of the bioavailability value derived from the Single Dose Administration (Acute) experiment using microswine oyer other bioavailability values derived has been addressed. In brief, the Phase I work was conducted to provide range-finding data for the Phase II work. The Phase I work could only yield relative bioavailability values. The choice of the Single Dose Administration (Acute) bioavailability value was based on a) the pig model is more suitable than rats as a representative of humans for soil bioavailability studies3, and b) greater statistical confidence Mid interpretive value was placed on the Single Dose Administration (Acute) microswine. Regarding the bioavailabiiity calculations, Dr. Ghantous has recalculated the bioavailability of lead using the subchronic feeding study-data. Following the guidance contained in "Gudinace for the Integrated Exposure Uptake Biokinetic Model for Lead in Children4" the data were re-evaluated by measuring areas under the blood lead curves (AUC) from time zero (just prior to dosing) to 24 hours, rather than extending the AUC calculation to infinity (as was done in the report)5. Recalculating the data in this manner avoids the complications observed in the subchronic study that were attributed to remobiiization of lead. The mean AUC for the i.v. group was 15.08 and the mean AUC for the oral group was 5.50. Thus the calculated bioavailabiiity is 23.8%. 6. The lack of evidence to indicate that the soil used in the bioavailabiiity studies is representative ofsoils throughout the site. This comment is best addressed by the remediation program scientists. 2 Lorenzana, R. M., Dawson, M., Kettere, M., Simon, J., Lowry, J., and Poppenga, R. (1995). Stable isotope method to determine bioavailabiiity of lead in swine. Toxicologist, abstract no. 716. 3 Weis, C. P., and LaVelle, J. M. (1991). Characteristics to consider when choosing an animal model for the study of lead bioavailabiiity. Chemical Speciation and Bioavailabiiity 3(134), 113-119 4 U. S. EPA. Guidance Manula for the Integrated Exposure Uptake Biokinetic Model for Lead in Children. 5 Welling, P. G. (1986). Multiple Dose Kinetics. In Pharmacokinetics Process and Mathematics, Chapter 12. DUP040000723 6/9/95 Page4 7. The lack ofquality assurance/quality control datafor the studies. This study was Conducted in full compliance with Good Laboratory Practices as stated on page 2 of the report. Minor protocol and GLP deviations are disclosed in Appendix B. The four deviations noted did not, as attested to in the report, affect the integrity of the report. DUP040000724 w x? & MM HH HH I, d,, tS T3 OS 9) >C 0H OJ-4 0O *<s*> OS OS as as irl i-> an) qas M '"O0> *T0a3 0rl ai-4 a *-> 0 005 00 Q40-J O40-i As) afH$0 .2 & 3I B* 4t>, =#= *** =#= 3 O 0) OS aOs OcnS aCaSS tess Hcnas Hcnas OS ts 0v) ac r-aSs rHas cnaS c(a9 4? nS ca Hmas g nJs ^ Ma aM sOeasSs (aaV e aids ->v0-0ma aes Cj Jal "Di>) -r-i Tc3 U Hns aos 3U IHd D<J 43J as as V as vs as ai vs as aS v5 as XI *-> XS m*-> bO C C bo C rt 5 D -H -O cSo3 ats* <oe<o& acs* o ce to to =tt= w~ w wo 05 STe.,te. lo/isfe Room Date a.m. Check .- *906&. ^ Jo* fuwt t/H/t ty&ko ) yUCsiZf J/lM> U/^CLku. ^~3 IMAL ROOM TASK ACCOUNTABILITY SHEET 'fUt, -^n YM/C?6r&6, J mr # j^yy^H # 2fiov Year t?T3 pOlau^ )__R_>f_M_7w. &t t.Vls&J&V0iiC?r`6>7- 3lu\dll\tf$.\5p \nj^_\$>/f\S7i}: i/{t, fqy ` Temperature (C) Humidity (40-60%) Time of Readings Recorder's initials Mortality/Moribundity checked Water availability checked/Bowls filled Animals fed Time <z oo p.m. Check 4^ -- 31 H zz 22. '23 bU> 3tT .15?- r* ' -- *on t h ( l;ao Cfr T c 2r 'T.y -ru %>W c \ cf- rf cf Iff- -- C -v c k cw T.n V.oO jm. ... ZiiXi. Temperature (C) Humidity (40-60%) Time of readings Recorder's initials Mortality/Moribundity checked Water availability checked/Bowls filled Animals fed Time Tasks (initial each item 2231 1 -00 3'o0 c\ r,7k/f coc Tfc \ bloo lzzr 22, 22 33 3ft 3 la vT'fi 1:00 2t 1020 i Co Kf4 KU C# fa mTF of ( f oS i:oe> PffO r*>i eft- c^5 r.Ofo 1 ;06 3-if: 3'?o ) $?) Room cleaned Cage change Room scrubbed 1 Animals rotated/Animalj Feeders cleaned and dr| Water bowls cleaned an< Ceiling tiles checked J o" j Rabbit room temperatur N/A = Not applicable, a Hygrothermograph usI b Temperature or humii contacted. i 7W cot 'C t - -- -- r* ft cf -- C ft i/h N/A N/A N/A 21-25 C). lits. date personnel Remarks/Footnotes: (j) O^Vxywvj2kJ-O 0^wwr>gJil uotl: T}iiS Acco^iTHitUr^ J/feT V*S *>' foT'iOT^ WS WT>L IfcjM 6j #* 0* l/Sfa Witnessed by: k ____ Date: \{sjf / DUP040000734