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MR-9905
DuPont Central Research and Development Haskell Laboratory for Toxicology and Industrial Medicine
TO:
Timothy Bingman
DuPont Chemicals
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PROJECT MR-9905 PARTI
April 28,1994
TITLE: ABSOLUTE BIOAVAILABILITY OF LEAD SOIL IN MICROSWINE: Soils Identified as Fort Madison and PLW-1
Authority is requested to conduct a study to determine the absolute bioavailability of soil lead in microswine- Details are in the study protocol, provided under a separate cover.
The full cost of the study is $47,000.00 and is distributed to cost centers 19,23, and 25.
The allocated cost of this study is $34,625.00. The allocated costs are to be decrementally billed over 5 months (June through December of 1994) as follows:
Cost Center 19 Cost Center 23 Cost Center 25
$30,000.00 3,925,00 700.00
(86%) (11%) (02%)
Allocated cost requested: $34,625.00
Previously authorized
$ 0.00
Expended through [date]
$ 0.00
General Ledger & Sub-Account to be
Charged
- S40/Z3
,,
,.
Forecast month and year
Experiment to start:
6/94
Experiment to end:
7/94
Final report to issue:
1/95
Request for study made by: T. Bingman
TESTING X
RESEARCH (Check appropriate classification)
CONSULTING.,
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:
Hanan N. Ghantous
Approved by:.
PLEASE RETURN AUTHORIZED COPY DIRECTLY TO: LAURA WALLS, FINANCIAL SERVICES HASKELL LABORATORY
^Authorized by:
N 26889
DuPont HLR 581-94
Study Title ABSOLUTE BIOAVAILABILITY OF LEAD-CONTAMINATED SOILS IN MICROSWINE
Laboratory Protect ID ^ Haskell Laboratory Report No. 581-94
Author Hanan N, Ghantous, Ph.D.
Study Completed on October 20,1994
Performing Laboratory E. I. du Pont de Nemours and Company
Elkton Road, P.O. Box 50 Newark, Delaware 19714-0050
Medical Research Project Nos. 9727-001,9740-001, and 9905-001
Page 1 of 101
DUP040004321
Absolute Bioavailability of Lead-Contaminated Soils in Mlcroswlne
______ ________________ DuPont HLR 581-94
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:
Hanan N. Ghantous, Ph.D. Research Toxicologist
Biochemical Toxicology and Risk Analysis
(date)
Company Representative: .
.. ' . . '
,,_______ (date)
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Absolute Bioavailability of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
GENERAL INFORMATION
Intravenous Administration
Substance Tested: 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 LeadCQ) acetate trihydrate Lead acetate Acetic acid, lead(2+) salt 20202 99.999% 6080-56-4
Oral Administration
Substance Tested: Synonyms/Codes:
Haskell Sample No.: Purity:
Composition: C.A.S. Registry No.:
Soils, Remington site, contaminated with lead H-20366 Lead-contaminated soil AEC-3-3 Soil, contaminated with lead 20366 N/A 1070 ppm lead in soil None Available
Substance Tested: Soils, Remington site, contaminated with lead Synonyms/Codes: H-20367
Lead-contaminated soil AEC-9-3
Soil, contaminated with lead
Haskell Sample No.: 20367 Purity: N/A
Composition: 1120 ppm lead in soil C.A.S. Registry No.: None Available
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Absolute Unavailability of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
GENERAL INFORMATION (Continued)
Substance Tested: Soils, Remington site, contaminated with lead Synonyms/Codes: H-20366
Lead-contaminated soil AEC-10-1
Soil, contaminated with lead
Haskell Sample No.: 20368 Purity: N/A
Composition 1210 ppm lead in soil C.A.S. Registry No.: None Available
Substance Tested: Synonyms/Codes:
Haskell Sample No.: Purity:
Composition C.A.S. Registry No.:
Soils, Pompton Lakes, contaminated with lead H-20383 Lead-contaminated soil PLW Soil with lead #PLW (Pompton Sample) 20383 N/A 803 ppm lead; 205 ppm mercury None Available
Substance Tested: Synonyms/Codes:
Haskell Sample No.: Purity:
Composition: C.A.S. Registry No.:
Soils, East Chicago, contaminated with lead H-20384 Lead-contaminated soil EC1 Soil with lead #EC1 (East Chicago Sample) 20384 N/A 3000 ppm lead; 235 ppm arsenic None Available
Substance Tested: Synonyms/Codes:
Haskell Sample No.: Purity:
Composition C.A.S. Registry No.:
Soils, Fort Madison, contaminated with lead H-20659 Lead-contaminated soil Fort Madison (FM) Lead-contaminated soil from Fort Madison (FM) 20659 N/A 500 ppm lead None Available
4 - DUP040004324
Absolute Bioavailability of Lead-Contsuninated Soils in Microswine
DuPont HLR 581-94
GENERAL INFORMATION (Continued)
Substance Tested: Synonyms/Codes:
Haskell Sample No.: Purity:
Composition C.A.S. Registry No.:
Soils, Pompton Lake, contaminated with lead H-20658 Lead-contaminated soil (PLW-1 or PLW1) Lead-contaminated soil (from Pompton Lake) 20658 N/A 4000 ppm lead 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.
Study Initiated/Completed: 10-11-93 / Report Issue Date (see cover page)
Experiments Initiated/Completed: 10-19-93 /11-08-93 (Groups A, B, C, and D) 11-30-93 / 12-17-93 (Groups E, F, and G) 06-14-94 / 07-01-94 (Groups H, I, and J)
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Absolute Bioavallabillty of Lead-Contamliiated Soils in Microswine
DuPont HLR581-94
TABLE OF CONTENTS
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT....................... 2
GENERAL INFORMATION.................................. ....................................... ................ 3
SUMMARY............................................ ............................................. ............................ 8
SIGNATURE PAGE...... ........................ .............................. ......................... ................ 9
QUALITY ASSURANCE DOCUMENTATION....... .................. ........................ 10
STUDY PERSONNEL............................................................. ...................... ..............11
INTRODUCTION........................................................... ....................... ......................13
OBJECTIVE............................... ................................ ............... ................................... 13
MATERIALS AND METHODS............ ............................................... ......................13
A. Test Substance........................................ ............................ ....................... .......13
B. Test Species............................................................ ........................ .................. 13
C. Animal Husbandry........... ......................... ......................... ........... .
14
D. Pretest Period....... .............. ........................... .................... ............................. 15
E. Assignment to Groups and Study Start............................. .............................. 15
F. Diet Preparation, Administration, and Sampling..............................................16
G. Dose Preparation, Administration, and Sampling......... ................ .................. 16
H. Blood Sampling............................ ................... .................................... .............16
I. Body Weights, Clinical Observations, and Mortality ..., ......................... . 17
J. Sacrifice of Animals ...... .................................... ................ ...................
17
K. Data Analysis........ ............................................. ........................ .................. 17
RECORDS AND SAMPLE RETENTION................................ ............................. ;.... 18
RESULTS AND DISCUSSION........................................................................ .
18
A. Diet Samples ....... ............... ,,................................... .................. .................... 18 B. Dosing Solutions...................................... .......................................... .......... . 18 C. Blood Lead Concentrations, Area Under the Curve, and
Absolute Bioavailability................................................. ........... ....................... 18 D. Body Weights and Clinical Observations.... ..............................,.....................20
CONCLUSION.,..,.......... ........................................... ................... ...................... ..........20
REFERENCES.......................... ................................. ......... ...... ....................... ........... 20
TABLES ............................................................................................... .............. ........21
1. Measured concentration of lead in intravenous and oral gavage dosing solutions................... ...................... ................................................. . 22
2. Miscellaneous lead concentrations..... .............. .................... ..................... . 23 3. Mean blood lead concentration....... ............................. .............. ...................... 24
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Absolute Bioavailabillty of Lead-ContaminatedSoils in Microswine
DuPont HLR 581-94
TABLE OF CONTENTS (Continued)
TABLES (Continued)
4. Absolute bioavailability of soil lead in microswine.,........................................26 5. Area under the blood concentration-time curve (AUC)............... ............... . 27
FIGURES ........................... ....................................... ................ .................................. 28
1. Mean blood lead concentration in male microswine administered Pb Acetate (Group C) or AEC 3-3 (Group A)........................................ ..........29
2. Mean blood lead concentration in male microswine administered Pb Acetate (Group C) or AEC 9-3 (Group B)..........--.................. ................ 29
3. Mean blood lead concentration in male microswine administered Pb Acetate (Group C) or AEC 10-1 (Group D).... .................................... ........30
4. Mean blood lead concentrationin male microswine administered Pb Acetate (Group C) or PLW (Group E)..........--........ ............................30
5. Mean blood lead concentration in male microswine administered Pb Acetate (Group C) or EC1 (Group G)............................................ .............31
6. Mean blood lead concentration in male microswine administered Pb Acetate (Group I) or FM (Group H).......................... ...................................31
7. Mean blood lead concentration in male microswine administered Pb Acetate (Group I) or PLWl (Group J)..........................................................32
APPENDICES.............................. .......... ............ ........................................................... 33
A. Protocol and protocol amendments................................ ................ ...............,...34
1. Protocol #1 and amendments (Groups A-D)____ ___ ____ 2. Protocol #2 and amendment (Groups E-G).... ............ 3. Protocol #3 and amendment (Groups H-J).......
35 47 57
B. GLP deviations.............................. ..................... ............ ........................ ,..,....,.67
C. Individual blood lead concentrations in microswine..............................68
D. Individual body weights of microswine...... ............ ......................................... 74
E. Individual clinical observations in microswine ............ ................... ................ 78
F. Sample Calculations...................... ................... ............. ............................ .......84
1. Sample calculation for the amount of lead in thedose_____ ___ 2. Sample calculation for area under the curve (AUC)..... ...............
85 86
G. Lead Analyses Reports.... ........,....,................ .................. ................... .............. 87
Experiment #1 (Groups A-D)........................................... ................... .......... ,...88 Experiment #2 (Groups E-G)..... ............................ ................... ,..............,..,.,...93 Experiment #3 (Groups H-J)........................................................ ..................... 98
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Absolute Bioavailability of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
SUMMARY
The objective of this study was to determine the absolute bioavailability of lead in test soil from contaminated sites. The bioavailabilities of lead from contaminated soils were measured by using a single dose administration to juvenile microswine.
Groups of 5 or 6 male juvenile microswine were orally administered (via gavage) test soil (approximately 17 mg lead/microswine). Three groups of 5 or 6 additional male juvenile microswine received an intravenous injection of lead acetate solution containing a lead dose equivalent to that of the oral (gavage) dose.
Blood was drawn from all groups of microswine 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 bioavailability was determined as the ratio of the area under the bloodlead versus time curve (AUC) for the oral and intravenous groups.
The absolute bioavailability results were:
Test Soil Administered
AEC3-3 AEC9-3 AEC 10-1
PLW EC1 FM PLW1
Absolute Bioavailability
28.52% 16.70% 24,12% 30.97% 31.72% 14.72% 9.18%
The apparent differences in oral bioavailability observed in this study could be related to a combination of factors, including the chemical form of lead in the test soil and the intrinsic soil properties.
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Absolute Bioavailability of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
MEDICAL RESEARCH PROJECT NO. 9727-001
Authored by:
Hanan N. Ghantous, Ph.D. Research Toxicologist
Biochemical Toxicology and Risk Analysis
Approved by:
Matthew S. Bogdanffy{Ph.D./ Manager
Biochemical Toxicology and Risk Analysis
Reviewed and Approved for Issue by Study Director:
Research Toxicologist Biochemical Toxicology and
Risk Analysis
Date Report Issued:
(o j
jyy
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DUP040004329
Absolute BioavailabiUty of Lead-Contaminated Soils in Mlcroswiue
DuPont HLR 581-94
(Haskell Numbers: 20202,20366,20367,20368,20383,20384,20558, and 20659)
Date(s) of Inspection: Conduct - 10/21,22,25,26/93,11/5,9/93,12/2,3,6,7,10/93, 1/11/94,6/13,14,17,21,24/94,7/1/94 Records, Report(s) -10/14/93,5/23/94,6/9,11,13,24/94, 8/15,18,19/94,9/13-16,20-22,29/94,10/6-8,10-13/94
Date(s) Findings Reported to: Study Director - 8/1,10,19/94,9/22,28,29/94,10/13/94 Management - 8/1,10/94,9/28/94,10/3,5,12,20/94
Reported by:
Robert C. Rhea Quality Assurance Auditor
(date)
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DUP040004330
Absolute Bioavailability of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
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: Benjamin Robinson, B.A.
Assisting Technicians: Steven C. Carpenter Gerald L. King
Technical Assistants:
A. Richard Conrad, Jr. Victor L. Cruz Roger B. Walls
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 (Jackson Lab):
Coordinator: Keith A. McCleary, Ph.D.
Technicians:
Billy R. Cooper Robert Eyberger (MR 9905-001 only) Ralph Postorivo Michael Sisitka
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DUP040004331
Absolute Bioavailability of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
The following individuals were responsible for quality assurance: Coordinator: Joseph C. Hamill Auditor: Robert C. Rhea, B.S. Deborah A. Vick, A.A.S.
The health status of the animals on study was assessed by the attending Laboratory Veterinarian, Charles E. Cover, V.M.D.
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Absolute Bioavailability of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
INTRODUCTION
In a recent study conducted at Haskell Laboratory/15 the absolute bioavailability of lead from a site's soil was measured in both juvenile rats and microsowine using either single doses or subchronic-dosingfollowed by single-dose exposures. Both rats and microswine were used in order to choose the correct animal model in the conduct of experimental investigations relevant to humans.
Based on the results of the above study, microswine appeared to provide a more conservative estimation of bioavailability than rats. In addition, because of difficulties encountered interpreting blood lead data resulting from a bioaccumulative effect of subchronic exposure, single-dose exposures provided the most meaningful results for measuring lead bioavailability.
Thus, microswine given a single-dose exposure are used in this study to measure the bioavailability of lead from contaminated soils from different sites.
OBJECTIVE
The objective of the study was to determine the absolute oral bioavailability of lead from soil in microswine following single oral (gavage) doses. These results will assist in the development of site-specific clean-up goals by providing soil-specific data on gastrointestinal absorption.
MATERIALS AND METHODS The original protocols and protocol amendments are in Appendix A.
A. Test Substance
Lead acetate trihydrate was supplied by Aldrich, Milwaukee, Wisconsin; the leadcontaminated soils were supplied by the sponsor. See the General Information section of this report for more information about lead acetate trihydrate and the lead-contaminated soils. Characterization and mineralogy data of the lead-contaminated soils are maintained by the sponsor.
B, Test Species Groups of juvenile male Yucatan microswine were received from Charles River Laboratories, Inc., Windham, Maine or Pittsfield, New Hampshire. Twenty were
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DUP040004333
Absolute BioavailabiUty of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
received on 10/05/93 (born August or September of 1993), 18 were received on 11/12/93 (born September or October of 1993), and 15 were received on 6/02/94 (born April or May of 1994). The day after arrival, all microswine were weighed. Body weights ranged from 2884 to 7813 g. The male Yucatan microswine was selected on the bases of its low body weight and its reported suitability as a model of soil-lead ingestion by children.
Groups of female Yucatan microswine were received during the pretest periods and used for oral gavage practice. These microswine were housed irithe same room with the male microswine, but were not considered part of the study; the females were sacrificed before the males were dosed.
C. Animal Husbandry
During the pretest period, microswine were housed according to the Guidefor Care and Use ofLaboratory AnimalsTM After assignment to groups, microswine were housed in stainless steel cages bedded with biodegradable shredded paper (Enviro Dri). To improve housing conditions, microswine were provided with recreational equipment (balls) in their cages. Occasionally, microswine were rewarded with Oreo cookies (to calm the animals after blood samples were collected).
The animal room was targeted at a temperature of 252C and a relative humidity of 50+10%. Sporadically, the temperature and relative humidity of the animal room were slightly below these ranges, but did not affect the study. The animal room was artificially illuminated (fluorescent light) on a 12-hour light/dark cycle with additional natural light entering through a window at one end of the room.
Haskell Laboratory has an animal health monitoring program. The following procedures were performed periodically:
Water samples were analyzed for total bacterial counts and the presence of coliforms, lead, and other contaminants,
Feed samples were analyzed for the presence of bacteria and fungi. Samples from freshly washed cages and cage racks were analyzed to assure
adequate sanitation by the cageWashers.
Haskell Laboratory uses certified animal feed. The feed is guaranteed by the manufacturer to meet specified nutritional requirements and to be free of a list of specified contaminants.
The animal health monitoring program is administered by the laboratory animal veterinarian. Data are maintained separately from study records and are not included in this report. Evaluation of these data did not indicate any conditions that affected the validity of the study.
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Absolute Bioavailabilily of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
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
Microswine were quarantined for at least 13 days. During the pretest period, 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%), Transfer to Teklad basal mix and sucrose was completed before study start. Microswine were provided deionized water ad libitum.
All microswine were weighed at least 4 times during the quarantine period and observed daily with respect to eating habits, weight gain, and any signs of disease or injury.
E, Assignment to Groups and Study Start
Microswine were divided by computerized, stratified randomization into groups as follows:
Date of Arrival
Experiment Number
Group Number
Method of Test Substance
TDa Per Group Administration Administered
10/05/93 10/05/93 10/05/93 10/05/93 11/12/93 11/12/93 11/12/93 06/02/94 06/02/94 06/02/94
1 1 1 1 2 2 2 3 3 3
A5 B5 C5 D5 E6 F6 G6 H5 I5 J5
Oral Gavage Oral Gavage Intravenous Oral Gavage Oral Gavage Intravenous51 Oral Gavage Oral Gavage Intravenous Oral Gavage
Soil AEC 3-3 Soil AEC 9-3 Lead Acetate Soil AEC 10-1 Soil PLW Lead Acetate Soil EC 1 SoilFM Lead Acetate Soil PLW1
a Group identification labels are for the purpose of reporting and do not correspond to the group identification in the study records.
b Due to technical error, the dosing solution was incorrectly prepared. Lead concentration data from this group will not be used in the report.
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Absolute Bioavailability of Lead-Contaminated Soils in Microswine
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F, Diet Preparation, Administration, and Sampling
Diets were prepared by combining Teklad basal mix (95%) with sucrose (5%), These ingredients were mixed in a high-speed mixer for 3 minutes. Diets were refrigerated until used or discarded.
Each microswine received 340 g diet per day. Samples of the Teklad basal mix and sucrose diet were sent to DuPont Chambers Works, Deepwater, New Jersey and analyzed for presence of lead,
G. Dose Preparation, Administration, and Sampling
Oral Dose. Microswine designated for oral (gavage) administration received a 30.0 or 40,0 mL suspension of test soil and deionized water. Each microswine was targeted to receive approximately 17 mg lead within the 30.0 or 40 mL dose. The suspensions were constantly stirred until all animals were dosed. The dose of some soils was diluted due to the consistency of the soil; however, the dilution was taken into consideration in calculating the dose of lead administered.
Oral dosing suspensions were analyzed for lead concentration (Table 1) and the measured values were used for calculating lead bioavailability.
Intravenous Dose. Microswine designated for intravenous administration received a 0.1 mL solution of lead acetate and sterile water via the ear vein. The solution was prepared to contain a lead dose equivalent to that contained in the oral (gavage) dose.
Dose group F is not included in this report because the dosing solution was not prepared correctly.
Intravenous dosing solutions were analyzed for lead concentration (Table 1) and the measured values were used for calculating lead bioavailability.
H. Blood Sampling
Approximately 0.2-0.5 mL of blood were drawn from the vena cava at 0.5, 1,2,4, 8,12, 24,48,72,96,120, 144,192, and 240 hours after dosing. All blood samples were stored frozen at -20C, then sent to DuPont Chambers Works for analysis.
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Absolute BioavailabiUty of Lead-Contaminated Soils In Microswine
DuPont HLR581-94
I. Body Weights, Clinical Observations, and Mortality
All microswine were weighed and carefully examined for clinical signs twice each week.
On test day 1, animal #1094 (Group B) was accidentally killed as a result of a dosing accident. In addition, it was noticed at the 4-hour blood collection time point that microswine #984 (Group A) was experiencing labored breathing. At the 24-hour blood collection time point, microswine #984 was still having difficulty breathing; it was sacrificed in extremis, and a necropsy was performed. Samples collected from these microswine were not evaluated or further used in the study.
j. Sacrifice of Animals
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 were collected, weighed, frozen at approximately -20C, and stored for potential future analysis.
K. 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.
Area under the blood lead Concentration versus time curve (AUC) for lead was determined by trapezoidal rule using the terminal elimination rate constant ((3) to extrapolate from die last blood concentration value (Figures 1-7),
Absolute bioavailability was determined as the ratio of area under the blood-lead concentration versus time curve for the oral/intxavenous groups in comparison to the dosage administered (Table 4).
% Absolute Bioavailability
AUC(oral) X. Dose(intravcnous) x 100
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.
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Absolute Bioavailability of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
RECORDS AND SAMPLE RETENTION
All original records are retained at Haskell Laboratory, Jackson Laboratory (DuPont Chambers Works), or at the DuPont Records Management Center, E. I. du Pont de Nemours and Company, Wilmington, Delaware.
RESULTS AND DISCUSSION
A- Diet Samples
Stability testing of the soils was not performed. U.S. EPA Publication SW-846, Methods for the Analysis ofSolid Waste, specifies a holding time for solid material samples of 6 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 and sucrose diets, Oreo cookies, swine bedding, and deionized water were also analyzed. Results are provided in Table 2. Results from these analyses demonstrated that no lead was present in any of the samples except the leadcontaminated soils.
B. 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.
C. Blood Lead Concentrations, Area Under the Curve, and Absolute Bioavailability
Mean blood lead concentrations are listed in Table 3. Individual blood lead concentrations are listed in Appendix C.
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Absolute BloavaUabUUy of Lead-Contaminated Soils in Microswine
DuPont HLR581-94
Individual animal AUCs were determined for the microswine and are reported in Table 5, The mean AUC for each group is as follows:
Experiment Number
1 1 1 1 2 2 2 3 3 3
Group ID
A B C D E F G H I J
Substance
Mean
Administered ue(hr)/mL
Standard Deviation
Soil AEC 3-3 Soil AEC 9-3 Lead Acetate Soil AEC 10-1 Soil PLW Lead Acetate Soil EC1 SoilFM Lead Acetate Soil PLW1
50.45
5.418
40.72
21.183
107.20
70.647
38.61
6.726
16.29*
7.126
(not used)
52.89
10.868
25.86**
6.594
28.06
8.674
49,83**
21.217
* Statistically significant difference from lead acetate (Group C) by Student t-test (p<0,05).
** Statistically significant difference from lead acetate (Group I) by Student t-test (p<0.05).
In Experiment #1 (Groups A, B, and D), the mean AUC of soils AEC 3-3, AEC 9-3, and AEC 10-1 were compared to the mean AUC of the intravenous dose (Group C), There were no statistically significant differences (Student t-test) between the AUCs of the oral (soils) and intravenous doses. The absolute oral bioavailability was 28,52% for AEC 3-3, 16.70% for AEC 9-3, and 24.12% for AEC 10-1.
In Experiment #2 (Groups E and G), the mean AUC of soils PLW and EC were also compared to the mean AUC of the intravenous dose from Group C. Group F, which received an intravenous dose, was not used in the calculations because the dose was prepared incorrectly. The Group C intravenous dose was chosen because that experiment was conductedjust 1 month earlier; Experiment #3 was not conducted until 7 months after Experiment #2. There was a significant difference (Student t-test) between the AUCs of the oral PLW soil and the intravenous dose (Group C). The absolute oral bioavailability was 30.97% for PLW and 31.72% for EC1.
In Experiment #3 (Groups H and J), the mean AUC of soils FM and PLW1 were compared to the mean AUC of the intravenous dose (Group I). There were statistically significant differences (Student t-test) between the AUCs of the oral (soils) and intravenous dose. The absolute oral bioavailability was 14.72% for FM and 9.18% for PLW1.
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Absolute BloavailabiUty of Lead-Contaminated Soils in Microswine
DuPont HLR 581-94
D, Body Weights and Clinical Observations
Exposure to lead produced no effects on body weights (Appendix D) or food consumption. No deaths or compound-related clinical observations of toxicity were observed in any group; however, two microswine died from dosing accidents (Appendix E).
CONCLUSION
The bioavailability of lead in soils following single-dose oral gavage administration to microswine were:
Test Soil
AEG 3-3 AEC9-3 AEC 10-1
PLW EC1 FM PLW1
Absolute Bioavailabilitv
28.52% 16.70% 24.12% ' 30.97% 31.72% 14.72%
9.18%.
The observed differences in oral bioavailabilities support the necessity to determine these values for each unique site mid to develop site-specific clean-up goals.
REFERENCES
1. Haskell Laboratory Report No, 671-93. Unpublished data.
2. U.S. Department of Health and Human Services, Public Health Service, National Institute of Health, Revised 1985. Guidefor the Use and Care ofLaboratory Animals.
-20-
DUP040004340
TABLES
DuPont HLR 581-94
- 21 -
DUP040004341
DuPont HLR 581-94
TABLE 1. Measured concentration of lead in intravenous and oral gavage dosing solutions.
Experimental Grouo A B C D E F G H I J
Route of Administration Oral Gavage Oral Gavage Oral Gavage Intravenous Oral Gavage Intravenous
Oral Gavage Oral Gavage Oral Gavage Intravenous
Substance Administered
AEC3-3 ABC 9-3 AEC 10-1 Pb Acetate PLW (not used) EC1 FM PLW1 Pb Acetate
Amount of Lead* 31.56 mg 43.51 mg 28.55 mg 19.12 mg 9.38 mg
Amount of Water 300 mL (deionized) 500 roL (deionized) 300 mL (deionized) 10 mL (sterile) 400 mL (deionized)
29.74 mg 11.01 mg 34.05 mg
1.76 mg
300 mL (deionized) 400 mL (deionized) 400 mL (deionized) 10 mL (sterile)
1 The amount of lead was calculated from the values of analyzed dosing solutions. See Appendix F for a sample calculation.
-22-
DUP040004342
TABLE 2, Miscellaneous lead concentrations.
DuPont HLR 581-94
TvDe of Sample
Concentration of Lead fue/mLY
Teklad Basal Chow + Sucrose
<1 ' <1
Cage Bedding
<1 73 15
Nitric Acida
<1 <1
Deionized Water
<1 0.071
Oreo Cookies
<15
a Nitric acid was used in the process to determine the concentration of lead in samples.
-23 DUP040004343
TABLE 3, Mean blood lead concentration (ixg/mL).
Collection Time Chrsi
predose 0.5 1 2 4 8 12 24 48 72 96 120 144 192 240
Group A
AF.C3-3
Mean
S.D,
0.02 0.23 0,31 0.51 0.49 0.20 0.22 0.42 0.33 0.14 0,12 0.07 0,12 0.07 0.05
0.03 0.15 0.17 0.22 0.11 0.11 0,08 0.13 0.15 0.07 0.05 0.02 0.05 0.02 0.00
Group B
ABC 9-3
Mean
S.D.
0.01 0.08 0.13 0.43 0.31 0.32 0.26 0.20 0.13 0.12 0,09 0.08 0.12 0.04
0.06
0.00 0.07 0.09 0.18 0.15 0.13 0.08 0.08 0.03 0.05 0.03 0.02 0.04 0.03 0.04
Collection Time fhrsl
predose 0.5 1 2 4 8 12 24 48 72 96 120 144 192 240
Group D
AEG 10-1
Mean
S.D.
0.01 0.06 0.12 0.15 0.42 0.27 0.24 0.26 0.19 0.16 0.13 0,08 0.07 0.06 0.05
0.02 0.06 0.10 0.13 0.29 0.10 0.08 0.08 0.03 0.05 0,03 0.03 0.03 0.02 0.01
Group E
PLW
Mean
S.D.
0.01 0.01 0.01 0,01 0.05 0.08 0.06 0,07 0.02 0.04 0.03 0.02 0.02 0,02 0.02
0.00 0.00 0.00 0.00 0.04 0.03 0.02 0.03 0.01 0.02 0,01 0.01 0.01 0.01 0.01
DuPont HLR 581-94
Group C
Lead Acetate
Mean
S.D.
0.01 3.75 3.57 1.86 0.87
0.95 0.65 0.54 0.33 0.29 0,29 0.23 0.18 0.16 0.14
0-00 4.95 4.71 1.50 0.75 0.47 0.41 0.33 0.26 0.16 0.15
0.13 0.07 0.10 0.10
Group G
EC1
Mean
S.D.
0.04 0.05 0.05 0.05 0.06 0.06
0.10 0.15 0.10 0.07 0.07 0.07 0.05 0.06 0.07
'
0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.04 0.02 0.02 0.01 0.02
0.02 0.01 0.01
-.24-
DUP040004344
DuPont HLR 581-94
TABLE 3 (continued). Mean blood lead concentration (ug/mL).
Collection Time thrs'l
predose 0.5 1 2 4 8 12 24 48 72 96 120 144 192 240
Group H
PM
Mean
S.D.
0.01 0.01 0.02 0.03 0.12 0.10 0.11 0.08 0.04 0.02 0.03 0.02 0.03 0.02 0.02
6.00
0.00 0.01 0.03 0.09 0.05 0.04 0.04 0.02 0.02 0.02 0.01 0.01 0.01 0.01
Group I
Lead Acetate
Mean
_S.D.
0.01 0.28 0.27 0.32 0.17 0.17 0.13 0.11 0:08 0.08 0.06 0.05 0.04 0.04 0.05
0.00 0.20 0.21 042 0.10 0.07 0.07 . 0.04 0.03 0.03 0.02 0.01 0.02 0.01 0.02
Group J
PLW1
Mean
S.D.
0.01 0.31 0.63 0.69 0.34 0.38 0.29 0.41 0,54 0.13 0.06 0.04 0.05 0.04 0,04
0.00 0.31 0.79 0.53 0.19 0.18 0.11 0.14 0.21
0.05 0.01 0.01 0.02 0.02 0.03
-25-
DUP040004345
DuPont HLR 581-94
TABLE 4, Absolute bioayailability of soli lead in microswine.
Substance Administered
AEG 3-3 AEC9-3 AEC 10-1
PLW ECl FM PLW1
Absolute Bioavailabilitv*
28.52% 16.70% 24.12% 30.97% 31.72% 14.72%
9.18%
"Absolute bioavailability was determined as the ratio of the area under the blood-lead concentration versus time curve for the oral/intravenous groups in comparison to the dosage size administered.
-26-
DUP040004346
DuPont HLR 581-94
TABLE 5, Area under the blood concentration-time curve fAUCI (ug(hr)/mL).
Test Substance
Animal #1
Animal #2
Animal #3
Animal #4
Animal #5'
Animal #6
AEC 3-3
57.63
44.56
AEC 9-3
32.55
Pb Acetate (Grp C) 96.13
27.11 214.34
AEC 10-1
32.91
40.88
PLW
14.75
14.37
Pb Acetate
(not used)
EC1
46.87
44.15
FM
18.31
19.50
Pb Acetate (Grp I) 36.31
14.83
PLW1
23,88
63.09
49.09 30.92 128.60 30.37
9.97
60.35 29.96 35.02 47.87
50.53 72.31 70.84 46,38 29.99
44,76 28.25 25.13 36.64
--
26.07 42.50 11.83
-98,46* 33,17 29.03 77.64
--
16.85
68.32
-H-* --
Mean
S,D.
50.45 40.72 107.20 38.61 16,29**
5.42 21.18 70.65
6.73 7.13
52.89 25.86* 28.06 49.83*
10.87 6,59 8.67
21.22
Note: For statistical evaluation, soils AEC 3-3,9-3,10-1, PLW and EC1 were compared to Pb Acetate (Grp C). Soils FM and PLW1 were compared to Pb Acetate (Grp 1). In addition, AUCs were multiplied by the dose value to determine statistical significance.
* Statistically different from lead acetate (p<0.05) by Student t-test.
a Value not used in calculation of the mean AUC nor the bioavailability of the test substance.
-27-
DUP040004347
FIGURES
DuPont HLR 581-94
-28 -
DUP040004348
FIGURES land2.
DuPont HLR 581-94
Figure 1. Mean blood lead concentration in male microswine administered Pb Acetate (Group C) or AEC 3-3 (Group A).
Figure 2. Mean blood lead concentration in male microswine administered Pb Acetate (Group C) or AEC 9-3 (Group B).
-29-
D U P040004349
FIGURES 3 and 4.
DwPontHLR 581-94
Figure 3, Mean blood lead concentration in male microswine administered Pb Acetate (Group C) or AEC10-1 (Group D).
Figure 4. Mean blood lead concentration in male microswine administered Pb Acetate (Group C) or PLW (Group E),
-30-
DUP040004350
FIGURES 5 AND 6.
DuPont HLR 581-94
Figure 5. Mean blood lead concentration in male microswine adiministered Pb Acetate (Group C) or EC1 (Group G).
Figure 6. Mean blood lead concentration in male microswine adiministered Pb Acetate (Group I) or FM (Group H),
-31
DUP040004351
FIGURE 7.
DuPont HLR 581-94
Blood Lead Concentration (ng/mL)
Figure 7. Mean blood lead concentration In male microswine adiministered Pb Acetate (Group I) or PLW1 (Group J).
-32 DUP040004352
APPENDICES
DuPont HLR 581-94
-33-
DUP040004353
APPENDIX A, Protocol and protocol amendments.
DuPont HLR 581-94
-34-
DUP040004354
DuPont HLR 581-94
Protocol #1: Soils ABC 3-3, AEC 9-3, and AEC10-1
ABSOLUTE BIOAVAILABILlTy OF SOIL LEAD IN MICROSWINE: SOILS AEC 3-3, AEC 9-3, AND AEC 10-1
MEDICAL RESEARCH PROJECT NO. 9727-001 PROTOCOL
N 26889.01
Stlnc-HaskeU Animal Welfare Committee No. CBT51-P
-35-
D U P040004355
DuPont HLR 581-94
Protocol #1: Soils AEC 3-3, AEC 9-3, and AEC10-1
Protocol Absolute BloarallabUlty ofSoil Lead fa Mkronrfae: Soli*AEC 3-3, AEC 9-3, and AEC 10-1
TABLE OF CONTENTS
page 2
OBJECTIVE.
.3
SPONSOR AND TEST FACILITY-
___ 3
STUDY DESIGN..
.3
MATERIALS AND METHODS.
.4
A. Test Materials.
.4
B. Test Species.
.4
C. Pretest Procedures.
.4
D. Assignment to Groups.
.5
E. Animal Husbandry. F. Diet Preparation and Analysis. G. Body Weights..
.5
.5 .5
H. DosingTM..TMTMTM.TMTM.................TMTMTM...TM..TM........TM.TM.....TM,,TM,,TM....TM.TM..6
L Blood SamplingTMTM. TMTM,,TM___________.._________ ____________-..........6
J. Sacrifice ofAnimals .................................... TM.,,........... ...... ...................................,, 6
K. Blood AnalysisTM.TMTM.TM...TM.TM...... TM..... .............. .................... ............ ............... .. 6
L. Data Analysis
M. Statistical Methods
SAFETY AND HOUSEKEEPING..TMTMTM.TM..................... -___________ __________7
RECORDS AND SAMPLE RETENTION. APPENDIX I.
.7
.8
A, Study Personnel .
B. Study Dates.
-36-
DUP040004356
DuPont HLR 581-94
Protocol #1: Solis AEC 3-3, AEC 9-3, and AEC10-1
Protocol Absolute BlouvaUabMIty ofSoli Lend la Mtenwwtae Sails AEC 3-3, AEC 9-3, nd AEC 10-1*
page 3
OBJECTIVE
The objective of this study is to determine the absolute bioavailability of lead from three different soils inmicroswine to aid in developing a site specific-clean-up goal.
SPONSOR AND TEST FACILITY
This study is sponsored by the Corporate Remediation Group ofDuPont Chemicals, E. L du Pont de Nemours and Company, Bellevue Corporate Center, Wilmington, Delaware. The study will be conducted at HaskellLaboratory forToxicology and Industrial Medicine, E. L du Pontde Nemours and Corapahy, Newark, Delaware, in accordance with all applicable Good Laboratory Practice Standards. The sponsor's approval was effective the date the sponsor signed the Medical Research (MR) project.
STUDY DESIGN
The study design is as follows:
twenty male microswine will be used a predosing blood sample will be drawn from each animal for levels of background
lead fifteen microswine will be dosed orally by gavage (S microswine/soil) with test
soils containing 520,570, or585 ppm lead (approximately 17 grams/microswine) five microswine will be given a single intravenous injection oflead acetate solution
containing lead-dose equivalent to that contained in the gavage dose approximately 0.5 ml ofblood will be drawn from each microswine at 0,5,1,2,4,
8,12,24,48,72,96,120,144,192, and 240 hours after dosing, blood will be collected in lead-free tubes containing heparin and stored at 4C until
analysis at sacrifice, liver and bone (both cortical and trabecular as well as whole femur)
will be collected. Weighed, and frozen at -20C
-37-
DUP040004357
DuPont HLR 581-94
Protocol #1: Soils AEC 3-3, AEC 9-3, and AEC10-1
Protocol Aiuotate Elo*rttbtUtj otSoll Lead la Mlonwurliw. SotoAEC3-3, AEC9-3, and AEC 10-1
pare 4
MATERIALS ANO METHODS
A, Test Materials
The test materials will be supplied by Aldrich, Milwaukee, Wisconsin. The lead acetate trihydeate will be assigned Haskell Laboratory sample no. 20202. The lead/soils will be assigned Haskell Laboratory sample number as follows: AEC 3-3 will be assigned no. 20366; AEC 9-3 will be assigned no. 20367; and AEC 10-1 will be assigned no. 20368. Characterization and mineralogy data of the test soils Will be maintained by the sponsor.
B. TestSpecies
Male Yucatan microswine, approximately 30 days ofage, with body weights in the range of approximately 3 to 6 kilograms will be acquired from Charles River Laboratories, Inc., Windham, Maine. The weanling Charles Rivermicroswine has been selected on the bases of its low body weight, its suitability as a model ofsoil-lead ingesrion by children, and its suitability with respect to longevity, hardiness, sensitivity, and low incidence of spontaneous disease.
C. Pretest Procedures
Microswine will be housed according to the Guide for Care and Use of Laboratory Animals (NIH Publications No. 86-23, Revised 1985).
The quarantine period will be approximately two weeks, during which microswine will be fed an optimized diet (once per day) ofmicroswine diet (Charies River) and transferred gradually to Teldad basal mix (95%) formicroswine. Microswine will be provided deionized water ad libitum, weighed three times, and observed with respect to eating habits,weight gain, and any signs ofdisease or injury.
Animals thatthe or are sacrificed in extremis during the quarantine period will be necropsied to cheek for the presence ofdisease. Animals Wifi 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 (he study room for pathogens.
-38-
DUP040004358
DuPont HLR 581-94
Protocol#!; Soils AEC 3-3, AEC 9-3, and AEC10-1
Protocol Absolute BlorlUblUtyofSoU Lead In Mlcrotwlnc: SoHiAEC3-3, AEC9-3, sod AEC 10*1
pigcS
Hie 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 according to age. Microswine will be housed individually. Each animal will be assigned its own Haskell animal number and permanently identified by a number marked on the microswine's ear.
E. Animal Husbandry
After assignment to groups, animals will be identified with a cage card and housed in stainless steel cages. All microswine will be provided optimized testdiets of approximately 340 graro/microswine/day. Deionized water will be provided ad libitum.
Animal rooms will be targeted at a tempeatureof 25 2C and a relative humidity of 50410%.
F. Diet Preparation and Analysis
During the test period, microswine in each group will be fed Swine Basal mix (95%) (TD 93034).
These diets will be custom formulated by their manufacturers so that each dietcontains complete normal nutrition foreach species. The diets will contain < 0.1 ug Pb/g.
Sucrose will be added to the diets and thoroughly mixed Cor a period of time adequate to assure homogeneous distribution in the diet Once prepared, diets will be refrigerated until used or discarded.
G. Body Weights infrequent weighing schedule.
warrantamore
-39-
DUP040004359
DuPont HLR 581-94
Protocol #1; Soils AEC 3-3, AEC 9-3, and AEC10-1
Protocol Absolute Blo*YUxblUty of Sod Lad la Microvwlac: Soils AEC 3*3,AECM) asset AEC 10-1
H. Dosing Microswine will be dosed intravenously via the ear vein. Sterile water will be used as a vehicle. Oral doses will be administered by gavage. Deionized water will be used as a vehicle. The route of administration of the lead-contaminated soil will be used by oral gavage since ingestion ofsoil lead activity presents one ofthe principle direct pathways forexposure to children. Intravenous administration of lead acetate will be performed in order to calculate area under the curve (AUC) and absolute bioavaliability oflead.
L Blood Sampling Approximately 0.5-1.0 ml of microswine venous blood will be drawn from each designated animal at 0.5,1,2,4,8,12,24,48,72,96,120,144,192, and 240 hours after dosing. Blood will be collected into a lead-free tube containing heparin and refrigerated until the time of preparation for analysis.
J. Sacrifice ofAnimals At the end of the experiment, the animals will be humanely euthanatized. Animals will be necropsied, liver and bone will be collected, weighed, frozen at approximately -20C, and stored for potential future analyses.
K. Blood Analysis
spectroscopy (GFAA), at DuPont Chambers Worts, E. L du Pont de Nemours and Company, Deepwater, New Jersey. Doting solutions, diets, and drinking water will also be analyzed.
-40-
DUP040004360
DuPont HLR 581-94
Protocol #1; Soils AEC 3-3, AEC 9-3, and AEC10-1
Protocol
,.
AbcolutcBtoaraQabUityorSoULoid LaMknuwiae SoilsAEC3-3,AEC $-3, andAEC 10-1
page 7
L. Data Analysis
K
The blood lead concentrations determined by atomic absorption will be used to calculate AUC (area under blood concentration versus timecurve) and absolute bioavailability of lead. Individual animal data as well as the mean for live animals in each group will be reported.
M. Statistical Methods
Body weights and body weightgain will be analyzed by one-way analysis ofvariance. Appropriate statistical analysis will be performed on mean AUC values between oral and intravenous administrations.
SAFETY AND HOUSEKEEPING
Good housekeeping practices will be used to avoid contamination of work areas and potential health hazards. Gloves will be worn when handling formulation, blood, animals, animal tissues, or analytical standards. Animal carcasses, feces, and diet mil be incinerated.
RECORDS AND SAMPLE RETENTION
AH original records will be retained at Haskell Laboratory or at the Records Management Center, E.Ldu Pont deNemours and Company, Wilmington, Delaware.
-41-
DUP040004361
DuPont HLR 581-94
Protocol #1: Solis AEC 3-3, AEC 9-3, and AEC XO-1
Protocol Abcolutc Bloaratlabltity ofSoil Lead la Mlcrotwlac: Solli AEC 3-3, AEC 9-3, and AEC 104
APPENDIX I
page!
A. Study Personnel
Study Sponsor: Corporate Remediation Group ofDuPont Chemicals E. I. du Pont de Nemours and Company Bellevue Corporation Center Wilmington, Delaware 19809
Contact: Timothy S. Bingman, D.A.B.T. Consultant (302) 571-7761
Study Director: Hanan N. Ghantous, PhJD. Research Toxicologist
B. Study Dates Proposed Experiment Start: October, 1993
Proposed Sponsor Approval: Date the Sponsor signed the MR. Project Proposed Experiment Completion: November, 1993
Proposed Report Issue Date: April, 1994
-43-
DUP040004362
DuPont HLR 581-94
Protocol #1; Soils AEC 3-3, AEC 9-3, and AEC10-1
Protocol Absolute B!o*YiulablKty ofSod Lead In Mkmyrluc Sods AEC 3-3, AEC 9-3, aod AEC 10-1
MEDICAL RESEARCH PROJECT NO. 9727-001
p*se9
Approved:
Jianan N. Ghantous, Ph.D. Study Director
(date)
Matthew S. Bogdanffy, 1 Manager
Biochemical Toxicology and Risk Analysis
jzk/m
(date)
cc: P.E. Ross T.S. Bingraan (REO-PH) K.McCIeaiy R.C.Rhea
-43-
DUP040004363
DuPont HLR 581-94
Protocol #1 (Amendment): Soils AEC 3-3, AEC 9-3, and AEC10-1
Protocol Amendmeat No. 1 Absolute BloaviUablUty ofSoil Lead la Mlcroswtne: Soils AEC 3-3, AEC 9-3, and AEC 10-1 (lap 1 fl
TO: MEDICAL RESEARCH PROJECTNO. 9727-001 Hie following changes arc made to the original protocol:
*>
page 4
MATERIALS AND METHODS C Pretest Procedures paragraph 4. sentence l
Delete '--and sampling non-study animals housedIn the study room Cor pathogens.' This study will not use non-study animals to test forpathogens.
pages
MATERIALSAND METHODS D. Assignment to Groups paragraph 1, sentence 1
. Delete sentence I. Replace with Tfcalthyaninials will be selected by computerized stratifiedrandomization such thatthere are no statistically significantdifferences among group body weight means.'
pageS
MATERIALSAND METHODS F. Diet Preparation andAnalysis paragraph X sentence I
Change '--normal nutrition foceaeft species.' to '--normal nutrition for this species.' This was a clerical error.
pageS
MATERIALSAND METHODS O. Body Weights paragraph 1
Add the followiog sentence: 'Concurrentwith the collection ofbody weights, microswine will be individually examined andclinicalobservationswill be recorded.'
Approved by:
l
Hanan N. Ghantous, Ph-D. Study Director
cc PJB-Ross T.S. Bingman
Matthew S. Bdgdanfl Manager/
BiochemicalToxicology a K.McCIeary Quality Assurance
(date)
'>/*--> /?3
(date)
-44-
D U P040004364
DuPont HLR 581-94
Protocol #1 (Amendment): Soils AEC 3-3, AEC 9-3, and AEC10-1
Protocol Amendment No. 2 Absolute BloxvalbblUlj ofSoil Lead In Microswinc: Soils AEC2*3,AEC 9-3, and AEC 10-1
page 1 of2
TO: MEDICAL RESEARCH PROJECT NO. 9727-001
October 18.1994
The original protocol is amended as follows;
1. Add the following to Materials andMethods, section F, Diet Preparation andAnalysis: The contaminants, ifany, In the sucrose to be used for this study were considered. Because the sucrose chosen for use was originally intended forhuman consumption, it was felt that any contaminants would be within acceptable ranges. However, the dim (containing sucrose) will be analyzed for lead content and the results from the analysis will be presented in the final report.' This addition, required by Good Laboratory Practices, was erroneously omitted from the original protocol.
2. The original protocol states that blood samples will be refrigerated until the time ofpreparation for analysis. This is changed to state that blood samples will be frozen (approximately -20*0 until preparation for analysis. This change was made because blood samples will bo held until all time points have been collected, then sent to Jackson Labs for analysis.
3. Add the following to Materials andMethods, section J, Sacrifice ofAnimals; `Animals that are sacrificed in extremis or accidentally killed will be necropsied and examined for cause ofdeath.' This addition is made to explain the procedure for animals that are sacrificed in extremis or accidentally killed.
4. The origmalprotocol stated that each microswine would receive an oral dose of test soil winch contained approximately 17 g lead. This is changed to state that each microswine would receive an Oral doseoftest soil whichcontained approximately 17 mg lead. This changeis madeto correct a typing error.
2. The amountoflead contained in each soil is changed from220,570, and 585 ppm to 1070,1120, and 1210 ppm for AEC 3-3, AEC 9-3, and AEC 10-1, respectively. This change was made based on data received afterthe sponsor seat thesoils for reanalysis of lead concentrations.
6, In protocol Amendment #1, reasons for two changes were not presented. Regarding the change describing animal grouping, random animal selection replaced selection by age in order to enhance the study design. Regarding the addition of animal-examination and recording ofclinical signs concurrent with the collection of body weights, this was always the intent and was erroneously not included in the original protocol.
7, The results of this study will be included in a final report written for Medical Research Projects (MRs) 9727,9740, and 9905. These three projects were absolute bioavailability studies of soil lead in microswine; each project used different test soils. This change is made in order to present the results more efficiently and in a timelier manner.
-45-
DUP040004365
DuPont HLR 581-94
Protocol #1 (Amendment): Soils AEC 3-3, AEG 9-3, and AEC10-1
Protocol Amendment No. 2 Absolute BlmvanabUUyofSoil Lead In Mfcroswtne: Soils ABC 3-3, ARC9-3, and AEC 10-1
page2of2
8. The intravenous dose of Pb Acetate administered in this study was intendeefto be used in comparison with thetest soils labeled AEC 3-3. AEC 9-3, and AEC 10-1, The dose ofthe Pb Acetate was calculated to beequivalent to the dose of lead received by animals orally garaged with the leadcontiining test soils. The intravenous dose of Pb Acetate used in MR9740 was designed to accomplish a similar task (at the same dose oflead per animal); however, due to technical error, the
Pb Acetate dose was incorrectly prepared. Therefore, the Pb Acetatedose group front MR 9727 will be used in comparison with the test soilsin MR 9740. Thischange is tnade in order to corrects mistake in a cost-effective manner.
Study Director
cc: T-S.Bingman
K.Mcdeaty Quality Assurance PJ3.R0SS
Biochemical Toxicology and Risk Analysis
4fiDUP040004366
Protocol #2: Soils PLW and EC1
DuPont HLR 581-94
ABSOLUTE BIOAVAILABXLrrSf OF SOIL LEAD IN MICROSWINE: SOILS PLW and EC1
MEDICAL RESEARCH PROJECT NO. 9740-001
PROTOCOL
SUne-HaskcU Animal Welfare Committee No. CDTS1-P -47-
DUP040004367
Protocol #2: Soils PLW and EC1
DuPont HLR 581-94
Protocol Absolute Btoaroilabllitir ofSoil Lend la Mlmmtac: Solti PLW .nd EC1
TABLE OF CONTENTS
page 2 ________
3-- **--** --w--*,,*--****e--****--*,-****M**e*->-----a--
SPONSOR AND TEST FACILITY...-------------------------- ----------------------------- ------------3
STUDY DESIGN______ ________________ ____________ ______ ____________________ 3
MATERIALS AND METHODS ~~~--
----------------------------------------------- 4
C. Pretest Procedures.......................-........ ......... .--_____ 4
D. Assignment to Groups............................. ...... --........................... ...............-__S
E. Animal Husbandry............................................ .-.m....~...~.....~.....m~........... 5
F. Diet Preparation and Analysts
............. S
J. Sacnfi.eeofAnimals
in.......
--.m....m..--h ..--6
K. Blood Anaiysis.WWM^Ww....^,,.......^.w...w..M-~....~~.~-.~..~.-~~v--6
L. Data Analysis m-w w *w w ..iw .w .w w ..i...--n-..wwwww.iwwwwww.w^e-wTM..-w 7
M. Statistical M6thod5MI...M..IW.M..i...W.MW*...WIWH--.HIM--HWWW..W^HMWWW*.H.WMM 7
SAFETY AND HOUSEKEEPING-------------------------------- --------...--------------------------7
RECORDS AND SAMPLE RETENTION
APPENDIX I
......------------------------------ ------- ----------------------------------------- 8
So StllCly JDSltCS
3
.48-
DUP040004368
Protocol #2: Soils PLW and EC1
DuPont HLR 581-94
Protocol Absolut* Bkwtv*U*UUUy ofSoil Lend U Mlcroovrlae: Soils PLW and-ECl
page 3
OBJECTIVE
The objective of (his study is to detenuine the absolute bioavailafeility of lead from two different soils in microswine to aid in developing a site specific-clean-up goal.
SPONSOR AND TEST FACILITY
This study is sponsored by the Corporate Remediation Group ofDuPont Chemicals, E. L du Pontde Nemours and Company, Bellevue Corporate Center, Wilmington, Delaware. The study will be conducted at Haskell Laboratory forToxicology and Industrial Medicine, E.L du Pont de Nemours and Company, Newark, Delaware, in accordance with all applicable Good Laboratory Practice Standards. The sponsor's approval was effective the dale the sponsor signed the Medical Research (MR) project
STUDY DESIGN The study design is as follows:
* eighteen male microswine will be used; a predosing blood sample will be drawn from each animal for levels ofbackground
soils containing803 or 3000 ppm lead (approximately 17 mg of lead/microswme); six microswine will be given a single intravenous injection of lead acetate solution
containing lead-dose equivalentto that contained in the gavage dose; approximately 0.5 ml of blood will be drawn from each mictoswine at 0.5,1,2,4,
8,12,24,48,72,96,120,144,192, and 240 hours after dosing; blood will be collected In lead-free tubes containing heparin and stored at
approximately -20C until analysis; and at sacrifice, liver and bone (both cortical and trabecular as well as whole femur)
will be collected, weighed, and frozen at approximately -20C.
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