OSWER 9285.7-77 May 2007 ESTIMATION OF RELATIVE BIOAVAILABILITY OF LEAD IN SOIL AND SOIL-LIKE MATERIALS USING IN VIVO AND IN VITRO METHODS Office of Solid Waste and Emergency Response U.S. Environmental Protection Agency Washington, DC 20460 ------- UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460 OKHCtOF SOI. 113 WASTU AND CMHRC1:NCV RESPONSE OSWER 9285.7-77 MEMORANDUM SUBJECT: Estimation of Relative Bioavailability of Lead in Sojl and Soil-like Materials Using In Vivo and In Vitro Methods FROM: James E. Woolford, Director SJZ~- ^ /W Office of Superfund Remediation and Technology Innovation TO: Superfund National Policy Managers, Regions 1-10 Regional Toxics Integration Coordinators (RTICs), Regions 1-10 Purpose This memorandum addresses an in vivo swine bioavailability bioassay and an in vitro bioaccessibility assay (further described in the attached document), which generally are scientifically sound and feasible methodologies for predicting the relative bioavailability (RBA) of lead in soil and soil-like materials. The Office of Superfund Remediation and Technology Innovation (OSRTI) believes that the Regions normally should consider these particular test methodologies to be validated methodologies for quantitative use in site-specific risk assessments. The use of the recommended in vitro methodology in site risk assessment is discussed in greater detail below. This memorandum and the document released by this memorandum (U.S. EPA, 2007a) provide technical and policy guidance to the U.S. Environmental Protection Agency (EPA) staff on making risk management decisions for contaminated sites. It also provides information to the public and to the regulated community on how EPA intends to exercise its discretion in implementing its regulations at contaminated sites. It is important to understand, however, that this memorandum and attached document do not substitute for statutes that EPA administers or their implementing regulations, nor is it a regulation itself. Thus, these documents do not impose legally-binding requirements on EPA, states, or the regulated community, and may not apply to a ------- particular situation based upon the particular circumstances. Rather, these documents suggest approaches that may be used at particular sites as appropriate, given site-specific circumstances. Background Over the past several years, considerable effort has been directed at developing validated laboratory methods for determining bioavailability of soil-borne lead, arsenic, and other metals, including the development of rapid screening tools (e.g., in vitro bioaccessibility tests). The availability of new methods has reinforced the need for additional guidance on evaluating bioavailability data and incorporating this information into site-specific risk assessments. Beginning in mid-2002, the Office of Solid Waste and Emergency Response initiated an intra- agency workgroup to respond to the need for additional guidance. A bioavailability workshop was held in April 2003 that brought together a diverse group of experts from academia, industry, and government to discuss and provide input to EPA on bioavailability issues. The information shared at the workshop was used to develop recommended criteria for evaluating the validation and regulatory acceptance of alternative bioavailability test methods (see U.S. EPA, 2007b). EPA has used these recommended criteria to evaluate two separate test methods for predicting the relative bioavailability of lead. The results of this evaluation are reflected in this memorandum and the attached technical support document which are intended to facilitate national consistency in the use of lead bioavailability information in site-specific human health risk assessments. The attached document reflects comments received from offices within the Office of Solid Waste and Emergency Response, the Regions, the Office of General Counsel, and from external peer reviewers. This document was also reviewed by the EPA Science Policy Council Steering Committee. Implementation ASSESSMENT OF LEAD BIOAVAILABILITY METHODS The attached document describes methodologies for predicting lead RBA in soil and soil- like materials using either an in vivo swine bioavailability bioassay or an in vitro bioaccessibility assay (IVBA). These two methodologies generally satisfy the recommended method validation and regulatory acceptance criteria discussed in the Guidance for Evaluating the Oral Bioavailability of Metals in Soils for Use in Human Health Risk Assessment (U.S. EPA, 2007b). Thus, Regions should consider both the in vivo and the in vitro methodologies described in the attachment as potentially appropriate regulatory methodologies for determining the relative bioavailability of lead for quantitative use in site-specific risk assessments. The in vitro methodology described in the attached document can provide a tool for characterizing site-specific RBA of lead in soil that is far less resource intensive than the in vivo model. A major advantage of utilizing this in vitro methodology may be that larger numbers of soil samples can be included in the characterization of soil lead bioaccessibility/bioavailability at a site. This typically would allow characterization of variability that might be associated with ------- location, proximity to sources of lead contamination, soil characteristics, or lead mineralogy at a site, which in turn could provide a more comprehensive assessment of site-specific RBA and greater confidence in lead risk estimates. The use of this in vitro method is also consistent with Agency objectives to reduce reliance on animal testing (U.S. EPA, 1999). Therefore, the Agency supports and encourages use of this methodology in appropriate circumstances, consistent With the recommended decision framework described in Figure 1 of U.S. EPA (2007b), and considering the following additional information: I. Quality assurance. The attachment describes in vivo and in vitro approaches for predicting soil lead RBA that have undergone extensive testing and evaluation. Detailed protocols for the assays and results of inter-laboratory comparisons of the data are available (U.S. EPA, 2007a, Casteel el a/., 2006, Drexler and Brattin, 2006). These protocols have been reviewed by the Agency for site-specific application and serve as the basis for inter-laboratory comparisons and quality assurance evaluations of results obtained with the assay that are submitted to the Agency in support of site-specific risk assessments. 2. Scientific validation status. As noted above, the methodologies described in the attached document generally meet the recommended criteria for acceptance of these toxicological test methods by EPA. It should be noted that an underlying assumption in the application of these assays is that the RBA predicted for juvenile swine provides an accurate estimate of the RBA in human children. Although this assumption has not been rigorously tested, extensive physiological studies support the use of swine over other potentially feasible laboratory species (e.g., rodents) for studies of absorption of lead from the gastrointestinal tract (U.S. EPA, 2007a; Weis and LaVelle, 1991). 3. Application to children and extrapolation to adults. The juvenile swine model, described in the attachment, has been utilized as an experimental methodology for predicting RBA in human children; therefore, the prediction equations for estimating RBA from results of the in vitro assay apply to human children (but see issues raised in item #2, above). While there is evidence to indicate that absolute bioavailability of soluble lead (e.g., in food or water) varies with age, the Agency is not aware at this time of information on the age-dependence (or independence) of the RBA for lead in soil. However, existing information on the development of gastric secretion in mammals indicates that gastric acid and pepsinogen production rates and acidity are lower in the neonate than in adults. A limitation in the availability of gastric acid, if it were to affect dissolution rates of soil-borne lead in the stomach at all, would be expected to lower RBA. Thus, it is conceivable that RBA for a given lead and soil matrix could be lower in children compared to adults (U.S. EPA, 2007a), introducing additional uncertainty into RBA estimates for adults that are derived from the methodology described in the attachment. 4. Sample lead concentration limits. The 19 samples tested in the in vitro - in vivo comparison described in the attached document ranged from 1,200-14,000 ppm lead. This validation range should be sufficient for most applications of the methodology. Although ------- there is no basis for predicting that errors would necessarily be introduced into the estimates of RBA if sample concentrations outside this range were used in the in vitro methodology, use of such samples without validating comparisons with results of the in vivo swine assay generally will introduce additional uncertainty into estimates of RBA. A further constraint on the lead concentration is noted in the attachment; sample concentrations used in the in vitro bioaccessibility assay should not exceed 50,000 ppm for relatively soluble forms of lead (i.e., lead acetate, lead oxide, lead carbonate), in order to avoid saturation of the extraction fluid. However, applications of the in vitro bioaccessibility assay to such high lead concentrations is unlikely to be relevant for improving risk management decisions; thus, this limitation is not likely to be a serious constraint for use of the methodology. Should additional data become available that would suggest modification of the above limits, the Agency will issue additional guidance. 5. Particle size. All samples tested in the in vitro - in vivo comparison described in the attached document were sieved through a 60 mesh screen which excluded particles greater than 250 um. Particle size can be expected to affect dissolution rates for lead that is embedded in particles and is known to affect absolute bioavailability of lead (U.S. EPA, 1986). Therefore, additional uncertainty typically will be associated with RBA estimates based on application of the in vitro assay to samples having particle sizes larger than 250 um. In general, humans are believed to ingest particles that are predominantly smaller than 250 um in diameter (Kissel et al, 1996; Sheppard and Evenden, 1994; Driver et a/,,1989; Duggan and Inskip, 1985; Que Hee, et al., 1985; Duggan, 1983), so measures of RBA on samples more coarse than this would usually not be considered relevant to risk assessment. Likewise, RBA estimates based on in vitro bioaccessibility assays of samples that have not been processed through a 60 mesh (or finer) sieve are generally not appropriate for quantitative use in site-specific risk assessments. 6. Soil mineralogy. Results of evaluations that are described in the attached document indicate that RBA of lead in soil-like materials typically can be reliably estimated using the in vitro assay and the associated regression equation relating in vitro bioaccessibility to in vivo RBA. At present, it appears that this equation should be widely appropriate, having been found to hold true for a wide range of different soil types and lead phases from a variety of different sites. However, most of the 19 samples included in the evaluation were collected from mining and milling sites, and it is plausible that some forms of lead that do not occur at this type of site might not follow the observed correlation. Thus, whenever a sample that contains an unusual and/or untested lead phase is evaluated by the in vitro bioaccessibility protocol, this should be identified as a potential source of uncertainty. In the future, as additional samples, having a wider variety of new and different lead forms, are tested by both in vivo and in vitro methods, the applicability of the method to a wider range of lead mineralogy and soil characteristics should be more clearly defined. The Agency encourages the collection and dissemination of such data as a means for further assessing uncertainties in the application of the assays for predicting site-specific RBA. Although mineralogy is among ------- the factors that influence RBA, soil mineralogy information alone does not provide the basis for substitution of bioavailability information for quantitative risk assessment. 7. Uncertainty in predicted RBA value. As noted above, the in vitro methodology for lead (U.S. EPA, 2007a) measures 1VBA for a test material, and converts this to an estimate of RBA by application of a mathematical formula. The resulting prediction of RBA should be thought of as the best estimate of the true RBA associated with that IVBA, but the actual RBA (if measured in vivo) might be either higher or lower than the prediction, due either to authentic inter-sample variability and/or to measurement error in RBA or IVBA. In general, the best estimate of RBA is the most appropriate value for use in the IEUBK mode}, but risk assessors and risk managers should use their professional judgment to decide if calculations using other values from within the RBA prediction interval should also be evaluated as part of an uncertainty analysis. OSRTI has established a "Bioavailability Committee," which will operate under EPA's Technical Review Work Group for Metals and Asbestos (TRW), to provide technical support to those engaged in human health risk assessment at contaminated sites. Part of the Committee's responsibilities will be to review new methods for assessing bioavailability of inorganic soil contaminants (i.e., new method validation). It is anticipated that the attached document normally will serve as a template for future submissions of methods to the Bioavailability Committee. In addition, the Bioavailability Committee of the TRW will compile and evaluate information on applications of bioavailability assessments in EPA site-specific risk assessments, with the objective of promoting consistent application of the framework described in U.S. EPA (2007b) across the EPA Regions. To facilitate collection of this information, the Regions are asked to report all site-specific risk assessment applications of the in vitro lead bioaccessibility methodology or in vivo juvenile swine model to the Bioavailability Committee. The Regions are also asked to contact Aaron Yeow (veow.aaron@eDa.uov) in OSRTI or Michael Beringer (beringer.michael@epa.gov) in Region 7 of the Bioavailability Committee for information on any other bioavailability assessment methodologies under consideration for use in site risk assessment. References Casteel, S.W., C.P. Weis, G.M. Henningsen, and W. J. Brattin. 2006. Estimation of Relative Bioavailability of Lead in Soil and Soil-Like Materials Using Young Swine. Environ Health Perspect 114:1162-1171. Drexler J. and W. Brattin W. 2006. (Submitted). A Validated In Vitro Procedure for Estimating the Relative Bioavailability of Lead. Driver, J.H., J.J.Konz, and O.K. Whitmyre. 1989. Soil adherence to human skin. Bull Environ Contam Toxicol 43(6): 814-820. ------- Duggan, MJ. 1983. Contribution of lead in dust to children's blood lead. Environ Health Perspect 50: 371-381. Duggan, M.J. and M.J. Inskip. 1985. Childhood exposure to lead in surface dust and soil: a community health problem. Public Health Rev 13(1-2): 1-54. Kissel, J.C., K.Y. Richter, and R.A. Fenske. 1996. Factors affecting soil adherence to skin in hand-press trials. Bull Environ Contam Toxicol 56(5): 722-728. Que Hee, S.S., B. Peace, C.S. Clark, J.R. Boyle, R.L. Bornschein, and P.B. Hammond. 1985. Evolution of efficient methods to sample lead sources, such as house dust and hand dust, in the homes of children. Environ Res 38(1): 77-95. Sheppard, S.C. and W.G. Evenden. 1994. Contaminant enrichment and properties of soil adhering to skin. JEnviron Qual 23(3): 604-613. U.S. EPA. 1989. Risk Assessment Guidance for Superfund, Volume I: Human Health Evaluation Manual (Part A). EPA/540/1-89/002. U.S. EPA. 1999. US Submission to Meeting of OECD Working Party on Existing Chemicals. February, 1999 HPV Chemical Human Health Testing: Animal Welfare Issues and Approaches. U.S. EPA. 2007a. Estimation of Relative Unavailability of Lead in Soil and Soil-like Materials Using In Vivo and In Vitro Methods. OSWER 9285.7-77. U.S. EPA. 2007b. Guidance for Evaluating the Oral Bioavailability of Metals in Soils for Use in Human Health Risk Assessment. OSWER 9285.7-80. Weis, C.P. and J.M. LaVelle. 1991. Characteristics to consider when choosing an animal model for the study of lead bioavailability. Chem. Spec. andBioavail. 3:113-19. Attachment cc: Susan Bodine, OSWER Barry Breen, OSWER Scott Sherman, OSWER Ed Chu, Land Revitatization Staff Debbie Dietrich, OEM Matt Hale, OSW David Lloyd, OBCR John Reeder, FFRRO Susan Bromm, OSRE Dave KHng, FFEO Mary-Kay Lynch, OGC Joanne Marinelli, Superfund Lead Region Coordinator, US EPA Region 3 ------- NARPM Co-Chairs ------- OSWER 9285.7-77 May 2007 ESTIMATION OF RELATIVE BIO AVAILABILITY OF LEAD IN SOIL AND SOIL-LIKE MATERIALS USING IN VIVO AND 77V VITRO METHODS Office of Solid Waste and Emergency Response U.S. Environmental Protection Agency Washington, DC 20460 ------- ACKNOWLEDGMENTS The work described in this report is the product of a team effort involving a large number of people. In particular, the following individuals contributed significantly to the findings reported here and the preparation of this report: PROGRAM SUPPORT U.S. Environmental Protection Agency (U.S. EPA) support for the development of this report was provided by Michael Beringer, U.S. EPA Region 7, Kansas City, KS; Jim Luey, U.S. EPA Region 8, Denver, CO; and Richard Troast, formerly with U.S. EPA Office of Superfund Remediation and Technology Innovation, Washington, DC. Contractor support to U.S. EPA was provided by Syracuse Research Corporation. IN VIVO STUDIES All of the in vivo studies described in this report were planned and sponsored by U.S. EPA, Region 8. The technical direction for all aspects of the in vivo portion of this project was provided by Christopher P. Weis, PhD, DABT, and Gerry M. Henningsen, DVM, PhD, DABT/DABVT. Mr. Stan Christensen provided oversight and quality assurance support for analyses of blood during the later studies performed in this program. AJJ of the in vivo studies described in this report were performed by Stan W. Casteel, DVM, PhD, DABVT, at the Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri, Columbia, Missouri. Dr. Casteel was supported by Larry D. Brown, DVM, MPH, Ross P. Cowart, DVM, MS, DACVIM, James R. Turk, DVM, PhD, DACVP, John T. Payne, DVM, MS, DACVS, Steven L. Stockham, DVM, MS, DACVP, and Roberto E. Guzman, DVM, MS. Analysis of biological samples (blood, tissues) was performed by Dr. Edward Hindenberger, of L.E.T., Inc, Columbia, Missouri. IN VITRO STUDIES Development of the method used to estimate in vitro bioaccessibility was performed primarily by John Drexler, PhD, at the University of Colorado, Boulder, with input and suggestions from a consortium of industry, academic, and governmental personnel organized by Mr. Michael V. Ruby at Exponent. Dr. Drexler also performed all of the electron microprobe and particle size analyses of the test materials evaluated in these studies. STATISTICAL ANALYSIS Dr. Timothy Barry, U.S. EPA National Center for Environmental Economics, provided on going support in the selection and application of the statistical methods used in dose-response curve-fitting and data reduction. In addition, Glenn Shaul and Lauren Drees at U.S. EPA's National Risk Management Research Laboratory provided several rounds of valuable review comments and constructive discussions regarding statistical methodology. OSWER 9285.7-77 ------- REVIEWERS A draft of this report was provided to three independent experts for external peer review and comment. This satisfies the Agency's requirements for peer review. These reviewers were: Paul Mushak, PB Associates, Durham, NC Michael Rabinowitz, Marine Biological Laboratory, Woods Hole, MA Rosalind Schoof, Integral Consulting, Inc., Mercer Island, WA The Agency has responded to the peer review comments, as appropriate. The comments and Agency responses are contained in a responsiveness summary that has been placed in the Administrative Record. OSWER 9285.7-77 ------- EXECUTIVE SUMMARY 1.0 INTRODUCTION Reliable analysis of the potential hazard to children from ingestion of lead in environmental media depends on accurate information on a number of key parameters, including the rate and extent of lead absorption from each medium ("bioavailability"). Bioavailability of lead in a particular medium may be expressed either in absolute terms {absolute bioavailability, ABA) or in relative terms (relative bioavailability, RBA). For example, if 100 micrograms (ug) of lead dissolved in drinking water were ingested and a total of 50 ug were absorbed into the body, the ABA would be 0.50 (50%). Likewise, if 100 u,g of lead contained in soil were ingested and 30 ug were absorbed into the body, the ABA for soil would be 0.30 (30%). If the lead dissolved in water was used as the frame of reference for describing the relative amount of lead absorbed from soil, the RBA would be 0.30/0.50, or 0.60 (60%). When reliable data are available on the absolute or relative bioavailability of lead in soil, dust, or other soil-like waste material at a site, this information can be used to improve the accuracy of exposure and risk calculations at that site. Based on available information in the literature on lead absorption in humans, the U.S. Environmental Protection Agency (U.S. EPA) estimates that relative bioavailability of lead in soil compared to water and food is about 60%. Thus, when the measured RBA in soil or dust at a site is found to be less than 60%, it may be concluded that exposures to and hazards from lead in these media at that site are probably lower than typical default assumptions. Conversely, if the measured RBA is higher than 60%, absorption of and hazards from lead in these media may be higher than usually assumed. This report summarizes the results of a series of studies performed by scientists in U.S. EPA Region 8 to measure the RBA of lead in a variety of soil and soil-like test materials using both in vivo and in vitro techniques. 2.0 IN VIVO STUDIES Basic Approach for Measuring RBA In Vivo The in vivo method used to estimate the RBA of lead in a particular test material compared to lead in a reference material (lead acetate) is based on the principle that equal absorbed doses of lead will produce equal increases in lead concentration in the tissues of exposed animals. Stated another way, RBA is the ratio of oral doses that produce equal increases in tissue burden of lead. Based on this, the technique for estimating lead RBA in a test material is to administer a series of oral doses of reference material (lead acetate) and test material (site soil) to groups of experimental animals, and to measure the increase in lead concentration in one or more tissues in the animals. For each tissue, the RBA is calculated by fitting an appropriate dose-response model to the data, and then solving the equations to find the ratio of doses that produce equal responses. The final estimate of RBA for the test material then combines the RBA estimates across the different tissues. OSWER 9285.7-77 ES-1 ------- Animal Exposure and Sample Collection All animals used in this program were intact male swine approximately 5 to 6 weeks of age. In general, exposure occurred twice a day for 15 days. Most groups were exposed by oral administration, with one group usually exposed to lead acetate by intravenous injection. Lead concentrations were measured in four different tissues: blood, liver, kidney, and bone. For blood, samples were collected from each animal at multiple times during the course of the study (e.g., days 0, 1,2, 3,4, 6, 9, 12, and 15), and the blood concentration integrated over time (commonly referred to as "area under the curve" or AUC) was used as the measure of blood lead response. For liver, kidney, and bone, the measure of response was the concentration of lead in these tissues on day 15. Calculation of RBA Based on testing several different types of dose-response models to the data, it was concluded that most dose-response curves for liver, kidney, and bone lead were well described by a linear model, and that most blood lead AUC data sets were well described by an exponential model: Liver. Kidney. Bone Blood AUC A UC = a + b • [\ - exp(-c • Dose)] where C,,a,,e is the concentration of lead in a given tissue; a, b, and c are the terms of the mathematic equation used to describe the shape of the curve; and Dose is the total daily administered dose of lead (ug/kg-day). Based on these models, RBA is calculated from the best model fits as follows: test material r. kidney, bone • ® reference material lesl material ad AUC ~ , reference material Results and Discussion RBA Values for Various Test Materials Table ES-1 lists the 19 different materials tested in this program and shows the RBA values estimated using each of the four alternative endpoints (blood AUC, liver, kidney, bone). OSWER 9285.7-77 ES-2 ------- Based on an analysis that indicated that each endpoint has approximately equal reliability, the point estimate for each test material is the mean of the four endpoint-specific values. Inspection of these RBA point estimates for the different test materials reveals that there is a wide range of values across different samples, both within and across sites. For example, at the California Gulch site in Colorado, RBA estimates for different types of material range from about 6% (Oregon Gulch tailings) to 105% (Fe/Mn lead oxide sample). This wide variability highlights the importance of obtaining and applying reliable RBA data in order help to improve risk assessments for lead exposure. Correlation of RBA with Mineral Phase Available data are not yet sufficient to establish reliable quantitative estimates of RBA for each of the different mineral phases of lead that are observed to occur in the test materials. However, multivariate regression analysis between point estimate RBA values and mineral phase content of the different test materials allows a tentative rank ordering of the phases into three semi-quantitative tiers (low, medium, or high RBA), as follows: Low Bioavailability Fe(M) Sulfate Anglesite Galena Pb(M) Oxide Fe(M) Oxide Medium Bioavailability Lead Phosphate Lead Oxide High Bioavailability Cerussite Mn(M) Oxide (M) = Metal 3.0 IN VITRO STUDIES Measurement of lead RBA in animals has a number of potential benefits, but is also rather slow and costly and may not be feasible in all cases. It is mainly for this reason that a number of scientists have been working to develop alternative in vitro procedures that may provide a faster and less costly alternative for estimating the RBA of lead in soil or soil-like samples. These methods are based on the concept that the rate and/or extent of lead solubilization in gastrointestinal fluid is likely to be an important determinant of lead bioavailability in vivo, and most in vitro tests are aimed at measuring the rate or extent of lead solubilization in an extraction solvent that resembles gastric fluid. The fraction of lead which solubilizes in an in vitro system is referred to as in vitro bioaccessibility (1VBA). Description of the Method The IVBA extraction procedure is begun by placing 1.0 g of test substrate into a bottle and adding 100 mL of extraction fluid (0.4 M glycine, pH 1.5). This pH is selected because it is similar to the pH in the stomach of a fasting human. Each bottle is placed into a water bath adjusted to 37°C, and samples are extracted by rotating the samples end-over-end for 1 hour. After 1 hour, the bottles are removed, dried, and placed upright on the bench top to allow the soil OSWER 9285.7-77 ES-3 ------- to settle to the bottom. A sample of supernatant fluid is removed directly from the extraction bottle into a disposable syringe and is filtered to remove any particulate matter. This filtered sample of extraction fluid is then analyzed for lead. Results Table ES-2 summarizes the in vitro bioaccessibility results for the set of 19 different test materials evaluated under the Phase II program. As seen, IVBA values span a considerable range (min of 4.5%, max of 87%), with a mean of about 55%. This variability among test materials indicates that the rate and extent of solubilization of lead from the solid test material into the extraction fluid do depend on the attributes of the test material, and that IVBA may be a useful indication of absorption in vivo (see below). Comparison of In Vivo and In Vitro Results In order for an in vitro bioaccessibility test system to be useful in predicting the in vivo RBA of a test material, it is necessary to establish empirically that a strong correlation exists between the in vivo and the in vitro results across many different samples. Figure ES-1 shows the best fit weighted linear regression correlation between the in vivo lead RBA estimates and the in vitro lead bioaccessibility estimates for each of the 19 test materials investigated during this program. The equation of the line is: RBA = 0.878-IVBA -0.028 (r2 = 0.924) These results indicate that the in vivo RBA of lead in soil-like materials can be estimated by measuring the IVBA and using the equation above to calculate the expected in vivo RBA. Actual RBA values may be either higher or lower than the expected value, as indicated by the 95% prediction interval shown in Figure ES-1. At present, it appears that this equation is likely to be widely applicable, having been found to hold true for a wide range of different soil types and lead phases from a variety of different sites. However, most of the samples tested have been collected from mining and milling sites, and it is plausible that some forms of lead that do not occur at this type of site might not follow the observed correlation. Thus, whenever a sample that contains an unusual and/or untested lead phase is evaluated by the in vitro bioaccessibility protocol, this should be identified as a potential source of uncertainty. In the future, as additional samples with a variety of new and different lead forms are tested by both in vivo and in vitro methods, the applicability of the method will be more clearly defined, 4.0 CONCLUSIONS The data from the investigations performed under this program support the following main conclusions: 1. Juvenile swine are believed to be a useful model for the evaluation of lead absorption in children and provide a reliable system for measuring the RBA of lead in a variety of soil and soil-like materials. OSWER 9285.7-77 ES-4 ------- 2. Each of the four different endpoints employed in these studies (blood AUC, liver, kidney, bone) to estimate RBA in vivo yield reasonable data, and the best estimate of the RBA value for any particular sample is the average across all four endpoint-specific RBA values. 3. There are clear differences in the in vivo RBA of lead between different types of test material, ranging from near zero to close to 100%. Thus, knowledge of the RBA value for different types of materials at a site can be very important in improving lead risk assessments at a site. 4. Available data support the view that certain types of lead minerals are well-absorbed (e.g., cerussite, manganese lead oxide), while other forms are poorly absorbed (e.g., galena, anglesite). However, the data are not yet sufficient to allow reliable quantitative calculation or prediction of the RBA for a test material based on knowledge of the lead mineral content alone. 5. In vitro measurements of bioaccessibility performed using the protocol described in this report correlate well with in vivo measurements of RBA, at least for 19 materials tested under this program. At present, the results appear to be broadly applicable, although further testing of a variety of different lead forms is required to determine if there are exceptions to the apparent correlation. OSWER 9285.7-77 ES-5 ------- TABLE ES-1. SUMMARY OF ESTIMATED RBA VALUES FOR TEST MATERIALS Experiment 2 3 4 5 6 7 8 9 11 12 Test Material Bingham Creek Residential Bingham Creek Channel Soil Jasper County High Lead Smelter Jasper County Low Lead Yard Murray Smelter Slag Jasper County High Lead Mill Aspen Berm Aspen Residential Midvale Slag Butte Soil California Gulch Phase 1 Residential Soil California Gulch Fe/Mn PbO California Gulch AV Slag Palmerton Location 2 Palmerton Location 4 Murray Smeller Soil NIST Painl Galena-enriched Soil California Gulch Oregon Gulch Tailings Blood ADC RBA 0.34 0.30 0.65 0.94 0.47 0.84 0.69 0.72 0.21 0.19 0.88 1.16 0.26 0.82 0.62 0.70 0.86 0.01 0.07 LB 0.23 0.20 0.47 0.66 0.33 0.58 0.54 0.56 0.15 0.14 0.62 0.83 0.19 0.61 0.47 0.54 0.66 0.00 0.04 UB 0.50 0.45 0.89 1.30 0.67 1.21 0.87 0.91 0.31 0.29 1.34 1.76 0.36 1.05 0.80 0.89 1.09 0.02 0.13 Liver RBA 0.28 0.24 0.56 1.00 0.51 0.86 0.87 0.77 0.13 0.13 0.75 0.99 0.19 0.60 0.53 0.58 0.73 0.02 0.11 LB 0.20 0.17 0.42 0.75 0.33 0.54 0.58 0.50 0.09 0.09 0.53 0.69 0.11 0.41 0.37 0.42 0.52 0.00 0.04 UB 0.39 0.34 0.75 1.34 0.88 1.47 1.39 1.21 0.17 0.19 1.12 1.46 0.32 0.91 0.79 0.80 1.03 0.04 0.21 Kidney RBA 0.22 0.27 0.58 0.91 0.31 0.70 0.73 0.78 0.12 0.15 0.73 1.25 0.14 0.51 0.41 0.36 0.55 0.01 0.05 LB 0.15 0.19 0.43 0.68 0.22 0.50 0.46 0.49 0.08 0.09 0.50 0.88 0.08 0.30 0.25 0.25 0.38 0.00 0.02 UB 0.31 0.37 0.79 1.24 0.46 1.02 1.26 1.33 0.18 0.22 1.12 1.91 0.25 0.91 0.72 0.52 0.78 0.02 0.09 Femur RBA 0.24 0.26 0.65 0.75 0.31 0.89 0.67 0.73 0.11 0.10 0.53 0.80 0.20 0.47 0.40 0.39 0.74 0.01 0.01 LB 0.19 0.21 0.52 0.60 0.23 0.69 0.51 0.56 0.06 0.04 0.33 0.51 0.13 0.37 0.32 0.31 0.59 -0.01 -0.04 UB 0.29 0.31 0.82 0.95 0.41 1.18 0.89 0.97 0.18 0.19 0.93 1.40 0.30 0.60 0.52 0.49 0.93 0.03 0.06 Point Estimate RBA 0.27 0.27 0.61 0.90 0.40 0.82 0.74 0.75 0.14 0.14 0.72 1.05 0.20 0.60 0.49 0.51 0.72 0.01 0.06 LB 0.17 0.19 0.43 0.63 0.23 0.51 0.48 0.50 0.07 0.06 0.38 0.57 0.09 0.34 0.29 0.29 0.44 0.00 -0.01 UB 0.40 0.36 0.79 1.20 0.64 1.14 1.08 1.04 0.24 0.23 1.07 1.56 0.31 0.93 0.72 0.79 0.98 0.03 0.15 LB = 5% Lower Confidence Bound UB = 95% Upper Confidence Bound ------- TABLE ES-2 IN VITRO BIOACCESSIBILITY VALUES Experiment 2 2 3 3 4 4 5 5 6 6 7 7 8 9 9 11 11 12 12 Test Material 1 2 1 2 1 2 1 2 1 2 1 2 1 1 2 1 2 1 3 Sample Bingham Creek Residential Bingham Creek Channel Soil Jasper County High Lead Smelter Jasper County Low Lead Yard Murray Smelter Slag Jasper County High Lead Mill Aspen Berm Aspen Residential Midvale Slag Butte Soil California Gulch Phase I Residential Soil California Gulch Fe/Mn PbO California Gulch AV Slag Palmerton Location 2 Palmerton Location 4 Murray Smelter Soil N 1ST Paint Galena-enriched Soil California Gulch Oregon Gulch Tailings In Vitro Bioaccessibility (%) (Mean ± Standard Deviation) 47.0 ±1.2 37.8 ±0.7 69.3 ±5.5 79.0 ± 5.6 64.3 ±7.3 85.3 ± 0.2 64.9 ± 1.6 71.4 ±2.0 17.4 ±0.9 22.3 ±0.6 65.1 ±1.5 87.2 ±0.5 9.4 ±1.6 63.6 ± 0.4 69.7 ±2.7 74.7 ± 6.8 72.5 ±2.0 4.5 ±1.2 11. 2 ±0.9 ------- FIGURE ES-1. RELATION BETWEEN RBA AND IVBA CO 95% Prediction Interval 0.878* VBA - 0.028 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 IVBA ------- TABLE OF CONTENTS 1.0 INTRODUCTION 1 1.1 Overview 1 1.2 Using Bioavailability Data to Improve Exposure Calculations for Lead 2 1.3 Overview of U.S. EPA's Program to Study Lead Bioavailability in Animals 3 1.4 Overview of Methods for Estimating Lead RBA In Vitro 3 2.0 IN VIVO STUDIES 4 2.1 Basic Approach for Measuring RBA In Vivo 4 2.2 Animal Exposure and Sample Collection 4 2.3 Preparation of Biological Samples for Analysis 5 2.4 Data Reduction 5 2.5 Results and Discussion 6 2.5.1 Effect of Dosing on Animal Health and Weight 6 2.5.2 Time Course of Blood Lead Response 6 2.5.3 Dose-Response Patterns 7 2.5.4 Estimation of ABA for Lead Acetate 7 2.5.5 Estimation of RBA for Lead in Test Materials 8 2.5.6 Effect of Food 9 2.5.7 Correlation of RBA with Mineral Phase 10 2.5.8 Quality Assurance 12 3.0 IN VITRO STUDIES 14 3.1 Introduction 14 3.2 In Vitro Method 14 3.2.1 Sample Preparation 14 3.2.2 Apparatus 14 3.2.3 Selection of IVBA Test Conditions 15 3.2.4 Summary of Final Leaching Protocol 16 3.2.5 Analysis of Extraction Fluid for Lead 17 3.2.6 Quality Control/Quality Assurance 17 3.3 Results and Discussion 18 3.3.1 IVBA Values 18 3.3.2 Comparison with In Vivo Results 19 4.0 REFERENCES 21 OSWER 9285.7-77 ------- LIST OF TABLES TABLE TITLE 2-1 Typical Feed Composition 2-2 Typical In Vivo Study Design 2-3 Description of Phase II Test Materials 2-4 Relative Lead Mass of Mineral Phases Observed in Test Materials 2-5 Matrix Associations for Test Materials 2-6 Particle Size Distributions for Test Materials 2-7 Estimated RBA Values for Test Materials 2-8 Grouped Lead Phases 2-9 Curve Fitting Parameters for Oral Lead Acetate Dose-Response Curves 2-10 Reproducibility of RBA Measurements 3-1 In Vitro Bioaccessibility Values LIST OF FIGURES FIGURE TITLE 2-1 Average Rate of Body Weight Gain in Test Animals 2-2 Example Time Course of Blood Lead Response 2-3 Dose Response Curve for Blood Lead AUC 2-4 Dose Response Curve for Liver Lead Concentration 2-5 Dose Response Curve for Kidney Lead Concentration 2-6 Dose Response Curve for Femur Lead Concentration 2-7 Estimated Group-Specific RBA Values 2-8 Correlation of Duplicate Analyses 2-9 Results for CDC Blood Lead Check Samples 2-10 Interlaboratory Comparison of Blood Lead Results 3-1 In Vitro Bioaccessibility Extraction Apparatus 3-2 Effect of Temperature, Time, and pH on IVBA 3-3 Precision of In Vitro Bioaccessibility Measurements 3-4 Reproducibility of In Vitro Bioaccessibility Measurements 3-5 RBA vs. IVBA 3-6 Prediction Interval for RBA Based on Measured IVBA OSWER 9285.7-77 11 ------- LIST OF APPENDICES APPENDIX TITLE A Evaluation of Juvenile Swine as a Model for Gastrointestinal Absorption in Young Children B Detailed Description of Animal Exposure C Detailed Methods of Sample Collection and Analysis D Detailed Methods for Data Reduction and Statistical Analysis E Detailed Dose-Response Data and Model Fitting Results F Detailed Lead Speciation Data for Test Materials OSWER 9285.7-77 111 ------- ACRONYMS AND ABBREVIATIONS °C Degrees Celsius jxg Microgram [Am Micrometer ABA Absolute bioavailability AF0 Oral absorption fraction AIC Akaike's Information Criterion AUC Area under the curve cc Cubic centimeter CDC Centers for Disease Control and Prevention dL Deciliter g Gram GLP Good Laboratory Practices HC1 Hydrochloric acid HOPE High density polyethylene ICP-AES Inductively Coupled Plasma-Atomic Emission Spectrometry ICP-MS Inductively Coupled Plasma-Mass Spectrometry IV Intravenous IVBA In vitro bioaccessibility kg Kilogram L Liter M Molar (M) Metal MDL Method detection limit mg Milligram mL Milliliter mm Millimeter N1ST National Institute of Standards and Testing Pb Lead PbAc Lead acetate ppm Parts per mil lion RBA Relative bioavailability RLM Relative lead mass OSWER 9285.7-77 IV ------- ACRONYMS AND ABBREVIATIONS (CONTINUED) rpm Revolutions per minute SOP Standard operating procedure SRM Standard Reference Material TAL Target Analyte List TCLP Toxicity Characteristic Leaching Procedure U.S. EPA U.S. Environmental Protection Agency OSWER 9285.7-77 ------- |