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

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              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

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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

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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

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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

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       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.

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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

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NARPM Co-Chairs

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                                     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

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                            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

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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

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                             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

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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

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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 37C, 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

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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

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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

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                             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

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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

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                    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

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                            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

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                                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

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                            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

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                    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

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                    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

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