BIOAVAILABILITY OF LEAD IN A SLAG SAMPLE
FROM THE MIDVALE SLAG NPL SITE
MIDVALE, UTAH
June 1998
II
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BIO AVAILABILITY OF LEAD IN A SLAG SAMPLE
FROM THE MIDVALE SLAG NPL SITE
MIDVALE, UTAH
June 1998
Stan W. Casteel, DVM, PhD, DABVT
Principal Investigator
Veterinary Medical Diagnostic Laboratory
College of Veterinary Medicine
University of Missouri, Columbia
Columbia, Missouri
Christopher P. Weis, PhD, DABT
Gerry M. Henningsen, DVM, PhD, DABT/DABVT
Eva Hoffman, PhD
Study Design and Technical Advisors
US Environmental Protection Agency
Region VIII
Denver, Colorado
William J. Brattin, PhD
Tracy L. Hammon, MS
Technical Consultants
ISSI Consulting Group, Inc.
Denver, Colorado
Document Control Number 04800-030-0166
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ACKNOWLEDGEMENTS
The work described in this report is the product of a team effort involving a number of
people. In particular, the authors would like to acknowledge the efforts and support of the
following:
Dr. John Drexler at the University of Colorado, Boulder, performed the electron microprobe
and particle size analyses of the test materials.
Dr. Dan Paschal at the Centers for Disease Control and Prevention (CDCP) provided samples
of blood for use as internal quality control samples, and also performed independent
preparation and analyses of blood lead samples from the study for interlaboratory
comparisons.
Mr. Stan Christensen of the USEPA has provided oversight and quality assurance support
regarding many aspects of the analytical phases of this study.
EPA's Environmental Services Division (ESD) performed the analyses of all of the samples
generated during this study, including blood, liver, kidney, bone, feed, water, and
miscellaneous other materials.
Ms. Regina Prevosto at Roy F. Weston provided quality assurance oversite and review, and
assisted in development of data organization and analysis protocols.
Mr. Gerald Almquist at Roy F. Weston provided overall program management for the
project, including management of subcontractors and coordination of interactions between
team members.
Ross P. Cowart, DVM, MS, University of Missouri, Columbia, provided expert evaluation of
the health of the animals on study
Roberto E. Guzman, DVM, MS, University of Missouri, Columbia, assisted with
dosing, feeding, sample collection and sample preparation
Matthew F. Starost, DVM, University of Missouri, Columbia,., assisted with dosing, feeding,
sample collection and sample preparation
James R. Turk, DVM, PhD, University of Missouri, Columbia, performed necropsy and
pathological examination of all animals
John T. Payne, DVM, MS, University of Missouri, Columbia, performed the surgery to
implant intravenous catheters and vascular access ports
Steven L. Stockham, DVM, MS, University of Missouri, Columbia, assessed clinical
pathology data.
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EXECUTIVE SUMMARY
A study using young swine as test animals was performed to measure the gastrointestinal
absorption of lead from a slag sample from the Midvale Slag National Priority List site in
Mid vale, Utah. Young swine were selected for use in the study primarily because the
gastrointestinal physiology and overall size of young swine are similar to that of young
children, who are the population of prime concern for exposure to lead.
The test material was collected from the northern portion of OU 2 at the Midvale Slag site.
The sample contained 7,900 ppm lead. Groups of 5 swine were given average oral doses of
9.5, 28.5, or 85.5 mg/kg-d of test material for 15 days. This corresponded to target average
doses of 75, 225, or 675 ug/kg/day of lead. Other groups of animals were given a standard
lead reference material (lead acetate) either orally at doses of 0, 75 or 225 ug Pb/kg-day, or
intravenously at a dose of 100 ug Pb/kg-day. The amount of lead absorbed by each animal
was evaluated by measuring the amount of lead in the blood (measured on days -4, 0, 1, 2,
3, 5, 7, 9, 12, and 15), and the amount of lead in liver, kidney and bone (measured on day
15 at study termination). The amount of lead present in blood or tissues of animals exposed
to test material was compared to that for animals exposed to lead acetate, and the results
were expressed as relative bioavailability (RBA). For example, a relative bioavailability of
50% means that 50% of the lead in test material was absorbed equally as well as lead from
lead acetate, and 50% behaved as if it were not available for absorption. Thus, if lead
acetate were 40% absorbed, the test material would be 20% absorbed.
The RBA results for the sample from the Midvale Slag site are summarized below:
Measurement
Endpoint
Blood Lead AUC
Liver Lead
Kidney Lead
Bone Lead
Estimated
RBA for Lead
0.20
0.08
0.08
0.09
Because the estimates of RBA based on blood, liver, kidney, and bone do not agree in all
cases, judgment must be used in interpreting the data. In general, we recommend greatest
emphasis be placed on the RBA estimates derived from the blood lead data. This is because
blood lead data are more robust and less susceptible to random errors than the tissue lead
data, so there is greater confidence in RBA estimates based on blood lead. In addition,
absorption into the central compartment is an early indicator of lead exposure, is the most
relevant index of central nervous system exposure, and is the standard measurement endpoint
in investigations of this sort. However, data from the tissue endpoints (liver, kidney, bone)
also provide valuable information. We consider the plausible range to extend from the RBA
based on blood AUC to the mean of the other three tissues (liver, kidney, bone). The
ES-1
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preferred range is the interval from the RBA based on blood to the mean of the blood RBA
and the tissue mean RBA. Our suggested point estimate is the mid-point of the preferred
range. These values are presented below:
RBA Estimate
Plausible Range
Preferred Range
Suggested Point Estimate
Value
0.08 - 0.20
0.14-0.20
0.17
These RBA estimates may be used to help assess lead risk at this site by refining the estimate
of absolute bioavailability (ABA) of lead in slag, as follows:
ABAslag = ABAsoluble • RBAslag
Available data indicate that fully soluble forms of lead are about 50% absorbed by a child.
Thus, the estimated absolute bioavailability of lead in the site sample is as follows:
Absolute
Bioavailability
of Lead
Plausible Range
Preferred Range
Suggested Point Estimate
Value
4%-lO%
7%-10%
8%
These absolute bioavailability estimates are appropriate for use in EPA's IEUBK model for
this site, although it is clear that there is both natural variability and uncertainty associated
with these estimates. This variability and uncertainty arises from several sources, including :
1) the inherent variability in the responses of different individual animals to lead exposure, 2)
uncertainty in the relative accuracy and applicability of the different measurement endpoints,
3) the extrapolation of measured RBA values in swine to young children, and 4) the potential
effect of food in the stomach on lead absorption. Thus, the values reported above are judged
to be reasonable estimates of typical lead absorption by children at this site, but should be
interpreted with the understanding that the values are not certain.
ES-2
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TABLE OF CONTENTS
1.0 INTRODUCTION 1
2.0 STUDY DESIGN 3
2.1 Test Material 3
2.2 Experimental Animals 8
2.3 Diet 8
2.4 Dosing 10
2.5 Collection of Biological Samples 10
2.6 Preparation of Biological Samples for Analysis 13
2.7 Lead Analysis 14
3.0 DATA ANALYSIS 15
3.1 Overview 15
3.2 Fitting the Curves 15
3.3 Responses Below Quantitation Limits 16
3.4 Quality Assurance 16
4.0 RESULTS 20
4.1 Blood Lead vs. Time 20
4.2 Dose-Response Patterns 20
4.3 Calculated RBA Values 26
4.4 Estimated Absolute Bioavailability in Children 27
4.5 Uncertainty 27
5.0 REFERENCES 29
APPENDIX TITLE
A DETAILED DATA SUMMARY
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TABLE
2-1
2-2
2-3
2-4
LIST OF TABLES
TITLE
PAGE
Metal Analysis of Test Material 4
Geochemical Characteristics of Test Material 6
Typical Feed Composition 11
Dosing Protocol 12
LIST OF FIGURES
FIGURE TITLE PAGE
2-1 Lead Minerals Observed in Test Material 5
2-2 Particle Size Distribution 7
2-3 Body Weights of Test Animals 9
3-1 Comparison of Duplicate Analyses 18
3-2 CDCP Check Samples 19
4-1 Group Mean Blood Lead by Day 21
4-2 Blood Lead Dose-Response 22
4-3 Bone Lead Dose-Response 23
4-4 Liver Lead Dose-Response 24
4-5 Kidney Lead Dose-Response 25
11
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BIOAVAILABILITY OF LEAD IN A SLAG SAMPLE
FROM THE MIDVALE SLAG NPL SITE
MIDVALE, UTAH
1.0 INTRODUCTION
Absolute and Relative Bioavailability
Bioavailability is a concept that relates to the absorption of chemicals and how absorption
depends upon the physical-chemical properties of the chemical and its medium (e.g., dust, soil,
rock, food, water, etc.) and the physiology of the exposed receptor. Bioavailability is normally
described as the fraction (or percentage) of a chemical which enters into the blood following an
exposure of some specified amount, duration and route (usually oral). In some cases,
bioavailability may be measured using chemical levels in peripheral tissues such as liver, kidney,
and bone, rather than blood. The fraction or percentage absorbed may be expressed either in
absolute terms (absolute bioavailability, ABA) or in relative terms (relative bioavailability,
RBA). Absolute bioavailability is measured by comparing the amount of chemical entering the
blood (or other tissue) following oral exposure to test material with the amount entering the
blood (or other tissue) following intravenous exposure to an equal amount of some dissolved
form of the chemical. Similarly, relative bioavailability is measured by comparing oral
absorption of test material to oral absorption of some fully soluble form of the chemical (e.g.,
either the chemical dissolved in water, or a solid form that is expected to fully dissolve in the
stomach). For example, if 100 ug of dissolved lead were administered in drinking water and
a total of 50 ug entered the blood, the ABA would be 0.50 (50%). Likewise, if 100 ug of lead
in soil were administered and 30 ug entered the blood, the ABA for soil would be 0.30 (30%).
If the lead dissolved in water were used as the reference substance for describing the relative
amount of lead absorbed from soil, the RBA would be 0.30/0.50 = 0.60 (60%). These values
(50% absolute bioavailability of dissolved lead and 30% absolute absorption of lead in soil) are
the values currently employed as defaults in EPA's IEUBK model.
It is important to recognize that simple solubility of a test material in water or some other fluid
(e.g., a weak acid intended to mimic the gastric contents of a child) may not be a reliable
estimator of bioavailabilitv due to the non-equilibrium nature of the dissolution and transport
processes that occur in the gastrointestinal tract (Mushak 1991). For example, transport of lead
across the gut may continuously shift the equilibrium of a poorly soluble lead compound in the
direction of dissolution. However, information on the solubility of lead in different materials
is useful in interpreting the importance of solubility as a determinant of bioavailability. To avoid
confusion, the term "bioaccessability" is used to refer to the relative amount of lead that
dissolves under a specified set of test conditions.
For additional discussion about the concept and application of bioavailability see Goodman et
al. (1990), Klaassen et al. (1996), and/or Gibaldi and Perrier (1982).
1
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Using Bioavailability Data to Improve Exposure Calculations for Lead
Data on bioavailability are important for evaluating exposure and potential health effects for a
variety of different types of chemicals. This investigation focused mainly on evaluating the
bioavailability of lead in various samples of soil or other solid materials from mining, milling
or smelting sites. This is because lead may exist, at least in part, as poorly water soluble
minerals (e.g., galena), and may also exist inside particles of inert matrix such as rock or slag
of variable size, shape and association. These chemical and physical properties may tend to
influence (usually decrease) the solubility (bioaccessability) and the absorption (bioavailability)
of lead when ingested.
When data are available on the bioavailability of lead in soil, dust, or other soil-like waste
material at a site, this information can often be used to improve the accuracy of exposure and
risk calculations at that site. The basic equation for estimating the site-specific ABA of a test
soil is as follows:
ABAsoil = ABAsoluble-RBAsoil
where:
ABAsoil = Absolute bioavailability of lead in soil ingested by a child
ABAsoiubie = Absolute bioavailability in children of some dissolved or fully soluble
form of lead
RBAsoil = RBA for soil measured in swine
Based on available information on lead absorption in humans and animals, the EPA estimates
that the absolute bioavailability of lead from water and other fully soluble forms of lead is
usually about 50% in children. Thus, when a reliable site-specific RBA value for soil is
available, it may be used to estimate a site-specific absolute bioavailability as follows:
ABAsoil = 50%-RBAsoi,
In the absence of site-specific data, the absolute absorption of-.lead from soil, dust and other
similar media is estimated by EPA to be about 30%. Thus, the default RBA used by EPA for
lead in soil and dust compared to lead in water is 30%/50% = 60%. When the measured RBA
in soil or dust at a site is found to be less than 60% compared to some fully soluble form of
lead, it may be concluded that exposures to and risks from lead in these media at that site are
probably lower than typical default assumptions. If the measured RBA is higher than 60%,
absorption of and risk from lead in these media may be higher than usually assumed.
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2.0 STUDY DESIGN
A standardized study protocol for measuring absolute and relative bioavailability of lead was
developed based upon previous study designs and investigations that characterized the young pig
model (Weis et al. 1995). The study was performed as nearly as possible within the spirit and
guidelines of Good Laboratory Practices (GLP: 40 CFR 792). Standard Operating Procedures
(SOPs) that included detailed methods for all aspects of the study were prepared, approved, and
distributed to all study members prior to the study. The generalized study design, quality
assurance project plan and all standard operating procedures are documented in a project
notebook that is available through the administrative record.
2.1 Test Material
The sample tested in this study was collected from 4 locations of Pile D (Water Quenched Slag)
located in the northern portion of Mid vale Slag Operable Unit 2. The composite was prepared
for administration to the animals by air drying (maximum temperature = 40°C) followed by
sieving through a nylon mesh to yield particles less than about 250 um. This was done because
it is believed that fine particles are most likely to adhere to the hands and be ingested by hand-
to-mouth contact, and are most likely to be available for absorption. Grinding was not
employed.
The sample was analyzed for metals using standard EPA Contract Laboratory program (CLP)
methods. The results are shown in Table 2-1.
The sample of test material was well mixed and analyzed by electron microprobe in order to
identify a) how frequently particles of various lead minerals were observed, b) how frequently
different types of mineral particles occur entirely inside particles of rock or slag ("included")
and how often they occur partially or entirely outside rock or slag particles ("liberated"), c) the
size distribution of particles of each mineral class, and d) approximately how much of the total
amount of lead in the sample occurs in each mineral type. This is referred to as "relative lead
mass". The results are summarized in Figure^2-1 and in Table 2-2.
As seen in Figure 2-1, the most common lead-bearing-particle types (i.e, those which are
observed most often) were slag, accounting for about 98% of all lead-bearing particles.
However, because the concentration of lead in slag is relatively low, this phase accounted for
only about 16% of the lead mass. The remainder of the lead occurred mainly in particles of
lead-arsenic oxide (33%), other lead-metal oxides (26%), native lead (15%) and galena (6%).
Figure 2-2 shows the distribution of the size of lead-bearing particles in the sample. As seen,
there was a fairly broad distribution of lead-bearing particle sizes, mainly ranging from 50-200
um. As noted above, small particles are often assumed to be more likely to adhere to the hands
and be ingested and/or be transported into the house. Further, small particles have larger
surface area-to-volume ratios than larger particles, and so may tend to dissolve more rapidly in
the acidic contents of the stomach than larger particles. Thus, small particles (e.g. less than 50-
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TABLE 2-1 METAL ANALYSIS OF TEST MATERIAL
Chemical
Aluminum
Antimony
Arsenic
Barium
Beryllium
Cadmium
Calcium
Chromium
Cobalt
Copper
Iron
Lead
Magnesium
Manganese
Mercury
Nickel
Potassium
Selenium
Silver
Sodium
Thallium
Vanadium
Zinc
Concentration2
(ppm)
10,075
74.2
591
605
0.55
24.4
90,100
136.5
32
1,280
196,000
7,895
5,935
1,580
0.77
< 0.31
4,055
38.5
< 0.11
7,845
7.8
< 10.1
31,850
Mean of analyses of original sample and a split; all values rounded to
two significant figures
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FIGURE 2-1 LEAD MINERALS OBSERVED IN SITE MATERIAL
o%
20%
40%
60%
80%
100%
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TABLE 2-2 GEOCHEMICAL CHARACTERISTICS OF TEST MATERIAL3
Mineral
Phase
Cerrusite
Fe Pb Oxide
Galena
Native Lead
Pb-As Oxide
Lead-Metal Oxide
Slag
Sulfosalts
Ferric-Lead Sulfate
Panicle Freq.(%)
Count-Based*
0.4
0.2
0.1
3.4
6.0
3.1
86.7
0.1
0.1
Length-
Weighted1'
0.07
0.04
0.08
0.12
0.82
0.31
98.5
0.02
0.01
Particle Size11 (urn)
ruin
JO
12
80
1
1
1
10
50
15
max
45
45
100
40
100
55
600
50
15
mean
22
26
90
4
16
12
131
50
15
Relative
Lead
Mass * (%)
3.8
0.3
5.7
15.4
32.6
25.9
16
0.4
0.1
* Samples were analyzed using an electron microprobe (JEOL 8600) to identify the number of panicles of each lead species present in the
sample and the panicle size (largest dimension) of each particle.
* Percentage of all lead-bearing particles of the mineral form shown
' Percentage of total length of all lead particles consisting of mineral form shown
11 Based on longest dimension of each particle
' Rough estimate of the percent of the total mass of lead present in each mineral form
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FIGURE 2-2 PARTICLE SIZE DISTRIBUTION
o
0>
5-9 10-19
2W9 50-99 100-149 150-199 200-249 >250
Particle Size (um)
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100 um) are thought to be of greater potential concern to humans than larger particles (e.g., 100-
250 um or larger).
Another property of lead particles that may be important in determining bioaccessability and/or
bioavailability is the degree to which they are partially or entirely free from surrounding matrix
("liberated"). Based on the measured frequency of each type of particle existing in a liberated
state, it can be calculated that of the total relative lead present in the samples, about 77% exists
in liberated particles, mainly in the form of lead-arsenic oxide and lead-metal oxide. These high
percentages of partially or entirely liberated grains may tend to increase the bioavailability of
lead in the sample.
2.2 Experimental Animals
Young swine were selected for use in these studies because they are considered to be a good
physiological model for gastrointestinal absorption in children (Weis and LaVelle 1991). The
animals were intact males of the Pig Improvement Corporation (PIC) genetically defined Line
26, and were purchased from Chinn Farms, Clarence, MO. The animals were held under
quarantine to observe their health for one week before beginning exposure to the test material.
To minimize weight variations between animals and groups, the number of animals purchased
from the supplier was six more than needed for the study, and the six animals most different in
body weight on day -4 (either heavier or lighter) were excluded from further study. The
remaining animals were assigned to dose groups at random. When exposure began, the animals
were about 5-6 weeks old (juveniles, weaned at 3 weeks) and weighed an average of about 10.9
kg. Animals were weighed every three days during the course of the study. The group mean
body weights over the course of the study are shown in Figure 2-3. As seen, on average,
animals gained about 0.5 kg/day, and the rate of weight gain was comparable in all groups.
All animals were housed in individual lead-free stainless steel cages. Each animal was examined
by a certified veterinary clinician (swine specialist) prior to being placed on study, and all
animals were examined daily by an attending veterinarian while on study. Any animal that
displayed significant signs of illness was given appropriate treatment, and was removed from
study if the illness could not be promptly controlled. (This only occurred rarely, and usually
only in animals with surgically-implanted venous catheters). Blood samples were collected for
hematological analysis on days -4, 7, and 15 to assist in clinical health assessments. In this
study, there were no animals that were judged by the principle investigator and the veterinary
clinician to be seriously ill, and no animals were removed from the study due to concerns over
poor health.
2.3 Diet
Animals provided by the supplier were weaned onto standard pig chow purchased from MFA
Inc., Columbia, MO. In order to minimize lead exposure from the diet, the animals were
gradually transitioned from the MFA feed to a special low-lead feed (guaranteed less than 0.2
ppm lead, purchased from Zeigler Brothers, Inc., Gardners, PA) over the time interval from day
8
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ra
o>
"55
Q>
o»
S
I
FIGURE 2-3 BODY WEIGHTS OF TEST ANIMALS
MIDVALE SLAG
-2
- Grp 1 —B— Grp 2 —ft— Grp 3 - - X• - • Grp 4 * • * • • Grp 5 --*•-• Grp 6 —6— Grp 10
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-7 to day -3, and this feed was then maintained for the duration of the study. The feed was
nutritionally complete and met all requirements of the National Institutes of Health-National
Research Council. The typical nutritional components and chemical analysis of the feed are
presented in Table 2-3. Typically, the feed contained approximately 5.7% moisture, 1.7% fiber,
and provided about 3.4 kcal of metabolizable energy per gram. Periodic analysis of feed
samples during this program indicated the mean lead level (treating non-detects at one-half the
quantitation limit of 0.05 ppm) was less than 0.05 ppm.
Each day every animal was given an amount of feed equal to 5 % of the mean body weight of
all animals on study. Feed was administered in two equal portions of 2.5% of the mean body
weight at each feeding. Feed was provided at 11:00 AM and 5:00 PM daily. Drinking water
was provided ad libitum via self-activated watering nozzles within each cage. Periodic analysis
of samples from randomly selected drinking water nozzles indicated the mean lead concentration
(treating non-detects at one-half the quantitation limit) was less than 2 ug/L.
2.4 Dosing
The protocol for exposing animals to lead is shown in Table 2-4. Animals were exposed to lead
for 15 days, with the dose for each day being administered in two equal portions given at 9:00
AM and 3:00 PM (two hours before feeding). Doses were based on measured group mean body
weights, and were adjusted every three days to account for animal growth. For animals exposed
by the oral route, dose material was placed in the center of a small portion (about 5 grams) of
moistened feed, and this was administered to the animals by hand. Most animals consumed the
dose promptly, but occasionally some animals delayed ingestion of the dose for up to two hours
(the time the daily feed portion was provided). These delays are noted in the data provided in
Appendix A, but are not considered to be a significant source of error. Occasionally, some
animals did not consume some or all of the dose (usually because the dose dropped from their
mouth while chewing). All missed doses were recorded and the time-weighted average dose
calculation for each animal was adjusted downward accordingly. Any animal that missed 5 or
more of the 30 total oral doses administered during the study was excluded from data analysis.
There were no animals that missed doses in this study.
For animals exposed by intravenous injection, doses were given via a vascular access port (VAP)
attached to an indwelling venous catheter that had been surgically implanted according to
standard operating procedures by a board-certified veterinary surgeon through the external
jugular vein to the cranial vena cava about 3 to 5 days before exposure began.
Actual mean doses, calculated from the administered doses and the measured body weights, are
also shown in Table 2-4.
2.5 Collection of Biological Samples
Blood
10
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TABLE 2-3 TYPICAL FEED COMPOSITION3
Nutrient Name
Protein
Arginine
Lysine
Methionine
Met+Cys
Tryptophan
Histidine
Leucine
Isoleucine
Phenylalanine
Phe+Tyr
Threonine
Valine
Fat
Saturated Fat
Unsaturated Fat
Linoleic 18:2:6
Linoleic 18:3:3
Crude Fiber
Ash
Calcium
Phos Total
Available Phosphorous
Sodium
Potassium
Amount II Nutrient Name
20.1021%
1.2070%
1.4690%
0.8370%
0.5876%
0.2770%
0.5580%
1.8160%
1.1310%
1.1050%
2.0500%
0.8200%
1.1910%
4.4440%
0.5590%
3.7410%
1.9350%
0.0430%
3.8035%
4.3347%
0.8675%
0.7736%
0.7005%
0.2448%
0.3733%
Chlorine
Magnesium
Sulfur
Manganese
Zinc
Iron
Copper
Cobalt
Iodine
Selenium
Nitrogen Free Extract
Vitamin A
Vitamin D3
Vitamin E
Vitamin K
Thiamine
Riboflavin
Niacin
Pantothenic Acid
Choline
Pyridoxine
Folacin
Biotin
Vitamin B12
Amount
0.1911%
0.0533%
0.0339%
20.471 9 ppm
118.0608ppm
135. 37 10 ppm
8. 1062 ppm
0.01 10 ppm
0.2075 ppm
0.3 196 ppm
60.2340%
5.1892kIU/kg
0.6486 klU/kg
87.2080 lU/kg
0.9089 ppm
9.1681 ppm
10.2290 ppm
30. 1147 ppm
19. 1250 ppm
1019. 8600 ppm
8.2302 ppm
2.0476 ppm
0.2038 ppm
23. 44 16 ppm
Nutritional values provided by Zeigler Bros., Inc.
11
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TABLE 2-4 DOSING PROTOCOL
Group2
1
2
3
4
5
6
10
Number
of
Animals
2
5
5
5
5
5
8
Dose
Material
Administered
None
Lead acetate
Lead acetate
Midvale Slag
Midvale Slag
Midvale Slag
Lead acetate
Exposure
Route
Oral
Oral
Oral
Oral
Oral
Oral
Intravenous
Lead Dose (ug Pb/kg-d)
Target
0
75
225
75
225
675
100
Actualb
0
76.5
252
77
228
713
102
Doses were administered in two equal portions given at 9:00 AM and 3:00 PM each
day. Doses were based on the mean weight of the animals in each group, and were
adjusted every three days to account for weight gain.
a Groups 7-9 not shown; data for samples from another site
b Calculated as the administered daily dose divided by the measured or extrapolated
daily body weight, averaged over days 0-14 for each animal and each group.
12
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Samples of blood were collected from each animal four days before exposure began (day -4),
on the first day of exposure (day 0), and on days 1, 2, 3, 5, 7, 9, 12, and 15 following the start
of exposure. All blood samples were collected by vena-puncture of the anterior vena cava, and
samples were immediately placed in purple-top Vacutainer® tubes containing EDTA as
anticoagulant. Blood samples were collected each sampling day beginning at 8:00 AM,
approximately one hour before the first of the two daily exposures to lead on the sampling day
and 17 hours after the last lead exposure the previous day. This blood collection time was
selected because the rate of change in blood lead resulting from the preceding exposures is
expected to be relatively small after this interval (LaVelle et al. 1991, Weis et al. 1993), so the
exact timing of sample collection relative to last dosing is not likely to be critical.
Following collection of the final blood sample at 8:00 AM on day 15, all animals were humanely
euthanized and samples of liver, kidney, and bone (the right femur) were removed and stored
in lead-free plastic bags for lead analysis. Samples of all biological samples collected were
archived in order to allow for later reanalysis and verification, if needed. All animals were also
subjected to detailed examination at necropsy by a certified veterinary pathologist in order to
assess overall animal health.
2.6 Preparation of Biological Samples for Analysis
Blood
One mL of whole blood was removed from the purple-top Vacutainer and added to 9.0 mL of
"matrix modifier", a solution recommended by the Centers for Disease Control and Prevention
(CDCP) for analysis of blood samples for lead. The composition of matrix modifier is 0.2%
(v/v) ultrapure nitric acid, 0.5% (v/v) Triton X-100, and 0.2% (w/v) dibasic ammonium
phosphate in deionized and ultrafiltered water. Samples of the matrix modifier were routinely
analyzed for lead to ensure the absence of lead contamination.
Liver and Kidney
One gram of soft tissue (liver or kidney) was placed in a lead-free screw-cap teflon container
with 2 mL of concentrated (70%) nitric acid and heated in an-oven to 90°C overnight. After
cooling, the digestate was transferred to a clean lead-free 10 mL volumetric flask and diluted
to volume with deionized and ultrafiltered water.
Bone
The right femur of each animal was removed and defleshed, and dried at 100°C overnight. The
dried bones were then placed in a muffle furnace and dry-ashed at 450°C for 48 hours.
Following dry ashing, the bone was ground to a fine powder using a lead-free mortar and pestle,
and 200 mg was removed and dissolved in 10.0 mL of 1:1 (v:v) concentrated nitric acid:water.
After the powdered bone was dissolved and mixed, 1.0 mL of the acid solution was removed
13
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and diluted to 10.0 mL by addition of 0.1% (m/v) lanthanum oxide (La2O3) in deionized and
ultrafiltered water.
2.7 Lead Analysis
Samples of biological tissue (blood, liver, kidney, bone) and other materials (food, water,
reagents and solutions, etc.) were arranged in a random sequence and provided to EPA's
analytical laboratory in a blind fashion (identified to the laboratory only by a chain of custody
tag number). Each sample was analyzed for lead using a Perkin Elmer Model 5100 graphite
furnace atomic absorption spectrophotometer. Internal quality assurance samples were run every
tenth sample, and the instrument was recalibrated every 15th sample. A blank, duplicate and
spiked sample were run every 20th sample.
All results from the analytical laboratory were reported in units of ug Pb/L of prepared sample.
The quantitation limit was defined as three-times the standard deviation of a set of seven
replicates of a low-lead sample (typically about 2-5 ug/L). The standard deviation was usually
about 0.3 ug/L, so the quantitation limit was usually about 0.9-1.0 ug/L (ppb). For prepared
blood samples (diluted 1/10), this corresponds to a quantitation limit of 10 ug/L (1 ug/dL). For
soft tissues (liver and kidney, diluted 1/10), this corresponds to a quantitation limit of 10 ug/kg
(ppb) wet weight, and for bone (final dilution = 1/500) the corresponding quantitation limit is
0.5 ug/g (ppm) ashed weight.
14
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3.0 DATA ANALYSIS
3.1 Overview
Studies on the absorption of lead are often complicated because some biological responses to lead
exposure may be non-linear functions of dose (i.e., tending to flatten out or plateau as dose
increases). The cause of this non-linearity is uncertain but might be due either to non-linear
absorption kinetics and/or to non-linear biological response per unit dose absorbed. When the
dose-response curve for either the reference material (lead acetate) and/or the test material is
non-linear, RBA is equal to the ratio of doses that produce equal responses (not the ratio of
responses at equal doses). This is based on the simple but biologically plausible assumption that
equal absorbed doses yield equal biological responses. Applying this assumption leads to the
following general methods for calculating RBA from a set of non-linear experimental data:
1. Plot the biological responses for individual animals exposed to a series of oral
doses of soluble lead (e.g., lead acetate). Find an equation which gives a smooth
best fit line through the observed data.
2. Plot the biological response for individual animals exposed to a series of doses
of test material. Find an equation which gives a smooth fit line through the
observed data.
3. Using the best fit equations for reference material and test material, calculate
RBA as the ratios of doses of test material and reference material which yield
equal biological responses. Depending on the relative shape of the best-fit lines
through the lead acetate and test material dose response curves, RBA may either
be constant (dose-independent) or variable (dose-dependent).
The principal advantage of this approach is that it is not necessary to understand the basis for
a non-linear dose response curve (non-linear absorption and/or non-linear biological response)
in order to derive valid RBA estimates. Also, it is important to realize that this method is very
general, as it will yield correct results even if one or both of the dose-response curves are linear.
In the case where both curves are linear, RBA is dose-independent and is simply equal to the
ratio of the slopes of the best-fit linear equations.
3.2 Fitting the Curves
There are a number of different mathematical equations which can yield reasonable fits with the
dose-response data sets obtained in this study. In selecting which equations to employ, the
following principles were applied: 1) mathematically simple equations were preferred over
mathematically complex equations, 2) the shape of the curves had to be smooth and biologically
realistic, without inflection points, maxima or minima, and 3) the general form of the equations
had to be able to fit data not only from this one study, but from all the studies that are part of
15
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this project. After testing a wide variety of different equations, it was found that all data sets
could be well fitted using one of the following three forms:
Linear (LIN): Response = a + b-Dose
Exponential (EXP): Response = a 4- c-(l-exp(-d-Dose))
Combination (LIN + EXP): Response = a 4- b-Dose 4- c-(l-exp(-d-Dose))
Although underlying mechanism was not considered in selecting these equations, the linear
equation allows fitting data that do not show evidence of saturation in either uptake or response,
while the exponential and mixed equations allow evaluation of data that appear to reflect some
degree of saturation in uptake and/or response.
Each dose-response data set was fit to each of the equations above. If one equation yielded a
fit that was clearly superior (as judged by the value of the adjusted correlation coefficient R2)
to the others, that equation was selected. If two or more models fit the data approximately
equally well, then the simplest model (that with the fewest parameters) was selected. In the
process of finding the best-fits of these equations to the data, the values of the parameters (a,
b, c, and d) were subjected to some constraints, and some data points (those that were outside
the 95 % prediction limits of the fit) were excluded. These constraints and outlier exclusion steps
are detailed in Appendix A (Section 3). In general, most blood lead AUC dose-response curves
were best fit by the exponential equation, and most dose-response curves for liver, kidney, and
bone were best fit by linear equations.
3.3 Responses Below Quantitation Limit
In some cases, most or all of the responses in a group of animals were below the quantitation
limit for the endpoint being measured. For example, this was normally the case for blood lead
values in unexposed animals (both on day -4 and day 0, and in control animals), and also
occurred during the early days in the study for animals given test materials with low
bioavailability. In these cases, all animals which yielded responses below the quantitation limit
were evaluated as if they had responded at one-half the_ quantitation limit.
3.4 Quality Assurance
A number of steps were taken throughout this study and the other studies in this project to
ensure the quality of the results. These steps are summarized below.
Duplicates
A randomly selected set of about 5 % of all samples generated during the study were submitted
to the laboratory in a blind fashion for duplicate analysis. The raw data are presented in
16
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Appendix A, and Figure 3-1 plots the results for blood (Panel A, upper) and for bone, liver and
kidney (Panel B, lower). As seen, there was good intra-laboratory reproduciblity between
duplicate samples for all tissues, with linear regression lines having a slope near 1.0, an
intercept near zero, and an R2 value equal to 1.00.
Standards
The Centers for Disease Control and Prevention (CDCP) provide a variety of blood lead "check
samples" for use in quality assurance programs for blood lead studies. Each time a group of
blood samples was prepared and sent to the laboratory for analysis, several CDCP check samples
of different concentrations were included in random order and in a blind fashion.
The results for the samples submitted during this study are presented in Appendix A, and the
values are plotted in Figure 3-2 (Panel A, upper). As seen, the analytical results obtained for
the check samples were generally good at all three concentrations, with mean results of 1.5 ug/L
for the low standards (nominal =1.7 ug/L), 4.7 ug/L for the middle standard (nominal = 4.8
ug/L), and 14.1 ug/L for the high standards (nominal = 14.9 ug/L).
Intel-laboratory Comparison
An interlaboratory comparison of blood lead analytical results was performed by sending a set
of 20 randomly selected whole blood samples from this study to CDCP for blind independent
preparation and analysis. The results are presented in Appendix A, and the values are plotted
in Figure 3-2 (Panel B, lower). As seen, the results of analyses by EPA's laboratory are
generally similar to those of CDCP, with a mean inter-sample difference of 0.16 ug/L. The
slope of the best-fit straight line through the data is 0.74 if all of the data points are included,
but is 0.86 if one data point (shown by an open diamond in Panel B) for which the CDPC result
(9.6 ug/L) was noticeably higher than the EPA result (6.6 ug/L) is excluded.
Data Audits and Spreadsheet Validation
All analytical data generated by EPA's analytical laboratory were validated prior to being
released in the form of a database file. These electronic data files were "decoded" (linking the
sample tag to the correct animal and day) using Microsoft's database system ACCESS® (Version
5 for Windows). To ensure that no errors occurred in this process, original downloaded
electronic files were printed out and compared to printouts of the tag assignments and the
decoded data. All spreadsheets used to manipulate the data and to perform calculations (see
Appendix A) were validated by hand-checking random cells for accuracy.
17
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FIGURE 3-1 COMPARISON OF DUPLICATE ANALYSES
Panel A
Blood Lead
y=1.00x + 0.10
R2 = 1.00
8 10
Duplicate Value (ug/dL)
120
Panel B
Tissue Lead
y = 0.94x + 1.04
R2 = 1.00
Duplicate Value (ug/L)
Blind random duplicates submitted at a 5% rate to EPA laboratories to provide
a measure of analytical precision (reproducibility)
18
-------
FIGURE 3-2 CDCP CHECK SAMPLES
_!
5
o>
m
a
o_
-
PANEL A
17 -
16 -
14 -
• LowS.0 «
B MedStd
• HighStd 11 '
Low Nominal 10 •
- - - Med Nominal 9 •
High Nominal 8 -
7
6
"^1
B 4 1 4 -
3 -
2 _
• 1 1 -
5 -3 -1
ANALYSIS OF CDCP BLOOD LEAD CHECK SAMPLES
- ' * 14.4 * ™-° « 14.4
* 13.5
* 11.7
B 6.1
, .r., " JL4 ffi.U> T,r - ,
B 3.3
A ? A 1 Q
: ]'°* 1
1 3 5 7 9 11 13 15 1
Study Day
7
PANEL B INTERLABORATORY COMPARISON BETWEEN EPA AND CDCP
5
s
B)
0)
tr
m
^
a.
All Points
y - 0.74x + 0.62
R2 - 0.91
4567
CDCP PbB Results (ug/dL)
19
-------
4.0 RESULTS
The following sections provide results based on the group means for each dose group
investigated in this study. Appendix A provides detailed data for each individual animal.
4.1 Blood Lead vs Time
Figure 4-1 shows the group mean blood lead values as a function of time during the study. As
seen, blood lead values began below quantitation limits (about 1 ug/dL) in all groups, and
remained below quantitation limits in control animals (Group 1). In animals given repeated oral
doses of lead acetate (Groups 2 and 3) or the Midvale Slag test material (Groups 4-6), blood
levels began to rise within 1-2 days, and tended to plateau by the end of the study (day 15). A
similar pattern was observed in animals exposed to lead acetate by intravenous injection (Group
10).
4.2 Dose-Response Patterns
Blood Lead
The measurement endpoint used to quantify the blood lead response was the area under the curve
(AUC) for blood lead vs time (days 0-15). This AUC was calculated using the trapezoidal rule
to estimate the AUC between each time point that a blood lead value was measured (days 0,1,
2, 3, 5, 7, 9, 12, and 15), and summing the areas across all time intervals in the study. The
detailed data and calculations are presented in Appendix A, and the results are shown graphically
in Figure 4-2. Each data point reflects the group mean exposure and group mean response, with
the variability in dose and response shown by standard error bars. The figure also shows the
best-fit equation through each data set.
As seen, the dose response pattern is non-linear for both the soluble reference material (lead
acetate, abbreviated "PbAc") and for the test material, with the dose response curves for the test
material being clearly lower than the curve for lead acetate.
Tissue Lead
The dose-response data for lead levels in bone, liver and kidney (measured at sacrifice on day
15) are detailed in Appendix A, and are shown graphically in Figures 4-3 through 4-5,
respectively. As seen, all of these dose response curves for tissues are fit by linear equations,
with the responses (slopes) for the test material being lower than for lead acetate.
20
-------
1)
ft
1
14
12 -
10
8
4 -
2
> . '" "nl
4 -2 (
FIGURE 4-1 Group Mean Blood Lead by Day
Midvale Slag
„*
/
* * *A
* ^•."A-"*-*1"'"''' ^lJL_
* */•*"" — ~~^-A +~~- — ~~^^ - ~ 8
-i* &^*^ » -°* * " " "°
,,^* n « "jftTL" "^^_— -— 3K X H=X
_* ^0^®*^JM — ^H»t-___ * ___— -x- — " — —
J^^HTiii (T^ i * ^
) 2 4 6 8 10 12 14 1
Study Day
6
• 1 control
- @ - 2 PbAc (75)
- A - 3 PbAc (225)
X4 Midvale f?51
V R MiHwnlp /*?9^^
— • — 6 Midvale (675)
- * - 10 IV (100)
-------
200
180 -
-t eft
lOU
UT
n 14°
3
S 120
I
U lnn
3 100
<
80
_l
Jen
60 -
m
40
20
FIGURE 4-2 BLOOD LEAD DOSE-RESPONSE
GROUP MEANS ±SEM
IV
Best Fit Eqn for PbAc
y=8.0+92*{1 -exp(-Q,0086))
\
\
y
/T
/ m f , -
(.---'
4
0
_. --
^^^
. - ' ' '
-^
..-
"n
75 150
BLOOD AUC
Best Fit Eqn for Midvale Slag
y=8.0+92*{1 -*xp(-Q.001 7X))
/
/
_/....
,---•""
» *
. - • "
• Avg PbAc
X Avg Midvale
*Avg IV
225 300 375 450 525 600
Lead Dose (ug Pb/kg-d)
f Ma i
• \ " NV ^^
675 750
825
-------
DU -
45
|35
1 30
O)
3 ?<>
1
* 20
m 15
10
5
n t
FIGURE 4-3 BONE LEAD DOSE-RESPONSE
GROUP MEANS ± SEM
1
i
•
Best Fit Eqn for PbAc
y=0.45+0.043X
K!
Best Fit Eqn lor Midvale Slag
y=0.45+0,0037X
/
/
*
• Avg PbAc
0 Avg Midvale
• Avg IV
•'• i '
75 150 225 300 375 450
Lead Dose (ug/kg-d)
525
600
675
750
825
-------
FIGURE 4-4 LIVER LEAD
DOSE-RESPONSE
GROUP MEANS ± SEM
1400
01 4nnn
S 1UUU
O)
5* 800 -
•o
3
| CAA
— i OUU
*
4nn
200
(
1
r IV
Best Fit Eqn for PbAc
y=54.4+2.05X
.^
0
'f\
'I
\
\
^
\
i
75
^
150
ill
A
•
LIVER
/
/
r
Best Fit Eqn for Midvale Slag
y=54.
4+0.1 72X
/
225 300 375
450 525
I I
• Avg PbAc
BAvg Midvale
• Avg IV
600
^ " 'iui i
675 750
825
Lead Dose (ug/kg-d)
-------
to
1400
1200
^ 1000
5* 800
1
T3
S 600
1
1 400
200
0
FIGURE 4-5 KIDNEY LEAD DOSE-RESPONSE
GROUP MEANS ± SEM
f W
Best Fit Eqn for PbAc
y=39.5-H.86X
/>
K^- ^ *
0
\
-~\^
S
~?^
1 i— — —
75 150
KIDNEY
A
225
/
-
Best Fit Eqr
y=39.5+0,1
• Avg PbAc
BAvg Midvale
• Avg IV
i for Midvale Slag
54X
300 375 450 525 600
Lead Dose (ug/kg-d)
J . JEI - I
675 750
825
-------
4.3 Calculated RBA Values
Relative bioavailability values were calculated for each test material for each measurement
endpoint (blood, bone, liver, kidney) using the method described in Section 3.0. The results are
shown below:
Measurement
Endpoint
Blood Lead AUC
Liver Lead
Kidney Lead
Bone Lead
RBA
Estimate
0.20
0.08
0.08
0.09
Recommended RBA Values
As shown above, there are four independent estimates of RBA (based on blood, liver, kidney,
and bone), and the values do not agree in all cases. In general, we recommend greatest
emphasis be placed on the RBA estimates derived from the blood lead data. There are several
reasons for this recommendation, including the following:
1) Blood lead calculations are based on multiple measurements over time, and so are
statistically more robust than the single measurements available for tissue
concentrations. Further, blood is a homogeneous medium, and is easier to
sample than complex tissues such as liver, kidney and bone. Consequently, the
AUC endpoint is less susceptible to random measurement errors, and RBA values
calculated from AUC data are less uncertain.
2. Blood is the central compartment and one of the first compartments to be affected
by absorbed lead. In contrast, uptake of lead into peripheral compartments (liver,
kidney, bone) depend on transfer from blood to the tissue, and may be subject to
a variety of toxicokinetic factors that could make bioavailability determinations
more complicated.
3. The dose-response curve for blood lead is non-linear, similar to the non-linear
dose-response curve observed in children (e.g., see Sherlock and Quinn 1986).
Thus, the response of this endpoint is known to behave similarly in swine as in
children, and it is not known if the same is true for the tissue endpoints.
4. Blood lead is the classical measurement endpoint for evaluating exposure and
health effects in humans, and the health effects of lead are believed to be
proportional to blood lead levels.
26
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However, data from the tissue endpoints (liver, kidney, bone) also provide valuable information.
We consider the plausible range to extend from the RBA based on blood AUC to the mean of
the other three tissues (liver, kidney, bone). The preferred range is the interval from the RBA
based on blood to the mean of the blood RBA and the tissue mean RBA. Our suggested point
estimate is the mid-point of the preferred range. These values are presented below:
RBA Estimate
Plausible range
Preferred range
Suggested Point Estimate
Value
0.08-0.20
0.14-0.20
0.17
4.4 Estimated Absolute Unavailability in Children
These RBA estimates may be used to help assess lead risk at this site by refining the estimate
of absolute bioavailability (ABA) of lead in slag, as follows:
ABAslag = ABA,
soluble
RBAsli
Available data indicate that fully soluble forms of lead are about 50% absorbed by a child
(USEPA 1991, 1994). Thus, the estimated absolute bioavailability of lead in the site sample is
calculated as follows:
ABAMidva]e Slag = 50 % • RBAMidvale Slag
Based on the RBA values shown above, the estimated absolute bioavailability in children is as
follows:
ABA Estimate __
Plausible range
Preferred range
Suggested Point Estimate
Value
.4%
7%
- 10%
- 10%
8%
4.5 Uncertainty
These absolute bioavailability estimates are appropriate for use in EPA's IEUBK model for this
site, although it is clear that there is both variability and uncertainty associated with these
estimates. This variability and uncertainty arises from several sources. First, differences in
physiological and pharmacokinetic parameters between individual animals leads to variability in
response even when exposure is the same. Because of this inter-animal variability in the
27
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responses of different animals to lead exposure, there is mathematical uncertainty in the best fit
dose-response curves for both lead acetate and test material. This in turn leads to uncertainty
in the calculated values of RBA, because these are derived from the two best-fit equations.
Second, there is uncertainty in how to weight the RBA values based on the different endpoints,
and how to select a point estimate for RBA that is applicable to typical site-specific exposure
levels. Third, there is uncertainty in the extrapolation of measured RBA values in swine to
young children. Even though the immature swine is believed to be a useful and meaningful
animal model for gastrointestinal absorption in children, it is possible that differences in stomach
pH, stomach emptying time, and other physiological parameters may exist and that RBA values
in swine may not be precisely equal to values in children. Finally, studies in humans reveal that
lead absorption is not constant even within an individual, but varies as a function of many
factors (mineral intake, health status, etc.). One factor that may be of special importance is time
after the last meal, with the presence of food tending to reduce lead absorption. The values of
RBA measured in this study are intended to estimate the maximum uptake that occurs when lead
is ingested in the absence of food. Thus, these values may be somewhat conservative for
children who ingest lead along with food. The magnitude of this bias is not known, although
preliminary studies in swine suggest the factor may be relatively minor.
28
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5.0 REFERENCES
Gibaldi, M. and Perrier, D. 1982. Pharmacokinetics (2nd edition) pp 294-297. Marcel Dekker,
Inc, NY, NY.
Goodman, A.G., Rail, T.W., Nies, A.S., and Taylor, P. 1990. The Pharmacological Basis
of Therapeutics (8th ed.) pp. 5-21. Pergamon Press, Inc. Elmsford, NY.
Klaassen, C.D., Amdur, M.O., and Doull, J. (eds). 1996. Cassarett and Doulls Toxicology:
The Basic Science of Poisons, pp. 190. McGraw-Hill, Inc. NY,NY
LaVelle, J.M., Poppenga, R.H., Thacker, B.J., Giesy, J.P., Weis, C., Othoudt R, and
Vandervoot C. 1991. Bioavailability of Lead in Mining Waste: An Oral Intubation Study in
Young Swine. In: The Proceedings of the International Symposium on the Bioavailabilitv and
Dietary Uptake of Lead. Science and Technology Letters 3:105-111.
Mushak, P. 1991. Gastro-intestinal Absorption of Lead in Children and Adults: Overview of
Biological and Biophysico-chemical Aspects. In: The Proceedings of the International
Symposium on the Bioavailabilitv and Dietary Uptake of Lead. Science and Technology Letters
3:87-104.
Sherlock, J.C., and Quinn, M.J. 1986. Relationship Between Blood Lead Concentration and
Dietary Intake in Infants: the Glasgow Duplicate Diet Study 1979-1980. Food Additives and
Contaminants 3:167-176.
USEPA 1991. Technical Support Document on Lead. United States Environmental Protection
Agency, Environmental Criteria and Assessment Office. ECAO-CIN-757.
USEPA 1994. Guidance Manual for the Integrated Exposure Uptake Biokinetic Model for Lead
in Children. United States Environmental Protection Agency, Office of Emergency and
Remedial Response. Publication Number 9285.7-15-1. EPA/540/R-93/081.
Weis, C.P. and LaVelle, J.M. 1991. Characteristics to consider when choosing an animal
model for the study of lead bioavailability. In: The Proceedings of the International Symposium
on the Bioavailability and Dietary Uptake of Lead. Science and Technology Letters 3:113-119.
Weis, C.P., Henningsen, G.M., Poppenga, R.H., and Thacker, B.J. 1993. Pharmacokinetics
of Lead in Blood of Immature Swine Following Acute Oral and Intravenous Exposure. The
Toxicologist 13(1): 175.
Weis, C.P., Poppenga, R.H., Thacker, B.J., Henningsen, G.M., and Curtis, A. 1995.
"Design of Pharmacokinetic and Bioavailability Studies of Lead in an Immature Swine
29
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Model." In: LEAD IN PAINT. SOIL. AND DUST: HEALTH RISKS. EXPOSURE
STUDIES. CONTROL MEASURES. MEASUREMENT METHODS. AND QUALITY
ASSURANCE. ASTM STP 1226, Michael E. Beard and S. D. Allen Iske, Eds., American
Society for Testing and Materials, Philadelphia, 1995.
30
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APPENDIX A
DETAILED DATA AND CALCULATIONS FOR
USEPA SWINE BIOAVAILABILITY STUDY
PHASE II, EXPERIMENT 6
MIDVALE SLAG NPL SITE
-------
APPENDIX A
DETAILED DATA SUMMARY
1.0 OVERVIEW
Performance of this study involved collection and reduction of a large number of data items.
All of these data items and all of the data reduction steps are contained in a Microsoft Excel
spreadsheet named "MIDVALE.XLS" that is available upon request from the administrative
record. This file is intended to allow detailed review and evaluation by outside parties of all
aspects of the study.
The following sections of this Appendix present printouts of selected tables and graphs from the
XLS file. These tables and graphs provide a more detailed documentation of the individual
animal data and the data reduction steps performed in this study than was presented in the main
text. Any additional details of interest to a reader can be found in the XLS spreadsheet.
2.0 RAW DATA AND DATA REDUCTION STEPS
2.1 Body Weights and Dose Calculations
Animals were weighed on day -1 (one day before exposure) and every three days thereafter
during the course of the study. Doses of lead for the three days following each weighing were
based on the group mean body weight, adjusted by addition of 1 kg to account for the expected
weight gain over the interval. After completion of the experiment, body weights were estimated
by interpolation for those days when measurements were not collected, and the actual
administered doses (ug Pb/kg) were calculated for each day and then averaged across all days.
If an animal missed a dose or was given an incorrect dose, the calculation of average dose
corrected for these factors. (There were no missed or wrong doses in this study). These data
and data reduction steps are shown in Tables A-l and A-2.
2.2 Blood Lead vs Time
Blood lead values were measured in each animal on days -4, 0, 1, 2, 3, 5, 7, 9, 12, and 15.
The raw laboratory data (reported as ug/L of diluted blood) are shown in Table A-3. These data
were adjusted as follows: a) non-detects were evaluated by assuming a value equal to one-half
the quantitation limit, and b) the concentrations in diluted blood were converted to units of ug/dL
in whole blood by dividing by a factor of 1 dL of blood per L of diluted sample. The results
are shown in the right-hand column of Table A-3. Figures A-l to A-3 plot the results for
individual animals organized by group and by day. Figure A-4 plots the mean for each dosing
group by day.
A-l
-------
After adjustment as above, values that were more than a factor of 1.5 above or below the group
mean for any given day were "flagged" by computer as potential outliers. These values are
shown in Table A-4 by cells that are shaded gray. Each data point identified in this way was
reviewed and professional judgement was used to decide if the value should be retained or
excluded. In order to avoid inappropriate biases, blood lead outlier designations were restricted
to values that were clearly aberrant from a time-course and/or dose-response perspective. Those
which were judged to warrant exclusion are shown by a heavy black box around the value. All
other flagged values were retained.
Rarely, a value not flagged by the computer was judged to be an outlier that should be excluded.
These are shown by unshaded cells surrounded by a heavy black box.
Table A-5 provided a discussion of the rationale used to decide if a blood lead value should be
designated as an outlier or not.
2.3 Blood Lead AUC
The area under the blood lead vs time curve for each animal was calculated by finding the area
under the curve for each time step using the trapezoidal rule:
AUC(di to dj) = 0.5*(ri+rj)*(dj-di)
where:
d = day number
r = response (blood lead value) on day i (ri) or day j (rj)
The areas were then summed for each of the time intervals to yield the final AUC for each
animal. These calculations are shown in Table A-6. If a blood lead value was missing (either
because of problems with sample preparation, or because the measured value was excluded as
an outlier), the blood lead value for that day was estimated by linear interpolation.
2.4 Liver, Kidney and Bone Lead Data
At sacrifice (day 15), samples of liver, kidney and bone (femur) were removed and analyzed for
lead. The raw data (expressed as ug Pb/L of prepared sample) are summarized in Table A-7.
These data were adjusted as follows: a) non-detects were evaluated by assuming a value equal
to one-half the quantitation limit, and b) the concentrations in prepared sample were converted
to units of concentration in the original biological sample by dividing by the following factors:
Liver: 0.1 kg wet weight/L prepared sample
Kidney: 0.1 kg wet weight/L prepared sample
Bone: 2 gm ashed weight/L prepared sample
A-2
-------
The resulting values are shown in the right-hand column of Table A-7.
3.0 CURVE FITTING
Basic Equations
A commercial curve-fitting program (Table Curve-2D™ Version 2.0 for Windows, available
from Jandel Scientific) was used to derive best fit equations for each of the individual dose-
response data sets derived above. A least squares regression method was used for both linear
and non-linear equations. As discussed in the text, three different user-defined equations were
fit to each data set:
Linear (LIN): Response = a + b-Dose
Exponential (EXP): Response = a + c-(l-exp(-d-Dose))
Combination (LIN+EXP): Response = a + b-Dose + c-(l-exp(-d-Dose))
Constraints
In the process of finding the best-fits of these equations to the data, the values of the parameters
(a, b, c, and d) were constrained as follows:
• Parameter "a" (the intercept, equal to the baseline or control value of the
measurement endpoint) was constrained to be non-negative and was forced in all
cases to be the same for the reference material (lead acetate) and the test
materials. This is because, by definition, all dose-response curves for groups of
animals exposed to different materials must arise from the same value at zero
dose. In addition, for blood lead data, "a" was constrained to be equal to the
mean of the control group ±20% (typically 7.5 ± 1.5 AUC units).
• Parameter "b" (the slope of the linear dose-response line) was constrained to non-
negative values, since all of the measurement endpoints evaluated are observed
to increase, not decrease, as a function of lead exposure.
• Parameter "c" (the plateau value of the exponential curve) was constrained to be
non-negative, and was forced to be the same for the reference material (lead
acetate) and the test material. This is because: 1) it is expected on theoretical
grounds that the plateau (saturation level) should be the same regardless of the
source of lead, and 2) curve-fitting of individual curves tended to yield values of
"c" that were close to each other and were not statistically different.
A-3
-------
• Parameter "d" (which determines where the "bend" in the exponential equation
occurs) was constrained to be greater than 0.0045 for the lead acetate blood lead
(AUC) dose-response curve. This constraint was judged to be necessary because
the weight of evidence from all studies clearly showed the lead acetate blood lead
dose response curve was non-linear and was best fit by an exponential equation,
but in some studies there were only two low doses of lead acetate used to define
the dose-response curve, and this narrow range data set could sometimes be fit
nearly as well by a linear as an exponential curve. The choice of the constraint
on "d" was selected to be slightly lower than the observed best-fit value of "d"
(0.006) when data from all lead acetate AUC dose-response curves from all of the
different studies in this program were used. This approach may tend to
underestimate relative bioavailability slightly in some studies (especially at low
doses), but use of the information gained from all studies is judged to be more
robust than basing fits solely on the data from one study.
In general, one of these models (the linear, the exponential, or the combination) usually yielded
a fit (as judged by the value of the adjusted correlation coefficient R2 and by visual inspection
of the fit of the line through the measured data points) that was clearly superior to the others.
If two or more models fit the data approximately equally well, then the simplest model (that with
the fewest parameters) was selected.
Outlier Identification
During the dose-response curve fitting process, all data were carefully reviewed to identify any
anomalous values. Typically, the process used to identify outliers was as follows:
Step 1 Any data points judged to be outliers based on information derived from analysis
of data across multiple studies (as opposed to conclusions drawn from within the
study) were excluded.
Step 2 The remaining raw data points were fit to the equation judged to be the most
likely to be the best fit (linear, exponential, or mixed). Table Curve 2-D was
then used to plot the 95% prediction limits around the best fit line. All data
points that fell outside the 95 % prediction limits were considered to be outliers
and were excluded.
Step 3 After excluding these points (if any), a new best-fit was obtained. In some cases,
data points originally inside the 95% prediction limits were now outside the
limits. However, further iterative cycles of data point exclusion were not
performed, and the fit was considered final.
A-4
-------
Curve Fit Results
Table A-8 lists the data used to fit these curves, indicating which endpoints were excluded as
outliers and why. Table A-9 shows the type of equation selected to fit each data set, and the
best fit parameters. The resulting best-fit equations for the data sets are shown in Figures A-5
to A-16. Values excluded as outliers are represented in the figures by the symbol " + ".
4.0 RESULTS -- CALCULATED RBA VALUES
The value of RBA for a test substance was calculated for a series of doses using the following
procedure:
1. For each dose, calculate the expected response to test material, using the best fit
equation through the dose-response data for that material.
2. For each expected response to test material, calculate the dose of lead acetate that
is expected to yield an equivalent response. This is done by "inverting" the dose-
response curve for lead acetate, solving for the dose that corresponds to a
specified response.
3. Calculate RBA at that dose as the ratio of the dose of lead acetate to the dose of
test material. For the situation where both curves are linear, the value of RBA
is the ratio of the slopes (the "b" parameters). In the case where both curves are
exponential and where both curves have the same values for parameters "a" and
"c", the value of RBA is equal to the ratio of the "d" parameters.
The results are summarized in Table A-10.
5.0 QUALITY ASSURANCE DATA
A number of steps were taken throughout this study and the other studies in this project to
ensure the quality of the results, including 5% duplicates, 5% standards, and a program of
interlaboratory comparison. These steps are detailed below.
Duplicates
Duplicate samples were prepared and analyzed for about 5 % of all samples generated during the
study. Table A-11 lists the first and second values for blood, liver, kidney, and bone. The
results are shown in Figure 3-1 in the main text.
Standards
The Centers for Disease Control and Prevention (CDCP) provide a variety of blood lead "check
samples" for use in quality assurance programs for blood lead studies. Each time a group of
A-5
-------
blood samples was prepared and sent to the laboratory for analysis, several CDCP check samples
of different concentrations were included. Table A-12 lists the concentrations reported by the
laboratory compared to the nominal concentrations indicated by CDCP for the samples submitted
during this study, and the results are plotted in Figure 3-2 (Panel A) in the main text.
Interlaboratory Comparison
An interlaboratory comparison of blood lead analytical results was performed by sending a set
of 15 randomly selected whole blood samples from this study to CDCP for independent analysis.
The data are presented in Table A-13, and the results are plotted in Figure 3-2 (Panel B) in the
main text.
A-6
-------
DISK INSTRUCTIONS
Enclosed is a disk entitled "MIDVALE.EXE". This disk contains all of the data items and all
of the data reduction steps for the Midvale site in a Microsoft Excel spreadsheet named
"MIDVALE.XLS". This file is intended to allow detailed review and evaluation by outside
parties of all aspects of the study. In order to conserve space and help guard against accidental
changes in the spreadsheet, all of the formulas and links present in the original spreadsheet used
by EPA have been "frozen". Thus, the values shown in the attached file represent the final
values employed by EPA. Due to the size of the file (approximately 2 MB), it has been
provided as a self-extracting zipped file. To extract the file from the enclosed disk to a location
on your hard drive, the following steps should be taken:
1) Go to the DOS Prompt
2) Change directory to desired destination directory (e.g., C:\data)
3) Place the source disk in the appropriate drive (e.g., A:)
4) At the DOS prompt (C:\data >) type "A:\MIDVALE" and press enter. This will
cause the MIDVALE.XLS file to extract from your source disk (A:) to your
destination directory (C:\data).
5) Open Microsoft Excel to view the unzipped file. Note that even though the
formulas have been frozen, the file remains quite large, so it is recommended that
the user have a minimum of 8 MB of RAM to facilitate use of this spreadsheet.
A-7
-------
Seme Study PTiise 1 £«p B
TABLE A-1 BODY WEIGHTS AND ADMINISTERED DOSES, BY DAY*
Body wtifltTts w«« mMs.trcdofldi.yi-t, 2, 5,8, 11, 14. W^ghtt fcf other iiy* «r«
on inatr Bi4w?oUf0nb«l#Mtim**siJridvidu*3.
Croup S3*
1 ei4
1 638
2 «13
2 624
2 633
2 <41
3 HE
3 844
3 151
3 153
3 654
4 119
4 623
4 828
4 631
4 HT
5 602
5 «28
5 640
5 650
6 603
6 61S
< 629
6 645
10 S04
10 107
10 512
10 «35
ID 932
10 842
10 Hi
Diy-1
aw ugPb
(kg) pafday
io.a o
9.5 g
11.7 0
10.6 0
122 0
9.7 0
9.6 0
8.6 0
IDS 0
101 a
101 0
11 0
11.3 0
1.6 0
115 0
102 0
10.1 0
105 0
8.8 0
103 0
9.5 0
It 0
9.2 0
116 0
96 0
124 0
9.9 0
fl.5 0
10.1 0
11 0
II 0
D«yO
BW ugR.
(kg) podgy
ii.c a
10.0 0
122 911
110 911
126 911
9.7 2732
100 2732
11.1 2732
10-6 2732
t3.5 2732
110 879
11.7 879
10.1 179
120 879
10.5 (79
1S.S 2475
106 2475
102 2475
15.8 2475
8.8 7S03
11.2 7603
9.6 7103
120 7803
100 1141
127 1141
100 1141
111 1141
10.5 1141
11.3 1141
90 1141
0«y1
EW ugPb
(ka> pcrtfey
114 0
10.5 0
12.6 911
11.5 911
13.1 lit
9.« 2732
105 2732
11.9 2732
11. 1 2732
10.9 2732
11.! 979
121 879
10.5 879
124 879
10.9 879
11.2 2475
10.4 2475
10* 2475
t.8 780
11.5 780
10.0 780
124 76Q
103 114
131 114
103 114
120 114
10.9 114
115 114
S.2 114
Drfl
BW ugPb
fto) ordtv
11.8 0
11 0
13.1 911
115 911
135 911
9.9 2732
109 2732
12.4 2732
11.6 2732
11.2 2732
11.1 879
12.5 679
11 879
12.9 979
11.2 879
11.5 2475
10.7 2475
11,1 2475
8.9 7803
11.7 7803
104 7803
129 7803
10.7 1141
134 1141
105 1141
123 1141
112 1141
119 1141
94 1141
Days
BW »BPb
fca) parday
12.2 0
114 0
134 992
12.2 992
131 992
B.B 2975
11.1 2975
12.7 2975
!2.t 2975
11.5 2975
11.4 958
129 951
11.3 956
134 956
11.« 956
11.9 2714
11.1 2714
11.4 !714
104 8S67
12.0 8667
10.8 8667
13.2 8687
11.1 1233
139 1233
110 1233
124 1233
114 1233
123 1233
97 1233
Day 4
BW ugPb
(kgj parday
12.6 0
11.9 0
138 992
12.5 992
14.1 992
110 992
8.8 2975
11.4 2975
130 2975
12.7 2975
11.8 2975
11.6 956
132 956
116 956
139 856
121 856
12.4 2714
11.6 2714
11.6 2714
10.8 8667
124 8667
11.2 8667
136 8667
11.4 1233
14,4 1233
114 1233
125 1233
117 1233
12.7 1233
9.9 1233
Days
BW ugPb
(kg) par day
13 0
12,3 0
141 992
129 992
144 992
114 992
9.7 2875
119 2975
133 2975
13.2 2975
12.2 2S75
11.9 956
135 956
11,9 956
144 958
12.5 958
12,8 2714
12 2714
119 2714
11.3 6667
12.7 8667
11J 8$S7
14 8687
11.9 1233
14,9 1233
119 1233
128 1233
11,9 1233
13,2 1233
10.2 1233
Day 8
BW ugPi
(ksrt parday
135 0
13.2 0
146 1062
13.2 1062
15.! 1062
119 1062
10.0 3186
12.1 3199
13.2 3186
13.1 3196
128 3186
121 1038
137 1038
12,3 1038
14.8 1038
129 1038
13.4 2157
124 2957
122 2957
11.8 9329
131 9329
122 9329
145 9329
12,3 1334
154 1334
124 1334
13! 1334
123 1334
140 1334
10.4 1334
D«y7
BW uJPb
(kg) pa>*y
149 0
14.0 0
152 1062
135 1062
15.1 1062
123 1062
10.2 3186
126 3181
132 3166
12.9 3186
134 3188
12.3 1039
139 1039
12.9 1038
141 1031
134 1031
13.4 2957
14.0 2957
12.7 2957
12.5 2957
12.4 8329
138 8329
128 9329
150 9329
12.9 1334
159 1334
129 1334
137 1334
12.8 1334
147 1334
10.6 1334
B«yl
BW ug Pb
fko) parday
145 0
149 0
157 1062
139 1062
16.$ 1062
12* 1062
10.5 3196
131 3196
131 S188
129 3196
14 3198
12,5 1038
141 1039
132 1036
151 1038
13S 1036
13.9 2957
14,8 2957
131 2957
12S Z957
12.8 8329
14 9329
134 9329
IS5 8329
13.4 1334
184 1334
134 1334
142 1334
132 1334
155 1334
108 1334
Bay 8
BW ygPb
(kg) wrrJay
151 0
14.8 0
161 182
14.3 192
16,8 182
13.3 182
10.9 3546
13.8 3546
14.2 3548
14.0 3548
14.5 3546
I2.B 1106
14.5 1108
13,5 1106
15.6 1106
143 1108
14.4 3258
1S.1 J2S8
13.6 3258
13.3 3258
145 10287
139 10287
15.9 10287
13.9 1481
169 1481
139 1481
141 1491
13.7 1481
15.9 1481
11.1 1481
Day 19
BW ujPb
(kg) pat day
156 0
15.0 0
16.6 182
14,9 182
17.1 182
139 162
113 35*8
14.1 3546
153 3548
15.1 3548
15,0 3546
13,2 11D6
149 1108
138 1108
162 1108
14.8 1108
14.9 3258
15,7 325S
14.0 3259
13,7 3258
15,1 10287
14.5 10297
16,4 10237
14.5 1481
17.4 14B1
14.3 1481
15 5 1491
14.3 1481
18.2 1481
114 1481
Dayll
BW iigpi
(kg) per dry
16.2 0
15 0
17 1182
152 1182
17,4 1182
143 1182
11.7 3546
14.6 3546
164 3546
163 3546
US 3546
15.2 1106
14.1 1106
167 1108
153 1106
15.4 3258
16.2 3258
14 5 3258
142 3258
15.6 10287
15 10287
16.6 10287
15 1481
17.9 1481
148 1481
161 1481
14.8 1481
18.6 1481
11.7 1481
Day 12
BW ug Pb
(kg) par day
13.1 0
15.5 0
17.7 1274
15.1 1274
11,0 1274
ISO 1274
12.4 3821
15,3 3821
17.1 3921
15,9 3821
18,1 3821
15.5 1167
14.6 1197
17.3 1187
15.6 1197
16.0 3596
19,8 3598
15.1 3596
14.7 3598
18.2 11232
15,6 11232
17.4 11232
15.5 1S26
18.5 1628
15.4 1628
18.7 162S
15.5 1828
17.3 1628
12.3 162$
Day 13
BW uqPb
(kg) par day
17.5 0
15.9 0
184 1274
16,3 1274
18.5 1274
15.7 1274
13.0 3821
159 3821
178 3821
17.4 S921
16.7 5821
158 1197
15.1 1197
17* 1197
18.3 1197
16.5 3596
17.5 3591
15.7 3594
15.1 359S
169 11232
1S.1 11232
19.1 11232
16.1 1626
19.2 1626
15.9 1626
172 1626
1 S.2 1626
19.1 1628
1 2.9 1626
Day 14
BW ugPb
(kg) par day
19.1 0
164 0
19.1 1274
16.9 1274
19.1 1274
184 1274
13.7 3821
166 3921
185 3921
19 3921
17.3 3921
182 97
15.6 97
184 97
16.8 97
17.1 3596
18.1 3596
16.3 3596
15.6 35S6
17.5 11232
16.7 11232
18.7 1 1232
16.6 1826
19.8 1626
16.5 1626
17.8 UK
16.8 1628
16.8 1626
13.5 1628
Day 15
BW ugpb
(kg) p»r day
18.7 0
16.9 0
199 0
17.5 0
18.7 0
171 0
14.4 0
17-3 0
19.2 0
18.6 0
17,9 0
185 0
16.1 0
19.0 0
17J 0
17.7 0
18,7 0
169 0
16,1 0
18.1 0
17,3 0
18.3 0
17 0
20 0
17 0
IB 0
17,8 0
185 0
141 0
I
-J
* Graups 7,8,19 iwi shown £dat* for t«mp!« from • diff»r«rt si
-------
TABLE A-2
Body Weight Adjusted Doses
(Dose tor Day/BW for Day)
Swine Sludy PtaM II E«p 8
Group ID H
1 614
1 638
2 613
2 624
2 630
2 639
2 641
3 618
3 644
3 651
3 653
3 654
4 819
4 823
4 628
4 631
4 647
5 602
5 605
5 628
5 640
5 850
6 603
e BIS
6 629
6 633
6 645
10 604
10 60S
10 807
10 812
10 825
10 832
10 642
DayO Day 1
0 0
0 0
74.8 721
82.5 79 4
774 757
72.1 697
920 90.1
281.6 2787
2722 261.0
2453 2321
257.7 246.1
259.3 251.4
79.7 79.4
751 72.6
87.3 83,4
73.5 707
834 809
235.7 2271
2552 2426
2285 221.6
2434 2372
2342 228.5
810.0 7989
694.8 660.5
6128 7803
7S5.1 738.5
650 3 629 3
114.5 110.4
108.7 104.7
89.6 87.3
113.7 111.2
97.0 94.8
109,0 105.3
1013 S90
1288 1240
Day 2 Day 3
0 0
0 0
69.5 73.8
76.5 81 3
740 78.9
674 71.8
88 4 93.0
27S.9 302,5
250.6 267.2
220.3 234,2
235.5 2452
243.9 2579
79.2 841
70.3 745
799 848
681 713
78. 5 82.1
219.0 233.3
231.3 245.9
215.2 227.4
2313 2437
223 0 238 7
788.2 636.0
6669 720.2
750.3 802.5
722.5 780.8
609.6 656.6
106.7 111.4
101.0 106.3
852 88.7
108.7 112.4
928 99.4
101.9 107.8
96.7 100.5
121.4 127.5
Day 4 Day 5
0 0
0 0
72,0 70.3
79.3 775
77.3 75.7
70.3 68.9
89.9 87.0
3046 308.6
261.7 258.4
2288 223.6
2348 225.3
250.7 243.8
821 803
726 70,8
824 80.3
68.7 664
792 78.4
2268 2206
2387 231.9
2194 212.0
234.6 226.1
233.3 228.0
800.0 767.0
700.8 6824
773.8 7472
7603 7408
637.3 819.1
1078 104.4
1036 101.0
85.6 82.7
107.8 103.6
98.6 976
105.6 103.6
668 934
124.1 120.8
0 0
0 0
726 700
807 7B.5
775 74.3
703 67.2
89 5 86.1
3197 311.3
283.3 252.9
240 8 242.0
2438 248.3
248 9 237.8
858 844
758 74.7
842 81.3
70.9 69.8
80.3 77.7
230,4 2212
243.7 235.3
220.6 211.2
239.1 2322
242.3 2365
788.3 754.3
710.3 687.6
764 8 728 8
7774 758,4
6433 621. S
108.1 103,7
105.3 1016
86.6 83.9
107.6 103,4
101.6 97.6
108. 1 104.5
95.5 90.5
128.2 125.8
0 0
676 73.3
76.4 82.5
71.3 77.4
64.4 70.4
83.0 88,9
303.4 325.3
243.2 2607
243.2 249.7
248,9 253,9
2276 2448
83.0 86.1
73.6 76,4
78,8 819
687 707
75.2 77.3
2127 226.3
227.4 240.7
2025 2153
2257 2401
231 0 245 6
7231 7601
668.3 707,8
6962 7383
740.4 7913
601.8 645.6
995 1063
98.1 1048
81.3 87.6
995 1068
939 99.9
101.0 107.9
86.0 93.4
123,5 133.4
0 0
71.3 695
80.0 77.6
75.6 73.9
691 67.9
85,7 82.7
3138 303.1
251.5 242.9
2318 216.2
2343 2175
236.4 2268
840 81.9
74.5 72.7
80.1 78.4
684 682
74.7 72.3
218.7 211.6
231.6 223.2
2080 2D1.1
232.2 224.7
237.2 229.4
726.1 685.1
682.8 859.4
711,1 685.8
7677 7454
628.5 612.3
1024 98.8
1010 975
85,1 82.8
103.3 100.1
95.8 92.0
103.8 100.1
912 89.2
129.9 126.6
0 0
71.9 692
80.8 78.0
77.0 74.6
70.9 88.7
849 81.1
308.9 293.1
250.3 239.8
223.4 214.6
226.5 2191
237.3 228.8
86.7 84.9
77.1 75.4
82.0 793
69,3 67.1
75,8 73.4
2252 2175
238.1 230.5
213.6 205.8
238.1 229.0
245.1 237.6
727,8 699.1
8919 6859
7215 6962
7818 752.1
644.3 621.7
104.7 101.2
102.1 97.6
87.7 84.8
1058 102.1
976 944
104.9 100.4
938 90.0
132.2 126.1
0
66.7
75.4
72.4
66.7
77.7
2789
230.2
206.5
212.3
220.8
831
739
767
65.1
71.3
210.3
223.3
198.6
220.6
2305
672.6
641 8
672.6
724.6
600.8
980
935
821
986
91.4
962
86.5
120.5
0.00
71.0
79.1
755
691
86,6
300.5
253.6
230.2
236.5
241.2
83.0
74.0
81.4
69.0
77.2
222.4
2360
2134
233.2
2347
756.4
684.0
7388
755.8
6282
105.2
101 8
854
1056
963
1040
93.6
126,1
0
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
95
105
101
92
116 102
134
113
102
105
107 112
99
108
92
103 103
99
105
95
104
104 101
112
101
109
112
93 106
105
102
85
106
96
104
94
126 102
I
00
* Groups 7, 8, S 9 not shown (data for samples from a different site)
-------
SWIM Study Ph«« II Exp 6
TABLE A - 3 RAW AND ADJUSTED BLOOD LEAD DATA
PHASE II EXPERIMENT 6 (Data not shown for groups 7,8, & 9)
pig number sample group material administered
dosage qualifier lab result (uq/L) dav
Adjusted Value fun/dLY
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
64B
614
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
62B
640
650
603
615
629
633
645
604
606
607
612
625
632
642
64B
614
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
B- 960 124
8-960163
8-960167
8-960153
8-960155
8-960141
8-960 158
8-960132
8-960120
8-960140
8-960172
8-960129
8-960136
8-960138
8-960145
8-960123
8-960157
8-960133
8-960147
8-960171
8-960152
8-960135
8-960121
8-960154
8-960161
8-960131
8-960148
8-960164
8-960122
8-960150
8-960125
8-960160
8-960173
8-960151
8-960126
8-960214
8-960229
8-960181
8-960213
8-960179
8-960222
8-960219
8-960193
8-960205
8-960 189
8-960226
8-960224
8-960227
8-960202
8-960200
8-960216
8-960209
8-96021B
8-9601B8
8-960183
8-960217
8-960221
8-960204
8-960201
8-9601 85
8-960195
8-960206
8-960225
8-96022B
S-960220
B-96019B
8-960208
8-960182
8-960191
B-960199
8-960277
8-960258
8-960268
8-960246
6-960283
8-960251
8-960242
8-960233
8-960262
8-960278
8-960261
8-96024B
8-960254
8-960231
8-960241
8-960260
8-960240
8-960237
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
control
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Mldvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
MiovateStag
IV
IV
IV
IV
IV
IV
IV
IV
control
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Mldvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
control
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
0
0
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
0
0
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
o
0
75
75
75
75
75 <
225
225
225
225
225
75
75
75
75
75
225
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
• .,
1
1
1
1
1
1
1
1
1.1
1
1
1
1
1
1
1.2
2.4
1.2
2.1
1
1.7
2.B
1.9
3.8
3
1
1
1
1
1
1
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
-4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
a
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
plg41.dat
pig41.dat
pig41.dat
pig41.dat
pt941.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pl941.dat
pig41.da1
pig41.dat
pig41.dat
pig41.dat
pjg41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig4l.dat
pig41.dat
pig41.oat
pig41.dat
pig41.dat
F» 941 Oat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
plg41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pcg41da1
pigit.dal
pig41.dat
pig41.dat
pig41.dat
pig41.dat
plg41.dat
pig41.dat
pig41.dat
p.g41.oat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
plg41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
BLOOD
8LQOO
BLOOD
BLOOO
8LOQO
BLQDD
SLQOO
BLOOD
BLOOD
8LOOO
BLOOD
BLOOO
8LOOO
BLOOD
stooo
BLOOO
BLOOD
8LQOO
BLOOD
BLOOO
BLOOD
BLOOP
8LOOO
BLOOD
BLOOD
BLOOD
BLOOD-
BLOOO
BLOOD
BLOOD
8LOOO
BLOCK)
BLOOD
8LQOO
BLOOD
BLOOD
8LOOO
BLOOO
BLOOD
BLQOO
BLOOO
BLOOO
BLQOO
BLOOD
BLOOO
BLOOD
BLOOO
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
8LOOO
BLOOD
BLOOD
8LOOO
BLOOD
BLOOO
BLOOD
BLOOD
8LOOO
BLOOD
BLOOD
BLOOO
BLOOD
BLOOO
BLOOD
BLOOD
BLOOO
BLOOD
BLOOD
BLOOD
BLQOO
BLOOD
BLOOO
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOO
BLOOD
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
05
0.5
0.5
1.1
0.5
0.5
0.5
0.5
0.5
0.5
1.2
24
1.2
2.1
0.5
1.7
2.8
1.9
3.8
3
1
0.5
05
0.5
0.5
0.5
A-9
-------
Swini Study PhiM II Exp 6
group material administered
dosage qualifier lab result (ug/L) day
MATRIX
Adjusted Value (ug/dL)* Notes
60S
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
648
614
636
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
648
614
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
614
638
613
624
8-960269
8-960253
8-960255
8-960282
8-960270
8-960230
8-960281
8-960252
8-960272
8-960249
8-960267
8-960274
8-960273
8-960232
8-960239
8-360243
8-960266
6-960308
8-960329
8-960298
8-960323
8-360300
8-960231
8-960332
8-960293
8-960312
8-960311
8-960327
6-960328
6-960319
6-960335
6-960304
6-960317
6-960297
6-960316
6-960322
8-960303
8-960330
6-960310
6-960321
6-960290
8-960337
8-960301
6-960305
6-960294
8-960306
6-960289
6-960296
8-960326
8-960324
6-960307
6-960334
8- 960389
6-960367
6-960394
8-960344
8-960350
8-960365
8-960340
6-960357
8-960351
6-960368
6-960363
6-960384
8-960354
8-960387
8-960378
8-960346
8-960385
8-960359
6-960366
6-960386
6-960393
8-960353
8-960383
6-960370
8-960391
6-960349
8-960355
6-960341
6-960392
6-960356
8-960376
8-960379
8-960360
6-960375
8-960347
8-960413
6-960435
6-960401
6-960415
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
1
1
2
2
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
control
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Stag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
control
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
control
control
PbAc
PbAc
225 <
225 <
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
0 <
0 <
75
75
75
75
75
225
225
225
225
225
75
75
75 <
75 <
75 <
225
225
225 <
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
0 <
0 <
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
0 <
0 <
75
75
1
1
1
1.1
3.8
1.2
1.9
1.2
1.7
6.6
7.5
8.2
9.2
8
6.6
7.3
B.4
1
1
3.4
29
1.2
2.6
1.5
3
4.3
2.1
7.1
2.7
1.6
2
1
1
1
1.7
2.2
1
1.3
1.8
5.5
3.8
3.3
2.3
2.2
9.5
104
9.4
9.7
11.3
8.6
6.6
12.5
1
1
4.1
3
1.8
2.9
2.1
3.7
5.4
3.3
6.5
4.4
1.4
1.9
1.4
1.2
1.4
2.3
2.4
2.1
2.4
2.2
5.1
3.4
4.1
3.5
3.6
10.5
11.1
8.9
10.3
11.5
9.7
9.9
11.8
1
4
3.4
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
5
5
5
5
pig41.dat
pig41.dat
plg41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
pig41.dat
plg41.dat
pig41.dat
pig41.dat
pig41.da1
pig41.dat
pig41.dat
pig41.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
plg44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
plg44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
plg44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44da1
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pi844.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pg44.dat
pig44.dat
pig44.da1
pig44.dat
pig44.dat
pig44.dat
pig44.dat
BLOOD
BLOOD
8LDOO
BLOOD
BLOOD
BLOOD
BLOOD
SLOOO
BLOOD
SLQOO
BLOOD
BLOOD
SLQOO
BLOOD
BLOOD
BLOOD
BLOOD
8U3OO
SLOOO
BLOOD
BLOOD
BLQOO
SUOOO
8LOOO
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
8LQOO
SLOOP
BLOOD
SLOOO
BLOOD
SLOOO
BLOOD
BLOOD
SLOOO
BLOOD
BLOOD
BLOOD
BLOOD
81000
BLOOD
BLOOD
SLOOO
BLOOD
BLOOD
SLOOO
BLOOD
BLOOD
BLOOD
91000
BLOOD
8LOOO
BLOOD
SLOOO
BLOOD
BLOOD
SLOOO
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
SLOOO
8LOOO
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
8LQOO
BLOOD
0.5
0.5
1
1.1
3.8
1.2
1.9
1.2
1.7
6.6
7.5
8.2
9.2
8
6.6
7.3
84
0.5
3.4
2.9
1.2
2.6
1.5
3
4.3
2.1
7.1
2.7
1.6
2
0.5
0.5
0.5
1.7
2.2
0.5
1.3
1.8
5.5
3.8
3.3
2.3
2.2
9.5
10.4
9.4
9.7
11.3
8.6
8.8
12.5
0.5
4.1
3
1.8
2.9
2.1
3.7
5.4
3.3
6.5
4.4
1.4
1.9
1.4
1.2
1.4
2.3
2.4
2.1
24
2.2
5.1
3.4
4.1
3.5
3.6
10.5
11.1
8.9
10.3
11.5
9.7
9.9
11.8
0.5
0.5
4
3.4
A-10
-------
Swwi* Study Phase II Exp 6
pig number
639
641
616
644
651
S53
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
648
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
64B
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
62B
640
650
603
615
629
sample
8-960410
8-960440
8-960420
8-960421
8-960418
8-960434
8-960397
8-960395
8-960443
8-960402
8-960409
8-960419
8-960433
8-960405
8-960445
8-960412
8-960446
8-960396
8-960398
8-960426
8-960422
8-960423
8-960442
8-960448
8-960449
8-960431
8-960399
8-960425
8-960406
8-960444
8-960456
8-960500
8-960484
8-960468
8-960440
8-960502
8-960460
8-960467
8-960492
8-960452
8-960462
8-960495
8-960461
8-960483
8-9604 B6
8-960463
8-960475
8-960482
8-960471
8-960476
8-960479
8-960503
8-960487
8-960454
8-960499
8-960470
8-960460
8-960504
8-960451
8-960465
8-960453
8-960472
8-9604B8
8-960498
8-96052B
8-960510
8-960537
8-960549
8-960530
8-960506
8-960516
8-960541
8-960539
8-960553
8-960536
8-960516
8-960557
8-960551
8-960532
8-960538
8-960521
8-960509
8-960556
8-960513
8-960507
8-960531
8-960559
8-960519
.group
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
material administered
PbAc
PhAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Stag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
dosage
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
0
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
0
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
qualifier lab result (ug/L)
2.7
4
2.1
52
6.5
5.7
7.6
4.9
1.9
2.3
1.9
1.7
1.3
2.8
3
2.6
2.4
2.7
6
5.8
6.1
6.1
4.7
13.2
. 12.3
11.1
12.6
13.3
11.8
12.8
15.6
< 1
5
2.8
2.5
2.8
3.2
6.5
6.3
1.6
7.9
5
5.6
1.6
1.2
1.3
1.3
3.5
2.2
1.9
2.6
1.7
3
5
6.4
15
4.6
13.5
4.9
11.7
12.3
15.5
11.6
12.2
< 1
< 1
4.3
3.2
3.4
4.9
3.8
4.1
1.1
7
B.7
6.2
2.6
1.9
1.3
1.6
2.3
4.5
2.7
2.3
2.2
2.1
4.7
4.8
3.2
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.da1
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
plg44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
Pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pi944.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
plg44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
Pig44.dat
Pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pi944.dat
pig44.dat
Pig44.dat
pig44.dat
pig44.dat
pig44.dat
Dig44
-------
Swin* Study Pha<« II E»p 6
633
645
604
606
607
612
625
632
642
648
614
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
648
614
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
B-960523
8-960556
8-960505
8-960554
8-960543
8-960535
8-960527
8-960508
8-960545
8-960515
8-960602
8-960578
8-960566
8-960608
8-960577
8-960560
8-960592
8-960594
8-960601
8-960574
8-960604
8-960580
8-960562
8-960600
8-960591
8-960584
8-960565
8-960571
8-960595
8-960589
8-960590
8-960599
8-960588
B-9605B1
8-960611
8-960607
8-960610
8-960603
8-960561
8-960612
8-960597
8-960613
8-960570
B-960583
8-960564
8-960628
8-960622
8-960626
8-960621
8-960666
8-960657
8-960642
8-960650
8-960656
8-960648
8-960625
8-960629
8-960643
8-960641
8-960630
8-960645
8-960633
8-960619
8-960627
8-960624
8-960618
8-960644
6-960640
8-960639
8-960652
8-960667
8-960651
8-960637
8-960635
8-960668
8-960665
8-960617
8-960653
6-960658
6
6
10
10
10
10
10
10
10
10
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
control
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
control
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
MioVale Slag
MioVale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
dosage
675
675
100
100
100
100
100
100
100
100
0
0
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
0
0
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
qualifier lab result (ug/L) day
5.9
6
12.3
13.1
11.9
13.4
13.7
12.2
13.8
15
< 1
< 1
4.5
5.7
2.9
5.2
6.1
5.8
7.2
6.3
7.9
5.8
38
3
1.6
1.8
11.3
2.9
3
1.8
3.1
6.2
6.4
6.1
6.1
5.4
12.4
12
13.1
13
13.3
10.9
13.5
12.7
1
6.7
6.2
4.6
4.7
4.5
5.1
9.3
8.1
8.1
8.2
36
3
2.9
1.7
2.1
4.1
2.2
3.8
2.2
2.5
5.9
5.3
6.9
5.3
5.4
15.5
13.7
12.4
13.8
13.8
11.5
14.7
17.2
9
9
9
9
9
9
9
9
9
9
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
12
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
"' " 15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
source file
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44dat
pig44.dat
pi844.dat
pig44.dat
pig44.oat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig4-4.dat
pig44.dat
pig44.dat
plg44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
plg44.dat
pig44.dat
pig44.dat
pig44.dat
pi944.dat
pig44.dat
pig44.dat
pig44.dat
pig44.dat
MATRIX Adjusted Value (ug/dL)' Notes
BLOOD
BLOOD
BLOOO
BLOOD
8LOOO
BLOOD
BLOOD
BLOOO
BLOOD
BLOOO
BLOOD
BIOOO
BLOOD
BLOOO
BLOOD
BLOOO
eiooo
BLOOD
8LQOO
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
SLQQO
BLOOD
BLOOD
8LOOO
BLOOD
BLQOO
8LOOO
BLOOD
BLOOD
BLOOD
BLOOD
BLOOO
BLOOD
BLQOO
8LOQ0
BLOOD
BLQOO
BLOOO
BLOOD
BLQOO
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
8LOOO
BLOOD
BLQOO
BLOOO
BLOOO
BLOOO
BLOOD
BLOOO
BLOOO
BLOOD
BLOOO
BLOOO
BLOOD
8LOOO
BLOOD
BLOOD
BLOOO
BLOOD
8LOOO
BLOOO
BLOOD
BLOOO
BLOOD
BLOOD
•BLOOO •
BLOOD
5.9
6
12.3
13.1
11.9
13.4
13.7
12.2
13.8
15
0.5
0.5
4.5
5.7
2.9
5.2
6.1
5.8
7.2
6.3
7.9
5.8
3.8
3
Clotted
1.6
1.8
11.3
2.9
3
1.8
3.1
6.2
6.4
6.1
6.1
54
12.4
12
13.1
13
13.3
10.9
13.5
12.7
1
6.7
62
46
4.7
4.5
5.1
9.3
8.1
8.1
8.2
3.6
3
2.9
1.7
2.1
4.1
2.2
3.8
2.2
2.5
5.9
5.3
6.9
5.3
5.4
15.5
13.7
12.4
13.8
13.8
11.5
14.7
17.2
Non-detects evaluated using 1/2 the ouanttation limit; laboratory resuNs (ug/L) converted to concentration in Wood (uo/dL) by dividing by CHition factor of 1 dL/L.
A-12
-------
Swine Study Phase II EOT 6
TABLE A-4 BLOOD LEAD OUTLIERS
Flagged Data Points
JOutliers
test
material
control
control
PbAc
PbAe
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
target
dosage
0
0
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
Actual
Dose* group
0.00 1
0.00 1
70.99 2
79.09 2
75.53 2
69.05 2
8665 2
300.50 3
253.58 3
230.18 3
236.49 3
241.19 3
B2.9B A
74.00 4
81.36 4
69.00 4
77.23 4
222.40 5
235.96 5
213.39 5
233.20 5
234.73 5
756.45 6
68396 6
738.80 6
75581 6
628.15 6
105.19 10
101.77 10
8541 10
105.64 10
96.30 10
104.02 10
93.59 10
126.06 10
_PJP*
614
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
648
-4
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1.1
0.5
0.5
0.5
0.5
1
0.5
0.5
1.2
2.4
1.2
2.1
0.5
1.7
2.8
1.9
3.8
3
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
1.1
3.8
1.2
1.9
1.2
1.7
6.6
7.5
8.2
9.2
8
6.6
7.3
8.4
2
0.5
0.5
3.4
2.9
1.2
2.6
1.5
3
4.3
2.1
r.t
2.7
1.6
2
0.5
0.5
0.5
1.7
2.2
0.5
1.3
1.8
S.&
3.8
3.3
2.3
2.2
9.5
10.4
94
9.7
11.3
8.6
8.8
12.5
BLOOD
3
0.5
0.5
4.1
3
1.8
2.9
2.1
3.7
5.4
3.3
6.5
4.4
1.4
1.9
1.4
1.2
1.4
2.3
2.4
2.1
2.4
2.2
5.1
3.4
4.1
3.5
3.6
10.5
11.1
8.9
10.3
11.5
9.7
9.9
11.8
LEAD (ug/dL) BY DAY
5
0.5
0.5
4
3.4
2.7
4
2.1
5.2
6.5
5.7
7.6
4.9
1.9
2.3
1.9
1.7
1.3
2.8
3
2.6
2.4
2.7
6
5.8
6.1
6.1
4.7
13.2
12.3
11.1
12.6
13.3
11.8
12.8
15.6
7
| 2.6 |
0.5
5
2.8
2.5
2.8
3.2
6.5
6.3 r
t iia |
?.9
5
{ S.6 |
1.6
1.2
1.3
1.3
3.5
2.2
1.9
2.6
1.7
3
5
6.4
[ jS |
4.6
13.5
[ 4,s 1
11.7
12.3
m5
11.6
12.2
p:;ip:i]
9
0.5
0.5
4.3
3.2
3.4
4.9
3.8
4.1
•ifMiBiMwiafaf
11
7
W
6.2
2.6
1.9
1.3
1.6
2.3
4.5
2.7
2.3
2.2
2.1
4.7
4.8
3.2
5.9
6
12.3
13.1
11.9
13.4
13.7
12.2
13.8
15
12
0.5
05
4.5
5.7
2.9
5.2
6.1
5.8
1 72
6.3
7.9
5.8
3.8
3
Clotted
1.6
1.8
| ^3. '|
2.9
3
L*
3.1
6.2
64
6.1
6.1
5.4
12.4
12
13.1
13
13.3
10.9
13.5
12.7
15
0.5
1
6.7
6.2
4.6
4.7
4.5
&1
9.3
8.1
8.1
8.2
3.6
3
2.9
1.7
2.1
4.1
2.2
3.8
2.2
2.5
5.9
5.3
6.9
5.3
5.4
15.5
13.7
12.4
13.8
13.8
11.5
14.7
17.2
* Average Time and Weight-Adjusted Dose for Each Pig
A-13
-------
Swine Study Phase II Exp 6
TABLE A-5 RATIONALE FOR PbB OUTLIER DECISIONS
OUTLIER
1
2
3
4
5
6
7
8
IDENTIFICATION
Day?
Group 1
Pig #614
Day?
Group 3
Pig #651
Day?
Group 4
Pig #619
Day?
Group 6
Pig # 633
Day?
Group 10
Pig # 606
Day?
Group 10
Pig # 648
Day 9
Group 3
Pig # 644
Day 12
Group 5
Pig # 602
RATIONALE
Based on comparison with responses by other animals in this group on this day, the response of animal
614 is notably higher. Therefore, this value is excluded and replaced with an interpolated value of 0.5
ug/dL.
Based on the time-trend for this animal, the PbB on day 7 is substantially lower than expected from the
PbB values measured before and after:
Day PbB
5 5.7
7 1.6
9 7.0
Also, based on comparison with responses by other animals in this group on this day, the response of
animal 65 1 is notably lower. Therefore, this value is excluded and replaced with an interpolated value
(6.35 ug/dL).
Based on the time-trend for this animal, the PbB on day 9 is substantially higher than expected from
the PbB values measured before and after:
Day PbB
5 1.9
7 5.6
9 2.6
Also, based on comparison with responses by other animals in this group on this day, the response of
animal 619 is notably higher. Therefore, this value is excluded and replaced with an interpolated value
(2.25 ug/dL).
Based on the time-trend for this animal, the PbB on day 9 is substantially higher than expected from
the PbB values measured before and after:
Day PbB
5 6.1
7 15.0
9 5.9
Also, based on comparison with responses by other animals in this group on this day, the response of
animal 633 is notably higher. Therefore, this value is excluded and replaced with an interpolated value
(6.0 ug/dL).
Based on the time-trend for this animal, the PbB on day 7 is substantially lower than expected from the
PbB values measured before and after:
Day PbB
5 12.3
7 4.9
9 13.1
Also, based on comparison with responses by other animals in this group on this day, the response of
animal 606 is notably lower. Therefore, this value is excluded and replaced with an interpolated value
(12.7 ug/dL).
Based on the time-trend for this animal, the PbB on day 7 is substantially lower than expected from the
PbB values measured before and after:
Day PbB
5 15.6
7 0.5
9 15.0
Also, based on comparison with responses by other animals in this group on this day, the response of
animal 648 is notably lower. Therefore, this value is excluded and replaced with an interpolated value
(15.3 ug/dL).
Based on the time-trend for this animal, the PbB on day 9 is substantially lower than expected from the
PbB values measured before and after:
Day PbB
7 6.3
9 1.1
12 7.2
Also, based on comparison with responses by other animals in this group on this day, the response of
animal 644 is notably lower. Therefore, this value is excluded and replaced with an interpolated value
(6.66 ug/dL).
Based on the time-trend for this animal, the PbB on day 9 is substantially higher than expected from
the PbB values measured before and after:
Day PbB
9 4.5
12 11.3
15 4.1
Also, based on comparison with responses by other animals in this group on this day, the response of
animal 602 is notably higher. Therefore, this value is excluded and replaced with an interpolated value
(4.3 ug/dL).
A-14
-------
Swine Study Phase II Exp 6
TABLE A-6 Area Under Curve Determinations
Calculated using interpolated values for missing or excluded data as noted in Table A-5
AUC (ug/dL-days) For Time Span Shown
group
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
Pi9#
614
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
648
0-1
0.50
0.50
0.85
1.70
0.85
1.30
0.50
1.10
1.65
1.20
2.15
1.75
0.75
0.75
0.50
0.50
0.50
0.50
0.50
0.50
0.75
0.80
2.15
0.85
1.20
0.85
1.10
3.55
4.00
4.35
5.15
4.25
3.55
3.90
4.45
1-2
0.50
0.50
2.30
2.65
1.20
2.35
1.00
2.35
3.55
2.00
5.45
2.85
1.30
1.25
0.50
0.50
0.50
1.10
1.35
0.50
1.15
1.45
4.65
2.50
2.60
1.75
1.95
8.05
8.95
8.80
9.45
9.65
7.60
8.05
10.45
2-3
0.50
0.50
3.75
2.95
1.50
2.75
1.80
3.35
4.85
2.70
6.80
3.55
1.50
1.95
0.95
0.85
0.95
2.00
2.30
1.30
1.85
2.00
5.30
3.60
3.70
2.90
2.90
10.00
10.75
9.15
10.00
11.40
9.15
9.35
12.15
3-5
1.00
1.00
8.10
6.40
4.50
6.90
4.20
8.90
11.90
9.00
14.10
9.30
3.30
4.20
3.30
2.90
2.70
5.10
5.40
4.70
4.80
4.90
11.10
9.20
10.20
9.60
8.30
23.70
23.40
20.00
22.90
24.80
21.50
22.70
27.40
5-7
1.00
1.00
9.00
6.20
5.20
6.80
5.30
11.70
12.80
12.05
15.50
9.90
4.15
3.90
3.10
3.00
2.60
6.30
5.20
4.50
5.00
4.40
9.00
10.80
12.50
12.10
9.30
26.70
25.00
22.80
24.90
28.80
23.40
25.00
30.90
7-9
1.00
1.00
9.30
6.00
5.90
7.70
7.00
10.60
12.96
13.35
16.60
11.20
4.85
3.50
2.50
2.90
3.60
8.00
4.90
4.20
4.80
3.80
7.70
9.80
9.60
11.90
10.60
25.80
25.80
23.60
25.70
29.20
23.80
26.00
30.30
9-12
1.50
1.50
13.20
13.35
9.45
15.15
14.85
14.85
20.79
19.95
24.90
18.00
9.60
7.35
5.10
4.80
6.15
13.20
8.40
7.95
6.00
7.80
16.35
16.80
13.95
18.00
17.10
37.05
37.65
37.50
39.60
40.50
34.65
40.95
41.55
12-15
1.50
2.25
16.80
17.85
11.25
14.85
15.90
16.35
24.75
21.60
24.00
21.00
11.10
9.00
7.50
4.95
5.85
12.60
7.65
10.20
6.00
8.40
18.15
17.55
19.50
17.10
16.20
41.85
38.55
38.25
40.20
40.65
33.60
42.30
44.85
AUC Total
(ug/dL-days)
7.50
8.25
63.30
57.10
39.85
57.80
50.55
69.20
93.25
81.85
109.50
77.55
36.55
31.90
23.45
20.40
22.85
48.80
35.70
33.85
30.35
33.55
74.40
71.10
73.25
74.20
67.45
176.70
174.10
164.45
177.90
189.25
157.25
178.25
202.05
A-15
-------
Swine Study Ph»« II Exp 6
TABLE A - 7 TISSUE LEAD DATA
PHASE II EXPERIMENT 6 {Data not shown for groups 7, 8, & 9)
pig number sample grouP material administered
doeage qualifier lab result (ug/L) day
MATRIX
Adjusted Value*
614
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
648
614
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
605
628
640
650
603
615
629
633
645
604
606
607
612
625
632
642
648
614
638
613
624
630
639
641
616
644
651
653
654
619
623
626
631
647
602
8-960839
8-960854
8-960833
8-960871
8-960863
8-960832
8-960872
8-960840
8-960870
8-960868
8-960825
8-960845
8-960862
8-960842
8-960874
8-960837
8-960841
8-960869
8-960846
8-960875
8-960849
8-960873
8-960865
8-960824
8-960848
8-960876
8-960859
8-960827
8-960826
8-960866
8-960855
8-960851
8-960829
8-960835
8-960858
8-960785
8-960797
8-960821
8-960814
8-960772
8-960786
8-960817
5-960823
8-960791
8-960799
8-960787
8-960805
8-960800
8-960782
8-960793
8-960812
8-960778
8-960775
8-960774
8-960819
8-960822
8-960776
8-960813
8-960792
8-960794
8-960779
8-960795
8-960771
8-960820
8-960802
8-960804
8-960B15
8-960783
8-960810
8-960790
8-960762
8-960752
8-960729
8-960755
B-960720
8-960724
8-960736
8-960753
8-960738
8-960721
8-960726
8-960766
8-960718
8-960742
8-960731
8-960735
8-960733
8-960744
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
1
1
2
2
2
2
2
3
3
3
3
3
4
4
4
4
4
5
control
corrtrol
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvate Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
control
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
control
control
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
0 <
0
75
75
75
75
75
225
225
225
225
225
75
75 <
75
75 <
75 <
225
225
225
225 <
225
675
675
675
675
675
100
100
100
100
100
100
100
100
0
0
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
0
0
75
75
75
75
75
225
225
225
225
225
75
75
75
75
75
225
2
7.6
6.4
8.9
6
3.7
6
13.2
35.8
21.6
26.1
19.1
3.8
2
3.9
2
2
5
1.2
10.9
2
5.1
10.8
2.2
64
3.3
7.6
73
71.3
75.7
130
82.8
76.3
58.3
104
4.7
152
22.8
18.4
14.2
20
16.7
30.1
72.5
39.9
66
62
10.1
7.3
4
6.7
4.5
11.2
10.5
4.8
6.4
68
18.9
11,3
197
164
17.1
122
109
148.2
123
133
106
135
135
7.2
118
16.6
154
17.6
16.6
16.2
33.5
56
73
86
55
6.9
7.1
4.6
4.1
4.3
9
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T960106F
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T951213K
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMUR
FEMi/R
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
MONEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KtDNEY
KIDNEY
KIDNEY
KIDNEY
KIDNEY
KtDNEY
KtDNEY
KIDNEY
KtDNEY
MONEY
KIDNEY
KIDNEY
KIDNEY
KtDNEY
UVER
UVER
UVE*
IMER
MVEfi
UVER
LIVER
UVER
UV£R
UVER
LIVER
LIVER
UVER
LWER
L1VER
OVER
UVER
UVER
0.5
3.8
3.2
4.45
4
1.85
4
6.6
17.9
10.8
13.05
9.55
1.9
0.5
1.95
0.5
0.5
2.5
0.6
5.45
0.5
2.55
5.4
1.1
3.2
1.65
3.8
36.5
35.65
37.85
65
41.4
38.15
29.15
52
47
1520
228
184
142
200
167
301
725
399
660
620
101
73
40
67
45
112
105
48
64
68
189
113
197
164
171
1220
1090
1482
1230
1330
1060
1350
1350
72
1180
166
154
176
166
162
335
560
730
860
550
69
71
46
41
43
90
A-16
-------
Swm» Study Phwi II Exp 6
pig number sample group material administered
dosage qualifier lab result (uq/L) day
Adjusted Value*
605
626
640
650
603
615
629
633
645
604
606
607
612
625
632
642
646
&-960746
&-960719
8-960749
8-960722
8-960759
8-960723
8-960758
8-960756
8-960734
8-960732
8-960764
8-960769
8-960725
8-960767
8-960763
8-960770
8-960741
5
5
5
5
6
6
6
6
6
10
10
10
10
10
10
10
10
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
IV
IV
IV
IV
IV
IV
IV
225
225
225
225
675
675
675
675
675
100
100
100
100
100
100
100
100
8.3
7
7.4
11.7
21.3
15.8
17.7
18.7
16.5
98
110
159
164
154
127
137
197
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
T960105L
LIVER
UVEft
(JVEfi
L1VER
UVER
i-TVER
LIVER
UVER
LIVER
MVER
OVER
UVER
UVER
UVER
LIVER
LIVER
UVER
83
70
74
117
213
158
177
187
165
980
1100
1590
1640
1540
1270
1370
1970
Non-detects evaluated using 1/2 the quantrtation limit. Laboratory results (ug/L) converted to tissue concentrations by dividing by sample dilution factors of
0.1 kg/L (liver, kidney) or 2 g/L (ashed bone). Final units are ug Pt*g wet weight (iver, kidney) or ug Pb/g ashed bone (femur).
A-17
-------
Swine Study Phase II Dtp 6
TABLE A-8 SUMMARY OF ENDPOINT OUTLIERS
| | Selected Outliers
test target Actual
control 0 0.00 1 614
control 0 0.00 1 638
PbAc 75 70.99 2 613
PbAc 75 79.09 2 624
PbAc 75 75.53 2 630
PbAc 75 69.05 2 639
PbAc 75 86.65 2 641
PbAc 225 300.50 3 616
PbAc 225 253.58 3 644
PbAc 225 230.18 3 651
PbAc 225 236.49 3 653
PbAc 225 241.19 3 654
MidvaleSlag 75 82.98 4 619
MidvaleSlag 75 74.00 4 623
MidvaleSlag 75 81.36 4 626
MidvaleSlag 75 69.00 4 631
Midvale S aq 75 77.23 4 647
MidvaleSlag 225 222.40 5 602
MidvaleSlag 225 235.96 5 605
MidvaleSlag 225 213.39 5 628
MidvaleSlag 225 233.20 5 640
Midvale Slag 225 23473 5 650
MidvaleSlag 675 756.45 6 603
MidvaleSlag 675 683.96 6 615
MidvaleSlag 675 738.80 6 629
MidvaleSlag 675 755.81 6 633
Midvale Slag 675 628.15 6 645
IV 100 105.19 10 604
IV 100 101.77 10 606
IV 100 85.41 10 607
IV 100 105.64 10 612
IV 100 96.30 10 625
IV 100 104.02 10 632
IV 100 93.59 10 642
Blood
7.5
8.3
63.3
57.1
39.9
57.8
50.6
69.2
93.3
81.9
109.5
77.6
36.6
31.9
23.5
20.4
22.9
48.8
35.7
33.9
30.4
33.6
74.4
71.1
73.3
74.2
67.5
176.7
174.1
164.5
177.9
189.3
157.3
178.3
202.1
Femur
3.8 |a1
3.2
4.45
4
1.85
4
6.6
17.9
10.8
13.05
9.55
1.9
0.5
1.95
0.5
0.5
2.5
0.6
5.45 jb
0.5
2.55
5.4
1.1
3.2
1.65
3.8
36.5
35.65
37.85
65
41.4
38.15
29.15
52
Liver
1180 |a1
166
154
176
166
162
335
560
730
860
550
69
71
46
41
43
90
83
70
74
117
213
158
177
187
165
980
1100
1590
1640
1540
1270
1370
1970
Kidney
1520 |a1
228
184
142
200
167
301
725
399
660
620
101
73
40
67
45
112
105
48
64
68
189
113
197
164
171
1220
1090
1482
1230
1330
1060
1350
1350
a a priori outlier determinations ...... _,
a1 - These two control values were excluded based on the fact that the values were abnormally high compared
~ to data from other studies, and were also higher than those for the low dose PbAc group
b Outside 95% Prediction Interval
A-18
-------
TABLE A-9 Best Curve Fit Parameters
Swine Study Phase II Exp 6
BLOOD
PbAc Curve -
BONE
PbAc Curve -
Linear
a
b
c
d
R2
Midvale Curve -
a
b
c
d
R2
8 a 0,45
b 0.043
92 c
0.0086 d
0.893 R2 0.727
Exp Midvale Curve • Linear
8 a 0.45
b 0.0037
92 c
0.0017 d
0.934 R2 0.332
Equations Used
EXP Y=a+c*(1-exp(-d"dose)|
LIN Y=a+b'dose
LIVER
PbAc Curve -
a
b
c
d
R2
Midvale Curve -
b
c
d
R2
Linear
54.4
2.052
0692
Linear
54.4
0.172
0.878
KIDNEY
PbAc Curve -
Linear
a
b
c
d
R2
Midvale Curve -
39.5
1.858
0.727
Linear
b
C
d
R2
39.5
0.154
0.796
-------
Swine Study Phase II Exp 6
TABLE A-10 Relative Bioavailability of Lead in Test Materials
Endpoint
Blood
Liver
Kidney
Bone
Test Material
Midvale
0.20
0.08
0.08
0.09
Definitions
Plausible Range:
Preferred Range:
Suggested Point Est:
RBA(Blood) to mean RBA for Tissues
RBA(Blood) to (RBA(Blood) + RBA(Tissues))/2
1/2(RBA(Blood) + (RBA(Blood)+RBA(Tissues))/2)
Relative Bioavailability
Plausible Range
Preferred Range
Point Estimate
Midvale
0.20 0.08
0.20 0.14
0.17
Absolute Bioavailability
Plausible Range
Preferred Range
Point Estimate
Midvale
10% 4%
10% 7%
8%
A-20
-------
Swine Study Phase II Exp 6
TABLE A-11 INTRALABORATORY DUPLICATES
RPD = Relative Percent Difference
RPD = 1QO*[Orig-Dupl/((Orig+Dup)/2
Non detects evaluated at 1/2 DL
Pig number
653
617
609
639
645
655
651
626
650
631
605
604
614
618
606
628
633
601
610
607
612
630
625
642
644
643
621
647
629
648
651
626
604
614
618
606
640
615
646
group
3
7
8
2
6
9
3
4
5
4
5
10
1
8
10
5
6
8
7
10
10
2
10
10
3
7
8
4
6
10
3
4
10
1
8
10
5
6
9
material administered
PbAc
Butte
Butte
PbAc
Midvale Slag
Butte
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Midvale Slag
IV
control
Butte
IV
Midvale Slag
Midvale Slag
Butte
Butte
IV
IV
PbAc
IV
IV
PbAc
Butte
Butte
Midvale Slag
Midvale Slag
IV
PbAc
Midvale Slag
IV
control
Butte
IV
Midvale Slag
Midvale Slag
Butte
dosage
225
75
225
75
675
675
225
75
225
75
225
100
0
225
100
225
675
225
75
100
100
75
100
100
225
75
225
75
675
100
225
75
100
0
225
100
225
675
675
day
-4
-4
-4
0
0
0
1
1
1
2
2
2
3
3
3
5
5
5
7
7
7
9
9
9
12
12
12
15
15
15
15
15
15
15
15
15
15
15
15
matrix
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
BLOOD
FEMUR
FEMUR
FEMUR
KIDNEY
KIDNEY
KIDNEY
LIVER
LIVER
LIVER
Duplicate Value*
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1.5
10.4
0.5
2.6
10.6
2.6
5.9
2.5
2
10.3
13.6
2.7
13.8
13.5
6.9
2.1
2.4
2
6.7
15.3
21.8
1
88
3.9
10.8
114
6.4
15.1
21.2
Original Value*
0.5
0,5
0.5
0.5
0.5
0.5
1.9
0.5
1.1
0.5
22
95
0.5
2.8
11.1
2.6
6.1
2.7
2
11.7
12.3
3.4
13.7
13.8
7.2
1.7
3.2
2.1
6.9
17.2
21.6
3.8
73
4.7
13.3
109
7.4
15.8
21.3
Average
0.5
0.5
0.5
0.5
0.5
0.5
1.2
0.5
0.8
0.5
1.85
9.95
0.5
2.7
10.85
2.6
6
2.6
2
11
12.95
3.05
13.75
13.65
7.05
1.9
2.8
2.05
6.8
16.25
21.7
2.4
80.5
4.3
12.05
111.5
6.9
15.45
21.25
RPD
0%
0%
0%
0%
0%
0%
117%
0%
75%
0%
38%
-9%
0%
7%
5%
0%
3%
8%
0%
13%
-10%
23%
-1%
2%
4%
-21%
29%
5%
3%
12%
-1%
117%
-19%
19%
21%
-4%
14%
5%
0%
Avg RPD
10% BLOOD
32% FEMUR
12% KIDNEY
6% LIVER
-------
Swine Study Phase II Exp 6
TABLE A-12 CDC STANDARDS
Sample ID
6.1
6.1
6.1
6.1
6.1
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.2
6.3
6.3
6.3
6.3
6.3
6.3
6.3
Averages
Day Q
-4
0
1
3
9
-4
0
1
2
5
7
12
15
2
3
5
7
9
12
15
Measured
Low Std Med Std
1
1.6
1
2
1.9
4.1
4.7
4.5
5.4
4.9
6.1
3.3
4.4
1.5 4.7
Hiah Std
14.9
14.4
15
13.5
14.6
11.7
14.4
Nominal
Cone
1.7
1.7
1.7
1.7
1.7
4.8
4.8
4.8
4.8
4.8
4.8
4.8
4.8
14.9
14.9
14.9
14.9
14.9
14.9
14.9
14.1
A-22
-------
Swine Study Phase II Exp 6
TABLE A-13 INTERLABORATORY COMPARISON
Tag
8-960158
8-960174
8-960208
8-960221
8-960249
8-960265
8-960313
8-960322
8-960370
8-960378
8-960401
8-960445
8-960452
8-960457
8-960511
8-960551
8-960577
8-960600
8-960618
8-960643
Pig
Number
641
617
625
650
604
609
634
605
615
626
613
628
653
601
618
626
630
623
640
619
Group
2
7
10
5
10
8
9
5
6
4
2
5
3
8
8
4
2
4
5
4
Material
Administered
PbAc
Butte
IV
Midvale Slag
IV
Butte
Butte
Midvale Slag
Midvale Slag
Midvale Slag
PbAc
Midvale Slag
RbAc
Butte
Butte
Midvale Slag
PbAc
Midvale Slag
Midvale Slag
Midvale Slag
Dosage
75
75
100
225
100
225
675
225
675
75
75
225
225
225
225
75
75
75
225
75
Qualifier
CDC
U
U
U
ESD
<
<
<
<
<
Result
CDC
0.6
0.6
0.6
9.6
1
3.3
1.7
4.1
1.2
3
2.3
7.9
2.7
3.6
1.3
4.2
3.3
2.8
4.3
ESD
1
1
1
6,6
1
3.2
2.2
3.4
1.4
4
2.6
7.9
2.9
3.3
1.3
2.9
3
2.2
3.6
RPD
50
50
50
-37
0
-3
26
-19
15
29
12
0
7
-9
0
-37
-10
-24
-18
I
to
GJ
-------
Swine Study Phase ii Exp 6
FIGURE A-1 PbAc and IV Groups by Day
Raw Data
I
m
.a
D.
-* 614
Hi 638
-A 613
-X 624
-X 630
-« 639
H 641
616
— - -644
*• --651
•• --653
*• --654
-X—604
-X—606
Hg> 607
-+ 612
—— 625
*—642
-648
-4
-2
-------
Swine Study Phase II Exp 6
FIGURE A-2 Midvale Groups by Day
Raw Data
m
£
—A 619
—K 623
—m—-626
—® 631
1 647
- * - •602
• - » - • 605
628
640
- «• - -650
B 603
—ft—615
^K 629
—X 633
—•$—645
-2
-------
Swine Study Phase II Exp 6
i
co
01
CQ
a
Qu
FIGURE A-3 Group Mean PbB By Day
Raw Data
1 control
@-2 PbAc 75
A-3 PbAc 225
4 Midvale 75
5 Midvale 22
6 Midvale 67
- * - 101V
-------
FIGURE A-4
THIS PAGE INTENTIONALLY LEFT BLANK
A-27
-------
FIGURE A-5 BEST FIT CURVE WITH 95% PREDICTION INTERVALS*
as
O
CO
125
100-
75-
50-
5 25 H
MATERIAL: PbAc
ENDPOINT: Blood Lead AUC
BEST FIT EQUATION: Y=a+c*(1-exp(-d*X))
100 200
Dose (ug Pb/kg-day)
300
Parameters
a
c
d
Value
8
92
0.0086
Std. Error
fixed value
fixed value
0.0012
95% Confidence Limits
_
—
0.0059
_
_
0.0113
\ Adj R2
0.893
Generated using Table Curve 2D v. 3.0. Outliers represented by"+".
A-28
-------
FIGURE A-6 BEST FIT CURVE WITH 95% PREDICTION INTERVALS*
v>
>N
TO
)
O
•o
TO
CD
•O
O
.0
CD
80
70-
60-
50-
40-
30-
20-
10-
MATERIAL: Midvale
ENDPOINT: Blood Lead AUC
BEST FIT EQUATION: Y=a+c*(1-exp{-d*X))
200 400
Dose (ug Pb/kg-day)
600
800
Parameters
a
c
d
Value
8
92
0.0017
Std. Error
fixed value
fixed value
0.0001
95% Confidence Limits
—
—
0.0015
—
~
0.002
Adj R
0.934
Generated using Table Curve 2D v. 3.0. Outliers represented by"+"
A-29
-------
FIGURE A-7 BEST FIT CURVE WITH 95% PREDICTION INTERVALS*
MATERIAL: PbAc
ENDPOINT: Bone Lead
BEST FIT EQUATION: Y=a+b*X
•o
0)
0)
co
ra
ra
•o
as
0)
0)
o
CO
17.5-
12.5-
100
200
Dose (ug Pb/kg-day)
300
Parameters
a
b
Value
0.45
0,043
Std. Error
fixed value
0.0053
95% Confidence Limits
_
0.031
_
0.055
AdjR2
0.727
Generated using Table Curve 2D v. 3.0. Outliers represented by"+"
A-30
-------
FIGURE A-8 BEST FIT CURVE WITH 95% PREDICTION INTERVALS*
0
MATERIAL: Midvale
ENDPOINT: Bone Lead
BEST FIT EQUATION: Y=a+b*X
200400
Dose (ug Pb/kg-day)
800
Parameters
a
b
Value
0.45
0.0037
Std. Error
fixed value
0.0007
95% Confidence Limits
-
0.0023
-
0.0052
[ Adj R2 0.332 |
Generated using Table Curve 2D v. 3.0. Outliers represented by"+".
-------
FIGURE A-9 BEST FIT CURVE WITH 95% PREDICTION INTERVALS*
MATERIAL: PbAc
ENDPOINT: Liver Lead
BEST FIT EQUATION: Y=a+b*X
1000
CD
I
0.
O)
en
o>
CD
100
200
Dose (ug Pb/kg-day)
300
Parameters
a
b
Value
54.4
2.05
Std. Error
fixed value
0.278
95% Confidence Limits
—
1.43
—
2.67
AdjFT
0.692
Generated using Table Curve 2D v. 3.0. Outliers represented by"+",
A-32
-------
FIGURE A-10 BEST FIT CURVE WITH 95% PREDICTION INTERVALS*
S
]3
0.
•a
CD
a>
250
200-
100-
50-
MATERIAL: Midvale
ENDPOINT: Liver Lead
BEST FIT EQUATION: Y=a+b*X
200 400
Dose (ug Pb/kg-day)
600
800
Parameters | Value
a
b
54.4
0.172
Std. Error
fixed value
0.012
95% Confidence Limits
—
0.147
_
0.197
I AdjR2 0.878 |
Generated using Table Curve 2D v. 3.0. Outliers represented by"+".
A-33
-------
FIGURE A-11 BEST FIT CURVE WITH 95% PREDICTION INTERVALS*
CD
800
700-
•Q 600 H
$
I* 500H
.O
0.
°> 400 H
300-
200-
100-
0
MATERIAL: PbAc
ENDPOINT: Kidney Lead
BEST FIT EQUATION: Y=a+b*X
100 200
Dose (ug Pb/kg-day)
300
Parameters
a
b
Value
39.5
1.86
Std. Error
fixed value
0.235
95% Confidence Limits
—
1.334
—
2.382
I AdjR2 0.727 j
Generated using Table Curve 20 v. 3.0. Outliers represented by"+".
A-34
-------
FIGURE A-12 BEST FIT CURVE WITH 95% PREDICTION INTERVALS*
co
j£
s
Q.
CO
o>
250
200-
150-
100-
§ 50i,
0.
MATERIAL: Midvale
ENDPOINT: Kidney Lead
BEST FIT EQUATION: Y=a+b*X
200 400
Dose (ug Pb/kg-day)
600
800
Parameters
a
b
Value
39.5
0.154
Std. Error
fixed value
0.015
95% Confidence Limits
—
0.121
—
0.186
AdjR2 0.796 j
Generated using Table Curve 2D v. 3.0. Outliers represented by"+".
A-35
------- |