United States
Environmental Protection
m\ Agency
EPA/600/R-23/375
January 2024
www.epa.gov/isa
Integrated Science
Assessment for Lead
Appendix 6: Immune System Effects
January 2024
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
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DISCLAIMER
This document has been reviewed in accordance with the U.S. Environmental Protection Agency
policy and approved for publication. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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DOCUMENT GUIDE
This Document Guide is intended to orient readers to the organization of the Lead (Pb) Integrated
Science Assessment (ISA) in its entirety and to the sub-section of the ISA at hand (indicated in bold). The
ISA consists of the Front Matter (list of authors, contributors, reviewers, and acronyms), Executive
Summary, Integrated Synthesis, and 12 appendices, which can all be found at https://assessments.epa.gov/
isa/document/&de id=3 5 9536.
Front Matter
Executive Summary
Integrated Synthesis
Appendix 1. Lead Source to Concentration
Appendix 2. Exposure, Toxicokinetics, and Biomarkers
Appendix 3. Nervous System Effects
Appendix 4. Cardiovascular Effects
Appendix 5. Renal Effects
Appendix 6. Immune System Effects
Appendix 7. Hematological Effects
Appendix 8. Reproductive and Developmental Effects
Appendix 9. Effects on Other Organ Systems and Mortality
Appendix 10. Cancer
Appendix 11. Effects of Lead in Terrestrial and Aquatic Ecosystems
Appendix 12. Process for Developing the Pb Integrated Science Assessment
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CONTENTS
DOCUMENT GUIDE 6-iii
LIST OF TABLES 6-v
LIST OF FIGURES 6-vi
ACRONYMS AND ABBREVIATIONS 6-vii
APPENDIX 6 IMMUNE SYSTEM EFFECTS 6-1
6.1 Introduction, Summary of the 2013 Pb ISA, and Scope of the Current Review 6-1
6.2 Scope 6-3
6.3 Immunosuppression 6-5
6.3.1 Epidemiologic Studies of Immunosuppression 6-5
6.3.2 Toxicological Studies of Immunosuppression 6-9
6.3.3 Integrated Summary of Immunosuppression 6-18
6.4 Sensitization and Allergic Responses 6-20
6.4.1 Epidemiologic Studies of Sensitization and Allergic Responses 6-21
6.4.2 Toxicological Studies of Sensitization and Allergic Responses 6-23
6.4.3 Integrated Summary of Sensitization and Allergic Responses 6-24
6.5 Autoimmunity and Autoimmune Disease 6-25
6.5.1 Epidemiologic Studies of Autoimmunity and Autoimmune Disease 6-25
6.5.2 Toxicological Studies of Autoimmunity and Autoimmune Disease 6-26
6.5.3 Integrated Summary of Autoimmunity and Autoimmune Disease 6-26
6.6 Biological Plausibility 6-27
6.6.1 Immunosuppression 6-29
6.6.2 Sensitization and Allergic Responses 6-31
6.7 Summary and Causality Determinations 6-32
6.7.1 Causality Determination for Immunosuppression 6-32
6.7.2 Causality Determination for Sensitization and Allergic Responses 6-37
6.7.3 Causality Determination for Autoimmunity and Autoimmune Disease 6-40
6.8 Evidence Inventories - Data Tables to Summarize Study Details 6-42
6.9 References 6-76
6-iv
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LIST OF TABLES
Table 6-1 Summary of evidence for a likely to be causal relationship between Pb exposure and
immunosuppression 6-35
Table 6-2 Summary of evidence that is suggestive of, but not sufficient to infer, a causal relationship
between Pb exposure and sensitization and allergic responses 6-39
Table 6-3 Summary of evidence that is inadequate to determine the presence or absence of a
causal relationship between Pb exposure and autoimmunity and autoimmune disease 6-41
Table 6-4 Epidemiologic studies of exposure to Pb and immunosuppression 6-42
Table 6-5 Animal toxicological studies of delayed-type hypersensitivity responses 6-51
Table 6-6 Animal toxicological studies of antibody response 6-52
Table 6-7 Animal toxicological studies of ex vivo white blood cell function 6-52
Table 6-8 Animal toxicological studies of immune organ pathology 6-54
Table 6-9 Animal toxicological studies of immunoglobulin levels 6-56
Table 6-10 Animal toxicological studies of immune organ weight 6-57
Table 6-11 Animal toxicological studies of white blood cell counts and differentials (spleen, thymus,
lymph node, bone marrow) 6-63
Table 6-12 Animal toxicological studies of white blood cell counts (hematology and subpopulations) 6-65
Table 6-13 Epidemiologic studies of exposure to Pb and sensitization and allergic response 6-66
Table 6-14 Animal toxicological studies of immediate-type hypersensitivity 6-73
Table 6-15 Epidemiologic studies of exposure to Pb and autoimmunity and autoimmune disease 6-74
Table 6-16 Animal toxicological studies of autoimmunity and autoimmune disease 6-75
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LIST OF FIGURES
Figure 6-1 Potential biological plausibility pathways for immunological effects associated with
exposure to Pb. 6-28
6-vi
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ACRONYMS AND ABBREVIATIONS
AQCD
anti-TT
BLL
BMI
BW
Cd
CD
CI
CMI
Con A
CR1
d
DNFB
DTH
e-waste
EDEN
EE
EGFP
ELISA
F
Fe
GFAAS
GM-CSF
hr
HBc
HBsAb
HBV
Hib
HLA-DR
HR
ICR
ICP-MS
IFN-y
Ig-
IL-
ILC
ILCP
ISA
ISO
KNHANES
Air Quality Criteria Document
anti-tetanus toxoid
blood lead level
body mass index
body weight
cadmium
cluster of differentiation
confidence interval
cell-mediated immune
Concanavalin A
complement receptor type 1
day(s)
1 -Fluoro-2,4-dinitrobenzene
delayed-type hypersensitivity
electronic-waste
Effect of Diet and Exercise on
Immunotherapy and the Microbiome
effect estimate
enhanced green fluorescent protein
enzyme-linked immunosorbent assay
female
iron
graphite furnace atomic absorption
spectrometry
granulocyte-macrophage colony-
stimulating factor
hour, hours
hepatitis B core
hepatitis B surface antigen
hepatitis B virus
Haemophilus influenzae type B
Major histocompatibility complex
(MHC) II cell surface receptor
hazard ratio
Institute for Cancer Research
inductively coupled plasma mass
spectrometry
interferon-gamma
immunoglobulin type -
interleukin type -
innate lymphoid cell
innate lymphoid cell progenitor
Integrated Science Assessment
isolation
Korea National Health and Nutrition
Examination Survey
In
M
MMR
M/F
min
mo
MRSA
MSSA
NHANES
NK
NO
NR
OR
Pb
PbONP
PCR
PECOS
PND
ppm
Q
ROS
RR
RSV
S/CO
SCORAD
SD
SES
SPT
STELLAR
T#
TDAR
Th2
TNF
Treg
TSLP
IT
tTG
WBC
wk
yr
vs.
natural log
male
measles, mumps, and rubella
male/female
minute(s)
month(s)
methicillin-resistant Staphylococcus
aureus
methicillin-sensitive Staphylococcus
aureus
National Health and Nutrition
Examination Survey
natural killer
nitric oxygen
not reported
odds ratio
lead
lead oxide nanoparticle
polymerase chain reaction
Population, Exposure, Comparison,
Outcome, and Study Design
postnatal day
parts per million
quartile
reactive oxygen species
relative risk
respiratory syncytial virus
signal to cut-off
scoring atopic dermatitis
standard deviation
socioeconomic status
skin prick test
Systemic Tracking of Elevated Lead
Levels and Remediation
tertile #
T cell dependent antibody response
T cell-derived helper cell 2
tumor necrosis factor
regulatory T cell
thymic stromal lymphopoietin
tetanus toxoid
tissue transglutaminase
white blood cell
week(s)
year(s)
versus
6-vii
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APPENDIX 6 IMMUNE SYSTEM EFFECTS
Causality Determinations for Pb Exposure and Immune System Effects
This appendix characterizes the scientific evidence that supports causality
determinations for lead (Pb) exposure and immune system effects. The types of studies
evaluated within this appendix are consistent with the overall scope of the ISA as
detailed in the Process Appendix (see Section 12.4). In assessing the overall evidence,
the strengths and limitations of individual studies were evaluated based on scientific
considerations detailed in Table 12-5 of the Process Appendix (Section 12.6.1). More
details on the causal framework used to reach these conclusions are included in the
Preamble to the ISA (U.S. EPA. 2015). The evidence presented throughout this
appendix supports the following causality conclusions:
Outcome Group Causality Determination
Immunosuppression Likely to be Causal
Sensitization and Allergic Suggestive of, but not sufficient to infer, a
Responses causal relationship
Autoimmunity and , , .
... J. Inadequate
Autoimmune Disease M
The Executive Summary, Integrated Synthesis, and all other appendices of this Pb ISA
can be found at https://assessments.epa.gov/isa/document/&deid=359536.
6.1 Introduction, Summary of the 2013 Pb ISA, and Scope of
the Current Review
The 2013 Integrated Science Assessment for Lead (hereinafter referred to as the 2013 Pb ISA)
issued causality determinations for the effects of Pb exposure on different aspects of the immune system
including atopic and inflammatory responses, decreased host resistance, and autoimmunity (U.S. EPA.
2013). It is not without precedent for a single chemical to exert both stimulatory and suppressive effects
on various immune parameters (IPCS. 2012). The evidence underpinning these causality determinations is
briefly summarized below.
The body of epidemiologic and toxicological evidence integrated across the 2013 Pb ISA
indicates a "likely to be causal" relationship between Pb exposure and increased atopic and inflammatory
conditions. This relationship is supported by evidence for associations of blood Pb levels (BLL) with
asthma and allergy in children and Pb-associated increases in immunoglobulin E (IgE) in children and
laboratory animals. Uncertainties in the epidemiologic evidence related to potential confounding by
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socioeconomic status (SES), smoking, or allergen exposure are reduced through consideration of the
evidence from experimental animal studies. The biological plausibility for the effects of Pb on IgE is
provided by consistent findings in animals with gestational or gestational-lactational Pb exposures, with
some evidence at BLL relevant to humans. These findings are supported by strong evidence of Pb-
induced increases in T cell-derived helper (Th)2 cytokine production and inflammation in animals (U.S.
EPA. 20.1.3).
Available toxicological evidence evaluated in the 2013 Pb ISA indicates a "likely to be causal"
relationship between Pb exposure and decreased host resistance. This conclusion was based primarily on
animal toxicological studies in which relevant Pb exposures decreased responses to antigens
(i.e., suppressed the delayed-type hypersensitivity (DTH) response and increased bacterial titers and
subsequent mortality in rodents). Further, evidence demonstrating biological plausibility, including
suppressed production of Thl cytokines and decreased macrophage function in animals support these
conclusions (U.S. EPA. 20.1.3).
The 2013 Pb ISA also included an evaluation of the epidemiologic and toxicological evidence for
Pb-induced autoimmunity. Only a few toxicological studies provided evidence for Pb-induced generation
of autoantibodies and the formation of neoantigens that could result in the development of autoantibodies
following Pb exposure. Considering the limited evidence at hand, the available studies were inadequate to
determine if there is a causal relationship between Pb exposure and autoimmunity ( 20.1.3).
This ISA determined causality for adverse effects of Pb exposure on the three different aspects of
the immune system. Accounting for recent toxicological and epidemiologic studies demonstrating that Pb
exposure decreases host resistance to infection, suppresses the DTH response in animals, and decreases
the vaccine antibody response in children, there is sufficient evidence to conclude that there is likely to be
a causal relationship between Pb exposure and immunosuppression. Recognizing that recent
epidemiologic studies do not provide evidence of an association between exposure to Pb and atopic
disease and consistent toxicological evidence that exposure to Pb alters physiological responses in
animals consistent with allergic sensitization, the body of evidence supports changing the causal
determination from likely to be causal to suggestive of a causal relationship between Pb exposure and
sensitization and allergic responses. Evidence for effects of Pb exposure on autoimmunity and
autoimmune disease are disparate and highly limited. For that reason, the body of evidence describing the
relationship between exposure to Pb and autoimmunity remains inadequate to determine if a causal
relationship exists.
The following sections provide an overview of study inclusion criteria for this appendix
(Section 6.2), summaries of recent health effects evidence (Sections 6.3, 6.4, and 6.5), a discussion of
biological plausibility (Section 6.6), and a discussion of the causality determination for Pb exposure and
immune system effects (Section 6.7, Table 6-1, Table 6-2, and Table 6-3).
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6.2 Scope
The scope of this appendix is defined by Population, Exposure, Comparison, Outcome, and Study
Design (PECOS) statements. The PECOS statement defines the objectives of the review and establishes
study inclusion criteria, thereby facilitating identification of the most relevant literature to inform the Pb
ISA.1 In order to identify the most relevant literature, the body of evidence from the 2013 Pb ISA was
considered in the development of the PECOS statements for this appendix. Specifically, well-established
areas of research; gaps in the literature; and inherent uncertainties in specific populations, exposure
metrics, comparison groups, and study designs identified in the 2013 Pb ISA inform the scope of this
appendix. The 2013 Pb ISA used different inclusion criteria than the current ISA, and the studies
referenced therein often do not meet the current PECOS criteria (e.g., due to higher or unreported
biomarker levels). Studies that were included in the 2013 Pb ISA, including many that do not meet the
current PECOS criteria, are discussed in this appendix to establish the state of the evidence prior to this
assessment. With the exception of supporting evidence used to demonstrate the biological plausibility of
Pb-associated effects on the immune system, recent studies were only included if they satisfied all of the
components of the following discipline-specific PECOS statements:
Epidemiologic Studies:
Population: Any human population, including specific populations or lifestages that might be at
increased risk of a health effect;
Exposure: Exposure to Pb2 as indicated by biological measurements of Pb in the body - with a
specific focus on Pb in blood, bone, and teeth; validated environmental indicators of Pb
exposure3; or intervention groups in randomized trials and quasi-experimental studies;
Comparison: Populations, population subgroups, or individuals with relatively higher versus
lower levels of the exposure metric (e.g., per unit or log unit increase in the exposure metric,
or categorical comparisons between different exposure metric quantiles);
Outcome: Immune system effects including but not limited to immunotoxicity, systemic
inflammation, and immune-based diseases; and
'The following types of publications are generally considered to fall outside the scope and are not included in the
ISA: review articles (which typically present summaries or interpretations of existing studies rather than bringing
forward new information in the form of original research or new analyses), Pb poisoning studies or clinical reports
(e.g., involving accidental exposures to very high amounts of Pb described in clinical reports that may be extremely
unlikely to be experienced under ambient air exposure conditions), and risk or benefits analyses (e.g., that apply
concentration-response functions or effect estimates to exposure estimates for differing cases).
2Recent studies of occupational exposure to Pb were considered insofar as they addressed a topic area that was
relevant to the National Ambient Air Quality Standards review (e.g., longitudinal studies designed to examine recent
versus historical Pb exposure).
3Studies that estimate Pb exposure by measuring Pb concentrations in particulate matter with a nominal mean
aerodynamic diameter less than or equal to 10 |im3 (PMio) and particulate matter with a nominal mean aerodynamic
diameter less than or equal to 2.5 |im3 (PM2 5) ambient air samples are only considered for inclusion if they also
include a relevant biomarker of exposure. Given that size distribution data for Pb-PM are fairly limited, it is difficult
to assess the representativeness of these concentrations to population exposure | Section 2.5.3 (U.S. EPA. 20.1. 3)].
Moreover, data illustrating the relationships of Pb-PMio and Pb-PM2 5 with BLLs are lacking.
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Study Design: Epidemiologic studies consisting of longitudinal and retrospective cohort studies,
case-control studies, cross-sectional studies with appropriate timing of exposure for the health
endpoint of interest, randomized trials, and quasi-experimental studies examining
interventions to reduce exposures.
Experimental Studies:
Population: Laboratory nonhuman mammalian animal species (e.g., mouse, rat, guinea pig,
minipig, rabbit, cat, dog) of any lifestage (including preconception, in utero, lactation,
peripubertal, and adult stages);
Exposure: Oral, inhalation, or intravenous treatment(s) administered to a whole animal (in
vivo) that results in a BLL of 30 (ig/dL or below;4'5
Comparators: A concurrent control group exposed to vehicle-only treatment or untreated
control;
Outcome: Immunological effects; and
Study Design: Controlled exposure studies of animals in vivo.
Consistent with this scoping, the following sections evaluate evidence for the effects of Pb
exposure on the immune system. In the 2013 Pb ISA, evidence for effects on the immune system was
organized into atopic and inflammatory responses, decreased host resistance, and autoimmunity.
Immunological evidence for this ISA is organized to reflect disease categories most relevant to Pb
exposure including immunosuppression (Section 6.3), sensitization and allergic responses (Section 6.4),
and autoimmunity and autoimmune diseases (Section 6.5). These categories encapsulate the immune-
related endpoints used in the 2013 Pb ISA while recognizing advances in the field of immunotoxicology.
The sections that follow focus on studies published since the completion of the 2013 Pb ISA. This
evidence is organized and weighed based on the World Health Organization's Guidance for
Immuno toxicity Risk Assessment for Chemicals ("IPCS. 2012). As detailed in this guidance, data from
endpoints observed in the absence of an immune stimulus (e.g., levels of serum immunoglobulins, white
blood cell (WBC) counts, WBC differentials, T cell subpopulations, immune organ weights) are not
sufficient on their own to draw a conclusion regarding immune hazard but may provide useful supporting
evidence, especially when evaluated in the broader context of functional data ("IPCS. 2012).
Consequently, the sections that follow are organized into two categories: the more informative measures
of immune system function and supporting immune system data. Study-specific details, including animal
4Pb mixture studies are included if they employ an experimental arm that involves exposure to Pb alone.
5This level represents an order of magnitude above the upper end of the distribution of U.S. young children's BLLs.
The 95th percentile of the 2011-2016 National Health and Nutrition Examination Survey distribution of BLL in
children (1-5 years; n = 2,321) is 2.66 (.ig/dL (CDC. 20.1.9') and the proportion of individuals with BLLs that exceed
this concentration varies depending on factors including (but not limited to) housing age, geographic region, and a
child's age, sex, and nutritional status.
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type, exposure concentrations, and exposure durations in experimental studies, and study design, exposure
metrics, and select results in epidemiologic studies are presented in evidence inventories in Section 6.8.
6.3 Immunosuppression
Immunosuppression can lead to the increased incidence and/or severity of infectious and
neoplastic diseases. Immunosuppressants may be identified using data generated from general toxicity
studies or through completion of dedicated immunotoxicity studies. In either case, evidence may be
collected from assays designed to assess the function of the immune system following xenobiotic
exposure or from supporting immune system endpoints.
6.3.1 Epidemiologic Studies of Immunosuppression
Epidemiologic studies relevant to immunosuppression generally include studies of viral and
bacterial infection and vaccine antibody response, as well as studies of WBCs and cytokines. A limited
number of epidemiologic studies evaluated in the 2013 Pb ISA ("U.S. EPA. 20.1.3') provided evidence of
associations between cord blood or blood Pb and viral and bacterial infection in children. However, these
studies were cross-sectional and did not include adjustment for potential confounders, limiting the
strength of conclusions that could be drawn about the effects of Pb exposure on viral or bacterial
infections. Cross-sectional studies of cell-mediated immunity reported consistent associations between
BLL and lower T cell abundance in children, while results from other studies on lymphocyte activation,
macrophages, neutrophils, and natural killer (NK) cells were generally inconsistent or not sufficiently
informative (e.g., cross-sectional study designs with limited or no consideration of potential confounding
and a lack of information on concentration-response relationships).
There have been a number of recent epidemiologic studies of immunosuppression, including
prospective birth cohorts and studies with lower mean or median BLL than those reviewed in the 2013 Pb
ISA, many with measures of central tendency <2 (ig/dL. The recent studies also apply more robust
statistical methods and consistently consider a wider range of potential confounders. In general, recent
studies provide consistent evidence that exposure to Pb is associated with greater susceptibility to
infection and a less robust vaccine antibody response. Additionally, a group of studies in the same
population provides some evidence of altered immune cells and cytokines in association with BLLs.
Measures of central tendency for BLLs used in each study, along with other study-specific details,
including study population characteristics and select effect estimates, are highlighted in Table 6-4. An
overview of the recent evidence is provided below.
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6.3.1.1 Host Resistance
While the 2013 Pb IS A (U.S. EPA. 20.1.3') evaluated a limited number of epidemiologic studies
that indicated an association between BLL and viral and bacterial infections in children, none of the
studies considered potential confounders and most analyzed populations with high mean BLLs (means
>10 (ig/dL). Recent studies expand the evidence base by examining populations with wider age-ranges
and much lower mean and median BLLs. The recent studies also adjust for a wide range of potential
confounders, including extensive consideration of SES factors.
Recent cross-sectional studies provide consistent evidence of associations between Pb exposure
and viral and bacterial infections, including Helicobacter Pylori, Toxoplasma Gondii, and hepatitis B
("Park et ah. 2020; Krueger and Wade. 2016). or susceptibility to antibiotic resistance measured via nasal
Staphylococcus aureus colonization (Eggers et ah. 20.1.8). In a National Health and Nutrition Examination
Survey (NHANES) analysis including children and adults, each 1 (ig/dL higher level of blood Pb was
associated with 8 to 10% higher odds of H. Pylori (odds ratio [OR]: 1.09 [95% confidence interval (CI):
1.05, 1.13]), T. Gondii (OR: 1.10 [95% CI: 1.06, 1.14]), and hepatitis B (OR: 1.08 [95% CI: 1.03, 1.13])
seropositivity in the U.S. population ("Krueger and Wade. 20.1.6). Positive associations were persistent, but
varied in magnitude across more specific age groups, including children under 13, participants aged 13 to
35, and adults >35 years old. The associations for H. Pylori were markedly stronger in magnitude for
children less than 13 years old compared with the other age groups, whereas the associations for T.
Gondii were slightly weaker in children. Additionally, in multipollutant models with cadmium (Cd), there
was no evidence to suggest additive or multiplicative interaction between Pb and Cd. Another cross-
sectional study of adults with abnormal lesions identified during endoscopy also reported that H. Pylori
infection rates were associated with increased BLL (Park et ah. 2020).
In addition to cross-sectional studies, a recent test-negative case-control study reported that peak
BLLs were associated with elevated influenza and respiratory syncytial virus (RSV) rates in children
<4 years old presenting with relevant symptomology (Feiler et ah. 2020). Test-negative case-control
study designs are often used in vaccine efficacy studies to control for healthcare seeking behaviors, but
for the intended purposes of this study, the design could bias results toward the null if the non-RSV and
influenza illnesses are also related to Pb-induced immune deficiencies. The results in the full population
were adjusted for fewer potential confounders (i.e., age, sex, race, ethnicity, insurance status, and season)
on account of missing variables, and the observed associations were null in a notably reduced sample
population (<25%) with expanded adjustment for confounders.
6.3.1.2 Antibody Responses
There were no studies evaluated in the 2013 Pb ISA (U.S. EPA. 20.1.3) that examined the
relationship between exposure to Pb and vaccine antibody response in children. There are a few recent
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studies that provide generally consistent evidence of Pb-related decreases in vaccine antibodies in
populations with low mean or median BLL.
In a birth cohort of vaccinated children in South Africa. irdo et al. (2020) reported that
each 1 (ig/dL higher level of blood Pb at age 1 was associated with 13% (95% CI: 2%, 26%) higher risk
of tetanus IgG titers below the protective limit at age 3.5 years. A key strength of this study was its
prospective nature and the timing of blood Pb measures that approximately coincided with vaccine
administration. The authors also examined measles and Haemophilus influenzae type B (Hib) IgG levels
but did not observe associations with BLL. Cross-sectional studies—including a large NHANES analysis
of children ages 6 to 17 years old ("Jusko et al.. 20.1.9) and another small study comparing kindergarten-
aged children in China living near an e-waste facility to those in a nearby community with similar
sociodemographic characteristics ("Xu et al.. 2015)—also provide evidence that higher BLLs are
associated with lower counts of virus-neutralizing antibodies. However, unlike the results from Pi
Lenardo et al. (2020). Jusko et al. (20.1.9) reported that higher BLLs were associated with lower counts of
anti-measles IgG antibodies, as well as anti-mumps antibodies. The authors observed a null association
with anti-rubella IgG levels. In the analysis in China. Xu et al. (2015) noted that geometric mean BLL
dropped precipitously between the 2 years of the study (>3 (.ig/dL). The authors conducted an analysis
stratified by the year of the study and observed lower anti-hepatitis B surface antigen (HBsAb) titers in
relation to higher BLLs in both years; however, the association was notably stronger in magnitude in the
year with higher geometric mean BLL (2011: -0.447 s/co [95% CI: -0.491, -0.403 s/co]; 2012: -0.366
s/co [95% CI: -0.404, -0.328 s/co] per 1 (ig/dL higher BLL). An important uncertainty in this analysis is
potential confounding by other contaminants present in the community. In contrast to the previously
discussed evidence, a birth cohort of vaccinated children in Bangladesh reported a positive association
between cord BLL and diphtheria and tetanus IgG antibodies at age 5 (Welch et al.. 2020). Notably, the
associations were null when the exposure metric was concurrent BLL rather than cord blood Pb.
6.3.1.3 White Blood Cells and Cytokines
Several epidemiologic studies evaluated in the 2013 Pb ISA (U.S. EPA. 2013) examined the
relationship between Pb exposure and changes in WBC populations (i.e., counts and phenotypes) and
cytokine levels. Although WBC counts and cytokine levels are commonly evaluated in epidemiologic
studies, these data can be challenging to interpret because (1) WBC populations are not particularly
sensitive indicators of immunotoxicity and (2) changes in cytokine levels can be associated with many
different types of tissues and toxicities, either as part of cell differentiation to different immune cell types
or including site-specific inflammation, which reflects an immune response to tissue injury but not
necessarily an effect on or impairment of immune function (Tarrant. 2010). For these reasons. WBC
populations and cytokine secretion data (in the absence of a stimulus) are not considered apical outcomes
for the purpose of identifying immune hazard, but rather as supporting evidence for understanding
mechanisms of immune disruption.
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In the 2013 Pb ISA, there was generally consistent evidence of positive associations between
BLLs and T cell counts in children, but epidemiologic evidence for other immune cell and cytokine
measures were uninformative due to cross-sectional study designs with limited or no consideration of
potential confounding and a lack of information on the concentration-response relationship. Recent
studies provide some evidence of associations between Pb exposure and immune cell and cytokine
abundance in children, though the number of studies examining overlapping immunological markers is
limited.
The majority of recent epidemiologic studies of WBCs and cytokines come from a group of
related, small cross-sectional studies evaluating a study population of kindergarten-aged children in
Guangdong, China living either near an e-waste facility or in a nearby community with otherwise similar
sociodemographic characteristics and pollutant exposures ("Chen et aL 2021; Zhang et ah. 2020; Huo et
ah. 20.1.9; Cao et ah. 20.1.8; Dai et ah. ). Across these studies, authors reported that BLLs were
positively associated with a number of biomarkers related to immunological function, including the
proinflammatory cytokines interleukin (IL)-1(3 ("Zhang et ah. 2020; Huo et ah. 2019). IL-12p70, and
interferon (IFN)-y (Huo et ah. 20.1.9) and pleiotropic cytokine IL-6 (Zhang et ah. 2020). Chronic
inflammation has the potential to contribute to the development of immunosuppression (Kanterman et ah.
20.1.2). In addition, higher BLLs were associated with differences in several other biomarkers of immune
system function including higher erythrocyte complement receptor type 1 (CR1) expression (Dai et ah.
20.1.7); a higher percentage of cluster of differentiation (CD)4+ central memory T cells (Cao et ah. 20.1.8);
higher neutrophil counts (Zhang et ah. 2020); higher counts of WBCs, neutrophils, and monocytes (Chen
et ah. 2021); a lower percentage of CD4 naive T cells (Cao et ah. 20.1.8); and lower levels of tumor
necrosis factor alpha (TNF)-a (Zhang et ah. 2020). The authors of these studies also noted some null
associations with BLLs. including CD3 . CD4 . and CDS cell counts (Cao etah. 20.1.8) and monocytes,
lymphocytes, IL-8, and IL-10 (Zhang et ah. 2020). Consistent with Chen et ah (2021). another cross-
sectional study in China with a similar design (e.g., kindergartners recruited from reference and control
communities with and without industrial exposure to Pb) reported null associations between BLL and
odds of lowerlower WBC counts (Li et ah. 20.1.8).
In the only recent study of an adult population, a small cross-sectional analysis of oil spill
response workers with low BLLs (mean: 1.82 (.ig/dL). Werder et ?0) observed associations between
higher BLLs and higher proinflammatory cytokines (i.e., IL-1(3 and IL-8) and pleiotropic cytokine IL-6
but not the proinflammatory cytokine TNF-a. This was generally consistent with the previously discussed
results in children, with the exception of IL-8 for which a null association was reported in children.
Notably, as highlighted in a stratified analysis to examine effect modification by obesity, the observed
associations are entirely driven by associations in obese participants Werder et ah (2020). For example,
each 1 (ig/dL higher level of blood Pb was associated with 72.8 pg/mL (95% CI: 36.9, 108.7 pg/mL)
higher IL-6 levels in the entire study population. However, in the stratified analysis, the association was
stronger in magnitude in obese participants (169.6 pg/mL [95% CI: 119.8, 219.4 pg/mL]) and null in non-
obese participants (-2.6 pg/mL [95% CI: -45.5, 40.3 pg/mL]).
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6.3.2
Toxicological Studies of Immunosuppression
Toxicological studies evaluated in the 2013 Pb ISA (U.S. EPA, 20.1.3) investigating Pb-induced
immunosuppression were derived from several lines of evidence including functional assays (i.e., host
resistance, antibody responses, DTH response, and ex vivo WBC function) and bolstered by various
forms of supporting immune system data. Some of these data were reviewed in the 2006 Pb Air Quality
Criteria Document (AQCD) (U.S. EPA, 2006). Based on these previous evaluations, there is clear
evidence that exposure to Pb decreases host resistance to bacterial infection and increases production of
some pathogen-specific antibody subtypes promoting the shift toward Th2-type immune responses. The
results of investigations of the T cell dependent antibody response were inconsistent, with one study
reporting a decrease in the antibody response (BLL not reported) and another showing no effect in mice
with high BLLs (i.e., 59-132 (.ig/dL). However, Pb has consistently been shown to suppress the DTH
response in animal models. The DTH assay has a long history of use in immunotoxicity testing and i s
considered one of the most predictive immunotoxicity tests available (Dietert et al., 20.1.0). Suppression
of the DTH response is a hallmark of Pb exposure. Exposure to Pb suppressed the DTH response in rats
(Chen et al., 2004; Bunn et al., 2001a; Bunn et al., 2001b; Chen et al., .1.999; Miller etah, .1.998) and
chickens (Lee et al., 2002; Lee et al., 200.1.) with BLLs relevant to this ISA (i.e., <30 (.ig/dL). The DTH
response was also suppressed by Pb exposures in other studies not reporting a BLL (Laschi-Loquerie et
al., .1.984; Faith et al., .1.979; Mtiller et al., .1.977) and in studies reporting BLLs outside the scope of this
ISA (Bunn et al., 2001c; McCabe et al., .1.999; Fandrich et al., .1.995).
Pb exposure also affected the functions of various WBCs under ex vivo conditions leading to (1)
suppression of Thl-mediated immunity (i.e., suppressed Thl cytokine production (e.g., IFN-y) and DTH
response); (2) altered macrophage function (e.g., increased reactive oxygen species [ROS] production,
decreased nitric oxygen [NO] production); and (3) reduced monocyte/macrophage phagocytosis. In
addition to assessing the effect of Pb on measures of immune system function, the effects of Pb exposure
on various immunotoxicology-related supporting immune system endpoints were also evaluated,
including (1) total serum immunoglobulins, (2) immune organ weight, (3) WBC number in the spleen,
thymus, lymph nodes, and bone marrow, and (4) WBC counts and subpopulation data collected from
blood samples. Generally, the number of these studies was limited and differences in study design and the
specific endpoints measured create challenges when interpreting these supporting immune system data.
Recent toxicological studies are limited in number and report on disparate outcomes, but
generally support evidence reported in the 2013 Pb ISA. Consistent with findings reported in the 2013 Pb
ISA, Pb exposure was again shown to suppress the DTH response. There are no recent toxicology studies
investigating the effects of Pb exposure on host resistance; however, there is some recent evidence that Pb
exposure altered the levels of some classes of antigen-specific antibodies in iron-deficient rats. Pb
exposure also reduced the total serum levels of some immunoglobulins in rats. As with the 2013 Pb ISA,
the effects of Pb on immune organ pathology and spleen weight were inconsistent. New to this ISA, a
recent study reported that Pb exposure decreased relative thymus weight. Differences in experimental
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design and the specific types of WBCs assessed complicate interpretation of data collected on the number
and relative abundance of the different types of WBCs in the spleen, thymus, lymph nodes, and bone
marrow following exposure to Pb. WBC counts and subpopulation data collected from hematological
investigations are similarly challenging to interpret.
6.3.2.1 Host Resistance
Available toxicological evidence evaluated in the 2013 review provides clear evidence that host
resistance to bacterial infection is compromised following Pb exposures, resulting in BLLs as low as
20 (.ig/dL. The 2013 Pb ISA (U.S. EPA. 2013) reported several rodent host resistance studies wherein
mortality was increased in pathogen-exposed animals that were also exposed to Pb through drinking
water. For example, various studies reported decreased clearance of bacteria and increased mortality
induced by Listeria monocytogenes in mice exposed postnatally to Pb acetate in drinking water for 3 to
8 weeks, resulting in BLLs ranging from 20-25 (.ig/dL (Dvatlov and Lawrence. 2002; Kim and Lawrence.
2000; Kishikawa et a ; wrence. .1.98.1). Other studies reported increased mortality from
Salmonella or Escherichia coli, or decreased clearance of Staphylococcus, in mice administered Pb
acetate or Pb nitrate via injection, resulting in BLLs relevant to the 2013 Pb ISA (Fernandez-Cabezudo et
ah. 2007; Bishavi and Sengupta. 2006; Cook et ah. .1.975; Hemphill et ah. .1.971; Selve et ah. 1966). In
addition to high BLL (i.e., 71-313 (ig/dL), increased mortality from viral infection was also reported in
mice and chickens administered Pb (mostly Pb acetate) for 4-10 weeks ("Gupta et ah. 2002; Exon et ah.
.1.979; Thind and Khan. .1.978). Further, evidence suggested a plausible mode of action involving
suppressed production of Thl cytokines (Fernandez-Cabezudo et ah. 2007; Lara-Teiero and Paroer.
2004). decreased macrophage function (Lodi et ah. 2 ; shavi and Sengupta. 2006; Chen et ah. .1.997;
Hilbertz et ah. .1.986; Castranova et ah. 1.980). and increased inflammation in animals (Miller et ah. .1.998;
Bavkov et ah. .1.996; Zelikoff et ah. .1.993).
There were no recent toxicology studies investigating the effects of Pb exposure on host
resistance that satisfied the PECOS criteria described in Section 6.2 available for this review.
6.3.2.2 Delayed-Type Hypersensitivity Responses
Antigen-specific cell-mediated immune (CMI) responses are a key component of host defense
mechanisms against virally infected cells, tumor cells, and certain fungal infections. The DTH assay is a
standard test for assessing CMI responses in animals (IPCS. 20.1.2). As noted in the 2013 Pb ISA.
suppressed DTH response is one of the most consistently reported immune effects associated with Pb
exposure in animals (U.S. EPA. 20.1.3). Suppression of the DTH response has been reported following
gestational (Chen et ah. 2004; Bunn et ah. 2001a; Bunn et ah. 2001b. c; Lee et ah. 2001; Chen et ah.
.1.999; Miller et ah. .1.998; Faith et ah. .1.979) and postnatal (McCabe et ah. .1.999; Laschi-Loauerie et ah.
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.1.984; Mulleretal.. 1977) exposures to Pb acetate resulting in BLLs ranging from 6.75 to >100 (.ig/dL) in
rats, mice and chickens (U.S. EPA. 20.1.3).
In a recent study, administration of Pb acetate in drinking water for 42 days (BLL = 18.48 (ig/dL)
significantly suppressed the DTH response in adult male Sprague Dawley rats ("Fang et ah. 20.1.2). To
explore the role of regulatory T cells (Tregs) in the DTH response, Fang et al. (20.1.2) employed a T cell
transfer model. Total CD4+ T cells and CD4+CD25- cells were collected from control and Pb-exposed
rats and then transferred to recipient rats that were subsequently challenged with l-Fluoro-2,4-
dinitrobenzene (DNFB) to induce a DTH response. The DTH response was diminished in rats receiving
CD4+ T cells from Pb-exposed rats compared with those receiving CD4+ cells from control animals.
Importantly, the effect was lost when Tregs were depleted from the pool of CD4+ cells transferred to the
recipient rats. These findings suggest that Tregs play a critical role in Pb-induced immune suppression
(Fang et al.. 20.1.2). Study-specific details, including animal species, strain, sex, and BLLs are highlighted
in Table 6-6.
6.3.2.3 Antibody Responses
The production of antigen-specific antibodies is a major defense mechanism of humoral immune
responses. Only one study reporting effects on antigen-specific antibody responses was evaluated in the
2013 Pb ISA (U.S. EPA. 20.1.3). In that study, Fernandez-Cabezudo i 307) reported no difference in
the serum levels of Salmonella-specific IgM following infection with a sublethal dose of Salmonella
(1.5 x 104 organisms/mouse) in control C3H/HeN mice and mice exposed to 10 mM Pb acetate in
drinking water for 16 weeks (resultant mean BLL: 106 (.ig/dL). However, compared with control mice,
mice exposed to Pb acetate had less IgG2a and more IgGl antibodies providing evidence for a shift
toward Th2-type immune responses resulting in decreased resistance to Salmonella enterica (Fernandez-
Cabezudo et al.. 2007). Studies describing effects of Pb exposure on the T cell dependent antibody
response (TDAR) were also reviewed in the 2013 Pb ISA. The TDAR is a comprehensive immune
function assay that integrates several aspects of immune responses. Thus, xenobiotic-induced alterations
in antigen processing and presentation, B and T cell interactions, antibody production, and isotype class
sw itching and modification have the potential to modify this defense mechanism (IPCS. 20.1.2). Results of
the TDAR response to sheep RBCs have been inconsistent. For example, the TDAR was significantly
decreased in mice exposed to Pb acetate through drinking water for 3 weeks, resulting in BLLs of
25.4 (ig/dL (Blaklev and Archer. .1.981). However, in a second drinking water study, the TDAR was
increased in 1 of 8 mouse strains (the other 7 strains were unaffected) evaluated following administration
of Pb acetate in drinking water for 8 weeks resulting in high BLL (mean range 59-132 (.ig/dL) (Mudzinski
et al.. .1.986).
In a recent study, adult Sprague Dawley rats (data from both sexes pooled) were fed either a
control diet or an iron-deficient diet for the duration of the experiment (Y athapu et al.. 2020). After
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confirming iron deficiency at 4 weeks, rats were administered Pb acetate in drinking water for 4 weeks.
At this time, a subset of mice was vaccinated with tetanus toxoid (TT). Rats received two booster doses
(2-week interval) before assessing antigen-specific antibody levels 2 weeks after the last booster dose.
Under these conditions, Pb acetate (BLL = 16.1 (ig/dL) had no effect on the levels of anti-TT-specific IgG
and IgM antibodies in the serum of rats that received the control diet whereas the levels of anti-TT-
specific IgM were decreased and those of IgG were unaffected in the serum of iron-deficient rats
("Yathapu et al.. 2020). Study-specific details, including animal species, strain, sex, and BLLs are
highlighted in Table 6-5.
6.3.2.4 Ex Vivo White Blood Cell Function
White blood cells are cells of the immune system involved in protecting the body from infectious
disease. These cells can be organized into two lineages—myeloid cells and lymphoid cells. Myeloid cells
(i.e., myelocytes) include neutrophils, eosinophils, mast cells, basophils, and monocytes. Lymphoid cells
(i.e., lymphocytes) include T cells, B cells, and NK cells. Xenobiotic-induced alterations in ex vivo WBC
function is considered clear evidence of immunosuppression (IPCS, 20.1.2). Ex vivo WBC function assays
are performed outside the body using immune cells collected from exposed individuals.
The 2013 Pb ISA reviewed the effects of Pb exposure on the functions of various WBCs under ex
vivo conditions indicating (1) a shift in lymphocyte cytokine production towards the production of Th2
cytokines (Heo et al., 2007; McCabe and Lawrence, .1.991), reduced number of Thl cells and Thl
cytokine levels (McCabe and Lawrence, .1.991), (2) increased dendritic cell induced Th2 cell proliferation
and cytokine production (Gao et al., 2007), and (3) reduced monocyte/macrophage phagocytosis (Lodi et
al., 2( : jsolaro et al., 2008; Deng and Poretz, 2001; Kowolenko et al., 1.991; Zhou et al., .1.985) and
decreased NO production (Fairer et al., 2008; Mishraet al., 2006; Bunn et al., 2001b; Lee et al., 2001;
Krocova et al., 2000; Chen et al., .1.997; Tian and Lawrence, .1.996; Tian and Lawrence, .1.995). No studies
on neutrophils and NK cells were reviewed in the 2013 Pb ISA.
A few PECOS-relevant papers evaluating the effects of Pb exposure on ex vivo WBC function
have been published since the 2013 Pb ISA. Fang et al. (20.1.2) reported that administration of Pb acetate
in drinking water for 42 days (BLL = 18.48 (ig/dL) had no effect on the suppressive properties of Tregs
isolated from adult male Sprague Dawley rats. In a second study, the effects of Pb administration on
Concanavalin A (Con A)-stimulated lymphocyte proliferation and cytokine production were investigated
(Yathapu et al., 2020). For this investigation, adult male and female Sprague Dawley rats were fed either
a control diet or an iron-deficient diet for the duration of the experiment. After confirming iron deficiency
at 4 weeks, the rats were administered Pb acetate in drinking water for 4 weeks. At this time, a subset of
rats was vaccinated with TT. Rats received two booster doses (2-week interval) before splenocytes were
collected 2 weeks after the last booster dose. Irrespective of vaccine status, Pb treatment
(BLL =16.1 (ig/dL) had no effect on Con A-stimulated proliferation of splenocytes collected from rats
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fed the control diet. However, when rats were fed an iron-deficient diet, Pb treatment (BLL = 41.6 (ig/dL)
increased Con A-stimulated splenocyte proliferation ("Yathapu et ah. 2020). Unfortunately, because of
incomplete reporting, data related to cytokine production by Con A-stimulated splenocytes reported by
Yathapu etal. (2020) are not interp re table. In addition, Cai et al. (20.1.8) measured cytokine levels directly
in blood and reported that, administration of Pb acetate drinking water (0.2%; BLL = 9.3 (ig/dL) for
84 days had no effect on erythropoietin, granulocyte-macrophage colony-stimulating factor (GM-CSF),
interleukin (IL)-6, and TNF-a levels in adult Sprague Dawley rats (data from sexes pooled). Study-
specific details, including animal species, strain, sex, and BLLs are highlighted in Table 6-7 and
Table 6-14.
6.3.2.5 Immune Organ Pathology
The 2013 Pb ISA did not report on the effects of Pb exposure on immune organ pathology (U.S.
EPA. 20.1.3). However, xenobiotic exposure can alter primary immune sites important for immune cell
maturation, including the bone marrow, liver, thymus, and Peyer's patches. Secondary lymphoid sites
(i.e., spleen, lymph nodes, tonsils) can also be affected by exposure to immunotoxicants. Data from these
endpoints are not sufficient on their own to draw a conclusion regarding immune hazard, but may provide
useful supporting evidence (IPCS. 20.1.2). Pb-induced alterations in immune organ pathology were not
addressed in the 2013 Pb ISA.
Since the 2013 Pb ISA, there have been three reports published that included an assessment of
immune organ pathology following exposure to Pb and that fit the PECOS criteria described in
Section 6.2. In the first study, Pb treatment induced changes in the spleen architecture of adult male
C57BJ mice exposed via drinking water (200 ppm; BLL = 21.6 (ig/dL) for 45 days. These changes
included increasing the amount of white pulp (qualitative) and decreasing the definition of the
germinative center of the inner peri-arteriolar lymphoid sheath, but the marginal zone was unaffected
(Corsetti et al.. 20.1.7). In a different study, inhalation of Pb oxide nanoparticles (1.23 x 106 x 10
particles/cm3, 24 hours/day for 6 weeks BLL 13.9 (ig/dL) had no effect on spleen pathology in two
experiments conducted in adult female Institute for Cancer Research (ICR) mice (Dumkova. et al.. 20.1.7).
Dumkova et al. (2020a) conducted another study with Pb oxide nanoparticles (68.6 x 106 particles/cm3,
24 hours/day for up to 6 weeks) in CD-I (ICR) mice that included histological analysis of the spleen, but
did not report their findings. Exposure to Pb oxide nanoparticles (0.95 6 x 106 particles/cm3, 24 hours/day
for 11 weeks, BLL =18.1 (.ig/dL) had no effect on spleen histopathology in CD-I (ICR) BR mice (Smutna
et al.. 2022). Study-specific details, including animal species, strain, sex, and BLLs are highlighted in
Table 6-8.
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6.3.2.6
Immunoglobulin Levels
Immunoglobulins (i.e., antibodies) are produced by plasma cells (i.e., differentiated B cells).
Immunoglobulins are a critical part of the immune response and act by recognizing and binding to
specific antigens such as bacteria and viruses leading to their destruction. Although immunoglobulin type
and quantity are easy to measure in serum, their levels are difficult to interpret in the absence of a
controlled immune challenge. For this reason, these data are not considered a predictive measure for
immunotoxicity and are most useful for supporting data collected from immune functional assays. The
2013 Pb ISA reviewed the effects of Pb exposure on total serum IgE in the context of immediate-type
hypersensitivity (Chen et ah, 2004; Snyder et ah, 2000; Miller et ah, .1.998; Heo et ah, .1.997; Heo et ah,
.1.996). In addition, the 2013 Pb ISA reviewed the effects of Pb exposure on total serum IgG subtypes
(Kasten-Jolly et ah, 20.1.0; Carey et ah, 2006; Gao et ah, 2006; Snyder et ah, 2000). While noting that the
BLLs were not relevant to human exposures, the 2013 Pb ISA described the observed effects as
inconsistent.
Since the 2013 Pb ISA, only one PECOS-re levant publication included an assessment of total
serum immunoglobulin levels following exposure to Pb. For this investigation, adult Sprague Dawley
(data from sexes pooled) were fed either a control diet or an iron-deficient diet for the duration of the
experiment. After confirming iron deficiency after 4 weeks, rats were administered Pb acetate in drinking
water for 4 weeks. At this time, a subset of mice was vaccinated with TT. Rats received two booster doses
(2-week interval) before splenocytes were collected 2 weeks after the last booster dose. Irrespective of
vaccine status, Pb treatment reduced mucosal IgA levels in rats fed the control diet (BLL =16.1 (ig/dL).
Under conditions of iron deficiency, Pb treatment further reduced mucosal IgA levels
(BLL = 41.6 (ig/dL). Total serum IgM and IgG were unchanged by Pb under all conditions evaluated
(Yathapu et ah, 2020). Study-specific details, including animal species, strain, sex, and BLLs are
highlighted in Table 6-9.
6.3.2.7 Immune Organ Weights
Changes in lymphoid organ weights (thymus, spleen, lymph node, or bone marrow) may indicate
immunotoxicity and are useful for supporting data collected on immune function. As reported in the 2013
Pb ISA, exposure to Pb increased relative spleen weight in mice and rats exposed to Pb acetate and Pb ion
in drinking water (U.S. EPA. 20.1.3). In the only available study, lymph node weight decreased following
exposure to Pb acetate (Institoris et ah. 2006). There were no studies that evaluated changes in thymus
weight reviewed in the 2013 Pb ISA. Several recent studies evaluating the effects of Pb exposure on
lymphoid tissues are described below, including one study describing effects on the thymus. Study-
specific details, including animal species, strain, sex, and BLLs are highlighted in Table 6-10.
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6.3.2.7.1
Thymus Weight
The thymus, which is essential for T cell development, is a critically important component of the
immune system; changes in thymus weight are a more sensitive indicator of immunotoxicity than changes
in spleen weight. Relative thymus weight was significantly decreased in juvenile Sprague Dawley rats
(data from sexes pooled) orally administered Pb acetate (1 or 10 mg/kg with BLL of 3.27 (ig/dL and
12.5 (ig/dL. respectively) for up to 25 days ("Graham et ah. 20.1.1). A second study performed by the same
laboratory using the same experimental design investigated the effects of oral administration of Pb acetate
(gavage) on relative thymus weight (Amos-Kroohs et aL 20.1.6). Because of incomplete reporting,
however, the effect of Pb on thymus weight could not be discerned and this element of the study was
rejected for study quality deficiencies.
6.3.2.7.2 Spleen Weight
The spleen has a prominent role in immune function, as well as serving as a reservoir for
monocytes. The effect of Pb administration via oral and inhalation routes in rats and mice has been
recently investigated. In juvenile Sprague Dawley rats (data from sexes pooled), relative spleen weight
was not affected following oral administration of Pb acetate (gavage, 1 or 10 mg/kg with BLL up to 3.27
and 12.5 (ig/dL. respectively) for up to 25 days ("Amos-Kroohs et aL 20.1.6; Graham et aL 20.1.1).
Absolute spleen weight, however, was decreased significantly following exposure to 10 mg/kg
(BLL =12.5 (ig/dL) Pb acetate (Graham et aL. 20.1. D. Similarly, spleen weight was unaffected in adult
male Wistar rats exposed to Pb acetate in drinking water (357 (ig/kg/day or 1607 (ig/kg/day with BLL of
1.77 ± 0.7 (ig/dL and 8.6 ± 2.9 (ig/dL, respectively) for 4 weeks (Wildemann et aL 2015). In the only
study investigating the effects of Pb exposure in mice, Pb acetate treatment significantly increased
relative spleen weight in adult male C57BJ mice exposed via drinking water (200 ppm,
BLL = 21.6 (ig/dL) for 45 days (Corsetti et aL 20.1.7).
Effects of Pb exposure through inhalation were inconsistent. Inhalation exposure to Pb oxide
nanoparticles (1.23 x 106 nanoparticles/cm3, BLL 13.9 (ig/dL) increased relative spleen weight in adult
female ICR mice exposed for 6 weeks, but the finding was not replicated in a duplicate experiment
performed as part of the same study (Dumkova. et aL 2017). In a second study performed by the same
lead investigator, inhalation exposure to a higher concentration of Pb oxide nanoparticles (2.23 x 106
nanoparticles/cm3) for a longer duration (i.e., 11 weeks) had no effect on relative spleen weight adult
female CD-I (ICR) BR mice with a BLL of 17.4 (ig/dL (Dumkova et aL 2020b). However, inhalation
exposure to Pb (II) nitrate nanoparticles (68.6 x 106 nanoparticles/cm3) decreased relative spleen weight
in adult female CD-l(ICR) BR mice exposed for 2 weeks (BLL = 4.0 (ig/dL), but the effect was not
observed at the 6 week or 11-week timepoints with BLL up to 8.5 (ig/dL (Dumkova et aL 2020a').
Similarly, exposure to Pb oxide nanoparticles (0.956 x 106 particles/cm3, 24 hours/day for 11 weeks,
BLL =18.1 (ig/dL) had no effect on relative spleen weight in CD-I (ICR) BR mice (Smutna et aL. 2022)
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6.3.2.8 White Blood Cell Counts and Differentials (Spleen, Thymus, Lymph node,
Bone Marrow)
Changes in WBC number and differentials collected from lymphoid organs may indicate
immunotoxicity and are useful for supporting data collected from immune function assays. Although
there were no data for WBC counts and differentials in lymphoid tissues reviewed in the 2013 Pb ISA,
several recent studies describing the effects of Pb exposure on lymphoid tissues are described below.
Study-specific details, including animal species, strain, sex, and BLLs are highlighted in Table 6-11.
6.3.2.8.1 Spleen
The effects of Pb exposure on spleen cellularity were investigated in three recent studies.
Administration of Pb acetate in drinking water (300 ppm; BLL = 18.48 (ig/dL) for 42 days significantly
increased the number of Tregs, reduced the absolute number of CD3+ cells and the percentage of CD4+ T
cells, but not the percentage CD8+ T cells in the spleens of adult male Sprague Dawley rats ("Fang et ah.
2012). In contrast, administration of Pb acetate in drinking water for 28 days had no effect on percentage
of CD4+ cells, but the percentage of CD8+ cells was significantly increased in the spleens of adult male
and female Sprague Dawley rats (BLL =16.1 (.ig/dL) (Yathapu et ah. 2020). Drinking water exposure to
Pb acetate (1250 ppm; BLL 4.7-41.3 (ig/dL) for 56 days decreased the number of innate lymphoid cells
(ILC), type 1 innate lymphoid cells (ILC1), NK- like ILC1 (NK-ILC1), type 2 innate lymphoid cells
(ILC2), and type 3 innate lymphoid cells (ILC3), but Pb had no effect on cell proliferation in vivo in
spleens collected from adult male and female (samples pooled) C57BL/6 mice (Zhu et ah. 2020).
6.3.2.8.2 Thymus
Pb acetate treatment had no effect on the total number of thymocytes or the number of thymic
CD4-/CD8- and CD4+CD8+ cells, but reduced the number of thymic CD4+CD8- cells by 25% and
slightly increased the number of CD4-CD8+ cells in adult male Sprague Dawley rats exposed via
drinking water (300 ppm; BLL = 18.48 (.ig/dL) for 42 days (Fang et ah. 20.1.2). Administration of Pb in
drinking water (300 ppm) for 42 days resulted in a 1.59-fold increase in the number of Tregs in the
thymus of adult male Sprague Daw ley rats exposed (Fang etah. 20.1.2). There are no other recent studies
meeting PECOS criteria available for this endpoint.
6.3.2.8.3 Lymph Node
Two recent studies investigated the effects of Pb exposure on lymph node cellularity.
Administration of Pb acetate in drinking water (300 ppm; BLL = 18.48 (ig/dL)) to adult male Sprague
Dawley rats for 42 days had no effect on the absolute number of CD8+ T cells but reduced the absolute
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number of CD3+ cells and CD4+ T cells and increased the number of Tregs in the lymph nodes (type not
specified) ("Fang et ah. 2012). Drinking water exposure to Pb acetate (1250 ppm; BLL 4.7-41.3 (.ig/dL)
for 56 days decreased the number of ILCs, ILCls, NK-like ILCls (NK-ILCls), ILC2s, and ILC3s in
cervical lymph nodes collected from adult male and female (samples pooled) C57BL/6 mice (Zhu et al..
2020).
6.3.2.8.4 Bone Marrow
Two recent studies investigated the effects of Pb exposure on populations of immune cells in
bone marrow. Administration of Pb acetate in drinking water (0.2%; BLL = 9.3 (ig/dL) for 84 days had no
effect on the number of CD90+CD45- pluripotent hematopoietic stem cells in bone marrow collected
from adult male and female Sprague Dawley rats (Cai et aL 2018). In a second study, administration of
Pb acetate in drinking water (1250 ppm; BLL 4.7-41.3 (ig/dL) for 56 days decreased the number of ILC
progenitors (ILCPs) and reduced number of ILCPs in the bloods of adult C57BL/6 mice (data from sexes
pooled) ("Zhu et al.. 2020). These data suggest that Pb exposure impaired mobilization of ILCP cells to the
periphery. In the same study, the number of ILCs, ILCls, NK-ILCls, ILC2s, and ILC3s in bone marrow
were reduced, but Pb had no effect on cell proliferation in vivo (Zhu et al.. 2020). Pb suppressed
proliferation of ILCP in bone marrow, however.
To determine if the increase in the number of ILCPs associated with Pb exposure was caused by
impeded differentiation, common lymphoid progenitors from the bone marrow of Pb-treated (1250 ppm,
56 days; BLL 4.7-41.3 (ig/dL) or control enhanced green fluorescent protein (EGFP) mice were
transplanted into Pb-treated or control B6 mice (Zhu et al.. 2020). Common lymphoid progenitors
collected from Pb-treated EGFP mice gave rise to more ILCs compared with common lymphoid
progenitors from control donors in both Pb-treated and control recipients. Furthermore, common
lymphoid progenitors from Pb-treated donors produced more mature ILCs in control recipients than in
Pb-treated recipients. These findings indicate that common lymphoid progenitors in Pb-treated mice could
differentiate into mature ILCs, however, the Pb-treated host environment impeded differentiation into
ILCPs.
6.3.2.9 White Blood Cell Counts (Hematology and Subpopulations)
Changes in WBC number and differentials in blood may indicate potential immunotoxicity and
are useful for supporting data collected on immune function. The 2013 Pb ISA reviewed one toxicology
study that described the effects of Pb exposure on WBC numbers in blood (Sharma et al.. 2010). In that
study, the total number of WBCs, lymphocytes and monocytes were reduced in male Swiss albino mice
treated with Pb nitrate (50 mg/kg/day) (Sharma et al.. 2010). The effect of Pb exposure on WBC counts
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and subpopulations in blood reported in four recent studies are described below. Study-specific details,
including animal species, strain, sex, and BLLs are highlighted in Table 6-12.
Administration of Pb acetate in drinking water (0.2%, BLL 30.9 ± 14.7 (ig/dL) for 1 day had no
effect on the number of WBC, lymphocytes and neutrophils in whole blood collected from adult male
Wistar rats ("Andielkovic et aL 2019). However, when Pb acetate was administered in drinking water
(200 ppm; BLL = 21.6 (.ig/dL) for 45 days consecutively, the numbers ofWBCs, neutrophils,
lymphocytes, and eosinophils decreased while the numbers of monocytes and basophils were unchanged
in blood collected from adult male C57BJ mice (Corsetti et aL 20.1.7). Changes in WBC number and
subpopulations were reported in a second study wherein the total number ofWBCs and the number of
CD4+ and CD8+ T cells were reduced in blood collected from male and female Sprague Dawley rats
(data from sexes pooled) following exposure to Pb acetate in drinking water (0.2%; BLL = 9.3 (ig/dL) for
84 days ("Cai et al.. 20.1.8). Additionally, exposure to Pb acetate (drinking water, 1250 ppm. BLL 4.7-
41.3 (ig/dL) for 56 days decreased the number of ILCs, type 1 innate lymphoid cells (ILC1), NK-like
ILC1 (NK-ILC1), type 2 innate lymphoid cells (ILC2), and type 3 innate lymphoid cells (ILC3). Pb
exposure additionally suppressed proliferation of ILCP in blood collected from adult male and female
(samples pooled) C57BL/6 mice ("Zhu et al.. 2020).
6.3.3 Integrated Summary of Immunosuppression
Toxicological evidence for Pb-induced immunosuppression is derived from several lines of
evidence including functional assays (i.e., host resistance, antibody responses, DTH response, and ex vivo
WBC function) that are bolstered by various forms of supporting immune system data including
immunoglobulin levels, immune organ weight, WBC counts and differentials (immune organs), and WBC
counts (hematology). Toxicological studies evaluated in the 2013 Pb ISA (U.S. EPA, 20.1.3) provide clear
evidence that host resistance to bacterial infection is compromised following Pb exposure. Evidence
available in 2013 also demonstrated that levels of antigen-specific IgM were unaffected in Pb-exposed
mice infected with Salmonella. However, levels of IgG2a were decreased and IgGl antibodies were
increased in these mice providing evidence for a shift toward Th2-type immune responses resulting in
decreased resistance to Salmonella. The potential for Pb exposure to result in immunosuppression was
further evaluated using the DTH assay. Based on a long history of use, the DTH assay is considered one of
the most predictive immunotoxicity tests available (Dietert et al., 20.1.0). Suppression of the DTH response
is a hallmark of Pb exposure and has been consistently reported in rats and chickens with PECO-relevant
BLLs as well as in other studies either not reporting BLLs or reporting BLLs outside the scope of this
ISA. The effects of Pb administration on the TDAR was also evaluated in the 2013 Pb ISA. Results from
these investigations were inconsistent with one study reporting a decrease in the antibody response (BLL
not reported) and another showing no effect in mice with high BLLs (i.e., 59-132 (.ig/dL). The effects of
Pb exposure on the functions of various WBCs under ex vivo conditions indicated that Pb exposure results
in (1) suppression of Thl-mediated immunity (i.e., suppressed Thl cytokine production [e.g., IFN-
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y] and DTH response); (2) altered macrophage function (e.g., increased ROS production, decreased NO
production); and (3) reduced monocyte/macrophage phagocytosis.
The 2013 Pb ISA also described toxicological evidence for effects of Pb exposure on various
supporting immune system endpoints (e.g., total serum immunoglobulins, immune organ weights, WBC
counts) that support data derived from immune function assays. Investigations of these endpoints are
limited in number, however, and due to differences in experimental design, challenging to interpret. For
example, inconsistent effects of Pb exposure on total serum IgE and IgG subtypes were described in the
2013 Pb ISA. Data reporting effects of Pb exposure on immune organ weight were limited to one study
reporting increased relative spleen weight and another study reporting decreased lymph node weight
following Pb exposure. Additional studies investigated the number and relative abundance of different
types of WBC in the spleen, thymus, lymph nodes and bone marrow following exposure to Pb, although
study design limitations and differences in the types of WBC assessed limit our ability to interpret these
data. In the only study reporting on WBC counts and subpopulation data collected in blood reviewed in
the 2013 Pb ISA, Pb exposure reduced the total number of WBC, lymphocytes, and monocytes.
The epidemiologic studies relevant to immunosuppression that were evaluated in the 2013 Pb
ISA (U.S. EPA, 20.1.3) were more limited in number than the available toxicological evidence base.
Irrespective, these studies indicated some evidence of an association between BLLs and viral and
bacterial infections in children. None of the studies considered potential confounders, however, and most
analyzed populations with higher BLLs (means >10 (ig/dL). As described in the 2013 Pb ISA, some
epidemiologic studies also examined the effects of Pb exposure on WBC populations and cytokine levels.
Evaluation of these provided generally consistent evidence of inverse associations between BLLs and T
cell abundance in children, though most associations were seen with high concurrent BLLs (>10 (.ig/dL).
These results were coherent with the toxicological evidence base. Studies examining macrophages,
neutrophils, and NK cells and lymphocyte activation (i.e., HLA-DR expression) were largely
uninformative because of limitations associated with consideration of potential confounders and a lack of
information on concentration-response relationship.
Since the 2013 Pb ISA, there have been several epidemiologic studies published investigating
aspects of immunosuppression. Recent studies investigating associations between Pb exposure and
decreased host resistance examine populations with wider age-ranges and much lower mean and median
BLLs than studies evaluated in the 2013 Pb ISA. Recent studies also adjust for a wide range of potential
confounders, including extensive consideration of SES factors. Cross-sectional and case-control studies
provide consistent evidence of associations between Pb exposure and viral and bacterial infections or
susceptibility to antibiotic resistance. Antibody response, an endpoint that was not examined in studies
evaluated in the 2013 Pb ISA, was investigated in several recent studies. Specifically, a birth cohort study
and a few cross-sectional studies demonstrate generally consistent evidence of an association between
higher BLLs and lower counts of virus-neutralizing antibodies. A group of epidemiologic studies
examining children in China living either near an e-waste facility or in a nearby community with
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otherwise similar sociodemographic characteristics and pollutant exposures provides evidence that BLLs
are associated with differences in (1) the percentage of CD4+ naive and CD4+ central memory T cells, (2)
proinflammatory cytokine levels (IFN-y, IL-1(3, IL-8, IL-10, IL-12p70, and TNF-a), (3) levels of the
pleiotropic cytokine IL-6, (4) levels of the anti-inflammatory cytokine IL-10, and (5) the number of
neutrophils and monocytes. A few of the studies also reported null associations between BLLs and CD3+,
CD4+ and CD8+ cell counts, monocytes, and lymphocytes. The only recent study of an adult population
reported similar increases in cytokine levels associated with BLLs.
Available recent studies of immune function generally support evidence reported in the previous
Pb ISA. There are no recent toxicology studies investigating the effects of Pb exposure on host resistance
available for this review, but the strength of evidence reviewed in 2013 Pb IS As demonstrating that host
resistance to bacterial infection is compromised following Pb exposure has not diminished. A recent,
study shows, exposure to Pb had no effect on levels of anti-TT-specific IgM and IgG antibodies in rats.
However, levels of anti-TT-specific IgM (but not IgG) were decreased in iron-deficient rats. Consistent
with findings reported in the 2013 Pb ISA, recent studies also show that Pb exposures suppress the DTH
response, a widely-accepted measure of immunosuppression. Assessment of the effects of Pb exposure on
ex vivo WBC function is limited to assessments of Con A-stimulated lymphocyte proliferation and direct
measurement of cytokines in blood. Pb treatment had no effect on Con A-stimulated proliferation of
splenocytes collected from rats, however, treatment increased Con A-stimulated splenocyte proliferation
in iron-deficient rats. Pb exposure had no effect on levels of erythropoietin, GM-CSF, IL-6, and TNF-a in
a single study performed in rats. Recent studies reporting on the effects of Pb exposure on immune organ
pathology were inconsistent, with one study reporting effects on spleen architecture and another showing
no effect. Pb exposure reduced total serum IgA immunoglobulins in rats fed a control diet and in iron-
deficient rats but had no effect on total serum IgM and IgG in rats fed either diet. Recent investigations
also include assessments of the effects of Pb exposure on immune organ weight. Relative thymus weight,
which was not evaluated in the 2013 Pb ISA, decreased following exposure to Pb. As with the 2013 Pb
ISA, the effects of Pb exposure on relative spleen weight were inconsistent, varying with dose, exposure
duration, and route of administration (oral versus inhalation). Similarly, because of differences in
experimental design and the specific types of WBCs assessed in each study, it is difficult to interpret data
collected on the number and relative abundance of the different types of WBCs in the spleen, thymus,
lymph nodes and bone marrow following exposure to Pb. WBC counts and subpopulation data collected
from hematology investigations are similarly challenging to interpret.
6.4 Sensitization and Allergic Responses
Hypersensitivity responses are the result of an over-reaction of the immune system.
Hypersensitivity reactions are organized into four different classes, types I. II. III. and IV (Murphy and
Weaver. 20.1.6). Irrespective of the type of response, all hypersensitivity responses develop in the same
two phases: sensitization and elicitation (or challenge). During the sensitization phase, the immune
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system is trained to respond to an otherwise innocuous antigen. This phase typically occurs without
symptoms. During the elicitation phase, the previously sensitized individual is re-exposed to the antigen
precipitating the symptoms of the allergic disease. Important for risk assessors, the concentration of the
sensitizing chemical required to elicit an allergic response is, in some cases, orders of magnitude lower
than the concentration required for sensitization. Consequently, preventing allergic sensitization from
developing in the first place is of paramount importance because dangerous, potentially life-threatening
allergic reactions can occur in response to exposure to a prohibitively-low concentration of the sensitizer.
6.4.1 Epidemiologic Studies of Sensitization and Allergic Responses
Epidemiologic studies of sensitization and allergic response generally cover studies of atopic
diseases, including asthma, rhinitis, and eczema, as well as studies examining cells and antibodies that
mediate these diseases, such as IgE and eosinophils. A limited number of studies evaluated in the 2013
Pb ISA ( 20.1.3) provide evidence of associations between exposure to Pb and asthma and
allergic sensitization. The strongest evidence comes from two prospective analyses, one investigating
incident asthma requiring medical care (Joseph et ah, 2005) and another examining allergic
hypersensitization via skin prick tests (SPTs) (Jedrychowski et ah, 20.1.1). Associations in both studies
were reported after adjustment for multiple confounders, including sex; birth weight; parity; maternal
age, education, and atopy; income; and prenatal and postnatal smoking exposure. Joseph et ah (2005)
observed associations between asthma incidence and BLLs >5 (ig/dL in white children (relative risk
[RR]: 2.7 [95% CI: 0.9, 8.1] compared with white children with BLL <5 (.ig/dL). In analyses restricted to
Black children, those with BLLs >10 (ig/dL had an elevated risk of incident asthma requiring medical
care (RR: 1.3 [95% CI: 0.6, 2.6] compared with children with BLLs <5 (.ig/dL). The effect estimates for
both groups were imprecise due to small numbers of children with asthma in the higher BLL categories
(five white children with BLLs >5 (ig/dL and nine Black children with BLLs >10 (ig/dL). Jedrychowski et
ah (20.1.1) also reported positive but imprecise associations (i.e., wide 95% CIs) between prenatal cord
BLLs and risk of positive SPT (rash/inflammatory reaction) to dust mite, dog, or cat allergen (RR: 2.3
[95% CI: 1.1, 4.6] for each 1 (ig/dL higher level of prenatal cord BLL). An additional prospective cohort
analysis reported an imprecise association between cord BLLs and prevalent asthma in children
(Rabinowitz et ah, .1.990), but did not adjust for potential confounders and had low participation rates
with no information on nonparticipants. These findings were supported by a cross-sectional study of cord
blood and blood Pb-associated prevalent asthma (Pugh Smith and Nriagu, 20.1.1). In addition to studies
examining atopic disease incidence or prevalence, the 2013 Pb ISA (U.S. EPA, 20.1.3) also includes
supporting evidence from population-based cross-sectional studies in children that reported associations
between BLL and elevated serum IgE. Notably, many of these studies had limited adjustment for
potential confounders and included populations with mean BLLs >5 (ig/dL.
There have been several recent epidemiologic studies of sensitization and allergic response,
including prospective birth cohorts and cross-sectional studies with mean or median BLLs <2 (ig/dL. In
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general, these recent studies provide little evidence of an association between exposure to Pb and atopic
disease, and inconsistent evidence for immunological biomarkers involved in hypersensitivity and allergic
response. Measures of central tendency for BLL used in each study, along with other study-specific
details, including study population characteristics and select effect estimates, are highlighted in
Table 6-13. An overview of the recent evidence is provided below.
Whereas epidemiologic evidence from the 2013 Pb ISA supported the presence of an association
between BLL and incident and prevalent asthma in children, evidence from a few recent studies at lower
BLL is not indicative of an association. Specifically, in a small prospective birth cohort in France, Pesce
et al. (202.1.) reported that neither BLL measured during pregnancy nor cord BLL at birth were associated
with incident parental-reported asthma attacks through 5 years of age. Notably, there was a low rate of
asthma in the study population, limiting the statistical power to detect an association. However, because
asthma can be difficult to diagnose in children under 5, asthma attacks may be the most reliable measure.
The odds of asthma development associated with maternal BLLs were slightly elevated in the highest
quartile of exposure compared to the lowest, but the reported OR(1.25 [95% CI: 0.71, 2.2]) was
imprecise and the authors did not adjust the estimates for multiple comparisons (i.e., two exposure metrics
and four outcomes). In a cross-sectional NHANES analysis including slightly older children (2-12 years
old), Wells et al. (20.1.4) also observed a null association between BLL and prevalent asthma.
Other recent epidemiologic studies of atopic disease are also generally consistent in reporting a
lack of an association with low levels of exposure to Pb. A few birth cohorts (Kim et al.. 20.1.9; Kim et al..
20.1.3) and a cross-sectional NHANES analysis including respondents of all ages (Wei et al.. 20.1.9) did not
observe associations between cord blood or BLL and eczema incidence or prevalence. While Pesce et al.
(2 reported a null association between maternal BLL and eczema in the aforementioned French birth
cohort, the authors did note substantially higher odds of eczema incidence for children in the higher
quartiles of cord blood Pb exposure compared with the lowest quartile. However, given the range of
outcomes examined (which included null associations for rhinitis and food allergy, in addition to asthma)
and the use of two exposure metrics (maternal blood and cord blood), the eczema results could be an
artifact of multiple testing. Consistent with Pesce et al. (2021). Mener et al. (20.1.5) also reported a null
association between BLL and food allergies in children. However, the authors noted 10% higher odds of
food allergy sensitization in adults per 1 (ig/dL higher BLL (95% CI: 1%, 20%). In a restricted cubic
spline model, the observed relationship was approximately linear across the range of lower BLLs
(<3 (ig/dL), with no evidence of a threshold.
Results from a limited number of recent epidemiologic studies of allergen-specific and non-
specific immunological biomarkers of hypersensitivity in adults are inconsistent. A cross-sectional Korea
National Health and Nutrition Examination Survey (KNHANES) analysis reported higher total IgE
concentrations associated with higher BLLs in adults (Kim et al.. 20.1.6). Notably, the observed
association was stronger in magnitude in respondents with house dust mite sensitization (10.4% [95% CI:
3.3%, 17.8%] per 1 (ig/dL higher BLL) compared with those without (3.5% [95% CI: -1.8%, 9.4%]). No
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other recent studies examined total IgE levels in adults, although Tsuii et al. (20.1.9) reported that BLLs
were not associated, or slightly negatively associated, with allergen-specific serum IgE concentrations in
pregnant women, including egg white, hose dust mite, Japanese cedar pollen, animal dander, and moth
allergens. The interpretation of the results is complicated, however, by timing of the exposure and
outcome, where IgE concentrations were measured earlier in pregnancy (first trimester) than BLL (second
or third trimester).
Recent epidemiologic studies of non-specific immunological biomarkers of hypersensitivity in
neonates and children also provide inconsistent evidence of an association with exposure to Pb. In a small
birth cohort in south Korea. Kim et al. (20.1.9) observed a cross-sectional association between higher cord
BLL and higher cord blood IL-13. In another cross-sectional analysis. Wells et al. (2014) reported that
each 1 (ig/dL higher level of BLL was associated with 10.3% (95% CI: 3.5%, 17.5%) higher serum total
IgE and 4.6% (95% CI: 2.4%, 6.8%) higher percent eosinophils. In contrast, results from a larger birth
cohort in Canada did not indicate higher odds of elevated cord blood IgE concentrations in relation to
higher average BLL across the first and third trimesters of pregnancy (Ashley-Martin et al.. 2015).
Further, the authors reported an inverse association between pregnancy BLL and odds of simultaneously
elevated cord blood IL-33 and thymic stromal lymphopoietin (TSLP).
6.4.2 Toxicological Studies of Sensitization and Allergic Responses
The 2013 Pb ISA reviewed evidence for the ability of Pb to induce immediate-type
hypersensitivity leading to the development of allergic asthma ( \. 2013). Available studies
reported that exposure to Pb increased lymph node cell proliferation, increased production of Th2
cytokines such as IL-4, increased total serum IgE antibody levels in serum, and misregulated
inflammation. Recent toxicological evidence is limited in number and reports on the effects of Pb
exposure on production of cytokines relevant to immediate-type hypersensitivity, as discussed below.
6.4.2.1 Immediate-Type Hypersensitivity
Immediate-type hypersensitivity (i.e., type I) responses are the result of the production of IgE
antibodies, which trigger an array of responses, including anaphylaxis, allergic rhinitis, allergic
conjunctivitis, food allergy, atopic eczema, and allergic asthma. As with other forms of hypersensitivity,
immediate-type hypersensitivity develops in two stages. During the sensitization phase, antigen is
presented to naive T cells by antigen-presenting cells which promotes differentiation to the Th2
phenotype and the formation of memory T cells. Memory-specific T cells interact with antigen-specific B
cells leading the production of antigen-specific IgE antibodies that bind to Fc receptors on the surface of
mast cells. Upon secondary exposure to the allergen, the antigen binds to mast cell-bound IgE, triggering
mast cell degranulation resulting in eosinophil recruitment, mucus production, reactive airways and,
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potentially, anaphylaxis (Janewav et al.. 2005'). There are no validated animal models for determining
whether a xenobiotic can cause immediate-type hypersensitivity. For that reason, the potential for a
chemical to cause immediate-type hypersensitivity is assessed using a weight of the evidence approach
where data from an array of experimental endpoints (total serum IgE, antigen-specific IgE, eosinophilia of
the lung, measures of lung function, etc.) are carefully integrated (IPCS. 2012).
As reviewed in the 2013 Pb ISA, toxicological evidence, and to a lesser extent epidemiologic
evidence, have supported the effects of Pb exposure on stimulating Th2 activity. Studies have reported
increased lymph node cell proliferation (Teiion et al.. 20.1.0; Carey et al.. 2006). increased production of
Th2 cytokines such as IL-4 (Fernandez-Cabezudo et al.. 2007; Iavicoli et al.. 2006; Chen et al.. 2004; Heo
et al.. .1.998; Miller etah. .1.998; Heo et al.. .1.997; Heo et al.. 1996). increased total serum IgE antibody
levels ("Snyder et al.. 2000; Miller etah. .1.998; Heo et al.. .1.997; Heo et al.. .1.996). and misregulated
inflammation (Lodi et al.. 20.1.1; Chettv et al.. 2005; Flohe et al.. 2002; Shabani and Rabbani. 2000; Miller
et al.. .1.998; Chen et al.. .1.997; Knowles and Donaldson. .1.997; Bavkov et al.. .1.996; Lee and Battles. .1.994;
ZelikoffetaL .1.993; Knowles and Donaldson. .1.990; Hilbertz et al.. .1.986; Castranova et al.. .1.980). These
endpoints comprise a well-recognized mode of action for the development and exacerbation of atopic and
inflammatory conditions such as asthma and allergy.
Only two recent toxicology studies investigated the effects of Pb exposure on production of
cytokines relevant to immediate-type hypersensitivity. In one of these studies, administration of Pb
acetate drinking water (300 ppm; BLL = 18.48 (ig/dL) for 42 days decreased IFN-y levels, but had no
effect on IL-10 levels (data not shown) in adult male Sprague Daw ley rats (Fang et al.. 2012). In addition,
administration of Pb acetate in drinking water (0.2%; BLL = 9.3 (ig/dL) for 84 days had no effect on
erythropoietin, GM-CSF, IL-6, and TNF-a levels in blood collected from Sprague Dawley rats (data from
sexes pooled) (Cai et al.. 2018). Study-specific details, including animal species, strain, sex and BLLs. are
highlighted in Table 6-14.
6.4.3 Integrated Summary of Sensitization and Allergic Responses
As review ed in the 2013 Pb ISA (U.S. EPA. 2013), toxicological evidence, and to a lesser extent
epidemiologic evidence, have supported the effects of Pb exposure on increased lymph node cell
proliferation, increased production of Th2 cytokines such as IL-4, increased total serum IgE antibody
levels in serum, and misregulated inflammation. Additionally, a limited number of longitudinal
epidemiologic studies evaluated in the 2013 Pb IS A (U.S. EPA. 2013) provide evidence of associations
between exposure to Pb and asthma (Joseph et al., 2005) and allergic sensitization (Jedrychowski et al.,
20.1.1). The associations in these studies are imprecise (i.e., wide 95% CIs), but are supported by cross-
sectional studies of cord blood and blood Pb-associated prevalent asthma and population-based cross-
sectional studies in children that reported associations between BLL and elevated serum IgE (U.S. EPA.
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20.1.3). Many of these cross-sectional studies had limited adjustment for potential confounders and
included populations with mean BLLs >5 (ig/dL.
Though limited in number, recent PECOS-relevant animal toxicological studies continue to
support the findings from the last review. Specifically, these studies consistently report effects of Pb on
sensitization and allergic responses including two studies of the effects of Pb exposure on production of
cytokines relevant to immediate-type hypersensitivity. In contrast, recent epidemiologic evidence is not
consistent with studies evaluated in the 2013 Pb ISA. Specifically, recent studies provide little evidence
of an association between exposure to Pb and atopic disease, and inconsistent evidence for
immunological biomarkers involved in hypersensitivity and allergic response. Similar to cohort studies
evaluated in the 2013 Pb ISA, recent longitudinal analyses are limited in number and have limited
statistical power because of small case numbers. Limited statistical power results in the reduced
likelihood of detecting a true effect and a reduced likelihood that an observed result reflects a true effect.
Whereas there was coherence between the animal toxicological and epidemiologic evidence evaluated in
the 2013 Pb ISA, the recent evidence is less coherent given the inconsistencies and null findings across
epidemiologic studies.
6.5 Autoimmunity and Autoimmune Disease
Autoimmunity is characterized by the reaction of autoreactive T lymphocytes or autoantibodies
against self-molecules (i.e., autoantigens). Depending on the etiology, autoimmunity may lead to the
development of autoimmune diseases such as rheumatoid arthritis and lupus. While the precipitating
event for the development of autoimmunity is often unknown, intrinsic factors (e.g., gene polymorphisms,
sex-related hormones, and age) and extrinsic factors (e.g., lifestyle, exposure to certain drugs, chemicals,
and infectious agents) are known to play a role in the induction, development, or exacerbation of
autoimmunity (IPCS. 20.1.2). Although animal models have been used to study a variety of autoimmune
diseases, there are currently no validated models to assess or identify chemicals that induce or exacerbate
autoimmune diseases ("IPCS. 20.1.2). Consequently, the potential to induce or exacerbate autoimmunity is
best investigated using atiered approach composed of multiple methods. The 2013 Pb ISA concluded the
available toxicological and epidemiologic studies were inadequate to infer that a causal relationship exists
between Pb exposure and the development of autoimmunity and autoimmune disease.
6.5.1 Epidemiologic Studies of Autoimmunity and Autoimmune Disease
A single epidemiologic study evaluated in the 2013 Pb ISA (U.S. EPA. 20.1.3) examined the
association between exposure to Pb and autoimmunity (El-Fawal etah. .1.999). While the authors reported
higher levels of autoantibodies in Pb-exposed battery workers, the analysis did not include adjustment for
important confounders (e.g., other occupational exposures) and included BLLs of 10-40 (ig/dL, much
higher than those found in the general population. Recent epidemiologic studies of autoimmunity are
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limited in number and examine disparate outcomes. Mean BLL used in each study, along with other
study-specific details, including study population characteristics and select effect estimates, are
highlighted in Table 6-15. An overview of the recent evidence is provided below.
Two recent population-based cross-sectional studies provide inconsistent evidence of associations
between exposure to Pb and autoimmune disorders ("too et ah. 20.1.9; Kamvcheva et al.. 20.1.7'). In an
NHANES analysis of seropositivity for Celiac Disease (i.e., tissue transglutaminase [tTg]-IgA),
Kamvcheva et al. (20.1.7) reported lower adjusted mean BLLs in children with Celiac Disease compared
with those without (-0.14 (ig/dL [95% CI: -0.27, -0.02 |ig/dL|). Associations were comparable in
magnitude, but less precise in adults (i.e., wider 95% CIs). Cross-sectional studies cannot establish
temporality and the nature of malabsorption in Celiac Disease makes it biologically plausible that the
disorder could result in reduced absorption of Pb rather than there being a protective effect of Pb
exposure. Another population-based study did not observe an association between BLL and rheumatoid
arthritis (too et al.. 20.1.9'). A notable limitation of this study is that it included children, while rheumatoid
arthritis primarily affects adults.
6.5.2 Toxicological Studies of Autoimmunity and Autoimmune Disease
As reported in the 2013 Pb ISA, evidence for the ability of Pb to induce autoimmunity is limited
( \. 20.1.3'). Only one study performed in rats showed the generation of autoantibodies following
Pb administration by a relevant route of exposure (i.e., dietary) (El-Fawal et al.. .1.999). Several other
studies utilized Pb exposure routes or doses that produced BLLs that are not relevant to humans (Hudson
et al.. 2003; Bunn et al.. 2000; Waterman et al.. .1.994). There is only one recent toxicology study that
investigates an endpoint directly related to the development of autoimmunity. In that study. Fang etal.
(20.1.2) reported that administration of Pb acetate in drinking water for 42 days (BLL = 18.48 (.ig/dL) had
no effect on the suppressive properties of Tregs isolated from adult male Sprague Dawley rats. Study-
specific details, including animal species, strain, sex, and BLLs are highlighted in Table 6-16.
6.5.3 Integrated Summary of Autoimmunity and Autoimmune Disease
An epidemiologic study evaluated in the 2013 Pb ISA (i 5- ! i*A. 20.1.3) observed an association
between higher BLLs and elevated autoantibodies, but the strength of conclusions that can be draw n from
this study is limited because it did not control for important confounders. Toxicological evidence
demonstrating that Pb exposure leads to autoimmunity is similarly limited. As discussed in the 2013 Pb
ISA (U.S. EPA. 20.1.3). one PECOS-relevant study and several other studies utilizing non-PECOS routes
of exposure and doses that produced BLLs that are not relevant to humans showed the generation of
autoantibodies following Pb administration. Recent epidemiologic studies of autoimmunity are limited in
number, examine disparate outcomes and provide inconsistent evidence of associations between exposure
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to Pb and autoimmune disorders. A recent toxicological study reported that Pb exposure had no effect on
the suppressive properties of Tregs, which are critical mediators of immune tolerance.
6.6 Biological Plausibility
This section describes biological pathways that potentially underlie effects on the function of the
immune system resulting from exposure to Pb. Figure 6-1 depicts the proposed pathways as a continuum
of upstream events, connected by arrows, that may lead to downstream events observed in epidemiologic
studies. Evidence supporting these proposed pathways was derived from Sections 6.3, 6.4, and 6.5 of
this ISA. evidence reviewed in the 2013 Pb ISA (U.S. EPA, 20.1.3), and recent evidence collected from
studies that may not meet the current PECOS criteria, but contain mechanistic information supporting
these pathways. This discussion of how exposure to Pb may lead to immune system effects contributes
to an understanding of the biological plausibility of epidemiologic results evaluated later in the ensuing
sections. Note that the structure of the Biological Plausibility section and the role of biological
plausibility in contributing to the weight-of-evidence analysis used in the 2013 Pb ISA are discussed
below.
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Altered dendric
cell function
Increase Th2
cytokines
{e.g., IL-4)
Antibody
production/secretion
Class switching
Increased IgE secretion
Allergic asthma
Pb
Exposure
h
Increase proinflammatory
cytokines
(e.g.,TNF-a)
Increase ROS production
Increase cell death
Decrease phagocyte function
Decrease chemotaxis
function
Decrease nitric oxide
production
Immunosuppression/
increased incidence of
infection
DTH = delayed-type hypersensitivity; IgE = immunoglobulin E; IFN-y = interferon-gamma; IL-4 = interleukin 4; ROS = reactive
oxygen species; Th2 = T helper; TNF-a = tumor necrosis factor alpha.
Note: The boxes above represent the effects for which there is experimental or epidemiologic evidence related to Pb exposure, and
the arrows indicate a proposed relationship between those effects. Solid arrows denote evidence of essentiality as provided, for
example, by an inhibitor of the pathway used in an experimental study involving Pb exposure. Dotted arrows denote a possible
relationship between effects. Shading around multiple boxes is used to denote a grouping of these effects. Arrows may connect
individual boxes, groupings of boxes, and individual boxes within groupings of boxes. Progression of effects is generally depicted
from left to right and color coded (white, exposure; green, initial effect; blue, intermediate effect; orange, effect at the population
level or a key clinical effect). Here, population-level effects generally reflect results of epidemiologic studies. When there are gaps in
the evidence, there are complementary gaps in the figure and the accompanying text below. The structure of the biological
plausibility sections and the role of biological plausibility in contributing to the weight-of-evidence analysis used in the 2024 Pb ISA
are discussed in Section 6.7.
Figure 6-1 Potential biological plausibility pathways for immunological
effects associated with exposure to Pb.
Immunotoxicity may be expressed as immunosuppression, unintended stimulation of immune
responses, hypersensitivity, or autoimmunity (IPCS. 2012). The World Health Organization's Guidance
for Immunotoxicity Risk Assessment for Chemicals (IPCS. 2012) describes best approaches for weighing
immunotoxicological data. Within this framework, data from endpoints observed in the presence of
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immune challenge (e.g., including effects on antibody responses, host resistance, and ex vivo WBC
function) are considered most informative whereas other measures collected in the absence of immune
stimulation (e.g., immune organ pathology, non-specific immunoglobulin levels, WBC counts,
lymphocyte subpopulations, T cell subpopulations, immune organ weights) are considered supporting
evidence. Careful review of the evidence base suggests that exposure to Pb has the potential to modulate
the immune system leading to immunosuppression and sensitization and allergic responses. Below,
evidence from peer-reviewed toxicology studies providing biological plausibility for Pb-associated
immunotoxicity is reviewed.
6.6.1 Immunosuppression
Immunosuppression can lead to the increased incidence and severity of infectious and neoplastic
diseases. Importantly, there are internationally validated animal models and human correlates (e.g., the
rodent DTH assay and the human tuberculin test) for assessing the potential for a chemical to induce
immunosuppression. Still, the potential for a chemical to suppress the function of the immune system is
best assessed using a weight of the evidence approach where data from an array of experimental
endpoints are carefully integrated (IPCS. 2012).
The initiating event that ultimately leads to Pb-induced immunosuppression is unknown.
However, Pb exposure has been shown to affect several indicators of immunosuppression including
decreased Thl cytokine production, production of other inflammatory mediators, decreased macrophage
function (chemotaxis and phagocytosis), and ultimately suppressed the DTH response (Figure 6-1).
Exposure to Pb has been convincingly shown to result in the skewing of T cell populations,
simultaneously promoting the formation of Th2 cells while suppressing the formation of Thl cells and
their cytokines including IFN-y that play key roles in cell-mediated immunity (Heo et al.. .1.996; Fochtman
etal.. 1969). Available evidence suggests that this phenomenon may involve Pb-induced effects on
dendritic cells, which promote skewing towards the Th2 phenotype ("Gao et aL. 2007'). Mitogen-
stimulated production of IFN-y was significantly lower in splenocytes collected from Pb-exposed mice
(Dvoroznakova and Jalcova. 20.1.3). IFN-y levels in serum were reduced in Pb-exposed mice (Aiouaoi et
al.. 2020). I FN -y is the primary cytokine that stimulates recruitment of macrophages associated to sites of
inflammation (Lee et al.. 2001; Chen et aL. .1.999). Relevant decrements in macrophage function
associated with Pb exposure have been reported, including decreased chemotaxis ("Lodi et al.. 20.1.1;
Bishavi and Sengupta. 2006) and phagocytosis (Lodi et aL. 20.1.1; Bussolaro et al.. 2008; Bishavi and
Sengupta. 2006; Hilbertz et al.. .1.986; Zhou et al.. .1.985; Castranova et aL. .1.980). Macrophages play a
vital role in cell-mediated immunity, which is often assessed using the DTH response when assaying
potential immunosuppressants. Pb exposure has been consistently shown to suppress the DTH response in
rodents with BLLs relevant to human exposures. Observations of a concomitant decrease in IFN-y
strengthen the link between Pb-induced inhibition of Thl functional activities and suppression of the
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DTH response ("Lee et ah. 2001; Chen et al.. .1.999'). Furthermore, the effects of Pb exposure on
macrophage PGE2 (Chettv et al.. 2005). decreased ROS production ("Chen et al.. .1.997; Hi 1 hertz et al..
.1.986; Castranova et al.. 1980). decreased NO production (Fairer et al.. 2008; Mishra et al.. 2006; Bunn et
al.. 2001b; Lee et al.. 2001; Krocova et al.. 2000; Chen et al.. .1.997; Tian and Lawrence. .1.996; Tian and
Lawrence. 1.995). and increased cell death (Metrvka et al.. 2021; Guam et al.. 2020; Choi et al.. 2018; Kerr
et al.. 2013) may contribute to decreased resistance to bacterial or viral infection (Hilbertz et al.. .1.986;
Castranova et al.. .1.980). Pb exposure has also been shown to increase levels of TNF-a, a
proinflammatory cytokine, secreted by LPS- stimulated mouse J774A. 1 macrophages (Luna et al.. 2012)
and human THP-1 monocytes through a mechanism involving ERK1/2 (Khan et al.. 20.1.1). As reviewed
in the 2006 Pb AQCD (U.S. EPA. 2006). Pb exposure also has the potential to reduce neutrophil
chemotaxis, phagocytosis, and respiratory oxidative burst, but the effect was not judged to be as strong as
what has been observed in relation to macrophages. Finally, decreased Thl signaling leading to
differences in IgG isotypes produced in response to S. enterica infection was implicated in impaired host
defense in mice (Fernandez-Cabezudo et al.. 2007).
While there is compelling evidence that Pb exposure can decrease host resistance to infection, the
effect may not be attributable to direct effects of Pb exposure on the immune system. Instead, decreased
host resistance may be the result of Pb acting on the microbiome. The microbiome is the body's gateway,
disruption of microbiome can have profound effects on xenobiotic processing, and resistance to pathogens
(Zhai et al.. 2020; Dietertand Silbergeld. 2015; Nriagu and Skaar. 2015). The human microbiome
comprises most of the cells and genes in the human body, and these cells are the first to be exposed to
environmental chemicals. The microbiome plays a key role in excretion levels, transport barriers
(e.g.. skin. lung, gut barriers), metabolism of xenobiotics (Zhai et al.. 2020; Dietert. 2018; Nriagu and
Skaar. 2015). In addition, changes in the composition of the microbiome follow ing exposure to
xenobiotics can affect the process of colonization resistance to pathogens which may lead to loss of
mucosal barrier function, elevated risk of infection, and the development of noncommunicable diseases
such as asthma (Huang et al.. 2020; Zhai et al.. 2020; Dietert. 2018; Nriagu and Skaar. 2015).
Importantly. Pb is know n to possess antimicrobial properties! Mi\ano et al.. 2007). As reviewed by Liu et
al. (2021). exposure to Pb has been shown to alter the diversity and relative composition of the gut
microbiota in several toxicology studies performed in laboratory animals. Our ability to interpret these
findings is limited, however, by the fact that the investigators conducting these studies either did not
measure BLL at all or, in the two studies that did, the BLL was not relevant to human exposure. In
addition to toxicological studies, a limited number of epidemiologic studies reported associations between
biomarkers of Pb exposure and altered gut microbiota diversity, including a birth cohort study (Sitarik et
al.. 2020) and a few cross-sectional analyses (Zeng et al.. 2022; Eggers et al.. 20.1.9). Further, the
possibility that the effects of Pb on the immune system are at least partly mediated by the microbiome is
supported by the capacity of certain probiotics to protect against Pb-induced toxicity (i.e., decreases BLL
and relieves Pb-induced intestinal barrier impairment) in mice (Zhai et al.. 2020). In rats, chelation
treatment reduced IL-4 production and IFN-y suppression induced by Pb (Chen et al.. .1.999). Similarly.
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Vitamin D supplementation was shown to reduce Pb-induced IL-4 in rats, but the concentration of IL-4
remained significantly elevated relative to control ("BaSalamah et aL 20.1.8').
6.6.2 Sensitization and Allergic Responses
Hypersensitivity responses (i.e., allergies) are the result of an over-reaction of the immune
system. Immediate-type hypersensitivity responses are the result of the production of IgE antibodies,
which trigger an array of responses including anaphylaxis, allergic rhinitis, allergic conjunctivitis, food
allergy, atopic eczema, and allergic asthma. Like with other forms of hypersensitivity, immediate-type
hypersensitivity, develops in two stages. During the sensitization phase, antigen is presented to naive T
cells by antigen-presenting cells, which promotes differentiation to the Th2 phenotype and the formation
of memory T cells. Memory-specific T cells interact with antigen-specific B cells leading the production
of antigen-specific IgE antibodies that bind to Fc receptors on the surface of mast cells. Upon secondary
exposure to the allergen, the antigen binds to mast cell-bound IgE, triggering mast cell degranulation
resulting in eosinophil recruitment, mucus production, reactive airways and, potentially, anaphylaxis
(Janewav et aL 2005). Importantly, there are no validated animal models for determining whether a
xenobiotic can cause allergic asthma. For that reason, the potential for a chemical to cause allergic asthma
is assessed using a weight of the evidence approach where data from an array of experimental endpoints
are carefully integrated (IPCS. 2012).
The initiating event that ultimately leads to allergic sensitization is called haptenation, the process
where sensitizing chemical binds to endogenous proteins leading to detection by the immune system and
ultimately allergic sensitization (Janewav et aL 2005). To date, there are no publications demonstrating
that Pb acts as a hapten. Pb exposure, however, is associated with other hallmarks of allergic
hypersensitivity and asthma including Th2 cytokine production, B cell activation, and production of IgE
antibodies that are central to these responses.
Exposure to Pb resulting in BLLs relevant to humans has been convincingly shown to result in
the skewing of T cell populations, simultaneously suppressing the formation of Thl cells while promoting
the formation of Th2 cells and cytokines that promote the development of allergic airway disease (Heo et
aL .1.996; Fochtman et aL .1.969). IL-4 is a key regulator of immune responses produced by Th2 cells.
This pleiotropic cytokine not only inhibits production of Thl cytokines, but also promotes B cell
activation, differentiation, proliferation and class switching leading to the production of IgE antibodies
(Dietert and Piepenbrink. 2006). Importantly, in most cases where Pb exposure was associated with
increased IgE levels, IL-4 levels were also elevated (Snyder et aL 2000; Chen et aL .1.999; Miller et aL
.1.998). IgE antibodies are a hallmark of immediate-type hypersensitivity responses that are responsible for
inducing allergic asthma (Janewav et aL 2005). In sensitized individuals, binding of allergen to antigen-
specific IgE antibodies on the surface of mast cells triggers mast cell degranulation and release histamine,
leukotrienes, and cytokines, which in turn, produce the inflammatory-related effects associated with
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asthma and allergy, i.e., airway responsiveness, mucus secretion, respiratory symptoms (Janewav et al..
2005). Consistent with this condition, inflammation was identified as a major immune-related effect of Pb
based on consistent toxicological evidence for Pb-induced increases in proinflammatory cytokines
(e.g., IL-4) and increased levels of PGE2 (Chettv et al.. 2005) and ROS production ("Chen et al.. .1.997;
Hilbertz et al.. .1.986; Castranova et al.. .1.980). decreased NO production (Fairer et al.. 2008; Mishra et al..
2006; Bunn et al.. 2001b; Lee et al.. 2001; Krocova et al.. 2000; Chen et al.. .1.997; Tian and Lawrence.
.1.996; Tian and Lawrence. 1.995). and increased cell death (Metrvka et al.. 2021; Guam et al.. 2020; Choi
et al.. 20.1.8; Kerr et al.. 20.1.3) that may also contribute to Pb-induced decreased resistance to bacterial or
viral infection (Hilbertz et al.. .1.986; Castranova et al.. .1.980).
6.7 Summary and Causality Determinations
The body of epidemiologic and toxicological evidence describes several effects of Pb exposure
on the immune system. The majority of this evidence predates this ISA. These effects can be traced back
to two major targets including T cells and macrophages promoting immunosuppression and sensitization
and allergic responses, respectively. In addition, a very limited number of studies report findings related
to autoimmunity. The sections that follow describe the evaluation of evidence for these three groups of
outcomes with respect to causality determinations for exposure to Pb using the framework described in
the Preamble to the ISA (U.S. EPA. 20.1.5). The key evidence, as it relates to the causal framework, is
outlined below, and summarized in Table 6-1, Table 6-2, and Table 6-3.
6.7.1 Causality Determination for Immunosuppression
The 2013 Pb ISA concluded "a causal relationship is likely to exist between Pb exposures and
decreased host resistance."(I S. EPA, 2013). This causality determination was primarily based on
consistent evidence that exposure to relevant BLLs suppresses the DTH response and increases bacterial
titers and subsequent mortality in rodents. For example, various studies reported decreased clearance of
bacteria and increased mortality induced by Listeria monocytogenes in mice exposed postnatally to Pb
acetate in drinking water for 3 to 8 weeks, resulting in BLL ranging from 20-25 (.ig/dL (Fernandez-
Cabezudo et al.. 2007; Dyatlov and Lawrence. 2002; Kim and Lawrence. 2000; Kishikawa et al., .1.997;
Lawrence, 1.981). Other studies reported increased mortality from Salmonella or E. coli, or decreased
clearance of Staphylococcus, in mice administered Pb acetate or Pb nitrate via injection resulting in BLL
relevant to the 2013 Pb ISA (Bishayi and Sengupta, 2006; Cook ; :mphill et al., 1.971; Selye
etah, .1.966). Although BLLs were high (i.e., 71 -3 13 (.ig/dL). increased mortality from viral infection was
also reported in mice and chickens administered Pb (mostly Pb acetate) for 4-
10 weeks (Gupta et al., 2002; Exon et al., .1.979; Thind and Khan. .1.978). Additional evidence for Pb-
induced immunosuppression comes from studies investigating the DTH response. Suppressed DTH
response is one of the most consistently reported immune effects associated with Pb exposure in animals
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( 20.1.3). Suppression of the DTH response has been reported following gestational (Chen et al.,
2004; Bunn et al., 2001a; Bunn et al., 2001b, c; Lee et al., 2001; Chen et al., .1.999; Miller etah, .1.998;
Faith et al., .1.979) and postnatal (McCabe et al., .1.999; Haneefetah, .1.995; Laschi-Loquerie et al., .1.984;
Mtilleretah, .1.977) exposures to Pb acetate resulting in BLLs ranging from 6.75 to >100 (ig/dL) in rats,
mice, chickens, and goats. Further, evidence suggested a plausible mode of action involving suppressed
production of Thl cytokines (e.g., IFN-y) (Fernandez-Cabezudo et al., 2007; Lara-Tejero and Pamer,
2004), and decreased macrophage function (Lodi et al., 20.1.1; Bishayi and Sengupta, 2006; Chen et al.,
.1.997; Hilbertz et al., .1.986; Castranova et al., .1.980). A limited number of epidemiologic studies reviewed
in the 2013 Pb ISA (U.S. EPA, 20.1.3) indicated an association between BLL and viral and bacterial
infections in children. None of the studies considered potential confounders, however, and most analyzed
populations with higher BLLs (means >10 (ig/dL). Cross-sectional studies of cell-mediated immunity
reported consistent associations between BLL and lower T cell abundance in children, while results from
other studies on lymphocyte activation, macrophages, neutrophils, and NK cells were generally
inconsistent or not sufficiently informative (e.g., cross-sectional study designs with limited or no
consideration of potential confounding, and a lack of information on concentration-response relationship).
Recent toxicological studies provide additional evidence for immunosuppression. Although there
were no recent studies directly investigating the effects of Pb exposure on host resistance, the ability of Pb
to alter antibody responses was investigated and provides evidence for immunosuppression. Yathapu et
al. (2020) showed that serum levels of anti-TT specific IgM antibodies were decreased while anti-TT
specific IgG levels were unaffected in rats exposed to Pb (BLL =16.1 (ig/dL) in drinking water.
Consistent with the 2013 Pb ISA, administration of Pb acetate in drinking water for 42 days
(BLL = 18.48 (ig/dL) significantly suppressed the DTH response in adult male Sprague Dawley rats
(Fang et al., 20.1.2). Additional supporting evidence for Pb-induced immunosuppression can be derived
from supporting immune system endpoints including (1) reduced non-specific mucosal IgA
immunoglobulins (but not IgM or IgG) in rats with BLLs of 16.1 (ig/dL (¥athapu et al., 2020) and (2)
reduced relative thymus weight in juvenile rats orally administered Pb (1 or 10 mg/kg with BLL of
3.27 (ig/dL and 12.5 (ig/dL, respectively) for up to 25 days (Graham et al., 20.1.1). Because of differences
in experimental design parameters and specific endpoints measured, effects of Pb exposure on immune
organ pathology, WBC counts and differentials, and WBC counts (hematology and subpopulations) are
challenging to interpret and, for that reason, do not support or refute evidence obtained from immune
function assays.
The relationship between Pb exposure and immunosuppression is further supported by recent
epidemiologic studies, which expand quantity and quality of the supporting immune system evidence
base evaluated in the 2013 Pb ISA. Recent case-control and cross-sectional studies provide consistent
evidence that BLLs are associated with greater susceptibility to viral and bacterial infection in children
and adults (Feileret al., 2020; Park et al., 2020; Kraeger and Wade, ) and reduced antibiotic
resistance in children, as measured by nasal Staphylococcus aureus colonization (Eggers et al., 20.1.8).
Associations were observed with mean, median, or geometric mean BLLs <3.5 (ig/dL. The evaluated
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studies used concurrent blood Pb measures, raising uncertainty regarding the temporal sequence between
Pb exposure and immunosuppression and the magnitude, timing, frequency, and duration of Pb exposures
that contributed to the observed associations. Recent studies also provide generally consistent evidence of
an inverse association between BLLs and vaccine antibodies in children with low mean or median BLLs,
including a birth cohort of vaccinated children in South Africa with median BLLs <2 (.ig/dL I)i Lenardo et
al. (2020). A strength of this analysis is that it establishes temporality between exposure and outcome.
Cross-sectional studies, including a large analysis of children ages 6 to 17 from the 1990-2004 NHANES
(Jusko et al.. 2019). are consistent with results from the prospective birth cohort. Notably, this study
includes many children who were born before the phaseout of leaded gasoline and were likely subject to
higher past exposures. Thus, there is uncertainty concerning the specific Pb exposure level, timing,
frequency, and duration contributing to the associations observed in this study.
In summary, the collective body of evidence indicates that there is likely to be a causal
relationship between Pb exposure and immunosuppression. The strongest evidence supporting a
'likely to be causal' relationship between Pb exposure and immunosuppression comes from toxicological
studies consistently demonstrating that Pb exposures suppress the DTH response and increase
susceptibility to bacterial infection in animals with BLLs < 30 |ag/dL. These toxicological studies are
coherent with recent case-control and cross-sectional epidemiologic studies providing consistent evidence
that higher BLLs are associated with greater susceptibility to viral and bacterial infection in children and
adults and lower antibiotic resistance in children. Though these epidemiologic studies used concurrent
blood Pb measures, raising uncertainty regarding the temporal sequence between Pb exposure and
immunosuppression, a smaller body of supporting epidemiologic studies provide evidence that prenatal
(mean < 4 |ig/dL). as well as concurrent (mean and/or medians < 2 |ig/dL). BLLs are associated with a
smaller vaccine antibody response. The two toxicological studies examining the animal correlate for the
human vaccine response (i.e., TDARto sheep red blood cells) reported mixed results. The biological
plausibility of Pb-induced immunosuppression is supported by toxicological studies demonstrating (1)
skewing of T cell populations, promoting Th2 cell formation and cytokine production, (2) decreased IFN-
y production, (3) decrements in macrophage function, (4) production of inflammatory mediators, and (5)
disruption of the microbiome.
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Table 6-1 Summary of evidence for a likely to be causal relationship between Pb exposure and
immunosuppression
Rationale for Causality
Determination3
Key Evidence"
Key References"
Pb Biomarker Levels Associated with
Effects0
Consistent evidence from
toxicological studies with
relevant exposures
investigating immune
functional endpoints
Oral Pb exposures increased bacterial Dvatlov and Lawrence (2002)
infection. Similar observations in several
other studies using non-PECOS routes of Fernandez-Cabezudo et al. (2007)
exposure and/or higher Pb exposures
Mean BLL:
20 [jg/dL after adult 16-wk exposure
25 [jg/dL after lactational exposure
Oral gestational Pb exposures
suppressed DTH response. Similar
observations in several other studies with
higher Pb exposures
Chen et al. (2004)
Bunn et al. (2001a)
Fang et al. (2012)
Mean BLL:
6.75 [jg/dL
25 [jg/dL
18.48 [jg/dL
Evidence from other
toxicological studies with
relevant exposures
investigating immune
functional endpoints
Oral Pb exposure decreased levels of
anti-TT-specific IgM, levels of anti-TT-
specific IgG were unaffected
Yathapu et al. (2020)
Mean BLL:
16.1 ± 5.5 [jg/dL
Supporting evidence from
toxicological studies with Oral Pb exposure decreased non-specific Yathapu et al. (2020)
relevant exposures supporting mucosa| |gA immunoglobulins
immune functional endpoints
Oral administration of Pb decreased
relative thymus weight in juvenile rats
Graham et al. (2011)
Mean BLL:
16.1 ± 5.5 [jg/dL
1 or 10 mg/kg exposure dose with BLL of
3.27 [jg/dL and 12.5 [jg/dL, respectively
Coherence from a small body A limited number of case-control and
of epidemiologic studies
demonstrating consistent
evidence of decreased host
resistance at low BLLs
cross-sectional studies reported
associations between concurrent BLLs
and:
Increased susceptibility to viral and
bacterial infection, and
Krueqer and Wade (2016)
Park et al. (2020)
Feiler et al. (2020)
Mean, Median, or Geometric Mean BLL
across studies:
1.4-3.15 [jg/dL
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Reduced antibiotic resistance
Eqqers et al. (2018)
Uncertainty regarding the temporal
sequence between Pb exposure and
immunosuppression and the magnitude,
timing, frequency, and duration of Pb
exposures that contributed to the
observed associations.
Coherence from a small body
of epidemiologic studies
demonstrating consistent
evidence of decreased
vaccine antibody response at
low BLLs
Jusko et al. (2019) Mean BLL: 1.4 (jg/dL
See Section 6.3.1.2
A limited number of prospective birth Pi Lenardo et al. (2020) Median BLL: 1.9 (jg/dL
cohort and cross-sectional studies
reported associations between BLLs and
decreased vaccine antibody response
Biological Plausibility Evidence that Pb (1) suppressed See Section 6.6
production of Th1 cytokines (i.e., IFN-y),
(2) decreased macrophage function, and
(3) increased inflammation in animals
anti-TT = anti-tetanus toxoid; BLL = blood lead level; DTH = delayed-type hypersensitivity; IgG = immunoglobulin G; IgM = immunoglobulin M; Pb = lead; PECOS = population,
exposure, comparator, outcome and study.
Based on aspects considered in judgments of causality and weight of evidence in causal framework in Table I and Table II of the Preamble to the ISAs (U.S. EPA. 2015).
'Describes the key evidence and references, supporting or contradicting, contributing most heavily to causality determination and, where applicable, to uncertainties or
inconsistencies. References to earlier sections indicate where the full body of evidence is described.
°Describes the Pb biomarker levels at which the evidence is substantiated.
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6.7.2
Causality Determination for Sensitization and Allergic Responses
The 2013 Pb ISA concluded "that a causal relationship is likely to exist between Pb exposures
and an increase in atopic and inflammatory conditions" (U.S. EPA. 20.1.3'). This causality determination
was made on the basis of a body of evidence integrated across epidemiologic and toxicological studies.
Epidemiologic evidence included a prospective analysis reporting associations between BLLs and asthma
incidence in children (Joseph et ah, 2005) and another longitudinal study that observed an association
between cord BLLs and immediate-type allergic responses in children that were detected clinically using
SPTs (Jedrychowski et ah, 20.1.1). Both studies had small sample sizes, however, and lacked precision
(i.e., had wide 95% CIs), which increases the likelihood of chance findings. An additional prospective
cohort analysis reported an imprecise association between cord BLLs and prevalent asthma in children
(Rabinowitz et ah, .1.990) but did not adjust for potential confounders. The associations observed in the
prospective analyses are supported by a cross-sectional study of BLL-associated parental-reported asthma
in children and population-based cross-sectional studies in children that reported associations between
BLLs and elevated serum IgE. Notably, many of the serum IgE studies had limited adjustment for
potential confounders and included population mean BLLs >5 (ig/dL. The epidemiologic findings are
coherent with a large body of toxicological studies that reported physiological responses in animals
consistent with the development of allergic sensitization, including increased lymph node cell
proliferation (Teijon et ah, 20.1.0; Carey et ah, 2006), increased production of Th2 cytokines such as IL-4
(Fernandez-Cabezudo et ah, 2007; Iavicoli et ah, 2006; Chen et ah, 2004; Heo et ah, .1.998; Miller et ah,
.1.998; Heo etah, .1.997; Heoetah, .1.996), increased total serum IgE antibody levels (Snyder et ah, 2000;
Miller et ah, .1.998; Heo et ah, .1.997; Heo et ah, .1.996), and misregulated inflammation (Lodi et ah, 20.1.1;
Chetty et ah, 2005; Flohe et ah, 2002; Shabani and Rabbani, 2000; Miller etah, .1.998; Chen et ah, .1.997;
Knowles and Donaldson, .1.997; Baykov et ah, .1.996; Lee and Battles, .1.994; Zelikoff et ah, .1.993; Knowles
and Donaldson, .1.990; Hilbertz et ah, .1.986; Castranova et ah, .1.980).
There have been several recent epidemiologic studies of sensitization and allergic response,
including prospective birth cohorts and cross-sectional studies with mean or median BLLs <2 (ig/dL. In
contrast to evidence presented in the 2013 Pb ISA (U.S. EPA, 20.1.3). the recent studies provide little
evidence of an association between exposure to Pb and atopic disease, and inconsistent evidence for
immunological biomarkers involved in sensitization and allergic response. Specifically, recent
epidemiologic studies of atopic disease, including analyses of prospective cohort studies examining of
asthma (Pesce et ah, 2021), eczema (Pesce et ah, 2021; Kim et ah, 20.1.9; Kim et ah, 2013), and food
allergies (Pesce et ah, 2021) were generally consistent in reporting a lack of an association in populations
with low mean BLLs. A considerable uncertainty in the evidence base is the limited number of children
with asthma in the cohort studies evaluated, both in recent studies and in the 2013 Pb ISA. This decreases
the statistical power to detect an association. Although less informative than the prospective cohort
studies due to a lack of temporality and a less relevant exposure window, recent cross-sectional NHANES
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analyses also reported null associations between childrens' BLLs and asthma (Wells et ah. 2014). eczema
("Wei et ah. 2019). and food allergies (Mener et ah. 20.1.5) in much larger study populations. Results from
recent epidemiologic studies of allergen-specific and non-specific immunological biomarkers of
hypersensitivity in children and adults were less consistent than the generally null results for atopic
diseases, providing inconsistent evidence in both children and adults.
Recent toxicological evidence for effects of Pb exposure on biomarkers of allergic disease is
sparse and limited to two reports investigating cytokine levels in blood. Decreased IFN-y, a Thl cytokine
known to play a role in the resolution of asthma, was reported in a recent study. Pb exposure had no effect
on the levels of other cytokines that have been reported to play a role in allergic disease (i.e., GM-CSF,
IL-6, IL-10, and TNF-a). However, the value of these data for hazard identification is limited by two
factors. Changes in cytokine levels (particularly when measured in blood) can be associated with many
different types of tissues and toxicities and may reflect an immune response to tissue injury but not
necessarily an effect on or impairment of immune function. For this reason, cytokine secretion data (in the
absence of a stimulus) are considered supporting evidence for understanding mechanisms of immune
disruption, not as apical data. In addition, the utility of these data is further diminished by the lack of
additional studies corroborating these findings.
In summary, the collective body of evidence is suggestive of, but not sufficient to infer, a
causal relationship between Pb exposure and sensitization and allergic responses. Whereas a few
small prospective studies reviewed in the 2013 Pb ISA supported the presence of an association between
BLLs and incident asthma in children, recent prospective epidemiologic studies provide little evidence of
an association between exposure to Pb and atopic disease in children and inconsistent evidence for
immunological biomarkers involved in sensitization and allergic response. The recent epidemiologic
studies add considerable uncertainty to the line of evidence that previously provided support for the
'likely to be causal' determination in the 2013 Pb ISA. Differences in study designs and exposure
concentrations do not appear to explain the inconsistency in results of the more recent studies compared
to studies reviewed in the 2013 Pb ISA. While the epidemiologic evidence base for sensitization and
allergic response is inconsistent, there is consistent toxicological evidence that exposure to Pb increased
lymph node cell proliferation, increased production of Th2 cytokines such as IL-4, increased total serum
IgE antibody levels in serum, and misregulated inflammation in studies reporting BLL relevant to this
ISA. Biological plausibility for the associations observed in some epidemiologic studies is provided by
toxicological evidence that Pb exposure (1) promotes the production of Th2 cells and cytokines including
IL-4 and (2) increases total serum IgE levels in studies utilizing non-relevant routes of administration
(i.e., injection) and in studies either reporting high BLLs or those not reporting BLLs at all.
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Table 6-2 Summary of evidence that is suggestive of, but not sufficient to infer, a causal relationship
between Pb exposure and sensitization and allergic responses
Rationale for Causality K F./iH~n_„b Kpv Rpforpnrpcb Pb Biomarker Levels Associated with
Determination3 *ey tviaence *ey Terences Effects0
Consistent evidence from other Increased IL-4 production, decreased IFN- Fernandez-Cabezudo et al. (2007)
toxicological studies with y production in mice administered Pb in
relevant exposures drinking water for 16 wk
investigating immune functional
endpoints Increased IL-4 production in mice exposed lavicoli et al. (2006)
prenatally and postnatally
Increased total serum IgE antibody in mice Snyder et al. (2000)
exposed prenatally and postnatally to
0.1 mM Pb acetate for 2 wk
Mean BLL: 5 or 10 mM with BLL of 20.5
and 106.2 [jg/dL, respectively
0.02, 0.06, 0.11, 0.2, 40.00, and 400.0 ppm
with mean BLL of 0.83, 1.23, 1.59, 1.97,
11.86, and 61.48 [jg/dL, respectively
Mean BLL: 25.3 pg/dL
Inconsistent epidemiologic A limited number of studies reported Joseph et al.
evidence for atopic disease positive but imprecise associations Puah Smith and Nriaau (2011)
provides limited coherence with between BLLs and asthma incidence and
toxicological evidence prevalence in children. Studies limited by
small number of cases
A limited number of recent studies with
lower BLLs reported null associations
between BLLs and asthma incidence and
prevalence in children
Generally null associations observed in
studies of other atopic diseases in
children, including eczema and food
allergies
Pesce et al. (2021)
Wells et al.
See Section 6.4.2
Associations observed in stratified analysis
for participants with BLLs >5 and >10 pg/dL
Mean cord BLL: 1.45 pg/dL
Geometric Mean BLL: 1.13 pg/dL
Mean/Median BLL across studies:
1.01-1.75 pg/dL
Biological Plausibility Evidence that Pb (1) promotes T cell See Section 6.6
skewing leading to the production of Th2
cells and cytokines including IL-4, (2)
increased IgE levels, and (3) increased
inflammation in animals
BLL = blood lead level; IFN-y = interferon-gamma; IgE = immunoglobulin E; IL-4 = interleukin 4; Pb = lead.
Based on aspects considered in judgments of causality and weight of evidence in causal framework in Table I and Table II of the Preamble to the ISAs (U.S. EPA. 2015).
'Describes the key evidence and references, supporting or contradicting, contributing most heavily to causality determination and, where applicable, to uncertainties or inconsistencies.
References to earlier sections indicate where the full body of evidence is described.
°Describes the Pb biomarker levels at which the evidence is substantiated.
6-39
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6.7.3
Causality Determination for Autoimmunity and Autoimmune Disease
In the 2013 Pb ISA, it was concluded "the evidence is inadequate to determine if there is a
causal relationship between Pb exposure and autoimmunity." (U.S. EPA. 20.1.3'). This causality
determination was reached based on evaluation of a limited body of evidence that does not sufficiently
inform Pb-induced generation of autoantibodies with relevant Pb exposures. While elevated levels of
autoantibodies were reported in a single study of Pb-exposed battery workers with BLLs (10-40 (ig/dL)
(El-Fawal etah, .1.999), the internal validity and relevance of this study to this ISA is uncertain because of
a lack of adjustment for important confounders. In the only toxicology study available for the 2013 Pb
ISA with BLLs relevant to humans, autoantibodies were detected in rats following dietary administration
of Pb resulting in BLLs of 11-50 (ig/dL (El-Fawal etah, .1.999).
Recent epidemiologic studies of autoimmunity are limited in number and examine disparate
outcomes (Joo et ah, 20.1.9; Kamycheva et ah, 20.1.7). Neither study observed evidence supporting an
association between Pb exposure and autoimmunity. Although Kamycheva et ah (20.1.7) reported an
inverse association between BLLs and seropositivity for Celiac Disease, the cross-sectional study design
does not preclude reverse causality, whereby the association may result from reduced absorption of Pb
rather than a protective effect of Pb exposure. Only one recent toxicology study was available for this
assessment. In that study. Fang et ah (20.1.2) reported that administration of Pb acetate in drinking water
for 42 days (BLL = 18.48 (ig/dL) had no effect on the suppressive properties of Tregs isolated from adult
male Sprague Dawley rats.
In summary, the collective body of evidence remains inadequate to infer the presence or
absence of a causal relationship between Pb exposure and autoimmunity and autoimmune disease.
This determination is based on the limited number of epidemiologic and toxicological studies and the
disparate outcomes examined therein, which make it difficult to draw conclusions about the nature of the
relationship. The evidence available to date does not indicate a relationship between exposure to Pb and
autoimmunity and autoimmune disease.
6-40
-------
Table 6-3 Summary of evidence that is inadequate to determine the presence or absence of a causal
relationship between Pb exposure and autoimmunity and autoimmune disease
Rationale for Causality
Determination3
Key Evidence"
Key References"
Pb Biomarker Levels Associated with
Effects0
Limited toxicological evidence
A study in rats shows generation of
El-Fawal et al, f 1999)
BLL: 11-50 pg/dL
for increased autoantibodies
autoantibodies with relevant adult-only
oral Pb exposure for 4 d. Several other
studies have Pb exposure concentrations
and/or exposure routes
(e.g., intraperitoneal) with uncertain
relevance to humans
Coherence from a limited
Evidence for increased autoantibodies in
El-Fawal et al. (1999)
BLL: 10-40 pg/dL
number of epidemiologic
studies for increased
autoantibodies at high BLLs
Pb-exposed workers with high BLL and
limited consideration for potential
confounding, including other workplace
exposures
Lack of coherence from
epidemiologic studies of
autoimmune disease
Limited number of epidemiologic studies
reported null or associations between
BLLs and autoimmune disease
Kamvcheva et al, (2017)
Joo et al, (2019)
Limited evidence for biological
plausibility
Administration of Pb for 42 d had no
effect on Treg activity in rats
Fana et al, (2012)
BLL: 18.48 pg/dL
BLL = blood lead level; d = day; Pb = lead; Treg = regulatory T cells.
Based on aspects considered in judgments of causality and weight of evidence in causal framework in Table I and Table II of the Preamble to the ISAs (U.S. EPA. 2015).
'Describes the key evidence and references, supporting or contradicting, contributing most heavily to causality determination and, where applicable, to uncertainties or
inconsistencies. References to earlier sections indicate where the full body of evidence is described.
°Describes the Pb biomarker levels at which the evidence is substantiated.
6-41
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6.8
Evidence Inventories - Data Tables to Summarize Study Details
Table 6-4 Epidemiologic studies of exposure to Pb and immunosuppression
Referent^ and Study study Popu|atjon
Exposure Assessment
Outcome
Confounders
Effect Estimates and
95% Clsa
Host Resistance
tEggers et al. (2018)
United States
2001-2004
Cross-Sectional
NHANES
n: 18626
General population;
>1 yr old
Blood
Blood Pb was measured in
venous whole blood using
GFAAS (2001-2002) and ICP-
MS (2003-2004)
Age at measurement:
>1 yr old
Median: 1.4 pg/dL
75th: 2.3 pg/dL
Maximum: 68.9 pg/dL
Q1
Q2
Q3
Q4
<0.91 pg/dL
0.91-1.4 pg/dL
1.41-2.3 pg/dL
>2.3 pg/dL
Prevalence of MRSA and
MSSA colonization
Colonization by S. aureus
tested using nasal swabs
and standard culture-
based procedures
Age at Outcome:
>1 yr old
Age, sex, race, income,
smoking, iron, calcium, and
Vitamin C
ORs
MRSA Colonization:
Q1: Reference
Q2
Q3
Q4
1.52 (0.83, 2.76)
1.56 (0.75, 3.24)
1.82 (0.81, 4.1)
MRSA Colonization:
Q1
Q2
Q3
Q4
Reference
1.07 (0.95, 1.21)
1.1 (0.94, 1.28)
0.91 (0.76, 1.09)
6-42
-------
Reference and Study
Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates and
95% Clsa
tKrueqer and Wade
United States
1999-2012
Cross-Sectional
NHANES
n: 18,425 (7. gondii)
17,389 (hepatitis B),
5,994 (H. pylori)
General population; >3
yr old (H. pylori), >6 yr
old (7. gondii and
HBV)
Blood
Blood Pb was measured in
venous whole blood using ICP-
MS
Age at measurement:
>3 yr old (H. pylori), >6 yr old
(7. gondii and HBV)
Geometric mean: 1.5 pg/dL
Seropositivity for 7. gondii,
H. pylori, and hepatitis B
Serum tested for 7. gondii
and H. pylori IgG
antibodies using an ELISA
and HBc ELISA was used
to detect total antibodies
against hepatitis B core
antigen
Age at Outcome:
>3 yr old (H. pylori), >6 yr
old (7. gondii and HBV)
Age, sex, race/ethnicity,
country of birth, family
income, self-reported health,
tap water source, household
crowding, NHANES cycle,
and use of illicit intravenous
drugs
ORs
H. pylori Seropositivity:
I.09 (1.05, 1.13)
T. gondii Seropositivity:
1.10 (1.06, 1.14)
Hepatitis B
Seropositivity:
1.08 (1.03, 1.13)
tFeiler et al. (2020)
Rochester, NY
United States
2012-2017
Case-control
n: 2,663 (full sample);
617 (reduced sample)
Test-negative case-
control study of
children <4 yr old
tested for
influenza/RSV
Blood
Blood Pb measured in venous or
capillary whole blood samples
using GFAAS. When multiple
measurements were available
Age at measurement:
Between 6 mo and 4 yr
Mean: NR
-60% of children had peak BLLs
<1 [jg/dL; 5% had peak BLLs
>5 [jg/dL
Influenza and RSV
diagnosis
Nasopharyngeal swab
samples tested for
influenza or RSV by PCR
Age at Outcome:
<4 yr old
Full sample: age, sex, race,
ethnicity, insurance status,
and respiratory season.
Reduced sample: Same as
full, plus maternal age,
parity, feeding type, maternal
smoking, and area-level
poverty, unemployment,
education, and housing built
before 1980
ORs
Influenza
<1 pg/dL: Reference
1-3: 1.52 (0.69, 3.37)
>3: 1.12 (0.45, 2.82)
RSV
<1 pg/dL: Reference
1-3: 0.97 (0.56, 1.66)
>3: 0.9 (0.5, 1.62)
6-43
-------
RefereDCesignnd Study Population Exposure Assessment
Outcome
Confounders
Effect Estimates and
95% Clsa
tPark etal. (2020)
n: 2625
Blood
H. pylori infection
Age, smoking, drinking, BMI,
and diabetes, exercise
ORs
Hwasun
South Korea
Patients >20 yr old
undergoing
Blood Pb measured in whole
blood using GFAAS
H. pylori infection
confirmed histologic
H. pylori Infection
2014-2016
gastrointestinal
Age at measurement:
examination using
endoscopy
>20 yr old
Giemsa staining of
Men: 1.05 (1.03, 1.08)
Women: 1.06 (1.00, 1.13)
Cross-sectional
Mean:
Men: 3.15 |jg/dL; Women:
abnormal lesions
identified during
endoscopy
2.19 [jg/dL
Age at Outcome:
>20 yr old
Vaccine Antibody Response
tDi Lenardo et al.
Venda Health
Blood
Measles, Tetanus, and H.
Maternal age, HIV status,
ORs for odds of being
(2020)
Examination of
influenzae type B IgG
duration of breast feeding
below protective cut
Mothers, Babies and
Blood Pb measured in triplicate
titers
point
Limpopo
their Environment
in whole blood using ICP-MS
South Africa
2012-2013
n: 425
Age at measurement:
1 yr
Serum IgG specific to
measles, tetanus, and Hib
Measles IgG Levels:
Women recruited
measured by ELISA
1.00 (0.77, 1.31)
Cohort
when presenting for
Median: 1.9 [jg/dL
delivery. Children were
75th: 2.8 pg/dL
Age at Outcome:
Tetanus IgG Levels:
excluded if they did not
3.5 yr
receive measles,
1.13 (1.02, 1.26)
tetanus, and Hib
immunizations
Hib IgG Levels:
0.99 (0.89, 1.11)
6-44
-------
Reference and Study
Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates and
95% Clsa
tJusko et al. (2019)
United States
1999-2004
Cross-Sectional
NHANES
n: 7005
General population;
children 6-17 yr old.
Percent unvaccinated
not reported. MMR
vaccine schedule
between 1999 and
2004 was:
1st dose: 12-18 mo;
2nd dose: 4-6 yr; and
Catch-up 2nd dose by
11-12 yr
Blood
Blood Pb was measured in
venous whole blood using ICP-
MS
Age at measurement:
6-17 yr old
Mean: 1.4 pg/dL
Median: 1.0 pg/dL
Measles, Mumps, and
Rubella Antibody Levels
Measles and Rubella
antigen-specific IgG
levels were determined
using an ELISA; Mumps
antigen-specific IgG
levels were determined
via Wampole Mumps IgG
test
Age at Outcome:
6-17 yr old
Sex, age, race/ethnicity,
family poverty-income ratio,
and NHANES cycle
% Change
Anti-Measles IgG Levels:
-2.75 (-5.10, -0.41)
Anti-Mumps IgG Levels:
-2.07 (-3.87, -0.24)
Anti-Rubella IgG Levels:
0.00 (-2.58, 2.65)
tWelch et al. (2020) n: 502
Munshiganj and
Pabna
Bangladesh
2008-2011 enrollment
(follow-up through 5 yr
of age)
Cohort
Pregnant women with
singleton pregnancies
recruited and children
followed through 5 yr
of age
Blood
Cord blood Pb measured using
ICP-MS; Blood Pb measure in
capillary samples using portable
Lead-Care II instruments
Age at measurement:
At birth, 20-40 mo and 4-5 yr
Median:
Pregnancy: 3.1 [jg/dL;
Toddler: 6.4 [jg/dL;
Early Childhood: 4.7 [jg/dL
Serum vaccine antibody
concentrations (diphtheria
and tetanus)
Serum diphtheria and
tetanus antibodies
measured using an ELISA
Age at Outcome:
5 yr old
Maternal education,
breastfeeding duration, and
child sex
% Change in Median
Antibody Concentration
Cord BLLs
Diphtheria:
0.97 (-1.11, 3.05)
Tetanus:
1.54 (-0.17, 3.24)
BLLs
75th:
Pregnancy: 5.6 [jg/dL;
Toddler: 10.0 pg/dL;
Early Childhood: 7.0 pg/dL
Diphtheria:
-0.96 (-3.26, 1.33)
Tetanus:
0.33 (-2.36, 3.02)
6-45
-------
Reference and Study
Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates and
95% Clsa
tXu eta!. (2015)
Shantou
China
2011-2013
Cross-sectional
n: 490
Hepatitis B vaccinated
children 3-7 yr old
from two kindergartens
(one near an e-waste
facility, and the other
in a matched reference
area)
Blood
Blood Pb measured in venous
whole blood using GFAAS
Age at measurement:
3-7 yr old
Geometric Mean:
Reference kindergarten:
6.05 [jg/dL; Exposed (e-waste)
kindergarten: 6.76 [jg/dL
Hepatitis B surface
antibody levels
Blood plasma HBsAb titer
was measured by ELISA
Age at Outcome:
3-7 yr old
Age and sex (areas matched
on traffic density, population,
SES, lifestyle, and cultural
background)
Change in HBsAb titers
(S/CO)
2011 Sample:
-0.45 (-0.49, -0.40)
2012 Sample:
-0.37 (-0.40, -0.33)
WBCs and Cytokines
tCao et al
Guiyu and Haojiang
China
2014
Cross-Sectional
n: 118
Children 3-7 yr old at
two kindergartens (one
near an e-waste
facility, and the other
in a matched reference
area)
Blood
Pb measured in venous whole
blood using GFAAS
Age at measurement:
3-7 yr
Median: Reference kindergarten:
3.6 [jg/dL
Exposed (e-waste) kindergarten:
5.1 [jg/dL
T cell subpopulations, IL-
2, IL-7, IL-15 levels
T cell subpopulations
measured in whole blood
using flow cytometry;
Serum cytokines
measured using the
ProcartaPlex Human
Cytokine Chemokine
Panel 1A
Age at Outcome:
3-7 yr
Age and sex (areas matched
on traffic density, population,
SES, lifestyle, and cultural
background)
Change in percentage
of T cells
CD4+ Tn
-0.59 (-1.07, -0.12)
CD4+ Tcm
0.49 (0.10, 0.88)
6-46
-------
Reference and Study
Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates and
95% Clsa
tChen et al. (2021)
Shantou
China
Nov.-Dec. 2018
Cross-sectional
n: 486
Pre-school children
(aged 2-6) from two
towns with similar
but different Pb
exposure
Blood
Blood Pb measured in venous
whole blood using GFAAS
Age at measurement:
2-6 yr
Median:
Exposed: 4.51 [jg/dL;
Reference: 3.98 [jg/dL
75th:
Exposed: 5.67 [jg/dL,
Reference: 4.84 [jg/dL
WBC, neutrophil, and
monocyte counts
WBCs, neutrophils, and
monocytes measured in
venous whole blood
Age at Outcome:
2-6 yr
Gender, age, BMI, e-waste
contamination w/ in 50 m of
residence, residence as
workplace, distance of
residence from road, family
member daily smoking,
monthly household income,
maternal work associated w/
e-waste, duration of outdoor
play, child contact w/ e-
waste, washing hands
before eating, nail biting
habit, chewing pencil habit,
yearly canned food
consumption, yearly
fruit/vegetable consumption,
yearly iron rich food
consumption, yearly marine
product consumption, and
yearly salted food
consumption
ln(WBC count)
0.006 (0.001, 0.012)
ln(Monocyte count)
0.006 (-0.001, 0.013)
ln(Neutrophil count)
0.009 (0, 0.018)
tDai et al. (2017)
Shantou
China
Cross-sectional
n: 484
Children 2-6 yr old
randomly sampled
from volunteers at two
kindergartens (one
near an e-waste
facility, and the other
in a matched reference
area)
Blood
Blood Pb measured in venous
whole blood using GFAAS
Age at measurement:
2-6 yr old
Q1
Q2
Q3
Q4
<3.78 [jg/dL
3.78-5.22 [jg/dL
5.23-7.00 [jg/dL
>7.00 [jg/dL
Erythrocyte CR1
expression measured
using flow cytometry
Age at Outcome:
2-6 yr old
Age, gender, paternal and
maternal education level,
and family income
Mean Difference in
Erythrocyte CR1
Expression
Q1
Q2
Q3
Q4
Reference
-0.07 (-0.23, 0.08)
-0.04 (-0.20, 0.11)
-0.16 (-0.32, -0.01)
6-47
-------
Reference and Study
Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates and
95% Clsa
tHuo et al. (2019)
Shantou
China
NR
Cross-sectional
n: 267
Children 2-7 yr old at
two kindergartens (one
near an e-waste
facility, and the other
in a matched reference
area)
Blood
Blood Pb measured in venous
whole blood using GFAAS
Age at measurement:
2-7 yr old
Median:
Reference kindergarten:
4.4 [jg/dL; Exposed (e-waste)
kindergarten: 6.5 [jg/dL
75th:
Reference kindergarten:
5.6 [jg/dL; Exposed (e-waste)
kindergarten: 8.2 [jg/dL
IFN-y, IL-113, and IL-
12p70
Serum cytokine measured
using the ProcartaPlex
Human Cytokine
Chemokine Panel 1A
Age at Outcome:
2-7 yr old
Age and sex (areas matched
on traffic density, population,
SES, lifestyle, and cultural
background)
Per natural log increase in
erythrocyte Pb
IL-ip pg/ml
0.08 (-0.01, 0.17)
IL-12p70 pg/ml
0.99 (0.53, 1.44)
ifn-y pg/mi
1.43 (0.57, 2.30)
tli et al. (2018)
Hubei and Hunan
Provinces
China
2012-2017
Cross-Sectional
Blood Lead
Intervention Program
n: 758
Children Ages 5-8 yr
recruited from 4
counties in 2
provinces. One county
in each province had
high environmental Pb
levels (battery plant
and mining)
Blood
Blood Pb measured in venous
whole blood using GFAAS
Age at measurement:
5-8 yr old
Geometric mean: 8.24 [jg/dL
75th: 13.51 pg/dL
90th: 18.77 pg/dL
95th: 21.82 pg/dL
WBC count
Hematological
parameters were
analyzed by an
automated hematology
analyzer (BC-5800;
Mindray, Shenzhen,
China) with quality control
processes.
Age at Outcome:
5-8 yr old
Age, gender, BMI,
environmental lead exposure
level, and serum iron, zinc,
and calcium
OR
Decreased WBC count
(<4 x 109/L)
1 (0.905, 1.105)
6-48
-------
RefereDCesignnd Study Population Exposure Assessment
Outcome
Confounders
Effect Estimates and
95% Clsa
tWerder et al. (2020) Gulf Long-Term
Follow-up Study
Pb measure in blood using solid- IL-6, IL-8, IL-113, TNF-a Age, race, alcohol
Gulf Region
United States
2012-2013
Cross-sectional
n: 214
Non-smoking >30 yr
old male oil spill
response workers and
oil spill safety trainees
with no history of liver
disease or heavy
alcohol use
phase micro-extraction with gas
chromatography/mass
spectrometry
Age at measurement:
>30
Mean: 1.82 [jg/dL
Cytokeratin 18 (CK18
M65 and CK18 M30)
Age at Outcome:
>30
consumption, serum
cotinine, BMI, diabetes
diagnosis, and education
pg/mL change (obese
participants)
IL-6
169.6 (119.8, 219.4)
IL-8
360.9 (246.2, 475.6)
IL-1 p
76.3 (63.6, 89.0)
TNF-p
1.1 (-1.5, 3.6)
6-49
-------
Reference and Study
Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates and
95% Clsa
tZhanq et al.
Shantou
China
Cross-sectional
n: 147
Children 3-7 yr old at
two kindergartens (one
near an e-waste
facility, and the other
in a matched reference
area)
Blood
Blood Pb measured in venous
whole blood using GFAAS
Age at measurement:
3-7 yr old
Median:
Reference kindergarten:
2.3 [jg/dL; Exposed (e-waste)
kindergarten: 3.7 [jg/dL
Neutrophils, monocytes,
lymphocytes, IL-113, IL-6,
IL-8, IL-10, and TNF-a
Immune cells measured
in whole blood using an
automated blood cell
analyzer; Serum
cytokines measured using
the ProcartaPlex Human
Cytokine Chemokine
Panel 1A
Age at Outcome:
3-7 yr old
Gender, age, BMI, e-waste
contamination w/ in 50 m of
residence, residence as
workplace, distance of
residence from road, family
member daily smoking,
maternal work associated w/
e-waste, child contact w/ e-
waste, washing hands
before eating, milk
consumption frequency, and
ventilation of house
Per natural log increase in
erythrocyte Pb
In(Neutrophils)
0.20 (0.00, 0.39)
In(Monocytes)
0.02 (-0.14, 0.18)
In(Lymphocytes)
-0.05 (-0.24, 0.16)
ln(IL-1b)
0.19 (-0.08, 0.45)
ln(IL-6)
0.33 (0.04, 0.62)
ln(IL-8)
0.05 (-0.28, 0.37)
ln(IL-10)
0.08 (-0.29, 0.44)
In(TNF-a)
-0.18 (-0.44, 0.08)
BLL = blood lead level; BMI = body mass index; CD = cluster of differentiation; CI = confidence interval; CK = cytokeratin; CR1 = complement receptor type 1; e-waste = electronic-waste;
ELISA = enzyme-linked immunosorbent assay; GFAAS = graphite furnace atomic absorption spectrometry; HBc = Hepatitis B core; HBsAb = Hepatitis B surface antigen; HBV = Hepatitis B
virus; Hib = Haemophilus influenzae type B; ICP-MS = inductively coupled plasma mass spectrometry; Ig- = immunoglobulin type; IL = interleukin type; IFN-g = interferon-gamma;
In = natural log; mo = month(s); MRSA = methicillin-resistant Staphylococcus aureus; NHANES = National Health and Nutrition Examination Survey; NR = not reported; OR = odds ratio;
Pb = lead; PCR = polymerase chain reaction; RSV = respiratory syncytial virus; S/CO = signal to cut-off; SES = socioeconomic status; SPT = skin prick test; TNF-a = tumor necrosis factor
alpha; WBC = white blood cell; yr = year(s).
aEffect estimates are standardized to a 1 |jg/dL increase in blood Pb level or a 10 |jg/g increase in bone Pb level, unless otherwise noted. For studies that report results corresponding to a
change in log-transformed Pb biomarkers, effect estimates are assumed to be linear within the 10th to 90th percentile interval of the biomarker and standardized accordingly.
fStudies published since the 2013 Pb ISA.
6-50
-------
Table 6-5
Animal toxicological studies of delayed-type hypersensitivity responses
Study
Species (Stock/Strain),
n, Sex
Timing of
Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported Endpoints
(pg/dL)a Examined
Fanq et al. (2012)
Rat (Sprague Dawley)
Control (vehicle), M,
n = 20
300 ppm Pb, M, n = 20
23-25 d to 65-
67 d
Dosing solutions were changed twice
per wk
4.48 |jg/dL for 0 ppm DTH
18.48 |jg/dL for
300 ppm - d 65-67
BLL = blood lead level; d = day; DTH = delayed-type hypersensitivity; M = male; MMR = measles, mumps, and rubella; Pb = lead; ppm = parts per million; wk = week
"If applicable, reported values for BLL were converted to
-------
Table 6-6 Animal toxicological studies of antibody response
Study Species (Stock/Strain), n, Timing of Exposure Exposure Details BLL as Reported Endpoints
Sex a H (Concentration, Duration) (HSJ'dL) Examined
Yathapu et al
Rat (Sprague Dawley)
Control (vehicle)
M/F, n = 32 (16/16)
PND 54- PND 82
Weanling rats (PND 21) were acclimated
to the facility for 5 days before being
divided into two groups (n = 16) to begin
a 28-day long Fe deficiency diet. After
28 days, the rats were exposed to Pb or
control diet (n = 16). At this point
(PND 82), blood was collected from rats
before immunization with TT (n = 8)
followed by two boosters administered in
2-wk intervals. Vaccine response was
evaluated 2 wk later
2.1 ± 1.0 pg/dL for
0 mg/4 mL/kg,
16.1 ± 5.5 pg/dL for
25 mg/4 mL/kg - PND 82,
Control diet
1.9 ± 0.7 pg/dL for
0 mg/4 mL/kg
41.6 ± 10.2 pg/dL for
25 mg/4 mL/kg - PND 82,
Iron deficiency diet
Vaccine
response,
Antigen-specific
antibodies
BLL = blood lead level; Fe = iron; M/F = male/female; Pb = lead; PND = postnatal day; TT = tetanus toxoid.
"If applicable, reported values for BLL were converted to mg/dL using WebPlot Digitizer (https://apps.automeris.io/wpd/) and are shown in parenthesis.
Table 6-7 Animal toxicological studies of ex vivo white blood cell function
Study
Species (Stock/Strain), n,
Sex
Timing of Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported
(Mg/dLf
Endpoints
Examined
Fang et al. (2012)
Rat (Sprague Dawley)
Control (vehicle), M, n = 20
300 ppm Pb, M, n = 20
23-25 d to 65-67 d
Dosing solutions were changed twice
per wk.
4.48 pg/dL for 0 ppm, Tregcell
18.48 pg/dL for suppression
300 ppm — d 65-67 assay
6-52
-------
Yathapu et al. (2020)
Rat (Sprague Dawley)
Control (vehicle), M/F, n = 32
(16/16)
500 ppm Pb, M/F, M/F,
n = 32 (16/16)
PND 54 - PND 82
Weanling rats (PND 21) were
acclimated to the facility for 5 days
before being divided into two groups
(n = 16) to begin a 28-day long Fe
deficiency diet. After 28 days, the rats
were exposed to Pb or control diet
(n = 16). At this point (PND 82), blood
was collected from rats before
immunization with TT (n = 8) followed
by two boosters administered in 2-wk
intervals. Vaccine response was
evaluated 2 wk later.
2.1 ± 1.0 pg/dL for
0 mg/4 mL/kg
16.1 ± 5.5 pg/dL for
25 mg/4 mL/kg -
PND 82, Control diet
1.9 ± 0.7 pg/dL for
0 mg/4 mL/kg
41.6 ± 10.2 pg/dL for
25 mg/4 mL/kg -
PND 82, Iron
deficiency diet
Spleen cell
proliferation
BLL = blood lead level; d = day; Fe = iron; M/F = male/female; Pb = lead; PND = postnatal day; ppm = parts per million; Treg = regulatory T cells; TT = tetanus toxoid; wk = week.
alf applicable, reported values for BLL were converted to mg/dL using WebPlot Digitizer (https://apps.automeris.io/wpd/) and are shown in parenthesis.
6-53
-------
Table 6-8
Animal toxicological studies of immune organ pathology
Study
Species (Stock/Strain), n,
Sex
Timing of
Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported
(ng/dL)a
Endpoints
Examined
Corsetti et al. (2017) Mouse (C57BJ)
Control (vehicle), M, n = 8
200 ppm Pb, M, n = 8
30-75 d
Mice were exposed via drinking water
for 45 consecutive days. Control
animals were exposed to drinking
water containing acetic acid (1 mL/L)
<5 [jg/dL for 0 ppm
21.6 |jg/dLfor200 ppm
Spleen
histopathology
Dumkova et al. (2017) Mouse (ICR) NR
Control (vehicle), F, n = 10
1.23 x 10s particles/cm3 Pb,
F, n = 10
Mice were exposed continuously
(24 hr/d, 7 d/wk) for 6 wk. Control
animals were exposed to the same air
as the treated group without the
addition of Pb nanoparticles. The
investigators pooled animals from two
independent experiments, each with
five animals per treatment
11 ng/g forO * 10s
particles/cm3 Pb
(1.166 [jg/dL)
132 ng/g for 1.23 * 10®
particles/cm3 Pb
(13.992 [jg/dL)
Spleen
histopathology
Dumkova et al. Mouse CD-1 (ICR) NR
{2020b) Control (vehicle), F, n = 10
(2 wk, 6 wk, 11 wk)
2.23 x 10® NPs/cm3 PbO
NP, F, n = 10 (2 wk, 6 wk,
11 wk)
2.23 x 10s NPs/cm3 PbO NP
recovery, F, n = 10 (6 wk
PbO NP, 5 wk clean air)
Mice (unknown age) were exposed to <3 ng/g for 0 PbO
clean air or PbO NPs 24 hr/d 7 d/wk
for 2 wk, 6 wk, or 11 wk. a recovery
group was exposed to PbO NPs for
6 wk and then clean air for 5 wk
(11 wk total)
NPs/cm3(<0.3 |jg/dL)
104 ng/g for 2.23 x 10®
N Ps/cm3 - 2 wk
(10.4 [jg/dL)
<3 ng/g for 0 PbO
N Ps/cm3 - 6 wk
(<0.3 [jg/dL)
148 ng/g for 2.23 * 10®
N Ps/cm3 - 6 wk
(14.8 [jg/dL)
Spleen
histopathology
<3 ng/g for 0 PbO
N Ps/cm3 -11 wk
(<0.3 [jg/dL)
6-54
-------
Species (Stock/Strain), n, Timing of Exposure Details BLL as Reported Endpoints
Sex Exposure (Concentration, Duration) (ng/dL)a Examined
Dumkova et al. MouseCD-1 (ICR)
{2020a] Control (vehicle), F, n = 10
68.6 |jg/m3 Pb, F, n = 10
174 ng/g for 2.23 * 10®
N Ps/cm3 -11 wk
(17.4 pg/dL)
<3 ng/g for 0 PbO
NPs/cm3 (<0.3 |jg/dL)
27 ng/g - recovery (6 wk
PbO NP, 5 wk clean air)
(2.7 pg/dL)
6-8 wk old mice
exposed for 3 d,
2 wk, 6 wk, or
11 wk
40 ng/g for 68.6 pg/m3 Pb
- 2 wk (4.0 pg/dL)
47 ng/g for 68.6 pg/m3 Pb
- 6 wk (4.7 pg/dL)
85 ng/g for 68.6 pg/m3 Pb
-11 wk (8.5 pg/dL)
10 ng/g for 68.6 pg/m3 Pb
- 6 wk exposure plus
5 wk clean air
(1.0 pg/dL)
Mice were exposed to Pb for 3 d,
2 wk, 6 wk, or 11 wk. To assess
recovery, a separate group of mice
were exposed for 11 wk followed by
5 wk of clean air. Control group was
exposed to filtered air
<0.3 ng/g for control at all Spleen
timepoints (d 3, 2 wk, histopathology
6wk, 11 wk) (<0.3 pg/dL)
31 ng/g for 68.6 pg/m3 Pb
-d 3 (3.1 pg/dL)
6-55
-------
Study
Species (Stock/Strain), n,
Sex
Timing of
Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported
(ng/dL)a
Endpoints
Examined
Smutna et al, (2022)
Mouse CD-1 (ICR)
Control (vehicle), F, n = 10
0.956 pg/m3 Pb, F, n = 10
6-8 wk old mice
exposed for 11 wk
Mice were exposed to Pb for
11 wk. Control group was exposed to
filtered air
<0.003 ± 0.001 ng/g for
control at 11 wk
(0.318 ±0.106 pg/dL)
0.171 ± 0.012 ng/g for
0.956 pg/m3 Pb -11 wk
(18.126 ± 1.272 pg/dL)
Spleen
histopathology
BLL = blood lead level; d = day; F = female; Pb = lead; PbO nanoparticles = lead oxide nanoparticles; ppm = parts per million; wk = week.
"If applicable, reported values for BLL were converted to mg/dL using WebPlot Digitizer (https://apps.autorneris.io/wpd/) and are shown in parenthesis.
Table 6-9 Animal toxicological studies of immunoglobulin levels
Study Species (Stock/Strain), n, Timing of Exposure Details BLL as Reported (ug/dL)a fndpoints
y Sex Exposure (Concentration, Duration) p ' Examined
Yathapu et al
(2020)
Rat (Sprague Dawley)
Control (vehicle)
M/F, n = 32 (16/16)
500 ppm Pb, M/F, n = 32
(16/16)
PND 54-PND 82
Weanling rats (PND 21) were
acclimated to the facility for 5 days
before being divided into two groups
(n = 16) to begin a 28-day long Fe
deficiency diet. After 28 days, the rats
were exposed to Pb or control diet
(n = 16). At this point (PND 82), blood
was collected from rats before
immunization with TT (n = 8) followed
by two boosters administered in 2- wk
intervals. Vaccine response was
evaluated 2 wk later.
2.1 ± 1.0 pg/dL for
0 mg/4 mL/kg - PND 82,
Control Diet
16.1 ± 5.5 pg/dL for
25 mg/4 mL/kg - PND 82,
Control diet
1.9 ± 0.7 pg/dL for
0 mg/4 mL/kg - PND 82, Iron
deficiency diet
Immunoglobulin
levels
41.6 ± 10.2 pg/dL for
25 mg/4 mL/kg - PND 82,
Iron deficiency diet
BLL = blood lead level; Fe = iron; M/F = male/female; Pb = lead; PND = postnatal day; TT = tetanus toxoid.
"If applicable, reported values for BLL were converted to mg/dL using WebPlot Digitizer (https://apps.automeris.io/wpdfl and are shown in parenthesis.
6-56
-------
Table 6-10
Animal toxicological studies of immune organ weight
Study
Species (Stock/Strain), n, Timing of
Sex Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported (|jg/dL)a
Endpoints
Examined
Amos-Kroohs et al.
Rat (Sprague Dawley)
Control (vehicle), M/F, n = 4
(2/2)
1 mg/kg Pb, M/F, n = 16
(8/8)
PND4-PND28
10 mg/kg Pb,
(8/8)
M/F, n = 16
Male and female rats were gavaged
every other day from PND 4 to
PND 10, 18, or 28. Starting on
PND 4, ISO offspring were isolated
from their dam individually for 4 hr.
Control animals remained with their
dam throughout this period. On
PND 11, 19, or 29, subsets within
each group were subjected to acute
stressor (shallow water stressor for 0,
30, or 60 min) or left
undisturbed. Control animals were
gavaged with vehicle containing
anhydrous sodium acetate (0.01 M)
1.19 |jg/dL for 0 mg/kg
2.73 |jg/dL for 1 mg/kg
9.15 |jg/dL for 10 mg/kg ¦
PND 29 w/o ISO stress
1.31 pg/dL for 0 mg/kg,
4.55 pg/dL for 1 mg/kg
17.1 pg/dL for 10 mg/kg ¦
PND 29 w/ ISO stress
Spleen weight,
Thymus
weight
Corsefti et al. (2017) Mouse (C57BJ)
Control (vehicle), M, n
200 ppm Pb, M, n = 8
d 30-d 75 Mice were exposed via drinking water <5 pg/dL for 0 ppm
for 45 consecutive days. Control
animals were exposed to drinking
water containing acetic acid (1 mL/L)
21.6 pg/dL for 200 ppm
Spleen weight
Dumkova et al. (2017) Mouse (ICR) NR
Control (vehicle), F, n = 10
1.23 x 10s particles/cm3 Pb,
F, n = 10
Mice were exposed continuously 11 ng/g forO * 10s Spleen weight
(24 h/d, 7 d/wk) for 6 wk. particles/cm3 Pb (1.166 pg/dL)
Control animals were exposed to the „„R
m . . i ... , 32 nq/q for 1 23 * 10s
same air as the treated group without a a 3
the addition of Pb nanoparticles.
The investigators pooled animals
from two independent experiments,
each with five animals per treatment
particles/cm3 Pb
(13.992 pg/dL)
6-57
-------
Study
Species (Stock/Strain), n, Timing of
Sex Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported (|jg/dL)a
Endpoints
Examined
Dumkova et al.
Mouse CD-1 (ICR) NR
Control (vehicle), F, n = 10
(2 wk, 6 wk, 11 wk)
2.23 x 10® NPs/cm3 PbO
NP, F, n = 10 (2 wk, 6 wk,
11 wk)
2.23 x 10® NPs/cm3 PbO
NP recovery, F, n = 10
(6 wk PbO NP, 5 wk clean
air)
Mice (unknown age) were exposed to
clean air or PbO NPs 24 hr/d, 7 d/wk
for 2 wk, 6 wk, or 11 wk. a recovery
group was exposed to PbO NPs for
6 wk and then clean air for 5 wk
(11 wk total)
<3 ng/g for 0 PbO NPs/cm3-
2 wk (<0.3 [jg/dL)
104 ng/g for 2.23 x 10®
NPs/cm3 - 2 wk (10.4 |jg/dL)
<3 ng/g for 0 PbO NPs/cm3 -
6wk (<0.3 |jg/dL)
148 ng/g for 2.23 * 10®
NPs/cm3 - 6 wk (14.8 |jg/dL)
<3 ng/g for 0 PbO NPs/cm3 -
11 wk (<0.3 [jg/dL)
Spleen weight
174 ng/g for 2.23 x 10®
NPs/cm3 - 11 wk (17.4 |jg/dL)
<3 ng/g for 0 PbO NPs/cm3
(<0.3 [jg/dL)
27 ng/g - recovery (6 wk PbO
NP, 5 wk clean air) (2.7 |jg/dL)
Dumkova et al.
Mouse CD-1 (ICR)
6-8 wk old mice
Mice were exposed to Pb for 3 d,
<0.3 ng/g for control at all Spleen weight
(2020a)
Control (vehicle), F, n = 10
exposed for 3 d,
2 wk, 6 wk, or 11 wk. To assess
timepoints (d 3, 2 wk, 6 wk,
2 wk, 6 wk, or
recovery, a separate group of mice
11 wk) (<0.3 [jg/dL)
68.6 |jg/m3 Pb, F, n = 10
11 wk
were exposed for 11 wk followed by
5 wk of clean air. Control group was
exposed to filtered air
31 ng/g for 68.6 |jg/m3 Pb -
d 3
(3.1 pg/dL)
40 ng/g for 68.6 pg/m3 Pb -
2 wk (4.0 pg/dL)
6-58
-------
Study Species (Stock/Strain), n, Timing of Exposure Details BLL as Reported (ug/dL)a fndpoints
Sex Exposure (Concentration, Duration) Examined
47 ng/g for 68.6 |jg/m3 Pb -
6 wk (4.7 [jg/dL)
85 ng/g for 68.6 |jg/m3 Pb -
11 wk (8.5 [jg/dL)
10 ng/g for 68.6 |jg/m3 Pb -
6 wk exposure plus 5 wk clean
air (1.0 |jg/dL)
Smutna et al, (2022) Mouse CD-1 (ICR)
6-8 wk old mice
Mice were exposed to Pb for 11 wk.
<0.003 ± 0.001 ng/g for control
Spleen
Control (vehicle), F, n = 10
exposed for
Control group was exposed to filtered
at 11 wk (0.318 ±0.106 pg/dL)
histopathology
11 wk
air
0.956 |jg/m3 Pb, F, n = 10
0.171 ± 0.012 ng/g for
0.956 pg/m3 Pb - 11 wk
(18.126 ± 1.272 pg/dL)
Graham et al. (2011) Rat (Sprague Dawley), PND 4-PND 28 Dosed every other day. Control 0.267 (jg/dL for 0 mg/kg, Spleen weight,
animals were gavaged with vehicle 3.27 [jg/dL for 1 mg/kg, Thymus
containing anhydrous sodium acetate 12.5 [jg/dL for 10 mg/kg - weight
(0.01 M) PND 29
6-59
-------
Study Species (Stock/Strain), n, Timing of Exposure Details BLL as Reported (ug/dL)a fndpoints
Sex Exposure (Concentration, Duration) Examined
Groups:
PND 11
Control (vehicle), M/F,
n = 192 (96/96)
1 mg/kg Pb, M/F, n = 192
(96/96)
10 mg/kg Pb, M/F, n = 191
(96/95)
PND 19
Control (vehicle), M/F,
n = 191 (96/95)
1 mg/kg Pb, M/F, n = 191
(96/95)
10 mg/kg Pb, M/F, n = 192
(96/96)
PND 29
Control (vehicle), M/F,
n = 192 (96/96)
1 mg/kg Pb, M/F, n = 192
(96/96)
10 mg/kg Pb, M/F, n = 192
(96/96)
Graham et al. (2011) Rat (Sprague Dawley)
PND 4-PND 28 Dosed every other day. Control
animals were gavaged with vehicle
containing anhydrous sodium acetate
(0.01 M)
0.267 [jg/dL for 0 mg/kg ¦
PND 29
3.27 |jg/dL for 1 mg/kg -
PND 29
Spleen weight,
Thymus
weight
12.5 |jg/dL for 10 mg/kg -
PND 29
6-60
-------
Study Species (Stock/Strain), n, Timing of Exposure Details BLL as Reported (ug/dL)a fndpoints
Sex Exposure (Concentration, Duration) Examined
Groups:
PND 11
Control (vehicle), M/F,
n = 192 (96/96)
1 mg/kg Pb, M/F, n = 192
(96/96)
10 mg/kg Pb, M/F, n = 191
(96/95)
PND 19
Control (vehicle), M/F,
n = 191 (96/95)
1 mg/kg Pb, M/F, n = 191
(96/95)
10 mg/kg Pb, M/F, n = 192
(96/96)
PND 29
Control (vehicle), M/F,
n = 192 (96/96)
1 mg/kg Pb, M/F, n = 192
(96/96)
10 mg/kg Pb, M/F, n = 192
(96/96)
6-61
-------
Study
Species (Stock/Strain), n, Timing of
Sex Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported (|jg/dL)a
Endpoints
Examined
Wildemann et al.
Rat (Wistar)
Control (vehicle), M, n = 6
NR
Control group provided tap water with
0.2% nitric acid
1.4 ± 1.2 |jg/L for
0 pg/kg/d (0.14 pg/dL)
Spleen weight
357 pg/kg/d Pb, M, n = 5
1607 pg/kg/d Pb, M, n = 5
17 ± 7 pg/L for
357 pg/kg/d (1.77 ± 0.7 pg/dL)
86 ± 29 pg/L for 1607 pg/kg/d
(0.14 pg/dL for 0 pg/kg/d,
1.77 ± 0.7 pg/dL for
357 pg/kg/d, 8.6 ±2.9 pg/dL
for 1607 pg/kg/d)
BLL = blood lead level; d = day; M = male; M/F = male/female; F = female; hr = hour; ISO = isolation, min = minute(s); NR = not reported; Pb = lead; PbO NPs = lead oxide
nanoparticles; PND = postnatal day; ppm = parts per million; w/o = without; wk = week.
"If applicable, reported values for BLL were converted to mg/dL using WebPlot Digitizer (https://apps.automeris.io/wpd/) and are shown in parenthesis.
6-62
-------
Table 6-11
Animal toxicological studies of white blood cell counts and differentials (spleen, thymus, lymph
node, bone marrow)
Study
Species (Stock/Strain),
n, Sex
Timing of Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported (|jg/dL)a
Endpoints
Examined
Cai et al, (2018)
Rat (Sprague Dawley)
Control (vehicle), M/F,
n = 5
0.2% Pb, M/F, n = 5
8-10 wk to 20-30 wk
Rats were 8-10 wk old when
acquired. Whether or not the rats
were allowed to acclimate to the
facility prior to study initiation was
not reported. The number of males
and females not reported.
Control animals received tap water
20.5 ± 0.68 pg/L for 0%
(2.2 ± 6.4 pg/dL)
87.4 ± 9.2 pg/L for 0.2%
(9.3 ± 0.98 pg/dL)
Bone marrow
cell counts
and
differentials
Fanq et al. (2012)
Rat (Sprague Dawley)
Control (vehicle), M,
n =20
300 ppm Pb, M, n = 20
23-25 d to 65-67 d
Dosing solutions were changed
twice per week
4.48 pg/dL for 0 ppm
18.48 pg/dL for 300 ppm - d
65-67
Thymus cell
counts and
differentials,
Spleen cell
counts and
differentials,
Lymph node
cell counts
and
differentials
Yathapu et al.
Rat (Sprague Dawley)
Control (vehicle), M/F,
n = 32 (16/16)
500 ppm Pb, M/F, n = 32
(16/16)
PND 54-PND 82
Weanling rats (PND 21) were
acclimated to the facility for 5 days
before being divided into two groups
(n = 16) to begin a 28-day long Fe
deficiency diet. After 28 d, the rats
were exposed to Pb or control diet
(n = 16). At this point (PND 82),
blood was collected from rats before
immunization with TT (n = 8)
followed by two boosters
administered in 2 wk intervals.
Vaccine response was evaluated
2 wk later
2.1 ± 1.0 pg/dL for
0 mg/4 mL/kg - PND 82,
Control diet
Spleen cell
counts and
differentials
16.1 ± 5.5 pg/dL for
25 mg/4 mL/kg - PND 82,
Control diet
1.9 ± 0.7 pg/dL for
0 mg/4 mL/kg - PND 82, Iron
deficiency diet
41.6 ± 10.2 pg/dLfor
25 mg/4 mL/kg - PND 82,
Iron deficiency diet
6-63
-------
Study
Species (Stock/Strain),
n, Sex
Timing of Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported (|jg/dL)a
Endpoints
Examined
Zhu et al. (2020)
Mouse (C57BL.6) 7-9 wk
Control (vehicle), M/F,
n = NR
125 ppm Pb, M/F, n = NR
1250 ppm Pb, M/F,
n = NR
Control animals were exposed to
drinking water containing sodium
acetate. The investigators specified
that an equal number of male and
female mice were used in the study,
but the number of animals used in
some analyses was not an even
number. Consequently, it is not
possible to determine sex
composition ofthe group and it
suggests there may have been
unreported attrition
0 [jg/dL for 0 ppm
4.7 ± 0.2 |jg/dL for 125 ppm
41.3 |jg/dL for 1250 ppm
Spleen cell
counts and
differentials,
Bone marrow
cell counts
and
differentials,
Lymph node
cell counts
BLL = blood lead level; d = day; M/F = male/female; NR = not reported; Pb = lead; PND = postnatal day; ppm = parts per million; wk = week; TT = tetanus toxoid.
"If applicable, reported values for BLL were converted to mg/dL using WebPlot Digitizer (https://apps.automeris.io/wpd/) and are shown in parenthesis.
6-64
-------
Table 6-12
Animal toxicological studies of white blood cell counts (hematology and subpopulations)
Study
Species (Stock/Strain),
n, Sex
Timing of Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported (|jg/dL)a
Endpoints
Examined
Andielkovic et al
Rat (Wistar)
Control (vehicle), M,
n = i
0.2% Pb, M, n = 6
NR
Rats (250 g), age at time of dosing not 24.9 ± 19 [jg/kg for 0 mg WBC counts,
reported, were exposed to a single
dose of 150 mg Pb/kg BW Pb acetate
via oral gavage. Control animals were
given "water"
Pb/kg BW (2.6 ± 2.0 pg/dL) WBC
subpopulations
291.2 ± 139 pg/kg for
150 mg Pb/kg BW
(30.9 ± 14.7 pg/dL)
Cai et al
Rat (Sprague Dawley)
Control (vehicle), M/F,
n = 5
0.2% Pb, M/F, n = 5
-10 wk to 20-30 wk
Rats were 8-10 wk old when acquired.
Whether or not the rats were allowed
to acclimate to the facility prior to study
initiation was not reported. The
number of males and females not
reported
Control animals received tap water
20.5 ± 0.68 pg/L for 0%
(2.2 ± 6.4 pg/dL)
87.4 ± 9.2 pg/L for 0.2%
(9.3 ± 0.98 pg/dL)
WBC counts
Corsefti et al. (2017)
Mouse (C57BJ)
Control (vehicle), M,
n = I
30-75 d
200 ppm Pb, M, n = 8
Mice were exposed via drinking water
for 45 consecutive days. Control
animals were exposed to drinking
water containing acetic acid (1 mL/L)
<5 pg/dL for 0 ppm
21.6 pg/dL for 200 ppm
WBC counts
Zhu et al. (2020)
Mouse (C57BL.6) 7-9 wk
Control (vehicle), M/F,
n = NR
125 ppm Pb, M/F, n = NR
1250 ppm Pb, M/F,
n = NR
Control animals were exposed to
drinking water containing sodium
acetate. The investigators specified
that an equal number of male and
female mice were used in the study,
but the number of animals used in
some analyses was not an even
number. Consequently, it is not
possible to determine sex composition
of the group and it suggests there may
have been unreported attrition
0 pg/dL for 0 ppm
4.7 ± 0.2 pg/dL for
125 ppm
41.3 pg/dL for 1250 ppm
WBC
subpopulations
BW = body weight; d = day; F = female; M = male; M/F = male/female; NR = not reported; Pb = lead; ppm = parts per million; WBC = white blood cell; wk = week.
"If applicable, reported values for BLL were converted to mg/dL using WebPlot Digitizer (https://apps.automeris.io/wpd/) and are shown in parenthesis.
6-65
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Table 6-13
Epidemiologic studies of exposure to Pb and sensitization and allergic response
Reference and
Study Design
Study Population
Exposure Assessment
Outcome Confounders
Effect Estimates
and 95% Clsa
tAshlev-Martin et al.
Maternal-Infant
Maternal/Cord Blood
IL-33, TSLP, and IgE Age
ORs per 10-fold
(2015)
Research on
increase in Pb
Environmental
Blood Pb was measured in whole
IL-33, TSLP, and IgE
Canada
Chemicals Study
blood using ICP-MS;
measured in cord blood
Elevated IL-33/TSLP
2008-2011
n: 1256
concentrations measured in the
plasma using a commercial
Cohort
Pregnant women
first and third trimester were
averaged to create an index of
antibody kit and ELISA.
0.72 (0.48, 0.95)
were recruited at
exposure throughout pregnancy
Age at Outcome:
Elevated IgE
<4 wk gestation.
Age at measurement:
At birth
Singleton non-pre-
First and third trimesters
0.98 (0.66, 1.3)
term births
Median: 0.62 pg/dL
Maximum: 4.14 pg/dL
Joseph et al. (2005) n: 4,634
Southeastern
Michigan
1994-1997
Enrollment
(Follow-up for 12 mo
after Pb screening)
Cohort
Children enrolled in
a managed care
organization.
Enrollment at 4 mo
to 3 yr
Blood
Blood Pb measured in venous
whole blood using GFAAS.
Age at measurement: 4 mo to 3 yr
Mean: 4.7 pg/dL (SD: 4.0)
Incident Asthma
Four or more asthma-
medication-dispensing
events in 12 mo or met one
or more of the following
within a 12-mo period:
emergency department visit
for asthma, hospitalization
for asthma, or four or more
outpatient visits for asthma
with at least two asthma-
medication-dispensing
events
Sex, birth weight, and
average annual income
available only at census
block level
HRs:
White children,
>5 vs. <5 pg/dL:
2.7 (0.9, 8.1)
Black children,
>10 vs. <5 pg/dL:
1.3 (0.6, 2.6)
6-66
-------
Reference and
Study Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates
(EEs) and 95% Clsa
tKimetal. (2019)
Cohort for
Childhood Origin of
Maternal/Cord Blood
Atopic Dermatitis and IL-13
Gender and parental
history of allergic diseases
EEs per unit increase
in In(Pb)
Seoul
Asthma and
Cord blood Pb measured using
IL-13 measured in cord
South Korea
Allergic Disease
ICP-MS
blood; diagnosis of atopic
HR Atopic Dermatitis
2007-2011
n: 331
Age at measurement:
dermatitis by pediatric
enrollment (at least
At birth
allergists, and atopic
0.96 (0.60, 1.53)
2 yr of follow-up)
Pregnant women
dermatitis scored using a
Cohort
enrolled in third
Median: 1.3 pg/dL
validated measure
In(SCORAD)
trimester, children
Maximum: 4.3 [jg/dL
(SCORAD)
followed at least
Atopic Dermatitis
2 yr
Age at Outcome:
At birth (IL-13), 6 mo, 12
mo, and 2 yr
Severity
1.11 (-2.65, 4.87)
IL-13 (pg/ml)
0.69 (0.11, 1.28)
+bKim et al. (2013)
Mothers' and
Children's
Maternal/Cord Blood
Atopic Dermatitis
Age, weight, history of
atopic disease, maternal
OR
South Korea
Environmental
Cord blood Pb measured using
Age at Outcome:
education, infant sex,
Atopic Dermatitis
2006-2009
Health Study
GFAAS
6 mo
family income, family size,
enrollment (follow-up
n: 637
Age at measurement: At birth
parity, duration of breast
1.05 (0.60, 1.81)
with infants 6 mo
feeding, passive smoking
after birth)
Singleton children
Mean: 1.01 pg/dL
during pregnancy, and
Cohort
of mothers enrolled
between weeks 12
and 28 of gestation
cord blood cadmium
tKimetal. (2016)
KNHANES
n: 2184
Blood
igE
Age, sex, urine cotinine,
mercury, and cadmium
% Change in Total
IgE (kU/L)
South Korea
Blood Pb was measured in
Serum total IgE (kU/L)
2010-2011
General population;
venous whole blood using GFAAS
measured by
Sensitization
Negative
Cross-Sectional
26-55 yr old
Age at measurement:
26-55 yr old
immunoradiometric assay
Age at Outcome:
3.5% (-1.8%, 9.4%)
Median: 2.14 pg/dL
26-55 yr old
Sensitization
75th: 2.82 pg/dL
Positive
10.4% (3.3%, 17.8%)
6-67
-------
Reference and
Study Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates
(EEs) and 95% Clsa
tMener et al, (2015)
NHANES
Blood
Immune System Effects
Age, sex, ethnicity, BMI,
ORs
n: 2,712 children;
exposure to tobacco
Increased sensitivity
United States
4,333 adults
Blood Pb was measured in
Food Allergen-Specific
smoke, asthma, musty
to food allergens
2005-2006
venous whole blood using ICP-MS
Serum IgE measured using
smell, presence of
Cross-Sectional
General population;
Age at measurement:
immunoassays
cockroaches, and
children 6-19 yr
>6 yr old
domestic animals living at
Children
old, adults >20 yr
Age at Outcome:
home, and year home
0.72 (0.48, 0.95)
old
Serum median:
>6 yr old
was built
Children: 0.87 [jg/dL;
Adults
Adults: 1.48 pg/dL
0.98 (0.66, 1.3)
75th:
Children: 1.31 pg/dL;
Adults: 2.34 pg/dL
6-68
-------
Reference and
Study Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates
(EEs) and 95% Clsa
tPesce et al. (2021)
Nancy and Poitier
France
2003-2006
Enrollment (Follow-
up to 8 yr of age)
Cohort
EDEN Birth Cohort
n: 651
Pregnant women
enrolled early in
pregnancy, children
followed through
8 yr of age
Maternal/Cord Blood
Maternal blood Pb measured
between 24 and 28 gestational
weeks using GFAAS; Cord blood
Pb measured at birth using
GFAAS
Age at measurement:
Prenatal
Mean:
Cord blood: 1.45 [jg/dL; Maternal
blood: 1.91 [jg/dL; Median: Cord
blood: 1.2 pg/dL; Maternal blood:
1.7 Mg/dL
Atopic Diseases
Parental questionnaires
using validated questions
from the International Study
on Asthma and Allergies in
Childhood
Age at Outcome:
4, 8, and 12 mo; 2,
and 5 yr; and 8 yr
3, 4,
Sex, Maternity Center,
BMI, maternal education,
parental smoking,
parental history of allergy,
maternal smoking in
pregnancy, birth weight,
gestational age at
delivery, type of delivery,
manganese, and
cadmium
ORs (Q4, Q1)
Maternal Blood
(>2.2 vs. <1.2):
Asthma
1.25 (0.71, 2.2)
Rhinitis
0.86 (0.51, 1.43)
Eczema
1.04 (0.73, 1.48)
Food Allergy
1.02 (0.51, 2.01)
75th:
Cord blood: 1.8 pg/dL;
Maternal blood: 2.2 [jg/dL
Cord Blood
(>1.8 vs. <0.9):
Asthma
0.74 (0.41, 1.33)
Rhinitis
0.64 (0.37, 1.11)
Eczema
1.35 (0.92, 1.98)
Food Allergy
0.57 (0.25, 1.34)
6-69
-------
Reference and
Study Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates
(EEs) and 95% Clsa
Puqh Smith and
Nriaqu (2011)
Saginaw, Ml
Cross-sectional
n: 356
Blood
Children residing in Blood Pb measured in venous
low-income and whole blood
minority
households
identified by the
Statewide Systemic
Tracking of
Elevated Lead
Levels and
Remediation
(STELLAR)
database
Prevalent Asthma
Parental report of asthma
diagnosis
Age, sex, income, stories
in unit, pet ownership,
cockroach problem,
persons in home, smoker
in home, clutter, highest
BLL at address, candles
or incense, months of
residency, housing tenure,
stove type, heating
source, air conditioning
type, peeling paint,
ceiling/wall damage,
housing age, water
dampness
OR
(>10 vs. <10 [jg/dL)
Asthma
7.5 (1.3, 42.9)
Rabinowitz et al.
Boston, MA
Enrollment: 1979-
1981
Follow-up: Unclear
Cohort
n: 159
Mother infant pairs
recruited from
Boston Hospital for
Women
Cord Blood
Cord blood Pb measured in
samples at birth using anodic
stripping voltammetry
Prevalent Eczema and
Asthma
Eczema and asthma
prevalence evaluated via
parental questionnaire
N/A
OR
(>10, vs. <10[jg/dL)
Eczema
1.0 (0.6, 1.6)
Asthma
1.3 (0.8, 2.0)
6-70
-------
Reference and
Study Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates
(EEs) and 95% Clsa
tTsuii eta!. (2019)
Japan
2011-2014
Cross-sectional
Japan Environment Blood, Maternal/Cord Blood
and Children's
Study
n: 14408
Blood Pb measure using ICP-MS
Age at measurement:
Second/third trimester
Mean: 6.44 ng/g
Q1: <4.79 ng/g
Q4: >7.42 ng/g
Allergen-Specific IgE
Allergen-specific serum IgE
measured using
immunological assays
Age at Outcome:
First trimester
Age, BMI, allergic
diseases, smoking during
pregnancy, smoking
habits of partner, alcohol
consumption during
pregnancy, pet ownership,
month of blood sample,
and geographic region
ORs (Q4, Q1)
House Dust Mite
Sensitization
0.91 (0.83, 1.01)
Japanese Cedar
Pollen Sensitization
1.04 (0.94, 1.15)
Animal Dander
Sensitization
0.99 (0.88, 1.12)
tWeietal. (2019)
United States
2005-2006
Cross-Sectional
NHANES
n: 4509
General population;
all ages
Blood
Eczema
Blood Pb was measured in Self-reported physician's
venous whole blood using ICP-MS diagnosis of eczema
Age at measurement:
>1 yr old Age at Outcome:
>1 yr old
Mean:
Ages >20: 1.75 [jg/dL;
<20: 1.24 [jg/dL
Adults
T1
0.18-1.09
pg/dL
T2
1.09-1.99
pg/dL
T3
2.00-26.4
pg/dL
Children
T1
0.18-0.77
pg/dL
T2
0.78-1.36
pg/dL
T3
1.37-55.3
pg/dL
Age, gender, ethnicity, ORs
education, poverty-income
ratio, smoking, alcohol
use, sleep, and BMI
T1: Reference
Eczema - Adults:
T2
T3
1.14 (0.75, 1.76)
1.09 (0.62, 1.92)
Eczema - Children:
T1
T2
T3
Reference
0.99 (0.62, 1.58)
0.90 (0.60, 1.35)
6-71
-------
Reference and
Study Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates
(EEs) and 95% Clsa
tWells et al.
United States
2005-2006
Cross-Sectional
NHANES
n: 1788
General population;
children 2-12 yrold
Blood
Blood Pb was measured in
venous whole blood using ICP-MS
Age at measurement:
2-12 yrold
Geometric Mean: 1.13 pg/dL
Immune System Effects
Serum total IgE),
Eosinophils (WBC
differential from complete
blood counts), Asthma
(parental/guardian
reported), Atopy (at least
one specific IgE >0.35
kU/L), Allergies
(parental/guardian
reported)
Age at Outcome:
2-12 yrold
Season, age, sex,
race/ethnicity, parental
education, presence of
smokers in the home,
prenatal smoke exposure,
BMI, presence of
cockroaches in the home,
and avoidance/removal of
pets
ORs
Asthma
1.01 (0.76,
Atopy
1.05 (0.93,
1.35)
1.18)
% Increase
Total IgE (kU/L)
10.3% (3.5%, 17.5%)
Percent Eosinophils
4.6% (2.4%, 6.8%)
BLL = blood lead level; BMI = body mass index; CI = confidence interval; EDEN = Effect of Diet and Exercise on Immunotherapy and the Microbiome; ELISA = enzyme-linked
immunosorbent assay; EEs = effect estimates; GFAAS = graphite furnace atomic absorption spectrometry; HR = hazard ratio; ICP-MS = inductively coupled plasma mass
spectrometry; Ig- = immunoglobulin type; IL- = interleukin type; KNHANES = Korea National Health and Nutrition Examination Survey; In = natural log; mo = month(s); N/A = not
applicable; NHANES = National Health and Nutrition Examination Survey; NR = not reported; OR = odds ratio; Pb = lead; Q = quartile; SCORAD = scoring atopic dermatitis;
SD = standard deviation; SES = socioeconomic status; T# = fertile #; TSLP = thymic stromal lymphopoietin; vs. = versus; WBC = white blood cell; wk = week; yr = year.
aEffect estimates are standardized to a 1 |jg/dL increase in blood Pb level or a 10 |jg/g increase in bone Pb level, unless otherwise noted. For studies that report results
corresponding to a change in log-transformed Pb biomarkers, effect estimates are assumed to be linear within the 10th to 90th percentile interval of the biomarker and standardized
accordingly.
fStudies published since the 2013 Pb ISA.
6-72
-------
Table 6-14
Animal toxicological studies of immediate-type hypersensitivity
Study
Species (Stock/Strain), Timing of Exposure Exposure Details BLL as Reported (Mg/dLf
n, Sex M (Concentration, Duration) K VMM '
Endpoints Examined
Cai et al.
Rat (Sprague Dawley)
Control (vehicle), M/F,
n = 5
0.2% Pb, M/F, n = 5
8-10 wk to 20-30 wk
Rats were 8-10 wk old
when acquired. Whether or
not the rats were allowed to
acclimate to the facility prior
to study initiation was not
reported. The number of
males and females not
reported.
Control animals received tap
water
20.5 ± 0.68 [jg/L for 0%
(2.2 ± 6.4 [jg/dL)
87.4 ± 9.2 |jg/L for 0.2%
(9.3 ± 0.98 [jg/dL)
Blood cytokine levels
Fang et al.
Rat (Sprague Dawley)
Control (vehicle), M,
n =20
300 ppm Pb, M, n = 20
23-25 d to 65-67 d Dosing solutions were
changed twice per week
4.48 |jg/dL for 0 ppm
18.48 [jg/dL for 300 ppm-d 65-
67
Blood cytokine levels
BLL = blood lead level; d = day(s); M = male; M/F = male/female; ppm = parts per million; wk = week(s).
"If applicable, reported values for BLL were converted to mg/dL using WebPlot Digitizer (https://apps.automeris.io/wpd/) and are shown in parenthesis.
6-73
-------
Table 6-15 Epidemiologic studies of exposure to Pb and autoimmunity and autoimmune disease
Reference and
Study Design
Study Population
Exposure Assessment
Outcome
Confounders
Effect Estimates and
95% Clsa
tJoo et al, (2019)
KNHANES
n: 32215
Blood
Rheumatoid Arthritis
Age, sex, SES, and
smoking status
Rheumatoid Arthritis
(OR)
South Korea
Blood Pb was measured
Self-reported physician
2008-2013
General population
in venous whole blood
diagnosis of rheumatoid
1.01 (0.89, 1.14)
Cross-sectional
using GFAAS
Age at measurement:
All ages
Mean: Rheumatoid
Arthritis: 2.38 |jg/dL;
Control: 2.44 |jg/dL
arthritis
Age at Outcome:
All ages
tKamycheva et al.
NHANES
Blood
Celiac Disease
Family income to poverty
Celiac Disease
(2017)
n: 3,643 children and 11,040
Seropositivity
ratio and race/ethnicity
Mean difference in BLL
adults
Blood Pb was measured
by CD seropositivity
status
United States
in venous whole blood
Serum tTG-lgA analyzed
2009-2012
General population, >6 yr old
using ICP-MS
with an ELISA
Cross-sectional
Age at measurement:
>6 yr
Age at Outcome:
Adults
>6 yr
-0.17 |jg/dL (-0.54, 0.20)
Mean: Non-Hispanic
white: 1.39 |jg/dL; other
race/ethnicity:
Children
1.47 |jg/dL
-0.14 |jg/dL (-0.27,
-0.02)
BLL = blood lead level; CD = cluster of differentiation; CI = confidence interval; ELISA = enzyme-linked immunosorbent assay; Ig- = immunoglobulin type; GFAAS = graphite furnace
atomic absorption spectrometry; ICP-MS = inductively coupled plasma mass spectrometry; KNHANES = Korea National Health and Nutrition Examination Survey;
NHANES = National Health and Nutrition Examination Survey; OR = odds ratio; Pb = lead; SES = socioeconomic status; tTG-lgA = tissue transglutaminase immunoglobulin A;
yr = year(s)
aEffect estimates are standardized to a 1 |jg/dL increase in blood Pb level or a 10 |jg/g increase in bone Pb level, unless otherwise noted. For studies that report results
corresponding to a change in log-transformed Pb biomarkers, effect estimates are assumed to be linear within the 10th to 90th percentile interval of the biomarker and standardized
accordingly.
fStudies published since the 2013 Pb ISA.
6-74
-------
Table 6-16
Animal toxicological studies of autoimmunity and autoimmune disease
Study
Species (Stock/Strain), Tjmjng of Exposure
Exposure Details
(Concentration, Duration)
BLL as Reported (|jg/dL)a
Endpoints Examined
Fanq et al,
(2012)
Rat (Sprague Dawley) 23-25 d to 65-67 d
Control (vehicle), M,
n =20
300 ppm Pb, M, n = 20
Dosing solutions were
changed twice per week
4.48 |jg/dL for 0 ppm
18.48 |jg/dL for 300 ppm-
d 65-67
Treg cell suppression assay
BLL = blood lead level; d = day; M = male; ppm = parts per million; Treg = regulatory T cell.
"If applicable, reported values for BLL were converted to mg/dL using WebPlot Digitizer (https://apps.automeris.io/wpd/) and are shown in parenthesis.
6-75
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