United States Environmental Protection Jf lkAgency EPA/600/R-23/061 March 2023 www.epa.gov/isa Integrated Science Assessment for Lead Appendix 7: Hematological Effects External Review Draft March 2023 Health and Environmental Effects Assessment Division Center for Public Health and Environmental Assessment Office of Research and Development U.S. Environmental Protection Agency External Review Draft DRAFT: Do not cite or quote ------- DISCLAIMER This document is an external review draft for peer review purposes only. This information is distributed solely for the purpose of predissemination peer review under applicable information quality guidelines. It has not been formally disseminated by the Environmental Protection Agency. It does not represent and should not be construed to represent any agency determination or policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. External Review Draft 7-ii DRAFT: Do not cite or quote ------- DOCUMENT GUIDE This Document Guide is intended to orient readers to the organization of the Lead (Pb) Integrated Science Assessment (ISA) in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 ://cfpub. epa. gov/ncea/isa/recordisplav .cfm?deid=3 57282. Front Matter Executive Summary Integrative 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 Process for Developing the Pb Integrated Science Assessment External Review Draft 7-iii DRAFT: Do not cite or quote ------- CONTENTS LIST OF TABLES 7-v LIST OF FIGURES 7-vi ACRONYMS AND ABBREVIATIONS 7-vii APPENDIX 7 HEMATOLOGICAL EFFECTS 7-1 7.1 Introduction, Summary of the 2013 Integrated Science Assessment, and Scope of the Current Review 7-1 7.1.1. Red Blood Cell Survival and Function 7-2 7.1.2. Heme Synthesis 7-2 7.2 Scope 7-3 7.3 Red Blood Cell Survival and Function 7-5 7.3.1. Epidemiologic Studies of Red Blood Cell Survival and Function 7-5 7.3.2. Toxicological Studies of Red Blood Cell Survival and Function 7-7 7.3.3. Integrated Summary of Red Blood Cell Survival and Function 7-9 7.4 Heme Synthesis 7-9 7.4.1. Epidemiologic Studies of Heme Synthesis 7-10 7.4.2. Toxicological Studies of Heme Synthesis 7-10 7.4.3. Integrated Summary of Heme Synthesis 7-11 7.5 Biological Plausibility 7-11 7.5.1. Decreased Red Blood Cell Survival and Function 7-13 7.5.2. Altered Heme Synthesis 7-14 7.6 Summary and Causality Determination 7-16 7.6.1. Causality Determination for Red Blood Cell Survival and Function 7-16 7.6.2. Red Blood Cell Survival and Function 7-16 7.6.3. Heme Synthesis 7-17 7.6.4. Causality Determination 7-18 7.7 Evidence Inventories—Data Tables to Summarize Study Details 7-24 7.8 References 7-32 External Review Draft 7-iv DRAFT: Do not cite or quote ------- LIST OF TABLES Table 7-1 Summary of evidence indicating a causal relationship between Pb exposure and hematological effects 7-19 Table 7-2 Epidemiologic studies of exposure to Pb and hematological effects 7-24 Table 7-3 Animal toxicological studies of Pb exposure and hematological effects 7-29 External Review Draft 7-v DRAFT: Do not cite or quote ------- LIST OF FIGURES Figure 7-1 Potential biological plausibility pathways for hematological effects associated with exposure to Pb 7-12 External Review Draft 7-vi DRAFT: Do not cite or quote ------- ACRONYMS AND ABBREVIATIONS ALAD 8-aminolevulinate dehydratase ALA aminolevulinic acid AQCD Air Quality Criteria Document BLL blood lead level BMI body mass index BW body weight Ca2+ calcium CAT catalase CI confidence interval e-waste electronic waste EPA Environmental Protection Agency EPO erythropoietin Fe2+ iron ion GFAAS graphite furnace atomic absorption spectroscopy GPx glutathione peroxidase GR glutathione reductase GSH glutathione GSSG glutathione disulfide Elb hemoglobin Hct hematocrit HSC hematopoietic stem cell OH hydroxyl radical h hour, hours H202 hydrogen peroxide ICP-MS inductively coupled plasma mass spectrometry KNHANES Korean National Health and Nutrition Examination Survey In natural logarithm M male M/F male/female MCH mean corpuscular hemoglobin MCHC mean corpuscular hemoglobin concentration MCV mean corpuscular volume MDA malondialdehyde Mg2+ magnesium ion mo month, months NAAQS National Ambient Air Quality Standards NCE normochromatic red blood cell NR not reported OR odds ratio Pb lead PCE polychromatic red blood cells NCE normochromatic red blood cells PCV packed cell volume PECOS Population, Exposure, Comparison, Outcome, and Study Design Pit platelets PND postnatal day PS phosphatidylserine Q quartile RBC red blood cell RDW red blood cell distribution width ROS reactive oxygen species SD Sprague Dawley SES socioeconomic status SOD superoxide dismutase O2- superoxide wk week, weeks yr year, years Zn zinc ZPP zinc-protoporphyrin External Review Draft 7-vii DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 APPENDIX 7 HEMATOLOGICAL EFFECTS Causality Determination for Pb Exposure and Hematological Effects This appendix characterizes the scientific evidence that supports the causality determination for lead (Pb) exposure and hematological 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 determination: Outcome Causality Determination Altered Heme Synthesis and Decreased Red Blood Causal Cell Survival and Function The Executive Summary, Integrated Synthesis, and all other appendices of this Pb ISA can be found at https://cfpub.epa.gov/ncea/isa/recordisplav.cfm?deid=357282. 7.1 Introduction, Summary of the 2013 Integrated Science Assessment, and Scope of the Current Review Hematology is a subgroup of clinical pathology concerned with the morphology, physiology, and pathology of blood and blood-forming tissues. Hematological measures, when evaluated with information on other biomarkers, are informative diagnostic tests for blood-forming tissues (i.e., bone marrow, spleen, and liver) and organ function. The 2013 Integrated Science Assessment for Lead (hereinafter referred to as the 2013 Pb ISA) issued causality determinations for hematological effects resulting from lead (Pb) exposure on red blood cell (RBC) survival and function and altered heme synthesis (U.S. EPA 2013). The evidence underpinning these causality determinations is summarized below. Given the interconnectedness of the effects of Pb on RBC survival and function and altered heme synthesis, this assessment presents a single causality determination for Pb exposure and hematological effects. This approach allows for a more holistic evaluation of inter-related health endpoints, including a discussion of how all individual lines of evidence contribute to the overall hematological effects causality determination. External Review Draft 7-1 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 7.1.1. Red Blood Cell Survival and Function The body of epidemiologic and toxicological evidence assessed in the 2013 Pb ISA indicates a "causal" relationship between Pb exposure and decreased RBC survival and function. Experimental animal studies demonstrate that relevant human blood Pb levels (BLLs) from oral and inhalation exposure alter several hematological parameters (e.g., RBC number), increase measures of oxidative stress (e.g., inhibition of antioxidant enzymes in RBCs), and increase cytotoxicity in RBC precursor cells. Some of these effects have been observed in animal toxicological studies with exposures resulting in BLLs of 2- 7 (ig/dL. Evidence of biologically plausible modes of action, including increased intracellular calcium 2+ concentrations [Ca ], decreased Ca2+/Mg2+ ATPase activity, and increased phosphatidylserine (PS) exposure leading to RBC destruction by macrophages, support these findings. Epidemiologic studies reported associations between exposure to Pb, BLL, and altered hematological endpoints, increased measures of oxidative stress, and altered hematopoiesis in adults and children. Although most of these studies are limited by their lack of rigorous methodology (i.e., correlations, t tests, or chi squared analyses), some studies in children did adjust for potential confounding factors, including age, sex, mouthing behavior, anemia, dairy product consumption, maternal age, education, employment, marital status, family structure, and socioeconomic status (SES)-related variables. Though limited in number, studies that adjusted for confounders also reported consistent associations between BLL and altered hematological parameters, strengthening their support for findings in experimental animals. Collectively, the strong evidence from toxicological studies supported by findings from mode of action and epidemiologic studies reviewed in the 2013 Pb ISA was sufficient to conclude that there is a causal relationship between Pb exposures and decreased RBC survival and function. 7.1.2. Heme Synthesis Available toxicological evidence evaluated in the 2013 Pb ISA indicated a causal relationship between Pb exposure and altered heme synthesis (U.S. EPA 2013). Altered heme synthesis is demonstrated by a small but consistent body of studies in adult animals, which report that exposures that result in BLLs relevant to humans (e.g., 10 (ig/dL) lead to decreased 5-aminolevulinate dehydratase (ALAD) and ferrochelatase activities. Supporting this evidence is a larger body of ecotoxicological studies that demonstrate decreased ALAD activity across a wide range of taxa exposed to Pb. Evidence of biologically plausible modes of action, including evidence that Pb acts directly on two enzymes involved in heme synthesis (ALAD and ferrochelatase), decreased RBC Hb concentration, measures of oxidative stress, and evidence that administration of antioxidants reduced the effects of Pb exposure on antioxidant enzymes, support these findings. Epidemiologic studies find associations in both adults and children between higher BLLs and decreased ALAD and ferrochelatase activities. Although most of these studies are limited by their lack of rigorous methodology and consideration of potential confounders, some studies in children did incorporate potential confounding factors (i.e., age, sex, urban/rural residence, External Review Draft 7-2 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 height, weight, BMI [body mass index]). Although limited in number, studies that adjusted for confounders also reported consistent associations between BLLs and decreased ALAD and ferrochelatase, strengthening support for the findings in the animal toxicological studies. Evidence for altered heme synthesis is also provided by a large body of toxicological and epidemiologic studies that report decreased hemoglobin (Hb) concentrations in association with Pb exposure or BLL. The 2013 Pb ISA concluded that, collectively, the strong evidence from toxicological and ecotoxicological studies—supported by findings from epidemiologic studies—is sufficient to conclude a causal relationship between Pb exposures and altered heme synthesis. This ISA determined causality for adverse effects of Pb exposure on altered heme Synthesis and decreased red blood cell survival and function. Recent evidence demonstrates that Pb exposures alter several hematological parameters, decrease enzyme activity related to heme synthesis, and increase RBC oxidative stress. Biological plausibility is provided by toxicological and epidemiologic studies demonstrating increased intracellular calcium concentrations, decreased Ca2+/Mg2+ ATPase activity, and increased PS exposure. Taken together, there is sufficient evidence to conclude that there is a causal relationship between Pb exposure and hematological effects, including altered heme synthesis and decreased RBC survival and function. The following sections provide an overview of the scope of the appendix (Section 7.2), evaluation of the scientific evidence relating Pb exposures and hematological effects (Sections 7.3 and 7.4), a discussion of biological plausibility (Section 7.5), and a summary section with the updated causality determination (Section 7.6, Table 7-1). The focus of these sections is on studies published since the completion of the 2013 Pb ISA (U.S. EPA 2013). Study-specific details, including animal 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 7.7. 7.2 Scope The scope of this section 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 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, 1 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). External Review Draft 7-3 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 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 hematological effects, 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 Pb1 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 exposure2; 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: Hematological effects including but not limited to disruption of heme synthesis and RBC survival and function; and 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 mg/dL or below;3,4 Comparators: A concurrent control group exposed to vehicle-only treatment or untreated control; 1 Recent studies of occupational exposure to Pb were only 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). 2 Studies 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 limited, it is difficult to assess the representativeness of these concentrations to population exposure [Section 2.5.3 (U.S. EPA 201311. Moreover, data illustrating the relationships of Pb-PMio and Pb-PM2 5 with BLLs are lacking. 3 Pb mixture studies are included if they employ an experimental arm that involves exposure to Pb alone. 4 This level is approximately 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 20191 and the proportion of individuals with BLL 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. External Review Draft 7-4 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Outcome: Hematological effects; and Study design: Controlled exposure studies of animals in vivo. 7.3 Red Blood Cell Survival and Function Toxicological and epidemiologic studies evaluated in the 2013 Pb ISA (U.S. EPA 2013) provided strong evidence that exposure to Pb affects a range of hematological outcomes related to RBC survival and function; which is consistent with epidemiologic evidence from the 2006 Pb Air Quality Criteria Document (AQCD) (U.S. EPA 2006b). demonstrating an association between high BLLs and anemia in children. Given the extensive evidence base at higher BLLs, the scope for this appendix focuses on toxicological studies conducted at lower exposure levels and epidemiologic studies in nonoccupational populations (as described in Section 7.2). Under the defined PECOS criteria, recent toxicological and epidemiologic studies provide additional support for Pb-related changes in Hb concentration and some other hematological measures of RBC survival and function. Below, recent evidence is reviewed in the context of evidence from past assessments. 7.3.1. Epidemiologic Studies of Red Blood Cell Survival and Function The epidemiologic evidence evaluated in the 2013 Pb ISA (U.S. EPA 2013) covered a range of measures related to RBC survival and function, including RBC counts and other hematological parameters, hematopoiesis, Ca2+/Mg2+ ATPase activity, PS exposure, and RBC oxidative stress. Specifically, epidemiologic studies provided evidence that elevated BLLs in children and adults are associated with altered hematological parameters (e.g., decreased RBC counts, Hb concentration, and hematocrit [Hct] and changes in mean corpuscular volume [MCV] and mean corpuscular hemoglobin [MCH]), increased measures of oxidative stress (e.g., altered antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)], decreased cellular glutathione (GSH), and increased lipid peroxidation), and altered hematopoiesis (e.g., decreased erythropoietin [EPO]). Notably, most of these epidemiologic studies are cross-sectional in design and conducted either in occupationally exposed populations or other populations with higher mean Pb exposures (i.e., BLLs >10 |J.g/dL). These studies were additionally limited by their lack of consideration of potential confounders, although some studies in children did adjust for a range of factors, including age, sex, mouthing behavior, anemia, dairy product consumption, maternal age, education, employment, marital status, family structure, and SES-related variables (Queirolo et al. 2010; Ahamed et al. 2007; Riddell et al. 2007). Studies that did account for potential confounders reported consistent associations between increased BLLs and decreased Hb levels, increased prevalence of anemia, and increased RBC oxidative stress (Sections 4.7.2.1 and 4.7.2.7 of the 2013 Pb ISA). As a whole, the cross-sectional study designs, higher exposures, and lack of rigorous statistical methodologies in many studies raise uncertainties in the epidemiologic evidence regarding the directionality of effects; the level, timing, frequency, and duration External Review Draft 7-5 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 of Pb exposure that contributed to the observed associations; and whether the observed associations are independent of potential confounders. Recent epidemiologic studies provide generally consistent evidence of associations between Pb exposures and decreased Hb levels in children. These associations are reported at lower BLLs than in studies included in the 2013 Pb ISA. Evidence for associations with other hematological parameters of RBC function and survival, as well as Hb levels in adults, is less robust. Consistent with the 2013 Pb ISA, most recent studies are cross-sectional analyses, which are unable to establish temporality between exposure and outcome. However, recent studies include populations with lower mean BLLs and more robust adjustment for potential confounders compared with studies included in the 2013 Pb ISA. 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 7-2. An overview of the recent evidence is provided below. The most common hematological parameter evaluated in recent epidemiologic studies is Hb levels. A number of cross-sectional studies of children in China observed decreases in Hb levels associated with increases in blood Pb or erythrocyte Pb levels (Guo et al. 2021; Kuang et al. 2020; Li et al. 2018; Liu et al. 2015; Liu et al. 2012). Kuang et al. (2020) and Li et al. (2018) also reported inverse associations between BLLs and MCH in children, which is a measure of average Hb concentration in a single erythrocyte. Notably, all of these studies had mean and/or median BLLs below 10 |ag/dL, including some below 5 (ig/dL (Guo et al. 2021; Kuang et al. 2020; Liu et al. 2012). While only a few studies attempted to account for potential confounding by SES (Kuang et al. 2020; Liu et al. 2015). others adjusted directly for iron deficiency (Li et al. 2018; Liu et al. 2012). which may be the direct mechanism by which SES could potentially confound the relationship between BLLs and Hb (i.e., via nutritional deficiency). Although the magnitude of the observed effect estimates was not directly comparable across studies, BLLs were negatively associated with Hb levels in all studies. Notably, Liu et al. (2012) reported that a 1 (ig/dL increase in BLLs was associated with a larger decrease in Hb levels when restricting their sample to children with BLLs less than 10 (ig/dL (-0.174 g/dL [95% confidence interval (CI): -0.27, -0.078 g/dL]) compared with the full sample (-0.096 g/dL [95% CI: -0.18, -0.012 g/dL]). In studies that examined quantiles of exposure, blood or erythrocyte Pb levels were only associated with Hb levels at the higher quantiles (Guo et al. 2021; Liu et al. 2015). For example, in a large hospital-based study in China, children with BLLs between 3.33 and 4.50 (ig/dL (quintile 4) and those with levels greater than 4.50 (ig/dL (quintile 5) had decreases in Hb relative to children with BLLs less than 1.61 (ig/dL (-0.49 g/L [95% CI: -0.94, -0.04 g/L] and -1.25 g/L [95% CI: -1.71, -0.78 g/L], respectively); however, null associations were reported for quintiles 2 (1.61-2.44 |a,g/dL) and 3 (2.44-3.33 (ig/dL) relative to the lowest quintile of exposure (Guo et al. 2021). While the clinical relevance of small mean decrements in Hb across exposure quintiles is unclear, Guo et al. (2021) also reported monotonic increases in the odds of anemia (defined as Hb levels below 110 g/L) in association with increasing blood Pb quintiles, with a 45% (95% CI: 26%, 67%) increase in the odds of anemia for children in the highest External Review Draft 7-6 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 quintile of exposure relative to the lowest quintile. Similarly, Li et al. (2018) observed a 5% (95% CI: 0%, 11%) increase in the odds of decreased Hb levels (<115 g/L) per 1 ng/dL increase in blood Pb. Recent studies of Pb exposure and Hb levels in adults are more limited in number and include overlapping study populations. In contrast to studies in children, Park and Lee (2013) reported increases in Hb associated with continuous increases in BLLs for adult participants of the 2008—2010 cycles of the Korean National Health and Nutrition Examination Survey (KNHANES). The analysis was stratified by sex and the observed associations were comparable for men and women. A similar study analyzed the same KNHANES cycles, but examined the population as a whole, rather than stratified by sex, and also categorized exposure into quartiles (Kim and Lee 2013). The authors noted similar positive associations between blood Pb and Hb levels. However, Kim and Lee (2013) observed inverse associations across exposure quartiles when correcting BLLs for Hct in order to estimate erythrocyte Pb. As described in the 2006 Pb AQCD (U.S. EPA 2006b). Pb exposure decreases Hct and MCV, meaning the negative effects of Pb can potentially decrease Pb levels in whole blood. In addition to studies examining the relationship between Pb exposure and Hb levels, a few recent cross-sectional studies evaluate associations between BLLs and other hematological parameters. In a group of children in China, including some living near a battery plant or a lead/-zinc mine, Li et al. (2018) reported increased odds of low RBC counts and low blood platelets (Pit) in association with increased BLLs. In contrast, Kuang et al. (2020) observed a positive association with increased RBC counts in a convenience sample of slightly older boys in Nanjing, China, with notably lower median BLLs (2.61 (ig/dL compared with 8.38 (ig/dL). In an adult population, a cohort of pregnant women in Durango, Mexico, La-Llave-Leon et al. (2015) also observed a positive cross-sectional association between RBC counts and BLLs. Given this small body of studies that examine diverse populations with varying BLLs, it is difficult to discern methodological or demographic factors contributing to the inconsistent results. 7.3.2. Toxicological Studies of Red Blood Cell Survival and Function As previously reported in the 2013 Pb ISA, epidemiologic evidence is coherent with experimental animal studies demonstrating that exposures via drinking water and oral gavage resulting in BLLs relevant to what was found in humans affect multiple hematological outcomes related to RBC survival and function (U.S. EPA 2013). Specifically, exposure to Pb has been shown to decrease RBC survival, either through direct effects on RBC membranes leading to increased fragility or through the induction of eryptosis and eventual phagocytosis by macrophages (U.S. EPA 2013). Some of these effects have been observed in animal toxicological studies with exposures resulting in 2-7 (ig/dL BLL. For example, Hb concentrations in plasma was significantly decreased in male mice exposed to Pb nitrate (50 mg/kg BW in drinking water for 40 days; BLL: 1.72 ± 0.02 (ig/dL) (Sharma et al. 2010). BLLs >100 (ig/dL were also associated with decreased RBC survival in laboratory animals. External Review Draft 7-7 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 The evidence is limited and conflicting for the observed effects of Pb exposure on hematopoiesis in rats and mice. For example, administration of Pb acetate (140, 250, or 500 mg/kg) via oral gavage once per week for 10 weeks decreased the number of polychromatic RBCs (PCE) and increased numbers of micronucleated PCEs in female rats (Celik et al. 2005). Increased micronucleated PCEs were reported in female and male rats exposed to Pb acetate in drinking water for 125 days, but decreased PCEs/normochromatic RBCs (NCEs) ratio was only observed in male rats (Alghazal et al. 2008). However, in mice exposed to Pb acetate (1 g/L in drinking water for 90 days), PCE increased, but PCE/NCE was unaffected (Marques et al. 2006). In addition, the 2013 Pb ISA reported that Pb exposure significantly decreases several hematological parameters. In studies reporting BLL relevant to this ISA, decreased RBC counts (Andiclkovic et al. 2019; Cai et al. 2018; Sharma et al. 2010). Hb concentration (Andiclkovic et al. 2019; Cai et al. 2018; Berrahal etal. 2011; Sharma etal. 2010; Baranowska-Bosiacka et al. 2009; Masso- Gonzalez and Antonio-Garcia 2009). and Hct (Andiclkovic et al. 2019; Masso-Gonzalez and Antonio- Garcia 2009; Masso et al. 2007) were reported in laboratory studies conducted in rats. In other studies in which BLLs were not reported, decreased RBC counts (Simsek et al. 2009; Marques et al. 2006; Lee et al. 2005). Hb (Wang et al. 2010b; Simsek et al. 2009; Lee et al. 2005). Hct (Molina etal. 2011; Marques et al. 2006; Lee et al. 2005). MCV (Wang et al. 2010b). MCH (Wang et al. 2010b; Simsek et al. 2009). and mean corpuscular hemoglobin concentration (MCHC) (Wang et al. 2010b; Simsek et al. 2009) were reported in laboratory studies conducted in rats and mice. Some toxicological studies found no evidence of hematological effects (Gautam and Flora 2010; Lee et al. 2006). Recent studies also report effects of Pb exposure on hematological parameters at BLLs relevant to this ISA. Administration of Pb acetate in drinking water (0.2%; BLL = 9.3 ± 0.98 (ig/dL) for 84 days resulted in RBC hemolysis, and significantly decreased RBC lifespan and number, and Hb levels, but had no effect on Pit number in blood collected from Sprague Dawley rats (Cai et al. 2018). In a different study, administration of Pb acetate in drinking water (0.150 mg/kg; BLL = 14.7 |ag/dL) for 1 day decreased RBCs, Pits, Hb concentration, and Hct, but had no effect on MCV, MCH an MCHC in whole blood collected from adult male Wistar rats (Andielkovic et al. 2019). Pb acetate treatment increased Hct and RBC distribution width, decreased MCHC, and had no effect on RBC number, Hb, MCV, and MCH in adult male C57BJ mice exposed via drinking water (200 ppm; BLL = 21.6 (ig/dL) for 45 days (Corsetti et al. 2017). The potential effects of Pb exposure in rats were also investigated through a combination of lactational and drinking water exposures. Beginning on postnatal day (PND) 1, dams were given drinking water containing Pb acetate (50 mg/L). On PND 21, male pups were subsequently administered Pb acetate (50 mg/L) in drinking water for an additional 40 or 65 days, at which time they were sacrificed. Hct was significantly reduced in mice exposed until PND 40 (BLL = 12.67 ± 1.68 (ig/dL) whereas Hct and Hb were significantly decreased at PND 65 (BLL = 7.49 ± 0.78 ug/dL) (Berrahal et al. 2011). Study- specific details, including animal species, strain, sex, and BLLs are highlighted in Table 7-3. External Review Draft 7-8 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 7.3.3. Integrated Summary of Red Blood Cell Survival and Function Experimental animal studies evaluated in the 2013 Pb ISA (U.S. EPA 2013) demonstrate that Pb exposures resulting in BLLs relevant to humans (i.e., <10 (ig/dL) alter several hematological parameters, increase measures of oxidative stress, and increase cytotoxicity in RBC precursor cells. While epidemiologic evidence synthesized in the last review was generally coherent with results from the animal studies, most of the epidemiologic studies evaluated are cross-sectional, were conducted in populations with higher mean Pb exposures (i.e., BLLs >10 (.ig/dL). did not thoroughly consider potential confounders, and lacked rigorous statistical methodology. As a result, there were considerable uncertainties in the epidemiologic evidence regarding the directionality of effects; the level, timing, frequency, and duration of Pb exposure that contributed to the observed associations; and whether the observed associations are independent of potential confounders. Though limited in number, recent PECOS-relevant animal toxicological studies continue to support the findings from the last review. Specifically, these studies consistently report the effects of Pb on hematological parameters, including mostly consistent evidence of a Pb-related decrease in Hb. Recent epidemiologic studies expand on the evidence presented in the 2013 Pb ISA and provide additional support for the experimental evidence. Although the recent studies are also cross-sectional, they include populations with much lower BLL means (<10 (ig/dL) and include more robust adjustment for potential confounding, addressing important uncertainties from the last review. The most consistent epidemiologic evidence indicates an association between BLLs and decreased Hb levels in children, which is coherent with the evidence from recent experimental animal studies. While the clinical relevance of small mean decrements in Hb is unclear, a few of the recent epidemiologic studies include analyses linking increased BLLs to increased prevalence of anemia. Recent epidemiologic studies of Hb levels in adults were more limited in number and less consistent than those in children. Additionally, of the relatively few studies examining RBC counts, the results were also inconsistent. 7.4 Heme Synthesis Toxicological and ecotoxicological studies evaluated in the 2006 Pb AQCD (U.S. EPA 2006b) and the 2013 Pb ISA (U.S. EPA 2013) provided strong evidence that exposure to Pb affects heme synthesis. A limited number of epidemiologic studies contributed compelling supporting evidence. Given the extensive evidence base at higher BLLs, the scope for this appendix focuses on toxicological studies conducted at lower exposure levels and epidemiologic studies in nonoccupational populations (as described in Section 7.2). Below, recent evidence is reviewed in the context of evidence from past assessments. External Review Draft 7-9 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 7.4.1. Epidemiologic Studies of Heme Synthesis The epidemiologic studies evaluated in the 2013 Pb ISA (U.S. EPA 2013) provided evidence that BLLs in children and adults are associated with decreased activity of enzymes involved in the heme synthesis pathway, including ALAD and ferrochelatase. Similar to studies of RBC function and survival, most studies on heme synthesis are cross-sectional in design and conducted either in occupationally exposed populations and/or in populations with higher mean Pb exposures (i.e., BLL >20 (ig/dL). These studies were additionally limited by their lack of consideration of potential confounders, although some studies adjusted for or consider potential confounding factors (i.e., age, sex, urban/rural residence, height, weight, BMI (Wang et al. 2010a; Ahamed et al. 2007; Ahamed et al. 2006)). Studies that did account for potential confounders reported consistent associations between increased BLLs and decreased ALAD activity (Section 4.7.3.1 of the 2013 Pb ISA). Evidence for altered heme synthesis is also provided by the epidemiologic studies discussed in Section 7.3.1 that report decreased Hb concentrations in association with increased Pb exposure or BLLs. Decreased RBC survival and hematopoiesis can be expected to occur simultaneously, and any effect on Hb levels is likely a combination of the two processes. Outside of the recent studies on Hb concentrations discussed in Section 7.3.1, there are no recent PECOS-relevant epidemiologic studies examining the relationship between Pb exposure and heme synthesis. 7.4.2. Toxicological Studies of Heme Synthesis Pb-induced alterations in heme synthesis occurring at BLLs relevant to this ISA (e.g., 10 |ag/dL) have been demonstrated convincingly by a small but consistent body of evidence. In brief, Pb exposure in rats inhibits several enzymes involved in heme synthesis, most notably ALAD, the enzyme that catalyzes the second, rate-limiting step in heme biosynthesis (Rendon-Ramirez et al. 2007; Teravama et al. 1986). Pb exposure has also been shown to inhibit ferrochelatase, a mitochondrial iron (Fe)-sulfur (S) containing 2+ enzyme that incorporates Fe into protoporphyrin IX to create heme (Rendon-Ramirez et al. 2007). Toxicological studies have found that Pb exposures result in increases in markers of oxidative stress. For example, Lee et al. (2005) reported increased RBC malondialdehyde (MDA), SOD and CAT levels accompanied by significant decreases in GSH and GPx in rats exposed to Pb (25 mg/kg) once a week for 4 weeks. In a second drinking water study performed in rats, administration of Pb acetate (750 mg/kg in drinking water for 11 weeks) resulted in decreased concentrations of plasma Vitamin C, Vitamin E, nonprotein thiol, and RBC-GSH, with simultaneous increased activity of SOD and GPx (Kharoubi et al. 2008). Effects on measures of oxidative stress were also observed in in vitro studies including increased MDA and decreased SOD and CAT in RBCs (Ciubar et al. 2007). and decreased glutathione reductase (GR) activity in human RBCs (Coban et al. 2007). and decreased GSH and increased glutathione disulfide (GSSG). External Review Draft 7-10 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 There were no recent toxicology studies investigating the effects of Pb exposure on heme synthesis that satisfied the PECOS criteria described in Section 7.2 available for this review. 7.4.3. Integrated Summary of Heme Synthesis A small number of animal toxicological studies evaluated in the 2013 Pb ISA provide consistent evidence that Pb exposures affect heme synthesis, including Pb-induced decreases in ALAD (Rendon- Ramirez et al. 2007; Teravama et al. 1986) and ferrochelatase activities (Rcndon-Ramircz et al. 2007). The toxicological evidence was supported by a larger body of ecotoxicological studies that demonstrate ALAD inhibition in Pb-exposed aquatic and terrestrial invertebrates and vertebrates (Sections 6.3.4.3, 6.4.5.2, 6.4.5.3, and 6.4.15.2 of the 2013 Pb ISA). Ecological evidence from previous reviews consistently observed Pb-induced ALAD inhibition in multiple species, including birds and fish (U.S. EPA 2013. 2006b). Some cross-sectional epidemiologic studies evaluated in previous reviews provide supporting evidence that concurrent BLLs are associated with decreased ALAD and ferrochelatase activities in both adults and children. The majority of these studies, however, are limited by the lack of rigorous methodology and consideration of potential confounding. Recent evidence provided by epidemiologic and toxicological studies of Pb exposure and Hb levels provides additional support for Pb-related impairment of heme synthesis. These studies are discussed in more detail in Section 7.3. 7.5 Biological Plausibility This section describes biological pathways that potentially underlie effects on hematology measures resulting from exposure to Pb. Figure 7-1 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 7.3 and 7.4 of this ISA, evidence reviewed in the 2013 Pb ISA (U.S. EPA 2013). and recent evidence collected from studies that may not meet the current PECOS criteria but contain mechanistic information supporting these pathways. Discussion of how exposure to Pb may lead to hematological effects contributes to an understanding of the biological plausibility of epidemiologic results. 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. External Review Draft 7-11 DRAFT: Do not cite or quote ------- Pb Exposure h Increased intracellular Ca2+ Increased phosphatidylserine exposure Altered hematopoiesis Cytotoxicity/senescence RBC precursor cells Decreased RBC survival Decreased RBC number & altered hematological endpoints Anemia Oxidative stress Decreased ferrochelatase activity Decreased ALAD activity Altered heme synthesis Decreased RBC Hb content Decreased RBC function ALAD = 5-aminolevulinate dehydratase; Ca2+ = calcium ion; Mg2+ = Magnesium ion; RBC = red blood cell. Note: The boxes 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 2022 Pb ISA are discussed in Section 7.6. Figure 7-1 Potential biological plausibility pathways for hematological effects associated with exposure to Pb External Review Draft 7-12 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Careful review of the available evidence indicates that exposure to Pb has the potential to modulate the hematological parameters leading to decreased RBC survival and function and altered heme synthesis. These deficits converge, promoting the development of anemia, a condition that occurs when the number of RBCs and/or the concentration of Hb in RBCs is abnormally low. Below, evidence from peer-reviewed toxicology studies providing biological plausibility for Pb-associated effects on hematological parameters is reviewed. 7.5.1. Decreased Red Blood Cell Survival and Function As described below, there is strong evidence that Pb impacts a series of hematological parameters along a cascade of events that results in decreased RBC function and survival, possibly leading to anemia (Figure 7-1). As reviewed in the 2013 Pb ISA, Pb uptake into human RBCs occurs through a passive anion transport mechanism, and once Pb is in the cell, little leaves (Bergdahl et al. 1997; Simons 1993; Simons 1986). While the precise mechanisms responsible for decreasing RBC lifespan and mobility are unknown, occupational Pb exposure has been shown to decrease intracellular free Ca+2 levels and decrease Ca2+/Mg2+ ATPase activity in RBCs in workers (Abam et al. 2008; Ouintanar-Escorza et al. 2007). These changes are associated with fragility and morphological alterations in RBCs in Pb-exposed workers. Pb-induced increases in intracellular Ca2+ levels also play a role in the activity of phospholipid scramblases and flippases in RBCs, increasing access to PS by tissue macrophages and triggering splenic sequestration and destruction of RBCs, leading to reduced numbers of RBCs in circulation (Jang et al. 2011). Importantly, Ahvavauch et al. (2018) showed that inhibiting Ca2+ increase stimulated by Pb results in decreased flippase activity and prevented destruction of RBCs. This pathway is depicted in Figure 7-1 by solid lines linking increased intracellular Ca2+ to increased PS exposure and decreased RBC survival. In addition, phagocytosis of Pb-exposed RBC by human renal proximal tubular cells was mediated by PS (Kwon and Chung 2016). Heme-regulated eIF2a kinase was shown to protect RBC from Pb-induced hemolytic stress in mice (Wang et al. 2015). Pb-induced hemolysis was also documented in other recent studies (Hossain et al. 2015; Mrugesh etal. 2011). Furthermore, Pb exposure reduced the number of RBCs and Hb levels in rats (Ibrahim et al. 2012). Consistent with the pattern seen in epidemiology studies, the effects of Pb exposure on RBC number and Hb level were more pronounced in 3-month-old Wistar rats than in adult animals (Daku et al. 2019). In addition, Cai et al. (2018) reported that Pb administration reduced RBC lifespan in mice. These findings support the conclusion that Pb alters RBC survival and function, consistent with the larger body of evidence showing measures of decreased hematological parameters (i.e., RBC number, Hb, Hct, MCV, and/or MCH) in children (Guo et al. 2021; Kuang et al. 2020; Li et al. 2018; Liu et al. 2015; Liu et al. 2012) and animals (Odo et al. 2020; Andielkovic et al. 2019; Cai et al. 2018; Corsetti et al. 2017; Lakshmi et al. 2013; Berrahal et al. 2011; Sharma et al. 2010; Wang et al. 2010b; Baranowska-Bosiacka et al. 2009; Masso-Gonzalez and Antonio- Garcia 2009; Simsek et al. 2009; Masso et al. 2007). External Review Draft 7-13 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Pb exposure also has the potential to disrupt normal hematopoiesis. Erythropoietin (EPO) is a glycoprotein hormone excreted by the kidney to promote the development of RBCs in bone marrow. As reviewed in the 2006 Pb AQCD, Pb exposure has been observed to alter EPO production in children (Graziano et al. 2004; Factor-Litvak et al. 1999; Factor-Litvak et al. 1998). Available data support the postulation that observed increases in EPO in younger children reflect bone marrow hyperactivity to counteract RBC destruction, whereas the lack of EPO elevation in older children may reflect a transitional period in which increasing renal and bone marrow toxicity leads to observed decreases in EPO later in life (U.S. EPA 2006a). In addition to altering levels of a key hormone involved in hematopoiesis, Pb exposure has the potential to alter hematopoiesis by causing cytotoxicity (Alghazal et al. 2008; Marques et al. 2006; Celik et al. 2005) and senescence (Cai et al. 2018; Nagano et al. 2015) of RBC precursors. Baktvbaeva (2011) reported that intraperitoneal injection of Pb acetate resulted in reduced bone marrow hematopoiesis in mice. Furthermore, Pb is known to reduce erythropoiesis, causing anemia in children (Dai et al. 2017; Ahamed et al. 2011). Altered EPO levels and RBC precursor cytotoxicity have the potential to alter the number of RBCs in circulation which may lead toto anemia. 7.5.2. Altered Heme Synthesis Although the mechanisms that could lead to Pb-induced anemia are not fully understood, as reviewed in EPA's 2006 AQCD (U.S. EPA 2006b). Pb is known to act directly on two enzymes involved in heme synthesis: ALAD and ferrochelatase. ALAD, a cytoplasmic enzyme requiring zinc (Zn) for enzymatic activity, catalyzes the rate-limiting step in heme biosynthesis. Inhibition of ALAD activity has been reported in adults (Wang et al. 2010a) and children (Dai et al. 2017; Wang et al. 2010a; Ahamed et al. 2007) as well as in animal toxicology studies reporting BLLs relevant to this ISA (i.e., 24.7 |ag/dL) (Rendon-Ramirez et al. 2007; Teravama et al. 1986). ALAD activity was also reduced in animal studies reporting BLLs higher than those meeting the PECOS criteria in this ISA (Mani et al. 2020; Velaga et al. 2014; Whittaker et al. 2011; Gautam and Flora 2010; Lee et al. 2005). Pb exposure has also been shown 2+ to inhibit ferrochelatase, a mitochondrial iron (Fe)-sulfur (S)containing enzyme that incorporates Fe into 2+ protoporphyrin IX to create heme (Rendon-Ramirez et al. 2007). Pb inhibits the insertion of Fe into the protoporphyrin ring and instead, Zn is inserted into the ring creating Zn-protoporphyrin (ZPP). Evidence for altered heme synthesis is supported by a large body of evidence collected from occupationally exposed adults (Ukaeiiofo et al. 2009; Khan et al. 2008; Patil et al. 2006; Karita et al. 2005). children (Oueirolo et al. 2010; Shah et al. 2010; Olivero-Verbel et al. 2007; Riddell et al. 2007). and Pb-exposed experimental animal models (Andielkovic et al. 2019; Cai et al. 2018; Berrahal et al. 2011; Baranowska- Bosiacka et al. 2009; Masso-Gonzalez and Antonio-Garcia 2009; Simsek et al. 2009; Rendon-Ramirez et al. 2007; Marques et al. 2006; Teravama et al. 1986) reporting decreased Hb concentrations in association with Pb exposure or increased BLLs. Further demonstrating the role Pb plays in the activity of these important enzymes, chelation therapy restored ALAD activity and reduced ZPP levels in blood harvested from rats exposed to Pb via drinking water for 90 days (Ataetal. 2018). External Review Draft 7-14 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Oxidative stress is caused by an imbalance between production and elimination of reactive oxygen species (ROS) in cells or tissues that exceed the capacity of antioxidant defense mechanisms. ROS are unstable, highly reactive molecules formed from molecular oxygen and include, for example, superoxide (O2), hydroxyl radical (OH), and hydrogen peroxide (H2O2). Although ROS play an important role in healthy biological systems (e.g., cell signaling, cellular differentiation, immune responses), unregulated ROS can cause direct and indirect damage to nucleic acids, proteins, and lipids leading to cytotoxicity, tissue injury, and even disruption of normal physiology (Autcn and Davis 2009). Oxidative stress is involved in both arms of the pathway leading to anemia shown in Figure 7-1, including effects on RBC MDA, SOD, CAT, GSH, and GPx levels (Kharoubi et al. 2008; Lee et al. 2005). Effects of Pb exposure on measures of oxidative stress—including MDA and decreased SOD, CAT, GR, and GSSG— were also observed in vitro (Ciubar et al. 2007; Coban et al. 2007). Supporting the role of oxidative stress in the development of anemia, administration of antioxidants reduced the effects of Pb exposure on levels of GSH (Alcaraz-Contreras et al. 2011). MDA (Alcaraz-Contreras et al. 2011) and Hb (Farooq et al. 2016; Saiitha etal. 2016; Sarkar et al. 2015; Eshginia and Marjani 2013) and reduced the effects of Pb exposure on SOD activity (Eshginia and Marjani 2013). ROS production (Nagano et al. 2015; Sarkar et al. 2015). hematopoietic stem cell (HSC) number (Nagano et al. 2015). HSC colony formation (Cai et al. 2018). HSC senescence markers (Cai et al. 2018; Nagano et al. 2015). RBC number (Farooq et al. 2016; Saiitha et al. 2016; Sarkar et al. 2015). and PS exposure (Sarkar et al. 2015) in animal studies. Conflicting evidence on the effects of antioxidant treatment on ALAD activity was reported in the literature (Saiitha et al. 2016; Alcaraz-Contreras et al. 2011). whereas the evidence for effects of Pb exposure on Hb levels was consistent; thus, a solid arrow connects oxidative stress to decreased RBC Hb content in Figure 7-1. Further demonstrating the role of oxidative stress in the development of anemia, decreased ALAD activity results in the accumulation of 5- aminolevulinic acid (5-ALA) in blood and urine, where it undergoes tautomerization and autoxidation. Oxidized 5-ALA leads to the generation of ROS (i.e., O2, OH, H2O2, and an aminolevulinic acid [ALA]) radicals (Hermes-Lima etal. 1991; Monteiro et al. 1991; Monteiro et al. 1989; Monteiro et al. 1986). Reflecting the strength of the evidence described above, the pathway connecting oxidative stress to altered hematopoiesis, cytotoxicity/senescence RBC precursor cells, and decreased RBC survival is depicted as a solid line. Decreased RBC Hb content and oxidative stress associated with Pb exposure have been demonstrated to alter RBC function. This conclusion is supported by direct evidence for binding of Pb to key enzymes in the heme synthesis pathway, Pb-induced oxidative stress resulting in decreased RBC Hb content and effects on hematopoiesis, RBC precursor cells, and RBC survival. Decreased RBC number coupled with impaired RBC function, if of sufficient magnitude, leads to Pb-induced anemia. External Review Draft 7-15 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 7.6 Summary and Causality Determination 7.6.1. Causality Determination for Red Blood Cell Survival and Function The 2013 Pb ISA presented causality determinations for two groups of hematological endpoints: heme synthesis and RBC survival and function. Although there are enzymes and hematological parameters that are distinct indicators of these processes, the potential biological plausibility pathways in which exposure to Pb may result in hematological effects demonstrate a spectrum of events, which can be challenging to attribute to a unique line of evidence (Figure 7-1). For example, altered heme synthesis can decrease Hb levels, which in turn has been shown to alter RBC function. Because of this interconnectedness, this assessment presents a single causality determination for Pb exposure and heme synthesis and RBC survival and function. This approach allows for a more holistic evaluation of inter- related health endpoints, including a discussion of how all individual lines of evidence contribute to the overall causality determination. The key evidence, as it relates to the causal framework, is outlined below, and summarized in Table 7-1. 7.6.2. Evidence for Red Blood Cell Survival and Function The 2013 Pb ISA concluded that there is a "causal relationship" between Pb exposure and decreased RBC survival and function (U.S. EPA 2013). This causality determination was made on the basis of a strong body of evidence from experimental animal studies demonstrating that Pb exposures alter several hematological parameters (e.g., Hb, Hct, MCV, MCH), induce oxidative stress (e.g., alter antioxidant enzyme activities [SOD, CAT, GPx], decrease cellular GSH, and increase lipid peroxidation), and increase cytotoxicity in RBC precursor cells in rodents exposed to various forms of Pb via drinking water and gavage resulting in BLLs < 30 |ag/dL (Molina etal. 2011; Baranowska-Bosiacka et al. 2009; Lee et al. 2005). Consistent results were observed in several additional studies in rodents that did not report BLLs. Epidemiologic evidence was coherent with results from the evaluated toxicological studies but was subject to more uncertainties. Notably, the epidemiologic evidence consisted of cross-sectional studies that were conducted in populations with higher mean Pb exposures (i.e., BLLs >10 (.ig/dL). did not thoroughly consider potential confounders, and lacked rigorous statistical methodology. These limitations precluded strong conclusions on the directionality of effects; the level, timing, frequency, and duration of Pb exposure that contributed to the observed associations; and whether the observed associations are independent of potential confounders. Although there were substantial uncertainties in the epidemiologic evidence, animal toxicological evidence established a clear basis for temporality of exposure to Pb and effects on RBCs. Additional support for these findings was provided by toxicological and epidemiologic studies demonstrating increased intracellular calcium concentrations, decreased Ca2+/Mg2+ ATPase activity, and increased PS exposure, establishing biologically plausibility for Pb-induced changes in RBC survival. External Review Draft 7-16 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Although limited in number, recent PECOS-relevant animal toxicological studies continue to support the findings from the last review. The most consistent evidence comes from studies that report decreased Hb levels in rodents following Pb exposures, resulting in BLLs ranging from 7.5 to 14.7 (ig/dL (Andiclkovic et al. 2019; Cai et al. 2018; Berrahal et al. 2011). Other recent toxicological studies noted Pb-induced decrements in Hct (Andiclkovic et al. 2019). packed cell volume (PCV) (Berrahal et al. 2011). and hematopoiesis (Andielkovic et al. 2019). Recent epidemiologic studies expand on the evidence presented in the 2013 Pb ISA and are coherent with the experimental evidence. Although the recent studies are also cross-sectional, they include populations with much lower BLL means (<10 (ig/dL) and include more robust adjustment for potential confounding, addressing important uncertainties from the last review. The most consistent epidemiologic evidence indicates an association between increased BLL and decreased Hb levels in children (Section 7.3.1), which is in line with the evidence from recent experimental animal studies. While the clinical relevance of small mean decrements in Hb across exposure quintiles is unclear, a few of the recent epidemiologic studies observed increases in the odds of prevalent anemia in children associated with increasing quantiles of BLLs (Guo et al. 2021; Li et al. 2018). Recent epidemiologic studies of Hb in adults were more limited in number and less consistent than those in children. Additionally, the relatively few studies examining RBC counts were also inconsistent. 7.6.3. Evidence for Heme Synthesis The 2013 Pb ISA concluded there is a "causal relationship" between Pb exposure and altered heme synthesis (U.S. EPA 2013). This determination was based on a small but consistent body of studies in adult animals reporting that Pb exposures via drinking water and gavage (resulting in BLLs relevant to this ISA) for 15 days to 9 months decreased ALAD (Rendon-Ramirez et al. 2007; Teravama et al. 1986) and ferrochelatase activities (Rendon-Ramirez et al. 2007). Notably, Rendon-Ramirez et al. (2007) observed effects on ALAD and ferrochelatase activities in albino Wistar rats at mean BLLs of 24.7 (ig/dL after Pb administration drinking water for 15 or 30 days. Supporting this toxicological evidence was a larger body of ecotoxicological studies that demonstrate altered heme synthesis in Pb-exposed aquatic and terrestrial invertebrates and vertebrates. Ecological evidence from previous reviews consistently observed Pb-induced ALAD inhibition in multiple species, including birds and fish (U.S. EPA 2013. 2006b). Cross-sectional epidemiologic studies provided supporting evidence that concurrent elevated BLLs are associated with decreased ALAD and ferrochelatase activities in both adults and children. However, the majority of these studies are limited by the lack of rigorous methodology and consideration of potential confounding. Although there were limitations in the epidemiologic evidence, some studies in children did control for or consider potential confounding, and effects in adults and children in these studies are coherent with effects observed in animal toxicological studies. The relationship between Pb exposure and altered heme synthesis was further supported by cross- sectional epidemiologic studies indicating that increased BLLs were associated with decreased Hb in children and occupationally exposed adults. These findings were consistent with several toxicological External Review Draft 7-17 DRAFT: Do not cite or quote ------- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 studies that observed decreased Hb levels in laboratory animals exposed to Pb. Decreased Hb levels can be a direct indicator of decreased heme synthesis. Recent PECOS-relevant studies are limited in number and focus mainly on Hb levels but continue to provide support for Pb-related alterations in heme synthesis. Notably, recent epidemiologic studies indicating an association between increased BLLs and decreased Hb include more robust statistical methods, expanded consideration of potential confounders, and populations with much lower BLLs than the studies included in the previous reviews (mean or median BLLs ranging from 3.04 to 8.38 j^ig/dL: Section 7.3.1). The recent epidemiologic evidence is coherent with recent toxicological studies, which observed Hb decrements in Pb-exposed mice (Andiclkovic et al. 2019; Cai et al. 2018). While the cross- sectional nature of the epidemiologic studies introduces uncertainty about the temporality of the exposure and outcome, animal toxicological evidence establishes clear temporality of exposure to Pb and altered heme synthesis. 7.6.4. Causality Determination In summary, there is coherent evidence across toxicological and epidemiologic studies that Pb exposures alter several hematological parameters, decrease enzyme activity related to heme synthesis, and increase RBC oxidative stress. Although all evaluated epidemiologic studies are cross-sectional, toxicological studies establish temporality between exposure to Pb and effects on heme synthesis and RBCs. Additionally, recent epidemiologic and animal studies continue to demonstrate evidence of a causal relationship at relevant BLLs, and recent epidemiologic studies address uncertainties from previous reviews by expanding adjustment for potential confounders and using more robust statistical methods (i.e., multivariable regression models). Because of the contribution of bone Pb levels to concurrent BLLs, associations with concurrent BLLs may reflect an effect of past and/or recent Pb exposures. Therefore, there is uncertainty regarding the timing, duration, and level of Pb exposure associated with observed hematological effects in children and adults. Biological plausibility for the observed associations is provided by toxicological and epidemiologic studies demonstrating increased intracellular calcium concentrations, decreased Ca2+/Mg2+ ATPase activity, and increased PS exposure, which collectively can lead to fragility, morphological alterations in RBCs, and RBC destruction. Taken together, there is sufficient evidence to conclude that there is a causal relationship between Pb exposure and hematological effects, including altered heme synthesis and decreased RBC survival and function. External Review Draft 7-18 DRAFT: Do not cite or quote ------- Table 7-1 Summary of evidence indicating a causal relationship between Pb exposure and hematological effects Rationale for Causality Determination3 Key Evidence" Key References" Pb Biomarker Levels Associated with Effects0 Red Blood Cell Survival and Function Large body of studies with generally consistent findings for decreased RBC survival and function in rodents: See Section 7.3.2 Mean BLLs (± SD): Decreased plasma Hb concentration Cai etal. (2018) Andielkovic et al. (2019) Berrahal etal. (2011) 9.3 ± 0.98 |ig/dl_ 29.0 ±43.1 |ig/dl_ 12.67 ± 1.68 |ig/dl_ PND 40; 7.49 ± 0.78 ug/dL PND 65 Consistent evidence from toxicological studies with relevant exposures Sharma et al. (2010) Masso-Gonzalez and Antonio-Garcia (2009) Baranowska-Bosiacka et al. (2009) 1.72 ±0.02 ug/dL 22.8 ± 0.50 ug/dL 7.11 ± 1.7 ug/dL Decreased Hct Andielkovic et al. (2019) Masso et al. (2007) Masso-Gonzalez and Antonio-Garcia (2009) Berrahal etal. (2011) 29.0 ± 14.7 ug/dL 22.8 ± 0.50 ug/dL 22.8 ± 0.50 ug/dL 12.67 ± 1.68 |ig/dl_ PND 40; 7.49 ± 0.78 ug/dL PND 65 Decreased RBCs Andielkovic et al. (2019) 29.0 ± 14.7 ug/dL External Review Draft 7-19 DRAFT: Do not cite or quote ------- Rationale for Causality Determination3 Key Evidence" Key References" Pb Biomarker Levels Associated with Effects0 Consistent evidence from multiple epidemiologic studies of children with relevant BLLs provides coherence with toxicological evidence. Cai etal. (2018) 9.3 ± 0.98 ng/dL Sharma et al. (2010) 1.72 ± 0.02 ng/dL Decreased PCV U.S. EPA (2013) Increased eryptosis U.S. EPA (2013) U.S. EPA (2006b) Decreased hematopoiesis U.S. EPA (2013) U.S. EPA (2006b) Increased oxidative stress In vitro: Ciubaretal. (2007) Coban et al. (2007) Shin etal. (2007) Cross-sectional studies provide support for Mean BLLs: experimental evidence with consistent Guo et al. (2021) 3.07-3.21 pg/dL associations between blood Pb and decreased in Hb in children. Recent studies Kuang et al. (2020) 3.04 pg/dL adjusted for a number of relevant potential u et g| f2Q18 38 ua/dL fMedian^ confounders, including age, sex, BMI, SES factors, and nutrition. Liu et al. (2015) 7.33 pg/dL Liu etal. (2012) 4.30 pg/dL (Median) External Review Draft 7-20 DRAFT: Do not cite or quote ------- Rationale for Causality Determination3 Key Evidence" Key References" Pb Biomarker Levels Associated with Effects0 Cross-sectional studies provide generally (U.S. EPA 2013) >10pg/dL consistent evidence for associations between blood Pb and altered hematological parameters (e.g., RBC counts, Hct, MCV, and MCH), measures of oxidative stress (e.g., SOD, CAT, GPx, GSH, and lipid peroxidation), and hematopoiesis (e.g., decreased erythropoietin). Evidence base limited by lack of adjustment for potential confounders and populations with higher BLLs. Evidence of increased [Ca2+]i and decreased See Section 7.5.2 Ca2+/Mg2+ATPase activity in the RBCs of exposed workers. [Ca2+]i levels highly correlated with blood Pb even among unexposed controls. [Ca2+]i levels increased in RBCs from healthy volunteers when exposed in vitro to Pb. PS exposure r [Ca2+]i associated with increased RBC fragility and alterations in RBC morphology. Consistent evidence from in vivo and in vitro studies that Pb exposure increases PS exposure on RBC membranes via modulation of[Ca2+]i concentrations. Increased PS exposure leads to eryptosis and phagocytosis by macrophages. Biological Plausibility Altered RBC membrane ion transport Heme Synthesis Consistent evidence in animals with relevant exposures A small, but consistent toxicology evidence base indicates decreased heme synthesis in rodents with relevant Pb concentrations and routes of exposure. Rendon-Ramirez et al. (2007) Teravama et al. (1986) BLL: 24.7 ±2.4 pg/dL Exposures: 500-5,000 ppm in drinking water, 15-30 days as adults Ahamed et al. (2006) Mean BLL: 7.40 and 13.27 pg/dL External Review Draft 7-21 DRAFT: Do not cite or quote ------- Rationale for Causality Determination3 Key Evidence" Key References" Pb Biomarker Levels Associated with Effects0 Coherence in a limited number of epidemiologic studies with relevant BLLs Cross-sectional studies that considered potential confounding by age, sex, urban/rural residence, height, weight, BMI found consistent associations with lower ALAD and ferrochelatase activities in children. Concurrent BLL associated with lower ALAD and higher ZPP in adults with consideration for potential confounding by age, sex, smoking status, and alcohol use. Ahamed et al. (2007) Wang et al. (2010a) BLL: >10 [jg/dL compared with <10 |jg/dL Mean BLL: 6.71 pg/dL Support from toxicological Consistent evidence in animals with relevant Baranowska-Bosiacka et al. (2009). and epidemiologic evidence for decreases in Hb, a direct marker of decreased heme synthesis Pb exposures for decreases in Hb levels. Consistent associations between concurrent BLLs and decreased Hb in children. Associations observed at low BLLs with thorough consideration of potential confounders. Sharma et al. (2010) Section 7.4.2 Guo et al. (2021) Kuana et al. (2020) Li et al. (2018) Liu et al. (2015) Liu et al. (2012) Adult animals: BLL 1.7 pg/dL after 40- day Pb exposure Mean BLLs: 3.07-3.21 pg/dL 3.04 pg/dL 8.38 pg/dL (Median) 7.33 pg/dL 4.30 pg/dL (Median) Biological Plausibility Altered Ion Status: Evidence that Pb See Section 7.5.2 competitively inhibits the binding ofZn ions necessary for ALAD activity. Pb also inhibits the incorporation of Fe2+ into protoporphyrin IX by ferrochelatase, resulting in Zn- protoporphyrin production. External Review Draft 7-22 DRAFT: Do not cite or quote ------- Rationale for Causality Determination3 Key Evidence" Key References" Pb Biomarker Levels Associated with Effects0 ALAD = S-aminolevulinate dehydratase; BLL = blood lead level; BMI = body mass index; Ca2+ = calcium; CAT = catalase; Fe2+ = iron; GPx = glutathione peroxidase; GSH = glutathione; Hb = hemoglobin; Hct = hematocrit; i = inorganic; MCV = mean corpuscular volume; Mg2+ = magnesium; Pb = lead; PCV = packed cell volume; PND = postnatal day; PS = phosphatidylserine; RBC = red blood cell; SES = socioeconomic status; SOD = superoxide dismutase; Zn = zinc; ZPP = Zn-protoporphyrin. "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. 1 External Review Draft 7-23 DRAFT: Do not cite or quote ------- 7.7 Evidence Inventories—Data Tables to Summarize Study Details Table 7-2 Epidemiologic studies of exposure to Pb and hematological effects Reference and Study Design Study Population Exposure Assessment Outcome Confounders Effect Estimates and 95% Clsa Children tGuoetal. (2021) Guangdong China 2014-2017 Cross-sectional Guangdong Women and Children's Hospital n: 17486 Children 0-5 yr old visiting hospital for routine health examination Blood BLLs were measured using atomic absorption spectrometry Age at Measurement: 0-5 yr Means: Males: 3.21 [jg/dL; Females: 3.07 [jg/dL Hb Hb (g/L) measured using an automated hematology analyzer Age at Outcome: 0-5 yr Age and sex Hb Mean Difference (g/L) Q1 (<1.61) Reference Q2 (1.61-2.44) -0.05 (-0.51, 0.40) Q3 (2.44-3.33) -0.02 (-0.48, 0.43 Q4 (3.33-4.50) -0.48 (-0.94, -0.04) Q5(>4.50) -1.25 (-1.71, -0.78) Anemia (OR) Q1 (<1.61) Reference Q2 (1.61-2.44) 1.08 (0.94, 1.23) Q3 (2.44-3.33) 1.16 (1, 1.33) Q4 (3.33-4.50) 1.25 (1.07, 1.43) Q5(>4.50) External Review Draft 7-24 DRAFT: Do not cite or quote ------- Reference and Study Design Study Population Exposure Assessment Outcome Confounders Effect Estimates and 95% Clsa 1.45 (1.26, 1.67) tKuana et al. (2020) Nanjing China 2012 Cross-sectional n: 395 Convenience sample of children 7-11 yrold Blood Blood Pb was measured in venous whole blood using ICP-MS Age at Measurement: 7-11 yr old Mean: 3.04 [jg/dL; Median: 2.61 [jg/dL Hematological Parameters RBC, Hb, Hct, MCV, MCH, and MCHC measured by a whole cell analyzer Age at Outcome: 7-11 yr old Picky eaters and passive smoking (age, gender, parents' education, and parents' occupation also considered) RBC Count (1012/L) Boys: 0.02 (-0.01, 0.04) Girls: 0.01 (-0.02, 0.03) Hb (g/L) Boys:-0.12 (-0.22, -0.02) Girls: -0.08 (-0.23, 0.07) Hct (%) Boys:-0.04 (-0.08, -0.01) Girls: -0.02 (-0.07, 0.02) MCV (fL) -0.04 (-0.06, -0.02) MCH (pg) -0.01 (-0.02, -0.00) MCHC (g/L) 0.26 (-0.64, 1.15) tLiuetal. (2012) Changzhou City China Cross-sectional China Jintan Child Cohort Study n: 140 Convenience sample of preschool age children Blood Blood Pb was measured in whole blood using GFAAS Age at Measurement: Median age: 3 yr old Median: 4.3 [jg/dL Maximum: 11.4 pg/dL Hb Hb measured in whole blood using a photoelectric colorimeter Age at Outcome: Median age: 3 yr old Age, sex, height, weight, iron deficiency Hb (g/dl_)* Full Population -0.096 (-0.18, -0.012) Blood Pb < 10 jjg/dL -0.174 (-0.27, -0.078) External Review Draft 7-25 DRAFT: Do not cite or quote ------- Reference and Study Design Study Population Exposure Assessment Outcome Confounders Effect Estimates and 95% Clsa tLi etal. (2018) Hubei and Hunan Provinces China 2012-2017 Cross-sectional Blood Lead Intervention Program n: 758 Children Ages 5-8 yr recruited from four counties in two provinces One county in each province had high environmental Pb levels (battery plant and mining) Blood Blood Pb was measured in venous whole blood using GFAAS Age at Measurement: 5-8 yr Median: 8.38 [jg/dL 75th 90th 95th 13.51 [jg/dL 18.77 [jg/dL 21.82 [jg/dL Hematological Parameters Hb, MCH, RBCs, and Pit measured in venous whole blood using an automated hematology analyzer Age at Outcome: 5-8 yr Age, sex, BMI, environmental Pb exposure level, and serum iron, zinc, and calcium ORs Decreased Hb (<115 g/L) 1.05 (1.00, 1.11) Decreased RBC (<4 x 1012/L for boys; <3.5 x 1012/L for girls) 1.11 (1.05, 1.16) Decreased Pit (<100 x 109/L) 1.11 (1.05, 1.16) Decreased MCH (<27 pg) 1.11 (1.05, 1.16) tLiuetal. (2015) n: 855 Guiyu, Chendian, and Chaonan China 2006-2011 Cross-sectional Children 3-7 yr old from e- waste processing area or control industrial areas without high environmental Pb exposures Blood Blood Pb was measured using GFAAS. Blood Pbwas divided by Hct as a fraction of the whole blood to estimate erythrocyte Pb Age at Measurement: 3-7 yr old Median: Blood Pb: 7.33 pg/dL; Erythrocyte Pb: 19.3 pg/dL Hb Hb, MCH, RBCs, and Pit measured in venous whole blood using an automated hematology analyzer Age at Outcome: 3-7 yr old Age, sex, residence area, and SES Hb (g/L) Mean Difference Q1 (5.98-13.52)* Reference Q2 (13.52-19.35)* -0.02 (-1.89, 1.52) Q3 (19.35-28.42)* -3.01 (-4.71, 1.31) Q4 (28.42-101.01)* -3.97 (-5.68, -2.27) *Erythrocyte Pb (pg/dL) Adults External Review Draft 7-26 DRAFT: Do not cite or quote ------- Reference and Study Design Study Population Exposure Assessment Outcome Confounders Effect Estimates and 95% Clsa tPark and Lee KNHANES Blood Hb Age, BMI, education, Hb (g/dL)* (2013) n: 4522 smoking and drinking Men Blood Pb was measured in Blood Hb (g/dL) status, and rural/urban 0.04 (0.03, 0.06) South Korea General population, >20 yr venous whole blood using measured using an residence 2008-2010 old GFAAS automated hematology Women Cross-sectional Age at Measurement: analyzer 0.04 (0.02, 0.06) >20 yr Age at Outcome: Geometric Means: >20 yr *Not standardized. Per Males: 2.46 |jg/dL; In(Pb) increase Females: 1.98 |jg/dL tKim and Lee KNHANES Blood Hb Sex, age, obesity, Hb (g/L) (2013) n: 5951 residence area, Mean Difference Blood Pb was measured in Hb measured in whole education level, Q1 (<1.73) South Korea General population, >20 yr whole blood using GFAAS. blood using an smoking and drinking 2008-2010 old Blood Pb was divided by Hct automated hematology status, serum ferritin, Reference Cross-sectional as a fraction of the whole analyzer and serum creatinine Q2 (1.73-2.31) blood to estimate erythrocyte 0.13 (0.03, 0.23) Pb Age at Outcome: Age at Measurement: >20 yr old Q3 (2.31-3.01) >20 yr old 0.33 (0.23, 0.42) Median: Q4 (>3.01) 0.42 (0.30, 0.53) Blood Pb: 2.31 pg/dL; Erythrocyte Pb: 5.4 pg/dL 75th: Q1 (<4.1)* Blood Pb: 3.01 pg/dL; Reference Erythrocyte Pb: 6.9 pg/dL Q2 (4.1-5.4) -0.06 (0.15, 0.03) Q3 (5.4-6.9) -0.06 (-0.15, 0.03) Q4 (>6.9) -0.14 (-0.25, -0.04) *Erythrocyte Pb (pg/dL) External Review Draft 7-27 DRAFT: Do not cite or quote ------- Reference and Study Design Study Population Exposure Assessment Outcome Confounders Effect Estimates and 95% Clsa tLa-Llave-Leon et n: 292 Blood Hematological BMI, gestational age, RBC Count (x 10s |jg/dL) al. (2015) Parameter age, parity, gestations, 0.034 (0.013, 0.056) Pregnant women, 14-41 yr Blood Pb was measured in and household monthly Durango old venous whole blood using RBC, Hb, Hct, MCV, income per person Mexico GFAAS MCH, and MCHC 2007-2008 Age at Measurement: measured using an Cross-Sectional 14-41 yr old Mean: 2.79 |jg/dL automated hematology analyzer Age at Outcome: 14-41 yr old BMI = body mass index; BW = body weight; CI = confidence interval; e-waste = electronic waste; GFAAS = graphite furnace atomic absorption spectroscopy; Hb = hemoglobin; Hct = hematocrit; ICP-MS = inductively coupled plasma mass spectrometry; KNHANES = Korean National Health and Nutrition Examination Survey; In = natural log; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; OR = odds ratio; Pb = lead; Pit = platelet; PND = postnatal day; Q = quartile; RBC = red blood cell; RDW = red blood cell distribution width; SES = socioeconomic status; yr = years. a Effect 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. External Review Draft 7-28 DRAFT: Do not cite or quote ------- Table 7-3 Animal toxicological studies of Pb exposure and hematological effects Study Exposure Details Species (Stock/Strain), n, Sex Timing of Exposure (Concentration, BLL as Reported (|jg/dL)a Duration) Endpoints Examined Berrahal etal. (2011) Rat (Wistar) Control (vehicle), M,n = 12-16 50 mg/L Pb, M, n = 12-16 PND 1 to PND21: Lactational PND 21 to PND 40 or PND 65: Drinking water Dams were given 50 mg/L Pb acetate in drinking water until weaning on PND 21. Male offspring received 50 mg/L Pb acetate in drinking water from PND 21 to PND 40 or PND 65. Control animals received tap water. PND 40: 1.76 ± 0.33 [jg/dL for 0 pg/dL 12.67 ± 1.68 [jg/dL for 50 mg/L PND 65: 2.06 ± 0.35 [jg/dL for 0 [jg/dL 7.49 ± 0.78 [jg/dL for 50 mg/L Hct, Hb Bashaetal. (2012) Rat (Wistar) Control (vehicle), M, n 0.2% Pb, M, n = 8 PND 1 to PND 21 Damns given Pb acetate in drinking water or Pb acetate containing water supplemented with 0.02% calcium, zinc, and iron. Control group received deionized water as vehicle (no supplement). Pups exposed through lactation. PND 45: 0.42 ± 0.04 [jg/dL for 0%, 52.5 ± 0.67 [jg/dL for 0.2%, 21.1 ± 1.12 pg/dL for 0.2% + supplementation PND 12 mo: 0.56 ± 0.08 [jg/dL for 0%, 16.4 ± 1.95 [jg/dL for 0.2%, 7.2 ± 0.56 [jg/dL for 0.2% + supplementation RBC, Hb PND 24 mo: 0.46 ± 0.02 [jg/dL for 0%, 12.2 ± 0.76 [jg/dL for 0.2%, 4.8 ± 0.5 [jg/dL for 0.2% for 0.2% + supplementation External Review Draft 7-29 DRAFT: Do not cite or quote ------- Study Species (Stock/Strain), n, Sex Timing of Exposure Exposure Details (Concentration, Duration) BLL as Reported (|jg/dL)a Endpoints Examined Zou etal. (2015) Mouse (ICR) Control (vehicle), M, n = 10 250 mg/L Pb, M, n = 10 3 wk exposure Rats received 250 mg/L Pb acetate in redistilled drinking water for 3 weeks. The rats were 30 d old when acquired, but the authors did not specify the age at the time of treatment. PND 58: 1.8 pg/dLforO mg/L 21.7 |jg/dL for 250 mg/L RBC, MCHC Corsetti etal. (2017) Mouse(C57BL.6) Control (vehicle), M, n 200 ppm Pb, M, n = 8 d 30 to d 75 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 RBC, Hb, Hct, MCV, MCH, MCHC, RDW %, Pit Andielkovic et al. (2019) Rat (Wistar) Control (vehicle), M, n 0.2% Pb, M, n = 6 NR Rats (250 g), age at time of dosing not reported, were exposed to a single dose of 150 mg Pb/kg BWPb acetate via oral gavage. Control animals were given "water." 24.9 ±1 9 ug/kg for 0 mg Pb/kg BW (2.6 ± 2.0 [jg/dL) 291.2 ± 139 |jg/kg for 150 mg Pb/kg BW (29.0 ± 14.7 [jg/dL) RBC, Hb, Hct, MCV, MCH, MCHC, Pit External Review Draft 7-30 DRAFT: Do not cite or quote ------- Study Species (Stock/Strain), n, Sex Timing of Exposure Exposure Details (Concentration, Duration) BLL as Reported (|jg/dL)a Endpoints Examined Cai etal. (2018) Rat (Sprague Dawley) Control (vehicle), M/F, n = 5 0.2% Pb, M/F, n = 5 8-10 wk to 20-22 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. The exposure period was 12 wk, assumed rats were exposed 7 d/wk for a total of 84 d. 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) Hb, Pit, Erythrocyte life span, RBC BLL = blood lead level; BW = body weight; d = day; Hb = hemoglobin; Hct = hematocrit; M = male; M/F = male/female; MCH = mean corpuscular hemoglobin; MCHC = mean corpuscular hemoglobin concentration; MCV = mean corpuscular volume; mo = month; NR = not reported; Pb = lead; Pit = platelet; PND = postnatal day; RBC = red blood cell; RDW = red blood cell distribution width; wk = weeks. alf applicable, reported values for BLL were converted to (xg/dL using WebPlot Digitizer (https://apps.automeris.io/wpd/) and are shown in parenthesis. External Review Draft 7-31 DRAFT: Do not cite or quote ------- 7.8 References Abam. E: Okediran. BS: Odukova. 00: Adamson. I; Ademuviwa. O. (2008). Reversal of ionoregulatory disraptions in occupational lead exposure by vitamin C. 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