4

Hazard Identification

The major goals of the Environmental Protection Agency (EPA) toxicologic review of inorganic arsenic are identification of the disease hazards associated with chronic exposure to arsenic and estimation of the potential disease risks when people are exposed for a portion of their lifetime. For hazard identification, EPA has outlined a process that involves approaches to screen and organize studies, a causality framework for evaluating cancer and noncancer effects, and an evidence framework for determining susceptible life stages and populations. Those approaches have been designed to incorporate recommendations made in other National Research Council reports that are applicable to all Integrated Risk Information System (IRIS) assessments; they are under review by another Research Council committee.

The present committee has focused on EPA’s plans for hazard identification that are specific to inorganic arsenic. EPA has indicated that its assessment of causality will be based primarily on studies in humans under the assumption that any health effects observed in such studies are relevant to humans, regardless of country of origin and regardless of whether the mode of action is understood. Animal and mechanistic data will provide supporting evidence with respect to biologic plausibility. If particular effects are observed only in animal studies, mechanistic data will be used to address questions about the relevance of the data to humans. If mode-of-action data are insufficient for determining relevance, EPA will assume that the effects are relevant to humans.

The sections that follow contain a preliminary survey of the scientific literature on what appear to be the most affected organ systems and focus on the epidemiologic evidence. The strengths and weaknesses of epidemiologic studies of cancer and noncancer effects were discussed in the committee’s workshop by Cantor (2013) and Steinmaus (2013), respectively. The committee also considered mode-of-action information that would inform dose–response analyses, particularly with respect to low to moderate exposure (see Box 5). Consideration was also given to factors that could increase susceptibility to the effects of inorganic arsenic. In approaching its task, the committee did not attempt to conduct systematic reviews of the literature for each organ system. That task lies ahead for EPA. Rather, the committee has attempted to focus on factors that will help to tailor such reviews and to inform decisions about performing dose–response analyses.

The committee notes that among the noncancer effects of inorganic arsenic exposure, particular attention should be paid to the category of diseases that occur with a high prevalence in the US population so that it can be determined whether exposure to inorganic arsenic may be contributing to the underlying burden of disease in the population. That category includes cardiovascular disease, respiratory disease, kidney disease, and diabetes. Adverse effects of early-life exposure should also be considered.

BOX 5 Concentration Descriptors Used in This Report

The terms used here to describe the degree of arsenic exposure are not necessarily the same as those used in the cited published studies. High exposure is used to denote exposure to concentrations of inorganic arsenic in drinking water at 100 μg/L or higher. Low to moderate exposure refers to water concentrations of less than 100 μg/L.



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4 Hazard Identification The major goals of the Environmental Protection Agency (EPA) toxicologic review of inorganic ar- senic are identification of the disease hazards associated with chronic exposure to arsenic and estimation of the potential disease risks when people are exposed for a portion of their lifetime. For hazard identifi- cation, EPA has outlined a process that involves approaches to screen and organize studies, a causality framework for evaluating cancer and noncancer effects, and an evidence framework for determining sus- ceptible life stages and populations. Those approaches have been designed to incorporate recommenda- tions made in other National Research Council reports that are applicable to all Integrated Risk Infor- mation System (IRIS) assessments; they are under review by another Research Council committee. The present committee has focused on EPA’s plans for hazard identification that are specific to in- organic arsenic. EPA has indicated that its assessment of causality will be based primarily on studies in humans under the assumption that any health effects observed in such studies are relevant to humans, re- gardless of country of origin and regardless of whether the mode of action is understood. Animal and mechanistic data will provide supporting evidence with respect to biologic plausibility. If particular ef- fects are observed only in animal studies, mechanistic data will be used to address questions about the relevance of the data to humans. If mode-of-action data are insufficient for determining relevance, EPA will assume that the effects are relevant to humans. The sections that follow contain a preliminary survey of the scientific literature on what appear to be the most affected organ systems and focus on the epidemiologic evidence. The strengths and weaknesses of epidemiologic studies of cancer and noncancer effects were discussed in the committee’s workshop by Cantor (2013) and Steinmaus (2013), respectively. The committee also considered mode-of-action infor- mation that would inform dose–response analyses, particularly with respect to low to moderate exposure (see Box 5). Consideration was also given to factors that could increase susceptibility to the effects of inorganic arsenic. In approaching its task, the committee did not attempt to conduct systematic reviews of the literature for each organ system. That task lies ahead for EPA. Rather, the committee has attempted to focus on factors that will help to tailor such reviews and to inform decisions about performing dose– response analyses. The committee notes that among the noncancer effects of inorganic arsenic exposure, particular at- tention should be paid to the category of diseases that occur with a high prevalence in the US population so that it can be determined whether exposure to inorganic arsenic may be contributing to the underlying burden of disease in the population. That category includes cardiovascular disease, respiratory disease, kidney disease, and diabetes. Adverse effects of early-life exposure should also be considered. BOX 5 Concentration Descriptors Used in This Report The terms used here to describe the degree of arsenic exposure are not necessarily the same as those used in the cited published studies. High exposure is used to denote exposure to concentrations of inorganic arsenic in drinking water at 100 µg/L or higher. Low to moderate exposure refers to water concentrations of less than 100 µg/L. 23

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24 Critical Aspects of EPA’s IRIS Assessment of Inorganic Arsenic SKIN DISEASES Arsenic-related skin conditions include two related conditions: preneoplastic or nonneoplastic skin lesions (henceforth referred to as skin lesions) and nonmelanoma skin cancers. The types of skin condi- tions and their risks are determined by dose, duration, and susceptibility to exposure. The epidemiologic features of the two conditions, especially as related to those determining factors, are described below. Skin Lesions Arsenical skin lesions predispose a person to some skin cancers and serve as an indicator of suffi- cient arsenic exposure for (or increased susceptibility to) the occurrence of other cancer and noncancer diseases (Ghosh et al. 2007). Owing to differences in arsenic metabolism and toxicity among species, ob- servational epidemiologic studies are preferred for determining the association between arsenic exposure and skin lesions. Skin lesions have a well-established dose–response relationship with arsenic in drinking water. ATSDR (2007) based its chronic minimal risk level of 0.0003 mg/kg-day on skin lesions. Skin lesions were reported to occur at concentrations as low as 10 µg/L in cross-sectional (Ahsan et al. 2006) and pro- spective cohort (Argos et al. 2011) studies. The association was confirmed at concentrations of 50–100 µg/L (Guha Mazumder 2003; Guo et al. 2006; Argos et al. 2011). However, other studies have found skin lesions to occur only at high concentrations (e.g., Haque et al. 2003). In a longitudinal study in Bangladesh, the odds of skin lesions were about 70% higher at water con- centrations of 50–100 µg/L than at less than 10 µg/L. The odds nearly doubled in those exposed at 100– 200 µg/L and nearly tripled in those exposed at 200 µg/L or higher (Argos et al. 2011). The association may be modified by sex (Chen et al. 2006), genetic variations (Ahsan et al. 2007), and diet (Pierce et al. 2011). The clear dose–response relationship persists when exposure is measured by way of urinary concen- tration of monomethyl arsenic (MMA), a metabolic intermediate of arsenic. A case–control study in Bangladesh found a dose–response relationship between the percentage of total urinary arsenic that was MMA and skin lesions in 594 cases and 1,041 population-based controls (Ahsan et al. 2007). A small Taiwanese case–control study of 26 skin-lesion patients with sex- and age-matched controls indicated that participants who had a high percentage of MMA (more than 15.5%) had an odds ratio (OR) of 5.5 com- pared with those who had a low percentage of MMA, and those with low dimethyl arsenic (DMA), less than 72.2%, had an OR of 3.25 compared with those who had high DMA (Yu et al. 2000). In a Mexican cross-sectional study of 104 people who lived near mining operations and contaminat- ed groundwater (76 exposed at 50 µg/L or more, 28 exposed at 10 µg/L or lower), residents who had skin lesions had urinary MMA concentrations of 7.5 µg/L whereas residents who did not have lesions had 4.8 µg/L (Valenzuela et al. 2005). By using pathway analysis to address the collinear relationships among urinary arsenic metabolites, Kile et al. (2011) have also shown that urinary MMA concentration is associ- ated with an increased risk of skin lesions in a case–control study. Skin Cancer Arsenic is an established skin carcinogen, first classified by the International Agency for Research on Cancer (IARC) in 1987 on the basis of observations of patients treated with the arsenical Fowler’s so- lution. A causal relation between arsenic in drinking water and skin cancer was confirmed in the 2012 IARC publication based on ecologic studies in Taiwan, primarily in the southwest region, where geologic arsenic is endemic (Wu et al. 1989). To date, almost all published studies linking arsenic exposure to skin cancer have found supportive evidence of nonmelanoma skin cancers (basal-cell and squamous-cell car- cinoma).

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Hazard Identification 25 The evidence of skin-cancer risk posed by high arsenic exposure appears to be well supported by cohort, case–control, and ecologic studies, with increasing arsenic exposure associated with increasing skin-cancer risk. A US population-based case–control study of skin cancers also reported evidence of an increase in invasive squamous-cell carcinoma in those who had the highest concentrations of toenail arse- nic (Karagas et al. 2001b). At lower arsenic exposure, some studies have been able to estimate the effect of arsenic exposure on skin-cancer risk although in general the findings from these studies have been less conclusive. The ASHRAM case–control study in Hungary, Romania, and Slovakia found an association between histolog- ically confirmed basal-cell carcinoma of the skin and drinking-water inorganic arsenic concentrations greater than 100 µg/L (Leonardi et al. 2012). The study suggested that at lower concentrations an OR of 1.18 was associated with each 10-µg/L increase in average lifetime water concentration, with the odds of basal-cell carcinoma increasing with increasing concentrations of inorganic arsenic. A case–control study in Slovakia showed statistically significantly higher concentrations of urinary arsenic metabolites in peo- ple who had nonmelanoma skin cancer than in controls (Ranft et al. 2003). Arsenic exposure was primari- ly from residential proximity to a coal-burning power plant. The urinary arsenic concentrations in the study were all less than 50 µg/L but showed an association with nonmelanoma skin-cancer status. Other studies of lower arsenic exposure have been unable to measure skin-cancer risk at the lower concentrations precisely because of methodologic and sample-size limitations. A Danish ecologic study of low exposure (primarily arsenic at less than 2 µg/L) was conducted with geographic information sys- tem (GIS) methods. No association was observed between water arsenic and nonmelanoma skin-cancer incidence (Baastrup et al. 2008). Aside from the extremely low concentrations, the study was limited by lack of histologic specificity in that basal-cell and squamous-cell carcinomas were grouped as one out- come. A small case–control study in Lagunera, Mexico, with 42 cases and 48 controls suggested an in- creased risk of nonmelanoma skin cancer, which was modified by the presence or absence of human pap- illomavirus (Rosales-Castillo et al. 2004). Exposure in the study was measured as cumulative exposure as determined by measurement of urinary arsenic and extrapolation to cumulative exposure after assessment of the participant’s residential history. A more recent US case–control study of squamous-cell carcinoma used sensitive methods for uri- nary arsenic detection. It reported a dose-related increase in total urinary arsenic after arsenobetaine was subtracted out (median = 4.76 µg/L) and a trend in each urinary fraction (inorganic arsenic, MMA, and DMA), with the association strongest for MMA (Gilbert-Diamond et al. 2013). A US case–control study of melanoma skin cancer found an increased risk starting at an toenail ar- senic concentration of 0.04 µg/g and showed evidence of a dose-related increase in risk above that con- centration (Beane Freeman et al. 2004). The risk was particularly strong in those who had a prior skin- cancer diagnosis. However, the study was limited by the use of colon-cancer cases as controls. Mode of Action Few studies have been able to elucidate the molecular or cellular basis of skin lesions or cancer di- rectly in skin tissues in human populations exposed to arsenic. Several in vitro and animal studies have attempted to examine molecular and cellular alterations in response to external exposure to arsenic at var- ious doses. Those studies have identified immune-related, oxidative stress–related, apoptotic, stem-cell, mitochondrial, and genomic alterations that may potentially underlie basal-cell and squamous-cell cancer development in response to arsenic stimuli (Yu et al. 2006; Kitchin and Conolly 2010; Lee et al. 2011; Liao et al. 2011; Tokar et al. 2011a; Zhao et al. 2012; Huang et al. 2013; Lee et al. 2013; Pei et al. 2013). The findings from the experimental studies are difficult to extrapolate directly to understand how arsenic induces skin lesions or tumor development in humans. However, to the extent that the studies involved exposures at relevant dose ranges and are compatible with the epidemiology, the evidence might be worth considering in the IRIS assessment to strengthen or refute results of analyses that are relevant to skin pa- thology.

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26 Critical Aspects of EPA’s IRIS Assessment of Inorganic Arsenic Although this section integrates discussions of skin lesions and skin cancers, the committee notes that although skin lesions are clinical markers of susceptibility to arsenic-related skin and internal cancers and other health outcomes, it is not clear whether skin lesions are direct precursors of most skin cancers in arsenic-exposed people. Despite the existing dogma, no published longitudinal studies have documented the transition from skin lesions to skin cancer in humans. Thus, it is important to evaluate modes of action underlying skin lesions and skin cancer carefully to understand how they are similar and different and their potential implications for dose–response analyses for these seemingly related end points. Key Considerations for the IRIS Assessment The committee recommends that EPA consider skin studies that have histologic specificity and that allow the separation, for example, of basal-cell carcinoma and squamous-cell carcinoma because current data suggest that they are influenced by arsenic exposure and could have different dose–response relation- ships. RESPIRATORY EFFECTS Lung Cancer Arsenic exposure via drinking water is an established lung carcinogen in humans. Associations have been consistently observed in highly exposed populations in Taiwan, Japan, Chile, Argentina, and the United States (Guo 2004; IARC 2004, 2012). And ecologic analyses in Chile suggest that in utero or ear- ly-life exposure increases the risk of arsenic-related lung-cancer mortality (Smith et al. 2006). Case–control and cohort studies in those and other regions examined risks at lower levels of expo- sure (reviewed in EFSA 2009; Steinmaus et al. 2013). For example, a hospital-based case–control study in Chile (151 cases and 419 controls) detected increased risks beginning with the category of average wa- ter concentrations of 10–29 µg/L which became statistically significant at 30–39 µg/L compared with 10 µg/L or lower (Ferreccio et al. 2000). In a population-based study of 306 lung-cancer cases and 604 con- trols, the ORs were specifically increased in those who drank water during the peak exposure periods (1958–1970), which was about 40 years or more before the diagnostic period of the cases (2007–2009); however, no test of latency or dose–response effects was performed (Steinmaus et al. 2013). In a preliminary case–control study in Argentina, there was evidence that the percentage of MMA and polymorphisms in cystathionine beta-synthase modified the risk of arsenic-induced lung cancer (Steinmaus et al. 2010). In Taiwan, a cohort study (139 lung-cancer cases) found a trend of increasing lung-cancer risk be- ginning at an arsenic concentration of 100 µg/L; less than 10 µg/L in water was the reference concentra- tion, and there was no observable association with 10-99 µg/L (Chen et al. 2004). In Bangladesh, a trend of an association between well-water arsenic concentration and risk of ma- lignant lung tumors, in particular squamous-cell tumors, was found in a pathology-based case-control study of smokers (3,223 cases, 1,588 controls); a limitation is that the study used people who had suspi- cious lung lesions (for example, inflammatory, tubular, or other disease) as controls (Mostafa et al. 2008). Each of those studies examined trends in estimates of relative risk associated with specific categories of drinking-water arsenic, however, and did not model continuous dose–response relationships. The one study that did examine a linear dose–response relationship was conducted on the basis of GIS exposure models in Denmark and found no association with lung cancer (409 cases) (Baastrup et al. 2008); however, the concentrations of arsenic in drinking water were less than 2 µg/L for most participants. Likewise, in an ecologic analysis in a population of Mormons in Utah (34 lung-cancer deaths) (Lewis et al. 1999), there were no observable associations compared with the US general population. However, such ecologic com- parisons at low exposure are not likely to be informative, because of misclassification and confounding. A case–control study in New Hampshire that measured people’s toenail arsenic as a biomarker (223 cases, 238

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Hazard Identification 27 controls) found an increased OR for small-cell or squamous-cell carcinoma of the lung at higher concentra- tions of arsenic, especially in those who had a history of lung disease (Heck et al. 2009a). In the Strong Heart Study of 3,932 American Indians 45-74 years old, baseline urinary arsenic concentrations were related to an increased hazard ratio for lung-cancer mortality of 1.56 (95% confidence interaval [CI] 1.02-2.39) over almost 20 years of followup; 78 lung-cancer deaths were observed. There was a linear trend of increas- ing lung-cancer mortality with increasing urinary arsenic concentrations with no evidence of a threshold (p = 0.04) (Garcia-Esquinas in press). Most recently, a large prospective cohort study in Japan observed a dose-dependent increase in lung-cancer incidence in relation to arsenic intake in food as measured through a food-frequency questionnaire (Sawada et al. 2013). The increase in risk was particularly pronounced in cig- arette-smokers. Mode of Action The mode of action of arsenic in lung carcinogenesis is not completely understood, but some pat- terns appear to be emerging. Although arsenic is not directly mutagenic, it has been shown to affect sev- eral oncogenic pathways that are relevant to lung cancer, including epigenetic, microRNA, gene expres- sion, and mitochondrial DNA alterations. In particular, three oncogenic pathways have been implicated in lung cancer, including those of the epidermal growth factor receptor (Biscardi et al. 1999; Simeonova and Luster 2002; Andrew et al. 2009a; Li et al. 2011; Stueckle et al. 2012; Sung et all. 2012), phosphoinosi- tide 3-kinase/Akt (Dong 2002; Gao et al. 2004; Zhang et al. 2006; Beezhold et al. 2011; Chen et al. 2012; Z. Wang et al. 2012), and Nrf2/Keap1 (X.J. Wang et al. 2007; Andujar et al. 2010; Zheng et al. 2012). Key Considerations for the IRIS Assessment There is evidence of a trend of increasing risk at higher levels of arsenic exposure, but relatively few studies at lower levels of exposure have examined dose–response relationships with lung cancer. It may be possible to model the dose–response relationships from the estimated relative risks associated with categories of exposure. Risks may involve cofactors (including genetic factors) and be modified by timing of exposure (for example, early in life) and may necessitate assessment of potential confounding by ciga- rette-smoking. Associations could be specific to histologic types of lung cancer. The ability to detect as- sociations at low levels of exposure requires measurement not only of water concentrations of arsenic but of biomarker concentrations that encompass all sources of exposure, including diet, that may be important especially when drinking-water arsenic concentrations are relatively low. Nonmalignant Respiratory Outcomes Arsenic’s noncancer pulmonary effects have been less well studied than its lung-cancer effects, but results of a number of human epidemiologic studies suggest deleterious effects of arsenic on a variety of nonmalignant pulmonary outcomes, including respiratory symptoms, airway epithelial damage, impaired pulmonary function, chronic obstructive pulmonary disease (COPD), and tuberculosis (Mazumder et al. 2000, 2005; Milton and Rahman 2002; Milton et al. 2003; Smith et al. 2006, 2011; Parvez et al. 2008; Rahman et al. 2011). Cross-sectional studies in Bangladesh and India report increased risks of clinical respiratory symp- toms in people who have skin lesions and are exposed to arsenic at concentrations greater than 500 µg/L (Mazumder et al. 2000; Milton and Rahman 2002). A prospective evaluation of the participants (11,746) in the Health Effects of Arsenic Exposure Longitudinal Study in Bangladesh revealed that those in the second quintile of water arsenic (7-40 µg/L) had a 27% increase in risk of respiratory symptoms com- pared with those in the lowest quintile (Parvez et al. 2010). In a subset of the same cohort (950), arsenic exposure was associated with significantly lower forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) in people exposed to water arsenic at concentrations above 97 µg/L than in those

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28 Critical Aspects of EPA’s IRIS Assessment of Inorganic Arsenic exposed at less than 19 µg/L (Parvez et al. 2013). The greatest effect of arsenic was evident in male smokers and people who had skin lesions. A handful of earlier studies in India and Pakistan also observed decreased pulmonary function in those exposed at greater than 250 µg/L (De et al. 2004; von Ehrenstein et al. 2005; Nafees et al. 2011). A retrospective cohort study in Chile reported that in utero arsenic exposure was associated with a decline in FEV1 and FVC in adulthood (Dauphine et al. 2011). In utero exposure to arsenic has also been linked with increased risk of acute respiratory infections in infants in a large cohort (1,551) in Bangladesh (Rahman et al. 2011). A cross-sectional study in India reported a 10-fold increase in the prevalence of bronchiectasis, a specific type of COPD, in people who had skin lesions (108) compared with those who did not (150) (Mazumder et al. 2005). An earlier ecologic study in the arsenic-endemic areas of Chile re- ported an excess of mortality from COPD in people 30–39 years old (especially women) who were ex- posed to a weighted average arsenic concentration of 570 µg/L during 1955–1969 (Smith et al. 1998). Higher mortality from chronic bronchiectasis was observed only in those who were exposed to arsenic during early childhood or in utero (Smith et al. 2006). The ecologic analysis captured mortality in 1989– 2000 in the two cohorts that were exposed to arsenic in early childhood or in utero before (1950–1957) or during (1958–1970) times when the peak exposure reached as high as 1,000 µg/L. Significantly higher risks of death from bronchiectasiswere observed in those who were exposed to arsenic during early child- hood (standardized mortality ratio [SMR] = 12.4; 95% CI 3.3–31.7) or in utero and early childhood (SMR = 46.2; 95% CI 21.1–87.7) (Smith et al. 2006). An ecologic study in Taiwan also reported higher mortali- ty from bronchitis in people living in areas with a high prevalence of blackfoot disease than in a reference population or the rest the country during the peak arsenic exposure (median 780 µg/L, 1971-1994) (Tsai et al. 1999). In addition to COPD and associated outcomes, arsenic exposure has been linked to chronic lung in- fections, especially pulmonary tuberculosis. In the same population in Chile discussed above, Smith and colleagues reported significantly higher mortality from pulmonary tuberculosis (RR = 2.1; 95% CI 1.7- 2.6) resulting from arsenic exposure (Smith et al. 2011). The risk mimicked the exposure history trend in the population (rising risk after chronic exposure and then falling back to normal after cessation of expo- sure). Although that novel finding is consistent with potential immunosuppression effects of arsenic, it has yet to be replicated in other large population studies. A recent study in the United States has reported an association between prenatal inorganic arsenic exposure and increased risk of respiratory disease, such as upper respiratory tract infections and colds (Farzan et al. in press). Mode of Action As discussed in the committee’s workshop by Lantz (2013), a substantial body of literature from both epidemiologic and animal studies of arsenic suggests impaired immune function, aberrant wound repair, and disrupted matrix and barrier function. A major consideration in elucidating the mode of action should be epidemiologic and animal studies that identify potential pathogenic mechanisms in response to low to mod- erate arsenic exposures. As indicated above, results of prospective epidemiologic studies suggest that arse- nic exposures are linked to chronic loss of lung defenses, such as secretion of CC16 protein from airway cells (Parvez et al. 2008, 2010). Subchronic or in utero exposures of mice to low to moderate concentrations of arsenic in drinking water (10–100 ppb) led to a decrease in immune gene expression and aberration in inflammatory protein expression (Kozul et al. 2009a; Ramsey et al. 2013a) that may make mice more sus- ceptible to airway inflammation (Kozul et al. 2009b). However, other mouse studies report that genes that sustain lung and vascular matrix, wound repair, and barrier function are compromised by arsenic exposure (Lantz et al. 2007, 2009; Hays et al. 2008; Petrick et al. 2009). Those changes in matrix and wound repair observed in mice correlate to changes observed in exposed humans and isolated human cells (Josyula et al. 2006; Lantz et al. 2007; Olsen et al. 2008). Increased matrix metalloproteinase-9 (MMP-9) expression and activity may be important biomarkers of inhibitory effects of arsenic on lung function (Josyula et al. 2006;

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Hazard Identification 29 Olsen et al. 2008), although they may also be portions of the overall change in inflammatory gene expres- sion. Another important consideration with respect to mode of action is whether in utero or perinatal expo- sure poses a significant risk of lung dysfunction or disease. Recent animal studies suggest that that may be the case (Lantz et al. 2007, 2009; Hays et al. 2008; Kozul et al. 2009b; Ramsey et al. 2013b), but few epi- demiologic studies have examined affects of in utero arsenic exposure on lung disease. The underlying mechanism of chronic inflammation, impaired immune function, and aberrant matrix maintenance is not clear but may involve arsenic-induced oxidative stress (Lantz and Hays 2006). Protection from oxidative stress can reduce arsenic-induced pulmonary inflammatory responses and injury in mouse models (Zheng et al. 2012; Tao et al. 2013). Key Considerations for the IRIS Assessment Despite a large number of studies that have demonstrated nonmalignant lung disease after exposure to a wide range of arsenic (40-1,000 µg/L), little information is available to determine a full dose- dependent relationship, especially at low to moderate doses. For the IRIS assessment, the challenge is to integrate information from different studies that focus on different types of nonmalignant respiratory out- comes in populations exposed at different doses and for different periods. Critical synthesis of the human population studies coupled with a coherent integration of mode of actions underlying different nonmalig- nant respiratory outcomes and phenotypes should be a focus for consideration by EPA. CARDIOVASCULAR DISEASE In 2012, a systematic review and meta-analysis of epidemiologic reports of arsenic-related cardio- vascular disease concluded that evidence from different countries consistently shows an association be- tween high chronic arsenic exposure and cardiovascular disease (Moon et al. 2012). (See Chapter 3 for comments on the usefulness of this systematic review for EPA’s IRIS assessment.) Until recently, the evaluation of the evidence on low to moderate exposure in drinking water (less than 100 µg/L) has been limited by the low quality of the available studies that support the role of arsenic as a cardiovascular- disease risk factor (Moon et al. 2012). However, a number of epidemiologic studies of arsenic exposures in large populations report dose–response relationships that are useful to evaluate if there is an increased risk of cardiovascular disease at low to moderate arsenic concentrations (Sohel et al. 2009; Wade et al. 2009; Y. Chen et al. 2011a) (see Table 1). The joint evaluation of the dose–response relationship across those studies can provide useful information for performing the IRIS assessment. Cardiovascular disease may be the most important noncancer disease risk posed by environmental arsenic exposures given the high burden of cardiovascular disease worldwide. Although the current studies suggest that cardiovascular disease risk is increased by low to moderate arsenic concentrations in drinking water and possibly food, there is a need to confirm the relationship. Inclusion of the most recently published data should provide an opportunity for establishing better dose– response relationships in the low-dose region. Ample human studies are available to establish dose– response relationships. However, results of a number of animal studies support the identification of cardi- ovascular disease risk and provide valuable information on mode of action that reduces the uncertainty regarding causality (see below). The focus should be on human studies that investigated coronary arterial disease, myocardial infarction, cardiovascular disease, and overall cardiovascular-disease mortality be- cause risk of these disease outcomes appears to be increased by low to moderate exposure. Peripheral ar- terial disease (such as blackfoot disease) can be excluded from the focus because few studies have inves- tigated the association with low to moderate arsenic exposure and the association with high exposure seems to be limited to populations that have poor nutrition (Tseng et al. 2005). Cerebrovascular disease can be included, but there may be few conclusive epidemiologic studies that had adequate exposure and

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TABLE 1 Large Cohort Studies of Overall Cardiovascular Disease and Arsenic Exposure Measured at the Individual Level and Reported in 30 Two or More Arsenic Categories in Populations with Arsenic in Drinking Water at Less than 150 µg/L End Point Arsenic Exposure No. Relative Evaluation of Effect Reference Population Ascertainment Outcome(s) Assessment Categories Cases Risk 95% CI Adjustment Factors Modification Y. Chen et Araihazar, Verbal autopsy, CVD Baseline <12 µg/L 43 1.00 Reference Age, sex, education, Stronger associations al. 2011aa Bangladesh medical records mortality individual well 12.1-62.0 51 1.21 0.80-1.84 BMI, smoking status in current smokers Ages 18-75 y water 62.1-148.0 41 1.24 0.80-1.93 Baseline urine <105.9 µg/g 44 1.00 Reference arsenic (µg/g 106.0-199.0 48 1.15 0.77-1.72 of creatinine) 199.1-351.8 54 1.56 1.03-2.38 Sohel et Matlab, Verbal autopsy CVD Household <10 g/L 129 1.00 Reference Age, sex, education, No differences by sex. al. 2009a Bangladesh mortality well levels 10-49 153 1.03 0.82-1.29 asset score (SES) Other subgroups were Ages ≥15 y 50-150 476 1.16 0.96-1.40 not reported. 50% men Wade et Inner Mongolia Verbal autopsy, CVD Household, <5 µg/L 44 1.00 Reference Age, sex, education, Not reported. al. 2009b (one village) medical-record mortality shared, or 5.1-20 26 1.07 0.64-1.78 smoking status, Ages: 0 to >80 y review community 20.1-100 72 1.22 0.82-1.82 drinking, farm work 50% men well levels Moon et Arizona, Hospitalization CVD Baseline urine <5.8 µg/g 265 1.00 Reference Age, sex, education, Stronger associations al. 2013 Oklahoma, N/S and death records, incidence arsenic (µg/g 5.8-9.7 297 1.14 0.95-1.35 BMI, smoking in participants from Dakota (Strong adjudication by of creatinine) 9.8-15.7 291 1.05 0.87-1.26 status, LDL-chol Arizona, participants Heart Study) physician panel >15.7 331 1.24 1.02-1.50 with diabetes, and Ages: 45-64 participants with 40.8% men CVD Baseline urine <5.8 µg/g 86 1.00 Reference DMA above the mortality arsenic (µg/g 5.8-9.7 95 1.12 0.83-1.52 median. of creatinine) 9.8-15.7 115 1.26 0.92-1.73 >15.7 143 1.65 1.20-2.27 This table is provided as an example that can be useful also for other end points (such as ischemic heart disease). Abbreviations: BMI, body-mass index; CI, confidence interval; CVD, cardiovascular disease; eGFR, estimated glomerular filtration rate; LDL-chol, low-density lipoprotein cholesterol; NR, not reported; SES, socioeconomic status. a Data on exposures greater than 150 µg/L are not presented. b Subset of residents exposed since 1990.

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Hazard Identification 31 outcome assessment at low to moderate exposure. Even in studies that had high exposures, the relation- ship of arsenic to cerebrovascular disease appears inconclusive. Finally, hypertension, although shown to have some relation to arsenic exposure (Abhyankar et al. 2012), is less of a priority inasmuch as there are few studies at low to moderate exposure, most of the evidence available is cross-sectional, and it is not a clinical cardiovascular end point. Mode of Action As with arsenic-induced cancers, there may be several plausible modes of action for arsenic-related cardiovascular conditions. If determination of the mode of action is needed, EPA should consider evi- dence and reviews that suggest that chronic inflammation and reactive oxygen species (ROS) formation are central to the pathogenesis of arsenic-induced cardiovascular disease. Chronic vascular inflammation is suggested by a mechanistic epidemiologic study (Wu et al. 2012) within a large prospective study of cardiovascular-disease mortality associated with arsenic (Y. Chen et al. 2011a). Chronic inflammation may be a derivative of a chronic increase in ROS, a known risk factor for cardiovascular disease. Animal studies have been effective in demonstrating that vascular remodeling occurs in response to low to mod- erate arsenic exposures (10-100 ppb) (reviewed by States et al. 2011) and that ROS generation, especially from NADPH oxidase activation, is essential in promoting vascular pathogenesis (Straub et al. 2008). Atherosclerosis is a primary mechanism in the etiology of arsenic-related ischemic heart disease, and ar- senic-induced atherogenesis has been demonstrated in a genetic mouse model of atherosclerosis after low to moderate exposure to arsenic, as discussed by Lantz (2013) in the committee’s workshop (also see Le- maire et al. 2011). In addition, atherogenic potential in mice may be enhanced by in utero arsenic expo- sures (Srivastava et al. 2007). Alternatively, other modes of action—such as interactions with protein thi- ols to change protein function, disruption of cardiac and vascular matrix after increased expression of matrix-degrading proteases (such as MMP-9) (Soucy et al. 2005; Hays et al. 2008; States et al. 2009; Wu et al. 2012), or interference with transcriptional regulation of metabolic and inflammatory genes (Pado- vani et al. 2010; States et al. 2011)—cannot be ruled out. However, those changes in gene regulation may act through or derive from upstream activation of ROS signaling. Key Considerations for the IRIS Assessment Ample epidemiologic reports find a causal association between arsenic exposure of humans and in- creased risk of cardiovascular disease and mortality. A review of current literature, including recent meta- analyses, provides support for identification of arsenic as a hazard for cardiovascular disease. The recent literature includes large prospective studies that provide an opportunity to establish dose–response rela- tionships for arsenic-induced cardiovascular disease. In evaluating the causal relationship between arsenic exposure and cardiovascular diseases, however, EPA should address potential uncertainties resulting from differences between the study population and the general population. Animal data and data that reveal potential modes of action demonstrate sensitivity of the cardiovascular system to arsenic exposures rele- vant to humans and support identification of modes of action. That can be useful for supporting causality in the epidemiologic studies, help to identify sensitive populations, and inform the dose–response analy- sis. BLADDER EFFECTS Arsenic is a known bladder carcinogen (IARC 2004, 2012). Assessment by IARC included ecologic studies of highly exposed populations in Taiwan, Chile, and Argentina and indicated higher mortality from bladder cancers in exposed than in nonexposed populations. In addition, ecologic studies in Taiwan reported increasing SMRs with higher categories of exposure on the basis of village median water con- centrations of arsenic, and associations with well-water arsenic concentrations were supported by evi-

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32 Critical Aspects of EPA’s IRIS Assessment of Inorganic Arsenic dence from cohort and case–control studies in Taiwan (IARC 2004, 2012). More recently, a case–control study in Chile found evidence of a dose-related increase in bladder-cancer incidence (Steinmaus et al. 2013). Those studies were important for establishing a causal relationship between arsenic exposure and bladder cancer, and, as described later in this report, those from Region II in Chile signal the importance of exposures early in life. Those studies, however, did not estimate the shape of the dose–response curve and cannot inform estimates of the dose–response relationship at lower exposures. There is supportive evidence that drinking-water arsenic could be affecting bladder-cancer occur- rence in the United States; this evidence should be carefully evaluated by EPA, particularly with respect to smoking, which is a known risk factor for bladder cancer (see Chapter 5). The studies include an eco- logic study of the proportion of private-well users that found correlations in New England where high arsenic concentrations in bedrock wells are known to occur (Ayotte et al. 2006). Case–control and cohort studies that detected effects at lower exposure tended to have poor statistical precision and mostly exam- ined dose–response relationships by using categories of exposure. For example, in a categorical analysis in a nested case–control study of male smokers (Michaud et al. 2004), the OR was over 2 in the highest tertile of toenail arsenic in long-term smokers (older than 45 years old), but this was not statistically sig- nificant. Likewise, a case–control study in New Hampshire found about a 2-fold risk of transitional-cell carcinoma of the bladder in the highest exposure category in smokers but with wide confidence intervals (Karagas et al. 2004). However, an analysis of the New Hampshire data that treated exposure as a contin- uous variable observed a dose–response relationship with increasing exposure in smokers, and a case– control study in Nevada reported more than a 2-fold OR of bladder cancer in smokers in the highest cate- gory of estimated cumulative intake of arsenic via drinking water, but it was evident only for an exposure period 40 years before diagnosis (Steinmaus et al. 2003). In an earlier US National Bladder Cancer Study, a linear trend in the ORs for bladder cancer was observed in smokers on the basis of index of arsenic exposure (drinking-water concentrations multiplied by number of years consumed), a trend present for the exposure window of 10–19 years before diagnosis (Bates et al. 1995). Fifty years or more of well-water consumption in a region of Argentina that had high contamination was associated with bladder cancer in a case–control study of smokers (Bates et al. 2004). Estimated drinking-water arsenic exposure (limited to up to 40 years before diagnosis), however, was un- related. A cohort study in Taiwan detected risks at arsenic concentration below 50 µg/L but was based on only a few cases (two men and five women exposed at less than10 µg/L and one man and three women at 10–49 µg/L) (Huang et al. 2008); thus, a dose–response analysis could not be performed. No excess risks were observed in association with the time-weighted average arsenic exposure from drinking water, even among smokers, in a case–control study in Michigan (Meliker et al. 2010). No association was also found in an ecologic analysis of a cohort study of bladder-cancer mortality in Mormons (a nonsmoking popula- tion) in Utah (Lewis et al. 1999) or in an analysis of cancer incidence in an Australian cohort (Hinwood et al. 1999). It is conceivable that lack of associations in studies at low exposure could be due to weak statis- tical power to detect the more modest effects that might be expected, especially in the cohort studies that had few bladder-cancer cases. Studies based on bladder-cancer mortality are inherently biased toward the null because bladder cancer is typically survivable. Mode of Action An emerging body of literature suggests that susceptibility to arsenic exposure may involve genetic factors, and studies have helped to inform understanding of the mechanism of action. Findings include interactions between arsenic exposure and polymorphisms in genes involved in DNA repair, cell cycle, xenobiotic and arsenic metabolism, and metal transport (e.g., Hsu et al. 2008; Andrew et al. 2009b; Kara- gas et al. 2012; Lesseur et al. 2012), and differences in risk depending on percentages or ratios of urinary metabolites (IARC 2012). These studies are discussed in greater detail in Chapter 5 (section on Genetics and Arsenic Metabolism and Toxicity). There is a vast literature on mechanistic studies related to arsenic tumorigenesis. Experimental de- signs range from detailed probing of specific molecular pathways to broad studies of genetic differences

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Hazard Identification 33 associated with carcinogenesis or cancer risk, including gene-expression studies and population-based studies of genomic changes. Potential modes of action have been proposed, such as cytotoxicity and re- generative repair, formation of reactive oxygen species and resulting oxidative stress and DNA damage, impaired DNA repair, dysregulation of signaling pathways that control the fate of cells (cycle progres- sion, proliferation, differentiation, and apoptosis), and perturbations of gene structure. Epidemiologic and experimental studies have shown genetic differences and epigenetic changes in various processes. A mode-of-actin analysis will need to evaluate the results of mechanistic studies. It will need to consider that, just as bladder cancer is not a single disease, there may be multiple means by which arsenic can cause bladder cancer. Which mode of action applies may depend on many factors, including the popula- tions or species in question (in the case of experimental studies), individual genetic differences, nutrition- al differences, magnitude of arsenic exposure, and the period of exposure (e.g., in utero, adult). Key Considerations for the IRIS Assessment There are a number of considerations in assessing the potential relationship between arsenic expo- sure and bladder cancer, especially at levels of exposure relevant to the US population. Evaluation of dose–response relationships may necessitate conversion of categories of exposures to fit continuous dose– response curves as was done in the European Food Safety Authority Assessment (EFSA 2009). Compli- cations in evaluating lower exposures include diet’s possible importance as a contributor to exposure and the need to incorporate considerations regarding smoking and other cofactors. It is crucial to assess expo- sure on an individual level and to include a biomarker and relevant cofactors where possible. A limitation of early ecologic studies of highly exposed populations that relied on mortality data is that bladder cancer is not typically fatal. In addition, timing and duration of exposure can influence the magnitude of the ef- fects of arsenic on bladder-cancer incidence. Associations were observed with more recent exposure in some case–control studies and exposure in the distant past in others. In summary, if possible, the IRIS assessment should focus on the studies of both recent and past exposure that examined bladder-cancer incidence (rather than mortality) and that examined susceptible groups of the population (on the basis of cofactor exposures or genetics) and dose–response relationships. RENAL EFFECTS Adverse effects on the kidneys have been found in different arsenic-exposed populations around the world, including the United States. Renal cancer is relatively rare, but chronic renal disease is common. Although reports vary in study design, have different extents of exposure, and use different outcome measures, they appear to concur that arsenic exposure causes renal disease in humans. Some examples are discussed below, but they are not meant to limit EPA’s evaluation of the literature. Renal Cancer Arsenic has been shown to cause cancer of tissues in the urinary system other than the bladder; for those, there may be different modes of action for cancer development. Broadly speaking, cancer of the urinary system is either renal-cell carcinoma or cancer of the urothelium (which occurs in the renal pelvis and ureters as well as the bladder). In International Classification of Diseases, Ninth Revision (ICD-9) diagnosis codes commonly used in epidemiologic studies of cancers associated with arsenic exposure, urothelial cancer is coded as a subtype of renal cancer rather than bladder cancer. As noted in the commit- tee’s workshop by Cohen (2013), most cancers of the urinary system occur in urothelial tissues, especially the bladder. Increased mortality from cancer of the kidney, including the renal pelvis and ureters (ICD-9, code 189), has been reported in studies in Argentina (Hopenhayn-Rich et al. 1998), Chile (Yuan et al. 2010),

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40 Critical Aspects of EPA’s IRIS Assessment of Inorganic Arsenic Early-life exposure (during pregnancy and infancy) was not associated with infant development as assessed at the age of 7 or 18 months by the Bayley Scales of Infant Development in a large cohort study conducted in Bangladesh (Tofail et al. 2009; Hamadani et al. 2010). However, in a followup study of the same children (more than 1,700), inverse associations between maternal urinary arsenic in pregnancy and verbal/full-scale IQ (Wechsler Preschool and Primary Scale of Intelligence) were found at the age of 5 years of age but only in girls (Hamadani et al. 2011). Associations were stronger with the cross-sectional measures of urinary arsenic, again only in girls. Results of a study of mice support that sexually dimor- phic finding: female pups were more vulnerable than males to the developmental effects of arsenic expo- sure with regard to locomotor activity and depletion of dopamine in the nucleus accumbens (Bardullas et al. 2009). Another study in Bangladesh indicated effects on motor function. Parvez et al. (2011) assessed motor function in 8- to 11-year-old children with the Bruininks-Oseretsky Test of Motor Proficiency and found an inverse association of total composite scores with water arsenic and with biomarkers of arsenic exposure. Whether the effects are peripheral or central nervous system effects is not clear. With respect to behavioral studies, Roy et al. (2011) conducted a cross-sectional study of school-age children (median age 7 years) in Mexico and found that urinary DMA concentrations in boys were associated with higher scores scores on the oppositional, cognitive problems and ADHD subscales of the teacher ratings on the Conners Comprehensive Behavior Rating Scales, although adjustment for cogni- tive-test results diminished the association. That might suggest that the behavioral findings were driven largely by cognitive deficits. Studies in Adults The relationship between arsenic exposure and neurotoxicity in adults is less well studied than in children. A community-based participatory research study conducted in rural elderly adults found that GIS-based groundwater arsenic exposure was significantly related to poorer scores in language, visuospa- tial skills, and executive functioning. Long-term low-level exposure to arsenic was also associated with poorer scores in global cognition and memory (O’Bryant et al. 2011). No biomarkers were collected. The effects of arsenic in elderly populations is a particular research need. Animal and in Vitro Experimental Studies A study by Rodríguez et al. (2002) showed learning and behavioral deficits in rats exposed prenatal- ly to arsenic via maternal drinking water. Increased spontaneous locomotor activity and increased errors in delayed alternation tasks (a test of memory and executive function) were found in arsenic-exposed off- spring compared with nonexposed controls. Animals in the exposed groups consumed arsenic at 2.93– 4.20 mg/kg per day, far more than any human study has reported except Dekeish et al. (2006) in Japan. (That report described the consequences of ingesting arsenic-contaminated infant formula, which led to a mass poisoning and reports of 130 fatalities. An IQ score of less than 85 was reported in 20.6% of survi- vors.) Animal studies have also shown a dose–response relationship between arsenic in the brain and arse- nic in drinking water—a demonstration that arsenic crosses the blood–brain barrier to the central nervous system (Rodríguez et al. 2002; Luo et al. 2009; Xi et al. 2009, 2010). In each of those animal studies, doses of arsenic were probably in the range of milligrams per kilogram per day, although not all studies reported water consumption rates. All studies involved concentrations of inorganic arsenic greater than 1,000 μg/L. Nagaraja and Desiraju (1994) found delayed acquisition and extinction of operant behaviors in rats exposed to sodium arsenate at 5 mg/kg per day in drinking water for 3 months. Increased concen- trations of arsenic in cerebellar tissues have been associated with impaired performance on the Morris water-maze test (working and spatial memory) in mice (Y. Wang et al. 2009). Those deficits might be due to increased oxidative toxicity and changes in neurotransmission.

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Hazard Identification 41 Increased neurotoxic oxidative stress (lowered concentrations of reduced glutathione and increased lipid peroxidation) in mouse brains after oral administration of arsenic trioxide was reported by Rao and Avani (2004) and Chaudhuri et al. (1999). An in vitro study demonstrated that neurite outgrowth was suppressed by sodium arsenite possibly because of induced neuronal apoptosis (Aung et al. 2013). Changes in concentrations of neurotransmitters—such as acetylcholine, dopamine, serotonin, and norepi- nephrine—have been found in the central nervous system after arsenic exposure (Nagaraja and Desiraju 1993, 1994; Chattopadhyay et al. 2002a,b). In studies using brain explants and neuronal cell cultures, neu- ral networking and increased reactive oxygen intermediates were altered after exposure to arsenic (Chat- topadhyay et al. 2002a). In rats exposed to arsenic during pregnancy, fetal brain neurons underwent apop- totic changes and neuronal necrosis (Chattopadhyay et al. 2002b). However, not all studies have demonstrated adverse effects of developmental exposure to inorganic arsenic, and null findings have been reported (Gandhi et al. 2012). There is a growing literature on inorganic-arsenic toxicity that uses doses more commonly encoun- tered in human exposure scenarios. The most commonly used drinking-water concentration is 50 μg/L, which is within the range seen in many epidemiologic studies. Martinez-Finley et al. (2008) focused on perinatal exposure to inorganic arsenic (defined as pregnancy through weaning at the age of 23 days). They reported that inorganic arsenic at 50 μg/L in drinking water was associated with increased depres- sive behaviors. (This period of exposure is parallel to a prenatal-exposure paradigm in humans, and preweaning mice are developmentally equivalent to a third-trimester human fetus.) Plasma corticosterone concentrations were about two-fold higher in the exposed group compared with controls. Hippocampal corticotropin-releasing-factor receptor binding was increased, and serotonin 5HT1A receptor binding was reduced. Overall, the results suggest that affected offspring might be predisposed to depressive-like be- havior because of disrupted regulatory interactions between the hypothalamic–pituitary–adrenal axis and the serotonergic system in the dorsal hippocampal formation. In a followup study by the same group (Martinez-Finley et al. 2009), mice exposed to inorganic arsenic perinatally took longer to acknowledge a novel object and demonstrated deficits in spatial memory on an eight-arm radial-maze task. Arseni- treat- ed animals had lower concentrations of glucocorticoid receptors in the brain coupled with higher plasma corticosterone; this suggests that the lower glucocorticoid receptor concentrations are a primary event that is followed by a compensatory increase in blood concentrations of corticosterone. A third paper by the group (Martinez-Finley et al. 2011) reported glucocorticoid-receptor–mediated transcriptional deficits in the mitogen-activated protein kinase/extracellular signal-related kinase pathway. That pathway plays a critical role in learning and memory formation, and consideration should be given to whether it could be an underlying cause of learning deficits associated with inorganic arsenic. Key Considerations for the IRIS Assessment A growing body of literature on both animals and humans suggests that low to moderation concen- trations of arsenic are associated with neurologic deficits, particularly in IQ tests in children. Animal stud- ies have demonstrated deficits in executive function, motor function, and spatial memory. Mechanisms appear related to oxidative-stress–induced apoptosis and effects on neurotransmitters. Human studies on arsenic neurotoxicity have been conducted primarily in children. Prenatal exposure did not predict per- formance on general developmental tests at the age of 18 months in Bangladeshi children but did predict performance at the age of 5 years; this either suggests a latent period for arsenic exposure in utero or sug- gests that subtle effects of arsenic exposure cannot be detected in the relatively insensitive psychometric tests used in toddlers and can be adequately assessed only at higher ages. Most studies have used IQ tests or other tests of general cognitive function and their subscales. No clear pattern has emerged with respect to nonverbal skills associated with inorganic arsenic exposure; however, deficits in verbal cognitive skills are the most common finding reported in epidemiologic studies (see Table 3).

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42 Critical Aspects of EPA’s IRIS Assessment of Inorganic Arsenic TABLE 3 Most Sensitive Neurodevelopmental End Points in Human Studies Study End Point Verbal Skills Hamadani et al. (2011) Verbal IQ Wasserman et al. (2011) Verbal IQ Von Ehrenstein et al. (2007) Vocabulary Wright et al. (2006) Verbal IQ, verbal learning, story memory Calderón et al. (2001) Verbal IQ Nonverbal Skills Wasserman et al. (2004, 2007) Performance IQ, processing speed Von Ehrenstein et al. (2007) Object assembly, picture completion Rosado et al. (2007) Visual–spatial abilities, digit span, memory, sequencing Parvez et al. (2011) Scores on Bruininks-Oseretsky Test of Motor Proficiency, including total motor composite, body coordination, fine manual control There is evidence of a dose–response relationship in animal studies, but the dose range used is or- ders of magnitude higher than what is typically assessed in human studies. A small body of literature that used 50 ppb during perinatal exposures has demonstrated measurable effects, but there has not been an attempt to delineate the dose–response relationship. Epidemiologic studies are not uniform in how they assess exposure. Studies have used a wide variety of exposure metrics, including concentrations in water, urine, hair, and blood. A compounding difficulty is that few studies are prospective and the dose–response relationship may depend on the timing and duration of exposure. One study that showed a clear dose– response relationship between water arsenic and IQ is the one by Wasserman et al. (2004) in Bangladesh. Lower full-scale and performance IQ scores were observed for each increase in the quartile of water arse- nic. Exposure was similar to that in the United States in the lowest quartiles (less than 5.5 ppb and 6–50 ppb). Water arsenic concentrations of 10 ppb and 50 µg/L were associated with raw score losses of 3.8 and 6.4 points, respectively, but these are not equivalent to IQ points, because the WISC-III has not been standardized in Bangladesh. Results of studies of behavior and more domain-specific tasks (such as exec- utive function and behavior) suggest possible adverse effects, but this aspect of neurotoxicity has been relatively understudied. Overall, more epidemiologic studies are needed to confirm animal findings. Mo- tor function has also been understudied, as have the effects of arsenic exposure on cognitive function in adults, especially the elderly. DIABETES Several epidemiologic studies have evaluated the association between arsenic exposure and the risk of diabetes. In 2011, the National Toxicology Program (NTP) comprehensively evaluated the epidemiologic and experimental evidence on arsenic and diabetes end points (Maull et al. 2012). After considering con- sistency among populations, the strength and temporality of the associations, and biologic plausibility, the NTP review concluded that existing human data provided “limited to sufficient” support of an association between chronic exposure to arsenic at high concentrations (150 µg/L or more in drinking water) and diabe- tes. Below 150 µg/L, concentrations that are more relevant for arsenic risk assessment, the NTP review judged that the evidence was “insufficient” to conclude that arsenic was associated with diabetes because of the lack of prospective studies, limitations in exposure and outcome assessments, and lack of adjustment for relevant confounders. Some studies, however, characterized by better measures of outcome and exposure were supportive of an association, including several cross-sectional studies in Mexico (Coronado-Gonzalez et al. 2007; Del Ra-

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Hazard Identification 43 zo et al. 2011). For dose–response considerations, the Mexican studies included water arsenic concentra- tions of less than 10 µg/L to greater than 100 µg/L (geometric mean 24.4 µg/L) (del Razo et al. 2011). An- other relevant study was conducted in pregnant women who lived in an area of Oklahoma in which at least 25% of drinking-water sources had arsenic concentrations greater than 10 µg/L (Ettinger et al. 2009). In that study, arsenic measured in blood and hair samples at delivery was associated with impaired glucose toler- ance during pregnancy, but the association with hair arsenic was not statistically significant (Ettinger et al. 2009). Evidence from National Health and Nutrition Examination Survey (Navas-Acien et al. 2008, 2009a; Steinmaus et al. 2009), although directly relevant to the US population, was considered limited in the NTP review because of challenges in interpreting urinary arsenic biomarkers in the presence of seafood intake (Navas-Acien et al. 2011). Since the publication of the NTP report (Maull et al. 2012), several studies have been published, in- cluding one cross-sectional study (Gribble et al. 2012) and two prospective studies (James et al. 2013; Kim et al. 2013) conducted in the United States. They were conducted in rural communities that had stable popu- lations and low migration rates from Arizona, Colorado, Oklahoma, North Dakota, and South Dakota. Arse- nic was measured at the individual level either in urine or in drinking water, diabetes was measured by using standardized outcome definitions, and the associations were adjusted for relevant risk factors, including body-mass index and sociodemographic factors. Table 4 provides the evidence from those studies as an il- lustration of important features of the studies that should be considered. Some recent studies have also re- ported that genetic variation in several genes (such as AS3MT, NOTCH2) could increase the risk of type 2 diabetes (Drobna et al 2013; Pan et al. 2013). Overall, recent epidemiologic studies support an association between arsenic at low to moderate concentrations (10–50 µg/L) and diabetes risk. They are directly rele- vant to evaluating a dose–response relationship for US populations and should be carefully reviewed by the IRIS program in evaluating diabetes as a relevant end point to be included quantitatively for arsenic risk assessment. Mode of Action An increasing number of experimental and mechanistic studies have evaluated the potential mode of action of arsenic in causing diabetes. The NTP review of the experimental evidence concluded that although as a whole the experimental literature was inconclusive, recent studies designed to evaluate diabetes-related end points generally supported a link between arsenic and diabetes (Maull et al. 2012). Diabetes could be related to the inhibition of insulin production by pancreatic β cells or the inhibition of basal or insulin- stimulated glucose uptake. Relevant mechanisms by which arsenic could affect β-cell function and insulin sensitivity include oxidative stress, glucose uptake and transport, gluconeogenesis, adipocyte differentiation, calcium signaling, and epigenetic effects (Diaz-Villasenor et al. 2007; Maull et al. 2012). In a genomewide study of peripheral blood lymphocytes in northern Mexico, moderate to high arsenic exposure was associat- ed with hypermethylation and hypomethylation of several diabetes-related genes (Smeester et al. 2011). Additional coexposures, especially to a high-fat diet, could be particularly important for US populations: experimental evidence on rodents suggests that arsenic interacts with a high-fat diet to induce glucose intol- erance without changes in plasma insulin concentrations (Paul et al. 2011). Key Considerations for the IRIS Assessment Results of recent epidemiologic evidence from studies conducted in Mexican and US populations, including prospective studies conducted in Arizona (Kim et al. 2013) and Colorado (James et al. 2013), support the association between low to moderate arsenic exposure and diabetes risk. The studies provide useful information for the IRIS assessment to use in evaluating the dose–response relationship at arsenic concentrations relevant for US populations. Mechanistic and animal evidence constitutes information that is relevant for evaluating the mode of action in arsenic-related diabetes. The NTP review, systematic re- views of the literature, and the possible conduct of dose–response meta-analyses should guide the inclu- sion of diabetes as a relevant health end point for quantitative arsenic risk assessment.

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TABLE 4 Recent Studies of Diabetes and Arsenic Measured at the Individual Level That Reported Two or More Arsenic Categoriesa 44 Exposure Diabetes Arsenic Categories, Relative Adjustment Evaluation of Effect Reference Design Population Definition Assessment µg/L C/NC Risk 95% CI Factors Modification Gribble et CS Arizona, Prevalent diabetes Baseline total <7.9 413/558 1.00 Reference Age, sex, BMI, Association was al. 2012 Oklahoma, N. (FPG ≥ 126 arsenic in 7.9–14.1 492/507 1.26 1.14–1.39 education, found mostly in those and S. Dakota mg/dL, OGTT ≥ population with 14.1–24.2 503/467 1.38 1.25– 1.52 smoking, with poor diabetes (Strong Heart 200 mg/dL, HbA1c low seafood ≥24.2 531/454 1.55 1.39– 1.72 alcohol, control and was stronger Study) ≥ 6.5%, or diabetes intake p trend <0.001 urinary in former smokers and Ages: 45-64 medication) creatinine in N/S Dakota years 40.8 % men Kim et al. Nested Arizona Incident diabetes Baseline total 6.6–15.3 1.00 Reference Age, sex, BMI, Not evaluated 2013 CC (Southwestern (OGTT ≥ 200 urine arsenic in 15.3–21.0 1.3 0.12 urinary (small sample) American mg/dL) population with 21.1–29.3 2.2 creatinine Indians) low seafood 29.4–123.1 1.3 Ages: ≥25 intake p trend years No sex data 0.1–4.5 1.00 Reference Baseline inorganic 4.6–6.9 2.4 0.06 arsenic (µg/g of 7.0–9.4 2.3 creatinine) 9.5–36.0 1.8 p trend James et Case- San Luis Incident diabetes Time-weighted 1–3 120 1.00 Reference Age, sex, Not evaluated al. 2013 CO Valley, (FPG ≥ 140 average arsenic in 4–7 148 1.11 0.82–1.95 race, income, (small sample) Colorado mg/dL, OGTT ≥ drinking water 8–19 139 1.42 0.94– 2.48 BMI, physical Ages: 20-74 200 mg/dL, or self- ≥20 141 1.55 1.00– 2.51 activity, years reported diagnosis p trend 0.04 smoking, 46% men and diabetes alcohol, medication) family history a The studies were conducted in populations with drinking water concentrations of arsenic less than 100 µg/L in the United States and were published after the NTP (2011) review. Abbreviations: BMI, body-mass index; CC, case–control; CO, cohort; CS, cross-sectional; C/NC, cases/noncases; FPG, fasting plasma glucose; HbA1c, hemo- hemoglobin A1C; OGTT, oral glucose tolerance test.

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Hazard Identification 45 EFFECTS ON LIVER, PROSTATE, AND PANCREAS Liver Arsenic is known to have potential carcinogenic and toxic effects on the liver (IARC 2004, 2012). Ecologic epidemiologic studies of populations in Taiwan and Chile have reported increased liver-cancer mortality in relation to arsenic exposure. In studies conducted in southwest Taiwan, Chen et al. (1986) reported an age- and sex-adjusted OR of 2.67 for liver-cancer mortality in those who had used well water that had high arsenic concentrations for 40 years or more compared with a relatively nonexposed area. In another study, they reported a dose–response relationship between arsenic concentrations in artesian well water and mortality associated with a number of cancers, including liver cancer in males, but the trend in females was not significant (Wu et al. 1989). In southwest Taiwan, in an area that had a median arsenic concentration in drinking water of 780 µg/L, Tsai et al. (1999) reported an increase in mortality due to liver cancer compared with local and national reference groups. Rivara et al. (1997) in a study in Antofa- gasta and Calama-Chuquicamata, Chile, determined that in an area that had high concentrations of arsenic in both the soil and water, some as high as 800 µg/L of drinking water, there was an increase in mortality (RR = 2.2) due to liver cancer in comparison with a region that had low concentrations of arsenic. Smith and co-workers (Smith et al. 1998; Liaw et al. 2008; Smith et al. 2012) also studied that population in An- tofagasta, Chile. In their initial study (Smith et al. 1998), they observed increases in mortality associated with bladder and lung cancer but no increase in overall liver-cancer mortality from arsenic. In a later study, they found increased liver-cancer mortality in those who were exposed in utero and when young during peak exposure periods (before a change in water treatment). Specifically, they found higher child- hood mortality from liver cancer than expected (RR = 10.6) (Liaw et al. 2008) and increased liver-cancer mortality in young adults (SMR = 2.5) (Smith et al. 2012), albeit on the basis of a small number of cases. Not all epidemiologic studies have identified increases in liver cancer in relation to arsenic expo- sure. In a study of cancer mortality associated with arsenic in the drinking water in Cordoba, Argentina, although increases in lung and renal cancer incidence were found to be dose-dependent, mortality from liver cancer was similar in all three arsenic-exposed (low, medium, and high) groups (Hopenhayn-Rich et al. 1998). Guo (2003) used data from the Taiwan National Cancer Registry on over 40,000 patients in 243 townships who had liver cancer to determine the distribution of hepatocellular carcinoma and cholangio- carcinoma. No difference in the distribution of cancer cell types was found between the townships with endemic arsenic intoxication and the other townships. The study also found no association between arse- nic and hepatocellular carcinoma. Baastrup et al. (2008) also found no increase in liver-cancer cases asso- ciated with arsenic in a Danish cohort study that had low drinking-water concentrations of arsenic (0.05– 25.3 µg/L). However, none of those studies examined timing of exposure. In studies of liver toxicity, Islam et al. (2011) measured arsenic concentrations in the hair and nails of 200 people in Bangladesh (which correlated with drinking-water concentrations) and found that indica- tors of liver toxicity (serum alkaline phosphatase, aspartate transaminase, and alanine transaminase) were significantly higher in the high-exposure group (over 50 µg/L in drinking water). Similarly, in a study of people in West Bengal, India, Das et al. (2012) found higher concentrations of bilirubin, alanine transam- inase, aspartate transaminase, and antinuclear antibodies (an indicator of autoimmune status) in the serum of people who lived in an area that had an average arsenic concentration of 203 µg/L in drinking water than in serum of nonexposed people. Arsenic and liver cancer have been studied in experimental animals. Waalkes et al. (2003, 2004a,b, 2006) found that male mice from dams exposed to sodium arsenite (0, 42.4, and 85 ppm) in drinking wa- ter on gestation days 8–18 had dose-dependent increases in hepatocellular-carcinoma incidence and mul- tiplicity. An increase in hepatocellular carcinomas was also found in male and female mice exposed to arsenic (0, 6, 12, and 24 ppm in drinking water) in utero and over their lifetime (Tokar et al. 2011b).

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46 Critical Aspects of EPA’s IRIS Assessment of Inorganic Arsenic Key Considerations for the IRIS Assessment There is some epidemiologic evidence that liver cancer may be associated with arsenic exposure, in particular with exposure early in life, but the total body of work is not completely coherent. There is also evidence of hepatocyte damage, but overall the studies are not conclusive, especially with respect to low to moderate arsenic exposure. Thus, liver disease is unlikely to have high priority for the IRIS assessment. Prostate The prostate is among the tissues potentially associated with arsenic toxicity, including cancer (IARC 2004). In a number of studies of prostate and other cancers in Taiwan, increased prostate-cancer mortality was noted with a dose-related gradient of arsenic in drinking water from wells (Chen et al. 1985; Wu et al. 1989; Chen and Wang 1990; Yang et al. 2008). Notably, in a cohort study of mortality from malignant neoplasms in 314 precincts and townships, an age-adjusted increase in mortality from prostate cancer was associated with arsenic concentrations in well water (Chen and Wang 1990). The au- thors also found that mortality from prostate cancer declined when the contaminated water sources were replaced (Yang et al. 2008); this supports a causal link. Lewis et al. (1999) examined the relationship be- tween arsenic in drinking water and prostate cancer in an ecologic analysis of a cohort of Mormon resi- dents of Utah and found an SMR of 1.45 and support of a dose–response relationship. In an ecologic study in Canada that evaluated areas that had soil concentrations of arsenic greater than 100 mg/kg and water concentrations greater than 10 µg/L, Hinwood et al. (1999) found an increase in prostate cancer (standardized incidence ratio 1.14) that was the only cancer significantly increased after stratification into exposure categories. In American Indians in Arizona and North and South Dakota who participated in the Strong Heart Study, baseline urinary arsenic concentrations (median 9.7 µg/g of creatinine; interquartile range 5.8–15.6) were associated with increased prostate-cancer mortality over almost 20 years of fol- lowup (Garcia-Esquinas et al. in press). However, associations with prostate cancer have not been consistently found in epidemiologic stud- ies. Rivara et al. (1997) examined mortality from prostate cancer in residents of two regions in Chile, one of which was exposed to high concentrations of arsenic in drinking water (some as high as 800 µg/L), and found an RR of 0.9. Baastrup et al. (2008) found no increase in mortality from prostate cancer due to ar- senic in a Danish drinking-water cohort study in which exposures ranged from 0.05 to 25.3 µg/L, alt- hough most participants were exposed at below 2 µg/L. Mode of Action A series of studies that used cell culture have also suggested that arsenic has an ability to transform normal cells into a cancer-like state. Achanzar et al. (2002) incubated human prostate epithelial cells (RWPE-1) in 5 µM arsenic and found a malignant transformation of the cells that produced epithelial-cell tumors when placed in nude mice. Benbrahim-Tallaa et al. (2005) found that that transformation was as- sociated with aberrant genomic DNA methylation and K-ras oncogene activation at the same arsenic con- centration. It was associated with Ras signaling activation (Benbrahim-Tallaa et al. 2007). Similarly, To- kar et al. (2010a) found that arsenic at 5 µM transformed the human prostate epithelial/progenitor cell line WPE-stem to a malignant cancer stem-cell–like phenotype. Singh et al. (2011) found that exposure of human prostate epithelial cells (RWPE-1) to arsenic caused nuclear DNA damage and mutations in mito- chondrial DNA and resulted in increased cell survival when arsenic concentrations were as low as 8 pM. They also found (Treas et al. 2012) that arsenic in combination with estrogen altered epigenetic regulato- ry-gene expression, global DNA methylation, and histone modification in those cells.

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Hazard Identification 47 Key Considerations for the IRIS Assessment There is some, albeit modest epidemiologic evidence of an association between arsenic and prostate cancer, including two studies in the United States. In vitro studies of arsenic suggest that a change in phe- notype may occur in isolated prostate cells exposed to arsenic. Given that evidence and the burden of prostate disease in US men and its associated health-care costs, the committee believes that the IRIS as- sessment of inorganic arsenic should at least consider the issue of prostate cancer. Pancreas Increased incidence of or mortality from pancreatic cancer in relation to arsenic-contaminated drink- ing water has not been reported in populations of Taiwan, Argentina, Chile, or Bangladesh that were ex- posed to high concentrations of arsenic (IARC 2004). However, increased pancreatic-cancer incidence was found in an ecologic comparison of children born before and after a 1955 episode of arsenic- contaminated milk powder (used for bottle-fed infants) in Japan (Yorifuji et al. 2010, 2011). Excess mor- tality from pancreatic cancer was observed in children younger than 5 years old during the contamination episode. The study was ecologic, and exposure occurred only during a limited period early in life, so an evaluation of a dose–response relationship was not possible. Recent studies at low to moderate exposure have also reported an association between arsenic and pancreatic cancer. In a hospital-based case–control study in Spain, the OR for pancreatic cancer when the highest and lowest quartiles of toenail arsenic con- centrations were compared (>0.106 vs ≤0.052 µg/g) was 2.02 (95% CI 108–3.78) (Amaral et al. 2012). In American Indians in Arizona and North and South Dakota who participated in the Strong Heart Study, baseline urinary arsenic concentrations (median 9.7 µg/g of creatinine, interquartile range 5.8–15.6) were associated with increased pancreatic-cancer mortality over almost 20 years of followup (Garcia-Esquinas et al. in press). Although limited because of uncertain exposure assessment, GIS analysis in Florida also noted higher pancreatic-cancer incidences in relation to residential proximity to wells that had arsenic concentrations above 10 µg/L (Liu-Mares et al. 2013). Although the mechanism has not been character- ized, one hypothesis is that arsenic increases the risk of diabetes (see section “Diabetes: above), which is a risk factor for pancreatic cancer. Mode of Action Studies of arsenic as a causative agent of pancreatic cancer are few. Results of studies of arsenic tri- oxide by Xue et al. (2009) indicated a dose-dependent effect on AR42J cells (rat pancreatic epithelial cell line). At low concentrations, it caused apoptosis; but at high concentrations (8 µM), it caused oncosis as identified by an increase in malignant changes in the cells detected with laser scanning confocal micros- copy. Key Considerations for the IRIS Assessment Although there is emerging evidence of potential carcinogenic effects of arsenic on the pancreas, it may be difficult for EPA to establish risks on the basis of available evidence. IMMUNE EFFECTS In the hazard assessment of inorganic arsenic, it is important to consider effects on the immune sys- tem. The immune system is required to protect against infection. Dysregulation of the immune system may influence cancer and cardiovascular-disease risk. Both innate and adaptive immune responses should be considered. Epidemiologic, rodent, and in vitro data all support a relationship between inorganic arse-

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48 Critical Aspects of EPA’s IRIS Assessment of Inorganic Arsenic nic exposure and immune system effects. The immunologic effects could include sensitization to expo- sure to viruses and bacteria; such effects have been seen in human populations exposed to arsenic. It is also important to consider the relation of adverse effects on the immune system to periods of developmental susceptibility, when the thymus plays a substantial role. In early pregnancy, for example, the immune system changes toward Th2 cytokine response, which protects the fetus from being recog- nized as foreign (Calleja-Agius and Brincat 2008). As a result, Th1-dependent cytokine production is suppressed, and the child is born with Th2-biased immune responses. After birth, the immune system re- quires maturation of the Th1 cytokine response to achieve effective resistance to diseases (Dietert and Zelikoff 2008). That series of prenatal and postnatal events seems to be more sensitive to toxic insult than is the adult immune system (Dietert 2009). Suppression of theTh1-dependent function increases suscepti- bility to infections and reduces response to childhood vaccination (Heilmann et al. 2006; Vorderstrasse et al. 2006). The hazard assessment of inorganic arsenic should include the following body of literature and inte- grate mode-of-action information where possible. Epidemiologic Evidence Results of several cross-sectional studies in both children and adults indicate that arsenic may be immunosuppressive. Results of recent studies indicate that arsenic is immunotoxic early in life when the thymus is an essential organ of the immune system. In a mother–child cohort in rural Bangladesh, thymus size, measured with ultrasonography, was inversely associated with maternal exposure to arsenic through drinking water, measured as the concentration of arsenic metabolites in urine in early and late pregnancy (range 5.5–1150 µg/L; n = 1,556) (Moore et al. 2009), which corresponded to similar concentrations in the drinking water (Vahter et al. 2006). Maternal urinary arsenic was also positively associated with diar- rhea and acute respiratory infections in the infants (n = 1,552) (Rahman et al. 2011). The findings suggest that in utero arsenic exposure impaired child thymus development and increased morbidity, probably via immunosuppression. The results of those studies are supported by those of a recent study conducted in the United States in which low prenatal inorganic arsenic exposure was associated with an increased risk of respiratory disease, such as upper respiratory tract infections and colds (Farzan et al. 2013). With respect to mode of action, it is important to consider the relationship of dose and time dependence of exposure to key immunologic events. In a followup of the Bangladeshi study (Ahmed et al. 2011), immune and inflammatory markers, measured with immunohistochemistry and multiplex cytokine assay, were assessed in 130 women. Sig- nificant positive associations between maternal urinary arsenic and placental markers of 8-oxoguanine (8- oxoG) and proinflammatory cytokines—such as interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNFα), and interferon-gamma (IFNγ)—were found. The 8-oxoG measured in the placenta was also posi- tively associated with the placental proinflammatory cytokines. Urinary arsenic in early gestation was inversely associated with CD3+ T cells in the placenta. In a study in Thailand, researchers examined the extent to which maternal arsenic exposure affects gene expression in the newborn by assessing cord-blood genomic signaling. The researchers monitored gene-expression profiles in a population of 32 newborns whose mothers experienced varied arsenic expo- sure during pregnancy. The investigators used genomewide gene-expression analyses to identify genes that were predictive of prenatal arsenic exposure in a later test population. Those genes could be reduced to a set of 11 transcripts that maintained the maximal predictive capacity to classify prenatal arsenic expo- sure. Analyzing the genes in the context of their protein–protein and protein–DNA interactions demon- strated that prenatal arsenic exposure was associated with genes involved in stress, inflammation, metal exposure, and apoptosis in the newborn (Fry et al. 2007). Many of those genes are key players in the im- mune response. Exposure in the study was determined on the basis of arsenic in toenail samples. A toe- nail-water comparison suggested that the cohort was exposed to high concentrations of inorganic arsenic as evidenced by toenail measurements of up to 68.63 µg/g. A dose–response relationship was observed

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Hazard Identification 49 between arsenic in toenails and the expression-level changes in the identified set of genes. Moreover, the genes shared binding sites for transcription factors that are known to be metal-responsive (such as metal- responsive transcription factor 1). Experimental Evidence Researchers examined mRNA and protein-expression changes in the lungs of mice that were chroni- cally exposed to arsenic in food or drinking water to evaluate whether immune modulation contributes arsenic-related disease risk in the lung (Kozul et al. 2009a). Groups of C57BL/6J mice were exposed to arsenic in drinking water or food at 10 or 100 ppb for 5–6 weeks. A whole-genome transcriptome profil- ing assay of the lungs revealed substantial alterations in the expression of genes involved in innate im- mune response and in cell adhesion and migration, channels, receptors, differentiation, and proliferation. The investigators further demonstrated that genes for IL-1β, IL-1 receptor, a number of toll-like receptors, and several cytokines and cytokine receptors were also altered (Kozul et al. 2009a). Following up on that work, the researchers investigated the effects of arsenic exposure on respirato- ry influenza A (H1N1) virus infection. C57BL/6J mice were exposed to arsenic at 100 μg/L in drinking water for 5 weeks and were then infected with influenza A/PuertoRico/8/34 (H1N1) virus by intranasal inoculation. Increased morbidity was observed in the mice, and higher titers of influenza were found in whole-lung homogenates. Several alterations in immune response were also found. For example, a de- crease in dendritic cells in the mediastinal lymph nodes was observed early in infection relative to nonex- posed controls (Kozul et al. 2009b). Acharya et al. (2010) investigated arsenic-induced carcinogenesis and effects on the immune system in a mouse model. Ethylnitrosourea was used to induce tumors in mice, and arsenic was used as a pro- moter. Effects on the immune system were assessed on the basis of cytokine (TNFα, IFNγ, IL-4, IL-6, IL- 10, and IL-12) production of lymphocytes and the specific apoptotic cascade in lymphocytes. Arsenic, especially in combination with ethylnitrosourea, induced marked neoplastic changes. Those findings were supported in histologic studies and by evidence of severe immune suppression that resulted from cytokine modulation and lymphocyte death. Key Considerations for the IRIS Assessment Taken together, the evidence of a relationship between exposure to inorganic arsenic and altered immune function warrants consideration of the immune system in the IRIS assessment. The immunologic changes seem to increase the risk of respiratory symptoms in particular. Those effects have been observed after both lower and higher exposures to inorganic arsenic, although few studies are available on low to moderate exposure. It is conceivable that such effects may contribute to impairmet in fetal and infant health and to detrimental health effects in adults. SUMMARY To help EPA set priorities among its efforts, the committee has created the following hierarchy of health end points of concern:  Tier 1: Evidence of a causal association determined by other agencies and/or in published sys- tematic reviews. o Lung, skin, and bladder cancer (Celik et al. 2008; IARC 2012). o Ischemic heart disease (Navas-Acien et al. 2005; C.H. Wang et al. 2007; Moon et al. 2012). o Skin lesions (ATSDR 2007).  Tier 2: Other priority outcomes.

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50 Critical Aspects of EPA’s IRIS Assessment of Inorganic Arsenic o Prostate and renal cancer. o Diabetes. o Nonmalignant respiratory disease. o Pregnancy outcomes (infant morbidity). o Neurodevelopmental toxicity. o Immune effects.  Tier 3: Other end points to consider. o Liver and pancreatic cancer. o Renal disease. o Hypertension. o Stroke. o Pregnancy outcomes (fetal loss, stillbirth, and neonatal mortality). The first tier consists of end points that have been identified by other agencies or in systematic reviews as having a causal association with inorganic arsenic. The second tier consists of other high-priority out- comes of concern. The last tier consists of end points for which the evidence appears to be less strong. These categorizations will be refined by EPA after it conducts its more comprehensive analysis. EPA’s draft plans indicate that a causal determination framework will be used to categorizethe evi- dence on the different end points into five possible categories: causal relationship, likely to be a causal relationship, suggestive of a causal relationship, inadequate to infer a causal relationship, and not likely to be a causal relationship. The committee supports that five-category approach and recommends that strength-of-evidence judgments be characterized with respect to the modified Bradford Hill criteria for causality. In evaluating the studies, it will be important to consider the timing of exposure with respect to life stage, duration of exposure, and the latent period for the health outcome. The assessment of causality will help EPA to set priorities among end points for later analysis of mode of action and dose–response relationships.