Evidence for Association of Human Bladder Cancer With Chlorination Disinfection By-Products

Transcription

1 Evidence for Association of Human Bladder Cancer With Chlorination Disinfection By-Products Web Report #4530 Subject Area: Water Quality

2 Evidence for Association of Human Bladder Cancer With Chlorination Disinfection By-Products

3 About the Water Research Foundation The Water Research Foundation (WRF) is a member-supported, international, 501(c)3 nonprofit organization that sponsors research that enables water utilities, public health agencies, and other professionals to provide safe and affordable drinking water to consumers. WRF s mission is to advance the science of water to improve the quality of life. To achieve this mission, WRF sponsors studies on all aspects of drinking water, including resources, treatment, and distribution. Nearly 1,000 water utilities, consulting firms, and manufacturers in North America and abroad contribute subscription payments to support WRF s work. Additional funding comes from collaborative partnerships with other national and international organizations and the U.S. federal government, allowing for resources to be leveraged, expertise to be shared, and broad-based knowledge to be developed and disseminated. From its headquarters in Denver, Colorado, WRF s staff directs and supports the efforts of more than 800 volunteers who serve on the board of trustees and various committees. These volunteers represent many facets of the water industry, and contribute their expertise to select and monitor research studies that benefit the entire drinking water community. Research results are disseminated through a number of channels, including reports, the Website, Webcasts, workshops, and periodicals. WRF serves as a cooperative program providing subscribers the opportunity to pool their resources and build upon each other s expertise. By applying WRF research findings, subscribers can save substantial costs and stay on the leading edge of drinking water science and technology. Since its inception, WRF has supplied the water community with more than $460 million in applied research value. More information about WRF and how to become a subscriber is available at

4 Evidence for Association of Human Bladder Cancer With Chlorination Disinfection By-Products Steve E. Hrudey University of Alberta, Edmonton, Alberta, T6G2G3 Canada Lorraine C. Backer Centers for Disease Control and Prevention, Atlanta, GA Andrew R. Humpage SA Water, 250 Victoria Square, Adelaide, SA 5000, Australia Stuart W. Krasner Metropolitan Water District of Southern California, Los Angeles, CA Dominique S. Michaud Tufts University School of Medicine, Boston, MA Lee E. Moore Epidemiologist, Bethesda, Maryland Philip C. Singer University of North Carolina, Chapel Hill, NC Benjamin D. Stanford Hazen and Sawyer, Raleigh, NC Jointly sponsored by: Water Research Foundation 6666 West Quincy Avenue, Denver, C and American Water Works Association (AWWA) Published by:

5 DISCLAIMER This study was funded by the Water Research Foundation (WRF) and the American Water Works Association (AWWA). WRF and AWWA assume no responsibility for the content of the research study reported in this publication or for the opinions or statements of fact expressed in the report. The mention of trade names for commercial products does not represent or imply the approval or endorsement of WRF or AWWA. This report is presented solely for informational purposes. The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Copyright 2015 by Water Research Foundation ALL RIGHTS RESERVED. No part of this publication may be copied, reproduced or otherwise utilized without permission. ISBN Printed in the U.S.A.

6 CNTENTS TABLES... vii FIGURES... ix FREWRD... xi ACKNWLEDGMENTS... xiii EXECUTIVE SUMMARY... xv CHAPTER 1: BACKGRUND... 1 History... 1 The Human Health Issue... 3 CHAPTER 2: BLADDER CANCER AS A MAJR PUBLIC HEALTH ISSUE... 5 Context Types of Bladder Cancer... 6 Risk Factors/Causes of Bladder Cancer... 7 CHAPTER 3: EVALUATIN F EVIDENCE FR BLADDER CANCER RISK ASSCIATIN WITH CXDBPs Evidence Available to Deliberations for the Stage 2 DBP Rule Evidence Available Since Deliberations for the Stage 2 DBP Rule Chevrier et al. (2004) Bove et al. (2007) Villanueva et al. (2007) Cantor et al. (2010) Costet et al. (2011) Jeong et al. (2012) Cancer Risk Assessment Comparisons CHAPTER 4: SUMMARY AND DISCUSSIN F FINDINGS Major Findings verview Summary of the Evidence Strengths and Limitations of the Evidence for Linking CxDBPs and Bladder Cancer Knowledge Gaps Evidence Supporting Better Risk Management CHAPTER 5: RESEARCH PTINS AND RECMMENDATINS Research ptions Recommended for Consideration Valuable Studies Currently Underway Lower Priority Research ptions v

7 REFERENCES...63 ABBREVIATINS AND ACRNYMS...75 Appendix A: asses of Chlorination DBPs Appendix B: Analytical Epidemiology Studies Addressing Urinary Bladder Cancer...87 Appendix C: Quantitative Cancer Risk Estimate from Toxicology Data to Compare with Epidemiological Risk Estimate...97 vi

8 TABLES 1.1 Drinking water guidelines for organic disinfection by-products (DBPs) Probability of Developing Bladder Cancer* over Selected Age Intervals by Sex in the United States IARC List of assifications by cancer sites with sufficient or limited evidence in humans, Volumes 1 to 109 (Last update: 18 February 2014) Analytical framework for judging strength of epidemiologic evidence Application of an analytical frame to a variety of risk factors for bladder cancer Comparison of risk estimates (R) of bladder cancer for males in Cantor et al. (1998): original analysis versus reanalysis Risk estimates (R) of bladder cancer for males in Cantor et al. (1998): new exposure categories in reanalysis Comparison of risk estimates (Rs) of bladder cancer for King and Marrett (1996): original analysis versus re-analysis Assignment of THM levels by process and number of plants over time Location, water quality and THM composition for Villanueva et al. (2007) as inferred from Villanueva et al. (2003b, 2006b) and Salas et al(2013) Location, water quality and Haloacetic Acid (HAA) composition for Villanueva et al. (2007) as inferred from Villanueva et al. (2003b, 2006b) and Salas et al. (2013) Bladder cancer odds ratio in relation to residential THM4 exposure (Cantor et al. 2010) for specified genotype comparisons...47 vii

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10 FIGURES 3.1 Trends in drinking water source over the years in all geographic study areas for controls in a hospital-based case-control study conducted in Spain, Trends in drinking water source over the years in geographic areas with high THM4 levels for controls in a hospital-based case-control study conducted in Spain, ix

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12 FREWRD The Water Research Foundation (WRF) is a nonprofit corporation dedicated to the development and implementation of scientifically sound research designed to help drinking water utilities respond to regulatory requirements and address high-priority concerns. WRF s research agenda is developed through a process of consultation with WRF subscribers and other drinking water professionals. WRF s Board of Trustees and other professional volunteers help prioritize and select research projects for funding based upon current and future industry needs, applicability, and past work. WRF sponsors research projects through the Focus Area, Emerging pportunities, and Tailored Collaboration programs, as well as various joint research efforts with organizations such as the U.S. Environmental Protection Agency and the U.S. Bureau of Reclamation. This publication is a result of a research project fully funded or funded in part by WRF subscribers. WRF s subscription program provides a cost-effective and collaborative method for funding research in the public interest. The research investment that underpins this report will intrinsically increase in value as the findings are applied in communities throughout the world. WRF research projects are managed closely from their inception to the final report by the staff and a large cadre of volunteers who willingly contribute their time and expertise. WRF provides planning, management, and technical oversight and awards contracts to other institutions such as water utilities, universities, and engineering firms to conduct the research. A broad spectrum of water supply issues is addressed by WRF's research agenda, including resources, treatment and operations, distribution and storage, water quality and analysis, toxicology, economics, and management. The ultimate purpose of the coordinated effort is to assist water suppliers to provide a reliable supply of safe and affordable drinking water to consumers. The true benefits of WRF s research are realized when the results are implemented at the utility level. WRF's staff and Board of Trustees are pleased to offer this publication as a contribution toward that end. Denise L. Kruger Chair, Board of Trustees Water Research Foundation Robert C. Renner, P.E. Executive Director Water Research Foundation xi

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14 ACKNWLEDGMENTS The Interdisciplinary Expert Panel met in Washington, D.C. on March 24 and 25, 2014 to review the evidence and worked with the Chair of the Panel to produce this consensus report. The Washington workshop was attended by Project Advisory Committee members Mike Hotaling, Edward Means, Stig Regli, Steve Via and Djanette Khiari. bservers at the workshop included Richard Weisman, EPA, Hannah Holsinger, EPA, Will Sander, (AAAS Fellow) EPA Alan Roberson, AWWA, and, EPA. xiii

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16 EXECUTIVE SUMMARY BJECTIVES The Water Research Foundation and the American Water Works Association commissioned an interdisciplinary expert panel (the authors of this report) to address three fundamental questions regarding disinfection by-products (DBPs): 1. What are the qualitative strengths and limitations of the evidence for known and/or unidentified chlorination disinfection by-products (CxDBPs) that inform causality of human urinary bladder cancer? 2. What critical knowledge gaps exist regarding human urinary bladder cancer risk in relation to drinking water chlorine and/or chloramine-based disinfection practice(s) in the United States? 3. Does the available evidence support further quantitative and qualitative analysis of CxDBP risk management strategies? Would this further analysis be more accurate and informative for guiding risk management than the available evidence being used to inform the Stage 2 DBP Rule (EPA 2006a)? BACKGRUND Forty years ago, Rook (1974) and Bellar et al. (1974) independently published the discovery that chlorination of natural organic matter in drinking water produced trihalogenated methanes (THMs), including chloroform, bromodichloromethane (BDCM), dibromochloromethane (DBCM) and bromoform (referred to hereafter as THM4). This discovery was followed by a cancer bioassay finding that chloroform was carcinogenic in rodents, leading to the banning of chloroform in a wide range of consumer products such as cough syrups, antihistamines, decongestants, and cosmetics (NTP 2014). This finding for chloroform was obtained by dosing rodents with near lethal levels of chloroform dissolved in corn oil, and the cancer response was subsequently shown to result from the experimental protocol (Jorgenson et al. 1985, Bull et al. 1986). Chloroform itself has now been judged to pose no cancer risk to humans via normal drinking water exposures (Butterworth 2006, Butterworth and Bogdanffy 1999, EPA 2000, EPA 2006a). Subsequently, hundreds of new DBPs were discovered with varying degrees of toxicity and potential cancer risk. DBPs are produced to some degree by any chemical disinfection process, but those produced by disinfection processes involving chlorine (i.e., including chloramination) have attracted the most attention. For the purposes of this report, DBPs produced by any disinfection process involving any chlorine (excluding chlorine dioxide) are collectively termed CxDBPs. CxDBPs do not necessarily contain chlorine, but do include non-halogenated nitrogenous DBPs like nitrosamines. Numerous cancer sites have also been evaluated for association with chlorinated drinking water (Mills et al. 1998), including two IARC reviews of the evidence for specific CxDBPs (chloramines, chloral, chloral hydrate, dichloroacetic acid, trichloroacetic acid and 3-chloro-4-[dichloromethyl]-5-hydoxy-2[5H]-furanone [MX] [IARC 2004], and bromochloroacetic acid, dibromoacetic acid, and dibromoacetonitrile [IARC 2013]). To date, epidemiologic evidence related to urinary bladder cancer has shown the greatest consistency xv

17 (EPA 2000), and bladder cancer was a key aspect of the cost-benefit analysis for the Stage 2 DBP Rule (EPA 2006b). Consequently, the focus of this report is specifically on reviewing and assessing the evidence for an association between exposure to CxDBPs from drinking water and human bladder cancer. Bladder cancer is an important public health issue, causing the fifth-highest number of estimated new U.S. cases of cancer in 2015 (American Cancer Society 2015) for both sexes, accounting for about 74,000 estimated new cases out of 1.66 million (4.5%) new cases for all cancer sites. Bladder cancer accounted for about 16,000 estimated deaths out of the 590,000 (2.7%) U.S. deaths for all cancer sites in The risk for bladder cancer rises exponentially for people over age 40. The U.S. median age at diagnosis is 73, with 91% of new cases incident after age 55, 72% after age 65 and 45% after age 75 (SEER 2015). In the United States, bladder cancer is currently three times more common in men than in women. The National Cancer Institute (2014) summarizes causes of bladder cancer as smoking, chemicals in the workplace, personal history of bladder cancer, certain cancer treatments, arsenic, and family history of bladder cancer. The American Cancer Society (2014) has concisely summarized its view on the causes/risk factors for bladder cancer: Smoking is the most well established risk factor for bladder cancer. Smokers risk of bladder cancer is approximately four-fold that of nonsmokers, and smoking is estimated to cause about half of all bladder cancer cases in both men and women. Workers in the dye, rubber, or leather industries, painters, and people who live in communities with high levels of arsenic in the drinking water also have increased risk. APPRACH A draft background paper was prepared by the panel chair, outlining the issues and available evidence related to the objectives of this project, and was provided to the expert panel 10 days before an in-person, two-day workshop, March 24 and 25, 2014, in Washington, D.C. In addition to the expert panel members, the workshop was attended by the WRF/AWWA Project Advisory Committee and three observers from the U.S. Environmental Protection Agency. The background paper for the panel workshop was developed through a comprehensive literature search of major databases, including Web of Science, Medline, EMBASE, Global Health, Toxline, Pollution Abstracts, Water Resource Abstracts, Cochrane Library, BISIS previews, and Scopus. The bibliographies in the relevant papers were also searched. Tracking citations of major relevant papers using Web of Science was also pursued. In addition to the literature search results, the background paper review was built upon and compared with coverage from several earlier reviews that included some of the objective topics (Hrudey 2008, Hrudey et al. 2003, IARC 2004, 2013, Mills et al. 1998, Sinclair et al. 2002, EPA 2006b). The objectives of this study were addressed during the workshop to generate discussion and work toward panel consensus on the issues. The available evidence being used in deliberations for the Stage 2 DBP Rule was briefly reviewed and taken as the starting point for reviewing newer, relevant evidence. Extensive notes were collected to translate the background paper into a first draft of this report. Subsequent drafts were circulated to the panel over the following months to arrive at a consensus report that was acceptable to all panel members. This report is the final consensus product of that consultation and review process. xvi

18 RESULTS/CNCLUSINS assification of exposure to CxDBPs for epidemiologic studies was considered in some detail because of the inherent challenges posed by estimating CxDBP exposures among individuals over several decades to address an estimated bladder cancer latency period, which typically exceeds 30 years. As long as associations are large enough to be demonstrated, even epidemiologic study results suffering from exposure misclassification can still provide useful evidence toward an evaluation of causation. However, from the perspective of those who must take actions to minimize potential health risks in drinking water, quantitatively inaccurate exposure assessment has serious implications, if used to inform regulation. The most common means of exposure quantification specific to CxDBPs that are subject to control measures are likely only a surrogate for something else (e.g. THM4 as a surrogate for an unknown causal agent). Consequently, there is no guarantee, without developing further knowledge, that aiming mitigation measures at controlling the surrogate will effectively or adequately control the unknown causal agent. In this situation, the most secure option is reducing precursors for THM4 formation, which may also reduce precursors for formation of the unknown CxDBPs. If the focus is on brominated compounds and THM4 is a surrogate, many of the brominecontaining DBPs will have different amenability to formation or removal than THM4. Conventional coagulation and advanced treatment processes, such as granular activated carbon (GAC), remove total organic carbon but not bromide, so the proportion of brominated DBPs (brominated THMs, brominated HAAs, etc.) can increase, even though total THM4 and the sum of the brominated DBPs are reduced (Summers et al. 1993, Watson et al. 2015). However, the formation of the most brominated species (e.g., bromoform) may be higher in the GAC effluent than in the GAC influent. If the focus is on HAAs, removal of the semi-volatile THM4 by aeration will not accurately represent removal of the non-volatile HAAs, because they have different physical-chemical properties. In either illustration, the behavior through treatment processes of brominated DBPs or HAAs will not be accurately represented by class sums like THM4. Equally important to any consideration of using evidence for supporting or revising any quantitative limits for specific CxDBPs, it is not clear what levels of those CxDBPs are implicated by the epidemiologic evidence. Epidemiologic exposure assessment is primarily concerned with assuring that the measures of exposure will provide accurate and consistent ranking from highest to lowest. Consequently, quantitative inaccuracy of absolute exposure does not necessarily affect the ability to discern an association between exposure and outcome. If true DBP exposures are under- or over-estimated, the level of CxDBPs associated with elevated cancer risk may not accurately reflect the actual exposure levels associated with the health outcome. The limitations of the exposure numbers that are obtained may seriously misinform attempts at setting appropriate quantitative limits for any CxDBP, according to those estimated exposure numbers. The evidence for associations between bladder cancer and CxDBPs are primarily reliant on 10 case-control studies (Appendix B), three with methodological and/or size limitations (Bove et al. 2007, Chevrier et al. 2004, McGeehin et al. 1993), and two based on the same original study with the most recent original case data collection from prior to 2001 (Villanueva et al. 2007, Cantor et al. 2010). The other case-control studies have cases mostly from a decade earlier, which may reflect effects of earlier, higher exposures prior to implementation of THM xvii

19 regulations. f the 10 case control studies, eight have suggested an association of bladder cancer with various indicators of exposure to CxDBPs or chlorinated surface waters. For certain exposures (e.g., dietary factors), case-control studies on bladder cancer have shown consistent and significant findings that have not been borne out subsequently in large, prospective cohort studies. This experience demonstrates the need for caution in prematurely adopting findings from the available number of large, well-executed case-control studies on bladder cancer and CxDBPs as certain evidence of a causal association. Such studies need to be replicated in other settings to provide much greater confidence in there being a causal association between CxDBP exposure and bladder cancer. Much better exposure and susceptibility assessments would ideally be a part of these newer studies. To specifically evaluate the impact of the Stage 2 DBP Rule on bladder cancer incidence in the United States, future studies would be required that accounted for both the lower CxDBP levels associated with the Rule implementation, and passage of sufficient time to account for bladder cancer latency. 1. Strengths and Limitations of the Evidence Linking CxDBPs and Bladder Cancer Strengths There are two cohort studies and 10 case-control studies using various indicators of exposure to CxDBPs or exposure to chlorinated surface water. Among the case-control studies, eight have suggested an association with bladder cancer, with significant Rs for men ranging from about 1.4 to 2.5 (Appendix B). There are two meta-analyses (Villanueva et al. 2003a, Costet et al. 2011) involving case-control studies that provide some consistency and support for an association of CxDBPs with bladder cancer. A pooled analysis (Villanueva et al. 2004) found similar associations. The Villanueva et al study based on the comparatively large Spanish casecontrol study from included explicit exposure categories of showering/bathing and swimming. bserved associations support a conceptual model that suggests inhalation/dermal exposure to CxDBPs may be at least as important for bladder cancer risk as ingestion. The Villanueva et al study also included explicit exposure to CxDBPs with a majority proportion of brominated species (Villanueva et al. 2003b). bserved associations are stronger than found previously in North American studies, suggesting that brominated species might be more important than their fully chlorinated counterparts. A conceptual model involving GSTT1 developed around several toxicological investigations by Pegram and colleagues (Cantor et al. 2010, Richardson et al. 2007, Pegram 2014 personal communication) combined with the findings of Villanueva et al. (2007) involve a number of plausible elements that collectively raise the conceptual model above that of simply being a possibility. However, the model currently falls short of providing a probable explanation of the epidemiologic observations. Cantor et al. (2010) evaluated a subset of the Villanueva et al. (2007) subjects according to a number of candidate genes with variations in single nucleotide polymorphisms (SNPs), some of which were selected with the conceptual model of Pegram and colleagues in mind. These results provide a possible mechanistic basis for the association, however the specific tie to THMs or other volatile DBPs needs to be xviii

20 Limitations established and the genetic susceptibility results require replication in an independent population. f the 10 case-control studies mentioned above, two are different analyses of the same case-control study (Cantor et al. 2010, Villanueva et al. 2007), and three (Bove et al. 2007, Chevrier et al and McGeehin et al. 1993) have size or methodological issues that reduce the weight of their findings as sources of replication for the purposes of judging consistency with regard to causal inference. The two meta-analyses (Villanueva et al. 2003a, Costet et al. 2011) have six casecontrol studies in common. The pooled analysis (Villanueva et al. 2004) includes three of the case-control studies among the six used in the two meta-analyses. The impact of a high proportion of brominated DBPs involved in Villanueva et al. (2007) was not found in the meta-analysis of Costet et al. (2011), although the other two European studies reported no information on brominated DBPs. The North American epidemiologic studies have examined waters without such high levels of bromide and brominated DBPs as found in the Spanish study (Villanueva et al. 2007, 2003b). THM4 (chloroform in particular) may be serving as a surrogate for an as-yet unidentified causal agent. CxDBP epidemiologic studies that used estimates of past THM levels, with the exception of the CxDBP exposure re-analyses performed by Amy et al. (2006), have estimated CxDBP exposure levels in the distant past by extrapolating back in time based on current or recent measurements. These extrapolations have not taken into account changes in treatment practices over time, such that past CxDBP exposures are likely underestimated. As a result, where associations with bladder cancer are found, those associations may be reported to occur at lower CxDBP exposures than were likely experienced. There is a quantitative discrepancy between the epidemiologic, cancer-site-specific risk estimates for bladder cancer from THM4 and what can be estimated by summing all the upper bound, all-cancer-site risk predictions from application of cancer slope factors for individual genotoxic CxDBPs. This analysis has not been conducted with all the emerging CxDBPs that may be of higher health concern than the regulated CxDBPs because most of the former do not have estimated cancer slope factors. Two genotoxic CxDBPs (MX, NDMA) that were included have cancer slope factors that are orders of magnitude more potent than the THMs or HAAs but they also occur at orders of magnitude lower concentrations than THM4 or HAA9. Many of the study results do not differentiate between THM4 being causal and THM4 being an indicator of another chemical that poses a risk for bladder cancer. The case for THM4 being causal for bladder cancer has not been made with the currently available evidence. xix

21 2. Knowledge Gaps THM4 has been evaluated in many epidemiologic studies because of the availability of these monitoring data. Using these studies, however, it is not possible to determine whether THM4 or some correlate is an etiologic factor associated with bladder cancer. For the genetic factors studied by Cantor et al. (2010), the specific tie to THMs or other volatile THMs needs to be established, and there is an absolute need for replication of results in another setting with independent populations before confidence can be placed in these findings. The differing views between Bull (2012) and Pegram (personal communication 2014) on the causal role of THMs according to the conceptual inhalation/dermal exposure model underlying Cantor et al. (2010) will not be resolved without additional direct human evidence (see discussion on Cancer Risk Assessment). Although there has been a major reduction in the U.S. population exposure to THM4 since the introduction of regulations in 1979, there has not been an analysis of how or if this decreasing THM4 exposure has had any population health benefit. 3. Evidence Supporting Better Risk Management The available new evidence does not clearly or adequately indicate what changes to DBP quantitative limits would be beneficial for the United States. The evidence opens possibilities of new hazard identification (e.g., brominated compounds, many of the other non-regulated DBPs, inhalation/dermal exposure). There are opportunities to inform a better understanding of CxDBP exposures, but any causative CxDBP agent(s) for bladder cancer have not yet been identified. RECMMENDATINS The panel considered a wide range of options and priorities for future research to address the limitations and gaps in knowledge. Research recommendations included: The most important data needed to advance the understanding of possible causation of human bladder cancer by CxDBPs is a comprehensive, statistically-powered, and welldocumented epidemiology study in a location with a range of CxDBP exposures from high to low, including a substantial proportion of brominated compounds, and designed to obtain the genetic marker information necessary to test hypotheses generated by Cantor et al. (2010). This study will need to have the best available exposure assessment approach that maximizes the use of water provider monitoring data, other sources of CxDBP and water quality data, and expert knowledge (from water quality/treatment professionals) of CxDBP inter-correlations and relationships, and should also be individualized to the maximum degree feasible. Such a comprehensive study will require a major investment, likely more than $10 million, and will take several years to complete. If substantial capital and operating costs for additional water treatment are proposed to further reduce CxDBPs beyond current regulatory levels, this research investment may be justified. Because of the inherent limitations of such studies, complete clarity of answers obtained cannot be assured, nor necessarily expected from another case-control study. xx

22 It would be valuable to undertake epidemiological studies in regions where a variety of different water treatment options (e.g., ozonation, chloramination, chlorine dioxide, etc.) producing lower levels of CxDBPs have been in place for extended periods. There would be some value in undertaking an ecological study in Australia where there are some large historical differences in exposure to CxDBPs (e.g., Melbourne only began chlorination in 1967 and has produced comparatively low levels of THMs since then, versus Adelaide and Perth, which both have high THMs with a substantial proportion of brominated species). This study would be a cost-effective step for generating and/or clarifying hypotheses about the role of various exposures. This would be a much smaller investment (<$1 million) than the full case-control study outlined above. There would also be some value in undertaking an ecological study in Canada where there are some large differences in exposure to CxDBPs (e.g., Winnipeg and Regina have had a long history of very high CxDBP exposures compared with other major cities in Canada, like Edmonton, which has used chloramination since the 1930s). Such a study would also be a cost-effective step for generating and/or clarifying hypotheses about the role of various exposures. There would be substantial value in performing more short-term experimental studies to look at inhalation and dermal uptake for various DBPs using valid, meaningful measures of response (e.g., urinary bladder epithelial cell micronuclei, SNPs, serum and urine levels of CxDBPs and metabolites, mutagenicity). These studies should consider what other classes of CxDBPs should be included and will need to have sufficient statistical power to address gender differences, bearing in mind that urinary measures are less confounded in males. These studies can be performed with smaller investments (<$1 million). The ongoing analysis of existing THM and HAA compliance monitoring data, and associated treatment to inform temporal (long-term) exposure in the United States, would be useful for interpreting results from epidemiologic studies, once any THM data reported in epidemiologic studies has been adjusted to better reflect actual exposures as done by Amy et al. (2006) for epidemiologic studies in Iowa and ntario. These would be comparatively low-cost analyses. The improved exposure assessment approach of Amy et al. (2006) could be applied to the Spanish study (Villanueva et al. 2007, Cantor et al. 2010), if the study investigators were open to collaboration to improve CxDBP exposure assessment and refine the observed outcomes. These would be comparatively low-cost analyses. A study in the Netherlands could take advantage of the large-scale natural experiment by which the use of chlorination of drinking water was largely eliminated decades ago. This would require securing access to the existing Dutch bladder cancer cohort data and could be relatively inexpensive. There may be benefits in evaluating the regional variations in risk in the Spanish bladder cancer study (Villanueva et al. 2007), given the large regional differences in THM4 levels. This would be a low-cost undertaking. RESEARCH PARTNERS American Water Works Association xxi

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24 CHAPTER 1 BACKGRUND HISTRY ver forty years ago, drinking water purveyors encountered an unexpected and profoundly important finding. The discovery and publication, first by Rook (1974) in the Netherlands and almost simultaneously and independently by Bellar et al. (1974) at the EPA, that chlorination of drinking water containing natural organic matter created readily detectable quantities of a group of halogenated contaminants that came to be known as the trihalomethanes 1 (THMs): chloroform, bromodichloromethane (BDCM), dibromochloromethane (DBCM) and bromoform. This momentous discovery has forever changed how water purveyors and water consumers view the safety of treated drinking water. The finding that chloroform caused cancer in long-term rodent bioassays (National Cancer Institute 1976) led to bans on using chloroform in a variety of consumer products such as cough syrups, antihistamines, decongestants and cosmetics (NTP 2014). These findings set in motion a series of regulatory processes to limit the levels of THMs and other subsequently discovered disinfection by-products (DBPs) that were allowed in drinking water (Table 1.1). Research soon documented that hundreds of DBPs (Appendix 1) were formed in drinking water by disinfection processes at levels ranging from hundreds of µg/l 2 (THMs) to fractions of ng/l 2 for nitrosamines. All chemical 3 disinfection processes, because they must be capable of inactivating microbial pathogens, involve using reactive chemical agents that inevitably produce DBPs, although the quantity and character of DBPs differ substantially among chemical disinfectants. Disinfection processes involving chlorine in some form remain the most common in the developed world partly because the efficacy 4 of chlorination provides the best assurance of disinfection across the greatest range of water quality scenarios, although many process modifications and options have been developed because of efforts to minimize DBP formation. After the THMs, the most prevalent forms of DBPs produced by chlorination processes are the haloacetic acids (HAAs). There are nine HAAs formed with the available combination of chlorine and bromine substitution. Collectively, DBPs formed by any disinfection process involving chlorine (including chloramine) can be termed as chlorination DBPs. Attention will also be focused on DBP species that contain bromine or possibly other halogens. Additionally, some DBPs (e.g., aldehydes, nitrosamines) produced by disinfection processes involving chlorine do not contain any chlorine or any halogen, for that matter. 1 The combination of these 4 compounds are often termed Total trihalomethanes or TTHM, but because we know that there are other less common tri-halogenated methanes (i.e. iodinated species), we will use the less ambiguous term THM4 to refer to these most prevalent trihalomethanes. 2 µg/l are approximately equal to parts per billion (ppb, 1 in 10 9 ), ng/l are parts per trillion (ppt, 1 in ) 3 The only physical disinfection process that is practical and effective at full scale is ultraviolet (UV) radiation. The energy imparted to water by UV causes reactions in a dose-dependent fashion leading to compositional changes that can result in DBP formation. 4 Ease of use, effective results and low cost make chlorination particularly attractive for smaller systems, which are often challenged financially. 1

25 Date 1 Table 1.1 Drinking water guidelines for organic disinfection by-products (DBPs) Jurisdiction Initiative (μg/l) Comment 1978 Health Canada THM4 350 MAC, guideline, not to exceed 1979 EPA, Safe Drinking Water Act THM4 100 MCL, regulation, running annual average (SDWA) 1984 World Health rganization(wh) Chloroform 30 MAC, guideline, not to exceed 1993 WH Chloroform TBM 100 DBCM 100 BDCM 60 TCP Australia, National Health & Medical Research Council (NHMRC) THM4 250 MCAA 150 DCAA 100 TCAA 100 CH 20 3 MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed 1996 Health Canada THM4 100 MAC, guideline, running annual average, quarterly samples 1998 EPA, SDWA THM4 80 MCL, regulation, system-wide running HAA to WH DCAA 50 DCAN DBAN 70 MCAA 20 TCAA EPA, SDWA THM4 80 HAA5 60 annual average, quarterly samples MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed MAC, guideline, not to exceed MCL, regulation, locational running annual average, quarterly samples - to include locations with the highest concentrations 2006 Health Canada BDCM 16 MAC, guideline, not to exceed 2006 WH NDMA 0.10 MAC, guideline, not to exceed 2008 Health Canada THAA (HAA5) MAC, guideline, running annual average, quarterly samples 2009 Health Canada BDCM New NTP cancer bioassay for BDCM withdrawn Health Canada NDMA 0.04 MAC, guideline, not to exceed 2011 Australia, NHMRC NDMA 0.10 MAC, guideline, not to exceed 2011 WH chloroform 300 μg/l 6 MAC, guideline, not to exceed 1 earliest date adopted. Later dates are only shown if the number has changed with later updates 2 the recognition that chloroform was not genotoxic led to a risk assessment that chloroform is a threshold carcinogen 3 a revision up to 80 µg/l has been provided for public consultation and the results of that consultation are currently under consideration before publication of the revised guideline. 4 MAC value increased because TCAA weight of evidence is judged not to be genotoxic 5 the result of a 2006 National Toxicology Program (NTP) bioassay on BDCM removed the basis for the risk calculation by finding no evidence of cancer risk for BDCM administered in drinking water. 6 MAC value increased because of a larger proportion of exposure assigned to water, noting that other uses of chloroform have decreased substantially since the previous MAC was set in

26 Accordingly, for the purposes of this report, the term CxDBPs will be used to represent all DBPs produced by any disinfection process involving chlorine (excluding chlorine dioxide), including chloramination. CxDBPs include DBPs with halogens other than chlorine (e.g. bromine), as well as DBPs formed by disinfection processes using chlorine that contain no halogens. The finding that chloroform was carcinogenic in rodents was obtained by dosing near lethal levels of chloroform dissolved in corn oil by daily gavage and the carcinogenic response was subsequently shown to result from the experimental protocol (Jorgenson et al. 1985, Bull et al. 1986, Butterworth et al., 1995). Chloroform has now been judged to pose no cancer risk to humans via normal drinking water exposures (Butterworth 2006, Butterworth and Bogdanffy 1999, EPA 2000, EPA 2006a, p.407). THE HUMAN HEALTH ISSUE Possible human health outcomes from exposure to CxDBPs via chlorinated drinking water have been subjected to considerable research investigation. A wide variety of health effects have been considered starting with cancer. While there is some evidence of non-cancer health effects, generally the epidemiology for other health outcomes has been inconsistent and generally less of a driver in guiding drinking water regulation. A wide variety of cancer sites have also been evaluated for association with exposure to chlorinated drinking water and CxDBPs, but only bladder cancer has shown any consistent association with such exposure. Therefore, this report will focus on the evidence on associations of human bladder cancer with exposure to CxDBPs. The risk management of drinking water exposure to CxDBPs is a classic case of risk trade-off because we know with a high degree of confidence that inadequate or lack of disinfection will virtually ensure waterborne disease outbreaks. Likewise, alternate disinfectants raise other risks (other DBPs, some inorganic) and challenges in cost and efficacy (especially for small systems). Considerable evidence over the past 40 years suggests that there is very likely no disinfection process or combination of processes that will be free of the formation of DBPs (Shah and Mitch 2012). 3

27

28 CHAPTER 2 BLADDER CANCER AS A MAJR PUBLIC HEALTH ISSUE CNTEXT Bladder cancer is an important public health issue causing the fifth highest number of estimated new U.S. cases of cancer in 2015 (American Cancer Society 2015) for both sexes, accounting for 74,000 estimated new cases out of 1.66 million (4.5%) new cases for all cancer sites. Bladder cancer accounted for 16,000 estimated deaths in 2015 out of the 590,000 (2.7%) U.S. deaths for all cancer sites in For the United States, bladder cancer is currently 3 times more common in men than women. For U.S. men it is the fourth most common cancer site (6.6% of all new male cases) and eighth most common cause of cancer death (3.5% of all male cancer deaths) in 2015 (American Cancer Society 2015). For U.S. women, it is the twelfth most common cancer site (2.3% of all new female cases) and the twelfth most common cause of death (1.6% of all female cancer deaths) in 2015 (American Cancer Society 2015). The probability of developing bladder cancer increases sharply with age (Table 2.1) and is three times higher in men than in women at all age categories. For reference, currently U.S. men have a lifetime risk of developing any type of cancer slightly less than 1 in 2 and U.S. women have a lifetime risk of developing any type of cancer slightly greater than 1 in 3 (American Cancer Society 2015). Table 2.1 Probability of Developing Bladder Cancer* over Selected Age Intervals by Sex in the United States Sex Birth to to to & lder Birth to Death Male 0.02% (1 in 5,035) 0.37% (1 in 272) 0.92% (1 in 109) 3.69% (1 in 27) 3.81% (1 in 26) Female 0.01% (1 in 12,682) 0.12% (1 in 864) 0.24% (1 in 410) 0.98% (1 in 106) 1.15% (1 in 87) *For bladder cancer, in situ and invasive cancer cases are included. For total cancer risk estimates totaling all other cancer sites, all basal and squamous skin cancers and in situ cancers are excluded. Source: data taken from American Cancer Society 2013a The risk for bladder cancer rises exponentially with age over 40. The U.S. median age at diagnosis is 73, with 91% of new cases incident after age 55, 72% after 65 and 45% after 75. (SEER 2015). Abdollah et al. (2013) reported that the age-standardized bladder cancer incidence in the United States increased from 21.0 to 25.5 / 100,000 person-years between 1973 and This study suggested virtually no significant progress against bladder cancer over this period and found minor fluctuations in incidence and mortality across all stages of bladder cancer but judged them to be of inconsequential clinical significance. In contrast, the American Cancer Society (2015) reported that 5-year survival rates for bladder cancer increased from 72% in to 79% in and 79% in These statistics are provided to assist in understanding the background about the nature of the disease and its prognosis. Because of the many variables involved, no valid inference can be drawn about such observations being related in any way to changes in drinking water CxDBP exposure. De aud et al. (2002) reported for the European Union that the annual incidence rate for 5

29 bladder cancer was 36 / 100,000 (M) and 10 / 100,000 (F), while the annual mortality rate was 13 / 100,000 (M) and 4 / 100,000 (F). About 71% of patients with bladder cancer were over 65 years of age. In men, the incidence increased with age from one per 100,000/ year in patients aged less than 45, to 25 per 100,000/ year in the age group 45 64, 87 per / year for aged and 129 per 100,000/ year for those older than 75 years of age. Recent international comparisons of bladder cancer statistics place age-standardized incident rates of bladder cancer for men in the United States in 20 th place at 19.6 cases per 100,000 behind Germany in 9 th place at 22.7 cases per 100,000, Spain in 6 th place at 26 cases per 100,000 and Belgium in 1 st place at 31.0 cases per 100,000 (Ferlay et al. 2012). For women, the rankings are United States in 17 th place at 5.1 cases per 100,000, Germany in 13 th place at 5.5 cases per 100,000, Canada in 10 th place at 5.6 cases per 100,000, Belgium in 4 th place at 6.2 cases per 100,000 and Hungary in 1 st place at 7.4 cases per 100,000. Because the numbers for women are much lower and are all very similar in the top 20, the rankings should not be overinterpreted, given inevitable uncertainties involved in comparing cancer statistics across countries. Spain does not appear in the top 20 for bladder cancer in women. The bladder cancer rates in different countries are provided only to illustrate that incidence does vary widely among countries and geographic regions. These rates are influenced by a range of risk factors and cannot be interpreted directly with regard to the presence of CxDBPs in tap water. TYPES F BLADDER CANCER Several sources have described and explained the common types of bladder cancer (American Cancer Society 2013b, CRUK 2013, Silverman et al. 2006, Jones 2010, Wai and Miller 2002). Transitional cell (urothelial) carcinoma (TCC) is by far the most common type of bladder cancer in the United States. Bladder cancers are also described based on how far the tumor has invaded into the wall (urothelium) of the bladder. Non-invasive, superficial or in-situ bladder cancers are limited to the cells lining the inner surface of the bladder (the transitional epithelium) but have not passed through the lamina propria into deeper tissue layers making these tumor types somewhat manageable. Invasive cancers grow into the muscle layer and are more likely to spread, making them inherently harder to treat and correspondingly more dangerous. ther more rare types of bladder tumors in the United States are the squamous cell carcinoma and adenocarcinoma. assification of bladder cancer tumor types continues to evolve. Squamous cell carcinomas comprise only about 1 to 2% of bladder cancers in the United States, but in the developing world, these are important invasive bladder cancer tumors because they are caused by a nematode infection (schistosomiasis) that is endemic in many of these countries. Adenocarcinomas are also mostly invasive, but they comprise only about 1% of bladder cancers found in the United States. The distinction between in situ and invasive bladder tumors has been difficult to determine on first diagnosis and the reporting of in situ tumors has increased dramatically since 1970, which may account for some of the observed increases in bladder cancer incidence (Silverman et al. 2006). The substantial growth of diagnoses of in situ bladder tumors places an increasing burden on healthcare resources because of the long-term need for testing and on-going monitoring of these patients with only about 20% ever going on to become invasive. In situ bladder tumors impose a serious negative quality of life and economic cost burden on many cases, even if they do not prove fatal (Sievert et al. 2009). 6

30 Key characteristics of bladder cancer concerning symptoms, detection, treatment and survival have been concisely presented by the American Cancer Society (2014): Signs and symptoms: The most common symptom is blood in the urine. ther symptoms may include increased frequency or urgency of urination and irritation during urination. Early detection: There is currently no screening method recommended for people at average risk. Bladder cancer is diagnosed by microscopic examination of cells from urine or bladder tissue and examination of the bladder wall with a cystoscope, a slender tube fitted with a lens and light that can be inserted through the urethra. These tests may be used to screen people at increased risk due to occupational exposure or certain bladder birth defects, or for follow up after bladder cancer treatment to detect recurrent or new tumors. Treatment: Surgery, alone or in combination with other treatments, is used in more than 90% of cases. Localized cancers may be treated by administering immunotherapy or chemotherapy directly into the bladder after surgery. More advanced cancers may require removal of the entire bladder (cystectomy). Chemotherapy, alone or with radiation before cystectomy, has improved treatment results. Timely follow-up care is extremely important because of the high rate of bladder cancer recurrence. Survival: For all stages combined, the 5-year relative survival rate is 78%. Survival declines to 71% at 10 years and 65% at 15 years after diagnosis. Half of all bladder cancer patients are diagnosed while the tumor is in situ (noninvasive, present only in the layer of cells in which the cancer developed), for which the 5-year survival is 97%. Patients with invasive tumors diagnosed at a localized stage have a 5-year survival rate of 71%; 35% of cancers are detected at this early stage. For regional and distant staged disease, 5-year survival is 35% and 5%, respectively. RISK FACTRS/CAUSES F BLADDER CANCER A primary objective of cancer research is to learn what can be done to prevent cancers because there are clear and practical limits to what can be achieved in treating cancers that have already been diagnosed. Being able to prevent cancer implies that we must understand something about what is causing cancer so that interventions may be designed to reduce or remediate known causes. The primary source of human evidence regarding cancer comes from epidemiological studies. These findings generally need to be supported by toxicology studies that can identify and characterize mechanisms by which agents are able to function as causes of the specific cancer in question, particularly if quantitative cancer risk issues are to be addressed. Replicated, positive associations in epidemiologic studies are almost always required to establish causality, but a key aspect to elaborate, before summarizing the evidence on causes of bladder cancer, concerns the challenge of obtaining evidence for causation when the associations between disease and putative cause are comparably weak (Boffeta 2010, Schlesselman 1987, Wynder 1987). f course weak is a relative term, but in this context, it refers to the magnitude of association, usually expressed as the relative risk (RR) or odds ratio (R) 5, that can be demonstrated for a 5 A common statistical evaluation of either the RR or the R is to determine whether it is statistically significant, typically expressed as whether or not the 95% confidence interval on the RR or R estimate includes the null value 7

31 given epidemiologic study. Boffeta (2010) cites Wynder (1987) as indicating that moderate or weak associations are those below about 3 (as R or RR), and argues that most strong epidemiologic associations for causes of cancer by this criterion have already been discovered. However, the sum of these strong association causes cannot explain all observed cancer occurrence suggesting that other causes with smaller effects must account for some substantial proportion of cancers. Consequently, Boffeta (2010) argues that remaining causes of cancer are likely to fall into the region of comparatively weak associations according to that designation. These authors explaining weak associations are not arguing that such epidemiologic evidence is weak in the sense of being necessarily invalid or unimportant, because a small (weak) but valid association for an exposure or risk factor that is pervasive in the population can correspond to a major public health issue. Rather, the point about weak associations is that the closer such associations get to the null value (1.0), the more subject those observed associations inevitably are to error caused by chance, bias and confounding. These errors can lead to both false negative and false positive errors. Therefore, an important consideration is the consistency between studies in different study populations; with more studies showing the same effect, confidence in the association being causal grows stronger. Consistency of study results is one of the key criteria for judging confidence in a causal association between exposure and disease (adford Hill 1965). Such confidence in the causal association is needed to assure that actions taken to manage exposure to a risk found with a weak association can reasonably be expected to reduce occurrence of cancer. These considerations also encounter the complexity of cancer causation such that complete causes (i.e. conditions that are able to cause cancer largely on their own and, if removed entirely, will completely remove any incremental cancer risk they contribute) are more likely to be able to demonstrate strong associations. Conditional causes, 6 on the other hand, are more likely to exhibit weaker associations. These factors will occur along a continuum, from complete causes to progressively weaker risk factors. The causes / risk factors for bladder cancer range from at least one strong causal factor to many weaker associations along this continuum. The American Cancer Society (2014) has concisely summarized its view on these causes / risk factors as: Smoking is the most well-established risk factor for bladder cancer. The risk of bladder cancer among smokers is approximately four-fold that among nonsmokers. Half of all bladder cancers in both men and women are attributed to smoking. Workers in the dye, rubber, leather, and aluminum industries, painters, people who live in communities with high levels of arsenic in the drinking water, and people with certain bladder birth defects also have an increased risk. The National Cancer Institute (2014) summarizes causes of bladder cancer as: Studies have found the following risk factors for bladder cancer: Smoking: Smoking tobacco is the most important risk factor for bladder cancer. Smoking of 1.0. In this report, for those RR or R values reported to be statistically significant will be noted by showing the RR or R in bold. 6 Conditional causes are conditions that must operate in concert with other factors to increase cancer risk and which, if removed entirely, may not correspondingly remove all the incremental cancer risk they appear to contribute. 8

32 causes most of the cases of bladder cancer. People who smoke for many years have a higher risk than nonsmokers or those who smoke for a short time. Chemicals in the workplace: Some people have a higher risk of bladder cancer because of cancer-causing chemicals in their workplace. Workers in the dye, rubber, chemical, metal, textile, and leather industries may be at risk of bladder cancer. Also at risk are hairdressers, machinists, printers, painters, and truck drivers. Personal history of bladder cancer: People who have had bladder cancer have an increased risk of getting the disease again. Certain cancer treatments: People with cancer who have been treated with certain drugs (such as cyclophosphamide) may be at increased risk of bladder cancer. In addition, people who have had radiation therapy to the abdomen or pelvis may be at increased risk. Arsenic: Arsenic increases the risk of bladder cancer. In some areas of the world, arsenic may be found at high levels in drinking water. However, the United States has safety measures limiting the arsenic level in public drinking water. Family history of bladder cancer: People with family members who have bladder cancer have a slightly increased risk of the disease. Many more detailed and rigorous reviews of causes and risk factors for bladder cancer have been published, including comparatively recent reviews by: Zlotta et al. (2010), Negri and LaVecchia (2007), Murta-Nascimento et al. (2007), Silverman et al. (2006) and Zeegers, et al. (2004). Bladder cancer was among the very first cancers to be attributed to a specific cause. A German physician first attributed bladder tumors to occupational exposures among aniline workers in Epidemiological evidence of this cause of bladder cancer was not rigorously provided until a comprehensive itish study by Case et al. (1954) demonstrated the occupational cancer risk of exposure to the dye chemicals: benzidene (4,4 diamino-biphenyl), α-naphthylamine and β-naphthylamine. A recent Italian follow-up study (Pira et al. 2010) of more than 58 years demonstrated that 56 bladder cancer cases among 590 long-term exposed dye-workers corresponded to a standardized mortality ratio (observed / expected, where null=1.0) of 16.5 (95% CI: 12.4 to 21.4). This outcome is an extraordinarily strong epidemiologic association which, taken together with substantial biological plausibility for the carcinogens involved, makes a compelling case for causation of bladder cancer. As noted above, tobacco use is the most pervasive exposure that demonstrates a strong causal association with bladder cancer (American Cancer Society 2014). Many studies have shown consistent, low uncertainty (i.e., narrow confidence intervals) bladder cancer risk associations (R or RR) with tobacco smoking, ranging from 2 to much greater than 4. In addition to containing a number of aromatic amine initiator (DNA-reactive) carcinogens (e.g., 4- aminobiphenyl) tobacco smoke includes a number of irritant chemicals (e.g., nicotine, acrolein) causing inflammation and other mechanisms that promote tumor growth (Zlotta et al. 2010). Positive dose-response relationships have been found for both the number of cigarettes smoked daily and number of years of smoking (Silverman et al. 2006). A meta-analysis by Zeegers et al. (2000) reported that tobacco use accounted for 50% of bladder cancer cases in men and 35% in women and that current cigarette smokers have a RR of 2.57 (95%CI: 2.20 to 3.00) compared with non-smokers. More recently, Freedman et al. (2011) have reported on follow-up of a large U.S. cohort (men=281,394; women 186,134) between 9

33 1995 and the end of 2006 to determine that 50% of bladder cancer in men and 52% of bladder cancer in women could be attributed to having ever smoked. Current smokers showed a relative risk (hazard ratio) of 4.06 (95%CI: 3.66 to 4.50). Although the bladder cancer risk drops for those who quit smoking, the decrease over time is not as dramatic as generally found for lung cancer. Freedman et al. (2011) found that former smokers still showed a relative risk (hazard ratio) for bladder cancer of 2.22 (95%CI: 2.03 to 2.44) indicating a substantial persistence of bladder cancer risk despite quitting smoking. These and other studies of bladder cancer etiology suggest that issues of latency, dose rate and duration are vitally important issues to consider. Despite the unequivocal association of tobacco smoking with bladder cancer, at least 50% of bladder cancers in men occur in non-smokers (Zlotta et al. 2010), some of whom may be exposed to second hand smoke (SHS), alternatively referred to as environmental tobacco smoke (ETS), although an association of bladder cancer with ETS is neither clear nor consistent. Van Hemelrijck et al. (2009) used levels of 4-aminobiphenyl, a compound classified as a human carcinogen by IARC and a suspected active agent for cancer causation by tobacco smoke, in the blood and urine of participants as a biomarker of exposure to SHS to then determine any association with bladder cancer. While elevated levels of 4-aminobiphenyl were found to be significantly associated with exposure to SHS, no association was found between SHS exposure and bladder cancer with a RR of 0.99 (95% CI: ). Similarly, Zeegers et al. (2002) concluded from a large prospective cohort study (120,852 adult subjects in the Netherlands) that current cigarette smokers have a 3-fold higher bladder cancer risk than non-smokers, and that exsmokers experience a 2-fold increased risk. About 50% of male bladder cancer and 20% of female bladder cancer was attributable to cigarette smoking, but environmental tobacco smoke (ETS) was not associated with increased bladder cancer risks. Exposure to a variety of occupational hazards, including those with unambiguous exposure to aromatic amines and related substances, has been evaluated for bladder cancer risk (IARC 1987, 2010, 2012). Table 2.2 summarizes the most recent categorization by IARC of those substances and occupations that have been judged to exhibit sufficient evidence of being carcinogenic to humans and those that have been formally evaluated by IARC and found to have limited evidence of being carcinogenic to humans in relation to bladder cancer. 10

34 Table 2.2 IARC List of assifications by cancer sites with sufficient or limited evidence in humans, Volumes 1 to 109 (Last update: 18 February 2014) Cancer site Carcinogenic agents with sufficient evidence in humans Agents with limited evidence in humans Urinary Bladder Aluminum production 4-Aminobiphenyl Arsenic and inorganic arsenic compounds Auramine production Benzidine Chlornaphazine Cyclophosphamide Magenta production 2-Naphthylamine Painting Rubber production industry Schistosoma haematobium Tobacco smoking ortho-toluidine X-radiation, gamma-radiation 4-Chloro-ortho-toluidine Coal-tar pitch Coffee Dry cleaning Engine exhaust, diesel Hairdressers and barbers (occupational exposure) Pioglitazone Printing processes Soot Textile manufacturing Tetrachloroethylene A sampling of research and review papers on these occupational exposures follows. An analysis by Golka et al. (2004) reported variable excess risks among dyers in the textile industries, painters (commercial, not artistic), varnish workers and hairdressers. Boffetta et al. (1997) reviewed exposures to polynuclear aromatic hydrocarbons (PAH) for a variety of cancer sites, including bladder cancer. Most studies suffered from small numbers of exposed cases; Rs were modest and none were statistically significant. Boffetta and Silverman (2001) performed a meta-analysis of studies evaluating bladder cancer and exposure to diesel exhaust and found the RR for truck drivers was 1.17 (95% CI: 1.06 to 1.29, 15 studies) and for bus drivers was 1.33 (95%CI: 1.22 to 1.45, 10 studies). A positive dose-response was suggested by 10 of 12 studies providing sufficient information and the RR for high diesel exposure was 1.44 (95%CI: 1.18 to 1.76). Kogevinas et al. (2003) estimated that about 5 10% of bladder cancers in European men could be attributed to occupational exposures, a lower estimate than that of an earlier large study in the United States in which 21 25% of bladder cancers were attributed to occupational exposure (Silverman et al 1989). More recently, Colt et al. (2011) performed a case-control study of 1,158 newly diagnosed bladder cancer cases and 1,402 population based controls (both 99% participation rates) in northern New England using lifetime occupational histories obtained by individual questionnaire to calculate R adjusted for age, race, Hispanic ethnicity, State, smoking status and employment in a high-risk occupation other than the one being analyzed. Men reporting use of metalworking fluids had a significantly elevated bladder cancer risk (68 cases, 53 controls R of 1.7 (95% CI: ) suggesting possible carcinogen exposure from these fluids. ver 40 occupations were evaluated, with significant findings of increased risk for bladder cancer observed for men engaged as: textile machine operators, automobile mechanics, plumbers, and landscape workers; and for women, engaged in: health services, cleaning and building services, electronic components manufacturing, and transportation equipment manufacturing. Arsenic has been accepted as a human carcinogen by IARC and is listed in Table 2.2 as a bladder carcinogen even though there is not a suitable animal model to verify the mechanism by which arsenic causes cancer. Arsenic has been generally accepted as causing bladder cancer for very high arsenic exposures by a drinking water exposure route, but it remains unclear whether 11

35 low dose arsenic exposure via drinking water increases the risk of bladder cancer. Christoforidou et al. (2013) have performed a recent systematic review of epidemiologic evidence concerning bladder cancer and arsenic in drinking water. ut of 99 papers addressing arsenic in drinking water, only 20 qualified for detailed review out of which they could identify six incident case-control studies and three incident cohort studies. Steinmaus et al. (2003) studied 181 bladder cancer cases (diagnosed 1994 to 2000) and 328 age- and gender-matched controls in the western United States and found no increased risks for arsenic intake greater than 80 μg/day, R = 0.94 (95% CI: 0.56 to 1.57). However, with exposures greater than 40 years among smokers, an R of 3.67 (95% CI: 1.43 to 9.42) was found for arsenic intake >80 μg/day (median intake: 177 μg/day). Steinmaus et al. (2003) concluded that this provided some evidence that smokers who ingest water with arsenic at concentrations close to 200 μg/day may be at increased risk of bladder cancer contributed by the arsenic exposure. Bates et al. (2004) found no evidence of association between bladder cancer and estimates of drinking water arsenic exposure in a case-control study in Argentina using 114 matched (age, sex, county) case-control pairs. nly the case of ever-smokers exposed to well water (presuming higher arsenic in well water) for more than 50 years produced a significant association, with an R = 2.5 (95% CI: 1.1 to 5.5). Meliker et al. (2010) performed a casecontrol study in Michigan with 411 bladder cancer cases (diagnosed 2000 to 2004) and 566 controls. Arsenic exposure history was estimated for 99% of the participant person-years. No increase in bladder cancer risk was found for time-weighted average lifetime exposure to >10 μg/l compared to a reference group exposed to <1 μg/l, yielding an R of 1.10 (95% CI: 0.65 to1.86). Even among ever-smokers, bladder cancer risks for those exposed to arsenic level >10 μg/l were not elevated when compared to the reference group exposed to arsenic concentration <1 μg/l, as shown by an R of 0.94 (95% CI: 0.50 to 1.78). Likewise, Karagis et al. (2004) performed a case-control study in New Hampshire, with 383 incident cases (diagnosed between 1994 and 1998) and 641 population controls. Well water arsenic was typically >10 μg/l. Individual arsenic exposure was assessed based on the biomarker of arsenic concentration in toenail clippings, which ranged from to μg/g among controls and μg/g among bladder cancer cases. There was no observed association for never-smokers and bladder cancer risk, but among smokers, bladder cancer risk was elevated, with an R of 2.17 (95% CI: 0.92 to 5.11) for the highest arsenic exposure compared with the lowest. Michaud et al. (2004) also used arsenic toenail concentrations as a biomarker for a nested case-control study with 280 bladder cancer cases and 293 controls, all male smokers aged drawn from a cohort study performed over 14 years in Finland with drinking water arsenic concentrations generally < 100 μg/l. Toenail arsenic concentrations were similar to the US studies (0.02 to 17.5 μg/g) and there was no association with bladder cancer among these male smokers comparing the lowest arsenic quintile with the highest showing an R of 1.13 (95% CI: 0.70 to 1.81). Total fluid intake as a factor in bladder cancer has also been inconsistent and controversial, although the variability of study quality may be a factor in the conflicting results. The premise that larger volumes of fluid flushing the bladder may reduce the contact duration of carcinogens in the bladder would suggest an inverse relationship of total fluid consumption with bladder cancer risk. However, if the consumed fluid is the source of the bladder carcinogens, higher volume consumption of such fluids could be expected to increase bladder cancer risk. In case-control studies, Vena et al. (1993) showed higher bladder cancer risk with higher total fluid consumption. Slattery et al. (1988) and uemmer et al. (1997) showed little consistent association of bladder cancer with total fluid consumption, and Villanueva et al. (2006a) found a 12

36 small (R of 1.08, 95% CI: ), but significant increase in bladder cancer risk in men for data pooled from six case-control studies. In contrast, Michaud et al. (2007) found an inverse association for bladder cancer risk for both total fluid consumption and tap water consumption for the Spanish Bladder Cancer case-control study 7, while Wilkens et al. (1996) showed a strong inverse relationship between total fluid consumption and bladder cancer risk for women, but not overall in men. Michaud et al. (1999) found a clear inverse association for bladder cancer in men in a 10-year follow-up of a large cohort study but a 22-year follow-up of this cohort found the inverse association had been attenuated (Zhou et al. 2012). For the Spanish Bladder Cancer case-control study (SBCS), 5 Silverman et al. (2008) approached the issue of carcinogen duration within the bladder from a different angle by finding an inverse trend in risk with increasing night-time voiding frequency in both men and women. A voiding frequency of at least two times per night was associated with a significant, 40 to 50% bladder cancer risk reduction. The protective effect of overnight urination was apparent across study participants with low, moderate and high water consumption. Furthermore, the bladder cancer risk associated with cigarette smoking was reduced by overnight urination. Current smokers who did not urinate at night showed an R of 7.0 (95% CI: ) relative to nonsmokers who did not urinate at night, while smokers who voided at least twice per night had a reduced R of 3.3 (95% CI: ) for the same reference comparison. Several other risk factors have shown convincing evidence of causing bladder cancer, including some medicines (Murta-Nascimento et al. 2007, Table 3): phenacetin (an analgesic), chlornaphazine (used for treatment of polycythemia and Hodgkin s disease) and cyclophosphamide (used for cancer chemotherapy and for treatment of some autoimmune diseases). Low-dose ionizing radiation is capable of causing bladder cancer and there is evidence of therapeutic pelvic radiation increasing bladder cancer risk (Murta-Nascimento et al. 2007). Chronic urinary tract infection caused by Schistosoma haematobium, a nematode worm that is endemic in large parts of the developing world, is associated with squamous cell carcinoma of the bladder in those regions, but is not a substantial risk factor in the United States unless cases appear by immigration of infected individuals. There is inconsistent evidence about other urinary tract infections being associated with bladder cancer (Murta-Nascimento et al. 2007). Likewise, the role of urinary tract stones has been controversial and is not a clearly established risk factor. Dietary factors have shown a diverse set of results regarding association with bladder cancer. LaVecchia and Negri (1996) reviewed seven studies considering fruit and vegetable consumption, finding that six of these studies showed a RR between 0.5 and 0.7 for the highest vs. lowest levels of consumption. Steinmaus et al. (2000) performed a meta-analysis of dietary factors for bladder cancer risk and found higher risk for diets low in fruit intake, with a RR of 1.40 (95% CI: 1.08 to 1.83), and slightly increased risks were associated with diets low in vegetable intake, with a RR of 1.16 (95% CI: 1.01 to 1.34). Elevated risks were also identified for diets high in fat intake, with a RR of 1.37 (95% CI: 1.16 to 1.62) but not for diets high in meat intake, with a RR of 1.08 (95% CI: 0.82 to 1.42). Michaud et al. (2000) evaluated dietary factors for the Health Professionals cohort of 51,529 men, yielding 320 cases of bladder cancer over 12 years of follow-up. They found no association with bladder cancer risk for total calorie or macronutrient intake, while finding an inverse association for vitamin E. 7 discussed later regarding Villanueva et al. (2007) and Cantor et al. (2010) 13

37 inkman et al. (2011) evaluated intake of various micronutrients in a Belgian casecontrol study of bladder cancer. The found a positive association for calcium intake, with an R of 1.77 (95% CI: ) for participants with the highest intake of phosphorus and lowest intake of vitamin D showing an R of 4.25 (95% CI: ). Adjustment was made for several potential confounders: age, sex, smoking characteristics, occupational exposure to polycyclic aromatic hydrocarbons (PAHs) and aromatic amines, and energy intake (kcal/day). Further analyses to account for consumption of alcohol, water, coffee, and tea did not appreciably change the results. Amaral et al. (2012) studied the potential protective effects of vitamin D against bladder cancer by measuring the serum levels of the most active metabolite (25-hydroxyvitamin D3) which has been shown to possess properties for regulating cell proliferation, apoptosis, cell adhesion at the tumor cell level, and modulation of the growth of new blood cells to support tumor growth (angiogenesis), invasion and metastasis. They found a statistically significantly increased risk of bladder cancer among subjects presenting severely deficient serum vitamin D levels vs. those with sufficient 25(H)D3 as an adjusted R = 1.83 (95% CI: 1.19 to 2.82; P =.006), that showed a much stronger effect in those with the dangerous muscle invasive tumors. Results were adjusted for age at interview, sex, region, smoking status, season of blood draw, body mass index, alcohol and calcium intake as previously associated with vitamin D, occupational exposure to aromatic amines and toenail arsenic. If this protective effect of serum vitamin D is established, this may signal a risk difference between rural and urban dwellers in relation to their comparative sun exposure (as a known factor in individual vitamin D levels) that has not typically been adjusted for directly when studies have performed adjustments for possible confounders. Zeegers et al. (2004) reviewed the strong evidence regarding tobacco use and bladder cancer relative to the weaker associations for beverage consumption and diet. For this purpose, they proposed evaluation criteria for judging the strength of the epidemiologic evidence, summarized in Table 2.3. This framework was applied to the literature they reviewed on bladder cancer risk to produce the analysis for these risk factors in Table 2.4. While the logic of this framework has merit, a key challenge for it to yield meaningful insight is how well the quality, not just the number of studies can be considered. For risk factors that have been subjected to many studies, there may be scope for screening studies to include only high quality studies. Table 2.3 Analytical framework for judging strength of epidemiologic evidence Number of epidemiologic studies showing consistent Associations (N study ) Supportive mechanistic evidence a No supportive mechanistic evidence b N study 15 Convincing Probable 5 N study <15 Probable Possible 1 N study <5 N study = 0 Possible No evidence Possible No evidence a Biologically plausible and supportive laboratory evidence b Biologically not plausible or no supportive laboratory evidence Source: Reprinted with permission from Springer Science+Business Media: World Journal of Urology; The Association Between Smoking, Beverage Consumption, Diet, and Bladder Cancer: A Systematic Literature Review; 21; 2004; ; M.P. Zeegers, E. Kellen, F. Buntinx, and P.A. van den andt; Table 1. 14

38 Table 2.4 Application of an analytical frame to a variety of risk factors for bladder cancer Evidence Convincing Probable Possible Substantially increased risk a Cigarette smoking - - Moderately increased risk b Slightly increased risk c Alcohol - Cigar smoking consumption Pipe smoking Environmental smoke No association d Coffee consumption Total fluid consumption Tea consumption Folate intake Vegetable consumption Vitamin A intake ** Vitamin C intake Slightly decreased risk e - Fruit consumption - Moderately decreased risk f - Selenium intake Vitamin E intake Substantially decreased risk g Convincing: 15 well designed epidemiologic studies showing consistent associations (N study ) and supportive mechanistic evidence, 2 Probable: 5 N study <15 and supportive mechanistic evidence, or N study 15 and no supportive mechanistic evidence. 3 Possible: 5 N study <15 and supportive mechanistic evidence, or 1 N study <5 comparing exposure vs. no exposure: a (RR 2.5), b (1.5 RR<2.5), c (1.2 RR<1.5), d (0.8 RR<1.2), e (0.7 RR<0.8), f (0.4 RR<0.7), g (RR<0.4), * for men; the risk related to alcohol consumption for women remains unclear, ** and carotenoids Source: Reprinted with permission from Springer Science+Business Media: World Journal of Urology; The Association Between Smoking, Beverage Consumption, Diet, and Bladder Cancer: A Systematic Literature Review; 21; 2004; ; M.P. Zeegers, E. Kellen, F. Buntinx, and P.A. van den andt; Table 1. Murta-Nascimento et al. (2007) reviewed evidence about the role of genetic susceptibility and impact on bladder cancer. Because of the accepted role of aromatic amines and the suspected role of PAHs in contributing to bladder cancer, differences in individual capacity of various enzymes involved in the metabolism of these compounds may confer different susceptibility. For example, the N-acetyltransferases (NATs) are involved in the bio-activation and detoxification of aromatic amines. The so-called slow-acetylator phenotype, NAT2 has been studied, including Garcia-osas (2005) who reported an R = 1.4 (95%CI: 1.2 to 1.7) for bladder cancer risk in NAT2 slow-acetylators for the Spanish Bladder Cancer case-control study 8 (1,134 cases, 1,131 controls) which agreed well with an updated meta-analysis of 31 case-control studies (5091 cases) showing an R = 1.4 (95%CI: 1.2 to 1.6). Moore et al. (2010) found that NAT2 slow acetylation status was not associated with risk among never, former or current smokers unless smoking intensity was considered. For slow acetylators compared with rapid acetylators in each category, those who ever smoked at least 40 cigarettes/day, bladder cancer risk was elevated among ever (R = 1.82; 95% CI: 1.14 to 2.91) and current heavy smokers (R = 3.16; 95% CI: 1.22 to 8.19). The elevated risk was not observed at lower smoking intensities. Evidence reviewed by Murta-Nascimento et al. (2007) on glutathione-s -transferases (GST), specifically GSTM1 and GSTT1, was reported as being mixed and inconclusive. ne 8 discussed later regarding Villanueva et al. (2007) and Cantor et al. (2010) 15

39 exception was found for GSTM1. Garcia-osas (2005) reported an R = 1.9 (95%CI: 1.4 to 2.7) for bladder cancer risk in those who were GSTM1 null in the Spanish Bladder Cancer casecontrol study 7 (1,134 cases, 1,131 controls) compared with an updated meta-analysis of 31 casecontrol studies (5072 cases) which showed an R = 1.5 (95%CI: 1.3 to 1.6) for this association of GSTM1 null with bladder cancer risk. Moore et al. (2010) found subjects with one or two inactive GSTM1 alleles (i.e. the null genotype) compared with those with two active copies which showed an R = 1.26 (95% CI: 0.85 to 1.88) and an R = 1.54 (95% CI: ), respectively. No effect of GSTM1 with smoking intensity was observed. GSTT1 inactive alleles were not associated with risk. 16

40 CHAPTER 3 EVALUATIN F EVIDENCE FR BLADDER CANCER RISK ASSCIATIN WITH CxDBPs EVIDENCE AVAILABLE T DELIBERATINS FR THE STAGE 2 DBP RULE A wide variety of health outcomes and several possible cancer risks have been assessed for an association with exposure to chlorination DBPS, but the evidence has remained inconsistent and inconclusive for major human health outcomes except for bladder cancer. Consequently, the focus of this report is on the evidence regarding CxDBPs and bladder cancer. The published analytical epidemiologic studies (those with any capability of informing the question of causation, cohort and incident case-control studies) are summarized in Appendix 2. The International Programme on Chemical Safety (ICPS 2000) of the World Health rganization reviewed two cohort studies (Wilkins and Comstock 1981, Doyle et al. 1997) and four incident case-control studies (Cantor et al. 1985, 1987, 1990 & Lynch et al. 1990, King and Marrett 1996, McGeehin et al. 1993, Freedman et al. 1997). This panel concluded for the epidemiologic evidence available at that time: The hypothesis of a causal relationship between consumption of chlorination by-products and the increased relative risk of any cancer remains an open question. There is insufficient epidemiological evidence to support a causal relationship between bladder cancer and exposures to chlorinated drinking-water, THMs, chloroform or other THM species. The International Agency for Research on Cancer (IARC) published reviews of this evidence in IARC (2004) reviewed seven incident case-control studies (Cantor et al. 1987, McGeehin et al. 1993, Vena et al. 1993, King and Marrett 1996, Freedman et al. 1997, Cantor et al and Koivusalo et al. 1998) and three cohort studies (Wilkins and Comstock 1981, Doyle et al and Koivusalo et al. 1997) published prior to The EPA relied primarily upon five of the incident case-control studies for assessing the health benefits of the Stage 1 DBP Rule (Cantor et al. 1987, McGeehin et al. 1993, King and Marrett 1996, Freedman et al and Cantor et al. 1998). For assessing the health benefits of the final Stage 2 DBP Rule, EPA (2006) also included consideration of a meta-analysis by Villanueva et al. (2003a) performed on six case-control studies (Cantor et al. 1987, McGeehin et al. 1993, Vena et al. 1993, King and Marrett 1996, Cantor et al and Koivusalo et al. 1998) and two cohort studies (Wilkins and Comstock 1981 and Doyle et al. 1997), and a pooled analysis by Villanueva et al. (2004) that included six case-control studies (Lynch et al. 1989, Cordier et al. 1993, King and Marrett 1996, Cantor et al. 1998, Koivusalo et al. 1998, and Porru an unpublished Italian study). The EPA (2006a) determined for its evidence review for setting the Stage 2 DBP Rule: Based on a collective evaluation of both the human epidemiology and animal toxicology data on cancer and reproductive and developmental health effects discussed below and in consideration of the large number of people exposed to chlorinated byproducts in 17

41 drinking water (more than 260 million), EPA concludes that (1) new cancer data since Stage 1 strengthen the evidence of a potential association of chlorinated water with bladder cancer and suggests an association for colon and rectal cancers, (2) current reproductive and developmental health effects data do not support a conclusion at this time as to whether exposure to chlorinated drinking water or disinfection byproducts causes adverse developmental or reproductive health effects, but do support a potential health concern, and (3) the combined health data indicate a need for public health protection beyond that provided by the Stage 1 DBPR. This finding was expanded to explain: Human epidemiology studies and animal toxicology studies have examined associations between chlorinated drinking water or DBPs and cancer. While EPA cannot conclude there is a causal link between exposure to chlorinated surface water and cancer, EPA believes that the available research indicates a potential association between bladder cancer and exposure to chlorinated drinking water or DBPs. The EPA concluded: verall, bladder cancer data provide the strongest basis for quantifying cancer risks from DBPs. These findings are reasonable for the evidence base that was available for precautionary regulatory purposes. In performing its review of the epidemiologic data, the EPA referred to causal criteria including: consistency, strength and specificity of association, temporality (exposure precedes outcome), a biological gradient (dose-response relationship), biological plausibility and coherence with multiple lines of evidence - criteria that can be traced to those originally proposed by adford-hill (1965). In addition, the EPA also considered the reliability of exposure data, statistical power and significance, and freedom from bias and confounding. We considered these factors and criteria in our following review of the more recent epidemiologic evidence for its strengths and limitations. EVIDENCE AVAILABLE SINCE DELIBERATINS FR THE STAGE 2 DBP RULE In 2013 IARC re-reviewed the evidence for some CxDBPs, determining that bromochloroacetic acid, dibromoacetic acid and dibromoacetonitrile are possibly carcinogenic to humans (Group 2B) based on insufficient data in humans and sufficient evidence in animals. IARC (2013) noted that epidemiologic studies have relied primarily on information related to ingestion of chlorinated drinking water rather than compound-specific exposure assessment. In April 2014, an IARC Advisory Panel reviewed substances nominated in response to a call for nominations on the IARC website (Straif et al. 2014). The Advisory Panel included disinfected water used for showering, bathing, swimming, or drinking among a total of more than 26 agents or groups of agents (including coffee, aspartame and nicotine) and 13 chemicals studied in recent cancer bioassays as high priorities for assessment by IARC Monographs for 2015 to The rationale for including disinfected water was based on ubiquitous exposure; extensive new mechanistic evidence of specific disinfection by-products, including molecular epidemiology studies published. IARC will use this advice when selecting agents for future Monograph assessments. 18

42 An experimental study was reported by Ranmuthugala et al. (2003), who recruited participants from three Australian communities. Bungendore, NSW, is a community of slightly over 2000 providing a non-chlorinated (zero THM) community water supply. Canberra, ACT, is Australia s national capital with a population of about 360,000 using a chlorinated water supply in which chloroform comprises over 90% of THM4. Adelaide, SA, South Australia s capital, is a community of about 1.2 million, with a chlorinated, high organic, high bromide water supply in which chloroform comprises only about 20% of THM4 (i.e. ~80% brominated THMs). These choices provided an excellent range of THM4 exposures from complete absence, to moderate with chloroform being the dominant THM (a common scenario) and high THM4 exposure with a high proportion of brominated THMs (a THM exposure suspected of greater potential for adverse human health effects). From a total cohort of 1087 households across the 3 communities, 353 males were recruited to the study, 348 completed the study and 228 (85 exposed, 143 unexposed) provided urine micronucleus slides suitable for scoring by the School of Public Health, University of California, Berkeley. Eligibility was restricted to men aged 30 to 65 years who had resided at their current address for at least 6 months, had never been diagnosed with any cancer other than skin cancer, and were not regular swimmers in chlorinated pools. Detailed individual exposure assessment was performed as an available dose, being the average concentration of four THM4 readings taken over the 2-week study period from each participant s individual home supply without adjustment for individual variation of intake. Intake dose was determined by adjusting each individual available dose for ingestion, inhalation and dermal exposure using participants water ingestion according to a 2-week fluid intake diary. Individual showering and bathing behavior was also recorded during the 2-week study period. Inhalation and dermal exposure were estimated according to the multi-route exposure model of Jo et al. (1990). The intake dose was determined as the sum of the ingestion, inhalation and dermal doses. Although swimming pool exposure was intentionally excluded from this study by excluding regular swimmers from participating, inhalation and dermal uptake were taken into account for showering and bathing data obtained over a 2-week interval for each participant, along with individual fluid consumption diaries for ingestion exposure, to estimate a total dose of THM4 and each individual THM. Ranmuthugala et al. (2003) evaluated a substantial range of THM4 exposures from zero for the unexposed group, to between 38 and 157 µg/l with data for each individual THM. Likewise, for the exposed group, the multimedia intake dose ranged from 3 to 469 µg/kg bw-d. Some apparent mathematical discrepancies appear in the reported summary (Table 1 of the paper) of available (water concentration) vs. intake (modeled uptake in µg/kg per day) for individual THM and THM4. A partial explanation might be that columns of median data that do not add up are reporting medians rather than averages. However, the range of THM4 exposures reported up to 469 µg/kg bw-d cannot be reconciled with the summarized upper bounds of the ranges for all individual THMs that sum to 290 µg/kg bw-d. Likewise, data were reported in Table 1 for BDCM in Adelaide, a THM of particular interest, but no BDCM results were reported in the final analyses. Results were reported for bromoform, which was observed in Adelaide at much lower levels than BDCM. Results were reported for AX (adsorbable organic halogen), but without any prior explanation. verall, Ranmuthugala et al. (2003) concluded: This study provides no evidence that trihalomethane concentrations, at the levels we investigated, are associated with DNA damage to bladder cells. This finding was based on the absence of any significant associations of 19

43 micronuclei occurrence with any measure of THM exposure, either as exposure concentration in tap water or individual modeled intake for THM4 or for individual THMs. The sensitivity of this study was seriously undermined by having a much higher occurrence of smokers in the community having no THM exposure. Ranmuthugala et al. (2003) concluded: However, micronuclei might not be sufficiently sensitive to serve as an indicator of carcinogenicity in a relatively small study. The small size of this study could also explain the lack of significant association between smoking and the frequency of micronuclei. The higher prevalence of micronuclei in our unexposed community might have been the result of the higher prevalence of cigarette smoking in this community compared with the other groups. The median occurrence of micronuclei (per 1000 normal cells) was much higher (2.25- and 9- fold) in the unexposed (no chlorination) community that also had and fold higher smoking prevalence than Canberra and Adelaide, respectively. However, differences in micronuclei occurrence between communities were not reflected in the overall percentage of micronuclei occurrence or in the range of micronuclei occurrence. verall, the results do not allow meaningful conclusions to be drawn for what was, conceptually, a promising study. Amy et al. (2006) performed an updated exposure assessment for the studies by Cantor et al. (1998) and King and Marrett (1996) with collaboration from the original investigators. Noting that exposure assessment is challenging in environmental epidemiology studies in general (Arbuckle et al. 2002), this study sought to apply expert knowledge about the formation of CxDBPs to better use available (current and historic) water quality and DBP data to model past exposures for these two major case-control studies. In addition to improving the exposure assessment for THMs (including bromine speciation), exposure assessments were developed for HAAs, which had not been done in the original studies. The report noted that exposure misclassification is inevitable in such studies that attempt to estimate exposures that occurred decades earlier, but that such misclassification will likely be non-differential (i.e. no difference between cases and controls) unless an unrecognized bias has occurred. In cases of nondifferential misclassification, in general, the effect will be to bias the magnitude of the observed R toward the null. The effort by Amy et al. (2006) expanded the scope of the exposure assessment, making better-informed predictions of historical THM4 occurrence for each of these studies, based on greater water quality detail from accessible site-specific monitoring databases. These approaches provided for significantly more discrimination at higher doses in the Cantor et al. study, leading to what was regarded as substantially improved exposure assessment. In the original Cantor et al. (1998) study, the assigned maximum average THM exposure was 74 µg/l, whereas in the reanalysis the maximum lifetime average exposure was 154 µg/l. While it cannot be firmly shown that these improvements in exposure assessment substantially reduced exposure misclassification, they did not substantially increase the observed R values for bladder cancer as might have been expected if non-differential misclassification of the hypothesized risk factor had occurred. A possible explanation of these results is that the exposure variables may be surrogates rather than causal agents. In the historical monitoring database, which was collected following the 1979 THM rule, the 75 th and 95 th percentile and maximum THM4 concentrations were 46, 96 and 219 µg/l, respectively (Amy et al. 2006). In the original analysis, the R values were significant for the 20

44 two highest assigned exposure categories, which included the 90 th and 95 th percentiles ( µg/l and >46.4 µg/l) for males. However, using the same cut-points for the categories, but based on more detailed and better-informed exposure estimates, only the highest category (>46.4 µg/l) remained statistically significant (Table 3.1). Table 3.1 Comparison of risk estimates (R) of bladder cancer for males in Cantor et al. (1998): original analysis versus reanalysis * Lifetime Cases / Controls riginal Analysis Cases / Controls Re-analysis Average THM4* / (reference) 347 / (reference) / ( ) 107 / ( ) / ( ) 156 / ( ) / / ( ) 1.67 ( ) 150 / / ( ) 1.32 ( ) 1.46 ( ) / ( ) 83 / 85 *Exposure categories based on 35 th, 60 th, 75 th, 90 th and 95 th percentiles in distribution among controls of lifetime average THM4 as estimated in the original analysis Source: data taken from Amy et al In the re-analysis that provided a new distribution of exposure, the 97.5 th percentile of lifetime average THM4 among controls was >96.1 g/l (Table 3.2), and it was only at this level that there was a significant R. By comparison, as noted above, in the re-analysis based on the original cutpoints of exposure (Table 3.1), only lifetime average THM4 exposure above 46.4 µg/l showed a significant R. This is an important difference given that the current U.S. regulatory level for THM4 is a maximum locational annual average of 80 µg/l and an operational target THM4 average of 80% of that limit, i.e., 64 µg/l (Roberson et al. 1995). The reclassification in Table 3.2 resulted in a wider range for estimated exposure, which, being based on the monitoring database, is more representative of the true THM4 exposure. The cutpoints used in Table 3.2 resulted in a smaller number of cases in the higher exposure categories, which also led to more imprecision in the point estimates for these categories. The Amy et al. (2006) re-analysis of the King and Marrett (1996) study showed lower bladder cancer risks, with wider confidence intervals, and without a consistent exposureresponse pattern for THM4 exposure (Table 3.3). These data are for men and women. Unlike Cantor et al. (1998), women showed higher bladder cancer risk than men in King and Marrett (1996). 21

45 Table 3.2 Risk estimates (R) of bladder cancer for males in Cantor et al. (1998): new exposure categories in reanalysis * Lifetime Average (50 yr) THM4* Cases / Controls Re-analysis / / / / (reference) 1.13 ( ) 1.04 ( ) 1.15 ( ) / ( ) > / ( ) *Exposure categories based on 35 th, 70 th, 90 th, th and 97.5 th percentiles in distribution among controls Source: data taken from Amy et al Table 3.3 Comparison of risk estimates (Rs) of bladder cancer for King and Marrett (1996): original analysis versus re-analysis Duration of exposure, Cases / Controls riginal Analysis Peak THM4 > 50 µg/l Cases / Controls Re-analysis Mean THM4 > 40 µg/l 10 years 253 / (reference) 593 / (reference) years years 226 / / ( ) 1.36 ( ) 23/ / ( ) 1.01 ( ) 35 years 54 / ( ) 43 / ( ) Source: data taken from Amy et al Finally, in terms of studies since the deliberations for implementing the Stage 2 DBP Rule, IARC (2013) has reviewed four newer (at least in terms of publication date) studies: Chevrier et al. (2004), Villanueva et al. (2007, 2006), Bove et al. (2007) and Cantor et al. (2010). Although all those individual studies considered by IARC and EPA noted above are summarized in Appendix 2, the following discussion will focus only on those studies published since EPA (2006) with a view to assessing any new insights and strength of evidence provided. Finally, the whole collection of valid, analytical studies will be discussed as a body of knowledge overall, in addition to highlighting what progress has been achieved since the analysis of health benefits was performed in preparation for the Stage 2 DBP Rule. Chevrier et al. (2004) Chevrier et al. (2004) [see Appendix 2] performed a somewhat unique study compared with all other analytical epidemiology studies addressing bladder cancer because they assessed drinking water exposure from water treatment plants disinfecting with ozone either alone or in combination with chlorination. This is important because ozone is known to produce substantially different DBPs, both qualitatively and quantitatively, compared with chlorination. nly the studies by McGeehin et al. (1993) and Zierler et al. (1988) included comparison of subjects drinking chloramine-disinfected surface water that produced lower and/or different DBPs than the CxDBPs primarily produced by chlorination. More such comparisons among disinfection process options would be useful to better inform options for managing CxDBP risks. McGeehin et al. (1993) was a comparatively small study (327 cases). Also, like a number of case-control studies on bladder cancer, it reported a significant increased risk for coffee consumption (R = 3.3; 95% CI: 1.8 to 6.0), after adjusting for smoking, a risk factor reported by a number of case-control studies that has more recently been judged to probably not be associated with bladder cancer (Zeegers et al. 2004). Zierler et al. (1988) was a death certificate- 22

46 based study rather than an incident case-control. Because bladder cancer has a comparatively low mortality rate and death certificate studies pose various other validity problems, not much weight can be given to Zierler et al. (1988). Chevrier et al. (2004) used data from an incident case-control study in France performed almost 20 years earlier ( ) with both cases and controls recruited from seven hospitals (three in Paris, four from other urban centers). In addition to the cases newly diagnosed during the 3-year window of recruitment, some cases diagnosed earlier (but after January 1, 1982) who were receiving treatment during the three-year window were also included. Controls were based on individuals admitted to the same hospital, matched for sex, age (±5 yrs) and area of residence. Controls were randomly selected for matching provided they did not have cancer, respiratory disease or any symptoms suggestive of bladder cancer. Chevrier et al. (2004) stated, The interviewers schedules constituted the only systematic reason for not interviewing case or control subjects. This seems to imply that some proportion of cases and controls were not interviewed, but no number is stated, nor whether those not interviewed were excluded. If they were not interviewed, it is not clear how the required information was obtained. Chevrier et al. (2004) did exclude all cases and controls for which it was not possible to characterize at least 70% of a 30-year drinking water exposure window. This criterion (along with excluding subjects under 30 or over 89, and with more than 2 years elapsed time between diagnosis and interview) resulted in sharply reducing the 765 cases (658M, 107F) and 765 controls (658M, 107F) to 281 eligible cases and 272 eligible controls. Given this large reduction in sample size and the attendant possibility of selection bias, Chevrier et al. (2004, Table 2) compared 49 potential confounders and socioeconomic characteristics of those remaining with those excluded to show the percentages with each characteristic for cases and controls compared with those included. As would be expected, some differences among included vs. excluded emerged in all categories with the greatest difference occurring in smoking status. Residential histories had been collected from 1,240 subjects covering the period from 1948 to 5 years before diagnosis for a total of 39,204 person-years from 928 municipalities. Water treatment information could only be obtained for 557 of the largest communities covering 22,881 person-years. Chevrier et al. (2004) estimated THM4 levels for 90% of these personyears as described below. Subjects (68) who reported a private well being used for more than half their residential history were excluded. Each utility described its treatment process according to a flow chart of up to 13 possible steps to assign THM4 levels by means of a Delphi 9 judgment process to develop the THM4 exposure categories in Table The Delphi Method is a structured, iterative procedure for eliciting opinions from a group of experts in a manner that develops consensus among participants. The accuracy of this procedure is obviously dependent on the relevant knowledge of the participating experts and the quality of evidence used to inform the experts. 23

47 Source of water Ground water Surface water Table 3.4 Assignment of THM levels by process and number of plants over time Number of Water Treatment Plants by Period No zonation With zonation Chlorination with respect to filtration Assigned THMs Pre- Post- (µg/l) no no no yes yes no yes yes no no no yes yes no yes yes Total Each water utility was judged on the characteristics above and process steps including first ph adjustment, flocculation, filtration with or without nitrification, activated carbon filtration, second ph adjustment, post chlorination or ozonation, third ph adjustment and distribution system chlorination. Source: Chevrier, C., B. Junod, and S. Cordier Does zonation of Drinking Water Reduce the Risk of Bladder Cancer? Epidemiology 15(5): Cases and controls were interviewed at each hospital. The questionnaire covered fluid intake, with information on individual intake of various types of drink, including bottled and tap water consumption. Tap water consumption was classified into either above or below 3.85 L per week. The comparison provided for those included vs. those excluded according to known water treatment history (Chevrier et al. 2004, Table 1); 52 % of included subjects ingested zero tap water. A further 23% of included cases and 24% of included controls consumed less than 3.85 L per week (<550 ml per day) of tap water. This study was opportunistic in using case-control data along with questionnaire data on tap water consumption from more than 15 years earlier, but the retrospective assignment of THM4 exposure level was based on historical records going back more than 40 years. Chevrier et al. (2004) reported numerous categories of stratification of results. The reference category, corresponding to <1 µg/l of THM4, was taken to be non-ozonated ground water (whether or not simultaneously chlorinated). This reference category included 5,313 person-years for cases and 4,871 person-years for controls, being more than 60% of the total of 16, 590 person-years of exposure for all categories in this study. nly 1.6% (0.2% for cases, 1.4% for controls) was classified as ozonated ground water (whether or not simultaneously chlorinated), 23.6% (12.2% for cases and 11.4% for controls) as chlorinated-only surface water, and 9.8% (4.5% for cases and 5.3% for controls) as simultaneously chlorinated and ozonated surface water. These low proportions result in comparatively low numbers of cases and controls in the THM4 exposure categories vs. the chosen reference category. Despite all the qualifiers, this study yielded some intriguing results. For men, considering duration of exposure to ozonated water in the highest duration category (10-30 years), adjusted for exposure to chlorinated surface water, they found the R = 0.32 (95% CI: 0.1 to 0.7), adjusted for average level of THM4 they found the R = 0.27 (95% CI: 0.1 to 0.6) and adjusted for cumulative level of THM4 (µg/l-yr) they found the R = 0.28 (95% CI: 0.1 to 0.7). Even though the numbers are very small (33 cases, 48 controls), these comparisons suggest a significant inverse relationship between duration of consuming ozonated water and bladder 24

48 cancer risk. For the chlorination comparisons, some significant associations were found. For men, for the estimated category 50 µg/l of THM4, the R was 3.73 (95% CI: 1.2 to 11) based on 46 cases and 36 controls. For men, for the estimated category 1500 µg/l-yr of cumulative THM4 exposure, the R was 3.49 (95% CI: 1.2 to 10.6) based on 45 cases and 35 controls. These substantial R values are statistically significant even though the confidence intervals are large because of the small numbers of cases and controls. The numbers of cases and controls for women are even smaller, making statistical significance unachievable for those Rs. The authors were realistic about avoiding over-interpretation of their study noting: ur estimation of the THM content of drinking water was based on the combinations of key steps in the treatment process at each plant and can only be considered a marker of the chemicals generated by chlorination rather than a specific measurement of THM. [Emphasis added] Furthermore, they stated: In conclusion, our results suggest that ozonation of drinking water could have beneficial effects and important public health implications. Possible bias cannot be ruled out, however, and the sample size is modest. These results make clear that further studies on the carcinogenic impact of disinfection byproducts should not be limited to the consequences of chlorination and that the impact of ozonation should be evaluated further in countries where ozone treatment has long been in use. Unfortunately, 10 years later, apparently no one has taken up the authors suggestion to perform additional studies on treatment alternatives to chlorine disinfection. Even more interesting are large-scale exposure scenarios in Berlin, Germany and much of the Netherlands where chlorination is not used for primary disinfection. Bove et al. (2007) Bove et al. (2007) [see Appendix 2] published a case-control study based on much older data originally reported by Vena et al. (1993). The older study reported fluid consumption data, but no attempt had been made to consider associations with CxDBPs in the tap water consumed. The new study used residential addresses for study participants in the Upstate New York Diet Study ( ) to estimate what Bove et al. (2007) describe as total 551 and individual THM levels in tap water. The total 551 levels refer to a sum of results from the 1990 EPA Method 551 that was designed to measure 11 halogenated organic compounds, including THMs, but also solvents such as carbon tetrachloride, tetrachloroethylene, 1,1,1-trichloroethane and trichloroethylene. IARC (2013) summarized these results from Bove et al. (2007) as total THMs, but total 551 would not necessarily be equal to total THM if other halogenated (non-dbp) contaminants were present. The retrospective exposure assessment based on THMs involved some consideration of residence time of water in the distribution system, which apparently played a large role in the results predicted for THMs, but insufficient details are described in the paper to provide an adequate basis to judge the merits of their approach or whether it may have introduced any systematic bias to the retrospective exposure assessment that provided the entire rationale for 25

49 publishing this paper based on the original epidemiologic study by Vena et al. (1993). The latter had no information on exposure to CxDBPs. The total water consumption classification in Vena et al. (1993) may have dominated the outcomes determined by Bove et al. (2007). Bove et al. (2007) also presented results for individual THMs and found several significant R values despite the study s small sample size: Chloroform o R=2.55 (95% CI: ) for 43 cases and 53 controls for the highest quartile of to µg/d vs. the reference quartile of µg/d. omodichloromethane (BDCM) o R=2.49 (95% CI: ) for 42 cases and 54 controls for the highest quartile of to µg/d vs. the reference quartile of 9.35 µg/d (21 cases, 76 controls). o R=2.20 (95% CI: ) for 35 cases and 62 controls for the second highest quartile of to µg/d vs. the reference quartile of 9.35 µg/d (21 cases, 76 controls). omoform o R=3.05 (95% CI: ) for 42 cases and 54 controls for the highest quartile of 1.16 to µg/d vs. the reference quartile of 0.43 µg/d (19 cases, 78 controls). o R=2.34 (95% CI: ) for 36 cases and 60 controls for the second highest quartile of 0.75 to 1.14 µg/d vs. the reference quartile of 0.43 µg/d (19 cases, 78 controls). o R=2.12 (95% CI: ) for 32 cases and 64 controls for the third highest quartile of 0.44 to 0.73 µg/d vs. the reference quartile of 0.43 µg/d (19 cases, 78 controls). No significant Rs were found for dibromochloromethane (DBCM). verall, these results did not attract much commentary by the IARC (2013) working group, other than the observation: The Working Group noted that there could be potential exposure misclassification due to the exposure assessment based on measurements of trihalomethanes conducted 20 years after the recruitment of study subjects. While this observation is certainly valid, the IARC working group misread some elements of Bove et al. (2007) (e.g., total 551 is not equal to total THM, results were reported in µg/d not µg/l). The finding of the highest study R result for bromoform that was reported to be present at a mean of 1.3 µg/l (min 0.12 µg/l, max 24 µg/l) is difficult to accept as implicating bromoform as the active agent (as opposed to being possibly a marker for something else). The low potency carcinogenic assessment of bromoform (EPA 2014a) would equate an upper bound lifetime cancer risk (all tumors) as only 0.33 x 10-6 for a mean exposure of 1.3 µg/l of bromoform in drinking water. This is far too low to be a factor in the reported R for bromoform unless it is only serving as a surrogate for other uncharacterized brominated DBPs. Likewise, the finding of a significant R for chloroform cannot be reconciled with chloroform being the active agent for causing cancer vs. chloroform being a marker for something else. The current carcinogen mechanistic understanding of chloroform is that it is a threshold carcinogen that only acts to produce tumors in the liver at very high doses, certainly much greater than the highest value of the reported range 192 µg/d (~2.7 µg/kg bw-d). By 26

50 comparison. The EPA (2014b) concludes with respect to chloroform that: A dose of 1x10-2 mg/kg-day (equal to the RfD) can be considered protective against cancer risk. In other words, the highest dose estimated for chloroform in Bove et al. (2007) is only about 27% of the reference dose (RfD) judged to be protective against cancer risk, and for a threshold carcinogenic mechanism there is judged to be a negligible cancer risk below the RfD. The median dose in the highest chloroform exposure category (mean is not reported by Bove et al. 2007) is ~1.7 µg/kg bw-d, about 17% of the RfD for chloroform. The finding of Bove et al. (2007) of an observed R of 2.55 (95% CI: ) for chloroform can only be rationalized if chloroform was acting as a surrogate for something else. Chloroform would not be expected to be a good surrogate for brominated DBPs. In summary, the main limitations evident in Bove et al. (2007) are the very small sample size, the limited explanation of how the retrospective exposure assessment was done, difficulty in reconciling the R observations for bromoform (except as a surrogate for uncharacterized brominated DBPs) and chloroform with what is known about cancer risk. Villanueva et al. (2007) A series of papers have been published by Villanueva and colleagues (Villanueva et al. 2003b, 2006b, 2007) related to a comparatively large (1219 cases, 1271 controls), hospital-based (18 participating hospitals from five distinct geographic regions in Spain), incident case-control study on bladder cancer (SBCS) performed between June 1998 and June 2001 (Villanueva et al [see Appendix 2]). This study provided a number of important advances over earlier epidemiologic studies on this subject, including some more specific exposure assessment, explicit inclusion of inhalation and bathing routes of exposure in addition to ingestion and inclusion of some water supplies with major proportions of brominated THM species. Controls were admitted to hospitals for hernias (37%), other abdominal surgery (11%), fractures (23%), other orthopedic problems (7%), hydrocele 10 (12%,), circulatory disorders (4%), dermatologic disorders (2%), ophthalmologic disorders (1%), and other diseases (3%). Eightyfour percent of the 1457 eligible cases and 87% of the 1465 eligible controls responded to the study questionnaire addressing socio-demographic characteristics, smoking habits, occupational, residential, and medical histories, and familial history of cancer. The smoking status of the population for cases and controls is noteworthy when considering the external validity of the Spanish results for application to the U.S. population. For the Spanish cases, 18% were never-smokers, 41.2% were former smokers and 40.8% were current smokers and for controls, 36.8% were never smokers, 40.5% former smokers and 22.7% were current smokers. Although these numbers were not subdivided according to gender, the proportion of male to female cases vs. controls was provided. Using this distribution to weight 2008 U.S. smoking statistics (American Lung Association 2014) shows U.S. combined data as 24% current smokers, about the same as for the controls, but much lower than the 40.8% current smokers among the cases for this study. All the R calculations were adjusted for smoking status. There is no evidence that residual confounding by smoking might explain the observed associations. 10 A pathological accumulation of fluid within a body cavity -the most common presentation is a fluidfilled sac causing swelling around a testicle in the scrotum caused by inflammation by infection (including sexually-transmitted diseases) or injury. 27

51 The research on CxDBPs took advantage of the SBCS that was exploring a number of possible risk factors, including hypotheses related to water consumption and use. Information gathered on water-related habits included: residential history from birth (all residences of at least 1 year); drinking water source at each residence (municipal / bottled / private well / other); average daily consumption, including water-based fluids (e.g., coffee, tea, and water); average frequency and duration of showering and bathing; and lifetime swimming in pools. The questionnaire of critical items covered residential history, drinking water source by residence (municipal / bottled / private well / other), frequency of showering and bathing, and whether the subject ever swam in indoor and outdoor pools and the number of times per year doing so. Controls were matched 1:1 to cases by gender, age group and residence area (Villanueva et al. 2007, 2006b). Villanueva et al. (2007) also explored using a biomarker of bladder exposure to mutagens by analyzing exfoliated bladder cells appearing in urine for micronuclei based on 92 female controls to obtain 72 that provided samples adequate for analysis, of which 44 provided information on THM exposure. Exposure data were obtained by contacting 200 local authorities and 150 water companies and results on annual average THM levels and water source history since 1920 (proportion of surface and ground water and the year when chlorination was initiated) were determined / estimated for 78.5% of the total study person-years of exposure. New THM data, using a single laboratory, was obtained for 113 samples between September and December 1999 to cover the geographic regions in the study (Villanueva et al. 2003b). However, DBP occurrence varies seasonally. Sampling in fall only will not represent year-round exposure (e.g., spring runoff, warmer temperatures in the summer). Moreover, there can be significant year-toyear variability. For example, Barcelona can experience serious droughts, during which the bromide level and the DBP levels (and extent of bromine speciation) rise substantially. Collecting new samples in only one type of water year (e.g., wet, normal, dry, critical) will not be representative of all types of water years. Assuming historical THM4 levels were unchanged for a constant water source, THM4 exposures were extrapolated back to This approach cannot be accurate for representing actual THM4 exposure over time. The levels of THM4 exposure estimated for many of the chlorinated water supplies is likely to seriously underestimate actual past THM4 exposures because actual THM4 exposures were most likely much higher before the discovery of THMs in chlorinated drinking water and their reported health effects, findings which led to lowering of THM4 concentrations to satisfy new drinking water guidance (Health Canada, EPA and WH) from the early 1980s followed by an EU Directive in 1998 (EU 1998). For groundwater supplies and surface waters with low levels of DBP precursors, it is likely that treatment did not change significantly over time because of THM concerns. Alternatively, for surface waters with significant levels of DBP precursors and/or those impacted by other micro-pollutants, treatment most likely has changed substantially over time. For example, one of the Barcelona treatment plants currently uses chlorine dioxide, ozone, and granular activated carbon (GAC) to control DBP formation and to remove various micropollutants. The unit processes in this advanced treatment plant were likely introduced over time 28

52 (their implementation staged over time). Because GAC removes natural organic matter (which includes DBP precursors) but not bromide, the efficacy of this plant to control DBP formation (and bromine speciation) would vary seasonally and from year-to-year, depending on bromide loading in the water supply. Thus, for the plants with the higher DBP precursor loadings, DBP exposure has undoubtedly varied substantially over time and cannot be accurately represented by one set of currently-derived values. After World War II, many water systems in Western Europe were privatized and treatment was significantly improved. Barcelona has a privatized water system. The major source of bromide in one of the Barcelona rivers is from mine discharges. The impact of pollution sources over time has also varied widely (Goslan et al. 2014, Harvey and Carlos de Armas 2010, Huerta-Fontela et al. 2008) Although there is most likely misclassification of THM4 exposure caused by this method of exposure assessment, recall bias is unlikely to be a concern in this study where the participants provided a residential history. The investigators independently assigned THM4 exposure based on reported residential history. THM4 ingestion exposure was estimated as µg/d based on a time-weighted average THM4 concentration at all residences reported for the individual beyond age 15 times the amount of water consumed according to questionnaire reporting. THM4 consumption was assumed to be zero for water from private wells, bottled sources or other non-municipal sources. Total tap water consumed included direct consumption of tap water and all beverages made with tap water including beverages made with boiled water, like coffee and tea. Duration of showering or bathing in minutes were combined with estimated THM4 to provide an exposure index intended to represent inhalation and dermal absorption from showering and bathing. Separate data were obtained for showering and bathing. These data are not shown, presumably because little difference was found. Swimming was represented as lifetime duration estimated in lifetime hours. dds ratios were adjusted for age, gender, smoking status (never / former / current), size of the municipality of longest residence until age 18 years, education, geographic area, and overall quality of the interview (reported by the interviewer). The analyses of residential THM levels were restricted to subjects for whom exposure information for at least 70 percent of the exposure window was available (n=1,572). The overall quality of interview was considered unsatisfactory or questionable for 93 subjects that were excluded, leaving data for a total of 707 cases and 772 controls for analysis. For ingestion of THMs, 1,770 subjects provided information covering at least 70 percent of the exposure window, but 579 subjects were excluded because volume of water they consumed was missing. f the remaining 1,191 subjects, 44 whose quality of interview was unsatisfactory were also excluded, resulting in a total of 577 cases and 570 controls. Regarding duration of exposure to chlorinated surface water, information on water source and chlorination status that covered at least 70 percent of the exposure window was available to provide 1,573 subjects. For the resulting subsets, data are not presented with regard to how included subjects compared with excluded subjects for other key variables. Similarly, for THM4 exposure during showering and bathing, only subjects with known THM4 levels for at least 70 percent of the exposure window (n=1,351) were considered. A further 71 subjects were excluded because of unsatisfactory or questionable interviews, and 74 were excluded because of missing data on showering and bathing, resulting in 546 cases and 660 controls for this part of the study. For the swimming in pools analysis, 44 subjects were excluded based on missing data about swimming in pools and 118 were excluded because of interview quality, to yield 787 cases and 963 controls for this analysis. Finally, for duration of swimming 29

53 in pools, 115 subjects were excluded because of missing data and 105 were excluded because of interview quality, to yield 750 cases and 915 controls for analysis. Villanueva et al. (2007) observed that those who reported that their longest residence until age 18 years was in a village had a lower risk of bladder cancer compared with those who lived in a metropolitan area as the reference (R=0.75; 95% CI: , n=451 for cases, n=543 for controls). A possible explanation might be that village water supplies were less likely to have been chlorinated compared with urban areas, suggesting a possible effect of childhood exposure on eventual bladder cancer outcome. No other discussion of this observation or possible implications was provided. Villanueva et al. (2007) provided a graphical display of their source water distribution among their categories of municipal, bottled, private well and other (e.g. springs) for all geographic areas studied in Figure 1. The estimated municipal annual mean THM level is shown on a second scale rising from <5 µg/l around 1950, reaching a level of about 45 µg/l in A similar depiction is provided in Figure 2, except that the proportions of water source and the estimated municipal annual mean THM4 level is shown on a second scale, rising from <5 µg/l around 1930, with a sharp increase from about 25 µg/l to about 60 µg/l between 1965 and 1970, then further increasing to around 80 µg/l from 1995 to These figures are useful for showing the overall temporal change in THM4 levels assumed for the municipal water supply category. According to the description of how exposure information and lifetime individual exposure indices were handled, these type of data were determined for each individual as a time-weighted average THM4 exposure, both as a THM4 concentration in µg/l and using individual information on water consumed, an individual timeweighted average for ingestion THM4 exposure (µg/d). Based on what is stated in the manuscript, it is assumed that these THM4 indices, calculated for each individual case and control, were what was used in determining the R calculations reported. What is not clear from the description of these two figures is how the profile of increasing municipal annual mean THM4 levels was determined in each figure. These figures appear to be composites for multiple municipalities. As individual municipalities adopted chlorination over time, the composite value for THM4 has increased accordingly. However, for plants that changed their treatment process over time and/or for those with source waters with significant variability in water quality over time, changes in THM4 formation were not considered in this analysis. How the individual municipalities were weighted for combination into the single lines shown in Figures 3.1 and 3.2 (i.e., according to flow, population-served, cases and controls served, or something else) is not described. Considering the municipality monitoring for THM4 that was done (Villanueva 2006b, 2003), to the extent that it was available, it is not clear how THM4 levels were assigned from the 123 study municipalities that did provide THM4 data (78.5% of total study person-years) to the other 21.5% of municipalities where data were not provided. Figures 3.1 and 3.2 also show that the proportion of subjects consuming bottled water increased substantially in all geographic areas over the years, especially in geographic areas with high THM levels (Figure 3.2). Consumption of municipal water decreased correspondingly, particularly in high-thm4 geographic areas. Regarding questionnaire responses addressing exposure Villanueva et al. (2007) concluded: Reporting bias is not likely to have occurred. Study subjects were unlikely to have had knowledge of possible links between water-related 30

54 exposures and the disease; therefore, differential response to these questions from cases and controls was improbable. A major finding of this study and central to the evidence warranting review was a clear, significant association of male bladder cancer with estimated average residential THM4, (expressed as µg/l), for men and a significant (p<0.01) dose-response trend. The cut points used for the ranges of THM4 are based on distributing the numbers of cases and controls approximately equally among the categories shown. Using the lowest THM4 exposure as the reference, <8 µg/l (137 cases, 172 controls): R = 1.0 > µg/l (140 cases, 158 controls): R = 1.53 (95% CI: 0.95 to 2.48) > µg/l (183 cases, 160 controls): R = 2.34 (95% CI: 1.36 to 4.03) >49 µg/l (158 cases, 180 controls): R = 2.53 (95% CI: 1.23 to 5.20) Women, with substantially fewer cases, showed results generally consistent with those for men, but the Rs were not significant. The evidence of an association based on duration of using chlorinated surface water in the residence (years) for men was similar. Using the shortest duration as the reference, 0 3 yr (135 cases, 173 controls): R = 1.0 >3 25 yr (156 cases, 155 controls): R = 2.26 (95% CI: 1.19 to 4.29) >25 30 yr (183 cases, 160 controls): R = 2.58 (95% CI: 1.33 to 5.01) >30 yr (158 cases, 180 controls): R = 2.21 (95% CI: 1.17 to 4.20) The trend for increasing duration was not significant (p=0.20) Women, with substantially fewer cases, showed similar Rs as those for men, but the Rs were not significant. An issue that emerges from these results is the finding of a significant and substantial R for exposure to chlorinated water between 3 and 25 years. ther studies on drinking water and bladder cancer have generally only seen significant associations for durations of exposure longer than 30 years. The distribution of cases and controls within this quartile is not evident, and therefore it is difficult to compare the consistency of these findings with the published literature. 31

55 Source: Villanueva, C.M., K.P. Cantor, J.. Grimalt, N. Malats, D. Silverman, A. Tardon, R. Garcia-osas, C. Serra, A. Carrato, G. Castano-Vinyals, R. Marcos, N. Rothman, F.X. Real, M. Dosemeci, and M. Kogevinas Bladder cancer and exposure to water disinfection. Am. J.Epidemiol. 165(2): Figure 3.1 Trends in drinking water source over the years in all geographic study areas for controls in a hospital-based case-control study conducted in Spain, Source: Villanueva, C.M., K.P. Cantor, J.. Grimalt, N. Malats, D. Silverman, A. Tardon, R. Garcia-osas, C. Serra, A. Carrato, G. Castano-Vinyals, R. Marcos, N. Rothman, F.X. Real, M. Dosemeci, and M. Kogevinas Bladder cancer and exposure to water disinfection. Am. J.Epidemiol. 165(2): Figure 3.2 Trends in drinking water source over the years in geographic areas with high THM4 levels for controls in a hospital-based case-control study conducted in Spain,