UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460

OFFICE OF THE ADMINISTRATOR SCIENCE ADVISORY BOARD

June 28, 2007

EPA-SAB-07-008

Honorable Stephen L. Johnson Administrator U.S. Environmental Protection Agency 1200 Pennsylvania Avenue, N.W. Washington, D.C. 20460

Subject: Advisory on EPA’s Assessments of Carcinogenic Effects of Organic and Inorganic Arsenic: A Report of the US EPA Science Advisory Board

Dear Administrator Johnson:

The U. S. Environmental Protection Agency’s (EPA) Office of Pesticide Programs (OPP), Office of Water (OW), a nd Office of Research and Development (ORD) coordinated the development of two scientific documents that address the carcinogenicity of Dimethylarsinic Acid (DMA V ) and inorganic arsenic (iAs). In response to an Agency request, the Science Advisory Board (SAB) convened an expert panel to review and comment on key scientific issues pres ented in these two documents, including: (a) the metabolism and toxic res ponses of arsenic speci es; (b) mode(s) of carcinogenic action; (c) data selection for dose-response assessment; and (d) approaches and methods for low-dose extrapolation for DMA V and iAs.

The SAB Panel supported the Agency’s conc lusion that on the ba sis of available data, human exposure to DMA V appears to result in a narrower spectrum of active metabolites than those expected in the meta bolic profile associated with exposure to iAs. Therefore, the Panel agreed with EPA th at, in the absence of human data on DMA V , the bladder tumor data from DMA V rat bioassays is better suited for DMA V cancer risk assessment than is epidemiology data from iAs exposure. The Panel, however, noted that there remain significant uncertainties associat ed with the use of animal data for DMA V cancer risk assessment due to the observed metabolic differences between rats andhumans. The Panel agreed with the Agency’s conclusion that DMA V -induced bladder cancer in rats, at high dose, is mediated by a cytotoxic mode of acti on, and that this MOA should be considered relevant to humans. However, the Panel concluded there are not sufficient data to support a reactive oxygenated species-media ted mode of direct genetic action for DMA V . The Panel supported the nonl inear approach for low dose extrapolation of DMA V and the use of uncertainty fact ors to account for interspecies differences and human variability for sens itive human populations, and concluded that presently there is no arsenic-specific informa tion that can inform the choice of specific values. This means that, at least for now, such choices must be based on more general considerations, including EPA’s science polic y judgment of the degree of precaution that it deems appropriate.

EPA concluded that the mechanisms by wh ich inorganic arsenic induces bladder cancer in humans are not yet known, but they are likely to be mediated by multiple modes of action. The Agency used a linear default approach for low dose extrapolation because it lacked a full understanding of the iAs modes of carcinogenic action. The Panel agreed that available human and animal data do not fully describe the shape of the iAs carcinogenic dose-response cu rve at low doses. Given the considerable uncertainties regarding low dose extrapolation, the Panel s upported the use of a linear cancer risk model for iAs as recommended by the National Research Council in it s 2001 report. The Panel also supported the use of the epidem iologic data on the Taiwanese population for estimating human cancer risk for iAs especia lly to identify the potential range of responses of human populations. However, th e Panel recognized limitations to these data, and that there is some evidence on iA s from animal toxicology, pharmacokinetics, and pharmacodynamics research, that suggests other than a linear bladder cancer doseresponse. The Panel urged the Agency to c onsider other epidemiol ogic studies from the U.S. and other countries, utilizing a uniform set of evaluative criter ia. The Panel also recommended sensitivity analyses be conducted to account for human variability in drinking water consumption rates, dietary in take of iAs from f ood, and certain other assumptions currently used in EPA’s assessm ent. The Panel made several suggestions for improvements in the currently applied risk model’s programming and documentation conventions.

Finally, the Panel believes there is a critical need for a continued research effort to strengthen EPA’s cancer risk assessment for DMA V and iAs. The scientific bases for the Panel’s conclusions and research recommendati ons are detailed thr oughout this report. We look forward to receiving your response to this review and we appreciate the

2opportunity to provide EPA with advice on th is important subject and stand ready to assist the Agency in any future effo rts in updating the assessment.

Sincerely,

/Signed/ /Signed/

Dr. M. Granger Morgan, Chair Dr. Genevieve Matanoski, Chair EPA Science Advisory Board EPA Science Advisory Board Arsenic Review Panel

3

### NOTICE

This report has been written as part of the activ ities of the EPA Science Advisory Board, a public advisory committee providing extramural scientific information and advice to the Administrator and other officials of the Environmental Protection Agency. The Board is structured to provide balanced, expert assessment of sc ientific matters related to problems facing the Agency. This report has not been reviewed for approval by the Ag ency and, hence, the contents of this report do not necessarily represent the views and policie s of the Environmental Protection Agency, nor of other agencies in the Executiv e Branch of the Federal governm ent, nor does mention of trade names or commercial products constitute a re commendation for use. Reports of the EPA Science Advisory Board are posted on the EPA Web site at: http://www.epa.gov/sab

.U.S. Environmental Protection Agency Science Advisory Board

Arsenic Review Panel

CHAIR Dr. Genevieve Matanoski , Professor, Department of Epid emiology, Johns Hopkins University, Baltimore, MD

MEMBERS Dr. H. Vasken Aposhian , Professor, Department of Molecu lar and Cell Biology, The University of Arizona, Tucson, AZ

Dr. Aaron Barchowsky , Associate Professor, Department of Environmental and Occupational Health, Graduate School of Public Healt h, University of Pittsburgh, Pittsburgh, PA

Dr. David Brusick , Retired, Convance Labs, Vienna, VA

Dr. Kenneth P. Cantor , Senior Investigator, Occupational and Environmental Epidemiology Branch, Division of Cancer Epidemiology and Ge netics, National Cancer Institute, Bethesda, MD

Dr. John (Jack) Colford , Associate Professor, Divisi on of Public Health, Biology & Epidemiology, School of Public Health, Un iversity of California, Berkeley, CA

Dr. Yvonne P. Dragan , Director of the Divisi on of Systems Toxicology (DST) and Chief of the Center for Hepatotoxicology, National Center for Toxicological Rese arch (NCTR), Food and Drug Administration's (FDA), Jefferson, AR

Dr. Sidney Green , Associate Professor, Department of Pharmacology, College of Medicine, Howard University, Washington, DC

Dr. Sioban Harlow , Professor, Department of Epid emiology, School of Public Health, University of Michigan, Ann Arbor, MI

Dr. Steven Heeringa , Research Scientist and Director, Statistical Design Group, Institute for Social Research (ISR), Universi ty of Michigan, Ann Arbor, MI

Dr. Claudia Maria Hopenhayn , Associate Professor, Department of Epidemiology, Markey Cancer Control Program, College of Public Health, Un iversity of Kentucky, Lexington, KY

Dr. James E. Klaunig , Professor and Director, Departme nt of Pharmacology and Toxicology, School of Medicine, Indiana University, Indianapolis, IN

iiDr. X. Chris Le , Professor, Department of Public Hea lth Sciences, Department of Chemistry & Department of Laboratory Medici ne & Pathology, University of Alberta, Edmonton, Alberta, Canada

Dr. Michele Medinsky , Toxicology Consultant, Toxcon, Durham, NC

Dr. Kenneth Portier , Program Director, Department of Statistics and Evaluation, American Cancer Society, Atlanta, GA

Dr. Barry Rosen , Professor and Chairman, Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI

Dr. Toby Rossman , Professor, Environmental Medicine , School of Medicine, New York University, Tuxedo, NY

Dr. Miroslav Styblo , Research Associate Professor, Depart ment of Nutrition and the Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina, Chapel Hill, NC

Dr. Justin Teeguarden , Senior Scientist, Pacific Northw est National Laboratory, Richland, WA

Dr. Michael Waalkes , Chief, Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer In stitute, National In stitute of Environmental Health Science, Research Triangle Park, NC

Dr. Janice Yager , Research Program Manager, Envir onment Department, Electric Power Research Institute, Palo Alto, CA

SCIENCE ADVISORY BOARD STAFF Mr. Thomas Miller , Designated Federal Officer, EPA Scie nce Advisory Board Staff Office

iiiU.S. Environmental Protection Agency Science Advisory Board

CHAIR Dr. M. Granger Morgan , Professor and Head, Department of Engineering and Public Policy, Carnegie Mellon University, Pittsburgh, PA

SAB MEMBERS Dr. Gregory Biddinger , Environmental Programs Coordi nator, ExxonMobil Biomedical Sciences, Inc, Houston, TX

Dr. James Bus , Director of External Technology, Toxi cology and Environmental Research and Consulting, The Dow Chemical Company, Midland, MI

Dr. Deborah Cory-Slechta , Director, Environmental and Occupational Health Sciences Institute, Robert Wood Johnson Medical School, Un iversity of Medicine and Dentistry of New Jersey and Rutgers State Un iversity, Piscataway, NJ

Dr. Maureen L. Cropper , Professor, Department of Economics, University of Maryland, College Park, MD

Dr. Virginia Dale , Corporate Fellow, Environmental Sc iences Division, Oak Ridge National Laboratory, Oak Ridge, TN

Dr. Kenneth Dickson , Professor, Institute of Applied Sciences, University of North Texas, Denton, TX

Dr. Baruch Fischhoff , Howard Heinz University Professor, Department of Social and Decision Sciences, Department of Engineering and Public Policy, Carnegie Mellon University, Pittsburgh, PA

Dr. James Galloway , Professor of Environmental Sciences, Environmental Sciences Department, University of Virginia, Charlottesville, VA

Dr. Lawrence Goulder , Shuzo Nishihara Professor of E nvironmental and Resource Economics, Department of Economics, Stanfo rd University, Stanford, CA

Dr. James K. Hammitt, Harvard Center for Risk Analys is, Harvard University, Boston, MA

Dr. Rogene Henderson , Scientist Emeritus, Lovelace Re spiratory Research Institute, Albuquerque, NM

Dr. James H. Johnson , Dean, College of Engineering, Ar chitecture & Com puter Sciences, Howard University, Washington, DC

ivDr. Agnes Kane, Dept. of Pathology and Laboratory Medi cine, Brown University, Providence, RI.

Dr. Meryl Karol , Associate Dean for Academic Affairs, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA

Dr. Catherine Kling , Professor, Department of Economics, Iowa State University, Ames, IA

Dr. George Lambert , Associate Professor and Director , Center for Child and Reproductive Environmental Health & Pediatric Clinical Rese arch Center, Departmentof Pediatrics, UMDNJRobert Wood Johnson Medical School/University of Medicine and Dentistry of New Jersey, New Brunswick, NJ

Dr. Jill Lipoti , Director, Division of Environmental Safe ty and Health, New Jersey Department of Environmental Protection, Trenton, NJ

Dr. Genevieve Matanoski , Professor, Department of Epid emiology, Johns Hopkins University, Baltimore, MD

Dr. Michael McFarland , Associate Professor, Department of Civil and Environmental Engineering, Utah State University, Logan, UT

Dr. Judith Meyer, Institute of Ecology, Univers ity of Georgia, Athens, GA.

Dr. Jana Milford , Associate Professor, Department of Mechanical Engineer ing, University of Colorado, Boulder, CO

Dr. Rebecca Parkin , Professor and Associate Dean, Envi ronmental and Occupational Health, School of Public Health and H ealth Services, The George Wa shington University, Washington, DC

Mr. David Rejeski , Foresight and Governance Project Di rector, Woodrow W ilson International Center for Scholars, Washington, DC

Dr. Stephen Roberts, Department of Physiological Sciences, University of Florida, Gainesville, FL.

Dr. Joan B. Rose , Professor and Homer Nowlin Chair for Water Research, Department of Fisheries and Wildlife, Michigan State University, E. Lansing, MI

Dr. Jerald Schnoor, Department of Civil and Environmental Engineering, University of Iowa, Iowa City, IA.

Dr. Kathleen Segerson , Professor, Department of Economics , University of Connecticut, Storrs, CT

vDr. Kristin Shrader-Frechette , O'Neil Professor of Philosophy- Concurrent Professor of Biological Sciences-and Director of the Center for Environmenta l Justice and Children's Health, Department of Biological Sciences and Philoso phy Department, University of Notre Dame, Notre Dame, IN

Dr. Philip Singer, Department of Environmental Sciences and Engineering, Un iversity of North Carolina, Chapel Hill, NC.

Dr. Robert Stavins , Albert Pratt Professor of Busine ss and Government, Environment and Natural Resources Program, John F. Kennedy Sc hool of Government, Harvard University, Cambridge, MA

Dr. Deborah Swackhamer , Professor, Division of Environm ental Health Sciences, School of Public Health, University of Minnesota, Minneapolis, MN

Dr. Thomas L. Theis , Professor and Director, Institute for Environmental Science and Policy, University of Illinois at Chicago, Chicago, IL

Dr. Valerie Thomas , Anderson Interface Associate Professo r of Natural Systems, School of Industrial and Systems Engi neering, Georgia Institute of Technology, Atlanta, GA

Dr. Barton H. (Buzz) Thompson, Jr. , Robert E. Paradise Professor of Natural Resources Law, Stanford Law School, and Director , Woods Institute for the Envir onment , Stanford University, Stanford, CA

Dr. Robert Twiss , Professor, University of California-Berkeley, Ross, CA

Dr. Terry F. Young , Consultant, Environmenta l Defense, Oakland, CA

Dr. Lauren Zeise , Chief, Reproductive and Cancer Hazard Assessment Section, California Environmental Protection Agency, Oakland, CA

vi

### TABLE OF CONTENTS

1. EXECUTIVE SUMMARY

....................................................................................................... 1

2. INTRODUCTION

............................................................................................................... .... 12 2.1 Process for Developing this Report and Structure of the Report

........................................ 12 2.2 Background

......................................................................................................................... 13

3. RESPONSE TO THE CHARGE

.............................................................................................. 17 3.1 Overview

............................................................................................................................. 17 3.2 Metabolism and Toxic Responses of Arsenic Species

....................................................... 17 3.2.1 Metabolism and pharmacokinetics

.............................................................................. 17 3.2.2 Response to mixtures of metabolites

........................................................................... 20 3.3 Modes of Carcinogenic Action for DMA V and Inorganic Arsenic..................................... 22 3.3.1. Mode of Action of DMA V

.......................................................................................... 22 3.3.2 Human relevance of animal DMA V MOA

................................................................... 26 3.3.3 Modes of carcinogenic action from exposures to Inorganic Arsenic

.......................... 27 3.4 Selection of Data for Dose-Response Assessment ............................................................. 34 3.4.1 Use of animal data for DMA V

..................................................................................... 34 3.4.2. Use of human epidemiological data from direct inorganic arsenic exposure

............. 36 3.5 Approaches to Low-Dose Extrapol ation for Inorganic Arsenic and DMA V ...................... 40 / 3.5.1 Mode of carcinogenic acti on understanding for DMA V III

........................................... 40 3.5.2 Implementation of the recommendations of the NRC

................................................. 43 3.5.3 EPA Model Re-Implementation

.................................................................................. 45 3.5.4 Available literature describing drinking water consumption rates

............................. 49 3.5.5 Selection of an estimate of diet ary intake of arsenic from food

................................. 51

REFERENCES

........................................................................................................................... R-1

APPENDIX A Charge to the EPA Science Advisory Board

..................................................... A-1

APPENDIX B Assignments to Charge-Specific Groups

........................................................... B-1

APPENDIX C Abbreviations

.................................................................................................... C-1

vii

### 1. EXECUTIVE SUMMARY

New information has been developed on the metabolism, pharmacokinetics (PK) and mode of carcinogenic action of arseni c and its methylated species and new epidemiology studies have been conducted on inorganic arsenic sin ce the publication of reviews by the National Res earch Council (NRC, 1999, 2001). EPA considered this new science in the development of the Office of Pesticide Programs’ (OPP) Draft Science Issue Paper: Mode of Action for Cacody lic Acid (Dimethylarsinic Acid) and Recommendations for Dose Response Extrapolation (USEPA OPP, 2005) and the Office of Water’s (OW) Draft Toxicologic Review of Inorganic Arsenic (USEPA OW, 2005). EPA’s Office of Research and Development (ORD ) further captured key scientific issues to be considered in its Issue Paper Cancer Risk Assessment for Organic Arsenical Herbicides: Comments on Mode of Action, Human Relevance and Implications for Quantitative Dose-Response Assessment (Appendix E of USEPA OPP, 2005, USEPA ORD, 2005). The Science Advisory Board (S AB) was asked to review these documents and offer advice on the metabolism, mode of action, dose-response, and approaches to low-dose extrapolation of cancer risk for Dimethylarsinic Acid (DMA V ) and inorganic arsenic (iAs). The full charge to the SAB is in Appendix A to this document.

In response to the Agency’s request, the SAB convened an expert Panel to provide advice to the Agency on these scie ntific issues. In responding to the EPA Charge, the Panel reviewed the EPA asse ssments mentioned above, and considered comments and information that members of the interested public provided during each of the Panel’s advisory meetings (during 2005 and 2006), and additional studies that are identified in the reference section of this report. The Panel considered expanding the Charge to include other health endpoints as sociated with arsenic and arsenic containing compounds. However, the Panel decided not to expand its activ ities beyond the EPA Charge that largely focused on bladder cance r and to some degree on lung cancer doseresponse issues. It is important to rec ognize that the Panel di d not conduct its own arsenic risk assessment. To do so would have required an updated literature search and exploration and resolution of many issues that are discusse d throughout this report. The Panel leaves the larger activity of completing a full risk assessment of all relevant health endpoints associated with arsenic, and ar senic containing compounds, to the Agency itself when it conducts its final arsenic assessments.

The Panel was organized into small groups of three to seven members to evaluate and respond to each specific charge question (s ee Appendix B to this report for a list of those members assigned to each charge questi on). The Panel’s response to each question reflects consensus, though not necessarily unanimous agreement, among Panel Members that addressed each specific charge questi on. In addition, all Panel Members had the opportunity to participate in meeting discussions of each ch arge question and each was able to provide written comments on all que stions during report drafting. Many Membersparticipated in this way and each response reflects adjustments that were considered to be appropriate by the specific charge group that led the Panel’s efforts for each question. In that manner, this advisory report provides th e Panel’s judgments on ea ch specific issue. This advice is intended to assist the Agency’s continued efforts to complete its assessments on various arsenicals. Th ere are many specific conclusions and recommendations on specific issues associatedwith each charge question, as well as recommendations for sensitivity analyses and additional research to answer many of the remaining questions on arsenic risk. The Panel’s advice on each charge question is discussed in the remainder of this Executive Summary and discussed in detail in Section 3 of this report.

1.1 Metabolism and Toxic Responses of Arsenic Species

Charge Question A1

EPA concluded that available in vivo and in vitro metabolism and pharmacokinetic studies in humans and laboratory an imals suggest that the efficiency of methylation reactions and cellu lar uptake varies with the arsenic compound administered exogenously. Most studies s uggest a predominantly one-way process in mammals and that after DMA V exposure, significant amounts of iAs III , iAs V , methylarsonous acid (MMA III ), or methylarsinic acid (MMA V ) are not expected at target tissues. EPA asked the SAB to comment on how best to consider the PK processes in cancer risk assessment based on data derived from di rect dimethylarsinic acid (DMA V ) exposure versus direct inorganic arsenic (iAs) exposure.

Summary Response

The Panel agreed that:

i) Metabolism of iAs appears to be a one-way process in which iAs is converted to monomethylarsenic (MMA ), dimethylarsenic (DMA), and in some species to trimethylarsenic (TMA) metabolites with arsenic in +3 or +5 oxidation states. Thus, signifi cant amounts of MMA or iAs are not expected to be found in tissues or urin e of rats or humans as a result of exposure to DMA V , although iAs may be present in human tissues or urine from other sources. ii) In contrast, exposure to iAs may re sult in production, tissue retention, and urinary excretion of a variety of tri- and pentavalent iAs and methylated arsenic species. iii) The uptake and reduction of DMA V to dimethylarsinous acid (DMA III ) are apparently critical step s in activation of DMA V – though it is not clear if, where and to what extent these proc esses occur in humans exposed to DMA V

. iv) The capacity to reduce DMA V to DMA III seems to exist in human tissues and the conversion of even a small amount of exogenous DMA V to DMA III is of toxicological concern.

2v) Given the differences in the metabolic pattern for iAs and DMA V , the Panel believes data derived from DMA V exposure, not from iAs exposure, is better suited for cance r risk assessment of DMA V

. vi) Significant uncertainties are associ ated with this approach. The toxicologic data on DMA V are mainly from rat studies, and considering several key differences between rats and humans in the metabolism of arsenic, these uncertainties should be considered in the assessment of DMA V cancer risk. Additional uncertainties include methylation and demethylation of arsenic compounds inhumans by intestinal bacteria, coexposures to other environmental cont aminants, deficiencies in nutrients, and malnutrition. vii) The physiologically based pharmacokinetic (PBPK) model under development by EPA may be a usef ul approach but it is not yet sufficiently robust to conduct in terspecies extrapolations. viii) EPA should continue developing the arsenic PBPK model and conducting research to obtain kinetic constants needed to describe rates of uptake, efflux, metabolism, and elimination of DMA V in rats and humans. ix) There is a need to validate such models for predicting tissue concentrations of active species re gardless of the source of arsenic
exposure.

Charge Question A2

EPA concluded that direct exposure to iAs III or iAs V is expected to result in a more complex mixture of toxic metabolites than with DMA V exposure given that mixtures of metabolites vary based on whic h chemical is administered exogenously. EPA expects a less complex mixture of metabolites following DMA V exposure than following iAs exposure. EPA further expects th at the tumorigenic profiles vary with the arsenical compound administered. For its DMA V assessment, EPA asked the SAB to comment on the use of data derived from rode nt exposures to orga nic arsenicals versus data derived from directhuman exposure to iAs.

Summary Response

The Panel agreed that:

i) Neither rodent laboratory data on organic arsenicals nor data from studies of human exposure to inor ganic arsenic provide an optimal basis for the assessment of DMA V exposure in humans because of differences between the metabolic profiles for inorgani c arsenic and DMA and because of interspecies differences in their metabolism. Despite these uncertainties, for now, the data from rodent exposures to DMA V appear to be the most reasonable approach for the DMA V assessment, though this approach has a significant degree of uncertain ty (see charge question A1). ii) The metabolism of iAs yields a wide spectrum of metabolites which are apparently not produced during the metabolism of DMA V

.

3iii) Production of iAs and MMA metabolites may be associated with specific toxic or cancer endpoints that are absent in DMA V exposure to rats or humans. iv) All published data on toxico logical responses to DMA V are from studies in rodents, mainly rats; no human data are available. As noted in the response to A1 above, these differe nces raise concerns for risk assessments based on these data.

1.2. Modes of Carcinogenic Action for DMA V and Inorganic Arsenic

Charge question B1

EPA’s approach to cancer risk assessme nt incorporates two key science policy assumptions when there are inadequate human data and it needs to rely on laboratory animal data: (a) animal tumor data are pr edictive of human cancer and (b) effects found at high experimental doses in animals predict human risk at lower exposure levels. Understanding a mode of action (MOA) for a ch emical can help to inform the agency about these assumptions and the most appr opriate approach to follow in low dose extrapolation. EPA asked the SAB to co mment on the scientif ic soundness of the postulated MOA for DMA V -induced bladder carcinogenesis in the rat.

Summary Response

The Panel concluded that:

i) There are adequate data to support an MOA for bladder carcinogenesis induced by high doses of DMA V in the rat and that MOA involves cytotoxicity to the bladder epithelium and increased, sustained regenerative proliferation as key events. ii) The rat metabolizes a significant fraction of exogenous DMA V to trimethylarsine oxide (TMA V O) and possibly trimethylarsine (TMA III ) and that these compounds cannot be exclude d as additional mediators of the necrotic cytotoxicity in th e bladder of exposed rats. iii) There are not sufficient data to invoke reactive oxygen species (ROS)- induced DNA damage as a key ev ent in the carci nogenic process associated with exposures to DMA V and DMA III

. iv) The Panel’s postulated MOA for DMA V is: a) Reductive metabolism of DMA V to DMA III

, b) High concentrations of DMA III (and possibly DMA V ) in urine cause urothelial cyto toxicity, and c) Continuous exposure and persistent stress-associated regenerative cell proliferation leads to genomic in stability, acquisition of genetic alterations, clonal expansion of alte red cells and eventually tumors. v) The Panel suggested several high priori ty research needs for this issue.

4Charge question B2

EPA concluded that their postulated MOA for DMA V induced bladder carcinogenesis in the rat would be relevant to humans as th ere are little or no data to suggest that key precursor events and ultim ately tumor formation would not occur in exposed humans if sufficient DMA III were present. EPA asked the SAB to comment on the relevance of the postulated key events to tumors in humans and how differences in humans and experimental animals should be accounted for in DMA V risk assessments.

Summary Response

The Panel concluded that:

i) If high enough concentrations of DMA V or DMA III were present in human urine or the bladder after exposure to DMA V it is plausible that a similar response would take place; however, no data are available to support or reject this assumption. ii) The suggested greater conversion of DMA V to TMA V O or possibly TMA III in rats vs. in humans, may contribute to induction of bladde r cancer in rats, however, the extent of th e contribution is unknown. iii) No studies have been conducted to determine whether the DMA V carcinogenic risk differs by life stag e, e.g., among the young, or elderly.

Charge Question B3

EPA concluded that iAs causes human cancer most likely by many different modes of action. This is based on the obs erved findings that iAs undergoes successive methylation steps in humans and results in th e production of a number of intermediate metabolic products and that each has its own toxicity. EPA asked the SAB to comment on the soundness of its conclusion.

Summary Response

The Panel concluded that:

i) Multiple modes of action may operate in carcinogenesis induced by iAs because there is simultaneous exposure to multiple metabolic products as well as multiple target organs and the composition of metabolites can differ in different organs. ii) Each arsenic metabolite has its ow n cytotoxic and genotoxic capability. iii) Inorganic arsenic (iAs III ) and its metabolites are not direct genotoxicants because these compounds do not direc tly react with DNA. However, iAS III and some of its metabolites can e xhibit indirect genotoxicity, induce aneuploidy, cause changes in DNA met hylation, and alter signaling and hormone action. In addition, iAs can act as a transplacental carcinogen and a cocarcinogen.

5iv) Studies of indirect genotoxicity stro ngly suggest the possibility of a threshold for arsenic carcinogenicity. However, the studies discussed herein do not show where such a threshold might be, nor do they show the shape of the dose-respon se curve at these low levels. In addition, a threshold has not been confirmed by ep idemiological studies. This issue is an extremely important area for research attention, and it is an issue that should be evaluated in EPA’s continuing risk assessment for iAs. v) Arsenic essentiality and the possibility of hormetic effects are in need of additional research to determin e how they would influence the determination of a threshold for specific arsenic-associated health endpoints.

1.3. Selection of Data for Dose-Response Assessment

Charge Question C1

In the absence of human data , EPA proposed to use the bladder tumor data from the DMA V rat bioassay for quantifying poten tial human cancer risk to DMA V . EPA asked the SAB to comment on the appropriatene ss of this approach. The SAB was also asked to comment on whether the iAs epidem iology data can be used to inform the DMA V dose-response assessment which is now base d on data derived from studies in rats dosed with DMA V

.
Summary Response

The Panel agreed that:

i) Given the lack of human data, the bladder tumor data from DMA V rat bioassays, are the most suitable data set for quantifying potential human cancer risk from DMA V . The Panel stated that the available data suggest that the uncertainty associated with extrapolation across forms of arsenic in the DMA V risk assessment would be greater than interspecies extrapolation. ii) The Panel strongly suggested that EPA’s DMA V assessment discuss the key uncertainties in using data from studies in rats to conduct human health risk assessments. Panel respon ses to charge questions A1 and C1 discuss issues that members consider ed important to discuss in EPA’s Science Issue Paper. These issues relate to the pharmacokinetic and pharmacodynamic similarities and differe nces between rats and humans in response to arsenic exposure, the use of rodent bladder tumor models in general, and issues in the use of r odent data for human risk assessment. iii) The Panel considers research on thes e issues to be a high priority. iv) The Panel concluded that without mo re detailed information on target tissue dosimetry for arsenic species, the iAs epidemiology data would be of limited use to inform the DMA V dose-response assessment derived

6from rat data with DMA V . Additional details are contained in the Panel’s response to charge question C1.

Charge Question C2

EPA reviewed the available epidemiologi c studies including those published since the NRC 2001 review for U.S. populations e xposed to inorganic arsenic via drinking
water. EPA concluded that the Taiwanese datase t remains the most appropriate choice for estimating cancer risk in humans. Th e SAB was asked to comment on the soundness of this conclusion and also on whether these da ta provide adequate ch aracterization of the impact of childhood exposure to iAs.

Summary Response

The Panel concluded that:

i) Because of various factors (e.g., size and statistical stability of the Taiwanese database relative to ot her studies, the reliability of the population and mortality count s, the stability of re sidential patterns, and the inclusion of long-term exposures), th is database remains, at this time, the most appropriate choice for es timating bladder cancer risk among humans, though the data have considerable limitations that should be described qualitatively or quantitatively to help inform risk managers about the strength of the conclusions. ii) There are other epidemiologic database s from studies of populations also exposed at high levels of arsenic, an d the Panel recommends that these be used to compare the unit risks at th e higher exposure levels that have emerged from the Taiwan data. iii) The Panel also suggests that publishe d epidemiology studies of US and other populations chronically exposed from 0.5 to 160 μg/L inorganic arsenic in drinking water be critically evaluated, using a uniform set of criteria and that the results from these evaluations be transparently documented in EPA’s assessment documen ts. If, after th is evaluation, one or more of these studies are shown to be of potenti al utility, the low-level studies and Taiwan data may be comp ared for concordance. Comparative analyses could lead to further insight s into the possible influence of these differences on population responses to arsenic in drinking water. iv) Regarding childhood exposure to iAs, it was the Panel’s view that, based on available data, it is not clear whet her children differ from adults with regard to their sensitivity to the carc inogenic effects of arsenic in drinking water. However, the possibility of a different response in degree or kind should not be ignored and n eeds to be investigated.

71.4 Approaches to Low-Dose Extrapolation for iAs and DMA V

Charge Question D1

EPA’s Guidelines for Carcinogen Risk Assessment underscore the importance of understanding the MOA as the basis for maki ng judgments on how to best extrapolate cancer risk at lower exposures. EPA concluded that available data on DMA V are not sufficient to support development of biologically -based models and therefore opted to use a default nonlinear low-dose extrapolation method. The SAB was asked to comment on the Agency’s scientific rationa le in support of this approach and how uncertainty should be incorporated into low-dose extrapolation.

Summary Response

The Panel concluded that:

i) Though there are adequate data to support the proposed EPA MOA, neither the MOA postulated by the Pa nel, nor those postulated by EPA’s ORD or OPP contain key events expect ed to be a linear function of dose of DMA V

. ii) Several processes important to some postulated key events would have non-linear components or are non- linear (e.g., saturable metabolic processes, cytotoxicity, formation of heritable alterati ons in DNA by ROS, cell proliferation, repair of ROS-induced DNA damage). iii) The linear approach would be cons istent with evidence for direct genotoxicity of DMA III/V ; however, it is generally accepted that DMA V is not directly genotoxic and neither DMA III nor DMA V react directly with DNA.

DMA iv) There are insufficient data to invoke ROS-induced DNA damage as a key event in the carcinogenic process as sociated with exposures to DMA V or III

. v) The nonlinear approach is more consistent with available DMA V data and current concepts of chemical carcinogenesis. vi) Uncertainty is best incorporated thro ugh the use of uncerta inty factors that capture pharmacokinetic and pharmacodynamic differences across species and differences associated with sensitive populations. vii) There are not sufficient data on comparative dosimetry in rats and humans to make any conclusive statemen ts about species differences in pharmacokinetics, though available data on uroepithelial cell cytotoxicity might allow EPA to assemble a case for pharmacodynamic equivalency. There is presently no arsenic-specific information that can inform the choice of uncertainty factors for sensitive human populations. Thus, at least for now, such choices must be based on more general considerations including EPA’s science policy judgmentof the degree of precaution that it deems appropriate.

8Charge Question D2

EPA determined that the most prudent approach for modeling cancer risk from iAs is to use a linear model because of the remaining uncertainties regarding the ultimate carcinogenic metabolites and whethe r mixtures of toxic metabolit es interact at the site(s) of action. EPA asked the SAB if it concurred with the selection of a linear model following the recommendations of the NRC (2001) to estimate cancer risk in light of the multiple modes of carcinogenic action for iAs.

Summary Response

The Panel concluded that:

i) Inorganic arsenic has the potential fo r a highly complex mode of action. ii) Until more is learned about the complex PK and PD properties of iAs and its metabolites there is not sufficient justification for the choice of a specific nonlinear form of th e dose-response relationship. iii) The NRC (2001) recommendation to base risk assessments on a linear dose response model that includes th e Southwestern Taiwan population as a comparison group seems the mo st appropriate approach. iv) The Panel also recommends that EPA perform a sensitivity analysis of the Taiwanese data with different expos ure metrics, with the subgroup of villages with more than one well measurement, and using a multiplicative model that includes a quadratic term for dose.

Charge Question D3

EPA employed the Microsoft Excel softwa re that was previously used by the NRC (2001) to project estimated cancer risks from iAs exposure. The SAB was asked to comment on the precision and accuracy of this program.

Summary Response:

The Panel concluded:

i) That the EPA program conformed to the NRC (2001) recommendation for modeling cancer hazard as a function of age and the average daily dose of exposure to arsenic through drinking water sources. ii) The panel did, however, identify and report to the EPA on two potential discrepancies in the data inputs and one computational error in the portion of the program that employs the BEIR-IV formula to evaluate excess lifetime cancer risk from arsenic exposure. iii) The panel made several suggestions for improvements in the model’s programming and documentation conven tions as well as recommendations for specific sensitivity analyses desi gned to test the robustness of the

9model to alternative formulations of the hazard function and aggregate population data inputs.

Charge Question D4

In calculating estimated cancer risk to the US general population from drinking water exposure to iAs, the EPA utilized epid emiologic data from Taiwan. EPA followed the NRC (2001) recommendations to account fo r the differences in the drinking water consumption rates for the Taiwanese populati on and U.S. populations. On the basis of more recent data (noted in USEPA, 2005b), EP A utilized water intake adjustments for 2 to 3.5 liters/day. EPA asked the SAB to recommend a drinking water value.

Summary Response

The Panel agreed that water consumption (v ia drinking as water, in beverages, or in cooking water) assumptions have a substant ial impact on the assessment of arsenic’s risk. However, the Panel did not recommend sp ecific values for EPA to use in evaluating dose-response in the Taiwanes e study nor for levels of e xposure in the U.S. population risk estimates. It did recommend that uncertain ty in this parameter be evaluated for both the Taiwanese study population and the U. S. populations at risk. The Panel recommended that EPA should:

i) Evaluate the impact of drinking wate r consumption rates associated with more highly exposed population grou ps with differing exposures and susceptibilities (e.g., children, pregnant women). ii) Incorporate variability parameters fo r individual water consumption into their analysis for dose-response in th e Taiwanese population as they have done for the U.S. population. iii) Conduct sensitivity analyses of the impact of using a range of consumption values for the Taiwanese population. iv) Provide a better justification for assu ming different consumption levels by gender or in the absence of such a justification, conduct additional sensitivity analyses to examine th e impact of equalizing the genderspecific consumption level. v) More fully articulate and document ho w different sources of water intake, as well as variability, are incorporatedinto the risk model (e.g. data for intake from beverages and cooking water).

Charge Question D5

As recommended by the NRC (2001) EP A considered the background dietary intake of iAs and incorporated adjustment va lues of 0, 10, 30, and 50 μg per day into the cancer modeling based on available new da ta. The SAB was asked to recommend a value for the background dietary intake of iAs for both the contro l population and study population of Southwestern Taiwan.

10Summary Response

The Panel agreed that arsenic levels in food are important considerations for EPA’s assessment of lung and bladder cancer ri sk associated with exposures to arsenic in drinking water. However, the Panel did not recommend a specific value for EPA to use in its base risk assessment. It did recomme nd a range of values for consideration by EPA in its sensitivity analysis and the Panel offered suggestions to EPA for additional analytical steps to clarify the impact of food levels of arse nic on dose-response and exposure as it revises its risk estimates. Th ese Panel recommendations include that EPA should:

i) Conduct sensitivity analyses using a ra nge of total arsenic food intake values from at least 50 to 100 μg per day to perhaps as high as 200 μg per day to assess the impact of this range of dietary intakes on risk of lung and bladder cancer from exposure via drinking water in the Taiwan cohort. ii) Not assume that the control population has an intake value of zero arsenic from food. iii) Apply greater rigor in thei r discussions of data used in these assessments (e.g., sources, methodological and analyt ical issues, bioavailability). iv) Give immediate research attention to the issue of arsenic bioavailability.

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### 2. INTRODUCTION

2.1. Process for Developing this Repo rt and Structure of the Report

In response to the Agency’s request (USEPA, 2005a), the SAB convened an expert Panel to review the Agency’s hazard and dose-response assessments for dimethylarsinic acid (DMA V ) and inorganic arsenic (iAs). The full EPA charge to the SAB is in Appendix A to this advisory report. The Panel was established in accordance with the SAB Panel Formation Process: Immediat e Steps to Improve Policies and Procedures (EPA-SAB-EC-COM-02-003). The Pane l held public telephone conference meetings and a face-to-face meeting to plan for and conduct its advisory activities. The Panel met on September 12-13, 2005 to discuss th e issues and deliber ate on its response to the charge questions. The Panel held three subsequent publi c telephone conference meetings on January 24, 2006, February 23, 2006 and February 28, 2006 (see GPO, 2005a; 2005b; 2005c; 2005d; 2005e; and 2005f). Th e Panel considered written comments and oral statements from the public during each of these advisory meetings. This was also the case for the chartered SAB’s pub lic telephone conference meeting held on November 27, 2006 to conduct a review of the draft Panel report (see GPO 2006). Written public comments submitted for considera tion at these meetings are available on the SAB web page.

Several individuals from the public request ed that the Panel consider broadening its charge to include other human health e ndpoints associated with exposure to arsenic and to undertake a quantitative dose-respons e assessment for arsenic induced bladder cancer. While the Panel recognized that there is a need for the Agency to conduct a complete and thorough review of all health effects endpoints associated with arsenic exposure, the Panel limited its advisory to is sues relevant to carcinogenicity of various arsenicals as requested by EPA. The Panel did not conduct a full risk assessment of the arsenicals of interest itself, because this was beyond the scope of the project and the resources available to the SAB to conduct such an analysis.

Throughout the Panel’s report writing a nd editing process, and during the Chartered SAB’s quality review, some Panel Memb ers, and some of the interested public, suggested that additions to lite rature subsequent to the Pane l’s deliberations be evaluated and referenced in the panel’s report. The Pane l did not add such studies to its discussions because its active deliberations were complete . The Panel’s advice to EPA considers the review of other relevant studi es to be within its suggesti on to EPA to evaluate other endpoints, and other studies, as the Agency completes its arsenic assessments.

This advisory report is st ructured according to the ch arge questions submitted by EPA. Subgroups of Panel members were assigned to focus on specific charge questions (see Appendix B), and each such group was responsible for leading the discussions for specific charge questions dur ing the public meetings a nd for drafting the written responses to the charge questions based upon th e Panel’s discussions and deliberations at the meetings. All Panel members were encour aged to participate in all discussions as well as to provide comments on the draft re sponses for each question during the entire

12advisory process. The Panel’s draft repor t (dated September 15, 2006) was reviewed by the chartered SAB and approved conditional to the Panel Chair making the series of clarifying editorial revi sions; that are now reflected in th is final report which has been approved by the Panel Chair and the SAB Chair.

2.2 Background

EPA’s Office of Research and Developm ent (ORD), the Office of Water (OW ) and the EPA Office of Pesticide Programs (O PP), requested that the EPA SAB evaluate certain components of the EPA draft asse ssment of potential human carcinogenicity associated with arsenic, and arsenic cont aining compounds. Information from the EPA request is summarized in the remainder of this section of the Panel’s report.

Inorganic arsenic (iAs) is found naturally in the environment and it is typically present in soil and water at some determinat e level. Human exposure to inorganic arsenic can come from drinking water, food, ai r and anthropogenic sources such as wood preservatives, industrial wast es, and certain pesticides containing organic arsenic.

Specific statutory mandates require th at EPA consider human health risks associated with arsenic and arsenic containing compounds. The Safe Drinking Water Act (SDWA) directs EPA to establish national st andards for arsenic containing compounds, among other contaminants, in public drinki ng water supplies. EPA’s Superfund and Resource Conservation and Recovery Act (RCRA) programs require the evaluation of exposure to arsenic containing compounds at locations undergoing clean up or remediation, and the Clean Air Act, require s EPA to set air emissions standards for certain sources of arsenic. EPA’s OPP evalua tes the exposure and health risks associated with arsenicals used as pesticides in the U.S. and under the mandate of the Food Quality Protection Agency (FQPA) is r eevaluating tolerances for arsenicals, and other pesticides. Tolerances are legal limits of pesticides on or in food or animal feed. Several organic arsenic containing herbicides are undergoing re registration and/or to lerance reassessment (e.g., cacodylic acid which is often referred to as dimethylarsinic acid or DMA V , and the monosodium, disodium, and calcium salts of methanearsonate acid --MSMA, DSMA, and CAMA, collectively as referred as MMA V ).

Arsenic, and arsenic containing com pounds, have been the focus of many EPA assessments as the above statutory authorities suggest. In addition, the National Research Council (NRC) of the National Academ y of Sciences (NAS) has conducted comprehensive health sciences reviews of ar senic on at least two occasions (NRC, 1999; NRC, 2001). EPA SAB Panels have also considered inorganic arsenic issues (US EPA SAB, 2000; USEPA SAB, 2001).

Since the 2001 NAS review, new informa tion has been developed on the mode of carcinogenic action, metabolism and pharmacokinetic s (PK) of arsenic and its methylated species, and new epidemiology studies have b een conducted on inorganic arsenic. EPA considered this new information in its hazar d characterization for to lerance assessment of dimethylarsinic acid (DMA V ) and methylarsonic acid (MMA V )(USEPA OPP, 2005 and

13USEPA ORD, 2005). EPA also develope d a revised hazard and dose response assessment for inorganic Arsenic (USEPA OW, 2005) which relies on the two NRC reviews and provides an updated human health effects and dose-response assessment for inorganic arsenic. In its charge to th e SAB, EPA asked for advice on the soundness of the major science conclusions in these tw o documents. These documents focus on the assessment of DMA V and inorganic arsenic carci nogenicity (more specifically, metabolism, mode of action, dose-response, a nd approaches to low- dose extrapolation of cancer risk (see the specific Charge questi ons in subsections 2.2.1 through 2.2.4 and in Appendix A to the report).

2.2.1 Metabolism and Toxic Responses of Arsenic Species

Charge Question A1. Metabolism and pharmacokinetics : Please comment on how pharmacokinetic processes are best consid ered regarding the use of data derived from direct DMA V exposure versus direct iAs expos ure for cancer risk assessment.

Charge Question A2. Response to mixtures of metabolites : Given the toxicological response profiles observed following direct exposures to iAs versus MMA V and DMA V , and the differences in human and rodent toxicologic responses to arsenicals, please comment on the use of data derived from rodent exposures to the organic arsenicals versus use of data derived from direct iAs human exposure, in the DMA V

assessment.

2.2.2 Modes of Carcinogenic Action for DMA V and Inorganic Arsenic

Charge Question B1. Mode of action of DMA V : Please comment on the sufficiency of evidence to establish the animal mode of carci nogenic action for DMA V

. Are the scientific c onclusions sound and cons istent with the ava ilable evidence on DMA V and the current state of know ledge for chemical carcinogene sis. Please comment on whether the key events in DMA’s mode of action are supported by th e available data. Specifically comment on the role of: a) reactive oxygen species in producing chromosomal damage and the strength of th e evidence supporting ox idative damage as a causal key event in DMA V /DMA III‘ s mode of carcinogenic acti on versus an associative event or a secondary consequence of cytotoxi city; b) cell proliferation and cytotoxicity and the strength of the evidence as causal key events in DMA V /DMA III‘ s mode of carcinogenic action versus associative or sec ondary events, and c) other potential modes of action that have substantial scientific support that may be contributing to the carcinogenicity of DMA.

Charge Question B2. Human relevance of animal DMA V MOA: Please comment on the relevance of the postulated ke y events (see B1) to tumors in humans. Please comment on how, if at all, differen ces in the human population vs. experimental animals should be accounted for in the risk assessment for DMA V . Please comment on the Agency’s conclusion that the young are likely to respond like the adult to the formation of bladder tumors following exposure to DMA.

14Charge Question B3. Modes of carcinogeni c action from exposure to inorganic arsenic: Please comment on the conclusion that the available data support the hypothesis that multiple modes of action may be operational following exposure to inorganic arsenic.

2.2.3 Selection of Data for Dose-Response Assessment

Charge Question C1. Use of animal data for DMA V : Please comment on the use of the bladder tumor data from the DMA V rat bioassay as the most suitable dataset for quantifying potential human cancer risk to DMA V , including the weight of evidence to support this conclusion. Please comment on wh ether the iAs epidemiology data can be used to inform the DMA V dose-response assessment deri ved from rat data with DMA V

. If so, please discuss how such information might be used.

Charge Question C2. Use of human ep idemiological data from direct iAs
exposure: Does the SAB agree that the Taiwanese dataset remains the most appropriate choice for estimating cancer risk in humans ? Please discuss the rationale for your response. Do these data provide adequate characterization of the impact of childhood exposure to iAs? Please discuss the rationale for your response.

2.2.4 Approaches to Low-Dose Extrapolation for Inorganic Arsenic and DMA V

Charge Question D1. Mode of carcinogenic action understanding for DMA V/III and implications for dose response extrap olation to estimate human cancer risk : Please comment on the scientific evidence and bi ological rationale in support of nonlinear versus linear low dose extrapolation approaches , which approach is more consistent with the available data on DMA V and current concepts of ch emical carcinogenesis, and how scientific uncertainty should most appropriately be incorporated into low-dose extrapolation.

Charge Question D2. Implementation of the recommendations of the NRC (2001): Does the panel concur with the sele ction of a linear model following the recommendations of the NRC (2001) to estimate cancer risk at this ti me? Please discuss your response in light of the highly complex mode of action for iAs with its metabolites.

Charge Question D3. EPA re-implemented the model presented in the NRC (2001) in the language R as well as in an Excel spreadsheet format. In addition, extensive testing of the re sulting code was conducted: Please comment upon precision and accuracy of the re-implementation of the model.

Charge Question D4. Evaluation of Available literature describing drinking water consumption rates for the southw estern Taiwanese study population : What drinking water value does the panel recommend for use in deri ving the cancer slope factor for inorganic arsenic?

15Charge Question D5. Selection of an estim ate of dietary intake of arsenic from food: What background dietary intake (of arsenic) value does the panel recommend for both the control population and study populati on of Southwestern Taiwan used in deriving the cancer slope fact or for inorganic arsenic?

16

### 3. RESPONSE TO THE CHARGE

3.1 Overview

The SAB Arsenic Review Panel was as ked to comment on the i) toxicity/ metabolic profile/bioavailability for differe nt arsenic species, ii) the Agency’s understanding of the mode of action of arsenic carcinogenesis and implications of that on dose response extrapolation for DMA V and inorganic arsenic, and iii) the implications of newer epidemiology studies as well as the 2001 National Research Council recommendations on modeling of the human cancer slope factor for inorganic arsenic. The SAB Panel’s advice is contained in sections 3.2 through 3.5 that follow.

3.2. Metabolism and Toxic Responses of Arsenic Species

3.2.1 Metabolism and pharmacokinetics (Charge Question A1)

EPA’s charge states that, “ Evidence from in vivo and in vitro metabolism and pharmacokinetic studies with humans and laborat ory animals suggests that the efficiency of the methylation reaction(s) and cellular uptake varies based on which arsenical compound is administered exogenously. Most available studies suggest that the metabolic process in most mammals is primarily a one-way process and that following direct exposure to DMA V significant amounts of [arsenite] (iAs III ), [arsenate] (iAs V ), [methylarsonous acid] (MMA III ), or [methylarsonic acid] (MMA V ) at the target tissue are not expected” (USEPA, 2005a). Charge Question A1 asks the SAB to “...comment on how pharmacokinetic processes are best cons idered regarding the use of data derived from direct DMA V exposure versus direct iAs exposure for cancer risk assessment.”

Charge questions A1 and A2 address expos ure to and the metabolic fate of DMA V associated organoarsenic-c ontaining herbicides. DMA V from these herbicides can be degraded by microorganisms, both in the enviro nment and in the intestinal tract, to yield a variety of methylated and inorganic arsenic species, which have specific metabolic fates and toxicities. The Panel’s responses to questions A1 and A2 do not take into consideration potential byproducts of the microbial degradation of DMA V in the environment. This is because EPA repres entatives stated during the September, 2005 Arsenic Review Panel meeting that the environmental conversion of DMA V from organoarsenic pesticid es, and the risk associated with exposures to these conversion products, will be addressed later by EPA in a separate document.

The panel agrees with the Agency’s reasoning behind this question which is summarized at the beginning of this subs ection (3.2.1). In mammalian tissues/cells (including human), the metabolism of inorgani c arsenic (iAs) appears to be a one-way process in which iAs is converted to MMA, DMA and in some species to TMA metabolites containing arsenic in +3 or +5 oxi dation states (Vahter, 1999; Thomas, et al., 2001). There is no evidence for demethylation of methylated arsenic species in either animal or human tissues, though as noted in the preceding paragraph and in subparagraph

17“d” below microbial transforma tion is possible in the intestine. However, this issue needs further investigation. While the step-w ise addition of methyl groups is likely a one-way process, a cycling between +3 and +5 arsenic species may occur at each of the methylation steps due to a spontaneous oxi dation of +3 species (Gong, et al., 2001; Aposhian, et al., 2003) and non-enzymatic (D elnomdedieu, et al., 1994; Scott et al., 1993) or enzymatic (Zakharayn and Apos hian, 1999; Radabaugh and Aposhian, 2000; Waters et al., 2004) reduction of +5 species . Given the likely one-way character of arsenic methylation, significant amounts of MMAor iAs are not anticipated as products of DMA V metabolism in either rat or human tissues or urine on the basis of available data.

DMA In contrast, exposure to iAs may resu lt in the production, tis sue retention, and urinary excretion of all the above iAs and methylated arsenic species. Based on data from rodent studies, both th e uptake and reduction of DMA V to DMA III are apparently critical steps in the activation of exogenous DMA V . It is not clear, where and to what extent (if at all) these processe s occur in humans exposed to DMA V , although it appears that uptake may be rate limiting for further metabolism of DMA V . DMA III is a urinary metabolite in individuals chronically exposed to iAs (Le et al., 2000; Aposhian, et al., 2000; Mandal, Ogra and Suzuki, 2001; Del Razo et al., 2001; Valenzuela, et al., 2005), indicating that the capacity to reduce DMA V to DMA III exists in human tissues. The Panel pointed out that even the conversi on of a small amount/fraction of exogenous DMA V to DMA III is of toxicological significance due to the significant toxicity of
III . Thus, strictly from the point of view of the metabolic pattern, data derived from DMA V exposure (in the rat), not from iAs exposure in humans, is better suited for cancer risk assessment of DMA V . However, this approach is uncertain because of specific metabolic differences between rats and hu mans, and other factors, including:

a) The uptake pathway or pathways for DMA V in humans is/are unidentified. The expressi on or properties of DMA V transporters may differ in rats and humans, leading to differences in uptake of DMA V in tissues and organs. b) Results of laboratory and epidemio logical studies suggest that the pattern for DMA V metabolism in rats is different from that in humans (Figure 1). Rats metabolize DMA V to DMA III , trimethylarsine oxide (TMA V O) (Yoshida et al., 1997; Yosh ida et al., 1998; Cohen et al., 2002), and possibly, tr imethylarsine (TMA III ) (Waters et al., 2004). DMA V , DMA III , and TMA V O are urinary metabolites of DMA V in the rat. In addition, TMA V O was also detected in urine of rats chronically exposed to iAs (Yoshida et al., 1998 ). In contrast, little or no TMA V O was found in human urine after a single dose of DMA V (Marafante et al, 1987; Buchet et al., 1981) or after acute (Mahieu, et al., 1981; Apostoli et al., 1997; Benramdane et al., 1999) or chronic exposures to iAs (Vahter, 1999; Thomas et al., 2001). These data suggest that the capacity to produce TMA V O from iAs or DMA V or to excrete TMA V O in urine is lower in humans compared to rats. Thus, while it is possible that the urinary TMA V/III metabolites significantly affect the

18overall toxic or cancerous outcomes in the bladder of rats exposed to DMA V

, the relative lack of these meta bolites in human urine suggests that the outcome in humans would not be as severe as in rats. However, because the suggested toxi city differences above reflect a very limited human data set, more research is needed to characterize the role of TMA V/III metabolites in bladder carcinogenesis induced in rats by chronic exposures to DMA V . Research is also needed to determine whether the apparent absence of these metabolites in humans is associated with a decreased susceptibility to the carcinogenic effects of DMA V

. c) Accumulation of DMA III in rat erythrocytes, due to a high-affinity for binding to hemoglobin (Lu et al., 20 04) contributes to a specific kinetic pattern for DMA V in rats. It is not clear how and to what extent this factor affects the yi eld and concentration of the active arsenic species (e.g., DMA III , TMA V O, or TMAs III ) in urine or in target tissues of rats and how lower accumulation in human erythrocytes would alter th e kinetic pattern for DMA V and toxic/cancerous outcomes of DMA V exposure in humans. d) Microorganisms, including intestinal bacteria, have a capacity to either methylate or demethylate arsenicals (Hall et al., 1997; Cullen et al., 1984; Cullen et al, 1989; Lehr et al., 2003; Bently and Chasten, 2002; Tamaki and Frankenberger, 1992; Mukhopadhyay et al, 2002; Ridley et al., 1977; Qin, et al., 2006). A lthough the patterns and extent of DMA V metabolism by human intestinal microflora are not known, it is possible that oral exposure to DMA V results in the absorption of a wide spectrum of arsenic metabolit es produced by bacteria in the gastrointestinal tract of exposed i ndividuals. In contrast, bacterial metabolism would not affect the absorption of DMA V after inhalation or dermal exposures. Thus, arsenic species found in tissues may differ with different routes of exposur e. Interspecies differences in endogenous intestinal bacteria may further complicate extrapolation from rats to humans. e) Additional factors may affect th e metabolic profiles for DMA V in humans, including co-exposures to other environmental contaminants, deficiencies of specific nutrients (e.g., selenium ) or malnutrition. For example, poor nutrition has been shown to induce expression of aquaglyceroporin-9 (AQP9), an iAs III /MMA III transporter (Liu et al., 2002; Liu et al., 2004; Liu et al., 200 6), 20-fold (Carbrey et al., 2003).

All the above concerns should be consid ered in the risk assessment of DMA V

exposure.

EPA’s briefing documents presented information on a physiologically based pharmacokinetic (PBPK) model for arsenic disposition and metabolism that is under development. PBPK modeling might be a us eful approach for integrating tissue and excreta concentrations of arsenic metabolit es resulting from exposure to the various

19forms of arsenic, including DMA V , in laboratory animals and humans. For now, the modeling work described by EPA is in the de velopmental stage and is not considered sufficiently robust to conduct in terspecies extrapolations. However, the Panel strongly encourages the Agency to proceed with PBPK model development, including laboratory studies to obtain the kinetic constants needed to descri be rates of uptake, efflux, metabolism, and elimination of DMA V in both rats and humans. When sufficiently validated, this model could simulate concen trations of active (t oxic or carcinogenic) metabolites in urine and bladder tissue following exposure to DMA V . This approach could be used for dose response analysis in canc er risk assessment. Such models must be validated for predicting tissue concentrations of active species regardless of the source of arsenic exposure.

3.2.2 Response to mixtures of me tabolites (Charge Question A2)

EPA’s Charge stated that, “ Tumorigenic profiles vary based on which arsenical compound is administered exogenously. In vivo and in vitro studies indicate that each of the arsenical compounds exhibit similarities and differences in their profiles of biological activities. Direct exposure to iAs III or iAs V is expected to result in more of a mixture of toxic metabolites than for direct exposure to DMA V ; the mixture of metabolites is expected to vary based on which chemical is administered exoge nously. The potential mixture of metabolites followi ng direct exposure to DMA V appears less complex as compared to iAs” (USEPA, 2005a). Charge Question A2 asks, “Given the toxicological response profiles observed following direct exposures to iAs versus MMA V and DMA V

, and the differences in human and rodent toxicologic responses to arsenicals, please comment on the use of data derived from rodent exposures to the organic arsenicals versus use of data derived from direct iAs human exposure, in the DMA V assessment.”

The Panel believes that neither rodent la boratory data on organic arsenicals nor data from studies of the results of human exposures to inorganic arsenic provide an optimal basis for the assessment of DMA V exposures in humans. This is because of the differences between the metabolic profiles fo r inorganic arsenic a nd DMA, and because of interspecies differences in the metabolism of both arsenicals. The panel agrees that using the data from rodent exposures to DMA V may, at this time, be the most reasonable approach for the DMA V assessment.

The reasoning behind this response is linke d to the answer to charge question A1 above (see section 3.2.1). The metabolism of iAs yields a wide spectrum of metabolites (Figure 1) some of which (iAs III/V , MMA III/V ) are apparently not produced during the metabolism of exogenous DMA V . The production of iAs a nd MMA metabolites may be associated with specific toxic or canc erous endpoints that are absent in DMA V exposure in rats or humans except when there is a si gnificant co-exposure to iAs as is often found in U.S. drinking water supplies, or in food or the environment. The Panel notes that there are no published data on toxi cological responses to DMA V in humans. The toxic and carcinogenic effects of DMA V have been examined only in rodents, mainly in rats.

20AsVO43-+ 2e­→AsIIIO33-3+→CH3AsVO32-­→CH3AsO22-+ CH3+→(CH3)2AsVO2- ­→3)2AsIIIO­3+→(CH3)3AsV­→(CH3)3AsIIIV) (iAs)(V) ()V)(III)(VO) (III)itelldi iilacidRat+ CH+ 2eIII+ 2e(CH+ CHO + 2e(iAsIIIMMAMMAIII(DMADMATMATMAarsenate arsen methy arsonic methy arsonous methylars nic dimethylarsinous trmethy arsinetrimethylarsineacidacidacidoxideHumanFigure 1. Schema of Inorganic Arsenic Metabolism in the Rat and Human:The metabolic pathway for inorganic arsenic inthe rat and human involves a stepwise addition of methyl groups to yield methylarsenic (MMA), dimethylarsenic (DMA), andtrimethylarsenic (TMA) metabolites that contain trivalent arsenic (AsIII) or pentavalent arsenic (AsV). Results of epidemiological andlaboratory studies suggest thatwhile MMA and DMA are products of this metabolicpathway in both ratsand humans, only ratsexcrete significant amounts of TMAVO in urine when exposed to inorganicarsenic, MMA or DMA. In addition,in vitromethylationof inorganic arsenic by recombinant rat, but not human arsenic (+3 oxidationstate) methyltransferase produces TMAIII.Althoughalternative pathways have been suggestedfor inorganic arsenic metabolism and additional methylated metabolites were found in theurine of rats and humans exposed to arsenicals, more research is needed to determinethe significance of these pathways or metabolitesfor inorganic arsenic or DMA metabolism in both species.21However, a significant degree of uncertainty is associated with this approach due to the metabolic differences between rats and humans and due to othe r factors, including those listed in the response to charge question A1 above. The differences in the production and urinary excretion of TMA III/V species that could affect the toxic and cancer outcomes of DMA V exposure are of a particular concern to this panel. TMA V O is a hepatocarcinogen in rats (Shen et al., 2003). TMA III is apparently more potent than DMA III in damaging purified DNA in in vitro systems (Andrews, et al., 2003). On the other hand, both TMA V O and TMA III are less acutely toxic or cytotoxic than DMA III (Yamauchi et al., 1990; Cullen, 2005; Sakurai et al., 1998; Ochi et al., 1994). The contribution of these two metabolites to cy totoxicity and carcinogenesis in the urinary bladder of rats exposed to DMA V remains unclear. This uncertainty should be properly addressed in the risk assessment for DMA V exposure in humans

.

3.3 Modes of Carcinogenic Action for DMA V and Inorganic Arsenic

3.3.1. Mode of Action of DMA V (Charge Question B1)

EPA’s Charge stated that, “When relying on laboratory animal data, two critical assumptions are made: (i) data on animal tumo rs are predictive of hu man cancer, and (ii) animal tumor effects found at high experime ntal doses predict human risk at lower exposures. An understanding of a chemical mode of carcinogenic actio n can help inform the above assumptions. In the case of DMA V , mode of action (MOA) data are available and were evaluated using the framework described in EPA’s cancer guidelines” (USEPA, 2005a). Charge Question B1 asks the SAB to “... comment on the sufficiency of evidence to establish the animal mode of carcinogenic action for DMA V

. Are the scientific conclusions sound and consistent with the available evidence on DMA V and the current state of knowledge for chem ical carcinogenesis?” In addition, the Charge asks the SAB
to “...comment on whether the key events in DMA’s mode of action are supported by the available data. Specifically comment on the role of: a) reactive oxygen species in producing chromosomal damage and the strength of the evidence supporting oxidative damage as a causal key event in DMA V /DMA III‘ s mode of carcinogenic action versus an associative event or a secondary consequence of cytotoxicity; b) cell proliferation and cytotoxicity and the strength of the ev idence as causal key events in DMA V /DMA III‘ s mode of carcinogenic action versus associative or secondary events, and c) other potential modes of action that have substantial scien tific support that may be contributing to the carcinogenicity of DMA.

The Panel concluded that there are ade quate data to support a MOA for bladder carcinogenesis induced by high doses of DMA V in the rat that involves cytotoxicity to the bladder epithelium and increased, sustained rege nerative proliferation as key events. The urine of DMA V -treated rats contains DMA III at levels that cause necrotic cytotoxicity in vitro, so it is reasonable to postulate that DMA III might mediate the necrotic cytotoxicity in the rat bladder. However, the rat (unlik e the human) metabolizes a significant fraction of exogenous DMA V to TMA V O (Cohen et al., 2002; Yosh ida et al., 1997, 1998) and

22possibly to TMA III (Waters et al., 2004). Thus, th ese compounds cannot be excluded as additional mediators of the necrotic cytotoxic ity in the bladder of rats exposed to DMA V

. The Panel thought that there are not su fficient data to invoke reactive oxygen species (ROS)-induced DNA damage as a ke y event in the carcinogenic process associated with exposures to DMA V or DMA III for the reasons discussed in the following paragraphs.

Chemically, neither oxidation of As III to As V nor reduction of As V to As III can produce an oxygen radical in the absence of other reactants. Ar senic can only undergo two-electron reduction (no free electron or radical to donate to oxygen). Although there are indirect sources of ROS that can partic ipate in arsenite-stimu lated cell signaling (e.g. stimulation of NADPH oxidase ; Smith et al., 2001) or arsenic trioxide-mediated apoptosis via mitochondrial collapse (Jing et al., 1999), these have not been demonstrated for DMA. Arsenic compounds could also in crease ROS by promoting an inflammatory response in many tissues. This may contribute to carcinogenesis, but it is not likely to be the primary MOA for carcinogenesis.

Much of the argument suggesting th at the mode of action of DMA V -induced bladder cancer involves ROS-induced chro mosome damage derives from studies on DMA III -induced DNA damage. Very high cy totoxic concentr ations of DMA III have been shown to induce DNA damage in cell-free system s and in intact cells (Mass et al., 2001), possibly via an ROS-mediated mechanism or by dimethylarsine (Yamanaka et al., 2003; Kitchin and Ahmad, 2003; Nesnow et al, 2002; Andrews et al., 2003). However, cellular genetic toxicology assays of DMA do not support an ROS-dependent mechanism. Neither DMA V nor DMA III is significantly mutagenic at loci which are known to detect oxidant DNA damage. Neither compound was mutagenic in the Ames Salmonella strain TA104, a strain developed primarily to aid in the detection of oxidative mutagens, nor

were 8 prophage induced (Kligerman et al., 200 3). Prophage induction (which depends on the E. coli SOS system, a system responsive to DNA damage) was readily detectable after treatment with other agents acting by oxidant mechanisms, such as bleomycin, carbon tetrachloride (+S9), hydrogen peroxi de, and iron compounds (Rossman et al., 1991).

DMA V was either negative or very weakly positive at extremely high doses in a number of other test systems which can de tect oxidative mutagens (Moore et al., 1997, Kligerman et al., 2003; Oya-Ohta et al., 1996). Treatment of Muta TM mouse with DMA V (10.6 mg/kg per day) IP for 5 days caused only a (not significant) 1.3-fold increase in lacZ mutations in the lung, but not in the bladder or bone marrow (Noda et al., 2002). DMA V presumably could be converted to DMA III in mouse liver. (Arsenite also gave negative results in this assay.)

The single study of DMA III in the mouse lymphoma assay (Kligerman et al., 2003), which detects clastogens as well as poi nt mutagens at the TK locus, showed no significant effect on mutant fract ion at concentrations <1.5 μM (38% survival) in the agar assay. “Significant” was defined as at le ast a 2-fold increase over control, but no

23statistical analysis was done, only a singl e assay is shown, and there is only one “significant” response, at 1.51 μM. (This assumes a background mutant fraction of between 38 and 50 X 10 -6 ). A microwell assay using the same cells showed a “significant” effect at 2.56 μM (9% survival).

Kligerman et al. (2003) argued that DMA III (and MMA III ) are basically clastogens and not point mutagens. The clastogenesis st udies suffer from the same problems as the mutagenesis studies: no statisti cal analysis and single data points at some doses. “Significant” (defined as a 2-fold increase) e ffects are seen only at toxic doses (toxicity data are not given but can be inferred from other mammalian cell data). In addition, since cytogenetic assays do not requir e cell survival for scoring, when clastogenic effects are seen in a population of cells with low survival, it is not possible to determine whether those cells with chromosome aberrations woul d be among the survivors, and thus capable of resulting in a tumor. Examination of ch romosomes in tumor and pre-tumor tissues in the rat bladder model might establish whet her specific chromosome aberrations are associated with DMA V -induced bladder cancers.

Even model clastogens such as ionizi ng radiation can also be detected as mutagens at single gene loci such as HPRT, a useful locus for studying mutations in vitro as well as in vivo (unlike TK). Thus, if DMA III was really a clastogen acting by ROS, it should cause increased deletion mutagenesis at the HPRT locus. More research on the ability of arsenic metabolites to induce gene mutations in vivo should be carried out.

The fact that (some) antioxidants blocked DMA V -induced bladder cancer in the rat does not provide ev idence as to the origin of th e oxidants nor where they act. Activation of NADPH oxidase (Smith et al ., 2001) or inhibition of GSH reductase (Styblo et al., 1997) could increase oxidants . Tissues subjected to continuing cellular assault produce a number of cytokines whose signaling may be modulated by oxidants (even in the absence of frank inflammation) . Nuclear factor-kappaB (NFkB), which is activated by low-dose arsenite via oxidants (Barchowsky et al., 1996), is thought to provide a link between inflammatory signaling and carcinogenesis, as well as providing survival signaling to block apoptosis in damage d cells that might otherwise die. NFkB is a transcriptional regulator of genes incl uding cyclooxygenase-2 (COX-2), which is also induced by arsenite (Trouba and Germolec, 2004). Research is needed to determine whether bladder cells undergoing stress- related proliferation in the rat DMA V carcinogenesis model show effects on NFkB and other signaling pathways, similar to those seen with arsenite.

Given the preponderance of scientific evid ence to date (which is reviewed above in this section), the principal MOA for DMA V does not appear to be mediated via the ROS-induced DNA damage pathway. Rather , the MOA is likely to be sustained cytotoxicity followed by genomic instability as a result of stress-re lated proliferation. Permanent genetic change is necessary fo r carcinogenesis, and it is unlikely that increased proliferation alone in the absence of increased genomic instability will result in the multiple changes needed to transform a nor mal cell into a tumor cell. The mechanism of cell killing by DMA V or DMA III is not known. Regardless of how the cells die, there

24is substantial evidence supporti ng the hypothesis that continual proliferation of surviving cells under conditions of stress results in ge nomic instability (Karpinets and Foy, 2005). For example, in the case of arsenite this would involve such factors as:

a) Inducing intracellular pro liferative signals and over-riding cell cycle checkpoints (reviewed in Rossman, 2003), b) Blocking DNA repair (reviewed in Rossman, 2003), c) Inhibiting GSH reductase (S tyblo et al., 1997) and thio redoxin reductase (Lin et al., 2001), d) Inducing stress-related survival signals to block apoptosis (Pi et al., 2005; Wu et al. 2005), e) Effects on thiols in tubulin and cyto skeletal proteins, interfering with microfilament function and cytoskeletal changes (Li et al., 1992; Ling et al., 2002; Ochi et al., 1999), f) Affecting DNA methylation le vels, (Chen et al., 2004), g) Inducing oxidant signaling (B archowsky et al., 1999), and h) Effects on hormone functi on (Bodwell et al., 2004).

More research should be carried out on cel ls undergoing stress-re lated proliferation in the rat bladder model to determine whethe r these same programs have come into play for DMA V . Changes in DNA methylat ion patterns have just been demonstrated in arsenic-associated human bla dder cancers (Marsit et al. 2006) , making this a priority for study in the rat model.

Live cells exposed to the c ontents of necrotic cells ma y experience additional stress signals similar to that seen in the “bysta nder effect” after ionizi ng radiation (Iyer and Lehnert, 2000) or via cytokines from inflam matory cells. Although there is no direct evidence to support this mechanis m in the rat bladder cancer mode l, it is of interest that heat-killed E. coli instilled into the bladder was f ound to increase bladder carcinogenesis by N-methyl-N-Nitrosourea (Yamamoto et al., 1992), presumably by an inflammatory mechanism. Research on this topic should be carried out both in vivo and in vitro

. Further, generation of low levels of oxidants from enzymatic sources (Smith et al., 2001) or possibly by uncoupling of m itochondrial oxidations (if DMA V can act in a manner similar to arsenate) may contribute to e ffects on cell signaling and transcriptional activation, as well as in crease oxidant DNA damage.

In summary, the Panel postulates that the mode of action for DMA V involves the following key events:

a) Reductive metabolism of DMA V to DMA III

. b) High concentrations of DMA III in urine cause urothelial cytotoxicity. Some toxicity may also be caused by DMA V itself. c) Continuous exposure and persistent st ress associated regenerative cell proliferation leads to genomic instabi lity, acquisition of ge netic alterations, clonal expansion of altered cel ls and eventually tumors.

25This MOA, as well as the original MOA suggested by EPA, depends upon prolonged extensive cytotoxicity in the bla dder. Without the c ontinual cytotoxicity, sustained stress-associated prol iferation would not occur. The tumor response in the rat bladder system is non-linear, as is the key event (i.e. the necessity for necrotic cytotoxicity). Since the MOA involves cytotoxicity, doses below those causing cytotoxicity would not be expected to cause tumors. The other events mentioned above would not be sufficient to cause tumors in the absence of the cytotoxicity and the resulting prolifer ative response.

3.3.2 Human relevance of animal DMA V MOA (Charge Question B2)

EPA states that, “There are little or no scie ntific data to suggest that if sufficient DMA III were present, key precursor events and ultimately tumor formation would not occur in humans directly exposed to DMA V ” Charge Question B2 asks the SAB to “...comment on the relevance of the postula ted key events (see B1) to tumors in humans...”and to comment on how, if at all, differences in the human population vs. experimental animals should be accounted for in the risk assessment for DMA V

. If high enough (cytotoxic) concentrations of DMA V or DMA III were present in the human urine or bladder after exposure to DMA V , it is plausible that a similar response (necrosis followed by regenerative, stress-a ssociated proliferation) would take place. However, no data are available to support or reject this assumption. No studies have been carried out on DMA V -induced bladder cancer in humans, so it is not known at this time whether there have been any cases. Con centrations high enough to cause necrosis in the bladder might be achievable in an industria l accident or deliberate poisoning. It is not clear whether a repeated or chronic exposure to DMA V from the environment could produce cytotoxic concentrations of critical metabolites in hu man urine. Even in the case of high exposure, the exposures would proba bly have to be repeated often enough to produce persistent necrosis and regene ration in order to cause cancer.

As already mentioned in Charge Question A1 above, DMA V is converted to TMA V O, and possibly TMA III , more efficiently by rats than by humans. TMA V O is a hepatocarcinogen in rats (Shen et al., 2003). TMA III is more potent than DMA III in damaging DNA in in vitro systems (Andrews et al, 2003). Thus, although acute toxicities of TMA V O and TMA III are lower than that of DMA III (Ochi et al., 1994; Sakurai and Kaise, 1998; Yamauchi et al., 1990), these me tabolites could contri bute to the MOA for DMA V -induced bladder cancer in rats. The ex tent of this contribution is unknown. However, it is possible that the rat data ove restimates the human risk for bladder cancers from DMA V

.

No studies have been done in either an imal models or in human populations to determine whether the young are at greater or lesser risk wi th regard to DMA V -induced carcinogenesis, or whether there is greater or lesser risk during any other life stages.

263.3.3 Modes of carcinogenic action from exposures to inorganic arsenic (Charge Question B3)

EPA stated that, “Inorganic arsenic (i As) undergoes successive methylation steps in humans, resulting in the in termediate production of iAs III , MMA V , MMA III , DMA V

, and DMA III . Each arsenical metabolite exhibits its own toxicity.” Charge Question B3 asks the SAB to “...comment on the conclusion that the available data support the hypothesis that multiple modes of action may be operational following exposure to inorganic arsenic.”

The Panel agrees that multiple modes of action may operate in carcinogenesis induced by inorganic arsenic. This is becau se there is simultaneous exposure to multiple metabolic products as well as multiple target organs. There are differences in metabolic capability and probably transport into and out of different organs for different metabolic products, so that the composition of the metabolit es can differ in different organs as well. Each of the metabolites has its own cytotoxic and genotoxic capability. In general, the pentavalent compounds are less cytotoxic and genotoxic than are the trivalent compounds.

In the remainder of this section of th e report, the Panel di scusses studies of indirect genotoxic effects asso ciated with iAs and/or its me tabolites, as well as the notion that iAs might have some beneficial effect s at very low doses. Taken together, these studies suggest the possi bility of a threshold. However, the Panel does not identify what the threshold might be, nor does it describe the shape of the dose-response curve, rather it leaves that to be addressed by EPA in its final assessment based on the outcome of EPA’s evaluation of the relevant literature. The Pa nel identifies this as an extremely important area for research attention.

In the strictest (and original) defin ition, genotoxic carcinogens, i.e., direct carcinogens (or their metabolites) dama ge DNA by covalently binding to DNA or intercalating into the DNA-helix. Indirect ge notoxicity occurs thr ough interactions with non-DNA targets leading to genotox ic effects. Non-DNA targets, (e.g. proteins) exist in many copies per cell, thus a single event is un likely to have any signi ficant consequences. This suggests a threshold for effects associated with such events. The modes of action of “non-genotoxic” carcinogens are numerous, an d can include regenerative cell growth following cytotoxic effects, modulation of metabolizing enzymes, inhibition of DNA repair, induction of peroxisome proliferati on, stimulation of oxidative stress or other signaling resulting in suppression of apoptosis, lo ss of cell cycle cont rol, and stimulation of proliferation. A number of indirect genotoxic events are listed in Table 1.

The genetic toxicology of iAs III has been previously reviewed (Rossman, 1998, 2003; Basu et al., 2001). The interpretation of the genotoxicity of arsenic compounds is difficult because of the very high cytotoxic co ncentrations used in many studies and the lack of analysis for statistical significance in many studies. Another part of the problem

27Table 1. Some potential mechanisms for indirect genotoxicity of Inorganic Arsenic (iAs)

Potential Mechanisms

Interference with DNA repair Interference with cell cycle control proteins Interference with DNA replication Blocking apoptosis of cells with DNA damage Interaction with nuclear proteins such as topoisomerases or spindle proteins Nuclease and protease rel ease from lysosomes or dead cells Protein denaturation l eading to genomic instability Production of or change in re active oxygen species leading to altered signaling Other changes in gene expression (e.g., COX-2; Trouba and Germolec, 2004) Interference with oxidative phosphorylation Changes in ionic concentration, pH, or osmolarity Altered DNA methylation

stems from inappropriate (or absent) asse ssment of toxicity of arsenic compounds (Komissarova et al., 2005). Low c oncentrations (even 1 μM) of iAs III can cause apoptosis in human cells that can only be detected 48 hours (or more) after exposure and cytotoxicity assays (other than clonal su rvival) are usually performed too soon after exposure to enable identificati on of apoptotic cells after arsenite exposure. Thus, many “positive” genotoxicity results (especially in cytogenetic assays ) could have been conducted too soon and thus they could be repo rted for dead or dying cells. In such cases, they would only be useful in describing a MOA if the cel ls continued to live and if the genotoxic effect noticed was consistent w ith effects seen in tumorigenesis studies in animals and in human tumors. In the absence of such tumorigenesis data, cell transformation studies can yi eld some insight into MOA.

Arsenite, i.e., iAs III (and other arsenicals) does not exhibit direct DNA binding and the inability of iAs III to induce the SOS system in E. coli is consistent with its lack of reaction with DNA (Rossman et al., 1984) . In mammalian cells, highly toxic concentrations of both inorgani c and organic arsenic compounds in vitro cause chromosome breakage, which some have at tributed to ROS-induced DNA strand breaks but which might be caused directly by othe r free radical species (Yamanaka and Okada, 1994; Andrews, et al. 2003). Cellular DNA strand breakage and clastogenicity are limited almost exclusively to trivalent sp ecies. Unlike many other carcinogens, iAs III is

28an extremely weak (or insignificant) mutagen at single gene loci such as HPRT or TK in mammalian cells (Rossman, 1998, 2003). However, iAs III (but not MMA III ) at low (nontoxic) concentrations can induce delayed indirect mutagenesis at the HPRT locus after >15 generations as a secondary result of genomic instability (Mure et al., 2003).

The argument has been made that ar senite is a clastogen that causes predominantly multilocus deletions, and that su ch deletions near the HPRT locus (which is located on a single active X chromosome) ma y be lethal, accounting for the lack of (or extremely weak) mutagenesis by arsenicals at the HPRT locus (Hei et al., 1998). However, molecular analysis of mutations inthe HPRT gene shows that large deletions (up to ~3.5 Mb) can be tolerated in the HPRT region of the human X chromosome (Nelson et al., 1995; Lippert et al., 1995). Despite this, attempts have been made to find genetic markers more likely to de tect large deletions. Also, iAs III was an insignificant mutagen, and only at toxic doses, in transgenic Chinese hamster G12 cells (a derivative of V79 cells) (Li and Rossman, 1991). Thes e cells can detect clastogens causing deletions in the single copy of the E. coli gpt gene inserted into Chinese hamster chromosome 1. Similar results (extremely weak mutagenesis at toxic arsenite concentrations) are seen in mouse lymphoma ce lls, which can tolerate deletions at the TK locus due to its autosomal location (M oore et al., 1997), at the transgenic gpt locus of AS52 Chinese hamster ovary cells (Meng and Hsie, 1996) and in A L cells, which are CHO-K1 cells containing a single copy of human chromosome 11 (Hei et al., 1998).

In vivo , iAs III induced micronuclei (MN) in mouse peripheral blood lymphocytes and in mouse bone marrow (Tinwell et al., 1991 ; Noda et al., 2002). Humans exposed to iAs III show increased MN and sometimes ch romosome aberrations in lymphocytes, exfoliated bladder epithelial cells, and buccal epithelial cells (revie wed in Basu et al., 2001). In vivo studies on genotoxic activity of methyl ated arsenic species are limited to a small number of studies in rodents. IP injections of high doses of DMA V induced a slight but insignificant increase in mutagenesis in the Muta TM Mouse lung, but not in bladder or bone marrow. Also, iAs III was negative in this assa y (Noda et al., 2002). High concentrations of DMA V administered orally to mice caused oxidative damage and DNA strand breaks in the lung ( not a target organ for DMA V carcinogenesis). The strand breaks were attributed to dimethylarsine, via the dime thylarsenic peroxy radical (CH 3 ) 2 AsOO· (Yamanaka and S. Okada, 1994). DMA V also induced aneuploidy in mouse bone marrow cells (Kashiwada et al., 1998).

As noted in the previous paragr aph, micronuclei are induced by iAs III in vivo , and MN frequency is increased in humans exposed to iAs III in drinking water. Because of this, it is important to consider the signi ficance of MN for MOA. MN are defined as small, round, DNA-containing cytoplasmic bodies formed during cell division by loss of either acentric chromatin fragments or w hole chromosomes. The two basic phenomena leading to the formation of MN are ch romosome breakage (d ouble strand breaks associated with clastogenesis) and dysfunc tion of the mitotic apparatus leading to aneuploidy (change in chromosome number fr om the normal diploid or haploid number other than an exact multiple). MN as a result of clastogenesis contain acentric chromosome or chromatid fragments while MN associated with aneuploidy contain

29whole chromosomes. Currentl y, the most widespread and re liable assay to identify whole chromosomes in MN is by fluorescent label of their kinetochores (with antibodies) or their centromeres (with DNA probes). Howeve r, only a few laboratories routinely use these techniques because they are very cost ly. In most studies, there is not enough information to determine whether the MN result from: 1) toxicity, 2) clastogenicity, or 3) non-dysjunction (leading to aneuplody). Also, MN in cells trigger apoptosis, so many cells with MN will have no progeny.

In a study of 18 arsenic-exposed individua ls (average 1,312 μg arsenic/L drinking water) and 18 matched controls (16 μg/L), the exposed group had a 1.65-fold increase in MN with acentric fragments (p=0.07) and a 1.37-fold increase in MN with whole chromosomes (p=0.15) (Moore et al., 1996). The combined difference (1.8-fold) was significant. Thus, exposure to iAs III induces MN by multiple mechanisms. In normal human fibroblasts, low dose, long term exposure to iAs III is aneugenic, inducing MN with whole chromosomes, but high dose, short te rm exposure is clast ogenic, inducing MN with chromosome fragments (Yih and Lee, 1999). Evidence supports an aneugenic role for iAs III in many other cells at concentrations lower than those causing chromosome aberrations (Kochhar et al., 1996; Vega et al., 1995; Ramirez et al., 1997; Huang and Lee, 1998; Moore et al., 1997; Sciandrello et al., 200 2). Of importance is the association of aneuploidy with malignant transfor mation induced by arsenite and DMA III (Ochi et al., 2004; Chien et al., 2004). Aneuploidy is an event that has a threshol d (Kirsch-Volders et al., 2002), whereas many people assume that clas togenesis does not (at least for ionizing radiation) even though repair of radi ation-induced DNA damage exists. The development of aneuploidy is a marker of genomic instability and is typical of many tumors. IP injection of DMA V in mice induced aneupl oidy, but no chromosome aberrations, in bone marrow (Kashiwada et al., 1998). (DMA V would be converted to DMA III in the mouse, so the active agent may be DMA III ). Bladder tumors in patients with high iAs III exposure showed higher levels of aneuploidy compared with other bladder tumors (Moore et al., 2002).

Genomic instability can result from changes in DNA methylation in iAs III -treated cells. The first report of arsenite induci ng DNA methylation change s was the increased cytosine methylation in the p53 promoter in human adenocarcinoma A549 cells (Mass and Wang, 1997). Later it was found that th ere was both hypo- and hypermethylation (of different genes) in human ki dney UOK cells treated with iAs III (Zhong and Mass, 2001). When SHE cells or rat liver TRL 1215 cells were transformed by iAs III , specific oncogenes were more highly expressed due to hypomethylation (Zhao et al., 1997; Takahashi et al., 2002) and there was evidence of decreased DNA methyltransferase activity (Zhao et al., 1997). These findi ngs are consistent with the usual DNA methylation changes observed in cancer, in wh ich global methylation is reduced but some gene specific promoter methylation is incr eased (Baylin and Herman, 2000). Arsenite (iAs III )-induced DNA hypomethylation and al tered gene expression has been demonstrated in mouse liver (Chen et al., 2004), in hepatocellular carcinoma derived from transplacental iAs III exposure (Waalkes et al., 2003), and in prostate epithelium where the hypomethylation was shown to activat e K-ras (Benbrahim-Ta llaa, et al., 2005). In a study of iAs III exposed individuals in India, in creased levels of hypermethylated p16

30and p53 gene promoters were seen in blood DNA (Chanda et al., 2006) . Methylated CpG sites are mutational hotspots (e.g. by a second agent), methylation ch anges affect gene expression, and hypomethylation leads to genomic instability. Low concentrations of iAs III induce delayed mutagenesis and chromosome aberrations that might be mediated by hypomethylation (Mure et al. 2003; Sciandrello et al., 20 04). This is a mechanism that might also explain transplacental carcinogenesis.

There are a number of non-genotoxic act ions of arsenite (and perhaps MMA III and DMA III ) that can contribute to the carcinogenic pr ocess. The role of ROS in (low dose) arsenic carcinogenesis is proba bly via signaling changes rath er than as a genotoxicant (otherwise, one would expect more mutagenesi s). This may contribute to carcinogenesis, but it is not likely the prim ary MOA for arsenic carcinogen esis. Cell signaling can be affected by arsenite via low levels of oxi dants that do not cause DNA damage (reviewed in Simeonova and Luster, 2004). Low iAs III concentrations increased oxidant signaling and oxidant-dependent activati on of nuclear factor kappaB (NFkB) in the absence of DNA damage in human endothelial cells (B archowsky et al., 1999). The increased oxidants appear to result from activat ion of membrane-bound NAD(P)H oxidase. Arsenite (iAs III )-induced signaling results in expressi on of inflammatory cytokines such as IL-8 that can mediate atherogenesis (Simeonova and Luster, 2004). Arsenite (iAs III ) also increases ROS by promoting an inflamma tory response in many tissues. In addition, there is redox chemistry involved in iAs III -dependent signaling in that arsenite binds to protein thiols (particularly vicinal thiols) to stimulate signaling cascades or affect DNA repair. Arsenite (iAs III ) interaction with thiols is a redox reaction, but oxygen radicals are not involved.

Exposure to low, non-toxic doses of iAs III enhances positive growth signaling (reviewed in Rossman 2003), whic h can readily contribute to hyperplastic pre-cancerous skin growth. Arsenite (iAs III ) can disrupt glucocorticoid r eceptor (GR) and other steroid signaling at very low doses, and it has been s uggested that these eff ects on GR may affect carcinogenesis (Kaltreider et al ., 2001). This may also affect other disease processes, such as cardiovascular disease, diabetes and ot her diseases that have been associated with iAs exposure, since GR and other steroid recep tors have been shown to be important in these diseases as well.

Animal studies indicate that for some organs, transplacental carcinogenesis after maternal exposure to iAs III occurs. This includes the formation in C3H mice of tumors of the lung and liver, target sites of potential human relevance, after exposure to arsenic in

utero . In addition, in utero arsenic induces tumors of the ovary and adrenal, sites not observed in humans to date. The C3H mouse was selected in these studies because it is, in general, sensitive to chemical carcinogene sis, although this strain shows spontaneous tumor formation in several tissues. Recen t work has shown that with gestational exposure to CD1 mice, inorganic arsenic is a complete carcinogen in the female offspring (Waalkes, et. al ., 2006). The CD1 mouse strain is noteworthy as having a well defined, low rate of spontaneous tumors. EPA’s revi sed assessment document should take note of this important development. Other studi es indicate that in skin, neither iAs III nor iAs V is a complete carcinogen, but they act as enhancers (cocarcinogens, sometimes mistakenly

31called “promoters”) with ot her agents. Arsenite (iAs III ) acts as a cocarcinogen with solar UV light (Rossman et al. 2001; Burns et al., 2004) and arsenate is cocarcinogenic with 9,10 dimethyl1-2-benzanthracene (Motiwale et al., 2005). Th is leaves open the possibility that a cocarcinogenic MOA may also operate for other organs, but this remains to be investigated.

Arsenite (iAs III )-enhanced UV carcinogenesis could result from acquired resistance to UV-induced apoptosis. Such resistance was recently demonstrated in human keratinocytes (HaCaT) treated with 0.1 μ M arsenite for 28 week s (Pi et al., 2005). In mouse keratinocytes, the repair of UV-i nduced 6-4 photoproducts was slowed by acute 5.0 μ M (24 hr) arsenite exposure, which also inhibited the UV-induced apoptosis as indicated by TUNEL flow cytometry and by re duction of caspase 3/7 activities (Wu, et al., 2005). Arsenite (iAs III ) also blocked UVB-induced apop tosis in human keratinocytes (Chen et al., 2005). One mechanism by which iAs III may perturb apoptotic pathways is by PI3K-mediated phosphorylati on of PKB (Akt). When PI 3K activity was inhibited by Wortmannin or LY294002, arsenic-induced apopto tic resistance was also blocked (Pi et al., 2005).

Table 2 lists some activities of iAs III (or its metabolites) that might explain how iAs III can act as a transplacental carcinoge n and a cocarcinogen but not a complete carcinogen in neonatal and older animals.

In the future, it will be important to determine whether the trivalent arsenic metabolites can induce tumors in vivo or transform or mutate keratinocytes and other major target cells of arsenic at biologically relevant concentrati ons. The mechanism of iAs-induced carcinogenesis is likel y to be different in different tissues, with contributions from all the various arsenic species present in that tissue.

There have been a number of reports claimi ng an essential role for iAs in various animal species (chick, goat, hamster, pig, ra t), but many of these reports exist only in abstracts or in meeting reports (reviewed in Uthus, 1992; Nielson, 1996). Reports in the peer-reviewed literature follow. A study of arsenic-deprived goats found muscle atrophy, reduction in oxidative enzymes and abnorma l mitochondria in muscle, possibly via disturbance of calcium metabolism (Schmidt et al., 1984). Studies by Uthus (1990, 1992) suggest that iAs has a role in methionine/met hyl metabolism. Arsenic (iAs)-deprived rats had decreased plasma taurine, hepatic polyamines, and S-adenosylmethionine decarboxylase (needed for polyamine synthesis) . Arsenic (iAs)-deprivation as well asiAs excess caused DNA hypomethylation in rat liver. The same effect was seen in Caco2 cells. Arsenic (iAs) deprivation or excess also increased the formation of aberrant colon crypts in rats treated with dimethyl hydrazine (Uthus and Davis, 2005), suggesting a cocarcinogenic effect. So far, no exact biochemical mechanism linking any iAs species with methionine/methyl metabolism has been found, but the fact that many laboratories have reported effects of iAs III on DNA methylation makes this an important area of study.

32Table 2. Activities of iAS III that may contribute to its cocarcinogenic and/or transplacental carcinogenic action

Activities

DNA repair inhibition Increased oxida nts (signaling changes) Gene dosage effects (aneuploidy, amplification) Altered DNA methylation Proliferative response Increased angiogenesis Effects on immune system (not discussed; see Vega et al ., 2004) Inhibition of apoptosis Hormonal effects Delayed mutagenesi s (not enough generations in vivo but maybe enough if transplacental or added to a genotoxic agent? )

Hormetic effects of iAs (beneficial eff ects at very low doses) have also been suggested and need further investigation even if arsenic should not be essential. In cell culture, subtoxic con centrations of iAs III reduced the levels of ROS in keratinocytes and fibroblasts by upregulating thioredoxin and glutathione reductase (Snow et al., 2005). Some DNA repair proteins are also upregulated. The same paper also describes protection of mice from skin tumors indu ced by dimethylbenzanthracine + phorbol 12­ tetradecanoate 13-acetate if the mice were given drinking water containing 0.2-2 ppb arsenate. Inorganic arsenic (iAs) has both positive and negative effects on the growth and function of blood vessels (Soucy et al., 2003, 2005; Kamat et al., 2005). Low concentrations fuel angiogenesis, while higher concentrations injure endothelial cells and promote the vessel dysfunctions seen in is chemic diseases and peripheral vascular diseases. Thus, iAs may provide improved va scularization and growth of normal tissues, which could reduce cardiovascular risks. However, this proc ess could also pose risks for iAs increasing the vascularization and growth of both atherosclerotic lesions (Simeonova and Luster, 2004) and tumors from a sec ondary source (Kamat et al., 2005). Mice drinking 10-250 ppb iAs III had increased metastases from transplacental tumors (Kamat et al., 2005). However, iAs at high doses has been used to destroy the tumor vasculature (Griffin et al., 2003).

While mechanistic studies sugge st that there should be a threshold for iAs bladder cancer, available data, and data from epidemio logical studies, are lacking or problematic with regard to low-dose effects. This critic ally important issue s hould be the subject of additional mechanistic and epidemiologic rese arch. This research should attempt to determine whether the suggested hormetic or be neficial effects of arsenic, for several endpoints at very low doses, ex ist more broadly and whether th ey apply to all life stages. If iAs is shown to be essential, i.e., nece ssary for certain life-sustaining functions or processes, then a threshold would not be at zero. If iAs would be shown to have hormetic effects, then the issue of a threshold would be less clear and while a threshold might be

33possible for one health endpoint, it might not exist for another iAs associated health endpoint. Research should also further illumi nate the shape of the dose response curve at low doses for the biological effects of arsenic.

3.4 Selection of Data for Dose-Response Assessment

3.4.1 Use of animal data for DMA V (Charge Question C1)

EPA’s Charge stated that, “A number of different rodent bioassays (standard bioassay, transgenic animals, susceptible rodent strains, initiation and promotion studies) are available on DMA V .” Charge Question C1 asks the SAB to “... comment on the use of the bladder tumor data from the DMA V rat bioassay as the most suitable dataset for quantifying potential human cancer risk to DMA V

, including the weight of evidence to support this conclusion.

The consensus of the panel is that, give n the lack of huma n data, the bladder tumor data from the DMA V rat bioassay is the most su itable data set for quantifying potential human cancer risk to DMA V . Given the differences in metabolic fates of DMA V and iAs, the use of human data from iAs exposure to predict risk from DMA V is not recommended. In this case, reliance on interspecies extrapol ation using the rat bioassay data is the best alternative.

This question indirectly raises the issue as to the largest source of uncertainty for DMA V risk assessment—conventional interspecies extrapolation or extrapolation across various forms of arsenic. The available material suggests that extrapolation across various forms of arsenic would lead to th e greatest degree of uncertainty in a risk assessment. Although the panel ag reed that use of the rat DMA V bioassay data is the preferred method, the panel also felt strongly that a discussion of the key uncertainties with using data from testing in rats to c onduct human risk assessment should be included in EPA’s Office of Pesticide Programs report “ Science Issue Paper: Model of Carcinogenic Action for Cacodylic Acid (Dimethylarsinic Acid, DMA V ) and Recommendations for Dose Response Extrapolation .” Issues that panel members consider important to discuss in EPA’s Scien ce Issue Paper are discussed in more detail below and in Section 3.2.1. These i ssues include the pharmacokinetic and pharmacodynamic similarities and differences between rats and humans in response to arsenic exposure (e .g., the role of TMA III/V species); the use of rodent bladder tumor models in general; shared MOAs w ith iAs due to the possibility of DMA V demethylation by intestinal bacteria (see sect ion 3.2.1); and issues related to in the use of rodent data for human risk assessment. The panel also r ecommends that the EPA consider applying the Human Relevance Framework (HRF) proposed by the International Life Sciences Institute-Risk Science Institute (ILSI-RSI) (Seed, et al, 2005) to the mode of action. Application of the framework or its elemen ts may assist EPA in evaluating the human relevance of the DMA V rat data. This framework has beenused to assess the relevance of rodent liver tumors to human cancer risk (Holsapple et al, 2006).

Several pharmacokinetic differences betw een rats and humans have been reported after arsenic exposure. For example, arsenic methylation in rat hepatocytes proceeds at a

34faster rate than in human hepatocytes (St yblo, et al., 1999). Additionally, rats have a considerably slower whole body clearance of DMA V compared with humans. This slower whole body clearance in rats result s from a significant portion of DMA being retained in the erythrocytes of rats (Vahter, et al., 1984). The affinity of rat hemoglobin to bind DMA III is 15 to 20 fold higher than that of human hemoglobin (Lu, et al, 2004). These differences in metabolism and pharmacokinetics may be consistent with a greater sensitivity of the rat to induc tion of bladder tumors by DMA V . However, without a more complete data set demonstrating that exposur e of the bladder epithe lium (urothelial cells) to DMA V metabolites (particularly DMA III ) is greater in rats than in humans for a given dose, the data are not sufficien t to support reducti on of interspecies uncertainty factors based on differences in pharmacokinetics. Clear ly, this is a high priority area of research with the potential to reduce uncertainty in the risk assessment of DMA V

. In the EPA Science Issue Paper cons ideration should be given to the pharmacodynamic similarities and differen ces between rats and humans and the relevance of the rat response to human risk assessment. Although data illustrating the mode of action for DMA V as a bladder carcinogen in rats seem quite convincing, it should be noted that rats are more sensitive to DMA V in carcinogenicity testing than are mice (Rossman, 2003; Arnold, et al., 2003). While the relative in vivo sensitivities of rats and humans to DMA III are unknown, it has been shown that in vitro rat and human urothelial cell lines are equally sensitive in terms of acute toxicity to DMA III in the micromolar range (Cohen et al., 2002). For arsenite, however, the rat MYP3 urothelial cell line showed toxicity at about one tenth (LC 50 of 0.4 μM) the concentration as did the human 1T1 urothelial cell line (LC 50 of 4.8 μM). As a result of the Panel’s analysis of the information on this key pharmacodynamic re sponse, urothelial ce ll cytotoxicity, the consensus was the EPA could explore a case for pharmacodynamic equivalency between the test species, rats, and humans from ex isting experimental data. Pharmacodynamic equivalency could be incorporated into the assessment as a reduction of the pharmacodynamic component of the interspecies uncertainty factor, which is 3, to a value of one. However, as discussed in the respons e to question D1, there remains considerable uncertainty due to limited comparative in vi vo data across species. The final EPA risk assessment should fully discuss the interspeci es similarities and differences and the implications for risk assessment as well as explore opportunities to reduce uncertainty factors.

EPA’s Science Issue Paper should discu ss similarities and differences between rats and humans in the development of bla dder tumors and how these differences impact interspecies extrapolation. St udies suggest that in rats it takes two or more years of continuous high dose exposure to DMA V to induce these tumors. Human bladder tumors are also late occurring. The Science I ssue Paper should specif ically discuss the similarities and differences in the time for induction of DMA V related tumors in rats with the pattern observed with humans and ar senic associated urinary bladder cancer.

EPA’s Science Issue Paper should also disc uss general issues associated with rat urinary bladder cancer. One such issue is the relationship between the induction of tumors and high concentrations of arsenic in the urine. Also , there is a need to address

35evidence that simple enhancement of prolifera tion is not associated with carcinogenesis in many tissues. Studies by Gur et al. (cited on page 97 of the DMA MOA Science Issue Paper, US EPA OPP, 2005) on the carcinogenicity of DMA V were never published and thus cannot be critical ly evaluated by the Panel. The Science Issue Paper notes that the Gur studies in rats and mice are key bioassay st udies. Reliance on these studies would be stronger if the studies had th e benefit of peer review.

The EPA’s Science Issue Paper expresses concern with the mouse transplacental model for inorganic arsenic becau se the strain of mice used (namely C3H) in the original two studies had a significant ra te of spontaneous tumors in several tissues that are also targets of arsenic. Recent follow-up work has shown that gestational exposure to inorganic arsenic in CD1 mice is a comple te carcinogen in the female offspring (Waalkes, et al ., 2006). The CD1 mouse strain is noteworthy as having a well defined, low rate of spontaneous tumors. The Scien ce Issue Paper should take note of this important development.

Charge Question C1.B also asks the SAB to “... comment on whether the iAs epidemiology data can be used to inform the DMA V dose-response assessment derived from rat data with DMA V . If so, please discuss how such information might be used. ” (See Appendix A).

The panel consensus was that without mo re detailed information on target tissue dosimetry of arsenic species th e iAs epidemiology data would be of limited use to inform the DMA V dose-response assessment derived from rat data with DMA V . Direct exposure to iAs elicits a different cascade of metabol ite concentrations with related differential kinetics compared to direct exposure to DMA V , therefore the iAs epidemiology data cannot reasonably be used to inform the DMA V dose-response assessment derived from rat data with DMA V . In the absence of specific information on target tissue levels, assumptions would have to be made regard ing the proportion of the iAs for human and DMA V for rodents that reaches the bla dder tissue as the toxic DMA species.

In principle, epidemiology data from iAs exposed humans could be used to inform the DMA assessment to the extent that the data might be able to address the appropriateness of interspecies extrapolation, specifically the relativ e sensitivities of rat and human to bladder cancer following arseni c exposure. However, as noted above, in order to be useful some information on target tissue dose of DMA following human exposure to iAs and rodent exposure to DMA V would be necessary. With both in vivo tumor indices (human and rodent) expressed in terms of the same tissue dose of relevant metabolites, rather than iAs or DMA V exposure levels, the relative sensitivities of the human and rodent c ould be assessed.

3.4.2. Use of human epidemiological data from direct iAs exposure (Charge Question C2)

EPA’s Charge states that, “ Since the NRC (2001) report on iAs, an additional body of literature has developed describing ep idemiology data from populations in the

36U.S. exposed to iAs in drinking water” (USEPA, 2005a). Charge Question 2 asks, “Does the SAB agree that the Taiwanese dataset remains the most appropriate choice for estimating cancer risk in humans? Please discuss the rationale for your response.”

For reasons noted below in this section, it is the Panel’s view that, at this time, the Taiwanese database remains the most appropr iate choice for EPA’s use in deriving the cancer unit risk for iAs. However, the Panel suggests that EPA also conduct adjunct analyses to test the robustness of results against their assumptions, determine the impact of variability in some parameters, compare the results against tho se from other data sets, and provide a transparent assessment of the available epidemiological data using a consistent set of criteria.

The Taiwanese dataset consists of populat ion and mortality data from 42 villages in southwest Taiwan for the years 1973-1986. Ar senic levels in wells from these villages were measured in 1964-1966. The database is one of the largest that has been evaluated for cancer risk relative to ar senic exposures. A total of almost 900,000 person years of follow-up were included, with 1,152 cancer deaths (637 males, 515 females). Among the cancer deaths were 181 due to bladder cance r (85 males, 96 females), 268 lung cancer (147 males, 121 females), and several hundred du e to other types of cancer. These data have been subject to several ecologic analys es, starting with the or iginal publications by Chen et al. (1988) and Wu et al. (1989), followed by furthe r analyses by Morales et al. (2000) and by the National Research Council (1999 and 2001).

Among the 42 villages, the arsenic c oncentration ranged from 10 to 934 ppb ( μ g/L). Twenty of these 42 villages used a single well. Among many of the 22 villages with multiple wells, many had wide variability in the measured arsenic level in their wells. Analyses using the full dataset give re sults comparable to results from a reduced dataset including only the villages with singl e wells, providing some confidence in the stability of the overall results (National Research Council, 1999). The Panel recognizes the limitations of the southwest Taiwan database, including its ecologic character, lack of smoking information, limited precision of exposure estimates, especially among villages with multiple wells, and the possible issue of compromised nutrition among segments of the exposed population. However, in view of the size and statistical stability of the database relative to other studies, the reliab ility of the population and mortality counts, the stability of reside ntial patterns, and the inclusion of long-term exposures, it is the Panel’s view that this database remains, at this time, the most appropriate choice for estimating cancer risk among humans. Supporting this view is the fact that the datasets from Taiwan have been subjected to many y ears of peer review as part of published studies.

Given the concerns regarding the use of the median well water concentrations in some of the 42 villages in southwest Taiwan that have more than a single measurement, the Panel recommends that EPA conduct a sensit ivity analysis. This should include the range of exposures in said villages to provide a range of risk estim ates. One alternative (suggested in response to D-3) is a full Mont e Carlo analysis in wh ich the individual well concentrations for 22 villages with multiple wells are taken into account. The Panel recognizes the difficulties with this approach including the issue of how to allocate cases to wells within villages. A simpler, but us eful first approach w ould be to test the

37sensitivity of the model fitting when arsenic concentrations for multiple-well villages are set to: 1) a low level concentration from the range for the village (10 th percentile, 20 th percentile); 2) the median (current procedure) ; and 3) a high level concentration from the village range (90 th percentile, 80 th percentile).

New studies have been published sin ce the NRC report in 2001 and the Panel considered some of these (and additional in formation provided by the public during this review process) in its evalua tion regarding the Taiwanese data sets that are the focus of this charge question. To be clear, the pa nel did not do an exha ustive review of all possible toxicologic and epidemiologic litera ture during its review . That was beyond the scope of the Panel’s charge. The Panel recognizes that this must be done in EPA’s final assessment and calls on the Agency to do so.

In view of the limitations of this databa se, the Panel recommends that the other relevant epidemiologic databases from studies of arsenic-exposed populations be used to compare the unit risks at high exposure levels th at emerge from the Taiwan data. Several of these studies had the adva ntage of data with excellent exposure assessment. In addition, some populations likely differed from the Taiwanese population with regard to their nutritional status. The accuracy and precision of exposure assessment is a major issue in all environmental epidemiologic studies, and in particular, in studies of arsenic in drinking water. Misclassification of exposure in such studies (when non-differential) can have a profound effect in atte nuating the magnitude of the observed risk. The excellence of exposure assessment is an especially str ong aspect of several studies from northern Chile, and the Panel recommends that the find ings of Smith et al. (1998) and of Ferreccio et al. (2000) be included by EP A in evaluation of other datasets as described below. In addition, arsenic exposures appear to be well characterized in cohort studies of Chiou et al.(2001) of transitional cell carci noma (mostly bladder cancers) and Chen et al. (2004) of lung cancer, from arsenic-expos ed cohorts in southwest and northeast Taiwan. The latter study also provides data on th e joint effects of arsenic and cigarette smoking in the Taiwanese population.

The accuracy of estimated long-term exposur es to arsenic is of concern for recent studies with water concentrations belo w 100 ppb. Misclassification of exposure may compromise their overall util ity in assessing concordance w ith risk estimates obtained from the Taiwan study. The Panel suggests that results on bladder cancer risk from published epidemiology studies of U.S. and other populations chronically exposed to arsenic levels ranging from 0.5 to 160 μg/L inorganic arsenic in drinking water, be critically evaluated. A sensitivity analysis to evaluate the potential impact of sources of bias in the low level case control and cohor t studies could be informative. Several arsenic epidemiology studies have the advant age of data with drinking water arsenic exposure levels ostensibly most relevant to the U.S. population [Bates, et al., 1995; Karagas et al., 2004; Lewis et al., 1999; Ku rttio et al., 1999; Stei nmaus, et al., 2003; Bates et al., 2004; Michaud et al., 2004; Chiou et al, 2001; Ferreccio et al., 2000]. Most of these populations have a nutritional and gene tic background similar to that of the U.S. or the studies were conducted in a U.S. popul ation. EPA should determine the potential utility of these studies in exploring overal l concordance of the cancer risk estimates

38derived from their data with risk estimate s obtained from extrapolation of the Taiwan data. The Panel suggests that if findings from a critical review of “low-level” studies indicate that some or all studi es are potentially of value in further analyses, that results from these studies should be explored in secondary analyses, particularly on bladder cancer risk, and compared with the main analys is for concordance. Analyses integrating health outcome information from a numbe r of epidemiology studies can result in improved statistical power and precision of the estimates; these factors represent an additional advantage of utiliz ing a larger dataset.

When reviewing these “low-level” studies (and the “high level” studies as well), EPA should consider at least the following i ssues: estimates of the level of exposure misclassification; temporal variability in a ssigning past arsenic levels from recent measurements; the extent of reliance on imputed exposure le vels; the number of persons exposed at various estimated levels of wate rborne arsenic; study response/participation rates; estimates of exposure variability; cont rol selection methods in case-control studies; and the resulting influence of these factors on the magnitude and stat istical stability of risk estimates. Most populations in the U. S. and many other countries differ from the Taiwanese population of inte rest in genetic background (e.g., genetic polymorphisms), dietary intake, and background exposure concentr ations to inorganic arsenic, and if one or more of these studies are s hown to be of potential utilit y, comparative analyses of the U.S. and Taiwan data may lead to further in sights into the possibl e influence of these differences on population responses to arsenic in drinking wate r. For compounds such as arsenic for which there are human data beyond the Taiwanese study on which human cancer risk has been based, data from the ot her investigations at high exposure levels (>150 μg/L) can be used to gauge the Taiwanese findings [Smith et al.(1998), Ferreccio et al.(2000), Chen et al.(2004) , Chiou et al.(2001)].

All of these studies, including those from Taiwan, Chile, Argentina and the U.S. as described above, should be judged by the sa me set of criteria, with the comparative assessment of those criteria across studies clearl y laid out in a tabular format. Some of the criteria have been listed in the prev ious paragraph. The relative strengths and weaknesses of each study need to be describe d in relation to each criterion. The caveats and assumptions used should be presented so that they are apparent to anyone who uses these data. Included in the risk assessme nt background document should be a complete and transparent treatment of variability within and among studies and how it affects risk estimates. The present lack of transparency in the application of the criteria in the process of study selection was pointed out by several panel members.

Charge Question C2 also asks, “Do these data provide adequate characterization of the impact of childhood expos ure to iAs? Please disc uss the rationale for your response.”

The Taiwanese data are inadequate to characterize the im pact of childhood exposure to inorganic arsenic with respect to carcinogenesis. That is, it is not clear whether children differ from adults with rega rd to their sensitivity to the carcinogenic effects of arsenic in drinking water. More data are needed to fully characterize the

39impact of transplacental exposures. However, data from the studies in southwestern Taiwan which include childhood exposures in th e calculation of life time dose show that in the population under 30 years of age ther e were no bladder can cer cases, and only 5 lung cancer cases but few cas es are actually expected in that age group. Childhood exposures are included in the lifetime dose estimates. Smith etal (1998) report the highest excessive risk for male lung cancer in the 30-39 year old age group, suggesting the importance of childhood exposure and ri sk and perhaps smoking behavior as young adults. For 533 women exposed to arsenic in drinking water from t ube wells at greater than 50 μg/L compared with those exposed at 50μg/L or less, findings suggest that there are significantly increa sed odds ratios for spontaneous a bortion, stillbirth and neonatal death (Milton et al., 2005). Another reproductive study in Chile, which followed over 800 pregnancies, found that pregnant women drinking water containing 40 μg/L gave birth to infants of lower birt h weight than a comparable gr oup drinking water containing very low arsenic concentrations (<1 μg /L) (Hopenhayn et al, 2003). Thus maternal exposure at moderately high levels may have toxic eff ects; the issue of childhood carcinogenic susceptibility has had only limited study.

3.5 Approaches to Low-Dose Extrapol ation for Inorganic Arsenic and DMA V

3.5.1 Mode of carcinogenic action understanding for DMA V/III and implications for dose-response extrapolation to estimate human cancer risk (Charge Question D1)

EPA’s Charge stated that, “The use of m ode of action data in the assessment of potential carcinogens is a ma in focus of EPA’s 2005 cancer guidelines. As stated in th[o]se guidelines ‘The approach to dose-res ponse assessment for a particular agent is based on the conclusion reached as to its potential mode(s) of action.’ Although a biologically-based model is the preferred a pproach to estimating cancer risk, there are insufficient data on DMA V to support development of such a model.” Charge Question D1 asks the SAB to “...comment on the scientific evi dence and biologi cal rationale in support of nonlinear versus lin ear low dose extrapolation approaches, which approach is more consistent with the available data on DMA V and current concepts of chemical carcinogenesis, and how scientific uncertainty should most appropriately be incorporated into low-dose extrapolation.”

3.5.1.1 Please comment on the scientif ic evidence and biological rationale in support of the no nlinear versus linear low dose extrapolation approaches

The Panel believes, based on the review of EPA’s analyses, that there are adequate data to support much of the EP A-postulated MOA for bladder carcinogenesis induced by high doses of DMA V in the rat. The MOA involves cytotoxicity of the bladder epithelium and increased, sustained regenerativ e proliferation, as key events. However, the Panel concluded that ther e are insufficient data to invoke ROS-induced DNA damage as a key event in the carcinogenic proce ss, associated with exposures to DMA V or DMA III (see Charge Question B1).

40The postulated MOA for DMA V is:

a) Reductive metabolism of DMA V to DMA III

. b) High concentrations of DMA III in urine cause urothelial cytotoxicity. Some toxicity may also be caused by DMA V itself. c) Continuous exposure and persistent, st ress associated, regenerative cell proliferation leads to genomic instabi lity, acquisition of ge netic alterations, clonal expansion of altered cel ls and eventually tumors.

Neither the MOA postulated here, nor those postulated by ORD or OPP (USEPA OPP, 2005; USEPA ORD, 2005), contain key events expected to be a linear function of dose. Reductive metabolism of DMA V is likely to be saturabl e and therefore non-linear but this does not necessarily im ply a threshold-based response. In vitro cytotoxicity of uroepithelial cells, using rat (MYP3) a nd human (1T1) bladder cell lines, occurs only at concentrations greater than ~0.35 μ M (rat, 0.38 μ M DMA III ; human 1T1 0.35 μ M DMA III ) (unpublished data 1 ). In vivo , cytotoxicity of the uroe pithelium occurred at the lowest tested dietary DMA V concentration (2 ppm), but the incidence and severity increased, and the latency decr eased significantly as a function of dose. Statistically significant increases in regenerative cell pr oliferation only occu r in rats at DMA V dietary concentrations greater than 40 ppm, again, a nonlinear or ap parent threshold response.

Even ROS production, and its interaction with DNA, would be expected to be linear at some low dose

, but nonlinear across the larger dose range. Formation of heritable alterations in DNA by ROS is believed to be a nonl inear or curvilinear effect (USEPA ORD, 2005) best represented by a qua dratic function with a low-dose linear component (USEPA OPP, 2005). The formation ra te of heritable alterations is a function of the rate of DNA damage and the rates th e various DNA repair processes and finally the rate of DNA misreplication (USEPA OPP, 2005). The latter being a function of cytotoxicity and regenerative cell pro liferation which in the case of DMA V , are also highly nonlinear functions of dose (USEPA OR D, 2005). With respect to repair of postulated ROS induced DNA damage, highly spec ific enzymatic systems that exist for

1 Personal communication from L. Arnold of Dr. Cohen’s Lab with the EPA SAB Designated Federal Officer; 4.4.2006. Samuel M. Cohen, M.D., Ph.D. Professor and Chair, Pathology and Microbiology Havlik-Wall Professor of Oncology University of Nebraska Medical Center, Text of the email is provided below: “During the process of determining the LC 50 for the various arsenicals we did develop some data concerning the no effect level especially in the MYP3 rat bladder cell line. We do not have as much data for the 1T1 human bladder cell line since we used con centrations that we had already determined caused cytotoxicity in the MYP3 cell line. We do have detailed data for DMA III for both cell lines since there was a very sharp drop between concentrations which had no effect on the cell viability and concentrations which were cytotoxic. In the MYP3 rat bladder cell line, DMA III concentrations of 0.38 μ M and below had no effect on the viability of MYP3 cells but at a concentration of 0.39 μ M DMA III the cell viability dropped to 69%. In the 1T1 human bladder cell line, DMA III concentrations of 0.35 μ M and below had no effect on viability but at 0.40 μ M the viability dropped to 76%. The following data show doses for other arsenicals at which there was no effect on cell viability however, the no effect dose may be somewhat higher but we do not have enough data points to determine an exact concentration. MYP3 rat bladder cell line:

Arsenite-0.05 μ M; Arsenate-1 μ M; MMA III -0.5 μ M; MMA V -1mM; DMA V -0.05mM; TMAO-0.1mM

41their repair (Slupphaug et al., 2003) prot ect the genome, whether from exogenous chemicals or the high levels of endogenous ROS induced DNA damage. These enzymatic repair processes are expected to be nonlinear processes. EPA’s position on a linear oxidative stress MOA induced by DMA V is likely not defensible and should not be used. The state of the science is overwhelmi ngly in favor of a nonlinear approach for the risk assessment of DMA V . In summary, the Panel’s opinion is that the available data support the nonlinear approach for th e low dose extrapolation of DMA V

. The linear approach would be consistent with evidence for direct genotoxicity of DMA III/V . There are no compelling da ta demonstrating that DMA III/V are directly genotoxic. It is generally accepted that DMA V is not directly genotoxic (not DNA reactive). This conclusion is well su pported by the data presented in the Science Issue Paper: Model of Carcinogenic Action for Ca codylic Acid (Dimethylarsinic Acid, DMA V ) and Recommendations for Dose Response Extrapolation , and in section 3.3.1 of this report.

3.5.1.2 Charge Question D1 further asks the SAB, “ Which approach is more consistent with the available data on DMA V and current concepts of chemical carcinogenesis,”

The non-linear approach is more consistent with the available data and current concepts of chemical carcinogenesis (see section 3.5.1.1, above).

3.5.1.3 Charge Question D1 asks the SAB, “ How [should] scientific uncertainty most appropriately be incorporated into low-dose extrapolation?”

After some discussion, the Panel viewed th is question from the perspective of the EPA’s RfC guidelines (USEPA, 19 94). Similar guidelines for the derivation of chemical specific uncertainty factors have been de veloped by the Intern ational Program for Chemical Safety (IPCS 2001). These guideline s provide an approach for incorporating uncertainty into risk assessments in the form of uncertainty factors. Uncertainties in the interspecies extrapolation of the rat dose-response data can be broadly grouped into a) those related to interspecies differences in pharmacokinetics, b) those related to interspecies differences in pharmacodynamics, and c) those associated with sensitive populations such as children and the elderl y. The default value for interspecies differences in pharmacokinetics is 3, the default for interspecies differences in pharmacodynamics is 3, and the default for se nsitive populations is 10, made up of two factors of 3 each, one for pharmacokineti c differences and one for pharmacodynamic differences.

While it was the opinion of the Panel that rats might deliver a higher dose of the proximate toxicant, DMA III to the bladder for a given dose of DMA V than humans, the Panel recognized that there was insufficient data on the comparative dosimetry in rats and humans to make any conclusive stat ements about species differences in pharmacokinetics. The possible role of microbial demethyl ation in humans is another

42potential issue for consideration. Therefore, the uncertainty factor for interspecies differences in pharmacokinetics should be 3, th e default value. However, there appears to be emerging data on DMA V kinetics which might be broughtto bear on the question and the agency is encouraged to consider th ese data with respect to pharmacokinetic differences between the species and the charac terization of this component of uncertainty in the dose response assessment.

As a result of the Panel’s analysis of the data for the key pharmacodynamic response, uroepithelial cell cy totoxicity, the consensus wa s the EPA could assemble a case for pharmacodynamic equivalency between the test species, rats, and humans from existing experimental data. Cohen et al. showed LC 50 s of 0.5 and 0.8 μ M DMA III for rat and human bladder epithelial cells lines (Cohen, 2002). In the context of EPA and International Program on Chemical Safety (IPCS) guidelines, th is finding could be incorporated in the assessment as a reduc tion of the pharmacodynamic component of the interspecies uncertainty factor (typically a default value of 3), to a value of one. However, this suggestion is based upon limited comparative in vitro data. The application of uncertainty f actors has also been addresse d in the Panel’s response to question C1. There is presently no arsenic-specific information that can inform the choice of uncertainty factors for sensitive human populations. For now, the choice of these factors must be based on more general c onsiderations, including EPA’s science policy judgment of the degree of precau tion that it deems appropriate.

3.5.2 Implementation of the recomm endations of the NRC (2001)(Charge Question D2)

EPA believes that the most prudent a pproach for modeling cancer risk from exposure to iAs is to use a linear model because there are significant remaining uncertainties regarding which of the meta bolite(s) may be the ultimate carcinogenic moiety and whether or not mixtur es of toxic metabolites intera ct at the site(s) of action” (USEPA, 2005A). EPA asked if the SAB concurs, for now , with the selection of a linear model to estimate cancer ri sk for inorganic arsenic [i.e., following the recommendations of the NRC (2001)]. EPA also asked that th e SAB discuss its response in light of the highly complex mode of action for iAs.

The Panel recognizes the potential for a highly complex mode of action of iAs and its metabolites, and until more is learned about the complex PK and PD properties of iAs and its metabolites there is not a sufficien t justification for the choice of a specific nonlinear form of the dose-respons e relationship. Therefore, based on information in this section and on the Panel’s understanding of the EPA’s 2005 Guidelines for Cancer Risk Assessment, the final recommendation of NRC (2001) to base current risk assessments on a linear dose response model that include s the SW Taiwan population as a comparison group, seems to be the most appropriate appr oach. Below, the Panel suggests that EPA conduct sensitivity analyses to addre ss uncertainties in this issue.

Existing epidemiologic studies have been mentioned in the response to charge question C2. These studies of different popula tions across different countries seem to

43support a possible linear dose -response between exposure from drinking water and internal cancer risks (particularly in Taiwa n, Chile and Argentina). These dose-response relationships are observed at higher exposure levels (>100 ppb). The Panel believes that because of limitations in the epidemiologi c studies conducted to date, that adequate human data at the lower range of iAs exposure is lacking.

Some recent studies have included populat ions with exposures in the lower range (<100 ppb), but they tend to be problematic for use in dose-response analysis for lower exposure levels. In particular, when studi es are based almost exclusively on low dose exposure populations (Lamm et al, 2004; Bate s et al, 2003; Steinmaus et al, 2003), they lack statistical power and the estimations of lo w dose risk tend to be unstable and to have a high degree of uncertainty. Some of these studies also have probl ems related to study design. For example, in the Lamm et al. (2004) ecological study, exposure assessment is highly problematic given that a single mediancounty-level exposure value is assigned to all the person-years contributed by each county in the analysis, even though it is not clear that these are the arsenic exposure values for a large number of residents within each county. A recent follow-up of the Taiwanese cohort reports a monotonic trend in lung cancer risk for exposure to arseni c levels ranging from <10 to 700 μ g/L, however this study also has limited power to examine the form of the dose-response relationship within the 10-100 μ g/L range (Chen et al 2004). There are no human data available that is adequate to characterize the shape of th e dose response curve below a given point of departure.

At present the experimental evidence on mode of action of inorganic arsenic supports a possible nonlinear dose-response at low exposure levels yet there is no clear indication of what shape a nonlinear dose-res ponse would take for application to human cancer risks at low exposures (<50 or < 100 ppb). In examining the dose-response relationships of arsenicals ininducing direct or indirect mutagenic responses (including effects thought to be clastogenic in nature), it is clear that effects are only seen at doses that induce cytotoxicity. This implies a th reshold (Rossman, T.G. 2003). Until more is learned about the complex properties and MOAs of iAs and its metabolites there is insufficient justification for the choice of a specific nonlinear form of the dose-response relationship. Under these circumstances, the EPA’s 2005 Guidelines for Cancer Risk Assessment are clear that linear extrapolation below the point of departure is the method to be used.

Although the EPA has chosen a linear m odel for the arsenic dose component of the hazard model for lung and bladder cancer, the Panel encourages the Agency to test the sensitivity of the assump tion of linearity by comparing its corresponding estimate of excess life risk to an alternative hazard model that has a dose contribution that is multiplicative and nonlinear in form (see question D3 for additional information).

In summary, the Panel recognizes the pot ential for a highly complex mode of action of iAs and its metabolites, but until more is learned about the complex PK and PD properties of iAs and its metabolites there is not sufficient justification for the choice of a specific nonlinear form of the dose-response relationship. Based on this and the EPA’s

442005 Guidelines for Cancer Risk Assessment, the final recommendation of NRC (2001) to base current risk assessments on a lin ear dose response model that includes the southwest Taiwan population as a compar ison group seems the most appropriate approach. However, the Panel also recommends performing a sensitivity analysis with different exposure metrics with the subgroup of villages with more than one well measurement (as discussed in responses to charge questions C2 and D3) and using a multiplicative model that includes a quadratic term for dose, as performed by NRC (2001) and as discussed in charge question D3.

3.5.3 EPA Model Re-Implementation (Charge Question D3)

The Charge states that, “EPA re-imple mented the model presented in the NRC (2001) in the language R as well as in an Excel spreadsheet format. In addition, extensive testing of the resulting code was conducted” (USEPA, 2005a). Charge Question D3 asks the SAB to “... comment upon precision and accuracy of the reimplementation of the model.”

Question D3 suggests that the estimati on of the dose-response model and the hazard assessment were originally programmed in the R language. Page 63 of the issue paper indicates that the Poisson hazard model was originally estimated in the R language (optim routine) but neither the main text of the paper nor its appendices provided any additional information. A cl arifying question from the panel through the Designated Federal Officer provided clarifyi ng information, stating that:

“The reference to the impl ementation in R in question D.3 is outdated, and should have been removed. This was an oversight on EPA's part. The model implementation in Excel is our implementa tion of record, and was used to prepare the results in the draft toxicological revi ew. We would ask the Panel to please review and comment only on the implem entation in Excel. (Background: EPA did originally implement its model in R. However we found that version to be not very transparent, and hard to debug. We then re-implemented the model in Excel, found and corrected some errors, and used that corrected vers ion to prepare the tox review. While Excel may not be th e best choice from the standpoint of numerical accuracy, it is greatly supe rior in the transparency of the implementation, and is powerful enough to perform the entire model calculation from start to finish, even including the nonlinear optimization. Once the Panel is satisfied that the implementation in Excel is correct and appropriate, then the model can be re-implemented in R or some other numerically superior language.)”

The Agency staff is to be commended for d eciding to test its original R-language version of the model program through a separa te implementation in Excel. The Excel version serves as a check of programming pe rformed in alternative systems (e.g. R, S) and provides transparency for review by non-specialists. For the calc ulations required in this model of hazard and excess risk, the Ex cel computations should provide sufficient numerical accuracy. If the EPA returns to another model program, it should begin with

45the original model formulas and not simply transcribe the programming from the existing Excel version of the model. As a debuggi ng and error-checking tool, comparisons of intermediate results from the two model impl ementations should be performed to verify the equivalence of the two model systems.

Overview of the EXCEL spreadsh eet implementation of the model : The Excel model implementation is described in Appendi x B (pages 105-106) of the Issue Paper. The Issue Paper (page 65 ) referenced a URL, www .epa.gov/waterscience.sab ; however this proved to be not available. EPA sta ff notified the panel of the correct address, http://epa.gov/waterscience/sab/

. The Issue Paper suggests that a listing of the variable and parameter input field is provided in Table B-3 but the current draf t of the Issue Paper did not include this table. (The fields in the spreadsheetmodel were interpreted by the Panel based on the description provided in the text of the Issue Paper and general understanding of the model f itting procedure employed.)

The spreadsheet model requires two Excel files and associated macros. The first of these is MCCancerfit.XLS. This workbook component of the model consists of eight worksheets in four pairs (e.g. fblad and MC fblad for female bladder cancer) that cover the two cancers of interest (lung and bladder) and gender (m ale, female). The initial worksheet (e.g. fblad) in each of the four can cer/gender pairs contains the input data for fitting the hazard model. The first step in the model fitting algorithm is to employ the Excel Solver to find initial values of a1, a2, a3 and β (Cells G2:G5) that maximize the Poisson likelihood under the following model:

2 λ

, = exp( a + a 2 ⋅ age + a 3 ⋅ age )

⋅ (1 + β

⋅ dose ) i dose 1 i i where :

age = the midpoint of a five-year age range, e.g. 22.5 for 20-24; i dose = the arsenic dose in ppb.

This is the model described by the EPA in the Issue Paper and is one of two models that appeared to provide best fit to the data based on the Akaike Information Criterion (NRC, 2001).

The second worksheet in each the four disease/gender pairs (e.g. MC fblad) is used in conjunction with the in itial starting values, generated by Solver and stored in Cell N2, to simulate the empirical Bayes posterior distribution of the model parameters based on a set of 1000 random perturbations of the coefficient vector (a1,a2,a3, β ) about the maximum likelihood estimates produced by Solver. The perturbation involves independent, random (uniform) dispersion of th e coefficient estimates in a relative range of +/- 10% about the point estimates generate d by Solver. Parameter draws outside this range are not performed since the posterior likelihood takes on a near zero value outside the boundaries +/- 10% of MLE. The corre sponding macro (e.g. mcfblad) is then invoked to apply the observed data and these pe rturbed coefficient values to establish the value of the posterior log-likelihood for eachof the 1000 draws. The empirical Bayes

46estimate of the slope parameter and its lower confidence limit are then estimated based on the mean and standard deviation of the simulated posterior distribution:

1000 

## ∑ b j ⋅ L j

b = j = 1 L

max 1000 

## ∑ L j

j = 1 L

max
⎧ 1000 ⎡ L j 2 ⎪ 1000 j = 1 ⎣ L

max ⋅ ( b j − b ) ⎥ ⎪ ⎪ 

## ∑ ⎢ ⎤ ⎫

sd b ⎬ () = sqrt ⎨

⋅ ⎦ ⎪

1000 ⎪ 999 L j ⎪ 

## ∑ ⎪ j = 1 L

max ⎪ ⎩ ⎭

and
, () = + 2

⋅ sd b ) UCL b b (

The estimated UCL(b) is then carried fo rward to the BEIR.IV computation of the excess lifetime risk in the BEIR.xls spreadsheet.

Based on its review, the Pane l noted that for the given data inputs, the empirical Bayes estimation algorithm programmed in th e MCCancerFit.xls spreadsheet does match the form of the model and the general description of the parameter fitting algorithm outlined in the Issue Paper.

As described in the Issue Paper, the EPA data inputs for at risk populations and cancer deaths agree with Morale s, et al. (2000). In genera l, the panel recommends that all tables of inputs for these models be publis hed in appendices to the Issue Paper or final risk assessment so that reviewers can indepe ndently reference and verify the critical inputs to the hazard and excess risk analysis.

The MCCancerft.xls spreadsheet in cludes an adjustment of 50 μ g/day of arsenic from food intake. Based on the formula prov ided on page 103 of the Issue Paper, the current model assumes a combined daily intake of 2 liters/day of cooking and drinking water. The Issue Paper suggests th at the current analysis uses 30 μ g/day. Although the Issue Paper notes the NRC (2001) finding that dietary intake had no significant effect on the estimated cancer slope factor, the apparent discrepancy between the value of 30
μ g/day cited in the Issue Paper and the 50 μ g/day value used in the spreadsheet model should be resolved. The model does not allo cate a food input of ar senic to the control population. This is a decision that presumes f ood-based intake of ar senic originates from cooking water only.

The second Excel workbook in the risk assessment model employs estimates of the dose response model parameter, β , and its upper bound to ev aluate excess lifetime risk under the BEIR-IV formula. The BEIR .xls workbook includes four worksheets, one

47for each cancer type by gender combination ( flung, mlung, fblad, mblad). The estimates of the linear dose response parameter a nd its estimated 95% UCL (see above) are manually pasted from the corresponding worksh eet in MCCancerFit.xls. The excess risk is computed in cell T15. Solver can be applied to the dose value in Cell T11 (not U10 as indicated on Page 105 in the Issue Paper) to esta blish the dose level required to produce a user-specified values of excess risk (i.e., ED 01 ).

The columns of each worksheet in the BEIR.xls spreadsheet incorporate data for a specific age range of the U.S. population. These columns are not labeled with the corresponding age range. Identifying labels should be applied to all rows and columns in these worksheets. By deduction, column 3 a pplies to individuals age 20-24, column 4 to age 25-29, etc. If this is co rrect, the Panel recommends that the entry in cell B3 of each of the four BEIR.xls spreadsheets be verifie d. It appears that th is mortality figure may apply to more than just the 20-24 year old population represented in Column 3. Referring to the data inputs for 20-24 year olds in th e flung spreadsheet in BEIR.xls, the population value is 9,423,000, all deaths are 18,121 and the baseline hazard is .00192. Moving over one column to the 25-29 y ear olds, the population is n early the same at 9,491,000, all deaths are 1580 and the baseline hazard is .00017—less than 1/10 th that for the previous five year age group.

The BEIR.xls spreadsheet implementation of the BEIR-IV excess risk calculation includes a 3-fold divisor to transform the risk to a U.S. population base (assuming exposure per kg is 3-fold higher in the SW Ta iwanese population). This scaling occurs in the calculation of the age-spec ific cancer hazard (Row 11). It should be documented and also should be a target for future sensitivity studies. Since this is a model parameter it should identified as a distinct input on the spreadsheet instead of simply embedded in the calculations.

The notation for the BEIR-IV formula on Page 102 in the Issue Paper does not distinguish between total survivorship (S i ) and survivorship adjusted for the added risk of cancer. However, the spreadsheet implementation of the model decomposes survival into the product of baseline survival and a survival factor that reflects excess cancer deaths due to the prior age group’s exposure to arse nic. Based on a versi on of the spreadsheet downloaded from the Office of Water website , calculation of cancer -specific survival (Row 13) appears to incorporate mortality thr ough age interval I, no t interval I-1 as it should. This should be checked. The calcula tion of baseline survival appears to be correct – the survival parameter at age inte rval I includes only mo rtality through the end of time period I-1. With this exception, calcu lation of Excess Risk follows the BEIR IV formula.

Following the series of checks and correc tions to the model listed above, the Panel encourages the Agency to extend its test ing of the model sensitivity to alternative models forms and model assumptions. Speci fic areas where the Panel felt additional sensitivity testing is warranted include:

48a) A Monte Carlo analysis in which the i ndividual well concentrations for 22 villages with multiple wells are taken in to account. The Panel recognizes the difficulties with this approach including the issue of how to allocate cases to wells within villages.

b) MCCancerFit.xls:

a. A test of the sensitivity of the m odel to the choice of the reference population (SW Taiwan). b. A test of the sensitivity of model results to the assumption that the reference population has 0 in take of arsenic via food.

c. A contrast of results for the linear dose model employed in this program to alternative hazard models that are multiplicative and nonlinear in form. For example, the following multiplicative, quadratic model is one of several that NRC(2001) found to have best fit to the data based on the Akaike Information Criterion (AIC):

2 2 λ

, = exp( a + a 2 ⋅ age + a 3 ⋅ age )

⋅ exp( β + β

⋅ dose + β

⋅ dose ) iC 1 i i 0 1 2

c) BEIR.xls

a. The Panel recommends a sensitivity analysis be conducted to investigate the effect of the age groupings used to estimate the baseline hazard and excess lifetime risk. In ad dition to the current practice of using 5-year intervals (e.g. 20-24, 25- 29, etc.), a logical choice is to test the sensitivity of the model re sults to using 10-year groupings (e.g. 20-29, 30-39...). b. The exposure/kg parameter used to transfer the dose/response model from the original SW Taiwanese population to a U.S. general population is a major driver in the co mputation of excess lifetime risk. In preparing its final risk assessment, the EPA should conduct a sensitivity analysis to determine precisely how much the choice of a factor of 3 impacts the final estimates of excess lifetime risk.

3.5.4 Available literature describing drinking water consumption rates for the Southwestern Taiwanese study population (Charge Question D4)

EPA, as well as the NRC (2001) state thatthe drinking water consumption rate, as well as variability of that rate in both U.S. and Taiwanese populations, are important factors to consider. EPA notes that in cal culating risk estimates for U.S. populations exposed to arsenic through drinking water, NRC used a drinking water consumption rate of 1 L/day for the U.S. population and two po ssible consumption rates for the Taiwanese population: 1 L/day (identical to the U.S. population) and 2.2 L/day with little or no supporting rationale. Since publication of NRC 2001, a number of new studies have become available and are summarized in the Cancer Slope Factor Workgroup Issue Paper. Agency reviews of the relevant lite rature suggest that th e mean drinking water

49(for the Taiwanese study population) consumpti on rate is between 1 to 4.6 L/day. EPA’s current cancer modeling includes water intake adjustments for 2.0 and 3.5 L/day” (USEPA, 2005a). Charge Question D4 asks what drinking water value the panel recommended for use in deriving the can cer slope factor for inorganic arsenic?

The Panel agrees that assumptions about water consumption levels in the U.S. and in Taiwan have a substantial impact on the risk assessment. Relative to U.S. consumption, overestimating water consumpti on in Taiwan decrease s potency estimates and underestimating consumption increases pot ency estimates. Evidence for gender differences in consumption is limited, but c onsiderable within-popul ation variability in consumption occurs (NRC, 2001). EPA should evaluate the impact of drinking water consumption rates associated with more highly exposed population groups with potentially different exposures and susceptibilities (e.g., children, pregnant women) in its arsenic exposure estimates as the Agency de termines the overall aff ects of drinking water consumption rates on arsenic risk.

U.S. water consumption data are obtained from comprehensive U.S. surveys including surveys by the U.S. Department of Agriculture (USDA) and as part of the National Health and Nutrition Examinati on Survey (NHANES) (as cited in USEPA, 2005), among others. These studies provide information on tap water consumption as well as water consumption attributable to other beverage consumption and consumption of food prepared with water containing arseni c. Estimates of mean daily drinking water consumption and total water consumption (i ncluding water used in food preparation) range from 1.0 to 2.8 L/day and fr om 1.2 to 3.2 L/day respectively.

In comparison, information on water-consumption in Taiwan derives from a small study by Yang and Blackwell and an EPA info rmal, anecdotal assessment (as cited in USEPA, 2005) that include only inform ation on drinking water consumption. Information on water consumption in South As ia, another world region with high arsenic levels in the water supply, is available fr om a large population based survey in India (Chowdhury et al., 2001 cited in EPA 2005) and a small study from Bangladesh (Watanabe et al., 2004). The South Asia n studies include information on water consumption associated with food prepar ation. Although similar in socioeconomic characteristics, the diet and climate differ in Taiwan and South Asia, with temperatures higher in South Asia. These studies report mean daily drinking water intake of 1 to 3.5 L, with an additional 1 L associated with food preparation.

The Panel recommends that:

a) EPA incorporate variability parameters for individual water consumption into their analysis for the Taiwanese populat ion as they have done for the U.S. population estimates as NRC recommended; b) Because assumptions about water consumption are an important source of variability in the risk estimates, EPA should conduct sensitivity analyses of the impact of using a range of c onsumption values for the Taiwanese population.

50c) Because data on gender differences in consumption in Taiwan are limited, a better justification for assuming differ ent consumption levels by gender is needed, particularly given the lack of sex difference in consumption in U.S. and observed in studies from other count ries (Watanabe et al., 2004). In the absence of such a justification, the pa nel recommends an additional sensitivity analysis to examine the impact of e qualizing the gender-specific consumption level. d) The source of data for intake from other beverages and cooking water needs to be more fully discussed and documented. Specifically, the document should more clearly articulate how different s ources of water intake are incorporated into the risk model including beverages other than water (e.g. green tea) and water used in food preparation. Cl arification of both the assumed consumption level and how water consum ption and consumption variability is introduced within the model is needed.

3.5.5 Selection of an estimate of di etary intake of arsenic from food (Charge Question D5)

EPA stated that, “The issue of intake of arsenic from food (e .g., dry rice, sweet potatoes) has been distinguished from the issue of intake of arsenic from drinking water. The NRC addressed the issue of arsenic infood by determining how sensitive the calculation of ED 01 was to the consumption rate. NRC found that changing the consumption rate from 50 μg/day to 30 μg/day did not change the calculated ED 01 significantly (about 1% differe nce). Since the publica tion of NRC 2001, a number of new studies have become available, summari zed in the Cancer Slope Factor Workgroup Issue Paper. EPA’s current cancer modeling includes dietary intake adjustments for 0, 10, 30, and 50 μg/day” (USEPA, 2005a).” Charge Question D5 asks the SAB ”... what background dietary arsenic inta ke value it recommends for both the control population and study population of Southwestern Taiwan (which is used in deriving the cancer slope factor for inorganic arsenic?)”

The Panel did not recommend a specific value for EPA to use; however, it did recommend a sensitivity analysis to assess the impact of a range of dietary intakes on risk from lung and bladder cancer ri sk associated with arsenic indrinking water used by this population (e.g. 50 to as high as 200 μg/day). Th e Panel stated that an intake of zero arsenic from food should not be assumed.

Three studies that summarize daily arseni c consumption as derived from food in areas of high arsenic intake are listed in Table 4 (USE PA OPP, 2005). Based on the NRC’s recommendations, EPA used a range of 30 -50 μg per day total arsenic intake from dry rice (uncooked) and dried yams in the di et of southeastern Taiwan that also was based on the work of Schoof et al. (1998) as list ed in this table. In materials presented and submitted to the Panel (Schoof, 2005), Dr. Sc hoof, stated her belief that the field had not been recently treated given the levels she found (7 to 8 ppm) but that seasonality and recent application of arsenicals should influe nce the levels of arsenic found in the field

51and in plants grown on those fields. Thus th e Schoof et al. (1998) da ta cited in Table 4 may underestimate the dietary arsenic intake from food in this population.

In the following paragraphs, the term total arsenic indicates the sum of all inorganic and organic arsenic species. The te rm inorganic arsenic as stated in published literature on analysis of arsenic in food generally refers to the sum of the inorganic arsenic species (iAs III and iAs V ). Unless specifically stated otherwise, the term organic arsenic indicates total organic arsenic co mpounds in food. In reference to seafood, arsenobetaine is generally the major organi c arsenic compound present when organic arsenic compounds are specifically identifie d in the analysis; other minor organic arsenicals may also be present. Th e methylated arsenic metabolites (MMA III MMA V

, DMA III , DMA V ) are organic arsenic compounds, how ever, they are not generally determined in food.

Daily intake of arsenic from food obs erved by Chowdhury et al. (2001) and Watanabe et al., (2004) sugge st total arsenic intakes ra nging from a mean of 120 to 285 μg/day from food in Bangladeshi and Indi an populations exposed to high levels of naturally occurring arsenic. Mean total arse nic intakes for males were shown to be 214 μg /day and for females 120 μg /day (Watan abe et al., 2004). In studies conducted in West Bengal in which both chemical analys is of food items and interviews for food intake were conducted to assess exposure , Roychowdhury et al., (2002) show daily dietary intakes from food for adults (based on 34 families in 5 villages) ranging from 171-189 μg/day and for children of about 10 years ranging from 91-101 μg/day. These figures are ranges of means for two different geographic areas – standa rd deviations were not published. Although these data are not deri ved specifically from the area of Taiwan studied, they indicate along w ith ancillary information presented here and elsewhere that dietary exposure from food in this geographic area may be higher than previously thought. Raw rice, a staple of the area, has been shown in other st udies to contain among the highest iAs values in food (Schoof, et al ., 1999) while for vegetables approximately 95% of total arsenic is organic arsenic (C howdhury et al., 2001). Variation in arsenic concentration and speciation occurs relative to rice cultivar (Williams et al., 2005). Duxbury et al. (2003) estimates that 30-85% of arsenic in ri ce is inorganic arsenic.

Diet is the largest source of total arsenic exposure in the U.S. relative to water and air exposures. Average intake is about 40 μg/day total arsenic (ATSDR, 2006) compared with the approximately five-fold higher tota l dietary arsenic intake observed in Asian studies cited in the foregoing paragraph. Th e estimated range of daily intake of total arsenic from food in the U.S. is reported at 2-92 μg/day (Tao and Bolger, 1999) while U.S. daily total intake of iAs at the 10th and 90th percentiles is estimated to be 1.8 to 11.4 μg/day for males and 1.3 to 9.4 μg/day for females (Meacher, et al, 2002). The U.S. dietary intake of inorganic ar senic is estimated to range from 1 to 20 μg/day (ATSDR, 2006). U.S. shellfish and other marine foods contain the hi ghest total arsenic concentrations and are the largest dietary sour ce (76% - 96%) of arsenic, however, most of the arsenic in seafood is present as the organic arseni c compound arsenobetaine, which is excreted rapidly and unchanged and doe s not appear to be harmful to humans (ATSDR, 2006). It is known, however, that fish may contai n some portion of iAs further

52pointing to the need for the sensitivity anal ysis described below. Certain seafood may also contain DMA that may al so contribute to background expo sure from food relative to water sources (Huang, et al., 2003).

It is clear that the adjustment for ba ckground iAs intake from food is extremely important given that the total exposure dose from all sources does likely matter in terms of toxicity and cancer inducti on and that the U.S. population likely has a considerably lower total arsenic intake from f ood than do populations in Asia.

The Panel recommends that a range of valu es from at least 50 to 100 μg/day and up to perhaps as high as 200 μg/day be run in a sensitivity analysis to assess the impact of this range of dietary intakes on risk of lung and bladder cancer from exposure via drinking water in this populat ion. The cancer risk model ne eds to be evaluated using a wider range of iAs food values above 50 μg/day to determine if there is a change in the arsenic cancer exposure-response slope as a resu lt. It also cannot be assumed that the control population has an intake of zero arsenic from food.

Such a sensitivity analysis of the impact of dietary arsenic upt ake using a range of data from high arsenic-exposed populations is unlikely to introduce larger uncertainty than the myriad dietary differences – protein deficiency, Se, Zn, folate deficiency etc. – between this Taiwanese populat ion and the U.S. population

Much greater rigor needs to be applie d in discussing and presenting documented data sources and making clear the basis on which assumptions are being made and the relative strength of those assumptions. Compar isons of the impact of differing levels of iAs intake from food between the exposed a nd reference population need to be made on the basis of comparative relative risk. Cl earer statements are needed on the data limitations of past daily dietar y arsenic intake for the Blac kfoot endemic area of Taiwan and for the reference population(s).

EPA needs to be aware of and includ e a discussion of methodological and analytical issues related to reported arsenic concentrations in food, because these values are dependent upon differential extraction proce sses and analytical procedures applied by diverse laboratories on a variet y of food stuffs. Only the ar senic extracted from food can be measured. More importantly, laboratory extraction procedures are not designed to equate with that portion of arsenic in food that is bioavail able. Thus, the arsenic value resulting from extraction and measurement is not necessarily related to the concentration that is bioavailable to humans from specific sample sources. There is an immediate need for thorough research on the bioava ilability of arsenic from food.

53

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R-16

### APPENDIX A

Charge to EPA Science Advisory Board Arsenic Review Panel July 25, 2005

Background : There are both natural and anthropogeni c sources of arsenic and arsenic containing compounds (or arsenicals). Exposur e to arsenicals can be through different environmental media including drinking water, food, soil, and air. EPA assesses and regulates the potential exposure and health risks associated with exposure to arsenic and arsenic containing compounds through several st atutory authorities. The Safe Drinking Water Act (SDWA), directs EPA to estab lish national standards for contaminants including arsenical compounds in public dr inking water supplies. EPA’s Superfund and Resource Conservation and Recovery Act (RCR A) programs evaluate exposure to arsenic compounds at sites selected for clean up or re mediation. Under the Clean Air Act, EPA’s Office of Air and Radiation sets emissions standards for sources of arsenic to air. These include standards based on c ontrol technology and those base d on risks to human health from inhalation of airborne arsenic or ingestion of arsenic arising from air sources. EPA’s Office of Pesticide Programs (OPP) ev aluates the exposure and health risks associated with arsenicals used as pesticides in the U.S. Under the mandate of the Food Quality Protection Agency (FQPA), EPA must reevaluate all pesticide food tolerances (the legal limits of pesticid es on/in food or animal feed) in the U.S. by August, 2006. There are several organic arsenic herbicides that are undergoing re registration and/or tolerance reassessment including cacodylic acid (referred to as di methylarsinic acid or DMA V ), monosodium, disodium, and calcium salts of methanearsonate acid (MSMA, DSMA, and CAMA, collectiv ely as referred as MMA V ). In 2003, most residential uses of chromated copper arsenate (CCA) as a wood preservative were cancelled.

The health effects of arse nicals have been the subj ect of two reviews by the National Research Council (NRC) of the Na tional Academy of Sciences (NAS) (NRC 1999; 2001). Since the 2001 NAS review, ther e has been substantial new information developed on the mode of carcinogenic acti on and metabolism and toxicokinetics for arsenic and its methylated species, and ne w epidemiology on inorganic arsenic. The Agency has considered this new science in re gards to the hazard characterization required for tolerance assessment of DMA V (and MMA V ) as described in the draft OPP Science Issue Paper: Mode of Action for Cac odylic Acid (Dimethylarsinic Acid) and Recommendations for Dose Response Extrapolation, and also in th e ORD Issue Paper - Cancer Risk Assessment for Organic Arse nical Herbicides: Comments on Mode of Action, Human Relevance and Implications for Quantitative Dose-Response Assessment (See Appendix E). In addition, the Agency has developed a revised hazard and dose response assessment/characteri zation of inorganic Arsenic (Toxicological review of inorganic arsenic i n Support of Summary Information on the Integrated Risk Information System (IRIS) ) which relies on the two NRC revi ews and provides an updated human health effects and dose-response assessment fo r inorganic arsenic. The Agency seeks comment and advice from the SAB on the scientific soundness of major science conclusions drawn in these two documents regarding the carcinogenic assessments of

A-1DMA V and inorganic arsenic and the appropriateness of the Ag ency’s application of its own Guidelines for Carcinogen Ri sk Assessment for arsenicals.

Overview of Science and Assessment Issues : Ingestion of inorgani c arsenic has been demonstrated to cause cancer of the sk in, lung, and urinary bladder in humans. Historically, standard chronic bioassays with exposure to inorganic arsenic in rodents have been negative for increased tumor form ation. There are, however, more recent studies at high doses, in transgenic animal s, and following transplacental exposures which have demonstrated cancer potential in rodent studies following exposure to inorganic arsenic. The NRC 1999 report advise s that the bladder and lung cancer human mortality data, particularly from the southw estern Taiwanese studies provide the best dose-response data for evaluating the long-term effects of ingestion of inorganic arsenic. In the 2001 NRC report, a number of recommenda tions were made to EPA to revise the oral cancer slope for inorgani c arsenic. Given the available database, and recognizing that the mode(s) of action by which inorganic ar senic causes cancer has not been fully established, the draft Toxicologi cal Review of Arsenic, cons istent with advice from the NRC uses linear low dose extrapolation to esti mate cancer risks from ingestion of arsenic at low dose and has addressed ma ny of the NRC recommendations.

In approaching the cancer assessment on the pesticide cacodylic acid (DMA V ), an organic arsenical, EPA has c onfronted a number of challenging issues. No human epidemiological information is available for DMA V . Rodent cancer bioassay data have shown that dietary administration of DMA V can result in bladder carcinogenesis in the rat. DMA, however, is a key urinary metabolite from exposure to inorganic arsenic. Thus, the question is raised regarding the extent the cancer epidemiology on inorganic arsenic may provide an appropr iate dataset or may inform the low dose extrapolation for the cancer risk associated with direct exposure to DMA V

. Available in vivo and in vitro pharmacokinetic, metabolism studies, and toxico logy studies were reviewed to address this issue. The draft OPP Science Issue Pape r states that the evidence indicates inorganic arsenic and DMA V have different pharmacokinetic and pharmacodynamic characteristics, EPA proposes to use the rat bioassay data on DMA V to estimate its cancer risk. The ORD Issue Paper (Appendix E of the OPP Science Issue Paper: Cancer Mode of Action of Cacodylic Acid (Dimethylarsini c Acid) and Recommendations for Dose Response Extrapolation) provides additional discussion on the MOA issues and perspective on the nexus betw een science issues for orga nic and inorganic arsenicals. T he use of mode of action data in the assessm ent of potential carcinogens is a main focus of EPA’s 2005 cancer guidelines. Mode of action data are available on DMA and were evaluated to guide the lo w dose extrapolation.

The Agency seeks comments and advice from the SAB on key science issues concerning (A) the metabolism and toxic respon ses of arsenic species, (B) the mode of action for carcinogenesis and implications for dose-response extrapolation for DMA V and inorganic arsenic, (C) the sele ction of data for dose-response, and (D) approaches to lowdose extrapolation. In addition, the Agency is requesting comment on the implications of newer epidemiology and the incorporation of the 2001 NRC recommendation on modeling the human cancer datafor inorganic arsenic.

A-2Issues and Charge Questions

A. Metabolism and Toxic Responses of Arsenic Species

A1. Metabolism and pharmacokinetics: Evidence from in vivo and in vitro metabolism and pharmacokinetic studies with humans and laboratory animals suggests that the efficiency of the met hylation reaction(s) and cellular uptake varies based on which arsenical compound is administered exogenously. Most available studies suggest that the metabolic process in most mammals is primarily a one-way process and that following direct exposure to DMA V significant amounts of iAs III , iAs V , MMA III , or MMA V at the target tissue are not expected.

Please comment on how pharmacokinetic processes are best considered regarding the use of data derived from direct DMA V exposure versus direct iAs exposure for cancer risk

assessment.

A2. Response to mixtures of metabolites: Tumorigenic profiles vary based on which arsenical compound is administered exogenously. In vivo and in vitro studies indicate that each of the arsenical compounds exhibit similarities and differences in their profiles of biological activities. Direct exposure to iAs III or iAs V is expected to result in more of a mixture of toxic metabolites than for direct exposure to DMAv; the mixture of metabolites is expected to vary based on which chemical is administered exogenously. The potential mixture of metabolites following direct exposure to DMA V appears less complex as compared to iAs.

Given the toxicological response profiles observed following direct exposures to iAs versus MMA v and DMA V , and the differences in human and rodent toxicologic responses to arsenicals, please comment on the use of data derived from rodent exposures to the organic arsenicals versus use of data derived from direct iAs human exposure, in the DMA V assessment.

B. Modes of Carcinogenic Action for DMA V and Inorganic Arsenic

B1. Mode of action of DMA v : When relying on laboratory animal data, two critical assumptions are made: (i) data on animal tumors are predictive of human cancer, and (ii) animal tumor effects f ound at high experimental doses predict human risk at lower exposures. An u nderstanding of a chemical mode of carcinogenic action can help inform the above assumptions. In the case of DMA V , mode of action (MOA) data are ava ilable and were evaluated using the framework described in EPA’s cancer guidelines.

Please comment on the sufficiency of evide nce to establish the animal mode of carcinogenic action for DMA V

. Are the scientific conclusions sound and consistent with the available evidence on DMA V and the current state of knowledge for chemical carcinogenesis.

Please comment on whether the key events in DMA’s mode of action are supported by the available data. Specifically comment on th e role of: a) reactive oxygen species in

A-3producing chromosomal damage and the strength of the evidence supporting oxidative damage as a causal key event in DMA V /DMA III‘ s mode of carcinogenic action versus an associative event or a secondary consequence of cytotoxicity; b) cell proliferation and cytotoxicity and the strength of the evidence as causal key events in DMA V /DMA III‘ s mode of carcinogenic action versus associative or secondary events, and c) other potential modes of action that have substantial scientific support that may be contributing to the carcinogenicity of DMA.

B2. Human relevance of animal DMA V MOA: There are little or no scientific data to suggest that if sufficient DMA III were present, key precursor events and ultimately tumor formation would not occur in humans directly exposed to DMA V

.
Please comment on the relevance of the postulated key events (see B1) to tumors in humans.

Please comment on how, if at all, differen ces in the human population vs. experimental animals should be accounted fo r in the risk a ssessment for DMA V

. There are little to no chemical specific da ta regarding an increased susceptibility of humans for bladder tumor developm ent during different life stages.

Please comment on the Agency’s conclusion that the young are likely to respond like the adult to the formation of bladder tumors following exposure to DMA.

B3. Modes of carcinogenic action fr om exposure to inorganic arsenic:

DMA Inorganic arsenic (iAs) undergoes succe ssive methylation steps in humans, resulting in the intermed iate production of iAs III , MMA V , MMA III , DMA V , and
III . Each arsenical metabolite exhibits its own toxicity.

Please comment on the conclusion that the available data support the hypothesis that multiple modes of action may be operational following exposure to inorganic arsenic.

C. Selection of Data for Dose-Response Assessment

C1. Use of animal data for DMA V : A number of different rodent bioassays (standard bioassay, transgenic animals, su sceptible rodent strains, initiation and promotion studies) are available on DMA V

. Please comment on the use of the bladder tumor data from the DMA V rat bioassay as the most suitable dataset for quantif ying potential human cancer risk to DMA V

, including the weight of evid ence to support this conclusion.

Please comment on whether the iAs epidemiol ogy data can be used to inform the DMA V dose-response assessment derived from rat data with DMA V . If so, please discuss how such information might be used. (See Appendix).

A-4C2. Use of human epidemiologi cal data from direct iAs exposure: Since the NRC (2001) report on iAs, an additi onal body of literature has developed describing epidemiology data from populat ions in the US exposed to iAs in drinking water.

Does the SAB agree that the Taiwanese dataset remains the most appropriate choice for estimating cancer risk in humans? Please discuss the rationale for your response.

Do these data provide adequate characterizati on of the impact of childhood exposure to iAs? Please discuss the rationale for your response.

D. Approaches to Low-Dose Extrapol ation for Inorganic Arsenic and DMA V

D1. Mode of carcinogenic action understanding for DMA V/III and implications for dose response extrapolat ion to estimate human cancer risk: The use of mode of action data in the assessm ent of potential carcinogens is a main focus of EPA’s 2005 cancer guidelines. As stated in these guidelines “The approach to dose-response assessment for a particular agent is based on the conclusion reached as to its potential mode(s) of action”. Although a biologicalbased model is the preferred approach to estimating cancer risk, there are insufficient data on DMA V to support development of such a model

. Please comment on the scientific eviden ce and biological rationale in support of nonlinear versus linear low dose extrapola tion approaches, which approach is more consistent with the available data on DMA V and current concepts of chemical carcinogenesis, and how scientific uncertainty should most appropriately be incorporated into low-dose extrapolation.

D2. Implementation of the reco mmendations of the NRC (2001): EPA has determined that the most prudent approach for modeling cancer risk from exposure to iAs is to use a linear model because there are significant remaining uncertainties regarding which of the metabolite(s) may be the ultimate carcinogenic moiety and whether or not mixtures of toxic metabol ites interact at the site(s) of action.

Does the panel concur with the selec tion of a linear model following the recommendations of the NRC (2001) to estimate cancer risk at this time? Please discuss your response in light of the highly complex mode of action for iAs with its metabolites.

D3. EPA re-implemented the model presented in the NRC (2001) in the language R as well as in an Excel spr eadsheet format. In addition, extensive testing of the resul ting code was conducted.

Please comment upon precision and accuracy of the re-implementation of the model.

D4. Available literature describing dr inking water consumption rates for the southwestern Taiwanese study population: NRC (2001) stated that the

A-5drinking water consumption rate, as well as variability of that rate in both US and Taiwanese populations, are important fact ors to consider. In calculating risk estimates for U.S. populations exposed to arsenic through drinking water, NRC used a drinking water consumption rate of 1 L/day for the US population and two possible consumption rates for the Taiwanes e population: 1 L/day (identical to the US population) and 2.2 L/day with lit tle or no supporting rationale. Since publication of NRC 2001, a number of new st udies have become available and are summarized in the Cancer Slope Factor Workgroup Issue Paper. Agency reviews of the relevant literature suggests that the mean drinking wate r (for the Taiwanese study population) consumption rate is be tween 1 to 4.6 L/day. EPA’s current cancer modeling includes water intake adjustments for 2.0 and 3.5 L/day.

What drinking water value does the panel recommend for use in deriving the cancer slope factor for inorganic arsenic?

D5. Selection of an estimate of di etary intake of arsenic from food: The issue of intake of arsenic from food (e.g., dry rice, sweet potatoes) has been distinguished from the issue of intake of arsenic from drinking water. The NRC addressed the issue of arsenic in food by determining how sensitive the calculation of ED 01 was to the consumption rate. NRC found that changing the consumption rate from 50 μg/day to 30 μg/day did not change the calculated ED 01 significantly (about 1% di fference). Since the publication of NRC 2001, a number of new studies have become avai lable, summarized in the Cancer Slope Factor Workgroup Issue Paper. EPA’s cu rrent cancer modeling includes dietary intake adjustments for 0, 10, 30, and 50 μg/day.

What background dietary intake (of arsen ic) value does the panel recommend for both the control population and study population of Southwestern Taiwan used in deriving the cancer slope factor for inorganic arsenic?

A-6

### APPENDIX B

Assignments to Charge-Specifi c Groups (revised on 8/24/05)

Issue A : Metabolism and Toxic Responses of Arsenic Species

Question A1 : Metabolism and Pharmacokinetics Dr. Aposhian Dr. Medinsky Dr. Le Dr. Rosen Dr. Styblo Dr. Hopenhayn

Question A2: Response to mixtures of metabolites Dr. Aposhian Dr. Medinsky Dr. Le Dr. Rosen Dr. Styblo Dr. Hopenhayn

Issue B : Modes of Carcinogenic Action for DMA and iAs

Question B1 : Mode of Action of DMA V Dr. Barchowsky Dr. Rossman Dr. Brusick Dr. Styblo Dr. Dragan Dr. Waalkes Dr. Klaunig

Question B2 : Human relevance of animal DMA V MOA Dr. Barchowsky Dr. Rossman Dr. Brusick Dr. Styblo Dr. Dragan Dr. Waalkes Dr. Klaunig

Question B3: Modes of carcinogenic action from exposure to inorganic arsenic Dr. Barchowsky Dr. Klaunig Dr. Brusick Dr. Rossman Dr. Dragan Dr. Waalkes

Issue C: Selection of Data for Dose-Response Assessment

Question C1 : Use of animal data for DMAV Dr. Cantor Dr. Green Dr. Medinsky Dr. Teeguarden Dr. Waalkes Dr. Yager

B-1Question C2: Use of human epidemiological data from direct iAs exposure Dr. Cantor Dr. Colford Dr. Harlow Dr. Hopenhayn Dr. Rosen Dr. Rossman Dr. Yager

Issue D : Approaches to Low-Dose Extrapolation for iAs and DMA V

Question D1 : Mode of Carcinogenic action understanding for DMAV/III and implications for dose response extrapolation to estimate human cancer risk Dr. Cantor Dr. Medinsky Dr. Colford Dr. Teeguarden Dr. Green Dr. Waalkes Dr. Klaunig

Question D2: Implementation of the recommendations of the NRC (2001) Dr. Colford Dr. Harlow Dr. Hopenhayn Dr. Heeringa Dr. Portier Dr. Rosen Dr. Rossman

Question D3 : EPA re-implementation of th e NRC (2001) model in language R and Excel spreadsheet. Dr. Heeringa Dr. Portier Dr. Teeguarden

Question D4 : Literature describing drinki ng water consumption rates for the southwestern Taiwanese study population Dr. Barchowsky Dr. Harlow Dr. Colford Dr. Yager

Question D5: Selection of an estimate for dietary intake of arsenic Dr. Aposhian Dr. Barchowsky Dr. Harlow Dr. Styblo Dr. Yager

B-2APPENDIX C

ABBREVIATIONS

Abbreviations Meaning ARP US EPA SAB Arse nic Review Panel As Arsenic BEIR Biological Effects of Ionizing Radiation CAMA Calcium salt of MMA V
CCA Chromated copper arsenate DMA III Dimethylarsinous acid DMA V Dimethylarsinic acid, Cacodylic Acid DSMA Disodium salt of MMA V
EPA US Environmental Protection Agency FIFRA Federal Insecticide, Fungicide, and Rodenticide Act FQPA The Food Quality Protection Act GPO US Government Printing Office iAs Inorganic arsenic iAs III Arsenite, Trivalent inorganic arsenic NFkB Nuclear factor-kappa B iAs V Arsenate, Pentavalen t inorganic arsenic MLE Maximum Likelihood Estimates MMA III Methylarsonous acid MMA V Methanearsonate acid, methylarsenic acid MN Micronuclei MOA Mode of Action MSMA Monosodium salt of MMA V
NAS National Academy of Sciences NHANES National Health and Nutrition Examination Survey NRC National Research Council of the NAS OPP US EPA Office of Pesticide Programs ORD US EPA Office of Research & Development OW US EPA Office of Water PBPK Physiologically Based Pharmacokinetic Models PD Pharmacodynamics PK Pharmacokinetics RCRA Resource Conservati on and Recovery Act ROS Reactive Oxygen Species SAB US EPA Science Advisory Board TMA III Trimethylarsine TMA V O Trimethylarsine oxide

C-1