to Inform EPA's Upcoming IRIS

Toxicological Review
of Inorganic Arsenic

SESSION 4:

Roundtable Discussion on
Planning and Scoping

Tuesday, January 8 &

Wednesday, January 9
RTP, North Carolina

Hosted by EPA's National Center
for Environmental Assessment


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Identifying Client Needs: Draft planning and
scoping summary discussion

What are client needs for the Toxicological Review
of iAs?

Discuss key themes from the workshop to be
considered during the development of the draft
Toxicological Review of iAs.

*>EPA

United States
Environmental Protection
Agency

Key Themes from Sessions 1-3 of the iAs Public

Stakeholder Workshop

Reeder Sams
U.S. EPA/ORD/NCEA
Research Triangle Park, NC

Office of Research and Development	Disclaimer: the views expressed in this presentation are those of the speaker

National Center for Environmental Assessment	and do not necessarily represent the views or policies oftheM.S. EPA.

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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e pp/x

v'criA	Key Themes from Introduction

United States
Environmental Protection
Agency

>	Partnership and outreach

>	Engagement of stakeholders, including at-
risk populations

>	Consideration of at-risk populations in scope

&EPA	Key Themes - Session 1

Applying Systematic Review

>	Process that is transparent and reproducible

>	Iterative process with combination of search
strategies

>	Relevancy - use previous iAs work to guide,
engage stakeholders grappling with iAs
issues

>	Set up key questions - e.g., focus on low-
dose effects?

>	Consider bias - e.g., negative data bias

>	Feasibility (time and resources)

Office of Research and Development

Natinnal Hpntpr fnr Fnvirnnmpntal iewecmpnt

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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oEPA	Key Themes - Session 2

United States
Environi
Agency

Environmental Protection	Hazard ID

>	Susceptibility factors - in utero,
smoking/carcinogens, diet, genetic variation,
metabolism

>	Effects low-level exposure is major question
and challenge for epi studies

>	Aggregate exposure; concern over
misclassification of exposure

&EPA	Key Themes - Session 2

United States
Environi
Agency

Environmental Protection	MOA

>	Consider a MOA analysis as an organizing
principle

>	Lack of speciation data at target tissues

>	Key biological processes/chemical stressors
that influence MOAs

>	Multiple MOAs and multiple targets - dose
plays important role in MOA

>	Aggregate exposure; concern over
misclassification of exposure

>	Complex metabolism

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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SEPA	Key Themes - Session 3

~z,~ Relevant Factors for D-R

>	Arsenic speciation and importance of
chemical nomenclature

>	Aggregate exposure (e.g., diet,
contamination ) vs. "sole source" exposure

>	Exposure in U.S. population

>	Uncertainty in the exposure - life stage, dose

>	Misclassification of exposure in studies

>	Biomarkers of exposure (proteomics,
epigenetics, transcriptomics) - utility for
D-R?

>	Bioavailability/bioaccumulation

Office of Research and Development

Matinnal rpntorfnr Pnuirnnmantal flccoccmont

&EPA	Key Themes - Session 3

United States	_	_	__ __

Approaches to D-R

>	Multi-tumor modeling - feasible in time-frame?

>	PBPK - challenge of animal-human
extrapolations, metabolism, and
polymorphisms

>	Factors impacting D-R: MOA/TK, iAs
speciation, exposure estimation, susceptible
populations (e.g. age, ethnicity)

>	Treat iAs speciation as a mixture for D-R?

>	Uncertainty - probability
distribution/sensitivity analyses, MOA
organization

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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SEPA	Key Themes - Session 3

irlM" Extrapolation Approaches to D-R

>	Need extrapolations to U.S. population, high
dose to low dose, and interspecies/in vitro
extrapolations - consider value added

>	Human PBPK model - simplify exposure to
"total load" of iAs?

>	Low dose extrapolation is to a lower
response level; a dose-response
characterization approach

Office of Research and Development

Matinnal rpntorfnr Pnuirnnmantal flccoccmont

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Key Themes From Sessions 1-3
Mike Waalkes

Key Themes From Sessions 1-3

• Early life as a critical period of susceptibility
to inorganic arsenic

-	Remarkable concordance between human and
rodent data in terms of carcinogenesis

•	Sensitivity and sites

•	Cannot be ignored

-	Basis not known

-	Other diseases need further exploration

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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Key Themes From Sessions 1-3

•	The role of biomethylation in inorganic
arsenic

-	Susceptibility factor

•	Poor tissue and target site dosimetry for
inorganic arsenic in humans

-	We do not know what gets where with regards
to target cells

•	Or what is generated where

•	Urinary metabolites poor substitute


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Identifying Client Needs: Draft planning and
scoping summary discussion

What are client needs for the Toxicological Review
of iAs?

Recommendations for revisions to the planning
and scoping summary to adequately
address stakeholder input.

EPA ARSENIC WORKSHOP







PRESENTATION TO U.S.
ENVIRONMENTAL PROTECTION AGENCY
RESEARCH TRIANGLE PARK, NC







JANUAR? 8-9, 2013









SAMUEL M. COHEN, M.D., PH.D.

DEPARTMENT OF PATHOLOGY AND MICROBIOLOGY
UNIVERSITY OF NEBRASKA MEDICAL CENTER
OMAHA, NE 6819 8-31 35

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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MODE OF ACTION

• Metabolism and kinetics



• Genotoxicity vs. non-genotoxicity



• Cellular responses



• Critical review of the literature



• Cancer and non-cancer





65

CLASSIC PATHWAY
FOR ARSENIC METABOLISM

Putative Pathway for Arsenic Metabolism

9	2e"	CH3*	o	2e-

M	-ie	—	II

OH—As—OH	~OH— jks—OH	*«-CH3—As—OH

J)H	OH	OH

L

As	As	MMA

CH3+	
-------
THIOLATED ARSENICALS

?

HO"^s

r oh

OH
iAsv

in-vitro

cecum

MU / OH
OH	S

iAsVSi H0-JL

.	MU / SH

-> O SH

!

i

^ / SH

OH
MMDTAV

-As-.

HO j ^SH
OH

iAsvS2

iAs S3 H3C"/S>

H.C'f

I H>C

/*Ab\

SH nun

/ SH

SH
WIMTTAV

DMDTA

Pinyayev et al., Chem. Res. Toxicol., 24:475-7,2011.

TOXICITY OT ARSENIC METABOLITES
IN UROTHELIUM IN VITRO,
EXPRESSED AS LC50 VALUES (fiM)

Cell Line

As111

Asv

MMA1"

MMAV

DMA111

DMAV

TMAO

DMMTAV*

MYP3
(Rat)

0.75

3.4

0.42

3000

0.38

600

1600

1.3

ITI

(Human)

8.3

34.6

0.9

2700

1.0

230

14000

1.4

* DMMTAV

- Dimethyl monothiol arsenic acid







Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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RECENT DEVELOPMENTS IN RESE ARCH
OF ARSENIC METABOLISM

Asll! methyl transferase (As3Mt):

•	Glutathione in reaction mixture has little effect on methylation

Strongly favors classical pathway (Does not support
Hayakama's proposed pathway)

•	DMA is poor substrate for human enzyme

Explains lack of TMAO formation in humans

Thiol analogs:

•	Formed primarily by a chemical reaction of oxyarsenicals with H2S

•	Primarily from Gl bacteria, but also in tissues

•	Rapidly transported into cells and rapidly converted chemically to
trivalent oxyarsenicals

Explains their high cytotoxicity compared to pentavalent
oxyarsenical analogs

69

INTRAMITOCHONDRIAL GRANULES IN
MOUSE UROTHELIUM

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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I NT RAC YT OPL ASM IC GRANULES IN UROTHELIAL
CELLS OF PML PATIENTS TREATED WITH As203







% %

€>

* 4* * -

& . { '•

•**•

% »



J -

¦> ./«

> * (* . /

¦ J' V /
/ *

•? ..-p





71

MODE OF ACTION OF DMAV

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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UROTHELIAL CYTOTOXICITY AND
PROLIFERATION INDUCED BY DMAV IN RATS



GENE EXPRESSION IN DI11J REN 1 HUMAN
CELL TYPES INDUCED BY ARSENICALS

HBECs (bronchial)

HEK001 (skin) 1T1 (bladder)



Cell death & survival

Cell death & survival



Cell growth & proliferation

Cell growth & proliferation



Cell-cell signaling

Cell-cell signaling



Cell movement

Cell movement



Cell development

DNA replication & repairs



Small molecule bioschemistry

Cell cycle





Cell assembly & organization





Lipid metabolism







74

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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RELEVANCE TO HUMANS

Clinical Manifestations and Arsenic Methylation After a Rare Subacute Arsenic Poisoning Accident
Y. Xu, Y. Wang, Q. Zheng, B. Li, X. Li, Y. Jin, X. Lv, G. Qu, and G. Sun.

Toxicol. Sciences, 10: 278-284, 2008.

Observations
High exposure
DMAV & TMAO in urine
Hematuria in 1/3 of exposed group

HUMAN CHRONIC BRONCHITIS	SKIN: ACTINIC KERATOSIS

IN VIVO CYTOTOXICITY OF



INORGANIC ARSENIC



• Threshold is 1 ppm elemental



• Rats and mice



• Males and females



• Arsenite and arsenate



• Detection method:



• Histopathology



• Labeling index



• Scanning electron microscopy



• Genomics

27

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicolbgicail Review of Inorganic Arsenic

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IN VITRO CYTOTOXICITY OF
INORGANIC ARSENIC

Threshold approximately 0.2 |jM

•	Urothelial cells (rats and humans)

•	Bronchial epithelial cells (humans)

•	Keratinocytes (humans and mice)

Less than 0.2 pM - Adaptive

•	0.2-1.0 pM - Cytotoxicity

•	Greater than 10 |jM - Lethal

ARSENIC RESEARCH
LITERATURE PRECAUTIONS (1/2)

•	Analytical methods

•	Arsenic form:

•	As111 vs. Asv

•	Inorganic vs. methylated vs. other organics

•	Concentrations (in vitro) and doses (in vivo):

•	Environmental drinking water concentration of 10 |jg/L
(0.2 |jM) is not systemic or tissue concentration

•	Many claim in vitro studies with "environmentally
relevant concentrations of 10 M9/L"

•	Concentrations of trivalents >10 |jM in vitro are
meaningless; lethal dose

78

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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ARSENIC RESEARCH
LITERATURE PRECAUTIONS (2/2)

Cell types:

•	Must use epithelial cells

•	Lung tiProblasts do not represent cell of origin for iung cancer

•	Malignant cell lines (e.g., osteosarcoma) have limited value
without corroboration

Primary cell vs. established cell lines:

•	Established cell lines have multiple differences compared to
normal, especially p53

in vitro vs. in vivo:

•	Trivalent arsenicals bound to rat hemoglobin

•	Oxidative stress in vitro (Gentry et al„ 2010)

•	No oxidative stress in vivo (Clewell et ai„ 2011) (Suzuki et al.,
submitted)	79

INTERACTION OF
ARSENICALS WITH RAT HEMOGLOBIN

In Vitro	In ViVo

As111 + Hb 		* As111	Hb Asv + Hb	* DMA111	Hb

MMA111 + Hb—»¦ MMAI!!—Hb MMAV + Hb DMA111	Hb

DMA111 + Hb-* DMA111—Hb DMAV + Hb —~ DMA111	Hb

Feeding Asv, MMAvor DMAvto rats produces DMAHi—Hb

80

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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BIOLOGICAL EFFECTS OF ARSENIC

81



Mode of Action for

Inorganic Arsenic: Working Hypothesis

Cancer



Non-Cancer

Ingestion of Arsenic

Conversion to Trivalent Arsenicals
(iAsln, AAMAIM, DMA"1)

Interaction with Critical
Cellular Sulfhydryl Groups

Threshold 		*

Cytotoxicity
Cell death—>.



Ingestion of Arsenic

'J'

Conversion to Trivalent Arsenicals
(iAsm, MMA"!, DMA"1)

Interaction with Critical





Cellular Sulfhydryl Groups

Threshold	»

Cytotoxicity —y Non-cancer
effects

Regenerative Proliferation

1





Carcinoma



82

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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PUBLIC STAKEHOLDER WORKSHOP TO
INFORM EPA's UPCOMING IRIS

TOXICOLOGICAL REVIEW OF
INORGANIC ARSENIC

Michele Roberts
Environmental Justice Health Alliance
January 9, 2013

Environmental Justice

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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Why other sources matter in
toxicological assessments

Studies show the need to protect the

Bell Curve

•	RECENTLY published in Environmental Health

Perspectives, "Beyond Uncertainty Factors:
Protecting the Bell Curve

•	"some individuals have predisposing risk
factors that make them uniquely sensitive to
the effects of these environmental stressors."

(Schmidt, 2012 at: http://ehp.niehs.nih.gov/2013/01/121-
a26/)

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Health professionals call for broad

reform

•	Dr. David Bellinger, Harvard Medical School
Professor stated: "The assessment of effect
modifiers should drive the study design. As it
stands now, analysis of potential modifiers is
usually something tacked on at the end of the
main study..."

•	Dr. Bellinger concludes that toxicological reviews
need to identify the most susceptible people and
quantify their added risk in order to make
appropriate management decisions.

Social Stressors

•	Poverty

-access to nutritional food

-access to health care and prenatal care

-housing conditions

•	Racism

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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Arsenic and Cardiovascular Disease

•	2012 systemic review on arsenic exposure and
hypertension found an increasing trend in the
odds of hypertension with increasing arsenic
exposure. (Abhyankar et. al, 2012)

•	Abhyankar found that "low" arsenic exposure
imposed a more than 50% increase in the risk of
hypertension.

•	Another study, Zhang et. al (2012) found
substantially elevated risk with low exposure to
arsenic.

Cardiovascular Disease Among African
Americans
Race/Ethnic Group Differences

Heart Disease Age-Adjusted Death Rates by Race/Ethnicity
California, 2000 -2008



2000

2001

2002

2003

2004

2005

2006

2007

2008

Black

351.2

347.3

347.1

331.3

304.5

302.1

301.8

280.4

262.2

Pacific Islander

317.3

306.0

241.2

269.4

230.1

216.4

215.0

220.1

219.0

White

251.8

248.2

240.2

236.4

218.1

211.1

206.6

196.2

187.3

Hispanic

184.8

177.0

165.4

163.3

150.3

148.5

143.8

130.1

127.4

American Indian

192.6

170.3

142.0

150.1

133.7

123.0

123.8

110.1

93.8

Asian

150.2

144.7

142.0

132.1

117.0

114.5

115.6

106.5

103.9

Two or More Races

34.1

25.5

22.7

64.7

58.7

58.0

58.2

60.9

65.8

_





*s^C'Sla









—Q— American Indian



O Two

or More Races



Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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Scientific Studies call for Reform

•	Study by researchers of the University of North
Carolina, (Mo et al, 2011) findings suggest the need for
an integrative mode of action in humans, with many
results supporting numerous other studies that have
found chronic arsenic exposure can cause cancer,
cardiovascular disease and diabetes.

•	Arsenic associated with diabetes mellitus,
cerebrovascular disease and other potentially fatal
diseases that are more prevalent in African Americans
(Meliker et al, 2007).

Conclusion and next steps

•	Arsenic exposure can exacerbate cardiovascular
disease as evidenced by a large body of science
and standard medical tests.

•	African Americans are at elevated risks of
preliminary and advanced stages of
cardiovascular disease and have higher mortality
rates from heart disease across the age span,
with young and middle aged having more
disproportionate elevations in death rates.

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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Conclusion and next steps

•	Evidence of arsenic's cardiovascular toxicity and
elevated risk among African Americans is
substantial persuasive, and sufficient to rely on in
risk assessments of arsenic.

•	EPA's evaluation of arsenic must take under
consideration and explicitly address the
disproportionate health burden exposure
imposes on African Americans.

•	The elevated risks must be incorporated in
quantitative evaluations of noncancer toxicity
and be addressed in public policy.

Thank you!

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Recommendations

Mike Waalkes

Recommendations

• In general, humans appear more sensitive than
rodents to inorganic arsenic

-	Clear with cancer

• Other diseases (?)

-	Should be considered in review

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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Recommendations

• Since inorganic arsenic is associated with
multiple, diverse diseases

- Review should keep an open mind to the possibility
of multiple modes of action

Recommendations

• There is a good evidence that inorganic arsenic
as well as various metabolites are toxicologically
active

- Review should keep an open mind to the possibility
of multiple species being active

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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Methods for Identifying, Evaluating,
and Synthesizing Literature

Public Comments

m

O
5

¦o ¦£*
-=
£0 Tf

* |
rtl «£

2	*

3	I

ii

0016

EC

o
s

ciam

0.005

0 000

NRC 2001
Risk Estimate

\

MORTALITY = 4.9>c10"3 4.7x1 O^xC,

Fig. 3

tODTlIlK

0	50	too	150	200	250

Cmea/i Mean County Arsenic Concentration (ng/L)

. While male bladder cancer lifednne mortality one by Mean, mric rancemriaticm ("U.I
with median arsenic le'-'eli of £-3 §igfl_ ill drinking wafer).

Public Stakeholder Workshop to Inform EPA's Upcoming IRIS Toxicological Review of Inorganic Arsenic

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SMRs for Bladder and Lung Cancers for Low and High Arsenic Villages
by Mean Village Well Water Arsenic Level (ug/L)

Low Arsenic

High Arsenic

— Linear (Low Arsenic)

1200
1100

W


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