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> **UNITED STATES ENVIRONMENTAL PROTECTION AGENCY**

**WASHINGTON D.C. 20460**

> **OFFICE OF THE ADMINISTRATOR**
>
> **SCIENCE ADVISORY BOARD**

December 19, 2008

EPA-SAB-08-009

The Honorable Stephen L. Johnson

Administrator

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue, N.W.

Washington, DC 20460

> Subject: Review of EPA's draft entitled "Toxicological Review of
> Acrylamide".

Dear Administrator Johnson:

In response to a request from EPA's Office of Research and Development
(ORD), the Science Advisory Board (SAB) convened an expert panel to
conduct a peer review of EPA\'s draft Integrated Risk Information System
(IRIS) assessment entitled, "*Toxicologic Review of Acrylamide*"
(December 2007). This draft document updates EPA's current evaluation of
the potential health effects of acrylamide.

The SAB was asked to comment on the hazard characterization and
dose-response assessment of acrylamide, including the Agency's selection
of the most sensitive non-cancer health endpoint, the use of a
physiologically-based toxicokinetic (PBTK) model, the derivation of a
proposed oral reference dose (RfD), an inhalation reference
concentration (RfC) for non-cancer endpoints, as well as the cancer
descriptor, oral slope factor, and inhalation unit risk for acrylamide.
The SAB Panel's report contains a number of recommendations that are
aimed at making the assessment more transparent and improving the
scientific bases for the conclusions presented. While a more detailed
description of the technical recommendations is contained in the body of
the report, the key points and recommendations are highlighted below:

-   The Panel agreed with the EPA's conclusion that based on the
    existing toxicity data base for acrylamide, neurotoxicity appears to
    be the most sensitive non-cancer endpoint, and therefore, the most
    appropriate for developing the RfD and RfC for non-cancer health
    effects. The Panel was concerned, however, that the RfD/RfC was
    derived from studies which were primarily designed as cancer
    bioassays and therefore did not include the most sensitive measures
    of neurotoxicity.

-   The Panel believed that the use of the benchmark dose methodology in
    this assessment was scientifically supportable, given the nature and
    robustness of the data sets available on the endpoint of concern.

-   The Panel supported the Agency's conclusions that exposure to
    acrylamide in animals leads to heritable gene mutations and that
    these results indicate that it may also pose a hazard to humans. The
    Panel further supported the Agency's conclusions that the available
    data on heritable gene mutations are not adequate to conduct a
    robust assessment of this endpoint at this time. The Panel urges
    further research on acrylamide-induced heritable germ cell
    mutations, given the serious nature of such effects.

-   The Panel concluded that the rationale and justification for
    acrylamide being a "*likely human carcinogen*" via a mutagenic
    mechanism was well described and the conclusion was scientifically
    supportable, although it should be further elaborated. While the
    Panel did consider available information regarding non-mutagenic
    MOAs (e.g., hormonal) as presented by public commenters, they did
    not find it to be compelling.

-   The Panel encouraged the Agency to use the two main chronic
    bioassays in rats for deriving the oral cancer slope factor and
    include an in depth discussion of the strengths and limitations of
    both studies.

-   The Panel commends EPA for using the PBTK model for developing the
    RfD, RfC and cancer slope factor for acrylamide. The Panel did
    however provide some recommendations to the Agency for improving the
    model as they revise their draft document. The Panel notes that the
    use of internal dose metrics combined with a fairly robust
    understanding of the mechanism of action may replace the use of the
    default interspecies factor for toxicokinetic differences. Internal
    dose may be derived using the PBTK model or through application of
    other pharmacokinetic approaches indicated in the Panel report.

-   The Panel agreed with the use of PBTK modeling to conduct dose-route
    extrapolation and commended the EPA for using the PBTK model to fill
    the gap created due to the absence of robust animal toxicology
    studies that would support the development of an RfC. In estimating
    the cancer slope factor and unit risk, human-rodent differences in
    toxicokinetics were taken into account with the PBTK model, whereas
    toxicodynamic differences were not, but should be, through the
    application of a standard factor.

-   Finally, the Panel agreed that the use of the age-dependent
    adjustment factors to adjust the unit risk for early life exposure
    is well justified and transparently and objectively described.

The SAB appreciates the opportunity to provide EPA with advice on this
important subject. Although cognizant of additional acrylamide studies
currently underway, the SAB urges EPA to move expeditiously to finalize
the IRIS document on acrylamide as the Agency considers relevant data
which has been published since the release of the draft assessment. We
look forward to receiving the Agency's response.

> Sincerely,

***/signed/*** ***/signed/***

Dr. Deborah L. Swackhamer, Chair Dr. Deborah Cory-Slechta, Chair

EPA Science Advisory Board SAB Acrylamide Review Panel

**NOTICE**

This report has been written as part of the activities 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 scientific matters
related to problems facing the Agency. This report has not been reviewed
for approval by the Agency and, hence, the contents of this report do
not necessarily represent the views and policies of the Environmental
Protection Agency, nor of other agencies in the Executive Branch of the
Federal government, nor does mention of trade names or commercial
products constitute a recommendation for use. Reports of the EPA Science
Advisory Board are posted on the EPA Web site at:
[http://www.epa.gov/sab]{.underline}.

**U.S. Environmental Protection Agency**

**Science Advisory Board**

**Acrylamide Review Panel**

**CHAIR**

**Dr. Deborah Cory-Slechta**, Professor, Department of Environmental
Medicine, School of Medicine and Dentistry, University of Rochester,
Rochester, NY

**PANEL MEMBERS**

**Dr. Alfred Branen**, Associate Vice President, University of Idaho,
Coeur d\'Alene, ID

**Dr. Daniel R. Doerge**, Research Chemist, National Center for
Toxicological Research, Food and Drug Administration, Jefferson, AR

**Dr. James S. Felton**, Senior Biomedical Scientist, University of
California, Lawrence Livermore National Laboratory, Livermore, CA

**Dr Timothy Fennell**, Senior Research Chemist, Drug Metabolism and
Pharmacokinetics, RTI International, Research Triangle Park, NC

**Dr. Penelope Fenner-Crisp**, Independent Consultant, North Garden, VA

**Dr. Jeffrey Fisher**, Professor, Department Environmental Health
Science, University of Georgia, Athens, GA

**Mr. Sean Hays**, President, Summit Toxicology, Allenspark, CO

**Dr. Steven Heeringa**, Director, Division of Surveys and Technologies,
Institute for Social Research, University of Michigan, Ann Arbor, MI

**Dr. Richard M. LoPachin**, Professor of Anesthesiology, Department of
Anesthesiology, Montefiore Medical Center, Albert Einstein College of
Medicine, Bronx, NY

**Dr. Lorelei Mucci**, Assistant Professor, Harvard Medical School,
Channing Laboratory, Boston, MA

**Dr. Jerry M. Rice**, Distinguished Professor, Department of Oncology,
Lombardi Cancer Center, Box 571465, Georgetown University Medical
Center, Washington, DC

**Dr. Dale Sickles**, Professor and Vice-Chair, Department of Cellular
Biology and Anatomy, Medical College of Georgia, Augusta, GA

**Dr. Gina Solomon**, Senior Scientist, Health and Environment Program,
Natural Resources Defense Council, San Francisco, CA

**Dr. Anne Sweeney**, Professor of Epidemiology, Commonwealth Medical
Education, The Commonwealth Medical College, Scranton, PA

**Dr. Lauren Zeise**, Chief, Reproductive and Cancer Hazard Assessment
Branch, Office of Environmental Health Hazard Assessment, California
Environmental Protection Agency, Oakland, CA

**SCIENCE ADVISORY BOARD STAFF**

**Dr. Suhair Shallal,** Designated Federal Officer, EPA Science Advisory
Board, Science Advisory Board Staff Office, Washington, DC

**U.S. Environmental Protection Agency**

**Science Advisory Board**

**CHAIR**

**Dr. Deborah L. Swackhamer***,* Professor, Co-Director of the Water
Resources Center,*\
*University of Minnesota School of Public, St. Paul, MN

**SAB MEMBERS**

**Dr. Gregory Biddinger**, Coordinator, Natural Land Management
Programs, Toxicology

and Environmental Sciences, ExxonMobil Biomedical Sciences, Inc.,
Houston, TX

**Dr. Thomas Burke**, Professor, Department of Health Policy and
Management, Johns

Hopkins Bloomberg School of Public Health, Johns Hopkins University,
Baltimore, MD

**Dr. James Bus**, Director of External Technology, Toxicology and
Environmental

Research and Consulting, The Dow Chemical Company, Midland, MI

**Dr. Deborah Cory-Slechta**, Professor, Department of Environmental
Medicine, School of

Medicine and Dentistry, University of Rochester, Rochester, NY

**Dr. Maureen L. Cropper**, Professor, Department of Economics,
University of Maryland,

College Park, MD

**Dr. Virginia Dale**, Corporate Fellow, Environmental Sciences
Division, Oak Ridge

National Laboratory, Oak Ridge, TN

**Dr. Kenneth Dickson**, Regents Professor, Department of Biological
Sciences, University

of North Texas, Aubrey, TX

**Dr. David A. Dzombak**, Walter J. Blenko Sr. Professor of
Environmental Engineering,

Department of Civil and Environmental Engineering, College of
Engineering, Carnegie

Mellon University, Pittsburgh, PA

**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, Department of Environmental Sciences,
University of

Virginia, Charlottesville, VA

**Dr. James K. Hammitt**, Professor, Center for Risk Analysis, Harvard
University, Boston,

MA

**Dr. Rogene Henderson**, Senior Scientist Emeritus, Lovelace
Respiratory Research

Institute, Albuquerque, NM

**Dr. James H. Johnson**, Professor and Dean, College of Engineering,
Architecture &

Computer Sciences, Howard University, Washington, DC

**Dr. Bernd Kahn**, Professor Emeritus and Director, Environmental
Radiation Center,

Nuclear and Radiological Engineering Program, Georgia Institute of
Technology, Atlanta,

GA

**Dr. Agnes Kane**, Professor and Chair, Department of Pathology and
Laboratory Medicine,

Brown University, Providence, RI

**Dr. Meryl Karol**, Professor Emerita, 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 of Pediatrics, Director,
Center for Childhood

Neurotoxicology, Robert Wood Johnson Medical School-UMDNJ, Belle Mead,
NJ

**Dr. Jill Lipoti**, Director, Division of Environmental Safety and
Health, New Jersey

Department of Environmental Protection, Trenton, NJ

**Dr. Michael J. McFarland**, Associate Professor, Department of Civil
and Environmental

Engineering, Utah State University, Logan, UT

**Dr. Judith L. Meyer**, Distinguished Research Professor Emeritus,
University of Georgia,

Lopez Island, WA

**Dr. Jana Milford**, Associate Professor, Department of Mechanical
Engineering,

University of Colorado, Boulder, CO

**Dr. M. Granger Morgan**, Lord Chair Professor in Engineering,
Department of

Engineering and Public Policy, Carnegie Mellon University, Pittsburgh,
PA

**Dr. Rebecca Parkin**, Professor and Associate Dean, Environmental and
Occupational

Health, School of Public Health and Health Services, The George
Washington University

Medical Center, Washington, DC

**Mr. David Rejeski**, Director, Foresight and Governance Project,
Woodrow Wilson

International Center for Scholars, Washington, DC

**Dr. Stephen M. Roberts**, Professor, Department of Physiological
Sciences, Director,

Center for Environmental and Human Toxicology, 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, East Lansing, MI

**Dr. James Sanders**, Director and Professor, Skidaway Institute of
Oceanography,

Savannah, GA

**Dr. Jerald Schnoor**, Allen S. Henry Chair Professor, Department of
Civil and

Environmental Engineering, Co-Director, Center for Global and Regional
Environmental

Research, University of Iowa, Iowa City, IA

**Dr. Kathleen Segerson**, Professor, Department of Economics,
University of Connecticut,

Storrs, CT

**Dr. Kristin Shrader-Frechette**, O\'Neil Professor of Philosophy,
Department of Biological

Sciences and Philosophy Department, University of Notre Dame, Notre
Dame, IN

**Dr. V. Kerry Smith**, W.P. Carey Professor of Economics, Department of
Economics,

W.P Carey School of Business, Arizona State University, Tempe, AZ

**Dr. Thomas L. Theis**, Director, Institute for Environmental Science
and Policy,

University of Illinois at Chicago, Chicago, IL

**Dr. Valerie Thomas**, Anderson Interface Associate Professor, School
of Industrial and

Systems Engineering, Georgia Institute of Technology, Atlanta, GA

**Dr. Barton H. (Buzz) Thompson, Jr.**, Robert E. Paradise Professor of
Natural Resources

Law at the Stanford Law School and Director, Woods Institute for the
Environment

Director, Stanford University, Stanford, CA

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

**Dr. Lauren Zeise**, Chief, Reproductive and Cancer Hazard Assessment
Branch, Office of

Environmental Health Hazard Assessment, California Environmental
Protection Agency,

Oakland, CA

**LIAISON MEMBERS**

**Dr. Steven Heeringa**, (FIFRA SAP), Research Scientist and Director,
Statistical Design Group,

Institute for Social Research (ISR), University of Michigan, Ann Arbor,
MI

**Dr. Melanie Marty**, (CHPAC Chair), Chief, Air Toxicology and
Epidemiology Branch, Office of

Environmental Health Hazard Assessment, California EPA, Oakland, CA

**SCIENCE ADVISORY BOARD STAFF**

**Mr. Thomas Miller**, Designated Federal Officer, EPA SAB, Washington,
DC

# TABLE OF CONTENTS

# 

# 

[EXECUTIVE SUMMARY 9](#executive-summary)

[INTRODUCTION 15](#introduction)

[RESPONSES TO THE CHARGE QUESTIONS
17](#responses-to-the-charge-questions)

[Question 1. 17](#__RefHeading___Toc217455199)

[Question 2. 19](#__RefHeading___Toc217455201)

[Question 3.
21](#charge-question-3.-please-comment-on-the-qualitative-discussion-of-acrylamides-heritable-germ-cell-effects-and-whether-the-discussion-is-clear-transparently-and-objectively-described-and-reflective-of-the-current-science.)

[Question 4.
22](#charge-question-4.-please-comment-on-whether-the-selection-of-the-friedman-et-al-1995-and-johnson-et-al-1986-studies-as-co-principal-studies-has-been-scientifically-justified.-although-epa-considers-friedman-et-al-and-johnson-et-al-to-be-co-principal-studies-the-final-quantitative-rfd-value-is-derived-only-from-the-johnson-study.-please-comment-on-this-aspect-of-the-epas-approach.-please-comment-on-whether-this-choice-is-transparently-and-objectively-described-in-the-document.-please-identify-and-provide-the-rationale-for-any-other-studies-that-should-be-selected-as-the-principal-studies.)

[Question 5. 24](#__RefHeading___Toc217455204)

[Question 6. 25](#__RefHeading___Toc217455205)

[Question 7. 27](#__RefHeading___Toc217455206)

[Question 8 28](#charge-question-8)

[Question 9.
34](#charge-question-9.-is-the-young-et-al-model-adequately-discussed-relative-to-structure-parameter-values-and-data-sets-used-in-the-model)

[Question 10.
34](#charge-question-10.-according-to-us-epas-rfc-methodology-1994-the-use-of-pbtk-models-is-assumed-to-account-for-uncertainty-associated-with-the-toxicokinetic-component-of-the-interspecies-uncertainty-factor-across-routes-of-administration.-does-the-use-of-the-pbtk-model-for-acrylamide-objectively-predict-internal-dose-differences-between-the-f344-rat-and-humans-is-the-use-of-the-model-scientifically-justified-and-does-the-use-of-the-pbtk-reduce-the-overall-uncertainty-in-this-estimate-compared-to-the-use-of-the-default-factor-are-there-sufficient-scientific-data-and-support-for-use-of-this-pbtk-model-to-estimate-interspecies-toxicokinetic-differences-and-to-replace-the-default-interspecies-factor-for-toxicokinetic-differences-i.e.-1012-is-the-remaining-uncertainty-factor-for-toxicodynamic-differences-scientifically-justified-appropriate-and-correctly-used)

[Question 11.
35](#charge-question-11.-please-comment-on-whether-the-pbtk-model-is-adequate-for-use-to-conduct-a-route-to-route-extrapolation-for-acrylamide-to-derive-an-rfc-in-the-absence-of-adequate-inhalation-animal-or-human-dose-response-data-to-derive-the-rfc-directly.-was-the-extrapolation-correctly-performed-and-sufficiently-well-documented)

[Question 12. 36](#__RefHeading___Toc217455211)

[Question 13. 36](#__RefHeading___Toc217455212)

[Question 14. 38](#__RefHeading___Toc217455213)

[Question 15. 39](#__RefHeading___Toc217455214)

[Question 16. 40](#__RefHeading___Toc217455215)

[Question 17.
41](#charge-question-17.-do-you-know-of-any-additional-data-or-analyses-that-would-improve-the-quantitative-characterization-of-the-dose-response-for-acrylamide-induced-heritable-germ-cell-effects-would-these-data-also-support-the-quantitative-characterization-of-total-male-mediated-reproduction-risks-to-offspring-i.e.-lethality-heritable-defect-if-data-are-not-available-do-you-have-any-recommendations-for-specific-needed-studies)

[Question 18. 42](#__RefHeading___Toc217455217)

[Question 19. 44](#__RefHeading___Toc217455218)

[Question 20. 46](#__RefHeading___Toc217455219)

[Question 21. 47](#__RefHeading___Toc217455220)

[Question 22. 50](#__RefHeading___Toc217455221)

[Question 23.
50](#charge-question-23.-please-comment-on-whether-auc-for-glycidamide-is-the-best-choice-of-the-dose-metric-in-estimating-human-equivalent-concentration-to-derive-the-oral-slope-factor.)

[Question 24. 51](#__RefHeading___Toc217455223)

[Question 25. 52](#__RefHeading___Toc217455224)

[Question 26. 53](#__RefHeading___Toc217455225)

[ABREVIATIONS 54](#abreviations)

[REFERENCES 55](#references)

[Appendix A Memorandum and Charge Questions
68](#__RefHeading___Toc217455228)

[Appendix B](#appendix-b) [Proposed Modes of Action (MOAs) for
Acrylamide Neurotoxicity
76](#proposed-modes-of-action-moas-for-acrylamide-neurotoxicity)

# 

# 

# 

# 

# 

# EXECUTIVE SUMMARY

This report was prepared by the Science Advisory Board (SAB) Acrylamide
Review Panel (the "Panel") in response to a request by EPA's Office of
Research and Development (ORD) to review the Draft IRIS Toxicological
Review of Acrylamide (hereafter referred to as the draft document). The
Panel deliberated on the charge questions (see Appendix A) during a
March 10-11, 2008 face-to-face meeting and discussed its draft report in
a subsequent conference call on July 16, 2008. The final draft of the
panel's report was reviewed and approved during a meeting of the
chartered SAB on October 28, 2008. There were 26 charge questions that
focused on the selection of the most sensitive non-cancer health
endpoint, the use of a PBTK model for the derivation of a proposed oral
reference dose (RfD), an inhalation reference concentration (RfC) for
non-cancer endpoints, as well as the cancer descriptor, oral slope
factor, and inhalation unit risk for acrylamide. The Panel encourages
the Agency to review relevant data which has been published since their
draft assessment was completed as they revise and finalize the IRIS
document.

This Executive Summary highlights the Panel's major findings and
recommendations as a result of their deliberations. The responses that
follow represent the views of the Panel.

## Selection of Endpoint

In the draft document, EPA identified neurotoxicity as the most
sensitive non-cancer effect from exposure to acrylamide. This endpoint
was based on an extensive database of animal and human studies. Other
endpoints were also considered, such as reproductive toxicity and
heritable germ cell effects. The Panel agreed that based on the existing
toxicity data base for acrylamide, neurotoxicity does appear to be the
most sensitive non-cancer endpoint, and therefore, the most appropriate
for developing the RfD and RfC for non-cancer effects from exposure to
acrylamide.

## Mechanism of Action

The Panel discussed two hypotheses regarding the mechanism of acrylamide
neurotoxicity. The Panel did not attempt to resolve the debate over a
definitive or single MOA for neurotoxicity; however, there was agreement
that the discussion of MOA is important for inclusion in the draft
document. The Panel found the separation of the discussion of MOA(s) for
neurotoxicity in two different sections of the document confusing and
recommended their incorporation into a single section. A more complete
presentation by the Panel of these MOAs has been appended (see Appendix
B) to this report for EPA's consideration as they revise their draft
document.

## Derivation of RfD

EPA's proposed RfD (0.003 mg/kg-day) for acrylamide is based on a
benchmark dose analysis of the dose-response relationship for
neurotoxicity in two chronic drinking water exposure bioassays using
Fischer 344 rats. Uncertainty factors and a PBTK model were used to
extrapolate the animal dose-response to a human equivalent dose-response
in the derivation of the RfD. The Panel afforded considerable discussion
to the question of whether the Friedman *et al.* (1995) and Johnson *et
al.* (1986) studies were the best choices for derivation of the
quantitative RfD (and RfC). The main concerns with these studies are
that they were primarily designed as cancer bioassays and therefore did
not include the most sensitive measures of neurotoxicity. Nevertheless,
the Panel agreed that the selected studies did have some important
strengths, including reasonable statistical power due to the relatively
large number of animals, chronic dosing, and the fact that the NOAELs
for the endpoint in the two studies were similar, implying some
precision in the effect estimate measured. Several Panel members noted
that the lack of sensitive functional/behavioral assessments is a
significant data gap that should be considered in the context of setting
a database uncertainty factor. Use of the benchmark dose methodology in
this assessment was deemed scientifically supported, given the nature
and robustness of the data sets available on the endpoint of interest.
The calculations and choices made were described clearly and at an
appropriate level of detail. The Panel suggested that EPA undergo the
exercise of generating an RfD from the Calleman study for purposes of
comparison with the RfD derived based on the animal data. This
comparison can serve as a type of sensitivity analysis, to help
determine whether the RfD based on the Johnson study appears to be
adequately health-protective despite the insensitive endpoint used in
that study.

## Heritable Germ Mutations

EPA's draft document concluded that data exist that reveal acrylamide's
capacity to induce heritable germ cell effects at doses somewhat above
those at which neurotoxicity has been observed, but that there are as
yet no studies providing an in-depth examination of dose- response or
identification of credible no-effect levels. The Panel supports the
Agency's conclusions that exposure to acrylamide in animals leads to
heritable gene mutations and that these results indicate that it may
also pose a hazard to humans. In addition, the Panel supports the
Agency's conclusions that the available data are not adequate to conduct
a robust assessment of this endpoint at this time. There is still
uncertainty about the mode of action of acrylamide and its metabolite,
glycidamide, in the induction of heritable genetic effects. The
potential for DNA adducts of glycidamide to play a role is an attractive
hypothesis for the mode of action. The Panel found the discussion in the
document on heritable germ cell effects useful and presented in a clear,
transparent manner reflective of the current science. However, the Panel
suggested that, given the serious consequences of heritable germ cell
effects, the considerable deficiencies of the database should be
identified and the significance of this endpoint emphasized.

## Physiologically-Based Toxicokinetic (PBTK) modeling

A physiologically-based toxicokinetic (PBTK) model originally developed
by Kirman *et al.* (2003), and recalibrated by EPA with more recent
kinetic and hemoglobin binding data in rats, mice, and humans, was used
in the derivation of the RfD. The PBTK model was used to extrapolate
from the animal dose-response relationship to derive a human equivalent
concentration. The Panel commends EPA for their efforts to adapt the
PBTK model of Kirman *et al.* (2003) for acrylamide and glycidamide,
recognizing that this was a complex and challenging task. The Panel
believes, though, that the documentation is not adequate to determine
whether the recalibrated Kirman model is appropriate for its intended
use. While the Panel considered that the model structure was reasonable,
the parameter estimates require greater justification. The Panel was
concerned about the ability of the model to adequately simulate the
kinetics of acrylamide and glycidamide. The Panel has proposed several
modifications to the PBTK model for making the estimates of internal
dose in rats needed for both the non-cancer and cancer assessments and
for calculating the Human Equivalent Dose (HED).

## Uncertainty Factors

EPA has proposed to use the default 10X uncertainty factors (UF) to
account for intraspecies (i.e., human) differences. The Panel concurred
with this choice, noting that there were insufficient data on
inter-individual differences, based upon lifestage, gender or genetic
characteristics, to support departing from the default. Consensus was
not achieved on the issue of the inclusion of an UF to account for
deficiencies in the existing database.

EPA has suggested that the acrylamide IRIS document include a Table that
lists points of departure for various endpoints to facilitate a Margin
of Exposure (MOE) evaluation by EPA's Regional or Program offices, or by
other end users of the assessment. The Panel recommends the inclusion of
such a table, to the extent possible, in all IRIS documents, which
provides information that may be used to conduct a variety of analyses.
Uses may include, for example, MOE analyses for specific endpoints of
interest and/or for other than lifetime durations of exposure and for
windows of increased susceptibility early in the life cycle, in addition
to the traditional lifetime focus. Agency risk assessments would benefit
from the inclusion of transparently-developed, peer-reviewed consensus
hazard values.

## Carcinogenicity 

The Panel believes that the rationale and justification for acrylamide
being a "*likely human carcinogen"* has been well described and the
conclusion is scientifically supportable based on the fact that it
produces tumors in rodents of both sexes, that there are multiple tumor
sites, and tumors are induced via multiple routes of exposure.
Acrylamide is also clearly and reproducibly carcinogenic in both rats
and mice. Nonetheless, the draft document can be improved by expanding
the discussion of biological plausibility and coherence beyond DNA
adducts. The weight of evidence supports a mutagenic mode of action for
carcinogenesis, and overall the rationale has been clearly and
objectively presented. During the advisory process, information was
presented by several members of the public for the Panel's
consideration. While the Panel did consider the information regarding
alternative, non-mutagenic MOAs, they did not find it to be compelling.
Significant biological support and data on any putative alternate MOAs
are not sufficient for either explaining cancer findings or quantifying
dose response relationships. More than one MOA may operate for a given
carcinogenic chemical, and the likelihood that more than a single MOA is
operative increases as levels of exposure increase.

EPA used two chronic drinking water exposure bioassays in Fischer 344
rats (Friedman *et al.*, 1995; Johnson *et al.*, 1986) to derive the
oral cancer slope factor, and to identify the tumors of interest for the
MOA discussion. The Panel agrees that the two chronic bioassays in F344
rats are the main studies to consider in dose response analysis, but the
rationale for using only the Friedman *et al.* study for derivation of
the oral cancer slope factor should be improved with the strengths and
limitations of both studies discussed in greater depth The use of the
Weibull-in-time multistage-in-dose analysis is a reasonable and
scientifically justifiable way to take into account the early mortality
in the high dose group in the male study. The decision not to employ
this analysis, in the case of the female because mortality across
treatment and control groups did not differ and the overall survival
appears to be fairly good, is also reasonable.

## Derivation of the RfC and IUR

The draft document used area under the curve (AUC) in the blood for the
putative genotoxic metabolite, glycidamide, as the dose metric for the
PBTK model analysis to derive the human equivalent concentration. The
Panel agreed that the AUC for glycidamide is the best choice for
estimating the human equivalent concentration to derive the oral slope
factor. One consideration in using this as the dose metric, however,
comes from some of the human studies in which variability is not
accounted for adequately. Consideration of additional human data can
provide an improved basis for adjustments for cross-species differences
in pharmacokinetics, as well as human variability in glycidamide
formation from acrylamide.

As with the RfC, EPA concluded that there were insufficient inhalation
data to derive an inhalation unit risk (IUR). The PBTK model was used in
a route-to-route extrapolation of the dose-response relationship from
the oral data, and to estimate the human equivalent concentration for
inhalation exposure to acrylamide. The Panel commended the EPA for using
the PBTK model to fill the gap resulting from the absence of robust
animal toxicology studies investigating neurotoxicity via the inhalation
route that would support the development of an RfC. The Panel agreed
that the absence of evidence for route of entry specific effects would
allow route-to-route extrapolation for deriving an RfC based on using
the PBTK model to calculate the human equivalent concentration (HEC).

*Age-dependent adjustment factors*

The Panel agreed that the recommendation to use the age-dependent
adjustment factors is well justified and transparently and objectively
described. Additionally the Panel believed that the discussion of
uncertainties is adequate, but that human variability could be more
completely addressed. There is no characterization of sensitive
populations, and this should be explored and discussed to a much greater
extent.

*PBTK model and uncertainty*

The Panel commends EPA for using the PBTK model for developing the RfD,
RfC and Cancer Slope Factors for acrylamide. The Panel notes that the
use of internal dose metrics combined with a fairly robust understanding
of the mechanism of action may replace the use of the default
interspecies uncertainty factor for toxicokinetic differences (i.e.,
10^1/2^) , but not the default interspecies uncertainty factor for
toxicodynamics. This uncertainty factor is still needed in deriving the
RfC and RfD. Further the Panel strongly encourages the Agency to move
forward with revising and finalizing their assessment.

*Additional Comments*

The Panel is aware that studies of acrylamide toxicity are ongoing in
the FDA. However, these studies will not be finalized or go through the
peer-review process for some time. Therefore, the Panel believed that
the draft risk assessment document should not be held-up awaiting those
results, but that the Agency should consider relevant data which has
been published since their draft assessment was released in December
2007 as they finalize the IRIS document on Acrylamide.

# 

# 

# 

# INTRODUCTION

This report was prepared by the Science Advisory Board (SAB) Acrylamide
Review Panel (the "Panel") in response to a request by EPA's Office of
Research and Development (ORD) to review the Draft Toxicological Review
of Acrylamide (hereafter referred to as the "draft document"). The IRIS
Toxicological Review of Acrylamide, released in December 2007, is a
compilation and summary of the available information on the potential
for cancer and non-cancer hazardous effects in humans from exposure to
acrylamide.

The SAB was asked to comment on (1) whether the document is logical,
clear and concise, (2) if the discussion is objectively and
transparently represented, and (3) if it presents an accurate synthesis
of the scientific evidence for non-cancer and cancer hazard. The SAB was
also asked to identify any additional relevant studies that should be
included in the evaluation of the non-cancer or cancer health effects of
acrylamide, or in the derivation of toxicity values. In addition, the
SAB was asked to provide advice on 26 specific charge questions related
to the derivation of a proposed oral reference dose (RfD) and an
inhalation reference concentration (RfC) for non-cancer endpoints, as
well as the cancer descriptor, oral slope factor, and inhalation unit
risk for acrylamide.

The Panel deliberated on the charge questions during a March 10-11,
2008, face-to-face meeting and discussed their draft report in a
subsequent conference call on July 16, 2008. The final draft of the
panel's report was reviewed and approved during a meeting of the
chartered SAB on October 28, 2008.

The responses that follow represent the views of the Panel. The charge
to the SAB is available in Appendix A.

# RESPONSES TO THE CHARGE QUESTIONS 

[]{#__RefHeading___Toc217455199 .anchor}**Charge Question 1.** ***Please
comment on the selection of neurotoxicity as the most appropriate choice
for the most sensitive endpoint (in contrast to reproductive toxicity,
heritable germ cell effects, or other endpoint) based upon the available
animal and human data.***

Based on the existing toxicity data base for acrylamide, neurotoxicity
does appear to be the most sensitive endpoint, and therefore, the most
appropriate for developing the (non-cancer) RfD and RfC. Animal studies
report microscopically-detected degeneration in peripheral nerve cells
at doses of 1-2 mg/kg day, as compared to levels of 3-13 mg/kg day to
detect impaired male reproductive performance. Animal studies provide a
clear mechanistic understanding whereby low-dose, subchronic exposure
leads to toxicity with concomitant nerve damage. Acrylamide has a direct
or indirect effect on the motor protein kinesin or nerve terminals,
producing damage in the peripheral and central nervous systems, which
leads to sensory and motor disease. Correspondingly, reports of
central-peripheral neuropathy, ataxia and muscle weakness in exposed
human cohorts have been documented since the early 1950's. Acute
occupational exposure to acrylamide can lead to an immediate neurologic
response, e.g., [sweating](http://en.wikipedia.org/wiki/Sweating),
[nausea](http://en.wikipedia.org/wiki/Nausea),
[myalgia](http://en.wikipedia.org/wiki/Myalgia),
[numbness](http://en.wikipedia.org/wiki/Numbness),
[paresthesia](http://en.wikipedia.org/wiki/Paresthesia), and weakened
legs and hands. Following termination of short term exposure, these
acute effects disappear.

There were issues of concern that should be noted:

1)  As detailed in the response to Question 4, the determination of
    accurate benchmark doses (e.g., LOAELs, NOAELs, RfDs) from the
    Friedman *et al.* (1995) and Johnson *et al.* (1986) studies may be
    compromised by their lack of functional testing of neurotoxicity and
    the use of a relatively insensitive measure, peripheral axonopathy,
    as the primary index neurotoxicity.

2)  There was concern that axonal degeneration observed under light
    microscopy was the endpoint chosen from the Friedman *et al.* (1995)
    and Johnson *et al.* (1986) studies for derivation of the RfD and
    RfC. Animal studies indicate that nerve terminal degeneration can
    occur prior to axonal degeneration at some doses. This would suggest
    that all of the cited studies, including the subchronic Burek study
    and the 2 year bioassay studies of sciatic nerve (Friedman *et
    al.*, 1995) and tibial nerve (Johnson *et al.*, 1986) axons, in
    looking at axonal degeneration, may have missed a preceding terminal
    degeneration at a lower dose, particularly as no specific mention of
    terminal degeneration is provided and functional/behavioral measures
    of neurotoxicity were not included.

3)  It should be noted that future studies may demonstrate effects of
    acrylamide exposure on male reproductive function, as currently
    evidenced in animal studies by increased pre- and post-implantation
    losses and decreased litter sizes, at even lower doses than those
    currently associated with neurotoxicity after acrylamide dosing in
    animal studies. The draft document states that "associations between
    human exposure to acrylamide and reproductive effects have not been
    reported" (p. 187 and p. 224); rather, these associations *have not
    been adequately studied*. The lack of human data is a major
    limitation in this regard. As noted in the draft document, data also
    exists that reveal acrylamide's capacity to induce heritable germ
    cell effects at doses somewhat above those at which neurotoxicity
    has been observed, but there are as yet no studies providing an
    in-depth examination of dose response or identification of credible
    no-effect levels. The heritable germ cell effects are very worrisome
    and deserve even more consideration, including perhaps the use of
    this endpoint to generate an independent RfD.

4)  Although still controversial and recognizing that cigarette smoke is
    a complex mixture made up of hundreds of compounds, there is growing
    evidence that supports an association between cigarette smoking, a
    known source of acrylamide exposure, and altered semen parameters,
    including concentration, morphology, motility, and DNA fragmentation
    (Richthoff *et al.*, 2008; Sepaniak *et al.*, 2006; Marinelli *et
    al.*, 2004). The lack of data regarding potential interactions
    between acrylamide and other exposures, including cigarette smoke,
    alcohol use, and cosmetics (another source of acrylamide exposure)
    has been cited as a major limitation in studies of human acrylamide
    exposure and adverse health effects (Rice 2005; draft document
    p.194; p. 224). The investigation of altered semen parameters among
    occupationally exposed males, controlling for smoking and alcohol
    consumption, should be a high priority.

[New References]{.underline}

Richthoff J, Elzanaty S, Rylander L, Hagmar L, Giwercman A. Association
between tobacco exposure and reproductive parameters in adolescent
males. Int J Androl 2008; 31:31-9.

##  Sepaniak S, Forges T, Foliguet B, Bene MC, Monnier-Barbarino P. The influence of cigarette smoking on human sperm quality and DNA fragmentation. Toxicol 2006; 223:54-60. 

Marinelli D, Gaspari L, Pedotti P, Taioli E. Mini-review of studies on
the effect of smoking and drinking habits on semen parameters. Toxicol
2004; 207:185-92.

[]{#__RefHeading___Toc217455201 .anchor}**Charge Question 2. *Please
comment on the discussion of mode of action for acrylamide-induced
neurotoxicity. ***

The Panel found the separation of the discussion of MOA(s) for
neurotoxicity in two different sections of the document (Section 4.6.1,
pages 123-124; and Section 4.7.3, pages 134-136) confusing and
recommends their incorporation into a single section.

Acrylamide is a member of the type-2 alkene chemical class, which
includes acrolein, methylvinyl ketone and methyl acrylate. A weight of
evidence evaluation of the current body of data now suggests that the
type-2 alkenes produce toxicity via a common molecular mechanism: i.e.,
formation of adducts with essential sulfhydryl thiolate groups on
proteins that play regulatory roles in cellular processes (LoPachin *et
al.*, 2007a,b, 2008a; reviewed in LoPachin and Barber, 2006b; LoPachin
*et al.*, 2008b).

Currently, there are two hypotheses regarding the mechanism of
acrylamide neurotoxicity: 1) Acrylamide/glycidamide inhibits fast axonal
transport by forming adducts with kinesin, the transport motor (reviewed
in Sickles *et al.*, 2002). 2) Acrylamide disrupts nerve nitric oxide
(NO) signaling at the nerve terminal (reviewed in LoPachin *et al.*,
2006a). The Panel did not attempt to resolve the debate over the MOA of
neurotoxicity. It is also possible that both MOAs may be pertinent, and
studies directly comparing the time course of the two proposed MOAs in a
single model have not been carried out. However, the Panel agreed that
the further delineation of MOAs will improve acrylamide risk assessment.
Both of the proposed MOAs suggest that visible axonal degeneration seen
with light microscopy is not likely to be the low-dose effect in the
causal pathway. Regardless, it should also be evident that substantial,
detailed molecular information is available regarding mechanisms of
acrylamide neurotoxicity and that these data should be included.

Thus, the following deficiencies in the draft document were identified
by the Panel:

1)  As drafted, the document's coverage of research findings is
    incomplete and does not adequately reflect the current molecular
    understanding of the mechanisms of acrylamide neurotoxicity.
    Moreover, information in the document regarding the hypothesized
    MOAs is not presented in a sufficiently transparent manner
    consistent with the Agency's guidance on identification of the key
    events leading to the effect of concern, i.e., use of the modified
    Bradford Hill criteria with respect to dose-response concordance,
    temporal relationship(s), strength, consistency, specificity of
    association and biological plausibility and coherence, as is done
    for carcinogenicity.

2)  There was insufficient discussion of acrylamide adduct chemistry and
    corresponding neuronal targets pertinent to understanding the MOAs.

3)  There was lack of a discussion of residual questions surrounding the
    respective roles of the parent toxicant, acrylamide, and its epoxide
    metabolite, glycidamide, in the production of neurotoxicity.

The Panel recommends that the Agency expand its discussion of the two
MOAs. Panel members provided more specific text that describes the two
proposed MOAs, and the Panel offers this text to EPA for consideration
in revising the acrylamide assessment. The text is given in Appendix B
of this report.

## Charge Question 3. Please comment on the qualitative discussion of acrylamide's heritable germ cell effects and whether the discussion is clear, transparently and objectively described, and reflective of the current science.

Discussion in the document of heritable germ cell effects, consisting of
five (5) heritable translocation studies, the two (2) specific locus
studies, two (2) studies on acrylamide transformation to glycidamide and
the importance of this metabolism to toxicity, is relevant and useful,
and is presented in a clear, transparent manner reflective of the
current science. However, the discussion is a linear description of germ
cell toxicity with little synthesis, analysis and scrutiny. While some
SAB members considered the presentation objective, some expressed
concerns over the lack of inclusion of all potential MOAs. Given the
serious consequences of heritable germ cell effects, the considerable
deficiencies of the database should be identified and the significance
of this endpoint emphasized.

The entire section is prefaced and summarized with the perspective that
DNA adduct formation and mutagenicity is the only operative mechanism
for heritable germ cell effects of acrylamide. While adducts can
certainly lead to these observations, there are alternative mechanisms
for discussion. Clastogenic mechanisms, as well as, mitotic spindle
defects are viable candidates for dominant lethal effects. There is a
wealth of acrylamide studies reporting these alternative mechanisms that
should be included in this discussion as well. They were briefly
outlined in the carcinogenicity section, but should also be identified
here. In regards to spindle defects, the effects of acrylamide on
kinesin motors involved in cell division should be added to the document
(Sickles *et al.*, 2007).

Adequate response data are lacking in the existing heritable germ cell
studies such that the shape of the dose response relationship cannot be
ascertained. However, in Tyl *et al.* (2000) dose responses are
identified - a NOAEL of 2 mg/kg/d and a LOAEL of 5 mg/kg/d for a 13 week
exposure. All of the dominant lethal studies were conducted at a dose of
50 mg/kg or higher and most with multiple exposures. The specific locus
studies were conducted at 50 mg/kg/d for 5 days (Russel *et al.*, 1991)
or with a single 100-125 mg/kg exposure (Ehling and Neuhauser-Klaus,
1992). The discrepancy between the negative results of Russel *et al.*
(1991) and the positive results of Ehling and Neuhauser-Klaus (1992) may
be dose-related or due to other factors. The fact that heritable
translocations appeared at high frequency at the lowest doses tested
implies that even lower doses may produce such effects.

However, in the absence of these data, the uncertainty should be
identified. As a consequence of these limitations in the database, there
is some uncertainty related to the RfD. The Panel unanimously agreed
that this is an extremely serious data gap that should be a top priority
for further study. Additional studies to address the aforementioned
database deficiencies in mechanisms and dose-responses would be
desirable.

The document requires correction in that the NTP/CERHR report was
published in February 2005, not 2004. Also, there appears to be a
discrepancy in the text (Pg 117 indicates the historical controls were
6%, yet on pg 116 in the discussion of the Adler *et al.* (1994) study,
the historical controls are listed as 5/9890 which is 0.05%).

## Charge Question 4. Please comment on whether the selection of the Friedman et al, 1995 and Johnson et al, 1986 studies as co-principal studies has been scientifically justified. Although EPA considers Friedman et al and Johnson et al to be co-principal studies, the final quantitative RfD value is derived only from the Johnson study. Please comment on this aspect of the EPA's approach. Please comment on whether this choice is transparently and objectively described in the document. Please identify and provide the rationale for any other studies that should be selected as the principal studies.

The Panel afforded considerable discussion to the question of whether
the Friedman *et al.* (1995) and Johnson *et al.* (1986) studies were
the best choices for derivation of the quantitative RfD (and RfC). The
main concerns with these studies included the fact that they were
primarily designed as cancer bioassays rather than for evaluation of
neurotoxicity. Specifically, the Panel contended that the endpoint of
axonal degeneration visible under light microscopy is an insensitive
measure of neurotoxicity. Alterations visible under electron microscopy
or functional/behavioral alterations would have provided more sensitive
endpoints.

Nevertheless, the Panel agreed that the selected studies did have some
important strengths, including reasonable statistical power due to the
relatively large number of animals, chronic dosing, and the fact that
the NOAELs for the endpoint in the two studies were similar, implying
some precision in the effect estimate measured. The Panel also noted
that there are no studies yet available which include the sensitive
functional/behavioral assessments that would be most desirable. Several
Panel members noted that this issue is a significant data gap that
should be considered in the context of setting a database uncertainty
factor.

With respect to the Burek *et al.* (1980) study, the Panel notes that
while the endpoint in this study (axolemmal invaginations under electron
microscopy) is a highly sensitive one for use in risk assessment, the
study was subchronic. One Panel member proposed that EPA consider
generating an RfD based on the data in Burek *et al.* (1980), but not
use a subchronic-to-chronic uncertainty factor given the existence of
the two chronic studies, to compare the resulting RfD to that based on
the less sensitive endpoint of axonal degeneration. Such a comparison
might begin to quantify the degree of potential under-estimate of risk
due to the less satisfactory choice of endpoint in the Johnson and
Friedman studies.

There was a brief discussion of the report of foot splay at 0.5 mg/kg in
F~0~ males in the Tyl *et al.* (2000a) two-generation reproductive
toxicity/dominant lethal mutation study. The use of this gross
functional endpoint could also serve as a point of departure, although
it was considered questionable because: it was only observed in the F~0~
generation, was found in control animals to some degree (raising
questions about the methodology used in the lab), and did not follow a
clear dose-response relationship. Overall, the Panel decided that the
Tyl study was not a good choice for derivation of the RfD.

The Panel also considered the option of deriving an RfD based on human
data. Both the Calleman *et al.* (1994) and the Hagmar *et al.* (2001)
studies contain sufficient data to allow the Agency to calculate an RfC
or potentially an RfD. In this regard, the Panel made the following
observations: (1) in general, it is preferable to use human data when
available; (2) the Calleman study included a measure of internal dose
(adduct levels) and a fairly sensitive measure of effect, thereby making
it appealing for risk assessment; (3) PBTK modeling could allow dose
extrapolation based on adduct levels, such that an ingested or inhaled
dose could be estimated for purposes of setting either an RfC or an RfD
from the data.

However, the Panel also cautioned that there are a number of drawbacks
to using the human studies, including the following: (1) the sample
sizes are small; (2) the samples mostly include young adult males; (3)
the healthy worker effect would tend to bias these studies (especially
the Calleman study) toward the null, since workers with significant
neurological symptoms would leave the workplace, thus selecting for
individuals with lower genetic susceptibilities; (4) the workers in each
study were exposed to other confounding neurotoxicants (acrylonitrile
and *N*-methylolacrylamide (NMA)), but this would tend to generate a
more conservative risk estimate because these other exposures would tend
to result in an over-estimate of the effect; and (5) the exposure
duration was relatively short and variable (1 month to 11.5 years in the
Calleman study with an average of 3 years, and 55 days in the Hagmar
study). In the end, the Panel suggested that EPA undergo the exercise of
generating an RfD from the Calleman study for purposes of comparison
with the RfD derived based on the animal data. The Panel stopped short
of recommending that the human RfD be used in place of the one in the
draft document, but instead saw this as a type of sensitivity analysis,
to help determine whether the RfD based on the Johnson study appears to
be adequately health-protective despite the insensitive endpoint used in
that study.

[]{#__RefHeading___Toc217455204 .anchor}**Charge Question 5. *Please
comment on the benchmark dose methods and the choice of response level
used in the derivation of the RfD, and whether this approach is
accurately and clearly presented. Do these choices represent the most
scientifically justifiable approach for modeling the slope of the
dose-response for neurotoxicity? Are there other response levels or
methodologies that EPA should consider? Please provide a rationale for
alternative approaches that should be considered or preferred to the
approach presented in the document. ***

Use of the benchmark dose methodology has become the preferred approach
and an acknowledged improvement over the historically traditional NOAEL
÷ UF procedure for the derivation of RfDs. Its application in this
instance is scientifically supported, given the nature and robustness of
the data sets available for the endpoint of interest. The calculations
and choices made were described clearly at an appropriate level of
detail.

EPA's Benchmark Dose guidance provides default criteria to be used for
selecting the benchmark response (BMR). For quantal data, an excess risk
of 10% is the default BMR, since the 10% response is at or near the
limit of sensitivity in most studies. In this case, even though the BMR
at 10% extra risk also was within the range of observation, the BMR~5~
was selected for the point of departure. The choice of a BMR~5~ makes
sense and is well-justified: (1) the 95% lower bound of the benchmark
dose (BMD), BMDL~5~, remained near the range of observation; (2) the 5%
extra risk level is supportable given the relatively large number of
animals used in the critical studies; and (3) the use of BMDL~5~ is
consistent with the Agency's technical guidance for BMD analysis which
allows flexibility in making such a choice. One of the strengths of the
Johnson study is that it is sufficiently large (i.e., numbers of
animals/group) to allow the lower 5% bound to be identified with
sufficient stability that it is usable for risk assessment purposes.
Therefore, it is reasonable to use that strength in the underlying data
set and choose this number. Such a choice is appropriately conservative
(i.e., public health protective).

While alternative approaches such as averaging the BMDLs from each of
the four data sets (Friedman and Johnson, male and female) rather than
using just the one for males in the Johnson study were discussed, the
Panel concluded that the steps described by the Agency in the draft
document represented the preferred approach.

[]{#__RefHeading___Toc217455205 .anchor}**Charge Question 6. *Please
comment on the selection of the uncertainty factors (other than the
interspecies uncertainty factor) applied to the point of departure (POD)
for the derivation of the RfD. For instance, are they scientifically
justified and transparently and objectively described in the document?
\[Note: This question does not apply to the interspecies uncertainty
factor which is addressed in the questions on the use of the PBTK model
(see PBTK model questions below)\] ***

The Agency has proposed to use a composite uncertainty factor (UF) of
30: 10X to represent human variability (10~H~) and 3X to reflect the
toxicodynamic component of the default interspecies uncertainty factor
(10~A~). The other half of the 10x interspecies UF, i.e., the 3X that
would otherwise account for interspecies differences in toxicokinetics,
is subsumed in the PBTK modeling.

Two points were raised about the use of 3X as a default to account for
interspecies toxicodynamic differences. First, it was noted that the
rodents are less sensitive to the neurotoxic effects of acrylamide than
humans. The Panel concluded that the application of a UF for
interspecies toxicodynamics was directionally correct. Second, there is
insufficient information available to define a chemical-specific factor
and the default factor of 3X UF for interspecies in toxicodynamics is
therefore appropriate. It was noted that recent International Programme
for Chemical Safety guidelines divide the default 10~A~ into 2.5X for
toxicodynamic differences and 4.0X for toxicokinetics differences, based
primarily upon a review of the literature published in 1993 -(WHO IPCS
2005. *Guidance Document for the Use of Data in Development of
Chemical-specific Adjustment Factors (CSAFs) for Interspecies
Differences and Human Variability in Dose/Concentration-Response
Assessment*). The use of the factor of 3 (or √10) is consistent with
current EPA practice: according to the recent EPA (2004) Staff Paper "a
default UF of 10 for interspecies variability that can now be reduced to
3 when animal data are dosimetrically adjusted to account for
toxicokinetics." The Staff paper cites the EPA (2002) RfD/RfC
methodology document. That document divides UFs "into toxicokinetic and
toxicodynamic components that have assigned default values of 3.16
(10^1/2^) each."

EPA has proposed to use the default 10X UF to account for intraspecies
(i.e., human) differences. The Panel concurred with this choice, noting
that there were insufficient data on interindividual differences, based
upon lifestage, gender or genetic characteristics, to support departing
from the default.

Consensus was not achieved on the issue of the inclusion on an UF to
account for deficiencies in the existing database that would confound
the derivation of the most scientifically-defensible RfD. EPA concluded
that an UF~D~ \> 1 was not necessary, arguing that the existing database
is sufficiently robust, even though they acknowledge there are some
unresolved issues that warrant further research: describing the MOA(s)
for neurotoxicity, the potential for behavioral or functional adverse
effects not detected in the assays to date, and the uncertainty that
heritable germ cell effects may occur at lower than previously reported
doses. Some Panel members agreed with EPA's position. One Panel member
noted that additional UFs were implicitly, if not explicitly,
incorporated into the RfD derivation. Using the output of the
log-logistic model applied to the data set for the male rats in the
Johnson study resulted in the lowest set of BMDs/BMDLs. According to one
Panel member, it was perhaps conferring an extra UF of \~2X. In
addition, using the BMDL~5~ as the POD, rather than the default
BMDL~10~, also could be seen as conferring an extra UF of \~2X.

Other Panel members, however, disagreed with the Agency's position
regarding the database UF, arguing that the remaining uncertainties have
major implications that could result in effects at significantly lower
doses and thus a lower RfD. Database deficiencies include the following:

1)  EPA had to rely on the observation of axonal degeneration visible by
    light microscopy, an endpoint which is not likely to be the most
    sensitive. EPA is using studies that were not designed to evaluate
    neurotoxicity robustly, e.g., histopathology coupled with systematic
    evaluation of functional or behavioral parameters at multiple time
    points with robust numbers of animals/treatment and robust number of
    treatment groups; these studies should be done in adult animals and
    in a developmental neurotoxicity study in order to determine whether
    or not critical lifestage differences exist;

2)  Both existing chronic studies were done in the rat, creating some
    remaining uncertainty about interspecies differences that is not
    addressed by the interspecies UF. Based upon the comparison of
    results from the Tyl *et al.* (2000) 2-generation study in rats and
    the Chapin *et al.* (1995) 2-generation study in mice, the NOAEL for
    (adult) neurotoxicity is essentially the same (0.5 mg/kg/day in rats
    vs. 0.8 mg/kg/day in mice), but the difference could potentially be
    driven by the dose spacing regimen rather than a true difference in
    response. The outcomes of long-term exposure in mice hold the
    possibility of yielding lower NOAELs/LOAELs/BMDs than
    observed/calculated from the rat data. If this were to occur, the
    RfD/RfC would be lower.

3)  The germ cell effects have not been fully explored and have major
    intergenerational implications if they do occur at dose levels lower
    than those for neurotoxicity. There is a lack of adequate data to
    define the dose response for heritable germ cell effects. While the
    existing data describe adverse effects at doses somewhat higher than
    those at which neurotoxicity was observed, BMD modeling of robust
    dose-response data may yield results competitive with/lower than the
    neurotoxicity BMDs/BMDLs.

[]{#__RefHeading___Toc217455206 .anchor}**Charge Question 7. *Please
provide any other comments on the derivation of the RfD and on the
discussion of uncertainties in the RfD. ***

**Acrylamide and Cumulative Risk Assessment**

While there were no additional comments on the derivation of the RfD
*per se*, the Panel did want to address the potential for cumulative
effects from exposure to acrylamide and other type-2 alkenes. The Food
Quality Protection Act (FQPA) of 1996 mandates EPA to consider the
"cumulative effects" of pesticides and other substances that have a
"common mechanism of toxicity" when setting, modifying or revoking
tolerances for food-use pesticides. Were acrylamide registered as a food
use pesticide, its activity as a type-2 alkene would support a
cumulative risk assessment of it and other chemicals in the class. From
a scientific standpoint and particularly from a public health
perspective, this class of chemicals should be subjected to a cumulative
risk assessment (e.g., see Wilkinson *et al.*, 2000). Evaluating the
cumulative effects of the type-2 alkenes is particularly germane since
human exposure is pervasive; i.e. chemicals in this class are used
extensively in the agricultural, chemical and manufacturing industries.
Furthermore, they are well-recognized environmental pollutants (e.g.,
acrolein, acrylonitrile), food contaminants (e.g., acrylamide, methyl
acrylate) and endogenous mediators of cellular damage (e.g., acrolein,
4-hydroxy-2-nonenal) (see LoPachin *et al.*, 2008b). Thus, the
application of standard approaches may result in RfDs and RfCs which
could be associated with risks in the population. At a minimum, a caveat
in this regard should be included in the acrylamide assessment document.

## Charge Question 8

***Use of the PBTK Model***

***A physiologically-based toxicokinetic (PBTK) model originally
developed by Kirman et al. (2003), and recalibrated by EPA with more
recent kinetic and hemoglobin binding data in rats, mice, and humans
(Boettcher et al., 2005; Doerge et al., 2005a,b; Fennell et al., 2005)
was used in the derivation of the RfD to extrapolate from the animal
dose-response relationship (observed in the co-principal oral exposure
studies for neurotoxicity) to derive a human equivalent concentration
(HEC). The HEC is the external acrylamide exposure level that would
produce the same internal level of parent acrylamide (in this case the
area under the curve \[AUC\] of acrylamide in the blood) that was
estimated to occur in the rat following an external exposure to
acrylamide at the level of the proposed point of departure, and related
to a response level of 5% (i.e., the BMDL~5~). The model results were
used in lieu of the default interspecies uncertainty factor for
toxicokinetics differences of 10^1/2^, which left a factor of 10^1/2^
(which is rounded to 3) for interspecies differences in
toxicodynamics.***

***With respect to the RfC, there are presently insufficient human or
animal data to directly derive an RfC for acrylamide. The PBTK model was
thus used to conduct a route-to-route extrapolation (oral-to-inhalation)
to derive an RfC based on the dose-response relationship observed in the
co-principal oral exposure studies for neurotoxicity. In this case, the
HEC was based on a continuous inhalation exposure to acrylamide in the
air that would yield the same AUC for the parent acrylamide in the blood
as that estimated for the rat following an external oral exposure to
acrylamide at the level of the proposed point of departure (i.e., the
BMDL~5~).***

***Please comment on whether the documentation for the recalibrated
Kirman et al. (2003) PBTK model development, evaluation, and use in the
assessment is sufficient to determine if the model was adequately
developed and adequate for its intended use in the assessment. Please
comment on the use of the PBTK model in the assessment, e.g., are the
model structure and parameter estimates scientifically supportable?***
***Is the dose metric of area-under-the-curve (AUC) for acrylamide in
the blood the best choice based upon what is known about the mode of
action for neurotoxicity and the available kinetic data? Please provide
a rationale for alternative approaches that should be considered or
preferred to the approach presented in the document.***

The Panel commends EPA for their efforts to adapt the PBTK model of
Kirman *et al.* (2003) for acrylamide and glycidamide, recognizing that
this was a complex and challenging task. The modified Kirman *et al.*
model was produced by changing the model initially described for the
rat, and adapting it to fit updated data published since the original
publication in 2003, and to describe toxicokinetics in humans. Three
major modifications were described - the partition coefficients for
glycidamide, the metabolic rate constants for oxidation and conjugation,
and the partition coefficients for acrylamide. The simulations of the
modified Kirman model were presented as tables containing comparisons of
AUC data, and the extent of metabolism of acrylamide to glycidamide, and
the extent of conjugation of each with glutathione. However, the Panel
had a number of concerns about the description of the model and its
parameterization. The Panel believed that the documentation is not
adequate to determine whether the recalibrated Kirman model is
appropriate for its intended use. Among the items that the Panel would
like to see to justify the performance of the model are: the model code;
graphical presentation of the data for time course simulations; and
graphical presentation of dose response simulated by the model. Side by
side comparisons of the model parameters for the rat and human could be
accomplished by combining Tables E-4 and E-6.

The Panel noted that the model with some changes has been described in a
manuscript published in 2007 by Walker *et al.* If life stage
considerations are planned for subsequent work, PBTK modeling is the
recommended tool for dosimetry estimates across life stages. The Panel
would like to see the model used to simulate or show the degree of
consistency with data published since 2005.

The Panel also noted that there have been additional studies of
acrylamide, its metabolites and adducts, with varying data quality, and
varying understanding of exposures. For example, exposures in smokers
are likely a composite of exposure from diet (oral) and smoke
(inhalation). There are possible ambiguities in assignment of acrylamide
and glycidamide metabolites (the acrylamide mercapturic acid sulfoxide
and the glycidamide mercapturic acids are isomeric, and need to be
resolved chromatographically for appropriate quantitation). The Panel
suggests that EPA review these reports for data quality and suitability,
and if appropriate use them in evaluation/refinement of the model.

The Panel noted discrepancies between the PBTK predicted and measured
critical dose metrics for the non-cancer (acrylamide AUC) or cancer
(glycidamide AUC) PODs following drinking water exposures in rats (see
table below).

  -------- ------------- ----------- -------------------- ------------------
                                     EPA PBTK Model       Tareke/Doerge
                                     Predictions          Measured Data
                                                          (2005, 2006)

  EGV      BMDL          Critical    Internal dose        Internal dose
           (mg/kg/day)   Dose Metric (uM-hr)              (uM-hr)

  RfD      0.27          AA_AUC      18.1                 4.2

  oral     0.3           GA_AUC      15.1                 4.7
  cancer                                                  
  -------- ------------- ----------- -------------------- ------------------

The draft document notes that the data of Doerge *et al.* (2005 a,b)
were available (page E-5), but it is not clear if the data were actually
considered in updating the model.

While the Panel concluded that the model structure was reasonable, the
parameter estimates require greater justification. The review notes
(Page E-18 last paragraph) that: "In comparing different versions of the
model, it was also noted that the model parameters were underdetermined,
that is, there is just not enough basic toxicokinetic data to derive a
unique set of optimal parameter values, given the number of "adjustable"
parameters in the current model."

The Panel was concerned about the ability of the model to adequately
simulate the kinetics of acrylamide and glycidamide. There is little
justification presented for the adjustment of parameters from the
original Kirman model. The method of optimization was not well
described. The comparisons provided between observed data and model
simulations are largely for AUC in tables. Thus it is difficult to
determine how the model would perform under the kind of tests usually
applied to a model, including the ability to fit kinetic data. Table E-4
indicates that while AUC for acrylamide and glycidamide can be simulated
reasonably well with the revised rat model, and AM-GSH is reasonably
close, the extent of metabolism to GA-GSH is overestimated by 3 fold by
the model. Approximately 40% of the urinary metabolites were reported as
GA-GSH (Fennell *et al.*, 2005), but the model simulates that 70% would
be derived from GA-GSH.

Table E-9 indicates that almost 50% of acrylamide is converted to
glycidamide in humans. The data reported in Fennell *et al.* (2005)
indicate approximately 13.5 % of the urinary metabolites were derived
from glycidamide. Some recent studies indicate a higher degree of
glycidamide formation from acrylamide, and substantial variation among
individuals in this formation (Vesper *et al.* 2008; Hartmann *et al.*
2008). The model simulations are based on the assumption that all of the
acrylamide not accounted for by excretion in urine by 24 hours is
converted to glycidamide. As noted above, there are data not modeled
that could greatly improve the model parameter estimates, using human
urine kinetic data for acrylamide, glycidamide and urinary metabolites
(e.g., Fennell *et al.* 2006; Hartmann *et al.* 2008;Vesper *et al.*
2006, 2008). Table E-7 cites the Ratio of GA-GSH to AA-GSH metabolite
excretion at low doses reported by Boettcher *et al.* (2005) as 0.206 as
a data point used for calibration. Yet the model simulation reports a
value of 0.733 (Table E-9). The half-life estimated for acrylamide in
the model is approximately 5.8 hours and the half-life estimated for
glycidamide is approximately 6.1 hours. The half life calculated from
urinary excretion rate for acrylamide in humans by Fennell *et al.*
(2006), who studied small groups of healthy infertile adult men, was
approximately half this, ranging from 3.13-3.49 hours. The issue of
adjusting the parameters for partition coefficients and the rates of
glutathione conjugation and oxidation is a serious one. It is possible
to simulate the same AUC in blood with different model parameters, but
with wildly different extents of metabolism and dose to the tissues for
acrylamide or glycidamide, by adjusting partition coefficients, and
metabolic rate constants. In other words, there may not be unique
solutions unless the full body of reported data can be used in model
verification. It is exceedingly important to carefully consider the
extent of metabolism as a key piece of information in making parameter
selections.

The description of the parameters and calibration for the human Kirman
model are generally presented clearly on pages E-17 and E-18. A possible
exception is the very general description of the "iterative process"
that was used to evaluate physiologically feasible options to best fit
the Fennell *et al.* (2005b) and Boettcher (2005) human data on adult
adduct levels and urinary metabolites. A rough comparison of the final
rat and human values suggests increased values for a number of tissue
binding and metabolic parameters in the human model. Many of these
parameters that changed from rat to human increased roughly by a factor
of 2 with the exception of the Cytochrome P-450 oxidation rate that
decreased by a factor of almost 2.1. It is not clear from the
description of the iterative process used to calibrate these values
whether the process was designed to force these parameters to move as
groups or exactly what logic was employed to adjust these multiple
parameters. The general logic behind the iterative testing of
permutations of values could be clarified here without going into
extreme detail.

An alternative approach that should be considered is a re-evaluation of
the revised PBTK model of Kirman *et al.* (2003). Determining how well
it simulates the more recent data and adjusting the metabolic parameters
as necessary is one approach. The Panel had an extensive discussion as
to whether the dose metric of area-under-the-curve (AUC) for acrylamide
in the blood was the best choice based upon what is known about the mode
of action for neurotoxicity and the available kinetic data. A variety of
opinions were expressed, ranging from the assertion that AUC for
acrylamide in blood was a suitable dose metric, to the belief that it
may not the best choice, but may be expedient. The best choice would be
to have compartments for the tissues of interest, and to model the
amount of acrylamide and/or glycidamide reaching the tissues. The Kirman
model and the modified Kirman model are both limited by the tissue
descriptions: liver, lung, blood and a single compartment for remaining
tissues.

There was extensive discussion among the Panel members about whether the
neurotoxicity of acrylamide could clearly be attributed to acrylamide
alone, to glycidamide, or to a mixed mode of action. This question was
raised in the review document (Page 136, last full paragraph). Therefore
the choice of acrylamide in blood as the dose metric may need to be
revisited as this question is clarified.

Several alternatives to the PBTK model exist for making the estimates of
internal dose in rats needed for both the non-cancer and cancer
assessments and for calculating the Human Equivalent Dose (HED). The
data available in Doerge *et al.* (2005) and Tareke *et al.* (2006)
provide measured serum acrylamide and glycidamide AUCs in rats exposed
at drinking water concentrations and resulting doses near the PODs.
Simple linear extrapolation could be used to calculate the critical
internal dose metrics. The hemoglobin adduct and other data available in
several recent publications (Fennell *et al.* 2005; Vesper *et al.*
2006, 2008; Hartmann *et al.* 2008) together provide a robust means of
estimating HEDs. The Panel also discussed the alternative approach of
using pharmacokinetic principles to interpret measurements of hemoglobin
adducts of acrylamide and glycidamide and thereby model glycidamide
formation.

The Panel also raised concerns about the population variability in the
metabolism and toxicokinetics of acrylamide, and how that could be
incorporated in the model. It was recognized that there are some high
quality human data sets that could be used for PBTK model development
(e.g. Fennell *et al.*, 2005, 2006). However, there are limitations with
the small number of selected subjects compared with the general
population, in describing the population variation. The Panel has
identified some studies that suggest variation in the extent of
metabolism of acrylamide to glycidamide (Vesper *et al.* 2006, 2008;
Hartmann *et al.* 2008), and differences in extent of conversion of
acrylamide to glycidamide in children (Heudorf *et al.*, 2008). There is
a need for a better understanding of exposure route differences,
inter-individual variation and life stage differences in the metabolism
of acrylamide to glycidamide, and their clearance. The Panel encourages
an evaluation of the available literature, and if possible, simulation
of human variability within the PBTK model.

## Charge Question 9. Is the Young et al model adequately discussed relative to structure, parameter values and data sets used in the model?

The Young *et al.* paper does not provide citations or values for many
of its physiological model parameters. This is an unusual situation for
a PBTK modeling paper. For chemical specific model parameter values, the
authors fitted the chemical specific model parameter values for each
administered dose, creating a model that is calibrated for each dose.
This results in an unwieldy model for use in risk assessment. The
preferred approach is to use all the administered dose groups and create
a model with one set of chemical specific model parameters that
describes all the toxicokinetic data sets. The model was based on the
use of linear terms to describe chemical specific reactions (e.g.,
binding, DNA adducts, and metabolism). This approach may not hold (and
non-linear terms will be needed) when developing one set of chemical
specific model parameters to describe the kinetics over a range of
doses.

***Do you agree with the conclusion that the recalibrated Kirman et al.
2003 model is the best for deriving toxicity values?***

In the opinion of the Panel, the recalibrated Kirman model was superior
to the Young *et al.* PBTK model. However, the Panel noted that the
recalibrated model requires updating to include new data sets in the rat
and human. The concerns described in Charge Question 8 need to be
addressed to use the recalibrated Kirman *et al.* 2003 model. The Panel
also noted that an approach to calculating internal doses at the
non-cancer and cancer PODs is available that relies on measured data
(and minimal linear extrapolation in a dose range that has been shown to
be linear) instead of the PBTK model. This approach also affords the
ability to calculate the HED corresponding with the critical internal
dose metrics associated with the PODs (see response to question 8). If
life stages are considered, the PBTK modeling or another pharmacokinetic
approach is the preferred approach for determining a HED or HEC.

## Charge Question 10. According to US EPA's RfC Methodology (1994), the use of PBTK models is assumed to account for uncertainty associated with the toxicokinetic component of the interspecies uncertainty factor across routes of administration. Does the use of the PBTK model for acrylamide objectively predict internal dose differences between the F344 rat and humans, is the use of the model scientifically justified, and does the use of the PBTK reduce the overall uncertainty in this estimate compared to the use of the default factor? Are there sufficient scientific data and support for use of this PBTK model to estimate interspecies toxicokinetic differences and to replace the default interspecies factor for toxicokinetic differences (i.e., 101/2)? Is the remaining uncertainty factor for toxicodynamic differences scientifically justified, appropriate and correctly used?

The Panel commends EPA for using the PBTK model for developing the RfD,
RfC and Cancer Slope Factors for acrylamide. The kinetics of acrylamide
are well characterized and thus the use of internal dose metrics that
are thought to represent the critical dose metrics for non-cancer
(neurotoxicity) and cancer (various tumor types) is a preferred approach
for extrapolating across species. The Panel agrees that the use of
internal dose metrics (calculated using the PBTK model or other
pharmacokinetic approaches alluded to above) combined with a fairly
robust understanding of the mechanism of action and thus the critical
dose metric replaces the use of the default interspecies factor for
toxicokinetic differences (i.e., 10^1/2^).

The Panel agreed with the use of the remaining UFs representing
interspecies differences in toxicodynamics and intraspecies variability
in both toxicokinetics and toxicodynamics.

## Charge Question 11. Please comment on whether the PBTK model is adequate for use to conduct a route-to-route extrapolation for acrylamide to derive an RfC in the absence of adequate inhalation animal or human dose-response data to derive the RfC directly. Was the extrapolation correctly performed and sufficiently well documented?

The Panel discussed the lack of inhalation toxicology and PK studies.
One Panel member who has conducted inhalation PK exposure studies noted
the difficulty with conducting controlled rodent exposure studies and
the difficulty in maintaining stable exposure concentrations because of
the low volatility of acrylamide and its propensity to sublime. The
Panel agreed with the use of PBTK modeling to conduct dose-route
extrapolation. Additionally, the Panel commends the EPA for using the
PBTK model to fill the gap resulting from the absence of robust animal
toxicology studies investigating neurotoxicity via the inhalation route
that would support the development of an RfC. The Panel agreed that the
absence of evidence for route of entry specific effects would allow
route-to-route extrapolation for deriving an RfC by using the PBTK model
to calculate the human equivalent concentration (HEC). This would yield
an equivalent internal dose (Acrylamide AUC) associated with those
achieved at the POD from the oral sentinel (Johnson *et al.*) studies.
The Panel noted that few inhalation PK studies exist to allow a robust
parameterization of the inhalation component of the PBTK model for
either rats or humans. Despite this, the Panel noted that acrylamide is
very water soluble and non-volatile, and the compound has a relatively
long half-life. Therefore, the absorption of acrylamide via inhalation
should be nearly complete, and first pass effects are negligible,
thereby making the pharmacokinetics of acrylamide via inhalation easy to
extrapolate from the oral case, using simple principles of
pharmacokinetics. The Panel agreed that the application of
pharmacokinetic approaches (e.g,. the use of the PBTK model) reduces
uncertainty associated with animal to human extrapolation and thus
warrants replacing the default UF associated with interspecies
extrapolation for pharmacokinetic differences as was done for deriving
the RfD.

The Panel noted that the air concentration one would derive using the
default approach (multiply the HED by body weight \[70 kg\] and dividing
by daily inhalation rate \[20 m^3^/day\] yielding 0.266 µg/m^3^) is very
similar to the HEC derived using the PBTK model (0.25 µg/m^3^).
Therefore, if the EPA also decides to provide an extrapolation based on
measured data (as described in the response to charge question 8), the
default approach of extrapolating from an absorbed oral dose to an
equivalent intake from the inhalation route (multiplying by 70 kg and
dividing by 20 m^3^/day) can be used with confidence to calculate the
RfC.

[]{#__RefHeading___Toc217455211 .anchor}**Charge Question 12. *Please
provide any other comments on the derivation of the RfC and on the
discussion of uncertainties in the RfC.***

The Panel has no further comments beyond those already discussed above.

[]{#__RefHeading___Toc217455212 .anchor}**Charge Question 13. *Would you
suggest that EPA include a Table that lists points of departure (e.g.,
NOAELs, BMDs, etc.) for various endpoints that could be used, in
conjunction with exposure assessments, to conduct a MOE analysis? ***

To the extent permitted by the available data, the Panel supports the
concept of the inclusion of a table in the IRIS acrylamide document that
provides information which could be used to conduct a variety of MOE
analyses for specific endpoints of interest and/or for other than
lifetime durations of exposure, in addition to the traditional lifetime
focus. In doing so the magnitude of the MOE that represents a negligible
risk should be reported for each point of departure tabulated.

Currently, for those environmental agents for which sufficient data
exist, IRIS documents will present the derivation of a Reference Dose
(RfD) and a Reference Concentration (RfC), as traditionally defined, to
be used in the assessment of scenarios which assume that long-term or
lifetime exposures are occurring to non-carcinogenic hazards.
Additionally, in those cases where the agent of interest has been shown
to have carcinogenic potential, an oral cancer slope factor (CSF) and/or
an inhalation unit risk (IUR) may be derived, in order to estimate
lifetime cancer risks. Whether or not this step is included is
determined by a weight-of-evidence evaluation of the body of evidence
supporting carcinogenic potential and an understanding, or lack thereof,
of the mode(s) of action by which the carcinogenic responses are
mediated. These four values (the RfD, RfC, CSF and IUR) are applicable
in situations where the assessment is focused on the general population
exposed over a lifetime, and may have more limited utility in the
assessment of specific subpopulations and/or less-than-lifetime exposure
durations.

EPA Program and Regional offices and other end-users of IRIS documents
often must develop risk assessments for specific populations and/or
less-than-lifetime exposure scenarios in order to carry out their
respective legislative and regulatory mandates. These risk assessments
would benefit from the inclusion of transparently-developed,
peer-reviewed consensus hazard values.

A comprehensive table would, for example, include NOAELs, LOAELs, BMDs
and BMDLs at the 1%, 5% and 10% risk levels (as the default) for those
studies deemed the most appropriate for the assessment of specific
endpoints and for acute, intermediate and long-term exposure scenarios,
data permitting. It is recognized that it will typically not be possible
to fill in every cell for every endpoint and all exposure durations of
interest and that a different BMD~R~/BMDL~R~ may better reflect the
study's results. Some EPA program offices have extensive experience in
the selection of study types and durations that best lend themselves to
the assessment of specific endpoints, exposure durations and
subpopulations.

For this draft acrylamide assessment, such a table would display the
relevant outcomes of a review of the reliable and well-performed studies
which evaluated the potential for neurotoxicity in the adult and
developing organism, reproductive toxicity including heritable germ
effects, developmental toxicity, and general systemic toxicity following
acute, intermediate and long-term exposure, as appropriate.

[]{#__RefHeading___Toc217455213 .anchor}**Charge Question 14. *Please
comment on the discussion of methods to quantitate the dose-response for
heritable germ cell effects as to whether it is appropriate, clear and
objective, and reflective of the current science. Has the uncertainty in
the quantitative characterization of the heritable germ cell effects
been accurately and objectively described? ***

*\[It should be noted that the section under review is 5.5 rather than
5.4. In addition, page 215 which includes figures 5-2 and 5-2a, was
inadvertently omitted in the draft EPA report and thus not available for
review by the Panel. Correction of this error, however, is not expected
to impact the recommendations of the Panel on this question as outlined
below.\]*

Although reservations were expressed about the lack of data to quantify
dose-response, it was the consensus of the Panel that the discussion of
the methods should be retained in the report. The report adequately
characterizes the current science, reflects historical attempts to
estimate these risks and notes that the quantitation methods are based
only on the Dearfield *et al.* (1995) publication. Concerns about the
validity of the data and methods are given throughout the section and it
is appropriately noted on page 217, " these uncertainties in the
assumptions and data gaps warrant further research to improve the
usefulness of the following quantitative estimates of risk of
acrylamide-induced heritable effects."

Some specific observations/recommendations/concerns are outlined below:

-   The parallelogram models were clearly described and the rationale
    for the decision to use the modified direct and doubling dose
    approach appears appropriate.

-   Clearly, there is considerable uncertainty regarding the validity of
    the underlying assumptions for these methods, and these methods may
    underestimate risk since they do not take into account all elements
    that may contribute to the risk.

-   The extrapolation of exposure is based on animal studies using high
    dosages (50 to 100 mg/kg or even higher)

-   The risk extrapolation factors (REFs; pg. 217) should be explained
    in more detail and information included on how each number is
    derived (range, etc).

-   In agreement with the report, given the differences in glycidamide
    production in different species, an REF of 1 for the metabolic and
    dose rate variability is likely incorrect. There appear to be
    significant dose-rate and species-dependent variations in acrylamide
    metabolism to glycidamide (e.g., see Barber *et al.*, 2001; Fennell
    and Friedman, 2005).

-   An REF for uncertainty in the mode of action was recommended since
    the doubling dose is dramatically higher when generated using
    specific locus studies which are clearly point mutations (53.1 mg/kg
    using Ehling and Neuhauser-Klaus, 1992) versus using heritable
    translocation data that could be based on clastogenic mechanisms
    (1.8, 3.3, 0.39 mg/kg for Shelby *et al.*, 1987, Adler *et al.*,
    1994 and Adler, 1990).

-   The implementation of the modified direct approach was difficult to
    understand when, in the absence of the number of human loci capable
    of mutating to dominantly expressed disease alleles, it was assumed
    to be 1000. Clarification of how this number was derived would be
    helpful (i.e. how do we know the number of mutable genes?).

-   In the doubling dose approach it was not clear how the four data
    sets, each of which used high acrylamide dosing rates without
    significant dose ranges, could accurately predict the number of new
    diseases in the offspring at low doses.

Lack of current research in this area is a major concern and little has
been done to update the research and data collection based on the
Dearfield *et al.* (1995) methods. The Panel is in agreement with the
report that recommends further research and data to fill the critical
data gaps and reduce uncertainties including gaps in interspecies
extrapolation factors, the quantitative relationship between genetic
alterations in germ cells and heritable disease, and the shape of the
low-dose response relationship. Research might include multiple dose
studies, including dose selection comparable to that employed in the
repeated dose studies which identified neurotoxicity as a critical
effect. It is also recommended that impacts on different cell types be
determined and that biomonitoring data be utilized in any models
developed.

[]{#__RefHeading___Toc217455214 .anchor}**Charge Question 15. *Please
comment on the scientific support for the hypothesis that heritable germ
cell effects are likely to occur at doses lower than those for
neurotoxicity? What on-going or future research might help resolve this
issue?***

The Panel unanimously agreed that germ cell-induced effects should be
taken very seriously, as their implications are highly significant from
a public health perspective. There is an absence of data on these
effects in lower dose ranges, making it very difficult to speculate
about the relevance of this endpoint at or below the dose levels that
cause neurotoxicity. Panelists did point out that heritable
translocations appeared with very high frequency at the lowest doses
tested (i.e., 5 x 40 mg/kg resulted in 24% translocation carriers,
Shelby *et al.*, 1987). The high frequency of germ cell effects at these
doses implies that these studies were far from identifying a LOAEL or
NOAEL, and that there would likely be germ cell effects at much lower
doses. However, the combination of lack of testing at lower doses, and
the narrow dose range in which testing has been done, makes it very
difficult to extrapolate down to a low dose range. The Panel agreed that
it is a high priority to extend the heritable translocation studies down
into lower dose ranges, and that this information would be very useful
for risk assessment once it is completed.

[]{#__RefHeading___Toc217455215 .anchor}**Charge Question 16. *The risks
of heritable germ cell effects (i.e., number of induced genetic diseases
per million offspring) for some estimated exposure in workers and the
population are presented in Table 5-11, and are based on the
quantitative methods and parameter estimates discussed in Section 5.4 of
the Toxicological Review. Please comment on whether or not the
quantitation of heritable germ effects should be conducted, the level of
uncertainty in the results, if Table 5-11 is useful for risk assessment
purposes, and if the RfD should be included in the Table as one of the
exposure levels.***

The Panel supports the Agency's conclusions that exposure to acrylamide
in animals leads to heritable gene mutations and that these results
indicate that it may also pose a hazard to humans. In addition, the
Panel supports the Agency's conclusions that the available data are not
adequate to conduct a robust assessment of this endpoint at this time.

The Panel's deliberations regarding quantifying heritable germ cell
mutations centered on the importance of including data such as those
presented in Table 5-14 (not Table 5-11, as noted in the final
question), the potential significance of these endpoints to human risk
assessment, and the paucity of new data developed since the Dearfield
*et al.* (1995) review upon which this section relied heavily (including
Table 5-14). A majority of Panel members were supportive of the
inclusion of this table in the document and for including the RfD and
RfC among the concentrations in the table as this would facilitate
comparison with the neurological endpoints. Suggestions also included
adding more information into the review regarding the role of CYP 2E1 in
the dominant lethal effects of acrylamide, which indicated a requirement
for metabolism to glycidamide. While the caveats from the Dearfield *et
al.* (1995) review were recapitulated in the document, the Panel
discussed the need to further elaborate the limitations in the
underlying data and to include reference to the new relevant studies
that pertain to uncertainty and dose-response.

## Charge Question 17. Do you know of any additional data or analyses that would improve the quantitative characterization of the dose-response for acrylamide-induced heritable germ cell effects? Would these data also support the quantitative characterization of "total" male-mediated reproduction risks to offspring (i.e., lethality + heritable defect)? If data are not available, do you have any recommendations for specific needed studies?

A concern raised by the Panel was that there is a lack of a suitable
data set for dose response assessment for acrylamide-induced heritable
germ cell effects. The majority of the studies reported have been
conducted in mice, using relatively high doses.

Using wild type and Cyp 2E1 knockout mice, it has been demonstrated that
oxidation of acrylamide to glycidamide is required for the dominant
lethal effect (Ghanayem *et al.*, 2005a) and for the induction of
erythrocyte micronuclei and DNA strand breaks in lymphocytes, liver and
lung using the Comet assay (Ghanayem *et al.*, 2005b). The greater
incidence of heritable translocation carriers in mice administered
glycidamide (Generoso *et al.*, 1996) compared with acrylamide (Adler
*et al.*, 1994) suggests that glycidamide plays a key role in the mode
of action for heritable genetic effects.

The risk equivalent factors (REFs, page 217) need to be updated. There
are profound differences between rats, mice and humans in the extent of
metabolism of acrylamide to glycidamide, and the relative internal dose
of acrylamide and glycidamide differs markedly between mice, rats and
humans. The extension of the physiologically-based pharmacokinetic
modeling approach to include the mouse should be a priority. The
blood-testis barrier is thought to contribute to the reduction of
internal dose in the testis compared with other tissues for ethylene
oxide (Fennell *et al.*, 2001). Testis should be included as a
compartment in the model. Data permitting, including the testis as a
compartment in the model could potentially improve the dose response
characterization for this endpoint.

In reviewing data needs (page 220), it is noted that "The estimates do
not take into account other potential genotoxic mechanisms such as
effects in spermatogonia stem cells, effects in female germ cells, or
induction of recessive mutations that would not appear in the first
generation, but could lead to additional adverse effects in subsequent
generations." Studies to examine the dose response for heritable genetic
effects, and the effect of long-term exposure to acrylamide are needed.

There is still uncertainty about the mode of action of acrylamide and
glycidamide in the induction of heritable genetic effects. The potential
for DNA adducts of glycidamide to play a role is an attractive
hypothesis for the mode of action. With respect to the possible role for
protamine modification in the generation of effects, there was extensive
Panel discussion concerning the potential of glycidamide to form adducts
with cysteine in proteins and peptides. Adducts to protamine from
acrylamide have been identified in late stage spermatids and suggested
to mediate the dominant lethal effects (Sega *et al.*, 1989). Whether
glycidamide will form similar protamine adducts has not been determined.
Kinesin motor proteins associated with cell division are an additional
site of potential action leading to heritable germ defects (Sickles *et
al.*, 2007) that requires future consideration. Both AA and GA inhibit
two kinesin motors associated with spindle formation and maintenance as
well as separation of chromosomes. Loss of fidelity of chromosomal
separation is related to aneuploidy, micronuclei formation and
instability of the genome. The motor protein inhibitions occur at
concentrations well below the occurrence of all heritable germ cell
effects. Furthermore, glycidamide is more potent than acrylamide.
Surveying populations occupationally exposed to acrylamide in
manufacturing plants was suggested as an approach for evaluation in
humans.

[]{#__RefHeading___Toc217455217 .anchor}**Charge Question 18. *Have the
rationale and justification for the cancer designation for acrylamide
been clearly described? Is the conclusion that acrylamide is a likely
human carcinogen scientifically supportable? ***

Yes, the rationale and justification have been clearly described,
although it should be further expanded (see below), and the conclusion
is scientifically supportable. Acrylamide is clearly and reproducibly
carcinogenic in both rats and mice. As outlined in the draft document,
it produced tumors at multiple sites in the rat in multiple chronic
studies, and was a skin tumor initiator in mice by multiple routes.
Therefore, in accordance with EPA's cancer guidelines, it is consistent
with the "likely human carcinogen" cancer descriptor.

To paraphrase the International Agency for Research on Cancer (IARC)
Monographs Preamble, in the absence of tumor data in humans it is both
reasonable and prudent to regard evidence of carcinogenicity in
experimental animals as evidence for a probable cancer hazard to humans.
This conclusion is consistent with both national and international
guidelines for carcinogenic hazard identification. The U.S. National
Toxicology Program (NTP) has long emphasized that chemicals that cause
tumors at multiple sites or in more than a single species are reasonably
anticipated to be human carcinogens. Both the NTP and IARC have placed
acrylamide in cancer classifications similar to that of EPA's "likely
human carcinogen" (This could be noted in the Toxicological Review).

When experimental exposure of rats or mice to known human carcinogens is
via diet or drinking water, tumor sites observed in those species do not
necessarily correspond to the same tumor sites in humans. Exposure to
chemicals that cause tumors of the mammary gland or the liver in mice or
rats, for example, does not necessarily correspond to increased cancer
risk specifically for female breast or liver in humans. The essential
point to be considered is that in any given case a tumor at these or any
other site(s) results from an MOA known to operate in humans, such as
somatic cell mutagenicity.

Primary CNS tumors as a group, which are discussed at considerable
length in the draft document, should be restored to the list of
experimental tumors produced by acrylamide and that are of interest for
the MOA discussion. The Panel cautions that the viruses that can cause
primary CNS tumors in hamsters and other non-human species are not
relevant to this discussion.

It should be emphasized that the spectrum of tumors consistently seen in
acrylamide-exposed rats is completely consistent with a DNA-reactive
MOA, based on published data about other substances that induce or
initiate the same kinds of neoplasms. The only agents known conclusively
to induce tumors of the brain and peritesticular mesothelium in rats are
all DNA-reactive, and in fact a single exposure to a direct-acting
mutagenic carcinogen has been observed to suffice for tumor induction at
either site. The concept that acrylamide acts by a mutagenic MOA is thus
supported by the spectrum of acrylamide-associated tumors that occur in
exposed rats and mice, as well as by the biotransformation pathway of
acrylamide *in vivo*.

Tumor initiation -- promotion data for mouse skin are perhaps not
sufficiently emphasized in the draft document. First, only DNA-reactive
chemicals or chemicals biotransformed to DNA-reactive metabolites are
established tumor initiators. As acrylamide is an initiator, and by
multiple routes of administration, it is a permissible inference that
acrylamide is also acting by a DNA-reactive MOA in mouse skin, as do
other initiators. It is most striking that, in mice, systemic exposure
to acrylamide is more effective for skin tumor initiation than direct
application to the skin. The order of efficiency, oral \> ip \> dermal
application, for initiation of TPA-promotable squamous cell papillomas
and carcinomas on mouse skin strongly supports the importance of
systemic exposure and post-hepatic distribution of a reactive metabolite
in the MOA for carcinogenicity at this site.

[]{#__RefHeading___Toc217455218 .anchor}**Charge Question 19. *Do you
agree that weight of the available evidence supports a mutagenic mode of
carcinogenic action, primarily for the acrylamide epoxide metabolite,
glycidamide (GA)? Has the rationale for this MOA been clearly and
objectively presented, and is it reflective of the current science? ***

A sound rationale and justification already supports the mutagenic mode
of action (MOA), and this evidence is further supported by additional
new data as described below. The weight of evidence supports a mutagenic
MOA, and overall the rationale for this MOA has been clearly and
objectively presented. Some improvements to the presentation are as
follows. The discussion of biological plausibility and coherence could
be expanded beyond DNA adducts and the human relevance section could be
somewhat more expansive without being repetitive. The argument on page
145 regarding the lack of relationship of cytogenetic damage to a
mutagenic MOA should be carefully re-considered, as the literature is
full of these correlations. Evidence for and against the arguments set
out should be carefully evaluated, and much better referencing included.
Reports from Bonassi and Hagmar are cited as supportive, yet
contradictory findings from the same authors supporting an alternative
argument could just as easily have been cited. The discussion includes
strong generalizations that may not hold up to close scrutiny.

There has been one published study to date that has examined biomarkers
of acrylamide exposure and human cancer risk. Olesen *et al.* (2008)
characterized hemoglobin adducts of acrylamide and glycidamide in a
case-control study of post-menopausal breast cancer. The authors found
no association between levels of glycidamide hemoglobin adducts and
breast cancer risk. Moreover, they found no overall association between
acrylamide adducts and risk. Upon adjustment for smoking status,
however, they observed a 2.7-fold (1.1-6.6) increased risk restricted to
ER+ breast cancer per 10-fold increase in acrylamide-hemoglobin level.
With respect to this study design, the authors did not match or restrict
the cases and controls on smoking status, which raises concern given the
very strong link between smoking and acrylamide adducts.
Interpretability of the Olesen study with respect to supporting the mode
of carcinogenic action should be taken cautiously.

For very high levels of acrylamide exposure, the animal and other
experimental data do support a mutagenic effect of acrylamide. It has
been questioned whether such a mechanism might also apply to lower doses
(and indeed, at the lowest doses to which humans are exposed), because
of uncertainty about whether the compensatory mechanisms are in place to
detoxify acrylamide. But data clearly indicate that glycidamide is
formed. There are the consistent observations in humans of
glycidamide-hemoglobin adducts (Bjellaas *et al.*, 2007; Chevolleau *et
al.*, 2007; Vesper *et al.*, 2006, 2007) or glycidamide urinary
metabolites (Urban *et al.*, 2006) , including children (Heudorf *et
al.* 2008), thus demonstrating the widespread internal exposure to the
putative mutagenic metabolite of acrylamide at ongoing low levels of
exposure in the general population.

The Panel did not consider the carcinogenicity to be hormonally-related.
The existing short-term mouse studies in SENCAR, ICR (skin) and A/J
(lung) show no such selectivity of carcinogenicity for hormonally
regulated tissues. Also, the Panel discussed the fact
acrylamide/glycidamide is not unique among DNA-reactive epoxides for
carcinogenic action in thyroid, peritesticular mesothelium, and mammary
tissue (e.g., glycidol, ethylene oxide). In addition, this argument does
not consider the CNS tumors observed in both chronic acrylamide cancer
bioassays, a site that was discussed by the Panel as representing strong
evidence for a DNA-damaging mechanism (cf. Rice, 2005). Finally, a
recent publication considered by the Panel of short-term exposures to
high doses of acrylamide in male F344 rats found essentially no evidence
for hormonal dysregulation in the hypothalamus-pituitary-thyroid axis
based on measurements of gene expression, neurotransmitters, hormones,
and histopathology (Bowyer *et al.*, 2008). Some studies of chronic low
dose exposure, such as the cohort study of acrylamide and
ovarian/endometrial cancers (Hogervorst *et al.*, 2007) and others (Khan
*et al.*, 1999) have shown positive associations with hormones. The
Panel encourages the Agency to review all relevant new data that has
been published since their completion of the current draft assessment as
they revise and finalize this IRIS document.

[]{#__RefHeading___Toc217455219 .anchor}**Charge Question 20. *Are there
other MOAs that should be considered? Is there significant biological
support for alternative MOAs for tumor formation, or for alternative
MOAs to be considered to occur in conjunction with a mutagenic MOA?
Please specifically comment on the support for hormonal pathway
disruption. Are data available on alternate MOAs sufficient to
quantitate a dose-response relationship?***

No, there is not significant biological support for MOA alternatives to
the mutagenic MOA, and data on any putative alternate MOAs are not
sufficient to quantify dose response relationships. It must be
emphasized that more than one MOA may operate for a given carcinogenic
chemical, and the likelihood that more than a single MOA is operative
increases as levels of exposure increase. Some well-documented non-DNA
reactive MOAs appear to be high-dose phenomena. These are often
important for understanding bioassay results in experimental animals,
and sometimes for high-exposure situations in human experience, but they
are usually less important because they represent negligible risks when
cumulative human exposures to these and similarly acting compounds fall
considerably below bioassay dosage levels. MOAs that can occur both in
experimental rodents and in humans and that operate both at bioassay
dosage levels in experimental animals and at lower levels as well, into
the human exposure range, are most significant for humans. In general,
for chemicals such as acrylamide where there is a compelling body of
data to support a DNA-reactive MOA via biotransformation to glycidamide,
the evidence for alternative or additional high-dose MOAs would have to
be convincing to explore alternative approaches to dose response and
risk assessment. One caveat that should be mentioned is that mutations
induced by acrylamide are observed following high doses. There are
similarly acting agents, such as methylmethanesulfonate (MMS) that
create N7-Guanine, the same DNA adducts, as does glycidamide yet show a
threshold for mutations. These data are consistent with robust repair
mechanisms for the specific type of DNA adducts produced by glycidamide
and MMS. However, it should also be noted that low dose exposures have
not been tested in animal mutation studies and NOAELs have not yet been
established. Therefore future research should include dose response
analyses to stringently test the relationship between DNA adducts and
mutations and gain a better understanding of the effects at lower doses.
The Agency should mention the finding of inhibition of kinesin motor
proteins as a newly-identified and potential site of action of AA or GA
in the production of carcinogenicity (Sickles *et al.*, 2007).

Occasionally high-dose or "unique rodent-specific" MOAs may be invoked
or postulated to discredit bioassay results as irrelevant to humans,
especially when such putative MOAs are observed uniquely in non-human
species. Such a postulated MOA needs to be very precisely defined and
its relevance thoroughly investigated and critically tested before the
postulated MOA is accepted by the biomedical and risk assessment
communities. Any MOA developed for a single substance is at best
speculative until a general pattern can be rigorously demonstrated for a
family of substances that operate via the same MOA. The hormonal
disruption MOAs proposed for acrylamide as tissue-specific alternatives
to a DNA-reactive MOA are highly speculative, are supported by at most
limited evidence, and do not meet this standard as noted in response to
charge question 19. The data are insufficient for characterizing
dose-response relationships for any of these proposed alternatives.

[]{#__RefHeading___Toc217455220 .anchor}**Charge Question 21. *Two
chronic drinking water exposure bioassays in Fischer 344 rats (Friedman
et al., 1995; Johnson et al., 1986) were used to derive the oral slope
factor, and to identify the tumors of interest for the MOA discussion.
Are the choices for the studies, tumors, and methods to quantify risk
transparent, objective, and reflective of the current science? Do you
have any suggestions that would improve the presentation or further
reduce the uncertainty in the derived values?***

The two chronic bioassay studies in F344 rats are the main studies to
consider in dose response analysis. Overall the document does a good job
discussing these studies, but the rationale for using only the Friedman
*et al.* study for derivation of the oral slope factor is problematic.
The strengths and limitations of both studies should be discussed in
greater depth. The text describes the Friedman *et al.* study as
"superior" and "larger and better designed" but the Panel does not agree
that this is the case, and recommends that both studies be subjected to
modeling for the purposes of deriving oral slope factors. The two
studies may have fairly similar oral slope factors. At a minimum,
estimates for the second study should also be presented to clarify the
impact of study selection in the uncertainty discussion.

The methods to quantify risk are transparently presented and reflective
of current science, with the exception that a factor to scale for
pharmacodynamic differences in potency between humans and animals has
not been applied. The development of unit risk based on HEC accounts for
the pharmacokinetic but not pharmacodynamic differences, and in such
situations EPA's 2005 *Guidelines for Carcinogen Risk Assessment* (p.
3-7) indicates inclusion of a pharmacodynamic factor be considered. The
potential human variability in cancer response attributable to human
pharmacokinetic variability in handling acrylamide should be discussed
qualitatively and analyzed quantitatively. Hemoglobin adduct data could
provide the basis for such an analysis. The assumption underlying the
modeling, that each and every individual of the same age exposed to the
same external dose faces the same risk of cancer, is inconsistent with
these data.

With respect to study selection, one of the reasons for not using the
Johnson study had to do with the rates of CNS tumors in this study,
particularly in the controls. The Friedman *et al.* study was designed
"to investigate whether glial tumors in the Johnson *et al.* study were
significant." But, as Rice (2005) points out, the histopathological
examination for glial tumors was incomplete. Only one-fifth of the 1.0
mg/kg-day dose females' spinal cords were subjected to histopathological
examination, even though one-third of the glial tumors in the Johnson
*et al.* study were seen in the spinal cord. The approach to the
evaluation of CNS tumors in Friedman *et al.* was seen by the Panel as a
significant study limitation.

Another improvement over the Johnson study noted in the document for the
Friedman *et al.* study was different and presumably better dose
intervals. The doses for males in the Friedman *et al.* and Johnson *et
al.* studies were the same, except Johnson *et al.* had one additional
lower dose group. The doses in Friedman for females were 1.0 and 3.0
mg/kg-day compared to 0.01, 0.1, 0.5 and 2.0 mg/kg-day for the Johnson
study. The Friedman study did extend the high end of the dose response
range for females and did offer a more complete dose response function
for thyroid tumors, employed somewhat larger dose groups (100 per group
and two control groups). But Johnson *et al.* did have 60 animals per
dose group, did provide a complete histopathological evaluation, and had
more dose groups than a standard bioassay.

Another limitation of the Friedman *et al.* study is that the degree of
histopathological examination of oral tissue is unclear. The Friedman
study does not tabulate findings for certain tumor sites seen in the
Johnson study, so quantitative comparisons are not possible and the
reader is not able to consider these sites or perform independent
evaluations regarding the significance of the findings. It appears EPA
may have the data needed to do the analysis since it was able to do a
time-dependent analysis for slope estimation using the Tegeris Lab
report. EPA could then look at the data and analyze as appropriate the
data for these sites.

A criticism about the possible impact of a sialodacryoadenitis virus on
tumor findings had been raised and was another reason given for using
the Friedman study. On the other hand, US FDA had raised some issues in
auditing the Friedman *et al.* study regarding environmental controls at
the lab facility and the possibility of some under-dosing of animals.
Ultimately both studies have strengths and weaknesses and on balance
neither seems clearly superior. Both are reasonably strong studies, and
thus oral slope estimates should be presented for both studies.

Some comments regarding details on tumor data presentation and analysis
in the EPA draft document follow:

Tests for dose-related trends should be conducted and presented for the
all tabulated sites. By Fisher's exact test, the mammary tumors in the
0.5 mg/kg-d group in the Friedman *et al.* study are significant
(p\<0.05). The statistics used in the draft document that correct for
intercurrent mortality should be re-checked. It appears this group has a
treatment-related finding and this should be noted and the discussion
that this group is devoid of treatment-related tumors (page 75) changed.
The clitoral gland findings in the Johnson *et al.* study stand out
because histology was done only on clitoral tissues observed with gross
masses. This is worth an explanatory footnote. Also given the approach
taken to collecting this tissue, the clitoral tumors in the 0.5 mg/kg
dose group also appear worthy of note. All four masses analyzed
indicated tumor compared to none in controls (p\<0.1). In the Friedman
*et al.* study, CNS tumors of glial origin should be combined for
analysis as was done by WHO 2006. Considering the findings of glial
tumors in females in the Johnson study, the dose related trend for both
sexes in the Friedman study, although falling a hair short of
statistical significance at the p [\<]{.underline} 0.05 level, provide
some evidence of a CNS glial cell effect in the Friedman study. This
should be discussed. Also, the extent of examination of oral tissue in
the Friedman study is unclear. Finally, the Friedman study employed two
control groups for the male rats that do not differ from one another.
For the statistical treatments, there is no apparent reason why these
groups should not be combined. The Toxicological Review did this for the
dose response analysis but may not have done the same for the pair-wise
comparisons.

The data choice for modeling to address the discrepancy between the
Friedman *et al.* and the Tegeris laboratory reporting of thyroid tumors
for the male noted in Appendix D of EPA's draft document was
appropriate. A final minor point, in the discussion of the confidence in
dose response analysis in chapter 6 (page 229), issues are raised that
seem better placed in the discussion of the hazard characterization.

[]{#__RefHeading___Toc217455221 .anchor}**Charge Question 22. *The
cancer slope factor (CSF) derivation includes an adjustment for early
mortality (i.e., time-to-tumor analysis). Is this adjustment
scientifically supported in estimating the risk from the 2-year bioassay
data for increased incidence of tumors in the rats?***

The use of the Weibull-in-time multistage-in-dose analysis is a
reasonable and scientifically justifiable way to take into account the
early mortality in the high dose group in the male study. The decision
not to employ this analysis in the case of the females is also
reasonable since mortality across treatment and control groups did not
differ and the overall survival appears to be fairly good.

## 

## Charge Question 23. Please comment on whether AUC for glycidamide is the best choice of the dose metric in estimating human equivalent concentration to derive the oral slope factor.

The Panel agreed that using the AUC for glycidamide is the best choice
for estimating the human equivalent concentration to derive the oral
slope factor. This decision was based on the strong evidence from
experimental results that the AUC was linearly correlated with adduct
levels in single/repeat dosing studies. There was agreement that
glycidamide is the more mutagenic metabolite based on experimental
studies. The Panel felt there was good documentation in the report
regarding the correlation between levels of DNA adducts and extent of
mutations *in vivo*. Moreover, the metabolic conversion of acrylamide to
glycidamide supports the MOA.

One consideration in using this as the dose metric, however, comes from
some of the human studies in which variability is not accounted for
adequately, specifically, inter-individual variation is not assessed and
that the value used for cross-species comparisons is based on small
numbers of healthy adult male humans. This is discussed at greater
length in response to Question 8. Consideration of additional human data
(e.g., Vesper *et al.*, 2006) to evaluate the degree humans form
glycidamide from acrylamide is clearly warranted. Such data may provide
the basis for comparing human acrylamide and glycidamide AUCs, using
methodology of Calleman, Bergmark and colleagues (Bergman *et al.*,
1991). This in turn can provide an improved basis for adjustments for
cross-species differences in pharmacokinetics, as well as human
variability in glycidamide formation from acrylamide.

[]{#__RefHeading___Toc217455223 .anchor}**Charge Question 24. *As with
the RfC, there were insufficient cancer inhalation data to derive an
inhalation unit risk (IUR). The PBTK model was used in a route-to-route
extrapolation of the dose-response relationship from the oral data, and
to estimate the human equivalent concentration for inhalation exposure
to acrylamide. Please comment on whether this extrapolation to derive
the inhalation unit risk was correctly performed and sufficiently well
documented.***

The response to this question is nearly identical to the response to
charge question #11. The Panel agreed with the use of PBTK modeling to
conduct dose-route extrapolation and commended the EPA for using the
PBTK model to fill the gap resulting from the absence of robust animal
toxicology studies investigating neurotoxicity via the inhalation route
that would support the development of an IUR. The Panel agreed that the
absence of evidence for route of entry specific effects would allow
route-to-route extrapolation for deriving an IUR based on using the PBTK
model to calculate the human equivalent concentration (HEC). This would
yield an equivalent internal dose (Glycidamide- AUC) associated with
those achieved at the point of departure from the oral sentinel (Johnson
*et al.*) studies. The Panel noted that few inhalation PK studies exist
to allow a robust parameterization of the inhalation component of the
PBTK model for either rats or humans. Despite this, the Panel noted that
acrylamide is very water soluble and non-volatile, and the compound has
a relatively long half life. Therefore, the absorption of acryalmide via
inhalation should be nearly complete, and first pass effects are
negligible, thereby making the pharmacokinetics of acrylamide via
inhalation easy to extrapolate from simple principles of
pharmacokinetics. The Panel agreed that the application of
pharmacokinetic approaches (e.g,, the use of the PBTK model) reduces
uncertainty associated with animal to human extrapolation and thus
warrants replacing the default uncertainty factor associated with
interspecies extrapolation for pharmacokinetic differences as was done
for deriving the RfD. The use of the PBTK model however does not address
cross-species differences in pharmacodynamics, which should be
considered, following the Agency's 2005 Guidelines for Carcinogen Risk
Assessment.

The Panel noted that the air concentration one would derive using the
default approach (multiply the HED by body weight \[70 kg\] and dividing
by daily inhalation rate \[20 m^3^/day\] yielding 0.266 µg/m^3^) is very
similar to the HEC derived using the PBTK model (0.25 µg/m^3^).
Therefore, if the EPA decides to also provide an extrapolation based on
measured data (as described in the response to charge question 8), the
default approach of extrapolating from an absorbed oral dose to an
equivalent intake from the inhalation route (multiplying by 70 kg and
dividing by 20 m^3^/day) can be used with confidence to calculate the
IUR.

[]{#__RefHeading___Toc217455224 .anchor}**Charge Question 25. *The
recommendation to use the age-dependent adjustment factors (ADAFs) is
based on the determination of a mutagenic MOA for carcinogenicity. Is
this recommendation scientifically justifiable and transparently and
objectively described?***

The recommendation to use the age-dependent adjustment factors is well
justified and transparently and objectively described. The Panel's
deliberations regarding quantitating age-dependent adjustment factors
(Section 5.4.6) followed from discussions of a mutagenic mode of action
for acrylamide and the typically enhanced sensitivity of fetal and
neonatal animals from exposure to such agents in accordance with EPA's
Supplemental Guidance for Assessing Susceptibility from Early Life
Exposure to Carcinogens (2005b). The Panel also discussed the value of
using the PBTK model to evaluate the effect of lifestage on CYP 2E1 and
glutathione levels that could modify internal exposure to glycidamide.
Such modeling results could be used to reduce the uncertainty associated
with lifestage extrapolations and the derivation of age-dependent
adjustment factors. Such efforts would be directed at pharmacokinetic
aspects of the age-dependent adjustment factors. Uncertainty regarding
pharmacodynamics would remain to be addressed by the age-dependent
adjustment factors.

[]{#__RefHeading___Toc217455225 .anchor}**Charge Question 26. *Please
provide any other comments on the CSF or IUR, and on the discussion of
uncertainties in the cancer assessment.***

The discussion of uncertainties is good, but human variability could be
addressed in greater length. It is unclear why in Table 5-13 the
consideration/approach is "Method used to protect sensitive
populations." There is no characterization of sensitive populations, and
this could be explored and discussed to a much greater extent.

Specifically, not enough attention was paid to consequences of
individual differences in metabolism and cancer risk. Both the CYP2E1
polymorphisms and glutathione transferase(s) (even though rodent data
suggests no role for this pathway) polymorphisms could be looked at in
human populations. The degree to which increased activity influences the
risk should be considered, including whether this might be tumor site
dependent. Also, much weight is put on the two chronic studies in the
Fischer344 rat. The limitations of not having another rodent species
should be discussed in more detail with respect to other carcinogens
where 2 species were evaluated and similar or different results were
found.

A factor to scale for toxicodynamic differences between humans and
animals was not included in the derivation of the CSF and IUR. The 2005
EPA Carcinogenic Risk Assessment Guidelines (see e.g., Guidelines pp
1-13 and 3-7) discusses how toxicodynamics can be addressed by such a
factor. The development of unit risk-based on HEC accounts for the
toxicokinetic but not toxicodynamic interspecies differences.

# 

# 

# 

# 

# 

# ABREVIATIONS

ADAF age-dependent adjustment factor

AM-GSH Acrylamide-Glutathione

AUC area under the curve

BMD benchmark dose

BMDL benchmark dose level

BMR benchmark response

CNS Central Nervous System

CSAF Chemical-specific Adjustment Factors

CSF Cancer slope factor

DNA Deoxyribonucleic Acid

EPA Environmental Protection Agency

FQPA Food Quality Protection Act

GA or Gly Glycidamide

GA-GSH Glycidamide-Glutathione

HEC Human Equivalent Concentration

IARC International Agency for Research on Cancer ()

IRIS Integrated Risk Information System

IUR inhalation unit risk

LOAEL Lowest Adverse Effect Level

MMS Methylmethanesulfonate

MOA mode of action

MOE Margin of Exposure

NMA N-Methylolacrylamide

NO Nitric Oxide

NOAEL No Adverse Effect Level

NTP/CERHR National Toxicology Program

PBPK physiologically-based pharmacokinetic

PBTK physiologically-based toxicokinetic

PK Pharmacokinetic

POD point of departure

RfC reference concentration

RfD reference dose

TP tumor promoter

UF uncertainty Factor

WHO World Health Organization

# REFERENCES 

Abou-Donia, MB, Ibrahim, SM, Corcoran, JJ, *et al.* (1993) Neurotoxicity
of glycidamide, an acrylamide metabolite, following i.p. injections in
rats. *J Toxicol Environ Health* 39:447--464.

Adler, I-D. (1990) Clastogenic effects of acrylamide in different
germ-cell stages of male mice. In: Biology of Mammalian Germ Cell
Mutagenesis, Banbury Report Vol. 34.

Adler, I-D, Reitmer, P, Schmöller, R, *et al.* (1994) Dose response for
heritable translocations induced by acrylamide in spermatids of mice.
*Mutat Res* 309:285--291.

Barber, D.S., Hunt, J.R., Ehrich, M.F., Lehning, E.J. and LoPachin, R.M.
(2001). Metabolism, toxicokinetics and hemoglobin adduct formation in
rats following subacute and subchronic acrylamide dosing.
*Neurotoxicology* 22, 341-353.

Barber, D.S. and LoPachin, R.M. (2004). Proteomic analysis of
acrylamide-protein adduct formation in rat brain synaptosomes. *Toxicol.
Appl. Pharmacol.* 201, 120-136.

Barber, D.S. Stevens, S. and LoPachin, R.M. (2007). Proteomic analysis
of rat striatal synaptosomes during acrylamide intoxication at a low
dose rate. *Toxicol. Sci.* 100, 156-167.

[]{#__RefHeading___Toc217455228 .anchor}**Appendix A Memorandum and
Charge Questions**

![](media/image2.png){width="6.747916666666667in"
height="7.527083333333334in"}

**Charge Questions**

**[Selection of Studies and Endpoints for the Oral Reference Dose
(RfD)]{.underline}**

In the draft document, the proposed most sensitive noncancer effect from
exposure to acrylamide is neurotoxicity. This endpoint is based on an
extensive database of animal and human studies. The next most sensitive
effect is reproductive toxicity, which was in the 3-5 fold higher
exposure range for a no effect response in animal studies. No human data
were identified for acrylamide related reproductive effects. Heritable
germ cell effects, a potentially serious noncancer effect, have been
observed in male mice, however, the lowest dose levels tested are
considerably higher (two orders of magnitude) than the doses where
neurotoxicity were observed, and there is uncertainty about the shape of
the low-dose-response relationship.

1.  Please comment on the selection of neurotoxicity as the most
    appropriate choice for the most sensitive endpoint (in contrast to
    reproductive toxicity, heritable germ cell effects, or other
    endpoint) based upon the available animal and human data.

2.  Please comment on the discussion of mode of action for
    acrylamide-induced neurotoxicity. Is the discussion clear,
    transparently and objectively described, and accurately reflective
    of the current scientific understanding?

3.  Please comment on the qualitative discussion of acrylamide's
    heritable germ cell effects and whether the discussion is clear,
    transparently and objectively described, and reflective of the
    current science.

**[Derivation of the Reference Dose (RfD)]{.underline}**

The proposed RfD (0.003 mg/kg-day) for acrylamide is based on a
benchmark dose analysis of the dose-response relationship for
neurotoxicity in two chronic drinking water exposure bioassays using
Fischer 344 rats. Uncertainty factors and a PBPK model are used to
extrapolate the animal dose-response to a human equivalent dose-response
in the derivation of the RfD.

4\. Please comment on whether the selection of the Friedman *et al.*,
(1995) and Johnson *et al.*, (1986) studies as co-principal studies has
been scientifically justified. Although EPA considers Friedman *et al.*
and Johnson *et al.* to be co-principal studies, the final quantitative
RfD value is derived only from the Johnson study. Please comment on this
aspect of EPA\'s approach. Please also comment on whether this choice is
transparently and objectively described in the document. Please identify
and provide the rationale for any other studies that should be selected
as the principal study(s).

5.  Please comment on the benchmark dose methods and the choice of
    response level used in the derivation of the RfD, and whether this
    approach is accurately and clearly presented. Do these choices
    represent the most scientifically justifiable approach for modeling
    the slope of the dose-response for neurotoxicity? Are there other
    response levels or methodologies that EPA should consider? Please
    provide a rationale for alternative approaches that should be
    considered or preferred to the approach presented in the document.

6.  Please comment on the selection of the uncertainty factors (other
    than the interspecies uncertainty factor) applied to the point of
    departure (POD) for the derivation of the RfD. For instance, are
    they scientifically justified and transparently and objectively
    described in the document? \[Note: This question does not apply to
    the interspecies uncertainty factor which is addressed in the
    questions on the use of the PBPK model (see PBPK model questions
    below)\]

7.  Please provide any other comments on the derivation of the RfD and
    on the discussion of uncertainties in the RfD.

**[Use of a PBPK Model in the Derivation of the RfD and the Inhalation
Reference Concentration (RfC)]{.underline}**

A physiologically-based toxicokinetic (PBTK) model originally developed
by Kirman *et al.* (2003), and recalibrated by EPA with more recent
kinetic and hemoglobin binding data in rats, mice, and humans (Boettcher
*et al.*, 2005; Doerge *et al.*, 2005a,b; Fennell *et al.*, 2005) was
used in the derivation of the RfD to extrapolate from the animal
dose-response relationship (observed in the co-principal oral exposure
studies for neurotoxicity) to derive a human equivalent concentration
(HEC). The HEC is the external acrylamide exposure level that would
produce the same internal level of parent acrylamide (in this case the
area under the curve \[AUC\] of acrylamide in the blood) that was
estimated to occur in the rat following an external exposure to
acrylamide at the level of the proposed point of departure, and related
to a response level of 5% (i.e., the BMDL~5~). The model results were
used in lieu of the default interspecies uncertainty factor for
toxicokinetics differences of 10^1/2^, which left a factor of 10^1/2^
(which is rounded to 3) for interspecies differences in toxicodynamics.

With respect to the RfC, there are presently insufficient human or
animal data to directly derive an RfC for acrylamide. The PBPK model was
thus used to conduct a route-to-route extrapolation (oral-to-inhalation)
to derive an RfC based on the dose-response relationship observed in the
co-principal oral exposure studies for neurotoxicity. In this case, the
HEC was based on a continuous inhalation exposure to acrylamide in the
air that would yield the same AUC for the parent acrylamide in the blood
as that estimated for the rat following an external oral exposure to
acrylamide at the level of the proposed point of departure (i.e., the
BMDL~5~).

8.  Please comment on whether the documentation for the recalibrated
    Kirman *et al.* (2003) PBTK model development, evaluation, and use
    in the assessment is sufficient to determine if the model was
    adequately developed and adequate for its intended use in the
    assessment. Please comment on the use of the PBTK model in the
    assessment, e.g., are the model structure and parameter estimates
    scientifically supportable? Is the dose metric of
    area-under-the-curve (AUC) for acrylamide in the blood the best
    choice based upon what is known about the mode of action for
    neurotoxicity and the available kinetic data? Please provide a
    rationale for alternative approaches that should be considered or
    preferred to the approach presented in the document.

9.  Is the Young *et al.* (2007) PBTK model adequately discussed in the
    assessment with respect to model structure, parameter values, and
    data sets used to develop the model? Do you agree with the
    conclusion (and supporting rationale) that the recalibrated Kirman
    *et al.* (2003) model (model structure and parameter values
    presented in the Toxicological Review) currently represents the best
    model to use in the derivation of the toxicity values?

10. According to US EPA's RfC Methodology (1994), the use of PBTK models
    is assumed to account for uncertainty associated with the
    toxicokinetic component of the interspecies uncertainty factor
    across routes of administration. Does the use of the PBTK model for
    acrylamide objectively predict internal dose differences between the
    F344 rat and humans, is the use of the model scientifically
    justified, and does the use of the PBTK reduce the overall
    uncertainty in this estimate compared to the use of the default
    factor? Are there sufficient scientific data and support for use of
    this PBTK model to estimate interspecies toxicokinetic differences
    and to replace the default interspecies factor for toxicokinetic
    differences (i.e., 10^1/2^)? Is the remaining uncertainty factor for
    toxicodynamic differences scientifically justified, appropriate and
    correctly used?

11. Please comment on whether the PBTK model is adequate for use to
    conduct a route-to-route extrapolation for acrylamide to derive an
    RfC in the absence of adequate inhalation animal or human
    dose-response data to derive the RfC directly. Was the extrapolation
    correctly performed and sufficiently well documented?

12. Please provide any other comments on the derivation of the RfC and
    on the discussion of uncertainties in the RfC.

**[Margin of Exposure (MOE) Analysis]{.underline}**

IRIS documents do not include exposure assessments, which precludes the
ability to conduct a Margin of Exposure (MOE) analysis. It has been
suggested, however, that the acrylamide assessment include a Table that
lists points of departure for various endpoints to facilitate a MOE
evaluation by EPA's Regional or Program offices, or by other end users
of the assessment.

13. Would you suggest that EPA include a Table that lists points of
    departure (e.g., NOAELs, BMDs, etc.) for various endpoints that
    could be used, in conjunction with exposure assessments, to conduct
    a MOE analysis?

**[Quantitating Heritable Germ Cell Effects]{.underline}**

The Toxicological Review includes a discussion of methods to quantitate
the risk for heritable germ cell effects (Section 5.4). The questions
below address the uncertainty and utility of the quantitative results.

14. Please comment on the discussion of methods to quantitate the
    dose-response for heritable germ cell effects as to whether it is
    appropriate, clear and objective, and reflective of the current
    science. Has the uncertainty in the quantitative characterization of
    the heritable germ cell effects been accurately and objectively
    described?

15. Please comment on the scientific support for the hypothesis that
    heritable germ cell effects are likely to occur at doses lower than
    those seen for neurotoxicity? What on-going or future research might
    help resolve this issue?

16. The risks of heritable germ cell effects (i.e., number of induced
    genetic diseases per million offspring) for some estimated exposure
    in workers and the population are presented in Table 5-11, and are
    based on the quantitative methods and parameter estimates discussed
    in Section 5.4 of the Toxicological Review. Please comment on
    whether or not the quantitation of heritable germ effects should be
    conducted, the level of uncertainty in the results, if Table 5-11 is
    useful for risk assessment purposes, and if the RfD should be
    included in the Table as one of the exposure levels.

17. Do you know of any additional data or analyses that would improve
    the quantitative characterization of the dose-response for
    acrylamide-induced heritable germ cell effects? Would these data
    also support the quantitative characterization of "total"
    male-mediated reproduction risks to offspring (i.e., lethality +
    heritable defect)? If data are not available, do you have any
    recommendations for specific needed studies?

**[Carcinogenicity of Acrylamide]{.underline}**

In accordance with EPA's 2005 *Guidelines for Carcinogen Risk
Assessment*
([www.epa.gov/iris/backgr-d.htm](http://www.epa.gov/iris/backgr-d.htm)),
acrylamide is described as *likely to be carcinogenic to humans* based
on: (1) significant increased incidences of thyroid tumors in male and
female rats, scrotal sac mesotheliomas in male rats, and mammary gland
tumors in female rats in two drinking water bioassays; (2) initiation of
skin tumors following oral, intraperitoneal, or dermal exposure to
acrylamide and the tumor promoter, TPA, in two strains of mice; and (3)
increased incidence of lung adenomas in another mouse strain following
intraperitoneal injection of acrylamide. Evidence from available human
studies is judged to be limited to inadequate.

The mechanisms by which acrylamide may cause cancer are poorly
understood, but EPA has determined that the weight of the available
evidence supports a mutagenic mode of carcinogenic action, primarily for
the acrylamide epoxide metabolite, glycidamide (GA). Other mode(s) of
action (MOA) have been proposed for the carcinogenicity of acrylamide,
but there is less support.

18. Have the rationale and justification for the cancer designation for
    acrylamide been clearly described? Is the conclusion that acrylamide
    is a likely human carcinogen scientifically supportable?

19. Do you agree that weight of the available evidence supports a
    mutagenic mode of carcinogenic action, primarily for the acrylamide
    epoxide metabolite, glycidamide (GA)? Has the rationale for this MOA
    been clearly and objectively presented, and is it reflective of the
    current science?

20. Are there other MOAs that should be considered? Is there significant
    biological support for alternative MOAs for tumor formation, or for
    alternative MOAs to be considered to occur in conjunction with a
    mutagenic MOA? Please specifically comment on the support for
    hormonal pathway disruption. Are data available on alternate MOAs
    sufficient to quantitate a dose-response relationship?

21. Two chronic drinking water exposure bioassays in Fischer 344 rats
    (Friedman *et al.*, 1995; Johnson *et al.*, 1986) were used to
    derive the oral slope factor, and to identify the tumors of interest
    for the MOA discussion. Are the choices for the studies, tumors, and
    methods to quantify risk transparent, objective, and reflective of
    the current science? Do you have any suggestions that would improve
    the presentation or further reduce the uncertainty in the derived
    values?

22. The cancer slope factor (CSF) derivation includes an adjustment for
    early mortality (i.e., time-to-tumor analysis). Is this adjustment
    scientifically supported in estimating the risk from the 2-year
    bioassay data for increased incidence of tumors in the rats?

23. The dose metric used in the PBTK model analysis to derive the human
    equivalent concentration was area under the curve (AUC) in the blood
    for the putative genotoxic metabolite, glycidamide. Please comment
    on whether AUC for glycidamide is the best choice of the dose metric
    in estimating the human equivalent concentration to derive the oral
    slope factor. If other dose metrics are preferable, please provide
    the scientific rationale for their selection.

24. As with the RfC, there were insufficient cancer inhalation data to
    derive an inhalation unit risk (IUR). The PBTK model was used in a
    route-to-route extrapolation of the dose-response relationship from
    the oral data, and to estimate the human equivalent concentration
    for inhalation exposure to acrylamide. Please comment on whether
    this extrapolation to derive the inhalation unit risk was correctly
    performed and sufficiently well documented.

25. The recommendation to use the age-dependent adjustment factors
    (ADAFs) is based on the determination of a mutagenic MOA for
    carcinogenicity. Is this recommendation scientifically justifiable
    and transparently and objectively described

26. Please provide any other comments on the CSF or IUR, and on the
    discussion of uncertainties in the cancer assessment.

# 

# 

# 

# 

# 

# Appendix B 

# Proposed Modes of Action (MOAs) for Acrylamide Neurotoxicity

The following text on the two proposed MOAs for acrylamide neurotoxicity
was written by one panel member. It is offered for the Agency's
consideration in writing the revised version of the acrylamide IRIS
document:

1.  Disruption of Nitric Oxide (NO) Signaling at the Nerve Terminal
    Hypothesis

Acrylamide is a conjugated α,β-unsaturated carbonyl derivative in the
type-2 alkene chemical class. Because electrons in pi orbitals of a
conjugated system are mobile, the α,β-unsaturated carbonyl structure of
acrylamide is characterized as a soft electrophile according to the
hard-soft, acid-base principle (reviewed in Pearson, 1967).
Characteristically, soft electrophiles will preferentially form
Michael-type adducts with soft nucleophiles, which in biological systems
are primarily sulfhydryl groups on cysteine residues (Hinson and
Roberts, 1992; LoPachin and DeCaprio, 2005). Free sulfhydryl groups can
exist in the reduced thiol-state or in the anionic thiolate-state and
recent research indicates that the highly nucleophilic thiolate is the
preferential adduct target for acrylamide (LoPachin *et al.*, 2007b; see
also Friedman *et al.*, 1995). Based on the pKa of cysteine (pH 8.5), at
physiological pH (7.4) the thiolate state exists only in unique protein
motifs called catalytic triads, where proton shuttling through an
acid-base pairing of proximal amino acids (e.g., aspartic acid and
lysine) regulates the protonation and deprotonation of the cysteine
sulfhydryl group. Indeed, both mass spectrometric and kinetic data have
demonstrated the selective adduction of cysteine residues on many
neuronal proteins (Barber and LoPachin, 2004; Barber *et al.*, 2007).
Furthermore, it is now recognized that the redox state or
nucleophilicity of cysteine sulfhydryl groups within catalytic triads
can determine the functionality of these proteins (reviewed in LoPachin
and Barber, 2006; Stamler *et al.*, 2001). In contrast to acrylamide,
the epoxide metabolite glycidamide (Gly), is a hard electrophile that
preferentially forms adducts with hard nucleophiles such as nitrogen,
carbon and oxygen. Nucleotide residues of DNA contain abundant hard
nucleophilic targets, which is consistent with the formation of
glycidamide adducts on adenine and guanine bases in
acrylamide-intoxicated animals (Doerge *et al.*, 2005; reviewed in
Besaratinia and Pfeifer, 2007).

Based on the observation that the processes affected (e.g.,
neurotransmitter release and storage) and corresponding kinetics (K~m~,
V~max~) were similar in synaptosomes exposed in vitro to acrylamide and
those isolated from acrylamide-intoxicated rats (Barber and LoPachin,
2004; LoPachin *et al.*, 2004, 2006), LoPachin and colleagues have
reasoned that the parent compound, acrylamide, is responsible for
neurotoxicity. Moreover, cysteine thiolate groups have clear regulatory
functions in many critical neuronal processes (LoPachin and Barber,
2006), whereas protein valine, lysine and histidine residues, which are
the likely hard nucleophilic targets for a hard electrophile such as
Gly, have unclear functional and therefore toxicological relevance.
Quantitative morphometric and silver stain analyses of PNS and CNS of
acrylamide-intoxicated animals have shown that axon degeneration was an
epiphenomenon related to dose-rate; i.e., degeneration occurred at lower
but not higher dose-rates. In contrast, nerve terminal degeneration
occurred regardless of dose-rate and in correspondence with the onset
and development of neurological deficits (Crofton *et al.*, 1996;
Lehning *et al.*, 1998, 2002a,b, 2003; reviewed in LoPachin *et al.*,
1994, 2002, 2003), suggesting the nerve terminals as a primary site of
action. Subsequent neurochemical studies showed that both in vitro and
in vivo acrylamide exposure produced early disruptions of
neurotransmitter release, reuptake and vesicular storage (Barber and
LoPachin, 2004; LoPachin *et al.*, 2004, 2006, 2007a). Further,
proteomic analyses indicated that the inhibition of presynaptic function
was due to the formation of cysteine adducts on proteins that regulate
neurotransmitter handling; e.g., Cys 264 of *N*-ethylmaleimide sensitive
factor, Cys 254 of v-ATPase (see Barber and LoPachin, 2004; Barber *et
al.*, 2007; Feng and Forgac, 1992; LoPachin *et al.*, 2007a,b, 2008b;
reviewed in LoPachin and Barber, 2006). The anionic sulfhydryl state,
which is only found in the catalytic triads of regulatory proteins, is
an acceptor for nitric oxide (NO) and, therefore, has lead to the
proposal that acrylamide-induced neurotoxicity results from disruption
of neuronal NO signaling (LoPachin and Barber, 2006; LoPachin *et al.*,
2008a).

2.  Fast Axonal Transport Disruption Hypothesis

Another proposed MOA is that both acrylamide and Gly inhibit the
movement of materials in fast axonal transport (Sickles *et al.*, 2002).
According to the "kinesin/axonal transport" hypothesis, toxicant
inhibition of kinesin could lead to reductions in the axonal delivery of
macromolecules that would eventually produce a deficiency of essential
proteins required to maintain axon structure and/or function. Distal
axons and nerve terminals are particularly vulnerable to transport
defects based upon an exceptionally large axonal volume (as much as 1000
times the volume of the neuron cell body) and the dependence of these
distal regions on long distance transport (100 fold longer length than
diameter of the cell body). This regional sensitivity is consistent with
the previously identified distal spatial distribution of
toxicant-induced damage (Cavanagh, 1964).

Microtubule motility assays using purified kinesin from bovine brain
identified a dose-dependent inhibition of kinesin as well as a less
sensitive effect on microtubules (Sickl*es et a*l., 1996). Preincubation
of either kinesin or taxol-stabilized microtubules produced a reduction
in the affinity between kinesin and microtubules, recognized as a
reduced number of microtubules bound or locomoting on an absorbed bed of
kinesin. Microtubules that were locomoting did so in a less directed or
staggering type of progression. The inhibitions were due to covalent
adduction, presumably through sulfhydryl alkylation, although adduction
of other amino acid residues such as valine was possible. The
non-neurotoxic analogue, propionamide had no effect. Other investigators
have identified kinesin inhibition by sulfhydryl reagents such as
N-ethylmaleimide and ethacrynic acid (Walk*er et a*l., 1997). As with
acrylamide, inhibition by these sulfhydryl reagents produced the
characteristic staggering movement of microtubules. The reaction was
slow and temperature dependent suggesting a sterically hindered cysteine
residue as an important adduct target. Additional studies have
demonstrated a comparable effect of glycidamide on kinesin (Sickles,
unpublished data). The predicted outcome of such an effect would be
reduced quantity of flow, precisely the outcome from several experiments
where rate of transport versus quantity could be discriminated (Sickles,
1989a; Sickles, 1989b; Ston*e et a*l., 1999).

Fast axonal transport has been studied in a variety of model systems
using diverse techniques. A comprehensive survey of acrylamide effects
on fast anterograde and retrograde axonal transport (Sickle*s et a*l.
,2002) revealed that all studies measuring fast transport within 24
hours of acrylamide exposure demonstrated significant reductions,
whereas longer postexposure delay was not associated with changes in
transport. Furthermore, a reduction in transport quantity (but not rate)
has been reported within 20 minutes of exposure. The duration of this
effect was 16 hours, with full recovery at 24 hours (Sickles, 1991).
Quantitation of transport after multiple dosings (i.e. 4, 7 or 10 doses)
had a similar effect on transport in the proximal sciatic nerve
(Sickles, 1991). The changes in transport were not due to an effect on
protein synthesis and exposure of only the axons confirmed that the
target was axonal (Sickles, 1989a; Sickles, 1992). Collectively, these
results suggested action on a target that is replaced via the fast
transport system, consistent with kinesin. The actions of acrylamide on
fast axonal transport were independent of effects on axonal
neurofilaments, as similar reductions were observed in wild-type and
transgenic mice lacking axonal neurofilaments (Sto*ne et a*l., 1999;
Sto*ne et a*l., 2000). The same results were observed using
radiolabelling of proteins in mouse optic nerves and differential
interference microscopy of isolated sciatic nerve axons. Other recent
studies have identified a parallel inhibition of retrograde axonal
transport by acrylamide (Sabri and Spencer, 1990), although it is
unclear whether this effect is due to inhibition of cytoplasmic dynein,
the retrograde axonal transport motor, or whether this is a result of
indirect effects of kinesin motor inhibition (Brad*y et a*l., 1990).

The predicted outcome from axonal transport compromise is a reduction in
vital macromolecules in the distal axons and an accumulation of
transported material within the axon. Morphological studies have
consistently identified accumulations of tubulovesicular profiles and
neurofilaments in axons of acrylamide-intoxicated animals (Spencer and
Schaumburg, 1991), which are morphological elements transported via
kinesin along microtubules. Other studies have identified reduced
synaptic vesicles in neuromuscular junctions (DeGrandchamp and Lowndes,
1990; DeGrandcha*mp et a*l., 1990). A reduction in GAP-43 in the
terminal neurites of cultured primary spinal cord neurons following
acrylamide exposure has been observed (Clarke and Sickles, 1996). Future
studies are required to quantitate reductions in specific axonal
compartments using a variety of neurotoxic and non-neurotoxic dosing
regimens in vivo to confirm the loss of physiologically or structurally
important macromolecules.

Additional supportive data for the axonal transport hypothesis comes
from several studies of kinesin knockouts as well as similarity to human
diseases. While most knockouts are lethal, low level mutations of
kinesin motors in Drosophila have identified an identical spatial
pattern of dysfunction and morphological similarity in axonal pathology
(G*ho et a*l., 1992; Hurd and Saxton, 1996) as with acrylamide
intoxication. The group of neurological disorders classified as
hereditary spastic paraplegias has a spatial pattern of ataxia,
spasticity and muscle weakness as observed with acrylamide intoxication.
Some of these types have been associated with mutations in kinesin
motors (Re*id et a*l., 2002), while others are the result of either
axonal or glial protein mutations. However, the common theme is
alteration in axonal transport (Reid, 2003; Gould and Brady, 2004).

Role of Acrylamide vs. Glycidamide

The respective adduct chemistries of acrylamide and glycidamide are well
understood and could have fundamental implications for neurotoxicity
regardless of the proposed mechanism; i.e., kinesin inhibition (Sickles
*et al.*, 2002) or blockade of NO signaling (LoPachin and Barber, 2006;
LoPachin *et al.*, 2008). Accordingly, an obvious data gap in the
current mechanistic understanding of acrylamide neurotoxicity, is the
relative roles of the parent compound and Gly. Thus, although early
research suggested that Gly produced neurotoxicity both in whole animal
(Abou-Donia *et al.*, 1993) and in vitro (Harris *et al.*, 1994) model
systems, other studies using similar models failed to find neurotoxic
effects associated with this metabolite (Brat and Brimijoin, 1993; Costa
*et al.*, 1992, 1995; Moser *et al.*, 1992). Clearly, resolving the
relative roles of acrylamide vs. glycidamide is an important issue that
will require more research. Although the adduct chemistry of these
toxicants has been reasonably defined, the precise molecular mechanisms
and sites of neurotoxicity are unknown.