YTAAP-12021; No.

of pages: 10; 4C:

Toxicology and Applied Pharmacology xxx (2011) xxx–xxx

Contents lists available at ScienceDirect

Toxicology and Applied Pharmacology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y t a a p

Utilizing toxicogenomic data to understand chemical mechanism of action in

risk assessment☆
Vickie S. Wilson a,⁎, Nagalakshmi Keshava b, Susan Hester a, Deborah Segal b, Weihsueh Chiu b,
Chad M. Thompson c, Susan Y. Euling b
a
National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711, USA
b
National Center for Environmental Assessment, Office of Research and Development, U.S. Environmental Protection Agency, 1200 Pennsylvania Ave., NW, Washington, DC 20460, USA
c
ToxStrategies, Inc., 23501 Cinco Ranch Blvd., Suite G265, Katy, TX 77494, USA

a r t i c l e i n f o a b s t r a c t

Article history: The predominant role of toxicogenomic data in risk assessment, thus far, has been one of augmentation of
Received 12 October 2010 more traditional in vitro and in vivo toxicology data. This article focuses on the current available examples of
Revised 25 January 2011 instances where toxicogenomic data has been evaluated in human health risk assessment (e.g., acetochlor and
Accepted 25 January 2011
arsenicals) which have been limited to the application of toxicogenomic data to inform mechanism of action.
Available online xxxx
This article reviews the regulatory policy backdrop and highlights important efforts to ultimately achieve
Keywords:
regulatory acceptance. A number of research efforts on specific chemicals that were designed for risk
Mechanism of action assessment purposes have employed mechanism or mode of action hypothesis testing and generating
Toxicogenomics strategies. The strides made by large scale efforts to utilize toxicogenomic data in screening, testing, and risk
Risk assessment assessment are also discussed. These efforts include both the refinement of methodologies for performing
toxicogenomics studies and analysis of the resultant data sets. The current issues limiting the application of
toxicogenomics to define mode or mechanism of action in risk assessment are discussed together with
interrelated research needs. In summary, as chemical risk assessment moves away from a single mechanism
of action approach toward a toxicity pathway-based paradigm, we envision that toxicogenomic data from
multiple technologies (e.g., proteomics, metabolomics, transcriptomics, supportive RT-PCR studies) can be
used in conjunction with one another to understand the complexities of multiple, and possibly interacting,
pathways affected by chemicals which will impact human health risk assessment.
Published by Elsevier Inc.

Introduction
The ability to assess a collection of biological changes in response to
exposure to environmental chemicals in toxicological evaluations and risk
assessments is an attractive promise of genomic and related “-omics”
technologies. If this potential was fully realized, toxicogenomic data could
contribute to identification of the multiple modes of action (MOA) and the
underlying detailed mechanistic steps involved in chemical toxicity. Omic
technologies have been successfully applied in screening and prioritiza-
tion programs, particularly in the pharmaceutical industry. For example,
microarrays, reverse transcriptase-polymerase chain reaction (rt-PCR),
proteomics, and metabolomics data have been used to inform mechanism
☆ Disclaimer: This article has been reviewed by the U.S. Environmental Protection
Agency and is approved for publication. The views expressed in this article are those of
the authors and do not necessarily reflect the policies of the U.S. Environmental
Protection Agency.
⁎ Corresponding author at: National Health and Environmental Effects Research
Laboratory, Office of Research and Development, U.S. Environmental Protection Agency,
MD-72, Research Triangle Park, NC 27711, USA. Fax: + 1 919 541 4017.
E-mail address: wilson.vickie@epa.gov (V.S. Wilson).
of action (Hamadeh et al., 2002a, 2002b) and, in general terms, screen for
adverse effects on human health of pharmaceutical drugs under
development. Screening and prioritization data can certainly complement
other toxicological evaluations in risk assessment especially as an aid to
early problem formulation. The goals of screening applications, however,
ultimately have a different purpose than risk assessment. In a screening
and prioritization context, genomic data interpretation can be less precise
than would be required for risk assessment. For example, a relatively high
false positive rate and low false negative rate in a genomic assay may be
acceptable for screening purposes (e.g., clinical screens, chemical
screening programs, early stages of drug development) because false
positives would be discovered and eliminated in subsequent “testing”
phases. Assays with high false positive rates and low false negative rates,
however, are not as useful when performing a risk assessment. Use of
genomic data in risk assessment requires that the data and statistical
analysis be reproducible and sensitive (with a low false positive rate) to
confirm genomic changes as well as to evaluate the magnitude of the
health risk associated with the genomic change after exposure. The more
firmly a change in gene expression or other cellular level alteration can be
linked to an adverse health outcome or malformation (e.g. phenotypic
anchoring), the more useful it is to risk assessment.

0041-008X/$ – see front matter. Published by Elsevier Inc.

doi:10.1016/j.taap.2011.01.017

Please cite this article as: Wilson, V.S., et al., Utilizing toxicogenomic data to understand chemical mechanism of action in risk assessment,
Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.01.017
2 V.S. Wilson et al. / Toxicology and Applied Pharmacology xxx (2011) xxx–xxx
In light of the recent National Academy of Science (NAS) report (NRC,
2007b) that developed a long-term vision for moving to a risk
assessment paradigm based on identifying toxicity pathways, it is
important to determine how to best utilize and/or mine toxicogenomic
data for risk assessment. The NAS report pointed to the strong potential
for genomic technologies to contribute to the risk assessment process
but acknowledged that neither its full impact has been realized nor has it
been broadly implemented in the field.
As definitions vary, the toxicogenomic terms used in both this article
and this special issue are described. -Omics is the broad discipline that
analyzes genome-wide interactions within a biological system. Various
subsets of -omics have developed including genomics, epigenomics,
transcriptomics, proteomics, and metabolomics. Genomics is the study
of an organism's DNA sequence (i.e., genome). Epigenomics or
epigenetics relates to the mechanisms by which parts of the genome
are switched off or on at strategic times and locations without actual
modification of gene sequence. Transcriptomics measures genome-wide
mRNA expression or, in other words, genes that have been transcribed at
a particular point in time (NRC, 2007a). Proteomics is the study of a
broad spectrum of proteins within an organism, which might include
their expression at a particular point in time, structural status (e.g.,
phosphorylated or dephosphorylated), functional states (i.e., specificity
and activity level), and their interactions with other cellular components
(Pandey and Mann, 2000). Metabolomics and metabonomics both study
low molecular weight (LMW) molecules that are produced as the net
result of cellular function (NRC, 2007a), but the former refers to the study
of LMW molecules within cells, whereas the latter refers to a more
systemic and complex change in tissues and body fluids (Ekins et al.,
2005). Toxicogenomics (TGx) is the application of any of these -omics-
based techniques to characterize responses to a toxic stimulus (NRC,
2007a).
One key strength of microarray and related -omics technologies is the
ability to generate information about thousands of genes or proteins (or
entire genomes or proteomes) in a single assay. Transcriptomics uses high
density and/or high-through-put methods of assessing mRNA expression
and new, second-generation microarrays are designed for the maximum
coverage of genes across the genome. However, the evaluation,
confirmation and interpretation of such a large, high density data set
presents additional challenges (Pettit et al., 2010). Specialized quantitative
real-time reverse transcriptase-polymerase chain reaction (qrt-PCR)-
based approaches that focus on specific genes or specialized sub-sets of
gene expression changes have also been developed. Qrt-PCR has
developed into a powerful, preferred confirmatory tool for quantifying
gene expression with improvements in sensitivity and specificity and
efficiency for signal detection.
Pathway-level analysis usually requires the data analyst or bioinfor-
matician to employ several tools to adequately mine the data set, as each
software tool provides a different perspective and evaluation of the data.
Bioinformatics is an overarching term referring to the creation of archival
databases, statistical algorithms, and specific analytical techniques for
both management and analysis of biological data. The primary goal of
bioinformatics is to increase the understanding of biological processes
through the application of computationally intensive techniques such as
pattern recognition and visualization of high-dimensional data (http://
www.ncbi.nlm.nih.gov/Tools/). Challenges to risk assessment include the
selection of appropriate data analysis tools as well as the interpretation of
the results.
The goal of this article is to review advances in the application of
toxicogenomic technologies to inform mechanism of action for risk
assessment, including strengths and limitations regarding the use of -omic
technologies and data analysis tools in risk assessment. As such, this article
focuses on the largely qualitative application of toxicogenomic data to
informing mechanism of action for risk assessment.
Further, toxicogenomic data have the potential to inform other
areas of risk assessment which are addressed in other articles within
this edition, including dose response assessment (Chiu et al., 2010-
Please cite this article as: Wilson, V.S., et al., Utilizing toxicogenomic data to understand chemical mechanism of action in risk assessment,
Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.01.017
this issue), intra-species (Mortensen and Euling, 2010—this issue) and
inter-species differences in MOA, and toxicokinetics. A number of issues,
however, must be surmounted before genomic technologies can be fully
utilized in risk assessment (Pettit et al., 2010) and, therefore, approaches
to using these datasets in a risk assessment scenario continue to be
explored (Hackett and Lesko, 2003; Boverhof and Zacharewski, 2006).
Use of toxicogenomic data in risk assessment
Standardization and validation of techniques
A significant challenge in the use of microarray technologies is the lack
of standards for presenting and exchanging such data. As a result, a
consortium of scientists from the Microarray Gene Expression Data
(MGED) Society developed and agreed upon the Minimum Information
Standards for Microarray Experiments (MIAME) for publications and
submission to public repositories (Brazma et al., 2001; Ball et al., 2004).
Since its inception, this database has become the “gold-standard” for
generating, analyzing, sharing, and publishing microarray-based data
(Table 1).
A more recent large-scale standardization effort is the MicroArray
Quality Control (MAQC) Consortium, a scientific community-wide
effort, spearheaded by FDA scientists, which brought together
researchers from government, industry, and academia to address
some of the issues of historical variability and to contribute to the
standardization of microarray procedures. This consortium in its first
two phases, through various experimental design modifications and
technical discussions, has contributed significantly towards providing
reproducible results within sites, between sites, and among the
various platforms (Canales et al., 2006; Casciano and Woodcock,
2006; Dix et al., 2006; Frueh, 2006; Guo et al., 2006; Ji and Davis, 2006;
Patterson et al., 2006; Shi et al., 2006; Shippy et al., 2006; Tong et al.,
2006). The group's goal is to reach consensus among stakeholders on
the best practices for the development and validation of predictive
models based on microarray gene expression as well as genotyping
data for personalized medicine and safety assessment (Table 1).
Briefly, in MAQC I, four pools of RNA samples were evaluated using
seven microarray platforms (6 commercially available and one
National Cancer Institute produced microarray spotted with Operon
oligonucleotides) by three independent laboratories per platform
with five replicates per RNA sample. The working list of genes was
refined to include 12,091 reference genes that were detected on each of
the six high density platforms. Generally there was good concordance
across the various platforms. For example, all one-color microarray
platforms (such as Affymetrix) had median coefficients of variation (CVs)
of 5–15% and a concordance rate of 80–95% for their qualitative detection
calls (detected or non-detected) between sample replicates. When
comparing variation within and between test sites, the one-color
platforms demonstrated 80−95% agreement (Shi et al., 2006). Further,
the results from three qrt-PCR-based platforms were evaluated against
five of the commercial microarray platforms. Linear regression analysis
indicated good agreement across the three qrt-PCR platforms with R2
values from 0.81 to 0.88. There was also very good correlation of fold
change data between microarray results and quantitative gene expression
results. Several sources for limited incongruence were identified such as
decreased sensitivity for low expression genes in the microarray platforms
as compared to the qrt-PCR-based technologies and differences in probe
location (Canales et al., 2006).
In MAQC-II the capabilities and limitations of various data analysis
methods were assessed. The information and conclusions have been
recently published in a series of articles in the Pharmacogenomics
Journal (Aug 2010) and Nature Biotechnology (Shi et al., 2010). The
third phase, MAQC-III aims to evaluate the technical performance of
next-generation sequencing platforms and phase IV, MAQC-IV, is
being planned with an objective of assessing inter-individual
 V.S. Wilson et al. / Toxicology and Applied Pharmacology xxx (2011) xxx–xxx
Table 1
Some of the major activities, including documents, tools, and workshops, in the field of toxicogenomics that have significantly impacted the acceptance and use of toxicogenomic
data in research and regulatory arenas.
Activity (source) Description
Research and resources
MicroArray Quality Control MAQC is a scientific community-wide research
Consortium (MAQC) (multi-stakeholder effort, led by U.S. FDA, including MAQC I, II, and III
scientific and regulatory community) (to date). The goal of the project is to assess
microarray study variability and develop
standards and quality measures in order for
microarray and next generation sequencing data
to be used in a regulatory context.
Application of toxicogenomics The document, aimed at regulatory decision-
technologies to predictive toxicology makers, presents the potential benefits and
and risk assessment (NRC) limitations to utilizing genomic data and
committee recommendations to address
challenges.
Toxicity testing in the 21st century: a The document presents a future vision of a
vision and a strategy (NRC) toxicity-pathway based toxicity testing and risk
assessment, representing a paradigm shift.
Policies and standards
Minimum Information About a Established to provide guidance for a minimum
Microarray Experiment (MIAME) amount and type of microarray information
(multistakeholder; The Functional needed to publish microarray papers. The goal
Genomics Data (FGED) Society) was to have uniformity among scientists for the
amount of microarray data needed for
publication. Currently, several public
repositories exist such as ArrayTrack™ at the
FDA (U.S.), ArrayExpress at the European
Bioinformatics Institute (EBI) (U.K.), GEO at
NCBI (U.S.), and CIBEX at DDBJ (Japan), which
are designed to accept, hold, and distribute
MIAME-compliant microarray data.
Guidance for industry to The guidance provides recommendations to
incorporate submission of industry with investigational new drug
pharmacogenomic data (FDA) applications, new drug applications, and
biologics license applications on when, during
product development and review processes,
the pharmacogenomic data should be
submitted; the submission format; and how
and in what cases the data will be used in
regulatory decision making.
Interim policy on genomics (EPA) Outlines the strengths and limitations to using
genomic data for EPA regulatory decision-
making. Encourages the submission and
evaluation of genomic data, when available, as
supporting material, particular for
characterizing a chemical's MOA.
Draft interim guidance for microarray- Document developed to respond to the need
based assays: data submission, quality, for a framework for analysis and acceptance
analysis, management, and training criteria for genomic data for use in regulatory
considerations (EPA) decision-making. The document covers the
performance of assays across genomic
platforms (e.g., reproducibility, sensitivity,
pathway analysis tools) and the criteria for
accepting genomic data for use in a risk
assessment (e.g., assay validity, biologically
meaningful response).
Please cite this article as: Wilson, V.S., et al., Utilizing toxicogenomic data to understand chemical mechanism of action in risk assessment,
Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.01.017
3
Key point(s) References
▪ MAQC I resulted in demonstration of http://www.fda.gov/ScienceResearch/
relatively high reproducibility within and BioinformaticsTools/
between laboratories and among microarray MicroarrayQualityControlProject/
platforms (but not different biological sample default.htm
preparations).
▪ MAQC II assessed predictive models among 36 MAQC Consortium (Shi et al., 2006)
teams, for microarray data analysis. The
conclusions are that model performance
depended on the endpoint and the team and that
the performance of models generated using
different methods were quite similar.
▪ The goal of MAQC-III (or Sequencing Quality MAQC Consortium (Shi et al., 2010)
[SEQC]) is to assess the performance of next-
generation sequencing platforms by developing
benchmark datasets with reference samples and
assessing advantages and limitations of various
RNA and DNA bioinformatic approaches.
Overall conclusions regarding risk NRC, 2007a
assessment include:
▪ The reproducibility issues surrounding
toxicogenomic tools have been largely resolved
but regulatory validation of technologies needed;
▪ Specialized bioinformatic, statistical, and
computational methods are needed;
▪ Potential of toxicogenomic data to improve
risk assessment is just beginning and is not
ready to replace current toxicity testing;
▪ Need to improve govt agencies' ability to
integrate toxicogenomic data into the
different risk assessment steps/areas by
investing in research and personnel.
Toxicity tests would be based on identifying NRC, 2007b
perturbations in key human toxicity pathways
from a combination of human in vitro tests and
computational tools.
The MIAME standards were developed in order http://www.mged.org/
to 1) allow for the interpretation of the results of Brazma et al., 2001
the experiment unambiguously; and 2) to
reproduce the experiment.
FDA is requiring for some cases, and accepting, in FDA, 2005; http://www.fda.gov/
other voluntary cases, genomic data for some downloads/RegulatoryInformation/
drug applications. Guidances/ucm126957.pdf
Genomic data can be used on a case-by-case basis U. S. EPA, 2002; http://www.epa.gov/
in a weight-of-evidence approach in EPA risk spc/pdfs/genomics.pdf
assessments.
Regarding submission of microarray data to EPA, U. S. EPA, 2007 http://www.epa.gov/
the document recommends that submissions spc/pdfs/
include sufficient information to allow an epa_interim_guidance_for_microarray-
independent reviewer to reconstruct how the based_assays-external-review_draft.
data were collected and analyzed and further, pdf
proposes a slight modification to MIAME format
for EPA submissions. Regarding data analysis, the
document presents a systematic approach, a
genomics Data Evaluation Record template to
present and organize data from genomics studies.
4 V.S. Wilson et al. / Toxicology and Applied Pharmacology xxx (2011) xxx–xxx
variability in response to treatment with an emphasis on idiosyncratic
adverse drug reactions.
The MAQC project evaluated intrasite, intersite and interplatform
differences, and has contributed much valuable information for
evaluation of TGx data. There are several issues of value to the risk
assessment process, however, that were not addressed. Because all
test sites used the same RNA samples, protocol, and generated
replicate data at approximately the same time (Shi et al., 2006), the
evaluation does not address different sample preparation methods,
variation in protocols, effect of time differences or other technical
variables. It also did not include more “biology-based” performance
metrics such as pathway analysis. Further, while providing valuable
information, the MAQC efforts are all technique and technology focused.
Little attention has been dedicated to other needs for the use of TGx data in
risk assessment such as study design including dose–response, length of
exposure and adequate statistical power especially at low doses. These
factors all currently limit the application of TGx data to the risk assessment
process.
The development of improved toxicogenomic tools for the
technologies and data analysis, including publicly-available resources,
has led to increased sensitivity and reproducibility of the resulting
toxicogenomic data, and as a result, improves the ability to evaluate
and interpret these data for risk assessment. For example, multiple
species now have complete genome sequence data available. The
availability of the DNA genome sequences for multiple species used in
toxicity testing and risk assessment should enhance the development
of more complete microarrays. The availability of the human genome
sequence allows for comparisons to be made between the function of
genes and pathways between test species and humans. Another
development that has improved toxicogenomic tools is the develop-
ment of “2nd” generation microarrays that represent a majority of the
species’ genes and microarrays that represent genes from particular
pathways or processes with enhanced capability to provide informa-
tion about altered gene expression and affected pathways. As
mentioned earlier, the development of quantitative RT-PCR allows
for assessment of the quantity and timing of expression of genes of
interest. There are numerous repositories for microarray data (e.g.,
Array Express) that are consistent with MIAME compliance that have
improved transparency of toxicogenomic data. Further, the develop-
ment and testing of data analysis tools, including publicly-available tools
(National Center for Biotechnology Information, U.S. National Library of
Medicine, NIH EntrezGene, http://www.ncbi.nlm.nih.gov/gene; NIEHS
Environmental Genome Project, http://www.niehs.nih.gov/research/
supported/programs/egp/; National Human Genome Research Institute,
http://www.genome.gov/; Wellcome Trust's The Human Genome,
http://genome.wellcome.ac.uk), have provided the scientific and regu-
latory communities with multiple tools for different applications, some
of which are more appropriate for risk assessment than others (see
(Ovacik et al., 2010—this issue).
Regulatory guidance
Many regulatory agencies such as the Food and Drug Administration
(FDA) and Environmental Protection Agency (EPA) are evaluating -omics-
generated data for risk assessment. An important benchmark in the use of
toxicogenomic technologies is its inclusion and acceptance in regulatory
submission packages and its use in the risk assessment process. While at
this point in time, there is no requirement for the submission of
toxicogenomic data to major regulatory agencies, many agencies
including the EPA and FDA encourage voluntary submission and have
drafted guidance documents for those submissions (FDA, 2007, 2009;
European Medicines Agency, 2009; U. S. EPA, 2009).
The EPA's Science Policy Council Interim Policy on Genomics instructs
EPA to use genomics data on a case-by-case basis in a weight-of-evidence
approach in risk assessment (U. S. EPA, 2002). Following the release of the
Interim Policy, the EPA's Science Policy Council (SPC) created a cross-EPA
Please cite this article as: Wilson, V.S., et al., Utilizing toxicogenomic data to understand chemical mechanism of action in risk assessment,
Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.01.017
Genomics Task Force that produced a white paper, Potential Implications of
Genomics for Regulatory and Risk Assessment Applications at EPA, which
identified four areas of oversight likely to be influenced by genomic data
and identified needs for (a) analysis and acceptance criteria for genomic
information in scientific and regulatory applications, (b) methods for
interpreting genomic information for risk assessment, and (c) deter-
mining a relationship between genomic changes and adverse outcomes
(U.S. EPA, 2004b). In response, the Genomics Technical Framework and
Training Workgroup, developed an Interim Guidance for Microarray-
Based Assays: Data Submission, Quality, Analysis, Management, and
Training Considerations (U. S. EPA, 2007). This document made specific
recommendations for the submission, quality assurance, analysis, and
management of genomics data in the context of current possible
applications.
The NRC report, Applications of Toxicogenomic Technologies to Predictive
Toxicology and Risk Assessment (NRC, 2007a) and Toxicity Testing in the 21st
Century: a Vision and a Strategy (NRC, 2007b) recommends a toxicity-
pathway perturbation approach to toxicity testing and risk assessment
and making full use of the rapidly evolving scientific understanding of
molecular pathways that maintain cell function and influence the causal
pathway to disease. The NAS vision (NRC, 2007b) to move to a process
that relies more on in vitro assays has been adopted by EPA (U. S. EPA,
2009).
Using toxicogenetics data to inform mechanism of action for risk
assessment purposes
Toxicogenomic data can be used in risk assessment of a given chemical
at multiple levels (e.g., toxicokinetic, toxicodynamic), depending on the
available data, to inform the exposure to outcome continuum. Using data
from multiple genomic approaches (genome, transcriptome, proteome,
and metabolome) is promising for defining the temporal steps in the
process of toxicity and for identifying precursor events. To do so, however,
would require the integration of multiple disciplines and require expertise
from multiple fields. Further, studies would need to be designed with risk
assessment requirements for dose response information and linkages to
adverse outcome. The predominant role of toxicogenomic data in risk
assessment thus far has been one of augmentation of other more
traditional in vitro and in vivo toxicology data. Nevertheless, considera-
tions and approaches for using toxicogenomic data sets in risk assessment
continue to be explored (Waters and Fostel, 2004; Chan and Theilade,
2005; Oberemm et al., 2005; Boverhof and Zacharewski, 2006; NRC,
2007a, 2007b; Thybaud et al., 2007; Daston, 2008; Carlson and Silkworth,
2009; Van Aggelen et al., 2010). This section provides an overview of how
toxicogenomic data, depending on the type, could potentially be used in
screening and prioritization programs and risk assessment to inform a
chemical's mechanism of action, defined here as toxicokinetics and
toxicodynamics steps (Fig. 1).
Toxicokinetics (TK) is the rate at which a chemical enters the body and
what happens to the chemical within the body. Pre-and post-exposure
information on gene and protein expression, protein function, and LMW
molecules (e.g., GSH) can provide important information about the
absorption, distribution, metabolism, and excretion (ADME) capabilities
of an organism or tissue. In this regard, -omics technologies are similar to
more traditional in vitro and in vivo studies. However, these newer
technologies can provide information on activities of key proteins such as
transporters and metabolic enzymes in tissues on a larger scale. For
example, high-throughput approaches have been used to broadly assess
metabolic differences between cell types of normal, malignant, and
immortalized phenotypes (Hedberg et al., 2001; Vondracek et al., 2002;
Staab et al., 2008). Furthermore, metabolomic/metabonomic data may
indicate depletion, consumption, or production of LMW molecules
involved in metabolism of a chemical. Such changes in some tissues, but
not others, may inform the disposition of a toxicant of interest. In addition,
pre- and post-exposure differences in transcripts, proteins, and LMW
molecules in vitro have potential to be useful for toxicokinetic
 V.S. Wilson et al. / Toxicology and Applied Pharmacology xxx (2011) xxx–xxx 5

Fig. 1. Potential uses of toxicogenomic data in understanding a chemical's mechanism of action for application to human health risk assessment of environmental chemicals. The
exposure–outcome continuum for environmental chemical exposures is presented. In between exposure and outcome are all of the steps of the mechanism of action, including TK as
well as TD steps. TGx data can inform one or more steps in this continuum depending on the available TGx data information. Arrows with “TGx” indicate the potential types of
information these data can provide. For example, data from direct TGx measures of gene expression changes after chemical exposure can inform ADME (e.g., by identifying metabolic
pathways/liver enzyme effects) which could be used, in turn to inform internal dose. Direct TGx measurement data can provide information about altered molecular events within TD. TGx
data associated with either exposure or a particular outcome can be used as a biomarker of exposure or outcome. TGx data from multiple species can be used to inform interspecies
differences. Or, if TGx data are only available for one species, sequence data for multiple species could be used to make inferences about interspecies differences in the mechanism of action.
TGx data from multiple individuals can inform intraspecies differences (see Mortensen and Euling, 2010—this issue). ADME, absorption, distribution, metabolism, and excretion; TD,
toxicodynamics; TK, toxicokinetics; TGx, toxicogenomic. This figure is adapted from US EPA (2009).

extrapolations to in vivo settings for risk assessment purposes (Kedderis
et al., 2006). Conceivably, toxicogenomic techniques could also be used to
similarly examine in vivo differences in metabolic competency across
species although, at the current level of understanding, these would not be
substitutes for traditional cross-species toxicokinetic evaluations.
Within humans, inter-individual differences in chemical or drug
disposition arise in part from genetic variability within the population.
Genetic polymorphisms in phase I and phase II enzymes can alter
responses to xenobiotics (Ginsberg et al., 2002, 2009a, 2009b, 2009c;
Neafsey et al., 2009a, 2009b; Walker et al., 2009; Ginsberg et al., 2010).
Copy number polymorphisms in phase I enzymes such as that seen with
the metabolic enzyme, CYP2D6, can result in some individuals being
characterized as either “poor” or “ultrarapid” metabolizers of certain
xenobiotics (Bodin et al., 2005; Buckley et al., 2005) Toxicogenomic
techniques can be used to assess the frequency of genetic and copy
number polymorphisms within populations. When the impact of these
polymorphisms on enzyme function is established, the information could
be used to characterize the differences in predicted internal dose across
the population, and potentially be used in probabilistic risk assessment.
Example uses of toxicogenomic data to inform mechanism of action in
EPA risk assessments
Toxicogenomic data have been used in several different ways for
informing mechanism of action. EPA has evaluated toxicogenomic data
qualitatively in two final assessments of environmental chemicals,
acetochlor and dimethylarsenic acid (DMA). The EPA's Office of Pesticide
Programs (OPP) Cancer Assessment Review Committee (CARC) evaluated
genomic data in the cancer assessment of acetochlor (U.S. EPA, 2004a).
OPP considered acetochlor, alachlor, and butachlor as a group with a
common mechanism of action based on the observation that exposure to
each of the three pesticides induced nasal turbinate tumors via
cytotoxicity and cell proliferation. Genomic data from studies with
olfactory mucosa from rats treated with alachlor, were used to support
cytotoxicity with regenerative cell proliferation as the MOA for acetochlor
in the assessment (Genter et al., 2002). The genomic data enabled the
identification of processes and pathways affected by alachlor exposure
including oxidative damage and the generation of reactive oxygen species,
as possible mechanisms underlying cytotoxicity (Genter et al., 2002).
Specifically, based on genomic analysis of alachlor-induced tumors in the
olfactory mucosa, a four-step carcinogenesis model was proposed.
Second, a recent EPA risk assessment of DMA evaluated available
genomic and toxicity studies as part of their evaluation (U.S. EPA, 2006).
The DMA data suggest that urothelial cytotoxicity and regeneration, key
events in rat bladder tumor formation, are relevant to humans. Pathway-
level analysis revealed that DMA affects both common and unique
pathways in the bladder transitional cells of rats and an immortalized
human cell line (non-availability of primary human cells influenced the
choice of an immortalized cell line (Sen et al., 2007). This UROtsa cell line
was derived from the normal urothelium lining the ureter and was
immortalized (simian virus (SV40) large T antigen). The authors
recognized that immortalized cell lines have inherent limitations, but in
lieu of primary cells, human immortalized cell lines can provide relevant
information. The finding of common pathways in the rat urothelium (Sen
et al., 2005) and human cells provides evidence of concordant biological
processes affected by DMA, thereby supporting the conclusion of
biological plausibility in humans. However, the finding of other pathways
indicated that there are some species differences in response. Thus, the
genomic data, in conjunction with toxicology studies, provided both
mechanism of action and human relevance information to the assessment.
In both cases, the toxicogenomic data informed the mechanism of action
in the weight of evidence analysis.
Hypothesis generation for mechanism of action and testing studies
Policy language (U. S. EPA, 2002) clearly indicates that EPA is
interested in utilizing toxicogenomic data in the risk assessment of
environmental chemicals. Some available toxicogenomic studies are being

Please cite this article as: Wilson, V.S., et al., Utilizing toxicogenomic data to understand chemical mechanism of action in risk assessment,
Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.01.017
6 V.S. Wilson et al. / Toxicology and Applied Pharmacology xxx (2011) xxx–xxx
evaluated and incorporated in ongoing EPA risk assessments, particularly
by providing information to further define a chemical's mechanism of
action. Conazoles, phenobarbital, and formaldehyde are examples of
environmental agents with toxicogenomic data that informs the
mechanism/mode of action. A comprehensive research study was
undertaken to examine the toxicity and transcriptional effects of
hepatotumorigenic conazoles, a group of fungicides used in agriculture
and medicine, in mouse (Ward et al., 2006; Wolf et al., 2006; Nesnow et al.,
2009). These papers, of gene transcriptional and in-life evaluation studies,
related the alterations at both the gene expression and pathway level to
tumorigenesis leading to the identity of potential modes of tumorigenic
action. The mouse studies of Ward et al. (2006) and Allen et al. (2006) are
briefly reviewed first. Ward et al. (2006) conducted a microarray study to
identify transcriptional changes provide clues for potential mechanisms of
tumorigenic action. Parallel in-life studies were conducted to measure
conventional toxicological endpoints (Allen et al., 2006). Briefly, CD-1
mice received triadimefon, propiconazole, or myclobutanil in the feed for
4, 30, or 90 days. Combining apical endpoints described in Allen et al.
(2006) to the transcriptional profiles defined in Ward et al. (2006)
allowed a richer understanding of conazole-induced toxicity. The three
conazoles shared common treatment-related responses for the following:
induction of hepatomegaly, high levels of hepatic pentoxyresorufin-O-
dealkylase activity, increased hepatic cell proliferation, and decreased
serum cholesterol. GeneChip arrays were used to generate the transcrip-
tional measurements. As expected all tested conazoles had effects on
nuclear receptors, however, a subset of altered genes and pathways
distinguished the three conazoles. In summary, while triadimefon,
propiconazole, and myclobutanil had similar non-genomic effects,
genomic analyses showed differences in their gene expression profiles.
Moreover, a series of potential key events leading to conazole-induced
hepatocarcinogenesis were proposed from the transcriptomic and
toxicological data. These data have the potential to support a future
conazole mechanism of action risk assessment review.
In a second mouse toxicogenomic study, Nesnow et al. (2009)
noted that some hepatotumorigenic conazoles display many of the
same hepatic toxicologic responses as the prototypical mouse liver
carcinogen phenobarbital (PB) and therefore the authors examined
whether these conazoles were like or unlike PB using toxicogenomics.
This study was relevant because epidemiologic studies on patients
who received long-term PB therapy showed no increase in the
incidence of hepatic cancer, even though their PB plasma levels were
close to those found in susceptible rodents (IARC, 2001). Chemicals
that are mouse liver tumorigens and produce PB-associated responses
in mouse liver are considered to be “PB-like” and thus their
tumorigenic responses have been viewed as not relevant to humans
(Holsapple et al., 2006). Therefore, it was important to compare the
molecular changes induced by conazoles to those change induced by
PB using detailed transcriptomic analyses. Nesnow et al., 2009 applied
transcriptional analyses to hepatic tissues from mice exposed to
tumorigenic doses of PB, propiconazole (Pro) or triadimefon (Tri)
(same genomic data generated in mouse liver from Ward et al., 2006).
This was done to probe whether gene changes induced by the
tumorigenic conazoles shared similarities or differences to those
generated by PB treatments. Mice were administered feed containing
PB, Pro or Tri for 4 and 30 days. Targeted transcriptomic analyses were
conducted at many levels including genes, canonical pathways,
toxicity pathways, and gene networks. In addition, subsets of genes
including cell cycle genes, transcription factors, and genes reported to
be associated with human hepatocellular cancer were compared. The
authors concluded that although PB and the two conazoles induced
mouse liver tumors and exhibited similar toxicological responses,
their transcriptional profiles were significantly different and thus
their mechanisms of tumorigenic action were highly likely to differ.
The authors posited that the transcriptional profiles of tissues exposed
to toxic chemicals inherently contain their mechanisms of toxicity.
The authors suggest, therefore, that the results of this study provided
Please cite this article as: Wilson, V.S., et al., Utilizing toxicogenomic data to understand chemical mechanism of action in risk assessment,
Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.01.017
a more scientifically defensible approach to determining whether the
mechanism of tumorigenesis of these pesticides are unlike PB, or like
PB, and thus potentially relevant or not relevant to humans. Moreover,
these data may be informative for the assessment of human relevance.
Another example of toxicogenomic studies that were used to
inform the MOA is a transcriptional study related to nasal carcinoge-
nicity that compared gene expression changes after formaldehyde vs.
after glutaraldehyde exposure (Hester et al., 2005). Human exposure
to formaldehyde and glutaraldehyde occurs in a number of workplace
and environmental settings. Formaldehyde is a known carcinogen in
the rat nose, while glutaraldehyde, though cytotoxic, is not known to
induce cancer. Both compounds induce similar tissue responses,
including inflammation and cellular damage, and cannot be distin-
guished by commonly used toxicological endpoints. Given that the
risk to humans is uncertain, a series of genomic studies were
undertaken at the U.S. EPA's Office of Research and Development
utilizing genomic profiling to discriminate between the two com-
pounds (Hester et al., 2003, 2005). The studies were conducted using
F344 rats. Treatments included 1-, 5-, or 28-day exposure by nasal
instillation of formaldehyde solution (400 mM) or glutaraldehyde
solution (20 mM). Total RNA was extracted from respiratory cells for
subsequent genomic profiling. Given that both aldehydes induced
similar acute and subchronic histopathology and given that early
aldehyde-induced lesions are microscopically similar, the authors
investigated whether transcriptional patterns using cDNA technology
could explain the different cancer outcomes.
Results showed that the carcinogenic formaldehyde could be
distinguished from the noncarcinogenic glutaraldehyde based on the
different patterns of genomic responses. Glutaraldehyde's lack of
carcinogenicity may be more related to the pattern of transcriptional
profiles showing greater toxicity from the observed lack of DNA-
repair, greater mitochondrial damage, and increased apoptosis
compared to profiles associated with formaldehyde exposure. These
two environmental chemical examples illustrate how microarray and
RT-PCR studies, by examining molecular pathway alterations associ-
ated with exposure to environmental chemicals, can be used to
generate and test mode and mechanism of action hypotheses to
further understand the mechanism of action that, in turn, could be
utilized in risk assessments.
New methods to analyze toxicogemomics data in risk assessment
As mentioned in earlier sections, to fully utilize toxicogenomic
data in understanding mechanism of action, new data analysis and
management methods are needed. This is in line with the recom-
mendations put forth in the NAS report Toxicity Testing in the 2st
Century (NRC, 2007b). The NAS report stated that in order to
implement its vision, regulatory agencies will need to utilize high
and medium-throughput assays, many of which provide -omics data.
The NAS report added that the development of methods to
understand the circuitry of toxicity pathways will involve the use of
“functional genomics techniques for integrating and interpreting
various data types…It will most likely require improved methods in
bioinformatics, systems biology, and computational toxicology” (page
129) (NRC, 2007b). To this end, several institutions have been
engaged in developing such methods and tools for analyzing
microarray data and merging data from genomics, quantitative
structure–activity relationship (QSAR), and other medium to high
throughput assays. This section highlights cutting edge techniques
under development at the EPA-funded Science To Achieve Results
(STAR) Computational Toxicology Research Centers.
The STAR New Jersey Research Center for Environmental Bioinfor-
matics and Computational Toxicology (ebCTC) is a research consor-
tium of the University of Medicine and Dentistry — Robert Wood
Johnson Medical School, Princeton University, Rutgers University, and
the FDA's Center for Toxicoinformatics. This center is developing tools
 V.S. Wilson et al. / Toxicology and Applied Pharmacology xxx (2011) xxx–xxx
and methods for the analysis or prediction of molecular events and
the evaluation of changes to genes and proteins in an organism
following exposure to toxicants (Ovacik et al., 2010—this issue). These
tools and methods include ebTrack and the environmental bioinfor-
matics Knowledge Base.
ebTrack, an integrated bioinformatics system for environmental
research and analysis, is being built upon the framework of the FDA's
ArrayTrack™ . ebTrack will provide (1) centralized data management,
(2) rapid data analysis capability via an intuitive user interface, and
(3) informed data interpretation through interfaces to relevant
knowledge bases of gene annotation, protein function and pathways
(Chen et al., 2009). By enhancing ArrayTrack™, which manages and
interprets microarray data, ebTrack will be more applicable to
environmental health research data and will not be limited to the
management and analysis of data from DNA microarray experiments
for humans, mice, and rats. In addition to gene expression data from
DNA microarrays, it will be able to handle proteomics data,
metabonomics data, chemical structure information, and in vitro/in
vivo toxicological data (Chen et al., 2009).
The environmental bioinformatics Knowledge Base is a comprehen-
sive compendium of tools, databases, and literature under development
by the Center. Currently, the Knowledge Base is undergoing alpha testing
(Chen et al., 2009). This Knowledge Base will support ebTrack, and will
eventually be a public resource. It will provide resources on research areas
that are relevant to the development of methods and tools for improving
linkages in the source-to-outcome paradigm, hazard identification and
quantitative risk assessment. Specifically, it will support the following
bioinformatics areas: physiomics/histomics, cytomics, metabolomics,
proteomics/interactomics, and genomics/transcriptomics (http://dido.
rutgers.edu:4000/enviroinformatics).
The STAR Carolina Environmental Bioinformatics Center (CEBC)
established to support the field of computational toxicology through
expertise in -omics and the “elucidation of genetic variation” (U.S. EPA,
2009), is developing many tools and methods to assist in the
interpretation of toxicogenomics. FastMap and SAFEGUI are two
examples. FastMap is a software package for gene Expression Quantitative
Trait Locus (eQTL) mapping (Gatti et al., 2009a). The increase in gene
expression and high-density genotype data has improved the ability of
eQTL mapping to identify genomic loci associated with phenotypic
variation in homozygous populations. However, existing tools cannot
accurately calculate associations between tens of thousands of transcripts
and thousands to millions of single nucleotide polymorphisms (SNPs).
FastMap organizes SNPs into what is called a “Hamming distance-based
tree” that reduces the number of arithmetic operations needed for such
calculations by taking advantage of the association between the SNP and
its parent SNP in the tree (Gatti et al., 2009a). The graphical user interface,
SAFEGUI, for the Significance Analysis of Function and Expression (SAFE)
software assesses the significance of biological categories in microarray
studies, while accounting for the effects of expression correlations among
mRNA transcripts. This is accomplished through the incorporation of
permutation and bootstrap algorithms that are better able to detect
differential expression across categories of genes (Gatti et al., 2009b).
Although only nearing the end of its second year, the STAR Carolina
Center for Computational Toxicology (CCCT), also located at the University
of North Carolina, is developing novel methods, including one for the
analysis of metabolomics data, Progressive Consensus Alignment of NMR
Spectra—PCANS (Staab et al., 2010). Just as microarrays and other
technological advances are enabling the evaluation of thousands of mRNA
transcripts, techniques have been developed that allow for quantitative
measurements of metabolites in a large number of biological samples.
However, when trying to measure large numbers of samples using NMR
spectroscopy, peak location can vary as a function of different sample
conditions. PCANS aligns NMR spectra, through the progressive integra-
tion of many pairwise comparisons (Staab et al., 2010). This approach
should improve the ability to analyze metabolomic data that will likely be
useful in evaluating the toxicity of chemicals.
Please cite this article as: Wilson, V.S., et al., Utilizing toxicogenomic data to understand chemical mechanism of action in risk assessment,
Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.01.017
7
The Texas-Indiana Virtual Star Center (TIVS), the newest STAR
computational toxicology center, is a collaborative effort between the
University of Houston, Texas A&M, and Indiana University. This center
began its research to develop methods and tools for assessing
developmental toxicity in November 2009. Through medium and
high throughput assays, utilizing mouse embryonic stem cells and
zebrafish, the Center plans to generate data that will be used to build
predictive in silico models for developmental toxicity that are also
relevant to humans. First, in silico models will be built to replicate
normal developmental processes and then they will be developed to
elucidate the mechanisms by which chemical exposures perturb the
pathways (U.S. EPA, 2009). The major challenge to be addressed by
the in silico models is how to move from events taking place on the
cellular level to tissue-level patterning (U. S. EPA, 2002).
Discussion
Current issues and research needs in utilizing toxicogenomics to define
mode and mechanism of action in chemical risk assessment
The genome-wide coverage of genomic technologies as compared
to the toxicological evaluations including histology, organ weights,
and single gene expression measures offers great potential for
advancing chemical risk assessment by providing a much richer
picture of the biological state of the an organism at the mechanistic
level across, for example, life stage, duration of exposure, and organs
and tissues. However, there are also a number of limitations to
applying the technology to human health chemical risk assessment.
Here we highlight some of the current issues and key research needs
in utilizing toxicogenomic data to define a chemical's mode or
mechanism of action within a risk assessment context, some of which
are related to regulatory acceptance of these data within a changing
risk assessment landscape.
The Health and Environmental Sciences Institute's (HESI) Committee
on the Application of Genomics to Mechanism-based Risk Assessment
multi-sector scientist survey identified three areas that toxicogenomic
data would have the greatest potential utility to risk assessment:
understanding biological mechanisms, biomarker candidates, and species
differences (Pettit et al., 2010). Regarding understanding biological
mechanism, which is the focus of this discussion, the survey identified
some key challenges, including the currently incomplete biological
context in which to understand toxicogenomic data and variability in
data interpretation. For instance, a particular chemical's toxicogenomic
data set may identify some genes lacking annotation; some genes with
data indicating involvement in multiple pathways; and pathways with
limited information about pathway relationships to one other and to the
toxicological outcome. This lack of information about the complex inter-
relationships among chemicals, genes, pathways, and toxicological
outcome is exacerbated by two other overarching problems: 1) data
interpretation variability; 2) insufficient data to determine causal
relationships between toxicogenomic effects and toxicological outcomes
(i.e., phenotypic anchoring). While these issues apply to the evaluation of
any data type, they present a particular challenge for toxicogenomic data
because of the high density and quantity of data.
With respect to data interpretation variability, because data are
generated from thousands of genes, the probability increases of
incorrectly interpreting the data for any particular gene and
generating either false-positive or false-negative results. Thus, many
algorithms such as multiple test corrections have been developed in
an attempt to control for this issue. Improved methods are greatly
needed, particularly those that err conservatively to further reduce
identifying genes and pathways by chance. On the other hand, there is
also the potential for missing larger patterns of more modest gene
changes that may be filtered out by stricter statistical cut-offs on
individual genes. Methodologies are currently being developed for
such “pathway-based” analyses to estimate the degree of toxicity
8 V.S. Wilson et al. / Toxicology and Applied Pharmacology xxx (2011) xxx–xxx
pathway perturbations, some of which do not involve “pre-filtering”
that may increase false negatives (see Ovacik et al., 2010—this issue).
However, no method or set of methods has reached sufficient
maturity and undergone sufficient testing for general regulatory
acceptance and the determination of biological significance of affected
genes or pathway remains a challenge. Further, data analysis and
interpretation methods considered appropriate for screening pur-
poses, which have been the focus of most existing toxicogenomic
applications and research, differs significantly from those appropriate
for risk assessment purposes. Thus, a key research need is the
development of data analysis and interpretation methods specifically
geared towards testing hypotheses related to risk assessment.
One example of a risk assessment need related to the interpreta-
tion of toxicogenomic data is the issue of establishing causation
between gene expression changes and toxicological outcomes. Part of
the difficulty stems from the fact that, even with traditional data,
different scientists may put the mechanism of action puzzle together
differently based on the same data set. However, with respect to
toxicogenomic data, variability in scientific judgment is exacerbated
by the complexity and incompleteness of the biological understanding
of how genes relate to one another, and how they relate to toxicity. In
addition, most studies are not specifically designed to test causation.
Ideally, experiments for establishing causation would involve evalu-
ation of genomic data and data on the toxicological outcome of
interest in the same, or at least the same set of, experimental animals.
Better yet would be longitudinal data in which early (in time)
genomic markers could be linked to subsequent development of a
toxicological outcome in the same experimental unit. Thus, stronger
experimental designs that include linkages to adverse toxicological
outcome are a key research need, as determining associations or
causal links between the alterations in gene expression and in vivo
toxicological endpoints is difficult with the currently available data.
Other concerns with the use of toxicogenomics in risk assessment
reach beyond the use of these data to establish a mechanism or mode
of action. While the MAQC I project results demonstrated good
reproducibility when using the same biological sample and platform,
other issues leading to variability in response (e.g., biological sample
preparation and intrinsic heterogeneity) remain to be addressed. To
resolve these issues, continued iterative and collaborative efforts
between research scientists of various institutions, industry that
develops and improves the technology, and risk assessors would be
beneficial. Additional concerns include the lack of available toxicoge-
nomic data for most environmental chemicals; lack of maturity of
some of the technologies for use as toxicology testing and risk
assessment assays (including the establishment of reproducibility,
sensitivity, and specificity); concern about whether there is a return
on such a large investment; and the need for criteria specifically for
inclusion of toxicogenomic data in risk assessment. The lack of
available toxicogenomic data is due in part to the newness and cost of
conducting toxicogenomic studies. Studies become even more costly
when designing specific comparative studies assessing multiple
tissues or time points, for example. While the cost has been declining
and may be less of an issue in the future, this limitation has resulted in
investigations with single-dose, single-time point of exposure or
fewer samples/replicates. For example, the dibutyl phthalate (DBP)
case study example discussed in this issue (Euling et al., 2010—this
issue) has such a limited data set. More complex study designs may
also be needed for MOA hypothesis testing, as described above.
In sum, toxicogenomics is continuing to be refined with new
methodologies being developed for both performing toxicogenomic
studies and analyzing data, particularly for microarray studies. A
number of major efforts have addressed some of the technical
challenges to providing reliable, reproducible data. The next step in
this regard is greater standardization in data analysis, particularly the
development of analyses geared towards risk assessment rather than
screening and prioritization. Examples of these include determining
Please cite this article as: Wilson, V.S., et al., Utilizing toxicogenomic data to understand chemical mechanism of action in risk assessment,
Toxicol. Appl. Pharmacol. (2011), doi:10.1016/j.taap.2011.01.017
the appropriate cutoffs for differentially expressed gene identification,
developing appropriate pathway analyses to estimate the degree of
toxicity pathway perturbations, and the determination of biological
significance of affected genes or pathway. Each of these requires
placing the data in a more biological context specifically related to
toxicity — a context for which the current state of knowledge remains
sparse. Therefore, a key parallel research need is developing a base of
knowledge as to the web of biological pathways, their interrelations,
and how they interact to the give occurrence to adverse effects. Part of
this effort also necessarily involves relating pathway perturbations to
human outcome data as opposed to experimental animal model data.
This can be accomplished only through enhanced collaboration
between toxicology and other biological sciences, particularly those
rich in human data.
Summary
Despite the challenges and limitations in utilizing toxicogenomic
data in risk assessment, these data can currently be informative to risk
assessment by corroborating the existing information base on mode
or mechanism of action. In addition, toxicogenomic data can provide
supportive information for the acceptance of a related critical effect,
including providing some information about human relevance. For
example, if the human relevance of a particular toxicological endpoint
considered the critical effect is debated, and toxicogenomic data
related to the endpoint indicates effects on pathways known to be
related to human disease, then the toxicogenomic data may provide
support for human relevance. This is one example of how toxicoge-
nomic data can be used in a weight-of evidence approach in a
qualitative manner to impact decisions within the risk assessment.
Data from well-designed microarray studies have been used in
hypothesis generating and testing of a mechanism of action and have
been used as supporting information for a particular mode of action in
some EPA risk assessments. Many challenges and research needs
remain in terms of experimental designs, data analysis, and
interpretation appropriate for risk assessment. Nonetheless, as
chemical risk assessment moves away from a single mechanism of
action approach toward a toxicity pathway-based paradigm, we
envision that toxicogenomic data from multiple technologies (e.g.,
proteomics, metabolomics, transcriptomics, supportive RT-PCR stud-
ies) can be used in conjunction with one another to understand the
complexities of TK and TD steps in multiple, and possibly interacting,
pathways affected by a single chemical or mixture in human health
risk assessment.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
The authors would like to thank US EPA scientists, Drs. Babasaheb
Sonawane, Sury Vulimiri, Doug Wolf, and William Ward, for their
helpful, constructive review and comments.
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