T18176_Cover.indd 1 4/18/09 3:41:39 

 

EPA/100/K-09/001 I March 2009 

www.epa.gov/osa 

The U.S. Environmental Protection 

Agency’s Strategic Plan for 

Evaluating the Toxicity of Chemicals 

Office of the Science Advisor 

Science Policy Council 
EPA 100/K-09/001 

March 2009 

The U.S. Environmental Protection Agency’s 

Strategic Plan for Evaluating 

the Toxicity of Chemicals 

Office of the Science Advisor 

Science Policy Council 

U.S. Environmental Protection Agency 

Washington, DC 20460 

Recycled/Recyclable 

Printed with vegetable-based ink on paper that 

contains a minimum of 50% post-consumer 

fiber and is processed chlorine free. 
DISCLAIMER 

Mention of trade names or commercial products does not constitute endorsement or 

recommendation for use. Notwithstanding any use of mandatory language such as "must" and 

"require" in this document with regard to or to reflect scientific practices, this document does not 

and should not be construed to create any legal rights or requirements. 

ii
AUTHORS AND CONTRIBUTORS 

Future of Toxicity Testing Workgroup Co-Chairs 

Michael Firestone, Office of Children’s Health Protection and Environmental Education, U.S. 

EPA 

Robert Kavlock, Office of Research and Development, U.S. EPA 

Hal Zenick, Office of Research and Development, U.S. EPA 

Science Policy Council Staff 

Melissa Kramer, Office of the Science Advisor, U.S. EPA 

Future of Toxicity Testing Workgroup Representatives 

Marcia Bailey, Region 10, U.S. EPA 

Arden Calvert, Office of the Chief Financial Officer, U.S. EPA 

Laurel Celeste, Office of General Counsel, U.S. EPA 

Vicki Dellarco, Office of Prevention, Pesticides, and Toxic Substances, U.S. EPA 

Scott Jenkins, Office of Air and Radiation, U.S. EPA 

Gregory Miller, Office of Policy, Economics, and Innovation, U.S. EPA 

Nicole Paquette, Office of Environmental Information, U.S. EPA 

Santhini Ramasamy, Office of Water, U.S. EPA 

William Sette, Office of Solid Waste and Emergency Response, U.S. EPA 

Other Contributors 

Katherine Anitole, Office of Prevention, Pesticides, and Toxic Substances, U.S. EPA 

Hugh Barton, Office of Research and Development, U.S. EPA 

Norman Birchfield, Office of the Science Advisor, U.S. EPA 

Michael Brody, Office of the Chief Financial Officer, U.S. EPA 

Rory Conolly, Office of Research and Development, U.S. EPA 

David Dix, Office of Research and Development, U.S. EPA 

Stephen Edwards, Office of Research and Development, U.S. EPA 

Andrew Geller, Office of Research and Development, U.S. EPA 

Karen Hamernik, Office of Prevention, Pesticides, and Toxic Substances, U.S. EPA 

Jean Holmes, Office of Prevention, Pesticides, and Toxic Substances, U.S. EPA 

Richard Judson, Office of Research and Development, U.S. EPA 

Thomas Knudsen, Office of Research and Development, U.S. EPA 

Julian Preston, Office of Research and Development, U.S. EPA 

Kathleen Raffaele, Office of the Science Advisor, U.S. EPA 

Ram Ramabhadran, Office of Research and Development, U.S. EPA 

James Samet, Office of Research and Development, U.S. EPA 

Patricia Schmieder, Office of Research and Development, U.S. EPA 

Banalata Sen, Office of Prevention, Pesticides, and Toxic Substances, U.S. EPA 

Imran Shah, Office of Research and Development, U.S. EPA 

Linda Sheldon, Office of Research and Development, U.S. EPA 

iii
John Vandenberg, Office of Research and Development, U.S. EPA 

Maurice Zeeman, Office of Prevention, Pesticides, and Toxic Substances, U.S. EPA 

External Peer Reviewers 

John R. Bucher, Ph.D., Associate Director, National Toxicology Program, National Institute of 

Environmental Health Sciences 

George Daston, Ph.D., Research Fellow, P&G 

Daniel Krewski, Ph.D., MHA, Professor and Director, McLaughlin Centre for Population Health 

Risk Assessment, University of Ottawa 

Martin Stephens, Ph.D., Vice President for Animal Research Issues, The Humane Society of the 

United States 

iv
TABLE OF CONTENTS
 

LIST OF FIGURES ..................................................................................................................... 

....................................................................................................................... 

............................................................................................................................... 

............................................................................................................................... 

..................................................................................... 

vi
 

LIST OF TABLES vi
 

ACRONYMS vii
 

1. Introduction 1
 

2. Regulatory Applications and Impacts 5
 

2.1 Chemical Screening and Prioritization ................................................................................. 

........................................................................... 

.......................................................................................................... 

.................... 

5 

2.2 Toxicity Pathway-Based Risk Assessment 5 

2.3 Institutional Transition 7 

3. Toxicity Pathway Identification and Chemical Screening and Prioritization 8
 

3.1 Strategic Goal 1: Toxicity Pathway Identification and Assay Development ..................... 

........................................................................... 

............................................................................ 

........................................................ 

10 

3.2 Strategic Goal 2: Chemical Prioritization 11 

4. Toxicity Pathway-Based Risk Assessment 12
 

4.1 Strategic Goal 3: Toxicity Pathway Knowledgebases 13 

4.2 Strategic Goal 4: Virtual Tissues, Organs, and Systems: Linking Exposure, Dosimetry, and 

Response ................................................................................................................................... 

........................... 

.......................................................................................................... 

............................................................................ 

....................................................................... 

14 

4.3 Strategic Goal 5: Human Evaluation and Quantitative Risk Assessment 16 

5. Institutional Transition 18
 

5.1 Strategic Goal 6: Operational Transition 18 

5.2 Strategic Goal 7: Organizational Transition 20 

5.3 Strategic Goal 8: Outreach.................................................................................................. 

............................................................................................................................. 

.......................................................................................... 

.................................................................................................................................... 

20 

6. Future Steps 23
 

Appendix: Other Related Activities 24
 

References 27
 

v
 

LIST OF FIGURES 

Figure 1. Toxicity Pathways ........................................................................................................... 

........................ 

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

........................................................................... 

....................................................................................... 

........................................................................................... 

...................................................... 

2 

Figure 2. Toxicity Pathways Target Multiple Levels of Biological Organization. 8 

Figure 3. ToxCastTM 1
 

Figure 4. Toxicity Pathways to Dose-Response 12 

Figure 5. Knowledgebase Development 14 

Figure 6. Relative (%) Emphasis of the Three Main Components of this Strategic Plan over its 

Expected 20-year Duration. 23 

LIST OF TABLES 

Table 1. Strategic Plan: Applications and Impacts 6 

vi
ACRONYMS 

ACToR Aggregated Computational Toxicology Resource 

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act 

FTTW Future of Toxicity Testing Workgroup 

HTS High Throughput Screening 

IRIS Integrated Risk Information System 

NRC National Research Council of the National Academies 

OPPTS Office of Prevention, Pesticides, and Toxic Substances 

ORD Office of Research and Development 

QSAR Quantitative Structure-Activity Relationship 

SAR Structure-Activity Relationships 

vii

1
 

 

1. INTRODUCTION 

EPA bases its regulatory decisions on a wide range of tools and information that represent the best 

available science. In some situations, where very limited or no animal toxicity data exist, EPA may 

use tools such as structure-activity relationships (SAR) and quantitative structure-activity 

relationship (QSAR) modeling, together with information on exposure to make decisions about 

priority setting and the need for further evaluation (e.g., for new chemicals in the toxics program, 

high production volume chemicals, and pesticide inerts). To establish regulatory standards, EPA 

relies heavily on toxicity testing to evaluate clinical or pathological effects in experimental animal 

models. As such, toxicity testing and related research is currently a multi-billion dollar activity that 

engages thousands of research scientists, risk assessors, and risk managers throughout the world. 

To that end, the historical path taken in toxicity testing of environmental agents has generally been 

either to make incremental modifications to existing tests or to add additional tests to cover 

endpoints not previously considered (e.g., developmental neurotoxicity). This approach has led 

over time to a continual increase in the number of tests, cost of testing, use of laboratory animals, 

and time to develop and review the resulting data. Moreover, the application of current toxicity 

testing and risk assessment approaches to meet existing, and evolving, regulatory needs has 

encountered challenges in obtaining data on the tens of thousands of chemicals to which people are 

potentially exposed and in accommodating increasingly complex issues (e.g., lifestage 

susceptibility, mixtures, varying exposure scenarios, cumulative risk, understanding mechanisms 

of toxicity and their implications in assessing dose-response, and characterization of uncertainty)1. 

While the challenges of such information gaps are great, the explosion of new scientific tools in 

computational, informational, and molecular sciences offers great promise to address these 

challenges and greatly strengthen toxicity testing and risk assessment approaches. Proven benefits 

have been demonstrated in allied fields such as medicine and pharmaceuticals. Although untapped, 

the potential application to toxicity testing and risk assessment has also been recognized by EPA as 

witnessed by the issuance of a series of papers that provided guidance on the use of genomic data.2 

To better anticipate the potential contribution of new technologies and scientific advances to issues 

associated with toxicity testing and risk assessment, EPA commissioned the National Research 

Council (NRC) in 2004 to review existing strategies (NRC, 2006) and develop a long range vision 

for toxicity testing and risk assessment (NRC, 2007). In the subsequent release of Toxicity Testing 

in the 21st Century: a Vision and a Strategy, a landmark transformation in toxicity testing and risk 

assessment is envisioned that focuses on “toxicity pathways.”3 This approach is based on the 

rapidly evolving scientific understanding of how genes, proteins, and small molecules interact to 

form molecular pathways that maintain cell function. The goal is to determine how exposure to 

environmental agents can perturb these pathways causing a cascade of subsequent key events 

1

 These limitations have been described more fully in A Review of the Reference Dose and Reference Concentration 

Processes: http://www.epa.gov/ncea/iris/RFD_FINAL[1].pdf 2

 Interim Policy on Genomics (2002): http://www.epa.gov/osa/spc/genomics.htm; Genomics White Paper (2004): 

http://www.epa.gov/osa/pdfs/EPA-Genomics-White-Paper.pdf; Interim Guidance for Microarray-Based Assays 

(2007): http://www.epa.gov/osa/spc/pdfs/epa_interim_guidance_for_microarray-based_assays-externalreview_draft.pdf. 

3

 Toxicity pathways are cellular response pathways that, when sufficiently perturbed, are expected to result in 

adverse health effects. 
leading to adverse health effects. This sequence of events is illustrated in Figure 1 wherein the 

introduction of an environmental stressor may trigger such a cascade. Successful application of 

these new scientific tools and approaches will inform and produce more credible decision making 

with an increased efficiency in design and costs and a reduction in animal usage. 

Source 

Fate/Transport 

Exposure 

Tissue Dose 

Biologic Interaction 

Perturbation
 

Biologic Normal
 

Inputs Biologic
 

Function
 

Early Cellular 

Toxicity Pathways: Cellular Changes
 response pathways that,
 

when sufficiently 

perturbed, are expected Adaptive Stress Cell 

to result in adverse health Responses Injury 

effects. Morbidity and 

Mortality 

Modified from NRC, 2007 

Figure 1. Toxicity Pathways. Toxicity pathways describe the 

processes by which perturbations of normal biological processes 

due to exposure to a stressor (e.g., chemical) produce changes 

sufficient to lead to cell injury and subsequent events (modified 

from NRC, 2007). 

Other agencies have also recognized

the need for this transformative shift, 

including the National Toxicology

Program in their Roadmap for the 

Future and the Food and Drug

Administration in their Critical Path 

Program. In anticipating the 

emergence, and potential, of this new 

scientific paradigm, EPA’s Office of 

Research and Development (ORD) and

some of the Agency’s regulatory 

programs have also begun to redirect 

resources in intramural and extramural 

research programs to “jump start” the 

process of transformation. For 

example, ORD created the National 

Center for Computational Toxicology4 

in 2006. Likewise, ORD National

Laboratories and Centers have also 

begun to incorporate these new scientific tools to better support the research being conducted 

under several of its multiyear research plans. Several ongoing projects address the use of in vitro 

assays in risk assessment and toxicity testing (e.g., Guyton, et al., 2008), and assessments under 

the Integrated Risk Information System (IRIS)5

 program are describing and evaluating published 

genomic data. EPA's Office of Prevention, Pesticides, and Toxic Substances (OPPTS) is also 

actively involved in the development and transition of computational toxicology tools into 

regulatory practice. OPPTS has developed a multi-year strategic plan to advance computational 

toxicology tools in its risk assessment and management paradigm. Current activities include 

assisting ORD by providing the necessary databases to support the development of models for 

efficiently and credibly predicting toxic potency and levels of exposure, beta testing the new 

computer models, training staff, and initiating plans for successful international coordination and 

stakeholder involvement. Furthermore, recognizing the need to partner to achieve the vision and 

goals laid out by the NRC, EPA recently signed a Memorandum of Understanding for research 

cooperation with the National Toxicology Program and the National Institutes of Health 

Chemical Genomics Center as a substantive step forward in building collaborations across sister 

federal agencies.6

 EPA is also working actively at the international level with programs such as 

the Organization for Economic Cooperation and Development (OECD) through the Molecular 

4 Computational toxicology is the application of mathematical and computer models and molecular biological 

approaches to improve the Agency’s prioritization of data requirements and risk assessments (from A Framework 

for a Computational Toxicology Research Program, EPA 600/R-03/065). 5

 http://cfpub.epa.gov/ncea/iris/index.cfm 

6

 http://www.epa.gov/comptox/articles/comptox_mou.html 

2
 
Screening Initiative, the Integrated Approaches for Testing and Assessment Workgroup, Test 

Guideline Committees, and the QSAR Expert Group to ensure global harmonization of any new 

approach that originates from the research program. A more complete listing of these 

collaborations may be found in the appendix. 

In response to the release of the NRC reports, EPA has established an intragency workgroup, the 

Future of Toxicity Testing Workgroup (FTTW), under the auspices of the Science Policy 

Council. The FTTW includes representatives from across the Agency, including the Regions and 

all major Program Offices. It has produced this current document, which will serve as a blueprint 

for ensuring a leadership role for EPA in pursuing the directions and recommendations presented 

in the 2007 NRC report. This document presents a strategy that is consistent with the NRC’s 

directions and recommendations. It presents the Agency’s vision of how to incorporate a new 

scientific paradigm and new tools into toxicity testing and risk assessment practices with everdecreasing reliance on traditional apical approaches. The overall goal of this strategy is to 

provide the tools and approaches to move from a near exclusive use of animal tests for predicting 

human health effects to a process that relies more heavily on in vitro assays, especially those 

using human cell lines. The topics to be covered include (1) the applications and impacts/benefits 

for various types of regulatory activities (Section 2), (2) the research to be conducted to facilitate 

the screening and prioritization of environmental agents (Section 3), (3) the implementation of a 

toxicity pathway-based approach to risk assessment (Section 4), and (4) the critical companion 

component, namely, the institutional transition that must occur before the changes can be fully 

implemented (Section 5). 

As described in Section 6, the workgroup recognizes that the full implementation of the vision 

set out in this strategy will require a significant investment of resources over a long period of 

time. The workgroup has identified a range of partners in this effort, and some planning on the 

relative role of these partners has begun, although the specific areas of work to be 

conducted/funded by EPA versus other partners needs further assessment. Decisions on the 

relative roles will have a significant impact on EPA resources required to implement the vision. 

Since the NRC charge and report centered on advancing toxicity testing for assessing human 

health effects of environmental agents, this strategic plan is presented primarily within that 

context. However, under environmental legislative mandates (e.g., the Toxic Substances Control 

Act; the Federal Insecticide, Fungicide, and Rodenticide Act; and the Clean Water Act), most 

EPA programs must regulate compounds to ensure both environmental and human health risks 

are properly managed. Since statutory language and/or resulting policy typically require single 

regulatory decisions for a chemical(s) that encompass environmental and human health risks at 

the same time, accelerated and cost effective approaches for both areas are critical to realize 

programmatic benefits. As in the human health arena, development and application of 

approaches described in this strategy apply to ecotoxicology and risk assessment as well. Notable 

progress is being made within EPA Laboratories and Centers on the development and use of 

toxicity pathway models and the creation of prioritization schemes, toxicology knowledgebases, 

and systems biology models in the field of environmental science. The bringing together of 

relevant disciplines to share data and integrate models is critical to fully achieve increased 

efficiency in toxicity testing and a reduction in animal usage for both human health and 

environmental risk assessment. Consequently, the Agency will be implementing this strategy in a 

manner that addresses both human health and ecological risk assessment. Future versions of the 

3
 
strategy will summarize progress made in advancing integrated testing and assessment capability 

and revisit remaining challenges. 

4
 
2. REGULATORY APPLICATIONS AND IMPACTS 

The research arising from implementation of this strategy will change the nature of the methods, 

models, and data that will inform the major components of the risk assessment process (i.e., 

hazard identification, dose response, exposure assessment, and risk characterization). Without 

attempting to be all-inclusive, Table 1 presents some of the major cross-office applications and 

impacts of these new scientific approaches, with more in-depth discussion of the planned work 

described in Sections 3-5. The three components of this strategic plan, namely, chemical 

screening and prioritization, toxicity pathway-based risk assessment, and institutional transition, 

are not independent elements but rather highly interactive and integrative efforts that will 

maximize the value and application of the research generated. 

2.1. Chemical Screening and Prioritization 

An ongoing need of several regulatory offices is to have tools to assist in chemical screening and 

prioritization, e.g., high production volume chemicals, air toxics, the drinking water Contaminant 

Candidate Lists, and Superfund chemicals. These programs consider anticipated exposure and 

hazard to select chemicals to evaluate in longer-term, whole-animal laboratory studies. An early 

use for data developed under the new paradigm will be as an efficient and cost effective screen 

for several types of chemical toxicity. Thus, risk assessors could use in silico (computer-based) 

technologies and structure/molecular/bioactivity profiling from diagnostic high-throughput/in 

vitro assays, along with predicted exposure/dose information, to predict chemicals most likely to 

cause hazards of concern for humans. This approach will also enable risk assessors to determine 

the specific effects, in vivo data, and exposures that would be most useful to assess, quantify, and 

manage. As the technology develops, EPA will be able to screen previously untested chemicals 

using libraries of chemical, molecular, biological, and toxicological data and models to identify 

the types of adverse effects that they are most likely to produce in standard animal bioassays. 

More importantly, EPA will be able to gain better insight into whether such effects would likely 

be manifest in humans under various exposure scenarios. As noted earlier, these needs are 

common to a number of federal agencies; discussions are underway to develop more common 

paradigms among federal agencies to facilitate data sharing. 

2.2. Toxicity Pathway-Based Risk Assessment 

The current approach to risk assessment includes uncertainties associated with (1) the human 

relevance of laboratory animal studies (species extrapolation), (2) the use of high doses in 

animals to estimate risk associated with lower environmental/ambient exposures (dose 

extrapolation), and (3) predicting the risk to susceptible populations. In recent years, the 

consideration of such issues has been better informed by the incorporation of information on 

potential modes of action through which toxicity may be expressed. The approach outlined 

earlier in Figure 1 focuses on perturbations in baseline biological processes that may lead down 

toxicity pathways to adverse health outcome(s). Combining this information with distributional 

data on population characteristics of exposure and dose (magnitude, frequency, and duration) 

provides a scientifically based approach for reducing the uncertainties associated with current 

risk assessments. By relying on a quantitative understanding of perturbations in toxicity 

pathways that lead to adverse health effects, the new approach to toxicity testing and risk 

assessment envisioned in this document will greatly increase EPA’s capacity to assess individual 

5
 
 

 

 

 

 

chemicals and their mixtures. The new approach will also increase EPA’s confidence that the 

Agency’s assessments adequately protect human health. Realization and acceptance of this new 

approach will likely encounter numerous challenges, but the effort is expected to ultimately lead 

to better protection of human health. 

Table 1. Strategic Plan: Applications and Impacts 

Toxicity Pathway 

Identification and 

Chemical Screening & 

Prioritization 

Toxicity Pathway-Based Risk Assessment Institutional Transition 

Issue 

Need to screen 10,000’s 

of chemicals for wide 

range of endpoints in a 

manner that considers 

toxicity pathways and the 

potential for human 

exposure. 

For many chemicals, the current approach 

relies on expensive animal testing that takes 

time to conduct and review. Limitations in the 

design of in vivo studies often prevent 

complete evaluation of all endpoints and 

hazard/risk scenarios of concern. 

Limited understanding of biological 

Implementing the new approach will 

require significant institutional investment 

in operational and organizational transition 

and in public outreach. 

mechanisms most often leads to uncertainty in 

assessing cumulative risk or extrapolating in 

vitro to in vivo or across doses, lifestages, 

species, or genetic diversity. 

Drivers 

Need to limit cost and 

animal usage, improve 

timeliness, and decrease 

uncertainty in testing 

decisions. 

New scientific understanding and tools in 

molecular, computational, and information 

sciences consistent with applications in allied 

areas such as medicine and pharmaceuticals 

represent a path forward. 

EPA lacks appropriate expertise and 

sufficient funding to fully and most 

efficiently utilize the new toxicity testing 

technologies when making regulatory 

decisions. 

New Approach 

Identification of toxicity 

pathways for key 

toxicological endpoints. 

Combine in silico and 

bioprofiles from HTS7 

along with QSAR 

approaches linked to 

animal study data. 

Reliance on increased understanding of how 

perturbations of biological processes at 

environmentally relevant concentrations 

trigger events (i.e., toxicity pathway(s)) that 

may lead to adverse health outcomes. 

Develop linked exposure/dose models to 

inform dosing levels for toxicity testing and 

inform risks. 

Fully adopting the new paradigm should 

be supported by mechanistically based 

proof-of-concept and verification studies. 

Further, such adoption will require 

additional training of existing staff and 

hiring new staff conversant in state-of-thescience knowledge in fields such as 

toxicology, biochemistry, bioinformatics, 

etc. 

Impact 

Offices would be better 

able to direct efforts and 

resources to chemicals 

with greatest potential 

risk. Significant increase 

in efficiency with marked 

reduction in cost for 

toxicity testing. 

More scientifically relevant data on which to 

base EPA’s regulatory decisions and/or 

impact analyses that rely on these risk 

assessments. 

A well informed public will have greater 

confidence as EPA greatly expands the 

number of chemicals assessed for possible 

risks and improves existing strategies for 

hazard and risk assessment! 

7 High-Throughput Screening (HTS) refers to robotic technologies developed by the pharmaceutical industry for 

drug development that enable the ability to evaluate the effects of hundreds to thousands of chemicals per day on 

molecular, biochemical or cellular processes. 

6 
2.3. Institutional Transition 

Implementing major changes in toxicity testing of environmental contaminants and incorporating 

new types of toxicity data into risk assessment will require significant institutional change 

involving: 

•	 Operational transition – how EPA will transition to the use of new types of data and 

models for toxicity testing and risk assessment; 

•	 Organizational transition – how EPA will deploy resources necessary to implement the 

new toxicity testing paradigm such as hiring of scientists with particular scientific 

expertise and training of existing scientific staff and risk managers; 

•	 Outreach – efforts by EPA to share information with the public and improve risk 

communication. 

The process of moving from research to regulatory acceptance for implementing new science 

related to toxicity testing will be an iterative and long-term effort (likely encompassing more 

than a decade). Essential to this iterative process will be the demonstration that the predictive 

nature of these new approaches is superior to that of our current practices for toxicity testing and 

risk assessment. It will be critical to begin activities geared toward regulatory acceptance early in 

the process of implementing this strategic plan. 

7
 
3.	 TOXICITY PATHWAY IDENTIFICATION AND CHEMICAL SCREENING 

AND PRIORITIZATION 

The advancements in biotechnology brought about by the sequencing of the human genome and 

the investment in high throughput screening tools to mine large chemical libraries for potential 

drugs have for the first time allowed a broad scale, unbiased examination of the molecular and 

cellular targets of chemicals. At this time, the examination of the relationships between the 

molecular and cellular targets of chemicals and the traditional endpoints of toxicity is at an early 

stage of development. Even upon characterization of these types of relationships, significant 

phenotypic data will be required to critically establish the role of toxicity pathways in evaluating 

hazards and risks. The great potential is that identification of a toxicity pathway and 

development of an in vitro bioassay for studying its chemical interactions will enable evaluation 

of the effects of thousands of chemicals in that pathway. Broadening this approach to the many 

toxicity pathways present in living systems allows a new avenue for identifying those chemicals 

that pose the greatest potential hazard. Knowledge of the toxicity pathways triggered by any one 

chemical will also allow targeting of specific in vivo tests to more fully characterize the potential 

hazard and risk. The identification of toxicity pathways for key target tissues, organs, and 

lifestages, and their linkage across levels of biological organization and exposure pathways and 

intensities are core elements of this strategy. 

As indicated in Figure 2, chemicals may interact with a single pathway (the blue chemical) or 

multiple pathways (the yellow chemical). Also, multiple pathways can lead to the same 

expression of toxicity in the target organ as signaling pathways converge on common elements. 

It is important to note that multiple 

mechanisms of action8

 for any 

particular adverse response likely 

exist, and that many environmental 

pollutants are likely to have multiple 

mechanisms of action. Two critical 

components of the toxicity pathway 

concept are (1) extending knowledge 

of molecular perturbations and cell 

signaling pathways to understand 

linkages between levels of biological 

organization and (2) extending 

knowledge of in vitro and in vivo 

markers relevant to adaptive changes and/or adverse outcomes (see Section 5). As the research 

moves forward, it will be important to capture quantitative relationships between the molecular 

events and the higher order changes. Demonstration of plausible connectivity along the 

mechanism of action from initiating event to adverse outcome will serve as the rationale for 

using data from subcellular or cell-based in vitro assays for not only chemical prioritization but 

also predictive risk assessment. As toxicity pathways are identified, relevant in vitro assays can 

8 Mode of action is defined as a sequence of key events and processes, starting with interaction of an agent with a 

cell, proceeding through operational and anatomical changes, and resulting in an adverse health effect. Mechanism 

of action implies a more detailed understanding and description of events, often at the molecular level, than is meant 

by mode of action. 

8
 

Biological Organization. 
 

be utilized and their results compared to in vivo studies as appropriate given the need to predict 

effects in humans or other species. While comparing responses to those in animal bioassays will 

be an early milestone of this strategy, the ultimate goal is the prediction of human risk. 

Therefore, efforts will shift towards that goal as experience with the approach increases. An 

added benefit to the toxicity pathway approach is that mixtures or their components could be 

evaluated in this manner, and as knowledge grows, it will be possible to predict where 

interaction with multiple toxicity pathways might be expected to lead to non-additive outcomes. 

This later activity will be an important outcome of the research highlighted in Section 4.2 

(Strategic Goal 4) that is focused on the development of virtual tissue models. As noted below, 

virtual tissue models will also provide a basis for predicting emergent properties of tissues by 

integrating knowledge of molecular and cellular behaviors obtained from reductionist in vitro 

approaches. 

In 2007, EPA launched ToxCastTM 9 in order to develop a cost-effective approach for prioritizing 

the toxicity testing of large numbers of chemicals in a short period of time. Using data from a 

broad range of state-of-the-art HTS bioassays developed in the pharmaceutical industry, 

ToxCastTM is building computational models to forecast the potential human toxicity of 

chemicals. Results from the HTS bioassays are being analyzed for signatures of bioactivity that 

correlate with known toxicities. These hazard predictions will provide EPA regulatory programs 

with science-based information helpful in prioritizing chemicals for more detailed toxicological 

evaluations, and lead to more efficient use of animal testing. 

The research described here focuses on two major strategic goals: 

1) Identification of toxicity pathways and deployment of in vitro assays to characterize the 

ability of chemicals to perturb those pathways in different biological contexts, and 

2)	 Implementation of ToxCastTM, with an initial focus on providing input for chemical 

prioritization, shifting over time to providing input for dose-response modeling. 

A key feature of ToxCastTM is the phased nature of implementation (see Strategic Goal 2, Section 

3.2), from proof of concept, to forward validation, and finally to reduction to practice. The 

number of chemicals will grow from the hundreds to the thousands, and the number of assays 

will change as experience and biology dictate. As the number of chemicals and breadth of 

toxicity pathways covered increase, ToxCastTM will improve as a unique resource to build chemoinformatic-based predictions of chemicals’ potential human toxicity. Such advancements should 

help promote improved QSAR models and data upon which to build virtual tissue models. 

Exposure science also plays a large role in this strategy. More simple and reliable screening 

models are needed that predict exposures to chemicals so that information from the full sourceto-outcome continuum is brought into consideration in the evaluation of chemicals – a critically 

important step for new chemicals that have not yet been released into the environment. Examples 

of such simple methods and models for new chemicals can be found at EPA's Sustainable 

Futures Initiative10. Additional such models should further evaluate exposure based on the life 

cycle of intended product use and the physical-chemical properties of the chemicals. This 

9

 http://www.epa.gov/ncct/toxcast/ 

10 http://www.epa.gov/oppt/sf/ 

9
 
 

research should include the expansion of computational chemistry methods to further predict 

exposures as well as methods to predict release into the environment during product life cycle. 

Several additional screening-level models are currently under development in Canada and 

Europe. Research in this area should be coordinated with these groups to facilitate an 

international approach for chemical screening. EPA should promote easy public access to all of 

these additional models through the Internet. 

3.1. Strategic Goal 1: Toxicity Pathway Identification and Assay Development 

The most systematic and extensive approach currently underway for screening and prioritization 

is EPA’s ToxCastTM. Fully implementing the proposed strategy for more efficient toxicity testing 

will utilize a combination of the more exploratory ToxCastTM chemical signature approach (see 

Strategic Goal 2), and the more hypothesis-driven approaches to elucidating toxicity pathways. 

Developing systems-based models will require comprehensive identification of the biological 

processes that can result in toxicity when they are perturbed by chemical exposures. Therefore, 

toxicity pathway identification and development of appropriate in vitro assays to characterize the 

dose-response and time course of perturbations to those pathways will be needed. Measurement 

of chemical form and concentration from in vitro assays will also be important in hypothesisdriven research that seeks to establish linkages between perturbations of toxicity pathways and 

adverse effects, as well as for establishing structure-activity relationships. These research goals 

will utilize a range of methods (e.g., transcriptomic, proteomic, metabolomic, cellular, and 

biochemical analyses) to identify toxicity pathways using in vivo and in vitro systems. The in 

vitro assays and toxicity pathways already included in the ToxCastTM project will be a part of this 

research, but additional assays providing greater coverage of relevant toxicity pathways will 

need to be developed. For example, developmental neurotoxicity key responses are known to 

include cell proliferation, apoptosis, differentiation (into different cell types and creating 

different functionality/architecture of a cell), neurite outgrowth, synaptogenesis, and myelination 

(Coeke et al., 2007; Lien et al., 2007), but the underlying molecular pathways are not yet 

completely identified. Through the informed use of newer “systems-based” approaches (Edwards 

& Preston, 2008), the flow of molecular regulatory information underlying the control of these 

cellular events can be characterized, classified, and modeled. To facilitate use in risk assessment, 

these studies will be coupled with mechanism of action-based studies, including animal and 

human components as described in Strategic Goal 4. 

Current priorities for research include developing in vitro assays for the key targets of chemicals 

in the environment for which limited knowledge is available (e.g., developmental neurotoxicity, 

immunotoxicity, reproductive toxicity) as well as for relatively well-characterized toxicity 

pathways such as stress response signaling. Studies representative of the full range of human 

variability will be necessary to characterize processes that may occur more readily in sensitive 

populations (e.g., asthmatics) or at certain lifestages (e.g., prenatal development). Additional 

emphasis needs to be placed on toxicities demonstrated to occur in humans. For example, clinical 

trials or post-marketing surveillance for pharmaceuticals, as well as molecular and genetic 

epidemiology studies, afford the opportunity to examine effects of chemicals already introduced 

into the environment that may not currently be well assessed by in vivo animal toxicity studies. 

Some of these pathways may be important for environmental chemicals with respect to human 

variability or exposure to complex mixtures. 

10
 
3.2. Strategic Goal 2: Chemical Prioritization 

Cancer 

ReproTox 

DevTox 

NeuroTox 

PulmonaryTox 

ImmunoTox 

in vitro testing in silico analysis 

HTS Bioinformatics/ 

-omics Machine Learning 

Figure 3. ToxCastTM is using a variety of HTS assays to develop bioactivity 

signatures that are predictive of effects in traditional toxicity testing approaches. 

This strategy extends approaches that are currently under development for EPA’s ToxCastTM 

program to include greater coverage of toxicity pathways and chemicals. The goal of the 

ToxCastTM program is to provide a comprehensive assessment of toxicity pathways for a 

relatively low cost per chemical (current estimates are in range of $20-25,000). ToxCastTM (see 

Figure 3) was 

designed to collect 

data from a wide 

range of in vitro 

assays, mostly 

mechanistic in nature, 

to prioritize which 

chemicals to test 

further and which in 

vivo studies were 

likely most important. 

This screening and 

prioritization approach 

provides a near-term benefit during an extended transition to the more comprehensive proposed 

vision. As more comprehensive descriptions of processes involved in toxicological responses 

become available, different assays may be identified to replace those in the initial ToxCastTM 

effort, and the relationship to in vivo studies will shift from prioritization to providing input for 

dose-response modeling. 

ToxCastTM is being developed in a phased manner. During FY08-09, substantial progress will be 

made on the first two phases of the ToxCastTM program (Dix et al., 2007; Kavlock et al., 2008). 

Phase I is a proof of concept involving 320 chemicals that have robust in vivo animal toxicity 

information. These chemicals have been profiled using over 400 high and medium throughput in 

vitro assays. From these in vitro bioactivity profiles, classifiers or signatures predictive of 

chemicals’ in vivo toxicity are being derived. Phase II will involve validation of the predictive 

bioactivity and expansion of the diversity of chemicals tested. Phase III is the most relevant to 

this strategic plan, as it would begin to apply the knowledge gained in Phases I and II to the tens 

of thousands of chemicals of concern to EPA regulatory offices. An adaptation of the approach 

to evaluate the hazardous properties of nanomaterials is also anticipated. 

11
 
4. TOXICITY PATHWAY-BASED RISK ASSESSMENT 

The goals of the proposed new strategy for toxicity testing include collecting mechanistic data, 

largely in vitro, for the purpose of predicting human risk from exposure to chemicals. Prediction 

of in vivo effects in humans requires a combination of measurements and computer modeling to 

link in vitro responses to tissue dosimetry to alterations in the structure and function of tissues 

and organs. A substantial challenge will be to address the range of human variability arising from 

differences in age, life stage, genetics, disease susceptibility, epigenetics, diet, disease status, and 

other factors that potentially influence or interact with toxicity pathways. 

The initial process for predicting human risk under this new approach could be summarized as 

(1) characterizing or predicting potential human exposures; (2) estimating the resulting chemical 

dosimetry (magnitude, frequency, and duration) for target pathways, tissues or organs; (3) 

measuring toxicity pathway response at doses consistent with human exposures; (4) predicting 

the in vivo human response resulting from pathway perturbations; (5) quantifying the range of 

human variability and susceptibility; and (6) validating predictions utilizing in vivo systems (e.g., 

laboratory animals, human data). In the current state of mechanistic toxicology (top row of 

Figure 4), chemicals are administered to the test animals (usually at high doses), a variety of 

 

R

Molecular Cellular Tissue
 

Chemicals Sensing Signaling Responses
 

Environmental 

C

A

X

R

α

AXRRα

Y

P

2

B

G

S

T

M

R

P

*

Virtual
 

Tissues
 

Knowledgebase Molecular Cellular Toxicity Dose-Response Networks Networks Pathways 

Figure 4. Toxicity Pathways to Dose-Response. The vertical arrows at each step in the process reflect the 

iterative nature of experimentation and modeling needed to gain full understanding of both the toxicity pathway 

determination and the relationship to normal biology. 

biochemical approaches are used to detect alterations in molecular pathways, the data are mined 

to describe the ensuing cellular alterations (e.g., oxidative stress damage, mitochondrial 

dysfunction), and tissue changes are confirmed at the level of morphology or function. The 

12
 
bottom row of the figure depicts the vision for future ways of assessing risk, which includes 

determining the key toxicity pathways, defining approaches for examining perturbations in 

molecular networks, and translating the results to responses at the cell, and ultimately tissue and 

organ level, using computational models of the relevant systems. The expectation is that 

assessments in the future will utilize data from in vitro studies, and the need for in vivo animal 

testing will be substantially reduced. However, until the state of science of this new approach has 

reached a level of confidence for use in regulatory decision making, the traditional approach to 

toxicity testing will continue into the foreseeable future. With time, we expect that it will be 

progressively augmented and ideally replaced by computational models that integrate the 

information generated from non-animal sources into predictive models of response based upon 

the underlying biology. The vertical arrows at each step in the process reflect the iterative nature 

of experimentation and modeling needed to gain full understanding of both the toxicity pathway 

determination and the relationship to unperturbed biology. One anticipated outcome of the 

development of virtual tissues will be an increased understanding of the role of metabolism and 

of intra- and inter-cellular signaling pathways. This understanding will lead to the development 

of improved in vitro systems that, for example, might include combined cell-based systems to 

provide metabolic competency or to better reflect the intercellular responses in heterogeneous 

tissues. 

As the transition progresses, it is important that increased emphasis will be placed on 

examination of exposure concentrations that are expected to occur in the environment. The key 

difference in future toxicity evaluations will be the transition to a focus on ways in which 

molecular pathways (as detected by in vitro models) are perturbed by chemical exposure 

throughout the range of exposures from environmental to the higher dose levels commonly used 

in contemporary toxicity studies. Dosimetry measurements coupled with computational 

modeling will be critical for predicting in vivo exposure levels of concern and for determining 

relevant in vitro concentrations. Some responses of targeted toxicity pathways can be evaluated 

in simpler cell culture models, whereas, in other cases, multiple in vitro assays may be necessary 

for the integration of multiple pathways that produce in vivo responses. These situations would 

require biologically based models for the responses as well as for chemical dosimetry in order to 

predict the integrated in vivo response. 

Implementing this new paradigm requires organization of existing scientific information; 

computational methods for exposure, chemical dosimetry, and perturbations of biological 

processes; and evaluation of the methods for risk assessment applications. The research program 

to implement this element of the strategy is defined by three goals: development of toxicity 

pathway and exposure knowledgebases; development of virtual tissues, organs, and systems; and 

evaluation of human relevance. 

4.1. Strategic Goal 3: Toxicity Pathway Knowledgebases 

The underlying basis of the 2007 NRC report is that there are a finite number of toxicity 

pathways (i.e., in the hundreds) that could be queried using in vitro assays to obtain insights into 

the ability of chemicals to perturb those pathways. It refers to several stress pathways (e.g., 

oxidative stress response) and notes the general listing of signaling pathways in a previous NRC 

report (2006). However, an inventory of toxicity pathways and their involvement in a variety of 

toxicological responses needs to be created. Likewise, from exposure science there needs to be a 

13
 
complementary effort focusing on those chemical properties and computational methods that 

could be used to reliably predict behaviors in the environment and exposures. This effort would 

include information on stability in the environment, likely routes for exposure, potential for 

bioaccumulation, and extent of metabolism. Therefore, a strategic goal is the development of a 

knowledgebase for toxicity pathways and exposure. Knowledgebases differ from traditional 

databases in the extent of integration of information and the inclusion of tools that can draw 

inferences from amongst the diverse elements. 

The knowledgebase would serve a variety of functions throughout the research and development 

effort associated with implementing this new approach to toxicity testing and will become a 

standard tool in the risk assessments of the future. ACToR (Figure 5), the Aggregated 

Computational 

Toxicology Resource 

under development in 

ORD, is an example of 

the needed approach of 

bringing together diverse 

types of information into 

a system where 

interrelationships of 

individual database 

elements (e.g., traditional 

toxicology, chemical 

structure information, 

high throughput 

screening data, molecular 

pathway analysis, chemical data repositories, peer reviewed published literature, and internal 

Agency databases) can be explored and utilized (Judson et al., 2008). Key steps in development 

of these knowledgebases include: (1) creating electronic repositories of existing toxicity 

information; (2) developing semantics for describing toxicity pathways; (3) automating pathway 

inference tools to aid in discovering mechanistic links between genomic information and 

molecular and cellular observations; and (4) creating a toolbox with a user-friendly interface to 

organize, access, and analyze toxicity pathway assay results. 

 

Figure 5. Knowledgebase Development. ACToR brings together a diverse set of 

currently unlinked resources available from internal and external sources into a 

system with a user friendly interface to readily mine and analyze toxicity data. 

4.2.	 Strategic Goal 4: Virtual Tissues, Organs, and Systems: Linking Exposure, 

Dosimetry, and Response 

Computational techniques relevant to this strategy fall into two general branches: knowledgediscovery (data-collection, mining, and analysis) represented in Strategic Goal 3, and dynamic 

computer simulation (mathematical modeling at various levels of detail) described in this 

section. The central premise of the latter approach is that critical effects of environmental agents 

on molecular-, cellular-, tissue-, and organ-level pathways can be captured by computational 

models that focus on the flow of molecular regulatory information (Knudsen & Kavlock, 2008). 

This information flow is influenced by genetic and environmental signals, with the net outcome 

being the emergent properties associated with baseline or abnormal collective cell behavior. 

Thus, computational systems modeling will be used to predict organ injury due to chemical 

exposure by simulating: (1) the dynamics and characteristics of exposure and dose, (2) the 

14
 
dynamics of perturbed molecular pathways, (3) their linkage with processes leading to alterations 

of cell state, and (4) the integration of the molecular and cellular responses into a physiological 

tissue model. By placing a strong emphasis on understanding the biology of the system and the 

key regulatory components, these virtual tissue models represent a significant opportunity to 

better understand the linkage between chemically induced alterations in toxicity pathways and 

effects at the organ level. This research represents an ambitious effort, conceivable for the first 

time due to the current technological advances. Virtual tissue and organ system models will 

initially include liver, cardiopulmonary function, selected immune system tissues, multi-organ 

endocrine axes, and developing embryonic tissues. Development of these virtual tissue and organ 

systems will require newly generated data to both fill data gaps identified within the iterative 

process and test the predictive nature of these virtual systems. Comparative studies should 

include pathways fundamentally reliant upon cell signaling (e.g., cell proliferation, apoptosis, 

cell adhesion), intermediary metabolism (e.g., glycolysis, oxygen utilization, fatty acid 

biosynthesis), differentiation-specific functions (e.g., extracellular matrix remodeling), and other 

categories as developed above (see Strategic Goal 1) to ensure that predictions are broadly 

applicable. The wealth of existing data from NTP assays, published reports, and previous EPA 

intramural studies will be leveraged wherever possible with additional experiments designed to 

fill data gaps. Such efforts will also help answer how well in vitro experimental systems 

represent the full range of diverse cells present in the human body, how variability observed in 

the human population can modify quantitative predictions of in vivo dose-response, how 

exposure conditions influence outcomes, and how well the virtual tissue models represent the 

underlying processes. 

Not all toxicity pathways are likely to be expressed in every tissue, and likewise not all tissues 

are likely to manifest adverse outcomes following chemical perturbation. Chemicals that affect 

the same toxicity pathway can do so via a number of different (and overlapping) mechanisms, 

and development of assays across toxicity pathways leading to the same outcome is a necessary 

component of the proposed strategy. Some toxicities are manifest only when multiple cell types 

and specific cell-cell interactions are present. Other toxicities may be dependent upon tissue 

geometry and three-dimensional architecture. Examples include signaling between hepatocytes 

and Kupffer cells, or the many forms of signaling between epithelial and mesenchymal cells. As 

such, developers of virtual cells, tissues, organs, and systems must always bear in mind the need 

to remain relevant to the processes critical to expressions of toxicity in vivo. Consistent with the 

NRC vision (2007), this need will likely entail a continued although decreasing role for in vivo 

systems for the foreseeable future. 

A premise of the new toxicity testing strategy is that computational methods combined with an 

understanding of biological and exposure processes can be used to develop a more efficient and 

accurate approach for predicting risks from many chemicals. On the exposure side, models have 

been developed and are available that predict fate and transport, environmental concentration, 

exposures, and doses. These models work at multiple scales; for multiple sources, routes, and 

pathways; and for multiple chemicals, although each model only addresses a single process or 

compartment. Research is needed so that such models can take into account weathering of 

contaminants, differences in bioavailability of contaminants, variations in exposures with age, 

and variability in exposures within populations. Research is also needed to combine these models 

across various scales to develop a linked source-to-outcome modeling framework, to evaluate the 

framework using multiple chemicals and exposure scenarios, and to improve the computational 

15
 
 

efficiency for the approach. Ultimately, these exposure models will be linked to the virtual tissue 

models for utilizing in vitro toxicity test results in quantitative risk assessments. Given the 

complexity of the challenges present in addressing each of these components, this effort 

represents a long-term goal of the strategy. However, efforts must begin now to put us on the 

path to achieving the ultimate vision of Toxicity Testing in the 21st Century (NRC, 2007). 

The derived computational models must accurately describe the processes and mechanisms that 

determine exposure and effect. They must have reliable input parameters in order to quantify 

these processes. On the exposure side, our current understanding of processes and factors for 

many classes of chemicals and pathways (i.e., dermal and incidental ingestion) is limited. New 

approaches will be evaluated that will allow us to address the most significant uncertainties. 

Relational databases populated with data on exposures, exposure factors, activity patterns, and 

biomarkers will be developed as described. Informatic approaches or applications of network 

theory could potentially be used to provide a better understanding of important exposures, as 

well as exposure/response relationships. In the 2007 NRC report, emphasis was placed on 

biomarkers and their role in relating real world exposure to in vivo and in vitro biological 

response. They were also proposed as primary indicators in surveillance programs for tracking 

predicted exposures and health outcomes. Because of this emphasis, novel approaches for using 

biomarkers and integrating them into new risk assessment approaches will be investigated for 

chemicals already existing in the human environment. Perhaps such biomarker data can be used 

to improve predictive exposure models that will be relied upon for new chemicals not yet 

introduced into the environment. 

4.3. Strategic Goal 5: Human Evaluation and Quantitative Risk Assessment 

The critical challenge of this new vision for toxicity testing using mechanistic in vitro assays, 

targeted in vitro or in vivo testing, and computational models is to demonstrate that it 

successfully and adequately predicts human toxicological responses. Proof of concept efforts 

need to address this challenge both retrospectively and prospectively. Existing human data from 

pharmaceutical and environmental studies will be used to the extent possible. Human data could 

come from a range of sources including case reports, epidemiological studies (e.g., from the 

National Children’s Study), and clinical trials. EPA has extensive experience obtaining human 

clinical data following exposure to the criteria air pollutants (e.g., ozone, particulate matter) and 

other chemicals (e.g., MTBE)11. Engagement of the pharmaceutical industry and the Food and 

Drug Administration to access toxicity findings from clinical trials of drugs that were 

successfully registered or that failed to be registered would be a desirable component of this 

effort. Limited data may be available for some nutrients or dietary supplements as well. 

Such efforts will help address the question of the extent to which key events (critical 

perturbations) that are predictive of health endpoints (e.g., cancer, immunosuppression, kidney 

disease) must be demonstrated or whether the perturbation of baseline biological processes 

sufficient to induce substantial cellular level response (e.g., a stress response) should be 

considered an adequate endpoint for risk assessment. Linking a specific pathway perturbation to 

11 All EPA conducted or supported research is subject to and must comply with EPA regulations on the protection of 

human subjects. See http://www.epa.gov/fedrgstr/EPA-GENERAL/2006/February/Day-06/g1045.htm; 

http://www.epa.gov/oamrtpnc/forms/1000_17a.pdf 

16
 
a particular target organ endpoint has the advantage of predicting outcomes that are already used 

in risk assessment, while alternative approaches raise issues of which endpoints should and 

should not be considered for risk assessment. This approach is relatively straightforward for 

some effects (e.g., hemolysis of red blood cells by EGBE, where the effect and the mechanism of 

action leading to it are qualitatively the same, even if quantitatively different). Linkage is more 

complicated for effects observed in animals that may predict human effects that are related, but 

not identical to, the outcomes in animals (e.g., developmental effects in an animal model may 

predict developmental effects in humans, but the exact manifestation might be different). On the 

other hand, as knowledge is gained about the interaction of chemicals with molecular targets, and 

this knowledge is combined with information on how perturbations of those targets are translated 

to responses in species-specific patterns (e.g., how activation of certain transcription factors lead 

to species-specific tissues responses), it will be increasingly possible to predict human outcomes 

from in vitro studies that identify mechanism of action. Clearly this aspect will need to be 

addressed on a case-by-case basis as we gain experience. 

To be most useful in evaluation of risk to humans, the pathway-based efforts should ideally be 

tied to a known mechanism of action, such as via the use of quantitative biologically based, doseresponse models. Understanding of the relevant mechanism of action will enable the 

identification of biomarkers for key event parameters (linked to toxicity pathways) that can be 

monitored in human studies for those chemicals already released into the environment at 

significant levels. These biomarkers could be measured in observational human studies to 

provide in vivo data to support the underlying pathway-based model. In addition, genetic 

susceptibility in humans identified via whole genome association studies will provide support for 

pathway-based models when genes critical for a key toxicity pathway are associated with 

susceptibility. Finally, the use of quantitative models requires estimation of uncertainty and 

variability in the predictions from in vitro assays and computational models. Formal methods for 

model evaluation are essential for demonstrating the success of this new approach to toxicity 

testing and risk assessment. 

17
 
 

5. INSTITUTIONAL TRANSITION 

Implementing major changes in toxicity testing of environmental contaminants and incorporating 

new types of toxicity data into risk assessment will require significant institutional changes. This 

section will touch upon three major thrusts of implementing institutional transition: operational 

transition, organizational transition, and outreach. 

5.1. Strategic Goal 6: Operational Transition 

Operational transition covers the technical aspects associated with EPA’s implementation of a 

new toxicity testing paradigm and associated changes in risk assessment. It will consider such 

disparate topics as the importance of grounding the science, ensuring consistency of approaches 

within EPA, and working with outside partners and issues associated with the use of new models 

and tools. 

The NRC “envision[s] a future in which tests based on human cell systems can serve as better 

models of human biologic responses than apical studies in different species.” Achieving such a 

future, however, will require substantial research to study and define various toxicity pathways. 

In evaluating possible options for the future of toxicity testing, the NRC eventually chose an 

option involving both in vitro and in vivo tests but based primarily upon human biology and the 

attendant use of substantially fewer animal studies that would be focused on mechanism and 

metabolism. Their vision for the next 10 to 20 years relies on understanding perturbations of 

critical cellular responses and the use of computational approaches for assessing hazard and risk. 

A paradigm shift in toxicity testing based on pathway perturbation will likely require significant 

methodological advances and future changes to EPA’s risk assessment guidelines. Although it is 

infeasible to denote a specific timeline for how long it will take to substantially complete the 

strategic goals associated with toxicity pathway identification, chemical screening and 

prioritization, and toxicity pathway-based risk assessment, this plan takes the view that advances 

are likely to be gradual over the next decade or two. The good news is that toxicity testing 

research efforts have already begun moving EPA and others towards the use of in silico 

technologies and high throughput testing systems. The speed at which we are able to complete 

this transition will depend on the availability of increased research funding. It is important to 

note that our understanding of toxicity pathways for some apical endpoints (e.g., hepatotoxicity) 

may be developed at a faster pace than others (e.g., neurotoxicity) thus, allowing more rapid 

introduction of newer high-throughput in vitro testing methods. 

Grounding the Science – From a broad regulatory perspective, data used by EPA to support 

regulatory decisions will be shaped by the statutory language covering the action, regulatory 

policies, and the resulting time and resources allocated to the assessment. Where appropriate, use 

of data should be consistent with the EPA guidance articulated in a number of science policy and 

guidance documents, including toxicity testing guidelines, risk assessment guidelines12, 

information quality guidelines13, and peer review guidance.14 

12 http://www.epa.gov/risk/guidance.htm 

13 http://www.epa.gov/quality/informationguidelines/ 

14 http://www.epa.gov/peerreview/pdfs/Peer%20Review%20HandbookMay06.pdf 

18
 
 

To implement this new paradigm, regulators, stakeholders, and the public will need to develop 

confidence that the data generated can be used effectively and that public health will continue to 

be protected. A step-wise implementation is envisioned: first, experience will be gained from 

proof of concept studies using data from chemicals (e.g., pesticides) with a large set of toxicity 

data developed using the current paradigm. Availability of both new and traditional types of data 

will allow extrapolation and comparison of results across methodologies. 

Optimally, early success stories that meet programmatic needs in specific areas such as 

mechanism of action analyses or cumulative risk assessments will demonstrate the broader 

applicability of computational toxicology within the Agency. Reliability of the testing paradigm 

will need to be evaluated via a comprehensive development and review process, involving public 

comment, harmonization with other agencies and international organizations, and peer review by 

experts in the field. Bringing new methods into regulatory practice will require several phases 

starting from the development of the science and technologies, to technology transfer and 

building the regulatory infrastructure, to incorporation of the new tools into decision making. 

Because this transformative paradigm will rely on new and complex science and will likely be 

surrounded by some controversy, an important part of regulatory acceptance will be to conduct 

research that will verify the approaches and models that will come to replace much of the way 

toxicity testing and risk assessments are conducted in the Agency today. An important 

component of the effort to develop new approaches to testing will be to translate the research 

into regulatory applications. 

Issues Associated With the Use of New Methods and Models – For this new paradigm to be 

successful, new methods and models should be thoroughly evaluated prior to their application 

and use in regulatory decision making. The computer-based models used by the Agency should 

be publicly available. Testing methods should be accompanied by documentation that describes 

(1) the method and its theoretical basis, (2) the techniques used to verify that the method is 

accurate, and (3) the process used to evaluate whether the method and the results are sufficient to 

provide an adequate basis for its use in regulatory decision making. Access to data to allow for 

third party independent replication of results, to the extent practicable, is essential. Such review 

is appropriate before the Agency relies on data from such a method.15 

Working With Outside Partners –The appendix provides details about the many outside parties 

EPA will need to partner with in order to implement this strategic plan including: 

•	 Other federal bodies such as the National Toxicology Program (NTP) and the NIH 

Chemical Genomics Center (NCGC), with whom EPA has a memorandum of 

understanding to collaborate; 

•	 The Interagency Coordinating Committee on the Validation of Alternative Methods 

(ICCVAM), which is made up of representatives from 15 federal agencies that generate 

or use toxicological data; 

•	 Foreign governmental parties and programs such as REACH, which is the new European 

Union Regulation on Registration, Evaluation, Authorization, and Restriction of 

Chemicals that went into effect June 1, 2007; 

15 See http://epa.gov/crem/library/CREMguidancedraft12_03.pdf 

19
 
•	 The OECD (Organization for Economic Co-Operation and Development), which 

represents over 30 countries in the Americas, Europe and Asia; 

•	 Academia; 

•	 Chemical industry; and 

•	 Non-governmental organizations. 

Case Study Development – Significant challenges, such as interpretation and communication of 

data obtained using new toxicity testing approaches, will emerge under a new paradigm for 

toxicity testing. A key feature of a successful communication strategy will be to develop case 

studies using new kinds of data that can serve as a basis to explore, evaluate, and most 

importantly explain hazard, dose-response, and exposure information in a risk assessment 

framework. Characterization of risk information, both qualitative and quantitative, in a manner 

suitable for communication to risk managers will be a significant challenge for the research and 

risk assessment community, but it will be crucial if the new toxicity testing paradigm is to reach 

its potential. 

5.2. Strategic Goal 7: Organizational Transition 

Organizational transition is meant to cover changes in direction over time with regard to 

deployment of human capital resources necessary to implement the new toxicity testing 

paradigm such as hiring of scientists with particular scientific expertise and training of existing 

scientific staff. For example, EPA has hired key new scientific staff and initiated training 

including three new training courses in genomics designed and implemented by EPA’s Risk 

Assessment Forum. Additional resources and training programs will be needed in both EPA’s 

research program as well as its regulatory and regional programs. 

As noted in Section 2, several intra-agency, interagency, and international activities are already 

underway to begin the transformation that will change the nature of toxicity data generated and 

how it is used to assess chemically induced risks to human health. Substantial funding will be 

needed to provide the scientific basis for creating new testing tools; to verify the utility of new 

testing tools including conducting peer review; to develop and standardize data-storage, dataaccess, and data-management systems; to evaluate predictive power for humans; and to improve 

the understanding of the implications of test results and how they can be applied in risk 

assessments used in environmental decision-making. 

EPA expects that the use of less expensive, high-throughput testing methods will allow for the 

generation of toxicity data for thousands of currently untested or under-tested chemicals. The 

availability of these new data will likely lead to the need for more staff to interpret the data for 

many more chemicals and manage their risks. Additionally, toxicity databases such as EPA’s 

IRIS and models used to assess risks may need to undergo substantial changes in the long term 

requiring future resources. 

5.3. Strategic Goal 8: Outreach 

Outreach consists of those efforts that will be used to help educate the public and stakeholders as 

well as improve risk communication. 

20
 
 

In reaching out to the public, it will be important to re-emphasize points made by EPA 

Administrator Carol Browner in a 1995 memorandum to senior Agency staff about the Agency’s 

policy related to its new Risk Characterization Program. This memorandum described the 

importance of adhering to the “core values of transparency, clarity, consistency, and 

reasonableness (which) need to guide each of us in our day-to-day work; from the toxicologist 

reviewing the individual (scientific) study, to the exposure and risk assessors, to the risk 

manager, and through to the ultimate decision-maker.” Further, “because transparency in 

decision-making and clarity in communication will likely lead to more outside questioning of our 

assumptions and science policies, we must be more vigilant about ensuring that our core 

assumptions and science policies are consistent and comparable across programs, well grounded 

in science, and that they fall within a ‘zone of reasonableness.’”16 

Stakeholder Involvement – Implementation of a paradigm shift in toxicity testing and related 

changes to risk assessment methods and practices will require a sustained effort over many years 

– remember that the NRC envisioned some 10 to 20 years to reach their goal. This transition to 

new methods and approaches will need to be transparent, including efforts to share information 

with both the public and risk managers. It will be critical to effectively communicate with 

stakeholders (the public, scientists, federal and state agencies, industry, the mass media, 

nongovernmental organizations) about the new tools and the overall program regarding its 

strengths, limitations, and uncertainties. One way to enhance stakeholder involvement and ensure 

cooperation is to hold periodic workshops where all parties can gather to share information and 

progress; another tool is for EPA to establish a web portal to detail advancements in the science 

and relate these to improvements in risk assessment methods and practice. 

Collaboration among different elements in the research community involved in relevant research 

on new testing approaches will be needed to take advantage of the new knowledge, technologies, 

and analytical tools as they are developed, and collaboration between research and regulatory 

scientists will be vital to ensure that the methods developed can be reliably used in risk 

assessments of various types (initially qualitative, but ultimately both qualitative and 

quantitative). Mechanisms for ensuring sustained communication and collaboration, such as data 

sharing, will also be needed. Independent review and evaluation of the new toxicity testing 

paradigm should be conducted to provide advice for midcourse corrections, weigh progress, 

evaluate new and emerging methods, and make any necessary refinements in light of new 

scientific challenges/advances. This may be accomplished using existing EPA mechanisms for 

peer review, e.g., through reviews by the Board of Scientific Counselors, the Science Advisory 

Board, and the FIFRA Scientific Advisory Panel. For testing that the Agency may wish to 

require, performance standards should be considered so that individual methods from any 

qualified source may be used. The NRC (2007) stressed that “in vitro tests would be developed 

not to predict the results of current [animal] apical toxicity tests but rather as [human] cell-based 

assays that are informative about mechanistic responses of human tissues to toxic chemicals. 

The [NRC] committee is aware of the implementation challenges that the new toxicity-testing 

paradigm would face.” Presumably, establishing regulatory confidence that the new approaches 

are robust and protective of human health will be at the forefront of future challenges for EPA 

and its partners. 

16 http://www.epa.gov/oswer/riskassessment/pdf/1995_0521_risk_characterization_program.pdf 

21
 
 

Risk Communication – Communicating with policy makers and the public is an important part 

of any risk management exercise. The complexity of the emerging toxicity testing paradigm and 

how new types of data and information will be used to assess risk will make communication of 

results challenging; consequently, the Agency must work to build public trust in the adopted 

technologies. As the science moves away from well-established animal models, a significant 

effort must be made to share information with risk assessors/managers and the public by clearly 

describing test results and methodologies in a transparent manner. A fundamental aspect of 

gaining public trust is transparency. Therefore, education and effective communication with 

stakeholders (the public, scientists, regulatory authorities, industry, the mass media, and 

nongovernmental organizations) on the strengths, limitations, and uncertainties of the new 

tools/paradigm will be critical. 

Given that these new methods will be less intuitive than looking for traditional effects in whole 

animal studies, communication strategies will be very important. At this time, much of EPA’s 

effort in this area is presented on the Agency’s National Center for Computational Toxicology 

Web site.17 As the new toxicity testing paradigm continues to evolve, the Agency will need to be 

vigilant in maintaining an interactive Web site to describe each individual assay or method in use 

and where it fits into the exposure-response continuum. 

When communicating about risk, it is important for the Agency to address the source, cause, 

variability, uncertainty, and the potential adversity of the risks, including the degree of 

confidence in the risk assessment methodology, the rationale for the risk management decision, 

and the options for reducing risk (U.S. EPA, 1995; U.S. EPA, 1998). EPA will continue to 

interact with stakeholders in order to develop and maintain effective informational tools. 

17 http://www.epa.gov/comptox/ 

22
 
6. FUTURE STEPS 

This strategic plan describes an ambitious and substantive change in the process by which 

chemicals are evaluated for their toxicity. The NRC (2007) suggested that such a transformation 

would require up to $100M per year in funding over a 10-20 year period to have a reasonable 

chance of reaching the goals. Even including the resources of sister agencies, the overall federal 

budget for the collaborative efforts does not approach the NRC proposed level of funding. 

Decision on the relative role of EPA vis-à-vis other partners will have a major impact on the 

resources that EPA needs to dedicate to this effort. These decisions will have to be made as the 

strategy is implemented. Explanation of these decisions, their rationale, and implications will be 

included in a subsequent implementation plan. 

Regardless of whatever level of funding is ultimately applied to the vision of a more efficient and 

effective chemical safety evaluation effort, translation of this strategy into research and activities 

related to operational and organizational change will require development of an implementation 

plan as well as periodic peer review of directions and progress. Representatives from those EPA 

organizations most involved and impacted by the new vision will play key roles in the 

implementation program. The Science Advisory Board and/or the Board of Scientific Counselors 

will play key roles in the scientific peer review of the program. As noted in Section 4, there will 

be a progression in the 

implementation efforts from an 

early focus on hazard 

identification to a growing 

emphasis on the use of toxicity 

pathway characterization in risk 

assessment. Support for 

institutional transitions is also 

expected to increase over time as 

the tools and technologies 

emerge out of the research 

programs and become available 

for regulatory use. Figure 6 

depicts one potential way that the 

level of effort of the three main 

activities involved in this strategy 

could change over time. 

23
 

% Effort

this Strategic Plan over its Expected 20-year Duration. 

Screening/P rioritization 

Toxici ty P athways in 

Ri sk Assessment 

Institutional Transi ti on 

2010 2015 2020 2025 

Year 
 

APPENDIX: OTHER RELATED ACTIVITIES 

Other US Government Activities 

The National Toxicology Program (NTP) at the National Institute of Environmental Health 

Sciences (NIEHS) coordinates toxicological testing programs within the Department of Health 

and Human Services18. Similar to EPA, NTP is developing the use of computational models, in 

vitro assays, and non-mammalian in vivo assays targeting key pathways, molecular events, or 

processes linked to disease or injury for incorporation into a transformed chemical testing 

paradigm. 

The NIH Chemical Genomics Center (NCGC) of the National Human Genome Research 

Institute conducts ultra high throughput screening assays as part of the NIH’s Molecular 

Libraries Initiative within the NIH Roadmap 

A Memorandum of Understanding19 was recently signed by EPA, the NTP, and the NCGC to 

collaborate on generating a comprehensive map of the biological pathways affected by 

environmental chemical exposures and use this map to predict how potential chemical toxicants 

will affect various types of cells, tissues, and individuals. The hope is to refine many of the 

toxicity tests performed on animals and eventually supplant them with in vitro testing and 

computational prediction (Collins et al., 2008). 

In 2004 the Food and Drug Administration (FDA) produced a report20 addressing the need to 

translate the rapid advances in basic biomedical sciences into new preventions, treatments and 

cures. FDA holds large databases of human, animal, and in vitro data for screening drug 

candidates for toxicity that may also be useful for screening environmental chemicals. The 

FDA’s National Center for Toxicological Research (NCTR) aims to develop methods for the 

analysis and integration of genomic, transcriptomic, proteomic, and metabolomic data to 

elucidate mechanisms of toxicity21. NCTR has coordinated the Microarray Quality Control 

(MAQC) project, with numerous partners including EPA (Shi et al., 2006). In addition, NCTR 

has provided its ArrayTrack database to EPA for storage of genomics data for research and 

possible regulatory use. 

The Interagency Coordinating Committee on the Validation of Alternative Methods 

(ICCVAM) was established by law in 2000 to promote development, validation, and regulatory 

acceptance of alternative safety testing methods. ICCVAM is made up of representatives from 15 

federal agencies that generate or use toxicological data. Emphasis is on alternative methods that 

will reduce, refine, and/or replace the use of animals in testing while maintaining and promoting 

scientific quality and the protection of human health and the environment22. The NTP 

Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) 

administers and provides scientific support for ICCVAM. ICCVAM/NICEATM evaluates test 

method submissions and nominations, prepares technical review documents, and organizes 

18 http://ntp.niehs.nih.gov/ntp/main_pages/NTPVision.pdf 19 http://www.epa.gov/ncct/articles/comptox_mou.html; 

20 http://69.20.19.211/oc/initiatives/criticalpath/whitepaper.html 21 http://www.fda.gov/nctr/overview/mission.htm 

22 http://iccvam.niehs.nih.gov/about/ni_QA.htm 

24
 
 

scientific workshops and peer review meetings. For example, ICCVAM/NICEATM recently 

released a report23 that describes two in vitro cytotoxicity tests that can be used for estimating 

starting doses for acute oral toxicity tests, thereby reducing the number of animals used. 

Related Activities by Foreign Governments 

A new European Union Regulation on Registration, Evaluation, Authorization, and 

Restriction of Chemicals (REACH) went into effect June 1, 2007. The main goals of REACH 

are (1) to improve the protection of human health and the environment from risks associated with 

chemicals in commerce and (2) to promote alternative test methods. REACH requires 

manufacturers and importers to demonstrate they have appropriately identified and managed the 

risks of substances produced or imported in quantities of one ton or more per year per company. 

The new European Chemicals Agency (ECHA)24 will manage the system databases, coordinate 

evaluation of chemicals, and run a public database of hazard information25. 

The European Centre for the Validation of Alternative Methods (ECVAM)26 coordinates 

the validation of alternative test methods in the European Union. ECVAM develops, maintains, 

and manages a database on alternative procedures and promotes the development, validation, and 

international recognition of alternative test methods. 

The Japanese Center for the Validation of Alternative Methods (JaCVAM) is part of the 

Japanese National Institute of Health Sciences. JaCVAM has conducted validation studies for 

alternative test methods and participates in international validation efforts27. 

The Korean Center for the Validation of Alternative Methods (KoCVAM) is a branch of 

NITR, the National Institute of Toxicological Research. NITR is collaborating with the Korean 

Society for Alternatives to Animal Experiments (KSAAE) to refine methods in acute oral, 

reproductive/development, genetic, and endocrine toxicity testing28. 

The Organization for Economic Co-Operation and Development (OECD) represents 30 

countries in the Americas (including the United States), Europe, and Asia. The OECD 

“Guidelines for the Testing of Chemicals” provides a collection of internationally harmonized 

testing methods for a number of toxicological endpoints using in vivo, in vitro, and even 

alternative approaches.29 Test guidelines can be updated to reflect scientific advances and the 

state of the science if member countries agree to do so. A few OECD workgroups and efforts 

address issues relevant to this EPA strategy, e.g., the OECD QSAR Toolbox30 and the joint 

OECD/IPCS (International Programme for Chemical Safety) Toxicogenomics Working Group, 

which has developed a proposal for a Molecular Screening Project, modeled after EPA’s 

ToxCastTM program. 

23 http://iccvam.niehs.nih.gov/methods/acutetox/inv_nru_tmer.htm 24 http://echa.europa.eu/reach_en.asp 

25 http://ec.europa.eu/environment/chemicals/reach/reach_intro.htm 26 http://ecvam.jrc.it/ 

27 http://www.nihs.go.jp/english/index.html 

28 http://wwwsoc.nii.ac.jp/jsaae/PARK.pdf 29 http://titania.sourceoecd.org/vl=856000/cl=23/nw=1/rpsv/periodical/p15_about.htm?jnlissn=1607310x 

30 http://www.oecd.org/document/23/0,3343,en_2649_37465_33957015_1_1_1_37465,00.html 

25
 
 

Academia 

Numerous U.S. academic researchers and centers are funded by NIH or EPA’s National Center 

for Environmental Research to develop assays and analysis methods that might be helpful to the 

goals of this EPA research strategy. This includes two Bioinformatics Centers funded by EPA in 

2006. 

The European Commission funds several large academic, government, and industry consortia 

that are conducting research that could lead to effective in vitro toxicity tests. The CASCADE 

Network of Excellence31 studies human health effects of chemical residues and contaminants in 

food and drinking water, designing assays to elucidate estrogen, testosterone, and thyroid 

hormone pathways for the development of mechanism- and disease-based test methods. The aim 

of the carcinoGENOMICS32 project is to develop in vitro methods for assessing the 

carcinogenic potential of compounds. ReProTect33 is optimizing an integrated set of 

reproductive/developmental tests for a detailed understanding of gametogenesis, steroidogenesis, 

and embryogenesis that can support regulatory decisions. 

Industry 

The European Partnership for Alternative Approaches to Animal Testing (EPAA)34 is a 

joint initiative from the European Commission and a number of companies and trade federations. 

Its purpose is to promote the development of alternative approaches to safety testing. The EPAA 

focuses on mapping existing research; developing new alternative approaches and strategies; and 

promoting communication, education, validation, and acceptance of alternative approaches. 

Non-Governmental Organizations (NGOs) 

The Comparative Toxicogenomics Database35 (CTD) elucidates molecular mechanisms by 

which environmental chemicals affect human disease. CTD includes manually curated data 

describing cross-species chemical–gene/protein interactions and chemical– and gene–disease 

relationships to illuminate molecular mechanisms underlying variable susceptibility and 

environmentally influenced diseases. These data will also provide insights into complex 

chemical–gene and protein interaction networks. 

The Johns Hopkins Center for Alternatives to Animal Testing36 supports the creation, 

development, validation, and use of alternatives to animals in research, product safety testing, 

and education. Similarly, AltTox.org37 provides information on non-animal methods for toxicity 

testing including a table38 that summarizes the alternative testing methods by endpoint that have 

been approved or endorsed internationally by at least one regulatory agency. 

31 http://www.cascadenet.org/ 

32 http://www.carcinogenomics.eu/
 33 http://www.reprotect.eu/
 34 http://ec.europa.eu/enterprise/epaa/index_en.htm
 35 http://ctd.mdibl.org/ 

36 http://altweb.jhsph.edu/index.htm
 37 http://www.alttox.org/about/ 

38 http://www.alttox.org/ttrc/validation-ra/validated-ra-methods.html 

26
 
 

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