Challenges on the Road to Pathway-based Predictive Toxicology

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Overarching Challenges

Challenges on the Road to Pathway-based Predictive Toxicology

Martin L. Stephens, Center for Alternatives to Animal Testing, and Nina S. Mak, Alternatives Research and Development Foundation

Published: June 11, 2014

About the Author(s)
Martin L. Stephens is a senior research associate at the Johns Hopkins Center for Alternatives to Animal Testing (CAAT), where he promotes new methods in toxicology and new approaches to assessing their performance. He serves as director of the Evidence-based Toxicology Collaboration, which seeks to translate evidence-based approaches from medicine to toxicology. Prior to joining Johns Hopkins University in 2011, Dr. Stephens was vice president for animal research issues at The Humane Society of the United States. He served on the National Academy of Sciences’ committee that produced the landmark report on Toxicity Testing in the 21st Century: A Vision and a Strategy. He currently serves on the Management Team of AltTox and on the board of directors of Chimp Haven, a congressionally mandated sanctuary for former laboratory chimpanzees. Dr. Stephens has received the Society of Toxicology Enhancement of Animal Welfare Award, the Doerenkamp-Zbinden Award, and the CAAT Recognition Award, for his contributions to animal protection and alternative methods. He has a Ph.D. in biology from The University of Chicago.

Martin Stephens, Ph.D.
Johns Hopkins Bloomberg School of Public Health,
Center for Alternatives to Animal Testing
615 N Wolfe St.
Baltimore MD 21205 USA

Nina Mak is a Program Consultant for the Alternatives Research & Development Foundation.  In addition to her work on alternatives in toxicology, she has also been a Research and Policy Analyst at the American Anti-Vivisection Society for 8 years, focused on animal biotechnology, IACUCs, the 3Rs, and other issues related to the use of animals in science.


Editor’s note: A new book co-edited by David Allen and Mike D. Waters, “Reducing, Refining and Replacing the Use of Animals in Toxicity Testing” (Royal Society of Chemistry, 2014), provides an overview of the state of technology and policy governing the use of alternative methods in toxicity testing. founder and Management Team member, Martin L. Stephens, along with co-author Nina S. Mak, contributed the opening chapter, “History of the 3Rs in Toxicity Testing: From Russell and Burch to 21st Century Toxicology.” We featured an excerpt of their chapter in a recent “Spotlight:” “Validation and Acceptance Status of Alternatives.”

Here we present the authors’ overview of the challenges that remain in fully implementing a pathway-based approach to predictive toxicology such as that outlined in the U.S. National Research Council’s (NRC) seminal 2007 report, “Toxicity Testing in the 21st Century: A Vision and Strategy.” References and footnotes available in the chapter text are not included in this brief excerpt (section 1.7, “Remaining Challenges”), which is published with the generous permission of the Royal Society of Chemistry.

In the decades following publication of Russell and Burch’s The Principles of Humane Experimental Technique in 1959, the emerging 3Rs community devoted much of its energy to the field of toxicology. An infrastructure for making progress was slowly and steadily developed. This included alternatives-based organizations and centers, journals, websites, laws, conferences, and other activity.

The 3Rs community also spearheaded the development of the scientific standards (e.g., validation) and worked with fellow toxicologists and others to improve the techniques (e.g., tissue culture) that facilitated progress. The result has been a progressive chipping away at traditional animal-based methods.

The publication of the NRC report on Toxicity Testing in the 21st Century in 2007 created a new point of reference for the 3Rs community, suggesting a new approach to replacing animal use in toxicology. Within the mainstream toxicology and public health communities, the NRC report engendered considerable enthusiasm for modernizing toxicology. Leading federal scientists began predicting a paradigm shift from in vivo– to in vitro-based methods (see Section 1.1). Many in the alternatives community have been seeking to facilitate this long-term effort. As pathway-based approaches are further elaborated, they can be incorporated into ongoing 3Rs efforts.

However, implementing pathway-based approaches to predictive toxicology raises a host of formidable challenges. These include developing fit-for-purpose assays to monitor pathway perturbations, distinguishing adverse responses in these assays from homeostatic responses, accounting for metabolism of the parent compound, and shifting to a safety-based risk assessment paradigm, rather than one based on hazard.

21st century toxicology efforts have the long-term goal of providing a new paradigm for safety assessment; however, for the time being, most of these efforts are being harnessed to predict the outcomes of traditional animal testing. Consequently, the first applications are supplementing rather than supplanting the current paradigm. We need to accelerate the transition to the new paradigm, based on human biology.

20th century validation processes will need to be adapted to 21st century toxicology. There are many reasons why the existing validation processes should not be deployed as is. First, pathway-based methods are not intended as one-to-one replacements of animal-based tests; multiple pathway-based assays, perhaps numbering in the hundreds, will be used to make predictions about individual chemicals. Second, pathway-based methods ultimately will be called on to mimic human biology, rather than to predict the results of animal testing – the standard assurance of relevance. Third, according to the NRC framework, pathway-based assays will be used to predict regions of safe exposures, rather than predict specific toxicities. And fourth, the science and technology of 21st century toxicology are changing too rapidly for an evaluation process that takes a year or more to complete.

Most of the existing validation principles may carry over and transcend a paradigm shift in testing, but the prevailing validation procedures will need to be translated to accommodate these new realities. Validation procedures must also somehow be speeded up to accommodate the new pace of change and be flexible enough to accommodate an ideal of continual improvement in testing methods. Some rethinking of validation has begun to occur in this context.

And finally, perhaps the biggest real-world challenge to further progress on the 3Rs in toxicology is tackling systemic and chronic toxicity testing. Some large-scale programs in Europe have been seeking to address elements of this challenge, such as REPROTECT (reproductive toxicity) and SEURAT-1 (repeat dose systemic toxicity testing), with coordination and guidance provided by the AXLR8 program. In the U.S., high-throughput testing is being used to identify biological signatures of chronic, systemic endpoints, such as in developmental and reproductive toxicity. Clearly, integrated testing strategies involving multiple types of testing and approaches (e.g., pathway-based testing, high-throughput testing, toxicogenomics, organ-on-a-chip platforms, virtual organs) will play a role. Also critical will be a realistic assessment of the limitations (as well as the strengths) of the current animal-based assays.

©2014 Martin L. Stephens and Nina S. Mak