The last resort requirement under REACH: From principle to practice.

REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) is a European Union regulation that aims to protect human health and the environment from the risks posed by chemicals. Article 25 clearly states that: "[i]n order to avoid animal testing, testing on vertebrate animals for the purposes of this Regulation shall be undertaken only as a last resort." In practice, however, the standard information requirements under REACH are still primarily filled using animal studies. This paper presents examples illustrating that animal testing is not always undertaken only as a last resort. Six over-arching issues have been identified which contribute to this: (1) non-acceptance of existing animal or non-animal data, (2) non-acceptance of read-across, (3) inflexible administrative processes, (4) redundancy of testing, (5) testing despite animal welfare concerns and (6) testing for cosmetic-only ingredients. We, members of the Animal-Free Safety Assessment (AFSA) Collaboration, who work together to accelerate the global adoption of non-animal approaches for chemical safety assessment, herein propose several recommendations intended to aid the European Commission, the European Chemicals Agency and registrants to protect human health and the environment while avoiding unnecessary animal tests - truly upholding the last resort requirement in REACH.

Analysis of health concerns not addressed by REACH for low tonnage chemicals and opportunities for new approach methodology.

In Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) the criterion for deciding the studies that must be performed is the annual tonnage of the chemical manufactured or imported into the EU. The annual tonnage may be considered as a surrogate for levels of human exposure but this does not take into account the physico-chemical properties and use patterns that determine exposure. Chemicals are classified using data from REACH under areas of health concern covering effects on the skin and eye; sensitisation; acute, repeated and prolonged systemic exposure; effects on genetic material; carcinogenicity; and reproduction and development. We analysed the mandated study lists under REACH for each annual tonnage band in terms of the information they provide on each of the areas of health concern. Using the European Chemicals Agency (ECHA) REACH Registration data base of over 20,000 registered substances, we found that only 19% of registered substances have datasets on all areas of health concern. Information limited to acute exposure, sensitisation and genotoxicity was found for 62%. The analysis highlighted the shortfall of information mandated for substances in the lower tonnage bands. Deploying New Approach Methodologies (NAMs) at this lower tonnage band to assess health concerns which are currently not covered by REACH, such as repeat and extended exposure and carcinogenicity, would provide additional information and would be a way for registrants and regulators to gain experience in the use of NAMs. There are currently projects in Europe aiming to develop NAM-based assessment frameworks and they could find their first use in assessing low tonnage chemicals once confidence has been gained by their evaluation with data rich chemicals.

Exposure considerations in human safety assessment: Report from an EPAA Partners' Forum.

Understanding and estimating the exposure to a substance is one of the fundamental requirements for safe manufacture and use. Many approaches are taken to determine exposure to substances, mainly driven by potential use and regulatory need. There are many opportunities to improve and optimise the use of exposure information for chemical safety. The European Partnership for Alternative Approaches to Animal Testing (EPAA) therefore convened a Partners' Forum (PF) to explore exposure considerations in human safety assessment of industrial products to agree key conclusions for the regulatory acceptance of exposure assessment approaches and priority areas for further research investment. The PF recognised the widescale use of exposure information across industrial sectors with the possibilities of creating synergies between different sectors. Further, the PF acknowledged that the EPAA could make a significant contribution to promote the use of exposure data in human safety assessment, with an aim to address specific regulatory needs. To achieve this, research needs, as well as synergies and areas for potential collaboration across sectors, were identified.

Use of New Approach Methodologies (NAMs) in regulatory decisions for chemical safety: Report from an EPAA Deep Dive Workshop.

New Approach Methodologies (NAMs) are considered to include any in vitro, in silico or chemistry-based method, as well as the strategies to implement them, that may provide information that could inform chemical safety assessment. Current chemical legislation in the European Union is limited in its acceptance of the widespread use of NAMs. The European Partnership for Alternative Approaches to Animal Testing (EPAA) therefore convened a 'Deep Dive Workshop' to explore the use of NAMs in chemical safety assessment, the aim of which was to support regulatory decisions, whilst intending to protect human health. The workshop recognised that NAMs are currently used in many industrial sectors, with some considered as fit for regulatory purpose. Moreover, the workshop identified key discussion points that can be addressed to increase the use and regulatory acceptance of NAMs. These are based on the changes needed in frameworks for regulatory requirements and the essential needs in education, training and greater stakeholder engagement as well the gaps in the scientific basis of NAMs.

Upholding the EU's Commitment to 'Animal Testing as a Last Resort' Under REACH Requires a Paradigm Shift in How We Assess Chemical Safety to Close the Gap Between Regulatory Testing and Modern Safety Science.

Animal use for testing chemicals under REACH continues to increase, despite advances in non-animal safety science during the past 15 years. The application of modern science and technology, and the use of 'next generation' weight-of-evidence assessment approaches, are embedded in EU guidance for establishing the safety of cosmetics and foods - and of the ingredients used in these products. However, this is still not the case for the regulation of chemicals. Under the new Chemicals Strategy for Sustainability, thought leaders in human health and environmental protection are calling on the European Commission to quickly embrace the benefits of modern and innovative non-animal safety science, in place of outdated animal testing, if the EU is to be a leader in safe and sustainable innovation under the European Green Deal transformational change ambitions. The European Commission also needs to enable companies to meet their legal obligation to only conduct animal testing as a last resort, by providing a more flexible, science-based and consistent regulatory framework for assuring chemical safety, which supports the integration of data from different sources. We are at a tipping point for closing the gap between regulatory chemicals testing and modern safety science. It is time to join forces, across policy makers, scientists, regulators and lawyers, to lead the paradigm shift needed to deliver what EU citizens want - namely, chemicals and products that are safe and sustainable, without resorting to animal testing.

Finding synergies for the 3Rs - Repeated Dose Toxicity testing: Report from an EPAA Partners' Forum.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) convened a Partners' Forum on repeated dose toxicity (RDT) testing to identify synergies between industrial sectors and stakeholders along with opportunities to progress these in existing research frameworks. Although RTD testing is not performed across all industrial sectors, the OECD accepted tests can provide a rich source of information and play a pivotal role for safety decisions relating to the use of chemicals. Currently there are no validated alternatives to repeated dose testing and a direct one-to-one replacement is not appropriate. However, there are many projects and initiatives at the international level which aim to implement various aspects of replacement, reduction and refinement (the 3Rs) in RDT testing. Improved definition of use, through better problem formulation, aligned to harmonisation of regulations is a key area, as is the more rapid implementation of alternatives into the legislative framework. Existing test designs can be optimised to reduce animal use and increase information content. Greater use of exposure-led decisions and improvements in dose selection will be beneficial. In addition, EPAA facilitates sharing of case studies demonstrating the use of Next Generation Risk Assessment applying various New Approach Methodologies to assess RDT.

International Harmonization and Cooperation in the Validation of Alternative Methods.

The development and validation of scientific alternatives to animal testing is important not only from an ethical perspective (implementation of 3Rs), but also to improve safety assessment decision making with the use of mechanistic information of higher relevance to humans. To be effective in these efforts, it is however imperative that validation centres, industry, regulatory bodies, academia and other interested parties ensure a strong international cooperation, cross-sector collaboration and intense communication in the design, execution, and peer review of validation studies. Such an approach is critical to achieve harmonized and more transparent approaches to method validation, peer-review and recommendation, which will ultimately expedite the international acceptance of valid alternative methods or strategies by regulatory authorities and their implementation and use by stakeholders. It also allows achieving greater efficiency and effectiveness by avoiding duplication of effort and leveraging limited resources. In view of achieving these goals, the International Cooperation on Alternative Test Methods (ICATM) was established in 2009 by validation centres from Europe, USA, Canada and Japan. ICATM was later joined by Korea in 2011 and currently also counts with Brazil and China as observers. This chapter describes the existing differences across world regions and major efforts carried out for achieving consistent international cooperation and harmonization in the validation and adoption of alternative approaches to animal testing.

New approach methodologies (NAMs): identifying and overcoming hurdles to accelerated adoption.

New approach methodologies (NAMs) can deliver improved chemical safety assessment through the provision of more protective and/or relevant models that have a reduced reliance on animals. Despite the widely acknowledged benefits offered by NAMs, there continue to be barriers that prevent or limit their application for decision-making in chemical safety assessment. These include barriers related to real and perceived scientific, technical, legislative and economic issues, as well as cultural and societal obstacles that may relate to inertia, familiarity, and comfort with established methods, and perceptions around regulatory expectations and acceptance. This article focuses on chemical safety science, exposure, hazard, and risk assessment, and explores the nature of these barriers and how they can be overcome to drive the wider exploitation and acceptance of NAMs. Short-, mid- and longer-term goals are outlined that embrace the opportunities provided by NAMs to deliver improved protection of human health and environmental security as part of a new paradigm that incorporates exposure science and a culture that promotes the use of protective toxicological risk assessments.

Next generation risk assessment for occupational chemical safety - A real world example with sodium-2-hydroxyethane sulfonate.

Next Generation Risk Assessment (NGRA) is an exposure-led approach to safety assessment that uses New Approach Methodologies (NAMs). Application of NGRA has been largely restricted to assessments of consumer use of cosmetics and is not currently implemented in occupational safety assessments, e.g. under EU REACH. By contrast, a large proportion of regulatory worker safety assessments are underpinned by toxicological studies using experimental animals. Consequently, occupational safety assessment represents an area that would benefit from increasing application of NGRA to safety decision making. Here, a workflow for conducting NGRA under an occupational safety context was developed, which is illustrated with a case study chemical; sodium 2-hydroxyethane sulphonate (sodium isethionate or SI). Exposures were estimated using a standard occupational exposure model following a comprehensive life cycle assessment of SI and considering factory-specific data. Outputs of this model were then used to estimate internal exposures using a Physiologically Based Kinetic (PBK) model, which was constructed with SI specific Absorption, Distribution, Metabolism and Excretion (ADME) data. PBK modelling indicated a worst-case plasma maximum concentration (Cmax) of 0.8 µM across the SI life cycle. SI bioactivity was assessed in a battery of NAMs relevant to systemic, reproductive, and developmental toxicity; a cell stress panel, high throughput transcriptomics in three cell lines (HepG2, HepaRG and MCF-7 cells), pharmacological profiling and specific assays relating to developmental toxicity (Reprotracker and devTOX quickPredict). Points of Departure (PoDs) for SI ranged from 104 to 5044 µM. Cmax values obtained from PBK modelling of occupational exposures to SI were compared with PoDs from the bioactivity assays to derive Bioactivity Exposure Ratios (BERs) which demonstrated the safety for workers exposed to SI under current levels of factory specific risk management. In summary, the tiered and iterative workflow developed here represents an opportunity for integrating non animal approaches for a large subset of substances for which systemic worker safety assessment is required. Such an approach could be followed to ensure that animal testing is only conducted as a "last resort" e.g. under EU REACH.

Ready for regulatory use: NAMs and NGRA for chemical safety assurance.

New approach methodologies (NAMs) that do not use experimental animals are, in certain settings, entirely appropriate for assuring the safety of chemical ingredients, although regulatory adoption has been slow. In this opinion article we discuss how scientific advances that utilize NAMs to certify systemic safety are available now and merit broader acceptance within the framework of next generation risk assessments (NGRA).

Computational chemistry, systems biology and toxicology. Harnessing the chemistry of life: revolutionizing toxicology. a commentary.

There is a continuing interest in, and increasing imperatives for, the development of alternative methods for toxicological evaluations that do not require the use of animals. Although a significant investment has resulted in some achievements, progress has been patchy and there remain many challenges. Among the most significant hurdles is developing non-animal methods that would permit assessment of the potential for a chemical or drug to cause adverse health effects following repeated systemic exposure. Developing approaches to address this challenge has been one of the objectives of the European Partnership for Alternative Approaches to Animal Testing (EPAA). The EPAA is a unique partnership between the European Commission and industry that has interests in all aspects of reducing, refining and replacing the use of animals (the '3Rs'). One possible strategy that emerged from a broad scientific debate sponsored by the EPAA was the opportunity for developing entirely new paradigms for toxicity testing based upon harnessing the increasing power of computational chemistry in combination with advanced systems biology. This brief commentary summarizes a workshop organized by the EPAA in 2010, that had the ambitious title of 'Harnessing the Chemistry of Life: Revolutionizing Toxicology'. At that workshop international experts in chemistry, systems biology and toxicology sought to map out how best developments in these sciences could be exploited to design new strategies for toxicity testing using adverse effects in the liver as an initial focus of attention. Here we describe the workshop design and outputs, the primary purpose being to stimulate debate about the need to align different areas of science with toxicology if new and truly innovative approaches to toxicity testing are to be developed.

A Next-Generation Risk Assessment Case Study for Coumarin in Cosmetic Products.

Next-Generation Risk Assessment is defined as an exposure-led, hypothesis-driven risk assessment approach that integrates new approach methodologies (NAMs) to assure safety without the use of animal testing. These principles were applied to a hypothetical safety assessment of 0.1% coumarin in face cream and body lotion. For the purpose of evaluating the use of NAMs, existing animal and human data on coumarin were excluded. Internal concentrations (plasma Cmax) were estimated using a physiologically based kinetic model for dermally applied coumarin. Systemic toxicity was assessed using a battery of in vitro NAMs to identify points of departure (PoDs) for a variety of biological effects such as receptor-mediated and immunomodulatory effects (Eurofins SafetyScreen44 and BioMap Diversity 8 Panel, respectively), and general bioactivity (ToxCast data, an in vitro cell stress panel and high-throughput transcriptomics). In addition, in silico alerts for genotoxicity were followed up with the ToxTracker tool. The PoDs from the in vitro assays were plotted against the calculated in vivo exposure to calculate a margin of safety with associated uncertainty. The predicted Cmax values for face cream and body lotion were lower than all PoDs with margin of safety higher than 100. Furthermore, coumarin was not genotoxic, did not bind to any of the 44 receptors tested and did not show any immunomodulatory effects at consumer-relevant exposures. In conclusion, this case study demonstrated the value of integrating exposure science, computational modeling and in vitro bioactivity data, to reach a safety decision without animal data.

Vision of a near future: Bridging the human health-environment divide. Toward an integrated strategy to understand mechanisms across species for chemical safety assessment.

There is a growing recognition that application of mechanistic approaches to understand cross-species shared molecular targets and pathway conservation in the context of hazard characterization, provide significant opportunities in risk assessment (RA) for both human health and environmental safety. Specifically, it has been recognized that a more comprehensive and reliable understanding of similarities and differences in biological pathways across a variety of species will better enable cross-species extrapolation of potential adverse toxicological effects. Ultimately, this would also advance the generation and use of mechanistic data for both human health and environmental RA. A workshop brought together representatives from industry, academia and government to discuss how to improve the use of existing data, and to generate new NAMs data to derive better mechanistic understanding between humans and environmentally-relevant species, ultimately resulting in holistic chemical safety decisions. Thanks to a thorough dialogue among all participants, key challenges, current gaps and research needs were identified, and potential solutions proposed. This discussion highlighted the common objective to progress toward more predictive, mechanistically based, data-driven and animal-free chemical safety assessments. Overall, the participants recognized that there is no single approach which would provide all the answers for bridging the gap between mechanism-based human health and environmental RA, but acknowledged we now have the incentive, tools and data availability to address this concept, maximizing the potential for improvements in both human health and environmental RA.

Non-animal approaches for consumer safety risk assessments: Unilever's scientific research programme.

Non-animal based approaches to risk assessment are now routinely used for assuring consumer safety for some endpoints (such as skin irritation) following considerable investment in developing and applying new methods over the past 20 years. Unilever's research programme into non-animal approaches for safety assessment is currently focused on the application of new technologies to risk assessments in the areas of skin allergy, cancer and general toxicity (including inhalation toxicity). In all of these areas, a long-term investment is essential to increase the scientific understanding of the underlying biological and chemical processes that we believe will ultimately form a sound basis for novel risk assessment approaches. Our research programme in these priority areas consists of in-house research as well as Unilever-sponsored academic research, involvement with EU-funded projects (e.g. Sens-it-iv, carcinoGENOMICS), participation in cross-industry collaborative research (e.g. COLIPA, EPAA) and ongoing involvement with other scientific initiatives on non-animal approaches to risk assessment (e.g. UK NC3Rs, US 'Human Toxicology Project' consortium).

Assuring consumer safety without animal testing: a feasibility case study for skin sensitisation.

Allergic Contact Dermatitis (ACD; chemical-induced skin sensitisation) represents a key consumer safety endpoint for the cosmetics industry. At present, animal tests (predominantly the mouse Local Lymph Node Assay) are used to generate skin sensitisation hazard data for use in consumer safety risk assessments. An animal testing ban on chemicals to be used in cosmetics will come into effect in the European Union (EU) from March 2009. This animal testing ban is also linked to an EU marketing ban on products containing any ingredients that have been subsequently tested in animals, from March 2009 or March 2013, depending on the toxicological endpoint of concern. Consequently, the testing of cosmetic ingredients in animals for their potential to induce skin sensitisation will be subject to an EU marketing ban, from March 2013 onwards. Our conceptual framework and strategy to deliver a non-animal approach to consumer safety risk assessment can be summarised as an evaluation of new technologies (e.g. 'omics', informatics), leading to the development of new non-animal (in silico and in vitro) predictive models for the generation and interpretation of new forms of hazard characterisation data, followed by the development of new risk assessment approaches to integrate these new forms of data and information in the context of human exposure. Following the principles of the conceptual framework, we have been investigating existing and developing new technologies, models and approaches, in order to explore the feasibility of delivering consumer safety risk assessment decisions in the absence of new animal data. We present here our progress in implementing this conceptual framework, with the skin sensitisation endpoint used as a case study.

A PGC-1α-Mediated Transcriptional Network Maintains Mitochondrial Redox and Bioenergetic Homeostasis against Doxorubicin-Induced Toxicity in Human Cardiomyocytes: Implementation of TT21C.

Chemical toxicity testing is fast moving in a direction that relies increasingly on cell-basedin vitroassays anchored on toxicity pathways according to the toxicity testing in the 21st century vision. Identifying points of departure (POD) via these assays and revealing their mechanistic underpinnings via computational modeling of the relevant pathways are critical and challenging steps. Here we used doxorubicin (DOX) as a prototype chemical to study mitochondrial toxicity in human AC16 cells. Mitochondrial toxicity has been linked to cardiovascular risk of DOX, which has limited its clinical use as an antitumor drug. Ourin vitrostudy revealed a well-defined POD concentration of DOX below which adaptive induction of proliferator-activated receptor-γ coactivator-1α (PGC-1α) -mediated mitochondrial genes, including NRF-1, MnSOD, UCP2, and COX1, concurred with negligible changes in mitochondrial superoxide and cytotoxicity. At higher DOX concentrations adversity became significant with elevated superoxide and suppressed ATP levels. A computational model was formulated to simulate the PGC-1α-mediated transcriptional network comprising multiple negative feedback loops that underlie redox and bioenergetics homeostasis in the mitochondrion. The model recapitulated the transition phase from adaptive to adverse responses, supporting the notion that saturated induction of PGC-1α-mediated gene network underpins POD. The model further predicts (follow-up experiments verified) that silencing PGC-1α compromises the adaptive function of the transcriptional network, leading to disruption of mitochondria and cytotoxicity at lower DOX concentrations. In summary, our study demonstrates that combining pathway-focusedin vitroassays and computational simulation of relevant biochemical network is synergistic for understanding dose-response behaviors in the low-dose region and identifying POD.

Non-cytotoxic concentrations of acetaminophen induced mitochondrial biogenesis and antioxidant response in HepG2 cells.

Mitochondrial dysfunction has been implicated in acute, severe liver injury caused by overdose of acetaminophen (APAP). However, whether mitochondrial biogenesis is involved is unclear. Here we demonstrated that mitochondrial biogenesis, as indicated by the amounts of mitochondrial DNA and proteins, increased significantly in HepG2 cells exposed to low, non-cytotoxic concentrations of APAP. This heightened response was accompanied by upregulated expression of PGC-1α, NRF-1 and TFAM, which are key transcriptional regulators of mitochondrial biogenesis. Additionally, antioxidants including glutathione, MnSOD, HO-1, NQO1, and Nrf2 were also significantly upregulated. In contrast, for HepG2 cells exposed to high, cytotoxic concentration of APAP, mitochondrial biogenesis was inhibited and the expression of its regulatory proteins and antioxidants were concentration-dependently downregulated. In summary, our study indicated that mitochondrial biogenesis, along with antioxidant induction, may be an important cellular adaptive mechanism counteracting APAP-induced toxicity and overwhelming this cytoprotective capacity could result in liver injury.

Aligning the 3Rs with new paradigms in the safety assessment of chemicals.

There are currently several factors driving a move away from the reliance on in vivo toxicity testing for the purposes of chemical safety assessment. Progress has started to be made in the development and validation of non-animal methods. However, recent advances in the biosciences provide exciting opportunities to accelerate this process and to ensure that the alternative paradigms for hazard identification and risk assessment deliver lasting 3Rs benefits, whilst improving the quality and relevance of safety assessment. The NC3Rs, a UK-based scientific organisation which supports the development and application of novel 3Rs techniques and approaches, held a workshop recently which brought together over 20 international experts in the field of chemical safety assessment. The aim of this workshop was to review the current scientific, technical and regulatory landscapes, and to identify key opportunities towards reaching these goals. Here, we consider areas where further strategic investment will need to be focused if significant impact on 3Rs is to be matched with improved safety science, and why the timing is right for the field to work together towards an environment where we no longer rely on whole animal data for the accurate safety assessment of chemicals.

Implementing Toxicity Testing in the 21st Century (TT21C): Making safety decisions using toxicity pathways, and progress in a prototype risk assessment.

Risk assessment methodologies in toxicology have remained largely unchanged for decades. The default approach uses high dose animal studies, together with human exposure estimates, and conservative assessment (uncertainty) factors or linear extrapolations to determine whether a specific chemical exposure is 'safe' or 'unsafe'. Although some incremental changes have appeared over the years, results from all new approaches are still judged against this process of extrapolating high-dose effects in animals to low-dose exposures in humans. The US National Research Council blueprint for change, entitled Toxicity Testing in the 21st Century: A Vision and Strategy called for a transformation of toxicity testing from a system based on high-dose studies in laboratory animals to one founded primarily on in vitro methods that evaluate changes in normal cellular signalling pathways using human-relevant cells or tissues. More recently, this concept of pathways-based approaches to risk assessment has been expanded by the description of 'Adverse Outcome Pathways' (AOPs). The question, however, has been how to translate this AOP/TT21C vision into the practical tools that will be useful to those expected to make safety decisions. We have sought to provide a practical example of how the TT21C vision can be implemented to facilitate a safety assessment for a commercial chemical without the use of animal testing. To this end, the key elements of the TT21C vision have been broken down to a set of actions that can be brought together to achieve such a safety assessment. Such components of a pathways-based risk assessment have been widely discussed, however to-date, no worked examples of the entire risk assessment process exist. In order to begin to test the process, we have taken the approach of examining a prototype toxicity pathway (DNA damage responses mediated by the p53 network) and constructing a strategy for the development of a pathway based risk assessment for a specific chemical in a case study mode. This contribution represents a 'work-in-progress' and is meant to both highlight concepts that are well-developed and identify aspects of the overall process which require additional development. To guide our understanding of what a pathways-based risk assessment could look like in practice, we chose to work on a case study chemical (quercetin) with a defined human exposure and to bring a multidisciplinary team of chemists, biologists, modellers and risk assessors to work together towards a safety assessment. Our goal was to see if the in vitro dose response for quercetin could be sufficiently understood to construct a TT21C risk assessment without recourse to rodent carcinogenicity study data. The data presented include high throughput pathway biomarkers (p-H2AX, p-ATM, p-ATR, p-Chk2, p53, p-p53, MDM2 and Wip1) and markers of cell-cycle, apoptosis and micronuclei formation, plus gene transcription in HT1080 cells. Eighteen point dose response curves were generated using flow cytometry and imaging to determine the concentrations that resulted in significant perturbation. NOELs and BMDs were compared to the output from biokinetic modelling and the potential for in vitro to in vivo extrapolation explored. A first tier risk assessment was performed comparing the total quercetin concentration in the in vitro systems with the predicted total quercetin concentration in plasma and tissues. The shortcomings of this approach and recommendations for improvement are described. This paper therefore describes the current progress in an ongoing research effort aimed at providing a pathways-based, proof-of-concept in vitro-only safety assessment for a consumer use product.

Pathway-based toxicity: history, current approaches and liver fibrosis and steatosis as prototypes.

The Human Toxicology Project Consortium (HTPC) was created to accelerate implementation of the science and policies required to achieve a pathway-based foundation for toxicology as articulated in the 2007 National Research Council report, Toxicity Testing in the 21st Century: a Vision and a Strategy. The HTPC held a workshop, "Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action," in January, 2013, in Baltimore, MD, to further the science of pathway-based approaches to liver toxicity. This review was initiated as a thought-starter for this workshop and has since been updated to include insights from the workshop and other activities occurring in 2013. The report of the workshop has been published elsewhere in this journal (Willett et al., 2014).