Current Trends of Practices in Nonclinical Toxicology: An Industry Survey.

The growth in drug development over the past years reflects significant advancements in basic sciences and a greater understanding of molecular pathways of disease. Benchmarking industry practices has been important to enable a critical reflection on the path to evolve pharmaceutical testing, and the outcome of past industry surveys has had some impact on best practices in testing. A survey was provided to members of SPS, ACT, and STP. The survey consisted of 37 questions and was provided to 2550 participants with a response rate of 24%. Most respondents (∼75%) came from the US and Europe. The survey encompassed multiple topics encountered in nonclinical testing of pharmaceuticals. The most frequent target indications were oncology (69%), inflammation (55%), neurology/psychiatry/pain (46%), cardiovascular (44%), and metabolic diseases (39%). The most frequent drug-induced toxicology issues confronted were hepatic, hematopoietic, and gastrointestinal. Toxicological effects that impacted the no observed adverse effect level (NOAEL) were most frequently based on histopathology findings. The survey comprised topics encountered in the use of biomarkers in nonclinical safety assessment, most commonly those used to assess inflammation, cardiac/vascular, renal, and hepatic toxicity as well as common practices related to the assessment of endocrine effects, carcinogenicity, genotoxicity, juvenile and male-mediated developmental and female reproductive toxicity. The survey explored the impact of regulatory meetings on program design, application of the 3 Rs, and reasons for program delays. Overall, the survey results provide a broad perspective of current practices based on the experience of the scientific community engaged in nonclinical safety assessment.

Analysis and reflection on the role of the 90-day oral toxicity study in European chemical risk assessment.

The 90-day toxicity study is one of the studies used in the safety assessment of food ingredients, medicines or other chemical substances. This paper reviews the current role of the 90-day oral toxicity study in European regulatory dossiers of chemicals by reviewing EU legislation and EU and OECD guidance documents. Regulatory provisions with regard to necessity, objectives and design of such 90-day toxicity studies vary between the different sectors addressed in this review. Most often the 90-day study is expected to be part of the standard test battery used for chemical risk assessment, without necessarily being a legal requirement and its objectives may vary between regulatory domains. Exceptions, when a 90-day study is not required are spelled out in the chemicals legislation and for food contact materials. The sectorial study design requirements of the 90-day toxicity study are very often embedded in the OECD TG 408 protocol. Differences in study objectives are not necessarily reflected in specific study designs. Considering the call for the reduction of using experimental animals for scientific purposes and the fact that a 90-day study may serve different purposes, consistency between the necessity to conduct such a study, its objectives and the study design to achieve these objectives may improve judicious use of laboratory animals. Thus there may be an opportunity to reflect and further optimise the design of in vivo toxicology studies, such as the 90-day study. This should be based on a systematic analysis of past studies and risk assessments.

Animal testing and its alternatives - the most important omics is economics.

For a long time, the discussion about animal testing vs its alternatives centered on animal welfare. This was a static warfare, or at least a gridlock, where life scientists had to take a position and make their value choices and hardly anyone changed sides. Technical advances have changed the frontline somewhat, with in vitro and in silico methods gaining more ground. Only more recently has the economic view begun to have an impact: Many animal tests are simply too costly, take too long, and give misleading results. As an extension and update to previous articles in this series written a decade ago, we reanalyze the economic landscape of especially regulatory use of animal testing and this time also consider respective alternative tests. Despite some ambiguity and data gaps, which we have filled with crude estimates, a picture emerges of globally regulated industries that are subject to stark geographic and sectorial differences in regulation, which determine their corresponding animal use. Both animal testing and its alternatives are industries in their own right, offering remarkable business opportunities for biotech and IT companies as well as contract research organizations. In light of recent revelations as to the reproducibility and relevance issues of many animal tests, the economic consequences of incorrect results and the reasons for still maintaining often outdated animal test approaches are discussed.

Advances in assessing ingredient safety.

The safety of food ingredients will be assessed in the 21st century by mixture of traditional methods, such as the "safe" dose concept, which is thought to be an accurate but imprecise estimation of dose below the population threshold for adverse effect, and contemporary methods, such as the Benchmark Dose (BMD), Chemical Specific Adjustment Factors (CSAF), physiologically-based pharmacokinetic models, and biologically-informed dose response modeling. New research on the horizon related to toxicology 21 may also improve these risk assessment methods, or suggest new ones. These traditional, contemporary and new methods and research will be briefly described.

Adverse Outcome Pathways can drive non-animal approaches for safety assessment.

Adverse Outcome Pathways (AOPs) provide an opportunity to develop new and more accurate safety assessment processes for drugs and other chemicals, and may ultimately play an important role in regulatory decision making. Not only can the development and application of AOPs pave the way for the development of improved evidence-based approaches for hazard and risk assessment, there is also the promise of a significant impact on animal welfare, with a reduced reliance on animal-based methods. The establishment of a useable and coherent knowledge framework under which AOPs will be developed and applied has been a first critical step towards realizing this opportunity. This article explores how the development of AOPs under this framework, and their application in practice, could benefit the science and practice of safety assessment, while in parallel stimulating a move away from traditional methods towards an increased acceptance of non-animal approaches. We discuss here the key areas where current, and future initiatives should be focused to enable the translation of AOPs into routine chemical safety assessment, and lasting 3Rs benefits.

Predicting the future: opportunities and challenges for the chemical industry to apply 21st-century toxicity testing.

Interest in applying 21st-century toxicity testing tools for safety assessment of industrial chemicals is growing. Whereas conventional toxicology uses mainly animal-based, descriptive methods, a paradigm shift is emerging in which computational approaches, systems biology, high-throughput in vitro toxicity assays, and high-throughput exposure assessments are beginning to be applied to mechanism-based risk assessments in a time- and resource-efficient fashion. Here we describe recent advances in predictive safety assessment, with a focus on their strategic application to meet the changing demands of the chemical industry and its stakeholders. The opportunities to apply these new approaches is extensive and include screening of new chemicals, informing the design of safer and more sustainable chemical alternatives, filling information gaps on data-poor chemicals already in commerce, strengthening read-across methodology for categories of chemicals sharing similar modes of action, and optimizing the design of reduced-risk product formulations. Finally, we discuss how these predictive approaches dovetail with in vivo integrated testing strategies within repeated-dose regulatory toxicity studies, which are in line with 3Rs principles to refine, reduce, and replace animal testing. Strategic application of these tools is the foundation for informed and efficient safety assessment testing strategies that can be applied at all stages of the product-development process.

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.

The long and winding road of progress in the use of in vitro data for risk assessment purposes: From "carnation test" to integrated testing strategies.

This paper introduces the special issue on quantitative in vitro-in vivo extrapolations (QIVIVE). It highlights important issues in the development of in vitro toxicology towards its implementation in toxicological risk assessment.

Strategic focus on 3R principles reveals major reductions in the use of animals in pharmaceutical toxicity testing.

The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level.

Role of toxicogenomics in the development of safe, efficacious and novel anti-microbial therapies.

Over the last two decades, occurrence of bacterial resistance to commonly used antibiotics has necessitated the development of safer and more potent anti-microbial drugs. However, the development of novel antibiotics is severely hampered by adverse side effects, such as drug-induced liver toxicity. Several antibacterial drugs are known to have the potential to cause severe liver damage. The major challenge in developing novel anti-microbial drugs is to predict, with certain amount of probability, the drug-induced toxicity during the pre-clinical stages, thus optimizing and reducing the time and cost of drug development. Toxicogenomics approach is generally used to harness the potential of genomic tools and to understand the physiological basis of drug-induced toxicity based on the in-depth analysis of Metagenomic data sets, i.e., transcriptional, translational or metabolomic profiles. Toxicogenomics, therefore, represents a new paradigm in the drug development process, and is anticipated to play an invaluable role in future to develop safe and efficacious medicines, by predicting the toxic potential of a new chemical entity (NCE) in early stages of drug discovery. This review examines the toxicogenomic approach in predicting the safety/toxicity of novel anti-microbial drugs, and analyses the promises, pitfalls and challenges of applying this powerful technology to the drug development process.

Incorporating new technologies into toxicity testing and risk assessment: moving from 21st century vision to a data-driven framework.

Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

Science and decisions: advancing toxicology to advance risk assessment.

In 2009, the National Research Council (NRC) released the latest in a series of advisory reports on human health risk assessment, titled Science and Decisions: Advancing Risk Assessment. This wide-ranging report made a number of recommendations related to risk assessment practice at the U.S. Environmental Protection Agency that could both influence and be influenced by evolving toxicological practice. In particular, Science and Decisions emphasized the scientific and operational necessity of a new approach for dose-response modeling; addressed the recurring challenge of defaults in risk assessment and the question of when research results can be used in place of defaults; and reinforced the value of cumulative risk assessment, which would require enhanced understanding of the joint influence of chemical and nonchemical stressors on health outcomes. The objective of this article is to summarize key messages from Science and Decisions, both as a stand-alone report and in comparison with another recent NRC report, Toxicity Testing in the 21st Century: A Vision and a Strategy. Although these reports have many conclusions in common and reinforce similar themes, there are important differences that merit careful consideration, such as the move away from apical endpoints in Toxicity Testing and the emphasis on benefit-cost analyses and related decision tools in Science and Decisions that would be strengthened by quantification of apical endpoints. Moving risk assessment forward will require toxicologists to wrestle with the implications of Science and Decisions from a toxicological perspective.

Implementation challenges for designing integrated in vitro testing strategies (ITS) aiming at reducing and replacing animal experimentation.

At the IVTIP (in vitro testing industrial platform) meeting of November 26th 2009 entitled 'Toxicology in the 21st century ('21C')--working our way towards a visionary reality' all delegates endorsed the emerging concept of the '21C' vision as the way forward to enable a thorough, reliable and systematic approach to future toxicity testing without the use of animals. One of the emerging concepts focused on integrating a defined number of tests modelling in vivo-relevant and well-characterised toxicity pathways representing mechanistic endpoints. At this meeting the importance of Integrated Testing Strategies (ITS) as tools towards reduction and eventually replacement of the animals currently used for hazard identification and risk assessment was recognised. A follow-up IVTIP Spring 2010 meeting entitled 'Integrated In Vitro Testing Strategies (ITS)--Implementation Challenges' was organised to address pending questions about ITS. This report is not a review of the ITS literature, but a summary of the discussions triggered by presented examples on how to develop and implement ITS. Contrasts between pharmaceutical and chemical industry, as well as a list of general but practical aspects to be considered while developing an ITS emerged from the discussions. In addition, current recommendations on the validation of ITS were discussed. In conclusion, the outcome of this workshop improved the understanding of the participants of some important factors that may impact the design of an ITS in function of its purpose (e.g., screening, or early decision making versus regulatory), the context in which they need to be applied (e.g., ICH guidelines, REACH) and the status and quality of the available tools. A set of recommendations of best practices was established and the importance of the applicability of the individual tests as well as the testing strategy itself was highlighted.

Toxicity testing in the 21 century: defining new risk assessment approaches based on perturbation of intracellular toxicity pathways.

The approaches to quantitatively assessing the health risks of chemical exposure have not changed appreciably in the past 50 to 80 years, the focus remaining on high-dose studies that measure adverse outcomes in homogeneous animal populations. This expensive, low-throughput approach relies on conservative extrapolations to relate animal studies to much lower-dose human exposures and is of questionable relevance to predicting risks to humans at their typical low exposures. It makes little use of a mechanistic understanding of the mode of action by which chemicals perturb biological processes in human cells and tissues. An alternative vision, proposed by the U.S. National Research Council (NRC) report Toxicity Testing in the 21(st) Century: A Vision and a Strategy, called for moving away from traditional high-dose animal studies to an approach based on perturbation of cellular responses using well-designed in vitro assays. Central to this vision are (a) "toxicity pathways" (the innate cellular pathways that may be perturbed by chemicals) and (b) the determination of chemical concentration ranges where those perturbations are likely to be excessive, thereby leading to adverse health effects if present for a prolonged duration in an intact organism. In this paper we briefly review the original NRC report and responses to that report over the past 3 years, and discuss how the change in testing might be achieved in the U.S. and in the European Union (EU). EU initiatives in developing alternatives to animal testing of cosmetic ingredients have run very much in parallel with the NRC report. Moving from current practice to the NRC vision would require using prototype toxicity pathways to develop case studies showing the new vision in action. In this vein, we also discuss how the proposed strategy for toxicity testing might be applied to the toxicity pathways associated with DNA damage and repair.

From alternative methods to a new toxicology.

Mechanistic toxicology has evolved by relying, to a large extent, on methodologies that substitute or complement traditional animal tests. The biotechnology and informatics revolutions of the last decades have made such technologies broadly available and useful, but regulatory toxicology has been slow to embrace these new approaches. Major validation efforts, however, have delivered the evidence that new approaches do not lower safety standards and can be integrated into regulatory safety assessments. Particularly in the EU, political pressures, such as the REACH legislation and the 7th Amendment to the cosmetic legislation, have prompted the need of new approaches. In the US, the NRC vision report calling for a toxicology for the 21st century (and its most recent adaptation by EPA for their toxicity testing strategy) have initiated a debate about how to create a novel approach based on human cell cultures, lower species, high-throughput testing, and modeling. Lessons learned from the development, validation, and acceptance of alternative methods support the creation of a new approach based on identified toxicity pathways. Conceptual steering and an objective assessment of current practices by evidence-based toxicology (EBT) are required. EBT is modeled on evidence-based medicine, which has demonstrated that rigorous systematic reviews of current practices and meta-analyses of studies provide powerful tools to provide health care professionals and patients with the current best scientific evidence. Similarly, a portal for high-quality reviews of toxicological approaches and tools for the quantitative meta-analyses of data promise to serve as door opener for a new regulatory toxicology.

Validation of innovative technologies and strategies for regulatory safety assessment methods: challenges and opportunities.

Advances in science and innovative technologies are providing new opportunities to develop test methods and strategies that may improve safety assessments and reduce animal use for safety testing. These include high throughput screening and other approaches that can rapidly measure or predict various molecular, genetic, and cellular perturbations caused by test substances. Integrated testing and decision strategies that consider multiple types of information and data are also being developed. Prior to their use for regulatory decision-making, new methods and strategies must undergo appropriate validation studies to determine the extent that their use can provide equivalent or improved protection compared to existing methods and to determine the extent that reproducible results can be obtained in different laboratories. Comprehensive and optimal validation study designs are expected to expedite the validation and regulatory acceptance of new test methods and strategies that will support improved safety assessments and reduced animal use for regulatory testing.

Reproductive environmental health.

PURPOSE OF REVIEW: There is heightened recognition that the environment is an important driver of human reproductive health. This article provides an overview of the nature and extent of the science in the field of reproductive environmental health and its implications for OB/GYN clinical practice. RECENT FINDINGS: Women of childbearing age incur ubiquitous contact to numerous toxic environmental contaminants. Even subtle perturbations caused by chemical exposures during critical and sensitive windows of development may lead to increased risks of disease and disability across the entire span of human life. The strength of the evidence is sufficiently high that leading scientists and clinicians have called for timely action to prevent harm. SUMMARY: OB/GYNs are uniquely poised to intervene in critical stages of human development (i.e., preconception and during pregnancy) to prevent harm. Efforts are underway to provide clinicians with the evidence-based foundation to develop recommendations for prevention. If adopted, current directions in toxicity testing, risk assessment and policy are likely to create important changes in how environmental chemicals are evaluated and regulated in the future. Together, these changes have the potential to assist in clinical assessment of patient risk and reductions in patient exposure to environmental contaminants linked to adverse reproductive health outcomes.