60 Years of the 3Rs symposium: Lessons learned and the road ahead.

When The Principles of Humane Experimental Technique was published in 1959, authors William Russell and Rex Burch had a modest goal: to make researchers think about what they were doing in the laboratory - and to do it more humanely. Sixty years later, their groundbreaking book was celebrated for inspiring a revolution in science and launching a new field: The 3Rs of alternatives to animal experimentation. On November 22, 2019, some pioneering and leading scientists and researchers in the field gathered at the Johns Hopkins Bloomberg School of Public Health in Bal-timore for the 60 Years of the 3Rs Symposium: Lessons Learned and the Road Ahead. The event was sponsored by the Johns Hopkins Center for Alternatives to Animal Testing (CAAT), the Foundation for Chemistry Research and Initiatives, the Alternative Research & Development Foundation (ARDF), the American Cleaning Institute (ACI), the International Fragrance Association (IFRA), the Institute for In Vitro Sciences (IIVS), John "Jack" R. Fowle III, and the Society of Toxicology (SoT). Fourteen pres-entations shared the history behind the groundbreaking publication, international efforts to achieve its aims, stumbling blocks to progress, as well as remarkable achievements. The day was a tribute to Russell and Burch, and a testament to what is possible when people from many walks of life - science, government, and industry - work toward a common goal.

William Russell and Rex Burch published their book The Principles of Humane Experimental Technique in 1959. The book encouraged researchers to replace animal experiments where it was possible, to refine experiments with animals in order to reduce their suffering, and to reduce the number of animals that had to be used for experiments to the minimum. Sixty years later, a group of pioneering and leading scientists and researchers in the field gathered to share how the publi­cation came about and how the vision inspired international collaborations and successes on many different levels including new laws. The paper includes an overview of important milestones in the history of alternatives to animal experimentation.

Community-based medication disposal pilot initiative in southwest tribal communities.

BACKGROUND: Misuse and abuse of prescription drugs including opioids has been a driving force behind the drug overdose epidemic plaguing communities across the USA for more than two decades. Medication accumulation in the home environment can contribute to this issue. However, research on proper disposal in rural communities is limited. For this project, an applied public health approach was used to raise awareness and improve prescription drug disposal practices by pilot testing prescription drug disposal systems in participating communities. METHODS: A community-based disposal project was facilitated with assistance from community partners. The project centered on distribution of drug deactivation bags in homes and medication drop boxes at multiple healthcare facilities. RESULTS: The team distributed 215 drug deactivation bags to 162 community households resulting in destruction of 8011 pills, 8 medicated dermal patches and 777 mL of liquid medication. A total of 4684 pounds of medication were collected and disposed of through healthcare facility drop boxes. CONCLUSION: The strategies identified are scalable and easy to replicate to meet any community's needs in reducing potential challenges of medication diversion.

A Systematic Review to Compare Chemical Hazard Predictions of the Zebrafish Embryotoxicity Test With Mammalian Prenatal Developmental Toxicity.

Originally developed to inform the acute toxicity of chemicals on fish, the zebrafish embryotoxicity test (ZET) has also been proposed for assessing the prenatal developmental toxicity of chemicals, potentially replacing mammalian studies. Although extensively evaluated in primary studies, a comprehensive review summarizing the available evidence for the ZET's capacity is lacking. Therefore, we conducted a systematic review of how well the presence or absence of exposure-related findings in the ZET predicts prenatal development toxicity in studies with rats and rabbits. A two-tiered systematic review of the developmental toxicity literature was performed, a review of the ZET literature was followed by one of the mammalian literature. Data were extracted using DistillerSR, and study validity was assessed with an amended SYRCLE's risk-of-bias tool. Extracted data were analyzed for each species and substance, which provided the basis for comparing the 2 test methods. Although limited by the number of 24 included chemicals, our results suggest that the ZET has potential to identify chemicals that are mammalian prenatal developmental toxicants, with a tendency for overprediction. Furthermore, our analysis confirmed the need for further standardization of the ZET. In addition, we identified contextual and methodological challenges in the application of systematic review approaches to toxicological questions. One key to overcoming these challenges is a transition to more comprehensive and transparent planning, conduct and reporting of toxicological studies. The first step toward bringing about this change is to create broad awareness in the toxicological community of the need for and benefits of more evidence-based approaches.

Prescription drug safe storage practices in Arizona tribal communities.

This paper provides an overview of an opioid poisoning prevention pilot project conducted in several American Indian/Alaskan Native communities using an applied public health approach. The intent of the project was to identify a prescription medication safeguarding option for use in the home environment. The authors engaged the target population to obtain their buy-in to select an intervention that was acceptable and appropriate for their needs. Focus groups and key informant interviews conducted in several tribal communities resulted in the selection of a heavy-duty, lockable storage box as the intervention. Through community-based partnerships, 55 boxes were installed in participating households. Along with the box, participants also received education on safe medication storage and disposal. At baseline, only 1% of the participants reported storing their medication securely. During a 60-day follow-up visit, 95% of the observed boxes were being used to store medications. Also at baseline, 31% of the participants reported a history of lost or stolen medications. There were no reported lost or stolen medications during the 60-day project period among the participants. During the follow-up visits, project staff also found the boxes being used to store other items valuable to the participants. Reportedly, having their medication and other valuables secured in one location provided a heightened feeling of security. Since the completion of this pilot project, several organisations and entities have replicated it in their communities.

Beyond the 3Rs: Expanding the use of human-relevant replacement methods in biomedical research.

This year marks the 60th anniversary of Russell and Burch's pioneering book, The Principles of Humane Experimental Technique. Their 3Rs framework has helped to inspire humane and scientific progress in experimental technique. However, it is time to update its strategic application. The 21st century has already seen the development of promising, high-tech non-animal models, such as organs-on-a-chip and computational approaches that, in our view, will replace animals as the default option in biomedical experimentation. How fast this transition will take place will depend on the pace at which these new models are optimized to reflect the biology of humans, rather than that of non-human animals. While the new methods are likely to reshape all areas in which animals are currently used in science, we particularly encourage their application in biomedical research, which accounts for the bulk of animals used. We call for the pursuit of a three-prong strategy that focuses on (1) advancing non-animal methods as replacements of animal experiments, (2) applying them to biomedical research, and (3) improving their relevance to human biology. As academics and scientists, we feel that educational efforts targeted at young scientists in training will be an effective and sustainable way to advance this vision. Our strategy may not promise an imminent end to the use of animals in science, but it will bring us closer to an era in which the 3Rs are increasingly perceived as a solution to a receding problem. Russell and Burch themselves surely would have welcomed these positive changes.

Adaptation of the Systematic Review Framework to the Assessment of Toxicological Test Methods: Challenges and Lessons Learned With the Zebrafish Embryotoxicity Test.

Systematic review methodology is a means of addressing specific questions through structured, consistent, and transparent examinations of the relevant scientific evidence. This methodology has been used to advantage in clinical medicine, and is being adapted for use in other disciplines. Although some applications to toxicology have been explored, especially for hazard identification, the present preparatory study is, to our knowledge, the first attempt to adapt it to the assessment of toxicological test methods. As our test case, we chose the zebrafish embryotoxicity test (ZET) for developmental toxicity and its mammalian counterpart, the standard mammalian prenatal development toxicity study, focusing the review on how well the ZET predicts the presence or absence of chemical-induced prenatal developmental toxicity observed in mammalian studies. An interdisciplinary team prepared a systematic review protocol and adjusted it throughout this piloting phase, where needed. The final protocol was registered and will guide the main study (systematic review), which will execute the protocol to comprehensively answer the review question. The goal of this preparatory study was to translate systematic review methodology to the assessment of toxicological test method performance. Consequently, it focused on the methodological issues encountered, whereas the main study will report substantive findings. These relate to numerous systematic review steps, but primarily to searching and selecting the evidence. Applying the lessons learned to these challenges can improve not only our main study, but may also be helpful to others seeking to use systematic review methodology to compare toxicological test methods. We conclude with a series of recommendations that, if adopted, would help improve the quality of the published literature, and make conducting systematic reviews of toxicological studies faster and easier over time.

Evidence for the outcomes and impact of clinical pharmacy: context of UK hospital pharmacy practice.

OBJECTIVES: The role of clinical pharmacists in hospitals has evolved and continues to expand. In the UK, outside of a few national policy drivers, there are no agreed priorities, measures or defined outcomes for hospital clinical pharmacy (CP). This paper aims to (1) highlight the need to identify and prioritise specific CP roles, responsibilities and practices that will bring the greatest benefit to patients and health systems and (2) describe systematic weaknesses in current research methodologies for evaluating CP services and propose a different approach. METHOD: Published reviews of CP services are discussed using the Economic, Clinical and Humanistic Outcomes framework. Recurring themes regarding study methodologies, measurements and outcomes are used to highlight current weaknesses in studies evaluating CP. RESULTS: Published studies aiming to demonstrate the economic, clinical or humanistic outcomes of CP often suffer from poor research design and inconsistencies in interventions, measurements and outcomes. This has caused difficulties in drawing meaningful conclusions regarding CP's definitive contribution to patient outcomes. CONCLUSION: There is a need for more research work in National Health Service (NHS) hospitals, employing a different paradigm to address some of the weaknesses of existing research on CP practice. We propose a mixed-methods approach, including qualitative research designs, and with emphasis on cost-consequence analyses for economic evaluations. This approach will provide more meaningful data to inform policy and demonstrate the contribution of hospital CP activities to patient care and the NHS.

A primer on systematic reviews in toxicology.

Systematic reviews, pioneered in the clinical field, provide a transparent, methodologically rigorous and reproducible means of summarizing the available evidence on a precisely framed research question. Having matured to a well-established approach in many research fields, systematic reviews are receiving increasing attention as a potential tool for answering toxicological questions. In the larger framework of evidence-based toxicology, the advantages and obstacles of, as well as the approaches for, adapting and adopting systematic reviews to toxicology are still being explored. To provide the toxicology community with a starting point for conducting or understanding systematic reviews, we herein summarized available guidance documents from various fields of application. We have elaborated on the systematic review process by breaking it down into ten steps, starting with planning the project, framing the question, and writing and publishing the protocol, and concluding with interpretation and reporting. In addition, we have identified the specific methodological challenges of toxicological questions and have summarized how these can be addressed. Ultimately, this primer is intended to stimulate scientific discussions of the identified issues to fuel the development of toxicology-specific methodology and to encourage the application of systematic review methodology to toxicological issues.

Evidence-Based Toxicology.

Evidence-based toxicology (EBT) was introduced independently by two groups in 2005, in the context of toxicological risk assessment and causation as well as based on parallels between the evaluation of test methods in toxicology and evidence-based assessment of diagnostics tests in medicine. The role model of evidence-based medicine (EBM) motivated both proposals and guided the evolution of EBT, whereas especially systematic reviews and evidence quality assessment attract considerable attention in toxicology.Regarding test assessment, in the search of solutions for various problems related to validation, such as the imperfectness of the reference standard or the challenge to comprehensively evaluate tests, the field of Diagnostic Test Assessment (DTA) was identified as a potential resource. DTA being an EBM discipline, test method assessment/validation therefore became one of the main drivers spurring the development of EBT.In the context of pathway-based toxicology, EBT approaches, given their objectivity, transparency and consistency, have been proposed to be used for carrying out a (retrospective) mechanistic validation.In summary, implementation of more evidence-based approaches may provide the tools necessary to adapt the assessment/validation of toxicological test methods and testing strategies to face the challenges of toxicology in the twenty first century.

The Emergence of Systematic Review in Toxicology.

The Evidence-based Toxicology Collaboration hosted a workshop on "The Emergence of Systematic Review and Related Evidence-based Approaches in Toxicology," on November 21, 2014 in Baltimore, Maryland. The workshop featured speakers from agencies and organizations applying systematic review approaches to questions in toxicology, speakers with experience in conducting systematic reviews in medicine and healthcare, and stakeholders in industry, government, academia, and non-governmental organizations. Based on the workshop presentations and discussion, here we address the state of systematic review methods in toxicology, historical antecedents in both medicine and toxicology, challenges to the translation of systematic review from medicine to toxicology, and thoughts on the way forward. We conclude with a recommendation that as various agencies and organizations adapt systematic review methods, they continue to work together to ensure that there is a harmonized process for how the basic elements of systematic review methods are applied in toxicology.

Guidance on assessing the methodological and reporting quality of toxicologically relevant studies: A scoping review.

Assessments of methodological and reporting quality are critical to adequately judging the credibility of a study's conclusions and to gauging its potential reproducibility. To aid those seeking to assess the methodological or reporting quality of studies relevant to toxicology, we conducted a scoping review of the available guidance with respect to four types of studies: in vivo and in vitro, (quantitative) structure-activity relationships ([Q]SARs), physico-chemical, and human observational studies. Our aims were to identify the available guidance in this diverse literature, briefly summarize each document, and distill the common elements of these documents for each study type. In general, we found considerable guidance for in vivo and human studies, but only one paper addressed in vitro studies exclusively. The guidance for (Q)SAR studies and physico-chemical studies was scant but authoritative. There was substantial overlap across guidance documents in the proposed criteria for both methodological and reporting quality. Some guidance documents address toxicology research directly, whereas others address preclinical research generally or clinical research and therefore may not be fully applicable to the toxicology context without some translation. Another challenge is the degree to which assessments of methodological quality in toxicology should focus on risk of bias - as in clinical medicine and healthcare - or be broadened to include other quality measures, such as confirming the identity of test substances prior to exposure. Our review is intended primarily for those in toxicology and risk assessment seeking an entry point into the extensive and diverse literature on methodological and reporting quality applicable to their work.

The usefulness of systematic reviews of animal experiments for the design of preclinical and clinical studies.

The question of how animal studies should be designed, conducted, and analyzed remains underexposed in societal debates on animal experimentation. This is not only a scientific but also a moral question. After all, if animal experiments are not appropriately designed, conducted, and analyzed, the results produced are unlikely to be reliable and the animals have in effect been wasted. In this article, we focus on one particular method to address this moral question, namely systematic reviews of previously performed animal experiments. We discuss how the design, conduct, and analysis of future (animal and human) experiments may be optimized through such systematic reviews. In particular, we illustrate how these reviews can help improve the methodological quality of animal experiments, make the choice of an animal model and the translation of animal data to the clinic more evidence-based, and implement the 3Rs. Moreover, we discuss which measures are being taken and which need to be taken in the future to ensure that systematic reviews will actually contribute to optimizing experimental design and thereby to meeting a necessary condition for making the use of animals in these experiments justified.

Green toxicology.

Historically, early identification and characterization of adverse effects of industrial chemicals was difficult because conventional toxicological test methods did not meet R&D needs for rapid, relatively inexpensive methods amenable to small amounts of test material. The pharmaceutical industry now front-loads toxicity testing, using in silico, in vitro, and less demanding animal tests at earlier stages of product development to identify and anticipate undesirable toxicological effects and optimize product development. The Green Chemistry movement embraces similar ideas for development of less toxic products, safer processes, and less waste and exposure. Further, the concept of benign design suggests ways to consider possible toxicities before the actual synthesis and to apply some structure/activity rules (SAR) and in silico methods. This requires not only scientific development but also a change in corporate culture in which synthetic chemists work with toxicologists. An emerging discipline called Green Toxicology (Anastas, 2012) provides a framework for integrating the principles of toxicology into the enterprise of designing safer chemicals, thereby minimizing potential toxicity as early in production as possible. Green Toxicology`s novel utility lies in driving innovation by moving safety considerations to the earliest stage in a chemical`s lifecycle, i.e., to molecular design. In principle, this field is no different than other subdisciplines of toxicology that endeavor to focus on a specific area - for example, clinical, environmental or forensic toxicology. We use the same principles and tools to evaluate an existing substance or to design a new one. The unique emphasis is in using 21st century toxicology tools as a preventative strategy to "design out" undesired human health and environmental effects, thereby increasing the likelihood of launching a successful, sustainable product. Starting with the formation of a steering group and a series of workshops, the Green Toxicology concept is currently spreading internationally and is being refined via an iterative process.

Consensus report on the future of animal-free systemic toxicity testing.

Since March 2013, animal use for cosmetics testing for the European market has been banned. This requires a renewed view on risk assessment in this field. However, in other fields as well, traditional animal experimentation does not always satisfy requirements in safety testing, as the need for human-relevant information is ever increasing. A general strategy for animal-free test approaches was outlined by the US National Research Council`s vision document for Toxicity Testing in the 21st Century in 2007. It is now possible to provide a more defined roadmap on how to implement this vision for the four principal areas of systemic toxicity evaluation: repeat dose organ toxicity, carcinogenicity, reproductive toxicity and allergy induction (skin sensitization), as well as for the evaluation of toxicant metabolism (toxicokinetics) (Fig. 1). CAAT-Europe assembled experts from Europe, America and Asia to design a scientific roadmap for future risk assessment approaches and the outcome was then further discussed and refined in two consensus meetings with over 200 stakeholders. The key recommendations include: focusing on improving existing methods rather than favoring de novo design; combining hazard testing with toxicokinetics predictions; developing integrated test strategies; incorporating new high content endpoints to classical assays; evolving test validation procedures; promoting collaboration and data-sharing of different industrial sectors; integrating new disciplines, such as systems biology and high throughput screening; and involving regulators early on in the test development process. A focus on data quality, combined with increased attention to the scientific background of a test method, will be important drivers. Information from each test system should be mapped along adverse outcome pathways. Finally, quantitative information on all factors and key events will be fed into systems biology models that allow a probabilistic risk assessment with flexible adaptation to exposure scenarios and individual risk factors.

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.

Mechanistic validation.

Validation of new approaches in regulatory toxicology is commonly defined as the independent assessment of the reproducibility and relevance (the scientific basis and predictive capacity) of a test for a particular purpose. In large ring trials, the emphasis to date has been mainly on reproducibility and predictive capacity (comparison to the traditional test) with less attention given to the scientific or mechanistic basis. Assessing predictive capacity is difficult for novel approaches (which are based on mechanism), such as pathways of toxicity or the complex networks within the organism (systems toxicology). This is highly relevant for implementing Toxicology for the 21st Century, either by high-throughput testing in the ToxCast/Tox21 project or omics-based testing in the Human Toxome Project. This article explores the mostly neglected assessment of a test's scientific basis, which moves mechanism and causality to the foreground when validating/qualifying tests. Such mechanistic validation faces the problem of establishing causality in complex systems. However, pragmatic adaptations of the Bradford Hill criteria, as well as bioinformatic tools, are emerging. As critical infrastructures of the organism are perturbed by a toxic mechanism we argue that by focusing on the target of toxicity and its vulnerability, in addition to the way it is perturbed, we can anchor the identification of the mechanism and its verification.

Evidence-based toxicology for the 21st century: opportunities and challenges.

The Evidence-based Toxicology Collaboration (EBTC) was established recently to translate evidence-based approaches from medicine and health care to toxicology in an organized and sustained effort. The EBTC held a workshop on "Evidence-based Toxicology for the 21st Century: Opportunities and Challenges" in Research Triangle Park, North Carolina, USA on January 24-25, 2012. The presentations largely reflected two EBTC priorities: to apply evidence-based methods to assessing the performance of emerging pathway-based testing methods consistent with the 2007 National Research Council report on "Toxicity Testing in the 21st Century" as well as to adopt a governance structure and work processes to move that effort forward. The workshop served to clarify evidence-based approaches and to provide food for thought on substantive and administrative activities for the EBTC. Priority activities include conducting pilot studies to demonstrate the value of evidence-based approaches to toxicology, as well as conducting educational outreach on these approaches.

Accelerating the development of 21st-century toxicology: outcome of a Human Toxicology Project Consortium workshop.

The U.S. National Research Council (NRC) report on "Toxicity Testing in the 21st century" calls for a fundamental shift in the way that chemicals are tested for human health effects and evaluated in risk assessments. The new approach would move toward in vitro methods, typically using human cells in a high-throughput context. The in vitro methods would be designed to detect significant perturbations to "toxicity pathways," i.e., key biological pathways that, when sufficiently perturbed, lead to adverse health outcomes. To explore progress on the report's implementation, the Human Toxicology Project Consortium hosted a workshop on 9-10 November 2010 in Washington, DC. The Consortium is a coalition of several corporations, a research institute, and a non-governmental organization dedicated to accelerating the implementation of 21st-century Toxicology as aligned with the NRC vision. The goal of the workshop was to identify practical and scientific ways to accelerate implementation of the NRC vision. The workshop format consisted of plenary presentations, breakout group discussions, and concluding commentaries. The program faculty was drawn from industry, academia, government, and public interest organizations. Most presentations summarized ongoing efforts to modernize toxicology testing and approaches, each with some overlap with the NRC vision. In light of these efforts, the workshop identified recommendations for accelerating implementation of the NRC vision, including greater strategic coordination and planning across projects (facilitated by a steering group), the development of projects that test the proof of concept for implementation of the NRC vision, and greater outreach and communication across stakeholder communities.