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 Baltimore 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 presentations 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.

Ethnopharmacological relevance: Atopic dermatitis (AD) is a common chronic inflammatory skin disorder and its prevalence is increasing in the last few decades. No treatment can cure the condition. Pregnancy often worsens the clinical manifestation. There are considerable interests in Chinese Herbal Medicine (CHM) as an alternative treatment for AD. A well tolerated CHM formula (Pentaherbs formulation, PHF) has been proven efficacious in improving life quality and reducing topical corticosteroid use in children with moderate-to-severe AD. However, safety data of PHF are not available.Aim of the study: Our study aimed to evaluate the safety of PHF and its 5 individual herbal extracts, including embryotoxicity by Embryonic Stem Cell Test (EST) and irritation by Skin Irritation Test (SIT).Materials and methods: Quality of 5 herbal extracts of PHF was confirmed by chromatography. In EST, mouse embryonic stem cell line (D3) and mouse fibroblast cell line (3T3) were used to study potential embryotoxicity. Three endpoints were assessed by concentration-response curves after 10 days' culture: 50% inhibition of D3 differentiation into beating cardiomyocytes (ID50D3), 50% cytotoxic effects on D3 (IC50D3) and on fibroblasts (IC503T3). A biostatistically based prediction model (PM) was applied to predict the embryotoxic potentials of each CHM. In SIT, epidermis equivalent commercially available kits (EpiDermTM) were used, and concentration-viability curves were obtained by MTT assay to detect skin irritations of each CHM.Results: Chemical authentication confirmed that 5 test herbal extracts contained their main active compounds. EST results indicated that the formula PHF and its individual CHMs were non-embryotoxic, except one CHM, Amur Corktree Bark (Huang Bai, Phellodendron chinense C.K.Schneid), was weakly embryotoxic. SIT results showed that cell viability was above 50% after treatment with different concentrations of all tested CHMs.Conclusions: Our in vitro tests provided preliminary evidence for safety of the formula PHF in embryonic stem cell test and skin irritation model, but PHF shall be cautiously used in pregnant women with AD. Further studies are needed to support its clinical application as an alternative treatment for AD, especially to the patients who plan for pregnancy or at lactation stages.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

This manuscript provides a review focused on embryonic stem cell-based models and their place within the landscape of alternative developmental toxicity assays. Against the background of the principles of developmental toxicology, the wide diversity of alternative methods using pluripotent stem cells developed in this area over the past half century is reviewed. In order to provide an overview of available models, a systematic scoping review was conducted following a published protocol with inclusion criteria, which were applied to select the assays. Critical aspects including biological domain, readout endpoint, availability of standardized protocols, chemical domain, reproducibility and predictive power of each assay are described in detail, in order to review the applicability and limitations of the platform in general and progress moving forward to implementation. The horizon of innovative routes of promoting regulatory implementation of alternative methods is scanned, and recommendations for further work are given.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Chinese herbal medicines (CHMs) have been widely used during pregnancy, but feto-embryo safety tests are lacking. Here we evaluated in vitro embryotoxicity tests (IVTs) as alternative methods in assessing developmental toxicity of CHMs. Ten CHMs were selected and classified as strongly, weakly and non-embryotoxic. Three well validated IVTs and prediction models (PMs), including embryonic stem cell test (EST), micromass (MM) and whole embryo culture (WEC), were compared. All strongly embryotoxic CHMs were predicted by MM and WEC PM2. While all weakly embryotoxic CHMs were predicted by MM and WEC PM1. All non-embryotoxic CHMs were classified by EST, MM, but over-classified as weakly embryotoxic by WEC PM1. Overall predictivity, precision and accuracy of WEC determined by PM2 were better than EST and MM tests. Compared with validated chemicals, performance of IVTs for CHMs was comparable. So IVTs are adequate to identify and exclude embryotoxic potential of CHMs in this training set.

The regulatory assessment of the complex endpoints, carcinogenicity and reproductive and developmental toxicity, still heavily relies on animal testing. This chapter reviews the alternative approaches that have been developed in these areas in the last decades to waive testing and reduce the number of animals used and summarises the lessons learned. The alternative approaches range from the individual test methods, such as the cell transformation assay for carcinogenicity and the embryotoxicity tests for reproductive toxicity, to reduction approaches, such as the International Conference on Harmonisation strategy for pharmaceuticals and the extended-one-generation assay. Currently, these areas are being revitalised due to the broad recognition of the shortcomings of current in vivo testing requirements and the current regulatory environment (e. g. the European Registration, Evaluation, Authorisation and Restriction of Chemicals and Cosmetics Regulations). More-recent developments aimed at a more human-relevant chemical assessment, which rely on the integration of different sources of information, are also described.

Regulatory toxicology has begun to embrace new hazard characterisation approaches, which can be integrated into regulatory safety assessments. The vision is to fundamentally change the way the safety of chemicals is assessed, by replacing or reducing the use of traditional animal experiments with a predictive toxicology that is based on a comprehensive understanding of how chemicals can cause adverse effects in humans (MoA and AOP), or adversely impact on the environment, and a ‘renewed’ awareness of the importance of the fate of the chemical in the body (kinetics) for the induction of adverse effects. Integrated Approaches to Testing and Assessment (IATA) will help evaluators to assess data from such novel methods consistently, and to understand their relevance for specific endpoints, to obtain reproducible, understandable and statistically-sound results. To position toxicity data generated by alternative methods in a regulatory context, the need for reliable methods has to be combined with a strategy for how to best integrate the resulting data with computational modelling and existing knowledge and information, in order to provide a prediction with sufficient confidence for acceptance in a safety assessment. The new concept proposed by the US Food and Drug Administration to replace formal validation with qualification for a specific context of use, seems to be the most promising way forward. Once a new model or assay is considered to be qualified for a specific context of use, industry and other stakeholders can use it for its qualified purpose during product development, and regulators should then be confident in applying it without needing to re-review the underlying data supporting validation and acceptance for specific purposes.

Regulatory toxicology has begun to embrace new hazard characterisation approaches, which can be integrated into regulatory safety assessments. The vision is to fundamentally change the way the safety of chemicals is assessed, by replacing or reducing the use of traditional animal experiments with a predictive toxicology that is based on a comprehensive understanding of how chemicals can cause adverse effects in humans (MoA and AOP), or adversely impact on the environment, and a ` renewed' awareness of the importance of the fate of the chemical in the body (kinetics) for the induction of adverse effects. Integrated Approaches to Testing and Assessment (IATA) will help evaluators to assess data from such novel methods consistently, and to understand their relevance for specific endpoints, to obtain reproducible, understandable and statistically-sound results. To position toxicity data generated by alternative methods in a regulatory context, the need for reliable methods has to be combined with a strategy for how to best integrate the resulting data with computational modelling and existing knowledge and information, in order to provide a prediction with sufficient confidence for acceptance in a safety assessment. The new concept proposed by the US Food and Drug Administration to replace formal validation with qualification for a specific context of use, seems to be the most promising way forward. Once a new model or assay is considered to be qualified for a specific context of use, industry and other stakeholders can use it for its qualified purpose during product development, and regulators should then be confident in applying it without needing to re-review the underlying data supporting validation and acceptance for specific purposes.

The Three Rs concept was welcomed in Austria, Germany and Switzerland, after the EEC had accepted Directive 86/609/EEC on the protection of animals used for experimental and other scientific purposes. However, it was implemented quite differently in each of the three countries. In 1985, Switzerland took the lead, because of generous funding by the Doerenkamp-Zbinden Foundation, by focusing on replacing the LD50 test, and in 2002, by establishing five chairs on alternatives in Germany, India, Switzerland, The Netherlands and the USA. In 1989, Germany established ZEBET at the Federal Health Institute (BGA) in Berlin, as the first national centre for alternatives, which significantly contributed to the international acceptance of the first in vitro OECD Test Guideline for local toxicity testing, for phototoxicity. Since 1992, the Austrian government has supported annual international Three Rs congresses in Linz, which are now organised by EUSAAT, the European Society for Alternatives to Animal Testing.

We reviewed six key peer-reviewed publications that assessed the need for one-year studies of pesticide toxicity in dogs. Each of the six papers took a different approach to comparing the value of one-year studies relative to three-month studies, and despite the adoption of different databases and approaches, each study reached the same conclusion: the recommended limit to the testing of pesticide toxicity in dogs should be three months. Therefore, the present review supports the conclusion that the routine inclusion of a one-year dog study should not be a mandatory requirement for the safety assessment of pesticides, since it is scientifically no longer justifiable. We recommend that the OECD should adapt a harmonized approach that is in line with the legislation in Europe and the USA.

We report the facile synthesis and the chemoresistive performances of villi-like WO3 nanostructures (VLWNs) which were fabricated by RF sputter with glancing angle deposition (GAD) mode. A fast deposition of GAD effectively forms self-assembled anisotropic nanostructures with high porosity and surface-to-volume ratio. The sensing tests were examined for NO2 detection in dry air and a mixture of reducing gases. As a result, these sensors at 200 °C exhibit an highly selective and sensitive NO2 detection down to 800 parts per trillion (ppt) level, and could also respond well to NO2 in the concentration range of 0.2–1 parts per million (ppm) without the interference of gas mixture. These results show that the enhanced sensing properties to NO2 are attributed to the highly efficient surface modulation by double potential barriers at nano-necks of WO3 nanostructures.

There is a fundamental change in thinking within the regulatory community due to a better understanding of the underlying biology of adverse effects to human health and the environment. The development of alternatives to use laboratory animals has become a priority. In addition, technological progress is impacting greatly on the amount of data available and on the ways to process and analyse it. Topics, such as identification of adverse outcome pathways (AOPs) and modes of action (MoA), together with integrated assessment and testing approaches (IATAs), represent fundamental tools for hazard identification and characterisation of a chemical. Complex endpoints cannot be predicted by a single standalone non-animal test; thus, a major challenge is the complex nature of biological systems. Microphysiological systems (MPS) will enable more complex in vitro human models that better simulate the organ's biology and function by combining different cell types in a specific three-dimensional configuration that simulates functional organs. The process of validation of new approaches needs to be considered in terms of efficiency and length. Regulators might still not have enough confidence to adopt and apply these new approaches: this phase is very challenging and the activities performed by assay developers are not yet addressing the regulatory requirements needs sufficiently. The IATAs provide a framework to consistently evaluate new approach data and could assist in understanding their relevance for specific endpoints. The data need to be reproducible, understandable and statistically sound: indeed, a major issue lies in the interpretation and integration of the results based on subjective assessment, which relies on expert judgement. A well-defined mechanistic characterisation is proposed as a way forward to ensure the relevance of new cell-based test systems. (C) 2016 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.

A further increase in the use of genetically-modified animals will be against the European Union policy to move away from the use of animals in biomedical research

The induction of teratoma in mice by the transplantation of stem cells into extra-uterine sites has been used as a read-out for cellular pluripotency since the initial description of this phenomenon in 1954. Since then, the teratoma assay has remained the assay of choice to demonstrate pluripotency, gaining prominence during the recent hype surrounding human stem cell research. However, the scientific significance of the teratoma assay has been debated due to the fact that transplanted cells are exposed to a non-physiological environment. Since many mice are used for a result that is heavily questioned, it is time to reconsider the teratoma assay from an ethical point of view. Candidate alternatives to the teratoma assay comprise the directed differentiation of pluripotent stem cells into organotypic cells, differentiation of cells in embryoid bodies, the analysis of pluripotency-associated biomarkers with high correlation to the teratoma forming potential of stem cells, predictive epigenetic footprints, or a combination of these technologies. Each of these assays is capable of addressing one or more aspects of pluripotency, however it is essential that these assays are validated to provide an accepted robust, reproducible alternative. In particular, the rapidly expanding number of human induced pluripotent stem cell lines, requires the development of simple, affordable standardized in vitro and in silico assays to reduce the number of animal experiments performed.