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.

Brazilian National Network of Alternative Methods (RENAMA) 10th Anniversary: Meeting of the Associated Laboratories, May 2022.

The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed.

The 19th FRAME Annual Lecture, November 2022: Safer Chemicals and Sustainable Innovation Will Be Achieved by Regulatory Use of Modern Safety Science, Not by More Animal Testing.

The decisions we make on chemical safety, for consumers, workers and the environment, must be based on the best scientific data and knowledge available. Rapid advances in biology, in cell-based technologies and assays, and in analytical and computational approaches, have led to new types of highly relevant scientific data being generated. Such data enable us to improve the safety decisions we make, whilst also enabling us to avoid animal testing. Stimulated by the UK and EU bans on animal testing for cosmetics, Next Generation Risk Assessment (NGRA) approaches, which integrate various types of non-animal scientific data, have been established for assessing the safety of chemical ingredients used in cosmetics and other consumer products. In stark contrast, the chemicals regulations in Europe and other parts of the world have not kept pace with modern safety science and regulators are now mandating even more animal testing. Urgently closing this science-regulation gap is essential to upholding the EU's legislative requirement that any animal testing is a last resort. The ongoing revisions of UK and EU chemicals strategy and regulations provide an opportunity to fundamentally change the design and assessment paradigm needed to underpin safe and more sustainable innovation, through applying the best science and tools available rather than continuing to be anchored in animal tests dating back many decades. A range of initiatives have recently been launched in response to this urgent need, in the UK as well as in the EU.

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.

Lessons from Toxicology: Developing a 21st-Century Paradigm for Medical Research.

Biomedical developments in the 21st century provide an unprecedented opportunity to gain a dynamic systems-level and human-specific understanding of the causes and pathophysiologies of disease. This understanding is a vital need, in view of continuing failures in health research, drug discovery, and clinical translation. The full potential of advanced approaches may not be achieved within a 20th-century conceptual framework dominated by animal models. Novel technologies are being integrated into environmental health research and are also applicable to disease research, but these advances need a new medical research and drug discovery paradigm to gain maximal benefits. We suggest a new conceptual framework that repurposes the 21st-century transition underway in toxicology. Human disease should be conceived as resulting from integrated extrinsic and intrinsic causes, with research focused on modern human-specific models to understand disease pathways at multiple biological levels that are analogous to adverse outcome pathways in toxicology. Systems biology tools should be used to integrate and interpret data about disease causation and pathophysiology. Such an approach promises progress in overcoming the current roadblocks to understanding human disease and successful drug discovery and translation. A discourse should begin now to identify and consider the many challenges and questions that need to be solved.

Assuring safety without animal testing: Unilever's ongoing research programme to deliver novel ways to assure consumer safety.

Assuring consumer safety without the generation of new animal data is currently a considerable challenge. However, through the application of new technologies and the further development of risk-based approaches for safety assessment, we remain confident it is ultimately achievable. For many complex, multi-organ consumer safety endpoints, the development, evaluation and application of new, non-animal approaches is hampered by a lack of biological understanding of the underlying mechanistic processes involved. The enormity of this scientific challenge should not be underestimated. To tackle this challenge a substantial research programme was initiated by Unilever in 2004 to critically evaluate the feasibility of a new conceptual approach based upon the following key components: 1.Developing new, exposure-driven risk assessment approaches. 2.Developing new biological (in vitro) and computer-based (in silico) predictive models. 3.Evaluating the applicability of new technologies for generating data (e.g. "omics", informatics) and for integrating new types of data (e.g. systems approaches) for risk-based safety assessment. Our research efforts are focussed in the priority areas of skin allergy, cancer and general toxicity (including inhaled toxicity). In all of these areas, a long-term investment is essential to increase the scientific understanding of the underlying biology and molecular mechanisms 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 in 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).

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.

The ECVAM international validation study on in vitro tests for acute skin irritation: report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test.

ECVAM sponsored a formal validation study on three in vitro tests for skin irritation, of which two employ reconstituted human epidermis models (EPISKIN, EpiDerm), and one, the skin integrity function test (SIFT), employs ex vivo mouse skin. The goal of the study was to assess whether the in vitro tests would correctly predict in vivo classifications according to the EU classification scheme, "R38" and "no label" (i.e. non-irritant). 58 chemicals (25 irritants and 33 non-irritants) were tested, having been selected to give broad coverage of physico-chemical properties, and an adequate distribution of irritancy scores derived from in vivo rabbit skin irritation tests. In Phase 1, 20 of these chemicals (9 irritants and 11 non-irritants) were tested with coded identities by a single lead laboratory for each of the methods, to confirm the suitability of the protocol improvements introduced after a prevalidation phase. When cell viability (evaluated by the MTT reduction test) was used as the endpoint, the predictive ability of both EpiDerm and EPISKIN was considered sufficient to justify their progression to Phase 2, while the predictive ability of the SIFT was judged to be inadequate. Since both the reconstituted skin models provided false predictions around the in vivo classification border (a rabbit Draize test score of 2), the release of a cytokine, interleukin-1alpha (IL-1alpha), was also determined. In Phase 2, each human skin model was tested in three laboratories, with 58 chemicals. The main endpoint measured for both EpiDerm and EPISKIN was cell viability. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1alpha release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. For EPISKIN, the sensitivity was 75% and the specificity was 81% (MTT assay only); with the combination of the MTT and IL-1alpha assays, the sensitivity increased to 91%, with a specificity of 79%. For EpiDerm, the sensitivity was 57% and the specificity was 85% (MTT assay only), while the predictive capacity of EpiDerm was not improved by the measurement of IL-1alpha release. Following independent peer review, in April 2007 the ECVAM Scientific Advisory Committee endorsed the scientific validity of the EPISKIN test as a replacement for the rabbit skin irritation method, and of the EpiDerm method for identifying skin irritants as part of a tiered testing strategy. This new alternative approach will probably be the first use of in vitro toxicity testing to replace the Draize rabbit skin irritation test in Europe and internationally, since, in the very near future, new EU and OECD Test Guidelines will be proposed for regulatory acceptance.

Working together to respond to the challenges of EU policy to replace animal testing.

This paper presents a personal perspective on efforts during the past 15 years to replace animal testing for assessing the safety of chemicals and products. It is based on an invited lecture--the FRAME Annual Lecture--given in October 2005, with the theme of "making progress by working together" (government-industry-academia-NGOs). Where we have achieved some successes, these have clearly been due to effective cooperation and collaboration between the relevant stakeholders. In recent times, there has not been this same level of active commitment and coordination. This needs to change, since, if we are to make good progress in the years to come in responding to the new challenges of the EU policy to replace animal testing, this will undoubtedly require us to work together, hopefully facilitated by effective leadership and coordination from the EU policy-makers themselves.

The Third FRAME Toxicity Committee: working toward greater implementation of alternatives in toxicity testing.

FRAME (the Fund for the Replacement of Animals in Medical Experiments; http://www. frame.org.uk) is a scientific charity, which has, for over 30 years, been advocating and conducting its own research on the application of the Three Rs (reduction, refinement and replacement) to animal experimentation. FRAME develops and validates scientifically based replacement alternative methods to facilitate their acceptance by scientists and regulators. As part of these activities, FRAME established a FRAME Toxicity Committee in 1979, and a report of its work was published in 1982, and discussed in the proceedings of a subsequent meeting, published in 1983. A Second Toxicity Committee formed in 1988, reported its work in 1990, which was discussed in the proceedings of a subsequent conference, published in 1991. The work of these committees was extremely successful and influential in laying the foundation for later activities in alternatives research. A Third FRAME Toxicity Committee was formed in 1999, since much progress had been achieved in the previous decade, especially with regard to the successful validation of several non-animal replacement methods and the start of their regulatory acceptance. Moreover, some new test methods are on the point of being validated, and many new techniques and discoveries are impacting on toxicity testing. Also, interest in reduction and refinement in toxicology has increased. However, there is considerable scope and need for the further implementation of the Three Rs in toxicity testing, especially due to recent plans for the large-scale testing of high-production volume, hormonally-active and existing chemicals, and the increasing use of transgenic animal models. The new committee comprises 18 experts from industry, academia, animal welfare, legislative and regulatory bodies, with one observer from the UK Government Home Office. The main objective is to review progress made in the application of the Three Rs in the development and safety evaluation of medicines, biologicals, cosmetics, agrochemicals and other products, as well as industrial chemicals, and to make recommendations as a basis for further sensible progress according to sound scientific and ethical criteria. The main committee is to be augmented by several working parties that will focus on specific scientific issues: 1) targeted risk assessment versus hazard identification; 2) data sharing; 3) endocrine disruption; and 4) carcinogenicity testing. The Committee is also to publish a status report on the current situation regarding alternatives in toxicity testing, based on the recommendations of the Second Toxicity Committee, and will organise a conference to discuss its overall conclusions and recommendations.

Update on the validation and regulatory acceptance of alternative tests for skin corrosion and irritation.

The European Centre for the Validation of Alternative Methods (ECVAM) has supported validation studies on in vitro tests for skin corrosion, resulting in the validities of four alternative tests being endorsed. The US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) has also evaluated the validity of these alternative methods for skin corrosion testing. In the European Union, a new Test Method on Skin Corrosion (B.40), incorporating the rat skin transcutaneous electrical resistance and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000. At an international level, two OECD Test Guidelines (430 and 431) on these alternative methods have been approved as of May 2002. To date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. ECVAM supported prevalidation studies on five in vitro tests for acute skin irritation during 1999-2001. These tests were based on human, pig and mouse skin. However, none of them met the criteria set for inclusion in a large-scale formal validation study. Following additional work on the test protocols and/or prediction models, it appears that several modified tests could now be ready for validation in 2003.

The feasibility of replacing animal testing for assessing consumer safety: a suggested future direction.

At present, we are unable to use much of the data derived from alternative (non-animal) tests for human health risk assessment. This brief Comment outlines why it is plausible that new paradigms could be developed to enable risk assessment to support consumer safety decisions, without the need to generate data in animal tests. The availability of technologies that did not exist 10 years ago makes this new approach possible. The approach is based on the concept that data and information derived from applying existing and new technologies to non-animal models can be interpreted in terms of harm and disease in man. A prerequisite is that similar data and information generated in a clinical setting are available to permit this "translation". The incorporation of this additional translation step should make it possible to use data and information generated in non-animal models as inputs to risk assessment. The new technologies include genomics, transcriptomics, proteomics and metabonomics. Their application to in vitro and human "models" enables large amounts of data to be generated very quickly. The processing, interpretation and translation of these data need to be supported by powerful informatics capabilities and statistical tools. The use of integrated "systems biology" approaches will further support the interpretation by providing better understanding of the underlying biological complexity and mechanisms of toxicity. Clinical medicine is using the opportunities offered by the new "omics" technologies to advance the understanding of disease. The application of these technologies in clinical medicine will generate massive amounts of data that will need processing and interpretation to allow clinicians to better diagnose disease and understand the patients' responses to therapeutic interventions. Support from clinical epidemiology will be essential. If these data and information can be made generally accessible in an ethical and legal way, they should also permit the "translation" of experimental non-animal data, so that they can then be used in risk assessment.

Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. The European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2.

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN and PREDISKIN, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999-2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.

ECVAM's activities in validating alternative tests for skin corrosion and irritation.

ECVAM has funded and managed validation studies on in vitro tests for skin corrosion, resulting in the validities of four in vitro tests being endorsed by the ECVAM Scientific Advisory Committee: the rat skin transcutaneous electrical resistance (TER) assay, two tests based on the use of commercial reconstituted human skin equivalents, EPISKIN and EpiDerm, and another commercially-produced test, CORROSITEX. In the European Union (EU), a new test method on skin corrosion (B.40), incorporating the rat skin TER and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000, thereby making the use of in vitro alternatives for skin corrosion testing of chemicals mandatory in the EU. At the recommendation of its Skin Irritation Task Force, ECVAM has funded prevalidation studies on five in vitro tests for acute skin irritation: EpiDerm, EPISKIN, PREDISKIN, the pig-ear test, and the mouse-skin integrity function test (SIFT). However, none of the tests met the criteria (set by the Management Team for the studies) for inclusion in a large-scale formal validation study. Thus, to date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. Following further work on the EPISKIN, EpiDerm and SIFT test protocols and/or prediction models after the completion of the prevalidation studies, it appears that the modified tests could meet the performance criteria defined for progression to a validation study. This will now be assessed independently by the ECVAM Skin Irritation Task Force, with the objective of taking a decision before the end of 2002 on whether to conduct a formal validation study.