In vitro and integrated in vivo strategies to reduce animal use in genotoxicity testing.

Scientific, financial, and ethical drivers have led to unprecedented interest in implementing human-relevant, mechanistic in vitro and in silico testing approaches. Further, as non-animal approaches are being developed and validated, researchers are interested in strategies that can immediately reduce the use of animals in toxicology testing. Here, we aim to outline a testing strategy for assessing genotoxicity beginning with standard in vitro methods, such as the bacterial reverse mutation test and the in vitro micronucleus test, followed by a second tier of in vitro assays including those using advanced 3D tissue models. Where regulatory agencies require in vivo testing, one demonstrated strategy is to combine genotoxicity studies traditionally conducted separately into a single test or to integrate genotoxicity studies into other toxicity studies. Standard setting organisations and regulatory agencies have encouraged such strategies, and examples of their use can be found in the scientific literature. Employing approaches outlined here will reduce animal use as well as study time and costs.

Breaking barriers to ethical research: An analysis of the effectiveness of nonhuman animal research approval in Canada.

In Canada, all institutions that conduct publicly funded, animal-based research are expected to comply with the standards of the Canadian Council on Animal Care (CCAC). The CCAC promotes the use of animal alternatives, and uses the "3Rs" principles of Replacement, Reduction, and Refinement as a guiding ethical framework. To ensure these standards are strictly enforced, internal ethics committees at each institution are tasked with creating "Animal Use Protocol" (AUP) forms to be filled out by researchers and evaluated by the committees.In this paper, we assess AUP forms from Canada's top research universities to identify the extent to which they conform to, or advance, the 3Rs framework. Our results show various deficiencies that call into question the quality of information elicited by these forms. To remedy this, we recommend that the CCAC assume responsibility for creating a standardized 3Rs section to be used on all AUP forms. In addition, proposal forms and experimental results for all research at CCAC-certified institutions should be digitized and uploaded into a national database. We argue that this would offer higher quality information for researchers at the experimental design stage, while strengthening the CCAC's mandate to be accountable to the Canadian public.

Recent efforts to elucidate the scientific validity of animal-based drug tests by the pharmaceutical industry, pro-testing lobby groups, and animal welfare organisations.

BACKGROUND: Even after several decades of human drug development, there remains an absence of published, substantial, comprehensive data to validate the use of animals in preclinical drug testing, and to point to their predictive nature with regard to human safety/toxicity and efficacy. Two recent papers, authored by pharmaceutical industry scientists, added to the few substantive publications that exist. In this brief article, we discuss both these papers, as well as our own series of three papers on the subject, and also various views and criticisms of lobby groups that advocate the animal testing of new drugs. MAIN TEXT: We argue that there still remains no published evidence to support the current regulatory paradigm of animal testing in supporting safe entry to clinical trials. In fact, the data in these recent studies, as well as in our own studies, support the contention that tests on rodents, dogs and monkeys provide next to no evidential weight to the probability of there being a lack of human toxicity, when there is no apparent toxicity in the animals. CONCLUSION: Based on these data, and in particular on this finding, it must be concluded that animal drug tests are therefore not fit for their stated purpose. At the very least, it is now incumbent on-and we very much encourage-the pharmaceutical industry and its regulators to commission, conduct and/or facilitate further independent studies involving the use of substantial proprietary data.

Phase 0, including microdosing approaches: Applying the Three Rs and increasing the efficiency of human drug development.

Phase 0 approaches, including microdosing, involve the use of sub-therapeutic exposures to the tested drugs, thus enabling safer, more-relevant, quicker and cheaper first-in-human (FIH) testing. These approaches also have considerable potential to limit the use of animals in human drug development. Recent years have witnessed progress in applications, methodology, operations, and drug development culture. Advances in applications saw an expansion in therapeutic areas, developmental scenarios and scientific objectives, in, for example, protein drug development and paediatric drug development. In the operational area, the increased sensitivity of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), expansion of the utility of Positron Emission Tomography (PET) imaging, and the introduction of Cavity Ring-Down Spectroscopy (CRDS), have led to the increased accessibility and utility of Phase 0 approaches, while reducing costs and exposure to radioactivity. PET has extended the application of microdosing, from its use as a predominant tool to record pharmacokinetics, to a method for recording target expression and target engagement, as well as cellular and tissue responses. Advances in methodology include adaptive Phase 0/Phase 1 designs, cassette and cocktail microdosing, and Intra-Target Microdosing (ITM), as well as novel modelling opportunities and simulations. Importantly, these methodologies increase the predictive power of extrapolation from microdose to therapeutic level exposures. However, possibly the most challenging domain in which progress has been made, is the culture of drug development. One of the main potential values of Phase 0 approaches is the opportunity to terminate development early, thus not only applying the principle of 'kill-early-kill-cheap' to enhance the efficiency of drug development, but also obviating the need for the full package of animal testing required for therapeutic level Phase 1 studies. Finally, we list developmental scenarios that utilised Phase 0 approaches in novel drug development.

Ten years of REACH - An animal protection perspective.

It has now been 11 years since the EU's new chemicals legislation (Regulation No. 1907/2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals [REACH]) came into force. Two important statements in the REACH Regulation in relation to animal testing and alternatives are: Article 1(1), which states that one of its purposes is to promote alternative methods; and Article 25(1), which states that animal testing should be used as a last resort. This review looks at the mechanisms that were put in place within REACH to achieve these aims and asks, not only if they are being implemented properly, but also if they have been sufficient. Whilst the chemical industry has heavily used data-sharing and read-across, this review concludes that nevertheless over 2.2 million animals have already been used in new tests for REACH registrations. This equates to an annual average of 275,000 animals; 58,000 more per year than the best-case estimate made by the European Commission in 2004. The use of in vitro and (Q)SAR approaches as standalone replacements for animal tests has been relatively low. The levels of funding for research into alternative methods remain low, and there are concerns over the speed of formal adoption of those that have been validated. In addition, there have been issues with the recognition that testing as a last resort and the promotion of alternative methods applies to all parties, including the Commission, Member States and the agency responsible, the European Chemicals Agency. This review provides ten recommendations for better implementation of these two key aspirations, as well as lessons to be learned for future similar legislation.

PUBLIC ATTITUDES TOWARDS THE USE OF ANIMALS IN BIOMEDICAL RESEARCH IN GEORGIA.

In different societies there are different opinions about moral and ethical aspects of animal testing in biomedical research. Many studies have been conducted worldwide since 1980s to evaluate public perception and attitude towards the animal research. In EU Eastern Neighboring Countries, including Georgia, ethical aspects of animal usage for biomedical experiments have not been well emphasized. There are no ethical-legal regulations on animal use for biomedical research in Georgia neither on national nor on institutional level. At the same time, public attitude concerning the animal testing for scientific research is unclear. The aim of the study has been to explore public attitudes towards the animal research for the first time in Georgia. In this survey quantitative and qualitative research methods had been used. A special questionnaire was developed and an individual interview method was utilized. Totally 750 interviews were conducted and 715 questionnaires were used for analysis. Codified questionnaires were included in the database and then analyzed with SPSS 27 software. The technique of cross-tabulation was used for the bivariate analysis. For most respondents (68.0%) animal use in Biomedical research is acceptable. 82,0% of respondents fully or partly agreed with the concept, that involvement of animals in experiments which comply to the principles of international norms and regulations (reduction of the number of animals, improving the experiment/lowering pain for animals, and using alternative methods) is acceptable. The results of the study have demonstrated that the attitude of the study sample towards the animal testing in biomedical research is influenced by age (Pearson Chi-Square=32.479, df=8, p<0.05; p=0.000), education (Pearson Chi-Square=27.850 df=12, p<0.05; p=0.006) and occupation (Pearson Chi-Square = 37.767 df=16, p<0.05; p=0.002). Gender, place of residence and experience with owning the pet/animal doesn't have statistically significant influence on the public attitude.  The results of our study highlight that animal testing in biomedical research is acceptable for the respondents involved in the study. However, most of the respondent's consider use of International regulations highly important.

[Animal experiment, can we replace?] / Expérimentation animale, peut-on s'en passer ?

Animal experiment is a subject of controversies. Some people, defenders of animals, think that it is not acceptable to use for scientific purposes at the risk of making them suffer or assert that the results obtained with animals are not transposable in the human beings. Others, in particular researchers in biology or medicine, think that the animal models are essential for the biomedical search. This confrontation of the opinions bases largely on an evolution of the place of animals in our society. The regulations authorize the use of animals for scientific purposes but oblige to make it under restrictive conditions. The application of 3Rs - replacement, reduction, and refinement - expressed in 1959 by Russel and Burch is an ethical guide to improve the welfare of animals in research. The alternative methods do not allow, in the present state of the knowledge, to answer all the scientific questions in biology and medicine research. They are, most of the time, complementary methods of the in vivo methods.

Opinion versus evidence for the need to move away from animal testing.

Science is based on facts and their discourse. Willingly or unwillingly, facts are mixed with opinion, i.e., views or judgments formed, not necessarily based on fact or knowledge. This is often necessary, where we have controversial facts or no definitive evidence yet, because we need to take decisions or have to prioritize. Evidence-based approaches aim at identifying the facts and their quality objectively and transparently; they are now increasingly embraced in toxicology, especially by employing systematic reviews, meta-analyses, quality scoring, risk-of-bias tools, etc. These are core to Evidence-based Toxicology. Such approaches aim at minimizing opinion, the "eminence-based" part of science. Animal experiments are the basis of a lot of our textbook knowledge in the life sciences, have helped to develop desperately needed therapies, and have made this world a safer place. However, they represent only one of the many possible approaches to accomplish all these things. Like all approaches, they come with shortcomings, and their true contribution is often overrated. This article aims to summarize their limitations and challenges beside the ethical and economical concerns (i.e., costs and duration as well as costs following wrong decisions in product development): they include reproducibility, inadequate reporting, statistical under-powering, lack of inter-species predictivity, lack of reflection of human diversity and of real-life exposure. Each and every one of these increasingly discussed aspects of animal experiments can be amended, but this would require enormous additional resources. Together, they prompt a need to engineer a new paradigm to ensure the safety of patients and consumers, new products and therapies.

Reproducibility Issues: Avoiding Pitfalls in Animal Inflammation Models.

In light of an enhanced awareness of ethical questions and ever increasing costs when working with animals in biomedical research, there is a dedicated and sometimes fierce debate concerning the (lack of) reproducibility of animal models and their relevance for human inflammatory diseases. Despite evident advancements in searching for alternatives, that is, replacing, reducing, and refining animal experiments-the three R's of Russel and Burch (1959)-understanding the complex interactions of the cells of the immune system, the nervous system and the affected tissue/organ during inflammation critically relies on in vivo models. Consequently, scientific advancement and ultimately novel therapeutic interventions depend on improving the reproducibility of animal inflammation models. As a prelude to the remaining hands-on protocols described in this volume, here, we summarize potential pitfalls of preclinical animal research and provide resources and background reading on how to avoid them.

E-cigarettes and the need and opportunities for alternatives to animal testing.

E-cigarettes have become within only one decade an important commodity, changing the market of the most mass-killing commercial product. While a few years ago estimates suggested that in the course of the 21st century one billion people would die prematurely from tobacco consumption, e-cigarettes continuously gaining popularity promise 10-30fold lower health effects, possibly strongly changing this equation. However, they still are not a harmless life-style drug. Acceptability simply depends on whether we compare their use to smoking or to not-smoking. In the absence of long-term follow-up health data of users, additional uncertainty comes from the lack of safety data, though this uncertainty likely only is whether they represent 3 or 10% of the risk of their combustible counterpart. This means that there is little doubt that they represent a prime opportunity for smokers to switch, but also that their use by non-smokers should be avoided where possible. The real safety concerns, however, are that e-cigarettes expose their users to many compounds, contaminants and especially flavors (more than 7,000 according to recent counts), which have mostly not been tested, especially not for long-term inhalation exposure. Neither the precautionary traditional animal testing nor post-marketing surveillance will offer us data of sufficient quality or sufficiently fast to support product development and regulatory decisions. Thus, alternative methods lend themselves to fill this gap, making this new product category a possible engine for new method development and its implementation and validation.

Ending the use of animals in toxicity testing and risk evaluation.

This article discusses the use of animals for the safety testing of chemicals, including pharmaceuticals, household products, pesticides, and industrial chemicals. It reviews changes in safety testing technology and what those changes mean from the perspective of industrial innovation, public policy and public health, economics, and ethics. It concludes that the continuing use of animals for chemical safety testing should end within the decade as cheaper, quicker, and more predictive technologies are developed and applied.

Los métodos alternativos: un reto para las administraciones y para los científicos / Alternative methods: a challenge for governments and scientists

La normativa actual que regula en el ámbito europeo la utilización de los animales con fines científicos establece con absoluta rotundidad que su prioridad absoluta es el fomento y la implantación de los enfoques alternativos a los métodos tradicionales de utilización de los animales. Insistimos sobre dos aspectos que consideramos relevantes. En primer lugar es necesario aclarar que los métodos alternativos no son solo aquellos en los que se alcanza el Reemplazo total de los animales, sino que también comprenden aquellos otros métodos y estrategias en las que se reduce el número de animales utilizados (Reducción) o se refinan las condiciones en los que éstos se utilizan y mantienen (Refinamiento). En segundo lugar quisiéramos animar a la comunidad científica a dar los pasos que sean necesarios para poder implementar también en el ámbito de las 3 erres el cambio de paradigma que los avances y el desarrollo que en el campo de la biología molecular y de sistemas están haciendo posible en toxicología. En el futuro es posible que algunas pruebas puedan consistir ya no tanto en analizar los efectos que determinadas sustancias tóxicas tienen sobre los animales, sino más bien en evaluar los cambios metabólicos que a nivel molecular son los que realmente causan los mencionados efectos sobre los animales (y lógicamente el ser humano) (AU)

Current European regulations on the use of animals for scientific purposes categorically states that the first priority is the development and implementation of alternative approaches to traditional methods using animals. We insist on two aspects that we consider relevant. First able it is necessary to clarify that alternative methods are not only those in which the total replacement of animals is reached, but also include those other methods and strategies in which the number of animals used decreases (reduction) or refine the conditions under which they are used and maintained (Refinement). Secondly, we would like to encourage the scientific community to take the steps necessary to also implement in the field of the 3Rs principle that progress and development in the field of molecular biology and systems are possible in toxicology. In the future it is possible that some studies may consist not so much as to analyze the effects that certain toxic substances have on the animals, but rather to evaluate metabolic changes at the molecular level that actually cause the effects on animals (and logically on humans) (AU)

Actividades de promoción y difusión de alternativas en España / Advocacy and outr each on alternatives in Spain

Se revisan las actividades de promoción y desarrollo de los métodos alternativos y complementarios a la experimentación animal en España. Se muestran las reuniones o jornadas que se han celebrado y organizado, incluyendo la constitución de la Red Española de Métodos Alternativos. Como foro de discusión para lograr un menor y más racional uso de los animales de experimentación, fomentando el desarrollo, validación y utilización de los métodos in vitro, tiene como fin conseguir el eficaz desarrollo de los procedimientos alternativos mediante el uso de los principios de reducción, refinamiento y reemplazo de los animales en la experimentación, lo que constituye la aplicación de las 3Rs. Se destaca el papel de la citada Red detallando información sobre la misma, sus miembros, la participación en numerosos cursos, seminarios y jornadas que se organizan por Universidades españolas y otras entidades, y la estrecha colaboración con el Ministerio de Sanidad, Servicios Sociales e Igualdad y el Ministerio de Agricultura Alimentación y Medio Ambiente, contando con observadores en su Junta Directiva (AU)

A revision of advocacy and development of alternative and complementary methods to animal testing in Spain was done. Meetings and conferences that have been organized, including the constitution of the Spanish Network for Alternative Methods, are shown. As a discussion forum for a lower and more rational use of experimental animals, promoting the development, validation and use of in vitro methods. The aims were the effective development of alternative procedures using the principles of reduction, refinement and replacement of animal use in experiments, which is the application of the 3Rs. The role of the aforementioned net is detailed, with the information about the network and their members. It has participated in many courses, seminars and conferences in the Spanish universities and other entities, maintaining a close collaboration with the Ministry of Health and the Ministry of Agriculture, Food and Environment, with observers included in the Board (AU)

Alternativas a los animales de laboratorio en la docencia / Alternatives to laboraroty animals in education

En este artículo se hace una revisión sobre la utilización de animales y de métodos alternativos en docencia y se constata que todavía se siguen utilizando animales, a pesar de los avances tecnológicos que permiten cada vez métodos mejores y más efectivos para reemplazar a los animales en las prácticas docentes. Así mismo se analizan las ventajas y las limitaciones que pueden presentar estos métodos y se hace una revisión bibliográfica de los últimos artículos publicados en este sentido. A pesar, de existir muchos docentes que utilizan métodos alternativos, no existen demasiados artículos que nos informen de la situación de las alternativas ni tampoco de los beneficios que aportan a los estudiantes (AU)

This paper is a review on the use of laboratory animals and alternatives methods in education. Laboratory animals are still used despite technological advances that allow better alternative methods and more effectives to replace animals in laboratory practices. Moreover, we analyze the advantages and limitations of these alternative methods and we review the literature of the last years in this field. Although, there are many teachers who use alternative methods, there are not too many papers giving information about the real situation of alternatives neither the benefits for students (AU)

[Reduction of animal experiments in experimental drug testing]. / Reduktion von Tierversuchen in der experimentellen Arzneimittelprüfung.

In order to ensure the quality of biomedical products, an experimental test for every single manufactured batch is required for many products. Especially in vaccine testing, animal experiments are traditionally used for this purpose. For example, efficacy is often determined via challenge experiments in laboratory animals. Safety tests of vaccine batches are also mostly performed using laboratory animals. However, many animal experiments have clear inherent disadvantages (low accuracy, questionable transferability to humans, unclear significance). Furthermore, for ethical reasons and animal welfare aspects animal experiments are also seen very critical by the public. Therefore, there is a strong trend towards replacing animal experiments with methods in which no animals are used ("replacement"). If a replacement is not possible, the required animal experiments should be improved in order to minimize the number of animals necessary ("reduction") and to reduce pain and suffering caused by the experiment to a minimum ("refinement"). This "3R concept" is meanwhile firmly established in legislature. In recent years many mandatory animal experiments have been replaced by alternative in vitro methods or improved according to the 3R principles; numerous alternative methods are currently under development. Nevertheless, the process from the development of a new method to its legal implementation takes a long time. Therefore, supplementary regulatory measures to facilitate validation and acceptance of new alternative methods could contribute to a faster and more consequent implementation of the 3R concept in the testing of biomedical products.