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The EU’s decision to ban animal testing for toxicity testing, has positively influenced the pace of developing New Approach Methodologies (NAMs). This development also supports replacing animal methods in other forms of risk assessment (RA), such as for oral-toxicity testing. This study aims to identify the hurdles and opportunities for validation and implementation of NAMs in the current EU’s chemical RA. Through conducting semi-structured interviews with 14 stakeholders, experiences and perspectives about the validation and implementation of NAMs in RA for orally ingested chemicals were analyzed. Stakeholders considered the use of NAMs for RA processes both a cultural and generational issue. Both were perceived as hurdles for reaching the next generation RA approach. The differing views on NAMs originated from experience and stakeholder positions, but communication and collaboration on developing future RA approaches could support overcoming this skepticism. Irrespectively of their background, all interviewees were generally optimistic that NAMs will support the development of more accurate and sustainable RA. This research highlights the need for the EU to adjust legislation and guidance documents to shift in testing requirements from the traditional overexposure approach to more predictive, mechanistic testing in RA, which will take time. This study, however, shows that—when all stakeholders engage in communication and confidence building—NAMs can already play an important role in reducing and refining animal testing.

This article tracks the transformation of beagle dogs from a common breed in mid-twentieth century American laboratories to the de jure standard in global toxicological research by the turn of the twenty-first. The breed was dispersed widely due to the expanding use of dogs in pharmacology in the 1950s and a worldwide crisis around pharmaceutical safety following the thalidomide scandal of the 1960s. Nevertheless, debates continued for decades over the beagle's value as a model of carcinogenicity, even as the dogs became legislated stand-ins for human beings in multiple countries. Situating beagles as a biocommodity, the article calls for more sustained attention to the "political economy" of laboratory organism breeding, use, and production. The story of American commercial breeder Marshall Farms offers insight into the role of for-profit companies in contemporary laboratory animal provision, as the article makes a case for the value of a global perspective on transnational corporations as key sites of scientific practice and collaboration.

The objective of this study was to systematically review the literature to determine the effect of combined hypothermia (HTH) and mesenchymal stem cell (MSC) therapy (administered during or immediately before or after HTH) compared with HTH alone on brain injury and neurobehavioural outcomes in animal models of neonatal hypoxic-ischaemic encephalopathy. Primary outcomes assessed were neuropathological measures and neurobehavioural measures of brain outcome. Secondary outcomes were brain protein proinflammatory cytokine status. Risk of bias (ROB) was assessed with the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) ROB assessment tool. Of 393 studies identified, 3 studies in postnatal day 7 (P7) male Sprague-Dawley rats met the inclusion criteria. Meta-analyses were undertaken for neuropathological measures (apoptotic cells, astrocytes, microglia), neurobehavioral measures (rotarod test and negative geotaxis), and proinflammatory cytokine levels. Two of the three studies scored low or unclear ROB across all measures. Treatment with HTH-MSCs together significantly improved astrocyte optical density by standardised mean difference (SMD) of 0.71 [95% confidence interval (CI) -1.14, -0.28]. No other measures showed significant differences. There is insufficient preclinical data to confirm the efficacy of combined HTH-MSC therapy over HTH alone. Future studies should utilise a reporting checklist such as in SYRCLE or Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines to improve reporting standards. Very few articles investigating the use of MSCs for the treatment of hypoxic-ischaemic encephalopathy are clinically relevant. Continuing to publish studies in models of hypoxic-ischaemic encephalopathy without the inclusion of HTH therapy does not progress the field towards improved clinical outcomes. This study shows that HTH and MSC therapy improves measures of astrogliosis. More studies are required to establish the efficacy of HTH and MSCs on measures of neuropathology and neurobehavior. The reporting of preclinical data in this space could be improved by using reporting checklists such as the SYRCLE or ARRIVE tools.

The EU Directive 2010/63/EU on the protection of animals used for scientific purposes and other EU regulations, such as REACH and the Cosmetic Products Regulation advocate for a change in the way toxicity testing is conducted. Whilst the Cosmetic Products Regulation bans animal testing altogether, REACH aims for a progressive shift from in vivo testing towards quantitative in vitro and computational approaches. Several endpoints can already be addressed using non-animal approaches including skin corrosion and irritation, serious eye damage and irritation, skin sensitisation, and mutagenicity and genotoxicity. However, for systemic effects such as acute toxicity, repeated dose toxicity and reproductive and developmental toxicity, evaluation of chemicals under REACH still heavily relies on animal tests. Here we summarise current EU regulatory requirements for the human health assessment of chemicals under REACH and the Cosmetic Products Regulation, considering the more critical endpoints and identifying the main challenges in introducing alternative methods into regulatory testing practice. This supports a recent initiative taken by the International Cooperation on Alternative Test Methods (ICATM) to summarise current regulatory requirements specific for the assessment of chemicals and cosmetic products for several human health-related endpoints, with the aim of comparing different jurisdictions and coordinating the promotion and ultimately the implementation of non-animal approaches worldwide. Recent initiatives undertaken at European level to promote the 3Rs and the use of alternative methods in current regulatory practice are also discussed.

To systematically review indexed literature related to the influence of mini-screw implant (MSI)-assisted intrusion on orthodontically induced inflammatory root resorption (OIIRR). Indexed databases were searched without time and language restrictions using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The inclusion criteria were: (a) original studies; (b) patients/subjects undergoing MSI-assisted intrusion; and (c) tomographic and/or histological assessment of OIIRR. Letters to the Editor, commentaries, case reports/series, reviews, and studies based on two-dimensional radiographic assessment of OIIRR were excluded. For experimental and clinical studies, the risk of bias assessment was performed using the Systematic Review Centre for Laboratory animal Experimentation risk of bias tool and the Risk of Bias in Non-randomized Studies of Interventions guidelines, respectively. The initial search yielded 453 studies, out of which 6 (3 clinical and 3 on animal-models) were included. The clinical studies were performed on males and females with a mean age ranging between 16.07 and 25.5 years. Duration of the clinical studies ranged from 3.8 to 9 months. The animal studies were performed on mini-pigs, rats, and dogs. The mean age in the studies on rats and mini-pigs was 2.76 and 18 months, respectively. In the study on canine models, mean age was not reported. In all studies, MSI-assisted intrusion was shown to cause OIIRR. Power analysis was performed in one study. All studies had a moderate risk of bias. MSI-assisted intrusion is a risk factor for OIIRR; however, from a clinical perspective, further well-designed and power-adjusted studies are needed.

Animal experiments have served to improve our knowledge on diseases and treatment approaches since ancient times. Today, animal experiments are widely used in medical, biomedical and veterinary research, and are essential means of drug development and preclinical testing, including toxicology and safety studies. Recently, great efforts have been made to replace animal experiments with in vitro organoid culture methods and in silico predictions, in agreement with the 3R strategy to “reduce, refine and replace” animals in experimental testing, as outlined by the European Commission. Here we present a mini-review on the development of animal testing, as well as on alternative in vitro and in silico methods, that may at least partly replace animal experiments in the near future.

The main aim is to identify, by means of different imaging modalities, the early bone changes in patients “at risk” and in stage 0 MRONJ. A search of the literature was performed on PubMed, Embase, Web of Science, and Cochrane Library databases, until June 9, 2020. No language or year restrictions were applied. Screening of the articles, data collection, and qualitative analysis was done. The Newcastle-Ottawa Scale (NOS) was used for observational studies, and the Systematic Review Centre for Laboratory Animal Experimentation’s (SYRCLE) risk of bias tool for the animal studies. A total of 1188 articles were found, from which 47 were considered eligible, whereas 42 were suitable for the qualitative analysis. They correspond to 39 human studies and 8 animal studies. Radiographic findings such as bone sclerosis, osteolytic areas, thickening of lamina dura, persisting alveolar socket, periapical radiolucency, thicker mandibular cortex, widening of the periodontal ligament space, periodontal bone loss, and enhancement of the mandibular canal were identified as early bone changes due to antiresorptive therapy. All those findings were also reported later in Stage 0 patients. The main limitations of these results are the lack of prospective data and comparisons groups; therefore, careful interpretation should be made. It is a fact that radiographic findings are present in antiresorptive-treated patients, but the precise timepoint of occurrence, their relation to the posology, and potential risk to develop MRONJ are not clear. The importance of a baseline radiographic diagnosis for antiresorptive-treated patients.

The COVID-19-inducing virus, SARS-CoV2, is likely to remain a threat to human health unless efficient drugs or vaccines become available. Given the extent of the current pandemic (people in over one hundred countries infected) and its disastrous effect on world economy (associated with limitations of human rights), speedy drug discovery is critical. In this situation, past investments into the development of new (animal-free) approach methods (NAM) for drug safety, efficacy, and quality evaluation can be leveraged. For this, we provide an overview of repurposing ideas to shortcut drug development times. Animal-based testing would be too lengthy, and it largely fails, when a pathogen is species-specific or if the desired drug is based on specific features of human biology. Fortunately, industry has already largely shifted to NAM, and some public funding programs have advanced the development of animal-free technologies. For instance, NAM can predict genotoxicity (a major aspect of carcinogenicity) within days, human antibodies targeting virus epitopes can be generated in molecular biology laboratories within weeks, and various human cell-based organoids are available to test virus infectivity and the biological processes controlling them. The European Medicines Agency (EMA) has formed an expert group to pave the way for the use of such approaches for accelerated drug development. This situation illustrates the importance of diversification in drug discovery strategies and clearly shows the shortcomings of an approach that invests 95% of resources into a single technology (animal experimentation) in the face of challenges that require alternative approaches. Electronic supplementary material The online version of this article (10.1007/s00204-020-02787-2) contains supplementary material, which is available to authorized users.

Animals have been used for the testing of new drugs and nanoparticles to determine their safety and effectiveness before application in humans. Numerous drugs and nanoparticles have been developed through animal tests, and development in medical nanotechnology is progressing rapidly. However, a good experiment requires many animals. The primary measures to reduce animal experiments include reducing the number of animals used in each experiment and replacing laboratory animals with inanimate, cellular, or inferior animals to reduce sacrifices. Developing nanoparticles using simulations rather than experiments may be another alternative. Instead of animals, human cells or animal-derived organs, tissues, or cells could be used in experiments. These methods allow rapid toxicity and efficacy testing at low cost. However, the disadvantage of these methods is that they cannot accurately replicate the complex interrelationships between human organs, biological reactions to specific routes of administration, or toxicity of substances resulting from metabolic processes. To overcome these shortcomings of in vitro tests, new technologies such as organoids and organ-on-chips are progressing. These can be used to quickly examine the efficacy of newly developed drugs and nanoparticles and can be useful in developing patient-tailored agents. These technologies offer some promise but it is still difficult to entirely replace animal experiments. However, the scope of methods to replace experimental animals is widening, suggesting the potential to refine, reduce, and ultimately replace animal testing.

Drug-induced liver injury (DILI) complicates safety assessment for new drugs and poses major threats to both patient health and drug development in the pharmaceutical industry. A number of human liver cell-based in vitro models combined with toxicogenomics methods have been developed as an alternative to animal testing for studying human DILI mechanisms. In this review, we discuss the in vitro human liver systems and their applications in omics-based drug-induced hepatotoxicity studies. We furthermore present bioinformatic approaches that are useful for analyzing toxicogenomic data generated from these models and discuss their current and potential contributions to the understanding of mechanisms of DILI. Human pluripotent stem cells, carrying donor-specific genetic information, hold great potential for advancing the study of individual-specific toxicological responses. When co-cultured with other liver-derived non-parenchymal cells in a microfluidic device, the resulting dynamic platform enables us to study immune-mediated drug hypersensitivity and accelerates personalized drug toxicology studies. A flexible microfluidic platform would also support the assembly of a more advanced organs-on-a-chip device, further bridging gap between in vitro and in vivo conditions. The standard transcriptomic analysis of these cell systems can be complemented with causality-inferring approaches to improve the understanding of DILI mechanisms. These approaches involve statistical techniques capable of elucidating regulatory interactions in parts of these mechanisms. The use of more elaborated human liver models, in harmony with causality-inferring bioinformatic approaches will pave the way for establishing a powerful methodology to systematically assess DILI mechanisms across a wide range of conditions.

The development of in vitro and ex vivo models to mimic human illness is important not only for scientific understanding and investigating therapeutic approaches but also to mitigate animal testing and bridge the inter-species translational gap. While in vitro models can facilitate high-throughput and cost-efficient evaluation of novel therapeutics, more complex ex vivo systems can better predict both desirable and adverse in vivo effects. Here we describe an ex vivo cultured human skin explant model in which we have characterized pathological tissue integrity, barrier function and metabolic stability over time. Our findings suggest that human skin can be successfully cultured for pharmacodynamic use up to and beyond 9 days without any adverse physiological consequence.

Using animal cells and tissues as precise measuring devices for developing new drugs presents a long-standing challenge for the pharmaceutical industry. Despite the very significant resources that continue to be dedicated to animal testing of new compounds, only qualitative results can be obtained. This often results in both false positives and false negatives. Here, we show how the effect of drugs applied to animal ventricular myocytes can be translated, quantitatively, to estimate a number of different effects of the same drug on human cardiomyocytes. We illustrate and validate our methodology by translating, from animal to human, the effect of dofetilide applied to dog cardiomyocytes, the effect of E-4031 applied to zebrafish cardiomyocytes, and, finally, the effect of sotalol applied to rabbit cardiomyocytes. In all cases, the accuracy of our quantitative estimates are demonstrated. Our computations reveal that, in principle, electrophysiological data from testing using animal ventricular myocytes, can give precise, quantitative estimates of the effect of new compounds on human cardiomyocytes.

COVID-19 has become one of the biggest health concern, along with huge economic burden. With no clear remedies to treat the disease, doctors are repurposing drugs like chloroquine and remdesivir to treat COVID-19 patients. In parallel, research institutes in collaboration with biotech companies have identified strategies to use viral proteins as vaccine candidates for COVID-19. Although this looks promising, they still need to pass the test of challenge studies in animal models. As various models for SARS-CoV-2 are under testing phase, biotech companies have bypassed animal studies and moved to Phase I clinical trials. In view of the present outbreak, this looks a justified approach, but the problem is that in the absence of animal studies, we can never predict the outcomes in humans. Since animal models are critical for vaccine development and SARS-CoV-2 has different transmission dynamics, in this review we compare different animal models of SARS-CoV-2 with humans for their pathogenic, immune response and transmission dynamics that make them ideal models for vaccine testing for COVID-19. Another issue of using animal model is the ethics of using animals for research; thus, we also discuss the pros and cons of using animals for vaccine development studies.

Drug-induced liver injury (DILI) constitutes one of the most frequent reasons of restricted-use warnings as well as withdrawals of drugs in postmarketing and poses an important concern for the pharmaceutical industry. The current hepatic in vivo and in vitro models for DILI detection have shown clear limitations, mainly for studies of long-term hepatotoxicity. For this reason, we here evaluated the potential of using Upcytes human hepatocytes (UHH) for repeated-dose long-term exposure to drugs. The UHH were incubated with 15 toxic and non-toxic compounds for up to 21 days using a repeated-dose approach, and, in addition to conventional examination of effects on viability, the mechanisms implicated in cell toxicity were also assessed by means of high-content screening. The UHH maintained the expression and activity levels of drug-metabolizing enzymes for up to 21 days of culture and became more sensitive to the toxic compounds after extended exposures, showing inter-donor differences which would reflect variability among the population. The assay also allowed to detect the main mechanisms implicated in the toxicity of each drug as well as identifying special susceptibilities depending on the donor. UHH can be used for a long-term repeated detection of DILI at clinically relevant concentrations and also offers key mechanistic features of drug-induced hepatotoxicity. This system is therefore a promising tool in preclinical testing of human relevance that could help to reduce and/or replace animal testing for drug adverse effects.

Regulatory ethical frameworks governing animal experimentation are a hallmark of modern biology. While most countries have ethical standards regarding the use of animals for scientific purposes, experiments involving insects are not included in these standards. With studies in recent years suggesting that insects may possess faculties akin to emotive states, there is growing discussion surrounding the ethical implications of scientific experimentation involving insects. This paper explores some of the current evidence for the ability of insects to experience emotive states and highlights how current formal animal experimentation ethics frameworks are unnecessary for governing the use of insects for scientific purposes. At its conclusion, this paper discusses ways in which scientists can, and should, uniformly maximise the welfare of insects used in their experiments in a way that is of benefit to their science as well as to the dignity and welfare of their study organisms.

Educational institutions have a responsibility not only to provide a solid theoretical background on scientific phenomena, but to also frame them within the wider social context and highlight their numerous ethical implications. It is fundamental that tomorrow’s scientists be encouraged to develop an informed and critical approach towards scientific issues that, as in the case of animal experimentation, bring undeniable advantages to our society while carrying highly controversial moral implications. However, despite the considerable social and scientific relevance of the use of animal models in biomedical research, there is a scarcity of scholarly literature exploring the topic from a pedagogical standpoint. The case study presented in this paper aims to investigate the effectiveness of role play simulation in promoting critical understanding of the use of animal models in biomedical research and its ethical implications. A simulation was set up in which first year undergraduate students took the role of delegates to a conference purportedly organised by the United Nations Ethics Office. The simulation included two electronic voting sessions, an open debate, and a keynote lecture. Post-session feedback indicated that most students found the simulation more engaging than a conventional lecture. A comparison of self-assessment questionnaires compiled at the start and at the end of the session indicated that students’ understanding of, and confidence in, the topic of animal experimentation had considerably increased after taking part in the simulation.

Breastfeeding women are excluded from clinical trials of psychoactive drugs because of ethical concerns. Animal testing, which often is predictive of adverse effects in humans, represents the only avenue available for assessing drug safety for human offspring exposed to drugs during lactation. I determined whether behavioral outcomes for children exposed during breastfeeding to antidepressants, anxiolytics, antipsychotics, anti-seizure medications, analgesics, sedatives, and marijuana can be predicted by rodent studies of offspring exposed to drugs during lactation. Animal data were available for only 10 of 80 CNS-active drugs canvassed. Behavioral deficits in adolescence or adulthood in rats and mice after various drug exposures during lactation included reductions in sexual behavior, increased anxiety, hyperactivity, and impaired learning and memory. Whether similar adverse effects will emerge in adulthood in children exposed to drugs during breastfeeding is unknown. Rodent research has the potential to forecast impairments in breastfed children long before information emerges from post-marketing reports and should be prioritized during preclinical drug evaluation by the FDA for new drugs and for drugs currently prescribed off-label for lactating women.

Since 2013, as ethics for animal experiments has been strengthened in the European Union, artificial skin models have attracted attention as an alternative to animal testing for assessing the safety and toxicity of products. Driven by regulatory authorities and industry demands, various artificial skin models have been developed by combining various biomaterials and human cells as well as using a variety of techniques, including freeze-drying, 3-D printing, electrospinning, and microfluidic system. Elaborately designed artificial skin models which closely mimic the human skin can be highly valuable and effective tools to replace in vivo animal tests for the evaluation of the safety and efficacy in the field of cosmetic and pharmaceutical industries as well as for the basic studies on cell to cell interactions, cell to extracellular matrix interactions, tissue formation and development. This review recapitulates diverse fabrication techniques for artificial skin models and their main applications.

Animal experiments have been widely conducted in the life sciences for more than a century, and have long been a subject of ethical and societal controversy due to the deliberate infliction of harm upon sentient animals. However, the harmful use of animals may also negatively impact the mental health of researchers themselves. We sought to evaluate the anxiety level of researchers engaged in animal use to analyse the mental stress from animal testing. The State Anxiety Scale of the State-Trait Anxiety Inventory (STAI) was used to evaluate how researchers feel when they conduct animal, as opposed to non-animal, based experiments (95 non-animal and 98 animal testing researchers). The Trait Anxiety Scale of STAI was employed to measure proneness to anxiety, namely the base trait of the researchers. Additionally, the information on sex, age, education, income, and total working periods was collected. While the Trait Anxiety scores were comparable (41.5 ± 10.9 versus 42.9 ± 10.1, p = 0.3682, t-test), the State Anxiety scores were statistically significantly higher for animal users than non-animal users (45.1 ± 10.7 versus 41.3 ± 9.4, p = 0.011). This trend was consistent for both male and female. Notably, younger animal testers (≤ 30 years of age) with less work experience (≤ 2 years) and lower income level (≤ 27,000 USD) exhibited higher anxiety scores, whereas these factors did not affect the anxiety level of non-animal users. The present study demonstrated that participation in animal experiments can negatively impact the mental health of researchers.

Nature Reviews Materials speaks to Donald Ingber, Founding Director of the Wyss Institute for Biologically Inspired Engineering at Harvard University, about the animal testing conundrum and the importance of human-relevant models in biomedical research.

The scientific literature is full of articles discussing poor reproducibility of findings from animal experiments as well as failures to translate results from preclinical animal studies to clinical trials in humans. Critics even go so far as to talk about a "reproducibility crisis" in the life sciences, a novel headword that increasingly finds its way into numerous high-impact journals. Viewed from a cynical perspective, Fett's law of the lab "Never replicate a successful experiment" has thus taken on a completely new meaning. So far, poor reproducibility and translational failures in animal experimentation have mostly been attributed to biased animal data, methodological pitfalls, current publication ethics and animal welfare constraints. More recently, the concept of standardization has also been identified as a potential source of these problems. By reducing within-experiment variation, rigorous standardization regimes limit the inference to the specific experimental conditions. In this way, however, individual phenotypic plasticity is largely neglected, resulting in statistically significant but possibly irrelevant findings that are not reproducible under slightly different conditions. By contrast, systematic heterogenization has been proposed as a concept to improve representativeness of study populations, contributing to improved external validity and hence improved reproducibility. While some first heterogenization studies are indeed very promising, it is still not clear how this approach can be transferred into practice in a logistically feasible and effective way. Thus, further research is needed to explore different heterogenization strategies as well as alternative routes toward better reproducibility in animal experimentation.

This review highlights selected effects of untreated pain and of widely used analgesics such as opioids, non-steroid anti-inflammatory drugs and antipyretics, to illustrate the relevance of carefully planned, appropriate and controlled analgesia for greater reproducibility in animal experiments involving laboratory rodents.

Good animal welfare is linked to the quality of research data derived from laboratory animals, their validity as models of human disease, the number of animals required to reach statistical significance and the reproducibility of in vivo studies. Identifying new ways of understanding and improving animal welfare, and promoting these in the scientific community, is therefore a key part of the work of the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs). Our strategy for animal welfare includes funding research to generate an evidence base to support refinements, office-led data sharing to challenge existing practices, events and networks to raise awareness of the evidence base, and the creation of online and other resources to support practical implementation of refinement opportunities.

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.

The hazard assessment of skin sensitizers relies mainly on animal testing, but much progress is made in the development, validation and regulatory acceptance and implementation of non-animal predictive approaches. In this review, we provide an update on the available computational tools and animal-free test methods for the prediction of skin sensitization hazard. These individual test methods address mostly one mechanistic step of the process of skin sensitization induction. The adverse outcome pathway (AOP) for skin sensitization describes the key events (KEs) that lead to skin sensitization. In our review, we have clustered the available test methods according to the KE they inform: the molecular initiating event (MIE/KE1)-protein binding, KE2-keratinocyte activation, KE3-dendritic cell activation and KE4-T cell activation and proliferation. In recent years, most progress has been made in the development and validation of in vitro assays that address KE2 and KE3. No standardized in vitro assays for T cell activation are available; thus, KE4 cannot be measured in vitro. Three non-animal test methods, addressing either the MIE, KE2 or KE3, are accepted as OECD test guidelines, and this has accelerated the development of integrated or defined approaches for testing and assessment (e.g. testing strategies). The majority of these approaches are mechanism-based, since they combine results from multiple test methods and/or computational tools that address different KEs of the AOP to estimate skin sensitization potential and sometimes potency. Other approaches are based on statistical tools. Until now, eleven different testing strategies have been published, the majority using the same individual information sources. Our review shows that some of the defined approaches to testing and assessment are able to accurately predict skin sensitization hazard, sometimes even more accurate than the currently used animal test. A few defined approaches are developed to provide an estimate of the potency sub-category of a skin sensitizer as well, but these approaches need further independent evaluation with a new dataset of chemicals. To conclude, this update shows that the field of non-animal approaches for skin sensitization has evolved greatly in recent years and that it is possible to predict skin sensitization hazard without animal testing.

The 3Rs guideline is the gold standard for ethics in animal experimentation. Two of those rules, namely refinement and reduction, require further improvement. The objective of this study was to define pathways to better compliance with these prerequisites. Two methods which move us in this direction are: (1) using small animal imaging techniques for pancreatic ductal adenocarcinoma (PDAC) follow-up and (2) reduction of the number of control animals included in a study of PDAC progression under treatment. Firstly, we used MicroCT scan to diagnose events showing PDAC progression prior to any clinical symptoms to thereby define more humane endpoints identifiable before any painful phenomenon is observed. Secondly, in order to test the hypothesis of using a reference control group in all preclinical studies of a new treatment of PDAC, we investigated the stability of the results obtained with the control groups in three successive identical studies comparing placebo and gemcitabine in tumor-bearing Lewis rats. Two imaging endpoints were found. The first was the observation of a liver metastasis assessing PDAC diffusion and, earlier than liver metastasis, the presence of bands of fluid along the flanks, with more or less a medial displacement of bowel and solid viscera, reflecting a peritoneal ascites. Results of the longitudinal follow-up of rats in the gemcitabine study revealed heterogeneity in the survival rate in the three control groups, as opposed to the survival rate in the three treated groups which did not differ statistically. As a result, the significance of improved survival with chemotherapy varied greatly according to the control group used for the comparison, ranging from no impact to a highly significant effect. The early detection by the means of animal imaging of one or more signs indicating the onset of a critical step in the development of the disease (e.g., ascites or/and metastasis) allows the researcher to prevent the occurrence of animal pain, thereby ensuring better animal welfare. However, using a single standard control group in an effort to use fewer animals for a given model runs such a significant risk of false results that it mars the entire study. Although reducing the number of animals in a study remains the gold standard of our experimental practice, in this case it would come at the price of a loss of validity of the results.

In the present study, we identified the effects of inhaled Pimpinella peregrina essential oil (1 and 3 %, for 21 continuous days) on scopolamine-induced memory impairment, anxiety, and depression in laboratory rats. Y-maze and radial arm-maze tests were used for assessing memory processes. Also, the anxiety and depressive responses were studied by means of the elevated plus-maze and forced swimming tests. The scopolamine alone-treated rats exhibited the following: decrease of the spontaneous alternation percentage in Y-maze test, increase of the number of working and reference memory errors in radial arm-maze test, along with decrease of the exploratory activity, the percentage of the time spent and the number of entries in the open arm within elevated plus-maze test and decrease of swimming time and increase of immobility time within forced swimming test. Inhalation of the P. peregrina essential oil significantly improved memory formation and exhibited anxiolytic- and antidepressant-like effects in scopolamine-treated rats. Our results suggest that the P. peregrina essential oil inhalation ameliorates scopolamine-induced memory impairment, anxiety, and depression. Moreover, studies on the P. peregrina essential oil may open a new therapeutic window for the prevention of neurological abnormalities closely related to Alzheimer's disease.

Background Animal models remain at that time a reference tool to predict potential pulmonary adverse effects of nanomaterials in humans. However, in a context of reduction of the number of animals used in experimentation, there is a need for reliable alternatives. In vitro models using lung cells represent relevant alternatives to assess potential nanomaterial acute toxicity by inhalation, particularly since advanced in vitro methods and models have been developed. Nevertheless, the ability of in vitro experiments to replace animal experimentation for predicting potential acute pulmonary toxicity in human still needs to be carefully assessed. The aim of the study was to evaluate the differences existing between the in vivo and the in vitro approaches for the prediction of nanomaterial toxicity and to find advanced methods to enhance in vitro predictivity. For this purpose, rats or pneumocytes in co-culture with macrophages were exposed to the same poorly soluble and poorly toxic TiO2 and CeO2 nanomaterials, by the respiratory route in vivo or using more or less advanced methodologies in vitro. After 24 h of exposure, biological responses were assessed focusing on pro-inflammatory effects and quantitative comparisons were performed between the in vivo and in vitro methods, using compatible dose metrics. Results For each dose metric used (mass/alveolar surface or mass/macrophage), we observed that the most realistic in vitro exposure method, the air-liquid interface method, was the most predictive of in vivo effects regarding biological activation levels. We also noted less differences between in vivo and in vitro results when doses were normalized by the number of macrophages rather than by the alveolar surface. Lastly, although we observed similarities in the nanomaterial ranking using in vivo and in vitro approaches, the quality of the data-set was insufficient to provide clear ranking comparisons. Conclusions We showed that advanced methods could be used to enhance in vitro experiments ability to predict potential acute pulmonary toxicity in vivo. Moreover, we showed that the timing of the dose delivery could be controlled to enhance the predictivity. Further studies should be necessary to assess if air-liquid interface provide more reliable ranking of nanomaterials than submerged methods. Electronic supplementary material The online version of this article (10.1186/s12989-018-0260-6) contains supplementary material, which is available to authorized users.

One impediment to breastfeeding is the lack of information on the use of many drugs during lactation, especially newer ones. The principles of drug passage into breastmilk are well established, but have often not been optimally applied prospectively. Commonly used preclinical rodent models for determining drug excretion into milk are very unreliable because of marked differences in milk composition and transporters compared to those of humans. Measurement of drug concentrations in humans remains the gold standard, but computer modeling is promising. New FDA labeling requirements present an opportunity to apply modeling to preclinical drug development in place of conventional animal testing for drug excretion into breastmilk, which should improve the use of medications in nursing mothers.

Objectives The current global rates of obesity and type 2 diabetes are staggering. In order to implement effective management strategies, it is imperative to understand the mechanisms of obesity-induced insulin resistance and diabetes. Macrophage infiltration and inflammation of the adipose tissue in obesity is a well-established paradigm, yet the role of macrophages in muscle inflammation, insulin resistance and diabetes is not adequately studied. In this systematic review, we will examine the evidence for the presence of macrophages in skeletal muscle of obese humans and mice, and will assess the association between muscle macrophages and insulin resistance. We will identify published studies that address muscle macrophage content and phenotype, and its association with insulin resistance. We will search MEDLINE/PubMed, EMBASE, and Web of Science for eligible studies. Grey literature will be searched in ProQuest. Quality assessment will be conducted using the Systematic Review Centre for Laboratory Animal Experimentation risk of bias Tool for animal studies. Results The findings of this systematic review will shed light on immune-metabolic crosstalk in obesity, and allow the consideration of targeted therapies to modulate muscle macrophages in the treatment and prevention of diabetes. The review will be published in a peer-reviewed journal and presented at conferences. Electronic supplementary material The online version of this article (doi:10.1186/s13104-017-2686-6) contains supplementary material, which is available to authorized users.

The goal is to compare these engineered organs to animal models, with an eye towards replacing animal testing.

There is an urgent need for the development of alternative methods to replace animal testing for the prediction of repeat dose chemical toxicity. To address this need, the European Commission and Cosmetics Europe have jointly funded a research program for 'Safety Evaluation Ultimately Replacing Animal Testing.' The goal of this program was the development of in vitro cellular systems and associated computational capabilities for the prediction of hepatic, cardiac, renal, neuronal, muscle, and skin toxicities. An essential component of this effort is the choice of appropriate reference compounds that can be used in the development and validation of assays. In this review, we focus on the selection of reference compounds for liver pathologies in the broad categories of cytotoxicity and lipid disorders. Mitochondrial impairment, oxidative stress, and apoptosis are considered under the category of cytotoxicity, while steatosis, cholestasis, and phospholipidosis are considered under the category of lipid dysregulation. We focused on four compound classes capable of initiating such events, i.e., chemically reactive compounds, compounds with specific cellular targets, compounds that modulate lipid regulatory networks, and compounds that disrupt the plasma membrane. We describe the molecular mechanisms of these compounds and the cellular response networks which they elicit. This information will be helpful to both improve our understanding of mode of action and help in the selection of appropriate mechanistic biomarkers, allowing us to progress the development of animal-free models with improved predictivity to the human situation.

Many immune-mediated diseases like inflammatory bowel disease, multiple sclerosis, type 1 diabetes, asthma, and food allergy appeared to have increased in frequency in developed countries in the latter part of the twentieth century. Reports from less developed countries suggest that the "epidemic" of immune-mediated diseases now is spreading into these regions as well. The "hygiene hypothesis" was developed to partly explain this phenomenon. It has been proposed that modern-day sanitary living has altered our exposure to organisms that provided protection from these diseases in the past. Alternations in the composition of our intestinal flora and fauna could play a role. Helminths are a group of worm-like parasitic organisms that have adapted to live in various regions of their hosts. Epidemiological and some clinical data suggest that these organisms can protect people from developing immune-mediated diseases. Animal experimentation has shown that helminths stimulate the production of regulatory cytokines, activate regulatory T cells, and induce regulatory dendritic cells and macrophages. This could be the mechanism by which they protect the host from these diseases. Early clinical studies also suggest that helminths may prove useful for treating immunological diseases. More sophisticated clinical studies are underway, testing live helminth agents as therapeutic agents. Also, a strong effort is ongoing to discover the agents produced by helminths that modulate host immune responses with an eye on developing new, highly effective immune modulatory therapeutic agent.

Burn injury is a severe form of trauma affecting more than two million people in North America each year. Burn trauma is not a single pathophysiological event but a devastating injury that causes structural and functional deficits in numerous organ systems. Due to its complexity and the involvement of multiple organs, in vitro experiments cannot capture this complexity nor address the pathophysiology. In the past two decades, a number of burn animal models have been developed to replicate the various aspects of burn injury; to elucidate the pathophysiology and explore potential treatment interventions. Understanding the advantages and limitations of these animal models is essential for the design and development of treatments that are clinically relevant to humans. This review paper aims to highlight the common animal models of burn injury in order to provide investigators with a better understanding of the benefits and limitations of these models for translational applications. While many animal models of burn exist, we limit our discussion to the skin healing of mouse, rat, and pig. Additionally, we briefly explain hypermetabolic characteristics of burn injury and the animal model utilized to study this phenomena. Finally, we discuss the economic costs associated with each of these models in order to guide decisions of choosing the appropriate animal model for burn research.

A strong argument for the practice of animal testing in medical research is the potential benefit to patients in getting improved pain relief, minimising morbidity and mortality. However, patients’ opinions on the ethics of animal testing are seldom sought, despite their role as principal stakeholders. This study compared the attitudes of patients and researchers on animal testing. Focus-group interviews were held with patients suffering from chronic inflammatory diseases, resulting in a questionnaire that was distributed January–May 2011. The questionnaire was posted to patient members of the Swedish Rheumatism Association (n=1195) and to all scientific experts serving on Ethical Review Boards in Sweden (n=364), with response rates of 65 per cent and 60 per cent, respectively. Results show that patients hold a positive stance towards animal testing, but with many caveats, and the level of support is comparable to those held by the general public found in national surveys. A clear majority of researchers were positive towards animal testing, and large statistical differences between patients and researchers were found regarding their attitudes towards testing animals commonly held as pets (P

Background Sensitive diagnostic tools are necessary for the detection of Mycobacterium suricattae infection in meerkats (Suricata suricatta) in order to more clearly understand the epidemiology of tuberculosis and the ecological consequences of the disease in this species. We therefore aimed to develop a cytokine release assay to measure antigen-specific cell-mediated immune responses of meerkats. Results Enzyme-linked immunosorbent assays (ELISAs) were evaluated for the detection of interferon-gamma (IFN-γ) and IFN-γ inducible protein 10 (IP-10) in meerkat plasma. An IP-10 ELISA was selected to measure the release of this cytokine in whole blood in response to Bovigam® PC-HP Stimulating Antigen, a commercial peptide pool of M. bovis antigens. Using this protocol, captive meerkats with no known M. suricattae exposure (n = 10) were tested and results were used to define a diagnostic cut off value (mean plus 2 standard deviations). This IP-10 release assay (IPRA) was then evaluated in free-living meerkats with known M. suricattae exposure, categorized as having either a low, moderate or high risk of infection with this pathogen. In each category, respectively, 24.7%, 27.3% and 82.4% of animals tested IPRA-positive. The odds of an animal testing positive was 14.0 times greater for animals with a high risk of M. suricattae infection compared to animals with a low risk. Conclusion These results support the use of this assay as a measure of M. suricattae exposure in meerkat populations. Ongoing longitudinal studies aim to evaluate the value of the IPRA as a diagnostic test of M. suricattae infection in individual animals. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0927-x) contains supplementary material, which is available to authorized users.

The development of pluripotent cells that enable stem cell research (SCR) without destroying human embryos is now a leading priority for science. Public and political controversies associated with human embryonic SCR experienced in the recent past should be alleviated if scientists no longer need to harvest cells from human embryos. This research suggests however additional issues needing attention in order to gain the public's trust and support: the use of mouse embryos and the commercialisation of research. Using a representative sample of 2,800 Australians, and an experimental telephone survey design, this research compared levels and predictors of public support for stem cell research across three cell source conditions: human embryo (HE), mouse embryo (ME) and induced pluripotent cells (iPSCs). The results revealed that the public were significantly more likely to support research using iPSCs than HE and ME cells and public compared to private research (regardless of the cell source). There was no significant difference in support for HE compared to ME research, but the former was viewed as more likely to lead to accessible health care benefits and to be associated with more trustworthy scientists. The results of a multimediation structural equation model showed that the primary reason support for SCR significantly dropped in a private compared to public context (i.e., the commercialisation effect) was because public scientists were trusted more than private scientists. This effect was consistent across all three SCR materials, suggesting that the use of mouse embryos or even iPSCs will not reduce the publics' concern with commercialised science. The implications these results have for public acceptance of stem cell and animal research are discussed in relation to possible solutions such as increasing public awareness of the regulation of animal research and benefit sharing.

In order to determine whether a newly developed implant material conforms to the requirements of biocompatibility, it must undergo rigorous testing. To correctly interpret the results of studies on implant material osseointegration, it is necessary to have a sound understanding of all the testing methods. The aim of this overview is to elucidate the methods that are used for the experimental evaluation of the osseointegration of implant materials. In recent decades, there has been a constant proliferation of new materials and surface modifications in the field of dental implants. This continuous development of innovative biomaterials requires a precise and detailed evaluation in terms of biocompatibility and implant healing before clinical use. The current gold standard is in vivo animal testing on well validated animal models. However, long-term outcome studies on patients have to follow to finally validate and show patient benefit. No experimental set-up can provide answers for all possible research questions. However, a certain transferability of the results to humans might be possible if the experimental set-up is carefully chosen for the aspects and questions being investigated. To enhance the implant survival rate in the rising number of patients with chronic diseases which compromise wound healing and osseointegration, dental implant research on compromised animal models will further gain importance in future.

Modern society faces an inherent dilemma. In our globalized society, we are spoilt for choice by an ever-increasing number of products, many of which are made of new materials and compound mixtures. At the same time, as consumers we got accustomed to the idea of a life minimized for risk, including our own exposure to chemicals from the environment or to compounds present in and released from everyday products. Chemical safety testing bridges these obviously diverging interests, and the corresponding legislation has hence been tremendously extended (e.g., introduction of the European legislation REACH in 2007). However, the underlying regulatory toxicology still relies mainly on animal testing, which is relatively slow, expensive, and ethically arguable. Meanwhile, recent years have seen a surge in efforts to develop alternative testing systems and strategies. Expectations are particularly high for the applicability of stem cells as test systems especially for developmental toxicity testing in vitro. For the first time in history, test systems can be based on differentiating cells and tissue progenitors in culture, thus bringing the 'vision of toxicity testing in the 21st century' a step closer.

A hard-won political victory for primate research is at risk of unravelling in pockets of Europe.

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Despite the prevalence of problem gamblers and the ethical issues involved in studying gambling behavior with humans, few animal models of gambling have been developed. When designing an animal model it is necessary to determine if behavior in the paradigm is similar to human gambling. In human studies, response latencies following winning trials and near win trials are greater than those following clear losses. Weatherly and Derenne (Anal Gambl Behav 1:79-89, 2007) investigated whether this pattern was found with rats working in an animal analogue of slot machine gambling. They found a similar pattern of response latencies but the subjects' behavior did not come under control of the visual stimuli signalling the different outcomes. The animal model of slot machine gambling we used addressed procedural issues in Weatherly and Derenne's model and examined whether reinforcer magnitude and the presence of near win trials influenced response latency and resistance to extinction. Response latencies of the six female Norway Hooded rats varied as a function of reinforcer magnitude and the presence of near-win trials. These results are consistent with prior research and with the idea that near win trials serve as conditional reinforcers.

The last decade has been marked by the withdrawal from the market of several medicines whose use in patients has been associated with the development of torsade de pointes (TdP), a potentially life-threatening polymorphic tachycardia. In a few cases, TdP can degenerate into ventricular fibrillation and lead to sudden death, thus constituting a real problem of public health. The recently finalized ICH S7B guideline defines the prolongation of the QT interval on the electrocardiogram as the best biomarker for predicting the torsadogenic risk of a given compound. However, a growing body of evidence suggests that drugs' torsadogenic potential may not necessarily be proportional to their ability to prolong the QT interval. It is a dynamic combination of multiple predisposing factors and components rather than a single particular event that can trigger this particular tachycardia. Following recommendations of the guideline, pharmaceutical companies have intensively implemented methodologies to assess the possible risk of QT prolongation and TdP in humans. The main problem in cardiac safety pharmacology is how best to combine the capabilities of different methodologies with their strengths and limitations in order to detect the potential of one molecular entity to induce a lethal arrhythmia of very low clinical incidence. This publication will review the current methodologies, focusing on the alternative methods to animal experimentation, including an overview of cardiac modeling.

Background Health care workers (HCW) often perform, promote, and advocate use of public funds for animal research (AR); therefore, an awareness of the empirical costs and benefits of animal research is an important issue for HCW. We aim to determine what health-care-workers consider should be acceptable standards of AR methodology and translation rate to humans. Methods After development and validation, an e-mail survey was sent to all pediatricians and pediatric intensive care unit nurses and respiratory-therapists (RTs) affiliated with a Canadian University. We presented questions about demographics, methodology of AR, and expectations from AR. Responses of pediatricians and nurses/RTs were compared using Chi-square, with P 40% of the time; thought that misleading AR results should occur

Stroke is the second most common cause of death in people over 45 years of age in Colombia and is the leading cause of permanent disability worldwide. Cerebral ischemia is a stroke characterized by decreased blood flow due to the occlusion of one or more cerebral arteries, which can cause memory problems and hemiplegia or paralysis, among other impairments. The literature contains hundreds of therapies (invasive and noninvasive) that exhibit a neuroprotective effect when evaluated in animal models. However, in clinical trials, most of these drugs do not reproduce the previously demonstrated neuroprotective property, and some even have adverse effects that had not previously been detected in animal experimentation. Statins are drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol synthesis. Several studies have shown that statin therapy in an animal model of focal cerebral ischemia reduces infarct volume, as well as markers of neurodegeneration, activates neuronal survival pathways, and improves performance on learning and memory tests. Given the implied therapeutic benefit and the limited understanding of the mechanism of action of statins in brain repair, it is necessary to address the biochemical and tissue effects of these drugs on synaptic proteins, such as NMDA receptors, synaptic adhesion proteins, and cytoskeletal proteins; these proteins are highly relevant therapeutic targets, which, in addition to giving a structural account of synaptic connectivity and function, are also indicators of cellular communication and the integrity of the blood–brain barrier, which are widely affected in the long term post-cerebral infarct but, interestingly, are protected by statins when administered during the acute phase.

The history of the regulation of animal research is essentially the history of the emergence of meaningful social ethics for animals in society. Initially, animal ethics concerned itself solely with cruelty, but this was seen as inadequate to late 20(th)-century concerns about animal use. The new social ethic for animals was quite different, and its conceptual bases are explored in this paper. The Animal Welfare Act of 1966 represented a very minimal and in many ways incoherent attempt to regulate animal research, and is far from morally adequate. The 1985 amendments did much to render coherent the ethic for laboratory animals, but these standards were still inadequate in many ways, as enumerated here. The philosophy underlying these laws is explained, their main provisions are explored, and future directions that could move the ethic forward and further rationalize the laws are sketched.