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New Approach Methodologies (NAMs) that employ artificial intelligence (AI) for predicting adverse effects of chemicals have generated optimistic expectations as alternatives to animal testing. However, the major underappreciated challenge in developing robust and predictive AI models is the impact of the quality of the input data on the model accuracy. Indeed, poor data reproducibility and quality have been frequently cited as factors contributing to the crisis in biomedical research, as well as similar shortcomings in the fields of toxicology and chemistry. In this article, we review the most recent efforts to improve confidence in the robustness of toxicological data and investigate the impact that data curation has on the confidence in model predictions. We also present two case studies demonstrating the effect of data curation on the performance of AI models for predicting skin sensitisation and skin irritation. We show that, whereas models generated with uncurated data had a 7–24% higher correct classification rate (CCR), the perceived performance was, in fact, inflated owing to the high number of duplicates in the training set. We assert that data curation is a critical step in building computational models, to help ensure that reliable predictions of chemical toxicity are achieved through use of the models.

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.

Across the spectrum of industrial sectors, including pharmaceuticals, chemicals, personal care products, food additives and their associated regulatory agencies, there is a need to develop robust and reliable methods to reduce or replace animal testing. It is generally recognised that no single alternative method will be able to provide a one-to-one replacement for assays based on more complex toxicological endpoints. Hence, information from a combination of techniques is required. A greater understanding of the time and concentration-dependent mechanisms, underlying the interactions between chemicals and biological systems, and the sequence of events that can lead to apical effects, will help to move forward the science of reducing and replacing animal experiments. In silico modelling, in vitro assays, high-throughput screening, organ-on-a-chip technology, omics and mathematical biology, can provide complementary information to develop a complete picture of the potential response of an organism to a chemical stressor. Adverse outcome pathways (AOPs) and systems biology frameworks enable relevant information from diverse sources to be logically integrated. While individual researchers do not need to be experts across all disciplines, it is useful to have a fundamental understanding of what other areas of science have to offer, and how knowledge can be integrated with other disciplines. The purpose of this review is to provide those who are unfamiliar with predictive in silico tools, with a fundamental understanding of the underlying theory. Current applications, software, barriers to acceptance, new developments and the use of integrated approaches are all discussed, with additional resources being signposted for each of the topics.

Recently developed high-throughput in vitro assays in combination with computational models could provide alternatives to animal testing. The purpose of the present study was to model the plasma, hepatic, and renal pharmacokinetics of approximately 150 structurally varied types of drugs, food components, and industrial chemicals after virtual external oral dosing in rats and to determine the relationship between the simulated internal concentrations in tissue/plasma and their lowest-observed-effect levels. The model parameters were based on rat plasma data from the literature and empirically determined pharmacokinetics measured after oral administrations to rats carried out to evaluate hepatotoxic or nephrotic potentials. To ensure that the analyzed substances exhibited a broad diversity of chemical structures, their structure-based location in the chemical space underwent projection onto a two-dimensional plane, as reported previously, using generative topographic mapping. A high-throughput in silico one-compartment model and a physiologically based pharmacokinetic (PBPK) model consisting of chemical receptor (gut), metabolizing (liver), central (main), and excreting (kidney) compartments were developed in parallel. For 159 disparate chemicals, the maximum plasma concentrations and the areas under the concentration-time curves obtained by one-compartment models and modified simple PBPK models were closely correlated. However, there were differences between the PBPK modeled and empirically obtained hepatic/renal concentrations and plasma maximal concentrations/areas under the concentration-time curves of the 159 chemicals. For a few compounds, the lowest-observed-effect levels were available for hepatotoxicity and nephrotoxicity in the Hazard Evaluation Support System Integrated Platform in Japan. The areas under the renal or hepatic concentration-time curves estimated using PBPK modeling were inversely associated with these lowest-observed-effect levels. Using PBPK forward dosimetry could provide the plasma/tissue concentrations of drugs and chemicals after oral dosing, thereby facilitating estimates of nephrotoxic or hepatotoxic potential as a part of the risk assessment.

CYP enzyme induction is a sensitive biomarker for phenotypic metabolic competence of in vitro test systems; it is a key event associated with thyroid disruption, and a biomarker for toxicologically relevant nuclear receptor-mediated pathways. This paper summarises the results of a multi-laboratory validation study of two in vitro methods that assess the potential of chemicals to induce cytochrome P450 (CYP) enzyme activity, in particular CYP1A2, CYP2B6, and CYP3A4. The methods are based on the use of cryopreserved primary human hepatocytes (PHH) and human HepaRG cells. The validation study was coordinated by the European Union Reference Laboratory for Alternatives to Animal Testing of the European Commission's Joint Research Centre and involved a ring trial among six laboratories. The reproducibility was assessed within and between laboratories using a validation set of 13 selected chemicals (known human inducers and non-inducers) tested under blind conditions. The ability of the two methods to predict human CYP induction potential was assessed. Chemical space analysis confirmed that the selected chemicals are broadly representative of a diverse range of chemicals. The two methods were found to be reliable and relevant in vitro tools for the assessment of human CYP induction, with the HepaRG method being better suited for routine testing. Recommendations for the practical application of the two methods are proposed., Highlights • Paper summarises the results of a multi-laboratory validation study. • Two human derived metabolically competent test systems used. • In vitro methods validated as reliable and relevant for CYP enzyme induction assessment. • Two in vitro methods can support regulatory chemical risk assessment with HepaRG-based method better suited for routine chemical testing. • CYP enzyme induction biomarker for thyroid and nuclear receptor-mediated disruption.

Laboratory measurements of intrinsic clearance support the development of TK models, with potential relevance to weight of evidence toxicity assessments of xenobiotics, including read-across, the concept of predictive estimation by data extrapolation between chemicals of similar structure (analogues). In this work a procedure with analytical method for determination of in vitro hepatic metabolic clearance, relevant to biotransformation toxicokinetic (TK) modelling, is presented. Cryopreserved primary human hepatocytes represent a suitable cells, due to their biological characteristics, for providing an in vitro model for simulating in vivo metabolic clearance. The experimental part considered an adequate sequential time-frame for collecting samples and controls for all chemicals tested, including centrifugation and aliquoting of the corresponding fractions until the instrumental session. For the first time, in vitro hepatocyte intrinsic clearance was measured for six analogue test chemicals: valproic acid, 2-ethyl caproic acid, octanoic acid, valeric acid, 2-methyl butyric acid and 2-trans pentenoic acid, during incubated cell culture exposure up to 2 h or 3.5 h. The time dependence of any metabolism was determined from analysis of the supernatant at intervals using a new developed analytical method for UPLC coupled with QTOF mass spectrometer. The chemicals could then be ranked by their relative intrinsic clearance. The analyses were reproducible, with coherence of the calculated in vitro intrinsic clearance between experiments.

Millions of experimental animals are widely used in the assessment of toxicological or biological effects of manufactured nanomaterials in medical technology. However, the animal consciousness has increased and become an issue for debate in recent years. Currently, the principle of the 3Rs (i.e., reduction, refinement, and replacement) is applied to ensure the more ethical application of humane animal research. In order to avoid unethical procedures, the strategy of alternatives to animal testing has been employed to overcome the drawbacks of animal experiments. This article provides current alternative strategies to replace or reduce the use of experimental animals in the assessment of nanotoxicity. The currently available alternative methods include in vitro and in silico approaches, which can be used as cost-effective approaches to meet the principle of the 3Rs. These methods are regarded as non-animal approaches and have been implemented in many countries for scientific purposes. The in vitro experiments related to nanotoxicity assays involve cell culture testing and tissue engineering, while the in silico methods refer to prediction using molecular docking, molecular dynamics simulations, and quantitative structure–activity relationship (QSAR) modeling. The commonly used novel cell-based methods and computational approaches have the potential to help minimize the use of experimental animals for nanomaterial toxicity assessments.

Recently, alternatives to animal testing have been used to evaluate skin sensitisers in cosmetic products. However, testing is still complicated and expensive. To develop a simpler, cost-effective and more accurate evaluation method for the skin sensitising chemicals, we employed cell-based and RT-PCR-based assay. Representative sensitiser specific gene expression in THP-1 cells was analysed by microarray. Gene ontology (GO) analysis revealed that 26 genes induced by the sensitisers were associated with immune function. First, seven of the 26 genes were chosen arbitrarily as candidate markers for our sensitisation assay. Then, THP-1 cells were exposed to 13 reference chemicals with known sensitising potential, and real-time RT-PCR assays targeting the candidate marker genes were performed. Among them, six markers were able to properly evaluate the sensitisation potential by classifying the gene induction rates with appropriate criteria. Especially, the results of the assay using TREM1 and TNFRSF12A gene markers showed 100% sensitivity and specificity. An existing test method, h-CLAT, requires a flow cytometer and is complicated to operate. In contrast, our method is relatively simpler and more cost-effective. Therefore, our method is a promising one to evaluate sensitising chemicals.

To develop and evaluate scientifically robust and innovative approaches for the safety assessment of chemicals across multiple regulatory sectors, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) has started a project to explore how to better use the available information, including that from existing animal studies. The aim is to minimize reliance on in vivo testing to avoid redundancy and to facilitate the integration of novel non-animal methods in the regulatory setting with the ultimate goal of designing sustainable testing strategies. In this thought-starter paper, we present a number of examples to illustrate and trigger further discussions within the scientific and regulatory communities on ways to extrapolate useful information for predicting toxicity from one toxicity endpoint to another or across endpoints based on mechanistic information.

The zebrafish embryo test has been discussed as an alternative test system to provide data on acute fish toxicity required by diverse regulations. A meta-analysis of zebrafish embryo acute toxicity (ZFET) data has revealed conflicting evidence that narcotic compounds (i.e. compounds with baseline toxicity) may exhibit weaker sensitivity in the ZFET if compared to the acute (adult) fish toxicity test (AFT). Therefore, six compounds with presumably narcotic or unknown mode of action, and for which a previous meta-analysis has indicated weaker sensitivity were experimentally analysed for their fish embryo acute toxicity and exposure concentrations were monitored. The data indicated that ZFET and AFT for the selected compounds revealed similar sensitivity and differences were in the range of species differences of the AFT.

There is a very high rate of withdrawal of new drugs during or even after clinical trials, due to lack of efficacy or adverse side-effects not identified during expensive and time-consuming preclin...

Skin sensitisation is the regulatory endpoint that has been at the centre of concerted efforts to replace animal testing in recent years, as demonstrated by the Organisation for Economic Co-operation and Development (OECD) adoption of five non-animal methods addressing mechanisms under the first three key events of the skin sensitisation adverse outcome pathway. Nevertheless, the currently adopted methods, when used in isolation, are not sufficient to fulfil regulatory requirements on the skin sensitisation potential and potency of chemicals comparable to that provided by the regulatory animal tests. For this reason, a number of defined approaches integrating data from these methods with other relevant information have been proposed and documented by the OECD. With the aim to further enhance regulatory consideration and adoption of defined approaches, the European Union Reference Laboratory for Alternatives to Animal testing in collaboration with the International Cooperation on Alternative Test Methods hosted, on 4-5 October 2016, a workshop on the international regulatory applicability and acceptance of alternative non-animal approaches, i.e., defined approaches, to skin sensitisation assessment of chemicals used in a variety of sectors. The workshop convened representatives from more than 20 regulatory authorities from the European Union, United States, Canada, Japan, South Korea, Brazil and China. There was a general consensus among the workshop participants that to maximise global regulatory acceptance of data generated with defined approaches, international harmonisation and standardisation are needed. Potential assessment criteria were defined for a systematic evaluation of existing defined approaches that would facilitate their translation into international standards, e.g., into a performance-based Test Guideline. Informed by the discussions at the workshop, the ICATM members propose practical ways to further promote the regulatory use and facilitate adoption of defined approaches for skin sensitisation assessments.

The in vitro EpiSkin™ test method was validated in 2007 by the European Union Reference Laboratory for alternatives to animal testing (EURL ECVAM) as a full replacement method for the Draize acute skin irritation test and adopted in the OECD Test Guideline 439 in 2009. Based on the EpiSkin™ technology, the production of a reconstructed epidermis model has been established and standardized in China. The evaluation of the in vitro skin irritation test method using this EpiSkin™ model produced in China was performed on a set of 45 chemicals. Good predictive capacity was obtained with 94% (n=17) for sensitivity, 75% (n=28) for specificity and 82% for accuracy. The accuracy of the included 20 OECD reference chemicals also met the OECD acceptance criteria, indicating that this testing method based on the EpiSkin™ model produced in China can be used as a stand-alone test method to predict skin irritation. The availability and validity of in vitro epidermis model and testing method are of great significance for extending the applications of non-animal alternative testing methods in China.

In vitro methods that can replace animal testing in the identification of skin sensitisers are now a reality. However, as cell culture and related techniques usually rely on animal-derived products, these methods may be failing to address the complete replacement of animals in safety assessment. The objective of this study was to identify the animal-derived products that are used as part of in vitro methods for skin sensitisation testing. Thus, a systematic review of 156 articles featuring 83 different in vitro methods was carried out and, from this review, the use of several animal-derived products from different species was identified, with the use of fetal bovine serum being cited in most of the methods (78%). The use of sera from other animals, monoclonal antibodies and animal proteins were also variously mentioned. While non-animal alternatives are available and methods free of animal-derived products are emerging, most of the current methods reported used at least one animal-derived product, which raises ethical and technical concerns. Therefore, to deliver technically and ethically better in vitro methods for the safety assessment of chemicals, more effort should be made to replace products of animal origin in existing methods and to avoid their use in the development of new method protocols.

We would like to express strong concerns about the publication by Rodrigues et al., entitled: “Cell-based assays seem not to accurately predict fish short-term toxicity of pesticides”, which was recently published in “Environmental Pollution” (2019, 252, pages 476–482, accepted May 07/2019, available online May 27/2019). The topic of the paper is of great interest to the toxicology community because it addresses the need to define alternatives to animals in chemical risk assessment. The authors collected a large amount of in vitro data on chemical testing and added some of their own – the entire data set being predominantly focused on mammalian cell systems - and then attempted a comparison with data for the same chemicals regarding their toxicity to fish. Unfortunately, the work presented is flawed in several ways, sending an undifferentiated, if not wrong, message. Because we fear that this publication can cause unjustified damage to the achievements already made and to the ongoing efforts of the growing community in academia, industry and regulation to further alternatives to animal testing, we wish to openly discuss our concerns.

Amid growing efforts to advance the replacement, reduction, and refinement of the use of animals in research, there is a growing recognition that in vitro testing of medical devices can be more effective, both in terms of cost and time, and also more reliable than in vivo testing. Although the technological landscape has evolved rapidly in support of these concepts, regulatory acceptance of alternative testing methods has not kept pace. Despite the acceptance by regulators of some in vitro tests (cytotoxicity, gene toxicity, and some hemocompatibility assays), many toxicity tests still rely on animals (irritation, sensitization, acute toxicity, reproductive/developmental toxicity), even where other industrial sectors have already abandoned them. Bringing about change will require a paradigm shift in current approaches to testing - and a concerted effort to generate better data on risks to human health from exposure to leachable chemicals from medical devices, and to boost confidence in the use of alternative methods to test devices. To help advance these ideas, stir debate about best practices, and coalesce around a roadmap forward, the JHU-Center for Alternatives to Animal Testing (CAAT) hosted a symposium believed to be the first gathering dedicated to the topic of in vitro testing of medical devices. Industry representatives, academics, and regulators in attendance presented evidence to support the unique strengths and challenges associated with the approaches currently in use as well as new methods under development, and drew next steps to push the field forward from their presentations and discussion. published

The 21st century paradigm for toxicology and the adverse outcome pathway concept envisage a future toxicology largely based on mechanistic in vitro assays and relying mainly on cellular models. In the skin sensitisation field, this concept was not intuitive at the beginning. Given the high structural diversity of skin sensitising molecules, classical receptor binding as the molecular initiating event in a cell-based assay could be excluded from the start, leaving the question of how cells could sense potential skin sensitising chemicals and be able to differentiate them from non-sensitisers. When we entered this field in 2006, we realised that, in another emerging field of toxicology, detailed work on the antioxidant/electrophile sensing pathway Keap1/Nrf2/ARE was being performed. We postulated that, based on their intrinsic electrophilicity, a large structural variety of skin sensitisers would activate this pathway. This was demonstrated in a preliminary pilot study with an existing, breast cancer-derived reporter cell line. Broader confirmation of this initial hypothesis then came from a multitude of genome-wide studies, in which sensitiser-induced changes to the transcriptome were investigated. The results showed that this regulatory pathway is indeed the most common regulatory pathway activated by sensitisers at the gene expression level, and the underlying event in keratinocytes has become formalised as a Key Event in the Organisation for Economic Co-operation and Development (OECD) Adverse Outcome Pathway for sensitisation. These studies led to the development of the KeratinoSens® assay, which became the first cell-based in vitro test for skin sensitisation to be endorsed by a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) statement and an OECD Test Guideline. More recently, a number of studies have further developed this approach into 3-D skin models. Here, we review the underlying mechanism and the development of the KeratinoSens assay. We also present data on the stability of the assay over time, which is a key requirement for a cell-based biological assay to be endorsed in a regulatory context.

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 assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.

Acute oral toxicity is determined for regulatory hazard classification or non-classification. The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) recommends the following modules for acute oral toxicity testing: a) the use of the in vitro 3T3 Neutral Red Uptake (NRU) test to identify substances not requiring classification and to estimate starting doses for in vivo acute oral toxicity studies; and b) the use of data from sub-acute toxicity studies to identify substances not requiring classification. However, the application of these modules in a regulatory context would require a predefined, validated and formally accepted testing strategy and data interpretation procedure, which are not available. Furthermore, the application of the 3T3 NRU assay for starting dose estimations could in fact increase the number of animals used. Finally, only very few substances exist for which data from sub-acute or other repeated dose studies are available, but data from acute studies are not. Therefore, in practice, the prediction of acute toxicity by using sub-acute toxicity data is generally irrelevant. It could even lead to a risk of overdosing in the range-finding study, which may result in the death of many or all of the animals used.

In modern toxicology, substantial efforts are undertaken to develop alternative solutions for in vivo toxicity testing. The adverse outcome pathway (AOP) concept could facilitate knowledge-based safety assessment of chemicals that does not rely exclusively on in vivo toxicity testing. The construction of an AOP is based on understanding toxicological processes at different levels of biological organisation. Here, we present the developed AOP for liver fibrosis and demonstrate a linkage between hepatic injury caused by chemical protein alkylation and the formation of liver fibrosis, supported by coherent and consistent scientific data. This long-term process, in which inflammation, tissue destruction, and repair occur simultaneously, results from the complex interplay between various hepatic cell types, receptors, and signalling pathways. Due to the complexity of the process, an adequate liver fibrosis cell model for in vitro evaluation of a chemical’s fibrogenic potential is not yet available. Liver fibrosis poses an important human health issue that is also relevant for regulatory purposes. An AOP described with enough mechanistic detail might support chemical risk assessment by indicating early markers for downstream events and thus facilitating the development of an in vitro testing strategy. With this work, we demonstrate how the AOP framework can support the assembly and coherent display of distributed mechanistic information from the literature to support the use of alternative approaches for prediction of toxicity. This AOP was developed according to the guidance document on developing and assessing AOPs and its supplement, the users’ handbook, issued by the Organisation for Economic Co-operation and Development.

Introduction The increasing incidence of mental illnesses and neurodegenerative diseases results in a high demand for drugs targeting the central nervous system (CNS). These drugs easily reach the CNS, have a high affinity for CNS targets, and are prone to cause seizures as an adverse drug reaction. Current seizure liability assessment heavily depends on in vivo or ex vivo animal models and is therefore ethically debated, labor intensive, expensive, and not always predictive for human risk. Areas covered The demand for CNS drugs urges the development of alternative safety assessment strategies. Yet, the complexity of the CNS hampers reliable detection of compound-induced seizures. This review provides an overview of the requirements of in vitro seizure liability assays and highlights recent advances, including micro-electrode array (MEA) recordings using rodent and human cell models. Expert opinion Successful and cost-effective replacement of in vivo and ex vivo models for seizure liability screening can reduce animal use for drug development, while increasing the predictive value of the assays, particularly if human cell models are used. However, these novel test strategies require further validation and standardization as well as additional refinements to better mimic the human in vivo situation and increase their predictive value.

Current alternatives to animal testing methods for skin irritation evaluation such as reconstructed human epidermis models are not fully representing physiological response caused by skin irritants. Skin irritation is physiologically induced by the dilation and increased permeability of endothelial cells. Thus, our objectives were to mimic physiological skin irritation using a skin-on-a-chip model and compare predictive capacities with a reconstructed human epidermis model to evaluate its effectiveness. To achieve our goals, the skin-on-a-chip model, consisting of three layers representing the epidermal, dermal and endothelial components, was adapted. Cell viability was measured using the OECD TG 439 protocol for test substance evaluation. The tight junctions of endothelial cells were also observed and measured to assess physiological responses to test substances. These parameters were used to physiologically evaluate cell-to-cell interactions induced by test substances and quantify model accuracy, sensitivity, and specificity. Based on in vivo data, the classification accuracy of twenty test substances using a dual-parameter chip model was 80%, which is higher than other methods. Besides, the chip model was more suitable for simulating human skin irritation. Therefore, it is important to note that the dual-parameter chip model possesses an enhanced predictive capacity and could serve as an alternative to animal testing for skin irritation.

A sizeable proportion of drug attrition is due to drug-induced seizures. Current available animal models frequently fail to predict human seizure liability. Therefore, there is a need for in vitro alternatives, preferably based on human-derived neurons to circumvent interspecies translation. The increasing number of commercially available human induced pluripotent stem cell (hiPSC)-derived neuronal models holds great promise for replacing rodent primary cultures. We therefore tested three different hiPSC-derived neuronal models for their applicability for in vitro seizure liability assessment. Using immunofluorescent staining and multi-well micro-electrode arrays we show that all models develop functional neuronal networks that exhibit spontaneous activity and (network) bursting behavior. Developmental patterns differ between the models, probably due to differences in model composition and seeding density. Nevertheless, neuronal activity and (network) bursting can be reproducibly modulated with the seizurogenic compounds strychnine, picrotoxin (PTX) and 4-aminopyridine (4-AP). However, the sensitivity and degree of chemical-induced effects differs between the models, which can likely be explained by differences in seeding density, maturation and different ratios of inhibitory and excitatory cell types. Importantly, compared to rat primary cortical neurons, the hiPSC-derived neuronal models were equally, or even better in the case of 4-AP, suited to detect seizurogenicity. Overall, our data indicate that hiPSC-derived neuronal models may in the future be used as a first screening tool for in vitro seizure liability assessment. However, before hiPSC-derived neuronal models can fully replace animal experiments, more compounds should be tested and the available models must be further characterized to fully understand their applicability.

There is a growing need for alternatives to animal testing to derive biokinetic data for evaluating both efficacy and safety of chemicals. One such alternative is bottom-up physiologically-based biokinetic (PBK) modeling which requires only in vitro data. The primary objective of this study is to develop and validate bottom-up PBK models of 3 HMG-CoA reductase inhibitors: rosuvastatin, fluvastatin and pitavastatin. Bottom-up PBK models were built using the Simcyp® Simulator by incorporating in vitro transporter and metabolism data (Vmax, Jmax, Km, CLint) obtained from the literature and proteomics-based scaling factors to account for differences in transporters expression between in vitro systems and in vivo organs. Simulations were performed for single intravenous, single oral and multiple oral dose of these chemicals. The results showed that our bottom-up models predicted systemic exposure (AUC0h-t), maximum plasma concentration (Cmax), plasma clearance and time to reach Cmax (Tmax) within two-fold of the observed data, with the exception of parameters associated with multiple oral pitavastatin dosing and single oral fluvastatin dosing. Additional middle-out simulations were performed using animal distribution data to inform tissue-to-plasma equilibrium distribution ratios for rosuvastatin and pitavastatin. This improved the predicted plasma-concentration time profiles but did not significantly alter the predicted biokinetic parameters. Our study demonstrates that quantitative proteomics-based mechanistic in vitro-to-in vivo extrapolation (IVIVE) could account for downregulation of transporters in culture and predict whole organ clearances without empirical scaling. Hence, bottom-up PBK modeling incorporating mechanistic IVIVE could be a viable alternative to animal testing in predicting human biokinetics.

Investigative Toxicology describes the de-risking and mechanistic elucidation of toxicities, supporting early safety decisions in the pharmaceutical industry. Recently, Investigative Toxicology has contributed to a shift in pharmaceutical toxicology, from a descriptive to an evidence-based, mechanistic discipline. This was triggered by high costs and low throughput of Good Laboratory Practice in vivo studies, and increasing demands for adhering to the 3R (Replacement, Reduction and Refinement) principles of animal welfare. Outside the boundaries of regulatory toxicology, Investigative Toxicology has the flexibility to embrace new technologies, enhancing translational steps from in silico, in vitro to in vivo mechanistic understanding to eventually predict human response. One major goal of Investigative Toxicology is improving preclinical decisions, which coincides with the concept of animal-free safety testing. Currently, compounds under preclinical development are being discarded due to the use of inappropriate animal models. Progress in Investigative Toxicology could lead to humanized in vitro test systems and the development of medicines less reliant on animal tests. To advance this field a group of 14 European-based leaders from the pharmaceutical industry founded the Investigative Toxicology Leaders Forum (ITLF), an open, non-exclusive and pre-competitive group that shares knowledge and experience. The ITLF collaborated with the Centre for Alternatives to Animal Testing Europe (CAAT-Europe) to organize an "Investigative Toxicology Think-Tank", which aimed to enhance the interaction with experts from academia and regulatory bodies in the field. Summarizing the topics and discussion of the workshop, this article highlights Investigative Toxicology's position by identifying key challenges and perspectives. published

The acute toxicity 'six-pack' is a suite of tests for hazard identification and risk assessment, primarily conducted for the classification and labelling of industrial chemicals and agrochemicals. The 'six-pack' is designed to provide information on health hazards likely to arise from short-term exposure to chemicals via inhalation, oral and dermal routes, including the potential for eye and skin irritation/corrosion and skin sensitization. The component tests of the 'six-pack' currently rely heavily on the use of experimental animals. In 2017, the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), together with the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM), and the US National Toxicology Program (NTP) Centre for the Evaluation of Alternative Methods (NICEATM) held a workshop entitled 'Towards Global Elimination of the Acute Toxicity 'Six-Pack'' to explore opportunities to use alternative (non-animal) methods for hazard identification and classification without compromising human or environmental safety. The Workshop included scientists from regulatory agencies and industrial organisations worldwide, and sought to gain a more detailed understanding of the barriers to the adoption of suitable animal-free alternatives at an international level. Among the issues addressed were: the recurring theme of validation and scientific credibility, as well as the need for international standards, an understanding of the limitations of each new/alternative method and characterisation against the variability of current animal methods. The practicality and cost of new tests was also an important consideration. However, the need for mutual acceptance, and global harmonization of requirements were thought to be the major hurdle to overcome to realise a vision of the eventual complete elimination of the current, animal test-based, acute toxicity 'six-pack'.

Changes in chemical regulations worldwide have increased the demand for new data on chemical safety. New approach methodologies (NAMs) are defined broadly here as including in silico approaches and in chemico and in vitro assays, as well as the inclusion of information from the exposure of chemicals in the context of hazard [European Chemicals Agency, " New Approach Methodologies in Regulatory Science ", 2016]. NAMs for toxicity testing, including alternatives to animal testing approaches, have shown promise to provide a large amount of data to fill information gaps in both hazard and exposure. In order to increase experience with the new data and to advance the applications of NAM data to evaluate the safety of data-poor chemicals, demonstration case studies have to be developed to build confidence in their usability. Case studies can be used to explore the domains of applicability of the NAM data and identify areas that would benefit from further research, development, and application. To ensure that this science evolves with direct input from and engagement by risk managers and regulatory decision makers, a workshop was convened among senior leaders from international regulatory agencies to identify common barriers for using NAMs and to propose next steps to address them. Central to the workshop were a series of collaborative case studies designed to explore areas where the benefits of NAM data could be demonstrated. These included use of in vitro bioassays data in combination with exposure estimates to derive a quantitative assessment of risk, use of NAMs for updating chemical categorizations, and use of NAMs to increase understanding of exposure and human health toxicity of various chemicals. The case study approach proved effective in building collaborations and engagement with regulatory decision makers and to promote the importance of data and knowledge sharing among international regulatory agencies. The case studies will be continued to explore new ways of describing hazard (i.e., pathway perturbations as a measure of adversity) and new ways of describing risk (i.e., using NAMs to identify protective levels without necessarily being predictive of a specific hazard). Importantly, the case studies also highlighted the need for increased training and communication across the various communities including the risk assessors, regulators, stakeholders (e.g., industry, non-governmental organizations), and the general public. The development and application of NAMs will play an increasing role in filling important data gaps on the safety of chemicals, but confidence in NAMs will only come with learning by doing and sharing in the experience.

We investigated the applicability of the ES-D3 cell differentiation assay combined with the in vitro BeWo transport model to predict the relative in vivo developmental toxicity potencies. To this purpose, the in vitro developmental toxicity of five antifungal compounds was investigated by characterizing their inhibitory effect on the differentiation of ES-D3 cells into cardiomyocytes. The BeWo transport model, consisting of BeWo b30 cells grown on transwell inserts and mimicking the placental barrier, was used to determine the relative placental transport velocity. The ES-D3 cell differentiation data were first compared to benchmark doses (BMDs) for in vivo developmental toxicity as derived from data reported in the literature. Correlation between the benchmark concentration for 50% effect (BMCd50) values, obtained in the ES-D3 cell differentiation assay, with in vivo BMD10 values showed a reasonable correlation (R2 = 0.57). When the ES-D3 cell differentiation data were combined with the relative transport rates obtained from the BeWo model, the correlation with the in vivo data increased (R2 = 0.95). In conclusion, we show that the ES-D3 cell differentiation assay is able to better predict the in vivo developmental toxicity ranking of antifungal compounds when combined with the BeWo transport model, than as a stand-alone assay.

A seminar and interactive workshop on “In silico Methods – Computational Alternatives to Animal Testing” was held in Berlin, Germany, organized by Annemarie Lang, Frank Butt- gereit and Andrea Volkamer at the Charite-Universitatsmedizin Berlin, on August 17-18, 2017. During the half-day seminar, the variety and applications of in silico methods as alternatives to animal testing were presented with room for scientific discus- sions with experts from academia, industry and the German fed- eral ministry (Fig. 1). Talks on computational systems biology were followed by detailed information on predictive toxicology in order to display the diversity of in silico methods and the potential to embrace them in current approaches (Hartung and Hoffmann, 2009; Luechtefeld and Hartung, 2017). The follow- ing interactive one-day Design Thinking Workshop was aimed at experts, interested researchers and PhD-students interested in the use of in silico as alternative methods to promote the 3Rs (Fig. 2). Forty participants took part in the seminar while the workshop was restricted to sixteen participants.

Alternatives to animal testing for quality control of biologicals have been a goal since 1959. Instituto Butantan has been developing such methods for quality control of biologicals for human use (vaccines and hyperimmune equine sera) for the last 13 years. In this paper we compare the modified ToBI test and the in vivo seroneutralization test to assess immunogenicity of diphtheria and tetanus vaccines and hyperimmune sera. Data from the last 10 years were statistically analyzed to compare the results for in vivo and in vitro titrations (diphtheria, n = 525 and tetanus, n = 455). The agreement between the tests depended on the serum titer range. For both diphtheria and tetanus components, the correlation and concordance coefficient was higher as the serum titer increased. Overall, the in vitro/in vivo titer ratio did not vary systematically over the range of measurements. These results indicate that although the in vitro ToBI test is not completely able to replace the in vivo serum titration, it is a useful tool to guide the tests during the production process, which can reduce the number of animals used for lot release.

Evaluating the safety of newly expressed proteins in genetically modified (GM) crops is conducted prior to commercialization to determine whether they could present a hazard upon consumption. A multicomponent, weight of evidence approach has been applied to individual proteins that has often included acute oral toxicology studies. Based on resources required to produce and purify the proteins, the number of animals necessary for these studies and the fact that no evidence of hazard has been observed for any of the proteins tested to date, it is questionable whether acute toxicology studies should be conducted for all proteins. This article reviews the chronology of the acute toxicology study from its origins into application for hazard assessment and classification of individual substances including proteins expressed in GM crops. It further proposes that a physiologic approach using cultured intestinal epithelial cell (IEC) line monolayers as an in vitro model of the gastrointestinal system provides results relevant to the hazard characterization of proteins when necessary. Benefits of this approach would include reduced quantities of proteins for testing and minimization or elimination of animal studies while maintaining confidence in the safety assessment process.

Rapidly evolving technological methods and mechanistic toxicological understanding have paved the way for new science-based approaches for the determination of chemical safety in support of advancing public health. Approaches including read-across, high-throughput screening, in silico models, and organ-on-a-chip technologies were addressed in a 2017 workshop focusing on how scientists can move effectively toward a vision for 21st century food safety assessments. The application of these alternative methods, the need for further development of standardized practices, and the interpretation and communication of results were addressed. Expert presentations encompassed regulatory, industry, and academic perspectives, and the workshop culminated in a panel discussion in which participants engaged experts about current issues pertaining to the application of alternative methods in toxicological testing for food safety assessments.

The sensitizing potential of chemicals is usually identified and characterized using in vivo methods such as the murine local lymph node assay (LLNA). Due to regulatory constraints and ethical concerns, alternatives to animal testing are needed to predict skin sensitization potential of chemicals. For this purpose, combined evaluation using multiple in vitro and in silico parameters that reflect different aspects of the sensitization process seems promising. We previously reported that LLNA thresholds could be well predicted by using an artificial neural network (ANN) model, designated iSENS ver.1 (integrating in vitro sensitization tests version 1), to analyze data obtained from two in vitro tests: the human Cell Line Activation Test (h-CLAT) and the SH test. Here, we present a more advanced ANN model, iSENS ver.2, which additionally utilizes the results of antioxidant response element (ARE) assay and the octanol-water partition coefficient (LogP, reflecting lipid solubility and skin absorption). We found a good correlation between predicted LLNA thresholds calculated by iSENS ver.2 and reported values. The predictive performance of iSENS ver.2 was superior to that of iSENS ver.1. We conclude that ANN analysis of data from multiple in vitro assays is a useful approach for risk assessment of chemicals for skin sensitization.

The Cosmetics Europe (formerly COLIPA) Genotoxicity Task Force has driven and funded three projects to help address the high rate of misleading positives in in vitro genotoxicity tests: The completed “False Positives” project optimized current mammalian cell assays and showed that the predictive capacity of the in vitro micronucleus assay was improved dramatically by selecting more relevant cells and more sensitive toxicity measures. The on-going “3D skin model” project has been developed and is now validating the use of human reconstructed skin (RS) models in combination with the micronucleus (MN) and Comet assays. These models better reflect the in use conditions of dermally applied products, such as cosmetics. Both assays have demonstrated good inter- and intra-laboratory reproducibility and are entering validation stages. The completed “Metabolism” project investigated enzyme capacities of human skin and RS models. The RS models were shown to have comparable metabolic capacity to native human skin, confirming their usefulness for testing of compounds with dermal exposure. The program has already helped to improve the initial test battery predictivity and the RS projects have provided sound support for their use as a follow-up test in the assessment of the genotoxic hazard of cosmetic ingredients in the absence of in vivo data.

Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1min for liquid chemicals or 24h for solid chemicals, and the post-exposure incubation periods were set at 24h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test.

The Japanese Center for the Validation of Alternative Methods (JaCVAM) was established in 2005 to promote the use of alternatives to animal testing in regulatory studies, thereby replacing, reducing, or refining the use of animals, according to the Three Rs principles. JaCVAM assesses the utility, limitations and suitability for use in regulatory studies, of test methods needed to determine the safety of chemicals and other materials. JaCVAM also organises and performs validation studies of new test methods, when necessary. In addition, JaCVAM co-operates and collaborates with similar organisations in related fields, both in Japan and internationally, which also enables JaCVAM to provide input during the establishment of guidelines for new alternative experimental methods. These activities help facilitate application and approval processes for the manufacture and sale of pharmaceuticals, chemicals, pesticides, and other products, as well as for revisions to standards for cosmetic products. In this manner, JaCVAM plays a leadership role in the introduction of new alternative experimental methods for regulatory acceptance in Japan.

Skin sensitization is the process by which a substance induces an allergic reaction following skin contact. The process has been described as an adverse outcome pathway (AOP), including several key events, from skin penetration and covalent protein binding, to keratinocyte activation, dendritic cell activation and T-lymphocyte proliferation. The in vitro assay KeratinoSens™ measures the activation of keratinocytes. It is fully accepted at a regulatory level (OECD TG 442d) and complies with a range of legislation including the EU Cosmetics Regulation, REACH, and the CLP Regulation. Currently, many in vitro methods use animal-derived components in their cell culture systems. Many stakeholders in the cosmetics industry have both scientific and ethical concerns relating to this issue and have stated a strong preference for fully human in vitro test systems. We have adapted the KeratinoSens™ method to animal product-free condi­tions, and carried out an in-house validation with 21 reference substances, including those listed in the Performance Standards associated with OECD TG442d. The modified method was shown to be equivalent to the Validated Ref­erence Method (VRM), with comparable values for accuracy (85.7%), sensitivity (84.6%) and specificity (87.5%), and all acceptance criteria being met. In Europe, data generated by the adapted method may be used in REACH submissions, and we are now seeking approval to list the adaptation in OECD TG 442d, enabling formal compliance with a range of global regulations.

The objective of the present work was to review the availability and predictive value of non-animal toxicokinetic approaches and to evaluate their current use in European risk evaluations of food contaminants, additives and food contact materials, as well as pesticides and medicines. Results revealed little use of quantitative animal or human kinetic data in risk evaluations of food chemicals, compared with pesticides and medicines. Risk evaluations of medicines provided sufficient in vivo kinetic data from different species to evaluate the predictive value of animal kinetic data for humans. These data showed a relatively poor correlation between the in vivo bioavailability in rats and dogs versus that in humans. In contrast, in vitro (human) kinetic data have been demonstrated to provide adequate predictions of the fate of compounds in humans, using appropriate in vitro-in vivo scalers and by integration of in vitro kinetic data with in silico kinetic modelling. Even though in vitro kinetic data were found to be occasionally included within risk evaluations of food chemicals, particularly results from Caco-2 absorption experiments and in vitro data on gut-microbial conversions, only minor use of in vitro methods for metabolism and quantitative in vitro-in vivo extrapolation methods was identified. Yet, such quantitative predictions are essential in the development of alternatives to animal testing as well as to increase human relevance of toxicological risk evaluations. Future research should aim at further improving and validating quantitative alternative methods for kinetics, thereby increasing regulatory acceptance of non-animal kinetic data.

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.

On 30 June 2011, the European Chemicals Agency published two reports, one on the functioning of the REACH system, the other on the use of alternatives to animal testing in compliance with that system. The data presented are based on information gained during the first registration period under the REACH system, which included high production volume chemicals and substances of very high concern, which have the most extensive information requirements. A total of 25,460 registration dossiers were received, covering 3,400 existing, so-called ‘phase-in’, substances, and 900 new, so-called ‘non-phase-in’, substances. Data sharing and the joint submission of data are reported to have worked successfully. In the registration dossiers for these substances, results from new animal tests were included for less than 1% of all the endpoints; testing proposals (required for ‘higher-tier’ information requirements) were submitted for 711 in vivo tests involving vertebrate animals. The registrants mainly used old, existing experimental data, or options for the adaptation (waiving) of information requirements, before collecting new information. For predicting substance toxicity, ‘read-across’ was the second most-used approach, followed by ‘weight-of-evidence’. In vitro toxicity tests played a minor role, and were only used when the respective test methods had gained the status of regulatory acceptance. All in all, a successful start to the REACH programme was reported, particularly since, in contrast to most predictions, it did not contribute to a significant increase in toxicity testing in animals.

The present paper aims at identifying strategies to increase the impact and applicability of alternative testing strategies in risk assessment. To this end, a quantitative and qualitative literature evaluation was performed on (a) current research efforts in the development of in vitro methods aiming for alternatives to animal testing, (b) the possibilities and limitations of in vitro methods for regulatory purposes and (c) the potential of physiologically-based kinetic (PBK) modeling to improve the impact and applicability of in vitro methods in risk assessment practice. Overall, the evaluation showed that the focus of state-of-the-art research activities does not seem to be optimally directed at developing in vitro alternatives for those endpoints that are most animal-demanding, such as reproductive and developmental toxicity, and carcinogenicity. A key limitation in the application of in vitro alternatives to such systemic endpoints is that in vitro methods do not provide so-called points of departure, necessary for regulators to set safe exposure limits. PBK-modeling could contribute to overcoming this limitation by providing a method that allows extrapolation of in vitro concentration-response curves to in vivo dose-response curves. However, more proofs of principle are required. (C) 2011 Elsevier Inc. All rights reserved.

Policy on the Humane Care and Use of Laboratory Animals and it supports educational programs and monitors compli­ ance. This is a very important part of what we do involving our assured institutions and the PHS funding components. It ensures the humane use of animals in our research, in our testing and training, and contributes to the quality of what we do in our research. NIH also supports animal model systems and alterna­ tives. I want to say a bit more about our intramural animal care and use program and extramural support of animal re­ search. Virtually all of our institutes that have an intramural component—24 of them—have animals in their research programs. Almost 50% of the NIH-funded grants and con­ tracts that we support involve animals. As this is a major part of the research program here at NIH it is very important that we have appropriate compliance and oversight of this pro­ cess. For the intramural program in 2009, we used 1.3 mil­ lion animals; 81% of them were mice but there were more than 20 different species—fish and frogs, rats, guinea pigs, rabbits, hamsters, nonhuman primates, birds, dogs, cats, pigs, and sheep. There’s a variety of animals supported here in the intramural program, and NIH has a number of resources available if you are looking for model organisms for bio­ medical research. Our website provides information on model organisms, both mammalian and nonmammalian models in­ cluding Arabidopsis, the mustard plant. We also have a number of programs that support the de­ velopment of nonmammalian models and the improvement of mammalian models. At the National Center for Research Resources (NCRR) the Biological Models and Materials Re­ search Program supports research to develop and broaden the utility of models including cell culture, nonmammalian model organisms, and nonbiological systems such as math­ ematical modeling or computer modeling. These are exam­ ples of our interest in alternatives to the use of animals. The NCRR Laboratory Animal Science Program supports animal research–related resources and research training through awards and grants contracts and cooperative agree­ ments. Through its Division of Comparative Medicine, NCRR awards animal research grants and resource grants to develop, characterize, and improve mammalian animal models for hu­ man disease and study. NIH is interested in both improving mammalian model systems and offering nonmammalian and nonanimal alternatives. The NLM has another resource, the Bibliography on Alternatives to Animal Testing (ALTBIB), which [includes] a search engine to identify resources on al­ ternatives to the use of live vertebrates in biomedical research and testing. On the ALTBIB web page you can identify the sources, put in keywords, and find available alternatives. Much of the program for today focuses on biomedical re­ search using animal models and how it has advanced knowledge to find prevention, cures, and treatments for the many serious diseases and conditions of humans and, of course, of animals.

For the assessment of genotoxic effects of cosmetic ingredients, a number of well-established and regulatory accepted in vitro assays are in place. A caveat to the use of these assays is their relatively low specificity and high rate of false or misleading positive results. Due to the 7th amendment to the EU Cosmetics Directive ban on in vivo genotoxicity testing for cosmetics that was enacted March 2009, it is no longer possible to conduct follow-up in vivo genotoxicity tests for cosmetic ingredients positive in in vitro genotoxicity tests to further assess the relevance of the in vitro findings. COLIPA, the European Cosmetics Association, has initiated a research programme to improve existing and develop new in vitro methods. A COLIPA workshop was held in Brussels in April 2008 to analyse the best possible use of available methods and approaches to enable a sound assessment of the genotoxic hazard of cosmetic ingredients. Common approaches of cosmetic companies are described, with recommendations for evaluating in vitro genotoxins using non-animal approaches. A weight of evidence approach was employed to set up a decision-tree for the integration of alternative methods into tiered testing strategies.

This report presents some of the results of a joint research project, sponsored by Defra and conducted by FRAME and Liverpool John Moores University, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This report focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for skin sensitisation testing. The manuscript reviews in vitro tests based on protein-ligand binding, dendritic/Langerhans cells and T-lymphocyte activation, and also the QSAR models and expert systems available for this endpoint. These tests are then incorporated into an integrated, decision-tree testing strategy, which also includes the Local Lymph Node Assay (in its original and new reduced protocols) and the traditional guinea-pig tests (which should only be used as a last resort). The aim of the strategy is to minimise the use of animals in testing for skin sensitisation, while satisfying the scientific and logistical demands of the EU REACH legislation.

This paper presents some results of a joint research project conducted by FRAME and Liverpool John Moores University, and sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity end-points associated with REACH. This paper focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for repeat dose (sub-acute, sub-chronic and chronic) toxicity testing. It reviews the limited number of in silico and in vitro tests available for this endpoint, and outlines new technologies which could be used in the future, e.g. the use of biomarkers and the ‘omics’ technologies. An integrated testing strategy is proposed, which makes use of as much non-animal data as possible, before any essential in vivo studies are performed. Although none of the non-animal tests are currently undergoing validation, their results could help to reduce the number of animals required for testing for repeat dose toxicity.

Liverpool John Moores University and FRAME recently conducted a research project sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This paper focuses on the prospects for using alternative methods (both in vitro and in silico) for mutagenicity (genotoxicity) and carcinogenicity testing — two toxicity endpoints, which, together with reproductive toxicity, are of pivotal importance for the REACH system. The manuscript critically discusses well-established testing approaches, and in particular, the requirement for short-term in vivo tests for confirming positive mutagenicity, and the need for the rodent bioassay for detecting non-genotoxic carcinogens. Recently-proposed testing strategies focusing on non-animal approaches are also considered, and our own testing scheme is presented and supported with background information. This scheme makes maximum use of pre-existing data, computer ( in silico) and in vitro methods, with weight-of-evidence assessments at each major stage. The need for the improvement of in vitro methods, to reduce the generation of false-positive results, is also discussed. Lastly, ways in which reduction and refinement measures can be used are also considered, and some recommendations are made for future research to facilitate the implementation of the proposed testing scheme.

The use of fish embryos is not regulated by current legislations on animal welfare and is therefore considered as a refinement, if not replacement of animal experiments. Fish embryos represent an attractive model for environmental risk assessment of chemicals since they offer the possibility to perform small-scale, high-throughput analyses. Beyond their application for determining the acute toxicity, fish embryos are also excellent models for studies aimed at the understanding of toxic mechanisms and the indication of possible adverse and long-term effects. Therefore, we have reviewed the scientific literature in order to indicate alternative applications of the fish embryo model with focus on embryos of the zebrafish. The analysis of the mode of action is important for the risk assessment of environmental chemicals and can assist in indicating adverse and long-term effects. Toxicogenomics present a promising approach to unravel the potential mechanisms. Therefore, we present examples of the use of zebrafish embryos to study the effect of chemicals on gene and protein patterns, and the potential implications of differential expression for toxicity. The possible application of other methods, such as kinase arrays or metabolomic profiling, is also highlighted. Furthermore, we show examples of toxicokinetic studies (bioconcentration, ABC transporters) and discuss limitations that might be caused by the potential barrier function of the chorion. Finally, we demonstrate that biomarkers of endocrine disruption, immune modulation, genotoxicity or chronic toxicity could be used as indicators or predictors of sub-acute and long-term effects. The zebrafish embryo represents a model with an impressive range of possible applications in environmental sciences. Particularly, the adaptation of molecular, system-wide approaches from biomedical research is likely to extend its use in ecotoxicology. Challenges for future research are (1) the identification of further suitable molecular markers as indicators of the mode of action, (2) the establishment of strong links between (molecular) effects in short-term assays in embryos and long-term (toxic) effects on individuals, (3) the definition of limitations of the model and (4) the development of tests that can be used for regulatory purposes.

Liverpool John Moores University and FRAME conducted a research project, sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This paper focuses on the prospects for the use of alternative methods (both in vitro and in silico) in developmental and reproductive toxicity testing. It considers many tests based on primary cells and cell lines, and the available expert systems and QSARs for developmental and reproductive toxicity, and also covers tests for endocrine disruption. Ways in which reduction and refinement measures can be used are also discussed, particularly the use of an enhanced one-generation reproductive study, which could potentially replace the two-generation study, and therefore considerably reduce the number of animals required in reproductive toxicity. Decision-tree style integrated testing strategies are also proposed for developmental and reproductive toxicity and for endocrine disruption, followed by a number of recommendations for the future facilitation of developmental and reproductive toxicity testing, with respect to human risk assessment.