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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.

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of harmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.

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.

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.

and hospitalization. Lancaster and colleagues discuss a case in which a patient died, who did not receive N-acetylcysteine according to the ‘200 μg/ml line’. Therefore, it became standard in the UK to treat any patient with serum APAP equivalent to only 100 μg/ml at 4 h after intoxication (Lancaster et al. 2015). However, because the majority of patients with an overdose do not show such serve consequences, the new UK standard means that one life is saved every 2 years associated with costs of 17.3 million euros per saved life. This underlines the urgent need of better predictive biomarkers (Lancaster et al. 2015). The article of Lancaster et al. (2015) is also of relevance for the field of alternative methods (Hammad 2013; Ghallab 2013, 2014a, b; Reif 2014a, b; Godoy 2011). Currently, intensive research activities are ongoing to establish in vitro systems particularly for hepatotoxicity (Grinberg et al. 2014; Ramaiahgari et al. 2014; Rodrigues et al. 2013; Schug et al. 2013), nephrotoxicity (Sanchez-Nino et al. 2014; Fujiki et al. 2013; 2014), neurotoxicity (Barbosa et al. 2014; Sisnaiske et al. 2014; Frimat et al. 2010) and developmental toxicity (Krug et al. 2013; Rempel et al. 2015; Balmer et al. 2014; Zimmer et al. 2014). For validation of in vitro systems with hepatocytes APAP represents a popular reference compound (Jennings et al. 2014; Hengstler et al. 2014). However, it can often be observed that in vitro studies used very high APAP concentrations in the range of 10 mM or even higher. The review of Lancaster et al. (2015) reminds us of the time-honored ‘200 μg/ml line’, which means that a serum concentration of 200 μg APAP/ml 4 h after oral uptake represents a threshold above which the risk of acute liver failure significantly increases. Considering the molecular weight of APAP of 151.16 g/ mol this corresponds to a threshold of 1.3 mM. It should also be considered that due to a half-life of 1–4 h in human, concentrations in vivo decrease much faster than in vitro. Acetaminophen (APAP) represents the leading cause of liver failure in Europe (Lancaster et al. 2015). Much effort has been invested to gain a better understanding of the molecular mechanisms of APAP hepatotoxicity (Saito et al. 2010; Kon et al. 2004; Hwang et al. 2015; Sjogren et al. 2014; Schyschka et al. 2013; Singh et al. 2013) and to identify biomarkers of APAP overdose (McGill et al. 2014; Beger et al. 2015). APAP is known to be metabolically activated to the reactive N-acetyl-p-benzoquinone imine (NAPQI), which forms protein adducts including mitochondrial proteins leading to mitochondrial oxidative stress (Ramachandran et al. 2013; Cohen et al. 1997). As a consequence c-jun N-terminal kinase is translocated to the mitochondria, which enhances generation of reactive oxygen species (Ramachandran et al. 2013; Hanawa et al. 2008; Saito et al. 2010). This may lead to opening of the mitochondrial membrane transition pore and cause a form of cell death which differs from classical apoptosis and has been named ‘necroptosis’ (Kon et al. 2004; Gujral et al. 2002; Ni et al. 2012). Although cytochrome c is released from the mitochondria, for so far unknown reasons apoptotic cell death is not induced (Gujral et al. 2002; Lawson et al. 1999; Williams et al. 2010). In a recent issue of the Archives of Toxicology, Lancaster and colleagues have published an updated review on diagnosis and clinical management of APAP-induced liver failure (Lancaster et al. 2015). One of the most urgent clinical research fields seems to be the identification of biomarkers that predict more reliably whether a patient is in need of treatment

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commissions Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 79 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 57 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

Qing et al. 2013; Doricakova et al. 2013; Ren et al. 2013). In recent years, a strong increase in the number of publications has been observed aiming to establish and improve in vitro systems (Ireno et al. 2014; Seeliger et al. 2013; Godoy 2011). The most cited example in Archives of Toxicology in 2013 was the successful development of embryonic stem cell-based test systems that recapitulate specific phases of human development during which the in vitro system can be exposed to the test compounds (Krug et al. 2013a). Several further studies based on in vitro systems with neurons or neuronal precursor cells have been published (van Thriel 2011; Krug et al. 2013b; Hoelting et al. 2013; Da Silva et al. 2013; Bolt 2013). A particularly high number of in vitro studies focus on hepatotoxicity (e.g., Hadi et al. 2013; Schaap et al. 2012; Burkhardt et al. 2012). Goals of these studies are to improve differentiated functions by 3D cultivation conditions (Messner et al. 2013; Schyschka et al. 2013) to study metabolites (Sierra-Santoyo et al. 2012; Watzek et al. 2013; Abdelhamid et al. 2013; Tolosa et al. 2013) and drug transporters hepatocytes (Wassermann et al. 2013). Moreover, zebrafish-based test systems have been studied as an alternative method to identify hepatotoxic compounds (Driessen et al. 2013; Scholz 2013). Compared to the high number of studies based on in vitro systems the fraction of in vivo studies published in the Archives of Toxicology has become relatively small (Rossato et al. 2013; Yu et al. 2013; Early et al. 2013; Cordova et al. 2013; Saito et al. 2013; Hammad et al. 2014). Moreover, studies systematically comparing the functions of in vitro systems to the in vivo situation are rare (Schug et al. 2013; Heise et al. 2012). This leads to a situation where huge sets of data including omics analyses are available but a limitation is that the in vivo relevance particularly the relationships to adverse effects remain unclear. Bridging this gap will be a major challenge for toxicological research in future. At the end of each year, the Archives of Toxicology analyzes the most intensively studied fields of research published in our journal. This includes particularly research topics where important progress beyond the state of the art has been achieved. Moreover, the editors analyze the topics to which most manuscripts have been submitted and where most citations have been counted, try to identify the present state of the art and give recommendations how further progress beyond the state of the art can be achieved. Of course, these analyses represent individual opinions and have no influence which manuscripts will be published in future; in this regard, only scientific quality is of relevance. Clearly, in vitro systems represent one of the most popular topics in our journal (Godoy et al. 2013; Azqueta and Collins 2013; Hessel et al. 2013; Jiang et al. 2013; Liu et al. 2013; Fraczek et al. 2013; Leist and Hartung 2013). This seems to represent a general trend in toxicological sciences (Weng et al. 2014; Waldmann et al. 2014; Karlsson et al. 2013; Onrubia et al. 2013; Lake et al. 2013; Houston et al. 2012; Hardelauf et al. 2011; Ilowski et al. 2011; Gabriel et al. 2012; Hammad et al. 2013) but also in other disciplines of biomedical research (Zellmer et al. 2010; Godoy et al. 2009, 2010a, b; Ilowski et al. 2010; Meyer et al. 2011). In the Archives of Toxicology, in vitro systems are used to study mechanisms of action of toxic compounds, a traditional strategy since decades (Chen et al. 2013; Qu et al. 2013

Alternative testing methods have become increasingly important within EU funding policy. The driving force behind this push for alternative testing methods is the urgent need for effective and robust alternative toxicity tests in order to make the tools available that are necessary to comply with the 3R-strategy (Replacement, Reduction, Refinement) that has been built into the European Cosmetic (Directive 2003/15/EC) and Chemical (REACH) (Regulation EC No 1907/2006) legislations. Indeed, a general European policy has to take into consideration the growing ethical concern of the public with respect to the use of experimental animals in the risk assessment process of consumer products. It is reasonable to believe that further development and strategic incorporation of recent progress in different areas of scientific research could lead to a better and more mechanistically based approach of studying toxicological problems. In line with this, the pharmaceutical industry is confronted with unexpected failures during the drug development process, even late during the clinical phase. These could not be properly anticipated. Reasons are in particular safety (hepatoand cardiotoxicity) as well clinical efficacy of the new chemical entities. Therefore, incorporation of novel alternative methods, human based and metabolically functional, is high on the priority list since the competitiveness of the European pharmaceutical industry is at stake. An overview of the different EU initiatives in the context of the 3Rs funded research is given in EUR22846 (European Commission 2008) and in the update EUR23886 (European Commission 2009). The EU offers four different funding schemes: (1) Collaborative/Integrated projects, which are medium/large-sized ambitious projects, usually involving several areas of expertise, (2) Specific Targeted Research Projects (STREP), representing smaller networks focussed on specific research issues, (3) SME-STREP, aimed at supporting research of smalland medium-sized enterprises (SMEs), and (4) Specific Support Actions (SSA), for scientific training of personal as well as organization of conferences, workshops and expert meetings (EUR22846, EUR23886). The research networks fall into the categories of high-throughput techniques, specific toxicity tests and organization of forums and workshops. T. Vanhaecke (&) S. Snykers V. Rogiers Department of Toxicology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium e-mail: tamara.vanhaecke@vub.ac.be

The development of alternative methods to experimental animals in safety studies is at the leading edge of the current toxicological research (Seiler et al. 2011; Driessen et al. 2013; Lilienblum et al. 2008; Krug et al. 2013; Aritomi et al. (2014); Dias da Silva et al. (2013); Guo et al. (2014); Knobeloch et al. (2012)). Relevant in vitro systems for hepatotoxicity studies are of particular importance (Godoy et al. 2013; Schyschka et al. 2013; Rodrigues et al. 2013; Tolosa et al. 2013; Messner et al. 2013; Hewitt et al. 2007; Ufelmann and Schrenk 2014). Therefore, the wellchosen set of compounds that represent known mechanisms of hepatotoxicity that are presented by Jennings et al. (2014) will facilitate the systematic establishment of in vitro systems for hepatotoxicity. Nevertheless, the bar could be raised even higher in the future. One pertinent question is how comprehensive testing of the recommended compounds brings us beyond the current state of the art. We will certainly learn whether the gold standard compounds are able to activate certain mechanisms of toxicity in the exposed cells. Specific patterns of deregulated genes or proteins will be associated with specific compounds and will provide evidence of the involved biological processes. At best, this strategy will allow hazard identification; however, toxicology requires quantitative information. To aid regulatory processes, in vitro systems must ideally predict in vivo doses that are safe and those that would result in adverse effects. Simply having the knowledge that a particular concentration (often very high) activates a characteristic mechanism of toxicity in vitro will not be sufficient. Despite intensive research in the field of alternative methods, some challenges are still currently underestimated. For example, a response induced by a compound in January 1, 2011, rang in the start of the European Union’s ambitious initiative to ‘replace in vivo repeated dose systemic toxicity testing.’ SEURAT-1, shortened for Safety Evaluation Ultimately Replacing Animal Testing, was launched as the first step in the gargantuan FP7 research initiative to change the way that chemical safety is assessed by providing more accurate in vitro testing methods and improved predictive tools. In the present issue of the Archives of Toxicology, the editors would like to specially highlight the review article by member scientists of SEURAT-1 who comprehensively summarized SEURAT-1 liver gold standard compounds (Jennings et al. 2014). The authors have selected a particularly useful set of compounds to support the development of in vitro systems for the identification of hepatotoxic compounds, while simultaneously reviewing their known mechanisms of action. Briefly, the following principles were addressed

compounds often lead to hyperplasia of hepatocytes. Therefore, the novel method presented by Hammad and colleagues provides a potentially powerful tool to quantify aspects of liver microarchitecture that could prove indispensable in toxicological pathology. Currently, much effort is being invested into the establishment of alternative methods to characterize organ toxicity (Godoy et al. 2009, 2013; Godoy 2011; Hammad et al. 2013, 2014; Hengstler et al. 2012; Hewitt et al. 2007; Schug et al. 2013; Rodrigues et al. 2013; Messner et al. 2013; Bolt 2013; Frey et al. 2014). However, it is also becoming more obvious that fully replacing animal experiments within the next decade may not be realistic (Ghallab 2013; Adler et al. 2011; Lilienblum et al. 2008; Campos et al. 2014). Therefore, methods to refine animal experiments should not be neglected, and the current standardized imaging and image processing techniques represent a plausible approach. Future experiments investigating diseased tissue as well as tissue after exposure to toxic compounds must show that the results obtained with tissue microarchitecture analyses can support histological diagnosis.

Drug metabolism represents a cutting edge topic in toxicology with a particular focus on toxification versus detoxification of xenobiotics (Oesch et al. 2014; Rodriguez-Mateos et al. 2014; Dohnal et al. 2014), contribution of drug metabolism to oxidative stress (Xiao et al. 2014; Moller et al. 2014; Wu et al. 2014), and regulation of metabolic activity (Juricek et al. 2014; Bitter et al. 2014; Lim et al. 2014; Braeuning 2014). Moreover, recent research focuses on integrating metabolic pathways into mathematical models in order to predict the metabolic capacity of tissues and organs under physiological and disease conditions (Drasdo et al. 2014a, b; Schliess et al. 2014; Hoehme et al. 2010). Finally, compromised xenobiotic metabolism represents one of the major problems in alternative methods and in vitro systems (Godoy et al. 2009, 2013, 2015; Pfeiffer et al. 2015; Heise et al. 2012; Ghallab 2013, 2014a, b; Stewart 2010). The present review of El-Sherbeni and ElKadi represents a must-read for anyone interested in these fields of research.

Every summer, the editors of the Archives of Toxicology analyse their most cited articles from the previous 2 years. Topping this year’s list as the most cited original article is the pharmacokinetic and biodistribution study on silica nanoparticles (Xie et al. 2010). This study demonstrates that silica nanoparticles are retained for more than 30 days in various organs of mice (Table 1). The high interest and citation rates of this systematic biodistribution study illustrate the importance and relevance of nanoparticles to the field of toxicology today. The second most cited original article is the study by Drobna et al. (2010) on the metabolism and transport of inorganic arsenic. The high interest generated by the results of this study is not surprising. Arsenic poisoning, as a result of drinking water from contaminated wells, represents one of the largest man-made catastrophes in recent history (Golka et al. 2010). A study on the mechanism of action of silver nanoparticles ranks third in this citation analysis (Table 1). In addition to original work, review articles were also included into our analysis. In the last 2 years, the most cited review, and coincidentally the most cited article, proposed a systems biology concept of an autocatalytic generation of oxidative stress, which is intensified by positive feedback loops and plays a central role in several degenerative diseases and toxic insults (Kell 2010). Following the review of Kell et al. is a comprehensive summary of the mechanisms of action and toxicological relevance of the mycotoxin, deoxynivalenol, also named ‘vomitoxin’ (Pestka 2010). The review of Adler et al. (2011) on in vitro systems ranks third, illustrating that the development of alternative methods currently represents a cutting-edge topic in toxicology. The present citation analysis does not, by any means, represent a ranking of the scientific quality of our articles. However, it identifies the fields of research that attract the most attention and, therefore, may be a sensitive indicator of how our discipline could evolve in the future.

During the last two decades, substantial efforts have been made towards the development and international acceptance of alternative methods to safety studies using laboratory animals. In the EU, challenging timelines for phasing out of many standard tests using laboratory animals were established in the seventh Amending Directive 2003/15/EC to Cosmetics Directive 76/768/EEC. In continuation of this policy, the new European Chemicals Legislation (REACH) favours alternative methods to conventional in vivo testing, if validated and appropriate. Even alternative methods in the status of prevalidation or validation, but without scientific or regulatory acceptance may be used under certain conditions. Considerable progress in the establishment of alternative methods has been made in some fields, in particular with respect to methods predicting local toxic effects and genotoxicity. In more complex important fields of safety and risk assessment such as systemic single and repeated dose toxicity, toxicokinetics, sensitisation, reproductive toxicity and carcinogenicity, it is expected that the development and validation of in silico methods, testing batteries (in vitro and in silico) and tiered testing systems will have to overcome many scientific and regulatory obstacles which makes it extremely difficult to predict the outcome and the time needed. The main reasons are the complexity and limited knowledge of the biological processes involved on one hand and the long time frame until validation and regulatory acceptance of an alternative method on the other. New approaches in safety testing and evaluation using “Integrated Testing Strategies” (ITS) (including combinations of existing data, the use of chemical categories/grouping, in vitro tests and QSAR) that have not been validated or not gained wide acceptance in the scientific community and by regulatory authorities will need a thorough justification of their appropriateness for a given purpose. This requires the availability of knowledge and experience of experts in toxicology. The challenging deadlines for phasing out of in vivo tests in the Cosmetics Amending Directive 2003/15/EC appear unrealistic. Likewise, we expect that the application of validated alternative methods will only have a small or moderate impact on the reduction of in vivo tests under the regimen of REACH, provided that at least the same level of protection of human health as in the past is envisaged. As a consequence, under safety aspects, it appears wise to consider established in vivo tests to be indispensable as basic tools for hazard and risk assessment with respect to systemic single and repeated dose toxicity, sensitisation, carcinogenicity and reproductive toxicity, especially regarding quantitative aspects of risk assessment such as NOAELs, LOAELs and health-related limit values derived from them. Based on the overall evaluation in this review, the authors are of the opinion that in the short- and mid-term, the strategy of the development of alternative methods should be more directed towards the refinement or reduction of in vivo tests. The lessons learnt during these efforts will provide a substantial contribution towards the replacement initiatives in the long-term.

Since some years, our journal initiated a series of review articles on cutting-edge topics in the field of toxicology. Ten of the most cited and downloaded articles are summarized in Table 1. The focus of the articles published in 2012 is oxidative stress, toxicity of zinc, amphetamines, pyrethroid insecticides, cisplatin and nanomaterials. Moreover, the role of estrogen receptors in human disease and alternative methods for skin sensitization have been covered. A key feature of our review articles is that our authors are not limited by strict word counts. Comprehensive manuscripts that cover an entire research field are encouraged. We hope that this type of review is of particular interest to our readers and we are looking forward to further contributions.

A dermal acute toxic class (ATC) method is presented with the use of significantly fewer animals in comparison with the classical dermal 50% lethal dose (LD50) test. The principle of the dermal ATC method is based on the oral and the inhalation ATC method. The method was developed for three fixed starting doses. Depending on the dermal LD50, the slope, the classification system and the starting dose on average 40 to 90% fewer animals will be used in comparison to at least 30 animals with the dermal LD50 test. The method was biometrically evaluated by using the Probit model for dose-response relationships. At present, there are eight different international classification systems based on dermal LD50 values. The test procedures and the calculations of the classification probabilities demonstrate that the dermal ATC method is a reliable alternative to the dermal LD50 test with the use of significantly fewer animals. Classification probabilities are presented for all classification systems currently in use, and expected numbers of experimental and of moribund/dead animals are demonstrated for the system of chemicals in the European Union for all three starting doses. The conclusion is justified that, similarly to the inhalation ATC method, there is no need to validate the dermal ATC method with the use of experimental animals.

An alternative to the oral LD50 test, the acute toxic class (ATC) method (oral), was validated with 20 substances in an international collaborative study with nine laboratories in five countries. The ATC method is a stepwise procedure with the use of three animals per step. It has been designed with three fixed doses (25, 200 and 2000 mg/kg). In general, this testing is sufficient for allocation to the toxicity classes of the majority of the international classification systems currently in use. The selection of testing at additional fixed doses (5, 50 and 500 mg/kg) may be considered if further refinement is necessary or for specific allocation to those international classification systems with a cut-off value of 5 mg/kg. On average, two to four steps are necessary to complete a test. With the ATC method substances can be ranked in a similar or even better manner than with an LD50 test but it uses up to 90% fewer animals, the average being 70% fewer. This also results in substantially fewer moribund/dead animals. The ATC method is based on biometric evaluations that, together with the experimental results, demonstrate that this method is a sensitive and reliable alternative to the LD50 test.

On the occasion of the 20th anniversary of the Center for Alternative Methods to Animal Experiments (ZEBET), an international symposium was held at the German Federal Institute for Risk Assessment (BfR) in Berlin. At the same time, this symposium was meant to celebrate the 50th anniversary of the publication of the book "The Principles of Humane Experimental Technique" by Russell and Burch in 1959 in which the 3Rs principle (that is, Replacement, Reduction, and Refinement) has been coined and introduced to foster the development of alternative methods to animal testing. Another topic addressed by the symposium was the new vision on "Toxicology in the twenty-first Century", as proposed by the US-National Research Council, which aims at using human cells and tissues for toxicity testing in vitro rather than live animals. An overview of the achievements and current tasks, as well as a vision of the future to be addressed by ZEBET@BfR in the years to come is outlined in the present paper.

A thorough understanding of which of the effects assessed in the in vivo Draize eye test are responsible for driving UN GHS/EU CLP classification is critical for an adequate selection of chemicals to be used in the development and/or evaluation of alternative methods/strategies and for properly assessing their predictive capacity and limitations. For this reason, Cosmetics Europe has compiled a database of Draize data (Draize eye test Reference Database, DRD) from external lists that were created to support past validation activities. This database contains 681 independent in vivo studies on 634 individual chemicals representing a wide range of chemical classes. A description of all the ocular effects observed in vivo, i.e. degree of severity and persistence of corneal opacity (CO), iritis, and/or conjunctiva effects, was added for each individual study in the database, and the studies were categorised according to their UN GHS/EU CLP classification and the main effect driving the classification. An evaluation of the various in vivo drivers of classification compiled in the database was performed to establish which of these are most important from a regulatory point of view. These analyses established that the most important drivers for Cat 1 Classification are (1) CO mean ≥ 3 (days 1–3) (severity) and (2) CO persistence on day 21 in the absence of severity, and those for Cat 2 classification are (3) CO mean ≥ 1 and (4) conjunctival redness mean ≥ 2. Moreover, it is shown that all classifiable effects (including persistence and CO = 4) should be present in ≥60 % of the animals to drive a classification. As a consequence, our analyses suggest the need for a critical revision of the UN GHS/EU CLP decision criteria for the Cat 1 classification of chemicals. Finally, a number of key criteria are identified that should be taken into consideration when selecting reference chemicals for the development, evaluation and/or validation of alternative methods and/or strategies for serious eye damage/eye irritation testing. Most important, the DRD is an invaluable tool for any future activity involving the selection of reference chemicals. Electronic supplementary material The online version of this article (doi:10.1007/s00204-016-1679-x) contains supplementary material, which is available to authorized users.