The topic of Integrated Testing Strategies (ITS) has attracted considerable attention, and not only because it is supposed to be a central element of REACH, the ambitious European chemical regulation effort. Although what ITSs are supposed to do seems unambiguous, i.e. speeding up hazard and risk assessment while reducing testing costs, not much has been said, except basic conceptual proposals, about the methodologies that would allow execution of these concepts. Although a pressing concern, the topic of ITS has drawn mostly general reviews, broad concepts, and the expression of a clear need for more research on ITS. Published research in the field remains scarce. Solutions for ITS design emerge slowly, most likely due to the methodological challenges of the task, and perhaps also to it its complexity and the need for multidisciplinary collaboration. Along with the challenge, ITS offer a unique opportunity to contribute to the Toxicology of the 21st century by providing frameworks and tools to actually implement 21st century toxicology data in the chemical management and decision making processes. Further, ITS have the potential to significantly contribute to a modernization of the science of risk assessment. Therefore, to advance ITS research we propose a methodical approach to their design and will discuss currently available approaches as well as challenges to overcome. To this end, we define a framework for ITS that will inform toxicological decisions in a systematic, transparent, and consistent way. We review conceptual requirements for ITS developed earlier and present a roadmap to an operational framework that should be probabilistic, hypothesis-driven, and adaptive. Furthermore, we define properties an ITS should have in order to meet the identified requirements and differentiate them from evidence synthesis. Making use of an ITS for skin sensitization, we demonstrate how the proposed ITS concepts can be implemented.

The efforts of the committee that produced the report on Toxicity Testing in the 21st Century reflected the need to look at the issue of assessing risks to humans from exposure to various chemicals through a lens of 21st century biology. The problem – determining if there is a risk of specific exposures – is as old as humanity; every generation brings its own perspective and tools for examining the problem and coming to answers and solutions. Bringing this generation’s tools to bear requires us to see the problem of chemical risk assessment in a different light, both in terms of testing of toxicity pathways in vitro and in the interpretation of the tests for estimating whether exposures will be safe. One key issue will be to assess when pathway perturbations are believed to be excessive, i.e., when they are deemed adverse. Redefinition of adversity based on in vitro testing will require a new perception of dose response functions as probabilities of failures, with multiple underlying processes acting sequentially and in parallel leading to failure at a cellular and an organism level. These dose response relationships for adversity will also require a computational systems biology approach for examining toxicity pathway dynamics and stress pathway overload. While the overall approach of defining adversity for in vitro endpoints and using this definition of adversity for risk assessment can be painted in broad brush strokes, as we have done here for DNA-reactive compounds, it will take implementation with a series of prototypes to show the process in practice.

Validation has become a primary driver of the evolution of toxicological methods. Agreement at OECD level currently requires validation of new approaches for consideration in test guideline development. Several examples of this exist. However, the toxicology in the 21st century movement, prompted by the 2007 NRC/NAS vision document, might lead to a revolutionary change in the toxicological toolbox. The challenge is whether the validation process, as it has been formalized over the last two decades, meets the needs for this paradigm shift.
The concept of evidence-based medicine (EBM) has emerged from clinical medicine, which retrospectively assesses the evidence of adequacy of a given approach. This is not typically done in prospective studies – the equivalent of validation studies might be multicenter randomized trials. Evidently, where such unambiguous evidence is available, no other assessment is necessary. EBM, however, has developed procedures, including meta-analysis, to collect and evaluate all the available evidence where no such definitive study is available. The recent successful introduction of retrospective validation, i.e. the collection and evaluation of existing evidence from various sources, represents a step in this direction. Here, we will explore new toxicological approaches via evidence-based toxicology (EBT).

“Endocrine disruption” is a public and political buzzword that has and is still receiving high media attention. Based on the latter, numerous tiered testing strategies have evolved that should ensure that humans will not run a health risk due to the voluntary or involuntary exposure to endocrine active compounds (EAS). An analysis of the currently available knowledge on EAS mediated endocrine disruption in humans demonstrates that there are very few EAS that causally induce endocrine disruptive effects. Conversely, the association EAS exposure with increased risk or incidences of endocrine disruptive effects in humans are difficult to reconcile with the results from animal studies. Consequently, the analysis of the traditional and historically grown tiered approach in EAS testing, often at very high doses or concentrations, demonstrates that the likelihood of detecting EAS with true potential for endocrine disruption in humans is very low, primarily due to inherent differences between the surrogate species and the human, and will provide for a high number of false-positives commensurate with low efficiency, high cost, and often violently disputed interpretations of what the data would mean for human risk assessment.
It is thus proposed that EAS testing for putative endocrine disruption in humans and qualitative and quantitative evaluation for risk assessment purposes should be entirely focused on human data, and derived from a combination of in silico and in vitro systems, PBPK modeling, metabonomic or genomic profiling of human tissue, realistic human EAS exposure, dose-effect principles and adverse effect scenarios, human patient or exposure cohort datasets, etc. Animals models should be used only where specific pathways in endocrine physiology and thus development and reproduction is nearly identical to the situation in the human, thereby guaranteeing that causal exposure and effect relationships in the animals can be extrapolated to the human.

On 8^th September 2010 the long process of revising the EU Directive for the protection of laboratory animals was concluded. Here a comparative evaluation of the new and old Directive is provided. While its ultimate goal is to replace the use of animals, the new Directive acknowledges that animals, including nonhuman primates, are still needed for scientific purposes today. Importantly, animals have an intrinsic value, which must be respected. There are some major advances for animal welfare, many of which had however already been common practice in the more progressive Member States. The new Directive prohibits new, more progressive legislation if not already in place and thus harmonises but also freezes the 27 Member States at a relatively high level. The revision was an important opportunity for the European Commission, on the one side to demonstrate its commitment to improve human health and safety by enabling animal testing and on the other side to improve animal health and welfare by setting minimum standards. By this Directive Europe is again taking a leading role in research and development for new non-animal tests and technologies by introducing a series of measures that strengthen the evaluation of the need of animal use in each case. It also represents a formal implementation of the 3Rs principle (Replacement, Reduction and Refinement of animal tests) put forward by Russel and Burch 1959.

Americans consider dogs and cats as household pets, but many are harmed and killed for teaching and training purposes, despite the availability of alternatives. A review of 92 U.S. public college and university Institutional Animal Care and Use Committee (IACUC) 2005-2007 records indicates that 52% are using live and dead dogs and cats, and 26% are using live dogs and cats in harmful teaching exercises in undergraduate life science, veterinary, and medical education. In specific cases, IACUCs are failing to minimize animal use and suffering in education as required by the Animal Welfare Act (AWA). Sources of dogs and cats for education include Class A and Class B dealers, and United States Department of Agriculture (USDA) 2005-2007 inspections reveal repeated violations and inhumane treatment. Regardless, dealers continue to sell thousands of dogs and cats, many whom were former pets, annually to universities for use in education. A growing number of universities, however, are changing their policies and replacing harmful animal use with pedagogically sound alternatives.

The development and application of new test systems and test methods is central to the progress of in vitro toxicology. In order to live up to the future challenges, it is important to use the vast knowledge of adjoining fields, such as cell biology or developmental biology, and to attract specialists from such areas to develop new methods. Not all of them bring with them the necessary experience and training required for the development of toxicological test systems. Thus, promising new biological test systems sometimes still require additional considerations to become successful. Sometimes even the referees of scientific journals and their editors seem to lack judgement guidelines for minimum performance standards. Here we provide a list of points to be considered for the establishment of a test method. The chapters range from the explanation of the meaning of positive controls, performance standards or signal-noise ratios to a discussion of statistical considerations, suitable solvents and data display formats. The considerations are simple and expressed in a non-mathematical format, with a strong focus on plausibility and common sense. The major intention of this article is to provide a compilation of important issues requiring consideration. Whether they apply to a specific system and whether action is required must be determined by individual judgement.