Search

Implementation of reliable methodologies allowing Reduction, Refinement, and Replacement (3Rs) of animal testing is a process that takes several decades and is still not complete. Reliable methods are essential for regulatory hazard assessment of chemicals where differences in test protocol can influence the test outcomes and thus affect the confidence in the predictive value of the organisms used as an alternative for mammals. Although test guidelines are common for mammalian studies, they are scarce for non-vertebrate organisms that would allow for the 3Rs of animal testing. Here, we present a set of 30 reporting criteria as the basis for such a guideline for Developmental and Reproductive Toxicology (DART) testing in the nematode Caenorhabditis elegans. Small organisms like C. elegans are upcoming in new approach methodologies for hazard assessment; thus, reliable and robust test protocols are urgently needed. A literature assessment of the fulfilment of the reporting criteria demonstrates that although studies describe methodological details, essential information such as compound purity and lot/batch number or type of container is often not reported. The formulated set of reporting criteria for C. elegans testing can be used by (i) researchers to describe essential experimental details (ii) data scientists that aggregate information to assess data quality and include data in aggregated databases (iii) regulators to assess study data for inclusion in regulatory hazard assessment of chemicals. [ABSTRACT FROM AUTHOR]

Data in a toxicity test are evaluated generally per parameter. Information on the response per animal in addition to per parameter can improve the evaluation of the results. The results from the six studies in rats, described in the paper by Kemmerling, J., Fehlert, E., Rühl-Fehlert, C., Kuper, C.F., Stropp, G., Vogels, J., Krul, C., Vohr, H.-W., 2015. The transferability from rat subacute 4-week oral toxicity study to translational research exemplified by two pharmaceutical immunosuppressants and two environmental pollutants with immunomodulating properties (In this issue), have been subjected to principal component analysis (PCA) and principal component discriminant analysis (PC-DA). The two pharmaceuticals azathioprine (AZA) and cyclosporine A (CSA) and the two environmental pollutants hexachlorobenzene (HCB) and benzo(a)pyrene (BaP) all modulate the immune system, albeit that their mode of immunomodulation is quite diverse. PCA illustrated the similarities between the two independent studies with AZA (AZA1 and AZA2) and CSA (CSA1 and CSA2). The PC-DA on data of the AZA2 study did not increase substantially the information on dose levels. In general, the no-effect levels were lower upon single parameter analysis than indicated by the distances between the dose groups in the PCA. This was mostly due to the expert judgment in the single parameter evaluation, which took into account outstanding pathology in only one or two animals. The PCA plots did not reveal sex-related differences in sensitivity, but the key pathology for males and females differed. The observed variability in some of the control groups was largely a peripheral blood effect. Most importantly, PCA analysis identified several animals outside the 95% confidence limit indicating high-responders; also low-to-non-responders were identified. The key pathology enhanced the understanding of the response of the animals to the four model compounds. [ABSTRACT FROM AUTHOR]

Exposure to chemicals may have an influence on the immune system. Often, this is an unwanted effect but in some pharmaceuticals, it is the intended mechanism of action. Immune function tests and in depth histopathological investigations of immune organs were integrated in rodent toxicity studies performed according to an extended OECD test guideline 407 protocol. Exemplified by two immunosuppressive drugs, azathioprine and cyclosporine A, and two environmental chemicals, hexachlorobenzene and benzo[a]pyrene, results of subacute rat studies were compared to knowledge in other species particular in humans. Although immune function has a high concordance in mammalian species, regarding the transferability from rodents to humans various factors have to be taken into account. In rats, sensitivity seems to depend on factors such as strain, sex, stress levels as well as metabolism. The two immunosuppressive drugs showed a high similarity of effects in animals and humans as the immune system was the most sensitive target in both. Hexachlorobenzene gave an inconsistent pattern of effects when considering the immune system of different species. In some species pronounced inflammation was observed, whereas in primates liver toxicity seemed more obvious. Generally, the immune system was not the most sensitive target in hexachlorobenzene-treatment. Immune function tests in rats gave evidence of a reaction to systemic inflammation rather than a direct impact on immune cells. Data from humans are likewise equivocal. In the case of benzo[a]pyrene, the immune system was the most sensitive target in rats. In the in vitro plaque forming cell assay (Mishell–Dutton culture) a direct comparison of cells from different species including rat and human was possible and showed similar reactions. The doses in the rat study had, however, no realistic relation to human exposure, which occurs exclusively in mixtures and in a much lower range. In summary, a case by case approach is necessary when testing immunotoxicity. Improvements for the translation from animals to humans related to immune cells can be expected from in vitro tests which offer direct comparison with reactions of human immune cells. This may lead to a better understanding of results and variations seen in animal studies. [ABSTRACT FROM AUTHOR]

The two-generation study (OECD TG 416) is the standard requirement within REACH to test reproductive toxicity effects of chemicals with production volumes >100 tonnes. This test is criticized in terms of scientific relevance and animal welfare. The Extended One Generation Reproductive Toxicity Study (EOGRTS), incorporated into the OECD test guidelines in 2011 (OECD TG 443) has the potential to replace TG 416, while using only one generation of rats and being more informative. However, its regulatory acceptance proved challenging. This article reconstructs the process of regulatory acceptance and use of the EOGRTS and describes drivers and barriers influencing the process. The findings derive from literature research and expert interviews. A distinction is made between three sub-stages; The stage of Formal Incorporation of the EOGRTS into OECD test guidelines was stimulated by retrospective analyses on the value of the second generation (F2), strong EOGRTS advocates, animal welfare concern and changing US and EU chemicals legislation; the stage of Actual Regulatory Acceptance within REACH was challenged by legal factors and ongoing scientific disputes, while the stage of Use by Industry is influenced by uncertainty of registrants about regulatory acceptance, high costs, the risk of false positives and the manageability of the EOGRTS. [ABSTRACT FROM AUTHOR]

Abstract: At the IVTIP (In Vitro Testing Industrial Platform) meeting of November 26th 2009 entitled ‘Toxicology in the 21st century (‘21C’) – working our way towards a visionary reality’ all delegates endorsed the emerging concept of the ‘21C’ vision as the way forward to enable a thorough, reliable and systematic approach to future toxicity testing without the use of animals. One of the emerging concepts focused on integrating a defined number of tests modelling in vivo-relevant and well-characterised toxicity pathways representing mechanistic endpoints. At this meeting the importance of Integrated Testing Strategies (ITS) as tools towards reduction and eventually replacement of the animals currently used for hazard identification and risk assessment was recognised. A follow-up IVTIP Spring 2010 meeting entitled ‘Integrated In Vitro Testing Strategies (ITS) – Implementation Challenges’ was organised to address pending questions about ITS. This report is not a review of the ITS literature, but a summary of the discussions triggered by presented examples on how to develop and implement ITS. Contrasts between pharmaceutical and chemical industry, as well as a list of general but practical aspects to be considered while developing an ITS emerged from the discussions. In addition, current recommendations on the validation of ITS were discussed. In conclusion, the outcome of this workshop improved the understanding of the participants of some important factors that may impact the design of an ITS in function of its purpose (e.g. screening, or early decision making versus regulatory), the context in which they need to be applied (e.g. ICH guidelines, REACH) and the status and quality of the available tools. A set of recommendations of best practices was established and the importance of the applicability of the individual tests as well as the testing strategy itself was highlighted. [Copyright &y& Elsevier]

Abstract: A workshop to reappraise the previous IWGT recommendations for photogenotoxicity testing [E. Gocke, L. Muller, P.J. Guzzie, S. Brendler-Schwaab, S. Bulera, C.F. Chignell, L.M. Henderson, A. Jacobs, H. Murli, R.D. Snyder, N. Tanaka, Considerations on photochemical genotoxicity: report of the International Workshop on Genotoxicity Test Procedures working group, Environ. Mol. Mutagen., 35 (2000) 173–184] was recently held as part of the 5th International Workshop on Genotoxicity Testing (IWGT) meeting in Basel, Switzerland (August 17–19, 2009). An Expert Panel was convened from regulatory, academic and industrial scientists (with several members serving on the original panel) and chaired by Dr Peter Kasper (BfArM, Germany). The aim of the workshop was to review progress made in photo(geno)toxicity testing over the past decade; a period which saw the introduction of several regulatory photosafety guidances in particular in Europe and the USA. Based on current regulatory guidelines a substantial proportion of compounds trigger the requirements for photosafety testing. Moreover, there has been growing concern within industry about the performance of the in vitro photosafety tests in the “real world” of compound development. Therefore, the expert group reviewed the status of the current regulatory guidance''s and the impact these have had on compound development in the context of the various triggers for photosafety testing. In addition, the performance of photogenotoxicity assays (old and new) was discussed, particularly in view of reports of pseudophotoclastogencity. The Expert Panel finished with an assessment of the positioning of photogenotoxicity testing within a photosafety testing strategy. The most significant conclusion made by the Expert Panel was that photogenotoxicity testing should no longer be recommended as part of the standard photosafety testing strategy. In addition, progress was made on the refinement of triggers for photosafety testing. For example, there was support for harmonisation of methods to determine the Molar Extinction Coefficient (MEC) and a consensus agreement that there should be no requirement for testing of compounds with a MEC<1000Lmol−1 cm−1. [Copyright &y& Elsevier]

Abstract: In November 2009 the In Vitro Testing Industrial Platform (IVTIP) organized a meeting entitled ‘Toxicology in the 21st century – working our way towards a visionary reality’. Participating delegates included scientists, key opinion leaders, developers and users of 3Rs-related tests and testing strategies. This paper summarizes the discussions with respect to the conditions required to move the vision towards an applicable reality. It should not be considered as a comprehensive review of technologies that could be relevant for moving the in vitro testing and risk assessment field forward. Overall, the US National Research Council (NRC) vision and strategy for toxicity testing in the 21st century was unanimously considered as the right approach to enable future toxicity testing without animal experimentation. Many elements of this vision were identified in the European initiatives aimed at the development of non-animal based methods. However, the need for concerted actions moving the current state-of-the-art towards a thorough, reliable and systematic approach to future toxicity testing was made evident by the discussions. Among the difficulties and hurdles on the way forward, the lack of physiologically relevant, metabolic competent and robust in vivo, ex vivo and in vitro models of both healthy and diseased people was frequently mentioned. In addition, there was a call for immediate implementation of emerging technologies and paradigms considered to be essential for transferring the vision into the reality of a toxicity-testing system assessing biologically significant perturbations in key pathways which are relevant for human biology. While the unique strengths of each of the available and emerging technologies was recognized, integration of available data and emerging technologies to integrated testing strategies (ITS) was highlighted as the preferred way forward. Method harmonization and standardization, as well as procedures and guidelines for putting together ITS, were urgently requested in order to facilitate proper implementation and acceptance. There was an urgent call for better coordination of the efforts that are ongoing or initiated in the 3Rs arena at national and international level. Education, training, communication and dissemination were addressed. It was recognised that the EPAA, through its ‘Platform for Communication and Dissemination’, has a very important and central role in this area. [Copyright &y& Elsevier]

The 3Rs concept, calling for replacement, reduction and refinement of animal experimentation, is receiving increasing attention around the world, and has found its way to legislation, in particular in the European Union. This is aligned by continuing high-level efforts of the European Commission to support development and implementation of 3Rs methods. In this respect, the European project called "ONTOX: ontology-driven and artificial intelligence-based repeated dose toxicity testing of chemicals for next generation risk assessment" was recently initiated with the goal to provide a functional and sustainable solution for advancing human risk assessment of chemicals without the use of animals in line with the principles of 21st century toxicity testing and next generation risk assessment. ONTOX will deliver a generic strategy to create new approach methodologies (NAMs) in order to predict systemic repeated dose toxicity effects that, upon combination with tailored exposure assessment, will enable human risk assessment. For proof-of-concept purposes, focus is put on NAMs addressing adversities in the liver, kidneys and developing brain induced by a variety of chemicals. The NAMs each consist of a computational system based on artificial intelligence and are fed by biological, toxicological, chemical and kinetic data. Data are consecutively integrated in physiological maps, quantitative adverse outcome pathway networks and ontology frameworks. Supported by artificial intelligence, data gaps are identified and are filled by targeted in vitro and in silico testing. ONTOX is anticipated to have a deep and long-lasting impact at many levels, in particular by consolidating Europe's world-leading position regarding the development, exploitation, regulation and application of animal-free methods for human risk assessment of chemicals. [ABSTRACT FROM AUTHOR]