in vitro testing

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The EpiOcular™ Eye Irritation Test is the Method of Choice for the In Vitro Eye Irritation Testing of Agrochemical Formulations:

Correlation Analysis of EpiOcular Eye Irritation Test and BCOP Test Data According to the UN GHS, US EPA and Brazil ANVISA Classification Schemes

Susanne N. Kolle, Maria Cecilia Rey Moreno, Winfried Mayer, Andrew van Cott, Bennard van Ravenzwaay and Robert Landsiede

The Bovine Corneal Opacity and Permeability (BCOP) test is commonly used for the identification of severe ocular irritants (GHS Category 1), but it is not recommended for the identification of ocular irritants (GHS Category 2). The incorporation of human reconstructed tissue model-based tests into a tiered test strategy to identify ocular non-irritants and replace the Draize rabbit eye irritation test has been suggested (OECD TG 405). The value of the EpiOcular™ Eye Irritation Test (EIT) for the prediction of ocular non-irritants (GHS No Category) has been demonstrated, and an OECD Test Guideline (TG) was drafted in 2014. The purpose of this study was to evaluate whether the BCOP test, in conjunction with corneal histopathology (as suggested for the evaluation of the depth of the injury) and/or the EpiOcular-EIT, could be used to predict the eye irritation potential of agrochemical formulations according to the UN GHS, US EPA and Brazil ANVISA classification schemes. We have assessed opacity, permeability and histopathology in the BCOP assay, and relative tissue viability in the EpiOcular-EIT, for 97 agrochemical formulations with available in vivo eye irritation data. By using the OECD TG 437 protocol for liquids, the BCOP test did not result in sufficient correct predictions of severe ocular irritants for any of the three classification schemes. The lack of sensitivity could be improved somewhat by the inclusion of corneal histopathology, but the relative viability in the EpiOcular-EIT clearly outperformed the BCOP test for all three classification schemes. The predictive capacity of the EpiOcular-EIT for ocular non-irritants (UN GHS No Category) for the 97 agrochemical formulations tested (91% sensitivity, 72% specificity and 82% accuracy for UN GHS classification) was comparable to that obtained in the formal validation exercise underlying the OECD draft TG. We therefore conclude that the EpiOcular-EIT is currently the best in vitro method for the prediction of the eye irritation potential of liquid agrochemical formulations.
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ESNATS Conference — The Use of Human Embryonic Stem Cells for Novel Toxicity Testing Approaches

Costanza Rovida, Manon Vivier, Bernward Garthoff and Jürgen Hescheler

The main achievements and results of the ESNATS project (Embryonic Stem Cell-based Novel Alternative Testing Strategies) were presented at the final project conference that was held on 15 September 2013, the day before the traditional EUSAAT (European Society for Alternatives to Animal Testing) Congress in Linz, Austria. The ESNATS project was an FP7 European Integrated Project, running from 2008 to 2013, the aim of which was to develop a novel toxicity testing platform based on embryonic stem cells (ESCs), and in particular, human ESC (hESCs), to accelerate drug development, reduce related R&D costs, and propose a powerful alternative to animal tests in the spirit of the Three Rs principles. Altogether, ESNATS offered the first proof of concept that hESCs can be used to create robust, reproducible and ready-to use test assays for predicting human toxicity. In the end, essentially five test systems were developed to an adequate level for entering possible pre-validation procedures. These methods are based on hESCs, and can be combined to study the possible effects, on the human embryo, of exposure to a chemical during the early stages of development. In addition to the presentations by the main project partners, external speakers were invited to give lectures on relevant topics, both in the field of neurotoxicity and, more generally, on the applicability of hESCs in the development of advanced in vitro tests.

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The ECVAM Prevalidation Study on the Use of EpiDerm for Skin Corrosivity Testing

Manfred Liebsch, Dieter Traue, Christa Barrabas, Horst Spielmann, Patricia Uphill, Susan Wilkins, Janet P. McPherson, Christiane Wiemann, Tanja Kaufmann, Martina Remmele and Hermann-Georg Holzhütter

In 1996 and 1997, ECVAM supported a formal validation study on in vitro methods for predicting skin corrosivity. Two of the in vitro tests included in the study employed human skin models, the Skin2 ZK1350 and EPISKIN models. In the ECVAM validation study, BASF, Huntingdon Life Sciences (HLS) and ZEBET tested the Skin2 human skin model, production of which ceased in October 1996, while the validation study was still in progress. Since both of the skin models had shown basic usefulness for corrosivity testing and, in particular, the EPISKIN corrosivity test had proved to be a scientifically valid test, the three laboratories decided to conduct a study to determine whether another commercially available human skin model, EpiDerm, could also be successfully used to predict skin corrosivity. The study was performed according to the ECVAM prevalidation scheme, to allow for refinement of the test protocol and the prediction model, as well as for independent assessment of the performance of the refined methodology in a final blind trial in the three laboratories. In phase I of the study, ZEBET (Laboratory 1) drafted a Standard Operating Procedure (SOP), including a prediction model (PM1), and the project plan for the study. It was a major task to simplify an existing EpiDerm test protocol, which used the time-course of cytotoxicity as its endpoint. To evaluate the predictivity of the simplified method, which used only a 3-minute exposure to test chemicals, 50 chemicals representing a wide spectrum of chemical entities were tested, revealing that the test sensitivity was too low (65%), whereas the specificity was very high (88%). In addition, acceptance criteria for the negative and positive controls were established. Before proceeding to the next phase of the study, ZEBET distributed a refined SOP, data-recording software and documentation sheets, which allowed Good Laboratory Practice (GLP)-compliant quality assurance for each assay. The main goal of phase II was to produce sufficient data to assess the reproducibility of the EpiDerm skin corrosivity test after transfer to Laboratory 2 (HLS). Repeated testing of several chemicals in both laboratories revealed excellent intralaboratory and interlaboratory reproducibility. In addition, chemicals classified as "non-corrosive" (NC) with a 3-minute exposure in phase I, were re-tested by ZEBET with extended exposure periods of 1 hour and 4 hours. The test sensitivity could be significantly increased, if chemicals classified NC with a 3- minute exposure were tested with a 1-hour exposure. Before proceeding to the final blind trial, a refined SOP was drafted, according to which all chemicals had to be tested with exposure times of 3 minutes and 1 hour, and data for these two exposure times were used in the refined hierarchical prediction model, PM2. In phase III, the blind trial, BASF (Laboratory 3) joined the study. ECVAM selected 24 chemicals from the test chemical set used in the ECVAM skin corrosivity validation study, and BIBRA International (UK) purchased, coded and distributed the chemicals. Each chemical was tested twice, independently, according to the principles of GLP, and coded data were submitted to the Humboldt University (Berlin, Germany) for biostatistical analysis. The analysis revealed that the final test protocol and the refined prediction model (PM2) provided a highly balanced prediction of 88% sensitivity and 86% specificity, which is regarded as the best predictivity an in vitro skin corrosivity test can be expected to achieve. In conclusion, the EpiDerm skin corrosivity test gives an excellent prediction for a wide spectrum of chemicals, and could be used within the context of the new Annex V (EU Dangerous Substances Directive) test method (human skin model assay) for skin corrosion. The results obtained were reproducible, both within and between laboratories, and showed that EpiDerm could be used for testing a wide range of chemicals (both liquids and solids), including organic acids and bases, neutral organics, inorganic acids and bases, electrophiles and phenols. The concordances between the skin corrosivity classifications derived from the in vitro data were very good, and the test was able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied
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Alternatives to Animal Testing in the Safety Evaluation of Products

Derek J. Knight and Damien Breheny

The conventional method for assessing the safety of products, ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals - including cosmetics - involves determining their toxicological properties by using experimental animals. The aim is to identify any possible adverse effects in humans by using these animal models. Providing safe products is undoubtedly of the utmost importance but, over the last decade or so, this aim has come into conflict with strong public opinion, especially in Europe, against animal testing. Industry, academia and the regulators have worked in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals required and the severity of the tests in which they are used. There is a long way to go before products can be evaluated without any animal studies, and it may be that this laudable aim is an impossible dream. Nevertheless, considerable progress has been made by using a combination of in vitro tests and the prediction of properties based on chemical structure. The aim of this review is to describe these important and worthwhile developments in various areas of toxicological testing, with a focus on the European regulatory framework for general industrial and household chemicals
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Transepithelial Resistance and Inulin Permeability as Endpoints in In Vitro Nephrotoxicity Testing

Tracey Duff, Simon Carter, Gemma Feldman, Gordon McEwan, Walter Pfaller, Pauline Rhodes, Michael Ryan and Gabrielle Hawksworth

Transepithelial electrical resistance (RT) and the flux of fluorescein isothiocyanate (FITC) across Madin Darby canine kidney (MDCK) strain 1 cells and porcine epithelial kidney (LLC-PK1) monolayers were compared between three laboratories for a range of nephrotoxins. The precision of the REMS AutoSampler was similar to that of the Ussing chamber and the ENDOHM® technique, but superior to using chopstick electrodes, for measurements of resistance. The nephrotoxins used were selective for the proximal tubule, and in all cases, LLC-PK1 cells were more sensitive than MDCK cells. In most cases, change in RT was a more sensitive indicator of damage than alterations in FITC flux. The REMS system provides high intra-plate precision for RT measurements and is a higher throughput system, which is applicable to screening for nephrotoxicity in vitro.
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Heterologous Expression of Mouse Cytochrome P450 2e1 in V79 Cells: Construction and Characterisation of the Cell Line and Comparison with V79 Cell Lines Stably Expressing Rat P450 2E1 and Human P450 2E1

Ulrike Bernauer, Hansruedi Glatt, Barbara Heinrich-Hirsch, Yungang Liu, Eva Muckel, Bärbel Vieth and Ursula Gundert-Remy

A V79 Chinese hamster cell line was constructed for stable expression of mouse cytochrome P450 2e1 (Cyp2e1), as an addition to the existing cell battery consisting of cell lines stably expressing rat CYP2E1 and human CYP2E1 (V79 Cell Battery). The aim was to establish a cell battery that offers the in vitro possibility of investigating species-specific differences in the toxicity and metabolism of chemicals representing substrates for CYP2E1. The newly established cell line (V79m2E1) effectively expressed Cyp2e1 in the catalytically active form. The expression of catalytically active CYP2E1 in V79m2E1 cells was maintained over several months in culture, as demonstrated by Western Blotting and chlorzoxazone (CLX) 6-hydroxylase activity. The cells exhibited CLX 6-hydroxylase activity with a Km of 27.8μM/l and Vmax of 40pmol/mg protein/minute, compared with a Km of 28.2/28.6μM/l and a Vmax of 130/60pmol/mg protein/minute from V79r2E1/V79h2E1 cells. Furthermore, the CYP2E1-dependent mutagenicity of N-nitrosodimethylamine could be demonstrated in the V79m2E1 cells. Therefore, the new cell battery permits the interspecies comparison of CYP2E1-dependent toxicity and of metabolism of chemicals between humans and the two major rodent species the rat and the mouse that are usually used in classical toxicity studies.
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The Case for Taking Account of Metabolism when Testing for Potential Endocrine Disruptors In Vitro

Robert D. Combes

Legislation in the USA, Europe and Japan will require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Such chemicals are known as endocrine disruptors (EDs), and will require extensive testing as part of the new European Union Registration, Evaluation and Authorisation of Chemicals (REACH) system for the risk assessment of chemicals. Both in vivo and in vitro tests are proposed for this purpose, and there has been much discussion and action concerning the development and validation of such tests. However, to date, little interest has been shown in incorporating metabolism into in vitro tests for EDs, in sharp contrast to other areas of toxicity testing, such as genotoxicity, and, ironically, such in vitro tests are criticised for not modelling in vivo metabolism. This is despite the existence of much information showing that endogenous and exogenous steroids are extensively metabolised by Phase I and Phase II enzymes both in the liver and in hormonally active tissues. Such metabolism can lead to the activation or detoxification of steroids and EDs. The absence of metabolism from these tests could give rise to false-positive data (due to lack of detoxification) or false-negative data (lack of activation). This paper aims to explain why in vitro assays for EDs should incorporate mammalian metabolising systems. The background to ED testing, the test methods available, and the role of mammalian metabolism in the activation and detoxification of both endogenous and exogenous steroids, are described. The available types of metabolising systems are compared, and the potential problems in incorporating metabolising systems into in vitro tests for EDs, and how these might be overcome, are discussed. It is recommended that there should be: a) an assessment of the intrinsic metabolising capacity of cell systems used in tests for EDs; b) an investigation into the relevance of using the prostaglandin H synthase system for metabolising EDs; and c) a feasibility study into the generation of genetically engineered mammalian cell lines expressing specific metabolising enzymes, which could also be used to detect EDs.
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Integrated Testing Strategies for Use in the EU REACH System

Christina Grindon, Robert Combes, Mark T.D. Cronin, David W. Roberts and John F. Garrod

Integrated testing strategies have been proposed to facilitate the process of chemicals risk assessment to fulfil the requirements of the proposed EU REACH system. Here, we present individual, decision- tree style, strategies for the eleven major toxicity endpoints of the REACH system, including human health effects and ecotoxicity. These strategies make maximum use of non-animal approaches to hazard identification, before resorting to traditional animal test methods. Each scheme: a) comprises a mixture of validated and non-validated assays (distinguished in the schemes); and b) decision points at key stages to allow the cessation of further testing, should it be possible to use the available information to classify and label and/or undertake risk assessment. The rationale and scientific justification for each of the schemes, with respect to the validation status of the tests involved and their individual advantages and limitations, will be discussed in detail in a series of future publications.
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Integrated Decision-tree Testing Strategies for Environmental Toxicity With Respect to the Requirements of the EU REACH Legislation

Christina Grindon, Robert Combes, Mark T.D. Cronin, David W. Roberts and John Garrod

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 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 environmental (aquatic) toxicity testing. The manuscript reviews tests based on fish cells and cell lines, fish embryos, lower organisms, and the many expert systems and QSARs for aquatic toxicity testing. Ways in which reduction and refinement measures can be used are also discussed, including the Upper Threshold Concentration — Step Down (UTC) approach, which has recently been retrospectively validated by ECVAM and subsequently endorsed by the ECVAM Scientific Advisory Committee (ESAC). It is hoped that the application of this approach could reduce the number of fish used in acute toxicity studies by around 65–70%. Decisiontree style integrated testing strategies are also proposed for acute aquatic toxicity and chronic toxicity (including bioaccumulation), followed by a number of recommendations for the future facilitation of aquatic toxicity testing with respect to environmental risk assessment.
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A Review of the Status of Alternative Approaches to Animal Testing and the Development of Integrated Testing Strategies for Assessing the Toxicity of Chemicals under REACH — A Summary of a DEFRA-funded Project Conducted by Liverpool John Moores University and FRAME

Christina Grindon, Robert Combes, Mark T.D. Cronin, David W. Roberts and John Garrod

Liverpool John Moores University and FRAME were recently awarded a DEFRA tender to conduct a review of the status of alternative approaches to animal testing, and to recommend further research with regard to the forthcoming European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The outcome of the project is summarised, including the prospects for in vitro and in silico testing, areas where reduction and refinement could be applied, and how decision-tree integrated testing strategies could be used to reduce the number of animals needed to fulfil the testing requirements of the REACH system. This paper is a prelude to a series of individual papers on detailed suggestions for applying non-animal methods to each of the major toxicity endpoints in REACH.
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