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The AR-CALUX® in vitro method is a reporter gene-based transactivation method where endocrine active chemicals with androgenic or anti-androgenic potential can be detected. Its primary purpose is for screening chemicals for further prioritization and providing mechanistic (endocrine mode of action) information, as defined by the Organisation of Economic Cooperation and Development (OECD) conceptual framework for the testing and assessment of endocrine-disrupting chemicals. This article describes the conduct and results of an international ring trial with 3 EU-NETVAL laboratories and the test method developer. It was organized by EURL ECVAM to validate the method by testing 46 chemicals. A very good reproducibility within and between laboratories was concluded (94.7–100% and 100% concordance of classification) with low within and between laboratory variability (less than 2.5% CV on EC50 values). Moreover, the variability is within the range of other validated, mechanistically similar methods. In comparison to the AR-reference list compiled by ICCVAM, an almost 100% concordance of classifications was obtained. This method allows the detection of the agonist and antagonist properties of a chemical. A specificity control test was developed during the validation study and added to the antagonist assay rendering the assay more specific. A comparison is made with the mechanistically similar methods AR-EcoScreen™ and 22Rv1/MMTV GR-KO TA. The AR-CALUX® method was approved for inclusion in the recently updated OECD test guideline TG458 which incorporates all 3 methods.

This chapter focuses on practical aspects of conducting prospective in vitro validation studies, and in particular, by laboratories that are members of the European Union Network of Laboratories for the Validation of Alternative Methods (EU-NETVAL) that is coordinated by the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). Prospective validation studies involving EU-NETVAL, comprising a multi-study trial involving several laboratories or "test facilities", typically consist of two main steps: (1) the design of the validation study by EURL ECVAM and (2) the execution of the multi-study trial by a number of qualified laboratories within EU-NETVAL, coordinated and supported by EURL ECVAM. The approach adopted in the conduct of these validation studies adheres to the principles described in the OECD Guidance Document on the Validation and International Acceptance of new or updated test methods for Hazard Assessment No. 34 (OECD 2005). The context and scope of conducting prospective in vitro validation studies is dealt with in Chap. 4 . Here we focus mainly on the processes followed to carry out a prospective validation of in vitro methods involving different laboratories with the ultimate aim of generating a dataset that can support a decision in relation to the possible development of an international test guideline (e.g. by the OECD) or the establishment of performance standards.

This chapter focuses on practical aspects of conducting prospective in vitro validation studies, and in particular, by laboratories that are members of the European Union Network of Laboratories for the Validation of Alternative Methods (EU-NETVAL) that is coordinated by the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). Prospective validation studies involving EU-NETVAL, comprising a multi-study trial involving several laboratories or “test facilities”, typically consist of two main steps: (1) the design of the validation study by EURL ECVAM and (2) the execution of the multi-study trial by a number of qualified laboratories within EU-NETVAL, coordinated and supported by EURL ECVAM. The approach adopted in the conduct of these validation studies adheres to the principles described in the OECD Guidance Document on the Validation and International Acceptance of new or updated test methods for Hazard Assessment No. 34 (OECD 2005). The context and scope of conducting prospective in vitro validation studies is dealt with in Chap. 4. Here we focus mainly on the processes followed to carry out a prospective validation of in vitro methods involving different laboratories with the ultimate aim of generating a dataset that can support a decision in relation to the possible development of an international test guideline (e.g. by the OECD) or the establishment of performance standards.

Until now, no in vitro methods for determining neurotoxic effects, on Phase I and Phase II biotransformation-driven metabolite formation or for the evaluation of the metabolism-mediated hazard of a chemical, have been validated. The current test guidelines are based on studies in vivo, involving animals exposed to the test substance. Novel in vitro testing instead of animal testing is required by Directive 86/609/EEC. In the EU White Paper on a Strategy for a Future Chemicals Policy, which may result in up to 20,000 chemicals being screened for toxicity, the use of non-animal test methods is seen as essential and is encouraged. The aim of the present work was to demonstrate the significance of novel technologies, including the use of genetically engineered cell lines and three-dimensional cell culture techniques for direct application in the regulatory hazard-assessment process. Furthermore, attempts were made to make in vitro toxicity tests for specific applications more-readily available for inclusion in the chemical hazard-assessment process, by exploiting advances made in the life sciences.

As an example of advanced testing in the field of metabolism in an industrial environment, the introduction of some novel approaches, including the use of genetically engineered cell lines for assessing CYP 2D6-related polymorphic effects is illustrated. In this paper, it is demonstrated that novel in vitro test systems can be developed by using these genetically engineered cell lines for evaluating the potential risks associated with proprietary drugs (especially if their metabolism depends to a high extent on CYP 2D6). Moreover, it is demonstrated that, by the use of these in vitro methods, issues such as polymorphism, for which no animal models are available, can be assessed in such a way that predictions can be made on adverse effects which, up to now, could only be detected during clinical trials. Through the use of these new biotechnological in vitro metabolism models, clinically relevant data can be obtained for a scientifically-based human risk assessment, and animal use can be reduced.

Publisher Summary Many sources exist that contain valuable information on toxicology testing methods comprising in vivo (use of whole animals) and in vitro (use of artificial environment outside of living organisms) testing methods. The information resources presented in this chapter relate to chemical substances and are collected to provide key guidance on available information on in vivo, in vitro, and other toxicological testing and assessment methods (e.g., in silico toxicological methods performed on computer or via computer simulation), on relevant databases, published literature, books and journals, teaching aids, and finally organizations, foundations, and testing laboratories involved in toxicity assessment. The current information is of relevance for research, applied sciences and policy implementation purposes, such as the implementation of the European Union's (EU's) chemicals policy REACH (Registration, Evaluation, Authorisation, and Restriction of CHemicals) or the GPSD (General Product Safety Directive). The provided information resources are recommended starting points when searching for information on testing methods, alternative testing methods and computational assessment methods.

Immunotoxicology is a relatively new field in toxicology, and is one of emerging importance, because immunotoxicity appears to contribute to the development of cancer, autoimmune disorders, allergies and other diseases. At present, there is a lack of human cell-based immunotoxicity assays for predicting the toxicity of xenobiotics toward the immune system in a simple, fast, economical and reliable way. Existing immunotoxicity tests are mainly performed in animals, although species differences favour human-based testing. Whole-blood cytokine release models have attracted increasing interest, and are broadly used for pharmacological in vitro and ex vivo studies, as well as for pyrogenicity testing. We have adapted those methods for immunotoxicity testing, to permit the potency testing of immunostimulants and immunosuppressants. Following stimulation with a lipopolysaccharide or staphylococcal enterotoxin B, monocytes and lymphocytes release interleukin-1β and interleukin-4, respectively. Thirty-one pharmaceutical compounds, with known effects on the immune system, were used to optimise and standardise the method, by analysing their effects on cytokine release. The in vitro results were expressed as IC50 values for immunosuppression, and SC4(fourfold increase) values for immunostimulation, and compared with therapeutic serum concentrations of the compounds in patients, and in vivo LD50 values from animal studies. The in vitro results correlated well with the in vivo data, so the test appears to reflect immunomodulation. Results were reproducible (CV = 20 ± 5%), and the method could be transferred to another laboratory (r2= 0.99). We therefore propose this method for further validation and for use in immunotoxicity testing strategies.

Xenobiotics undergong bioactivation by CYP450 enzymes form reactive metabolites that may exert direct metabolism-mediated toxicity. An in vitro model was developed to study the direct toxic effects that follow the metabolic activation of chemicals. The model uses monolayer cultures of genetically engineered NIH-3T3 or V79 cells that express individual human or rat CYP450 isoforms, respectively. Following exposure to 1,3-dichloropropanol or cyclophosphamide, basal cytotoxicity endpoints, including neutral red uptake and Alamar Blue™ reduction were used to assess changes in cell number and functional viability resulting from the formation of metabolites. Cell lines that express cytochrome P450 enzymes metabolised the test compounds, leading to increased toxicity compared with that observed in the control cell line. The use of specific inhibitors confirmed that the formation of reactive metabolites was CYP450-isoform dependent. These results indicate that a panel of genetically engineered cell lines expressing various cytochrome P450 enzyme isoforms can be used to reveal measurable metabolising capabilities, and could become a useful tool for the detection and possible determination of CYP450 isoforms in human liver metabolism-mediated toxicity.

The generation of biologically active complement split products through the direct reaction of microorganisms with complement proteins is one of the earliest events of the defence reaction in humans. Complement activation develops within minutes, which highly corresponds with the onset of a febrile reaction after exposure to pyrogens. The possibility of the use of complement activation in human plasma as an indicator of pyrogen contamination has been tested. Additionally, the co-stimulatory effect of complement activation on tumor necrosis factor-alpha (TNF-α) production by blood-separated macrophages exposed to lipopolysaccharide (LPS) has been demonstrated. As an indicator of complement activation in test samples, the concentration of the iC3b fragment was measured by using an ELISA system based on neoantigen formation. The 3-h exposure time has been identified as optimal for the test. The variability between iC3b concentrations in untreated control samples obtained from seven unrelated healthy donors was less than 10%, while after activation by 100 ng/ml LPS, it increased to 13%. The lower detection limit has been identified as 10 pg/ml LPS. As the complement test is not affected by drug–cell interactions or cell viability, the test can be used in situations where tested formulations contain active substances, which interfere with a cell-based test. We conclude that a test based on the detection of complement activation in human plasma should be considered as a valuable element of an in vitro pyrogenicity testing battery along with a cell-based assay.