Report of the EPAA–ECVAM Workshop on the Validation of Integrated Testing Strategies (ITS)

Abstract

The use of Integrated Testing Strategies (ITS) permits the combination of diverse types of chemical and toxicological data for the purposes of hazard identification and characterisation. In November 2008, the European Partnership for Alternative Approaches to Animal Testing (EPAA), together with the European Centre for the Validation of Alternative Methods (ECVAM), held a workshop on Overcoming Barriers to Validation of Non-animal Partial Replacement Methods/Integrated Testing Strategies, in Ispra, Italy, to discuss the extent to which current ECVAM approaches to validation can be used to evaluate partial replacement in vitro test methods (i.e. as potential ITS components) and ITS themselves. The main conclusions of these discussions were that formal validation was only considered necessary for regulatory purposes (e.g. the replacement of a test guideline), and that current ECVAM approaches to validation should be adapted to accommodate such test methods (1). With these conclusions in mind, a follow-up EPAA–ECVAM workshop was held in October 2009, to discuss the extent to which existing validation principles are applicable to the validation of ITS test methods, and to develop a draft approach for the validation of such test methods and/or overall ITS for regulatory purposes. This report summarises the workshop discussions that started with a review of the current validation methodologies and the presentation of two case studies (skin sensitisation and acute toxicity), before covering the definition of ITS and their components, including their validation and regulatory acceptance. The following main conclusions/recommendations were made: that the validation of a partial replacement test method (for application as part of a testing strategy) should be differentiated from the validation of an in vitro test method for application as a stand-alone replacement, especially with regard to its predictive capacity; that, in the former case, the predictive capacity of the whole testing strategy (rather than of the individual test methods) would be more important, especially if the individual test methods had a high biological relevance; that ITS allowing for flexible and ad hoc approaches cannot be validated, whereas the validation of clearly defined ITS would be feasible, although practically quite difficult; and that test method developers should be encouraged to develop and submit to ECVAM not only full replacement test methods, but also partial replacement methods to be placed as parts of testing strategies. The added value from the formal validation of testing strategies, and the requirements needed in view of regulatory acceptance of the data, require further informed discussion within the EPAA forum on the basis of case studies provided by industry.

Evaluation of anIn VitroHuman Dermal Sensitization Test for Use with Medical Device Extracts

“…25 In recent years ECVAM and OECD have been collaborating on a document that incorporates key AOP elements into an ITS; this work should be completed in 2016. 26 Also, the U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the NTP Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) have been providing expertise and advice to ECVAM and Cosmetics Europe concerning their ongoing evaluations of alternative test methods and testing strategies for the identification of skin sensitizers. 27 To date, ECVAM has validated three in vitro sensitization methods: a direct peptide reactivity assay (DPRA) 28,29 ; KeratinoSensÔ, an ARE-Nrf2 luciferase assay 30,31 ; and the human cell line activation test (h-CLAT), which measures the upregulation of CD86 and CD54 markers of DC activation in THP-1 cells.…”

“…[26][27][28][29][30][31][32][33][34][35] In these assays (with the exception of DPRA), the solubility of a test material in aqueous solutions is critical for the performance of the assay, making assessment of nonpolar extracts nearly impossible. In contrast, the SenCeeTox assay evaluates a test substance's electrophilic nature by measuring interactions with and covalent binding to GSH, as well as the induction of genes in the ARE/Keap1/Nrf, AhR/ARNT/XRE, and Nrf1/MTF/MRE pathways.…”

Integrated testing strategies for safety assessments

“…For example, two In Vitro testing Industrial Platform workshops were summarized stating (De Wever et al, 2012): “ As yet, there is great dispute among experts on how to represent ITS for classification, labeling, or risk assessments of chemicals, and whether or not to focus on the whole chemical domain or on a specific application. The absence of accepted Weight of Evidence (WoE) tools allowing for objective judgments was identified as an important issue blocking any significant progress in the area .” Similarly, the ECVAM/EPAA workshop concluded (Kinsner-Ovaskainen et al, 2012): “ Despite the fact that some useful insights and preliminary conclusions could be extracted from the dynamic discussions at the workshop, regretfully, true consensus could not be reached on all aspects .”…”

“…We would respectfully disagree, as validation certainly is desirable for other uses, but it should be tailored to the use scenario and the available resources. The follow-up workshop (Kinsner-Ovaskainen et al, 2012) did not go much further with regard to recommendations for validation: “ Firstly, it was agreed that the validation of a partial replacement test method (for application as part of a testing strategy) should be differentiated from the validation of an in vitro test method for application as a stand-alone replacement. It was also agreed that any partial replacement test method should not be any less robust, reliable or mechanistically relevant than standalone replacement methods.…”

The role ofin vitromethods as alternatives to animals in toxicity testing

“…This report summarizes the workshop discussions that started with a review of the current validation methodologies and the presentation of two case studies (i.e., skin sensitization and acute toxicity), before covering the definition of ITS and their components, including their validation and regulatory acceptance. The following conclusions and recommendations were made: i) the validation of a partial replacement test method (for application as part of a testing strategy) should be differentiated from the validation of an in vitro test method for application as a stand-alone replacement, especially with regard to its predictive capacity; ii) in the former case, the predictive capacity of the whole testing strategy (rather than of the individual test methods) would be more important, especially if the individual test methods had a high biological relevance; iii) ITS allowing for flexible and ad hoc approaches cannot be validated, whereas the validation of clearly defined ITS would be feasible, although practically quite difficult and iv) test method developers should be encouraged to develop and submit to the ECVAM not only full replacement test methods but also partial replacement methods to be placed as parts of testing strategies [5]. A subsequent workshop was held on 'ITS and their impact on the implementation of 3Rs' and identified a number of criteria that ITS should meet if data generated are to be accepted in regulatory compliance procedures [6].…”