EURL ECVAM Recommendation on Non-Animal-Derived Antibodies

Affinity reagents are binding molecules that have a high specificity for their unique target (antigen). They are crucial tools for research, diagnostics, therapeutic and regulatory applications. Based on their recognition properties and binding specificity, protein-based antibodies are currently still the most important tools for the specific detection of proteins or other molecules. The development and production of monoclonal and polyclonal antibodies as well as other affinity reagents is still involving animals despite the availability of technologies that do not entail the use of animals. In line with the legal requirements of EU Directive 2010/63/EU on the protection of animals used for scientific purposes, animals should not be used in procedures, where a non-animal alternative exists, which provide the same or higher level of information as obtained from animal procedures. For this reason, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) mandated its Scientific Advisory Committee (ESAC) to review the available evidence and deliver an opinion on the scientific validity of antibodies and non-antibody affinity reagents produced using animal-free technologies. The review focused on non-animal-derived antibodies generated by phage-display technology since this is the most mature technology and already widely used. Taking into consideration the available evidence, the ESAC endorsed an opinion on the suitability of existing animal-free technologies to produce affinity reagents with equal or better quality (purity, activity, specificity, affinity, stability, reproducibility) than that offered by antibodies produced using the conventional animal-based methods. In addition, ESAC commented on the scientific benefits of using animal-free affinity reagents and assessed whether there are any production and/or application scenarios for which these are not fit-for-purpose and animal-derived antibodies are still indispensable. This science-for-policy report describes the EURL ECVAM recommendations on non-animal-derived antibodies developed on the basis of the ESAC Opinion (Annex 1) and its detailed Working Group Report (Annex 2).JRC.F.3-Chemicals Safety and Alternative Method

Retrospective analysis of the Draize test for serious eye damage/eye irritation: importance of understanding the in vivo endpoints under UN GHS/EU CLP for the development and evaluation of in vitro test methods

For more than two decades, scientists have been trying to replace the regulatory in vivo Draize eye test by in vitro methods, but so far only partial replacement has been achieved. In order to better understand the reasons for this, historical in vivo rabbit data were analysed in detail and resampled with the purpose of (1) revealing which of the in vivo endpoints are most important in driving United Nations Globally Harmonized System/European Union Regulation on Classification, Labelling and Packaging (UN GHS/EU CLP) classification for serious eye damage/eye irritation and (2) evaluating the method’s within-test variability for proposing acceptable and justifiable target values of sensitivity and specificity for alternative methods and their combinations in testing strategies. Among the Cat 1 chemicals evaluated, 36–65 % (depending on the database) were classified based only on persistence of effects, with the remaining being classified mostly based on severe corneal effects. Iritis was found to rarely drive the classification (<4 % of both Cat 1 and Cat 2 chemicals). The two most important endpoints driving Cat 2 classification are conjunctiva redness (75–81 %) and corneal opacity (54–75 %). The resampling analyses demonstrated an overall probability of at least 11 % that chemicals classified as Cat 1 by the Draize eye test could be equally identified as Cat 2 and of about 12 % for Cat 2 chemicals to be equally identified as No Cat. On the other hand, the over-classification error for No Cat and Cat 2 was negligible (<1 %), which strongly suggests a high over-predictive power of the Draize eye test. Moreover, our analyses of the classification drivers suggest a critical revision of the UN GHS/EU CLP decision criteria for the classification of chemicals based on Draize eye test data, in particular Cat 1 based only on persistence of conjunctiva effects or corneal opacity scores of 4. In order to successfully replace the regulatory in vivo Draize eye test, it will be important to recognise these uncertainties and to have in vitro tools to address the most important in vivo endpoints identified in this paper.JRC.I.5-Systems Toxicolog

ECVAM Technical Report on the Status of Alternative Methods for Cosmetics Testing (2008-2009)

The ECVAM technical report presents the progress made in the development and validation of alternative methods for the human health effects relevant to the Cosmetics Directive. It provides an update on the activities described by ECVAM in 2005 , 2006 and 2007 . The report intends to present the latest scientific and technical developments in the field during 2008-2009. As required by Directive 2003/15/EC, the seventh amendment to Directive 76/768/EEC, developments in refinement and reduction methods are also described (EU, 2003). Most successes in the development of alternative methods are in acute local toxicity and short-term testing, such as e.g. skin and eye irritation/corrosion, phototoxicity and skin penetration The test methods consuming a high number of animals, however, are in long-term testing and systemic toxicity, such as e.g. reproductive toxicity and repeated dose toxicity. In these complex fields, several research initiatives are ongoing. However full replacement approaches are still lacking.JRC.DG.I.3-In-vitro method

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2013-April 2014)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches since the last report published in April 2013. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and international acceptance of alternative methods and approaches. R&D activities are ongoing for the complex endpoints where the toxicological processes and the mechanistic understanding have not been sufficiently elucidated yet and for which 3Rs solutions are more difficult to find. On the other hand, good progress In the validation and regulatory acceptance is made in areas where non-animal alternative methods have been developed and validated and where the focus lies in an intelligent combination/ integration of the various non-animal approaches.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2015)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches and their dissemination since the last report published in June 2014. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and regulatory/international acceptance of alternative methods and approaches and dissemination activities. R&D activities within large European or International consortia continued in toxicity areas where 3Rs solutions are more difficult to find due to the underlying complexity of the area. On the other hand, toxicity areas where promising non-animal approaches have been developed, their validation and regulatory acceptance/international adoption could be progressed. Particular emphasis was given to the best and most intelligent combination and integration of these different non-animal approaches to ultimately obtain the required information without resorting to animal testing.JRC.I.5-Systems Toxicolog

Alternative methods for regulatory toxicology – a state-of-the-art review

This state-of-the art review is based on the final report of a project carried out by the European Commission’s Joint Research Centre (JRC) for the European Chemicals Agency (ECHA). The aim of the project was to review the state of the science of non-standard methods that are available for assessing the toxicological and ecotoxicological properties of chemicals. Non-standard methods refer to alternatives to animal experiments, such as in vitro tests and computational models, as well as animal methods that are not covered by current regulatory guidelines. This report therefore reviews the current scientific status of non-standard methods for a range of human health and ecotoxicological endpoints, and provides a commentary on the mechanistic basis and regulatory applicability of these methods. For completeness, and to provide context, currently accepted (standard) methods are also summarised. In particular, the following human health endpoints are covered: a) skin irritation and corrosion; b) serious eye damage and eye irritation; c) skin sensitisation; d) acute systemic toxicity; e) repeat dose toxicity; f) genotoxicity and mutagenicity; g) carcinogenicity; h) reproductive toxicity (including effects on development and fertility); i) endocrine disruption relevant to human health; and j) toxicokinetics. In relation to ecotoxicological endpoints, the report focuses on non-standard methods for acute and chronic fish toxicity. While specific reference is made to the information needs of REACH, the Biocidal Products Regulation and the Classification, Labelling and Packaging Regulation, this review is also expected to be informative in relation to the possible use of alternative and non-standard methods in other sectors, such as cosmetics and plant protection products.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2016)

Replacement, Reduction and Refinement of animal testing is anchored in EU legislation. Alternative non-animal approaches facilitate a shift away from animal testing. Cell-based methods and computational technologies are integrated to translate molecular mechanistic understanding of toxicity into safety testing strategies.JRC.F.3-Chemicals Safety and Alternative Method

Analysis of the Local Lymph Node Assay (LLNA) variability for assessing the prediction of skin sensitisation potential and potency of chemicals with non-animal approaches

The knowledge of the biological mechanisms leading to the induction of skin sensitisation has favoured in recent years the development of alternative non-animal methods. During the formal validation process, results from the Local Lymph Node Assay (LLNA) are generally used as reference data to assess the predictive capacity of the non-animal tests. This study reports an analysis of the variability of the LLNA for a set of chemicals for which multiple studies are available and considering three classification schemes: POS/NEG, GHS/CLP and ECETOC. As the type of vehicle used in a LLNA study is known to influence to some extent the results, two analyses were performed: considering the solvent used to test the chemicals and without considering the solvent. The results show that the number of discordant classifications increases when chemical is tested in more than one solvent. Moreover, it can be concluded that study results leading to classification in the strongest classes (1A and EXT) seem to be more reliable than those in the weakest classes. This study highlights the importance of considering the variability of the reference when evaluating non-animal tests.JRC.I.5-Systems Toxicolog

ECVAM Bottom-Up/Top-Down Testing Approach: Testing Strategy to Reduce/Replace the Draize Eye Test and Validation/Regulatory Acceptance of In Vitro Assays: Current Status

To reduce and/or replace the Draize test, testing schemes combining strengths of particular in vitro assays were proposed during a 2005 ECVAM expert meeting. The testing scheme proposes, based on expected irritancy of the test substance, a Bottom-Up approach, beginning with test methods that accurately identify non-irritants, or a Top-Down approach, beginning with test methods that accurately identify severe irritants before progression of further in vitro testing. Furthermore as its core activity, ECVAM participated in the retrospective validation of and has peer reviewed scientific validity of four organotypic assays, and undertook retrospective validation of four cell function/cytotoxicity assays. The BCOP and ICE organotypic assays were ICCVAM and ESAC endorsed as scientifically valid for identifying severe irritants, and OECD Test Guidelines are under adoption. NRR, FL and CM cell function/cytotoxicity assays were recommended by an ECVAM Validation Management Group for identification of non-irritants or severe eye irritants in the Bottom-Up/Top-Down approaches. These assays were peer reviewed by ESAC during 2009. Finally, a joint ECVAM-COLIPA prospective validation study was initiated in 2008 to evaluate two Reconstructed human Tissue assays to discriminate non-classified materials from eye irritants, based on the proposed test strategies. The ultimate goal is to combine validated in vitro assays, based on their performances and applicability domains, to define the most suitable testing strategy to classify substances for eye irritation potential and ultimately replace the Draize test. This manuscript presents the proposed testing scheme and provides details on the validation/regulatory status of in vitro assays for use in this scheme.JRC.I.2-Validation of Alternative Method

Two Novel Prediction Models Improve Predictions of Skin Corrosive Sub-categories by Test Methods of OECD Test Guideline No. 431

Alternative test methods often use prediction models (PMs) for converting endpoint measurements into predictions. Two PMs are used in the OECD Test Guideline No. 431 (TG431) on skin corrosion. One is specific to EpiSkin™ test method, whereas EpiDerm™, SkinEthic™RHE and epiCS® share a common PM. These four methods are Reconstructed human Epidermis models wherein several cell viability values are measured. Their PMs allow translating those values into sub-categories of corrosive chemicals, Category 1A (Cat1A) and a combination of Categories 1B/1C (Cat1BC), and identifying non-corrosive chemicals. EpiSkin™'s PM already results in sufficiently accurate predictions. The common PM of the three others accurately identifies all corrosive chemicals but, regarding their sub-categorization, an important fraction of Cat1BC chemicals (40-50%) is over-predicted as Cat1A. This paper presents a post-hoc analysis of validation data on a consistent set of n=80 chemicals. It investigates (i) why this common PM causes these over-predictions and (ii) how two novel PMs that we developed (PMvar1 and PMvar2) improve the predictive capacity of these methods. PMvar1 is based on a two-step approach; PMvar2 is based on a single composite indicator of cell viability. Both showed a marked greater capacity to predict Cat1BC, and Cat1A correct predictions remain at least at the same level of EpiSkin™. We suggest revising TG431, and its performance standards, to include the novel PMs in view of improving the predictive capacity of its in vitro skin corrosion methods.JRC.I.5-Systems Toxicolog