The usefulness of integrated strategy approaches in replacing animal experimentation

It is currently well accepted that in general, more than one method is necessary to allow the full replacement of an animal experimentation. These so called partial replacement methods can be used within integrated strategy approaches that combine different methods and information sources. A number of integrated strategy approaches were implemented within recent years in different areas of safety and regulatory toxicology.Moreover, latest advances in biomedical research and bioengineering provide a major opportunity to make use of in vitro human-based and/or three-dimensional complex models that can contribute to achieve more physiologically-relevant models. Examples herein describe currently existing integrated strategy frameworks aiming at full or partial replacement purposes and/or at gaining mechanistic insights. Furthermore, a general concept is provided on how 3R methods might be integrated in a strategy approach in order to ensure that animal experimentation is conducted only as a last resort

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

ESAC Opinion on the Scientific Validity of the AR-CALUX Test Method

ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientific issues. Its main role is to conduct independent peer review of validation studies of alternative test methods and to assess their scientific validity for a given purpose. The committee reviews the appropriateness of study design and management, the quality of results obtained and the plausibility of the conclusions drawn. ESAC peer reviews are formally initiated with a EURL ECVAM Request for ESAC Advice, which provides the necessary background for the peer-review and establishes its objectives, timelines and the questions to be addressed. The peer review is normally prepared by specialised ESAC Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions' and 'Working Group Reports' at the end of the peer review. ESAC may also issue Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM but not directly related to a specific alternative test method. The ESAC Opinion expressed in this report relates to the peer-review of the AR-CALUX in vitro test method.JRC.F.3-Chemicals Safety and Alternative Method

ESAC Opinion on the Scientific Validity of the Bioelution Test Method: ESAC Opinion No. 2019-03 of 2 December 2019

ESAC, the EURL ECVAM Scientific Advisory Committee, advises EURL ECVAM on scientific issues. Its main role is to conduct independent peer review of validation studies of alternative test methods and to assess their scientific validity for a given purpose. The committee reviews the appropriateness of study design and management, the quality of results obtained and the plausibility of the conclusions drawn. ESAC peer reviews are formally initiated with a EURL ECVAM Request for ESAC Advice, which provides the necessary background for the peer-review and establishes its objectives, timelines and the questions to be addressed. The peer review is normally prepared by specialised ESAC Working Groups. ESAC's advice to EURL ECVAM is formally provided as 'ESAC Opinions' and 'Working Group Reports' at the end of the peer review. ESAC may also issue Opinions on other scientific issues of relevance to the work and mission of EURL ECVAM but not directly related to a specific alternative test method. The ESAC Opinion expressed in this report relates to the peer-review of the Bioelution in vitro test method.JRC.F.3-Chemicals Safety and Alternative Method

t4 Workshop Report: Integrated Testing Strategies (ITS) for Safety Assessment

Integrated testing strategies (ITS), as opposed to single definitive tests or fixed batteries of tests, are expected to efficiently combine different information sources in a quantifiable fashion to satisfy an information need, in this case for regulatory safety assessments. With increasing awareness of the limitations of each individual tool and the development of highly targeted tests and predictions, the need for combining pieces of evidence increases. The discussions that took place during this workshop, which brought together a group of experts coming from different related areas, illustrate the current state of the art of ITS, as well as promising developments and identifiable challenges. The case of skin sensitization was taken as an example to understand how possible ITS can be constructed, optimized and validated. This will require embracing and developing new concepts such as adverse outcome pathways (AOP), advanced statistical learning algorithms and machine learning, mechanistic validation and “Good ITS Practices”.JRC.I.5-Systems Toxicolog

Alternative (non-animal) Methods for Cosmetics Testing: Current Status and Future Prospects

This 335 pages monograph was prepared in the context of the 7th Amendment of the Cosmetics Directive for establishing the timetables for phasing out animal testing. It is the result of a work that involved 75 scientists representing 14 stakeholders. The experts have evaluated for the 11 human health effects of concern: (1) which alternative methods are currently available, (2) what is their current status, (3) the time that would be necessary to achieve validation of each test method, and the time necessary to achieve replacement of the animal test. Attached are the table of contents, the overview, and the executive summary of the monograph.JRC.I.2-Validation of biomedical testing method

Ocular Toxicity

The assessment of ocular toxicity, (i.e., eye irritation and serious eye damage) is important to ensure the safety of products and their components used in our daily life. In several EU legislations related to chemicals and products, the generation of information on eye irritation and serious eye damage represents a standard requirement. The traditional eye irritation test, also known as the Draize eye test. is performed on albino rabbits. Ethical and scientific considerations as well as legal requirements in EU legislations have triggered the development and validation of alternative methods to the Draize test. Since no in vitro test taken individually can nowadays fully replace the Draize eye test, testing schemes combining strengths of particular in vitro assays were proposed The conceptual framework of testing strategies based on a Bottom-Up or Top-Down approach, are described in this chapter. In addition, a summary of the most promising in vitro test methods as well as their validation and regulatory acceptance status is given. Finally some strategic proposals on the way forward in this area are made.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