Chantra Eskes

A roadmap for the development of alternative (non-animal) methods for systemic toxicity testing - t4 report

Systemic toxicity testing forms the cornerstone for the safety evaluation of substances. Pressures to move from traditional animal models to novel technologies arise from various concerns, including: the need to evaluate large numbers of previously untested chemicals and new products (such as nanoparticles or cell therapies), the limited predictivity of traditional tests for human health effects, duration and costs of current approaches, and animal welfare considerations. The latter holds especially true in the context of the scheduled 2013 marketing ban on cosmetic ingredients tested for systemic toxicity. Based on a major analysis of the status of alternative methods (Adler et al., 2011) and its independent review (Hartung et al., 2011), the present report proposes a roadmap for how to overcome the acknowledged scientific gaps for the full replacement of systemic toxicity testing using animals. Five whitepapers were commissioned addressing toxicokinetics, skin sensitization, repeated-dose toxicity, carcinogenicity, and reproductive toxicity testing. An expert workshop of 35 participants from Europe and the US discussed and refined these whitepapers, which were subsequently compiled to form the present report. By prioritizing the many options to move the field forward, the expert group hopes to advance regulatory science.

A proposed eye irritation testing strategy to reduce and replace in vivo studies using Bottom–Up and Top–Down approaches

In spite of over 20 years of effort, no single in vitro assay has been developed and validated as a full regulatory replacement for the Draize Eye Irritation test. However, companies have been using in vitro methods to screen new formulations and in some cases as their primary assessment of eye irritation potential for many years. The present report shows the outcome of an Expert Meeting convened by the European Centre for the Validation of Alternative Methods in February 2005 to identify test strategies for eye irritation. In this workshop test developers/users were requested to nominate methods to be considered as a basis for the identification of such testing strategies. Assays were evaluated and categorized based on their proposed applicability domains (e.g., categories of irritation severity, modes of action, chemical class, physicochemical compatibility). The analyses were based on the data developed from current practice and published studies, the ability to predict depth of injury (within the applicable range of severity), modes of action that could be addressed and compatibility with different physiochemical forms. The difficulty in predicting the middle category of irritancy (e.g. R36, GHS Categories 2A and 2B) was recognized. The testing scheme proposes using a Bottom-Up (begin with using test methods that can accurately identify non-irritants) or Top-Down (begin with using test methods that can accurately identify severe irritants) progression of in vitro tests (based on expected irritancy). Irrespective of the starting point, the approach would identify non-irritants and severe irritants, leaving all others to the (mild/moderate) irritant GHS 2/R36 categories.

The value of alternative testing for neurotoxicity in the context of regulatory needs

Detection and characterisation of chemical-induced toxic effects in the central and peripheral nervous system represent a major challenge for employing newly developed technologies in the field of neurotoxicology. Precise cellular predictive test batteries for chemical-induced neurotoxicity are increasingly important for regulatory decision making, but also the most efficient way to keep costs and time of testing within a reasonable margin. Current in vivo test methods are based on behavioural and sensory perturbations coupled with routine histopathological investigations. In spite of the empirical usefulness of these tests, they are not always sensitive enough and often, they do not provide information that facilitates a detailed understanding of potential mechanisms of toxicity, thus enabling predictions. In general, such in vivo tests are unsuitable for screening large number of agents. One way to meet the need for more powerful and comprehensive tests via an extended scientific basis is to study neurotoxicity in specific cell types of the brain and to derive generalised mechanisms of action of the toxicants from such series of experiments. Additionally, toxicokinetic models are to be developed in order to give a rough account for the whole absorption, distribution, metabolism, excretion (ADME) process including the blood-brain barrier (BBB). Therefore, an intensive search for the development of alternative methods using animal and human-based in vitro and in silico models for neurotoxic hazard assessment is appropriate. In particular, neurotoxicology represents one of the major challenges to the development of in vitro systems, as it has to account also for heterogeneous cell interactions of the brain which require new biochemical, biotechnological and electrophysiological profiling methods for reliable alternative ways with a high throughput.

A feasibility study developing an integrated testing strategy assessing skin irritation potential of chemicals

The regulatory assessment of chemical safety is still driven by hazard testing in animals. The Registration, Evaluation, Authorisation and restriction of Chemicals (REACH) legislation and new technologies require a shift in the way in which safety assessments are conducted. Integrated testing strategies (ITSs) help in providing such a framework. Many of the ITS building blocks are already in use, but the concepts for their integration and application in a regulatory setting have yet to be fully implemented. This paper describes a feasibility study investigating how a combination of in silico, in vitro, and in vivo information could be applied in the assessment of skin irritation hazard. Therefore, a database of 100 existing and new chemicals was compiled. A number of strategies, both animal-free and inclusive of animal data were constructed and subsequently evaluated considering predictive capacities, severity of misclassifications and testing costs. Comparison of constructed ITS based on these assessment parameters identified best performing strategies for chemical classification. However, defining the in vivo test as the reference test limited the evaluation of the ITS inclusive of animal data. This study demonstrated that ITS can be constructed, evaluated and compared in a systematic fashion. To promote ITS, further guidance on construction and multi-parameter evaluation need to be developed.

Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations

Validated in vitro methods for skin corrosion and irritation were adopted by the OECD and by the European Union during the last decade. In the EU, Switzerland and countries adopting the EU legislation, these assays may allow the full replacement of animal testing for identifying and classifying compounds as skin corrosives, skin irritants, and non irritants. In order to develop harmonised recommendations on the use of in vitro data for regulatory assessment purposes within the European framework, a workshop was organized by the Swiss Federal Office of Public Health together with ECVAM and the BfR. It comprised stakeholders from various European countries involved in the process from in vitro testing to the regulatory assessment of in vitro data. Discussions addressed the following questions: (1) the information requirements considered useful for regulatory assessment; (2) the applicability of in vitro skin corrosion data to assign the corrosive subcategories as implemented by the EU Classification, Labelling and Packaging Regulation; (3) the applicability of testing strategies for determining skin corrosion and irritation hazards; and (4) the applicability of the adopted in vitro assays to test mixtures, preparations and dilutions. Overall, a number of agreements and recommendations were achieved in order to clarify and facilitate the assessment and use of in vitro data from regulatory accepted methods, and ultimately help regulators and scientists facing with the new in vitro approaches to evaluate skin irritation and corrosion hazards and risks without animal data.

Suitability of histopathology as an additional endpoint to the Isolated Chicken Eye Test for classification of non-extreme pH detergent and cleaning products

A.I.S.E. investigated the suitability of histopathological evaluations as an additional endpoint to the regulatory adopted ICE in vitro test method (OECD TG 438) to identify non-extreme pH detergent and cleaning products that require classification as EU CLP/UN GHS Category 1 (serious eye damage). To this aim, a total of 30 non-extreme pH products covering the range of in vivo classifications for eye irritation, and representing various product categories were tested. Epithelium vacuolation (mid and lower layers) and erosion (at least moderate) were found to be the most relevant histopathological effects induced by products classified in vivo as Category 1. Histopathology criteria specifically developed for non-extreme pH detergent and cleaning products were shown to correctly identify materials classified as Category 1 based on in vivo persistent effects, and to significantly increase the overall sensitivity of the standard ICE prediction model for Category 1 identification (to 75%) whilst maintaining a good concordance (73%). In contrast, use of EU CLP additivity approach for classification of mixtures was considerably less predictive, with a concordance of only 27%, and 100% over-predictions of non-Category 1 products. As such, use of histopathology as an addition to the ICE test method was found suitable to identify EU CLP/UN GHS Category 1 non-extreme pH detergent and cleaning products and to allow a better discrimination from Category 2 products.

Validation study on the Ocular Irritection® assay for eye irritation testing

Both a prospective and a retrospective validation study were undertaken to assess the suitability of the Ocular Irritection assay to discriminate ocular hazards as defined by the OECD and UN Globally Harmonized System (UN GHS) for classification. The primary focus of the study was to evaluate the usefulness of the Ocular Irritection assay to reliably discriminate chemicals not requiring classification (UN GHS non-classified), from classified chemicals (UN GHS Categories 1 and 2). Furthermore a post-hoc evaluation was carried out to evaluate the usefulness of the assay to discriminate chemicals inducing serious eye damage (UN GHS Category 1) from other classes. The prospective validation study was conducted between 2009 and 2012 following internationally agreed principles. A set of 56 coded test chemicals for which quality and/or peer-reviewed in vivo data were available were used to obtain prospective data on the assays reliability (reproducibility within and between laboratories) and relevance (predictive capacity). The assay showed good within-laboratory variability, transferability including to a naïve laboratory, and between-laboratory concordance of classifications (82% for the discrimination of non-classified from classified chemicals, and 83% for the discrimination of Category 1 from other classes). The obtained prospective data were then combined with existing data on the Ocular Irritection collected from various sources, totaling 88 chemicals with parallel in vivo and in vitro data to obtain a comprehensive assessment of the test method performances. The enlarged dataset comprised 43 non-classified, 25 Category 2 and 20 Category 1 chemicals according to the UN GHS classification. When used for the identification of UN GHS non-classified versus classified materials (based on the existing cut-off of 12.5) the Ocular Irritection assay showed an overall a sensitivity of 93% and a specificity of 58%. An evaluation on possible reasons for misclassification identified some organic functional groups (acrylate, carboxamide and cycloalkene) to correlate with the observed mispredictions. If these functional groups were excluded from the Ocular Irritection applicability domain, the obtained dataset (n=79 chemicals distributed as 41 UN GHS Classified and 38 Non-Classified chemicals) had an overall sensitivity of 98%, and specificity of 63%, which is in line with currently adopted test methods. When used for the identification of UN GHS Category 1 versus other categories (based on the existing cut-off of 30.0) the Ocular Irritection assay showed an overall specificity of 81% and a sensitivity of 50% which is again in line with currently adopted test methods. The Ocular Irritection assay appeared therefore as a useful test method to predict chemicals not requiring classification for eye hazards according to the UN GHS classification system. Furthermore the method was found suitable to identify serious/irreversible eye damage (UN GHS Category 1). The detailed documentation and results of the study have been submitted to an internationally recognized validation centre for peer-review.

Suitability of the isolated chicken eye test for classification of extreme pH detergents and cleaning products.

A.I.S.E. investigated the suitability of the regulatory adopted ICE in vitro test method (OECD TG 438) with or without histopathology to identify detergent and cleaning formulations having extreme pH that require classification as EU CLP/UN GHS Category 1. To this aim, 18 extreme pH detergent and cleaning formulations were tested covering both alkaline and acidic extreme pHs. The ICE standard test method following OECD Test Guideline 438 showed good concordance with in vivo classification (83%) and good and balanced specificity and sensitivity values (83%) which are in line with the performances of currently adopted in vitro test guidelines, confirming its suitability to identify Category 1 extreme pH detergent and cleaning products. In contrast to previous findings obtained with non-extreme pH formulations, the use of histopathology did not improve the sensitivity of the assay whilst it strongly decreased its specificity for the extreme pH formulations. Furthermore, use of non-testing prediction rules for classification showed poor concordance values (33% for the extreme pH rule and 61% for the EU CLP additivity approach) with high rates of over-prediction (100% for the extreme pH rule and 50% for the additivity approach), indicating that these non-testing prediction rules are not suitable to predict Category 1 hazards of extreme pH detergent and cleaning formulations.

International Harmonization and Cooperation in the Validation of Alternative Methods

The development and validation of scientific alternatives to animal testing is important not only from an ethical perspective (implementation of 3Rs), but also to improve safety assessment decision making with the use of mechanistic information of higher relevance to humans. To be effective in these efforts, it is however imperative that validation centres, industry, regulatory bodies, academia and other interested parties ensure a strong international cooperation, cross-sector collaboration and intense communication in the design, execution, and peer review of validation studies. Such an approach is critical to achieve harmonized and more transparent approaches to method validation, peer-review and recommendation, which will ultimately expedite the international acceptance of valid alternative methods or strategies by regulatory authorities and their implementation and use by stakeholders. It also allows achieving greater efficiency and effectiveness by avoiding duplication of effort and leveraging limited resources. In view of achieving these goals, the International Cooperation on Alternative Test Methods (ICATM) was established in 2009 by validation centres from Europe, USA, Canada and Japan. ICATM was later joined by Korea in 2011 and currently also counts with Brazil and China as observers. This chapter describes the existing differences across world regions and major efforts carried out for achieving consistent international cooperation and harmonization in the validation and adoption of alternative approaches to animal testing.

Advanced Good Cell Culture Practice for human primary, stem cell-derived and organoid models as well as microphysiological systems

A major reason for the current reproducibility crisis in the life sciences is the poor implementation of quality control measures and reporting standards. Improvement is needed, especially regarding increasingly complex in vitro methods. Good Cell Culture Practice (GCCP) was an effort from 1996 to 2005 to develop such minimum quality standards also applicable in academia. This paper summarizes recent key developments in in vitro cell culture and addresses the issues resulting for GCCP, e.g. the development of induced pluripotent stem cells (iPSCs) and gene-edited cells. It further deals with human stem-cell-derived models and bioengineering of organo-typic cell cultures, including organoids, organ-on-chip and human-on-chip approaches. Commercial vendors and cell banks have made human primary cells more widely available over the last decade, increasing their use, but also requiring specific guidance as to GCCP. The characterization of cell culture systems including high-content imaging and high-throughput measurement technologies increasingly combined with more complex cell and tissue cultures represent a further challenge for GCCP. The increasing use of gene editing techniques to generate and modify in vitro culture models also requires discussion of its impact on GCCP. International (often varying) legislations and market forces originating from the commercialization of cell and tissue products and technologies are further impacting on the need for the use of GCCP. This report summarizes the recommendations of the second of two workshops, held in Germany in December 2015, aiming map the challenge and organize the process or developing a revised GCCP 2.0.