Conceptual basis for the development of guidance for the use of biomarkers of effect in regulatory risk assessment of chemicals.

This Scientific Report was carried out in the context of the self-task mandate (M-2023-00097) of the EFSA's Scientific Committee on 'Guidance on the use of biomarkers of effect in regulatory risk assessment of chemicals'. In the first phase, the project on biomarkers of effect started with a feasibility study (EFSA-Q-2024-00128), with the intention to look closer at definitions and descriptions of biomarkers of effect, as well as to explore several concepts related to the context of application and other scientific principles to be further considered for its development. In addition, relevant activities, initiatives and knowledge in this area were collected and analysed within a complementary mapping study.

Risks for animal and human health related to the presence of polychlorinated naphthalenes (PCNs) in feed and food.

EFSA was asked for a scientific opinion on the risks for animal and human health related to the presence of polychlorinated naphthalenes (PCNs) in feed and food. The assessment focused on hexaCNs due to very limited data on other PCN congeners. For hexaCNs in feed, 217 analytical results were used to estimate dietary exposures for food-producing and non-food-producing animals; however, a risk characterisation could not be performed because none of the toxicological studies allowed identification of reference points.

The inter-laboratory validation study of EpiSensA for predicting skin sensitization potential.

The Epidermal Sensitization Assay (EpiSensA) is a reconstructed human epidermis (RhE)-based gene expression assay for predicting the skin sensitization potential of chemicals. Since the RhE model is covered by a stratified stratum corneum, various kinds of test chemicals, including lipophilic ones and pre-/pro-haptens, can be tested with a route of exposure akin to an in vivo assay and human exposure. This article presents the results of a formally managed validation study of the EpiSensA that was carried out by three participating laboratories.

Application of evidence-based methods to construct mechanism-driven chemical assessment frameworks.

The workshop titled “Application of evidence-based methods to construct mechanism-driven chemical assessment frameworks” was co-organized by the Evidence-based Toxicology Collaboration and the European Food Safety Authority (EFSA) and hosted by EFSA at its headquarters in Parma, Italy on October 2 and 3, 2019. The goal was to explore integration of systematic review with mechanistic evidence evaluation. Participants were invited to work on concrete products to advance the exploration of how evidence-based approaches can support the development and application of adverse outcome pathways (AOP) in chemical risk assessment.

Inter-laboratory performance of ICE histopathology scoring to identify UN GHS Category 1 surfactants and non-extreme pH detergents.

The inter-laboratory performance of Isolated Chicken Eye (ICE) histopathology scoring was assessed for predicting EU CLP/UN GHS Cat. 1 surfactants. Furthermore, the predictive capacity of ICE histopathology was evaluated for the combined dataset of surfactants and existing data for non-extreme pH (2 < pH < 11.

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.

Value and limitation of bioassays to support the application of the threshold of toxicological concern to prioritise unidentified chemicals in food contact materials.

Some of the chemicals in materials used for packaging food may leak into the food, resulting in human exposure. These include so-called Non-intentionally Added Substances (NIAS), many of them being unidentified and toxicologically uncharacterized. This raises the question of how to address their safety.

Alternatives to Carbon Dioxide-Taking Responsibility for Humanely Ending the Life of Animals.

Carbon dioxide (CO) is commonly used to kill rodents. However, a large body of research has now established that CO is aversive to them. A multidisciplinary symposium organized by the Swiss Federal Food Safety and Veterinary Office discussed the drawbacks and alternatives to CO in euthanasia protocols for laboratory animals.

The HaCaT/THP-1 Cocultured Activation Test (COCAT) for skin sensitization: a study of intra-lab reproducibility and predictivity.

The Cocultured Activation Test (COCAT) consists of cocultured HaCaT (human keratinocyte cell line) and THP-1 cells (surrogate of antigen presenting cells). Individually, these cell lines are used to address key event 2 and 3 of the skin sensitization Adverse Outcome Pathway (AOP). Their exposure in coculture was found to have the potential to increase their response to sensitizing chemicals, enable the detection of pro-haptens and support the identification of skin sensitization potency.

Workshop on the validation and regulatory acceptance of innovative 3R approaches in regulatory toxicology - Evolution versus revolution.

At a joint workshop organized by RIVM and BfR, international experts from governmental institutes, regulatory agencies, industry, academia and animal welfare organizations discussed and provided recommendations for the development, validation and implementation of innovative 3R approaches in regulatory toxicology. In particular, an evolutionary improvement of our current approach of test method validation in the context of defined approaches or integrated testing strategies was discussed together with a revolutionary approach based on a comprehensive description of the physiological responses of the human body to chemical exposure and the subsequent definition of relevant and predictive in vitro, in chemico or in silico methods. A more comprehensive evaluation of biological relevance, scientific validity and regulatory purpose of new test methods and assessment strategies together with case studies that provide practical experience with new approaches were discussed as essential steps to build up the necessary confidence to facilitate regulatory acceptance.

One science-driven approach for the regulatory implementation of alternative methods: A multi-sector perspective.

EU regulations call for the use of alternative methods to animal testing. During the last decade, an increasing number of alternative approaches have been formally adopted. In parallel, new 3Rs-relevant technologies and mechanistic approaches have increasingly contributed to hazard identification and risk assessment evolution.

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.

Workshop on acceleration of the validation and regulatory acceptance of alternative methods and implementation of testing strategies.

This report describes the proceedings of the BfR-RIVM workshop on validation of alternative methods which was held 23 and 24 March 2017 in Berlin, Germany. Stakeholders from governmental agencies, regulatory authorities, universities, industry and the OECD were invited to discuss current problems concerning the regulatory acceptance and implementation of alternative test methods and testing strategies, with the aim to develop feasible solutions. Classical validation of alternative methods usually involves one to one comparison with the gold standard animal study.

Good cell culture practices &in vitro toxicology.

Good Cell Culture Practices (GCCP) is of high relevance to in vitro toxicology. The European Society of Toxicology In Vitro (ESTIV), the Center for Alternatives for Animal Testing (CAAT) and the In Vitro Toxicology Industrial Platform (IVTIP) joined forces to address by means of an ESTIV 2016 pre-congress session the different aspects and applications of GCCP. The covered aspects comprised the current status of the OECD guidance document on Good In Vitro Method Practices, the importance of quality assurance for new technological advances in in vitro toxicology including stem cells, and the optimized implementation of Good Manufacturing Practices and Good Laboratory Practices for regulatory testing purposes.

Evolving the Principles and Practice of Validation for New Alternative Approaches to Toxicity Testing.

Validation is essential for the translation of newly developed alternative approaches to animal testing into tools and solutions suitable for regulatory applications. Formal approaches to validation have emerged over the past 20 years or so and although they have helped greatly to progress the field, it is essential that the principles and practice underpinning validation continue to evolve to keep pace with scientific progress. The modular approach to validation should be exploited to encourage more innovation and flexibility in study design and to increase efficiency in filling data gaps.

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.

Introduction.

Alternative approaches to animal testing are gaining momentum with an increasing number of test methods obtaining international acceptance, thanks in large part to the validation efforts conducted on these assays. The principles and process of validation were first established in the 1990s in Europe and USA, and further gained international recognition ensuring the broader acceptance of alternative test methods at a regulatory level. If these principles were successful in pioneering the regulatory acceptance of alternative methods for less complex endpoints, an evolution of concepts is needed to embrace emerging technologies and the increased complexity of endpoints.

Brazilian Center for the Validation of Alternative Methods (BraCVAM) and the process of validation in Brazil.

The need for the creation of a Brazilian centre for the validation of alternative methods was recognised in 2008, and members of academia, industry and existing international validation centres immediately engaged with the idea. In 2012, co-operation between the Oswaldo Cruz Foundation (FIOCRUZ) and the Brazilian Health Surveillance Agency (ANVISA) instigated the establishment of the Brazilian Center for the Validation of Alternative Methods (BraCVAM), which was officially launched in 2013. The Brazilian validation process follows OECD Guidance Document No.

Physician assistant program directors' attitudes, practices, and plans regarding financial compensation to clinical sites.

Purpose: Some physician assistant (PA) program directors believe paying clinicians and administrators for clinical sites is fair and necessary, while others regard such practices as undermining traditional altruistic motivations for precepting. The purpose of this study was to assess PA program directors' attitudes on this topic and describe current practices and future plans regarding compensation to clinical sites.

Methods: A cross-sectional descriptive survey was sent to directors of PA programs with continuing and provisional accreditation status in 2012.

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

A.I.S.

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.

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.

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.

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.