Report of the First ONTOX Stakeholder Network Meeting: Digging Under the Surface of ONTOX Together With the Stakeholders.

The first Stakeholder Network Meeting of the EU Horizon 2020-funded ONTOX project was held on 13-14 March 2023, in Brussels, Belgium. The discussion centred around identifying specific challenges, barriers and drivers in relation to the implementation of non-animal new approach methodologies (NAMs) and probabilistic risk assessment (PRA), in order to help address the issues and rank them according to their associated level of difficulty. ONTOX aims to advance the assessment of chemical risk to humans, without the use of animal testing, by developing non-animal NAMs and PRA in line with 21st century toxicity testing principles. Stakeholder groups (regulatory authorities, companies, academia, non-governmental organisations) were identified and invited to participate in a meeting and a survey, by which their current position in relation to the implementation of NAMs and PRA was ascertained, as well as specific challenges and drivers highlighted. The survey analysis revealed areas of agreement and disagreement among stakeholders on topics such as capacity building, sustainability, regulatory acceptance, validation of adverse outcome pathways, acceptance of artificial intelligence (AI) in risk assessment, and guaranteeing consumer safety. The stakeholder network meeting resulted in the identification of barriers, drivers and specific challenges that need to be addressed. Breakout groups discussed topics such as hazard versus risk assessment, future reliance on AI and machine learning, regulatory requirements for industry and sustainability of the ONTOX Hub platform. The outputs from these discussions provided insights for overcoming barriers and leveraging drivers for implementing NAMs and PRA. It was concluded that there is a continued need for stakeholder engagement, including the organisation of a 'hackathon' to tackle challenges, to ensure the successful implementation of NAMs and PRA in chemical risk assessment.

Inhibitors of SARS-CoV-2 main protease: Biological efficacy and toxicity aspects.

The emergence of the highly contagious respiratory disease, COVID-19, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a significant global public health concern. To combat this virus, researchers have focused on developing antiviral strategies that target specific viral components, such as the main protease (Mpro), which plays a crucial role in SARS-CoV-2 replication. While many compounds have been identified as potent inhibitors of Mpro, only a few have been translated into clinical use due to the potential risk-benefit trade-offs. Development of systemic inflammatory response and bacterial co-infection in patients belong to severe, frequent complications of COVID-19. In this context, we analysed available data on the anti-inflammatory and antibacterial activities of the SARS-CoV-2 Mpro inhibitors for possible implementation in the treatment of complicated and long COVID-19 cases. Synthetic feasibility and ADME properties were calculated and included for better characterisation of the compounds' predicted toxicity. Analysis of the collected data resulted in several clusters pointing to the most prospective compounds for further study and design. The complete tables with collected data are attached in Supplementary material for use by other researchers.

The Safety Assessment of Cosmetic Perfumes by Using In Chemico and In Vitro Methods in Combination with GC-MS/MS Analysis.

Animal testing has been prohibited for the safety assessment of cosmetic ingredients or finished products. Thus, alternative non-animal methods, followed by confirmatory clinical studies on human volunteers, should be used as the sole legally acceptable approach within the EU. The safety assessment of cosmetic products requires the involvement of multiple scientific disciplines, including analytical chemistry and biomedicine, as well as in chemico, in vitro and in silico toxicology. Recent data suggest that fragrance components may exert multiple adverse biological effects, e.g. cytotoxicity, skin sensitisation, (photo)genotoxicity, mutagenicity, reprotoxicity and endocrine disruption. Therefore, a pilot study was conducted with selected samples of fragrance-based products, such as deodorant, eau de toilette and eau de parfum, with the aim of integrating results from a number of alternative non-animal methods suitable for the detection of the following toxicological endpoints: cytotoxicity (with 3T3 Balb/c fibroblasts); skin sensitisation potential (in chemico method, DPRA); skin sensitisation potential (LuSens in vitro method, based on human keratinocytes); genotoxicity potential (in vitro Comet assay with 3T3 Balb/c cells); and endocrine disruption (in vitro YES/YAS assay). The presence of twenty-four specific known allergens in the products was determined by using GC-MS/MS. The strategies for estimation of the NOAEL of a mixture of allergens, which were proposed by the Scientific Committee on Consumer Products in their 'Opinion on Tea tree oil' document and by the Norwegian Food Safety Authority in their 'Risk Profile of Tea tree oil' report, were used as models for the NOAEL estimation of the mixtures of allergens that were identified in the individual samples tested in this study.

The "Big Three" in biocompatibility testing of medical devices: implementation of alternatives to animal experimentation-are we there yet?

Biocompatibility testing ensures the safety of medical devices by assessing their compatibility with biological systems and their potential to cause harm or adverse reactions. Thus, it is a critical part of the overall safety evaluation process for medical devices. Three primary types of biocompatibility tests-cytotoxicity, irritation, and sensitisation assessment-are standard for nearly all medical devices. However, additional biocompatibility tests, such as genotoxicity, systemic toxicity, hemocompatibility, and implantation studies, may also be necessary, depending on the device's nature and intended use. The testing is partly conducted in vitro, but the industry still heavily relies on animal experiments. Compared to other industrial sectors, implementing alternatives in medical device biocompatibility testing has been notably slower. This delay can be attributed to the absence of specific validation processes tailored to medical devices and the resulting hesitation regarding the predictive capacity of these alternative methods despite their successful applications in other domains. This review focuses on the progress and obstacles to implementing new approach methodologies in the areas of cytotoxicity, irritation and sensitisation testing of medical devices. While challenges persist in adopting these innovative methods, the trend towards embracing alternatives remains robust. This trend is driven by technological advancements, ethical considerations, and growing industrial interest and support, all collectively contributing to advancing safer and more effective medical devices.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

Safer chemicals using less animals: kick-off of the European ONTOX project.

The 3Rs concept, calling for replacement, reduction and refinement of animal experimentation, is receiving increasing attention around the world, and has found its way to legislation, in particular in the European Union. This is aligned by continuing high-level efforts of the European Commission to support development and implementation of 3Rs methods. In this respect, the European project called "ONTOX: ontology-driven and artificial intelligence-based repeated dose toxicity testing of chemicals for next generation risk assessment" was recently initiated with the goal to provide a functional and sustainable solution for advancing human risk assessment of chemicals without the use of animals in line with the principles of 21st century toxicity testing and next generation risk assessment. ONTOX will deliver a generic strategy to create new approach methodologies (NAMs) in order to predict systemic repeated dose toxicity effects that, upon combination with tailored exposure assessment, will enable human risk assessment. For proof-of-concept purposes, focus is put on NAMs addressing adversities in the liver, kidneys and developing brain induced by a variety of chemicals. The NAMs each consist of a computational system based on artificial intelligence and are fed by biological, toxicological, chemical and kinetic data. Data are consecutively integrated in physiological maps, quantitative adverse outcome pathway networks and ontology frameworks. Supported by artificial intelligence, data gaps are identified and are filled by targeted in vitro and in silico testing. ONTOX is anticipated to have a deep and long-lasting impact at many levels, in particular by consolidating Europe's world-leading position regarding the development, exploitation, regulation and application of animal-free methods for human risk assessment of chemicals.

Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Qualitative and Quantitative Analysis of Certain Aspects of the Cytotoxic and Genotoxic Hazard of Hospital Wastewaters by Using a Range of In Vitro Assays.

Health care facilities and hospitals generate significant amounts of wastewater which are released into the sewage system, either after a preliminary treatment or without any further treatment. Hospital wastewater may contain large amounts of hazardous chemicals and pharmaceuticals, some of which cannot be eliminated entirely by wastewater treatment plants. Moreover, hospital effluents may be loaded with a plethora of pathogenic microorganisms or other microbiota and microbiome residues. The need to monitor hospital effluents for their genotoxic hazard is of high importance, as detailed information is scarce. DNA-based information can be acquired directly from samples through the application of various molecular methods, while cell-based biomonitoring assays can provide important information about impaired cellular pathways or mechanisms of toxicity without prior knowledge of the identity of each toxicant. In our study, we evaluated samples of chlorinated hospital wastewater discharged into the sewage system after this disinfection process. The assessment of cytotoxicity, genotoxicity and mutagenicity of the hospital effluents was performed in vitro by using a broad battery of biomonitoring assays that are relevant for human health effects. All the tested hospital wastewater samples could be classified as potentially genotoxic, and it is concluded that the microbiota present in hospital wastewater might contribute to this genotoxic potential.

Sensitization potential of medical devices detected by in vitro and in vivo methods.

Medical devices must be tested before marketing in accordance with ISO EN 10993-10 in order to avoid skin sensi­tization. This standard predominantly refers to the in vivo test but does not exclude the use of in vitro methods that have been sufficiently technically and scientifically validated for medical device testing. It is foreseen that, due to the complexity of the sensitization endpoint, a combination of several methods will be needed to address all key events occurring in the sensitization process. The objective of this pilot study was to evaluate the sensitization potential of selected medical devices using a combination of in chemico (DPRA, OECD TG 442C) and in vitro (LuSens, OECD TG 442D) methods in comparison with the in vivo (LLNA DA, OECD TG 442A) method and to suggest a possible testing strategy for the safety assessment of medical device extracts. Overall, one of the 42 tested samples exhibited positive results in all employed test methods, while 33 samples were predicted as non-sensitizing in all three performed methods. This study demonstrated good agreement between in vitro and in vivo results regarding non-sensitizing samples; however, some discrepancies in positive classification were recorded. A testing strategy is suggested in which negative results are accepted and any positive results in the in chemico or in vitro tests are followed up with a third in vitro test and evaluated in accordance with the "2 out of 3 approach". This strategy may reduce and replace animal use for testing the sensitization potential of medical devices.

Standardised Reconstructed Skin Models in Toxicology and Pharmacology: State of the Art and Future Development.

Three-dimensional (3D) reconstructed human skin (RhS) models featuring fully-differentiated characteristics of in vivo human epidermis have been known for almost 40 years. In this chapter, the topic of commercial in vitro tissue models is described, taking RhS models as an example. The need for highly standardised models is evident for regulatory testing purposes, e.g. the classification and labelling of chemicals and formulations, as well as for pharmacology-oriented research and drug development. Following the standardisation of RhS model production by commercial developers, international validation studies and regulatory acceptance, 3D RhS models are now used globally in both industrial and academic research laboratories. Industrial production of standardised 3D RhS models involves GMP-compliant processes together with ISO 9001 documentation in order to control and ensure reproducibility and quality. Key biological, functional, and performance features that are addressed in industrial production include barrier properties, histological and immunohistochemical characterisation, lipid profile characterisation, and tissue viability before and after transport. An up-to-date survey of commercial RhS tissue producers and the regulatory acceptance status of major safety, hazard, and efficacy assays currently available to chemical and pharmaceutical industries is presented in this chapter. Safety and ethical concerns related to the use of human tissue in the industrial production of RhS models are discussed. Finally, innovative approaches to the production of standardised 3D RhS models including automated production, development of more representative 3D RhS models using advanced additive manufacturing tools, microfluidics technologies, and bioprinting are presented. The future outlook for 3D RhS models includes a prevalence of high-quality models which will be fabricated by end-users rather than commercial producers. These will overcome problems with shipments and customs clearance that many users still face when buying RhS from overseas commercial suppliers. Open-source technologies and commercial components for "do-it-yourself" RhS will significantly change the skin model market as well as regulatory acceptance of open-source models during the next decade. All of these developments and improvements will together allow more widespread use of in vitro RhS models for broader application as animal replacements in areas ranging from industrial and regulatory toxicology and pharmacology, to drug development and personalised medicine.

Tissue-on-a-Chip: Microphysiometry With Human 3D Models on Transwell Inserts.

Microphysiometry has proved to be a useful tool for monitoring the energy metabolism of living cells and their interactions with other cells. The technique has mainly been used for monitoring two-dimensional (2D) monolayers of cells. Recently, our group showed that it is also possible to monitor the extracellular acidification rate and transepithelial electrical resistance (TEER) of 3D skin constructs in an automated assay maintaining an air-liquid interface (ALI) with a BioChip extended by 3D-printed encapsulation. In this work, we present an optimized multichannel intestine-on-a-chip for monitoring the TEER of the commercially available 3D small intestinal tissue model (EpiIntestinalTM from MatTek). Experiments are performed for 1 day, during which a 60 min cycle is repeated periodically. Each cycle consists of three parts: (1) maintain ALI; (2) application of the measurement medium or test substance; and (3) the rinse cycle. A cytotoxic and barrier-disrupting benchmark chemical (0.2% sodium dodecyl sulfate) was applied after 8 h of initial equilibration. This caused time-dependent reduction of the TEER, which could not be observed with typical cytotoxicity measurement methods. This work represents a proof-of-principle of multichannel time-resolved TEER monitoring of a 3D intestine model using an automated ALI. Reconstructed human tissue combined with the Intelligent Mobile Lab for In vitro Diagnostic technology represents a promising research tool for use in toxicology, cellular metabolism studies, and drug absorption research.

Toxicological testing of a photoactive phthalocyanine-based antimicrobial substance.

The aim of the study was toxicological testing of an innovative and efficient antimicrobial agent based on photoactive phthalocyanine (Pc) derivative. A promising Aluminium phthalocyanine (AlPc) with efficient and stable antimicrobial effects was subjected to a battery of toxicological tests to avoid local and systemic toxicity hazard. In compliance with the current European legislation restricting the use of experimental animals, the methods comprised exclusively in vitro procedures based on cellular and tissue models of human origin or mimicking human tissues. The battery of toxicological tests to identify local toxicity included skin corrosion/irritation, eye irritation, and phototoxicity. The basic systemic toxicity tests included acute toxicity, skin sensitization, genotoxicity, and endocrine disruption. The results showed that AlPc induced skin and eye irritation, exhibited borderline sensitization potential and mutagenic potential in one test strain of the Ames test, which was not confirmed in the chromosome aberration test. The AlPc was found to be phototoxic. The results from the cytotoxicity test designed for acute oral toxicity estimation were not conclusive, the acute toxicity potential has to be determined by conventional tests in vivo. Regarding endocrine disruption, no agonistic activity of the AlPc on human estrogen receptor α, nor human androgen receptor was observed. The skin penetration/absorption test revealed that the AlPc has not penetrated into the dermis and receptor fluid, confirming no risk of systemic exposure via the bloodstream.