3Rs implementation in veterinary vaccine batch-release testing: Current state-of-the-art and future opportunities. A webinar and workshop report.

Regulatory authorities require veterinary batch-release testing to confirm vaccine potency and safety, but these tests have traditionally relied on large numbers of laboratory animals. Advances in vaccine research and development offer increasing opportunities to replace in vivo testing, and some stakeholders have made significant progress in incorporating 3Rs elements in quality control strategies. A three-part event series entitled "3Rs Implementation in Veterinary Vaccine Batch-Release Testing: Current state-of-the-art and future opportunities" was jointly organized by the Animal-Free Safety Assessment Collaboration, HealthforAnimals, and the International Alliance of Biological Standardization. Two webinars and a workshop aimed to outline the state-of-the-art non-animal approaches for veterinary batch-release testing. The events included information on the state of the deletion of obsolete safety testing and the current initiatives implemented by European, North American, and Asian-Pacific stakeholders on 3Rs implementation and regulatory acceptance. The events contributed to a better understanding of the barriers to 3Rs implementation. Participants highlighted the need for open communication, continued collaboration between stakeholders, and international harmonization of regulatory requirements to help accelerate acceptance. Despite the challenges, the countries represented at this three-part event have shared their commitments to advancing the acceptance of alternative methods.

The future of pyrogenicity testing: Phasing out the rabbit pyrogen test. A meeting report.

The rabbit pyrogen test (RPT) was the benchmark for pyrogenicity testing, but scientific advancements have provided innovative and humane methods, such as the in vitro monocyte-activation test (MAT). However, transitioning from the RPT to the MAT has been challenging. The European Directorate for the Quality of Medicines & HealthCare, the Council of Europe, and the European Partnership for Alternative Approaches to Animal Testing jointly hosted an international conference entitled "The future of pyrogenicity testing: phasing out the rabbit pyrogen test". The conference aimed to show how the European Pharmacopoeia intends to remove the RPT from its texts by 2026, facilitate the use of MAT, and identify gaps in the suppression of RPT. The events contributed to a better understanding of the barriers to RPT replacement and acceptance of in vitro alternatives. Participants comprised stakeholders from Asia, Europe, and North America, including vaccine developers, contract laboratories, and regulators. Participants shared their replacement strategies and experiences with MAT implementation. They emphasised the need for continued cooperation between stakeholders and stressed the importance of international harmonisation of regulatory requirements to help accelerate MAT acceptance outside Europe. Despite the challenges, the willingness to eliminate the unnecessary use of RPT was common across all participants.

Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals: Responses from a survey of National Control Laboratories and National Regulatory Authorities.

The UK National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) is reviewing World Health Organization (WHO) manuals, guidelines and recommendations for vaccines and biotherapeutics to identify the extent to which animal-based testing methods are described. The aim is to recommend where updates to these documents can lead to an increased and more harmonised adoption of 3Rs principles (i.e. Replacement, Reduction and Refinement of animal tests) in the quality control and batch release testing requirements for vaccines and biotherapeutics. Improved adoption of 3Rs principles and non-animal testing strategies will help to reduce the delays and costs associated with product release testing. Developing recommendations that are widely applicable by both the manufacturers and national regulatory authorities for vaccines and biological therapeutics globally requires a detailed understanding of how different organisations view the opportunities and barriers to better integration of the 3Rs. To facilitate this, we developed and distributed a survey aimed at individuals who work for national regulatory authorities (NRAs) and/or national control laboratories (NCLs). In this paper, we present the key findings from this survey and how these will help inform the recommendations for wider integration of 3Rs approaches by WHO in their guidance documents applicable to the quality control and batch release testing of vaccines and biotherapeutics.

Ciliary Beat Frequency: Proceedings and Recommendations from a Multi-laboratory Ring Trial Using 3-D Reconstituted Human Airway Epithelium to Model Mucociliary Clearance.

The use of reconstituted human airway (RHuA) epithelial tissues to assess functional endpoints is highly relevant in respiratory toxicology, but standardised methods are lacking. In June 2015, the Institute for In Vitro Sciences (IIVS) held a technical workshop to evaluate the potential for standardisation of methods, including ciliary beat frequency (CBF). The applicability of a protocol suggested in the workshop was assessed in a multi-laboratory ring study. This report summarises the findings, and uses the similarities and differences identified between the laboratories to make recommendations for researchers in the absence of a validated method. Two software platforms for the assessment of CBF were used - Sisson-Ammons Video Analysis (SAVA; Ammons Engineering, Clio, MI, USA) and ciliaFA (National Institutes of Health, Bethesda, MD, USA). Both were utilised for multiple read temperatures, one objective strength (10×) and up to four video captures per tissue, to assess their utility. Two commercial RHuA tissue cultures were used: MucilAir™ (Epithelix, Geneva, Switzerland) and EpiAirway™ (MatTek, Ashland, MA, USA). IL-13 and procaterol were used to induce CBF-specific responses as positive controls. Further testing addressed the impact of tissue acclimation duration, the number of capture fields and objective strengths on baseline CBF readings. Both SAVA and ciliaFA reliably collected CBF data. However, ciliaFA failed to generate accurate CBF measurements above ∼10 Hz. The positive controls were effective, but were subject to inter-laboratory variability. CBF endpoints were generally uniform across replicate tissues, objective strengths and laboratories. Longer tissue acclimation increased the percentage active area, but had minimal impact on CBF. Taken together, these findings support the development and validation of a standardised CBF measurement protocol.

Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals.

Animal testing has long been integral to the development of biologicals, including vaccines. The use of animals can provide important information on potential toxicity, insights into their mechanism of action, pharmacokinetics and dynamics, physiologic distribution, and potency. However, the use of these same methods is often adopted into the post-licensure phase of the product life cycle for the monitoring of product qualities, such as potency or safety, as part of their routine batch release. The UK National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) and the World Health Organization (WHO) are collaborating on a project to review animal-based testing methods described in WHO manuals, guidelines and recommendations for biologicals to identify where updates can lead to a more harmonised adoption of 3Rs principles (i.e. Replacement, Reduction, and Refinement of animal tests) in batch release testing requirements. An international working group consisting of more than 30 representatives from pharmaceutical and biotechnology companies, national control laboratories and regulatory bodies is performing this review. This project aims to address concerns about inconsistencies in the guidance for the scientifically justified use of animal methods required for the post-licensure quality control and batch release testing of biologicals, and the near absence of recommendations for the application of 3Rs principles within the relevant guidelines. Improved adoption of 3Rs principles and non-animal testing strategies will help to reduce the delays and costs associated with product release testing and help support faster access to products by the global communities who need them most urgently.

Devising a Tier-based Skin Sensitisation Screening Strategy for Personal Care and Cosmetic Products.

Concerns regarding the use of potential skin sensitisers in personal care and cosmetic products continue to grow. The goal of this study was to develop a proof-of-concept tier-based screening strategy for the assessment of skin sensitisation potential by using non-animal methodologies. As a case example, this screening framework was applied to three WEN® by Chaz Dean cleansing conditioners. The first tier of testing utilised the Organisation for Economic Co-operation and Development (OECD) Quantitative Structure Activity Relationship Toolbox profiler to evaluate the skin sensitisation potential of individual ingredients within the formulation; a literature review was performed on the substances that generated in silico alerts. Tier 2 testing utilised the OECD in chemico Test Guideline (TG) 442C to evaluate these substances. Tier 3 testing adapted OECD TG442C to evaluate the formulated product. The literature review on the four substances that generated in silico alerts revealed that they were not sensitising at the concentrations reported in the formulated products. Tier 2 testing demonstrated that these substances were not sensitising at the concentrations tested. Finally, Tier 3 testing revealed that the evaluated cleansing conditioners had low mean percentage peptide depletion at the concentrations tested. Together, the results obtained suggest that the products tested are unlikely to induce skin sensitisation under the given experimental conditions. These findings are in agreement with other in vitro and clinical studies. The proposed tier-based testing approach may be used as a conceptual framework for the prospective safety screening of other personal care and cosmetic products. However, to establish the validity of the proposed testing strategy, further studies must be performed, including comparisons with established models.

Integrative Strategy of Testing Systems for Identification of Endocrine Disruptors Inducing Metabolic Disorders-An Introduction to the OBERON Project.

Exposure to chemical substances that can produce endocrine disrupting effects represents one of the most critical public health threats nowadays. In line with the regulatory framework implemented within the European Union (EU) to reduce the levels of endocrine disruptors (EDs) for consumers, new and effective methods for ED testing are needed. The OBERON project will build an integrated testing strategy (ITS) to detect ED-related metabolic disorders by developing, improving and validating a battery of test systems. It will be based on the concept of an integrated approach for testing and assessment (IATA). OBERON will combine (1) experimental methods (in vitro, e.g., using 2D and 3D human-derived cells and tissues, and in vivo, i.e., using zebrafish at different stages), (2) high throughput omics technologies, (3) epidemiology and human biomonitoring studies and (4) advanced computational models (in silico and systems biology) on functional endpoints related to metabolism. Such interdisciplinary framework will help in deciphering EDs based on a mechanistic understanding of toxicity by providing and making available more effective alternative test methods relevant for human health that are in line with regulatory needs. Data generated in OBERON will also allow the development of novel adverse outcome pathways (AOPs). The assays will be pre-validated in order to select the test systems that will show acceptable performance in terms of relevance for the second step of the validation process, i.e., the inter-laboratory validation as ring tests. Therefore, the aim of the OBERON project is to support the organization for economic co-operation and development (OECD) conceptual framework for testing and assessment of single and/or mixture of EDs by developing specific assays not covered by the current tests, and to propose an IATA for ED-related metabolic disorder detection, which will be submitted to the Joint Research Center (JRC) and OECD community.

Achieving scientific and regulatory success in implementing non-animal approaches to human and veterinary rabies vaccine testing: A NICEATM and IABS workshop report.

This two-day workshop, co-sponsored by NICEATM and IABS-NA, brought together over 60 international scientists from government, academia, and industry to advance alternative methods for human and veterinary Rabies Virus Vaccine (RVV) potency testing. On day one, workshop presentations focused on regulatory perspectives related to in vitro potency testing, including recent additions to the European Pharmacopoeia (5.2.14) that provide a scientific rationale for why in vivo methods may be less suitable for vaccine quality control than appropriately designed in vitro methods. Further presentations reviewed the role of the consistency approach to manufacturing and vaccine batch comparison to provide supportive data for the substitution of existing animal-based methods with in vitro assays. In addition, updates from research programs evaluating and validating RVV glycoprotein (G) quantitation by ELISA as an in vitro potency test were presented. On the second day, RVV stakeholders participated in separate human and veterinary vaccine discussion groups focused on identifying potential obstacles or additional requirements for successful implementation of non-animal alternatives to the in vivo potency test. Workshop outcomes and proposed follow up activities are discussed herein.

Reflections on the progress towards non-animal methods for acute toxicity testing of chemicals.

The acute toxicity 'six-pack' is a suite of tests for hazard identification and risk assessment, primarily conducted for the classification and labelling of industrial chemicals and agrochemicals. The 'six-pack' is designed to provide information on health hazards likely to arise from short-term exposure to chemicals via inhalation, oral and dermal routes, including the potential for eye and skin irritation/corrosion and skin sensitization. The component tests of the 'six-pack' currently rely heavily on the use of experimental animals. In 2017, the UK National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), together with the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM), and the US National Toxicology Program (NTP) Centre for the Evaluation of Alternative Methods (NICEATM) held a workshop entitled 'Towards Global Elimination of the Acute Toxicity 'Six-Pack'' to explore opportunities to use alternative (non-animal) methods for hazard identification and classification without compromising human or environmental safety. The Workshop included scientists from regulatory agencies and industrial organisations worldwide, and sought to gain a more detailed understanding of the barriers to the adoption of suitable animal-free alternatives at an international level. Among the issues addressed were: the recurring theme of validation and scientific credibility, as well as the need for international standards, an understanding of the limitations of each new/alternative method and characterisation against the variability of current animal methods. The practicality and cost of new tests was also an important consideration. However, the need for mutual acceptance, and global harmonization of requirements were thought to be the major hurdle to overcome to realise a vision of the eventual complete elimination of the current, animal test-based, acute toxicity 'six-pack'.

The 21st Century movement within the area of skin sensitization assessment: From the animal context towards current human-relevant in vitro solutions.

In a regulatory context, skin sensitization hazard and risk evaluations of manufactured products and their ingredients (e.g. cosmetics) are mandatory in several regions. Great efforts have been made within the field of 21st Century Toxicology to provide non-animal testing approaches to assess the skin allergy potential of materials (e.g. chemicals, mixtures, nanomaterials, particles). Mechanistic understanding of skin sensitization process through the adverse outcome pathway (AOP) has promoted the development of in vitro methods, demonstrating accuracies superior to the traditional animal testing. These in vitro testing approaches are based on one of the four AOP key events (KE) of skin sensitization: formation of immunogenic hapten-protein complexes (KE-1 or the molecular initiating event, MIE), inflammatory keratinocyte responses (KE-2), dendritic cell activation (KE-3), and T-lymphocyte activation and proliferation (KE-4). This update provides an overview of the historically used in vivo methods as well as the current in chemico and in cell methods with and without OECD guideline designations to analyze the progress towards human-relevant in vitro test methods for safety assessment of the skin allergenicity potential of materials. Here our focus is to review 96 in vitro testing approaches directed to the KEs of the skin sensitization AOP.

Skin toxicology and 3Rs-Current challenges for public health protection.

Driven by the fast paced development of complex test systems in vitro, mass spectrometry and omics, we finally have the tools to unravel the molecular events that underlie toxicological adversity. Yet, timely regulatory adaptation of these new tools continues to pose major challenges even for organs readily accessible such as skin. The reasons for this encompass a need for conservatism as well as the need of tests to serve an existing regulatory framework rather than to produce scientific knowledge. It is important to be aware of this in order to align regulatory skin toxicity with the 3R principles more readily. While most chemical safety testing is still based on animal data, regulatory frameworks have seen a strong push towards non-animal approaches. The endpoints corrosion, irritation, sensitisation, absorption and phototoxicity, for example, can now be covered in vitro with the corresponding test guidelines (TGs) being made available by the OECD. However, in vitro approaches tend to be more reductionist. Hence, a combination of several tests is usually preferable to achieve satisfying predictivity. Moreover, the test systems and their combined use need to be standardised and are therefore subject not only to validation but also to the ongoing development of so-called integrated approaches to testing and assessment (IATAs). Concomitantly, skin models are being refined to deliver the complexity required for increased applicability and predictivity. Given the importance of regulatory applicability for 3R-derived approaches to have a long-lasting impact, this review examines the state of regulatory implementation and perspectives, respectively.

Functional cardiotoxicity assessment of cosmetic compounds using human-induced pluripotent stem cell-derived cardiomyocytes.

There is a large demand of a human relevant in vitro test system suitable for assessing the cardiotoxic potential of cosmetic ingredients and other chemicals. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we have already established an in vitro cardiotoxicity assay and identified genomic biomarkers of anthracycline-induced cardiotoxicity in our previous work. Here, five cosmetic ingredients were studied by the new hiPSC-CMs test; kojic acid (KJA), triclosan (TS), triclocarban (TCC), 2,7-naphthalenediol (NPT), and basic red 51 (BR51) based on cytotoxicity as well as ATP assays, beating rate, and genomic biomarkers to determine the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC). The LOEC for beating rate were 400, 10, 3, >400, and 3 µM for KJA, TS, TCC, NPT, and BR51, respectively. The corresponding concentrations for cytotoxicity or ATP depletion were similar, with the exception of TS and TCC, where the cardiomyocyte-beating assay showed positive results at non-cytotoxic concentrations. Functional analysis also showed that the individual compounds caused different effects on hiPSC-CMs. While exposure to KJA, TS, TCC, and BR51 induced significant arrhythmic beating, NPT slightly decreased cell viability, but did not influence beating. Gene expression studies showed that TS and NPT caused down-regulation of cytoskeletal and cardiac ion homeostasis genes. Moreover, TS and NPT deregulated genomic biomarkers known to be affected also by anthracyclines. The present study demonstrates that hiPSC-CMs can be used to determine LOECs and NOECs in vitro, which can be compared to human blood concentrations to determine margins of exposure. Our in vitro assay, which so far has been tested with several anthracyclines and cosmetics, still requires validation by larger numbers of positive and negative controls, before it can be recommended for routine analysis.

Cell death mechanisms of the anti-cancer drug etoposide on human cardiomyocytes isolated from pluripotent stem cells.

Etoposide (ETP) and anthracyclines are applied for wide anti-cancer treatments. However, the ETP-induced cardiotoxicity remains to be a major safety issue and the underlying cardiotoxic mechanisms are not well understood. This study is aiming to unravel the cardiotoxicity profile of ETP in comparison to anthracyclines using physiologically relevant human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). Using xCELLigence real-time cell analyser (RTCA), we found that single high dose of ETP induces irreversible increase in hPSC-CMs beating rate and decrease in beating amplitude. We also identified 58 deregulated genes consisting of 33 upregulated and 25 downregulated genes in hPSC-CMs after ETP treatment. Gene ontology (GO) and pathway analysis showed that most upregulated genes are enriched in GO categories like positive regulation of apoptotic process, regulation of cell death, and mitochondria organization, whereas most downregulated genes were enriched in GO categories like cytoskeletal organization, muscle contraction, and Ca2+ ion homeostasis. Moreover, we also found upregulation in 5 miRNAs (has-miR-486-3p, has-miR-34c-5p, has-miR-4423-3p, has-miR-182-5p, and has-miR-139-5p) which play role in muscle contraction, arginine and proline metabolism, and hypertrophic cardiomyopathy (HCM). Immunostaining and transmission electron microscopy also confirmed the cytoskeletal and mitochondrial damage in hPSC-CMs treated with ETP, as well as noticeable alterations in intracellular calcium handling and mitochondrial membrane potential were also observed. The apoptosis inhibitor, Pifithrin-α, found to protect hPSC-CMs from ETP-induced cardiotoxicity, whereas hPSC-CMs treated with ferroptosis inhibitor, Liproxstatin-1, showed significant recovery in hPSC-CMs functional properties like beating rate and amplitude after ETP treatment. We suggest that the damage to mitochondria is a major contributing factor involved in ETP-induced cardiotoxicity and the activation of the p53-mediated ferroptosis pathway by ETP is likely the critical pathway in ETP-induced cardiotoxicity. We also conclude that the genomic biomarkers identified in this study will significantly contribute to develop and predict potential cardiotoxic effects of novel anti-cancer drugs in vitro.

The Fibrotic Phenotype of Human Precision-Cut Lung Slices Is Maintained after Cryopreservation.

Human precision-cut lung slices (hPCLS) prepared from fibrotic lungs recapitulate the pathophysiological hallmarks of fibrosis. These hallmark features can also be induced by treating non-fibrotic hPCLS with a fibrotic cocktail (FC). As a result, the fibrotic and fibrosis-induced hPCLS are rapidly emerging as preferred models for disease modeling and drug discovery. However, current hPCLS models are limited by tissue viability in culture, as they are usually only viable for one week after harvesting. Here, we demonstrate that the fibrotic hPCLS can be cryopreserved, stored for months, and then thawed on demand without loss of hPCLS viability or protein content for 14 days post-thawing. Cryopreservation also preserves the pro-fibrotic potential of non-fibrotic hPCLS. Specifically, when we treated the thawed non-fibrotic hPCLS with an FC, we observed significant pro-fibrotic cytokine secretion and elevated tissue stiffness. These pro-fibrotic changes were inhibited by the small-molecule tyrosine kinase inhibitor, Nintedanib. Taken together, our work indicates that a feasible solution to prolong the pre-clinical utility of fibrotic and fibrosis-induced hPCLS is cryopreservation. We anticipate that cryopreserved hPCLS will serve as an advantageous predictive model for the evaluation of pro-fibrotic pathways during acute and chronic toxicity testing.

The role of extracellular vesicles in non-small-cell lung cancer, the unknowns, and how new approach methodologies can support new knowledge generation in the field.

Extracellular vesicles (EVs) are nanosized particles released from most human cell types that contain a variety of cargos responsible for mediating cell-to-cell and organ-to-organ communications. Current knowledge demonstrates that EVs also play critical roles in many aspects of the progression of Non-Small-Cell Lung Cancer (NSCLC). Their roles range from increasing proliferative signalling to inhibiting apoptosis, promoting cancer metastasis, and modulating the tumour microenvironment to support cancer development. However, due to the limited availability of patient samples, intrinsic inter-species differences between human and animal EV biology, and the complex nature of EV interactions in vivo, where multiple cell types are present and several events occur simultaneously, the use of conventional preclinical and clinical models has significantly hindered reaching conclusive results. This review discusses the biological roles that EVs are currently known to play in NSCLC and identifies specific challenges in advancing today's knowledge. It also describes the NSCLC models that have been used to define currently-known EV functions, the limitations associated with their use in this field, and how New Approach Methodologies (NAMs), such as microfluidic platforms, organoids, and spheroids, can be used to overcome these limitations, effectively supporting future exciting discoveries in the NSCLC field and the potential clinical exploitation of EVs.

RE-Place: A Unique Project Collecting Expertise on New Approach Methodologies.

By applying "New Approach Methodologies (NAMs)" based on innovative technologies such as computer modeling, high throughput testing, omics, and sophisticated cell cultures, the use of experimental animals in the life sciences can be reduced or sometimes even completely avoided. Stimulating NAMs may benefit from a bottom-up approach, i.e., local initiatives mapping the available NAMs and promoting their use. An example of such an initiative in Belgium is the RE-Place project, which collects the available NAMs in one central database, and links this knowledge with the names of experts and research centers. To this extent, a template was created to collect the information of interest in a fast and consistent manner. Based on this template, a web-based application was developed to facilitate the entry of information, which was evaluated in a pilot study by experts in the field of NAMs. After integration of their feedback, a revised version of the RE-Place online tool was launched to the public. Aspects such as user-friendliness, quality of submitted information, protection of personal data and Intellectual Property (IP) rights were all considered in the development process. Hurdles like incentives for collaboration were also taken into account. Information submitted with the online tool is directly integrated in the RE-Place open access database. By consulting the database, scientists from various disciplines can easily identify the different types of NAMs and the experts using them in Belgium. As such, the RE-Place database contributes to building trust in the use of NAMs and stimulating their use and regulatory uptake.

Use of LUCS (Light-Up Cell System) as an alternative live cell method to predict human acute oral toxicity.

LUCS (Light-Up Cell System) is a new live cell test that allows assessment of a cell's homeostasis and its alteration by a toxic agent. To evaluate the effectiveness of LUCS as an alternative test method for acute oral toxicity, we compared EC50s determined in HepG2 cells treated with 53 chemicals selected from the ACuteTox EU database with corresponding human blood LC50s derived from human acute poisoning cases. Linear regression analysis showed that LUCS results predict human data to 69 %. Rodent oral LD50s and LUCS EC50s were then correlated to human LC50s using shared data sets. Linear regression analyses comparing LUCS and animal data clearly showed that LUCS always predicts human toxicity better than animal data do. These successful prediction values prompted us to simplify the LUCS test, adapting it to regulatory and high throughput applications, resulting in a new protocol with consistent dose-response profiles and EC50s. This study demonstrates that the LUCS test method could be relevant for assessing human acute oral toxicity with a simplified protocol adapted to commercially available fluorescence readers. We suggest that this new alternative method can be used for acute systemic toxicity testing in combination with other tests under European REACH and other regulations, wherever pertinent alternative methods are still lacking.

Qualification of a non-animal vaginal irritation method admitted as nonclinical assessment model (NAM) in the Incubator Phase of the United States Food and Drug Administration (US FDA) Medical Devices Development Tool (MDDT).

The U.S. Food and Drug Administration (FDA) Center for Devices and Radiological Health (CDRH) classifies personal lubricants as Class II medical devices. Because of this status and the nature of body contact common to personal lubricants, CDRH reviewers routinely recommend a standard biocompatibility testing battery that includes: an in vivo rabbit vaginal irritation (RVI) test; an in vivo skin sensitization test, such as the guinea pig maximization test (GPMT); and an in vivo acute systemic toxicity test using mice or rabbits. These tests are conducted using live animals, despite the availability of in vitro and other non-animal test methods that may be suitable replacements. The only test included in the biocompatibility battery currently conducted using in vitro assay(s) is cytotoxicity. FDA's recently launched Predictive Toxicology Roadmap calls for the optimization of non-animal methods for the safety evaluation of drugs, consumer products and medical devices. In line with these goals, a Consortium comprising the Institute for In Vitro Sciences, Inc. (IIVS), industry, the Consumer Healthcare Products Association (CHPA), and the PETA International Science Consortium (PETA-ISC) is qualifying the use of an in vitro testing method as replacement for the RVI test. Participating companies include manufacturers of personal lubricants and those interested in the advancement of non-animal approaches working collaboratively with the FDA CDRH to develop an in vitro testing approach that could be used in place of the RVI in pre-market submissions. Personal lubricants and vaginal moisturizers with diverse chemical and physical properties (e.g., formulation, viscosity, pH, and osmolality) in their final undiluted form will be the focus of the program. In vitro vaginal irritation data generated using commercially available human reconstructed vaginal tissue model(s) will be paired with existing in vivo RVI data and analyzed to develop a Prediction Model for the safety assessment of these products. This research plan has been accepted into the FDA CDRH Medical Device Development Tools (MDDT) program as a potential non-clinical assessment model (NAM). The proposed NAM aligns with the goals of the recently launched FDA Roadmap to integrate predictive toxicology methods into safety and risk assessment with the potential to replace or reduce the use of animal testing.

The impact of precision uncertainty on predictive accuracy metrics of non-animal testing methods.

The ability of non-animal methods to correctly predict the outcome of in vivo testing in repeated applications is referred to as precision. Due to dichotomising continuous read-outs into discrete "positive/negative" hazard data, non-animal methods can reveal discordant classifications if results are sufficiently close to a defined classification threshold. This paper explores the impact of precision uncertainty on the predictive accuracy of non-animal methods. Using selected non-animal methods for assessing skin sensitisation hazard as case study examples, we explore the impact of precision uncertainty separately and in combination with uncertainty due to varying composition and size of experimental samples. Our results underline that discrete numbers of non-animal methods' sensitivity, specificity and concordance are of limited information for evaluations of non-animal testing methods' predictivity. Instead, information on the variability, and the upper and lower limits of accuracy metrics, should be provided to ensure a transparent assessment of testing methods' predictivity, and to allow for a meaningful comparison of the predictivity of non-animal methods with that of animal tests.

In vitro assessment of skin sensitization, photosensitization and phototoxicity potential of commercial glyphosate-containing formulations.

This study evaluated the applicability of a modified Direct Peptide Reactivity Assay (DPRA) (OECD N° 442C, 2015) through the 10-fold reduction of reaction volume (micro-DPRA, mDPRA) for skin sensitization evaluation of six commercial glyphosate-containing formulations. In addition, another modification of DPRA was proposed by adding a UVA (5J/cm2) irradiation step, namely photo-mDPRA, to better characterize (photo)sensitizer materials. The phototoxicity profile of pesticides was also evaluated using the 3T3 Neutral Red Uptake Phototoxicity Test (3T3-NRU-PT) (OECD N° 432, 2004). The mDPRA could represent an environmentally acceptable test approach, since it reduces costs and organic waste. Peptide depletion was greater in photo-mDPRA and changed the reactivity class of each test material, in comparison to mDPRA. Thus, the association of mDPRA with photo-mDPRA was better for correctly characterizing human (photo)sensitizer substances and pesticides. In general, cysteine depletion was greater than that of lysine for all materials tested in both mDPRA and photo-mDPRA. Furthermore, while 3T3-NRU-PT is unable to predict (photo)sensitizers, it was capable of correctly identifying the phototoxic potential of the tested agrochemical formulations. In conclusion, mDPRA plus photo-mDPRA and 3T3-NRU-PT seem to be preliminary non-animal test batteries for skin (photo)sensitization/phototoxicity assessment of chemicals, agrochemical formulations and their ingredients.