Technical evaluation and standardization of the human thyroid microtissue assay.

The success and sustainability of U.S. EPA efforts to reduce, refine, and replace in vivo animal testing depends on the ability to translate toxicokinetic and toxicodynamic data from in vitro and in silico new approach methods (NAMs) to human-relevant exposures and health outcomes. Organotypic culture models employing primary human cells enable consideration of human health effects and inter-individual variability but present significant challenges for test method standardization, transferability, and validation. Increasing confidence in the information provided by these in vitro NAMs requires setting appropriate performance standards and benchmarks, defined by the context of use, to consider human biology and mechanistic relevance without animal data. The human thyroid microtissue (hTMT) assay utilizes primary human thyrocytes to reproduce structural and functional features of the thyroid gland that enable testing for potential thyroid-disrupting chemicals. As a variable-donor assay platform, conventional principles for assay performance standardization need to be balanced with the ability to predict a range of human responses. The objectives of this study were to (1) define the technical parameters for optimal donor procurement, primary thyrocyte qualification, and performance in the hTMT assay, and (2) set benchmark ranges for reference chemical responses. Thyrocytes derived from a cohort of 32 demographically diverse euthyroid donors were characterized across a battery of endpoints to evaluate morphological and functional variability. Reference chemical responses were profiled to evaluate the range and chemical-specific variability of donor-dependent effects within the cohort. The data-informed minimum acceptance criteria for donor qualification and set benchmark parameters for method transfer proficiency testing and validation of assay performance.

Replacing animal-derived components in in vitro test guidelines OECD 455 and 487.

The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly relevant for human- and environment-related risk assessment. To examine the effects on a mechanism-specific level, standardized cell-based in vitro methods are widely applied. However, these methods include animal-derived components like fetal bovine serum (FBS) or rat-derived liver homogenate fractions (S9-mixes), which are a source of variability, reduced assay reproducibility and ethical concerns. In our study, we evaluated the adaptation of the cell-based in vitro OECD test guidelines TG 487 (assessment of genotoxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ERα-CALUX® and GeneBLAzer™ ERα-UAS-bla GripTite™ (for OECD TG 455) were investigated for growth in a chemically defined medium without the addition of FBS. Secondly, the biotechnological S9-mix ewoS9R was implemented in comparison to the induced rat liver S9 to simulate in vivo metabolism capacities in both OECD test guidelines. As a model compound, Benzo[a]pyrene was used due to its increased genotoxicity and endocrine activity after metabolization. The metabolization of Benzo[a]Pyrene by S9-mixes was examined via chemical analysis. All cell lines (A549, ERα-CALUX® and GeneBLAzer™ Erα-UAS-bla GripTite™) were successfully cultivated in chemically defined media without FBS. The micronucleus assay could not be conducted in chemically defined medium due to formation of cell clusters. The methods for endocrine activity assessment could be conducted in chemically defined media or reduced FBS content, but with decreased assay sensitivity. The biotechnological ewoS9R showed potential to replace rat liver S9 in the micronucleus in FBS-medium with A549 cells and in the ERα-CALUX® assay in FBS- and chemically defined medium. Our study showed promising steps towards an animal-component free toxicity testing. After further improvements, the new methodology could lead to more reproducible and reliable results for risk assessment.

Rethinking agrochemical safety assessment: A perspective.

Agrochemical safety assessment has traditionally relied on the use of animals for toxicity testing, based on scientific understanding and test guidelines developed in the 1980s. However, since then, there have been significant advances in the toxicological sciences that have improved our understanding of mechanisms underpinning adverse human health effects. The time is ripe to 'rethink' approaches used for human safety assessments of agrochemicals to ensure they reflect current scientific understanding and increasingly embrace new opportunities to improve human relevance and predictivity, and to reduce the reliance on animals. Although the ultimate aim is to enable a paradigm shift and an overhaul of global regulatory data requirements, there is much that can be done now to ensure new opportunities and approaches are adopted and implemented within the current regulatory frameworks. This commentary reviews current initiatives and emerging opportunities to embrace new approaches to improve agrochemical safety assessment for humans, and considers various endpoints and initiatives (including acute toxicity, repeat dose toxicity studies, carcinogenicity, developmental and reproductive toxicity, exposure-driven approaches, inhalation toxicity, and data modelling). Realistic aspirations to improve safety assessment, incorporate new technologies and reduce reliance on animal testing without compromising protection goals are discussed.

The SCCS Notes of Guidance for the testing of cosmetic ingredients and their safety evaluation, 11th revision, 30-31 March 2021, SCCS/1628/21.

The "SCCS Notes of Guidance for the Testing of Cosmetic Ingredients and Their Safety Evaluation, 11 th Revision" (SCCS/1628/21) contains relevant and updated information on the different aspects of testing and safety evaluation of cosmetic substances in Europe. The emphasis is on cosmetic ingredients for which a concern has been expressed for human health. Indirectly, the Guidance also provides some advice on the safety of finished products. A general aim is to improve harmonised compliance with the current cosmetic EU legislation, Regulation (EC) No 1223/2009, for which animal testing and marketing bans fully apply from 2013 onwards. This means that no in vivo testing of ingredients or finished products is allowed in Europe for the purpose of cosmetics. For this reason, the SCCS has closely followed the progress made in regard to the development and validation of alternative replacement methods, also referred to as new approach methodology (NAM). The "SCCS Notes of Guidance" are regularly revised and updated in order to incorporate progress made and experience gained over time, in particular on the use of NAMs, and the new methods and data that became available since previous revision (SCCS/1602/18) formed the basis of the current (11 th) Revision.

Regulation of Sun Protection Products in the EU.

Unlike more "traditional" cosmetic products, sunscreens do not sit inertly on the skin, providing a simple decorative effect. Their recognized and important contribution to public health has led many regions in the world to treat them as drugs or special cosmetics. Against the trend at that time, in 1976, the EU legislator already took a conscious decision to treat and regulate sunscreens as fast-moving consumer products. Since then, the EU Cosmetics Directive/Regulation balances the need for strict safety and efficacy requirements, with need for rapid innovation and easy consumer availability. Whilst the EU Regulation considers that "all cosmetic products are equal," sunscreens are clearly "more equal." In several areas of the legislation, specific requirements or guidance for sunscreen products have been introduced over the years. Whilst staying in the overall spirit of the legislation, these requirements take into account the specificity of sunscreens with regard to ingredient safety (positive list for UV filters), product safety assessment (photostability, deliberate exposure to UV light), minimum efficacy (UVA/UVB), efficacy testing (standardized test methods) and labelling (clear use instructions, non-misleading information to consumers). The article presents the history of the EU Cosmetics Regulation, its main requirements, where applicable, and specific considerations relating to sunscreens are highlighted and explained.

Application of the 3Rs principles in the development of pharmaceutical generics.

Although the 3Rs are broadly applied in nonclinical testing, a better appreciation of the 3Rs is needed in the field of differentiated or value-added pharmaceutical generics because the minor changes in formulation, dosage form, indication, and application route often do not require additional safety testing. The US FDA and the EU EMA have comprehensive regulations for such drugs based on quality, therapeutic equivalence, and safety guidelines. However, no scientific publications on how the concept of replacement and reduction from 3Rs principles can be applied in the safety assessment of differentiated generics were found in the public domain. In this review, we discuss the application of 3Rs in nonclinical testing requirements for differentiated generics. Practical examples are provided in the form of case studies from regulated markets. We highlight the need for utilization of existing data to establish equivalence (differentiated generic vs innovator) in efficacy and safety. The case studies indicate that data requirements from animal experiments have been reduced to a large extent in some major markets without compromising quality and safety. In this context, we also highlight the problem that on a global scale, a true reduction of animal experiments will only be achieved when all countries adopt similar practices.

Enhancing between-facility reproducibility of the SH test as an in vitro skin sensitization test by the improved test method.

There has been an increased demand to eliminate animal experiments and to replace the experiments with alternative tests for assessing the safety of cosmetics. The SH test is an in vitro skin sensitization test that evaluates the protein binding abilities of a test substance. Skin sensitization must be evaluated by multiple test methods. The SH test uses the same cell line and measuring instruments as the human Cell-Line Activation Test (h-CLAT), which is one of the test methods used to evaluate different key events and is listed in the OECD test guidelines. There are cost advantages to usher the SH test into facilities that are already running the h-CLAT. The SH test is conducted only at a facility that has developed the SH test because studies on the between-facility reproducibility and validity have not been performed. Therefore, to verify the transferability of the SH test and the between-facilities reproducibility, we evaluated the reproducibility of the SH test results at three facilities, including the development facility. After an initial round of testing, the protocol was refined as follows to improve reproducibility among the three facilities: i) determine the optimum pH range, ii) change the maximum applicable concentration of water-soluble substances, and iii) define the appropriate dispersion conditions for evaluating hydrophobic substances. These refinements markedly enhanced the between-facility reproducibility (from 76.0% to 96.0%) for the 25 substances evaluated in this study. This study confirmed that the SH test is an effective skin sensitization test method with high technical transferability and between-facility reproducibility.

Analysis of variability in the rabbit skin irritation assay.

The in vivo rabbit test is the benchmark against which new approach methodologies for skin irritation are usually compared. No alternative method offers a complete replacement of animal use for this endpoint for all regulatory applications. Variability in the animal reference data may be a limiting factor in identifying a replacement. We established a curated data set of 2624 test records, representing 990 substances, each tested at least twice, to characterize the reproducibility of the in vivo assay. Methodological deviations from guidelines were noted, and multiple data sets with differing tolerances for deviations were created. Conditional probabilities were used to evaluate the reproducibility of the in vivo method in identification of U.S. Environmental Protection Agency or Globally Harmonized System hazard categories. Chemicals classified as moderate irritants at least once were classified as mild or non-irritants at least 40% of the time when tested repeatedly. Variability was greatest between mild and moderate irritants, which both had less than a 50% likelihood of being replicated. Increased reproducibility was observed when a binary categorization between corrosives/moderate irritants and mild/non-irritants was used. This analysis indicates that variability present in the rabbit skin irritation test should be considered when evaluating nonanimal alternative methods as potential replacements.

Application of the 3R principles: Vertebrae as an additional source of murine bone-marrow cells.

Experimental murine models are an essential tool in the field of bone marrow (BM) transplantation research. Therefore, numerous mice are required to obtain a sufficient number of BM cells, which is in contrast with the Reduction principle of the 3R principles. The selection of the cell source and the isolation protocol are therefore critical in obtaining a sufficient yield of cells for experiments. Nowadays, the vertebrae are already used as an extra source of BM cells to enrich the number of isolated cells from the long bones and ilia (LBI), when needed. Yet, little is known if BM cells from LBI and vertebrae share the same characteristics and can be pooled together for further analysis. Therefore, in this study, we aimed to compare the quantity and characteristics of haematopoietic and stromal cell lines in the BM from the LBI and vertebrae. To count haematopoietic and mesenchymal stem/stromal progenitors, colony-forming unit assays were performed. To determine the expansion capacity of mesenchymal stem/stromal cells (MSCs), cultivation of MSCs and measurement of the expression of surface markers by flow cytometry was performed. The characterisation and enumeration of immune cell populations was also performed by flow cytometry. Here, we show that the vertebrae are a comparable source of BM cells to the LBI regarding the analysed parameters.

A cross-industry collaboration to assess if acute oral toxicity (Q)SAR models are fit-for-purpose for GHS classification and labelling.

This study assesses whether currently available acute oral toxicity (AOT) in silico models, provided by the widely employed Leadscope software, are fit-for-purpose for categorization and labelling of chemicals. As part of this study, a large data set of proprietary and marketed compounds from multiple companies (pharmaceutical, plant protection products, and other chemical industries) was assembled to assess the models' performance. The absolute percentage of correct or more conservative predictions, based on a comparison of experimental and predicted GHS categories, was approximately 95%, after excluding a small percentage of inconclusive (indeterminate or out of domain) predictions. Since the frequency distribution across the experimental categories is skewed towards low toxicity chemicals, a balanced assessment was also performed. Across all compounds which could be assigned to a well-defined experimental category, the average percentage of correct or more conservative predictions was around 80%. These results indicate the potential for reliable and broad application of these models across different industrial sectors. This manuscript describes the evaluation of these models, highlights the importance of an expert review, and provides guidance on the use of AOT models to fulfill testing requirements, GHS classification/labelling, and transportation needs.

Animal testing for vaccines. Implementing replacement, reduction and refinement: challenges and priorities.

Transition to in vitro alternative methods from in vivo in vaccine release testing and characterization, the implementation of the consistency approach, and a drive towards international harmonization of regulatory requirements are most pressing needs in the field of vaccines. It is critical for global vaccine community to work together to secure effective progress towards animal welfare and to ensure that vaccines of ever higher quality can reach the populations in need in the shortest possible timeframe. Advancements in the field, case studies, and experiences from Low and Middle Income Countries (LMIC) were the topics discussed by an international gathering of experts during a recent conference titled "Animal Testing for Vaccines - Implementing Replacement, Reduction and Refinement: Challenges and Priorities". This conference was organized by the International Alliance for Biological Standardization (IABS), and held in Bangkok, Thailand on December 3 and 4 2019. Participants comprised stakeholders from many parts of the world, including vaccine developers, manufacturers and regulators from Asia, Europe, North America, Australia and New Zealand. In interactive workshops and vibrant panel discussions, the attendees worked together to identify the remaining barriers to validation, acceptance and implementation of alternative methods, and how harmonization could be promoted, especially for LMICs.

Analysis and reflection on the role of the 90-day oral toxicity study in European chemical risk assessment.

The 90-day toxicity study is one of the studies used in the safety assessment of food ingredients, medicines or other chemical substances. This paper reviews the current role of the 90-day oral toxicity study in European regulatory dossiers of chemicals by reviewing EU legislation and EU and OECD guidance documents. Regulatory provisions with regard to necessity, objectives and design of such 90-day toxicity studies vary between the different sectors addressed in this review. Most often the 90-day study is expected to be part of the standard test battery used for chemical risk assessment, without necessarily being a legal requirement and its objectives may vary between regulatory domains. Exceptions, when a 90-day study is not required are spelled out in the chemicals legislation and for food contact materials. The sectorial study design requirements of the 90-day toxicity study are very often embedded in the OECD TG 408 protocol. Differences in study objectives are not necessarily reflected in specific study designs. Considering the call for the reduction of using experimental animals for scientific purposes and the fact that a 90-day study may serve different purposes, consistency between the necessity to conduct such a study, its objectives and the study design to achieve these objectives may improve judicious use of laboratory animals. Thus there may be an opportunity to reflect and further optimise the design of in vivo toxicology studies, such as the 90-day study. This should be based on a systematic analysis of past studies and risk assessments.

Building confidence in skin sensitisation potency assessment using new approach methodologies: report of the 3rd EPAA Partners Forum, Brussels, 28th October 2019.

Skin sensitising substances that induce contact allergy and consequently risk elicitation of allergic contact dermatitis (ACD) remain an important focus regarding the replacement of animal experimentation. Current in vivo methods, notably the local lymph node assay (LLNA) refined and reduced animal usage and led to a marked improvement in hazard identification, characterisation and risk assessment. Since validation, regulatory confidence in the LLNA approach has evolved until it became the first choice assay in most regulated sectors. Currently, hazard identification using the LLNA is being actively replaced by a toolbox of non-animal approaches. However, there remains a need to increase confidence in the use of new approach methodologies (NAMs) as replacements for LLNA sensitiser potency estimation. The EPAA Partners Forum exchanged the current state of knowledge on use of NAMs in various industry sectors and regulatory environments. They then debated current challenges in this area and noted several ongoing needs. These included a requirement for reference standards for potency, better characterisation of applicability domains/technical limitations of NAMs, development of a framework for weight of evidence assessments, and an increased confidence in the characterisation of non-sensitisers. Finally, exploration of an industry/regulator cross-sector user-forum on skin sensitisation was recommended.

Second-phase validation study of an alternative developmental toxicity test using mouse embryonic stem cell-derived embryoid bodies.

The embryoid body test (EBT) is a developmental toxicity test method that measures the size of embryoid bodies (EBs) and the viability of mouse embryonic stem cells (mESCs) and fibroblasts (3T3 cells). The previous pre-validation study confirmed the high accuracy (above 80%) of EBT using 26 coded test chemicals. This second-phase validation study assessed the inter-laboratory reproducibility (5 chemicals in common) and predictive capacity (10 chemicals in each laboratory) test using the coded test chemicals at three laboratories. For the prediction model, the accuracy is increased when more data is accumulated. Therefore, we updated the prediction model and analyzed the results of the second year with the newly created-prediction model. Statistical analysis of the inter-laboratory reproducibility test results indicated that accuracy, sensitivity, and specificity were 87%, 78%, and 100%, respectively. The results of the statistical analysis of the predictive capacity test showed an accuracy of 80%, sensitivity of 78%, and specificity of 81%. In conclusion, the EBT can accurately classify various embryotoxicants within a short period and with relatively little effort. Therefore, EBT can be used as a good way to test developmental toxicity.

Collaborative study for the validation of cell line assays for in-process toxicity and antigenicity testing of Clostridium septicum vaccine antigens - Part 1.

Large numbers of mice are used in testing during the production of Clostridial vaccines. Previous work has indicated that cell line assays could replace mouse tests for certain aspects of this testing. Replacement assays have been developed for the testing of the toxins and toxoids of several clostridial species but none of these assays have been assessed in an international collaborative study. Under the common aegis of the European Partnership for Alternative Approaches to Animal Testing (EPAA) and of the European Directorate for the Quality of Medicines & HealthCare (EDQM), collaborative study BSP130 was initiated to evaluate Vero cell based alternative methods to the current mouse tests used to measure the toxicity of Clostridium septicum toxin (the minimum lethal dose (MLD) test), the freedom from toxicity of C. septicum toxoid (the MLD test) and the antigenicity of C. septicum toxoid (the total combining power (TCP) test). The principal aims of BSP130 were to determine the repeatability and reproducibility of the in vitro assays and to demonstrate concordance of the proposed in vitro and current in vivo TCP and MLD tests. 11 laboratories from 7 countries participated in the collaborative study and each tested 6 toxins and 6 toxoids. The participants' Vero cell lines were up to 1 000 times more sensitive than the mouse strains. The MLD assay in mice and on Vero cells generally ranked the toxins in a similar order in most of the laboratories. The TCP assay in mice and on Vero cells also generally ranked the toxoids in a similar order in most of the laboratories. The results demonstrate that the repeatability and reproducibility of the in vitro Vero cell based assays are no worse than that of the in vivo assays and that they are easily transferable to other laboratories. The concordance correlations between the in vivo and in vitro methods were for the MLD assays ρc=0.961 (log-transformed values) and ρc=0.921 (non-log-transformed values) and for the TCP assays ρc=0.968 (log-transformed values) and ρc=0.980 (non log-transformed values). These correlations are excellent showing that the Vero cell assays can be used as alternatives to the mouse tests for the assessment of C. septicum toxin MLD and toxoid TCP values. This study can be used by vaccine manufacturing companies as a guide for applying the same approach to other clostridial toxins and toxoids.

New approach methodologies (NAMs) for human-relevant biokinetics predictions. Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.

The Ex Vivo Skin Model as an Alternative Tool for the Efficacy and Safety Evaluation of Topical Products.

The development of alternative approaches for safety and efficacy testing that avoid the use of animals is a worldwide trend, which relies on the improvement of current models and tools so that they better reproduce human biology. Human skin from elective plastic surgery is a promising experimental model to test the effects of topically applied products. As the structure of native skin is maintained, including cell population (keratinocytes, melanocytes, Langerhans cells and fibroblasts) and dermal matrix (containing collagen, elastin, glycosaminoglycans, etc.), it most closely matches the effects of substances on in vivo human skin. In this review, we present a collection of results that our group has generated over the last years, involving the use of human skin and scalp explants, demonstrating the feasibility of this model. The development of a test system with ex vivo skin explants, of standard size and thickness, and cultured at the air-liquid interface, can provide an important tool for understanding the mechanisms involved in several cutaneous disorders.

Mechanistic Similarities between 3D Human Bronchial Epithelium and Mice Lung, Exposed to Copper Oxide Nanoparticles, Support Non-Animal Methods for Hazard Assessment.

The diversity and increasing prevalence of products derived from engineered nanomaterials (ENM), warrants implementation of non-animal approaches to health hazard assessment for ethical and practical reasons. Although non-animal approaches are becoming increasingly popular, there are almost no studies of side-by-side comparisons with traditional in vivo assays. Here, transcriptomics is used to investigate mechanistic similarities between healthy/asthmatic models of 3D air-liquid interface (ALI) cultures of donor-derived human bronchial epithelia cells, and mouse lung tissue, following exposure to copper oxide ENM. Only 19% of mouse lung genes with human orthologues are not expressed in the human 3D ALI model. Despite differences in taxonomy and cellular complexity between the systems, a core subset of matching genes cluster mouse and human samples strictly based on ENM dose (exposure severity). Overlapping gene orthologue pairs are highly enriched for innate immune functions, suggesting an important and maybe underestimated role of epithelial cells. In conclusion, 3D ALI models based on epithelial cells, are primed to bridge the gap between traditional 2D in vitro assays and animal models of airway exposure, and transcriptomics appears to be a unifying dose metric that links in vivo and in vitro test systems.