60 Years of the 3Rs symposium: Lessons learned and the road ahead.

When The Principles of Humane Experimental Technique was published in 1959, authors William Russell and Rex Burch had a modest goal: to make researchers think about what they were doing in the laboratory - and to do it more humanely. Sixty years later, their groundbreaking book was celebrated for inspiring a revolution in science and launching a new field: The 3Rs of alternatives to animal experimentation. On November 22, 2019, some pioneering and leading scientists and researchers in the field gathered at the Johns Hopkins Bloomberg School of Public Health in Bal-timore for the 60 Years of the 3Rs Symposium: Lessons Learned and the Road Ahead. The event was sponsored by the Johns Hopkins Center for Alternatives to Animal Testing (CAAT), the Foundation for Chemistry Research and Initiatives, the Alternative Research & Development Foundation (ARDF), the American Cleaning Institute (ACI), the International Fragrance Association (IFRA), the Institute for In Vitro Sciences (IIVS), John "Jack" R. Fowle III, and the Society of Toxicology (SoT). Fourteen pres-entations shared the history behind the groundbreaking publication, international efforts to achieve its aims, stumbling blocks to progress, as well as remarkable achievements. The day was a tribute to Russell and Burch, and a testament to what is possible when people from many walks of life - science, government, and industry - work toward a common goal.

William Russell and Rex Burch published their book The Principles of Humane Experimental Technique in 1959. The book encouraged researchers to replace animal experiments where it was possible, to refine experiments with animals in order to reduce their suffering, and to reduce the number of animals that had to be used for experiments to the minimum. Sixty years later, a group of pioneering and leading scientists and researchers in the field gathered to share how the publi­cation came about and how the vision inspired international collaborations and successes on many different levels including new laws. The paper includes an overview of important milestones in the history of alternatives to animal experimentation.

Impedance-based in vitro eye irritation testing enables the categorization of diluted chemicals.

Products containing chemicals with eye irritation potential need to be labeled with the respective hazard symbol. To avoid the testing of numerous dilutions of chemicals on animals, their labeling is directed by a theoretical approach. In this report, a previously described in vitro tissue model of the cornea based on human epithelial cells was used for eye irritation testing of dilutions. As a sensitive and non-destructive method to analyze the barrier function of the epithelium, impedance spectroscopy was applied. Moreover, the morphology and viability of the epithelial models were assessed. We tested four chemicals that, neatly, cause severe damage to the eye: tetrahydrofuran, acetic acid, diethylethanolamine, and benzalkonium chloride. With our test method, we were able to determine the concentrations of the chemicals which are critical for the integrity of the cornea. The threshold was < 0.1% for the most and > 5% for the least toxic substance. The described test system is not only an alternative for animal models but also for the theoretical examination of the hazard potential of diluted chemicals. By using the advantages of tissue engineering and non-destructive analysis tools, we can achieve more precise and safer labeling of the eye irritation potential of products.

Development of a hierarchical support vector regression-based in silico model for the prediction of the cysteine depletion in DPRA.

Topical and transdermal treatments have been dramatically growing recently and it is crucial to consider skin sensitization during the drug discovery and development process for these administration routes. Various tests, including animal and non-animal approaches, have been devised to assess the potential for skin sensitization. Furthermore, numerous in silico models have been created, providing swift and cost-effective alternatives to traditional methods such as in vivo, in vitro, and in chemico methods for categorizing compounds. In this study, a quantitative structure-activity relationship (QSAR) model was developed using the innovative hierarchical support vector regression (HSVR) scheme. The aim was to quantitatively predict the potential for skin sensitization by analyzing the percent of cysteine depletion in Direct Peptide Reactivity Assay (DPRA). The results demonstrated accurate, consistent, and robust predictions in the training set, test set, and outlier set. Consequently, this model can be employed to estimate skin sensitization potential of novel or virtual compounds.

Computational application of internationally harmonized defined approaches to skin sensitization: DASS App.

BACKGROUND: Chemically induced skin sensitization, or allergic contact dermatitis, is a common occupational and public health issue. Regulatory authorities require an assessment of potential to cause skin sensitization for many chemical products. Defined approaches for skin sensitization (DASS) identify potential chemical skin sensitizers by integrating data from multiple non-animal tests based on human cells, molecular targets, and computational model predictions using standardized data interpretation procedures. While several DASS are internationally accepted by regulatory agencies, the data interpretation procedures vary in logical complexity, and manual application can be time-consuming or prone to error. RESULTS: We developed the DASS App, an open-source web application, to facilitate user application of three regulatory testing strategies for skin sensitization assessment: the Two-out-of-Three (2o3), the Integrated Testing Strategy (ITS), and the Key Event 3/1 Sequential Testing Strategy (KE 3/1 STS) without the need for software downloads or computational expertise. The application supports upload and analysis of user-provided data, includes steps to identify inconsistencies and formatting issues, and provides predictions in a downloadable format. CONCLUSION: This open-access web-based implementation of internationally harmonized regulatory guidelines for an important public health endpoint is designed to support broad user uptake and consistent, reproducible application. The DASS App is freely accessible via https://ntp.niehs.nih.gov/go/952311 and all scripts are available on GitHub ( https://github.com/NIEHS/DASS ).

Examining animal testing for risk assessment: A WC-12 workshop report.

In toxicology and regulatory testing, the use of animal methods has been both a cornerstone and a subject of intense debate. To continue this discourse a panel and audience representing scientists from various sectors and countries convened at a workshop held during the 12th World Congress on Alternatives and Animal Use in the Life Sciences (WC-12). The ensuing discussion focused on the scientific and ethical considerations surrounding the necessity and responsibility of defending the creation of new animal data in regulatory testing. The primary aim was to foster an open dialogue between the panel members and the audience while encouraging diverse perspectives on the responsibilities and obligations of various stakeholders (including industry, regulatory bodies, technology developers, research scientists, and animal welfare NGOs) in defending the development and subsequent utilization of new animal data. This workshop summary report captures the key elements from this critical dialogue and collective introspection. It describes the intersection of scientific progress and ethical responsibility as all sectors seek to accelerate the pace of 21st century predictive toxicology and new approach methodologies (NAMs) for the protection of human health and the environment.

Development of an in silico evaluation system that quantitatively predicts skin sensitization using OECD Guideline No. 497 ITSv2 defined approach for skin sensitization classification.

The Integrated Testing Strategy version 2 (ITSv2) Defined Approach, which is a reliable skin sensitization hazard and multi-step risk assessment method, does not support quantitative risk assessment such as local lymph node assay EC3 values. In this study, we developed a high-performance in silico evaluation system that quantitatively predicts the EC3 values of chemical substances by combining the ITSv2 Defined Approach for hazard identification (ITSv2 HI) with machine learning models. This system uses in chemico/in vitro test data, molecular descriptors, and distance information based on read-across concepts as explanatory variables. The system achieves an R2 value of 0.617 on external-validation data. Substances misclassified in ITSv2 HI are considered to have properties that do not match the correspondence between tests expressing the adverse outcome pathway assumed in the ITSv2 Defined Approach and skin sensitization. Therefore, ITSv2 HI is assumed to be correct within the applicability domains of this system. When using only substances within the applicability domains to reconstruct CatBoost models, the R2 value reached 0.824 on the external-validation data, representing an improvement in system performance. The results demonstrate the utility of explanatory variables that reflect the read-across concept and the advantages of integrating multiple prediction methods.

Reconstructed human epidermis-based testing strategy of skin sensitization potential and potency classification using epidermal sensitization assay and in silico data.

The hazards and potency of skin sensitizers are traditionally determined using animal tests such as the local lymph node assay (LLNA); however, significant progress has been made in the development of non-animal test methods addressing the first three mechanistic key events of adverse outcome pathway in skin sensitization. We developed the epidermal sensitization assay (EpiSensA), which is a reconstructed human epidermis-based assay, by measuring four genes related to critical keratinocyte responses during skin sensitization. Four in vitro skin sensitization test methods (EpiSensA, direct peptide reactivity assay [DPRA], KeratinoSens™, and human cell line activation test [h-CLAT]) were systematically evaluated using 136 chemicals including lipophilic chemicals and pre/pro-haptens, which may be related to assay-specific limitations. The constructed database included existing and newly generated data. The EpiSensA showed a broader applicability domain and predicted the hazards with 82.4% and 78.8% accuracy than LLNA and human data. The EpiSensA could detect 76 out of 88 sensitizers at lower concentrations than the LLNA, indicating that the EpiSensA has higher sensitivity for the detection of minor sensitizing constituents. These results confirmed the potential use of the EpiSensA in evaluating a mixture of unknown compositions that can be evaluated by animal tests. To combine different information sources, the reconstructed human epidermis-based testing strategy (RTS) was developed based on weighted multiple information from the EpiSensA and TImes MEtabolism Simulator platform for predicting Skin Sensitization (TIMES-SS; RTSv1) or Organization for Economic Cooperation and Development (OECD) QSAR Toolbox automated workflow (RTSv2). The predictivities of the hazards and Globally Harmonized System (GHS) subcategories were equal to or better than the defined approaches (2 out of 3, integrated testing strategy [ITS]v1, and ITSv2) adopted as OECD Guideline 497.

[Alternative methods to animal testing, present and future]. / Les méthodes alternatives à l'expérimentation animale, présent et futur.

Alternative methods to animal testing are used in fundamental and clinical research, for the realization of studies for regulatory purposes, and also screening operations in the development of new molecules. They are based on in vitro (cell models) or in silico (mathematical models) replacement methods. They have been largely promoted by the 3Rs rule (Replace, Reduce, Refine) which aims at regulating animal experimentation. For biomedical research, these different methods are valuable tools for better understanding the physiology of organisms and the mechanisms of the effects of chemicals and physical agents on them.

Title: Les méthodes alternatives à l'expérimentation animale, présent et futur. Abstract: Les méthodes alternatives à l'expérimentation animale sont utilisées en recherche fondamentale et clinique, pour la réalisation d'études à visée réglementaire et d'opérations de criblage en matière de développement de nouvelles molécules. Elles reposent sur des procédures de remplacement in vitro (modèles cellulaires) ou in silico (modèles mathématiques). Les méthodes alternatives ont été largement promues par la règle des 3R (Remplacer, Réduire, Raffiner) qui vise à encadrer l'expérimentation animale. Dans le domaine de la recherche, ces différentes méthodes sont des outils précieux qui permettent de mieux comprendre la physiologie des organismes et les mécanismes d'action des agents chimiques et physiques sur ces derniers.

Prediction of skin sensitization using machine learning.

As global awareness of animal welfare spreads, the development of alternative animal test models is increasingly necessary. The purpose of this study was to develop a practical machine-learning model for skin sensitization using three physicochemical properties of the chemicals: surface tension, melting point, and molecular weight. In this study, a total of 482 chemicals with local lymph node assay results were collected, and 297 datasets with 6 physico-chemical properties were used to develop Random Forest (RF) model for skin sensitization. The developed model was validated with 45 fragrance allergens announced by European Commission. The validation results showed that RF achieved better or similar classification performance with f1-scores of 54% for penal, 82% for ternary, and 96% for binary compared with Support Vector Machine (SVM) (penal, 41%; ternary, 81%; binary, 93%), QSARs (ChemTunes, 72% for ternary; OECD Toolbox, 89% for binary), and a linear model (Kim et al., 2020) (41% for penal), and we recommend the ternary classification based on Global Harmonized System providing more detailed and precise information. In the further study, the proposed model results were experimentally validated with the Direct Peptide Reactivity Assay (DPRA, OECD TG 442C approved model), and the results showed a similar tendency. We anticipate that this study will help to easily and quickly screen chemical sensitization hazards.

Applicability of the DPRA on mixture testing: challenges and opportunities.

The in chemico direct peptide reactivity assay (DPRA) is validated to assess protein reactivity of chemical compounds, relating to the molecular initiating event of skin sensitization induction. According to OECD TG 442C, the DPRA is technically applicable to test multi-constituent substances and mixtures of known composition, even though limited experimental data are publicly available. First, we assessed the DPRA's predictive capability for individual substances, but at concentrations other than the recommended 100 mM, i.e., based on the LLNA EC3 concentration (Experiment A). Next, the applicability of the DPRA to test unknown mixtures was assessed (Experiment B). Here, the complexity of unknown mixtures was reduced to mixtures containing either two known skin sensitizers with varying potencies, or a combination of a skin sensitizer with a non-skin sensitizer, or multiple non-sensitizers. Experiments A and B revealed that one extremely potent sensitizer (oxazolone) was incorrectly classified as a non-sensitizer when tested at its low EC3 concentration of 0.4 mM instead of the suggested molar excess conditions of 100 mM (Experiments A). For binary mixtures tested in experiments B, the DPRA was able to distinguish all skin sensitizers and the strongest skin sensitizer in the mixture was determinant for the overall peptide depletion of a sensitizer. In conclusion, we confirmed that the DPRA test method can be used efficiently for well-known characterized mixtures. However, when deviating from the recommended testing concentration of 100 mM, caution should be taken in case of negative results, limiting the DPRA's applicability for mixtures of unknown composition.

REACH out-numbered! The future of REACH and animal numbers.

The EU's REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) Regulation requires animal testing only as a last resort. However, our study (Knight et al., 2023) in this issue reveals that approximately 2.9 million animals have been used for REACH testing for reproductive toxicity, developmental toxicity, and repeated-dose toxicity alone as of December 2022. Currently, additional tests requiring about 1.3 million more animals are in the works. As compliance checks continue, more animal tests are anticipated. According to the European Chemicals Agency (ECHA), 75% of read-across methods have been rejected during compliance checks. Here, we estimate that 0.6 to 3.2 million animals have been used for other endpoints, likely at the lower end of this range. The ongoing discussion about the grouping of 4,500 regis-tered petrochemicals can still have a major impact on these numbers. The 2022 amendment of REACH is estimated to add 3.6 to 7.0 million animals. This information comes as the European Parliament is set to consider changes to REACH that could further increase animal testing. Two proposals currently under discussion would likely necessitate new animal testing: extending the requirement for a chemical safety assessment (CSA) to Annex VII substances could add 1.6 to 2.6 million animals, and the registration of polymers adds a challenge comparable to the petrochemical discussion. These findings high-light the importance of understanding the current state of REACH animal testing for the upcoming debate on REACH revisions as an opportunity to focus on reducing animal use.

4.2 million and counting The animal toll for REACH systemic toxicity studies.

The EU's chemicals regulation, REACH, requires that most chemicals in the EU be evaluated for human health and ecosystem risks, with a mandate to minimize use of animal tests for these evaluations. The REACH process has been ongoing since about 2008, but a calculation of the resulting animal use is not publicly available. For this reason, we have undertaken a count of animals used for REACH. With EU legislators set to consider REACH revisions that could expand animal testing, we are releasing results for test categories counted to date: reproductive toxicity tests, developmental toxicity tests, and repeat-ed-dose toxicity tests for human health. The total animal count as of December 2022 for these categories is about 2.9 million. Additional tests involving about 1.3 million animals are currently required by a final proposal authorization or compliance check but not yet completed. The total, 4.2 million, for just these three test categories exceeds the original European Com-mission forecast of 2.6 million for all REACH tests. The difference is primarily because the European Commission estimate excluded offspring, which are most of the animals used for REACH. Other reasons for the difference are extra animals included in tests to ensure sufficient survive to meet the minimum test requirement; dose range-finding tests; extra test animal groups, e.g., for recovery analysis; and a high rejection rate of read-across studies. Given higher than forecast animal use, the upcoming debate on proposed REACH revisions is an opportunity to refocus on reducing animal numbers in keeping with the REACH mandate.

Development of an in chemico high-throughput screening method for the identification of skin sensitization potential.

It is well established that chemical-peptide conjugation represents the molecular initiating event (MIE) in skin sensitization. This MIE has been successfully exploited in the development of in chemico peptide reactivity assays, with the Direct Peptide Reactivity Assay (DPRA) being validated as a screening tool for skin sensitization hazard as well as an OECD test guideline. This test relies on the use of a high-performance liquid chromatography/ultraviolet detection method to quantify chemical-peptide conjugation through measurement of the depletion of two synthetic peptides containing lysine or cysteine residues, which is labor-intensive and time-consuming. To improve assay throughput, sensitivity, and accuracy, we have developed a spectrophotometric assay for skin sensitization potential based on MIE measurement-the ProtReact assay. ProtReact is also a cheaper, faster, simpler, and more accessible alternative for the DPRA, giving comparable results. A set of 106 chemicals was tested with ProtReact and the peptide depletion values compared with those reported for the DPRA. The predictive capacity of both assays was evaluated with human reference data. ProtReact and DPRA assays show similar predictive capacities for hazard identification (75% and 74%, respectively), although ProtReact showed a higher specificity (86% versus 74%, respectively) and lower sensitivity (69% versus 73%). Overall, the results show that ProtReact assay described here represents an efficient, economic, and accurate assay for the prediction of skin sensitization potential of chemical haptens.

Recent Advances in Drug Discovery Toxicology.

Major advances in scientific discovery and insights that stem from the development and use of new techniques and models can bring remarkable progress to conventional toxicology. Although animal testing is still considered as the "gold standard" in traditional toxicity testing, there is a necessity for shift from animal testing to alternative methods regarding the drug safety testing owing to the emerging state-of-art techniques and the proposal of 3Rs (replace, reduce, and refine) towards animal welfare. This review describes some recent research methods in drug discovery toxicology, including in vitro cell and organ-on-a-chip, imaging systems, model organisms (C. elegans, Danio rerio, and Drosophila melanogaster), and toxicogenomics in modern toxicology testing.

Alternatives to animal testing in toxicity testing: Current status and future perspectives in food safety assessments.

The development of alternative methods to animal testing has gained great momentum since Russel and Burch introduced the "3Rs" concept of Reduction, Refinement, and Replacement of animals in safety testing in 1959. Several alternatives to animal testing have since been introduced, including but not limited to in vitro and in chemico test systems, in silico models, and computational models (e.g., [quantitative] structural activity relationship models, high-throughput screens, organ-on-chip models, and genomics or bioinformatics) to predict chemical toxicity. Furthermore, several agencies have developed robust integrated testing strategies to determine chemical toxicity. The cosmetics sector is pioneering the adoption of alternative methodologies for safety evaluations, and other sectors are aiming to completely abandon animal testing by 2035. However, beyond the use of in vitro genetic testing, agencies regulating the food industry have been slow to implement alternative methodologies into safety evaluations compared with other sectors; setting health-based guidance values for food ingredients requires data from systemic toxicity, and to date, no standalone validated alternative models to assess systemic toxicity exist. The abovementioned models show promise for assessing systemic toxicity with further research. In this paper, we review the current alternatives and their applicability and limitations in food safety evaluations.

Off to a good start? Review of the predictivity of reactivity methods modelling the molecular initiating event of skin sensitization.

The assessment of skin sensitizing properties of chemicals has moved away from animal methods to new approach methodologies (NAM), guided by qualitative mechanistic understanding operationalized in an adverse outcome pathway (AOP). As with any AOP, the molecular initiating event (MIE) of covalent binding of a chemical to skin proteins is particularly important. This MIE has been modelled by several test methods by measuring the reaction of a test chemical with model peptides in chemico. To better understand the similarities and differences, a data repository with publicly available data for the direct peptide reactivity assay (DPRA), amino acid derivative reactivity assay (ADRA) and kinetic DPRA (kDPRA), as well as the peroxidase peptide reactivity assay (PPRA) was assembled. The repository comprises 260 chemicals with animal and human reference data, data on four relevant physicochemical properties, and between 161 to 242 test chemical results per test method. First, an overview of the experimental conditions of the four test methods was compiled allowing to readily compare them. Second, data analyses demonstrated that the test methods' predictivity was consistently reduced for poorly watersoluble chemicals and that the DPRA and ADRA can be used interchangeably. It also revealed new categorization thresholds for the DPRA and ADRA that are potentially relevant for strategic uses. In summary, a detailed assessment of reactivity test methods is provided, highlighting their potential and limitations. The results presented are intended to stimulate scientific discussion around test methods modelling the MIE of the skin sensitization AOP.

Animal-derived products in science and current alternatives.

More than fifty years after the 3Rs definition and despite the continuous implementation of regulatory measures, animals continue to be widely used in basic research. Their use comprises not only in vivo experiments with animal models, but also the production of a variety of supplements and products of animal origin for cell and tissue culture, cell-based assays, and therapeutics. The animal-derived products most used in basic research are fetal bovine serum (FBS), extracellular matrix proteins such as Matrigel™, and antibodies. However, their production raises several ethical issues regarding animal welfare. Additionally, their biological origin is associated with a high risk of contamination, resulting, frequently, in poor scientific data for clinical translation. These issues support the search for new animal-free products able to replace FBS, Matrigel™, and antibodies in basic research. In addition, in silico methodologies play an important role in the reduction of animal use in research by refining the data previously to in vitro and in vivo experiments. In this review, we depicted the current available animal-free alternatives in in vitro research.

Evaluation of MTT reducers and strongly colored substances in the Short Time Exposure test method for assessing eye irritation potential.

The Short Time Exposure (STE) test evaluates eye irritation potential using a 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT assays may underpredict results for some substances that directly reduce MTT (i.e., MTT reducers) or interfere with absorbance because of their strong color (i.e., strongly colored substances). Based on previous research, we selected 25 substances as MTT reducers. Of these, 13 were expected to be MTT reducers at 5% dilution (5% MTT reducers) of the STE test condition. These 13 substances were then tested to determine whether the results were interfered from direct MTT reduction. Those 5% MTT reducers that were classified as irritants based on in vivo data were identified as irritants by the STE test. In addition, the low cell viability results at 5% dilution suggested that direct MTT reduction had not occurred. Next, the remaining 5% MTT reducers that were classified as non-irritants based on in vivo data were identified as non-irritants by the STE test. We then examined two strongly colored substances. One was classified as an irritant based on in vivo data and was confirmed as an irritant by the STE test. The other was classified as a non-irritant by the STE test. This was further evaluated using a medium that did not contain MTT; the result indicated that it was a non-irritant correctly. In conclusion, the STE test is useful for evaluating eye irritation potential without the drawback of underprediction for MTT reducers and strongly colored substances.

[New Approach Method for drug discovery and development aimed at regulatory acceptance].

Recently, the importance of safety assessment based on mechanism of action for drug discovery has been emphasized, and international organizations are increasingly requesting safety assessment using non-animal test methods (Hereafter referred to as alternative method) that are not based on animal experiments using human-derived cells or tissues. However, it is clear that the variety of phenomena captured in animal studies cannot be covered by a stand-alone alternative method, as has been developed in the past, and there are some cases that are not intended to assess human toxicity based on comparison with the data of animal experiments. Expectations are therefore growing for the use of the New Approach Method (NAM) in drug discovery. In this article, we summarize the current status of alternative methods for reproductive toxicity testing and the regulatory acceptance of safety assessments by the Microphysiological system (MPS) as examples regarding NAM.

In vitro, ex vivo, and in vivo models for dental pulp regeneration.

Based on the concept of tissue engineering (Cells-Scaffold-Bioactive molecules), regenerative endodontics appeared as a new notion for dental endodontic treatment. Its approaches aim to preserve dental pulp vitality (pulp capping) or to regenerate a vascularized pulp-like tissue inside necrotic root canals by cell homing. To improve the methods of tissue engineering for pulp regeneration, numerous studies using in vitro, ex vivo, and in vivo models have been performed. This review explores the evolution of laboratory models used in such studies and classifies them according to different criteria. It starts from the initial two-dimensional in vitro models that allowed characterization of stem cell behavior, through 3D culture matrices combined with dental tissue and finally arrives at the more challenging ex vivo and in vivo models. The travel which follows the elaboration of such models reveals the difficulty in establishing reproducible laboratory models for dental pulp regeneration. The development of well-established protocols and new laboratory ex vivo and in vivo models in the field of pulp regeneration would lead to consistent results, reduction of animal experimentation, and facilitation of the translation to clinical practice.