Hybrid non-animal modeling: A mechanistic approach to predict chemical hepatotoxicity.

Developing mechanistic non-animal testing methods based on the adverse outcome pathway (AOP) framework must incorporate molecular and cellular key events associated with target toxicity. Using data from an in vitro assay and chemical structures, we aimed to create a hybrid model to predict hepatotoxicants. We first curated a reference dataset of 869 compounds for hepatotoxicity modeling. Then, we profiled them against PubChem for existing in vitro toxicity data. Of the 2560 resulting assays, we selected the mitochondrial membrane potential (MMP) assay, a high-throughput screening (HTS) tool that can test chemical disruptors for mitochondrial function. Machine learning was applied to develop quantitative structure-activity relationship (QSAR) models with 2536 compounds tested in the MMP assay for screening new compounds. The MMP assay results, including QSAR model outputs, yielded hepatotoxicity predictions for reference set compounds with a Correct Classification Ratio (CCR) of 0.59. The predictivity improved by including 37 structural alerts (CCR = 0.8). We validated our model by testing 37 reference set compounds in human HepG2 hepatoma cells, and reliably predicting them for hepatotoxicity (CCR = 0.79). This study introduces a novel AOP modeling strategy that combines public HTS data, computational modeling, and experimental testing to predict chemical hepatotoxicity.

A Novel Ex Vivo Blinking Method for Comparing the Corneal Residence Time of New Shampoo Formulations.

In the cosmetics sector, many products such as shampoos have a probability of accidental ocular exposure during their routine use. One very specific safety parameter is the residence time of the substance on the corneal surface, as prolonged exposure may cause injury. In this study, we developed a system that simulates corneal exposure to blinking and tear flow, for comparing the corneal clearance times of viscous detergent formulations. The Ex Vivo Eye Irritation Test (EVEIT), which uses corneal explants from discarded rabbit eyes from an abattoir, was used as the basis for the new system. To simulate blinking, we developed a silicone wiping membrane to regularly move across the corneal surface, under conditions of constant addition and aspiration of fluid, to mimic tear flow. Six shampoo formulations were tested and were shown to differ widely in their corneal clearance time. Three groups could be identified according to the observed clearance times (fast, intermediate and slow); the reference shampoo had the shortest clearance time of all tested formulations. With this new system, it is now possible to investigate an important physicochemical parameter, i.e. corneal clearance time, for the consideration of ocular safety during the development of novel cosmetic formulations.

KoCVAM-led development of phototoxicity alternative test method using reconstructed human epidermis model (KeraSkin™).

Phototoxicity is an acute toxic reaction induced by topical skin exposure to photoreactive chemicals followed by exposure to environmental light and thus chemicals that absorb UV are recommended to be evaluated for phototoxic potential. There are currently three internationally harmonized alternative test methods for phototoxicity. One of them is the in vitro Phototoxicity: RhE Phototoxicity test method (OECD TG498). Korean center for the Validation of Alternative Methods (KoCVAM) developed an in vitro phototoxicity test method using a KeraSkin™ reconstructed human epidermis model (KeraSkin™ Phototoxicity Assay) as a 'me-too' test method of OECD TG498. For the development and optimization of KeraSkin™ Phototoxicity Assay, the following test chemicals were used: 6 proficiency chemicals in OECD TG498 (3 phototoxic and 3 non-phototoxic), 6 reference chemicals in OECD Performance Standard No. 356 (excluding the proficiency test chemicals, 3 phototoxic and 3 non-phototoxic) and 13 additional chemicals (7 phototoxic and 6 non-phototoxic). Based on the test results generated from the test chemicals above, the overall predictive capacity of KeraSkin™ Phototoxicity Assay was calculated. In particular, the assay exhibited 100 % accuracy, 100 % sensitivity, and 100 % specificity. Therefore, it fulfills the requirements to be included as a 'me-too' test method in OECD TG498.

An in vitro assay for toxicity testing of Clostridium perfringens type C ß-toxin.

Introduction: Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the ß-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the ß-toxin. Methods: Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for ß-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. Results: The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the ß-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Conclusions: Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

The Accomplishments of KoCVAM in the Development and Implementation of Alternative Methods in Korea.

The Korean Center for the Validation of Alternative Methods (KoCVAM), which promotes the Three Rs principles and the use of alternative methods in Korea, has been operating within the Toxicological Screening and Testing Division of the Ministry of Food and Drug Safety (MFDS) since 2009. KoCVAM has exchanged opinions and information on the development and validation of non-animal alternative test methods as part of the International Cooperation on Alternative Test Methods (ICATM), and provided input into draft OECD Test Guidelines (TGs). Several Korean laws (e.g. the Cosmetics Act) encourage the use of alternative test methods for chemical testing and assessment. To promote and support the use of alternative test methods in the country, KoCVAM has published information and provided training on the national guidelines, which are based on the OECD TGs. In addition, KoCVAM has held annual training workshops on alternative test methods, to help Korean research institutions (including GLP test facilities) to implement them. In addition, by helping to develop and validate alternative test methods that were adopted in OECD TG 442B, TG 492 and TG 439, KoCVAM has contributed to the enhanced competitiveness of Korean industry on the worldwide stage.

Benchmarking of BMDC assay and related QSAR study for identifying sensitizing chemicals.

The Bone-Marrow derived Dendritic Cell (BMDC) test is a promising assay for identifying sensitizing chemicals based on the 3Rs (Replace, Reduce, Refine) principle. This study expanded the BMDC benchmarking to various in vitro, in chemico, and in silico assays targeting different key events (KE) in the skin sensitization pathway, using common substances datasets. Additionally, a Quantitative Structure-Activity Relationship (QSAR) model was developed to predict the BMDC test outcomes for sensitizing or non-sensitizing chemicals. The modeling workflow involved ISIDA (In Silico Design and Data Analysis) molecular fragment descriptors and the SVM (Support Vector Machine) machine-learning method. The BMDC model's performance was at least comparable to that of all ECVAM-validated models regardless of the KE considered. Compared with other tests targeting KE3, related to dendritic cell activation, BMDC assay was shown to have higher balanced accuracy and sensitivity concerning both the Local Lymph Node Assay (LLNA) and human labels, providing additional evidence for its reliability. The consensus QSAR model exhibits promising results, correlating well with observed sensitization potential. Integrated into a publicly available web service, the BMDC-based QSAR model may serve as a cost-effective and rapid alternative to lab experiments, providing preliminary screening for sensitization potential, compound prioritization, optimization and risk assessment.

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.

Revolutionizing developmental neurotoxicity testing - a journey from animal models to advanced in vitro systems.

Developmental neurotoxicity (DNT) testing has seen enormous progress over the last two decades. Preceding even the publication of the animal-based OECD test guideline for DNT testing in 2007, a series of non-animal technology workshops and conferences (starting in 2005) shaped a community that has delivered a comprehensive battery of in vitro test methods (IVB). Its data interpretation is covered by a very recent OECD test guidance (No. 377). Here, we aim to overview the progress in the field, focusing on the evolution of testing strategies, the role of emerging technologies, and the impact of OECD test guidelines on DNT testing. In particular, this is an example of a targeted development of an animal-free testing approach for one of the most complex hazards of chemicals to human health. These developments started literally from a blank slate, with no proposed alternative methods available. Over two decades, cutting-edge science enabled the design of a testing approach that spares animals and enables throughput for this challenging hazard. While it is evident that the field needs guidance and regulation, the massive economic impact of decreased human cognitive capacity caused by chemical exposure should be prioritized more highly. Beyond this, the claim to fame of DNT in vitro testing is the enormous scientific progress it has brought for understanding the human brain, its development, and how it can be perturbed.

Developmental neurotoxicity (DNT) testing predicts the hazard of exposure to chemicals to human brain development. Comprehensive advanced non-animal testing strategies using cutting-edge technology can now replace animal-based approaches to assess this complex hazard. These strategies can assess large numbers of chemicals more accurately and efficiently than the animal-based approach. Recent OECD test guidance has formalized this battery of in vitro test methods for DNT, marking a pivotal achievement in the field. The shift towards non-animal testing reflects both a commitment to animal welfare and a growing recognition of the economic and public health impacts associated with impaired cognitive function caused by chemical exposures. These innovations ultimately contribute to safer chemical management and better protection of human health, especially during the vulnerable stages of brain development.

Public views of animal testing and alternatives in chemical risk assessment.

Next-Generation Risk Assessment (NGRA) aims to implement New Approach Methodologies (NAMs) into risk assessment and to rely on new in vivo testing in animals only as a last resort. However, various technical and regulatory hurdles impede their regulatory implementation. Assumptions about the public's expectations could act as barriers to the acceptance of NAMs. This study aimed at investigating public views of animal testing and potential alternatives, namely in vitro and in silico testing. An online survey was conducted (N = 965). The results suggest that people make trade-offs, as they experience negative affect regarding in vivo testing, which partly might explain their openness regarding certain alternatives. In vitro tests were attributed the highest ability to determine harmful effects of chemicals for different endpoints, followed by in vivo and in silico tests. Our results further showed that many people accept chemicals to be only tested with alternatives, with highest acceptance for household consumer products, food contact material or building materials and less accepting for medicines and foods. This article addresses potential challenges that might arise from public perceptions and thus, contributes to the bottom-up initiatives to overcome the hurdles to the implementation of NAMs in regulatory risk assessment.

GARD™skin and GARD™potency: A proof-of-concept study investigating applicability domain for agrochemical formulations.

Several New Approach Methodologies (NAMs) for hazard assessment of skin sensitisers have been formally validated. However, data regarding their applicability on certain product classes are limited. The purpose of this project was to provide initial evidence on the applicability domain of GARD™skin and GARD™potency for the product class of agrochemical formulations. For this proof of concept, 30 liquid and 12 solid agrochemical formulations were tested in GARDskin for hazard predictions. Formulations predicted as sensitisers were further evaluated in the GARDpotency assay to determine GHS skin sensitisation category. The selected formulations were of product types, efficacy groups and sensitisation hazard classes representative of the industry's products. The performance of GARDskin was estimated by comparing results to existing in vivo animal data. The overall accuracy, sensitivity, and specificity were 76.2% (32/42), 85.0% (17/20), and 68.2% (15/22), respectively, with the predictivity for liquid formulations being slightly higher compared to the solid formulations. GARDpotency correctly subcategorized 14 out of the 17 correctly predicted sensitisers. Lack of concordance was justifiable by compositional or borderline response analysis. In conclusion, GARDskin and GARDpotency showed satisfactory performance in this initial proof-of-concept study, which supports consideration of agrochemical formulations being within the applicability domain of the test methods.

Rodent tests for psychiatric drugs get a rethink.

Under pressure to ditch a controversial swim test for depression, scientists look for more precise measures.

Advancing ocular safety research: A comprehensive examination of benzocaine acute exposure without animal testing.

Benzocaine is a widely employed local anaesthetic; however, there is a notable dearth of preclinical and clinical evidence regarding its safety in ophthalmological products. To address this, a comprehensive strategy incorporating in silico and in vitro methodologies was proposed for assessing benzocaine's ocular toxicity without animal testing. To collect the in silico evidence, the QSAR Toolbox (v4.5) was used. A single exposure to two benzocaine concentrations (2% and 20%) was evaluated by in vitro methods. Hen's Egg Chorioallantoic Membrane Test (HET-CAM) was performed to evaluate the effects on the conjunctiva. To study corneal integrity, Short Time Exposure test (STE) and Bovine Corneal Opacity and Permeability (BCOP) assay, followed by histopathological analysis, were carried out. Results from both in silico and in vitro methodologies categorize benzocaine as non-irritating. The histopathological analysis further affirms the safety of using benzocaine in eye drops, as no alterations were observed in evaluated corneal strata. This research proposes a useful combined strategy to provide evidence on the safety of local anaesthetics and particularly show that 2% and 20% benzocaine solutions do not induce eye irritation or corneal damage, supporting the potential use of benzocaine in the development of ophthalmic anesthetic products.

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.

Ph. Eur. testing for histamine and depressor substances using guinea-pigs and cats: the end of an era. Strategy for removal of animal tests for histamine and depressor substances and their vestiges from the Ph. Eur.

For more than 50 years, in vivo assays have been used for testing pharmaceutical product safety due to their assumed ability to broadly detect potential unidentified contaminants. As part of these in vivo tests, the animal tests for depressor substances and histamine have been described in the European Pharmacopoeia since its first edition in 1977. Both tests measure the effect of histamine and histamine-like substances, using guinea-pigs and cats respectively. In 2024, the Histamine (2.6.10) general chapter is referenced in the Production section of four monographs and 10 monographs have variations of a sentence on designing the manufacturing process to eliminate or minimise substances lowering blood pressure in this same section, without referencing the chapter. The Depressor substances (2.6.11) chapter is referenced only in the Histamine (2.6.10) chapter as a next step if the histamine test is invalid. A historical search was performed and it has shown that the tests for histamine and for depressor substances were introduced by different groups of experts in an inconsistent way at different times, and for different reasons, leading to non-harmonised approaches across monographs. The control of histamine and other depressor substances has been the subject of numerous debates where their use was questioned. During these discussions, reports on positive cases or batches failing the test for histamine or depressor substances were anecdotal. In addition, in vivo tests can be considered non-specific, very variable, time-consuming, costly and ethically doubtful. More importantly, the majority of in vivo methods originate from a time when good manufacturing practice was not widely used and formal method validation requirements were not yet established. In view of the above, the removal of all references to animal tests for histamine or depressor substances from all texts still referring to them is proposed. Since the sentences in the Production section referring to the control of "substances lowering blood pressure", "vasoactive substances" or "hypotensive substances" appeared as remainders of the animal test without further guarantee of safety, it will also be proposed to remove all these sentences from the concerned monographs. Ultimately, the suppression of general chapters 2.6.10 and 2.6.11 from the Ph. Eur. is envisaged. Independently from the above, it is also envisaged to elaborate a new general chapter Histamine in active substances (2.5.47) to include physicochemical or immunochemical methods enabling the detection of histamine. This new text would aim at supporting manufacturers in their histamine control strategy following the inclusion of precaution statements in the general monograph on Products of fermentation (1468); these statements had been added in Ph. Eur. Supplements 9.6 and 10.4, following adverse events related to a GMP issue with gentamicin sulfate. This strategy has been endorsed by the European Pharmacopoeia Commission at its 177th Session in November 2023. The concerned monographs would be a subject of public consultation in Pharmeuropa 36.2 (April 2024).

Phase-out planning for animal experimentation.

Since the late 2010s, the idea of phase-out planning for animal experimentation (PPAE) has come to the foreground of political debates, but central notions and arguments are understood differently by different participants and stand in need of clarification. This article draws on public communications on ten political projects related to PPAE to propose a philosophical explication of PPAE and to articulate proponents' central moral argument. According to the argument, the phase-out of animal experimentation is morally desirable and planned interventions are both necessary and sufficient to achieve it. The normative and descriptive premises of the argument are stated and discussed, flagging questions that need answering for a more thorough assessment of the argument. This results in a series of seven action points for researchers and stakeholders of phase-out planning for animal experimentation. The overall goal is to enable an open and productive discussion about PPAE in public, political, and academic settings.

In recent years, a new demand has entered the political arena: that the phase-out of animal experimentation should be planned. But it is important to understand exactly what this means. This article draws on ten documents from governments, parliaments, and NGOs to tease out what they mean by "planning the phase-out of animal experimentation." It also discusses the main argument that is advanced in favor of phase-out planning and highlights seven gaps in our knowledge that we should try to fill in order to move the discussion forward. In sum, the article is the first to explicitly define phase-out planning for animal experimentation and to directly discuss its pros and cons from a philosophical point of view. This is helpful in avoiding misunderstandings and talking past each other, enabling an open and productive debate.

A novel three-dimensional Nrf2 reporter epidermis model for skin sensitization assessment.

Skin sensitization assessment has progressed from the use of animal models towards the application of New Approach Methodologies (NAMs). Several skin sensitization NAMs are accepted for regulatory use, but a majority relies on submerged in vitro cell cultures that limit their applicability domain, posing challenges for testing hydrophobic chemicals and mixtures. A newly developed three-dimensional (3D) Nrf2 reporter epidermis model for skin sensitization assessment is reported. This NAM may help to overcome these limitations. The NAM combines the in vivo-like biology and exposure conditions of 3D epidermis models with the reliability, convenience, and cost-effectiveness of secreted reporter gene technology. The Keap1-Nrf2-ARE pathway was chosen as the reporter gene read-out, as it is induced by most skin sensitizers and already adopted in OECD Test guideline 442D. Immortalized human primary keratinocytes (Ker-CT) were stably transfected with the pIGB-Nrf2-SEAP vector to construct a Nrf2 reporter cell line. Ker-CT Nrf2 reporter cells showed negligible basal expression of the Secreted Embryonic Alkaline Phosphatase (SEAP) reporter, which was induced 13.5-fold by exposure to the skin sensitizer cinnamic aldehyde (CA). Co-exposure to CA and the Nrf2 inhibitor glucocorticoid clobetasol propionate significantly suppressed the CA-induced SEAP expression, confirming dependance of the SEAP expression on Nrf2 activation. Using air-liquid interface and animal constituent free culture conditions, the Ker-CT Nrf2 reporter cells differentiated to stratified 3D epidermis models with an in vivo-like skin architecture and functional skin barrier. Evaluation of a Ker-CT Nrf2 reporter cell-based 2D assay by testing 10 conventional reference chemicals showed a predictive accuracy for skin sensitization potential of 80% and 70% compared to LLNA and human data in two independent laboratories and a high intra- and interlaboratory reproducibility. Moreover, the 3D epidermis models predicted 3 sensitizing and 2 non-sensitizing reference chemicals correctly in a first proof-of-concept study. Further investigations foresee the testing of additional chemicals, including hydrophobic compounds and mixtures to confirm the potential of the 3D epidermis models to broaden the applicability domain for NAM-based skin sensitization assessment.

The skin sensitization adverse outcome pathway: exploring the role of mechanistic understanding for higher tier risk assessment.

For over a decade, the skin sensitization Adverse Outcome Pathway (AOP) has served as a useful framework for development of novel in chemico and in vitro assays for use in skin sensitization hazard and risk assessment. Since its establishment, the AOP framework further fueled the existing efforts in new assay development and stimulated a plethora of activities with particular focus on validation, reproducibility and interpretation of individual assays and combination of assay outputs for use in hazard/risk assessment. In parallel, research efforts have also accelerated in pace, providing new molecular and dynamic insight into key events leading to sensitization. In light of novel hypotheses emerging from over a decade of focused research effort, mechanistic evidence relating to the key events in the skin sensitization AOP may complement the tools currently used in risk assessment. We reviewed the recent advances unraveling the complexity of molecular events in sensitization and signpost the most promising avenues for further exploration and development of useful assays.

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.

Exploring rabbit as a nonrodent species for general toxicology studies.

To avoid adverse events in humans, toxicity studies in nonclinical species have been the foundation of safety evaluation in the pharmaceutical industry. However, it is recognized that working with animals in research is a privilege, and conscientious use should always respect the 3Rs: replacement, reduction, and refinement. In the wake of the shortages in routine nonrodent species and considering that nonanimal methods are not yet sufficiently mature, the value of the rabbit as a nonrodent species is worth exploring. Historically used in vaccine, cosmetic, and medical device testing, the rabbit is seldom used today as a second species in pharmaceutical development, except for embryo-fetal development studies, ophthalmic therapeutics, some medical devices and implants, and vaccines. Although several factors affect the decision of species selection, including pharmacological relevance, pharmacokinetics, and ADME considerations, there are no perfect animal models. In this forum article, we bring together experts from veterinary medicine, industry, contract research organizations, and government to explore the pros and cons, residual concerns, and data gaps regarding the use of the rabbit for general toxicity testing.