Differences in the anatomy and physiology of the human and rat respiratory tracts and impact on toxicological assessments.

Inhalation is a critical route through which substances can exert adverse effects in humans; therefore, it is important to characterize the potential effects that inhaled substances may have on the human respiratory tract by using fit for purpose, reliable, and human relevant testing tools. In regulatory toxicology testing, rats have primarily been used to assess the effects of inhaled substances as they-being mammals-share similarities in structure and function of the respiratory tract with humans. However, questions about inter-species differences impacting the predictability of human effects have surfaced. Disparities in macroscopic anatomy, microscopic anatomy, or physiology, such as breathing mode (e.g., nose-only versus oronasal breathing), airway structure (e.g., complexity of the nasal turbinates), cell types and location within the respiratory tract, and local metabolism may impact inhalation toxicity testing results. This review shows that these key differences describe uncertainty in the use of rat data to predict human effects and supports an opportunity to harness modern toxicology tools and a detailed understanding of the human respiratory tract to develop testing approaches grounded in human biology. Ultimately, as the regulatory purpose is protecting human health, there is a need for testing approaches based on human biology and mechanisms of toxicity.

Use of human predictive patch test (HPPT) data for the classification of skin sensitization hazard and potency.

Since the 1940s, patch tests in healthy volunteers (Human Predictive Patch Tests, HPPTs) have been used to identify chemicals that cause skin sensitization in humans. Recently, we reported the results of a major curation effort to support the development of OECD Guideline 497 on Defined Approaches (DAs) for skin sensitization (OECD in Guideline No. 497: Defined Approaches on Skin Sensitisation, 2021a. https://doi.org/10.1787/b92879a4-en ). In the course of this work, we compiled and published a database of 2277 HPPT results for 1366 unique test substances (Strickland et al. in Arch Toxicol 97:2825-2837, 2023. https://doi.org/10.1007/s00204-023-03530-3 ). Here we report a detailed analysis of the value of HPPT data for classification of chemicals as skin sensitizers under the United Nations' Globally Harmonized System of Classification and Labelling of Chemicals (GHS). As a result, we propose the dose per skin area (DSA) used for classification by the GHS to be replaced by or complemented with a dose descriptor that may better reflect sensitization incidence [e.g., the DSA causing induction of sensitization in one individual (DSA1+) or the DSA leading to an incidence of induction in 5% of the tested individuals (DSA05)]. We also propose standardized concepts and workflows for assessing individual HPPT results, for integrating multiple HPPT results and for using them in concert with Local Lymph Node Assay (LLNA) data in a weight of evidence (WoE) assessment. Overall, our findings show that HPPT results are often not sufficient for deriving unambiguous classifications on their own. However, where they are, the resulting classifications are reliable and reproducible and can be integrated well with those from other skin sensitization data, such as the LLNA.

Defined approaches to classify agrochemical formulations into EPA hazard categories developed using EpiOcularTM reconstructed human corneal epithelium and bovine corneal opacity and permeability assays.

Many sectors have seen complete replacement of the in vivo rabbit eye test with reproducible and relevant in vitro and ex vivo methods to assess the eye corrosion/irritation potential of chemicals. However, the in vivo rabbit eye test remains the standard test used for agrochemical formulations in some countries. Therefore, two defined approaches (DAs) for assessing conventional agrochemical formulations were developed, using the EpiOcularTM Eye Irritation Test (EIT) [Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 492] and the Bovine Corneal Opacity and Permeability (OECD TG 437; BCOP) test with histopathology. Presented here are the results from testing 29 agrochemical formulations, which were evaluated against the United States Environmental Protection Agency's (EPA) pesticide classification system, and assessed using orthogonal validation, rather than direct concordance analysis with the historical in vivo rabbit eye data. Scientific confidence was established by evaluating the methods and testing results using an established framework that considers fitness for purpose, human biological relevance, technical characterisation, data integrity and transparency, and independent review. The in vitro and ex vivo methods used in the DAs were demonstrated to be as or more fit for purpose, reliable and relevant than the in vivo rabbit eye test. Overall, there is high scientific confidence in the use of these DAs for assessing the eye corrosion/irritation potential of agrochemical formulations.

A database of human predictive patch test data for skin sensitization.

Critical to the evaluation of non-animal tests are reference data with which to assess their relevance. Animal data are typically used because they are generally standardized and available. However, when regulatory agencies aim to protect human health, human reference data provide the benefit of not having to account for possible interspecies variability. To support the evaluation of non-animal approaches for skin sensitization assessment, we collected data from 2277 human predictive patch tests (HPPTs), i.e., human repeat insult patch tests and human maximization tests, for skin sensitization from 1555 publications. We recorded protocol elements and positive or negative outcomes, developed a scoring system to evaluate each test for reliability, and calculated traditional and non-traditional dose metrics. We also traced each test result back to its original report to remove duplicates. The resulting database, which contains information for 1366 unique substances, was characterized for physicochemical properties, chemical structure categories, and protein binding mechanisms. This database is publicly available on the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods website and in the Integrated Chemical Environment to serve as a resource for additional evaluation of alternative methods and development of new approach methodologies for skin sensitization assessments.

International regulatory uses of acute systemic toxicity data and integration of new approach methodologies.

Chemical regulatory authorities around the world require systemic toxicity data from acute exposures via the oral, dermal, and inhalation routes for human health risk assessment. To identify opportunities for regulatory uses of non-animal replacements for these tests, we reviewed acute systemic toxicity testing requirements for jurisdictions that participate in the International Cooperation on Alternative Test Methods (ICATM): Brazil, Canada, China, the European Union, Japan, South Korea, Taiwan, and the USA. The chemical sectors included in our review of each jurisdiction were cosmetics, consumer products, industrial chemicals, pharmaceuticals, medical devices, and pesticides. We found acute systemic toxicity data were most often required for hazard assessment, classification, and labeling, and to a lesser extent quantitative risk assessment. Where animal methods were required, animal reduction methods were typically recommended. For many jurisdictions and chemical sectors, non-animal alternatives are not accepted, but several jurisdictions provide guidance to support the use of test waivers to reduce animal use for specific applications. An understanding of international regulatory requirements for acute systemic toxicity testing will inform ICATM's strategy for the development, acceptance, and implementation of non-animal alternatives to assess the health hazards and risks associated with acute toxicity.

Human cell-based in vitro systems to assess respiratory toxicity: a case study using silanes.

Inhalation is a major route by which human exposure to substances can occur. Resources have therefore been dedicated to optimize human-relevant in vitro approaches that can accurately and efficiently predict the toxicity of inhaled chemicals for robust risk assessment and management. In this study-the IN vitro Systems to PredIct REspiratory toxicity Initiative-2 cell-based systems were used to predict the ability of chemicals to cause portal-of-entry effects on the human respiratory tract. A human bronchial epithelial cell line (BEAS-2B) and a reconstructed human tissue model (MucilAir, Epithelix) were exposed to triethoxysilane (TES) and trimethoxysilane (TMS) as vapor (mixed with N2 gas) at the air-liquid interface. Cell viability, cytotoxicity, and secretion of inflammatory markers were assessed in both cell systems and, for MucilAir tissues, morphology, barrier integrity, cilia beating frequency, and recovery after 7 days were also examined. The results show that both cell systems provide valuable information; the BEAS-2B cells were more sensitive in terms of cell viability and inflammatory markers, whereas MucilAir tissues allowed for the assessment of additional cellular effects and time points. As a proof of concept, the data were also used to calculate human equivalent concentrations. As expected, based on chemical properties and existing data, the silanes demonstrated toxicity in both systems with TMS being generally more toxic than TES. Overall, the results demonstrate that these in vitro test systems can provide valuable information relevant to predicting the likelihood of toxicity following inhalation exposure to chemicals in humans.

Replacement of in vivo leptospirosis vaccine potency testing in the United States.

The U.S. Department of Agriculture (USDA) regulates the potency testing of leptospirosis vaccines, which are administered to animals to protect against infection by Leptospira bacteria. Despite the long-term availability of in vitro test methods for assessing batch potency, the use of hamsters in lethal in vivo batch potency testing persists to varying degrees across leptospirosis vaccine manufacturers. For all manufacturers of these products, data collected from public USDA records show an estimated 40% decline in the annual use of hamsters from 2014 to 2020, with an estimated 55% decrease in the number of hamsters expected to have been used in leptospirosis vaccine potency tests (i.e., those in USDA Category E). An estimated 49,000 hamsters were used in 2020, with about 15,000 hamsters in Category E specifically. Based on this assessment, additional efforts are needed to fully implement in vitro batch potency testing as a replacement for the in vivo batch potency test. We propose steps that can be taken collaboratively by the USDA Center for Veterinary Biologics (CVB), manufacturers of leptospirosis vaccines, government agencies, and non-governmental organizations to accelerate broader use of the in vitro approach.

Mixtures-Inclusive In Silico Models of Ocular Toxicity Based on United States and International Hazard Categories.

Computational modeling grounded in reliable experimental data can help design effective non-animal approaches to predict the eye irritation and corrosion potential of chemicals. The National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) has compiled and curated a database of in vivo eye irritation studies from the scientific literature and from stakeholder-provided data. The database contains 810 annotated records of 593 unique substances, including mixtures, categorized according to UN GHS and US EPA hazard classifications. This study reports a set of in silico models to predict EPA and GHS hazard classifications for chemicals and mixtures, accounting for purity by setting thresholds of 100% and 10% concentration. We used two approaches to predict classification of mixtures: conventional and mixture-based. Conventional models evaluated substances based on the chemical structure of its major component. These models achieved balanced accuracy in the range of 68-80% and 87-96% for the 100% and 10% test concentration thresholds, respectively. Mixture-based models, which accounted for all known components in the substance by weighted feature averaging, showed similar or slightly higher accuracy of 72-79% and 89-94% for the respective thresholds. We also noted a strong trend between the pH feature metric calculated for each substance and its activity. Across all the models, the calculated pH of inactive substances was within one log10 unit of neutral pH, on average, while for active substances, pH varied from neutral by at least 2 log10 units. This pH dependency is especially important for complex mixtures. Additional evaluation on an external test set of 673 substances obtained from ECHA dossiers achieved balanced accuracies of 64-71%, which suggests that these models can be useful in screening compounds for ocular irritation potential. Negative predictive value was particularly high and indicates the potential application of these models in a bottom-up approach to identify nonirritant substances.

IVIVE: Facilitating the Use of In Vitro Toxicity Data in Risk Assessment and Decision Making.

During the past few decades, the science of toxicology has been undergoing a transformation from observational to predictive science. New approach methodologies (NAMs), including in vitro assays, in silico models, read-across, and in vitro to in vivo extrapolation (IVIVE), are being developed to reduce, refine, or replace whole animal testing, encouraging the judicious use of time and resources. Some of these methods have advanced past the exploratory research stage and are beginning to gain acceptance for the risk assessment of chemicals. A review of the recent literature reveals a burst of IVIVE publications over the past decade. In this review, we propose operational definitions for IVIVE, present literature examples for several common toxicity endpoints, and highlight their implications in decision-making processes across various federal agencies, as well as international organizations, including those in the European Union (EU). The current challenges and future needs are also summarized for IVIVE. In addition to refining and reducing the number of animals in traditional toxicity testing protocols and being used for prioritizing chemical testing, the goal to use IVIVE to facilitate the replacement of animal models can be achieved through their continued evolution and development, including a strategic plan to qualify IVIVE methods for regulatory acceptance.

Application of Defined Approaches for Skin Sensitization to Agrochemical Products.

Skin sensitization testing is a regulatory requirement for safety evaluations of pesticides in multiple countries. Globally harmonized test guidelines that include in chemico and in vitro methods reduce animal use, but no single assay is recommended as a complete replacement for animal tests. Defined approaches (DAs) that integrate data from multiple non-animal methods are accepted; however, the methods that comprise them have been evaluated using monoconstituent substances rather than mixtures or formulations. To address this data gap, we tested 27 agrochemical formulations in the direct peptide reactivity assay (DPRA), the KeratinoSens™ assay, and the human cell line activation test (h-CLAT). These data were used as inputs to evaluate three DAs for hazard classification of skin sensitization potential and two DAs for potency categorization. When compared to historical animal results, balanced accuracy for the DAs for predicting in vivo skin sensitization hazard (i.e., sensitizer vs. nonsensitizer) ranged from 56 to 78%. The best performing DA was the "2 out of 3 (2o3)" DA, in which the hazard classification was based on two concordant results from the DPRA, KeratinoSens, or h-CLAT. The KE 3/1 sequential testing strategy (STS), which uses h-CLAT and DPRA results, and the integrated testing strategy (ITSv2), which uses h-CLAT, DPRA, and an in silico hazard prediction from OECD QSAR Toolbox, had balanced accuracies of 56-57% for hazard classification. Of the individual test methods, KeratinoSens had the best performance for predicting in vivo hazard outcomes. Its balanced accuracy of 81% was similar to that of the 2o3 DA (78%). For predicting potency categories defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (GHS), the correct classification rate of the STS was 52% and that of the ITSv2 was 43%. These results demonstrate that non-animal test methods have utility for evaluating the skin sensitization potential of agrochemical formulations as compared to animal reference data. While additional data generation is needed, testing strategies such as DAs anchored to human biology and mechanistic information provide a promising approach for agrochemical formulation testing.

U.S. Federal Agency interests and key considerations for new approach methodologies for nanomaterials.

Engineered nanomaterials (ENMs) come in a wide array of shapes, sizes, surface coatings, and compositions, and often possess novel or enhanced properties compared to larger sized particles of the same elemental composition. To ensure the safe commercialization of products containing ENMs, it is important to thoroughly understand their potential risks. Given that ENMs can be created in an almost infinite number of variations, it is not feasible to conduct in vivo testing on each type of ENM. Instead, new approach methodologies (NAMs) such as in vitro or in chemico test methods may be needed, given their capacity for higher throughput testing, lower cost, and ability to provide information on toxicological mechanisms. However, the different behaviors of ENMs compared to dissolved chemicals may challenge safety testing of ENMs using NAMs. In this study, member agencies within the Interagency Coordinating Committee on the Validation of Alternative Methods were queried about what types of ENMs are of agency interest and whether there is agency-specific guidance for ENM toxicity testing. To support the ability of NAMs to provide robust results in ENM testing, two key issues in the usage of NAMs, namely dosimetry and interference/bias controls, are thoroughly discussed.

Guidance document on Good Cell and Tissue Culture Practice 2.0 (GCCP 2.0).

Good Cell and Tissue Culture Practice (GCCP) 2.0 is an updated guidance document from GCCP 1.0 (published by ECVAM in 2005), which was developed for practical use in the laboratory to assure the reproducibility of in vitro (cell-based) work. The update in the guidance was essential as cell models have advanced dramatically to more complex culture systems and need more comprehensive quality management to ensure reproducibility and high-quality scientific data. This document describes six main principles to consider when performing cell culture including characterization and maintenance of essential characteristics, quality management, documentation and reporting, safety, education and training, and ethics. The document does not intend to impose detailed procedures but to describe potential quality issues. It is foreseen that the document will require further updates as the science and technologies evolve over time.

Application of Defined Approaches to Assess Skin Sensitization Potency of Isothiazolinone Compounds.

Introduction: Isothiazolinones (ITs) are widely used as antimicrobial preservatives in cosmetics and as additives for preservation of consumer and industrial products to control bacteria, fungi, and algae. Although they are effective biocides, they have the potential to produce skin irritation and sensitization, which poses a human health hazard. In this project, we evaluated nonanimal defined approaches (DAs) for skin sensitization that can provide point-of-departure estimates for use in quantitative risk assessment for ITs. Materials and Methods: The skin sensitization potential of six ITs was evaluated using three internationally harmonized nonanimal test methods: the direct peptide reactivity assay, KeratinoSens™, and the human cell line activation test. Results from these test methods were then applied to two versions of the Shiseido Artificial Neural Network DA. Results: Sensitization hazard or potency predictions were compared with those of the in vivo murine local lymph node assay (LLNA). The nonanimal methods produced skin sensitization hazard and potency classifications concordant with those of the LLNA. EC3 values (the estimated concentration needed to produce a stimulation index of three, the threshold positive response) generated by the DAs had less variability than LLNA EC3 values, and confidence limits from the DAs overlapped those of the LLNA EC3 for most substances. Conclusion: The application of in silico models to in chemico and in vitro skin sensitization data is a promising data integration procedure for DAs to support hazard and potency classification and quantitative risk assessment.

Human-relevant approaches to assess eye corrosion/irritation potential of agrochemical formulations.

There are multiple in vitro and ex vivo eye irritation and corrosion test methods that are available as internationally harmonized test guidelines for regulatory use. Despite their demonstrated usefulness to a broad range of substances through inter-laboratory validation studies, they have not been widely adopted for testing agrochemical formulations due to a lack of concordance with parallel results from the traditional regulatory test method for this endpoint, the rabbit eye test. The inherent variability of the rabbit test, differences in the anatomy of the rabbit and human eyes, and differences in modelling exposures in rabbit eyes relative to human eyes contribute to this lack of concordance. Ultimately, the regulatory purpose for these tests is protection of human health, and, thus, there is a need for a testing approach based on human biology. This paper reviews the available in vivo, in vitro and ex vivo test methods with respect to their relevance to human ocular anatomy, anticipated exposure scenarios, and the mechanisms of eye irritation/corrosion in humans. Each of the in vitro and ex vivo methods described is generally appropriate for identifying non-irritants. To discriminate among eye irritants, the human three-dimensional epithelial and full thickness corneal models provide the most detailed information about the severity of irritation. Consideration of the mechanisms of eye irritation, and the strengths and limitations of the in vivo, in vitro and ex vivo test methods, show that the in vitro/ex vivo methods are as or more reflective of human biology and less variable than the currently used rabbit approach. Suggestions are made for further optimizing the most promising methods to distinguish between severe (corrosive), moderate, mild and non-irritants and provide information about the reversibility of effects. Also considered is the utility of including additional information (e.g. physical chemical properties), consistent with the Organization for Economic Cooperation and Development's guidance document on an integrated approach to testing and assessment of potential eye irritation. Combining structural and functional information about a test substance with test results from human-relevant methods will ensure the best protection of humans following accidental eye exposure to agrochemicals.

Retrospective analysis of dermal absorption triple pack data.

Dermal toxicity is driven by the ability of a substance to penetrate the skin. The "triple pack" approach, which combines in vivo rat, in vitro rat, and in vitro human data, is used to calculate an estimated human dermal absorption factor (DAF). To assess the feasibility of deriving a DAF using only in vitro data, we retrospectively evaluated agrochemical formulations to compare the DAF derived from each individual method to the DAF generated from the triple pack approach. For most of the formulations evaluated, the in vitro rat method generated a similar or higher DAF value than the in vivo method. Absorption through in vitro human skin was similar to or less than that observed in rat skin for all formulations. For most of the formulations, the human in vitro method provided a similar or higher estimate of dermal absorption than the triple pack approach. For human health risk assessment, in vitro assays using human skin would be preferable, as they would be directly relevant to the species of interest and avoid overestimation of dermal absorption using rat models. However, rat in vitro studies would still have utility in the absence of human in vitro data. In vitro rat data provide estimates of dermal absorption that are at least as protective as in vivo rat data and thus could also be considered adequate for use in estab­lishing DAFs. The comparisons presented support potentially using in vitro data alone for DAF derivation for human health risk assessment of pesticides.

Conserved Role of the Large Conductance Calcium-Activated Potassium Channel, KCa1.1, in Sinus Node Function and Arrhythmia Risk.

BACKGROUND: KCNMA1 encodes the α-subunit of the large-conductance Ca2+-activated K+ channel, KCa1.1, and lies within a linkage interval for atrial fibrillation (AF). Insights into the cardiac functions of KCa1.1 are limited, and KCNMA1 has not been investigated as an AF candidate gene. METHODS: The KCNMA1 gene was sequenced in 118 patients with familial AF. The role of KCa1.1 in normal cardiac structure and function was evaluated in humans, mice, zebrafish, and fly. A novel KCNMA1 variant was functionally characterized. RESULTS: A complex KCNMA1 variant was identified in 1 kindred with AF. To evaluate potential disease mechanisms, we first evaluated the distribution of KCa1.1 in normal hearts using immunostaining and immunogold electron microscopy. KCa1.1 was seen throughout the atria and ventricles in humans and mice, with strong expression in the sinus node. In an ex vivo murine sinoatrial node preparation, addition of the KCa1.1 antagonist, paxilline, blunted the increase in beating rate induced by adrenergic receptor stimulation. Knockdown of the KCa1.1 ortholog, kcnma1b, in zebrafish embryos resulted in sinus bradycardia with dilatation and reduced contraction of the atrium and ventricle. Genetic inactivation of the Drosophila KCa1.1 ortholog, slo, systemically or in adult stages, also slowed the heartbeat and produced fibrillatory cardiac contractions. Electrophysiological characterization of slo-deficient flies revealed bursts of action potentials, reflecting increased events of fibrillatory arrhythmias. Flies with cardiac-specific overexpression of the human KCNMA1 mutant also showed increased heart period and bursts of action potentials, similar to the KCa1.1 loss-of-function models. CONCLUSIONS: Our data point to a highly conserved role of KCa1.1 in sinus node function in humans, mice, zebrafish, and fly and suggest that KCa1.1 loss of function may predispose to AF.

Mind the gaps: Prioritizing activities to meet regulatory needs for acute systemic lethality

Efforts are underway to develop and implement nonanimal approaches which can characterize acute systemic lethality. A workshop was held in October 2019 to discuss developments in the prediction of acute oral lethality for chemicals and mixtures, as well as progress and needs in the understanding and modeling of mechanisms of acute lethality. During the workshop, each speaker led the group through a series of charge questions to determine clear next steps to progress the aims of the workshop. Participants concluded that a variety of approaches will be needed and should be applied in a tiered fashion. Non-testing approaches, including waiving tests, computational models for single chemicals, and calculating the acute lethality of mixtures based on the LD50 values of mixture components, could be used for some assessments now, especially in the very toxic or non-toxic classification ranges. Agencies can develop policies indicating contexts under which mathematical approaches for mixtures assessment are acceptable; to expand applicability, poorly predicted mixtures should be examined to understand discrepancies and adapt the approach. Transparency and an understanding of the variability of in vivo approaches are crucial to facilitate regulatory application of new approaches. In a replacement strategy, mechanistically based in vitro or in silico models will be needed to support non-testing approaches especially for highly acutely toxic chemicals. The workshop discussed approaches that can be used in the immediate or near term for some applications and identified remaining actions needed to implement approaches to fully replace the use of animals for acute systemic toxicity testing.